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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2487SUPPORTING INFORMATION

Chemical polyglycosylation and nanolitre detection enables single-molecule recapitulation of bacterial sugar export

Lingbing Kong1, Andrew Almond2, Hagan Bayley1,*, Benjamin G. Davis1,*

1Department of Chemistry, University of Oxford, Chemical Research Laboratory, Mansfield Road, OX1 3TA, Oxford, UK;

2Faculty of Life Sciences, The University of Manchester, Carys Bannister Building, Dover St, Manchester, M13 9PL, England, UK.

Table of Contents

Supplementary Figure S1: Planar bilayer setup for single channel recording experiment. 3Supplementary Figure S2. Synthesis of disaccharide donors 4 and 7. 4Supplementary Figure S3. Synthesis of compounds 15 (a), 17 (b), 16 (c) and 8. 5Supplementary Figure S4. “2+2” glycosylation of dissacharide donor 4 and disaccharide acceptor 5. 6Supplementary Figure S5. “2+2” glycosylation of dissacharide donor 7 and disaccharide acceptor 8. 7Supplementary Figure S6. Synthesis of tetrasaccharide ‘monomers’ by “2+2” glycosylation. 8Supplementary Figure S7. HPLC traces for the purification of compounds protected K30-2n=2 and protected K30-2n=3. 9Supplementary Figure S8: DIB-Fusion setup (a) and the process of “Droplet Fusion” technique (b). 10Supplementary Figure S9. Interaction between K30-2n=1 and mutant Wza pore ∆P106-A107 at the trans and cis side. 11Supplementary Figure S10. Interaction of mutant pore ΔP106-A107 with K30-2n=1 at pH 4.3. 13Supplementary Figure S11. Interaction between mutant Wza pore ∆P106-A107 and hydrolyzed natural K30. 14Supplementary Figure S12. Comparison of CPS purification by gel extraction and by ultracentrifugation. 15Supplementary Figure S13. 1H NMR spectra of CPS before and after acid hydrolysis.

16Supplementary Figure S14. Overlaid HSQC (red) and coupled HSQC (green) NMR spectra of extracted K30 CPS after acid hydrolysis for 4 hours. 17Supplementary Figure S15. Representative events of K30-1n=1 interacting with the Wza pore ΔP106-A107. 18Supplementary Figure S16. Representative events of K30-2n=1 interacting with the Wza pore ΔP106-A107. 19Supplementary Figure S17. Histograms of dwell time of K30-2n=2 and K30-2n=3 in the Wza pore ΔP106-A107. 20Supplementary Figure S18. Representative events of K30-2n=2 interacting with the Wza pore ΔP106-A107. 21

SUPPORTING INFORMATION

Chemical polyglycosylation and nanoliter detection allows single-molecule recapitulation of bacterial sugar export

Lingbing Kong1, Andrew Almond2, Hagan Bayley1,*, Benjamin G. Davis1,*

1Department of Chemistry, University of Oxford, Chemical Research Laboratory, Mansfield Road, OX1 3TA, Oxford, UK;

2Faculty of Life Sciences, The University of Manchester, Carys Bannister Building, Dover St, Manchester, M13 9PL, England, UK.

Table of Contents

Supplementary Figure S1: Planar bilayer setup for single channel recording experiment. 3Supplementary Figure S2. Synthesis of disaccharide donors 4 and 7. 4Supplementary Figure S3. Synthesis of compounds 15 (a), 17 (b), 16 (c) and 8. 5Supplementary Figure S4. “2+2” glycosylation of dissacharide donor 4 and disaccharide acceptor 5. 6Supplementary Figure S5. “2+2” glycosylation of dissacharide donor 7 and disaccharide acceptor 8. 7Supplementary Figure S6. Synthesis of tetrasaccharide ‘monomers’ by “2+2” glycosylation. 8Supplementary Figure S7. HPLC traces for the purification of compounds protected K30-2n=2 and protected K30-2n=3. 9Supplementary Figure S8: DIB-Fusion setup (a) and the process of “Droplet Fusion” technique (b). 10Supplementary Figure S9. Interaction between K30-2n=1 and mutant Wza pore ∆P106-A107 at the trans and cis side. 11Supplementary Figure S10. Interaction of mutant pore ΔP106-A107 with K30-2n=1 at pH 4.3. 13Supplementary Figure S11. Interaction between mutant Wza pore ∆P106-A107 and hydrolyzed natural K30. 14Supplementary Figure S12. Comparison of CPS purification by gel extraction and by ultracentrifugation. 15Supplementary Figure S13. 1H NMR spectra of CPS before and after acid hydrolysis. 16Supplementary Figure S14. Overlaid HSQC (red) and coupled HSQC (green) NMR spectra of extracted K30 CPS after acid hydrolysis for 4 hours. 17Supplementary Figure S15. Representative events of K30-1n=1 interacting with the Wza pore ΔP106-A107. 18Supplementary Figure S16. Representative events of K30-2n=1 interacting with the Wza pore ΔP106-A107. 19Supplementary Figure S17. Histograms of dwell time of K30-2n=2 and K30-2n=3 in the Wza pore ΔP106-A107. 20Supplementary Figure S18. Representative events of K30-2n=2 interacting with the Wza pore ΔP106-A107. 21

© 2016 Macmillan Publishers Limited. All rights reserved.


SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2487

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Supplementary Figure S19. Representative events of K30-2n=3 interacting with the Wza pore ΔP106-A107. 22Supplementary Figure S20. Transport of am8γCD through Wza pore ∆P106-A107 in the DIB-Fusion droplet system. 23Supplementary Figure S21. Frequency of interaction events of synthetic K30 substrates interacting with the Wza pore ΔP106-A107 at pH 7.5. 24Supplementary Figure S22. Pore radii of the Wza pore ΔP106-A107. 25Supplementary Methods 26General experimental information 26General polyglycosylation procedures 26Preparation and extraction of K30 CPS 27Hydrolysis of extracted K30 CPS 28Single-channel planar bilayer recording 29Droplet interface bialyer-fusion system for single channel recording 29Analysis of the single-channel recording data 30Molecular dynamics simulation 30Calculation of relative electrophoretic mobilities 31Experimental procedures and compound characterization 33References 63NMR spectra of compounds 65




3

Supplementary Figures

Supplementary Figure S1: Planar bilayer setup for single channel recording

experiment.

a) overall system; b) recording chamber.




4

Supplementary Figure S2. Synthesis of disaccharide donors 4 and 7.

Reagents and conditions: (a) PhCH(OMe)2, CSA, DMF, 70%. (b) BnBr, DCM,

n-Bu4NHSO4, NaOH, aq. reflux, 35% 21, 23% 10. (c) 10, 11, TMSOTf, DCM, -

20°C. (d) 80% AcOH aq. 60°C, 6h, 75%. (e) Ac2O, pyr., 93%. (f) NIS, Tf2O,

DCM/water (100:1, v/v), 62%. (g) CCl3CN, DBU, DCM, 45% 4, 31% 7.




5

Supplementary Figure S3. Synthesis of compounds 15 (a), 17 (b), 16 (c) and 8.

Reagents and conditions:

a) (a) FmocCl, pyr., overnight. (b) Ac2O, pyr., (c) Et3N, DCM, 57% for three

steps. (d) i. 14, TMSOTf, DCM, -20 °C, 81%. ii. NaOMe, MeOH, 72 %.

b) (a) NaOMe, MeoH, 85%. (b) NaH, BnBr, DMF, 64%. (c) i. NIS, TFA,

DCM/water, 1h, CNCCl3, DCM, DBU, 56% for two steps. (d) TMSOTf,

HSCHMe2, DCM, 65%. (e) Pd/C, H2, 86%. (f) butanedione, CSA, CH(OMe)3,

DCM, 35%. (g) TBSOTf, 2,6-lutidine, DCM, 99%.

c) (a) 80% AcOH aq. 70°C, 3h. (b) Ac2O, pyr., 69% for two steps. (c) i. NIS,

TFA, acetone/water, 1h, CNCCl3, DCM, DBU, 71% for two steps. (d) TMSOTf,

DCM, 17, -20°C. (e) TFA/water (9:1, v/v), 29% for two steps. (f) TBSOTf, 2,6-

lutidine, DCM, 100%.




6

Supplementary Figure S4. “2+2” glycosylation of dissacharide donor 4 and

disaccharide acceptor 5.

Reagents and conditions: (a) TMSOTf, DCM, -20°C, 35% 3 and 26% 37.




7

Supplementary Figure S5. “2+2” glycosylation of dissacharide donor 7 and

disaccharide acceptor 8.

Reagents and conditions: (a) TMSOTf, DCM, -20°C, 45% 19, 16% 38, 6% 39,

1% 24. (b) i. TFA/water 9/1 (v/v); ii, Ac2O, pyr., 76% over two steps.




8

Supplementary Figure S6. Synthesis of tetrasaccharide ‘monomers’ by “2+2”

glycosylation.

Reagents and conditions: (a) TMSOTf, DCM, -20°C, MS 4Å, 35%. (b) i. 80%

AcOH aq. 70°C, 6h, 77%; ii.1 N NaOH, MeOH, 2 h, 95%; iii. Pd/C, H2,

water/THF, 63%. (c) TMSOTf, DCM, -20°C, MS 4 Å. (d) i. TFA/water 9/1 (v/v);

ii, Ac2O, pyr., 35% over two steps. (e) Pd/C, H2, 64%. (f) 0.5 N NaOH, MeOH-

DCM (1:1, v/v), overnight, 100%.




9

Supplementary Figure S7. HPLC traces for the purification of compounds

protected K30-2n=2 and protected K30-2n=3.

A C-18 reverse phase column (Phenomenex, Cat. no. 00D-4375-P0-AX) was used

with water and acetonitrile. a) HPLC trace for separating pure protected K30-2n=2,

the dominant fraction in the dimeric region, and crude protected K30-2n=3, the

dominant fraction in the trimeric or polymeric region which was further purified as

follows. b) HPLC trace for further purification of protected K30-2n=3.




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Supplementary Figure S8: DIB-Fusion setup (a) and the process of “Droplet

Fusion” technique (b).

To maintain the channel, the distance between the cis and trans electrodes was kept

constant. At the same time, the distance between the substrate droplet and the trans

droplet is lowered to drive the fusion process.




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Supplementary Figure S9. Interaction between K30-2n=1 and mutant Wza pore

∆P106-A107 at the trans and cis side.

(a) DIB-Fusion droplet setup. The substrate droplet was moved towards either cis or

trans droplet. The distance between the cis and trans electrodes was kept constant.

Using trans droplet as the droplet for fusion with the substrate as example, the

distance between the sugar droplet and the trans droplet is lowered to drive the fusion

process. (b) Schematic and actual image of the DIB system where a Wza pore was in

the bilayer. (c) Electrical recording trace of the Wza pore in 2 M KCl, buffered with 5

mM HEPES, pH 7.5. (d) Schematic and actual images of a substrate droplet with

K30-2n=1 (40 mM in the 2 M KCl buffer) was moved and attached to the trans droplet.

(e) The trans droplet and the substrate was forced to fused so that K30-2n=1 in trans

side became 20 mM. (f) Electrical recording trace of the Wza pore (∆P106-A107)




12

after addition of K30-2n=1 to trans in the 2 M KCl buffer. (g) Schematic and actual

images of a substrate droplet with K30-2n=1 (40 mM in the 2 M KCl buffer) was

moved and attached to the cis droplet. (h) The cis droplet and the substrate was forced

to fuse so that K30-2n=1 in cis side became 20 mM as well. (i) Electrical recording

trace of the Wza pore (∆P106-A107) after addition of K30-2n=1 to both trans and cis

in the 2 M KCl buffer. The volume of each fresh droplet was ~0.2 µL. Dashed line =

0 pA.




13

Supplementary Figure S10. Interaction of mutant pore ΔP106-A107 with K30-

2n=1 at pH 4.3.

a) Single-channel trace of ΔP106-A107 after it was introduced from cis droplet (0.2

µL). b) Single-channel trace after a third droplet (0.2 µL) with GlcA in 2 M KCl

(buffered with 5 mM HEPES, 100 µM EDTA, 200 µM DTT, pH 4.3) was moved

towards the cis one and fused to it, making it the same pH as in Supplementary Fig.

S9. c) Single-channel trace after a K30-2n=1 droplet (0.4 µL) in 2 M KCl (buffered

with 5 mM HEPES, 100 µM EDTA, 200 µM DTT, 40 mM K30-2n=1, pH 4.3) was

moved to and fused with the cis one to make K30-2n=1 concentration to 20 mM. The

applied electrical potential was +15 mV. The sampling rate is 5 k Hz and the signal

was filtered at a frequency of 1 kHz. Dashed line = 0 pA.




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Supplementary Figure S11. Interaction between mutant Wza pore ∆P106-A107

and hydrolyzed natural K30.

Representative events obtained from hydrolysed, extracted natural K30 CPS

interacting with the Wza pore ΔP106-A107 at +25 mV. K30 CPS obtained after 30

min hydrolysis (50 µg/mL). See also Supplementary Figures S12-14 for extraction

and hydrolysis. The sampling rate is 5 k Hz and the signal was filtered at a frequency

of 1 kHz. Dashed line = 0 pA.




15

Supplementary Figure S12. Comparison of CPS purification by gel extraction

and by ultracentrifugation.

Ultracentrifugation proved to be ineffective; instead, pure K30 CPS was obtained by

gel extraction




16

Supplementary Figure S13. 1H NMR spectra of CPS before and after acid

hydrolysis.

The acidic hydrolysis of extracted, natural K30 CPS was monitored by 1H NMR.

Gelation among intact K30 CPS led to weak signals at 0 min but these intensified and

sharpened with the progression of hydrolysis. Detailed NMR analysis of the 240 min

hydrolyzed K30 CPS gave a ratio of mannosyl : galactosyl/glucuronyl as 1 : 3,

matching with that of the expected structure (see also Supplementary Fig. S14).




17

Supplementary Figure S14. Overlaid HSQC (red) and coupled HSQC (green)

NMR spectra of extracted K30 CPS after acid hydrolysis for 4 hours.




18

Supplementary Figure S15. Representative events of K30-1n=1 interacting with

the Wza pore ΔP106-A107.

(a) K30-1n=1 (10 mM), at +10 mV in the 300mM KCl buffer. (b) K30-1n=1 (10 mM),

at +15 mV in the 300mM KCl buffer. (c) K30-1n=1 (10 mM), at +25 mV in the

300mM KCl buffer. (d) K30-1n=1 (10 mM), at +35 mV in the 300mM KCl buffer.

Dashed line = 0 pA.




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(a) K30-2n=1 (100 mM), at +15 mV in the 300mM KCl buffer. (b) K30-2n=1 (100 mM),

at +25 mV in the 300mM KCl buffer. (c) K30-2n=1 (100 mM), at +35 mV in the

300mM KCl buffer. (d) K30-2n=1 (100 mM), at +50 mV in the 300mM KCl buffer.

Dashed line = 0 pA.




20

Supplementary Figure S17. Histograms of dwell time of K30-2n=2 and K30-2n=3 in


(a) K30-2n=2 (5 mM), +15 mV. (b) K30-2n=2 (5 mM), +25 mV. (c) K30-2n=2 (5

mM), +35 mV. (d) K30-2n=2 (5 mM), +50 mV. (e) K30-2n=2 (5 mM), +75 mV. (f)

K30-2n=3 (1 mM), +15 mV. (g) K30-2n=3 (1 mM), +25 mV. (h) K30-2n=3 (1 mM), +35

mV. (i) K30-2n=3 (1mM), +50 mV. (j) K30-2n=3 (1mM), +75 mV.




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(a) K30-2n=2 (5 mM), at +25 mV in the 300 mM KCl buffer. (b) K30-2n=2 (5 mM), at

+35 mV in the 300 mM KCl buffer. (c) K30-2n=2 (5 mM), at +50 mV in the 300 mM

KCl buffer. (d) K30-2n=2 (5 mM), at +75 mV in the 300 mM KCl buffer. (e) K30-2n=2

(5 mM), at +100 mV in the 300 mM KCl buffer. Dashed line = 0 pA.




22



(a) K30-2n=3 (1 mM), at +15 mV in the 300mM KCl buffer. (b) K30-2n=3 (1 mM), at

+25 mV in the 300mM KCl buffer. (c) K30-2n=2 (1 mM), at +35 mV in the 300mM

KCl buffer. (d) K30-2n=3 (1 mM), at +50 mV in the 300mM KCl buffer. (e) K30-2n=3

(1 mM), at +75 mV in the 300mM KCl buffer. (f) An example of irreversible block

by K30-2n=3 (1 mM), at +100 mV in the 300mM KCl buffer. Reversal of the applied

potential (-100 mV) is required for the dissociation of the K30-2n=3 molecule from the

pore. Dashed line = 0 pA.




23

Supplementary Figure S20. Transport of am8γCD through Wza pore ∆P106-

A107 in the DIB-Fusion droplet system.

a) Schematic view of the transport of am8γCD through Wza pore ∆P106-A107 from

the cis side to the trans side. b) Lifetime of am8γCD•Wza pore ∆P106-A107 versus

applied potential in the 2 M KCl buffer at pH 7.5. The sampling rate is 5 k Hz and the

signal was filtered at a frequency of 1 k Hz.




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Supplementary Figure S21. Frequency of interaction events of synthetic K30

substrates interacting with the Wza pore ΔP106-A107 at pH 7.5.

The frequency of all interaction events when normalized by concentration (directly

proportional to ‘on’ rate; see Supplementary Methods for the calculation of relative

electrophoretic mobilities) was greater for greater displayed overall charge, e.g., K30-

2n=3 > K30-2n=2 > K30-2n=1 = K30-1n=1, measured at pH 7.5 where the carboxylic acid

group of every repeating 4-mer unit would be expected to be negatively charged.

Whilst transported sugars K30-2n=2 > K30-2n=1 = K30-1n=1 displayed no significant

voltage dependency, non-transported sugar K30-2n=3 displayed a moderate

dependency.




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Supplementary Figure S22. Pore radii of the Wza pore ΔP106-A107.

The pore radii were determined with HOLE.




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Supplementary Methods

General experimental information

Melting points were recorded on a Kofler hot block and are uncorrected. Proton

nuclear magnetic resonance spectra were recorded on a Bruker DPX 200 (200 MHz),

Bruker DPX 400 (400 MHz), Bruker DQX 400 (400 MHz), Bruker AC 500 (500

MHz) or Bruker AV700 (700 MHz) spectrometer. Carbon nuclear magnetic

resonance spectra were recorded on a Bruker DPX 200 (50 MHz), Bruker DQX 400

(100 MHz) spectrometer or on a Bruker AC 500 (125 MHz) with a 13C cryoprobe

(125 MHz). Spectra were fully assigned using a combination of COSY, HSQC,

coupled HSQC, HMBC and DEPT 135. All chemical shifts were quoted on the scale

in ppm using residual solvent as the internal standard. Coupling constants (J) are

reported in hertz (Hz). Infrared spectra were recorded on a Bruker Tensor 27 Fourier

Transform spectrophotometer with absorption maxima recorded in wavenumbers

(cm-1). Oils were analyzed as thin films. Low resolution mass spectra were recorded

on an LCT Premier XE using electrospray ionization (ES). High resolution mass

spectra were recorded on a Bruker micrOTOF. Specific rotations were measured on a

Perkin Elmer 241 polarimeter with a pathlength of 1.0 dm with concentrations (c)

given in g/100 mL. Thin layer chromatography (TLC) was carried out on Merk

Kieselgel 60F254 precoated aluminum backed plates and visualized with a

combination of the following: 254 nm UV lamp; aqueous KMnO4 (5% KMnO4 in 1

M NaOH); aqueous phosphomolybdic acid and Ce(IV) (2.5% phosphomolybdic acid

hydrate, 1% cerium(IV) sulfate hydrate, and 6% H2SO4); ammonium molybdate (5%

in 2M H2SO4); and/or sulfuric acid (2 M in ethanol/water 1:1). Flash column

chromatography was carried out with Fluka Kiegselgel 60 220-440 mesh silica gel.

THF and CH2CL2 were dried through a column of Al2O3. Other anhydrous solvents

were purchased from Aldrich or Fluka and stored over molecular sieves (MS)

(<0.005% H2O). All other solvents were used as supplied (analytical or HPLC grade),

without further purification. Petrol refers to the fraction of petroleum ether boiling in

the range 40-60 ºC. Brine refers to a saturated solution of sodium chloride.

General polyglycosylation procedures

Tetrasaccharide monomer 6 (30 mg, 22 µmol, 1.0 eq.) and NIS (6 mg, 26.4 µmol, 1.2

eq.) were dried in vacuo overnight in separate 5 mL flasks. CH2Cl2 with activated 4 Å




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MS (1 mL) was added individually into the two flasks. After stirring for half an hour

at RT, TMSOTf (1 µL, 5.5 µmol, 0.25 equiv) was added into the NIS flask. The

flasks were immersed into acetone-dry ice mixture at -30 ℃. The suspension of NIS

in CH2Cl2 with activated 4 Å MS was transferred via canula into the flask of 6 to

initiate the reaction. After maintaining the temperature of the reaction flask at -30 ℃

for 3 hours, Et3N (10 µL) in CH2Cl2 (0.5 mL) with activated 4 Å MS was added to

quench the reaction at -30 ℃. MS was filtered and the solution was diluted with

CH2Cl2 (40 mL). Aqueous Na2S2O3 solution (10 %, 50 mL) was added under

turbulent stirring. After 20 min, the lower organic layer was separated, dried over

MgSO4 and filtered. The solvent was removed on a rotary evaporator to give a crude

oligomerisation product. Reverse-phase HPLC (Supplementary Fig. S7) was used to

separate the oligosaccharides/ polysaccharides. MALDI analysis of every fraction

identified which oligomer/polymer the fraction is. If the purity of a fraction was good

(at least >85% pure based on HPLC analysis) and the quantity was sufficient (at

least >0.2 mg), further analysis (NMR, IR, optical rotation, etc.) was carried out.

Then full deprotection of this fraction, i.e. deacetylation, was performed to afford the

pure free K30 oligosaccharide/polysaccharide. Among the fractions, one major

octasaccharide (protected K30-2n=2, 1mg, 3%) and one major dodecasaccharide

(protected K30-2n=3, 0.5 mg, 1%) from the mixture were separated. More

specifically, the fraction eluted at retention time 8.1 min in Supplementary Fig. S7a

gave octasaccharide protected K30-2n=2 as a white solid and the fraction eluted at

retention time 11.0 min in Supplementary Fig. S7a and then at retention time 13.6

min in Supplementary Fig. S7b gave dodecasaccharide protected K30-2n=3 as a

white solid. After characterizations, the K30 oligosaccharides/polysaccharides were

treated with a sodium hydroxide solution to afford the desired K30 fragments in pure

forms (refer to specific K30 oligosaccharide/polysaccharide for detailed experimental

procedures).

Preparation and extraction of K30 CPS

E. coli strain E69 was grown in six 2 L flasks containing 1 L Luria Broth medium at

37 °C for 20 hours to OD ~ 3.0. The cells were collected by centrifugation at 8,000 ×

g in 600 mL centrifuge bottles for 30 min and transferred to two 50 mL centrifuge

tubes, followed by centrifugation at 20,000 × g for 10 min. The supernatant was




28

removed and the pellet (35 g) was resuspended in 10 mL PBS buffer supplemented

with 20 mM MgCl2. Phenol (30 mL) was added to each tube. The two tubes were

incubated at 65 °C for 10 min. After a quick spin, the mixture was placed on ice.

After centrifugation at 20,000 × g for 5 min, the upper aqueous phase containing

polysaccharides was then transferred to two 50 mL Falcon tubes, followed by

washing with chloroform twice (5 mL each). The aqueous phase was separated and

any residual chloroform in it was blown away by nitrogen. The solution was then

transferred to two Vivaspin 20 MWCO 10000 centrifugal concentrators and

concentrated by centrifugation at 1,500 × g, followed by washing with water and

repetition of centrifugation for multiple times. The polysaccharide solution was then

lyophilized to dryness (0.9 g). The dried polysaccharides powder was dissolved in

water (2.3 % or 1.0%) and subjected to ultracentrifugation at 100,000 × g at 4 °C for

16 h. It was proven that this approach was ineffective to obtain pure K30 CPS

(Supplementary Fig. S12). To improve the purity of K30 CPS, the polysaccharide

solution (2.3%, 180 µL, 4.1 mg polysaccharides inside) was mixed with equal volume

of Laemmli sample buffer and loaded to a CriterionTM XT 4-12% Bis-Tris precast gel.

The gel was run at 35-40 V at 4°C overnight with 1 × XT-MOPS running buffer. The

region of the gel containing K30 CPS, as indicated by the protein marker >100 k, was

cut out, transferred to two 50 mL Falcon tubes and crushed in 10 mL water. After

overnight on a rotary tube mixer at room temperature, the gel fragments were

removed by passing the suspension through a syringe filter with 0.22 µm pore size.

The solution was transferred to two Vivaspin 20 MWCO 10000 centrifugal

concentrators and concentrated by centrifugation at 1,500 x g, followed by washing

with water and repetition of centrifugation for multiple times. The K30 CPS in the

upper compartment was diluted in water (10 mL) and transferred to two 15 mL Falcon

tubes and lyophilized to dryness. Approximately ~1.5 mg (~36% yield) pure K30 CPS

was obtained from one gel (Supplementary Fig. S12).

Hydrolysis of extracted K30 CPS

Extracted K30 CPS (0.30 mg) obtained in this way was dissolved in D2O (600 µL) to

obtain a 0.5 mg/mL solution of K30 CPS. Sulfuric acid-d2 (Sigma, 96-98%, Cat. NO:

176769) was diluted to a 40% solution in D2O. The diluted sulfuric acid-d2 solution

(12 µL) was added to the K30 CPS solution in a NMR tube to obtain a 150 mM

solution of sulfuric acid-d2. After a quick spin, the solution was heated up to 95 °C in




29

a water bath. After 10, 30, 60, 180 and 240 min, the solution was rapidly cooled to RT

and measured by NMR, respectively. NaOD (Sigma, Cat. No: 372072, 40%, ~18 µL)

was added to neutralise the solution to pH 7. The hydrolysis for durations of 10 and

30 min followed by neutralisation was repeated, respectively, to obtain the 10 min-

hydrolysed and 30 min-hydrolysed samples. The 0, 10, 30, 240 min-hydrolysed

samples were lyophilized to dryness and stored at -20 °C for later use.

Single-channel planar bilayer recording

The interaction of hydrolyzed K30 CPS, i.e. 0, 10, 30 or 240 min-hydrolyzed K30

CPS, with the Wza pore ΔP106-A107 was measured in a 200 µL planar bilayer

system (Supplementary Fig. S1).

Droplet interface bialyer-fusion system for single channel recording

The droplet interface bilayer setup used in this report is a DIB-Fusion system. Two

micromanipulators (NMN-21, Narishige) held one electrode each1-5, while the third

one (MC-36, Narishige) was used to fuse a substrate droplet to either the cis or trans

droplet. The Ag/AgCl electrodes were prepared by dipping silver wires (0.1 mm in

diameter, Aldrich) fused to HDP20 crimp pin contacts into a sodium hypochlorite

solution (Fisher Scientific) under positive potential for 30 min. The Ag/AgCl

electrode tips were further coated with a 50-100 µm layer of 3 % (w/v) agarose in 2

M KCl or 300 mM KCl, buffered with 5 mM HEPES, 100 µM EDTA and 200 µM

DTT, pH 7.5. This agarose layer ensured the droplet hung properly in the oil during

an experiment.

The electric recording with this DIB-Fusion setup was carried out in a 0.5 ml Perspex

bath filled with 0.4 ml of DPhPC in hexadecane solution (1 mg/ml). The ~0.2 µL

droplets were generated by attaching the volume of buffer through a P2 pipette to the

electrodes in the oil bath. For high salt (2M KCl) experiments (Supplementary Fig.

S9-10), the solution for protein droplets was prepared by diluting a ΔP106-A107

sample from IVTT 40 times with 2 M KCl containing DPhPC (1mg/ml), buffered

with 5 mM HEPES, 100 µM EDTA and 200 µM DTT, pH 7.5. The substrate droplet

with K30-2n=1 were prepared by dissolving white solid K30-2n=1 in the same 2 M

KCl buffer containing DPhPC, while the 2 M KCl buffer at pH 4.3 was prepared by

adjusting the pH through the addition of a 1M D-glucuronic acid (GlcA) solution in




30

water. The bilayer was created by moving a protein/cis droplet (ground) and a

buffer/trans droplet into proximity after 5 min of incubation. On formation of a

bilayer, the capacitance was measured and adjusted to around 200 ± 50 pF in the

mode of WHOLE-CELL(β=1). A positive voltage was applied from the trans side to

assist the insertion of the protein into the bilayer. The distance between the cis and

the trans electords was kept constant. The distance between the substrate droplet and

either the cis or the trans droplet was lowered until the two droplets attached to each

other and fused into one droplet.

For the DIB-Fusion system used for the detection of interaction between mutant Wza

pore ΔP106-A107 and compound K30-2n=1, K30-1n=1, K30-2n=2 or K30-2n=3 in low

salt buffer, the solution for protein droplets was prepared by diluting a ΔP106-A107

sample from IVTT 40 times with 300 mM KCl containing DPhPC (1mg/ml),

buffered with 5 mM HEPES, pH 7.5. The substrate droplet with K30-2n=1, K30-1n=1,

K30-2n=2 or K30-2n=3 were prepared seperately by dissolving the corresponding

substrate in the 300 mM KCl buffer, followed by adjusting the pH to 7.5 with 1 M

KOH in water. The procedures to form bilayer and add substrates are the same as

above.

Analysis of the single-channel recording data

The data were acquired with a sampling rate of 5 k Hz and filtered at a frequency of 1

k Hz. The traces were Gaussian filtered at 1000 Hz with Clampfit 10.0. Some long

events needed to be further filtered to be recognised as individual events. For the

scatter plots (Figs. 6a-d) and the plot of frequency of events (Supplementary Fig.

S21), the events with lifetimes < 1 ms were filtered off during Single-Channel Search;

for the lifetime-voltage plots (Fig. 6e, Supplementary Fig. S20) the events with

lifetimes < 5 ms were filtered off during Single-Channel Search. The frequency of

interaction events for each single pore was calculated by: [(Number of events after

addition of sugar substrate)-(Number of events before addition of sugar

substrates)]/concentration (mM)/time(min).

Molecular dynamics simulation

Molecular modeling of K30 CPS oligomers K30-2n=1, K30-2n=2, and K30-2n=3 as

their reducing sugars was performed using the GLYCAM06 force-field topologies




31

and parameters for carbohydrates.6 Each oligosaccharide was built using AMBER127

and then added to cubic boxes of TIP3P water,8 resulting in side lengths in the range

4.1-5.1 nm (containing 1,500-3,500 water molecules). Solute atoms were positioned

at least 1.2 nm from the solvent box edge and charge neutral assemblies were

achieved by adding the appropriate number of explicit Na+ ions. Following initial

conjugate-gradient energy minimization (1,000 steps), each assembly was heated to

298 K and equilibrated in the NPT ensemble for 20 ns prior to 10 µs unbiased NVT

production molecular dynamics, performed using ACEMD software9 and GeForce

GTX 980 graphics processing unit hardware (Nvidia Corp., Santa Clara, USA), as

described previously.10,11Atomic coordinate data for the oligosaccharides were saved

at 10 ps intervals over the full 10 µs for analyses (water molecule and ion coordinate

data were discarded). Radii of gyration for each oligosaccharide were computed at

every step from complete 10 µs trajectories (i.e., one million datasets) using the

standard equation and plotted (Fig. 6j). Coordinates for each oligosaccharide were

also extracted at every 1 µs for each oligosaccharide, aligned and overlaid (by

minimizing the mean square distance between all atoms), and visualized (Fig. 6k).

Calculation of relative electrophoretic mobilities

Both the electrophoretic mobility and the mobility of the electroosmotic flow

contribute to the apparent electrophoretic mobility of a charged solute. In the DIB-

Fusion system, the mobility of the electroosmotic flow in the bulk solution could be

omitted. The apparent electrophoretic mobility equals approximately the

electrophoretic mobility for the charged solute. It could be described by the Stokes-

Einstein equation12:

where µa, µep and µeo are the apparent electrophoretic mobility and the electrophoretic

mobility of the solute with an effective charge z, and the mobility of the

electroosmotic flow, respectively, ƞ is the viscosity of the solution and rh is the

hydrodynamic radius. The electrical channel recording data were acquired with K30-

2n=3, K30-2n=2 and K30-2n=1 under low mM concentrations, i.e. 1 mM, 5 mM and ≥

10 mM, respectively. Higher molecular weight (Mw) and higher mass concentration

both lead to higher viscosity of the sugar solution.13 To simplify the calculation for

comparison, the viscosities (ƞ) of these sugar solutions were assumed to be equal. In




32

addition, the ratios of rg/rh (rg refers to the radius of gyration) were also assumed to be

equal for these sugar solutions.14,15 Therefore, the ratio of electrophoretic mobility of

two charged sugar substrates here could be simplified as:

The rg values for K30 sugar substrates K30-2n=3, K30-2n=2 and K30-2n=1 are taken

from Fig. 6j as 7.5, 7.0 and 4.8 nm, respectively. The ratios of the (apparent)

electrophoretic mobility of these substrates under the same applied electric potential

could then be calculated as:

Therefore, the relative (apparent) electrophoretic mobilities for the sugar substrates

are:




33

Experimental procedures and compound characterization

β-D-glucuronosyl-(1→3)-α-D-galactopyransyl-(1→3)-α-D-mannopyranosyl-(1→ 3)-isopropyl-1-thio-β-D-galactopyranoside (K30-1n=1)

Compound 3 (50 mg, 34 µmol) was dissolved in 80% AcOH (10 ml) in a 50 mL flask. The solution was heated up to 60 °C and the reaction was kept for 4 h with stirring. The solvent was then removed on a rotary evaporator. The residue was subjected to silica gel chromatography (ethyl acetate: methanol = 9: 1) to afford a glassy tetra-ol compound (34 mg, 77%). The compound (31.5 mg, 25 µmol) was dissolved in methanol (5 mL). 1N NaOH (5 mL) was added. The reaction was kept with stirring for 1h. IR120 (H+) resin was added to quench the reaction by adjusting the pH to ~4-5. The resin was filtered and the filtrate was concentrated on a rotary evaporator. The residue was subjected to silica gel chromatography (water: 2-propanol: ethyl acetate = 1: 4: 4) to afford a colorless glassy carboxylic acid (19 mg, 95%). The acid (16.5 mg, 20 µmol, 1.0 eq.) was dissolved in THF-water (1: 1, 6 mL). Pd/C (10.5 mg, 10 µmol, 0.5 eq.) was added to the solution. A balloon of Ar was used to exchange the air in the flask. Afer 10 mins, the Ar balloon was replaced with a balloon of H2 to exchange Ar with H2. The reaction was kept for overnight with stirring. Pd/C was filtered over Celite. The solvent was removed on a rotary evaporator. The residuc was subjected to silica gel chromatography (water: 2-propanol: ethyl acetate = 1: 2: 2) to afford the desired deprotected tetrasaccharide 1 or K30-1n=1 as a colorless solid (9.2 mg, 63%): Rf 0.35 (water: 2-propanol: EtOAc = 1: 2: 2); [α] D

20 = +95.6 (c 0.25, H2O); m.p. 234 °C (water); νmax (transmission, KBr, pellet)/cm-1 3400 (νO-H); HRMS m/z (ES¯) [calculated for C27H45O21S¯ 737.2180; found 737.2189]; Full assignment of all hydrogen atoms of 1 or K30-1n=1 (700 MHz). Chemical shift (ppm)

Peak shape Numbers of hydrogen atoms (H)

Coupling constant (Hz)

Assignment

1.23 dd 6 4.7, 6.9 SCH(CH3)2 3.19 m 1 \ SCH 3.33 m 1 \ H-2IV

3.43 m 2 \ H-5IV, H-3IV 3.53 t 1 9.8 H-2I 3.58-3.74 m 8 \ H-5I, H-6Ia&b, H-5IV,

H-6IIIa&b, H-3I, H-6IIa 3.77-3.84 m 2 \ H-6IIb, H-4II 3.88 m 1 \ H-5II 3.92 dd 1 3.8, 10.4 H-2III 3.96 dd 1 3.1, 9.5 H-3II 3.98-4.04 m 2 \ H-5III, H-3III 4.13 dd 1 1.9, 3.1 H-2II 4.14 d 1 3.1 H-4I 4.18 d 1 3.1 H-4III 4.51 d 1 10.1 H-1I (JCH 155 Hz) 4.60 d 1 7.9 H-1IV (JCH 163 Hz) 4.97 d 1 1.3 H-1II (JCH 171 Hz) 5.23 d 1 3.8 H-1III (JCH 172 Hz)




34

Full assignment of all carbon atoms of 1 or K30-1n=1 (125 MHz). Position Chemical

shift (ppm) Position Chemical



shift (ppm)

C-1I 84.9 C-1II 95.9 C-1III 100.5 C-1IV 103.7 C-2I 68.1 C-2II 69.9 C-2III 67.7 C-2IV 73.1 C-3I 77.4 C-3II 78.2 C-3III 79.3 C-3IV 75.3 C-4I 64.5 C-4II 65.9 C-4III 68.9 C-4IV 71.8 C-5I 78.5 C-5II 72.7 C-5III 71.3 C-5IV 76.2 C-6I 61.0 C-6II 60.7 C-6III 61.4 C-6IV 176.0

Note: SCH(CH3)2, 35.1 ppm; SCH(CH3)2, 23.0, 23.1. Isopropyl 2-O-(β-D-galactopyransyl)-3-O-[(β-D-glucuronosyl)-α-D-galacto-pyranosyl]-1-thio-β-D-mannopyranoside (K30-2n=1)

Compound 6 (38 mg, 28 µmol, 1.0 eq.) was dissolved in MeOH-CH2Cl2 (3 ml, 1:1). 0.5 N NaOH aq. (1 ml, 0.5 mmol, 18 eq.) was added. The reaction was kept for overnight with stirring. The reaction was quenched by the addition of IR120 (H+) to reach a pH of 3-4. The resin was filtered. The solvent was evaporated on a rotary evaporator. The residue was subjected to silica chromatography (water: 2-propanol: EtOAc = 1:2:2) to afford K30-2n=1 as a white crystalline solid (18 mg, 100%). Full assignment of all hydrogen atoms of K30-2n=1 (700 MHz).

Chemical shift (ppm)

Peak shape

Numbers of hydrogen atoms (H)

Coupling constant (Hz)

Assignment

1.22 2d 6 3.6, 3.2 SCH(CH3)2 3.15 m 1 \ SCH 3.42-3.48 m 3 \ H-5II, H-3IV, H-5IV 3.51-3.57 m 3 \ H-6aI, H-5I, H-2I 3.58 dd 1 3.4, 9.9 H-3I 3.61-3.72 m 5 \ H-6aIII, H-4IV, H-6aI,

H-6bIII, H-6aII 3.79 t 1 9.9 H-4II 3.84 dd 1 2.0, 12.4 H-6bII 3.86 d 1 3.4 H-4I 3.87-3.93 m 3 \ H-3III, H-2III, H-3II 4.16 d 1 3.0 H-4III 4.17 dd 1 3.6, 7.8 H-5III 4.37 d 1 3.2 H-2II 4.62 d 1 7.7 H-1IV (JCH 162 Hz) 4.70 d 1 7.8 H-1I (JCH 164 Hz) 4.87 s 1 \ H-1II (JCH 154 Hz) 5.34 d 1 3.7 H-1III (JCH 175 Hz)




35

Full assignment of all carbon atoms of K30-2n=1 (125 MHz). Position Chemical




shift (ppm) C-1I 103.2 C-1II 83.1 C-1III 99.6 C-1IV 104.1 C-2I 75.2 C-2II 78.5 C-2III 67.6 C-2IV 73.1 C-3I 72.5 C-3II 76.6 C-3III 79.7 C-3IV 75.2 C-4I 68.5 C-4II 67.0 C-4III 69.5 C-4IV 76.7 C-5I 71.6 C-5II 80.7 C-5III 71.2 C-5IV 71.8 C-6I 61.3 C-6II 60.6 C-6III 61.6 C-6IV 176.0 Note: SCH(CH3)2, 36.5 ppm; SCH(CH3)2, 22.8, 23.0. Rf 0.22 (water: 2-propanol: EtOAc = 1: 2: 2) and 0.44 (water: 2-propanol: EtOAc = 1: 1: 1); [α] D

20 = +81.2 (c 0.25, H2O); mp. 204-206˚C (water); νmax (transmission, KBr, pellet)/cm-1 3400 (νO-H); HRMS m/z (ES¯) [calculated for C27H45O21S¯ 737.2180; found 737.2188]. 4-«2-O-‹3-O-{2-O-(β-D-galactopyransyl)-3-O-[3-O-(β-D-glucuronosyl)-α-D-gala-ctopyransyl]-β-D-galactopyranosyl}-α-D-mannopyranosyl›-3-O-[(β-D-glucuro-nosyl)-α-D-galactopyransyl]-α-D-mannopyranosylamino»-4-oxobutanoic acid (K30-2n=2)

Compound protected K30-2n=2 (1 mg, 0.35 µmol, 1.0 eq.) was dissolved in MeOH (0.1 mL) and CH2Cl2 (0.1 mL). Sodium hydroxide aqueous solution (0.1 mL, 0.5 N, 50 µmmol, 143 eq.) was added, resulting in a homogenous solution. The reaction was kept for 1 day. IR120 (H+) resin was added to quench the reaction by adjusting the pH until 4-5. The resin was filtered and the solvent was removed on a rotary evaporator. Water (25 mL) and ethyl acetate (25 mL) were added to the residue under turbulent stirring. The lower aqueous layer was separated and the solvent was removed on rotary evaporator. The residue was dried in vacuo to afford compound K30-2n=2 as a white solid (0.5 mg, 100 %): Rf 0.24 (water: 2-propanol: EtOAc = 1: 1: 1); [α] D

20 = +64.0 (c 0.025, H2O); νmax (ATR)/cm-1 3387 (νO-H), 2924 (νCOO-H), 1659, 1599 (νN-H), 1411 (νC-H), 1130 (νC-N), 1083, 1040 (νC-O); 1H NMR (700 MHz, D2O) δ 2.40-2.51 (m, 4H, COCH2CH2CO), 3.25 (t, 1H, J 8.6 Hz, H-2IV(1)), 3.26 (t, 1H, J 8.6 Hz, H-2IV(2)), 3.36 (m, 2H, H-3IV(1), H-5IV(1)), 3.37 (m, 2H, H-3IV(2), H-5IV(2)), 3.39 (dd, 1H, J 7.7, 9.9 Hz, H-2I(2)), 3.40 (m, 1H, H-5II(1)), 3.41 (m, 2H, H-6I(1)a, H-6I(2)a), 3.44 (bs, 1H, H-5I(2)), 3.45 (dd, 1H, J 3.3, 9.9 Hz, H-3I(2)), 3.46 (s, 1H, H-5I(1)), 3.50 (dd, 1H, J 7.7, 9.7 Hz, H-2I(1)), 3.51 (m, 6H, H-6I(1)b, H-6I(2)b, H-6III(1)a & b, H-6III(2)a & b), 3.57 (dd, 1H, J 3.3, 9.9 Hz, H-3I(1)), 3.62 (m, 2H, H-6II(1)a&b), 3.67 (m, 2H, H-




36

6II(2)a&b), 3.73 (d, 1H, J 3.4 Hz, H-4I(2)), 3.76 (t, 1H, J 9.8 Hz, H-4II(1)), 3.78 (m, 2H, H-5II(2), H-4II(2)), 3.81 (d, 1H, J 9.2 Hz, H-4IV(1)), 3.82 (bs, 2H, H-2III(2), H-3III(2)), 3.83 (bs, 2H, H-2III(1), H-3III(1)), 3.83 (d, 1H, J 9.2 Hz, H-4IV(2)), 3.98 (dd, 1H, J 3.7, 9.7 Hz, H-3II(1)), 3.99 (d, 1H, J 3.1 Hz, H-4I(1)), 4.00 (s, 1H, H-4III(1)), 4.01 (s, 1H, H-4III(2)), 4.02 (dd, 1H, J 3.5, 8.6 Hz, H-3II(2)), 4.05 (m, 2H, H-5III(1), H-5III(2)), 4.09 (dd, 1H, J 1.5, 3.4 Hz, H-2II(1)), 4.13 (dd, 1H, J 1.2, 3.4 Hz, H-2II(2)), 4.31 (d, 1H, J 7.7 Hz, H-1I(2)), 4.34 (d, 1H, J 7.8 Hz, H-1I(1), 1JC-H = 161 Hz), 4.58 (d, 1H, J 7.9 Hz, H-1IV(1), 1JC-H = 164 Hz), 4.62 (d, 1H, J 7.9 Hz, H-1IV(2), 1JC-H = 164 Hz), 4.97 (s, 1H, H-1II(2), 1JC-H = 170 Hz), 5.29 (d, 1H, J 1.7 Hz, H-1III(2), 1JC-H = 174 Hz), 5.32 (d, 1H, J 1.7 Hz, H-1III(1), 1JC-H = 174 Hz), 5.38 (s, 1H, H-1II(1), 1JC-H = 165 Hz); 13C NMR (176 MHz, D2O) δ 28.8, 30.0 (COCH2CH2CO), 59.9 (C-6II(2)), 60.1 (C-6II(1)), 61.2 (C-6I(2)), 61.5 (C-6I(1)), 61.56 (C-6III(2)), 61.62 (C-6III(1)), 64.4 (C-4I(1)), 66.4 (C-4II(2)), 66.5 (C-4II(1)), 67.5 (C-2III(1)), 67.6 (C-2III(2)), 68.6 (C-4I(2)), 69.1 (C-2I(1)), 69.5 (C-4III(1), C-4III(2)), 70.6 (C-2I(2)), 71.2, 71.3 (C-5III(2), C-5III(1)), 71.36, 71.37 (C-5IV(1), C-5IV(2)), 72.6 (C-5II(2)), 72.7 (C-3I(2)), 72.9 (C-2IV(2), C-2IV(1)), 73.77, 73.80 (C-5II(1), C-3II(1)), 74.2 (C-3II(2)), 74.60, 74.62 (C-4IV(1), C-4IV(2)), 74.99, 75.03, 75.19 (C-3IV(1), C-3IV(2), C-5I(1), C-5I(2)), 76.2 (C-2II(1)), 76.4 (C-3I(1)), 76.6 (C-1II(1)), 77.0 (C-2II(2)), 79.7 (C-3III(2)), 79.8 (C-3III(1)), 94.2 (C-1II(2)), 99.9 (C-1III(1)), 100.0 (C-1III(2)), 101.6 (C-1I(1)), 102.1 (C-1I(2)), 104.1 (C-1IV(2)), 104.2 (C-1IV(1)), 172.8, 172.9 (C-6IV(1), C-6IV(2)), 175.6 (NHCOCH2CH2COOH), 176.9 (NHCOCH2CH2COOH); HRMS m/z (ES+) [calculated for ½(C52H81NO45

2-) 719.7046; found 719.7045]. 4-‹‹‹‹2-O-‹‹‹3-O-«2-O-‹3-O-{2-O-(β-D-galactopyransyl)-3-O-[3-O-(β-D-glucuronosyl)-α-D-galactopyransyl]-α-D-mannopyranosyl}-β-D-galactopyranosyl›-3-O-[(β-D-glucuronosyl)-α-D-galactopyransyl]-α-D-mannopyranosyl»-β-D-galactopyranosyl›››-3-O-(β-D-glucuronosyl)-α-D-galactopyransylamino››››-4-oxobutanoic acid (K30-2n=3)

Compound protected K30-2n=3 (0.5 mg, 0.13µmol) was dissolved in MeOH (0.1 mL) and CH2Cl2 (0.1 mL). Sodium hydroxide aqueous solution (0.1 mL, 0.5 N, 50 µmmol, 385 eq.) was added, resulting in an homogenous solution. The reaction was kept for 2 days. IR120 (H+) resin was added to quench the reaction by adjusting the pH until 4-5.




37

The resin was filtered and the solvent was removed on a rotary evaporator. Water (25 mL) and ethyl acetate (25 mL) were added to the residue under turbulent stirring. The lower aqueous layer was separated and the solvent was removed on rotary evaporator. The residue was dried in vacuo to afford compound K30-2n=3 as a glassy solid (0.25 mg, 100 %): Rf 0.24 (water: 2-propanol: EtOAc = 1: 1: 1); νmax (ATR)/cm-1 3200(νO-

H), 2931 (νCOO-H), 1697 (νC=O), 1590, 1657 (νN-H), 1412 (νC-H), 1194 (νC-N), 1075, 1037 (νC-O); HRMS m/z (ES+) [calculated for ½(C76H121NO66

2-) 1050.7999; found 1050.7979]. 1H NMR (700 MHz, D2O) δ 3.25 (t, 1H, J 8.6 Hz, H-2IV(1)), 3.26 (t, 1H, J 8.6 Hz, H-2IV(2)), 3.35 (m, 3H, H-5IV(1), H-5IV(2), H-5IV(3)), 3.35 (m, 3H, H-3IV(1), H-3IV(2), H-3IV(3)), 3.38 (dd, 1H, J 7.7, 9.9 Hz, H-2I(3)), 3.40 (m, 1H, H-5II(1)), 3.40-3.45 (m, 3H, H-6I(1)a, H-6I(2)a, H-6I(3)a), 3.43-3.47 (m, 7H, H-5I(1), H-5I(2), H-5I(3), H-5I(2), H-3I(3), H-2I(1), H-2I(2)), 3.50-3.58 (m, 9H, H-6I(1)b, H-6I(2)b, H-6I(3)b, H-6III(1)a & b, H-6III(2)a & b, H-6III(3)a & b), 3.58 (m, 2H, H-3I(1), H-3I(2)), 3.59-3.68 (m, 6H, H-6II(1)a&b, 6II(2)a&b, 6II(3)a&b), 3.70 (m, 3 H, H-4IV(1), H-4IV(2), H-4IV(3)), 3.74 (d, 1H, J 3.1 Hz, H-4I(3)), 3.75 (m, 2H, H-5II(2), H-5II(3)), 3.77 (m, 2H, H-4II(1), H-4II(2)), 3.81 (m, 6H, 3III(1), 3III(2), 3III(3), 2III(1), 2III(2), 2III(3)), 3.98-4.08 (m, 11 H, H-3II(1), H-4I(1), H-4I(2), H-3II(2), H-3II(3), H-4III(1), H-4III(2), H-4III(3), H-5III(1), H-5III(2), H-5III(3)), 4.09 (d, 1H, J 2.5 Hz, H-2II(1)), 4.13 (d, 1H, J 2.9 Hz, H-2II(2)), 4.15 (d, 1H, J 3.4 Hz, H-2II(3)), 4.31 (d, 1H, J 7.7 Hz, H-1I(3), 1JC-H = 162 Hz), 4.34 (d, 1H, J 7.7 Hz, H-1I(1), 1JC-H = 163 Hz), 4.36 (d, 1H, J 7.8 Hz, H-1I(2), 1JC-H = 163 Hz), 4.53-4.59 (3d, 3H, J 7.6, 6.4, 7.8 Hz, H-1IV(1), H-1IV(2), H-1IV(3), 1JC-H = 164, 163, 164 Hz), 4.98 (s, 1H, H-1II(2), 1JC-H = 172 Hz), 4.99 (s, 1H, H-1II(3), 1JC-H = 169 Hz), 5.19 (bs, 2H, H-1III(2), H-1III(3), 1JC-H = 176, 175 Hz), 5.22 (bs, 1H, H-1III(1), 1JC-H = 173 Hz), 5.38 (s, 1H, H-1II(1), 1JC-H = 164 Hz); 13C NMR (176 MHz, D2O) δ 59.6, 59.8, 59.9 (C-6II(1), C-6II(2), C-6II(3)), 61.10, 61.13, 61.4 (C-6I(1), C-6I(2), C-6I(3)), 61.43, 61.45, 61.50 (C-6III(1), C-6III(2), C-6III(3)), 64.1 (C-4I(1), C-4I(2)), 66.1, 66.3 (C-4II(1), C-4II(2)), 67.4 (C-2III(1), C-2III(2), C-2III(3)), 68.4 (C-4I(3)), 68.9 (C-2I(1), C-2I(2)), 69.3 (C-4III(1), C-4III(2), C-4III(3)), 70.3 (C-2I(3)), 71.1, 71.2 (C-5III(1), C-5III(2), C-5III(3)), 71.4 (C-5IV(1), C-5IV(2), C-5IV(3)), 72.3 (C-5II(2), C-5II(3)), 72.4 (C-3I(3)), 72.8 (C-2IV(1), C-2IV(2), C-2IV(3)), 73.3 (C-3II(1)), 73.5 (C-5II(1)), 73.8 (C-3II(2), C-3II(3)). 74.8, 74.9, 75.0 (C-5I(1), C-5I(2), C-5I(3)), 74.9 (C-3IV(1), C-3IV(2), C-3IV(3)), 75.2 (C-4IV(1), C-4IV(2), C-4IV(3)), 75.9 (C-2II(1)), 76.5 (C-1II(1)), 76.1 (C-3I(1), C-3I(2)), 76.7 (C-2II(3)), 76.8 (C-2II(2)), 79.7 (C-3III(1), C-3III(2), C-3III(3)), 93.9 (C-1II(3)), 94.0 (C-1II(2)), 99.7 (C-1III(1)), 99.8 (C-1III(2), C-1III(3)), 101.4 (C-1I(1)), 101.8 (C-1I(2)), 102.0 (C-1I(3)), 103.97, 104.02, 104.06 (H-1IV(1), H-1IV(2), H-1IV(3)). Quaternary carbons and carbons with chemical shifts lower than 50 were not observable in the spectra acquired. Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3) 2-O-benzyl-4,6-acetyl-α-D-galactopyransyl-(1→3)-4,6-O-benzylidene-α-D-mannopyranosyl-(1→3)-isopro-pyl-2-O-acetyl-4,6-O-benzylidene-1-isopropylthio-β-D-galactopyranoside (3)

Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3)-2-O-benzyl-4,6-acetyl-α-D-galactopyransyl-(1→2)-4,6-O-benzylidene-α-D-mannopyranosyl-(1→3)-isopro-pyl-2-O-acetyl-4,6-O-benzylidene-1-isopropylthio-β-D-galactopyranoside (37)




38

4 (132 mg, 0.13 mmol, 1.0 eq.) and 5 (82 mg, 0.13 mmol, 1.0 eq.) were dissolved in anhydrous CH2Cl2 (7 mL) with activated 4 Å MS (MS) under an argon atmosphere. The solution was cooled to -20 ˚C. TMSOTf (4.2 µL, 23 µmol, 0.18 eq.) was added. The mixture was stirred under these conditions for 30 min. The reaction was quenched by the addition of Et3N. The solvent was evaporated on a rotary evaporator and the residue was subjected to silica chromatography (petroleum: EtOAc = 1.2: 1) to afford 3 as a colorless glassy solid (67 mg, 35%) and 37 as the other colorless glassy solid (50 mg, 26%). Compound 3: Rf 0.40 (petroleum: EtOAc = 1: 2); [α] D

20 = + 48.7 (c 1.0, CHCl3); νmax (transmission, KBr, pellet)/cm-1 3385 (νO-H), 3063, 3028 (νC-Harom), 2924 (νC-Haliph), 1738 cm-1 (νC=O); 1H NMR (500 MHz, CDCl3) δ 1.27 (d, 3H, J 6.6 Hz, CH3), 1.39 (d, 3H, J 6.62 Hz, CH3), 1.74, 2.07, 2.15 (3s, 9H, 3CH3), 3.28 (m, 1H, SCH), 3.48 (s, 1H, H-5I), 3.67 (dd, 1H, J 3.8, 9.8 Hz, H-2III), 3.70 (s, 3 H, OCH3), 3.81-3.93 (m, 2H, H-5II, H-6aII), 3.93 (m, 2H, H-3I, H-6aIII), 3.99-4.06 (m, 4H, H-6bIII, H-2II, H-6aI, H-4II), 4.09 (d, 1H, J 11.4 Hz, CH2Ph-a), 4.13-4.20 (m, 2H, H-3II, H-5III), 4.26 (d, 1H, J 9.8 Hz, H-6bII), 4.29 (d, 1H, J 11.7 Hz, CH2Ph-b), 4.37 (d, 1H, J 11.0 Hz, H-6bI), 4.39 (s, 1H, H-4I), 4.52 (d, 1H, J 9.8 Hz, H-1I), 5.11 (s, 1H, H-1II), 5.27 (d, 1H, J 7.3 Hz, H-1IV), 5.31 (at, 1H, J 9.9 Hz, H-2I), 5.37 (d, 1H, J 3.8 Hz, H-1III), 5.38 (s, 1H, PhCH), 5.43-5.51 (m, 2H, H-4III, H-2IV), 5.54 (s, 1H, PhCH), 5.68 (at, 1H, J 9.6 Hz, H-4IV), 5.80 (at, 1H, J 9.3 Hz, H-3IV), 6.80-8.00 (m, 30 H, 30 x Ar-H); 13C NMR (125 MHz, CDCl3) δ 20.2, 20.5, 21.0 (3 CH3), 23.5, 24.8 (2 CH3), 34.5 (SCH), 52.9 (OCH3), 62.7 (C-6III), 63.5 (C-5II), 67.5 (C-5III, C-2I), 68.6 (C-6II), 69.3 (C-6I), 69.6 (C-4II), 69.8 (C-5I), 70.0 (C-4IV), 70.8 (C-2II), 71.1 (CH2Ph), 71.3 (C-4I), 71.8 (C-2IV), 71.9 (C-3IV), 72.1 (C-4II), 72.4 (C-3III, C-5IV), 73.8 (C-3I), 75.5 (C-2III), 78.6 (C-3II), 83.1 (C-1I), 95.7 (C-1II), 96.8 (C-1III), 100.4 (C-1IV), 101.3, 102.4 (2 PhCH), 125.3, 126.1, 126.4, 126.9, 128.1, 128.2, 128.3, 128.4, 129.0, 129.6, 129.7, 129.8, 134.4, 136.4, 137.0, 137.3, 137.4, 137.8 (36 x Ar-C), 164.7, 165.0, 165.6, 167.3, 169.1, 170.0, 171.3 (6 CO); HRMS m/z (ES+) [calculated for C76H80NaO27S+ 1479.4500; found 1479.4641. Compound 37: Rf 0.70 (petroleum: EtOAc = 1: 2); [α] D

20 = +49.9 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 3504 (νO-H), 3065 (νC-Harom), 2977, 2960, 2925 (νC-Haliph), 1733 cm-1 (νC=O OAc); 1H NMR (500 MHz, CDCl3): 1.27 (d, 3H, J 6.6 Hz, CH3), 1.39 (d, 3H, J 6.6 Hz, CH3), 2.07, 2.16, 2.18 (3s, 9H, 3CH3), 3.15 (m, 1H, SCH), 3.47 (s, 1H, H-5I), 3.67 (dd, 1H, J 3.8, 9.8 Hz, H-2III), 3.73-3.90 (m, 5H, H-3II, H-2II, H-5II, H-6aII, H-3I), 3.97 (dd, 1H, J 3.0, 8.5 Hz, H-3II), 4.00 (dd, 2H, J 1.2, 6.2 Hz, H-6III), 4.05 (d, 1H, J 12.3 Hz, H-6aI), 4.16 (at, 1H, J 6.0 Hz, H-5III), 4.25 (m, 2H, H-3III, H-6bII), 4.28 (d, 1H, J 11.6 Hz, CH2Ph-a), 4.37 (m, 3H, H-6bI, H-5IV, CH2Ph-b), 4.45 (d, 1H, J 3.3 Hz, H-4I), 4.51 (d, 1H, J 9.8 Hz, H-1I), 4.72 (d, 1H, J 3.8 Hz, H-1III), 5.12 (s, 1H, H-1II), 5.33 (d, 1H, J 7.4 Hz, H-1IV), 5.40 (t, 1H, J 9.8 Hz, H-2I), 5.52 (s, 1H, PhCH), 5.55 (s, 1H, PhCH), 5.58 (m, 2H, H-4III, H-2IV), 5.72 (t, 1H, J 9.6 Hz, H-4IV), 5.92 (t, 1H, J 9.5 Hz, H-3IV), 7.0-8.0 (m, 30H, 30 x Ar-H); 13C NMR (125 MHz, CDCl3) δ 20.8, 21.1, 21.2 (3 CH3), 23.7, 25.0 (2 CH3), 34.7 (SCH), 53.1 (OCH3), 61.9




39

(C-6III), 63.8 (C-5II), 67.4 (C-5III), 67.8 (C-2I), 68.5 (C-3II), 68.6 (C-6II), 69.5 (C-6I), 69.6 (C-4III), 69.8 (C-5I), 70.2 (C-4IV), 71.2 (C-4I), 72.1 (C-2IV), 72.2 (H-3IV), 73.0 (C-5IV), 73.8 (CH2Ph), 74.1 (C-3I, C-3III), 76.3 (C-2III), 79.6 (C-3II), 81.1 (C-2II), 83.2 (C-1I), 95.0 (C-1II), 100.7 (C-1IV), 100.8 (C-1III), 101.2, 102.4 (2 PhCH), 126.4, 128.32, 128.36, 128.43, 128.5, 128.6, 128.7, 128.8, 128.9, 129,16, 129.19, 129.3, 129.7, 129.9, 133.5, 133.6, 137.1, 137.3, 137.7 (36 x Ar-C), 164.5, 165.2, 165.8, 167.4, 169.5, 170.0, 170.7 (7 CO); HRMS (M+Na)+ calcd. 1479.4500; found 1479.4009. Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3)-2-O-benzyl-4,6-acetyl-1-thio-α-D-galactopyransyl trichloroacetimidate (4)

Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3) 2-O-benzyl-4,6-acetyl-1-thio-β-D-galactopyransyl trichloroacetimidate (7)

Compound 24 (0.12 g, 0.014 mmol, 1.0 eq.) was dissolved in CH2Cl2 (4 mL) at 0 ˚C. CCl3CN (2 mL, 20 mmol, 1428 eq.) and DBU (10 µL, 0.06 mmol, 4.3 eq.) were added. The solution was stirred at 0 °C for 45 min and was then allowed to warm to RT, followed by stirring for a further 1.5 h. The solution was concentrated and the residue was subjected to silica chromatography (Petroleum: EtOAc = 2 :1) to afford compound 4 as a colorless glass (72 mg, 54%) and compound 7 as a foam (41 mg, 31%). Compound 4: Rf 0.80 (petroleum: EtOAc = 1: 2); [α] D

20 = +51.6 (c 0.55, CDCl3); νmax (transmission, thin film)/cm-13330 (νN-H), 3063, 3028 (νC-Harom), 2956 (νC-Haliph), 1738 cm-1 (νC=O OAc), 1602 (νC-H), 1259 (νC-N); 1H NMR (400 MHz, CDCl3) δ 2.03, 2.17 (2s, 3H each, 2 OAc), 3.73 (s, 3H, OCH3), 3.91 (dd, 1H, J 3.8, 9.8 Hz, H-2I), 3.98 (dd, 1H, J 7.3, 11.6 Hz, H-6aI), 4.17 (dd, 1H, J 5.3, 11.6 Hz, H-6b), 4.30 (d, 1H, J 8.8 Hz, H-5II), 4.32-4.44 (m, 4H, H-3I, H-5I, PhCH2), 5.31 (d, 1H, J 7.3 Hz, H-1II), 5.55 (dd, 1H, J 7.3, 9.3 Hz, H-2II), 5.71 (at, 1H, J 9.6 Hz, H-3II), 5.83 (at, 1H, J 9.5 Hz, H-4I), 6.37 (d, 1H, J 3.5 Hz, H-1I), 7.09-8.00 (m, 20 H, 20 x Ar-H), 8.59 (NH); 13C NMR (CDCl3) δ 20.6, 20.7 (2 OAc), 52.9 (OCH3), 62.2 (C-6I), 69.1 (C-4I), 69.6 (C-3I), 70.1 (C-4II), 71.8 (C-2II), 72.1 (C-3II), 72.8 (C-5I), 73.1 (CH2Ph), 73.5 (C-5II), 75.7 (C-2I), 91.1 (CCl3), 94.1 (C-1I), 100.7 (C-1II), 127.4, 127.8, 128.4, 128.4, 128.5, 129.7, 129.8, 133.3, 133.4, 133.4 (24 x Ar-C), 160.9 (CNH), 164.8, 165.0, 165.6, 167.2, 170.0, 170.5 (6 CO); HRMS m/z (ES+) [calculated for C49H51Cl3N3O17

+ 1058.2279; found 1058.2286]. Compound 7: Rf 0.30 (petroleum: EtOAc = 3: 2); [α] D

20 = + 39.8 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 3331 (νN-H), 3063, 3028 (νC-Harom), 2960 (νC-Haliph), 1738 cm-1 (νC=O), 1601 (νC-H), 1255 (νC-N); 1H NMR (500 MHz, CDCl3) δ 2.08, 2.20 (2s, 2 × 3H, 2 × OAc), 3.71 (m, 4H, OMe and H-5II), 3.86 (at, 1H, J 8.8 Hz, H-2II), 4.01 (at, 1H, J 6.4 Hz, H-6aI), 4.08-4.22 (m, 3H, H-6bI, H-3II, H-5I), 4.43 (d, 1H, J 10.6 Hz, PhCH2-a), 4.76 (d, 1H, J 10.6 Hz, PhCH2-b), 5.31 (d, 1H, J 7.6 Hz, H-1I), 5.50-5.58 (m, 2H, H-2I, H-4II), 5.71 (t, 1H, J 9.6 Hz, H-4I), 5.78 (d, 1H, J 8.3 Hz, H-




40

1II), 5.83 (at, 1H, J 9.4 Hz, H-3I), 7.0-8.0 (m, 20 H, 20 x Ar-H), 8.72 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 20.7, 20.8 (2OAc), 52.9 (COOMe), 61.6 (C-6), 68.7 (C-4II), 70.0 (C-4I), 71.8, 72.0, 72.1 (C-2I, C-3I, C-5I), 72.8 (C-5I), 75.0 (PhCH2), 76.1 (C-3II), 78.3 (C-2II), 98.0 (C-1I), 100.4 (C-1II), 125.3 (CCl3), 127.5, 127.9, 128.2, 128.3, 128.4, 128.5, 128.7, 128.8, 129.0, 129.6, 129.8, 133.3, 133.4, 133.4, 137.5 (24 x Ar-H), 161 (CNH), 164.7, 165.0, 165.6, 167.0, 170.0, 170.6 (6 C=O); HRMS m/z (ES+) [calculated for C47H44Cl3NNaO17

+ 1022.1567, found 1022.1567]. 4,6-O-benzylidene-α-D-mannopyranosyl-(1→3)-isopropyl-2-O-acetyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (5)

Compound 15 (0.19 g, 0.36 mmol, 1.0 eq.) and PhCH(OMe)2 (0.065 g, 0.43 mmol, 1.2 eq.) were dissolved in DMF (10 ml) in a water-ice bath. CSA (0.017 g, 0.072 mmol, 0.02 eq.) was added. The mixture was allowed to warm to RT and kept at RT for 48 h. The reaction was quenched by the addition of Et3N to adjust the pH to ~7. The solution was then diluted with EtOAc (100 mL) and washed with brine. The upper layer was separated and dried over dry MgSO4 and filtered. The solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (Petroleum : EtOAc = 1: 4) to afford compound 5 as a white crystalline solid (0.11 g, 50 %): Rf 0.20 (petroleum: EtOAc = 1: 2); [α] D

20 = +15.7 (c 1.05, acetone); m.p. 232-234˚C; νmax (transmission, thin film)/cm-1 3418 (νO-H), 3064, 3037 (νC-Harom), 2962, 2924, 2868 (νC-Haliph), 1739 cm-1 (νC=O); 1H NMR (400 MHz, acetone) δ 1.25 (d, 1H, J 0.8, 6.8 Hz, CH3), 1.35 (d, 1H, J 6.6 Hz, CH3), 2.11 (s, 3H, CH3), 3.27 (m, 1H, SCH), 3.76 (s, 1H, H-5I), 3.79-3.90 (m, 3H, H-3II, H-6aII, H-6bII), 3.87 (d, 1H, J 3.5 Hz, H-4II), 3.95 (t, 1H, J 9.1 Hz, H-2II), 4.14 (m, 1H, H-3I), 4.16-4.27 (m, 3H, H-6aI, H-6bI, H-5II), 4.68 (d, 1H, J 3.3 Hz, H-4I), 4.77 (d, 1H, J 10.6 Hz, H-1I), 5.13 (s, 1H, H-1II), 5.30 (t, 1H, J 9.8 Hz, H-2I), 5.62, 5.72 (2s, 2H, 2 CHPh), 7.31-7.61 (m, 10 H, 10 x Ar-H); 13C NMR (100 MHz, acetone) δ 20.8 (OAc), 23.6, 24.5 (2 CH3), 34.7 (SCH), 64.4 (C-3II), 68.6 (C-2I), 68.9, 69.5 (C-6I, C-6II), 70.1 (C-5I), 71.7 (C-4II), 71.9 (C-4I), 73.9 (C-3I), 79.4 (C-2II), 83.1 (C-1I), 96.7 (C-1II), 100.9, 102.3 (2 CHPh), 126.7, 126.8, 128.3, 128.4, 129.0, 129.1,138.8,139.2 (12 x Ar-C), 169.4 (CO); HRMS m/z (ES+) [calculated for C31H38NaO11S+ 641.2027; found 641.2044]. Isopropyl-2-O-(2,4,6-O-acetyl-β-D-galactopyransyl)-3-O-[2-O-benzyl -3-O-(methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl) -4,6-O-acetyl -α-D-galacto-pyransyl]- 4,6-O-acetyl-1-thio-β-D-mannopyranoside (6)

Compound 40 (500 mg, 0.33 mmol, 1.0 eq.) was dissolved in EtOH (15 mL) and EtOAc (15 mL) in a 100 mL flask. Pd/C (1.52 g, 1.4 mmol, 4.2 eq.) was added under an atmosphere of argon. A bollon of H2 was used to exchange argon in the flask. The




41

reaction was kept for 2 days with stirring, The Pd/C was filtered over Celite and the solvent was removed on a rotary evaporator to afford compound 6 as a white crystalline solid (304 mg, 64 %): Rf 0.33 (petroleum: EtOAc = 1: 2); [α] D

20 = + 21.4 (c 0.5, CHCl3); mp. 121-123 ˚C; νmax (transmission, thin film)/cm-1 3525 (νO-H), 2961 (νC-Haliph), 1739 cm-1 (νC=O OAc); 1H NMR (400 MHz, CDCl3) δ 1.30 (dd, 6H, J 6.8, 13.1 Hz, SCH(CH3)2), 2.00 (s, 3H, OCH3), 2.06 (bs, 9H, 3 × OCH3), 2.07 (s, 3H, OCH3), 2.20 (s, 3H, OCH3), 2.46 (s, 3H, OCH3), 3.01 (d, 1H, J 9.1 Hz, 3-OHI), 3.14 (m, 1H, SCH), 3.53 (s, 3H, COOMe), 3.58 (m, 1H, H-5II), 3.66 (d, 1H, J 11.1 Hz, H-2III), 3.72 (dd, 1H, J 3.0, 9.8 Hz, H-3II), 3.81 (bs, 1H, H-3I), 3.95 (at, 1H, J 6.1 Hz, H-5I), 3.99 (dd, 1H, J 7.1, 11.4 Hz, H-6aIII), 4.06-4.25 (m, 5H, H-6bIII, H-6a&bI, H-6a&bII), 4.36 (m, 2H, H-5IV, H-3III), 4.45 (at, 1H, J 6.7 Hz, H-5III), 4.47 (d, 1H, J 2.8 Hz, H-2II), 4.68 (s, 1H, H-1II), 4.88 (d, 1H, J 3.8 Hz, H-1III), 4.91 (d, 1H, J 7.6 Hz, H-1I), 4.96 (at, 1H, J 8.8 Hz, H-2I), 5.19 (t, 1H, J 9.8 Hz, H-4II), 5.40-5.56 (m, 4H, H-4I, H-2IV, H-4III, H-4IV), 5.58 (d, 1H, J 7.8 Hz, H-1IV), 5.94 (t, 1H, J 9.5 Hz, H-3IV), 7.23-8.09 (m, 15H, 15 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 20.3, 20.7, 20.7, 20.8, 21.0 (7 OCH3), 23.3, 23.6 (2 CH3 of SCH(CH3)2), 36.2 (SCH), 52.9 (COOMe), 61.8 (C-6II), 62.4 (C-6III), 63.0 (C-6I), 67.5 (C-5III), 67.8 (C-4II), 69.3 (C-2III), 69.6 (C-4III), 70.0 (C-4I), 70.3 (C-3I), 70.4 (C-4IV), 72.0 (C-2IV), 72.1 (C-3IV + C-3III), 72.8 (C-5IV), 73.0 (C-5I), 73.1 (C-2II), 73.3 (C-2I), 76.3 (C-5II), 77.8 (C-3II), 83.0 (C-1II), 98.0 (C-1I), 100.1 (C-1IV), 100.7 (C-1III), 128.3, 128.3, 128.4, 128.8, 128.9, 129.5, 129.7, 129.8, 129.9, 133.1, 133.2, 133.4 (18 x Ar-C), 165.2, 165.5, 168.6, 170.1, 170.2, 170.4, 170.5, 170.6, 170.7, 171.2, 171.4 (11 CO); HRMS m/z (ES+) [calculated for C63H74NaO31S+ 1381.3838; found 1381.3803]. 2,4,6-O-acetyl-3-benzy-β-D-galactopyransyl-(1→2)-6-O-tert-butyldimethylsilyl- Isopropyl-1-thio-β-D-mannopyranoside (8)

Compound 36 (2.22 g, 3.6 mmol, 1.0 eq.) was dissolved in dry dichloromethane (80 mL). 2,6-lutidine (0.64 mL, 5.5 mmol, 1.5 eq.) and TBSOTf (0.82 mL, 3.6 mmol, 1.0 eq.) were added. The reaction was kept for overnight with stirring. The solvent was removed on a rotary evaporator and the residual was subjected to silica chromatography (petrol: ethyl acetate 4 : 5) to afford compound 8 as a white solid (2.7 g, 100%): Rf 0.17 (petroleum: EtOAc = 1: 1); [α] D

20 = + 10.8 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 3460 (νO-H), 3063, 3028 (νC-Harom), 2960 (νC-Haliph), 1738 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3) δ 0.08 (d, 6 H, J 8.1 Hz, CH3SiCH3), 0.88 (s, 9H, SiC(CH3)3), 1.27 (at, 6H, J 6.7 Hz, SCHMe2), 2.09, 2.10, 2.16 (3s, 9H, 3OAc), 3.09 (m, 1H, SCH), 3.28 (m, 1H, H-5II), 3.45 (dd, 1H, J 2.5, 9.3 Hz, H-3II), 3.56 (dd, 1H, J 3.3, 10.1 Hz, H-3I), 3.62 (t, 1H, J 9.3 Hz, H-4II), 3.77-3.91 (m, 3H, H-5I, H-6Ia,b), 3.93 (d, 1H, J 2.27 Hz, H-2II), 4.06-4.23 (m, 2H, H-6IIa,b), 4.39 (d, 1H, J 12.4 Hz, PhCH2-a), 4.47 (d, 1H, J 8.1 Hz, H-1I), 4.60 (s, 1H, H-1II), 4.68 (d, 1H, J 12.1 Hz, PhCH2-b), 5.15 (dd, 1H, J 8.1, 9.8 Hz, H-2I), 5.48 (d, 1H, J 3.0 Hz, H-4I), 7.2-7.4 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ -5.5, -5.4 (CH3SiCH3),




42

18.2 (SiC(CH3)3), 20.7, 20.8, 21.5 (3 × OAc), 23.7, 24.0 (SCH(CH3)2), 25.8 (C(CH3)3), 35.8 (SCH), 61.9 (C-6II), 64.6 (H-6I), 65.7 (C-4I), 70.1 (C-2I), 70.5 (C-4II), 71.4 (PhCH2, C-5I), 74.4 (C-3II), 76.5 (C-3I), 79.5 (C-5II), 82.9 (C-1II), 83.8 (C-2II), 102.5 (C-1I), 127.8, 128.4, 137.3 (6 x Ar-H), 169.3, 170.3, 170.7 (3C=O); HRMS m/z (ES+) [calculated for C34H54NaO13SSi+ 753.2947; found 753.2947. Isopropyl-2-O-benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (10)

Isopropyl-3-O-benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (21)

20 (5.0 g, 15.3 mmol, 1.0 eq.), benzyl bromide (1.76 mL, 17 mmol, 1.0 eq.) and n-Bu4NHSO4 (4.6 g, 17 mmol, 1.0 eq.) were dissolved in a suspension of CH2Cl2 (460 mL) and an aqueous NaOH (38 mL, 30%). The suspenstion was heated under reflux overnight and then cooled to RT. The organic layer was separated, washed with water, saturated aqueous NaHCO3 solution and brine. The solvent was removed on a rotary evaporator. After carrying out 4 equivalent spots of reaction, the residues were collected and subjected to silica chromatography (petroleum: EtOAc: CH2Cl2 = 5: 1: 5) to afford 21 (9.1 g, 35%) as a syrup and 10 (6.0 g, 23%) as a crystalline colorless solid. Compound 10: Rf 0.62 (petroleum: EtOAc = 1: 2) and 0.20 (petrol: EtOAc: CH2Cl2 = 5: 1: 5); [α] D

20 = -31.2 (c 1.0, CHCl3); m.p. 41-42˚C; νmax (transmission, thin film)/cm-

1 3444 (νO-H), 3060, 3033 (νC-Harom), 2961 (νC-Haliph); 1H NMR (400 MHz, CDCl3) δ 1.37 (d, 3H, J 6.8 Hz, CH3), 1.40 (d, 3H, J 6.8 Hz, CH3), 2.61 (d, 1H, J 8.1 Hz, OH), 3.31 (m, 1H, SCH), 3.43 (d, 1H, J 1.01 Hz, H-5), 3.64 (t, 1H, J 9.35 Hz, H-2), 3.77 (m, 1H, H-3), 4.02 (dd, 1H, J 1.8, 12.4 Hz, H-6a), 4.23 (dd, 1H, J 1.0, 3.8 Hz, H-4), 4.33 (dd, 1H, J 1.5, 12.4 Hz, H-6b), 4.52 (d, 1H, J 9.6 Hz, H-1), 4.78 (d, 1H, J 10.6 Hz, CH2Ph-a), 4.95 (d, 1H, J 10.6 Hz, CH2Ph-b), 5.55 (s, 1H, CHPh), 7.25-7.60 (m, 10H, 10 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 23.8, 24.2 (2CH3), 34.9 (SCH), 69.3 (C-6), 69.8 (C-5), 74.3 (C-3), 75.6 (CH2), 75.9 (C-4), 78.9 (C-2), 84.1 (C-1), 126.5, 127.8, 128.3, 128.4, 129.3, 137.6, 138.3 (12 x Ar-C); HRMS m/z (ES+) [calculated for C23H28NaO5S+ 439. 1550; found 439.1549]. Compound 21: Rf 0.67 (petroleum: EtOAc = 1: 2) and 0.34 (petrol: EtOAc: CH2Cl2 = 5: 1: 5); [α] D

20 = -8.0 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 3371 (νO-H), 2960 (νC-Haliph); 1H NMR (400 MHz, CDCl3) δ 1.34 (d, 3H, J 6.8 Hz, SCH(CH3)2), 1.39 (d, 3H, J 6.8 Hz, SCH(CH3)2), 2.59 (d, 1H, J 1.5 Hz, OH-2), 3.31 (m, 1H, SCH), 3.39 (s, 1H, H-5), 3.51 (dd, 1H, J 3.3, 9.1 Hz, H-3), 3.98 (dd, 1H, J 1.5, 12.4 Hz, H-6a), 4.05 (at, 1H, J 9.4 Hz, H-2), 4.19 (d, 1H, J 3.3 Hz, H-4), 4.31 (d, 1H, H-6b), 4.42 (d, 1H, J 9.6 Hz, H-1), 4.79 (s, 2H, PhCH2-a&b), 5.46 (s, PhCH), 7.25 (m, 5 H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 24.0, 24.5 (SCH(CH3)2), 34.5 (SCH), 68.4 (C-2), 69.4 (C-6), 70.1 (C-5), 71.5 (PhCH2), 73.6 (C-4), 80.3 (C-3), 85.5 (C-1),




43

101.3 (PhCH), 126.4, 127.9, 128.1, 128.5, 129.0 (6 x Ar-C); HRMS m/z (ES+) [calculated for C23H28NaO5S+ 439. 1550; found 439.1551]. 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (Intermediate 1)16

β-D-glucopyranose pentaacetate (63.3 g, 354 mmol, 1.0 eq.) was dissolved in a solution of HBr in AcOH (250 mL, 33 %) at 0 ˚C, followed by warming up to RT. The reaction was kept at RT for overnight at 0 ˚C. The solution was then washed with saturated NaHCO3 and 1M HCl aqueous solution, followed by drying with MgSO4 and filtering. The solvent was removed and the residue was subjected to silica gel chromatography to afford Intermediate 1 as a white crystalline solid (52 g, 79 %): Rf 0.25 (petroleum: EtOAc = 3: 1); [α] D

20 = +203 (c 1.0, CHCl3); m.p. 81-83˚C; 1H NMR (400 MHz, CDCl3) δ 2.03, 2.05, 2.09, 2.10 (4s, 12H, 4 × COCH3), 4.13 (dd, 1H, J 2.0, 12.6 Hz, H-6a), 4.25-4.36 (m, 2H, H-5, H-6b), 4.83 (dd, 1H, J 4.0, 10.1 Hz, H-2), 5.16 (at, 1H, J 9.9 Hz, H-4), 5.55 (at, 1H, J 9.7 Hz, H-3), 6.61 (d, 1H, J 4.0 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ 20.4-20.8 (4 CH3), 60.9 (C-6), 67.2 (C-5), 70.2 (C-4), 70.6 (C-2), 72.1 (C-3), 86.6 (C-1), 169.4, 169.8, 169.8,170.5 (4 CO); LRMS m/z (ES+) 412 ([M + H]+, 100%). 2,3,4,6-tetra-O-acetyl-benzyl-β-D-glucopyranoside (Intermediate 2)16

Intermediate 1 (52 g, 126 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (320 mL) with activated 4Å MS. The suspension was kept under stirring for 20 min under the atmosphere of argon. Benzyl alcohol (42 mL, 399 mmol, 3.2 eq.) and silver carbonate (62.4g, 227 mmol, 1.8 eq.) were added. After 24 h at RT, the MS was filtered over a pad of Celite. The solvent was removed on a rotary evaporator. The residue was subjected to silica gel chromatography (petroleum: ethyl acetate= 4:1) to afford Intermediate 2 as a white crystalline solid (33 g, 60 %): Rf 0.38 (petroleum: EtOAc = 2: 1); [α] D

20 = -41.8 (c 1.0, CHCl3); m.p. 67-69˚C; 1H NMR (400 MHz, CDCl3) δ 2.00, 2.01, 2.02, 2.11 (4s, 12H, 4 × COCH3), 3.68 (m, 1H, H-5), 4.17 (dd, 1H, J 2.5, 12.4 Hz, H-6a), 4.28 (dd,1H, J 4.5, 12.1 Hz, H-6b), 4.55 (d, 1H, J 7.8 Hz, H-1), 4.63 (d, 1H, J 12.4 Hz, CH2Ph-a), 4.90 (d, 1H, J 12.1 Hz, CH2Ph-b), 5.04-5.21 (m, 3H, H-2, H-4, H-3), 7.26-7.38 (m, 5H, 5 x Ar-H). 13C NMR (100 MHz, CDCl3) δ 20.6, 20.7, 20.8 (CH3), 61.9 (C-6), 68.4 (C-4), 70.8 (CH2Ph), 71.3 (C-2), 71.8 (C-5), 72.8 (C-3), 99.3 (C-1), 127.8, 128.1, 128.5, 136.7 (6 x Ar-C), 169.3, 169.4, 170.3, 170.7 (4 CO); LRMS m/z (ES+) 461 ([M + Na]+, 100%). Benzyl β-D-glucopyranoside (Intermediate 3)16

Intermediate 2 (32 g, 72 mmol, 1.0 eq.) was dissolved in anhydrous MeOH (500 mL) under the asmosphere of argon at RT. Sodium methoxide (250 mg, 3.7 mmol, 0.05 eq.) was added. The reaction was kept under stirring for 20h. The reaction mixture was then quenched by the addition of IR 120 (H+) to adjust the pH to ~7. The resin




44

was filtered off and the solvent was removed on a rotatory evaporator. The residue was subjected to silica gel chromatography (ethyl acetate: MeOH = 50: 6) to afford Intermediate 3 as a white crystalline solid (19.0 g, 96%): Rf 0.11 (EtOAc); [α] D

20 = -49.4 (c 1.0, CHCl3); m.p. 108-110˚C (MeOH); 1H NMR (400 MHz, CDCl3) δ 3.24-3.41 (m, 4H, H-2, H-3, H-4, H-5), 3.71 (dd, 1H, J 5.8, 12.1 Hz, H-6a), 3.91 (dd, 1H, J 2.0, 11.9 Hz, H-6b), 4.37 (d, 1H, J 7.8 Hz, H-1), 4.68 (d, 1H, J 11.9 Hz, CH2Ph-a), 4.95 (d, 1H, J 11.9 Hz, CH2Ph-b). 7.24-7.46 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 61.8 (C-6), 70.7, 74.1, 77.0, 77.1 (C-2, C-3, C-4, C-5), 102.3 (C-1), 127.7, 128.2, 128.3, 138.1 (6 x Ar-C); LRMS m/z (ES+) 329 ([M + CH3CN+NH4]+, 100%). Benzyl-β-D-glucopyranosiduronic acid (Intermediate 4)16

Intermediate 3 (19.0 g, 70 mmol, 1.0 eq.) was dissolved in THF (285 mL). The solution was cooled to 0 ˚C. Saturated aqueous NaHCO3 (285 mL), KBr (0.8 g, 6.7 mmol), TEMPO (400 mg, 2.6 mmol, 0.04 eq.) and NaOCl (380 mL, 10 %, 616 mmol, 8.8 eq.) were added. After 30 minutes at 0 °C, a further portion of NaOCl (380 mL, 10 %, 616 mmol, 8.8 eq.) was added. The reaction was kept for 30 more minutes at 0 °C with stirring and then another 23 h at RT. The reaction mixture was acidified to reach pH 4-5 by the addition of 1N HCl. The solvent were removed on a rotary evaporator. The residue was dissolved in MeOH (200 mL) and the insoluble salt was filtered off. The solvent was removed on a rotary evaporator. After repeating this three times, most of the salt was removed. The residue was subjected to silica chromatography (EtOAc: MeOH = 50:11) to afford Intermediate 4 as a yellow solid (19.4 g, 97%): Rf 0.31 (water: 2-propanol: EtOAc = 1: 3: 3); [α] D

20 = -61.9 (c 1.0, MeOH) (Lit: -74 (c=0.5, water))16; 1H NMR (400 MHz, MeOD) δ 3.30-3.35 (m, 1H, H-2), 3.43 (t, 1H, J 8.8 Hz, H-3), 3.49 (at, 1H, J 9.1 Hz, H-4), 3.62 (d, 1H, J 9.6 Hz, H-5), 4.40 (d, 1H, J 7.8 Hz, H-1), 4.68 (d, 1H, J 11.6 Hz, CH2Ph-a), 5.02 (d, 1H, J 11.6 Hz, CH2Ph-b), 7.24-7.46 (m, 5H, 5 x Ar-H). 13C NMR (100 MHz, MeOD) δ 71.0 (CH2Ph), 72.6 (C-4), 73.9 (C-2), 75.1 (C-5), 76.7 (C-3), 102.3 (C-1), 127.7, 128.3, 138.0 (6 x Ar-C), 175.6 (COOH); LRMS m/z (ES¯) 283 ([M-H]¯,100%). Methyl 2-O-benzyl-β-D-glucopyranoside (Intermediate 5)16

Intermediate 4 (19.0 g, 68 mmol, 1.0 eq.) was dissolved in anhydrous MeOH (1250 mL) under the atmosphere of argon. IR 120 (H+

, 2.0 g) was added. The reaction was kept overnight under stirring. The resin was then filtered and the solvent was removed on a rotary evaporator. The residue was dissolved in CHCl3. The solution was washed with water, dried over anhydrous MgSO4 and filtered. The solvent was removed on a rotary evaporator. Then the residue was subjected to silica chromatography (EtOAc) to afford Intermediate 5 as a white solid (11.5 g, 56%): Rf 0.22 (EtOAc); [α] D

20 = -69.1 (c 1.0, MeOH) (Lit: -84 (c=1.0, CHCl3))17; 1H NMR (400 MHz, MeOD) δ 3.33 (at, 1H, J 6.8 Hz, H-2), 3.39 (t, 1H, J 9.1 Hz, H-3), 3.58 (t, 1H, J 8.9 Hz, H-4), 3.80 (s, 3H, CH3), 3.86 (d, 1H, J 9.8 Hz, H-5), 4.44 (d, 1H, J 7.6 Hz, H-1), 4.63 (d, 1H, J 11.6 Hz, H-6a), 4.88 (d, 1H, J 4.9 Hz, H-6b), 7.25-7.45 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, MeOD) δ 51.9 (CH3), 71.2 (CH2), 72.2 (C-4), 73.8 (C-2), 75.8 (C-5), 76.3 (C-




45

3), 102.8 (C-1), 127.8, 128.2, 128.3 137.8 (6 x Ar-C), 170.3 (CO); LRMS m/z (ES+) 321 ([M + Na]+, 100%). Methyl 2,3,4-tri-O-benzoyl-α-D-glucopyranoside (Intermediate 6)16

Intermediate 5 (0.45 g, 1.5 mmol, 1.0 eq.) was dissolved in pyridine (2 mL). BzCl (0.7 mL, 6.0 mmol, 4.0 eq.) was added dropwisely and the solution was kept at RT overnight under stirring. The mixture was diluted with CH2Cl2 and washed with 1N HCl, water and saturated aqueous NaHCO3, The organic layer was dried over magnesium sulfate. The solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (petroleum: ethyl acetate = 4: 1) to give Intermediate 6 as a white crystalline solid (0.9 g, 98 %): Rf 0.88 (EtOAc); [α] D

20 = -25.6 (c 1.0, CHCl3); m.p. 152-154˚C; 1H NMR (400 MHz, CDCl3) δ 4.34 (d, 1H, J 10.1 Hz, H-1), 4.73 (d, 1H, J 12.6 Hz, H-6a), 4.87 (m, 1H, H-5), 5.00 (d, 1H, J 12.4 Hz, H-6b), 5.63 (at, 1H, J 8.3 Hz, H-4), 5.72 (t, 1H, J 9.3 Hz, H-2), 5.86 (t, 1H, J 9.2 Hz, H-3), 7.18-7.97 (m, 20 H, 20 x Ar-H); LRMS m/z (ES+) 633 ([M + Na]+, 100%). Methyl 2,3,4-tri-O-benzoyl-α-D-glucupyranose (Intermediate 7)16

Intermediate 6 (1.5 g, 2.5 mmol, 1.0 eq.) was dissolved in EtOH (15 mL) and EtOAc (15 mL) in a 250 mL flask. The mixture was thoroughly degassed and filled with an atmosphere of argon. Catalytic Pd-C (50 mg, 10 %, 0.05 mmol, 0.02 eq.) was added. H2 was applied to exchange the argon and filled the atmosphere of the flask. The reaction was kept overnight under stirring. The hydrogen was vented and the mixture was filtered. The solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (petroleum: ethyl acetate = 2: 1) to afford Intermediate 7 (1.23 g, 96 %) as an α/β mixture (α : β =5.6 : 1): Rf 0.53 (petroleum: EtOAc = 1: 1); LRMS m/z (ES+) 538 ([M + NH4]+, 100%); α: 1H NMR (400 MHz, CDCl3) δ 3.66 (s, 3H, OCH3), 4.89 (d, 1H, J 9.6 Hz, H-5), 5.35 (dd, 1H, J 3.5, 10.1 Hz, H-2), 5.68 (t, 1H, J 9.6, H-4), 5.88 (d, 1H, J 3.5Hz, H-1), 6.27 (t, 1H, J 9.60 Hz, H-3), 7.25-8.05 (15 H, 15 x Ar-H); 13C NMR (100 MHz, MeOD) δ 52.9 (OCH3), 68.6 (C-5), 69.4 (C-3), 70.0 (C-4), 71.6 (C-2), 90.5 (C-1), 128.4, 128.5, 129.8, 129.9, 130.0, 133.3, 133.4, 133.5 (18 x Ar-C), 165.4, 165.7, 165.8 (3CO), 168.6 (COOMe). β: 4.42 (d, 1H, J 9.6 Hz, H-5), 5.13 (d, 1H, J 9.6 Hz, H-1), 5.42 (dd, 1H, J 9.4 Hz, H-2), 5.72 (d,1H, J 9.4 Hz, H-4), 5.99 (t, 1H, J 9.4 Hz, H-3). Methyl 2,3,4-tri-O-benzoyl-α-D-glucuronosyl trichloroacetimidate (11)16

Intermediate 7 (0.85 g, 1.6 mmol, 1.0 eq.) was dissolved in CH2Cl2 (4 mL). DBU (47 µl, 0.31 mmol, 0.2 eq.) and CCl3CN (1.6 mL, 16 mmol, 10 eq.) were added. The reaction was kept for 30 min under stirring. The solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (petroleum: ethyl acetate = 3: 1) to afford compound 11 as a white solid (1.35 g, 96 %): Rf 0.71




46

(petroleum: EtOAc = 1: 1); [α] D20 = +49.5 (c 1.0, CHCl3) (Lit.: +59 (c 1, CHCl3))18;

m.p. 110-112˚C; 1H NMR (400 MHz, CDCl3) δ 3.70 (s, 3H, CH3), 4.78 (d, 1H, J 10.1 Hz, H-5), 5.65 (dd, 1H, J 3.5, 10.1 Hz, H-2), 5.77 (at, 1H, J 10.0 Hz, H-4), 6.30 (at, 1H, J 10.0 Hz, H-3), 6.92 (d, 1H, J 3.28 Hz, H-1), 7.25-8.10 (m, 15H, 15 x Ar-H), 8.70 (NH); 13C NMR (100 MHz, CDCl3) δ 53.1 (CH3), 69.3 (C-3), 69.6 (C-4), 70.2 (C-2), 70.9 (C-5), 90.5 (CCl3), 92.9 (C-1), 128.4, 128.5, 128.6, 128.7, 129.8, 129.9, 133.4, 133.6 (18 x Ar-C), 160.3 (CNH), 165.3, 165.5, 167.2 (4 CO); LRMS m/z (ES+) 726 ([M + MeCN+ NH4]+, 100%). Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3)-isopropyl-2-O-benzyl-4,6-acetyl-1-thio-β-D-galactopyranoside (12)

Compound 23 (0.36 g, 0.45 mmol, 1.0 eq.) and Ac2O (1 mL, 10.6 mmol, 24 eq.) were dissolved in pyridine (2 mL). The reaction was kept overnight under stirring. The solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (petroleum: ethyl acetate = 2: 1) to afford compound 12 as a colorless glass (0.37 g, 93 %): Rf 0.64 (petroleum: EtOAc = 1: 1); [α] D

20 = +36.8 (c 1.0, CDCl3); νmax (transmission, thin film)/cm-1 3442 (νO-H), 3064, 3033 (νC-Harom), 2960, 2926, 2887 (νC-Haliph), 1738 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3) δ 1.33 (at, 6H, J 6.2 Hz, 2 CH3), 2.07, 2.18 (2 × s, 6H, 2 × OAc), 3.20 (m, 1H, SCH), 3.56 (t, 1H, J 9.5 Hz, H-2I), 3.70 (s, 3H, 1 CH3), 3.82 (t, 1H, J 6.4 Hz, H-5I), 4.06 (dd, 1H, J 3.5, 9.3 Hz, H-3I), 4.08-4.19 (m, 3H, H-6aI, H-6bI, H-5II), 4.39 (d, 1H, J 10.1 Hz, CH2Ph-a), 4.51 (d, 1H, J 9.8 Hz, H-1I), 4.73 (d, 1H, J 10.1 Hz, CH2Ph-b), 5.27 (d, 1H, J 7.6 Hz, H-1II), 5.50-5.57 (m, 2H, H-4I, H-2II), 5.71 (at, 1H, J 9.6 Hz, H-4II), 5.83 (t, 1H, J 9.2 Hz, H-3II), 7.00-8.00 (m, 20H, 20 x Ar-H); 13C NMR (CDCl3) δ 20.7, 20.8, (2 OAc), 23.7, 23.9 (2 CH3), 35.9 (SCH), 52.9 (OCH3), 62.6 (C-6I), 69.4 (C-4I), 70.1 (C-4II), 71.9 (C-2II), 72.2 (C-3II), 72.8 (C-5II), 74.8 (C-5I), 75.3 (CH2Ph), 77.8 (C-3I), 78.5 (C-2I), 84.8 (C-1I), 100.4 (C-1II), 128.0, 128.2, 128.4, 125.4, 128.5, 128.7, 128.7, 129.0, 129.6, 129.8, 133.3, 133.4, 133.5, 137.7 (24 x Ar-C), 164.7, 165.0, 165.7, 167.0, 170.2, 170.6 (6 CO); HRMS m/z (ES+) [calculated for C50H57N2O16S+ 973.3423; found 973.3425]. Isopropyl-2-O-acetyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (13)

Compound 26 (6.0 g, 10 mmol, 1.0 eq.) was dissolved in CH2Cl2 (100 mL). Et3N (2.5 mL, 18 mmol, 1.8 eq.) was added at RT. The reaction was kept for 20h under stirring. The solvent was removed on a rotary evaporator. The residue was subjected to silica gel chromatography (petroleum: ethyl acetate = 5: 2) to give compound 13 as a white crystalline solid (3.0 g, 81%): Rf 0.22 (petroleum: EtOAc = 1: 1); [α] D

20 = -28.4 (c 1.0, CHCl3); m.p. 154-156˚C; νmax (transmission, thin film)/cm-1 3447 (νO-H), 3066, 3035 (νC-Harom), 2966, 2866 (νC-Haliph), 1735 cm-1 (νC=O OAc); 1H NMR (400 MHz, CDCl3) δ 1.28 (d, 3H, J 6.9 Hz, CH3), 1.39 (d, 3H, J 6.6 Hz, CH3), 2.13 (s, 3H, OAc), 2.54 (d, 1H, J 11.0 Hz, OH), 3.29 (m, 1H, SCH), 3.50 (d, 1H, J 1.3 Hz, H-5), 3.75 (m, 1H, H-




47

3), 4.04 (dd, 1H, J 1.9, 12.6 Hz, H-6a), 4.26 (dd, 1H, J 0.9, 3.8 Hz, H-4), 4.35 (dd, 1H, J 1.6, 12.3 Hz, H-6b), 4.49 (d, 1H, J 9.8 Hz, H-1), 5.17 (t, 1H, J 9.8 Hz, H-2), 5.54 (s, 1H, CHPh), 7.37-7.53 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 21.1 (OAc), 23.6, 23.8 (2CH3), 34.5 (SCH), 69.2 (C-6), 69.9 (C-5), 70.6 (C-2), 72.6 (C-3), 75.7 (C-4), 82.7 (C-1), 101.6 (CHPh), 126.5, 128.3, 129.4, 137.3 (6 x Ar-C), 170.4 (CO); HRMS m/z (ES+) [calculated for C18H24NaO6S+ 391.1186; found 391.1185]; Anal. Calcd for C18H24O6S: C, 58.68; H, 6.57; Found: C, 58.86; H, 6.60. D-Mannose pentaacetate (Intermediate 8)19

Mannose (10.7 g, 55mmol, 1.0 eq.) was dissolved in pyridine (150 mL). Acetic anhydride (100 mL, 885 mmol, 16 eq.) was added. The reaction was kept at RT for 12 h under stirring. The solvent was removed on a rotary evaporator. The residue was diluted with EtOAc (800 mL) and washed with aqueous copper (II) sulfate (4%, 800 mL), saturated aqueous NaHCO3 (800 mL), brine (800 mL), dried over MgSO4 and filtered. The solvent was removed on a rotary evaporator and the residue was dried in vacuo to afford Intermediate 8 as a syrup (21.0 g, 91 %, α/β 11:1) mixture: Rf 0.20 (petroleum: EtOAc = 2: 1); LRMS m/z (ES+) 413 ([M + Na]+, 100%),; α anomer: 1H NMR (400 MHz, CDCl3) δ 2.00, 2.06, 2.09, 2.16, 2.17 (5s, 15 H, 5 OAc), 4.04 (m, 1H, H-5), 4.08 (dd, 1H, J 2.5, 12.4 Hz, H-6a), 4.28 (dd, 1H, J 4.8, 12.4 Hz, H-6b), 5.25 (at, 1H, J 2.4 Hz, H-2), 5.34 (m, 2H, H-3, H-4), 6.08 (d, 1H, J 2.0 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ 20.5-20.9 (5 OAc), 62.1 (C-6), 65.5, 68.7 (C-3, C-4), 68.3 (C-2), 70.6 (C-5), 90.6 (C-1), 168.1, 169.6, 170.0 (CO). D-mannose-tetra-acetate (Intermediate 9)19

Intermediate 8 (54.0 g, 138 mmol, 1.0 eq.) was dissolved in DMF (120 mL) at room temperature. Ammonium carbonate (14.5 g, 146 mmol, 1.06 eq.) was added. The suspension was kept at RT for 1 day under turbulent stirring. The reaction mixture was diluted with EtOAc (800 mL) and washed with water (800 mL) and then brine (800 mL), dried by MgSO4 and filtered. The solvent was removed on a rotary evaporator to give the hemiacetal derivative D-mannose-tetra-acetate (Intermediate 9) as a syrup (41.0 g, 85 %, α/β 11:1): Rf 0.30 (petroleum: EtOAc = 1: 1); LRMS m/z (ES+) 366 ([M + NH4]+, 100%). α anomer: 1H NMR (400 MHz, CDCl3) δ 1.99, 2.04, 2.10, 2.15 (4s, 12 H, 4 OAc), 4.08-4.30 (m, 3H, H-6a, H-5, H-6b), 5.23 (d, 1H, J 1.5 Hz, H-1), 5.25-5.34 (m, 2H, H-2, H-4), 5.43 (dd, 1H, J 3.3, 10.1 Hz, H-3); 13C NMR (100 MHz, CDCl3) δ 20.5-21.0 (4 OCH3), 62.6 (C-6), 66.2 (C-4), 68.3 (C-5), 68.9 (C-3), 70.2 (C-2), 92.2 (C-1), 169.9, 170.0, 170.2, 170.9 (CO). 2,3,4,6,-tetra-O-acetyl-α-D-mannopyranosyl trichloroacetimidate (14) 19




48

Intermediate 9 (7.6 g, 20 mmol, 1.0 eq.) and trichloroacetonitrile (12 mL, 120 mmol, 6.0 eq.) were dissolved in CH2Cl2 (12 mL). DBU (0.4 mL, 2.7 mmol, 0.13 eq.) was added at 0 ˚C in an ice-water bath. The reaction was kept for 2 h under stirring and the temperature was allowed to warm up to RT. The solvent was removed on a rotary evaporator and the residue was subjected to silica gel column chromatography (petroleum: ethyl acetate, 1:1) to afford compound 14 (8.2 g, 73%): Rf 0.55 (petroleum: EtOAc = 1: 1); [α] D

20 = +49.5 (c 1.0, CHCl3) (Lit.: +53 (c=1.0, CHCl3))19; 1H NMR (400 MHz, CDCl3) δ 2.01, 2.07, 2.08, 2.20 (4s, 12 H, 4 OAc), 4.16 (dd, 1 H, J 2.5, 12.3 Hz, H-6a), 4.18-4.22 (m, 1H, H-5), 4.28 (dd, 1H, J 4.7, 12.0 Hz, H-6b), 5.37-5.43 (m, 2H, H-3, H-4), 5.47 (m, 1H, H-2), 6.28 (d, 1H, J 1.9 Hz, H-1), 8.79 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 20.6, 20.7, 20.8 (4 × CH3), 62.0 (C-6), 65.4 (C-3), 67.8 (C-2), 68.8 (C-4), 71.2 (C-5), 90.5 (CCl3), 94.5 (C-1), 160.0 (OCNH), 169.6, 169.7, 169.8, 170.6 (4 CO); LRMS m/z (ES+) 516 ([M + Na]+, 100%). 2,3,4,6,-tetra-O-acetyl-α-D-mannopyranosyl-(1→3)-Isopropyl-2-O-acetyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (15)

14 (2.8 g, 7.6 mmol, 2.0 eq.) and 13 (1.4 g, 3.8 mmol, 1.0 eq.) were dissolved in anhydrous CH2Cl2 (200 mL) with activated 4 Å MS. TMSOTf (0.3 mL) was added at -20 ˚C under an argon atmosphere. The mixture was stirred under these conditions for 30 min, then quenched with Et3N. The solvent was evaporated, and the residue was purified by silica gel column chromatography (Petroleum: Ethyl acetate 3: 2) to afford the protected dissachride (2.1 g, 81%), which (1.8 g, 2.6 mmol) was dissolved in anhydrous MeOH (25 ml) and reacted under argon and at room temperature with a catalytic amount of sodium methoxide (100 mg) for 9 h. The reaction mixture was neutralized by addition of resin IR120 (H+), filtered, and the solvent was evaporated. Purification of the crude residue by chromatography on silica gel (Ethyl acetate-methanol, v/v 8:1) afforded 15 as a white crystalline solid (1.0 g, 72 %): Rf 0.12 (EtOAc); [α] D

20 = +39.9 (c 1.0, CHCl3); m.p. 142-144˚C; νmax (transmission, thin film)/cm-1 3385 (νO-H), 3063, 3037 (νC-Harom), 2960, 2887 (νC-Haliph), 1734 cm-1 (νC=O OAc); 1H NMR (400 MHz, MeOD) δ 1.28 (d, 3H, J 7.1 Hz, CH3), 1.36 (d, 3H, J 6.6 Hz, CH3), 2.12 (s, 3H, CH3), 3.26 (m, 1H, SCH), 3.58-3.87 (m, 3H, H-3II, H-4 II, H-5

II), 3.61 (s, 1H, H-5I), 3.75 (d, 1H, J 11.4 Hz, H-6aII), 3.89 (d, 1H, J 11.1 Hz, H-6bII), 4.10 (dd, 1H, J 3.5, 9.9 Hz, H-3I), 4.15 (dd, 1H, J 1.5, 12.4 Hz, H-6aI), 4.24 (dd, 1H, J 1.5, 12.6 Hz, H-6bI), 4.57 (d, 1H, J 3.0 Hz, H-4), 4.70 (d, 1H, J 10.1Hz, H-1I), 5.05 (d, 1H, J 1.5 Hz, H-1II), 5.23 (t, 1H, J 9.8 Hz, H-2I), 5.63 (s, 1H, CHPh), 7.30-7.60 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, MeOD) δ 20.3 (OAc), 23.2, 23.9 (2 CH3), 35.0 (SCH), 62.1 (C-6II), 67.2, 70.1, 71.2, 74.2 (C-5I, C-3II, C-4II, C-5II), 68.7 (C-2I), 69.5 (C-6I), 71.0 (C-2II), 71.7 (C-4I), 73.4 (C-3I), 83.2 (C-1I), 95.8 (C-1II), 101.2 (CHPh), 126.5, 128.1,138.6 (6 x Ar-C), 170.8 (CO); HRMS m/z (ES+) [calculated for C24H34NaO11S+ 553.1714; found 553.1715].




49

3-O-benzyl-2, 4, 6-O-Tri-O-acetyl-α-D-galactopyranosyl trichloroacetimidate (16)

Compound 35 (9.0 g, 20 mmol, 1.0 eq.) was dissolved in acetone (60 mL) and water (3 mL). NIS (5.4 g, 24 mmol, 1.2 eq.) and TFA (0.15 mL, 2.0 mmol, 0.1 eq.) were added at 0 ºC. The reaction was kept for 1 hour under stirring and the temperature was allowed to warm to RT. The reaction was quenched by the addition of aqueous solution of Na2S2O3 (100 mL, 10%). Then EtOAc (500 mL) and CH2Cl2 were added under stirring. The upper organic layer was separated, dried over magnesium sulfate and filtered. The solvent was removed on a rotary evaporator and residue was subjected to silica chromatography (petrol: ethyl acetate = 5: 4) to afford the hydrolyzed intermediate as syrup (7.9 g, 100 %). The syrup (7.9 g, 20 mmol, 1.0 eq.) was then dissolved in CH2Cl2. DBU (0.3 mL, 2.0 mmol, 0.1 eq.) and CNCCl3 (100 ml, 1 mol, 50 eq.) were added at 0 ºC. After 2 h, the solvent was removed on a rotary evaporator and the residue was subjected to silica chromatography (petrol: ethyl acetate = 3: 1) to afford compound 16 as a syrup (8.83 g, 80%): Rf 0.66 (petroleum: EtOAc = 1: 1); [α] D

20 = + 108.6 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 2.01, 2.05, 2.17 (3s, 9 H, 3 OCH3), 4.04 (dd, 1H, J 3.5, 10.4 Hz, H-3), 4.07 (dd, 1H, J 6.8, 11.4 Hz, H-6a), 4.23 (dd, 1H, J 6.1, 11.4 Hz, H-6b), 4.36 (at, J 6.4 Hz, H-5), 4.50 (d, 1H, J 11.9 Hz, PhCH2-a), 4.75 (d, 1H, J 11.9 Hz, PhCH2-b), 5.28 (dd, 1H, J 3.5, 10.4 Hz, H-2), 5.67 (d, 1H, J 2.5 Hz, H-4), 6.58 (d, 1H, J 3.5 Hz, H-1), 7.14-7.38 (m, 5H, 5 x Ar-H), 8.60 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 20.6, 20.7, 20.8 (3 OCH3), 61.9 (C-6), 66.6 (C-4), 68.7 (C-2), 69.5 (C-5), 71.6 (PhCH2), 72.4 (C-3), 90.9 (CCl3), 93.9 (C-1), 127.9, 128.0, 128.2, 128.3, 129.0, 137.3 (6 x Ar-C), 160.8 (-CNH-), 170.1, 170.2, 170.4 (3C=O); HRMS m/z (ES+) [calculated for C21H24Cl3NNaO9

+ 562.0409; found 562.0405]. Isopropyl-tert-butyldiphenylsilyl-(2’S,3’S)-methyl-3,4-O-(2’,3’-dimethoxybu-tane-2’,3’-diyl)-1-thio-β-D-mannopyranoside (17)

Compound 33 (5.15 g, 14.6 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (40 mL). 2,6-lutidine (2.54 mL, 21.8 mmol, 1.5 eq.) and TBSOTf (3.52 mL, 15.3 mmol, 1.05 eq.) were added at RT. The reaction was kept overnight under stirring. The solvent was then removed on a rotary evaporator and the residue was subjected to silica chromatography (petrol: ethyl acetate = 6: 1) to afford compound 33 as a white crystalline solid (6.7 g, 99 %): Rf 0.66 (petroleum: EtOAc = 2: 1); [α] D

20 = + 74.5 (c 1.0, CHCl3); m.p. 85-87˚C; νmax (transmission, thin film)/cm-1 3449 (νO-H), 2955 (νC-

Haliph); 1H NMR (400 MHz, CDCl3) δ 0.06 (d, 6H, J 12.3 Hz, -Si(CH3)2), 0.87 (s, 9H, -C(CH3)3), 1.25-1.38 (m, 12H, 2CCH3 of BDA and SCH(CH3)2), 2.28 (d, 1H, J 3.3 Hz, OH), 3.19 (m, 1H, SCH), 3.23, 3.27 (2s, 6H, 2OCH3 of BDA), 3.42 (m, 1H, H-5), 3.75 (dd, 1H, J 3.0, 10.4 Hz, H-3), 3.78 (dd, 1H, J 5.6, 11.4Hz, H-6a), 3.88 (dd, 1H, J 1.8, 11.4 Hz, H-6b), 3.95-4.05 (m, 2H, H-2, H-4), 4.72 (s, 1H, H-1); 13C NMR (100 MHz, CDCl3) δ -5.46, -5.13 (-Si(CH3)2), 17.6, 17.7 (2CCH3 of BDA), 18.2 (-Si(CH3)2C(CH3)3), 23.6, 23.9 (-S(CH3)2), 25.8 (-Si(CH3)2C(CH3)3), 35.0 (SCH), 47.8,




50

48.0 (2 –CCH3(OCH3)2), 61.8 (C-6), 62.8 (C-4), 70.9 (C-2), 71.7 (C-3), 79.6 (C-5), 83.3 (C-1), 99.7, 100.3 (-CCH3(OCH3)2); HRMS m/z (ES+) [calculated for C21H42NaO7SSi+ 489.2313; found 489.2313]. Isopropyl-2-O-(2,4,6-O-acetyl-3-O-benzyl-β-D-galactopyransyl)-3-O-[2-O-benzyl -3-O-(methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl)-4,6-O-acetyl-α-D-galacto-pyransyl]-6-O-tert-butyldimethylsilyl -1-thio-β-D-mannopyranoside (19)

Isopropyl-2-O-(2,4,6-O-acetyl-3-O-benzyl-β-D-galactopyransyl)-3-O-[2-O-benzyl -4-O-(methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl)-4,6-O-acetyl-α-D-galacto-pyransyl]-6-O-tert-butyldimethylsilyl -1-thio-β-D-mannopyranoside (38)

Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3) 2-O-benzyl-4,6-acetyl-1-N-trichloroacetyl-α-D-galactopyransylamine (39)

Compound 7 (43 mg, 43 µmol, 1.0 eq.) and compound 8 (30 mg, 41 µmol, 0.95 eq.) were dissolved in anhydrous CH2Cl2 (3 ml) with activated 4 Å MS. After stirring for 30 min at -30 °C, TMSOTf (0.4 µL, 2.2 µmol, 0.05 eq.) was added. The reaction was kept at -20 to -30 °C under stirring. Et3N (4 µL) was added to quench the reaction. MS was filtered and the solvent was removed on a rotary evaporator. The residue was subjected to silica gel chromatography (petrol: ethyl acetate 2:1, 7:4, 3:2, 5:4 and 1:1) to afford 19 (30 mg, 45%), 38 (11 mg, 16%), 39 (2.7 mg, 6%) and 24 (1.0 mg, 2%). Compound 19: Rf 0.45 (petroleum: EtOAc = 3: 2); [α] D

20 = +23 (c 0.8, CHCl3); νmax (transmission, thin film)/cm-1 3502 (νO-H), 3064, 3028 (νC-Harom), 2957, 2925, 2854 (νC-Haliph), 1736 cm-1 (νC=O OAc), 1251, 1225, 1092, 1070 (νC-O); 1H NMR (500 MHz, CDCl3) δ 0.00 (s, 3H, Si(CH3)2C(CH3)3), 0.01 (s, 3H, Si(CH3)2C(CH3)3), 0.81 (s, 9H, Si(CH3)2C(CH3)3), 1.18 (d, 3H, J 6.8 Hz, SCH(CH3)2), 1.22 (d, 3H, J 6.8 Hz, SCH(CH3)2), 2.00, 2.019, 2.028, 2.038, 2.061, 2.079 (5s, 3H each, 5COCH3), 2.99 (m, 1H, SCH(CH3)2), 3.23 (ddd, 1H, J 4.2, 6.7, 9.4 Hz, H-5II), 3.34 (dd, 1H, J 2.6, 9.4 Hz, H-3II), 3.54 (s, 3H, COOCH3), 3.55 (dd, 1H, J 3.5 10.3 Hz, H-3I), 3.60-3.70 (m, 3H, H-6aII, H-4II, H-2III), 3.79 (m, 2H, H-6bII, H-5I), 3.86 (dd, 1H, J 7.9, 11.9 Hz, H-6aIII), 3.90-3.98 (m, 2H, H-6aI, H-2II), 4.20 (dd, 1H, J 3.7, 11.8 Hz, H-6bIII), 4.25 (dd, 1H, J 7.4 10.9 Hz, H-6bI), 4.33 (dd, 1H, J 3.5 10.0 Hz, H-3III), 4.34-4.43 (m, 4H, PhCH2-




51

a&bIII, PhCH2-aI, H-5IV), 4.45 (dd, 1H, J 3.7, 7.8 Hz, H-5III), 4.48 (s, 1H, H-1II), 4.64

(m, 2H, H-1I, PhCH2-bI), 4.93 (d, 1H, J 3.8 Hz, H-1III), 5.10 (dd, 1H, J 8.0, 10.3 Hz, H-2I), 5.27 (d, 1H, J 7.9 Hz, H-1IV), 5.48 (dd, 1H, J 0.9, 3.4 Hz, H-4II), 5.50 (dd, 1H, J 0.8, 3.5 Hz, H-4III), 5.53 (dd, 1H, J 7.7 0.7 Hz, H-H-2IV), 5.59 (t, 1H, J 9.7 Hz, H-4IV), 5.87 (t, 1H, J 9.6 Hz, H-3IV), 6.98-7.89 (m, 25 H, 5 Ph); 13C NMR (125 MHz, CDCl3) δ -5.42, -5.38 (Si(CH3)2C(CH3)3), 18.2 (Si(CH3)2C(CH3)3), 20.74, 20.78, 20.85, 20.91, 21.5 (5 COCH3), 23.5, 24.1 (SCH(CH3)2), 25.8 (Si(CH3)2C(CH3)3), 36.8 (SCH(CH3)2), 52.7 (COOCH3), 61.6 (C-6I), 63.8 (C-6III), 64.5 (C-6II), 66.0 (C-4I), 67.3 (C-5III), 68.5 (C-4II), 69.7 (C-4III), 70.3 (C-4IV), 70.5 (C-2I), 71.1 (C-5I), 71.5 (PhCH2

I), 72.0 (C-2IV), 72.3 (C-3IV), 72.6 (C-5IV), 73.5 (PhCH2III), 75.2 (C-2III), 75.8

(C-3III), 76.6 (C-3I), 78.8 (C-2II), 80.6 (C-5II), 82.9 (C-1II, 1JCH 149 Hz), 83.6 (C-3II),

100.0 (C-1IV, 1JCH 163 Hz), 100.7 (C-1III

, 1JCH 171 Hz), 100.8 (C-1I, 1JCH 161 Hz),

127.6, 127.8, 128.29, 128.34, 128.40, 128.43, 128.53, 129.8, 133.3, 133.4, 137.6, 137.8 (5 Ph), 165.1, 165.2, 165.6, 167.1, 169.7, 170.4,170.6, 170.7, 170.8 (9 CO); HRMS m/z (ES+) [calculated for C79H96NaO29SSi+ 1591.54194; found 1591.54847]. Compound 38: Rf 0.30 (petroleum: EtOAc = 3: 2); [α] D

20 = +31 (c 0.3, CHCl3); νmax (transmission, thin film)/cm-1 3441 (νO-H), 2927, 2855 (νC-Haliph), 1742 cm-1 (νC=O OAc), 1251, 1225, 1094, 1070 (νC-O); 1H NMR (500 MHz, CDCl3) δ 0.02 (s, 3H, Si(CH3)2C(CH3)3), 0.00 (s, 3H, Si(CH3)2C(CH3)3), 0.81 (s, 3H, Si(CH3)2C(CH3)3), 1.19 (d, 3H, J 6.6 Hz, SCH(CH3)2), 1.21 (d, 3H, J 6.7 sHz, SCH(CH3)2), 1.90, 2.02, 2.03, 2.06, 2.13 (5s, 3H each, 5COCH3), 3.03 (m, 1H, SCH(CH3)2), 3.26 (m, 1H, H-5III), 3.51 (dd, 1H, J 3.4, 10.1 Hz, H-3I), 3.62 (s, 3H, COOCH3), 3.62-3.68 (m, 4H, H-6aII, H-4II, H-3II, H-2III), 3.76-3.81 (m, 2H, H-6bII, H-5I), 3.82 (d, J 2.2 Hz, H-2II), 3.96 (dd, J 7.1, 11.1 Hz, H-6aIII), 4.00-4.10 (m, 3H, H-6bIII, H-6aI, H-6bI), 4.13 (m, 1H, H-5III), 4.15-4.21 (m, 3H, H-5IV, PhCH2-aIII, H-3III), 4.34 (d, 1H, J 12.3 Hz, PhCH2-aI), 4.39 (d, 1H, J 8.0 Hz, H-1I), 4.47 (s, 1H, H-1II), 4.62 (d, 2H, J 11.3 Hz, PhCH2-bIII, PhCH2-bI), 5.07 (dd, 1H, J 8.1, 10.0 Hz, H-2I), 5.29 (d, 1H, J 7.5 Hz, H-1IV), 5.42 (dd, 1H, J 0.8, 3.5 Hz, H-4I), 5.43-5.47 (m, 2H, H-4III, H-2IV), 5.61 (t, 1H, J 9.6 Hz, H-4IV), 5.77 (at, 1H, J 9.4 Hz, H-3IV), 5.86 (d, 1H, J 3.9 Hz, H-1III), 7.14-7.89 (m, 25 H, 5 Ph); 13C NMR (125 MHz, CDCl3) δ -5.22 (Si(CH3)2C(CH3)3), -5.20 (Si(CH3)2C(CH3)3), 18.4 (Si(CH3)2C(CH3)3), 20.61, 20.63, 20.7, 20.8, 20.9 (5 COCH3), 23.7 (SCH(CH3)2), 24.0 (SCH(CH3)2), 25.9 (Si(CH3)2C(CH3)3), 35.8 (SCH(CH3)2), 52.8 (COOCH3), 61.9 (C-6I), 62.3 (C-6III), 63.9 (C-6II), 65.7 (H-4I), 67.3 (C-5III), 69.8 (C-4III), 70.0 (C-2I), 70.3 (C-4IV), 71.4 (C-5I), 71.5 (PhCH2

I), 71.9 (C-5IV), 72.0 (C-2IV), 72.17 (PhCH2

III), 72.22 (C-3IV), 72.5 (C-4II), 72.8 (C-3III), 75.2 (C-3II), 76.5 (C-2III), 76.7 (C-3I), 79.8 (C-5II), 82.6 (C-1II, JCH 150 Hz), 83.9 (C-2II), 96.0 (C-1III, 1JCH 176 Hz), 100.4 (C-1IV, JCH 168 Hz), 102.6 (C-1I, 1JCH 150 Hz), 127.8, 129.9, 128.2, 128.36, 128.40, 128.42, 128.48, 129.7, 129.8, 129.9, 133.2, 133.3, 133.4, 137.4, 137.8 (5 Ph), 164.8, 165.1, 167.2, 169.1, 170.0, 170.5, 170.6 (9 CO); HRMS m/z (ES+) [calculated for C79H96NaO29SSi+ 1591.54194; found 1591.54325]. Compound 39: Rf 0.49 (petroleum: EtOAc = 3: 2); [α] D

20 = +36 (c 0.07, CHCl3); νmax (transmission, thin film)/cm-1 2956, 2923, 2853 (νC-Haliph), 1735 cm-1 (νC=O OAc), 1260, 1093 (νC-O); 1H NMR (500 MHz, CDCl3) δ 1.96 (COCH3), 2.09 (COCH3), 3.64 (COOCH3), 3.82 (dd, 1H, J 5.3, 9.0 Hz, H-2I), 3.92-3.99 (m, 3H, H-4I, H-5I, H-




52

6aI), 4.12 (q, 1H, H-6bI), 4.20 (d, 1H, J 11.5 Hz, PhCH2-a), 4.23 (d, 1H, J 9.9 Hz, H-5II), 4.37 (d, 1H, J 11.5 Hz, PhCH2-b), 5.11 (d, 1H, J 7.3 Hz, H-1II), 5.28 (t, 1H, J 5.3 Hz, H-1I), 5.41 (dd, 1H, J 1.1, 3.5 Hz, H-4I), 5.48 (dd, 1H, J 7.2, 9.2 Hz, H-2II), 5.65 (t, 1H, J 9.7 Hz, H-4II), 5.81 (at, 1H, J 9.4 Hz, H-3II), 7.10 (d, 1H, J 5.4 Hz, NH), 7.01-7.06 and 7.20-7.90 (m, 20 H, 4 Ph); 13C NMR (125 MHz, CDCl3) δ 20.6 (COCH3), 20.7 (COCH3), 53.0 (COOCH3), 61.8 (C-6I), 68.2 (C-4I), 68.5 (C-5I), 69.9 (C-4II), 71.9 (C-3II), 72.0 (C-3II), 72.9 (C-5II), 73.6 (PhCH2), 74.2 (C-3I), 74.4 (C-2I), 92.2 (COCCl3), 100.6 (C-1II), 128.0, 128.41, 128.48, 128.59, 128.64, 128.8, 129.7, 129.8, 133.47, 133.51, 133.53, 136.4 (4 Ph), 161.9 (COCCl3), 164.8, 165.1, 165.7, 167.1, 170.0, 170.7 (6 COOR); HRMS m/z (ES+) [calculated for C47H44Cl3NNaO17

+ 1022.15670; found 1022.15599]. Isopropyl 4, 6-di-O-benzylidene-1-thio-β-D-galactopyranoside (20)

Isopropyl 1-thio-β-D-galactopyranoside 9 (IPTG, 10.0 g, 42.0 mmol, 1.0 eq.) and PhCH(OMe)2 (7.69 g, 48.4 mmol, 1.2 eq.) were dissolved in DMF (100 mL) in a 500 mL flask at 0 ˚C. CSA (1.95 g, 8.4 mmol, 0.2 eq.) was added. The temperature was allowed to warm to RT. The reaction was kept for 48 hours with stirring at RT and was then quenched by the addition of triethylamine (2 mL). DMF was removed on a rotary evaporator. The residue was diluted with ethyl acetate (300mL) and then washed with brine. The upper organic fraction was dried over MgSO4, and filtered. The solvent was removed on a rotary evaporator. The residue was subjected to silica flash column chromatography (1:1 Petroleum ether: Ethyl acetate) to give 20 as a white crystalline solid (9.6 g, 70%): Rf 0.15 (petroleum: EtOAc = 1: 2); [α] D

20 = -89.9 (c 1.0, CHCl3) (Lit. -62,)20; m.p. 176-178˚C; 1H NMR(400 MHz, CDCl3) δ 1.36 (d, 3H, J 6.8 Hz, CH3), 1.39 (d, 3H, J 6.8 Hz, CH3) 3.29 (m, 1H, SCH), 3.51 (d, 1H, J 1.3 Hz, H-5), 3.69 (dd, 1H, J 3.5, 9.1 Hz, H-3), 3.78 (t, 1H, J 9.3 Hz, H-2), 4.04 (dd, 1H, J 1.8, 12.4 Hz, H-6a), 4.26 (dd, 1H, J 1.0, 3.8 Hz, H-4), 4.34 (dd, 1H, J 1.5, 12.4 Hz, H-6b), 4.42 (d, 1H, J 9.3 Hz, H-1), 5.54 (s, 1H, PhCH), 7.35-7.55 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 24.0, 24.4 (2 CH3), 35.0 (SCH), 69.4 (C-6), 70.1, 70.1 (C-2, C-5), 74.0 (C-3), 75.6 (C-4), 85.5 (C-1), 101.5 (PhCH), 126.5, 128.3, 129.3, 137.5 (6 x Ar-C); LRMS m/z (ES+) 349 ([M + Na]+, 100%); LRMS m/z (ES+) 349 ([M + Na]+, 100%); Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3)-isopropyl-2-O-benzyl-1-thio-β-D-galactopyranoside (23)

11 (1.17 g, 1.75 mmol) and 10 (0.73 g, 1.75 mmol) were dissolved in anhydrous CH2Cl2 (55 mL) with activated 4Å molecular sieves. TMSOTf (120 µL, 0.66 mmol, 0.38 eq.) was added at -20 °C under an atmosphere of argon. The reaction was kept for 1 h under stirring and then quenched with Et3N. The solvent was removed on a rotary evaporator and the residue was subjected to silica chromatography (petroleum: ethyl acetate =2: 1) to afford compound 22 as a foam (1.35 g, 84 %). Compound 22 (0.53 g, 1.15 mmol, 1.0 eq.) was dissolved in aqueous 80 % HOAc (7 mL) at 60 °C




53

for overnight and then co-evaporated with toluene. The residue was subjected to silica chromatography (petroleum: EtOAc = 2: 5) to afford compound 23 as a colorless glass (0.36 g, 75 %): Rf 0.16 (petroleum: EtOAc = 1: 1); [α] D

20 = +11.3 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 3512 (νO-H), 3065, 3033, 3010 (νC-Harom), 2960, 2925, 2866 (νC-Haliph), 1734 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3) δ 1.31 (d, 6H, J 6.6 Hz, 2 CH3), 3.21 (m, 1H, SCH), 3.58 (t, 1H, J 5.3 Hz, H-5I), 3.63 (t, 1H, J 9.3 Hz, H-2I), 3.68 (s, 3H, CH3), 3.81 (dd, 1H, J 4.3, 11.6 Hz, H-6aI), 3.91 (dd, 1H, J 2.8, 9.1 Hz, H-3I), 4.00 (dd, 1H, J 6.8, 11.9 Hz, H-6bI), 4.21 (d, 1H, J 2.3 Hz, H-4I), 4.29 (d, 1H, J 9.3 Hz, H-5II), 4.38 (d, 1H, J 10.4 Hz, CH2Ph-a), 4.48 (d, 1H, J 9.8 Hz, H-1I), 4.70 (d, 1H, J 10.4 Hz, CH2Ph-b), 5.31 (d, 1H, J 7.6 Hz, H-1II), 5.64 (at, 1H, J 8.3 Hz, H-2II), 5.71 (at, 1H, J 9.4 Hz, H-4II), 5.90 (at, 1H, J 9.2 Hz, H-3II), 7.15-8.00 (m, 20H, 20 x Ar-H); 13C NMR (CDCl3) δ 23.9, 24.0 (2 CH3), 35.4 (SCH), 53.1 (CH3), 62.6 (C-6I), 69.3 (C-4I), 70.0 (C-4II), 71.7, 71.9 (C-2II, C-3II), 72.5 (C-5II), 75.3 (CH2Ph), 77.8 (C-2I), 77.9 (C-5I), 82.5 (C-3I), 84.7 (C-1I), 101.0 (CHPh), 127.7, 127.8, 128.3, 128.4,128.5, 128.5, 128.7, 128.7, 129.8, 129.8 (24 x Ar-C), 165.2, 165.6, 167.3 (4 CO). Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3)-isopropyl-2-O-benzyl-4,6-acetyl-1-hydroxyl-α/β-D-galactopyranose (24)

Compound 12 (0.2 g, 0.2 mmol, 1.0 eq.) was dissolved in CH2Cl2 (2 mL) and H2O (0.02 mL) at RT. NIS (62 mg, 0.28 mmol, 1.4 eq.) and Tf2O (5.2 µL, 31.2 µmol, 0.16 eq.) in CH2Cl2 (3 mL) was added. After 2h, Na2S2O3 aqueous solution (10%, 10 mL) was added under turbulent stirring. The layers were separated and the organic layer was washed with saturated NaHCO3, dried over MgSO4 and filtered. The residue was subjected to silica chromatography (petroleum: ethyl acetate = 3: 2) to afford an α & β mixture of 24 as a colorless glass (135 mg, 72%): Rf 0.15 (petroleum: EtOAc = 1: 1); IR (thin film): 3453 (νO-H), 3064, 3032 (νC-Harom), 2955 (νC-Haliph), 1737 cm-1 (νC=O); HRMS m/z (ES+) [calculated for C47H51N2O17

+ 915.3182; found 915.3188]. α anomer: 1H NMR (400 MHz, CDCl3) δ 2.05, 2.16 (2s, 2 × 3H, 2 × CH3), 3.69-3.71 (m, 4H, H-2I, OAc), 3.94-4.16 (m, 2H, H-6aI, H-6bI), 4.25-4.39 (m, 3 H, PhCH2-a and H-5II,H-5I), 4.50 (d, 1H, J 11.9 Hz, PhCH2-b), 5.08 (d, 1H, J 3.5 Hz, H-1I), 5.28 (d, 1H, J 7.6 Hz, H-1II), 5.53 (d, 1H, J 6.8 Hz, H-4I), 5.57 (m, 1H, H-2II), 5.72 (at, 1H, J 9.5 Hz, H-4II), 5.90 (t, 1H, J 9.5 Hz, H-3II), 7.10-8.00 (m, 20H, 20 x Ar-H); 13C NMR (CDCl3) δ 20.7, 20.8 (2OAc), 52.9 (CH3), 62.7 (C-6I), 67.1 (C-5I), 70.0 (C-4I), 70.2 (C-4II), 71.3 (C-2II), 71.9 (C-3II), 72.8(C-5II), 73.5 (CH2Ph), 76.6 (C-2I), 91.5 (C-1I), 100.8 (C-1II), 127-139 (24 x Ar-C), 164.8, 165.1, 165.7, 167.0, 170.1, 170.7 (6 CO); Isopropyl-3-O-Fmoc-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (25)

Compound 20 (7.50 g, 23 mmol, 1.0 eq.) was dissolved in pyridine (43 mL). FmocCl (7.2 g, 28 mmol, 1.2 eq.) and DMAP (375 mg, 3.1 mmol, 0.13 eq.) were added to the solution in an ice-water bath. The reaction was kept at RT for 22 h under stirring. The




54

solvent was removed on rotary evaporator. The residue was subjected to silica column chromatography (petroleum: ethyl acetate = 11 : 4) to give 25 (7.5 g, 50 %) as a white foam: Rf 0.65 (petroleum: EtOAc = 1: 1); [α] D

20 = +14.6 (c 1.0, CHCl3) (Lit. +31)20; 1H NMR(400 MHz, CDCl3) δ 1.37 (d, 3H, J 6.8 Hz, CH3), 1.41 (d, 3H, J 6.8 Hz, CH3) 2.55 (d, 1H, J 2.0 Hz, OH), 3.32 (m, 1H, SCH), 3.55 (d, 1H, J 1.0 Hz, H-5), 4.04 (dd, 1H, J 1.8, 12.6 Hz, H-6a), 4.14 (q, 1H, J 7.1 Hz, H-2), 4.31 (t, 1H, J 7.6 Hz, CH), 4.35 (dd, 1H, J 1.3, 12.4 Hz, H-6b), 4.46 (m, 1H, H-4), 4.50 (d, 1H, J 9.8 Hz, H-1), 4.78 (dd, 1H, J 3.5, 9.6 Hz, H-3), 5.52 (s, 1H, CHPh), 7.20-7.80 (m, 13H, 13 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 24.1, 24.5 (2s, 2 CH3), 35.1 (SCH), 46.6 (CH), 66.8 (C-2), 69.2 (CH2), 69.8 (C-5), 70.2 (C-6), 73.5 (C-4), 78.5 (C-3), 85.9 (C-1), 101.0 (CHPh), 120.4, 125.3, 126.3, 127.2, 127.9, 128.2, 129.1, 137.6, 141.3, 143.4, 154.6 (18 x Ar-C); LRMS m/z (ES+) 570 ([M + Na]+, 100%). Isopropyl-2-O-acetyl-3-O-Fmoc-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (26)

Compound 25 (6.0 g, 11 mmol, 1.0 eq.) was dissolved in pyridine (20 mL). Ac2O (60 mL, 63.5 mmol, 5.8 eq.) was added. The reaction was kept for 2h at RT under stirring. The solvent was removed on a rotary evaporator. The residue was subjected to silica gel chromatography (petroleum: ethyl acetate = 11 : 4) to afford 26 (6.33 g, 98 %) as a white foam: Rf 0.85 (petroleum: EtOAc = 1: 1); [α] D

20 = +26.2 (c 1.0, CDCl3); νmax (transmission, thin film)/cm-1 3607, 3017 (νC-Harom), 2963, 2926, 2867 (νC-Haliph), 1750 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3): δ 1.29 (d, 3H, J 6.8 Hz, CH3), 1.41 (d, 3H, J 6.8 Hz, CH3), 2.09 (s, 3H, COCH3), 3.34 (m, 1H, SCH), 3.54 (d, 1H, J 1.0 Hz, H-5), 4.04 (dd, 1H, J 1.8, 12.4 Hz, H-6a), 4.27 (t, 1H, J 7.6 Hz, CH), 4.36 (dd, 1H, J 1.0, 12.4 Hz, H-6b), 4.35-4.46 (m, 2H, CH2), 4.49 (dd, 1H, J 0.8, 3.8 Hz, H-4), 4.57 (d, 1H, J 9.9 Hz, H-1), 4.88 (dd, 1H, J 3.5, 10.1 Hz, H-3), 5.53 (at, 1H, J 9.9 Hz, H-2), 7.20-7.80 (m, 13H, 13 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 21.0, 23.6, 25.0 (3 CH3), 34.7 (SCH), 46.5 (CH), 66.9 (C-2), 69.2 (C-6, CH2), 69.6 (C-5), 70.3 (C-4), 73.4 (C-3), 83.2 (C-1), 101.2 (CHPh), 120.1, 125.2, 125.3, 126.5, 127.2, 128.0, 128.2, 129.2, 137.4, 141.3, 143.1, 143.2 (3Ph), 154.4, 169.4 (CO); HRMS m/z (ES+) [calculated for C33H34NaO8S+ 613.1867; found 613.1873]. Isopropyl-2,3,4,6-tetra-O-acetyl-1-thio-α & β-D-mannopyranoside (27)

Intermediate 8 (20 g, 51 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (80 mL). HSCHMe2 (7.2 mL, 78 mmol, 1.5 eq.) and BF3.Et2O (13 mL, 102 mmol, 2.0 eq.) were added. The mixture was stirred at RT for 2h. CH2Cl2 (100 mL) was added to dilute the mixture, which was neutralized by saturated NaHCO3 aqueous solution. The organic phase was sepearated, dried over MgSO4 and filtered. The solvent was removed on a rotary evaporator. The residue was subjected to silica gel chromatography (petroleum: ethyl acetate = 3: 1) to afford 14.6 g (70%) of the α




55

product (27α) as an oil and 3.5 g (17 %) of the β product (27β) as white solid: Rf 0.59 (petroleum: EtOAc = 1: 1); LRMS m/z (ES+) 424 ([M + NH4]+, 100%). α anomer: [α] D

20 = +85.5 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 2963, 2929, 2890 (νC-Haliph), 1751 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3) δ 1.32 (d, 3H, J 3.3 Hz, CH3), 1.34 (d, 3H, J 3.5 Hz, CH3), 1.99, 2.05, 2.09, 2.17 (4s, 12 H, 4 × CH3), 3.08 (m, 1H, SCH), 4.07 (dd, 1H, J 3.0, 12.9 Hz, H-6a), 4.32 (dd, 1H, J 5.6, 12.4 Hz, H-6b), 4.42 (m, 1H, H-5), 5.24 (dd, 1H, J 3.3, 9.9 Hz, H-3), 5.27-5.34 (m, 2H, H-2, H-4), 5.35 (d, 1H, J 1.5 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ 20.7, 21.0, 23.4, 23.7 (4 CH3), 36.5 (SCH), 62.4 (C-6), 66.4 (C-4), 68.9, 69.5 (C-3, C-5), 71.5 (C-2), 81.9 (C-1), 169.8, 170.0, 170.6 (CO). β anomer: [α] D

20 = -51.5 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 2964, 2930, 2669 (νC-Haliph), 1751 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3) δ 1.30 (d, 3H, J 6.8 Hz, CH3), 1.33 (d, 3H, J 6.8 Hz, CH3), 1.98, 2.04, 2.06, 2.18 (4s, 12 H, 4 × OCH3), 3.18 (m, 1H, SCH), 3.70 (m, 1H, H-5), 4.13 (dd, 1H, J 2.5, 12.1 Hz, H-6a), 4.26 (dd, 1H, J 6.3, 12.1 Hz, H-6b), 4.81 (d, 1H, J 1.0 Hz, H-1), 5.08 (dd, 1H, J 3.5, 10.1 Hz, H-3), 5.24 (t, 1H, J 10.1 Hz, H-4), 5.51 (dd, 1H, J 1.0, 3.5 Hz, H-2); 13C NMR (100 MHz, CDCl3) δ 20.5, 20.6, 20.7, 20.8 (4 OAc), 23.4, 23.9 (2 CH3), 36.1 (SCH), 62.9 (C-6), 66.0 (C-4), 70.8 (C-2), 72.0 (C-3), 76.4 (C-5), 81.9 (C-1); 169.6, 170.1, 170.2, 170.7 (CO). Isopropyl-1-thio-α-D-mannopyranoside (28)

27 (12.5 g, 30.8 mmol, 1.0 eq.) was dissolved in anhydrous MeOH (100 mL) under an atmosphere of argon at RT. Catalytic amount of sodium methoxide (100 mg, 1.85 mmol, 0.06 eq.) was added. After 1 h, the reaction was quenched by neutralizing the pH to ~7 with IR 120 (H+). The resin was filtered and the residue was subjected to silica gel chromatography (ethyl acetate : MeOH = 8: 1) to afford 28 as a white foam (6.2 g, 85 %): Rf 0.17 (EtOAc: MeOH = 8: 1); [α] D

20 = +124.8 (c 1.0, MeOH); νmax (transmission, thin film)/cm-1 3300 (νO-H); 1H NMR (400 MHz, CDCl3) δ 1.31 (d, 3H, J 6.8 Hz, CH3), 1.33 (d, 3H, J 6.6 Hz, CH3), 3.11 (m, 1H, SCH), 3.60-3.72 (m, 2H, H-4, H-5), 3.75 (dd, 1H, J 5.3, 11.9 Hz, H-6a), 3.82 (dd, 1H, J 2.5, 11.8 Hz, H-6b), 3.91 (m, 1H, H-2), 3.93 (dd, 1H, J 2.3, 5.3 Hz, H-3), 5.31 (d, 1H, J 1.5 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ 23.0, 23.2 (2 CH3), 35.4 (SCH), 61.8 (C-6), 67.8, 72.3 (C-4, C-5), 73.0 (C-2), 73.9 (C-3), 84.6 (C-1); HRMS m/z (ES+) [calculated for C9H18NaO5S+ 261.0767; found 261.0767]. 2,3,4,6-tetra-O-benzyl-1-isopropyl-thio-α-D-mannopyranoside (29)

Compound 28 (40.7 g, 171 mmol, 1.0 eq.) was dissolved in anhydrous DMF (400 mL). NaH (34.2 g, 855 mmol, 5.0 eq.) was added. After 1h, BnBr (91.4 mL, 769 mmol, 4.5 eq.) was added dropwise at 0 ºC. The reaction was kept overnight under




56

stirring. The reaction was quenched by the addition of methanol (100 mL). The solvent was removed on a rotary evaporator. Water (500 mL) and CH2Cl2 (500 mL) were added to the residues under turbulent stirring. The lower organic layer was separated, dried over MgSO4 and filtered. The solvent was removed on a rotary evaporator and the residue was subjected to silica chromatography (petrol: ethyl acetate = 9: 1) to afford compound 29 as a syrup (66 g, 65%): Rf 0.73 (petroleum: EtOAc = 7: 1); [α] D

20 = + 10.2 (c 1.0, CHCl3); m.p. 93-95 ˚C; νmax (transmission, thin film)/cm-1 3063, 3028 (νC-Harom), 2960 (νC-Haliph); 1H NMR (400 MHz, CDCl3) δ 1.31 (d, 3H, J 6.6 Hz, SCH(CH3)2), 1.36 (d, 3H, J 6.8 Hz, SCH(CH3)2), 3.18 (SCH), 3.51 (m, 1H H-5), 3.64 (dd, J 2.8, 9.3 Hz, H-3), 3.72 (dd, 1H, J 6.3, 10.9 Hz, H-6a), 3.81 (dd, 1H, J 1.0, 10.6 Hz, H-6b), 3.91 (t, 1H, J 9.6 Hz, H-4), 4.01 (d, 1H, J 2.3 Hz, H-2), 4.62 (s, 1H, H-1), 4.55-5.00 (m, 8H, 4 PhCH2-), 7.15-7.55 (m, 20H, 20 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 23.8, 24.1 (SCH(CH3)2), 35.8 (SCH), 69.9 (C-6), 72.2, 73.4, 74.9, 75.2 (4PhCH2, C-4), 80.1 (C-2), 83.7 (C-1), 84.5 (C-3), 127.4, 127.5, 127.5, 127.7, 127.8, 128.2, 128.2, 128.3, 128.4, 138.1, 138.2, 138.4, 138.5 (24 x Ar-C); HRMS m/z (ES+) [calculated for C37H46NO5S+ 616.3091; found 616.3090. 2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (30)

Compound 29 (56.9 g, 95 mmol, 1.0 eq.) was dissolved in acetone (400 mL) and water (20 mL). NIS (25.3 g, 112 mmol, 1.2 eq.) and TFA (0.7 mL, 9.1 mmol, 0.10 eq.) were added at 0 ºC. The reaction was kept under stirring for 1 hour. Then Na2S2O3 (200 mL, 10%) was added to quench the reaction, followed by extraction with CH2Cl2 (700 mL) and water (700 mL). The organic layer was separated, dried over magnesium sulfate and filtered. The solvent was removed and the residue was subjected to silica chromatography (petrol: ethyl acetate = 9:4) to afford the hydrolyzed intermediate (51 g, 94 mmol, 1.0 eq.), which was further dissolved in CH2Cl2 (400 mL). DBU (1.4 mL, 9.4 mmol, 0.1 eq.) and CNCCl3 (20 mL, 200 mmol, 2.1 eq.) were added at 0 ºC. The temperature of the solution was allowed to warm to RT. After 2 h, the solvent was removed on a rotary evaporator and the residue was subjected to silica chromatography (petrol: ethyl acetate 8: 1) to afford compound 30 as a syrup (60 g, 94 % in two steps): Rf 0.83 (petroleum: EtOAc = 3: 1); [α] D

20 = + 32.1 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 3.78 (d, 1H, J 10.9 Hz, H-6a), 3.88 (dd, 1H, J 4.3, 11.1 Hz, H-6b), 3.92 (bs, 1H, H-2), 3.98 (dd, 1H, J 3.3, 9.8 Hz, H-3), 4.02 (dd, 1H, J 3.0, 9.8 Hz, H-5), 4.21 (t, 1H, J 9.6 Hz, H-4), 4.54-5.00 (m, 8H, 4 PhCH2-), 6.42 (s, 1H, H-1), 7.15-7.50 (m, 20H, 20 x Ar-H), 8.57 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 68.8 (C-6), 72.3, 72.6, 73.4, 75.4 (4PhCH2), 73.5 (C-2), 74.2 (C-4), 74.8 (C-5), 78.9 (C-3), 91.0 (CCl3), 96.1 (C-1), 127.5, 127.7, 127.8, 127.9, 128.0, 128.2, 128.3, 128.4, 128.4,138.0, 138.1, 138.3, 138.4 (24 x Ar-C), 160.4 (C=NH); LRMS m/z (ES+) 706 ([M + Na]+, 100%).




57

Isopropyl-1-thio-β-D-mannopyranoside (32)

Compound 30 (60 g, 88 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (300 mL) with activated 4 Å MS. HSCHMe2 (16mL, 176 mmol, 2.0 eq.) and TMSOTf (1.6 mL, 8.8 mmol, 0.1 eq.) were added. The reaction was kept at -30 ˚C for 1h under stirring. Et3N (2 mL) was then added to quench the reaction. MS was filtered through a pad of Celite and the solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (petrol: ethyl acetate 9:1), followed by crystallisation to afford protected monosaccharide 31 as a white crystalline solid (34 g, 65%). Compound 31 (34 g, 57 mmol, 1.0 eq.) was dissolved in EtOAc (200 mL) and EtOH (200 mL) in a 1 L flask. Pd/C (10.0 g, 60%, 57 mmol, 1.0 eq.) was added under an atmosphere of argon. H2 was applied to exchange argon and fill the flask. The reaction was kept overnight at RT under stirring. Pd/C was filtered and the solvent was removed on rotary evaporator. The residue was subjected to silica chromatography (MeOH: ethyl acetate 1: 8) to afford compound 32 as a white solid (11.6 g, 86 %): Rf 0.14 (EtOAc: MeOH = 8: 1); [α] D

20 = -67.3 (c 1.0, MeOH); νmax (transmission, thin film)/cm-1 3300 (νO-H); 1H NMR (400 MHz, CDCl3) δ 1.31 (d, 3H, J 6.9 Hz, CH3), 1.32 (d, 3H, J 6.7 Hz, CH3), 3.20 (m, 1H, SCH), 3.27 (m, 1H, H-5), 3.51 (dd, 1H, J 3.3, 9.3 Hz, H-3), 3.60 (t, 1H, J 9.6 Hz, H-4), 3.72 (dd, 1H, J 5.6, 11.9 Hz, H-6a), 3.85 (dd, 1H, J 2.5, 12.4 Hz, H-6b), 5.81 (d, 1H, J 3.3 Hz, H-2), 4.79 (s, 1H, H-1); 13C NMR (100 MHz, MeOH) δ 23.2, 23.4 (SCHMe2), 35.1 (SCH), 61.9 (C-6), 67.3 (C-4), 73.3 (C-2), 75.3 (C-3), 81.3 (C-5), 84.1 (C-1); HRMS m/z (ES+) [calculated for C9H18NaO5S+ 261.0767; found 261.0768]. Isopropyl-(2’S,3’S)-methyl-3,4-O-(2’,3’-dimethoxybutane-2’,3’-diyl)-1-thio-β-D-mannopyranoside (33)

To compound 32 (11.0 g, 46 mmol, 1.0 eq.) and CSA (1.7 g, 7.3 mmol, 0.16 eq.) in anhydrous methanol (100 mL) was added CH(OMe)3 (42.6 mL, 390 mmol, 8.5 eq.) and butane 2,3-dione (12.8 mL, 146 mmol, 3.2 eq.). The reaction mixture was heated under reflux overnight. The solvent was then removed on a rotary evaporator and the residue was subjected to silica chromatography (petrol: ethyl acetate = 4: 5), followed by crystallisation with diethyl ether to afford compound 33 as a white crystalline solid (5.2 g, 35 %): Rf 0.69 (EtOAc); [α] D

20 = + 92.7 (c 1.0, CHCl3); m.p. 147-149 ˚C; νmax (transmission, thin film)/cm-1 3449 (νO-H), 2955 (νC-Haliph); 1H NMR (400 MHz, CDCl3) δ 1.25-1.35 (m, 12H, 2CCH3 of BDA and SCH(CH3)2), 2.18 (at, 1H, J 6.3 Hz, OH-6), 2.57 (d, 1H, J 1.8 Hz, OH-2), 3.16 (m, 1H, SCH), 3.23, 3.27 (2s, 6H, 2OCH3 of BDA), 3.49 (m, 1H, H-5), 3.71-3.81 (m, 2H, H-3, H-6a), 3.83-3.91 (m, 1H, H-6b), 4.01 (s, 1H, H-2), 4.07 (at, 1H, J 10.0 Hz, H-4), 4.75 (s, 1H, H-1); 13C NMR (100 MHz, CDCl3) δ 17.6, 17.7 (2 × OCH3), 23.6, 23.9 (-S(CH3)2), 35.2 (SCH), 47.8, 48.1 (2 × OCH3), 61.7 (C-6), 63.2 (C-4), 71.1 (C-2), 71.3 (C-3), 78.3 (C-5), 83.6 (C-1), 99.8, 100.4 (-CCH3(OCH3)2).




58

3-O-benzyl-1-thio-β-D-galactopyranoside (34)

Compound 21 (10.0 g, 24 mmol, 1.0 eq.) was dissolved in 80% acetic acid (100 mL). The reaction was kept at 70 ºC for 3 hours under stirring. The solvent was then removed on a rotary evaporator. The residue was crystallized in EtOAc-methanol to afford the tri-hydroxyl compound 34 as a white crystalline solid (7.1 g, 90%): Rf 0.10 (EtOAc); [α] D

20 = -13.2 (c 0.5, CHCl3); νmax (transmission, thin film)/cm-1 3371 (νO-H), 2960 (νC-Haliph); 1H NMR (400 MHz, CDCl3) δ 1.22 (at, 6H, J 6.7 Hz, SCH(CH3)2), 3.13 (m, 1H, SCH), 3.25 (dd, 1H, J 3.0, 9.1 Hz, H-3), 3.33 (t, 1H, J 6.1 Hz, H-5), 3.41- 3.59 (m, 3H, H-6a&b, H-2), 3.96 (s, 1H, H-4), 4.32 (d, 1H, J 9.6 Hz, H-1), 4.52-4.73 (m, 4H, OH-4, OH-6, PhCH2-a&b), 5.12 (d, 1H, J 5.8 Hz, OH-2), 7.21-7.45 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 24.6, 24.7 (SCH(CH3)2), 34.4 (SCH), 61.3 (C-6), 65.7 (C-4), 69.8 (C-2), 71.0 (PhCH2), 79.7 (C-5), 83.3 (C-3), 86.0 (C-1), 128.0, 128.4, 128.9, 139.9 (6 x Ar-C); HRMS m/z (ES+) [calculated for C16H24NaO5S+ 351.1237; found 351.1241]. 3-O-benzyl-2, 4, 6-O-tri-O-acetyl-1-thio-β-D-galactopyranoside (35)

To compound 34 (7.0 g, 21 mmol, 1.0 eq.) in pyridine (50 mL) was added acetic anhydride (10 mL, 106 mmol, 5.0 eq.). The reaction was kept overnight under stirring. The solvent removed on a rotary evaporator and the residue was subjected to silica chromatography (petrol: ethyl acetate = 5: 2) to afford compound 35 as white solid (9.0 g, 93 %): Rf 0.63 (petroleum: EtOAc = 1: 1); [α] D

20 = + 50.1 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 2960 (νC-Haliph) 1746 cm-1, (νC=O OAc); 1H NMR (400 MHz, CDCl3) δ 1.28 (d, 3H, J 6.8 Hz, CH3), 1.29 (d, 3H, J 6.7 Hz, CH3), 2.01, 2.04, 2.13 (3s, 9H, 3 OCH3), 3.15 (m, 1H, SCH), 3.56 (dd, 1H, J 3.5, 9.6 Hz, H-3), 3.80 (at, 1H, J 6.4 Hz, H-5), 4.14 (d, 2H, J 6.6 Hz, H-6-a&b), 4.40 (d, 1H, J 12.4 Hz, PhCH2-a), 4.47 (d, 1H, J 10.1 Hz, H-1), 4.68 (d, 1H, J 12.4 Hz, PhCH2-b), 5.12 (t, 1H, J 9.8 Hz, H-2), 5.54 (d, 1H, J 3.3 Hz, H-4), 7.21-7.35 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 20.7, 20.8, 20.9 (3 OCH3), 23.7, 24.0 (SCH(CH3)2), 35.4 (SCH), 62.2 (C-6), 66.1 (C-4), 69.0 (C-2), 71.2 (PhCH2), 74.5 (C-5), 76.8 (C-5), 77.6 (C-3), 83.7 (C-1), 127.8, 127.9, 128.4 137.4 (6 x Ar-C), 169.4, 170.4, 170.5 (3C=O); HRMS m/z (ES+) [calculated for C22H30NaO8S+ 477.1554; found 477.1554]. 3-O-benzyl-2, 4, 6-O-tri-O-acetyl-β-D-galactopyranosyl-(1→2)-isopropyl-1-thio-β- D-mannopyranoside (36)

Compound 16 (6.3 g, 17.9 mmol, 1.2 eq.) and compound 17 (7.74 g, 14.4 mmol, 1.0 eq.) were dissolved in CH2Cl2 (80 mL) with activated 4 Å MS. After stirring for 30 min at -30 ºC, TMSOTf (0.12 mL, 0.66 mmol, 0.05 eq.) was added. The reaction was




59

kept for 30 min at -20 to -30 °C and then quenched by the addition of Et3N (0.25 mL). MS was fillered over Celite and the solvent was removed on a rotary evaporator. The residue was subjected to silica chromatography (petrol: ethyl aceate = 7: 2) to afford a dissaccharide, to which was added TFA (27 mL) and Water (3.0 mL) at RT. The reaction was kept for 30 min under stirring. The solvent was then removed on a rotary evaporator at RT. The residue was subjected to silica gel chromatography (petrol: ethyl acetate = 3 : 1) to afford compound 36 as a white solid (2.3 g, 29% in two steps): Rf 0.68 (EtOAc: MeOH = 6: 1); [α] D

20 = + 13.8 (c 1.0, CHCl3); νmax (transmission, thin film)/cm-1 3449 (νO-H), 2960 (νC-Haliph), 1746 cm-1 (νC=O); 1H NMR (400 MHz, CDCl3) δ 1.30 (d, 3H, J 6.8 Hz, CH3), 1.33 (d, 3H, J 6.7 Hz, CH3), 2.09, 2.11, 2.17 (3s, 9H, 3OAc), 3.11 (m, 1H, SCH), 3.33 (m, 1H, H-5II), 3.53 (d, 1H, J 8.6 Hz, H-3II), 3.59 (dd, 1H, J 3.5, 10.1 Hz, H-3I), 3.78 (at, 1H, J 9.6 Hz, H-4II), 3.82-3.92 (m, 3H, H-5I, H-6II), 3.95 (d, 1H, J 1.8 Hz, H-2II), 4.14-4.22 (m, 2H, H-6I), 4.41 (d, 1H, J 12.1 Hz, PhCH2-a), 4.46 (d, 1H, J 8.1 Hz, H-1I), 4.65 (s, 1H, H-1II), 4.68 (d, 1H, J 12.1 Hz, PhCH2-b), 5.12 (at, 1H, , J 8.5 Hz, H-2I), 5.50 (d, 1H, J 3.0 Hz, H-4I), 7.22-7.38 (m, 5H, 5 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 20.6, 20.7, 21.6 (3s, 3OAc), 23.6, 23.9 (2s, SCH(CH3)2), 35.8 (SCH), 61.9 (C-6I), 62.2 (C-6II), 65.7 (C-4I), 68.4 (C-4II), 70.3 (C-2I), 71.5 (C-5I, PhCH2), 74.1 (C-3II), 76.3 (C-3I), 79.8 (C-5II), 82.9 (C-1II), 83.7 (C-2II), 102.5 (C-1I), 127.8, 127.9, 128.4, 137.3 (6 x Ar-C), 170.0, 170.4, 170.9 (3C=O); HRMS m/z (ES+) [calculated for C28H40NaO13S+ 639.2082; found 639.2079]. Isopropyl-2-O-(2,4,6-O-acetyl-3-O-benzyl-β-D-galactopyransyl)-3-O-[2-O-benzyl -3-O-(methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl)-4,6-O-acetyl-α-D-galacto-pyransyl]- 4,6-O-acetyl-1-thio-β-D-mannopyranoside (40)

Compound 19 (1.88 g, 1.2 mmol) was dissolved in TFA (2.7 mL) and water (0.3 ml). After 10 min at RT, the solvent was removed on a rotary evaporator. The residue was subjected to silica gel chromatography (petrol: ethyl acetate = 2:3) to afford the di-ol tetrasaccharide (1.34 g, 0.92 mmol, 1.0 eq.), which was dissolved in pyridine (20 mL). Ac2O (2 mL, 21 mmol, 23 eq.) was added. The reaction was kept overnight under stirring. The solvent was removed on a rotary evaporator and the residue was subjected to silica gel chromatography (petrol: ethyl acetate = 1: 1) to afford compound 40 as a glassy solid (1.4 g, 76 % in two steps): Rf 0.38 (petroleum: EtOAc = 1: 2); [α] D

20 = +10.2 (c 1.0, CHCl3); m.p. 174-176 ˚C; νmax (transmission, thin film)/cm-1 3063, 3028 (νC-Harom), 2960 (νC-Haliph), 1738 cm-1 (νC=O OAc); 1H NMR (400 MHz, CDCl3) δ 1.26 (d, 3H, J 6.8 Hz, SCH(CH3)2), 1.31 (d, 3H, J 6.8 Hz, SCH(CH3)2), 1.48 (s, 3H, COCH3), 2.08, 2.09, 2.10, 2.12, 2.16, 2.17 (6s, 18H, 6 COCH3), 3.05 (m, 1H, SCH), 3.54 (m, 1H, H-5II), 3.59 (s, 3H, COOMe), 3.61 (dd, 1H, J 3.3, 10.4 Hz, H-3I), 3.66 (dd, 1H, J 2.3, 9.4 Hz, H-3II), 3.78 (dd, 1H, J 3.3, 10.1 Hz, H-2III), 3.88 (t, 1H, J 6.3 Hz, H-5I), 4.0-4.17 (m, 4H, H-6II-a & b, H-6III-a, H-6I-a), 4.25-4.40 (m, 4H, H-2II, PhCH2(1)-a, H-6I-b, H-6III-b), 4.42-4.52 (m, 4H, PhCH2(1)-b, PhCH2(2)-a, H-3III, H-5IV), 4.61 (q, 1H, J 3.3, 5.8 Hz, H-3II), 4.63 (s, 1H, H-1II), 4.71




60

(d, 1H, J 12.4 Hz PhCH2(2)-b), 4.84 (d, 1H, J 8.1 Hz, H-1I), 4.91 (d, 1H, J 3.5 Hz, H-1III), 5.15 (at, 1H, J 8.7 Hz, H-4II), 5.20 (dd, 1H, J 8.3, 10.1 Hz, H-2I), 5.26 (d, 1H, J 7.8 Hz, H-1IV), 5.53-5.61 (m, 3H, H-4I, H-2IV, H-4III), 5.64 (at, 1H, J 9.7 Hz, H-4IV), 5.92 (at, 1H, J 9.5 Hz, H-3IV), 6.90-7.98 (m, 25H, 25 x Ar-H); 13C NMR (100 MHz, CDCl3) δ 20.2, 20.6. 20.7, 20.8, 20.9, 21.0, 21.1 (7COCH3), 23.2, 23.9 (2 CH3 of SCH(CH3)2), 37.2 (SCH), 52.5 (COOMe), 61.9, 63.4, 64.2 (C-6I, C-6II, C-6III), 66.1 (C-4I), 67.4 (C-5III, C-4II), 70.1, 70.3, 70.4 (C-2I, C-4IV, C-5I and PhCH2(1)), 71.4, 71.9, 72.4, 72.6, 73.8, 75.0, 76.3, 76.9, 79.9, 82.6 (C-1II), 99.4 (C-1I), 100.3 (C-1IV), 100.6 (C-1III), 127.0, 127.3, 127.7, 128.2, 128.3, 128.4, 128.5, 128.8, 128.9, 129.0, 129.7, 129.8, 133.2, 133.3, 137.7, 138.1, (30 x Ar-C), 165.1, 165.2, 165.5, 167.0, 169.3, 169.6, 170.4, 170.6, 170.7, 170.8 (11 C=O); HRMS m/z (ES+) [calculated for C77H86NaO31S+ 1561.4766; found 1561.4741]. 1-«2-O-‹2,4,6-O-acetyl-3-O-{isopropyl-2-O-(2,4,6-O-acetyl-β-D-galactopyran-syl)-3-O-[2-O-benzyl-3-O-(methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl)-4,6-O-acetyl-α-D-galacto-pyransyl]-4,6-O-acetyl-1-thio-β-D-mannopyranosyl}-α-D-mannopyranosyl›-3-O-[(methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl)-4,6-O-acetyl-α-D-galacto-pyransyl]-4,6-O-acetyl-α-D-mannopyranosyl»-1H-pyrrol-2,5-dione (protected K30-2n=2)

Compound 6 (30 mg, 22 µmol, 1.0 eq.) and NIS (6 mg, 26.4 µmol, 1.2 eq.) were dried in vacuo overnight in separate 5 mL flasks. CH2Cl2 with activated 4 Å MS (1 mL) was added individually into the two flasks. After stirring for half an hour at RT, TMSOTf (1 µL, 5.5 µmol) was added into the NIS flask. The flasks were immersed into acetone-dry ice mixture at -30 ˚C. The suspension of NIS in CH2Cl2 with activated 4 Å MS was transferred via canula into the flask of 6 to initiate the reaction. After maintaining temperature of the reaction flask at -30 ˚C for 3 hours, Et3N (10 µL) in CH2Cl2 (0.5 mL) with activated 4 Å MS was added to quench the reaction at -30 ℃. MS was filtered and the solution was diluted with CH2Cl2 (40 mL). Aqueous Na2S2O3 solution (10 %, 50 mL) was added under turbulent stirring. After 20 min, the lower organic layer was separated, dried over MgSO4 and filtered. The solvent was removed on a rotary evaporator to give a crude oligomerisation product. HPLC (Supplementary Fig. S7) was used to isolate one major octasaccharide (protected K30-2n=2, 1mg, 3%) and one major dodecasaccharide (protected K30-2n=3, 0.5 mg, 1%) from the mixture. Specifically, the fraction eluted at retention time 8.1 min in Supplementary Fig. S7a gave octasaccharide protected K30-2n=2 as a white solid (1 mg, 3 %) and the fraction eluted at retention time 11.0 min in Supplementary Fig.




61

S7a and then at retention time 13.6 min in Supplementary Fig. S7b gave dodecasaccharide protected K30-2n=3 as a white solid. protected K30-2n=2: Rf 0.28 (petroleum: EtOAc = 1: 4); [α] D

20 = +18.0 (c 0.05, CHCl3); νmax (ATR)/cm-1 3416 (νO-H), 2960 (νCOO-H), 1736 (νC=O), 1371 (νC-H),1070 (νC-N); 1H NMR (500 MHz, CDCl3) δ 1.94, 2.01, 2.03, 2.04, 2.05, 2.06, 2.07, 2.10, 2.12, 2.13, 2.18, 2.43 (12s, 42H, 14 COCH3), 2.78 (m, 4H, COCH2CH2CO), 3.53 (OCH3), 3.62-3.80 (m, 7H, H-2III(1), H-3II(1), H-5II(2), H-5III(2), OCH3), 3.82-3.87 (m, 2H, H-3I(2), H-3I(1)), 3.87-4.34 (m, 20H, H-2II(1), H-2III(2), H-3II(2), H-5I(2), H-5II(1), H-6I(1)a&b, H-6II(1)a&b, H-6III(1)a&b, H-6I(2)a&b, H-6II(2)a&b, H-6III(2)a&b), 4.33-4.40 (m, 5H, H-5IV(1), H-5IV(2), H-5III(1), H-5I(1), H-5II(1)), 4.49 (d, 1H, J 8.0 Hz, H-1I(2)), 4.65 (d, 1H, J 7.7 Hz, H-1I(1)), 4.83 (d, 1H, J 3.8 Hz, H-1III(1)), 4.87 (m, 1H, H-2I(2)), 4.98 (dd, 1H, J 7.9, 10.4 Hz, H-2I(1)), 5.09 (m, 2H, H-1III(2), H-4II(1)), 5.14 (s, 1H, H-1II(1)), 5.33 (d, 1H, J 3.1 Hz, H-4I(2)), 5.39 (d, 1H, J 7.6 Hz, H-1IV(2)), 5.40-5.46 (m, 4H, H-4I(1), H-2IV(1), H-4II(2), H-4III(1)), 5.47-5.59 (m, 5H, H-2IV(2), H-4IV(1), H-4III(2), H-1II(2), H-1IV(1)), 5.67 (t, 1H, J 9.6 Hz, H-4IV(2)), 5.93 (m, 2H, H-3IV(1), H-3IV(2)), 7.22-7.55 (m, 30 H, 30 x Ar-H); 13C NMR (125 MHz, CDCl3) δ 20.3, 20.4, 20.5, 20.6, 20.63, 20.65, 20.68, 20.72, 20.78, 20.84, 20.89, 20.93 (14 COCH3), 27.8 (COCH2CH2CO), 52.7 (2 OCH3), 60.6, 60.8, 61.1, 61.6, 62.1, 62.2 (C-6I(1), C-6 II(1), C-6 III(1), C-6 I(2), C-6 II(2), C-6 III(2)), 64.8 (C-4I(2)), 66.90, 66.93, 66.97, 67.7 (C-4II(1)), 68.3, 68.5, 68.67, 68.70, 68.78, 69.0, 69.21, 69.22, 69.9, 70.2, 70.4, 70.7, 71.11, 70.13, 71.19, 71.21, 71.26, 71.59, 72.02, 72.06, 72.13, 72.78, 73.21, 73.5, 74.3, 75.5, 76.0, 92.6 (C-1II(1)), 97.2 (C-1I(1)), 98.4 (C-1II(2), C-1III(2)), 99.8 (C-1IV(1)), 100.6 (C-1IV(2)), 100.7 (C-1III(1)), 100.9(C-1I(2)), 128.1, 128.29, 128.32, 128.38, 128.43, 128.48, 128.50, 128.79, 128.81, 128.84, 128.88, 129.1, 129.4, 129.7, 129.8, 129.8, 133.13, 133.22, 133.27, 133.33, 133.38 (36 x Ar-C), 165.07, 165.08, 165.3, 165.4, 165.5, 165.6, 167.1, 168.4, 168.7, 169.8, 169.9, 170.0, 170.3, 170.43, 170.45, 170.54, 170.57, 170.60, 170.7,171.0, 171.5 (22 CO), 175. 6 (COCH2CH2CO); HRMS m/z (ES+) [calculated for C124H137NNaO64

+ 2886.7389; found 2686.8305]. Methyl 2,3,4-tri-O-benzoyl-β-D-glucuronosyl (1→3)-2-O-benzyl-4,6-acetyl-β-D-galactopyransyl-(1→3)-4,6-O-benzylidene-α-D-mannopyranosyl-(1→3)-isopro-pyl-2-O-acetyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (3β)

4 (40 mg, 40 µmol, 1.0 eq.) and 5 (24 mg, 40 µmol, 1.0 eq.) were dissolved in anhydrous CH2Cl2 (2 mL) with activated 4 Å MS (MS) under an argon atmosphere. The solution was cooled to -20 ˚C. BF3⋅Et2O (2 µL, 16 µmol, 0.4 eq.) was added. The mixture was stirred under these conditions for 30 min. The reaction was quenched by the addition of Et3N. The solvent was evaporated on a rotary evaporator and the residue was subjected to silica chromatography (Petroleum : Ethyl acetate = 1 : 1.8) to afford the tetrasaccharide 3β as a white solid (46 mg, 82%): Rf 0.35 (petroleum: EtOAc = 1: 2); νmax (transmission, thin film)/cm-1 3385 (νO-H), 3063, 3028 (νC-Harom), 2924 (νC-Haliph), 1738 cm-1 (νC=O OAc); 1H NMR (500 MHz, MeOD) δ 1.30 (d, 3H, J 7.3 Hz, CH3), 1.38 (d, 3H, J 6.6 Hz, CH3), 1.95, 2.07, 2.12, (3s, 9H, 3CH3), 3.28 (m, 1H, SCH), 3.54 (m, 1H, H-2III), 3.63 (s, 1H, H-5I), 3.67 (s, 3H, OCH3), 3.75 (m, 1H, H-5III), 3.79-3.88 (m, 3H, H-6aIII, H-4II,H-6II), 3.96 (brs, 1H, H-2II), 4.02-4.11 (m, 5H, H-3II, H-5II, H-6III, H-3III, H-3I), 4.13 (d, 1H, J 12.9 Hz, H-6aI), 4.20-4.27 (m, 2H, H-




62

6I, H-6II), 4.40 (d, 1H, J 12.6 Hz, CH2Ph-a), 4.53 (d, 1H, J 3.1 Hz, H-4I), 4.62 (d, 1H, J 7.6 Hz, H-1III), 4.71 (d, 1H, J 11.0 Hz, CH2Ph-b), 4.75 (d, 1H, J 10.1 Hz, H-1I), 5.08 (s, 1H, H-1II), 5.29 (t, 1H, J 9.8 Hz, H-2I), 5.38 (d, 1H, J 7.9 Hz, H-1IV), 5.42 (at, 1H, J 4.1 Hz, H-4III), 5.45 (at, 1H, J 8.2 Hz, H-2IV), 5.59 (at, 1H, J 8.3 Hz, H-4IV), 5.61 (s, 1H, PhCH), 5.93 (t, 1H, J 9.6 Hz, H-3IV), 7.10-8.00 (m, 30 H, 30 x Ar-H); 13C NMR (125 MHz, MeOD) δ 20.8, 21.5 (2 CH3), 24.2, 25.0 (2 CH3), 36.0 (SCH), 53.3 (OCH3), 63.2 (C-6III), 65.6 (C-5II), 69.7 (C-2I), 69.8 (C-6II), 69.8 (C-2II), 70.4 (C-6I), 70.9 (C-4III), 71.1 (C-5I), 71.7 (C-4IV), 72.1 (C-5III), 72.8 (C-2IV), 73.2 (C-2IV), 73.5 (C-5IV), 73.8 (C-3IV), 74.8, 76.3, 78.7, 79.4 (C-3II, C-5II, C-3I, C-3III), 76.0 (CH2Ph), 80.2 (C-2III), 84.1 (C-1I), 97.7 (C-1II), 101.6 (C-1IV), 102.1 (C-1III), 102.1, 103.3 (2 PhCH), 127.5, 127.6, 128.7, 129.0, 129.2, 129.4, 129.5, 129.6, 129.7, 130.0, 130.6, 130.7, 134.6, 134.7, 134.8, 139.1, 139.5, 139.6 (36 x Ar-C), 166.5, 166.6, 167.0, 169.1, 171.4, 172.2, 172.4 (7 CO); HRMS (M+Na)+ calcd. 1479.4500; found 1479.4559.




63

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16 Coutant, C. & Jacquinet, J.-C. 2-Deoxy-2-trichloroacetamido-D-glucopyranose derivatives in oligosaccharide synthesis: from hyaluronic acid




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to chondroitin 4-sulfate trisaccharides. J. Chem. Soc., Perkin Trans. 1, 1573-1581 (1995).

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Compound K30-1n=1 1H spectrum




Compound K30-1n=1 13C spectrum




Compound K30-2n=2 1D TOCSY spectra




Compound K30-2n=2 HSQC spectrum




HSQC alignment of compound K30-2n=2 (black) and K30-2n=3 (red)




Compound 3 1H spectrum




Compound 3 13C spectrum




Compound protected K30-2n=2 1H spectrum




Compound protected K30-2n=2 13C spectrum




Compound 3β 1H spectrum




Compound 3β 13C spectrum


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