I. Stereochemical Control of Chiral Assembly and Liquid ...

Syracuse University Syracuse University

SURFACE SURFACE

Chemistry - Dissertations College of Arts and Sciences

5-2013

I. Stereochemical Control of Chiral Assembly and Liquid Crystal I. Stereochemical Control of Chiral Assembly and Liquid Crystal

Phase Formation of Nonamphiphilic Molecules in Water II. Control Phase Formation of Nonamphiphilic Molecules in Water II. Control

Bacterial Quorum Sensing and Quorum Sensing-mediated Bacterial Quorum Sensing and Quorum Sensing-mediated

Activities Using Small Unnatural Molecules Activities Using Small Unnatural Molecules

Sijie Yang Syracuse University

Follow this and additional works at: https://surface.syr.edu/che_etd

Part of the Chemistry Commons

Recommended Citation Recommended Citation Yang, Sijie, "I. Stereochemical Control of Chiral Assembly and Liquid Crystal Phase Formation of Nonamphiphilic Molecules in Water II. Control Bacterial Quorum Sensing and Quorum Sensing-mediated Activities Using Small Unnatural Molecules" (2013). Chemistry - Dissertations. 197. https://surface.syr.edu/che_etd/197

This Dissertation is brought to you for free and open access by the College of Arts and Sciences at SURFACE. It has been accepted for inclusion in Chemistry - Dissertations by an authorized administrator of SURFACE. For more information, please contact [email protected].

https://surface.syr.edu/

https://surface.syr.edu/che_etd

https://surface.syr.edu/cas

https://surface.syr.edu/che_etd?utm_source=surface.syr.edu%2Fche_etd%2F197&utm_medium=PDF&utm_campaign=PDFCoverPages

http://network.bepress.com/hgg/discipline/131?utm_source=surface.syr.edu%2Fche_etd%2F197&utm_medium=PDF&utm_campaign=PDFCoverPages

https://surface.syr.edu/che_etd/197?utm_source=surface.syr.edu%2Fche_etd%2F197&utm_medium=PDF&utm_campaign=PDFCoverPages

mailto:[email protected]

ABSTRACT

The primary goal of this research is to utilize organic synthesis as a tool to prepare small

molecules that find potential application in different areas including colloidal and material science,

biological chemistry, and medicinal chemistry.

Chapter 1 describes the interpretation of the conformation of nonamphiphilic mesogen disodium

cromoglycate (5 DSCG) when it exists as part of an assembly in water. The study of thermodynamic

incompatibility and miscibility suggest that a previously proposed model for the assembly of

5 DSCG may be applicable to nonamphiphilic organic dyes and other mesogens.

Chapter 2 presents a study of stereochemical control on assembly and liquid crystal formation by

nonamphiphilic molecules. Three stereoisomers of a disodium chromonyl carboxylate derivative,

5 DSCG-diviol, were designed and synthesized. The chiral stereoisomers formed chiral nematic

liquid crystals while the achiral counterpart did not form any kind of liquid crystals. The hydrated

assemblies of chiral 5 DSCG-diviol were able to interact with each other across a 6 nm separation in

aqueous environment and the chirality information was transmitted through achiral medium.

Chapter 3 describes the synthesis and biological studies of a class of bicyclic brominated

furanones. These molecules interacted with quorum sensing in an opportunistic pathogen P.

aeruginosa. Some representative compounds in this class inhibited quorum sensing-controlled

activities such as biofilm formation and virulence factor production, which were key factors in the

pathogenicity of the bacteria. These compounds exhibited significant reduction in the toxicity of

human neuroblastoma SK-N-SH and did not inhibit bacterial growth. Furthermore, one compound,

6-BBF, significantly improved P. aeruginosa clearance in the lungs of mice in an

immunocompromised pneumonia mouse model in vivo.

Chapter 4 reports the synthesis of a library of squarylated homoserine lactones (SHLs) as

analogues to the natural autoinducers N-acyl homoserine lactones (AHL) in Gram-negative bacteria.

These SHLs were shown to have no or minimal impact of the growth of P. aeruginosa and V.

fischeri, but maintain the abilities to modulate quorum sensing and inhibit biofilm formation.

Primary studies of structural activity relationship revealed that the alkyl chain length was critical to

activities of SHLs. These SHLs are promising candidates as modulators of other AHL-mediated QS

systems.

I. Stereochemical Control of Chiral Assembly and Liquid Crystal Phase Formation of

Nonamphiphilic Molecules in Water

II. Control Bacterial Quorum Sensing and Quorum Sensing-mediated Activities Using

Small Unnatural Molecules

by

Sijie Yang

B.S., Wuhan University, Wuhan, China, 2005

M.Phil., Syracuse University, Syracuse 2008

Dissertation

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Chemistry.

Syracuse University

May 2013

v

ACKNOWLEDGEMENT

This dissertation would not have been possible without the guidance and support of many

people.

I would like to express my sincere gratitude to my advisor Professor Yan-Yeung Luk for

providing me an opportunity to work on the diverse, inspiring and challenging projects.

Professor Luk’s enthusiasm for science and drive for novel ideas have been a constant source of

inspiration through my years in his laboratory. I thank him for his continuous support and

valuable guidance during the course of my graduate studies.

I would like to thank all the members of my defense committee, Professors Pepling, Chaiken,

Chisholm, Dabrowiak and Sponsler for finding time out of their very busy schedule to be on my

defense committee. I’m grateful to them for critically reviewing of my PhD dissertation. My

special thanks to Professor Dabrowiak, who generously helped me with comments and

suggestions during my writing of this dissertation. I would also like to thank Professors

Hougland, Korendovych and Loh for generously allowing me to use various equipments in their

laboratories.

Each of my chapters involves extensive collaborations with fellow graduate students,

postdoctoral associates and other personnel. I thank Dr. Debjyoti Bandyopadhyay, Fei Cheng,

Dr. Dawei Cui, Dr. Junjie Han, Dr. Deborah Kerwood, Gauri Shetye, Nisha Varghese, Dr. Bing

Wang, Dr. Guirong Wang, and Dr. Stephen Wilkens. These works wouldn’t have been as fruitful

without their help and I never stopped learning from working and discussing science with them. I

also thank my other colleagues from Luk laboratory, Dr. Deepali Prashar, Dr. Karen Simon,

Nischal Singh and Luke Nye for being great friends in and outside the laboratory. Their company

vi

enabled the smooth progression of research during my graduate study. I have truly enjoyed

working and spending time with them.

I would also like to thank other faculty members who helped me during my graduate study.

These include but not limit to Dr. Nancy Totah, Dr. Christopher Boddy, Dr. Roger Hahn, and Dr.

Daniel Clark. Their guidance and critics during group meetings and problem sessions made me a

better organic chemist.

During my time in Syracuse University, I have made a few wonderful friends, whose

friendship is the treasure of my life. I thank Dr. Laura Bateman, Dr. Troy Lam, Dakin Sharum,

Dr. Alexander Augatis, Michael Robinson, Dr. Dennis Viernes and Lydia Choi, for all their

support, help and laughs. My special thanks to Laura and Troy, whose continuous support and

love helped me through my toughest time in graduate school and my life. Laura is not only a

sister I never had but also a role model that motivates me for my life.

I would also like to thank the wonderful staffs at the Department of Chemistry, Cathy, Jodi,

Deb and Joyce, for all their help during my years at Syracuse University.

My sincere gratitude also goes to my roommates and sisters during these years. To Dr. Nan

Qin, Dr. Yi Shi, and Dr. Bing Deng, for your understanding, encouragement and sharing laugh

and tears. You’ve made my life away from home so colorful.

Finally, I would like to thank my parents Xiaoying Qi and Xianqiao Yang, who planted the

seed for the love of science and knowledge in my heart since I was a kid, and whose

unconditional love and support enable me to reach where I am today.

vii

TABLE OF CONTENTS

Chapter 1 Page

Molecular Conformation and Self-assembly of Nonamphiphilic Liquid Crystals in Water…...- 1 -

1.1 Background and Significance ............................................................................................ - 1 -

1.1.1 Lyotropic, thermotropic, and nonamphiphilic liquid crystals ................................. - 1 -

1.1.2 Different assembly models of nonamphiphilic liquid crystals ................................. - 4 -

1.2 Results and Discussion ...................................................................................................... - 7 -

1.2.1 Possible molecular conformation and assembly formation mechanism of 5 DSCG in

water ........................................................................................................................ - 7 -

1.2.2 Promote the assembly formation of nonamphiphilic mesogens by thermodynamically

incompatible polymers .......................................................................................... - 12 -

1.2.3 Investigate assembly model of organic dye-based nonamphiphilic molecule by

miscibility .............................................................................................................. - 15 -

1.3 Conclusion and Perspectives............................................................................................ - 20 -

1.4 Experimental Section ....................................................................................................... - 20 -

Chapter 2

Stereochemical Control of Nonamphiphilic Liquid Crystals: Effect of Chiral Mesogens and

Dopants on Assemblies Separated by Six Nanometers of Aqueous Solvents.……………….- 23 -

2.1 Background and Significance .......................................................................................... - 23 -

2.1.1 Cholesteric liquid crystal phase ............................................................................ - 23 -

2.1.2 Chiral chromonic liquid crystals ........................................................................... - 24 -

2.2 Results and Discussion .................................................................................................... - 25 -

viii

2.2.1 Design and synthesis of the stereoisomers of 5 DSCG-diviols ............................. - 25 -

2.2.2 Stereochemistry controls the formation of liquid crystals ..................................... - 28 -

2.2.3 Stereoisomers of 5 DSCG-diviols exhibit different polymorphism ....................... - 30 -

2.2.4 Conformations of stereoisomers are different ....................................................... - 31 -

2.2.5 Circular dichroism indicates a chiral conformation ............................................. - 34 -

2.2.6 Cryogenic transmission electron microscopy reveals early-staged and crossed

assemblies by chiral mesogens .............................................................................. - 37 -

2.2.7 Nonuniform alignment formed by 5 DSCG-(R,R)-diviol ...................................... - 43 -

2.2.8 Homeotropic alignment (fingerprint texture) of chiral nematic phase of

nonamphiphilic liquid crystals .............................................................................. - 49 -

2.3 Conclusions and perspectives .......................................................................................... - 65 -

2.4 Experimental Section ....................................................................................................... - 66 -

Chapter 3

Bicyclic Brominated Furanones: A New Class of Quorum Sensing Modulators that Inhibit

Biofilm Formation with Reduced Toxicity and Improved Bacterial Clearance in

vivo….….…………………………………………………………………………………...- 133 -

3.1 Background and Significance ........................................................................................ - 133 -

3.2.1 Quorum sensing and autoinducers ...................................................................... - 133 -

3.2.2 Biofilm formation and consequences .................................................................. - 137 -

3.2.3 Use quorum sensing modulators to control biofilm formation ........................... - 140 -

3.2.4 Other biofilm inhibitors ....................................................................................... - 141 -

3.2.5 Quorum sensing and biofilm inhibitors with furanone moiety ............................ - 142 -

ix

3.2 Results and Discussion .................................................................................................. - 146 -

3.2.1 Synthesis of BBFs ................................................................................................ - 146 -

3.2.2 Inhibition of E.coli biofilm formation by BBFs ................................................... - 147 -

3.2.3 Cytotoxicity study of BBFs on the growth of E. coli ........................................... - 149 -

3.2.4 Inhibition of P. aeruginosa biofilm formation by BBFs ...................................... - 150 -

3.2.5 Cytotoxicity study of BBFs on the growth of P.aeruginosa ................................ - 156 -

3.2.6 Study of synergistic effect between BBFs and tobramycin .................................. - 157 -

3.2.7 Modulation of the quorum sensing in P. aeruginosa by BBFs ............................ - 160 -

3.2.8 Effect on elastase B production in P. aeruginosa by BBFs ................................. - 165 -

3.2.9 Improved bacterial clearance by 6-BBF in vivo ................................................. - 167 -

3.2.10 Cytotoxicity study of BBFs on human cells ......................................................... - 169 -

3.3 Conclusion and Perspectives.......................................................................................... - 173 -

3.4 Experimental Section ..................................................................................................... - 174 -

3.4.1 Biological studies ................................................................................................ - 174 -

3.4.2 Chemical synthesis and characterization ............................................................ - 181 -

Chapter 4

Modulation of Quorum Sensing in Gram-negative Bacteria by Squarylated Homoserine

Lactones...…………………………………………………………………………………...- 192 -

4.1 Background and Significance ........................................................................................ - 192 -

4.1.1 AHL-based quorum sensing in Gram-negative bacteria ..................................... - 192 -

4.1.2 Modulation of LuxI/LuxR-type quorum sensing system ...................................... - 194 -

x

4.1.3 Biofilm inhibitors containing squarate moiety .................................................... - 198 -

4.2 Results and Discussion .................................................................................................. - 201 -

4.2.1 Synthesis of SHLs ................................................................................................ - 201 -

4.2.2 Cytotoxicity study of SHLs on the growth of P. aeruginosa ................................ - 202 -

4.2.3 Modulation of quorum sensing in P. aeruginosa by SHLs .................................. - 203 -

4.2.4 Inhibition of P. aeruginosa biofilm formation by SHLs ...................................... - 206 -

4.2.5 Cytotoxicity study of SHLs on the growth of V. fischeri ...................................... - 207 -

4.2.6 Modulation of quorum sensing in V. fischeri by SHLs ........................................ - 209 -

4.3 Conclusion and Perspectives.......................................................................................... - 210 -

4.4 Experimental Section ..................................................................................................... - 211 -

References ............................................................................................................................... - 248 -

xi

LIST OF FIGURES

Chapter 1 Page

Figure 1.1 Illustrations of the molecular order in liquid, liquid crystal, and crystalline solid state

of matter. ..................................................................................................................................... - 2 -

Figure 1.2 Examples of thermotropic and lyotropic mesogens. ................................................. - 3 -

Figure 1.3 Examples of nonamphiphilic mesogens. ................................................................... - 4 -

Figure 1.4 Stacking models for nonamphiphilic mesogens. ....................................................... - 5 -

Figure 1.5 NMR spectroscopy of 1.20 wt% (~ 24 mM) 5 DSCG in water at 25 oC. (A)

1H NMR

with the linker region enlarged. (B) Partial NOESY spectrum showing the cross peaks between

proton a and proton e/e . ............................................................................................................. - 8 -

Figure 1.6 Possible conformations of 5 DSCG in water at 1.20 wt% (~ 24 mM) at 25 oC. (A)

Staggered. (B) Eclipsed. ............................................................................................................. - 9 -

Figure 1.7 Temperature effect on chemical shifts of protons in 1.20 wt% (~24 mM) 5 DSCG (in

water). NMR of 0.45 wt% (~ 8.8 mM) 5 DSCG in water at 25 oC is shown for comparison. . - 10 -

Figure 1.8 Effect of temperature on the chemical shifts of protons a-d in 5 DSCG (1.20 wt% in

water). ....................................................................................................................................... - 12 -

Figure 1.9 Two possible pathways of the LC formation promoted by addition of polymer. ... - 13 -

Figure 1.10 1H NMR of SSY mixed with ~ 8 wt% of PAAm (10K) in D2O at 25

oC. ............ - 14 -

Figure 1.11 1H NMR of 5 MSCG mixed with ~ 8 wt% of PAAm (10K) in D2O at 25

oC. ..... - 15 -

Figure 1.12: Asymmetric H-stacking model of Sunset Yellow FCF. Aggregations formed by five

SSY molecules are shown. ........................................................................................................ - 16 -

Figure 1.13: Optical images of organic dyes mixed with different concentrations of mono-

charged molecule viewed between crossed polarizers.............................................................. - 18 -

xii

Figure 1.14: Optical images of SY dye mixed with Allura Red AC or Acid Red 13 at different

concentrations viewed between cross polarizers ...................................................................... - 19 -

Chapter 2

Figure 2.1 Schematic representation of the cholesteric liquid crystal phase. ........................... - 24 -

Figure 2.2 The structures of the chiral isomers 5 DSCG-(R,R)-diviol 1a, 5 DSCG-(S,S)-diviol 1b,

and achiral isomer 5 DSCG-meso-diviol 1c. ............................................................................ - 25 -

Figure 2.3 Optical images (cross polarizers) of 12.0 wt % 5 DSCG-(R,R)-diviol 1a (A),

5 DSCG-(S,S)-diviol 1b (B), and 5 DSCG-meso-diviol 1c (C) in water sandwiched between two

glass slides (spacer: 13-15 µm) ................................................................................................. - 29 -

Figure 2.4 Optical images (cross polarizers) of 18.0 wt% 5 DSCG-(R,R)-diviol 1a, 18.1 wt%

5 DSCG-(S,S)-diviol 1b, and 18.0 wt% 5 DSCG-meso-diviol 1c in water when freshly prepared

(A, B, and C, respectively), and when aged at ambient temperature overnight in sealed vials (D,

E, and F, respectively) .............................................................................................................. - 31 -

Figure 2.5 ROESY of 0.503 wt% of 5 DSCG -(S,S)-diviol 1b and 0.503 wt% of 5 DSCG-meso-

diviol 1c in D2O at 25 oC. ......................................................................................................... - 33 -

Figure 2.6 ROESY spectrum of 0.503 wt% 5 DSCG -(S,S)-diviol 1b and 5 DSCG-meso-diviol

1c in D2O at 25 oC. .................................................................................................................... - 34 -

Figure 2.7 UV spectrum of 5 DSCG-(R,R)-diviol 1a (dot line) and CD spectra of 5 DSCG-(R,R)-

diviol 1a (solid line) and 5 DSCG-(S,S)-diviol 1b (dash line) in water (8 × 10-4

M) at 5 oC. . - 36 -

Figure 2.8 Temperature-dependent CD spectra of 5 DSCG-(R,R)-diviol 1a in water (8 × 10-4

M).

................................................................................................................................................... - 36 -

Figure 2.9 1H NMR of 5 DSCG-(S,S)-diviol 1b from 0.02 wt% (0.4 mM) to 3.5 wt% (72 mM) in

water. ......................................................................................................................................... - 37 -

xiii

Figure 2.10 Cryogenic transmission electronic microscopic image of 5.5 wt% (488) 5 DSCG

(inside the curved edge) ............................................................................................................ - 38 -

Figure 2.11 Cryogenic transmission electronic microscopic image of 5.0 wt% (587) 5 DSCG-

(R,R)-diviol 1a (inside the curved edge). .................................................................................. - 39 -


(inside the curved edge) ............................................................................................................ - 39 -


(R,R)-diviol 1a(inside the curved edge). ................................................................................... - 40 -

Figure 2.14 Cryo-TEM images of 5.5 wt% of 5 DSCG, and 5.0 wt% of 5 DSCG-(R,R)-diviol 1a.

................................................................................................................................................... - 41 -

Figure 2.15 Cryogenic transmission electronic microscopic images of 8.2 wt% of 5 DSCG and

8.8 wt% of 5 DSCG-(R,R)-diviol 1a. ........................................................................................ - 42 -

Figure 2.16 (A). Geometry of gold deposition on glass slides at an oblique angle from the surface

normal. (B). Scheme of self-assembly molecules (SAMs) formed by HS(CH2)10(OCH2CH2)3OH.

(C). Schematic representation of uniform alignment of threads of 5 DSCG liquid crystal on

SAMs supported by gold films deposited at 45o from the surface normal of the glass slides.

Optical images (cross polarizers) of (D) 16.8 wt% 5 DSCG, (E) 14.1 wt% 5 DSCG mixed with

14.5 wt% xylitol, (F) 13.4 wt% 5 DSCG mixed with a 16.2 wt% racemic mixture of D- and L-

mannitol, (G) 13.4 wt% 5 DSCG doped with 16.2 wt% D-mannitol, and (H) 18.0 wt% 5 DSCG-

(R,R)-diviol 1a in water sandwiched between obliquely deposited gold films supporting

HS(CH2)10(O CH2CH2)3OH ...................................................................................................... - 45 -

Figure 2.17 Illustrations of the molecular assemblies in (A) homogeneous or (B) homeotropic

alignment of chiral nematic liquid crystal phase on surface. .................................................... - 47 -

xiv

Figure 2.18 Optical images (cross polarizers) of 13.5 wt% 5 DSCG doped with 16.5 wt% D-

mannitol in water sandwiched between obliquely deposited gold films supporting HS(CH2)10(O

CH2CH2)3OH with different thickness of spacers .................................................................... - 47 -

Figure 2.19 (A) Schematic representation of a wedge cell composed of

HS(CH2)10(OCH2CH2)3OH SAMs supported by gold films. Optical images (cross polarizers) of

13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol when the thickness of the cell is (B) 13

µm, (C) 296 µm, (D) 555µm, and (E) 778 µm. Scale bar = 76 µm .......................................... - 51 -

Figure 2.20 Optical images (cross polarizers) of (A) 13.4 wt% 5 DSCG mixed with 16.2 wt% D-

mannitol and (B) 18.0 wt% 5 DSCG-(R,R)-diviol when the samples were sandwiched between

obliquely deposited gold films supporting HS(CH2)10(O CH2CH2)3OH in a 1 mm thick cell. - 52 -

Figure 2.21 Optical images with detailed rotations of 13.4 wt% 5 DSCG mixed with 16.2 wt%

D-mannitol in a wedge cell composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold

films when the thickness of the cell ranges from 13 to 222 µm. .............................................. - 53 -



films when the thickness of the cell ranges from 296 to 778 µm. ............................................ - 54 -


D-mannitol in a wedge cell composed of plain glass slides when the thickness of the cell ranges

from 13 to 778 µm. ................................................................................................................... - 55 -

Figure 2.24 Optical images of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol in a wedge

cell composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold films when the cell

thickness is (A) 555 µm, (B) 593 µm, and (C) 778 µm. ........................................................... - 56 -

xv

Figure 2.25 Optical images (cross polarizers) of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-

mannitol viewed in a wedge cell with (A) 27 mm and (B) 17 mm cell length. Both of the wedge

cells are composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold films and the spacer

is 13 µm on one end and 1mm on the other .............................................................................. - 57 -

Figure 2.26 Schematic representation of splay deformation of liquid crystals in a wedge cell of

(A) small angle and (B) large angle. ......................................................................................... - 58 -

Figure 2.27 Schematic representation of chiral information transmitted through water molecules

if the molecular assemblies (thread model) are separated by pure wate ................................... - 60 -


if the molecular assemblies (H-stacking model) are separated by pure water .......................... - 61 -


with free solvated chiral mesogens between the molecular assemblies (thread model) ........... - 62 -


with free solvated chiral mesogens between the molecular assemblies (H-stacking model). .. - 63 -

Figure 2.31 Schematic representation of chiral information transmitted through chiral mesogens

between the molecular assemblies (thread model). .................................................................. - 64 -


between the molecular assemblies (H-stacking model). ........................................................... - 64 -

Chapter 3

Figure 3.1 Five stages of biofilm development. ..................................................................... - 138 -

Figure 3.2 Examples of brominated furanones isolated form extracts of Delisea pulchra. ... - 143 -

Figure 3.3 Examples of synthetic brominated furanones........................................................ - 144 -

Figure 3.4 Structures of 5-BBF 15, 6-BBF 16 and 7-BBF 17. ............................................... - 146 -

xvi

Figure 3.5 The effect of brominated furanones on biofilm formation by E.coli RP437

(pRSH103). Representative confocal laser scanning microscopy (CLSM) images of biofilm

formed by E.coli RP437 (pRSH103) in the (A) absence and presence of 200 µM (B) 3, (C) 5-

BBF 15, (D) 6-BBF 16 and (E) 7-BBF 17. ............................................................................ - 148 -

Figure 3.6 Quantification of biofilm formation by E.coli RP437 (pRSH103) in the absence and

presence of 200 µM brominated furanones ............................................................................ - 149 -

Figure 3.7 Growth curve of E.coli RP437 in the absence and presence of 200 µM brominated

furanones ................................................................................................................................. - 150 -

Figure 3.8 The effect of brominated furanones on biofilm formation by P. aeruginosa.

Representative confocal laser scan microscopy (CLSM) images of biofilm formed by PAO1-

GFP (expresses green fluorescence on plasmid pSMC2) (A) in the absence of agents, and in the

presence of 400 µM (B) 2, (C) 5-BBF 15, (D) 6-BBF 16, and (E) 7-BBF 17........................ - 152 -

Figure 3.9 Quantification of biofilm formation by PAO1-GFP in the absence and presence of 400

µM brominated furanones ....................................................................................................... - 153 -

Figure 3.10 P. aeruginosa biofilm formation dose-response curves with IC50 values for (A) 5-

BBF 15, (B) 6-BBF 16, and (C) 7-BBF 17 ............................................................................. - 155 -

Figure 3.11 Growth curves of P. aeruginosa PAO1 in the absence (control) and presence of 400

µM brominated furanones. ...................................................................................................... - 157 -

Figure 3.12 Viability of cells within the biofilm treated with and without 6-BBF 16 after

exposure to tobramycin ........................................................................................................... - 159 -

Figure 3.13 GFP expression by PAO-JP2 (plasI-LVAgfp) in the presence of 1 µM 3-oxo-C12-

HSL alone (control) or 1 µM 3-oxo-C12-HSL plus various concentration of 5-BBF 15, 6-BBF

16, 7-BBF 17 or BF 3. ............................................................................................................ - 161 -

xvii

Figure 3.14 GFP expression by PAO1 (plasI-LVAgfp) in the absence or presence of 5-BBF 15,

6-BBF 16, 7-BBF 17 or BF 3 at 50, 150, and 300 µM ........................................................... - 162 -

Figure 3.15 GFP expression by PAO-JP2 (prhlI-LVAgfp) in the presence of 1 µM 3-oxo-C12-

HSL and 10 µM C4-HSL alone (control) or 1 µM 3-oxo-C12-HSL and 10 µM C4-HSL plus

various concentration of 5-BBF 15, 6-BBF 16, 7-BBF 17, or BF 3 ....................................... - 164 -

Figure 3.16 GFP expression by PAO1 (prhlI-LVAgfp) in the absence or presence of 5-BBF 15,

6-BBF 16, 7-BBF 17, or BF 3 at 50, 150, and 300 µM .......................................................... - 165 -

Figure 3.17 Elastase B activity produced by P. aeruginosa PAO-JP2 in the presence of 5 µM 3-

oxo-C12-HSL and 10 µM C4-HSL alone (control) or 5 µM 3-oxo-C12-HSL and 10 µM C4-HSL

plus 300 µM BBFs or 5 µM PAI1 and 10 µM PAI2 plus various concentration of 6-BBF 16

(insert) ..................................................................................................................................... - 167 -

Figure 3.18 Effect of 6-BBF on the bacterial clearance of mouse lung in vivo ...................... - 168 -

Figure 3.19 Survival (%) of human neuroblastoma SK-N-SH cells at 0, 24, and 48 h after 1 h

treatment of 100 µM 5-BBF 15, 6-BBF 16, 7-BBF 17 and BF 3, respectively...................... - 170 -

Figure 3.20 Survival (%) of SK-N-SH when treated with 100 µM 5-BBF 15, 6-BBF 16, 7-BBF

17 and BF 3 for 2, 4, and 6 h .................................................................................................. - 171 -


treatment of 400 µM 5-BBF 15, 6-BBF 16, 7-BBF 17 and BF 3, respectively...................... - 172 -

Chapter 4

Figure 4.1 General chemical structure of N-acyl homoserine lactones .................................. - 193 -

Figure 4.2 Venn diagrams showing the structures of selective most potent LuxR-type protein

antagonists and agonists identified and their selectivities for different LuxR-type proteins from

A. tumefaciens (TraR), V. fischeri (LuxR), and P. aeruginosa (LasR)................................... - 197 -

xviii

Figure 4.3 Chemical structures of squarate-based AHL analogues examined by Narasimhan et al.

on inhibitory activities against biofilm formation by E. coli RP437 ...................................... - 199 -

Figure 4.4 Chemical structures of squarate-based AHL analogues examined by Bandyopadhyay

et al. on inhibitory activities against biofilm formation by E. coli RP437 ............................. - 200 -

Figure 4.5 The library of SHLs used in this study .................................................................. - 201 -


µM SHLs ................................................................................................................................. - 203 -


HSL alone (control) or 150 µM SHLs. ................................................................................... - 205 -


HSL alone (control) or 1 µM 3-oxo-C12-HSL plus different concentrations of SHLs. ......... - 205 -

Figure 4.8 P. aeruginosa biofilm formation in the presence of 300 µM SHLs ...................... - 207 -

Figure 4.9 Growth curves of V. fischeri VCW2G7 in the presence of 3 µM 3-oxo-C6-HSL alone

(control) or 3 µM 3-oxo-C6-HSL plus 10 µM SHLs. ............................................................ - 208 -

Figure 4.10 Luminescence expression by V. fischeri VCW2G7 in the presence of 3 µM 3-oxo-

C6-HSL alone (control) or 3 µM 3-oxo-C6-HSL plus 10 µM SHLs. .................................... - 210 -

xix

LIST OF SCHEMES

Chapter 1 Page

Scheme 1.1………………………………………………………………………………….... - 6 -

Chapter 2

Scheme 2.1…………………………………………………………………………………....- 26 -

Scheme 2.2………………………………………………………………………………........- 27 -

Chapter 3

Scheme 3.1………………………………………………………………………………..…- 146 -

Chapter 4

Scheme 4.1………………………………………………………………………………..…- 201 -

xx

LIST OF TABLES

Chapter 3 Page

Table 3.1…………………………………………………………………………………….- 134 -

- 1 -

Chapter 1

Molecular Conformation and Self-assembly of Nonamphiphilic Liquid Crystals in Water

Summary

NMR spectroscopy was used to interpret the conformation of nonamphiphilic mesogen disodium

cromoglycate (5 DSCG) when it exists as part of an assembly in water. In a 1.2 wt% sample,

5 DSCG forms thread assembly, possibly via an isodesmic mechanism. The validity of the thread

nature of nonamphiphilic organic dyes was also studied. Polyacrylamide promoted assembly

formation by 5 DSCG at concentrations below liquid crystal formation. The same phenomenon was

observed for another nonamphiphilic mesogen Sunset Yellow FCF (SSY) but not for 5 MSCG,

which does not form liquid crystal at any concentration. While 1H NMR alone was not sufficient to

show the potential thread nature of the assembly by SSY, a miscibility test further supported this

model.

1.1 Background and Significance

1.1.1 Lyotropic, thermotropic, and nonamphiphilic liquid crystals

Liquid crystal (LC) is a state of matter which has properties between the conventional liquid

and crystalline solid. Although a liquid crystal may flow like liquids, its molecules do exhibit a

certain degree of orientational order (Figure 1.1). The study of liquid crystals began in 1888

when Friedrich Reinitzer noted that cholesteryl benzoate had two distinct melting points. The

substance changed from a solid to a hazy liquid at the first melting point and then turned into a

clear liquid as the temperature increased further.1

- 2 -

Figure 1.1 Illustrations of the molecular order in liquid, liquid crystal, and crystalline solid state

of matter.

There are two main types of liquid crystals: thermotropic liquid crystals, the order of which is

temperature-dependent, and lyotropic liquid crystals, the order of which is both solvent-

dependent and temperature-dependent. Some of the earliest discovered liquid crystals are

thermotropic. Within a certain temperature range, cooperative ordering of the mesogens

(molecules that form LC) exhibited the LC phase. At too high temperature, the sample becomes

isotropic liquid; at too low temperature, the sample turns into crystalline solids. One such

example is para-azoxyanisole (Figure 1.2). Lyotropic liquid crystals exhibit liquid-crystalline

properties within certain concentration ranges. Typical compounds that form lyotropic liquid

crystals are amphiphilic; they consist of two immiscible hydrophobic and hydrophilic parts

within the same molecule and form micelles in solution above a critical concentration (critical

micelle concentration, or CMC). As the concentration increases further, the assemblies of the

micelles become ordered and lyotropic liquid crystalline phases form. There is a minimum

temperature for a given lyotropic liquid crystal, termed Krafft emperature, below which micelles

Liquid Liquid crystal Crystalline solid

Nematic phase Smectic A phase

- 3 -

will not form and the liquid crystal phase will not occur. Common examples of such molecules

are the salts of fatty acids, many of which are used in soap or detergents (Figure 1.2).

Figure 1.2 Examples of thermotropic and lyotropic mesogens.

Over the past forty years, a new fascinating class of lyotropic liquid crystals has become an

active field of research.2 These molecules structurally differ from the conventional amphiphilic

mesogens of lyotropic liquid crystals in that they are usually rigid, disk-like molecules having

aromatic cores with polar groups at the periphery, instead of flexible, rod-like molecules having

aliphatic chains with ionic groups at one end. They do not possess a critical micelle

concentration and the aggregation takes place at all concentrations, the process of which is

described as isodesmic, meaning that the energy advantage in adding a molecule to an aggregate

is independent of aggregate size. One of the most extensively studied example in this class is

disodium cromoglycate (5 DSCG) (Figure 1.3), an anti-asthmatic drug marketed under the trade

name INTAL in UK and Chromolyn in US.3 This special class of liquid crystals extends to other

drugs,4 dyes,

5-12 and nucleic acids (Figure 1.3),

13,14 and is also termed “chromonic liquid

crystals” (CLCs), not only because of the chromone moiety in 5´DSCG, but also because of the

connotations of both color (for dyes) and chromosomes (for nucleic acids).15

The existence of

nonamphiphilic liquid crystals has been known for some time. In 1915, Sandquist described a

system of 9-bromo-phenanthrene-3-sulfonic acid in aqueous solution that showed

NN

OO

O

O

O

Na

p-azoxyanisole

(thermotropic)

sodium dodecanoate

(lyotropic)

- 4 -

nonamphiphilic liquid crystal properties.16

5 DSCG was initially studied by Woodard and co-

workers in the 1970s17,18

and later investigated more extensively by Lydon and co-workers,4,19,20

and further by others.21-28

Figure 1.3 Examples of nonamphiphilic mesogens.

Nonamphiphilic liquid crystals have potential for a number of new applications, such as

preparation of optically anisotopic films,29

biosensing,30

controlled drug delivery,2

microptatterning,31

and nanofabrication.32

Understanding the assembly formation and the

structure-property relationships is important for designing novel nonamphiphilic mesogens,

controlling formation of nonamphiphilic LC and optimizing their properties.

1.1.2 Different assembly models of nonamphiphilic liquid crystals

Like in the case of many other nonamphiphilic LCs,10,33

there are two mesophases of

5 DSCG.18

The one formed at lower concentration shows optical textures typical for

thermotropic nematic phases and was termed the N phase. The second mesophases formed at

higher concentrations is characteristic of the amphiphile middle (hexagonal) phase and was

termed the M phase. The assembly structures of 5 DSCG in the two mesophases have been

studied by many and are still not known with certainty. The discoverers Hartshorne and

Woodard proposed a simple stacking model -- the planar molecules are arranged with their

O O

O

OO

OO

O

O

O

OHNa Na

NH

N

N

O

NH2N

O

OH

OP

OH

O

HO

OH

N

N

NaO3S

SO3Na

disodium chromoglycate

(5´DSCG)

Sunset Yellow FCF

(SSY)

deoxyguanosine-5´-monophosphate

(dGMP)

- 5 -

planes parallel or approximately parallel for the N phase and the planar discs of molecules

aggregate in cylindrical stacks which pack in a hexagonal lattice for the M phase.18

Later, a new

model was reported without any experimental results, proposing a hollow square columns of

5 DSCG filled with water.19

This paper was retracted by the author after a few years,15

even

though it is still cited in the literature sometimes.

In addition to 5 DSCG, the assembly behavior has been studied for other nonamphiphilic

molecules, including organic dyes, such as Sunset Yellow FCF (SSY), Blue 27, Direct Blue 67,

and Violet 20.10-12,34-37

The original H-stacking model for the nematic phase of these liquid

crystals is a continuous subject for discussion.15,17,18,38

For instance, a J-stacking model was

proposed,7,39-41

in which the molecules stack on an offset position by some fixed distance from

each other, instead of directly on top of each other (“H” type) (Figure 1.4).

Figure 1.4 Stacking models for nonamphiphilic mesogens.

Recently, our group reported a novel model for the assembly of nonamphiphilic molecule

5 DSCG in water, which showed that maintaining a nonamphiphilic molecular structure was

crucial to form a liquid crystal phase. This model consists of threads, instead of columns, of

5 DSCG molecules. Within each thread, the salt bridges formed between two molecules stacked

on another layer of aromatic rings, instead of simply aggregating to form molecular columns via

H-stacking J-stacking

- 6 -

π-π interaction of the aromatic rings (Scheme 1.1). Each thread is solvated with a hydration shell

of water.42

A mesogen mixing experiment was designed to validate the thread model. The

addition of mono-charged molecules attenuated or destroyed the liquid crystals phase of 5 DSCG

by acting as salt bridge terminators rather than column stackers. In contrast, adding divalent-

charged molecules retained the liquid crystal phases of 5 DSCG, suggesting two charged units in

the molecule are necessary to maintain a stable assembly and further demonstrating the thread

model.

Scheme 1.1 Thread model for the assembly of 5 DSCG in water. The drawing was adapted from

literature with modification.42

In another recent study by a previous group memeber, using self-assembled monolayers of

functionalized alkanethiols supported by anisotropic gold films with nanometer-scale

topography, it was demonstrated that uniform alignment of liquid crystals formed by hydrated

5 DSCG and Sunset Yellow molecules over a large surface area.43

This uniform alignment of

liquid crystals cannot be interpreted by the stacking assembly, but rather, by the thread assembly.

Hydration shell

O O

OO

O O

OH

O

O

O

O

Na Na

Salt bridges stacked on aromatic rings

+-+ - +-+ -

… …+-+ - +-+ -

-- --

… …+-+ - +-+ -

-- --+ ++ + ++

- 7 -

While oil-in-water emulsions (hydrophobic-hydrophilic separations) explain many

phenomena in conventional colloidal chemistry and novel fabrication methods,44-47

entirely water

soluble nonamphiphilic molecules can also exhibit phase separations in water. Water-in-water

emulsions are usually generated by mixing water-soluble and structurally different polymers, but

it was also revealed that some small nonamphiphilic molecules, such as 5 DSCG, can exhibit

phase separation with a non-ionic polymer when mixed in water.48-50

Later on, our group

demonstrated how mixing thermodynamically incompatible molecules, could promote non-

covalent polymerization and liquid crystal formation, as long as the assembly formed by

nonamphiphilic molecules was isodesmic in nature.51

1.2 Results and Discussion

1.2.1 Possible molecular conformation and assembly formation mechanism of 5 DSCG in water

We first used 1D and 2D NMR spectroscopy to investigate the conformation of 5 DSCG in

water. We noticed a clear splitting pattern for the protons at the linker region of 5 DSCG for a

1.20 wt% sample at 25 oC, which enabled us to calculate the coupling constants of these protons

and therefore the dihedral angles. Protons e and e on the methylene group at the linker are both

doublet of doublet, with coupling constants of 10.0 and 5.0 Hz; proton f on the methine group is

a quintet, with a coupling constant of 5.0 Hz (Figure 1.5A). According to the Karplus

relationship of dihedral angles and coupling constant,52,53

the dihedral angle of He-C-C-Hf and

He -C-C-Hf are the same (or very similar). In the spectrum of 2D Nuclear Overhauser effect

spectroscopy (NOESY), cross peaks between aromatic proton a and methylene protons e/e were

observed while no cross peak between proton a and proton f was present (Figure 1.5B). Because

the cross peaks connect resonances from nuclei that are spatially close to each other, it is very

- 8 -

likely that proton a is further away from proton f than from proton e/e . Based on the above data,

we proposed two possible conformations of 5 DSCG molecules in 1.20 wt% sample in water at

25 oC. If the dihedral angles of both He-C-C-Hf and He -C-C-Hf are 60 degree (Figure 1.6A),

5 DSCG is in a staggered conformation, meaning proton f is at the maximum distance from the

chromone moiety (abbreviated as “OR” in the figure). If the dihedral angles of both He-C-C-Hf

and He -C-C-Hf are 120 degree (Figure 1.6B), 5 DSCG is in an eclipsed conformation, and

proton f and the chromone moiety are in closest proximity. Even though the staggered

conformation is normally the conformational energy minimum, the repulsion between the lone

pairs on the carbonyl groups from the two “wings” of the chromone may disfavor this

conformation. The conformation of 5 DSCG under this condition is not known with certainty.

Figure 1.5 NMR spectroscopy of 1.20 wt% (~ 24 mM) 5 DSCG in water at 25 oC. (A)

1H NMR

with the linker region enlarged. (B) Partial NOESY spectrum showing the cross peaks between

proton a and proton e/e .

f

e/e’ e’/e

5.0

Hz5.0

Hz

10.0

Hz

10.0

Hz

e+e’

f

b

ca

d

A B

- 9 -

Figure 1.6 Possible conformations of 5 DSCG in water at 1.20 wt% (~ 24 mM) at 25 oC. (A)

Staggered. (B) Eclipsed.

When 5 DSCG exists as monomer in water, free rotation of the C-C bond in the linker region

should enable both protons e and e to experience identical chemical environment and therefore

appear as a doublet split by proton f in the 1H NMR. However, this is not the case observed. The

different chemical shift of protons e and e that resulted from different chemical environment

could only come from a more “locked” conformation, possibly in an assembly. We therefore

studied the effect of temperature on the chemical shift of protons in 5 DSCG (1.20 wt% in

water). If assembly exists at this concentration at 25 oC, heating might cause the assembly to

dissociate and proton peaks should shift. As shown in Figure 1.7, when the temperature of 1.20

wt% 5 DSCG sample was increased from 298 K to 363 K, all the proton peaks shifted downfield

(Figure 1.7). When the concentration decreased from 1.20 wt% to 0.45 wt%, all the peaks also

shifted downfield. In addition, the fine splitting pattern of protons e/e was lost, suggested a more

O

O

O

NaO2C

O

O

O

NaO2C

H

H

H

H

e'e

e

e'f

a

a

b

b

c

cd

d

H

OH

O

O

O

NaO2C

O

O

O

NaO2C

H

H

H

H

HHO

c

c

d

d

b

b

a

a

e

e

e'

e'

f

Red bonds are in the plane.

Blue bond points up out of the plane.

Green bond points down out of the plane.HO

H

R'

H

OR

H

f

e'e staggered

A

eclipsedOR

HH e'e

f H

HO

R'

B

- 10 -

“dynamic” conformation. These observations are in accordance with 5´DSCG forming a higher

order assembly structure in water. We also note that if 5 DSCG molecules formed assembly in

an “H-stacking”, the aromatic protons a-c would have experienced more deshielding due to the

aromatic ring current and would have shifted downfield instead of upfield upon assembly

formation. The observed upfield shift upon assembly formation (or downfield shift upon

assembly dissociation) by 5´DSCG suggested that the aromatic rings stack in an “off-set”

fashion, which agrees with the thread model.42

Figure 1.7 Temperature effect on chemical shifts of protons in 1.20 wt% (~24 mM) 5 DSCG (in

water). NMR of 0.45 wt% (~ 8.8 mM) 5 DSCG in water at 25 oC is shown for comparison. The

0.45 wt% 298 K

1.20 wt% 298 K

1.20 wt% 308 K

1.20 wt% 318 K

1.20 wt% 328 K

1.20 wt% 343 K

1.20 wt% 353 K

1.20 wt% 363 K

b c a d

f

e or e’e’ or e

HOD

- 11 -

chemical shift of the HOD peak is highly temperature-dependent and the HOD peaks at various

temperatures were calibrated to that reported in literature.54

The dependence of the chemical shift of the 5 DSCG aromatic protons on temperature was

analyzed in order to investigate the possible mechanism for the assembly formation in water

(Figure 1.8). A sudden change in the curve would suggest a cooperative self-assembly

mechanism while a smooth sigmoidal curve would suggest an isodesmic mechanism.55,56

Within

the temperature range tested, no obvious sigmoidal trend was observed for protons a-d; no

sudden change suggestive of cooperative mechanism was observed, either. It is very possible that

the solvent water limits the temperature range that could be tested and thus the plateau for a

sigmoidal curve could not be reached.57

However, the gradual, smooth increase of chemical shift

values upon heating indicates the assembly formation is more likely isodesmic than cooperative.

- 12 -

Figure 1.8 Effect of temperature on the chemical shifts of protons a-d in 5 DSCG (1.20 wt% in

water).

1.2.2 Promote the assembly formation of nonamphiphilic mesogens by thermodynamically

incompatible polymers

In a recent study, our group demonstrated LC formation of 5 DSCG molecules promoted by

addition of polymers based on thermodynamic incompatibility.51

A question arises naturally: Does

the presence of these polymers promote the assembly of 5 DSCG molecules in water directly into

liquid crystal phase (Figure 1.9, Path A) or does the presence of these polymers promote the

assembly formation by 5 DSCG before reaching the liquid crystal phase (Figure 1.9, Path B)?

7.56

7.57

7.58

7.59

7.6

7.61

7.62

7.63

7.64

298 308 318 328 338 348 358 368

δo

f H

b(p

pm

)

Temperature (K)

6.96

6.98

7

7.02

7.04

7.06

7.08

7.1

7.12

7.14

7.16

298 308 318 328 338 348 358 368

δo

f H

c(p

pm

)

Temperature (K)

6.52

6.54

6.56

6.58

6.6

6.62

6.64

6.66

6.68

6.7

6.72

298 308 318 328 338 348 358 368δ

of

Hd

(pp

m)

Temperature (K)

6.89

6.9

6.91

6.92

6.93

6.94

6.95

6.96

6.97

6.98

6.99

298 308 318 328 338 348 358 368

δo

f H

a(p

pm

)

Temperature (K)

- 13 -

Figure 1.9 Two possible pathways of the LC formation promoted by addition of polymer.

1H NMR experiments were performed to investigate at what stage the polymers promote the

liquid crystal formation of 5 DSCG in water. At ambient temperature, 5 DSCG forms LC above

~ 10-11wt%17

and SSY forms LC above ~ 25 wt%.11

We added 8 wt% polyacrylamide (PAAm,

molecular weight 10K) to aqueous 5 DSCG or SSY samples at concentrations far below the LC

formation concentrations. Upon concentration increase of SSY, the broadening of proton peaks

was a lot more significant in the presence of ~ 8 wt % of PAAm than in the absence of PAAm

(Figure 1.10). Similar phenomenon was also observed for 5 DSCG (data not shown). These

results indicated that the self-assembly by 5 DSCG or SSY was promoted by PAAm before

liquid crystal phase formed. On the contrary, for 5 -monosodium cromoglycate (5 MSCG),

which does not form LC at any concentration, the broadening of proton peaks was not much

different as the concentration increased, in the absence or presence of ~ 8 wt % of PAAm (10K)

(Figure 1.11). The similar self-assembly promoted by polymer suggested that like 5 DSCG, the

SSY may also form assembly in LC phases that can be explained by thread model.

dimer

oligomer

trimer

non-covalent polymerIsodesmic

assembly

Randomly aligned

non-covalent polymersLiquid crystal

Path B: A higher ordered

aggregation of assemblies forms

LC upon addition of polymer

Path A: LC forms directly

upon addition of polymer

- 14 -

Figure 1.10 1H NMR of SSY mixed with ~ 8 wt% of PAAm (10K) in D2O at 25

oC.

SSY PAAm (10K)

2.9 wt%

15.1 wt%

19.8 wt%

2.9 wt%

15.1 wt%

19.8 wt%

~ 8 wt%

~ 8 wt%

~ 8 wt%

0

0

0

- 15 -

Figure 1.11 1H NMR of 5 MSCG mixed with ~ 8 wt% of PAAm (10K) in D2O at 25

oC.

1.2.3 Investigate assembly model of organic dye-based nonamphiphilic molecule by miscibility

The exact assembly structures of organic dye-based nonamphiphilic lyotropic liquid crystals

are more difficult to determine than 5 DSCG liquid crystals, because unlike 5 DSCG molecules,

the organic dyes, such as SSY, lack a two-fold symmetry. Sunset Yellow dye can exist as two

tautomers, either as hydroxyl azo form or NH hydrazone form. NMR and X-ray results indicate

that the NH hydrazone form predominantly exists in solution.34,37,58,59

Thus, even in an H-

stacking assembly, a head-to-tail assembly was possible to facilitate electron-rich rings stacking

on electron-deficient rings, as is shown in a recent theoretical study (Figure 1.11).58

On the

contrary, a simple face-to-face stacking is impossible because of electrostatic repulsion.34

5´MSCG PAAm (10K)

0.43 wt%

1.3 wt%

2.2 wt%

0.43 wt%

1.3 wt%

2.2 wt%

~ 8 wt%

~ 8 wt%

~ 8 wt%

0

0

0

- 16 -

Figure 1.12: Asymmetric H-stacking model of Sunset Yellow FCF. Aggregations formed by five

SSY molecules are shown.

To further prove that the “thread model”42

not only applies to the assembly of 5 DSCG, but

also to other nonamphiphilic molecules in general, we chose three commercially available food

dyes as the mesogens. Allura Red AC and Acid Red 13 were chosen because of their similar

structures to that of SSY. Mono-charged molecule sodium 2-naphthol-6-sulfonate hydrate

resembles a portion of SSY and is a potential thread terminator (Figure 1.12). To our best

knowledge, the lowest concentration at which Allura Red AC and Acid Red 13 forms a liquid

crystal phase has not been reported. We prepared aqueous solutions of Allura Red AC and Acid

Red 13 at various concentrations and determined that at or above 22 wt %, both Allura Red AC

and Acid Red 13 displayed birefringence under crossed polarizers at ambient temperature. Below

this concentration, isotropic solutions were observed.

Mono-charged molecule sodium 2-naphthol-6-sulfonate was mixed with the three dyes

mentioned above at various concentrations (Figure 1.13). With low concentration of the added

- 17 -

mono-charged molecule (0.20 wt %) in SSY (30 wt %), the birefringence was reduced. As the

concentration of added mono-charged molecule increased to 0.80 wt%, the birefringence became

even less. At 1.51 wt % or higher of the added mono-charged molecule, no birefringence was

observed. At 5.19 wt % or higher of the added molecule, precipitate formed. When the mono-

charged molecule was added to Allura Red AC (22 wt %) and Acid Red 13 (22 wt %), even very

small amount of the addition of the mesogen (0.23 wt % and 0.26 wt %) destroyed the liquid

crystal phases completely. The results mentioned above indicated that the “thread model”

proposed for 5 DSCG42

could also be true for the LC phase formed by Sunset Yellow, Allura

Red AC, and Acid Red 13. Upon addition, sodium 2-naphthol-6-sulfonate acted as a salt bridge

terminator and attenuated or even annihilated the liquid crystal phase of these nonamphiphilic

dyes.

- 18 -

Figure 1.13: Optical images of organic dyes mixed with different concentrations of mono-

charged molecule sodium 2-naphthol-6-sulfonate hydrate viewed between crossed polarizers. a

Mixed with SY dye. b Mixed with Allura Red AC.

c Mixed with Acid Red 13. Scale bar = 152

µm.

When di-charged molecules Allura Red AC or Acid Red 12 was added at 5~6 wt% to a 30

wt% SSY sample, birefringence was retained (Figure 1.14 A and B). This phenomenon was

expected for the thread model because the di-charged molecules could function as thread

connectors. When the concentration of Allura Red AC or Acid Red 13 increased further,

precipitated formed due to relatively lower solubility of these molecules in water. When the

concentration of SSY was lowered below the nematic liquid crystal concentration, mixing di-

SSY (30 wt%) Allura Red AC (22 wt%) Acid Red 13 (22 wt%)

0.00

0.20a, 0.23b, 0.26c

0.80

1.51

Sodium 2-naphthol-6-

sulfonate hydrate (wt%)

O

N

NaO3SSunset Yellow

O

N

NaO3SAcid Red 13

O

N

NaO3SAllura Red

OH

NaO3S

sodium 2-naphthol-6-sulfonate hydrate

N SO3Na

HN

H

SO3Na

N

H

SO3Na

MeO

Me

- 19 -

charged molecule Allura Red AC or Acid Red 13 resulted in similar birefringence observed for

pure SSY. For example, when the concentration of SSY was reduced to as low as 14.7 wt %,

addition of 15.3 wt % Allura Red AC resulted in birefringence resembled to that formed by 30

wt % SY dye (Figure 1.14 C). Similar birefringence was observed when 14.0 wt % SSY sample

was mixed with 14.1 wt % Acid Red 13 (Figure 1.14 D). These results suggest that Allura Red

AC and Acid Red 13 not only promoted LC phase of SSY but also were thermodynamically

compatible with SSY. Therefore, the presence of Allura Red AC or Acid Red 13 promoted the

LC formation of SSY by elongating the salt bridge and further proved the validity of the thread

model.

Figure 1.14: Optical images of SY dye mixed with Allura Red AC or Acid Red 13 at different

concentrations viewed between cross polarizers. The orientation of the bars shown above the

SSY Allura Red AC

29.7 wt% 5.5 wt%

SSY Acid Red 13

29.9 wt% 5.0 wt%

SSY Allura Red AC

14.7 wt% 15.3 wt%

SSY Acid Red 13

14.0 wt% 14.1 wt%

A

B

C

D

- 20 -

images indicates the rotation angle of the samples under the cross polarizers. Scale bar = 152

µm.

1.3 Conclusion and Perspectives

In summary, 1D and 2D NMR spectroscopy revealed that 5 DSCG self-assembles into

hydrated threads via a possible isodesmic mechanism in a 1.2 wt% sample in water at 25 oC.

Similar to that by 5 DSCG, the assembly formation by nonamphiphilic molecule Sunset Yellow

FCF can also be promoted by thermodynamically incompatible polymer. In addition, miscibility

test showed that mono-charged molecule that is structurally similar to SSY annihilate LC

formation by SSY while di-charged molecules retain and promote LC formation. These results

suggest that the thread model proposed for 5 DSCG may in general explain the assembly

formation by nonamphiphilic dyes and other nonamphiphilic mesogens.

1.4 Experimental Section

Chemicals

Sunset Yellow FCF was purchased from Aldrich (Milwaukee, WI). Acid Red 13 and Allura Red AC

were purchased from TCI America. All aqueous solutions were prepared with deionized water with

a resistivity of 18.2 MΩ cm (MilliQ system, Millipore, Bedford, MA).

General procedure for preparation of optical cells and birefringence characterization

An aqueous solution of Sunset Yellow FCF (30.08 wt %) and Acid Red 13 (1.97 wt %) was prepared

and aged in a vial for 12 h to allow complete dissolution. The sample was assembled in a sandwiched

optical cell composed of two glass microscope slides with one sheet of Saran Wrap® (13-15 μm) to

afford a spacer. The sheet of Saran Wrap® was punched to create a hole to accommodate the sample

to be sandwiched between the glass slides. The sample was loaded between the slides and sealed

- 21 -

with binder clips on each side immediately to prevent the water from evaporating. The sample was

viewed and recorded between crossed and parallel polarizers on an Olympus BX51 polarizing

microscope.

General procedure for NMR study

1H spectra were recorded on a Bruker Advance DPX-300 spectrometer. Chemical shifts are reported

in ppm, using tetramethylsilane as the internal standard. NOESY spectra were recorded on a Bruker

Advance DRX-500 spectrometer.

- 22 -

Chapter 2

Stereochemical Control of Nonamphiphilic Liquid Crystals: Effect of Chiral Mesogens and

Dopants on Assemblies Separated by Six Nanometers of Aqueous Solvents

Summary

Unlike conventional thermotropic and lyotropic liquid crystals, the chromonic liquid

crystals consist of hydrated assemblies of nonamphiphilic molecules that are aligned with a

separation of about 6 nm between assemblies in an aqueous environment. This separation raises

the question of what the assembly structure would be for a chiral nematic (or cholesteric) phase

of this class of liquid crystals as the assemblies need to interact with each other that are about 6

nm apart. Here, we report the synthesis of three stereoisomers of disodium chromonyl

carboxylate derivative, 5 DSCG-diviol, and the correlation between the molecular structure, bulk

assembly and liquid crystal formation. Circular dichroism indicated a chiral conformation with

bisignate Cotton effect. Nuclear Overhauser Effect in proton NMR spectroscopy revealed

conformations that are responsible for liquid crystal formation. Cryogenic transmission electron

microscopy showed that chiral 5 DSCG-diviols form assemblies with crossings. We observed

that the chiral isomers formed chiral nematic liquid crystals while the achiral isomer 5 DSCG-

meso-diviol did not form any kind of liquid crystals. While generic 5 DSCG in water aligns

uniformly on SAMs supported by obliquely deposited gold films, 5 DSCG-(R,R)-diviol 1a

exhibited nonuniform alignment of on the same surfaces. Fingerprint texture of 18 wt%

5 DSCG-(R,R)-diviol can be observed with thick cell. Together, these results suggest that the

hydrated assemblies of chiral molecule 5 DSCG-(R,R)-diviol 1a interact with each other while

being separated by relatively large distance (6 nm) in water, causing a lyotropic chiral nematic

liquid crystal phase. These studies suggest that hydrated assemblies of chiral 5 DSCG-diviol can

- 23 -

interact with each other across a 6 nm separation in an aqueous environment, exhibiting a liquid

crystal phase with features that may indicate some type of chiral organization.


2.1.1 Cholesteric liquid crystal phase

The cholesteric liquid crystal phase is also known as chiral nematic liquid crystal phase. The

first materials exhibiting this phase were cholesterol derivatives and hence the name. Nowadays

there are many different types of chiral materials that exhibit cholesteric phase and most of them

have no resemblance to cholesterol. Cholesteric liquid crystals (LCs) can be visualized as a stack

of very thin nematic layers within which molecules have the same orientational orders but no

positional order. The directors in each layer twist with respect to those above and below and

rotate around a perpendicular direction (helical axis) and form a helix.60

The distance over which

the directors complete a full 360o rotation is called a pitch, p (Figure 2.1). The pitch can vary

with temperature, or introduction of other molecules, and the magnitude may range from several

hundred of nanometers to micrometers, depending on the chemical compositions. Thermotropic

cholesteric phases normally have pitches of a few hundred nanometers.61-63

Lyotropic cholesteric

phases have a broader range of pitch, from a few micrometers to a few hundred micrometers.22,64-

67 Various materials form cholesteric liquid crystal phase, such as cholesteryl benzoate,

68

hydroxypropyl cellulose,69

DNA,66

and even a suspension of viruses.70

Cholesteric liquid crystal phase can be formed by chiral mesogens alone or can be induced

by doping an achiral lyotropic or thermotropic nematic phase with a small amount of chiral

molecules.71-74

The molecular asymmetry is amplified in the supramolecular helix structure.

- 24 -

Figure 2.1 Schematic representation of the cholesteric liquid crystal phase.

2.1.2 Chiral chromonic liquid crystals

There have been many reports on cholesteric liquid crystals formed by either conventional

thermotropic75-79

or lyotropic65,66,72,80-82

mesogens. The mechanism of how a chiral nematic phase

arises for lyotropic liquid crystals is still poorly understood.60,72,83-85

The most intriguing aspect is

that as lyotropic liquid crystals are formed by organized assemblies of molecules in a solvent, the

effect of chiral dopants or chiral mesogens must transmit across the solvent between assemblies,

and pivot the orientation of the assemblies as a whole continuously throughout the sample.72,85

How the chiral assemblies interact with the solvent molecules surrounding them and influence

the neighboring assemblies over distance is unclear. There are few reports on chiral mesogens

that form chromonic liquid crystal phase, but several reports examined chromonic liquid crystal

phases influenced by chiral dopants that have an interaction over 6 nm between the molecular

assemblies.22,23,64,86

To investigate how stereochemistry controls the assembly structure and liquid crystal

formation, we designed and synthesized three stereoisomers of derivatives of disodium

chromonyl carboxylates (5 DSCG-diviol) which posses a diviol linker (Figure 2.2), and

demonstrated a strong correlation between molecular structures and assembly properties, as well

½ helical pitch

- 25 -

as the ability of the molecules to form liquid crystals. We also present evidence for a liquid

crystal phase that requires chemical communication across a distance of 5-6 nm of the aqueous

solvent, and is perhaps different from the conventional cholesteric liquid crystal phase.

Figure 2.2 The structures of the chiral isomers 5 DSCG-(R,R)-diviol 1a, 5 DSCG-(S,S)-diviol

1b, and achiral isomer 5 DSCG-meso-diviol 1c.


2.2.1 Design and synthesis of the stereoisomers of 5 DSCG-diviols

The generic 5 DSCG exhibits a wide range of liquid crystal properties in water that are

unmatched by conventional lyotropic liquid crystals that are made of amphiphilic

molecules.12,38,42

Several other fused aromatic dye-based molecules also form assemblies and

chromonic liquid crystals in water, but 5 DSCG forms liquid crystal phases at lower

concentration with higher birefringence compared to other dye-based molecules.12

To explore the

structural requirement for chromonic liquid crystal phase formation, our group recently screened

O

O O

OH OH

O

O

O

NaO2C CO2Na

5'DSCG-(R,R)-diviol 1a

O

O O

OH OH

O

O

O

NaO2C CO2Na

5'DSCG-(S,S)-diviol 1b

O

O O

OH OH

O

O

O

NaO2C CO2Na

5'DSCG-meso-diviol 1c

- 26 -

a series of dichromonyl molecules, and found an optically inactive mixture of stereoisomers that

exhibited polymorphism, of which upon rapid cooling resulted in a nematic liquid crystal phase

whereas aging at ambient temperature resulted in precipitation. This mixture consists of three

stereoisomers: a pair of enantiomers (52%), 5 DSCG-(R,R)-diviol 1a and 5 DSCG-(S,S)-diviol

1b; and an achiral compound (48%), 5 DSCG-meso-diviol 1c.42

The precipitates obtained from

the above sample after aging overnight were comprised of a mixture of all three stereoisomers,

and thus spontaneous resolution87,88

was not achieved. To study the effect of stereochemistry on

assembly and liquid crystal formation, we synthesized each stereoisomer individually.

The synthesis of the chiral isomers 5 DSCG-(R,R)-diviol and 5 DSCG-(S,S)-diviol was

accomplished using the same route (Scheme 2.1), but started with L-(-)-arabitol and D-(+)-

arabitol, respectively. For brevity, the synthesis of 5 DSCG-(R,R)-diviol 1a is described below.

2,4-Dihydroxypentane-1,5-diyl bis-(2,4,6-triisopropyl-1-benzenesulfonate) 6a was obtained in 5

steps starting from L-(-)-arabitol.89,90

Regioselective acetonization of L-(-)-arabitol afforded

diacetal 2a, which reacted with 1,1 -thiocarbonyldiimidazole to give compound 3a.

Deoxygenation of 3a lead to compound 4a and deacetionization afforded (2R,4R)-pentane-

1,2,4,5-tetraol 5a, which was then converted to bis-sulfonate 6a. Acetonization of bis-sulfonate

6a provided acetal 7a followed by iodination that produced diiodide 8a. Deacetionization of 8a

yielded diiodo diol 9a which was treated directly with excess of 2,6-dihydroxyacetophenone to

give the substitution adduct 10a. A two-step cyclization sequence from modified conditions91

built the chromonyl ring of diester 11a. First, hydroxyl ketone 10a was added to a mixture of

sodium methoxide and dimethyl oxalate in a solvent mixture of Et2O and MeOH (2:1 by volume)

and the reaction mixture was refluxed overnight. Acidification of the reaction mixture gave a

yellow precipitate. Second, the yellow precipitate was treated with concentrated hydrochloric

- 27 -

acid under reflux in methanol to give dichromonyl ester 11a. We note that the use of this mixed

solvent system was critical for obtaining useful yields. Basic hydrolysis of dichromonyl ester 11a

with stoichiometric amount of sodium hydroxide provided the final product 5 DSCG-(R,R)-

diviol 1a.

Scheme 2.1 Synthesis of 5 DSCG-(R,R)-diviol 1a and 5 DSCG-(S,S)-diviol 1b.a

aTPS = Triisopropylbenzenesulfonyl.

Using xylitol as the starting material to attain desired meso stereochemistry, 5 DSCG-meso-

diviol was synthesized with a modified synthetic route from that for the chiral stereoisomers

(Scheme 2.2) because the same route was not effective to prepare the meso isomer. The

preparation of tetraol 5c was essentially the same to that of 5a/5b. However, the reaction of

tetraol 5c and TPSCl only provided mono-protection product. There was also a trace amount of

byproduct due to the cyclization of the mono-protection product. The different steps included

- 28 -

acetyl-bromination of tetraol 5c to give bromoacetate 6c,92

epoxidation under basic conditions to

generate diepoxide 7c that was purified by bulb-to-bulb distillation,93

and iodination of diepoxide

7c to give diiodo diol 8c. Compound 8c was used to obtain the final product 1c under the same

reaction conditions as that for the chiral isomers. We note that while the chiral diester 11a or 11b

(for the chiral isomers) precipitated out from the reaction mixture, most of the achiral diester 10c

remained in the solution (Scheme 2.2).

Scheme 2.2 Synthesis of 5 DSCG-meso-diviol 1c.

2.2.2 Stereochemistry controls the formation of liquid crystals

To examine the potential liquid crystal properties, samples of each stereoisomer in water

were sandwiched between two microscope glass slides with one sheet of Saran Wrap® (13-15

µm thick) as a spacer. A square hole of ~0.5 x 0.5 cm was cut in the sheet of Saran Wrap®

to host

the sample. Binder clips were applied on each side to prevent the evaporation of solvent.

Between cross polarizers, 5 DSCG-(R,R)-diviol 1a exhibited birefringence at a concentration as

- 29 -

low as 12.0 wt% (with a mole ratio of water to mesogen of 226:1) at ambient temperature, about

21 °C (Figure 2.3 A). At the same concentration (12.0 wt%), 5 DSCG-(S,S)-diviol 1b showed

liquid crystals phase at 18 °C (Figure 2.3 B). We believed that this small difference in phase

behavior between the (R,R)- and (S,S)- isomers may be caused by the miniscule difference in

purity such as small amounts of salts that are beyond the measurement of NMR. Salts are known

to change the nematic-isotropic transition temperature of liquid crystals formed by 5 DSCG.28

The achiral stereoisomer 5 DSCG-meso-diviol 1c was a mixture of isotropic solution and a small

amount of insoluble solid at the same concentration (12.0 wt%) (Figure 2.3 C). Furthermore,

rapid cooling did not induce liquid crystal formation in this sample. This result indicates that

5 DSCG-meso-diviol 1c is either less capable or incapable of forming liquid crystals, which

likely contributes to the fact that the mixture of all three stereoisomers requires a lower

temperature or a higher concentration than the individual chiral stereoisomers to form a liquid

crystal phase. We note that 5 DSCG-meso-diviol 1c has lower water solubility than the chiral

isomers 5 DSCG-(R,R)-diviol 1a and 5 DSCG-(S,S)-diviol 1b.

Figure 2.3 Optical images (cross polarizers) of 12.0 wt % 5 DSCG-(R,R)-diviol 1a (A),

5 DSCG-(S,S)-diviol 1b (B), and 5 DSCG-meso-diviol 1c (C) in water sandwiched between two

glass slides (spacer: 13-15 µm). Samples were freshly prepared by dissolving solid in water at

ambient temperature. Scale bar = 76 µm.

A

5 DSCG-meso-diviol5 DSCG-(S,S)-diviol5 DSCG-(R,R)-diviol

B C

- 30 -

2.2.3 Stereoisomers of 5 DSCG-diviols exhibit different polymorphism

To explore liquid crystal formation without any cooling, we prepared 18.0 wt% 5 DSCG-

(R,R)-diviol 1a and 18.1 wt % 5 DSCG-(S,S)-diviol 1b (Figure 2.4 A and B). Both of these

chiral 5 DSCG-diviols exhibited birefringence over the entire sample prepared at ambient

temperature. In contrast, 5 DSCG-meso-diviol 1c at this concentration resulted in an isotropic

solution with insoluble solids (Figure 2.4 C). Rapid cooling of this sample did not induce liquid

crystal formation. After aging overnight at ambient temperature in vials sealed with parafilm to

prevent evaporation, we observed that the chiral and meso 5 DSCG-divol resulted in different

assemblies. The sample of 18.0 wt% 5 DSCG-(R,R)-diviol 1a became a mixture of liquid

crystals with small precipitates, whereas 18.1 wt% 5 DSCG-(S,S)-diviol 1b remained a mixture

of liquid crystals and isotropic solution (Figure 2.4 D and E). Under the same conditions, 18.0

wt% of 5 DSCG-meso-diviol 1c in water remained a mixture of isotropic solution and aggregates

(Figure 2.4 F). In comparison, the polymorphism of the individual enantiomer was different from

that observed in the earlier work of an optically inactive mixture of all three stereoisomers (a

mixture of 52% racemic mixture and 48% meso isomer): a freshly prepared sample of 18.0 wt%

of mesogens transitioned from an isotropic solution to liquid crystal phase by rapid cooling to 15

oC without observable precipitates, whereas isothermal aging gives precipitates in isotropic

solution. Together, these results suggest that, for the optically inactive mixture,42

the liquid

crystal phase represents a local energy minimum, and the precipitation in isotropic solution is of

a global minimum in the energy profile.

- 31 -

Figure 2.4 Optical images (cross polarizers) of 18.0 wt% 5 DSCG-(R,R)-diviol 1a, 18.1 wt%

5 DSCG-(S,S)-diviol 1b, and 18.0 wt% 5 DSCG-meso-diviol 1c in water when freshly prepared

(A, B, and C, respectively), and when aged at ambient temperature overnight in sealed vials (D,

E, and F, respectively). The samples are sandwiched between two glass slides with a spacer of

13-15 µm thick. Scale bar = 76 µm.

2.2.4 Conformations of stereoisomers are different

Diastereomers may or may not be of grossly different conformation. In proton NMR spectra,

differences in the chemical shifts of the diviol linker in the chiral and meso 5 DSCG-diviol

suggest that the conformations between these diastereomers are different from each other. To

access the conformations of the diastereomers and their differences, we used two-dimensional

NMR spectroscopy to characterize the conformations of the (S,S)-isomer 1b and the meso isomer

1c. Because the meso isomer has a lower solubility than the (S,S)-isomer 1b in water, we believe

that even with a concentration at which both molecules appear to dissolve in D2O, the dynamics

- 32 -

of the two molecules in solution may not be the same. For this reason, we focused on using

ROESY to characterize the structure to avoid the potential null of NOE signals due to the

dynamics of the molecules.94 We observed large differences in the NOE correlations in the

ROESY spectra for the diviol bridge region between the two diastereomers in D2O (Figure 2.5).

For 0.503 wt% of 5 DSCG-(S,S)-diviol 1b, NOE between protons g and e (and e ), and between

g and f were observed with similar peak intensities. This result suggests that these protons (g and

e/e , and g and f) may be in close proximity by similar distances. In contrast, for 5 DSCG-meso-

diviol 1c at the same wt%, protons g and g on the same methylene group were in different

chemical environment, and thus exhibited different chemical shifts. More importantly, one of the

“g” protons showed weak NOE correlation with only one of the “e” protons. The other “g”

proton exhibited NOE correlation with both of the “e” protons, with strong and weak NOE signal

intensities, respectively (Figure 2.6). These NOEs indicate that one of the “g” protons was in

close proximity to only one of the “e” protons, whereas the other “g” proton was close in space

to both of the “e” protons. These results suggest that the diviol linker region in the meso isomer

is twisted through certain preferred bond rotation angle. Together, these spacing of protons in the

two diastereomers suggests that 5 DSCG-(S,S)-diviol 1c likely adopts an overall “stretched”

conformation while 5 DSCG-meso-divol 1c is more of a “folded” conformation. These

conformational differences also appear to be consistent with the low solubility of the meso

isomer 1c and the formation of thread assemblies by the (S,S)-isomer 1b.

- 33 -

Figure 2.5 ROESY of 0.503 wt% of 5 DSCG -(S,S)-diviol 1b and 0.503 wt% of 5 DSCG-meso-

diviol 1c in D2O at 25 oC.

5'DSCG-(S,S)-diviol 5'DSCG-meso-diviol

f e/e'

g

f e/e'

g or g '

e/e'

f

d

bc

a

O

O O

OH OH

O

O

O

H H g

O

O O

ONa Na

- 34 -

Figure 2.6 ROESY spectrum of 0.503 wt% 5 DSCG -(S,S)-diviol 1b and 5 DSCG-meso-diviol

1c in D2O at 25 oC.

2.2.5 Circular dichroism indicates a chiral conformation

Circular dichroism (CD) spectra were collected to examine the chirality in chiral 5 DSCG-

diviol at feasible concentrations (0.1 to 0.8 mM). The two enantiomers of 5 DSCG-diviol

showed CD spectra with mirror signals having minimum and maximum at 274 nm and 339 nm

(Figure 2.7). Considering the UV absorption at 256 and 325 nm, bisignate Cotton effects in the

e/e'

f

d

bc

a

O

O O

OH OH

O

O

O

H H g

O

O O

ONa Na

5'DSCG-(S,S)-diviol 5'DSCG-meso-diviol

acb

f

e or e’e’ or ed

g or g’

g’ or gacb

f

e or e’e’ or e g

d

ac

b

d

e or e’e’ or e

f

g

ac

b

d

e or e’e’ or e

f

g’ or g

g or g’

- 35 -

π-π* transition were observed in the CD spectra for 5´DSCG-(R,R)-diviol 1a (8 × 10-4

M) with a

positive effect at 339 nm and a negative effect at 274 nm (Figure 2.7). Although bisignate Cotton

effect may suggest the presence of ordered chiral assembly,95,96

temperature-dependence study

(Figure 2.8) showed insignificant decrease in the intensity of CD signal as the temperature

increased from 5 oC to 85

oC. Furthermore, in a concentration-dependent study of the absorbance

by 5 DSCG-(R,R)-diviol 1a in water, a near constant molar absorptivity was observed at 325 nm

in the concentration ranging from 5 µM to 1.6 mM using 1 mm- and 1 cm-path curvettes (data

not shown). Together, these results suggest that at this concentration range (<1.6 mM) the

molecules are mostly in the monomeric state, and that the chiral conformation is stable at

relatively high temperature. However, in a study of concentration-dependent chemical shift in

NMR, we observed significant peak broadening and an upfield chemical shift in the proton NMR

of 5 DSCG-(R,R)-diviol 1a in D2O as the concentration was increased from 0.02 wt% (400 µM)

to 3.5 wt% (72 mM) (Figure 2.9). The peak broadening resulted from a significant reduction in

the molecular mobility due to aggregation and the upfield chemical shift is in accordance with

the thread model assembly in which the aromatic rings stack in an “off-set” fashion (also see

Section 1.2.1). Considering the upper limit of concentration that could be detected by the CD

spectrometer used, it is plausible to suggest that chiral assemblies of 5 DSCG-(R,R)-diviol 1a

indeed form at relatively high concentrations (at least at above 20 mM).

- 36 -

Figure 2.7 UV spectrum of 5 DSCG-(R,R)-diviol 1a (dot line) and CD spectra of 5 DSCG-(R,R)-

diviol 1a (solid line) and 5 DSCG-(S,S)-diviol 1b (dash line) in water (8 × 10-4

M) at 5 oC.

Spectra were taken in a 1 mm path length cuvette.

Figure 2.8 Temperature-dependent CD spectra of 5 DSCG-(R,R)-diviol 1a in water (8 × 10-4

M).

Spectra were taken in a 1 mm path length cuvette.

- 37 -

Figure 2.9 1H NMR of 5 DSCG-(S,S)-diviol 1b from 0.02 wt% (0.4 mM) to 3.5 wt% (72 mM) in

water.

2.2.6 Cryogenic transmission electron microscopy reveals early-staged and crossed assemblies

by chiral mesogens

Cryogenic transmission electron microscopy (cryo-TEM) provides valuable, direct

information of the complex nanostructures and microstructures in the solution state. It has been

used to characterize the size and shape of peptides, DNA, lipids, virus, as well as the self-

0.5 wt% (~10 mM)

3.5 wt% (65 mM) 5´DSCG-(S,S)-

diviol

0.1 wt% (~2.2 mM)

1.0 wt% (~20 mM)

cc

0.02 wt% (~0.4 mM)

1.5 wt% (~30 mM)

3.0 wt% (~61 mM)

3.5 wt% (~72 mM)

b c a d f e&e´ g

0.05 wt% (~1.0 mM)

HOD

- 38 -

assembly of polymers, surfactants, and liquid crystals.28,97

To access the assembly structure of

the chiral mesogens, we compared the cryo-TEM images of the 5 DSCG-(R,R)-diviol 1a and the

generic 5 DSCG at concentrations below and above which assemblies form (Figure 2.10-2.13).


(inside the curved edge). (Mole ratio) indicates water/5 DSCG.

- 39 -


(R,R)-diviol 1a (inside the curved edge). (Mole ratio) indicates water/5 DSCG-(R,R)-diviol.


(inside the curved edge). (Mole ratio) indicates water/5 DSCG.

- 40 -


(R,R)-diviol 1a(inside the curved edge). (Mole ratio) indicates water/5 DSCG-(R,R)-diviol.

A past study of small angle neutron scattering showed that the assembly structure started to

appear for 5 DSCG at about 5-6 wt%.42

Thus, we chose 5 DSCG (5.5 wt%, mole ratio of water

molecules to mesogens is 488) and 5 DSCG-(R,R)-diviol 1a (5.0 wt%, mole ratio of water to

mesogen is 587) to compare the early stage features of the molecular assembly. We note that the

sample of chiral mesogen 5 DSCG-(R,R)-diviol 1a had a lower concentration than the one with

achiral mesogen 5 DSCG. Interestingly, assemblies were more visible in the 5 DSCG-(R,R)-

diviol 1a samples, whereas mostly blocks of assemblies with about 5-7 nm gap were observed in

the 5 DSCG samples (Figure 2.14). These results indicate that the chiral mesogen 5 DSCG-

(R,R)-diviol 1a form assemblies at a lower concentration than that by 5 DSCG. At these

concentrations, a liquid crystal phase was not observed for either of the samples, and the

supramolecular assembly structures appeared to be connected by “blocks” of assemblies.

- 41 -

Although supramolecular assemblies were visible in the 5 DSCG-(R,R)-diviol 1a samples, they

were randomly oriented, consistent with a sample of isotropic solution at this concentration.

Figure 2.14 Cryo-TEM images of 5.5 wt% of 5 DSCG, and 5.0 wt% of 5 DSCG-(R,R)-diviol 1a.

Numbers in the parentheses indicate mole ratio of water and mesogens in the sample. The red

dash lines indicate the thread-like assembly. Scale bar = 10 nm.

As the concentration of 5 DSCG-(R,R)-diviol 1a increased to 8.8 wt%, the assemblies were

more elongated and more aligned with each other than those observed in samples at lower

concentrations (Figure 2.15). This elongation of the assemblies as the concentration increases is

consistent with the notion of an isodesmic assembly,35,56

for which there is no critical

concentration for assembly but the observed blocks of assemblies continue to grow to form

assembly. The cryo-TEM images also showed that the aligned assembly structures were

separated by about 6 nm, and the alignment between the assemblies was uniform over at least

hundreds of nanometers. Interestingly, there were more crossings between the assemblies formed

- 42 -

by 5 DSCG-(R,R)-diviol 1a than those formed by 5 DSCG (Figure 2.15), and that the chiral

mesogen exhibited large curvature in the assemblies.

Figure 2.15 Cryogenic transmission electronic microscopic images of 8.2 wt% of 5 DSCG and

8.8 wt% of 5 DSCG-(R,R)-diviol 1a. Numbers in the parentheses indicate mole ratio of water

and mesogens in the sample. Scale bar = 50 nm.

The polymorphism of liquid crystal phases and precipitates under different condition, and the

6 nm-separation between the threads as revealed by cryo-TEM suggest that the assemblies in

liquid crystals are likely hydrated by solvent water, which prevent aggregation and precipitation

for the chiral isomers. This hydration implies a competition between assemblies getting solvated

and aggregating with other assemblies. Whereas aggregation of mesogens leads to insoluble

precipitation, hydration of assemblies leads to liquid crystal formation. For the polymorphism

shown in the optically inactive mixture, we believe that the achiral mesogen is co-precipitating

with the chiral stereoisomers, but rapid cooling can condense the readily available water

- 43 -

molecules around the newly formed assembly to insulate the assemblies from aggregating, and

thus facilitate liquid crystal formation.

2.2.7 Nonuniform alignment formed by 5 DSCG-(R,R)-diviol

We note that this chiral mesogen in water at the concentrations studied did not give a

conventional fingerprint texture of the classical cholesteric liquid crystal phase.98

In addition,

without a magnetic field alignment, we did not observe the fingerprint texture of achiral 5’DSCG

mixed with small chiral dopants.22

To examine the liquid crystal phases comprised of chiral

mesogens, we used a surface that can align the generic 5 DSCG liquid crystal uniformly over a

large area and explored if a uniform alignment could also be obtained for the liquid crystals

formed by the chiral 5 DSCG-(R,R)-diviol 1a. Any chiral phase of a liquid crystal will not give a

uniform single-colored texture over the entire liquid crystal sample because of the continuous

change in the molecular orientation in the chiral nematic phase. Thus, a surface that can align a

nematic phase uniformly99,100

is a useful tool to determine whether a chiral nematic phase exists

or not. We have recently reported uniform alignment of 5 DSCG liquid crystal on surfaces

presenting self-assembled monolayers (SAMs) of functionalized alkanethiols supported on gold

films that were deposited onto glass slides at an incident angle oblique from the surface normal

of the glass slides. When sandwiched between two such surfaces, hydrated molecular assemblies

of 5 DSCG and Sunset Yellow dye aligned in parallel to the surface of the SAMs, and uniformly

in a preferred direction over the entire sample.43

For classical cholesteric liquid crystal samples, the characteristic fingerprint texture can often

be observed by chiral mesogens or by achiral mesogens mixed with a chiral dopant. Here, we

also compared the liquid crystal formed by chiral 5’DSCG-diviol 1a and by achiral 5’DSCG

- 44 -

mixed with a small chiral molecule. In this work, using the same surfaces (two SAMs of

HS(CH2)10(OCH2CH2)3OH on gold films that were deposited with a 45° incident angle),43

16.8

wt% 5 DSCG in water gave a uniform alignment (Fig 2.16 D), which exhibited a strong

modulation in the intensity of transmitted light when the sample is rotated with respect to either

one of the crossed polarizers. Uniform alignment was observed as expected for 14.1 wt%

5 DSCG mixed with 14.5 wt% achiral molecule xylitol (Figure 2.16 E). When 13.4 wt%

5 DSCG was mixed with a racemic mixture of D- and L-mannitol (8.1 wt% of each), uniform

alignment of liquid crystal phase was observed (Figure 2.16 F). On the contrary, a nonuniform

alignment was observed for a sample containing 13.4 wt% 5 DSCG doped with 16.2 wt%

enantiomerically pure D-mannitol (Figure 2.16 G). Two areas of different colors that switched

between bright and dark were observed as the sample was rotated between the cross polarizers.

Using the liquid crystal cell with the same monolayers, the 5 DSCG-(R,R)-diviol 1a (18.0

wt%) liquid crystal sample also exhibited non-uniform alignments with two domains of different

orientations (Figure 2.16 H). We note that all the samples were warmed to above liquid crystal-

isotropic transition temperature to remove potential effect of viscosity or flow on the liquid

crystal alignment. Because of the 6 nm solvent separation between the assemblies, this technique

cannot predict if the neighboring assemblies of the chiral molecules can twist relative to each

other in the aqueous solution. However, uniform alignment of liquid crystal is not obtained for

chiral lyotropic mesogens. This result suggests this liquid crystal phase may have chiral features,

perhaps different from those of the classical cholesteric phase.

- 45 -

Figure 2.16 (A). Geometry of gold deposition on glass slides at an oblique angle from the surface

normal. (B). Scheme of self-assembly molecules (SAMs) formed by HS(CH2)10(OCH2CH2)3OH.

(C). Schematic representation of uniform alignment of threads of 5 DSCG liquid crystal on

16.8 wt% (141)

5 DSCG

D

14.1 wt% (141) 5'DSCG +

14.5 wt% (41) xylitol

E

S

O

O

O

S

O

O

O

S

O

O

O

HO HO HO

Au

Au

S(CH2)10(OCH2CH2)3OH45o Au

S(CH2)10(OCH2CH2)3OH45o Au

A B C

45o

13.4 wt% (149) 5'DSCG +

8.1 wt% (88) D-mannitol + 8.1 wt% (88) L-mannitol

13.4 wt% (149) 5'DSCG +

16.2 wt% (44) D-mannitol

18.0 wt% (141)

5 DSCG-(R,R)-diviol

G

F

H

- 46 -

SAMs supported by gold films deposited at 45o from the surface normal of the glass slides.

Optical images (cross polarizers) of (D) 16.8 wt% 5 DSCG, (E) 14.1 wt% 5 DSCG mixed with

14.5 wt% xylitol, (F) 13.4 wt% 5 DSCG mixed with a 16.2 wt% racemic mixture of D- and L-

mannitol, (G) 13.4 wt% 5 DSCG doped with 16.2 wt% D-mannitol, and (H) 18.0 wt% 5 DSCG-

(R,R)-diviol 1a in water sandwiched between obliquely deposited gold films supporting

HS(CH2)10(O CH2CH2)3OH. Numbers in the parentheses indicate mole ratio of water and

mesogens in the sample. Scale bar = 152 µm. The arrows indicate direction of gold deposition

projected onto the glass slides, and the orientation of the sample relative to the cross polarizers.

Between two surfaces, cholesteric liquid crystal can align homogeneously or

homeotropically. In the homogeneous (planar) alignment, liquid crystals twist as they propagate

away from the surface, and all the liquid crystals align parallel relative to the surface. In this

case, the pitch is not observable (Figure 2.17 A). In the homeotropic alignment, the surface

tends to align the liquid crystal perpendicular to the surface, and the liquid crystals twist as they

propagate along the surface. In this case, the pitch can be observed (Figure 2.17 B). For a

sample containing 13.5 wt% 5'DSCG doped with 16.5 wt% mannitol, as the thickness of liquid

crystal cell (the spacer) increases, the liquid crystal becomes brighter, and more importantly, the

modulation of light changing from dark to bright is significantly reduced (Figure 2.18). This

result is consistent with a cholesteric phase on a planar-alignment surface (Figure 2.17 A).

- 47 -

Figure 2.17 Illustrations of the molecular assemblies in (A) homogeneous or (B) homeotropic

alignment of chiral nematic liquid crystal phase on surface.

Figure 2.18 Optical images (cross polarizers) of 13.5 wt% 5 DSCG doped with 16.5 wt% D-

mannitol in water sandwiched between obliquely deposited gold films supporting HS(CH2)10(O

CH2CH2)3OH with different thickness of spacers. Scale bar = 152 µm. The arrows indicate

direction of gold deposition projected onto the glass slides, and the orientation of the sample

relative to the cross polarizers.

In a conventional cholesteric phase created by doping thermotropic liquid crystals with a

small amount of chiral molecules, the chiral effect from the small molecule dopants is

transmitted over large distances – up to hundreds of micrometers.101,102

If one considers the

0 0

A B

Chiral nematic phase on homogenous

(planar) alignment surface.

Chiral nematic phase on

homeotropic alignment surface.

½ h

elic

al p

itch

½ helical pitch

~13 µm x 1 ~13 µm x 2 ~13 µm x 3 ~13 µm x 4 ~13 µm x 5Thickness

of spacer

- 48 -

nematic liquid crystal as the solvent for the chiral dopants, then the dopants are changing the

entire organization of the solvent molecules. In our case, the assemblies are separated by water

molecules – a medium that comprised of hydrogen bond networks. As the assemblies require

strong hydration by water molecules to prevent themselves from coalescing together, at the

boundary between the domains, the water molecules are likely organized to facilitate the

interaction between the assemblies.

It is important to note that there are precedents of organized water structures.103-109

For

example, the sucrose density gradients in water that are commonly used for purification of

biomolecules are stable over a long period of time.103,104

In such a gradient solution, water

molecules between the sucrose molecules are dynamically different across the gradient.105

In a

recent study using molecular dynamics simulation, water dynamics in the solvation shell of

proteins have shown to be different from bulk water. The terahertz spectroscopy data suggested

an influence on the correlated water network motion beyond two nm between neighboring

proteins.107

Our recent work also demonstrated that the stereochemistry of polyol diastereomers

on surfaces impacted their ability to resist protein adsorption, mammalian cell adhesion and

biofilm formation.109

As the atomic composition is the same for those surfaces, this resistance to

biofouling is likely due to the templated aqueous solvent structure at the interfaces. Surface force

studies on bioinert SAMs by Grunze and coworkers measured force effect as far as more than 10

nm from the surfaces.110,111

Finally, using chiral lyotropic liquid crystals as solvents,

discrimination of enantiomers can be observed in NMR spectra. These studies suggest that the

solvent is promoting a preferred orientation of the solutes.106,108

The formation of mesophases for conventional thermotropic liquid crystals (such as 5CB) is

primarily a result of anisotropic dispersion forces between molecules.60,112

In the present study,

- 49 -

hydrogen bonding appears to be the main factor that governs the interaction between assemblies

and the basis for mediating liquid crystal threads to influence each other in an aqueous

environment up to 6 nm apart. Together with the anisotropic interactions at meso-scale, liquid

crystal materials create complex and interesting assembly structures.113

With the potential of

structuring the organization of water molecules, new assemblies in water can be explored.

2.2.8 Homeotropic alignment (fingerprint texture) of chiral nematic phase of nonamphiphilic

liquid crystals

Homeotropic alignment is difficult to obtain for nonamphiphilic chiral nematic liquid

crystals. In a nematic liquid crystal, the homeotropic alignment on a surface occurs when the

directors of the mesogens are perpendicular to the surface. For a chiral nematic (or cholesteric)

phase, an entirely homeotropic alignment for all mesogens is impossible, as the director of each

neighboring layer continue to twist with a certain angle. It turns out that homeotropic alignment

is very difficult to attain for the achiral nonamphiphilic lyotropic liquid crystal. To date (2013),

there is not yet a report on homeotropic alignment of this class of liquid crystal (5 DSCG or

other dyes). Homeotropic alignment of conventional thermotropic liquid crystals, however, is

easily attainable and is routinely prepared by controlling the surface chemistry and structure. The

reason for this “homeotropic” difficulty for nonamphiphilic lyotropic liquid crystal is not clear.

From a macroscopic point of view, any long thread-like structure, wire-like or spaghetti-like or

else, does not stand vertical to a surface presumably due to weight. At molecular level, the

interaction between the long axis of the assembly and the surface is of close to infinitely larger

area than between the point of the assembly and the surface. For this reason, the interaction of

- 50 -

the long axes of the assemblies and the surface over a large area outweighs the interaction

between the endpoint of the assemblies and the surface.

In a homeotropic alignment of chiral nematic liquid crystals the helical axis is oriented

parallel to a surface, with directors in the planes of assembly continuously rotate along the

surface. For this surface alignment for chiral nonamphiphilic liquid crystals, some vertical (and

thus homeotropic alignment) assembly alignment on a surface is inevitable. Because the surface

control strongly favors planar alignment for thread or assembly-based lyotropic liquid crystals,

we seek to reduce the surface effect by increasing the cell thickness to achieve fingerprint

texture, which provides additional, and likely stronger evidence, for the existence of a lyotropic

chiral nematic phase.

Attaining fingerprint texture of nonamphiphilic chiral lyotropic liquid crystals. To explore

the existence of chiral nematic phase, we study the effect of cell thickness and liquid crystal

alignment by preparing a sandwiched sample of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-

mannitol in a wedge cell composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold

films. In the wedge cell, the thickness of the cell continuously increases from the thin end (13

µm) to the thick end (1 mm) (Figure 2.19 A). We observe that with thin cell thickness, planar

alignment is observed as the color of the optical images does not change much with rotation

(Figure 2.19 B and C). However, as the cell thickness increases to 555 um, fingerprint region

began to appear (Figure 2.19 D). As the cell thickness increases further, fingerprint region is

observed over a larger area (Figure 2.19 E). The helical pitch of the sample of 13.4 wt% 5 DSCG

mixed with 16.2 wt% D-mannitol observed in this wedge cell is 25~28 µm. These results are

consistent with the notion that with thin spacer, the surface alignment is dominant. As the

thickness of liquid crystal cell increases, the liquid crystal is less influenced by the surface, and

- 51 -

the twist-induced (twist from the chiral nematic) vertical or homeotropic alignment becomes

possible.

Figure 2.19 (A) Schematic representation of a wedge cell composed of

HS(CH2)10(OCH2CH2)3OH SAMs supported by gold films. Optical images (cross polarizers) of

13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol when the thickness of the cell is (B) 13

µm, (C) 296 µm, (D) 555µm, and (E) 778 µm. Scale bar = 76 µm. The arrows indicate direction

of gold deposition projected onto the glass slides, and the orientation of the sample relative to the

cross polarizers.

With this interpretation, we examined if fingerprint texture for this liquid crystal can be

achieved in a uniformly thick cell. Using a cell composed of SAM and spacers of 1 mm to

provide a uniform and thick spacing, we also obtained fingerprint texture for sample of 18 wt%

13 µm

1 mm

27 mm

Thickness of

the cell

13 µm

296 µm

555 µm

778 µm

Wedge cell

S(CH2)10(OCH2CH2)3OHSurface is 45o Au

Pitch ~ 25 µm

Pitch ~ 28 µm

B

C

D

E

A

- 52 -

5 DSCG-(R,R)-diviol, which is a strong evidence that the liquid crystal phase formed is chiral

nematic (Figure 2.20 A). It is interesting to find that the liquid crystal phase of 13.4 wt%

5 DSCG mixed with 16.2 wt% D-mannitol has a longer helical pitch (17 ~19 µm) than that of

the chiral mesogen (10 µm) (Figure 2.20 B). Considering the distance between neighboring

thread assembly is ~ 6nm as evidenced in the cryo-TEM, we could calculate the degree of

rotation between neighboring assemblies in each case using Equation 2.1. P represents the

helical pitch. In the sample of 18 wt% 5 DSCG-(R,R)-diviol, each thread-like assembly twists

~0.22 degree with respect to the neighboring ones and that angle is 0.13 degree in the case of

13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol.

Eqn 2.1

Figure 2.20 Optical images (cross polarizers) of (A) 13.4 wt% 5 DSCG mixed with 16.2 wt% D-

mannitol and (B) 18.0 wt% 5 DSCG-(R,R)-diviol when the samples were sandwiched between

- 53 -

obliquely deposited gold films supporting HS(CH2)10(O CH2CH2)3OH in a 1 mm thick cell.

Schematic representations of the distance and the twisting angle between two neighboring

assemblies are shown below the images.

Optical images with detailed rotations of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-

mannitol in a wedge cell (composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold

films) with different cell thickness are shown in Figure 2.21 and 2.22.



films when the thickness of the cell ranges from 13 to 222 µm.

- 54 -



films when the thickness of the cell ranges from 296 to 778 µm.

The above results are all on surfaces that provide uniform planar alignment of liquid crystals

(both thermotropic and this class of nonamphiphilic lyotropic liquid crystals). The observed

homeotropic alignment is caused by the splay strain and facilitated by the large thickness (see

below). To show that the chiral nematic phase forms on any surface for this class of liquid

crystal, we used a plane glass slides and wedge cells. Optical images with detailed rotations of

- 55 -

13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol in a wedge cell (composed of plain glass

slides) with different cell thickness are shown in Figure 2.23


D-mannitol in a wedge cell composed of plain glass slides when the thickness of the cell ranges

from 13 to 778 µm.

- 56 -

For the planar alignment on the thin end, there are more domains on the plain glass slides

than on the SAMs supported by obliquely deposited gold films. This result is consistent with

uniform alignment on SAMs and random alignment on plane glass. As the samples are rotated

between the cross polarizers, the color did not change in either samples. These results are

consistent with a homogeneous planar alignment and random planar alignment of the chiral

nematic liquid crystal phase on oblique gold-supported SAMs and on plane glasses, respectively.

The helical pitch of the sample of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol is not

uniform. For example, when cell thickness is 555, 593, and 778 µm, the helical pitch is 25, 22,

and 28 µm, respectively (Figure 2.24). The correlation between the size of the pitch and cell

thickness is not yet clear.

Figure 2.24 Optical images of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol in a wedge

cell composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold films when the cell

thickness is (A) 555 µm, (B) 593 µm, and (C) 778 µm.

Splay strain-induced homeotropic alignment. It is interesting that the “critical thickness” of

the wedge cell at which fingerprint region begins to show and the size of the helical pitch vary

with the shape of the wedge cell. For example, when a sample of 13.4 wt% 5 DSCG mixed with

16.2 wt% D-mannitol was viewed in a 27 mm wedge cell with an angle of 2.12o composed of

HS(CH2)10(OCH2CH2)3OH SAMs supported on gold films, the critical thickness that the

76 µm

Helical pitch ~ 28 µmHelical pitch ~ 22 µm

Helical pitch ~ 25 µm

A B C

- 57 -

fingerprint region begins to show is ~555 µm and the helical pitch observed is ~25 µm (Figure

2.25 A). When the same sample was viewed in a 17 mm long wedge cell with an angle of 3.18o,

the critical thickness is ~222 µm and the helical pitch observed is ~18 µm (Figure 2.25 B).

Figure 2.25 Optical images (cross polarizers) of 13.4 wt% 5 DSCG mixed with 16.2 wt% D-

mannitol viewed in a wedge cell with (A) 27 mm and (B) 17 mm cell length. Both of the wedge

cells are composed of HS(CH2)10(OCH2CH2)3OH SAMs supported by gold films and the spacer

is 13 µm on one end and 1mm on the other. We note that the wedge cell with 1 mm thickness

spacer provide an asymmetry triangle rather than isosceles triangle.

Examining the fingerprint texture of the liquid crystal in Figure 2.21 A, the longest

undisrupted lines (by defects) spanned a distance of > 849 µm, which is larger than the critical

thickness 555 µm at which the fingerprint texture started to appear. This result indicates that the

threads aligned perpendicular to the surface are shorter than the threads parallel to the surface in

13 µm

1 mm

S(CH2)10(OCH2CH2)3OHSurface is 45o Au

> 849 µm

A

>1028µm

B

Thickness of wedge cellDistance from the thin end 15 mm

555 µm

4 mm

222 µm

Helical pitch 25 µm 18 µmAngle of wedge cell 2.12o 3.18o

13 µm

1 mm

2.12o 3.18o

17 mm27 mm

A B

- 58 -

the fingerprint domains of the liquid crystals. Interestingly, a more wedged cell (wedge angle

3.18o) caused the fingerprint texture (and thus homeotropic alignment) to occur at a relatively

small thickness (222 µm) whereas a less wedged cell (wedge angle 2.12o) caused the fingerprint

to occur at 555 µm thickness. As a larger wedge angle causes more splay strain in liquid crystals,

this result suggests that although the thickness of the cell is important, the strong splay strain can

also induce a forced homeotropic alignment of chiral nematic (i.e. fingerprint texture). As two

neighboring assemblies are splayed, the energy for a homeotropic alignment to fill up the void

space between the splayed assemblies is lowered (Figure 2.26 B).

Figure 2.26 Schematic representation of splay deformation of liquid crystals in a wedge cell of

(A) small angle and (B) large angle.

Significance of chiral nematic phase created by assemblies of chiral mesogens in water

versus achiral mesogens mixed with chiral molecules in water. In comparison with conventional

chiral nematic for thermotropic liquid crystals, the key question for a chiral nematic version of

lyotropic liquid crystal is how the chiral information is being transmitted between the assemblies

that are separated by a rather large volume of water molecules, more than 80% by weight. In

conventional thermotropic liquid crystal, the mesogens are in van der Waals contact; the chiral

information is transmitted through dispersion interaction between molecules and manifested by

θsθL

A B

- 59 -

molecules twisting relative to each other continuously throughout each domain in the liquid

crystal sample. In lyotropic liquid crystals, the assemblies are not in direct van der Waals contact

with each other. There are two scenarios. One is chiral mesogen in water, the other is achiral

mesogen mixed with a chiral molecule in water. We discuss each separately and first, the case of

chiral mesogen in water.

We believe that there are three possible modes for chemical information transmission

between the assemblies of chiral mesogens in water. First, the assemblies are separated by pure

water, and there is no or negligible amount of chiral mesogens. For both isodesmic and

cooperative assemblies, this case is probable when the self-association constant is high. In this

case chiral information is transmitted through an achiral solvent medium (Figure 2.27). For a

rough estimate in our case, the two neighboring assemblies are separated by at least 14 to 18

water molecules (Figure 2.27 and 2.28). This number is estimated by assuming a linear hydrogen

bonding arrangement between the water molecules.

- 60 -


if the molecular assemblies (thread model) are separated by pure water. The number of water

molecules between neighboring thread-like assemblies is estimated.

HO

H

H

OH

HO

H

H

OH

O

O O

OH OH

O

O

O

NaO2C CO2Na

… …+-+ - +-+ -

-- --

… …+-+ - +-+ -

-- --+ ++ + ++

3.4 Å

60 Å

7.0 Å 0.96 Å

1.97 Å

+-+ - +-+ -

Number of water molecules between

neighboring thread-like assemblies

In the thread model

HO

H

H

OH

HO

H

H

OH

…

…

- 61 -


if the molecular assemblies (H-stacking model) are separated by pure water. The number of

water molecules between neighboring assemblies is estimated.

Second, there may be some amount of chiral mesogens solvated as individual free molecules

in the aqueous solvent between the threads (Figure 2.29 and 2.30). These molecules may

function as a chiral dopant to twist the molecular assemblies. This scenario is possible, but the

transmission of chiral information is still mediated by the solvent molecules because the free

molecules are still solvated.

In the H-stacking model

O

O O

OH OH

O

O

O

NaO2C CO2Na

60 Å

18 Å



HO

H

H

OH

HO

H

H

OH

0.96 Å

1.97 Å

HO

H

H

OH

HO

H

H

OH

…

…

- 62 -


with free solvated chiral mesogens between the molecular assemblies (thread model). The

number of water molecules between neighboring thread-like assemblies is estimated.

HO

H

H

OH

HO

H

H

OH

O

O O

OH OH

O

O

O

NaO2C CO2Na

… …+-+ - +-+ -

-- --

… …+-+ - +-+ -

-- --+ ++ + ++

3.4 Å

60 Å

7.0 Å 0.96 Å

1.97 Å

+-+ - +-+ -



In the thread model

HO

H

H

OH

HO

H

H

OH

…

…

+-+ -

- 63 -


with free solvated chiral mesogens between the molecular assemblies (H-stacking model). The

number of water molecules between neighboring assemblies is estimated.

Third, the chiral mesogens branch out (or grow out) of the assemblies and connect to the

neighboring ones (Figure 2.31 and 2.32). Through this connection, the chiral information is

somehow transmitted and manifested as twisting neighboring assemblies. This scenario is

unlikely because such a cross-linking network will cause gel formation instead of a fluid phase.


O

O O

OH OH

O

O

O

NaO2C CO2Na

60 Å

18 Å



HO

H

H

OH

HO

H

H

OH

0.96 Å

1.97 Å

HO

H

H

OH

HO

H

H

OH

…

…

- 64 -


between the molecular assemblies (thread model).


between the molecular assemblies (H-stacking model).

In the case of achiral mesogens mixed with chiral molecules in water, the transmission of

chemical information is not likely through pure water as the concentration of the chiral

molecules are high. However, because the chiral molecules are small molecules, the transmission

O

O O

OH OH

O

O

O

NaO2C CO2Na

… …+-+ - +-+ -

-- --

… …+-+ - +-+ -

-- --+ ++ + ++

+-+ - +-+ -In the thread model

… …+-+ - +-+ -

-- --

… …+-+ - +-+ -

-- --+ ++ + ++


O

O O

OH OH

O

O

O

NaO2C CO2Na

- 65 -

of the chemical information must still go through many molecular interactions between the chiral

molecules and the solvent water molecules, and eventually the neighboring assemblies. In an

aqueous solution, hydrogen bonds are ubiquitous. It is important to note that one does not know

the contribution from the dispersion force which is present regardless the types of functional

groups. What types of the molecular interactions are contributing and enabling the chiral

information transmission is a still an open question.

2.3 Conclusions and perspectives

In conclusion, enantiomers of 5 DSCG-(R,R)-diviol 1a and 5 DSCG-(S,S)-diviol 1b, and

achiral 5 DSCG-meso-diviol 1c were individually synthesized. Nuclear Overhauser Effect in

proton NMR spectroscopy revealed that the 5 DSCG-meso-divol 1c adopted a more “folded”

conformation while the chiral 5 DSCG-diviol had a more “stretched” conformation. Circular

dichroism suggested a stable chiral molecular conformation by 5 DSCG-(R,R)-diviol 1a. Proton

NMR revealed significant upfield chemical shift and peak broadening around 72 mM. Cryo-

TEM showed visible assembly at 5 wt% (~103 mM), and that chiral stereoisomer 5 DSCG-

(R,R)-diviol 1a forms more elongated assembly at low concentration in water, and exhibited

more crossings between the assemblies at high concentration than those shown by generic

5 DSCG in water. Chiral stereoisomers formed chiral nematic liquid crystal phases while the

meso isomer did not any liquid crystal phase. While generic 5 DSCG in water aligns uniformly

on SAMs supported by obliquely deposited gold films, 5 DSCG-(R,R)-diviol 1a exhibited

nonuniform alignment of on the same surfaces. Splay strain-induced homeotropic alignment of

liquid crystals formed by 13.4 wt% 5 DSCG mixed with 16.2 wt% D-mannitol. Fingerprint

texture of 18 wt% 5 DSCG-(R,R)-diviol can be observed with thick cell. Together, these results

suggest that the hydrated assemblies of chiral molecule 5 DSCG-(R,R)-diviol 1a interact with

- 66 -

each other while being separated by relatively large distance (6 nm) in water, causing a lyotropic

chiral nematic liquid crystal phase. Further works include investigate the assembly structure of

chiral 5 DSCG-diviol at higher concentration than already studied using cuvette with shorter

path length, and further investigate the mechanism and chiral features of the assembly thus

formed.


Chemicals.

All reagent grade starting materials were obtained from commercial supplies (Sigma-Aldrich,

TCI, Alfa Aesar, and Acros) and used as received. Anhydrous solvents were purchased from

Sigma-Aldrich. Water used to prepare all buffers and solutions had resistivity of 18 MΩ cm

(Millipore, Billerica, MA).

General procedure.

All air sensitive reactions were performed in oven dried glassware under an atmosphere of argon

unless otherwise notified. Analytical thin layer chromatography was performed on EM silica gel

60 F254 glass plates (0.25 mm). Visualization of analytical thin layer chromatography was

achieved using UV absorbance (254 nm), KMnO4, and ceric ammonium molybdate stains. Flash

column chromatography was performed using SiliaFlash P60 silica gel (40-60 Å) from SiliCycle,

Inc (Quebec City, Quebec, Canada). 1D 1H and

13C NMR spectra were recorded on a Bruker

Advance DPX-300 spectrometer. Chemical shifts are reported in ppm, using tetramethylsilane as

the internal standard. ROESY spectra were acquired at a temperature of 300 K recorded on a

Bruker AVANCE-II NMR spectrometer equipped with a 5 mm TCI CryoProbe with z-gradient

operating at a frequency of 600.13 MHz for 1H. For 2D ROESY experiments,

114 a total of 320 t1

increments of 2048 complex points with 16 scans each were collected. The ROESY spin lock

- 67 -

was set to 250 ms for all experiments. NMR data were processed using Bruker TOPSPIN 3.0

software by setting forward linear prediction to 640 points and zero filled to 2048 points for F1

dimension.Mass spectra were measured using a MAT 95 XP mass spectrometer, carried out by

the Mass Spectroscopy Facility at Indiana University

Circular dichroism.

Wavelength scan and thermal study of chiral 5 DSCG-diviols were performed on an AVIV 420

CD spectrometer (AVIV Biomedical, Inc., Lakewood, NJ) using a 1 mm path length cuvette. For

thermal study, the temperature was increased at a rate of 2 oC/min and samples were equilibrated

for 60 s prior to each data collection.

UV.

UV measurements were performed on an Agilent 8453 spectrometer (Agilent Technologies,

Santa Clara, CA) using a 1mm or 1cm path length cuvette at ambient temperature.

Cleaning of glass substrates.109

Substrates used for gold films were Fisher’s Finest premium microscope slides purchased from

Fisher Scientific (Pittsburgh, PA). Prior to gold deposition, the glass slides were cleaned with

Piranha solution. The slides were soaked in Piranha solution (7 parts of 35% aqueous hydrogen

peroxide solution and 3 parts of concentrated sulfuric acid) for 45 min at 70 oC. Warning!

Piranha solution is extremely corrosive and can potentially detonate when mixed with significant

amounts of oxidizable materials. It is advised to neutralize the Piranha solution with sodium

hydroxide before disposal. After cooling to ambient temperature, the Piranha solution was

poured off and the glass slides rinsed 20 times sequentially with deionized water, followed by 10

times with ethanol, and then 10 times with methanol. The cleaned slides were dried individually

with a stream of nitrogen gas and kept in an 80 oC oven overnight.

- 68 -

Deposition of Gold Films.

Deposition of gold films was done following literature procedure.115

Semitransparent gold films

were deposited onto the glass substrate using an electron beam evaporation system (Thermionics,

Port Townsend, WA). A layer of titanium (~70 Å) was deposited first to enhance the adhesion of

the gold. A layer of gold (~280 Å) was then deposited at an oblique angle of 45o to the surface

normal of the substrate. The rate of deposition was kept at 0.2 Å/s for both gold and titanium and

the pressure was maintained at no higher than 2 × 10-6

Torr throughout the deposition.

Preparation of SAMs on obliquely deposited gold films.

Gold films deposited on microscope glass slides were cut into strips (8 mm × 2.5 mm) along the

direction of gold deposition. The strips were rinsed with 200 proof ethanol (3 ×), dried with a

stream of nitrogen gas and then immersed in ethanolic solutions containing 2 mM of thiols

overnight (~15 h). Each strip was rinsed with 200 proof ethanol (3 ×), dried with a stream of

nitrogen gas and used immediately.

Assembly of optical cells and characterization of birefringence.

Aqueous solutions of 5 DSCG and 5 DSCG-diviols were prepared by dissolution in deionized

water in vials. The vials were subjected to vortex mixing for at least 1 min before use. The liquid

crystal samples were assembled in a sandwiched cell composed of two microscope glass slides or

gold films supporting SAMs with one sheet of Saran Wrap® (13 – 15 µm) inserted in between as

a spacer. A square hole was cut on the sheet of Saran Wrap®

to accommodate the liquid crystal

sample. The liquid crystal samples were loaded between the slides in the square hole and sealed

with binder clips on each side to prevent the evaporation of solvent. The samples were viewed

under crossed polarizers on an Olympus BX51 polarizing microscope and were rotated form an

arbitrary starting point to record the birefringence.

- 69 -

Sample preparation for cryo-TEM.

The samples were prepared by applying ~5 µL droplet of the sample on the microperforated

cryo-TEM grid, followed by blotting the grid with a filter paper to produce a thin layer. The grid

was then rapidly plunged into liquid ethane that had been cooled to near liquid nitrogen

temperature to freeze the sample.

General information of synthesis.


unless otherwise notified. Analytical thin layer chromatography was performed on EM silica gel

60 F254 glass plates (0.25 mm). Visualization of analytical thin layer chromatography was

achieved using UV absorbance (254 nm), KMnO4, and ceric ammonium molybdate stains. Flash

column chromatography was performed using SiliaFlash P60 silica gel (40-60 Å) from SiliCycle,

Inc (Quebec City, Quebec, Canada). 1D 1H and

13C NMR spectra were recorded on a Bruker

Advance DPX-300 spectrometer. 1H chemical shifts are reported in ppm, downfield from

tetramethylsilane using residual CHCl3 as the internal standard (δ 7.26 ppm). 13

C chemical shifts

are reported in ppm, downfield from tetramethylsilane relative to CDCl3 (δ 77.0 ppm), DMSO-d6

(δ 39.5 ppm) and CD3CN (δ 1.94 ppm). ROESY spectra were acquired at a temperature of 300 K

recorded on a Bruker AVANCE-II NMR spectrometer equipped with a 5 mm TCI CryoProbe

with z-gradient operating at a frequency of 600.13 MHz for 1H. For ROESY experiments,

114 a

total of 320 t1 increments of 2048 complex points with 16 scans each were collected. The

ROESY spin lock was set to 250 ms for all experiments. NMR data were processed using

Bruker TOPSPIN 3.0 software by setting forward linear prediction to 640 points and zero filled

to 2048 points for F1 dimension. Mass spectra were measured using a MAT 95 XP mass

spectrometer, carried out by the Mass Spectroscopy Facility at Indiana University.

- 70 -

Synthetic procedures and spectral data.

OO O

O

OH

2a

1,2:4,5-bis-acetal 2a: To a stirred suspension of L-arabitol (3.008 g, 19.37 mmol) in anhydrous

THF (48 mL) was added 2,2-dimethoxypropane (5.1 mL, 41 mmol) at ambient temperature. The

reaction mixture was refluxed for 15 min and then L-(-)-camphorsulfonic acid (461.2 mg, 1.946

mmol) was added at refluxing temperature. After refluxing for another 5 minutes, the reaction

was quenched with 2 M NaOH (11 mL) at refluxing temperature. The solvent was removed

under reduced pressure and residue was extracted with diethyl ether (11 mL × 3). The combined

organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The

colorless oil residue was dissolved in anhydrous methylene chloride (48 mL) and treated with

Et3N (2.9 mL). The reaction mixture was then brought to reflux and succinic anhydride (488.9

mg, 4.837 mmol) was added. After refluxing for 5 h, the reaction was quenched with saturated

aqueous NaHCO3 (7.0 mL) at refluxing temperature. The mixture was cooled to ambient

temperature and the organic layer was dried over MgSO4, filtered, and concentrated under

reduced pressure. Flash column chromatography (SiO2; hexane:ethyl acetate, 5:1 to 3:1) provided

1,2:4,5-bis-acetal 2a: (3.0925 g, 69 %) as a pale yellow oil. TLC Rf = 0.48 (hexane:ethyl acetae,

1:1). 1H NMR (300 MHz, CDCl3): δ 4.26 (ddd, 1H, J = 6.6, 6.6, 4.2 Hz), 4.13 (m, 2H), 4.04 (dd,

1H, J = 10.8, 5.4 Hz), 3.98 (m, 1H), 3.92 (dd, 1H, J = 8.3, 6.6 Hz), 3.43(m, 1H), 2.29 (d, 1H, J =

6.3 Hz), 1.45 (s, 3H), 1.41 (s, 3H), 1.39 (s, 3H), 1.36 (s, 3H).

OO O

O

OS

N

3a

N

- 71 -

Imidazolyl thiocarbonyl derivative 3a: To a stirred solution of 1,2:4,5-bis-acetal 2a (2.241 g,

9.657 mmol) in anhydrous 1,2-dichloroethane (32 mL) was added 1,1’-thiocarbonyl diimidazole

(2.249 g, 11.36 mmol) at ambient temperature. The resulting brown solution was refluxed for 7 h

and then cooled to ambient temperature. The solvent was removed under reduced pressure. Flash

column chromatography (SiO2; hexane:ethyl acetate 3:1) provided imidazolyl thiocarbonyl

derivative 3a (3.17 g, 96 %) as a yellow oil. TLC Rf = 0.36 (hexane:ethyl acetate, 1:1). 1H NMR

(300 MHz, CDCl3): δ 8.34 (m, 1H), 7.63 (m, 1H), 7.03 (m, 1H), 5.88 (dd, 1H, J = 5.7, 3.3 Hz),

4.45(m, 2H), 4.08 (m, 3H), 3,83 (dd, 1H, J = 9.0, 6.0 Hz), 1.39 (s, 3H), 1.33 (s, 9H); 13

C NMR

(75 MHz，CDCl3): δ 184.5, 137.0, 131.0, 118.0, 109.7, 109.6, 80.9, 74.8, 74.5, 65.8, 65.2, 26.3,

26.1, 25.0, 24.9. HRMS: Cacld. for (M + H)+: 343.1328, found: 343.1314.

OO O

O

4a

1,2:4,5-bis-acetal 4a: Benzoyl peroxide (202.9 mg, 0.821 mmol) was added to a stirred solution

of imidazolyl thiocarbonyl derivative 3a (1.444 g, 4.223 mmol) in triethylsilane (25 mL) at

refluxing temperature. The reaction mixture was refluxed for 2 h during which similar amount of

benzoyl peroxide was added after 30 min, 60 min, and 90 min, respectively. The solvent was

then removed under reduced pressure. Flash column chromatography (SiO2; hexane:ethyl

acetate, 3:1) provided 1,2:4,5-bis-acetal 4a as a light yellow oil which contained some aromatic

impurities and triethyl silane but was good enough to be carried on. TLC Rf = 0.65 (hexane:ethyl

acetate, 1:1).

HO OH

OH OH

5a

(2R,4R)-pentane-1,2,4,5-tetraol 5a: 0.5 M H2SO4 (5.4 mL, 2.7 mmol) was added to a stirred

solution of 1,2:4,5-bis-acetal 4a (1.508 g, impure) in ethanol (5.4 mL) at ambient temperature.

- 72 -

The reaction mixture was refluxed for 4.5 h and then BaCO3 was added to adjust the pH to 7.

The resulting milky mixture was filtered and the white solid thus collected was heated with

MeOH (5 mL) at 50 oC for 10 min. The combined filtrate was concentrated under reduced

pressure. Flash column chromatography (SiO2; DCM:MeOH, 7:3) provided (2R,4R)-pentane-

1,2,4,5-tetraol 5a (546.7 mg, 95 % over 2 steps) as a white solid. TLC Rf = 0.25 (DCM:MeOH,

7:3). 1H NMR (300 MHz, MeOH-d4):

δ 3.85 (m, 2H), 3.47 (m, 4H), 1.52 (dd, 2H, J = 7.1, 5.6

Hz); 13

C NMR (75 MHz, MeOH-d4): δ 70.1, 67.9, 38.1. HRMS: Cacld. for (M + Na)+:

159.0633, found: 159.0631.

TPSO OTPS

OH OH

6a

(2R,4R)-2,4-Dihydroxypentane-1,5-diyl Bis-(2,4,6-triisopropyl-1-benzenesulfonate) 6a: To a

stirred solution of (2R,4R)-Pentane-1,2,4,5-tetraol 5a (92.2 mg, 0.678 mmol) in anhydrous

pyridine (0.55 mL) was added 2,4,6-triisopropyl benzenesulfonyl chloride (529.2 mg, 1.695

mmol) at 0 oC. The pale yellow cloudy mixture was stirred at ambient temperature for 13 h and

then the solvent removed under reduced pressure. Flash column chromatography (SiO2;

hexane:acetone, 3:1) provided (2R,4R)-2,4-Dihydroxypentane-1,5-diyl bis-(2,4,6-triisopropyl-1-

benzenesulfonate) 6a (325.5 mg, 72 %) as a white fluffy solid. TLC Rf = 0.70 (hexane:ethyl

acetate, 1:1). 1H NMR (300 MHz, CDCl3): δ 7.20 (s, 4H), 4.23 (m, 2H), 4.10 (m, 6H), 3,98 (dd,

2H, J = 10.2, 6.9 Hz), 2.92 (septet, 2H, J = 6.9 Hz), 2.87 (br, 2H), 1.64 (t, 2H, J = 5.7 Hz), 1.27

(d, 24H, J = 6.9 Hz), 1.26 (d, 12H, J = 6.9 Hz).

TPSO OTPS

O O

7a

Acetonide 7a: To a stirred solution of (2R,4R)-2,4-Dihydroxypentane-1,5-diyl bis-(2,4,6-

triisopropyl-1-benzenesulfonate) 6a (472.8 mg, 0.708 mmol) in acetone (7.1 mL) was added 2,2-

- 73 -

dimethoxypropane (0.89 mL, 7.1 mmol) and p-toluenesulfonic acid (18.2 mg, 0.094 mmol) at

ambient temperature. The resulting colorless solution was stirred at ambient temperature

overnight (23 h). The reaction mixture was quenched with aqueous saturated NaHCO3 (8 mL)

and the solvent removed under reduced pressure. The residue was extracted with methylene

chloride (4 mL × 3) and the combined organic layer dried over Na2SO4, filtered, and

concentrated under reduced pressure. Flash column chromatography (SiO2; hexane:ethyl acetate,

3:1) provided acetonide 7a (473.4 mg, 94 %) as a pale yellow solid. TLC Rf = 0.64 (hexane:ethyl

acetate, 3:1). 1H NMR (300 MHz, CDCl3): δ 7.19 (s, 4H), 4.13 (septet, 4H, J = 6.9 Hz), 4.03 (m,

6H), 2.92 (septet, 2H, J = 6.9 Hz), 1.66 (t, 2H, J = 7.7 Hz), 1.26 (d, 36H, J = 6.9 Hz), 1.22 (s,

6H); HRMS: Cacld. for (M + Na)+: 731.3627, found: 731.3651.

I I

O O

8a

Diiodide 8a: To a stirred solution of (2R,4R)-2,4-Dihydroxypentane-1,5-diyl bis-(2,4,6-

triisopropyl-1-benzenesulfonate) 7a (938.1 mg, 1.325 mmol) in 2-butanone (13 mL) was added

sodium iodide (1.985 g, 13.24 mmol) followed by anhydrous pyridine (0.54 mL, 6.7 mmol) at

ambient temperature. The resulting light yellow suspension was refluxed overnight (25 h). The

reaction mixture was cooled to ambient temperature and the solvent was removed under reduced

pressure. The residue was treated with water (10 mL), extracted with ethyl acetate (10 mL × 4),

dried over MgSO4, filtered and concentrated under reduced pressure. Flash column

chromatography (SiO2; hexane to hexane:ethyl acetate, 15:1) provided diiodide 8a (484.7 mg, 92

%) as a colorless oil. TLC Rf = 0.55 (hexane:ethyl acetate, 10:1). 1H NMR (300 MHz, CDCl3): δ

3.85 (m, 2H), 3.18 (m, 4H), 1.78 (t, 2H, J = 3.3 Hz), 1.39 (s, 6H); 13

C NMR (75 MHz，CDCl3):

δ 101.6 (C), 66.9 (CH), 38.6 (CH2), 24.6 (CH3), 8.9 (CH2).

- 74 -

I I

OH OH

9a

Diiodo diol 9a: To a stirred solution of diiodide 8a (100.9 mg, 0.255 mmol) in THF (2.5 mL)

was added 1N HCl (2.6 mL) at ambient temperature. The reaction mixture was stirred at ambient

temperature for 45 min. NaHCO3 (powder) was added to adjust pH to 7 and then the organic

solvent was removed under reduced pressure. The residue was extracted with ethyl acetate (3 mL

× 4), dried over MgSO4, filtered and concentrated under reduced pressure. Diiodo diol 9a was

obtained as a white solid which was in good enough quality to be carried on without further

purification. TLC Rf = 0.14 (hexane:ethyl acetate, 3:1). 1H NMR (300 MHz, CDCl3): δ 3.94 (m,

2H), 3.39 (dd, 2H, J = 10.2, 4.5 Hz), 3,28 (dd, 2H, J = 10.2, 6.9 Hz), 2.54 (d, 2H, J = 4.5 Hz),

1.86 (dd, 2H, J = 6.3, 5.4 Hz); 13

C NMR (75 MHz, CDCl3): δ 68.4, 41.6, 15.0.

O O

HO OH

O O

OH OH

10a

Hydroxyacetophenone 10a: To a stirred solution of diiodo diol 9a (90.7 mg, 0.255 mmol,

assuming quantative yield from previous step) in 2-propanol (5.1 mL) was added 2,6-

dihydroxyacetophenone (240.3 mg, 1.532 mmol) at ambient temperature. The resulting yellow

solution was heated to reflux and a mixture of KOH (118.5 mg, 1.901 mmol) in water (0.20 mL)

and 2-propanol (0.26 mL) was added. The reaction mixture was refluxed for 24 h, cooled to

ambient temperature, and then concentrated under reduced pressure. The dark brown residue was

treated with ethyl acetate (5 mL) and filtered. The filtrate was concentrated under reduced

pressure. Flash column chromatography (SiO2; hexane:ethyl acetate, 5:1 to 3:1 to 1:1 to 1:2)

provided hydroxyacetophenone 10a (87.9 mg, 85 %) as a light yellow solid. TLC Rf = 0.24

(hexane:ethyl acetate, 1:2). 1H NMR (300 MHz, DMSO-d6): δ 12.21 (br, 2H), 7.34 (t, 2H, J =

- 75 -

8.3 Hz), 6.56 (d, 2H, J = 8.4 Hz), 6.49 (d, 2H, J = 8.1 Hz), 4.97 (d, 2H, J = 5.4 Hz), 4.08 (m,

2H), 3.99 (m, 4H), 2.62 (s, 6H), 1.64 (t, 2H, J = 6.3 Hz); HRMS: Cacld. for (M + Na)+:

427.1369, found: 427.1354.

O

O O

OH OH

O

O

O

MeO2C CO2Me11a

Diester 11a: To a freshly prepared NaOMe solution (206.8 mg of sodium cube in 18 mL of

anhydrous MeOH) was added anhydrous diethyl ether (36 mL) followed by dimethyl oxalate

(1.065 g, 8.931 mmol). After the solid disappeared, hydroxylacetophenone 10b (360.3 mg, 0.892

mmol) was added to the reaction mixture. The resulting yellow solution was refluxed for 24 h,

cooled to ambient temperature, and then concentrated under reduced pressure. The yellow solid

residue was treated with water (45 mL) and acidified with concentrated HCl (0.20 mL) until pH

~ 2. The yellow precipitated was filtered off, dissolved in anhydrous MeOH, and treated with

concentrated HCl (2.7 mL). The reaction mixture was refluxed for 40 min and then cooled to

ambient temperature. The pale brown precipitate was filter off and found to be pure to be carried

on (191.7 mg, 40 %). TLC Rf = 0.10 (ethyl acetate:ethanol, 9:1). 1H NMR (300 MHz, DMSO-

d6): δ 7.73 (t, 2H, J = 8.4 Hz), 7.22 (d, 2H, J = 8.4 Hz), 7.09 (d, 2H, J = 8.4 Hz), 6.75 (s, 2H),

4.07 (m, 6H), 3.92 (s, 6H), 3.50 (br, 2H), 1.78 (t, 2H, J = 6.0 Hz); 13

C NMR (75 MHz, DMSO-

d6): δ 176.6, 160.6, 158.6, 157.1, 150.1, 135.4, 115.4, 114.8, 110.4, 109.7, 74.4, 65.2, 53.4, 37.2.

HRMS: Cacld. for (M + Na)+: 563.1165, found: 563.1149.

O

O O

OH OH

O

O

O

NaO2C CO2Na1a

5 DSCG-(R,R)-diviol 1a: To a stirred suspension of diester 11a (101.5 mg, 0.188 mmol) in

ethanol (9.4 mL) was added aqueous NaOH solution (2.5 M, 0.15 mL, 0.38 mmol) at ambient

- 76 -

temperature. The reaction mixture was refluxed for 10 min, cooled to ambient temperature, and

then at 0 oC for 20 min. After filtration and drying under high vacuum for 24 h, 1a was obtained

as brown solid (91.2 mg, 89%). 1H NMR (300 MHz, D2O): δ 7.60 (t, 2H, J = 8.4 Hz), 7.08 (d,

2H, J = 8.4 Hz), 6.89 (d, 2H, J = 8.4 Hz), 6.64 (s, 2H), 4.37 (m, 2H), 4.13 (m, 4H), 1.97 (t, 2H, J

= 6.2 Hz); 13

C NMR (75 MHz, D2O): δ 181.4, 165.1, 157.2, 157.2, 156.2, 135.2, 113.0, 112.0,

110.6, 108.2, 73.1, 65.5, 35.5. HRMS: Cacld. for (M + Na)+: 579.0483, found: 579.0491.

OO O

O

OH

2b

1,2:4,5-bis-acetal 2b:116

To a stirred suspension of D-arabitol (3.004 g, 19.55 mmol) in

anhydrous THF (49 mL) was added 2,2-dimethoxypropane (5.1 mL, 41 mmol) at ambient

temperature. The reaction mixture was refluxed for 15 min and then L-(-)-camphorsulfonic acid

(463.7 mg, 1.956 mmol) was added at refluxing temperature. After refluxing for another 3

minutes, the reaction was quenched with 2 M NaOH (11 mL) at refluxing temperature. The

solvent was removed under reduced pressure and residue was extracted with diethyl ether (11

mL × 3). The combined organic layer was dried over MgSO4, filtered, and concentrated under

reduced pressure. The colorless oil residue was dissolved in anhydrous methylene chloride (49

mL) and treated with Et3N (3.0 mL). The reaction mixture was then brought to reflux and

succinic anhydride (504.6 mg, 4.891 mmol) was added. After refluxing for 6.5 h, the reaction

was quenched with saturated aqueous NaHCO3 (8.0 mL) at refluxing temperature. The mixture

was cooled to ambient temperature and the organic layer was dried over MgSO4, filtered, and


5:1 to 3:1) provided 1,2:4,5-bis-acetal 2b: (2.7085g, 60 %) as a pale yellow oil. TLC Rf = 0.49

(hexane:ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3): δ 4.26 (ddd, 1H, J = 6.6, 6.6, 4.2 Hz),

- 77 -

4.13 (m, 2H), 4.04 (dd, 1H, J = 10.8, 5.4 Hz), 3.98 (m, 1H), 3.92 (dd, 1H, J = 8.3, 6.6 Hz),

3.43(m, 1H), 2.29 (d, 1H, J = 6.3 Hz), 1.45 (s, 3H), 1.41 (s, 3H), 1.39 (s, 3H), 1.36 (s, 3H).

OO O

O

OS

N

N3b

Imidazolyl thiocarbonyl derivative 3b: To a stirred solution of 1,2:4,5-bis-acetal 2b (2.678 g,

11.54 mmol) in anhydrous 1,2-dichloroethane (38 mL) was added 1,1’-thiocarbonyl diimidazole

(2.8955g, 14.62 mmol) at ambient temperature. The resulting brown solution was refluxed for 7

h and then cooled to ambient temperature. The solvent was removed under reduced pressure.

Flash column chromatography (SiO2; hexane:ethyl acetate 3:1 to 1:1) provided imidazolyl

thiocarbonyl derivative 3b (3.8679 g, 98 %) as a yellow oil. TLC Rf = 0.25 (hexane:ethyl acetate,

1:1). 1H NMR (300 MHz, CDCl3): δ 8.34 (m, 1H), 7.63 (m, 1H), 7.03 (m, 1H), 5.88 (dd, 1H, J =

5.7, 3.3 Hz), 4.45(m, 2H), 4.08 (m, 3H), 3,83 (dd, 1H, J = 9.0, 6.0 Hz), 1.39 (s, 3H), 1.33 (s, 9H);

13C NMR (75 MHz，CDCl3): δ 184.5, 137.0, 131.0, 118.0, 109.7, 109.6, 80.9, 74.8, 74.5, 65.8,

65.2, 26.3, 26.1, 25.0, 24.9. HRMS: Cacld. for (M + H)+: 343.1328, found: 343.1313.

O

O O

O

4b

1,2:4,5-bis-acetal 4b: Benzoyl peroxide (560.0 mg, 2.266 mmol) was added to a stirred solution

of imidazolyl thiocarbonyl derivative 3b (3.8679 g, 11.31 mmol) in triethylsilane (56 mL) at

refluxing temperature. The reaction mixture was refluxed for 2 h during which similar amount of

benzoyl peroxide was added after 30 min, 60 min, and 90 min, respectively. The solvent was

then removed under reduced pressure. Flash column chromatography (SiO2; hexane to

hexane:ethyl acetate, 30:1 to 15:1) provided 1,2:4,5-bis-acetal 4b (~2.42 g) as a light yellow oil

- 78 -

which contained some aromatic impurities and triethyl silane but was good enough to be carried

on. TLC Rf = 0.65 (hexane:ethyl acetate, 1:1).

HO OH

OH OH

5b

(2S,4S)-pentane-1,2,4,5-tetraol 5b:117

0.5 M H2SO4 (14 mL, 7.0 mmol) was added to a stirred

solution of 1,2:4,5-bis-acetal 4b (~2.42 g, 11.1 mmol, impure) in ethanol (14 mL) at ambient

temperature. The reaction mixture was refluxed for 4 h and then BaCO3 was added to adjust the

pH to 7. The resulting milky mixture was filtered and the white solid thus collected was heated

with MeOH (15 mL) at 50 oC for 10 min. The combined filtrate was concentrated under reduced

pressure. Flash column chromatography (SiO2; DCM:MeOH, 7:3) provided (2S,4S)-pentane-

1,2,4,5-tetraol 5b (960.5 mg, 62 % over 2 steps) as a white solid. TLC Rf = 0.25 (DCM:MeOH,

7:3). 1H NMR (300 MHz, MeOH-d4): δ 3.85 (m, 2H), 3.47 (m, 4H), 1.52 (dd, 2H, J = 7.1, 5.6

Hz).

TPSO OTPS

OH OH

6b

(2S,4S)-2,4-Dihydroxypentane-1,5-diyl Bis-(2,4,6-triisopropyl-1-benzenesulfonate) 6b:117

To

a stirred solution of (2S,4S)-pentane-1,2,4,5-tetraol 5b (85.3 mg, 0.627 mmol) in anhydrous

pyridine (0.63 mL) was added 2,4,6-triisopropyl benzenesulfonyl chloride (490.5 mg, 1.571

mmol) at 0 oC. The pale yellow cloudy mixture was stirred at ambient temperature for 10 h and

then the solvent removed under reduced pressure. Flash column chromatography (SiO2;

hexane:acetone, 3:1) provided (2S,4S)-2,4-dihydroxypentane-1,5-diyl bis-(2,4,6-triisopropyl-1-

benzenesulfonate) 6a (293.3 mg, 70 %) as a white fluffy solid. TLC Rf = 0.77 (hexane:ethyl

acetate, 1:1). 1H NMR (300 MHz, CDCl3): δ 7.20 (s, 4H), 4.23 (m, 2H), 4.10 (m, 6H), 3,98 (dd,

- 79 -

2H, J = 10.2, 6.9 Hz), 2.92 (septet, 2H, J = 6.9 Hz), 2.87 (br, 2H), 1.64 (t, 2H, J = 5.7 Hz), 1.27

(d, 24H, J = 6.9 Hz), 1.26 (d, 12H, J = 6.9 Hz).

TPSO OTPS

O O

7b

Acetonide 7b: To a stirred solution of (2S,4S)-2,4-Dihydroxypentane-1,5-diyl bis-(2,4,6-

triisopropyl-1-benzenesulfonate) 6b (1.115 g, 1.670 mmol) in acetone (17 mL) was added 2,2-

dimethoxypropane 2.1 mL, 17 mmol) and p-toluenesulfonic acid (36.5 mg, 0.189 mmol) at

ambient temperature. The resulting colorless solution was stirred at ambient temperature

overnight (25 h). The reaction mixture was quenched with aqueous saturated NaHCO3 (17 mL)

and the solvent removed under reduced pressure. The residue was extracted with methylene

chloride (17 mL × 3) and the combined organic layer dried over Na2SO4, filtered, and


3:1) provided acetonide 7b (2.099 g, 80 %) as a white solid. TLC Rf = 0.73 (hexane:ethyl

acetate, 3:1). 1H NMR (300 MHz, CDCl3): δ 7.19 (s, 4H), 4.08 (m, 10H), 2.92 (septet, 2H, J =

6.9 Hz), 1.65 (t, 2H, J = 7.8 Hz), 1.27 (d, 12H, J = 7.5 Hz), 1.26 (d, 24H, J = 6.9 Hz), 1.23 (s,

6H) ); 13

C NMR (75 MHz，CDCl3): δ 153.7, 150.8, 129.3, 123.8, 101.0, 70.1, 64.5, 34.2, 30.0,

29.6, 24.7, 24.3, 23.5. Cacld. for (M+Na)+: 731.3627, found: 731.3610.

I I

O O

8b

Diiodide 8b: To a stirred solution of (2S,4S)-2,4-Dihydroxypentane-1,5-diyl bis-(2,4,6-

triisopropyl-1-benzenesulfonate) 7b (1.343 g, 1.897 mmol) in 2-butanone (19 mL) was added

sodium iodide (2.815 g, 18.78 mmol) followed by anhydrous pyridine (0.77 mL, 9.5 mmol) at

ambient temperature. The resulting light yellow suspension was refluxed overnight (22 h). The

- 80 -

reaction mixture was cooled to ambient temperature and the solvent was removed under reduced

pressure. The residue was treated with water (30 mL), extracted with ethyl acetate (30 mL × 3),

dried over MgSO4, filtered and concentrated under reduced pressure. Flash column

chromatography (SiO2; hexane to hexane:ethyl acetate, 15:1) provided diiodide 8b (1.632 g, 85

%) as a light yellow oil. TLC Rf = 0.53 (hexane:ethyl acetate, 10:1). 1H NMR (300 MHz,

CDCl3): δ 3.85 (m, 2H), 3.18 (m, 4H), 1.78 (t, 2H, J = 3.3 Hz), 1.39 (s, 6H); 13

C NMR (75 MHz

，CDCl3): δ 101.6 (C), 66.9 (CH), 38.6 (CH2), 24.6 (CH3), 8.9 (CH2).

I I

OH OH

9b

Diiodo diol 9b: To a stirred solution of diiodide 8b (972.0 mg 2.455 mmol) in THF (25 mL) was

added 1N HCl (25 mL) at ambient temperature. The reaction mixture was stirred at ambient

temperature for 2 h. NaHCO3 (powder) was added to adjust pH to 7 and then the organic solvent

was removed under reduced pressure. The residue was extracted with ethyl acetate (20 mL × 3),

dried over MgSO4, filtered and concentrated under reduced pressure. Diiodo diol 9b (1.339 g, 92

%) was obtained as a white solid which was in good enough quality to be carried on without

further purification. TLC Rf = 0.14 (hexane:ethyl acetate, 3:1). 1H NMR (300 MHz, CDCl3): δ

3.94 (m, 2H), 3.39 (dd, 2H, J = 10.2, 4.5 Hz), 3,28 (dd, 2H, J = 10.2, 6.9 Hz), 2.53 (d, 2H, J =

5.1 Hz), 1.86 (dd, 2H, J = 6.3, 5.4 Hz); 13

C NMR (75 MHz，CDCl3): δ 68.4, 41.6, 15.0. HRMS:

Cacld. for M+: 355.8765, found: 355.8768.

O O

HO OH

O O

OH OH

10b

Hydroxyacetophenone 10b: To a stirred solution of diiodo diol 9b (651.1 mg, 1.829 mmol) in 2-

propanol (37 mL) was added 2,6-dihydroxyacetophenone (1.7231 g, 10.99 mmol) at ambient

- 81 -

temperature. The resulting yellow solution was heated to reflux and a mixture of KOH (843.8

mg, 13.53 mmol) in water (1.0 mL) and 2-propanol (1.9 mL) was added. The reaction mixture

was refluxed for 22 h, cooled to ambient temperature, and then concentrated under reduced

pressure. The dark brown residue was treated with ethyl acetate (50 mL) and filtered. The

collected solid was washed with ethyl acetate and the combined filtrate was concentrated under

reduced pressure. Flash column chromatography (SiO2; hexane:ethyl acetate, 1:1 to 1:2 to 1:5,

and then ethyl acetate) provided hydroxyacetophenone 10b (g, 35 %) as a light yellow solid.

TLC Rf = 0.18 (hexane:ethyl acetate, 1:2). 1H NMR (300 MHz, DMSO-d6): δ 12.21 (s, 2H), 7.34

(t, 2H, J = 7.8 Hz), 6.56 (d, 2H, J = 7.8 Hz), 6.50 (d, 2H, J = 7.8 Hz), 4.97 (br, 2H), 4.11 (m,

2H), 3.99 (m, 4H), 2.62 (s, 6H), 1.64 (m, 2H); 13

C NMR (75 MHz, DMSO-d6): δ 204.4, 161.1,

159.5, 134.9, 113.2, 109.5, 103.0, 73.7, 65.2, 38.1, 33.5. HRMS: Cacld. for (M + Na)+:

427.1369, found: 427.1354.

O

O O

OH OH

O

O

O

MeO2C CO2Me11b

Diester 11b: To a freshly prepared NaOMe solution (206.8 mg of sodium cube in 18 mL of

anhydrous MeOH)was added anhydrous diethyl ether (36 mL) followed by dimethyl oxalate

(1.0653 g, 8.931 mmol). After the solid disappeared, hydroxylacetophenone 10b (360.3 mg,

0.892 mmol) was added to the reaction mixture. The resulting yellow solution was refluxed for

24 h, cooled to ambient temperature, and then concentrated under reduced pressure. The yellow

solid residue was treated with water (45 mL) and acidified with concentrated HCl (0.20 mL)

until pH ~ 2. The yellow precipitated was filtered off, dissolved in anhydrous MeOH, and treated

with concentrated HCl (2.7 mL). The reaction mixture was refluxed for 40 min and then cooled

to ambient temperature. The pale brown precipitate was filter off and found to be pure to be

- 82 -

carried on (191.7 mg, 40 %). TLC Rf = 0.10 (ethyl acetate:ethanol, 9:1). 1H NMR (300 MHz,

DMSO-d6): δ 7.73 (t, 2H, J = 8.4 Hz), 7.22 (d, 2H, J = 8.4 Hz), 7.09 (d, 2H, J = 8.4 Hz), 6.75 (s,

2H), 4.85 (d, 2H J = 4.8 Hz), 4.07 (m, 6H), 3.92 (s, 6H), 1.78 (t, 2H, J = 6.0 Hz); 13

C NMR (75

MHz, DMSO-d6): δ 176.6, 160.6, 158.6, 157.1, 150.1, 135.4, 115.4, 114.8, 110.4, 109.7, 74.4,

65.2, 53.4, 37.2. MS: Cacld. for (M + Na)+: 563.1165, found: 563.1178.

O

O O

OH OH

O

O

O

NaO2C CO2Na1b

5 DSCG-(S,S)-diviol 1b: To a stirred suspension of diester 11b (120.5 mg, 0.223 mmol) in

ethanol (11 mL) was added aqueous NaOH solution (2.5 M, 0.18 mL, 0.45 mmol) at ambient

temperature. The reaction mixture was refluxed for 10 min, cooled to ambient temperature, and

then at 0 oC for 20 min. After filtration and drying under high vacuum for 24 h, 1b was obtained

as brown solid (108.1 mg, 87%). 1H NMR (300 MHz, D2O): δ 7.59 (t, 2H, J = 8.4 Hz), 7.06 (d,

2H, J = 8.7 Hz), 6.87 (d, 2H, J = 8.4 Hz), 6.62 (s, 2H), 4.38 (m, 2H), 4.11 (m, 4H), 1.97 (t, 2H, J

= 6.2 Hz); 13

C NMR (75 MHz, D2O): δ 181.4, 165.1, 157.2, 157.1, 156.2, 135.2, 113.0, 112.0,

110.5, 108.1, 73.1, 65.5, 35.4. HRMS: Cacld. for (M + Na)+: 579.0483, found: 579.0491.

OO O

O

OH

2c

1,2:4,5-bis-acetal 2c:90

To a stirred suspension of xylitol (2.590 g, 16.85 mmol) in anhydrous

THF (48 mL) was added 2,2-dimethoxypropane (4.6 mL, 37 mmol) at ambient temperature. The

reaction mixture was refluxed for 15 min and then L-(-)-camphorsulfonic acid (398.7 mg, 1.682

mmol) was added at refluxing temperature. After refluxing for another 5 minutes, the reaction

was quenched with 2 M NaOH (10 mL) at refluxing temperature. After removing the majority of

the solvent under reduced pressure, the aqueous layer was extracted with diethyl ether (10 mL ×

- 83 -

3). The combined organic layer was dried over MgSO4, filtered, and concentrated under reduced

pressure. The colorless oil residue was dissolved in anhydrous DCM (50 mL) and Et3N (2.5 mL)

was added. The reaction mixture was then brought to reflux and succinic anhydride (427.6 mg,

4.203 mmol) was added. After refluxing for another hour, the reaction was quenched with

saturated aqueous NaHCO3 (5 mL) at refluxing temperature. The mixture was cooled to ambient

temperature and the organic layer was dried over MgSO4, filtered, and concentrated under

reduced pressure. Flash column chromatography (SiO2; diethyl ether:hexane) provided 1,2:4,5-

bis-acetal 5: (1.720 g, 44 %) as a pale yellow oil. TLC Rf = 0.23 (diethyl ether:hexane, 1:1). 1H

NMR (300 MHz, CDCl3): δ 4.18 (ddd, 2H, J = 6.6, 6.6, 5.1 Hz), 4.07 (dd, 2H, J = 8.1, 6.6 Hz),

3,89 (dd, 2H, J = 8.3, 6.8 Hz), 3.60 (dt, 1H, J = 6.0, 5.1 Hz), 2.44 (d, 1H, J = 6.0 Hz), 1.45 (s,

6H), 1.38 (s, 6H).

OO O

O

OS

N

3cN

Imidazolyl thiocarbonyl derivative 3c: To a stirred solution of 1,2:4,5-bis-acetal 5 (265.2 mg,

1.143 mmol) in anhydrous 1,2-dichloroethane (4.1 mL) was added N,N -thiocarbonyl

diimidazole (396.1 mg, 2.000 mmol) at ambient temperature. The resulting brown solution was

refluxed for 4 h and then cooled to ambient temperature. The solvent was removed under

reduced pressure. Flash column chromatography (SiO2; hexane:ethyl acetate) provided

imidazolyl thiocarbonyl derivative 6 (371.8 mg, 95 %) as a yellow oil. TLC Rf = 0.30 (diethyl

ether:hexane, 3:2). 1H NMR (300 MHz, CDCl3): δ 8.38 (t, 1H, J = 0.9 Hz), 7.68 (t, 1H, J = 1.5

Hz), 7.06 (dd, 1H, J = 1.8, 0.9 Hz), 5.84 (t, 1H, J = 5.4 Hz), 4.13 (m, 4H), 3.92 (dd, 2H, J = 8.9,

5.9 Hz), 1.43 (s, 6H), 1.35 (s, 6H). HRMS: Cacld. for (M + H): 343.1328, found: 343.1312.

- 84 -

OO O

O

4c

1,2:4,5-bis-acetal 4c:118

Benzoyl peroxide (53. 4 mg, 0.216 mmol) was added to a stirred

solution of imidazolyl thiocarbonyl derivative 6 (371.8 mg 1.087 mmol) in triethylsilane (8.0

mL) at refluxing temperature. The reaction mixture was refluxed for 2h during which similar

amount of benzoyl peroxide was added after 30 min, 60 min, and 90 min, respectively. The

solvent was then removed under reduced pressure. Flash column chromatography (SiO2;

hexane:ethyl acetate) provided 1,2:4,5-bis-acetal 7 (199.7 mg, 85 %) as a yellow oil. TLC Rf =

0.71 (diethyl ether:hexane, 1:1). 1H NMR (300 MHz, CDCl3): δ 4.19 (m, 2H), 4.09 (dd, 2H, J =

8.1, 6.0 Hz), 3.62 (dd, 2H, J = 7.8, 7.4 Hz), 2.01 (m, 1H), 1.79 (dt, 1H, J = 13.8, 6.0 Hz), 1.42 (s,

6H), 1.36 (s, 6H).

HO OH

OH OH

5c

(2S,4R)-pentane-1,2,4,5-tetraol 5c: 0.5 M H2SO4 (1.0 mL) was added to a stirred solution of

1,2:4,5-bis-acetal 7 (170.0 mg, 0.787 mmol) in ethanol (1 mL) at ambient temperature. The

reaction mixture was refluxed for 3.5 h and then BaCO3 was added to adjust the pH to 7. The

resulting milky mixture was refluxed for another 10 min, cooled to ambient temperature, and

then filtered. The solid was treated with MeOH (5 mL) and the mixture was stirred at 50 oC for

10 min. Filtered again and washed the solid with another 5 mL of MeOH. The combined filtrated

was concentrated under reduced pressure. Flash column chromatography (SiO2; DCM:MeOH)

provided (2S,4R)-pentane-1,2,4,5-tetraol 5c (119.4 mg, 97 %) as a white solid. TLC Rf = 0.01

(ethyl acetate). 1H NMR (300 MHz, MeOH-d4): δ3.82 (m, 4H), 3.49 (m, 2H), 1.73 (dt, 1H, J =

14.1, 4.8 Hz), 1.53 (dt, 1H, J = 14.1, 8.1 Hz); 13

C NMR (75 MHz，MeOH-d4): δ 71.7 (CH),

67.4 (CH2), 37.7 (CH2).

- 85 -

Br Br

OAc OAc

6c

Bromoacetate 6c: To a stirred suspension of (2S,4R)-pentane-1,2,4,5-tetraol 5c (597.7 mg, 4.395

mmol) in anhydrous 1,4-dioxane (11 mL) was added acetyl bromide (0.80 mL, 10 mmol)

dropwise at 0 oC. The reaction mixture was stirred at ambient temperature overnight (22 h) and

then the solvent was removed under reduced pressure. The residue was taken up in diethyl ether

(20 mL) and washed with saturated aqueous NH4Cl. The aqueous layer was extracted with

diethyl ether (20 mL × 3), dried over MgSO4, filtered, and concentrated under reduced pressure.

1H NMR of the crude indicated the presence of the desired product bromoacetate 6c, which was

unstable and carried on immediately without further purification.

O O

7c

Diepoxide 7c: The crude bromoacetate 6c obtained from previous step was dissolved in

anhydrous diethyl ether (44 mL) and then cooled to 0 oC. Freshly ground KOH (904.7 mg, 14.51

mmol) was added to the reaction mixture in one portion. The reaction mixture was then warmed

to ambient temperature while stirred vigorously. Another portion of freshly ground KOH (905.8

mg, 14.53 mmol) was added to the reaction mixture after 1 h at 0 oC. After stirring at ambient

temperature for 5 h, the reaction mixture was filter through a pad of MgSO4. The filtrate was

carefully distilled from an ice-water bath under reduce pressure with the receiving flask

immersed in a bath at -78 oC. The pale yellow oil left in the distilling flask was in good enough

purity to be carried on. TLC Rf = 0.40 (hexane:ethyl acetate, 3:1).

I I

OH OH

8c

- 86 -

Diiodo diol 8c: The crude diepoxide 7c obtained from previous step was dissolved in anhydrous

acetonitrile (11 mL). To the reaction mixture was added sodium iodide (1.352 g, 9.022 mmol)

followed by cerium (III) chloride heptahydrate (1.643 g, 4.409 mmol) at ambient temperature.

The resulting yellow suspension was refluxed for 2 h and then cooled to ambient temperature.

The reaction mixture was washed with water (15 mL), extracted with methylene chloride (15 mL

× 3), dried over MgSO4, filtered, and then concentrated. The crude yellow oil was carried on

without further purification. TLC Rf = 0.15 (hexane:ethyl acetate, 3:1).

O O

HO OH

O O

OH OH

9c

Hydroxyacetophenone 9c: To a stirred solution of the crude diiodo diol 8c in 2-propanol (88

mL) was added 2,6-dihydroxyacetophenone (3.616 g, 23.05 mmol) at ambient temperature. The

resulting yellow solution was heated to reflux and a mixture of KOH (1.7024 g, 27.31 mmol) in

water (1.5 mL) and 2-propanol (4.4 mL) was added. The reaction mixture was refluxed for 15 h,

cooled to ambient temperature, and then concentrated under reduced pressure. Flash column

chromatography (SiO2; hexane:ethyl acetate, 1:1 to 1:2 to 1:5 to 1:8, and then ethyl acetate)

provided hydroxyacetophenone 9c (438.5 mg, 25 % over 4 steps) as a light yellow solid. TLC Rf

= 0.26 (hexane:ethyl acetate, 1:2). 1H NMR (300 MHz, CDCl3): δ 12.2 (s, 2H), 7.34 (t, 2H, J =

8.3 Hz), 6.56 (d, 2H, J = 8.4 Hz), 6.49 (d, 2H, J = 8.1 Hz), 5.02 (d, 2H, J = 4.8 Hz), 4.05 (m,

6H), 2.61 (s, 6H), 1.83 (m, 1H), 1.71 (m, 1H); 13

C NMR (75 MHz, DMSO-d6): δ 204.4, 161.0,

159.5, 134.9, 113.2, 109.5, 103.0, 73.0, 66.0, 37.8, 33.4. HRMS: Cacld. for M+: 404.1466,

found: 404.1458.

O

O O

OH OH

O

O

O

MeO2C CO2Me10c

- 87 -

Diester 10c: To a freshly prepared NaOMe solution (202.3 mg of sodium cube in 17.6 mL of

anhydrous MeOH) was added anhydrous diethyl ether (35.2 mL) followed by dimethyl oxalate

(1.060 g, 8.886 mmol). After the solid disappeared, hydroxylacetophenone 9c (355.7 mg, 0.880

mmol) was added to the reaction mixture. The resulting yellow solution was refluxed for 24 h,

cooled to ambient temperature, and then concentrated under reduced pressure. The yellow solid

residue was treated with water (45 mL) and acidified with concentrated HCl (0.22 mL) until pH

~ 2. The yellow precipitated was filtered off, dissolved in anhydrous MeOH, and treated with

concentrated HCl (2.7 mL). The reaction mixture was refluxed for 1.5 h, cooled to ambient

temperature and then 0 oC for 1 h. The brown precipitate was filter off and found to be the

desired product diester 10c (8.6 mg, 2 %). However, the majority of the diester 10c remained in

the filtrate as oily substance. The filtrate was concentrated to about half the volume and carried

on without further purification. 1H NMR (300 MHz, DMSO-d6): δ 7.71 (t, 2H, J = 8.4 Hz), 7.20

(d, 2H, J = 8.4 Hz), 7.08 (d, 2H, J = 8.4 Hz), 6.73 (s, 2H), 4.91 (br, 2H), 4.08 (m, 6H), 3.92 (s,

6H), 2.03 (m, 1H), 1.81 (m, 1H). HRMS: Cacld. for (M + Na)+: 563.1165, found: 563.1167.

O

O O

OH OH

O

O

O

NaO2C CO2Na1c

5 DSCG-meso-diviol 1c: To the filtrate from the previous step was added aqueous NaOH

solution (2.5 M, 5.0 mL, 13 mmol) at ambient temperature. Some pale brown precipitate formed

and the pH of the liquid was approximately 1. The reaction mixture was warmed to reflux and

treated with NaOH pellet in 10 portions. The resulting suspension was refluxed for 1.5 h, cooled

to ambient temperature, and then 0 oC for 30 min. The pale brown precipitate was collected by

centrifugation and dried under vacuum overnight. 5 DSCG-meso-diviol 1c was obtained as pale

brown powder (332.3 mg, 73% over 2 steps). 1H NMR (300 MHz, D2O): δ 7.58 (t, 2H, J = 8.4

- 88 -

Hz), 7.06 (d, 2H, J = 8.4 Hz), 6.91 (d, 2H, J = 8.4 Hz), 6.60 (s, 2H), 4.25 (m, 4H), 4.09 (m, 2H),

2.36 (m, 1H), 1.92 (m, 1H); 13

C NMR (75 MHz, D2O): 181.1, 165.1, 157.0, 156.9, 156.2, 135.2,

113.1, 112.1, 110.7, 108.4, 72.5, 65.9, 37.6. HRMS Cacld. for (M + Na)+: 579.0491, found:

579.0471.

- 89 -

OO O

O

OH

2a

- 90 -

OO O

O

OS

N

N 3a

- 91 -

OO O

O

OS

N

N 3a

- 92 -

HO OH

OH OH

5a

- 93 -

HO OH

OH OH

5a

- 94 -

O OS S

O

O

O

O

OH OH

6a

- 95 -

O OS S

O

O

O

O

OO

7a

- 96 -

O OS S

O

O

O

O

OO

7a

I I

O O

8a

- 97 -

I I

O O

8a

- 98 -

I I

OH OH

9a

- 99 -

I I

OH OH

9a

- 100 -

O O

OH OH

OH OH

OO

10a

- 101 -

O O

OH OH

O O

OO

CO2Me CO2Me11a

- 102 -

O O

OH OH

O O

OO

CO2Me CO2Me11a

- 103 -

O O

OH OH

O O

OO

CO2Me CO2Me11a

O

O O

OH OH

O

O

O

NaO2C CO2Na1a

- 104 -

O

O O

OH OH

O

O

O

NaO2C CO2Na1a

- 105 -

O O

OH OH

O O

OO

CO2Na CO2Na12a

RR

OO O

O

OH

2b

- 106 -

OO O

O

OS

N

3bN

- 107 -

OO O

O

OS

N

3bN

- 108 -

HO OH

OH OH

5b

- 109 -

TPSO OTPS

OH OH

6b

- 110 -

O OS S

O

O

O

O

OO

7b

- 111 -

O OS S

O

O

O

O

OO

7b

- 112 -

I I

O O

8b

- 113 -

I I

O O

8b

- 114 -

I I

OH OH

9b

- 115 -

I I

OH OH

9b

- 116 -

I I

OH OH

9b

O O

OH OH

OH OH

OO

10b

- 117 -

O O

OH OH

OH OH

OO

10b

- 118 -

O O

OH OH

OH OH

OO

10b

O O

OH OH

O O

OO

CO2Et CO2Et11b

- 119 -

O O

OH OH

O O

OO

CO2Et CO2Et11b

- 120 -

O

O O

OH OH

O

O

O

NaO2C CO2Na1b

- 121 -

O

O O

OH OH

O

O

O

NaO2C CO2Na1b

- 122 -

O O

OH OH

O O

OO

CO2Na CO2Na12b

S S

OO O

O

OH

2c

- 123 -

OO O

O

OS

N

3cN

- 124 -

OO O

O

OS

N

3cN

O

O O

O

4c

- 125 -

HO OH

OH OH

5c

- 126 -

HO OH

OH OH

5c

- 127 -

O O

OH OH

OH OH

OO

9c

- 128 -

O O

OH OH

OH OH

OO

9c

- 129 -

O O

OH OH

OH OH

OO

9cO O

OH OH

O O

OO

CO2Me CO2Me10c

- 130 -

O O

OH OH

O O

OO

CO2Me CO2Me10c

O

O O

OH OH

O

O

O

NaO2C CO2Na1c

- 131 -

O

O O

OH OH

O

O

O

NaO2C CO2Na1c

- 132 -

OO O

O

OH

2a

OO O

O

OS

N

N 3a

OO O

O

OS

N

N 3a

OO O

O

4a

HO OH

OH OH

5a

O OS S

O

O

O

O

OO

7bI I

O O

8b

I I

OH OH

9b

I I

OH OH

9b

I I

OH OH

9b

O O

OH OH

OH OH

OO

10b

O O

OH OH

OH OH

OO

10b

O O

OH OH

O O

OO

CO2Et CO2Et11b

O O

OH OH

O O

OO

CO2Et CO2Et11b

O O

OH OH

O O

OO

CO2Et CO2Et11b

O O

OH OH

O O

OO

CO2Na CO2Na12b

S SO O

OH OH

O O

OO

CO2Na CO2Na12b

S S

OO O

O

OH

2c

OO O

O

OS

N

3cN

OO O

O

OS

N

3cN

O

O O

O

4c

HO OH

OH OH

5c

O O

OH OH

OH OH

OO

9c

O

O O

OH OH

O

O

O

NaO2C CO2Na1c

- 133 -

Chapter 3

Bicyclic Brominated Furanones: A new Class of Quorum Sensing Modulators that Inhibit

Biofilm Formation with Reduced Toxicity, and Improved Bacterial Clearance in vivo

Summary

Both natural and synthetic brominated furanones are known to inhibit biofilm formation by

bacteria, but their toxicity to mammalian or human cells is often not reported. Here, we designed

and synthesized a new class of bicyclic brominated furanones (BBFs) that contained only one

bromide group with controlled regiochemistry. This class of molecules exhibited significant

reduction in the toxicity to human neuroblastoma SK-N-SH cells and did not inhibit bacterial (E.

coli and P. aeruginosa) growth, but retained the inhibitory activity towards biofilm formation

and virulence factor expression of bacteria. A representative compound 6-BBF also increased the

susceptibility of P. aeruginosa in biofilms to the antibiotic tobramycin. All BBFs exhibited

antagonistic activities in the lasI quorum sensing circuit, while only 5-BBF showed agonistic

activity for the rhlI quorum sensing circuit. Furthermore, 6-BBF significantly improved P.

aeruginosa clearance in the lung of mice in an immunocompromised pneumonia mouse model in

vivo. This study suggests that structural variation of brominated furanones can be designed for

targeted biological activities.


3.2.1 Quorum sensing and autoinducers

A number of bacteria species use small molecule signals to coordinate gene expression in a

cell-density dependent manner. This mechanism of sophisticated intercellular communication is

- 134 -

termed quorum sensing (QS).119,120

Quorum-sensing bacteria intracellularly synthesize small

diffusible molecules (autoinducers or AIs) that are released into the surroundings. The

extracellular concentration of AIs increases as the number of cells in a bacterial colony increases.

Upon reaching a threshold concentration, AIs bind to cognate receptors within the bacterial cells,

triggering a signal transduction cascade that results in a population-wide alteration in gene

expression. Therefore, the response of bacterial population of AIs facilitates multicellular

activities, such as bioluminescence, virulence factors expression, swarming, biofilm formation,

production of antibiotics, conjugation and sporulation.121

The first QS system studied was that in

the marine bacterium Vibrio fischeri which produces luminescence at high but not low cellular

densities.122

Although originally thought to be limited to a small number of marine bacterial

species, it is now widely recognized that QS is a common part of regulatory machinery utilized

by many bacterial species.120,123

A number of AIs have been identified, among which N-acyl

homoserine lactones (AHLs) are the most common signals used by Gram-negative bacteria and

oligopeptides by Gram-positive bacteria. A furanosyl borate diester (autoinducer-2 or AI-2) is

produced and used by both Gram-negative and Gram-positive bacteria and is proposed to enable

interspecies communication.124

Other small molecule signals have also been associated with QS

in Gram-negative bacteria, such as 2-heptyl-3-hydroxy-4-quinolone (PQS) used by P.

aeruginosa.125,126

One of the best-understood bacterial QS systems to date is that mediated by AHLs, which are

composed of a homoserine lactone ring (HSL) and an acyl chain. The types of AHLs produced

vary greatly among bacterial species (Table 3.1). Some species such as Agrobacterium

tumefaciens127

and Erwinia carotovara128

produce mainly a single type of AHL, N-(3-oxo-

octanoyl)-L-homoserine lactone (3-oxo-C8-HSL) for Agrobacterium tumefaciens and N-(β-

- 135 -

ketocaproyl)-L-homoserine lactone (3-oxo-C6-HSL) for Erwinia carotovara, respectively. Other

species such as Pseudomonas aeruginosa has two AHL synthases, LasI and RhlI, which produce

N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL)129

and N-butanoyl-L-homoserine

lactone (C4-HSL)130

, respectively. In addition, some of the AHLs are produced by multiple

species. For example, 3-oxo-C6-HSL is produced by both Erwinia carotovara and Vibrio

fischeri.122

- 136 -

Table 3.1 Representative AHL systems and corresponding bacteria. (Adapted and modified from

the review by Spring and co-workers.131

)

aAHL abbreviation used in this chapter.

bQscR is an orphan receptor that responds to 3-oxo-C12-

HSL produced by LasI. cThis system is distinct from the typical V. fischeri as the genes coding

for LuxM and LuxN are not homologs with LuxI and LuxR.

Bacterium AHL Nomenclaturea LuxI/R homologs

3-oxo-C8-HSL TraI/R

C8-HSL CepI/R

C6-HSL CviI/R

3-oxo-C6-HSL ExpI/R; CarI/R

3-oxo-C12-HSL LasI/R; QscRb

C4-HSL RhlI/R

3-oxo-C12-HSL PpuI/R

3-oxo-C6-HSL

SpnI/SpnR

C6-HSL

3-oxo-C6-HSL LuxI/R

C8-HSL AinS/R

3-hydroxy-C4-HSLGenerated by LuxM,

detected by LuxNc

Agrobacterium

tumeaciens

Burkholderia

cenocepacia

Chromobacterium

violaceum

Erwinia

carotovara

Pseudonmonas

aeruginosa

Pseudonmonas

putida

Serratia

marcescens AS-1

Vibrio

fischeri

Vibrio

harveyic

NH

O

OO

O

NH

O

O

O

NH

O

O

O

NH

O

OO

O

NH

O

OO

O

NH

O

O

O

NH

O

OO

O

NH

O

OO

O

NH

O

OOH

O

NH

O

O

O

NH

O

OO

O

NH

O

O

O

- 137 -

3.2.2 Biofilm formation and consequences

Biofilms are surface-attached sessile communities of microorganisms enclosed in

extracellular polymeric substance (EPS). Approximately 65-80% of microbial infections

occurring in human body are biofilm-related according to the National Institute of Health (NIH)

estimation.132

Biofilms are associated with many diseases including tooth decay, catheter

infections, endocarditis, otitis media, chronic prostatitis, periodontal disease, chronic urinary

tract infections, and osteomyelitis,133-135

and cause enormous burden and detrimental effects in

medical and industrial settings.136,137

For example, the nosocomial infections caused and

perpetuated by the persistent bacterial biofilm colonization of hospital facilities place a $10

billion burden on the U.S. healthcare system annually.138

The formation of biofilm is regulated by multiple genes in the quorum sensing systems,

which results in highly complex biofilm structures on the surface of microbes.123,139,140

There are

several stages of biofilm formation (Figure 3.1).141

After the initial reversible attachment of

planktonic cells to a surface and irreversible attachment of the cells by secreting extracellular

polymeric matrix, quorum sensing triggers further development of biofilm and maturation.

Finally, the dispersion allows the detachment of cells from the biofilm and colonization of new

surfaces. The bacteria reside in the complex structure of biofilm are often up to 1000-fold more

tolerant to antibiotics and biocides than planktonic bacteria.133,142-144

- 138 -

Figure 3.1 Five stages of biofilm development: (1) initial reversible attachment of planktonic

cells, (2) irreversible attachment of cells, (3) early development of biofilm, (4) maturation of

biofilm, and (5) dispersion and release of cells.145

[Citation: Monroe D (2007) Looking for

Chinks in the Armor of Bacterial Biofilms. PLoS Biol 5(11): e308.

Doi:10.1371/journal.pbio.0050307; Image Credit: D. Davies; Copyright: ©2007 Don Monroe.

This is an open-access article distributed under the terms of the Creative Commons Attribution

License, which permits unrestricted use, distribution, and reproduction in any medium, provided

the original author and source are credited].

There are several mechanisms of biofilm resistance to antimicrobial compounds, which vary

with the species of bacteria present in the biofilm and the antimicrobials used.133,144

One of the

mechanisms is the failure of an agent to penetrate the biofilm. It has been suggested that the

exopolysaccharide matrix, one of the characteristics of biofilms, can be an initial barrier and

- 139 -

limit the transportation of the agents to the cells within the biofilm by either reacting with or

sorption to the compound. For example, Hoyle et al. formed P. aeruginosa biofilms on one side

of a dialysis membrane and found that the biofilms was a diffusion barrier to piperacillin.146

Bacteria cells experience different nutrient and oxygen availability at different locations in the

biofilms, both due to consumption by cells on the periphery of biofilm and by reduced diffusion

of nutrients and oxygen deep into the biofilm.147

At the surface of the biofilm, sufficient nutrients

and oxygen allow bacteria to grow actively. However, nutrients and oxygen are more limited

deeper in the biofilms, and therefore bacteria grow more slowly or not at all. Brown et al. have

suggested that other environmental factors can also cause a general stress response by bacteria

reside in the biofilm that results in slow growth. These factors include heat shock, cold shock,

pH changes and many chemical agents.148

The consequent heterogeneous metabolic activity of

cells accounts for an increase in resistance to antibiotics.149-151

Quorum sensing (QS) has also

been found to play a role in the resistance of biofilms to antimicrobials. Davies et al.

demonstrated that a lasI mutant of P. aeruginosa formed flat, undifferentiated biofilms which

were sensitive to the biocide sodium dodecyl sulfate (SDS) while the wild-type biofilms were

not. In addition, the mutant biofilms appeared normal when grown in the presence of the

autoinducer 3-oxo-C12-HSL (naturally produced by LasI synthase in wild-type).139

Similarly, a

double mutant (lasR and rhlR) of P. aeruginosa forms biofilms that are more susceptible to

killing by hydrogen peroxide and antibiotic tobramycin than the wild-type biofilms.152

Biofilms have also shown increased resistance to host defense systems. In bronchopulmonary

P. aeruginosa infections in cystic fibrosis (CF) patients, bacteria mainly exist in the biofilm

mode and persist despite an intact host immune defense and frequent antibiotic treatment. Jensen

- 140 -

et al. showed a reduced activation of a complex of blood serum proteins of the immune system in

biofilm-grown P. aeruginosa compared with planktonic cells.153

3.2.3 Use quorum sensing modulators to control biofilm formation

Controlling the formation of biofilm has been challenging because inhibition of biofilm

formation and dispersion of already formed biofilm are difficult.154

One rational approach to

control biofilm formation is to interfere with the chemical communication that results in a

quorum sensing between bacteria, which is one of the key events leading to the biofilm

formation.121,139 Existing antibiotics generally inhibit bacterial cellular processes that are

essential for survival and thus exerts an evolutionary selective pressure for drug-resistant

mutations.155

Although quorum sensing is essential for bacterial group behaviors such as biofilm

formation and production of virulence factors, it is not essential for survival.156

Thus inhibiting

quorum sensing may inhibit biofilm formation and attenuate pathogenicity without leading to the

development of drug-resistance.

The QS circuit in V. fischeri, the first discovered AHL-mediated system of its kind, is

composed of the autoinducer synthase LuxI, which is responsible for the synthesis of AHL

autoinducer, and the receptor LuxR, which is a transcriptional activator and upon binding to the

cognate autoinducer, promotes transcription of the luciferase structural operon luxCDABE and

the production of more AHL autoinducer.157-159

This circuit is the basic paradigm for AHL-based

QS systems in Gram-negative bacteria. The majority of autoinducer synthases and receptors

subsequently discovered in other Gram-negative bacteria are termed LuxI-type and LuxR-type

proteins, respectively. Even though several bacteria species have been demonstrated to have a

more complicated system for cell-to-cell signaling which involves more than one AHL signaling

- 141 -

molecules, and even other types of AIs, the general principles of AHL-based QS apply to the

majority of Gram-negative bacteria.

The strategies applied to inhibit QS can be generally separated into three categories. The first

is to interfere with the AHL production by targeting LuxI-type synthase protein. The second is to

interfere with the AHL signaling molecules themselves. And the majority of work focuses on the

third category that is to discover agents which can interact with the LuxR-type receptors. In this

chapter, I will mainly discuss existing examples and our efforts to synthesize and identify small

molecules as quorum sensing inhibitors (QSIs).

One of the most significant efforts lies in the design and synthesis of mimics to natural AHL

autoinducers. Several synthetic autoinducer analogs have been reported to induce or inhibit

quorum sensing of Gram-negative bacteria. Suga and coworkers reported autoinducer analogues

that reduced the production of virulence factors and biofilm formation by inhibiting quorum

sensing in P. aeruginosa.160,161

They also found that the agonist/antagonist activity of these

molecules could be tuned by structural modifications. Blackwell and coworkers reported a series

of systematic and comprehensive studies of synthesis of range of non-natural AHLs which were

shown to inhibit quorum sensing.162-167

Some of these AHLs were found to selectively modulate

one, two, or three LuxR-type proteins, namely LuxR in V. fischeri, TraR in A. tumefaciens, and

LasR in P. aeruginosa. Moreover, a few synthetic AHLs show moderate to high inhibition of

biofilm formation by the opportunistic pathogen P. aeruginosa.162,167

3.2.4 Other biofilm inhibitors

By dissecting the natural products instead of mimicking natural AHL autoinducers, Melander

and coworkers reported a library of 2-aminoimidazole derivatives that can inhibit and disperse

- 142 -

biofilm formation by one or multiple species, including Escherichia coli,168,169

Pseudomonas

aeruginosa,169,170

Acinetobacter baumannii,169

Bordetella bronchiseptica,171

Enterococcus

faecium,171

Streptococcus mutans,172

and methicillin-resistant Staphylococcus aureus

(MRSA).173

Davies et al. discovered that a fatty acid produced by P. aeruginosa is able to induce

dispersion of biofilms formed by a number of other bacterial species.174

Chemical library

screening has also been utilized to discover biofilm formation inhibitors.175-178

3.2.5 Quorum sensing and biofilm inhibitors with furanone moiety

Brominated furanones (BFs) were first isolated in 1970s from a red marine alga Delisea

pulchra largely unfouled in nature,179,180

found on the south-eastern coast of Australia. They are

released on the surface of the alga and inhibit the colonization by both prokaryotes and

eukaryotes,181,182

and are proposed to be an evolutionary response to the adverse effects of AHL-

driven colonization. Brominated furanones differ on the extent of bromination, both on the

furanone ring and on the exocyclic alkene, as well as on the substitution of the alkyl chain with

hydroxy or acetoxy functionality (Figure 3.2). There are apparent structural similarities between

brominated furanones and AHLs: both groups of compounds consist of a relatively polar “head”

and a non-polar aliphatic carbon “tail”. Studies have shown that some of the natural brominated

furanones are capable of inhibiting biofilm formation and swarming of both Gram-positive and

Gram-negative bacteria.183,184

These findings have led to the efforts to chemically modify the

furanone scaffold to identify the key components in the structure for biological activity and to

discover potent modulators of biological activities.

- 143 -

Figure 3.2 A few examples of brominated furanones isolated form extracts of Delisea pulchra.

A few brominated furanones have thus been synthesized and studied along with natural

brominated furanones for their activity to modulate quorum sensing and biofilm formation

(Figure 3.3). Both natural and synthetic brominated furanones were reported to control the

expression of AHL-dependent phenotypes.185

As a result, many brominated furanones have been

shown to inhibit a wide range of quorum sensing-controlled activities, including

bioluminescence,186-189

swarming,186,190-194

biofilm formation, 183,192,194-201

production of

prodigiosin,193,202

and production of virulence factor.195,203-206

Brominated furanones are also

reported to restore the antibiotic tolerance of persister cells.207

To give a few examples, Hentzer

et al. used in vitro studies to demonstrate that brominated furanone 1 inhibit QS-controlled

reporter genes and virulence factor expression. Although this compound did not inhibit the

formation of biofilms, it did affect biofilm architecture.195

In a subsequent study, brominated

furanone 2 was shown to not only inhibit virulence factor expression of P. aeruginosa both in

vitro and in vivo but also increased the susceptibility of P. aeruginosa biofilms to SDS and

antibiotic tobramycin. Moreover, in a mouse pulmonary infection model, compound 2

accelerated clearances of infecting bacteria from the lungs by the host.208

Wu et al. found that

both brominated furanones 1 and 2 were able to accelerate bacterial clearance in the lungs of

infected mice and reduced the severity of lung pathology. In mice treated with lethal P.

aeruginosa infections, treatment of compound 1 significantly prolonged the survival time.209

In

an effort to investigate the important structural elements responsible for biological activities of

OO

Br

Br

OO

Br

Br

OO

BrOO

Br

Br

OH OAc

OO

BrOO

Br

OH OAc

- 144 -

brominated furanones, Han et al. discovered potent inhibitors 3-5 for E.coli biofilms, together

with inactive compounds 6 and 7, as well as compounds 8 and 9, which are toxic to the growth

of strain E. coli RP437.198

Janssens et al. reported that brominated furanone 10 inhibit flagella

biosynthesis and biofilm formation by Salmonella enterica and increased the susceptibility of

Salmonella biofilms to antibiotics without affecting currently known QS systems in

Salmonella.210

A couple of adamantine tethered brominated furanones 13 and 14, together with

some of the synthesis intermediate 11 and 12, were tested for their cytotoxicity and inhibitory

activity of biofilm formation of the strain E. coli RP437.211

All the molecules tested in this work

showed moderate inhibition of biofilm formation without having significant impact on the

growth of bacteria under the conditions tested.

Figure 3.3 Examples of synthetic brominated furanones.

The mechanism of how brominated furanones interfere with quorum sensing is still unclear.

Manefield et al. demonstrated that brominated furanones may inhibit QS by binding to and

inducing degradation of the LuxR-type protein.185

Defoirdt et al. indicated that a natural

brominated furanone from Delisea pulchra blocks QS in V. harveyi by disabling the LuxR

OO

Br

OO

BrOO

BrOO

BrOO

Br

Br

OO

Br

Br

Br

OO

Br

Br

OO

Br

Br

OO

Br

Br

OO

Br

BrBr

Br

Br Br

BrBr

Br

Br

O

OH

OO

Br

OOO

Br

OO

Br

OO

Br

1 2 43 5

6 7 98 10

11 12 13 14

Br

- 145 -

protein to bind to the promoter sequence of QS-regulated genes.212

It is also proposed that

brominated furanones may disrupt AHL-mediated QS by competitively binding to the receptor

site.186,191,195,213-215

A natural brominated furanone from Delisea pulchra was suggested to inhibit

QS of E .coli via AI-2.184,216

In this work, we aim to develop new structures of inhibitors of biofilm formation that are

nonmicrobicidal to bacteria and nontoxic to mammalian cells which should have potent use in

clinics. Han et al. designed and studied a library of structurally closely related synthetic

brominated furanones and suggested the most important structural element of this class of

molecules to be a conjugated exocyclic vinyl bromide on the furanone ring as in compounds 3-5.

Supporting this hypothesis, brominated furanones 6 and 7 lack the conjugated exocyclic vinyl

bromide and were inactive. Brominated furanones 8 and 9 which bear monosubstituted bromides

on an exocyclic methyl group were found to be toxic to the growth of E. coli cells.198

Based on

these findings, we speculated that a bicyclic version of brominated furanones which retain the

conjugated exocyclic vinyl bromide in the furanone moiety could potentially reduce their toxicity

while retaining the biofilm inhibitory activities. Therefore, we designed a new class of bicyclic

brominated furanones (BBFs), 5-BBF 15, 6-BBF 16, and 7-BBF 17, with [3,3,0], [4,3,0], and

[5,3,0]-fused ring structures, respectively (Figure 3.4). Compared to the known brominated

furanones (will use “BF” from now on for brevity), such as 2208

and 3,198

the fused bicyclic

systems bear only one bromo-substitution, and introduce bulkier but semi-rigid cyclic

hydrocarbon skeletons into the molecules that can potentially increase the binding and selectivity

to the receptor proteins.

- 146 -

Figure 3.4 Structures of 5-BBF 15, 6-BBF 16 and 7-BBF 17.


3.2.1 Synthesis of BBFs

Bicyclic brominated furanones, 5-BBF 15, 6-BBF 16, and 7-BBF 17, were synthesized via a

similar route starting with keto acids. We describe the longest synthesis sequence that was used

to make 7-BBF in Scheme 1. Under basic conditions, coupling of cycloheptanone and dimethyl

carbonate provided methyl 2-oxo-1-cycloheptanecarboxylate 19,217

followed by acetoacetic ester

synthesis to give 2-oxocycloheptaneacetic acid 20218

. This intermediate was then subjected to

bromination and dehydration to build the fused ring framework, followed by elimination to

obtain the conjugated final product without isolating the intermediates (Scheme

3.1).219,220

Brominated furanones 2221

and 3198

were also synthesized to compare their toxicities

and biofilm inhibition activities to BBFs. Compound 2 was synthesized by Dr. Debjyoti

Bandyopadhyay in my research group.

OO

Br

O

O

Br

OO

Br

5-BBF 15 6-BBF 16 7-BBF 17

- 147 -

Scheme 3.1 Synthesis of BBFsa

aReagents and conditions: (a) Br2, CH2Cl2, 0 °C to rt; (b) P2O5, DCM, 0

oC to reflux; (c) Et3N,

DCM, 0 °C to reflux; (d) LiOH, THF/H2O (9:4), 23 h, 1M HCl (aq.), rt; (e) NaH, benzene, rt to

85 °C; (f) ethyl bromoacetate, K2CO3, acetone, rt to reflux, 16 h; (g) 6 M HCl (aq.), AcOH, rt to

reflux, 2 d.

3.2.2 Inhibition of E.coli biofilm formation by BBFs

We first evaluated the inhibitory activity of BBFs on biofilm formation by strain E.coli

RP437 (pRSH103), which constantly expresses red fluorescence and enables easy visualization

of the biofilm using confocal laser scanning microscopy (CLSM). BF 3 was a known biofilm

inhibitor to this strain198

at ~200 µM and was used as a positive control. Biofilms were grown on

sterile steel coupons in the absence or presence of 200 µM brominated furanones for 24 h before

visualization. Appropriate amount of DMSO was added to the non-BF treated control to

eliminate solvent effect. E. coli RP437 (pRSH103) biofilms grown in the presence of the

brominated furanones all displayed less fluorescence than the DMSO control. The formation of

O

Bra b c

HO

OO

HO

OO

Br

Br

O

Br

OO

Br

15

d a-cEtO

OO

HO

OO

OO

Br

1618

MeO OMe

O

O

MeO

O

O OO

O

Br

HO

O

1719 20

a-ce f,g

10% over 3 steps87% over 3 steps

3% over 3 steps

16% over 5 steps

- 148 -

biofilm was quantified from fluorescence images using COMSTAT software.222

Biomass, mean

thickness, and surface area values were calculated from 4 random locations on the steel coupons

as 4 replicates and were normalized by that of the DMSO control. Biomass represents the overall

volume of the biofilm. Mean thickness is the average thickness of the biofilm. Surface area is the

area summation of all biomass voxel surfaces exposed to the background. It is noteworthy that 6-

BBF 16 results in the least amount of biofilm formation (based on quantified biomass) of this

strain (31.3 ± 2.5%) (P < 0.01) compared with 5-BBF 15 (56.3 ± 5.0%) (P < 0.05) and 7-BBF 17

(49.7 ± 2.4%) (P < 0.01) and even less than that treated with BF 3 (49.1 ± 8.4%) (P < 0.05).

Figure 3.5 The effect of brominated furanones on biofilm formation by E.coli RP437

(pRSH103). Representative confocal laser scanning microscopy (CLSM) images of biofilm

formed by E.coli RP437 (pRSH103) in the (A) absence and presence of 200 µM (B) 3, (C) 5-

Control

5-BBF 6-BBF 7-BBF

3

A B

C D E

OO

Br

OO

Br

O

O

Br

OO

Me Br

Br

- 149 -

BBF 15, (D) 6-BBF 16 and (E) 7-BBF 17. The control is supplemented with the same amount

(0.4%) of DMSO as present in the brominated furanone-treated conditions. Scale bar = 100 µm.

Figure 3.6 Quantification of biofilm formation by E.coli RP437 (pRSH103) in the absence and

presence of 200 µM brominated furanones. Biomass, mean thickness, and surface area were

quantified from fluorescence image using COMSTAT software.222

Z-Stack images from four

different locations were used. Values are normalized by that of the brominated furanone-free

control and represent the means ± standard deviation from 4 replicates. Significant differences in

the biofilm formation from the BF-free control are indicated by asterisks: *, P < 0.05; **, P <

0.01.

3.2.3 Cytotoxicity study of BBFs on the growth of E. coli

We conducted cytotoxicity study of BBFs on the growth of E. coli to investigate if the

observed biofilm inhibition was due to cytotoxicity. Bacteria cells were allowed to grow in the

0

10

20

30

40

50

60

70

80

90

100

5-BBF 6-BBF 7-BBF BF8

Re

lative

bio

film

fo

rma

tio

n(%

)

Biomass

Mean thickness

Surface area

3

*

**

***

*

*

*

*

- 150 -

absence or presence of 200 µM BBFs and the OD600 values were measured every 2 h for the first

12 h and then after 24 h. BF 3 was used as a positive control (Figure 3.7). None of the BBFs

exhibited obvious impact on the growth of E. coli RP437. The growth curve of bacteria in the

presence of BF 3 deviated from that of the control for up to 8 h and then the OD600 values were

essentially the same to the control after then. Therefore, BBFs all showed moderate to good

biofilm inhibition of E. coli RP437 at concentrations that do not inhibit the growth of bacteria

cells.

Figure 3.7 Growth curve of E.coli RP437 in the absence and presence of 200 µM brominated

furanones. Values are normalized by that of the BF-free control and represent the means ±

standard deviation from 6 replicates.

3.2.4 Inhibition of P. aeruginosa biofilm formation by BBFs

P. aeruginosa is an opportunistic pathogen and the most common Gram-negative bacterium

found in nosocomial and life-threatening infections of immunocompromised patients, including

0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10 12 14 16 18 20 22 24

OD

600

Time (h)

control

5-BBF

6-BBF

7-BBF

BF83

- 151 -

cystic fibrosis (CF),223

cancer,224

and AIDS225

patients. The airways of CF children are colonized

by P. aeruginosa soon after birth; an infection is initiated followed by massive immune response

from the host that causes severe damage to the lung tissues.226

This infection is reoccurring and

develops into a serious chronic infection that resists antibiotic therapies by 10 years of age.227

The presence of biofilm and the resistance to antimicrobial therapy appear to be the main reasons

for the persistence of P. aeruginosa infections.133

Controlling and eradicating P. aeruginosa

biofilm is therefore a significantly clinically related issue.

We used the wild type PAO1-GFP that constitutively expresses green fluorescent proteins

(GFP)228

to grow biofilm on steel coupons with and without BBFs, and monitored the biofilm

formation directly by using confocal fluorescent microscopy. BF 2 has been reported to inhibit

biofilm formation by P. aeruginosa and was thus used as a positive control.208

All three BBFs

showed moderate to good inhibitory activity against biofilms formed by P. aeruginosa PAO1 at

400 µM, with 6-BBF 16 provided more inhibition than that by 5-BBF 15 and 7-BBF 17. BF 2

reduced biofilm almost completely (Figure 3.8). To evaluate the biofilm inhibition more

quantitatively, we also calculated the biomass, mean thickness and surface area of P. aeruginosa

biofilms formed with and without agents by using COMSTAT software (Figure 3.9). The results

were normalized by the BF-free control. Compound 6-BBF 16 exhibited the strongest inhibition

as it reduced the biofilm formation by 71% (P < 0.01), followed by 5-BBF 15 at 53% (P < 0.01)

and 7-BBF 17 at 50% (P < 0.05). The mean thickness of biofilm was reduced by 6-BBF 16 to

60% (P < 0.01) and by 7-BBF 17 to 74% (P < 0.05). We note that the mean thickness of biofilm

treated by 5-BBF 15 did not seem to reduce. One possibility is that the biofilm becomes more

“fluffy” in the presence of 400 µM 5-BBF 15. The surface areas of biofilm in the presence of all

three BBFs were about 60% of that formed in the absence of BFs (P < 0.05).

- 152 -

Figure 3.8 The effect of brominated furanones on biofilm formation by P. aeruginosa.

Representative confocal laser scan microscopy (CLSM) images of biofilm formed by PAO1-

GFP (expresses green fluorescence on plasmid pSMC2) (A) in the absence of agents, and in the

presence of 400 µM (B) 2, (C) 5-BBF 15, (D) 6-BBF 16, and (E) 7-BBF 17. The control is

supplemented with the same amount (0.8%) of DMSO as present in the brominated furanone-

treated conditions. Scale bar = 50 µm.

2

OO

Br

Br

Control

5-BBF

OO

Br

6-BBF

OO

Br

7-BBF

O

O

Br

A B

C D E

- 153 -

Figure 3.9 Quantification of biofilm formation by PAO1-GFP in the absence and presence of 400

µM brominated furanones. Biomass, mean thickness, and surface area were quantified from

fluorescence image using COMSTAT software. Z-Stack images from four different locations

were used. Values are normalized by that of the BF-free control and represent the means ±

standard deviation from 4 replicates. Significant differences in biofilm formation from BF-free

control are indicated by asterisks: *, P < 0.05; **, P < 0.01.

We followed up with a dose-dependence study on inhibitory activity of BBFs on P.

aeruginosa biofilm using a colorimetric assay in 96-well plates employing crystal violet (CV)

(Figure 3.10).170

IC50 values obtained from the dose-response curves were more than 400 µM for

5-BBF 15, and 145.8 µM and 139.7 µM for 6-BBF 16 and 7-BBF 17, respectively. These results

suggest that 6-BBF 16 and 7-BBF 17 are stronger biofilm inhibitors than 5-BBF 15 for P.

aeruginosa. We note that the percentage of relative biofilm formation in the presence of 400 µM

BBFs obtained from confocal laser scan microscopy and that from the CV assay did not match

perfectly. This discrepancy most likely comes from the different experimental conditions and

0

20

40

60

80

100

120

140

1 2 3 4

Re

lative

pe

rce

nta

ge

(%

)

Biomass

Mean thickness

Surface area

5-BBF 6-BBF 7-BBF 2

**

**

***

**

**

** ** **

- 154 -

procedures used in the two different assays. However, the general trend that 6-BBF and 7-BBF

showed more inhibitory activity against Pseudomonas biofilm formation is similar in both

assays.

- 155 -

Figure 3.10 P. aeruginosa biofilm formation dose-response curves with IC50 values for (A) 5-

BBF 15, (B) 6-BBF 16, and (C) 7-BBF 17. Values are normalized by that of the BF-free control

0

20

40

60

80

100

120

0 100 200 300 400 500

Re

lative

bio

film

fo

rma

tio

n (%

)

Concentration (µM)

0

20

40

60

80

100

120

0 100 200 300 400 500

Re

lative

bio

film

fo

rma

tio

n (%

)

Concentration (µM)

0

20

40

60

80

100

120

0 100 200 300 400 500

Re

lative

bio

film

fo

rma

tio

n

Concentration (µM)

IC50 > 400 µM

IC50 = 145.8 µM

IC50 = 139.7 µM

OO

Br

5-BBF 15

OO

Br

6-BBF 16

OO

Br

7-BBF 17

A

B

C

- 156 -

and represent the means ± standard deviation from 4 replicates. Data shown is a representative of

at least three separate experiments.

3.2.5 Cytotoxicity study of BBFs on the growth of P.aeruginosa

The toxicity of brominated furanones to the growth of bacteria is difficult to predict based on

structures. For example, BF 3 does not inhibit the growth of E. coli, whereas similar structures

do.198

Here, we compared the toxicity of BBFs and BF 2 to the growth of P. aeruginosa. At 400

µM, none of the three BBFs showed dramatic inhibition to the growth of P. aeruginosa PAO1

(Figure 3.11), and bacteria grown in the presence of BBFs reached the same optical density

(OD600) as those in the absence of BBFs (control) after 24 h. At the same concentration and

under the same conditions, BF 2 completely inhibited bacterial growth (P < 0.01). Therefore,

BBFs all inhibited P. aeruginosa biofilm at concentrations that did not affect the bacterial

growth. Together with other reports,198,210,229

these results suggest that small structural

modifications of brominated furanones can have substantial impact on their activities.230

In

particular, the toxicity and biological activity of brominated furanones is highly sensitive to

small variation in the structure whereas the biofilm inhibition activity may be quite general for

most of the structures. We note that all three BBFs appear to be more stable than BF 2 and 3.

Bacteria culture containing BF 2 and 3 became pinkish after incubating for ~3 h and the

coloration deepens over time, which may indicate decomposition. However, BBFs did not show

any color change even after 72 h of incubation under the same condition.

- 157 -


µM brominated furanones. Values represent the means ± standard deviation from six replicates.

Significant differences in the optical density from BF-free control are indicated by asterisks: **,

P < 0.01.

3.2.6 Study of synergistic effect between BBFs and tobramycin

As our results indicated that bicyclic brominated furanones were effective at inhibiting

biofilm formation with very low toxicity, we studied the effect of combined use of antibiotics

and these biofilm inhibitors on killing P. aeruginosa within the biofilm. We choose tobramycin

because of its clinical relevance, superior effectiveness against Pseudomonas than other

antibiotics, and wide use in treating Pseudomonas-related diseases.231

We applied 6-BBF 16 at

200 µM, a relatively low concentration at which the inhibition of biofilm formation is still

effective without killing bacteria cells. Tobramycin was initially applied at 2.0 µg/mL as

commonly used in clinics to one-day-old biofilm. However, tobramycin alone at this

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18 20 22 24

OD

600

Time (h)

control

5-BBF

6-BBF

7-BBF

2

**

2

- 158 -

concentration was highly effective at killing bacteria (> 95%) in an in vitro setting. After

concentration screening, we used 0.25 µg/mL of tobramycin and one-day-old biofilm to study

the synergistic effect in the presence of 6-BBF 16. Either 200 µM 6-BBF 16 or 0.25 µg/mL

tobramycin alone resulted in only a mild decrease in the colony forming units (CFUs) of P.

aeruginosa PAO1 biofilm cells (14.6 ± 7.4% and 24.1 ± 6.8%, respectively) (P < 0.05). The

combination of consecutive treatment of 6-BBF 16 and tobramycin resulted in significant

decrease of viable cell counts in the PAO1 biofilm (70.5 ± 1.6%) (P < 0.05) (Figure 3.12). Only

less than half log drop was observed for the viable counts in our case whereas a drop of more

than one magnitude would be more useful in practice. This result suggests that treatment with 6-

BBF 16 slightly increased the susceptibility of PAO1 biofilm to tobramycin in vitro. A past study

has used a LIVE/DEAD BacLight Bacterial Viability staining kit to show that BF 2 (10 µM)

significantly increased the susceptibility of bacteria in P. aeruginosa biofilms to tobramycin (100

µg/mL).208

However, biofilms used in this study was grown under different conditions (ABtrace

minimal medium in flow chamber compared to LB broth in 24-well plate in our case). It has

been shown that the susceptibility of the P. aeruginosa biofilms toward tobramycin can be model

and strain dependent.232

We note that the dosage used in our study is lower than that normally

used in clinical studies, about 1.7-2.0 µg/mL.233

- 159 -

Figure 3.12 Viability of cells within the biofilm treated with and without 6-BBF 16 after

exposure to tobramycin. Preformed 24 h-old biofilms grown on steel coupons were treated with

200 µM 6-BBF 16 for 24 h and then with and without 0.25 µg/mL tobramycin for another 24 h.

Two independent experiments were performed, with four replicates per experiment. Values

represent the means ± standard error of a total of eight replicates. Significant differences in the

CFU from the control are indicated by asterisks: *, P < 0.05.

The mechanism of how the presence of 6-BBF 16 increases the effectiveness of antibiotics in

a synergistic way is not yet clear. One possible explanation is that 6-BBF 16 inhibits quorum

sensing, leading to a reduction of biofilm formation, which reduces the number of biofilm-

associated bacteria that bear high resistance to antibiotics. Changes in the biofilm structure may

also facilitate the transport of antibiotics to bacteria in the biofilm. In addition, quorum sensing

inhibitors may also lead to a reduction in persister formation, as a recent study have shown that

quorum sensing molecules (not inhibitors) can increase the persister formation by P.

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

control 200 µM 6-BBF 0.25 µg/mL TOB combine

Lo

gC

FU

(m

L-1

)

6-BBF

Tobramycin

-

-

-

++

+

+

-

*

- 160 -

aeruginosa.234

Alternatively, these bicyclic brominated furanones may directly target the proteins

that are required to maintain the persisters,235

and subsequently increase the susceptibility of the

persister bacteria to antibiotics.

3.2.7 Modulation of the quorum sensing in P. aeruginosa by BBFs

To investigate whether BBFs inhibit biofilm formation via interference with QS, we studied

the agonistic and antagonistic effects of these BBFs on the two quorum sensing systems in P.

aeruginosa.139

There are two identified AHL-mediated quorum sensing circuits in P.

aeruginosa.195

The las circuit involves autoinducer 3-oxo-C12-HSL and the rhl circuit utilizes

autoinducer C4-HSL.

A double-knockout reporter strain PAO-JP2236

(ΔlasIΔrhlI) harboring the plasmid plasI-

LVAgfp237

was used to evaluate the las quorum sensing system. When bacteria are growing in

the presence of exogenously introduced autoinducer 3-oxo-C12-HSL or analogues, LasR

receptor binds to the AI and activates transcription of the lasI promoter which controls green

fluorescence protein (GFP) expression. The GFP production is quantified by correcting the

measured fluorescence signal for cell density as shown in Equation 3.1. “RFUsample” is the

relative fluorescence unit measured by the plate reader. “No AI” represents bacteria culture

grown in the presence of no AI analogue, supplemented with the same amount of DMSO present

in the SHL stocks.

(3.1)

The antagonism assay was conducted with competition of BBFs against 1 µM natural AI 3-

oxo-C12-HSL (a concentration within the range produced by wild type P. aeruginosa).130,160-162

All BBFs decreased fluorescence signals as the concentration was increased from 50 to 150 to

- 161 -

300 µM, indicating inhibition of GFP expression (Figure 3.13). Of all the BBFs, 5-BBF 15

appeared to be the strongest inhibitor at all concentrations tested. In the presence of 50 µM

agents, the relative fluorescence was 68.5 ± 4.4% (P <0.001), 85.0 ± 14.2%, and 72.9 ± 1.5% (P

<0.01) for bacteria treated with 5-BBF 15, 6-BBF 16, and 7-BBF 17, respectively. At 300 µM,

the GFP expression in the presence of 5-BBF 15 was reduced to about 27.6 ± 4.7% (P <0.05) as

compared to control with only 1 µM of 3-oxo-C12-HSL, while 6-BBF 16 and 7-BBF 17 reduced

the GFP expression to 55.2 ± 17.8% (P < 0.01) and 39.8 ± 6.2% (P <0.05), respectively. BF 3

showed similar inhibitory activity to 5-BBF 15.


HSL alone (control) or 1 µM 3-oxo-C12-HSL plus various concentration of 5-BBF 15, 6-BBF

16, 7-BBF 17 or BF 3. Fluorescence signals were corrected for cell density as shown in Equation

3.1 and the results were normalized to the BF-free control. Values represent the means ±

standard deviation from four replicates. Data shown is a representative of duplicate experiments.

Significant differences in the GFP expression from the control are indicated by asterisks: *, P <

0.05; **, P < 0.01; ***, P < 0.001.

0

10

20

30

40

50

60

70

80

90

100


Re

lative

flu

ore

sce

nce

(%

)

50 µM

150 µM

300 µM

3

**

******

**

**

**

****

**

*

- 162 -

Similar results were obtained using the wild type strain PAO1 harboring plasmid plasI-

LVAgfp (Figure 3.14). Both 6-BBF 16 and 7-BBF 17 decreased fluorescent signals as the

concentration was increased from 50 to 150 to 300 µM (Figure 3.15). At 300 µM , the GFP

production in the presence of 6-BBF 16 or 7-BBF 17 was 78 ± 1.1% (P < 0.05) and 69 ± 4.0% (P

< 0.001), respectively, compared to that of the BF-free control. BF 3 showed similar inhibitory

activity to 5-BBF 15 in this strain, too, with 46 ± 4.8% (P < 0.001) GFP expression at 300 µM,

compared to that of the BF-free control (P < 0.001). However, BF 3 is in general more toxic than

6-BBF 16 and 7-BBF 17. Compound 5-BBF inhibited the GFP production slightly at 50 µM and

150 µM, and reduced the GFP expression by approximately half at 300 µM. These results

suggest that the BBFs are weak to moderate antagonists of LasR protein within the concentration

range of 50 to 300 µM.

Figure 3.14 GFP expression by PAO1 (plasI-LVAgfp) in the absence or presence of 5-BBF 15,

6-BBF 16, 7-BBF 17 or BF 3 at 50, 150, and 300 µM. Fluorescence signals were corrected for

cell density as shown in Equation 3.1and the results were normalized to the BF-free control.

0

20

40

60

80

100

120


Re

lative

flu

ore

sce

nce

(%

)

50 µM

150 µM

300 µM

3

*

***

***

***

**

*

- 163 -

Values represent the means ± standard deviation from four replicates. Data shown is a

representative of at least three separate experiments. Significant differences in the GFP

expression from the control are indicated by asterisks: *, P < 0.05; ***, P < 0.001.

We also tested the effects of BBFs on the quorum sensing system of rhlI using PAO-JP2

(prhlI-LVAgfp) in the presence of 1 µM 3-oxo-C12-HSL and 10 µM C4-HSL (Figure 3.15).160

Surprisingly, 5-BBF 15 promoted, instead of suppressed, the GFP expression significantly as its

concentration was increased. At 50 µM and 150 µM, 5-BBF 15 increased the GFP expression by

16.4 ± 11.8% and 62.2 ± 14.2% (P < 0.001), respectively. When the concentration was increased

to 300 µM, the fluorescence was increased by 154 ± 18.6% (P < 0.001) compared to the control.

On the contrary, 6-BBF 16, 7-BBF 17 or BF 3 did not show observable increase in green

fluorescence. Similar results were also obtained when reporter strain PAO1 (prhlI-LVAgfp) was

used (Figure 3.16). At 150 µM and 300 µM, 5-BBF 15 increased the GFP expression by 82.6 ±

4.4% and 165 ± 17.6%, respectively (P < 0.01). On the contrary, 6-BBF 16, 7-BBF 17 and BF 3

had no effect on the GFP expression at all concentrations tested. These results suggest that 5-

BBF is a strong agonist of RhlR, while 6-BBF 16 and 7-BBF 17 are not.

- 164 -

Figure 3.15 GFP expression by PAO-JP2 (prhlI-LVAgfp) in the presence of 1 µM 3-oxo-C12-

HSL and 10 µM C4-HSL alone (control) or 1 µM 3-oxo-C12-HSL and 10 µM C4-HSL plus

various concentration of 5-BBF 15, 6-BBF 16, 7-BBF 17, or BF 3. Fluorescence signals were

corrected for cell density as shown in Equation 3.1 and the results were normalized to the BF-

free control. Values represent the means ± standard deviation from four replicates. Data shown is

a representative of at least three separate experiments. Significant differences in the GFP

expression from the control are indicated by asterisks: ***, P < 0.001.

0

50

100

150

200

250

300


Re

lative

flu

ore

sce

nce

(%

)

50 µM

150 µM

300 µM

3

***

***

- 165 -

Figure 3.16 GFP expression by PAO1 (prhlI-LVAgfp) in the absence or presence of 5-BBF 15,

6-BBF 16, 7-BBF 17, or BF 3 at 50, 150, and 300 µM. Fluorescence signals were corrected for

cell density as shown in Equation 3.1 and the results were normalized to the BF-free control.

Values represent the means ± standard deviation from four replicates. Data shown is a

representative of at least three separate experiments. Significant differences in the GFP

expression from the control are indicated by asterisks: ***, P < 0.001.

3.2.8 Effect on elastase B production in P. aeruginosa by BBFs

Another bioactivity of P. aeruginosa that is also controlled by quorum sensing (las

pathway)238

is the production of the virulence factor metalloprotease elastase B. This highly toxic

virulence factor facilitates the invasion and destruction of host tissues,239

induces inflammatory

responses from the host240

and also signals the biofilm formation.241

Thus elastase B is a

potential target for developing antimicrobial agents. Molecules that inhibit virulence factor

production without bactericidal action would place little selection pressure on the bacteria strains

0

50

100

150

200

250

300


Re

lative

flu

ore

sce

nce

(%

)

50 µM

150 µM

300 µM

3

***

***

***

- 166 -

for the emergence of resistance and therefore have been proposed as a class of second-generation

antibiotics.242

BF 2, for example, was reported to inhibit the production of virulence factors and

to increase the bacterial susceptibility to tobramycin.208

Another synthetic BF 1 was also shown

to reduce the production of virulence factors, including protease and chitinase.195

Because BBFs

appeared to be potent antagonists of LasR, we also tested their ability to inhibit elastase B

production. A colormeric assay that utilized elastin-congo red substrate was conducted to

measure the elastase B activity produced by P. aeruginosa PAO-JP2. Natural autoinducers 3-

oxo-C12-HSL and C4-HSL were added at 5 µM and 10 µM, respectively, for consistent

induction of elastase B activity in this strain.160

At 300 µM, all BBFs significantly reduced the

production of elastase B by ~80% (P < 0.05) (Figure 3.17). In a dose-dependent study, the

elastase B activity in the presence of 50 µM 6-BBF 16 was not significantly different from the

non-treated control. But the inhibitory activity of 6-BBF 16 increased as the concentration

increased up to 300 µM (Figure 3.17, insert). Because the elastase B production is controlled by

las quorum sensing pathway, these results are consistent with BBFs being antagonists of LasR in

P. aeruginosa.

- 167 -

Figure 3.17 Elastase B activity produced by P. aeruginosa PAO-JP2 in the presence of 5 µM 3-

oxo-C12-HSL and 10 µM C4-HSL alone (control) or 5 µM 3-oxo-C12-HSL and 10 µM C4-HSL

plus 300 µM BBFs or 5 µM PAI1 and 10 µM PAI2 plus various concentration of 6-BBF 16

(insert). Values were normalized to that of the brominated furanone-free control and represent

the means ± standard deviation from four replicates. Significant differences in the elastase B

activity from the control are indicated by asterisks: *, P < 0.05.

3.2.9 Improved bacterial clearance by 6-BBF in vivo

A more clinically-related method to evaluate the in vivo function of a QSI compound is by

means of a pulmonary infection model.243

To further explore the potential utility of BBFs in vivo,

we used a SP-A/D KO mouse model244

that lacked the expression of two host defense proteins

SP-A and SP-D in the lung and thus increased the mouse’s susceptibility to P. aeruginosa

infection. This model mimics microenvironments of the lung in the pulmonary

immunocompromised patients, who have a high susceptibility to lung infections. Four

0

20

40

60

80

100

120

control 5-BBF 6-BBF 7-BBF

Re

lative

ela

sta

se

B a

ctivity (

%)

0

10

20

30

40

50

60

70

80

90

100

50 µM 150 µM 200 µM 300 µM

Re

lative

ela

sta

se

B

activity (%

)

* *

- 168 -

independent experiments with a total of 68 age and gender matched mice were performed. The

results showed that the average CFU in mice treated with 6-BBF at 200 µM was significantly

lower (P < 0.05) than that in mice of the control group. The average CFU in the 6-BBF-treated

group and control group were (4.1 ± 0.29) × 104 CFU per lung and (18.6 ± 5.9) × 10

4 CFU per

lung, respectively, 48 hours after infection (Figure 3.18). Our results from this in vivo model

demonstrated that 6-BBF 16 can improve bacterial clearance compared to the controls (without

6-BBF treatment). The mechanisms underlying this increase clearance may be through the

inhibition of biofilm formation of bacteria by 6-BBF and/or increase of phagocytic efficiency of

bacteria by alveolar macrophages and neutrophils in the lung.

Figure 3.18 Effect of 6-BBF on the bacterial clearance of mouse lung in vivo. Each SP-A/D KO

mouse was infected with 50 µl (containing 1 × 107 CFU of wilt-type P. aeruginosa )

intratracheally. Bacterial suspension contained either saline as the control group or 200 µM 6-

BBF 16. The mice were sacrificed 48 hours after infection and CFUs of each lung were

determined. The data showed one representative experiment (n= 8 mice for each group) of four

independent experiments and values are means ± standard deviation. Significant differences in

the bacterial clearance from the control are indicated by asterisk: *, P < 0.05. This experiment of

6-BBF

- 169 -

mouse model was performed by Dr. Osama A. Abdel-Razek from Dr. Guirong Wang’s research

group at Upstate Medical University (SUNY).

3.2.10 Cytotoxicity study of BBFs on human cells

To avoid invoking bacterial resistance,173,209,245,246

nonmicrobicidal agents that do not kill

bacteria or influence bacterial growth are being sought. However, for any potential therapeutic

development, the toxicity issue of the molecules towards mammalian or human cells must be

addressed. Although many natural and non-natural brominated furanones inhibit biofilm

formation by a wide range of bacteria, few studies have addressed their toxicity to mammalian or

human cells.230,247

Here, we compare the toxicity of BBFs to a nonmicrobicidal brominated

furanone, BF 3, against human neuroblastoma SK-N-SH. Cells were allowed to attach to and

grow in 96-well tissue culture-treated microtiter plates for 24 h, after which they were treated

with at 100 µM of each agent (5-BBF 15, 6-BBF 16, 7-BBF 17 and BF 3) for 1 h, and then the

agent-containing medium was replaced with fresh medium. After the 1-h treatment of agents, the

cells were then allowed to recover for 0, 24, and 48 h (recovery time), at which time the number

of live cells was determined by the CCK 8 assay.248

Survival (%) was calculated based on cells

without any agent treatment as shown in Equation 3.2. The sample absorbance values were

obtained from the wells containing cells and brominated furanones, and the control absorbance

values were obtained from BF-free wells containing cells and DMSO.

(3.2)

At 100 µM, BF 3 exhibited moderate toxic effect toward human neuroblastoma SK-N-SH

cells after 0 h of recovery; about 50.3 ± 9.3% of cells were alive compare to those not treated

- 170 -

with agents (P < 0.001). None of the three BBFs showed any obvious toxicity at this time

(Figure 3.19). As the culture time prolonged after agent-treatment, survival (%) of cells treated

with 5-BBF 15 and BF 3 both decreased significantly, to 21.4 ± 3.6% (P < 0.001) and less than

5% (1.9 ±0.3%, P < 0.01), respectively, after 48 h. In contrast, cells treated with 6-BBF 16 and

7-BBF 17 did not show any decrease in the number of live cells, but rather a slight increase of

cells treated with 6-BBF 16 after 48 h of agent-treatment. These results suggest that 6-BBF 16

and 7-BBF 17 are not particularly toxic to this cell line under the conditions studied, whereas 5-

BBF 15 and BF 3 are.


treatment of 100 µM 5-BBF 15, 6-BBF 16, 7-BBF 17 and BF 3, respectively. Values are

normalized by that of the SHL-free control as shown in Equation 3.2 and represent the means ±

standard deviation from six replicates. Significant differences in the survival% from BF-free

control are indicated by asterisks: **, P < 0.01; ***, P < 0.001.

0

20

40

60

80

100

120


%S

urv

iva

l aft

er o

ne

ho

ur tr

ea

tme

nt 0h

24h

48h

***

**

***

**

**

3

- 171 -

To further explore the toxic effect of 6-BBF and 7-BBF, we increased the exposure time to

2, 4, 6 h to the human cells. The A450 was measured immediately after incubation with the

agents. The results showed that the survival (%) decreased as the exposure time prolonged for

cells treated with 5-BBF 15 and BF 3, and was lower than that for 1h agent treatment. This result

further confirms that these two compounds are toxic to this cell line. The survival (%) for cells

treated with 6-BBF 16 remained the same after 2, 4, and 6 h treatment, and the survival (%)

decreased by ~ 20% for cells treated with 7-BBF 17 (Figure 3.20). These results suggest that 6-

BBF 16 and 7-BBF 17 are not as toxic as BF 3 to the cells line studied.

Figure 3.20 Survival (%) of SK-N-SH when treated with 100 µM 5-BBF 15, 6-BBF 16, 7-BBF

17 and BF 3 for 2, 4, and 6 h. Values are normalized by that of the SHL-free control as shown in

Equation 3.2 and represent the means ± standard deviation from six replicates. Significant

differences in the survival (%) with 2 h exposure time are indicated by asterisks: *, P < 0.05.

0

10

20

30

40

50

60

70

80

90

100


%S

urv

iva

l aft

er d

iffe

ren

t dru

g

tre

atm

en

t tim

e

2h

4h

6h

3

*

- 172 -

Cytotoxicity of BBFs against this human neuroblastoma cell line SK-N-SH was also

evaluated at the highest concentration tested for biofilm inhibition (400 µM) following the same

protocols. After the 1 h treatment of agents and 0 h recovery time, BF 3 exhibited the strongest

toxic effect toward human neuroblastoma SK-N-SH cells; 29.0 ± 14.1 % of cells were alive

compare to those not treated with agents (P < 0.001). 5-BBF 15 and 6-BBF 16 showed mild

toxicity (~ 76% cell survival for both), and 7-BBF 17 resulted in ~ 44% of cell survival (Figure

3.21). At 24 h and 48 h of recovery time, survival (%) for cells treated with 5-BBF 15 dropped to

~ 21% and ~ 6%, respectively. For cells treated with BF 3, almost no live cell remained (< 2%)

after 24 and 48 h. For 7-BBF 17, cell survival dropped to ~ 7 % after 24 h of recovery time, but

appears to increase to 13 % after 48 h. In contrast, 6-BBF 16 exhibited the least toxic effect to

this human cell line. After 24 h and 48 h of recovery time, about 50 % survival was observed for

cells treated with 6-BBF 16.


treatment of 400 µM 5-BBF 15, 6-BBF 16, 7-BBF 17 and BF 3, respectively. Values represent

0

10

20

30

40

50

60

70

80

90

100


%S

urv

iva

l aft

er o

ne

ho

ur tr

ea

tme

nt 0 h

24 h

48 h

***

*** ***

******

*******

**

**

3

- 173 -

the means ± standard deviation from six replicates. Significant differences between BF-treated

conditions in the survival% and BF-free control at each time point are indicated by asterisks: *, P

< 0.05; **, P < 0.01; ***, P < 0.001.


The bicyclic brominated furanones are a new class of synthetic brominated furanones with

increased chemical stability and significant reduction in toxicity to human cancer cells compared

to the known compound 3. These BBFs inhibit biofilm formation by E. coli and P. aeruginosa

and inhibit production of virulence factor elastase B in P. aeruginosa at nonmicrobicidal

concentrations. At 200 µM, the representative molecule 6-BBF 16 moderately increased the

susceptibility of Pseudomonas biofilm to tobramycin. These biological activities are probably

realized through interfering with quorum sensing in bacteria. The relative high IC50 of biofilm

inhibition of these brominated furanones plague their use in pharmaceutical area. However, they

may still be useful in treatment of external wound infections. This work indicates a complex and

sensitive structure-bioactivity relationship for brominated furanones. Future work would include

studying the ability of BBFs to modulate QS in other species such as V. fischeri and A.

tumefaciens. Using X-ray crystal structures of a few LuxR-type proteins bound to their cognate

AHL ligands as a guidance, one could design and tune the structure of brominated furanones and

other potential quorum sensing modulators. Computer simulated binding of candidates to the

active site of LuxR-type receptors would help to predict and understand the activity of these

molecules.

- 174 -


3.4.1 Biological studies

Chemicals.

Minimum essential medium with Eagle’s salts and L-glutamine (EMEM) was obtained from

Mediatech (Herndon, VA). Trypan blue stain was purchased from Sigma-Aldrich (Milwaukee,

WI). Cell counting kit 8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc.

(Gaithersburg, MD). Water used for preparing all buffers and solutions had resistivity of 18 MΩ

cm (Millipore, Billerica, MA).

Bacteria strains and growth media.

Double-knockout mutants of Pseudomonas aeruginosa, PAO-JP2 (plasI-LVAgfp) and PAO-

JP2 (prhlI-LVAgfp) were kindly provided by Dr. Helen E. Blackwell (University of Wisconsin-

Madison) with permission by Dr. Babara H. Iglewski (University of Rochester Medical

Center).236,249

Plasmids plasI-LVAgfp and prhlI-LVAgfp were kindly provided by Dr. Hiroaki

Suga (The University of Tokyo). Escherichia coli RP437 250

and E. coli RP437 with pRSH103

plasmid (constitutively expresses red fluorescence proteins) 251

were kindly provided by Dr.

Dacheng Ren (Syracuse University). E. coli RP437 and E.coli RP437 (pRSH103) were grown in

Luria-Bertani (LB) broth (containing 10 µg/mL of tetracycline for E.coli RP437 (pRSH103)) at

37 oC. P. aeruginosa PAO1-BAA-47 (wild type) from ATCC and PAO1-GFP (constitutively

expresses green fluorescence proteins on plasmid pSMC2) 228

were grown in LB broth

(containing 300 µg/mL of carbenicillin for PAO1-GFP) at 37 oC. All the other P. aeruginosa

strains were grown in M9 minimal medium containing 300 µg/mL of carbenicillin.

Stock solutions of brominated furanones.

- 175 -

Stock solutions of all brominated furanones (BFs) (50 mM) were prepared in DMSO,

sterilized by filtering through a 0.2 µm syringe filter, and stored at -20 °C in sealed vials.

Appropriate amount of DMSO was added to controls in all assays to eliminate solvent effect.

The amount of DMSO in all cases was no more than 0.8%.

Confocal laser scanning microscopy (CLSM) analysis of biofilms.

Biofilms were grown on 316 stainless steel coupons (ca. 3/8 in. × 3/8 in., McMaster-Carr,

Elmhurst, IL) with or without BBFs in a 24-well microplate. The plate was wrapped in a Saran

wrap and incubated at 37 oC for 24 h without shaking. Each steel coupon was then washed gently

by dipping into 0.9% NaCl saline buffer 3 times (fresh saline buffer was used for each dipping)

and then placed upside down on a microscope cover glass (50 x 24mm, No. 2, Fisher Scientific,

Pittsburgh, PA). The biofilms were visualized using a Zeiss LSM 710 Confocal Laser Scanning

Microscope (Carl Zeiss, Jena, Germany). The biofilms formed by strain E.coli RP437

(pRSH103) were visualized by excitation with a HeNe laser at 543 nm and fluorescent emission

was detected with a LP 560 nm emission filter. A 488 nm laser line was used to visualize

biofilms formed by PAO1-GFP. Z-stacks from four randomly picked spots were taken for each

steel coupon. Quantification analysis of biomass, mean thickness, and surface area of the

biofilms formed in the absence and presence of brominated furanones were obtained from

fluorescence image using COMSTAT software 222

. Values are normalized by that of the BF-free

control.

Crystal violet biofilm assay in 96-well plates.

An overnight bacterial culture was subcultured at an optical density (OD600) of 0.1 in LB

broth. Appropriate amount of stock solutions of BBFs in DMSO were added to each well. The

outermost wells of the 96-well polystyrene microtiter plates (Costar 3370) were filled with 100

- 176 -

µL of sterile autoclaved water to prevent uneven evaporation. The plates were wrapped in Saran

wrap and incubated under stationary conditions at 37 oC for 24 h. The media was then discarded

and the plates were washed with deionized water (125 µL × 1) and dried for 30 min. Biofilms

were stained with 100 µL of 0.1% aqueous solution of crystal violet for 30 min at room

temperature. The crystal violet was discarded and then the plates were washed with deionized

water (125 µL × 3) and dried again for 30 min. The remaining stained cells were solubilized with

200 µL of 95% EtOH and 125 µL of the solubilized CV stain was transferred to a new

polystyrene microtiter dish. The amount of biofilm formation was determined by measure the

absorbance at 540 nm. The relative biofilm formation was determined using the following

equation: relative biofilm formation (%) = (A540 sample – A540 medium)/(A540 control – A540

medium) × 100. The sample absorbance values were obtained from the wells containing bacteria

and brominated furanones, and the control absorbance values were obtained from BF-free wells

containing bacteria and DMSO.

Growth curve of planktonic bacteria.

An overnight bacterial culture was diluted and grown to an OD600 of 0.05. The subculture

was aliquoted into 96-well plates at 200 µL per well. Appropriate amount of stock solutions of

brominated furanones in DMSO was added to each well. The outermost wells of the 96-well

plates were filled with 200 µL of sterile autoclaved water to prevent uneven evaporation. The

plates were incubated at 37 oC with shaking (250 rpm). The OD600 readings were acquired

aseptically at 0, 2, 4, 6, 8, 10, 12, and 24 h using Biotek ELx800 TM

absorbance microplate reader

(BioTek Instruments, Inc., Winooski, VT) using Gen5TM

data analysis software. The growth

curves were plotted as OD600 values (mean ± SD) versus time from six replicates.

Reporter gene assay for P. aeruginosa.162,167,195

- 177 -

An overnight culture of PAO1 (plasI-LVAgfp) and PAO1 (prhlI-LVAgfp) in M9 medium

(300 µg/mL carbenicillin) was grown from a single colony picked from an LB agar plate

supplemented with 300 µg/mL carbenicillin. The cell culture was diluted and grew to an OD600

of 0.1 in M9 medium containing 300 µg/mL of carbenicillin. Bacteria culture (200 µL) was

added to each well of a polystyrene 96-well microplate (Costar 3370, Corning Incorporated,

Corning, NY) containing appropriate amount of natural autoinducers with and without

brominated furanones. The plate was incubated at 37 oC for 24 h in a rotary shaking incubator

(250 rpm). The culture from each well was then transferred to a 96-well plate with black wall

(µClear, 655096, Greiner Bio-One North America, Inc., Monroe, NC). The fluorescence (an

excitation wavelength of 500 nm and an emission wavelength of 540 nm) and OD absorbance

(OD600) in each well was measured by Synergy H1 multi-mode microplate reader with a Gen5

data analysis software. Background signals from M9 medium were deducted from all samples.

Determination of combined effect of BBFs and tobramycin.

An overnight culture of P. aeruginosa PAO1 in LB broth was diluted 1:100 and grew to

OD600 of 0.1. The cell culture was inoculated on to 316 stainless steel coupons (ca. 3/8 in. x 3/8

in.) in a 24-well plate. After 24 h of biofilm formation at 37 oC, the old medium was then

replaced by fresh LB broth that contained 200 µM BBF and then the biofilms were allowed to

grow for another 24 h. Subsequently, the medium was replaced by fresh LB broth that contained

0.25 µg/mL tobramycin and then the biofilms were allowed to grow for another 24 h. Each steel

coupon was then washed gently by dipping into saline buffer (0.9% NaCl) 3 times and placed in

2 mL of saline buffer. The coupons were sonicated for 1 min and vortexed for 1 min; this process

was repeated twice to release biofilm cells 252

. The resulting cell suspensions were serially

- 178 -

diluted in saline buffer, spread on LB agar plates, and incubated at 37 oC for 24 h to count the

number of CFU.

Evaluating the production of virulence factor in the presence of BBFs.

The production of virulence factor elastase B by P. aeruginosa was measured as described

previously.160,253

Bacteria were grown overnight in PTSB media (5% Peptone, 0.1% Tryptic Soy

Broth) at 37 oC, diluted and grown to midlog phase, and subcultured to an OD600 of 0.05. The

culture was then added to test tubes containing BBFs at the desired final concentrations. The

tubes were incubated for 24 h at 37 oC with shaking (250 rpm). Culture supernatants were

recovered by centrifugation at 3000 rpm (Galaxy 5D centrifuge, VWR, Radnor, PA) for 10 min

at room temperature and then passed through a 0.45 µm PVDF syringe filter (Santa Cruz

Biotechnology, Inc., Santa Cruz, CA). A 100 µL aliquot of the supernatant was added to 900 µL

of Elastin-Congo red (ECR) buffer (100 mM Tris-HCl, 1 mM CaCl2, pH 7.2) containing 4.5 mg

of Elastin-Congo red and incubated for 24 h at 37 oC with shaking (250 rpm). After incubation,

0.2 mL of 0.12 M EDTA was added to stop the reaction. Insoluble ECR was removed by

centrifugation and the absorbance of the supernatant (OD490) was measured. Elastase B activity

was represented by OD490 of the samples treated with BBFs minus the OD490 of the bacteria-free

samples.

Mice (The protocols for SP-A/D KO mouse model, lung infection, tissue collection, lung

bacteriology and bronchoalveolar lavage were performed and described by Dr. Osama A. Abdel-

Razek from Dr. Guirong Wang’s research group at Upstate Medical University (SUNY)).

SP-A and SP-D double knockout (SP-A/D KO) mice with C57BL/6 background were used

for this study. The original SP-A/D KO mice were kindly provided by Dr. Samuel. Hawgood

(The University of Califonia San Francisco) and these mice had been backcrossed at least 10

- 179 -

generations against a C57BL/6 background 244

. Mice used in this study were bred in the animal

core facility at SUNY Upstate Medical University under pathogen-free conditions. All animal

experiments were conducted in accordance with the Institutional Animal Care and Use

Committee guidelines of SUNY Upstate Medical University and the National Institutes of Health

guidelines on the use of laboratory animals.

Lung infection model.

P. aeruginosa strain PAO1- BAA-47 (wild type) from frozen stocks were streaked onto LB

agar plates and incubated at 37°C for 24 hours, and a single colony was picked up and

transferred to a flask contains 20 mL of LB Broth and incubated at 37°C for 13 hours with

shaking at 250 rpm. The optical density at 600 nm (OD600) was measured and it is usually about

2.1 by this time. The bacterial cells were recovered by centrifugation, resuspended in saline and

diluted to an optical density of 0.6 at OD600. One mL of this solution was estimated to contain 2

× 109 CFU. The solution was diluted 10 times with saline for use. Based on our preliminary data,

a 50 µl (containing 1 × 107 CFU) of the diluted bacterial solution was used to inject each mouse

intratracheally. The tracheal delivery of [50 µl (1 × 107 CFU)/ mouse] was accomplished by

anesthetizing mice with 30 µl of the mixture of ketamine:xylazine (100 mg/kg:10 mg/kg). In 6-

BBF-treated group bacterial suspension was mixed with 6-BBF (final concentration to 200 µM

of 6-BBF). In control group bacterial suspension was added with same volume of saline. Forty

eight hours after lung infection the mice were sacrificed.

Tissue Collection.

After anesthetizing mice with 30 µl of the mixture of ketamine: xylazine (100 mg/kg:10

mg/kg), a large abdominal incision was made and the intestine was turned to the left side of the

- 180 -

inferior vena cava and aorta were cut using iris scissors and the animal was bleed and then lung

tissue was harvested or was lavaged.

Lung Bacteriology.

Mice from both 6-BBF-treated group and controls were studied for quantitative bacteriology.

The left half of the lung of each mouse was removed aseptically and homogenized in 1 ml of

sterile saline and 100 µl of appropriately serial diluted lung homogenates were plated on LB

agar, and incubated at 37°C for 24 hours after which bacterial colonies were counted. CFUs were

determined using the Quantity One colony-counting software (Bio-Rad, Hercules, CA).

Bronchoalveolar Lavage (BAL).

Mice from both 6-BBF-treated group and controls were prepared for BAL. After

anesthetizing mice with 30 µl of the mixture of ketamine/xylazine (90mg/kg/10mg/kg,

respectively), the animal was bleed and then the lung was lavaged 3 times each with 0.5 ml

saline. Then 100 µl of diluted BAL Fluid with saline was plated on LB agar plate, and incubated

at 37°C for 16 hours. Bacterial CFUs were determined using the Quantity One colony-counting

software (Bio-Rad, Hercules, CA).

Cytotoxicity Assay for human cells.

All cell cultures were maintained in a humidified incubator, at 37 °C, with 5% CO2

atmosphere. Human neuroblastoma SK-N-SH cells (a donation from Bonnie B. Toms at SUNY

Upstate Medical University) were cultured in 96-well plates with 100 µL of culture medium

(EMEM + 10% FBS, 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2.0 mM L-glutamine).

Each well contained 100 µL of cell suspension with a concentration of 5×104

cells/mL. The

viability of SK-N-SH was determined with a hemacytometer using standard trypan blue protocol.

For cytotoxicity assay, the colorimetric cell counting assay (CCK 8 assay) was used as described

- 181 -

previously.248

In brief, after being plated, the SK-N-SH cells were allowed to adhere to the

bottom of the wells for 24 h. The medium was then replaced with fresh ones (supplemented with

DMSO to eliminate solvent effect for the negative control) with or without brominated

furanones. After 1 h, the BF-containing media were removed and the cells were washed twice

with fresh culture medium without DMSO. The cells were then allowed to recover for 0, 24, and

48 h (recovery time), at which time the number of live cells was determined by the CCK 8

assay.248

The survival (%) was calculated using the equation: survival (%) = (A450 sample – A450

medium)/(A450 control – A450 medium) × 100. The sample absorbance values were obtained from

the wells containing cells and brominated furanones, and the control absorbance were obtained

from BF-free wells containing cells and DMSO.

Statistical analysis.

Experimental data were analyzed by SigmaStat 3.5 software (Systat Software, Inc., San Jose,

CA) and presented as means ± standard error. Two-group comparisons were performed using

Student’s t test. A P value of <0.05 was considered to be statistically significant.

3.4.2 Chemical synthesis and characterization

General procedure for synthesis.

All air sensitive reactions were performed in oven dried glassware under an atmosphere of

argon unless otherwise notified. All reagent grade starting materials were obtained from

commercial supplies and used as received. Anhydrous solvents were purchased from Sigma-

O

BrBr2, DCM

0 oC to RT

P2O5, DCM

0 oC to reflux

Et3N, DCM

0 oC to reflux

HO

OO

HO

OO

Br

Br

O

Br

OO

Br

n = 1: 5-BBF

n = 2: 6-BBF

n = 3: 7-BBF

n n n

n

- 182 -

Aldrich. Analytical thin layer chromatography was performed on EM silica gel 60 F254 glass

plates (0.25 mm). Visualization of analytical thin layer chromatography was achieved using UV

absorbance (254 mm), KMnO4, and ceric ammonium molybdate stains. Flash column

chromatography was performed using SiliaFlash P60 silica gel (40-60 Å) from SiliCycle, Inc. 1D

1H and

13 C NMR spectra were recorded on a Bruker Advance DPX-300 spectrometer.

1H

chemical shifts are reported in ppm, downfield from tetramethylsilane using residual CHCl3 as

the internal standard (δ 7.26 ppm). 13

C chemical shifts are reported in ppm, downfield from

tetramethylsilane relative to CDCl3 (δ 77.0 ppm). Mass spectra were measured using a MAT 95

XP mass spectrometer, carried out by the Mass Spectroscopy Facility at Indiana University.

OO

Br

5-BBF 15

5-BBF 15: Bromine (0.79 mL, 15 mmol) was added dropwise to a solution of 2-

oxocyclopentaneacetic acid (1.13 g, 7.69 mmol) in anhydrous methylene chloride (7.7 mL) at 0

oC. The reaction mixture was stirred at 0

oC for 40 min and then ambient temperature for 100

min. The resulting solution was washed with water (10 mL) followed by aqueous 1M Na2S2O3

solution (10 mL) and then extracted with methylene chloride (10 mL × 3). The combined organic

layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude oil was

taken up in anhydrous methylene chloride (30 mL) and cooled to 0 oC, followed by addition of

P2O5 (2.65 g, 18.6 mmol) in portions. The reaction mixture was stirred at 0 oC for 30 min,

allowed to warm to ambient temperature and then stirred under reflux for 2 h. The solid thus

formed was filtered off and the filtrate concentrated under reduced pressure. The crude oil was

dissolved in anhydrous methylene chloride (15 mL), cooled to 0 oC and treated with anhydrous

- 183 -

Et3N (1.12 mL, 8.0 mmol). The reaction mixture was stirred at ambient temperature for 30 min

and then under reflux for 2 h. After cooled to ambient temperature, the reaction mixture was

washed with aqueous saturated NH4Cl and extracted with methylene chloride (10 mL × 3). The

combined organic layer was dried over MgSO4, filtered and concentrated under reduced

pressure. Flash chromatography (SiO2, hexane:ethyl acetate, gradient) provided 5-BBF 15 (54.3

mg, 3% over 3 steps) as off white solid. TLC Rf = 0.29 (hexane:ethyl acetate, 4:1). 1H NMR (300

MHz, CDCl3): δ 5.67 (t, 1H, J = 0.9 Hz), 3.12 (t, 2H, J = 4.2 Hz), 2.95 (td, 2H, J = 3.9, 1.5 Hz).

HRMS: Cacld. for M+: 199.9467, found: 199.9466 (96.3%), 201.9442 (100.0%).

OO

Br

6-BBF 16

6-BBF 16: To a mixture of ethyl 2-cyclohexaneacetate (2.00 g, 10.6 mmol) in THF (72 mL) and

water (32 mL) was added lithium hydroxide (688.0 mg, 34.4 mmol). The reaction mixture was

stirred vigorously at ambient temperature overnight. The reaction mixture was concentrated to

remove THF and acidified with hydrochloric acid until pH was one. The mixture was extracted

with ethyl acetate (30 mL × 3), washed with brine, dried over MgSO4, filtered and concentrated

under reduced pressure. 2-Oxocyclohexaneacetic acid 18 was obtained as a light yellow crude oil

that was carried on to the next step without further purification. Bromine (1.14 mL, 22.3 mmol)

was added dropwise to a solution of crude 2-oxocyclohexaneacetic acid 18 (1.65 g, 10.6 mmol)

in anhydrous methylene chloride (10.6 mL) at 0 oC. The reaction mixture was stirred at 0

oC for

30 min and then ambient temperature for 2 h. The resulting solution was washed with water (6

mL) followed by aqueous 1M Na2S2O3 solution (6 mL) and then extracted with methylene

chloride (6 mL × 3). The combined organic layer was dried over MgSO4, filtered and

concentrated under reduced pressure. The crude oil was taken up in anhydrous methylene

- 184 -

chloride (42 mL) and cooled to 0 oC, followed by addition of P2O5 (3.67 g, 25.9 mmol) in

portions. The reaction mixture was stirred at 0 oC for 30 min, allowed to warm to ambient

temperature and then stirred under reflux for 2 h. The solid thus formed was filtered off and the

filtrate concentrated under reduced pressure. The crude oil was dissolved in anhydrous

methylene chloride (26 mL), cooled to 0 oC and treated with anhydrous Et3N (1.50 mL, 10.8

mmol). The reaction mixture was stirred at ambient temperature for 30 min and then under reflux

for 1 h. After cooled to ambient temperature, the reaction mixture was washed with aqueous

saturated NH4Cl and extracted with methylene chloride (18 mL × 3). The combined organic

layer was dried over MgSO4, filtered and concentrated under reduced pressure. Flash

chromatography (SiO2, hexane:ethyl acetate, gradient) provided 6-BBF (350.5 mg, 16% over 4

steps) as off white solid. TLC Rf = 0.66 (hexane:ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3):

δ 5.83 (s, 1H), 2.79 (t, 2H, J = 6.0 Hz), 2.73 (td, 2H, J = 6.0, 1.2 Hz), 2.00 (m, 2H); 13

C NMR

(75 MHz, CDCl3): δ 168.5, 154.9, 148.3, 111.0, 107.4, 34.0, 24.0, 23.6. HRMS: Cacld. for M+:

213.9624, found: 213.9626 (100.0%), 215.9596 (99.6%).

OO

Br

7-BBF 17

7-BBF 17: To a stirred suspension of NaH (5.10 g, 60% suspension in mineral oil, 128 mmol, 15

mL × 2 hexane washed) in anhydrous benzene (80 mL) was added dimethyl carbonate (7.6 mL,

90 mmol) via syringe. The reaction was stirred under reflux for 1 h. A solution of

cycloheptanone (4.3 mL, 36 mmol) in anhydrous benzene (6.0 mL) was then added via cannula

and the reaction mixture was stirred under reflux for 3 h. The heterogeneous mixture was coloed

to ambient temperature and then quenched with acetic acid (8 mL). The mixture was diluted with

- 185 -

water (200 mL), extracted with ethyl acetate (50 mL × 4), washed with brine, dried over MgSO4,

filtered and concentrated under reduced pressure. Methyl 2-oxo-1-ccloheptanecarboxylate 19

was obtained as yellow oil that was carried on to the next step without further purification. To a

solution of crude methyl 2-oxo-1-ccloheptanecarboxylate 19 in acetone (100 mL) was added

potassium carbonate (23.2 g, 168 mmol) followed by ethyl bromoacetate (3.8 mL, 34 mmol).

The reaction mixture was stirred under reflux overnight (~17 h). The suspension was cooled to

ambient temperature and concentrated under reduced pressure to remove ~ half of the acetone.

The residue was diluted with water (50 mL), extracted with diethyl ether (50 mL × 3), washed

with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The resulting

yellow oil was treated with a solution of hydrochloric acid (27 mL, 6M) and acetic acid (27 mL).

The reaction mixture was stirred under reflux for 2 days and cooled to ambient temperature. The

mixture was diluted with water (50 mL), extracted with methylene chloride (30 mL × 5), washed

with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Flash

chromatography (SiO2, methylene chloride:ethyl acetate, gradient) provided 2-

oxocycloheptaneacetic acid 20 (5.35 g, 87% over 3 steps) as a light yellow oil. TLC Rf = 0.31

(methylene chloride:ethyl acetate, 1:1). The identity of this compound was confirmed by

comparing 1H NMR data with that previously reported.

218 Bromine (0.93 mL, 18 mmol) was

added dropwise to a solution of 2-oxocycloheptaneacetic acid 20 (1.47 g, 8.66 mmol) in

anhydrous methylene chloride (8.7 mL) at 0 oC. The reaction mixture was stirred at 0

oC for 30

min and then ambient temperature for 2 h. The resulting solution was washed with water (5 mL)

followed by aqueous 1M Na2S2O3 solution (5 mL) and then extracted with methylene chloride (5

mL × 3). The combined organic layer was dried over MgSO4, filtered and concentrated under

reduced pressure. The crude oil was taken up in anhydrous methylene chloride (35 mL) and

- 186 -

cooled to 0 oC, followed by addition of P2O5 (3.00 g, 21.1 mmol) in portions. The reaction

mixture was stirred at 0 oC for 40 min, allowed to warm to ambient temperature and then stirred

under reflux for 2 h. The solid thus formed was filtered off and the filtrate concentrated under

reduced pressure. The crude oil was dissolved in anhydrous methylene chloride (22 mL), cooled

to 0 oC and treated with anhydrous Et3N (1.50 mL, 8.93 mmol). The reaction mixture was stirred

at ambient temperature for 30 min and then under reflux for 1 h. After cooled to ambient

temperature, the reaction mixture was washed with aqueous saturated NH4Cl and extracted with

methylene chloride (15 mL × 3). The combined organic layer was dried over MgSO4, filtered

and concentrated under reduced pressure. Flash chromatography (SiO2, hexane:ethyl acetate,

gradient) provided 7-BBF (194.8 mg, 10% over 3 steps) as off white solid. TLC Rf = 0.62

(hexane:ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3): δ 6.01 (s, 1H), 2.98 (t, 2H, J = 6.0 Hz),

2.82 (td, 2H, J = 6.0, 1.5 Hz), 1.91 (m, 2H), 1.81 (m, 2H); 13

C NMR (75 MHz, CDCl3): δ 168.0,

157.0, 148.2, 117.2, 115.4, 39.5, 29.3, 27.8, 23.9. HRMS: Cacld. for M+: 227.9780, found:

227.9785 (97.3%), 229.9766 (100.0%).

- 187 -

OO

Br

5-BBF 15

- 188 -

OO

Br

6-BBF 16

- 189 -

OO

Br

6-BBF 16

- 190 -

OO

Br

7-BBF 17

- 191 -

OO

Br

7-BBF 17

- 192 -

Chapter 4

Modulation of Quorum Sensing in Gram-negative Bacteria by Squarylated Homoserine

Lactones

Summary

An expanded library of squarylated homoserine lactones (SHLs) were designed and

synthesized as analogues to the natural AHLs. Together with some of the existing SHLs, these

compounds were evaluated for their activity to modulate the quorum sensing (QS) systems in

two Gram-negative bacteria, an opportunistic pathogen P. aeruginosa and a symbiont V. fischeri.

None of the SHLs were identified as agonists to LasR in P. aeruginosa or LuxR in V. fischeri at

the concentrations tested, except for octyl-SHL, which showed weak agonistic activity to LasR at

150 µM. The SHLs were weak to moderate antagonist to LasR in at 150 and 300 µM. All except

one SHL inhibited LuxR, with the alkyl-SHLs showing more inhibition in general than the aryl-

SHLs. The cytotoxicity study suggested that the SHLs have no or minimal inhibition to the

growth of these two bacteria species and the inhibition of QS or biofilm formation by P.

aeruginosa were not due to bactericidal effect. Primary investigation of structural activity

relationship indicated that alkyl chain length is critical to activity of SHLs. These SHLs are

promising candidates as modulators of other AHL-mediated QS systems.


4.1.1 AHL-based quorum sensing in Gram-negative bacteria

N-Acyl homoserine lactones (AHLs) are the most common class of signaling molecules used

by Gram-negative bacteria in their intercellular communication called quorum sensing (QS).123

This signaling process allows the bacteria to coordinate gene expression in response to

NH

O

O

O

R1

R2

R1 = CH3, CH3(CH2)n, or CH3(CH2)nCH=CH(CH2)mR2 = H, O, or OH

- 193 -

population density. Once the concentration of the signaling molecules reaches a threshold, the

bacteria undergo a lifestyle switch from that of a signal cell to that of a multicellular group,

initiating the gene expression that benefit the colony as a whole. These signaling molecules are

called autoinducers (AIs). There have been a number of AHLs identified in various bacteria

species (Table 3.1) but they are all composed of a homoserine lactone ring (HSL) with an acyl

chain (Figure 4.1). The AHLs may vary in acyl chain length (generally 4 to 18 carbons) and the

degree of oxidation at the AHL C3 position,254

and may have different degree of unsaturation on

the acyl chain.255

Some species produced mainly a single type of AHL while others produce

multiple AHLs.

Figure 4.1 General chemical structure of N-acyl homoserine lactones.254

The first QS system studied was that in the marine bacterium Vibrio fischeri. This QS system

is composed of the autoinducer synthase LuxI, which is responsible for the synthesis of

autoinducer (AI) 3-oxo-C6-HSL, and the receptor protein LuxR, which is a transcriptional

activator and upon binding to the autoinducer, promotes transcription of the luciferase structural

operon luxCDABE and the production of more AHL autoinducer.122,157-159

This principle of

AHL-based QS applies to the majority of Gram-negative bacteria. The autoinducer synthases and

receptors in other Gram-negative bacteria are termed LuxI-type and LuxR-type proteins,

respectively.

NH

O

O

O

R1

R2

R1 = CH3, CH3(CH2)n, or CH3(CH2)nCH=CH(CH2)mR2 = H, O, or OH

- 194 -

4.1.2 Modulation of LuxI/LuxR-type quorum sensing system

As discussed in Chapter 3, one of the most commonly used strategies to control QS is to

interfere with the binding of AIs to the LuxR-type receptors. There have been a number of

attempts to discover small molecules that can potentially interact with the LuxR-type receptors

and therefore modulate QS and QS-controlled bioactivities in bacteria. One powerful method is

the high-throughput screen which has been utilized to identify small molecules as inhibitors of

quorum sensing or biofilm formation.175-177

For example, ursolic acid was identified from 13000

compounds purified from plants as a biofilm inhibitor that inhibits biofilm formation by E. coli,

P. aeruginosa, and V. harveyi without being toxic.175

A rapid screening of 4509 compounds

revealed that ferric ammonium citrate inhibited biofilm formation by P. aeruginosa PA14 in a

dose-dependent manner.176

The human kind has long been utilizing plants and animals and fungus as traditional

medicines and we are still learning from Mother Nature as new technology allows us to identify

the active components in the therapy. We are therefore able to design and synthesize mimics of

the identified natural products which do not exist in nature, with similar or even higher biological

activities. One of the most successful approaches is based on the synthesis of mimics of naturally

occurring bacterial signaling molecules (e.g. AHLs).

The first a few AHL analogue studies were published by groups led by Eberhard,256

Greenberg,257

and Iglewski.258

They explored the agonistic and antagonistic activities of

synthetic AHL mimics against LuxR in V. fischeri or LasR in P. aeruginosa. These researchers

demonstrated that: (1). In general, the acyl chain length is critical for the activity of AHL

analogues. (2). For species whose natural AHLs possess a 3-oxo group, the presence of this

group in synthetic AHL analogues is important for activity, but not essential. The removal of the

- 195 -

3-oxo group most often results in reduced agonistic activity or results in antagonistic activity

rather than agonistic activity. (3). Replacement of the lactone ring in AHL analogues with γ-

thiolactone normally retains the same overall activity but switching to a γ-lactam is not tolerant.

In a series of studies reported by Doutheau and co-workers on AHL analogues and their

activities against LuxR, the authors found that medium-sized, branched acyl derivatives were

agonists of LuxR while phenyl derivatives were antagonists of LuxR.259

Moreover, they made a

group of AHLs in which the carbonyl group of the amide bond was replaced with a sulfonyl

group and determined that the sulfonyl AHLs with acyl chains close in length to 3-oxo-C6-HSL

and shorter were the strongest inhibitors of LuxR. None of the sulfonyl AHLs showed agonistic

activity against LuxR.260

In another report by the same group, it was shown that replacing the

amide in AHL with a urea resulted in compounds that displayed antagonistic but not agonistic

activities against LuxR.261

Suga and co-workers prepared a library of 96 AHL mimics, in which the lactone ring was

replaced with various heterocycles and carbocycles that could hydrogen-bond at the same

position as the carbonyl in the parent γ-lactone.160,161

They concluded that a five- or six-

membered ring with a hydrogen bond acceptor was necessary to activate LasR in P. aeruginosa

by AHL analogues. AHL mimics that contain an aromatic ring in place of the γ-lactone with a

hydrogen bond acceptor present could inhibit LasR. The authors speculate that compounds with

aromaticity at the AHL lactone ring position cause a conformational change of LasR upon

binding which then presumably reduces the ability of LasR to bind DNA, and therefore inhibits

transcription.262

The most comprehensive and systematic work to investigate the structure activity

relationship (SAR) for synthetic AHL analogues to date was done by Blackwell and co-

- 196 -

workers.263

Using a microwave-assisted, solid-phase route, these researchers synthesized more

than 100 AHL analogues and evaluated their activity to modulate QS across three Gram-negative

bacteria species: the symbiont V. fischeri and the pathogens P. aeruginosa and A.

tumefaciens.162,163,166,264

Their studies revealed that several of the AHL analogues exhibited

antagonistic activity in all three species examined, while other analogues were only active in one

or two species. Broad-spectrum agonists with high activity were not identified (Figure 4.2).

Several of the AHL analogues appeared to be agonist in one strain but antagonist in another.

Very interestingly, a few of these ligands were antagonists at lower concentrations but were able

to activate the transcriptional regulators at higher concentrations. From these studies, Blackwell

and co-workers summarized several SAR trends, a few of which are congruent with those found

by other groups previous reports as discussed above.256-258,260

In addition, the authors noted that:

(1). The TraR protein was the most sensitive to the length of the AHL acyl chains. Assuming the

synthetic AHLs target the same ligand-binding site on TraR compared to the natural AHL, this

finding suggests that the TraR may have a sterically crowded ligand-binding site, which is in

accordance with the X-ray crystal structure of TraR. (2). On the contrary, the LasR protein was

the most tolerant of varying AHL acyl chain length, functionality on the acyl chain, and the

stereochemistry of the homoserine lactone ring. This finding implies that LasR has a larger

ligand-binding site than TraR, which is in accordance with the X-ray crystal structure of LasR.

(3). The LuxR protein was most strongly activated by AHLs containing a phenylacetanoyl group

in the acyl chain with electron-withdrawing group in the 3-position, while was most strongly

inhibited by AHLs with acyl chain length of 6 to 14 carbons that had a 3-oxo group. These works

not only supply a highly efficient chemical tool to develop and expand new libraries of QS

- 197 -

modulators but also provide broad insights into the structural basis for QS mediated by AHL

signals in Gram-negative bacteria.

Figure 4.2 Venn diagrams showing the structures of selective most potent LuxR-type protein

antagonists and agonists identified and their selectivities for different LuxR-type proteins from

A. tumefaciens (TraR), V. fischeri (LuxR), and P. aeruginosa (LasR). Ligands in the

intersections of the circles have significant selectivity for two or more proteins.163

NA: No

applicable ligands identified.

Br

R

RMe

4

F3C R

RMe

8

MeR

O

10O

R

F3CO

OR

O2N R

NH

O

O

O

R =

Antagonists

Agonists

TraR LuxR

LasR

TraR

LasR

LuxR

MeR

O

8 O

R

MeR

O

10

MeR

O

2MeR

O

4 O2N R

NA

NA

NANA

- 198 -

4.1.3 Biofilm inhibitors containing squarate moiety

AHLs are prone to hydrolysis either at the lactone ring due to elevated pH or the activity of

acyl-HSL lactonases produced by strains of Agrobacterium, Arthrobacter, Bacillus, and

Klebsiella, or at the acyl-amide linkage catalyzed by acyl-HSL acylases produced by strains of

Pseudomonas, Ralstonia, and Variovorax.265

In addition, the flexible structures of the acyl chains

provide numerous possible conformations which may reduce the affinity of these molecules to

the receptor proteins. Taking these facts into consideration, Narasimhan et al. designed and

synthesized a group of squarylated homoserine lactones (SHLs) in which the homoserine lactone

is attached to a squarate moiety, which bears saturated or unsaturated aliphatic chains.266

Most

SHLs were prepared in a racemic form except for vinyl-SHL, which was prepared in both

enantiomerically pure forms (4 and 5) and the racemic form (3). Together with four squarylated

esters and two squarylated amides, these squarate-based AHL analogues were tested for their

inhibitory activity against biofilm formation by E.coli RP437 (Figure 4.3). Vinyl-SHL 3 and

butyl-SHL 6 were the most potent biofilm inhibitors among the molecules tested, with ~50%

inhibition at 200 µM. No different biofilm inhibitory activity was observed for the L- and D-

vinyl-SHLs and both resulted in only half biofilm inhibition compared to the racemic form.

- 199 -

Figure 4.3 Chemical structures of squarate-based AHL analogues examined by Narasimhan et al.

on inhibitory activities against biofilm formation by E. coli RP437. Figure adapted and modified

from Ph. D. thesis by Sri Kamesh Narasimhan.266

Bandyopadhyay et al. added three alkyl SHLs and three aryl SHLs to the library and

evaluated the ability of the new compounds to inhibit biofilm formation by E.coli RP437 (Figure

4.4).211

Butyl-SHL 6 was used as a control. In general, the aromatic SHLs appeared to exhibit

stronger biofilm inhibition, with the most potent compound tolyl-SHL 18 resulting in ~55%

inhibition while the least potent compound ethyl-SHL 14 showing ~34% inhibition.

Squarylated homoserine lactones (SHLs)

O O

O O

O O

O

O O

O O

O O

O O

7 8 9 10

O O

O NH

O O

NH

11 12

Squarylated esters

Squarylated amides

O O

O NH

O

O O

NH

O

O O

NH

O

O O

NH

O

O O

NH

O

1 2 3 4 5

NH

O O

O

6 OO O O O O

- 200 -

Figure 4.4 Chemical structures of squarate-based AHL analogues examined by Bandyopadhyay

et al. on inhibitory activities against biofilm formation by E. coli RP437.Note

Figure adapted and

modified from Ph. D. thesis by Debjyoti Bandyopadhyay.211

Although E. coli is not capable of producing AHL molecules because it lacks an AHL

synthase encoding gene, it does possess a LuxR-type receptor protein, called SdiA, and can

respond to AHL signals produced by other bacteria.267,268

The two publications described above

did not directly study the ability of SHLs to modulate QS in E. coli. However, since biofilm

formation is known to be regulated by AHL-based QS,139

it is possible that the SHLs inhibit

E.coli biofilm formation by binding to the LuxR-type receptor (e.g. SdiA) in E. coli.211

The most common Gram-negative bacterium found in nosocomial and life-threatening

infections of immunocompromised patients is the opportunistic pathogen P. aeruginosa. These

infections are very persistent because of the presence of Pseudomonal biofilm and the resistance

to antimicrobial therapy. Controlling the QS in P. aeruginosa may lead to inhibition or

eradication of P. aeruginosa biofilm and therefore is a significantly clinically related issue.

There are two identified AHL-mediated QS circuits in P. aeruginosa. In the las circuit,

autoinducer synthase LasI is responsible for the production of the AHL signal 3-oxo-C12-HSL

while in the rhl circuit, the RhlI protein is responsible for the production of a second AHL signal,

NH

O O

O

O

NH

O O

O

OS

16

O O

NH

O

O

17 18

NH

O O

O

O

NH

O O

O

O

NH

O O

O

O

NH

O O

O

O13 14 6 15

Squarylated homoserine lactones (SHLs)

- 201 -

C4-HSL. In the effort of evaluating the ability of synthetic AHLs to stimulate the expression of

the gene for elastase, one of many biological activities controlled by AHL-mediated QS in P.

aeruginosa, Passador et al. demonstrated that the length of the acyl side chain of the AHL

analogues is the most critical factor of activity.258

Therefore we designed the synthesis of SHLs

with chain length similar to that in the natural AI 3-oxo-C12-HSL (8 and 12 carbons for SHLs 19

and 20, respectively) (Scheme 4.1). Introduction of aromatic substituent to the structure of AHL

analogues leads to agonists and antagonists with increased activities.162,163,264

In addition to the

aryl-substituted SHLs 16-18 in hand, we also designed m-xylyl-SHL 21, which has more

substituents on the benzene ring. SHL 6, 13, and 15-21 constitute a library of candidates as

modulators for QS in P. aeruginosa (Figure 4.5).

Figure 4.5 The library of SHLs used in this study.Note.


4.2.1 Synthesis of SHLs

The synthesis route used to prepare SHLs was adapted and modified from that previously

reported by Narasimhan266

and Bandyopadhyay (Scheme 4.1).211

Briefly, 1,2-addition of various

alkylating reagents at one of the carbonyl groups of 3,4-dibutoxy-3-cyclobutene-1,2-dione 9

NH

O O

O

O

NH

O O

O

O

NH

O O

O

O13 6 15

NH

O O

O

O19

NH

O O

O

O20

NH

O O

O

O

NH

O O

O

OS

16

O O

NH

O

O

17 18

NH

O O

O

O

21

Alkyl-SHLs

Note: Methyl-SHL 13, butyl-SHL 6, and tolyl-SHL 17 were synthesized by Debjyoti Bandyopadhyay. Phenyl-SHL

16 and the precursor for tolyl-SHL 17 were synthesized by Nisha Varghese. All the other SHLs were synthesized

by Sijie Yang. Department of Chemistry, Syracuse University, Syracuse NY.

- 202 -

gave cyclobutenols 22-25, which yielded cyclobutenediones 26-30 upon treatment with

concentrated hydrochloric acid. The alkylating reagents were Grignard reagents or organolithium

reagent made from corresponding commercially available bromides. The cyclobutenediones 26-

30 then reacted with α-amino-γ-butyrolactone hydrobromide in the presence of triethylamine and

provided the desired SHLs, namely hexyl-SHL 15, thienyl-SHL 18, octyl-SHL 19, dodecyl-SHL

20, and xylyl-SHL 21. All the SHLs precipitated out from the reaction solvent EtOH, and did not

need further purification by column chromatography.

Scheme 4.1 Synthesis of SHLsa

aN/A indicates the shown compound was not obtained in purified form and a yield was not

available.

4.2.2 Cytotoxicity study of SHLs on the growth of P. aeruginosa

The increasing occurrence of drug-resistant bacteria strains and lack of new antibiotics pose a

significant challenge in treating diseases. It is essential to develop quorum sensing modulators

that do not exert evolutionary selective pressure on bacteria by inhibiting bacterial survival. We

first evaluate the cytotoxicity of SHLs on the growth of P. aeruginosa. Optical density values of

bacteria culture grown in the presence of 300 µM SHLs were measured at 600 nm (OD600) every

2 h for the first 12 h and then after 24h of incubation. Bacteria grown in the absence of SHLs

Sijie’s SHLs

O O

BuO OBuTHF, -78 oC to 0 oC

then NH4Cl (aq.)

conc. HCl, DCM, rtHO O

BuO OBu

O

BuO O EtOH, Et3N rt, overnight

OH3N

OBrNH

O

O

O O

RMgBr or RLin

n

n

9R = n-hexyl, 22 (44%) n-octyl, 23 (48%)

n-dodecyl, 24 (45%) thienyl, 25 (21%)

m-xylyl, (N/A)

R = n-hexyl, 15 (53%) n-octyl, 19 (65%)

n-dodecyl, 20 (30%) thienyl, 18 (60%) m-xylyl, 21 (65%)

R = n-hexyl, 26 (65%) n-octyl, 27 (80%)

n-dodecyl, 28 (21%) thienyl, 29 (59%)

m-xylyl, 30 (27% over 2 steps)

- 203 -

were used as the control (Figure 4.5). The same amount of DMSO introduced from the SHL

stocks was added to the control to eliminate solvent effect. None of the SHLs tested resulted in

any deviation of OD values from that of the SHL-free control and therefore these SHLs do not

inhibit the growth of P. aeruginosa PAO1under the conditions used.


µM SHLs. Values represent the means ± standard deviation from four replicates.

4.2.3 Modulation of quorum sensing in P. aeruginosa by SHLs

A double-knockout reporter strain PAO-JP2 (ΔlasIΔrhlI)236

harboring the plasmid plasI-

LVAgfp237

was used to evaluate the effect of SHLs on the las QS system in P. aeruginosa. When

bacteria are growing in the presence of exogenously introduce autoinducer 3-oxo-C12-HSL or

functional analogues, LasR receptor protein binds to the AI, activating transcription of the lasI

promoter and thus the expression of green fluorescence protein (GFP). The GFP production is

quantified by correcting the measured fluorescence signal for cell density as shown in Equation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 2 4 6 8 10 12 14 16 18 20 22 24

OD

600

Time (h)

control

Methyl-SHL

n-Butyl-SHL

n-Hexyl-SHL

n-Octyl-SHL

n-Dodecyl-SHL

Phenyl-SHL

Tolyl-SHL

Thienyl-SHL

m-Xylyl-SHL

- 204 -

4.1. “RFUsample” is the relative fluorescence unit measured by the plate reader. “No AI”

represents bacteria culture grown in the presence of no AI analogue, supplemented with the same

amount of DMSO present in the SHL stocks.

(4.1)

In the agonism assay, bacteria cultures grown in the presence of 150 µM SHLs were

compared to that grown in the presence of 1 µM natural AI 3-oxo-C12-HSL (a concentration

within the range produced by wild type P. aeruginosa).130,160-162

Only octyl-SHL 19 was able to

activate GFP production by a measurable amount, ~20% that of the control (1 µM 3-oxo-C12-

HSL). None of the other SHLs showed any activation at this concentration (Figure 4.6).

The antagonism assay was conducted with competition of 150 or 300 µM SHLs against 1

µM 3-oxo-C12-HSL (Figure 4.7). As the concentration increased from 150 to 300 µM, almost all

of the SHLs resulted in reduced GFP expression. The SHLs appeared to be weak to moderate

antagonists to LasR at the concentrations tested. The most potent antagonists were octyl-SHL 19

and tolyl-SHL 17, which showed ~35% and 42% inhibition, respectively.

- 205 -


HSL alone (control) or 150 µM SHLs. Fluorescence signals were corrected for cell density as

shown in Equation 4.1 and the results were normalized to the control. Values represent the

means ± standard deviation from four replicates. Data shown is a representative of at least three

separate experiments.


HSL alone (control) or 1 µM 3-oxo-C12-HSL plus different concentrations of SHLs.

0

20

40

60

80

100

120

Re

lative

flu

ore

sce

nce

(%

)

0

20

40

60

80

100

120

140

Re

lative

flu

ore

sce

nce

(%

)

150 µM

300 µM

- 206 -

Fluorescence signals were corrected for cell density as shown in Equation 4.1 and the results

were normalized to the control. Values represent the means ± standard deviation from four

replicates. Data shown is a representative of at least three separate experiments

4.2.4 Inhibition of P. aeruginosa biofilm formation by SHLs

Biofilm formation by P. aeruginosa in the presence of 300 µM SHLs was quantified using a

colorimetric assay in 96-well plates empolying crystal violet (CV) (Figure 4.8). The amount of

biofilm formation was determined indirectly by measuring the absorbance of CV at 540 nm after

workup (see experimental section for details) and normalized by that of the SHL-free control as

shown in Equation 4.2. The SHL-free control was supplemented with the same amount of

DMSO present in the SHL stocks to eliminate solvent effect.

(4.2)

In contrast to the trend that aryl-SHLs showed more biofilm inhibition than alkyl-SHLs for

E.coli as observed by Bandyopadhyay,211

alkyl-SHLs seemed to have stronger inhibitory

activities than aryl-SHLs against biofilm formation by P. aeruginosa. This difference may be

due to different structure at the binding site of receptors SdiA (in E. coli) and LasR (in P.

aeruginosa). Moreover, a clear structure activity relationship was observed for the alkyl-SHLs.

Within the range of one to eight carbons in the alkyl side chain, the biofilm inhibitory activity

increased as the chain length increased. Octyl-SHL 19, which possess the chain length most

similar to the natural AI 3-oxo-C12-HSL, resulted in the most inhibition, ~76% compared to the

control. We note that dodecyl-SHL 20 did not seem to be completely soluble in the bacterial

- 207 -

culture at the concentration tested. All of the aryl-SHLs were moderate biofilm inhibitors, with

~50% inhibition in all cases.

Figure 4.8 P. aeruginosa biofilm formation in the presence of 300 µM SHLs. Values are

normalized by that of the SHL-free control as shown in Equation 4.2 and represent the means ±

standard deviation from 4 replicates. Data shown is a representative of at least three separate

experiments. Significant differences in the biofilm formation from the SHL-free control are

indicated by asterisks: ***, P < 0.001.

4.2.5 Cytotoxicity study of SHLs on the growth of V. fischeri

Blackwell and co-workers discovered a few AHL analogues that exhibited antagonistic

activities against LuxR-type proteins in more than one bacteria species.163,165,166

We were also

interested in if any of our SHLs was capable of activating or inhibiting LuxR-type proteins in

more than one species. We selected the V. fischeri for this purpose. The QS system in V. fischeri

is representative of all AHL-mediated QS systems in Gram-negative bacteria and the natural AI

0

20

40

60

80

100

120R

ela

tive

bio

film

fo

rma

tio

n (%

)

******

***

***

**

****** ***

***

- 208 -

3-oxo-C6-HSL is structurally similar to the 3-oxo-C12-HSL produced by P. aeruginosa. We

expected at least one or more of the SHLs with shorter alkyl side chain should show some

activity for the LuxR protein.

The cytotoxicity of the SHLs to the V.fischeri was first studied. The strain VCW2G7 we used

lacks the luxI gene that encodes the AI synthase LuxI and does not produce the natural AI 3-oxo-

C6-HSL, but possesses the native lux operon that expresses luminescence.269

The EC50 of 3-oxo-

C6-HSL was determined to be 3 µM.163

We used this concentration as the control and compared

the growth curves of bacteria culture in the presence of 3 µM 3-oxo-C6-HSL plus 10 µM SHLs

to the control (Figure 4.9). At 10 µM, none of the SHLs except methyl-SHL 13 had negative

impact on the growth of bacteria. Methyl-SHL 13 caused about ~20% reduction in OD600 values

than the control throughout the experiment. Therefore, the SHLs had no or very little effect on

the growth of V. fischeri.

Figure 4.9 Growth curves of V. fischeri VCW2G7 in the presence of 3 µM 3-oxo-C6-HSL alone

(control) or 3 µM 3-oxo-C6-HSL plus 10 µM SHLs. Values represent the means ± standard

deviation from four replicates.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8 10 12 14 16 18 20 22 24

OD

600

Time (h)

control

Methyl-SHL

n-Butyl-SHL

n-Hexyl-SHL

n-Octyl-SHL

n-Dodecyl-SHL

Phenyl-SHL

Tolyl-SHL

Thienyl-SHL

m-Xylyl-SHL

- 209 -

4.2.6 Modulation of quorum sensing in V. fischeri by SHLs

The ability of SHLs to modulate the QS in V. fischeri was evaluated similarly to that in P.

aeruginosa except luminescence was measured instead of fluorescence. The luminescence

expression is quantified as shown in Equation 4.3. “RLUsample” is the relative luminescence unit

measured by the plate reader. “No AI” represents bacteria culture grown in the presence of no AI

analogue, supplemented with the same amount of DMSO present in the SHL stocks.

(4.3)

In the agonism assay, 10 µM SHLs were compared to 3 µM 3-oxo-C6-HSL and none of the

SHLs showed measurable agonistic activity (data not shown). When 10 µM SHLs competed

with 3 µM 3-oxo-C6-HSL in the antagonism assay (Figure 4.10), hexyl-SHL 15 and octyl-SHL

19 were the most potent antagonists identified, as expected, with ~90% and >99% reduction in

luminescence expression, respectively. Moreover, the alkyl-SHLs in general resulted in less

luminescence expression than aryl-SHLs. We note that while m-xylyl-SHL 21 (two methyl

substitution on the benzene ring) appeared to be a strong antagonist (23% relative fluorescence

signal of the control), tolyl-SHL 17 (one methyl substitution on the aromatic ring) appeared to be

a weak agonist (112% relative luminescence expression of the control). This difference in

activity is likely caused by structural difference and the specific mechanism is under

investigation.

- 210 -

Figure 4.10 Luminescence expression by V. fischeri VCW2G7 in the presence of 3 µM 3-oxo-

C6-HSL alone (control) or 3 µM 3-oxo-C6-HSL plus 10 µM SHLs. Luminescence signals were

corrected for cell density as shown in Equation 4.3 and the results were normalized to the

control. Values represent the means ± standard deviation from four replicates. Data shown is a

representative of at least three separate experiments. Significant differences in the luminescence

expression from the SHL-free control are indicated by asterisks: ***, P < 0.001.


An expanded library of squarylated homoserine lactones were designed and synthesized as

analogues to the natural AHLs. The SHLs were weak to moderate antagonist to LasR in P.

aeruginosa at 150 and 300 µM while only the octyl-SHL 19 was a weak agonist to LasR at 150

µM. Although none of the SHLs were capable of activating LuxR in V. fischeri at 10 µM, all

except one SHL inhibited LuxR, with the alkyl-SHLs showing more inhibition in general than

the aryl-SHLs. Octyl-SHL 19 was the most potent inhibitor of biofilm formation by P.

0

20

40

60

80

100

120

140

Re

lative

lum

ine

sce

nce

(%

)

*** ***

******

***

***

- 211 -

aeruginosa among all SHLs tested. Primary investigation of structural activity relationship

indicated that alkyl chain length is critical to the activity of SHLs. Future works include study of

the ligand-receptor binding between SHLs to LuxR-type receptor proteins by computationally

docking to elucidate the mechanism of observed structural activity relationship. This

computational analysis should provide important guidance in the in silico design of new QS

modulators of LuxI/LuxR-type QS systems in Gram-negative bacteria.


Bacteria strains and growth media.

Double-knockout mutants of Pseudomonas aeruginosa, PAO-JP2 (plasI-LVAgfp) was kindly

provided by Dr. Helen E. Blackwell (University of Wisconsin-Madison) with permission by Dr.

Babara H. Iglewski (University of Rochester Medical Center).236,249

P. aeruginosa PAO1-BAA-

47 (wild type) from ATCC and PAO-JP2 (plasI-LVAgfp) were grown in Luria-Bertani (LB)

broth (containing 300 µg/mL of carbenicillin for PAO-JP2 (plasI-LVAgfp)) at 37 oC. V. fischeri

VCW2G7 was grown in LB salt media (LBS) at ambient temperature. LBS media contained 2%

NaCl, 1% tryptone, 0.5% yeast extract, 0.3% (v/v) glycerol, and 50 mM Tris-HCl buffer (pH =

7.5).163

Stock solutions of SHLs.

Stock solutions of all SHLs (200 µM to 5 mM) were prepared in DMSO, sterilized by filtering

through a 0.2 µm syringe filter, and stored at -20 °C in sealed vials. Appropriate amount of

DMSO was added to controls in all assays to eliminate solvent effect. The amount of DMSO in

all cases was no more than 0.2%.

Growth curve of planktonic bacteria, reporter gene assay for P. aeruginosa, and crystal violet

biofilm assay in 96-well plates.

- 212 -

These experiments were performed as described in Chapter 3.

Reporter gene assay for V. fischeri.

An overnight culture of V. fischeri VCW2G7 in LBS broth was grown from a single colony

picked from an LBS agar. The cell culture was diluted by 10 times and aliquoted (200 µL per

well) to a polystyrene 96-well microplate (Costar 3370, Corning Incorporated, Corning, NY)

containing appropriate amount of natural autoinducers with and without SHLs. The plate was

incubated at ambient temperature (~21 oC) for 5~6 h in a rotary shaking incubator. The culture

from each well was then transferred to a 96-well plate with white wall (µClear, 655095, Greiner

Bio-One North America, Inc., Monroe, NC). The luminescence and OD absorbance (OD600) in

each well was measured by Synergy H1 multi-mode microplate reader with a Gen5 data analysis

software.

General procedure for synthesis.


unless otherwise notified. All reagent grade starting materials were obtained from commercial

supplies and used as received. Anhydrous solvents were purchased from Sigma-Aldrich.

Analytical thin layer chromatography was performed on EM silica gel 60 F254 glass plates (0.25

mm). Visualization of analytical thin layer chromatography was achieved using UV absorbance

(254 mm), KMnO4, and ceric ammonium molybdate stains. Flash column chromatography was

performed using SiliaFlash P60 silica gel (40-60 Å) from SiliCycle, Inc. 1H and

13 C NMR

spectra were recorded on a Bruker Advance DPX-300 spectrometer. 1H chemical shifts are

reported in ppm, downfield from tetramethylsilane using residual CHCl3 as the internal standard

(δ 7.26 ppm). 13

C chemical shifts are reported in ppm, downfield from tetramethylsilane relative

to CDCl3 (δ 77.0 ppm), (δ 39.5 ppm) and CD3CN (δ 1.94 ppm). Mass spectra were measured

- 213 -

using a MAT 95 XP mass spectrometer, carried out by the Mass Spectroscopy Facility at Indiana

University.

O

O O

OH

22

To a stirred solution of 3,4-butoxy-3-cyclobutene-1,2-dione (269.0 mg, 1.165 mmol) in

anhydrous THF (12 mL) was added hexylmagnesium bromide (2.0 M solution in diethyl ether,

0.61 mL, 1.2 mmol) dropwise at -78 oC. The reaction mixture was stirred at -78

oC for 4 h and

another portion of hexylmagnesium bromide (2.0 M solution in diethyl ether, 0.30 mL, 0.60

mmol) was added. After stirring at -78 oC for 1 h further, the reaction mixture was poured into a

separatory funnel containing 5% NH4Cl (14 mL) and diethyl ether (14 mL). The aqueous layer

was extracted with diethyl ether (14mL × 3) and the combined organic layer was washed with

brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Column

chromatography (SiO2; hexane:ethyl acetate, 50:1 to 3:1, gradient) provided alcohol 22 (161.5

mg, 44%) as a colorless oil. TLC Rf = 0.20 (hexane:ethyl acetae, 5:1). 1H NMR (300 MHz,

CDCl3) δ 4.35 (m, 2H), 4.19 (td, 2H, J = 6.6, 1.8 Hz), 3.68 (br, 1H), 1.76 (m, 4H), 1.59 (m, 2H),

1.38 (m, 4H), 1.25 (m, 8H), 0.93 (t, 3H, J = 7.5 Hz), 0.89 (t, 3H, J = 7.4 Hz), 0.83 (t, 3H, J = 6.5

Hz); 13

C NMR (75 MHz, CDCl3): δ 187.6, 167.9, 132.6, 86.4, 72.8, 70.5, 32.6, 31.7, 31.5, 31.4,

29.3, 24.9, 22.4, 18.7, 18.6, 13.9, 13.6, 13.5.

O O

O

26

To a stirred solution of alcohol 22 (138.5 mg, 0.444 mmol) in anhydrous methylene chloride (3.0

mL) was added one drop of concentrated hydrochloric acid (12 M) at ambient temperature. The

reaction mixture was stirred at ambient temperature for 20 min and concentrated under reduced

pressure. Column chromatography (SiO2; hexane:ethyl acetate, 50:1 to 15:1, gradient) provided

- 214 -

3-butoxy-4-hexyl-3-cyclobutene-1,2-dione 26 (68.7 mg, 65%) as a colorless oil. TLC Rf = 0.71

(hexane:ethyl acetae, 3:1). 1H NMR (300 MHz, CDCl3): δ 4.73 (t, 2H, J = 6.6 Hz), 2.60 (t, 2H, J

= 7.5 Hz), 1.80 (quintet, 2H, J = 7.0 Hz), 1.67 (m, 2H), 1.45 (m, 2H), 1.31 (m, 6H), 0.98 (t, 3H, J

= 7.4 Hz), 0.88 (t, 3H, J = 6.6 Hz); 13

C NMR (75 MHz, CDCl3): δ 198.6, 195.5, 194.3, 184.5,

74.3, 31.8, 31.3, 29.2, 25.7, 25.0, 22.4, 18.5, 14.0, 13.5.

NH

O O

O

O15

α-Amino-γ-butylrolactone hydrobromide (55.6 mg, 0.299 mmol) was added to a stirred solution

of 3-butoxy-4-hexyl-3-cyclobutene-1,2-dione 26 (58.7 mg, 0.247 mmol) in ethanol (2.5 mL).

The resulting suspension was then treated with triethylamine (41 µL, 0.30 mmol). After stirring

at ambient temperature for 2.5 h, the solvent was removed under reduced pressure. The crude

was washed with diethyl ether (3 mL), dissolved in a mixture of water (3 mL) and ethyl acetate

(3 mL), and extracted with ethyl acetate (3 mL × 2). The combined organic layer was dried over

MgSO4, filtered, and concentrated under reduced pressure. Hexyl-SHL 15 was obtained as a pale

yellow solid (34.5 mg, 53%). TLC Rf = 0.20 (hexane:ethyl acetae, 1:2).

1H NMR (300 MHz,

CD3CN): δ 7.11 (br, 1H), 5.00 (b, dd, J = 20.4, 7.8 Hz, 1H), 4.42 (t, 1H, J = 7.8 Hz), 4.28 (m,

1H), 2.68 (m, 1H), 2.54 (t, 2H, J = 7.5 Hz), 2.34 (m, 1H), 1.62 (m, 2H), 1.32 (m, 6H), 0.91 (t,

3H, J = 7.2 Hz) ; 13

C NMR (75 MHz, CD3CN): δ 195.2, 193.0, 186.0, 175.9, 175.8, 66.8, 53.9,

32.5, 30.9, 30.3, 27.0, 25.9, 23.6, 14.7 ; HRMS: Cacld. for M+: 265.2309, found: 265.1310.

O

O O

OH

23

A soltuion of 1-bromooctane (1.2 mL, 7.0 mmol) in anhydrous THF (4.5 mL) was added to a

stirred suspension of freshly ground magnesium turnings (201.3 mg, 8.39 mmol) in anhydrous

THF (1.0 mL) dropwise at ambient temperature. The mixture was stirred at ambient temperature

- 215 -

for 5 h and then added to a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (0.77 mL, 3.5

mmol) in anhydrous THF (70 mL) via cannula at -78 oC. The reaction mixture was stirred at this

temperature for 4 h and then quenched with saturated aqueous NH4Cl. The aqueous layer was

extracted with diethyl ether (25 mL × 3), dried over MgSO4, filtered, and concentrated under

reduced pressure. Column chromatography (SiO2; hexane:ethyl acetate, 20:1 to 5:1, gradient)

provided alcohol 23 (569.6 mg, 48%) as a colorless oil. TLC Rf = 0.23 (hexane:ethyl acetae,

5:1). 1H NMR (300 MHz, CDCl3): δ 4.31 (m, 2H), 4.14 (m, 2H), 1.71 (m, 4H),

1.55 (m, 2H),

1.35 (m, 4H), 1.18 (m, 13H), 0.88 (t, 3H, J = 7.5 Hz), 0.85 (t, 3H, J = 7.5 Hz), 0.79 (t, 3H, J =

6.6 Hz); 13

C NMR (75 MHz, CDCl3): δ 187.8, 168.0, 132.4, 86.2, 72.7, 70.3, 32.5, 31.6 (2 C),

31.3, 29.5, 29.2, 29.0, 24.9, 22.4, 18.6, 18.5, 13.8, 13.5, 13.4.

O O

O

27


mL) was added two drops of concentrated hydrochloric acid (12 M) at ambient temperature. The



3-butoxy-4-octyl-3-cyclobutene-1,2-dione 27 (209.9 mg, 80%) as a colorless oil. TLC Rf = 0.54

(hexane:ethyl acetae, 5:1). 1H NMR (300 MHz, CDCl3): δ 4.73 (t, 2H, J = 6.6 Hz), 2.59 (t, 2H, J

= 7.5 Hz), 1.79 (m, 2H), 1.68 (m, 2H), 1.45 (m, 2H), 1.26 (m, 10H), 0.97 (t, 3H, J = 7.5 Hz),

0.89 (t, 3H, J = 6.6 Hz); 13

C NMR (75 MHz, CDCl3): δ 198.6, 195.6, 194.3, 184.5, 74.3, 31.8,

31.7, 29.5, 29.0 (2 C), 25.7, 25.0, 22.6, 18.5, 14.0, 13.5. HRMS: Cacld. for M+: 266.1882, found:

266.1868.

NH

O O

O

O19

- 216 -


of 3-butoxy-4-octyl-3-cyclobutene-1,2-dione (209.9 mg, 0.789 mmol) in ethanol (7.9 mL). The

resulting suspension was then treated with triethylamine (0.13 mL, 0.94 mmol). After stirring at

ambient temperature for 1.5 h, the solvent was removed under reduced pressure. The crude was

washed with diethyl ether (10 mL), dissolved in a mixture of water (10 mL) and ethyl acetate (10

mL), and extracted with ethyl acetate (10 mL × 2). The combined organic layer was dried over

MgSO4, filtered, and concentrated under reduced pressure. Octyl-SHL 19 was obtained as a

white solid (150.3 mg, 65%). TLC Rf = 0.41 (ethyl acetae).

1H NMR (300 MHz, CD3CN): δ 6.99

(br, 1H), 5.00 (td, 1H, J = 11.7, 9.0 Hz), 4.42 (t, 1H, J = 9.0 Hz), 4.28 (m, 1H), 2.69 (m, 1H),

2.54 (t, 2H, J = 7.5 Hz), 2.32 (m, 1H), 1.63 (m, 2H), 1.28 (m, 10H), 0.88 (t, 3H, J = 6.6 Hz) 13

C

NMR (75 MHz, CD3CN): δ 195.2, 193.0, 186.0, 176.0, 175.8, 67.0, 66.9, 54.0, 32.9, 31.0, 30.7,

30.3, 27.1, 26.0, 23.7, 14.8. HRMS: Cacld. for M+: 293.1627, found: 293.1624.

O

O O

OH

24

A soltuion of 1-bromododecane (1.23 mL, 4.97 mmol) in anhydrous THF (4.0 mL) was added to

a stirred suspension of freshly ground magnesium turnings (148.3 mg, 6.18 mmol) in anhydrous

THF (0.5 mL) dropwise at ambient temperature. The mixture was stirred at ambient temperature

for 5 h and then added to a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (0.55 mL, 2.5

mmol) in anhydrous THF (45 mL) via cannula at -78 oC. The reaction mixture was stirred at this

temperature for 2 h and then quenched with saturated aqueous NH4Cl. The aqueous layer was

extracted with diethyl ether (10 mL × 3), dried over MgSO4, filtered, and concentrated under


provided alcohol 24 (407.5 mg, 45%) as a pale yellow oil. TLC Rf = 0.27 (hexane:ethyl acetae,

5:1). 1H NMR (300 MHz, CDCl3): 4.27 (m, 2H), 4.10 (td, 2H, J = 6.6, 1.8 Hz), 3.88 (br, 1H),

- 217 -

1.67 (m, 4H), 1.51 (m, 2H), 1.30 (m, 4H), 1.15 (m, 20H), 0.85 (t, 3H, J = 7.2 Hz), 0.82 (t, 3H, J

= 7.5 Hz), 0.77 (t, 3H, J = 6.9 Hz).

O O

O

28


mL) was added three drops of concentrated hydrochloric acid (12 M) at ambient temperature.

The reaction mixture was stirred at ambient temperature for 20 min and concentrated under


provided 3-butoxy-4-dodecyl-3-cyclobutene-1,2-dione 28 (69.5 mg, 21%) as a pale yellow oil.

TLC Rf = 0.62 (hexane:ethyl acetae, 5:1). 1H NMR (300 MHz, CDCl3): δ 4.73 (t, 2H, J = 6.6

Hz), 2.60 (t, 2H, J = 7.5 Hz), 1.80 (m, 2H), 1.67 (m, 2H), 1.46 (m, 2H), 1.25 (m, 18H), 0.98 (t,

3H, J = 7.5 Hz), 0.88 (t, 3H, J = 6.6 Hz); 13

C NMR (75 MHz, CDCl3): δ 198.8, 195.5, 194.3,

184.5, 74.3, 31.9, 31.8, 29.6 (3C), 29.5, 29.4, 29.3, 29.1, 25.7, 25.0, 22.7, 18.5, 14.1, 13.5.

HRMS: Cacld. for M+: 322.2508, found: 322.2505.

NH

O O

O

O20


of 3-butoxy-4-dodecyl-3-cyclobutene-1,2-dione 28 (69.5 mg, 0.216 mmol) in ethanol (2.2 mL).

The resulting suspension was then treated with triethylamine (0.37 mL, 0.27 mmol). After

stirring at ambient temperature for 1 h, the solvent was removed under reduced pressure. The

crude was washed with diethyl ether (5 mL), dissolved in a mixture of water (2 mL) and ethyl

acetate (2 mL), and extracted with ethyl acetate (2 mL × 2). The combined organic layer was

dried over MgSO4, filtered, and concentrated under reduced pressure. Octyl-SHL 20 was

obtained as a white solid (22.3 mg, 30%). TLC Rf = 0.77 (ethyl acetae).

1H NMR (300 MHz,

- 218 -

CD3CN): δ 7.06 (br, 1H), 5.00 (td, 1H, J = 11.7, 9.0 Hz), 4.42 (t, 1H, J = 6.0 Hz), 4.28 (m, 1H),

2.68 (m, 1H), 2.54 (t, 2H, J = 7.5 Hz), 2.33 (m, 1H), 1.63 (m, 2H), 1.27 (m, 18H), 0.88 (t, 3H, J

= 6.6 Hz); 13

C NMR (75 MHz, CD3CN): δ 195.0, 192.6, 185.6, 175.7, 175.5, 118.4, 66.5, 53.6,

32.7, 30.7, 30.4 (3C), 30.3, 30.1, 30.0, 26.8, 25.7, 23.5, 14.5. HRMS: Cacld. for M+: 349.2253,

found: 349.2251.

O

O O

OH

S

25

To a stirred solution of 2-bromothiophene (52 µL, 0.53 mmol) in anhydrous THF (7.0 mL) was

added n-butyllithium (2.5 M solution in hexane, 0.21 mL, 0.53 mmol) dropwise at -78 oC. The

reaction mixture was stirred at -78 oC for 20 min and then added to a stirred solution of 3,4-

dibutoxy-3-cyclobutene-1,2-dione (111.8 mg, 0.484 mmol) in anhydrous THF (17 mL) via

cannula at -78 oC. The reaction mixture was stirred at -78

oC for 20 min and then poured into a

sepatorary funnel containing 5% NH4Cl (15 mL) and diethyl ether (5 mL). The aqueous layer

was extracted with diethyl ether (5mL × 3) and the combined organic layer was washed with


chromatography (SiO2; hexane:ethyl acetate, 20:1 to 5:1, gradient) provided alcohol 25 (31.5 mg,

21%) as a colorless oil. TLC Rf = 0.38 (hexane:ethyl acetae, 5:1). 1H NMR (300 MHz, CDCl3): δ

7.30 (dd, 1H, J = 5.1, 1.2 Hz), 7.10 (dd, 1H, J = 3.6, 1.2 Hz), 7.01 (dd, 1H, J = 5.1, 3.6 Hz),

4.39 (m, 2H), 4.30 (t, 2H, J = 6.6 Hz), 3.53 (br, 1H), 1.70 (m, 4H), 1.43 (m, 4H), 0.95 (t, 3H, J =

7.2 Hz), 0.93 (t, 3H, J = 7.2 Hz).

O O

O

S29


mL) was added one drop of concentrated hydrochloric acid (12 M) at ambient temperature. The

- 219 -



3-butoxy-4-thienyl-3-cyclobutene-1,2-dione 29 (14.1 mg, 59%) as needle-like yellow crystals.

1H NMR (300 MHz, CDCl3): δ 7.91 (dd, 1H, J = 3.6, 1.2 Hz), 7.81 (dd, 1H, J = 5.1, 1.2 Hz),

7.28 (dd, 1H, J = 5.1, 3.6 Hz), 4.92 (t, 2H, J = 6.6 Hz), 1.90 (m, 2H), 1.53 (m, 2H), 1.02 (t, 3H, J

= 7.2 Hz).

NH

O O

O

OS

18


of 3-butoxy-4-thienyl-3-cyclobutene-1,2-dione 29 (14.1 mg, 0.060 mmol) in ethanol (2.0 mL).

The resulting suspension was then treated with triethylamine (11 µL, 0.079 mmol). After stirring

at ambient temperature for 2 h, the precipitate was collected by filtration. Diethyl ether (8.0 mL)

was added to the filtrate and precipitate thus formed was collected by filtration. The combined

solid was washed with pentane (5 mL) and dried under vacuum. Thienyl-SHL 18 was obtained

as a pale yellow solid (9.4 mg, 60%). TLC Rf = 0.35 (hexane:ethyl acetae, 1:3).

1H NMR (300

MHz, DMSO-d6): δ 9.35 (d, 1H, J = 4.5 Hz), 8.04 (d, 1H, J = 4.8 Hz), 7.87 (d, 1H, J = 3.6 Hz),

7.39 (t, 1H, J = 4.2 Hz), 5.27 (m, 1H), 4.48 (t, 1H, J = 8.9 Hz), 4.32 (m, 1H), 2.29 (m, 1H), 2.41

(m, 1H); 13

C NMR (75 MHz, DMSO-d6): δ 190.3, 186.6, 176.8, 174.6, 157.7, 132.2, 128.8,

128.8, 128.7, 65.6, 52.9, 29.3. HRMS: Cacld. for M+: 263.0247, found: 263.0240.

O O

O

30

To a stirred solution of 5-bromo-m-xylene (0.45 mL, 3.2 mmol) in anhydrous THF (15 mL) was

added n-butyllithium (2.5 M solution in hexane, 1.2 mL, 3.0 mmol) dropwise at -78 oC. The

- 220 -

reaction mixture was stirred at -78 oC for 20 min and then added to a stirred solution of 3,4-

dibutoxy-3-cyclobutene-1,2-dione (0.24 mL, 1.1 mmol) in anhydrous THF (35 mL) via cannula

at -78 oC. The reaction mixture was stirred at -78

oC for 45 min and then poured into a

sepatorary funnel containing 5% NH4Cl (15 mL) and diethyl ether (15 mL). The aqueous layer

was extracted with diethyl ether (15 mL × 3) and the combined organic layer was washed with


chromatography (SiO2; hexane:ethyl acetate, 10:1 to 3:1, gradient) provided a mixture of mono-

and di-subsituted product. The mixture was dissolved in DCM (5 mL) and treated with 2 drops

of concentration hydrochloric acid (12 M). The reaction mixture was concentrated under reduced

pressure after stirred at ambient temperature for 45 min. Column chromatography (SiO2;

hexane:ethyl acetate, 5:1) provided 3-butoxy-4-(3,5-dimethyl-phenyl)-3-cyclobutene-1,2-dione

30 (41.5 mg, 27% over 2 steps) as a light yellow oil. TLC Rf = 0.52 (hexane:ethyl acetae, 5:1).

1H NMR (300 MHz, CDCl3): δ 7.67 (s, 2H),

7.19 (s, 1H), 4.93 (t, 2H, J = 6.6 Hz), 2.39 (s, 6H),

1.92 (quintet, 2H, J = 7.2Hz), 1.53 (m, 2H), 1.03 (t, 3H, J = 7.5 Hz); 13

C NMR (75 MHz,

CDCl3): δ 194.5, 193.1, 192.6, 174.2, 138.8, 134.6, 127.6, 125.3, 75.1, 32.0, 21.2, 18.6, 13.6.

HRMS: Cacld. for M+: 258.1256, found: 258.1252.

NH

O O

O

O

21


of 3-butoxy-4-(3,5-dimethyl-phenyl)-3-cyclobutene-1,2-dione 30 (41.5 mg, 0.161 mmol) in

ethanol (1.6 mL). The resulting suspension was then treated with triethylamine (27 µL, 0.20

mmol). After stirring at ambient temperature for 1 h, the precipitate was collected by filtration.

Diethyl ether (10 mL) was added to the filtrate and precipitate thus formed was collected by

- 221 -

filtration. The combined solid was washed with pentane (5 mL) and dried under vacuum. Xylyl-

SHL 21 was obtained as a pale yellow solid (29.8 mg, 65%). TLC Rf = 0.75 (ethyl acetae).

1H

NMR (300 MHz, DMSO-d6): δ 9.26 (br, 1H), 7.62 (s, 1H), 7.18 (s, 1H), 5.32 (m, 1H), 4.48 (t,

1H, J = 8.7 Hz), 4.37 (m, 1H), 2.70 (m, 1H), 2.43 (m, 1H), 2.34 (s, 6H); 13

C NMR (75 MHz,

DMSO-d6): δ 192.6, 188.9, 179.1, 174.7, 163.0, 138.4, 132.6, 128.7, 123.9, 65.4, 52.9, 29.3,

20.8. for M+: 285.1001, found: 285.0955.

O O

O

O

To a stirred solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (0.22 mL, 1.0 mmol) and 1-

phenyl-1-(trimethylsilyloxy)ethylene (0.42 mL, 2.0 mmol) in anhydrous DCM (4.0 mL) was

added titanium tetrachloride (0.11 mL, 1.0 mmol) at -15 oC. After stirring at this temperature for

20 min, another portion of 1-phenyl-1-(trimethylsilyloxy)ethylene (0.21 mL, 1.0 mmol) and

titanium tetrachloride (0.05 mL, 0.5 mmol) was added. The reaction mixture was stirred for

another 20 min and quenched by pouring into cold water. The aqueous layer was extracted with

DCM (5 mL × 3), dried over MgSO4, filtered, and concentrated under reduced pressure.

O O

NH

O

O

O


of crude (124.8 mg, 0.552 mmol) in ethanol (5.5 mL). The resulting suspension was then treated

with triethylamine (77 µL, 0.56 mmol). After stirring at ambient temperature for 1 h, the

precipitate was collected by filtration. Diethyl ether (10 mL) was added to the filtrate and

precipitate thus formed was collected by filtration. The combined solid was washed with pentane

(5 mL) and dried under vacuum. Phenylethyl-SHL was obtained as a pale yellow solid (55 mg,

- 222 -

33%). TLC Rf = 0.18 (hexane:ethyl acetate, 1:1).

1H NMR or

13C NMR either indicate a mixture

or rotamers and/or decomposed products during the time of NMR experiment. This batch of

compound showed no activity in preliminary biofilm or reporter gene assay.

- 223 -

O

O O

OH

22

- 224 -

O

O O

OH

22

- 225 -

O O

O

26

- 226 -

O O

O

26

- 227 -

NH

O O

O

O15

- 228 -

NH

O O

O

O15

- 229 -

O

O O

OH

23

- 230 -

O

O O

OH

23

- 231 -

O O

O

27

- 232 -

O O

O

27

- 233 -

NH

O O

O

O19

- 234 -

NH

O O

O

O19

- 235 -

O

O O

OH

24

- 236 -

O O

O

28

- 237 -

O O

O

28

- 238 -

NH

O O

O

O20

- 239 -

NH

O O

O

O20

- 240 -

O

O O

OH

S

25

- 241 -

O O

O

S29

- 242 -

NH

O O

O

OS

18

- 243 -

NH

O O

O

OS

18

- 244 -

O O

O

30

- 245 -

O O

O

30

- 246 -

NH

O O

O

O

21

- 247 -

O O

NH

O

O

- 248 -

References

(1) Collings, P. J. H., Michael Introduction to Liquid Crystals: Chemistry and Physics Taylor

and Francis: Bristiol, PA, 1997.

(2) Lydon, J. Chromonic review. J. Mater. Chem. 2010, 20, 10071-10099.

(3) Cox, J. S. G. Disodium cromoglycate (FPL 670) ("Intal"). Specific inhibitor of reaginic

antibody-antigen mechanisms. Nature 1967, 216, 1328-1329.

(4) Attwood, T. K.; Lydon, J. E. Lyotropic mesophase formation by anti-asthmatic drugs. Mol.

Cryst. Liq. Cryst. 1984, 108, 349-357.

(5) Sadler, D. E.; Shannon, M. D.; Tollin, P.; Young, D. W.; Edmondson, M.; Rainsford, P.

Lyotropic liquid-crystalline mesophases and a novel, solid physical form of some water-

soluble, reactive dyes. Liq. Cryst. 1986, 1, 509-520.

(6) Tiddy, G. J. T.; Mateer, D. L.; Ormerod, A. P.; Harrison, W. J.; Edwards, D. J. Highly

ordered aggregates in dilute dye-water systems. Langmuir 1995, 11, 390-393.

(7) Harrison, W. J.; Mateer, D. L.; Tiddy, G. J. T. Liquid-Crystalline J-Aggregates Formed by

Aqueous Ionic Cyanine Dyes. J. Phys. Chem. 1996, 100, 2310-2321.

(8) von Berlepsch, H.; Boettcher, C.; Daehne, L. Structure of J-aggregates of pseudoisocyanine

dye in aqueous solution. J. Phys. Chem. B 2000, 104, 8792-8799.

(9) von Berlepsch, H.; Boettcher, C. Network superstructure of pseudoisocyanine J-aggregates

in aqueous sodium chloride solution revealed by cryo-transmission electron microscopy. J.

Phys. Chem. B 2002, 106, 3146-3150.

(10) Ruslim, C.; Matsunaga, D.; Hashimoto, M.; Tamaki, T.; Ichimura, K. Structural

characteristics of the chromonic mesophases of C.I. Direct Blue 67. Langmuir 2003, 19,

3686-3691.

- 249 -

(11) Horowitz Viva, R.; Janowitz Lauren, A.; Modic Aaron, L.; Heiney Paul, A.; Collings Peter,

J. Aggregation behavior and chromonic liquid crystal properties of an anionic monoazo

dye. Phys. Rev. E 2005, 72, 041710.

(12) Nastishin, Y. A.; Liu, H.; Schneider, T.; Nazarenko, V.; Vasyuta, R.; Shiyanovskii, S. V.;

Lavrentovich, O. D. Optical characterization of the nematic lyotropic chromonic liquid

crystals: Light absorption, birefringence, and scalar order parameter. Phys. Rev. E 2005, 72,

041711.

(13) Mariani, P.; Mazabard, C.; Garbesi, A.; Spada, G. P. A study of the structure of the

lyomesophases formed by the dinucleoside phosphate d(GpG). An approach by x-ray

diffraction and optical microscopy. J. Am. Chem. Soc. 1989, 111, 6369-6373.

(14) Mariani, P.; Spinozzi, F.; Federiconi, F.; Amenitsch, H.; Spindler, L.; Drevensek-Olenik, I.

Small Angle X-ray Scattering Analysis of Deoxyguanosine 5'-Monophosphate Self-

Assembling in Solution: Nucleation and Growth of G-Quadruplexes. J. Phys. Chem. B

2009, 113, 7934-7944.

(15) Lydon, J. A personal history of the early days of chromonics. Liq. Cryst. Today 2007, 16,

13-27.

(16) Sandqvist, H. Anisotropic aqueous solution. Ber. Dtsch. Chem. Ges. 1915, 48, 2054-2055.

(17) Cox, J. S. G.; Woodard, G. D.; McCrone, W. C. Solid-state chemistry of cromolyn sodium

(disodium cromoglycate). J. Pharm. Sci. 1971, 60, 1458-1465.

(18) Hartshorne, N. H.; Woodard, G. D. Mesomorphism in the system disodium chromoglycate-

water. Mol. Cryst. Liquid Cryst. 1973, 23, 343-368.

(19) Lydon, J. E. New models for the mesophases of disodium cromoglycate (INTAL). Mol.

Cryst. Liq. Cryst. 1980, 64, 19-24.

- 250 -

(20) Turner, J. E.; Lydon, J. E. Chromonic mesomorphism: the range of lyotropic discotic

phases. Mol. Cryst. Liq. Cryst., Lett. Sect. 1988, 5, 93-99.

(21) Goldfarb, D.; Labes, M. M.; Luz, Z.; Poupko, R. Orientational order in the lyomesophases

of the disodium cromoglycate-water system by deuterium, oxygen-17, and sodium-23

NMR. Mol. Cryst. Liq. Cryst. 1982, 87, 259-279.

(22) Lee, H.; Labes, M. M. Lyotropic cholesteric and nematic phases of disodium cromoglycate

in magnetic fields. Mol. Cryst. Liq. Cryst. 1982, 84, 137-157.

(23) Goldfarb, D.; Moseley, M. E.; Labes, M. M.; Luz, M. Determination of pitch in a

cholesteric DSCG-water lyomesophase by NMR techniques. Mol. Cryst. Liq. Cryst. 1982,

89, 119-135.

(24) Perahia, D.; Goldfarb, D.; Luz, Z. Sodium-23 NMR in the lyomesophases of disodium

cromoglycate. Mol. Cryst. Liq. Cryst. 1984, 108, 107-123.

(25) Goldfarb, D.; Luz, Z.; Spielberg, N.; Zimmermann, H. Structural and orientational

characteristics of the disodium/cromoglycate-water mesophases by deuterium NMR and x-

ray diffraction. Mol. Cryst. Liq. Cryst. 1985, 126, 225-246.

(26) Hui, Y. W.; Labes, M. M. Structure and order parameter of a nematic lyotropic liquid

crystal studied by FTIR spectroscopy. J. Phys. Chem. 1986, 90, 4064-4067.

(27) Nastishin Yu, A.; Liu, H.; Shiyanovskii, S. V.; Lavrentovich, O. D.; Kostko, A. F.;

Anisimov, M. A. Pretransitional fluctuations in the isotropic phase of a lyotropic chromonic

liquid crystal. Phys. Rev. E 2004, 70, 051706.

(28) Kostko, A. F.; Cipriano, B. H.; Pinchuk, O. A.; Ziserman, L.; Anisimov, M. A.; Danino, D.;

Raghavan, S. R. Salt Effects on the Phase Behavior, Structure, and Rheology of Chromonic

Liquid Crystals. J. Phys. Chem. B 2005, 109, 19126-19133.

- 251 -

(29) Sergan, T.; Schneider, T.; Kelly, J.; Lavrentovich, O. D. Polarizing-alignment layers for

twisted nematic cells. Liq. Cryst. 2000, 27, 567-572.

(30) Shiyanovskii, S. V.; Schneider, T.; Smalyukh, I. I.; Ishikawa, T.; Niehaus, G. D.; Doane, K.

J.; Woolverton, C. J.; Lavrentovich, O. D. Real-time microbe detection based on director

distortions around growing immune complexes in lyotropic chromonic liquid crystals. Phys.

Rev. E 2005, 71, 020702.

(31) Tam-Chang, S.-W.; Helbley, J.; Carson, T. D.; Seo, W.; Iverson, I. K. Template-guided

organization of chromonic liquid crystals into micropatterned anisotropic organic solids.

Chem. Commun. 2006, 503-505.

(32) Park, H.-S.; Agarwal, A.; Kotov, N. A.; Lavrentovich, O. D. Controllable side-by-side and

end-to-end assembly of Au nanorods by lyotropic chromonic materials. Langmuir 2008, 24,

13833-13837.

(33) Tam-Chang, S.-W.; Iverson, I. K.; Helbley, J. Study of the Chromonic Liquid-Crystalline

Phases of Bis-(N,N-diethylaminoethyl)perylene-3,4,9,10-tetracarboxylic Diimide

Dihydrochloride by Polarized Optical Microscopy and 2H NMR Spectroscopy. Langmuir

2004, 20, 342-347.

(34) Park, H.-S.; Kang, S.-W.; Tortora, L.; Nastishin, Y.; Finotello, D.; Kumar, S.; Lavrentovich,

O. D. Self-Assembly of Lyotropic Chromonic Liquid Crystal Sunset Yellow and Effects of

Ionic Additives. J. Phys. Chem. B 2008, 112, 16307-16319.

(35) Maiti, P. K.; Lansac, Y.; Glaser, M. A.; Clark, N. A. Isodesmic self-assembly in lyotropic

chromonic systems. Liq. Cryst. 2002, 29, 619-626.

- 252 -

(36) Prasad, S. K.; Nair, G. G.; Hegde, G.; Jayalakshmi, V. Evidence of Wormlike Micellar

Behavior in Chromonic Liquid Crystals: Rheological, X-ray, and Dielectric Studies. J. Phys.

Chem. B 2007, 111, 9741-9746.

(37) Edwards, D. J.; Jones, J. W.; Lozman, O.; Ormerod, A. P.; Sintyureva, M.; Tiddy, G. J. T.

Chromonic liquid crystal formation by Edicol Sunset Yellow. J. Phys. Chem. B 2008, 112,

14628-14636.

(38) Lydon, J. Chromonic mesophases. Curr. Opin. Colloid Interface Sci. 2004, 8, 480-490.

(39) Eckhardt, H.; Bose, A.; Krongauz, V. A. Formation of molecular H- and J-stacks by the

spiropyran-merocyanine transformation in a polymer matrix. Polymer 1987, 28, 1959-1964.

(40) Merino, G.; Heine, T.; Seifert, G. The induced magnetic field in cyclic molecules. Chem.--

Eur. J. 2004, 10, 4367-4371.

(41) Gonzalez-Rodriguez, D.; Janssen, P. G. A.; Martin-Rapun, R.; De Cat, I.; De Feyter, S.;

Schenning, A. P. H. J.; Meijer, E. W. Persistent, Well-Defined, Monodisperse, pi-

Conjugated Organic Nanoparticles via G-Quadruplex Self-Assembly. J. Am. Chem. Soc.

2010, 132, 4710-4719.

(42) Wu, L.; Lal, J.; Simon, K. A.; Burton, E. A.; Luk, Y.-Y. Nonamphiphilic Assembly in

Water: Polymorphic Nature, Thread Structure, and Thermodynamic Incompatibility. J. Am.

Chem. Soc. 2009, 131, 7430-7443.

(43) Simon, K. A.; Burton, E. A.; Cheng, F.; Varghese, N.; Falcone, E. R.; Wu, L.; Luk, Y.-Y.

Controlling Thread Assemblies of Pharmaceutical Compounds in Liquid Crystal Phase by

Using Functionalized Nanotopography. Chem. Mater. 2010, 22, 2434-2441.

(44) Kerker, M.; Editor Colloid and Interface Science, Vol. 2: Aerosols, Emulsions, and

Surfactants, 1976.

- 253 -

(45) Poulin, P.; Stark, H.; Lubensky, T. C.; Weitz, D. A. Novel colloidal interactions in

anisotropic fluids. Science 1997, 275, 1770-1773.

(46) Brake, J. M.; Daschner, M. K.; Luk, Y.-Y.; Abbott, N. L. Biomolecular Interactions at

Phospholipid-Decorated Surfaces of Liquid Crystals. Science 2003, 302, 2094-2098.

(47) Han, Y.; Cheng, K.; Simon, K. A.; Lan, Y.; Sejwal, P.; Luk, Y.-Y. A Biocompatible

Surfactant with Folded Hydrophilic Head Group: Enhancing the Stability of Self-Inclusion

Complexes of Ferrocenyl in a beta -Cyclodextrin Unit by Bond Rigidity. J. Am. Chem. Soc.

2006, 128, 13913-13920.

(48) Tolstoguzov, V. Foods as dispersed systems. Thermodynamic aspects of composition-

property relationships in formulated food. J. Therm. Anal. Calorim. 2000, 61, 397-409.

(49) Clark, A. H.; Gidley, M. J.; Richardson, R. K.; Ross-Murphy, S. B. Rheological studies of

aqueous amylose gels: the effect of chain length and concentration on gel modulus.

Macromolecules 1989, 22, 346-351.

(50) Kasapis, S.; Morris, E. R.; Norton, I. T.; Gidley, M. J. Phase equilibria and gelation in

gelatin/maltodextrin systems - Part II: Polymer incompatibility in solution. Carbohydr.

Polym. 1993, 21, 249-259.

(51) Simon, K. A.; Sejwal, P.; Falcone, E. R.; Burton, E. A.; Yang, S.; Prashar, D.;

Bandyopadhyay, D.; Narasimhan, S. K.; Varghese, N.; Gobalasingham, N. S.; Reese, J. B.;

Luk, Y.-Y. Noncovalent Polymerization and Assembly in Water Promoted by

Thermodynamic Incompatibility. J. Phys. Chem. B 2010, 114, 10357-10367.

(52) Karplus, M. Vicinal proton coupling in nuclear magnetic resonance. J. Am. Chem. Soc.

1963, 85, 2870-2871.

- 254 -

(53) Minch, M. J. Orientational dependence of vicinal proton-proton NMR coupling constants:

The Karplus relationship. Concepts Magn. Reson. 1994, 6, 41-56.

(54) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory

solvents as trace impurities. J. Org. Chem. 1997, 62, 7512-7515.

(55) Martin, R. B. Comparisons of Indefinite Self-Association Models. Chem. Rev. 1996, 96,

3043-3064.

(56) Smulders, M. M. J.; Nieuwenhuizen, M. M. L.; de Greef, T. F. A.; van der Schoot, P.;

Schenning, A. P. H. J.; Meijer, E. W. How to Distinguish Isodesmic from Cooperative

Supramolecular Polymerisation. Chem.--Eur. J. 2010, 16, 362-367.

(57) Ponnuswamy, N.; Pantos, G. D.; Smulders, M. M. J.; Sanders, J. K. M. Thermodynamics of

Supramolecular Naphthalenediimide Nanotube Formation: The Influence of Solvents, Side

Chains, and Guest Templates. J. Am. Chem. Soc. 2012, 134, 566-573.

(58) Chami, F.; Wilson, M. R. Molecular Order in a Chromonic Liquid Crystal: A Molecular

Simulation Study of the Anionic Azo Dye Sunset Yellow. J. Am. Chem. Soc. 2010, 132,

7794-7802.

(59) Renshaw, M. P.; Day, I. J. NMR Characterization of the Aggregation State of the Azo Dye

Sunset Yellow in the Isotropic Phase. J. Phys. Chem. B 2010, 114, 10032-10038.

(60) Hiltrop, K. In Chirality in Liquid Crystals; Kitzerow, H.-S., Bahr, C., Eds.; Springer:

Seacaucus, N. J., 2001; Ch 14.

(61) Tseng, S.-L.; Valente, A.; Gray, D. G. Cholesteric liquid crystalline phases based on

(acetoxypropyl)cellulose. Macromolecules 1981, 14, 715-719.

(62) Kasuya, S.; Sasaki, S.; Watanabe, J.; Fukuda, Y.; Uematsu, I. Thermotropic cholesteric

mesophases in copoly(γ-n-alkyl L-glutamate)s. Polym. Bull. 1982, 7, 241-248.

- 255 -

(63) Watanabe, J.; Krigbaum, W. R. Copolyesters exhibiting a thermotropic cholesteric phase. J.

Polym. Sci., Polym. Phys. Ed. 1985, 23, 565-574.

(64) Lee, H.; Labes, M. M. Helical twisting power of amino acids in a nematic lyophase. Mol.

Cryst. Liq. Cryst. 1984, 108, 125-132.

(65) Dorfler, H. D.; Gopfert, A. Comparison of structures and properties of lyotropic cholesteric

phases induced by center and axial chiral compounds. Colloid Polym. Sci. 2000, 278, 1085-

1096.

(66) Van Winkle, D. H.; Davidson, M. W.; Chen, W. X.; Rill, R. L. Cholesteric helical pitch of

near persistence length DNA. Macromolecules 1990, 23, 4140-4148.

(67) Do Aido, T. M. H.; Alcantara, M. R.; Felippe, O., Jr.; Pereira, A. M. G.; Vanin, J. A.

Carbohydrates as inducers in cholesteric lyotropic mesophases. Mol. Cryst. Liq. Cryst. 1990,

185, 61-66.

(68) Reinitzer, F. Beiträge zur Kenntniss des Cholesterins. Monatshefte für Chemie 1888, 9,

421-441.

(69) Werbowyj, R. S.; Gray, D. G. Ordered phase formation in concentrated

hydroxypropylcellulose solutions. Macromolecules 1980, 13, 69-73.

(70) Tombolato, F.; Ferrarini, A.; Grelet, E. Chiral nematic phase of suspensions of rodlike

viruses: left-handed phase helicity from a right-handed molecular helix. Phys Rev Lett 2006,

96, 258302.

(71) Buckingham, A. D.; Ceasar, G. P.; Dunn, M. B. Addition of optically active compounds to

nematic liquid crystals. Chem. Phys. Lett. 1969, 3, 540-541.

- 256 -

(72) Dorfler, H.-D. Chirality, twist and structures of micellar lyotropic cholesteric liquid crystals

in comparison to the properties of chiralic thermotropic phases. Adv. Colloid Interface Sci.

2002, 98, 285-340.

(73) Pieraccini, S.; Ferrarini, A.; Spada Gian, P. Chiral doping of nematic phases and its

application to the determination of absolute configuration. Chirality 2008, 20, 749-759.

(74) Pieraccini, S.; Masiero, S.; Ferrarini, A.; Piero Spada, G. Chirality transfer across length-

scales in nematic liquid crystals: fundamentals and applications. Chem. Soc. Rev. 2011, 40,

258-271.

(75) Krigbaum, W. R.; Ciferri, A.; Asrar, J.; Toriumi, H.; Preston, J. A polyester forming a

thermotropic cholesteric phase. Mol. Cryst. Liq. Cryst. 1981, 76, 79-91.

(76) Billard, J.; Blumstein, A.; Vilasagar, S. Phase diagrams of cholesteric and nematic

thermotropic liquid-crystalline polymers. Mol. Cryst. Liq. Cryst. 1982, 72, 163-169.

(77) Chiellini, E.; Galli, G. Chiral liquid crystal polymers. 6. Preparation and properties in

solution and in bulk of optically active thermotropic copolyesters. Macromolecules 1985,

18, 1652-1658.

(78) Dorset, D. L. Thermotropic mesomorphism of cholesteryl myristate. An electron diffraction

study. J. Lipid Res. 1985, 26, 1142-1150.

(79) Hara, H.; Satoh, T.; Toya, T.; Iida, S.; Orii, S.; Watanabe, J. Cholesteric liquid-crystalline

polyesters. 1. Cholesteric liquid-crystalline copolyesters based on poly(chloro-1,4-

phenylene trans-1,4-cyclohexanedicarboxylate). Macromolecules 1988, 21, 14-19.

(80) Bartusch, G.; Doerfler, H. D.; Hoffmann, H. Behavior and properties of lyotropic-nematic

and lyotropic-cholesteric phases. Prog. Colloid Polym. Sci. 1992, 89, 307-314.

- 257 -

(81) Toledano, P.; Figueiredo Neto, A. M.; de Sant'Ana, Z. A. Phase diagrams of lyotropic

cholesteric fluids. Phys. Rev. E 2000, 61, 486-492.

(82) Dorfler, H.-D.; Gopfert, A.; Gorgens, C. Structures and properties of induced lyotropic

cholesteric phases. Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. A 2001, 367, 517-527.

(83) Yu, L. J.; Saupe, A. Liquid crystalline phases of the sodium decylsulfate/decanol/water

system. Nematic-nematic and cholesteric-cholesteric phase transitions. J. Am. Chem. Soc.

1980, 102, 4879-4883.

(84) von Minden, H. M.; Vill, V.; Pape, M.; Hiltrop, K. Sugar Amphiphiles as Revealing

Dopants for Induced Chiral Nematic Lyotropic Liquid Crystals. J. Colloid Interface Sci.

2001, 236, 108-115.

(85) Dawin, U. C.; Dilger, H.; Roduner, E.; Scheuermann, R.; Stoykov, A.; Giesselmann, F.

Chiral Induction in Lyotropic Liquid Crystals: Insights into the Role of Dopant Location

and Dopant Dynamics. Angew. Chem., Int. Ed. 2010, 49, 2427-2430.

(86) Lavrentovich, M.; Sergan, T.; Kelly, J. Planar and twisted lyotropic chromonic liquid

crystal cells as optical compensators for twisted nematic displays. Liq. Cryst. 2003, 30, 851-

859.

(87) Walba, D. M.; Korblova, E.; Shao, R.; Maclennan, J. E.; Link, D. R.; Glaser, M. A.; Clark,

N. A. A ferroelectric liquid crystal conglomerate composed of racemic molecules. Science

2000, 288, 2181-2184.

(88) Takezoe, H. Spontaneous achiral symmetry breaking in liquid crystalline phases. Top.

Curr. Chem. 2012, 318, 303-330.

- 258 -

(89) Linclau, B.; Boydell, A. J.; Clarke, P. J.; Horan, R.; Jacquet, C. Efficient De-

Symmetrization of "Pseudo"-C2-Symmetric Substrates: Illustration in the Synthesis of a

Disubstituted Butenolide from Arabitol. J. Org. Chem. 2003, 68, 1821-1826.

(90) Fiori, K. W.; Puchlopek, A. L. A.; Miller, S. J. Enantioselective sulfonylation reactions

mediated by a tetrapeptide catalyst. Nat. Chem. 2009, 1, 630-634.

(91) Walenzyk, T.; Carola, C.; Buchholz, H.; Koenig, B. Chromone derivatives which bind to

human hair. Tetrahedron 2005, 61, 7366-7377.

(92) Terauchi, T.; Terauchi, T.; Sato, I.; Tsukada, T.; Kanoh, N.; Nakata, M. Synthetic studies

on altohyrtins (spongistatins): synthesis of the C15-C28 (CD) spiroacetal portion.

Tetrahedron Lett. 2000, 41, 2649-2653.

(93) Rychnovsky, S. D.; Griesgraber, G.; Zeller, S.; Skalitzky, D. J. Optically pure 1,3-diols

from (2R,4R)- and (2S,4S)-1,2:4,5-diepoxypentane. J. Org. Chem. 1991, 56, 5161-5169.

(94) Bauer, C. J.; Frenkiel, T. A.; Lane, A. N. A comparison of the ROESY and NOESY

experiments for large molecules, with application to nucleic acids. J. Magn. Reson. 1990,

87, 144-152.

(95) Langeveld-Voss, B. M. W.; Beljonne, D.; Shuai, Z.; Janssen, R. A. J.; Meskers, S. C. J.;

Meijer, E. W.; Bredas, J.-L. Investigation of exciton coupling in oligothiophenes by circular

dichroism spectroscopy. Adv. Mater. 1998, 10, 1343-1348.

(96) Besenius, P.; Portale, G.; Bomans, P. H. H.; Janssen, H. M.; Palmans, A. R. A.; Meijer, E.

W. Controlling the growth and shape of chiral supramolecular polymers in water. Proc.

Natl. Acad. Sci. U. S. A. 2010, 107, 17888-17893, S17888/17881-S17888/17819.

- 259 -

(97) Zhong, S.; Pochan, D. J. Cryogenic Transmission Electron Microscopy for Direct

Observation of Polymer and Small-Molecule Materials and Structures in Solution. Polym.

Rev. 2010, 50, 287-320.

(98) Okoshi, K.; Sakajiri, K.; Kumaki, J.; Yashima, E. Well-Defined Lyotropic Liquid

Crystalline Properties of Rigid-Rod Helical Polyacetylenes. Macromolecules 2005, 38,

4061-4064.

(99) Gupta, V. K.; Abbott, N. L. Design of surfaces for patterned alignment of liquid crystals on

planar and curved substrates. Science 1997, 276, 1533-1536.

(100) Luk, Y.-Y.; Tingey, M. L.; Hall, D. J.; Israel, B. A.; Murphy, C. J.; Bertics, P. J.; Abbott,

N. L. Using liquid crystals to amplify protein-receptor interactions: Design of surfaces

with nanometer-scale topography that present histidine-tagged protein receptors. Langmuir

2003, 19, 1671-1680.

(101) Gottarelli, G.; Hibert, M.; Samori, B.; Solladie, G.; Spada, G. P.; Zimmermann, R.

Induction of the cholesteric mesophase in nematic liquid crystals: mechanism and

application to the determination of bridged biaryl configurations. J. Am. Chem. Soc. 1983,

105, 7318-7321.

(102) Green, M. M.; Zanella, S.; Gu, H.; Sato, T.; Gottarelli, G.; Jha, S. K.; Spada, G. P.;

Schoevaars, A. M.; Feringa, B.; Teramoto, A. Mechanism of the Transformation of a Stiff

Polymer Lyotropic Nematic Liquid Crystal to the Cholesteric State by Dopant-Mediated

Chiral Information Transfer. J. Am. Chem. Soc. 1998, 120, 9810-9817.

(103) Brakke, M. K. Density gradient centrifugation: a new separation technique. J. Am. Chem.

Soc. 1951, 73, 1847-1848.

- 260 -

(104) Svensson, H.; Valmet, E. Large-scale density-gradient electrophoresis. II. A simple

experimental technique for securing perfectly stable zones and full utilization of the

separation capacity of a density-gradient column. Sci. Tools 1959, 6, 13-17.

(105) Seraydarian, K.; Mommaerts, W. F. Density gradient separation of sarcotubular vesicles

and other particulate constituents of rabbit muscle. J. Cell. Biol. 1965, 26, 641-656.

(106) Sarfati, M.; Lesot, P.; Merlet, D.; Courtieu, J. Theoretical and experimental aspects of

enantiomeric differentiation using natural abundance multinuclear NMR spectroscopy in

chiral polypeptide liquid crystals. Chem. Commun. 2000, 2069-2081.

(107) Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Heugen, U.; Gruebele, M.; Leitner, D. M.;

Havenith, M. An extended dynamical hydration shell around proteins. Proc. Natl. Acad.

Sci. U. S. A. 2007, 104, 20749-20752.

(108) Lesot, P.; Lafon, O.; Zimmermann, H.; Luz, Z. Enantiodiscrimination in Deuterium NMR

Spectra of Flexible Chiral Molecules with Average Axial Symmetry Dissolved in Chiral

Liquid Crystals: The Case of Tridioxyethylenetriphenylene. J. Am. Chem. Soc. 2008, 130,

8754-8761.

(109) Bandyopadhyay, D.; Prashar, D.; Luk, Y.-Y. Anti-Fouling Chemistry of Chiral

Monolayers: Enhancing Biofilm Resistance on Racemic Surface. Langmuir 2011, 27,

6124-6131.

(110) Feldman, K.; Haehner, G.; Spencer, N. D.; Harder, P.; Grunze, M. Probing Resistance to

Protein Adsorption of Oligo(ethylene glycol)-Terminated Self-Assembled Monolayers by

Scanning Force Microscopy. J. Am. Chem. Soc. 1999, 121, 10134-10141.

- 261 -

(111) Skoda, M. W. A.; Schreiber, F.; Jacobs, R. M. J.; Webster, J. R. P.; Wolff, M.; Dahint, R.;

Schwendel, D.; Grunze, M. Protein Density Profile at the Interface of Water with

Oligo(ethylene glycol) Self-Assembled Monolayers. Langmuir 2009, 25, 4056-4064.

(112) Wulf, A. Distribution function theory of nematic liquid crystals. J. Chem. Phys. 1971, 55,

4512-4519.

(113) Poulin, P.; Stark, H.; Lubensky, T. C.; Weitz, D. A. Novel colloidal interactions in

anisotropic fluids. Science 1997, 275, 1770-1773.

(114) Bax, A.; Davis, D. G. Practical aspects of two-dimensional transverse NOE spectroscopy.

J. Magn. Reson. 1985, 63, 207-213.

(115) Gupta, V. K.; Abbott, N. L. Azimuthal anchoring transition of nematic liquid crystals on

self-assembled monolayers formed from odd and even alkanethiols. Phys. Rev. E 1996, 54,

R4540-R4543.

(116) Francisco, C. G.; Leon, E. I.; Martin, A.; Moreno, P.; Rodriguez, M. S.; Suarez, E.

Reductive Fragmentation of Carbohydrate Anomeric Alkoxy Radicals. Synthesis of

Alditols with Potential Utility as Chiral Synthons. J. Org. Chem. 2001, 66, 6967-6976.

(117) Attwood, S. V.; Barrett, A. G. M. A convenient procedure for the deoxygenation and

homologation of D-ribose derivatives. J. Chem. Soc., Perkin Trans. 1 1984, 1315-1322.

(118) Xie, Z. F.; Sakai, K. Preparation of a chiral building block based on 1,3-syn-diol using

Pseudomonas fluorescens lipase and its application to the synthesis of a hunger modulator.

Chem. Pharm. Bull. 1989, 37, 1650-1652.

(119) Fuqua, W. C.; Winans, S. C.; Greenberg, E. P. Quorum sensing in bacteria: the LuxR-LuxI

family of cell density-responsive transcriptional regulators. J. Bacteriol. 1994, 176, 269-

275.

- 262 -

(120) Bassler, B. L.; Losick, R. Bacterially speaking. Cell 2006, 125, 237-246.

(121) Camilli, A.; Bassler, B. L. Bacterial Small-Molecule Signaling Pathways. Science 2006,

311, 1113-1116.

(122) Eberhard, A.; Burlingame, A. L.; Eberhard, C.; Kenyon, G. L.; Nealson, K. H.;

Oppenheimer, N. J. Structural identification of autoinducer of Photobacterium fischeri

luciferase. Biochemistry 1981, 20, 2444-2449.

(123) Bassler, B. L. Small talk: Cell-to-cell communication in bacteria. Cell 2002, 109, 421-424.

(124) Waters, C. M.; Bassler, B. L. Quorum sensing: Cell-to-cell communication in bacteria.

Annu. Rev. Cell Dev. Biol. 2005, 21, 319-346.

(125) Pesci, E. C.; Milbank, J. B.; Pearson, J. P.; McKnight, S.; Kende, A. S.; Greenberg, E. P.;

Iglewski, B. H. Quinolone signaling in the cell-to-cell communication system of

Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 11229-11234.

(126) Deziel, E.; Lepine, F.; Milot, S.; He, J.; Mindrinos, M. N.; Tompkins, R. G.; Rahme, L. G.

Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role

for 4-hydroxy-2-heptylquinoline in cell-to-cell communication. Proc. Natl. Acad. Sci. U. S.

A. 2004, 101, 1339-1344.

(127) Zhang, L.; Murphy, P. J.; Kerr, A.; Tate, M. E. Agrobacterium conjugation and gene

regulation by N-acyl-L-homoserine lactones. Nature 1993, 362, 446-448.

(128) Bainton, N. J.; Bycroft, B. W.; Chhabra, S. R.; Stead, P.; Gledhill, L.; Hill, P. J.; Rees, C.

E.; Winson, M. K.; Salmond, G. P.; Stewart, G. S.; et al. A general role for the lux

autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene

1992, 116, 87-91.

- 263 -

(129) Pearson, J. P.; Gray, K. M.; Passador, L.; Tucker, K. D.; Eberhard, A.; Iglewski, B. H.;

Greenberg, E. P. Structure of the autoinducer required for expression of Pseudomonas

aeruginosa virulence genes. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 197-201.

(130) Pearson, J. P.; Passador, L.; Iglewski, B. H.; Greenberg, E. P. A second N-acylhomoserine

lactone signal produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 1995,

92, 1490-1494.

(131) Galloway, W. R. J. D.; Hodgkinson, J. T.; Bowden, S. D.; Welch, M.; Spring, D. R.

Quorum sensing in Gram-negative bacteria: Small molecule modulation of AHL and AI-2

quorum sensing pathways. Chem. Rev. 2011, 111, 28-67.

(132) Davies, D. Understanding biofilm resistance to antibacterial agents. Nat. Rev. Drug

Discovery 2003, 2, 114-122.

(133) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial biofilms: a common cause of

persistent infections. Science 1999, 284, 1318-1322.

(134) Parsek, M. R.; Singh, P. K. Bacterial biofilms: An emerging link to disease pathogenesis.

Annu. Rev. Microbiol. 2003, 57, 677-701.

(135) Musk, D. J., Jr.; Hergenrother, P. J. Chemical countermeasures for the control of bacterial

biofilms: effective compounds and promising targets. Curr. Med. Chem. 2006, 13, 2163-

2177.

(136) Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; Lappin-Scott, H. M.

Microbial biofilms. Annu. Rev. Microbiol. 1995, 49, 711-745.

(137) Donlan, R. M. Biofilm formation: A clinically relevant microbiological process. Clin.

Infect. Dis. 2001, 33, 1387-1392.

- 264 -

(138) Smith, K.; Hunter, I. S. Efficacy of common hospital biocides with biofilms of multi-drug

resistant clinical isolates. J. Med. Microbiol. 2008, 57, 966-973.

(139) Davies, D. G.; Parsek, M. R.; Pearson, J. P.; Iglewski, B. H.; Costerton, J. W.; Greenberg,

E. P. The involvement of cell-to-cell signals in the development of a bacterial biofilm.

Science 1998, 280, 295-298.

(140) Stoodley, P.; Sauer, K.; Davies, D. G.; Costerton, J. W. Biofilms as complex differentiated

communities. Annu. Rev. Microbiol. 2002, 56, 187-209.

(141) Hunt, J. A. A short history of soap. The Pharmaceutical Journal 1999, 263, 985-989.

(142) Allison, C.; Lai, H. C.; Gygi, D.; Hughes, C. Cell differentiation of Proteus mirabilis is

initiated by glutamine, a specific chemoattractant for swarming cells. Mol. Microbiol.

1993, 8, 53-60.

(143) Anwar, H.; Dasgupta, M. K.; Costerton, J. W. Testing the susceptibility of bacteria in

biofilms to antibacterial agents. Antimicrob. Agents Chemother. 1990, 34, 2043-2046.

(144) Mah, T.-F. C.; O'Toole, G. A. Mechanisms of biofilm resistance to antimicrobial agents.

Trends Microbiol. 2001, 9, 34-39.

(145) Monroe, D. Looking for chinks in the armor of bacterial biofilms. PLoS Biol. 2007, 5,

e307.

(146) Hoyle, B. D.; Alcantara, J.; Costerton, J. W. Pseudomonas aeruginosa biofilm as a

diffusion barrier to piperacillin. Antimicrob. Agents Chemother. 1992, 36, 2054-2056.

(147) Nguyen, D.; Joshi-Datar, A.; Lepine, F.; Bauerle, E.; Olakanmi, O.; Beer, K.; McKay, G.;

Siehnel, R.; Schafhauser, J.; Wang, Y.; Britigan, B. E.; Singh, P. K. Active starvation

responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science

2011, 334, 982-986.

- 265 -

(148) Brown, M. R.; Barker, J. Unexplored reservoirs of pathogenic bacteria: protozoa and

biofilms. Trends Microbiol. 1999, 7, 46-50.

(149) Brown, M. R.; Allison, D. G.; Gilbert, P. Resistance of bacterial biofilms to antibiotics: a

growth-rate related effect?, The Journal of Antimicrobial Chemotherapy 1988, 22, 777-

780.

(150) Evans, D. J.; Allison, D. G.; Brown, M. R.; Gilbert, P. Susceptibility of Pseudomonas

aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth

rate. J. Antimicrob. Chemother. 1991, 27, 177-184.

(151) Hannedouche, S.; Zhang, J.; Yi, T.; Shen, W.; Nguyen, D.; Pereira, J. P.; Guerini, D.;

Baumgarten, B. U.; Roggo, S.; Wen, B.; Knochenmuss, R.; Noel, S.; Gessier, F.; Kelly, L.

M.; Vanek, M.; Laurent, S.; Preuss, I.; Miault, C.; Christen, I.; Karuna, R.; Li, W.; Koo, D.

I.; Suply, T.; Schmedt, C.; Peters, E. C.; Falchetto, R.; Katopodis, A.; Spanka, C.; Roy, M.

O.; Detheux, M.; Chen, Y. A.; Schultz, P. G.; Cho, C. Y.; Seuwen, K.; Cyster, J. G.; Sailer,

A. W. Oxysterols direct immune cell migration via EBI2. Nature 2011, 475, 524-527.

(152) Bjarnsholt, T.; Jensen, P. O.; Burmolle, M.; Hentzer, M.; Haagensen, J. A.; Hougen, H. P.;

Calum, H.; Madsen, K. G.; Moser, C.; Molin, S.; Hoiby, N.; Givskov, M. Pseudomonas

aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes

is quorum-sensing dependent. Microbiology 2005, 151, 373-383.

(153) Jensen, E. T.; Kharazmi, A.; Garred, P.; Kronborg, G.; Fomsgaard, A.; Mollnes, T. E.;

Hoiby, N. Complement activation by Pseudomonas aeruginosa biofilms. Microb. Pathog.

1993, 15, 377-388.

(154) Kaplan, J. B. Biofilm dispersal: mechanisms, clinical implications, and potential

therapeutic uses. J. Dent. Res. 2010, 89, 205-218.

- 266 -

(155) von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Haebich, D. Antibacterial natural

products in medicinal chemistry - exodus or revival?, Angew. Chem., Int. Ed. 2006, 45,

5072-5129.

(156) Rasmussen, T. B.; Givskov, M. Quorum-sensing inhibitors as anti-pathogenic drugs. Int. J.

Med. Microbiol. 2006, 296, 149-161.

(157) Engebrecht, J.; Nealson, K.; Silverman, M. Bacterial bioluminescence: isolation and

genetic analysis of functions from Vibrio fischeri. Cell 1983, 32, 773-781.

(158) Engebrecht, J.; Silverman, M. Identification of genes and gene products necessary for

bacterial bioluminescence. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 4154-4158.

(159) Engebrecht, J.; Silverman, M. Nucleotide sequence of the regulatory locus controlling

expression of bacterial genes for bioluminescence. Nucleic Acids Res. 1987, 15, 10455-

10467.

(160) Smith, K. M.; Bu, Y.; Suga, H. Induction and Inhibition of Pseudomonas aeruginosa

Quorum Sensing by Synthetic Autoinducer Analogs. Chem. Biol. 2003, 10, 81-89.

(161) Smith, K. M.; Bu, Y.; Suga, H. Library screening for synthetic agonists and antagonists of

a Pseudomonas aeruginosa autoinducer. Chem. Biol. 2003, 10, 563-571.

(162) Geske, G. D.; Wezeman, R. J.; Siegel, A. P.; Blackwell, H. E. Small Molecule Inhibitors

of Bacterial Quorum Sensing and Biofilm Formation. J. Am. Chem. Soc. 2005, 127, 12762-

12763.

(163) Geske, G. D.; O'Neill, J. C.; Miller, D. M.; Mattmann, M. E.; Blackwell, H. E. Modulation

of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated

homoserine lactones in multiple species and new insights into their mechanisms of action.

J. Am. Chem. Soc. 2007, 129, 13613-13625.

- 267 -

(164) Praneenararat, T.; Geske, G. D.; Blackwell, H. E. Efficient Synthesis and Evaluation of

Quorum-Sensing Modulators Using Small Molecule Macroarrays. Org. Lett. 2009, 11,

4600-4603.

(165) McInnis, C. E.; Blackwell, H. E. Design, synthesis, and biological evaluation of abiotic,

non-lactone modulators of LuxR-type quorum sensing. Bioorg. Med. Chem. 2011, 19,

4812-4819.

(166) Geske, G. D.; Mattmann, M. E.; Blackwell, H. E. Evaluation of a focused library of N-aryl

L-homoserine lactones reveals a new set of potent quorum sensing modulators. Bioorg.

Med. Chem. Lett. 2008, 18, 5978-5981.

(167) Frei, R.; Breitbach, A. S.; Blackwell, H. E. 2-Aminobenzimidazole derivatives strongly

inhibit and disperse Pseudomonas aeruginosa biofilms. Angew. Chem. Int. Ed. 2012, 51,

5226-5229.

(168) Reed, C. S.; Huigens, R. W., III; Rogers, S. A.; Melander, C. Modulating the development

of E. coli biofilms with 2-aminoimidazoles. Bioorg. Med. Chem. Lett. 2010, 20, 6310-6312.

(169) Bunders, C. A.; Richards, J. J.; Melander, C. Identification of aryl 2-aminoimidazoles as

biofilm inhibitors in Gram-negative bacteria. Bioorg. Med. Chem. Lett. 2010, 20, 3797-

3800.

(170) Huigens, R. W., III; Richards, J. J.; Parise, G.; Ballard, T. E.; Zeng, W.; Deora, R.;

Melander, C. Inhibition of Pseudomonas aeruginosa Biofilm Formation with

Bromoageliferin Analogues. J. Am. Chem. Soc. 2007, 129, 6966-6967.

(171) Richards, J. J.; Melander, C. Small molecule approaches toward the non-microbicidal

modulation of bacterial biofilm growth and maintenance. Anti-Infect. Agents Med. Chem.

2009, 8, 295-314.

- 268 -

(172) Liu, C.; Worthington, R. J.; Melander, C.; Wu, H. A new small molecule specifically

inhibits the cariogenic bacterium Streptococcus mutans in multispecies biofilms.

Antimicrob. Agents Chemother. 2011, 55, 2679-2687.

(173) Rogers, S. A.; Huigens, R. W., III; Cavanagh, J.; Melander, C. Synergistic effects between

conventional antibiotics and 2-aminoimidazole-derived antibiofilm agents. Antimicrob.

Agents Chemother. 2010, 54, 2112-2118.

(174) Davies, D. G.; Marques, C. N. H. A fatty acid messenger is responsible for inducing

dispersion in microbial biofilms. Journal of Bacteriology 2009, 191, 1393-1403.

(175) Ren, D.; Zuo, R.; Gonzalez Barrios, A. F.; Bedzyk, L. A.; Eldridge, G. R.; Pasmore, M. E.;

Wood, T. K. Differential gene expression for investigation of Escherichia coli biofilm

inhibition by plant extract ursolic acid. Appl. Environ. Microbiol. 2005, 71, 4022-4034.

(176) Musk, D. J.; Banko, D. A.; Hergenrother, P. J. Iron Salts Perturb Biofilm Formation and

Disrupt Existing Biofilms of Pseudomonas aeruginosa. Chem. Biol. 2005, 12, 789-796.

(177) Junker, L. M.; Clardy, J. High-throughput screens for small-molecule inhibitors of

Pseudomonas aeruginosa biofilm development. Antimicrob. Agents Chemother. 2007, 51,

3582-3590.

(178) Musk, D. J., Jr.; Hergenrother, P. J. Chelated iron sources are inhibitors of Pseudomonas

aeruginosa biofilms and distribute efficiently in an in vitro model of drug delivery to the

human lung. J. Appl. Microbiol. 2008, 105, 380-388.

(179) Kazlauskas, R.; Murphy, P. T.; Quinn, R. J.; Wells, R. J. A new class of halogenated

lactones from the red alga Delisea fimbriata (Bonnemaisoniaceae). Tetrahedron Lett. 1977,

37-40.

- 269 -

(180) de Nys, R.; Wright, A. D.; Konig, G. M.; Sticher, O. New halogenated furanones from the

marine alga Delisea pulchra (cf. fimbriata). Tetrahedron 1993, 49, 11213-11220.

(181) Maximilien, R.; de Nys, R.; Holmström, C.; Gram, L.; Givskov, M.; Crass, K.; Kjelleberg,

S.; Steinberg, P. D. Chemical mediation of bacterial surface colonisation by secondary

metabolites from the red alga Delisea pulchra. Aquat. Microb. Ecol. 1998, 15, 233-246.

(182) Dworjanyn, S. A.; De Nys, R.; Steinberg, P. D. Localisation and surface quantification of

secondary metabolites in the red alga Delisea pulchra. Mar. Biol. (Berlin) 1999, 133, 727-

736.

(183) Ren, D.; Sims, J. J.; Wood, T. K. Inhibition of biofilm formation and swarming of Bacillus

subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone. Lett. Appl.

Microbiol. 2002, 34, 293-299.

(184) Ren, D.; Sims, J. J.; Wood, T. K. Inhibition of biofilm formation and swarming of

Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone. Environ.

Microbiol. 2001, 3, 731-736.

(185) Manefield, M.; Rasmussen, T. B.; Henzter, M.; Andersen, J. B.; Steinberg, P.; Kjelleberg,

S.; Givskov, M. Halogenated furanones inhibit quorum sensing through accelerated LuxR

turnover. Microbiology 2002, 148, 1119-1127.

(186) Givskov, M.; de Nys, R.; Manefield, M.; Gram, L.; Maximilien, R.; Eberl, L.; Molin, S.;

Steinberg, P. D.; Kjelleberg, S. Eukaryotic interference with homoserine lactone-mediated

prokaryotic signalling. J. Bacteriol. 1996, 178, 6618-6622.

(187) Defoirdt, T.; Crab, R.; Wood, T. K.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Quorum

sensing-disrupting brominated furanones protect the gnotobiotic brine shrimp Artemia

- 270 -

franciscana from pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio

parahaemolyticus isolates. Appl. Environ. Microbiol. 2006, 72, 6419-6423.

(188) Estephane, J.; Dauvergne, J.; Soulere, L.; Reverchon, S.; Queneau, Y.; Doutheau, A. N-

Acyl-3-amino-5H-furanone derivatives as new inhibitors of LuxR-dependent quorum

sensing: Synthesis, biological evaluation and binding mode study. Bioorg. Med. Chem.

Lett. 2008, 18, 4321-4324.

(189) Wang, W.; Morohoshi, T.; Ikeda, T.; Chen, L. Inhibition of Lux quorum-sensing system

by synthetic N-acyl-L-homoserine lactone analogous. Acta Biochim. Biophys. Sin. 2008,

40, 1023-1028.

(190) Gram, L.; De Nys, R.; Maximilien, R.; Givskov, M.; Steinberg, P.; Kjelleberg, S.

Inhibitory effects of secondary metabolites from the red alga Delisea pulchra on swarming

motility of Proteus mirabilis. Appl. Environ. Microbiol. 1996, 62, 4284-4287.

(191) Rasmussen, T. B.; Manefield, M.; Andersen, J. B.; Eberl, L.; Anthoni, U.; Christophersen,

C.; Steinberg, P.; Kjelleberg, S.; Givskov, M. How Delisea pulchra furanones affect

quorum sensing and swarming motility in Serratia liquefaciens MG1. Microbiology 2000,

146, 3237-3244.

(192) Ren, D.; Sims, J. J.; Wood, T. K. Inhibition of biofilm formation and swarming of

Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone. Environ.

Microbiol. 2001, 3, 731-736.

(193) Tao, Y.; Morohoshi, T.; Kato, N.; Ikeda, T.; Zhuang, H. The function of SpnR and the

inhibitory effects by halogenated furanone on quorum sensing in Serratia marcescens AS-1.

Weishengwu Xuebao 2008, 48, 391-397.

- 271 -

(194) Guo, J.; Li, B.; Liu, W.; Chen, W. Inhibitory effects of brominated 2(5H)-furanones on

biofilms of Pseudomonas aeruginosa. Shengming Kexue Yanjiu 2009, 13, 38-41.

(195) Hentzer, M.; Riedel, K.; Rasmussen, T. B.; Heydorn, A.; Andersen, J. B.; Parsek, M. R.;

Rice, S. A.; Eberl, L.; Molin, S.; Hoiby, N.; Kjelleberg, S.; Givskov, M. Inhibition of

quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone

compound. Microbiology 2002, 148, 87-102.

(196) Hume, E. B. H.; Baveja, J.; Muir, B.; Schubert, T. L.; Kumar, N.; Kjelleberg, S.; Griesser,

H. J.; Thissen, H.; Read, R.; Poole-Warren, L. A.; Schindhelm, K.; Willcox, M. D. P. The

control of Staphylococcus epidermidis biofilm formation and in vivo infection rates by

covalently bound furanones. Biomaterials 2004, 25, 5023-5030.

(197) Lonn-Stensrud, J.; Petersen, F. C.; Benneche, T.; Schele, A. A. Synthetic bromated

furanone inhibits autoinducer-2-mediated communication and biofilm formation in oral

streptococci. Oral Microbiol. Immunol. 2007, 22, 340-346.

(198) Han, Y.; Hou, S.; Simon, K. A.; Ren, D.; Luk, Y.-Y. Identifying the important structural

elements of brominated furanones for inhibiting biofilm formation by Escherichia coli.

Bioorg. Med. Chem. Lett. 2008, 18, 1006-1010.

(199) Janssens, J. C. A.; Steenackers, H.; Robijns, S.; Gellens, E.; Levin, J.; Zhao, H.; Hermans,

K.; De Coster, D.; Verhoeven, T. L.; Marchal, K.; Vanderleyden, J.; De Vos, D. E.; De

Keersmaecker, S. C. J. Brominated furanones inhibit biofilm formation by Salmonella

enterica serovar typhimurium. Appl. Environ. Microbiol. 2008, 74, 6639-6648.

(200) Vestby, L. K.; Lonn-Stensrud, J.; Moeretroe, T.; Langsrud, S.; Aamdal-Scheie, A.;

Benneche, T.; Nesse, L. L. A synthetic furanone potentiates the effect of disinfectants on

Salmonella in biofilm. J. Appl. Microbiol. 2010, 108, 771-778.

- 272 -

(201) Zhang, L.; Wang, S.; Zhou, X.; Xu, Y. Effect of quorum sensing inhibitor brominated

furanone on formation of Porphyromonas gingivalis biofilm. Huaxi Kouqiang Yixue Zazhi

2011, 29, 469-472.

(202) Morohoshi, T.; Shiono, T.; Takidouchi, K.; Kato, M.; Kato, N.; Kato, J.; Ikeda, T.

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-

acylhomoserine lactone. Appl. Environ. Microbiol. 2007, 73, 6339-6344.

(203) Manefield, M.; Welch, M.; Givskov, M.; Salmond, G. P. C.; Kjelleberg, S. Halogenated

furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and

exoenzyme virulence factor production in the phytopathogen Erwinia carotovora. FEMS

Microbiol. Lett. 2001, 205, 131-138.

(204) Jones, M. B.; Jani, R.; Ren, D.; Wood, T. K.; Blaser, M. J. Inhibition of Bacillus anthracis

growth and virulence gene expression by inhibitors of quorum-sensing. J. Infect. Dis. 2005,

191, 1881-1888.

(205) Tinh, N. T. N.; Linh, N. D.; Wood, T. K.; Dierckens, K.; Sorgeloos, P.; Bossier, P.

Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth

rate of gnotobiotically cultured rotifer Brachionus plicatilis. J. Appl. Microbiol. 2007, 103,

194-203.

(206) Liu, H.-B.; Lee, J.-H.; Kim Jung, S.; Park, S. Inhibitors of the Pseudomonas aeruginosa

quorum-sensing regulator, QscR. Biotechnol. Bioeng. 2010, 106, 119-126.

(207) Pan, J.; Bahar, A. A.; Syed, H.; Ren, D. Reverting antibiotic tolerance of Pseudomonas

aeruginosa PAO1 persister cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-

2(5H)-one. PLoS One 2012, 7, e45778.

- 273 -

(208) Hentzer, M.; Wu, H.; Andersen, J. B.; Riedel, K.; Rasmussen, T. B.; Bagge, N.; Kumar, N.;

Schembri, M. A.; Song, Z.; Kristoffersen, P.; Manefield, M.; Costerton, J. W.; Molin, S.;

Eberl, L.; Steinberg, P.; Kjelleberg, S.; Hoiby, N.; Givskov, M. Attenuation of

Pseudomonas aeruginosa virulence by quorum sensing inhibitors. Embo J. 2003, 22, 3803-

3815.

(209) Wu, H.; Song, Z.; Hentzer, M.; Andersen, J. B.; Molin, S.; Givskov, M.; Hoiby, N.

Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in

Pseudomonas aeruginosa lung infection in mice. J. Antimicrob. Chemother. 2004, 53,

1054-1061.

(210) Janssens, J. C. A.; Steenackers, H.; Robijns, S.; Gellens, E.; Levin, J.; Zhao, H.; Hermans,

K.; De, C. D.; Verhoeven, T. L.; Marchal, K.; Vanderleyden, J.; De, V. D. E.; De, K. S. C.

J. Brominated furanones inhibit biofilm formation by Salmonella enterica serovar

typhimurium. Appl. Environ. Microbiol. 2008, 74, 6639-6648.

(211) Bandyopadhyay, D. Controlling biofouling by surface engineering and molecular

inhibition, Ph. D. Dissertation, Syracuse University, Syracuse, 2011.

(212) Defoirdt, T.; Miyamoto, C. M.; Wood, T. K.; Meighen, E. A.; Sorgeloos, P.; Verstraete,

W.; Bossier, P. The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-

furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by

decreasing the DNA-binding activity of the transcriptional regulator protein luxR. Environ.

Microbiol. 2007, 9, 2486-2495.

(213) Manefield, M.; De Nys, R.; Kumar, N.; Read, R.; Givskov, M.; Steinberg, P.; Kjelleberg,

S. Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine

- 274 -

lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor

protein. Microbiology 1999, 145, 283-291.

(214) Hentzer, M.; Givskov, M. Pharmacological inhibition of quorum sensing for the treatment

of chronic bacterial infections. J. Clin. Invest. 2003, 112, 1300-1307.

(215) Kjelleberg, S. S., P.; Givskov, M.; Gram, L.; Manefield, M.; de Nys R. Do marine natural

products interfere with prokaryotic AHL regulatory systems?, Aquat. Microb. Ecol. 1997,

13, 85-93.

(216) Ren, D.; Bedzyk, L. A.; Ye, R. W.; Thomas, S. M.; Wood, T. K. Differential gene

expression shows natural brominated furanones interfere with the autoinducer-2 bacterial

signaling system of Escherichia coli. Biotechnol. Bioeng. 2004, 88, 630-642.

(217) Trost, B. M.; Ferreira, E. M.; Gutierrez, A. C. Ruthenium- and Palladium-Catalyzed Enyne

Cycloisomerizations: Differentially Stereoselective Syntheses of Bicyclic Structures. J. Am.

Chem. Soc. 2008, 130, 16176-16177.

(218) Holloway, C. A.; Muratore, M. E.; Storer, R. l.; Dixon, D. J. Direct Enantioselective

Bronsted Acid Catalyzed N-Acyliminium Cyclization Cascades of Tryptamines and

Ketoacids. Org. Lett. 2010, 12, 4720-4723.

(219) Sorg, A.; Brueckner, R. Total synthesis of xerulinic acid. Angew. Chem., Int. Ed. 2004, 43,

4523-4526.

(220) Sorg, A.; Siegel, K.; Brueckner, R. Stereoselective syntheses of dihydroxerulin and

xerulinic acid, anti-hypocholesterolemic dyes from the fungus Xerula melanotricha. Chem.-

-Eur. J. 2005, 11, 1610-1624.

(221) Sorg, A.; Siegel, K.; Brueckner, R. A novel access to γ-alkylidenebutenolides: Sequential

Stille couplings of dibromomethylenebutenolides. Synlett 2004, 321-325.

- 275 -

(222) Heydorn, A.; Nielsen, A. T.; Hentzer, M.; Sternberg, C.; Givskov, M.; Ersboll, B. K.;

Molin, S. Quantification of biofilm structures by the novel computer program COMSTAT.

Microbiology 2000, 146, 2395-2407.

(223) Campana, S.; Taccetti, G.; Ravenni, N.; Masi, I.; Audino, S.; Sisi, B.; Repetto, T.; Doring,

G.; de Martino, M. Molecular epidemiology of Pseudomonas aeruginosa, Burkholderia

cepacia complex and methicillin-resistant Staphylococcus aureus in a cystic fibrosis center.

J. Cystic Fibrosis 2004, 3, 159-163.

(224) Van Delden, C.; Iglewski, B. H. Cell-to-cell signaling and Pseudomonas aeruginosa

infections. Emerg. Infect. Dis. 1998, 4, 551-560.

(225) Afessa, B.; Green, B. Bacterial pneumonia in hospitalized patients with HIV infection: the

Pulmonary Complications, ICU Support, and Prognostic Factors of Hospitalized Patients

with HIV (PIP) Study. Chest 2000, 117, 1017-1022.

(226) Burns, J. L.; Gibson, R. L.; McNamara, S.; Yim, D.; Emerson, J.; Rosenfeld, M.; Hiatt, P.;

McCoy, K.; Castile, R.; Smith, A. L.; Ramsey, B. W. Longitudinal assessment of

Pseudomonas aeruginosa in young children with cystic fibrosis. The Journal of infectious

diseases 2001, 183, 444-452.

(227) Garo, E.; Eldridge, G. R.; Goering, M. G.; Pulcini, E. D.; Hamilton, M. A.; Costerton, J.

W.; James, G. A. Asiatic acid and corosolic acid enhance the susceptibility of

Pseudomonas aeruginosa biofilms to tobramycin. Antimicrob. Agents Chemother. 2007, 51,

1813-1817.

(228) Bloemberg, G. V.; O'Toole, G. A.; Lugtenberg, B. J. J.; Kolter, R. Green fluorescent

protein as a marker for Pseudomonas spp. Appl. Environ. Microbiol. 1997, 63, 4543-4551.

- 276 -

(229) Wright, A. D.; de Nys, R.; Angerhofer, C. K.; Pezzuto, J. M.; Gurrath, M. Biological

activities and 3D QSAR studies of a series of Delisea pulchra (cf. fimbriata) derived natural

products. J. Nat. Prod. 2006, 69, 1180-1187.

(230) Kuehl, R.; Al-Bataineh, S.; Gordon, O.; Luginbuehl, R.; Otto, M.; Textor, M.; Landmann,

R. Furanone at subinhibitory concentrations enhances staphylococcal biofilm formation by

luxS repression. Antimicrob. Agents Chemother. 2009, 53, 4159-4166.

(231) Karney, W.; Holmes, K. K.; Turck, M. Comparison of five aminocyclitol antibiotics in

vitro against Enterobacteriaceae and Pseudomonas. Antimicrob. Agents Chemother. 1973,

3, 338-342.

(232) Brackman, G.; Cos, P.; Maes, L.; Nelis, H. J.; Coenye, T. Quorum sensing inhibitors

increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo.

Antimicrob. Agents Chemother. 2011, 55, 2655-2661.

(233) VanDevanter, D. R.; Geller, D. E. Tobramycin administered by the TOBI® Podhaler® for

persons with cystic fibrosis: a review. Medical Devices: Evidence and Research 2011, 4,

179-188.

(234) Moker, N.; Dean, C. R.; Tao, J. Pseudomonas aeruginosa increases formation of

multidrug-tolerant persister cells in response to quorum sensing signaling molecules. J.

Bacteriol. 2010, 192, 1946-1955.

(235) Lewis, K. Persister cells. Annu. Rev. Microbiol. 2010, 64, 357-372.

(236) Pesci, E.; Pearson, J. P.; Seed, P. C.; Iglewski, B. H. Regulation of las and rhl quorum

sensing in Pseudomonas aeruginosa. J. Bacteriol. 1997, 179, 3127-3132.

- 277 -

(237) De Kievit, T. R.; Gillis, R.; Marx, S.; Brown, C.; Iglewski, B. H. Quorum-sensing genes in

Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl. Environ.

Microbiol. 2001, 67, 1865-1873.

(238) Gambello, M. J.; Iglewski, B. H. Cloning and characterization of the Pseudomonas

aeruginosa lasR gene, a transcriptional activator of elastase expression. Journal of

Bacteriology 1991, 173, 3000-3009.

(239) Tamura, Y.; Suzuki, S.; Kijima, M.; Takahashi, T.; Nakamura, M. Effect of proteolytic

enzyme on experimental infection of mice with Pseudomonas aeruginosa. The Journal of

veterinary medical science / the Japanese Society of Veterinary Science 1992, 54, 597-599.

(240) Kon, Y.; Tsukada, H.; Hasegawa, T.; Igarashi, K.; Wada, K.; Suzuki, E.; Arakawa, M.;

Gejyo, F. The role of Pseudomonas aeruginosa elastase as a potent inflammatory factor in

a rat air pouch inflammation model. FEMS Immunol. Med. Microbiol. 1999, 25, 313-321.

(241) Kamath, S.; Kapatral, V.; Chakrabarty, A. M. Cellular function of elastase in

Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in

alginate synthesis. Mol. Microbiol. 1998, 30, 933-941.

(242) Travis, J.; Potempa, J. Bacterial proteinases as targets for the development of second-

generation antibiotics. Biochim. Biophys. Acta 2000, 1477, 35-50.

(243) Rasmussen, T. B.; Givskov, M. Quorum sensing inhibitors: A bargain of effects.

Microbiology 2006, 152, 895-904.

(244) Hawgood, S.; Ochs, M.; Jung, A.; Akiyama, J.; Allen, L.; Brown, C.; Edmondson, J.;

Levitt, S.; Carlson, E.; Gillespie Anne, M.; Villar, A.; Epstein Charles, J.; Poulain Francis,

R. Sequential targeted deficiency of SP-A and -D leads to progressive alveolar

- 278 -

lipoproteinosis and emphysema. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002, 283,

L1002-1010.

(245) Smith, A. W. Bacterial resistance to antibiotics. Hugo Russell's Pharm. Microbiol. (8th

Ed.) 2011, 217-229.

(246) Neu, H. C. The crisis in antibiotic resistance. Science 1992, 257, 1064-1072.

(247) Lowery, C. A.; Abe, T.; Park, J.; Eubanks, L. M.; Sawada, D.; Kaufmann, G. F.; Janda, K.

D. Revisiting AI-2 Quorum Sensing Inhibitors: Direct Comparison of Alkyl-DPD

Analogues and a Natural Product Fimbrolide. J. Am. Chem. Soc. 2009, 131, 15584-15585.

(248) Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura, Y.; Ueno, K. A combined

assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt,

neutral red and crystal violet. Biol. Pharm. Bull. 1996, 19, 1518-1520.

(249) De Kievit, T. R.; Gillis, R.; Marx, S.; Brown, C.; Iglewski, B. H. Quorum-sensing genes in

Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl. Environ.

Microbiol. 2001, 67, 1865-1873.

(250) Parkinson, J. S.; Houts, S. E. Isolation and behavior of Escherichia coli deletion mutants

lacking chemotaxis functions. J. Bacteriol. 1982, 151, 106-113.

(251) Bevis, B. J.; Glick, B. S. Rapidly maturing variants of the Discosoma red fluorescent

protein (DsRed). Nat. Biotechnol. 2002, 20, 83-87.

(252) Hou, S.; Liu, Z.; Young, A. W.; Mark, S. L.; Kallenbach, N. R.; Ren, D. Effects of Trp-

and Arg-containing antimicrobial-peptide structure on inhibition of Escherichia coli

planktonic growth and biofilm formation. Appl. Environ. Microbiol. 2010, 76, 1967-1974.

- 279 -

(253) Calfee, M. W.; Coleman, J. P.; Pesci, E. C. Interference with Pseudomonas quinolone

signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa. Proc.

Natl. Acad. Sci. U. S. A. 2001, 98, 11633-11637.

(254) Churchill, M. E. A.; Chen, L. Structural basis of acyl-homoserine lactone-dependent

signaling. Chem. Rev. 2011, 111, 68-85.

(255) Thiel, V.; Kunze, B.; Verma, P.; Wagner-Doebler, I.; Schulz, S. New Structural Variants

of Homoserine Lactones in Bacteria. ChemBioChem 2009, 10, 1861-1868.

(256) Eberhard, A.; Widrig, C. A.; McBath, P.; Schineller, J. B. Analogs of the autoinducer of

bioluminescence in Vibrio fischeri. Arch. Microbiol. 1986, 146, 35-40.

(257) Schaefer, A. L.; Hanzelka, B. L.; Eberhard, A.; Greenberg, E. P. Quorum sensing in Vibrio

fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol.

1996, 178, 2897-2901.

(258) Passador, L.; Tucker, K. D.; Guertin, K. R.; Journet, M. P.; Kende, A. S.; Iglewski, B. H.

Functional analysis of the Pseudomonas aeruginosa autoinducer PAI. J. Bacteriol. 1996,

178, 5995-6000.

(259) Reverchon, S.; Chantegrel, B.; Deshayes, C.; Doutheau, A.; Cotte-Pattat, N. New synthetic

analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional

regulators involved in bacterial quorum sensing. Bioorg. Med. Chem. Lett. 2002, 12, 1153-

1157.

(260) Castang, S.; Chantegrel, B.; Deshayes, C.; Dolmazon, R.; Gouet, P.; Haser, R.; Reverchon,

S.; Nasser, W.; Hugouvieux-Cotte-Pattat, N.; Doutheau, A. N-Sulfonyl homoserine

lactones as antagonists of bacterial quorum sensing. Bioorg. Med. Chem. Lett. 2004, 14,

5145-5149.

- 280 -

(261) Frezza, M.; Castang, S.; Estephane, J.; Soulere, L.; Deshayes, C.; Chantegrel, B.; Nasser,

W.; Queneau, Y.; Reverchon, S.; Doutheau, A. Synthesis and biological evaluation of

homoserine lactone derived ureas as antagonists of bacterial quorum sensing. Bioorg. Med.

Chem. 2006, 14, 4781-4791.

(262) Whitehead, N. A.; Barnard, A. M. L.; Slater, H.; Simpson, N. J. L.; Salmond, G. P. C.

Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 2001, 25, 365-404.

(263) Geske, G. D.; O'Neill, J. C.; Blackwell, H. E. Expanding dialogues: From natural

autoinducers to non-natural analogs that modulate quorum sensing in Gram-negative

bacteria. Chem. Soc. Rev. 2008, 37, 1432-1447.

(264) Geske, G. D.; O'Neill, J. C.; Blackwell, H. E. N-Phenylacetanoyl-L-homoserine lactones

can strongly antagonize or superagonize quorum sensing in Vibrio fischeri. ACS Chem.

Biol. 2007, 2, 315-319.

(265) Wang, Y.-J.; Leadbetter, J. R. Rapid acyl-homoserine lactone quorum signal

biodegradation in diverse soils. Appl. Environ. Microbiol. 2005, 71, 1291-1299.

(266) Narasimhan, S. K. Structured unnatural molecules: Inducing molecular folding, enabling

matured mammalian cell adhesion and inhibiting bacterial biofilm formation, Ph. D.

Dissertation, Syracuse University, Syracuse, NY, 2010.

(267) Lindsay, A.; Ahmer, B. M. M. Effect of sdiA on biosensors of N-acylhomoserine lactones.

J. Bacteriol. 2005, 187, 5054-5058.

(268) Van Houdt, R.; Aertsen, A.; Moons, P.; Vanoirbeek, K.; Michiels, C. W. N-acyl-L-

homoserine lactone signal interception by Escherichia coli. FEMS Microbiol. Lett. 2006,

256, 83-89.

- 281 -

(269) Lupp, C.; Urbanowski, M.; Greenberg, E. P.; Ruby, E. G. The Vibrio fischeri quorum-

sensing systems ain and lux sequentially induce luminescence gene expression and are

important for persistence in the squid host. Mol. Microbiol. 2003, 50, 319-331.

- 282 -

CURRICULUM VITAE

Name of Author: Sijie Yang

Place of Birth: Wuhan, Hubei, China

Date of Birth: November 13, 1983

Education

Ph. D. in Chemistry, Department of Chemistry, Syracuse University, Syracuse, NY (Degree

expected May, 2013)

M. Phil. in Chemistry, Department of Chemistry, Syracuse University, Syracuse, NY. 2008

B. S. in Chemistry, Department of Chemistry, Wuhan University, Wuhan, Hubei, China 2005

Awards

Outstanding Teaching Assistant: William D. Johnson Award, Syracuse University, 2009.

People’s Scholarship, Wuhan University, 2002-2004

Teaching Experience

Teaching assistant, Department of Chemistry, Syracuse University, Syracuse NY, 2006-2011.

Recitation Instructor of Organic Chemistry I (Fall 2010)

- Developed question-centered lessons with worksheet to create student involvement.

- Aided lecture professor in proctoring and grading exams.

Laboratory Instructor of Organic Chemistry I and II (Spring 2007 – Spring 2010)

- Assisted students during laboratory experiments, demonstrating laboratory techniques

and developing their problem-solving skills.

- Evaluated students’ success by grading laboratory reports.

Laboratory Instructor of General Chemistry I (Fall 2006)

- Assisted students during laboratory experiments, demonstrating laboratory techniques

and developing their problem-solving skills.

- Evaluated students’ success by grading laboratory reports.

Research Experience

- 283 -

Research assistant, Department of Chemistry, Syracuse University, Syracuse NY, 2007-2013.

Publications

Simon, K. A., Sejwal, P., Falcone, E. R., Burton, E. A., Yang, S., Prashar, D.,

Bandyopadhyay, D., Narasimhan, S. K., Varghese, N., Gobalasingham, N. S., Reese, J. B.,

and Luk, Y.-Y. Noncovalent polymerization and assembly in water promoted by

thermodynamic incompatibility. J. Phys. Chem. B, 2010, 114, 10357–10367.

Yang, S.; Wang, B.; Cui, D.; Kerwood, D.; Wilkens, S.; Han, J.; and Luk, Y.-Y.

Stereochemical Control of Nonamphiphilic Lyotropic Liquid Crystals: Interactions between

Assemblies Separated by Six Nanometers of Aqueous Solvents. In preparation.

Yang, S.; Abdel-Razek, O. A.; Cheng, F.; Bandyopadhyay, D.; Sheth, G.; Wang, G.; and

Luk, Y.-Y. Bicyclic Brominated Furanones: A new Class of Biofilm Inhibitors with Reduced

Toxicity and Improved Bacterial Clearance in vivo. In preparation.

Presentations

Yang, S., Cheng, Fei., and Luk, Y.-Y. Controlling bacterial behavior by bicyclic brominated

furanones with reduced toxicity. 243rd ACS National Meeting & Exposition, San Diego, CA,

2012.

Yang, S., Bandyopadhyay, D., Kerwood, D., Wang, B., and Luk, Y.-Y. Direct evidences and

structural characterizations of the thread assembly formed by nonamphiphilic lyotropic liquid

crystals. 243rd ACS National Meeting & Exposition, San Diego, CA, 2012.

Yang, S., Cui, D., Kerwood, D., Wilkens, S., Simon, K., Han, J., and Luk, Y.-Y.

Stereochemistry of mesogens controls the formation of nonamphiphilic lyotropic Liquid

crystals. 243rd ACS National Meeting & Exposition, San Diego, CA, 2012.

Yang, S., Totah, N. I. Total synthesis of neoliacinic acid: approaches toward the construction

of the bicyclic core. Alliance for Graduate Education and the Professoriate. 2nd Annual

Academic Excellence Symposium. Syracuse University, Syracuse, NY, 2008.

I. Stereochemical Control of Chiral Assembly and Liquid ...

Documents

Transcript of I. Stereochemical Control of Chiral Assembly and Liquid ...