Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

Carbohydrate Research 338 (2003) 2629Á2651

/

www.elsevier.com/locate/carres

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide

fragments of the capsular polysaccharide of Streptococcus

pneumoniae type 14

John A.F. Joosten, Bart J. Lazet, Johannis P. Kamerling,* Johannes F.G. Vliegenthart

Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH

Utrecht, The Netherlands

Received 16 April 2003; accepted 17 June 2003

Abstract

The chemo-enzymatic synthesis is described of b-

O(CH₂)₆NH₂(1), b- -Glcp-(106)-[b- -Galp-(104)]-b- -GlcpNAc-(10

-Galp-(104)-b- -GlcpNAc-(103)-b- -Galp-(104)-b- -Glcp-(10O(CH₂)₆NH₂(3), and b-

3)-b- -Galp-(104)-b- -Glcp-(106)-[b- -Galp-(104)]-b- -GlcpNAc-(10O(CH₂)₆NH₂(4), representing fragments of the re-

peating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5Á8 were

D

-Glcp-(10

/

6)-[b-

D

-Galp-(10

/

4)]-b-

4)-b-

-Galp-(10

D

-GlcpNAc-(10

-Glcp-(10O(CH₂)₆NH₂(2), b-

4)-b- -GlcpNAc-(10

/

3)-b-

D

-Galp-(10

/

D

/

D

/

D

/

3)-b-

D

-Galp-(10

/

D

/

D

/

D

/

D

/

D

/

D

/

D

/

D

/

D

/

D

/

D

/

synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk b-1,4-galactosyltransferase as a catalyst.

The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal

models.

Keywords: Carbohydrates; Streptococcus pneumoniae; Oligosaccharide synthesis

1. Introduction

gate vaccines against S. pneumoniae serotypes have been

introduced.⁷

The CPS of S. pneumoniae type 14 is built up from the

The encapsulated bacterium Streptococcus pneumoniae

is a major cause of life-threatening diseases such as otitis

media, pneumonia, meningitis, bacteraemia, and septi-

caemia,^1,2mainly due to a growing resistance towards

antibiotics.^3,4Vaccination with the available 23-valent

capsular polysaccharide (CPS) vaccines⁵offers protec-

tion in healthy adults, but they are ineffective in the

most important high-risk groups, such as infants, small

children, immuno-compromised patients, and the el-

derly.⁶Conjugation of S. pneumoniae carbohydrate

antigens to a protein carrier results in a T-cell dependent

neoglycoconjugate antigen that gives an efficient im-

mune response in the high-risk groups,⁷as have been

shown for other bacteria.^8,9Currently, neoglycoconju-

tetrasaccharide repeating unit 0

b- -GlcpNAc-(103)-b- -Galp-(10

In earlier reports we have described the chemo-

/

6)-[b-

D

-Galp-(10

/

4)]-

D

10

/

D

/

4)-b-

D

-Glcp-(10

/

.

enzymatic synthesis of a spacer-containing tetrasacchar-

ide fragment of the CPS of S. pneumoniae type 14,

corresponding with one repeating unit {b-

4)-b- -Glcp-(106)-[b- -Galp-(104)]-b-

D

-Galp-(10

/

D

/

D

/

D-GlcpNAc},

as well as mimics of the CPS.^11,12The tetrasaccharide-

containing neoglycoconjugate (CRM₁₉₇as carrier pro-

tein) showed particularly promising immunological data

when tested in mice models.¹³Based on these results, it

was decided to synthesize a series of longer oligosac-

charide fragments of the CPS for more detailed im-

munological studies. Recently, we have described the

chemo-enzymatic synthesis of four oligosaccharides

with a 6-aminohexyl spacer, varying in length between

one and two repeating units.^14,15Here, we report the

chemo-enzymatic synthesis of another four oligosac-

* Corresponding author. Tel.: ꢀ

/

31-30-2533479; fax: ꢀ31-

/

30-2540980.

E-mail address: j.p.kamerling@chem.uu.nl (J.P. Kamerling).

doi:10.1016/S0008-6215(03)00292-1

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J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

charides, containing the same spacer. The whole series

of eight spacered oligosaccharides will form an excellent

panel to investigate the structural parameters influen-

cing immunogenicity.

phthalimido-b-

methane at 0 8C, using 1 equivalent silver trifluoro-

methanesulfonate (AgOTf) as catalyst, gave

D

-glucopyranoside (10)¹¹in dichloro-

a

disaccharide 11 in 62% yield. A small amount of a side

product, the acetylated acceptor, was also isolated. Side

product formation was much higher when using 10%

trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a

2. Results and discussion

catalyst (0 8C: 34% 11, 53% side product; ꢁ40 8C: 50%

/

11, 25% side product). De-allylation of 11 by using

palladium(II)chloride, sodium acetate, and acetic acid in

For the organic synthesis of the structurally closely

related compounds 5Á8, a series of broadly applicable

/

an ultrasonic bath (0/12, 70%), followed by imidation

building blocks, 9, 13, 24, and 37, were designed.

Condensation of these key building blocks with appro-

priate acceptor or donor building blocks gave, after

of the anomeric center, using trichloroacetonitrile with

1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a catalyst,

gave disaccharide donor 13 (76%). Coupling of 13 to 6-

deprotection, the aimed linear oligosaccharides (5Á8),

/

which were used as acceptor substrates for b-1,4-

galactosyltransferase (EC 2.4.1.22) from bovine milk

azidohexyl 4-O-acetyl-2,6-di-O-benzyl-b-

D-galactopyra-

noside (14)¹⁴at ꢁ

/

70 8C in dichloromethane, using 10%

to give the desired title compounds 1Á4 (Scheme 1).

/

trimethylsilyl trifluoromethanesulfonate as a catalyst,

gave trisaccharide 21 (73%).

In the first step of an alternative route to trisaccharide

2.1. Synthesis of tetrasaccharide fragment 1

21, 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-

pyranosyl trichloroacetimidate (15)¹⁷was coupled to

galactose acceptor 14, at ꢁ70 8C in dichloromethane,

using 10% trimethylsilyl trifluoromethanesulfonate as a

catalyst, to give disaccharide 16 in 86% yield. Mild de-

O-acetylation of 16, using sodium methoxide at pH 8,

D-gluco-

The linear trisaccharide backbone 5 was prepared via

two different routes (Scheme 2). For the first route,

disaccharide donor 13 was needed. Coupling of 2,3,4,6-

tetra-O-acetyl-a-D-glucopyranosyl trichloroacetimidate

(9)¹⁶to allyl 2-deoxy-3,4-di-O-p-methylbenzoyl-2-

/

Scheme 1. Overview of 6-aminohexyl-spacered oligosaccharides 1Á/4, representing fragments of the repeating unit of the S.

pneumoniae serotype 14 capsular polysaccharide.

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2631

Scheme 2. Synthesis of the linear trisaccharide backbone 5: (a) 1 equiv AgOTf, CH₂Cl₂, 0 8C, 62%; (b) Pd(II)Cl₂, NaOAc, AcOH,

70%; (c) Cl₃CCN, DBU, CH₂Cl₂, 76%; (d) 10% TMSOTf, CH₂Cl₂, ꢁ70 8C, 73%; (e) 10% PdꢀC, H₂, water, tert-BuOH, aq 25%

NH₃/10% PdꢀC, AcOH, 69%; (f) NaOMe (pH 10), MeOH, CH₂Cl₂; (g) NH₂CH₂CH₂NH₂, 1-BuOH, 80 8C; (h) pyridine, Ac₂O,

80% over three steps; (i) NaOMe (pH 9), MeOH, CH₂Cl₂, 97%; (j) 10% TMSOTf, CH₂Cl₂, ꢁ40 8C, 56%; (k) 10% TMSOTf,

CH₂Cl₂, ꢁ70 8C, 86%; (l) NaOMe (pH 8), MeOH, CH₂Cl₂, 91%; (m) t-BdPSiCl, DMAP, Et₃N, CH₂Cl₂, pyridine, 83%; (n) p-

/

mBzCl, pyridine, CH₂Cl₂, 90%; (o) AcCl, MeOH, toluene, 0 8C, 81%.

gave 17 (91%) with a retention of the acetyl group at

galactose O-4. Selective introduction of a tert-butyldi-

phenylsilyl group (tBdPSi) at the primary hydroxyl

function of 17 using tert-butyldiphenylsilyl chloride

and a catalytic amount of 4-dimethylaminopyridine

tion (mBz) of the two remaining hydroxyl groups, using

p-methylbenzoyl chloride in pyridine, gave 19 (90%).

Finally, removal of the tert-butyldiphenylsilyl group

under mild acidic conditions afforded disaccharide

acceptor 20 in 81% yield. A minor side product, the

O-de-acetylated analogue of 20, was isolated also (14%

(DMAP) (0

/

18, 83%), followed by p-methylbenzoyla-

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J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

yield). Coupling of glucose donor 9 to disaccharide 20 in

dichloromethane at ꢁ40 8C, using 10% trimethylsilyl

in the presence of a-lactalbumin (lactose synthase

complex).^{18 1}H NMR data of 5 and 1, derived from

2D TOCSY and ROESY measurements, are presented

in Tables 1 and 2, respectively.

/

trifluoromethanesulfonate as a catalyst, gave trisacchar-

ide 21 in 56% yield. O-De-acylation of 21 using sodium

methoxide at pH 10, followed by N-de-phthaloylation

using 1,2-diaminoethane in 1-butanol at 80 8C, and

subsequent N,O-acetylation using acetic anhydride in

pyridine yielded 22 in 80% yield over three steps. The O-

acetylation step was carried out to facilitate chromato-

graphic purification. O-De-acetylation of 22 with so-

dium methoxide at pH 9 gave crude 23, and after

Table 1

500 MHz H NMR data (TOCSY, ROESY) of 5 at 300 K (in

ppm)

1

Proton

d_H

Gal GlcNAc II

I

Glc

III

reduction of the azido function using 10% Pdꢀ

in the presence of ammonia, and subsequent debenzyla-

tion using 10% PdꢀC and H₂in the presence of acetic

acid, linear trisaccharide backbone 5 was obtained in a

yield of 69%. Tetrasaccharide 1 was synthesized in 54%

yield by the transfer of galactose from UDP-galactose to

/

C and H₂

/

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.37 4.70

3.57 3.76

3.72 3.56

4.15 n.d. ^a

n.d. 3.61

n.d. 4.22

n.d. 3.89

4.50

3.31

3.50

3.39

3.48

3.92

3.72

O-4 of the N-acetyl-b-D-glucosamine residue of 5 by

using bovine milk b-1,4-galactosyltransferase as a cata-

lyst (Scheme 3). As a result of the enzymatic galactosy-

lation of the terminal glucose residue, the previously

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

1.39Á/1.41 (4

H)

described pentasaccharide 6-aminohexyl b-

pyranosyl-(104)-b- -glucopyranosyl-(106)-[b-

lactopyranosyl-(104)]-2-acetamido-2-deoxy-b- -gluco-

pyranosyl-(103)-b-

-galactopyranoside (47)¹⁴was

obtained as a side product (35%) (Scheme 3). So far,

the enzymatic galactosylation of glucose by b-1,4-

galactosyltransferase has only been shown to proceed

D

-galacto-

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

1.64Á

H)

2.96

/

1.65 (4

/

D

/

D

-ga-

/

D

CH₂ND₂

OCH₂(CH₂)₅ND₂

NDCOCH₃

/

D

3.68, 3.92

2.04

a

n.d., not determined.

Scheme 3. Synthesis of tetrasaccharide 1 and pentasaccharide 47: (a) 1.4 equiv UDP-Gal, aq 50 mM sodium cacodylate buffer (pH

7.5), 3 U b-1,4-galactosyltransferase, 14 U alkaline phosphatase, 37 8C, 54% (1); 35% (47).

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2633

Table 2

500 MHz H NMR data (TOCSY, ROESY) of 1 at 300 K (in ppm)

1

Proton

d_H

Gal I ^a

GlcNAc II

Glc III

Gal IV ^b

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.38

3.55

3.70

4.16

n.d. ^c

n.d.

4.72

3.81

3.73

3.87

3.73

4.28

3.95

4.52

3.32

3.50

3.39

3.47

3.86

3.72

4.54

3.54

3.67

3.92

n.d.

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

CH₂ND₂

1.39Á

1.63Á

2.96

/

1.41 (4 H)

/1.64 (4 H)

OCH₂(CH₂)₅ND₂

NDCOCH₃

3.67, 3.92

2.03

a

Gal(b1-O(CH₂)₆NH₂).

Gal(b1-4)GlcNAc.

n.d., not determined.

b

c

2.2. Synthesis of pentasaccharide fragment 2

lyst gave tetrasaccharide 30 (58%). O-De acylation of 30

using sodium methoxide at pH 10, followed by N-de-

phthaloylation using 1,2-diaminoethane in 1-butanol at

80 8C, and subsequent N,O-acetylation using acetic

anhydride in pyridine yielded 31 in 88% yield over three

steps. O-De-acetylation of 31 with sodium methoxide at

pH 10 gave crude 32. After reduction of the azido

The linear tetrasaccharide backbone 6 was prepared via

two different routes (Scheme 4). In the first route,

condensation of disaccharide donor 13 with 6-azido-

hexyl (4-O-acetyl-2,6-di-O-benzyl-b-

D-galactopyrano-

syl)-(10

/

4)-2,3,6-tri-O-benzyl-b-

D

-glucopyranoside

70 8C, using 10%

(24)¹⁴in dichloromethane at ꢁ

/

function using 10% Pdꢀ

/

C and H₂in the presence of

trimethylsilyl trifluoromethanesulfonate as a catalyst,

afforded tetrasaccharide 30 in 83% yield. An alternative

route to tetrasaccharide 30 involved the condensation of

glucose donor 9 (see Scheme 2) with trisaccharide

acceptor 6-azidohexyl (2-deoxy-3,4-di-O-p-methylben-

ammonia, and subsequent debenzylation using 10% Pdꢀ

/

C and H₂in the presence of acetic acid, the linear

tetrasaccharide backbone 6 was obtained in a yield of

81%. Pentasaccharide 2 was synthesized in 65% yield by

the transfer of galactose from UDP-galactose to O-4 of

zoyl-2-phthalimido-b-

D

-glucopyranosyl)-(10

-galactopyranosyl)-(10

-glucopyranoside (29). As a first

/

3)-(4-O-

the N-acetyl-b-

D-glucosamine residue of 6 by using

bovine milk b-1,4-galactosyltransferase as a catalyst

(Scheme 5). As a digalactosylated side product the

acetyl-2,6-di-O-benzyl-b-

D

/

4)-

2,3,6-tri-O-benzyl-b-

D

step in the synthesis of 29, glucosamine donor 15¹⁷(see

Scheme 2) was coupled to lactose acceptor 24 in

previously described hexasaccharide 6-aminohexyl b-

galactopyranosyl-(104)-b- -glucopyranosyl-(106)-[b-

-galactopyranosyl-(104)]-2-acetamido-2-deoxy-b-

glucopyranosyl-(103)-b- -galactopyranosyl-(104)-b-

D-

/

D

/

dichloromethane at ꢁ

/

70 8C, using 10% trimethylsilyl

D

/

D-

trifluoromethanesulfonate as a catalyst, to give trisac-

charide 25 in 97% yield. Mild O-de-acetylation of 25,

using sodium methoxide at pH 8, gave 26 (78%), with a

retention of the acetyl group at galactose O-4. Selective

introduction of a tert-butyldiphenylsilyl group at the

primary hydroxyl function of 26 using tert-butyldiphe-

nylsilyl chloride and a catalytic amount of 4-dimethyla-

/

D

/

D

-glucopyranoside (48)¹⁴(6%; Scheme 5) was obtained.

¹H NMR data of 6 and 2, derived from 2D TOCSY and

ROESY measurements, are presented in Tables 3 and 4,

respectively.

2.3. Synthesis of tetrasaccharide fragment 3

minopyridine ( 0

/

27, 84%), followed by p -

methylbenzoylation of the two remaining hydroxyl

groups, using p-methylbenzoyl chloride in pyridine

gave 28 (91%). Finally, selective removal of the silyl

group, using a 1:1 mixture of 1.0 M TBAF in THF and

AcOH at pH 6, gave 29 in 89% yield. Coupling of donor

The linear trisaccharide backbone 7 was prepared via

two different routes (Scheme 6). For the first route,

trisaccharide donor 37 was needed. Debenzylation of

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

pyranosyl)-(103)-(4-O-acetyl-2,6-di-O-benzyl-b-

lactopyranosyl)-(104)-1,2,3,6-tetra-O-benzyl-b-

copyranoside (34),¹⁵using 10% Pdꢀ

C and H₂in the

D

-gluco-

-ga-

-glu-

/

D

9 to acceptor 29 in dichloromethane at ꢁ

10% trimethylsilyl trifluoromethanesulfonate as a cata-

/

40 8C, using

/

D

/

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J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

Scheme 4. Synthesis of the linear tetrasaccharide backbone 6: (a) 10% TMSOTf, CH₂Cl₂, ꢁ

tert-BuOH, aq 25% NH₃/10% PdꢀC, AcOH, 81%; (c) NaOMe (pH 10), MeOH, CH₂Cl₂; (d) NH₂CH₂CH₂NH₂, 1-BuOH, 80 8C; (e)

pyridine, Ac₂O, 88% over three steps; (f) NaOMe (pH 10), MeOH, CH₂Cl₂, quantitative; (g) 10% TMSOTf, CH₂Cl₂, ꢁ40 8C, 58%;

(h) NaOMe (pH 8), MeOH, CH₂Cl₂, 78%; (i) t-BdPSiCl, DMAP, Et₃N, CH₂Cl₂, pyridine, 84%; (j) p-mBzCl, pyridine, CH₂Cl₂,

91%; (k) 1.0 M TBAF in THF, AcOH (pH 6), 89%; (l) 10% TMSOTf, CH₂Cl₂, ꢁ70 8C, 97%.

/

70 8C, 83%; (b) 10% Pdꢀ

/

C, H₂, water,

/

presence of acetic acid, followed by O-acetylation using

acetic anhydride in pyridine afforded 35 in 66% yield

over two steps. Selective O-de-acetylation of the anome-

ric center of 35, using hydrazinium acetate in N,N-

the yield. O-De-acetylation of 38, using sodium meth-

oxide (pH 10), followed by N-de-phthaloylation using

1,2-diaminoethane in 1-butanol at 80 8C, and subse-

quent N,O-acetylation using acetic anhydride in pyri-

dine yielded 39 (77% over three steps). De-O-acetylation

dimethylformamide (0

/

36, 89%), and subsequent imi-

dation using trichloroacetonitrile with 1,8-diazabicy-

clo[5.4.0]undec-7-ene as a catalyst gave trisaccharide

donor 37 in 69% yield. Condensation of 37 with 6-azido-

1-hexanol (33) in dichloromethane at 0 8C, using 1

equivalent silver trifluoromethanesulfonate as a catalyst,

gave trisaccharide 38 in 22% yield only. The low yield

was due to orthoester formation, and coupling attempts

at different temperatures and/or applying trimethylsilyl

trifluoromethanesulfonate as a catalyst did not improve

of 39 with sodium methoxide at pH 10 (0

reduction of the azido function using 10% Pdꢀ

/

40, 55%), and

/C and H₂

in the presence of ammonia, gave the linear trisaccharide

1

backbone 7 (83%). H NMR data of 40 and 7, derived

from 2D TOCSY and ROESY measurements, are

presented in Tables 5 and 6, respectively. In an alter-

native route to linear backbone 7, trisaccharide 25 was

O-de-acetylated using sodium methoxide (pH 10),

followed by N-de-phthaloylation using 1,2-diami-

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2635

Scheme 5. Synthesis of pentasaccharide 2 and hexasaccharide 48: (a) 1.4 equiv UDP-Gal, aq 50 mM sodium cacodylate buffer (pH

7.5), 3 U b-1,4-galactosyltransferase, 14 U alkaline phosphatase, 37 8C, 65% (2); 6% (48).

41 (78% over three steps). De-O-acetylation of 41 with

sodium methoxide (pH 10) gave crude 42, and after

Table 3

500 MHz H NMR data (TOCSY, ROESY) of 6 at 300 K (in

ppm)

1

reduction of the azido function of 42 using 10% Pdꢀ

and H₂in the presence of ammonia, and subsequent

debenzylation using 10% PdꢀC and H₂in the presence

/C

Proton

d_H

/

of acetic acid, linear trisaccharide backbone 7 was

obtained in a yield of 68%. Tetrasaccharide 3 was

synthesized in 94% yield by the transfer of galactose

Glc Gal GlcNAc Glc

I ^a

II

III

IV ^b

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.47 4.43 4.69

3.29 3.58 3.76

3.63 3.72 3.57

3.63 4.15 n.d. ^c

3.60 n.d. 3.61

3.97 n.d. 4.20

n.d. 3.88

4.49

3.30

3.49

3.38

3.47

3.92

3.72

from UDP-galactose to O-4 of the N-acetyl-b-

D-gluco-

samine residue of 7 by using bovine milk b-1,4-

galactosyltransferase as a catalyst (Scheme 7). 2D

TOCSY and ROESY measurements confirmed the

structure of 3 (Table 7). Chemical- and chemo-enzy-

matic syntheses of 7 containing other functionalities at

the anomeric center have been described earlier.¹⁹

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

1.39Á/1.41

(4 H)

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

1.64Á

(4 H)

2.95

/

1.65

2.4. Synthesis of hexasaccharide fragment 4

CH₂ND₂

OCH₂(CH₂)₅ND₂

NDCOCH₃

To obtain the linear tetrasaccharide backbone 8, as a

first step trisaccharide donor 37 (see Scheme 6) was

coupled with 6-azidohexyl 2-deoxy-3,4-di-O-p-methyl-

3.68, 3.92

2.03

a

benzoyl-2-phthalimido-b-

D

-glucopyranoside

(43)¹⁵

Glc(b1-O(CH₂)₆NH₂).

Glc(b1-6)GlcNAc.

n.d., not determined.

b

(Scheme 8) in dichloromethane at 0 8C, using 15%

trimethylsilyl trifluoromethanesulfonate as a catalyst,

to give tetrasaccharide 44 in 34% yield. O-De-acylation

of 44 using sodium methoxide (pH 10), followed by N-

de-phthaloylation using 1,2-diaminoethane in 1-butanol

at 90 8C, and subsequent N,O-acetylation using acetic

c

noethane in 1-butanol at 80 8C, and subsequent N,O-

acetylation using acetic anhydride in pyridine afforded

2636

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

Table 4

500 MHz H NMR data (TOCSY, ROESY) of 2 at 300 K (in ppm)

1

Proton

d_H

Glc I ^a

Gal II ^b

GlcNAc III

Glc IV ^c

Gal V ^d

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.48

3.29

3.64

n.d. ^e

n.d.

4.43

3.59

3.72

4.16

n.d.

4.71

3.81

3.73

3.87

3.73

4.27

3.95

4.53

3.33

3.50

3.40

3.46

3.86

3.73

4.52

3.54

3.67

3.93

n.d.

3.95

3.78

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

CH₂ND₂

1.39Á

1.64Á

2.96

/

1.41 (4 H)

1.65 (4 H)

/

OCH₂(CH₂)₅ND₂

NDCOCH₃

3.67, 3.92

2.03

a

Glc(b1-O(CH₂)₆NH₂).

b

Gal(b1-4)Glc.

c

Glc(b1-6)GlcNAc.

Gal(b1-4)GlcNAc.

n.d., not determined.

d

e

anhydride in pyridine afforded 45 (86% over three

steps). O-De-acetylation of 45 using sodium methoxide

at 40 8C. Column chromatography was performed on

Silica Gel 60 (E. Merck, 0.063Á0.200 mm). Optical

rotations were measured with a PerkinÁElmer 241

/

(pH 10) (0

/

46, 79%), and reduction of the azide

/

function using 10% Pdꢀ

/C and H₂in the presence of

polarimeter, using a 10 cm, 1 mL cell. ¹H NMR spectra

were recorded at 300 K with a Bruker AC 300 (300

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

the d_Hvalues are given in ppm relative to the signal for

internal Me₄Si (d_H0, CDCl₃) or internal acetone (d_H

2.225, D₂O). ¹³C 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_C76.9, CDCl₃) or internal acetone (d_C30.89,

ammonia, gave the linear tetrasaccharide backbone 8 in

82% yield. Hexasaccharide 4 was synthesized in 76%

yield by the transfer of galactose from UDP-galactose to

O-4 of the N-acetyl-b-D-glucosamine residues of 8 by

using bovine milk b-1,4-galactosyltransferase as a cata-

lyst (Scheme 9). H NMR data of 46, 8, and 4, derived

from 2D TOCSY and ROESY measurements, are

1

presented in Tables 8Á/10, respectively.

Conjugation of the oligosaccharides 1Á/4 to CRM₁₉₇

D₂O). Two-dimensional ¹HÁ¹

and 100 ms) and ROESY (mixing time 300 ms), and

¹HÁ¹³

C correlated HSQC NMR spectra were recorded

/

H TOCSY (mixing times 7

(cross reactive material) and immunological studies are

in progress.

/

at 300 K with a Bruker AMX 500 spectrometer. Exact

masses were measured by nano electrospray time-of-

flight mass spectrometry (positive-ion mode) using a

Micromass LCToF mass spectrometer at a resolution of

5000 FWHM. Gold-coated capillaries were loaded with

3. Experimental

3.1. General methods

1 mL of sample (conc 20 mM) dissolved in 1:1 MeCNÁ

/

water with 0.1% formic acid. Pentafluorophenylalanine

was added as internal standard. The capillary voltage

was set at 1500 V and the cone voltage was set at 30 V.

All chemicals were of reagent grade, and were used

without further purification. Reactions were monitored

by TLC on Silica Gel 60 F₂₅₄(E. Merck); after

examination under UV light, compounds were visua-

lized by heating with 10% (v/v) ethanolic H₂SO₄, orcinol

(2 mg/mL) in 20% (v/v) methanolic H₂SO₄, or ninhydrin

3.2. Allyl (2,3,4,6-tetra-O-acetyl-b-

(106)-2-deoxy-3,4-di-O-p-methylbenzoyl-2-

phthalimido-b- -glucopyranoside (11)

D-glucopyranosyl)-

/

(1.5 mg/mL) in 38:1.75:0.25 1-BuOHÁ

/

waterÁ/AcOH. In

D

the work-up procedures of reaction mixtures, organic

solutions were washed with appropriate amounts of the

indicated aqueous solutions, then dried (MgSO₄or

Na₂SO₄), and concentrated under diminished pressure

A soln of allyl 2-deoxy-3,4-di-O-p-methylbenzoyl-2-

phthalimido-b-

-glucopyranoside (10)¹¹(0.15 g, 0.26

mmol) and 2,3,4,6-tetra-O-acetyl-a- -glucopyranosyl

D

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2637

Scheme 6. Synthesis of the linear trisaccharide backbone 7: (a) 10% Pdꢀ/C, H₂, EtOH, EtOAc, AcOH; (b) pyridine, Ac₂O, 66% over

two steps; (c) hydrazinium acetate, DMF, 89%; (d) Cl₃CCN, DBU, CH₂Cl₂, 69%; (e) 1 equiv AgOTf, CH₂Cl₂, 0 8C, 22%; (f)

NaOMe (pH 10), MeOH, CH₂Cl₂; (g) NH₂CH₂CH₂NH₂, 1-BuOH, 80 8C; (h) pyridine, Ac₂O, 77% over three steps; (i) NaOMe (pH

10), MeOH, CH₂Cl₂, 55%; (j) 10% Pdꢀ

/

C, H₂, water, tert-BuOH, aq 25% NH₃, 83%; (k) 10% Pdꢀ

/

C, H₂, water, tert-BuOH, aq 25%

NH₃/10% PdꢀC, AcOH, 68%; (l) NaOMe (pH 10), MeOH, CH₂Cl₂; (m) NH₂CH₂CH₂NH₂, 1-BuOH, 80 8C; (n) pyridine, Ac₂O,

/

78% over three steps; (o) NaOMe (pH 10), MeOH, CH₂Cl₂, 99%.

trichloroacetimidate (9)¹⁶(0.16 g, 0.32 mmol) in dry

˚

CH₂Cl₂(7.5 mL), containing 4 A molecular sieves (0.1

ROESY): d 2.00, 2.01, and 2.08 (3 s, 6,3,3 H, 4 COCH₃),

2.23 and 2.31 (2 s, each 3 H, 2 COC₆H₄CH₃), 3.71 (m, 1

H, H-5^II), 3.83 (dd, 1 H, J_5,6b7.9, J_6a,6b10.8 Hz, H-6b^I),

4.23 (dd, 1 H, J_5,6b4.6, J_6a,6b12.1 Hz, H-6b^II), 4.36 (m, 1

g), was stirred under Ar for 0.5 h. After cooling to 0 8C,

AgOTf (77 mg, 0.3 mmol) was added and the mixture

was stirred for 1.5 h. After filtration, the soln was

washed with aq 10% NaHSO₃, aq satd NaHCO₃, and

water, dried (MgSO₄), filtered, and concentrated. Low-

H, OCHHCHꢁ

Hz, H-2^I), 4.69 (d, 1 H, J_1,27.9 Hz, H-1^II), 5.39 (t, 1 H,

H-4^I), 5.57 (d, 1 H, H-1^I), 5.77 (m, 1 H, OCH₂CH ꢁ

CH₂), 6.22 (dd, 1 H, J_3,49.4 Hz, H-3^I), 7.02 and 7.14

(2 d, each 2 H, Phth), 7.61Á7.80 (2 m, 8 H, 2

COC₆H₄CH₃); ¹³C NMR (75.5 MHz, CDCl₃): d 20.2Á

20.4 (COCH₃), 21.2 and 21.3 (2 COC₆H₄CH₃), 54.6 (C-

/

CH₂), 4.54 (dd, 1 H, J_1,28.4, J_2,310.5

/

pressure column chromatography (10:10

EtOAc) of the residue gave 11, isolated as a white foam

(0.14 g, 62%); R_f0.65 (1:1 tolueneÁ 48 (c

EtOAc); [a]²_D⁰

1, CHCl₃); H NMR (500 MHz, CDCl₃; 2D TOCSY,

/

4:1 tolueneÁ

/

ꢁ

/

1

2638

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

Table 5

500 MHz ¹H NMR data (TOCSY, ROESY) of 40 at 300 K (in

ppm)

3.3. (2,3,4,6-Tetra-O-acetyl-b-

6)-2-deoxy-3,4-di-O-p-methylbenzoyl-2-phthalimido-a,b-

-glucopyranose (12)

D

-glucopyranosyl)-(10

/

D

Proton

d_H

To a soln of 11 (0.41 g, 0.45 mmol) in AcOH (15 mL)

were added Pd(II)Cl₂(0.36 g, 2.03 mmol) and NaOAc

(0.31 g, 3.78 mmol), and the mixture was kept overnight

in an ultrasonic bath. After filtration over hyflo, the soln

was diluted with CH₂Cl₂then washed with water, aq

satd NaHCO₃, and water, dried (MgSO₄), filtered, and

concentrated. Low-pressure column chromatography

Glc I Gal II GlcNAc III

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.48 4.43

3.29 3.60

3.65 3.72

n.d. ^a4.15

3.60 n.d.

3.97 n.d.

3.80 n.d.

4.68

3.76

3.58

3.47

3.89

3.76

(4:10

isolated as a slightly yellow foam (0.27 g, 70%); R_f

0.45 (1:1 tolueneÁ

EtOAc); ¹H NMR b-product (300

/

1:1 tolueneÁEtOAc) of the residue gave 12,

/

O(CH₂)₂(CH₂)₂(CH₂)₂N₃

OCH₂CH₂(CH₂)₂CH₂CH₂N₃

CH₂N₃

1.39Á

1.63Á

3.32

3.62, 3.92

2.04

/

1.42 (4 H)

1.65 (4 H)

MHz, CDCl₃): d 2.02, 2.04, 2.05, and 2.12 (4 s, each 3

H, 4 COCH₃), 2.27 and 2.34 (2 s, each 3 H, 2

COC₆H₄CH₃), 4.02 (dd, 1 H, J_5,6a1.9, J_6a,6b11.2 Hz,

H-6a^I), 4.20 (dd, 1 H, J_5,6b4.7, J_6a,6b12.2 Hz, H-6b^II),

4.43 (dd, 1 H, J_1,28.4, J_2,310.7 Hz, H-2^I), 4.65 (d, 1 H,

J_1,27.5 Hz, H-1^II), 4.96 (dd, 1 H, J_2,38.6 Hz, H-2^II), 5.42

(t, 1 H, H-4^I), 5.74 (d, 1 H, H-1^I), 6.23 (dd, 1 H, J_3,49.2

/

OCH₂(CH₂)₅N₃

NDCOCH₃

a

n.d., not determined.

Hz, H-3^I), 7.04 and 7.15 (2 d, each 2 H, Phth), 7.60Á

/

7.81

(2 m, 8 H, 2 COC₆H₄CH₃); ¹³C NMR (75.5 MHz,

2^I), 53.2, 61.6, and 69.8 (C-6^I, C-6^II, OCH₂CHꢁ

/

CH₂),

68.1, 69.9, 70.7, 71.0, 71.6, 72.6, and 73.6 (C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II), 96.9 and 100.5 (C-1^I, C-1^II),

CDCl₃): d 20.3Á20.4 (COCH₃), 21.2 and 21.3 (2

/

COC₆H₄CH₃), 56.1 (C-2^I), 61.7 and 68.3 (C-6^I, C-6^II),

68.1, 69.7, 70.7, 71.2, 71.5, 72.5, and 73.3 (C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II), 92.4 and 100.5 (C-1^I, C-1^II),

117.5 (OCH₂CHꢁ

COC₆H₄CH₃), 168.9Á

C₄₇H₄₉NO₁₈(M, 915.295): [Mꢀ

/

CH₂), 165.0 and 165.2 (2

170.2 (COCH₃); HRMS data of

NH₄]^ꢀfound 933.335,

/

165.0 and 165.2 (2 COC₆H₄CH₃), 169.2Á

(COCH₃); HRMS of C₄₄H₄₅NO₁₈(M, 875.263): [Mꢀ

H]^ꢀfound 876.273, calcd 876.271.

/170.4

calcd 933.329.

/

3.4. (2,3,4,6-Tetra-O-acetyl-b-

6)-2-deoxy-3,4-di-O-p-methylbenzoyl-2-phthalimido-a,b-

-glucopyranosyl trichloroacetimidate (13)

D

-glucopyranosyl)-(10

/

D

Table 6

500 MHz H NMR data (TOCSY, ROESY) of 7 at 300 K (in

ppm)

1

To a soln of 12 (0.37 g, 0.42 mmol) in dry CH₂Cl₂(15

mL) were added, at 0 8C, trichloroacetonitrile (0.2 mL,

2.1 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (60

mL, 0.041 mmol), and the mixture was stirred for 1.5 h,

then concentrated. Column chromatography (2:1

Proton

d_H

Glc Gal II

I

GlcNAc

III

tolueneÁEtOAc) of the residue gave 13, isolated as a

/

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.47 4.43

3.29 3.59

3.64 3.73

3.64 4.15

3.59 n.d. ^a

3.98 n.d.

3.80 n.d.

4.68

3.74

3.56

3.46

3.90

3.77

slightly yellow foam (0.33 g, 76%); R_f0.64 (1:1 tolueneÁ

/

EtOAc); ¹H NMR b-product (300 MHz, CDCl₃): d

1.99, 2.03, and 2.09 (3 s, 6,3,3 H, 4 COCH₃), 2.27 and

2.34 (2 s, each 3 H, 2 COC₆H₄CH₃), 3.62 (m, 1 H, H-

5^II), 3.86 (dd, 1 H, J_5,6b6.8, J_6a,6b11.8 Hz, H-6b^I), 4.70

(d, 1 H, J_1,27.8 Hz, H-1^II), 5.15 (t, 1 H, H-3^II), 5.50 (t, 1

H, H-4^I), 6.32 (t, 1 H, H-3^I), 6.79 (d, 1 H, J_1,28.8 Hz, H-

1^I), 7.05 and 7.15 (2 d, each 2 H, Phth), 7.63Á

/

7.81 (2 m,

8 H, 2 COC₆H₄CH₃), 8.78 (s, 1 H, NH); ¹³C NMR (75.5

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

1.39Á

(4 H)

/

1.41

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

1.63Á

(4 H)

2.95

/1.65

MHz, CDCl₃): d 20.4Á20.5 (COCH₃), 21.3 and 21.4 (2

/

COC₆H₄CH₃), 53.7 (C-2^I), 61.7 and 67.3 (C-6^I, C-6^II),

68.2, 69.3, 70.5, 70.9, 71.6, 72.7, and 75.0 (C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II), 93.4 and 100.2 (C-1^I, C-1^II),

CH₂ND₂

OCH₂(CH₂)₅ND₂

NDCOCH₃

3.58, 3.83

2.03

160.2

(OC(NH)CCl₃),

165.0

170.3 (COCH₃).

and

165.3

(2

a

n.d., not determined.

COC₆H₄CH₃), 169.1Á

/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2639

Scheme 7. Synthesis of tetrasaccharide 3: (a) 1.5 equiv UDP-Gal, aq 50 mM sodium cacodylate buffer (pH 7.5), 2.5 U b-1,4-

galactosyltransferase, 14 U alkaline phosphatase, 37 8C, 94%.

3.5. 6-Azidohexyl (3,4,6-tri-O-acetyl-2-deoxy-2-

phthalimido-b- 3)-4-O-acetyl-2,6-

-glucopyranosyl)-(10

di-O-benzyl-b- -galactopyranoside (16)

Low-pressure column chromatography (10:10

tolueneÁEtOAc) of the residue gave 16, isolated as a

white foam (1.28 g, 86%); R_f0.44 (2:1 tolueneÁEtOAc);

[a]²_D⁰38 (c 1, CHCl₃); ¹H NMR (300 MHz, CDCl₃): d

1.22Á1.24 (m, 4 H, 2 CH₂), 1.37Á1.42 (m, 2 H, CH₂),

1.48Á1.52 (m, 2 H, CH₂), 1.82, 2.00, 2.03, and 2.08 (4 s,

/

4:1

D

/

D

/

ꢀ

/

A soln of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-

D-

/

glucopyranosyl trichloroacetimidate (15)¹⁷(1.09 g, 1.88

/

mmol) and 6-azidohexyl 4-O-acetyl-2,6-di-O-benzyl-b-

D

each 3 H, 4 COCH₃), 3.07 (t, 2 H, CH₂N₃), 3.84 (m, 1 H,

OCHH), 4.48 (d, 2 H, OCH₂C₆H₅), 4.56 (d, 1 H,

OCHHC₆H₅), 5.16 (t, 1 H, H-4^II), 5.38 (d, 1 H, J_3,43.5,

-galactopyranoside (14)¹⁴(0.84 g, 1.47 mmol) in dry

˚

CH₂Cl₂(50 mL), containing 4 A molecular sieves (1 g),

J_4,5

(dd, 1 H, J_2,310.7, J_3,49.1 Hz, H-3^II); ¹³C NMR (75.5

20.5 (COCH₃), 25.3, 26.2, 28.4,

B

/

1 Hz, H-4^I), 5.59 (d, 1 H, J_1,28.3 Hz, H-1^II), 5.77

was stirred under Ar for 1 h. After cooling to ꢁ70 8C,

/

TMSOTf (34 mL, 0.19 mmol) was added and the mixture

was stirred for 2 h, during which period the temperature

was allowed to reach room temperature (rt). The

mixture was neutralized with Et₃N, filtered, washed

with water, dried (MgSO₄), filtered, and concentrated.

MHz, CDCl₃): d 20.2Á

/

and 29.2 (4 CH₂), 51.0 (CH₂N₃), 54.7 (C-2^II), 61.2, 68.9,

69.8, 73.4, and 73.9 (C-6^I, C-6^II, 2 OCH₂C₆H₅, OCH₂),

68.6, 69.5, 70.4, 71.6, 72.6, 78.1, and 78.5 (C-2^I, C-3^I, C-

Table 7

500 MHz H NMR data (TOCSY, ROESY) of 3 at 300 K (in ppm)

1

Proton

d_H

Glc I

Gal II ^a

GlcNAc III

Gal IV ^b

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.48

3.29

3.63

3.60

3.62

3.96

3.78

4.44

3.58

3.72

4.15

n.d. ^c

n.d.

4.71

3.78

3.73

3.58

3.93

3.85

4.48

3.54

3.67

3.93

n.d.

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

CH₂ND₂

1.39Á

1.63Á

2.99

/

1.42 (4 H)

/1.70 (4 H)

OCH₂(CH₂)₅ND₂

NDCOCH₃

3.68, 3.92

2.03

a

Gal(b1-4)Glc.

Gal(b1-4)GlcNAc.

n.d., not determined.

b

c

2640

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

Scheme 8. Synthesis of the linear tetrasaccharide backbone 8: (a) 15% TMSOTf, CH₂Cl₂, 0 8C, 34%; (b) NaOMe (pH 10), MeOH,

CH₂Cl₂; (c) NH₂CH₂CH₂NH₂, 1-BuOH, 90 8C; (d) pyridine, Ac₂O, 86% over three steps; (e) NaOMe (pH 10), MeOH, CH₂Cl₂,

79%; (f) 10% PdÁC, H₂, water, tert-BuOH, aq 25% NH₃, 82%.

/

4^I, C-5^I, C-3^II, C-4^II, C-5^II), 97.9 and 103.3 (C-1^I, C-1^II),

169.1Á170.4 (COCH₃); HRMS of C₄₈H₅₆N₄O₁₆(M,

944.369): [Mꢀ

NH₄]^ꢀfound 962.399, calcd 962.403.

mixture was stirred for 3 h, then neutralized with Dowex

50ꢂ

8 (H^ꢀ), filtered, and concentrated. Column chro-

matography (1:2 tolueneÁEtOAc) of the residue gave

17, isolated as a colourless glass (1.01 g, 91%); R_f0.36

/

1

(1:3 tolueneÁ

(500 MHz, CDCl₃; 2D TOCSY, ROESY): d 1.13Á

(m, 4 H, 2 CH₂), 1.31Á1.37 (m, 2 H, CH₂), 1.39Á

/

EtOAc); [a]²_D⁰

ꢀ58 (c 1, CHCl₃); H NMR

/

3.6. 6-Azidohexyl (2-deoxy-2-phthalimido-b-

glucopyranosyl)-(103)-4-O-acetyl-2,6-di-O-benzyl-b-

galactopyranoside (17)

D-

/

1.17

/

D-

/

1.44

(m, 2 H, CH₂), 2.06 (COCH₃), 3.06 (t, 2 H, CH₂N₃),

4.00 (dd, 1 H, J_1,28.5, J_2,310.5 Hz, H-2^II), 4.21 (t, 1 H,

H-3^II), 4.24 (d, 1 H, J_1,27.7 Hz, H-1^I), 4.10 and 4.41 (2

To a soln of 16 (1.28 g, 1.35 mmol) in CH₂Cl₂(5 mL)

and MeOH (2 mL) was added NaOMe (pH 8). The

Scheme 9. Synthesis of hexasaccharide 4: (a) 3.2 equiv UDP-Gal, aq 50 mM sodium cacodylate buffer (pH 7.5), 5 U b-1,4-

galactosyltransferase, 30 U alkaline phosphatase, 37 8C, 76%.

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2641

Table 8

500 MHz H NMR data (TOCSY, ROESY) of 46 at 300 K (in ppm)

1

Proton

d_H

GlcNAc I ^a

Glc II

Gal III

GlcNAc IV ^b

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.52

3.69

3.54

n.d. ^c

3.62

4.22

3.89

4.56

3.38

3.65

3.62

3.99

3.81

4.44

3.59

3.72

4.16

n.d.

4.69

3.56

3.59

n.d.

3.48

3.90

3.77

O(CH₂)₂(CH₂)₂(CH₂)₂N₃

OCH₂CH₂(CH₂)₂CH₂CH₂N₃

CH₂N₃

1.35Á

1.57Á

3.33

/

1.37 (4 H)

/1.61 (4 H)

OCH₂(CH₂)₅N₃

NDCOCH₃

3.62, 3.91

2.04 (2)

a

GlcNAc(b1-O(CH₂)₆N₃).

GlcNAc(b1-3)Gal.

n.d., not determined.

b

c

d, each 1 H, OCH₂C₆H₅), 4.46 (s, 2 H, OCH₂C₆H₅),

5.36 (d, 1 H, H-1^II), 5.49 (d, 1 H, J_3,43.3, J_4,5

1 Hz, H-

3.7. 6-Azidohexyl (6-O-tert-butyldiphenylsilyl-2-deoxy-

2-phthalimido-b- 3)-4-O-acetyl-

-glucopyranosyl)-(10

2,6-di-O-benzyl-b- -galactopyranoside (18)

B

/

D

/

4^I); ¹³C NMR (75.5 MHz, CDCl₃): d 20.9 (COCH₃),

25.2, 26.1, 28.3, and 29.1 (4 CH₂), 51.1 (CH₂N₃), 56.6

(C-2^II), 61.3, 68.7, 69.8, 73.4, and 73.8 (C-6^I, C-6^II, 2

OCH₂C₆H₅, OCH₂), 70.4, 70.7, 71.0, 72.1, 75.8, 77.7,

and 79.9 (C-2^I, C-3^I, C-4^I, C-5^I, C-3^II, C-4^II, C-5^II), 99.2

and 103.3 (C-1^I, C-1^II), 171.4 (COCH₃); HRMS of

D

To a soln of 17 (0.18 g, 0.23 mmol) in CH₂Cl₂(5 mL)

and Py (0.4 mL) were added 4-dimethylaminopyridine

(20 mg, 0.18 mmol), Et₃N (70 mL), and tert-butyldiphe-

nylsilyl chloride (70 mL, 0.27 mmol). The mixture was

stirred for 4 h, then poured into ice water, extracted with

CH₂Cl₂, washed with aq satd NaHCO₃, dried (MgSO₄),

C₄₂H₅₀N₄O₁₃(M, 818.337): [Mꢀ

Na]^ꢀfound 841.332,

calcd 841.327.

/

Table 9

500 MHz H NMR data (TOCSY, ROESY) of 8 at 300 K (in ppm)

1

Proton

d_H

GlcNAc I ^a

Glc II Gal III

GlcNAc IV ^b

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.49

3.68

3.53

3.52

3.60

4.21

3.88

4.54

3.38

3.66

3.59

3.97

3.81

4.44

3.60

3.72

4.15

n.d. ^c

n.d.

4.69

3.76

3.57

3.46

3.90

3.77

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

CH₂ND₂

1.32Á

/

1.36 (4 H)

1.52Á

/

1.55 (2 H), 1.62Á1.65 (2 H)

/

2.97

3.56, 3.83

2.02, 2.04

OCH₂(CH₂)₅ND₂

NDCOCH₃

a

GlcNAc(b1-O(CH₂)₆NH₂).

GlcNAc(b1-3)Gal.

n.d., not determined.

b

c

2642

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

Table 10

500 MHz H NMR data (TOCSY, ROESY) of 4 at 300 K (in ppm)

1

Proton

d_H

GlcNAc I ^aGlc II Gal III ^b

GlcNAc IV ^cGal V ^dGal VI ^e

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

4.53

3.72

n.d. ^f

3.83

3.72

4.29

3.95

4.56

3.37

3.67

3.62

4.00

3.83

4.44

3.59

3.72

4.16

n.d.

4.71

4.53

3.55

3.67

3.93

n.d.

4.48

3.55

3.67

3.92

n.d.

3.80

3.73

3.58

3.95

3.85

O(CH₂)₂(CH₂)₂(CH₂)₂ND₂

OCH₂CH₂(CH₂)₂CH₂CH₂ND₂

CH₂ND₂

1.36Á

/

1.38 (4 H)

1.54Á

/

1.57 (2 H), 1.64Á1.67 (2 H)

/

2.99

3.68, 3.92

2.03 (2)

OCH₂(CH₂)₅ND₂

NDCOCH₃

a

GlcNAc(b1-O(CH₂)₆NH₂).

b

Gal(b1-4)Glc.

c

GlcNAc(b1-3)Gal.

d

Gal(b1-4)GlcNAc(b1-O(CH₂)₆NH₂).

Gal(b1-4)GlcNAc(b1-3)Gal.

n.d., not determined.

e

f

filtered, and concentrated. Low-pressure column chro-

matography (5:101:1 tolueneÁEtOAc) of the residue

gave 18, isolated as a colourless glass (0.19 g, 83%); R_f

chloride (60 mL, 0.48 mmol) in dry CH₂Cl₂(1 mL). The

mixture was stirred for 4 h at rt, then poured into ice

water, extracted with CH₂Cl₂, washed with aq satd

NaHCO₃, dried (MgSO₄), filtered, and concentrated.

/

1

0.53 (1:1 tolueneÁ

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY): d 1.05

[s, 9 H, SiC(CH₃)₃], 1.19Á1.22 (m, 4 H, 2 CH₂), 1.37Á

1.40 (m, 2 H, CH₂), 1.46Á1.48 (m, 2 H, CH₂), 1.96 (s, 3

/

EtOAc); [a]²_D⁰

ꢀ

/

98 (c 1, CHCl₃); H

Column chromatography (5:1 tolueneÁ

residue gave 19, isolated as a colourless syrup (0.21 g,

90%); R_f0.39 (5:1 tolueneÁ 18 (c 1,

EtOAc); [a]²_D⁰

CHCl₃); ¹H NMR (500 MHz, CDCl₃; 2D TOCSY,

ROESY): d 1.06 [s, 9 H, SiC(CH₃)₃], 1.23Á1.26 (m, 4 H,

2 CH₂), 1.38Á1.41 (m, 2 H, CH₂), 1.52Á1.54 (m, 2 H,

/EtOAc) of the

/

ꢀ

/

H, COCH₃), 3.07 (t, 2 H, CH₂N₃), 3.33 (dd, 1 H, J_1,2

7.9, J_2,39.8 Hz, H-2^I), 3.37 (m, 1 H, OCHH), 3.78 (dd, 1

H, J_3,43.5 Hz, H-3^I), 3.82 (m, 1 H, OCHH), 4.04 (dd, 1

H, J_1,28.3, J_2,311.0 Hz, H-2^II), 4.17 and 4.53 (2 d, each 1

H, OCH₂C₆H₅), 4.20 (d, 1 H, H-1^I), 4.37 (t, 1 H, H-3^II),

4.38 and 4.43 (2 d, each 1 H, OCH₂C₆H₅), 5.33 (d, 1 H,

/

CH₂), 2.04 (s, 3 H, COCH₃), 2.18 and 2.28 (2 s, each 3

H, 2 COC₆H₄CH₃), 3.07 (t, 2 H, CH₂N₃), 3.64 (t, 1 H,

H-6a^I), 3.83 (d, 1 H, J_6a,6b9.4 Hz, H-6b^II), 3.89 (m, 1 H,

OCHH), 4.00 (dd, 1 H, J_2,39.6, J_3,43.5 Hz, H-3^I), 4.24

and 4.65 (2 d, each 1 H, OCH₂C₆H₅), 4.30 (d, 1 H, J_1,2

7.7 Hz, H-1^I), 4.43 and 4.49 (2 d, each 1 H, OCH₂C₆H₅),

4.53 (dd, 1 H, J_1,28.3, J_2,310.7 Hz, H-2^II), 5.56 (t, 1 H,

J_4,5

B

1 Hz, H-4^I), 5.41 (d, 1 H, H-1^II); ¹³C NMR (75.5

/

MHz, CDCl₃): d 19.0 [SiC(CH₃)₃], 20.5 (COCH₃), 25.3,

26.2, 28.4, and 29.2 (4 CH₂), 26.6 [SiC(CH₃)₃], 51.0

(CH₂N₃), 56.6 (C-2^II), 64.5, 68.9, 69.8, 73.3, and 73.9 (C-

6^I, C-6^II, 2 OCH₂C₆H₅, OCH₂), 70.1, 71.0, 72.5, 73.6,

74.8, 76.4, and 78.6 (C-2^I, C-3^I, C-4^I, C-5^I, C-3^II, C-4^II,

C-5^II), 98.0 and 103.4 (C-1^I, C-1^II), 170.0 (COCH₃);

H-4^II), 5.59 (d, 1 H, J_4,5

B

1 Hz, H-4^I), 5.84 (d, 1 H, H-

/

1^II), 6.31 (dd, 1 H, J_3,49.2 Hz, H-3^II), 6.97 and 7.08 (2 d,

each 2 H, Phth); ¹³C NMR (75.5 MHz, CDCl₃): d 19.1

[SiC(CH₃)₃], 20.5 (COCH₃), 21.4 and 21.5 (2

COC₆H₄CH₃), 25.5, 26.3, 28.5, and 29.4 (4 CH₂), 26.6

[SiC(CH₃)₃], 51.1 (CH₂N₃), 55.5 (C-2^II), 63.0, 69.1, 69.8,

73.4, and 73.9 (C-6^I, C-6^II, 2 OCH₂C₆H₅, OCH₂), 69.9,

70.3, 70.8, 72.8, 75.1, 76.4, and 78.7 (C-2^I, C-3^I, C-4^I, C-

5^I, C-3^II, C-4^II, C-5^II), 98.2 and 103.6 (C-1^I, C-1^II), 164.9

and 165.5 (2 COC₆H₄CH₃), 169.7 (COCH₃); HRMS of

HRMS of C₅₈H₆₈N₄O₁₃Si (M, 1056.455): [Mꢀ

/

NH₄]^ꢀ

found 1074.496, calcd 1074.489.

3.8. 6-Azidohexyl (6-O-tert-butyldiphenylsilyl-2-deoxy-

3,4-di-O-p-methylbenzoyl-2-phthalimido-b-

glucopyranosyl)-(103)-4-O-acetyl-2,6-di-O-benzyl-b-

galactopyranoside (19)

D-

/

D-

C₇₄H₈₀N₄O₁₇Si (M, 1292.538): [Mꢀ

/

NH₄]^ꢀfound

To a soln of 18 (0.19 g, 0.19 mmol) in dry Py (4 mL) was

added dropwise, at 0 8C, a soln of p-methylbenzoyl

1310.584, calcd 1310.573.

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2643

3.9. 6-Azidohexyl (2-deoxy-3,4-di-O-p-methylbenzoyl-2-

phthalimido-b- 3)-4-O-acetyl-2,6-

-glucopyranosyl)-(10

di-O-benzyl-b- -galactopyranoside (20)

was allowed to reach rt, then neutralized with Et₃N,

filtered, and concentrated. Column chromatography

D

/

D

(4:1 tolueneÁ

/

EtOAc) of the residue gave 21, isolated

as a white foam (0.13 g, 73%); R_f0.54 (2:1 tolueneÁ

/

1

To a soln of acetyl chloride (0.80 mL, 9.70 mmol) in dry

MeOH (15 mL) was added, at 0 8C, a soln of 19 (0.88 g,

0.71 mmol) in dry toluene (3 mL). The mixture was

stirred for 4 h at 0 8C, then co-concentrated with

EtOAc); [a]²_D⁰

CDCl₃): d 1.23Á

H, CH₂), 1.47Á1.52 (m, 2 H, CH₂), 1.95, 1.98, 2.02, 2.05,

ꢀ

/

88 (c 1, CHCl₃); H NMR (300 MHz,

/

1.25 (m, 4 H, 2 CH₂), 1.39Á1.43 (m, 2

/

and 2.09 (5 s, each 3 H, 5 COCH₃), 2.27 and 2.34 (2 s,

each 3 H, 2 COC₆H₄CH₃), 3.11 (t, 2 H, CH₂N₃), 3.38

(dd, 1 H, J_1,27.8, J_2,39.5 Hz, H-2^I), 3.42 (m, 1 H,

OCHH), 4.07 (dd, 1 H, J_5,6b4.4, J_6a,6b12.3 Hz, H-6b^III),

4.10 and 4.55 (2 d, each 1 H, OCH₂C₆H₅), 4.36 (d, 1 H,

H-1^I), 4.44 (dd, 1 H, J_1,28.2, J_2,310.8 Hz, H-2^II), 4.47

and 4.56 (2 d, each 1 H, OCH₂C₆H₅), 4.70 (d, 1 H, J_1,2

8.0 Hz, H-1^III), 4.95 (dd, 1 H, J_2,39.6 Hz, H-2^III), 5.01 (t,

1 H, H-4^III), 5.21 (t, 1 H, H-3^III), 5.36 (t, 1 H, H-4^II),

toluene. Column chromatography (4:1 tolueneÁ

of the residue gave 20, isolated as a colourless syrup

(0.61 g, 81%); R_f0.44 (2:1 tolueneÁ 98 (c

EtOAc); [a]²_D⁰

1, CHCl₃); H NMR (500 MHz, CDCl₃; 2D TOCSY,

ROESY): d 1.12Á1.17 (m, 4 H, 2 CH₂), 1.32Á1.35 (m, 2

H, CH₂), 1.40Á1.44 (m, 2 H, CH₂), 2.18 (s, 3 H,

/EtOAc)

/

ꢀ

/

1

/

COCH₃), 2.26 and 2.33 (2 s, each 3 H, 2 COC₆H₄CH₃),

3.05 (CH₂N₃), 3.34 (m, 1 H, OCHH), 3.49 (dd, 1 H, J_1,2

7.7, J_2,39.4 Hz, H-2^I), 3.51 (d, 2 H, J_6a,6b5.8 Hz, H-6a^I,

H-6b^I), 3.82 (m, 1 H, OCHH), 3.90 (m, 1 H, H-5^II), 4.21

and 4.45 (2 d, each 1 H, OCH₂C₆H₅), 4.27 (d, 1 H, H-

1^I), 4.48 (s, 2 H, OCH₂C₆H₅), 4.51 (dd, 1 H, J_1,28.3, J_2,3

10.7 Hz, H-2^II), 5.43 (t, 1 H, H-4^II), 5.60 (d, 1 H, J_3,43.5,

5.55 (d, 1 H, J_3,43.5, J_4,5

1^II), 6.24 (dd, 1 H, J_3,49.1 Hz, H-3^II); ¹³C NMR (75.5

MHz, CDCl₃): d 20.4Á20.8 (COCH₃), 21.4 and 21.5 (2

B

/

1 Hz, H-4^I), 5.68 (d, 1 H, H-

/

COC₆H₄CH₃), 25.5, 26.3, 28.5, and 29.3 (4 CH₂), 51.2

(CH₂N₃), 55.3 (C-2^II), 61.5, 68.0 (2 C), 69.9, 73.4, and

73.9 (C-6^I, C-6^II, C-6^III, 2 OCH₂C₆H₅, OCH₂), 67.9,

69.7, 69.9, 70.3, 71.5, 71.6, 71.8, 72.7, 74.5, 77.2, and

78.5 (C-2^I, C-3^I, C-4^I, C-5^I, C-3^II, C-4^II, C-5^II, C-2^III, C-

3^III, C-4^III, C-5^III), 98.4, 100.9, and 103.6 (C-1^I, C-1^II, C-

J_4,5

B

1 Hz, H-4^I), 5.72 (d, 1 H, H-1^II), 6.13 (dd, 1 H, J_3,4

/

9.2 Hz, H-3^II); ¹³C NMR (75.5 MHz, CDCl₃): d 21.0

(COCH₃), 21.4 and 21.5 (2 COC₆H₄CH₃), 25.3, 26.2,

28.4, and 29.2 (4 CH₂), 51.1 (CH₂N₃), 55.0 (C-2^II), 61.5,

70.0, 73.6, 68.8, and 73.9 (C-6^I, C-6^II, 2 OCH₂C₆H₅,

OCH₂), 69.3, 70.1, 70.8, 72.4, 75.2, 77.5, and 81.5 (C-2^I,

C-3^I, C-4^I, C-5^I, C-3^II, C-4^II, C-5^II), 99.4 and 103.5 (C-

1^I, C-1^II), 165.2 and 165.4 (2 COC₆H₄CH₃), 171.4

(COCH₃); HRMS of C₅₈H₆₂N₄O₁₅(M, 1054.421):

1^III), 165.1 and 165.4 (2 COC₆H₄CH₃), 169.1Á

170.5

/

(COCH₃); HRMS of C₇₂H₈₀N₄O₂₄(M, 1384.516): [Mꢀ

/

Na]^ꢀfound 1407.495, calcd 1407.506.

3.11. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-D-

[Mꢀ

/

NH₄]^ꢀfound 1072.461, calcd 1072.455.

glucopyranosyl)-(10

deoxy-b-

-glucopyranosyl)-(10

benzyl-b- -galactopyranoside (22)

/

6)-(2-acetamido-3,4-di-O-acetyl-2-

D

/

3)-4-O-acetyl-2,6-di-O-

3.10. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-

glucopyranosyl)-(106)-(2-deoxy-3,4-di-O-p-

methylbenzoyl-2-phthalimido-b-

-glucopyranosyl)-(10

-galactopyranoside

D-

D

/

D

/

To a soln of 21 (90 mg, 64.9 mmol) in MeOH (8 mL) and

CH₂Cl₂(2 mL) was added NaOMe (pH 10), and the

mixture was stirred for 4 h, then neutralized with Dowex

50ꢂ

8 (H^ꢀ), filtered, and concentrated. To a soln of the

3)-4-O-acetyl-2,6-di-O-benzyl-b-

D

(21)

/

(a) A soln of 2,3,4,6-tetra-O-acetyl-a-

D-glucopyranosyl

trichloroacetimidate (9)¹⁶(0.40 g, 0.82 mmol) and 20

(0.48 g, 0.45 mmol) in dry CH₂Cl₂(20 mL), containing 4

residue in 1-BuOH (30 mL) was added 1,2-diami-

noethane (6 mL), and the mixture was stirred overnight

at 80 8C, then co-concentrated with toluene, EtOH, and

CH₂Cl₂. A soln of the residue in Py (30 mL) and Ac₂O

(30 mL) was stirred overnight, then co-concentrated

with toluene, EtOH and CH₂Cl₂. Column chromato-

˚

A molecular sieves (0.5 g), was stirred under Ar for 0.5

h. After cooling to ꢁ40 8C, TMSOTf (14 mL, 0.077

/

mmol) was added, and the mixture was stirred for 2.5 h,

during which period the temperature was allowed to

reach rt, then neutralized with Et₃N, filtered, and

graphy (1:2 tolueneÁ

isolated as a colourless syrup (60 mg, 80%); R_f0.27 (1:2

tolueneÁ

EtOAc); [a]²_D⁰88 (c 1, CHCl₃); ¹H NMR (500

MHz, CDCl₃; 2D TOCSY, ROESY): d 1.32Á1.39 (m, 4

H, 2 CH₂), 1.47Á1.54 (m, 2 H, CH₂), 1.59Á1.63 (m, 2 H,

/EtOAc) of the residue gave 22,

concentrated. Column chromatography (4:1 tolueneÁ

EtOAc) of the residue gave 21, isolated as white foam

(0.35 g, 56%).

/

ꢁ

/

(b) A soln of 13 (0.13 g, 0.13 mmol) and 6-azidohexyl

4-O-acetyl-2,6-di-O-benzyl-b-

CH₂), 1.59, 1.95, 1.96, 1.99, 2.06 and 2.07 (6 s,

3,3,3,6,3,6 H, 7 COCH₃, NHCOCH₃), 3.18 (t, 2 H,

CH₂N₃), 3.63 (m, 1 H, H-5^III), 3.71 (dd, 1 H, J_5,6b6.8,

J_6a,6b12.3 Hz, H-6b^II), 3.80 (dd, 1 H, J_5,6a1.5 Hz, H-

6a^II), 4.19 (dd, 1 H, J_5,6b4.2, J_6a,6b12.5 Hz, H-6b^III),

4.43 (d, 1 H, J_1,28.1 Hz, H-1^III), 4.47 and 4.56 (2 d, each

1 H, OCH₂C₆H₅), 4.55 and 5.04 (2 d, each 1 H,

D-galactopyranoside

(14)¹⁴(56 mg, 0.11 mmol) in dry CH₂Cl₂(3 mL),

˚

containing 4 A molecular sieves (0.1 g), was stirred

under Ar for 0.5 h. After cooling to ꢁ70 8C, TMSOTf

/

(2.2 mL, 0.013 mmol) was added, and the mixture was

stirred for 2.5 h, during which period the temperature

2644

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

OCH₂C₆H₅), 4.65 (d, 1 H, J_1,28.1 Hz, H-1^I), 4.73 (d, 1

H, J_1,28.3 Hz, H-1^II), 4.83 (t, 1 H, H-4^II), 4.89 (t, 1 H,

H-2^II), 4.99 (t, 1 H, H-2^III), 5.09 (t, 1 H, H-4^III), 5.25 (t, 1

syltransferase (3 U). The mixture was incubated for 20 h

at 37 8C, then water (100 mL) was added. UDP-galactose

was removed using a Dowex 1ꢂ

8 (Cl^ꢁ) column with

/

H, H-3^III), 5.47 (d, 1 H, J_3,43.5, J_4,5

B

/

1 Hz, H-4^I); ¹³C

20.7 (COCH₃), 22.8

water as eluent. The eluate was concentrated, and the

residue applied to a Bio-Gel P-2 column eluted with aq

0.1 M NH₄HCO₃at a flow rate of 40 mL/h. The

appropriate fractions were freeze-dried to give 1 (7.2 mg,

NMR (75.5 MHz, CDCl₃): d 20.4Á

/

(NHCOCH₃), 25.5, 26.3, 28.6, and 29.4 (4 CH₂), 51.2

(CH₂N₃), 54.1 (C-2^II), 61.5, 68.2 (2 C), 69.8, 73.4, and

74.4 (C-6^I, C-6^II, C-6^III, 2 OCH₂C₆H₅, OCH₂), 68.0,

69.0, 69.5, 71.6 (2 C), 72.1, 72.4, 72.6, 73.8, 76.9, and

79.9 (C-2^I, C-3^I, C-4^I, C-5^I, C-3^II, C-4^II, C-5^II, C-2^III, C-

3^III, C-4^III, C-5^III), 100.6, 101.2, and 103.4 (C-1^I, C-1^II,

54%) and 47 (5.2 mg, 35%); R_f0.20 (2:1:1 AcOHÁ

BuOHÁ 38 (c 0.5, water); HRMS of

water); [a]²_D⁰

C₃₂H₅₈N₂O₂₁(M, 806.353): [Mꢀ

H]^ꢀfound 807.352,

/1-

/

ꢁ

/

1

calcd 807.361. For H NMR data, see Table 2.

C-1^III), 169.1Á

(M, 1144.458): [Mꢀ

1162.492.

/

170.6 (COCH₃); HRMS of C₅₄H₇₂N₄O₂₃

/

NH₄]^ꢀfound 1162.489, calcd

3.14. 6-Azidohexyl (3,4,6-tri-O-acetyl-2-deoxy-2-

phthalimido-b-

di-O-benzyl-b-

benzyl-b- -glucopyranoside (25)

D

-glucopyranosyl)-(10

/

3)-(4-O-acetyl-2,6-

4)-2,3,6-tri-O-

D

-galactopyranosyl)-(10

/

3.12. 6-Aminohexyl b-

acetamido-2-deoxy-b-

galactopyranoside (5)

D

-glucopyranosyl-(10

/

6)-2-

D

D-glucopyranosyl-(103)-b-

/

D-

A soln of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-

D-

glucopyranosyl trichloroacetimidate (15)¹⁷(0.48 g, 0.77

To a soln of 22 (50 mg, 43.6 mmol) in CH₂Cl₂(1 mL)

and MeOH (1 mL) was added NaOMe (pH 9). The

mixture was stirred for 3 h, then neutralized with Dowex

mmol) and 6-azidohexyl (4-O-acetyl-2,6-di-O-benzyl-b-

D

-galactopyranosyl)-(10

/

4)-2,3,6-tri-O-benzyl-b-

D-glu-

50ꢂ

as a white solid (36 mg). To a soln of 23 in tert-BuOH (8

C (100 mg)

/

8 (H^ꢀ), filtered, and concentrated, giving crude 23

copyranoside (24)¹⁴(0.62 g, 0.64 mmol) in dry CH₂Cl₂

˚

(15 mL), containing 4 A powdered molecular sieves (0.5

g), was stirred under Ar for 0.5 h. After cooling to

mL) and water (8 mL) were added 10% Pdꢀ

/

and 3 drops of aq 25% NH₃. The mixture was stirred for

3 h under H₂after which NH₃was removed by bubbling

ꢁ70 8C, TMSOTf (14 mL, 0.077 mmol) was added, and

/

the mixture was stirred for 2.5 h, during which period

the temperature was allowed to reach rt, then neutra-

lized with Et₃N, filtered over hyflo, washed with water,

dried (MgSO₄), filtered, and concentrated. Column

with N₂, then 10% Pdꢀ

/

C (60 mg) and 3 drops of AcOH

were added, and the stirring under H₂was continued

overnight. The mixture was loaded on a short Dowex

50ꢂ

8 (H^ꢀ) column, which was first eluted with water

to remove contaminations, then with aq 10% NH₄OH to

/

chromatography (2:1 tolueneÁ/EtOAc) of the residue

gave 25, isolated as a colourless foam (0.87 g, 97%); R_f

1

0.50 (2:1 tolueneÁ

NMR (300 MHz, CDCl₃): d 1.33Á

1.51Á1.54 (m, 2 H, CH₂), 1.58Á1.60 (m, 2 H, CH₂), 1.82,

/

EtOAc); [a]²_D⁰

ꢁ

/

28 (c 1, CHCl₃); H

give 5, isolated as a white solid after lyophilization (19

water); [a]²_D⁰

mg, 69%); R_f0.34 (2:1:1 AcOHÁ

/

1-BuOHÁ

/

/1.37 (m, 4 H, 2 CH₂),

ꢁ

/

28 (c 1, water); ¹³C NMR (75.5 MHz, D₂O): d 22.9

/

(NDCOCH₃), 25.3, 26.0, 27.9, and 29.2 (4 CH₂), 40.3

(CH₂ND₂), 56.4 (C-2^II), 61.5, 61.7, 69.4, and 71.1 (C-6^I,

C-6^II, C-6^III, OCH₂), 69.0, 70.4 (2 C), 70.5, 73.8, 74.3,

75.4 (2 C), 76.5, 76.7, and 83.1 (C-2^I, C-3^I, C-4^I, C-5^I, C-

3^II, C-4^II, C-5^II, C-2^III, C-3^III, C-4^III, C-5^III), 103.3,

103.5, and 103.7 (C-1^I, C-1^II, C-1^III), 175.7

2.02, and 2.06 (3 s, 3,6,3 H, 4 COCH₃), 3.03 (m, 1 H, H-

5^I), 3.18 (t, 2 H, CH₂N₃), 3.49 (dd, 1 H, J_5,6a4.1, J_6a,6b

11.0 Hz, H-6a^I), 3.58 (dd, 1 H, J_2,39.5, J_3,43.5 Hz, H-

3^II), 3.87 (t, 1 H, H-4^I), 4.15 (d, 1 H, OCHHC₆H₅), 4.41

(d, 1 H, OCHHC₆H₅), 4.48 (d, 1 H, OCHHC₆H₅), 4.65

and 4.88 (2 d, each 1 H, OCH₂C₆H₅), 4.67 and 4.81 (2 d,

each 1 H, OCH₂C₆H₅), 5.16 (t, 1 H, H-4^III), 5.38 (d, 1 H,

(NDCOCH₃); HRMS of C₂₆H₄₈N₂O₁₆(M, 644.300):

1

[Mꢀ

/

H]^ꢀfound 645.306, calcd 645.308. For H NMR

J_4,5

(dd, 1 H, J_2,310.7, J_3,49.2 Hz, H-3^III); ¹³C NMR (75.5

MHz, CDCl₃): d 20.0Á20.3 (COCH₃), 25.3, 26.1, 28.4,

B

/

1 Hz, H-4^II), 5.53 (d, 1 H, J_1,28.3 Hz, H-1^III), 5.78

data, see Table 1.

/

and 29.2 (4 CH₂), 50.9 (CH₂N₃), 54.6 (C-2^III), 61.3, 67.4,

67.9, 69.3, 72.2, 73.1, 74.0, 74.5, and 74.7 (C-6^I, C-6^II, C-

6^III, 5 OCH₂C₆H₅, OCH₂), 68.6, 69.5, 70.2, 71.4, 72.2,

74.3, 75.3, 78.4, 78.9, 81.2, and 82.2 (C-2^I, C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-4^III, C-5^III), 97.9,

101.4, and 103.1 (C-1^I, C-1^II, C-1^III), 169.0, 169.3, 169.7,

and 170.3 (4 COCH₃); HRMS of C₇₅H₈₄N₄O₂₁(M,

3.13. 6-Aminohexyl b-

D

-glucopyranosyl-(10

4)]-2-acetamido-2-deoxy-b-

3)-b- -galactopyranoside (1)

/

6)-[b-

D

-

galactopyranosyl-(10

/

D

-

glucopyranosyl-(10

/

D

To a soln of 5 (9.9 mg, 15.35 mmol) in aq 50 mM sodium

cacodylate buffer pH 7.5 (700 mL), containing 5 mM

MnCl₂, bovine serum albumin (BSA) (0.5 mg), and

NaN₃(0.02%), were added alkaline phosphatase (14 U),

UDP-galactose (13 mg, 21.3 mmol), and b-1,4-Galacto-

1376.562): [Mꢀ

/

NH₄]^ꢀfound: 1394.548, calcd

1394.597.

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2645

3.15. 6-Azidohexyl (2-deoxy-2-phthalimido-b-

glucopyranosyl)-(103)-(4-O-acetyl-2,6-di-O-benzyl-b-

galactopyranosyl)-(104)-2,3,6-tri-O-benzyl-b-

glucopyranoside (26)

D

-

each 1 H, OCH₂C₆H₅), 4.40 (m, 1 H, H-3^III), 4.43 (d, 1

H, OCHHC₆H₅), 4.62 and 4.87 (2 d, each 1 H,

OCH₂C₆H₅), 4.66 and 4.81 (2 d, each 1 H, OCH₂C₆H₅);

¹³C NMR (75.5 MHz, CDCl₃): d 19.0 [SiC(CH₃)₃], 20.5

(COCH₃), 25.5, 26.3, 28.5, and 29.4 (4 CH₂), 26.6

[SiC(CH₃)₃], 51.1 (CH₂N₃), 56.6 (C-2^III), 64.8 (C-6^III),

67.7 (C-6^I), 68.1 (C-6^II), 69.4 (OCH₂), 70.1 (C-4^II), 70.9

(C-3^III), 72.5 (C-5^II), 73.9 (C-4^III), 74.5 (C-5^III), 74.6 (C-

5^I), 72.9, 73.2, 74.2, 74.7, and 74.9 (5 OCH₂C₆H₅), 75.5

(C-4^I), 77.2 (C-3^II), 79.1 (C-2^II), 81.4 (C-2^I), 82.4 (C-3^I),

98.3 (C-1^III), 101.7 (C-1^II), 103.3 (C-1^I), 169.7 (COCH₃);

/

D-

/

D-

To a soln of 25 (0.85 g, 0.62 mmol) in CH₂Cl₂(10 mL)

and MeOH (20 mL) was added NaOMe (pH 8). The

mixture was stirred for 3 h, then neutralized with Dowex

50ꢂ

matography (3:10

gave 26, isolated as a colourless syrup (0.60 g, 78%); R_f

/

8 (H^ꢀ), filtered, and concentrated. Column chro-

/

1:1 tolueneÁEtOAc) of the residue

/

1

EtOAc); [a]²_D⁰

ꢀ38 (c 1, CHCl₃); H

/

HRMS of C₈₅H₉₆N₄O₁₈(M, 1488.648): [Mꢀ

/

H]^ꢀfound

0.35 (1:3 tolueneÁ

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY): d

1.33Á1.36 (m, 4 H, 2 CH₂), 1.50Á1.53 (m, 2 H, CH₂),

1.57Á1.60 (m, 2 H, CH₂), 2.07 (s, 3 H, COCH₃), 2.98 (m,

/

1489.638, calcd 1489.657.

/

1 H, H-5^I), 3.17 (t, 2 H, CH₂N₃), 4.02 (dd, 1 H, J_1,28.3,

J_2,310.8 Hz, H-2^III), 4.08 and 4.21 (2 d, each 1 H,

OCH₂C₆H₅), 4.20 (d, 1 H, J_1,28.1 Hz, H-1^I), 4.22 and

4.42 (2 d, each 1 H, OCH₂C₆H₅), 4.24 and 4.37 (2 d,

each 1 H, OCH₂C₆H₅), 4.31 (d, 1 H, J_1,27.7 Hz, H-1^II),

4.64 and 4.81 (2 d, each 1 H, OCH₂C₆H₅), 4.66 and 4.87

(2 d, each 1 H, OCH₂C₆H₅), 5.37 (d, 1 H, H-1^III), 5.52

3.17. 6-Azidohexyl (6-O-tert-butyldiphenylsilyl-2-deoxy-

3,4-di-O-p-methylbenzoyl-2-phthalimido-b-

glucopyranosyl)-(103)-(4-O-acetyl-2,6-di-O-benzyl-b-

galactopyranosyl)-(104)-2,3,6-tri-O-benzyl-b-

glucopyranoside (28)

D-

/

D-

/

D-

(d, 1 H, J_3,43.5, J_4,5

B

/

1 Hz, H-4^II); ¹³C NMR (75.5

To a soln of 27 (0.12 g, 0.083 mmol) in dry Py (5 mL)

was added dropwise, at 0 8C, a soln of p-methylbenzoyl

chloride (27 mL, 0.21 mmol) in dry CH₂Cl₂(1 mL). The

mixture was stirred for 18 h at rt, then poured into ice

water, extracted with CH₂Cl₂, washed with aq satd

NaHCO₃, dried (MgSO₄), filtered, and concentrated.

MHz, CDCl₃): d 20.9 (COCH₃), 25.5, 26.3, 28.6, and

29.4 (4 CH₂), 51.2 (CH₂N₃), 56.7 (C-2^III), 61.0, 67.7,

67.8, 69.4, 72.9, 73.4, 74.1, 74.7, and 74.9 (C-6^I, C-6^II, C-

6^III, 5 OCH₂C₆H₅, OCH₂), 70.5 (2 C), 70.9, 71.9, 74.5,

75.7, 75.8, 78.4, 80.4, 81.5, and 82.5 (C-2^I, C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-4^III, C-5^III), 99.2,

101.7, and 103.4 (C-1^I, C-1^II, C-1^III), 171.1 (COCH₃);

Column chromatography (8:1 tolueneÁ

residue gave 28, isolated as a white solid (0.13 g, 91%);

R_f0.68 (4:1 tolueneÁ 38 (c 1, CHCl₃);

EtOAc); [a]²_D⁰

¹H NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY,

HSQC): d 1.06 [SiC(CH₃)₃], 1.32Á1.36 (m, 4 H, 2 CH₂),

1.48Á1.51 (m, 2 H, CH₂), 1.58Á1.62 (m, 2 H, CH₂), 2.10

/EtOAc) of the

HRMS of C₆₉H₇₈N₄O₁₈(M, 1250.531): [Mꢀ

/

NH₄]^ꢀ

/

ꢁ

/

found 1268.537, calcd 1268.565.

/

3.16. 6-Azidohexyl (6-O-tert-butyldiphenylsilyl-2-deoxy-

2-phthalimido-b- 3)-(4-O-acetyl-

-glucopyranosyl)-(10

2,6-di-O-benzyl-b- -galactopyranosyl)-(104)-2,3,6-tri-

O-benzyl-b- -glucopyranoside (27)

(COCH₃), 2.17 and 2.34 (2 s, each 3 H, 2 COC₆H₄CH₃),

3.13 (t, 2 H, CH₂N₃), 3.16 (m, 1 H, H-5^I), 3.43 (dd, 1 H,

J_1,28.3, J_2,39.2 Hz, H-2^II), 3.59 (m, 1 H, H-5^II), 3.63

(dd, 1 H, J_5,6b3.5, J_6a,6b10.8 Hz, H-6b^I), 4.18 (d, 1 H,

OCHHC₆H₅), 4.24 (d, 1 H, OCHHC₆H₅), 4.31 (d, 1 H,

J_1,28.3 Hz, H-1^I), 4.32 and 4.52 (2 d, each 1 H,

OCH₂C₆H₅), 4.58 (dd, 1 H, J_1,28.3, J_2,310.7 Hz, H-

2^III), 4.73 and 4.87 (2 d, each 1 H, OCH₂C₆H₅), 4.69 and

4.97 (2 d, each 1 H, OCH₂C₆H₅), 5.81 (d, 1 H, H-1^III),

6.37 (t, 1 H, H-3^III); ¹³C NMR (75.5 MHz, CDCl₃): d

18.7 [SiC(CH₃)₃], 20.3 (COCH₃), 21.0 and 21.3

(COC₆H₄CH₃), 25.3, 26.0, 28.3, and 29.2 (4 CH₂),

26.3 [SiC(CH₃)₃], 50.9 (CH₂N₃), 55.2 (C-2^III), 62.6 (C-

6^III), 67.4 (C-6^I), 67.9 (C-6^II), 69.2 (OCH₂), 69.5 (C-4^III),

70.1 (C-4^II), 70.6 (C-3^III), 72.5 (C-5^II), 72.7, 73.0, 74.1,

74.4, and 74.5 (5 OCH₂C₆H₅), 74.5 (C-5^I), 75.2 (2 C) (C-

4^I, C-5^III), 77.0 (C-3^II), 78.9 (C-2^II), 81.3 (C-2^I), 82.2 (C-

3^I), 98.2 (C-1^III), 101.6 (C-1^II), 103.1 (C-1^I), 164.6 and

165.2 (2 COC₆H₄CH₃), 169.2 (COCH₃); HRMS of

D

/

D

/

D

To a soln of 26 (0.30 g, 0.24 mmol) in CH₂Cl₂(3 mL)

and Py (0.2 mL) were added 4-dimethylaminopyridine

(9 mg, 0.072 mmol), Et₃N (0.2 mL), and tert-butyldi-

phenylsilyl chloride (73 mL, 0.29 mmol). The mixture

was stirred for 20 h, then poured into ice water,

extracted with CH₂Cl₂, washed with aq satd NaHCO₃,

dried (MgSO₄), filtered, and concentrated. Column

chromatography (1:1 tolueneÁ/EtOAc) of the residue

gave 27, isolated as a colourless syrup (0.30 g, 84%); R_f

1

0.42 (1:1 tolueneÁ

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY,

HSQC): d 1.04 [s, 9 H, SiC(CH₃)₃], 1.33Á1.38 (m, 4

1.59 (m, 2 H,

/

EtOAc); [a]²_D⁰

ꢀ

/

88 (c 1, CHCl₃); H

/

H, 2 CH₂), 1.48Á

/

1.55 (m, 2 H, CH₂), 1.58Á

/

CH₂), 1.97 (s, 3 H, COCH₃), 3.05 (m, 1 H, H-5^I), 3.16 (t,

2 H, CH₂N₃), 3.51 (t, 1 H, H-6b^I), 3.63 (dd, 1 H, J_2,39.8,

J_3,43.2 Hz, H-3^II), 4.04 (dd, 1 H, J_1,28.3, J_2,311.0 Hz,

H-2^III), 4.12 (d, 1 H, OCHHC₆H₅), 4.16 and 4.35 (2 d,

C

1725.741, calcd 1725.740.

₁₀₁H₁₀₈N₄O₂₀Si (M, 1724.736): [Mꢀ

/

H]^ꢀfound

2646

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

3.18. 6-Azidohexyl (2-deoxy-3,4-di-O-p-methylbenzoyl-

2-phthalimido-b- 3)-(4-O-acetyl-

-glucopyranosyl)-(10

2,6-di-O-benzyl-b- -galactopyranosyl)-(104)-2,3,6-tri-

O-benzyl-b- -glucopyranoside (29)

residue gave 30, isolated as colourless syrup (0.20 g,

83%).

D

/

D

/

(b) A soln of 9¹⁶(36.5 mg, 74.2 mmol) and 29 (65 mg,

˚

D

43.6 mmol) in dry CH₂Cl₂(1 mL), containing 4 A

molecular sieves (40 mg), was stirred under Ar for 0.5 h.

A soln of 28 (0.10 g, 60.2 mmol) in 1.0 M TBAF in THF

(1 mL) and AcOH (1 mL) (pH 6) was stirred for 1 h at

0 8C followed by 4 h at rt. After the addition of CH₂Cl₂,

the soln was washed with water and aq satd NaCl, dried

(Na₂SO₄), filtered, and concentrated. Column chroma-

After cooling to ꢁ40 8C, TMSOTf (1.4 mL, 7.7 mmol)

/

was added, and the mixture was stirred for 3 h, during

which period the temperature was allowed to reach rt.

The mixture was neutralized with Et₃N, filtered, washed

with water, dried (MgSO₄), filtered, and concentrated.

tography (4:1 tolueneÁ

isolated as a colourless syrup (80 mg, 89%); R_f0.34 (4:1

tolueneÁ

EtOAc); [a]²_D⁰28 (c 1, CHCl₃); ¹H NMR (500

MHz, CDCl₃; 2D TOCSY, ROESY): d 1.31Á1.33 (m, 4

H, 2 CH₂), 1.49Á1.52 (m, 2 H, CH₂), 1.55Á1.59 (m, 2 H,

/

EtOAc) of the residue gave 29,

Column chromatography (3:1 tolueneÁ

residue gave 30, isolated as a colourless syrup (46 mg,

58%); R_f0.56 (2:1 tolueneÁ 18 (c 1,

EtOAc); [a]²_D⁰

CHCl₃); ¹H NMR (500 MHz, CDCl₃; 2D TOCSY,

ROESY): d 1.34Á1.37 (m, 4 H, 2 CH₂), 1.52Á1.55 (m, 2

H, CH₂), 1.60Á1.63 (m, 2 H, CH₂), 1.85, 1.94, 2.00, 2.01,

/

EtOAc) of the

/

ꢁ

/

ꢀ

/

CH₂), 2.14 (s, 3 H, COCH₃), 2.25 and 2.33 (2 s, each 1

H, 2 COC₆H₄CH₃), 2.95 (m, 1 H, H-5^I), 3.18 (t, 2 H,

CH₂N₃), 3.55 (dd, 1 H, J_2,39.6, J_3,43.5 Hz, H-3^II), 3.66

(dd, 1 H, J_5,6b5.5, J_6a,6b10.8 Hz, H-6b^III), 3.81 (m, 1 H,

OCHH), 3.85 (t, 1 H, H-4^I), 3.92 (m, 1 H, H-5^III), 4.18

and 4.23 (2 d, each 1 H, OCH₂C₆H₅), 4.19 (d, 1 H, J_1,2

7.7 Hz, H-1^I), 4.23 and 4.45 (2 d, each 1 H, OCH₂C₆H₅),

4.29 and 4.38 (2 d, each 1 H, OCH₂C₆H₅), 4.34 (d, 1 H,

J_1,27.7 Hz, H-1^II), 4.49 (dd, 1 H, J_1,28.5, J_2,310.6 Hz,

H-2^III), 4.66 and 4.81 (2 d, each 1 H, OCH₂C₆H₅), 4.67

and 4.90 (2 d, each 1 H, OCH₂C₆H₅), 5.49 (t, 1 H, H-

/

and 2.11 (5 s, each 3 H, 5 COCH₃), 2.25 and 2.33 (2 s,

each 3 H, 2 COC₆H₄CH₃), 3.17 (m, 1 H, H-5^I), 3.18 (t, 2

H, CH₂N₃), 3.37 (dd, 1 H, J_1,28.1, J_2,39.7 Hz, H-2^II),

3.52 (ddd, 1 H, J_4,510.1, J_5,6a2.4, J_5,6b4.2 Hz, H-5^IV),

4.09 and 4.38 (2 d, each 1 H, OCH₂C₆H₅), 4.24 and 4.35

(2 d, each 1 H, OCH₂C₆H₅), 4.27 (d, 1 H, J_1,27.7 Hz, H-

1^I), 4.28 and 4.50 (2 d, each 1 H, OCH₂C₆H₅), 4.64 and

4.89 (2 d, each 1 H, OCH₂C₆H₅), 4.68 (d, 1 H, J_1,28.1

Hz, H-1^IV), 4.69 and 4.84 (2 d, each 1 H, OCH₂C₆H₅),

4.94 (dd, 1 H, J_2,39.9 Hz, H-2^IV), 4.98 (t, 1 H, H-3^IV),

5.19 (t, 1 H, H-4^IV), 5.40 (dd, 1 H, J_4,59.9, J_3,49.2 Hz,

4^III), 5.59 (d, 1 H, J_4,5

B

1 Hz, H-4^II), 5.70 (d, 1 H, H-

/

1^III), 6.16 (dd, 1 H, J_3,49.4 Hz, H-3^III); ¹³C NMR (75.5

MHz, CDCl₃): d 20.3 (COCH₃), 21.0 and 21.5

(COC₆H₄CH₃), 25.6, 26.4, 28.6, and 29.4 (4 CH₂),

51.2 (CH₂N₃), 55.0 (C-2^III), 61.3, 67.7, 68.0, 69.5, 72.9,

73.5, 74.2, 74.7, and 74.8 (C-6^I, C-6^II, C-6^III, 5

OCH₂C₆H₅, OCH₂), 69.0, 70.1, 70.7, 72.0, 74.5, 75.0,

75.7, 78.2, 81.5, 81.8, and 82.5 (C-2^I, C-3^I, C-4^I, C-5^I, C-

2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-4^III, C-5^III), 99.3, 101.7,

and 103.4 (C-1^I, C-1^II, C-1^III), 165.2 and 165.5

H-4^III), 5.59 (d, 1 H, J_3,43.7, J_4,5

B

1 Hz, H-4^II), 5.68 (d,

/

1 H, J_1,28.1 Hz, H-1^III), 6.28 (dd, 1 H, J_2,310.8 Hz, H-

3^III); ¹³C NMR (75.5 MHz, CDCl₃): d 20.3, 20.4, 20.5,

and 20.6 (2 C) (5 COCH₃), 21.4 (2 C) (2 COC₆H₄CH₃),

25.6, 26.3, 28.6, and 29.4 (4 CH₂), 51.2 (CH₂N₃), 55.3

(C-2^III), 61.4, 67.4, 67.9, 68.2, 69.5, 72.8, 73.2, 74.4, 74.7,

and 75.1 (C-6^I, C-6^II, C-6^III, C-6^IV, 5 OCH₂C₆H₅,

OCH₂), 62.5, 67.8, 69.7, 69.8, 70.2, 71.4, 71.6, 72.5,

74.5, 75.0, 76.3, 77.8, 79.0, 81.6, and 82.5 (C-2^I, C-3^I, C-

4^I, C-5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-4^III, C-5^III,

C-2^IV, C-3^IV, C-4^IV, C-5^IV), 98.4, 101.0, 102.1, and 103.3

(COC₆H₄CH₃),

C₈₅H₉₀N₄O₂₀(M, 1486.614): [Mꢀ

1487.621, calcd 1487.623.

171.0

(COCH₃);

HRMS

of

/

H]^ꢀfound

(C-1^I, C-1^II, C-1^III

,

C-1^IV), 165.1 and 165.3

(COC₆H₄CH₃), 169.0Á

C₉₉H₁₀₈N₄O₂₉(M, 1816.709): [Mꢀ

/

169.8 (COCH₃); HRMS of

NH₄]^ꢀfound

/

3.19. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-

glucopyranosyl)-(106)-(2-deoxy-3,4-di-O-p-

methylbenzoyl-2-phthalimido-b-

-glucopyranosyl)-(10

3)-(4-O-acetyl-2,6-di-O-benzyl-b- -galactopyranosyl)-

(104)-2,3,6-tri-O-benzyl-b- -glucopyranoside (30)

D-

1834.719, calcd 1834.743.

/

D

/

3.20. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-

glucopyranosyl)-(106)-(2-acetamido-3,4-di-O-acetyl-2-

deoxy-b- 3)-(4-O-acetyl-2,6-di-O-

-glucopyranosyl)-(10

benzyl-b- 4)-2,3,6-tri-O-benzyl-

-galactopyranosyl)-(10

b- -glucopyranoside (31)

D-

D

/

D

/

D

/

(a) A soln of 13 (0.27 g, 0.26 mmol) and 24¹⁴(0.23 g,

D

˚

0.24 mmol) in dry CH₂Cl₂(8 mL), containing 4 A

molecular sieves (0.2 g), was stirred under Ar for 0.5 h.

To a soln of 30 (0.20 g, 0.11 mmol) in MeOH (3.5 mL)

and CH₂Cl₂(1.5 mL) was added NaOMe (pH 10), and

the mixture was stirred for 4 h, then neutralized with

After cooling to ꢁ70 8C, TMSOTf (5 mL, 0.026 mmol)

/

was added, and the mixture was stirred for 3 h, during

which period the temperature was allowed to reach rt.

The mixture was neutralized with Et₃N, filtered, washed

with water, dried (MgSO₄), filtered, and concentrated.

Dowex 50ꢂ

8 (H^ꢀ), filtered, and concentrated. To a

/

soln of the residue in 1-BuOH (50 mL) was added 1,2-

diaminoethane (10 mL), and the mixture was stirred

overnight at 80 8C, then co-concentrated with toluene,

Column chromatography (3:1 tolueneÁEtOAc) of the

/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2647

EtOH, and CH₂Cl₂. A soln of the residue in Py (30 mL)

and Ac₂O (30 mL) was stirred for 4 h, then co-

concentrated with toluene, EtOH, and CH₂Cl₂. Column

2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-4^III, C-5^III, C-2^IV, C-3^IV,

C-4^IV, C-5^IV), 103.4, 104.2, 104.3, and 104.4 (C-1^I, C-1^II,

C-1^III, C-1^IV), 176.3 (NDCOCH₃); HRMS of

chromatography (1:1 tolueneÁ/EtOAc) of the residue

C₃₂H₅₈N₂O₂₁(M, 806.353): [Mꢀ

H]^ꢀfound 807.367,

calcd 807.361. For H NMR data, see Table 3.

/

1

gave 31, isolated as a colourless glass (0.16 g, 88%); R_f

1

0.45 (1:3 tolueneÁ

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY): d

1.36Á1.38 (m, 4 H, 2 CH₂), 1.52Á1.56 (m, 2 H, CH₂),

1.63Á1.65 (m, 2 H, CH₂), 1.91, 1.94, 1.95, 1.98, 2.00,

/

EtOAc); [a]²_D⁰

ꢁ58 (c 1, CHCl₃); H

/

3.22. 6-Aminohexyl b-

galactopyranosyl-(104)]-2-acetamido-2-deoxy-b-

glucopyranosyl-(103)-b- -galactopyranosyl-(10

glucopyranoside (2)

D

-glucopyranosyl-(10

/

6-[b-D-

/

D-

/

D

/4)-b-D-

2.04, 2.07, and 2.12 (8 s, each 3 H, 7 COCH₃,

NHCOCH₃), 3.19 (t, 2 H, CH₂N₃), 3.73 (dd, 1 H, J_5,6b

7.5, J_6a,6b11.6 Hz, H-6b^III), 3.95 (t, 1 H, H-4^I), 3.99 (dd,

1 H, J_5,6a1.8, J_6a,6b12.3 Hz, H-6a^IV), 4.16 (dd, 1 H, J_5,6b

4.2 Hz, H-6b^IV), 4.27 and 4.48 (2 d, each 1 H,

OCH₂C₆H₅), 4.34 (d, 1 H, J_1,27.7 Hz, H-1^I), 4.42 and

4.55 (2 d, each 1 H, OCH₂C₆H₅), 4.55 (d, 1 H, J_1,27.7

Hz, H-1^II), 4.56 (d, 1 H, OCHHC₆H₅), 4.63 (d, 1 H, J_1,2

7.9 Hz, H-1^IV), 5.21 (t, 1 H, H-3^IV), 5.48 (d, 1 H, J_3,43.5,

To a soln of 6 (11.4 mg, 14.12 mmol) in aq 50 mM

sodium cacodylate buffer pH 7.5 (700 mL), containing 5

mM MnCl₂, BSA (0.5 mg), and NaN₃(0.02%), were

added alkaline phosphatase (14 U), UDP-galactose (12

mg, 19.66 mmol), and b-1,4-galactosyltransferase (3 U).

The mixture was incubated for 20 h at 37 8C then water

(100 mL) was added. UDP-Galactose was removed using

J_4,5

B

/

1 Hz, H-4^II), 7.14Á

/

7.37 (m, 25 H, 5 OCH₂C₆H₅);

a Dowex 1ꢂ

8 (Cl^ꢁ) column with water as eluent. The

/

¹³C NMR (75.5 MHz, CDCl₃): d 20.3Á

/20.5 (COCH₃),

eluate was concentrated, and the residue applied to a

Bio-Gel P-2 column eluted with aq 0.1 M NH₄HCO₃at

a flow rate of 40 mL/h. The appropriate fractions were

freeze-dried to give 2 (9.0 mg, 65%) and 48 (1.0 mg, 6%);

22.6 (NHCOCH₃), 25.4, 26.2, 28.5, 29.3 (4 CH₂), 51.0

(CH₂N₃), 54.1 (C-2^III), 61.4, 67.5, 67.9, 68.1, 69.4, 73.0,

73.5, 74.6 (2 C), and 74.9 (C-6^I, C-6^II, C-6^III, C-6^IV, 5

OCH₂C₆H₅, OCH₂), 67.8, 69.3, 69.9, 71.3, 71.4, 71.9,

72.3, 72.4, 73.5, 75.0, 76.2, 77.4, 80.3, 81.5, and 82.2 (C-

2^I, C-3^I, C-4^I, C-5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-

4^III, C-5^III, C-2^IV, C-3^IV, C-4^IV, C-5^IV), 100.6, 101.2,

R_f0.23 (2:1:1 AcOHÁ

water); HRMS of C₃₈H₆₈N₂O₂₆(M, 968.406): [Mꢀ

found 969.418, calcd 969.414. For H NMR data, see

Table 4.

/

1-BuOHÁ

/

water); [a]²_D⁰

ꢁ

/

18 (c 0.5,

/

H]^ꢀ

1

101.8, and 103.2 (C-1^I, C-1^II, C-1^III, C-1^IV), 168.9Á

(COCH₃, NHCOCH₃); HRMS of C₈₁H₁₀₀N₄O₂₈(M,

1576.652): [Mꢀ

NH₄]^ꢀfound 1594.647, calcd 1594.686.

/

170.5

3.23. (3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-

glucopyranosyl)-(103)-(2,4,6-tri-O-acetyl-b-

galactopyranosyl)-(104)-1,2,3,6-tetra-O-acetyl-a,b-

glucopyranose (35)

D

-

/

D-

/

D

-

3.21. 6-Aminohexyl b-

acetamido-2-deoxy-b-

galactopyranosyl-(10

D

-glucopyranosyl-(10

-glucopyranosyl-(103)-b-

4)-b- -glucopyranoside (6)

/

6)-2-

D

/

D-

/

D

To a soln of (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-

3)-(4-O-acetyl-2,6-di-O-ben-

-galactopyranosyl)-(104)-1,2,3,6-tetra-O-ben-

-glucopyranose (34)¹⁵(1.5 g, 1.12 mmol) in

b-

D

-glucopyranosyl)-(10

/

To a soln of 31 (155 mg, 98.2 mmol) in CH₂Cl₂(4 mL)

and MeOH (2 mL) was added NaOMe (pH 10). The

mixture was stirred for 2 h, then neutralized with Dowex

zyl-b-

D

/

zyl-b-

D

EtOH (45 mL) and EtOAc (45 mL) was added 10%

C (1.78 g) and 2 drops of AcOH, and the mixture

was stirred under H₂for 8 h, then filtered over hyflo,

50ꢂ

as a white solid (126 mg). To a soln of 32 in tert-BuOH

C (200

/

8 (H^ꢀ), filtered, and concentrated, giving crude 32

Pdꢀ

/

(4 mL) and water (2 mL) were added 10% Pdꢀ

/

and concentrated. Column chromatography (7:1

mg) and 3 drops of aq 25% NH₃. The mixture was

stirred for 3 h under H₂after which NH₃was removed

CH₂Cl₂ÁMeOH) of the residue gave a white solid which

/

was dissolved in Py (75 mL) and Ac₂O (75 mL), and the

soln was stirred overnight, then co-concentrated with

toluene, EtOH and CH₂Cl₂. Low-pressure column

by bubbling with N₂, then 10% Pdꢀ

/

C (100 mg) and 3

drops of AcOH were added, and the stirring under H₂

was continued overnight. The mixture was loaded on a

chromatography (7:10

/

1:1 tolueneÁEtOAc) of the re-

/

short Dowex 50ꢂ

8 (H^ꢀ) column, which was first eluted

with water to remove contaminations, then with aq 10%

NH₄OH to give 6, isolated as a white solid after

/

sidue gave 35, isolated as a white foam (0.95 g, 66%); R_f

1

0.45 (1:2 tolueneÁ

/

EtOAc); [a]²_D⁰

ꢁ178 (c 1, CHCl₃); H

/

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY): d 4.46

(t, 1 H, H-6b^III), 4.80 (m, 1 H, H-2^II), 4.93 (dd, 0.5 H,

J_1,23.6, J_2,310.3 Hz, H-2^Ia), 4.98 (t, 0.5 H, H-2^Ib), 5.13

(t, 0.5 H, H-3^Ib), 5.18 (m, 1 H, H-4^III), 5.63 (d, 0.5 H,

J_1,28.3 Hz, H-1^Ib), 5.74 (m, 1 H, H-3^III), 6.19 (d, 0.5 H,

lyophilization (65 mg, 81%); R_f0.26 (2:1:1 AcOHÁ

/

1-

BuOHÁ

/

water); [a]²_D⁰28 (c 1, water); ¹³C NMR (75.5

ꢁ

/

MHz, D₂O): d 23.6 (NDCOCH₃), 26.0, 26.7, 28.5, and

29.9 (4 CH₂), 41.0 (CH₂ND₂), 57.1 (C-2^III), 61.6, 62.2,

62.5, 70.2, and 71.9 (C-6^I, C-6^II, C-6^III, C-6^IV, OCH₂),

69.7, 71.1 (2 C), 71.5, 74.3, 74.6, 75.0, 75.9, 76.1, 76.2,

76.4, 77.2, 77.4, 79.9, and 83.4 (C-2^I, C-3^I, C-4^I, C-5^I, C-

H-1^Ia), 7.77 Á

₄₆H₅₅NO₂₇(M, 1053.296): [Mꢀ

1071.329, calcd 1071.330.

/

7.84 (m, 4 H, Phth); HRMS of

NH₄]^ꢀfound

C

/

2648

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

3.24. (3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-

glucopyranosyl)-(103)-(2,4,6-tri-O-acetyl-b-

galactopyranosyl)-(104)-2,3,6-tri-O-acetyl-a-

glucopyranosyl trichloroacetimidate (37)

D

-

2.03, 2.09, 2.10, and 2.15 (9 s, each 3 H, 9 COCH₃), 3.24

(t, 2 H, CH₂N₃), 3.67 (t, 1 H, H-4^I), 4.09 (dd, 1 H, J_5,6b

3.5, J_6a,6b12.3 Hz, H-6b^III), 4.14 (dd, 1 H, J_1,28.3, J_2,3

10.9 Hz, H-2^III), 4.26 (d, 1 H, J_1,28.0 Hz, H-1^II), 4.48

(dd, 1 H, J_5,6a2.6 Hz, H-6a^III), 5.06 (dd, 1 H, H-3^I), 5.19

(dd, 1 H, J_3,49.1 Hz, H-4^III), 5.74 (dd, 1 H, H-3^III),

/

D-

/

D-

To a soln of 35 (714 mg, 0.68 mmol) in dry DMF (10

mL) was added hydrazinium acetate (67 mg, 0.73

mmol). The mixture was stirred for 3 h, then co-

concentrated with toluene, EtOH and CH₂Cl₂. Low-

7.72Á

/

7.75 and 7.79Á

/

7.81 (2 m, each 2 H, Phth); ¹³C

20.6 (COCH₃), 25.3,

NMR (75.5 MHz, CDCl₃): d 20.3Á

/

26.3, 28.6, and 29.1 (4 CH₂), 51.2 (CH₂N₃), 54.5 (C-2^III),

60.8, 61.5, 62.0, and 69.7 (C-6^I, C-6^II, C-6^III, OCH₂),

68.7, 68.8, 70.0, 70.6, 71.0, 71.5, 71.8, 72.4, 72.6, and

75.5 (2 C) (C-2^I, C-3^I, C-4^I, C-5^I, C-2^II, C-3^II, C-4^II, C-

5^II, C-3^III, C-4^III, C-5^III), 97.6 and 100.4 (2 C) (C-1^I, C-

pressure column chromatography (3:10

/

1:1 tolueneÁ

/

EtOAc) of the residue gave 36, isolated as a white solid

(609 mg, 89%). To a soln of 36 (519 mg, 0.51 mmol) in

dry CH₂Cl₂(20 mL) was added, at 0 8C, trichloroace-

tonitrile (4.9 mL, 49 mmol) and 1,8-diazabicy-

clo[5.4.0]undec-7-ene (0.26 mL, 1.25 mmol). The

mixture was stirred under Ar for 1.5 h at rt, then

concentrated. Low-pressure column chromatography

1^II, C-1^III), 168.5Á

C₅₀H₆₄N₄O₂₆(M, 1136.380): [Mꢀ

1159.358, calcd 1159.370.

/

171.6 (COCH₃); HRMS of

/

Na]^ꢀfound

(7:10

/

2:1 tolueneÁEtOAc) of the residue gave 37,

/

isolated as a slightly yellow solid (407 mg, 69%); R_f

1

EtOAc); [a]²_D⁰

ꢁ88 (c 1, CHCl₃); H

/

3.26. 6-Azidohexyl (2-acetamido-3,4,6-tri-O-acetyl-2-

deoxy-b- 3)-(2,4,6-tri-O-acetyl-b-

-glucopyranosyl)-(10

-galactopyranosyl)-(104)-2,3,6-tri-O-acetyl-b-

glucopyranoside (39)

0.28 (1:2 tolueneÁ

/

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY): d 1.79,

1.84, 1.97, 1.99, 2.03, 2.08, 2.10, 2.12, and 2.15 (9 s, each

3 H, 9 COCH₃), 3.96 (m, 1 H, H-5^I), 4.14 (dd, 1 H, J_1,2

8.3, J_2,311.0 Hz, H-2^III), 4.30 (d, 1 H, J_1,28.1 Hz, H-1^II),

4.36 (dd, 1 H, J_5,6a1.8, J_6a,6b12.1 Hz, H-6a^I), 4.51 (dd, 1

H, J_5,6a2.6, J_6a,6b12.3 Hz, H-6a^III), 4.81 (dd, 1 H, J_2,3

9.9 Hz, H-2^II), 5.00 (dd, 1 H, J_1,23.5, J_2,310.1 Hz, H-2^I),

5.19 (dd, 1 H, J_3,49.2, J_4,59.9 Hz, H-4^III), 5.41 (t, 1 H,

D

/

D

/

D-

To a soln of 38 (22 mg, 21 mmol) in CH₂Cl₂(0.5 mL)

and MeOH (3 mL) was added NaOMe (pH 10), and the

mixture was stirred for 2 h, then neutralized with Dowex

50ꢂ

8 (H^ꢀ), filtered, and concentrated. To a soln of the

/

H-3^I), 5.76 (dd, 1 H, H-3^III), 6.43 (d, 1 H, H-1^I), 7.73Á

7.75 (m, 4 H, Phth), 8.65 (s, 1 H, OC(NH)CCl₃); ¹³C

20.6 (COCH₃), 54.5

/

residue in 1-BuOH (10 mL) was added 1,2-diami-

noethane (2 mL), and the mixture was stirred overnight

at 80 8C, then co-concentrated with toluene, EtOH and

CH₂Cl₂. A soln of the residue in Py (10 mL) and Ac₂O

(10 mL) was stirred overnight, then co-concentrated

with toluene, EtOH, and CH₂Cl₂. Column chromato-

NMR (75.5 MHz, CDCl₃): d 20.3Á

/

(C-2^III), 60.8, 61.5, and 62.0 (C-6^I, C-6^II, C-6^III), 68.6,

68.7, 69.2, 69.8 (2 C), 70.0, 70.7, 71.0, 71.8, 74.9, and

75.5 (C-2^I, C-3^I, C-4^I, C-5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-

3^III, C-4^III, C-5^III), 92.8, 97.6, and 100.6 (C-1^I, C-1^II, C-

graphy (3:1 CH₂Cl₂Á

isolated as a white solid (17 mg, 77%); R_f0.22 (3:1

CH₂Cl₂Á

acetone); [a]²_D⁰68 (c 1, CHCl₃); ¹H NMR

(500 MHz, CDCl₃; 2D TOCSY, ROESY): d 1.25Á

(m, 4 H, 2 CH₂), 1.33Á1.36 (m, 2 H, CH₂), 1.57Á

/

acetone) of the residue gave 39,

1^III), 161.8 (OC(NH)CCl₃), 168.5Á

171.6 (COCH₃).

/

ꢀ

/

/1.28

3.25. 6-Azidohexyl (3,4,6-tri-O-acetyl-2-deoxy-2-

phthalimido-b- 3)-(2,4,6-tri-O-

-glucopyranosyl)-(10

acetyl-b- 4)-2,3,6-tri-O-acetyl-

-galactopyranosyl)-(10

b- -glucopyranoside (38)

/

/1.59

D

/

(m, 2 H, CH₂), 1.90, 2.00, 2.01, 2.02, 2.08, 2.09, 2.10,

2.11, and 2.12 (9 s, 3,3,6,3,3,3,3,3,3 H, 9 COCH₃,

NHCOCH₃), 3.46 (m, 1 H, OCHH), 3.59 (m, 1 H, H-

5^I), 3.66 (m, 1 H, H-5^III), 4.87 (dd, 1 H, J_1,28.1, J_2,39.6

Hz, H-2^I), 5.16 (t, 1 H, H-3^I), 5.32 (d, 1 H, J_3,42.4,

D

/

D

A soln of 37 (0.16 g, 0.14 mmol) and 6-azido-1-hexanol

33 (23 mg, 0.16 mmol) in dry CH₂Cl₂(4 mL), containing

˚

4 A powdered molecular sieves (0.1 g), was stirred under

Ar for 0.5 h. After cooling to 0 8C, AgOTf (35 mg, 0.14

mmol) was added, and the mixture was stirred for 1.5 h

at 0 8C, then at rt for 2 h. After neutralization with

Et₃N, the mixture was filtered over hyflo, washed with

water, dried (MgSO₄), filtered, and concentrated. Col-

J_4,5

J_NH,27.5 Hz, NHCOCH₃); ¹³C NMR (75.5 MHz,

CDCl₃): d 20.5Á20.8 (NHCOCH₃, COCH₃), 25.3,

26.3, 28.6, and 29.2 (4 CH₂), 51.2 (CH₂N₃), 56.1 (C-

2^III), 61.1, 61.5, 62.1, and 69.7 (C-6^I, C-6^II, C-6^III

B

/

1 Hz, H-4^II), 5.47 (t, 1 H, H-3^III), 5.60 (d, 1 H,

/

,

OCH₂), 68.7, 68.8, 71.0, 71.1 (2 C), 71.5, 71.7, 72.5,

72.7, 75.7, and 75.9 (C-2^I, C-3^I, C-4^I, C-5^I, C-2^II, C-3^II,

C-4^II, C-5^II, C-3^III, C-4^III, C-5^III), 99.5, 100.5, and 100.6

umn chromatography (1:2 tolueneÁ

residue gave 38, isolated as a white solid (34 mg, 22%);

R_f0.41 (1:2 tolueneÁ

EtOAc); [a]²_D⁰28 (c 1, CHCl₃); ¹H

NMR (300 MHz, CDCl₃): d 1.26Á1.40 (m, 6 H, 3 CH₂),

1.52Á1.60 (m, 2 H, CH₂), 1.58, 1.78, 1.84, 1.97, 1.99,

/EtOAc) of the

(C-1^I,

NHCOCH₃); HRMS of C₄₄H₆₄N₄O₂₅(M, 1048.386):

[Mꢀ

Na]^ꢀfound 1071.397, calcd 1071.375.

C-1^II,

C-1^III),

168.5Á/171.4

(COCH₃,

/

ꢀ

/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2649

3.27. 6-Azidohexyl 2-acetamido-2-deoxy-b-

glucopyranosyl-(103)-b- -galactopyranosyl-(10

glucopyranoside (40)

D

-

3.29. 6-Aminohexyl 2-acetamido-2-deoxy-b-

glucopyranosyl-(103)-b- -galactopyranosyl-(10

glucopyranoside (7)

D-

/

D

/

4)-b-

D-

/

D

/4)-b-D-

(a) To a soln of 41 (75 mg, 58.5 mmol) in CH₂Cl₂(1 mL)

and MeOH (2 mL) was added NaOMe (pH 10).

The mixture was stirred for 1.5 h, then neutralized

To a soln of 39 (15 mg, 14 mmol) in CH₂Cl₂(0.5 mL)

and MeOH (2 mL) was added NaOMe (pH 10). The

mixture was stirred for 2 h, then neutralized with Dowex

with Dowex 50ꢂ

8 (H^ꢀ), filtered, and concentrated,

giving crude 42 as a white solid (65 mg). To a soln of

42 in tert-BuOH (15 mL) and water (10 mL) were

/

50ꢂ

matography (1:1 CH₂Cl₂Á

40, isolated as a white solid (5.3 mg, 55%); R_f0.81 (1:2

CH₂Cl₂Á 48 (c 0.5, water); HRMS data

MeOH); [a]²_D⁰

of C₂₆H₄₆N₄O₁₆(M, 670.290): [Mꢀ

H]^ꢀfound 671.300,

/

8 (H^ꢀ), filtered, and concentrated. Column chro-

/MeOH) of the residue gave

added 10% Pdꢀ

/

C (150 mg) and 3 drops of aq 25%

/

ꢁ

/

NH₃. The mixture was stirred for 3 h under H₂

after which NH₃was removed by bubbling with N₂,

/

1

calcd 671.302. For H NMR data, see Table 5.

then 10% Pdꢀ

/

C (100 mg) and 3 drops of AcOH were

added, and the stirring under H₂was continued over-

night. The mixture was loaded on a short Dowex 50ꢂ8

/

3.28. 6-Azidohexyl (2-acetamido-3,4,6-tri-O-acetyl-2-

(H^ꢀ) column, which was first eluted with water to

remove contaminations, then with aq 10% NH₄OH

to give 7, isolated as a white solid after lyophilization

(25 mg, 68%).

deoxy-b-

benzyl-b-

b- -glucopyranoside (41)

D

-glucopyranosyl)-(10

/

3)-(4-O-acetyl-2,6-di-O-

D

-galactopyranosyl)-(10

/

4)-2,3,6-tri-O-benzyl-

D

(b) To a soln of 40 (5.3 mg, 7.90 mmol) in tert-BuOH

To a soln of 25 (0.21 g, 0.15 mmol) in MeOH (4 mL) and

CH₂Cl₂(2 mL) was added NaOMe (pH 10), and the

mixture was stirred for 3 h, then neutralized with Dowex

(3 mL) and water (2 mL) was added 10% Pdꢀ

/C (40 mg)

and 2 drops of aq 25% NH₃. The mixture was stirred for

20 h under H₂, after which NH₃was removed by

bubbling with N₂, then filtered over cotton and con-

centrated. Chromatography of the residue on a Bio-Gel

P-2 column eluted with 0.1 M NH₄HCO₃, and sub-

sequent lyophilization yielded 7, isolated as a white solid

50ꢂ

8 (H^ꢀ), filtered, and concentrated. To a soln of the

/

residue in 1-BuOH (30 mL) was added 1,2-diami-

noethane (6 mL), and the mixture was stirred overnight

at 80 8C, then co-concentrated with toluene, EtOH, and

CH₂Cl₂. A soln of the residue in Py (30 mL) and Ac₂O

(30 mL) was stirred for 48 h, then co-concentrated with

toluene, EtOH, and CH₂Cl₂. Column chromatography

(4.2 mg, 83%); R_f0.34 (2:1:1 AcOHÁ

/

1-BuOHÁwater);

/

[a]²_D⁰28 (c 1, water); ¹³C NMR (75.5 MHz, D₂O): d

ꢁ

/

23.0 (NDCOCH₃), 25.4, 26.1, 28.3, and 29.3 (4 CH₂),

40.4 (CH₂ND₂), 56.5 (C-2^III), 60.9, 61.3, 61.7, and 71.2

(C-6^I, C-6^II, C-6^III, OCH₂), 69.1, 70.5, 70.8, 73.6, 74.4,

75.3, 75.6, 75.7, 76.5, 79.3, and 82.8 (C-2^I, C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-3^III, C-4^III, C-5^III), 102.8,

103.6, and 103.7 (C-1^I, C-1^II, C-1^III), 175.7

(1:1 tolueneÁ/EtOAc) of the residue gave 41, isolated as a

colourless glass (0.15 g, 78%); R_f0.39 (1:1 tolueneÁ

/

1

EtOAc); [a]²_D⁰

CDCl₃; 2D TOCSY, ROESY): d 1.36Á

CH₂), 1.51Á1.56 (m, 2 H, CH₂), 1.61Á

ꢁ

/

28 (c 1, CHCl₃); H NMR (500 MHz,

1.42 (m, 4 H, 2

1.64 (m, 2 H,

/

CH₂), 1.51, 1.94, 1.99, 2.02, and 2.05 (5 s, each 3 H, 4

COCH₃, NHCOCH₃), 3.18 (t, 2 H, CH₂N₃), 3.72 (dd, 1

H, J_5,6b4.1, J_6a,6b11.0 Hz, H-6b^I), 3.98 (t, 1 H, H-4^I),

4.26 and 4.42 (2 d, each 1 H, OCH₂C₆H₅), 4.33 (d, 1 H,

J_1,27.9 Hz, H-1^I), 4.38 (d, 1 H, OCHHC₆H₅), 4.49 (d, 1

H, J_1,27.7 Hz, H-1^II), 4.72 and 4.86 (2 d, each 1 H,

OCH₂C₆H₅), 4.74 and 4.98 (2 d, each 1 H, OCH₂C₆H₅),

5.01 (t, 1 H, H-4^III), 5.09 (t, 1 H, H-3^III), 5.25 (d, 1 H,

(NDCOCH₃); HRMS of C₂₆H₄₈N₂O₁₆(M, 644.300):

1

[Mꢀ

/

H]^ꢀfound 645.311, calcd 645.308. For H NMR

data, see Table 6.

3.30. 6-Aminohexyl b-

D

-galactopyranosyl-(10

-glucopyranosyl-(103)-b-

4)-b- -glucopyranoside (3)

/

4)-2-

acetamido-2-deoxy-b-

D

/

D-

galactopyranosyl-(10

/

D

J_2,NH9.2 Hz, NHCOCH₃), 5.40 (d, 1 H, J_3,43.3, J_4,5

Hz, H-4^II), 7.18Á7.36 (m, 25 H, 5 OCH₂C₆H₅); ¹³C

NMR (75.5 MHz, CDCl₃): d 20.3Á20.5 (COCH₃), 22.5

B1

/

To a soln of 7 (8.2 mg, 12.71 mmol) in aq 50 mM sodium

cacodylate buffer pH 7.5 (600 mL), containing 5 mM

MnCl₂, BSA (0.5 mg), and NaN₃(0.02%), were added

alkaline phosphatase (14 U), UDP-galactose (11.5 mg,

18.84 mmol), and b-1,4-galactosyltransferase (2.5 U).

The mixture was incubated for 20 h at 37 8C then water

(200 mL) was added. UDP-Galactose was removed using

/

(NHCOCH₃), 25.4, 26.2, 28.5, and 29.3 (4 CH₂), 51.0

(CH₂N₃), 54.3 (C-2^III), 61.6, 67.9, 68.1, 69.4, 73.0, 73.2,

74.4, 74.6, and 74.8 (C-6^I, C-6^II, C-6^III, 5 OCH₂C₆H₅,

OCH₂), 68.3, 71.4 (2 C), 72.3, 72.6, 74.7, 75.8, 78.9, 79.5,

81.4, and 82.3 (C-2^I, C-3^I, C-4^I, C-5^I, C-2^II, C-3^II, C-4^II,

C-5^II, C-3^III, C-4^III, C-5^III), 100.9, 101.7, and 103.3 (C-

1^I, C-1^II, C-1^III), 168.9, 169.4, 169.5, 170.3, and 170.4 (4

COCH₃, NHCOCH₃); HRMS of C₆₉H₈₄N₄O₂₀(M,

a Dowex 1ꢂ

8 (Cl^ꢁ) column with water as eluent. The

/

eluate was concentrated, and the residue applied to a

Bio-Gel P-2 column eluted with aq 0.1 M NH₄HCO₃at

a flow rate of 40 mL/h. The appropriate fractions were

1288.567): [Mꢀ

NH₄]^ꢀfound 1306.629, calcd 1306.600.

/

2650

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á2651

/

freeze-dried to give 3 (9.6 mg, 94%); R_f0.22 (2:1:1

AcOHÁ1-BuOHÁ 18 (c 0.5, water);

water); [a]²_D⁰

HRMS data of C₃₂H₅₈N₂O₂₁(M, 806.353): [Mꢀ

H]^ꢀ

found 807.352, calcd 807.361. For H NMR data, see

Table 7.

Dowex 50ꢂ

8 (H^ꢀ), filtered, and concentrated. To a

/

ꢁ

/

soln of the residue in 1-BuOH (25 mL) was added 1,2-

diaminoethane (5 mL), and the mixture was stirred

overnight at 90 8C, then co-concentrated with toluene,

EtOH and CH₂Cl₂. A soln of the residue in Py (10 mL)

and Ac₂O (10 mL) was stirred overnight, then co-

concentrated with toluene, EtOH and CH₂Cl₂. Column

/

1

3.31. 6-Azidohexyl (3,4,6-tri-O-acetyl-2-deoxy-2-

phthalimido-b-

acetyl-b-

-galactopyranosyl)-(10

b- 6)-2-deoxy-3,4-di-O-p-

-glucopyranosyl)-(10

methylbenzoyl-2-phthalimido-b- -glucopyranoside (44)

D

-glucopyranosyl)-(10

/

3)-(2,4,6-tri-O-

chromatography (3:1 CH₂Cl₂Á

gave 45, isolated as a white foam (48 mg, 86%); R_f0.44

(2:1 CH₂Cl₂Á

acetone); [a]²_D⁰108 (c 1, CHCl₃); ¹H

NMR (300 MHz, CDCl₃): d 1.25Á1.34 (m, 4 H, 2 CH₂),

1.59Á1.64 (m, 4 H, 2 CH₂), 1.90, 1.93, 2.00, 2.01, 2.02,

/

acetone) of the residue

D

/

4)-(2,3,6-tri-O-acetyl-

D

/

ꢁ

/

D

/

To a soln of 37 (100 mg, 87 mmol) and 43 (70 mg, 104

2.08, 2.09, 2.10, 2.11, and 2.12 (10 s, 3,3,3,12,3,3,3,3,3,3

H, 11 COCH₃, 2 NHCOCH₃), 3.46 (m, 1 H, OCHH),

4.12 (dd, 1 H, J_5,6b5.1, J_6a,6b11.7 Hz, H-6b^II), 4.44 (dd,

˚

mmol) in dry CH₂Cl₂(3 mL), containing 4 A molecular

sieves (200 mg), was added, under Ar at 0 8C, TMSOTf

(2.5 mL, 12.96 mmol). The mixture was stirred for 1 h at

0 8C, followed by 1 h at rt, then neutralized with Et₃N,

filtered over hyflo, washed with water, dried (MgSO₄),

filtered, and concentrated. Low-pressure column chro-

1 H, J_5,6a

1^II), 4.61 (d, 1 H, J_1,28.3 Hz, H-1^I), 5.12 (t, 1 H, H-3^II),

5.24 (t, 1 H, H-3^I), 5.32 (d, 1 H, J_3,43.5, J_4,5

1 Hz, H-

4^III); ¹³C NMR (75.5 MHz, CDCl₃): d 20.5Á

20.7

B

/

1 Hz, H-6a^II), 4.56 (d, 1 H, J_1,27.8 Hz, H-

B

/

matography (4:10

gave 44, isolated as a white solid (49 mg, 34%); R_f0.58

(1:2 tolueneÁ

EtOAc); [a]²_D⁰118 (c 1, CHCl₃); ¹H

NMR (500 MHz, CDCl₃; 2D TOCSY, ROESY): d

1.12Á1.20 (m, 4 H, 2 CH₂), 1.25Á1.32 (m, 2 H, CH₂),

1.49Á1.53 (m, 2 H, CH₂), 1.78, 1.83, 1.96, 2.02, 2.03,

/

2:1 tolueneÁEtOAc) of the residue

/

(COCH₃), 23.2 (2 C) (2 NHCOCH₃), 25.4, 26.3, 28.6,

and 29.2 (4 CH₂), 51.2 (CH₂N₃), 54.8 and 56.1 (C-2^I, C-

2^IV), 61.0, 61.5, 61.9, 68.2, and 69.4 (C-6^I, C-6^II, C-6^III,

C-6^IV, OCH₂), 68.6, 68.8, 69.2, 70.9, 71.1 (2 C), 71.4,

71.7, 72.3, 72.5, 72.8, 73.4, 75.6, and 75.9 (C-3^I, C-4^I, C-

5^I, C-2^II, C-3^II, C-4^II, C-5^II, C-2^III, C-3^III, C-4^III, C-5^III,

C-3^IV, C-4^IV, C-5^IV), 99.5, 100.4 (2 C), and 100.6 (C-1^I,

/

ꢁ

/

2.04, 2.08, 2.10, and 2.14 (9 s, each 3 H, 9 COCH₃), 2.27

and 2.33 (2 s, each 3 H, 2 COC₆H₄CH₃), 3.64 (t, 1 H, H-

4^II), 4.09 (dd, 1 H, J_5,6b3.1, J_6a,6b12.1 Hz, H-6b^IV), 4.14

(dd, 1 H, J_1,28.3, J_2,310.8 Hz, H-2^IV), 4.24 (d, 1 H, J_1,2

8.1 Hz, H-1^III), 4.57 (d, 1 H, J_1,27.9 Hz, H-1^II), 4.78 (dd,

1 H, J_2,39.9 Hz, H-2^III), 4.85 (dd, 1 H, J_2,39.2 Hz, H-

2^II), 5.02 (t, 1 H, H-3^II), 5.19 (t, 1 H, H-4^IV), 5.44 (d, 1

H, J_1,28.5 Hz, H-1^I), 5.75 (dd, 1 H, J_3,49.2 Hz, H-3^IV),

6.16 (dd, 1 H, J_2,310.7, J_3,49.2 Hz, H-3^I); ¹³C NMR

C-1^II,

NHCOCH₃); HRMS of C₅₆H₈₁N₅O₃₂(M, 1335.486):

[Mꢀ

NH₄]^ꢀfound 1353.547, calcd 1353.520.

C-1^III

,

C-1^IV),

169.0Á/170.7

(COCH₃,

/

3.33. 6-Azidohexyl 2-acetamido-2-deoxy-b-

glucopyranosyl-(103)-b- -galactopyranosyl-(10

glucopyranosyl-(106)-2-acetamido-2-deoxy-b-

glucopyranoside (46)

D-

(75.5 MHz, CDCl₃): d 20.2Á20.6 (COCH₃), 21.4 (2 C)

/

D

/4)-b-D-

(2 COC₆H₄CH₃), 25.3, 26.0, 28.4, and 28.9 (4 CH₂),

51.0 (CH₂N₃), 54.4 and 54.8 (C-2^I, C-2^IV), 60.8, 61.5,

62.0, 68.2, and 69.6 (C-6^I, C-6^II, C-6^III, C-6^IV, OCH₂),

68.6, 68.7, 69.9, 70.0, 70.5, 70.8, 70.9, 71.3, 71.7, 72.4,

72.6, 74.1, and 75.4 (2 C) (C-3^I, C-4^I, C-5^I, C-2^II, C-3^II,

C-4^II, C-5^II, C-2^III, C-3^III, C-4^III, C-5^III, C-3^IV, C-4^IV, C-

5^IV), 97.5, 98.0, and 100.5 (2 C) (C-1^I, C-1^II, C-1^III, C-

/

D-

To a soln of 45 (48 mg, 35.9 mmol) in CH₂Cl₂(1 mL)

and MeOH (8 mL) was added NaOMe (pH 10). The

mixture was stirred for 3 h, then neutralized with Dowex

50ꢂ

matography (1:2 CH₂Cl₂Á

46, isolated as a white solid (25 mg, 79%); R_f0.62 (1:4

CH₂Cl₂Á

MeOH); [a]²_D⁰68 (c 1, water); ¹³C NMR

/

8 (H^ꢀ), filtered, and concentrated. Column chro-

MeOH) of the residue gave

1^IV), 165.1 and 165.4 (2 COC₆H₄CH₃), 168.4Á

170.6

/

(COCH₃); HRMS of C₆₈H₈₁N₅O₃₄(M, 1151.476): [Mꢀ

/

NH₄]^ꢀfound 1169.524, calcd 1169.510.

/

ꢁ

/

(75.5 MHz, D₂O): d 22.4 (2 C) (NDCOCH₃), 24.9, 25.8,

28.2, and 28.7 (4 CH₂), 51.4 (CH₂N₃), 55.8 (C-2^I, C-2^IV),

60.3, 60.7, 61.1, 68.8, and 70.7 (C-6^I, C-6^II, C-6^III, C-6^IV,

OCH₂), 68.6, 70.0 (2 C), 70.2, 73.0, 73.8, 73.9, 74.5, 75.1

(3 C), 75.9, 78.7, and 82.2 (C-3^I, C-4^I, C-5^I, C-2^II, C-3^II,

C-4^II, C-5^II, C-2^III, C-3^III, C-4^III, C-5^III, C-3^IV, C-4^IV, C-

5^IV), 101.3, 102.8, 103.0, and 103.1 (C-1^I, C-1^II, C-1^III,

C-1^IV), 174.6 and 175.1 (2 NHCOCH₃); HRMS of

3.32. 6-Azidohexyl (2-acetamido-3,4,6-tri-O-acetyl-2-

deoxy-b-

-galactopyranosyl)-(10

glucopyranosyl)-(106)-2-acetamido-3,4-di-O-acetyl-2-

deoxy-b- -glucopyranoside (45)

D

-glucopyranosyl)-(10

/

3)-(2,4,6-tri-O-acetyl-b-

D

/

4)-(2,3,6-tri-O-acetyl-b-

D-

/

D

To a soln of 44 (56 mg, 63 mmol) in CH₂Cl₂(0.45 mL)

and MeOH (0.55 mL) was added NaOMe (pH 10), and

the mixture was stirred for 2 h, then neutralized with

C₃₄H₅₉N₅O₂₁(M, 873.370): [Mꢀ

/

Na]^ꢀfound 896.359,

calcd 896.360. For H NMR data, see Table 8.

1

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2629Á

/2651

2651

3.34. 6-Aminohexyl 2-acetamido-2-deoxy-b-

glucopyranosyl-(103)-b- -galactopyranosyl-(10

glucopyranosyl-(106)-2-acetamido-2-deoxy-b-

glucopyranoside (8)

D

-

of building blocks 13 and 20. This work was ﬁnancially

supported by the EU, project BIO CT 95-0138.

/

D

/

4)-b-D-

/

D-

To a soln of 46 (10 mg, 11.44 mmol) in tert-BuOH (2

mL) and water (2 mL) was added 10% PdꢀC (50 mg)

and 1 drop of aq 25% NH₃. The mixture was stirred for

20 h under H₂, then acidified with Dowex 50ꢂ

8 (H^ꢀ)

References

/

1. Centers for Disease Control and Prevention. Prevention of

pneumococcal disease: recommendations of the Advisory

Committee on Immunization Practices (ACIP), MMWR

/

(pH 4) and loaded on a short column of Dowex 50ꢂ8

/

(H^ꢀ). Elution with water, to remove contaminants,

followed by 10% NH₄OH, and subsequent lyophiliza-

tion yielded 8, isolated as a white solid (8 mg, 82%); R_f

Morb. Mortal. Wkly. Rep. 1997, 46, 1Á24.

2. Fine, M. J.; Smith, M. A.; Carson, C. A.; Mutha, S. S.;

/

Sankey, S. S.; Weissfeld, L. A.; Kapoor, W. N. J. Am.

0.27 (2:1:1 AcOHÁ

/

1-BuOHÁ

/

water); [a]²_D⁰

ꢁ28 (c 0.5,

/

Med. Assoc. 1996, 275, 134Á

3. Doern, G. V.; Brueggemann, A. B.; Huynh, H.; Wingert,

E.; Rhomberg, P. Emerg. Infect. Dis. 1999, 5, 757Á765.

4. Klugman, K. P. Clin. Microbiol. Rev. 1990, 3, 171Á196.

5. Robbins, J. B.; Austrian, R.; Lee, C. J.; Rastogi, S. C.;

Schiffman, G.; Henrichsen, J.; Makala, P. H.; Broome, C.

/

141.

water); ¹³C NMR (75.5 MHz, D₂O): d 22.8 (2 C)

(NDCOCH₃), 25.3, 25.9, 27.4, and 29.0 (4 CH₂), 40.1

(CH₂ND₂), 56.2 and 56.3 (C-2^I, C-2^IV), 60.7, 61.2, 61.6,

69.3, and 71.2 (C-6^I, C-6^II, C-6^III, C-6^IV, OCH₂), 69.0,

70.4 (2 C), 70.7, 73.4, 74.2 (2 C), 75.0, 75.4, 75.6 (2 C),

76.3, 79.1, and 82.6 (C-3^I, C-4^I, C-5^I, C-2^II, C-3^II, C-4^II,

C-5^II, C-2^III, C-3^III, C-4^III, C-5^III, C-3^IV, C-4^IV, C-5^IV),

101.9, 103.3, 103.5, and 103.6 (C-1^I, C-1^II, C-1^III, C-1^IV),

175.1 and 175.6 (2 NDCOCH₃); HRMS of

/

¨

V.; Facklam, R. R.; Tiesjema, R. H.; Parke, J. C., Jr. J.

Infect. Dis. 1983, 148, 1136Á1159.

Ortqvist, A.; Hedlund, J.; Burman, L.-A.; Elbel, E.;

Hofer, M.; Leinonen, M.; Lindblad, I.; Sundelof, B.;

/

˚

¨

.

6.

¨

Kalin, M. Lancet 1998, 351, 399Á403.

/

C₃₄H₆₁N₃O₂₁(M, 847.379): [Mꢀ

/

H]^ꢀfound 848.390,

calcd 848.387. For H NMR data, see Table 9.

7. Rennels, M. B.; Edwards, K. M.; Keyserling, H. L.;

Reisinger, K. S.; Hogerman, D. A.; Madore, D. V.;

Chang, I.; Paradiso, P. R.; Malinoski, F. J.; Kimura, A.

1

3.35. 6-Aminohexyl b-

acetamido-2-deoxy-b-

galactopyranosyl-(10

-galactopyranosyl-(10

glucopyranoside (4)

D

-galactopyranosyl-(10

-glucopyranosyl-(103)-b-

4)-b-

-glucopyranosyl-(10

/

4)-2-

Pediatrics 1998, 101, 604Á

8. Adams, W. G.; Deaver, K. A.; Cochi, S. L. J. Am. Med.

Assoc. 1993, 269, 221Á226.

/

611.

D

/

D-

/

D

/

6)-[b-

/

D

/

4)]-2-acetamido-2-deoxy-b-D-

9. Richmond, P.; Borrow, R.; Miller, E.; Clark, S.; Sadler,

F.; Fox, A.; Begg, N.; Morris, R.; Cartwright, K. J. Infect.

Dis. 1999, 179, 1569Á

/

1572.

10. Lindberg, B.; Lonngren, J.; Powell, D. A. Carbohydr. Res.

To a soln of 8 (10.8 mg, 12.73 mmol) in aq 50 mM

sodium cacodylate buffer pH 7.5 (600 mL), containing 5

mM MnCl₂, BSA (0.5 mg), and NaN₃(0.02%), were

added alkaline phosphatase (30 U), UDP-galactose (25

mg, 40.96 mmol), and b-1,4-galactosyltransferase (5 U).

The mixture was incubated for 20 h at 37 8C, then water

(200 mL) was added. UDP-Galactose was removed using

¨

1977, 58, 177Á

11. Niggemann, J.; Kamerling, J. P.; Vliegenthart, J. F. G.

Bioorg. Med. Chem. 1998, 6, 1605Á1612.

12. Niggemann, J.; Kamerling, J. P.; Vliegenthart, J. F. G. J.

Chem. Soc. Perkin Trans. 1 1998, 3011Á3020.

/

186.

/

13. Mawas, F.; Niggemann, J.; Jones, C.; Corbel, M. J.;

Kamerling, J. P.; Vliegenthart, J. F. G. Infect. Immun.

a Dowex 1ꢂ

8 (Cl^ꢁ) column with water as eluent. The

/

2002, 70, 5107Á

14. Michalik, D.; Vliegenthart, J. F. G.; Kamerling, J. P. J.

Chem. Soc. Perkin Trans. 1 2002, 1973Á1981.

/

5114.

eluate was concentrated, and the residue applied to a

Bio-Gel P-2 column eluted with aq 0.1 M NH₄HCO₃at

a flow rate of 40 mL/h. The appropriate fractions were

freeze-dried to give 4 (11.4 mg, 76%); R_f0.15 (2:1:1

/

15. Joosten, J. A. F.; Kamerling, J. P.; Vliegenthart, J. F. G.

Carbohydr. Res. (People BAV020 same issue).

16. Schmidt, R. R.; Stumpp, M. Liebings Ann. Chem. 1983,

AcOHÁ

/

1-BuOHÁ

/

water); [a]²_D⁰

ꢁ

/

18 (c 1, water); HRMS

of C₄₆H₈₁N₃O₃₁(M, 1171.485): [Mꢀ

1172.503, calcd 1172.492. For H NMR data, see Table

10.

/

H]^ꢀfound

1249Á

17. Grundler, G.; Schmidt, R. R. Carbohydr. Res. 1985, 135,

203Á218.

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Acknowledgements

We thank C. Versluis (Utrecht University) for recording

the HRMS spectra, and N. Matsouki for the synthesis

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Article Doi

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

DOI: 10.1016/S0008-6215(03)00292-1

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Full text of DOI:10.1016/S0008-6215(03)00292-1

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