TOWARD REAGENTLESS ELECTROCHEMICALLY ... - SFU's Summit

TOWARD REAGENTLESS ELECTROCHEMICALLY

ADDRESSABLE MICROARRAYS: SYNTHESIS OF

SUITABLE MONOMERS AND ANCHOR MOLECULES

byFrederick F.R.M. Chesneau

BSc.Hons., Aston University, 2004

A THESIS SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

in the School

of

Chemistry

© Frederick F.R.M. Chesneau 2008

SIMON FRASER UNIVERSITY

Spring 2008

All rights reserved. This work may not be

reproduced in whole or in part, by photocopy

or other means, without the permission of the author.

Name:

Degree:

Title of Thesis:

Examining Committee:

Chair

Date Defended/Approved:

APPROVAL

Frederick F.R.M. Chesneau

Master of Science

Toward Reagentless Electrochemically AddressableMicroarrays: Sythesis of Suitable Monomers andAnchor Molecules

Dr. Zuo-Guang YeProfessor, Department of Chemistry

Dr. David J. VocadloSenior SupervisorAssistant Professor, Department of Chemistry

Dr. Hua-Zhong(Hogan) YuSupervisorAssociate Professor, Department of Chemistry

Dr. Melanie A. O'NeillSupervisorAssistant Professor, Department of Chemistry

Dr. Nancy R. FordeInternal ExaminerAssistant Professor, Department of Physics

March 31, 2008

11

SIMON PRASER UNIVERSITYLIBRARY

Declaration ofPartial Copyright Licence

The author, whose copyright is declared on the title page of this work, has grantedto Simon Fraser University the right to lend this thesis, project or extended essayto users of the Simon Fraser University Library, and to make partial or singlecopies only for such users or in response to a request from the library of any otheruniversity, or other educational institution, on its own behalf or for one of its users.

The author has further granted permission to Simon Fraser University to keep ormake a digital copy for use in its circulating collection (currently available to thepublic at the "Institutional Repository" link of the SFU Library website<www.lib.sfu.ca> at: <http://ir.lib.sfu.calhandle/1892/112>) and, without changingthe content, to translate the thesis/project or extended essays, if technicallypossible, to any medium or format for the purpose of preservation of the digitalwork.

The author has further agreed that permission for multiple copying of this work forscholarly purposes may be granted by either the author or the Dean of GraduateStudies.

It is understood that copying or publication of this work for financial gain shall notbe allowed without the author's written permission.

Permission for public performance, or limited permission for private scholarly use,of any multimedia materials forming part of this work, may have been granted bythe author. This information may be found on the separately cataloguedmultimedia material and in the signed Partial Copyright Licence.

While licensing SFU to permit the above uses, the author retains copyright in thethesis, project or extended essays, including the right to change the work forsubsequent purposes, including editing and publishing the work in whole or inpart, and licensing other parties, as the author may desire.

The original Partial Copyright Licence attesting to these terms, and signed by thisauthor, may be found in the original bound copy of this work, retained in theSimon Fraser University Archive.

Simon Fraser University LibraryBurnaby, BC, Canada

Revised: Fall 2007

Abstract

Combinatorial chemistry facilitates the synthesis of large libraries of compounds. However,

the screening of such libraries is time-consuming and remains a challenging problem. In

this thesis we propose that an array of gold electrodes could be used for the generation of

large combinatorial libraries of oligomers with defined sequences. The ability to address

individual electrodes in a controlled manner through directed electrochemical deprotection

of suitably designed molecules, facilitates the generation of spatially addressable libraries

that can be readily deconvoluted. Here, we outline this electrochemical strategy, which

involves electrochemical deprotection of surface-bound amines. Once deprotected, these

amines can be elaborated using monomeric units containing a suitable electrophile and an

amine protected by an electrochemically protected group. This process can be repeated to

generate the desired oligomers and should be amenable to such arrays. Enabling studies on

the electrochemical modification of monolayers of a diaryldisulfide molecule that inform on

optimising device design will also be presented.

iii

To my parents, my sisters and my late cousin Henry.

iv

v

"Impossible n 'est pas fraw;ais"

Napoleon Bonaparte

Acknowledgments

I would like to thank Dr. David J. Vocadlo for giving me the opportunity to work on

various aspects of chemistry, from chemical synthesis to biochemistry and electrochemistry.

Thanks go to Dr. Byron D. Gates and Pr. Neil R. Branda for their advice during my time

working on the Nanoparticles for Medecine project in 4DLabs. I would also like to thank all

my coworkers on the Nanoparticles for Medecine project for educting me on nanoparticles

and Scott Yuzwa for showing me the ropes in the biochemistry lab. Special thanks go

to Aleksandra Debowski for her patience during cell experiments for that project. More

related to the project described in this thesis, thanks go to Dr. Hogan Yu for letting me

use his laboratory space to perform both FT-IR and cyclic voltammetry experiments and

his advice, all the members of the Yu lab that I have befriended. And of course, I thank my

parents for allowing me to be where I am today and my banker for lending me the funds

that made it all possible.

vi

List of Abbreviations

General terms

3D

Calcd.

CM

conc.

(d)

8+

8

DNA

ELISA

equiv.

Expt.

expt

expts

LG

three dimensional

calculated

core

concentrated

decomposed

partially positive

partially negative

DeoxyriboNucleic Acid

Enzyme-Linked ImmunoSorbant Assay

equivalent

experimental

experiment

experiments

leaving group

vii

m.p.

PGM

pH

Lt.

SAM

SAMS

t

T

temp.

melting point

protecting group

potential Hydrogen

negative log of the acid ionization constant (Ka)

room temperature

self-assembled monolayer

self-assembled monolayers

time

temperature

temperature

Units of measure

A Angstrom

A area

°C degree Celsius

cm centimeter

g gram

M mole per liter

mg milligram

mM millimole per liter

J1m micrometer

viii

m% mole percent

ml milliliter

JLl microliter

MD megaohm

mol mole

mV millivolt

mV/s millivolt per second

MW molecular weight

s second

v/v volume by volume

V volt

wt. weight

Energy

~Go

kJ

Electron transfer

(J

free energy at standard conditions

free energy of activation

energy of activation

kilojoule

attenuation factor

IX

ko

kET

Electrochemistry

C

n

Q

preexponential factor

rate constant of electron transfer

reorganisational energy

Distance between the electron donor and acceptor

position of the electron acceptor

position of the electron donor

Coulombs

double-layer capacitance

Surface coverage

width at half peak height

Efficiency of the nth coupling

Peak potential

Faraday's constant

peak current

number of electrons involved in a redox process

charge

charge after nth deprotection cycle

charge after 1st deprotection cycle

x

Chemicals

4-ATP

Au(100)

Au(llO)

Au(l11)

Au(200)

Au(220)

Au(311)

BOC

BOC2 0

Cdiamine

C53

COOBt

DCC

DCU

DMSO

EDC.HCI

Et

EtOH

Fmoc

HOBt

4-aminothiophenol

gold 100 plane

gold 110 plane

gold 111 plane

gold 200 plane

gold 220 plane

gold 311 plane

tert-butyloxycarbonyl

di-tert-butyl dicarbonate

concentration of diamine

concentration of 53

N-hydroxybenzotriazole ester

dicyclohexylearbodimide

dicyclohexy1urea

dimethylsulfoxide

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride

ethyl

ethanol

(9H-fluoren-9-yl)methyl carbamate

N-hydoxybenzotriazole

xi

HQ hydroquinone

Me methyl

MeOH methanol

Pd/C 5% palladium on charcoal

R random group

RS- thiolate

TBDMS tert-butyldimethylsily1

TFA trifluoroacetic acid

THF tetrahydrofuran

Techniques

CHN

CV

E.A.

GC/MS

HPLC

MALDI-TOF

NMR

carbon, hydrogen, nitrogen

cyclic voltammetry

elemental analysis

gas chromatography / mass spectrometry

high performance liquid chromatography

matrix-assisted laser desorption/ionization-time of flight

nuclear magnetic resonance

br - broad

d - doublet

dd - doublet of doublet

xii

IH-NMR

13C-NMR

TLC

Rf

ddd - doublet of doublet of doublet

<5 - chemical shift

J - coupling constant

m - multiplet

MHz - megahertz

ppm - parts per million

q - quadruplet

s - singlet

t - triplet

proton nuclear magnetic resonance

carbon 13 nuclear magnetic resonance

thin layer chromatography

retention factor

xiii

Contents

Approval ii

Abstract iii

Dedication iv

Quotation v

Acknowledgments vi

List of Abbreviations vii

Contents xiv

List of Tables xx

List of Figures xxii

List of Schemes xxvii

1 Introduction 1

xiv

Combinatorial Chemistry or Rational Design? .

Spatially addressable arrays for chemical synthesis

1.1

1.2

1.1.1

1.1.2

1.2.1

1.2.2

1.2.3

Rational Design

Combinatorial Chemistry

Well plates . . . . . . . . . . . . . .

Photochemically addressable arrays

Electrochemically addressable arrays

1

2

3

7

7

8

8

1.3 Scope of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12

1.3.1 Built-in deconvolution . . . . . . . . . . . . . . . . 12

1.3.2 High throughput, synthesis and efficiency . . . . . . . . . . . . . . .. 13

1.3.3 General Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.3.4 Archiving of the combinatorial libraries . . . . . . . . . . . . . . . .. 16

1.3.5 Design principles " 17

1.3.5.1 Electrode design . . . . . . . . . . . . . .. 17

1.3.5.2 Monolayers for microarray design 18

1.3.5.3 Monomer design . . . . . . . . . . . . . . . . . . . . . . . .. 20

1.3.6 Critical evaluation of the array design . . . . . . . . . . . . . . . . " 20

2 Design and synthesis of monomers 22

2.1 General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.1.1 Unnatural amino acids in the synthesis and design of protein-like

structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22

xv

2.1.2 Guiding principles for designing the synthetic routes . . . . . . . . . . 26

2.2 Rational design of the monomer. . . . . . . . . . . . . . . . . . . . . . . . .. 27

2.2.1 Selection of the leaving group .............. 28

2.2.1.1 Mixed anhydrides as leaving groups . . . . . . . . . . . . .. 29

2.2.1.2 Reactive esters as leaving groups. . . . . . . . . . . . . . .. 34

2.2.2 The protecting group 35

2.2.3 The core. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36

2.3 Amino benzoic acid series . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 38

2.3.1 First approach . . 39

2.3.1.1 Retrosynthetic analysis and general synthetic scheme 39

2.3.2 Second approach . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.3.2.1 Retrosynthetic analysis and general synthetic scheme (Scheme

2.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 43

2.3.2.2 Synthesis of the chloroformate intermediate (2) . . . . . . . . 44

2.3.2.3 Synthesis of the carbamate 13 . . . . . . . . . . . . . . . .. 48

2.3.2.4 Coupling of N-hydroxybenzotriazole (HOBt)-Synthesis of 16 50

2.3.2.5 Testing the reactivity of the aniline monomers to aniline nu-

cleophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 50

2.3.3 Experimental . . . . 53

2.3.3.1 Solvents and chemicals 53

2.3.3.2 Characterisation......................... 53

XVI

2.4 Aminomethylbenzoic series 61

2.4.1 Retrosynthetic analysis 62

2.4.1.1 First approach 62

2.4.1.2

2.4.1.3

Second approach

General Synthetic scheme

.................... 66

............ 67

2.4.1.4 Synthesis of carbamate intermediate 31 . . . . . . . . . . . . 67

2.4.1.5 Synthesis of benzylamine carbamate 33 ..... 69

2.4.1.6 Selective debenzylation - synthesis of 37-39 and 59 . . . . . 73

2.4.1.7 Coupling of N-hydroxybenzotriazole-synthesis of monomers

40-42 78

2.4.1.8 Conclusion............................ 79

2.4.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . 81

2.4.2.1 Solvents and chemicals 81

2.4.2.2 Characterisation......................... 81

3 Self-assembled monolayers 88

3.1 A brief introduction to self-assembled monolayers . . . . . . . . . . . . . . .. 88

3.2 Effect of the molecular structure of the thiols on the formation of monolayers 91

3.2.1 Mercaptoalkane self-assembled monolayers . . . . . . . . . . . . . . 91

3.2.2 Self-assembled monolayers from amide-containing mercaptoalkanes 93

3.2.3 Arylthiol self-assembled monolayers 94

3.2.4 SAMS from thiols and disulfides .. . . . . . . . . . . . . . . . . . .. 96

xvii

3.2.5 Functional monolayers ~ the head group . . . . . . . . . . . . . . . .. 98

3.3 Electrochemistry of self-assembled monolayers . 99

3.3.1

3.3.2

General electrochemistry of self-assembled monolayers

Electron-transfer - from bulk to single molecules to SAMS

· 100

· 101

3.3.2.1 Influence of the monolayer thickness, arrangement and molec-

ular identity . . . . . . . . . . . . . . . . . . . . . . . .. . 104

3.3.2.2 Effect of the environment on the rate of electron transfer . 105

3.4 Synthesis and testing of anchor molecules · 109

3.4.1 Thioaniline-based anchor ..... · 109

3.4.1.1

3.4.1.2

Synthesis of disulfide 44 .

Growth of monolayers of disulfide 44 and thiol 45

· 111

· 112

3.4.1.3 Cyclic Voltammetry studies of monolayers formed from disul-

fide 44 . . . . . . . . . . . . . . . . . . . . . . . . . . 114

3.4.2 Amide-containing dialkyldisulfides as anchors - Synthesis of 55 129

3.4.2.1

3.4.2.2

Retrosynthetic analysis . . . .

Synthesis of diesterdisulfide 53

· 129

· 129

3.4.2.3 Synthesis of 55 via coupling of 1,6-diaminohexane to 53 . 130

3.4.2.4 Synthesis of 55 via coupling of monoamine 63 to diester-

3.5

disulfide 53 .

General experimental .

· 131

· 140

3.5.1 Synthesis ...

xviii

· 140

3.5.1.1

3.5.1.2

Solvents and chemicals

Characterisation

· 140

· 140

3.5.2 Monolayers . . . . . . . . · 141

3.5.2.1

3.5.2.2

3.5.2.3

Substrate preparation

Preparation of self-assembled monolayers

Characterisation

141

141

· 142

3.5.3 Experimental . . . . . . . · 143

4 Future Work

4.1 Coupling monomers to anchor molecules on a gold surface

148

· 148

4.1.1 Testing the surface chemistry . . . . . . . . . . . .

4.1.1.1 Reaction of monomers with amine-terminated self-assembled

monolayers . . . . . . . . .

· 148

· 149

4.1.1.2 Cyclic voltammetry studies · 151

4.2 Testing the array . . . . . . . . . . . . . . . · 153

A Visual index of compounds

Bibliography

xix

156

161

List of Tables

2.1 Protection of the carboxylic acid of aminobenzoic acids 42

2.2 Effect of the temperature, concentration and base on the yield of isolated

carbamate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47

2.3 Synthesis of aniline-based monomers ................... 50

2.4 Synthesis of the carbamate intermediate 31 69

2.5 Effect of the concentration, solvent, temperature and ratio of reactants and

reagents on the yield of 33. . . . . . . . . . . . . . . . . . . . . . . . . . . 70

2.6 Effect of the position of the substituent on the yield of benzyl carbamate 73

2.7 Hydrogenolysis of various benzylamine carbamates . . . . . . . . . . . . . 79

3.1 Table of FT-IR bands of 44 in KBr and as a monolayer on polycrystalline gold116

3.2 Data from CV scans of monolayers of 44 on various gold surfaces 120

3.3 Surface coverage and molecular area for various monolayers on polycrystalline

3.4

gold surfaces .

FT-IR bands and assignments for diamine 55

xx

. 121

. 139

4.1 Testing a 4 electrode array. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

xxi

List of Figures

1.1

1.2

1.3

1.4

1.5

1.6

1.7

Two rationally designed drugs. . . . . . . . . . . . . . . . . . . .

Two general strategies for carrying out combinatorial chemistry .

The split and mix strategy. . . . . . . . . . . . . . . . . .

Enzyme inhibitors designed using combinatorial chemistry

A 96 well plate . . . . . . . . . . . .

A photochemically addressable array

Spatially addressable electrochemical arrays

2

4

5

6

8

9

9

1.8 An electrochemically addressable array. . . . . . . . . . . . . . . . . . . . .. 13

1.9 Transacylation . . . . . . . . . . . . . . ............. 14

1.10 Hydroquinone as amide and carboxyl protecting group. . . . . . . . . . . .. 15

1.11 Proposed oligomerisation scheme . . . . . . . . . . . . . . . . . . . . . . . .. 16

1.12 Effect of the differences in monolayer composition on the oxidation behaviour

of a gold surface as reflected by cyclic voltammetry study . . . . . . . . . .. 18

1.13 The two main types of electrochemically addressable arrays . . . . . . . . .. 19

2.1 The 20 commonly occurring natural o:-amino acids . . . . . . . . . . . . . .. 23

xxii

2.2 Some unnatural amino acids. . . . . . . . . . . . . . . . . . . . . . . . . . .. 25

2.3 Examples of foldamers .... .......... " 26

2.4 Two unnatural peptides containing 3-aminobenzoic acid . . . . . . . . . . .. 26

2.5 General design of the monomeric units . . 28

2.6 Desired reactivity of the monomeric unit with amine groups . . . . . . . . .. 28

2.7 Mixed benzoic acid anhydrides .. . . . . . . . . . . . . . . . . . . . . . . .. 30

2.8 1H-NMR of benzoic pivalic anhydride and its possible coupling products with

4-bromoaniline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.9 Mixed anhydrides of benzoic acid and chlorinated benzoic acids. . . . . . .. 32

2.10 Electron density map of benzoic 2,4-dichlorobenzoic anhydride 32

2.11 1H-NMR spectra of benzoic dichloro- and trichlorobenzoic anhydrides and

their reaction products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 33

2.12 Some reactive esters . .

2.13 Thiol displacement at the gold surface

...................... 34

................. 35

2.14 Hydroquinone as a protecting group for amines 37

2.15 Some commercially available aminobenzoic acids . . . . . . . . . . . . . . . . 38

2.16 An oligomer of 3-aminobenzoic acid 39

2.17 Synthetic scheme for the synthesis of carbamate 7 . . . . . . . . . . . . . . . 42

2.18 Proton NMR of the undesired byproduct 57 . . . . . . . . . . . . . . . . . . . 43

2.19 Setup for high dilution reactions . . . . . . . . . . . . . . . . . . . . . . . .. 46

xxiii

2.20 Structure of the phosgene-pyridine complex at various temperatures in methy-

lene chloride. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

2.21 Aniline derivatives used to test 15 . . . .. 51

2.22 Reaction of (R)-l-phenylethanamine with aniline-based monomer 15 . . . . . 52

2.23 A few commercially available aminomethylbenzoic acids . . . . . . . . . . .. 61

2.24 Oligomers of aniline- and benzylamine-based amino acids 62

2.25 Structure of 4-(ammoniomethyl)benzoate 62

2.26 Some commercially available carboxybenzaldehydes. . . . . . . . . . . . . .. 67

2.27 Mechanism of benzyl carbamate formation. . . . . . . . . . . . . . . . . . .. 72

2.28 1H-NMR spectra of the products isolated from reactions of 3-carboxybenzaldehyde

with either 31 or 32 to generate 38 as the expected product. . . . . . . . .. 75

2.29 Proposed mechanism for the hydrogenolysis of O-benzyl alcohols 77

2.30 Proposed mechanism for the hydrogenolysis of N-benzylcarbamate 59 77

2.31 The various benzylamine monomers synthesised. . . . . . . . . . . . . 80

3.1 Schematic representation of a molecule capable of forming a monolayer. .. 89

3.2 Simplified growth mechanism of Self-assembled monolayers of thiols on gold 90

3.3 Cyclic voltammogram of short chain and long chain aminoalkyl thiol mono-

layers on polycrsytalline gold surfaces. . . . . . . . . . . . . . . . . . . . . .. 92

3.4 The different mercaptoalkanes capable of forming SAMS on gold . . . . . .. 93

3.5 Self-assembled monolayers from alkylthiols. . . . . . . . . . . . . . . . . . .. 94

3.6 Self-assembled monolayers from amide-containing alkylthiols. . . . . . . . .. 95

XXIV

3.7 Self-assembled monolayers from 4-aminothiophenol . . . . . . . . . . . . . .. 96

3.8 Self-assembled monolayers from various thiophenols . ..... 97

3.9 Three common functional headgroups for self-assembled monolayers ..... 98

3.10 Schematic representation of the double layer at an electrode surface

3.11 Marcus diagram of electron transfer .

3.12 Donor-acceptor pairs for SAMS adsorbed on an electrode

3.13 Effect of the amide linkage on the order of the monolayer

3.14 The resonance forms of aniline. . . . . . . .

3.15 The resonance forms of 4-aminothiophenol .

101

.102

.103

. 108

. 110

110

3.16 FT-IR spectra of monolayers formed from anchor molecules 44 and 45. 113

3.17 FT-IR spectra of monolayer of 44 on polycrystalline gold and of 44 in KBr . 115

3.18 Selection rules for the reflection of the electrical component of an infrared

light off a metal surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

3.19 Cyclic voltammogram of a monolayer of disulfide 44 on polycrystalline gold . 119

3.20 Arrangement of thiols on different gold surfaces 122

3.21 Comparison ofthe FT-IR spectra of a monolayer of 44 before and after cyclic

voltammetry. . . . . . . . . . . . . . . . . . . . . . . . . . .

3.22 Cyclic voltammogram of a monolayer of 4-aminothiophenol

3.23 Coupling of 53 with 1,6-diaminohexane

3.24 Coupling of 53 to 1,6-diaminohexane .

3.25 1Hand 13C NMR spectra of 63 ....

xxv

123

125

131

132

134

3.26 1H-NMR spectrum of 54 . ...

3.27 1Hand 13C NMR spectra of 55

3.28 Structures of the disulfides used by Bilewicz and coworkers

136

138

. 139

3.29 X-ray diffraction pattern of a gold slide obtained from EMF corporation . 142

4.1 Reaction of a monomer with an amine-terminated monolayer 150

4.2 Capping of unreacted surface amines after reaction of a monomer with an

4.3

4.4

amine-terminated monolayer

Possible coupling efficiency (Ecn ) profiles.

Schematic of a 4 electrode array

XXVI

152

153

. 155

List of Schemes

2.1 Synthesis of mixed benzoic acid anhydrides . . . . . . . . . . . . . . . . . ., 29

2.2 First retrosynthetic analysis of the aniline monomers 40

2.3 Protection of the acid terminus of 4-aminobenzoic acid . . . . . . . . . . . . . 40

2.4 Retrosynthetic approach to the aniline monomers involving a chloroformate

intermediate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 44

2.5 Synthetic scheme for the aniline series of monomers. . . . . . . . . . . . . . . 45

2.6 Formation of the carbonate byproduct in solution .. . 46

2.7 First retrosynthetic approach to the benzylamine monomers 63

2.8 Different ways to protect the acid terminal of benzylamine amino acids. 64

2.9 Synthesis of benzylamine carbamates via chloroformate intermediates 65

2.10 Second retrosynthetic approach to the benzylamine monomers. . . . . . . .. 66

2.11 Benzylamines . . .... 68

2.12 Reaction scheme for the synthesis of compound 38 via carbamate 32 . . . . . 74

2.13 Hydrogenolysis of benzyl ether 36 76

3.1 Synthesis of aniline-based anchors 111

xxvii

3.2 Retrosynthetic analysis of disulfide 55 . . . . . . . . . . . . . . . . . . 130

3.3 Synthetic scheme for the synthesis of N-Boc-1,6-diaminohexane (63) . 133

3.4 Synthetic scheme for the synthesis of 54 . 133

3.5 Synthetic scheme for the synthesis of 55

xxviii

. 135

Chapter 1

Introduction

1.1 Combinatorial Chemistry or Rational Design?

Chemistry has evolved from being an obscure science for a special few to a science that is

central to the society in which we live today. It is chemists that study, design and synthesise

the materials and molecules that are essential to our eveyday life. From plastics to complex

drugs, the influence of the chemist can be felt everywhere we look. Discoveries are not

always made just because we plan them, many of humanity's greatest breakthroughs have

been made by chance. Chemistry is no different and two main approaches now coexist for

the design of materials and molecules: rational design, which is a directed approach, and

combinatorial chemistry, which is a less targeted strategy.

1

CHAPTER 1. INTRODUCTION

a. b.

2

Figure 1.1: Two rationally designed drugs: a. Dorzolamide, a carbonic anhydrase inhibitorfor topical ophthalmic use. b. Imatinib, a tyrosine kinase inhibitor used to treat certaintypes of cancer (e.g. chronic myelogenous leukemia (CML), gastrointestinal stromal tumors(GISTs))

1.1.1 Rational Design

Traditionally, molecules and materials have been designed and synthesised with a solid

knowledge of their intended function. This approach is termed rational design. The phar-

maceutical industry, for instance, has been using this approach to discover and synthesise

new drugs for some time (Figure 1.1).1,2 Despite in silica approaches playing an ever greater

part in the rational design and structural refinement of new drugs,2 5 the rational approach

is not high throughput and designing and refining each possible candidate is still a lengthy

process.

With the number of possible organic molecules with a molecular weight less than 500

Daltons being estimated to be between 1060 and 10100 ,6 it is obviously a challenge to find

molecules having the most desirable properties fulfilling a certain need. There is conse-

quently a need to synthesise vast quantities of molecules in a more efficient manner to more

quickly identify lead compounds and also more speedily refine lead candidates. Combinato-

rial chemistry is one strategy that is now widely used to address this challenge.


1.1.2 Combinatorial Chemistry

3

General approach Combinatorial chemistry was first introduced in the 1980's7 and has

since emerged as an efficient way to quickly synthesise large numbers of molecules or mate

rials. In combinatorial chemistry, unlike in rational design, less emphasis is placed on the

details of the synthetic scheme and the absolute structure of the final products. Simple and

reliable reaction conditions are preferred and multiple reactant and reagent combinations are

used to yield a large variety of structurally diverse molecules or materials in a short period

of time. However, combinatorial techniques do not yield large amounts of final products,

rather focusing on providing a small amount of many different compounds for subsequent

screening. Despite this shortcoming the same library can be still be screened against many

potential targets thus maximising its usefulness. Both solution and solid phase variations

of combinatorial chemistry are commonly employed today (Figure 1.2).7

The introduction of solid supports in the form of resins by Merrifield in 19638 opened

the way to solid phase synthesis, notably, to solid phase combinatorial synthesis. By using

a solid support (generally polymer beads), it is easy to synthesise, handle and purify large

libraries. For these reasons, the solid phase approach is generally preferred over the solution

phase strategy. In addition to being used to generate traditional small molecules such

as lead enzyme inhibitors9 12 (Figure 1.4), solid phase combinatorial chemistry has been

successfully employed for the synthesis of biomolecules such as peptides and DNA.8,13,14

One of the most widely used solid phase techniques is the split and mix strategy in

which a pool of beads is split into several pots, each of which are then reacted with different


r=Cr-"O

---~---.----

-----,-CB

ASBO OA. ,'.' ~-.

a.

B~

...._-_ ..

-,--~,-

b.

4

Figure 1.2: Two general strategies for carrying out combinatorial chemistry: a. Solutionphase. b. Solid phase on beads.

reagents. 7 Afterwards, they are pooled together and randomly split again into separate

pools for a second round of reactions. This operation is repeated as many times as is

deemed necessary to generate the library of interest (Figure 1.3).

Solid phase techniques suffer from several drawbacks related to the solid support itself

and to the methodology. The supports are usually polymer beads which have to be insol-

uble in the reaction solvent but still need to be permeable enough for the solvent to reach

the inside of the bead and ensure delivery of the reactants and reagents inside the bead,

thereby guarantying uniform and maximum reaction accross the bead. Therefore, the choice


00000000

I II

I I

00 00 00 00-I I II I

-0 00-099 0

I

Figure 1.3: The split and mix strategy.

5

of solvents available for reactions is limited. As well, beads are usually polymerised with

their reactive groupa already present and hence limited accessibility of the reactive groups

attached to the polymer bead is a key issue that governs the sample loading of such supports.

"The reactive group is the group via which a building block is attached to the bead. e.g. for a Merrificlrlresin, it would be a benzyl chloride moiety.

CHAPTER 1. INTRODUCTION 6

a.

o

b. c.

Figure 1.4: Enzyme inhibitors designed using combinatorial chemistry. a. 3-(Amidoalkyl)and 3-(aminoalkyl)-2-arylindole derivatives. 12 b. Dysidiolide-derived phosphatase inhibitor. lO c. N-(Substituted)glycine peptoids.u

Screening of combinatorial libraries Screening combinatorial libraries is akin to screen-

ing mixtures; the mixture itself might be active but only a few components may be responsi-

ble for the observed activity. Identifying these components in a combinatorial mixture which

is composed of hundreds to millions of compounds requires a process known as deconvolu-

tion. Deconvolution of combinatorial libraries is defined as the method used to determine

the structural identity of the active compound in a given combinatorial library. When

screening large combinatorial mixtures there always exists the possibility that identification

of the active compound(s) will fail.15 In traditional combinatorial chemistry the choice of

pooling and deconvolution techniques will greatly influence the outcome of the screening

process. 15 It is therefore understandable that optimisation of the deconvolution step has

been the focus of intense research. 15 18 In order to track the reaction sequence used on a par-

ticular bead, combinatorial chemists often rely on the use of chemical tags which are added


with the reaction mixture at every step of the reaction sequence. 19 Deconvolution of such

libraries is cumbersome, often requiring the use of techniques such as HPLC, GCjMS, mass

spectromet ry16 or an iterative strategy that is limited to relatively small libraries « 1024

compounds).l8 Being able to spatially address a library makes the deconvolution process

easier by eliminating the randomness inherent to combinatorial libraries and so facilitating

deconvolution. Three common types of spatially addressable arrays are presented below.

1.2 Spatially addressable arrays for chemical synthesis

1.2.1 Well plates

Well plates are extensively used in the biological sciences and chemistry (Figure 1.5).20

Well plates have been used in combinatorial chemistry as a way of enabling two-dimensional

screening by spatially fixing the different reactions in a matrix format.2l Each well contains

a solution with the desired reactants and chemistry is carried out in the wells. Such a setup

is unsuitable for peptide synthesis and screening. A method called peptides-on-pins was

designed to alleviate this problem by immobilising the peptide chain.22 In this method, the

growing peptides are fixed on pins that are dipped in each individual well containing the

necessary reagents for peptide coupling. The peptides thus synthesised can then be probed

by a standard ELISA assay (Enzyme-Linked ImmunoSorbent Assay).


2 3 4 5 6 7 8 9 10 11 12

A 000 0000B 00000000000cOOOOOOOOOOOD 00000000000E 00000000000F 00000000000GOOOOOOOOOOOH 00000000000

Figure 1.5: A 96 well plate.

1.2.2 Photochemically addressable arrays

8

Photochemical methods have been used to prepare microarrays for several years and, more

recently, DNA and peptide microarrays have been prepared in this way.23 25 In the photo-

chemical approach, light is shone through a mask onto the array and the light-exposed area

undergoes photochemical modification (Figure 1.6). In this way reactive groups protected

by photocleavable groups can be selectively deprotected to generate predetermined patterns

(Figure 1.6). New molecules can be coupled onto the freed reactive groups and the cycle

repeated as many times as necessary (Figure 1.6).

1.2.3 Electrochemically addressable arrays

Electrochemically addressable arrays (EAA) have mainly been used for synthesising libraries

of small molecules and catalysts in solution26,28 32 and for patterning proteins onto elec-

trode surfaces.27,33,34 EAA's have seldom been used for the synthesis of small molecules or


hv

-!!H-~LG ~LG ~LG ~LG

NH NH NH NH~ ~ ~ ~

I

~LG

A~LG ~LG ~LG ~LG ( ~LG ~LG

-----;.~ NH NH2 NH NH -----l.~ NH NH NH NH~~ ~ ~ ~~~~

1 II....- ----J

jhv

--UU-..

~LG ~LG

A C~LG ( ~LG )NH NH NHHN

§ * * *

~LG

A~LG ( ~LG ~LGNH NH NH NH

* * § *I

Figure 1.6: A photochemically addressable array. The reaction sequence is shown.

a. b.

Figure 1.7: Spatially addressable electrochemical arrays. a. Arrays for solution phasesynthesis of small molecules.26 b. Arrays for immobilisation of biomolecules.27 Plain lines:functionalised. Dashed lines: non-functionalised


oligomers in solid phase. However, attempts have been made to use traditional chemistry

localised around an electrode35 38 but most of these approaches rely on the use of confining

agents to keep the electrochemically generated reagents in proximity to the electrode area.

The reaction times employed in these studies are very long (12 to 18 hours per coupling)

and, as such, these approaches are poorly suited for high throughput synthesis of combina

torial libraries. The approach taken by Egeland and coworkers is different since they use

an array of electrodes (primary surface) to pattern a secondary surface that carries amine

moieties protected by an acid labile group.39,40 Although this technique yields very well

defined patterns on the secondary surface, it relies on the electrochemical generation of acid

in the vicinity of the electrodes to selectively deprotect the area of interest. The acid thus

generated can lead to the degradation of the patterned surface in a matter of seconds. More

recently, Beyer et al. proposed a technique to alleviate the problems caused by the electro

chemical generation of acid as well as improve the coupling efficiency.41 They proposed to

deliver O-pentafluorophenyl esters of N-Fmoc-amino acids (Fmoc = (9H-fluoren-9-yl)methyl

carbamate) mixed in beads of N,N-diphenylformamide to specific sites of an array by guiding

the beads using electric fields. Once the particles have reached their intended targets, they

are all melted at once and subjected to standard Merrifield coupling conditions. Although

large libraries could be synthesised this way, the long coupling time and need for complex

machines to spray the beads are clear disadvantages of this method. Therefore, a real need

exists for simpler approaches to making large peptide libraries. The current limitations

and shortcomings of the techniques outlined above demonstrate the need to develop arrays


that do not use reagents such as acid or catalysts to effect amino acid coupling. We term

such arrays reagentless arrays. Indeed, no reagentless electrochemically addressable array

for chemical synthesis has been developed to date. This is especially critical in peptide

synthesis since some commonly used protecting groups are incompatible with the reagents

generated in the arrays mentioned in this paragraph and therefore degradation of the array

quality can ensue.37,39,40


1.3 Scope of the thesis

12

In this thesis will be presented the foundation work for the design of electrochemically

addressable reagentless arrays for the screening and deconvolution of oligomers. The main

focus of this thesis will be the design and synthesis of various molecules we believe to be

necessary for the development of such electrochemically addressable arrays.

1.3.1 Built-in deconvolution

Even though combinatorial chemistry enables the chemist to synthesise vast quantities of

molecules very quickly, it typically fails to satisfactorily address the issue of library decon

volution (Section 1.1.2). This issue is especially critical when synthesising molecules such

as peptides, since the composition and the precise order of the building-blocks are critical

pieces of information. Even the use of chemical tags in combinatorial chemistry does not

enable one to easily decode the order of the building-blocks in the final compound. By

fixing the various peptides (or oligomers) on a templated solid surface, such as an array of

gold electrodes, the order of addition of the building-blocks is controlled and known in real

time; effectively eliminating the need for further deconvolution of the array (Figure 1.8).

This feature of the array is termed built-in or spatial deconvolution. Indeed, as soon as the

array is probed and a positive response is identified, the precise sequence of the oligomer is

known. In figure 1.8, if oligomer 3 is found to be active, the ABA oligomer is immediately

identified as the active species; both AAB and BAA have been eliminated as possible

hits. The synthesis of large quantities of compounds is not the aim of this project (only


31

2

13

Figure 1.8: An electrochemically addressable array. 1. Oligomer AAB. 2. Oligomer BAA.3. Oligomer ABA.

nanomolar quantities of product can potentially be synthesised on a given electrode). The

focus is rather on developing a strategy for rapid synthesis, screening and deconvolution of

combinatorial libraries of oligomers allowing rapid identification of lead compounds.

1.3.2 High throughput, synthesis and efficiency

The array being developed here is conceived from the ground up with high throughput and

low material usage in mind. This means that each step (synthesis, deconvolution, surface

reaction, electrochemistry, etc... ) has to be as efficient and reliable as possible. This will be

reflected, in this particular thesis, in the evaluation of the utility of the materials themselves

and the development of effective syntheses to obtain these materials.

1.3.3 General Strategy

Traditional synthetic methods make use of the chemical properties of the reactants and

products (i.e. chemical reactivity, acidity, basicity, etc... ) in order to successfully effect a


a.

¢OR<±>JLQOR 0

1'-':::: i.1 I

ii. ¢ R10t

OR+~

OH 0 0

b.

60RQ1'-':::: iii. + R10tOR

~

OH OH

Figure 1.9: Transacylation. a. Electrochemical transacylation. i. -2 e- u. R 10H. b.traditional transacylation iii. R 10H, base.

transformation. Electrochemistry, on the other hand, makes use of the electronic properties

of the reactants (i.e. add or remove electrons to a molecule) to facilitate reactions. 42 45

For instance, by simply applying a potential across a solution of acylated hydroquinone,

it is possible to carry out a transacylation reaction under very mild conditions by making

use of the electrooxidation of hydroquinone42 (Figure 1.9). If electrochemistry were not

used, the very same reaction would typically require strongly basic conditions that could be

incompatible with other functionalities present on the product or the reactant.

Various electrochemically active groups have been used to protect various functional

groups such as amines, carboxylic acids and alcohols. 34 ,47 51 Hydroquinone has been suc-

cessfully used as an electrochemically cleavable protecting group for both amide27 ,33,34 and

carboxylic acid46 terminated self-assembled monolayers on gold but not amines (Figure

1.10). We propose to use hydroquinone as the basis for an electrochemically addressable

a.


oHN-'< 0

~N~-o-O~ S ·"H 0 , h OH

O~~O~

15

Figure 1.10: Hydroquinone as amide and carboxyl protecting group. a. Biotin is protectedby a hydroquinone diimide. Oxidation of the hydroquinone moiety frees the biotin whichis then available for binding by streptavidin.27 b. The carboxyl group is protected as ahydroquinone ester. Oxidation of the hydroquinone moiety frees the carboxylic acid whichis then free for further chemical modification, in this case attachement of a biotin moietyvia an amide linkage.46

reagentless electrode array for the synthesis, screening and deconvolution of oligomer li-

braries (Figure 1.11). The array we aim to generate will rely on the use of self-assembled

monolayers terminated with amine functionalities on gold electrodes (Section 1.3.5.2). In

this array, hydroquinone would be oxidised to quinone by applying a voltage to only the se-

lected electrodes. The resulting unstable intermediate is hydrolysed to give a carbamic acid

and 1,4-benzoquinone upon hydrolysis. The carbamic acid in turn decomposes rapidly to

generate CO2 and the free amine.45 ,52,53 The liberated amine is then available for reaction

with an activated acylating agent bearing a protected amine thus extending the oligomeric


chain. Since the new monomer can also be electrochemically deprotected, this cycle can, in

principle, be repeated as many times as necessary until the desired oligomer is obtained.

HO HO HO HO HO 0 HO HO

to oto to to be to toOJ

NH.1, .1, ~ .1, .1, 1. ~ oJ .1, JH

o NH 0 NH 0 NH 0 NH 0 NH NH 0 NH 0 NH

I ..L. ~ ..L. I r ..L. J. ..L. I I ..L. ..L. ..I. I

I ~

"h"h p"~ ~ 0,

.1, J )o NH 0 NH 0 NH

r ..L. ..L. ..I. I

Figure 1.11: Proposed oligomerisation scheme. Applied potential shown by twisted arrow.a. -2 e-. b. - Benzoquinone. c. - CO2 . d. Monomer, R = phenyl, alkyl.

1.3.4 Archiving of the combinatorial libraries

The amount of chemical data generated by combinatorial methods is immense and therefore

archiving of combinatorial libraries is essential if such techniques are to be used for cost- and

time-effective research and development. The method of generating combinatorial libraries

proposed in this thesis makes it possible to archive their output very easily. The spatial

resolution is a great tool to ease the archiving process as is the fact that the molecules in

the libraries will remain attached to metal surfaces. We estimate that a single 1 cm by

1 cm array of electrodes can support the synthesis of up to 10, 000 different compoundsb .4o

We envisage that such a system would be controlled by a computer54 56 which could then

bEstirnate based on electrodes 100 /-Lrn by 100 /-Lrn and 10 /-Lrn spacing between electrodes.


archive the data associated with the array (deprotectionjaddition sequences, cycle times,

deprotection voltages, addition of extra reagents, etc... ). Further discussion on these

details is beyond the scope of this thesis.

1.3.5 Design principles

1.3.5.1 Electrode design

The array will be composed of gold electrodes on an insulated medium spaced by a few

micrometers. Gold has been chosen since it forms oxide-free surfaces that are amenable to

self-assembled monolayer (SAM) formation. As well, the body of literature on self-assembled

monolayers on gold is vast which should facilitate our progress and aid interpretation of data

generated during research efforts.57 59 However, gold electrodes oxidise easily when poten-

tials in the vicinity of 1 V are used (Figure 1.12). This limit is important since it will play

a central role in the design of the electrochemically active monomers (see Sections 1.3.5.3

and 2.2) and the fatigueC behaviour of the final device. Fatigue is defined here as a com-

bination of electrode surface and self-assembled monolayer degradation. The composition

of the monolayer can also significantly influence the oxidation behaviour of the gold surface

(Figure 1.12) and as such will also playa key role in the overall performance of the device.

CFatigue is herein defined as the possible number of electrochemical cycles that one electrode can undergobefore the integrity of the device (electrode and SAM) is compromised.


1-amino-10-mercaptodecane monolayer

1.00.50.0

-~

-100 +--_----.----.->"'-----.--~-___,.Q.5

2iO 1-&re9dd

;ro-1-a-ri~1Q.1rercaptcxrore-cy.;tanine

1~

100

~- ~-

Pdential (V)

Figure 1.12: Effect of the differences in monolayer composition (1-amino-10mercaptodecane and cystamine SAMs, shown on the right) on the oxidation behaviourof a gold surface as reflected by cyclic voltammetry study. The data presented in this figureis actual experimental data. See Chapter 3.1 for a discussion on monolayer composition.Measurements done in 0.1 M sodium phosphate dibasic (pH 9.02). Potentials reportedagainst AgIAgCl13 M AgCI. Note: The cyclic voltammogram observed here for the baregold surface is reproducible when using this buffer and differs from that typically observedin strongly acidic solutions.

1.3.5.2 Monolayers for microarray design

The formation of self-assembled monolayers (SAMs) on metal substrates merely requires

that the metal be immersed in a millimolar solution of the desired organic molecule. 6o The

quality of the monolayers can easily be improved by a variety of techniques such as thermal

curing. 57 The ease of formation of self-assembled monolayers is a definite advantage in

the fabrication of microscopic arrays since it eliminates the need for careful microscopic

alignment typically required for the fabrication of such devices. 61 SAMs can easily be

modified both in composition and functionality (Section 3.1) making them ideally suited

for building addressable microarrays.27,:33,46,62 The monola.yers should a.llow for optimal

CHAPTERl. INTRODUCTION

~I

19

Figure 1.13: The two main types of electrochemically addressable arrays. a. With polymeroverlayer.37 b. Self-assembled monolayer-based array.

electron transfer from the electrode to the redox protecting group (Sections 2.2.2 and 3.3).

The efficiency of the electron transfer can be judged by the efficiency of the deprotection

of protected surface-bound amines. In other words, the rate of electron transfer will be

judged optimal within this system if all surface amines are found to be deprotected within

a reasonable time-frame « 1 min), without degradation of the monolayer.

To date, only peptides up to 10 monomers in length have been successfully synthesised

using an electrochemically addressable array using polymer overlayers as the solid phase.37

The use of suitably designed self-assembled monolayers as the solid phase may allow one to

overcome this limit by avoiding acid-catalysed degradation of the polymer overlayer used

in the previously mentioned microarrays during repeated electrochemical cycles (Figure

1.13).37 Several thiol containing molecules for self-assembled monolayers and preliminary

elecrochemical data will be presented (Section 3.4).


1.3.5.3 Monomer design

20

The new type of array being developed here requires a novel series of molecules (or building

blocks) to work with and therefore new amino acid based monomeric units were developed

for future use in the reagentless electrochemically addressable microarrays that we envision.

Two main series of monomers based on benzoic acid cores were designed. Their design

principles, synthesis and use will be presented in Chapter 2.

1.3.6 Critical evaluation of the array design

It is our belief that these arrays will ultimately be superior to previously available photo

chemical and electrochemical arrays. Our approach involves no reagents for oligomerisation

and provides this method with a clear advantage over any existing spatially addressable elec

trochemical or photochemical approach.24,25,37,41 As well, the use of monolayers gives us a

unique control over the performance of the device by allowing us to easily change the prop

erties of the self-assembled monolayers used on the surface of the array's electrodes (Section

1.3.5.2). By fixing the oligomers on an electrode surface, we can easily test those oligomers

by electrical feedback for folding or binding of an analyte of interest. Eventhough typical

electrode microarrays use electrodes 100 pm wide, we expect that further miniaturisation of

the electrode would be possible without significant degradation of the signal-to-noise ratio.

Variation in the shape of the electrodes may also help improve the signal-to-noise ratio.

However, the array design presented in this thesis does not allow for the synthesis of large


quantities of materials with at most less than a nanomole of oligomer per crn2 .d This implies

that if a suitable oligomer is found, traditional peptide chemistry will be required to scale

up the oligomer of interest.Even though the potential required for deprotection will be mild

(Section 2.2.2), electrochemical side-reactions cannot be ruled out. Furthermore, the high

concentration of amine nucleophiles at the surface may degrade the monomers in solution

(Chapter 4). This may be prevented by diluting the surface amines by simply modifying

the composition of the monolayers. Finally, incomplete deprotection of surface arnines may

result if the electron transfer from the electrode surface to the redox center at the tip of

the growing oligomer (Section 3.3.1) is not fast enough. This may lead to decreased yields

of oligomer and the formation of multiple length of oligorners on the same electrode. A full

discussion of the limitations touched upon above is beyond the scope of this thesis but these

issues should be addressed in the future.

dCalculated by assuming 25 A2 surface area per molecule givng 0.7 nmol.cm -2.

Chapter 2

Design and synthesis of monomers

Large libraries of oligomers can be synthesised for screening purposes using the approach

mentioned in Section 1.3. The novel approach described in this thesis requires suitable

monomeric units to be designed and synthesised. Since the surface of the arrays will display

both hydroxyl (present as part of the hydroquinone protecting group) and amine groups

(present after electrochemical deprotection), the reactivity of the monomers must be tuned

so as to enable selective discrimination of amines over alcohols (Figure 1.11).

2.1 General considerations

2.1.1 Unnatural ammo acids m the synthesis and design of protein-like

structures

Unnatural amino acids are non-genetically coded amino acids. They are useful for introduc

ing functionality into peptides that goes beyond that of the 20 commonly occuring natural

22

CHAPTER 2. DESIGN AND SYNTHESIS OF MONOMERS 23

~OHNH2

alanine

--<"'NH2HO-{

ovaline

O):0H HO 0 o3:H' ~~H'~I}, ..

o NNH2 Hphenylalanine proine OH

isoleucineleucine

HO~(OH~H2 tOH HO~S,HO~OH

H2N'"NH2 0 0 0 SH NH2

aspartic acid glutamic acid cysteine methionine

H ~H2

H2NI(N~OH

NH 0

arginine

~H2

H2N~(OH

o 0glutamine

HO~NH2NH2 0

asparagine

0HO~Q){oH ldOH- \ NH2 0 NH2 NH2

N HN NH2H histidine

lysine

tryptophan

OH

tOHH2N:('Q

~"NH'HO 0 ~ I OH HO H2N'"OH

tyrosine 0threonine

serine

Figure 2.1: The 20 commonly occurring natural a-amino acids.


amino acids (Figure 2.1). Given their close resemblance to the naturally occurring amino

acids and the possibility for introduction via biological incorporation or other methods,

a-amino acids bearing unnatural substituents (Figure 2.2) are usually used to expand the

repertoire of functional groups.63 Unnatural amino acids are not only used to introduce new

functionality but also to control the three-dimensional structure of the peptide in which they

are incorporated. ,a-amino acids and aromatic amino acids (Figure 2.2) have been used for

this purpose.63 ,a-amino acids are useful since they form unique and predictable secondary

structures differing from that formed by similar a-amino acids. They allow for more con

trolled studies of the folding properties of peptides.63 Pomerantz and coworkers64 have

suggested that ,a-peptide self-assembled monolayers provide a convenient path to "rational

engineering of surfaces in which chemical groups are presented in precise and predictable ar

rangements". Aromatic amino acids are usually employed to provide rigidity to the peptide

backbone.63,65,66 For instance, 3-aminobenzoic acid has been incorporated in the design of

cyclic phosphoester binders67 and artificial ion channels.65 Both classes of amino acids are

the building blocks of a large family of compounds termed foldamers (Figure 2.3).68 70 As

discussed above unnatural amino acids enable us to synthesise peptides tailored exactly to

our specific needs both in structure and in function. In this thesis we will be concerned with

aromatic unnatural amino acids.

Proteins are polymeric macromolecules made out of amino acid monomers that are highly

evolved to perform specific tasks. The precise arrangement of the amino acids both in se

quence and in space is what confers function to proteins. If one could quickly synthesise a


oOH

~yvO=~=O

HN

H2NlyO

OH

~,'R'H2N _1

o OH~O

HO

a-amino acids

H2N,~J.OHTV

y-amino acids

~OH

HO 0HO NH2

OH

sugar amino acids

J3-amino acids

t2~J.t) OH

Il-amino acids

Figure 2.2: Some unnatural amino acids.

large number of oligomers or polymers reminiscent of proteins but based on unnatural amino

acids that are soluble in organic solvents, a whole new class of catalytically efficient com-

pounds could be devised. The synthesis of unnatural oligomers has mainly been pursued by

traditional synthetic methods68 77 that, despite being conceptually simple, are cumbersome

and time consuming. Although such approaches are appropriate for the synthesis of limited

numbers of oligomers, generating and screening large libraries of compounds by this method

is impractical. The approach described in Section 1.3.3 could solve this problem although

it is not amenable to large scale synthesis. Many of the unnatural amino acids proposed for

making of such oligomers contain aromatic cores that provide a convenient synthetic lever

thanks to their ready availability and ease with which they can be chemically modified.

They also exhibit a host of interesting electronic properties owing to their structures. Most


a. b.

Figure 2.3: Examples of foldamers. a. A ,B-peptide. b. A foldamer containing 3aminobenzoic acid. 3-aminobenzoic acid bolded.

Figure 2.4: Two unnatural peptides containing 3-aminobenzoic acid.

importantly, for our particular application, they can conduct electricity. This property has

previously been exploited for the construction of molecular wires.78 88

2.1.2 Guiding principles for designing the synthetic routes

These syntheses were developed with scalability and possible industrial applications in mind.

In order to enable potentially interested parties to use this technology, and in the event that

the molecules to be used would not be commercially available, the following points were

considered as critical.

CHAPTER 2. DESIGN AND SYNTHESIS OF MONOMERS

• Simple chemistry applicable to the synthesis of many different compounds

• As few purification steps as possible (avoid chromatography)

• Scalability

• High yielding

• Diversity (one precursor, many final compounds)

27

In this chapter, the design and synthesis of the first generation of monomeric units will be

presented.

2.2 Rational design of the monomer

For the purpose of discussion, a monomer is defined as a single molecule based on an amino

acid core and comprising the following parts: a leaving group (LG), a core (eM) and

an electrochemically-cleavable protecting group (PGM). These monomeric units are highly

functionalised molecules sporting an activated carboxylic acid, a carbamate and a phenol

functionality as a minimum. The activated carboxylic acid allows for the attachment of

the monomers to the growing oligomers through reaction with free surface-bound amine

functionalities. The amine group of the monomer will be protected as a carbamate contain

ing hydroquinone (Section 2.2.2). The highly functionalised nature of the monomeric units

poses several synthetic challenges that will be described in the following sections.


Figure 2.5: General design of the monomeric units. Blue: leaving group, Black: core, Red:protecting group.

Figure 2.6: Desired reactivity of the monomeric unit with amine groups.

2.2.1 Selection of the leaving group

The leaving group (LG) will simply allow coupling to form covalent amide linkages between

the free surface-bound amines and the monomers in solution. Since the surface of the array

will display both hydroxyl and amine groups (Figure 1.10), the leaving group needs to be

chosen so as to enable complete selectivity for amines over alcohols. This selectivity is also

necessary to avoid cross reaction of the monomer with its own phenol moiety in solution.

In addition, the amine should react faster with the electrophilic center bearing the leaving

group than with the carbamate moiety in order to avoid undesirable cross reaction between

surface amines and the monomeric units (Figure 2.6). To this end, various mixed anhydrides

and reactive esters were investigated as potential leaving groups.


o d~~I-: OH + RJlCI triethylamine. I -: O~V CH2CI2, r.t. <'...,

Scheme 2.1: Synthesis of mixed benzoic acid anhydrides.

2.2.1.1 Mixed anhydrides as leaving groups

29

Influence of the steric bulk of the putative leaving group Anhydrides were the first

type of leaving group investigated. Even though anhydrides inherently offer two electrophilic

centers for attack it was hypothesised that adding a bulky group on the terminal end of

the anhydride (R) could hinder reactivity at the CO-b carbonyl center and therefore favour

attack at CO-a (Figure 2.7). In order to test this hypothesis, several anhydride derivatives of

benzoic acid were prepared by coupling of benzoic acid with the corresponding acid chlorides

in presence of triethylamine (Scheme 2.1). To prove the reactivity of these derivatives, 4-

bromoaniline was used since it has similar electronics and reactivity as the proposed aniline

containing monomers. In our hands no aminolysis of the adamantyl derivative could be

observed even in the presence of a large excess of bromoaniline. This result suggests that

adamantane may hinder attack at both the CO-a and CO-b carbonyl centers or the anilines

may simply not be nucleophilic enough to attack'under these conditions.

The isovaleryl mixed anhydride gave a similar result to the adamantyl derivative. How-

ever, the only coupling product of the reaction of benzoic pivalic anhydride with 4-bromoaniline

was identified by 1 H-NMR spectroscopy as N-(4-bromophenyl)pivalamide (Figure 2.8d) in-

stead of the expected N-(4-bromophenyl)benzamide (Figure 2.8b). In this example, benzoic


CO-a CO-b

R = Me, t-Bu. Ada,CH(CH2CH3n

a. b.

Figure 2.7: Mixed benzoic acid anhydrides. R = adamantyl, acetyl, t-butyl and isovaleryl.a. General structure of mixed anhydrides of benzoic acid. b. Possible products of mixedanhydride aminolysis.

acid (pKa 4.2) is seen to be a better leaving group than pivalic acid (pKa 5.1), consistant

with the ability of benzoic acid to stabilise a negative charge better than pivalic acid through

resonance structures. As well, it is clear that the t-butyl group is not bulky enough to direct

attack at the correct carbonyl center. It also suggests that if mixed anhydrides are to be

used as leaving groups, sterics cannot be the sole factor considered when designing a suitable

leaving group. Close attention must also be paid to the electronic factors at playas well to

ensure that the nucleophile and leaving group reactivities are matched.

Influence of the electronic structure of the putative leaving group Mixed an-

hydrides of benzoic acid and dichloro- (Figure 2.9a and 2.10) and trichlorobenzoic acid

(Figure 2.9b) were prepared as described above in order to study if improving the leaving

group ability of the putative leaving group would lead to the coupling of p-bromoaniline at


a.

cf°~b.

~yO, LLJI

j,

j i i i i i i i I I I9 6 7 6 5 4 2 10 8 6 4 2 0

c.

.D'\kd.

d 0• 1# 0 L

IJI I I I I I "0 I' , I I I

10 6 6 4 2 09 6 7 6 5 4 3 2

31

Figure 2.8: 1H-NMR of benzoic pivalic anhydride and its possible coupling products with4-bromoaniline. a. 1H-NMR of the benzoic pivalic anhydride in CDCI3 . b. Calculated 1HNMR of the coupling product of N-(4-bromophenyl)benzamide. c. Calculated IH-NMR ofN-(4-bromophenyl)pivalamide. d. 1H-NMR of the of the coupling product of 4-bromoanilineand benzoic pivalic anhydride in CDCb. Calculations performed using Chemdraw Ultra10.0.

the CO-a center as desired. Reaction of benzoic 2,4-dichlorobenzoic anhydride and benzoic

2,4,6-trichlorobenzoic anhydride with p-bromoaniline in methylene chloride was followed by

TLC (30/1; toluene / ethyl acetate). After leaving both reactions to proceed overnight a

new product having the same Rf was detected in both reactions. In the case of the reaction

of benzoic 2,4-dichlorobenzoic anhydride with p-bromoaniline, no benzoic acid release could

be observed by thin-layer chromatography, which hinted that attack of p-bromoaniline at the

CO-a center to form the desired N-(4-bromophenyl)benzamide product was occming (Fig-

me 2.11b). However, in the case of benzoic 2,4,6-trichlorobenzoic anhydride, benzoic acid

CHAPTER 2. DESIGN AND SYNTHESIS OF iVIONOMERS 32

a.

d'onCI CI

b.

Figure 2.9: Mixed anhydrides of benzoic acid and chlorinated benzoic acids. a. Benzoic2,4-dichlorobenzoic anhydride. b. Benzoic 2,4,6-trichlorobenzoic anhydride.

Figure 2.10: Electron density map of benzoic 2,4-dichlorobenzoic anhydride. Calculationdone using the ArguLab 4.0 soft-ware with UFF molecular mechanics set. Red = Highelectron density, White = low electron density.

release was observed in addition to the new product. These observations indicate either re-

action of p-bromoaniline is reacting at the CO-b center or the benzoic 2,4,6-trichlorobenzoic

anhydride is degrading during the reaction. To isolate the product both of the reaction

mixtures were combined and the common spot isolated using flash silica column chromatog-

raphy (9/1 toluene / ethyl acetate). The 1H-NMR spectrum of the isolated product (Figure

2.11e) was not conclusive and the use of anhydrides was abandoned.

In our hands, neither sterically shielding the CO-b center nor varying the electronic


I i I I10 ppm 8 10 ppm 8

c. 0 DB'CI~~

9.

I I10 ppm 8

d. 0 0

~,

I10

ppm

i8

I10

ppm

Figure 2.11: 1H-NMR spectra of benzoic dichloro- and trichlorobenzoic anhydrides andtheir reaction products. a. Calculated lH-NMR spectrum of N-(4-bromophenyl)benzamide.b. Calculated 1H-NMR spectrum of N-(4-bromophenyl)-2,4,6-trichlorobenzamide. c. Calculated 1H-NMR spectrum of N-(4-bromophenyl)-2,4-dichlorobenzamide. d. Calculated 1HNMR spectrum of benzoic 2,4-dichlorobenzoic acid. e. Experimental l H-NMR spectrum inCDCl3 of the isolated coupling products of the reactions between p-bromoaniline and benzoic 2,4-dichlorobenzoic anhydride and p-bromoaniline and benzoic 2,4,6-trichlorobenzoicacid.


a.

~ P R\J\S-ob. c.

Figure 2.12: Some reactive esters. a. 2,5-dioxopyrrolidin-l-yl benzoate. b. S-phenylbenzothioate. c. IH-benzo[d] [1,2,3]triazol-l-yl benzoate.

structure of the leaving group lead to the formation of the desired coupling product.

2.2.1.2 Reactive esters as leaving groups

Reactive esters were synthesised in order to address the shortcomings of the mixed anhy-

drides discussed in Section 2.2.1.1. Reactive esters have the advantage of offering only a

single centre for reaction with amines (Figure 2.12) and as such, only one amide coupling

product is expected. However, the selectivity of the ester must still be tuned so as to favour

reaction with amines over alcohols.

Thioesters (Figure 2.12b) are known to be highly reactive towards amines and have been

used for peptide ligations.89 However, they were quickly dismissed since the liberated thiol

could subsequently displace the molecules adsorbed on the surface of the gold electrode

which would lead to degradation of the monolayer and eventually failure of the device

(Figure 2.13).

Both N-hydroxysuccinimide (Figure 2.12a) and N-hydroxybenzotriazole (HOBt, Figure

2.12c) esters have also been used as reactive intermediates for peptide coupling90 93 and were

considered as potential leaving groups. Although under the coupling conditions envisaged


-Figure 2.13: Thiol displacement at the gold surface. 4-aminothiophenol is replaced bythe thiophenol released from the reaction of S-phenyl benzothioate with a neighbouring4-aminothiophenol monolayer component.

(coupling of primary amines in the presence of phenol groups) N-hydroxysuccinimide could

be an appropriate choice, N-hydroxybenzotriazole was chosen since it has been shown that

its benzoate ester derivatives can be reacted with anilines in solution and are completely

inert to phenols unless triethylamine is added to the reaction mixture.94

2.2.2 The protecting group

The protecting group is used to restrict reactivity of amines bound to the growing oligomer

chain. Using an electrochemically labile protecting group facilitates the generation of a spa-

tially addressable array in the following way. Reaction of both surface-bound and solution

phase protected amines with electrophiles is prevented until an electrical potential is applied

and the resulting benzoquinone is hydrolysed off of the amine (Figure 1.10). Once the nu-

cleophilic amine group on the monolayer is unmasked, its reaction with monomers bearing

reactive N-hydroxybenzotriazole esters in the solution phase should allow extension of the

oligomer chain via formation of amide bonds as explained in Section 1.3.3 (Figure 1.11).


Repetition of this sequence should permit further extension of the oligomer chain. The

hydroquinone protecting group can be cleanly and efficently removed under mild electro

chemical conditions (low potential). Although a host of electrochemically labile protecting

groups such as nosyl and tosyl groups have been used to protect amines, the electrochemical

cleavage of such groups can lead to the formation of side products. 50,51 The electrochem

ically cleavable cinnamyloxycarbonyl group has been used to protect amines,49 however,

the high negative potentials « -2.45 V) required for deprotection can potentially lead to

undesirable side products. The hydroquinone protecting group has been exploited by Kwak

and coworkers to protect the urea group of biotin so as to make an addressable array for

binding of streptavidin (a biotin binding protein).27 The electrochemical behaviour of hy

droquinone has been widely studied in a wide variety of solvents27,95 100 and its oxidation

potential was found to be low (0.5 V in aqueous solution). Similar to the cinnamyl group,

amines can be protected as hydroquinone carbamates. Given the suggestive results of Kwak

et ai. 27 and the well established electrochemistry of hydroquinone, the hydroquinone pro

tecting group was chosen as the electrochemically labile group to protect the primary amine

as a carbamate (Figure 2.14).

2.2.3 The core

The core of the monomeric units is key since it is the part of the monomer that will constitute

the main part of the oligomer after electrodeprotection (loss of the hydroquinone moiety

and liberation of the amine), coupling (cleavage of the reactive ester) and formation of the

amide linkage. It is the core that will make oligomers unique in sequence and confer their


H

Q0

o=(

fNH

~1 1a. b.

37

Figure 2.14: Hydroquinone as a protecting group for amines. a. Free amine. The amine canreact with HOBt esters and growth of the oligomer chain is possible. b. Amine protectedas a carbamate of hydroquinone. The amine is blocked and can not react with reactiveesters; no growth of the oligomer chain is possible. The carbamate is shown in red and thehydroquinone in bold.

functional properties. Unnatural amino acids based on benzoic acid cores were chosen since

the problems associated with traditional peptide coupling, such as loss of configuration at

the a-carbon, are non-existent for these systems.91 Benzoic acid derivatives, or appropriate

precursors, are also commercially available and can be modified using established chemistry.

Furthermore, we were encouraged in our approach by the previous work done using aromatic

thiols as molecular wires since it would be beneficial for chain extension if these oligomers

could conduct electrons from the surface to the electrochemically labile protecting group at

the terminus of the oligomer. 79 ,86,87,101,102 The combination of all these factors convinced us

that benzoic acid based amino acids were good candidates as building blocks for reagentless

electrochemically addressable arrays. Both aniline- and benzylamine-containing monomers

were synthesised.


~NH2 ~NH2

HO~ HO~o 0

~NH2H0nYo CI

F»"'" NH2

HO 1.0-F

o FH

NH2

HO I b'1o OH

/?CC"'"NH2

HO I bCI

o

Figure 2.15: Some commercially available aminobenzoic acids.

2.3 Amino benzoic acid series

Amino benzoic acids are attractive starting materials since many are commercially available

(Figure 2.15) and they can undergo facile chemical transformations (electrophilic aromatic

substitution and nucleophilic aromatic substitution) allowing further diversification. Fur-

thermore, since the synthesis of oligomers we envision requires electrochemical deprotection

of the growing amino terminus, which extends away from the electrode surface, it is desirable

that the monomers we design can conduct electricity. Aromatic compounds can conduct

electricity owing to their molecular structure, a property that has previously been exploited

to construct molecular wires. 78 88 Oligomers composed of aminobenzoic acids are expected


Figure 2.16: An oligomer of 3-aminobenzoic acid. The extended conjugation is shown inbold.

to efficiently conduct current due to their extended conjugation (Figure 2.16) and are there-

fore expected to allow for efficient electron transfer (Section 3.3.1). Aminobenzoic acids

are therefore good candidates as starting materials for the synthesis of monomeric units for

electrochemically promoted oligomerisation. In this section the approach to the synthesis

of such monomers will be presented.

2.3.1 First approach

The critical step in making any of the monomers depicted in this thesis is the formation of

the carbamate moiety. Two main ways of doing so are via formation of either an isocyanate

intermediate or a chloroformate intermediate. In this section, we will be concerned with the

use of an isocyanate intermediate, which we term the isocyanate approach.

2.3.1.1 Retrosynthetic analysis and general synthetic scheme

Monomer 16 could be obtained from the deprotection of 7 followed by coupling with N-

hydroxybenzotriazole (Scheme 2.2). 7 could be synthesised from isocyanate 56 which in

turn could be obtained from sHyl ester 4.


Q~~,N

z===?9~0

?"I~

OH

16

I .......~O-Sl

)=1'04-HN;;0 ===>h\=( 1+

O-S~

7 56 4

Scheme 2.2: First retrosynthetic analysis of the aniline monomers.

a. ..

Scheme 2.3: Protection of the acid terminus of 4-aminobenzoic acid. a. Methylene chloride,morpholine, TBDMS-CI, O°C.

Protection of the C-terminus In solution the predominant form of aminobenzoic acid

derivatives is a zwitterionic species, which is insoluble in most aprotic organic solvents

(Scheme 2.3). To fully solubilise these amino acids in methylene chloride at subzero tempera-

tures, protection at the C-terminus of the aminobenzoic acid derivatives using t-butyldimethylsilyl

chloride was carried out. This protection also ensured that the acid terminus could not react

with triphosgene.103

tert-Butyldimethylsilyl 3-aminobenzoate 4 was synthesised by modification of a litera-

ture procedure developed for preparing related compounds. 104 Reaction of 4-aminobenzoic


acid (1 equiv.) with tert-butyldimethylsilyl chloride (1.2 equiv.) in the presence of mor

pholine (2.2 equiv.) in methylene chloride at O°C for 18 hours, followed by extraction of

the reaction mixture with water and brine, afforded 4 in excellent yield (Table 2.1). The

identity of silyl ester 4 was established by IH-NMR, 13C-NMR and FT-IR spectroscopy.

Using this procedure, however, a small amount of tert-butyldimethylsilanol byproduct aris

ing from the decomposition of 4 and/or hydrolysis of tert-butyldimethylsilyl chloride was

consistently observed in the isolated material. 4 was not purified further, however, since

its presence was not found to be deleterious to the synthesis of 7 and the silanol byproduct

could easily be removed after subsequent steps. A series of C-protected amino acids was

made and is presented in Table 2.1. The present discussion will focus on molecules derived

from compounds 3-5 with an emphasis on compounds derived from 4. With 4 in hand, we

could now proceed with synthesising carbamate 7.

Attempted synthesis of carbamate 7 The synthesis of carbamate 7 was first at

tempted via isocyanate intermediate 56. 56 was synthesised by adding a solution of 4

(1 equiv.) in methylene chloride to a solution of triphosgene (0.33 equiv.) and triethy

lamine (2.3 equiv.) in methylene chloride at O°C (Figure 2.17). With isocyanate 56 in hand

we attempted the synthesis of carbamate 7 by in-situ dropwise addition of a solution of

4-(tert-butyldimethylsilyloxy)phenol (1, 1 equiv.) and triethylamine (1.1 equiv.) in methy

lene chloride to a solution of 56 in methylene chloride. Surprisingly however, addition of

two molecules of 1 to 56 was observed and compound 57 was obtained instead of the desired

product (Figure 2.18). In an effort to circumvent this problem, 1 was added in the absence


R,

H'N~~~ R3

4

# R1 R2 R3 R4 % producta % SiOHa yield (%)

3 H H eOOH H 95 5 87

4 H eOOH H H 99 92

5 OMe H eOOH H 92 8 96

60 Me eOOH H H 85 15 90

61 el H eOOH OMe 83 17 87

62 Me H eOOH H 88 12 63

a. ratio of isolated matarials datermined by NMR.

Table 2.1: Protection of the carboxylic acid group of aminobenzoic acids.

42

,/ / 0/fi'O~NH2 triphosgen;

I triethylamineo

4

,/ / 0/fi'O~N=C=O •V triethylamine

56

Figure 2.17: Synthetic scheme for the synthesis of carbamate 7.

of base, however, formation of 7 was still not observed. Consequently, the synthesis of 7

was attempted via a route involving a chloroformate intermediate.

2.3.2 Second approach

As explained in Section 2.3.1, the critical step in making the monomers is the formation

of the carbamate moiety. In this section, we will be concerned with making this functional

group via a chloroformate intermediate.


H13 HI

43

1.00 0.50

Solvent peak

0.00

H5

o ~~5HI/I"" I( I ""'jSi'OV 0 H7 # H9QH10

/ H4 H5 IH2 H3 .0 Hll

57 O'S/~/ '/ .......

H12 H13

Acetone I Acetic acid

I10.0

Impurity

I5.0

Figure 2.18: IH-NMR of undesired byproduct 57 in CDCb at 400 MHz. Top inset: Expansion of the 1.2 to 0 ppm region. Bottom inset: Expansion of the 8.5 to 6.5 ppm region.

2.3.2.1 Retrosynthetic analysis and general synthetic scheme (Scheme 2.4)

Aniline monomer 16 could be formed by coupling of N-hydroxybenzotriazole to the free

benzoic acid derivative 13 which, in turn, could be obtained by catalysed acid hydrolysis of

the silyl protecting groups off from compound 7. Reaction of the chloroformate intermediate

2 with tert-butyldimethylsilyl-4-aminobenzoate 4 would afford 7. Finally, chloroformate 2

could be obtained from the reaction of alcohol 1 with an appropriate phosgene equivalent.

With their synthetic scheme in mind the aniline series of monomers was prepared as


r(YO[(~y~ ~ 0 I ~

HO

HO 0

13

2

44

Scheme 2.4: Retrosynthetic approach to the aniline monomers involving a chloroformateintermediate.

outlined in Scheme 2.5. The following discussion will focus on the synthesis of compound 16.

All compounds in the aniline series of monomers (Table 2.3) were synthesised by following

the same procedure.

2.3.2.2 Synthesis of the chloroformate intermediate (2)

All reactions were conducted using high-dilution of reactants in dry methylene chloride at

sub-zero temperatures (Figure 2.19). This choice ofreaction conditions stems from the high

reactivity of triphosgene toward hydroxyl groups and the need to avoid self-condensation of

1. The reactions were performed using basic conditions in order to neutralise any hydrochlo-

ric acid released by the reaction of triphosgene with alcohol 1 that could result in the loss of

the silyl protecting group. The reaction was carried out by dropwise addition of a solution

of 1 to a solution of triphosgene and an appropriate base using an apparatus like the one

shown in Figure 2.19. Attempts to isolate the chlorofomate intermediate were unsuccessful


O OH TBDMS0'QI a. ITBDMSO h- - ~ oJlCI

1

~OH

N8t- () ~ • e.~N 0

o H16

b.

2

r91r0H

NHO () ~ • d.

~~ 0o

13 9

Scheme 2.5: Synthetic scheme for the aniline series of monomers. a. N2, dry CH2Ch,triphosgene, pyridine. b. 4, r.t.. c. TFA, MeOH. d. 1% HCl, CH3CN. e. HOBt, EDC·HCl,dry CH3CN.

and it was decided to react 2 in-situ with the appropriate amino acid derivative. Both the

base used and the temperature at which the reaction was performed were found to have

profound effects on the yield of the desired compound 7.

Effect of the base on the formation of chloroformate (2) The successful formation

of chloroformate 2 was gauged by the isolated yield of carbamates 7 or 9. Synthesis of 2 was

first attemped using triethylamine (pKa 11) as base. At -78°C the phenolate species formed

by the deprotonation of 1 appeared to react instantaneously with triphosgene, as determined

by 1H-NMR spectroscopy, to form the carbonate byproduct 20. Furthermore, addition

of triethylamine to either the solution of alcohol 1 or to the triphosgene solution prior

to dropwise mixing yielded exclusively 20 (Scheme 2.6). The formation of this undesired

material was observed over a wide range of temperatures ranging from -78°C to O°C (Table


Alcohol

46

Triphosgene

Figure 2.19: Setup for high dilution reactions.

..

+ +-~- -~~

o~ 9 ~o

~AJJo 0

20

1+ 2 rN.HC1

~

Scheme 2.6: Formation of the carbonate byproduct in solution

2.2).

Unfortunately, carbonate 20 did not react further with the protected amino acid 4

even at elevated temperatures of up to 50°C. The use of the weaker base pyridine (pKa

5.3) in place of triethylamine, however, was found to diminish the formation of 20 and

appeared instead to favour the formation of the desired chloroformate 2. However, the yield

of the carbonate byproduct was still high (29%) and therefore optimisation by altering the

R-N

H2

trip

hosg

ene

base

yiel

d(%

)c

exp

t#eq

uiv.

cone

.(m

M)

equi

v.co

ne.

(mM

)eq

uiv.

cone

.(m

M)

equi

v.co

ne.

(mM

)na

me

tem

p.(O

C)

time

(h)

OC

ON

H2

20O

the

r

18

188

1.2

510.

43

84

351

trie

thyl

amin

e0

18-

79

b2

15

21.

16

00.

430

3.6

25

2tr

ieth

ylam

ine

oto

20

48

N/A

4

3b

138

2.3

47

1.2

46

6.2

26

0py

ridi

ne-7

82

416

13

4b

14

02.

244

1.2

487.

62

40

pyri

dine

-78

1815

NlA

25

Sb

111

41

560.

44

23.

336

pyri

dine

-68

841

(34

29(2

9)

NlA

(37)

6b

118

01

78

0.4

642.

327

9py

ridi

ne-2

02

4SO

dN

lA

781

18

01

78

0.4

136

2.3

841

pyri

dine

-17

186

2e

8.R

-NH

2=

3b.

R-N

H,

=4

c.yi

eld

(%o

ftot

alw

eig

hto

fiso

late

dco

mp

ou

nd

)d

.9e.

10

N/A

:n

ota

vaila

ble

or

notm

easu

red

Tab

le2.

2:E

ffec

tof

the

tem

pera

ture

,co

ncen

trat

ion

and

base

onth

eyi

eld

ofis

olat

edca

rbam

ate.

Th

eco

ncen

trat

ions

stat

edar

eth

ein

itia

lco

ncen

trat

ions

prio

rto

drop

wis

em

ixin

g.O

CO

NH

2re

fers

toth

eca

rbam

ate

coup

ling

prod

uct,

eith

er6

for

R-N

H2

=3

or7

for

R-N

H2

=4.

@ ~ "tl ~ ::0 ~ t1 ~ < ~ @ CJ':J ~ ~ tri CJ':J (jj ~ a < o ~ ~ Cf:J

,j::o

.-.:

J


temperature was attempted.

48

Effect of the temperature on the formation of chloroformate 2 When the reaction

was performed at -78°C, it was observed that the yield of the final compound 7 was lower

than at -20°C and concomitantly, the yield of 20 increased. Notably, at -78°C a yellow

solid was observed to accumulate in solution over time. This solid was not isolated but

its observation is consistent with the formation and known solubility of phosgene-pyridine

complexes in methylene chloride.1°5 This phosgene adduct contains two pyridines bound

through a C2-N bound (Figure 2.20a); the adduct undergoes reversion to its components

at temperatures in excess of -30°C. This solid was suggested to have similar reactivity

as phosgene itself.1°5 At lower temperature (-78°C) the adduct in Figure 2.20 is the re

active species whereas at higher temperature (-20°C) triphosgene is the reactive species.

The higher yields of carbonate observed at the lower temperature (Table 2.2) lead us to

hypothesise that the phosgene-pyridine adduct is more reactive than triphosgene alone and

therefore all subsequent reactions were carried out at -20°C.

2.3.2.3 Synthesis of the carbamate 13

With chloroformate 2 in hand, we proceeded to synthesise carbamate 7. As explained

in Section 2.3.2.2, 2 was not isolated and tert-butyldimethylsilyl 3-aminobenzoate 4 (1.0

equiv.) was reacted in-situ with 2 (1.0 equiv.) in the presence of pyridine (1.0 equiv.)

to afford 7. Carbamate 7 could not be reliably purified by flash column chromatography

since it degraded to its free-acid derivative 9 during chromatography. We surmise that the


a.

b. Jl ~O"'N CI + ~ J

.0 CI- N

o

a~CI_

eCI'I ~

Figure 2.20: Structure of the phosgene-pyridine complex at various temperatures in methylene chloride. a. Structure of the phosgene-pyridine complex at T < -30D e. b. Equilibriumof the phosgene-pyridine complex at T > -30 De.

silica gel is acidic enough to hydrolyse the labile tert-butyldimethylsilyl ester of 7. The

presence of the carbamate moiety precluded the use of basic conditions such as potassium

fluoride and tetrabutylamonium fluoride to remove both silyl groups. It is known, however,

that N-arylcarbamates are not as sensitive to acidic conditions and therefore acid-catalysed

deprotection of the silyl ester and silyl ether was investigated.45

The labile tert-butyldimethylsilyl ester of 7 was cleaved by reacting 7 (1.0 equiv.) with

trifluoroacetic acid (10% vIv) in methanol at room temperature overnight. This cleanly

afforded carboxylic acid 9 in 60% yield over three steps after silca gel flash column chro-

matography. Notably, under these deprotection conditions, transesterification to form the

methyl ester of 9 was not observed. Finally, reaction of 9 with a catalytic amount of

hydrochloric acid in acetonitrile cleanly afforded carbamate 13 in 95% yield.


f)°"~UR'I b 0 I bHO R3

EDC.HCI HOSt# R1 R2 R3 (equiv.) (equiv.) yield (%)

15 H H COOSt 1.67 1.15 60

16 H COOSt H 1.08 1.00 97

17 OMe H COOSt 1.55 1.05 71

Table 2.3: Synthesis of aniline-based monomers.

2.3.2.4 Coupling of N-hydroxybenzotriazole (HOBt)-Synthesis of 16

50

With carbamate 13 in hand, we proceeded with the synthesis of monomer 16. Several

coupling agents were considered for making the activated ester. The widely used dicy-

clohexylcarbodiimide (DCC) dehydrating agent was first considered. However, dicyclo-

hexyl urea (DCD) was found to consistently contaminate 16 in significant amounts and

was difficult to remove. Reacting N-hydroxybenzotriazole (1.1 equiv.) and benzoic acid

13 (1 equiv.) in dry acetonitrile in the presence of the dehydrating agent l-Ethyl-3-[3-

dimethylaminopropyl]carbodiimide hydrochloride (EDC·HCI, 1.4 equiv.) cleanly afforded,

after aqueous workup, 16 in 97% yield from 13 or 55% overall yield from 1 (Table 2.3).

2.3.2.5 Testing the reactivity of the aniline monomers to aniline nucleophiles

The monomers synthesised thus far were tested for reactivity with various amines.

Reaction of aniline monomers with aniline derivatives Monomer 15 was reacted

with a variety of aniline derivatives at room temperature in tetrahydrofuran (Figure 2.21).


Figure 2.21: Aniline derivatives used to test 15.

No reaction was observed and no coupling product was isolated regardless of which aniline

was used as nucleophile. The addition of pyridine (1 equiv.) was not found to help the

reaction to proceed. This result can be rationalised by the low pKa and consequently low

nucleophilicity of aniline derivatives stemming from the delocalisation of the lone pair on

the nitrogen atom of the phenyl ring. N-methylpyrrolidone was used previously with great

success as a solvent for this type of reaction94 and was therefore tried here but no coupling

products could be isolated. As a consequence of these shortcomings in desired reactivity

this series of molecules are not ideally suited for use as components of the oligomers for the

electrochemical array strategy. They can, however, be used as capping agents in order to

terminate the oligomers and prevent further growth and could prove useful in this regard

to generate titrable functional groups at the end of the oligomer. For our purpose, however,

amino benzoic acids are not adequate monomers and therefore benzylamines were tested as

potential nucleophiles.

Reaction of aniline monomers with benzylamine derivatives Benzylamines do not

suffer from the resonance problems that afflict anilines (pKa 9.3) and are therefore much

better nucleophiles. The efficient reaction of (R)-l-phenylethanamine with 15 in tetrahy-

drofuran at room temperature shows that benzylamines are nucleophilic enough to couple


8 2 10 11

3

7

DMSO

4

16

12

T ~,,-- T T ITT T ~,

~ ~ 9 5 B ~ 5i , , , i i , , i , i i i i I I

10.1 10.2 10 •.s ,.• ,.. '.2 U L6 ~. '.2 7.' 7•• 7.• 7.2 ...U(ppm)

5

1T I~

Figure 2.22: 1H-NMR in DMSO-d6 of the coupling product of the reaction between (R)I-phenylethanamine with aniline-based monomer 15.

with N-hydroxybenzotriazole esters (Figure 2.22). The reaction was complete in 1.5 hours

at room temperature and the carbamate moiety was not cleaved under these conditions.

Conclusion Therefore, while the aniline monomers cannot likely be used in the reagentless

electrochemical strategy they could be used as capping agents to terminate the growth

of the oligomer and thereby generate titrable surface amines. Furthermore, the observed

coupling of (R)-l-phenylethanamine with 15 suggested that benzylamine derivatives would

be much more useful for reagentless electrochemical arrays since they have more appropriate


reactivity.

2.3.3 Experimental

2.3.3.1 Solvents and chemicals

53

All solvents were obtained from Caledon and used without purification except for triethy

lamine, pyridine and tetrahydrofuran which were obtained from Anachemia and distilled be

fore use. All deuterated solvents were obtained from Cambridge Isotope Laboratories, Inc..

Silica gel 60 was obtained from EMD (product # 9385 - 3). Trifluoroacetic acid and hydro

quinone were obtained from Sigma-Aldrich. l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide

hydrochloride was obtained from Fluka. All other chemicals were obtained from Aldrich.

All chemicals were used without purification.

2.3.3.2 Characterisation

All FT-IR measurements of molecules were made on a Bomem M-B series spectrometer. All

molecules were measured as a KBr pellet or as films deposited from chloroform on a sodium

chloride disc. Each spectrum is the average of 32 or 64 measurements. NMR spectra eH

and 13C) were acquired on a Varian Inova 500 MHz spectrometer or a Brueker Avance 600

MHz spectrometer. Melting points were taken using a Electrothermal Mel Temp@ melting

point apparatus and were not corrected. CHN elemental analysis was performed on a Carlo

Erba Model 1106 CHN analyzer at Simon Fraser University.


4-(tert-butyldimethylsilyloxy)phenol (1) 1 was synthesised using a modified litera

ture procedure. 106 A solution of tert-butyl-dimethylsilyl chloride (2.92 g, 19.4 mmol, 1.07

equiv.) in dry methylene chloride (20 ml) was added dropwise to a stirred solution of hydro

quinone (1.99 g, 18.1 mmol, 1 equiv.) and imidazole (2.68 g, 39.3 mmol, 2.17 equiv.) in dry

methylene chloride (35 ml) at O°C. The resulting suspension changed colour from colourless

to pink and was stirred overnight. The reaction mixture was washed with water (3x25 ml)

and the aqueous layer back-extracted with methylene chloride (3x25 ml). The combined

organic extracts were dried over sodium sulphate and the solvent removed in-vacuo. The

residue was purified by flash column chromatography (8/1 Hexanes / Ethyl acetate) to af

ford a colourless oil that crystallised upon standing (1.66 g, 41%). IH-NMR (CDCl3, 400

MHz) J (ppm) 6.70 (m, 4H), 0.97 (s, 9H), 0.16 (s, 6H); m.p. 53-55°C, lit. 56-57°C.

General Procedure for the Synthesis of tert-butyldimethylsilyl aminobenzoates

To a solution of tert-butylsimethylsilyl chloride (2.03 g, 13.5 mmol, 1.0 equiv.) and mor

pholine (2.98 g, 34.2 mmol, 2.53 equiv.) in dry methylene chloride (40 ml) at O°C was

added 4-aminobenzoic acid (2.15 g, 15.7 mmol, 1.16 equiv.). The reaction mixture was

stirred overnight, the volume made up to 100 ml with methylene chloride and washed with

water (3 x 35 ml). The organic layer was dried over sodium sulfate and the solvent removed

in-vacuo to yield a white solid in yields ranging from 84 to 92 %.

tert-butyldimethylsilyl 4-aminobenzoate (3) 1H-NMR (400 MHz, CDCl3) J (ppm)

7.85 (ABq, J = 8.8 Hz, 2H), 6.65 (ABq, J = 8.8 Hz, 2H), 1.01 (s, 9H), 0.35 (s, 6H); 13C-NMR

(100 MHz, CDCl3) J (ppm) 166.68, 150.79, 132.20, 121.12, 113.73, 25.71, 17.82, -4.68; IR


(KBr) 1915.68 cm-i , 1669.27 cm-i (C=O).

55

tert-butyldimethylsilyl 3-aminobenzoate (4) 1H-NMR (400 MHz, CDC13) 6 (ppm)

7.43 (dt, J= 8, 1.2 Hz, 1H), 7.34 (m, 1H), 7.21 (t, J=8 Hz, 1H), 6.86 (ddd, J= 7.9, 2.4,0.8

Hz, 3H), 1.02 (s, 9H), 0.36 (s, 6H); i3C-NMR (100 MHz, CDC13) 6 (ppm) 191.51, 129.19,

120.38, 119.41, 116.26, 25.68, -4.77; IR (KBr) 1669.27 cm-i (C=O).

tert-butyldimethylsilyI4-amino-3-methoxybenzoate (5) iH-NMR (500 MHz, CDCI3)

6 (ppm) 7.48 (dd, 1H, J =8,2 Hz), 7.40 (s, 1H, J = 2 HZ), 6.79 (d, 1H, J = 8.5 Hz), 3.91

(s, 3H), 1.01 (s, 9H), 0.35 (s, 6H); m.p. 143-146°C.

(tert-butyldimethylsilyI 3- ((4- (tert-butyldimethylsilyloxy)phenoxy)-

carbonylamino)benzoate (7) To a solution of 1 (2.01 g, 8.98 mmol, 1.0 equiv.) in dry

methylene chloride (120 ml) was added dropwise to a solution of triphosgene (1.01 g, 3.41

mmol, 0.38 equiv.) in dry methylene chloride (80 ml) with dry pyridine (1.7 ml, 21.0 mmol,

2.34 equiv.) at -10°C. The reaction mixture was allowed to warm up to room temperature

and a solution of 13 (2.36 g, 9.39 mmol, 1.05 equiv.) in dry methylene chloride (100 ml) was

added dropwise at room temperature. The reaction mixture was washed with 1% aqueous

hydrochloric acid, water and brine (50 ml each). The organic layer was dried over sodium.sulfate and the solvent removed in-vacuo to yield an orange oil (8.9 g, quantitative) which

was used without purification. iH-NMR (400 MHz, CDC13) 6 (ppm) 7.89 (br. s, 2H), 7.77

(dt, J = 4,1.2 Hz, 1H), 7.42 (t, J = 8 Hz, 1H), 7.07 (br. s, 1H), 7.04 (ABq, J = 8.2 Hz, 2H),

6.83 (ABq, J = 8.2 Hz, 2H), 1.02 (s, 9H), 0.98 (s, 9H), 0.38 (s, 6H), 0.20 (s, 6H); i3C-NMR


(100 MHz, CDCI3) 6 (ppm) 166.03, 153.31, 144.39, 137.67, 132.49, 129.34, 125.44, 122.30,

120.53,25.69,28.65,18.17,17.86, -4.47, -4.76; IR (KBr) 1748.4 cm-1 (C=O), 1547.6 cm-1

(HQ); E.A. Calcd. C 62.24, H 7.83, N 2.79, Expt. C 62.42, H 7.89, N 2.54; m.p. 97-98°C.

General Procedure for the Synthesis of (tert-butyldimethylsilyloxy)phenoxy)

carbonylamino)benzoic acids A solution of 7 in 10% methanolic solution of trifluo

roacetic acid (100 ml) was stirred overnight. When the reaction was complete by TLC the

solvent was removed in-vacuo and the pink residue taken up in ethyl acetate (150 ml) and

washed with 1% aqueous hydrochloric acid (3 x 50 ml). The organic layer was dried over

sodium sulfate and the solvent removed in-vacuo. The residue was purified by flash column

chromatography (2/1 ethyl acetate / hexanes with 1% acetic acid) yielding a white solid in

yields from 50 to 66% over three steps.

3-( (4-(tert-butyldimethylsilyloxy)phenoxy)carbonylamino)benzoic acid (9) 1H

NMR (500 MHz, CDCI3) 6 (ppm) 8.07 (br. s, 1H), 7.84 (d, J = 7 Hz, 2H), 7.46 (t, J = 8

Hz, 1H), 7.07 (br. s, 1H), 7.05 (m, 3H), 6.83 (ABq, J = 8.5 Hz, 2H), 0.99 (s, 9H), 0.20 (s,

6H); 13C-NMR (100 MHz, CDCI3) 6 (ppm) 144.37, 137.86, 130.01, 129.48, 125.52, 122.31,

120.56, 25.66, -4.47; IR (KBr) 1725.4 cm-1 (C=O), 1551.5 cm-1 (HQ); E.A. Calcd. C

61.99, H 6.50, N 3.61, Expt. C 61.71, H 6.61, N 3.45; m.p. 191-192°C.

4-((4-(tert-butyldimethylsilyloxy)phenoxy)carbonylamino)benzoic acid (11) IH_

NMR (500 MHz, CDCI3) 6 (ppm) 8.09 (ABq, J = 8.5 Hz, 2H), 7.56 (ABq, J = 8.5 Hz, 2H),

7.13 (br. s, 1H), 7.05 (ABq, J = 9 Hz, 2H), 6.84 (ABq, J = 9 Hz, 2H), 0.99 (s, 9H), 0.20 (s,


6H); 13C-NMR (125 MHz, CDCh) 6 (ppm) 171.15, 153.47, 144.20, 142.46, 131.72, 124.29,

122.27, 120.60, 117.80, 25.64, 18.17, -4.48; IR (KBr) 1754.6 cm-1 (C=O), 1529.4 cm-1

(HQ); E.A. Calcd. C 61.99 H 6.50 N 3.61 Expt. C 62.15 H 6.67 N 3.87; ; m.p. >220°C.

4- ((4- (tert-butyldimethylsilyloxy)phenoxy)carbonylamino)-3-methoxybenzoic acid

(10) IH-NMR (500 MHz, Acetone-d6) 6 (ppm) 8.75 (s, 1H), 8.28 (s, 1H), 7.80 (dd, J =

8.5, 2 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 7.12 (ABq, J = 9 Hz, 2H), 6.91 (ABq, J = 9

Hz, 2H), 4.02 (s, 3H), 1.0 (s, 9H), 0.23 (s, 6H); 13C-NMR (125 MHz, Acetone-d6) 6 (ppm)

168.27, 154.88, 153.70, 146.99, 129.27, 127.82, 124.82, 124.52, 122.16, 112.02, 57.59, 27.00,

19.74, -3.39; IR (KBr) 1748.4 cm-1 (C=O), 1675.5 cm-1 (C=O), 1535.5 cm-1 (HQ); E.A.

Calcd. C 60.41, H 6.52, N 3.35 Expt. C 60.68, H 6.81, N 3.58; m.p. >220°C.

General Procedure for the Synthesis of ((4-hydroxyphenoxy)carbonylamino)

benzoic acids Concentrated hydrochloric acid (6 ml, 2.9% HCI vIv) was added to a

solution of 10 (0.18 g, 0.44 mmol, 1 equiv.) in 95% acetonitrile (70 ml). The reaction mixture

was stirred overnight. The solvent was reduced in-vacuo and the reaction mixture taken up

in ethyl acetate (100 ml) and the organic layer was washed with 1% aqueous hydrochloric

acid (30 ml) and water (30 ml). The organic layer was dried over sodium sulfate and the

solvent removed in-vacuo. The residue was purified by flash column chromatography (1/1

ethyl acetate / hexanes with 1% acetic acid) affording a white solid in yields between 90

and 97%.


3-((4-hydroxyphenoxy)carbonylamino)benzoic acid (13) IH-NMR (500 MHz, Acetone

d6) J (ppm) 9.26 (br. s, 1H), 8.33 (s, 1H), 7.86 (dd, J = 8, 1.5 Hz, 1H), 7.74 (d, J = 7.5

Hz, 1H), 7.47 (t, J = 8 Hz, 1H), 7.04 (ABq, J = 8.5 Hz, 2H), 6.84 (ABq, J = 8.5 Hz, 2H);

13C-NMR (125 MHz, Acetone-d6) J (ppm) 168.35, 156.72, 154.10, 145.40, 141.18, 133.17,

130.86, 125.92, 124.52, 121.35, 117.29; IR (KBr) 1711.8 cm-1 (C=O), 1505.1 cm-1 (HQ);

E.A. Calcd. C 61.54 H 4.06 N 5.13 Expt. C 61.40 H 4.27 N 4.85; m.p. 218(d)OC.

4- ( (4-hydroxyphenoxy)carbonylamino)benzoic acid (12) 1H-NMR (500 MHz, Acetone

d6) J (ppm) 9.42 (br. s, 1H), 8.02 (ABq, J = 9 Hz, 2H), 7.73 (ABq, J = 8.5 Hz, 2H), 7.04

(ABq, J = 9 Hz, 2H), 6.85 (ABq, J = 9 Hz, 2H); 13C-NMR (125 MHz, Acetone-d6) J (ppm)

168.39, 156.85, 153.94, 145.32, 145.15, 132.65, 126.87, 124.49, 119.48, 117.36; IR (KBr)

1711.79 cm-1 (C=O), 1505.11 cm-1 (HQ); m.p. >220°C.

4-((4-hydroxyphenoxy)carbonylamino)-3-methoxybenzoic acid (14) IH-NMR (500

MHz, Acetone-d6) J (ppm) 8.75 (s, 1H), 8.23 (s, 1H), 7.80 (dd, J = 8.5, 2 Hz, 1H), 7.16

(d, J = 8.5 Hz, 1H), 7.05 (ABq, J = 9 Hz, 2H), 6.85 (ABq, J = 9 Hz, 2H), 4.02 (s, 3H);

13C-NMR (125 MHz, Acetone-d6) J (ppm) 168.29, 156.76, 153.91, 145.51, 129.36, 127.74,

124.81, 124.47, 117.32, 111.99, 57.59; IR (KBr) 1693.7 cm-1 (C=O), 1511.3 cm-1 (HQ);

E.A. Calcd. C 59.41, H 4.32, N 4.62, Expt. C 59.28, H 4.22, N 4.65; m.p. >260°C.

General procedure for the synhesis of 1H-benzo[d][1,2,3]triazol-1-yl ((4-hydroxy

phenoxy)carbonylamino)benzoates To a stirred solution of 1-Ethyl-3-(3-dimethyl-


aminopropyl)carbodiimide hydrochloride (32.3 mg, 0.17 mmol, 1.55 equiv.) and N-hydroxy-

benzotriazole (15.5 mg, 0.12 mmol, 1.05 equiv.) in dry acetonitrile (10 ml) was added

dropwise a solution of 14 (33.5 mg, 0.11 mmol, 1.0 equiv.) in dry acetonitrile (20 ml) at

room temperaturea . Once the reaction was complete by TLC the solvent was reduced in-

vacuo to 2 ml and the reaction mixture dissolved in ethy acetate (100 ml). The organic

layer was washed with water (20 ml), dried over sodium sulfate and the solvent was removed

in-vacuo. The residue was recrystallised from ethyl acetate / hexanes yielding a white solid

in yields ranging from 60 to 97% (see Table 2.3).

1H-benzo[d] [1,2,3]triazol-1-yl 3-( (4-hydroxyphenoxy)carbonylamino)benzoate (16)

IH-NMR (500 MHz, Acetone-d6) 8 (ppm) 9.52 (s, 1H), 8.62 (s, 1H), 8.40 (br. s, 1H), 8.12

(dt, J = 8.1, 1 Hz, 1H), 8.07 (m, 1H), 8.03 (m, 1H), 7.88 (dt, J = 8.5, 1 Hz, 1H), 7.69 (m,

2H), 7.55 (td, J = 8, 1 Hz, 1H);

1H-benzo[d] [1,2,3]triazol-1-yI4-( (4-hydroxyphenoxy)carbonylamino)benzoate (15)

IH-NMR (500 MHz, Acetone-d6) 8 (ppm) 9.74 (s, IH), 8.42 (s, IH), 8.31 (ABq, J = 8.5 Hz,

2H), 8.12 (d, J = 8.5 Hz, 1H), 7.94 (ABq, J = 8.5 Hz, 2H), 7.84 (d, J = 8 Hz, 1H), 7.68 (t,

J = 7.5 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.08 (ABq, J = 8.5 Hz, 2H), 7.88 (ABq, J = 8.5

Hz, 2H); 13C-NMR (500 MHz, Acetone-d6) 8 (ppm) 164.35, 153.89, 147.83, 145.29, 145.2,

134.02, 130.79, 130.75, 126.48, 121.88, 120.31, 120.24, 120.06, 117.41, 110.87; E.A. Calcd.

C 61.54, H 3.62, N 14.35, Expt. C 61.31, H 3.60, N 14.01; m.p. 206(d)OC.

"When the compound was found to be only partially soluble in acetonitrile, an equal volume of ethylacetate was added to solubilise the compound


IH-benzo[d] [1,2,3]triazol-l-yl 4-( (4-hydroxyphenoxy) carbonylamino)-3-methoxy

benzoate (17) 1H-NMR (500 MHz, Acetone-d6) 8 (ppm) 8.98 (8, IH), 8.53 (8, IH), 8.34

(8, IH), 8.10 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.5 Hz, IH), 7.66 (t, J = 7.5 Hz, IH), 7.53

(t, J = 7.5 Hz, IH), 7.39 (d, J = 8.5 Hz, IH), 7.06 (ABq, J = 9 Hz, 2H), 6.85 (ABq, J =

9 Hz, 2H), 4.13 (8, 3H); 13C-NMR (500 MHz, MeOH-d4) 8 (ppm) 163.88, 156.41, 144.73,

144.61, 123.62, 120.81, 117.73, 116.67, 112.32, 112.23, 57.0; IR (KBr) 1774.4 cm-1 (C=O),

1506.51 cm-1 (HQ); E.A. Calcd. C 60.00, H 3.84, N 13.33, Expt. C 59.86, H 3.73, N 13.60;

m.p. 173(d)OC.


o

H2N~OH

Figure 2.23: A few commercially available aminomethylbenzoic acids.

2.4 Aminomethylbenzoic serIes

Aminomethylbenzoic acids are a class of 6-amino acids that are attractive building blocks

since they contain an amine that is much more nucleophilic than anilines. We therefore find

them to be well suited to coupling with N-hydroxybenzotriazole benzoates both in solution

and on surfaces. Additionally, many are commercially available (Figure 2.23) and many

more are readily accessible using established synthetic methods. One drawback however, is

that aminomethylbenzoic acid-based monomers will break conjugation within the peptide

backbone (Figure 2.24). One compensating advantage is that these monomers will allow

greater flexibility in the backbone of the oligomers than aminobenzoic acid-based monomers.

This may allow for folding of the oligomers in the self-assembled monolayers. The following

sections will describe our approach to making the benzylamine monomers.


b. thOHN

r"\ ° ~~;, - ~ ~HN1Y~ - HN-l

62

Figure 2.24: Oligomers of aniline- and benzylamine-based amino acids. a. Oligomer ofaniline-based monomer 17. b. Oligomer of benzylamine-based monomer 41. Conjugationshown in bold.

rY"NH2HO~ "',;======...

o

Figure 2.25: Structure of 4-(ammoniomethyl)benzoate.

2.4.1 Retrosynthetic analysis

2.4.1.1 First approach

We first attempted to extend the chemistry established for making aniline containing car-

bamates to benzylamines (Scheme 2.7). N-hydroxybenzotriazole ester 40 could be obtained

from the coupling of carboxylic acid 37 with N-hydroxybenzotriazole. 37 would be obtained

from the deprotection of the acid and phenol moieties of 28. Coupling of benzylamine-based

amino acid derivative 26 to chloroformate 2 would afford 28.


40 37

28

Scheme 2.7: First retrosynthetic approach to the benzylamine monomers.

Protection of the carboxyl group of benzylamine-based amino acids Using 4-

aminomethylbenzoic acid presented several challenges. First, the molecule exists in a zwit-

terionic state which makes it insoluble in the various organic solvents tested including methy-

lene chloride and tetrahydrofuran (Figure 2.25). Addition of a strong base such as triethy-

lamine to deprotonate the amine moiety and therefore solubilise 4-aminomethylbenzoic acid

was unsuccessful. The use of triethylamine as a solvent was also not successful. Therefore,

protection of the carboxylic acid was judged to be necessary for working with this com-

pound (Scheme 2.8). Second, the highly nucleophilic benzylamine moiety limits the choice

of protecting groups for the carboxylic acid since the protecting group must be stable to

primary amines.


b. r:7il1

rIY"'"~3.eO-~~• ~Ol~ 0"=1

o27

e erlY"NH3 -.CI

/O~

o26

Scheme 2.8: Different ways to protect the acid terminal of benzylamine amino acids. a.TBDMS-CI (1 equiv.), morpholine (3 equiv.), methylene chloride. b. 1:1 benzyl alcohol /toluene, p-toluene sulfonic acid (1.2 equiv.), reflux. c. 95% MeOH, HCI(g).

Protection of the carboxylic acid was first attempted using tert-butyldimethylsilyl chlo-

ride in a manner analogous to how 3, 4 and 5 were protected (Scheme 2.8a). This ap-

proach, however, did not yield the expected silyl ester, likely due to the insolubility of

4-aminomethylbenzoic acid under these reaction conditions. It was then decided to pro-

tect 4-aminomethylbenzoic acid as an ester which could be easily synthesised from reaction

of 4-aminomethylbenzoic acid with the corresponding alcohol in the presence of a strong

Br¢nsted acid (Scheme 2.8, reactions b and c). The benzyl ester of 4-aminomethylbenzoic

acid (27) was prepared by reacting 4-aminomethylbenzoic acid in a 1:1 mixture of benzyl

alcohol and toluene in the presence of tosic acid monohydrate (1.2 equiv.) at reflux (180°C).

The water was collected throughout the reaction using a Dean-Starke apparatus. The diffi-

cult purification of 27 and the modest yields obtained (51%) prompted us to turn to alkyl


-+---Si_

I

°UOJLC1

2L~

N

)28

Scheme 2.9: Synthesis of benzylamine carbamates via chloroformate intermediates.

esters instead. The methyl ester of 4-(ammoniomethyl)benzoate was synthesised by react-

ing 4-(ammoniomethyl)benzoate in methanol saturated with Hel gas at room temperature

(Figure 2.8). The product (26) precipitated out of solution and filtration afforded 26 as a

white solid in 80% yield.

Synthesis of carbamate 28 The carbamate moiety was prepared in a similar manner

to that described in Section 2.3.2.3 (Scheme 2.9). 26 only partially dissolved in methylene

chloride at concentrations of 14 mM and afforded carbamate 28 as a white solid in 29% yield.

Subsequent efforts to deprotect the methyl ester using Lewis acids such as aluminium trichlo-

ride lO7 and trimethylsilyl iodidelO8,109 or hydrochloric acid in aqueous dimethyformamide

were unsuccessful. It has been observed that the cleavage of carbamates, and especially

benzyl carbamates, using trimethylsilyl iodide was more facile than that of esters. 108 The

substrates usually used in the studies cited above are simple substrates such as methyl ben-

zote or p-methyl toluate for which few side-reactions are expected. However, 28 is more

functionalised and multiple side-reactions can occur such as cleavage of the carbamate moi-

ety. Metal coordination to the phenol ether instead of the methyl ester is also expected. As


40 33

29 30 31

Scheme 2.10: Second retrosynthetic approach to the benzylamine monomers.

a result, this approach was abandoned and a new synthetic route was designed.

2.4.1.2 Second approach

The chemistry established for the aniline series of monomers appeared to be poorly suited

to the synthesis of benzylamine derivatives and thus a new approach to the synthesis of

benzylamine-based monomers was proposed (Scheme 2.10). Monomer 40 would be ob-

tained from precursor 37 which in turn could be obtained from benzyl ether 33. 33 could

be obtained from carbamate 31 and 4-carboxy-benzaldehyde. Carbamate 31 would be syn-

thesised from isocyanate 30 which could be obtained from commercially available 29. This

approach will be discussed in greater detail in Section 2.4.1.3. This route has great potential

for the divergent synthesis of a large number of monomers since the main intermediate 31

can be synthesised in large quantities and is so stable that it can be stored for prolonged


°z'<:::: OH

Ih-

HO 0

~oHO 0

~HO~

HO 0

Figure 2.26: Some commercially available carboxybenzaldehydes.

periods of time, unlike chloroformate 2. Carboxybenzaldehyde compounds are also commer-

cially available (Figure 2.26) and many more are synthetically available from iodobenzoic

acids. 110

2.4.1.3 General Synthetic scheme

The general synthetic scheme is presented in Scheme 2.11. The discussion presented there-

after is directed towards the synthesis of 40. However, comments regarding the various

derivatives synthesised will be made whenever appropriate.

2.4.1.4 Synthesis of carbamate intermediate 31

Carbamate 31 was prepared in two steps from commercially available 29 by adapting liter-

ature procedures.111 113 Phenol 29 was first converted to 1-(benzyloxy)-4-cyanatobenzene

(30) by reaction with cyanogen bromide (BrCN, 1.1 equiv.) and triethylamine (1.1 equiv.)


('Y0H

~BnO

29

a. ..~O =N

BnO~

30

°O

O~b... I NH2

BnO .0

31

0°'(° 0°'(° 0°'(°~'A9~ HO I A 9~ HO I A 9~

c. d. e.31 .. ... ... P'IP'I P'I

~ ~~

COOH COOH COOBt

33 37 40

Scheme 2.11: Synthetic scheme for the benzylamine series of monomers. a. BrCN, TEA,Et20. b. CF3COOH, CCl4 , c. CF3COOH, Et3SiH, CH3CN, 4-carboxybenzaldehyde, reflux.d. Pd/C, H2 , MeOH. e. EDC·HCl, HOBt, CH3CN.

in diethyl ether at room temperature for 18 hours. Filtration of the precipitated triethy-

lamine hydrobromide followed by washing the organic layer with water afforded 30 as a

white solid in 81% yield. Refluxing 30 in carbon tetrachloride in the presence of trifluo-

roacetic acid (3.2 equiv.) for 18 hours afforded 31 as a white solid in 94% yield. 31 can

dehydrate back to 29 if the concentration of both 30 and trifluoroacetic acid are too high.

The yield of 31 can be lowered by as much as 25% due to this process. However, the ratio

of 30 to trifluoroacetic acid does not appear to have a marked influence on the yield of 31

(Table 2.4).


30 TFAexpt# equiv. cone. (mM) equiv. cone. (mM) temp. (OC) time (h) yield (%)

1 1B 2.2 b 70 18 69

2 22 3.2 71 75 18 94

3 179 4 714 80 24 78

4 81 4 320 80 48 70

5 1c 4.3 d 80 18 75

B. " 30 = 0.33 mmol b. "TFA= 0.71 mmol c. " 30 = 6.63 mmol d. "TFA= 28.59 mmol

Table 2.4: Synthesis of the carbamate intermediate 31. When concentration values werenot available, the number of moles was given instead.

2.4.1.5 Synthesis of benzylamine carbamate 33

33 was synthesised via reductive N-alkylation of carbamate 31 by modifying literature pro-

cedures for related compounds. 1l4 31 was refluxed in acetonitrile with 4-carboxy benzalde-

hyde, trifluoroacetic acid and triethylsilane (Scheme 2.11). No matter what the substitution

pattern on the benzaldehyde ring, the products precipitated out of solution making purifi-

cation of these compounds simple. Both the concentration of the reactants and reagents

and the position of the substituents proved to have an effect on the yield of product (Table

2.5).

Effects of solvent and concentration of reagents and reactants on the yield of 33

The discussion that follows and all experiment numbers cited in this paragraph relate to

table 2.5. Neither tetrahydrofuran (THF, expt 10) nor toluene (expts 1 and 2) proved to be

good solvents for this reaction and acetonitrile was found to give much better yields (expts

0 ~ 'i:l ~ ~ !'=I

31C

HO

Eta

SiH

TF

A~

exp

t#eq

uiv.

cone

.(m

M)

equi

v.co

ne.

(mM

)eq

uiv.

cone

.(m

M)

equi

v.co

ne.

(mM

)S

olve

ntte

mp.

(DC

)tim

e(h

)yi

eld

(%)

~ G32

132

3.1

973.

210

0T

olue

ne12

096

-< ~

22.

972

125

2.9

722.

972

Tol

uene

120

120

4~

32.

823

18.

32

.924

2.8

23A

ceto

nitr

ile80

67

44en ~

41.

714

51

852.

925

03.

327

0A

ceto

nitr

ile65

-70

2459

~ ~5

1.3

103

182

6.1

502

5.9

482

Ace

toni

trile

7524

70en

aen

61.

0316

71

162

4.1

669

4.1

667

Ace

toni

trile

67

1853

~7

3.5

194

156

739

211

.463

8A

ceto

nitr

ile20

to80

42

98b

~ 08

1.35

225

116

614

.511

5712

.499

0A

ceto

nitr

ile80

then

20

18th

en18

64~ 0

92.

914

71

5012

.663

025

1250

Ace

toni

trile

JT

HF

204

84

9~ ~

10

2.8

450

116

02

.84

50

2.9

460

TH

F2

018

en

8.yi

eld

=31

%af

ter

1ho

urb.

yiel

d=2

9%af

ter

6ho

urs

Tab

le2.

5:E

ffec

to

fth

eco

ncen

trat

ion,

solv

ent,

tem

per

atu

rean

dra

tio

ofre

acta

nts

and

reag

ents

onth

eyi

eld

of3

3.

-:r o


3-9). Yields obtained with acetonitrile were all in excess of 44% (expts 3-9) compared to

only 4% with toluene (expts 2 and 3). Not only the concentration of both triethylsilane and

trifluoroacetic acid (expts 3 and 7), but also the ratio of triethylsilane to trifluoroacetic acid

appeared to influence the yield of 33 (expts 6 and 7). It was also found that the yield of 33

increased when a large excess of 31, with respect to the aldehyde, was used. A ratio of 31

to the aldehyde of between 2 and 3 was found to be optimal. When all of the aldehyde in

solution was consumed (determined by TLC), more aldehyde was added still in a 2-3 to 1.

This operation was repeated until the carbamate was fully used up as evidenced by TLC.

By this method compounds 33-36 were obtained in 56 to 86% yield (Table 2.5).

Effect of the substituents on the yield of 33 It was found that when the carboxylic

acid group was in the ortho position relative to the aldehyde, the reaction proceeded rapidly

(Table 2.6). This effect is believed to arise for two reasons. Firstly, the positioning of the

acid may allow intramolecular catalysis of the dehydration step (third step) in the reaction

mechanism (Figure 2.27) thus pushing the equilibrium towards the imine product. Rapid

accumulation of the imine intermediate 58 may hasten the overall reaction if this is the

rate-determining step of the overall process. The resulting imine is reduced by an ionic

hydrogenation reaction.1l4,1l5 In this reaction, the nitrogen in 58 is first protonated, forming

a carbonium ion at the benzylic position. This cation is susceptible to attack by the hydride

source thus forming 33. It is therefore possible to see the advantage of a carboxylic acid

group at the ortho position relative to the aldehyde.

Consistent with the existence of an effective intramolecular hydrogen bond, the ortho


H~_(£J'H oo~ H+7' ~ ~Q I -

o ~ 4'NAO:::""H '-! \

H

72

33

Figure 2.27: Mechanism of benzyl carbamate formation.

positioning of the carboxylic acid group in 36 also leads to increased solubility in acetoni-

trile and ethyl acetate when compared to the structurally related compounds 33, 34 and

35. Since purification was effected by recrystallisation from acetonitrile or ethyl acetate,

the higher solubility of 36 in those solvents may explain the lower overall yield obtained

even though the Thin Layer Chromatography (TLC) analysis of the reaction shows rapid,

complete and clean conversion to the desired product (Table 2.6). In general, however, the

presence of a carboxylic acid moiety on the ring is very well tolerated under these condi-

tions. This observation is in agreement with previous studies by DuM et al. that showed

that both benzylamides and benzylcarbamates of phenol could be prepared under similar

conditions,u4 The presence of a hydroxyl substituent at the ortho position with respect to


o~~~o-o-o NH ~-/; Ra

# R 1 R 2 R 3 31 eHO Et3SIH TFA time (h) yield (%)

33 H H eOOH 1.3 5.9 6.1 24 70

34 H eOOH H 0.95 4 3.8 18 86

35 OH H eOOH 1.1 3.9 4.1 18 65

36 eOOH H H 0.94 3.4 3.3 8 56

Table 2.6: Effect of the position of the substituent on the yield of benzyl carbamate.

the aldehyde was also found to be well tolerated. This tolerance to substitution, combined

with the proven stability of fluorine substituted compounds under ionic hydrogenation con-

ditions1l4 should allow for the synthesis of diverse modular building blocks having various

substitution patterns.

2.4.1.6 Selective debenzylation - synthesis of 37-39 and 59

The following discussion will focus on the preparation of carbamate 38 although the other

compounds in this series were prepared in the same way. 38 could be obtained either from

coupling of 32 (resulting by hydrogenolysis of 31) with 3-carboxybenzaldehyde (Scheme

2.12) or by hydrogenolysis of benzyl ether 33.

Synthesis of carbamate 38 via 4-hydroxyphenyl carbamate (32) 32 was easily

obtained in quantitative yield by reacting benzyl ether 31 in methanol in the presence of

either PdjC (21 wt%) or PdCb (10 wt%) under an atmosphere of hydrogen for 18 hours


¢NH' 0 0

l ~OH~~~oo1 : PdlC 9N~..

1"-'::..

(;MeOH CF3COOH 1"-'::

hEt3SiH b

OH CH3CN OH

31 32 38

Scheme 2.12: Reaction scheme for the synthesis of compound 38 via carbamate 32.

74

(Scheme 2.12). As we expected, under these conditions the carbamate moiety of 32 was left

untouched. 32 was then coupled to 3-carboxybenzaldehyde using the reaction conditions

described earlier (Section 2.4.1.5). Even after purification (flash column chromatography

on silica gel) significant quantities of an unidentified impurity were observed by 1H-NMR

(Figure 2.28). This impurity, however, was not observed when 38 was obtained from hy-

drogenolysis of 34 (Figure 2.28) allowing convenient and reliable access to this material. We

therefore used this route to prepare compound 38 routinely.

Synthesis of carbamate 38 via benzyl ether 34 Although 34 contains both O-benzyl

and N-benzyl groups, the O-benzyl moiety can be selectively cleaved by hydrogenolysis since

N-benzyl moieties are known to be considerably more stable to these conditions. llB This

proved to be true for the series of 34, 35 and 36. When benzyl ethers 34, 35 and 36

were hydrogenolysed in a 2 : 1 mixture of methanol and ethanol in the presence of 10-15%

PdCl2 only the free phenol product was observed in yields of "-J 95% for compounds 34 and

35 and none of the undesired p-methyl benzoic acid product expected from the cleavage of

(al

@ ~ 'i:I t;5 ~ ~

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

IB.

OO

7.5

07

.00

6.5

06

.00

5.5

05

.00

4.5

0

~ ......

G < ~ ~ CJ:l ~ ~ ~ Ci) o ~ 6 < o ~ ~ CJ:l

y~

oN

(:)W

N~

b 8y~

N

~~

yy

yy

y~

~0

8lil

~o

0~

0>0>

<:>CD

CD...

.

L,-

-Jyy

/ ---J..J~I~~-W~

I,

LrJ

LL

,--J

yy

yy

yyy

yy~

0o

0~

~0

~~

!='

0N

'"<0

<0<:>

<:>0>

0>CD

~N

<:>01

CD01

001

....~

N01

....CD

(bl

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

II

IB

.OO

7.5

07

.00

6.5

06

.00

5.5

05

.00

4.5

0

Fig

ure

2.28

:1H

-NM

Rsp

ectr

ao

fth

epr

oduc

tsis

olat

edfr

omre

acti

ons

of3-

carb

oxyb

enza

ldeh

yde

wit

hei

ther

31

or3

2to

gene

rate

38

asth

eex

pect

edpr

oduc

t.N

MR

spec

tra

run

inac

eton

e-d6

at50

0M

Hz.

a.C

oupl

ing

of3

2w

ith

3ca

rbox

yben

zald

ehyd

e.b.

Hyd

roge

noly

sis

of34

.

~ 01


Q~o~Jl.o NH Pd,c

~ MeoHIBoH

H~o

Ho)Oo

36 59 32

Scheme 2.13: Hydrogenolysis of benzyl ether 36.

the benzylic N-C bond was observed. However, in the case of 36 when hydrogenolysis was

attempted, a mixture of compounds was obtained (Scheme 2.13). Based on the IH-NMR

spectrum of this mixture, one product could be identified as 32, however the hydrogenolysis

product 59 (Scheme 2.13) could not be identifed. The rate of hydrogenolysis reactions are

known to increase in the presence of Br~lnsted acids.ll7 It is therefore reasonable to speculate

that the carboxyl group of 36 could act as an intramolecular acid catalyst for the cleavage

of the otherwise fairly stable benzylic N-C bond. The mechanism for the hydrogenolysis

of O-benzyl alcohols is not yet clearly understood, however, the mechanism proposed by

Mitsui et al. is shown in Figure 2.29.118 ,119

Considering this mechanism, it seems possible that the intramolecular acid acts to fa-

cilitate cleavage of the O-C bond by acting as a general acid catalyst. Therefore, if oxygen

is replaced by nitrogen, protonation of the nitrogen by the carboxyl moiety could poten-

tially result in an increased rate of cleavage of the N-C bond (Figure 2.30). Given these

complications the synthesis of 36 and 59 was not pursued further.


*

* catalyst surface

~ ~7~:-~A/* , '"*

Bl

.. ~*

jH,

~H

Figure 2.29: Proposed mechanism for the hydrogenolysis of O-benzyl alcohols. 1l8,119

HO

chN-{-o-OH~ ~~~-o-OH -.~~J~-o-ooo ~ H:, 0 , 0

o O-H 0 o-H

59

.fr--~OH

o

• catalyst surface

+ --

Figure 2.30: Proposed mechanism for the hydrogenolysis of N-benzylcarbamate 59.


Influence of the solvent and substitution pattern on the rate of hydrogenolysis

All O-benzyl compounds were insufficiently soluble in methanol, one of the best solvents for

hydrogenolysis. Addition of ethanol to generate in a 1/2 ratio of methanol to ethanol both

solubilised the compounds and enabled the reactions to proceed rapidly (Table 2.7). Notably,

the substitution pattern on the ring at the acid end of the compounds seemed to affect the

rate of cleavage of the benzylic O-C bond. Compared to compounds 34 and 35, the rate

of cleavage is dramatically reduced for the hydrogenolysis of 33 when palladium chloride is

used as the catalyst. Hydrogenolysis of 33 over Pd/C was not complete after 6 hours in

methanol-tetrahydrofuran (entry 1, table 2.7). Switching to a 9/1 mixture of ethanol-ethyl

acetate allowed for near quantitative conversion of 33 to 37 using Pd/C (entry 2, table 2.7).

Although tetrahydrofuran is a very good solvent for all O-benzyl compounds, it appears

detrimental for the efficient hydrogenolysis of 33. This observation is expected since protic

solvents are known to be better solvents for hydrogenolysis. So, increasing the amount of

protic solvent should increase the rate of the reaction as is seen for entries 1 and 3.

2.4.1.7 Coupling of N -hydroxybenzotriazole-synthesis of monomers 40-42

Monomers 40 to 42 (Figure 2.31) were synthesised using the same method developed for

the aniline series, the only change being that tetrahydrofuran was used as solvent for the

free acids (37-39). All compounds were reliably obtained in high yields (95 to 97%) as

white solids after recrystallisation from ethyl acetate / hexanes.


H. He

6'Cc

H

•

H0'CcHd

-:? I h- OlNH catalystI h- 1

• o NH

~I » H2 »I":::R1 h- RJ R1 h- RJ

R2R2

33 R1 =H. R2 =H, R 3 =eOOH 37 R1 =H, R2 =H. R 3 =eOOH34 R1 =H, R2 =eOOH, R3 =H 38 R1 =H. R2 =eOOH, R3 =H35 R1 =OH, R2 =H. R 3 =eOOH 39 R1 = OH. R2 = H, R 3 =eOOH

reactant

expt# # cone. (mM) solvent (1/2) catalyst (wt%) time (h) yield (%)a

33 35 MeOH/THF Pd/C (10) 6 40

2 33 N/A EtOH 1EtOAcb

Pd/C (23) 7 95e

3 33 N/A MeOH 1EtOH PdC'2 (10) 2 54

4 33 N/A MeOH 1EtOH PdCI2 (10) 15 82

5 34 10 MeOH 1EtOH PdC'2 (11) 2.25 97

6 35 9 MeOH 1EtOH PdCI2 (11) 2 95

a. yield calculated by NMR using the ratio of the Integrations of protons (Ha + Hb) and (He + Hd).NMR spectra run in Aeetone-d6 at 500 MHz.b. ratio 9/1.

e. isolated yield.

79

Table 2.7: Hydrogenolysis of various benzylamine carbamates. NjA: data not available.

2.4.1.8 Conclusion

A versatile and divergent synthetic route to IH-benzo[dl[1,2,3]triazol-l-yl n-(((4-hydroxy-

phenoxy)carbonylamino)methyl)benzoates was developed. In this method the formation

of the carbamate moiety, a critical step in the synthesis of all the compounds presented

in this chapter, was effected by reductive alkylation of a common carbamate precursor:

4-(benzyloxy)phenyl carbamate 31). Hydrogenolysis of the resulting benzyl ether (com-

pounds 33-35) yields the free phenol product (compounds 37~39) which can then be


~ro9,"::::b

o"QI"::::b

OH

40

q?o

o"'QI"::::b

OH

41

~t"~0I"::::

b OH

o"QI"::::b

OH

42

Figure 2.31: The various benzylamine monomers synthesised.

converted in high yields to their N-hydroxybenzotriazole benzoates (compounds 40-42).

All the reactants used in this synthesis were commercially available in multigram quanti-

ties and only compound 31 needed to be synthesised for use. Due to the ready availabil-

ity of the starting materials, the simple chemistry, scalability and high reactivity towards

N-hydroxybenzotriazole benzoates, the aminomethylbenzoic acid series of monomers (com-

pounds 40-42) appears ideally suited for use in reagent less electrochemical arrays. Testing is

underway to assess their reactivity with self-assembled monolayers of mercaptoalkyl amines

on polycrystalline gold surfaces.


2.4.2 Experimental


81

All solvents were obtained from Caledon and used without purification except for triethy

lamine, pyridine and tetrahydrofuran which were obtained from Anachemia and distilled be

fore use. All deuterated solvents were obtained from Cambridge Isotope Laboratories, Inc..

Silica gel 60 was obtained from EMD (product # 9385 - 3). Trifluoroacetic acid and hydro

quinone were obtained from Sigma-Aldrich. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide

hydrochloride was obtained from Fluka. All other chemicals were obtained from Aldrich.

All chemicals were used without purification.




chloride disc. Each spectrum is the average of 32 or 64 measurements. NMR spectra eH





1-(benzyloxy)-4-cyanatobenzene (30) A solution of 4-(benzyloxy)phenol (29, 2.06 g,

10.3 mmol, 1 equiv.) and triethylamine (1.4 ml, 10.4 mmol, 1.01 equiv.) in diethyl ether (60

ml) was added dropwise to a solution of cyanogen bromide (1.14 g, 10.8 mmol, 1.05 equiv.)


in diethyl ether (100 ml) at -10°C. The triethylamine hydrobromide precipitate was filtered

off and the filtrate was washed with 1% hydrochloric acid and dried over sodium sulfate. The

solvent was removed in-vacuo. The residue was purified by flash column chromatography

(9/1 Toluene / Ethyl acetate) yielding a white solid (1.89 g, 81%). IH-NMR (500 MHz,

Acetone-d6) J (ppm) 7.48 (d, J = 7 Hz, 2H), 7.40 (t, J = 7 Hz, 2H), 7.35 (m, 3H), 7.17

(ABq, J = 9.5 Hz, 2H), 5.17 (s, 2H); 13C-NMR (125 MHz, Acetone-d6) J (ppm) 159.21,

148.84, 138.75, 130.33, 129.80, 129.49, 118.38, 118.28, 72.05; IR (KBr) 2285.67 cm-1 (C=

N), 2238.4 cm-1 (C= N), 1501.71 cm-1 (HQ); E.A. Calcd. C 74.65, H 4.92, N 6.22, Expt.

C 74.59, H 5.11, N 6.12; m.p. 65 - 67°C.

4-(benzyloxy)phenyl carbamate (31) A solution of 30 (0.56 g, 2.5 mmol, 1 equiv.)

and trifluoroacetic acid (0.7 ml, 10.0 mmol, 10 equiv.) in carbon tetrachloride (14 ml) was

refluxed for 18 hours. The reaction mixture was cooled down to room temperature and

the solvent removed in-vacuo. The residue was recrystallised from ethyl acetate / hexanes

yielding a white solid (0.48 g, 78%). IH-NMR (500 MHz, Acetone-d6) J (ppm) 7.48 (d, J

= 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7 Hz, 1H), 7.03 (ABq, J = 9 Hz, 2H),

6.98 (ABq, J = 9 Hz, 2H), 6.40 (br. s, 1H), 6.11 (br. s, 1H), 5.11 (s, 2H); 13C-NMR (125

MHz, Acetone-d6) J (ppm) 157.81, 147.12, 139.36, 130.25, 129.59, 129.39, 124.51, 116.83,

71.74; IR (KBr) 1763.65 cm-1 (C=O), 1709.34 cm-1 (C=O), 1509.94 cm-1 (HQ); m.p.

141 - 143°C.

General procedure for the synthesis of «(4-(benzyloxy)phenoxy)carbonylamino)

methyl)-benzoic acids Triethylsilane (100 J.lI, 0.63 mmol, 3.96 equiv.) and trifluoroacetic


acid (40 jiJ, 0.58 mmol, 3.63 equiv.) were added to a solution of 31 (111.0 mg, 0.45 mmol,

2.81 equiv.) and 4-carboxybenzaldehyde (23.3 mg, 0.16 mmol, 1.0 equiv.) in acetonitrile (2

ml) at room temperature. The reaction mixture was then refluxed for 6 hours after which

time a white precipitate was observed. Additional triethylsilane (70 Itl, 0.44 mmol, 2.77

equiv.), trifluoroacetic acid (32 Itl, 0.46 mmol, 2.9 equiv.) and 4-carboxybenzaldehyde (14.6

mg, 0.097 mmol, 0.61 equiv.) and acetonitrile (1 ml to account for loss during reflux) were

added to the reaction mixture. After 15 hours triethylsilane (70 Itl, 0.44 mmol, 2.77 equiv.),

trifluoroacetic acid (25 Itl, 0.36 mmol, 2.27 equiv.) and 4-carboxybenzaldehyde (11.3 mg,

0.075 mmol, 0.47 equiv.) and acetonitrile (0.5 ml to account for loss during reflux) were

again added to the reaction mixture. When the reaction was complete by TLC, the white

solid was filtered off and rinsed twice with acetonitrile. The filtrate was evaporated under

reduced pressure and the residue recrystallised from ethyl acetate I hexanes to yield a white

solid in yields from 56 to 86%.

4-(((4-(benzyloxy)phenoxy)carbonylamino)methyl)-benzoic acid (33) 1H-NMR

(500 MHz, Acetone-d6) 15 (ppm) 8.02 (d, J = 8 Hz, 2H), 7.51 (ABq, J = 8 Hz, 2H),

7.48 (ABq, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.33 (m, 2H), 7.07 (ABq, J = 8.5

Hz, 2H), 7.00 (ABq, J = 9 Hz, 2H), 5.11 (s, 2H), 4.58 (br. s, 0.2H), 4.50 (d, J = 6 Hz,

1.8H); 13C-NMR (125 MHz, Acetone-d6) 15 (ppm) 168.36, 131.65, 130.26, 129.60, 129.40,

129.12, 124.44, 116.90, 111.63,71.76; IR (KBr) 1722.83 cm-1 (C=O), 1702.21 cm-1 (C=O),


m.p. 220-222°C.


3-(((4-(benzyloxy)phenoxy)carbonylamino)methyl)-benzoic acid (34) 1H-NMR

(500 MHz, Aeetone-d6) 6 (ppm) 8.06 (8, 1H), 7.95 (d, J = 8 Hz, 1H), 7.65 (d, J = 7

Hz, IH), 7.49 (m, 3H), 7.38 (m, 4H), 7.06 (ABq, J = 9 Hz, 2H), 6.99 (ABq, J = 9 Hz, 2H),

5.11 (8, 2H), 4.49 (d, J = 6 Hz, 2H); 13C-NMR (150 MHz, Aeetone-d6) 6 (ppm) 168.53,

157.90, 157.05, 147.16, 141.99, 139.36, 133.80, 130.48, 130.37, 130.27, 130.18, 129.61, 129.41,

124.42, 116.92, 71.78, 46.06; IR (KBr) 1693.7 em-1 (C=O), 1498.9 em-1 (HQ); E.A. Caled.

with 0.3 H20b C 69.03, H 5.16, N 3.66, Expt. C 69.27, H 4.75, N 3.87; m.p. 186-188°C.

4-(((4-(benzyloxy)phenoxy)carbonylamino)methyl)-3-hydroxybenzoic acid (35)

IH-NMR (500 MHz, Aeetone-d6) 6 (ppm) 7.55 (m, 2H), 7.48 (m, 2H), 7.40 (m, 3H), 7.32

(t, J = 7 Hz, 1H), 7.26 (t, J = 6.5 Hz, 1H), 7.07 (ABq, J = 9 Hz, 2H), 6.99 (ABq, J = 9

Hz, 2H), 5.11 (8, 2H), 4.46 (d, J = 6.5 Hz, 2H); 13C-NMR (150 MHz, Aeetone-d6) 6 (ppm)

168.39,158.01,157.53,156.79,147.15,139.33, 132.72, 132.49, 130.57, 130.27, 129.63, 129.42,

124.44, 122.81, 118.05, 116.95, 71.79, 41.76; IR (KBr) 1670.9 em-1 (C=O), 1500.7 em-1

(HQ); E.A. Calcd. with 0.5 H20 C 65.67, H 5.01, N 3.48, Expt. C 65.68, H 4.70, N 3.76;

m.p. 220(d)OC.

2-(((4-(benzyloxy)phenoxy)carbonylamino)methyl)-benzoic acid (36) IH-NMR

(500 MHz, Aeetone-d6) 6 (ppm) 8.10 (8, 1H), 7.84 (m, 2H), 7.75 (t, J = 7.5 Hz, 1H), 7.49

(d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.14 (ABq, J = 8 Hz,

2H), 7.04 (m, 3H), 5.13 (8, 2H); 13C-NMR (150 MHz, Aeetone-d6) 6 (ppm) 170.10, 158.41,

bRepeated on two different samples from two different reactions with different times on high vacuum.Both gave the similar result


147.38, 146.53, 139.30, 136.36, 132.41, 130.32, 129.68, 129.44, 129.24, 126.63, 125.52, 124.42,

117.09,85.14,71.83; IR (KBr) 1746.0 cm-I (C=O), 1505.6 cm-I (HQ); m.p. 172 - 174°C.

General procedure for the synthesis of (((4-hydroxyphenoxy)carbonylamino)

methyl)benzoic acids Palladium chloride (5.7 mg, 10.9% w/w) was added to a solution

of 35 (52.2 mg, 0.13 mmol) in methanol/ethanol (5 / 15 ml). The reaction flask was sealed

with a rubber septum and purged three times with hydrogen. The reaction mixture was

then stirred under hydrogen atmosphere until the reaction was complete by TLC (2 to 15

hours). The reaction mixture was filtered over celite. The solvent was removed in vacuo to

afford a white solid in yields from 82 to 97%.

3-(((4-hydroxyphenoxy)carbonylamino)methyl)benzoic acid (38) IH-NMR (500

MHz, Acetone-d6) 6 (ppm) 8.3 (br. s, IH), 8.07 (s, IH), 7.96 (d, J = 7.5 Hz, IH), 7.49

(d, J = 7.5 Hz, IH), 7.32 (t, J = 6 Hz, IH), 6.96 (ABq, J = 9 Hz, 2H), 6.80 (ABq, J

= 9 Hz, 2H), 4.48 (d, J = 6 Hz, 2H); I3C-NMR (125 MHz, Acetone-d6) 6 (ppm) 168.69,

157.23, 156.27, 145.93, 141.85, 133.81, 132.43, 130.42, 130.28, 130.12, 124.29, 117.13,45.95;

IR (KBr) 3333.2 em-I, 1698.5 em-I, 1498.9 em-I, 1197.5 em-I.

4-(((4-hydroxyphenoxy)carbonylamino)methyl)-3-hydroxybenzoic acid (39) IH_

NMR (500 MHz, Acetone-d6) 6 (ppm) 9.0 (s, IH), 8.3 (s, IH), 7.55 (m, 2H), 7.41 (d, J =

8.5 Hz, IH), 7.24 (t, J = 6 Hz, IH), 6.96 (ABq, J = 9 Hz, 2H), 6.80 (ABq, J = 9 Hz, 2H),

4.46 (d, J = 6.5 Hz, 2H); I3C-NMR (125 MHz, Acetone-d6) 6 (ppm) 168.51, 157.82, 156.71,

156.38,145.89,132.52,132.47,130.61,124.31, 122.80, 118.05, 117.16,41.73; IR (KBr) 3335.2


4- ( ((4-hydroxyphenoxy)carbonylamino)methyI)benzoic acid (37)

86

General procedure for the synthesis of IH-benzo[d][I,2,3]triazol-l-yl n

(((4-hydroxyphenoxy)carbonylamino)methyl)benzoates A solution of 39 (40 mg,

0.10 mmol, 1.0 equiv.) in distilled tetrahydrofuran (2 ml) was added dropwise to a stirred

solution of N-hydroxybenzotriazole (27.6 mg, 0.20 mmol, 2.0 equiv.) and l-Ethyl-3-(3

dimethyllaminopropyl)carbodiimide hydrochloride (41.9 mg, 0.22 mmol, 2.2 equiv.) in ace

tonitrile (2 ml). The reaction mixture was stirred at room temperature (20°C) for 1 hour.

The volume was reduced in-vacuo and the remaining solution taken up in ethyl acetate (100

ml) and washed with water (2 x 30 ml) and brine (30 ml). The organic layer dried over

sodium sulfate and the solvent removed in-vacuo affording a white solid in yields from 82

to 97%.

IH-benzo[d] [1,2,3]triazol-l-yl 3-( ((4-hydroxyphenoxy)carbonylamino)methyl)

benzoate (41) IH-NMR (500 MHz, Acetone-d6) <5 (ppm) 8.32 (s, IH), 8.22 (d, J = 7.5

Hz, IH), 8.12 (d, J= 8.5 Hz, IH), 7.88 (m, 2H), 7.67 (m, 2H), 7.54 (t, J = 8 Hz, IH), 7.45

(t, J = 6.5 Hz, IH), 6.97 (ABq, J = 9 Hz, 2H), 6.80 (ABq, J = 9 Hz, 2H), 4.58 (d, J = 6.5

Hz, 2H); 13C-NMR (150 MHz, Acetone-d6) <5 (ppm) 164.77, 157.27, 156.40, 145.95, 145.22,

143.21,136.52,131.21,131.01,130.66,127.08, 126.86, 124.51, 121.91, 121.76, 117.22, 117.14,

110.87.


IH-benzo[d:1 [1,2,3]triazol-1-yl 4-( ((4-hydroxyphenoxy)carbonylamino)methyl)-3

hydroxybenzoate (42) IH-NMR (500 MHz, Aeetone-d6) 0 (ppm) 8.11 (d, J = 8.5 Hz,

1H), 7.84 (m, 2H), 7.76 (8, IH), 7.65 (m, 2H), 7.53 (t, J = 8 Hz, 1H), 7.37 (t, J = 6.1

Hz, 1H), 6.99 (ABq, J = 9 Hz, 2H), 6.82 (ABq, J = 9 Hz, 2H), 4.55 (d, J = 6.2 Hz, 2H);

I3C-NMR (125 MHz, Aeetone-d6) 0 (ppm) 164.62, 157.80, 157.27, 156.49, 145.94, 145.22,

136.06, 131.21, 130.79, 126.86, 124.35, 123.84, 121.83, 118.40, 117.22, 110.82; IR (NaCl)

3308.7 em-I, 1791.0 em-I, 1715.9 em-I, 1500.7 em-I, 1200.2 em-I.

1H-benzo[d:1 [1,2,3]triazol-1-yl 4-( ((4-hydroxyphenoxy)carbonylamino)methyl)

benzoate (40) IH-NMR (500 MHz, Aeetone-d6) 0 (ppm) 8.26 (8, IH), 8.21 (t, J = 6 Hz,

1H), 7.96 (d, J = 8.5 Hz, 1H), 7.91 (ABq, J = 8.5 Hz, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.54

(t, J = 8 Hz, 1H), 7.41 (m, 3H), 6.88 (ABq, J = 8.5 Hz, 2H), 6.71 (ABq, J = 8.5 Hz, 2H),

4.31 (d, J = 6.5 Hz, 2H); I3C-NMR (125 MHz, Aeetone-d6) 0 (ppm) 167.30, 155.45, 154.47,

144.60, 143.39, 129.58, 129.53, 127.93, 127.46, 127.18, 124.68, 122.64, 119.18, 115.48, 109.71,

43.82.

Chapter 3

Self-assembled monolayers

3.1 A brief introduction to self-assembled monolayers

For a more complete view on self-assembled monolayers, the reader is directed to two review

articles by Love et at. 59 and Ulman. 57

Self-assembled monolayers (SAMS) are one molecule-thick layers of molecules that sponta

neously form at certain interfaces. Amphifunctional molecules are typically used to form

such self-assembled monolayers. These molecules have one part exhibiting a strong affin

ity to a surface and another that exhibits little or no affinity for the same surface (Figure

3.1).120

Self-assembled monolayers on various solid surfaces are simply made by incubating the

surface in a millimolar solution of the amphifunctional molecule in an appropriate solvent.

Monocomponent (composed of one type of amphifunctional molecules) and multicomponent

88

CHAPTER 3. SELF-ASSEMBLED MONOLAYERS

Head group- monolayer surface properties- monolayer surface reactions- monolayer surface modification

Spacer unil4 interchain interactions

89

Surface-active group• chemisorption

Figure 3.1: Schematic representation of a molecule capable of forming a monolayer. Thevarious physical and chemical properties associated with different parts of the amphifunctional molecule are shown.

(composed of more than one type of amphifunctional molecules) monolayers can be fabri-

cated in this manner. The ease of fabrication and ease of modification of SAMS makes them

attractive for building devices and surface engineering.27,33,34,46,57,58,121 124 From this point

on, we will concentrate on the self-assembled monolayers of sulfur-containing amphifunc-

tional molecules on gold surfaces.

Eventhough the mechanism and kinetics of formation of thiol monolayers on Au(l11)

have been extensively studied,125 134 the exact mechanism of assembly remains poorly un-

derstood. At thiol concentrations close to 1 mM, two distinct kinetic adsorption regimes can

be observed (Figure 3.2).57 One is a very fast process (few mnutes) in which the amphifunc-

tional molecules (Figure 3.1) adsorb on the surface in a disordered fashion and the second,

is a much slower process (few hours), during which the monolayers order themselves so as

to maximize the intermolecular interactions of the spacer groups. The first regime is con-

centration dependant and governed by the surface-active group-surface interaction whereas

CHAPTER 3. SELF-ASSEMBLED MONOLAYERS 90

/11///1 Aul/II/II

1f; / I 11; Au /1 J'/III

b. chemisorption

RS-Au

Figure 3.2: Simplified growth mechanism of Self-assembled monolayers of thiols on gold. a.The thiols adsorb randomly on the surface before ordering themselves in order to maximisethe interchain interactions. b. Energy diagram of the self-assembly process. ~G~dSoTPtion =

Free energy change of the adsorption process.

the second regime depends more on the intrinsic level of disorder of the spacer unita .57

The adsorption of thiols on gold is an exothermic process with a net change in free

energy of about ~G~dSoTPtion = -26 kJ /mol. 135 Studies suggest that S-H bond cleavage is

not the rate determining step.136 138

aSmaller spacer units will generate more disorder due to the reduced van der Waals interactions betweenaliphatic spacer units.


3.2 Effect of the molecular structure of the thiols on the for-

mation of monolayers

The nature of the thiol employed for making a monolayer dictates, in part, the physical

(friction, adhesion, electrical, etc... ) and chemical (reactivity, layer-by-layer assembly,

etc... ) properties of the SAMS.57,139 143 Both the head group and the spacer unit can

impact the final structure and reactivity of the monolayer. The degree to which the surface

is protected from the environment of the bulk solutionb is also dependant upon the nature

of the monolayer (Figure 3.3).57

The various classes of thiols used for self-assembled monolayers will be presented below.

3.2.1 Mercaptoalkane self-assembled monolayers

A wide variety of mercaptoalkanes are able to form self-assembled monolayers on gold (Fig-

ure 3.4). Of these, thiols and disulfides have been the most intensely investigated.57,139

For this discussion we will focus on alkanethiols since these are the most studied systems.

Disulfides will be discussed, where appropriate, in the rest of this thesis.

On Au(lll) substrate, the S· .. S distance is typically 4.5 to 5 A, this spacing accounts,

in part, for the arrangement of the spacer units. Alkanethiols are typically tilted by about

32° with respect to the surface normal and 55° about the molecular axis with the molecule

in an all-trans conformation. 139 Self-assembled monolayers made from alkanehiols such as

bThe degree to which the surface is protected from the environment is qualitatively determined by comparing the cyclic voltammogram of a monolayer-protected surface to that of bare gold. The more similar thecyclic voltammogram the less protected the surface is deemed to be.


250

200

150

100

:<"-

50

·50

-100

1

- Bare gold I--1-alTino-llJ.mercaptodecane

~CystalT1lne-.-

CystamIne

ssssssI I I I I I

I

.(l.S 0.0Potential M

os 1.0

Figure 3.3: Cyclic voltammogram of short chain and long chain aminoalkyl thiol monolayerson polycrsytalline gold surfaces. Black: cyclic voltammogram of a bare gold surface givenas basis for comparison. Blue: the short chain cystamine aminoalkyl thiol forms a loosemonolayer and as a result the cyclic voltammogram of a gold surface modified with such amonolayer resembles that of a bare gold surface. Current can flow through the monolayerwith ease. Red: the long chain cystamine aminoalkyl thiol forms a compact monolayer andas a result the cyclic voltammogram of a gold surface modified with such a monolayer differsgreatly from that of a bare gold surface. Very little current flows through the monolayer.The data presented in this figure is actual eperimental data. Reference electrode: AgAgCl13M NaCl, scan rate = 50 mVIs, 0.1 M sodium phosphate buffer (pH 0.2) as electrolyte. Note:The cyclic voltammogram observed here for the bare gold surface is reproducible when usingthis buffer and differs from that typically observed in strongly acidic solutions.


HS

a.

~ ~o"S~

o '0-d.

~s-sb.

~So

e.

c.

-S}-o

S

f.

Figure 3.4: The different mercaptoalkanes capable offorming SAMS on gold. a. Alkanethiolb. Dialkyl disulfide c. Dialkyl sulfide d. Bunte salt e. Alkyl thioacetate f. Alkyl xanthate.

dodecanethiol are mainly stabilised by maximized van del' Waals interactions between the

carbon and hydrogen atoms in the spacer units. The shorter the alkane thiol (4 to 8 carbons),

the weaker the van del' Waals interactions, and therefore the less rigid and the more fluid

the monolayer becomes. The high number of possible van del' Waals contacts in the longer

alkane thiols (8 to 20 carbons) solidifies and stabilises the monolayer (Figure :3.5).134

3.2.2 Self-assembled monolayers from amide-containing mercaptoalkanes

The introduction of an amide group into the spacer unit allows hydrogen bonding between

spacer units of adjacent monolayer components. The increased strength of the interchain

interaction increases the thermal stability of the monolayer. 144 The amide sublayer is well

ordered, however, the amide linkage has a deleterious effect on the packing of the alkyl


a. b.

Figure 3.5: Self-assembled monolayers from alkylthiols. a. van der Walls contacts shownas dashed lines. b. Orientation of the all-trans carbon chain on a Au(lll) surface. Chainshown as bold black line in gray circle.

chains that are further from the surface when compared to similar alkanethiols (Figure

3.6).145 The bulkiness of the amide group compared to the methylene group consequently

leads to more dilute monolayers when compared to monolayers of alkanethiols of similar

length. 146 Both the potential disorder and less densely packed nat me of such monolayers

may allow the design of mOllolayers with an increased probability of efficient couplings with

the monomers presented in Chapter 2 as compared to more compact aminoalkyl mecaptan-

based monolayers. The synthesis of these molecules will be presented later on in this chapter.

3.2.3 Arylthiol self-assembled monolayers

Unlike aliphatic thiols, aromatic thiols or arylthiols are strongly anisotropic. The inter-

molecular interactions (IT-IT, H-IT, etc ... ) are also stronger than the van der Waals forces that


R R R R R R R R R

~R

~0 ~H~o~__H~o/HNro--

,HN

~of r f r r s r r s s rI I I

I I I

a. b.

Figure 3.6: Self-assembled monolayers from amide-containing alkylthiols. a. Self-assembledmonolayers from amide-containing alkylthiols. b. Self-assembled monolayer of a similaralkanethiol without amide functionality.

hold together SAMS made of alkanethiols (Figure 3.7).149 151 These factors give arylthiol

self-assembled monolayers unique structural147 as well as electronic152 properties. Various

structural features of the monomer can have significant effects on the properties of the re-

suIting SAMS. For example, more aromatic rings within the monolayer component increase

the quality of the monolayer by the increasing the number of favorable 7r interactions (Figure

3.8).147 The introduction of a methylene unit between the sulfur atom and the phenyl ring

allows more flexibility in the orientation of the aromatic thiol at the surface and consequently

more favorable intermolecular interactions and a better packed monolayer. 149,153 Altering

the substituents on the arylthiol may alter its adsorption kinetics (by altering the acidity

of the thiol thus making ita harder or softer ligand), surface packing (by changing the

electron density of the arylthiol) and electronic properties (additional electron withdrawing

or donating groups can induce a dipole).148 Whereas alkanethiol monolayers exhibit high

resistivity, aromatic thiol monolayers differ in that they have high electrical conductivity.152


;rH-WH;r;ry--¢-y-ys s s SI I I I

a. b.

Figure 3.7: Self-assembled monolayers from 4-aminothiophenol. a. The different 1r interactions. Top: 1r-1r stacking. Bottom: 1r-H interaction b. A 3D representation of a possibleorientation of 4-aminothiophenol molecules on a gold surface. The proposed structure isconsistent with literature. 147,148 3D representation obtained with Arguslab 4.0 software foran energy minimized structure of 4-aminothiophenol. Semi-empirical calculations at theAMI level.

This property is currently being investigated for use in molecular electronics.86 ,87,lOl The

synthesis of a diamino-diaryl disulfide molecule and its electrochemical characterisation will

be presented later in this chapter.

3.2.4 SAMS from thiols and disulfides

As mentioned previously (Section 3.1 and 3.2.1), self-assembled monolayers on gold are often

made from either di~ulfidc- or thiol-containing compounds. The kinetics of adsorption of


00s s s s s sI I I I I I

a. b. c.

Figure 3.8: Self-assembled monolayers from various thiophenols. The stability of the monolayer increases with increasing conjugation. a. Thiophenol. b. p-biphenyl mercaptan. c.p-terphenyl mercaptan.

octadecanethiol and dioctadecane disulfide in ethanol were found to be indistinguishable.154

This observation suggests that neither the cleavage of the RS-H bond, nor that of the RS-SR

bond is rate-determining. More speculative is the fact that the species being adsorbed on

the surface are in both cases thiolates. 138,154 However, the equations for the adsorption

reactions, assuming that thiolates are the surface-bound species are as followsc .

1RS - H + Au~ ~ RS-Au+ . AU~_l + "2H2 LlG~dsoPtion = -26kJjmolj RS- (3.1)

RS - SR + Au~ ----7 RS- Au+ . AU~_l LlG~dsoPtion = -58kJjmoljRS- (3.2)

"Data found for octadecanethiol and octadecane disulfide1:J5


H

HO

R

o

a.

~R'

°1O-R"

"1 HN

°1~....

b. c.

Figure 3.9: Three common functional headgroups for self-assembled monolayers. a. Hydroquinone is easily oxidized to quinone to allow for subsequent Diels-Alder chemistry.156b. Amines allow for the coupling of reactive esters. 157 c. Carboxylic acids can be convertedto esters using EDC·HCI.

Consistent with the view that the thiolate is the bound species regardless of whether a

disulphide or a thiol is used is that monolayers formed from dialkyl disulfides have very sim-

ilar electrochemical,155 structural and physical 138,154 properties as compared to monolayers

formed from alkanethiols. We will take advantage of this fact later on when designing the

synthesis of some of the anchor molecules used in this thesis.

3.2.5 Functional monolayers - the head group

Many applications of SAMs take advantage of the unique chemical, physical and electro-

chemical properties of self-assembled monolayers with functional head groups containing


various functional groups such as carboxylic acids, amines, hydroquinone and many other ex

amples (Figure 3.9).130,158 160 F'unctionalised SAMs have been used in the fabrication of de

vices such as gas sensors,161 biosensors,158,162,163 surfaces for protein attachment27,33,34,48,164

and arrays for oligomer synthesis. 37

The chemistry typically used in solution can be used for the reactions between func

tionalised self-assembled monolayers with appropriately reactive solute molecules (Figure

3.9) .62,158,165 However, SAMs exhibit some properties that allow one to perform some

chemistry that would be otherwise very difficult in solution. For instance, amide bonds

can be made simply by pressing a silicon stamp soaked with a solution of an amine on

a monolayer of carboxylic acid-terminated alkanethiols143 The close proximity and high

local concentration of amines enables the reaction to proceed without traditional peptide

coupling reagents.

3.3 Electrochemistry of self-assembled monolayers

The electrochemical and electron transfer behaviour are critical parameters of the systems

that we wish to study and develop. The structure of the monolayers (anchors only or anchors

with monomers attached) will have a direct impact on the overall performance of the device.

In this section a simplified view of electrochemistry and electron-transfer in self-assembled

monolayers is presented to provide some background for the reader.


3.3.1 General electrochemistry of self-assembled monolayers

100

For a full account the reader is directed to two reviews by Bard et al. 166 and Speiser167 and

a book by Kuznetsov. 168

The electrochemical behaviour of interfaces in general, and self-assembled monolayers in

particular, has been the subject of intense scrutiny for most of the 20th century.166,169,170

When carrying out an electrochemical reaction, one needs to take into account not only the

solvent, the electrolyte (nature and concentration) and the electrode materials, but also the

nature of the electrode / electrolyte interface. The interface is usually depicted as a double

layer of closely packed ions near the electrode surface and a more diffuse layer above (Figure

3.10). The double layer can sometimes be as thick as several A.167 The electrical double

layer is dependent on the surface roughness, pH and nature of the electrolyte. 171 ,172 Giesbers

and coworkers171 have shown that the nature of the double layer on a gold substrate was

pH dependant. d The width of the double layer was found to be 80 A at pH 3, 30-40 A at

pH 4 and much larger at higher pH.

The capacitance associated with the double layer is called the double layer capacitance

(Cdd and can be used to estimate how ordered a monolayer ise and, consequently, its

permeability to either the electrolyte ions and / or the redox center. 174 Redox centers

in SAMS that undergo either reversible or irreversible electrochemical reactions have been

used to selectively immobilise proteins. 27 ,33,34,46,156 These applications and optimal electron

dThe values quoted here were determined by AFM force measurements between a gold surface and anAFM tip-bound silica particle at various pH17

!

"The more ordered the monolayer, the smaller the double layer capacitance. 17:J


I

-8:0 8

8: 0 8 0

-8 o 80

-8 0 80

8 0 8a. b.

101

Figure 3.10: Schematic representation of the double layer at an electrode surface. a. Acondensed layer of ions of opposite sign to the net charge at the electrode surface is foundnear the electrode surface. This layer is sometimes termed the Helmholtz layer. b. A morediffuse layer of ions of different charges is found on top of the Helmholtz layer. This layeris sometimes termed the Gouy-Chapman layer.

transfer is defined as a rate of electron transfer fast enough so as to not limit the function

of the proposed device.

3.3.2 Electron-transfer - from bulk to single molecules to SAMS

The modern theory of electron transfer has been formulated by Marcus (Figure 3.11).175,176

This model applies well to a wide range of donor-acceptor pairs and has been supported by

a wealth of experimental data. 177,178 Although this model has been formulated for a donor

and an acceptor separated by a medium such as a solvent, it is a good starting point for

understanding the electron-transfer processes in self-assembled monolayers.


a.

kET

b.

Energy I~Gacl = (Ie + ~GQ? /4lel Donor (D)

I- - - - -II •

X o QQA xA

Nuclear Coordinate

Acceptor (A)

102

Figure 3.11: Marcus diagram of electron transfer. a. Marcus diagram. Plot of the rateconstant governing electron transfer (kET) against the free energy at standard conditions(~GO). The rate is maximum when the reorganisational energy (A) is ~ero. b. Energydiagram of the donor-acceptor pair. b.Gact = energy of activation for the electron transferto occur, QDA = distance between the donor and acceptor. XD = nuclear coordinate of thedonor. XA = nuclear coordinate of the acceptor.


Acceptor -~..~

a,

Donor

b.

Figure 3,12: Donor-acceptor pairs for SAMS adsorbed on an electrode. a. Electrode isthe donor, acceptor is fixed on the monolayer. b. Electrode is the acceptor, the donor isembedded in the monolayer.

In the case of a monolayer of a thiol building-block on gold, the spacer unit can be

considered as the medium separating the donor-acceptor pair. The gold electrode and the

electroactive part of the thiol are the donor-acceptor pair, but which component is which

part of the pair depends on the case (Figure 3.12). For a solute-impermeable monolayer174

electron transfer can only occur through the orbitals of the spacer since the electrolyte can

not reach the electrode surface. Electron transfer dynamics through such a monolayer is

therefore dominated by both the molecular structure and the arrangement of molecules in

the monolayer (both their specific orientation defined by their structures as well as their

relative conformations), 78


3.3.2.1 Influence of the monolayer thickness, arrangement and molecular iden

tity

When measuring the rate of electron transfer within a monolayer, the monolayer should be

well ordered and defect free so as to make sure that the mass transport of the electrolyte

to the electrode surface does not contribute to electron transfer between the electrode and

the redox species in solution.174 It is well known that the rate of electron-transfer decays

exponentially with increasing donor-acceptor distance according to equation 3.3. 179

kET = ko exp( -(3dD,A) (3.3)

kET is the rate constant governing electron transfer, ko is a preexponential factor, dD,A

is the donor-acceptor distance and (3 (A -1) is an attenuation factor that is dependant on

the structure of the molecule making up the self-assembled monolayer under study. This

(3 parameter has been measured for a wide variety of alkane179 182 ((3 = 0.87-1.04 A-I)

and polyaromatic thiols ((3 = 0.42-0.61 A-I ).179,180,183 For comparison, DNA179 has a (3

value between 0.1 and 0.4 A-I when intercalated probes are used and proteins184 have an

average (3 value of 1.1 A-I. The lower value of (3 for polyaromatic thiols suggests that

conjugation along such molecules helps the electron-transfer process.179 It was found that

the attenuation factor (3 for mercaptooligo(phenylethynylene) was between the expected

values for the coplanar (where all molecular orbitals are in the same plane, (3 = 0.43 A-I)

and perpendicular ring geometries (where the molecular orbitals of one ring lie in one plane

and the molecular orbitals of the adjacent ring lie in a second plane that is perpendicular to


the first, (3 = 1.00 A-I) .170 This observation suggests that the conformation of the molecules

inside the monolayer is an important factor regulating efficient electron transfer.

The oligomers that could be formed by the molecules presented in this thesis are expected

to have (3 values between that of polyaromatic and alkane thiol monolayers ((3 = 0.42-1.04

A-I) tending toward the lower end. Interestingly, Kai and coworkers185 have recently found

low (3 values for self-assembled monolayers of long helical peptides (24mer) compared to

SAMs of shorter helical peptides ((3 = 0.02-0.04 A-I). They concluded that an electron

hopping mechanism via the amide bounds of the backbone was likely in the case of the

longer peptides. It is possible that the oligomers produced by this method may exhibit a

similar behaviour.

3.3.2.2 Effect of the environment on the rate of electron transfer

Effect of the environment around the redox center To illustrate by way of exam

ple, studies on ferrocene-containing SAMS on gold electrodes showed that the environment

around the redox center dramatically influenced the charge transfer between the electrode

surface and the ferrocene redox center. 181 ,186 When the ferrocene unit was buried in longer

alkanethiols, making it less available to the electrolyte, the rate of electron transfer between

ferrocene and the gold surface was reduced 200 fold as compared to a situation where a

ferrocene unit was exposed to the electrolyte solution. 186

The rate of electron transfer in mixed monolayers composed of mercaptooligo(phenyl

ethynylene) and either a functionalised alkanethiol (OH or COOH head groups, nc = 9 or

15) or 4-(phenylethynyl)benzenethiol was found to be independent of the diluent, showing


that electron transfer occurred mainly though the mercaptooligo(phenylethynylene) units

and that alkanethiols contribute very little to the electron transfer process in this system.17°

Effect of amide groups on the rate of electron transfer The introduction of amide

groups near the electrode surface increases the rate of electron transfer in ferrocene-derivatized

alkanethiol mixed monolayers. 174 Although Bilewicz et al. 174 imply that the H-bonding abil-

ity of the amide groups is solely responsible for the increased rate of electron-transfer, an

increase in the disorder of the monolayer above the amide sublayer (Figure 3.13) may also

be an important factor. The increased disorder of the top alkyl layer enables the electrolyte

to approach closer to the electrode surface, thereby facilitating electron transfer between

the electrode surface and the electrolyte. This view is supported by their own data since a

drop in double layer capacitancef (C dl ) of approximately half is observed when the amide

sublayer is capped with (CH2h3 instead of (CH2ho. 174 Also, in the case of the shorter

chain length, it is possible that the electrolyte participates more to the electron transfer

than in the case of the longer chain length due to increased disorder in the capping layer.

Electron transfer through SAMS proceeds via electron tunneling from the gold electrode

surface to the redox center.l87 Electron transfer in peptides and proteins has been shown to

proceed via electron tunneling at high rates184 and the presence of multiple amide linkages

in the spacer of the components of certain SAMS improves the rate of electron transfer

through the monolayer. A 70 fold increase in the rate of electron transfer was observed in

the case of mercaptooligoglycine monolayers as compared to the corresponding alkanethiol

fThe double-layer capacitance depends directly on the accessible electrode area(http://electrochem.cwru.edu/ed/encycl/).


(Figure 3.13).188 Notably, it has been established that the tunneling pathway in proteins is

a function of covalent and hydrogen bonds as well as through-space contacts. 184 It is rea

sonable to assume that similar factors are at play in the electron transfer through SAMS.

Such factors are critical for us as we contemplate the design of the monolayer component of

the Reagentless Electrochemically Addressable Array (RECAA) that we envision and are

therefore considered below.


a.

c.

b.

d.

Figure 3.13: Effect of the amide linkage on the rate of electron transfer. a. Amide linkageclose to the surface and short chain after the amide linkage. The disorder is high abovethe amide linkage. Highest rate of electron transfer. b. Alkanethiol monolayer. Highorder due to maximum van der Waals contacts between the chains. Lowest rate of electrontransfer. c. Amide linkage close to the surface and long chain after the amide linkage. Thedisorder is reduced above the amide linkage due to the increased van der Waals interactions.Intermediate rate of electron transfer. d. Oligoglycine monolayers. The amide linkages allowfor a rate of electron transfer greater than that of alkanethiol monolayers.


3.4 Synthesis and testing of anchor molecules

109

An anchor molecule is henceforth defined as a sulfur-containing organic molecule used to

form a primary self-assembled monolayer (SAM) on a gold electrode surface. Unmodified

SAMS are composed of sulfur-containing molecules having no other functional groups. As

explained in Section 1.3, in the strategy that is the focus of this thesis, surface amines are

the reactive groups and are critical since they allow controlled coupling and are therefore

a key requirement for anchor molecules. An important feature of the anchor molecule is

that it should not be affected by repeated electrochemical cycles (Section 3.1). As explained

in Sections 3.2 and 3.3, the composition of the anchor monolayer will profoundly affect the

reactions at the surface as well as the electron transfer through the monolayer. Therefore, the

anchor molecule must be carefully designed. Both thioanilines and amide-containing alkyl

thiols were investigated in this thesis as potential anchors. In order to address the issue

of spatial resolution of the array down to the first amide coupling, some anchor molecules

were synthesised with their amine group protected by a carbamate moiety as shown in

Scheme 3.1. Some details of their synthesis and preliminary results on their use in forming

monolayers will be presented.

3.4.1 Thioaniline-based anchor

The lone pair of the nitrogen atom of aniline derivatives is engaged in resonance and as

such, anilines have low pKa's (pKa 4) and are poor nucleophiles in solution (Figure 3.14).

However, it has been shown that the pKa of the amine group of 4-aminothiophenol (4-ATP)


Figure 3.14: The resonance forms of aniline.

Figure 3.15: The resonance forms of 4-aminothiophenol

increases significantly if 4-ATP forms a monolayer on the surface of a gold substrate.157,189

Strikingly, Xiao and coworkers observed no difference in reactivity between cystamine and

4-ATP on gold surfaces. 157 With a pKa of 6.9 ± 0.5 189,190 surface-bound 4-ATP should be

considered sufficiently reactive for reaction with esters via its amino group. This increase

in pKa can be understood by looking at the nature of the gold-sulfur bond. The sulfur

atom in thiols and disulfides exhibits partial negative charge character.120,191 Considering

the resonance forms of 4-aminothiophenol (Figure 3.15), it is easily seen that once bound to

gold, resonance form B would be destabilised leading to an increase of the pKa of the amino

group of 4-aminothiophenol. Since 4-ATP had increased reactivity once bound on SAMS,

disulfide 44 was synthesised as a potential anchor molecule.


's-Q'-so,on a. lB)S(~ J. b.

Iflu" \ ~- b -flBu'" " I • •

~ OH ~ 0 CI a HN-Q-s

22 43

I"Q-f d. "Q-f•

°H~SH o HN-Q-S

245 44

III

Scheme 3.1: Synthesis of aniline-based anchors. a. Triphosgene, dry CH2CI2, dry pyridine,N2,-20°C b. 4-aminophenyldisulfide, dry CH2Ch, N2, -20°C c. 1% HCI/MeOH d. Zn,AcOH, reflux.

3.4.1.1 Synthesis of disulfide 44

The synthetic scheme for 44 and 45 was similar to that employed for the aniline monomers

presented in Section 2.3 and is outlined in Scheme 3.1. A solution of 4-(tert-butyldimethyl-

silyloxy)phenol (1, 0.5 g, 2.2 mmol, 1.0 equiv.) in dry methylene chloride (45 mL) was

added dropwise to a solution of triphosgene (0.5 g, 1.7 mmol, 0.8 equiv.) at -20°C in dry

methylene chloride (200 mL) in the presence of dry pyridine (1.2 ml, 14.4 mmol, 6.5 equiv.)

to yield carbamoyl chloride 2. To this reaction mixture a solution of 4-aminophenyldisulfide

(0.4 g, 1.6 mmol, 0.7 equiv.) in dry methylene chloride (45 mL) was added dropwise.

After the reaction was complete as judged by TLC, the reaction mixture was washed with


water (3 x 50 mL) and brine (50 mL) and dried over sodium sulfate. The solvent was

then removed in-vacuo. The residue was purified by flash column chromatography yielding

compound 43 as a yellow oil which crystallisied upon standing (60% yield). Disilyl ether 43

was characterised by IH-NMR, 13C-NMR, FT-IR spectroscopy and CHN elemental analysis.

The removal of the t-butyldimethylsilyl protecting groups of 43 was effected by reacting 43

in a 1% solution of hydrochloric acid in methanol at room temperature for three hours. The

final compound 44 was isolated as an off-white solid in 95% yield from silyl ether 43 and

was characterised by IH-NMR, 13C-NMR and FT-IR spectroscopy. The simplicity and well

established chemistry used in this synthetic sequence makes it amenable to preparation of

the target on gram scale. Disulfide 44 can easily be reduced to its thiophenol equivalent

45 by refluxing in acetic acid in presence of zinc dust (Scheme 3.1). Reducing 44 to 45,

however, is not necessary since it has been shown that disulfides produce monolayers that

are indistinguishable from those formed by the corresponding thiols. 138 Disulfide 44 was

therefore used in the studies presented in this chapter.

3.4.1.2 Growth of monolayers of disulfide 44 and thiol 45

The formation of monolayers of both 44 and 45 was evaluated by Fourier-Transform In

frared spectroscopy. The next two sections will present some preliminary results on the

formation and electrochemical behaviour of monolayers of disulfide 44 and thiophenol 45

on polycrystalline gold surfaces.

The infrared spectra of monolayers prepared from disulfide 44 closely resemble that of

monolayers prepared from thiophenol45 (Figure 3.16). Moreover, the spectra of monolayers

@ >: '"tl ~ ~ ~

/c.

~ ~ ~ ~ ~ t:""l ~ ~ a :< a t:""l~ ~ f]

c.b.a.

1710

.0

T15

00W

ave

nu

mb

er

(em

'1)

1000

...,

-I

II

II

II

II

I~

3500

3550

3600

3650

3700

3750

3800

Wa

ven

um

be

r(e

m-1

)

0.00

0.02

2l c: ~ 5l .0 «

1500

1000

0.00

0.02

1l c: ~ U> .0 «

Fig

ure

3.16

:F

T-I

Rsp

ectr

aof

mon

olay

ers

form

edfr

oman

chor

mol

ecul

es4

4an

d45

.a.

FT

-IR

spec

tru

mo

fa

mon

olay

ero

f4

4af

ter

incu

bati

onin

a1

mM

etha

noli

cso

luti

onof

44

for

48ho

urs.

b.F

T-I

Rsp

ectr

um

of

am

onol

ayer

of

44

afte

rin

cuba

tion

ina

1m

Met

hano

lic

solu

tion

of

44

for

18ho

urs.

c.F

T-I

Rsp

ectr

um

ofa

mon

olay

erof

45

afte

rin

cuba

tion

ina

1m

Met

hano

lic

solu

tion

of4

5fo

r18

hour

s.In

set:

Exp

ansi

ono

fth

e80

0to

1800

cm-1

regi

on.

Dif

fere

ntgo

ldsu

rfac

esw

ere

used

for

form

ing

each

mon

olay

erst

udie

din

this

figu

re.

..... ..... ""


of 44 and 45 were in close agreement with the FT-IR spectrum of 44 in KBr (Figure 3.17).

The amide I band (1710.0 cm-1)192 is weak in the FT-IR spectra of monolayers of 44 and

45 (Figure 3.16). However, the amide II (1530.8 cm-1) and the amide III band (1232.7

cm-1) are stronger (Table 3.1).

Considering the selection rule for an infrared radiation at a surface (Figure 3.18), the

very weak C=O stretch and the strong amide II and amide III bands suggest that the

carbonyl group lies perpendicular to the plane of incident light, and therefore parallel to

the gold surface. Furthermore, the stretching vibration of the hydroquinone ring at 1508.9

cm-1 is strong and sharp,27 suggesting that the hydroquinone ring lies mainly in the plane

of incident light (perpendicular to the surface). The sharp benzene ring stretching band at

1592.7 cm -1 was assigned to the thioaniline ring190 and also suggests that it lies mainly in

the plane of incident light. The formation of the monolayers was reproducible between gold

electrode surfaces, however, further studies would be required to verify these suppositions.

3.4.1.3 Cyclic Voltammetry studies of monolayers formed from disulfide 44

Monolayers of disulfide 44 on polycrystalline gold surfaces were studied by cyclic voltam

metry at a scan rate of 50 mV/s or 100 mV/s using 0.1 M sodium phosphate monobasic /

sodium phosphate dibasic buffer (pH 7.1) as electrolyte (Figure 3.19).

Irreversible modification of monolayers of disulfide 44 The cyclic voltammogram

obtained for a monolayer of 44 cycled between -0.1 and 1 V reveals a broad oxidation peak

(E) centered at 0.479 V on the anodic wave of the first scan (black line) that is followed


a.

0.01

1508.9

0.01

0.00

1000

1084.5

1228.9

1500

1530.8

1592.3\

3000 2500 2000Wavenumber (em-1 )

4000 3500 3000 2500 2000 1500 1000

Wavenumber (em -1 )

3500

0.005

3738.0

8c:

~~ 0.005

b.

...1:-:----------3O\iO.-..----.-..--..-......------::c2000

::c.=--------·"1000--

Wavenumber (em -1)

Figure 3.17: FT-IR spectra of monolayer of 44 on polycrystalline gold and of 44 in KBr.a. FT-IR spectrum of a monolayer of 44 on polycrystalline gold. Inset: FT-IR spectrumbefore smoothing and baseline correction. b. FT-IR spectrum of 44 in KBr.


WavenumberA relative A Bond Assignment

(cm·1)

1012.3a 0.0015 0.21 C-O stretching

1181.3a 0.0013 0.19 (O=)C-O-C stretching

1203.3 0.0028 0.40

1232.7b 0.0031 0.44 N-H bending (Amide III)

1309.8a 0.00091 0.13 CoN stretching (Amide III)

1396.5 0.0010 0.14

1508.ge 0.0070 1.00 C=C stretching (HQ)

1529.5" 0.0020 0.29 C-N, N-H bending, stretching (Amide II)

1592.7d 0.0022 0.31 C=C stretching

1710.0' 0.00052 0.07 C=O stretching (Amide I)

3735.8 0.0013 0.19 NH2stretching

116

Table 3.1: Table of FT-IR bands of 44 in KBr and as a monolayer on polycrystalline gold.Assignments according to: a. Ferguson et al.;192 b. Ahn and Kimi193 c. Kim et al.i 27 d. Yuet al. 190

by a rapid rise in measured current after 0.8 V (Figure 3.19a). The absence of peak for

oxidation process E in the anodic wave of the second scan and the shift of the peak position

with scan rate (Table 3.2) provide good evidence that the oxidation process is irreversible.

The observed irreversibility of the oxidation process is in good agreement with related

systems reported in the literature and corresponds to the oxidation of hydroquinone to

benzoquinone (Figure 3.19).27,33,34,46 This oxidation is followed by hydrolysis of the oxonium

intermediate and subsequent loss of carbon dioxide resulting from rapid degradation of the

carbamic acid45 to yield 4-aminothiophenol and benzoquinone (Figure 3.19). The absence of

CHAPTER 3. SELF-ASSEj\lIBLED MONOLAYERS

p-polarisation

J \

117

s-polarisalion

Figure 3.18: Selection rules for the reflection of the electrical component of an infraredlight off of a metal surface. The sum of the electrical component vectors is shown on theright. A red point denotes a sum of zero. Top: p-polarised light, the electrical componentvector is within the plane of incident light. The phase of the reflected vector is shiftedby 900 with respect to the incident vector therefore, the sum of the incident and reflectedelectrical component vectors result in an electric field at the surface. Bottom: s-polarisedlight, the electrical component vector is rotated 900 with respect to the plane of incidentlight. The phase of the reflected vector is shifted by 1800 with respect to the incident vector,therefore, the sum of the incident and reflected electrical component vectors does not resultin an electric field at the surface.

a hydroquinone oxidation peak on the second scan proves that the removal of hydroquinone

from monolayers of disulfide 44 is clean and efficient. The reduction wave of the first scan

shows a broad reduction peak centered at 0.487 V which was shifted to 0.479 V in the second

scan. This peak has not been assigned, however it should be noted that when the monolayer

is cycled from a to 0.8 V this peak is not present in the cyclic voltammogram suggesting that

the reduction peak at 0.487 V is related to electrochemical processes occurring at voltages


over 0.8 V (Figure 3.19b.). It is likely possibility that the gold surface is oxidised at potentials

over 0.8 V. As well, we expect the monolayer to be modified above this potential. A more

detailed explanation will be presented in the following paragraphs.

Surface coverage and peak potential

fO=~nFA

(3.4)

The surface coverage can be derived from equation 3.4 where fa is the coverage in mol/cm2 ,

Q is the charge or peak area in C, n is the number of electrons involved in the process, F is

the Faraday constant (96,485.34 C/mol) and A is the working electrode areag in cm2. The

peak areas were determined using the GPES software assuming a straight baseline.

Both the peak potential for the redox process E (E p ), the width at half peak height

(Ep1 / 2) and the surface coverage (fa) of disulfide 44 were reproducibly observed using two

different electrodes and were fairly constant for a given electrode (Table 3.2, electrode 1) but

varied between electrodes (Table 3.2, electrodes 1 and 2). The surface coverage and area per

molecule (Table 3.3) of monolayers of 44 is larger than that of SAMs of 4-arninothiophenol

and comparable to both SAMs of 3-aminothiophenol and H2Q(CH2h2SH. Approximating

44 to a square, the distance between two molecules of 44 is between 5.2 and 6.3 A. This

value is slightly higher than the value for a typical (J3 x j3)R30° packing arrangement of

long chain alkyl thiols on a Au(111) surface (approximately 5 A).

gThe area was determined by mesuring the diameter of the working electrode area that was exposed tothe electrolyte solution. The formula for the area of a circle (A = 7fr2) was then used to calculate the area.


a.

0.00010

0.00008

O.D0Ca3

0.rxxxJ4

0.00002

ooסס0.0

-0.00002

0.0 0.2 0.4 0.6 0.8 1.0Potential M

¢ ¢°l,A( ° 0yOH

ONH -rL -e°2ONH ONH,"" H,o ""I ""I

1 1 1

0.80.602 0.4

Potential M

b.

0.00010

000008

0.00003

000Xl4

?- 0.00002

0./XXXXl

-0.00002

-D.00Xl400

Figure 3.19: Cyclic voltammogram of a monolayer of disulfide 44 on polycrystalline gold.Reference electrode: AgIAgCl13 IvI NaCI, scan rate = 50 mVIs, 0.1 M sodium phosphatebuffer (pH 7.1) as electrolyte. Cyclic voltammogram presented is representative of the dataobtained for at least 10 monolayers of 44. Top: Cycle from -0.1 to 1.0 V. Middle: Oxidationof 44 leads to the generation of 4-aminothiophenol via formation of benzoquinone and atransient carbamic acid. Bottom: Cycle from a to 0.8 V. The dip in current at 0.2 V couldnot be reproduced and was therefore attributed to experimental error.


Electrode Ep

(V) A (cm 2 ) Q(10'5 C) E,fl (V) ip

(10·5A) r O(1 0.10 moll em 2 )

0.479 0.38 4.476 0.123 1.699 6.10

0.479 0.38 4.241 0.121 1.649 5.78

2 0.517 0.38 3.055 0.150 a 2.220 4.17

a. Measured graphically. Method checked for electrode 1. The value oblained for elec1rode 1 by the graphical method and usingthe GPES software was 0.121 in both cases.

120

Table 3.2: Data from CV scans of monolayers of 44 on various gold surfaces. E (V) = peakpotential in volts, A (cm2) = working electrode area in cm2 , Q (10-5 C) = peak area inCoulombs, E1/ 2 (V) = width at half peak height in volts, ip = peak current in Ampere, rO

= surface coverage in moljcm2 . Reference electrode: AgIAgCl13 M NaCl, scan rate = 50mV/s (electrodes 1 & 3) or 100 mV (electrode 2), 0.1 M sodium phosphate buffer (pH 7.1)as electrolyte.

The width at half peak height for both electrodes is larger than the expected valueh

of E1/ 2 = 45.3 mV. 195 The gold electrodes used in this study exhibit multiple crystallo-

graphic textures (Figure 3.29). The main structures are Au(lll), Au(200) which is equiv-

alent to Au(lOO) and Au(220), which is equivalent to Au(llO). Thiols arrange differently

on each crystallographic surface (Figure 3.20). It has been shown that rnonolayers of 4-

pyridinethiolate on Au(l11) and Au(lOO) surfaces give rise to very different cyclic voltam-

mograms. 196 It is therefore quite possible that the many arrangements of 44 on the vari-

ous crystallographic textures are partially responsible for the broadening of the oxidation

peak. Moreover, the interactions between highly concentrated redox centers in undiluted

self-assembled monolayers have been shown to influence the shape of the cyclic voltammo-

gram. 197 199 Since the monolayers used in this study were undiluted SAMS of disulfide 44,

interactions between the redox centers cannot be ruled out. More studies would be required

hExpected value is 90.6/n mV where n is the number of electrons involved in the process.


Molecule ro Area per molecule(10- '0 mol/em') (A'/moleeule)

2-ATP 3.4 48.9

3-ATP 7.3 22.8

4-ATP 9.2 18.1

Thiophenol 2.8 59.3

H,QCH,SH 3.2 51.6

H,Q(CH,).SH 4.7 35.3

H,Q(CH,)"SH 5.8 28.9

44 6.1 27.2

44 5.8 28.7

44 4.2 40

121

Table 3.3: Surface coverage and molecular area for various monolayers on polycrystallinegold surfaces. Aminothiophenol and thiophenol data from Batz et al. 194 Data obtainedby reductive desorption of monolayers of aminothiophenols and thiophenol. Data for thealiphatic thiols from Hong and Park. 173 Values for the surface coverage calculated using theintegration of the redox peak of hydroquinone assuming a 2e-, 2H+ process. The values for44 were obtained in the same way as Hong and Park. Area per molecule values calculatedas follows: Coverage converted to mollA2 . This value was then multiplied by Avogadro'snumber (rD,I). The final value was obtained by taking the reciprocal of rD,I.

to definitely define the basis for the behaviour of these monolayers. Consistent and repro-

ducible surface coverage of electrodes is critical for generating reliable devices and should

not be neglected when fabricating an array of electrodes since variability could limit the use

of such arrays.


••

a. b.

E] • Thiol

0/. Gold alom

Unit cell

c.

Figure 3.20: Arrangement of thiols on different gold surfaces. a. Thiols on Au(l11). b.Thiols on Au(llO). c. Thiols on Au(100).

Electrochemically promoted modification of monolayers of disulfide 44 A gold

electrode modified with a monolayer of 44 was characterised by cyclic voltammetry (2 cy-

cles of-O.l V to 1 V to -0.1 V) in 0.1 M sodiumphosphate buffer (pH 7.1) and then kept

at a potential of 0.6 V for 60 s in an effort to ensure that all of 44 had been converted

to 4-aminothiophenol. The resulting monolayer was then rinsed thoroughly with doubly

deionised water and anhydrous ethanol and then characterised by FT-IR spectroscopy (Fig-

ure 3.21).

The main hydroquinone ring stretching band at 1509.0 cm-1 in the FT-IR spectrum

of the unmodified monolayer of disulfide 44 is absent from the FT-IR spectrum of the

modified monolayer of 44 where it is replaced by a broad absorption band at 1466.4 em-1.

0.00

03

CJa.

0.00

02

~1

50

9.0

C.

0.01

0..,

Q0.

0001

'l::l

$

~'c

0.00

8-l

::l

0.ססOO

~~

1013

~t

0"'(

0~

-0.0

001

W

~:e

10

47

.0

""']D~

15

31

.0«

?""

I1

23

0.2

!15

93

.7-0

.000

2C

f) t.TJ~

1310

.2

A/6

05

2t--<

0.0

04

-1

20

4.V

\1

14

39

.4-0

.000

3'"!

j1

01

3.2

10

85

.5---

_.I ~

0.0

02

+-'

./'"

,"u

v'tJ

'-V"\

....

1\;

'J

v~""'\

-o.O

C04

Cf)

Cf) t.TJ

-0.0

005

15

08

.8

~0

.00

0I

Ii

II

iI

II

II

800

000

1000

1100

1200

1300

1400

1500

1600

1700

1800

800

000

1000

1100

1200

1300

1400

1500

1600

1700

1800

t--<W

aven

urrt

:er

(em

")W

aven

urrt

ler

(an

")

~0.

0000

8..,

d.:s:

0.00

006

-I0

b.0.

0000

4:<

0.13

5..,

0$

0CX

XX

l2t--< ~

'c

~0

13

0-I

DN

H2

'T-~OOOOOO

''''t:I

~~

1l-o

.CX

XX

l2C

f):'l

<l:c O

J-o

.CXX

Xl4

-e~t8

~63

10

58

.5

i...

""..

...

It16

80

.40 V> .0 <(

-0.0

0006

822.

30.

120

15

64

.71

65

7.3

-0.0

0008

01

15

-I-I

.../

"-0

.000

10

-0.0

0012

~1

26

2.0

1100

1200

1300

1400

1500

1600

1700

1800

II

II

II

1000

800

000

1000

1100

1200

1300

1400

1500

1600

1700

1800

Wa

ven

um

be

r(e

m")

Wav

enLn

t>er

(an

-,)

Fig

ure

3.21

:C

ompa

riso

nof

the

FT

-IR

spec

tra

ofa

mon

olay

erof

44

befo

re(a

and

c)an

daf

ter

(ban

dd)

cycl

icvo

ltam

met

ry.

Sp

ectr

aac

quir

edat

are

solu

tion

of2

cm-I

.a.

FT

-IR

spec

tru

mof

am

onol

ayer

of4

4on

gold

befo

recy

clic

volt

amm

etry

.N

oba

seli

neco

rrec

tion

.b.

FT

-IR

spec

tru

mof

am

onol

ayer

of4

4on

gold

afte

rcy

clic

volt

amm

etry

.N

oba

seli

neco

rrec

tion

.c.

Sec

ond

deri

vati

veof

the

FT

-IR

spec

tru

msh

own

inpa

nel

a.d.

Sec

ond

deri

vati

veof

the

FT

-IR

spec

tru

msh

own

inpa

nel

b.

......

tv eN


This suggests that the hydroquinone group has been cleaved from 44 and t.he modified

monolayer is expected to now be a monolayer of 4-aminothiophenol instead. The weak

band at 1588.8 cm -1 and the shoulder at 1492.6 cm -1 (benzene ring stretching bands)

suggest that 4-aminothiophenol is now present at the gold surface, since these values have

been previously reported for monolayers of 4-aminothiophenol on gold. 190 However, the

high absorbance of the modified monolayer of 44 suggests a multilayer structure or, as a

minimum, a structure that is not a strict monolayer of 4-aminothiopheno1. 200

From the second derivative of the FT-IR spectrum of a monolayer of 44 before cyclic

voltammetry (Figure 3.21c) three main features can be clearly identified; a band at 1203.2

(C-O stretch), one at 1508.8 (hydroquinone ring stretch) and one at 1592.7 cm- 1 (benzene

ring stretch) with the peak at 1508.8 cm-1 being the dominant feature. The corresponding

bands, and relative intensities, are also observed in the FT-IR spectrum (Figure 3.21a). The

high level of noise in the second derivative trace of the FT-IR spectrum of the monolayer

of 44 after cyclic voltammetry (Figure 3.21d) complicates the identification of detailed

features. However, three main bands were identified at 876.3, 1181.0 and 1262.0 cm -1 with

the peak at 1262.0 cm-1 being the dominant feature. The band at 1262.0 cm-1 was assigned

to the C-N stretching band. The three bands mentioned above (876.3, 1181.0 and 1262.0

cm -1) were also present in the FT-IR spectrum of a monolayer of 4-aminothiophenol on gold

reported by Yu et al.. 190 More studies, beyond these preliminary efforts, would be required

to ascertain the structure of the monolayer after the cyclic voltammetry experiment.


a.

--Scan 1--Scan 2--Scan 3--Scan 4--ScanS

0.0003 0.00004

g-

0.0002 000002

0.0001 0.4 0\3 0.8

$ Potential M-

OOסס.0

-0.0001

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

PotenUal M

b.

- e

NK/ . NH/ d~+0- (-f ")J -~-.... ).J_.-.... . #

--L ...L ...L

Figure 3.22: Cyclic voltammogram of a monolayer of 4-aminothiopheno!. a. Cyclic voltammogram of a monolayer of 4-aminothiophenol in O.03M sodium phosphate buffer (pH 7.1)against AgIAgCl13 M NaC!. b. Previously proposed mechanism of the redox behaviour of4-aminothiophenol at the gold electrode surface. 201 ,202


Cyclic voltammetry of monolayers of 4-aminothiophenol on gold electrodes Mono-

layers of 4-aminothiophenol on polycrystalline gold surfaces were analysed by cyclic voltam-

metry (0.03 M sodium phosphate buffer, pH 7.1) in an attempt to clarify the results observed

in the FT-IR spectrum of monolayers made of 44 after electrochemical modification (Figure

3.22). The oxidation peaks A (1.05 V) and B (0.527 V) have been associated in the liter-

ature with the oxidation of 4-aminothiophenol to the 4-aminothiophenol radical cation and

ammonium hydroquinone respectively.201,202 Peaks C (0.402 V) and D (-0.315 V) could

not be assigned by refering to the literature. However, the voltammograms obtained were in

good agreement with previous studies involving 4-aminothiopheno1.201 203 The mechanism

proposed for the electrochemical behaviour of 4-aminothiophenoI2ol ,203 (Figure 3.22b) al-

lows for the possible radical polymerisation of 4-aminothiophenol on the gold surface. This

polymerisation could be the basis for an increase in layer thickness and hence an increase

in the intensity of the FT-IR signal of the thin film as observed in the FT-IR spectrum of

a monolayer of 44 after cyclic voltammetry (Figure 3.21) relative to the monolayer before

cyclic voltammetry.i However, further studies would be required to verify this preliminary

study and to fully understand the fate of the monolayer during the cyclic voltammetry exper-

iment. Regardless of the issues presented above, to the best of our knowledge, these results

are the first studies of electrochemical deprotection of self-assembled protected-amines at

electrode surfaces and reveal that we can carry out this reaction reproducibly.

iThe intensity of the FT-IR signal of thin films has been shown to increase with an increase in thicknessof the thin films. 20o


Circumventing the degradation of the deprotected monolayers As explained above,

self-assembled monolayers of 4-aminothiophenol can be degraded if the potential employed

is above 0.8 V (Ag IAgCl13 M NaCI reference electrode). However, the deprotection poten

tial of the hydroquinone carbamate of disulfide 44 was found to be in the vicinity of 0.5 V.

In most of the studies presented in this thesis the potential was varied up to 1 V. When the

maximum potential is kept under 0.8 V the monolayer, both the electrode surface and the

monolayer appear to be unmodified (Figure 3.19b). During device operation, the potential

applied would be kept in the vicinity of the deprotection potential of the hydroquinone car

bamate meaning that the degradation of the monolayers of 4-aminothiophenol observed in

our experiments would likely not be significant. As such, disulfide 44 can still be considered

as monolayer components for device fabrication.

Conclusion Both cyclic voltammetry and FT-IR data suggests that the hydroquinone

carbamate in monolayers of 44 is rapidly and irreversibly cleaved cleanly and efficiently

yielding benzoquinone, carbon dioxide and a significantly modified monolayer of 44. Cyclic

voltammetry of monolayers of 4-aminothiophenol (4-ATP) shows an oxidation peak in the

expected region for oxidation of hydroquinone to benzoquinone (0.520 V vs 0.479 to 0.532

V). It is therefore reasonable to propose that the monolayer will be irreversibly modified be

yond the desired product (a monolayer of 4-ATP). This suggests that such a monolayer might

not be useful for the fabrication of an electrochemical array as described in Section 1.3.5.

The degradation of the monolayer appears to be severe after cyclic voltammetry experiment

and device failure could occur after only a few cycles since the desired 4-aminothiophenol


monolayer has already undergone undesirable modification after only one CV cycle. How

ever, cyclic voltammograms where the maximum oxidising potential has been lowered to 0.8

V do not show the same degradation (Figure 3.19) peaks. As a consequence, monolayers

of disulfide 44 could be used for device fabrication if the hydroquinone carbamate depro

teet ion potential is kept below 0.8 V. However, further studies would be necessary to fully

understand the behaviour of these monolayers.


3.4.2 Amide-containing dialkyldisulfides as anchors - Synthesis of 55

129

As explained in Sections 3.2.2 and 3.3.2.2, the introduction of amide groups into the spacer

units of thiols or disulfides can significantly improve the efficiency of electron transfer and

alter various other properties of the monolayers. Since electron transfer through the mono

layer is critical to the development of electrochemical arrays of the type proposed here,

a method for synthesising amide-containing diaminoalkyldisulfides was devised in order to

enable testing of various amide-containing linkers as anchors.

3.4.2.1 Retrosynthetic analysis

Two main routes were identified for the synthesis of disulfide 55 (Scheme 3.2). 55 could

be obtained either by direct coupling of 1,6-diamine with 53 (Route 1) or by coupling of

64 with 53 followed by removal of the tert-butyloxycarbonyl (BOC) group under acidic

conditions (Route 2). The aim in this section is only to demonstrate the ease with which

such molecules can be prepared. The generality of the chemistry presented below allows for

variation in the position of the amide linkage within the anchor molecule.

3.4.2.2 Synthesis of diesterdisulfide 53

Diester 53 was synthesised by modifying a literature procedure.204 A solution of 4,4'-

dithiodibutyric acid (0.48 g, 1.99 mmol, 1 equiv.), 1-Ethyl-3-(3-dimethyllaminopropyl)

carbodiimide hydrochloride (0.95 g, 4.96 mmol, 2.49 equiv.) and N-hydroxysuccinimide

(0.50 g, 4.31 mmol, 2.17 equiv.) in dry acetonitrile (20 ml) and dry tetrahydrofuran (40

ml) was stirred at room temperature for 20 hours. The solvent was reduced in-vacuo and


Route 1:>

2

54

53

130

Scheme 3.2: Retrosynthetic analysis of disulfide 55.

the remaining solution taken up in ethyl acetate (100 ml) and washed with water (50 ml)

and brine (20 ml). The organic layer was dried over sodium sulfate and the solvent removed

in-vacuo to afford a white solid in 90% yield. This solid was identified as 53 by 1H-NMR

and FT-IR spectroscopy.

3.4.2.3 Synthesis of 55 via coupling of 1,6-diaminohexane to 53

When reacting a diamine with diacid 53, one has to be concerned with the possibility of

forming the undesired cyclic product (Figure 3.23). In order to minimise the probability of

forming the cyclic product, a dilute solution of 53 (CS3 = 6 - 16 mM) in either chloroform,

methanol or acetonitrile was added dropwise to a solution of 1,6-diaminohexane in the

same solvent (Cdiamine = 61 - 117 mM). The products obtained appear as a mixture, one

of which is a macrocyclic compound (Figure 3.24). This approach is therefore of limited


A ):(0~N-h 0~ H2N~f\ti.:

o 0 ( '; -0

5-5

53

o "'l, ~r-'<N-0 NH H~ 0H~ ~ -----------..

o 0 ) 0 0 0

5-5 5-5

Cyclic product

\"'l,'< 0"

5-555

Dithio-diamine product

Figure 3.23: Coupling of 53 with 1,6-diaminohexane. A distribution of both cyclicand dithio-diamine products is expected. Dilute conditions and addition 53 to 1,6diaminohexane was predicted to favour the formation of 55 and disfavour the dashed reaction to form the cyclic product.

use for producing amide-containing dialkyldisulfides, even with a diamine chain-length of 6

carbons. A second approach via monoprotected diamines is investigated in the next section.

3.4.2.4 Synthesis of 55 via coupling of monoamine 63 to diesterdisulfide 53

Synthesis of 63 tert-butyl 6-aminohexylcarbamate (63) was synthesised by modifying a

literature procedure (Scheme 3.3).205 In a typical procedure 1,6-diaminohexane (3.34 g, 28.8

mmol, 4.99 equiv.) was dissolved in chloroform (100 ml) and the solution was cooled to DoC.


'*

'*

132

'-T-'2

I3.0

1l:~ '*'*

'-T-' '-T-'2 2

I2.5

ppm

'-T-'2

I2.0

'-T-' '-T-'2 2

I1.5

I3.0 2.5

l"'~00"

s-s55

I2.0ppm

I1.5

Figure 3.24: Coupling of 53 to 1,6-diaminohexane. Top: Proton NMR spectrum of thepartially purified cyclic product in MeOH-d4 at 500 MHz. Bottom: Proton NMR spectrumof the isolated mixture of the coupling product of 53 and 1,6-diaminohexane in MeOH-d4at 500 MHz. Signals arising from impurities are marked with a star.


H2N~NH25 equiv.

tBu'OJLOJloABu1 equiv.

~

CHCl s' O°C to r.t., 24 hr

Scheme 3.3: Synthetic scheme for the synthesis of N-Boc-1,6-diaminohexane (63).

53

TEA (2 equiv.)CH2CI2

20°C54

Scheme 3.4: Synthetic scheme for the synthesis of 54.

A solution of di-tert-butyl dicarbonate (BOC2 0, 1.26 g, 5.76 mmol, 1 equiv.) in chloroform

(10 mL) was added dropwise under vigorous stirring. Once the addition was complete, the

reaction was stirred at room temperature for 24 hours after which time a precipitate had

formed (assumed to be the diprotected by-product by analogy to the study of Krapcho and

coworkers.205 ) After filtration of the precipitate the solvent was removed in-vacuo. The

residue was taken up in ethyl acetate (100 ml) and washed with half-saturated brine (3 x 50

ml). The organic layer was dried over sodium sulphate and the solvent removed in-vacuo

yielding 63 as a colourless oil (0.8 g, 66%). The IH-NMR spectrum of 63 is in agreement

with the literature205 and its 13C-NMR spectrum is in agreement with its structure (Figure

3.25, 9 signals expected and 9 signals observed).


I

6I

5I I4 3

ppm

I

2Io

63

Acetone

I fJ,

I

I IIi i i

60 40 20ppm

I Ii I i I i I I i I i I i I I I I I

200 180 160 140 120 100 80 60 40 20 0ppm

Figure 3.25: IH and 13C NMR spectra of 63. Top: IH-NMR spectrum of 63 in Acetoned6 at 500 MHz. Bottom: 13C-NMR spectrum of 63 in Acetone-d6 at 125 MHz. Inset:Expansion of the 60-20 ppm region.


{S~~4~t"B)2

54 55

Scheme 3.5: Synthetic scheme for the synthesis of 55.

Synthesis of diamide disulfide 54 With monoamine 63 in hand, we proceeded to syn-

thesise 54 (Scheme 3.4). A solution of 63 (0.11 g, 0.26 mmol, 1.0 equiv.) and triethylamine

(77 J.d, 0.56 mmol, 2 equiv.) in methylene chloride (10 ml) was added to solution of 53

(0.13 g, 0.61 mmol, 2.35 equiv.) in methylene chloride (10 ml) while vigorously stirring the

mixture at room temperature. The reaction was stirred at room temperature for 5 hours

after addition was completed. The reaction mixture was then washed with 3% aqueous

hydrochloric acid (10 ml) and brine (10 ml) and dried over sodium sulfate. The solvent was

removed in-vacuo to yield 54 as a white solid in 92% yield. The 1H-NMR spectrum of the

product is consistent with the expected structure 54 (Figure 3.26).

Synthesis of anchor 55 With 54 in hand we proceeded to remove the BOC groups to

yield 55 (Scheme 3.5). Trifluoroacetic acid (0.5 ml, 5% v/v, 7.1 mmol, 29.8 equiv.) was

added to a solution of 54 (0.15 g, 0.24 mmol, 1.0 equiv.) in methylene chloride (10 ml) at

room temperature. The reaction mixtre was stirred for 5 hours and the solvent removed

in-vacuo yielding an orange oil in quantitative yield that was characterised by 1H-NMR,

13C-NMR and FT-IR spectroscopy. The loss of the BOC signal at 1.39 ppm in the 1H-NMR

spectrum of the obtained oil, when compared to 54 the signals expected for 55, suggests that


54Acetone

ppm

Acetone

136

ppm

Io

Figure 3.26: IH-NMR spectrum of 54 in acetone-d6 at 500 MHz. Inset: Expansion of the3.5 to 1.0 ppm region.

both amines have been fully deprotected. All the expected signals for the methylene protons

are present in the 1H-NMR spectrum (Figure 3.27a). Two broad signals at 13.6 and 12.0 ppm

totaling 0.6 integration units j are observed that we hypothesise to correspond to the three

ammonium protons. Two multiplets at 8.34 and 7.73 ppm totaling 1.21 integration units

were also observed but these peaks could not be assigned. The reaction product contained

jSignals integrated with respect to the signal at 1.8 ppm. The reference signal is assigned a value of 2integration units.


small amounts of other identifiable impurities, notably, 2-methyl-2-propanol (peaks at 2.09

and 1.28 ppm, 5 m% estmated by integration).

The quadruplet at 160.46 ppm (C=O, Jl = 36 Hz) in the 13C-NMR spectrum of 55

suggests the trifluoroacetic acid salt of the product has been isolated.k

The FT-IR spectrum of the final product shows bands characteristic of a primary am

monium ion at 3298.6, 3093.2, 1550.6 and 1130.0 cm-1 j the trifluoroacetate anion at 1635.9

and 1195.0 cm-1; and amide 1670.9 cm-1 (Amide I).

It was concluded from IH-NMR, 13C-NMR and FT-IR spectra that the isolated oil was

the desired product 55.

Conclusion We have shown that amide-containing dialkyldisulfides could be conveniently

synthesised in two steps from dithiodiester 53 as trifluoroacetic acid salts in excellent yields.

This method is amenable to a wide variety of diamines and dithiodiesters allowing for the

variation of the position of the amide linkage within the alkyl chain of the anchor. Such

molecules should readily allow the modulation of the electronic and structural properties of

self-assembled monolayers as described in Sections 3.2.2 and 3.3.2.2. Compound 55 is also

the first of a new series of compounds never before synthesised. It is, however, analogous to

the amide-containing dialkyldisulfides prepared by Bilewicz and coworkers (Figure 3.28).174

kThe splitting of the carbon signals at 160.46 ppm is due to the strong coupling of 19F to 13C.


a.Acetone

I , , i j U i

14.0 13.0 12.0 9.0 8.0ppm

7 6 5 4ppm

3 2 o

Acetone

I

c.

55

60 50 40 30 20ppm

b.

o2060ppm

U_._"T",---.----,.,----.----,,....-.--r-..--,----r--I,',.r(-...,....--,----,----,---.------,

155 200 180 160 140 120

i

115

160ppm

ppm

i

120

165

Figure 3.27: IH and 13C NMR spectra of 55. a. IH-NMR spectrum of 55 in acetone-d6at 500 MHz. Inset: The 14 to 7.5 ppm region. b. 13C-NMR spectrum of 55 in acetone-d6at 125 MHz, 512 scans. c. 13C-NMR: alkyl carbon region (20 to 60 ppm). d. 13C-NMR:The quadruplet for the tertiary carbon of trifluoroacetic acid is likely in the noise of thespectrum (J~pparent = 91 Hz). e. 13C-NMR: The quadruplet characteristic of the carbonylcarbon of trifluoroacetic acid (Jl = 36 Hz). Signals arising from impurities are denoted witha star.


C=O Symmetrical stretching

Bond Assignment

NH; (N-H) Symmetrical stretching

CH2 (C-H) Symmetrical stretching

Not assigned

RockingCH2 (C-H)

C-N Symmetrical stretching

COO· Asymmetrical stretching

NH3

+ (N-H) Symmetrical stretching

CH2 (C-H) Asymmetrical stretching

NH3+ (N-H) Symmetrical bending

CH2

(C-H) Symmetrical scissoring

CF3 (C-F) Symmetrical stretching

WavenumberIntensity

(ern")

3298.6 medium

3093.2 medium

2937.9 strong

2862.8 strong

1670.9 strong

1635.9 strong

1550.6 medium

1435.4 weak

1195.0 strong

1130.0 strong

794.4 medium

714.2 (d) medium

Table 3.4: FT-IR bands and assignments for diamine 55. FT-IR spectrum acquired as afilm deposited from a chloroform solution of 55 on a sodium chloride disk. Number of scans= 16, resolution = 4 cm -1.

Figure 3.28: Structures of the disulfides used by Bilewicz and coworkers.174 a. Disulfidediamide with an alkane overlayer, n = 9 or 12. b. Sidulfide diamide with an alkane overlayerand an acetamide head group.


3.5 General experimental

3.5.1 Synthesis


140

Hexanes, ethyl acetate and diethyl ether were obtained from Caledon and used without

purification. Triethylamine, pyridine and tetrahydrofuran were obtained from Anachemia

and were distilled before use. All deuterated solvents were obtained from Cambridge Iso

tope Laboratories, Inc.. Silica gel 60 was obtained from EMD (product # 9385 - 3). Di

tert-butyldicarbonate, 4-aminothiophenol, potassium thioacetate, 4-aminophenyl disulfide,

cystamine dihydrochloride, tert-butyl-dimethylsilyl chloride, 4-dithiodibutyric acid and N

hydroxysuccinimide were obtained from Aldrich. Triftuoroacetic acid, hydroquinone and

lithium aluminium hydride were obtained from Sigma-Aldrich. 1,6-diaminohexane was ob

tained from Fluka. 5-bromopentanenitrile, 6-bromohexanenitrile and 7-bromoheptanenitrile

were obtained from TCI America. All chemicals were used without purification.




chloride disc. Each spectrum is the average of 32 or 64 measurements. NMR spectra (lH






3.5.2 Monolayers

141

Only 18.3 MO water and 99% ethanol were used for rinsing of the modified and unmodified

gold slides. All buffers made using 18.3 MO water. Concentrated sulfuric acid was obtained

from Anachemia and 30% hydrogen peroxide solution was obtained from Caledon.

3.5.2.1 Substrate preparation

Glass slides coated with a 50 A chromium underlayer and aI, 000 A gold layer were

obtained from EMF corporation (product # CA134, Figure 3.29) and cut to between 1.5

and 2.0 cm in length by 2.54 cm wide. Prior to monolayer deposition, each gold slide was

cleaned in piranha solution (3 : 1 conc. H2S04 /30 % H2 0 2 ) at 90°C for 5 minutes in a

teflon container and rinsed with copious amounts of 18.3 MO water. The slides were then

rinsed with copious amounts of anhydrous ethanol, dried under a stream of dry nitrogen,

and stored in clean teflon containers until required for use.

3.5.2.2 Preparation of self-assembled monolayers

A cleaned gold slide was immersed in a 1 mM ethanolic solution of the corresponding thiol or

disulfide for one to two days. The slide was then rinsed with copious amounts of anhydrous

ethanol and dried under a stream of dry nitrogen. The modified plates were stored in clean

teflon containers until required for use.


9000

111

IlOOO

7000

IlOOO

5000 200

I 220

r 4000

311

:JOOO

2000 311 420

1000

20 40 60 BO 100 12026

Figure 3.29: X-ray diffraction pattern of a gold slide obtained from EMF corporation. Thepattern observed here is consistent with polycrystalline gold. Reference crystallogrphic dataobtained from the Inorganic Crystal Structural Database (ICSD).


Cyclic Voltammetry Cyclic voltammetry measurements were performed on a Echo

Chemie BV j.lAutolab-type II instrument in the Cyclic Voltammetry staircase normal mode

using a three electrode cell with an AgIAgCl13 M NaCI reference electrode, a platinum wire

counter electrode with the modified gold slides as working electrode, and 0.1 M phosphate

buffer as the electrolyte. The exposed surface of the working electrode was 0.38 cm2 . The

measurements were performed with the GPES v. 4.9 software and the peak area, the width

at half peak, and the peak height values were obtained using this software. The data was


plotted using Origin v. 6.1.

143

Fourier transform infrared spectroscopy All FT-IR measurements of monolayers

were performed using a ThermoNicolet Nexus-IR 560 spectrometer equipped with a mer

cury cadmium telluride (MCT) detector and a KBr beam splitter fitted with a specular

reflectance accessory and a polariser set at 0° (polariser situated before the sample). Dry

air was purged through the instrument chamber for at least 2 h before each measurement

and a strong air flow was maintained while taking the measurement. Each measurement

consisted of 1024 scans acquired using the single beam setting at 2 cm-1 resolution. A back

ground spectrum was collected and subtracted from the measured monolayer spectrum. The

resulting spectrum was subjected to baseline correction and smoothed as appropriate using

the OMNIC software (v. 6.0a). The resulting data was plotted using Origin v. 6.1. Un

less stated otherwise, peak-picking the spectra was done on immediately after background

substraction and before any baseline correction or smoothing.

3.5.3 Experimental

bis(4-(tert-butyldimethylsilyloxy)phenyl) 4,4-disulfanediylbis(4,1-pheneylene)

dicarbamate (43) Triphosgene (0.5 g, 1.7 mmol, 0.8 equiv.) and dry pyridine (1.2 ml,

14.4 mmol, 6.5 equiv.) were added to dry methylene chloride (200 ml) under nitrogen and

cooled to -20°C. A solution of 1 (0.5 g, 2.2 mmol, 1.0 equiv.) in dry methylene chloride

(45 ml) was added dropwise over 45 minutes. A solution of 4-aminophenyl disulphide (0.4

g, 1.6 mmol, 0.7 equiv.) in dry methylene chloride (45 ml) was then added dropwise over


20 minutes. The resulting solution was stirred overnight and washed with a 1% aqueous

hydrochloric acid, water and brine (50 ml each). The organic extracts were dried over

sodium sulphate and the solvent evaporated in-vacuo to afford a yellow oil (quantitative)

which was then purified by two flash chromatography columns (30/1 toluene / ethyl acetate

followed by 5/1, hexanes / ethyl acetate) as a light yellow solid (0.5 g, 60%). IH-NMR (500

MHz, CDCI3) & (ppm) 7.42 (m, 8H), 7.02 (d, J = 9 Hz, 4H), 6.91 (s, 2H), 6.83 (d, J = 9 Hz,

4H), 0.98 (s, 18H), 0.20 (s, 12H); 13C-NMR (125 MHz, CDCI3) & (ppm) 153.3, 151.7, 144.3,

137.3, 131.7, 130.5, 122.3, 120.5, 119.1, 25.6, 18.2, -4.5; IR (KBr) 1725.35 cm-1 (C=O),


m.p. 7275°C; .

bis(4-hydroxyphenyl) 4,4'-disulfanediylbis(4,1-phenylene)dicarbamate (44) A so

lution of 43 (0.5 g, 0.67 mmol, 1.0 equiv.) in MeOH/H20/HCI (95:4:1, 50 ml) was stirred

for 3 hours at room temperature. The solvent was evaporated in-vacuo and the residue

taken up in ethyl acetate (200 ml) and washed with water and brine (50 ml each). The

organic extract was dried over sodium sulfate and the solvent removed in-vacuo affording

an off-white solid (0.33 g, 95%). IH-NMR (500 MHz, DMSO-d6) & (ppm) 10.26 (s, 2H), 9.40

(s, 2H), 7.48 (m, 8H), 6.99 (ABq, J = 8.5 Hz, 4H), 6.75 (ABq, J = 8.5 Hz, 4H); 13C-NMR

(125 MHz, DMSO-d6) & (ppm) 154.4, 151.8, 142.0, 138.6, 131.0, 128.6, 123.1, 122.2, 115.0;

IR (KBr) 1748.49 cm-1 (C=O), 1501.23 cm-1 (HQ); E.A. Calcd. C 59.99, H 3.87, N 5.38,

Expt. C 59.62, H 3.76, N 5.21; m.p. 194(d)OC.


4-hydroxyphenyl 4-mercaptophenylcarbamate (45) To a solution of 44 (49.6 mg,

0.096 mmol, 1.0 equiv.) in glacial acetic acid (5 ml) was added zinc dust (0.51 g, 7.9 mmol,

81.9 equiv.). The mixture was refluxed for 7 hours at 90°C and filtered through celite and

washed with toluene. Toluene was added and the solvent removed in-vacuo to afford an

off-white solid (48 mg, 97%). IH-NMR (500 MHz, DMSO-d6) 0 (ppm) 11.94 (br. s, IH),

9.81 (br. s, IH), 9.38 (s, IH), 7.17 (m, 4H), 6.96 (ABq, J = 8.5 Hz, 2H), 6.74 (ABq, J = 9

Hz,2H)

bis(2,5-dioxopyrrolidin-l-yl) 4,4'-dithiodibutanoate (53) A solution of 4,4'-dithio

dibutyric acid (0.48 g, 1.99 mmol, 1 equiv.), l-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide

hydrochloride (0.95 g, 4.96 mmol, 2.49 equiv.) and N-hydroxysuccinimide (0.50 g, 4.31

mmol, 2.17 equiv.) in dry acetonitrile (20 ml) and dry tetrahydrofuran (40 ml) was stirred

at room temperature for 20 hours. The solvent was reduced in-vacuo and the remaining

solution taken up in ethyl acetate (100 ml) and washed with water (50 ml) and brine (20

ml). The organic layer was dried over sodium sulfate and the solvent removed in-vacuo to

afford 53 as a white solid in (0.77 g, 90%). 1H-NMR (500 MHz, CDCl3) 0 (ppm) 2.82 (br. s,

8H), 2.75 (m, 8H), 2.14 (m, 4H); 13C-NMR (125 MHz, Acetone-d6) 0 (ppm) 171.50, 170.25,

38.07, 30.90, 27.25, 25.90; IR (KBr) 1733.51 cm-1 (C=O). E.A. Calcd. C 44.44, H 4.66, N

6.48, Expt. C 44.65, H 4.71, N 6.50; m.p. 115~116°C.

tert-butyl 6-aminohexylcarbamate (63) 63 was synthesised using a literature proce

dure. 206 A solution of di-tert-butyl dicarbonate (1.26 g, 5.76 mmol, 1 equiv.) in chloroform

(10 ml) was added dropwise to a a solution of 1,6-diaminohexane (3.34 g, 28.8 mmol, 4.99


equiv.) in chloroform (100 ml) at O°C under vigorous stirring. After the reaction was

complete by TLC the solvent was removed in-vacuo. The residue was taken up in ethyl

acetate and washed three times with half-saturated brine and the organic layer was dried

over sodium sulfate. Removal of the solvent in-vacuo afforded a colourless oil (1.2 g, 97%).

IH-NMR (500 MHz, Acetone-d6) 5 (ppm) 5.96 (br. s, IH), 3.15 (t, J = 7 Hz, 2H), 3.05 (q,

J = 7 Hz, 2H), 2.84 (br. s, IH), 1.44 (m, 17H); 13C-NMR (125 MHz, Acetone-d6) 5 (ppm)

157.62, 79.17, 52.72, 42.03, 32.67, 31.85, 30.83, 29.64, 28.97, 28.44.

4,4'-disulfanediylbis(N-(6-(tert-butyl carbamate)hexyl)butanamide) (54) A so

luttion of 63 (0.11 g, 0.26 mmol, 1.0 equiv.) in methylene chloride (10 ml) was added

dropwise to a stirred solution of 53 (0.13 g, 0.61 mmol, 2.35 equiv.) in methylene chloride

(10 ml) at room temperature. The solution was stirred for 5 hours at room temperature.

The volume was made to 100 ml with methylene chloride and the reaction mixture was

washed with 3% aqueous hydrochloric acid and brine (30 ml each). The organic layer was

dried over sodium sulfate and the solvent removed in-vacuo to afford a white solid (0.15 g,

92%). IH-NMR (500 MHz, Acetone-d6) 5 (ppm) 7.18 (br. s, 2H), 5.98 (br. s, 2H), 3.18 (dd,

J = 6.9, 12.7 Hz, 4H), 3.05 (dd, J = 6.7, 13.1 Hz, 4H), 2.74 (t, J = 7 Hz, 4H), 2.28 (t, J =

7.2 Hz, 4H), 1.48 (m, 9H), 1.39 (s, 19H), 1.33 (m, 8H); 13C-NMR (125 MHz, Acetone-d6)

5 (ppm) 173.12, 157.70, 79.27, 41.93, 40.59, 39.89, 36.01, 31.82, 31.44, 31.34, 29.68, 28.25,

28.12,26.95; IR (KBr) cm-1 (C=O). E.A. Calcd. C 56.75, H 9.21, N 8.82, Expt. C 56.54,

H 9.15, N 8.40; m.p. 89-90°C.


4,4'-disulfanediylbis(N-(6-aminiumhexyl)butanamide trifluoroacetate.) (55) Tri

fluoroacetic acid (0.5 ml, 5% v/v, 7.1 mmol, 29.8 equiv.) was added to a solution of 54 (0.15

g, 0.24 mmol, 1.0 equiv.) in methylene chloride (10 ml) at room temperature. The reaction

mixture was stirred for 5 hours. Toluene was added (5 ml) and the solvent was removed

in-vacuo affording an orange oil (166.5 mg, quant.). IH-NMR (500 MHz, Aeetone-d6) 6

(ppm) 3.79 (m, 2H), 3.18 (m, 4H), 2.73 (m, 4H), 2.56 (m, 4H), 2.33 (m, 4H), 1.97 (m, 4H),

1.80 (m, 4H), 1.45 (m, 6H); 13C-NMR (125 MHz, Aeetone-d6) 6 (ppm) 172.71, 159.9 (q, J

= 36 Hz), 117.12 (q, J = 91 Hz), 47.59, 39.94, 39.03, 38.06, 34.44, 27.51, 26.30, 25.49; IR

(KBr) 1670.9 em-I (C=O), 1195.0 em-I.

Chapter 4

Future Work

The work presented in this thesis has laid the foundations for studying the feasability of

reagentless electrochemically addressable arrays by providing the materials that we believe

to be necessary to our vision. The present chapter will outline various experiments that

we envision would serve to test the feasability of reagent less electrochemically addressable

arrays.

4.1 Coupling monomers to anchor molecules on a gold sur

face

4.1.1 Testing the surface chemistry

In Chapter 3.4.1 we have shown that monolayers formed from 4-aminothiophenol derivatives

are unsuitable for use as anchors in microarrays, hence monolayers formed from either

148

CHAPTER 4. FUTURE WORK 149

alkyl amines such as cystamine or l-amino-lO-mercaptodecane, or 4,4'-disulfanediylbis(N-(6

aminiumhexyl)butanamide trifluoroacetate (55) could be used in order to study the coupling

of the monomers synthesised in Chapter 2. Given the availability of equipment, the materials

needed for this experiment are the following:

• polycrystalline gold electrode

• alkyl amine (cystamine, l-amino-lO-mercaptodecane) or 4,4'-disulfanediylbis(N-(6

aminiumhexyl)butanamide trifluoroacetate (55)

• monomers from Chapter 2.4

• ethanol (99%)

• ethanol adjusted to pH 12 for deposition of the alkyl amines

• tetrahydrofuran (THF) for dissolving the monomers.

We propose to study the monolayers before and after reaction with the monomers using

cyclic voltammetry.

4.1.1.1 Reaction of monomers with amine-terminated self-assembled monolay-

ers

Amine-terminated self-assembled monolayers will be reacted with THF solutions of a suit

able monomer ranging in concentration from 10 to 100 mM. Two possible reactions can

occur at the surface (Figure 4.1). Either the monomer will couple with the amine mono

layer via its reactive benzotriazole or it is degraded by cleavage of its carbamate due to


Figure 4.1: Reaction of a monomer with an amine-terminated monolayer. It is expectedthat not all surface amines will react with the monomer. Once on the surface, the carbamatemoiety of the monomer could be cleaved by another surface amine. This could have aninfluence on the coupling efficiency (see below).

the high concentration of amines at the surface. Given what is known about the relative

reactivities of these two groups, the formation of the amide linkage between surface amines

and the monomers is expected to be much faster than the cleavage of the carbamate.

To verify that coupling has occurred, the presence of a hydroquinone oxidation peak in

the cyclic voltammogram of the monolayers after modification will provide good evidence


supporting the predicted reactivity to form the desired amide linkages. However, it is

anticipated that if the carbamate moiety reacts with the surface amines to form a urea

moiety, the cyclic voltammogram should not exhibit the oxidation peak characteristic of the

oxidation of hydroquinone since the hydroquinone group would have already been displaced.

As such, a negative response, characterised by the absence of hydroquinone oxidation peak

in the cyclic voltammogram, could be interpreted as either a lack of coupling or that the

carbamate moiety was cleaved. Other tests such as MALDI-TOF or HPLC determination

of the surface species after thiol exchange should be carried out to verify the outcome. In

order to limit the probability of hydrolytic cleavage of the carbamate moiety at the surface,

all solvents used in this experiment should be dry. After each coupling event, it may be best

if all the remaining free amines were capped using a capping agent such as the one shown

in Figure 4.2.

4.1.1.2 Cyclic voltammetry studies

Integration of the hydroquinone oxidation peak in the cyclic voltammogram of a monolayer

of mercaptoalkylamines after reaction with the first monomer will provide the base value for

evaluating the efficiency of monomer coupling to surface-amine. If all amines are acylated

by a monomer, the integration value for the second coupling should match that of the first.

The coupling efficiency can then be formulated as follows:

(4.1)


Figure 4.2: Capping of unreacted surface amines after reaction of a monomer with an amineterminated monolayer. Capping the unreacted amines prevents further surface chemistry.This could have a beneficial influence on the coupling efficiency (see below).

where Qn is the integration of the hydroquinone oxidation peak from the nth cyclic voltam-

mogram and Q1 is the integration of the hydroquinone oxidation peak from the first cyclic

voltammogram. Its value is expected to always be lower than one. Repeating such measure-

ments for a significant number of coupling events (5 to 10) could yield a coupling efficiency

profile with respect to the distance of the protected amine from the electrode surface as

reflected by the number of couplings (Figure 4.3).

01234567Number of cycles

-------CHAPTER 4. FUTURE WORK


a.

~ 1.0

.~'-'Iew01

.!:::a.:::>

80.0-+--r--r-.,r--.--or--.---.--


c.

>. 1.0'-'l:III'0Iew01

.!:::a.:::>

8O.O-+....,...~--.,-r--,....--.---.--

b.

~ 1.0l:.!!!~w01.!:::a.:::>

80.0 -+....,...~-.,-.---or--.---.--

o 1 234 567Number of cycles

d.

153

Figure 4.3: Possible coupling efficiency (Ecn ) profiles. a. E Cn is close to 100% everytime. b.Ecn decreases linearly with increasing distance from the electrode surface. c. ECn decreasesin a slow exponential manner. d. ECn decreases sharply for the first coupling events andslowly after that.

4.2 Testing the array

Having tested the electrochemical process on a single electrode, we envisage using a 4 elec-

trode array, as a proof of concept, to test the feasibility of developing a reagentless electro-

chemically addressable array (Figure 4.4). A potential would be applied to, at most, three

of the electrodes per cycle, thus only exposing amines on these electrodes. Then, all four

electrodes would be incubated in a solution of a given monomer simultaneously. The process

would be repeated from between 3 and 5 times varying only the electrode(s) to which the

potential is applied. Table 4.1 presents an example of this experiment. The efficiency of each


Cycle #

y

2 n

3 n

4 n

# units

MW 346.4

Electrode Q2 3 4

Y Y Y

oA~01"0y y y

2" 2" ~""In y y I: OHn n y

o ""'NH o NH

2 3 4 ~ ~479.6 612.7 745.8 j j

a. b.

c. d.

Table 4.1: Testing a 4 electrode array. Monomers are reacted on a primary cystaminemonolayer. y = an electrical potential is applied to the electrode, amines are generated atthe surface. n = no electrical potential is applied to the electrode, alcohol at the surface.a. Oligomer on electrode 1 just after one coupling event. b. Oligomer on electrode 2 aftertwo coupling events. c. Oligomer on electrode 3 after three coupling events. d. Oligomeron electrode 4 after four coupling events. It is, of course, simple to use different monomericunits to generate different sequence oligomers.


Side view

-- --~--+-

-

-

;-0----:-=-1 :

I I1 _

1-

0----;

-=- ~ :I I1 _

Top view

;-0----;: ~-=- -I 11 _

;-0----;: ~-=- -I I1 _

-

-

Bottom view

Figure 4.4: Schematic of a 4 electrode array. Side view: The black rectangles represent aconductive surface that would come in contact with the gold surface.

coupling event could be determined for each electrode as presented in Section 4.1.1.2. The

data collected could be used to assess the consistency of the monolayers between electrodes

by comparing the coupling efficiencies (EcJ of each electrodes between cycles (e.g. compare

cycle 1, Ecl' of electrode 1 with cycle 1, Ecl' of electrode 2). A clear extension would be

to use different monomeric units to generate oligomers with different sequences. In order to

determine the structure of oligomers on each electrode, the modified monolayers could be

analysed by MALDI-TOF or by HPLC after thiol exchange.

Appendix A

Visual index of compounds

156

APPENDIX A. VISUAL INDEX OF COMPOUNDS 157

0(0H

~?-Si-

T

p-{HHN

)=0

4 5

9

12 13 14

[(1 O-~i+N PoAo

¢ 0)=0

q-~l+/°;Si-f19 20

;}O HO~

>=> >=>HN HN)=0 )=0

o 0

Q QOH OH

11

/opO

HN)=0

o

QOH

18

10

~,~,N-N

o

Ort'IVHN

)=0o

QOH

17

2322

n-~~_N~

Z¢~O

-?I::::::,...

OH

16

21

8

~~N-N

HO Q?-O=0 pOHN HN

)=0 )=0o 0

Q QOH OH

15

76

APPENDIX A. VISUAL INDEX OF COMPOUNDS

q. 9'2H'~1 2H'HOT'

I~

QHN .0

pO NH2

6(0, ~ 1.0 HN

1.0 $0H)r=O O? ° U ~I (;00 :::::-...

QI

h

:tOH

24 25 26 27 28 29

158

HX ~ HXY y ° YOH 9):0I ~ 1 ~ I 1 ~ I h

h ~ .0

OH 6 6 6 630 31 32 33 34 35 36

9~ H~ Q ,R~z~~I: I: 0 ~ ~9

N" NI~ ?oN-:::-N I~~ OH I~

h .0 OH

HN "

°"9 °"9o~ °9 °"9 o"¢~ I~ I~I h ~ .0 I~I~~

OH OH OH h

OHOH OH

37 38 39 40 41 42


~/-Si

\

0 pH pHP ~ ~ ~ ~

°)=0 °)=0 °)=0HN HN HN

Q Q Qr I r I

SH2 2

43 44 45 63

)(~NS

46

)(S~'-':N

47

)(~NS

48

)(S~N49

)(~NS

50 51

HS~NH2

52

532

° °S~t.N~N)l.OX:H H

542

55


56

60

H

9I~~..-::

oJ¢I~~

658

HO

rr o-o-~OH~N-{ -o

59

References

1. Kresge, A. J. Ace. Chem. Res. 1987, 20, 364-370.

2. Greer, J.; Erickson, J. W.; Baldwin, J. J.; Varney, M. D. J. Med. Chem. 1994, 37,1035-1054.

3. Bowen, J. P.; Charifson, P. S.; Fox, P. C.; Kontoyanni, M.; Miller, A. B.; Schnur, D.;Stewart, E. L.; Vandyke, C. J. Clin. Pharma. 1993, 33, 1149-1164.

4. Schneider, G. Curro Med. Chem. 2002, 9, p2095 - 2101.

5. Schneider, G.; Fechner, U. Nat. Rev. Drug Discov. 2005, 4, 649-663.

6. Dobson, C. M. Nature 2004, 432, 824-828.

7. Furka, A. Drug Dev. Res. 1995, 36, 1-12.

8. Merrifield, R B. J. Am. Chem. Soc. 1963, 85, 2149-2154.

9. Brahm, D.; Metzger, S.; Bhargava, A.; MIler, 0.; Lieb, F.; Waldmann, H. Angew.Chem., Int. Ed. 2002, 41, 307-311.

10. Brohm, D.; Philippe, N.; Metzger, S.; Bhargava, A.; Muller, 0.; Lieb, F.; Waldmann, H.J. Am. Chem. Soc. 2002,124,13171--13178.

11. Zuckermann, R N.; Martin, E. J.; Spellmeyer, D. C.; Stauber, G. B.; Shoemaker, K. R;Kerr, J. M.; Figliozzi, G. M.; Goff, D. A.; Siani, M. A.; et al., J. Med. Chem. 1994,37, 2678-2685.

12. Willoughby, C. A.; Hutchinsa, S. M.; Rosauera, K. G.; Dhara, M. J.; Chapmana, K. T.;Chicchib, G. G.; Sadowskib, S.; Weinbergc, D. H.; Pateld, S.; Malkowitzb, L.;Di Salvoc, J.; Pacholokb, S. G.; Chengc, K. Biorg. Med. Chem. Lett. 2002, 12, 93-96.

13. Namuswe, F.; Goldberg, D. P. Chem. Commun. 2006, 2326-2328.

14. Hughes, M. D.; Zhang, Z.; Sutherland, A. J.; Andrew, J.; Santos, F.; Albert, F.;Hine, A. V. Nucl. Acids Res. 2005, 33, e32.

15. Konings, D.; Wyatt, J.; Ecker, D.; Freier, S. J. Med. Chem. 1996, 39, 2710-2719.

161

REFERENCES 162

16. Griffey, R H.; An, H. Y.; Cummins, L. L.; Gaus, H. J.; Haly, B.; Herrmann, R;Cook, P. D. Tetrahedron 1998, 54,4067-4076.

17. Patek, M.; Safar, P.; Smrcina, M.; Wegrzyniak, E.; Bjergarde, K; Weichsel, A.;Strop, P. J. Comb. Chem. 2004, 6,43-49.

18. Erb, E.; Janda, K; Brenner, S. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11422-11426.

19. Porco, J. A. Comb. Chem. High Throughput Screening 2000, 3, 93-102.

20. Bryan, M. c.; Fazio, F.; Lee, H. K; Huang, C. Y.; Chang, A.; Best, M. D.;Calarese, D. A.; Blixt, 0.; Paulson, J. C.; Burton, D.; Wilson, 1. A.; Wong, C. H.J. Am. Chem. Soc. 2004, 126, 8640-8641.

21. Beeler, A. B.; Su, S.; Singleton, C. A.; Porco, J. A. J. Am. Chem. Soc. 2007, 129,1413-1419.

22. Pirrung, M. C. Chem. Rev. 1997, 97, 473-488.

23. Baldi, P.; Hatfield, G. W. DNA microarrays and gene expression; Cambridge University Press, 2002.

24. Pellois, J. P.; Zhou, X.; Srivannavit, 0.; Zhou, T.; Gulari, E.; Gao, X. Nature Biotech.2002, 20, 922-926.

25. Gao, X.; Zhou, X.; Gulari, E. Proteomics 2003, 3, 2135-2141.

26. Yudin, A. K; Siu, T. CUrT. Opin. Chem. Biol. 2001, 5, 269-272.

27. Kim, K; Yang, H.; Jon, S.; Kim, E.; Kwak, J. J. Am. Chem. Soc. 2004, 126, 1536815369.

28. Siu, T.; Li, W.; Fradkin, L. E.; Yudin, A. K Abstracts of Papers of the AmericanChemical Society 2001, 222, U124-U124.

29. Sljukic, B.; Baron, R; Salter, C.; Crossley, A.; Compton, R G. Anal. Chim. Acta2007, 590, 67-73.

30. Sullivan, M. G.; Utomo, H.; Fagan, P. J.; Ward, M. D. Anal. Chem. 1999, 71, 43694375.

31. Siu, T.; Li, W.; Yudin, A. K J. Comb. Chem. 2001, 3, 554-558.

32. Siu, T.; Li, W.; Yudin, A. K J. Comb. Chem. 2000, 2, 545-549.

33. Kim, K; Hwang, J.; Seo, 1.; Youn, T. H.i Kwak, J. Chem. Commun. 2006,4723-4725.

34. Kim, K; Yang, H.; Kim, E.; Han, Y. B.; Kim, Y. T.; Kang, S. H.; Kwak, J. Langmuir2002, 18, 1460-1462.

REFERENCES 163

35. Chen, C.; Nagy, G.; Walker, A. V.; Maurer, K; McShea, A.; Moeller, K D. J. Am.Chem. Soc. 2006, 128, 16020~16021.

36. Cheng, C. C.; Chu, Y. H. J. Comb. Chem. 1999, 1,461-466.

37. Maurer, K; McShea, A.; Strathmann, M.; Dill, K J. Comb. Chem. 2005, 7,637-640.

38. Tian, J.; Maurer, K; Tesfu, E.; Moeller, K D. J. Am. Chem. Soc. 2005, 127, 13921393.

39. Egeland, R; Marken, F.; Southern, E. Anal. Chem. 2002, 74, 1590~1596.

40. Egeland, R D.; Southern, E. M. Nucl. Acids Res. 2005, 33, e125-.

41. Beyer, M.; Nesterov, A.; Block, 1.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.;Torralba, G.; Hausmann, M.; Trunk, D.; Lindenstruth, V.; Bischoff, F.R; Stadler, V.;Breitling, F. Science 2007, 318, 1888-.

42. Johnson, R W.; Bednarski, M. D.; O'Leary, B. F.; Grover, E. R Tetrahedron Lett.1981, 22, 3715-3718.

43. Johnson, R W.; Grover, E. R; MacPherson, L. J. Tetrahedron Lett. 1981, 22, 37193720.

44. Johnson, R W.; Grover, E. R; Macpherson, L. J.; Goldman, K Abstracts of Papersof the American Chemical Society 1981, 181, 58-0rgn.

45. Johnson, S. L.; Morrison, D. L. J. Am. Chem. Soc. 1972, 94, 1323-&.

46. Kim, K; Jang, M.; Yang, H.; Kim, E.; Kim, Y. T.; Kwak, J. Langmuir 2004, 20,3821-3823.

47. Barnhurst, L. A.; Wan, Y.; Kutateladze, A. G. Org. Lett. 2000, 2, 799--801.

48. Dondapati, S. K; Montornes, J. M.; Sanchez, P. L.; Acero Sanchez, J. L.;O'Sullivan, C.; Katakis, 1. Electroanalysis 2006, 18, 1879-1884.

49. Hansen, J.; Freeman, S.; Hudlicky, T. Tetrahedron Lett. 2003, 44, 1575--1578.

50. Lebouc, A.; Martin, P.; Carlier, R; Simonet, J. Tetrahedron 1985,41, 1251-1258.

51. Mairanovsky, V. G. Angew. Chem. Int. Ed. Engl. 1976, 15,281-292.

52. Aresta, M.; Ballivet-Tkatchenko, D.; Dell 'Amico, D. B.; Boschi, D.; Calderazzo, F.;Labella, L.; Bonnet, M. C.; Faure, R; Marchetti, F. Chem. Commun. 2000,1099--1100.

53. Khanna, R K; Moore, M. H. Spectrochim. Acta, Part A 1999, 55, 961-967.

54. Rohwedder, J.; Pasquini, C. Analyst 1998, 123, 1641-1648.

REFERENCES

55. Rohwedder, J.; Pasquini, C. Analyst 1998, 123, 1861-1866.

56. Freire, R.; Rohwedder, J.; Pasquini, C. Analyst 1999, 124, 1657~1660.

57. Ulman, A. Chem. Rev. 1996, 96, 1533-1554.

58. Schwartz, D. K. Ann. Rev. Phys. Chem. 2001, 52, 107-137.

164

59. Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev.2005, 105, 1103-1169.

60. Badia, A.; Lennox, R. B.; Reven, L. Ace. Chem. Res. 2000, 33, 475-81.

61. Tender, L. M.; Opperman, K. A.; Hampton, P. D.; Lopez, G. P. Adv. Mater. 1998,10,73-75.

62. Devaraj, N. K.; Dinolfo, P. H.; Chidsey, C. E. D.; Collman, J. P. J. Am. Chem. Soc.2006, 128, 1794~1795.

63. Ishida, H.; Inoue, Y. Rev. Hetero. Chem. 1999, 19, 79-142.

64. Pomerantz, W.; Cadwell, K.; Hsu, Y.-J.; Gellman, S.; Abbott, N. Chem. Mater. 2007,19, 4436-4441.

65. Ishida, H.; Qi, Z.; Sokabe, M.; Donowaki, K.; Inoue, Y. J. Org. Chem. 2001, 66,2978-2989.

66. Kluczyk, A.; Popek, T.; Kiyota, T.; de Macedo, P.; Stefanowicz, P.; Lazar, C.; Konishi, Y. Curro Med. Chem. 2002, 9, 1871-1892.

67. Ishida, H.; Suga, M.; Donowaki, K.; Ohkubo, K. J. Org. Chem. 1995, 60, 5374-5375.

68. Gellman, S. H. Ace. Chem. Res. 1998, 31, 173-180.

69. Hue, 1. Eur. J. Org. Chem. 2004, 17-29.

70. Jiang, H.; Leger, J. M.; Dolain, C.; Guionneau, P.; Hue, 1. Tetrahedron 2003, 59,8365-8374.

71. Appella, D. H.; Christianson, L. A.; Karle, 1. L.; Powell, D. R.j Gellman, S. H. J. Am.Chem. Soc. 1996, 118, 13071-13072.

72. Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. Rev. 2001,101, 3893-4011.

73. Khan, A.; Kaiser, C.; Hecht, S. Angew. Chem. Int. Ed. 2006, 45, 1878-1881.

74. Krauthauser, S.; Christianson, L. A.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc.1997,119,11719-11720.

REFERENCES 165

75. Sanford, A. R.; Yamato, K; Yang, X. W.j Yuan, L. H.; Han, Y. H.j Gong, B. Eur. J.Biochem. 2004, 271, 1416-1425.

76. Smaldone, R. A.j Moore, J. S. J. Am. Chem. Soc. 2007, 129, 5444-5450.

77. Yi, H. P.; Shao, X. B.; Hou, J. L.; Li, C.; Jiang, X. Kj Li, Z. T. New J. Chem.2005,29, 1213-1218.

78. Benniston, A. C.; Harriman, A. Chem. Soc. Rev. 2006, 35, 169-179.

79. Cai, L. T.; Yao, Y. X.; Yang, J. P.; Price, D. W.; Tour, J. M. Chem. Mater. 2002, 14,2905-2909.

80. Emberly, E. G.; Kirczenow, G. Phys. Rev. B 2001, 64, 235412-1-235412-8.

81. Ke, S. H.; Baranger, H. V.; Yang, W. T. J. Chem. Phys. 2005, 122,074704-1-0747048.

82. Lambert, C.; Kriegisch, V. Langmuir 2006, 22, 8807-8812.

83. Li, X. L.; He, J.; Hihath, J.; Xu, B. Q.; Lindsay, S. M.; Tao, N. J. J. Am. Chem. Soc.2006, 128, 2135-2141.

84. Park, S. H.j Pistol, C.; Ahn, S. J.; Reif, J. H.; Lebeck, A. R.; Dwyer, C.; LaBean, T. H.Angew. Chem. Int. Ed. 2006, 45, 735-739.

85. Stoermer, R. L.; Cederquist, K B.; McFarland, S. Kj Sha, M. Y.; Penn, S. G.; Keating, C. D. J. Am. Chem. Soc. 2006, 128, 16892-16903.

86. Tour, J. M. Ace. Chem. Res. 2000, 33,791-804.

87. Tour, J. M.j Jones, L.; Pearson, D. L.; Lamba, J. J. S.; Burgin, T. P.; Whitesides, G. M.jAllara, D. L.j Parikh, A. N.; Atre, S. V. J. Am. Chem. Soc. 1995, 117,9529-9534.

88. Zimbovskaya, N.; Gumbs, G. Appl. Phys. Lett. 2002, 81, 1518-1520.

89. Hodgson, D. R. W.; Sanderson, J. M. Chem. Soc. Rev. 2004, 33, 422-430.

90. Cline, G. W.; Hanna, S. B. J. Am. Chem. Soc. 1987, 109, 3087-3091.

91. Carpino, L. A.; Elfaham, A.; Albericio, F. J. Org. Chem. 1995, 60, 3561-3564.

92. Anderson, G.; Zimmerman, J.; Callahan, F. J. Am. Chem. Soc. 1964, 86, 1839-1842.

93. Anderson, G.; Zimmerman, J.; Callahan, F. J. Am. Chem. Soc. 1963, 85,3039.

94. Veda, M.; Oikawa, H.; Teshirogi, T. Synthesis 1983, 11,908-909.

95. Bensalah, N.; Gadri, A.; Canizares, P.; Saez, C.; Lobato, J.; Rodrigo, M. Environ. Sci.Technol. 2005, 39, 7234--7239.

REFERENCES

96. Eggins, B. R J. Chem. Soc. D-Chem. Commun. 1969, 1267-&.

97. Eggins, B. R; Chambers, J. Q. J. Chem. Soc. D-Chem. Commun. 1969,232-&.

98. Parker, V. D. Electrochim. Acta 1973, 18, 519-524.

99. Parker, V. D. J. Chem. Soc. D-Chem. Commun. 1969, 716-717.

100. Parker, V. D.; Eberson, L. J. Chem. Soc. D-Chem. Commun. 1970, 1289-1290.

101. James, D.; Tour, J. Aldrichimica Acta 2006, 39,47-56.

166

102. Takami, T.; Delamarche, E.; Michel, B.; Gerber, C.; Wolf, H.; Ringsdorf, H. Langmuir1995, 11,3876-3881.

103. Cotaraca, L.; Delogu, P.; Nardelli, A.; Sunjic, V. Synthesis 1996, 553-576.

104. Claussen, R.; Rabatic, B.; Stupp, S. J. Am. Chem. Soc. 2003, 125, 12680-12681.

105. King, J. A.; Donahue, P.; Smith, J. E. J. Org. Chem. 1988, 53, 6145-6147.

106. Lopez-Pelegrin, J.; Wentworth, P.; Sieber, F.; Metz, W.; Janda, K. J. Ory. Chem.2000, 65, 8527-8531.

107. Akiyama, T.; Hirofuji, H.; Hirose, A.; Ozaki, S. Synth. Commun. 1994, 24,2179-2185.

108. Olah, G. A.; Narang, S. C.; Gupta, B. G. B.; Malhotra, R J. Org. Chem. 1979, 44,1247-1251.

109. Morita, T.; Okamoto, Y; Sakurai, H. Chem. Commun. 1978, 874-875.

110. Cacchi, S.; Fabrizi, G.; Goggiamani, A. J. Comb. Chem 2004, 6, 692--694.

111. Grigat, E.; Putter, R. Chem. Ber. Rec. 1964, 97, 3018-&.

112. Martin, D. Chem. Ber. Rec. 1965, 98, 3286-&.

113. Kaupp, G.; Schmeyers, J.; Boy, J. Chem.-Eur. J. 1998, 4, 2467-2474.

114. DuM, D.; Scholte, A. A. Tetrahedron Lett. 1999, 40, 2295-2298.

115. Chatgilialoglu, C.; Ferreri, C.; Lucarini, M. J. Org. Chem. 1993, 58, 249-251.

116. Organic reactions; Adams, R, Blatt, A. H., Cope, A. C., McGrew, F., Niemann, C.,Snyder, H. R, Eds.; Wiley, 1953; pp 275-276.

117. Baltzly, R.; Buck, J. S. J. Am. Chem. Soc. 1943, 65, 1984-1992.

118. Mitsui, S.; Kudo, Y.; Kobayashi, M. Tetrahedron 1969, 25, 1921-1927.

119. Mitsui, S.; Imaizumi, S.; Esashi, Y. Bull. Chem. Soc. Jpn. 1970, 43, 2143-2152.

REFERENCES 167

120. Sellers, H.; Ulman, A.; Shnidman, Y.; Eilers, J. E. J. Am. Chem. Soc. 1993, 115,9389-9401.

121. Aoki, H.; Buhlmann, P.; Umezawa, Y. J. Electroanal. Chem. 1999, 473, 105-112.

122. Bunker, B. C.; Huber, D. L.; Kushmerick, J. G.; Dunbar, T.; Kelly, M.; Matzke, C.;Cao, J. G.; Jeppesen, J. 0.; Perkins, J.j Flood, A. H.; Stoddart, J. F. Langmuir 2007,23,31-34.

123. Chow, E.; Gooding, J. J. Electroanalysis 2006, 18, 1437-1448.

124. Chow, E.; Wong, E. L. S.; Pascoe, 0.; Hibbert, D. B.; Gooding, J. J. Anal. Bioanal.Chem. 2007, 387, 1489-1498.

125. Mikulski, P.; Herman, L.; Harrison, J. Langmuir 2005, 21, 12197-12206.

126. Shon, Y. S.; Lee, S.; Colorado, R.; Perry, S. S.; Lee, T. R. J. Am. Chem. Soc. 2000,122, 7556-7563.

127. Kim, Y. T.; Mccarley, R. L.; Bard, A. J. Langmuir 1993, 9, 1941-1944.

128. Woodward, J. T.; Doudevski, 1.; Sikes, H. D.; Schwartz, D. K. J. Phys. Chem. B 1997,101, 7535-7541.

129. Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112,558-569.

130. Wolf, H.; Ringsdorf, H.; Delamarche, E.; Takami, T.; Kang, H.; Michel, B.; Gerber, C.;Jaschke, M.; Butt, H. J.; Bamberg, E. J. Phys. Chem. 1995, 99, 7102-7107.

131. Delamarche, E.; Michel, B.; Gerber, C.; Anselmetti, D.; Guntherodt, H. J.; Wolf, H.;Ringsdorf, H. Langmuir 1994, 10, 2869-2871.

132. Delamarche, E.; Michel, B.; Kang, H.; Gerber, C. Langmuir 1994, 10, 4103-4108.

133. Poirier, G. E. Chem. Rev. 1997, 97, 1117-1127.

134. Poirier, G. E.; Tarlov, M. J. Langmuir 1994, 10, 2853-2856.

135. Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 12528-12536.

136. Grunze, M. Phys. Scr. 1993, T49B, 711-717.

137. Sellers, H. Surf. Sci. 1993, 294, 99-107.

138. Biebuyck, H. A.; Bian, C. D.; Whitesides, G. M. Langmuir 1994, 10, 1825-1831.

139. Mullen, T. J.; Dameron, A. A.; Andrews, A. M.; Weiss, P. S. Aldrichimica Acta 2007,40, 21-31.

140. Houston, J. E.; Kim, H. 1. Ace. Chem. Res. 2002, 35, 547-553.

REFERENCES

141. Choi, 1. S.; Chi, Y. S. Angew Chern Int Ed Engl 2006, 45, 4894-7.

142. Chechik, V. Annu. Rep. Prog. Chern., Sect. B. 2006, 102, 357-376.

143. Chechik, V.; Crooks, R M.; Stirling, C. J. M. Adv. Mater. 2000, 12, 1161-1171.

168

144. Valiokas, R.; Ostblom, M.; Svedhem, S.; Svensson, S.; Liedberg, B. J. Phys. Chern. B2002, 106, 10401-10409.

145. Clegg, R; Hutchison, J. J. Am. Chern. Soc. 1999, 121, 5319-5327.

146. Fabris, L.; Antonello, S.; Armelao, L.; Donkers, R; Polo, F.; Toniolo, c.; Maran, F. J.Am. Chern. Soc. 2006, 128,326-336.

147. Sabatani, E.; Cohenboulakia, J.; Bruening, M.; Rubinstein, 1. Langmuir 1993, 9,2974-2981.

148. Ulman, A. Ace. Chern. Res. 2001, 34, 855-863.

149. Tao, Y-T.; Wu, C.-C.; Eu, J.-Y; Lin, W.-L.; Wu, K-C.; Chen, C.-h. Langmuir 1997,13, 4018-4023.

150. Jin, Q.; Rodriguez, J. A.; Li, C. Z.; Darici, Y.; Tao, N. J. Surf. Sci. 1999,425, 101-111.

151. Xu, B. Q.; Tao, N. J. J. Science 2003, 301, 1221-1223.

152. Kaefer, D.; Witte, G.; Cyganik, P.; Terfort, A.; Woll, C. J. Am. Chern. Soc. 2006,128, 1723-1732.

153. Cyganik, P.; Buck, M.; Strunskus, T.; Shaporenko, A.; Wilton-Ely, J. D. E. T.;Zharnikov, M.; Woll, C. J. Am. Chern. Soc. 2006, 128, 13868-13878.

154. Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5, 723-727.

155. Kolega, R R.; Schlenoff, J. B. Langmuir 1998, 14, 5469-5478.

156. Yousaf, M. N.; Mrksich, M. J. Am. Chern. Soc. 1999, 121, 4286-4287.

157. Xiao, S. J.; Wieland, M.j Brunner, S. J. Colloid Interface Sci. 2005, 290, 172-183.

158. Chaki, N. K; Vijayamohanan, K Biosens. Bioelectron. 2002, 17, 1-12.

159. Liu, Y. J.; Navasero, N. M.; Yu, H. Z. Langmuir 2004, 20, 4039-4050.

160. Tagowska, M.; Mazur, M.; Krysinski, P. Synth. Met. 2004, 140, 29-35.

161. Pasquinet, E.; Bouvier, C.; Thery-Merland, F.; Hairault, L.; Lebret, B.; Methivier, C.;Pradier, C. M. J. Colloid Interf. Sci. 2004, 272, 21-27.

162. Davis, F.; Higson, S. P. J. Biosens. Bioelectron. 2005, 21, 1-20.

REFERENCES 169

163. Wink, T.; vanZuilen, S. J.; Bult, A.; vanBennekom, W. P. Analyst 1997, 122, R43R50.

164. Chen, 1.; Howarth, M.; Lin, W.; Ting, A. Y. Nat. Methods 2005, 2, 99-104.

165. Niemz, A.; Jeoung, E.; Boal, A. K.; Deans, R; Rotello, V. M. Langmuir 2000, 16,1460~1462.

166. Bard, A. J.; Abruna, H. D.; Chidsey, C. E.; Faulkner, L. Rj Feldberg, S. W.; Itaya, K.;Majda, M.; Melroy, 0.; Murray, R W.; et al., J. Phys. Chem. 1993, 97, 7147-7173.

167. Speiser, B. Curro Org. Chem. 1999,3, 171-191.

168. Kuznetsov, A. Charge transfer in physics, chemistry and biology - Physical mechanismsof elementary processes and an introduction to the theory; Gordon and Breach, 1995;pp 291-333.

169. Adams, D. M. et al. J. Phys. Chem. B 2003, 107, 6668-6697.

170. Sachs, S. B.; Dudek, S. P.; Hsung, R. P.; Sita, 1. R.; Smalley, J. F.; Newton, M. D.;Feldberg, S. W.; Chidsey, C. E. D. J. Am. Chem. Soc. 1997, 119, 10563-10564.

In. Giesbers, M.; Kleijn, J. M.; Cohen Stuart, M. A. J. Colloid Inter! Sci. 2002, 248,88-95.

172. Barten, D.; Kleijn, J.; Duval, J.; van Leeuwen, H.; Lyklema, J.; Cohen Stuart, M.Langmuir 2003, 19, 1133- 1139.

173. Hong, H.-G.; Park, W. Langmuir 2001, 17, 2485-2492.

174. Bilewicz, R; Sek, S.; Zawisza, 1. Russ. J. Electrochem. 2002,38,29-38.

175. Marcus, R. A. J. Chem. Phys. 1956, 24, 966-978.

176. Marcus, R A.; Sutin, N. Biochim. Biophys. Acta, Rev. Bioenerg. 1985, 811, 265-322.

177. Clegg, A. D.; Rees, N. V.; Klymenko, O. V.; Coles, B. A.; Compton, R G. Journal ofElectroanalytical Chemistry 2005, 580, 78-86.

178. Clegg, A. D.; Rees, N. V.; Klymenko, O. V.; Coles, B. A.; Compton, R. G.ChemPhysChem 2004, 5, 1234-1240.

179. Holmlin, R; Haag, R.; Chabinyc, M.; Ismagilov, R; Cohen, A.; Terfort, A.; Rampi, M.;Whitesides, G. J. Am. Chem. Soc. 2001, 123, 5075-5085.

180. Wold, D.; Haag, R; Rampi, M.; Frisbie, C. J. Phys. Chem. B 2002, 106, 2813-2816.

181. Finklea, H.; Liu, 1.; Ravenscroft, M.; Punturi, S. J. Phys. Chem. 1996, 100, 1885218858.

REFERENCES

182. Protsailo, L. V.; Fawcett, W. R. Electrochim. Acta 2000, 45, 3497-3505.

170

183. Wakamatsu, S.; Fuji, S.; Akiba, D.; Fujihira, M. Jpn. J. Appl. Phys. 2006, 45, 27362742.

184. Winkler, J. R. Curro Opin. Chem. Biol. 2000,4, 192-198.

185. Kai, M.; Takeda, K; Morita, T.; Kimura, S. Journal of Peptide Science 2008, 14,192-202.

186. Sumner, J.; Creager, S. J. Phys. Chem. B 2001, 105, 8739-8745.

187. Newton, M. D.; Smalley, J. F. Phys. Chem. Chem. Phys. 2007, 9, 555-572.

188. Sek, S.; Moszynski, R.; Sepiol, A.; Misicka, A.; Bilewicz, R. J. Electroanal. Chem.2003, 550-551, 359-364.

189. Bryant, M. A.; Crooks, R. M. Langmuir 1993, 9, 385-387.

190. Yu, H. Z.; Xia, N.; Zhang, J.; Liu, Z. F. J. Eleetroanal. Chem. 1998,448, 119-124.

191. Zhong, C.-J.; Brush, R.; Anderegg, J.; Porter, M. Langmuir 1999, 15, 518-525.

192. Ferguson, M.; Low, E.; Morris, J. Langmuir 2004, 20, 3319-3323.

193. Ahn, S. J.; Kim, K B. Kor. Chem. Soc. 1998, 19, 888-891.

194. Batz, V.; Schneeweiss, M. A.; Kramer, D.; Hagenstrom, H.; Kolb, D. M.; Mandler, D.J. Eleetroanal. Chem. 2000, 491, 55-68.

195. Electroanalytical chemistry; Bard, A., Rubinstein, 1., Eds.; Marcel Dekker, Inc., 1996;Vol. 19, p 242.

196. Yoshimoto, S.; Sawaguchi, T.; Mizutani, F.; Taniguchi, 1. Electrochem. Commun.2000,2, 39-43.

197. Brown, A. P.; Anson, F. C. J. Electroanal. Chem. 1978, 92, 133-145.

198. Matsuda, H.; Aoki, K; Tokuda, K J. Electroanal. Chem. 1987, 211, 1-13.

199. Matsuda, H.; Aoki, K; Tokuda, K J. Electroanal. Chem. 1987, 211, 15-32.

200. Fourier Transform Infrared Spectroscopy; Ferraro, J., Basile, L., Eds.; Academic Press,Inc., 1985.

201. Hayes, W. A.; Shannon, C. Langmuir 1996, 12, 3688-3694.

202. Raj, C. R.; Kitamura, F.; Ohsaka, T. Langmuir 2001, 11, 7378-7386.

REFERENCES 171

203. Lukkari, J.; Kleemola, K.j Meretoja, M.; Ollonqvist, T.; Kankare, J. Langmuir 1998,14, 1705-1715.

204. Grubisha, D.; Lipert, R.; Park, H.-Y.; Driskell, J.; Porter, M. Anal. Chem. 2003, 75,5936--5943.

205. Krapcho, A. P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559-2564.

206. Dardonville, C.; Fernandez-Fernandez, C.; Gibbons, S.-L.; Ryan, G. J.; Jagerovic, N.;Gabilondo, A. M.; Meana, J. J.; Callado, L. F. Bioorg. Med. Chem. 2006, 14, 65706580.

TOWARD REAGENTLESS ELECTROCHEMICALLY ... - SFU's Summit

Documents

Transcript of TOWARD REAGENTLESS ELECTROCHEMICALLY ... - SFU's Summit