Syntheses of the 3- and 4-thio analogues of 4-nitrophenyl 2-acetamido-2-deoxy-β-d-gluco- and galactopyranoside

Carbohydrate Research 342 (2007) 2212–2222

Syntheses of the 3- and 4-thio analogues of 4-nitrophenyl

2-acetamido-2-deoxy-b-^D-gluco- and galactopyranoside

Hong-Ming Chen and Stephen G. Withers^*

Department of Chemistry, The University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada V6T 1Z1

Received 20 February 2007; received in revised form 16 May 2007; accepted 21 May 2007

Available online 2 June 2007

Abstract—The syntheses of 4-nitrophenyl b-glycosides of the 3-thio and 4-thio analogues of the two principal 2-acetamido-2-deoxy-

hexoses found in living systems, GlcNAc and GalNAc, are described. While synthesis of the 4-thio analogues could be achieved via

nucleophilic displacements of sulfonate derivatives with thioacetate, problems with neighbouring group acetamido participation

necessitated the use of sulfamidate intermediates for the 3-thio analogues. These 3- and 4-thio analogues are employed in the

chemo-enzymatic synthesis of thio-oligosaccharide analogues of structures present in glycosaminoglycans, glycoproteins and

glycolipids.

Keywords: 4-ThioGlcNAc; 4-Thio-GalNAc; 3-ThioGlcNAc; 3-ThioGalNAc; Sulfamidate; Nucleophilic displacement; Thioglycosides; Glycosidase

inhibitors

1. Introduction

has been an increasing interest in the use of enzymatic

coupling approaches. However, regular glycosyl trans-

ferases are only rarely found to be capable of forming

thioglycosides and then only slowly.⁷Likewise glycosi-

dases and glycosynthases are also unhelpful. However

a new class of mutant glycosidases that selectively

assemble thioglycosides was recently unveiled: the thio-

glycoligases.^8–13In addition, eﬀorts to evolve glycosyl

transferases to synthesise thioglycosides are also bearing

fruit.¹⁴Thus the way is now open to the assembly of

thioglycosides on the surface of complex biomolecules.

Such strategies still, however, require the chemical

synthesis of thiosugar acceptors. In order to apply this

approach to the synthesis of thioglycoside analogues

of glycosaminoglycans, gangliosides and glycoproteins,

the 3- and 4-thio analogues of N-acetyl-^D-glucosamine

and N-acetyl-^D-galactosamine were required. More spe-

ciﬁcally, analogues containing an aryl substituent at the

anomeric centre were needed so that the binding of these

thiosugar acceptors to the thioglycoligases was opti-

mised, thus facilitating the monitoring of reactions and

puriﬁcations by TLC and HPLC. The choice of a 4-

nitrophenyl derivative in particular was dictated by its

proven value in thioglycoligase and glycosynthase

Oligosaccharide analogues that are resistant towards

enzymatic hydrolysis have proved to be very useful as

glycosidase inhibitors and have found application in

studies of enzyme mechanisms especially in the crystal-

lographic analysis of complexes of glycosidases with

substrate analogues. Particularly useful in this regard

have been thioglycosides: such sulfur analogues are

otherwise tolerated by most biological systems and are

also less susceptible to acid-catalysed hydrolysis.^1,2They

therefore have potential as therapeutics, especially in the

form of stable antigens for use as glycoconjugate

vaccines, or as more metabolically stable versions of

therapeutic glycoproteins.^3–6The synthesis of such thio-

glycosides typically requires, as a ﬁrst step, the synthesis

of the appropriate thio acceptor sugar, which is then

coupled with the appropriate glycosyl donor.¹Since

such chemical couplings are not compatible with the

assembly of thioglycosides on protein surfaces, there

*

Corresponding author. Tel.: +1 604 822 3402; fax: +1 604 822

8869; e-mail: withers@chem.ubc.ca

doi:10.1016/j.carres.2007.05.023

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

2213

couplings¹⁵and by the fact that the 4-nitrophenyl

deoxythio-N-acetyl-^D-hexosaminides generated could

also serve as substrates for the assay and screening of

hexosaminidases capable of tolerating a sulfur substitu-

ent at their 3- or 4-positions. In addition, the disaccha-

ride thioglycosides obtained after enzymatic coupling

might then serve as valuable substrates for the assay

or screening of endo-hexosaminidases.

The replacement of a sugar hydroxyl by a thiol is typ-

ically achieved by nucleophilic displacement of activated

alcohols of the opposite stereochemistry to that required

in the thiosugar product, usually via the intermediacy of

sulfonate derivatives.^1,16,17Such an approach proved

suitable, in our hands, for the synthesis of the 4-substi-

tuted derivatives. However, it was considerably compli-

cated by neighbouring group participation in the case of

substitution at the 3-position of N-acetyl-^D-glucosamine

and N-acetyl-^D-galactosamine, resulting in the incorrect

products. The use of cyclic sulfamidates as temporary

protecting/activating groups nicely obviates these prob-

lems and provided good access to the desired ana-

logues.^18–20

indeed involved incorporation of the 4-nitrophenyl

group at the beginning and then use of synthetic routes

compatible with this entity. The 4-nitrophenyl glyco-

sides of N-acetyl-^D-glucosamine and N-acetyl-D-galac-

tosamine were both synthesised via the initial

formation of the acylated glycosyl chloride using acetyl

chloride, then direct glycosylation with 4-nitrophenol

and anhydrous K₂CO₃in acetone, followed by deprotec-

tion under Zemplen conditions. This approach²¹is quite

straightforward and scalable and gives reasonable yields

of product, though somewhat diminished for the galac-

to-derivative by the surprising water solubility of the

protected galactosyl chloride intermediate.

2.1. Syntheses of 4-thio-N-acetylhexosaminides

Syntheses of the two derivatives in which the 4-position

was substituted by sulfur were achieved from the 4-

nitrophenyl glycoside of opposite conﬁguration at C-4

via diﬀerential protection of the 3- and 6-hydroxyls with

ester protecting groups, activation as the triﬂate and dis-

placement using potassium thioacetate. Final deprotec-

tion was performed under Zemplen conditions with

the inclusion of dithiothreitol (DTT) during workup

while working under reasonably oxygen-free conditions

to avoid the otherwise rapid oxidation to disulﬁde

derivatives.

2. Results and discussion

Due to the perceived limitations in reagents and ap-

proaches imposed by the presence of a 4-nitrophenyl

group at the anomeric centre prior to installation of

the thiol group, initial attempts to synthesise the 3-

and 4-thio analogues of N-acetyl-^D-glucosamine and

N-acetyl-^D-galactosamine were based upon the use of

an allyl protecting group at the anomeric centre during

thiol substitution, with subsequent replacement of the

allyl by the desired 4-nitrophenyl group. Unfortunately

this led to considerable complications, particularly that

of elimination chemistry for the 3-thiosugars in their

hemiacetal forms. In fact the strategy that was successful

2.2. Synthesis of 4-nitrophenyl 2-acetamido-2-deoxy-4-

thio-b-^D-galactopyranoside (8)

N-Acetyl-^D-glucosamine was reacted with acetyl chlo-

ride at room temperature to give a mixture of the fully

acetylated a-chloride (2) and a-per-O-acetate (3) of Glc-

NAc in a ratio of about 3:2 (¹H NMR) (Scheme 1). This

mixture was used directly for the glycosylation reaction

with 4-nitrophenol and anhydrous K₂CO₃in HPLC-

grade acetone, and subsequently deacetylated to give 5

OH

OAc

O

1

AcO

HO

AcO

2

O

AcO

OpNP

OH

+

NHAc

AcHN

1

NHAc

3

Cl

4

OAc

2

3

SH

OH

SAc

OPiv

OH

O

HO

PivO

O

6

OpNP

5

OpNP

HO

4

OpNP

PivO

HO

NHAc

8

NHAc

6

7

5

Scheme 1. Reagents and conditions: (1) AcCl, rt. (2) K₂CO₃, acetone, 4-nitrophenol, 55 ꢁC; 44% (two steps). (3) CHCl₃, CH₃OH, NaOCH₃, 97%. (4)

CH₂Cl₂, pyridine, pivaloyl chloride, 0 ꢁC, 91%. (5) CH₂Cl₂, pyridine, Tf₂O, 0 ꢁC; DMPU, KSAc, rt; 77%; (6) CH₃OH, NaOCH₃; DTT, H₂O; 37%.

2214

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

in 43% overall yield.²¹Selective protection of the hydro-

xyl groups at C-3 and C-6 was achieved using trimethyl-

acetyl (pivaloyl) chloride at 0 ꢁC to yield the required

partially protected intermediate 6.⁸The introduction

of sulfur at C-4 was performed via a nucleophilic dis-

placement on the triﬂate derivative of compound 6

(without its isolation) using potassium thioacetate. The

ester protecting groups were removed using sodium

methoxide in methanol and the disulﬁde formed be-

tween 2 equiv of product was reduced with DTT to give

the desired product, which was fully characterised. The

overall yield of the ﬁnal steps was unfortunately com-

promised due to diﬃculties in the removal of the piva-

loyl groups.

The equivalent reaction sequence, but starting from

N-acetyl-^D-galactosamine, was attempted as the logical

route to the synthesis of 4-nitrophenyl 2-acetamido-2-

deoxy-4-thio-b-^D-glucopyranoside. While the thioace-

tate was successfully introduced at C-4, all attempts at

deprotection, sadly, were unsuccessful, hence the follow-

ing route.

subsequent deprotection were accomplished using the

same procedures as those used for the preparation of

the 4-thio-GalNAc derivative.

2.4. Synthesis of 4-nitrophenyl 2-acetamido-2-deoxy-3-

thio-b-^D-glucopyranoside (20)

As noted in Section 1, due to severe problems with

neighbouring group participation from the 2-acetamide,

resulting in the formation of a 2,3-oxazoline, a com-

pletely diﬀerent synthetic strategy was used for the syn-

thesis of 4-nitrophenyl 2-acetamido-2-deoxy-3-thio-b-^D-

glucopyranoside 20. This strategy used a cyclic sulfami-

date as a temporary protecting/activating group, allow-

ing the introduction of the thioacetate at C-3 via a

nucleophilic displacement.^18–20The main problems

encountered with this approach related to the solubili-

ties of compounds 14–18, which were found to be poor

in any organic solvents except Me₂SO, DMF and pyri-

dine. Fortunately, most of the intermediates could be

precipitated by the addition of other organic solvents,

and the impurities remained in the mother liquor, mak-

ing this, ultimately, an advantage. Protection of the 4-

and 6-hydroxyls of 4-nitrophenyl N-acetyl-b-^D-glucos-

aminide 5 with a benzylidene gave 14 in which only

the 3-hydroxyl was unprotected.²⁵Reaction of the mes-

ylate of 14 with sodium acetate in reﬂuxing methoxyeth-

anol and water gave the allo-derivative 15,^18,26which

was reacted with 1,1⁰-sulfonyldiimidazole and sodium

hydride in dry THF, followed by acetylation to aﬀord

the acylated sulfamidate 17. Introduction of the thio-

acetate at C-3 by nucleophilic displacement gave com-

pound 18, which was precipitated by the addition of

acetone and petroleum ether. This product contained a

trace of impurity that was very diﬃcult to separate from

the desired compound 18. In order to further purify

compound 18 the benzylidene protecting group was

hydrolysed in acetic acid and water, and the resulting

residue was acetylated to give 19. Compound 19 was sol-

uble in most polar solvents, and easily puriﬁed by ﬂash

2.3. Synthesis of 4-nitrophenyl 2-acetamido-2-deoxy-4-

thio-b-^D-glucopyranoside (13)

Due to the aforementioned diﬃculty in removal of piva-

loyl protecting groups, an alternative strategy to the syn-

thesis of thiosugar 13 that avoids the use of pivaloyl

groups was developed (Scheme 2). 4-Nitrophenyl N-

acetyl-^D-glucosaminide 5 was temporarily protected at

the 4- and 6-positions using an isopropylidene group,

the free 3-OH, was benzoylated and then the isopropyl-

idene hydrolysed to give the mono-O-benzoyl derivative

9.²²Selective benzoylation of the primary hydroxyl of 9

was performed by reaction with benzoyl cyanide in

methylene chloride and pyridine to give the di-O-ben-

zoyl derivative 10.²³Inversion of stereochemistry at

the 4-hydroxyl was achieved by treatment of the triﬂate

of 10 with NaNO₂in DMF to give the dibenzoyl galac-

to-derivative 11.²⁴Introduction of the thiol at C-4 and

OBz

OH

2

O

1

O

HO

BzO

HO

BzO

HO

OpNP

NHAc

10

5

9

3

HO

OH

OBz

O

HS

HO

4

5

AcS

BzO

OpNP

BzO

NHAc

13

12

11

Scheme 2. Reagents and conditions: (1) DMF, 2,2-dimethoxypropane, 4-TsOH, rt; pyridine, benzoyl chloride, 0 ꢁC; AcOH, H₂O, 70 ꢁC; 84% (three

steps). (2) CH₂Cl₂, pyridine, benzoyl cyanide, rt, 86%. (3) CH₂Cl₂, pyridine, Tf₂O, 0 ꢁC; DMF, NaNO₂, rt; 79%. (4) CH₂Cl₂, pyridine, Tf₂O, 0 ꢁC;

DMPU, KSAc, rt; 50%. (5) CH₃OH, NaOCH₃; DTT, H₂O; 76%.

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

2215

OH

O

Ph

O

OpNP

O

OpNP

HO

1

O

2

O

HO

NHAc

5

OH

14

15

3

O

Ph

O

Ph

O

OpNP

O

Ph

OpNP

O

OpNP

NHAc

O

4

5

AcS

O

NH

NAc

18

S

17

16

O

6

OAc

OH

O

OpNP

NHAc

7

AcO

AcS

OpNP

NHAc

HO

HS

19

20

Scheme 3. Reagents and conditions: (1) DMF, benzaldehyde dimethyl acetal, 4-TsOH, 50 ꢁC, 96%. (2) CH₂Cl₂, pyridine, MsCl; 2-methoxyethanol,

H₂O, NaOAc, reﬂuxed; 63%. (3) THF, NaH, 1,1⁰-sulfonyldiimidazole, 82%. (4) CH₂Cl₂, pyridine, AcCl, 80%. (5) DMF, KSAc; THF, H₂O, H₂SO₄;

84%. (6) AcOH, H₂O, 60 ꢁC; pyridine, Ac₂O, rt; 81%. (7) CH₃OH, NaOCH₃, rt, 100%.

column chromatography. Deprotection of the fully acet-

ylated thiosugar 19 was easily performed under Zemplen

conditions, with no complication from disulﬁde forma-

tion (Schemes 3 and 4).

nucleophilic displacement reaction of 16 was complete

in a matter of hours at room temperature, when the sus-

pension of 30 and potassium thioacetate was stirred in

DMF overnight at room temperature, no product was

detected. Raising the reaction temperature above

100 ꢁC resulted, instead, in displacement of the 4-nitro-

phenyl substituent from the anomeric centre to give the

1-thioacetate. The best temperature discovered for this

reaction was around 65 ꢁC, though this still yielded

some by-products, which were diﬃcult to separate from

31. Consequently, crude 31 was hydrolysed and acety-

lated to give compound 32 and the per-O-acetate of

1,3-dithio-GalNAc, which were readily puriﬁed by ﬂash

column chromatography. The desired product 33 was

then obtained by standard Zemplen deacetylation with-

out signiﬁcant disulﬁde formation.

2.5. Synthesis of 4-nitrophenyl 2-acetamido-2-deoxy-3-

thio-b-^D-galactopyranoside (33)

The same overall strategy as that used for the 3-thio-

GlcNAc derivative, involving a sulfamidate intermedi-

ate, was applied in this sequence. Apart from the

previously mentioned surprising water solubility of the

acylated GalNAc chloride 23, the only surprise was in

the unreactive nature of the mesylate and tosylate deriv-

atives of 26. They were found to be so stable that no

reaction occurred, even upon reaction with sodium ace-

tate in methoxyethanol under reﬂux for two days. Con-

sequently triﬂate activation was employed, the triﬂate

derivative 27 proving to be surprisingly stable. Indeed

it was puriﬁed by ﬂash column chromatography and

then only slowly reacted with NaNO₂in DMF over

4 days to give compound 28. There is clearly a large dif-

ference of reactivity of these triﬂates since, in the case of

the gluco-derivative, the triﬂate had to be used directly

in the next step. However, the origin of this reactivity

diﬀerence is unclear.

3. Experimental

3.1. General

All chemicals were obtained from the Sigma Chemical

Co. unless otherwise noted. Methylene chloride and pyr-

idine were dried over CaH₂and distilled prior to use.

˚

DMF was dried over 4 A molecular sieves. MeOH was

Compound 30 was prepared from 28 in a similar man-

ner to that described for 16, but the reactivity of these

two compounds was again very diﬀerent. While the

dried over magnesium and distilled prior to use. Analyt-

ical thin-layer chromatography (TLC) was performed

on aluminium-backed sheets of Silica Gel 60F₂₅₄(E.

2216

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

OAc

OH

OAc

O

1

O

2

O

OpNP

+

AcO

HO

AcO

OH

AcO

AcHN

NHAc

AcHN

OAc

Ph

NHAc

Cl

21

22

23

24

Ph

3

O

OH

O

OH

O

5

4

OpNP

O

OpNP

HO

TfO

NHAc

25

27

26

Ph

6

Ph

O

OpNP

7

O

8

OpNP

NH

O

NAc

O

NHAc

S

OH

30

28

29

Ph

9

O

HO

O

OH

OAc

O

OpNP

NHAc

11

O

OpNP

10

OpNP

HS

AcS

NHAc

33

32

31

Scheme 4. Reagents and conditions: (1) AcCl, rt. (2) Acetone, K₂CO₃, 4-nitrophenol, 50 ꢁC; 29% (two steps). (3) CH₃OH, NaOCH₃, rt, 100%. (4)

DMF, 4-TsOH, benzaldehyde dimethyl acetal, 50 ꢁC, 92%. (5) CH₂Cl₂, pyridine, Tf₂O, 0 ꢁC. (6) DMF, NaNO₂, rt, 4 days; 50% (two steps). (7) THF,

NaH, 1,2⁰-sulfonyldiimidazole, rt, 79%. (8) CH₂Cl₂, pyridine, AcCl, rt, 98%. (9) DMF, KSAc, 65 ꢁC, 85%. (10) AcOH, H₂O, 70 ꢁC, 67%. (11)

CH₃OH, NaOCH₃, 98%.

Merck) of thickness 0.2 mm. The plates were visualised

using UV light (254 nm) and or/ by exposure to 10%

ammonium molybdate in 2 M H₂SO₄followed by char-

ring. Flash column chromatography was performed

using silicycle Silicagel 60. The NMR spectra were re-

corded at Bruker AV-400 MHz or AV-300 MHz spec-

trometers. The optical rotations were recorded on a

JASCO P-1010 Polarimeter. Elemental analysis was car-

ried out at the University of British Columbia microan-

alytical laboratory. Mass spectra were carried out by

using a PE-Sciex API 300 triple quadrupole mass spec-

trometer equipped with an electrospray ionisation

(ESI) ion source.

(7.5 mL) was added dropwise trimethylacetyl chloride

(1.0 mL, 8.12 mmol, 2.9 equiv) at 0 ꢁC with stirring.

The reaction mixture was stirred for a further period

of 2 h at 0 ꢁC, quenched with MeOH, the solvent evap-

orated under diminished pressure and the residue redis-

solved in CH₂Cl₂(100 mL). The soln was washed with

satd aq NaHCO₃and brine, dried over MgSO₄, ﬁltered

and concentrated. The resulting residue was puriﬁed by

ﬂash column chromatography (4:1 and 3:1 petroleum

ether–acetone) to give 6 as white crystals in 92% yield

22

(1.3 g). ½aꢀ_Dꢁ33.7 (c 1.0, acetone). ¹H NMR

(400 MHz, CDCl₃): d 8.11 (m, 2H, Ar–H), 7.09 (m,

2H, Ar–H), 6.34 (d, 1H, J 4.4 Hz, NHAc), 5.45 (d,

1H, J_1,28.2 Hz, H-1), 5.40 (dd, 1H, J_2,310.0, J_3,4

9.5 Hz, H-3), 4.44 (dd, 1H, J_5,6a1.9, J_6a,6b12.2 Hz, H-

6a), 4.27 (dd, 1H, J_5,6b6.5 Hz, H-6b), 4.14 (dd, 1H,

H-2), 3.93 (m, 1H, H-5), 3.58 (dd, 1H, J_4,59.3 Hz, H-

4), 1.88 (s, 3H, CH₃CO), 1.20 [s, 9H, (CH₃)₃CCO],

1.18 [s, 9H, (CH₃)₃CCO]. ¹³C NMR (100 MHz, CDCl₃):

d 179.6, 179.1, 170.5 (C@O); 161.7, 142.8, 125.6, 116.5

3.2. Preparation of 4-nitrophenyl 2-acetamido-2-deoxy-4-

thio-b-^D-galactopyranoside (8)

3.2.1. 4-Nitrophenyl 2-acetamido-2-deoxy-3, 6-di-O-piva-

loyl-b-^D-glucopyranoside (6). To a soln of 5²¹(0.95 g,

2.76 mmol) in dry CH₂Cl₂(4.7 mL) and dry pyridine

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

2217

(Ar–C); 97.8, 74.3, 74.1, 69.4, 63.5, 54.1; 39.0, 38.9

[(CH₃)₃CCO]; 27.1, 27.0 [(CH₃)₃CCO]; 23.2 (CH₃CO).

HRESIMS: calcd for [C₂₄H₃₄N₂O₁₀+Na]⁺: 533.2111.

Found m/z: 533.2103.

5.16 (d, 1H, J_1,28.3 Hz, H-1), 4.26 (dd, 1H, J_2,3

10.3 Hz, H-2), 4.00–3.70 (m, 4H, H-3, H-5, H-6a and

H-6b), 3.48 (dd, 1H, J_3,44.1 Hz, J_4,51.3 Hz, H-4), 1.97

(s, 3H, CH₃CO). ¹³C NMR (100 MHz, CD₃OD): d

174.2 (C@O), 163.6, 144.1, 126.7, 117.7 (Ar–C); 100.9,

76.8, 71.9, 63.4, 53.9, 45.4 (C-4); 23.0 (CH₃CO). HRE-

SIMS: calcd for [C₁₄H₁₈N₂O₇S+Na]⁺: 381.0732. Found

m/z: 381.0721. Anal. Calcd for C₁₄H₁₈N₂O₇S: C, 46.92;

H, 5.06; N, 7.82. Found: C, 46.84; H, 5.07; N, 8.22.

3.2.2. 4-Nitrophenyl 2-acetamido-4-S-acetyl-2-deoxy-3,6-

di-O-pivaloyl-4-thio-b-^D-galactopyranoside (7). A soln

of 6 (8.45 g, 16.57 mmol) in dry CH₂Cl₂(100 mL) and

dry pyridine (27 mL) was stirred and cooled to 0 ꢁC

under argon, to which was added dropwise triﬂuoro-

methanesulfonic anhydride (6.0 mL, 35.68 mmol,

2.2 equiv). The reaction mixture was stirred for 1 h at

0 ꢁC, diluted with CH₂Cl₂(100 mL), washed with icy

1 M HCl, satd aq NaHCO₃and brine, dried over

MgSO₄, ﬁltered, concentrated and dried under dimin-

ished pressure to give a brown foam. To the brown foam

were added DMPU (40 mL) and KSAc (5.6 g,

49.12 mmol, 3 equiv), and the mixture was stirred for

1.5 h under argon at rt, diluted with mixed solvents

(350 mL, 1:1 EtOAc–Et₂O). The organic phase was

washed with water (5 · 200 mL), dried over MgSO₄, ﬁl-

tered and concentrated. The resulting residue was puri-

ﬁed by ﬂash column chromatography (4:1 petroleum

ether–acetone) to aﬀord a brown solid, which was

recrystallised from ethanol to give yellowish product 7

3.3. Preparation of 4-nitrophenyl 2-acetamido-2-deoxy-4-

thio-b-^D-glucopyranoside (13)

3.3.1. 4-Nitrophenyl 2-acetamido-3-O-benzoyl-2-deoxy-b-

D-glucopyranoside (9). To a soln of compound 5

(5.65 g, 16.52 mmol) in dry DMF (65 mL) were added

4-toluenesulfonic acid monohydrate (60 mg) and

dimethoxypropane (8 mL, 65.15 mmol, 3.9 equiv). The

reaction mixture was stirred overnight at rt, neutralised

with Amberlite IR-45 (OH) ion exchange resin, ﬁltered,

evaporated and dried under diminished pressure to give

a residue. To the residue were added dry pyridine

(60 mL) and benzoyl chloride (6 mL, 51.66 mmol,

3.1 equiv) at 0 ꢁC, and the mixture was stirred for

1.5 h at 0 ꢁC. The mixture was poured into icy water,

stirred for 0.5 h, and extracted with CHCl₃

(3 · 200 mL). The organic phase was washed with icy

1 M HCl, satd aq NaHCO₃and brine, then evaporated.

To the resultant syrup were added acetic acid (70 mL)

and water (70 mL), and the mixture stirred for 3 h at

70 ꢁC and concentrated. The product was puriﬁed by

ﬂash column chromatography (2:1 petroleum ether–ace-

tone) to give 9 as a white foam in 84% overall yield

22

in 77% yield (7.2 g). ½aꢀ_Dꢁ31.4 (c 1.0, acetone). ¹H

NMR (400 MHz, CDCl₃): d 8.07 (m, 2H, Ar–H), 7.04

(m, 2H, Ar–H), 6.10 (d, 1H, J 4.4 Hz, NHAc), 5.54

(dd, 1H, J_2,310.0 Hz, J_3,44.3 Hz, H-3), 5.32 (d, 1H,

J_1,28.3 Hz, H-1), 4.38–4.05 (m, 5H, H-2, H-4, H-5, H-

6a and H-6b), 2.36 (s, 3H, CH₃CO), 1.90 (s, 3H,

CH₃CO), 1.17 [s, 9H, (CH₃)₃CCO], 1.09 [s, 9H,

(CH₃)₃CCO]. ¹³C NMR (100 MHz, CDCl₃): d 192.9,

177.9, 177.7, 170.4 (C@O); 161.6, 142.8, 125.6, 116.5

(Ar–C); 98.8, 72.3, 69.5, 63.8, 52.9, 45.8; 38.9, 38.7

[(CH₃)₃CCO]; 30.7 (CH₃CO); 27.1, 26.8 [(CH₃)₃CCO];

23.2 (CH₃CO). HRESIMS: calcd for [C₂₆H₃₆N₂O₁₀S+

Na]⁺: 591.1988. Found m/z: 591.2003.

22

(6.19 g). ½aꢀ_D+29.3 (c 0.5, acetone). ¹H NMR

(400 MHz, Me₂CO-d₆): d 8.20 (m, 2H, Ar–H), 8.01 (m,

2H, Ar–H), 7.60 (m, 1H, Ar–H), 7.49 (m, 2H, Ar–H),

7.26 (m, 2H, Ar–H), 5.63 (d, 1H, J_1,28.4 Hz, H-1),

5.45 (dd, 1H, J_2,310.6 Hz, J_3,49.0 Hz, H-3), 4.92 (d,

1H, J 5.3 Hz, NHAc), 4.29 (ddd, 1H, H-2), 3.89–3.77

(m, 5H, H-4, H-5, H-6a, H-6b and OH), 2.06 (s, 3H,

CH₃CO). ¹³C NMR (75 MHz, DMSO-d₆): d 169.4,

165.6 (C@O); 162.0, 142.1, 133.3, 130.0, 129.4, 128.6,

125.9, 116.7 (Ar–C); 97.6, 77.1, 76.4, 67.5, 60.1, 53.2;

22.7 (CH₃CO). HRESIMS: calcd for [C₂₁H₂₂N₂O₉+

Na]⁺: 469.1223. Found m/z: 469.1235.

3.2.3. 4-Nitrophenyl 2-acetamido-2-deoxy-4-thio-b-^D-

galactopyranoside (8). To a suspension of 7 (0.9 g,

1.59 mmol) in dry MeOH (50 mL) was bubbled argon

for 0.5 h at rt, and then sodium methylate soln (5.4 M,

0.8 mL, 4.32 mmol, 2.7 equiv) was added. The reaction

mixture was stirred under argon for 48 h at rt, neutra-

lised with Amberlite IR-120H ion exchange resin, ﬁl-

tered, washed with MeOH and concentrated to 10 mL.

To this soln was added DTT (1.2 g in 50 mL of distilled

water) with stirring, under argon for another 0.5 h and

the mixture was stirred overnight at rt. The solvents

were evaporated to give a residue that was dissolved in

DMF, loaded onto silica gel and puriﬁed by ﬂash col-

umn chromatography (15:1 and 9:1 CHCl₃–CH₃OH)

to give 8 as a white powder in 37% yield (210 mg).

3.3.2. 4-Nitrophenyl 2-acetamido-3,6-di-O-benzoyl-2-

deoxy-b-^D-glucopyranoside (10). To a soln of com-

pound 9 (6.54 g, 14.66 mmol) in dry CH₂Cl₂(250 mL)

and dry pyridine (25 mL) was added benzoyl cyanide

(3.27 g, 24.96 mmol, 1.7 equiv) at rt under argon, and

the reaction mixture was stirred overnight at rt and

quenched with MeOH. The solvents were evaporated

to give a white solid, and the solid was recrystallised

from chloroform and MeOH (1:1) to give compound

10 (6.05 g). The mother liquor was concentrated and

22

½aꢀ_Dꢁ27.9 (c 0.5, MeOH). ¹H NMR (400 MHz,

CD₃OD): d 8.19 (m, 2H, Ar–H), 7.18 (m, 2H, Ar–H),

2218

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

puriﬁed by ﬂash column chromatography (2:1 petro-

leum ether–acetone) to give a second portion of product

ing residue was puriﬁed by ﬂash column chromatogra-

phy (2:1 petroleum ether–EtOAc) to give 12 (300 mg,

22

1

(0.88 g). The yield of the reaction was 86% (6.93 g). ½aꢀ_D

50%). ½aꢀ_D+38.8 (c 0.5, acetone). H NMR (400 MHz,

CDCl₃): d 8.05–7.90 (m, 6H, Ar–H), 7.60 (m, 2H, Ar–

H), 7.44 (m, 4H, Ar–H), 7.06 (m, 2H, Ar–H), 6.06 (d,

1H, J 8.4 Hz, NHAc), 5.75 (dd, 1H, J_2,3= J_3,4

10.5 Hz, H-3), 5.60 (d, 1H, J_1,28.1 Hz, H-1), 4.76 (br

d, 1H, H-6a), 4.46 (dd, 1 H, J_5,6b7.3 Hz, J_6a,6b

11.8 Hz, H-6b), 4.30 (m, 2H, H-2 and H-5), 3.99 (dd,

1H, J_4,511.0 Hz, H-4), 2.24 (s, 3H, CH₃CO), 1.81 (s,

3H, CH₃CO). ¹³C NMR (100 MHz, CDCl₃): d 192.7,

170.7, 166.4, 165.9 (C@O); 161.4, 142.8, 133.8, 133.6,

129.9, 129.7, 129.5, 128.7, 128.6, 128.5, 125.6, 116.6

(Ar–C); 97.7, 73.4, 71.4, 63.8, 56.1, 44.5, 30.8, 23.3

(CH₃CO). HRESIMS: calcd for [C₃₀H₂₈N₂O₁₀S+Na]⁺:

631.1362. Found m/z: 631.1350.

+13.7 (c 0.5, acetone). ¹H NMR (400 MHz, Me₂SO-d₆):

d 8.14 (d, 1H, OH), 8.07 (m, 2H, Ar–H), 7.99 (m, 4H,

Ar–H), 7.63 (m, 2H, Ar–H), 7.53 (m, 4H, Ar–H), 7.23

(m, 2H, Ar–H), 5.89 (d, 1H, J 5.9 Hz, NHAc), 5.56 (d,

1H, J_1,28.5 Hz, H-1), 5.31 (dd, 1H, J_2,310.0 Hz, J_3,4

9.5 Hz, H-3), 4.64 (br d, 1H, H-6a), 4.44 (dd, 1H, J_5,6b

6.7 Hz, J_6a,6b11.8 Hz, H-6b), 4.17 (m, 2H, H-2 and H-

4), 3.79 (m, 1H, H-5), 1.66 (s, 3H, CH₃CO). ¹³C NMR

(100 MHz, Me₂SO-d₆): d 169.4, 165.5, 165.4 (C@O);

161.5, 142.0, 133.5, 133.3, 129.8, 129.5, 129.4, 129.3,

128.7, 128.6, 125.6, 116.7 (Ar–C); 97.1, 76.0, 73.8,

68.2, 63.6, 53.1; 22.6 (CH₃CO). HRESIMS: calcd for

[C₂₈H₂₆N₂O₁₀+Na]⁺: 573.1485. Found m/z: 573.1484.

3.3.3. 4-Nitrophenyl 2-acetamido-3,6-di-O-benzoyl-2-

deoxy-b-^D-galactopyranoside (11). A soln of com-

pound 10 (1.1 g, 2.00 mmol) in dry CH₂Cl₂(14 mL)

and dry pyridine (3 mL) was stirred under argon and

cooled to 0 ꢁC, to which was added dropwise Tf₂O

(0.7 mL, 4.16 mmol, 2.1 equiv). The mixture was stirred

for 1 h at 0 ꢁC, diluted with CH₂Cl₂, washed with icy

1 M HCl, satd aq NaHCO₃and brine, dried over

MgSO₄, ﬁltered, evaporated and dried under diminished

pressure to give a brown foam. To the brown foam were

added dry DMF (20 mL) and NaNO₂(1.4 g), and the

mixture was stirred for 1.5 h at rt. The mixture was

diluted with ethyl acetate (200 mL), washed with 1 M

HCl, satd aq NaHCO₃and brine, dried over MgSO₄,

ﬁltered and concentrated. The resulting residue was

puriﬁed by ﬂash column chromatography (2:1, 3:2 and

1:1 petroleum ether–acetone) to give 11 in 79% yield

3.3.5. 4-Nitrophenyl 2-acetamido-2-deoxy-4-thio-b-^D-

glucopyranoside (13). The deprotection of 12 (1.25 g,

1.86 mmol) in dry MeOH (60 mL) as described for 8

gave a residue, which was puriﬁed by ﬂash column chro-

matography (CHCl₃–CH₃OH = 12:1) to give 13 as a

22

white powder in 76% yield (450 mg). ½aꢀ_Dꢁ9.1 (c 0.5,

MeOH). ¹H NMR (400 MHz, CD₃OD): d 8.20 (m,

2H, Ar–H), 7.16 (m, 2H, Ar–H), 5.26 (d, 1H, J_1,2

8.5 Hz, H-1), 3.95 (dd, 1H, J_5,6a2.0 Hz, J_6a,6b12.3 Hz,

H-6a), 3.90 (dd, 1H, J_2,310.0 Hz, H-2), 3.84 (dd, 1H,

J_5,6b4.9 Hz, H-6b), 3.61 (ddd, 1H, H-5), 3.53 (dd, 1H,

H-3), 2.88 (dd, 1H, J_3,4= J_4,510.3 Hz, H-4), 1.97 (s,

3H, CH₃CO). ¹³C NMR (100 MHz, CD₃OD): d 173.9

(C@O), 163.6, 144.0, 126.7, 117.7 (Ar–C); 99.8, 79.8,

76.6, 63.0, 58.4, 43.6, 22.9 (CH₃CO). HRESIMS: calcd

for [C₁₄H₁₈N₂O₇S+Na]⁺: 381.0732. Found m/z:

381.0728. Anal. Calcd for C₁₄H₁₈N₂O₇S: C, 46.92; H,

5.06; N, 7.82. Found: C, 47.14; H, 5.18; N, 8.13.

22

(0.87 g). ½aꢀ_D+39.4 (c 0.5, acetone). ¹H NMR

(400 MHz, Me₂SO-d₆): d 8.10–7.90 (m, 8H, Ar–H,

NHAc, OH), 7.63 (m, 2H, Ar–H), 7.55 (m, 4H, Ar–

H), 7.22 (m, 2H, Ar–H), 5.47 (d, 1H, J_1,28.5 Hz, H-1),

5.09 (dd, 1H, J_2,311.0 Hz, J_3,43.0 Hz, H-3), 4.70–4.30

(m, 4H, H-2, H-5, H-6a and H-6b), 4.23 (br d, 1H, H-

4), 1.73 (s, 3H, CH₃CO). ¹³C NMR (100 MHz,

Me₂SO-d₆): d 169.7, 165.5, 165.4 (C@O); 161.7, 141.9,

133.5, 133.4, 129.7, 129.6, 129.4, 129.3, 128.7, 128.6,

125.5, 116.6 (Ar–C); 97.6, 74.4, 72.7, 64.9, 63.7, 48.7,

22.8 (CH₃CO). HRESIMS: calcd for [C₂₈H₂₆N₂O₁₀+

Na]⁺: 573.1485. Found m/z: 573.1479.

3.4. Preparation of 4-nitrophenyl 2-acetamido-2-deoxy-3-

thio-b-^D-glucopyranoside (20)

3.4.1. 4-Nitrophenyl 2-acetamido-4,6-O-benzylidene-2-

deoxy-b-^D-glucopyranoside (14). Compound 5 (2.2 g,

6.43 mmol), anhydrous DMF (30 mL), benzaldehyde di-

methyl acetal (1.16 mL, 1.2 equiv) and 4-toluenesulfonic

acid monohydrate (90 mg) were placed in a 100 mL

round bottom ﬂask. This was attached to a Buchi evap-

¨

orator under diminished pressure, then rotated in a

water bath at 50 ꢁC, so that DMF reﬂuxed in the vapour

duct. The suspension was rotated for 3 h until a clear

soln formed, then progress of the reaction was checked

by TLC. The mixture was poured into satd aq NaHCO₃

(200 mL), and stirred for 0.5 h at rt. The white precipi-

tate was ﬁltered and washed with water and diethyl

ether, then dried under diminished pressure to give com-

3.3.4. 4-Nitrophenyl 2-acetamido-4-S-acetyl-3,6-di-O-

benzoyl-2-deoxy-4-thio-b-^D-glucopyranoside (12). The

triﬂate of 11 (0.55 g, 1.00 mmol) was prepared as

described above. The brown foam was treated with

DMPU (4 mL) and KSAc (0.34 g, 2.98 mmol,

3.0 equiv), and the mixture was stirred for 1 h at rt.

The mixture was diluted with mixed solvents (100 mL,

1:1 EtOAc–Et₂O), washed with water (5 · 100 mL),

dried over MgSO₄, ﬁltered and evaporated. The result-

1

pound 14 in 96% yield (2.67 g). H NMR (400 MHz,

Me₂CO-d₆): d 8.22 (m, 2H, Ar–H), 7.51 (m, 2H, Ar–

H), 7.38 (m, 3H, Ar–H), 7.28 (m, 2H, Ar–H), 5.67

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

2219

(s, 1H, CHPh), 5.63 (d, 1H, J_1,28.3 Hz, H-1), 4.32 (dd,

1H, J_5,6a4.1 Hz, J_6a,6b9.4 Hz, H-6a), 4.10 (m, 1H, H-5),

3.98 (dd, 1H, J_5,6b8.6 Hz, H-6b), 3.83 (dd, 1H, J_4,5

9.9 Hz, H-4), 3.77 (ddd, 1H, H-2), 3.67 (dd, 1H,

J_2,3= J_3,49.0 Hz, H-3), 1.89 (s, 3H, CH₃CO). ¹³C

NMR (75 MHz, Me₂SO-d₆): d 170.0 (C@O); 162.4,

142.6, 138.2, 129.5, 128.6, 126.9, 126.4, 117.2 (Ar–C);

101.3 (CHPh); 99.0, 81.3, 70.7, 68.2, 66.7, 56.4, 23.6

(CH₃CO). HRESIMS: calcd for [C₂₁H₂₂N₂O₈+Na]⁺:

453.1274. Found m/z: 453.1269.

7.39 (m, 3H, Ar–H), 7.11 (m, 2H, Ar–H), 5.60 (d, 1H,

J_1,27.3 Hz, H-1), 5.59 (s, 1H, CHPh), 5.31 (dd,

1H, J_2,34.1 Hz, H-3), 5.25 (br s, 1H, NHAc), 4.47 (dd,

1H, J_5,6a5.2 Hz, J_6a,6b10.6 Hz, H-6a), 4.15 (ddd, 1H,

H-5), 4.10 (br dd, 1H, H-2), 3.99 (dd, 1H, J_3,42.7 Hz,

J_4,59.6 Hz, H-4), 3.78 (t, 1H, J_5,6b10.4 Hz, H-6b).

ESIMS: calcd for [C₁₉H₁₈N₂O₉S+Na]⁺: 473.1. Found

m/z: 473.0.

3.4.4. 4-Nitrophenyl 2-acetamido-4,6-O-benzylidene-2-

deoxy-2,3-sulfamido-b-^D-allopyranoside (17). To a sus-

pension of 16 (0.88 g, 1.96 mmol) in CH₂Cl₂(40 mL)

and pyridine (2 mL) at rt was added dropwise AcCl

(0.33 mL, 4.64 mmol, 2.4 equiv), and the mixture was

stirred for 1 h at rt. The reaction mixture was diluted

with CH₂Cl₂(150 mL), washed with water and brine,

dried over MgSO₄, ﬁltered and concentrated to give a

residue. To the residue were added EtOAc and petro-

leum ether and the precipitate was ﬁltered and washed

with petroleum ether to give 710 mg of product 17.

The mother liquor was concentrated and puriﬁed by

ﬂash column chromatography (250:1 and 100:1

CH₂Cl₂-EtOAc) to give a second portion of product

(60 mg). Totally 770 mg (80%) of 17 was obtained as a

3.4.2. 4-Nitrophenyl 2-acetamido-4,6-O-benzylidene-2-

deoxy-b-^D-allopyranoside (15). To a suspension of 14

(2.17 g, 5.05 mmol) in CH₂Cl₂(20 mL) at rt was added

dry pyridine (20 mL) under argon. The suspension was

stirred for 5 min, and MsCl (1.2 mL, 15.5 mmol,

3 equiv) was added dropwise. The reaction mixture

was stirred for 19 h at rt, quenched with MeOH and

evaporated to give a yellow residue. To the yellow resi-

due was added MeOH (40 mL), and the suspension was

stirred for 0.5 h at rt. The precipitate was ﬁltered and

washed with MeOH to give the mesylate as a solid

(1.90 g), which was suspended in 10:1 2-methoxyetha-

nol–water (60 mL), and reacted with sodium acetate

(2.5 g) at 130 ꢁC for 8 h. The mixture was cooled to rt,

and the solvents were evaporated to give a residue, to

which was added distilled water (300 mL), and the sus-

pension stirred for 0.5 h at rt. The white precipitate

was ﬁltered and washed with water and diethyl ether

1

white solid. H NMR (400 MHz, CDCl₃): d 8.22 (m,

2H, Ar–H), 7.48 (m, 2H, Ar–H), 7.39 (m, 3H, Ar–H),

7.09 (m, 2H, Ar–H), 5.61 (s, 1H, CHPh), 5.57 (d, 1H,

J_1,26.1 Hz, H-1), 5.34 (dd, 1H, J_2,34.8 Hz, J_3,42.7 Hz,

H-3), 5.04 (br t, 1H, H-2), 4.47 (dd, 1H, J_5,6a4.9 Hz,

J_6a,6b10.6 Hz, H-6a), 4.24 (ddd, 1H, J_4,59.9 Hz, H-5),

4.15 (dd, 1H, H-4), 3.77 (dd, 1H, J_5,6b10.2 Hz, H-6b),

2.49 (s, 3H, CH₃CO). ESIMS: calcd for

[C₂₁H₂₀N₂O₁₀S+Na]⁺: 515.1. Found m/z: 515.2.

1

to give 15 in 63% yield (1.37 g). H NMR (400 MHz,

Me₂CO-d₆): d 8.23 (m, 2H, Ar–H), 7.50 (m, 2H, Ar–

H), 7.38 (m, 3H, Ar–H), 7.29 (m, 2H, Ar–H), 5.71 (s,

1H, CHPh), 5.56 (d, 1H, J_1,28.6 Hz, H-1), 4.40–4.26

(m, 4H, H-2, H-3, H-5, H-6a), 3.86 (dd, 1H, J_5,6b

3.0 Hz, J_6a,6b9.3 Hz, H-6b), 3.81 (dd, 1H, J_3,4= J_4,5

9.9 Hz, H-4), 1.94 (s, 3H, CH₃CO). ¹³C NMR

(75 MHz, Me₂SO-d₆): d 169.5 (C@O); 162.6, 142.5,

138.2, 129.4, 128.6, 126.9, 126.3, 117.3 (Ar–C); 101.2

(CHPh), 97.3, 78.5, 68.6, 67.7, 63.7, 53.3; 23.2 (CH₃CO).

HRESIMS: calcd for [C₂₁H₂₂N₂O₈+Na]⁺: 453.1274.

Found m/z: 453.1279.

3.4.5. 4-Nitrophenyl 2-acetamido-3-S-acetyl-4,6-O-benz-

ylidene-2-deoxy-3-thio-b-^D-glucopyranoside (18). To a

soln of sulfamidate 17 (0.77 g, 1.57 mmol) in dry DMF

(50 mL) was added KSAc (0.89 g, 7.8 mmol, 5 equiv),

and the mixture was stirred for 1 h at rt, after which

the solvent was evaporated under diminished pressure

to give a yellowish solid. The solid was suspended in

THF (20 mL), and treated with 6 mL of THF–H₂SO₄–

H₂O (5 mL:220 lL:70 lL) for 0.5 h. The mixture was

extracted with CH₂Cl₂(400 mL) and the organic phase

washed with satd aq NaHCO₃and brine, dried over

MgSO₄, ﬁltered and concentrated to give a residue.

The product was precipitated by the addition of acetone

and petroleum ether and ﬁltered to give 600 mg of 18.

The mother liquor was puriﬁed by ﬂash column chroma-

tography (20:1 CH₂Cl₂–acetone) to give a second por-

tion of product (43 mg). In total 643 mg (84%) of 18

3.4.3. 4-Nitrophenyl 4,6-O-benzylidene-2-deoxy-2,3-sul-

famido-b-^D-allopyranoside (16). To a suspension of 15

(1.35 g, 3.14 mmol) in THF (50 mL) at rt was added

NaH (0.8 g, 60% in mineral oil, 20 mmol, 6.4 equiv).

After 15 min, a soln of 1,1⁰-sulfonyldiimidazole (1.5 g,

7.58 mmol, 2.4 equiv) in THF (30 mL) was added drop-

wise and the mixture was stirred for 20 h at rt. The reac-

tion was quenched with MeOH and the suspension

ﬁltered through a short pad of Celite column that was

washed with methylene chloride. The solvents were

evaporated, and the resulting residue was puriﬁed by

ﬂash column chromatography (100:1 CH₂Cl₂–CH₃OH)

22

was obtained as a yellowish solid. ½aꢀ_Dꢁ15.4 (c 0.5, ace-

tone). ¹H NMR (400 MHz, CDCl₃): d 8.19 (m, 2H, Ar–

H), 7.44 (m, 2H, Ar–H), 7.36 (m, 3H, Ar–H), 7.05 (m,

2H, Ar–H), 5.75 (d, 1H, NHAc), 5.53 (s, 1H, CHPh),

5.23 (d, 1H, J_1,28.1 Hz, H-1), 4.38 (m, 2H, H-2,

1

to give 16 in 82% yield (1.14 g). H NMR (400 MHz,

CDCl₃): d 8.23 (m, 2H, Ar–H), 7.48 (m, 2H, Ar–H),

2220

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

H-6a), 3.93 (dd, 1H, J_2,3= J_3,411.4 Hz, H-3), 3.82–3.68

(m, 2H, H-5 and H-6b), 3.67 (dd, 1H, J_4,58.3 Hz, H-4),

2.37 (s, 3H, CH₃CO), 1.93 (s, 3H, CH₃CO).

¹³C NMR (75 MHz, Me₂SO-d₆): d 194.1 (S-acetyl),

169.3 (C@O); 161.6, 142.2, 137.3, 128.9, 128.1, 126.0,

125.8, 116.7 (Ar–C); 100.6 (CHPh); 99.0, 77.3, 69.3,

67.6, 52.1, 47.2; 30.6, 22.6 (CH₃CO). HRESIMS: calcd

for [C₂₃H₂₄N₂O₈S+Na]⁺: 511.1151. Found m/z:

511.1152.

CD₃OD): d 173.8 (C@O); 163.7, 144.2, 126.8, 117.8

(Ar–C); 100.9, 81.1, 72.7, 62.7, 57.1, 48.9, 22.9

(CH₃CO). HRESIMS: calcd for [C₁₄H₁₈N₂O₇S+Na]⁺:

381.0732. Found m/z: 381.0722. Anal. Calcd for

C₁₄H₁₈N₂O₇S: C, 46.92; H, 5.06; N, 7.82. Found: C,

46.56; H, 5.20; N, 8.04.

3.6. Preparation of 4-nitrophenyl 2-acetamido-2-deoxy-3-

thio-b-^D-galactopyranoside (33)

3.4.6. 4-Nitrophenyl 2-acetamido-3-S-acetyl-4,6-di-O-

acetyl-2-deoxy-3-thio-b-^D-glucopyranoside (19). A sus-

pension of 18 (0.3 g, 0.6 mmol) in acetic acid (8 mL)

and water (2 mL) was stirred for 3.5 h at 60 ꢁC. The sol-

vents were then evaporated to give a yellowish solid,

which was dried under diminished pressure. To the solid

were added pyridine (5 mL) and Ac₂O (5 mL), and the

mixture was stirred overnight at rt. The mixture was

poured into icy water, stirred for 0.5 h, extracted with

CH₂Cl₂(200 mL) and the organic phase washed with

1 M HCl, satd aq NaHCO₃and brine, dried over

MgSO₄, ﬁltered and concentrated. The resulting residue

was puriﬁed by ﬂash column chromatography (1:1 and

1:2 petroleum ether–EtOAc) to give 19 in 81% overall

yield (241 mg). ½aꢀ_Dꢁ10.4 (c 1.0, acetone). H NMR

(400 MHz, CDCl₃): d 8.17 (m, 2H, Ar–H), 6.92 (m,

2H, Ar–H), 5.71 (d, 1H, J 8.9 Hz, NHAc), 5.30 (d,

1H, J_1,27.9 Hz, H-1), 5.09 (dd, 1H, J_3,410.5 Hz, J_4,5

9.8 Hz, H-4), 4.26 (m, 2H, H-2, H-6a), 4.13 (dd, 1H,

J_5,6b2.1 Hz, J_6a,6b12.2 Hz, H-6b), 3.96 (m, 2H, H-3

and H-5), 2.37 (s, 3H, CH₃CO), 2.05 (s, 6H,

2 · CH₃CO), 1.93 (s, 3H, CH₃CO). ¹³C NMR

(75 MHz, CDCl₃): d 196.0 (S-acetyl), 170.7, 170.5,

169.5 (C@O); 161.7, 143.1, 125.9, 116.7 (Ar–C); 99.7,

75.2, 66.8, 62.5, 54.2, 47.6; 30.9, 23.4, 20.9, 20.8

(CH₃CO). HRESIMS: calcd for [C₂₀H₂₄N₂O₁₀S+Na]⁺:

507.1049. Found m/z: 507.1048.

3.6.1. 4-Nitrophenyl 2-acetamido-3,4,6-tri-O-acetyl-2-

deoxy-b-^D-galactopyranoside (24). A mixture of 21

(4.1 g, 18.55 mmol) and acetyl chloride (20 mL) was

stirred overnight at rt, diluted with CH₂Cl₂(200 mL)

and poured into icy water. The organic phase was

washed with icy cold NaHCO₃and brine, dried over

MgSO₄, concentrated, and the solid dried under dimin-

ished pressure. ¹H NMR analysis of the white solid

showed that it was a mixture of 2-acetamido-3,4,6-tri-

O-acetyl-2-deoxy-a-^D-galactopyranosyl chloride (23,

ꢂ60%) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-

a-^D-galactopyranose (22, ꢂ40%). To this solid were

added acetone (50 mL, HPLC-grade), 4-nitrophenol

(6.0 g, 43.17 mmol) and K₂CO₃(8.0 g, 57.97 mmol),

and the suspension was stirred for 1 h at 50 ꢁC. After

evaporation, the residue was dissolved in CH₂Cl₂

(300 mL), washed with satd aq NaHCO₃and brine,

dried over MgSO₄, ﬁltered and concentrated. The

resulting residue was crystallised from MeOH to give

22

1

24 in 29% yield (2.5 g). H NMR (400 MHz, CDCl₃):

d 8.17 (m, 2H, Ar–H), 7.08 (m, 2H, Ar–H), 5.66 (d,

1H, J 8.4 Hz, NHAc), 5.48 (d, 1H, J_1,28.4 Hz, H-1),

5.47–5.43 (m, 2H, H-3 and H-4), 4.25–4.10 (m, 4H, H-

2, H-5, H-6a and H-6b), 2.16 (s, 3H, CH₃CO), 2.05 (s,

3H, CH₃CO), 2.03 (s, 3H, CH₃CO), 1.94 (s, 3H,

CH₃CO). ¹³C NMR (75 MHz, CDCl₃): d 171.0, 170.7,

170.6, 170.4 (C@O); 161.8, 143.1, 125.9, 116.8 (Ar–C);

98.2, 71.6, 69.5, 66.8, 61.9, 51.6, 23.6, 20.9 (3C)

(CH₃CO). ESIMS: calcd for [C₂₀H₂₄N₂O₁₁+Na]⁺:

491.1. Found m/z: 491.1.

3.5. 4-Nitrophenyl 2-acetamido-2-deoxy-3-thio-b-^D-

glucopyranoside (20)

Into a soln of compound 19 (160 mg, 0.33 mmol) in dry

MeOH (15 mL) was bubbled argon for 0.5 h, and then

sodium methylate soln (5.4 M, 0.2 mL, 1.08 mmol,

3.3 equiv) was added. The soln was stirred for 0.5 h at

rt, neutralised with Amberlite IR-120H ion exchange re-

sin, ﬁltered and washed with MeOH and concentrated.

The product was precipitated by addition of acetone

and petroleum ether to give a white solid in quantitative

yield. ½aꢀ_Dꢁ24.1 (c 0.5, MeOH). H NMR (400 MHz,

CD₃OD): d 8.14 (m, 2H, Ar–H), 7.10 (m, 2H, Ar–H),

5.14 (d, 1H, J_1,28.1 Hz, H-1), 3.89 (dd, 1H, H-2), 3.84

(dd, 1H, J_5,6a1.7 Hz, H-6a), 3.67 (dd, 1H, J_5,6b5.6 Hz,

J_6a,6b12.2 Hz, H-6b), 3.47 (m, 1H, H-5), 3.34 (dd, 1H,

J_3,4= J_4,59.7 Hz, H-4), 2.97 (dd, 1H, J_2,39.9 Hz,

H-3), 1.91 (s, 3H, CH₃CO). ¹³C NMR (75 MHz,

3.7. 4-Nitrophenyl 2-acetamido-2-deoxy-b-^D-galacto-

pyranoside (25)

Compound 24 (2.0 g, 4.27 mmol) was deacetylated as

described for 20 to give 25 in quantitative yield. ¹H

NMR (400 MHz, Me₂SO-d₆): d 8.20 (m, 2H, Ar–H),

7.77 (d, 1H, J 9.0 Hz, NHAc), 7.16 (m, 2H, Ar–H),

5.14 (d, 1H, J_1,28.4 Hz, H-1), 4.83 (d, 1H, J 6.2 Hz,

OH), 4.71 (d, 1H, J 8.8 Hz, OH), 4.70 (d, 1H, J

10.2 Hz, OH), 4.03 (m, 1H, H-2), 3.75–3.52 (m, 5H,

H-3, H-4, H-5, H-6a and H-6b), 1.80 (s, 3H, CH₃CO).

¹³C NMR (75 MHz, Me₂SO-d₆): d 169.7 (C@O);

162.4, 141.7, 125.8, 116.6 (Ar–C); 98.8, 75.9, 71.0,

67.4, 60.3, 51.7, 23.1 (CH₃CO). HRESIMS: calcd for

[C₁₄H₁₈N₂O₈+Na]⁺: 365.0961. Found m/z: 365.0961.

22

1

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

2221

3.7.1. 4-Nitrophenyl 2-acetamido-4,6-O-benzylidene-2-

deoxy-b-^D-galactopyranoside (26). Compound 26 was

prepared as described for 14 by the treatment of com-

pound 24 (1.74 g, 5.09 mmol) with benzaldehyde di-

methyl acetal and 4-toluenesulfonic acid monohydrate

in DMF. The product was obtained as a white solid in

92% yield (2.01 g). ¹H NMR (400 MHz, Me₂SO-d₆):

d 8.22 (m, 2H, Ar–H), 7.83 (d, 1H, J 8.7 Hz,

NHAc), 7.51 (m, 2H, Ar–H), 7.41 (m, 3H, Ar–H),

7.24 (m, 2H, Ar–H), 5.65 (s, 1H, CHPh), 5.32 (d,

1H, J_1,28.4 Hz, H-1), 5.16 (br s, 1H, OH), 4.24–3.81

(m, 6H, H-2, H-3, H-4, H-5, H-6a and H-6b), 1.82

(s, 3H, CH₃CO). ¹³C NMR (75 MHz, Me₂SO-d₆): d

169.7 (C@O); 162.2, 141.8, 138.5, 128.7, 128.0, 126.3,

125.8, 116.6 (Ar–C); 99.8 (CHPh), 99.4, 75.0, 69.1,

68.3, 66.5, 51.5, 23.1 (CH₃CO). HRESIMS: calcd for

[C₂₁H₂₂N₂O₈+Na]⁺: 453.1274. Found m/z: 453.1263.

2H, Ar–H), 7.39 (m, 3H, Ar–H), 7.13 (m, 2H, Ar–H),

5.67 (s, 1H, CHPh), 5.57 (d, 1H, J_1,28.1 Hz, H-1),

5.11 (dd, 1H, J_2,33.1 Hz, J_3,44.4 Hz, H-3), 5.04 (d,

1H, J 5.2 Hz, NHAc), 4.47 (m, 1H, H-5), 4.46 (dd,

1H, J_5,6a1.3, J_6a,6b12.7 Hz, H-6a), 4.22 (dd, 1H, J_5,6b

1.8 Hz, H-6b), 4.14 (dt, 1H, H-2), 4.00 (br d, 1H, H-

4). ¹³C NMR (75 MHz, CDCl₃): 163.2, 143.5, 139.6,

129.7, 129.0, 127.3, 126.5, 117.5 (Ar–C); 101.5 (CHPh);

96.9, 76.9, 70.2, 69.8, 66.9, 57.0. HRESIMS: calcd for

[C₁₉H₁₈N₂O₉S+Na]⁺: 473.0631. Found m/z: 473.0634.

3.7.4. 4-Nitrophenyl 2-acetamido-4,6-O-benzylidene-2-

deoxy-2,3-sulfamido-b-^D-gulopyranoside

(30). Com-

pound 29 (276 mg, 0.61 mmol) was acetylated and puri-

ﬁed as described for 17 to yield compound 30 as a white

1

solid (297 mg, 98%). H NMR (300 MHz, CDCl₃): d

8.23 (m, 2H, Ar–H), 7.50 (m, 2H, Ar–H), 7.41 (m, 3H,

Ar–H), 7.11 (m, 2H, Ar–H), 5.68 (s, 1H, CHPh), 5.53

(d, 1H, J_1,27.7 Hz, H-1), 5.15 (dd, 1H, J_2,32.4, J_3,4

4.5 Hz, H-3), 5.04 (m, 1H, H-2), 4.48 (m, 1H, H-5),

4.47 (dd, 1H, J_5,6a1.4 Hz, H-6a), 4.23 (dd, 1H, J_5,6b

1.8 Hz, J_6a,6b13.0 Hz, H-6b), 4.02 (br d, 1H, H-4),

2.49 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, Me₂CO-d₆):

162.1, 144.3, 138.9, 130.0, 129.0, 127.3, 126.7, 117.8

(Ar–C); 101.8 (CHPh); 99.2, 79.6, 70.5, 68.9, 67.7,

58.1, 23.1 (CH₃CO). HRESIMS: calcd for

[C₂₁H₂₀N₂O₁₀S+Na]⁺: 515.0736. Found m/z: 515.0742.

3.7.2. 4-Nitrophenyl 2-acetamido-4,6-O-benzylidene-2-

deoxy-b-^D-gulopyranoside (28).

A

mixture of 26

(2.1 g, 4.88 mmol), anhydrous CH₂Cl₂(60 mL) and

pyridine (12 mL) was cooled to 0 ꢁC under argon, and

to this soln was added, dropwise, Tf₂O (2.5 mL,

14.87 mmol, 3 equiv) with stirring. The reaction mixture

was stirred for another period of 1.5 h at 0 ꢁC, diluted

with CH₂Cl₂(250 mL), washed with icy 1 M HCl, satd

aq NaHCO₃and brine, and dried over MgSO₄. The sol-

vent was evaporated to give a residue, which was dried

under diminished pressure to aﬀord a yellowish foam

(27). To this foam were added dry DMF (65 mL) and

NaNO₂(3.6 g, 52.17 mmol), and the suspension was

stirred for 4 days at rt under argon. The reaction mix-

ture was diluted with CH₂Cl₂(500 mL), washed with

icy 1 M HCl, satd aq NaHCO₃, dried over MgSO₄, ﬁl-

tered and concentrated. The resulting residue was puri-

ﬁed by ﬂash column chromatography (20:1 CH₂Cl₂–

CH₃OH = 20:1) to give a yellowish foam from an ether

solution of which the product was precipitated by the

addition of acetone and petroleum. ¹H NMR

(400 MHz, CDCl₃): d 8.20 (m, 2H, Ar–H), 7.52 (m,

2H, Ar–H), 7.38 (m, 3H, Ar–H), 7.11 (m, 2H, Ar–H),

6.08 (d, 1H, J 7.3 Hz, NHAc), 5.80 (d, 1H, J_1,28.7 Hz,

H-1), 5.61 (s, 1H, CHPh), 4.39–4.12 (m, 5H, H-2, H-3,

H-5, H-6a and H-6b), 2.05 (s, 3H, CH₃CO). ¹³C NMR

(75 MHz, CDCl₃): d 172.2 (C@O); 162.2, 142.7, 137.7,

129.4, 128.5, 126.4, 125.7, 116.7 (Ar–C); 101.0 (CHPh),

97.2, 75.7, 69.8, 69.4, 66.3, 50.8; 23.4 (CH₃CO). HRE-

SIMS: calcd for [C₂₁H₂₂N₂O₈+Na]⁺: 453.1274. Found

m/z: 453.1280.

3.7.5. 4-Nitrophenyl 2-acetamido-3-S-acetyl-4,6-O-benz-

ylidene-2-deoxy-3-thio-b-^D-galactopyranoside (31).

A

mixture of compound 30 (142 mg, 0.29 mmol) and

KSAc (165 mg, 1.45 mmol, 5 equiv) in dry DMF

(10 mL) was heated to 65 ꢁC under argon and stirred

overnight. The solvent was evaporated under diminished

pressure to give a yellow residue, which was suspended

in THF (4 mL), and acid (1.5 mL, 5 mL:220 lL:70 lL

THF–H₂SO₄–H₂O) was added. The suspension was stir-

red for 0.5 h at rt, diluted with CH₂Cl₂(75 mL), washed

with satd aq NaHCO₃and brine, dried over MgSO₄, ﬁl-

tered and concentrated to give crude product. The prod-

uct was puriﬁed by ﬂash column chromatography (30:1

and 20:1 CH₂Cl₂–acetone) (31, 120 mg, 85% with a trace

22

of impurity). ½aꢀ_Dꢁ12.0 (c 0.5, acetone). ¹H NMR

(400 MHz, CDCl₃): d 8.14 (m, 2H, Ar–H), 7.51 (m,

2H, Ar–H), 7.38 (m, 3H, Ar–H), 7.07 (m, 2H, Ar–H),

5.84 (d, 1H, J 9.0 Hz, NHAc), 5.56 (s, 1H, CHPh),

5.42 (d, 1H, J_1,28.1 Hz, H-1), 4.43 (dt, 1H, H-2), 4.33

(br d, 1H, J_6a,6b12.4 Hz, H-6a), 4.22 (dd, 1H, J_2,3

12.0 Hz, J_3,43.0 Hz, H-3), 4.16 (d, 1H, H-4), 4.09 (br

d, 1H, H-6b), 3.84 (m, 1H, H-5), 2.40 (s, 3H, CH₃CO),

1.92 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, Me₂SO-d₆): d

196.8 (S-acetyl), 170.6 (C@O); 162.1, 142.9, 137.3, 129.4,

128.5, 126.4, 125.9, 116.9 (Ar–C); 101.3 (CHPh); 100.2,

74.9, 69.1, 68.9, 50.1, 46.4; 30.8, 23.6 (CH₃CO). HRE-

SIMS: calcd for [C₂₃H₂₄N₂O₈S+Na]⁺: 511.1151. Found

m/z: 511.1162.

3.7.3. 4-Nitrophenyl 4,6-O-benzylidene-2-deoxy-2,3-sul-

famido-b-^D-gulopyranoside

(29). Compound

28

(340 mg, 0.79 mmol) was converted to its sulfamidate

and puriﬁed as described for 16 to yield compound 29

as

(300 MHz, CDCl₃): d 8.23 (m, 2H, Ar–H), 7.50 (m,

a

yellowish solid (280 mg, 79%). ¹H NMR

2222

H.-M. Chen, S. G. Withers / Carbohydrate Research 342 (2007) 2212–2222

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3.7.6. 4-Nitrophenyl 2-acetamido-3-S-acetyl-4,6-di-O-

acetyl-2-deoxy-3-thio-b-^D-galactopyranoside (32). Com-

pound 31 (320 mg) was hydrolysed, acetylated and puri-

ﬁed as described for 19 to yield compound 32 (215 mg,

22

1

68%) as a white solid. ½aꢀ_D+9.4 (c 0.5, acetone). H

NMR (400 MHz, CDCl₃): d 8.20 (m, 2H, Ar–H), 7.09

(m, 2H, Ar–H), 5.59 (d, 1H, J 9.0 Hz, NHAc), 5.40 (d,

1H, J_3,42.6 Hz, H-4), 5.33 (d, 1H, J_1,28.1 Hz, H-1),

4.35 (dt, 1H, J_2,312.2 Hz, H-2), 4.24–4.06 (m, 4H, H-

3, H-5, H-6a and H-6b), 2.38 (s, 3H, CH₃CO), 2.18

(s, 3H, CH₃CO), 2.08 (s, 3H, CH₃CO), 1.96 (s,

3H, CH₃CO). ¹³C NMR (75 MHz, CDCl₃): d 195.3

(S-acetyl), 170.7, 170.5, 170.1 (C@O); 161.8, 143.0,

125.8, 116.7 (Ar–C); 99.9, 74.0, 68.3, 62.3, 50.5, 46.6;

30.7, 23.4, 20.8, 20.7 (CH₃CO). HRESIMS: calcd for

[C₂₀H₂₄N₂O₁₀S+Na]⁺: 507.1049. Found m/z: 507.1058.

3.7.7. 4-Nitrophenyl 2-acetamido-2-deoxy-3-thio-b-^D-

galactopyranoside (33). Compound 32 (115 mg,

0.24 mmol) was deacetylated as described for 20 to yield

22

compound 33 as a white solid (83 mg, 98%). ½aꢀ_D+30.9

1

(c 0.5, MeOH). H NMR (400 MHz, CD₃OD): d 8.12

14. Aharoni, A.; Thieme, K.; Chiu, C. P.; Buchini, S.; Lairson,

L. L.; Chen, H.; Strynadka, N. C.; Wakarchuk, W. W.;

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15. Hancock, S. M.; Vaughan, M. D.; Withers, S. G. Curr.

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17. Elhalabi, J.; Rice, K. G. Carbohydr. Res. 2002, 337, 1935–

1940.

(m, 2H, Ar–H), 7.10 (m, 2H, Ar–H), 5.10 (d, 1H, J_1,2

8.2 Hz, H-1), 4.03 (dd, 1H, J_2,311.8 Hz, H-2), 3.79 (d,

1H, H-4), 3.73–3.65 (m, 3H, H-5, H-6a and H-6b),

3.07 (dd, 1H, J_3,42.5 Hz, H-3), 1.87 (s, 3H, CH₃CO).

¹³C NMR (75 MHz, Me₂SO-d₆): d 169.3 (C@O),

162.2, 141.8, 125.8, 116.6 (Ar–C); 99.7, 78.2, 67.9,

60.5, 52.0, 44.8; 22.9 (CH₃CO). HRESIMS: calcd for

[C₁₄H₁₈N₂O₇S+Na]⁺: 381.0732. Found m/z: 381.0743.

Anal. Calcd for C₁₄H₁₈N₂O₇S: C, 46.92; H, 5.06; N,

7.82. Found: C, 46.73; H, 5.08; N, 8.18.

18. Aguilera, B.; FernandezMayoralas, A.; Jaramillo, C.

Tetrahedron 1997, 53, 5863–5876.

´

19. Calvo-Asın, J. A.; Calvo-Flores, F. G.; Exposito-Lopez, J.

´

M.; Hernandez-Mateo, F.; Garcıa-Lopez, J. J.; Isac-

´

Garcıa, J.; Santoyo-Gonzalez, F.; Vargas-Berenguel, A.

J. Chem. Soc., Perkin Trans. 1 1997, 1079–1082.

20. Melenedz, R. E.; Lubell, W. D. Tetrahedron 2003, 59,

2581–2616.

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We thank the Natural Sciences and Engineering Re-

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Article Doi

Syntheses of the 3- and 4-thio analogues of 4-nitrophenyl 2-acetamido-2-deoxy-β-d-gluco- and galactopyranoside

DOI: 10.1016/j.carres.2007.05.023

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Full text of DOI:10.1016/j.carres.2007.05.023

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