Synthesis and conjugation of oligosaccharide analogues of fragments of the immunoreactive glycan part of the circulating anodic antigen of the parasite Schistosoma mansoni

DOI: 10.1039/b410241j

Source and publish data:

Organic and Biomolecular Chemistry p. 2972 - 2987 (2004)

Update date:2022-08-04

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Authors:

De Sonza, Adriana Carvalho

Kuil, Joeri

Elizabeth

Maljaars

Halkes, Koen M.

Vliegenthart, Johannes F. G.

Kamerling, Johannis P.

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Article abstract of DOI:10.1039/b410241j

The gut-associated circulating anodic antigen (CAA) is one of the major excretory antigens produced by the parasite Schistosoma mansoni. The immunoreactive part of CAA is a threonine-linked polysaccharide composed of long stretches of the unique repeating

Full text of DOI:10.1039/b410241j

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A R T I C L E

Synthesis and conjugation of oligosaccharide analogues of fragments

of the immunoreactive glycan part of the circulating anodic antigen

of the parasite Schistosoma mansoni

Adriana Carvalho de Souza, Joeri Kuil, C. Elizabeth P. Maljaars, Koen M. Halkes,

Johannes F. G. Vliegenthart and Johannis P. Kamerling*

Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis

Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.

E-mail: j.p.kamerling@chem.uu.nl

Received 6th July 2004, Accepted 16th August 2004

First published as an Advance Article on the web 22nd September 2004

The gut-associated circulating anodic antigen (CAA) is one of the major excretory antigens produced by the

parasite Schistosoma mansoni. The immunoreactive part of CAA is a threonine-linked polysaccharide composed

of long stretches of the unique repeating disaccharide →6)-[b-D-GlcpA-(1→3)]-b-D-GalpNAc-(1→. Previously,

using surface plasmon resonance and ELISA techniques, it has been shown that some anti-CAA IgM monoclonal

antibodies (MAbs) also recognize members of a series of bovine serum albumin (BSA)-coupled synthetic di- to

penta-saccharide fragments of the CAA glycan. To generate information on the molecular level about the glycan

specificity of the relevant IgM MAbs, two series of oligosaccharides related to the CAA disaccharide epitope were

synthesized, and coupled to BSA. The first three analogues, b-D-GlcpA-(1→3)-b-D-GlcpNAc-(1→O), b-D-GlcpNAc-

(1→6)-[b-D-GlcpA-(1→3)]-b-D-GlcpNAc-(1→O), and b-D-GlcpA-(1→3)-b-D-GlcpNAc-(1→6)-[b-D-GlcpA-(1→

3)]-b-D-GlcpNAc-(1→O), wherein the native b-D-GalpNAc moiety was replaced by b-D-GlcpNAc, were synthesized

to investigate the specificity of the selected MAbs to the carbohydrate backbone of CAA. The second series of

analogues, b-D-Glcp6S-(1→3)-b-D-GalpNAc-(1→O), b-D-GalpNAc-(1→6)-[b-D-Glcp6S-(1→3)]-b-D-GalpNAc-(1→

O), and b-D-Glcp6S-(1→3)-b-D-GalpNAc-(1→6)-[b-D-Glcp6S-(1→3)]-b-D-GalpNAc-(1→O), wherein the native b-

D-GlcpA moiety was replaced by b-D-Glcp6S, was synthesized to evaluate the importance of the type/nature of the

charge of CAA for the MAb recognition.

this polysaccharide chain may be responsible for the absolute

specificity of the CAA immunodiagnostic assays. In a previous

study, a panel of monoclonal antibodies (MAbs) raised against

S. mansoni adult worm antigens was screened for recognition of

synthetic di- to penta-saccharide fragments of the CAA polysac-

charide, multivalently presented as bovine serum albumin (BSA)

conjugates.^11,12The results showed that several MAbs, especially

of the IgM class, already recognized the disaccharide unit b-D-

GlcpA-(1→3)-b-D-GalpNAc-(1→O). In order to understand in

molecular detail the specificity of the anti-carbohydrate MAbs,

we synthesized two series of structures (Fig. 1) related to the

CAA epitope, and conjugated these oligosaccharides to BSA,

using squaric diester chemistry. The first series of synthetic

analogues, b-D-GlcpA-(1→3)-b-D-GlcpNAc-(1→O) (1), b-D-

GlcpNAc-(1→6)-[b-D-GlcpA-(1→3)]-b-D-GlcpNAc-(1→O)

(2), and b-D-GlcpA-(1→3)-b-D-GlcpNAc-(1→6)-[b-D-GlcpA-

(1→3)]-b-D-GlcpNAc-(1→O) (3), has the native b-D-GalpNAc

residue replaced by b-D-GlcpNAc to evaluate the importance

of the hydroxyl function HO4 in the carbohydrate backbone

for the MAb recognition. In the other series of structures,

b-D-Glcp6S-(1→3)-b-D-GalpNAc-(1→O) (4), b-D-GalpNAc-

(1→6)-[b-D-Glcp6S-(1→3)]-b-D-GalpNAc-(1→O) (5), and

b-D-Glcp6S-(1→3)-b-D-GalpNAc-(1→6)-[b-D-Glcp6S-(1→

3)]-b-D-GalpNAc-(1→O) (6), the native b-D-GlcpA moiety was

replaced by b-D-Glcp6S in order to investigate the influence of

the nature of charge on the MAb recognition.

Introduction

One of the most prevalent tropical diseases is schistosomiasis,

otherwise known as bilharzia, which is caused by a parasitic

blood fluke of the genus Schistosoma. The intriguing and

complex life cycle of this worm involves several parasitic

stages in the intermediate (fresh-water snails) and definitive

host, alternated by two larvae stages.¹Owing to the regional

occurrence of the intermediate host, the disease is limited to

tropical and subtropical areas, where an estimated 200 million

people are infected and suffer from the debilitating effects of

this disease.²The control strategies against schistosomiasis are

normally built on the results of diagnosic tests. The microscopic

demonstration of the parasite’s eggs on faeces or in urine is

the more widespread tool for the diagnosis of Schistosoma

infections in epidemic areas.

In recent years a variety of immunological techniques have

been described in the literature as alternative techniques to faecal

or urinary egg counts.^3–6An early and strong humoral immune

response to schistosomes is directed to glycan epitopes of a

number of circulating antigens, in particular the gut-associated

circulating anodic antigen (CAA) and circulating cathodic

antigen (CCA).³Moreover, several studies have demonstrated

a strong correlation between antigen levels and the number

of adult worms,^7,8and that antigen levels decreased rapidly

following successful chemotherapy.⁸For the assessment of a

cure and for the diagnosis of active infections in endemic areas,

the method of choice is the determination of CAA or CCA in

the serum or urine of infected subjects.⁶So far, a specificity of

virtually 100% was found at the ELISA demonstration of CAA

in serum, while for CCA, false positive results were occasionally

observed.⁹

Results and discussion

Synthesis of oligosaccharide analogues 1–3 containing b-D-

GlcpNAc instead of b-D-GalpNAc moieties

In the synthetic routes to target oligosaccharides 1–3, four

earlier reported monosaccharide building blocks were used,

namely, 7, 8, 11, and 14. In contrast to our earlier approach of

preparing fragments of the native CAA glycan, which included a

The major immunogenic character of CAA is carried by

an O-linked polysaccharide chain composed of the unique

disaccharide repeating unit →6)-[b-D-GlcpA-(1→3)]-b-D-

GalpNAc-(1→.¹⁰The uniqueness of the primary structure of

2 9 7 2

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Fig. 1 Target oligosaccharides 1–6.

time-consuming C6 oxidation step at later stages of the synthesis

to generate glucuronic acid units, here, methyl 2,3,4-tri-O-acetyl-

a,b-D-glucopyranosyluronate trichloroacetimidate 8 has been

chosen.

86%; Scheme 2). Saponification of 12 with 3 M aq. NaOH in

5:1 methanol–water, followed by dephthaloylation with 1,2-di-

aminoethane in n-butanol at 90 °C, and subsequent N-acetyla-

tion using acetic anhydride in methanol at 0 °C, rendered allyl

glycoside 13 (42%). Elongation of the allyl spacer of 13 with

cysteamine resulted in target trisaccharide 2 (48%).

For the synthesis of tetrasaccharide 3, disaccharide donor

17 was prepared (Scheme 3). Coupling of donor 8¹⁴with

acceptor 4-methoxyphenyl 2-deoxy-4,6-O-isopropylidene-2-

phthalimido-b-D-glucopyranoside 14,²⁰using trimethylsilyl tri-

flate as a promoter, followed by removal of the isopropylidene

group under acidic conditions, gave disaccharide 15 (71%). As

mentioned already for 9, this condensation reaction also pro-

ceeds via orthoester formation and subsequent conversion into

the desired adduct. The good yield obtained here is probably

due to a different protection of the anomeric center of acceptor

14. After conventional acetylation of the HO4 and HO6 groups

of 15 (→16, quantitative), oxidative removal of the anomeric

4-methoxyphenyl group, using ammonium cerium(IV) nitrate,²¹

followed by imidation¹⁹of the hemiacetal, resulted in disaccha-

ride donor 17 (63%).

For the synthesis of disaccharide 1, acceptor allyl 2-deoxy-

4,6-O-isopropylidene-2-phthalimido-b-D-glucopyranoside 7¹³

was coupled with donor methyl 2,3,4-tri-O-acetyl-a,b-D-gluco-

pyranosyluronate trichloroacetimidate 8,¹⁴using trimethylsilyl

triflate as a promoter (Scheme 1). After removal of the

isopropylidene group under acidic conditions,¹⁵disaccharide 9

was obtained in a moderate yield (31%). The main side reaction

during the condensation is the formation of an orthoester

intermediate that could not be completely converted into the

desired product. Alternative attempts, such as using different

temperatures, promoter concentrations, and protecting groups

for the donor, did not improve the yield. For deprotection of

the disaccharide 9, the methyl ester and acetyl groups were

saponified with 3 M aq. NaOH in 5:1 methanol–water.¹⁴

Subsequent dephthaloylation was carried out with 1,2-diamino-

ethane in n-butanol at 90 °C,¹⁶and the formed product was N-

acetylated with acetic anhydride in methanol at 0 °C¹⁷to give the

fully deprotected allyl glycoside 10 (52%). Finally, 10 was reacted

with cysteamine hydrochloride¹⁸under radical conditions (UV-

irradiation) to afford the amino-spacer-containing disaccharide

1 (76%).

Regioselective coupling of disaccharide donor 17 with

disaccharide acceptor 9 (Scheme 4), using trimethylsilyl triflate

as a promoter, gave tetrasaccharide 18 (90%). Saponification

of 18 with 3 M aq. NaOH in 5:1 methanol–water, followed by

dephthaloylation and N-acetylation afforded allyl glycoside 19

(42%). Finally, 19 was elongated with cysteamine under UV-

light, to obtain the amino-spacer-containing tetrasaccharide 3

(56%).

For the synthesis of trisaccharide 2, 3,4,6-tri-O-acetyl-2-

deoxy-2-phthalimido-b-D-glucopyranosyl trichloroacetimidate

11¹⁹was regioselectively-coupled with disaccharide acceptor

9, at 0 °C, using trimethylsilyl triflate as a promoter (→12,

Scheme 1 Reagents and conditions: a, (i) 7, 8 (2 equiv.), TMSOTf, CH₂Cl₂, 30 min, 0 °C/2 h, rt; (ii) water, TFA, CH₂Cl₂, overnight, 31% over two

reaction steps; b, (i) 3 M aq. NaOH, 5:1 MeOH–water, 3 h; (ii) 1:2 1,2-diaminoethane–n-butanol, overnight, 90 °C; (iii) acetic anhydride, MeOH, 3 h,

0 °C, 52% over three reaction steps; c, cysteamine hydrochloride, UV-light, water, 2 h, 76%.

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 7 3

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Scheme 2 Reagents and conditions: a, 9, 11 (1.5 equiv.), TMSOTf, CH₂Cl₂, 1 h, 0 °C, 86%; b, (i) 3 M aq. NaOH, 5:1 MeOH–water, 3 h; (ii) 1:2

1,2-diaminoethane–n-butanol, overnight, 90 °C; (iii) acetic anhydride, MeOH, 3 h, 0 °C, 42% over three reaction steps; c, cysteamine hydrochloride,

UV-light, water, 2 h, 48%.

Scheme 3 Reagents and conditions: a, (i) 14, 8 (1.5 equiv.), TMSOTf, CH₂Cl₂, 45 min, 0 °C/30 min, rt; (ii) water, TFA, CH₂Cl₂, 1 h, 71% over two

reaction steps; b, 1:1 pyridine–acetic anhydride, CH₂Cl₂, overnight, quantitative; c, (i) CAN, 1:1:1 toluene–acetonitrile–water, 45 min; (ii) trichloro-

acetonitrile, DBU, CH₂Cl₂, overnight, 63% over two reaction steps. MP = C₆H₄OCH₃.

Scheme 4 Reagents and conditions: a, 9, 17 (1.5 equiv.), TMSOTf, CH₂Cl₂, 1 h, 0 °C, 90%; b, (i) 3 M aq. NaOH, 5:1 MeOH–water, 3 h; (ii) 1:2

1,2-diaminoethane–n-butanol, overnight, 90 °C; (iii) acetic anhydride, MeOH, 3 h, 0 °C, 42% over three reaction steps; c, cysteamine hydrochloride,

UV-light, water, 2 h, 56%.

Synthesis of oligosaccharide analogues 4–6 containing b-D-

D-glucopyranosyl trichloroacetimidate 20¹⁵with acceptor

Glcp6S instead of b-D-GlcpA moieties

4-methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-

b-D-glucopyranoside 21²²using trimethylsilyl triflate as a

promoter, followed by the removal of the benzylidene group

under acidic conditions,¹⁵afforded disaccharide 22 (78%)

(Scheme 5). It should be noted that the use of an isopropyl-

idene instead of a benzylidene protecting group gives rise

to much lower yields. A tert-butyldiphenylsilyl group was

selectively introduced at HO6 of 22 using tert-butyldiphenylsilyl

chloride in the presence of 2:1 pyridine–triethylamine and a

catalytic amount of 4-dimethylaminopyridine, to give 23 in

In the synthetic routes to target oligosaccharides 4, 5, and 6

(Fig.1),thedisaccharide4-methoxyphenyl(6-O-levulinoyl-2,3,4-

tri-O-p-toluoyl-b-D-glucopyranosyl)-(1→3)-4-O-acetyl-6-O-

tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-b-D-galactopyranoside

24 was used as a central building block, easy to transform into

either a donor or an acceptor. The levulinoyl group at HO6

of the b-D-glucose residue can be selectively removed, and

subsequently sulfated to prepare the desired mimic structures.

Coupling of donor 6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-a-

2 9 7 4

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

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91% yield.²³At this stage, the b-D-glucosamine residue was

transformed into the desired b-D-galactosamine residue by

epimerization of the HO4 function.¹⁶To this end, 23 was treated

with trifluoromethanesulfonic anhydride in the presence of

pyridine and a catalytic amount of 4-dimethylaminopyridine.

The S_N2 displacement of the introduced triflate group at O4

using tetrabutylammoniun acetate in DMF resulted in the

desired 4-O-acetylated disaccharide 24 (70%).

For the synthesis of disaccharide 4, the anomeric 4-methoxy-

phenyl group was oxidatively removed using ammonium

cerium(IV) nitrate, and subsequent imidation of the hemiacetal

yielded disaccharide donor 25 (61%). Coupling of 25 with

5-azidopentanol, using trimethylsilyl triflate as a promoter

resulted in the azido-spacer-containing disaccharide 26 (85%;

Scheme 6). Removal of the tert-butyldiphenylsilyl group, under

neutral conditions, using tetrabutylammonium fluoride (→27,

91%),²⁴followed by acetylation under conventional condi-

tions gave disaccharide 28 (83%). After selective removal of

the levulinoyl group using hydrazine acetate (→29, 91%),^25,26

sulfation of the liberated HO6 group was accomplished using

the sulfur trioxide trimethylamine complex (→30, 50%).²⁷

Dephthaloylation/deacylation with ethanolic 33% methylamine

(7 days), and N-acetylation with acetic anhydride in methanol at

0 °C,¹⁶yielded azido-spacer-containing disaccharide 31 (76%).

Finally, catalytic hydrogenation of the azido group of 31 using

10% palladium on charcoal and sodium borohydride¹¹gave the

amino-spacer-containing disaccharide 4 (71%).

this moderate yield can be explained by the loss of acceptor

during coupling due to in situ O-acetyl migration from HO4 to

HO6. Delevulinoylation of 33 using hydrazine acetate (→34,

74%), followed by sulfation of the generated HO6 group

yielded sulfated trisaccharide 35 in 82% overall yield. Finally,

dephthaloylation/deacylation followed by N-acetylation af-

forded the azido-spacer-containing trisaccharide 36 (74%), of

which the azido group was selectively hydrogenated to give the

amino-spacer-containing trisaccharide 5 (89%).

In order to synthesize tetrasaccharide 6, disaccharide donor

38 was prepared from disaccharide building block 24 in a

two-step reaction sequence (Scheme 8). Removal of the tert-

butyldiphenylsilyl group of 24 using tetrabutylammonium

fluoride was directly followed by conventional acetylation of the

generated HO6 function (→37, 84%). Oxidative removal of the

anomeric 4-methoxyphenyl group with ammonium cerium(IV)

nitrate, followed by imidation gave 38 in 58% overall yield.

Condensation of 27 with 38 in the presence of trimethylsilyl

triflate afforded tetrasaccharide 39 (70%). Treatment of 39 with

hydrazine acetate (→40, 75%), sulfation of the two generated

HO6 groups with the sulfur trioxide trimethylamine complex

(→41, 77%), and dephthaloylation/deacylation followed by

N-acetylation gave the disulfated azido-spacer-containing

tetrasaccharide 42 (62%). Finally, catalytic hydrogenation

of the azido group yielded the amino-spacer-containing

tetrasaccharide 6 (71%).

For the synthesis of trisaccharide 5, disaccharide accep-

tor 27 was coupled with donor 3,4,6-tri-O-acetyl-2-deoxy-2-

phthalimido-b-D-galactopyranosyl trichloroacetimidate 32,²⁸

using trimethylsilyl triflate as a promoter, to yield trisaccharide

33 (58%; Scheme 7). As the 6-O-acetylated variant of 27 is the

main side product isolated from this condensation reaction,

Preparation of neoglycoconjugates BSA-1–BSA-6

Compounds 1–6 were conjugated to pre-treated bovine

serum albumin (BSA)¹¹using diethyl squarate as a linker.

Reaction of the amine functions of 1–6 with diethyl

squarate was performed in ethanol–50 mM sodium phosphate

Scheme 5 Reagents and conditions: a, (i) 21, 20 (1.5 equiv.), TMSOTf, CH₂Cl₂, 30 min; (ii) water, TFA, CH₂Cl₂, 78% over two reaction steps; b, 2:1 pyr-

idine–Et₃N, catalytic DMAP, CH₂Cl₂, TBDPSCl, overnight, 91%; c, (i) trifluoromethanesulfonic anhydride, pyridine, catalytic DMAP, CH₂Cl₂, 30 min,

0 °C/5 h, rt; (ii) TBAA, DMF, 2 h, 70% over two reaction steps; d, (i) CAN, 1:1:1 toluene–acetonitrile–water, 2 h; (ii) trichloroacetonitrile, DBU, CH₂Cl₂,

3 h, 61% over two reaction steps. Lev = COCH₂CH₂COCH₃; MP = C₆H₄OCH₃; Ph = C₆H₅; Tol = COC₆H₄CH₃; TBDPS = (CH₃)₃CSi(C₆H₅)₂.

Scheme 6 Reagents and conditions: a, 25, 5-azidopentanol (2 equiv.), TMSOTf, CH₂Cl₂, 30 min, 85%; b, 1 M TBAF in THF, HOAc, pH 7, 2 h,

0 °C/4 h, rt, 91%; c, 1:1 pyridine–acetic anhydride, overnight, 83%; d, NH₂NH₂·HOAc, EtOH, toluene, 2 h, 91%; e, SO₃·NMe₃, DMF, 48 h, 50 °C,

50%; f, (i) 33% NH₂Me in EtOH, 7 d; (ii) acetic anhydride, MeOH, 3 h, 0 °C, 76% over two reaction steps; g, 0.05 M aq. NaOH, 10% Pd–C, NaBH₄,

water, 45 min, 71%. Lev = COCH₂CH₂COCH₃; Tol = COC₆H₄CH₃; TBDPS = (CH₃)₃CSi(C₆H₅)₂.

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 7 5

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Scheme 7 Reagents and conditions: a, 27, 32 (1.5 equiv.), TMSOTf, CH₂Cl₂, 15 min, 0 °C, 58%; b, NH₂NH₂·HOAc, EtOH, toluene, 2 h, 74%;

c, SO₃·NMe₃, DMF, 48 h, 50 °C, 82%; d, (i) 33% NH₂Me in EtOH, 7 d; (ii) acetic anhydride, MeOH, 3 h, 0 °C, 74% over two reaction steps; e, 0.05 M

aq. NaOH, 10% Pd–C, NaBH₄, water, 1 h, 89%. Lev = COCH₂CH₂COCH₃; Tol = COC₆H₄CH₃.

Scheme 8 Reagents and conditions: a, (i) 1 M TBAF in THF, HOAc, pH 7, 1 h, 0 °C/overnight, rt; (ii) 1:1 pyridine–acetic anhydride, overnight, 84%

over two reaction steps; b, CAN, 1:1:1 toluene–acetonitrile–water, 2 h; (ii) trichloroacetonitrile, DBU, CH₂Cl₂, 16 h, 58% over two reaction steps;

c, 27, 38 (1.5 equiv.), TMSOTf, CH₂Cl₂, 15 min, 70%; d, NH₂NH₂·HOAc, EtOH, toluene, 2 h, 75%; e, SO₃·NMe₃, DMF, 48 h, 50 °C, 77%; f, (i) 33%

NH₂Me in EtOH, 7 d; (ii) acetic anhydride, MeOH, 3 h at 0 °C, 62% over two reaction steps; g, 0.05 M aq. NaOH, 10% Pd–C, NaBH₄, water, 1 h, 71%.

Lev = COCH₂CH₂COCH₃; MP = C₆H₄OCH₃; Tol = COC₆H₄CH₃; TBDPS = (CH₃)₃CSi(C₆H₅)₂.

buffer (pH 7.2).^29,30The obtained squarate-linker-containing

disaccharides 43 and 46 were purified by solid phase extraction

on a C-18 Extract-Clean™ column. However, purification of the

larger saccharides (44, 45, 47, and 48) needed another protocol;

here, column chromatography on silica gel (7.5:1.5:1.0 EtOAc–

MeOH–water) was used. The isolated squarate-linker-contain-

ing saccharides 43–48 were directly-coupled to BSA in 0.1 M

sodium bicarbonate buffer at pH 9.0 (Fig. 2).

Experimental

General procedures

All chemicals were of reagent grade, and were used without

further purification. Reactions were monitored by TLC on

Silica Gel 60F₂₅₄(Merck); after examination under UV-light,

compounds were visualized by heating with 10% methanolic

H₂SO₄, orcinol (2 mg cm⁻³) in 20% methanolic H₂SO₄, or

ninhydrin (1.5 mg cm⁻³) in 38:1.75:0.25 1-BuOH–H₂O–HOAc.

In the work-up procedures of reaction mixtures, organic

solutions were washed with appropriate amounts of the

indicated aqueous solutions, then dried with MgSO₄, and

concentrated under reduced pressure at 30–50 °C on a water

bath. Column chromatography was performed on Silica Gel

60 (Merck, 0.040–0.063 mm). Optical rotations were measured

with a Perkin-Elmer 241 polarimeter, using a 10 cm, 1 cm³cell.

¹H NMR spectra were recorded at 300 K with a Bruker AC 300

(300 MHz) or a Bruker AMX 500 (500 MHz) spectrometer;

As already observed in previous studies,^11,31the conjugation

yield of an oligosaccharide to BSA decreases as its size increases.

The average number of carbohydrate fragments incorporated

in BSA was measured using MALDI-TOF MS analysis by

determination of the center of the distribution of the single-

charged molecular ion (Fig. 3). The neoglycoconjugates

BSA-1–BSA-6 have been applied to a panel of MAbs against

Schistosoma mansoni antigens in immunoreactivity studies,

using ELISA and surface plasmon resonance detection. The

results of this work will be published elsewhere.

2 9 7 6

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

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Fig. 2 Neoglycoconjugates BSA-1–BSA-6.

DE Pro (Applied Biosystems) instrument in the reflector mode

at a resolution of 5000 FWHM. 2,4-Dihydroxybenzoic acid in

H₂O (5 mg cm⁻³) was used as a matrix. A ladder of maltose

oligosaccharides (G3–G13) was added as internal standard.

Allyl (methyl 2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate)-

(1→3)-2-deoxy-2-phthalimido-b-D-glucopyranoside 9

solution of

allyl 2-deoxy-4,6-O-isopropylidene-2-

phthalimido-b-D-glucopyranoside¹³(7; 0.55 g, 1.41 mmol)

and methyl 2,3,4-tri-O-acetyl-a,b-D-glucopyranosyluronate

trichloroacetimidate¹⁴(8; 1.35 g, 2.82 mmol) in dry CH₂Cl₂

(20 cm³), containing activated molecular sieves (4 Å, 1 g), was

stirred for 1 h at rt, then TMSOTf (69 mm³, 0.35 mmol) was

added at 0 °C. The mixture was stirred for 30 min at 0 °C and

2 h at rt, when TLC (95:5 CH₂Cl₂–acetone) showed the forma-

tion of a new product (R_f= 0.56). After neutralization with dry

pyridine and filtration, the solution was washed with 10% aq.

NaCl, dried, filtered, and concentrated. To a solution of the

residue in CH₂Cl₂(20 cm³) and water (0.1 cm³) was added TFA

(1 cm³). The mixture was stirred overnight, when TLC (95:5

CH₂Cl₂–acetone) showed the removal of the isopropylidene

group to be complete (R_f= 0.24). Then, the mixture was washed

with saturated aq. NaHCO₃, dried, filtered, and concentrated.

Column chromatography (9:1 CH₂Cl₂–acetone) of the residue

gave 9 (0.3 g, 31%), isolated as a yellow foam; [a]²_D⁰−16 (c 0.5 in

CHCl₃); d_H(300 MHz; CDCl₃) 1.93, 1.99, and 2.16 (each 3 H,

3 × s, 3 × Ac), 3.54 (1 H, m, H-5), 3.67 (1 H, br t, H-4), 3.76

(3 H, s, COOCH₃), 3.96 (1 H, m, H-6), 4.01 and 4.22 (each 1 H,

2 × m, OCH₂CHCH₂), 4.05 (1 H, d, J_{H-4,H-5}9.6 Hz, H-5),

4.22 (1 H, dd, J_H-1,H-28.5, J_H-2,H-310.8, H-2), 4.46 (1 H, d, J_{H-1,H-2}

7.8, H-1), 4.53 (1 H, dd, J_H-3,H-48.1, H-3), 4.87 (1 H, br t, H-2),

5.05 (1 H, d, H-1), 5.64 (1 H, m, OCH₂CHCH₂), 7.78 and 7.86

(each 2 H, 2 × m, Phth); d_C(75.5 MHz; CDCl₃) 20.4 (COCH₃),

53.2 (COOCH₃), 54.9 (C-2), 63.0 (C-6), 69.9 (OCH₂CHCH₂),

68.7, 70.6, 71.0, 71.4, 71.9, 75.5, and 81.8 (C-3, C-4, C-5, C-

2, C-3, C-4, and C-5), 97.4 and 100.0 (C-1 and C-1), 117.4

(OCH₂CHCH₂), 133.5 (OCH₂CHCH₂), 123.6 and 134.6

[N(CO)₂C₆H₄]; high resolution MALDI-TOF MS, m/z found

M+Na 688.185, C₃₀H₃₅NNaO₁₆requires 688.185.

Fig. 3 MALDI-TOF MS spectra: (a) BSA (top), BSA-1 (n = 5.8,

c.e. 58%), BSA-2 (n = 5.0, c.e. 50%), and BSA-3 (n = 3.2, c.e. 32%);

(b) BSA (top), BSA-4 (n = 5.9, c.e. 59%), BSA-5 (n = 4.0, c.e. 40%), and

BSA-6 (n = 3.0, c.e. 30%). n = oligosaccharide loading; c.e. = coupling

efficiency.

d_Hvalues are given in ppm relative to the signal for internal

Me₄Si (d_H= 0, CDCl₃) or internal acetone (d_H= 2.22, D₂O). ¹³

NMR spectra (APT, 75.5 MHz) were recorded at 300 K with a

Bruker AC 300 spectrometer; d_Cvalues are given in ppm relative

to the signal of CDCl₃(d_C= 77.1, CDCl₃) or internal acetone

(d_C= 30.9, D₂O). Two-dimensional ¹H–¹H TOCSY (mixing

times 7 and 100 ms) and ¹H–¹³C correlated HSQC spectra were

recorded at 300 K with a Bruker AMX 500 spectrometer. Exact

masses were measured by matrix-assisted laser desorption

ionization time-of-flight mass spectrometry using a Voyager-

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 7 7

View Article Online

gave 12 (70 mg, 86%), isolated as a white solid; [a]²_D⁰−7 (c 0.2

in CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 1.51,

1.80, 1.87, 1.93, 1.96, and 2.07 (each 3 H, 6 × s, 6 × Ac), 3.21

(1 H, dd, J_H-3,H-49.8 Hz, J_H-4,H-58.3 Hz, H-4), 3.43 (1 H, m, H-5),

3.65 (1 H, dd, J_H-5,H-6b6.3, J_H-6a,H-6b11.0, H-6b), 3.67 and 3.87

(each 1 H, 2 × m, OCH₂CHCH₂), 3.69 (3 H, s, COOCH₃),

3.83 (1 H, m, H-5), 3.88 (1 H, d, J_{H-4,H-5}9.9, H-5), 3.97 (1 H,

dd, J_H-1,H-28.4, J_H-2,H-310.7, H-2), 4.12 (1 H, dd, J_{H-5,H-6a}2.3,

J_{H-6a,H-6b}12.2, H-6a), 4.20 (1 H, dd, J_H-5,H-6a1.4, H-6a), 4.22

(1 H, d, J_{H-1,H-2}8.0, H-1), 4.28 (1 H, br t, H-3), 4.29 (1 H, m,

H-6b), 4.30 (1 H, dd, J_{H-1,H-2}8.6, J_{H-2,H-3}10.9, H-2), 4.63 (1 H,

dd, J_{H-2,H-3}9.5, H-2), 4.78 (1 H, d, H-1), 4.86 and 4.90 (each

1 H, 2 × m, OCH₂CHCH₂), 4.92 (1 H, br t, H-3), 5.00 (1 H,

br t, H-4), 5.11 (1 H, dd, J_{H-3,H-4}9.2, J_{H-4,H-5}10.1, H-4), 5.38

(1 H, d, H-1), 5.40 (1 H, m, OCH₂CHCH₂), 5.76 (1 H, dd,

H-3), 7.69 and 7.78 (each 4 H, 2 × m, 2 × Phth); d_C(125 MHz;

CDCl₃) 18.8, 19.3, 19.4, 19.5, 19.6, and 19.8 (6 × COCH₃),

52.2 (COOCH₃), 53.7 (C-2), 53.8 (C-2), 61.0 (C-6), 67.6 (C-

4), 68.0 (OCH₂CHCH₂), 68.1 (C-4), 68.5 (C-6), 68.6 (C-4),

69.7 (C-2), 69.8 (C-3), 70.2 (C-5), 70.8 (C-5), 70.9 (C-3),

74.1 (C-5), 80.4 (C-3), 95.8 (C-1), 97.9 (C-1), 98.7 (C-1), 116.4

(OCH₂CHCH₂), 132.4 (OCH₂CHCH₂), 122.7 and 133.4

[N(CO)₂C₆H₄]; high resolution MALDI-TOF MS, m/z found

M+Na 1105.286, C₅₀H₅₄N₂NaO₂₅requires 1105.291.

Allyl (b-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-

deoxy-b-D-glucopyranoside 10

To a solution of 9 (28 mg, 42 lmol) in 5:1 MeOH–water (3 cm³)

was added, at 0 °C, 3 M aq. NaOH (1 cm³). The mixture was

stirred for 3 h at room temperature, neutralized with Dowex

50 X 8 H⁺resin, filtered, and concentrated. A solution of the

residue in 2:1 n-butanol–1,2-diaminoethane (6 cm³) was stirred

overnight at 90 °C, then co-concentrated with toluene. To a

solution of the residue in dry MeOH (5 cm³) was added, at 0 °C,

acetic anhydride (100 mm³). The mixture was stirred for 3 h,

then concentrated. Size-exclusion chromatography (Bio-Gel

P-2, 100 mM NH₄HCO₃) gave 10 (9.4 mg, 52%), isolated after

lyophilizationfromwater,asawhiteamorphouspowder;[a]_D²⁰−96

(c 0.6 in water); d_H(500 MHz; D₂O; 2D TOCSY and HSQC) 2.02

(3 H, s, NAc), 3.34 (1 H, br t, H-2), 3.45 (1 H, m, H-5), 3.49 and

3.50 (each 1 H, 2 × br t, H-3 and H-4), 3.52 (1 H, dd, J_H-3,H-4

8.2 Hz, J_H-4,H-59.8 Hz, H-4), 3.72 (1 H, d, J_{H-4,H-5}9.3, H-5), 3.73

(1 H, br t, H-3), 3.76 (1 H, dd, J_H-5,H-6b5.5, J_H-6a,H-6b12.4, H-6b),

3.84 (1 H, dd, J_H-1,H-28.6, J_H-2,H-310.2, H-2), 3.91 (1 H, dd, J_H-5,H-6a

2.2, H-6a), 4.16 and 4.33 (each 1 H, 2 × m, OCH₂CHCH₂), 4.46

(1 H, d, J_{H-1,H-2}7.8, H-1), 4.58 (1 H, d, H-1), 5.25 and 5.29 (each

1 H, 2 × m, OCH₂CHCH₂), 5.90 (1 H, m, OCH₂CHCH₂);

d_C(125 MHz; D₂O) 22.9 (NDCOCH₃), 55.1 (C-2), 61.5 (C-6),

69.5 (C-4), 71.2 (OCH₂CHCH₂), 72.4 and 76.2 (C-3 and C-

4), 73.5 (C-2), 76.3 (C-5), 76.5 (C-5), 83.7 (C-3), 100.7 (C-1),

103.7 (C-1), 119.0 (OCH₂CHCH₂), 134.1 (OCH₂CHCH₂);

high resolution MALDI-TOF MS, m/z found M+H 438.164,

C₁₇H₂₈NO₁₂requires 438.161.

Allyl (2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1→6)-[(b-D-

glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-b-D-

glucopyranoside 13

To a solution of 12 (26 mg, 24 lmol) in 5:1 MeOH–water

(3 cm³) was added, at 0 °C, 3 M aq. NaOH (1 cm³). The mix-

ture was stirred for 3 h at room temperature, neutralized with

Dowex 50 X 8 H⁺resin, filtered, and concentrated. A solution

of the residue in 2:1 n-butanol–1,2-diaminoethane (6 cm³) was

stirred overnight at 90 °C, then co-concentrated with toluene.

To a solution of the residue in dry MeOH (5 cm³) was added,

at 0 °C, acetic anhydride (100 mm³). The mixture was stirred for

3 h, then concentrated. Size-exclusion chromatography (Bio-Gel

P-2, 100 mM NH₄HCO₃) gave 13 (6.5 mg, 42%), isolated after

lyophilization from water, as a white amorphous powder; [a]_D²⁰–9

(c 0.3 in water); d_H(500 MHz; D₂O; 2D TOCSY and HSQC) 2.04

and 2.05 (each 3 H, 2 × s, 2 × NAc), 3.34 (1 H, br t, H-2), 3.45

(1 H, br t, H-4), 3.46 (1 H, m, H-5), 3.50 (1 H, br t, H-3), 3.51

(1 H, br t, H-4), 3.52 (1 H, br t, H-4), 3.54 (1 H, m, H-5), 3.55

(1 H, br t, H-3), 3.72 (1 H, br t, H-3), 3.73 (1 H, d, H-5), 3.74

(1 H, br t, H-2), 3.75 and 3.93 (each 1 H, 2 × m, 2 × H-6), 3.77

and 4.20 (each 1 H, 2 × m, 2 × H-6), 3.80 (1 H, br t, H-2), 4.13

and 4.30 (each 1 H, 2 × m, OCH₂CHCH₂), 4.45 (1 H, d, J_{H-1,H-2}

7.8, H-1), 4.52 (1 H, d, J_{H-1,H-2}8.4, H-1), 4.56 (1 H, d, J_H-1,H-2

8.6, H-1), 5.26 and 5.30 (each 1 H, 2 × m, OCH₂CHCH₂),

5.88 (1 H, m, OCH₂CHCH₂); d_C(125 MHz; D₂O) 22.8 and

23.0 (2 × NDCOCH₃), 55.1 (C-2), 56.3 (C-2), 61.4 (C-6),

69.1 (C-4), 69.4 (C-6), 70.5 (C-4), 71.0 (OCH₂CHCH₂),

72.4 (C-3), 73.4 (C-2), 74.5 (C-3), 74.8 (C-5), 75.5 (C-4),

76.3 (C-5), 76.5 (C-5), 83.8 (C-3), 100.5 (C-1), 102.6 (C-1),

103.7 (C-1), 119.1 (OCH₂CHCH₂), 134.0 (OCH₂CHCH₂);

High resolution MALDI-TOF MS, m/z found M+ H641.236,

C₂₅H₄₁N₂O₁₇requires 641.241.

3-(2-Aminoethylthio)propyl (b-D-glucopyranosyluronic acid)-

(1→3)-2-acetamido-2-deoxy-b-D-glucopyranoside 1

A solution of 10 (5 mg, 11.4 lmol) and cysteamine hydrochloride

(7.1 mg, 62.7 lmol) in water (1 cm³) was irradiated for 2 h in a

quartz vial, using a VL-50C Vilber Lourmat UV Lamp. The

mixture was loaded directly on to a size-exclusion column (Bio-

Gel P-2, 100 mM NH₄HCO₃), yielding 1 (4.5 mg, 76%), isolated

after lyophilization from water as a white, amorphous powder;

[a]²_D⁰−93 (c 0.3 in water); d_H(500 MHz; D₂O; 2D TOCSY and

HSQC) 1.85 [2 H, m, OCH₂CH₂CH₂S(CH₂)₂ND₂], 2.03 (3 H, s,

NAc), 2.62 [2 H, br t, O(CH₂)₂CH₂S(CH₂)₂ND₂], 2.84 and 3.23

[each 2 H, 2 × br t, O(CH₂)₃S(CH₂)₂ND₂], 3.33 (1 H, dd, J_{H-1,H-2}

8.0 Hz, J_{H-2, H-3}8.6 Hz, H-2), 3.46 (1 H, m, H-5), 3.48 (1 H, br

t, H-4), 3.50 (1 H, br t, H-3), 3.52 (1 H, br t, H-4), 3.69 and

3.98 [each 1 H, 2 × m, OCH₂(CH₂)₂S(CH₂)₂ND₂], 3.72 (1 H, d,

J_{H-4,H-5}9.6, H-5), 3.74 (1 H, br t, H-3), 3.75 (1 H, dd, J_H-5,H-6b

5.7, J_H-6a,H-6b12.4, H-6b), 3.81 (1 H, dd, J_H-1,H-28.5, J_H-2,H-310.4,

H-2), 3.91 (1 H, dd, J_H-5,H-6a2.1, H-6a), 4.47 (1 H, d, H-1),

4.52 (1 H, d, H-1); d_C(125 MHz; D₂O) 23.0 (NDCOCH₃), 27.7

[O(CH₂)₂CH₂S(CH₂)₂ND₂],28.9and39.1[O(CH₂)₃S(CH₂)₂ND₂],

29.3 [OCH₂CH₂CH₂S(CH₂)₂ND₂], 55.3 (C-2), 61.5 (C-6), 69.4

[OCH₂(CH₂)₂S(CH₂)₂ND₂], 69.5 (C-4), 72.4 (C-3), 73.5 (C-2),

76.1 (C-4), 76.2 (C-5), 76.6 (C-5), 83.3 (C-3), 101.9 (C-1), 103.6

(C-1); high resolution MALDI-TOF MS, m/z found M+Na

537.163, C₁₉H₃₄N₂NaSO₁₂requires 537.173.

Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-

b-D-glucopyranosyl)-(1→6)-[(methyl

2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate)-(1→3)]-2-deoxy-

2-phthalimido-b-D-glucopyranoside 12

3-(2-Aminoethylthio)propyl (2-acetamido-2-deoxy-b-D-

glucopyranosyl)-(1→6)-[(b-D-glucopyranosyluronic acid)-(1→

3)]-2-acetamido-2-deoxy-b-D-glucopyranoside 2

A solution of 9 (50 mg, 75 lmol) and 3,4,6-tri-O-acetyl-2-deoxy-

2-phthalimido-b-D-glucopyranosyl trichloroacetimidate¹⁹(11;

65 mg, 112 lmol) in dry CH₂Cl₂(2 cm³), containing activated

molecular sieves (4 Å, 0.1 g), was stirred for 1 h at room tempera-

ture, then TMSOTf (1.5 mm³, 7.5 lmol) was added at 0 °C. The

mixture was stirred for 1 h at 0 °C, when TLC (9:1 CH₂Cl₂–ace-

tone) showed the formation of a new product (R_f= 0.45). After

neutralization with dry pyridine and filtration, the solution was

washed with 10% aq. NaCl, dried, filtered, and concentrated.

Column chromatography (9:1 CH₂Cl₂–acetone) of the residue

A solution of 13 (3 mg, 4.7 lmol) and cysteamine hydrochloride

(3 mg, 26 lmol) in water (1 cm³) was irradiated for 2 h in a quartz

vial, using a VL-50C Vilber Lourmat UV Lamp. The mixture

was loaded directly on to a size-exclusion column (Bio-Gel P-2,

100 mM NH₄HCO₃), yielding 2 (1.6 mg, 48%), isolated after ly-

ophilization from water as a white, amorphous powder; [a]_D²⁰−11

(c 0.1 in water); d_H(500 MHz; D₂O; 2 D TOCSY and HSQC)

1.84 [2 H, m, OCH₂CH₂CH₂S(CH₂)₂ND₂], 2.01 and 2.06 (each

3 H, 2 × s, 2 × NAc), 2.61 [2 H, br t, O(CH₂)₂CH₂S(CH₂)₂ND₂],

2 9 7 8

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

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2.81 and 3.17 [each 2 H, 2 × br t, O(CH₂)₃S(CH₂)₂ND₂], 3.34

(1 H, br t, H-2), 3.45 (1 H, m, H-5), 3.46 (1 H, br t, H-4),

3.50 (1 H, br t, H-3), 3.51 (1 H, br t, H-4), 3.52 (1 H, br t,

H-4), 3.54 (1 H, br t, H-3), 3.55 (1 H, m, H-5), 3.67 and 3.94

[each 1 H, 2 × m, OCH₂(CH₂)₂S(CH₂)₂ND₂], 3.71 (1 H, br t,

H-3), 3.73 (1 H, d, H-5), 3.74 (1 H, br t, H-2), 3.75 (1 H, m,

H-6b), 3.76 (1 H, br t, H-2), 3.79 (1 H, m, H-6b), 3.92 (1

H, dd, J_{H-5,H-6a}1.8 Hz, J_{H-6a,H-6b}12.4 Hz, H-6a), 4.19 (1 H,

dd, J_H-5,H-6a1.2, J_H-6a,H-6b11.3, H-6a), 4.46 (1 H, d, J_{H-1,H-2}7.8,

H-1), 4.51 (1 H, d, J_H1,H28.3, H-1), 4.52 (1 H, d, J_{H-1,H-2}8.4,

H-1); d_C(125 MHz; D₂O) 23.0 and 23.1 (2 × NDCOCH₃), 27.8

[O(CH₂)₂CH₂S(CH₂)₂ND₂], 29.2 [OCH₂CH₂CH₂S(CH₂)₂ND₂],

29.5 and 39.2 [O(CH₂)₃S(CH₂)₂ND₂], 55.2 (C-2), 56.3 (C-2),

61.4 (C-6), 69.1 (C-4), 69.2 [OCH₂(CH₂)₂S(CH₂)₂ND₂], 69.4

(C-6), 70.6 (C-4), 72.4 (C-3), 73.5 (C-2), 74.5 (C-3), 74.8 (C-

5), 76.0 (C-4), 76.5 (C-5), 76.6 (C-5), 83.6 (C-3), 101.8 (C-1),

102.8 (C-1), 103.7 (C-1); high resolution MALDI-TOF MS,

m/z found M+Na 740.231, C₂₇H₄₇N₃NaO₁₇S requires 740.251.

(1 H, d, J_{H-1,H-2}8.0, H-1), 4.45 (1 H, dd, J_H-1,H-28.4, J_H-2,H-310.7,

H-2), 4.69 (1 H, br t, H-3), 4.70 (1 H, br t, H-2), 4.85 (1 H, br

t, H-3), 4.98 (1 H, br t, H-4), 5.09 (1 H, br t, H-4), 5.40 (1 H,

d, H-1), 6.62 and 6.68 (each 2 H, 2 × m, C₆H₄OCH₃), 7.74 and

7.82 (each 2 H, 2 × m, Phth); d_C(125 MHz; CDCl₃) 20.1, 20.3,

20.4, 20.5, and 20.7 (5 × COCH₃), 52.6 and 55.5 (COOCH₃and

C₆H₄OCH₃), 55.4 (C-2), 62.1 (C-6), 68.5 (C-4), 69.5 (C-4), 71.1

(C-2), 72.0 (C-3), 72.2 (2 C) (C-5 and C-5), 75.5 (C-3), 97.6 (C-

1), 100.0 (C-1), 114.4 and 118.7 (C₆H₄OCH₃), 123.7 and 134.7

[N(CO)₂C₆H₄]; high resolution MALDI-TOF MS, m/z found

M+Na 838.218, C₃₈H₄₁NNaO₁₉requires 838.217.

(Methyl 2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate)-(1→

3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl

trichloroacetimidate 17

To a solution of 16 (0.23 g, 0.28 mmol) in 1:1:1 toluene–

acetonitrile–water (15 cm³) was added ammonium cerium(IV)

nitrate (1.5 g, 2.8 mmol). The two-phase mixture was

vigorously stirred for 45 min, when TLC (95:5 CH₂Cl₂–

acetone) showed the disappearance of 16 and the appearance

of a lower moving spot (R_f= 0.09). After dilution with EtOAc,

the organic phase was washed with saturated aq. NaHCO₃

and 10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (95:5 CH₂Cl₂–acetone) of the residue gave

the hemiacetal intermediate isolated as a yellow solid. To a

solution of the hemiacetal (0.15 g, 0.20 mmol) in dry CH₂Cl₂

(4 cm³) and trichloroacetonitrile (0.21 cm³, 2.0 mmol) was

added, at 0 °C, 1,8-diazabicyclo[5.4.0]undec-7-ene (3.4 mm³,

20 lmol). The mixture was stirred overnight, when TLC (95:5

CH₂Cl₂–acetone) showed the formation of a new product

(R_f= 0.30). After concentration, column chromatography

(95:5 CH₂Cl₂–acetone) of the residue yielded 17 (0.15 g, 63%),

isolated as a yellow foam; [a]²_D⁰+18 (c 1 in CHCl₃); d_H(500 MHz;

CDCl₃; 2D TOCSY and HSQC) 1.86, 1.91, 1.95, 2.11, and 2.14

(each 3 H, 5 × s, 5 × Ac), 3.73 (3 H, s, COOCH₃), 3.87 (1 H, d,

J_{H-4,H-5}10.0 Hz, H-5), 3.97 (1 H, m, H-5), 4.18 (1 H, dd, J_H-5,H-6a

2.2, J_H-6a,H-6b12.4, H-6a), 4.33 (1 H, dd, J_H-5,H-6b4.3, H-6b), 4.34

(1 H, d, J_{H-1,H-2}8.0, H-1), 4.59 (1 H, dd, J_H-1,H-28.9, J_H-2,H-310.7,

H-2), 4.76 (1 H, dd, J_{H-2,H-3}9.5, H-2), 4.84 (1 H, dd, J_H-3,H-49.2,

H-3), 4.93 (1 H, br t, H-3), 5.05 (1 H, br t, H-4), 5.20 (1 H, br t,

H-4), 6.29 (1 H, d, H-1), 7.80 and 7.86 (each 2 H, 2 × m, Phth),

8.57 [1 H, s, OC(NH)CCl₃]; d_C(125 MHz; CDCl₃) 20.2, 20.3,

20.6, 20.7, and 20.8 (5 × COCH₃), 52.7 (COOCH₃), 54.6 (C-2),

61.9 (C-6), 68.3 (C-4), 69.6 (C-4), 71.3 (C-2), 72.1 (C-3), 72.5

(C-5), 73.0 (C-5), 75.4 (C-3), 93.7 (C-1), 100.2 (C-1), 123.9 and

135.0 [N(CO)₂C₆H₄].

4-Methoxyphenyl (methyl 2,3,4-tri-O-acetyl-b-D-

glucopyranosyluronate)-(1→3)-2-deoxy-2-phthalimido-b-D-

glucopyranoside 15

A solution of methyl 2,3,4-tri-O-acetyl-a,b-D-glucopyrano-

syluronate trichloroacetimidate¹⁴(8; 0.75 g, 1.65 mmol) and

4-methoxyphenyl2-deoxy-4,6-O-isopropylidene-2-phthalimido-

b-D-glucopyranoside²⁰(14; 0.5 g, 1.1 mmol) in dry CH₂Cl₂

(10 cm³), containing activated molecular sieves (4 Å, 0.8 g), was

stirred for 45 min at room temperature, then TMSOTf (54 mm³,

0.30 mmol) was added at 0 °C. The mixture was stirred for 45 min

at 0 °C and 30 min at rt, when TLC (95:5 CH₂Cl₂–acetone)

showed the formation of a new product (R_f= 0.33). After

neutralization with dry pyridine and filtration, the solution was

washed with 10% aq. NaCl, dried, filtered, and concentrated. To

a solution of the residue in CH₂Cl₂(20 cm³) and water (0.1 cm³)

was added TFA (1 cm³), and the mixture was stirred for 1 h,

when TLC (9:1 CH₂Cl₂–acetone) confirmed the removal of the

isopropylidene function (R_f= 0.31). The mixture was washed

with saturated aq. NaHCO₃, dried, filtered, and concentrated.

Column chromatography (9:1 CH₂Cl₂–acetone) of the residue

gave 15 (0.34 g, 71%), isolated as a colorless glass; [a]²_D⁰+16 (c

0.7 in CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC)

1.60, 1.94, and 2.00 (each 3 H, 3 × s, 3 × Ac), 3.64 (1 H, m, H-5),

3.70 and 3.76 (each 3 H, 2 × s, COOCH₃and C₆H₄OCH₃), 3.73

(1 H, br t, H-4), 3.86 (1 H, dd, J_H-5,H-6b5.5 Hz, J_H-6a,H-6b11.9 Hz,

H-6b), 4.03 (1 H, dd, J_H-5,H-6a3.4, H-6a), 4.07 (1 H, d, J_{H-4,H-5}9.9,

H-5), 4.45 (1 H, dd, J_H-1,H-28.6, J_H-2,H-310.6, H-2), 4.49 (1 H, d,

J_{H-1,H-2}7.8, H-1), 4.59 (1 H, dd, J_H-3,H-48.1, H-3), 4.89 (1 H, dd,

J_{H-2,H-3}10.7, H-2), 5.07 (1 H, br t, H-3), 5.15 (1 H, br t, H-4),

5.54 (1 H, d, H-1), 6.70 and 6.76 (each 2 H, 2 × m, C₆H₄OCH₃),

7.78 and 7.87 (each 2 H, 2 × m, Phth); high resolution MALDI-

TOF MS, m/z found M+Na 754.201, C₃₄H₃₇NNaO₁₇requires

754.196.

Allyl (methyl 2,3,4-tri-O-acetyl-b-D-

glucopyranosyluronate)-(1→3)-(4,6-di-O-acetyl-2-deoxy-

2-phthalimido-b-D-glucopyranosyl)-(1→6)-[(methyl

2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate)-(1→3)]-2-deoxy-

2-phthalimido-b-D-glucopyranoside 18

A solution of 9 (50 mg, 75 lmol) and 17 (96 mg, 112 lmol)

in dry CH₂Cl₂(2 cm³), containing activated molecular sieves

(4 Å, 0.1 g), was stirred for 1 h at rt, then TMSOTf (2.32 mm³,

11.2 lmol) was added at 0 °C. The mixture was stirred for 1 h

at 0 °C, when TLC (9:1 CH₂Cl₂–acetone) showed the forma-

tion of a new product (R_f= 0.42). After neutralization with

dry pyridine and filtration, the solution was washed with 10%

aq. NaCl, dried, filtered, and concentrated. Column chromato-

graphy (9:1 CH₂Cl₂–acetone) of the residue afforded 18 (90 mg,

90%), isolated as a white amorphous powder; [a]²_D⁰−16 (c 1.5 in

CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 1.59,

1.86, 1.90, 1.94, 1.95, 2.00, 2.12, and 2.13 (each 3 H, 8 × s,

8 × Ac), 3.19 (1 H, dd, J_H-3,H-49.6 Hz, J_H-4,H-58.4 Hz, H-4), 3.40

(1 H, m, H-5), 3.65 (1 H, dd, J_H-5,H-6b6.0, J_H-6a,H-6b11.0, H-6b),

3.69 and 3.93 (each 1 H, 2 × m, OCH₂CHCH₂), 3.72 and 3.76

(each 3 H, 2 × s, 2 × COOCH₃), 3.77 (1 H, m, H-5), 3.86 (1 H,

d, J_{H-4,H-5}9.9, H-5), 3.94 (1 H, d, J_{H-4,H-5}10.1, H-5), 3.96

4-Methoxyphenyl (methyl 2,3,4-tri-O-acetyl-b-D-

glucopyranosyluronate)-(1→3)-4,6-di-O-acetyl-2-deoxy-2-

phthalimido-b-D-glucopyranoside 16

To a solution of 15 (0.53 g, 0.72 mmol) in dry CH₂Cl₂(7.5 cm³)

and dry pyridine (7.5 cm³) was added acetic anhydride (7.5 cm³).

The mixture was stirred overnight, when TLC (9:1 CH₂Cl₂–

acetone) showed the reaction to be completed (R_f= 0.77).

Then, the mixture was washed with saturated aq. NaHCO₃

and 10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (95:5 CH₂Cl₂–acetone) of the residue yielded

16 (0.58 g, 100%), isolated as a white foam; [a]²_D⁰+10 (c 1 in

CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 1.81,

1.84, 1.87, 2.02, and 2.07 (each 3 H, 5 × s, 5 × Ac), 3.64 and

3.65 (each 3 H, 2 × s, COOCH₃and C₆H₄OCH₃), 3.79 (1 H, d,

J_{H-4,H-5}10.1 Hz, H-5), 3.80 (1 H, m, H-5), 4.11 (1 H, dd, J_H-5,H-6a

2.5, J_H-6a,H-6b12.2, H-6a), 4.21 (1 H, dd, J_H-5,H-6b5.1, H-6b), 4.24

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 7 9

View Article Online

(1 H, dd, J_H-1,H-28.6, J_H-2,H-310.7, H-2), 4.18 (1 H, dd, J_{H-5,H-6a}

2.5, J_{H-6a,H-6b}12.2, H-6a), 4.21 (1 H, dd, J_H-5,H-6a0.8, H-6a), 4.26

(1 H, dd, H-6b), 4.27 (1 H, d, J_{H-1,H-2}8.0, H-1), 4.28 (1 H, br

t, H-3), 4.30 (1 H, d, J_{H-1,H-2}8.1, H-1), 4.32 (1 H, dd, J_{H-1,H-2}

8.4, J_{H-2,H-3}10.8, H-2), 4.72 (1 H, dd, J_{H-2,H-3}9.7, H-2), 4.73

(1 H, br t, H-3), 4.76 (1 H, dd, J_{H-2,H-3}9.2, H-2), 4.80 (1 H,

d, H-1), 4.91 (1 H, br t, H-3), 4.94 and 5.00 (each 1 H, 2 × m,

OCH₂CHCH₂), 4.99 (1 H, br t, H-3), 5.05 (1 H, br t, H-4),

5.08 (1 H, br t, H-4), 5.09 (1 H, d, H-1), 5.10 (1 H, br t, H-4),

5.51 (1 H, m, OCH₂CHCH₂), 7.75, 7.82, and 7.89 (2 H, 4 H,

and 2 H, 3 × m, 2 × Phth); d_C(125 MHz; CDCl₃) 19.8, 20.2,

20.4 (3 C), 20.5, and 20.8 (2 C) (8 × COCH₃), 52.7 and 53.3

(2 × COOCH₃), 54.7 (C-2), 55.6 (C-2), 62.3 (C-6), 68.7 (2 C)

(C-4 and C-4), 69.0 (OCH₂CHCH₂), 69.2 (C-6), 69.4 (C-4),

69.6 (C-4), 70.7 (C-2), 71.2 (C-2), 71.5 (C-5), 71.8 (C-3),

72.0 (C-5), 72.2 (C-3), 72.4 (C-5), 74.8 (C-5), 75.7 (C-3), 81.4

(C-3), 96.9 (C-1), 99.0 (C-1), 99.8 (C-1), 100.2 (C-1), 117.6

(OCH₂CHCH₂), 133.4 (OCH₂CHCH₂), 123.8 and 134.8

[N(CO)₂C₆H₄]; high resolution MALDI-TOF MS, m/z found

M+Na 1379.358, C₆₁H₆₈N₂NaO₃₃requires 1379.360.

and H-5), 3.76 and 3.93 (each 1 H, 2 × m, 2 × H-6), 3.77

(1 H, br t, H-2), 3.78 and 4.20 (each 1 H, 2 × m, 2 × H-6),

4.46 (1 H, d, J_{H-1,H-2}7.0 Hz, H-1), 4.48 (1 H, d, J_{H-1,H-2}

7.5, H-1), 4.50 (1 H, d, J_{H-1,H-2}8.0, H-1), 4.57 (1 H, d,

J_H-1,H-28.5, H-1); d_C(125 MHz; D₂O) 22.9 (NDCOCH₃), 27.7

[O(CH₂)₂CH₂S(CH₂)₂ND₂], 29.1 [OCH₂CH₂CH₂S(CH₂)₂ND₂],

29.2 and 39.1 [O(CH₂)₃S(CH₂)₂ND₂], 55.1 (C-2), 55.2 (C-2),

61.3 (C-6), 69.1 [OCH₂(CH₂)₂S(CH₂)₂ND₂], 69.3 (C-6), 73.5 (2

C) (C-2 and C-2), 76.5 (2 C) (C-5 and C-5), 83.3 (C-3), 83.6

(C-3), 101.9 (C-1), 102.7 (C-1), 103.6 (2 C) (C-1 and C-1);

high resolution MALDI-TOF MS, m/z found M+Na 916.252,

C₃₃H₅₅N₃NaO₂₃S requires 916.284.

4-Methoxyphenyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-

b-D-glucopyranosyl)-(1→3)-2-deoxy-2-phthalimido-b-D-

glucopyranoside 22

A solution of 6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-a-D-gluco-

pyranosyl trichloroacetimidate¹⁵(20; 2.33 g, 3.0 mmol) and

4-methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-

b-D-glucopyranoside²²(21; 1.00 g, 2.0 mmol) in dry CH₂Cl₂

(50 cm³), containing activated molecular sieves (4 Å, 1 g),

was stirred for 45 min, then TMSOTf (56 mm³, 0.3 mmol)

was added at 0 °C. The mixture was stirred for 30 min, when

TLC (95:5 CH₂Cl₂–acetone) showed the formation of a new

product (R_f= 0.42). After neutralization with dry pyridine

and filtration, the solution was washed with 10% aq. NaCl,

dried, filtered, and concentrated. To a solution of the residue in

CH₂Cl₂(50 cm³) and water (0.5 cm³) was added TFA (1.5 cm³),

then the mixture was stirred for 4 h, when TLC (9:1 CH₂Cl₂–ac-

etone) showed the formation of a new product (R_f= 0.38).

The mixture was washed with saturated aq. NaHCO₃, dried,

filtered, and concentrated. Column chromatography (9:1

CH₂Cl₂–acetone) of the residue gave 22 (1.57 g, 78%), isolated

as a white foam; [a]_D²⁰+33 (c 0.5 in CHCl₃); d_H(500 MHz; CDCl₃;

2D TOCSY and HSQC) 2.21, 2.23, 2.32, and 2.33 [each 3 H,

4 × s, 3 × COC₆H₄CH₃and CO(CH₂)₂COCH₃], 2.65 and 2.79

[each 2 H, 2 × m, CO(CH₂)₂COCH₃], 3.64 (1 H, m, H-5), 3.68

(3 H, s, C₆H₄OCH₃), 3.79 (1 H, dd, J_H-3,H-48.1 Hz, J_H-4,H-59.6 Hz,

H-4), 3.87 and 4.00 (each 1 H, 2 × m, 2 × H-6), 4.09 (1 H, m,

H-5), 4.21 (1 H, dd, J_{H-5,H-6b}7.8, J_{H-6a,H-6b}12.2, H-6b), 4.37

(1 H, dd, J_{H-5,H-6a}2.3, H-6a), 4.44 (1 H, dd, J_H-1,H-28.6, J_H-2,H-3

10.8, H-2), 4.68 (1 H, dd, J_H-3,H-48.1, H-3), 4.83 (1 H, d, J_{H-1,H-2}

8.0, H-1), 5.38 (1 H, br t, H-4), 5.41 (1 H, dd, J_{H-2,H-3}9.8,

H-2), 5.48 (1 H, d, H-1), 5.72 (1 H, br t, H-3), 6.67 (4 H, m,

C₆H₄OCH₃), 6.84, 6.96, 7.13, 7.33, 7.52, and 7.74 (each 2 H,

6 × d, 3 × COC₆H₄CH₃); d_C(125 MHz; CDCl₃) 21.6, 21.7 (2 C),

and 29.9 [3 × COC₆H₄CH₃and CO(CH₂)₂COCH₃], 27.8 and

37.9 [CO(CH₂)₂COCH₃], 54.8 (C-2), 55.7 (C₆H₄OCH₃), 62.9

(C-6), 63.1 (C-6), 69.1 (C-4), 70.7 (C-4), 71.7 (C-2), 72.3 (C-

3), 72.6 (C-5), 75.9 (C-5), 82.8 (C-3), 97.6 (C-1), 101.6 (C-1),

114.6 and 118.2 (C₆H₄OCH₃), 123.8 and 133.7 [N(CO)₂C₆H₄],

129.0, 129.1, 129.4, 129.8 (2 C), and 130.0 (COC₆H₄CH₃); high

resolution MALDI-TOF MS, m/z found M+Na 1052.332,

C₅₆H₅₅NNaO₁₈requires 1052.332.

Allyl (b-D-glucopyranosyluronic acid)-(1→3)-(2-acetamido-2-

deoxy-b-D-glucopyranosyl)-(1→6)-[(b-D-glucopyranosyluronic

acid)-(1→3)]-2-acetamido-2-deoxy-b-D-glucopyranoside 19

To a solution of 18 (30 mg, 22 lmol) in 5:1 MeOH–water

(3 cm³) was added, at 0 °C, 3 M aq. NaOH (1 cm³). The mix-

ture was stirred for 3 h at rt, neutralized with Dowex 50 X 8 H⁺

resin, filtered, and concentrated. A solution of the residue in

2:1 n-butanol–1,2-diaminoethane (6 cm³) was stirred overnight

at 90 °C, then co-concentrated with toluene. To a solution of

the residue in dry MeOH (5 cm³) was added, at 0 °C, acetic

anhydride (100 mm³). The mixture was stirred for 3 h, then

concentrated. Size-exclusion chromatography (Bio-Gel P-2,

100 mM NH₄HCO₃) gave 19 (7.2 mg, 42%), isolated after

lyophilization from water, as a white amorphous powder;

[a]²_D⁰−33 (c 0.4 in water); d_H(500 MHz; D₂O; 2D TOCSY and

HSQC) 2.00 and 2.05 (each 3 H, 2 × s, 2 × NAc), 3.34 (2 H,

br t, H-2 and H-2), 3.73 (2 H, br t, H-3 and H-3), 3.76 (2 H,

br d, H-5 and H-5), 3.76 and 4.23 (each 1 H, 2 × m, 2 × H-

6), 3.77 and 3.93 (each 1 H, 2 × m, 2 × H-6), 3.81 (1 H, br t,

H-2), 3.89 (1 H, br t, H-2), 4.14 and 4.32 (each 1 H, 2 × m,

OCH₂CHCH₂), 4.47 (1 H, d, J_{H-1,H-2}7.8 Hz, H-1), 4.50

(1 H, d, J_{H-1,H-2}7.9, H-1), 4.56 (1 H, d, J_{H-1,H-2}7.8, H-1),

4.57 (1 H, d, J_H-1,H-28.5, H-1), 5.28 and 5.30 (each 1 H, 2 × m,

OCH₂CHCH₂), 5.91 (1 H, m, OCH₂CHCH₂); d_C(125 MHz;

D₂O) 22.7 and 23.0 (2 × NDCOCH₃), 55.1 (2 C) (C-2 and C-

2), 61.4 (C-6), 69.4 (C-6), 71.1 (OCH₂CHCH₂), 73.5 (2 C)

(C-2 and C-2), 76.4 (2 C) (C-5 and C-5), 83.7 (2 C) (C-3 and

C-3), 100.5 (C-1), 102.4 (C-1), 103.7 (2 C) (C-1 and C-1),

119.1 (OCH₂CHCH₂), 134.0 (OCH₂CHCH₂); high resolu-

tion MALDI-TOF MS, m/z found M+H 817.280, C₃₁H₄₉N₂O₂₃

requires 817.273.

3-(2-Aminoethylthio)propyl (b-D-glucopyranosyluronic acid)-

(1→3)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1→

6)-[(b-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-

deoxy-b-D-glucopyranoside 3

4-Methoxyphenyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-

phthalimido-b-D-glucopyranoside 23

solution of 19 (3 mg, 3.7 lmol) and cysteamine

hydrochloride (3 mg, 26 lmol) in water (1 cm³) was irradiated for

2 h in a quartz vial, using a VL-50C Vilber Lourmat UV Lamp.

The mixture was loaded directly on to a size-exclusion column

(Bio-Gel P-2, 100 mM NH₄HCO₃), yielding 3 (1.8 mg, 56%),

isolated after lyophilization from water as a white, amorphous

powder; [a]²_D⁰−35 (c 0.1 in water); d_H(500 MHz; D₂O; 2D TOCSY

and HSQC) 1.85 [2 H, br t, OCH₂CH₂CH₂S(CH₂)₂ND₂],

2.02 and 2.03 (each 3 H, 2 × s, 2 × NAc), 2.61 [2 H, br t,

O(CH₂)₂CH₂S(CH₂)₂ND₂], 2.82 and 3.18 [each 2 H, 2 × br t,

O(CH₂)₃S(CH₂)₂ND₂], 3.34 (2 H, br t, H-2 and H-2), 3.66

and 3.94 [each 1 H, 2 × m, OCH₂(CH₂)₂S(CH₂)₂ND₂], 3.72

(1 H, br t, H-3), 3.77 (1 H, br t, H-3), 3.73 (2 H, br d, H-5

To a solution of 22 (1.3 g, 1.26 mmol) in dry CH₂Cl₂(50 cm³),

containing pyridine (3.6 cm³), triethylamine (1.8 cm³), and a

catalytic amount of DMAP, was added tert-butyldiphenylsilyl

chloride (0.96 cm³, 3.79 mmol). The mixture was stirred over-

night, when TLC (95:5 CH₂Cl₂–acetone) showed the reaction to

be completed (R_f= 0.82). After dilution with EtOAc, the solu-

tion was washed with saturated aq. NaHCO₃and water, dried,

filtered, and concentrated. Column chromatography (toluene →

9:1 CH₂Cl₂–acetone) of the residue yielded 23 (1.46 g, 91%),

isolated as a white foam; [a]²_D⁰+19 (c 1.5 in CHCl₃); d_H(500 MHz;

CDCl₃; 2D TOCSY and HSQC) 1.06 [9 H, s, SiC(CH₃)₃(C₆H₅)₂],

2.10, 2.22, 2.32, and 2.33 [each 3 H, 4 × s, 3 × COC₆H₄CH₃and

2 9 8 0

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

View Article Online

CO(CH₂)₂COCH₃], 2.57 [4 H, m, CO(CH₂)₂COCH₃], 3.65 (3 H,

s, C₆H₄OCH₃), 3.71 (2 H, m, H-5 and H-4), 3.93 (1 H, dd, J_H-5,H-6b

6.1 Hz, J_H-6a,H-6b11.0 Hz, H-6b), 4.06 (1 H, m, H-5), 4.10 (1 H,

dd, J_H-5,H-6a1.7, H-6a), 4.19 (1 H, dd, J_{H-5,H-6b}7.7, J_{H-6a,H-6b}12.0,

H-6b), 4.34 (1 H, dd, J_{H-5,H-6a}2.3, H-6a), 4.46 (1 H, dd, J_H1,H2

8.4, J_H-2,H-310.8, H-2), 4.67 (1 H, dd, J_H-3,H-47.3, H-3), 4.83 (1 H,

d, J_{H-1,H-2}7.8, H-1), 5.36 (1 H, br t, H-4), 5.40 (1 H, dd, J_{H-2,H-3}

9.8, H-2), 5.43 (1 H, d, H-1), 5.71 (1 H, br t, H-3), 6.59 and 6.78

(each 2 H, 2 × m, C₆H₄OCH₃), 6.85, 6.96, 7.12, 7.27, 7.52, and

7.73 (each 2 H, 6 × d, 3 × COC₆H₄CH₃), 7.38 and 7.71 [6 H and

4 H, 2 × m, SiC(CH₃)₃(C₆H₅)₂]; d_C(125 MHz; CDCl₃) 21.5, 21.6

(2 C), and 29.7 [3 × COC₆H₄CH₃and CO(CH₂)₂COCH₃], 26.8

[SiC(CH₃)₃(C₆H₅)₂], 27.8 and 37.8 [CO(CH₂)₂COCH₃], 54.9 (C-

2), 55.6 (C₆H₄OCH₃), 62.8 (C-6), 63.6 (C-6), 69.1 (C-4), 70.0

(C-4), 71.6 (C-2), 72.4 (C-3), 72.5 (C-5), 77.0 (C-5), 83.2 (C-3),

97.7 (C-1), 101.6 (C-1), 114.5 and 118.4 (C₆H₄OCH₃), 123.8 and

133.6 [N(CO)₂C₆H₄], 127.8, 129.6, and 135.8 [SiC(CH₃)₃(C₆H₅)₂],

128.9, 129.0, 129.3, 129.7 (2 C), and 129.9 (COC₆H₄CH₃); high

resolution MALDI-TOF MS, m/z found M+Na 1290.456,

C₇₂H₇₃NNaO₁₈Si requires 1290.450.

(3.73 g, 6.8 mmol). The two-phase mixture was stirred for 2 h,

when TLC (95:5 CH₂Cl₂–acetone) showed the disappearance of

24. The mixture was diluted with EtOAc, and the organic phase

was washed with saturated aq. NaHCO₃and 10% aq. NaCl,

dried, filtered, and concentrated. Column chromatography

(95:5 CH₂Cl₂–acetone) of the residue gave the hemiacetal in-

termediate, isolated as a yellow, amorphous solid. To a solution

of the hemiacetal (0.60 g, 0.5 mmol) in dry CH₂Cl₂(5 cm³) and

trichloroacetonitrile (0.55 cm³, 5 mmol) was added, at 0 °C, 1,8-

diazabicyclo[5.4.0]undec-7-ene (8.4 mm³, 0.05 mmol). After 3 h,

the mixture was concentrated and the residue was subjected to

column chromatography (95:5 CH₂Cl₂–acetone), yielding 25

(0.57 g, 61%), isolated as a yellow foam; d_H(500 MHz; CDCl₃;

2D TOCSY and HSQC) 1.04 [9 H, s, SiC(CH₃)₃(C₆H₅)₂],

2.13, 2.16, 2.24, and 2.34 [3 H, 3 H, 3 H, and 6 H, 4 × s,

3 × COC₆H₄CH₃, COCH₃, and CO(CH₂)₂COCH₃], 2.64 [4 H,

m, CO(CH₂)₂COCH₃], 3.77 (2 H, m, 2 × H-6), 3.91 (1 H, m, H-

5), 4.11 (1 H, m, H-5), 4.20 (1 H, dd, J_{H-5,H-6b}5.1 Hz, J_{H-6a,H-6b}

12.3 Hz, H-6b), 4.32 (1 H, dd, J_{H-5,H-6a}2.8, H-6a), 4.74 (1 H,

dd, J_H-1,H-29.0, J_H-2,H-310.8, H-2), 4.78 (1 H, d, J_{H-1,H-2}7.5, H-1),

5.00 (1 H, dd, J_H-3,H-43.3, H-3), 5.27 (1 H, dd, J_{H-2,H-3}9.9, H-2),

5.44 (1 H, br t, H-4), 5.65 (1 H, br t, H-3), 5.74 (1 H, d, H-4),

6.07 (1 H, d, H-1), 6.88, 6.99, 7.13, 7.33, 7.56, and 7.74 (each

2 H, 6 × d, 3 × COC₆H₄CH₃), 7.40 and 7.65 [6 H and 4 H, 2 × m,

SiC(CH₃)₃(C₆H₅)₂], 8.48 [1 H, s, OC(NH)CCl₃].

4-Methoxyphenyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-4-O-acetyl-6-O-tert-butyldiphenylsilyl-

2-deoxy-2-phthalimido-b-D-galactopyranoside 24

To a solution of 23 (1.4 g, 1.1 mmol) in dry CH₂Cl₂(50 cm³),

containing pyridine (1.18 cm³) and a catalytic amount of

DMAP, was added slowly at 0 °C a solution of trifluoro-

methanesulfonic anhydride (1.1 cm³, 6.6 mmol) in dry CH₂Cl₂

(6 cm³). The mixture was stirred for 30 min at 0 °C and for 5 h

at rt, when TLC (95:5 CH₂Cl₂–acetone) showed the triflation to

be complete (R_f= 0.55). The mixture was washed with saturated

aq. NaHCO₃and 10% aq. NaCl, dried, filtered, and concen-

trated. To a solution of the residue in dry DMF (50 cm³) was

added tetrabutylammonium acetate (1.32 g, 4.35 mmol). After

2 h, when TLC (95:5 CH₂Cl₂–acetone) showed the formation

of a new product (R_f= 0.51), the mixture was co-concentrated

with toluene. A solution of the residue in CH₂Cl₂(50 cm³) was

washed with 10% aq. NaCl, dried, filtered, and concentrated.

Column chromatography (95:5 CH₂Cl₂–acetone) of the residue

afforded 24 (1.0 g, 70%), isolated as a yellow foam; [a]_D²⁰+18 (c 0.6

in CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 1.05

[9 H, s, SiC(CH₃)₃(C₆H₅)₂], 2.12, 2.13, 2.24, 2.32, and 2.33 [each

3 H, 5 × s, 3 × COC₆H₄CH₃, COCH₃, and CO(CH₂)₂COCH₃],

2.58 [4 H, m, CO(CH₂)₂COCH₃], 3.64 (3 H, s, C₆H₄OCH₃), 3.76

(1 H, dd, J_H-5,H-6b7.5 Hz, J_H-6a,H-6b10.9 Hz, H-6b), 3.81 (1 H,

dd, J_H-5,H-6a4.3, H-6a), 3.89 (1 H, m, H-5), 4.02 (1 H, m, H-5),

4.17 (1 H, dd, J_{H-5,H-6b}5.0, J_{H-6a,H-6b}12.1, H-6b), 4.31 (1 H, dd,

J_{H-5,H-6a}2.8, H-6a), 4.73 (1 H, dd, J_H-1,H-28.4, J_H-2,H-311.2, H-2),

4.78 (1 H, d, J_{H-1,H-2}8.0, H-1), 4.92 (1 H, dd, J_H-3,H-43.3, H-3),

5.25 (1 H, dd, J_{H-2,H-3}9.8, H-2), 5.42 (1 H, br t, H-4), 5.52

(1 H, d, H-1), 5.61 (1 H, br t, H-3), 5.62 (1 H, br d, H-4), 6.58

and 6.79 (each 2 H, 2 × m, C₆H₄OCH₃), 6.88, 6.98, 7.11, 7.35,

7.55, and 7.74 (each 2 H, 6 × d, 3 × COC₆H₄CH₃), 7.40 and 7.52

[6 H and 4 H, 2 × m, SiC(CH₃)₃(C₆H₅)₂]; d_C(125 MHz; CDCl₃)

20.9, 21.5, 21.6 (2 C), and 29.8 [3 × COC₆H₄CH₃, COCH₃, and

CO(CH₂)₂COCH₃], 26.8 [SiC(CH₃)₃(C₆H₅)₂], 27.8 and 37.8

[CO(CH₂)₂COCH₃], 52.6 (C-2), 55.6 (C₆H₄OCH₃), 62.4 (C-6),

63.1 (C-6), 69.0 (C-4), 69.3 (C-4), 71.8 (C-2), 72.2 (C-5), 72.5

(C-3), 74.8 (C-3), 75.4 (C-5), 97.9 (C-1), 101.4 (C-1), 114.5

and 118.3 (C₆H₄OCH₃), 123.5 and 134.0 [N(CO)₂C₆H₄], 127.8,

129.7, and 135.7 [SiC(CH₃)₃(C₆H₅)₂], 128.9, 129.0, 129.2, 129.8,

129.9, and 130.0 (COC₆H₄CH₃); high resolution MALDI-TOF

MS, m/z found M+Na 1332.445, C₇₄H₇₅NNaO₁₉Si requires

1332.460.

A solution of 25 (256 mg, 0.19 mmol) and 5-azidopentanol

(49 mg, 0.38 mmol) in dry CH₂Cl₂(2 cm³), containing acti-

vated molecular sieves (4 Å, 0.3 g), was stirred for 30 min,

then TMSOTf (5.1 mm³, 0.028 mmol) was added at 0 °C, and

the mixture was stirred for 30 min. After neutralization with

pyridine and filtration, the solution was washed with 10% aq.

NaCl, dried, filtered, and concentrated. Column chromato-

graphy (95:5 CH₂Cl₂–acetone) of the residue gave 26 (213 mg,

85%), isolated as a glass; [a]²_D⁰−5 (c 2 in CHCl₃); d_H(500 MHz;

CDCl₃; 2D TOCSY and HSQC) 0.99, 1.25, 1.37, and 2.83 [each

2 H, 4 × m, OCH₂(CH₂)₄N₃], 1.03 [9 H, s, SiC(CH₃)₃(C₆H₅)₂],

2.11, 2.14, 2.17, and 2.32 [3 H, 3 H, 3 H, and 6 H, 4 × s,

3 × COC₆H₄CH₃, COCH₃, and CO(CH₂)₂COCH₃], 2.54 and

2.60 [each 2 H, 2 × m, CO(CH₂)₂COCH₃], 3.29 and 3.78 [each

1 H, 2 × m, OCH₂(CH₂)₄N₃], 3.79 (2 H, m, 2 × H-6), 3.90

(2 H, m, H-5 and H-5), 4.19 (1 H, dd, J_{H-5,H-6b}4.8 Hz, J_{H-6a,H-6b}

12.0 Hz, H-6b), 4.30 (1 H, dd, J_{H-5,H-6a}2.7, H-6a), 4.46 (1 H,

dd, J_H-1,H-28.4, J_H-2,H-311.1, H-2), 4.77 (1 H, dd, J_{H-1,H-2}7.5,

H-1), 4.82 (1 H, dd, J_H-3,H-43.0, H-3), 5.00 (1 H, d, H-1), 5.25

(1 H, dd, J_{H-2,H-3}9.6, H-2), 5.43 (1 H, br t, H-4), 5.64 (1 H,

br t, H-3), 5.65 (1 H, br d, H-4), 6.88, 6.99, 7.13, 7.38, 7.55,

and 7.73 (each 2 H, 6 × d, 3 × COC₆H₄CH₃), 7.42 and 7.67

[6 H and 4 H, 2 × m, SiC(CH₃)₃(C₆H₅)₂]; d_C(125 MHz; CDCl₃)

20.9, 21.5, 21.7 (2 C), and 29.8 [3 × COC₆H₄CH₃, COCH₃, and

CO(CH₂)₂COCH₃], 23.0, 28.3, 28.7, and 51.1 [OCH₂(CH₂)₄N₃],

26.8 [SiC(CH₃)₃(C₆H₅)₂], 27.9 and 38.0 [CO(CH₂)₂COCH₃], 52.8

(C-2), 62.5 (C-6), 62.8 (C-6), 69.0 [OCH₂(CH₂)₄N₃], 69.1 (C-4),

69.2 (C-4), 71.8 (C-2), 72.2 (C-5), 72.5 (C-3), 74.8 (C-5), 74.9

(C-3), 98.7 (C-1), 101.4 (C-1), 123.5 and 133.8 [N(CO)₂C₆H₄],

127.8, 129.7, and 135.8 [SiC(CH₃)₃(C₆H₅)₂], 128.9 (2 C), 129.2,

129.8, 129.9, and 130.0 (COC₆H₄CH₃); high resolution MALDI-

TOF MS, m/z found M+Na 1337.490, C₇₂H₇₈N₄NaO₁₈Si requires

1337.498.

5-Azidopentyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-4-O-acetyl-2-deoxy-2-phthalimido-b-D-

galactopyranoside 27

To 26 (100 mg, 76 lmol) was added a 1 M TBAF solution in

THF (5 cm³), before use neutralized at 0 °C with HOAc. The

mixture was stirred for 2 h at 0 °C and for 4 h at rt, when TLC

(95:5 CH₂Cl₂–acetone) showed the disappearance of 26 and the

formation of a new product (R_f= 0.52). After concentration,

a solution of the residue in EtOAc was washed with water and

10% aq. NaCl, dried, filtered, and concentrated. Column chro-

matography (95:5 CH₂Cl₂–acetone → acetone) of the residue

5-Azidopentyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-4-O-acetyl-6-O-tert-butyldiphenylsilyl-

2-deoxy-2-phthalimido-b-D-galactopyranoside 26

To a solution of 24 (0.90 g, 0.68 mmol) in 1:1:1 toluene–aceto-

nitrile–water (60 cm³) was added ammonium cerium(IV) nitrate

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 8 1

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afforded 27 (74 mg, 91%), isolated as a white glass; [a]²_D⁰+2 (c 0.3

in CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 0.98,

1.25, 1.40, and 2.85 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 2.13,

2.16, 2.22, 2.24, and 2.26 [each 3 H, 5 × s, 3 × COC₆H₄CH₃,

COCH₃, and CO(CH₂)₂COCH₃], 2.54 and 2.70 [each 2 H,

2 × m, CO(CH₂)₂COCH₃], 3.23 and 3.70 [each 1 H, 2 × m,

OCH₂(CH₂)₄N₃], 3.44 (1 H, dd, J_H-5,H-6b8.5 Hz, J_H-6a,H-6b11.8 Hz,

H-6b), 3.61 (1 H, dd, J_H-5,H-6a4.6, H-6a), 3.69 (1 H, m, H-5),

3.86 (1 H, m, H-5), 4.08 (1 H, dd, J_{H-5,H-6b}5.6, J_{H-6a,H-6b}12.1,

H-6b), 4.22 (1 H, dd, J_{H-5,H-6a}2.3, H-6a), 4.45 (1 H, dd, J_H-1,H-2

8.5, J_H-2,H-311.2, H-2), 4.79 (1 H, d, J_{H-1,H-2}7.8, H-1), 4.81 (1 H,

dd, J_H-3,H-43.3, H-3), 4.93 (1 H, d, H-1), 5.23 (1 H, dd, J_{H-2,H-3}

9.8, H-2), 5.32 (1 H, br t, H-4), 5.51 (1 H, br d, H-4), 5.59 (1 H,

br t, H-3), 6.75, 6.90, 7.05, 7.24, 7.48, and 7.66 (each 2 H, 6 × d,

3 × COC₆H₄CH₃); d_C(125 MHz; CDCl₃) 21.0, 21.5, 21.6 (2 C),

and 29.9 [3 × COC₆H₄CH₃, COCH₃, and CO(CH₂)₂COCH₃],

22.9, 28.2, 28.7, and 51.0 [OCH₂(CH₂)₄N₃], 27.8 and 38.0

[CO(CH₂)₂COCH₃], 52.6 (C-2), 60.2 (C-6), 62.4 (C-6), 68.8 (C-

4), 69.4 [OCH₂(CH₂)₄N₃], 69.8 (C-4), 71.6 (C-2), 72.3 (C-5),

72.5 (C-3), 73.6 (C-5), 75.6 (C-3), 98.9 (C-1), 101.7 (C-1), 123.4

and 133.7 [N(CO)₂C₆H₄], 128.9, 129.0, 129.3, 129.5, 129.7, and

129.9 (COC₆H₄CH₃); high resolution MALDI-TOF MS, m/z

found M+Na 1099.376, C₅₆H₆₀N₄NaO₁₈requires 1099.380.

and 3.70 [each 1 H, 2 × m, OCH₂(CH₂)₄N₃], 3.56 and 3.66 (each 1

H, 2 × m, 2 × H-6), 3.72 (1 H, m, H-5), 3.93 (1 H, m, H-5), 4.06

(2 H, m, 2 × H-6), 4.44 (1 H, dd, J_H-1,H-28.7 Hz, J_H-2,H-311.0 Hz,

H-2), 4.74 (1 H, dd, J_H-3,H-43.4, H-3), 4.81 (1 H, d, J_{H-1,H-2}8.0, H-

1), 4.92 (1 H, d, H-1), 5.21 (1 H, br t, H-2), 5.27 (1 H, br t, H-4),

5.60 (1 H, br t, H-3), 5.65 (1 H, br d, H-4), 6.72, 6.89, 7.06, 7.23,

7.45, and 7.68 (each 2 H, 6 × d, 3 × COC₆H₄CH₃); d_C(125 MHz;

CDCl₃) 20.7, 21.3, 21.5, 21.6, and 21.7 (3 × COC₆H₄CH₃and

2 × COCH₃), 22.9, 28.3, 28.7, and 51.1 [OCH₂(CH₂)₄N₃], 52.4

(C-2), 61.6 (C-6), 62.0 (C-6), 68.8 (C-4), 69.3 [OCH₂(CH₂)₄N₃],

69.4 (C-4), 71.2 (C-5), 71.7 (C-2), 72.6 (C-3), 75.3 (C-5), 76.2

(C-3), 98.8 (C-1), 102.2 (C-1), 123.3 and 133.6 [N(CO)₂C₆H₄],

128.9, 129.0, 129.3, 129.5, 129.7, and 130.0 (COC₆H₄CH₃); high

resolution MALDI-TOF MS, m/z found M+Na 1043.354,

C₅₃H₅₆N₄NaO₁₇requires 1043.353.

5-Azidopentyl (sodium b-D-glucopyranosyl 6-sulfate)-(1→3)-2-

acetamido-2-deoxy-b-D-galactopyranoside 31

To a solution of 29 (95 mg, 93 lmol) in DMF (5 cm³) was

added the sulfur trioxide trimethylamine complex (515 mg,

3.67 mmol). The mixture was stirred for 48 h at 50 °C, when

TLC (9:1 CH₂Cl₂–methanol) showed the complete conversion

of 29 into non-sodiated 30 (R_f= 0.20). After quenching the

reaction with MeOH (10 cm³), the solution was co-concentrated

with toluene. A solution of the residue in CH₂Cl₂(50 cm³)

was washed with saturated aq. NaHCO₃, dried, filtered, and

concentrated. The residue was dissolved in MeOH (10 cm³),

containing Dowex 50W X 8 Na⁺resin, and stirred for 15 min,

then filtered and concentrated. Column chromatography (95:5

CH₂Cl₂–MeOH) of the residue gave 30 (52 mg, 50%), isolated

as a white, amorphous, powder; d_H(500 MHz; CDCl₃) 1.01,

1.22, and 2.85 [2 H, 4 H, and 2 H, 3 m, OCH₂(CH₂)₄N₃], 2.07,

2.19, 2.23, 2.32, and 2.36 (each 3 H, 5 × s, 3 × COC₆H₄CH₃and

2 × COCH₃), 3.33 and 3.78 [each 1 H, 2 × m, OCH₂(CH₂)₄N₃],

4.04 (2 H, m, H-5 and H-5), 4.14 and 4.24 (each 1 H, 2 × m,

2 × H-6), 4.29 and 4.59 (each 1 H, 2 × m, 2 × H-6), 4.48 (1 H,

dd, J_H-1,H-28.6 Hz, J_H-2,H-311.2 Hz, H-2), 4.80 (1 H, d, J_{H-1,H-2}7.6,

H-1), 4.91 (1 H, dd, J_H-3,H-42.9, H-3), 5.03 (1 H, d, H-1), 5.30

(1 H, br t, H-2), 5.50 (1 H, br t, H-4), 5.64 (1 H, br t, H-3), 5.88

(1 H, br d, H-4), 6.85, 6.92, 7.10, 7.35, 7.52, and 7.75 (each 2 H,

6 × d, 3 × COC₆H₄CH₃).

A solution of 30 (52 mg, 46 lmol) in ethanolic 33% CH₃NH₂

(5 cm³) was stirred for 7 days, during which time the mixture

was three times concentrated and fresh ethanolic 33% CH₃NH₂

(5 cm³) was added. After co-concentration with toluene, to a

solution of the residue in dry MeOH at 0 °C was added acetic

anhydride (100 mm³). The mixture was stirred for 3 h at 0 °C,

then concentrated. Size-exclusion chromatography (Bio-Gel P-

2, 100 mM NH₄HCO₃) of the residue afforded 31 (21 mg, 76%),

isolated after lyophilization from water, as a white, amorphous

powder; [a]_D²⁰−14 (c 1 in water); d_H(500 MHz; D₂O; 2D TOCSY

and HSQC) 1.40, 1.59, and 3.33 [2 H, 4 H, and 2 H, 3 × m,

OCH₂(CH₂)₄N₃], 2.03 (3 H, s, NAc), 3.31 (1 H, br t, H-2), 3.45

(1 H, br t, H-4), 3.46 (1 H, br t, H-3), 3.61 and 3.92 [each 1 H,

2 × m, OCH₂(CH₂)₄N₃], 3.62 (1 H, m, H-5), 3.68 (1 H, m, H-5),

3.78 (2 H, m, 2 × H-6), 3.83 (1 H, dd, J_H-2,H-311.0 Hz, J_H-3,H-43.1

Hz, H-3), 3.99 (1 H, br t, H-2), 4.18 (1 H, br d, H-4), 4.19 and 4.30

(each 1 H, 2 × m, 2 × H-6), 4.48 (1 H, d, J_H-1,H-28.9, H-1), 4.50 (1

H, d, J_{H-1,H-2}8.1, H-1); d_C(125 MHz; D₂O) 22.9 (NDCOCH₃),

23.2, 28.3, 28.8, and 51.8 [OCH₂(CH₂)₄N₃], 51.9 (C-2), 61.8 (C-6),

67.9 (C-6), 68.7 (C-4), 70.0 (C-3), 70.8 [OCH₂(CH₂)₄N₃], 73.5

(C-2), 74.2 (C-5), 75.7 (C-5), 76.1 (C-4), 81.0 (C-3), 102.1 (C-

1), 105.1 (C-1); high resolution MALDI-TOF MS, m/z found

M+Na 616.157, C₁₉H₃₃N₄Na₂O₁₄S requires 616.151.

5-Azidopentyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-

b-D-galactopyranoside 28

A solution of 27 (144 mg, 0.134 mmol) in 1:1 pyridine–

acetic anhydride (10 cm³) was stirred overnight, when

TLC (95:5 CH₂Cl₂–acetone) showed the acetylation to be

complete (R_f= 0.62). After co-concentration with toluene, a

solution of the residue in CH₂Cl₂was washed with saturated

aq. NaHCO₃, dried, filtered, and concentrated. Column

chromatography (95:5 CH₂Cl₂–acetone) of the residue gave 28

(125 mg, 83%), isolated as a white solid; [a]²_D⁰+9 (c 1 in CHCl₃);

d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 1.00, 1.22, 1.34,

and 2.81 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 2.01, 2.12, 2.14,

and 2.25 [3 H, 3 H, 6 H, and 6 H, 4 × s, 3 × COC₆H₄CH₃,

2 × COCH₃, and CO(CH₂)₂COCH₃], 2.55 and 2.72 [each

2 H, 2 × m, CO(CH₂)₂COCH₃], 3.28 and 3.72 [each 1 H, 2 × m,

OCH₂(CH₂)₄N₃], 3.84 (1 H, m, H-5), 3.92 (1 H, m, H-5), 4.02

(1 H, dd, J_H-5,H-6b7.2 Hz, J_H-6a,H-6b11.6 Hz, H-6b), 4.07 (1 H,

dd, J_{H-5,H-6b}5.2, J_{H-6a,H-6b}12.2, H-6b), 4.17 (1 H, dd, J_H-5,H-6a

5.1, H-6a), 4.31 (1 H, dd, J_{H-5,H-6a}2.5, H-6a), 4.42 (1 H, dd,

J_H-1,H-28.7, J_H-2,H-311.3, H-2), 4.71 (1 H, d, J_{H-1,H-2}8.0, H-1),

4.76 (1 H, dd, J_H-3,H-43.2, H-3), 4.93 (1 H, d, H-1), 5.18 (1 H, dd,

J_{H-2,H-3}9.8, H-2), 5.35 (1 H, br t, H-4), 5.53 (1 H, br d, H-4),

5.57 (1 H, br t, H-3), 6.80, 6.91, 7.05, 7.28, 7.48, and 7.66 (each

2 H, 6 × d, 3 × COC₆H₄CH₃); d_C(125 MHz; CDCl₃) 20.8, 21.0,

21.5, 21.6 (2 C), and 29.8 [3 × COC₆H₄CH₃, 2 × COCH₃, and

CO(CH₂)₂COCH₃], 23.0, 28.2, 28.6, and 51.1 [OCH₂(CH₂)₄N₃],

28.0 and 38.0 [CO(CH₂)₂COCH₃], 52.5 (C-2), 62.2 (C-6), 62.8

(C-6), 69.0 (C-4), 69.2 (C-4), 69.3 [OCH₂(CH₂)₄N₃], 71.6 (C-

5), 71.7 (C-2), 72.2 (C-5), 72.5 (C-3), 74.7 (C-3), 98.7 (C-1),

101.4 (C-1), 123.5 and 133.7 [N(CO)₂C₆H₄], 128.9, 129.0, 129.2,

129.6, 129.8, and 129.9 (COC₆H₄CH₃); high resolution MALDI-

TOF MS, m/z found M+Na 1141.370, C₅₈H₆₂N₄NaO₁₉requires

1141.390.

5-Azidopentyl (2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-

(1→3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-D-

galactopyranoside 29

To a solution of 28 (125 mg, 0.112 mmol) in EtOH (10 cm³) and

toluene (3 cm³) was added hydrazine acetate (51 mg, 0.56 mmol).

The mixture was stirred for 2 h, then concentrated. Column chro-

matography (95:5 CH₂Cl₂–acetone) of the residue yielded 29

(104 mg, 91%), isolated as a white glass; [a]_D²⁰+2 (c 1 in CHCl₃);

d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 0.99, 1.20, 1.33,

and 2.78 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 2.01, 2.16, 2.22, 2.23,

and2.28(each3H, 5 × s, 3 × COC₆H₄CH₃and2 × COCH₃), 3.25

5-Aminopentyl (sodium b-D-glucopyranosyl 6-sulfate)-(1→3)-2-

acetamido-2-deoxy-b-D-galactopyranoside 4

A solution of 31 (8.4 mg, 14 lmol) in 0.05 M aq. NaOH

(1.0 cm³) was added dropwise to a suspension of 10% Pd–C

(2 mg) and NaHB₄(8.0 mg) in water (0.5 cm³). The suspension

2 9 8 2

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

View Article Online

was stirred for 45 min, when TLC (6:2.5:1.5 EtOAc–MeOH–

water) showed the disappearance of 31. After filtration through

Celite, size-exclusion chromatography (Bio-Gel P-2, 100 mM

NH₄HCO₃) gave 4 (5.7 mg, 71%), isolated after lyophilization

from water, as a white, amorphous powder; [a]²_D⁰−11 (c 0.4 in

water); d_H(500 MHz; D₂O; 2D TOCSY and HSQC) 1.42, 1.61,

1.69, and 3.01 [each 2 H, 4 × m, OCH₂(CH₂)₄ND₂], 2.03 (3 H,

s, NAc), 3.34 (1 H, br t, H-2), 3.46 (1 H, br t, H-4), 3.47 (1 H,

br t, H-3), 3.62 and 3.92 [each 1 H, 2 × m, OCH₂(CH₂)₄ND₂],

3.65 (1 H, m, H-5), 3.68 (1 H, m, H-5), 3.79 (2 H, m, 2 × H-

6), 3.84 (1 H, dd, J_H-2,H-311.0 Hz, J_H-3,H-43.2 Hz, H-3), 4.00

(1 H, dd, J_H-1,H-28.6, H-2), 4.18 (1 H, dd, J_{H-5,H-6b}6.2, J_{H-6a,H-6b}

11.2, H-6b), 4.19 (1 H, br d, H-4), 4.32 (1 H, dd, J_{H-5,H-6a}2.2,

H-6a), 4.49 (1 H, d, H-1), 4.51 (1 H, d, J_{H-1,H-2}8.0, H-1);

d_C(125 MHz; D₂O) 22.8 (NDCOCH₃), 22.8, 27.0, 28.7, and 40.0

[OCH₂(CH₂)₄ND₂], 52.0 (C-2), 61.9 (C-6), 68.0 (C-6), 68.6 (C-

4), 70.0 (C-3), 70.3 [OCH₂(CH₂)₄ND₂], 73.4 (C-2), 74.3 (C-5),

75.7 (C-5), 76.2 (C-4), 80.9 (C-3), 102.2 (C-1), 105.0 (C-1);

high resolution MALDI-TOF MS, m/z found M+Na 593.166,

C₁₉H₃₅N₂Na₂O₁₄S requires 593.160.

230 lmol). The mixture was stirred for 2 h, then concentrated.

Column chromatography (9:1 CH₂Cl₂–acetone) of the residue

yielded 34 (48mg, 74%), isolatedasaglass;[a]_D²⁰+5(c1inCHCl₃);

d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 0.81, 1.09, 1.11,

and 2.69 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 1.77, 2.01, 2.14,

2.15, 2.18, 2.21, and 2.28 (each 3 H, 7 × s, 3 × COC₆H₄CH₃and

4 × COCH₃), 2.96 and 3.29 [each 1 H, 2 × m, OCH₂(CH₂)₄N₃],

3.49 (1 H, m, H-6b), 3.50 and 3.65 (each 1 H, 2 × m, 2 × H-6),

3.66 (1 H, m, H-5), 3.77 (1 H, m, H-5), 3.88 (1 H, dd, J_H-5,H-6a

2.6 Hz, J_H-6a,H-6b10.7 Hz, H-6a), 4.01 (1 H, m, H-5), 4.14 (2 H,

m, 2 × H-6), 4.31 (1 H, dd, J_H-1,H-28.6, J_H-2,H-311.2, H-2), 4.44

(1 H, dd, J_{H-1,H-2}8.4, J_{H-2,H-3}11.5, H-2), 4.63 (1 H, dd, J_H-3,H-4

3.4, H-3), 4.73 (1 H, d, J_{H-1,H-2}8.3, H-1), 4.75 (1 H, d, H-1),

5.16 (1 H, dd, J_{H-2,H-3}10.0, H-2), 5.25 (1 H, br t, H-4), 5.28

(1 H, d, H-1), 5.39 (1 H, br d, J_{H-3,H-4}3.1, H-4), 5.51 (1 H, br

d, H-4), 5.57 (1 H, br t, H-3), 5.71 (1 H, dd, H-3), 6.70, 6.89,

7.06, 7.21, 7.44, and 7.68 (each 2 H, 6 × d, 3 × COC₆H₄CH₃),

7.30 and 7.75 (each 4 H, 2 × m, 2 × Phth); d_C(125 MHz; CDCl₃)

19.4, 19.7, 19.8, 20.2, 20.4, 20.5, and 20.6 (3 × COC₆H₄CH₃and

4 × COCH₃), 21.9, 27.2, 28.6, and 50.0 [OCH₂(CH₂)₄N₃], 50.3

(C-2), 51.4 (C-2), 60.2 (C-6), 60.4 (C-6), 65.7 (C-4), 66.9 (C-

6), 67.0 (C-3), 67.6 (C-4), 67.7 [OCH₂(CH₂)₄N₃], 68.9 (C-4),

69.9 (C-5), 70.6 (C-2), 71.5 (C-3), 72.3 (C-5), 74.3 (C-5), 74.9

(C-3), 97.0 (C-1), 97.3 (C-1), 101.0 (C-1), 121.6, 122.6, 132.4,

and 133.3 [2 × N(CO)₂C₆H₄], 127.8, 127.9, 128.1, 128.4, 128.7,

and 128.9 (COC₆H₄OCH₃); high resolution MALDI-TOF MS,

m/z found M+Na 1418.415, C₇₁H₇₃N₅NaO₂₅requires 1418.449.

5-Azidopentyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-

b-D-galactopyranosyl)-(1→6)-[(6-O-levulinoyl-

2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-(1→

3)]-4-O-acetyl-2-deoxy-2-phthalimido-b-D-galactopyranoside 33

Asolutionof 27(92mg,86lmol)and3,4,6-tri-O-acetyl-2-deoxy-

2-phthalimido-b-D-galactopyranosyl trichloroacetimidate²⁸(32;

74.3 mg, 128 lmol) in dry CH₂Cl₂(5 cm³), containing activated

molecular sieves (4 Å, 0.5 g), was stirred for 30 min at rt, then

TMSOTf (2.3 mm³, 12.8 lmol) was added at 0 °C. The mixture

was stirred for 15 min at 0 °C, when TLC (9:1 CH₂Cl₂–acetone)

showed the formation of 33 (R_f= 0.50). After neutralization

with pyridine and filtration, the solution was washed with

10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (9:1 CH₂Cl₂–acetone) of the residue rendered

33 (74 mg, 58%), isolated as a glass; [a]²_D⁰+2 (c 1 in CHCl₃);

d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 0.83, 1.00, 1.12,

and 2.72 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 1.76, 2.02, 2.11,

2.13, 2.14, 2.16, 2.25, and 2.26 [each 3 H, 8 × s, 3 × COC₆H₄CH₃,

4 × COCH₃, CO(CH₂)₂COCH₃], 2.51 and 2.68 [each 2 H,

2 × m, CO(CH₂)₂COCH₃], 2.94 and 3.25 [each 1 H, 2 × m,

OCH₂(CH₂)₄N₃], 3.51 (1 H, dd, J_H-5,H-6b8.6 Hz, J_H-6a,H-6b10.9 Hz,

H-6b), 3.77 (2 H, m, H-5 and H-5), 3.90 (1 H, dd, J_H-5,H-6a2.0,

H-6a), 4.02 (1 H, br t, H-5), 4.08 (1 H, dd, J_{H-5,H-6b}4.9, J_{H-6a,H-6b}

12.2, H-6b), 4.14 (2 H, m, 2 × H-6), 4.19 (1 H, dd, J_{H-5,H-6a}2.8,

H-6a), 4.30 (1 H, dd, J_H-1,H-28.6, J_H-2,H-311.3, H-2), 4.44 (1 H,

dd, J_{H-1,H-2}8.4, J_{H-2,H-3}11.4, H-2), 4.63 (1 H, dd, J_H-3,H-43.4,

H-3), 4.65 (1 H, d, J_{H-1,H-2}8.0, H-1), 4.75 (1 H, d, H-1), 5.15

(1 H, dd, J_{H-2,H-3}9.8, H-2), 5.28 (1 H, d, H-1), 5.32 (1 H, br t,

H-4), 5.37 (1 H, br d, H-4), 5.40 (1 H, br d, J_{H-3,H-4}3.4, H-4),

5.53 (1 H, br t, H-3), 5.72 (1 H, dd, H-3), 6.78, 6.90, 7.05, 7.24,

7.46, and 7.65 (each 2 H, 6 × d, 3 × COC₆H₄CH₃), 7.41 and

7.72 (each 4 H, 2 × m, 2 × Phth); d_C(125 MHz; CDCl₃) 19.5,

19.6, 19.7, 19.8, 19.9, 20.5, 20.6, and 29.0 [3 × COC₆H₄CH₃,

4 × COCH₃, and CO(CH₂)₂COCH₃], 21.9, 27.1, 27.6, and 50.0

[OCH₂(CH₂)₄N₃], 26.9 and 36.9 [CO(CH₂)₂COCH₃], 50.4 (C-2),

51.4 (C-2), 60.2 (C-6), 61.2 (C-6), 65.7 (C-4), 66.9 (C-3), 67.6

[OCH₂(CH₂)₄N₃], 67.7 (C-4), 67.8 (C-6), 68.7 (C-4), 69.8 (C-5),

70.6 (C-2), 71.2 and 72.7 (C-5 and C-5), 71.4 (C-3), 73.4 (C-

3), 97.3 (C-1), 97.2 (C-1), 100.3 (C-1), 121.8, 122.4, 132.7, and

133.3 [2 × N(CO)₂C₆H₄], 127.8, 127.9, 128.2, 128.5, 128.7, and

128.9 (COC₆H₄OCH₃); high resolution MALDI-TOF MS, m/z

found M+Na 1516.456, C₇₆H₇₉N₅NaO₂₇requires 1516.486.

5-Azidopentyl (2-acetamido-2-deoxy-b-D-galactopyranosyl)-

(1→6)-[(sodium b-D-glucopyranosyl 6-sulfate)-(1→3)]-2-

acetamido-2-deoxy-b-D-galactopyranoside 36

To a solution of 34 (43 mg, 30.8 lmol) in DMF (3 cm³) was

added the sulfur trioxide trimethylamine complex (173 mg,

1.22 mmol). The mixture was stirred for 48 h at 50 °C, when

TLC (9:1 CH₂Cl₂–MeOH) showed the complete conversion of

34 into non-sodiated 35 (R_f= 0.38). After quenching the reac-

tion with MeOH (10 cm³), the solution was co-concentrated with

toluene. A solution of the residue in EtOAc (50 cm³) was washed

with saturated aq. NaHCO₃and 10% aq. NaCl, dried, filtered,

and concentrated. The residue was dissolved in MeOH (10 cm³),

containing Dowex 50W X 8 Na⁺resin, and stirred for 15 min,

then filtered and concentrated. Column chromatography (9:1

CH₂Cl₂–MeOH) of the residue gave 35 (38 mg, 82%), isolated as

a white, amorphous powder; d_H(300 MHz; CDCl₃) 0.90, 1.20, and

2.80 [2 H, 4 H, and 2 H, 3 × m, OCH₂(CH₂)₄N₃], 1.83, 2.05, 2.17,

2.26, 2.33, 2.34, and 2.38 (each 3 H, 7 × s, 3 × COC₆H₄CH₃and

4 × COCH₃), 3.19 and 3.50 [each 1 H, 2 × m, OCH₂(CH₂)₄N₃],

3.51 and 3.99 (each 1 H, 2 × m, 2 × H-6), 3.78 (1 H, m, H-5),

4.17 (2 H, m, 2 × H-6), 4.36 (2 H, m, 2 × H-6), 4.47 (1 H, dd,

J_{H-1,H-2}8.4 Hz, J_{H-2,H-3}11.4 Hz, H-2), 4.73 (1 H, d, J_{H-1,H-2}7.7,

H-1), 4.81 (1 H, dd, J_H-2,H-311.3, J_H-3,H-42.8, H-3), 4.87 (1 H, d,

J_H-1,H-28.5, H-1), 5.26 (1 H, d, H-1), 5.38 (1 H, br d, H-4), 5.55

(1 H, br t, H-4), 5.56 (1 H, br t, H-3), 5.77 (1 H, dd, J_{H-3,H-4}

3.3, H-3), 5.83 (1 H, br d, H-4), 6.87, 6.99, 7.19, 7.35, 7.49, and

7.72 (each 2 H, 6 × d, 3 × COC₆H₄CH₃), 7.46 and 7.76 (each

4 H, 2 × m, 2 × Phth).

A solution of 36 (33 mg, 22 lmol) in ethanolic 33% CH₃NH₂

(5 cm³) was stirred for 7 days, during which time the mixture

was three times concentrated and fresh ethanolic 33% CH₃NH₂

(5 cm³) was added. After co-concentration with toluene, to a

solution of the residue in dry MeOH at 0 °C was added acetic

anhydride (100 mm³). The mixture was stirred for 3 h at 0 °C,

then concentrated. Size-exclusion chromatography (Bio-Gel

P-2, 100 mM NH₄HCO₃) of the residue afforded 36 (13 mg,

74%), isolated after lyophilization from water, as a white,

amorphous powder; [a]_D²⁰+6 (c 0.2 in water); d_H(500 MHz; D₂O;

2D TOCSY and HSQC) 1.39, 1.61, and 3.33 [2 H, 4 H, and

2 H, 3 × m, OCH₂(CH₂)₄N₃], 2.01 and 2.02 (each 3 H, 2 × s,

2 × NAc), 3.34 (1 H, br t, H-2), 3.44 (1 H, br t, H-4), 3.45 (1

H, br t, H-3), 3.58 and 3.88 [each 1 H, 2 × m, OCH₂(CH₂)₄N₃],

5-Azidopentyl (3,4,6-tri-O-acetyl-2-deoxy-

2-phthalimido-b-D-galactopyranosyl)-(1→

6)-[(2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-(1→3)]-4-O-

acetyl-2-deoxy-2-phthalimido-b-D-galactopyranoside 34

To a solution of 33 (70 mg, 47 lmol) in EtOH (5 cm³) and

toluene (1.5 cm³) was added hydrazine acetate (21.3 mg,

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 8 3

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2.20, 2.24, 2.26, and 2.33 [3 H, 3 H, 3 H, 3 H, and 6 H, 5 × s,

3 × COC₆H₄CH₃, 2 × COCH₃, and CO(CH₂)₂COCH₃], 2.63

and 2.77 [each 2 H, 2 × m, CO(CH₂)₂COCH₃], 3.68 (3 H, s,

C₆H₄OCH₃), 3.91 (1 H, m, H-5), 4.10 (1 H, m, H-5), 4.26

(1 H, dd, J_H-5,H-6a4.3 Hz, J_H-6a,H-6b11.1 Hz, H-6a), 4.38 (1 H, dd,

J_{H-5,H-6a}2.8, J_{H-6a,H-6b}13.6, H-6a), 4.74 (1 H, dd, J_H-1,H-28.5,

J_H-2,H-311.3, H-2), 4.80 (1 H, d, J_{H-1,H-2}7.8, H-1), 4.93 (1 H, dd,

J_H-3,H-43.4, H-3), 5.27 (1 H, dd, J_{H-2,H-3}10.0, H-2), 5.42 (1 H, br

t, H-4), 5.48 (1 H, d, H-1), 5.64 (1 H, br t, H-3), 5.66 (1 H, br

d, H-4), 6.66 and 6.74 (each 2 H, 2 × m, C₆H₄OCH₃), 6.88, 6.99,

7.13, 7.37, 7.54, and 7.73 (each 2 H, 6 × d, 3 × COC₆H₄CH₃);

d_C(125 MHz; CDCl₃) 20.9, 21.1, 21.6, 21.7 (2 C), and 29.8

[3 × COC₆H₄CH₃, 2 × COCH₃, and CO(CH₂)₂COCH₃], 28.0

and 38.1 [CO(CH₂)₂COCH₃], 52.4 (C-2), 55.7 (C₆H₄OCH₃), 62.2

(C-6), 62.7 (C-6), 68.9 (C-4), 69.2 (C-4), 71.7 (C-2), 71.9 (C-

5), 72.3 (C-5), 72.5 (C-3), 74.6 (C-3), 98.0 (C-1), 101.6 (C-1),

114.5 and 118.6 (C₆H₄OCH₃), 123.6 and 133.8 [N(CO)₂C₆H₄],

128.9, 129.0, 129.2, 129.7, 129.8, and 130.0 (COC₆H₄CH₃);

high resolution MALDI-TOF MS, m/z found M+Na 1136.365,

C₆₀H₅₉NNaO₂₀requires 1136.353.

3.62 (1 H, m, H-5), 3.68 (1 H, m, H-5), 3.72 (1 H, dd, J_{H-2,H-3}

10.8 Hz, J_{H-3,H-4}3.4 Hz, H-3), 3.76 and 3.80 (each 1 H, 2 × m,

2 × H-6), 3.78 and 4.05 (each 1 H, 2 × m, 2 × H-6), 3.82 (1 H,

m, H-5), 3.84 (1 H, dd, J_H-2,H-310.9, J_H-3,H-43.2, H-3), 3.89 (1 H,

br t, H-2), 3.93 (1 H, br d, H-4), 3.98 (1 H, dd, J_H-1,H-28.6, H-

2), 4.16 (1 H, br d, H-4), 4.17 (1 H, dd, J_{H-5,H-6b}5.8, J_{H-6a,H-6b}

11.3, H-6b), 4.30 (1 H, dd, J_{H-5,H-6a}2.1, H-6a), 4.45 (1 H, d,

H-1), 4.46 (1 H, d, J_{H-1,H-2}8.4, H-1), 4.50 (1 H, d, J_{H-1,H-2}7.8,

H-1); d_C(125 MHz; D₂O) 22.9 (NDCOCH₃), 23.2, 28.4, 28.8,

and 51.8 [OCH₂(CH₂)₄N₃], 51.8 (C-2), 53.1 (C-2), 61.7 (C-6),

67.9 (C-6), 68.5 (C-4), 69.0 (C-4), 70.1 (C-3), 70.5 (C-6), 70.7

[OCH₂(CH₂)₄N₃], 71.7 (C-3), 73.5 (C-2), 74.2 (C-5), 74.3 (C-

5), 75.8 (C-5), 76.2 (C-4), 80.7 (C-3), 101.8 (C-1), 102.6 (C-

1), 104.9 (C-1); high resolution MALDI-TOF MS, m/z found

M+Na 822.229, C₂₇H₄₆N₅Na₂O₁₉S requires 822.230.

5-Aminopentyl (2-acetamido-2-deoxy-b-D-galactopyranosyl)-

(1→6)-[(sodium b-D-glucopyranosyl 6-sulfate)-(1→3)]-2-

acetamido-2-deoxy-b-D-galactopyranoside 5

A solution of 36 (5 mg, 6.3 lmol) in 0.05 M aq. NaOH (1.0 cm³)

was added dropwise to a suspension of 10% Pd–C (0.9 mg) and

NaHB₄(4.0 mg) in water (0.5 cm³). The suspension was stirred

for 1 h, when TLC (6:2.5:1.5 EtOAc–MeOH–water) showed

the disappearance of 36. After filtration through Celite, size-

exclusion chromatography (Bio-Gel P-2, 100 mM NH₄HCO₃)

gave 5 (4.3 mg, 89%), isolated after lyophilization from water,

as a white, amorphous powder; [a]²_D⁰+2 (c 0.1 in water);

d_H(500 MHz; D₂O; 2D TOCSY and HSQC) 1.42, 1.60, 1.67,

and 2.98 [each 2 H, 4 × m, OCH₂(CH₂)₄ND₂], 2.01 and 2.02

(each 3 H, 2 × s, 2 × NAc), 3.31 (1 H, br t, H-2), 3.43 (1 H, br

t, H-4), 3.45 (1 H, br t, H-3), 3.59 and 3.88 [each 1 H, 2 × m,

OCH₂(CH₂)₄ND₂], 3.62 (1 H, m, H-5), 3.67 (1 H, m, H-5), 3.72

(1 H, dd, J_{H-2,H-3}11.0 Hz, J_{H-3,H-4}3.5 Hz, H-3), 3.75 and 3.80

(each 1 H, 2 × m, 2 × H-6), 3.76 (1 H, m, H-5), 3.82 and 4.04

(each 1 H, 2 × m, 2 × H-6), 3.83 (1 H, dd, J_H-2,H-310.6, J_H-3,H-43.4,

H-3), 3.90 (1 H, br t, H-2), 3.93 (1 H, br d, H-4), 3.98 (1 H, dd,

J_H-1,H-28.4, H-2), 4.16 (1 H, br d, H-4), 4.17 (1 H, dd, J_{H-5,H-6b}

6.0, J_{H-6a,H-6b}11.1, H-6b), 4.31 (1 H, dd, J_{H-5,H-6a}1.8, H-6a),

4.44 (1 H, d, H-1), 4.46 (1 H, d, J_{H-1,H-2}8.5, H-1), 4.49 (1 H, d,

J_{H-1,H-2}8.0, H-1); d_C(125 MHz; D₂O) 22.7, 27.1, 28.7, and 40.0

[OCH₂(CH₂)₄ND₂], 22.8 and 22.9 (2 × NDCOCH₃), 51.8 (C-2),

53.1 (C-2), 61.7 (C-6), 67.9 (C-6), 68.5 (C-4), 69.0 (C-4), 70.1

(C-4), 70.4 [OCH₂(CH₂)₄ND₂], 70.5 (C-6), 71.7 (C-3), 73.4 (C-

2), 74.2 (C-5), 74.3 (C-5), 75.8 (C-5), 76.2 (C-3), 80.6 (C-3),

101.9 (C-1), 102.6 (C-1), 104.8 (C-1); high resolution MALDI-

TOF MS, m/z found M+Na 796.233, C₂₇H₄₈N₃Na₂O₁₉S requires

796.240.

(6-O-Levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-(1→

3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-D-galactopyranosyl

trichloroacetimidate 38

To a solution of 37 (0.52 g, 0.47 mmol) in 1:1:1 toluene–

acetonitrile–water (30 cm³) was added ammonium cerium(IV)

nitrate (2.56 g, 4.7 mmol). The two-phase mixture was

stirred for 2 h, when TLC (95:5 CH₂Cl₂–acetone) showed the

disappearance of 37. The mixture was diluted with EtOAc,

and the organic phase was washed with saturated aq. NaHCO₃

and 10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (95:5 CH₂Cl₂–acetone) of the residue gave

the hemiacetal intermediate, isolated as an orange foam. To a

solution of the hemiacetal (0.38 g, 0.38 mmol) in dry CH₂Cl₂

(3 cm³) and trichloroacetonitrile (0.41 cm³, 3.8 mmol) was

added, at 0 °C, 1,8-diazabicyclo[5.4.0]undec-7-ene (6.7 mm³,

38 lmol). The reaction was stirred for 16 h, then concen-

trated. Column chromatography (95:5 CH₂Cl₂–acetone) of

the residue yielded 38 (0.32 g, 58%), isolated as a yellow foam;

[a]²_D⁰+29 (c 1 in CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and

HSQC) 2.08, 2.21, 2.24, 2.28, 2.32, and 2.33 [each 3 H, 6 × s,

3 × COC₆H₄CH₃, 2 × COCH₃, and CO(CH₂)₂COCH₃], 2.63

and 2.80 [each 2 H, 2 × m, CO(CH₂)₂COCH₃], 3.92 (1 H, m,

H-5), 4.11 (1 H, dd, H-6b), 4.14 (1 H, dd, H-6b), 4.19 (1 H, br

t, H-5), 4.29 (1 H, dd, J_H-5,H-6a5.1 Hz, J_H-6a,H-6b11.3 Hz, H-6a),

4.39 (1 H, dd, J_{H-5,H-6a}2.6, J_{H-6a,H-6b}12.1, H-6a), 4.76 (1 H,

dd, J_H-1,H-29.0, J_H-2,H-311.3, H-2), 4.80 (1 H, d, J_{H-1,H-2}7.8, H-

1), 5.01 (1 H, dd, J_H-3,H-43.4, H-3), 5.27 (1 H, dd, J_{H-2,H-3}9.9,

H-2), 5.43 (1 H, br t, H-4), 5.65 (1 H, br t, H-3), 5.69 (1 H,

br d, H-4), 6.29 (1 H, d, H-1), 6.87, 6.99, 7.13, 7.36, 7.56, and

7.74 (each 2 H, 6 × d, 3 × COC₆H₄CH₃), 8.42 [OC(NH)CCl₃];

d_C(125 MHz; CDCl₃) 20.8, 21.0, 21.6, 21.7 (2 C), and 29.8

[3 × COC₆H₄CH₃, 2 × COCH₃, and CO(CH₂)₂COCH₃], 28.0

and 38.1 [CO(CH₂)₂COCH₃], 51.4 (C-2), 62.2 (C-6), 62.3 (C-

6), 68.9 (C-4), 69.0 (C-4), 71.6 (C-2), 72.3 (C-5), 72.4 (C-3),

72.7 (C-5), 74.1 (C-3), 94.4 (C-1), 101.5 (C-1), 123.3 and 133.8

[N(CO)₂C₆H₄], 128.9, 129.1, 129.2, 129.6, 129.8, and 130.0

(COC₆H₄CH₃).

4-Methoxyphenyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-

b-D-galactopyranoside 37

To 24 (0.75 g, 0.57 mmol) was added a 1 M TBAF solution in

THF (15 cm³), before use neutralized at 0 °C with HOAc. The

mixture was stirred for 1 h at 0 °C and overnight at rt, when TLC

(95:5 CH₂Cl₂–acetone) showed the disappearance of 24 and the

formation of a new product (R_f= 0.29). After concentration,

a solution of the residue in EtOAc was washed with water

and 10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (95:5 CH₂Cl₂–acetone) of the residue gave the

free HO6 intermediate isolated as a yellow foam. A solution

of the free HO6 intermediate (0.54 g, 0.50 mmol) in 1:1

pyridine–acetic anhydride (20 cm³) was stirred overnight, when

TLC (95:5 CH₂Cl₂–acetone) showed the reaction to be complete

(R_f= 0.79). After co-concentration with toluene, a solution of

the residue in CH₂Cl₂was washed with saturated aq. NaHCO₃

and 10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (95:5 CH₂Cl₂–acetone) of the residue gave

37 (0.53 g, 84%), isolated as a yellow foam; [a]²_D⁰+31 (c 0.5 in

CHCl₃); d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 2.07,

5-Azidopentyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-

b-D-glucopyranosyl)-(1→3)-(4,6-di-O-acetyl-2-

deoxy-2-phthalimido-b-D-galactopyranosyl)-(1→

6)-[(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-

(1→3)]-4-O-acetyl-2-deoxy-2-phthalimido-b-D-

galactopyranoside 39

A solution of 27 (74 mg, 68 lmol) and 38 (117.3 mg, 102 lmol)

in dry CH₂Cl₂(3 cm³), containing activated molecular sieves

(4 Å, 0.1 g), was stirred for 45 min at rt, then TMSOTf

(2.0 mm³, 10.2 lmol) was added at 0 °C. The mixture was

2 9 8 4

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

View Article Online

stirred for 15 min, when TLC (9:1 CH₂Cl₂–acetone) showed

the formation of 39 to be complete (R_f= 0.41). After neutral-

ization with pyridine and filtration, the solution was washed

with 10% aq. NaCl, dried, filtered, and concentrated. Column

chromatography (9:1 CH₂Cl₂–acetone) of the residue rendered

39 (100 mg, 70%), isolated as a glass; [a]²⁰+3 (c 1 in CHCl₃);

d_H(500 MHz; CDCl₃; 2D TOCSY and HS^DQC) 0.84, 1.00, 1.12,

and 2.74 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 2.12, 2.14, 2.20,

2.21, 2.22, 2.23, 2.24, 2.30, 2.31, and 2.33 [3 H, 3 H, 3 H, 3 H,

3 H, 3 H, 3 H, 3 H, 3 H, and 6 H, 10 × s, 6 × COC₆H₄CH₃,

3 × COCH₃, and 2 × CO(CH₂)₂COCH₃], 2.57 and 2.75 [each

4 H, 2 × m, 2 × CO(CH₂)₂COCH₃], 2.78 and 3.14 [each 1 H,

2 × m, OCH₂(CH₂)₄N₃], 3.34 (1 H, dd, J_H-5,H-6b8.7 Hz, J_H-6a,H-6b

10.9 Hz, H-6b), 3.77 (1 H, m, H-5), 3.84 (1 H, m, H-5), 3.91

(1 H, m, H-5), 3.93 (1 H, m, H-6a), 3.99 (1 H, m, H-5), 4.08

and 4.22 (each 1 H, 2 × m, 2 × H-6), 4.13 and 4.24 (each 1 H,

2 × br t, 2 × H-6), 4.15 (1 H, dd, J_{H-5,H-6b}5.2, J_{H-6a,H-6b}12.2,

H-6b), 4.32 (1 H, dd, J_H-1,H-28.6, J_H-2,H-311.3, H-2), 4.36 (1 H,

dd, J_{H-5,H-6a}2.1, H-6a), 4.46 (1 H, dd, J_{H-1,H-2}8.4, J_{H-2,H-3}11.2,

H-2), 4.67 (1 H, dd, J_H-3,H-43.2, H-3), 4.71 (2 H, br d, H-1 and

H-1), 4.76 (1 H, d, J_{H-1,H-2}7.8, H-1), 4.85 (1 H, dd, J_{H-3,H-4}3.2,

H-3), 5.02 (1 H, d, H-1), 5.20 (1 H, dd, J_{H-1,H-2}7.9, J_{H-2,H-3}9.9,

H-2), 5.23 (1 H, dd, J_{H-2,H-3}10.0, H-2), 5.37 (1 H, br t, H-4),

5.40 (1 H, br t, H-4), 5.41 (1 H, br d, H-4), 5.59 (1 H, br t, H-

3), 5.61 (1 H, br t, H-3), 5.62 (1 H, br d, H-4), 6.83, 6.84, 6.96,

6.98, 7.11, 7.12, 7.29, 7.31, 7.52, 7.53, 7.71, and 7.73 (each 2 H,

12 × d, 6 × COC₆H₄CH₃); d_C(125 MHz; CDCl₃) 20.8, 20.9, 21.0

(2 C), 21.6 (2 C), 21.7 (3 C), and 29.9 (2 C) [6 × COC₆H₄CH₃,

3 × COCH₃, and 2 × CO(CH₂)₂COCH₃], 23.0, 28.3, 29.8, and

51.1 [OCH₂(CH₂)₄N₃], 27.9 and 38.1 [2 × CO(CH₂)₂COCH₃],

52.4 (C-2), 52.5 (C-2), 62.3 (C-6), 62.4 (C-6), 62.6 (C-6), 68.5

[OCH₂(CH₂)₄N₃], 68.8 (C-6), 68.9 (C-4), 69.0 (C-4), 69.3 (C-

4), 69.8 (C-4), 71.6 (C-5), 71.7 (2 C) (C-2 and C-2), 72.2 (2 C)

(C-5 and C-5), 72.5 (2 C) (C-3 and C-3), 73.7 (C-5), 74.5 (C-

3), 75.0 (C-3), 98.3 (C-1), 98.6 (C-1), 101.4 (2 C) (C-1 and C-

1), 123.1, 123.3, and 133.7 [N(CO)₂C₆H₄], 128.9, 129.0, 129.2,

129.6, 129.8, and 130.0 (COC₆H₄CH₃); high resolution MALDI-

TOF MS, m/z found M+Na 2088.691, C₁₀₉H₁₁₁N₅NaO₃₆requires

2088.691.

(6 × COC₆H₄CH₃and 3 × COCH₃), 22.9, 28.3, 29.7, and 51.1

[OCH₂(CH₂)₄N₃], 52.3 (C-2), 52.5 (C-2), 61.4 (C-6), 61.5 (C-

6), 61.9 (C-6), 68.2 (C-6), 68.6 [OCH₂(CH₂)₄N₃], 68.7 (2 C) (C-

4 and C-4), 69.3 (C-4), 70.1 (C-4), 71.2 (C-5), 71.7 (2 C) (C-2

and C-2), 72.7 (2 C) (C-3 and C-3), 73.4 (C-5), 75.3 (C-5),

75.4 (C-5), 75.8 (C-3), 75.9 (C-3), 98.3 (C-1), 98.4 (C-1), 102.0

(C-1), 102.2 (C-1), 123.0, 123.3, and 133.5 [N(CO)₂C₆H₄],

128.9, 129.0, 129.2, 129.6, 129.8, and 130.0 (COC₆H₄CH₃);

high resolution MALDI-TOF MS, m/z found M+Na 1892.647,

C₉₉H₉₉N₅NaO₃₂requires 1892.617.

5-Azidopentyl (sodium b-D-glucopyranosyl 6-sulfate)-(1→3)-

(2-acetamido-2-deoxy-b-D-galactopyranosyl)-(1→6)-[(sodium

b-D-glucopyranosyl 6-sulfate)-(1→3)]-2-acetamido-2-deoxy-b-D-

galactopyranoside 42

To a solution of 40 (53 mg, 28 lmol) in DMF (3 cm³) was

added the sulfur trioxide trimethylamine complex (157 mg,

1.12 mmol). The mixture was stirred for 48 h at 50 °C, when

TLC (9:1 CH₂Cl₂–MeOH) showed the complete conversion of

40 into non-sodiated 41 (R_f= 0.16). After quenching the reac-

tion with MeOH (10 cm³), the solution was co-concentrated with

toluene. A solution of the residue in EtOAc (50 cm³) was washed

with saturated aq. NaHCO₃and 10% aq. NaCl, dried, filtered,

and concentrated. The residue was dissolved in MeOH (10 cm³),

containing Dowex 50 W X 8 Na⁺resin, and stirred for 15 min,

then filtered and concentrated. Column chromatography (9:1

CH₂Cl₂–MeOH) of the residue gave 41 (45 mg, 77%), isolated

as a white, amorphous powder; d_H(300 MHz; CDCl₃) 0.90,

1.13, and 2.82 [2 H, 4 H, and 2 H, 3 × m, OCH₂(CH₂)₄N₃], 2.21,

2.23, 2.25, 2.30, and 2.34 (3 H, 3 H, 6 H, 6 H, and 9 H, 5 × s,

6 × COC₆H₄CH₃and 3 × COCH₃), 3.01 and 3.35 [each 1 H,

2 × m, OCH₂(CH₂)₄N₃], 3.52 (1 H, dd, J_H-5,H-6b8.4 Hz, J_H-6a,H-6b

10.5 Hz, H-6b), 3.84 (1 H, dd, J_H-5,H-6a2.0, H-6a), 3.92 (1 H, m,

H-5), 4.42 (1 H, dd, J_{H-1,H-2}8.5, J_{H-2,H-3}11.2, H-2), 4.75 (1 H,

d, J_{H-1,H-2}7.8, H-1), 4.78 (1 H, d, J_H-1,H-28.5, H-1), 4.81 (1 H, d,

J_{H-1,H-2}7.7, H-1), 4.92 (1 H, dd, J_{H-2,H-3}11.3, J_{H-3,H-4}3.2, H-3),

5.14 (1 H, d, H-1), 5.18 (1 H, dd, J_{H-2,H-3}9.6, H-2), 5.22 (1 H,

dd, J_{H-2,H-3}9.5, H-2), 5.34 (1 H, br t, H-4), 5.37 (1 H, br t, H-

4), 5.58 (1 H, br d, J_H-3,H-43.4, H-4), 5.63 (1 H, br t, H-3), 5.67

(1 H, br t, H-3), 5.80 (1 H, br d, H-4), 6.87, 6.90, 7.00, 7.15,

7.31, 7.33, 7.54, and 7.73 (2 H, 2 H, 4 H, 4 H, 2 H, 2 H, 4 H, and

4 H, 8 × s, 6 × COC₆H₄CH₃).

A solution of 41 (35 mg, 22 lmol) in ethanolic 33% CH₃NH₂

(5 cm³) was stirred for 7 days, during which time the mixture

was three times concentrated and fresh ethanolic 33% CH₃NH₂

(5 cm³) was added. After co-concentration with toluene, to a

solution of the residue in dry MeOH at 0 °C was added acetic

anhydride (100 mm³). The mixture was stirred for 3 h at 0 °C,

then concentrated. Size-exclusion chromatography (Bio-Gel P-

2, 100 mM NH₄HCO₃) of the residue afforded 42 (11 mg, 62%),

isolated after lyophilization from water, as a white, amorphous

powder; [a]²_D⁰−11 (c 0.6 in water); d_H(500 MHz; D₂O; 2D TOCSY

and HSQC) 1.40, 1.59, and 3.30 [2 H, 4 H, and 2 H, 3 × m,

OCH₂(CH₂)₄N₃], 2.01 and 2.02 (each 3 H, 2 × s, 2 × NAc), 3.32

(2 H, br t, H-2 and H-2), 3.45 (4 H, m, H-3, H-3, H-4, and

H-4), 3.57 and 3.88 [each 1 H, 2 × m, OCH₂(CH₂)₄N₃], 3.62

(2 H, m, H-5 and H-5), 3.69 (1 H, m, H-5), 3.78 (2 H, m,

2 × H-6), 3.81 and 4.06 (each 1 H, 2 × m, 2 × H-6), 3.82 (1 H,

m, H-5), 3.85 (2 H, br t, H-3 and H-3), 3.99 (1 H, br t, H-2),

4.01 (1 H, br t, H-2), 4.15 (1 H, br d, J_H-3,H-43.2 Hz, H-4), 4.19

and 4.30 (each 2 H, 2 × m, 2 × H-6 and 2 × H-6), 4.19 (1 H,

br d, J_{H-3,H-4}3.4, H-4), 4.45 (1 H, d, J_H-1,H-28.5, H-1), 4.50

(1 H, d, J_{H-1,H-2}7.9, H-1), 4.51 (1 H, d, J_{H-1,H-2}7.8, H-1),

4.53 (1 H, d, J_{H-1,H-2}8.5, H-1); d_C(125 MHz; D₂O) 23.0 (2 C)

(2 × NDCOCH₃), 23.2, 28.3, 28.8, and 51.8 [OCH₂(CH₂)₄N₃],

51.8 (2 C) (C-2 and C-2), 61.9 (C-6), 67.8 (2 C) (C-6 and C-

6), 68.6 (C-4), 68.9 (C-4), 70.0 (C-3 and C-3), 70.3 (C-6), 70.6

[OCH₂(CH₂)₄N₃], 73.4 (2 C) (C-2 and C-2), 74.2 (C-5), 74.3 (2

C) (C-5 and C-5), 75.6 (C-5), 76.2 (2 C) (C-4 and C-4), 80.7

(2 C) (C-3 and C-3), 101.8 (C-1), 102.2 (C-1), 104.9 (C-1),

5-Azidopentyl (2,3,4-tri-O-p-toluoyl-b-D-

glucopyranosyl)-(1→3)-(4,6-di-O-acetyl-2-

deoxy-2-phthalimido-b-D-galactopyranosyl)-(1→

6)-[(2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-(1→3)]-4-O-

acetyl-2-deoxy-2-phthalimido-b-D-galactopyranoside 40

To a solution of 39 (90 mg, 43 lmol) in EtOH (10 cm³) and

toluene (3 cm³) was added hydrazine acetate (20 mg, 215 lmol).

The mixture was stirred for 2 h, then concentrated. Column

chromatography (9:1 CH₂Cl₂–acetone) of the residue yielded 40

(60 mg, 75%), isolated as a white glass; [a]²_D⁰+5 (c 0.2 in CHCl₃);

d_H(500 MHz; CDCl₃; 2D TOCSY and HSQC) 0.88, 1.05, 1.12,

and 2.72 [each 2 H, 4 × m, OCH₂(CH₂)₄N₃], 2.11, 2.20, 2.22,

2.27, 2.28, 2.31, and 2.33 (3 H, 3 H, 6 H, 3 H, 3 H, 3 H, and 6 H,

7 × s, 6 × COC₆H₄CH₃and 3 × COCH₃), 2.89 and 3.21 [each

1 H, 2 × m, OCH₂(CH₂)₄N₃], 3.43 (1 H, dd, J_H-5,H-6b8.4 Hz,

J_H-6a,H-6b10.7 Hz, H-6b), 3.56 (1 H, dd, J_{H-5,H-6b}6.1, J_{H-6a,H-6b}

12.3, H-6b), 3.60 (1 H, dd, J_{H-5,H-6b}6.4, J_{H-6a,H-6b}12.9, H-6b),

3.71 (2 H, m, H-6a and H-6a), 3.73 (1 H, m, H-5), 3.76 (1 H,

m, H-5), 3.79 (1 H, m, H-5), 3.88 (1 H, dd, J_H-5,H-6a2.1, H-6a),

4.00 (1 H, m, H-5), 4.15 (2 H, m, 2 × H-6), 4.33 (1 H, dd, J_H-1,H-2

8.6, J_H-2,H-311.2, H-2), 4.50 (1 H, dd, J_{H-1,H-2}8.6, J_{H-2,H-3}11.1,

H-2), 4.65 (1 H, dd, J_H-3,H-43.5, H-3), 4.71 (1 H, d, H-1), 4.79

(1 H, d, J_{H-1,H-2}8.0, H-1), 4.82 (1 H, dd, J_{H-3,H-4}3.6, H-3),

4.86 (1 H, d, J_{H-1,H-2}7.8, H-1), 5.02 (1 H, d, H-1), 5.21 (1 H,

dd, J_{H-2,H-3}10.0, H-2), 5.25 (1 H, dd, J_{H-2,H-3}9.9, H-2), 5.31

(1 H, br t, H-4), 5.33 (1 H, br t, H-4), 5.53 (1 H, br d, H-4), 5.63

(1 H, br t, H-3), 5.65 (1 H, br t, H-3), 5.72 (1 H, br d, H-4),

6.75, 6.78, 6.94, 7.13, 7.27, 7.50, 7.51, 7.74, and 7.75 (2 H, 2 H,

4 H, 4 H, 4 H, 2 H, 2 H, 2 H, and 2 H, 9 × d, 6 × COC₆H₄CH₃);

d_C(125 MHz; CDCl₃) 20.7, 21.2, 21.3, 21.6 (4 C), and 21.8 (2 C)

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 8 5

View Article Online

105.0 (C-1); high resolution MALDI-TOF MS, m/z found

M+Na 1086.254, C₃₃H₅₅N₅Na₃O₂₇S₂requires 1086.222.

stirring for 16 h, column chromatography (7.5:1.5:1.0 EtOAc–

MeOH–water) of the mixture yielded 45, isolated as a glass. The

pure, elongated saccharide was used directly for the preparation

of neoglycoconjugate BSA-3.

5-Aminopentyl (sodium b-D-glucopyranosyl 6-sulfate)-(1→3)-

(2-acetamido-2-deoxy-b-D-galactopyranosyl)-(1→6)-[(sodium

b-D-glucopyranosyl 6-sulfate)-(1→3)]-2-acetamido-2-deoxy-b-D-

galactopyranoside 6

5-N-(3,4-Dione-2-ethoxycyclobutene)aminopentyl (sodium b-

D-glucopyranosyl 6-sulfate)-(1→3)-2-acetamido-2-deoxy-b-D-

galactopyranoside 46

A solution of 42 (5 mg, 4.7 lmol) in 0.05 M aq. NaOH (0.5 cm³)

was added dropwise to a suspension of 10% Pd–C (0.7 mg) and

NaHB₄(2.7 mg) in water (0.5 cm³). The suspension was stirred

for 1 h, when TLC (6:2.5:1.5 EtOAc–MeOH–water) showed

the disappearance of 42. After filtration through Celite, size-ex-

clusion chromatography (Bio-Gel P-2, 100 mM NH₄HCO₃) gave

6 (3.5 mg, 71%), isolated after lyophilization from water, as a

white, amorphous powder; [a]²_D⁰−4 (c 0.2 in water); d_H(500 MHz;

D₂O; 2D TOCSY and HSQC) 1.41, 1.60, 1.66 and 2.99 [each

2 H, 4 × m, OCH₂(CH₂)₄ND₂], 2.01 (6 H, s, 2 × NAc), 3.31 (2 H,

br t, H-2 and H-2), 3.45 (4 H, m, H-3, H-3, H-4, and H-4),

3.59 and 3.87 [each 1 H, 2 × m, OCH₂(CH₂)₄ND₂], 3.62 (2 H, m,

H-5 and H-5), 3.69 (1 H, m, H-5), 3.78 (2 H, m, 2 × H-6),

3.83 and 4.03 (each 1 H, 2 × m, 2 × H-6), 3.83 (1 H, m, H-5),

3.84 (2 H, br t, H-3 and H-3), 3.99 (1 H, br t, H-2), 4.00 (1 H, br

t, H-2), 4.15 (1 H, br d, J_H-3,H-43.2 Hz, H-4), 4.18 and 4.30 (each

2 H, 2 × m, 2 × H-6 and 2 × H-6), 4.18 (1 H, br d, J_{H-3,H-4}

3.2, H-4), 4.45 (1 H, d, J_H-1,H-28.5, H-1), 4.50 (1 H, d, J_{H-1,H-2}

8.0, H-1), 4.52 (1 H, d, J_{H-1,H-2}8.1, H-1), 4.53 (1 H, d, J_{H-1,H-2}

8.6, H-1); d_C(125 MHz; D₂O) 22.9 (2 C) (2 × NDCOCH₃), 22.8,

27.0, 28.7, and 40.0 [OCH₂(CH₂)₄ND₂], 51.8 (2 C) (C-2 and

C-2), 61.9 (C-6), 67.9 (2 C) (C-6 and C-6), 68.6 (C-4), 68.8

(C-4), 70.0 (C-3 and C-3), 70.4 (C-6), 70.5 [OCH₂(CH₂)₄ND₂],

73.5 (2 C) (C-2 and C-2), 74.2 (C-5), 74.3 (2 C) (C-5 and

C-5), 75.6 (C-5), 76.1 (2 C) (C-4 and C-4), 80.5 (2 C) (C-3

and C-3), 101.9 (C-1), 102.3 (C-1), 104.8 (C-1), 104.9 (C-1);

high resolution MALDI-TOF MS, m/z found M+Na 1060.193,

C₃₃H₅₇N₃Na₃O₂₇S₂requires 1060.231.

To a solution of 4 (0.5 mg, 0.9 lmol) in 50 mM sodium

phosphate buffer (100 mm³, pH 7.2) was added a solution of

diethyl squarate (0.26 mm³, 3.6 lmol) in EtOH (100 mm³). After

stirring for 16 h, EtOH was evaporated by flushing with N₂,

and the residue in water was loaded on a C-18 Extract-Clean™

column. After elution of remaining 4 with water (3 × 3 cm³),

46 was eluted with MeOH (3 × 3 cm³), then concentrated in

vacuo. The pure, elongated saccharide was used directly for the

preparation of neoglycoconjugate BSA-4.

5-N-(3,4-Dione-2-ethoxycyclobutene)aminopentyl (2-

acetamido-2-deoxy-b-D-galactopyranosyl)-(1→6)-[(sodium

b-D-glucopyranosyl 6-sulfate)-(1→3)]-2-acetamido-2-deoxy-b-D-

galactopyranoside 47

To a solution of 5 (1.0 mg, 1.2 lmol) in 50 mM sodium phos-

phate buffer (100 mm³, pH 7.2) was added a solution of diethyl

squarate (0.4 mm³, 2.4 lmol) in EtOH (100 mm³). After stirring

for 16 h, column chromatography (7.5:1.5:1.0 EtOAc–MeOH–

water) of the mixture yielded 47, isolated as a glass. The pure,

elongated saccharide was used directly for the preparation of

neoglycoconjugate BSA-5.

5-N-(3,4-Dione-2-ethoxycyclobutene)aminopentyl (sodium

b-D-glucopyranosyl 6-sulfate)-(1→3)-(2-acetamido-2-deoxy-

b-D-galactopyranosyl)-(1→6)-[(sodium b-D-glucopyranosyl 6-

sulfate)-(1→3)]-2-acetamido-2-deoxy-b-D-galactopyranoside 48

To a solution of 6 (1.0 mg, 1.0 lmol) in 50 mM sodium

phosphate buffer (100 mm³, pH 7.2) was added a solution of

diethyl squarate (0.28 mm³, 2.0 lmol) in EtOH (100 mm³). After

stirring for 16 h, column chromatography (7.5:1.5:1.0 EtOAc–

MeOH–water) of the mixture yielded 48, isolated as a glass. The

pure, elongated saccharide was used directly for the preparation

of neoglycoconjugate BSA-6.

3-[2-N-(3,4-Dione-2-ethoxycyclobutene)aminoethylthio]propyl

(b-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-b-

D-glucopyranoside 43

To a solution of 1 (1 mg, 1.9 lmol) in 50 mM sodium phosphate

buffer (100 mm³, pH 7.2) was added a solution of diethyl

squarate (0.56 mm³, 3.8 lmol) in EtOH (100 mm³). After stirring

for 16 h, EtOH was evaporated by flushing with N₂, and the

residue in water was loaded on a C-18 Extract-Clean™ column.

After elution of remaining 1 with water (3 × 3 cm³), 43 was

eluted with MeOH (3 × 3 cm³), then concentrated in vacuo. The

pure, elongated saccharide was used directly for the preparation

of neoglycoconjugate BSA-1.

General procedure for the conjugation of elongated saccharides

43–48 to BSA

For a target oligosaccharide incorporation of about 15 mol mol⁻¹

BSA, to a solution of an elongated saccharide (43–48, 10 equiv.

based on BSA) in 0.1 M NaHCO₃buffer (0.5 mg cm⁻³, pH 9.0)

was added a solution of pre-treated BSA¹¹(20 mg cm⁻³) in 0.1 M

NaHCO₃buffer. After stirring for 3–5 days, the mixtures were

loadedontoa30kDaNalgenecentrifugalfilter, andwashedwith

water (6 × 15 cm³). After lyophilization from water, the degree

of incorporation of saccharides 43–48 in neoglycoconjugates

BSA-1–BSA-6, respectively, was determined by MALDI-TOF

MS analysis. Samples (0.1 mg cm⁻³1:1 acetonitrile–water) were

mixed on the target plate in a ratio of 1:1 with the matrix 3,5-

dimethoxy-4-hydroxycinnamic acid (10 mg cm⁻³) in 1:1 aceto-

nitrile–water containing 0.1% TFA.

3-[2-N-(3,4-Dione-2-ethoxycyclobutene)aminoethylthio]propyl

(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1→6)-[(b-D-

glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-b-D-

glucopyranoside 44

To a solution of 2 (1 mg, 1.4 lmol) in 50 mM sodium phosphate

buffer (100 mm³, pH 7.2) was added a solution of diethyl

squarate (0.4 mm³, 2.4 lmol) in EtOH (100 mm³). After stirring

for 16 h, column chromatography (7.5:1.5:1.0 EtOAc–MeOH–

water) of the mixture yielded 44, isolated as a glass. The pure,

elongated saccharide was used directly for the preparation of

neoglycoconjugate BSA-2.

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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

View Article Online

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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 7 2 – 2 9 8 7

2 9 8 7

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