Development of specific inhibitors for heparin-binding proteins based on the cobra cardiotoxin structure: An effective synthetic strategy for rationally modified heparin-like disaccharides and a trisaccharide

Carbohydrate

RESEARCH

Carbohydrate Research 340 (2005) 355–372

Development of speciﬁc inhibitors for heparin-binding proteins

based on the cobra cardiotoxin structure: an eﬀective

synthetic strategy for rationally modiﬁed heparin-like

disaccharides and a trisaccharide

Weijun Ke,^aDennis M. Whitﬁeld,^a,*Jean-Robert Brisson,^aGary Enright,^b

Harold C. Jarrell^aand Wen-guey Wu^c

^aInstitute for Biological Sciences, National Research Council, Ottawa, ON, Canada K1A 0R6

^bSteacie Institute for Molecular Sciences, National Research Council, Ottawa, ON, Canada K1A 0R6

^cDepartment of Life Science, National Tsing Hua University, Hsinchu 30043, Taiwan

Received 15 September 2004; accepted 30 November 2004

Available online 13 January 2005

Abstract—Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identiﬁed by NMR

spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding

studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six dif-

ferentially protected monosaccharide building blocks (three ^L-iduronic acids and three D-glucosamines) were prepared. These

include a ^L-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a-L-idopyranosyluronate

˚

trichloroacetimidate for which a single-crystal X-ray structure was determined to have M_r= 576.79, a = 9.3098(11) A a = 90ꢀ,

3

˚

b = 10.3967(12) A b = 90ꢀ, c = 28.026(3) A c = 90ꢀ, V = 2712.7(6) A , P2₁2₁2₁, Z = 4, l = 0.71073 A, and R = 0.0378 for 7586

observed reﬂections. It shows that the molecular structure of the donor is in the ¹C₄conformation with signiﬁcant 1,3-diaxial inter-

actions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and

N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the tem-

porary protecting group to protect the hydroxyl for elongation.

Keywords: Glycosylations; L-Iduronic acid building blocks; Heparin-like oligosaccharides; 1,3-Diaxial interactions; Cobra cardiotoxin

1. Introduction

shown to inhibit protein kinase C activity in the pres-

ence of phosphatidylserine,⁴contain weak anticoagulant

activity, and act as a potentiator of platelet aggrega-

tion.⁵There is also evidence to suggest a correlation

between cytolytic and antiplatelet activity.⁶

Glycosaminoglycans (GAGs) represent the saccharide

moieties of proteoglycans (PG) that occur abundantly

in all tissues, on cell surfaces, and in the extracellular

matrix (ECM).^7,8They perform a myriad of physiological

functions and are extensively sulfated, repeating copoly-

mers of hexosamine and hexuronic acid.^9,10Heparin and

heparan sulfate are highly sulfated glycosaminoglycans

(GAGs) involved in many important biological

Cardiotoxins (CTXs) from cobra venom are all b-sheet,

slightly curved, highly basic polypeptides, which are

capable of inducing general cytotoxic eﬀects on many

cell types.^1,2They also cause severe tissue necrosis and

local gangrene in humans.³Many workers are attracted

by the abundance of positive charge on CTXs and are

interested in studying its eﬀect on various biochemical

processes involving anionic molecules. CTX has been

*

Corresponding author. Tel.: +1 613 993 5265; fax: +1 613 952 9092;

e-mail: dennis.whitﬁeld@nrc-cnrc.gc.ca

doi:10.1016/j.carres.2004.11.029

356

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

functions such as blood coagulation, cell growth and

diﬀerentiation, angiogenesis, etc.¹¹Recently, glycosami-

noglycans (GAGs) have been suggested to be potential

targets for cobra cardiotoxin (CTX) action.^12–14The

interaction of GAGs, such as high-molecular-weight

heparin, with CTXs not only can induce aggregation

of CTXs molecules, but also can promote its penetration

into the phospholipid monolayer under physiological

ionic conditions.¹²The molecular basis of the CTX–

GAG interaction, however, remains elusive.

We have recently initiated a collaborative project

aimed at understanding at the molecular level the bind-

ing speciﬁcity of GAGs to the CTXs in order to be able

to identify speciﬁc inhibitors. For performing structure–

activity relationship studies in this ﬁeld, we designed and

synthesized a variety of monosaccharide building

blocks, which could be coupled to form oligosaccharides

that would serve to determine the key structural features

necessary for binding to the cobra cardiotoxin proteins

(CTXs). Since heparin is heavily O- and N-sulfated,

these building blocks must allow for selective sulfation.

At the outset of this program it was not known, which

sulfates or carboxylates were important for binding to

the CTXs. During the project a heparin disaccharide-

binding site on the convex side of CTX was identiﬁed

by NMR spectroscopy and molecular modeling.¹⁵The

N-sulfate of ^D-glucosamine as well as the 2-sulfate of

L-iduronic acid were established as key binding points.

However, the role of the 6-sulfate of ^D-glucosamine

and the carboxylate of ^L-iduronic acid were not estab-

lished. The former makes little or no contact with the

protein in the current model, whereas the carboxylateꢀs

role cannot be determined because the uronic acid of

the disaccharide used for testing has a 4,5-double bond

and is not the biologically relevant ^L-iduronic acid.

Moreover, it is not clear if the disaccharide-binding site

is for an a-^L-iduronic acid-(1!4)-D-glucosamine (pI-

doA(a1,4)pGlcN) disaccharide or the frame-shifted

pGlcN(a1,4)pIdoA disaccharide. Binding studies of

Figure 1. Disaccharides varying in the degree and position of O- and

N-sulfation.

Figure 2. Trisaccharide used for testing the sequence of the binding

disaccharide.

2. Results and discussion

Our synthetic approach was based on preparing and

assembling uronic acid monomers and glucosamine

monomers with various patterns of orthogonal protec-

tive groups. In our synthetic strategy, the pivaloyl group

and the benzyl group were used as permanent protecting

groups for hydroxyl functions. The levulinoyl group was

used as the temporary protecting group, which was used

to protect the hydroxyl group for elongation. The acetyl

group was also used as a temporary protecting group,

which was used to protect the hydroxyl destinated to

be O-sulfated.

a

suitably sulfated trisaccharide, pGlcN(a1,4)pI-

doA(a1,4)pGlcN, should settle this question.

It has often been diﬃcult to determine the oligosac-

charide sequence and sulfation pattern of a heparin or

heparin sulfate fragment required for activation or

deactivation of a given protein.¹⁶For this reason it is

highly desirable to develop eﬀective syntheses of this

heparin-like oligosaccharide chains with deﬁned size, se-

quence, and charge distribution to be used in the inter-

action studies. In this paper, we report a convenient

synthetic strategy the eﬀectiveness of which is illustrated

by the preparation of two known¹⁷heparin-like disac-

charides 1 and 2 (Fig. 1) and an eﬃcient synthesis of a

heparin-like trisaccharide 3 (Fig. 2). The biological

properties of the disaccharides and the trisaccharide

are being investigated and will be discussed in detail

elsewhere.

2.1. Preparation of glycosyl acceptors

Two monosaccharide acceptors were prepared (Scheme

1). The known acceptor methyl 6-O-acetyl-2-azido-3-

O-benzyl-2-deoxy-a-^D-glucopyranoside 6 was obtained

by selective acetylation of diol 4 using acetyl chloride

in pyridine.¹⁸This 6-O-acetyl group was used as a tem-

porary hydroxy protecting group that would be O-sul-

fated in the end product. Selective pivaloylation of

diol 4 gave a new acceptor methyl 2-azido-3-O-benzyl-

2-deoxy-6-O-pivaloyl-a-^D-glucopyranoside (5) in 72%

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

357

Scheme 1. Reagents and conditions: (a) AcCl, Py, CH₂Cl₂, ꢀ78 ꢀC, 2 h; (b) PivCl, Py, CH₂Cl₂, ꢀ78 ꢀC, 15 min.

yield. The reaction could be completely ﬁnished in

15 min. The pivaloyl group on the 6-position was used

as a permanent hydroxy protecting group.

the methyl ester 8 with 9:1 CF₃COOH–H₂O aﬀorded

the ^L-iduronic acid derivative 9.²²Then by regio- and

stereoselective silylation with dimethylthexylsilyl chloride

(TDSCl) and imidazole in dichloromethane at ꢀ20 ꢀC,

the b-silyl glycoside 10 was obtained from 9 (Scheme

2.2. Preparation of glycosyl donors

1

2).²³Analysis of the H NMR (chloroform-d) spectrum

Four diﬀerentially protected donors were prepared. The

synthetic route of two ^L-iduronic acid derivatives, do-

nors 17 and 18 for preparing disaccharides, are shown

in Scheme 2. Literature and experimentation showed

that a convenient route to ^L-iduronic acid building

blocks was to start from the commercially available

crystalline 1,2-O-isopropylidene-6,3-^D-glucuronolactone

7.^19–21Taking advantage of an O-5 to O-3 migration

side reaction noted in Ref. 19, we could prepare multi-

gram quantities of methyl ester 8 under the catalysis of

the organic base triethylamine at 0 ꢀC. Treatment of

of the key intermediate 10 indicated a preferred ¹C₄con-

formation for this compound (J_1,21.0 Hz, J_4,51.6 Hz

(⁴C₁: J_4,5> 4.2 Hz)^20a). Although the OH-2 and OH-4

of the intermediate 10 have similar reactivities, the use

of catalytic scandium triﬂate allowed for acylation to

give a mixture of O-4, O-2 monoacylates, and a diacyl-

ate.²⁴Pivaloylation of the key intermediate 10 with

1.1 equiv of pivaloic anhydride at 10 ꢀC for 1.5 h gave

a mixture of O-4 pivaloate 11, O-2 pivaloate 13, dipival-

oate 14, and traces of unreacted diol 10. The mixture

could be separated on a MPLC chromatography

Scheme 2. Reagents and conditions: (a) 90% TFA, rt, 3 h; (b) imidazole, CH₂Cl₂, ꢀ20 ꢀC, O/N; (c) Piv₂O, Sc(OTf)₃, CH₂Cl₂, 10 ꢀC, 1 h; (d) Ac₂O,

Sc(OTf)₃, CH₂Cl₂, rt, O/N; (e) CH₃COOH, Bu₄NF, THF, 10–15 ꢀC, 5 h; (f) CCl₃CN, DBU, CH₂Cl₂, 0 ꢀC, 1 h.

358

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

column eluting with 10:1 hexanes–EtOAc, and the pure

pivaloates 11, 13, and 14 could be obtained in the ratio

of 10:7:5. Then, under the catalysis of Sc(OTf)₃at room

temperature overnight, acetylation of 11 gave a mixture

of a anomer and b anomer of the corresponding O-2

acetylated silyl glycoside 12. Silica gel chromatography

aﬀorded pure a anomer and b anomer (5:8 a:b). Depro-

tection of glycoside 12 (including a anomer and b ano-

mer) with CH₃COOH and Bu₄NF in THF at 10–15 ꢀC

furnished the intermediate 15, which was then trans-

formed into trichloroacetimidate 17 in 55% yield by

standard protecting group manipulations with CCl₃CN

under the catalysis of DBU at 0 ꢀC. The O-2 acetylated

donor 17 allows for the preparation of O-2 sulfated ^L-

iduronic acid-containing oligosaccharides in the end

product. ¹H NMR analysis of building block 17 revealed

elongation building block for the synthesis of trisacchar-

ide 3 (Scheme 3). Using TESOTf as reagent, a triethyl-

silyl group could be added on the 2-O-position of the

diol silylate 10 selectively under the conditions of base

2,6-lutidine and CH₂Cl₂at 0 ꢀC, yielding 58% of sugar

19.²⁵The 4-O-position of the sugar 19 was then pro-

tected by a levulinoyl group in 64% yield. After that,

compound 20 was treated with Ac₂O under the catalysis

of Sc(OTf)₃at room temperature, resulting in more than

90% of sugar 21. Glycoside 21 was then deprotected

with CH₃COOH and Bu₄NF in THF at room tempera-

ture to give the intermediate 22. Standard protecting

group manipulations allowed for the formation of the

chain extension donor, trichloroacetimidate 23, in 55%

yield by treatment of intermediate 22 with CCl₃CN

under the catalysis of DBU at 0 ꢀC.

The single-crystal X-ray structure of the a anomer,

(methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a-^L-ido-

pyranosyluronate) trichloroacetimidate of ^L-iduronic

acid building block 23 was obtained at room tempera-

ture by slow evaporation of a 3:1 hexanes–CH₂Cl₂

solvent mixture. The molecular structure and the num-

bering scheme for the atoms are shown in Figure 3.

Crystal data and structure reﬁnement parameters are

given in Table 1; atomic coordinates and equivalent iso-

tropic displacement parameters are shown in Table 2;

and selected bond lengths, bond angles, and torsion an-

gles are given in Table 3.

1

a C₄conformation as judged by the coupling constant

(J_1,2< 1.0 Hz, J_4,52.3 Hz). Interestingly, a strong NOE

between H-1 and H-2, but not to H-5, was observed in

the NOESY experiment, showing that donor 17 has an

a conﬁguration. In a similar way, another donor 18

could be obtained from the tripivaloate 14 via the inter-

mediate 16. ¹H NMR analysis of 18 also revealed a pre-

dominant ¹C₄conformation (J_1,23.1 Hz, J_4,53.5 Hz, J_2,3

4.4 Hz, J_3,44.6 Hz). This L-iduronic acid building block

can be used to make only N-sulfated or N-acetylated oli-

gosaccharides as well as for testing the glycosylation

chemistry. Other variants can easily be constructed from

10.

The diﬀraction data show that the molecular structure

1

Another donor, ^L-iduronic acid building block 23,

(methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a,b-^L-

idopyranosyluronate) trichloroacetimidate, is the key

of the donor is in a C₄conformation (0.841 C₄, 0.004

B_1,4, and 0.188 ²S_O).²⁶This conformation has two

strong 1,3-diaxial interactions between the substituents

Scheme 3. Reagents and conditions: (a) TESOTf, 2,6-lutidine, CH₂Cl₂, 0 ꢀC, 1.5 h; (b) Lev₂O, DMAP, CH₂Cl₂, rt, 26 h; (c) Ac₂O, Sc(OTf)₃, CH₂Cl₂,

rt, 1 h; (d) CH₃COOH, Bu₄NF, THF, rt, O/N; (e) CCl₃CN, DBU, CH₂Cl₂, 0 ꢀC, 1 h.

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

359

Table 2. Atomic coordinates (·10⁴) and equivalent isotropic displace-

2

3

˚

ment parameters (A · 10 ) for 23

x

y

z

U(eq)

Cl(14)

Cl(15)

Cl(16)

O(5)

397(1)

10700(1)

8947(1)

8087(1)

6639(1)

7387(1)

5107(1)

4595(1)

4043(1)

4864(1)

6544(1)

3813(1)

1979(1)

2881(2)

6431(1)

9306(1)

6820(1)

5563(1)

4458(1)

4422(1)

5757(1)

8656(1)

9080(1)

5677(2)

5092(2)

4175(2)

4265(2)

3332(2)

5663(2)

3948(2)

2786(1)

2575(2)

3587(2)

3647(2)

4685(2)

5746(2)

4729(2)

9135(1)

9877(1)

9139(1)

8280(1)

9051(1)

8716(1)

9251(1)

8073(1)

7721(1)

9098(1)

9816(1)

8297(1)

7045(1)

7442(1)

8678(1)

8735(1)

8970(1)

8917(1)

8425(1)

8269(1)

8964(1)

9266(1)

8817(1)

8530(1)

9705(1)

38(1)

27(1)

31(1)

19(1)

22(1)

20(1)

21(1)

28(1)

32(1)

34(1)

38(1)

46(1)

32(1)

25(1)

19(1)

20(1)

22(1)

24(1)

36(1)

21(1)

22(1)

34(1)

27(1)

34(1)

28(1)

31(1)

37(1)

23(1)

36(1)

1290(1)

ꢀ652(1)

2854(1)

2405(1)

5186(1)

1688(1)

3204(1)

94(1)

O(11)

O(21)

O(31)

O(41)

O(52)

O(221)

O(321)

O(421)

O(451)

O(511)

N(121)

C(1)

6583(1)

3160(1)

3016(2)

2569(2)

1541(2)

2873(2)

3444(2)

3934(2)

2861(2)

2161(2)

1680(2)

2139(2)

847(2)

Figure 3. The structure and atom numbering scheme of L-iduronic

acid building block 23.

C(2)

C(3)

Table 1. Crystal data and structure reﬁnement parameters for 23

C(4)

C(5)

Empirical formula

Formula weight

Temperature

C₂₁H₂₈Cl₃NO₁₁

576.79

C(12)

C(13)

C(22)

C(23)

C(32)

C(33)

C(34)

C(35)

C(36)

C(42)

C(43)

C(44)

C(45)

C(46)

C(51)

C(53)

125(2) K

0.71073

6459(2)

7645(2)

1991(2)

690(2)

Wavelength

Crystal system

Space group

Unit cell dimensions

Orthorhombic

P2₁2₁2₁

˚

10035(1)

10450(1)

10215(1)

9771(1)

8050(1)

7695(1)

7308(1)

6995(1)

6626(1)

7757(1)

7254(1)

a = 9.3098(11) A a = 90ꢀ

˚

920(2)

639(3)

b = 10.3967(12) A b = 90ꢀ

˚

c = 28.026(3) A c = 90ꢀ

3

˚

ꢀ701(2)

3563(2)

4755(2)

4866(2)

3547(2)

3497(3)

1121(2)

ꢀ575(2)

Volume

Z

2712.7(6) A

4

D_calcd

1.412 Mg/m³

Absorption coeﬃcient

F(000)

Crystal size

0.394 mm^ꢀ1

1200

0.35 · 0.18 · 0.05 mm³

1.45–29.60ꢀ

h Range for data collection

Index ranges

ꢀ12 6 h 6 12, ꢀ14 6 k 6

14, ꢀ38 6 l 6 38

34,069

U(eq) is deﬁned as one third of the trace of the orthogonalized U^ij

Reﬂections collected

tensor.

Independent reﬂections

Completeness to h = 29.60

Reﬁnement method

7586 [R(int) = 0.0378]

99.8%

Full-matrix least-squares on F²

7586/0/438

1.009

Data/restraints/parameters

Goodness-of-ﬁt on F²

Final R indices [I > 2r(I)]

R indices (all data)

imidate (27) is a nonreducing terminal building block

used for preparing trisaccharide 3 (Scheme 4). This

building block could be synthesized from the known

starting material 24.¹⁸Pivaloylation of 24 with pivaloyl

chloride in pyridine at 45 ꢀC for 48 h under the catalysis

of small amount of DMF aﬀorded the sugar 25, which

was deprotected with N₂H₄ÆHOAc in DMF at room

temperature to give the intermediate 26. Standard pro-

tecting group manipulation allowed for the formation

of the donor 27 by treatment of intermediate 26 with

CCl₃CN under the catalysis of Cs₂CO₃at 0 ꢀC for 1 h.

R₁= 0.0332, wR₂= 0.0702

R₁= 0.0419, wR₂= 0.0730

0.02(3)

Absolute structure parameter

ꢀ3

˚

Largest diﬀerence peak and hole 0.270 and ꢀ0.173 e A

at C-1 and C-3, and between C-2 and C-4. In the previ-

ously reported structure of methyl (methyl 2,3,4-tri-O-

acetyl-a-^L-idopyranosyl)uronate, the C-2 and C-4

groups twisted away from each other to avoid such 1,3

interactions.^20aAll three ester carbonyls and the C@N

of the imidate are oriented syn to their respective sugar

ring methines. The ester at C-6 is planar with respect to

C-5–O-5. This solid-state structure of 23 conﬁrms all the

stereochemical and regiochemical selective reactions

used in its synthesis.

2.3. Preparation of disaccharides

To test the protecting group strategy and the glycosyla-

tion chemistry, coupling of acceptor 5 with donor 17

aﬀorded disaccharide 28 in 85% yield at ꢀ20 ꢀC under

promotion by TESOTf (Scheme 5). However, during

The last donor 6-O-acetyl-2-azido-3-O-benzyl-2-

deoxy-4-O-pivaloyl-a-^D-glucopyranosyl trichloroacet-

360

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

Table 3. Selected bond lengths, bond angles, and torsion angles of

donor 23

˚

Bond lengths [A]

O(5)–C(1)

O(5)–C(5)

C(1)–C(2)

C(2)–C(3)

C(3)–C(4)

C(4)–C(5)

C(5)–C(51)

1.3995(18)

1.4271(19)

1.533(2)

1.530(2)

1.525(2)

1.522(2)

1.526(2)

Bond angles [ꢀ]

C(1)–O(5)–C(5)

O(5)–C(1)–C(2)

C(3)–C(2)–C(1)

C(4)–C(3)–C(2)

C(5)–C(4)–C(3)

O(5)–C(5)–C(4)

114.03(11)

113.22(12)

113.82(12)

112.66(12)

111.33(13)

110.72(12)

Scheme 4. Reagents and conditions: (a) PivCl, DMF, Py, 45 ꢀC, 48 h;

(b) N₂H₄ÆHOAc, DMF, rt, 2.5 h; (c) CCl₃CN, Cs₂CO₃, CH₂Cl₂, 0 ꢀC,

1 h.

Torsion angles [ꢀ]

Ring

C(1)–C(2)–C(3)–C(4)

C(2)–C(3)–C(4)–C(5)

C(3)–C(4)–C(5)–O(5)

C(4)–O(5)–C(5)–C(1)

C(5)–O(5)–C(1)–C(2)

O(5)–C(1)–C(2)–C(3)

39.47(17)

ꢀ45.29(17)

55.64(16)

0.1 M DBU under the same conditions did not eﬀec-

tively lead to the deacetylated disaccharide 32. Fortu-

nately, treatment of disaccharide 31 with 7.5% HCl in

methanol at room temperature overnight²⁶did result

in the deacetylated disaccharide 32 in a good yield of

81%. O-Sulfation of both disaccharides 29 and 32 with

SO₃ÆNMe₃complex in DMF at 60 ꢀC overnight led to

disaccharides 30 (yield of 88%) and 33 (yield of 83%),

respectively. Next, the esters of 30 was cleaved by a

cocktail of reagents (LiOH, H₂O₂followed by NaOH)

in aqueous tetrahydrofuran (THF). The products were

puriﬁed by chromatography on Sephadex LH-20, elut-

ing with 1:1 MeOH–H₂O. The O-benzyl group was then

removed with concomitant N₃to NH₂reduction by 10%

Pd/C and H₂. These amines were treated with SO₃Æpyr-

idine in water at pH 9.8. The products were passed

through a Bio-Gel P-2 column, eluting with H₂O. In this

way by using a series of well-known reactions (saponiﬁ-

cation, hydrogenation, and N-sulfation),²⁹the O-sul-

fated disaccharides 30 was converted into the target

heparin-like disaccharide 1. The overall yield of 1 from

30 was 17%. Following the same route, we obtained 2

from 33 in an overall yield of 10%. Obviously, the yield

was very low. After a long investigation of the synthesis

of heparin-like disaccharide 2 from the O-sulfated disac-

charides 33, we found that the step of saponiﬁcation and

the following chromatographic process on Sephadex

LH-20 greatly inﬂuenced the overall yield of disacchar-

ide 2. We often got complex ¹H NMR spectra after

chromatographic puriﬁcation, and sometimes two or

ꢀ62.17(16)

55.90(16)

ꢀ43.95(17)

Side chains

C(5)–O(5)–C(1)–O(11)

C(12)–O(11)–C(1)–O(5)

C(1)–O(11)–C(12)–N(121)

O(5)–C(1)–C(2)–O(21)

C(22)–O(21)–C(2)–C(1)

C(2)–O(21)–C(22)–O(221)

C(1)–C(2)–C(3)–O(31)

C(32)–O(31)–C(3)–C(2)

C(3)–O(31)–C(32)–O(321)

C(2)–C(3)–C(4)–O(41)

C(42)–O(41)–C(4)–C(3)

C(4)–O(41)–C(42)–O(421)

C(3)–C(4)–C(5)–C(51)

O(5)–C(5)–C(51)–O(511)

C(53)–O(52)–C(51)–C(5)

ꢀ63.51(15)

ꢀ73.08(15)

ꢀ9.2(2)

70.15(15)

78.87(16)

ꢀ1.3(2)

ꢀ78.28(15)

ꢀ81.04(15)

4.9(2)

69.95(15)

76.38(17)

2.6(2)

174.14(13)

ꢀ5.1(2)

179.43(15)

coupling of acceptor 6 with donor 17 at ꢀ20 ꢀC under

the same reaction conditions, disaccharide 31 could

not be obtained. Only by raising the temperature to

ꢀ15 to ꢀ12 ꢀC, could the disaccharide 31 be obtained

in a satisfactory yield of 64%. The key to the deprotec-

tion of disaccharides is selective removal of the O-acetyl

groups in the presence of O-pivaloyl groups. When

treating disaccharide 28 with 7.5% HCl in methanol²⁷

in order to selectively remove the O-acetyl group, the

reaction was slow and the yield was low because of

migration of the pivaloyl group from O-4 of iduronic

acid to O-2. To ensure that the O-acetate of disaccharide

28 could be cleaved in an eﬀective way in the presence of

the O-pivaloate, a methanol solution of 28 was treated

at ꢀ20 ꢀC with 0.1 M DBU.²⁸The expected deacetylated

disaccharide 29 was then obtained in the yield of 55%.

However, treatment of the other disaccharide 31 with

1

more methoxy peaks were found in the same H NMR

spectrum. To solve this problem, puriﬁcation on Sepha-

dex LH-20 was avoided, and the crude product after the

work-up of the saponiﬁcation reaction was used to do

the next hydrogenation reaction directly. In this way,

we were able to obtain acceptable yields of disaccharide

2 (52% overall yield from 33).

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

361

˚

Scheme 5. Reagents and conditions: (a) TESOTf, 4 A MS, CH₂Cl₂, ꢀ20 ꢀC, 3 h; (b) 0.1 M DBU/MeOH, ꢀ20 ꢀC, 3 h; (c) SO₃ÆNMe₃, DMF, 60 ꢀC,

O/N; (d) 7.5% HCl–MeOH, 0 ꢀC!rt, O/N; (e) H₂O₂, LiOH, THF, NaOH–H₂O, rt, 36 h; (f) 10% Pd/C, H₂, t-BuOH–H₂O, rt, O/N; (g) SO₃Æpyridine,

NaOH–H₂O, pH 9.8, rt, 3 h.

2.4. Preparation of the trisaccharide

tion reaction as there was always some salt mixed with

the product after the column, though we got a very clean

¹H NMR spectrum. In order to get the pure heparin-like

trisaccharide 3 without salt, the mixture was passed

through a Sephadex G-10 column eluting with 10%

aqueous ethanol. The pure target trisaccharide 3 was

then obtained in an overall yield of 47% from 38 (see

Scheme 6).²⁸

Acceptor 6 was coupled with the elongation building

block 23 under the promotion of TESOTf in CH₂Cl₂

˚

and 4 A MS at ꢀ15 to ꢀ10 ꢀC to give disaccharide 34

in 75% yield. The levulinoyl group was cleanly removed

with hydrazine to give alcohol 35, which was then cou-

pled with nonreducing terminal building block 27 under

˚

the promotion of TESOTf in CH₂Cl₂and 3 A MS at

In conclusion, a simple and convenient synthetic

route for preparing ^L-iduronic acid building blocks

starting from readily available 1,2-O-isopropylidene-

6,3-^D-glucuronolactone was developed in less than

nine steps. Based on this synthetic route, a modiﬁed

and eﬃcient synthetic method for preparing an ^L-idu-

ronic acid building block from the key intermediate

methyl dimethylthexylsilyl 3-O-pivaloyl-b-^L-idopyra-

nosiduronate was described. The single-crystal X-ray

structure of the elongation building block (methyl 2-

O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a-^L-idopyranosyl-

uronate) trichloroacetimidate was obtained and

showed that the molecular structure of the building

block is in a ¹C₄conformation. Two heparin-like

disaccharides and one heparin-like trisaccharide with

diﬀerent sulfation patterns have been prepared by

using the ^L-iduronic acid building blocks developed

by the above synthetic routes. Thus, an eﬀective

ꢀ20 ꢀC to give trisaccharide 36 in 40% yield. Treatment

of 36 with 7.5% HCl in methanol led to a good yield

(84%) of the triol 37. The triol 37 was then reacted with

SO₃ÆNMe₃complex in DMF at 60 ꢀC overnight. The

product 38 was puriﬁed by chromatography on Sepha-

dex LH-20 eluting with methanol. The esters of 38 were

then cleaved by a cocktail of reagents (LiOH, H₂O₂

followed by NaOH) in aqueous tetrahydrofuran

(THF). We did this saponiﬁcation reaction the same

way as that of the synthesis of the heparin-like disaccha-

ride 2. The crude product was directly used to do the

next hydrogenation reaction without puriﬁcation on

Sepladex LH-20. The O-benzyl group was removed with

concomitant N₃to NH₂reduction to give 40. These

amines were treated with SO₃Æpyridine in water at

pH 9.5. However, a Bio-Gel P-2 column was not eﬀec-

tive for the puriﬁcation of the residue from the N-sulfa-

362

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

˚

Scheme 6. Reagents and conditions: (a) TESOTf, 4 A MS, CH₂Cl₂, ꢀ15 to ꢀ10 ꢀC, 3 h; (b) N₂H₄ÆHOAc, 2:1 EtOH–toluene, rt, 1 h; (c) TESOTf, 3 A

MS, CH₂Cl₂, ꢀ20 ꢀC, 1 h; (d) 7.5% HCl–MeOH, 0 ꢀC!rt, O/N; (e) SO₃ÆNMe₃, DMF, 50 ꢀC, O/N; (f) H₂O₂, LiOH, THF, NaOH–H₂O, rt, 36 h;

(g) 10% Pd/C, H₂, t-BuOH–H₂O, rt, O/N; (h) SO₃Æpyridine, NaOH–H₂O, pH 9.5, rt, 3 h.

synthetic strategy was developed for preparing heparin-

like oligosaccharide chains with deﬁned size, sequence

and charge distribution.

recorded on Varian-500 (125.75 MHz), Varian-400

(100.55 MHz), or Varian-200 (50.32 MHz) instruments.

Optical rotations were measured at 21 2 ꢀC in a 1-dm

cell on a Perkin–Elmer 341 polarimeter. Gel-permeation

chromatography was performed using Sephadex LH-20,

Sephadex G-10, and Bio-Gel P-2. Thin-layer chroma-

tography (TLC) was performed on precoated plates of

Silica Gel 60-F₂₅₄(E. Merck, Darmstadt) and visualized

with 1:20 H₂SO₄–H₂O, followed by heating. Unless

otherwise stated, column chromatography was per-

formed on Silica Gel 60 (230–400 mesh, E. Merck).

All solvents and reagents were puriﬁed and dried

according to standard procedures.

3. Experimental

3.1. General methods

The ¹H NMR spectra were obtained on Varian-500

(500 MHz) or Varian-400 (400 MHz) instruments with

tetramethylsilane or the residue signal of the solvent as

the internal standard. The ¹³C NMR spectra were

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

363

3.2. Methyl 2-azido-3-O-benzyl-2-deoxy-6-O-pivaloyl-a-

D-glucopyranoside (5)

J_5,41.6 Hz, H-5), 3.87 (m, 1H, H-4), 3.77 (s, 3H,

OCH₃), 3.59 (m, 1H, H-2), 3.14 (br, 1H, OH), 1.63

(m, 1H, CH), 1.20 (s, 9H, 3CH₃, Piv), 0.85 (m, 12H,

4CH₃, TDS), 0.25 (s, 3H, SiCH₃), 0.19 (s, 3H, SiCH₃);

¹³C NMR (50.32 MHz, CDCl₃): d 175.88 (C@O, Piv),

168.35 (C@O, ester), 93.49, 74.64, 69.00, 68.63, 66.99

(C-1, C-5, C-3, C-2, C-4), 52.24 (OCH₃), 38.75 (O@C–

C, Piv), 33.93 (CH), 27.02 (3CH₃, Piv), 24.98 (Si–C–

C), 20.16, 19.89, 18.54, 18.34 (4 · CH₃, TDS), ꢀ2.02,

ꢀ3.69 (2 · CH₃, 2 · SiCH₃). FABMS: Calcd for

C₂₀H₃₈O₈Si [M+H]⁺: m/z 435.2. Found: m/z 435.2.

Anal. Calcd for C₂₅H₄₆O₉Si: C, 57.89; H, 8.94. Found:

C, 58.19; H, 9.32.

Methyl 2-azido-3-O-benzyl-2-deoxy-a-^D-glucopyrano-

side (460 mg, 1.47 mmol) was dissolved in CH₂Cl₂

(3.4 mL) and pyridine (3.4 mL) and cooled to ꢀ78 ꢀC

(dry ice and acetone), then pivaloyl chloride (0.67 mL,

3.6 equiv) was added at ꢀ78 ꢀC. After 15 min, the reac-

tion was stopped by solvent removal and puriﬁed on a

ﬂash column chromatography eluting with 2:1 hex-

anes–EtOAc. The ﬁnal product 5 420 mg (72%) was ob-

tained as a colorless syrup: [a]_D+52.8 (c 2.44, CHCl₃);

¹H NMR (400 MHz, CDCl₃): d 7.38 (m, 5H, Ph), 4.92

(d, 1H, J_a,b11.1 Hz, CHaPh), 4.82 (d, 1H, J_b,a

11.1 Hz, CHbPh), 4.79 (d, 1H, J_1,23.5 Hz, H-1), 4.43

(dd, 1H, J_6a,54.7 Hz, J_6a,6b12.3 Hz, H-6a), 4.25 (dd,

1H, J_6b,52.3 Hz, J_6b,6a12.3 Hz, H-6b), 3.81 (dd, 1H,

J_3,41.4 Hz, J_3,210.1 Hz, H-3), 3.76 (m, 1H, H-5), 3.43

(m, 4H, OCH₃, H-4), 3.34 (dd, 1H, J_2,13.5 Hz, J_2,3

10.1 Hz, H-2), 2.69 (s, 1H, OH), 1.22 (s, 9H, 3CH₃,

Piv); ¹³C NMR (125.75 MHz, CDCl₃): d 179.15 (C@O,

Piv), 137.86, 128.66, 128.23, 128.14 (Ph), 98.68, 79.73

(C-1, C-3), 75.32 (CH₂Ph), 70.82, 70.05, 63.13, 62.94

(C-4, C-5, C-2, C-6), 55.21 (OCH₃), 38.91 (O@C–C,

Piv), 27.16 (CH₃, Piv); Anal. Calcd for C₁₉H₂₇N₃O₆:

C, 58.00; H, 6.92; N, 10.68. Found: C, 58.57; H, 7.23;

N, 10.71. HRMS Calcd for C₁₉H₂₇N₃O₆[MꢀH]⁺:

392.1821. Found: 392.1796.

3.5. Methyl (dimethylthexylsilyl 3,4-di-O-pivaloyl-b-^L-

idopyranosid)uronate (11), methyl (dimethylthexylsilyl

2,3-di-O-pivaloyl-b-^L-idopyranosid)uronate (13), and

methyl (dimethylthexylsilyl 2,3,4-tri-O-pivaloyl-b-^L-ido-

pyranosid)uronate (14)

The colorless syrup 10 (3.2 g, 7.6 mmol) was dissolved in

dry CH₂Cl₂(70 mL) at 10 ꢀC, followed by addition of

pivalic anhydride (1.7 mL, 1.1 equiv). Approximately

15 min later, Sc(OTf)₃(190 mg, 0.05 equiv) was added.

After 1 h, a TLC in 5:1 toluene–EtOAc showed that

the reaction was complete. The solvent phase was

washed with satd aq NaHCO₃(2 · 25 mL) then dried

over anhydrous Na₂SO₄. The organic phase was concen-

trated and dried overnight. The mixture was puriﬁed by

ﬂash column chromatography using 6:1 hexanes–EtOAc

as eluant. Products 11 1.26 g (32%), 13 0.86 g (22%), and

14 0.7 g (15%) were obtained (10:7:5 11:13:14); 11: [a]_D

3.3. Methyl 3-O-pivaloyl-a,b-^L-idopyranosuronate (9)

The syrup 8 (22.0 g, 66.4 mmol) was dissolved in 90%

TFA (210 mL) and stirred for 3 h at room temperature

under argon. The reaction was stopped by solvent

removal. The product was coevaporated with toluene

and then was put on the high-vacuum line. Crude prod-

uct 9 (19.0 g) was obtained, which was used to do the

next step directly without further puriﬁcation.

1

+2.1 (c 0.42, CHCl₃); H NMR (400 MHz, CDCl₃): d

5.18 (br t, 1H, H-3), 4.99 (m, 2H, H-1, H-4), 4.49 (d,

1H, J_5,42.4 Hz, H-5), 3.73 (s, 3H, OCH₃), 3.51 (br m,

1H, H-2), 2.41 (br, 1H, OH), 1.65 (m, 1H, CH), 1.22

(s, 9H, 3CH₃, Piv), 1.18 (s, 9H, 3CH₃, Piv), 0.88 (m,

12H, 4CH₃, TDS), 0.25 (s, 3H, SiCH₃), 0.20 (s, 3H,

SiCH₃); ¹³C NMR (50.32 MHz, CDCl₃): d 176.49,

175.64 (2 · C@O, 2 · Piv), 167.23 (C@O, ester), 94.26,

73.15, 68.90, 67.82, 66.68 (C-1, C-5, C-3, C-2, C-4),

52.25 (OCH₃), 38.88 (O@C–C, Piv), 38.83 (O@C–C,

Piv), 34.01 (CH), 27.13 (3CH₃, Piv), 26.93 (3CH₃, Piv),

25.13 (Si–C–C), 20.31, 19.99, 18.63, 18.41 (4 · CH₃,

TDS), ꢀ1.98 (CH₃, SiCH₃), ꢀ3.37 (CH₃, SiCH₃);

FABMS: Calcd for C₂₅H₄₆O₉Si [M+H]⁺: m/z 519.3.

3.4. Methyl (dimethylthexylsilyl 3-O-pivaloyl-b-^L-idopyr-

anosid)uronate (10)

The colorless syrup 9 (19.0 g, 65 mmol) was dissolved in

dry MeCN (95 mL), followed by addition of imidazole

(11.0 g, 2.5 equiv). The reaction mixture was then cooled

to ꢀ20 ꢀC, followed by addition of TDSCl (16.6 mL,

1.3 equiv). The reaction was let run overnight at

ꢀ20 ꢀC under argon. The reaction completion was veri-

ﬁed with a TLC in 3:1 hexanes–EtOAc. Then the reac-

tion was stopped by solvent removal. The solid was

dissolved in CH₂Cl₂, ﬁltered, and concentrated. The

crude product was puriﬁed by ﬂash column chromatog-

raphy using 4:1 hexanes–EtOAc as eluant to give 10

10.0 g (35%) as a colorless syrup: [a]_D+27.2 (c 1.0,

CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 5.22 (br t,

1H, H-3), 4.93 (d, 1H, J_1,21.0 Hz, H-1), 4.32 (d, 1H,

1

Found: m/z 519.3. 13: [a]_D+131.8 (c 1.0, CHCl₃); H

NMR (400 MHz, CDCl₃): d 5.54 (br t, 1H, J_3,47.8 Hz,

J_3,28.4 Hz, H-3), 5.53 (d, 1H, J_1,24.3 Hz, H-1), 5.06

(dd, 1H, J_2,14.3 Hz, J_2,38.4 Hz, H-2), 4.66 (dd, 1H,

J_4,51.0 Hz, J_4,37.8 Hz, H-4), 4.02 (br s, 1H, H-5), 3.80

(s, 3H, OCH₃), 2.93 (br, 1H, OH), 1.60 (m, 1H, CH),

1.22 (s, 9H, 3CH₃, Piv), 1.18 (s, 9H, 3CH₃, Piv), 0.84

(m, 12H, 4CH₃, TDS), 0.10 (s, 3H, SiCH₃), 0.06 (s,

3H, SiCH₃); ¹³C NMR (50.32 MHz, CDCl₃): d 178.35

(C@O, Piv), 177.61 (C@O, Piv), 172.75 (C@O, ester),

364

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

93.36, 76.47, 75.38, 74.11, 69.17 (C-1, C-2, C-4, C-3, C-

5), 52.85 (OCH₃), 38.74 (O@C–C, Piv), 38.55 (O@C–C,

Piv), 33.92 (CH), 27.12 (3CH₃, Piv), 26.92 (3CH₃, Piv),

24.72 (Si–C–C), 19.98, 19.88, 18.52, 18.47 (4 · CH₃,

TDS), ꢀ2.62, ꢀ3.15 (2 · CH₃, 2 · SiCH₃); HRMS:

Calcd for C₂₅H₄₆O₉Si [MꢀH]⁺: m/z 517.2833. Found:

m/z 517.2719. 14: [a]_Dꢀ31.6 (c 1.18, CHCl₃); ¹H

NMR (400 MHz, CDCl₃): d 5.34 (d, 1H, J_1,23.7 Hz,

H-1), 5.18 (br t, 1H, J_3,44.1 Hz, J_3,25.3 Hz, H-3), 5.14

(br t, 1H, J_4,53.9 Hz, J_4,34.1 Hz, H-4), 4.84 (d, 1H,

J_5,43.9 Hz, H-5), 4.76 (dd, 1H, J_2,13.7 Hz, J_2,35.3 Hz,

H-2), 3.73 (s, 3H, OCH₃), 1.61 (m, 1H, CH), 1.22,

1.16 (2 · s, 27H, 9CH₃, 3 · Piv), 0.84 (m, 12H, 4CH₃,

TDS), 0.16 (s, 3H, SiCH₃), 0.15 (s, 3H, SiCH₃); ¹³C

NMR (50.32 MHz, CDCl₃): d 176.87, 176.83, 176.44

(3 · C@O, 3 · Piv), 168.36 (C@O, ester), 93.46, 70.87,

68.94, 68.04, 68.02 (C-1, C-2, C-3, C-5, C-4), 52.27

(OCH₃), 38.80, 38.70 (overlap, 3 · O@C–C, 3 · Piv),

33.82 (CH), 27.20, 27.09, 27.08 (3 · 3CH₃, 3 · Piv),

24.82 (Si–C–C), 20.01, 19.95, 18.50, 18.47 (4 · CH₃,

TDS), ꢀ2.42, ꢀ3.21 (2 · CH₃, 2 · SiCH₃); FABMS:

Calcd for C₃₀H₅₄O₁₀Si [M+H]⁺: m/z 603.36. Found:

m/z 603.28.

1H, J_1,22.6 Hz, H-1), 5.10 (br t, 1H, H-3), 5.07 (br t,

1H, H-4), 4.87 (d, 1H, J_5,43.3 Hz, H-5), 4.70 (br t, 1H,

H-2), 3.75 (s, 3H, OCH₃), 2.03 (s, 3H, CH₃, Ac), 1.63

(m, 1H, CH), 1.21 (s, 9H, 3CH₃, Piv), 1.18 (s, 9H,

3CH₃, Piv), 0.86 (m, 12H, 4CH₃, TDS), 0.19 (s, 3H,

SiCH₃), 0.17 (s, 3H, SiCH₃); ¹³C NMR (50.32 MHz,

CDCl₃): d 176.69, 176.45 (2 · C@O, 2 · Piv), 169.33

(C@O, Ac), 168.50 (C@O, ester), 92.93, 69.43, 67.46,

67.16, 67.10 (C-1, C-2, C-3, C-4, C-5), 52.40 (OCH₃),

38.81, 38.78 (2 · O@C–C, 2 · Piv), 33.82 (CH), 27.06,

26.97 (2 · 3CH₃, 2 · Piv), 25.04 (Si–C–C), 20.85 (CH₃,

Ac), 20.04, 20.01, 18.48 (4CH₃, TDS), ꢀ2.46, ꢀ3.21

(2 · CH₃, 2 · SiCH₃); FABMS: Calcd for C₂₇H₄₈O₁₀Si

[M+H]⁺: m/z 561.3. Found: m/z 561.4. Anal. Calcd for

C₂₇H₄₈O₁₀Si: C, 57.83; H, 8.63. Found: C, 57.94; H, 9.13.

3.7. Methyl 2-O-acetyl-3,4-di-O-pivaloyl-a,b-^L-idopyr-

anosuronate (15)

The colorless syrup 12 (1.0 g, 1.8 mmol) was dissolved in

dry THF (13.5 mL) and cooled to 0 ꢀC, followed by

addition of HOAc (0.3 mL, 3.0 equiv) and Bu₄NF

(2.7 mL, 1.5 equiv, 1.0 M solution in THF). The reac-

tion mixture was stirred with the temperature gradually

increasing to 15 ꢀC. The reaction was checked by TLC

using 1:3 hexanes–EtOAc. The resulting orange solution

was ﬁltered through a bed of silica gel with 1:3 hexanes–

EtOAc to give three fractions of 200 mL each, and then

the three fractions were combined and concentrated.

The crude product was puriﬁed by ﬂash column chro-

matography using 1:2 hexanes–EtOAc as eluant to give

product 15 (0.73 g, 98%), which was used directly in the

next step.

3.6. Methyl (dimethylthexylsilyl 2-O-acetyl-3,4-di-O-

pivaloyl-a,b-^L-idopyranosid)uronate (12)

The colorless syrup 11 (430 mg, 0.8 mmol) was dissolved

in dry CH₂Cl₂(7.5 mL) followed by addition of Ac₂O

(0.8 mL, 10.0 equiv) then Sc(OTf)₃(20 mg, 0.05 equiv)

was added. The mixture was stirred at room temperature

overnight. The solvent phase was washed with H₂O

(2 · 5 mL) then dried over anhydrous Na₂SO₄. The or-

ganic phase was concentrated. The mixture was puriﬁed

by ﬂash column chromatography using 10:1 hexanes–

EtOAc as eluant. The a anomer 100 mg (22%) and b ano-

mer 160 mg (34%) of the product 12 was obtained (5:8

3.8. Methyl 2,3,4-tri-O-pivaloyl-a,b-^L-idopyranosuronate

(16)

1

a:b); a anomer: [a]_D+38.5 (c 1.01, CHCl₃); H NMR

The colorless syrup 14 (1.1 g, 1.9 mmol) was dissolved in

dry THF (13 mL) and cooled to 0 ꢀC, followed by addi-

tion of HOAc (0.32 mL, 3.0 equiv) and Bu₄NF (2.8 mL,

1.5 equiv, 1.0 M solution in THF). The reaction mixture

was stirred at room temperature overnight. The reaction

was checked by TLC using 2:1 hexanes–EtOAc. The

resulting orange solution was ﬁltered through a bed of

silica gel with 1:3 hexanes–EtOAc to give three fractions

of 250 mL each, and then the three fractions were com-

bined and concentrated. The crude product was puriﬁed

by ﬂash column chromatography using 1:2 hexanes–

EtOAc as eluant to give product 16 (0.81 g, 94%), which

was used directly in the next step.

(400 MHz, CDCl₃): d 5.12 (br t, 1H, J_2,11.8 Hz, J_2,3

3.3 Hz, H-2), 5.03 (d, 1H, J_1,21.8 Hz, H-1), 4.98 (m,

1H, J_4,52.5 Hz, H-4), 4.90 (m, 1H, H-3), 4.49 (d, 1H,

J_5,42.5 Hz, H-5), 3.73 (s, 3H, OCH₃), 2.06 (s, 3H, CH₃,

Ac), 1.61 (m, 1H, CH), 1.23 (s, 9H, 3CH₃, Piv), 1.19 (s,

9H, 3CH₃, Piv), 0.85 (m, 12H, 4CH₃, TDS), 0.22 (s,

3H, SiCH₃), 0.15 (s, 3H, SiCH₃); ¹³C NMR

(50.32 MHz, CDCl₃): d 176.67, 175.28 (2 · C@O,

2 · Piv), 169.40 (C@O, Ac), 167.17 (C@O, ester), 93.08,

72.86, 67.90, 66.72, 66.08 (C-1, C-5, C-2, C-3, C-4),

52.21 (OCH₃), 38.81 (2 · O@C–C, 2 · Piv), 33.92 (CH),

27.10 (3CH₃, Piv), 26.95 (3CH₃, Piv), 25.00 (Si–C–C),

20.87 (CH₃, Ac), 20.11, 19.89, 18.55, 18.41 (4 · CH₃,

TDS), ꢀ2.01, ꢀ3.33 (2 · CH₃, 2 · SiCH₃); FABMS:

Calcd for C₂₇H₄₈O₁₀Si [MꢀOTDS]⁺: m/z 401.2. Found:

m/z 401.3. Anal. Calcd for C₂₇H₄₈O₁₀Si: C, 57.83; H,

8.63. Found: C, 57.74; H, 8.86; b anomer: [a]_Dꢀ52.9 (c

3.9. Methyl 2-O-acetyl-3,4-di-O-pivaloyl-a-^L-idopyr-

anosyluronate trichloroacetimidate (17)

Product 15 (1.25 g, 3.0 mmol) was dissolved in dry

CH₂Cl₂(22 mL) and cooled in an ice bath. Then,

1

1.01, CHCl₃); H NMR (400 MHz, CDCl₃): d 5.39 (d,

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

365

CCl₃CN (3.0 mL, 10.0 equiv) was added and allowed to

cool down, followed by the addition of DBU (106.6 lL).

The reaction mixture was stirred for 1 h, and TLC in 3:2

hexanes–EtOAc showed that the reaction was complete.

The crude product was puriﬁed by ﬂash column chro-

matography using 6:1 hexanes–EtOAc as eluant to give

the ﬁnal product 17 0.92 g (55%): [a]_Dꢀ49.7 (c 1.88,

addition of HOAc (0.33 mL, 3.0 equiv) and Bu₄NF

(3.0 mL, 1.5 equiv, 1.0 M solution in THF). Then the

temperature was gradually increased to room tempera-

ture. The reaction mixture was stirred overnight.

The reaction was checked by TLC using a mixture of

1:3 hexanes–EtOAc. The resulting orange solution was

ﬁltered through a bed of silica gel with 1:3 hexanes–

EtOAc to give three fractions of 200 mL each, and

then the three fractions were combined and concen-

trated. The crude product was puriﬁed by ﬂash col-

umn using 1:2 hexanes–EtOAc as eluant to give

product 22 (0.55 g, 64%), which was used directly in

the next step.

1

CHCl₃); H NMR (400 MHz, CDCl₃): d 8.86 (s, 1H,

NH), 6.49 (s, 1H, 1-H), 5.13 (m, 2H, H-3, H-4), 4.97

(m, 1H, H-2), 4.89 (d, 1H, J_5,42.3 Hz, 5-H), 3.74 (s,

3H, OCH₃), 2.07 (s, 3H, CH₃, Ac), 1.23 (s, 9H, 3CH₃,

Piv), 1.18 (s, 9H, 3CH₃, Piv); ¹³C NMR (50.32 MHz,

CDCl₃): d 176.48, 176.26 (2 · C@O, 2 · Piv), 168.92

(C@O, Ac), 167.33 (C@O, ester), 159.80 (C@NH),

94.00 (C-1), 90.62 (CCl₃), 68.12 (C-5), 66.12 (overlap,

C-2, C-3, C-4), 52.62 (OCH₃), 38.89, 38.85 (2 · O@C–

C, 2 · Piv), 27.08, 26.93 (2 · 3CH₃, 2 · Piv), 20.72

(CH₃, Ac); FABMS: Calcd for C₂₁H₃₀Cl₃NO₁₀

[MꢀOTCI]⁺: m/z 401.2. Found: m/z 401.3. Anal. Calcd

for C₂₁H₃₀Cl₃NO₁₀: C, 44.81; H, 5.37; N, 2.49. Found:

C, 44.65; H, 5.58; N, 2.53.

3.12. Methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a,b-

L-idopyranosyluronate trichloroacetimidate (23)

The product 22 (390 mg, 0.91 mmol) was dissolved in

dry CH₂Cl₂(6.6 mL) and cooled in an ice bath. Then,

CCl₃CN (0.91 mL, 10.0 equiv) was added, followed by

the addition of DBU (32 lL). The reaction mixture

was stirred for 1.0 h at 0 ꢀC, and TLC using 1:2 hex-

anes–EtOAc showed that the reaction was complete.

The mixture was evaporated, and the residue was puri-

ﬁed on a MPLC column eluting with 2:1:1 hexanes–

EtOAc–CH₂Cl₂to give the a anomer (290 mg, 58%)

and the b anomer (79 mg, 15%) of the product 23 as col-

3.10. Methyl 2,3,4-tri-O-pivaloyl-a-^L-idopyranosyluron-

ate trichloroacetimidate (18)

The product 16 (0.81 g, 1.8 mmol) was dissolved in dry

CH₂Cl₂(13 mL) and cooled in an ice bath. Then

CCl₃CN (1.8 mL, 10.0 equiv) was added and allowed

to cool down, followed by the addition of DBU

(62.7 lL). The reaction mixture was stirred for 1 h,

and TLC in 2:1 hexanes–EtOAc showed that the reac-

tion was complete. The crude product was puriﬁed by

ﬂash column chromatography using 6:1 hexanes–EtOAc

as eluant to give the ﬁnal product 18 (0.66 g, 63%); [a]_D

1

orless oils. a Anomer: [a]_Dꢀ68.5 (c 1.0, CHCl₃); H

NMR (400 MHz, CDCl₃): d 8.86 (s, 1H, NH), 6.50 (br

s, 1H, H-1), 5.41 (m, 1H, H-3), 5.14 (br t, 1H, J_4,5

2.3 Hz, H-4), 4.99 (m, 1H, H-2), 4.88 (d, J_5,42.3 Hz,

1H, H-5), 3.79 (s, 3H, OCH₃), 2.76 (m, 2H, CH₂,

Lev), 2.58 (m, 2H, CH₂, Lev), 2.17 (s, 3H, CH₃, Lev),

2.14 (s, 3H, CH₃, Ac), 1.24 (s, 9H, 3CH₃, Piv); ¹³C

NMR (50.32 MHz, CDCl₃): d 205.80 (C@O, Lev),

176.32 (C@O, Piv), 171.10 (O@C, Lev), 169.09 (C@O,

Ac), 167.39 (C@O, ester), 159.93 (N@C–CCl₃), 94.16

(C-1), 90.65 (CCl₃), 68.04, 66.67, 66.15, 65.82 (C-5, C-

4, C-2, C-3), 52.80 (OCH₃), 38.84 (O@C–C, Piv), 37.50

(CH₂, Lev), 29.72 (CH₃, Lev), 27.61 (CH₂, Lev), 26.99

(3CH₃, Piv), 20.67 (CH₃, Ac); FABMS: Calcd for

C₁₉H₂₇O₁₀[MꢀOTCI]⁺: m/z 415.2. Found: m/z 415.2.

b Anomer: [a]_D+9.5 (c 1.0, CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d 8.68 (s, 1H, NH), 6.22 (d, 1H,

J_1,22.3 Hz, H-1), 5.41 (br t, 1H, J_3,24.7 Hz, H-3), 5.23

(dd, 1H, J_2,12.3 Hz, J_2,34.7 Hz, H-2), 5.14 (br t, 1H,

J_4,52.9 Hz, H-4), 4.71 (d, J_5,42.9 Hz, 1H, H-5), 3.80

(s, 3H, OCH₃), 2.72 (m, 2H, CH₂, Lev), 2.57 (m, 2H,

CH₂, Lev), 2.18 (s, 3H, CH₃, Lev), 2.12 (s, 3H, CH₃,

Ac), 1.24 (s, 9H, 3CH₃, Piv); ¹³C NMR (50.32 MHz,

CDCl₃): d 205.90 (O@C–CH₃, Lev), 175.71 (C@O,

Piv), 171.29 (O@C–O, Lev), 169.45 (C@O, Ac), 166.54

(O@C, ester), 160.45 (N@C–CCl₃), 94.32 (C-1), 90.33

(CCl₃), 73.05, 66.98, 66.82, 65.32 (C-5, C-4, C-3, C-

2), 52.76 (OCH₃), 38.86 (O@C–C, Piv), 37.52 (CH₂,

Lev), 29.74 (CH₃, Lev), 27.68 (CH₂, Lev), 27.01

1

ꢀ57.3 (c 1.0, CHCl₃); H NMR (400 MHz, CDCl₃): d

8.81 (s, 1H, NH), 6.51 (d, 1H, J_1,23.1 Hz, H-1), 5.26

(br t, 1H, H-3), 5.21 (br t, 1H, H-4), 5.07 (br t, 1H, H-

2), 4.89 (d, 1H, J_5,43.5 Hz, H-5), 3.75 (s, 3H, OCH₃),

1.22 (s, 9H, 3CH₃, Piv), 1.21 (s, 9H, 3CH₃, Piv), 1.19

(s, 9H, 3CH₃, Piv); ¹³C NMR (50.32 MHz, CDCl₃): d

176.94, 176.83, 176.45 (3 · C@O, 3 · Piv), 167.53

(C@O, ester), 160.12 (C@NH), 94.68 (C-1), 90.62

(CCl₃), 69.17, 67.83, 67.53, 67.11 (C-5, C-3, C-2, C-4),

52.53 (OCH₃), 38.86, 38.80, 38.78 (3 · O@C–C,

3 · Piv), 27.06 (9CH₃, 3 · Piv); FABMS: Calcd for

C₂₂H₃₅O₉[MꢀOTI]⁺: m/z 443.2. Found: m/z 443.3.

Anal. Calcd for C₂₄H₃₆Cl₃NO₁₀: C, 47.65; H, 6.00; N,

2.32. Found: C, 48.05; H, 6.33; N, 2.31.

The syntheses of compounds 19, 20, and 21 are de-

scribed in Ref. 25.

3.11. Methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a,b-

L-idopyranosuronate (22)

The colorless syrup 21 (1.14 g, 1.98 mmol) was dissolved

in dry THF (15 mL) and cooled to 0 ꢀC, followed by

366

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

(3CH₃, Piv), 20.59 (CH₃, Ac); FABMS: Calcd for

C₁₉H₂₇O₁₀[MꢀOTCI]⁺: m/z 415.1604. Found: m/z

415.1613.

Colorless and transparent crystals of the donor,

methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-a-^L-ido-

pyranosyluronate) trichloroacetimidate, the a anomer

of 23, were obtained by slow evaporation of a saturated

3:1 hexanes–CH₂Cl₂solution.

(1.6 g, 10.0 equiv). The reaction mixture was stirred for

1 h, and TLC in 2:1 hexanes–EtOAc showed that the

reaction was complete. The mixture was ﬁltered and

evaporated. The crude product was puriﬁed on a

MPLC column eluting with 8:1 hexanes–EtOAc to give

the product 27 (160 mg, 58%) as a colorless oil:

[a]_D+56.2 (c 1.0, CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d 8.79 (s, 1H, NH), 7.36–7.28 (m, 5H, Ph),

6.46 (d, 1H, J_1,23.5 Hz, H-1), 4.81 (d, 1H, J_a,b

10.7 Hz, CHaPh), 4.72 (d, 1H, J_b,a10.7 Hz, CHbPh),

4.16–4.10 (m, 3H, H-6a, H-6b, H-5), 4.04 (br t, 1H, H-

3), 3.79 (dd, 1H, J_2,13.5 Hz, J_2,310.1 Hz, H-2), 2.06

(s, 3H, CH₃, Ac), 1.21 (s, 9H, 3CH₃, Piv); ¹³C NMR

(50.32 MHz, CDCl₃): d 176.65 (C@O, Piv), 170.43

(C@O, Ac), 160.37 (C@NH), 136.98, 128.36, 127.87,

127.49 (Ph), 94.30 (C-1), 90.69 (CCl₃), 77.76 (C-3),

74.84 (CH₂Ph), 70.94, 69.03, 62.51, 61.52 (C-5, C-4, C-

2, C-6), 38.94 (O@C–C, Piv), 27.12 (3CH₃, Piv), 20.75

(CH₃, Ac); HRMS: Calcd for C₂₀H₂₆N₃O₆[MꢀTCI]⁺:

404.1822. Found: 404.1905. Anal. Calcd for

C₂₂H₂₇Cl₃N₄O₇: C, 46.70; H, 4.81; N, 9.90. Found: C,

47.34; H, 5.15; N, 10.03.

3.13. 1,6-Di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-

pivaloyl-a,b-^D-glucopyranose (25)

Sugar 24 (0.64 g, 1.68 mmol) was dissolved in pyridine

(10 mL). The temperature was raised to 45 ꢀC. Then

DMF (0.5 mL) and PivCl (1.04 mL, 5.0 equiv) were

added. After 24 h another batch of PivCl (5.0 equiv)

was added, and after a further 24 h of continuously stir-

ring, the mixture was diluted with CH₂Cl₂(50 mL),

washed twice with H₂O (2 · 20 mL). The organic phase

was dried with Na₂SO₄and ﬁltered, and the solvent was

evaporated. The crude product was puriﬁed on a MPLC

column eluting with 8:1 hexanes–EtOAc to give the

1

product 25 (0.32 g, 41%) as a colorless oil; H NMR

(400 MHz, CDCl₃): d 7.36–7.27 (m, 5Ha, 5Hb, Phs),

6.28 (d, 1Ha, J_1,23.7 Hz, H-1a), 5.52 (d, 1Hb, J_1,2

8.4 Hz, H-1b), 5.23 (br t, 1Ha, J_4,39.6 Hz, H-4a), 4.80

(d, 1Ha, J_a,b10.7 Hz, CHaPha), 4.69 (d, 1Ha, J_b,a

10.7 Hz, CHbPha), 4.17 (dd, 1Ha, J_6a,54.6 Hz, J_6a,6b

12.6 Hz, H-6aa), 4.01 (m, 2Ha, H-6ba, H-5a), 3.94 (br

t, 1Ha, J_3,49.6 Hz, J_3,29.9 Hz, H-3a), 3.69 (dd, 1Ha,

J_2,13.7 Hz, J_2,39.9 Hz, H-2a), 2.21 (s, 3Ha, CH₃a,

Aca), 2.08 (s, 3Ha, CH₃a, Aca), 1.20 (s, 9Ha, Piva);

HRMS: Calcd for C₂₂H₂₉N₃O₈M⁺: 463.1955. Found:

463.1995.

3.16. Methyl (methyl 2-O-acetyl-3,4-di-O-pivaloyl-a-^L-

idopyranosyluronate)-(1!4)-2-azido-3-O-benzyl-2-

deoxy-6-O-pivaloyl-a-^D-glucopyranoside (28)

Glucosamine 5 (44 mg, 0.1 mmol) and ^L-iduronic acid 17

(94 mg, 1.5 equiv) were coevaporated with toluene and

then dissolved in anhyd CH₂Cl₂(4 mL). Freshly acti-

˚

vated powdered molecular sieves (35 mg, 4 A) were then

added. The mixture was stirred for half an hour at

ꢀ20 ꢀC, then TESOTf (12.6 lL, 0.5 equiv) was added

dropwise, and 3 h later N,N-ethyldiisopropylamine

(25 lL, 1.0 equiv) was added. The mixture was ﬁltered

through a pad of Celite, and the solvent was removed

under reduced pressure. Puriﬁcation on a MPLC column

eluting with (5:1 toluene–EtOAc) aﬀorded the disaccha-

ride 28 75 mg (85%) as a colorless syrup: [a]_D+1.5 (c

3.14. 6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-pival-

oyl-a,b-^D-glucopyranose (26)

Sugar 25 (0.28 g, 0.61 mmol) was dissolved in DMF

(10 mL). Then (73 mg, 1.3 equiv) of N₂H₄ÆHOAc

was added. The mixture was stirred at room temperature

for 2.5 h. After that time, the mixture was diluted with

CH₂Cl₂(30 mL) and washed with H₂O (3 · 10 mL).

The organic layer was dried over Na₂SO₄and ﬁltered.

The solvent was evaporated. The crude product was

puriﬁed on a MPLC column eluting with 3:1 hexanes–

EtOAc to give product 27 (205 mg, 80%) as a colorless

oil that was directly used in the next step.

1

0.66, CHCl₃); H NMR (400 MHz, CDCl₃): d 7.37 (m,

5H, Ph), 5.34 (d, 1H, J_{1 ,2}4.7 Hz, H-1⁰), 5.22 (br t, 1H,

0

H-3⁰), 5.08 (dd, 1H, H-4⁰), 4.90 (dd, 1H, J_{2 ,3}1.8 Hz,

0

J_{2 ,1}4.7 Hz, H-2⁰), 4.84 (br t, 2H, CHaPh, CHbPh),

0

4.76 (br t, 2H, J_1,23.7 Hz, J_{5 ,4}5.5 Hz, H-5⁰, H-1), 4.54

(d, 1H, J_6a,6b11.3 Hz, H-6a), 4.12 (dd, 1H, J_6a,54.5 Hz,

J_6a,6b11.3 Hz, H-6b), 3.87 (br t, 1H, H-3), 3.80 (m, 2H,

H-4, H-5), 3.46 (s, 3H, OCH₃, ester), 3.42 (s, 3H,

OCH₃), 3.39 (dd, 1H, J_2,13.7 Hz, J_2,39.7 Hz, H-2),

2.05 (s, 3H, CH₃, Ac), 1.22 (s, 9H, 3CH₃, Piv), 1.20 (s,

9H, 3CH₃, Piv), 1.14 (s, 9H, 3CH₃, Piv); ¹³C NMR

0

3.15. 6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-pival-

oyl-a-^D-glucopyranosyl trichloroacetimidate (27)

(100.55 MHz, CDCl₃):

d

177.76, 176.67, 176.39

(3 · C@O, 3 · Piv), 169.31 (C@O, ester), 168.17 (C@O,

Ac), 137.55, 128.32, 127.62 (Ph), 98.23 (C-1), 97.99 (C-

1⁰), 78.10, 77.33 (C-3, C-5), 74.58 (CH₂Ph), 69.22 (C-4),

69.05, 68.88 (C-2⁰, C-5⁰), 67.86, 67.67 (C-3⁰, C-4⁰),

63.40, 61.72 (C-2, C-6), 55.40 (OCH₃, ester), 52.05

The product 26 (205 mg, 0.49 mmol) was dissolved in

dry CH₂Cl₂(3 mL) and cooled in an ice bath. Then,

CCl₃CN (0.5 mL, 10.0 equiv) was added and allowed

to cool down, followed by the addition of Cs₂CO₃

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

367

3.9 Hz, J_{4 ,3}5.1 Hz, H-4⁰), 4.90 (d, 1H, J_a,b10.8 Hz,

CHaPh), 4.83 (d, 1H, J_b,a10.8 Hz, CHbPh), 4.79 (d,

0

(OCH₃), 38.93, 38.80 (overlap, 3 · O@C–C, 3 · Piv),

27.25, 27.06, 26.97 (3 · 3CH₃, 3 · Piv), 20.74 (CH₃,

Ac); FABMS: Calcd for C₃₈H₅₅N₃O₁₅[M+Na]⁺: m/z

816.4. Found: m/z 816.4.

1H, J_{5 ,4}3.9 Hz, H-5⁰), 4.74 (d, 1H, J_1,23.5 Hz, H-1),

4.44 (dd, 1H, J_6a,51.6 Hz, J_6a,6b12.1 Hz, H-6a), 4.40

0

(dd, 1H, J_{2 ,1}3.9 Hz, J_{2 ,3}4.5 Hz, H-2⁰), 4.34 (dd, 1H,

J_6b,54.1 Hz, J_6b,6a12.1 Hz, H-6b), 3.89 (m, 3H, H-3,

H-5, H-4), 3.42 (s, 3H, OCH₃), 3.39 (m, 4H, H-2,

0

3.17. Methyl (methyl 3,4-di-O-pivaloyl-a-^L-idopyranosyl-

uronate)-(1!4)-2-azido-3-O-benzyl-2-deoxy-6-O-pival-

oyl-a-^D-glucopyranoside (29)

OCH₃, ester), 2.91 (d, 9H, J_CH,NH 5.1 Hz, 3CH₃,

3

NMe₃), 1.23 (s, 9H, 3CH₃, Piv), 1.20 (s, 9H, 3CH₃,

Piv), 1.13 (s, 9H, 3CH₃, Piv); ¹³C NMR (100.55 MHz,

CDCl₃): d 178.24, 177.05 (overlap, 3 · C@O, 3 · Piv),

168.40 (C@O, ester), 137.77, 128.27, 127.61, 127.51

(Ph), 99.02 (C-1⁰), 98.27, 78.05, 76.86 (C-1, C-3, C-4),

74.30 (CH₂Ph), 72.95 (C-2⁰), 69.08 (C-5), 68.11, 67.64,

67.33 (C-5⁰, C-4⁰, C-3⁰), 63.44, 62.46 (C-2, C-6), 55.29

(OCH₃, ester), 51.80 (OCH₃), 45.57 (3 · CH₃, NMe₃),

38.83, 38.82, 38.72 (3 · O@C–C, 3 · Piv), 27.18, 27.11,

26.88 (3 · 3CH₃, 3 · Piv); FABMS: Calcd for

C₃₉H₆₂N₄O₁₇[M+NMe₃]⁺: m/z 949.5. Found: m/z 949.8.

The disaccharide 28 (154 mg, 0.2 mmol) was dissolved in

anhyd MeOH (31 mL), and DBU (460.6 lL) was added

at ꢀ20 ꢀC. About 3 h later, the solution was diluted with

CH₂Cl₂(350 mL), washed with 5% HCl (2 · 35 mL), and

then washed with H₂O (3 · 35 mL). The organic phase

was dried over anhyd Na₂SO₄. Then the solvent was re-

moved under reduced pressure. Puriﬁcation on a MPLC

column eluting with 6:1 toluene–EtOAc aﬀorded the

deacetylated disaccharide 29 (80 mg, 55%) as a colorless

oil: [a]_D+3.5 (c 0.54, CHCl₃); ¹H NMR (400 MHz,

0

CDCl₃): d 7.35 (m, 5H, Ph), 5.40 (d, 1H, J_{1 ,2}6.0 Hz,

H-1⁰), 5.29 (br t, 1H, H-3⁰), 5.12 (dd, 1H, J_{4 ,5}5.9 Hz,

0

3.19. Methyl 2-O-sulfo-a-^L-idopyranosyluronic acid-

(1!4)-2-deoxy-2-sulfamido-a-^D-glucopyranoside triso-

dium salt (1)

J_{4 ,3}8.2 Hz, H-4⁰), 4.92 (d, 1H, J_a,b10.3 Hz, CHaPh),

4.78 (d, 1H, J_b,a10.3 Hz, CHbPh), 4.75 (d, 1H, J_1,2

3.5 Hz, H-1), 4.68 (d, 1H, J_{5 ,4}5.9 Hz, H-5⁰), 4.65 (dd,

1H, J_6a,51.6 Hz, J_6a,6b12.1 Hz, H-6a), 4.18 (dd, 1H,

J_6b,55.3 Hz, J_6b,6a12.1 Hz, H-6b), 3.88 (dd, 1H, J_3,2

10.1 Hz, J_3,410.4 Hz, H-3), 3.83 (ddd, 1H, J_5,6a1.6 Hz,

J_5,6b5.3 Hz, J_5,410.0 Hz, H-5), 3.73 (dd, 1H, J_4,5

0

3.19.1. Saponiﬁcation. The O-sulfated product 30

(120 mg, 135 lmol) was dissolved in THF (16 mL) and

cooled to ꢀ5 ꢀC. H₂O₂(6 mL, 30%) and aq LiOH

(2.8 mL, 0.7 M) were added dropwise. The reaction mix-

ture was stirred for 1 h at ꢀ5 ꢀC, then 2 h at 0 ꢀC. After

20 h stirring at room temperature, MeOH (13 mL), then

aq NaOH (3.6 mL, 4 N), were added dropwise at 0 ꢀC,

and the solution was left stirring for 1 h at 0 ꢀC. After that

time, the stirring was prolonged overnight at room tem-

perature. After neutralization with 6 N HCl and extrac-

tion with CH₂Cl₂, the organic layer was washed with

acidiﬁed aq Na₂S₂O₃(pH 3.5), the water phase was com-

bined and neutralized and then concentrated. The white

solid was extracted with 1:1 CH₂Cl₂–MeOH, then the sol-

vent phase was concentrated, and the residue was puriﬁed

on Sephadex LH-20 using 1:1 MeOH–H₂O as eluant.

0

10.0 Hz, J_4,310.4 Hz, H-4), 3.61 (dd, 1H, J_{2 ,1}6.0 Hz,

J_{2 ,3}8.0 Hz, H-2⁰), 3.54 (s, 3H, OCH₃, ester), 3.41 (s,

3H, OCH₃), 3.39 (dd, 1H, J_2,13.5 Hz, J_2,310.1 Hz, H-

2), 1.69 (br, 1H, OH), 1.22 (s, 9H, 3CH₃, Piv), 1.21

(s, 9H, 3CH₃, Piv), 1.14 (s, 9H, 3CH₃, Piv); ¹³C NMR

0

(50.32 MHz,

CDCl₃):

178.35,

177.68,

176.69

(3 · C@O, 3 · Piv), 168.81 (C@O, ester), 137.61,

128.31, 127.95, 127.68 (Ph), 101.45 (C-1⁰), 98.14 (C-1),

78.59, 78.44 (C-4, C-3), 75.12 (CH₂Ph), 71.68, 70.34,

70.15 (C-2⁰, C-3⁰, C-5⁰), 69.88 (C-5), 68.29 (C-4⁰),

63.31, 62.43 (C-2, C-6), 55.27 (OCH₃, ester), 52.03

(OCH₃), 38.96, 38.89, 38.81 (3 · O@C–C, 3 · Piv),

27.22, 27.13, 27.02 (3 · 3CH₃, 3 · Piv); FABMS: Calcd

for C₃₆H₅₃N₃O₁₄[M+Na]⁺: m/z 774.3. Found: m/z 774.7.

3.19.2. Hydrogenolysis. A solution of the compound ob-

tained above in 1:1 t-BuOH–H₂O (3 mL) was treated with

H₂(50 bar) in the presence of Pd/C catalyst (120 mg, 10%)

overnight. The mixture was ﬁltered and concentrated. The

residue was puriﬁed on Sephadex LH-20 using 1:1

MeOH–H₂O as eluant to give product (15.0 mg).

3.18. Methyl (methyl 2-O-trimethylaminesulfonato-3,4-

di-O-pivaloyl-a-^L-idopyranosyluronate)-(1!4)-2-azido-3-

O-benzyl-2-deoxy-6-O-pivaloyl-a-^D-glucopyranoside (30)

A solution of the deacylated disaccharide 29 (115 mg,

0.15 mmol) and SO₃ÆNMe₃complex (213 mg, 10.0 equiv)

in dry DMF (15 mL) was stirred overnight at 60 ꢀC.

After the reaction was ﬁnished, the solution was concen-

trated to ꢁ2 mL. Chromatography on Sephadex LH-20

using MeOH gave product (120 mg, 88%): [a]_D+13.0 (c

3.19.3. N-Sulfation. The product obtained above

(15 mg, 30 lmol) was dissolved in H₂O (6 mL). SO₃Æ

pyridine (23 mg, 145 lmol) was added in ﬁve portions

at t = 0; 0.5; 1.0; 1.5; 2.0; and 2.5 h, the pH being

maintained at pH 9.8 by addition of 4 N aq NaOH.

After 3 h, the reaction mixture was concentrated and

1

1.0, CHCl₃); H NMR (400 MHz, CDCl₃): d 9.72 (br,

NH, HN⁺Me₃), 7.34 (m, 5H, Ph), 5.34 (d, 1H, J_{1 ,2}

layered onto

with H₂O and product

a

Bio-Gel P-2 column and eluted

14.0 mg (78%) was

0

3.9 Hz, H-1⁰), 5.32 (br t, 1H, H-3⁰), 5.02 (br t, 1H, J_{4 ,5}

1

0

368

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

obtained; ¹H NMR (500 MHz, D₂O): d 5.12 (d, 1H,

0 ꢀC. The reaction mixture was gradually warmed up

to room temperature and let stand overnight. The solu-

tion was diluted with CH₂Cl₂(450 mL), washed with

satd aq NaHCO₃(35 mL), and then washed with H₂O

(3 · 35 mL). The organic phase was dried over anhy-

drous Na₂SO₄. Then the solvent was removed under

reduced pressure. Puriﬁcation on a MPLC column elut-

ing with 3:1 toluene–EtOAc aﬀorded the deacetylated

disaccharide 32 (63 mg, 81%): [a]_D+3.4 (c 1.0, CHCl₃);

¹H NMR (500 MHz, CDCl₃): d 7.35 (m, 5H, Ph), 5.47

J_{1 ,2}2.3 Hz, H-1⁰), 5.02 (d, 1H, J_1,23.6 Hz, H-1), 4.75

0

(d, 1H, J_{5 ,4}2.5 Hz, H-5⁰), 4.23 (br t, 1H, H-2⁰), 4.02

0

(br t, 1H, H-3⁰), 3.97 (d, 1H, J_{4 ,5}3.9 Hz, H-4⁰), 3.89

(br t, 1H, H-6a), 3.84 (br t, 1H, J_6b,52.2 Hz, H-6b),

3.76 (m, 1H, H-5), 3.71 (br t, 1H, J_4,38.6 Hz, H-4),

3.66 (br t, 1H, J_3,210.1 Hz, H-3), 3.41 (s, 3H, OCH₃),

3.26 (br t, 1H, H-2); MALDI-TOFMS: Calcd for

C₁₃H₂₀NO₁₇Na₃S₂[M+NH₄]^ꢀ: m/z 613.02. Found: m/z

613.33.

0

(d, 1H, J_{1 ,2}5.5 Hz, H-1⁰), 5.20 (br t, 1H, H-3⁰), 5.14

(br t, 1H, 4⁰-H), 4.92 (d, 1H, J_a,b10.8 Hz, CHaPh),

0

3.20. Methyl (methyl 2-O-acetyl-3,4-di-O-pivaloyl-a-^L-

idopyranosyluronate)-(1!4)-6-O-acetyl-2-azido-3-O-

benzyl-2-deoxy-a-^D-glucopyranoside (31)

0

4.79 (m, 2H, CHbPh, H-1), 4.76 (d, 1H, J_{5 ,4}5.3 Hz,

H-5⁰), 3.98 (br t, 1H, J_4,59.3 Hz, 4-H), 3.85 (m, 3H, H-

6a, H-3, H-6b), 3.69 (br d, 1H, H-5), 3.59 (br t, 1H, H-

2⁰), 3.43 (m, 7H, 2OCH₃, H-2), 1.22 (s, 9H, 3CH₃, Piv),

1.14 (s, 9H, 3CH₃, Piv); ¹³C NMR (50.32 MHz, CDCl₃):

d 177.95, 176.56 (2 · C@O, 2 · Piv), 168.50 (C@O, es-

ter), 137.78, 128.23, 127.46 (Ph), 101.00 (C-1⁰), 98.53,

78.62, 77.00 (C-1, C-3, C-4, overlap), 74.55 (CH₂Ph),

71.18 (C-2⁰), 71.12 (C-5), 70.58, 69.72, 68.12 (C-3⁰, C-

5⁰, C-4⁰), 63.51, 61.12 (C-2, C-6), 55.35 (OCH₃, ester),

52.09 (OCH₃), 38.90, 38.78 (2 · O@C–C, 2 · Piv),

27.11, 26.99 (2 · 3CH₃, 2 · Piv); FABMS: Calcd for

C₃₁H₄₅N₃O₁₃[M+Na]⁺: m/z 690.29. Found: m/z 690.23.

Glucosamine precursor 6 (145 mg, 0.41 mmol) and ^L-

iduronic acid derivative 17 (348 mg, 1.5 equiv) were

coevaporated with toluene and then dissolved in anhyd

CH₂Cl₂(15 mL), and freshly activated powdered

˚

molecular sieves (240 mg, 4 A) were added. The mixture

was stirred for half an hour at ꢀ20 ꢀC, then TESOTf

(46.7 lL, 0.5 equiv) was added dropwise, then

stirred at ꢀ10 to ꢀ15 ꢀC. After 3 h N,N-DIPEA

(72 lL, 1.0 equiv) was added, the mixture was ﬁltered

through a pad of Celite, and the solvent was removed

under reduced pressure. Puriﬁcation on a MPLC

column eluting with 5:1 toluene–EtOAc aﬀorded the

disaccharide 31 (199 mg, 64%) as a colorless syrup:

[a]_D+11.5 (c 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃):

3.22. Methyl (methyl 2-O-trimethylaminesulfonato-3,4-

di-O-pivaloyl-a-^L-idopyranosyluronate)-(1!4)-2-azido-3-

O-benzyl-2-deoxy-6-O-trimethylaminesulfonato-a-^D-

glucopyranoside (33)

d 7.36 (m, 5H, Ph), 5.37 (d, 1H, J_{1 ,2}4.6 Hz, H-1⁰), 5.18

0

(br t, 1H, H-3⁰), 5.08 (dd, 1H, J_{4 ,5}4.6 Hz, J_{4 ,3}5.9 Hz,

0

H-4⁰), 4.86 (m, 3H, CHaPh, H-2⁰, CHbPh), 4.80 (d, 1H,

A solution of the deacylated disaccharide 32 (60 mg,

90 lmol) and SO₃ÆNMe₃complex (250 mg, 20.0 equiv)

in dry DMF (8.7 mL) was stirred overnight at 60 ꢀC.

After the reaction was ﬁnished, the solution was concen-

trated to ꢁ2 mL. Chromatography on Sephadex LH-20

using MeOH as eluant gave product 33 (70 mg, 83%):

[a]_Dꢀ0.03 (c 0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃):

d 8.83 (br s, 2 · NH, 2 · HN⁺Me₃), 7.23 (m, 5H, Ph),

5.35 (s, 1H, H-1⁰), 5.20 (s, 1H, H-3⁰), 4.93 (s, 1H, H-

4⁰), 4.90 (s, 1H, H-5⁰), 4.89 (d, 1H, J_a,b11.5 Hz,

CHaPh), 4.79 (d, 1H, J_1,23.5 Hz, H-1), 4.76 (d, 1H,

J_b,a11.5 Hz, CHbPh), 4.34 (s, 1H, H-2⁰), 4.23 (s, 2H,

H-6a, H-6b), 4.05 (br t, 1H, H-4), 3.82 (br t, 1H, H-3),

3.73 (br d, 1H, H-5), 3.46 (dd, 1H, J_2,13.5 Hz, J_2,3

10.1 Hz, H-2), 3.38 (s, 3H, OCH₃), 3.16 (s, OCH₃, ester),

2.96 (s, 18H, 6CH₃, 2 · NMe₃), 1.25 (s, 9H, 3CH₃, Piv),

1.07 (s, 9H, 3CH₃, Piv); ¹³C NMR (50.32 MHz, CDCl₃):

d 176.60, 176.11 (2 · C@O, 2 · Piv), 168.40 (C@O, es-

ter), 137.52, 128.16, 127.22, 126.43, 126.33 (Ph), 98.31

(C-1), 97.85 (C-1⁰), 77.88, 73.58 (C-3, C-4), 73.02

(CH₂Ph), 69.52 (C-5), 69.38, 66.66 (C-2⁰, C-4⁰), 66.21

(overlap, C-3⁰, C-5⁰), 65.25, 63.25 (C-6, C-2), 55.44

(OCH₃, ester), 51.68 (OCH₃), 45.79 (overlap, 6CH₃,

2 · NMe₃), 38.76, 38.61 (2 · O@C–C, 2 · Piv), 27.18,

26.88 (2 · 3CH₃, 2 · Piv); FABMS: Calcd for

J_{5 ,4}4.6 Hz, H-5⁰), 4.77 (d, 1H, J_1,23.7 Hz, H-1), 4.50

(dd, 1H, J_6a,52.0 Hz, J_6a,6b12.3 Hz, H-6a), 4.18 (dd,

1H, J_6a,6b3.9 Hz, J_6b,6a12.3 Hz, H-6b), 3.89 (m, 2H,

H-3, H-4), 3.79 (dd, 1H, H-5), 3.44 (s, 3H, OCH₃, ester),

3.44 (m, 1H, H-2), 3.42 (s, 3H, OCH₃), 2.12 (s, 3H, CH₃,

Ac), 2.05 (s, 3H, CH₃, Ac), 1.21 (s, 9H, 3CH₃, Piv), 1.14

(s, 9H, 3CH₃, Piv); ¹³C NMR (100.55 MHz, CDCl₃):

d 176.56, 176.25 (2 · C@O, 2 · Piv), 170.30 (C@O,

ester), 169.24, 168.21 (C@O, Ac), 137.50, 128.23,

127.50, 127.39 (Ph), 98.36 (C-1), 97.62 (C-1⁰), 77.92,

76.60 (C-4, C-3), 74.20 (CH₂Ph), 68.84 (C-2⁰), 68.78

(C-5), 68.67, 67.72, 67.50 (C-5⁰, C-3⁰, C-4⁰), 63.16,

61.71 (C-2, C-6), 55.44 (OCH₃, ester), 51.99 (OCH₃),

38.73 (overlap, 2 · O@C–C, 2 · Piv), 26.98, 26.90

(2 · 3CH₃, 2 · Piv), 20.83, 20.70 (2 · CH₃, 2 · Ac);

FABMS: Calcd for C₃₅H₄₉N₃O₁₅[MꢀH]⁺: m/z 750.3.

Found: m/z 750.4.

0

3.21. Methyl (methyl 3,4-di-O-pivaloyl-a-^L-idopyranosyl-

uronate)-(1!4)-2-azido-3-O-benzyl-2-deoxy-a-^D-gluco-

pyranoside (32)

Disaccharide 31 (87 mg, 0.12 mmol) was dissolved in an-

hyd MeOH (30 mL), and AcCl (2.3 mL) was added at

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

369

C₃₇H₆₃N₅O₁₉S₂[M+NMe₃+H]⁺: m/z 1005.4. Found: m/

z 1005.5.

The reaction was stopped by adding N,N-DIPEA

(60 lL, 1.0 equiv). The mixture was ﬁltered through a

pad of Celite, and the solvent was removed under reduced

pressure. Puriﬁcation on a MPLC column eluting with

5:2 toluene–EtOAc aﬀorded the disaccharide 34 195 mg

3.23. Methyl (2-O-sulfo-a-L-idopyranosyluronic acid)-

(1!4)-2-deoxy-2-sulfamido-6-O-sulfo-a-^D-glucopyran-

oside tetrasodium salt (2)

1

(75%) as a colorless syrup: [a]_D+8.2 (c 1.0, CHCl₃); H

NMR (400 MHz, CDCl₃): d 7.34 (m, 5H, Ph), 5.35 (d,

3.23.1. Saponiﬁcation. The O-sulfated product 33

(57 mg, 60 lmol) was dissolved in THF (2.7 mL) and

cooled to ꢀ5 ꢀC. H₂O₂(1.3 mL, 30%) and aq LiOH

(0.59 mL, 0.7 M) were added dropwise. The reaction

mixture was stirred for 1 h at ꢀ5 ꢀC, then for 2 h at

0 ꢀC. After 20 h stirring at room temperature, MeOH

(2.36 mL) then aq NaOH (0.65 mL, 4 N) were added

dropwise at 0 ꢀC, and the solution was left stirring for

1 h at 0 ꢀC. After that time, the stirring was prolonged

overnight at room temperature. After neutralization

with 1 N HCl, the water phase was concentrated in

vacuo under 30 ꢀC.

1H, J_{1 ,2}4.7 Hz, H-1⁰), 5.19 (br t, 1H, H-3⁰), 5.06 (br t,

1H, H-4⁰), 4.86 (m, 3H, CHaPh, H-2⁰, CHbPh), 4.76

(br d, 2H, H-5⁰, H-1), 4.48 (br d, 1H, J_6a,6b12.4 Hz, H-

6a), 4.18 (dd, 1H, J_6b,53.8 Hz, J_6b,6a12.4 Hz, H-6b),

3.91 (br t, 1H, J_4,39.3 Hz, H-4), 3.87 (br t, 1H, J_3,4

9.3 Hz, H-3), 3.78 (br t, 1H, H-5), 3.51 (s, 3H, OCH₃, es-

ter), 3.41 (m, 4H, J_2,13.7 Hz, H-2, OCH₃), 2.68 (m, 2H,

CH₂, Lev), 2.50 (m, 2H, CH₂, Lev), 2.16 (s, 3H, CH₃,

Lev), 2.11 (s, 3H, CH₃, Ac), 2.08 (s, 3H, CH₃, Ac), 1.21

(s, 9H, 3CH₃, Piv); ¹³C NMR (50.32 MHz, CDCl₃): d

205.68 (C@O, Lev), 176.57 (C@O, Piv), 171.19 (C@O,

Lev), 170.48 (C@O, ester), 169.42, 168.30 (2 · C@O,

2 · Ac), 137.64, 128.34, 127.62, 127.53 (Ph), 98.43 (C-

1), 97.75 (C-1⁰), 78.05, 76.63 (C-3, C-4), 74.53 (CH₂Ph),

68.95 (C-2⁰), 68.85 (C-5), 68.67, 68.18, 67.69 (C-5⁰, C-4⁰,

C-3⁰), 63.31, 61.73 (C-2, C-6), 55.46 (OCH₃), 52.23

(OCH₃, ester), 38.74 (O@C–C, Piv), 37.38 (CH₂, Lev),

29.71 (CH₃, Lev), 27.53 (CH₂, Lev), 26.92 (3CH₃, Piv),

20.79, 20.67 (2 · CH₃, 2 · Ac); HRMS: Calcd for

C₃₅H₄₇N₃O₁₆[M+K]⁺: 804.2592. Found: 804.2689.

0

3.23.2. Hydrogenolysis. A solution of the residue ob-

tained above in 3:2 t-BuOH–H₂O (5 mL) was treated

with H₂(50 bar) in the presence of Pd/C catalyst

(80 mg, 10%) for overnight. The mixture was ﬁltered

and concentrated. The residue was puriﬁed on Sephadex

LH-20 using 1:1 MeOH–H₂O as eluant to give product

(20 mg).

3.23.3. N-Sulfation. The product obtained above

(20 mg, 33.6 lmol) was dissolved in water (5 mL).

SO₃Æpyridine (24 mg, 151 lmol) was added in ﬁve por-

tions at t = 0; 0.5; 1.0; 1.5; 2.0; and 2.5 h, the pH being

maintained at pH 9.8 by addition of 4 N aq NaOH.

After 3 h, the reaction mixture was neutralized with

1 N HCl and concentrated. The residue was layered

onto a Bio-Gel P-2 column eluted with H₂O, and prod-

3.25. Methyl (methyl 2-O-acetyl-3-O-pivaloyl-a-^L-ido-

pyranosyluronate)-(1!4)-6-O-acetyl-2-azido-3-O-benzyl-

2-deoxy-a-^D-glucopyranoside (35)

A solution of N₂H₄ÆHOAc (230 mg, 2.5 mmol) was

added to a mixture of disaccharide 34 (190 mg,

0.25 mmol) in 2:1 EtOH–toluene (48 mL), and the

resulting reaction mixture was stirred at room tempera-

ture for 1 h. The solvents were then evaporated in

vacuo, and the residue was diluted with CH₂Cl₂. The

mixture was then ﬁltered, and the solvent was removed

under reduced pressure. Puriﬁcation on a MPLC col-

umn eluting with 2:1 hexanes–EtOAc aﬀorded com-

pound 35 (113 mg, 68%) as a colorless oil: [a]_D+38.6

1

uct 2 was obtained (22 mg, 94%): H NMR (400 MHz,

D₂O): d 5.16 (br s, 1H, H-1⁰), 5.04 (d, 1H, J_1,23.5 Hz,

H-1), 4.74 (d, 1H, J_{5 ,4}2.7 Hz, H-5⁰), 4.34 (br t, 2H,

H-6a, H-6b), 4.26 (br t, 1H, H-2⁰), 4.05 (br t, 1H, H-

3⁰), 4.00 (m, 2H, H-4⁰, H-5), 3.75 (br t, 1H, H-4), 3.69

(br t, 1H, H-3), 3.44 (s, 3H, OCH₃), 3.30 (dd, 1H, J_2,1

3.5 Hz, J_2,39.9 Hz, H-2); MALDI-TOFMS: Calcd for

C₁₃H₁₉NNa₄O₂₀S₃[M+K]^ꢀ: m/z 735.8891. Found: m/z

735.0661.

0

1

(c 1.0, CHCl₃); H NMR (500 MHz, CDCl₃): d 7.35

(m, 5H, Ph), 5.12 (d, 1H, J_{1 ,2}3.3 Hz, H-1⁰), 4.99 (br

t, 1H, H-3⁰), 4.86 (d, 1H, J_a,b11.4 Hz, CHaPh), 4.84

(d, 1H, J_b,a11.4 Hz, CHbPh), 4.83 (br t, 1H, H-2⁰),

0

3.24. Methyl (methyl 2-O-acetyl-4-O-levulinoyl-3-O-

pivaloyl-a-^L-idopyranosyluronate)-(1!4)-6-O-acetyl-2-

azido-3-O-benzyl-2-deoxy-a-^D-glucopyranoside (34)

0

4.79 (d,1H, J_1,23.7 Hz, H-1), 4.74 (d, 1H, J_{5 ,4}3.1 Hz,

H-5⁰), 4.48 (dd, 1H, J_6a,51.9 Hz, J_6a,6b12.5 Hz, H-6a),

4.20 (dd, 1H, J_6b,53.9 Hz, J_6b,6a12.5 Hz, H-6b), 3.92

(m, 1H, H-4⁰), 3.87 (br t, 1H, H-4), 3.86 (br t, 1H, H-

3), 3.80 (m, 1H, H-5), 3.49 (s, 3H, OCH₃, ester), 3.44

(dd, 1H, J_2,13.7 Hz, J_2,39.7 Hz, H-2), 3.43 (s, 3H,

Glucosamine 6 (121 mg, 0.34 mmol) and ^L-iduronic acid

derivative 23 (290 mg, 1.5 equiv) were coevaporated with

toluene and then dissolved in anhyd CH₂Cl₂(13 mL),

and freshly activated powdered molecular sieves

OCH₃), 2.89 (d, 1H, J_{OH-4 ,H-4}11.0 Hz, OH-4⁰), 2.10

(s, 6H, 2CH₃, 2 · Ac), 1.25 (s, 9H, 3CH₃, Piv); ¹³C

NMR (50.32 MHz, CDCl₃): d 177.40 (C@O, Piv),

170.33 (C@O, ester), 168.86 (overlap, 2 · C@O,

0

˚

(200 mg, 4 A) were added. The mixture was stirred for

0.5 h at ꢀ20 ꢀC, then TESOTf (39 lL, 0.5 equiv) was

added dropwise and stirred at ꢀ15 to ꢀ10 ꢀC for 3 h.

370

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

2 · Ac), 137.72, 128.25, 127.46, 127.05 (Ph), 98.49 (C-1),

98.07 (C-1⁰), 77.98, 76.79 (C-3, C-4), 73.93 (CH₂Ph),

70.04, 69.94 (C-3⁰, C-5⁰), 68.88 (C-5), 68.34, 68.00 (C-

2⁰, C-4⁰), 63.40, 61.87 (C-2, C-6), 55.55 (OCH₃), 52.28

(OCH₃, ester), 38.89 (O@C–C, Piv), 27.06 (3CH₃, Piv),

20.90, 20.82 (2 · CH₃, 2 · Ac); FABMS: Calcd for

C₃₀H₄₁N₃O₁₄[M+Na]⁺: m/z 690.2. Found: m/z 690.2.

3.27. Methyl (2-azido-3-O-benzyl-2-deoxy-a-^D-glucopyr-

anosyl)-(1!4)-(methyl 3-O-pivaloyl-a-^L-idopyranosyluron-

ate)-(1!4)-2-azido-3-O-benzyl-2-deoxy-a-^D-glucopyr-

anoside (37)

Trisaccharide 36 (80 mg, 75.7 lmol) was dissolved in an-

hyd MeOH (19.6 mL), and AcCl (1.51 mL) was added at

0 ꢀC. The reaction mixture was gradually allowed to

warm to room temperature and left overnight. The reac-

tion was followed by TLC in 1:1 hexanes–EtOAc until it

was completed. The solution was diluted with cold

CH₂Cl₂(420 mL), washed with satd aq NaHCO₃

(39 mL), and then washed with H₂O (3 · 39 mL). The

organic phase was dried over anhydrous Na₂SO₄. The

solvent was then removed under reduced pressure. Puri-

ﬁcation on a MPLC eluting with 5:4:1 hexanes–EtOAc–

CH₂Cl₂aﬀorded the deacetylated trisaccharide 37 60 mg

3.26. Methyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-

D-glucopyranosyl)-(1!4)-(methyl 2-O-acetyl-3-O-pival-

oyl-a-^L-idopyranosyluronate)-(1!4)-6-O-acetyl-2-azido-

3-O-benzyl-2-deoxy-a-^D-glucopyranoside (36)

Glucosamine derivative 27 (153 mg, 0.27 mmol) and

disaccharide 35 (120 mg, 0.18 mmol) were coevaporated

with toluene and then dissolved in anhyd CH₂Cl₂

(11 mL), and freshly activated powdered molecular

1

˚

sieves (150 mg, 3 A) were added. The mixture was stir-

(84%) as a colorless oil: [a]_D+34.9 (c 1.0, CHCl₃); H

red for 0.5 h at ꢀ20 ꢀC, then TESOTf (20.4 lL,

0.5 equiv) was added dropwise. The reaction was stirred

for 1 h and then stopped by adding N,N-DIPEA

(30.7 lL, 1.0 equiv). The mixture was ﬁltered through

a pad of Celite, and the solvent was removed under

reduced pressure. Puriﬁcation on a MPLC column elut-

ing with 7:1:1 toluene–EtOAc–CH₂Cl₂aﬀorded the tri-

saccharide 36 (80 mg, 40%) as a colorless syrup: [a]_D

NMR (500 MHz, CDCl₃): d 7.33 (m, 10H, 2 · Ph),

5.31 (br s, 1H, H-1^I), 5.18 (d, 1H, J_{1G ,2G}3.7 Hz, H-

0

1^G), 5.16 (m, 1H, H-3^I), 4.98 (br t, 1H, H-4^G), 4.84

(m, 4H, CHaPh^G, CHbPh^G, H-5^I, H-1^G), 4.75 (d, 1H,

0

J_{aG ,bG}10.8 Hz, CHaPh^G), 4.68 (d, 1H, J_{bG ,aG}

0

10.8 Hz, CHbPh^G), 4.09 (br s, 1H, H-4^I), 4.01 (br t,

0

1H, J_4G,5G9.5 Hz, H-4^G), 3.87 (m, 3H, H-3^G, H-3^G,

0

H-6a^G), 3.81 (br d, 1H, H-6b^G), 3.68 (d, 1H, J_5G,4G

0

1

9.5 Hz, H-5^G), 3.63 (dd, 1H, J_{2G ,1G}3.7 Hz, H-2^G),

0

+47.2 (c 1.0, CHCl₃); H NMR (500 MHz, CDCl₃): d

7.32 (m, 10H, 2 · Ph), 5.32 (m, 2H, H-1^I, H-3^I), 5.17

3.61 (br d, 1H, H-2^I), 3.57 (d, 1H, J_6aG,6bG10.8 Hz,

0

(br t, 1H, H-4^G), 5.08 (d, 1H, J_{1G ,2G}3.7 Hz, H-1^G),

4.89 (d, 1H, J_aG,bG11.3 Hz, CHaPh^G), 4.83 (d, 1H,

H-6a^G), 3.46 (m, 2H, H-2^G, H-5^G), 3.43 (m, 4H,

OCH₃, H-6b^G), 3.36 (s, 3H, OCH₃, ester), 1.28 (s, 9H,

3CH₃, Piv), 1.17 (s, 9H, 3CH₃, Piv); ¹³C NMR

(50.32 MHz, CDCl₃): d 178.37, 177.19 (2 · C@O,

2 · Piv), 169.06 (C@O, ester), 137.96, 137.13, 128.44,

0

J_bG,aG11.3 Hz, CHbPh^G), 4.82 (br t, 1H, H-2^I), 4.79

0

(d, 1H, H-1^G), 4.72 (d, 1H, J_{aG ,bG}10.4 Hz, CHaPh^G),

0

4.70 (br t, 1H, H-5^I), 4.60 (d, 1H, J_{bG ,aG}10.4 Hz,

0

CHbPh^G), 4.50 (dd, 1H, J_6aG,5G1.7 Hz, J_6aG,6bG

12.5 Hz, H-6a^G), 4.22 (dd, 1H, J_6bG,5G4.0 Hz, J_6bG,6aG

128.31, 127.93, 127.47, 127.39, 126.75 (2 · Ph), 101.49

0

(C-1^I), 98.67 (C-1^G), 95.98 (C-1^G), 78.16, 78.09 (C-3^G₀,

0

12.5 Hz, H-6b^G), 4.09 (dd, 1H, J_{6aG ,5G}4.2 Hz,

C-3^G), 75.94 (C-4^G), 75.41, 73.28 (CH₂Ph^G, CH₂Ph^G),

0

J_{6bG ,6aG}12.6 Hz, H-6a^G), 4.04 (m, 2H, J_{6bG ,6aG}

71.64 (C-4^I), 71.28 (C-5^G), 71.11 (C-5^G), 70.25 (C-4^G),

0

12.6 Hz, H-6b^G, H-4^I), 3.84 (m, 5H, H-5^G, H-3^G, H-

68.24, 67.24 (C-5^I₀, C-2^I, C-3^I), 63.42 (C-2^G), 63.32 (C-

2^G), 61.16 (C-6^G), 60.61 (C-6^G), 55.44 (OCH₃, ester),

52.08 (OCH₃), 38.95, 38.88 (2 · O@C–C, 2 · Piv),

27.10, 27.08 (2 · 3CH₃, 2 · Piv); FABMS: Calcd for

C₄₄H₆₀N₆O₁₇[M+Na]⁺: m/z 967.4. Found: m/z 967.3.

Anal. Calcd for C₄₄H₆₀N₆O₁₇: C, 55.92; H, 6.40; N,

8.89. Found: C, 56.08; H, 6.22; N, 8.44.

3^G, H-4^G, H-5^G), 3.51 (s, 3H, OCH₃, ester), 3.43 (m,

0

5H, J_{2G ,1G}3.5 Hz, H-2^G, H-2^G, OCH₃), 2.12 (s, 3H,

CH₃, Ac), 2.11 (s, 3H, CH₃, Ac), 2.05 (s, 3H, CH₃,

Ac), 1.25 (s, 9H, 3CH₃, Piv), 1.17 (s, 9H, 3CH₃, Piv);

¹³C NMR (50.32 MHz, CDCl₃): d 176.48 (overlap,

2 · C@O, 2 · Piv), 170.47, 170.37, 169.53 (3 · C@O,

3 · Ac), 168.54 (C@O, ester), 137.88, 137.02, 128.36,

0

128.24, 127.84, 127.48, 127.41, 127.09 (2 · Ph), 98.49

3.28. Methyl (2-azido-3-O-benzyl-2-deoxy-6-O-trimeth-

ylaminesulfonato-a-^D-glucopyranosyl)-(1!4)-(methyl 3-

O-pivaloyl-2-O-trimethylaminesulfonato-a-^L-idopyr-

anosyluronate)-(1!4)-2-azido-3-O-benzyl-2-deoxy-6-O-

trimethylaminesulfonato-a-^D-glucopyranose (38)

0

(C-1^G), 98.04 (overlap, C-1^I, C-1^G), 78.08, 77.31 (C-

0

3^G, C-4^G₀), 77.20 (C-3^G), 74.39 (CH₂Ph^G), 73.95

0

(CH₂Ph^G), 72.60, 69.68 (C-4^I, C-5^I), 69.14 (C-4^G,

0

C-5^G), 68.89 (C-2^I, C-5^G), 67.88 (C-3^I), 63.31 (C-2^G),

0

62.52 (C-2^G), 61.93 (C-6^G), 61.48 (C-6^G),

55.53 (OCH₃), 52.12 (OCH₃, ester), 38.95, 38.89

(2 · O@C–C, 2 · Piv), 27.12 (overlap, 6CH₃, 2 · Piv),

20.91, 20.77, 20.69 (3 · CH₃, 3 · Ac); FABMS: Calcd

for C₅₀H₆₆N₆O₂₀[M+Na]⁺: m/z 1093.4. Found: m/z

1093.3.

A solution of the deacetylated trisaccharide 37 (27 mg,

30 lmol) and SO₃ÆNMe₃complex (119 mg, 30.0 equiv)

in dry DMF (4 mL) was stirred overnight at 60 ꢀC.

After the reaction was ﬁnished, the solution was con-

centrated to ꢁ2 mL. Chromatography on Sephadex

W. Ke et al. / Carbohydrate Research 340 (2005) 355–372

371

LH-20 using MeOH as eluant gave product 38 (34 mg,

1

on Sephadex LH-20 again using 1:1 MeOH–H₂O to give

product (22 mg).

86%): [a]_D+36.4 (c 1.0, CHCl₃); H NMR (400 MHz,

CDCl₃): d 8.83 (br s, 2 · NH, 2 · HN⁺Me₃), 7.39–7.23

(m, 10H, 2 · Ph), 5.58 (br t, 1H, J_3I,4I4.5 Hz, H-3^I),

3.29.3. N-Sulfation. The product obtained above

(22 mg, 25.6 lmol) was dissolved in H₂O (4 mL).

SO₃Æpyridine (36 mg, 226 lmol) was added in ﬁve por-

tions at t = 0; 0.5; 1.0; 1.5; 2.0; and 2.5 h, the pH being

maintained at pH 9.5 by addition of 1 N aq NaOH.

After 3 h, the reaction mixture was neutralized with

1 N HCl and concentrated to ꢁ1 mL. Chromatography

on a Sephadex G-10 column using 1:9 EtOH–H₂O as

eluant aﬀorded product 3 (22 mg, 81%); ¹H NMR

5.43 (d, 1H, J_1I,2I2.5 Hz, H-1^I), 5.18₀(d, 1H, J_{1G ,2G}

0

3.3 Hz, H-1^G), 5.05 (br t, 1H, H-4^G), 4.91 (m, 1H,

CHaPh^G, H-5^I), 4.82 (m, CHbPh^G, H-1^G), 4.79 (d,

0

1H, J_{aG ,bG}11.1 Hz, CHbPh^G), 4.64 (d, 1H, J_{bG ,aG}

0

11.1 Hz, CHbPh^G), 4.42 (m, 2H, H-6a^G, H-2^I), 4.29

(d, 1H, J_6bG,6aG10.9 Hz, H-6b^G), 4.10 (br t, 1H, H-

0

4^I), 4.02 (m, 3H, H-3^G, H-6a^G, H-6b^G), 3.91 (m, 4H,

0

H-4^G, H-5^G, H-5^G, H-3^G), 3.55 (dd, 1H, J_{2G ,1G}

0

3.5 Hz, J_{2G ,3G}10.3 Hz, H-2^G), 3.49 (s, 3H, OCH₃),

3.47 (dd, 1H, J_2G,1G3.5 Hz, J_2G,3G10.0 Hz, H-2^G),

3.44 (s, 3H, OCH₃, ester), 2.93 (s, 27H, 9CH₃,

3 · NMe₃), 1.30 (s, 9H, 3CH₃, Piv), 1.18 (s, 9H,

3CH₃, Piv); ¹³C NMR (50.32 MHz, CDCl₃): d 178.38,

178.21 (2 · C@O, 2 · Piv), 170.37 (C@O, ester),

0

(400 MHz, D₂O): d 5.46 (d, 1H, J_{1G ,2G}3.7 Hz, H-

0

1^G), 5.27 (s, 1H, H-1^I), 5.03 (d, 1H, J_1G,2G3.5 Hz,

H-1^G), 4.90 (d, 1H, J_5I,4I1.8 Hz, H-5^I), 4.38 (m, 2H,

0

H-3^I, H-6a^G), 4.35 (br s, 2H, H-2^I, H-6b^G), 4.31 (dd,

1H, J_6aG,5G4.9 Hz, J_6aG,6bG11.5 Hz, H-6a^G), 4.23 (m,

2H, H-6b^G, H-4^I), 4.07 (m, 1H, H-5^G), 3.99 (m, 2H,

0

H-5^G, H-3^G), 3.90 (br t, 1H, J_{3G ,4G}9.8 Hz, H-3^G),

139.71, 139.31, 129.43, 129.32, 128.74, 128.67, 128.51,

0

128.18 (2 · Ph), 100.₀08 (C-1^G), 99.90 (C-1^G), 98.09

3.81 (br t, 1H, J_4G,3G9.4 Hz, H-4^G), 3.59 (br t, 1H,

0

(C-1^I), 79.04 (C-3^G, C-3^G), 75.81 (C-4^G), 75.36

J_{4G ,3G}9.8 Hz, H-4^G), 3.48 (s, 3H, OCH₃), 3.42 (dd,

0

(CH₂Ph^G), 74.01 (C-2^I), 73.65 (CH₂Ph^G), 73.33 (C-

1H, J_2G,3G1.2 Hz, J_2G,1G3.5 Hz, H-2^G), 3.40 (dd, 1H,

0

4^I), 71.24 (C-4^G), 70.96₀(C-5^G, C-5^G), 70.08, 68.45

J_{2G ,3G}1.1 Hz, J_{2G ,1G}3.7 Hz, H-2^G); ¹³C NMR

0

(C-5^I, C-3^I), 67.28 (C-6^G, C-6^G), 64.44 (C-2^G), 63.93

(C-2^G), 55.92 (OCH₃, ester), 53.13 (OCH₃), 50.42,

50.00, 49.58, 49.15, 48.73, 48.30, 47.88, 45.91

(3 · NMe₃), 40.13, 40.00 (2 · O@C–C, 2 · Piv), 27.85,

27.74 (2 · 3CH₃, 2 · Piv).

(50.32 MHz, D₂O–acetone): d 175.84 (C@O), 99.55 (C-

0

1^I), 96.55 (C-1^G), 91.91 (C-1^G), 77.35 (C-4^G), 73.63

0

(C-2^I), 71.08 (C-4^I, C-3^G), 69.90 (C-3^G), 69.90 (C-5^G),

0

69.61, 69.40 (C-5^G, C-4^G), 68.01 (C-5^I), 66.81 (C-6^G),

65.19 (C-6^G), 63.55 (C-3^I), 55.97 (OCH₃), 54.78 (C-

0

2^G, C-2^G); MALDI-TOFMS Calcd for C₁₉H₂₈N₂O₃₀-

Na₆S₅[Mꢀ2 · SO₃Na]^ꢀ: m/z 855.98. Found: m/z

855.91.

3.29. Methyl (2-deoxy-2-sulfamido-6-O-sulfo-a-^D-gluco-

pyranosyl)-(1!4)-(2-O-sulfo-a-^L-idopyranosyluronic

acid)-(1!4)-2-deoxy-2-sulfamido-6-O-sulfo-a-^D-gluco-

pyranoside hexasodium salt (3)

Acknowledgements

3.29.1. Saponiﬁcation. The O-sulfated product 38

(75 mg, 55 lmol) was dissolved in THF (3.0 mL) and

cooled to ꢀ5 ꢀC. H₂O₂(1.43 mL, 30%) and aq LiOH

(0.66 mL, 0.7 M) were added dropwise. The reaction mix-

ture was stirred for 1 h at ꢀ5 ꢀC, then 2 h at 0 ꢀC. After

20 h stirring at room temperature, MeOH (2.65 mL),

then aq NaOH (0.77 mL, 4 N) were added dropwise at

0 ꢀC, and the solution was left stirring for 1 h at 0 ꢀC.

After that time, the stirring was prolonged overnight

at room temperature. After neutralization with 1 N

HCl, the water phase was concentrated in vacuo under

30 ꢀC.

This research was supported by NSC Grant 89-2311-B-

007-045 and an NSC–NRC Joint Research Project

Grant (N-001). Mr. Ken Chan and Dr. Jianjun Li are

thanked for the FABMSs and Lisa D. Morrison is

thanked for obtaining the HRMSs and MALDI-TOF-

MSs. Ms. Suzon Larocque is thanked for her assistance

in performing the NMR studies. This is NRC paper

#42497.

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