Synthesis of linear-type chondroitin clusters having a C8 spacer between disaccharide moieties and enzymatic transfer of D-glucuronic acid to the artificial glycans

www.elsevier.nl/locate/carres

Carbohydrate Research 332 (2001) 41–51

Synthesis of linear-type chondroitin clusters having a C₈

spacer between disaccharide moieties and enzymatic

transfer of

D-glucuronic acid to the artiﬁcial glycans

Jun-ichi Tamura,^a,c,* Hirofumi Urashima,^a,cKazunori Tsuchida,^bHiroshi Kitagawa,^b

Kazuyuki Sugahara^b

^aDepartment of En6ironmental Sciences, Faculty of Education and Regional Sciences, Tottori Uni6ersity,

Tottori 680-8551, Japan

^bDepartment of Biochemistry, Kobe Pharmaceutical Uni6ersity, Higashinada-ku, Kobe 658-8558, Japan

^cCREST, Japan Science and Technology Corporation (JST), Japan

Received 16 November 2000; accepted 28 February 2001

Abstract

Newly designed linear-type glycoclusters were synthesized which involve a chondroitin repeating disaccharide

ligand and a hydrophobic octyl ether spacer. The spacer mimics the corresponding disaccharide unit. Repeating

elongation of the pseudo-tetrasaccharide that was derived from the common cluster unit [8)-octyl-(13)-b-

NAc-(14)-b- -GlcA-(1] allowed the syntheses of up to the pseudo-decasaccharide analog of chondroitin. An

enzymatic -GlcA transfer at the non-reducing end of the synthesized artiﬁcial glycans by GlcATase II was observed.

D-Gal-

D

Keywords: Chondroitin; Glycocluster; Linear-type glycocluster; Spacers; Glycosyl transfer

1. Introduction

clusters are required in order to understand

the carbohydrate–protein interactions at the

molecular level.

Many artiﬁcial carbohydrate clusters (glyco-

clusters) have already been synthesized, in-

volving different shapes, spacer lengths and

saccharide moieties.^1,2These efforts have

proved valuable in the identiﬁcation of the

so-called ‘cluster effect’ involving aggregated

ligands and ﬂexible spacers in the modulation

of the interaction between carbohydrates and

proteins.¹By using simpliﬁed glycoclusters, it

is anticipated that carbohydrate-based drugs

that inhibit bacterial and viral infections can

be developed. Thus, structurally deﬁned glyco-

Major glycoclusters can be classiﬁed into

two categories based on their shapes: the den-

drimer (A) and pendant (B) types as shown in

Scheme 1. Commonly, these ligands locate at

the end of spacers extending from the core.

We now report the synthesis of novel linear-

type glycoclusters mimicking chondroitin sul-

fate that is a member of the GAGs. These

glycoclusters include a hydrophobic spacer be-

tween the carbohydrate ligand as depicted in

Scheme 1(C). There is a decisive difference

from the above dendrimer- and pendant-type

glycoclusters. Two target linear-type glyco-

clusters (1 and 2) mimicking the hexa- and

decasaccharides of chondroitin, respectively,

* Corresponding author. Tel./fax: +81-857-315108.

E-mail address: jtamura@fed.tottori-u.ac.jp (J. Tamura).

PII: S0008-6215(01)00071-4

42

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

are shown in Scheme 2. To more easily syn-

thesize these cluster molecules, we ﬁrst

planned to avoid any chemical sulfation.

Some linear-type glycoclusters have already

been synthesized. In 1984, Jegge and Lehmann

reported the synthesis of a pseudo-mal-

amide-type spacer.⁵Contrary to these linear-

type glycoclusters, our glycocluster is suitably

designed for elongation like natural polysac-

charides. Thus, higher biological activities are

expected by increasing the number of ligands.

totrioside.³They replaced the middle

D

-glu-

cose moiety of a

D

-maltotrioside with an

2. Results and discussion

acyclic hydrophilic spacer. This artiﬁcial

pseudo-maltotrioside competitively inhibited

The targeted glycoclusters, 1 and 2 (Scheme

2), were synthesized as follows. To effectively

elongate the cluster molecules, we designed a

common disaccharide unit 9 having an 8-

the hydrolysis of p-nitrophenyl a- -mal-

D

totrioside by porcine a-amylase. In 1990, Leh-

mann and Petry synthesized a spacer-modiﬁed

glycoconjugate having an aliphatic C₁₀spacer

levulinoyloxyoctyl ether at O-3 of the

D

-Gal-

between the b- -GlcNAc moieties that were

D

NAc residue as shown in Scheme 3. The

levulinoyl protecting group of the known dis-

accharide 3⁶was removed with hydrazine ace-

tate to give 4 in 84% yield, thus allowing the

introduction of a C₈moiety at the liberated

hydroxyl group of 4. In 1999, Neda et al.

reported a similar synthesis of a glycocluster

having a C₃spacer between the monosaccha-

ride moieties by employing 2-(3-bromopro-

poxy)tetrahydro-2H-pyran.⁷The alkylation

with 8-bromooctanol (1.5 equiv) and sodium

hydride (1.5 equiv) at −500 °C for 5 h

afforded the desired compound 5 without af-

fecting the acyl and silyl groups. The residual

4 was recovered in 38% yield. A higher tem-

perature and excess amount of sodium hy-

dride decreased the yield of 5 and the recovery

of the starting 4. The hydroxyl group of the

octyl moiety was then quantitatively protected

as a levulinoyl ester. The silyl group of 6 was

also quantitatively removed by using tetra-n-

butylammonium ﬂuoride and acetic acid as

previously reported.⁶The resulting C-6 alco-

hol 7 was converted to a carboxylic acid via a

Swern oxidation followed by further oxidation

mediated with sodium chlorite. The carboxylic

acid was ﬁnally esteriﬁed with (trimethylsi-

lyl)diazomethane to yield the methyl ester 8.

The total yield was 70% in these three steps.

The azide of 8 was reduced by the Lindlar

catalyzed hydrogenolysis and the following

conventional acetylation gave the acetamide 9

in 96% yield (two steps). Having in hand the

key disaccharide 9, we converted it to the

corresponding glycosyl donor 11 and acceptor

12. Protecting groups capping the head and

tail of 9 (MP and Lev) were chemoselectively

part of the biantennary core of the N-linked

glycoprotein.⁴This so-called head-on-type gly-

cocluster, which contains ligands (b- -Glc-

D

NAc) glycosylated with 1,10-decanediol, acted

as a substrate for galactosyltransferase from

bovine milk. Recently, the Kusumoto’s group

published the synthesis of glycoclusters with a

bis(disaccharide) structure linked through an

Scheme 1. Some typical artiﬁcial glycoclusters: dendrimer-

type (A) and pendant-type (B) glycoclusters have ligands at

the end of spacers. The linear-type glycocluster that we pro-

pose incorporate ligands between the spacers as in (C).

Scheme 2.

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

43

Scheme 3. Abbreviations: MP, 4-MeOC₆H₄; MBz, 4-MeC₆H₄CO; TBDPS, t-BuPh₂Si; Lev, MeCO(CH₂)₂CO; Piv, t-BuCO.

removed to give 10 and 12, respectively, both

in 95% yields. The hemiacetal 10 was then

converted to the corresponding imidate 11 in

88% yield. These disaccharide moieties were

coupled by employing trimethylsilyl triﬂ-

uoromethanesulfonate in dichloromethane to

give the tetrasaccharide 13 in 69% yield. It is

noteworthy that the 4-methylbenzoyl group

completely controlled the newly generated

anomeric conﬁguration of 13 as b during the

coupling reaction. The levulinoyl group of 13

was then removed as above in 70% yield.

Thus, we have regio- and stereoselectively pre-

pared the di- and tetrasaccharide acceptors, 12

and 14.

follows: (i) The levulinoyl group of 18 was

removed prior to the other deprotections; (ii)

benzylidene acetals were hydrolyzed in the

presence of camphorsulfonic acid; (iii) methyl

uronates were then exchanged with lithium

salts using aqueous lithium hydroxide and the

remaining acyl groups were removed by hy-

drolysis with aqueous sodium hydroxide.

Thus, the targeted compounds (1 and 2) were

obtained in 29 and 56% yields as sodium salts,

respectively

(four

and

three

steps,

respectively).

We also examined the enzymatic incorpora-

tion of a

D

-GlcA residue into the glycoclus-

In order to protect the cluster-end, we cou-

pled the corresponding disaccharide donors

15⁶and 17 with the acceptors 12 and 14,

respectively, as shown in Scheme 4. As for the

synthesis of 13, the protected pseudo-hexa-

and decasaccharides 18 and 19 were stereose-

lectively obtained in 75 and 48% yields, re-

spectively. Compared to the elongation results

for the synthesis of the natural-type chon-

droitin oligosaccharide, the coupling yields

substantially increased.^8†Finally, all the pro-

tecting groups of 18 and 19 were removed as

^†Coupling of the corresponding donor (disaccharide) and

acceptor (tetrasaccharide) afforded the hexasaccharide in 41%

yield. These saccharides possess azide and TBDMS groups

instead of acetamide and MP, respectively.

Scheme 4.

44

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

Table 1

Incorporation of

triple-stage quadrupole mass spectrometer

D

-[¹⁴C]GlcA into various acceptors by glu-

(Finnigan MAT TSQ 700) equipped with a

FAB ion source. Silica-gel chromatography,

analytical TLC and preparative TLC (PTLC)

were done on a column of Silica Gel 60 (E.

Merck) or glass plates coated with Silica Gel

F₂₅₄(E. Merck), respectively. Gels for size-ex-

clusion chromatography (Sephadex LH-20,

LH-60 and Biobeads S-X1) were purchased

from Pharmacia and BIORAD, respectively.

Molecular sieves (MS) were purchased from

GL Science, Inc. and activated at 180 °C un-

der diminished pressure prior to use. All reac-

tions in organic solvents were performed

under dry Ar atmosphere. As a usual work-

up, the organic phase of the reaction mixture

was washed with aq NaHCO₃, brine and dried

over anhyd MgSO₄.

curonosyltransferase II

Acceptor

D

-[¹⁴C]GlcA

(dpm)

1:

2:

D

-GalNAc-

GalNAc- -GlcA)-MP

-GalNAc- -GlcA-(C₈-

-GlcA)₂-MP

D

-GlcA-(C₈-

D

-

120

D

50

GalNAc-

D

-GalNAc-(

D

-GlcA-

D

-GalNAc)

-GalNAc)₂

-GalNAc)₅

200

14,000

98,000

ters, 1 and 2, using GlcATase II⁹partially

puriﬁed from fetal bovine serum. This enzyme

speciﬁcally transfers the

4-Methoxyphenyl O-(2-azido-4,6-O-benzyli-

D

-GlcA residue to

dene-2-deoxy-i-

6-O-tert-butyldiphenylsilyl-2,3-di-O-(4-methyl-

benzoyl)-i- -glucopyranoside (4).—To a solu-

tion of 4-methoxyphenyl O-(2-azido-4,6-O-

benzylidene-2-deoxy-3-O-levulinoyl-b- -galac-

topyranosyl)-(14)-6-O-tert-butyldiphenyl-

-gluco-

D

-galactopyranosyl)-(14)-

O-3 of b- -GalNAc in the repeating disacchar-

D

ide region. These results were compared with

those of the chondroitin oligosaccharides as

shown in Table 1. It proved that 1 and 2 were

D

able to accept

D

-GlcA with a similar speciﬁc-

ity as that found for the shorter chondroitin

repeating oligosaccharide units.

silyl-2,3-di-O-(4-methylbenzoyl)-b-

D

pyranoside (3)⁶(705.4 mg, 621.9 mmol) in

toluene (1 mL) and EtOH (5 mL) was added

H₂NNH₂·AcOH (570 mg, 6.19 mmol) with

stirring for 70 min. The volatiles were re-

moved under diminished pressure and the

residue was eluted from a column of gel per-

meation (LH-20, 1:1 CHCl₃–MeOH) to give 4

(571.3 mg) in 89% yield as a syrup: [h]_D

In conclusion, we have synthesized newly

designed linear-type glycoclusters, 1 and 2,

having a hydrophobic spacer between the dis-

accharide moieties. We replaced the chon-

droitin repeating disaccharide with a spacer

involving the hydrophobic octyl ether. The

repeating elongation of the cluster unit al-

lowed the syntheses of a pseudo-decasaccha-

ride analog of chondroitin showing the

possible synthesis of longer linear-type glyco-

clusters. In addition, it proved that the glyco-

clusters, 1 and 2, were able to enzymatically

1

+9.8° (c 0.41, CHCl₃); H NMR (CDCl₃): l

7.93–6.72 (m, 27 H, Ph), 5.76 (dd, 1 H, J_2,3

9.76, J_3,49.51 Hz, H-3^I), 5.61 (dd, 1 H, J_1,2

7.81 Hz, H-2^I), 5.32 (s, 1 H, PhCH), 5.14 (d,

1 H, H-1^I), 4.55 (d, 1 H, J_1,28.05 Hz, H-1^II),

4.41 (t, 1 H, J_4,59.51 Hz, H-4^I), 4.24 (dd, 1 H,

J_5,6a2.93, J_gem11.96 Hz, H-6a^I), 4.14 (dd, 1 H,

H-6b^I), 3.95 (d, 1 H, J_3,43.66 Hz, H-4^II), 3.89

(d, 1 H, H-6a^II), 3.73–3.72 (m, 5 H, H-5^I,

H-6b^II, OMe), 3.49 (dd, 1 H, J_2,310.00 Hz,

H-2^II), 3.41–3.39 (m, 1 H, H-3^II), 3.09 (bs, 1

H, H-5^II), 2.48 (d, 1 H, J_3,OH9.76 Hz, OH),

2.36, 2.19 (2 s, 3 H×2, 2 PhMe), 1.09 (s, 9 H,

tert-Bu). Anal. Calcd for C₅₈H₆₁N₃O₁₃Si: C,

67.23; H, 5.93; N, 4.06. Found: C, 67.46; H,

6.18; N, 3.94.

accept a

end.

D

-GlcA residue at the non reducing-

3. Experimental

General methods.—Optical rotations were

measured at 2293 °C with a HORIBA polar-

1

imeter. H NMR assignments were conﬁrmed

by two-dimensional HH COSY experiments

with a JEOL LA 400 MHz spectrometer. The

FAB mass spectra were measured with a

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

45

1

syrup: [h]_D+20.5° (c 1.10, CHCl₃); H NMR

(CDCl₃): l 7.88–6.71 (m, 27 H, Ph), 5.76 (dd,

1 H, J_2,39.76, J_3,49.51 Hz, H-3^I), 5.59 (dd, 1

H, J_1,27.81 Hz, H-2^I), 5.30 (s, 1 H, PhCH),

5.13 (d, 1 H, H-1^I), 4.49 (d, 1 H, J_1,28.29 Hz,

H-1^II), 4.35 (t, 1 H, J_4,59.51 Hz, H-4^I), 4.28

(dd, 1 H, J_5,6a3.15, J_gem11.71 Hz, H-6a^I), 4.15

(d, 1 H, H-6b^I), 4.08–4.01 (m, 3 H, H-4^II,

CH₂O), 3.86 (d, 1 H, J_gem11.47 Hz, H-6a^II),

3.78–3.53 (m, 3 H, H-5^I, H-6b^II, 1/2 CH₂O),

3.75 (s, 3 H, OMe), 3.65 (dd, 1 H, H-2^II),

3.45–3.37 (m, 1 H, 1/2 CH₂O), 3.10 (dd, 1 H,

J_2,310.25, J_3,43.42 Hz, H-3^II), 2.95 (bs, 1 H,

H-5^II), 2.76–2.54 (m, 4 H, 2 CH₂), 2.35, 2.28,

(2 s, 3 H×2, 3 PhMe), 2.19 (s, 3 H, COMe),

1.60–1.25 (m, 12 H, 6 CH₂), 1.09 (s, 9 H,

tert-Bu). Anal. Calcd for C₇₁H₈₃N₃O₁₆Si: C,

67.53; H, 6.64. Found: C, 67.23; H, 7.01.

4-Methoxyphenyl O-[2-azido-4,6-O-benzyli-

dene - 2 - deoxy - 3 - O - (8 - hydroxyoctyl) - i -

galactopyranosyl] - (14) - 6 - O - tert - butyldi-

phenylsilyl-2,3-di-O-(4-methylbenzoyl)-i-

D

-

D

-

glucopyranoside (5).—To a solution of 4 (2.36

g, 2.28 mmol) in DMF (20 mL) cooled to

−50 °C, NaH (50%, 140 mg, 3.5 mmol) was

added with stirring. 8-Bromooctanol (0.6 mL,

3.5 mmol) was added to the reaction mixture 1

h later, and then the temperature was raised

gradually up to 0 °C within 5 h. The reaction

was quenched with ice and extracted with

EtOAc. The organic phase was treated as

described in general methods. The volatiles

were removed under diminished pressure and

the residue was eluted from a column of silica

gel (50:1–5:1 toluene–toluene–EtOAc) to give

5 (1.19 g, 53%) as a syrup together with

residual 4 (865.5 mg, 38%): [h]_D+19.8° (c

1

1.11, CHCl₃); H NMR (CDCl₃): l 7.88–6.71

dene-2-deoxy-3-O-(8-le6ulinoyloxyoctyl)-i-

galactopyranosyl]-(14)-2,3-di-O-(4-methyl-

benzoyl)-i- -glucopyranoside (7).—To a solu-

D

-

(m, 27 H, Ph), 5.75 (dd, 1 H, J_2,39.76, J_3,49.51

Hz, H-3^I), 5.59 (dd, 1 H, J_1,27.81 Hz, H-2^I),

5.30 (s, 1 H, PhCH), 5.13 (d, 1 H, H-1^I), 4.49

(d, 1 H, J_1,28.29 Hz, H-1^II), 4.35 (t, 1 H, J_4,5

9.51 Hz, H-4^I), 4.28 (dd, 1 H, J_5,6a3.17, J_gem

11.71 Hz, H-6a^I), 4.15 (d, 1 H, H-6b^I), 4.01 (d,

1 H, J_3,43.42 Hz, H-4^II), 3.86 (d, 1 H, J_gem

10.25 Hz, H-6a^II), 3.76–3.57 (m, 5 H, H-5^I,

H-6b^II, 3/2 CH₂O), 3.75 (s, 3 H, OMe), 3.61

(dd, 1 H, H-2^II), 3.45–3.36 (m, 2 H, 1/2

CH₂O, OH), 3.10 (dd, 1 H, J_2,310.25 Hz,

H-3^II), 2.95 (bs, 1 H, H-5^II), 2.36, 2.21 (2 s, 3

H×2, 2 PhMe), 1.58–1.25 (m, 12 H, 6 CH₂),

1.11 (s, 9 H, tert-Bu). Anal. Calcd for

C₆₆H₇₇N₃O₁₄Si: C, 68.07; H, 6.68; N, 3.61.

Found: C, 68.13; H, 7.01; N, 3.28.

D

tion of 6 (2.902 g, 2.299 mmol) in THF (30

mL) were added a 1 M solution of TBAF in

THF (11.0 mL, 11.0 mmol) and HOAc (1.3

mL, 23 mmol) with stirring. After 4 days, the

volatiles were removed and the residue was

diluted with CHCl₃and brine. The organic

phase was treated as described in general

methods and the residue was eluted from a

column of silica gel (10:1–1:1 toluene–

EtOAc) to give 7 (1.847 g, 80%) as a syrup:

[h]_D+38° (c 0.98, CHCl₃); ¹H NMR

(CDCl₃): l 7.86–6.76 (m, 17 H, Ph), 5.79 (t, 1

H, J_2,3=J_3,49.51 Hz, H-3^I), 5.54 (dd, 1 H, J_1,2

7.81 Hz, H-2^I), 5.31 (s, 1 H, PhCH), 5.18 (d,

1 H, H-1^I), 4.33 (d, 1 H, J_1,28.29 Hz, H-1^II),

4.27 (t, 1 H, J_4,59.51 Hz, H-4^I), 4.09 (d, 1 H,

J_3,42.93 Hz, H-4^II), 4.05–4.01 (m, 4 H, H-

6ab^I, CH₂O), 3.79 (dt, 1 H, J_5,6a4.39, J_5,6b2.39

Hz, H-5^I), 3.75 (s, 3 H, OMe), 3.70 (dd, 1 H,

H-2^II), 3.68–3.37 (m, 5 H, H-6ab^II, CH₂O,

OH), 3.25 (dd, 1 H, J_2,310.25 Hz, H-3^II), 2.93

(bs, 1 H, H-5^II), 2.76–2.55 (m, 4 H, 2 CH₂),

2.35, 2.28, (2 s, 2 H×3, 2 PhMe), 2.19 (s, 3

H, COMe), 1.55–1.25 (m, 12 H, 6 CH₂). Anal.

Calcd for C₅₅H₆₅N₃O₁₆: C, 64.50; H, 6.40; N,

4.10. Found: C, 64.71; H, 6.56; N, 3.90.

4-Methoxyphenyl O-[2-azido-4,6-O-benzyli-

dene-2-deoxy-3-O-(8-le6ulinoyloxyoctyl)-i- -

D

galactopyranosyl] - (14) - 6 - O - tert - butyldi-

phenylsilyl-2,3-di-O-(4-methylbenzoyl)-i-

D

-

glucopyranoside (6).—To a solution of 5

(452.2 mg, 388.3 mmol) in Py (5 mL) were

added a solution of 1 M levulinic anhydride in

1,2-(CH₂Cl)₂(2.0 mL) and a catalytic amount

of DMAP with stirring. After 4 h, an excess of

ice was added to the reaction mixture. The

reaction mixture was diluted with CHCl₃. The

organic phase was treated as described in gen-

eral methods. The residue obtained was eluted

from a column of silica gel (25:1–10:1 tolu-

ene–EtOAc) to give 6 (470.9 mg, 96%) as a

Methyl [2-azido-4,6-O-benzylidene-2-deoxy-

3-O-(8-le6ulinoyloxyoctyl)-i- -galactopyran-

D

46

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

in EtOAc (1 mL) and the mixture was stirred

under H₂atmosphere overnight. Insoluble ma-

terials were removed on Celite and the ﬁltrate

was concentrated under diminished pressure.

The residue was acetylated (Ac₂O-Py) and the

crude materials was puriﬁed on a column of

silica gel (tolueneꢀ5:1ꢀ1:5 toluene–EtOAc)

to give 9 (23.6 mg, 96%) as a syrup: [h]_D

osyl]-(14)-[4-methoxyphenyl

2,3-di-O-(4-

uronate

methylbenzoyl)-i- -glucopyranosid]

D

(8).—Dimethyl sulfoxide (1.3 mL, 18.3 mmol)

was added dropwise within 3 min to a 1 M

solution of (COCl)₂in CH₂Cl₂(9.2 mL) at

−78 °C with stirring. After 5 min, a solution

of 7 (1.892 g, 1.847 mmol) in CH₂Cl₂(20 mL)

was added within 3 min and stirring was

continued for 30 min. Then, Et(i-Pr)₂N (6.6

mL, 36.8 mmol) diluted with the same volume

of CH₂Cl₂was added to the reaction mixture

and the cooling bath was removed 5 min later.

The reaction mixture was stirred for more 25

min and diluted with CH₂Cl₂, washed with 1

M HCl, brine, aq NaHCO₃and brine again.

The volatiles were removed under diminished

pressure and the residue was diluted in tert-

BuOH (25 mL). To the solution were added

2-methyl-2-butene (10 mL), water (25 mL),

NaHPO₄·2 H₂O (2.5 g), and NaClO₂(2.5 g).

The mixture was stirred overnight and the

volatiles were removed under diminished pres-

sure. The residue was dissolved in toluene (9

mL) and MeOH (3 mL), and an excess of

Me₃SiCHN₂(n-hexane solution) was added to

the solution. The reaction mixture was con-

centrated and the residue was eluted from a

column of silica gel (5:1–1:1 toluene–EtOAc)

to give 8 (1.498 g, 77%) as a syrup: [h]_D

1

+42.2° (c 1.36, CHCl₃); H NMR (CDCl₃): l

7.88–6.75 (m, 17 H, Ph), 5.77 (t, 1 H, J_2,3=

J_3,48.78 Hz, H-3^I), 5.64 (d, 1 H, J 6.83 Hz,

NH), 5.53 (dd, 1 H, J_1,27.32 Hz, H-2^I), 5.33

(s, 1 H, PhCH), 5.23 (d, 1 H, J_1,28.05 Hz,

H-1^II), 5.21 (d, 1 H, H-1^I), 4.56 (t, 1 H, J_4,5

9.03 Hz, H-4^I), 4.28 (d, 1 H, H-5^I), 4.22 (dd, 1

H, J_2,38.78 Hz, H-3^II), 4.05 (d, 1 H, J_3,45.37

Hz, H-4^II), 4.04–4.01 (m, 2 H, CH₂O), 3.86 (d,

1 H, J_gem12.20 Hz, H-6a^II), 3.78, 3.74 (2 s, 3

H×2, 2 OMe), 3.64 (dd, 1 H, H-6b^II), 3.57–

3.24 (m, 3 H, H-2^II, CH₂O), 2.96 (bs, 1 H,

H-5^II), 2.75–2.54 (m, 4 H, 2 CH₂), 2.34, 2.29,

(2 s, 3 H×2, 2 PhMe), 2.18 (s, 3 H, COMe),

1.97 (s, 3 H, NAc), 1.60–1.25 (m, 12 H, 6

CH₂). Anal. Calcd for C₅₈H₆₉NO₁₈·0.5 H₂O:

C, 64.66; H, 6.56; N, 1.30. Found: C, 64.69;

H, 6.56; N, 1.30.

Methyl [2-acetamido-4,6-O-benzylidene-2-

deoxy-3-O-(8-le6ulinoyloxyoctyl)-i- -galacto-

D

pyranosyl]-(14)-2,3-di-O-(4-methylbenzoyl)-

-glucopyranuronate (10).—To a solution of 9

1

+25.3° (c 1.20, CHCl₃); H NMR (CDCl₃): l

D

7.86–6.75 (m, 17 H, Ph), 5.79 (t, 1 H, J_2,3=

J_3,49.03 Hz, H-3^I), 5.59 (dd, 1 H, J_1,27.32 Hz,

H-2^I), 5.30 (s, 1 H, PhCH), 5.23 (d, 1 H,

H-1^I), 4.51 (t, 1 H, J_4,59.03 Hz, H-4^I), 4.35 (d,

1 H, H-5^I), 4.33 (d, 1 H, J_1,28.05 Hz, H-1^II),

4.09–4.03 (m, 3 H, H-4^II, CH₂O), 3.83, 3.75 (2

s, 3 H×2, 2 OMe), 3.74–3.55 (m, 3 H, H-

6ab^II, 1/2 CH₂O), 3.36 (dd, 1 H, H-2^II), 3.44–

3.38 (m, 1 H, 1/2 CH₂O), 3.15 (dd, 1 H, J_2,3

10.49, J_3,43.66 Hz, H-3^II), 3.00 (bs, 1 H,

H-5^II), 2.75–2.54 (m, 4 H, 2 CH₂), 2.35, 2.28,

(2 s, 3 H×2, 2 PhMe), 2.19 (s, 3 H, COMe),

1.59–1.26 (m, 12 H, 6 CH₂). Anal. Calcd for

C₅₆H₆₅N₃O₁₇·0.8 H₂O: C, 63.05; H, 6.31; N,

3.94. Found: C, 63.19; H, 6.34; N, 3.64.

(420.4 mg, 393.5 mmol) in MeCN (10 mL) and

water (2 mL) was added cerium (IV) ammo-

nium nitrate (650 mg, 1.19 mmol) with stirring

at 0 °C. After 1.5 h, CHCl₃was added to the

reaction mixture. The organic phase was

washed with brine and dried over HgSO₄. The

volatiles were removed under diminished pres-

sure and the residue was eluted from a column

of silica gel (10:1–1:2 toluene–EtOAc) to give

1

10 (290.1 mg, 77%) as a syrup: H NMR

(CDCl₃): l 7.91–7.08 (m, 13 H, Ph), 6.05 (t, 1

H, J_2,3=J_3,49.03 Hz, H-3^I), 5.72 (d, 1 H, J

6.58 Hz, NH), 5.68 (m, 1 H, H-1^I), 5.34 (s, 1

H, PhCH), 5.25 (d, 1 H, J_1,28.05 Hz, H-1^II),

5.10 (dd, 1 H, J_1,2B1.0 Hz, H-2^I), 4.68 (d, 1

H, J_4,59.27 Hz, H-5^I), 4.42 (dd, 1 H, H-4^I),

4.24 (m, 1 H, H-3^II), 4.06–4.01 (m, 3 H, H-4^II,

CH₂O), 3.92 (d, 1 H, J_gem12.20 Hz, H-6a^II),

3.81 (s, 3 H, COOMe), 3.77–3.42 (m, 2 H,

H-6b^II, 1/2 CH₂O), 3.34–3.17 (m, 2 H, H-2^II,

1/2 CH₂O), 2.96 (s, 1 H, H-5^II), 2.75–2.54 (m,

Methyl [2-acetamido-4,6-O-benzylidene-2-

deoxy-3-O-(8-le6ulinoyloxyoctyl)-i- -galacto-

D

pyranosyl]-(14)-[4-methoxyphenyl 2,3-di-O-

(4-methylbenzoyl)-i- -glucopyranosid] uronate

D

(9).—The Lindlar catalyst (100 mg) was

added to a solution of 8 (24.3 mg, 23.1 mmol)

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

47

NH), 5.53 (dd, 1 H, J_1,27.08 Hz, H-2^I), 5.33

(s, 1 H, PhCH), 5.23 (d, 1 H, J_1,28.05 Hz,

H-1^II), 5.21 (d, 1 H, H-1^I), 4.56 (dd, 1 H, J_4,5

9.03 Hz, H-4^I), 4.30 (d, 1 H, H-5^I), 4.22 (dd, 1

H, J_2,310.98, J_3,43.17 Hz, H-3^II), 4.05 (d, 1 H,

H-4^II), 3.86 (d, 1 H, J_gem12.20 Hz, H-6a^II),

3.78, 3.73 (2 s, 3 H×2, 2 OMe), 3.69–3.23

(m, 5 H, H-6b^II, 2 CH₂O, OH), 2.97 (bs, 1 H,

H-5^II), 2.33, 2.29, (2 s, 3 H×2, 2 PhMe), 1.95

(s, 3 H, NAc), 1.65–1.26 (m, 12 H, 6 CH₂).

Anal. Calcd for C₅₃H₆₃NO₁₆·H₂O: C, 64.41;

H, 6.64. Found: C, 64.09; H, 6.44.

4 H, 2 CH₂), 2.33, 2.28, (2 s, 3 H×2, 2

PhMe), 2.18, (s, 3 H, COMe), 1.97 (s, 3 H,

NAc), 1.61–1.25 (m, 12 H, 6 CH₂). Anal.

Calcd for C₅₁H₆₃NO₁₇·H₂O: C, 62.49; H, 6.70;

N, 1.43. Found: C, 62.28; H, 6.63; N, 1.73.

Methyl [2-acetamido-4,6-O-benzylidene-2-

deoxy-3-O-(8-le6ulinoyloxyoctyl)-i-

pyranosyl] - (14) - [2,3 - di - O - (4 - methylben-

zoyl)-h- -glucopyranosyl trichloroacetimidate]

D

-galacto-

D

uronate (11).—To a solution of 10 (226.7 mg,

235.6 mmol) in CH₂Cl₂(5 mL) were added

CCl₃CN (120 mL) and DBU (18 mL) with

stirring at 0 °C. After 1 h, the reaction mixture

was puriﬁed on a column of silica gel (5:1–1:3

toluene–EtOAc) to give 11 (228.7 mg, 88%) as

a syrup. A small amount of the product was

further puriﬁed over PTLC (1:5 toluene–

Methyl [2-acetamido-4,6-O-benzylidene-2-

deoxy-3-O-(8-le6ulinoyloxyoctyl)-i- -galacto-

D

pyranosyl]-(14)-[methyl 2,3-di-O-(4-methyl-

benzoyl)-i- -glucopyranosyluronate]-(18)-

octyl-(13)-[2-acetamido-4,6-O-benzylidene-

2 - deoxy - i - - galactopyranosyl] - (14) - [4-

methoxyphenyl 2,3-di-O-(4-methylbenzoyl)-i-

-glucopyranosid] uronate (13).—To a solu-

D

1

EtOAc): [h]_D+87.4° (c 1.03, CHCl₃); H

D

NMR (CDCl₃): l 8.59 (s, 1 H, NH), 7.90–

7.80 (m, 4 H, Ph), 7.33–7.08 (m, 9 H, Ph),

6.75 (d, 1 H, J_1,23.66 Hz, H-1^I), 6.13 (brt, 1

H, J 9.52 Hz, H-3^I), 5.63 (d, 1 H, J 7.07 Hz,

NH), 5.38 (dd, 1 H, J_2,310.0 Hz, H-2^I), 5.33

(s, 1 H, PhCH), 5.29 (d, 1 H, J_1,28.29 Hz,

H-1^II), 4.60 (d, 1 H, J_4,510.25 Hz, H-5^I), 4.48

(dd, 1 H, J_3,49.42 Hz, H-4^I), 4.25 (dd, 1 H, J_2,3

10.49, J_3,43.91 Hz, H-3^II), 4.05–4.01 (m, 3 H,

H-4^II, CH₂O), 3.86–3.76 (m, 1 H, H-6a^II), 3.82

(s, 3 H, COOMe), 3.61–3.53 (m, 2 H, H-6b^II,

1/2 CH₂O), 3.33 (m, 1 H, H-2^II), 3.31–3.17

(m, 1 H, 1/2 CH₂O), 2.86 (s, 1 H, H-5^II),

2.75–2.54 (m, 4 H, 2 CH₂), 2.33, 2.28, (2 s, 3

H×2, 2 PhMe), 2.19, (s, 3 H, COMe), 2.00

(s, 3 H, NAc), 1.58–1.25 (m, 12 H, 6 CH₂).

This compound was used for glycosylation

without further puriﬁcation.

D

tion of 11 (72.4 mg, 65.4 mmol) and 12 (49.4

mg, 50.9 mmol) in CH₂Cl₂(3 mL) was added

MS AW300 (100 mg). This mixture was

stirred for 30 min at rt, and then cooled to

−20 °C. To this solution was added a solu-

tion of 0.15 M Me₃SiOTf in CH₂Cl₂(1.0 mL,

0.15 mmol) with stirring and the reaction tem-

perature was gradually raised to rt. After stir-

ring overnight, an excess of Et₃N, CH₂Cl₂,

and brine were added. Insoluble materials

were ﬁltered on Celite. The organic phase was

treated as described in general methods. The

crude materials were subjected to a gel perme-

ation chromatography (LH-60, 1:1 CHCl₃–

MeOH) to give 13 (67.3 mg) in 68% yield as a

1

syrup: [h]_D+72.0° (c 0.15, CHCl₃); H NMR

Methyl [2-acetamido-4,6-O-benzylidene-2-

(CDCl₃): l 7.88–6.75 (m, 30 H, Ph), 5.80 (dd,

1 H, J_2,38.54, J_3,49.03 Hz, H-3^I), 5.70 (dd, 1

H, J_2,39.27, J_3,49.03 Hz, H-3^III), 5.61 (d, 1 H,

J 6.83 Hz, NH), 5.59 (d, 1 H, J 7.07 Hz, NH),

5.53 (dd, 1 H, J_1,27.32 Hz, H-2^I), 5.31, 5.31 (2

s, 1 H×2, 2 PhCH), 5.28 (t, 1 H, J_1,27.32 Hz,

H-2^III), 5.22 (d, 1 H, J_1,28.54 Hz, H-1^IV), 5.21

(d, 1 H, H-1^I), 5.17 (d, 1 H, J_1,28.29 Hz,

H-1^II), 4.69 (d, 1 H, H-1^III), 4.56 (t, 1 H, J_4,5

8.57 Hz, H-4^I), 4.42 (t, 1 H, J_4,59.03 Hz,

H-4^III), 4.28 (d, 1 H, H-5^I), 4.18–4.16 (m, 2 H,

H-3^II, 3^IV), 4.14 (d, 1 H, H-5^III), 4.08–4.01 (m,

6 H, H-4^II, 4^IV, 2 CH₂O), 3.86–3.24 (m, 10 H,

H-2^II, 2^IV, 6ab^II, 6ab^IV, 2 CH₂O), 3.82, 3.78,

3.74 (3 s, 3 H×2, 3 OMe), 2.95 (bs, 1 H,

deoxy - 3 - O - (8 - hydroxyoctyl) - i -

D

- galacto-

pyranosyl]-(14)-[4-methoxyphenyl 2,3-di-O-

(4-methylbenzoyl)-i- -glucopyranosid]uronate

D

(12).—To a solution of 9 (136.4 mg, 127.7

mmol) in EtOH (10 mL) and toluene (2 mL)

was added H₂NNH₂·AcOH (120 mg, 1.30

mmol) with stirring for 3 h. The volatiles were

removed under diminished pressure and the

residue was eluted from a column of gel per-

meation (LH-20, 1:1 CHCl₃–MeOH) to give

12 (117.6 mg) in 95% yield as a syrup: [h]_D

1

+43° (c 0.52, CHCl₃); H NMR (CDCl₃): l

7.88–6.74 (m, 17 H, Ph), 5.78 (t, 1 H, J_2,3=

J_3,48.78 Hz, H-3^I), 5.69 (d, 1 H, J 6.83 Hz,

48

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

H-5^IV), 2.89 (bs, 1 H, H-5^II), 2.74–2.54 (m, 4

H, 2 CH₂), 2.34, 2.31, 2.28, 2.27 (4 s, 3 H×4,

4 PhMe), 2.22, (s, 3 H, COMe), 1.98, 1.95 (2

s, 3 H×2, 2 NAc), 1.47–0.96 (m, 12 H, 6

CH₂). Anal. Calcd for C₁₀₄H₁₂₄N₂O₃₀: C,

65.25; H, 6.54; N, 1.46. Found: C, 65.08; H,

6.73; N, 1.88.

glucopyranosyl trichloroacetimidate] uronate

(17).—To a solution of 4-methoxyphenyl (2-

acetamido-4,6-O-benzylidene-2-deoxy-3-O-pi-

valoyl-b-

D

-galactopyranosyl)-(14)-[methyl

-glucopyran-

2,3-di-O-(4-methylbenzoyl)-b-

D

osid] uronate (16)¹⁰(499.7 mg, 539.7 mmol) in

MeCN (10 mL) and water (2 mL) was added

cerium(IV) ammonium nitrate (900 mg, 1.64

mmol) with stirring at 0 °C. After 3 h, the

reaction mixture was treated as described for

the synthesis of 10 and was eluted from a

column of silica gel (3:1–1:4 toluene–EtOAc)

to give the corresponding hemiacetal (326.7

mg, 74%). This hemiacetal was diluted with

CH₂Cl₂(5 mL) and CCl₃CN (0.2 mL). To this

solution was added DBU (30 mL) at 0 °C with

stirring. After 1 h, the reaction mixture was

directly chromatographed on a column of sil-

ica gel (5:1–4:3 toluene–EtOAc) to give 17

(305.3 mg, 79%) as a syrup. A small amount

of the product was puriﬁed over PTLC (1:5

toluene–EtOAc): [h]_D+96.6° (c 1.31,

Methyl [2-acetamido-4,6-O-benzylidene-2-

deoxy - 3 - O - (8 - hydroxyloctyl) - i -

pyranosyl]-(14)-[methyl 2,3-di-O-(4-methyl-

benzoyl)-i- -glucopyranosyluronate]-(18)-

octyl-(13)-[2-acetamido-4,6-O-benzylidene-

2 - deoxy - i - - galactopyranosyl] - (14) - [4-

methoxyphenyl 2,3-di-O-(4-methylbenzoyl)-i-

-glucopyranosid] uronate (14).—To a solu-

D

- galacto-

D

tion of 13 (122.9 mg, 64.20 mmol) in EtOH

(5mL) and toluene (1 mL) was added

H₂NNH₂·AcOH (60.0 mg, 651 mmol) with

stirring for 2.5 h. The volatiles were removed

under diminished pressure and the residue was

diluted with EtOAc and brine. The organic

phase was washed ﬁrst with cold 1 M HCl,

and was treated as described in general meth-

ods. The crude materials were eluted from a

column of silica gel (1:1–1:10 toluene–

EtOAc) to give 14 (81.1 mg) in 70% yield as a

1

CHCl₃); H NMR (CDCl₃): l 8.53 (s, 1 H,

NH), 7.83–7.05 (m, 13 H, Ph), 6.66 (d, 1 H,

J_1,23.66 Hz, H-1^I), 6.06 (t, 1 H, J 9.76 Hz,

H-3^I), 5.30 (m, 1 H, NH), 5.30 (dd, 1 H, H-2^I),

5.24 (s, 1 H, PhCH), 5.10 (dd, 1 H, J_2,310.74,

J_3,43.76 Hz, H-3^II), 4.89 (d, 1 H, J_1,28.30 Hz,

H-1^II), 4.53 (d, 1 H, J_4,59.76 Hz, H-5^I), 4.40 (t,

1 H, H-4^I), 4.05 (d, 1 H, H-4^II), 3.80 (m, 1 H,

H-2^II), 3.77 (m, 1 H, H-6a^II), 3.76 (s, 3 H,

COOMe), 3.50 (dd, 1 H, J_5,6b1.71, J_gem12.44

Hz, H-6b^II), 2.82 (s, 1 H, H-5^II), 2.26, 2.25 (2

s, 3 H×2, 2 PhMe), 1.87 (s, 3 H, NAc), 1.07

(s, 9 H, tert-Bu). This compound was used for

glycosylation without further puriﬁcation.

Methyl (2-acetamido-4,6-O-benzylidene-2-

1

syrup: [h]_D+58° (c 0.34, CHCl₃); H NMR

(CDCl₃): l 7.88–6.74 (m, 30 H, Ph), 5.77 (t, 1

H, J_2,3=J_3,49.03 Hz, H-3^I), 5.70 (dd, 1 H, J_2,3

9.03, J_3,49.51 Hz, H-3^III), 5.63 (d, 1 H, J 6.59

Hz, NH), 5.61 (d, 1 H, J 6.59 Hz, NH), 5.53

(dd, 1 H, J_1,27.07 Hz, H-2^I), 5.31, 5.31 (2 s, 1

H×2, 2 PhCH), 5.28 (dd, 1 H, J_1,27.32 Hz,

H-2^III), 5.22 (d, 1 H, J_1,28.54 Hz, H-1^IV), 5.21

(d, 1 H, H-1^I), 5.16 (d, 1 H, J_1,28.30 Hz,

H-1^II), 4.69 (d, 1 H, H-1^III), 4.56 (dd, 1 H, J_4,5

8.54 Hz, H-4^I), 4.42 (dd, 1 H, J_4,59.03 Hz,

H-4^III), 4.29 (d, 1 H, H-5^I), 4.20–4.16 (m, 2 H,

H-3^II, 3^IV), 4.15 (d, 1 H, H-5^III), 4.03, 4.03 (2d,

2 H, H-4^II, 4^IV) 3.82–3.26 (m, 17 H, H-2^II, 2^IV,

H-6ab^II, H-6ab^IV, OH, 4 CH₂O), 3.82, 3.78,

3.74 (3 s, 3 H×3, 3 OMe), 2.95 (bs, 1 H,

H-5^IV), 2.89 (bs, 1 H, H-5^II), 2.34, 2.31, 2.28,

2.27 (4 s, 3 H×4, 4 PhMe), 1.98, 1.95 (2 s, 3

H×2, 2 NAc), 1.68–0.87 (m, 12 H, 6 CH₂).

Anal. Calcd for C₉₉H₁₁₈N₂O₃₀·3 H₂O: C,

63.57; H, 6.69. Found: C, 63.50; H, 6.40.

Methyl (2-acetamido-4,6-O-benzylidene-2-

deoxy-3-O-le6ulinoyl-i-

(14)-[methyl 2,3-di-O-(4-methylbenzoyl)-i-

-glucopyranosyluronate]-(18)-octyl-(13)-

[2-acetamido-4,6-O-benzylidene-2-deoxy-i-

galactopyranosyl] - (14) - [4 - methoxyphenyl

2,3-di-O-(4-methylbenzoyl)-i- -glucopyran-

D

-galactopyranosyl)-

D

-

D

osid] uronate (18).—To a solution of methyl

(2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-

levulinoyl-b- -galactopyranosyl)-(14)-[2,3-

D

di-O-(4-methylbenzoyl)-b- -glucopyranosyl

D

trichloroacetimidate] uronate (15,⁶144.4 mg,

147.6 mmol) and 12 (117.6 mg, 121.2 mmol) in

CH₂Cl₂(5 mL) was added MS AW300 (400

mg). This mixture was stirred for 1 h at rt,

deoxy - 3 - O - pi6aloyl - i -

(14) - [2,3 - di - O - (4 - methylbenzoyl) - i -

D

- galactopyranosyl)-

D

-

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

49

puriﬁed on a column of gel permeation (LH-

20, 1:1 CHCl₃–MeOH) and the fractions con-

taining the pseudo-hexasaccharide were

concentrated and diluted with THF (5 mL).

To the stirred solution was added 1.25 M

LiOH (0.35 mL) at 0 °C. The volatiles were

removed after 2 h. The residue was diluted

with 1:3 CH₂Cl₂–MeOH (4 mL), and 0.5 M

NaOH (2 mL) was added. The reaction mix-

ture was stirred overnight and neutralized

with HOAc. All the solvents were removed

and the residue was puriﬁed on a column of

gel permeation (LH-20, water) to give 1 as an

amorphous hygroscopic powder (7.6 mg, 29%

and then cooled to −20 °C. To this was

added a solution of Me₃SiOTf (22 mL, 121.6

mmol) in CH₂Cl₂(1 mL) with stirring and the

reaction temperature was gradually raised to

rt. After 24 h, the reaction mixture was

worked up as described in the synthesis of 13.

The crude materials were puriﬁed by gel per-

meation (LH-60, 1:1 CHCl₃–MeOH) and sil-

ica-gel chromatography (3:1–1:4 toluene–

EtOAc, 1:1 EtOAc–MeOH) to give 18 (161.6

mg) in 75% yield as a syrup: [h]_D+62.0° (c

1

0.20, CHCl₃); H NMR (CDCl₃): l 7.87–6.75

(m, 30 H, Ph), 5.78 (dd, 1 H, J_2,38.78, J_3,48.54

Hz, H-3^I), 5.72 (t, 1 H, J_2,3=J_3,49.03 Hz,

H-3^III), 5.58 (d, 1 H, NH), 5.53 (dd, 1 H, J_1,2

7.07 Hz, H-2^I), 5.47 (d, 1 H, NH), 5.29, 5.27

(2 s, 1 H×2, 2 PhCH), 5.23 (dd, 1 H, J_1,27.56

Hz, H-2^III), 5.21 (d, 1 H, J_1,28.05 Hz, H-1^II),

5.20 (d, 1 H, H-1^I), 5.12 (dd, 1 H, J_2,38.05 Hz,

H-3^IV), 4.88 (d, 1 H, J_1,28.05 Hz, H-1^IV), 4.69

(d, 1 H, H-1^III), 4.56 (t, 1 H, J_4,58.54 Hz,

H-4^I), 4.40 (dd, 1 H, J_3,49.03, J_4,59.27 Hz,

H-4^III), 4.29 (d, 1 H, H-5^I), 4.17–4.01 (m, 4 H,

H-3^II, 4^II, 4^IV, 5^III), 3.86–3.27 (m, 10 H, H-2^II,

2^IV, 6ab^II, 6ab^IV, 2 CH₂O), 3.83, 3.78, 3.74 (3

s, 3 H×3, 3 OMe), 2.94 (bs, 1 H, H-5^IV), 2.89

(bs, 1 H, H-5^II), 2.68–2.50 (m, 4 H, 2 CH₂),

2.35, 2.31, 2.28 (3 s, 3 H×4, 4 PhMe), 2.04 (s,

3 H, COMe), 1.96, 1.94 (2 s, 3 H×2, 2 NAc),

1.68–1.26 (m, 12 H, 6 CH₂). Anal. Calcd for

C₉₆H₁₀₈N₂O₃₁·2 H₂O: C, 63.21; H, 6.21; N,

1.54. Found: C, 62.91; H, 5.76; N, 1.95.

1

from 18): [h]_D−16.7° (c 0.18, water); H

NMR (D₂O): l 6.98–6.84 (m, 4 H, Ph), 4.87

(d, 1 H, J_1,27.81 Hz, H-1^I), 4.37 (d, 1 H, J_1,2

8.30 Hz, H-1^II), 4.35 (d, 1 H, J_1,28.30 Hz,

H-1^IV), 4.31 (d, 1 H, J_1,27.82 Hz, H-1^III), 3.99

(d, 1 H, J_3,42.93 Hz, H-4^II), 3.79 (d, 1 H, J_3,4

2.93 Hz, H-4^IV), 3.78–3.73 (m, 1 H, 1/2

CH₂O), 3.77 (dd, 1 H, H-2^II), 3.75 (dd, 1 H,

H-2^IV), 3.72–3.67 (m, 4 H, H-6^II,IV), 3.71, 3.69

(2 d, 2 H, H-5^I,III), 3.68 (s, 3 H, OMe), 3.64

(dd, 1 H, H-4^I), 3.63, 3.63 (2 bs, 2 H, H-5^II,IV),

3.62 (dd, 1 H, H-4^III), 3.60 (m, 2 H, CH₂O),

3.58 (dd, 1 H, H-3^I), 3.56 (dd, 1 H, H-3^IV),

3.48 (t, 1 H, J_2,37.81 Hz, H-2^I), 3.45 (t, 1 H,

J_2,3=J_3,49.27 Hz, H-3^III), 3.34 (dd, 1 H,

H-3^II), 3.31–3.24 (m, 1 H, 1/2 CH₂O), 3.19

(dd, 1 H, H-2^III), 1.91, 1.91 (2 s, 3 H×2, 2

NAc), 1.49–1.17 (m, 12 H, 6 CH₂); FAB⁻MS:

m/z 1009.5, [M−H]⁻; FAB⁺MS: m/z 1033.5,

[M+Na]⁺, 1077.4, [M+3Na−2H]⁺.

Sodium (2-acetamido-2-deoxy-i-

topyranosyl)-(14)-(sodium i- -glucopyran-

osyluronate) - (18) - octyl - (13) - [2 - aceta-

mido-2-deoxy-i- -galactopyranosyl]-(14)-

(4-methoxyphenyl i- -glucopyranosid] uronate

D

-galac-

D

Methyl (2-acetamido-4,6-O-benzylidene-2-

D

deoxy - 3 - O - pi6aloyl - i - - galactopyranosyl)-

D

(14)-[methyl 2,3-di-O-(4-methylbenzoyl)-i-

-glucopyranosyluronate]-(18)-octyl-(13)-

[2-acetamido-4,6-O-benzylidene-2-deoxy-i-

galactopyranosyl]-(14)-[methyl 2,3-di-O-(4-

methylbenzoyl) - i - - glucopyranosyluronate]-

(18) - octyl - (13) - [2 - acetamido - 4,6 - O-

benzylidene-2-deoxy-i- -galactopyranosyl]-

(14)-[4-methoxyphenyl 2,3-di-O-(4-methyl-

benzoyl)-i- -glucopyranosid] uronate (19).—

(1).—To a solution of 18 (34.2 mg, 19.2 mmol)

in EtOH (5 mL) and toluene (1 mL) was

added H₂NNH₂·AcOH (18 mg, 0.20 mmol)

with stirring. The volatiles were removed un-

der diminished pressure 1 h later and the

residue was eluted from a column of gel per-

meation (LH-60, 1:1 CHCl₃–MeOH). Frac-

tions containing the pseudo-hexasaccharide

were concentrated and diluted with 1:1

CH₂Cl₂–MeOH (1 mL). Camphorsulfonic

acid (22 mg, 95 mmol) was added and the

solution was stirred for 24 h. Then an excess

of Et₃N was added and the reaction mixture

was concentrated to dryness. The residue was

D

-

D

To a solution of 17 (66.6 mg, 37.9 mmol) and

14 (68.8 mg, 37.9 mmol) in CH₂Cl₂(3 mL) was

added MS AW300 (100 mg). This mixture was

stirred for 30 min at rt, and then cooled to

−20 °C. To this was added a solution of

Me₃SiOTf (6.0 mL, 33 mmol) in CH₂Cl₂(1 mL)

50

J. Tamura et al. / Carbohydrate Research 332 (2001) 41–51

and diluted with THF (2 mL). To the stirred

solution was added 1.25 M LiOH (0.3 mL) at

0 °C. The volatiles were removed after 1 h.

The residue was diluted with 1:3 CH₂Cl₂–

MeOH (2 mL), and 0.5 M NaOH (0.3 mL)

was added. The reaction mixture was stirred

overnight and neutralized with HOAc. Fol-

lowing the puriﬁcation process described for

the synthesis of 1, the pseudo-decasaccharide

2 was obtained as an amorphous hygroscopic

powder (7.2 mg, 56% from 19): [h]_D−9 ° (c

with stirring and the reaction temperature was

gradually raised to rt. After 24 h, the reaction

mixture was worked up as described in the

synthesis of 13. The crude materials were

puriﬁed on a column of gel permeation (LH-

60, 1:1 CHCl₃–MeOH) and then by silica-gel

chromatography (2:1–1:4 toluene–EtOAc) to

give 19 (42.9 mg) in 43% yield as a white

1

powder: [h]_D+63.6° (c 0.28, CHCl₃); H

NMR (CDCl₃): l 7.88–6.74 (m, 43 H, Ph),

5.77 (t, 1 H, J_2,3=J_3,48.78 Hz, H-3^I), 5.72 (t,

1 H, J_2,3=J_3,49.03 Hz, H-3^III), 5.70 (dd, 1 H,

J_2,38.72, J_3,49.27 Hz, H-3^V), 5.59, 5.59 (m, 2

H, 2 NH), 5.53 (dd, 1 H, J_1,27.32 Hz, H-2^I),

5.31 (d, 1 H, J 8.78 Hz, NH), 5.31, 5.30, 5.29

(3 s, 1 H×3, 3 PhCH), 5.27–5.23 (m, 2 H,

H-2^III, H-2^V), 5.22 (d, 1 H, J_1,28.54 Hz, H-

1^IV), 5.21 (d, 1 H, H-1^I), 5.16 (d, 1 H, J_1,28.29

Hz, H-1^II), 5.04 (dd, 1 H, J_2,311.22, J_3,43.42

Hz, H-3^VI), 4.81 (d, 1 H, J_1,28.05 Hz, H-1^VI),

4.69 (d, 1 H, J_1,27.56 Hz, H-1^III), 4.69 (d, 1 H,

J_1,27.56 Hz, H-1^V), 4.56 (t, 1 H, J_4,59.03 Hz,

H-4^I), 4.42 (t, 1 H, J_4,58.78 Hz, H-4^III), 4.39

(t, 1 H, J_4,59.51 Hz, H-4^V), 4.28 (d, 1 H,

H-5^I), 4.19–3.86 (m, 5 H, H-2^VI, 3^II, 3^IV, 5^III,

5^V), 4.08 (d, 1 H, H-4^VI), 4.03 (d, 1 H, J_3,42.93

Hz, H-4^IV), 4.00 (d, 1 H, J_3,42.93 Hz, H-4^II),

3.86–3.23 (m, 16 H, H-2^II, 2^IV, 6ab^II, 6ab^IV,

6ab^VI, 4 CH₂O), 3.82, 3.82, 3.78, 3.74 (4 s, 3

H×4, 4 OMe), 2.94 (bs, 1 H, H-5^IV), 2.90 (bs,

1 H, H-5^II), 2.87 (bs, 1 H, H-5^VI), 2.34, 2.31,

2.31, 2.31, 2.28, 2.27 (6 s, 3 H×6, 6 PhMe),

1.96, 1.96, 1.92 (3 s, 3 H×3, 3 NAc), 1.39–

0.85 (m, 12 H, 6 CH₂), 1.09 (s, 9 H, tert-Bu).

1

0.09, water); selected H NMR data (D₂O): l

6.99–6.84 (m, 4 H, Ph), 4.92 (d, 1 H, J_1,28.54

Hz, H-1^I), 4.41–4.35 (m, 5 H, H-1^{II,III,IV,V,VI}),

4.00, 3.98 (m, 2 H, H-4^II,IV), 3.68 (s, 3 H,

OMe), 1.91, 1.90, 1.87 (3 s, 3 H×3, 3 NAc),

1.49–1.13 (m, 24 H, 12 CH₂); FAB⁻MS: m/z

1516.7, [M−H]⁻; FAB⁺MS: m/z 1540.5,

[M+Na]⁺, 1606.4, [M+4Na−3H]⁺.

Enzymatic transfer of GlcA.—Incubation

mixtures contained the following constituents

in a total volume of 20 mL: 3 nmol of 1 or 2,

14.3 mM UDP-

-[¹⁴C]GlcA [150 nCi (240,000

D

dpm)], 171 mM ATP-2Na, 50 mmol NaOAc

buffer, pH 5.6, 10 mM MnCl₂and 12 mL of

enzyme solution. They were incubated at

37 °C for 12 h. The products were fractionated

by gel ﬁltration chromatography on a column

(1×30 cm) of Superdex Peptide^®equilibrated

and eluted with 0.25 M NH₄HCO₃, 7% n-

PrOH. Fractions (0.5 mL each) were collected

at a rate of 0.4 mL/min and analyzed for

radioactivity. The results are summarized in

Table 1.

Sodium (2-acetamido-2-deoxy-i-

topyranosyl)-(14)-(sodium i- -glucopyran-

osyluronate) - (18) - octyl - (13) - (2 - aceta-

mido-2-deoxy-i- -galactopyranosyl)-(14)-

(sodium i - - glucopyranosyluronate) - (18)-

octyl - (13) - [2 - acetamido - 2 - deoxy - i -

galactopyranosyl]-(14)-(4-methoxyphenyl i-

-glucopyranosid] uronate (2).—To a solution

D

-galac-

D

Acknowledgements

D

We thank Dr S. Kurono (RIKEN) for the

mass spectra measurement. This study was

supported by the Kato Memorial Bioscience

Foundation. The work at Kobe Pharmaceuti-

cal University was supported, in part by the

Science Research Promotion Fund from the

Japan Private School Promotion Foundation.

D

-

D

of 19 (20.3 mg, 7.76 mmol) in 1:1 CH₂Cl₂–

MeOH (1 mL) was added camphorsulfonic

acid (18 mg, 77 mmol) followed by stirring for

29 h. Then, an excess of Et₃N was added and

the reaction mixture was concentrated to dry-

ness. The residue was chromatographed on a

column of gel permeation (LH-20, 1:1

CHCl₃–MeOH) and the fractions containing

the pseudo-decasaccharide were concentrated

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Synthesis of linear-type chondroitin clusters having a C8 spacer between disaccharide moieties and enzymatic transfer of D-glucuronic acid to the artificial glycans

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