Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2007

S1

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2007

Supporting Information

for

Synthesis, Conformational Studies, Binding Assessment and Liposome

Insertion of a Thioether-Bridged Mimetic of the Antigen GM3-Ganglioside

Lactone.

Lucio Toma, Emanuela Di Cola, Andrea Ienco, Laura Legnani, Carlotta Lunghi, Gloriano

Moneti, Barbara Richichi, Sandra Ristori, Daniela Dell’Atti, and Cristina Nativi*

Computational Methods page S2

Crystal data for compound 6 S4

Liposome preparation and characterization S5

Synthesis of spiro derivative 6 S8

Synthesis of compounds 9 – 13 S9

Synthesis of compound 3 S13

Synthesis of compounds 14 – 15 S14

Synthesis of compound 17 S16

Synthesis of compound 7 S17

Synthesis of compounds 18 – 19 S18

Synthesis of compound 8 S18

Synthesis of compound 2 S22

Binding evaluation of thioether 2 to WGA Selectin S23

References S28

S2

Computational Methods

All the calculations were performed with GAUSSIAN03 program package. The

exploration of the conformational space of compounds 1a and 2 was carried out through

DFT calculations using the B3LYP hybrid functional. Rotation around single bonds of the

substituents and ring inversions were considered as degrees of confor-mational freedom

to give the starting geometries, which were optimized through en-ergy minimizations at the

B3LYP/6-311+G(2df,p) level for S and 6-311+G(d,p) level for the other atoms. The

energies of the conformers so obtained were recalculated with the solvent polarizable

continuum model PCM, as implemented in the Gaussian 03 package, to take into account

the influence of water on the conformational beha-viour of the molecules.

Cartesian coordinates of the global minimum conformations

Compound 1a

C 2.050507 -1.640594 -0.378213 C 1.962576 -0.611977 0.735002 C 3.373933 -0.247006 1.187070 C 4.209438 -0.625036 -1.040037 C 2.843204 -1.097885 -1.566446 H 1.462835 0.300704 0.416593 H 2.565449 -2.529649 0.010006 H 4.769693 -1.514720 -0.711486 H 3.010409 -1.918078 -2.279725 H 3.910580 -1.137436 1.564246 O 4.062694 0.277651 0.056788 C 5.029977 0.077381 -2.107436 H 4.942429 -0.480130 -3.048693 H 4.623250 1.080645 -2.257587 O 6.388926 0.114614 -1.673357 H 6.838750 0.836343 -2.120317 O 2.178760 -0.018884 -2.196960 H 1.242644 -0.243856 -2.281301 O 0.750065 -2.063724 -0.819033 O 1.216575 -1.200725 1.823946 C -0.351191 -1.717735 -0.012545 C 0.035169 -1.755153 1.501060 O -0.669089 -2.246411 2.342970 O -0.711038 -0.390664 -0.382513 O 3.296909 0.729401 2.157626 C 4.542141 1.030124 2.789071 H 5.256331 1.442830 2.072891 H 4.322867 1.766681 3.560532 H 4.965403 0.131939 3.255638 C -1.924403 0.127261 0.188353 H -1.848311 0.130036 1.281934 C -3.109928 -0.764735 -0.247394 H -3.201675 -0.691382 -1.335651 C -2.848214 -2.228694 0.143186 H -2.875290 -2.317977 1.231675 C -1.476920 -2.691944 -0.353908

S3

H -1.256107 -3.680873 0.049330 H -1.486463 -2.762060 -1.446934 C -2.061166 1.591345 -0.270904 H -2.893549 2.017418 0.305367 O -3.886354 -3.092950 -0.313291 H -3.895222 -3.091723 -1.277787 N -4.377794 -0.333431 0.336600 H -4.704629 -0.841107 1.145293 C -5.269164 0.468034 -0.304422 O -5.006290 1.039833 -1.361788 C -6.620138 0.631838 0.359299 H -6.699871 0.119346 1.319322 H -6.815544 1.696251 0.500643 H -7.385800 0.244427 -0.316504 C -0.802275 2.404193 0.047517 H 0.009191 2.057983 -0.603469 C -1.012996 3.890900 -0.204594 H -1.221021 4.059424 -1.264464 H -1.857237 4.251744 0.397686 O -2.303345 1.714102 -1.660703 H -3.251774 1.545965 -1.805014 O -0.473285 2.168269 1.414428 H 0.232213 2.784817 1.646545 O 0.201743 4.537548 0.200499 H 0.091542 5.490068 0.138187 Compound 2

C -1.524369 0.351903 -0.967745 C -1.015019 -0.779480 -0.466348 C -1.816203 -1.749567 0.360434 H -1.706022 -2.749880 -0.084384 C -3.301054 -1.371810 0.329059 H -3.710312 -1.697628 -0.639602 O -3.459499 0.036100 0.455962 C -4.106479 -2.047290 1.424321 H -3.778811 -3.089895 1.520170 H -3.913369 -1.536031 2.370913 O 0.274754 -1.244118 -0.632033 C 1.268545 -0.411552 -1.262062 C 2.413615 -1.361911 -1.632419 H 3.147887 -0.801432 -2.215337 S -0.649542 1.446915 -2.035139 O 1.712079 0.597077 -0.394527 C 2.385771 0.193066 0.811046 H 1.668166 -0.314830 1.467548 C 3.542898 -0.773702 0.544019 H 3.842933 -1.208248 1.505283 C 3.091949 -1.911130 -0.376577 H 2.392716 -2.546202 0.178204 C 0.679670 0.290893 -2.489365 H 1.454943 0.883112 -2.975026 H 0.306874 -0.459374 -3.189359 H 2.022109 -2.175365 -2.249845 O -5.485117 -1.984399 1.058736 H -6.020889 -2.093194 1.848797 O -1.365625 -1.793112 1.719529

S4

C -2.958049 0.738432 -0.664844 H -3.603151 0.511180 -1.536189 O -2.987159 2.101168 -0.401395 C -4.299196 2.654917 -0.303362 H -4.834944 2.243760 0.555082 H -4.167442 3.728595 -0.178065 H -4.871299 2.460644 -1.219384 O 4.634356 -0.055838 -0.024286 H 5.266767 -0.712563 -0.342523 O 4.277299 -2.653794 -0.687867 H 4.062114 -3.371113 -1.292444 C 2.834103 1.493737 1.500347 H 3.486148 2.043276 0.816880 C 1.661832 2.410771 1.880740 H 0.857190 1.824054 2.345939 H 2.034136 3.114164 2.627157 O 3.528966 1.156354 2.705273 H 4.454776 1.014440 2.484077 O 1.178770 3.189648 0.800110 H 0.804121 2.601861 0.131055 H -0.420656 -1.976327 1.713773 Crystal data for compound 6:

A summary of the crystal and structural refinement are available from PDB. A

colourless crystal of 6 was chosen and mounted on a Philips PW1100 diffractometer

equipped with Cu radiation (λ=1.54178) at room temperature. The cell was obtained by a

least squared refinement of 25 reflection in the 2θ range of 11.5°-13°. The intensity were

corrected for Lorenz and polarization effect and semiempirical absorption corrections (ψ

scan). During the data collection three standard reflection were monitored every 2 hours.

The atomic scattering factors are those reported by Cromer and Waber.[S1] The structure

was solved by direct methods using the SIR97 program.[S2] The refinements were made by

full matrix least squared on all F2 data using SHELXL97.[S3] Anisotropic thermal parame-

ters were allotted to all non-hydrogen atoms. While ∆F Fourier maps allowed the localiza-

tion of the hydrogen atoms, these were introduced in their calculated position with a ther-

mal parameters 20% larger than those of the respective carbon atoms in order to have a

better data/parameters ratio. The molecular drawing was made using the ORTEP-III pro-

gram for Windows.[S4] The computational work was performed using the WINGX inter-

face.[S5]

S5

Figure S1. X-ray structure of spiro derivative 6

Liposome preparation and characterization

Liposome were prepared by the extrusion method.[S6] Briefly, a lipid film of 1,2-di-

oleoyl-sn-Glycero-3-Phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-

ethanolamine (DOPE) in 1:1 molar ratio or 1,2-dioleoyl-sn-glycero-3-phosphocholine

(DMPC) was obtained by solvent evaporation from the appropriate quantities of 2×10-2 M

stock solutions in chloroform. Upon rehydration with MilliQ grade water and vigorous vor-

texing, multilamellar vesicles were formed and submitted to eight cycles of freeze and

thaw (liquid nitrogen/water bath at 50°C). Final downsize and conversion to unilamellar

vesicles was obtained by extrusion through 200 nm polycarbonate membranes (27 pass-

ages) with the LiposoFast apparatus, Avestin, Ottawa, CA.

Size measurements were performed by dynamic light scattering (DLS) with a Coul-

ter sub-micron particle analyzer, model N4SD, equipped with a 4 mW helium-neon laser

(632.8 nm) and 90° detector. The autocorrelation function of the scattered light was ana-

lyzed by unimodal analysis, that assumes a log Gaussian distribution of the sample size[S7]

and by SDP (Size Distribution Processor, based on the algorithm CONTIN[S8]) analysis.

These methods allowed to obtain mean diameter and polydispersity index of plain and

drug-loaded liposomes.

Small-angle X-ray scattering (SAXS) experiments were performed at the ID02

beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The

wavelength (λ) of the incident X-rays was = 0.995 Å. Two sample-detector distances were

used (3 m and 1 m) and a total scattering vector q range of 0.05-5 nm-1 was covered (q =

4π/λ sin (θ/2), where θ is the scattering angle). The measured SAXS profiles were reported

S6

to the absolute scale using a Lupolene standard and the normalized 2D images were

radially averaged by integration of the intensity over the azimuthal angle Φ.[S9]

Pure liposomes showed a mean diameter of 160 ± 20 nm and 190 ± 20 nm, for the

DMPC and DOPC/DOPE formulations, respectively. These values were in line with the

pore size of the membranes used for the extrusion procedure. The polydispersity index

was fairly low, namely 0.15 for both types of liposomes. Insertion of thioether 3 caused this

value to increase in the case of DMPC liposomes (Table S1), while for DOPC/DOPE lipo-

some variations were very small, i.e. approximately in the range of experimental error, as

reported in the text. This behaviour could be rationalized with the help of results obtained

by SAXS.

Table S1. Mean size and polydispersity index of plain and thioether 3 loaded liposomes.

Mean diameter (nm) Polydispersion

Pure DMPC liposomes 160 ± 20 0.15

DMPC liposomes + 3 (10 :1) 340 ± 20 0.40

DMPC liposomes + 3 (5 :1) 380 ± 40 0.45

Figure S2 reports the SAXS diagrams of DMPC and with different GM3-thioether 3

content. The scattering profile of pure DMPC liposomes showed the characteristic of mo-

nolameller vesicles and was simulated with the form factor of a single bilayer, according to

a well established procedure.[S10, S11]

where ρc, ρh, tc and th are the electronic density and the thickness of the hydrophobic and

hydrophilic layer, respectively. ρs is the electronic density of the solvent and Σ the interface

extension per unit volume. A Gaussian distribution of the bilayer hydrophobic thicknesses

(with half-width at half maximum δtc) was assumed to take into account a small polydisper-

sity. The values obtained for the thickness and electron density, listed in Table S2, were

consistent with data previously reported in the literature.[S12, S13]

Upon GM3 thioether addition, pronounced Bragg peaks were observed in the SAXS

profiles of DMPC liposomes, whose intensity increased progressively with host molecule

content. This indicated that correlation among bilayer was established and oligolamellar

onion-like structures were formed.[S14] From the maxima q values, a repeating distance of

2ch

e,se,h

c

e,he,c4)]

2tt2

qsin()()2t

qsin()[(q4

)q(I+

ρ−ρ+ρ−ρΣπ

=

S7

D = 6.34 nm for the concentric shells was calculated ( 2π i/qm,i, where qm,i is the maximum q

value of the its Bragg peak).

Figure S2. SAXS diagrams of DMPC liposomes in the pure form and with different loading of thio-

ether 3. Slight deviation from the theoretical curve of monolamellar vesicles, in this context, might

be attributed to a very small fraction of oligolamellar structure.

Contrarily to the DMPC case, the SAXS diagrams of pure DOPC/DOPE liposomes

showed that the tendency to form oligolamellar structures was reduced upon GM3 thio-

ether insertion, as reported in the text. The electron density and thickness parameters of

the monolamellar curve which broadly reproduces the SAXS profile of pure DOPC/DOPE

liposomes (shown in Figure 3 in the text) are listed in the second line of Table S3.

Table S3. Best fit parameters for the monolamellar modelling of selected liposome

systems

Σ [cm-1] ρc [cm-2] ρh [cm-2] tc [Å] δtc [Å] rh [Å]

Pure DMPC liposomes 4.34×104 7.4×1010 1.31×1011 25.5 3.9 8.0

monolamellar fraction in

DOPC/DOPE liposomes + 3

(2:1 mol:mol)

4.84×104 7.8×1010 1.28×1011 27.5 3.0 6.9

(*) From the best fit Σ value and from the lipid concentration, a mean surface/polar head could be calculated. The obtained values are 72 Å2/molecule and 80 Å2/molecule in the pure DMPC liposomes and GM3-thioether loaded DOPC/DOPE liposomes, respectively. These values are quite reasonable and confirm the assumption made in the modelling of these systems

S8

The above findings are expected to be of relevance for the carrier properties of

GM3-thioether loaded liposomes, since it is known that monolamellar aggregates are able

to load a larger cargo than multilamellar liposomes. However, in vitro and in vivo

experiments have shown that monolamellar liposomes are less resistant toward clearance

and opsonisation with respect to multilamellar liposomes.[S15, S16]

Experimental Procedure

Synthesis of spiro derivative 6?:

A suspension of lactone (0.908 g, 2.70 mmol) and IBX (5.26 g, 18.6 mmol) in AcOEt (27

mL) was stirred at 80°C for 3h. The reaction mixture was cooled to 0°C then the white solid

obtained was filtered through a pad of Celite. The filtrate was concentrated to dryness to

give 4.79 g of a crude mixture of β-keto-δ-lactone (741 mg, 2.21 mmol, 82%, on the basis

of NMR spectra), and benzoic acid (275mg, 1.10 mmol).

To an ice-cooled solution of crude ketolactone (4.37 mmol) in dry CHCl3 (5 mL), phthalimi-

dosolfenyl chloride (930 mg, 4.37 mmol) was slowly added; after 5 min the ice bath was

removed. The reaction mixture was stirred at RT until the complete disappearance of start-

ing material (TLC analysis, 10 min). The crude solution of the phthalimidosolfenyl deriva-

tive formed was passed through a column with a cation-exchanger resin (Amberlite 200)

O

O O

OPivB nOO

H O O

OPivB nOIB X

A cO Et

OO

OBn OPiv

SO

OO

OO

OO

O O

OPivBnO

CHCl3rt, 15 min

O

O OS

OPivNSCl

O

O

N O

O

+ HCl

Py, K2CO3

OO

O

OO

BnO

Amb 200 4

6

6

O

O

OBn OPiv

SO

OO

OO

O1

5

2-2'3

4

67-7'

1'a, 1'b

a

bc

d ef-f'

S9

and then the neutralized suspension obtained was added to a suspension of K2CO3 (278

mg, 2.02 mmol) and 4 (850 mg, 3.148 mmol) in dry pyridine (4 mL) at 50°C. The mixture

was stirred for 30 min then a second, freshly prepared, suspension of solfenyl derivative

(prepared starting from 3.89 mmol of ketolactone and 3.89 mmol of phtalimidosulfenyl

chloride) was added. The reaction mixture was stirred at 50°C for 16 h then diluted with

CH2Cl2 and filtered. The filtrate was washed with a saturated solution of NH4Cl (4 x 20 mL)

and dried over Na2SO4. Concentration of the solvent under vacuum gave a crude which

was purified by a flash column chromatography on silica gel (petroleum ether/EtOAc 2:1)

to give unreacted 4 (250 mg, 0.925 mmol) as pale yellow oil, (petroleum ether/EtOAc 3:2),

6 (990 mg, 50%) as white solid, (petroleum ether: EtOAc 1:3). m.p.: 186-187 °C; [α]D25

+8.32 (c 0.33, CHCl3); 1H NMR (400MHz, CDCl3): δ 7.36-7.29 (m, 5H), 4.87-4.85 (A part of

an AB system, JAB = 11.6 Hz, 1H, CH2Ph), 4.68-4.65 (M part of an AMXY system, JMA =

7.6 Hz, JMX = JMY = 2.8 Hz, 1H, H-3), 4.56-4.52 (m, 1H, H-e), 4.55-4.52 (B part of an AB

system, JAB = 11.6 Hz, 1H, CH2Ph), 4.41-4.37 (m, 2H, H-f, H-4), 4.31-4.26 (m, 2H, H-f’, H-

6), 4.07- 4.05 (A part of an ABX system, JAB = 8.8 Hz, JAX = 1.2 Hz, 1H, H-7), 4.032- 4.01

(B part of an ABX system, JAB = 8.8 Hz, JBX = 1.2 Hz, 1H, H-7’), 3.90 (d, Jde = 2.8 Hz, 1H,

H-d), 3.84 (dd, J = 2.0 Hz, J = 6.4 Hz, 1H, H-5), 3.33-3.29 (A part of an AB system, JAB =

13.2 Hz, 1H, H-1’a), 2.83-2.80 (B part of an AB system, JAB = 13.2 Hz, 1H, H-1’b), 2.52-

2.47 (X part of an AMXY system, JXM = 2.8 Hz, JXY = 15.6 Hz, 1H, H-2), 2.10-2.06 (Y part

of an AMXY system, JYM = 3.2 Hz, JYX = 15.6 Hz, 1H, H-2’), 1.46 (s, 3H, C(CH3)2), 1.35 (s,

3H, C(CH3)2), 1.31 (s, 3H, C(CH3)2), 1.28 (s, 3H, C(CH3)2), 1.17 (s, 9H, C(CH3)3); 13C NMR

(50MHz, CDCl3): δ 178.1, 162.6, 156.1, 136.9, 128.49 (2C), 128.45 (2C), 128.1, 109.7,

109.1, 102.3, 95.9, 76.3, 73.8, 72.9, 72.1 (2C), 70.2, 70.0, 65.8, 62.1, 38.7, 35.2, 33.4,

27.0, 26.3, 25.9, 25.4, 24.6; ESI-MS 657.3 [M+Na]+, 673.2 [M+K]+.

Synthesis of peracetyl derivative 9:

CH3CO2H/H2O70/3060°C3h

Ac2O

6 9

O

OOBn OPiv

SO

OO

OO

O O

OOBn OPiv

SO

OHHO

OHOH

O O

OOBn OPiv

SO

OAcAcO

AcOOAc

OPy

A solution of 6 (84 mg, 0.132 mmol) in 2 mL of CH3CO2H:H2O / 70:30 was stirred at 60°C

for 3 h then toluene (2 mL) was added. Concentration of the solvent under vacuum gave a

S10

crude which was dissolved in CH2Cl2 (3 mL) and treated with pyridine (0.8 mL), acetic

anhydride (0.4 mL) and a catalytic amount DMAP. The mixture was stirred for 1 h at RT

then diluted with CH2Cl2, washed with a saturated solution of NH4Cl (2 x 10 mL) and dried

over Na2SO4. The solvent was removed under vacuum and the crude purified by column

chro-matography on silica gel (petroleum ether:EtOAc 3:2) to afford 9 (55 mg, 0.076 mmol,

58% over two steps) as pale oil. [α]D25 +47.9 (c 0.34, CHCl3); 1H NMR (200MHz, CDCl3): δ

7.39-7.30 (m, 5H), 5.43 (m, 2H), 5.098-5.018 (m, 1H), 4.73(A2 system, 2H), 4.64 (dt, J =

2.8 Hz, J = 6.6 Hz, 1H), 4.51-4.31 (m, 3H), 4.15-4.01 (m, 2H), 3.91 (dd, J = 4.4 Hz, J =

12.4 Hz, 1H), 3.06-3.00 (A part of an AB system, JAB = 13.2 Hz, 1H) 2.93-2.86 (B part of

an AB system, JAB = 13.2 Hz, 1H), 2.129-2.02 (m, 2H), 2.09 (s, 3H), 2.02 (s, 3H), 1.96 (s,

3H), 1.76 (s, 3H), 1.22 (s, 9H); 13C NMR (50MHz, CDCl3): δ 177.6, 170.3,169.9, 169.7,

169.3, 161.8, 153.8, 136.0, 128.6, 128.5, 128.3, 103.4, 95.5, 75.76, 75.70, 71.3, 69.2,

67.4, 66.1, 64.1, 61.7, 61.3, 38.8, 33.5, 27.3, 20.8, 20.7, 20.6, 20.3; ESI-MS 745.3

[M+Na]+.

Synthesis of compound 10:

TIPSOTf

CH2Cl2

10

CH3CO2H/H2O70/3060°C

3h6

O

OOBn OPiv

SO

OO

OO

O O

OOBn OPiv

SO

OHHO

HOOH

O O

OOBn OPiv

SO

OTIPSTIPSO

TIPSO OH

O

Py

A solution of 6 (684 mg, 1.078 mmol) in 12 mL of a mixture CH3CO2H:H2O / 70:30 was

stirred at 60°C for 3 h, then toluene (15 mL) was added. Concentration of the solvent

under vacuum gave a crude which was purified by flash chromatography on silica gel

(AcOEt/MeOH 15:1) to afford the unprotected derivative (473 mg, 0.853 mmol) which

was dissolved in dry CH2Cl2 (1.5 mL) and treated with pyridine (1.5 mL) and TIPSOTf

(0.920 mL, 3.408 mmol). The mixture was stirred at 0°C for 25 min then diluted with

CH2Cl2 (30 mL) and washed with a saturated solution of NH4Cl (3 x 30 mL), dried over

Na2SO4 and concentrated under vacuum. The crude was purified by column chromatog-

raphy on silica gel (petroleum ether:EtOAc 7:1) to afford 10 (460 mg, 0.449 mmol, 83%) as

pale yellow oil. [α]D25 +23.12 (c 1.24, CHCl3); 1H NMR (400MHz, CD3CN): δ 7.37-7.28 (m,

5H, Ph), 4.74-4.71 (A part of an AB system, JAB = 11.2 Hz, 1H, CH2Ph), 4.17-4.67 (X part

S11

of an ABX, Je-d = 2.8 Hz, JXA = JXB = 6.0 Hz, 1H, H-e), 4.61-4.60 (B part of an AB system,

JAB = 11.2 Hz, 1H, CH2Ph), 4.38-4.33 (A part of ABX system, , JAX = 6.0 Hz, JAB = 11.2 Hz

1H, H-f ), 4.34-4.30 (m, 1H, H-3), 4.33-4.28 (B part of an ABX system, JBX = 6.0 Hz, JAB =

11.2 Hz, 1H, H-f’), 4.15 (bs, 1H, H-4), 4.14-4.09 (X part of an ABX system, JXA = 2.4 Hz,

JXB = J6-5 = 8.0 Hz, 1H, H-6), 4.08 (d, Jd-e = 2.8 Hz, 1H, H-d), 3.94-3.91 (A part of an ABX ,

JAX = 2.4 Hz, JAB = 10.8 Hz, 1H, H-7), 3.48 (bd, J5-6 = 8.4 Hz, 1H, H-5), 3.38-3.34 (B part of

an ABX system, JBX = 8 Hz, JAB = 10.8 Hz, 1H, H-7’), 3.03 (d, JOH-4 = 2 Hz, 1H, OH), 2.99-

2.95 (A part of an AB system, JAB = 13.6 Hz, 1H, H-1’a), 2.94-2.91 (B part of an AB system,

JAB = 13.6 Hz, 1H, H-1’b), 2.13-2.07 (X part of an AMXY system, JXM = 10.8 Hz, JXY = 13.2

Hz 1H, H-2), 2.06-2.02 (Y part of an AMXY system, JYB = 5.4 Hz, JYX = 12.8 Hz, 1H, H-2’),

1.2 (s, 9H, C(CH3)3), 1.22-1.06 (m, 54H, Si-CH(CH3)2), 1.01-0.99 (m, 9H, Si-CH(CH3)2); 13C NMR (50MHz, CDCl3): δ 178.3, 162.6, 156.1, 136.9, 128.9 (2C), 128.6 (2C), 127.9,

101.8, 98.3, 76.1, 74.0, 73.4, 73.1, 71.0, 67.8, 67.3, 65.7, 61.8, 36.8, 36.3, 33.5, 27.1 (3C),

18.29 (4C), 18.20 (4C), 18.0 (6C), 17.9 (4C), 12.7 (3C),12.3 (3C), 11.8 (3C); ESI-MS 1023

[M+H]+, 1045 [M+Na]+.

Synthesis of compound 11:

1110

DIBAl-H

CH2Cl2-40°C to 10°C

O

OOBn OPiv

SO

OTIPSTIPSO

TIPSO OH

O O

OOBn OH

SO

OTIPSTIPSO

TIPSO OH

OH

A solution of 10 (770 mg, 0.753 mmol) in dry CH2Cl2 (10 mL) was cooled at -40°C and a

solution 1.5M in toluene of DIBAl-H (1.75 mL, 2.63 mmol) was added dropwise. The solu-

tion was slowly warmed to 10°C and the reaction mixture stirred for 5 h. After this time 0.5

mL of MeOH and a solution of potassium sodium tartrate .4 H2O (H2O, 20%, 20 mL) were

added; the mixture was diluted with CH2Cl2 (30 mL) and stirred for 20 min at RT. The or-

ganic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude

was purified by column chromatography on silica gel [petroleum ether:EtOAc2:1(+0.1%

NEt3)] to give 11 (424 mg, 0.451 mmol, 60%) as an oily mixture of anomers.

Selected for 11: 1H NMR (400MHz, CDCl3): δ 7.34-7.27 (m, 5H+5H), 5.42 (d, J = 4.0 Hz,

1Hα-an), 5.23 (d, J = 10.0 Hz, 1Hβ-an), 4.77-4.74 (A part of an AB system, JAB = 11.6 Hz,

1Hα-an), 4.63-4.60 (A part of an AB system, JAB = 12.0 Hz, 1Hβ-an), 4.53-4.50 (B part of an

S12

AB system, JAB = 11.6Hz, 1Hα-an), 4.13-4.38 (B part of an AB system, JAB = 12.0 Hz, 1Hβ-

an), 3.04 (bs, 1H, OH), 2.96-2.93 (A part of an AB system, JAB = 12.8 Hz, 1H), 2.91-2.88 (B

part of an AB system, JAB = 12.8 Hz, 1H); ESI-MS 963.4 [M+Na]+.

Synthesis of compound 12:

12

Cl3CCN

DBUCH2Cl2

11

O

OOBn OH

SO

OTIPSTIPSO

TIPSO OH

OH O

OOBn OH

SO

OTIPSTIPSO

TIPSO OH

O

NH

CCl3

A solution of 11 (100 mg, 0.106 mmol) in dry CH2Cl2 (4 mL) was cooled to 0°C and tri-

cloroacetonitrile (254 µl, 1.702 mmol) and DBU (12.5 µl, 0.125 mmol) were added. After

1 h at 0°C the solution was concentrated under vacuum. The residue was dissolved in

CH2Cl2, filtered through silica gel (eluant: petroleum ether/EtOAc 6:1). The filtrate was

concentrated to dryness to give 12 (100 mg, 87%) which was used without any other

purification.

Synthesis of compounds 13 and 13a:

12

O

OOBn OH

SO

OTIPSTIPSO

TIPSO OH

O

NH

CCl3

13

13a

C14H29OH

TMSOTfCH2Cl2MS 4A

O

OOBn OH

SO

OTIPSTIPSO

TIPSO OH

OC14H29

O

OOBn OR

SO

ORRO

RO OR

OC14H29

+

A mixture of 12 (277, 0.255 mmol), tetradecanol (163.0 mg, 0.763mmol) and molecular

sieves (4 Å) in dry CH2Cl2 (9 mL) was stirred for 20 min at RT. The reaction mixture was

cooled to -40°C, treated with 17 µL of triflic acid (0.1 M in CH2Cl2) and stirred for 10 min at

-40°C. Then 51 µL of triflic acid (0.1 M solution in CH2Cl2) were also added in two portions.

S13

After 1 h the suspension was neutralized with NEt3, filtered through a pad of Celite and the

filtrate concentrated to dryness. The crude was purified by flash chromatography on silica

gel [petroleum ether:EtOAc 7:1 (+0.1% NEt3)] to give the α-glycosyl derivative 13 (23 mg,

0.020 mmol) and an inseparable mixture of partially desilylated glycosyl derivatives 13a

(100 mg). Compound 13 was totally characterized.

Compound 13: 1H NMR (400MHz, CD3CN): δ 7.34-7.27 (m, 5H, Ph), 4.91 (s, 1H, H-a),

4.73-4.70 (A part of an AB system, JAB = 11.2 Hz, 1H, CH2Ph), 4.58-4.55 (B part of an AB

system, JAB = 11.2 Hz, 1H, CH2Ph), 4.34-4.29 (M part of an AMXY system, J = 2.8 Hz, J =

6.4 Hz, J = 9.6 Hz, 1H, H-3), 4.13-4.07 (m, 3H), 3.97 (A part of an ABX system, JAX = 2 Hz,

JAB = 10.8 Hz, 1H, H-7), 3.74 (d, Jd-e = 2.4 Hz, 1H, H-d), 3.72-3.68 (m, 1H, CH2X), 3.66-

3.64 (at, J = 6.4 Hz, 2H, H-f, H-f’), 3.53 (bd, J5-6 = 8.4 Hz, H-5), 3.49-3.43 (m, 1H, CH2X),

3.39 (B part of an ABX system, JBX = 7.6 Hz, JAB = 10.8 Hz, 1H, H-7’), 2.95-2.92 (A part of

an AB system, JAB = 13.2 Hz, 1H, H-1’a), 2.91-2.88 (B part of an AB system, JAB = 13.2 Hz,

1H, H-1’b), 2.90 (dd, J = 0.8 Hz, J = 2.4 Hz, 1H), 2.76 (t, J = 6.0 Hz, OH), 2.0-1.98 (m, 2H,

H-2a, H-2b), 1.6-1.53 (m, 2H, CH2Y), 1.29 (s, 22H, CH2 chain), 1.1-1.04 (m, 63H, Si-

(CH(CH3)6)3), 0.90 (t, J = 7.2 Hz, CH3 chain); 13C NMR (50MHz, CDCl3): δ 141.3, 137.6,

128.5 (2C), 128.0, 127.9 (2C), 105.9, 96.4, 94.9, 73.9, 73.4, 72.3, 72.1, 71.2, 68.4, 68.1,

67.3, 66.4, 62.2, 36.9, 33.7, 31.9, 29.67 (2C), 29.62 (2C), 29.59 (2C), 29.54, 29.4, 29.3,

26.0, 22.6, 18.3 (4C), 18.2 (4C), 18.05 (4C), 18.01 (6C), 14.0, 12.8 (3C), 12.2 (3C), 11.9

(3C); ESI-MS 1159.8 [M+Na]+.

Synthesis of compound 3:

13

13a

THF

TBAF

3

O

OOBn OH

SO

OTIPSTIPSO

TIPSO OH

OC14H29

O

OOBn OR

SO

ORRO

RO OR

OC14H29

O

OOBn OH

SO

OHHO

HO OH

OC14H29

To a solution of 13 and 13a (123 mg) in THF (3 mL), 110 mg of TBAF (0.345 mmol) were

added at room temperature. The reaction mixture was stirred until no trace of starting ma-

S14

terial was revealed (TLC analysis). The solution was diluted with AcOEt and washed with a

saturated solution of NH4Cl (2x20mL), dried over Na2SO4 and concentrated to dryness.

The crude was purified by flash chromatography on silica gel [petroleum ether:EtOAc 7:1

(+ 0.1% NEt3)] to afford 3 (30 mg, 0.044 mmol, 18% over two steps) as glassy solid. [α]D25

+40.3 (c 0.202, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.40-7.31 (m, 5H, Ph), 5.02 (s, 1H,

H-a), 4.83-4.80 (A part of an AB system, JAB = 10.4 Hz, 1H, CH2Ph), 4.62-4.56 (m, 3H),

4.51-4.46 (m, 1H), 3.94 (m, 1H, H-3), 3.88-3.71 (m, 4H), 3.64-3.57 (m, 2H), 3.28 (br s, 1H,

OH), 3.06-3.03 (A part of an AB system, JAB = 12.8 Hz, H-1’a), 2.93-2.90 (br part of an AB

system, JAB = 12.8 Hz, 1H, H-1’b), 2.93 (br s, 1H, OH), 2.64 (br s, 1H, OH), 2.24 (br s, 1H,

OH), 2.04-1.99 (X part of an AMXY system, JXM = 5.2 Hz, JXY = 12.8 Hz, 1H, H-2), 1.9 (br

s, 1H, OH), 1.81 (Y part of an AMXY system, JYM = JYX = 11.6 Hz, 1H, H-2’), 1.63-1.58 (m,

2H, CH2Y), 1.2 (s, 22H, CH2 chain), 0.87 (t, J = 8 Hz, 3H, CH3 chain); 13C NMR (50MHz,

CDCl3): δ 139.5, 136.5, 128.9 (2C), 128.8 (2C), 128.7, 108.9, 95.8, 92.5, 76.0, 72.8, 70.1,

68.8, 68.4, 68.2, 66.9, 66.3, 66.1, 61.9, 37.0, 34.0, 31.8, 29.6 (2C), 29.6 (2C), 29.59,

29.50, 29.4, 29.38, 29.33, 26.1, 22.6, 14.1; ESI-MS 691.4 [M+Na]+, 707.3 [M+K]+;

elemental analysis calcd (%) for C35H56O10S: (668.88) C 62.85; H 8.44; O 23.92; found: C

62.75; H 8.56; O 24.01

Synthesis of compound 14:

6

O

OOBn OPiv

SO

OO

OO

O

14

H2N NH2

NH

MeONaMeOH

.HCl

O

OOBn OH

SO

OO

OO

O

To a solution of guanidine hydrochloride (175 mg, 1.83 mmol) in MeOH (3.66 mL), MeONa

(82 mg, 1.52 mmol) was added. The suspension was stirred for 30 min at RT then the

white solid obtained filtered and slowly added to solution of 6 (388 mg, 0.611 mmol) in

CH2Cl2:MeOH / 2:1 (10 mL). The reaction mixture was stirred overnight then neutralized

with a solution of HCl 10% in MeOH and the solvent was removed under vacuum. The

crude was purified by flash chromatography on silica gel (petroleum ether/EtOAc 1:2) to

give 14 (233 mg, 70%) as a white solid. M.p.: 72-75 °C; [α]D25 +1.15 (c 0.695, CHCl3); 1H

NMR (400MHz, CDCl3): δ 7.39-7.28 (m, 5H, Ph), 4.87-4.84 (A part of an AB system, JAB =

S15

12Hz, 1H, CH2Ph), 4.67-4.63 (M part of an AMXY system JMA = 7.6 Hz, JMX = JMY = 2.8

Hz, 1H, H-3), 4.55-4.52 (br part of an AB system, JAB = 12Hz, 1H, CH2Ph), 4.04-4.36 (m,

2H, H-4, H-e) 4.30-4.26 (M part of an A2M system, J6-5 = 6.8 Hz, JMA = 5.6 Hz, 1H, H-6),

4.03-4.02 (A part of an A2M system, JAM = 5.6 Hz, 2H, H-7, H-7’), 3.94 (d, Jd-e = 2Hz, 1H,

H-d), 3.94-3.88 (m, 1H, H-f), 3.80 (dd, J5-6 = 6.8 Hz, J5-4 = 2 Hz, 1H, H-5), 3.68 (add, Jf’-f =

11.6 Hz, Jf’-e = 5.6 Hz, 1H, H-f’), 3.30-3.27 (A part of an AB system , JAB = 13.2 Hz, 1H, H-

1’a), 2.83-2.80 (B part of an system JAB = 13.2 Hz, 1H, H-1’b), 2.94-2.44 (X part of an

AMXY system, JXY = 16 Hz, JXA = 3.2 Hz, 1H, H-2), 2.3 (bs, 1H, OH), 2.09-2.05 (Y part of

an AMXY system, JYX = 16 Hz, JYA = 3.2 Hz, 1H, H-2’), 1.46 (s, 3H, C(CH3)2), 1.35 (s, 3H,

C(CH3)2), 1.31 (s, 3H, C(CH3)2), 1.28 (s, 3H, C(CH3)2); 13C NMR (50MHz, CDCl3): δ 163.2,

156.5, 137.0, 128.7 (2C), 128.5 (2C), 128.3, 109.6, 109.2, 102.0, 95.6, 78.9, 73.7, 73.0,

72.1 (2C), 70.3, 69.6, 66.0, 60.8, 35.1, 33.5, 26.4, 25.9, 25.3, 24.6; ESI-MS 573.18

[M+Na]+, 589.09 [M+K]+.

Synthesis compound 15:

14 15

CHCl3MeOH

HCl 10%

O

OOBn OH

SO

OO

OO

O O

OOBn OH

SO

OHHO

OHOH

O

To a ice-cooled solution of 14 (450 mg, 0.817 mmol) in CHCl3:MeOH / 2:1 (12 mL), 1.2 mL

of HCl solution (10% in MeOH) were added. The reaction mixture was stirred overnight at

RT then neutralized with NEt3 and the solvent removed under reduced pressure. The

crude was purified by flash chromatography on silica gel (EtOAc:MeOH 8:1) to give 15

(366 mg, 95%) as a white solid. Compound 15 was fully characterized as peracetyl deriv-

ative 16.

15

Ac2O Py

CH2Cl2

16

O

OOBn OH

SO

OHHO

OHOH

O O

OOBn OAc

SO

OAcAcO

AcOOAc

O

S16

To a stirred solution of 15 (366 mg, 0.778 mmol) in CH2Cl2 (3 mL), pyridine (1 mL), acetic

anhydride (0.5 mL) and DMAP (catalytic amount) were added. The reaction mixture was

stirred at RT overnight then diluted with CH2Cl2 and washed with a saturated solution of

NH4Cl (2 x 15 mL). The organic phase was dried over Na2SO4 and concentrated to dry-

ness. The crude product was purified by column chromatography on silica gel (petroleum

ether/EtOAc 1:1) to give 16 (480 mg, 91%) as a white solid. M.p.: 78-80 °C; [α]D25 +6.8 (c

0.6, CHCl3); 1H NMR (400MHz, CDCl3): δ 7.41-7-31 (m, 5H, Ph), 5.41-5.37 (M part of an

AMXY system, JMX = 12.0 Hz, JMY = 5.6 Hz, JMA = 3.2 Hz, 1H, H-3), 5.32 (m, 1H, H-4),

5.09-5.05 (X part of an ABX system J6-5 = 7.6 Hz, JXA = 4.0 Hz, JXB = 2.0 Hz, 1H, H-6),

4.77-4.74 (A part of an AB system, JAB = 10.8 Hz, 1H, CH2Ph), 4.72-4.69 (B part of an AB

system, JAB = 10.8 Hz, 1H, CH2Ph), 4.65-4.61 (X part of an ABX system, JXA = JXB = 6.4

Hz, Je-d = 2.8 Hz, 1H, H-e), 4.45-4.40 (A part of an ABX system, JAB = 11.6 Hz, JAX = 6.8

Hz, 1H, H-f), 4.41-4.37 (A part of an ABX system, JAB = 12.4 Hz, JAX = 2.4 Hz, 1H, H-7),

4.35-4.31 (B part of an ABX system, JAB = 11.6 Hz, JBX = 6.4 Hz, 1H, H-f’), 4.09 (dd, Jd-e =

2.8 Hz, 1H, H-d), 4.09-4.06 (dd, J5-6 = 6.8 Hz, J5-4 = 1.2 Hz, 1H, H-5), 3.96-3.92 (B part of

an ABX system, JAB = 12.4 Hz, JBX = 4.4 Hz, 1H, H-7’), 3.03-3.00 (A part of an AB system,

JAB = 13.2 Hz, 1H, H-1’a), 2.91-2.88 (B part of an AB system, JAB = 13.2 Hz, 1H, H-1’b),

2.16-2.05 (m, 2H, H-2, H-2’), 2.15 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.02

(s, 3H, CH3), 1.97 (s, 3H, CH3); 13C NMR (50MHz, CDCl3): δ 170.1, 169.9, 169.7, 169.5,

169.2, 161.7, 154.0, 135.9, 128.5 (2C), 128.4, 128.3 (2C), 103.0, 95.6, 75.5, 75.3, 70.8,

69.2, 67.3, 65.9, 64.0, 61.6, 61.4, 33.4 (2C), 20.7, 20.67, 20.62 (2C), 20.2; ESI-MS 703.2

[M+Na]+, 719.2 [M+K]+; ESI-Full ms: 703.16783 [M+Na]+, 719.14168 [M+K]+.

Synthesis of compound 17:

FeCl3

CH2Cl2

16 17

O

OOBn OAc

SO

OAcAcO

AcOOAc

O O

OOH OAc

SO

OAcAcO

AcOOAc

O

To a ice-cooled solution of 16 (180 mg, 0.264 mmol) in CH2Cl2 (15 mL), FeCl3 (428 mg,

2.64 mmol) was added under Ar atmosphere. The reaction mixture was warmed to RT and

stirred for 1h, then diluted with CH2Cl2 and washed with water. The organic layer was dried

over Na2SO4 and concentrated to dryness. The crude was purified by column chromatog-

raphy on silica gel (CH2Cl2:Acetone 10:1) to give 17 (114 mg, 73%) as a glassy solid;

S17

[α]D25 +79 (c 0.365, CHCl3); 1H NMR (400MHz, CDCl3): δ 5.15-5.46 (M part of AMXY

system, JMX = JMY = 8.0 Hz, JMA = 2.8 Hz, 1H, H-3), 5.42-5.41 (A part of an AMXY system,

JAM = 2.8 Hz, J4-5 = 1.6 Hz, 1H, H-4), 5.07-5.03 (X part of an ABX system, JXA = JXB = 3.2

Hz, J 6-5 = 10.0 Hz, 1H, H-6), 4.58-4.51 (m, 2H, H-e, H-f), 4.45 (A part of ABX system, JAB

= 12.4 Hz, JAX = 3.2 Hz, 1H, H-7), 4.38-4.34 (B part of an ABX system, JAB = 10.4 Hz, JBX

= 5.6 Hz, 1H, H-f’), 4.25 (dd, J5-6 = 10.0 Hz, J5-4 = 1.6 Hz, 1H, H-5), 4.18-4.14 (B part of

ABX system, JAB = 12.4 Hz, JBX = 3.2 Hz, 1H, H-7’) 4.14 (d, Jd-e = 3.2 Hz, 1H, H-d), 3.08-

3.04 (A part of AB system, JAB = 13.2 Hz, 1H, H-1’a), 2.97-2.94 (B part of an AB system,

JAB = 13.2 Hz, 1H, H-1’b), 2.62 (bs, 1H, OH), 2.13 (s, 3H, CH3), 2.11-2.09 (m, 2H, H-2, H-

2’), 2.09 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.01 (s, 3H, CH3), 1.98 (s, 3H, CH3); 13C NMR

(50MHz, CDCl3): δ 70.9, 170.7, 169.9, 169.8, 169.4, 162.2, 155.1, 102.9, 94.5, 76.3, 69.4,

67.5, 66.0, 64.3, 64.2, 61.9, 61.7, 33.9, 33.4, 20.8 (2C), 20.7 (3C); ESI-MS 613.08

[M+Na]+, 629.0 [M+K]+ .

Synthesis of compound 7:

17

O

OOH OAc

SO

OAcAcO

AcOOAc

OCH2Cl2

O

OOAc OAc

SO

OAcAcO

AcOOAc

O

7

Ac2O Py

DMAP

To a stirred solution of 17 (114 mg, 0.193 mmol) in CH2Cl2 (3 mL), pyridine (0.5 mL), ace-

tic anhydride (0.25 mL) and DMAP (catalytic amount) were added. The reaction mixture

was stirred at RT overnight then diluted with CH2Cl2 and washed with a saturated solution

of NH4Cl (2 x 15 mL). The organic phase was dried over Na2SO4 and concentrated to dry-

ness. The crude product was purified on column chromatography on silica gel (CH2Cl2/

acetone 10:1) to give 7 (120 mg, 99%) as a white solid. M.p.: 174-175 °C; [α]D25 +25.11 (c

0.36, CHCl3); 1H NMR (400MHz, CDCl3): δ 5.66 (d, Jd-e = 2.8 Hz, 1H, H-d), 5.33 (m, 1H, H-

4), 5.32-5.27 (M part of an AMXY system, JMY = 11.6 Hz, JMX = 5.2 Hz, JMA = 3.2 Hz, 1H,

H-3), 5.14-5.09 (X part of an ABX system, J6-5 = 10 Hz, JXB = 4.8 Hz, JXA = 2.4 Hz, 1H, H-

6), 4.82-4.78 (X part of ABX system, JXA = JBX = 6.8 Hz, Je-d = 2.8 Hz, 1H, H-e), 4.38-4.34

(A part of an ABX system, JAB = 12.4 Hz, JAX = 2.4 Hz, 1H, H-7), 4.34-4.30 (A part of an

ABX system, JAB = 11.2 Hz, JAX = 6.4 Hz, 1H, H-f), 4.23-4.19 (B part of an ABX system,

JAB = 11.6 Hz, JBX = 6.8 Hz, 1H, H-f’), 4.06-4.02 (B part of an ABX system, JAB = 12.4 Hz,

S18

JBX = 5.2 Hz, 1H, H-7’), 3.84 (dd, J5-6 = 10.0 Hz, J5-4 = 1.2 Hz, 1H, H-5), 3.06-3.02 (A part

of an AB system , JAB = 13.6 Hz, 1H, H-1’a), 2.95-2.92 (B part of an AB system, JAB = 13.6

Hz, 1H, H-1’b), 2.16 (s, 3H, CH3), 2.14-2.03 (m, 2H, H-2, H-2’), 2.08 (s, 3H, CH3), 2.05 (s,

3H, CH3), 2.01 (s, 3H, CH3), 1.96 (s, 6H, CH3); 13C NMR (50MHz, CDCl3): δ 169.9, 169.8,

169.7, 169.3, 169.1, 168.8, 161.1, 151.3, 105.1, 95.3, 74.1, 69.5, 67.0, 65.8, 64.0, 63.3,

62.0, 60.4, 33.3, 33.1, 20.6, 20.5 (3C), 20.4, 20.3; ESI-MS 655.2 [M+Na]+, 671.1 [M+K]+.

Synthesis of compound 18:

O

OOAc OAc

SO

OAcAcO

AcOOAc

O

7

CH2Cl2

Red-Al

O

OOAc OAc

SO

OAcAcO

AcOOAc

OH

18

-78°C

A solution of 7 (123 mg, 0.194 mmol), in dry CH2Cl2 (5.8 mL) was cooled to -78°C and a

freshly prepared Red/Al solution (990 µL), which was prepared by adding ethanol (103 µL)

to a ice-cooled solution of 3.4M Red-Al (500 µL) in dry toluene (1 mL), was added drop-

wise. After 1h at -78°C, 2 drops of MeOH and a solution of potassium sodium tartrate.

4 H2O (20% H2O, 20 mL) were added. Then the mixture was diluted with CH2Cl2 (20 mL)

and stirred for 10 min at RT. The organic phase was dried over Na2SO4 and concentrated

to dryness. The crude was purified by flash column chromatography on silica gel (petrole-

um ether/EtOAc 2:3) to give 18 as an oily mixture of anomers (115 mg, 93%).

Selected for 18: M.p.: 90-92 °C; 1H NMR (400MHz, CDCl3): δ 5.43-5.4 (m, 2H), 5.33 (dd, J

= 1.4 Hz, J = 2.8 Hz, 1H), 5.30-5.21 (m, 1H), 4.67-4.59 (td, J = 2.2 Hz, J = 6.6 Hz, 1H),

3.59 (d, J = 4.0 Hz, 1H); 13C NMR (50MHz, CDCl3): δ 170.8, 170.3, 170.0, 169.5, 169.4

(2C), 137.8, 109.4, 93.2 (C-anom. β), 93.1, 90.1 (C-anom. α), 68.0, 67.4, 67.3, 66.4, 64.4,

642, 62.0, 61.8, 33.8, 33.6, 20.79 (3C), 20.7 (3C); ESI-MS 657.1 [M+Na]+, 673.1 [M+K]+.

Synthesis of compound 8, method A

Synthesis of compound 19: A solution of 18 (85 mg, 0.134 mmol) in dry CH2Cl2 (5 mL) was

cooled to 0°C and DBU (20 µL, 0.201 mmol) and tricloroacetonitrile (400 µL, 2.68 mmol)

were added. After 1 h at 0 °C the solution was concentrated to dryness. The residue was

dissolved in CH2Cl2 and then filtered on silica gel (petroleum ether/EtOAc 2:3) to give 93

S19

mg of a mixture of 18 and 19 in a 7.8:1 ratio. Compound 19 was partially separated from

the mixture and totally character-ized.

O

OOAc OAc

SO

OAcAcO

AcOOAc

OH

18

O

OOAc OAc

SO

OAcAcO

AcOOAc

O

19

NH

CCl3

Cl3CCN

CH2Cl2DBU

Compound 19: [α]D25 +28.81 (c 0.2, CHCl3); 1H NMR (400MHz, CDCl3): δ 7.12 (d, JNH-a =

7.2 Hz, 1H, NH), 5.82 (d, Ja-NH = 7.2 Hz, 1H, H-a), 5.46 (d, Jd-e = 2.4 Hz, H-d), 5.36-5.35 (A

part of an AMXY system, J4-5 = 1.6 Hz, JAM = 2.8 Hz, 1H, H-4), 5.29-5.24 (m, 1H, H-3),

5.15-5.11 (X part of on ABX system, JXA = 2.4 Hz, JXB = 3.2 Hz, J6-5 = 10.0 Hz, 1H, H-6),

4.38-4.34 (X part of an ABX system, Je-d = 2.4 Hz, JXA = 6.0 Hz, JXB = 7.6 Hz, 1H, H-e),

4.32-4.28 (A part of an ABX system, JAX = 2.4 Hz, JAB = 12.4 Hz, 1H, H-7), 4.26-4.22 (B

part of an AB system, JBX = 3.0 Hz, JAB = 13 Hz, 1H, H-7’), 4.23-4.19 (A part of an ABX

system, JAX = 6.0 Hz, JAB = 11.6 Hz, 1H, H-f), 4.11-4.07 (B part of an ABX system, JBX =

7.2 Hz, JAB = 11.6 Hz, 1H, H-f’), 3.96 (dd, J5-4 = 1.2 Hz, J5-6 = 10.0 Hz, 1H, H-5), 3.06-3.02

(A part of an AB system, JAB = 12.8 Hz, 1H, H-1’a), 2.99-2.96 (B part of an AB system, JAB

= 12.8 Hz, 1H, H-1’b), 2.19 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.11-1.99 (m, 2H, H-2, H-2’),

2.09, 2.02 (2s, 6H, 2CH3), 2.01 (s, 3H, CH3), 1.98 (s, 3H, CH3); 13C NMR (50MHz, CDCl3):

δ 170.7, 170.5, 170.1, 170.2, 169.7, 169.6, 161.5, 138.8, 106.7, 98.3, 93.4, 76.2, 69.1,

68.4, 67.4, 66.3, 64.3, 63.8, 61.8, 61.4, 33.7, 33.5, 20.7 (3C), 20.6 (3C); ESI-MS 799.9

[M+Na]+, 816.1 [M+K]+.

Synthesis of compound 8

O

OOAc OAc

SO

OAcAcO

AcOOAc

O NH

CCl3CH2Cl2

C14H29OHTMSOTf

19

O

OOAc OAc

SO

OAcAcO

AcOOAc

OC14H29

8

A solution of 18 and 19 (7.8:1 ratio, 93 mg) and C14H29OH (50 mg, 0.232 mmol) in dry

CH2Cl2 (4 mL) was cooled to -30°C and 243 µL of TMSOTf (0.1 M in CH2Cl2) were added.

The reaction mixture was stirred for 30 min at -30°C, then 50 µL of TMSOTf (0.1 M in

S20

CH2Cl2) were added. After 30 min at –30° C the solution was neutralized with NEt3 and

concentrated to dryness. The crude was purified by flash column chromatography on silica

gel (petroleum ether/EtOAc 1:1) to give 8 (20 mg, 18% over two steps) and unreacted 18

(27 mg, 0.042 mmol).

O

OAcO

OAc

S

O

OAcAcOAcO OAc

1

52-2'

3

4

67-7'

1'a, 1'b

a

bc

d ef-f'

O CH3

x

y

[α]D25 +27.7 (c 0.155, CHCl3); 1H NMR (400MHz, CDCl3): δ 5.38 (d, Jd-e = 2.8 Hz, 1H, H-d),

5.35-5.34 (A part of an AMXY system, J4-5 = 1.6 Hz, JAM = 3.2 Hz, 1H, H-4), 5.28-5.23 (M

part of an AMXY system, JMA = 2.8 Hz, JMX = JMY = 8.4 Hz, 1H, H-3), 5.14-5.10 (X part of

an ABX system, JXA = 2.8 Hz, J6-5 = 9.6 Hz, 1H, H-6), 4.99 (s, 1H, H-a), 4.52-4.48 (X part

of an ABX system, Je-d = 2.4 Hz, JXA = 5.6 Hz, JXB = 7.2 Hz, 1H, H-e), 4.26-4.22 (A part of

an ABX system, JAX = 3.6 Hz, JAB = 12.4 Hz, 1H, H-7), 4.22-4.19 (B part of an ABX

system, JBX = 4.4 Hz, JAB = 11.0 Hz, 1H, H-7’), 4.20-4.16 (A part of an ABX system, JAX =

6.0 Hz, JAB = 11.6 Hz, 1H, H-f), 4.12-4.07 (B part of an ABX system, JBX = 7.2 Hz, JAB =

11.2 Hz, H-f’), 3.98 (dd, J5-4 = 1.6 Hz, J5-6 = 10.0 Hz, 1H, H-5), 3.75-3.67 (m, 1H, CH2X),

3.53-3.45 (m, 1H, CH2X), 3.03-3.00 (A part of an AB system, JAB = 12.8 Hz, 1H, H-1’a),

2.98-2.95 (B part of an AB system, JAB = 12.8 Hz, 1H, H-1’b), 2.17 (s, 3H, CH3), 2.11 (s,

3H, CH3), 2.03 (s, 3H, CH3), 2.003 (s, 3H, CH3), 2.005 (s, 3H, CH3), 1.98-1.97 (m, 2H, H-2,

H-2’), 1.96 (s, 3H, CH3), 1.64 (m, 1H, CH2Y), 1.36-1.24 (bs, 23H, CH2), 0.87 (t, J = 6.8 Hz,

3H, CH3 chain); 13C NMR (50MHz, CDCl3): δ 170.7, 170.1, 170.0, 169.5 (3C), 137.3,

109.1, 95.7, 93.0, 69.0, 68.0, 67.48, 67.47, 66.46, 64.4, 62.0, 61.8, 33.9, 33.7, 29.7, 29.6

(4C), 29.47 (3C), 29.42 (3C), 26.2, 22.7, 20.79 (3C), 20.71 (3C), 14.2 ; ESI-MS 853.1

[M+Na]+, 869.1 [M+K]+; ESI-Full ms 853.3678 [M+Na]+, 869.3407 [M+K]+.

Synthesis of compound 8 method B

Synthesis of compound 20: A solution of 18 (100 mg, 0.157 mmol), in dry CH2Cl2 (3.60

mL) was cooled to -40°C and 82 µL of DAST (0.630 mmol) were added. After 30’ at -40°C,

2 drops of MeOH and a satu-rated solution of NaHCO3 (10 mL) were added. Then the

mixture was diluted with CH2Cl2 (20 mL) and stirred for 10 min at RT. The organic phase

S21

O

OOAc OAc

SO

OAcAcO

AcOOAc

OH

18

O

OOAc OAc

SO

OAcAcO

AcOOAc

F

20

DAST

CH2Cl2

was washed with brine (1 x 10 mL) dried over Na2SO4 and concentrated to dryness. The

crude was purified by flash column chromatography on silica gel (petroleum ether/EtOAc

2:3) to give 20 (86 mg, 0.135 mmol, 87%) as glassy solid. [α]D25 + 12.7 (c 1.095, CH2Cl2); 1H NMR (400MHz, CDCl3): δ 5.75 (d, Ja-F = 57.6 Hz, 1H, H-1), 5.49-5.47 (dd, Jd-e = 2.4 Hz,

Jd-F = 3.8 Hz, 1H, H-d), 5.36-5.35 (A part of an AMXY system, J4-5 = 1.6 Hz, JAM = 2.8 Hz,

1H, H-4), 5.30-5.24 (M part of an AMXY system, JMA = 3.2 Hz, JMX = 6.8 Hz, JMY = 10.4 Hz,

1H, H-3), 5.14-5.11 (X part of an ABX system, JXA = JXB = 2.4 Hz, J6-5 = 9.6 Hz, 1H, H-6),

4.63-4.60 (m, 1H, H-e), 4.32-4.28 (A part of an ABX system, JAX = 3.6 Hz, JAB = 12.4 Hz,

1H, H-7), 4.27-4.23 (A part of an ABX system, JAX = 6.0 Hz, JAB = 11.6 Hz, 1H, H-f), 4.19-

4.16 (B part of an ABX system, JBX = 2.4 Hz, JAB = 12.8 Hz, 1H, H-7’), 4.16-4.12 (B part of

an ABX system, JBX = 7.2 Hz, JAB = 11.6 Hz, 1H, H-f’), 3.93 (dd, J5-4 = 1.6 Hz, J5-6 = 10.0

Hz, 1H, H-5), 3.06-3.02 (A part of an AB system, JAB = 13.2 Hz, 1H, H-1’a), 3.01-2.98 (B

part of an AB system, JAB = 13.2 Hz, 1H, H-1’b), 2.18 (s, 3H, CH3), 2.11 (s, 3H, CH3), 2.10

(s, 3H, CH3), 2.07 (s, 3H, CH3), 2.06-1.99 (m, 2H, H-2, H-2’), 2.01 (s, 3H, CH3), 1.98 (s,

3H, CH3); 13C NMR (50MHz, CDCl3): δ 170.4, 170.1, 169.9, 169.3 (2C), 169.1, 138.8 (d,

JC-F = 27.4 Hz, 1C), 106.8 (d, JC-F = 121.2 Hz, 1C), 102.8 (d, J = 218.5 Hz, 1C), 93.4, 69.7,

68.2, 67.2, 66.2, 64.2, 63.4, 61.8, 61.06, 33.7, 33.6, 20.7 (6C); ESI-MS 659.1 [M+Na]+,

675.1 [M+K]+.

Synthesis of compound 8: A solution of 20 (86 mg, 0.135 mmol) and C14H29OH (58 mg,

0.270 mmol), in dry CH2Cl2 (15.0 mL) was cooled to -40°C and Cp2HfCl2 (92 mg, 0.243

mmol) and AgOTf (134 mg, 0.522 mmol) were added. After 40 min at –40° C the solution

was neutralized with NEt3 and filtered throught a pad of Celite and the filtrate was washed

with a saturated solution of NaHCO3 (1 x 10 mL) then with brine (1 x 10 mL). The organic

phase was dried over Na2SO4 and concentrated to dryness to give 130mg of crude. The

crude was purified by flash column chromatography on silica gel to give 8 (47 mg, 42%),

(petroleum ether/EtOAc 2:1) and 15.6 mg of a secondary product.

S22

O

OOAc OAc

SO

OAcAcO

AcOOAc

F CH2Cl2

C14H29OHCp2HfCl2

20

O

OOAc OAc

SO

OAcAcO

AcOOAc

OC14H29

8

AgOTf

Synthesis of compound 2:

O

OOH OH

SO

OHHO

OHOH

OC14H29

O

OOAc OAc

SO

OAcAcO

AcOOAc

OC14H29

8 2

MeONa

MeOH

To a stirred solution of 8 (19 mg, 0.022 mmol) in CHCl3/MeOH 1:1 (1.6 mL), 285 µL of 0.05

M solution of MeONa in MeOH were added. The mixture was stirred for 5 h, then the pH

was adjusted to neutrality with HCl (10% in MeOH). Evaporation of the solvent under vacu-

um gave a crude which was purified by flash chromathography on silica gel (EtOAc/MeOH

6:1) to give 2 (10 mg, 77%) as a glassy solid. [α]D25 +55.58 (c 0.104, MeOH); 1H NMR

(400MHz, CD3OD): δ 4.95 (s, 1H, H-a), 4.16-4.11 (M part of an AMXY system, JMA = 2.8

Hz, JMX = 7.6 Hz, JMY = 9.6 Hz, 1H, H-3), 4.08-4.05 (X part of an ABX system, J = 2.0 Hz, J

= 5.2 Hz, J = 6.4 Hz, 1H, H-e), 4.017-4.012 (A part of an AMXY system J4-5 = 0.4 Hz, JAM =

2.4 Hz, 1H, H-4), 3.93 (dd, J5-4 = 0.8 Hz, J5-6 = 9.6 Hz, 1H, H-5), 3.81-3.60 (m, 7H, H-d, H-

6, H-7, H-7’, H-f, H-f’, CH2X), 3.50-3.44 (m, 1H, CH2X), 3.03-3.00 (A part of an AB system,

JAB = 13.2 Hz, 1H, H-1’a), 2.98-2.95 (B part of an AB system, JAB = 13.2 Hz, 1H, H1’b),

1.93-1.90 (m, 2H, H-2, H-2’), 1.59-1.54 (m, 2H, CH2Y), 1.40-1.25 (m, 23H, CH2 chain), 0.89

(t, J = 6.4 Hz, 3H, CH3); 13C NMR (50MHz, CD3OD): δ 144.0, 108.7, 98.5, 94.5, 74.7, 73.0,

70.6, 70.2, 68.6, 68.4, 66.7, 65.0, 63.1, 38.4, 35.5, 31.63 (4C), 31.60 (4C), 31.5, 31.3,

28.1, 24.6, 15.3; ESI-MS 601.2 [M+Na]+, 617.2 [M+K]+; elemental analysis calcd (%) for

C28H50O10S: (578.76) C 58.11; H 8.71; O 27.64; found C 58.34; H 8.86; O 27.68.

S23

Binding evaluation of thioether 2 to WGA selectin

The binding interaction of thioether 2 and WGA (Wheat Germ Agglutinin) was evaluated by

using a quartz-crystal microbalance (QCM). These data were then compared to those ob-

tained with the GM3-ganglioside.[S17] GM3-ganglioside and WGA (MW = 43200) were pur-

chased from Sigma-Aldrich.

Quartz crystals (9.5 MHz AT-Cut, 14 mm) with gold evaporated (42.6 mm2 area) on both

sides (International Crystal Manufacturing, USA) were used. The resonance frequency va-

riations caused by changes in mass on the crystal (according to Sauerbrey’s equation[S18])

was continuously recorded by the quartz crystal analyzer QCA 922 by SEIKO, and read

directly by a computer connected to the ins trument.

Before using, the electrode surface of the quartz crystals was cleaned by dipping it in a

boiling solution of H2O2 (33%): NH3 (33%): milliQ water (1:1:5) for 10 min, then washed

with milliQ water and used immediately. The freshly cleaned crystal was dipped overnight

into an unstirred 1mM solution of 1-dodecanethiol at room temperature and in the dark.

The crystal was then washed with milliQ water, and treated with a 10 mM solution of 6-

mercaptohexan-1-ol for 1 h. [S19]

Preparation of GM3-ganglioside probe monolayer

A solution of GM3-ganglioside (50 µL of a 10 ng/µL solution) in CHCl3:CH3OH (4:1) was

dropped on the gold crystal surface, previously modified with a monolayer of 1-dodecane-

thiol, outside the cell. The monolayer formation was monitored by the quartz crystal ana-

lyzer QCA 922 by SEIKO.[S20] After the evaporation of the organic solvent under nitrogen

flow, the electrode was housed in a methacrylate cell and washed with PBS until the fre-

quency was stable (Figure S2).

S24

9474000

9475000

9476000

9477000

9478000

9479000

9480000

9481000

9482000

9483000

0 200 400 600 800 1000 1200 1400 1600

time (sec)

Fre

quen

cy (

Hz)

?F= -694Hz

AC

B

9474000

9475000

9476000

9477000

9478000

9479000

9480000

9481000

9482000

9483000

0 200 400 600 800 1000 1200 1400 1600

time (sec)

Fre

quen

cy (

Hz)

?F= -694Hz

AC

B

Figure S2. Sensorgram recorded during GM3-ganglioside immobilisation on the gold modified

surface of the crystal. A): frequency signal recorded in air. B): frequency signal during GM3-

ganglioside dropping . C): frequency signal after organic solvent evaporation.

QCM measurements

In order to evaluate the molecular recognition with WGA selectin, 200 µL of two different

concentrations of WGA were added to the sensor surface previously modified with the gly-

cosphingolipide GM3-ganglioside monolayer. The interaction was monitored for 30 min.

The solution was then removed and the surface washed with the PBS buffer to eliminate

the unbound proteins. The analytical signal was given by the difference between the fre-

quency recorded after washing with PBS and the frequency displayed before the interac-

tion. This experiment was performed with 10 and 50 ppm of WGA. Figure S3 shows the

sensorgram (frequency versus time) recorded during the experiments. The experiments

were performed in triplicate.

S25

9471500

9471600

9471700

9471800

9471900

9472000

9472100

9472200

9472300

9472400

9472500

1 501 1001 1501 2001 2501 3001 3501 4001 4501 5001 5501 6001

time (sec)

Fre

quen

cy (

Hz)

WGA 10ppm?F = -32Hz

WGA 50ppm?F = -93Hz

A

B C

D

E F

G

9471500

9471600

9471700

9471800

9471900

9472000

9472100

9472200

9472300

9472400

9472500

1 501 1001 1501 2001 2501 3001 3501 4001 4501 5001 5501 6001

time (sec)

Fre

quen

cy (

Hz)

WGA 10ppm?F = -32Hz

WGA 50ppm?F = -93Hz

A

B C

D

E F

G

Figure S3. Sensorgram recorded during interaction GM3-ganglioside-WGA (10 ppm and 50 ppm).

A): baseline in PBS buffer. B): injection of WGA 10ppm. C): washing with PBS buffer. D): baseline

in PBS buffer. E): injection of WGA 50 ppm. F): washing with PBS buffer. G): baseline in PBS buf-

fer. Analytical signal was given by ?F (FD-FA and FG-FD).

To test the specificity of the surface, 200 µL of a 100 ppm solution of a non specific protein

(Bovine Serum Albumine, BSA) were added (Figure S4). The negligible frequency shift

demonstrates the specificity of the sensor surface.

9475600

9475650

9475700

9475750

9475800

9475850

9475900

0 200 400 600 800 1000 1200 1400 1600

T i m e ( s e c )

Fre

quen

cy (

Hz) BSA 100ppm

?F= 0Hz

A

BC

D

9475600

9475650

9475700

9475750

9475800

9475850

9475900

0 200 400 600 800 1000 1200 1400 1600

T i m e ( s e c )

Fre

quen

cy (

Hz) BSA 100ppm

?F= 0Hz

A

BC

D

Figure S4. Sensorgram recorded during the interaction between GM3-ganglioside and BSA (100

ppm). A) baseline in PBS buffer. B) injection of BSA 100 ppm. C) washing with PBS buffer. D)

baseline in PBS buffer. Analytical signal was given by ?F (FD-FA).

S26

Preparation of thioether 2 probe monolayer

A solution of thioether 2 (50 µL of a 10 ng/µL solution) in CHCl3:CH3OH (4:1) was dropped

on the gold crystal surface, outside the cell, previously modified with a monolayer of 1-do-

decanethiol. The monolayer formation was monitored by the quartz crystal analyzer QCA

922 by SEIKO. After evaporation of the organic solvent by nitrogen flow, the electrode was

housed in a methacrylate cell and washed with buffer until the frequency was stable (Fig-

ure S5).

9472500

9473500

9474500

9475500

9476500

9477500

9478500

0 100 200 300 400 500 600 700 800 900 1000

Time (sec)

Fre

qu

ency

(H

z)

?F= -781 Hz

A

B C

9472500

9473500

9474500

9475500

9476500

9477500

9478500

0 100 200 300 400 500 600 700 800 900 1000

Time (sec)

Fre

qu

ency

(H

z)

?F= -781 Hz

A

B C

Figure S5. Sensorgram recorded during the immobilisation of thioether 2 on the gold modified

surface of the crystal. A): frequency signal recorded in air. B): frequency signal recorded during

thioether 2 dropping. C): frequency signal after organic solvent evaporation.

QCM measurements

In order to evaluate the molecular recognition with WGA selectin, 200 µL of two different

concentrations of WGA were added to the sensor surface previously modified with a thio-

ether 2 monolayer. The interaction was monitored for 30 min. The solution was then re-

moved and the surface washed with the PBS buffer to eliminate the unbound proteins. The

analytical signal was given by the difference between the frequency recorded after wash-

ing with PBS and the frequency displayed before the interaction. This experiment was per-

formed with 10 and 50 ppm solution of WGA. Figure S6 shows the sensorgram (frequency

versus time) recorded during the experiments. The experiments were performed in tripli-

cate.

S27

9474980

9475000

9475020

9475040

9475060

9475080

9475100

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Time (sec)

Fre

quen

cy (

Hz)

WGA 10 ppm?F= -5 Hz

WGA 50 ppm?F= -25 Hz

A

B C

D

E F

G

9474980

9475000

9475020

9475040

9475060

9475080

9475100

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Time (sec)

Fre

quen

cy (

Hz)

WGA 10 ppm?F= -5 Hz

WGA 50 ppm?F= -25 Hz

A

B C

D

E F

G

Figure S6. Sensorgram recorded during the interaction between thioether 2 and WGA (10 ppm

and 50 ppm). A) baseline in PBS buffer. B) injection of WGA 10 ppm. C) washing with PBS buffer.

D) baseline in PBS buffer. E) injection of WGA 50ppm. F) washing with PBS buffer. G) baseline in

PBS buffer. Analytical signal was given by ?F (FD-FA and FG-FD).

To test the specificity of the surface, a 100 ppm solution of BSA were added (Figure S7).

The negligible frequency shift demonstrates the specificity of the sensor surface.

9474920

9474940

9474960

9474980

9475000

9475020

9475040

9475060

9475080

9475100

0 500 1000 1500 2000 2500 3000

Time (sec)

Fre

quen

cy (

Hz)

BSA 100 ppm?F= 0 Hz

A

B C

D

9474920

9474940

9474960

9474980

9475000

9475020

9475040

9475060

9475080

9475100

0 500 1000 1500 2000 2500 3000

Time (sec)

Fre

quen

cy (

Hz)

BSA 100 ppm?F= 0 Hz

A

B C

D

Figure S7. Sensorgram recorded during the interaction between thioeher 2 and BSA (100 ppm).

A) baseline in PBS buffer. B) injection of BSA 100 ppm. C) washing with PBS buffer. D) baseline

in PBS buffer. Analytical signal was given by ?F (FD-FA).

The interaction of compound 2 on the surface of the crystal with WGA was inhibited by the

presence of N-acetyl-D-glucosamine (GlcNAc). No decrease of frequency was observed

S28

after the addition of the protein (WGA 50 ppm) in the presence of a 10 mM solution of N-

acetyl-D-glucosamine in phosphate buffer (Figure S8). This confirms the selectivity of the

interaction.

9478000

9478050

9478100

9478150

9478200

9478250

2500 3000 3500 4000 4500 5000

Time (sec)

Fre

quen

cy (

Hz)

WGA 50 ppm + GlcNAc 10mM

?F= 0 Hz

A

B C

D

9478000

9478050

9478100

9478150

9478200

9478250

2500 3000 3500 4000 4500 5000

Time (sec)

Fre

quen

cy (

Hz)

WGA 50 ppm + GlcNAc 10mM

?F= 0 Hz

A

B C

D

Figure S8. Sensorgram recorded during the interaction between thioeher 2 and WGA (50 ppm) in

the presence of GlcNAc 10 mM. A) baseline in PBS buffer. B) injection of a solution of GlcNAc 10

mM and BSA 100 ppm. C) washing with PBS buffer. D) baseline in PBS buffer. Analytical signal

was given by ?F (FD-FA).

References:

[S1] D. T. Cromer, J. T. Waber; Acta Crystallogr. 1965, 18, 104.

[S2] A. Altomare, M. C. Burla, M. Cavalli, G. L. Cascarano, C. Giavovazzo, A. Gagliardi,

A. G. G. Moliterni, G. Polidori, R. Spagna; J. Appl. Cryst. 1999, 32, 115.

[S3] G.M. Sheldrick Program SHELXL97 (crystal structure refinement), University of Göttingen,

Germany, 1997.

[S4] ORTEP-III, M. N. Burnett, C. K. Johnson, Report ORNL-6895, Oak Ridge National Labora-

tory, Oak Ridge, USA, 1996; see: L. J. Farrugia; J. Appl. Chem. 1997, 30, 565.

[S5] L. J. Farrugia; J. Appl. Chem. 1999, 32, 837.

[S6] R. C. MacDonald, R. I. MacDonald, B. P. Menco, K. Takeshta, N. K. Subbarao, L. R. Hu,

Biochim. Biophys. Acta, 1991, 1061, 297.

[S7] D. E. Koppel, J. Chem. Phys. 1972, 57, 4814.

[S8] S. W. Provencher, Comp. Phys. Comm. 1982, 27, 213.

[S9] D. Pontoni, T. Narayanan, A. R. Rennie, J. Appl. Cryst. 2002, 35, 207.

S29

[S10] L. Cantù, M. Corti, E. Del Favero, M. Dubois, T. N. Zemb, J. Phys. Chem. B, 1998, 102,

5737.

[S11] S. Ristori, J.Oberdisse, I. Grillo, A. Donati, O. Spalla, Biophys. J. 2005, 88, 535.

[S12] T. J. McIntosh in Molecular Description of Biological Membranes by Computer-aided

Conformational Analysis, CRC Press, Boca Rton, 1990.

[S13] N. Kucerka, Y. Liu, N. Chu, H. I. Petrache, S. Tristram-Nagle, F.J. Nagle, Biophys. J. 2005

88, 2626.

[S14] T. Frühwirth, G. Fritz, N. Freiberg, O. Glatter, J. Appl. Cryst. 2004, 37, 703,

[S15] D. D. Lasic, D. Papahadjopoulos in Medical applications of liposomes, Elsevier, New York,

1998.

[S16] G. Gregoriadis, B. McCormack, M. Obrenovic, R. Saffie, B. Zadi, Y. Perrie; Methods 1999,

19, 156.

[S17] T. Sato, M. Ishii, T. Terabayashi, Y. Kawanishi, Y. Okahata, Chem. Lett. 1997, 669, and

references therein.

[S18] G. Sauerbrey, Z. Physik, 1959, 155, 206.

[S19] S. Tombelli, M. Minunni, M. Mascini, Methods, 2005, 37, 48.

[S20] L. Becucci, M. R. Moncelli, R. Guidelli, Langmuir, 2003, 19, 3386.