Synthesis and utility of sulfated chromogenic carbohydrate model substrates for measuring activities of mucin-desulfating enzymes

Carbohydrate Research 337 (2002) 1095–1111

www.elsevier.com/locate/carres

Synthesis and utility of sulfated chromogenic carbohydrate model

substrates for measuring activities of mucin-desulfating enzymes

Keith Clinch,^a,* Gary B. Evans,^aRichard H. Furneaux,^aPhillip M. Rendle,^a

Phillippa L. Rhodes,^bAnthony M. Roberton,^b,* Douglas I. Rosendale,^bPeter C. Tyler,^a

Damian P. Wright^b

^aIndustrial Research Ltd., Graceﬁeld Road, PO Box 31-310, Lower Hutt, New Zealand

^bSchool of Biological Sciences, The Uni6ersity of Auckland, Pri6ate Bag 92019, Auckland, New Zealand

Received 6 November 2001; received in revised form 5 March 2002; accepted 11 April 2002

This paper is dedicated to Professor Joachim Thiem, Institut fu¨r Organische Chemie, Universita¨t Hamburg, to

mark his 60th birthday

Abstract

A chromogenic substrate, 4-nitrophenyl 2-acetamido-2-deoxy-b-D-glucopyranoside 6-sodium sulfate was synthesized and used

in combination with b-N-acetylhexosaminidase for detection of the sulfatase, MdsA, by release of 4-nitrophenol. MdsA was

originally isolated from the bacterium Pre6otella strain RS2 and is believed to be involved in desulfation of sulfomucins, major

components of the mucus barrier protecting the human colon surface. The exo nature of the MdsA sulfatase was indicated by its

inability to de-esterify the disaccharide 4-nitrophenyl b-

6-sodium sulfate. This latter compound was prepared from monosaccharide precursors by two different methods, the shorter

requiring just six steps from 4-nitrophenyl 2-acetamido-2-deoxy-b- -glucopyranoside and giving an overall yield of 26.4%. The

syntheses of 4-nitrophenyl b- -galactopyranoside 3-triethylammonium sulfate and 6-triethylammonium sulfate and their use in

D

-galactopyranosyl-(14)-2-acetamido-2-deoxy-b-D-glucopyranoside

D

combination with b-galactosidase as chromogenic substrates for detecting Bacteroides fragilis sulfatases with different speciﬁcities

Keywords: Chromogenic sugar sulfates; Disaccharide sulfate; Monosaccharide sulfate; exo-Sulfatase; Sulfomucin

speciﬁc regions of the GI tract—particularly in those

1. Introduction

regions heavily colonized by bacteria, such as the

mouth and colon.¹Desulfation by bacterial enzymes is

The gastrointestinal (GI) tract is covered by a mucus

thought to be one of the rate-limiting steps in the

barrier at its mucosal surface, and the main structural

components of the barrier are the glycoprotein

‘mucins’, the carbohydrate portions of which are

oligosaccharides containing

degradation of the sulfomucin barrier.^1–4

There are different ways in which the mucin oligosac-

charide chains can be sulfated, three of the major

D-galactose, L-fucose, 2-

structural motifs being 2-acetamido-2-deoxy-

ranose 6-sulfate (GlcNAc 6-OSO₃H) (internal and ter-

minal), -galactopyranose 6-sulfate (Gal 6-OSO₃H)

(terminal) and -galactopyranose 3-sulfate (Gal 3-

D-glucopy-

acetamido-2-deoxy- -glucose, 2-acetamido-2-deoxy-D-

D

galactose, and N-acetylneuraminic acid. Importantly,

some of these oligosaccharides may carry sulfate ester

groups. Mucins containing higher levels of sulfate are

generally referred to as sulfomucins and are secreted in

D

OSO₃H) (terminal).^5,6Enzymes that desulfate model

substrates containing these moieties include a Pre6otella

strain RS2 enzyme called MdsA. It can partially desul-

fate rat gastric mucin and is speciﬁc for GlcNAc 6-

OSO₃H residues.^7,8It is not known whether MdsA acts

on external and/or internal GlcNAc 6-OSO₃H units of

the mucin oligosaccharide chains, both of which are

* Corresponding authors. Fax: +64-4-5690055 (K.C.); fax:

+64-9-3737416 (A.M.R.).

E -mail addresses : k.clinch@irl.cri.nz (K. Clinch),

t.roberton@auckland.ac.nz (A.M. Roberton).

PII: S0008-6215(02)00104-0

1096

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

found.^9,10Internal GlcNAc 6-OSO₃H entities generally

by puriﬁcation on ion exchange resin.¹⁴In the present

work it was made by standard methods from the

known 4-nitrophenyl 2-acetamido-3,4-di-O-acetyl-2-

have a b-D-galactopyranosyl unit attached at O-3 or

O-4.¹¹For sulfated galactose residues, the galactose-3-

sulfatase(s) and galactose-6-sulfatase(s) from Bac-

teroides fragilis can de-esterify appropriate model

substrates.¹²

deoxy-b-

For the preparation of the

based substrate 2, the known 4-nitrophenyl 4,6-O-iso-

propylidene-b-

-galactopyranoside,¹⁶made by the

kinetically controlled acetonation of 4-nitrophenyl b-D-

D

-glucopyranoside.¹⁵

D

-galactose 3-sulfate-

This paper describes (i) chemical syntheses of the

D

4-nitrophenyl b-

D

-glycopyranosides of GlcNAc 6-

OSO₃Na and b-

D

-Gal-(14)-GlcNAc 6-OSO₃Na (1

galactopyranoside (Gal-ONP), was used. The acetal

was also converted into the thermodynamically pre-

ferred 3,4-isopropylidene isomer¹⁷for the preparation

and 4, respectively) and potential use of these substrates

by the MdsA enzyme; and (ii) syntheses of the

analogous b-

D

-glycopyranosides of Gal 3-OSO₃Et₃NH

of the

D

-galactoside 6-sulfate 3. In both cases the tin

and Gal 6-OSO₃Et₃NH (2 and 3, respectively) and their

cleavage by B. fragilis sulfatases.

activation-sulfation technique¹⁸was used to introduce

the sulfate esters, and in each instance the protecting

acetal group was removed by selective, acid-catalysed

hydrolysis. The yield of sulfates 2 and 3 were 39 and

41%, respectively, from the glycoside acetals. Alterna-

tively, 6-sulfate 3 was obtained in 44% yield by direct

sulfation of Gal-ONP. Compound 2 has also previously

been prepared by direct stannylenation-sulfation of

Gal-ONP (77%), and in the same work ester 3 was

made from the glycoside in \80% yield by selective

enzymatic de-O-sulfation of 4-nitrophenyl b-D-

galactopyranoside 3,6-disulfate,^19,20which is the major

product of direct selective disulfation.

For reasons that will be alluded to below, a success-

ful route to the lactosamine-based 4 appeared to lie in

forming the aryl glycosidic bond at a late stage in the

synthesis, and glycosyl chloride 12 was selected as a key

precursor (Scheme 1)—particularly since 2,3,4,6-tetra-

O-acetyl-b-

D

-galactopyranosyl-(14)-2-acetamido-

2. Results and discussion

3,6-di-O-acetyl-2-deoxy-a-

D

-glucopyranosyl chloride²¹

has been efﬁciently converted into its b-linked 4-nitro-

phenyl glycoside by treatment with Amberlyst A-26

(4-nitrophenoxide) resin.²²Recently, Lay et al.²³de-

scribed the synthesis of thioglycoside 5 and found it to

be subject to highly selective glycosylation at O-4. We

therefore followed this guidance and prepared the cor-

responding 6-O-(4-methoxybenzyl) (PMB) ether 6 from

Syntheses of substrates 1–4.—Compound 1, which is

commercially available,¹³has been synthesized previ-

ously by direct sulfation of 4-nitrophenyl 2-acetamido-

2-deoxy-b-

D

-glucopyranoside (GlcNAc–ONP) followed

phenyl

2-deoxy-2-tetrachlorophthalimido-1-thio-b-D-

glucopyranoside.²³In this work the latter compound

was prepared by de-esterifying the corresponding 3,4,6-

triacetate using Ellervik and Magnusson’s method for

deacetylating tetrachlorophthalimido derivatives.²⁴Gly-

cosylation of compound 6 with 2,3,4,6-tetra-O-acetyl-a-

D

-galactopyranosyl trichloroacetimidate^25,26in CH₂Cl₂

with boron triﬂuoride etherate (BF₃·OEt₂) as catalyst

gave the b-(14)-linked disaccharide 7 in 59% yield.

No a-linked or O-3-bonded isomers were detected, but

a slight amount of PMB ether bond cleavage occurred.

Attempts to form a 4-nitrophenyl glycoside from the

3-acetate 8 of compound 7 by treatment with 4-nitro-

phenol in the presence of the thiophilic promoters

N-iodosuccinimide (NIS)–triﬂic acid, NIS–trimethylsi-

lyl triﬂate, NIS–silver triﬂate (AgOTf) or methyl-

sulfenyl bromide²⁷–AgOTf led mainly to a rapid loss of

Scheme 1.

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1097

the PMB ether group notwithstanding the ﬁnding that

such ethers can survive glycosylation conditions involv-

ing the use of thiophilic activators.²⁸When the N-tetra-

chlorophthaloyl group of compound 8 was replaced by

N-acetyl upon treatment with 1,2-diaminoethane, then

acetic anhydride and pyridine, and the product 9 was

tested with bromine^29,30or chlorine³¹in dichloro-

methane solution in order to form the glycosyl bromide

or chloride, some loss of the PMB ether group was

again observed. Removal of the ether protecting group

of 9 by treatment with triﬂuoroacetic acid (TFA) in the

presence of thiophenol gave crude alcohol 10, which

was silylated to give the fully protected 11 conversion

of which on treatment with chlorine in dichloromethane

proceeded rapidly and efﬁciently to give the required

Glycosylation of 4-nitrophenol with crude chloride

12 under phase transfer-catalysed conditions afforded

b-

-lactosaminide 13 (¹H NMR, CDCl₃, l 5.21, d, J_1,2

D

6.1 Hz, H-1) in 56% yield. Removal of the 6-O-silyl

ether was accomplished in high yield with HF–pyridine

to produce 14 which was sulfated to give 15 and

de-O-acetylated under standard conditions to afford 4

(¹H NMR D₂O, l 4.49, d, H-6_a; 4.35, dd, H-6_b; ¹³C

NMR D₂O, l 67.5, C-6). Compound 14 was also

de-O-acetylated to give the known 16,^22,32the desul-

fated analogue of 4, which was used as a positive

standard in the Pre6otella sulfatase assays.

The preparation of sulfate 4 from phenyl 2-deoxy-2-

tetrachlorophthalimido-1-thio-b-D-glucopyranoside by

this route required 11 steps and proceeded in an overall

yield of just 2.8%. The potentially shorter route to

glycosylating agent 12 using the 6-O-TBDMS ether

analogue of 6 as starting material was not viable;

treatment of the O-unprotected thioglycoside under

standard silylating conditions (TBDMSCl–imidazole–

DMF, TBDMSCl–pyridine or TBDMSOTf–2,6-

dimethylpyridine–CH₂Cl₂) gave none of the desired

product—presumably because of the base sensitivity of

the N-protecting group.

a-

D

-chloride 12 (¹H NMR, CDCl₃, l 6.17, d, J_1,23.7

Hz, H-1).

Taking advantage of the high selectivity of glycosyla-

tion at O-4 of 6-O-TBDPS-substituted-N-acetylglu-

cosaminide derivatives, and in particular the speciﬁc

access it affords to the N-acetyllactosamine framework

in a new, simple and efﬁcient way,³³we developed an

abbreviated route to compound 14 (Scheme 2). When

4-nitrophenyl glucosaminide 17, made by selective sub-

stitution of GlcNAc–ONP,³⁴was glycosylated with

tetra-O-acetyl-a-D-galactopyranosyl trichloroacetimi-

date^25,26with BF₃·OEt₂as promotor it gave, following

acetylation, disaccharide derivative 18 (50% from the

triol). Desilylation of 18 with HF–Py afforded the

previously prepared 14, the cleavage of the bulkier

TBDPS ether however requiring substantially longer

time than did that of the corresponding TBDMS ether

13. By this abbreviated route, sulfate 4 was obtained in

six steps from GlcNAc–ONP in 26.4% overall yield.

In the course of the development of the above routes

to compound 4, various others were explored brieﬂy

and several points of chemical signiﬁcance were noted.

Compound 20, made by highly selective reductive ring

opening of the acetal group of compound 19 (Scheme

3), was tested as a glycosyl acceptor, but it failed to

Scheme 2.

react with tetra-O-benzoyl-a-D-galactopyranosyl bro-

mide in the presence of AgOTf as promoter or with the

corresponding acetylated trichoroacetimidate together

with BF₃·OEt₂. Likewise allyl ether 23, made by the

sequence 4-nitrophenyl 2-acetamido-2-deoxy-4,6-O-(4-

methoxybenzylidene)-b-

2223 did not react under glycosylating conditions

with tetra-O-acetyl-a- -galactopyranosyl bromide or

trichloroacetimidate. Attempts to make the allyl ether

D

-glucopyranoside³⁵

21

D

Scheme 3.

1098

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

23 from the corresponding 3-allyl carbonate 25 (which

was made from the 3,4-diol 24) by heating in THF

solution with Pd(PPh₃)₄were unsatisfactory, the re-

quired compound 23 being obtained in only 19% yield

together with the 4-substituted allyl isomer 26 (24%)

and the 2,3-diallyl ether 27 (14%).

deoxy-a-

D

-glucopyranoside with tetra-O-benzoyl-a-D-

glucopyranosyl bromide.⁴⁴The product in this latter

case was assigned the structure with the introduced

glucosyl substituent bonded to the amide nitrogen atom

on the grounds of the absence and presence of ex-

changeable NH and OH resonances, respectively, in the

¹H NMR spectrum. This evidence, however, does not

preclude the possibility that glycosylation had occurred

at the oxygen atom of the enolic form of the acetamido

group. Similarly, in the present work it was concluded

that the acetamido group of chloroacetate 31 had un-

dergone glycosylation to give the incompletely charac-

terized compound 32. NMR evidence of its

dechloroacetylated derivative 33 did not permit clear

distinction between the O- and N-linked possibilities,

but chemical evidence given below favours the former.

During work with the primary chloroacetates 30–32,

their chloroacetyl groups were cleaved when small sam-

ples were either left in methanol at ambient temperature

overnight or heated under reﬂux brieﬂy in this solvent.

This may represent an advantageous way of removing

this protecting group which is normally cleaved with

the aid of acid or base catalysts or with such reagents as

thiourea.⁴⁵By the uncatalysed method, methanolysis of

chloroacetate 31 gave the known⁴⁶4,6-diol 34, and

analogously 32 produced 33 (Scheme 4). In the latter

case, however, monosaccharide derivative 31 was some-

times isolated instead of the disaccharide diol 33 indi-

cating the reactive nature of the inter-sugar linkage in

32. This reactivity was also reﬂected by failed attempts

to de-esterify compound 33 under Zemple´n conditions.

36

Although 2-acetamido-2-deoxy-D-glucopyranosides

having ester groups at C-3 can be difﬁcult to glycosy-

late at O-4, sometimes requiring speciﬁc adaptations for

efﬁcient reaction,^33,37the occasional report of such di-

rect substitution has appeared.³⁸On the other hand,

analogues with ether groups at C-3 can be efﬁciently

glycosylated^33,39and the lack of response of compounds

20 and 23 to glycosylation must presumably be ascribed

to the combined bulks of the protecting groups on O-3

and O-6.

Since

6-O-substituted-2-deoxy-2-phthalimido-D-

glucopyranosides can be glycosylated preferentially at

O-4,^33,40–42this approach was also considered for the

preparation of compound 4. 4-Nitrophenyl-3,4,6-tri-O-

acetyl-2-deoxy-2-phthalimido-b- -glucopyranoside (28)

D

was made from the corresponding glycosyl acetate,⁴³

but Zemple´n de-esteriﬁcation to give triol 29 was inefﬁ-

cient, suggesting that the base-catalysed transformation

of the N-phthaloyl group to N-acetyl, which this ap-

proach to compound 4 would require, could prove

problematical.

1

In this case, TLC and H NMR analysis of the crude

products indicated the presence of GlcNAc–ONP and

galactose. On the basis of the fragility of the inter-unit

bond of compound 32 the acetimidate structure with

glycosylation on the amido oxygen atom is favoured.

Attempts to explore the nature of the inter-unit bond

by crystallographic methods were frustrated by ‘twin-

ning’ of the crystals of compound 33.

When the 3-benzoate 31, made by stannylation–

chloroacetylation of GlcNAc–ONP³⁴to give the 6-ester

30 (Scheme 4) followed by 3-selective benzoylation, was

glycosylated, reaction did not occur at the C-4 hydroxyl

group as expected, but at the ambident acetamido

group, apparently in like manner to that observed on

glycosylation of benzyl 2-acetamido-3,6-di-O-acetyl-2-

Substrate speciﬁcity of recombinant Prevotella sulfa-

tase MdsA^7,8.—The substrate speciﬁcity of MdsA was

tested using

D-glucose 6-sodium sulfate (Glc 6-

OSO₃Na) and sulfates 1 and 4 together with and with-

out added auxiliary glycosidases (Aspergillus oryzae

extract, Sigma G 7138, 1993 catalogue, which has b-

galactosidase and b-N-acetylhexosaminidase activities).

It was expected that enzymic desulfation of 1 would

render it a substrate for the b-N-acetylhexosaminidase

with the consequent release of 4-nitrophenol. Similarly,

enzymic desulfation of 4 and sequential galactosidase

and b-N-acetylhexosaminidase action should release 4-

nitrophenol. From the results given in Table 1 and

those obtained in control experiments, the following

conclusions were drawn about the sulfatase speciﬁcity:

(i) there was a tenfold difference between the rates of

Scheme 4.

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1099

Table 1

Reactions of

mM) and 4-nitrophenyl b-

catalysed by MdsA sulfatase

D

-glucose 6-sodium sulfate (16 mM), 4-nitrophenyl 2-acetamido-2-deoxy-b-

D-glucopyranoside 6-sodium sulfate (1) (1

-galactopyranosyl-(14)-2-acetamido-2-deoxy-b- -glucopyranoside 6-sodium sulfate (4) (1 mM)

D

Substrate

Sulfatase reaction time

(min)

Glycosidase

added ^a

Product

quantiﬁed

Products observed ^b

Speciﬁc activity ^c

920

Glc 6-SO₃Na

30

20

60

no

yes

no

yes

glucose

ND

2511

267

0

1

nitrophenol

nitrophenol, GlcNAc,

358

GlcNAc–ONP (trace)

1

4

60

20

60

no

yes

no

yes

no

nitrophenol

GlcNAc–ONP

ND

Gal (trace)

ND

6

0

^aAsperigillus oryzae extract (Sigma G7138, 1993 catalogue) containing b-galactosidase and b-N-acetylhexosaminidase activities.

For sulfate 1, incubations were carried out in the presence of the sulfatase and glycosidases. For compound 4, the substrate was

incubated ﬁrst with the sulfatase, the reaction was terminated, and the glycosidases were added prior to a second incubation.

^bBy paper chromatography after 60 min reaction; ND, not determined; GlcNAc–ONP, 4-nitrophenyl 2-acetamido-2-deoxy-b-

D

-glucopyranoside; Gal,

D-galactose.

^cnmol product min⁻¹(mg sulfatase protein)⁻¹

.

product formation from Glc 6-OSO₃Na and ester 1, but

as the concentrations of substrates and reaction condi-

tions were different, we cannot speculate on the rea-

son(s) for the variation; (ii) the 6-ester (1) was not a

substrate for the auxiliary glycosidases, but following

its desulfation by MdsA, it was cleaved by this enzyme

preparation to give GlcNAc, 4-nitrophenol, and sulfate.

The reaction catalysed by the sulfatase precedes glyco-

sidic bond cleavage (although the relative rates of the

two reactions were not determined), since the action of

MdsA on ester 1 without the auxiliary glycosidases was

shown to give GlcNAc–ONP (by paper chromatogra-

phy), and only a trivial amount of free 4-nitrophenol, if

any; and (iii) disaccharide sulfate 4 did not react in the

presence of the sulfatase to give the unesteriﬁed com-

pound 16 since, after attempted desulfation of 4, the

auxiliary glycosidases failed to liberate 4-nitrophenol.

The auxiliary glycosidases by themselves did release

4-nitrophenol, Gal and GlcNAc from compound 16.

To conﬁrm that compound 4 did not react in the

presence of the sulfatase, the concentrations of the

auxiliary glycosidases were increased up to tenfold

without change to the initial observations (data not

shown). These results, obtained largely by quantitative

colorimetric methods, were conﬁrmed by paper chro-

matographic examination of the reaction products.

The assay results using glycoside esters 1 and 4

(Table 1) indicate that the sulfatase MdsA desulfates

the GlcNAc 6-OSO₃Na moiety when it is terminal, but

Substrate speciﬁcity of the B. fragilis sulfatases.—

Two galactose-6-sulfatase and two galactose-3-sulfatase

isozymes were partly-puriﬁed by DEAE chromatogra-

phy from the B. fragilis cell free extract. Galactose-3-

sulfatase and galactose-6-sulfatase activities (Table 2)

were assayed by release of 4-nitrophenol from 4-nitro-

phenylgalactoside sulfates 2 or 3, respectively, as sub-

strates in combination with the same auxiliary

glycosidase mixture as used in the Pre6otella case

above, although only the b-galactosidase activity was

required in this case. Auxiliary glycosidases alone did

not cleave 4-nitrophenol from the sulfated substrates.

Gal-ONP was shown by paper chromatography to be

the product formed from the action of each of the

partially-puriﬁed sulfatases on their own, conﬁrming

that the B. fragilis enzymes were sulfatases rather than

glycosidases. Each of the sulfatase enzymes was sub-

strate speciﬁc. This is consistent with the observation

by Salyers et al.⁴⁷that the predominant Gram-negative

bacteria from the colon often possess more than one

isozyme catalysing a single step in polysaccharide

degradation. The above data represent another example

of this phenomenon, the purpose of which may be to

circumvent metabolic disruption by mutation to a criti-

cal gene.

The B. fragilis cell-free extract alone was capable of

releasing 4-nitrophenol from the sulfated substrates,

presumably because it contains sulfatase and galactosi-

dase activities. The natural substrates for these galac-

tose-3-sulfatases and galactose-6-sulfatases are not

known precisely, but since B. fragilis is able to use

mucin for growth,⁴⁸we speculate that they may be

not when it is substituted at the 4 position by b-D-Gal,

i.e., when it is internal within an oligosaccharide chain.

Thus, the enzyme appears to be an ‘exo-sulfatase’.

1100

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

involved in desulfating oligosaccharide chains that ter-

minate in Gal 6-OSO₃H or Gal 3-OSO₃H, such groups

being chain-terminating.²Possible conﬁrmation will

have to await the isolation of pure enzymes and their

testing with appropriate mucins.

It is anticipated that the activity of mucin-desulfating

enzymes in diverse bacteria present in the digestive tract

should be measurable using the model substrates de-

scribed above, and assays for bacterial sulfatases that

de-esterify saccharide moieties containing GlcNAc 6-

OSO₃H, Gal 3-OSO₃H, and Gal 6-OSO₃H in faeces of

patients with colon diseases, in dental plaque of pa-

tients with oral diseases and in pulmonary sputum from

patients with lung diseases, could lead to important

new means of assessing speciﬁc bacterial involvement in

these conditions.^3,49,50

Me₄Si, l 0) or D₂O (internal acetone l 2.23) or at 75.5

MHz (¹³C) for solutions in CDCl₃(centre line l 77.0),

(CD₃)₂SO (centre line l 39.7) or D₂O (internal acetone,

1

l 31.5). Assignments of H and ¹³C resonances were

based on 2D (¹H–¹H DQF-COSY, ¹H–¹³C HSQC) and

DEPT experiments. The ¹³C spectra gave unambiguous

data on the numbers of protons bonded to each carbon

atom; these are expressed as s, d, t, and q being the

multiplicities expected in C,H undecoupled spectra.

High-resolution +ve FABMS determinations were

performed on a VG70-250S (VG Analytical) double

focussing, magnetic sector mass spectrometer equipped

with a standard liquid secondary caesium ion or

sodium ion gun in a glycerol or nitrobenzyl alcohol

matrix. IR data were recorded on a Perkin–Elmer 1600

series FTIR spectrometer.

4-Nitrophenyl 2-acetamido-2-deoxy-i-

oside 6-sodium sulfate (1).—To a solution of 4-nitro-

phenyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-b- -gluco-

D-glucopyran-

D

3. Experimental

pyranoside¹⁵(302 mg, 0.71 mmol), in dry DMF (12

mL) was added SO₃·pyridine (450 mg, 1.42 mmol), and

the mixture was stirred at ambient temperature for 2 h.

Methanol (0.2 mL) was added and the volatiles were

removed to leave a colourless oil which was dissolved in

MeOH (26 mL) and NaOMe–MeOH solution (2.0 mL,

1 M) was added. The mixture was stirred at ambient

temperature for 1.5 h (during which time a precipitate

formed), neutralized with Amberlite IRC 50 (H⁺) resin,

water was added to dissolve the precipitate, and the

resin was ﬁltered off and the volatiles were evaporated.

Chromatography [5:4:1 CH₂Cl₂–MeOH–NH₄OH (d

0.88)] afforded the title compound 1 as the ammonium

salt which was converted into its sodium salt by passage

through a Dowex 50X8 100 (Na⁺) resin column (10×

2.5 cm) eluted with water. The fractions containing

sodium salt 1 were evaporated and the resulting solid

was suspended in MeOH (1.0 mL), centrifuged (3000

rpm, 5 min) and the MeOH decanted off. This process

General chemical methods.—Melting points were

measured on a Reichert hot stage microscope and are

uncorrected. Optical rotations were determined with a

Perkin–Elmer 241 polarimeter and are in units of 10⁻¹

deg cm²g⁻¹(conventionally °). TLC was performed on

glass or aluminium backed Silica Gel 60 F254 (E.

Merck) with detection by UV absorption and/or by

heating after dipping in (NH₄)₆Mo₇O₂₄·6 H₂O (5 g) and

Ce(SO₄)₂(100 mg) in 5% aq H₂SO₄(100 mL) solution.

Chromatography (ﬂash column) was performed on

Scharlau or Merck Silica Gel 60 (40–60 mm). Chro-

matography solvents were distilled prior to use. Anhy-

drous solvents were those commercially available.

Organic solutions were dried over MgSO₄and evapo-

rated under reduced pressure. All air-sensitive reactions

were performed under argon. NMR spectra were

recorded on a Bruker AC300E spectrometer at 300

MHz (¹H) for solutions in CDCl₃, (CD₃)₂SO (internal

Table 2

Reactions of the 3-sulfate (2) and the 6-sulfate (3) of 4-nitrophenyl b-

D-galactopyranoside catalysed by B. fragilis sulfatases

Enzyme

Sample

Substrate Speciﬁc

activity ^agalactosidase

Presence of endogenous Products formed with sulfatase ^c

Without

With

glycosidase ^bglycosidase ^b

Galactose 3-sulfatase Cell free extract

Galactose 3-sulfatase DEAE fraction a

Galactose 3-sulfatase DEAE fraction c

Galactose 6-sulfatase Cell free extract

Galactose 6-sulfatase DEAE fraction b

Galactose 6-sulfatase DEAE fraction d

2

3

11.49

223.7

130.7

7.37

407.5

111.9

yes

no

yes

no

trace

ND

Gal-ONP

ND

Gal-ONP

Gal, nitrophenol

ND

Gal, nitrophenol

^anmol 4-Nitrophenol released min⁻¹(mg enzyme protein)⁻¹

^bAsperigillus oryzae extract (Sigma G7138, 1993 catalogue) containing b-galactosidase and b-N-acetylhexosaminidase activities.

^cBy paper chromatography; ND, not determined; Gal-ONP, 4-nitrophenyl b-

-galactopyranoside; Gal, -galactose.

.

D

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1101

was repeated twice and 1 was obtained as a colourless

solid after drying (228 mg, 72%); [h]²_D³−31° (c 0.57,

water); IR (CHBr₃mull): w 3693–3170 (s), 1643 (s),

zene (100 mL) and the mixture heated under reﬂux in a

Dean–Stark apparatus for 1 h. After cooling to ambi-

ent temperature, the volatiles were evaporated and the

1

1585 (s), 1510 (s), 1349 (s), 1248 (s), 1071 (s) cm⁻¹; H

residue dissolved in CH₂Cl₂(100 mL). b-D-Galactose

NMR (D₂O): l 8.12 (d, 2 H, J 9.2 Hz, ArH), 7.17 (d,

2 H, J 9.2 Hz, ArH), 5.32 (d, 1 H, J_1,28.4 Hz, H-1),

4.43 (dd, 1 H, J_6a,6b11.4, J_5,6a1.8 Hz, H-6a), 4.26 (dd,

1 H, J_5,6b, 5.8 Hz, H-6b), 4.07 (dd, 1 H, J_2,39.8 Hz,

H-2) 3.95 (ddd, 1 H, H-5), 3.74 (t, 1 H, H-3), 3.64 (t, 1

H, H-4), 2.04 (s, 3 H, Ac); ¹³C NMR (D₂O): l 176.2 (s),

162.9 (s), 143.9 (s), 127.4 (d), 117.8 (d), 99.9 (d, C-1),

75.4 (d, C-5), 74.5 (d, C-3), 70.7 (d, C-4), 68.2 (t, C-6),

56.5 (d, C-2), 23.4 (q, Me). FABMS: m/z Calcd. for

pentaacetate (20.0 g, 51.2 mmol) was added followed by

BF₃·OEt₂(9.5 mL, 74.97 mmol) and the mixture stirred

for 5.5 h at ambient temperature, left at 4 °C overnight,

and stirred a further 6 h at ambient temperature. It was

washed with aq NaOH (1 M), water, dried, and evapo-

rated and dissolved in CH₃CN. The solution was

washed with hexanes, evaporated, and the residue crys-

tallized from EtOH to give 4-nitrophenyl 2,3,4,6-tetra-

O-acetyl-b-

D

-galactopyranoside (16.5 g, 69%),

a

C₁₄H₁₈N₂NaO₁₁S

445.0548.

[M+H]⁺

445.0529.

Found:

portion (10.0 g, 21.3 mmol) of which was dissolved in

CH₂Cl₂(20 mL) and MeOH (150 mL). Aqueous

sodium hydroxide solution (5 mL, 1 M) was added and

the mixture stirred for 2 h at ambient temperature,

neutralized with Dowex 50X8 100 (H⁺) resin, ﬁltered,

the ﬁltrate was evaporated and the residue crystallized

from EtOH (5.19 g, 81%); mp 183–184 °C, lit. 181–

182 °C (EtOH);⁵¹[h]_D²⁰−86° (c 1.12, water), lit. [h]²_D⁰

−74.7° (c 9.0, water).⁵¹

4-Nitrophenyl i-D-galactopyranoside 3-triethylammo-

nium sulfate (2).—Dibutyltin(IV) oxide (610 mg, 2.45

mmol) was added to a solution of 4-nitrophenyl 4,6-O-

isopropylidene-b-D

-galactopyranoside¹⁶(830 mg, 2.43

mmol) in toluene (40 mL) and the mixture was heated

under reﬂux in a Dean–Stark apparatus for 1 h. The

toluene was evaporated and the residue dissolved in

DMF (20 mL), cooled to 0 °C, SO₃·pyridine (406 mg,

2.55 mmol) added, the mixture was stirred at ambient

temperature overnight and the volatiles were evapo-

rated. Excess 9:1 EtOH–Et₃N (15 mL) was added and

evaporated and the residue was chromatographed

(60:20:1 toluene–EtOH–Et₃N) to give 4-nitrophenyl

4-Nitrophenyl i-D-galactopyranoside 6-triethylammo-

nium sulfate (3).

Method A. To a solution of 4-nitrophenyl b-D-

galactopyranoside (1.00 g, 3.32 mmol), in dry DMF (10

mL) at −10 −15 °C, SO₃·pyridine (550 mg, 3.46

mmol) was added and the mixture was allowed to warm

to ambient temperature overnight. The solvent was

evaporated and the residue chromatographed

(200:100:1100:100:1 EtOAc–EtOH–Et₃N) to give

sulfate 3 as a colourless foam (700 mg, 44%); [h]_D²⁰

−54° (c 0.625, MeOH); TLC (100:100:1 toluene–

EtOH–Et₃N): R_f0.14; IR (neat): w 3658–3145 (br,s),

1590 (s), 1515 (s), 1494 (s), 1339 (s), 1248 (s), 1077 (s),

4,6-O-isopropylidene-b-D-galactopyranoside 3-triethy-

lammonium sulfate as a colourless solid (870 mg, 69%).

TLC (20:20:1 toluene–EtOH–Et₃N) R_f0.37. To a sus-

pension of this product (800 mg, 1.53 mmol) in EtOH

(30 mL) was added Amberlyst A15 (H⁺) resin (1.00 g)

and the mixture was stirred for 2 h at ambient temper-

ature then ﬁltered. Excess Et₃N (2 mL) was added and

evaporated then the residue chromatographed twice

(20:20:1 toluene–EtOH–Et₃N then 90:10:1 CH₂Cl₂–

MeOH–Et₃N) to give 2 as a colourless gum (420 mg,

57%); [h]²_D¹−34° (c 2.4, MeOH); TLC (20:20:1 tolu-

ene–EtOH–Et₃N): R_f0.26, (90:10:1 CH₂Cl₂–MeOH–

Et₃N): R_f0.24; IR (neat): w 3669–3145 (s), 1590 (s),

1510 (s), 1488 (s), 1339 (s), 1248 (s), 1072 (s), 986 (s)

1

1034 (s), 997 (s) cm⁻¹; H NMR (CD₃OD): l 8.23 (d,

2 H, J 9.2 Hz, ArH), 7.28 (d, 2 H, J 9.3 Hz, ArH), 5.02

(d, 1 H, J_1,27.7 Hz, H-1), 4.26–4.13 (m, 2 H, H-6a,

H-6b), 4.07 (br.t, 1 H, J_5,6a=J_5,6b6.2 Hz, H-5), 3.96 (d,

1 H, J_3,43.3 Hz, H-4), 3.83 (dd, 1 H, J_2,39.7 Hz, H-2),

3.62 (dd, 1 H, H-3), 3.20 (q, 6 H, J 7.3 Hz, CH₃CH₂N),

1.30 (t, 9 H, CH₃CH₂N); ¹³C NMR (CD₃OD): l 163.9

(s), 143.8 (s), 126.6 (d), 117.8 (d), 102.2 (d, C-1), 74.8

(d, C-5), 74.5 (d, C-3), 71.9 (d, C-2), 69.9 (d, C-4), 67.8

(t, C-6), 48.0 (t, CH₃CH₂N), 9.2 (q, CH₃CH₂N).

FABMS: m/z Calcd. for C₁₈H₃₁N₂O₁₁S [M+H]⁺

483.1649 Found: 483.1665.

1

cm⁻¹; H NMR (CD₃OD): l 8.21 (d, 2 H, J 9.2 Hz,

ArH), 7.26 (d, 2 H, J 9.3 Hz, ArH), 5.15 (d, 1 H, J_1,2

7.6 Hz, H-1), 4.39–4.33 (m, 2 H, H-4, H-3), 4.01 (dd, 1

H, J_2,39.3 Hz, H-2), 3.90–3.70 (m, 3 H, H-6a, H-6b,

H-5), 3.22 (q, 6 H, J 7.3 Hz, CH₃CH₂N), 1.31 (t, 9 H,

CH₃CH₂N); ¹³C NMR (CD₃OD): l 163.8 (s), 143.8 (s),

126.6 (d), 117.8 (d), 102.0 (d, C-1), 81.6 (d, C-3), 76.9

(d, C-5), 70.3 (d, C-2), 68.3 (d, C-4), 62.2 (t, C-6), 48.0

(t, CH₃CH₂N), 9.2 (q, CH₃CH₂N). FABMS: m/z

Calcd. for C₁₈H₃₁N₂O₁₁S [M+H]⁺483.1649. Found:

483.1639.

Method B. Dibutyltin(IV) oxide (270 mg, 1.08 mmol)

was added to a solution of 4-nitrophenyl 3,4-O-iso-

propylidene-b-D

-galactopyranoside¹⁷(330 mg, 0.97

mmol) in toluene (10 mL) and the mixture was heated

under reﬂux in a Dean–Stark apparatus until clear (2

h). The toluene was evaporated and the residue sus-

pended in DMF (4 mL), cooled to 0 °C then

SO₃·pyridine (172 mg, 1.08 mmol) was added. After a

few minutes most solids had dissolved. The mixture was

4-Nitrophenyl i-D-galactopyranoside.—Bis[tributyl-

tin(IV)] oxide (19.6 mL, 38.5 mmol) was added to a

solution of 4-nitrophenol (10.7 g, 76.9 mmol) in ben-

1102

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

stirred overnight at ambient temperature, the volatiles

were evaporated and the residue was chromatographed

(60:20:1 toluene–EtOAc–Et₃N) to give 4-nitrophenyl

Phenyl (2,3,4,6-tetra-O-acetyl-i-

(14)-2-deoxy-6-O-(4-methoxybenzyl)-2-tetrachloro-

phthalimido-1-thio-i- -glucopyranoside (7).—A solu-

D-galactopyranosyl)-

D

3,4-O-isopropylidene-b-

D

-galactopyranoside 6-triethy-

tion of BF₃·OEt₂in CH₂Cl₂(170 mL, 1 M) was added

lammonium sulfate (340 mg, 67%). A sample (60 mg,

0.115 mmol) was dissolved in EtOH (10 mL), Am-

berlyst A15 (H⁺) resin (1.00 g) was added and the

mixture was stirred at ambient temperature overnight.

After ﬁltration and evaporation of the solvent the

residue was chromatographed (100:100:1 toluene–

EtOH–Et₃N) to give sulfate 3 as a colourless foam (34

mg, 61%), identical to that made by method A.

to a solution of 6 (2.05 g, 3.10 mmol) and tetra-O-ace-

tyl-a-D

-galactopyranosyl trichloroacetimidate^25,26(2.30

g, 4.67 mmol) in anhyd CH₂Cl₂(8 mL) and hexanes (8

mL) at −30 °C. The mixture was stirred for 1.5 h

during which time a colourless precipitate formed. Et₃N

(1.0 mL) was added, the mixture was warmed to ambi-

ent temperature and the volatiles were evaporated.

Chromatography (3:74:6 EtOAc–hexanes) afforded

disaccharide derivative 8 as a yellow foam which on

trituration with EtOH gave an amorphous solid (1.80 g,

59%); [h]²_D¹+27° (c 1.32, CHCl₃); TLC (3:2 EtOAc–

Phenyl

2-deoxy-6-O-(4-methoxybenzyl)-2-tetra-

chlorophthalimido-1-thio-i-

D

-glucopyranoside

(6).—

Sodium methoxide in MeOH (14.5 mL, 1 M)²⁴was

1

added to a solution of phenyl 3,4,6-tri-O-acetyl-2-de-

hexanes): R_f0.41; H NMR (CDCl₃): l 7.42–7.39 (m, 2

oxy-2-tetrachlorophthalimido-1-thio-b-

D

-

H, ArH), 7.29–7.20 (m, 5 H, ArH), 6.92 (dd, 2 H, J

6.8, 2.0 Hz, ArH), 5.54 (d, 1 H, J_1,210.4 Hz, H-1), 5.33

(d, 1 H, J_3%,4%3.1 Hz, H-4%), 5.16 (dd, 1 H, J_2%,3%10.4, J_1%,2%

8.0 Hz, H-2%), 4.95 (dd, 1 H, H-3%), 4.54 (d, 1 H, J 11.6

Hz, CH_aAr), 4.52–4.43 (m, 2 H, H-1%, CH_bAr), 4.35

(br. t, 1 H, after D₂O exchange became a dd, J_2,310.3,

J_3,48.0 Hz, H-3), 4.23 (t, 1 H, H-2), 4.07–4.04 (m, 2 H,

H-6a%, H-6b%), 4.03 (d, 1 H, J_OH,30.9 Hz, exchanged

with D₂O, OH), 3.91 (br.t, 1 H, J_5%,6a%ꢀJ_5%,6b%ꢀ6.2 Hz,

H-5%), 3.83 (s, 3 H, OMe), 3.76–3.63 (m, 4 H, H-6a,

H-6b, H-5, H-4), 2.12, 2.00, 1.98, 1.97, (4s, 12 H, 4 Ac);

¹³C NMR (CDCl₃): l 170.5 (s), 170.0 (s), 169.9 (s),

169.0 (s), 163.4 (s), 162.5 (s), 159.4 (s), 140.3 (s), 132.6

(d), 131.7 (s), 130.1 (s), 129.8 (s), 129.5 (d), 128.9 (d),

128.0 (d), 127.3 (s), 114.0 (d), 101.5 (d), 82.9 (d), 81.5

(d), 78.3 (d), 73.4 (t), 71.3 (d), 70.7 (d), 70.4 (d), 68.7

(d), 67.7 (t), 66.8 (d), 61.4 (t), 55.9 (d), 55.3 (q), 20.7 (q),

20.53 (q), 20.45 (q). FABMS: m/z Calcd. for

C₄₂H₄₁Cl₄CsNO₁₆S [M+Cs]⁺: 1119.9955. Found:

1119.9928.

glucopyranoside²³(7.60 g, 11.4 mmol) in MeOH (875

mL) and CH₂Cl₂(15 mL) at 40–45 °C. The solution

was stirred for 15 min, acidiﬁed to ꢀpH 4 with Dowex

50X8 100 (H⁺) resin and ﬁltered. Evaporation of the

ﬁltrate afforded phenyl 2-deoxy-2-tetrachlorophthal-

imido-1-thio-b-

D

-glucopyranoside²³

as

a

cream

coloured solid (5.62 g, 91%), a portion (4.30 g, 7.98

mmol) of which was suspended in benzene (600 mL),

(Bu₃Sn)₂O (4.47 mL, 8.78 mmol) was added and the

mixture was heated under reﬂux in a Dean–Stark ap-

paratus for 16 h. After cooling, concentration to ꢀ120

mL and the addition of Bu₄NI (3.54 g, 9.58 mmol) and

4-methoxybenzyl chloride (1.6 mL, 11.97 mmol), the

mixture was heated to 95 °C for 2 h, further chloride (1

mL) was added and heating was continued for 2 h

during which time all solids had dissolved. A third

portion of the chloride (2.5 mL) and Bu₄NI (3.00g)

were added and heating at 95 °C with stirring was

continued for a further 16 h. The volatiles were evapo-

rated and chromatography (9:1 toluene–acetone) of the

residue gave the ether 6 as a yellow foam which on

heating to 85 °C under vacuum for 30 min became a

yellow glass (1.99 g, 38%); [h]²_D⁰+24° (c 1.00, CHCl₃);

TLC (4:1 toluene–acetone): R_f0.58; ¹H NMR (CDCl₃):

l 7.38–7.35 (m, 2 H, ArH), 7.25–7.20 (m, 5 H, ArH),

6.89 (d, 2 H, J 8.6 Hz, ArH), 5.55 (d, 1 H, J_1,210.1 Hz,

H-1), 4.55, 4.49 (ABq, 2 H, J 11.5 Hz, CH₂Ar), 4.37–

4.30 (m, 1 H, H-3), 4.22 (t, 1 H, H-2), 3.86–3.77 (m, 4

H, H-6a, OMe), 3.73 (dd, 1 H, J_6a,6b10.3, J_5,6b4.3 Hz,

H-6b), 3.67–3.55 (m, 2 H, H-5, H-4), 3.15 (br.s, 1 H,

exchanged with D₂O, OH), 2.54 (br.s, 1 H, exchanged

with D₂O, OH); ¹³C NMR (CDCl₃): l 163.5 (br. s),

162.9 (br.s), 159.4 (s), 140.3 (s), 132.3 (d), 131.9 (s),

129.6 (s), 129.4(d), 128.9 (d), 127.9 (d), 127.2 (br.s),

113.9 (d), 83.1 (d, C-1), 77.9 (d, C-4 or C-5), 73.5 (d,

C-4 or C-5), 73.4, (t, CH₂Ar), 72.2 (d, C-3), 70.0 (t,

C-6), 56.0 (d, C-2), 55.3 (q, OMe). FABMS: m/z Calcd.

for C₂₈H₂₂Cl₄NO₇S (M-H)⁺: 655.9871. Found:

655.9840.

Phenyl (2,3,4,6-tetra-O-acetyl-i-

(14)-3-O-acetyl-2-deoxy-6-O-(4-methoxybenzyl)-2-

tetrachlorophthalimido-1-thio-i- -glucopyranoside (8).

D-galactopyranosyl)-

D

—A solution of 7 (1.85 g, 1.87 mmol) in pyridine (10

mL) and Ac₂O (5 mL) was left to stand at ambient

temperature for 16 h then poured into ice-water and

extracted with CH₂Cl₂(3×30 mL). The combined

extracts were washed with aq satd NaHCO₃, water,

dried and evaporated to a yellow foam. Chromatogra-

phy (2:3 EtOAc–hexanes) gave 8 as a colourless foam

(1.62 g, 84%); [h]¹_D⁹+31° (c 1.43, CHCl₃); TLC (1:1

1

EtOAc–hexanes): R_f0.44; H NMR (CDCl₃): l 7.42–

7.39 (m, 2 H, ArH), 7.31–7.22 (m, 5 H, ArH), 6.94 (dd,

2 H, J 6.7, 2.0 Hz, ArH), 5.65 (d, 1 H, J_1,210.6 Hz,

H-1), 5.61 (dd, 1 H, J_2,310.0, J_3,48.9 Hz, H-3), 5.28 (d,

1 H, J_3%,4%3.1, H-4%), 5.02 (dd, 1 H, J_2%,3%10.4, J_1%,2%7.9

Hz, H-2%), 4.86 (dd 1 H, H-3%), 4.68, 4.46 (ABq, 2 H, J

11.6 Hz, CH₂Ar), 4.53 (d, 1 H, H-1%), 4.27 (t, 1 H, H-2),

4.04–3.98 (m, 3 H, H-6a%, H-6b%, H-4), 3.83 (s, 3 H,

OMe), 3.76 (br.s, 2 H, H-6a, H-6b), 3.69–3.60 (m, 2 H,

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1103

H-5%, H-5), 2.11 2.04, 1.96, 1.95, 1.89, (5s, 15 H, 5 Ac);

¹³C NMR (CDCl₃): l 170.5 (s), 170.3 (s), 170.2 (s),

170.1 (s), 168.8 (s), 163.1 (s), 162.5 (s), 159.5 (s), 140.7

(s), 140.4 (s), 133.1 (d), 131.1 (s), 130.1 (s), 129.9 (s),

129.6 (d), 129.0 (d), 128.3 (d), 127.2 (s), 126.9 (s), 114.0

(d), 100.4 (d), 82.5 (d), 79.0 (d), 74.9 (d), 73.3 (t), 72.1

(d), 71.0 (d), 70.5 (d), 69.1 (d), 67.2 (t), 66.8 (d), 60.8 (t),

55.3 (q), 54.8 (d), 20.6 (q), 20.5 (q). FABMS: m/z

Calcd. for C₄₄H₄₄Cl₄NO₁₇S [M+H]⁺: 1030.1084.

Found: 1030.1030.

20.7 (q), 20.6 (q), 20.5 (q). FABMS: m/z Calcd. for

C₃₀H₄₀NO₁₅S [M+H]⁺: 686.2119. Found: 686.2101.

Phenyl (2,3,4,6-tetra-O-acetyl-i-

(14)-2-acetamido-3-O-acetyl-6-O-tert-butyldimethyl-

silyl-2-deoxy-1-thio-i- -glucopyranoside (11).—2,6-

D-galactopyranosyl)-

D

Dimethylpyridine (0.19 mL, 1.64 mmol) was added to a

solution of 6-ol 10 (560 mg, 0.82 mmol) in CH₂Cl₂(6

mL) and the mixture was cooled to −20 °C. TBDM-

SOTf (0.28 mL, 1.23 mmol) was added dropwise, the

mixture stirred for 30 min, warmed to ambient temper-

ature and stirred for a further 15 min. The reaction

mixture was diluted with EtOAc (50 mL) and washed

with aq HCl (10%), aq satd NaHCO₃, dried and the

solvent was evaporated to leave a gum. Chromatogra-

phy (4:1 toluene–acetone) gave silyl ether 11 as a

colourless foam (521 mg, 79%); [h]²_D²−26° (c 1.13,

Phenyl (2,3,4,6-tetra-O-acetyl-i-D-galactopyranosyl)-

(14)-2-acetamido-3-O-acetyl-2-deoxy-1-thio-i-

D

-

glucopyranoside (10).—Dry 1,2-diaminoethane (0.37

mL, 5.5 mmol) was added to a solution of 8 (1.13 g,

1.10 mmol) in dry EtOH (30 mL, denatured with 5%

PrⁱOH) and the mixture was stirred at 60 °C for 4 h.

Coevaporation several times with toluene gave a

residue which was dissolved in pyridine (30 mL) and

Ac₂O (15 mL) and left at ambient temperature for 2

days. The solvent was evaporated and the residue dis-

solved in EtOAc (50 mL). The solution was washed

with aq HCl (10%), aq satd NaHCO₃, water, and dried

and the solvent was evaporated. Chromatography

(4:17:3 toluene–acetone) gave crude 9 as a bright

yellow foam (530 mg). ¹H NMR (CDCl₃) indicated it to

be approximately 90% pure. Treatment of 7 (1.78 g,

1.80 mmol) with 1,2-diaminoethane (0.6 mL, 9.00

mmol) in a similar way to that used for compound 8

and then Ac₂O–pyridine followed by chromatography

(50:50:150:50:2 toluene–EtOAc–MeOH) also gave

acetamido compound 9 (941 mg) as a pale yellow foam

of approximately 90–95% purity. To a solution of

crude 9 (810 mg, ꢀ1.01 mmol) in CH₂Cl₂(30 mL) was

added PhSH (15 mL) and TFA (10 mL) and the

mixture was left to stand at ambient temperature for 15

min. The volatiles were evaporated and the residue

ﬁltered through a small plug of SiOH (eluted with 1:1

EtOAc–hexanes then 85:15 EtOAc–MeOH) to give

crude alcohol 10 as a yellow gum after evaporation.

Chromatography (50:50:150:50:2 toluene–EtOAc–

MeOH) gave pure 10 as a colourless foam (603 mg,

87%); [h]²_D⁰−22° (c 1.11, CHCl₃); TLC (50:50:3 tolu-

ene–EtOAc–MeOH): R_f0.36; ¹H NMR (CDCl₃): l

7.48–7.38 (m, 2 H, ArH), 7.35–7.22 (m, 3 H, ArH),

5.85 (d, 1 H, J_NH,29.6 Hz, exchanged with D₂O, NH),

5.34 (d, 1 H, J_3%,4%3.0 Hz, H-4%), 5.13–5.03 (m, 2 H,

H-2%, H-3), 4.99 (dd, 1 H, J_2%,3%10.4 Hz, H-3%), 4.75 (d,

1 H, J_1,210.4 Hz, H-1), 4.61 (d, 1 H, J_1%,2%7.7 Hz, H-1%),

4.19–4.04 (m, 3 H, H-6a%, H-6b%, H-2), 3.96–3.73 (m, 3

H, H-5%, H-6a, H-4), 3.72 (dd, 1 H, J_6a,6b12.3, J_5,6b3.1

Hz, H-6b), 3.42 (br.dt, 1 H, H-5), 2.12, 2.06, 2.05, 2.04,

1.98, 1.96, (6s, 19 H, 6 Ac, OH); ¹³C NMR (CDCl₃): l

171.1 (s), 170.3 (s), 170.1 (s), 170.0 (s), 169.2 (s), 132.9

(s), 131.8 (d), 129.0 (d), 127.8 (d), 101.0 (d), 86.9 (d),

79.0 (d), 74.8 (d), 74.4 (d), 70.9 (d), 70.6 (d), 69.3 (d),

66.8 (d), 60.9 (t), 60.7 (t), 53.0 (d), 23.2 (q), 20.8 (q),

1

CHCl₃); TLC (7:3 toluene–acetone): R_f0.32; H NMR

(CDCl₃): l 7.50–7.45 (m, 2 H, ArH), 7.29–7.26 (m, 3

H, ArH), 5.54 (d, 1 H, J_NH,29.5 Hz, exchanged with

D₂O, NH), 5.35, (d, 1 H, J_3%,4%3.2 Hz, H-4%), 5.08 (dd,

1 H, J_2%,3%10.3, J_1%,2%7.9 Hz, H-2%), 5.01 (t, 1 H J_3,4=J_2,3

9.1 Hz, H-3), 4.93 (dd, 1 H, H-3%), 4.66 (d, 1 H, H-1%),

4.62 (d, 1 H, J_1,210.2 Hz, H-1), 4.15–4.01 (m, 3 H,

H-6a%, H-6b%, H-2), 3.94 (t, 1 H, H-4), 3.91–3.77 (m, 3

H, H-5%, H-6a, H-6b), 3.34 (dt, 1 H, J_5,6a1.9 Hz, H-5),

2.13, 2.00, 1.98, 1.96 (4s, 12 H, 4 Ac), 2.05 (s, 6 H, 2

Ac), 0.93 (s, 9 H, Si-Bu^t), 0.12, 0.10 (2s, 6 H, 2 Si-Me);

¹³C NMR (CDCl₃): l 171.1 (s), 170.3 (s), 170.14 (s),

170.05 (s), 169.9 (s), 169.0 (s), 133.2 (s), 132.4 (d), 128.9

(d), 127.8 (d), 100.2 (d), 87.0 (d), 79.6 (d), 74.2 (d), 73.8

(d), 71.1 (d), 70.7 (d), 69.3 (d), 66.9 (d), 61.2 (t), 61.1 (t),

52.9 (d), 25.9 (q), 23.3 (q), 20.8 (q), 20.7 (q), 20.6 (q),

20.5 (q), 18.3 (s), −5.0 (q), −5.3 (q). FABMS: m/z

Calcd. for C₃₆H₅₄NO₁₅SSi [M+H]⁺: 800.2984. Found:

800.2961.

4-Nitrophenyl (2,3,4,6-tetra-O-acetyl-i-

ranosyl)-(14)-2-acetamido-3-O-acetyl-6-O-tert-butyl-

dimethylsilyl-2-deoxy-i- -glucopyranoside (13).—A so-

D-galactopy-

D

lution of Cl₂in CH₂Cl₂(0.81 mL, 0.42 mmol, from a

stock solution of 37 mg mL⁻¹of Cl₂in CH₂Cl₂) was

added to a solution of 11 (280 mg, 0.35 mmol) in

CH₂Cl₂(3 mL) and the mixture was left at ambient

temperature for 15 min. The solvent was evaporated to

give crude glycosyl chloride 12 as a pale yellow gum. ¹H

NMR (CDCl₃l 6.17, 1 H, J_1,23.7 Hz, H-1). 4-Nitro-

phenol (97 mg, 0.71 mmol), aq K₂CO₃(1 mL, 1 M) and

Bu₄NHSO₄(140 mg, 0.35 mmol) were added to a

solution of this chloride in CH₂Cl₂(2 mL) and the

mixture was stirred vigorously for 16 h. The mixture

was diluted with CH₂Cl₂(20 mL) and the organic phase

washed with water (3×5 mL), dried and the volatiles

were evaporated. Chromatography (4:1 toluene–ace-

tone) gave aryl glycoside 13 as a colourless foam (161

mg, 56%); [h]²_D¹−42° (c 1.23, CHCl₃); TLC (7:3 tolu-

1

ene–acetone): R_f0.32; H NMR (CDCl₃): l 8.19 (d, 2

H, J 9.2 Hz, ArH), 7.05 (d, 2 H, J 9.2 Hz, ArH), 6.00

1104

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

4-Nitrophenyl (2,3,4,6-tetra-O-acetyl-i-

ranosyl)-(14)-2-acetamido-3-O-acetyl-2-deoxy-i-

D-galactopy-

(d, 1 H, J_NH,29.2 Hz, exchanged with D₂O, NH), 5.39

(d, 1 H, J_3%,4%3.1 Hz, H-4%), 5.21 (d, 1 H, J_1,26.1 Hz

H-1), 5.18–5.12 (m, 2 H, H-2%, H-3), 5.01 (dd, 1 H, J_2%,3%

10.5 Hz, H-3%), 4.60 (d, 1 H, J_1%,2%7.8 Hz, H-1%), 4.33

(br.q, 1 H, after D₂O exchange became a dd, J_2,37.9

Hz, H-2), 4.20–4.07 (d, 2 H, J 7.0 Hz, H-6a%, H-6b%),

4.01 (t, 1 H, J_4,5=J_3,46.7 Hz, H-4), 3.95 (dd, 1 H, J_6a,6b

10.4, J_5,6a4.2 Hz, H-6a), 3.87 (t, 1 H, H-5%), 3.72–3.59

(m, 2 H, H-6b, H-5), 2.16, 2.11, 2.08, 2.06, 2.02, 1.99,

(6s, 18 H, 6 Ac), 0.88 (s, 9 H, Si-Bu^t), 0.01 (s, 3 H,

Si-Me), 0.00 (Si-Me+Si-Me₄); ¹³C NMR (CDCl₃): l

170.6 (s), 170.3 (s), 170.1 (s), 170.0 (s), 169.4 (s), 161.6

(s), 142.8 (s), 125.7 (d), 116.5 (d), 100.3 (d, C-1%), 98.0

(d, C-1), 75.9 (d, C-5), 73.0 (d, C-4), 71.3 (d, C-2% or

C-3), 71.0 (d, C-5%), 70.7 (d, C-3%), 69.3 (d, C-2% or C-3),

66.8 (d, C-4%), 61.2 (t, C-6), 61.1 (t, C-6%), 51.7 (d, C-2),

25.8 (q), 23.2 (q), 20.9 (q), 20.8 (q), 20.6 (q), 20.5 (q),

20.4 (q), 18.2, (s, Si-Bu^t), −5.3 (q, Si-Me), −5.4 (q,

Si-Me). FABMS: m/z Calcd. for C₃₆H₅₃N₂O₁₈Si [M+

H]⁺: 829.3063. Found: 829.3057.

D

-

glucopyranoside 6-sodium sulfate (15).—Sulfur triox-

ide·Me₃N (292 mg, 2.1 mmol) was added to a solution

of 14 (250 mg, 0.35 mmol) in dry DMF (6 mL) and the

mixture was heated to 40 °C for 3 h. MeOH (1 mL) was

added and the volatiles were removed. The residue was

dissolved in MeOH and passed through a column

(10×2.5 cm) of Dowex 50X8 100 (Na⁺) resin eluted

with MeOH. The fractions containing 15 were evapo-

rated and the residue was chromatographed (9:1

CH₂Cl₂–MeOH), to afford sulfate 15 as a colourless

amorphous solid (225 mg, 79%); [h]²_D⁰−21° (c 1.24,

1

MeOH); TLC (9:1 CH₂Cl₂–MeOH): R_f0.1; H NMR

(CD₃OD): l 8.22 (d, 2 H, J 9.2 Hz, ArH), 7.22 (d, 2 H,

J 9.3 Hz, ArH), 5.38 (d, 1 H, J_3%,4%3.4 Hz, H-4%), 5.35 (d,

1 H, J_1,28.3 Hz, H-1), 5.27–5.18 (m, 1 H, H-3), 5.13

(dd, 1 H, J_2%,3%10.3 Hz, H-3%), 5.03 (dd, 1 H, J_1%,2%7.8

Hz, H-2%), 4.88 (d, 1 H, H-1%), 4.37 (d, 1 H, J_6a,6b10.8

Hz, H-6a), 4.28–4.07 (m, 5 H, H-6a%, H-6b%, H-6b,

H-5%, H-2), 4.01–3.87 (m, 2 H, H-5, H-4), 2.13, 2.12,

2.07, 2.05, 1.92, 1.90 (6s, 18 H, 6 Ac); ¹³C NMR

(CDCl₃): l 173.5 (s), 172.0 (s), 171.9 (s), 171.6 (s), 171.4

(s), 163.2 (s), 144.1 (s), 126.7 (d), 117.8 (d), 101.5 (d,

C-1%), 99.1 (d, C-1), 76.3 (d, C-4 or C-5), 74.9, (d, C-4

or C-5), 74.0 (d, C-3), 72.6 (d, C-3%), 71.7 (d, C-5%), 70.5

(d, C-2%), 68.8 (d, C-4%), 66.5 (t, C-6), 62.3 (t, C-6%), 55.0

(d, C-2), 22.7 (q), 21.0 (q), 20.8 (q), 20.6 (q), 20.4 (q).

FABMS: m/z Calcd. for C₃₀H₃₈N₂NaO₂₁S [M+H]⁺:

817.1586. Found: 817.1566.

4-Nitrophenyl (2,3,4,6-tetra-O-acetyl-i-D-galactopy-

ranosyl)-(14)-2-acetamido-3-O-acetyl-2-deoxy-i-

D

-

glucopyranoside (14).—Hydrogen ﬂuoride–pyridine

(0.3 mL, 65–75%) was added to a solution of 13 (140

mg, 0.17 mmol) in THF (2 mL) contained in a plastic

vial at 0 °C. The mixture was warmed to ambient

temperature and left for 1.5 h, diluted with EtOAc (30

mL) and the solution was washed with aq satd

NaHCO₃, dried and the volatiles were evaporated.

Chromatography (3:2 toluene–acetone) afforded the

6-hydroxy compound 14 as a colourless amorphous

solid (112 mg, 92%); [h]²_D¹−25° (c 0.71, CHCl₃). TLC

4-Nitrophenyl i-

D

-galactopyranosyl-(14)-2-acet-

amido-2-deoxy-i- -glucopyranoside (16).—To a solu-

D

tion of 14 (20 mg, 0.028 mmol) in MeOH (2 mL) was

added NaOMe in MeOH solution (0.1 mL, 1 M) and

the mixture was left at ambient temperature for 2 h.

Dilution with MeOH (5 mL), acidiﬁcation to ꢀpH 4

with Dowex 50X8 100 (H⁺) resin, ﬁltration and evapo-

ration gave a colourless crystalline solid (12 mg, 86%);

[h]²_D¹−6° (c 0.47, Me₂SO), lit. [h]_D−4° (c 0.50,

Me₂SO).^{22 1}H NMR ([CD₃]₂SO) in agreement with

literature data;²²mp 207–209 °C (MeOH), lit. 213 °C

(MeOH);³²[h]_D²¹−24° (c 0.74, water), lit. [h]²_D⁵−18° (c

1.0, water);^{32 1}H and ¹³C NMR (D₂O) in agreement

with literature data.³²

1

(7:3 toluene–acetone): R_f0.16; H NMR (CDCl₃): l

8.19 (d, 2 H, J 9.2 Hz, ArH), 7.07 (d, 2 H, J 9.2 Hz,

ArH), 5.91 (d, 1 H, J_NH,29.2 Hz, exchanged with D₂O,

NH), 5.38 (d, 1 H, J_3%,4%3.0 Hz, H-4%), 5.25–5.19 (m, 2

H, H-3, H-1), 5.14 (dd, 1 H, J_2%,3%10.5, J_1%,2%7.8 Hz,

H-2%), 5.03 (dd, 1 H, H-3%), 4.64 (d, 1 H, H-1%), 4.33 (q,

1 H, after D₂O exchange became a dd, J_2,39.7, J_1,27.7

Hz, H-2), 4.18–4.09 (m, 2 H, H-6a%, H-6b%), 4.03 (t, 1

H, J_4,5=J_3,48.6 Hz, H-4), 3.98–3.87 (m, 2 H, H-6a,

H-5%), 3.83–3.75 (m, 1 H, after D₂O exchange became a

dd, J_6a,6b12.3, J_5,6b3.3 Hz, H-6b), 3.63 (dt, 1 H, H-5),

2.11, 2.09, 2.08, 2.07, 2.00, 1.98 (6s, 18 H, 6 Ac), 1.87

(dd, 1 H, J_OH,6a8.5, J_OH,6b4.9 Hz, exchanged with

D₂O, OH-6); ¹³C NMR (CDCl₃): l 171.1 (s), 170.4 (s),

170.0 (s), 169.9 (s), 169.6 (s), 161.5 (s), 142.9 (s), 125.8

(d), 116.3 (d), 100.9 (d), 98.2 (d), 75.7 (d), 74.35 (d),

72.40 (d), 70.7 (d), 69.5 (d), 66.7 (d), 60.8 (t), 60.5 (t),

52.9 (d), 23.2 (q), 20.9 (q), 20.7 (q), 20.6 (q), 20.5 (q),

20.3 (q). FABMS: m/z Calcd. for C₃₀H₃₉N₂O₁₈[M+

H]⁺: 715.2198. Found: 715.2204. Similar treatment of

the TBDPS ether (18) (720 mg, 0.76 mmol) with HF–

pyridine (2.0 mL) for 48 h also gave compound 14 (460

mg, 85%).

4-Nitrophenyl i-

D

-galactopyranosyl-(14)-2-acet-

amido-2-deoxy-i- -glucopyranoside 6-sodium sulfate

D

(4).—To a solution of compound 15 (192 mg, 0.24

mmol) in MeOH (15 mL) was added NaOMe in MeOH

(0.19 mL, 1 M) and the mixture was left at ambient

temperature for 2 h. Acidiﬁcation to ꢀ pH 4 with

Dowex 50X8 100 (H⁺) resin, ﬁltration and evaporation

gave a syrup that was dissolved in MeOH and passed

through a Dowex 50X8 100 (Na⁺) resin column (10×

2.5 cm) eluted with MeOH. Fractions containing the

title compound were evaporated. Chromatography

(11.7:5:1 CH₂Cl₂–MeOH–water) followed by passage

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1105

through a plug of Biogel P-2 resin (water) gave sulfate

salt 4 as a colourless solid (111 mg, 78%); [h]²_D¹−37° (c

0.62, water); TLC (11.7:5:1 CH₂Cl₂–MeOH–water): R_f

0.28; IR (CHBr₃mull): w 3659–3159 (br, s), 1653 (s),

cooled to −30 °C and BF₃·OEt₂(0.135 mL, 1.10

mmol) was added dropwise over 10 min. After 1.5 h

Et₃N (1.0 mL) was added and the mixture warmed to

ambient temperature and the volatiles were evaporated.

Chromatography (7:3 toluene–acetone) gave a mono-

hydroxy product (556 mg) (R_f0.22) which was dis-

solved in pyridine (10 mL) and Ac₂O (5 mL) and left at

ambient temperature for 48 h. The volatiles were evap-

orated and the residue was dissolved in EtOAc, washed

with aq HCl (10%), aq satd NaHCO₃, dried and the

volatiles again evaporated. Chromatography (7:3

EtOAc–hexanes) gave 18 as a colourless foam (560 mg,

53%); [h]²_D¹−31° (c 1.32, CHCl₃); TLC (4:1 EtOAc–

1

1590 (s), 1515 (s), 1344 (s), 1248 (s), 1072 (s) cm⁻¹; H

NMR (D₂O): l 8.25 (d, 2 H, J 9.2 Hz, ArH), 7.21 (d,

2 H, J 9.2 Hz, Ar), 5.36 (d, 1 H, J_1,28.4 Hz, H-1), 4.57

(d, 1 H, J_1%,2%7.7 Hz, H-1%), 4.49 (d, 1 H, J_6a,6b11.0 Hz,

H-6a), 4.35 (dd, 1 H, J_5,6b4.8 Hz, H-6b), 4.16–4.04 (m,

2 H, H-5, H-2), 3.95 (d, 1 H, J_3%,4%,3.3 Hz, H-4%),

3.92–3.84 (m, 2 H, H-4, H-3), 3.83–3.72 (m, 3 H,

H-6a%, H-6b%, H-5%), 3.70 (dd, 1 H, J_2%,3%10.0 Hz, H-3%),

3.53 (dd, 1 H, H-2%), 2.03 (s, 3 H, Ac); ¹³C NMR (D₂O):

l 176.1 (s), 162.8 (s), 143.8 (s), 127.3 (d), 117.7 (d),

103.9 (d, C-1%), 99.7 (d, C-1), 78.6 (d, C-4), 76.6 (d,

C-5%), 74.2 (d, C-5), 73.7 (d, C-3%), 73.2 (d, C-3), 72.2 (d,

C-2%), 69.9 (d, C-4%), 67.5 (t, C-6), 62.3 (t, C-6%), 56.1

(d, C-2), 23.4 (q, Me). FABMS: m/z Calcd. for

1

hexanes): R_f0.52; H NMR (CDCl₃): l 8.14 (d, 2 H, J

9.1 Hz, ArH), 7.64–7.57 (m, 4 H, ArH), 7.45–7.26 (m,

4 H, ArH), 7.12 (t, 2 H, J 7.6 Hz, ArH), 7.03 (d, 2 H,

J 9.1 Hz, ArH), 5.98 (d, 1 H, J_NH,29.1 Hz, exchanged

with D₂O, NH), 5.37 (d, 1 H, J_3%,4%3.3 Hz, H-4%),

5.22–5.15 (m 2 H, H-3, H-1), 5.11 (dd, 1 H, J_2%,3%10.5,

J_1%,2%7.9 Hz, H-2%), 4.99 (dd, 1 H, H-3%), 4.73 (d, 1 H,

H-1%), 4.39 (q, 1 H, after D₂O exchange became a dd,

J_2,38.7, J_1,26.9 Hz, H-2), 4.21 (t, 1 H, J_4,5=J_3,47.7 Hz,

H-4), 4.14 (d, 2 H, J 6.6 Hz, H-6a%, H-6b%), 3.99 (dd, 1

H, J_6a,6b11.3, J_5,6a3.6 Hz, H-6a), 3.85–3.73 (m, 2 H,

H-5%, H-6b), 3.65–3.54 (m, 1 H, H-5), 2.11, 2.07, 2.06,

1.84 (4s, 12 H, 4 Ac), 2.02 (s, 6 H, 2 Ac), 1.05 (s, 9 H,

Si-Bu^t); ¹³C NMR (CDCl₃): l 171.0 (s), 170.3 (s), 169.9

(s), 169.1 (s), 161.6 (s), 142.7 (s), 135.7 (d), 135.3 (d),

133.0 (s), 131.8 (s), 130.1 (d), 129.9 (d), 127.9 (d), 127.6

(d), 125.6 (d), 116.5 (d), 100.2 (d, C-1%), 98.0 (d, C-1),

75.5 (d, C-5), 73.2 (d, C-4), 71.7 (d, C-3), 70.9 (d, C-5%),

70.8 (d, C-3%), 69.4 (d, C-2%) 66.8 (d, C-4%), 61.3 (t, C-6),

61.1 (t, C-6%), 52.3 (d, C-2), 26.8 (q), 23.2 (q), 20.9 (q),

20.6 (q), 20.5 (q), 20.3 (q), 19.2 (s, Si-Bu^t). FABMS:

m/z Calcd. for C₄₆H₅₇N₂O₁₈Si [M+H]⁺: 953.3376.

Found: 953.3362.

C₂₀H₂₈N₂NaO₁₆S

607.1040.

[M+H]⁺:

607.1057.

Found:

4-Nitrophenyl 2-acetamido-6-O-tert-butyldiphenylsi-

lyl-2-deoxy-i- -glucopyranoside (17).—tert-Butyl-

chlorodiphenylsilane (1.96 mL, 7.53 mmol) was added

-glucopyra-

D

to 4-nitrophenyl 2-acetamido-2-deoxy-b-

D

noside³⁴(2.00 g, 5.8 mmol) in pyridine (30 mL) and the

solution was stirred at ambient temperature for 16 h.

The solvent was evaporated and the residue dissolved in

EtOAc then washed with aq HCl (10%), aq satd

NaHCO₃, dried and the solvent evaporated. Chro-

matography (19:1 CH₂Cl₂–MeOH) gave the silyl ether

17 as a colourless foam (3.21 g, 95%); [h]²_D⁰−69° (c

1

1.01, CHCl₃); TLC (9:1 CH₂Cl₂–MeOH): R_f0.43; H

NMR (CDCl₃): l 8.07 (d, 2 H, J 9.2 Hz, ArH), 7.62 (d,

4 H, J 6.7 Hz, ArH), 7.42–7.25 (m, 6 H, ArH), 7.07 (d,

2 H, J 9.2 Hz, ArH), 5.86 (br.d, 1 H, J_NH,26.0 Hz,

exchanged with D₂O, NH), 5.30 (d, 1 H, J_1,28.1 Hz,

H-1), 4.29 (d, 1 H, J 3.1 Hz, exchanged with D₂O, OH),

4.03 (dd, 1 H, J_6a,6b11.0, J_5,6a2.8 Hz, H-6a), 3.96–3.82

(m, 2 H, H-6b, H-3), 3.81–3.71 (m, 1 H, after D₂O

exchange became a dd, J_2,310.1 Hz, H-2), 3.70–3.55

(m, 2 H, H-5, H-4), 2.97 (s, 1 H, exchanged with D₂O,

OH), 2.07 (s, 3 H, Ac), 1.05 (s, 9 H, Si-Bu^t); ¹³C NMR

(CDCl₃): l 172.6 (s), 161.7 (s), 142.6 (s), 135.6 (d), 135.5

(d), 133.0 (s), 132.8 (s), 129.8 (d), 127.7 (d), 125.7 (d),

116.4 (d), 97.9 (d), 76.7 (d), 75.1 (d), 71.5 (d), 63.8 (t),

56.8 (d), 26.8 (q), 23.5 (q), 19.2 (s). FABMS: m/z Calcd.

for C₃₀H₃₇N₂O₈Si [M+H]⁺: 581.2319. Found:

581.2320.

4-Nitrophenyl

silyl-2-deoxy-4,6-O-(4-methoxybenzylidene)-i-

2-acetamido-3-O-tert-butyldimethyl-

-gluco-

D

pyranoside (19).—Imidazole (2.19 g, 32.2 mmol) and

TBDMSCl (2.42 g, 16.1 mmol) were added to a solu-

tion of 4-nitrophenyl 2-acetamido-2-deoxy-4,6-O-(4-

methoxybenzylidene)-b-D

-glucopyranoside³⁵(4.90 g,

10.7 mmol) in dry DMF (10 mL) and the mixture was

left at ambient temperature for 16 h then heated at

40 °C for 3 h. The solvent was evaporated and the

residue dissolved in EtOAc. The solution was washed

with water, dried and the solvent evaporated. Chro-

matography (3:74:6 EtOAc–hexanes) gave silyl ether

19 as a colourless foam (3.1 g, 61%); mp (EtOAc–hex-

anes, needles) 138–140 °C; [h]²_D¹−25° (c 0.79, CHCl₃);

4-Nitrophenyl (2,3,4,6-tetra-O-acetyl-i-

ranosyl)-(14)-2-acetamido-3-O-acetyl-6-O-tert-butyl-

diphenylsilyl-2-deoxy-i- -glucopyranoside (18).—Pow-

D-galactopy-

1

D

TLC (2:3 EtOAc–hexanes): R_f0.56; H NMR (CDCl₃):

,

dered 4 A molecular sieve (3.0 g) was added to a

l 8.19 (d, 2 H, J 9.2 Hz, ArH), 7.40 (d, 2 H, J 8.7 Hz,

ArH), 7.06 (d, 2 H, J 9.2 Hz, ArH), 6.89 (d, 2 H, J 8.8

Hz, ArH), 5.83 (d, 1 H, J_1,28.3 Hz, H-1), 5.78 (d, 1 H,

J_NH,28.6 Hz, exchanged with D₂O, NH), 5.47 (s, 1 H,

CHAr), 4.48 (t, 1 H, J_3,4=J_2,39.2 Hz, H-3), 4.42–4.23

solution of diol 17 (636 mg, 1.10 mmol) and tetra-O-

acetyl-a-

D

-galactopyranosyl

(812 mg, 1.65 mmol) in dry CH₂Cl₂(30 mL). The

mixture was stirred at ambient temperature for 2 h,

trichloroacetimidate^25,26

1106

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

(m, 1 H, H-6a), 3.81 (s, 3 H, OMe), 3.79–3.65 (m, 2 H,

H-6b, H-5), 3.53–3.42 (m, 2 H, H-4, H-2), 1.98 (s, 3 H,

Ac), 0.85 (s, 9 H, Si-Bu^t), 0.03, −0.02 (2s, 6 H, 2

Si-Me); ¹³C NMR (CDCl₃): l 170.6 (s), 161.8 (s), 160.2

(s), 142.9 (s), 129.5 (s), 127.6 (d), 125.8 (d), 116.6 (d),

113.6 (d), 101.9 (d, CHAr), 97.7 (d, C-1), 82.1 (d, C-4),

70.9 (d, C-3), 68.5 (t, C-6), 66.5 (d, C-5), 59.4 (d, C-2),

55.3 (q, OMe), 25.8 (q), 23.7 (q), 18.2 (s, Si-Bu^t), −4.1

(q, Si-Me), −5.0 (q, Si-Me). FABMS: m/z Calcd. for

C₂₈H₃₉N₂O₉Si [M+H]⁺: 575.2425. Found: 575.2373.

4-Nitrophenyl 2-acetamido-3-O-tert-butyldimethylsil-

D

-glucopyranoside³⁵(3.06 g, 6.65 mmol) in dry DMF

(30 mL) and the mixture was stirred at ambient temper-

ature for 2 h. Dilution with 9:1 CH₂Cl₂–MeOH (50

mL) and ﬁltration through Celite followed by removal

of the solvent gave a residue that was extracted with

hot 9:1 CH₂Cl₂–MeOH (4×50 mL). The extracts were

combined, the solvent was removed and chromatogra-

phy of the further residue (99:119:1 CH₂Cl₂–MeOH)

gave, after recrystallization from a large volume of

acetone, allyl ether 21 as a colourless solid (1.21 g,

36%); mp 285–287 °C; [h]²_D¹−3° (c 0.67, Me₂SO); TLC

(19:1 CH₂Cl₂–MeOH): R_f0.48; ¹H NMR ([CD₃]₂SO): l

8.21 (d, 2 H, J 9.2 Hz, ArH), 8.09 (d, 1 H, J_NH,28.9 Hz,

NH), 7.37 (d, 2 H, J 8.7 Hz, ArH), 7.25 (d, 2 H, J 9.2

Hz, ArH), 6.95 (d, 2 H, J 8.7 Hz, ArH), 5.90–5.77 (m,

1 H, CH₂CHꢀCH₂), 5.64 (s, 1 H, CHAr), 5.45 (d, 1 H,

J_1,28.3 Hz, H-1), 5.22 (dd, 1 H, J_trans17.3, J_gem1.7 Hz,

yl-2-deoxy-6-O-(4-methoxybenzyl)-i-D-glucopyrano-

,

side (20).—Powdered 3A molecular sieve (15 g) was

added to a solution of 19 (3.00 g, 5.2 mmol) and

52

NaCNBH₃(1.64 g, 26.1 mmol) in dry DMF (40 mL)

and the mixture was cooled to 0 °C. A solution of TFA

(3.99 mL, 51.7 mmol) in DMF (30 mL) was added

dropwise over 30 min, and the mixture was stirred for

2 h then at ambient temperature for 4 h. TLC analysis

indicated the presence mainly of starting material and a

trace of the required title compound. An excess of

NaCNBH₃(4.2 g) and TFA (6 mL) in DMF (6 mL)

was added in portions over a period of 2 days but TLC

indicated little change in the ratio of the two compo-

nents. The mixture was ﬁltered into aq satd NaHCO₃

and the solution was extracted with CHCl₃(5×50

mL). The combined organic extracts were washed with

water (20 mL), dried and evaporated. Chromatography

(2:31:1 EtOAc–hexanes) gave 6-ether 20 together

with 15–20% of the 4-O-PMB regioisomer as an insep-

arable mixture (420 mg, 14%). Crystallization from

EtOH–water gave the pure title compound 20 as a

colourless solid (240 mg, 8%); mp 79–81 °C; [h]_D²¹

−35° (c 0.87, CHCl₃); TLC (1:1 EtOAc–hexanes): R_f

0.47; ¹H NMR (CDCl₃): l 8.15 (d, 2 H, J 9.2 Hz, ArH),

7.22 (d, 2 H, J 8.6 Hz, ArH), 7.05 (d, 2 H, J 9.2 Hz,

ArH), 6.86 (d, 2 H, J 8.6 Hz, ArH), 5.67 (d, 1 H, J_NH,2

7.8 Hz, exchanged with D₂O, NH), 5.66 (d, 1 H, J_1,27.9

Hz, H-1), 4.52, 4.43 (ABq, 2 H, J 11.6 Hz, CH₂Ar),

4.17 (dd, 1 H, J 9.4, 8.1 Hz, H-3), 3.81 (s, 3 H, OMe),

3.77–3.66 (m, 3 H, H-6a, H-6b, H-5), 3.59–3.46 (m, 2

H, H-4, H-2), 2.42 (d, 1 H, J_OH,43.2 Hz, exchanged

with D₂O, OH-4), 1.97 (s, 3 H, Ac), 0.91 (s, 9 H,

Si-Bu^t), 0.14, 0.09 (2s, 6 H, 2 Si-Me); ¹³C NMR

(CDCl₃): l 170.4 (s), 162.0 (s), 159.4 (s), 142.6 (s), 129.7

(s), 129.4 (d), 125.7 (d), 116.5 (d), 113.9 (d), 97.2 (d),

74.5 (d), 73.9 (d), 73.3 (t), 73.0 (d), 69.7 (t), 57.8 (d),

55.3 (q), 25.8 (q), 23.7 (q), 18.2 (s), −4.2 (q), −4.7 (q).

FABMS: m/z Calcd. for C₂₈H₄₀CsN₂O₉Si [M+Cs]⁺:

709.1557. Found: 709.1584.

CH₂CHꢀCHH), 5.10 (dd,

1

H, J_cis10.5 Hz,

CH₂CHꢀCHH), 4.27–4.18 (m, 2 H, CHHCHꢀCH₂,

H-6a), 4.09 (dd, 1 H, J 13.4, 5.4 Hz, CHHCHꢀCH₂),

4.00–3.86 (m, 1 H, H-2), 3.80–3.70 (m, 4 H, H-6b,

H-5, H-4, H-3), 3.76 (s, 3 H, OMe), 1.82 (s, 3 H, Ac);

¹³C NMR ([CD₃]₂SO): l 170.5 (s), 162.8 (s), 160.6 (s),

143.2 (s), 136.5 (d), 131.0 (s), 128.4 (d), 126.9 (d), 117.7

(d), 117.0 (t), 114.5 (d), 101.3 (d), 99.3 (d), 81.5 (d), 78.9

(d), 73.3 (t), 68.6 (t), 66.9 (d), 56.2 (q), 23.9 (q).

FABMS: m/z Calcd. for C₂₅H₂₉N₂O₉[M+H]⁺:

501.1873. Found: 501.1894.

4-Nitrophenyl 2-acetamido-3-O-allyl-6-O-tert-butyl-

dimethylsilyl-2-deoxy-i- -glucopyranoside (23).—Ace-

D

tyl chloride (0.5 mL) was added to MeOH (100 mL)

followed by compound 21 (1.12 g, 2.24 mmol) and the

solution left 30 min at ambient temperature. The neu-

tralized (Amberlyst A21 resin) solution on removal of

the solvent gave 4-nitrophenyl 2-acetamido-3-O-allyl-2-

deoxy-b- -glucopyranoside (22) as a cream coloured

D

solid that was washed with Et₂O and dried (600 mg,

1

70%); H NMR ([CD₃]₂SO): l 8.52 (d, 2 H, J 9.2 Hz,

ArH), 7.95 (d, 1 H, J_NH,29.0 Hz, NH), 7.18 (d, 2 H, J

9.2 Hz, ArH), 5.93–5.81 (m, 1 H, CH₂CHꢀCH₂), 5.33

(d, 1 H, J_OH,45.7 Hz, OH-4), 5.24–5.18 (m, 2 H, trans

CH₂CHCHH, H-1), 5.08 (dd, 1 H, J_cis11.8, J_gem1.4

Hz, CH₂CHꢀCHH), 4.62 (t, 1 H, J_OH,65.5 Hz, OH-6),

4.28 (dd, 1 H, J 13.2, 5.3 Hz, CHHCHꢀCH₂), 4.10 (dd,

1 H, CHHCHꢀCH₂), 3.83–3.69 (m, 2 H, H-6a, H-2),

3.54–3.35 (m, 4 H, H-6b, H-5, H-4, H-3), 1.80 (s, 3 H,

Ac). To a solution of 22 (699 mg, 1.83 mmol) in dry

DMF (3 mL) was added imidazole (199 mg, 2.93 mmol)

and TBDMSCl (331 mg, 2.20 mmol). The mixture was

stirred for 45 min at ambient temperature and the

solvent evaporated. Chromatography (7:3 EtOAc–hex-

anes) gave the title compound 23 as a colourless foam

(650 mg, 72%); [h]²_D¹−33° (c 1.17, CHCl₃); TLC (3:2

4-Nitrophenyl 2-acetamido-3-O-allyl-2-deoxy-4,6-O-

(4-methoxybenzylidene)-i-

D

-glucopyranoside

(21).—

Barium hydroxide octahydrate (1.47 g, 4.66 mmol),

BaO (4.38 g, 28.6 mmol), and allyl bromide (1.12 mL,

12.98 mmol) were added to a solution of 4-nitrophenyl

2-acetamido-2-deoxy-4,6-O-(4-methoxybenzylidene)-b-

1

EtOAc–hexanes) R_f0.30; H NMR (CDCl₃): l 8.17 (d,

2 H, J 9.2 Hz, ArH), 7.06 (2 H, J 9.2 Hz, ArH),

6.06–5.89 (m, 1 H, CH₂CHꢀCH₂), 5.75–5.70 (m, 2 H,

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1107

after D₂O exchange became a d, 1 H, J_1,28.0 Hz, H-1,

NH), 5.31 (dd, 1 H, J_trans17.2, J_gem1.5 Hz, CH₂-

CHꢀCHH), 5.22 (br.d, 1 H, J_cis10.4 Hz, CH₂CHꢀ

CHH), 4.33 (dd, 1 H, J 12.7, 5.8 Hz, CHHCHꢀCH₂),

4.22 (dd, 1 H, CHHCHꢀCH₂), 4.06 (dd, 1 H, J_2,310.0,

J_3,48.1 Hz, H-3), 3.94–3.80 (m, 2 H, H-6a, H-6b),

3.71–3.55 (m, 2 H, H-5, H-4), 3.47 (q, 1 H, after D₂O

exchange became a dd, H-2), 2.97 (d, 1 H, J_OH,42.0 Hz,

exchanged with D₂O, OH-4), 1.99 (s, 3 H, Ac), 0.89 (s,

9 H, Si-Bu^t), 0.06 (s, 3 H, Si-Me), 0.05 (s, 3 H, Si-Me);

¹³C NMR (CDCl₃): l 170.7 (s), 161.9 (s), 142.7 (s),

134.8 (d), 125.7 (d), 117.5 (t), 116.6 (d), 97.2 (d), 79.9

(d), 75.2 (d), 73.5 (t), 72.9 (d), 64.1 (t), 56.7 (d), 25.8 (q),

23.6 (q), 18.2 (s), −5.5 (q). FABMS: m/z Calcd. for

C₂₃H₃₇N₂O₈Si [M+H]⁺: 497.2319. Found: 497.2346.

4-Nitrophenyl 2-acetamido-6-O-tert-butyldimethylsi-

H-1), 5.37 (dd, 1 H, J_trans17.2, J_gem1.0 Hz, CH₂-

CHꢀCHH), 5.30 (dd, 1 H, J_cis10.4, CH₂CHꢀCHH),

5.20 (dd, 1 H, J_2,310.5, J_3,49.1 Hz, H-3), 4.72–4.59 (m,

2 H, CH₂CHꢀCH₂), 4.03 (q, after D₂O exchange be-

came a dd, 1 H, H-2), 3.96–3.88 (m, 2 H, after D₂O

exchange became a d, J 5.0 Hz H-6a, H-6b), 3.82 (dt, 1

H, J_4,53.9 Hz, after D₂O exchange became a t, H-4),

3.75–3.66 (m, 1 H, H-5), 3.28 (d, 1 H, J_OH,43.9 Hz,

OH-4), 1.93 (s, 3 H, Ac), 0.83 (s, 9 H, Si-Bu^t), 0.05, 0.04

(2s, 6 H, 2 Si-Me); ¹³C NMR (CDCl₃): l 170.7 (s),

161.8 (s), 155.6 (s), 142.9 (s), 131.1 (d), 125.7 (d), 119.3

(t), 116.7 (d), 97.8 (d), 78.5 (d), 75.3 (d), 70.6 (d), 69.1

(t), 63.8 (t), 54.8 (d), 25.8 (q), 23.4 (q), 18.2 (s), −5.5

(q). FABMS: m/z Calcd. for C₂₄H₃₇N₂O₁₀Si [M+H]⁺:

541.2218. Found: 541.2210.

4-Nitrophenyl 2-acetamido-3-O-allyl-6-O-tert-butyl-

lyl-2-deoxy-i-

mg, 9.25 mmol) and TBDMSCl (1.06 g, 7.01 mmol)

D

-glucopyranoside (24).—Imidazole (630

dimethylsilyl-2-deoxy-i-

trophenyl 2-acetamido-4-O-allyl-6-O-tert-butyldime-

thylsilyl-2-deoxy-i- -glucopyranoside (26) and 4-nitro-

phenyl 2-acetamido-2,3-di-O-allyl-6-O-tert-butyldime-

thylsilyl-2-deoxy-i- -glucopyranoside (27).—Freshly

prepared Pd(PPh₃)₄(10 mg) was added to a solution of

carbonate 25 (625 mg 1.16 mmol) in THF (2 mL,

freshly distilled from LAH), and the mixture was

heated under reﬂux for 20 min then the solvent was

evaporated. Chromatography (2:3 EtOAc–hexanes)

gave the following products in order of elution:

D

-glucopyranoside (23), 4-ni-

were added to a solution of 4-nitrophenyl 2-acetamido-

D

2-deoxy-b-D

-glucopyranoside³⁴(2.00 g, 5.84 mmol) in

dry DMF (9 mL) and the mixture was stirred at

ambient temperature for 30 min, then the solvent was

evaporated. Chromatography (93:7 CH₂Cl₂–MeOH)

gave silyl ether 24 as a colourless amorphous solid (2.65

g, quant.); [h]²_D⁰−29° (c 0.56, MeOH); TLC (9:1

D

1

CH₂Cl₂–MeOH): R_f0.37; H NMR ([CD₃]₂SO): l 8.17

(d, 2 H, J 9.2 Hz, ArH), 7.82 (d, 1 H, J_NH,28.9 Hz,

exchanged with D₂O, NH), 7.16 (d, 2 H, J 9.2 Hz,

ArH), 5.22–5.11 (m, 3 H, after D₂O exchange became

a d, 1 H, J_1,28.4 Hz, H-1, OH-4, OH-3), 3.90 (d, 1 H,

J_6a,6b10.2 Hz, H-6a), 3.75–3.65 (m, 2 H, H-6b, H-2),

3.49–3.40 (m, 2 H, H-5, H-3), 3.24–3.16 (m, 1 H, after

D₂O exchange became a t, J_4,5=J_3,49.3 Hz, H-4), 1.82

(s, 3 H, Ac), 0.83 (s, 9 H, Si-Bu^t), −0.01 (s, 3 H,

Si-Me), −0.05 (s, 3 H, Si-Me); ¹³C NMR ([CD₃]₂SO):

l 169.6 (s), 162.4 (s), 142.0 (s), 125.7 (d), 116.9 (d), 98.5

(d), 77.3 (d), 74.0 (d), 70.0 (d), 62.6 (t), 55.4 (d), 25.9

(q), 23.2 (q), 18.1 (s), −5.1 (q), −5.2 (q). FABMS:

m/z Calcd. for C₂₀H₃₃N₂O₈Si [M+H]⁺: 457.2006.

Found: 457.2004.

Diallyl ether 27 as a colourless solid (90 mg, 14%);

mp (EtOAc–hexanes, ﬁne needles) 147–148 °C; [h]_D²⁰

−38° (c 1.02, CHCl₃); TLC (3:2 EtOAc–hexanes): R_f

0.62; ¹H NMR (CDCl₃): l 8.16 (d, 2 H, J 9.2 Hz, ArH),

7.07 (2 H, J 9.2 Hz, ArH), 6.02–5.85 (m, 3 H, after

D₂O exchange became a m, 2 H, 2×CH₂CHꢀCH₂,

NH), 5.57 (d, 1 H, J_1,26.9 Hz, H-1), 5.29 (d, 2 H, J_trans

17.2 Hz, 2×CH₂CHꢀCHH), 5.20 (d, 2 H, J_cis10.3 Hz,

2×CH₂CHꢀCHH), 4.31–4.14 (m,

4

H, 2×

CH₂CHꢀCH₂), 4.01–3.91 (m, 2 H, H-6a, H-3), 3.74–

3.59 (m, 3 H, H-6b, H-5, H-2), 3.50 (t, 1 H, J_3,4=J_4,5

7.6 Hz, H-4), 1.99 (s, 3 H, Ac), 0.86 (s, 9 H, Si-Bu^t),

–0.01, –0.04 (2s, 6 H, 2 Si-Me); ¹³C NMR (CDCl₃): l

170.4 (s), 162.0 (s), 142.6 (s), 134.7 (d), 134.5 (d), 125.7

(d), 117.2 (t), 116.6 (d), 97.0 (d), 78.6 (d), 76.7 (d), 76.5

(d), 73.3 (t), 73.0 (t), 62.2 (t), 55.2 (d), 25.8 (q), 23.6 (q),

18.2 (s), −5.3 (q), −5.4 (q). FABMS: m/z Calcd. for

C₂₆H₄₁N₂O₈Si [M+H]⁺: 537.2632. Found: 537.2603.

4-Nitrophenyl 2-acetamido-3-O-allyloxycarbonyl-6-

O-tert-butyldimethylsilyl-2-deoxy-i-D-glucopyranoside

(25).—Allyl chloroformate (1.45 mL, 11.03 mmol) was

added dropwise to a solution of diol 24 (2.40 g, 5.26

mmol) in pyridine (80 mL) and THF (40 mL) at

−60 °C. The mixture was stirred for 2 h then allowed

to warm to −10 °C and left for 16 h then warmed to

ambient temperature and the volatiles were evaporated.

Chromatography (19:14:1 toluene–acetone) gave the

carbonate 25 as a colourless gum which solidiﬁed on

standing overnight (675 mg, 24%); mp (EtOAc–hex-

anes) 97–98 °C; [h]_D¹⁹−28° (c 0.84, CHCl₃); TLC (4:1

toluene–acetone): R_f0.22; ¹H NMR (CDCl₃): l 8.16 (d,

2 H, J 9.2 Hz, ArH), 7.08 (2 H, J 9.2 Hz, ArH), 6.03 (d,

1 H, J_NH,28.5 Hz, exchanged with D₂O, NH), 6.00–

5.86 (m, 1 H, CH₂CHꢀCH₂), 5.46 (d, 1 H, J_1,28.2 Hz,

1

The 3-allyl ether 23 (110 mg, 19%) gave identical H

and¹³C NMR data to those of the previously prepared

sample.

The 4-allyl ether 26 as a colourless foam (140 mg,

24%); [h]²_D⁰−66° (c 1.6, CHCl₃); TLC (3:2 EtOAc–

1

hexanes): R_f0.13; H NMR (CDCl₃): l 8.14 (d, 2 H, J

9.2 Hz, ArH), 7.08 (2 H, J 9.2 Hz, ArH), 6.01–5.88 (m,

2 H, after D₂O exchange became a m, 1 H, CH₂-

CHꢀCH₂, NH), 5.32–5.23 (m, 2 H, trans CH₂CHꢀ

CHH, H-1), 5.20 (d, 1 H, J_cis10.4 Hz, CH₂CHꢀCHH),

4.35 (dd, 1 H, J 12.6, 5.7 Hz, CHHCHꢀCH₂), 4.22 (dd,

1108

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1 H, CHHCHꢀCH₂), 4.01 (dt, 1 H, J_2,3=J_3,4ꢀ8.4,

J_3,OH3.6 Hz, after D₂O exchange became a t, partially

overlapped with H-6a, H-3), 3.94 (dd, 1 H, J_6a,6b11.3,

J_5,6a2.1 Hz, H-6a), 3.86–3.75 (m, 2 H, H-6b, H-2), 3.72

(d, 1 H, exchanged with D₂O, OH-3), 3.57–3.51 (m, 1

H, H-5), 3.42 (t, 1 H, J_4,58.4 Hz, H-4), 2.06 (s, 3 H,

Ac), 0.87 (s, 9 H, Si-Bu^t), 0.01, –0.02 (2s, 6 H, 2

Si-Me); ¹³C NMR (CDCl₃): l 172.2 (s), 162.0 (s), 142.5

(s), 134.8 (d), 125.7 (d), 117.3 (t), 116.6 (d), 98.0 (d),

77.8 (d), 76.8 (d), 75.0 (d), 73.6 (t), 62.3 (t), 57.1 (d),

25.8 (q), 23.4 (q), 18.3 (s), −5.3 (q), −5.4 (q).

FABMS: m/z Calcd. for C₂₃H₃₇N₂O₈Si [M+H]⁺:

497.2319. Found: 497.2318.

4.22–4.03 (m, 2 H, H-3, H-2), 3.82–3.77 (m, 1 H,

H-6a), 3.67–3.57 (m, 2 H, H-6b, H-5), 3.44–3.34 (m, 1

H, H-4); ¹³C NMR ([CD₃]₂SO): l 168.2 (br.s), 167.8

(br.s), 161.5 (s), 142.3 (s), 134.9 (d), 131.2 (s), 125.9 (d),

123.5 (br.s), 116.7 (d), 95.3 (d), 77.9 (d), 70.7 (d), 70.2

(d), 60.4 (t), 56.9 (d). FABMS: m/z Calcd. for C₂₀

H₁₈N₂NaO₉]M+Na]⁺: 453.0910. Found: 453.0892.

4-Nitrophenyl

oxy-i- -glucopyranoside (30).—Dibutyltin(IV) oxide

(218 mg, 0.88 mmol) was added to a solution of 4-ni-

trophenyl 2-acetamido-2-deoxy-b-

-glucopyranoside³⁴

2-acetamido-6-O-chloroacetyl-2-de-

D

(300 mg, 0.88 mmol) in dry MeOH (15 mL) and the

mixture was heated under reﬂux until a clear solution

was obtained (2 h). The solvent was evaporated and the

residue suspended in dioxane (10 mL) and DMF (5

mL). Chloroacetyl chloride (0.21 mL, 2.64 mmol) was

added, the mixture became clear and after 15 min,

MeOH (3.0 mL) was added and the volatiles were

evaporated. Chromatography (19:19:1 CH₂Cl₂–

MeOH) gave the 6-ester 30 as a colourless solid which

was washed with CH₂Cl₂, hexanes and dried (243 mg,

66%); mp (acetone–Et₂O) 216–217 °C; [h]²_D⁰−34° (c

4-Nitrophenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-

imido-i-

etherate (0.18 mL, 1.45 mmol) was added to a solution

of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-b-

D

-glucopyranoside (28).—Boron triﬂuoride

D

-

glucose⁴³(1.4 g, 2.93 mmol) and 4-nitrophenol (816 mg,

5.87 mmol) in CH₂Cl₂(25 mL) and the mixture was

stirred at ambient temperature for 15 h. Further

BF₃·OEt₂(0.54 mL) and 4-nitrophenol (408 mg) were

added and stirring was continued for a further 21 h.

Dichloromethane (50 mL) was added and the solution

was washed with aq satd NaHCO₃, water, dried, and

the solvent evaporated. Chromatography (3:72:3

EtOAc–hexane) gave glycoside 28 as a colourless gum

which crystallized on addition of MeOH (704 mg, 44%,

needles); mp 172–173 °C; [h]¹_D⁹+77° (c 0.8, CHCl₃);

1

0.72, Me₂SO); TLC (4:1 CH₂Cl₂–MeOH): R_f0.63; H

NMR (4:1 CDCl₃–[CD₃]₂SO): l 8.18 (d, 2 H, J 9.2 Hz,

ArH), 7.65 (m, after D₂O exchange became a s, NH,

CHCl₃), 7.11 (d, 2 H, J 9.2 Hz, ArH), 5.29 (d, 1 H, J_1,2

8.3 Hz, H-1), 5.13 (d, 1 H, J_OH,44.5 Hz, exchanged with

D₂O, OH-4), 4.90 (d, 1 H J_OH,3, 4.4 Hz, exchanged with

D₂O, OH-3), 4.59 (dd, 1 H, J_6a,6b11.8, J_5,6a1.8 Hz,

H-6a), 4.32 (dd, 1 H J_5,6b6.9 Hz, H-6b), 4.15 (s, 2 H,

CH₂Cl), 3.88 (q, 1 H, after D₂O exchange became a dd

J_2,310.2 Hz, H-2), 3.77–3.66 (m, 2 H, H-5, H-3), 3.45

(dt, 1 H, after D₂O exchange became a dd, J 9.6, 8.7

Hz, H-4), 1.96 (s, 3 H, Ac); ¹³C NMR ([CD₃]₂SO): l

169.6 (s), 167.3 (s), 162.1 (s), 142.1 (s), 125.9 (d), 116.7

(d), 98.3 (d), 74.1 (d), 73.6 (d), 70.2 (d), 64.7 (t), 55.3

(d), 41.3 (t), 23.2 (q). FABMS: m/z Calcd. for

C₁₆H₂₀ClN₂O₉[M+H]⁺: 419.0857. Found: 419.0865.

4-Nitrophenyl 2-acetamido-3-O-benzoyl-6-O-chloro-

1

TLC (1:1 EtOAc–hexanes): R_f0.42; H NMR (CDCl₃):

l 8.15 (d, 2 H, J 9.3 Hz, ArH), 7.88–7.85 (m, 2 H,

ArH), 7.78–7.74 (m, 2 H, ArH), 7.02 (d, 2 H, J 9.3 Hz,

ArH), 6.14 (d, 1 H, J_1,28.5 Hz, H-1), 5.88 (dd, 1 H, J_2,3

10.6, J_3,49.3 Hz, H-3), 5.25 (t, 1 H, H-4), 4.65 (dd 1 H,

H-2), 4.35 (dd, 1 H, J_6a,6b12.3, J_5,6a5.5 Hz, H-6a), 4.21

(dd, 1 H, J_5,6b2.3 Hz, H-6b), 4.12–4.06 (m, 1 H, H-5),

2.11, 2.07, 1.90, (3s, 9 H, 3 Ac); ¹³C NMR (CDCl₃): l

170.4 (s), 170.0 (s), 169.4 (s), 167.6 (br.s), 160.9 (s),

143.3 (s), 134.6 (d), 131.2 (s), 125.7 (d), 123.8 (d), 116.8

(d), 95.4 (d), 72.5 (d), 70.4 (d), 68.6 (d), 61.9 (t), 54.3

(d), 20.7 (q), 20.6 (q), 20.3 (q). FABMS: m/z Calcd. for

C₂₆H₂₄CsN₂O₁₂[M+Cs]⁺: 689.0384. Found: 689.0360.

acetyl-2-deoxy-i-

D

-glucopyranoside

(31).—Benzoyl

chloride (0.52 mL, 4.5 mmol) was added dropwise over

10 min to a solution of chloroacetate 30 (900 mg, 2.15

mmol) in pyridine (20 mL) and THF (5 mL) at

−60 °C. The mixture was stirred for 1.5 h, water (3.0

mL) was added and the solution was allowed to warm

to ambient temperature. The solvent was evaporated,

the residue dissolved in EtOAc and the solution was

washed with 10% aq HCl, aq satd NaHCO₃, water and

dried. The solvent was evaporated to low volume and

the concentrate ﬁltered through a plug of SiOH

(EtOAc) and evaporated to a gum which was crystal-

lized from EtOAc–hexanes to afford diester 31 as a

colourless solid (696 mg). The mother liquors were

evaporated and chromatographed (1:17:3 EtOAc–

hexanes) to give further product (total 844 mg, 79%);

mp 195–196 °C; [h]_D²¹+34° (c 0.6, Me₂SO); TLC (7:3

4-Nitrophenyl 2-deoxy-2-phthalimido-i-D-glucopy-

ranoside (29).—Sodium methoxide in MeOH (2 mL, 1

M) was added to a solution of triacetate 28 (690 mg

1.24 mmol) in a mixture of THF (15 mL) and MeOH

(15 mL) and after 2 h at ambient temperature the

mixture was acidiﬁed to ꢀpH 6 with Dowex 50X8 100

(H⁺) resin, ﬁltered and the solvent was evaporated.

Chromatography (19:1 CH₂Cl₂–MeOH) gave triol 29

as a colourless solid (190 mg, 37%); mp (MeOH) 271–

272 °C; [h]¹_D⁹−51° (c 0.64, MeOH); TLC (19:1

1

CH₂Cl₂–MeOH): R_f0.2; H NMR ([CD₃]₂SO): l 8.14

(d, 2 H, J 9.2 Hz, ArH), 8.00–7.77 (m, 4 H, ArH), 7.17

(d, 2 H, J 9.2 Hz, ArH), 5.93 (d, 1 H, J_1,28.0 Hz, H-1),

5.61 (d, 1 H, J_OH,34.7 Hz, OH-3), 5.36 (d, 1 H, J_OH,4

5.7 Hz, OH-4), 4.71 (t, 1 H, J_OH,65.9 Hz, OH-6),

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

1109

1

EtOAc–hexanes): R_f0.55; H NMR ([CD₃]₂SO): l 8.23

2.00 (t, 1 H J_OH,66.4 Hz, exchanged with D₂O, OH-6);

¹³C NMR (CDCl₃): l 166.8 (s), 165.8 (s), 165.4 (s),

165.0 (s), 163.0 (s), 161.5 (s), 142.9 (s), 133.6 (d), 133.4

(d), 133.2 (d), 130.0 (d), 129.8 (d), 129.7 (d), 129.4 (s),

129.1 (d), 129.0 (d), 128.7 (d), 128.5 (d), 128.4 (d), 128.3

(d), 125.8 (d), 116.1 (d), 100.2 (d, C-1), 92.3 (d, C-1%),

77.8 (d, C-3), 76.7 (d, C-5), 71.7 (d, C-3%), 71.3 (d, C-5%),

69.1 (d, C-4), 69.0 (d, C-2%), 67.7 (d, C-4%), 62.8 (d, C-2),

62.2 (t, C-6), 60.7 (t, C-6%), 15.7 (q, Me). FABMS: m/z

Calcd. for C₅₅H₄₉N₂O₁₈[M+H]⁺: 1025.2980. Found:

1025.3024. Anal. Calcd for C₅₅H₄₈N₂O₁₈: C, 64.45; H,

4.72; N, 2.73. Found: C, 64.18; H, 4.81; N, 2.71.

4-Nitrophenyl 2-acetamido-3-O-benzoyl-2-deoxy-i-

(d, 2 H, J 9.1 Hz, ArH), 8.08 (d, 1 H, J_NH,29.2 Hz,

NH), 7.96 (d, 2 H, J 7.3 Hz, ArH), 7.67 (t, 1 H, J 7.5

Hz, ArH), 7.54 (t, 2 H, J 7.7 Hz, ArH), 7.24 (d, 2 H, J

9.1 Hz, ArH), 5.80 (d, 1 H, J_OH,46.1 Hz, OH-4), 5.50

(d, 1 H, J_1,28.5 Hz, H-1), 5.28 (t, 1 H, J_3,4=J_2,39.7 Hz,

H-3), 4.49 (d, 1 H, partially overlapped with ABq at

4.45, J_6a,6bꢀ12 Hz, H-6a), 4.45 (ABq, partially over-

lapped with d at 4.49, 2 H, J 15.5 Hz, CH₂Cl), 4.31 (dd,

1 H, J_5,6b6.0 Hz, H-6b), 4.17 (q, 1 H, H-2), 4.00–3.96

(m, 1 H, H-5), 3.70 (dt, 1 H, J_4,56.1 Hz, H-4), 1.66 (s,

3 H, Ac); ¹³C NMR ([CD₃]₂SO): l 169.5 (s), 167.3 (s),

165.6 (s), 161.8 (s), 142.3 (s), 133.4 (d), 130.0 (s), 129.5

(d), 128.8 (d), 126.0 (d), 116.9 (d), 97.5 (d), 76.0 (d),

73.9 (d), 67.9 (d), 64.3 (t), 53.2 (d), 41.3 (t), 22.8 (q).

FABMS: m/z Calcd. for C₂₃H₂₄ClN₂O₁₀[M+H]⁺:

523.1120. Found: 523.1106.

D

-glucopyranoside (34).—Diester 31 (50 mg, 0.096

mmol) was heated in reﬂuxing MeOH (6 mL) for 6 h.

The solvent was evaporated and the residue chro-

matographed (7:3 EtOAc–hexanes) to give 34 as a

colourless solid (40 mg, 93%); mp 160–161 °C

(EtOAc–Et₂O–hexanes), lit. 163–165 °C (EtOAc–

Et₂O);⁴⁶[h]_D²⁰−4° (c 0.81, pyridine) lit. [h]²_D⁰−5° (c

1.12, pyridine);^{46 1}H NMR ([CD₃]₂SO): l 8.22 (d, 2 H,

J 9.2 Hz, ArH), 8.04 (d, 1 H, J_NH,29.3 Hz, exchanged

with D₂O, NH), 7.96 (d, 2 H, J 7.2 Hz, ArH), 7.66 (t,

1 H, J 7.4 Hz, ArH), 7.53 (t, 2 H, J 7.7 Hz, ArH), 7.24

(d, 2 H, J 9.3 Hz, ArH), 5.54 (br.s, 1 H, exchanged with

D₂O, OH-4), 5.46 (d, 1 H, J_1,28.5 Hz, H-1), 5.25 (br.t,

1 H, J_2,3ꢀJ_3,4ꢀ10.2 Hz, H-3), 4.73 (br.t, 1 H, J

ꢀ5.8 Hz, exchanged with D₂O, OH-6), 4.12 (q, 1 H,

H-2), 3.75 (br.dd, 1 H, J_6a,6bꢀ10.8, J_5,6aꢀ5.0 Hz,

H-6a), 3.67–3.53 (m, 3 H, H-6b, H-5, H-4), 1.65 (s, 3

H, Ac); ¹³C NMR ([CD₃]₂SO): l 169.5 (s), 165.7 (s),

162.1 (s), 142.2 (s), 133.4 (d), 130.2 (s), 129.5 (d), 128.8

(d), 126.0 (d), 116.9 (d), 97.8 (d), 77.2 (d), 76.6 (d), 67.7

(d), 60.3 (t), 53.4 (d), 22.8 (q).

Prevotella sulfatase assays.—MdsA sulfatase was ex-

pressed from a recombinant plasmid system in Bac-

teroides thetaiotaomicron. The expression system was

similar to that previously described,⁸except that a

DNA sequence encoding a hexahistidine motif was

added at the C-terminus of the mdsA gene, so that the

expressed MdsA terminated in (his)₆. This enabled a

one-step puriﬁcation from periplasm by batch adsorp-

tion and elution from a Ni-NTA agarose resin using

standard methods.⁵⁴The MdsA speciﬁc activity [nmol

glucose formed min⁻¹(mg enzyme)⁻¹] was determined

using Glc 6-OSO₃Na desulfation with glucose being

assayed as previously described.^7,8A unit of MdsA

sulfatase has been deﬁned as the amount of enzyme

required to release 1 mmol of glucose min⁻¹under

standard assay conditions.⁷Desulfation rates of 4-nitro-

phenyl glycoside substrates containing GlcNAc 6-

OSO₃Na were measured by determining the

4-nitrophenol released by added auxiliary glycosidases

(A. oryzae extract, Sigma G7138, 1993 catalogue, which

has b-galactosidase and b-N-acetylhexosaminidase ac-

tivities), as described below.

Glycosylation of alcohol 31 and dechloroacetylation of

the product 32 (33).—Tetra-O-benzoyl-a-

D-galactopy-

ranosyl bromide⁵³(2.00 g, 3.03 mmol) and powdered 4

,

A molecular sieve (3.0 g) were added to a solution of

the alcohol 31 (794 mg, 1.52 mmol) in dry CH₂Cl₂(20

mL) and dry toluene (4 mL). The mixture was stirred

for 30 min then cooled to −20 °C. A solution of

AgOTf (1.17 g, 4.55 mmol) and 2,4,6-collidine (0.38

mL, 2.89 mmol) in CH₂Cl₂(20 mL) and toluene (4 mL)

was added dropwise over 15 min. Stirring was carried

out for 1.5 h, 10% aq Na₂S₂O₃(20 mL) was added and

the mixture warmed to ambient temperature and

ﬁltered through Celite. The ﬁltrate was diluted with

CH₂Cl₂(20 mL) and washed with water, dried and the

volatiles were evaporated. Chromatography (49:1

CH₂Cl₂–MeOH) gave crude chloroacetate 32 as a foam

(1.01 g). TLC (24:1 CH₂Cl₂–MeOH) R_f0.55. Crude 32

was dissolved in MeOH (15 mL) and heated under

reﬂux for 45 min, cooled and the solvent was evapo-

rated. Chromatography (4:1 toluene–acetone) gave the

4,6-diol 33 as a colourless amorphous solid (785 mg).

Recrystallization from EtOH then from EtOAc–hex-

anes gave colourless ﬁne needles; mp 140–142 °C; [h]_D¹⁸

+91° (c 1.4, CHCl₃); TLC (19:1 CH₂Cl₂–MeOH): R_f

0.53; IR (CHBr₃mull): w 3690–3145 (br, s), 1723 (s),

1595 (m), 1515 (m), 1493 (m), 1451 (m), 1339 (m), 1317

(m), 1269 (br, s), 1178 (m), 1071 (br, s), 1028 m) cm⁻¹

;

¹H NMR (CDCl₃): l 8.07–7.94 (m, 8 H, ArH), 7.76 (d,

2 H, J 7.5 Hz, ArH), 7.71 (d, 2 H, J 7.4 Hz, ArH),

7.62–7.50 (m, 3 H, ArH), 7.49–7.34 (m, 8 H, ArH),

7.19 (t, 4 H, J 7.4 Hz, ArH), 6.83 (d, 2 H, J 9.2 Hz,

ArH), 6.22 (d, 1 H, J_1%,2%8.4 Hz, H-1%), 5.96 (d, 1 H, J_3%4%

3.1 Hz, H-4%), 5.79 (dd, 1 H, J_2%,3%10.3 Hz, H-2%), 5.61

(dd, 1 H, H-3%), 5.46 (t, 1 H, J_3,4=J_2,39.4 Hz, H-3),

5.19 (d, 1 H, J_1,27.5 Hz, H-1), 4.37 (dd, 1 H, J_6a%,6b%

10.5, J_5,6a%5.0 Hz, H-6a%), 4.30–4.25 (m, 1 H, H-5%),

4.10–3.89 (m, 4 H, H-6b%, H-6a, H-6b, H-4), 3.85 (dd,

1 H, H-2), 3.79–3.74 (m, 1 H, H-5), 2.95 (d, 1 H, J_OH,4

4.7 Hz, exchanged with D₂O, OH-4), 2.10 (s, 3 H, Ac),

1110

K. Clinch et al. / Carbohydrate Research 337 (2002) 1095–1111

Compound 1 (1 mM) was incubated for 0, 20, and 60

buffer (20 mM, pH 7.4) desalted by dialysis against

buffer, and the preparations were chromatographed on

DEAE Sepharose. Fractions containing two separate

galactose-3-sulfatase isozymes (fractions a and c, Table

2) and two separate galactose-6-sulfatase isozymes

(fractions b and d, Table 2) were identiﬁed by use of the

chromogenic substrates 2 and 3. Desulfation was de-

tected by incubating the substrate (1 mM) in Tris

chloride buffer (20 mM, pH 7.4) containing auxiliary

glycosidase mixture (7.3 units of b-galactosidase plus

0.6 units of b-N-acetylhexosaminidase) and 0.025 mL

of a column fraction in a ﬁnal volume 0.08 mL at 37 °C

for 20 min, terminating the reaction by adding 0.5 M

glycine buffer (0.92 mL) to pH 9.6 and measuring the

absorption of the released 4-nitrophenol at 410 nm.

Ascending paper chromatography was carried out on

the products formed from esters 2 and 3 after incuba-

tion with sulfatase-containing fractions from B. fragilis.

The incubations were performed as above, except that

they were carried out for 120 min with 2 or 180 min

with 3, and the auxiliary glycosidase mixture was omit-

ted unless indicated. The chromatography solvent was

2:3:1 BuⁿOH–AcOH–NH₄OH (1 M).⁵⁵Free 4-nitro-

phenol and its glycosides were visualized under ultra

violet light, and reducing sugars were detected by the

silver nitrate dip protocol.⁵⁶

min with Tris–Cl buffer (37.5 mM, pH 7.4), b-mercap-

toethanol (3mM), MdsA sulfatase (0.0061 units), and

auxiliary glycosidases (7.3 units of b-galactosidase and

0.6 units of b-N-acetylhexosaminidase), in a ﬁnal reac-

tion volume of 0.08 mL. After incubation at 37 °C,

reactions were terminated by addition of 0.5 M glycine

buffer, pH 9.6 (0.92 mL) to raise the pH, and released

4-nitrophenol was measured at 410 nm. Controls with-

out auxiliary glycosidases or without sulfatase were also

carried out.

Compound 4 (1 mM) was incubated for 0, 20, and 60

min with Tris–Cl buffer, 37.5 (mM, pH 7.4), b-mercap-

toethanol (3 mM) and MdsA sulfatase (0.0061 units) in

a total volume of 0.07 mL. The sulfatase was then

inactivated by heating for 2 min in a boiling water bath.

After cooling, auxiliary glycosidases (0.01 mL contain-

ing 7.3 units of b-galactosidase and 0.6 units of b-N-

acetylhexosaminidase) were added, and

a second

incubation continued for 30 min at 37 °C. It was termi-

nated by addition of 0.5 M glycine buffer, pH 9.6, (0.92

mL) to raise the pH. The free 4-nitrophenol was mea-

sured at 410 nm. Controls without glycosidases were

carried out.

Control experiments were conducted to ensure that

the activities of the auxiliary glycosidases used were

adequate. 4-Nitrophenyl b-

D

-galactopyranosyl-(14)-

2-acetamido-2-deoxy-b- -glucopyranoside (16, 1 mM)

D

Acknowledgements

was incubated with the auxiliary glycosidase mixture

(7.3 units of b-galactosidase plus 0.6 units b-N-acetyl-

hexosaminidase) and 37.5 mM Tris–Cl buffer, ﬁnal

volume 0.05 mL, for 110 min at 37 °C. Paper chro-

matography was used to detect products. Compound 4

(1 mM) was incubated with the auxiliary glycosidase

extract (7.3 units of b-galactosidase plus 0.6 units b-N-

acetylhexosaminidase) in 37.5 mM Tris Cl buffer in a

ﬁnal volume 0.05 mL, for 110 min at 37 °C. Ten times

higher levels of auxiliary glycosidases and extended

times of incubation were also used in other experiments

with compound 4 to ensure they were not a limiting

factor in product formation.

The authors thank Robin Ferrier (Industrial Re-

search Ltd.) for assistance with preparation of the

manuscript, Herbert Wong (Industrial Research Ltd.)

for recording NMR data, John Allen, Hort Research,

Palmerston North, New Zealand for MS measure-

ments, Marianne Dick, University of Otago, New

Zealand for combustion analysis data, and the New

Zealand Foundation for Research Science and Technol-

ogy for ﬁnancial support. D.P.W. was a recipient of the

Auckland University Maori and Paciﬁc Islander Doc-

toral Scholarship. D.I.R. was a recipient of a New

Zealand Lottery Health Doctoral Scholarship. The bio-

chemical work was supported by the Auckland Medical

Research Foundation.

The b-glycosidase activities present in the auxiliary

glycosidase extract were determined using 4-nitro-

phenyl-b-

D

-galactopyranoside or 4-nitrophenyl 2-acet-

-glucopyranoside (1 mM), Tris–Cl

amido-2-deoxy-b-

D

buffer, (37.5 mM, pH 7.4) and 4-nitrophenol produc-

tion determined after pH adjustment to 9.6, as above.

B. fragilis sulfatase assays.—B. fragilis ATCC 25285

was cultured and harvested as previously described.¹²

Cell free extracts were made by 5×10 s of sonication

(18 microns amplitude) with 30 s cooling on ice be-

tween sonications, with diisopropyl ﬂuorophosphate

(0.002%, w/v) added to minimize proteinase activity.¹²

Cell debris was removed by centrifugation, the proteins

precipitating between 40 and 90% ammonium sulfate

saturation were selected, dissolved in Tris chloride

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Article Doi

Synthesis and utility of sulfated chromogenic carbohydrate model substrates for measuring activities of mucin-desulfating enzymes

DOI: 10.1016/S0008-6215(02)00104-0

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Full text of DOI:10.1016/S0008-6215(02)00104-0

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