Modular synthesis of heparin oligosaccharides

FULL PAPER

Modular Synthesis of Heparin Oligosaccharides

Herna¬n A. Orgueira,^{[a, b]}Alessandra Bartolozzi,^{[a, c]}Peter Schell,^{[a, d]}

Remy E. J. N. Litjens,^{[a, e]}Emma R. Palmacci,^[a]and Peter H. Seeberger*^[a]

Abstract: A general, modular strategy

for the first completely stereoselective

synthesis of defined heparin oligosac-

charides is described. Six monosacchar-

ide building blocks (four differentially

protected glucosamines, one glucuronic

and one iduronic acid) were utilized to

prepare di- and trisaccharide modules in

a fully selective fashion. Installation of

the a-glucosamine linkage was control-

led by placing a conformational con-

straint on the uronic acid glycosyl ac-

ceptors thereby establishing a new con-

cept for stereochemical control.

Combination of disaccharide modules

to form trans-uronic acid linkages was

completely selective by virtue of C2

participating groups. Coupling reactions

between disaccharide modules exhibited

sequence dependence. While the union

of many glucosamine uronic acid dis-

accharide modules did not meet any

problems, certain sequences proved not

accessible. Elaboration of glucosamine

uronic acid disaccharide building blocks

to trisaccharide modules by addition of

either one additional glucosamine or

uronic acid allowed for stereoselective

access to oligosaccharides as demon-

strated on the example of a hexasac-

charide resembling the ATIII-binding

sequence. Final deprotection and sulfa-

tion yielded the fully synthetic heparin

oligosaccharides.

Keywords: carbohydrates ¥ glycosy-

lation ¥ heparin ¥ oligosaccharides

Introduction

OSO₃

O

Proteoglycans are complex protein carbohydrate assemblies

that consist of a core protein and one or more covalently

attached glycosaminoglycan chains.^[1]These linear polysac-

charides range in length from ꢀ20 to 200 disaccharide repeat

units, each composed of an amino sugar and an uronic acid

moiety (Scheme 1). Heparin-like glycosaminoglycans

(HLGAGs) are the most acidic naturally occurring biopol-

ymers. These complex polysaccharides, found in the extra

O₃SO

OSO₃

O

O₃SHN

O

HO

O₃SO

OH

CO₂

O₃SHN

O₂C

O

HO

OH

Scheme 1. Schematic view of heparin.

[a] Prof. Dr. P. H. Seeberger, Dr. H. A. Orgueira, Dr. A. Bartolozzi,

Dr. P. Schell, R. E. J. N. Litjens, E. R. Palmacci

Department of Chemistry

cellular matrix, play a key role in regulating the biological

activity of several proteins in the coagulation cascade along

with many other processes of biomedical importance includ-

ing growth factor interactions, virus entry, and angiogenesis.^[2]

Heparin, isolated from the mast cells of pigs, is currently

produced in multi-ton quantities and used in a variety of

medical applications.^[3]Most prominent is the use of heparin

as an anticoagulant in heart disease where it has served as a

therapeutic agent since the late 1930s. The heterogeneity of

heparin results in many severe side effects, making this

inexpensive drug dangerous and necessitates close monitor-

ing.^[4]

The heparin-antithrombin III (AT-III) interaction is re-

sponsible for heparin×s anticoagulant activity and is the only

system where the exact sequence of heparin that associates

with the protein has been identified. Extensive structure

activity studies using synthetic oligosaccharides^[5]as well as

NMR^[6]and X-ray crystallography^[7]have been performed.

Massachusetts Institute of Technology

77 Massachusetts Avenue, Cambridge, MA 02139 (USA)

Fax : (1) 617-253-2979

E-mail: seeberg@mit.edu

[b] Dr. H. A. Orgueira

Current Address: ArQule Inc., 19 Presidental Way

Wobrun, MA, 01801 (USA)

[c] Dr. A. Bartolozzi

Current Address: SurfaceLogix Inc.

50 Soldier Field Place, Brighton, MA, 02135 (USA)

[d] Dr. P. Schell

Current Address: NeoGenesis, 840 Memorial Drive

Cambridge, MA, 02139 (USA)

[e] R. E. J. N. Litjens

Current Address: Leiden University

Gorlaeus Laboratories, Leiden (The Netherlands)

Supporting information for this article is available on the WWW under

http://www.chemeurj.org/ or from the author.

140

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Chem. Eur. J. 2003, 9, No. 1

140 169

Based on these studies, a concerted drug development effort

has been undertaken and resulted in the development of a

synthetic pentasaccharide heparin analogue for use in hu-

mans.^[8]With the exception of the AT-III heparin interac-

tion, the structure and function relationship of HLGAGs is

still very poorly understood due to the complexity and

heterogeneity of these polymers. Defined HLGAG oligosac-

charides constitute valuable molecular tools to gain a detailed

understanding of the sequences of HLGAGs responsible for

binding to a particular protein and modulating its biological

activity.^[1]Determination of the structure activity relation-

ships of HLGAGs creates an opportunity for the discovery of

novel therapeutic interventions for a host of disease states.

Over the past two decades, a variety of synthetic methods

directed at the preparation of HLGAG oligosaccharides have

been disclosed^[9]and heroic total syntheses have resulted in

the assembly of AT-III-binding HLGAG oligosaccharides.^[10]

More recently, longer oligosaccharide HLGAG analogues

exhibiting impressive biological activity have been prepared

using simplified syntheses.^[11]Still, the procurement of specific

HLGAG sequences required a new total synthesis for each

oligosaccharide.

A modular, highly convergent synthetic approach for the

rapid assembly of defined HLGAG oligosaccharide sequen-

ces and eventually even libraries of defined glycosaminogly-

cans and non-natural analogues would be ideal. Such an

approach requires careful consideration of the many synthetic

challenges presented by the great diversity of native struc-

tures. The sulfation patterns mandate the placement of

specific protecting groups in all positions to carry sulfates

and different protection on hydroxyls that remain unaltered.

The amine portion of the glu-

matographic steps. Finally, the preparation of iduronic acid

monosaccharides requires lengthy synthetic procedures. A

successful synthetic strategy will have to find appropriate

solutions to all these challenges, enable high convergency

without compromising generality and ideally provide a

modular approach that can be readily transferred to the

automated solid-phase assembly of defined HLGAG struc-

tures.

Results and Discussion

Synthetic strategy: In contemplating the challenges and

rewards of a synthetic strategy for the rapid assembly of

defined HLGAG oligosaccharide sequences we designed a

modular, highly convergent synthetic plan. A set of four

glucosamine monosaccharide building blocks exhibiting pro-

tecting group patterns representative of the different forms of

sulfation found in native structures will be derived from

glucosamine. Glucuronic and iduronic acid monomers will be

prepared through a common route from diacetone glucose.

Coupling of glucosamine and uronic acid monomers will

furnish a set of disaccharide modules. Introduction of a new

method to control the stereoselectivity of glycosylation

reactions by conformationally constraining the uronic acid

acceptor will render the desired disaccharide modules with

complete selectivity.^[14]These disaccharides can be further

modified by protecting group manipulations to allow for

variable substitution of the amino group. A simple two step

elongation cycle involving the union of disaccharide units will

result in larger heparin oligosaccharides (Scheme 2). The

cosamine component has been

found to be acetylated, sulfated

and found to exist as the free

amine,^[12]thus requiring a pro-

tecting group scheme that al-

lows for the differentiation of

this position.

OSO₃

O

255 million decasaccharides

5.3 million octasaccharides

11592 hexasaccharides

COOH

O

O₃SO

OSO₃

O

AcHN

O

HO

O

HO

OH

AcHN

O

OSO₃

HO

COOH

OPG¹

O

In addition to the installation

of a host of protective groups,

48 modified

disaccharide

building blocks

PGO

PG¹O

COOMe

O

PG⁴N

the creation of the glycosidic

linkages making up the back-

bone of HLGAGs poses several

challenges. The use of uronic

acid derivatives as glycosidating

agents has received little atten-

tion^[13]and is often circumven-

ted^{[9b d, 10b, c]}due to the inherent

low reactivity imposed by the

C5 ester. Stereocontrol during

the fashioning of the a-glucos-

amine linkage is difficult as

anchimeric assistance cannot

be exploited, thus resulting in

the formation of mixtures of

glycosides.^[9d]Separation of

such anomeric mixtures often

necessitates very difficult chro-

OPG¹

O

X

PG²O

OPG³

PGO

OPG¹

O

PG¹O

COOMe

O

N

PG²O

³O

O

X

PG¹O

OPG¹

N₃

OPG³

O

PGO

PG¹O

COOMe

8 disaccharide

building blocks

32 trisaccharide building blocks

N

PG²O

O

³O

X

OPG³

OPG²

O

OPG²

O

COOMe

O

1glucuronic acid

building block

PGO

X

PG¹O

PG²O

Y

PG²O

N₃

OPG¹

O

OPG

OPG¹

O

PGO

PG²O

1 iduronic acid

building block

X

PGO

OPG

COOMe

PG¹O

Y

PG²O

N₃

4 glucosamine building blocks

Scheme 2. Retrosynthetic analysis of

glycosaminoglycans.

a general, modular approach to the preparation of heparin-like

Chem. Eur. J. 2003, 9, No. 1

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141

P. H. Seeberger et al.

FULL PAPER

overall scheme can also accom-

modate trisaccharide modules

prepared by coupling of mono-

saccharides and disaccharides.

Established procedures facili-

tate the final stages of the syn-

thesis as removal of temporary

acetate protecting groups fol-

lowed by sulfation of the ex-

posed hydroxyls and final de-

protection followed by N-sulfa-

tion will yield the target

molecules.^{[9b, c]}

AcO

O

HO

Ph

b

a

c or d

OTBS

O

HO

OTBS

OH

AcO

HO

N₃

NH₂

3

2

1

AcO

O

HO

RO

g, then h,

i or j

R²

TBSO

RO

OTBS

e

N₃

6: R = Bn (88%, 2 steps)

7: R = Ac (89%, 2 steps)

O

10: R = Bn; R²= F (85%, 2 steps)

11: R = Bn; R²= OC(NH)CCl₃

(92%, 2 steps)

12: R = Ac; R²= Br (71%, 2 steps)

13: R = Ac; R²= F (92%, 2 steps)

14: R = Ac; R²= OC(NH)CCl₃

(85%, 2 steps)

Ph

O

OTBS

RO

N₃

4: R= Bn

5: R= Ac

BnO

g, h

f

Preparation of the monosac-

charide building blocks: The

O

TBSO

RO

R²

O

HO

RO

OTBS

N₃

first challenge to be tackled in

trying to reduce such a general

approach to HLGAG synthesis

8: R = Bn (85%)

9: R = Ac (91%)

15: R = Bn; R²= F (89%, 2 steps)

16: R = Bn; R²= OC(NH)CCl₃

(67%, 2 steps)

17: R = Ac; R²= OC(NH)CCl₃

to practice is the procurement

of large amounts of differen-

(87%, 2 steps)

tially protected monosacchar-

ide building blocks. In order to

access large quantities of all

monosaccharides, a convergent

Scheme 3. Synthesis of differentially protected glucosamine monosaccharide building blocks. a) 1. TfN₃, H₂O,

K₂CO₃, CH₂Cl₂, MeOH, CuSO₄; 2. Ac₂O, pyridine, DMAP; 3. NH₃, MeOH, THF; 4. TBSCl, imidazole, CH₂Cl₂,

57% (four steps); b) 1. NaOMe, MeOH; 2. PhCH(OMe)₂, pTsOH, CH₃CN, 86% (two steps); c) BnBr, Ag₂O,

4 ä molecular sieves, CH₂Cl₂, 95%; d) Ac₂O, DMAP, pyridine, 95%; e) 1. TFA (60% aq.), CH₂Cl₂; 2. AcCl,

collidine, À408C; f) TES, TFA, CH₂Cl₂; g) 1. TBSOTf, lutidine, CH₂Cl₂; 2. TBAF, AcOH, THF; h) NCCCl₃,

DBU, CH₂Cl₂; i) DAST, CH₂Cl₂, 08C; j) SOBr₂, imidazole, THF.

synthesis from inexpensive

starting materials is needed that

ides will be required to mark the non-reducing end of the

target oligosaccharide. A 4-O-benzyl ether was readily

introduced by dibenzylation of diol 18 followed by trans-

formation into glycosyl trichloroacetimidate 20 (Scheme 4).

can be performed on large scale with a minimal number of

chromatographic steps. While a host of synthetic methods for

the preparation of glucosamine donors had been explored

previously^[15]we decided to focus on an approach that allows

access to all glucosamine monomers from common advanced

intermediates. To identify the building blocks most suitable

for installation of the desired a-glucosamine linkage, different

anomeric leaving groups replaced an anomeric silyl ether

during the late stages of the synthesis. A set of glucosamine

building blocks was prepared from glucosamine 1 (Scheme 3).

Conversion of the 2-amino group into the corresponding

azide, necessary for a-selective glycosylations, was followed

by acetylation and anomeric silylation to afford crystalline 2

in 57% yield over four steps.^[16]Deacetylation and installation

of the 4,6-benzylidene acetal furnished common precursor 3.

Benzylation of the 3-hydroxyl to fashion 4 or acetylation to

afford 5 was followed by either removal of the 4,6-benzyli-

dene protection and placement of 6-acetates to provide 6 and

7 or selective opening of the benzylidene acetal to furnish 8

and 9. These maneuvers provided access to the skeleton of

glucosamine building blocks containing 4-O-silyl ethers as

temporary protecting groups. The TBS groups will be

removed later during the preparation of oligosaccharides

utilizing disaccharide modules.

AcO

a

b

O

HO

OTBS

2

HO

N₃

18

AcO

O

N

BnO

c

NH

CCl₃

O

BnO

OTBS

N₃

³O

19

20

Scheme 4. Synthesis of a glucosamine monosaccharide building block as

non-reducing end terminus. a) 1. NaOMe, MeOH; 2. AcCl, collidine,

À408C, 93% (two steps); b) BnBr, Ag₂O, 4 ä molecular sieves, CH₂Cl₂,

80%; c) 1. THF, AcOH, TBAF; 2. CCl₃CN, DBU, CH₂Cl₂, 88% (two

steps).

Although we focused our efforts exclusively on cap 20 as a

proof of principle, three other cap building blocks with

different permutations of acetates and benzyl groups can be

synthesized in the same fashion.

After establishing a route for the preparation of glucos-

amine building blocks from a common precursor, a reliable

and efficient path for the synthesis of uronic acid building

blocks was needed. Traditionally, the preparation of iduronic

acid has been particularly difficult since no direct precursor

can be obtained from natural sources.^[17]Efficiency, scalability

and the avoidance of excessive chromatography is mandatory

for the procurement of large amounts of these starting

materials. Under this premise, we developed a route to

With the desired protecting group patterns in place we

turned our attention to the installation of different anomeric

leaving groups. Less reactive glycosyl fluorides 10, 13, and 15,

glycosyl bromide 12 exhibiting intermediate reactivity, and

highly reactive glycosyl trichloroacetimidates 11, 14, 16 and 17

were prepared for couplings with uronic acid acceptors.

In addition to glucosamine units that act as acceptors during

oligosaccharide formation, glucosamine ™cap∫ monosacchar-

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Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

differentially protected glucuronic acid and iduronic acid

monosaccharides via a common intermediate (Scheme 5).

Commercially available diacetone glucose 21 was converted

to crystalline glucuronic acid furanoside 22 through an eight-

step procedure that was easily scalable to 100 g starting

material and did not require purification of any intermedi-

ates.^[18]Access to iduronic acid furanoside 24 was readily

achieved by inversion of the C5 stereocenter of the triflate

derived from 22. Treatment of 22 and 24 with trifluoroacetic

acid resulted in deprotection and formation of the uronic acid

pyranosides 23 and 25.

separations. Couplings involving C2-azido glucosamine tri-

chloroacetimidate 11 and fluoride donors (10, 15^[22]) with

glucuronic acid acceptor 26^[22]yielded anomeric mixtures of 27

and 28 (Scheme 6). These results were in agreement with

previous studies involving other glucuronic acid accep-

tors.^{[10a,c, 23]}Interestingly, when the C5 epimer of glucuronic

acid, iduronic acid 29,^[17b]served as glycosyl acceptor,

exclusively a-glucosamine linkages were obtained.^{[5b, 14, 24]}

Contrary to findings reported by others,^[17b]glycosylation of

29 was not completely regioselective but afforded trisacchar-

ide by concomitant glycosylation of the C2 hydroxyl group.

The marked differences in the stereoselectivity of these

coupling reactions prompted us to investigate how the

observed effect could be harnessed to establish the desired

linkage with complete stereoselectivity. Electronic effects^[25]

and conformational constraint^[26]of the glycosidating agent

have been utilized to control the stereochemical outcome of

glycosylations. Manipulation of the steric and electronic

nature of the glycosyl acceptor to direct glycosylations had

received less attention.

CO₂Me

O

CO₂Me

O

HO

LevO

BnO

O

a

c

BnO

HO

O

22

24

21

COOMe

b

O

HO

BnO

OH

23

OBn

O

The conformation of the different acceptors suggested a

possible explanation for the different behavior of glucuronic

and iduronic acid acceptors. Glucuronic acid preferentially

d then b

MeO₂C

OH

24

OH

4

adopts a C₁conformation with an equatorial C4 hydroxyl

25

1

group. Iduronic acid in contrast adopts either a C₄confor-

mation or a skewed boat ²S₀conformation in oligosaccharide

Scheme 5. Synthesis of glucuronic acid and iduronic acid monosaccharide

building blocks. a) 1. NaH, BnBr, THF, Bu₄NI; 2. aq. HOAc (66%), 40 8C;

3. TBSCl, DMAP, CH₂Cl₂, pyridine; 4. Ac₂O, DMAP, pyridine; 5. HF/

pyridine, THF; 6. TEMPO (cat.), KBr, Bu₄NBr, NaHCO₃, NaOCl, CH₂Cl₂/

H₂O; 7. 4m NaOH, MeOH; 8. MeI, KHCO₃, DMF, 65% (eight steps); b)

TFA (90% aq.), quant; c) 1. Tf₂O, pyridine, CH₂Cl₂; 2. LevONa, DMF,

808C, 82% (two steps); d) N₂H₄, HOAc, pyridine, 91%.

sequences depending upon the substituents on the ring.^{[27, 28]}

1

In the C₄conformation the C4 hydroxyl occupies an axial

position.^{[19, 29]}Apparently, the conformation is responsible for

the observed a-selectivity of glycosylations without anchi-

meric assistance.

Based on these observations we engineered a conforma-

tional constraint into glycosyl acceptors to control the stereo-

chemistry during disaccharide formation (Scheme 7).^[14]NMR

Disaccharide formation–Stereocontrol of glycosylation re-

actions by conformational locking of the glycosyl acceptor:

With both differentially protect-

Glucuronic acid acceptors

ed glucosamine as well as glucur-

onic and iduronic monosacchar-

AcO

TBSO

NH

CCl₃

AcO

TBSO

CO₂Me

O

a

CO₂Me

O

ides at hand, a set of disaccharide

building blocks was to be assem-

bled by creation of an a-glucos-

amine linkage. While trans-gly-

cosidic linkages are readily in-

stalled with complete stereo-

selectivity by virtue of a C2

participating group using a host

of anomeric leaving groups, ster-

eoselective formation of cis-gly-

cosides is generally more difficult

since anchimeric assistance can-

not be exploited.^[19]

In order to form a-glucosa-

mine linkages that are ubiquitous

in nature,^[20]the C2 amino group

of glucosamine is typically

masked in form of a nonpartici-

pating azide.^{[9, 21]}Anomeric se-

lectivities vary greatly depending

upon the coupling partners in-

volved and often require difficult

O

HO

+

O

BnO

O

BnO

O

BnO

N₃

OBz

N₃

OBz

27: α/β=3/1 (57%)

26

11

R¹O

TBSO

R¹O

TBSO

CO₂Me

O

b

O

+

O

BnO

F

BnO

OR²

N₃

10: R¹= Ac

15: R¹= Bn

27: R¹= Ac; R²= Bz; α/β=7/1 (77%)

28: R¹= Bn; R²= Bz; α/β=5/1 (43%)

Iduronic acid acceptors

OBn

AcO

NH

TBSO

AcO

OBn

O

MeO₂C

O

OTDS

MeO₂C

a

O

OTDS

TBSO

AcO

+

O

CCl₃

OH

N₃

OH

N₃

O

14

29

30: completely α selective (47%)

30: completely α selective (30%)

AcO

b

O

TBSO

AcO

+

29

F

N₃

13

Scheme 6. Synthesis of disaccharides using uronic acid acceptors. a) TBSOTf, CH₂Cl₂, À788C ! room

temperature; b) AgClO₄, SnCl₂, Et₂O, 4 ä molecular sieves, 08C ! room temperature.

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P. H. Seeberger et al.

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analysis of glucuronic acid (31,

33) carrying cyclic isopropyli-

dene protecting groups re-

vealed a ¹C₄conformation as

judged by the coupling con-

stants (J_4,5ꢀ3.5 Hz (⁴C₁: J_4,5

>8 Hz) and J_1,2ꢀ2.7 Hz). Dif-

ferentially protected glucuronic

acid monomers (31, 33) were

prepared from triol 23 by

formation of isopropylidene^[30]

or cyclopentylidene^[31]acetals

upon reaction with 2-methoxy-

propene or methoxycyclopen-

tene under kinetic control

(Scheme 7). The corresponding

furanoside by-products (32, 34)

were also obtained and were

recycled by cleavage of the

acetal protective group and re-

submission to 1,2-acetal forma-

tion. With conformationally

constrained glucuronic acid ac-

ceptors 31 and 33 in hand, the

stereochemical outcome of the

coupling reactions was investi-

Glucuronic acid acceptors

AcO

TBSO

BnO

CO₂Me

OBn

O

11

or

10

31

or

33

a (if 11)

O

AcO

TBSO

BnO

c or d

+

CO₂Me

O

or

b (if 10)

O

N

O

³O

OH

R

BnO

N₃

O

OH

R

39: R= Me (86% via a; 80% via b)

41 (81% from 39;

90% from 40)

40: R=

(80% via a; 79% via b)

AcO

BnO

CO₂Me

OBn

AcO

BnO

NH

CCl₃

O

AcO

BnO

31

or

a

O

c or d

BnO

+

CO₂Me

O

N₃

O

R

N₃

33

OH

BnO

N₃

O

OH

20

R

42: R= Me (83%)

43: R=

44 (84% from 42;

81% from 43)

(82%)

CO₂Me

OBn

O

R¹O

TBSO

R²O

R¹O

TBSO

R²O

NH

CCl₃

e

O

+

31

O

N₃

O

14: R¹= Ac, R₂= Ac

16: R¹= Bn, R₂= Bn

17: R¹= Bn, R₂= Ac

45: R¹= Ac, R²= Ac (92%)

46: R¹= Bn, R²= Bn (77%)

47: R¹= Bn, R²= Ac (71%)

Scheme 8. Synthesis of disaccharides using ™locked∫ glucuronic acid acceptors. a) TBSOTf, 4 ä molecular sieves,

CH₂Cl₂, À788C ! room temperature; b) AgClO₄, SnCl₂, Et₂O, 4 ä molecular sieves, 08C ! room temperature;

c) dichloroacetic acid (75% aq.); d) dichloroacetic acid (50% aq.); e) TBSOTf, 4 ä molecular sieves, CH₂Cl₂,

À258C ! room temperature.

gated (Scheme 8). Both glycosyl trichloroacetimidates (e.g.

11) and glycosyl fluorides (e.g. 10) resulted exclusively in the

formation of a-linked disaccharides 39 and 40 in very good

In addition to their use as molecular locks 1,2-acetals are

convenient for differential protection of monosaccharides and

were applied to iduronic acid acceptors. In an analoguos

fashion to glucuronic acid, differentially protected iduronic

acid 35 and 37 were prepared from the triol 25. As expected,

1

CO₂Me

35 and 37 adopted a C₄conformation. The corresponding

CO₂Me

O

OBn

O

HO

furanoside by-products 36 and 38 were recycled analogously

to 32 and 34 (Scheme 7). Coupling of glycosyl donors 11, 14,

16 and 17 with iduronic acid acceptors 35 and 37 furnished

disaccharides 48 52 (Scheme 9).

After the cyclic acetal protecting groups served their

purpose, they were readily removed to yield disaccharide

diols 41, 44, 53, and 54 (Schemes 8 and 9). Complete a-

a or b

O

HO

OH

O

+

BnO

OH

R

O

OH

O

R

O

23

R

32: R = Me (32%)

34: R =

31: R = Me (48%)

33: R =

(57%)

(29%)

CO₂Me

OBn

O

HO

a or b

Iduronic acid acceptors

MeO₂C

OH

O

MeO₂C

OH

O

+

BnO

O

R

O

OH OH

OBn

O

R¹O

TBSO

R²O

R¹O

TBSO

R²O

R

O

NH

CCl₃

MeO₂C

O

35

or

37

25

R

a

O

R

+

R

O

36: R = Me (20%)

38: R =

35: R = Me (68%)

37: R =

N₃

O

N₃

R

11: R¹= Ac; R²= Bn

14: R¹= Ac; R²= Ac

16: R¹= Bn; R²= Bn

17: R¹= Bn; R²= Ac

48: R¹= Ac; R²= Bn; R = Me (91%)

49: R¹= Ac; R²= Ac; R = Me (90%)

50: R¹= Ac; R²= Ac; R=

(56%)

(18%)

Scheme 7. Installation of 1,2-acetal protecting groups as molecular locks of

uronic acid monosaccharides. a) 2-methoxypropene, DMF, CSA; b) me-

thoxycyclopentene, DMF, CSA.

(88%)

51: R¹= Bn; R²= Bn; R = Me (92%)

52: R¹= Bn; R²= Ac; R = Me (88%)

OBn

O

AcO

TBSO

R²O

MeO₂C

O

OH

b

yield upon coupling with glucuronic acid acceptors. The

nature of the cyclic protecting group (isopropylidene or

cyclopentylidene) and the anomeric leaving group had no

influence on the selectivity of the coupling reaction. Sub-

sequently, glycosyl trichloroacetimidates were employed in

couplings since they performed slightly better than glycosyl

fluorides and disaccharides 42, 43, 45 47 were formed.

OH

N₃

O

53: R²= Bn (92%)

54: R²= Ac (89% from 49)

(88% from 50)

Scheme 9. Synthesis of disaccharides using ™locked∫ iduronic acid accept-

ors. a) TBSOTf, 4 ä molecular sieves, CH₂Cl₂, À308C ! room temper-

ature; b) dichloroacetic acid (60% aq.).

144

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Heparin Oligosaccharides

140 169

selectivity of the coupling reac-

tions greatly simplified access

to the nine disaccharide mod-

ules (39, 42, 45 47, 48, 49, 51,

52) needed for heparin assem-

bly and purification of the re-

action products.

Glucuronic acid disaccharide donors

AcO

RO

BnO

O

a

b, c

CO₂Me

O

41

44

N₃

O

OMCA

BnO

55: R = TBS (96%)

56: R = Bn (93%)

d, then

a, e or f

After the a-glucosamine

linkages had been stereoselec-

tively formed, the resulting di-

saccharides had to be converted

into competent glycosylating

agents. In accordance with our

modular assembly strategy for

heparin oligosaccharides, dif-

ferent C2 participating groups

had to be introduced in the

uronic acid portion. To account

for the presence of uronic acid

C2 hydroxyl or sulfate groups,

participating protective groups

orthogonal to acetates were

needed. Levulinoyl (Lev),^[32]al-

lyloxycarbonate (Alloc),^[33]and

monochloroacetate (MCA)^[34]

groups were installed in a vari-

ety of disaccharides. These

groups can be replaced by per-

manent benzyl ether protection

AcO

RO

BnO

AcO

O

N

g, c

RO

BnO

CO₂Me

O

CO₂Me

N₃

NH

CCl₃

O

³O

BnO

OTBS

R'O

O

OR'

57: R = TBS, R' = MCA

59: R = TBS, R' = Lev (81%)

(64% from 55; 36% from 61)

58: R = Bn, R' = MCA (68% from 56)

64: R = TBS, R' = Lev (92%)

65: R = TBS, R' = Alloc (68%)

66: R = Bn, R' = Lev (85%)

60: R = TBS, R' = Alloc (76%)

61: R = TBS, R' = MCA (82%)

62: R = Bn, R' = Lev (80%)

63: R = Bn, R' = Alloc (73%)

67: R = Bn, R' = Alloc (83%)

Iduronic acid disaccharide donors

NH

OBn

O

OBn

O

AcO

TBSO

MeO₂C

O

AcO

MeO₂C

O

b, c

OAc

O

CCl₃

h

TBSO

RO

53

54

RO

OAc

N₃

O

N₃

O

70: R = Ac (67%)

71: R = Bn (75%)

68: R = Ac (95%)

69: R = Bn (99%)

Scheme 10. Preparation of uronic acid containing disaccharide building blocks. a) (MCA)₂O, CH₂Cl₂, DMAP,

pyridine; b) BnNH₂, Et₂O, 0 8C; c) NCCCl₃, DBU, CH₂Cl₂, 08C; d) TBSCl, imidazole, CH₂Cl₂, 08C;

e) (LevO)₂O, DMAP, CH₂Cl₂; f) AllocCl, DMAP, CH₂Cl₂; g) TBAF, HOAc, THF; h) Ac₂O, CH₂Cl₂, DMAP,

pyridine.

at the appropriate stage of the synthesis. The selective

introduction of 2-hydroxyl protective groups was accomplish-

ed via two different routes (Scheme 10). Diacetylation of

disaccharide diols 41 and 44 using monochloroacetyl chloride

and selective cleavage of the anomeric MCA group from 55

and 56 was followed by conversion to the corresponding

glycosyl trichloroacetimidates 57 and 58.^[35]For protecting

groups that do not allow for selective anomeric cleavage,

anomeric silylation,^[36]protection of the 2-hydroxyl group,

desilylation and preparation of the glycosyl trichloroacetimi-

dates furnished disaccharide modules 64 67. Synthesis of

iduronic acid containing disaccharide modules 70 and 71 were

accomplished from 53 and 54.

for further functionalization was identified; 2) uronic acid

glycosyl donors were employed to avoid difficult late stage

d, 10b, c, 24e]

oxidations;^[9b

3) products of the modular oligosac-

charide assembly carry protecting group patterns analogous

to previous total syntheses to enable established deprotection

and sulfation protocols.^{[9b, 10a, 37]}

We began oligosaccharide assembly by preparing the

reducing end. n-Pentenyl glycosides served to protect the

reducing terminus during oligosaccharide synthesis mimick-

ing an octenediol linker used in automated solid-phase

oligosaccharide synthesis.^[38]Furthermore the n-pentenyl

group can function as glycosidating agent or may be converted

into convenient handles for attachment to proteins or

surfaces.^[39]Three reducing end modules (73, 75, and 77) were

obtained by coupling disaccharide glycosyl trichloroacetimi-

dates 64, 57, 65, and n-pentenyl alcohol followed by removal

of the silyl protecting group from the C4 hydroxyl moiety

(Scheme 11).

Oligosaccharide assembly: Convergent routes to monosac-

charide building blocks and selective a-glucosamine glycoside

formation by conformational locking of uronic acid acceptors

provided ready access to a set of key disaccharide modules.

The use of di- and trisaccharide

building blocks in the assembly

of defined heparin tetra- and

hexasaccharides was the next

goal of our investigation.

Several key issues had to be

addressed to enable our ap-

proach based on the assembly

of di- and trisaccharide mod-

ules. 1) Aversatile reducing end

moiety mimicking the solid-

phase situation and allowing

AcO

TBSO

BnO

AcO

RO

BnO

O

N

CO₂Me

O

CO₂Me

O

a

N₃

³O

NH

CCl₃

O

BnO

R'O

O

72: R = TBS, R' = Lev ( 75%)

73: R = H, R' = Lev (85%)

74 R = TBS, R' = MCA (70%)

75: R = H, R' = MCA (85%)

76: R = TBS, R' = Alloc (66%)

77: R = H, R' = Alloc (85%)

64: R' = Lev

57: R' = MCA

65: R' = Alloc

b

Scheme 11. Synthesis of reducing end disaccharides. a) 4-penten-1-ol, TMSOTf, CH₂Cl₂, 08C; b) HF/pyridine,

HOAc, THF.

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145

P. H. Seeberger et al.

succeed.^[40]Tetrasaccharide 83 containing

3-O-benzyl

ether was employed as acceptor to explore if a 3-O-acetate

protecting group was responsible for the failure to

couple. Again, the desired union to form hexasaccharide 87

did not occur. Even benzylation of the 4-hydroxyl group of 81

failed!

FULL PAPER

Oligosaccharide assembly using disaccharide modules: Next,

the elongation of heparin oligosaccharide sequences by

combining disaccharide building blocks with reducing end

modules was explored. Coupling of disaccharide donors, 70

and 71, with reducing end modules, 73 and 75, furnished

tetrasaccharides 78 80 in excellent yield. Removal of the silyl

ether protecting group from the C4 hydroxyl group of 78 80

produced tetrasaccharides 81 83 as acceptors for further

elongation (Scheme 12).

a

To investigate the low reactivity of the C4 hydroxyl group of

the tetrasaccharide acceptors and rationalize the unexpected

results observed during the coupling of di- and tetrasacchar-

ides we performed model coupling experiments involving

acceptors containing the sequence GlcN-IdoA. The reaction

of glucosamine trichloroacetimidate 90 and iduronic acid 35

afforded disaccharide 91. Removal of the isopropylidene

group produced 92 before anomeric silylation, acetylation and

selective removal of the levulinoyl group afforded disacchar-

ide acceptor 93. The coupling between glucuronic acid

trichloroacetimidate 94 and GlcN-IdoA acceptor 93 did not

afford the desired trisaccharide, but rather the starting

materials were recovered (Scheme 14). Similarly, no penta-

saccharide product was obtained when disaccharide donor 97

and trisaccharide 98 where treated under glycosidation

conditions (Scheme 15).

AcO

HO

BnO

MeO₂C

OBn

O

N

O

AcO

NH

+

CO₂Me

O

N₃

TBSO

O

³O

CCl₃

R¹O

O

AcO

BnO

OR²

70: R¹= Bn

71: R¹= Ac

73: R²= Lev

75: R²= MCA

AcO

MeO₂C

O

OBn

O

N

a

AcO

O

BnO

CO₂Me

O

RO

N₃

R¹O

³O

AcO

O

BnO

OR²

1

To determine if the C₄conformation of the iduronic acid

78: R = TBS; R¹= Ac; R²= Lev (88%)

unit is responsible for the poor coupling behavior of the GlcN-

79: R = TBS; R¹= Ac; R²= MCA (91%)

80: R = TBS; R¹= Bn; R²= Lev (86%)

IdoA sequence, we prepared disaccharide 101 containing a

b

1

glucuronic acid locked in the C₄conformation (Scheme 15).

81: R = H; R¹= Ac; R²= Lev (86%)

82: R = H; R¹= Ac; R²= MCA (85%)

83: R = H; R¹= Bn; R²= Lev (75%)

Glycosylation of 101 with glucuronic acid trichloroacetimi-

date 94 and iduronic acid trichloroacetimidate 103 afforded

the expected coupling products 102 and 104 in good yield

(Scheme 16). Based on these results, the conformation of the

acceptor itself is not responsible of the low reactivity of the C4

hydroxyl group of GlcN-IdoA. Although we are currently not

able to explain the low reactivity of the C4 hydroxyl group of

the GlcN-IdoA sequence, these results clearly illustrated that

our initial strategy had to be amended to allow for the

incorporation of GlcN-IdoA sequences in the assembly of

bigger structures.

Scheme 12. Synthesis of tetrasaccharides by coupling of two disaccharides.

a) TMSOTf, CH₂Cl₂, À258C; b) HF/pyridine, AcOH, THF.

Encouraged by the success of the couplings between

disaccharides, the preparation of defined heparin hexasac-

charides by addition of a further disaccharide module was

attempted. Disaccharides 58, 66, and 67 containing different

C2 protecting groups, were employed in coupling with the

tetrasaccharides 81 and 82 under a variety of glycosylation

conditions (Scheme 13). The formation of the hexasacchar-

ides 84 and 86 was never detected in appreciable yield. Not

unexpectedly, activation of monochloroacetate protected

donor 58 afforded hexasaccharide orthoester 88. Treatment

of 88 with acid to procure rearranged product 84 did not

Oligosaccharide assembly using trisaccharide modules: Based

on the observations outlined above, the modular synthesis of

heparin oligosacharides was expanded to draw from di- and

trisaccharide modules to access all possible structures. The

overall strategy remains efficient as the linkage between

AcO

a

AcO

O

MeO₂C

OBn

BnO

O

NH

CCl₃

81

82

83

O

N

AcO

BnO

AcO

O

CO₂Me

O

CO₂Me

O

+

N

³O

BnO

CO₂Me

O

N₃

O

N₃

O

R²O

³O

BnO

AcO

OR¹

O

OR¹

BnO

OR³

58: R¹= MCA

66: R¹= Lev

67: R¹= Alloc

84: R¹= MCA; R²= Ac; R³= MCA

85: R¹= Lev; R²= Ac; R³= Lev

86: R¹= Alloc; R²= Ac; R³= Lev

87: R¹= Lev; R²= Bn; R³= Lev

O

AcO

BnO

CO₂Me

O

N₃

O

BnO

AcO

MeO₂C

OAc

OBn

O

a

O

58

82

+

BnO

CO₂Me

O

AcO

O

Cl

N₃

N

³O

H

AcO

O

BnO

OMCA

88 (30%)

Scheme 13. Attempted synthesis of a hexasaccharide by coupling of tetra- and disaccharides. a) TMSOTf, CH₂Cl₂, À258C.

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146

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Heparin Oligosaccharides

140 169

trisaccharide modules by pre-

paring tetra- and hexasacchar-

ides, installation of the desired

sulfation patterns had to be

demonstrated. From the outset

of the synthesis acetates

marked positions to be sulfated

and benzyl ethers designated

free hydroxyl groups. Thus, late

stage manipulations could fol-

AcO

LevO

BnO

AcO

LevO

BnO

c

a

b

O

HO

BnO

NH

CCl₃

OTBS

N₃

O

6

89

90

OBn

O

OBn

O

OH

OBn

AcO

MeO₂C

O

AcO

OTBS

AcO

MeO₂C

O

MeO₂C

O

e

O

d

O

HO

BnO

LevO

BnO

LevO

BnO

OAc

93

O

OH

N₃

O

N₃

91

O

N₃

O

92

OBn

O

low precedent from earlier total

CO₂Me

O

CO₂Me

O

AcO

MeO₂C

O

NH

CCl₃

BzlO

OTBS

f

c, 37]

syntheses.^{[9b, 10a}

Still, the

BzlO

93

+

O

BnO

BzlO

O

OAc

BzlO

placement of different C2 pro-

tection groups on uronic acid

donors had to be illustrated.

Tetrasaccharides 81 and 82

served to demonstrate the final

deprotection and sulfation

steps (Scheme 19). Selective re-

moval of the monochloroace-

tate group in 81^[41]and levuli-

noyl group in 82^[32]was ach-

ieved in high yield. Permanent

protection of the free hydroxyl

group was readily accomplished

by benzylation to furnish 110.

Alternatively, after saponifica-

tion of 111 the unprotected

hydroxyl groups can be sulfated

by reaction with Et₃NSO₃.^[9-

N₃

O

94

Scheme 14. Preparation of disaccharides to investigate the sequence dependence of coupling reactions. a) Lev₂O,

DMAP, CH₂Cl₂, 96%; b) 1. THF, AcOH, TBAF; 2. NCCCl₃, DBU, CH₂Cl₂, 87% (two steps); c) 35, TBSOTf, 4 ä

molecular sieves, CH₂Cl₂, À788C ! room temperature, 83%; d) dichloroacetic acid (60% aq.), 91%;

e) 1. TBSCl, imidazole, CH₂Cl₂, À158C; 2. Ac₂O, DMAP, pyridine, CH₂Cl₂; 3. NH₂NH₂¥ H₂O, AcOH, pyridine,

60% (three steps); f) TMSOTf, CH₂Cl₂, À208C.

AcO

LevO

BnO

AcO

a

b

O

HO

BnO

90

O

N₃

95

96

NH

OBn

O

AcO

LevO

BnO

MeO₂C

O

CCl₃

c

92

OAc

N₃

O

97

b, 10a c, 37]

Cleavage of all benzyl

NH

OBn

O

MeO₂C

O

AcO

LevO

BnO

MeO₂C

O

OBn

O

ether protective groups and

selective N-sulfation furnished

fully functionalized heparin tet-

CCl₃

AcO

HO

AcO

N₃

O

d

O

BnO

OAc

O

BnO

AcO

N₃

O

N₃

97

98

c]

rasaccharide 112.^{[9b, 10a}

OAc

MeO₂C

O

e

OBn

O

OBn

O

AcO

N₃

O

MeO₂C

O

BnO

Conclusion

O

BnO

AcO

N_{3 O}

OAc

AcO

N₃

LevO

In summary, we have developed

a general, modular strategy for

the first completely stereoselec-

tive synthesis of defined hepa-

rin oligosaccharides. Di- and

BnO

Scheme 15. Synthesis of trisaccharide models to investigate coupling reactions. a) 4-penten-1-ol, BF₃¥ Et₂O,

CH₂Cl₂/hexane, À788C ! room temperature, 75%; b) NH₂NH₂¥ H₂O, AcOH, pyridine, 91%; c) 1.Ac₂O, DMAP,

pyridine, CH₂Cl₂; 2. BnNH₂, Et₂O, 0 8C; 3. NCCCl₃, DBU, CH₂Cl₂, 08C, 87% (three steps); d) 1. 96, TMSOTf,

CH₂Cl₂, À208C; 2. NH₂NH₂¥ H₂O, AcOH, pyridine, 45% (two steps); e) 97, TMSOTf, CH₂Cl₂, À458C ! room

temperature.

trisaccharide modules were de-

rived from just six monosac-

glucosamine and iduronic acid can be established selectively

and in high yield.^[14]Trisaccharide modules were readily

accessed from the set of disaccharides as exemplified by the

synthesis of 105 and 107 (Scheme 17). The resulting trisac-

charides can be readily converted into glycosyl acceptors (e.g.

106) or glycosylating agents (e.g. 108) for the modular

assembly of oligosaccharides. This approach was successfully

demonstrated for the synthesis of hexasaccharide 109 by

coupling trisaccharide modules 106 and 108 in 62% yield

(Scheme 18).

charide building blocks in a fully selective fashion by placing a

conformational constraint on uronic acid glycosyl acceptors.

We demonstrated a new concept for stereochemical control of

a-glucosamine glycoside formation. Locking the conforma-

tion of the glucuronic acid acceptor allowed for completely

selective preparation of the desired cis-glycosides. This

innovation greatly simplified the key step in the preparation

of disaccharide building blocks. Combination of disaccharide

modules to form trans-uronic acid linkages proved also

completely selective by virtue of C2 participating groups.

Coupling reactions between modules exhibited sequence

dependence. Trisaccharide modules allowed for stereoselec-

tive access to oligosaccharides as demonstrated on the

Sulfation and final deprotection: After establishing a general

synthesis of heparin oligosaccharides based on di- and

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147

P. H. Seeberger et al.

FULL PAPER

MeO₂C

O

OBn

O

BnO

LevO

BnO

c, d

a,b

O

NH

CCl₃

O

8

HO

BnO

O

N₃

O

N₃

O

100

101

MeO₂C

OBn

O

CO₂Me

BnO

O

BnO

e

O

BzlO

O

94

+

101

O

BzlO

N₃

O

102

MeO₂C

O

OBn

O

NH

CCl₃

BnO

OBn

O

OBn

O

j

MeO₂C

TBSO

O

BnO

f, g, h, i

MeO₂C

TBSO

O

35

O

N₃

O

OAc

103

AcO

104

Scheme 16. Investigation of the sequence dependence of coupling reactions. a) 1. Lev₂O, DMAP, CH₂Cl₂, 98%; b) 1. THF, AcOH, TBAF; 2. NCCCl₃, DBU,

CH₂Cl₂, 08C, 86% (two steps); c) 31, TBSOTf, CH₂Cl₂, À258C ! room temperature, 89%; d) NH₂NH₂¥ H₂O, AcOH, pyridine, 77%; e) TMSOTf, CH₂Cl₂,

À208C ! room temperature, 59%; f) 1. TBSOTf, lutidine, CH₂Cl₂, 98%; g) dichloroacetic acid (60% aq.), 92%; g) Ac₂O, DMAP, pyridine, CH₂Cl₂, 98%;

h) BnNH₂, Et₂O, 0 8C, 75%; i) NCCCl₃, DBU, CH₂Cl₂, 08C, 92%; j) 101, TMSOTf, CH₂Cl₂, À258C ! room temperature, 71%.

AcO

HO

BnO

AcO

MeO₂C

RO

MeO₂C

TBSO

OBn

O

OBn

O

N

O

a

O

NH

BnO

+

CO₂Me

O

CO₂Me

O

N₃

O

BnO

O

³O

BnO

CCl₃

AcO

O

AcO

O

OLev

105: R = TBS

106: R = H

99

73

b

AcO

O

BnO

O

AcO

HO

AcO

OR

O

AcO

BnO

CO₂Me

O

c

AcO

O

AcO

OLev

CO₂Me

O

N₃

O

NH

O

BnO

+

N₃

OTBS

O

N₃

BnO

LevO

N₃

O

CCl₃

66

7

107: R = TBS

108: R = OC(NH)CCl₃

d

Scheme 17. Synthesis of trisaccharides by coupling mono- and disaccharides. a) TMSOTf, CH₂Cl₂, À258C, 93%; b) HF/pyridine, AcOH, THF, 82%;

c) TMSOTf, CH₂Cl₂, À258C, 63%; d) 1. TBAF, AcOH, THF; 2. NCCCl₃, DBU, CH₂Cl₂, 08C, 87% (two steps).

AcO

MeO₂C

OBn

O

N

AcO

BnO

a

AcO

O

CO₂Me

O

BnO

108

106

+

CO₂Me

O

N₃

O

N₃

AcO

³O

BnO

AcO

O

BnO

OLev

109

Scheme 18. Synthesis of a hexasaccharide by coupling trisaccharides. a) TMSOTf, CH₂Cl₂, À258C, 62%.

AcO

MeO₂C

AcO

MeO₂C

OBn

O

OBn

O

N

O

N

AcO

HO

O

AcO

HO

O

BnO

a, b

CO₂Me

O

BnO

CO₂Me

O

N₃

AcO

³O

BnO

AcO

³O

AcO

O

BnO

OBn

OR

81: R = MCA

82: R = Lev

110

c,d

OSO₃H

O

HOOC

O

AcO

OBn

MeO₂C

OH

O

HO₃SO

O

AcO

O

e, f, g, h

O

HO

BnO

COOH

O

CO₂Me

O

N₃

HO₃SHN

N

NHSO₃H

AcO

³O

O

OSO₃H

AcO

O

HO

BnO

( )₄

OSO₃H

OH

111

112

Scheme 19. Protecting group modifications, sulfation and final deprotection of a tetrasaccharide. a) Thiourea, DMF, pyridine, room temperature, 24 h, 90%;

b) BnBr, Ag₂O, 4 ä molecular sieves, CH₂Cl₂, room temperature, overnight, 76%; c) Ac₂O, pyridine, quant.; d) NH₂NH₂¥ H₂O, pyridine, AcOH, 90%;

e) 1. LiOH (0.7m aq.), H₂O₂(50% aq.), THF overnight; 2. 4m NaOH, room temperature overnight, 82% (two steps); f) Et₃NSO₃, DMF, 508C, overnight,

50%; g) H₂, Pd/C, EtOH, water, quant.; h) Py ¥ SO₃, H₂O, 60%.

148

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Heparin Oligosaccharides

140 169

EtOAc 7:3) afforded a colorless oil (mixture of a/b) (5.6 g, 0.015 mmol,

17%). The spectral data was in agreement with the reported data.^[16b]

example of a hexasaccharide. Final deprotection and sulfation

yielded the fully synthetic heparin oligosaccharides.

1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-a/b-d-glucopyranose

(30.2 g,

Utilizing the synthetic approach presented here, we are now

preparing heparin oligosaccharides to elucidate the exact

structures responsible for protein interactions involved in

growth factor signaling and viral entry. This general strategy is

expected to allow for the synthesis of heparin oligosaccharides

on solid support using a repetitive coupling/deprotection

cycle. The assembly of other classes of glycosaminoglycans of

medical importance such as hyaluronic acid and chondroitin

has come within reach. Investigations in these areas are

currently underway and will be reported in due course.

80 mmol) was coevaporated twice with toluene and dissolved in THF and

methanol (7:3, 300 mL). The solution was cooled to 08C and gaseous

anhydrous ammonia was bubbled through at a modest rate. After 15 min,

nitrogen was bubbled through the solution to remove excess ammonia and

the solvent was removed in vacuo to afford a brown oil. The residue was

coevaporated twice with toluene and dissolved in CH₂Cl₂(150 mL).

Imidazole (10.9 g, 160 mmol) and tert-butyldimethylsilyl chloride (13.3 g,

88 mmol) were added. After 2 h, the mixture was diluted with EtOAc,

washed with water, 1n HCl (2 Â ) and water. The organic layer was dried

over MgSO₄, filtered, and the solvent was removed in vacuo. Crystalliza-

tion from ethanol afforded 2 (29.8 g, 67 mmol, 84%) as colorless crystals.

¹H NMR (300 MHz, CDCl₃): d 5.00 4.90 (m, 2H), 4.63 (d, J 7.6 Hz,

1H), 4.20 (dd, J 5.9, 12.1 Hz, 1H), 4.09 (dd, J 2.6, 12.1 Hz, 1H), 3.70

3.64 (m, 1H), 3.48 3.40 (m, 1H), 2.10 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H),

0.95 (s, 9H), 0.15 (s, 6H). The spectral data was in agreement with the

reported data.^[42]

Experimental Section

tert-Butyldimethylsilyl 2-azido-4,6-O-benzylidene-2-deoxy-b-d-glucopyra-

noside (3): Compound 2 (36.9 g, 82.8 mmol) was dissolved in methanol

(300 mL) and NaOMe (25% in MeOH, 5.4 mL) was added. After 15 min,

DOWEX-50 acidic resin was added and the mixture was stirred until the

pH reached 6. The DOWEX resin was filtered off and the solvent was

removed in vacuo to afford a yellow oil. The residue was coevaporated

twice with toluene and dissolved in acetonitrile (400 mL). Benzaldehyde

dimethyl acetal (24.8 mL, 165 mmol) and p-toluenesulfonic acid monohy-

drate (400 mg, 2.1 mmol) were added. After stirring overnight at room

temperature, triethylamine (5 mL) was added and the solvents evaporated.

Flash chromatography on silica gel (hexanes/EtOAc 95:5 ! 9:1) afforded 3

(29.0 g, 71.2 mmol, 86%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃): d

7.55 7.45 (m, 2H), 7.45 7.38 (m, 3H), 5.52 (s, 1H), 4.65 (d, J 7.65 Hz,

1H), 4.29 (dd, J 4.9, 10.4 Hz, 1H), 3.77 (t, J 10.1 Hz, 1H), 3.60 3.30 (m,

4H), 2.91 (s, 1H), 1.00 (s, 9H), 0.19 (s, 6H). The spectral data was in

agreement with the reported data.^[43]

General methods: All chemicals used were reagent grade and used as

supplied except where noted otherwise. Anhydrous methanol (MeOH) and

dimethylformamide (DMF) were purchased from Aldrich in SureSeal

bottles. Dichloromethane (CH₂Cl₂), diethyl ether, toluene and tetrahydro-

furan (THF) were purchased from J. T. Baker (Cycletainer) and passed

through a neutral alumina column prior to use. Pyridine, 2,4,6-collidine,

and acetonitrile (CH₃CN) were heated under reflux over calcium hydride

and distilled prior to use. Analytical thin-layer chromatography was

performed on E. Merck silica gel 60 F₂₅₄glass plates (0.25 mm).

Compounds were visualized by dipping the plates in a cerium sulfate-

ammonium molybdate solution followed by heating. Liquid column

chromatography was performed using forced flow of the indicated solvent

on Silicycle 230 400 mesh (60 ä pore diameter) silica gel. ¹H NMR

spectra were obtained on a Varian VXR-500 (500 MHz) or a Varian-300

(300 MHz) spectrometer and are reported in parts per million (d) relative

to CHCl₃(7.27 ppm). Coupling constants (J) are reported in Hertz.

¹³C NMR spectra were obtained on a Varian VXR-500 (125 MHz) or a

Varian-300 (75 MHz) spectrometer and are reported in d relative to CDCl₃

(77.23 ppm) as an internal reference.

tert-Butyldimethylsilyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-b-

d-glucopyranoside (4): tert-Butyldimethylsilyl 2-azido-4,6-O-benzylidene-

2-deoxy-b-d-glucopyranoside (3; 28.1 g, 68.97 mmol) was dissolved in

CH₂Cl₂(250 mL). Powdered, freshly activated 4 ä molecular sieves (45 g)

and benzyl bromide (20.5 mL, 172 mmol) were added and the mixture was

stirred for 30 min. Silver(i)oxide (47 g, 203 mmol) was added and the

reaction vessel was covered in aluminum foil to exclude light. After 8 h, the

reaction mixture was filtered through Celite and the filtrate was concen-

trated in vacuo. Flash chromatography on silica (hexanes/EtOAc 50:1)

tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-b-d-glucopyra-

noside (2): Preparation of TfN₃: CH₂Cl₂(250 mL) was added to a solution

of NaN₃(59.5 g, 0.92 mol) in water (150 mL) at 08C. The mixture was

stirred vigorously and treated with trifluoromethanesulfonic anhydride

(31.0 mL, 0.19 mol) over a period of 3 h at 08C. After the complete addition

of trifluoromethanesulfonic anhydride, the reaction mixture was stirred at

08C for 2.5 h. The aqueous phase was extracted with CH₂Cl₂(2 Â 100 mL)

and the combined organic layers washed with saturated Na₂CO₃and saved

for use in the next step. [Caution: TfN₃is explosive when not in solution!]

afforded

4

(32.6 g, 65.5 mmol, 95%) as a colorless solid. ¹H NMR

(300 MHz, CDCl₃): d 7.60 7.28 (m, 10H), 5.51 (s, 1H), 4.98 (d, J

11.5 Hz, 1H), 4.84 (d, J 11.5 Hz, 1H), 4.63 (d, J 7.5 Hz, 1H), 4.33 (dd,

J 5.0, 9.4 Hz, 1H), 3.89 3.73 (m, 2H), 3.60 3.35 (m, 3H), 0.98 (s, 9H),

0.17 (s, 6H). The spectral data was in agreement with the reported data.^[43]

CuSO₄(140 mg, 0.88 mmol) and K₂CO₃(19.2 g, 0.14 mol) were added to a

solution of glucosamine hydrochloride (1) (20.0 g, 0.092 mol) in water

(300 mL). Methanol (600 mL) was added to the reaction mixture followed

by the addition of the TfN₃solution. Methanol was added until the solution

was homogeneous (ꢀ300 mL). The clear blue solution was allowed to stir

for 24 h at room temperature. Glycine (70 g) was added and the reaction

mixture was again allowed to stir for 24 h. The glycine was filtered off and

the solvent was removed in vacuo to afford a brown oil. The oil was taken

up in pyridine (95 mL), cooled to 08C and DMAP (ꢀ30 mg) and acetic

anhydride (86 mL, 0.91 mol) were added. The solution was stirred for 12 h

at room temperature. The reaction was quenched with saturated NaHCO₃

and the aqueous phase was extracted with CH₂Cl₂(3 Â 1000 mL). The

organic phase was dried over MgSO₄, filtered and the solvent was removed

in vacuo to yield a brown oil. Warm ethanol was added until the solution

was homogeneous. The resulting solution was cooled to À208C and a white

precipitate formed. Cold water was then added, the white precipitate was

filtered and washed with water and cold ethanol to afford 1,3,4,6-tetra-O-

acetyl-2-azido-2-deoxy-b-d-glucopyranose (23.8 g, 0.063 mol, 68%) as a

tert-Butyldimethylsilyl 3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-b-

d-glucopyranoside (5): Compound 3 (9.5 g, 23.3 mmol) was dissolved in

CH₂Cl₂(150 mL) and pyridine (21 mL), DMAP (280 mg, 2.3 mmol) and

acetic anhydride (10 mL, 106 mol) were added. The reaction mixture was

stirred overnight, water was added and stirred for 1 h. The organic layer

was extracted with water, 1n HCl, water and saturated NaHCO₃. The

organic phase was dried over MgSO₄, filtered and the solvent was removed

in vacuo. Flash chromatography on silica gel (hexanes/EtOAc 50:1 ! 5:1)

afforded 5 (9.96 g, 22.1 mmol, 95%) as a colorless crystalline solid. [a]_D²⁴

À72.1 (c 0.99, CH₂Cl₂); IR (thin film): nÄ 2111, 1751, 1370, 1222, 1099,

841 cm^À1; ¹H NMR (300 MHz, CDCl₃): d 7.45 7.33 (m, 5H), 5.49 (s, 1H),

5.13 (dd, J 9.7, 9.9 Hz, 1H), 4.72 (d, J 7.6 Hz, 1H), 4.31 (dd, J 4.9,

10.5 Hz, 1H), 3.80 (dd, J 10.2, 10.3 Hz, 1H), 3.65 (dd, J 9.4, 9.5 Hz, 1H),

3.49 (ddd, J 4.9, 9.6, 9.6 Hz, 1H), 3.42 (dd, J 7.5, 10.1 Hz, 1H), 2.14 (s,

3H), 0.95 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): d

169.9, 136.9, 129.3, 128.4, 126.3, 101.7, 97.8, 78.9, 71.1, 68.7, 67.4, 66.8, 25.8,

21.2, 18.2, À4.1, À4.9; FAB MS: m/z: calcd for C₂₁H₃₁N₃O₆Si: 449.1982;

1

colorless solid. H NMR (300 MHz, CDCl₃): d 5.55 (d, J 8.6 Hz, 1H),

found: 449.1876 [M] .

5.15 5.00 (m, 2H), 4.31 (dd, J 4.6, 12.5 Hz, 1H), 4.08 (dd, J 2.1,

12.5 Hz, 1H), 3.75 (ddd, J 2.1, 4.4, 6.3 Hz, 1H), 3.65 3.72 (m, 1H), 2.20

(s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H); IR (thin film): nÄ 2959,

2112, 1747 cm^À1. Flash chromatography of the mother liquor (hexanes/

tert-Butyldimethylsilyl 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-b-d-gluco-

pyranoside (6): Compound 4 (32.6 g, 65.5 mmol) was dissolved in CH₂Cl₂

(1.5 L) and trifluoroacetic acid (60% aq., 54 mL) was added. The resulting

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0149 $ 20.00+.50/0

149

P. H. Seeberger et al.

FULL PAPER

mixture was stirred vigorously at room temperature for 8.5 h and saturated

NaHCO₃was added carefully. After phase separation, the aqueous layer

was extracted with CH₂Cl₂. The combined organic layers were dried over

Na₂SO₄, filtered and the solvents were removed in vacuo. Flash chroma-

tography on silica (hexanes/EtOAc 4:1 ! 1:1) afforded tert-butyldimethyl-

silyl 2-azido-3-O-benzyl-2-deoxy-b-d-glucopyranoside (25 g, 61 mmol,

92%) as a colorless oil. [a]_D²⁴ À30.4 (c 1.00, CH₂Cl₂); IR (thin film):

nÄ 3415, 2110, 1361, 1079 cm^À1; ¹H NMR (300 MHz, CDCl₃): d 7.44 7.30

(m, 5H), 4.97 (d, J 11.4 Hz, 1H), 4.72 (d, J 11.4 Hz, 1H), 4.58 (d, J

7.5 Hz, 1H), 3.84 (dd, J 3.7, 11.8 Hz, 1H), 3.75 (dd, J 4.8, 11.8 Hz, 1H),

3.59 (dd, J 8.7, 9.5 Hz, 1H), 3.36 3.26 (m, 2H), 3.22 (dd, J 8.6, 9.9 Hz,

1H), 0.96 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): d

138.2, 128.9, 128.34, 128.26, 97.5, 82.5, 75.3, 75.2, 70.7, 68.5, 62.8, 25.8, 18.1,

À4.0, À4.9; FAB MS: m/z: calcd for C₁₉H₃₁N₃O₅Si: 409.2033; found:

CH₂Cl₂and phase separation, the aqueous phase was extracted with

CH₂Cl₂. The combined organic phases were dried over MgSO₄, filtered and

the solvents were removed in vacuo. Flash chromatography on silica gel

(hexanes/EtOAc 8:1 ! 6:1) afforded 8 (3.1 g, 6.2 mmol, 85%) as a colorless

oil. [a]²_D⁴ À33.9 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 3472, 2111, 1257,

1113, 1069 cm^À1; ¹H NMR (300 MHz, CDCl₃): d 7.45 7.28 (m, 10H), 4.93

(d, J 11.4 Hz, 1H), 4.77 (d, J 11.4 Hz, 1H), 4.61 (d, J 12.1 Hz, 1H),

4.56 (d, J 12.1 Hz, 1H), 4.55 (d, J 7.5 Hz, 1H), 3.73 (d, J 4.8 Hz, 2H),

3.65 (dd, J 8.5, 9.7 Hz, 1H), 3.46 3.39 (m, 1H), 3.33 (dd, J 7.5, 10.0 Hz,

1H), 3.23 (dd, J 8.5, 10.0 Hz, 1H), 0.95 (s, 9H), 0.18 (s, 6H); ¹³C NMR

(75 MHz, CDCl₃): d 138.3, 137.9, 128.8, 128.6, 128.23, 128.16, 127.9, 127.8,

97.4, 82.5, 75.2, 74.1, 73.9, 72.2, 70.6, 68.3, 25.9, 18.3, À4.0, À5.0; FAB MS:

m/z: calcd for C₂₆H₃₇N₃O₅Si: 499.2502; found: 499.2513. The spectral data

was in agreement with the reported data.^[43]

409.2029 [M] . The spectral data was in agreement with the reported

tert-Butyldimethylsilyl 3-O-acetyl-2-azido-6-O-benzyl-2-deoxy-b-d-gluco-

pyranoside (9): Compound 5 (42.0 mg, 0.093 mmol) and triethylsilane

(75 mL, 0.47 mmol) were dissolved in CH₂Cl₂(930 mL) under argon at 08C

and trifluoroacetic acid (36 mL, 0.47 mmol) was added dropwise over 6 min.

The reaction mixture was slowly warmed to room temperature, stirred for

3 h and quenched with saturated NaHCO₃. After addition of CH₂Cl₂and

phase separation, the aqueous phase was extracted three times with

CH₂Cl₂. The combined organic phases were dried over MgSO₄, filtered and

the solvents were removed in vacuo. Flash chromatography on silica gel

(hexanes/EtOAc 8:1 ! 6:1) furnished 9 (38.1 mg, 91%) as a colorless oil.

[a]²_D⁴ À21.6 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 3434, 2111, 1749,

data.^[44]

Acetyl chloride (4.5 mL, 63.3 mmol) was added dropwise to a solution of

tert-butyldimethylsilyl 2-azido-3-O-benzyl-2-deoxy-b-d-glucopyranoside

(25 g, 61 mmol) in 2,4,6-collidine (110 mL) under nitrogen at À408C.

After stirring at À408C overnight, water was added. The mixture was

poured into EtOAc and extracted with 1n HCl, brine and saturated

NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and the

solvents were removed in vacuo to afford 6 (26.5 g, 58.7 mmol, 96%) as a

colorless solid. [a]²_D⁴ À27.1 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 3482,

2110, 1743, 1255 cm^À1; ¹H NMR (300 MHz, CDCl₃): d 7.43 7.30 (m, 5H),

4.96 (d, J 11.4 Hz, 1H), 4.73 (d, J 11.4 Hz, 1H), 4.55 (d, J 7.6 Hz, 1H),

4.34 4.27 (m, 2H), 3.50 3.38 (m, 2H), 3.33 (dd, J 7.6, 9.9 Hz, 1H), 3.21

(dd, J 8.3, 9.9 Hz, 1H), 2.59 2.46 (brs, 1H), 2.09 (s, 3H), 0.96 (s, 9H),

0.18 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d 171.5, 138.1, 128.8, 128.31,

128.28, 97.4, 82.2, 75.3, 73.8, 70.3, 68.3, 63.5, 25.8, 21.1, 18.3, À4.1, À5.0;

1

1252 cm^À1; H NMR (300 MHz, CDCl₃): d 7.40 7.28 (m, 5H), 4.80 (dd,

J 9.1 Hz, 10.3 Hz, 1H), 4.61 (d, J 7.7 Hz, 1H), 4.61 4.57 (m, 2H), 3.75

(dd, J 1.6, 4.9 Hz, 1H), 3.69 (ddd, J 3.5, 9.3, 9.3 Hz, 1H), 3.53 3.45 (m,

1H), 3.36 (dd, J 7.7, 10.3 Hz, 1H), 3.00 (d, J 3.7 Hz, 1H), 2.18 (s, 3H),

0.95 (s, 9H), 0.178 (s, 3H), 0.175 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): d

171.5, 137.7, 128.6, 128.0, 127.8, 97.3, 75.4, 74.4, 73.9, 71.1, 70.3, 66.1, 25.8,

21.3, 18.2, À4.1, À5.0; FAB MS: m/z: calcd for C₂₁H₃₃N₃O₆Si: 451.2138;

FAB MS : m/z: calcd for C₂₁H₃₃N₃O₆Si: 451.2138; found: 451.2135 [M] .

tert-Butyldimethylsilyl 3,6-di-O-acetyl-2-azido-2-deoxy-b-d-glucopyrano-

found: 451.2131 [M] .

side (7): Compound

5 (9.5 g, 21.1 mmol) was dissolved in CH₂Cl₂

(500 mL) and trifluoroacetic acid (60% aq., 17 mL) was added. The

resulting mixture was stirred vigorously at room temperature overnight and

saturated NaHCO₃was added carefully. After phase separation, the

aqueous layer was extracted with CH₂Cl₂. The combined organic layers

were dried over Na₂SO₄, filtered and the solvents were removed in vacuo.

Flash chromatography on silica gel (hexanes/EtOAc 4:1 ! 1:1) afforded

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a/b-

d-glucopyranosyl fluoride (10a): tert-Butyldimethylsilyl trifluormethane-

sulfonate (3.4 mL, 14.8 mmol) was added to a solution of compound 6

(5.15 g, 11.4 mmol) and 2,6-lutidine (3.3 mL, 28.3 mmol) in CH₂Cl₂(50 mL)

at À208C. The reaction was allowed to warm to room temperature and stir

for 1 h. The mixture was poured into EtOAc and the aqueous layer was

extracted with 1n HCl, brine and saturated NaHCO₃. The organic layer

was dried over Na₂SO₄, filtered and the solvents were removed in vacuo.

Flash chromatography on silica gel (hexanes/EtOAc 199:1 ! 98:2) afford-

ed tert-butyldimethylsilyl 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldi-

methylsilyl-2-deoxy-b-d-glucopyranoside (5.8 g, 10.3 mmol, 90%) as a

colorless oil. [a]²_D⁴ 36.1 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 2110, 1748,

1112 cm^À1; ¹H NMR (300 MHz, CDCl₃): d 7.40 7.28 (m, 5H), 4.93 (d, J

11.1 Hz, 1H), 4.71 (d, J 11.1 Hz, 1H), 4.56 (d, J 7.6 Hz, 1H), 4.42 (dd,

J 2.2, 11.6 Hz, 1H), 4.07 (dd, J 6.6, 11.6 Hz, 1H), 3.59 (dd, J 8.3,

9.5 Hz, 1H), 3.45 (ddd, J 2.2, 6.6, 9.5 Hz, 1H), 3.34 (dd, J 7.6, 9.9 Hz,

1H), 3.20 (dd, J 8.3, 9.9 Hz, 1H), 2.08 (s, 3H), 0.94 (s, 9H), 0.89 (s, 9H),

0.164 (s, 3H), 0.160 (s, 3H), 0.05 (s, 3H), 0.03 (s, 3H); ¹³C NMR (75 MHz,

CDCl₃): d 170.8, 138.4, 128.5, 127.7, 127.6, 97.4, 83.0, 75.2, 74.6, 71.3, 69.2,

63.6, 26.1, 25.8, 21.1, 18.3, À3.5, À4.1, À4.6, À5.0; FAB MS: m/z: calcd for

tert-butyldimethylsilyl

3-O-acetyl-2-azido-2-deoxy-b-d-glucopyranoside

(7.25 g, 20 mmol, 95%) as

a

colorless solid. [a]²_D⁴ À26.4 (c 1.00,

CH₂Cl₂); IR (thin film): nÄ 3387, 2112, 1748, 1254 cm^À1

;

¹H NMR

(500 MHz, CDCl₃): d 4.77 (dd, J 9.2, 9.5 Hz, 1H), 4.64 (d, J 7.6 Hz,

1H), 3.90 (dd, J 3.7, 11.9 Hz, 1H), 3.81 (dd, J 4.9, 11.9 Hz, 1H), 3.66 (dd,

J 9.5, 9.5 Hz, 1H), 3.41 3.36 (m, 1H), 3.34 (dd, J 7.6, 10.4 Hz, 1H),

3.08 2.92 (brs, 1H), 2.15 1.92 (brs, 1H), 2.19 (s, 3H), 0.94 (s, 9H), 0.174

(s, 3H), 0.168 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) d 172.2, 97.2, 76.0,

75.8, 70.1, 66.2, 62.6, 25.7, 21.2, 18.1, À4.1, À5.0; FAB MS: m/z: calcd for

C₁₄H₂₇N₃O₆Si: 361.1669; found: 361.1677 [M] .

Acetyl chloride (1.5 mL, 21.1 mmol) was added slowly to a solution of tert-

butyldimethylsilyl

3-O-acetyl-2-azido-2-deoxy-b-d-glucopyranoside

(7.25 g, 20 mmol) in 2,4,6-collidine (47 mL) under nitrogen at À408C.

After stirring at À408C overnight, water was added. The mixture was

poured into EtOAc and extracted with 1n HCl, brine and saturated

NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and the

solvents were removed in vacuo. Flash chromatography on silica gel

(hexanes/EtOAc 5:1 ! 4:1) afforded 7 (7.59 g, 18.8 mmol, 94%) as a

colorless solid. [a]²_D⁴ À31.6 (c 1.00, CH₂Cl₂); IR (thin film on NaCl): nÄ

C₂₇H₄₇N₃O₆Si₂: 565.3003; found: 565.3011 [M] .

tert-Butyldimethylsilyl 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldime-

thylsilyl-2-deoxy-b-d-glucopyranoside (4.0 g, 7.07 mmol) was dissolved in

THF (60 mL) and cooled to 08C. Glacial acetic acid (500 mL, 8.7 mmol) and

TBAF (1m in THF, 8.2 mL, 8.2 mmol) were added simultaneously. After

30 min, the mixture was poured into Et₂O (200 mL) and washed three times

with brine. The organic layer was dried over Na₂SO₄, filtered and the

solvents were removed in vacuo. The residue was coevaporated with

toluene, dissolved in anhydrous THF (20 mL) and cooled to À308C. DAST

(1.2 mL, 9.08 mmol) was added dropwise and the mixture was stirred for

5 min at À308C and 30 min at room temperature. The reaction mixture was

cooled to À308C and anhydrous methanol (500 mL) was added. After

warming to room temperature, the mixture was poured into EtOAc

(300 mL) and washed with saturated NaHCO₃, water and brine. The

organic layer was dried over Na₂SO₄, filtered and the solvents were

removed in vacuo. Flash chromatography on silica gel (hexanes/EtOAc

95:5) afforded a mixture (5:1) of 10a and 10b (3.0 g, 6.62 mmol, 94%) as a

crystalline solid. 10a: ¹H NMR (300 MHz, CDCl₃): d 7.45 7.28 (m, 5H),

3459, 2112, 1747, 1233, 1042, 841 cm^À^{1 1}H NMR (300 MHz, CDCl₃): d

;

4.81 4.74 (m, 1H), 4.62 (d, J 7.6 Hz, 1H), 4.37 4.31 (m, 2H), 3.75

3.46 (m, 2H), 3.53 (dd, J 7.7, 10.3 Hz, 1H), 3.13 3.09 (m, 1H), 2.18 (s,

3H), 2.10 (s, 3H), 0.94 (s, 9H), 0.17 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d

171.8, 171.5, 97.2, 75.5, 74.3, 69.8, 66.0, 63.4, 25.8, 21.2, 21.1, 18.2, À4.2,

À5.0; FAB MS: m/z: calcd for C₁₆H₂₉N₃O₇Si: 403.1775; found: 403.1779

[M] .

tert-Butyldimethylsilyl 2-azido-3,6-di-O-benzyl-2-deoxy-b-d-glucopyrano-

side (8): Compound

4 (3.6 g, 7.24 mmol) and triethylsilane (6.5 mL,

43.4 mmol) were dissolved in anhydrous CH₂Cl₂(70 mL) under nitrogen

at 08C and trifluoroacetic acid (3.3 mL, 43.4 mmol) was added dropwise

over 5 min. The reaction mixture was slowly warmed to room temperature,

stirred for 5 h and quenched with saturated NaHCO₃. After addition of

150

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Heparin Oligosaccharides

140 169

5.68 (dd, J 2.7, 52.7, 1H), 4.92 (d, J 11.0 Hz, 1H), 4.84 (d, J 11.0 Hz,

1H), 4.45 (dd, J 1.9, 12.1 Hz, 1H), 4.11 (dd, J 4.7, 12.1 Hz, 1H), 4.04

3.94 (m, 1H), 3.83 3.71 (m, 2H), 3.74 3.36 (m, 1H), 2.11 (s, 3H), 0.92 (s,

9H), 0.06 (s, 3H), 0.05 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): d 170.7, 137.7,

128.5, 127.8, 127.5, 107.6, 104.6, 80.1, 75.6, 73.14, 73.08, 70.4, 64.3, 64.0, 62.5,

26.1, 21.1, 18.3, À3.4, À4.7. 10b: ¹H NMR (300 MHz, CDCl₃): d 7.45 7.28

(m, 5H), 5.68 (dd, J 2.7, 52.7 Hz, 1H), 4.92 (d, J 11.0 Hz, 1H), 4.84 (d,

J 11.0 Hz, 1H), 4.45 (dd, J 1.9, 12.1 Hz, 1H), 4.11 (dd, J 4.7, 12.1 Hz,

1H), 4.04 3.94 (m, 1H), 3.83 3.71 (m, 2H), 3.74 3.36 (m, 1H), 2.11 (s,

3H), 0.92 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): d

170.7, 137.7, 128.6, 128.5, 128.2, 127.9, 127.6, 109.6, 106.7, 82.4, 82.3, 75.4,

74.8, 74.7, 70.2, 66.6, 66.3, 62.8, 26.0, 21.1, 18.2, À3.5, À4.7.

were removed in vacuo. The crude material was evaporated three times

with toluene, dried under vacuum for 1 h and dissolved in THF (17 mL).

The resulting solution was added to a suspension of SOBr₂(760 mL,

9.6 mmol) and imidazole (585 mg, 8.6 mmol) in anhydrous THF (55 mL) at

08C. The resulting suspension was stirred for 1 h, diluted with anhydrous

Et₂O, filtered over a pad of florisil and ground Na₂S₂O₃and concentrated to

furnish 12 as a yellow solid (2.1 g, 4.5 mmol, 76%) which was used without

further purification. [a]²_D⁴ 5.8 (c 1.00, CHCl₃); IR (thin film): nÄ 2929,

2859, 2113, 1746, 1473, 1235, 1006 cm^À^{1 1}H NMR (500 MHz, CDCl₃): d

;

6.40 (d, J 3.9 Hz, 1H), 5.43 (dd, J 8.8, 10.4 Hz, 1H), 4.41 (dd, J 2.1,

12.5 Hz, 1H), 4.17 4.11 (m, 2H), 3.87 (t, J 8.8 Hz, 1H), 3.58 (dd, J 3.9,

10.4 Hz, 1H), 2.17 (s, 3H), 2.10 (s, 3H), 0.86 (s, 9H), 0.07 (s, 3H), 0.06 (s,

3H); ¹³C NMR (100 MHz, CDCl₃): d 170.6, 169.6, 88.1, 75.1, 73.7, 68.6,

63.4, 61.9, 25.8, 21.5, 20.9, 18.1, À3.8, À4.8; FAB MS: m/z: calcd for

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a/b-

d-glucopyranosyl trichloroacetimidate (11): tert-Butyldimethylsilyl 6-O-

acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-b-d-gluco-

pyranoside (1.16 g, 2.05 mmol) was dissolved in anhydrous THF (20 mL)

and cooled to 08C. Glacial acetic acid (146 mL, 2.56 mmol) and TBAF (1m

in THF) (2.25 mL, 2.25 mmol) were added simultaneously. After 30 min,

the mixture was poured into Et₂O (200 mL) and washed three times with

brine. The organic layer was dried over Na₂SO₄, filtered and the solvents

were removed in vacuo. The residue was dissolved in CH₂Cl₂(50 mL) and

cooled to 08C. Trichloroacetonitrile (3.1 mL, 30.9 mmol) and DBU (30 mL,

0.2 mmol) were added and the mixture was stirred for 1 h at 08C and

concentrated in vacuo. Flash chromatography on silica gel (hexanes/EtOAc

85:15) afforded a mixture of 11a and 11b (27/73) (1.12 g, 1.88 mmol, 92%)

as a colorless oil. 11a: [a]²_D⁴ 118.6 (c 1.69, CH₂Cl₂); IR (thin film): nÄ

C₁₆H₂₈BrN₃O₇Si: 465.0930; found: 465.0940 [M] .^[45]

3,6-O-Acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a/b-d-glucopyr-

anosyl fluoride (13): tert-Butyldimethylsilyl-3,6-O-acetyl-2-azido-4-O-tert-

butyldimethylsilyl-2-deoxy-a/b-d-glucopyranoside (202 mg, 0.39 mmol)

was dissolved in THF (4 mL) and cooled to 08C. Glacial acetic acid

(25 mL, 0.43 mmol) and TBAF (1m, in THF, 0.43 mL, 0.43 mmol) were

added simultaneously. After 30 min, the mixture was poured into Et₂O

(200 mL) and washed three times with brine. The organic layer was dried

over Na₂SO₄, filtered and the solvent were removed in vacuo. The residue

was coevaporated with toluene, dissolved in anhydrous CH₂Cl₂(3 mL) and

cooled to À308C. DAST (0.06 mL, 0.45 mmol) was added dropwise and the

mixture was stirred for 5 min at À308C and 30 min at room temperature.

The reaction mixture was cooled to À308C and anhydrous methanol

(0.1 mL) was added. After warming to room temperature, the mixture was

poured into EtOAc (100 mL) and washed with saturated NaHCO₃, water

and brine. The organic layer was dried over Na₂SO₄, filtered and the

solvent were removed in vacuo. Flash chromatograpy on silica gel

(hexanes/EtOAc 9:1) afforded an anomeric mixture (6:1) of 13 (145 mg,

92%) as a crystalline solid. FAB MS: m/z: calcd for C₁₆H₂₈FN₃O₆Si:

3344, 2954, 2929, 2857, 2110, 1745, 1674, 1255, 1069 cm^À^{1 1}H NMR

;

(500 MHz, CDCl₃): d 8.75 (s, 1H), 7.41 7.28 (m, 5H), 6.44 (d, J 3.4 Hz,

1H), 4.92 (d, J 11.3 Hz, 1H), 4.86 (d, J 11.0 Hz, 1H), 4.40 (dd, J 12.2,

2.1 Hz, 1H), 4.08 (dd, J 12.2, 4.6 Hz, 1H), 3.95 3.99 (m, 1H), 3.77 3.82

(m, 2H), 3.66 3.71 (m, 1H), 2.06 (s, 3H), 0.91 (s, 9H), 0.06 (s, 3H), 0.05 (s,

3H); ¹³C NMR (125 MHz, CDCl₃): d 170.8, 160.9, 137.9, 128.5, 127.8,

127.6, 94.8, 80.5, 75.5, 73.3, 70.8, 63.7, 62.7, 26.1, 21.0, 18.2, À3.5, À4.8; FAB

MS: m/z: calcd for C₂₃H₃₃Cl₃N₄O₆Si: 594.1235; found: 594.1219. 11b:

[a]²_D⁴ 43.6 (c 1.11, CH₂Cl₂); IR (thin film): nÄ 3329, 2928, 2857, 2113,

1745, 1676, 1063 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 8.74 (s, 1H), 7.48

7.28 (m, 5H), 5.66 (d, J 8.2 Hz, 1H), 4.94 (d, J 11.3 Hz, 1H), 4.79 (d, J

11.3 Hz, 1H), 4.42 (dd, J 11.9, 2.4 Hz, 1H), 4.13 (dd, J 12.2, 4.9 Hz, 1H),

3.77 (dd, J 9.5, 8.5 Hz, 1H), 3.69 (dd, J 9.8, 8.2 Hz, 1H), 3.60 (ddd, J

9.5, 4.9, 2.4 Hz, 1H), 3.36 (dd, J 9.5, 8.5 Hz, 1H), 2.08 (s, 3H), 0.90 (s, 9H),

0.05 (s, 3H), 0.04 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.9, 161.2,

138.1, 128.6, 127.9, 127.6, 97.1, 83.4, 75.5, 75.4, 70.4, 66.2, 62.8, 26.0, 21.1, 18.2,

À3.5, À4.8; FAB MS: m/z: calcd for C₂₃H₃₃Cl₃N₄O₆Si: 594.1235; found:

405.1731; found: 405.1758 [M] .

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a/b-d-gluco-

pyranosyl trichloroacetimidate (14): TBAF (1.0m in THF, 1.4 mL) and

glacial acetic acid (80 mL, 5.9 mmol) were added dropwise to a solution of

tert-butyldimethylsilyl 3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-

2-deoxy-b-d-glucopyranoside (590 mg, 1.14 mmol) in THF (12 mL) under

nitrogen at 08C. The reaction mixture was warmed to room temperature,

stirred for 1.5 h and quenched with saturated NaHCO₃solution. After

extracting three times with CH₂Cl₂, the combined organic phases were

dried over MgSO₄, filtered and the solvents were removed in vacuo. The

crude material was dried by coevaporation three times with toluene and

under vacuum for 1 h and dissolved in CH₂Cl₂(25 mL). Trichloroacetoni-

trile (1.3 mL, 12.50 mmol) and freshly activated 4 ä powdered molecular

sieves (300 mg) were added and the mixture was stirred for 30 minutes at

room temperature. After cooling to 08C, DBU (30 mL, 0.2 mmol) was

added and the temperature was allowed to rise to room temperature. After

1 h, the mixture was filtered through Celite and the solvents were removed

in vacuo. Flash chromatography on silica gel (hexanes/EtOAc 85:15)

afforded 14a (437 mg, 0.80 mmol, 70%) and 14b (94 mg, 0.17 mmol, 15%).

14a: ¹H NMR (500 MHz, CDCl₃): d 8.81 (s, 1H, NH), 6.44 (d, J 3.6 Hz,

1H), 5.43 (dd, J 8.8, 10.4 Hz, 1H), 4.41 (dd, J 1.9, 12.0 Hz, 1H), 4.12

3.99 (m, 2H), 3.87 (t, J 9.1 Hz, 1H), 3.58 (dd, J 3.6, 10.7 Hz, 1H), 2.17 (s,

3H), 2.01 (s, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H); ¹³C NMR

(125 MHz, CDCl₃) d 170.5, 169.7, 160.8, 94.7, 72.77, 69.1, 62.5, 61.5, 25.9,

594.1222 [M] .

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl bromide (12): tert-Butyldimethylsilyl trifluoromethanesulfonate

(4.1 mL, 17.9 mmol) was added to a solution of compound 7 (4.70 g,

11.65 mmol) and 2,6-lutidine (3.5 mL, 30 mmol) in CH₂Cl₂(25 mL) at

À208C. The reaction was allowed to warm to room temperature and stirred

for 1 h. The mixture was poured into EtOAc and extracted with 1n HCl,

brine and saturated NaHCO₃. The organic layer was dried over Na₂SO₄,

filtered and solvents removed in vacuo. Flash chromatography on silica gel

(hexanes/EtOAc 199:1 ! 98:2) afforded tert-butyldimethylsilyl 3,6-di-

O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-b-d-glucopyranoside

(5.61 g, 10.8 mmol, 93%) as a colorless oil. [a]²_D⁴ À3.1 (c 1.00, CH₂Cl₂);

IR (thin film): nÄ 2111, 1750, 1363, 1221 cm^À1

;

¹H NMR (300 MHz,

CDCl₃): d 4.87 (dd, J 8.8, 10.4 Hz, 1H), 4.63 (d, J 7.6 Hz, 1H), 4.39

(dd, J 2.2, 11.7 Hz, 1H), 4.08 (dd, J 6.2, 11.7 Hz, 1H), 3.67 (dd, J 9.1,

9.2 Hz, 1H), 3.54 3.46 (m, 1H), 3.27 (dd, J 7.6, 10.4 Hz, 1H), 2.14 (s,

3H), 2.08 (s, 3H), 0.92 (s, 9H), 0.83 (s, 9H), 0.15 (s, 3H), 0.14 (s, 3H), 0.05

(s, 3H), 0.04 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): d 170.7, 169.9, 97.1, 74.6,

74.5, 69.6, 66.8, 63.1, 25.80, 25.75, 21.6, 21.1, 18.2, 18.1, À3.9, À4.3, À4.6,

À5.0; FAB MS: m/z: calcd for C₂₂H₄₃N₃O₇Si₂: 517.2639; found: 517.2635

1

21.6, 21.0, 18.2, À3.8, À4.6. 14b: H NMR (500 MHz, CDCl₃): d 8.81 (s,

1H), 5.73 (d, J 8.2 Hz, 1H), 5.03 (dd, J 8.8, 9.8 Hz, 1H), 4.40 (dd, J

2.1, 11.9 Hz, 1H), 4.13 (dd, J 4.2, 11.9 Hz, 1H), 3.87 (t, J 9.5 Hz, 1H),

3.58 (m, 2H), 2.17 (s, 3H), 2.08 (s, 3H), 0.84 (s, 9H), 0.05 (s, 3H), 0.04 (s,

3H); ¹³C NMR (125 MHz, CDCl₃) d 170.7, 169.9, 160.8, 96.6, 75.3, 68.8,

64.1, 62.4, 25.9, 25.8, 25.8, 21.5, 21.1, 18.1, À3.9, À4.8.

[M] .

2-Azido-3,6-di-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a/b-d-gluco-

pyranosyl fluoride (15): A mixture of tetrabutylammonium fluoride (1.0m

in THF, 203 mL) and glacial acetic acid (12 mL, 0.203 mmol) was added

dropwise to a solution of tert-butyldimethylsilyl 3,6-di-O-benzyl-4-O-tert-

butyldimethylsilyl-2-deoxy-a/b-d-glucopyranosyide (104 mg, 0.169 mmol)

in THF (1.7 mL) under nitrogen at 08C. The reaction mixture was warmed

to room temperature, stirred for 1.5 h and quenched with brine. After

dilution with CH₂Cl₂, the two phases were separated. The organic phase

TBAF (1.0m in THF, 6.4 mL) and glacial acetic acid (350 mL, 5.9 mmol)

were added dropwise to a solution of tert-butyldimethylsilyl 3,6-di-O-

acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-b-d-glucopyranoside

(2.90 g, 5.6 mmol) in THF (60 mL) under nitrogen at 08C. The reaction

mixture was warmed to room temperature, stirred for 1.5 h and quenched

with saturated NaHCO₃. After extracting three times with CH₂Cl₂, the

combined organic phases were dried over MgSO₄, filtered and the solvents

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0151 $ 20.00+.50/0

151

P. H. Seeberger et al.

FULL PAPER

was dried over MgSO₄, filtered and the solvent was removed under reduced

pressure. The crude material was coevaporated three times with toluene

and dried under vacuum for 1 h. CH₂Cl₂(1.4 mL) was added and a solution

of DAST (29 mL, 0.22 mmol) in CH₂Cl₂(300 mL) was added dropwise to the

reaction mixture. After stirring at room temperature for 2.5 h, the reaction

mixture was diluted with CH₂Cl₂and washed with water. The organic phase

was dried over Na₂SO₄and after filtration the solvent was removed under

reduced pressure. Column chromatography on silica gel (hexanes/EtOAc

20:1) afforded 15 (80.1 mg, 89%) as a colorless oil. FAB MS: m/z: calcd for

97.2, 76.4, 74.9, 73.5, 69.1, 68.5, 67.0, 25.9, 25.8, 21.7, 18.2, 18.1, À3.9, À4.1,

À4.5, À5.0; FAB MS: m/z: calcd for C₂₇H₄₇N₃O₆Si₂: 565.3003; found:

565.3011 [M] .

tert-Butyldimethylsilyl 6-O-benzyl-2-azido-3-O-acetyl-4-O-tert-butyldime-

thylsilyl-2-deoxy-b-d-glucopyranoside (0.170 g, 0.30 mmol) was dissolved

in anhydrous THF (3 mL) and cooled to 08C. Glacial acetic acid (20 mL,

0.35 mmol) and TBAF (1m in THF) (330 mL, 0.33 mmol) were added

simultaneously. After 30 min, the mixture was poured into Et₂O (50 mL)

and washed three times with brine. The organic layer was dried over

Na₂SO₄, filtered and the solvents were removed in vacuo. The residue was

dissolved in CH₂Cl₂(3 mL) and cooled to 08C. Trichloroacetonitrile

(770 mL, 7.67 mmol) and DBU (5 mL, 0.03 mmol) were added and the

mixture was stirred for 1 h at 08C and concentrated in vacuo. Flash

chromatography on silica gel (hexanes/EtOAc 85:15) afforded a mixture of

17a and 17b (2.7:1) (0.160 g, 0.27 mmol, 89%) as a colorless oil. 17a

¹H NMR (500 MHz, CDCl₃): d 8.77 (s, 1H, NH), 7.36 7.27 (m, 5H), 6.50

(d, J 3.4 Hz, 1H), 5.43 (dd, J 7.9, 10.7 Hz, 1H), 4.58 (d, J 11.9 Hz, 1H),

4.51 (d, J 11.9 Hz, 1H), 4.01 3.95 (m, 2H), 3.75 (dd, J 3.3, 11.3 Hz,

1H), 3.65 (dd, 1H), 3.48 (dd, J 3.4, 10.4 Hz, 1H), 2.17 (s, 3H), 0.84 (s,

9H), 0.07 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): d 170.0, 161.0, 138.1,

128.5, 127.8, 127.7, 95.2, 74.7, 73.6, 73.1, 68.7, 68.0, 61.7, 25.9, 21.6, 18.2, À4.0,

À4.6; 17b ¹H NMR (500 MHz, CDCl₃): d 8.78 (s, 1H), 7.34 7.27 (m, 5H),

5.76 (d, J 8.5 Hz, 1H), 5.03 (dd, J 8.8, 10.0 Hz, 1H), 4.64 (d, J 12.2 Hz,

1H), 4.53 (d, J 12.3 Hz, 1H), 3.91 (t, J 9.1 Hz, 1H), 3.68 3.58 (m, 4H),

2.16 (s, 3H), 0.82 (s, 9H), 0.06 (s, 3H), 0.04 (s, 3H).

C₂₆H₃₆FN₃O₄Si: 501.2459; found: 501.2439 [M] .

2-Azido-3,6-di-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-d-glucopyra-

nosyl trichloroacetimidate (16): tert-Butyldimethylsilyl trifluoromethane-

sulfonate (244 mL, 1.06 mmol) was added under argon at room temperature

to a solution of compound 8 (353.7 mg, 0.708 mmol) and 2,6-lutidine

(206 mL, 1.77 mmol) in CH₂Cl₂(800 mL). The reaction mixture was stirred

for 1 h and quenched with saturated NaHCO₃. After addition of CH₂Cl₂

and phase separation the aqueous phase was extracted four times with

CH₂Cl₂. The combined organic phases were dried over MgSO₄, filtered and

solvents removed in vacuo. Flash chromatography on silica gel (hexanes/

EtOAc 25:1) afforded tert-butyldimethylsilyl 2-azido-3,6-di-O-benzyl-4-O-

tert-butyldimethylsilyl-2-deoxy-b-d-glucopyranoside (420 mg, 97%) as a

colorless solid. [a]²_D⁴ 31.1 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 2109,

1472, 1360, 1107, 1066 cm^À1; ¹H NMR (300 MHz, CDCl₃): d 7.40 7.28 (m,

5H), 4.93 (d, J 11.1 Hz, 1H), 4.72 (d, J 11.1 Hz, 1H), 4.64 (d, J

12.2 Hz, 1H), 4.58 (d, J 7.7 Hz, 1H), 4.53 (d, J 12.2 Hz, 1H), 3.78

3.64 (m, 2H), 3.59 (dd, J 5.5, 10.8 Hz, 1H), 3.44 3.39 (m, 1H), 3.34 (dd,

J 8.5, 9.8 Hz, 1H), 3.19 (dd, J 8.5, 9.8 Hz, 1H), 0.96 (s, 9H), 0.87 (s, 9H),

0.20 (s, 3H), 0.19 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H); ¹³C NMR (75 MHz,

CDCl₃): d 138.7, 138.5, 128.50, 128.46, 127.63, 127.60, 97.5, 83.4, 76.6, 75.0,

73.5, 71.0, 69.3, 69.2, 26.1, 25.8, 18.23, 18.19, À3.6, À4.0, À4.6, À5.0; FAB

tert-Butyldimethylsilyl-6-O-acetyl-2-azido-2-deoxy-b-d-glucopyranoside

(18): Compound 2 (1.23 g, 2.77 mmol) was dissolved in methanol (10 mL).

NaOMe (25% in methanol, 170 mL) was added. After 15 min DOWEX-50

acidic resin was added and the mixture was stirred until the pH reached 6.

The DOWEX resin was filtered off and the solvent was removed under

reduced pressure to afford a yellow oil. The residue was coevaporated twice

with toluene, dissolved in 2,4,6-collidine (7 mL), cooled to À408C and

acetyl chloride (196 mL, 2.74 mmol) was added. After stirring the reaction

mixture for 3 h, a second portion of acetyl chloride (42 mL, 0.6 mmol) was

added. The mixture was stirred for another 1 h at À408C and for 1 h at

room temperature and then quenched with saturated NaHCO₃. After

addition of CH₂Cl₂and phase separation the aqueous phase was extracted

three times with CH₂Cl₂. The combined organic phases were dried over

MgSO₄, filtered and the solvents were removed in vacuo. Flash chroma-

tography (hexanes/EtOAc 4:1) on silica afforded 18 (933 mg, 2.58 mmol,

93%) as a colorless syrup. [a]_D²⁴ À7.0 (c 1.00, CHCl₃); IR (thin film):

MS: m/z: calcd for C₃₂H₅₁N₃O₅Si₂: 613.3367; found: 613.3359 [M] .

tert-Butyldimethylsilyl 2-azido-3,6-di-O-benzyl-4-O-tert-butyldimethylsil-

yl-2-deoxy-b-d-glucopyranoside (0.121 mg, 0.197 mmol) was dissolved in

anhydrous THF (1.5 mL) and cooled to 08C. Glacial acetic acid (20.0 mL,

0.256 mmol) and TBAF (1.0m in THF, 240 mL, 0.240 mmol) were added

simultaneously. After 30 min, the mixture was poured into EtOAc (50 mL)

and washed with sat. NaHCO₃. The organic layer was dried over Na₂SO₄,

filtered and the solvents were removed in vacuo. The residue was dissolved

in CH₂Cl₂(1 mL) and cooled to 08C. Trichloroacetonitrile (1.0 mL) and

DBU (5 mL, 0.03 mmol) were added and the mixture was stirred for 1 h at

08C, diluted with CH₂Cl₂(30 mL), passed through a plug of silica and

concentrated in vacuo. Flash chromatography on silica gel (hexanes/EtOAc

5:1) afforded 16a and 16b (87.5 mg, 0.137 mmol, 69%) as an inseparable 1:1

nÄ 3412, 2929, 2958, 2111, 1741, 1463, 1370, 1257, 1177 cm^À1

;

¹H NMR

(500 MHz, CDCl₃): d 4.55 (d, J 7.5 Hz), 4.38 4.26 (m, 2H), 4.13 (brs,

2H), 3.45 3.20 (m, 4H), 2.06 (s, 3H), 0.93 (s, 9H), 0.15 (s, 3H), 0.14 (s,

3H); ¹³C NMR (125 MHz, CDCl₃): d 171.9, 97.3, 74.5, 73.8, 70.6, 68.2,

63.7, 25.7, 21.0, 18.1, À3.5, À4.6; FAB MS: m/z: calcd for C₁₄H₂₇N₃O₆Si:

1

mixture. H NMR (500 MHz, CDCl₃) d 8.76 (s, 1H), 8.73 (s, 1H), 7.40

7.27 (m, 15H), 6.48 (d, J 3.6 Hz, 1H), 8.24 (d, J 8.2, 1H), 4.94 (d, J

11.6 Hz, 1H), 4.91 (d, J 11.9 Hz, 1H), 4.85 (d, J 11.0 Hz, 1H), 4.78 (d,

J 11.3 Hz, 1H), 4.66 (d, J 12.2 Hz, 1H), 4.60 (d, J 12.2 Hz, 1H), 4.53

(d, J 12.5 Hz, 1H), 4.50 (d, J 11.9 Hz, 1H), 3.95 3.90 (m, 1H), 3.86

(appt, J 9.0 Hz, 1H), 3.81 3.73 (m, 3H), 3.71 3.64 (m, 5H), 3.60 3.57

(m, 2H), 3.38 (app t, J 9.0 Hz, 1H), 0.88 (s, 9H), 0.87 (s, 9H), 0.06 0.02

(m, 12H); ¹³C NMR (125 MHz, CDCl₃) d 161.2, 161.0, 138.4, 138.3, 138.1,

128.5, 128.5, 128.5, 127.8, 127.7, 127.6, 97.1, 95.2, 83.5, 80.6, 77.7, 75.4, 75.2,

73.3, 70.6, 70.4, 68.4, 66.3, 63.9, 26.2, 26.1, 18.2, 18.2, À3.5, À3.6, À4.6.

361.1669; found: 361.1680 [M] .

tert-Butyldimethylsilyl-6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-b-d-

glucopyranoside (19): Compound 18 (7.1 g, 19.64 mmol) was dissolved in

CH₂Cl₂(100 mL). Powdered, freshly activated 4 ä molecular sieves (20 g)

and benzyl bromide (12 mL, 100 mmol) were added and this mixture

stirred for 30 min. Silver(i)oxide (26.4 g, 114 mmol) was added and light was

excluded from the reaction mixture. After 48 h, the reaction mixture was

filtered over Celite and the filtrate was concentrated under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 97:3)

afforded 19 (8.5 g, 15.7 mmol, 80%) as a colorless oil. [a]²_D⁴ À4.7 (c

1.40, CH₂Cl₂); IR (thin film): nÄ 3031, 2955, 2858, 2109, 1745, 1454, 1252,

6-O-Benzyl-2-azido-3-O-acetyl-4-O-tert-butyldimethylsilyl-2-deoxy-a/b-

d-glucopyranosyl trichloroacetimidate (17): tert-Butyldimethylsilyl trifluor-

omethanesulfonate (9.5 mL, 0.041 mmol) was added to a solution of

compound 9 (12.4 mg, 0.027 mmol) and 2,6-lutidine (8.0 mL, 0.069 mmol)

in CH₂Cl₂(200 mL) under argon at room temperature. The reaction mixture

was stirred for 1 h and quenched with saturated NaHCO₃. After addition of

CH₂Cl₂and phase separation, the aqueous phase was extracted three times

with CH₂Cl₂. The combined organic phases were dried over MgSO₄,

filtered and the solvents were removed in vacuo. Flash chromatography on

silica gel (hexanes/EtOAc 30:1) afforded tert-butyldimethylsilyl 3-O-

acetyl-2-azido-6-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-b-d-gluco-

pyranoside (15.3 mg, 98%) as a colorless solid. [a]²_D⁴ À11.3 (c 0.71,

CH₂Cl₂); IR (thin film): nÄ 2109, 1752, 1473, 1221, 1107, 899 cm^À1; ¹H NMR

(300 MHz, CDCl₃): d 7.38 7.29 (m, 5H), 4.87 (dd, J 9.0, 10.5 Hz, 1H),

4.65 (d, J 12.4 Hz, 1H), 4.64 (d, J 7.8 Hz, 1H), 4.53 (d, J 12.4 Hz, 1H),

3.79 (dd, J 9.3, 9.1 Hz, 1H), 3.70 3.60 (m, 2H), 3.45 3.38 (m, 1H), 2.15

(s, 3H), 0.95 (s, 9H), 0.82 (s, 9H), 0.18 (s, 3H), 0.17 (s, 3H), 0.06 (s, 3H),

0.03 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) d 170.1, 138.3, 128.5, 127.7, 127.6,

1

1042, cm^À1; H NMR (500 MHz, CDCl₃): d 7.43 7.29 (m, 10H), 4.95 (d,

J 11.0 Hz, 1H), 4.89 (d, J 11.0 Hz, 1H), 4.82 (d, J 10.7 Hz, 1H), 4.61

(d, J 11.0 Hz, 1H), 4.56 (d, J 7.6 Hz, 1H), 4.36 (dd, J 11.9, 1.5 Hz, 1H),

4.17 (dd, J 11.9, 5.8 Hz, 1H), 3.55 3.49 (m, 2H), 3.47 3.43 (m, 1H), 3.38

(dd, J 9.8, 7.6 Hz, 1H), 2.06 (s, 3H), 0.98 (s, 9H), 0.20 (s, 3H), 0.19 (s, 3H);

¹³C NMR (125 MHz, CDCl₃): d 170.8, 138.0, 137.7, 128.7, 128.6, 128.2,

128.2, 128.1, 97.3, 83.1, 77.7, 75.7, 75.2, 73.2, 68.8, 63.2, 25.8, 21.0, 18.2, À4.2,

À5.1; FAB MS: m/z: calcd for C₂₈H₃₉N₃O₆Si: 541.2608; found: 541.2606

[M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a/b-d-glucopyranosyl

tri-

chloroacetimidate (20): Glacial acetic acid (100 mL, 1.75 mmol) and TBAF

(1m in THF, 1.55 mL, 1.55 mmol) were added simultaneously to a solution

of 19 (756 mg, 1.4 mmol) in anhydrous THF (15 mL) at 08C. After 30 min

this mixture was poured into Et₂O (150 mL) and extracted three times with

152

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Heparin Oligosaccharides

140 169

brine. The organic layer was dried over Na₂SO₄, filtered and the solvents

were removed in vacuo. The residue was dissolved in anhydrous CH₂Cl₂

(50 mL) and cooled in an ice bath. Trichloroacetonitrile (2.1 mL, 21 mmol)

and DBU (21 mL, 0.14 mmol) were added. After 45 min, the solvents were

removed in vacuo. Flash chromatography on silica gel (hexanes/EtOAc

85:15 ! 8:2) afforded a mixture (58:42) of 20a and 20b (708 mg, 1.24 mmol,

88%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): d 8.76 (s, 1H, NH),

7.44 7.26 (m, 10H, arom. H), 6.42 (d, J 3.4 Hz, 1H), 5.64 (d, J 8.2 Hz,

1H), 4.97 4.86 (m, 4H), 4.64 4.60 (m, 1H), 4.35 4.24 (m, 2H), 4.10 4.06

(m, 1H), 3.73 3.57 (m, 2H), 2.03 (s, 3H).

3.8 Hz, 1H), 3.76 (s, 3H), 3.34 (d, J 9.1 Hz, 1H), 1.50 (s, 3H), 1.34 (s, 3H).

The spectral data was in agreement with the reported data.^[18]

Methyl 3-O-benzyl-d-glucopyranosyluronate (23): Compound 22 (3.43 g,

10.14 mmol) was dissolved in 90% aqueous trifluoroacetic acid (20 mL)

and stirred for 15 min at room temperature. The solvent was evaporated

and the residue coevaporated twice with water and twice with toluene to

afford 23, which was used without further purification. The spectral data

was in agreement with the reported data.^[18]

Methyl 3-O-benzyl-1,2-O-isopropylidene-5-O-levulinoyl-b-l-idofuranosy-

luronate (24): A solution of trifluoromethanesulfonic anhydride (13 mL)

in CH₂Cl₂(250 mL) was added dropwise to a mixture of pyridine (13 mL)

and CH₂Cl₂(132 mL) at À208C. The mixture was allowed to warm to

À108C and a solution of compound 22 (12 g, 35.5 mmol) in CH₂Cl₂

(123 mL) was added dropwise. After 1 h at À108C, the mixture was

poured into ice cold water containing NaHCO₃and stirred for 1 h. The

organic layer was washed with 3% HCl, water, dried over MgSO₄, filtered

and the solvents were removed in vacuo. Sodium levulinate (9.8 g,

71 mmol) was added to a solution of the crude residue in DMF (65 mL)

and the resulting mixture was stirred overnight at 808C and cooled to room

temperature. After dilution with EtOAc, the mixture was washed with

water and the organic phase was dried over MgSO₄, filtered and the

solvents were removed in vacuo. Flash chromatography on silica gel

(toluene/EtOAc 9:1 ! 7:3) afforded 24 (12.8 g, 29.3 mmol, 82%) as an

amorphous solid. [a]²_D⁴ À4.2 (c 1, CHCl₃); IR (thin film): nÄ 2512, 1751,

1718, 1025 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.36 7.28 (m, 5H), 5.97

(d, J 3.9 Hz, 1H), 5.53 (d, J 7.0 Hz, 1H), 4.66 4.62 (m, 3H), 4.50 (d, J

11.4 Hz, 1H), 4.16 (d, 1H), 3.69 (s, 3H), 2.73 2.63 (m, 4H), 2.12 (s, 3H),

1.59 (s, 3H), 1.34 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 205.3, 171.9,

168.5, 137.2, 128.6, 128.4, 128.0, 112.7, 105.1, 83.1, 82.7, 72.5, 70.9, 52.8, 38.0,

30.0, 28.0, 27.3, 26.8; FAB MS: m/z: calcd for C₂₂H₂₈O₉: 436.1733; found:

Methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glucofuranosyluronate (22):

1,2:5,6-Di-O-isopropylidene-a-d-glucofuranose (21; 52.06 g, 200 mmol)

was dissolved in THF (500 mL) and NaH (60% in mineral oil, washed

with pentanes) (9.6 g, 240 mmol) was added in portions. After the evolution

of hydrogen ceased, tetrabutylammonium iodide (500 mg, 1.35 mmol) and

benzyl bromide (25 mL, 210 mmol) were added and the mixture stirred for

10 h at room temperature. Water was added slowly to the reaction mixture

and the organic solvents were removed in vacuo. The aqueous phase was

extracted three times with EtOAc. The combined organic phases were

dried over Na₂SO₄, filtered through a plug of silica gel and the solvents

were removed in vacuo.

Aqueous acetic acid (66%, 300 mL) was added to the resulting oil and the

mixture was stirred for 14 h at room temperature and for 6 h at 408C. After

removal of the solvents, the remaining residue was dissolved in CH₂Cl₂and

extracted with saturated NaHCO₃. After phase separation, the aqueous

layer was extracted with CH₂Cl₂. The combined organic phases were dried

over Na₂SO₄, filtered and the solvents were removed in vacuo.

The residual oil was dissolved in CH₂Cl₂(750 mL) and pyridine (80 mL),

before DMAP (3.5 g, 28.6 mmol) and tert-butyldimethylsilyl chloride (32 g,

212 mmol) were added. After stirring at room temperature for 19 h, the

mixture was extracted with water, 1n HCl, brine and saturated NaHCO₃.

The organics were dried over Na₂SO₄, filtered and the solvents were

removed in vacuo. The residue was dissolved in anhydrous pyridine

(170 mL) and DMAP (1 g, 8.2 mmol) was added. The mixture was cooled

to 08C and acetic anhydride (38 mL, 403 mmol) was added dropwise. After

stirring overnight at room temperature, the solvents were removed in

vacuo. The residue was dissolved in EtOAc and extracted with water, 1n

HCl, brine and saturated NaHCO₃. The organic layer was dried over

Na₂SO₄, filtered and the solvents were removed in vacuo.

436.1742 [M] .

Methyl 3-O-benzyl-l-idopyranosyluronate (25): Hydrazine hydrate

(7.3 mL, 146 mmol) was added to a solution of compound 23 (12.8 g,

29.3 mmol) in pyridine/acetic acid (3:2, 290 mL) at 08C. After 15 min,

acetone (1.2 mL) was added, and the mixture was stirred at room

temperature for 15 min. After removal of the solvents in vacuo, the crude

product was dissolved in aqueous trifluoroacetic acid (90%, 70 mL). The

mixture was stirred for 15 min, the solvents removed in vacuo and

coevaporated twice with water to give a white solid, which was recrystal-

lized from ethyl acetate/hexanes to afford 25 (8.0 g, 26.8 mmol, 91%).

Analytical data was in agreement with reported data.^[18]

The residue (91.2 g, max. 195 mmol) was dissolved in THF (300 mL) and

cooled to 08C. To this solution, HF/pyridine (24 mL) in pyridine (80 mL)

was added and stirred overnight at room temperature. The reaction

mixture was poured into water and extracted three times with EtOAc. The

combined organic phases were extracted with water, 1n HCl, water,

saturated NaHCO₃, dried over Na₂SO₄, filtered and the solvents were

removed in vacuo.

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

deoxy-a/b-d-glucopyranosyl)-(1 ! 4)-methyl 2-O-benzoyl-3-O-benzyl-b-

d-glucopyranosyluronate (27)

By coupling 11 and 26: Compounds 11 (27.0 mg, 0.045 mmol) and 26

(16.1 mg, 0.035 mmol) were coevaporated three times with toluene and

dissolved in CH₂Cl₂(1 mL). Freshly activated powdered 4 ä molecular

sieves (100 mg) were added and the mixture was stirred at room temper-

ature for 1 h. The reaction mixture was cooled to À788C and tert-

butyldimethylsilyl trifluoromethanesulfonate (2.0 mL, 0.009 mmol) was

added dropwise. The reaction mixture was warmed to room temparature

over 2.5 h. Triethylamine (0.5 mL) was added, the mixture was filtered

through a pad of Celite and the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 95:5)

afforded 27 (18 mg, 0.02 mmol, 57%) as a yellow oil.

The residue was dissolved in CH₂Cl₂(400 mL) and TEMPO (800 mg,

5.1 mmol) was added. A mixture of saturated NaHCO₃(700 mL), water

(200 mL), KBr (2.25 g, 18.9 mmol) and tetrabutylammonium bromide

(3.45 g, 10.7 mmol) was added and the resulting mixture was cooled to 08C.

With vigorous stirring, bleach (6.15% sodium hypochlorite, 700 mL) was

added in 100 mL portions every 10 min. After complete addition, stirring

was continued for 30 min and methanol was added until the mixture was

decolorized. After 20 min, the aqueous layer was extracted with CH₂Cl₂

followed by the dropwise addition of conc. HCl to pH 1. The aqueous layer

was extracted three times with CH₂Cl₂. The organic layers were dried over

Na₂SO₄, filtered and the solvents were removed in vacuo.

By coupling 10 and 26: Compounds 10 (57.3 mg, 0.126 mmol) and 26

(82.4 mg, 0.175 mmol) were coevaporated three times with toluene,

dissolved in Et₂O (4.0 mL) and cooled to 08C. Freshly activated powdered

4 ä molecular sieves (140 mg), SnCl₂(28.4 mg, 0.152 mmol) and AgClO₄

(31.5 mg, 0.152 mmol) were added to this mixture. After stirring for 90 min

at 08C, the mixture was warmed to 128C over 22 h, filtered through a pad of

Celite, washed with sat. NaHCO₃and brine. The organic layer was dried

over Na₂SO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 25:1 ! 1:1)

afforded 27 (87.7 mg, 0.097 mmol, 77%) as a pale yellow oil. ¹H NMR

(300 MHz, CDCl₃): d 8.06 8.03 (m, 2H), 7.92 7.89 (m, 2H), 7.61 7.04

(m, 13H), 5.71 5.59 (m, 1H), 5.57 (d, J 3.5 Hz), 5.36 (dd, J 8.6, 7.3 Hz),

5.23 (dd, J 8.6, 7.6 Hz), 4.93 4.60 (m, 7H), 4.48 (d, J 7.8 Hz), 4.40 4.25

(m, 2H), 4.15 3.80 (m, 7H), 3.65 3.18 (m, 5H), 2.11 (s, 3H, CH₃), 1.98

1.84 (m, 2H), 1.67 1.53 (m, 2H), 0.91 (s, 9H), 0.90 (s, 9H), 0.01 (s, 3H).

The residue was dissolved in methanol (280 mL), 4n NaOH (42 mL) was

added and the mixture was stirred overnight at room temperature. The

mixture was acidified with conc. HCl and extracted five times with CH₂Cl₂.

The combined organic phases were dried over Na₂SO₄, filtered and the

solvents were removed in vacuo.

The residue was dissolved in anhydrous DMF (200 mL) and powdered

KHCO₃(27.4 g, 274 mmol) and methyl iodide (17 mL, 273 mmol) were

added. After stirring at room temperature overnight, the mixture was

poured into Et₂O and extracted twice with water, saturated Na₂SO₃, and

water. The organic phase was dried over Na₂SO₄, filtered and the solvents

were removed in vacuo to afford 22 (44 g, 130 mmol, 65%) as a slightly

yellow oil. ¹H NMR (300 MHz, CDCl₃): d 7.40 7.27 (m, 5H), 6.04 (d, J

3.9 Hz, 1H), 4.70 4.52 (m, 4H), 4.41 (dd, J 6.2, 3.8 Hz, 1H), 4.16 (d, J

Chem. Eur. J. 2003, 9, No. 1

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153

P. H. Seeberger et al.

FULL PAPER

n-Pentenyl (2-azido-3,6-di-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-

a/b-d-glucopyranosyl)-(1 ! 4)-methyl 2-O-benzoyl-3-O-benzyl-b-d-gluco-

pyranosyluronate (28): Tin(ii) triflate (140 mg, 0.336 mmol) was added to

freshly activated powdered 4 ä molecular sieves (100 mg) under argon in a

glove box. Et₂O (2.0 mL) was added and the reaction mixture was cooled to

À108C before di-tert-butylpyridine (64 mg, 0.33 mmol) was added to the

reaction. Compound 15 (25.6 mg, 0.051 mmol) and 26 (31.8 mg,

0.068 mmol) were coevaporated three times with toluene and dissolved

in Et₂O (1.0 mL). The resulting solution was added dropwise to the reaction

mixture at À108C. The reaction was stirred for one hour at À108C and

then for three hours at room temperature. After dilution with CH₂Cl₂and

filtration through a pad of Celite, the solution was quenched with sat.

NaHCO₃. The organic layer was dried over Na₂SO₄and the solvent was

removed under reduced pressure. Flash chromatography on silica gel

(hexanes/EtOAc 20:1 ! 3:1) afforded 28 (21.1 mg, 0.022 mmol, 43%) as a

pale yellow oil. ¹H NMR (300 MHz, CDCl₃): d 7.92 7.89 (m, 2H), 7.60

7.15 (m, 8H), 5.71 5.60 (m, 1H), 5.59 (d, J 3.6 Hz), 5.38 (t, J 7.8 Hz),

5.26 (t, J 7.5 Hz), 4.84 4.47 (m, 9H), 4.39 4.23 (m, 1H), 4.14 4.01 (m,

2H), 3.93 3.56 (m, 8H), 3.52 3.34 (m, 2H), 3.28 (dd, J 3.6, 10.2 Hz),

3.19 (at, J 8.7 Hz, 1H), 2.02 1.93 (m, 2H), 1.66 1.54 (m, 2H), 0.88 (s,

9H), 0.86 (s, 9H), 0.05 (s, 3H, CH₃), 0.03 (s, 3H, CH₃).

3H); ¹³C NMR (125 MHz, CDCl₃): d 170.3, 137.2, 128.6, 128.2, 127.8,

111.4, 94.4, 75.8, 75.5, 73.7, 72.4, 67.0, 52.2, 28.0, 25.9; FAB MS: m/z: calcd

for C₁₇H₂₂O₇: 338.1366; found: 338.1377 [M] . Further elution (hexanes/

EtOAc 7:3) afforded 32 (1.1 g, 32%) as a colorless oil.

Methyl

3-O-benzyl-1,2-O-cyclopentylidene-a-d-glucopyranosyluronate

(33) and methyl 3-O-benzyl-1,2-O-cyclopentylidene-a-d-glucofuranosylur-

onate (34): Compound 23 (2.94 g, 9.87 mmol) was dissolved in DMF

(10 mL) and methoxycyclopentene (7.1 g, 72 mmol) and cooled to 08C. A

solution of (1S)-()-camphorsulfonic acid (244 mg, 1.05 mmol) in DMF

(2 mL) was added and stirring was continued at 08C for 1 h and at room

temperature overnight. Methanol (5 mL) was added and the mixture was

stirred for 30 min at room temperature. Triethylamine (3 mL) was added

and the mixture was concentrated. The residue was dissolved in Et₂O and

washed with water and twice with brine. The organic layer was dried over

Na₂SO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography on silica gel (toluene/EtOAc 98.5:1.5)

afforded 33 (2.05 g, 57%) as a colourless oil. [a]²_D⁴ 33.9 (c 1.48,

CH₂Cl₂); IR (thin film on NaCl): nÄ 3510, 3032, 2955, 2873, 1750, 1454,

1436, 1206 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.37 7.27 (m, 5H), 5.84

(d, J 2.8 Hz, 1H), 4.66 (d, J 11.6 Hz, 1H), 4.57 (d, J 11.9 Hz, 1H), 4.52

(d, J 4.3 Hz, 1H), 4.23 4.19 (m, 1H), 4.04 (dd, J 3.0, 2.4 Hz, 1H), 4.00

(dd, J 3.4, 3.1 Hz, 1H), 3.63 (s, 3H), 3.40 (d, J 9.5 Hz, 1H), 2.14 2.08

(m, 1H), 1.92 1.86 (m, 1H), 1.79 1.62 (m, 6H); ¹³C NMR (125 MHz,

CDCl₃): d 170.5, 137.3, 128.6, 128.2, 127.8, 94.2, 75.9, 75.0, 74.2, 72.4, 67.4,

52.3, 37.7, 36.8, 23.5, 23.2; FAB MS: m/z: calcd for C₁₉H₂₄O₇: 364.1522;

Dimethylthexylsilyl (3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-

2-deoxy-a-d-glucopyranosyl)-(1! 4)-methyl 3-O-benzyl-b-d-idopyranosyl-

uronate (30)

By coupling 14 and 29: Compound 14 (100 mg, 0.18 mmol) and 29 (119 mg,

0.27 mmol) were coevaporated with toluene (3 Â ) and dissolved in CH₂Cl₂

(50 mL). Freshly activated powdered 4 ä molecular sieves (100 mg) were

added and the mixture was stirred at room temperature for 1 h. The

reaction mixture was cooled to À788C and tert-butyldimethylsilyl trifluor-

omethanesulfonate (72 mL, 0.314 mmol) was added dropwise. The mixture

was warmed to room temperature over 2.5 h. Triethylamine (3 mL) was

added, the mixture was filtered through a pad of Celite and the solvent was

removed under reduced pressure. Flash chromatography on silica gel

(toluene/EtOAc 99:1 ! 9:1) afforded 30 (71 mg, 0.09 mmol, 47%), both as

a colorless foam.

found: 364.1534 [M] . Further elution (toluene/EtOAc 9:1) afforded 34

(1.04 g, 2.85 mmol, 29%) as a colorless oil. [a]²_D⁴ 0.7 (c 1.37, CH₂Cl₂);

IR (thin film on NaCl): nÄ 3470, 2955, 2875, 1738, 1455, 1208 cm^À1

;

¹H NMR (400 MHz, CDCl₃): d 7.37 7.27 (m, 5H), 5.98 (d, J 4.0 Hz,

1H), 4.66 (d, J 11.5 Hz, 1H), 4.63 4.52 (m, 3H), 4.43 (dd, J 6.0, 4.0 Hz,

1H), 4.16 (d, J 4.0 Hz, 1H), 3.73 (s, 3H), 3.36 (brs, 1H), 2.00 1.93 (m,

1H), 1.86 1.76 (m, 1H), 1.74 1.60 (m, 6H); ¹³C NMR (100 MHz, CDCl₃):

d 173.1, 136.9, 128.7, 128.3, 128.1, 121.9, 105.3, 83.3, 82.7, 80.1, 72.8, 70.1,

52.5, 37.1, 36.7, 23.4, 23.2; FAB MS: m/z: calcd for C₁₉H₂₄O₇: 364.1522;

found: 364.1530 [M] .

Methyl 3-O-benzyl-1,2-O-isopropylidene-b-l-idopyranosyluronate (35):

Methyl 3-O-benzyl-l-idopyranosyluronate (25; 1.7 g, 5.70 mmol) was

dissolved in DMF (6 mL) and 2-methoxypropene (10.7 mL, 114 mmol)

was added. The mixture was cooled at 08C and (1S)-()-camphorsulfonic

acid (132 mg, 0.57 mmol) in DMF (2 mL) was added dropwise under

stirring. The mixture was stirred for 6 h at 08C, then methanol (2 mL) was

added and stirred for 30 min at 08C before the reaction was quenched by

addition of triethylamine After dilution with EtOAc, the solution was

washed with water. The organic layer was dried over MgSO₄and

evaporated. Flash chromatography on silica gel (toluene/EtOAc 99:1 !

96:4) afforded 35 (1.3 g, 3.84 mmol, 68%) as a yellow oil. [a]²_D⁴ À29.5

By coupling 13 and 29: Compound 13 (100 mg, 0.250 mmol) and 29

(154 mg, 0.350 mmol) were coevaporated three times with toluene,

dissolved in Et₂O (1.5 mL) and cooled to 08C. Freshly activated powdered

4 ä molecular sieves (100 mg), SnCl₂(48 mg, 0.250 mmol) and AgClO₄

(52 mg, 0.250 mmol) were added to this mixture. After stirring for 5 h at

08C the mixture was warmed to 128C over 22 h, filtered through a pad of

Celite, washed with sat. NaHCO₃and brine. The organic layer was dried

over Na₂SO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography on silica gel (toluene/EtOAc 99:1 ! 9:1)

afforded 30 (60 mg, 0.07 mmol, 30%) as a colorless foam. ¹H NMR

(500 MHz, CDCl3): d 7.38 7.35 (m, 5H), 5.19 (dd, J 8.0, 10.8 Hz, 1H),

5.11 (d, J 3.6 Hz, 1H), 5.04 (d, J 0.8 Hz, 1H), 4.61 (d, 3H), 4.37 (d, 1H),

4.13 4.06 (m, 2H), 3.82 (s, 3H), 3.81 3.71 (m, 3H), 3.63 (brs, 1H), 3.24

(dd, J 3.3, 10.4 Hz, 1H), 2.80 (brs, 1H), 2.12 (s, 3H), 2.10 (s, 3H), 1.98 (s,

3H), 0.90 0.84 (m, 7H), 0.24 (s, 3H), 0.18 (s, 3H), 0.05 (s, 3H), 0.03 (s,

3H); ¹³C NMR (125 MHz, CDCl₃): d 170.6, 169.5, 169.4, 137.2, 128.7,

128.3, 127.8, 94.5, 73.9, 73.1, 72.9, 72.6, 70.6, 69.8, 69.0, 68.5, 62.6, 61.8, 52.5,

34.3, 25.8, 25.2, 21.6, 21.2, 20.6, 20.3, 18.9, 18.7, 18.1, À1.7, À3.0, À3.7, À4.7;

(c 1.00, CHCl₃); IR (thin film on NaCl): nÄ 2937, 1764, 1374, 843 cm^À1

;

¹H NMR (500 MHz, CDCl₃): d 7.40 7.30 (m, 5H), 5.35 (d, J 1.9 Hz,

1H), 4.71 (d, J 11.7 Hz, 1H), 4.63 (d, J 11.7 Hz, 1H), 4.49 (s, 1H), 4.12

4.10 (m, 1H), 4.0 (d, J 1.8 Hz, 1H), 3.97 (dd, J 1.9, 3.7 Hz, 1H), 3.80 (s,

3H), 3.12 (d, J 11.6 Hz, 1H), 1.63 (s, 3H), 1.38 (s, 3H); ¹³C NMR

(125 MHz, CDCl₃): d 169.4, 137.1, 128.8, 128.5, 128.0, 112.1, 96.5, 75.5,

73.4, 72.9, 72.2, 67.4, 52.6, 28.4, 25.7; FAB MS: m/z: calcd for C₁₇H₂₂O₇:

338.1365; found: 338.1357. Further elution (toluene/EtOAc 9:1) afforded

36 (382 mg, 20%) as a colorless oil.

FAB MS : m/z: calcd for C₃₈H₆₃N₃O₁₃Si₂: 825.3899; found: 825.3888 [M] .

Methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glucopyranosyluronate (31):

Compound 23 (3.02 g, 10.14 mmol) was dissolved in DMF (10 mL) and

2-methoxypropene (10 mL, 100 mmol) was added and cooled to 08C. A

solution of (1S)-()-camphorsulfonic acid (230 mg, 1 mmol) in DMF

(2 mL) was added and stirring was continued at 08C for 1 h and at room

temperature overnight. Methanol (15 mL) was added and the mixture was

stirred for 3 h at room temperature. Triethylamine (3 mL) was added and

the mixture was concentrated. The residue was dissolved in Et₂O and

washed with water and twice with brine. The organic layer was dried over

Na₂SO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 9:1) afforded

31 (1.65 g, 4.88 mmol, 48%) as a colorless oil. [a]²_D⁴ 22.3 (c 1.13,

CH₂Cl₂); IR (thin film on NaCl): nÄ 3520, 3037, 2987, 1752, 1455, 1169,

1105 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.34 7.26 (m, 5H), 5.87 (d, J

2.7 Hz, 1H), 4.63 (d, J 11.9 Hz, 1H), 4.54 (d, J 11.6 Hz, 1H), 4.54 (d, J

3.7 Hz, 1H), 4.24 4.20 (m, 1H), 4.11 4.09 (m, 1H), 4.00 (dd, J 3.1,

3.4 Hz, 1H), 3.58 (s, 3H), 3.51 (d, J 10.4 Hz, 1H), 1.60 (s, 3H), 1.37 (s,

Methyl 3-O-benzyl-1,2-O-cyclopentylidene-b-l-idopyranosyluronate (37)

and methyl 3-O-benzyl-1,2-O-cyclopentylidene-b-l-idofuranosyluronate

(38): A mixture of (1S)-()-camphorsulfonic acid (77 mg, 0.33 mmol)

and methoxycyclopentylidene (3.3 g, 33.5 mmol) in DMF (2 mL) was

cooled to 08C. A solution of 25 (1.0 g, 3.35 mmol) in DMF (1 mL) was

added dropwise under stirring. The mixture was stirrred for 3 h at 08C and

overnight at room temperature, after which methanol (2 mL) was added

and stirred 30 minutes. The reaction was quenched by adding triethylamine.

After dilution with EtOAc, the solution was washed with water and dried

over MgSO₄. After filtration the solvent was removed under reduced

pressure and the syrup was purified by flash chromatography on silica gel

(toluene/EtOAc 99:1 ! 98:2) to yield 37 (684 mg, 1.88 mmol, 56%) as a

yellow syrup. [a]²_D⁴ 10.9 (c 1.00, CHCl₃); IR (thin film on NaCl): nÄ

3534, 2954, 2111, 1764, 1437, 1336, 1120 cm^À1; ¹H NMR (500 MHz, CDCl₃):

d 7.40 7.30 (m, 5H), 5.35 (d, J 1.9 Hz, 1H), 4.70 (d, J 11.7 Hz, 1H),

4.63 (d, J 11.7 Hz, 1H), 4.50 (s, 1H), 4.11 4.09 (m, 1H), 4.07 (d, J

154

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Heparin Oligosaccharides

140 169

1.6 Hz, 1H), 3.88 (dd, J 2.1, 3.6 Hz, 1H), 3.80 (s, 3H), 3.10 (d, J 11.9 Hz,

1H), 2.14 2.08 (m, 1H), 2.23 2.16 (m, 1H), 1.86 1.61 (m, 6H), ¹³C NMR

(125 MHz, CDCl₃): d 169.4, 137.2, 128.8, 128.5, 128.0, 121.5, 96.1, 75.7,

73.5, 72.9, 72.1, 67.4, 52.6, 38.3, 36.6, 23.5, 23.4; FAB MS: m/z: calcd for

warmed to 128C over 8 h. The mixture was filtered through a pad of Celite,

washed with sat. NaHCO₃and brine. The organic layer was dried over

Na₂SO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 95:5 ! 9:1)

afforded 40 (693 mg, 0.87 mmol, 79%) as a colorless foam. [a]²_D⁴ 87.5

(c 1.20, CH₂Cl₂); IR (thin film on NaCl): nÄ 3025, 2954, 2849, 2105, 1746,

1455, 1250 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.40 7.27 (m, 10H), 5.74

(d, J 4.0 Hz, 1H), 5.15 (d, J 3.4 Hz, 1H), 4.87 (d, J 11.0 Hz, 1H), 4.79

(d, J 11.0 Hz, 1H), 4.70 (d, J 11.6 Hz, 1H), 4.67 (d, J 11.9 Hz, 1H),

4.59 (d, J 6.7 Hz, 1H), 4.38 (dd, J 2.1, 11.9 Hz, 1H), 4.26 (dd, J 3.1,

6.7 Hz, 1H), 4.14 (dd, J 3.4, 4.0 Hz, 1H), 4.08 4.04 (m, 2H), 3.89 3.84

(m, 1H), 3.74 (dd, J 8.5, 10.4 Hz, 1H), 3.71 (s, 3H), 3.64 (dd, J 9.2,

8.9 Hz, 1H), 3.24 (dd, J 3.7, 10.4 Hz, 1H), 2.08 2.00 (m, 5H), 1.79 1.62

(m, 6H), 0.89 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H); ¹³C NMR (125 MHz,

CDCl₃): d 171.0, 170.2, 138.1, 137.3, 128.7, 128.5, 128.2, 128.0, 127.8, 127.5,

120.6, 98.0, 95.5, 80.0, 75.7, 75.3, 75.2, 74.0, 72.3, 71.4, 71.2, 71.2, 63.6, 62.9,

52.6, 37.0, 36.9, 26.0, 23.8, 23.3, 21.1, 18.1, À3.5, À4.8; FAB MS: m/z: calcd

C₁₉H₂₄O₇: 364.1522; found: 364.1530 [M] .

Further elution (toluene/EtOAc 9:1) afforded 38 (219 mg, 0.6 mmol, 18%)

as a colorless oil. [a]²_D⁴ À13.9 (c 1.00, CHCl₃); IR (thin film on NaCl):

nÄ 3485, 2954, 2874, 1738, 1453, 1338, 1120 cm^À1

;

¹H NMR (500 MHz,

CDCl₃): d 7.38 7.30 (m, 5H), 5.97 (d, J 4.1 Hz, 1H), 4.74 (d, J

11.6 Hz, 1H), 4.64 (dd, J 1.2, 4.1 Hz, 1H), 4.57 4.50 (m, 2H), 4.53 (d,

J 11.4 Hz, 1H), 4.20 (dd, J 1.5, 4.7 Hz, 1H), 3.74 (s, 3H), 3.30 (d, J

3.3 Hz, 1H), 2.00 1.91 (m, 1H), 1.81 1.65 (m, 6H); ¹³C NMR (100 MHz,

CDCl₃): d 172.3, 137.0, 128.7, 128.3, 128.1, 122.2, 105.1, 83.5, 83.0, 80.4,

72.5, 70.1, 52.8, 37.3, 37.1, 23.4, 23.3; FAB MS: m/z: calcd for C₁₉H₂₄O₇:

364.1522; found: 364.1534 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glu-

copyranosyluronate (39)

for C₄₀H₅₅N₃O₁₂Si: 797.3555; found: 797.3578 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-d-syluronate (41): Compound

39 (1.68 g, 2.18 mmol) was dissolved in dichloroacetic acid (75% aqueous,

20 mL) and stirred at room temperature for 1 h. The reaction mixture was

added slowly to sat. NaHCO₃and extracted three times with CH₂Cl₂. The

organic layer was dried over Na₂SO₄and after filtration the solvent was

removed under reduced pressure. Flash chromatography on silica gel

(hexanes/EtOAc 1:1) afforded 41 (1.29 g, 1.86 mmol, 81%) colorless foam.

Compound 40 (1.19 g, 1.49 mmol) was dissolved in dichloroacetic acid

(50% aqueous, 15 mL) and stirred at room temperature for 2 h. The

reaction mixture was added slowly to sat. NaHCO₃and extracted three

times with CH₂Cl₂. The organic layer was dried over Na₂SO₄and after

filtration the solvent was removed under reduced pressure. Flash chroma-

tography on silica gel (hexanes/EtOAc 1:1) afforded 41 (980 mg,

1.34 mmol, 90%) as a colorless foam. FAB MS: m/z: calcd for C₃₅H₄₉N₃O_12-

By coupling 11 and 31: Compounds 11 (1.87 g, 3.14 mmol) and 31 (850 mg,

2.51 mmol) were coevaporated three times with toluene and dissolved in

CH₂Cl₂(50 mL). Freshly activated powdered 4 ä molecular sieves (1 g)

were added and the mixture was stirred at room temperature for 1 h. The

reaction mixture was cooled to À788C and tert-butyldimethylsilyl trifluor-

omethanesulfonate (72 mL, 0.314 mmol) was added dropwise. The mixture

was warmed to room temperature over 2.5 h. Triethylamine (3 mL) was

added, the mixture was filtered through a pad of Celite and the solvent was

removed under reduced pressure. Flash chromatography on silica gel

(hexanes/EtOAc 85:15) afforded 39 (1.68 g, 2.18 mmol, 86%) as a colorless

foam.

By coupling 10 and 31: Compounds 10 (540 mg, 1.19 mmol) and 31 (362 mg,

1.07 mmol) were coevaporated three times with toluene, dissolved in Et₂O

(20 mL) and cooled to 08C. To this mixture freshly activated 4 ä molecular

sieves (1 g), SnCl₂(229 mg, 1.21 mmol) and AgClO₄(250 mg, 1.21 mmol)

were added. This mixture was stirred for 90 min at 08C, then warmed to

128C over 22 h. The mixture was filtered through a pad of Celite, washed

with sat. NaHCO₃and brine. The organic layer was dried over Na₂SO₄and

after filtration the solvent was removed under reduced pressure. Flash

chromatography on silica gel (hexanes/EtOAc 9:1) afforded 39 (660 mg,

0.86 mmol, 80%) as a colorless foam. [a]²_D⁴ 97.2 (c 2.55, CH₂Cl₂); IR

(thin film on NaCl): nÄ 3032, 2953, 2930, 2858, 2106, 1746, 1455, 1372,

Si: 731.3086; found: 731.3107 [M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glucopyranosyluronate (42):

Compound 20 (946 mg, 1.65 mmol) and 31 (451 mg, 1.33 mmol) were

coevaporated three times with toluene and dissolved in CH₂Cl₂(30 mL).

Freshly activated powdered 4 ä molecular sieves (700 mg) were added and

the mixture was stirred at room temperature for 1 h. The mixture was

cooled to À788C and tert-butyldimethylsilyl trifluoromethanesulfonate

(38 mL, 0.165 mmol) was added dropwise. The reaction mixture was

warmed to room temperature and stirred overnight. Triethylamine

(3 mL) was added and the mixture was filtered through a pad of Celite

and the solvent was removed under reduced pressure. Flash chromatog-

raphy (hexanes/EtOAc 8:2) afforded 42 (828 mg, 1.11 mmol, 83%) as a

colorless oil. [a]²_D⁴ 58.0 (c 1.53, CH₂Cl₂); IR (thin film on NaCl): nÄ

1

1250 cm^À1; H NMR (500 MHz, CDCl₃): d 7.40 7.27 (m, 10H), 5.78 (d,

J 3.7 Hz, 1H), 5.17 (d, J 3.7 Hz, 1H), 4.88 (d, J 11.0 Hz, 1H), 4.78 (d,

J 11.0 Hz, 1H), 4.68 (s, 2H), 4.59 (d, J 6.1 Hz, 1H), 4.38 (dd, J 2.1,

11.9 Hz, 1H), 4.25 4.21 (m, 2H), 4.09 4.05 (m, 2H), 3.89 3.85 (m, 1H),

3.74 (dd, J 8.5, 10.4 Hz, 1H), 3.71 (s, 3H), 3.63 (dd, J 8.5, 9.8 Hz, 1H),

3.25 (dd, J 3.7, 10.4 Hz, 1H), 2.08 (s, 3H), 1.63 (s, 3H), 1.39 (s, 3H), 0.89 (s,

9H), 0.02 (s, 3H), 0.00 (s, 3H); ¹³C NMR (MHz, CDCl₃): d 170.9, 170.1,

138.1, 137.3, 128.6, 128.4, 128.2, 128.0, 127.7, 127.5, 111.0, 98.2, 95.7, 80.0, 76.0,

75.6, 75.1, 73.9, 72.3, 71.9, 71.3, 71.2, 63.6, 62.9, 52.5, 27.6, 26.0, 25.9, 21.0,

18.1, À3.6, À4.8; FAB MS: m/z: calcd for C₃₈H₅₃N₃O₁₂Si: 771.3399; found:

1

3029, 2938, 2017, 1743, 1454 cm^À1; H NMR (500 MHz, CDCl₃): d 7.41

7.27 (m, 15H), 5.72 (d, J 4.0 Hz, 1H), 5.14 (d, J 3.7 Hz, 1H), 4.90 (d, J

10.7 Hz, 1H), 4.87 (d, J 10.7 Hz, 1H), 4.86 (d, J 11.0 Hz, 1H), 4.70 (s,

2H), 4.59 (d, J 11.0 Hz, 1H), 4.48 (d, J 7.3 Hz, 1H), 4.30 (dd, J 2.4,

11.9 Hz, 1H), 4.28 4.22 (m, 3H), 4.06 (at, J 3.7 Hz, 1H), 4.00 3.92 (m,

2H), 3.73 (s, 3H), 3.54 (dd, J 8.8, 10.1 Hz, 1H), 3.30 (dd, J 3.7, 10.4 Hz,

1H), 2.05 (s, 3H), 1.62 (s, 3H), 1.39 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d

171.4, 170.7, 138.4, 138.2, 137.9, 129.3, 129.2, 128.8, 128.7, 128.7, 128.6,

128.5, 111.7, 98.3, 96.7, 80.5, 78.5, 77.1, 76.3, 76.1, 75.7, 74.4, 72.8, 72.1, 70.6,

771.3386 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-cyclopentylidene-a-d-

glucopyranosyluronate (40)

By coupling 11 and 33: Compounds 11 (870 mg, 1.46 mmol) and 33 (425 mg,

1.17 mmol) were coevaporated three times with toluene and dissolved in

CH₂Cl₂(20 mL). Freshly activated powdered 4 ä molecular sieves

(500 mg) were added and the mixture was stirred at room temperature

for 30 min. The mixture was cooled to À258C and tert-butyldimethylsilyl

trifluoromethanesulfonate (33 mL, 0.146 mmol) was added dropwise. The

reaction mixture was warmed to room temperature and stirred overnight.

Triethylamine (3 mL) was added, the mixture was filtered through a pad of

Celite and the solvent was removed under reduced pressure. Flash

chromatography on silica gel (hexanes/EtOAc 95:5 ! 9:1) afforded 40

(744 mg, 0.93 mmol, 80%) as a colorless foam.

63.9, 63.2, 53.2, 28.0, 26.5, 21.5; FAB MS: m/z: calcd for C₃₉H₄₅N₃O₁₂

747.3003; found: 747.3001 [M] .

:

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl

3-O-benzyl-1,2-O-cyclopentylidene-a-d-glucopyranosyluronate

(43): Compound 20 (855 mg, 1.50 mmol) and 33 (436 mg, 1.20 mmol) were

coevaporated three times with toluene and dissolved in CH₂Cl₂(20 mL).

Freshly activated powdered 4 ä molecular sieves (250 mg) were added and

the mixture was stirred at room temperature for 1 h. The mixture was

cooled to À208C and tert-butyldimethylsilyl trifluoromethanesulfonate

(100 mL, 0.44 mmol) was added dropwise. The reaction mixture was stirred

for 2.5 h at À208C. Triethylamine (3 mL) was added, the mixture was

filtered through a pad of Celite and the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 85:15 ! 8:2)

afforded 43 (760 mg, 0.98 mmol, 82%) as a colorless oil. [a]²_D⁴ 62.3 (c

By coupling 10 and 33: Compounds 10 (610 mg, 1.35 mmol) and 33 (402 mg,

1.10 mmol) were coevaporated three times with toluene, dissolved in Et₂O

(20 mL) and cooled to 08C. To this mixture freshly activated powdered 4 ä

molecular sieves (1 g), SnCl₂(260 mg, 1.37 mmol) and AgClO₄(290 mg,

1.40 mmol) were added. The mixture was stirred for 8 h at 08C, then

1.07, CH₂Cl₂); IR (thin film on NaCl): nÄ 2948, 2107, 1743, 1454, 1362 cm^À1

;

Chem. Eur. J. 2003, 9, No. 1

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155

P. H. Seeberger et al.

FULL PAPER

¹H NMR (500 MHz, CDCl₃): d 7.40 7.27 (m, 15H), 5.69 (d, J 4.0 Hz,

1H), 5.12 (d, J 3.7 Hz, 1H), 4.90 (d, J 10.7 Hz, 1H), 4.87 (d, J 10.7 Hz,

1H), 4.86 (d, J 11.3 Hz, 1H), 4.70 (s, 2H), 4.60 (d, J 11.0 Hz, 1H), 4.48

(d, J 7.3 Hz, 1H), 4.31 (dd, J 2.1, 12.2 Hz, 1H), 4.28 4.24 (m, 2H), 4.18

(dd, J 3.7, 4.0 Hz, 1H), 4.06 (at, J 3.4 Hz, 1H), 4.01 3.93 (m, 2H), 3.74

(s, 3H), 3.55 (dd, J 8.8, 10.0 Hz, 1H), 3.29 (dd, J 3.7, 10.4 Hz, 1H),

2.09 2.03 (m, 5H), 1.79 1.68 (m, 6H); ¹³C NMR (125 MHz, CDCl₃): d

171.4, 170.7, 138.4, 138.2, 137.9, 129.3, 129.2, 128.8, 128.7, 128.7, 128.6,

128.5, 121.2, 98.1, 96.4, 80.5, 78.5, 76.8, 76.1, 76.0, 75.7, 74.5, 72.8, 71.6, 70.6,

63.8, 63.2, 53.2, 37.4, 37.3, 24.4, 23.8, 21.5; FAB MS: m/z: calcd for

(t, J 3.0 Hz, 1H), 3.85 3.82 (m, 1H), 3.77 3.71 (m, 2H), 3.67 3.64 (A

part of ABX, J 4.5, 10.5 Hz, 1H), 3.63 3.61 (m, 4H), 3.27 (dd, J 3.5,

10.0 Hz, 1H), 1.64 (s, 3H), 1.40 (s, 3H), 0.82 (s, 9H), 0.02 (s, 3H), À0.02 (s,

3H); ¹³C NMR (125 MHz, CDCl₃): d 170.2, 138.2, 137.4, 128.7, 128.5,

128.4, 128.2, 128.0, 127.7, 127.8, 127.6, 127.5, 111.1, 98.6, 95.5, 80.2, 75.7, 75.6,

74.8, 73.9, 73.5, 72.7, 72.4, 72.3, 71.0, 68.7, 63.7, 52.4, 27.8, 26.1, 18.2, À3.58,

À4.68; ES MS: m/z: calcd for C₄₃H₅₇N₃NaO₁₁Si: 842.3654; found: 842.3638

[MNa] .

3-O-Acetyl-2-azido-6-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-l-glu-

copyranosyluronate (47): Compound 17 (49 mg, 0.09 mmol) and 31

(23 mg, 0.07 mmol) were coevaporated three times with toluene and

dissolved in CH₂Cl₂(0.7 mL). Freshly activated powdered 4 ä molecular

sieves (23 mg) were added and the mixture was stirred at room temperature

for 1 h. The mixture was cooled to À258C and tert-butyldimethylsilyl

trifluoromethanesulfonate (0.1m in CH₂Cl₂, 68 mL, 0.007 mmol) was added

dropwise. The reaction mixture was warmed to room temperature and

stirred for 30 minutes. Triethylamine was added and the mixture was

filtered through a pad of Celite and the solvent was removed under reduced

pressure. Flash chromatography (hexanes/EtOAc 4:1) afforded 47 (37 mg,

0.05 mmol, 71%) as a colorless oil. [a]²_D⁴ 93.3 (c 1.7, CHCl₃); IR (thin

C₄₁H₄₇N₃O₁₂: 773.3160; found: 773.3179 [M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

methyl-3-O-benzyl-d-glucopyranosyluronate (44): From 42: Compound 42

(315 mg, 0.42 mmol) was dissolved in dichloroacetic acid (75% aqueous,

4 mL) and stirred at room temperature for 2 h. The reaction mixture was

added slowly to sat. NaHCO₃and extracted three times with CH₂Cl₂. The

organic layer was dried over Na₂SO₄and after filtration the solvent was

remove under reduced pressure. Flash chromatography on silica gel

(hexanes/EtOAc 1:1) afforded 44 (250 mg, 0.35 mmol, 84%) as a colorless

foam. From 43: Compound 43 (836 mg, 1.08 mmol) was dissolved in

dichloroacetic acid (50% aqueous, 12 mL) and stirred at room temperature

for 2 h. The reaction mixture was added slowly to sat. NaHCO₃and

extracted three times with CH₂Cl₂. The organic layer was dried over

Na₂SO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography (hexanes/EtOAc 1:1) afforded 44 (619 mg,

film on NaCl): nÄ 2928, 2110, 1751, 1224 cm^À1

;

¹H NMR (500 MHz,

CDCl₃): d 7.37 7.26 (m, 10H), 5.77 (d, J 4.0 Hz, 1H), 5.34 (dd, J 7.5,

10.5, 1H), 5.23 (d, J 3.5 Hz, 1H), 4.69 4.66 (A part of AB system, J

11.5 Hz, 1H), 4.66 4.64 (B part of AB system, J 11.5 Hz, 1H), 4.62 (d,

J 6.5 Hz, 1H), 4.58 4.56 (A part of AB system, J 12.5 Hz, 1H), 4.53

4.50 (B part of AB system, J 12.5 Hz, 1H), 4.24 (dd, J 4.0, 6.0 Hz, 1H),

4.20 (t, J 3.5 Hz, 1H), 4.10 (t, J 3.0 Hz, 1H), 3.88 3.80 (m, 2H), 3.73

3.70 (A part of ABX, J 3.5, 10.5 Hz, 1H), 3.63 (s, 3H), 3.62 3.60 (B part

of ABX, J 2.0, 10.5 Hz, 1H), 2.97 (dd, J 3.5, 10.5 Hz, 1H), 2.14 (s, 3H),

1.62 (s, 3H, CH₃), 1.38 (s, 3H, CH₃), 0.81 (s, 9H), 0.04 (s, 3H, CH₃), 0.03 (s,

3H, CH₃); ¹³C NMR (125 MHz, CDCl₃): d 170.3, 170.1, 138.1, 137.4, 128.7,

128.5, 128.2, 128.0, 127.8, 127.7, 111.2, 98.4, 95.8, 76.3, 75.8, 73.9, 73.6, 72.9,

72.3, 71.9, 69.1, 68.2, 61.4, 52.5, 27.6, 26.1, 25.8, 21.6, 18.2, À4.05, À4.66; ES

0.87 mmol, 81%) as a colorless foam. FAB MS: m/z: calcd for C₃₆H₄₁N₃O₁₂

707.2690; found: 707.2678 [M] .

:

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-l-glucopyra-

nosyluronate (45): Compound 14 (215 mg, 0.39 mmol) and 31 (102 mg,

0.30 mmol) were coevaporated three times with toluene and dissolved in

CH₂Cl₂(3 mL). Freshly activated powdered 4 ä molecular sieves (100 mg)

were added and the mixture was stirred at room temperature for 1 h. The

mixture was cooled to À258C and tert-butyldimethylsilyl trifluorometha-

nesulfonate (0.1m in CH₂Cl₂, 300 mL, 0.030 mmol) was added dropwise.

The reaction mixture was warmed to room temperature and stirred for

30 minutes. Triethylamine (3 mL) was added and the mixture was filtered

through a pad of Celite and the solvent was removed under reduced

pressure. Flash chromatography (hexanes/EtOAc 4:1) afforded 45 (201 mg,

0.279 mmol, 92%) as a colorless oil. [a]²_D⁴ 90 (c 0.7, CHCl₃); IR (thin

MS: m/z: calcd for C₃₈H₅₃N₃NaO₁₂Si: 794.3281; found: 794.3275 [MNa] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-l-ido-

pyranosyluronate (48):

A mixture of 11 (1.38 g, 2.32 mmol) and 35

(627 mg, 1.9 mmol) was coevaporated three times with toluene and dried

under vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(20 mL) and

was stirred for 30 min at room temperature under argon in the presence of

freshly activated powdered 4 ä molecular sieves (800 mg). After cooling

the mixture to À308C, tert-butyldimethylsilyl trifluoromethanesulfonate

(1m in CH₂Cl₂, 230 mL, 0.23 mmol) was added dropwise. The mixture was

warmed to room temperature over 1 h. Triethylamine (4 mL) was added,

the mixture was filtered through a pad of Celite and the solvent was

removed under reduced pressure. The residue was purified by silica gel

column chromatography (toluene/EtOAc 95:5 ! 92:8) to yield 48 (1.30 g,

1.68 mmol, 91%) as a colorless glass. [a]²_D⁴ 93.3 (c 1, CHCl₃); IR (thin

film on NaCl): nÄ 2890, 2100, 1740, 1020 cm À 1; ¹H NMR (500 MHz,

CDCl₃): d 7.38 1.20 (m, 10H), 5.28 (d, J 2.4 Hz, 1H), 4.93 (d, J

3.0 Hz, 1H), 4.76 (d, J 11.0 Hz, 1H), 4.65 (d, J 11.0 Hz, 1H), 4.61 (s,

2H), 4.41 4.36 (m, 2H), 4.13 (t, J 2.1 Hz, 1H), 3.99 (s, 1H), 3.95 (dd, J

3.0, 12.2 Hz, 1H), 3.91 (m, 2H), 3.74 3.71 (m, 1H), 3.71 (s, 3H), 3.61 3.59

(m, 2H), 3.30 (dd, J 3.3, 9.5 Hz, 1H), 2.08 (s, 3H), 1.98 (s, 3H), 1.57 (s,

3H), 1.33 (s, 3H), 0.78 (s, 9H), À0.10 (s, 6H); ¹³C NMR (125 MHz, CDCl₃):

d 170.8, 169.0, 138.2, 137.1, 128.8, 128.4, 128.0, 127.9, 127.7, 127.6, 112.2,

98.1, 97.1, 80.1, 97.3, 75.4, 75.2, 73.5, 72.9, 72.8, 71.5, 71.3, 70.6, 68.9, 64.6,

62.5, 52.5, 28.3, 26.3, 26.0, 21.1, 18.1, À3.6, À5.0; FAB MS: m/z: calcd for

1

film on NaCl): nÄ 2932, 2110, 1747, 1372, 1221 cm^À1; H NMR (500 MHz,

CDCl₃): d 7.35 7.26 (m, 5H), 5.73 (d, J 4.0 Hz, 1H), 5.32 (dd, J 8.5,

10.5 Hz, 1H), 5.19 (d, J 4.0 Hz, 1H), 4.68 4.66 (A part of AB system, J

11.5 Hz, 1H), 4.65 4.62 (B part of AB system, J 11.5 Hz, 1H), 4.55 (d,

J 6.5 Hz, 1H), 4.38 4.35 (A part of ABX, J 2.0, 12.0 Hz, 1H), 4.22 (dd,

J 4.0, 6.5 Hz, 1H), 4.19 (t, J 3.0 Hz, 1H), 4.10 4.06 (m, 2H), 3.92 3.88

(m, 1H), 3.70 (t, J 9.0 Hz, 1H), 3.68 (s, 3H), 2.95 (dd, J 3.5, 11.0 Hz,

1H), 2.13 (s, 3H), 2.06 (s, 3H), 1.59 (s, 3H), 1.35 (s, 3H), 0.82 (s, 9H), 0.04 (s,

3H), 0.03 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.7, 170.2, 170.0,

137.3, 128.6, 128.2, 128.0, 111.2, 98.1, 95.9, 76.3, 75.7, 74.0, 72.5, 72.3, 71.6,

70.8, 69.4, 62.7, 61.3, 52.5, 27.4, 25.9, 25.7, 21.5, 20.9, À3.96, À4.82; ES MS:

m/z: calcd for C₃₃H₄₉N₃NaO₁₃Si: 746.2927; found: 746.2901 [M À Na] .

2-Azido-3,6-di-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-l-glucopyra-

nosyluronate (46): Compound 16 (39 mg, 0.06 mmol) and 31 (16 mg,

0.05 mmol) were coevaporated three times with toluene and dissolved in

CH₂Cl₂(0.7 mL). Freshly activated powdered 4 ä molecular sieves (16 mg)

were added and the mixture was stirred at room temperature for 1 h. The

mixture was cooled to À258C and tert-butyldimethylsilyl trifluorometha-

nesulfonate (0.1m in CH₂Cl₂, 48 mL, 0.005 mmol) was added dropwise. The

reaction mixture was warmed to room temperature and stirred for

30 minutes. Triethylamine was added and the mixture was filtered through

a pad of Celite and the solvent was removed under reduced pressure. Flash

chromatography (hexanes/EtOAc 6:1) afforded 46 (31 mg, 0.04 mmol,

77%) as a colorless oil. [a]²_D⁴ 86.5 (c 1.1, CHCl₃); IR (thin film on

C₃₈H₅₃N₃O₁₂Si: 771.3399; found: 771.3415 [M] .

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-b-l-idopyra-

nosyluronate (49): A mixture of 14 (500 mg, 0.91 mmol) and 35 (241 mg,

0.71 mmol) was coevaporated three times with toluene and dried under

vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(15 mL) and was

stirred for 30 min at room temperature under argon in the presence of

freshly activated powdered 4 ä molecular sieves (400 mg). After cooling

the mixture to À308C, tert-butyldimethylsilyl trifluoromethanesulfonate

(0.1m in CH₂Cl₂, 1 mL, 0.1 mmol) was added dropwise. The mixture was

warmed to 08C over 1 h. Triethylamine (1.8 mL) was added, the mixture

was filtered through a pad of Celite and the solvent was removed under

NaCl): nÄ 2928, 2105, 1751, 837 cm^À1

;

¹H NMR (500 MHz, CDCl₃): d

7.39 7.25 (m, 15H), 5.83 (d, J 3.5 Hz, 1H), 5.19 (d, J 3.5 Hz, 1H),

4.89 4.86 (A part of AB system, J 11.0 Hz, 1H), 4.78 4.76 (B part of AB

system, J 11.0 Hz, 1H), 4.69 (d, J 5.0 Hz, 1H), 4.66 (s, 2H), 4.58 4.55

(A part of AB system, J 12.0 Hz, 1H), 4.51 4.49 (B part of AB system,

J 12.0 Hz, 1H), 4.24 (dd, J 3.5, 5.0 Hz, 1H), 4.20 (t, J 3.5 Hz, 1H), 4.09

156

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0156 $ 20.00+.50/0

Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

reduced pressure. The residue was purified by silica gel column chroma-

tography (toluene/EtOAc 95:5 ! 92:8) to yield 49 (463 mg, 0.64 mmol,

90%) as a colorless glass. [a]_D²⁴ 87.4 (c 1.00, CHCl₃); IR (thin film on

NaCl): nÄ 2880, 2086, 1731, 1440, 1054 cm^À1; ¹H NMR (500 MHz, CDCl₃):

d 7.38 7.32 (m, 5H), 5.34 (d, J 2.1 Hz, 1H), 5.32 5.36 (dd, 1H), 4.93 (d,

J 3.0 Hz, 1H), 4.70 (d, J 11.6 Hz, 1H), 4.67 (d, J 11.6 Hz, 1H), 4.50

(dd, J 2.1, 12.5 Hz, 1H), 4.42 (d, J 1.5 Hz, 1H), 4.27 (t, J 2.1 Hz, 1H),

4.06 4.02 (m, 2H), 3.98 3.94 (m, 2H), 3.80 3.74 (m, 1H), 3.79 (s, 3H),

3.17 (dd, J 10.7, 3.4 Hz, 1H), 2.08 (s, 3H), 2.00 (s, 3H), 1.64 (s, 3H), 1.39 (s,

3H), 0.81 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d

170.6, 169.8, 169.1, 137.3, 128.8, 128.4, 128.1, 112.5, 99.1, 97.3, 75.7, 74.8,

73.9, 73.0, 72.8, 71.3, 71.3, 68.9, 62.4, 62.3, 52.7, 28.1, 26.2, 25.7, 21.5, 21.1,

18.0, À3.9, À4.9; FAB MS: m/z: calcd for C₃₃H₄₉N₃O₁₃Si: 723.3035; found:

in CH₂Cl₂, 84 mL, 0.084 mmol) was added dropwise. The mixture was

warmed to room temperature over 1 h. Triethylamine was added, the

mixture was filtered through a pad of Celite and the solvent was removed

under reduced pressure. The residue was purified by silica gel column

chromatography (toluene/EtOAc 95:5 ! 92:8) to yield 52 (50 mg,

0.065 mmol, 88%) as a colorless glass. [a]²_D⁴ 85 (c 1.0, CHCl₃); IR

(thin film on NaCl): nÄ 2940, 2106, 1740, 1766, 1017 cm^À1

;

¹H NMR

(500 MHz, CDCl₃): d 7.38 7.26 (m, 10H), 5.32 (m, 2H), 4.97 (d, J

3.0 Hz, 1H), 4.69 (d, J 11.6 Hz, 1H), 4.65 (d, J 11.9 Hz, 1H), 4.60 (d, J

12.2 Hz, 1H), 4.46 (d, J 11.9 Hz, 1H), 4.42 (d, J 1.5 Hz, 1H), 4.29 (t, J

2.7 Hz, 1H), 4.07 (s, 1H), 3.98 (s, 1H), 3.91 3.86 (m, 2H), 3.71 (dd, J 2.4,

11.0 Hz, 1H), 3.68 (s, 3H), 3.63 (dd, J 1.8, 10.4 Hz, 1H), 3.18 (dd, J 10.4,

3.0 Hz, 1H), 2.12 (s, 3H), 1.65 (s, 3H), 1.39 (s, 3H), 0.81 (s, 9H), À0.04 (s,

3H), 0.03 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.0, 169.0, 138.2,

137.3, 128.7, 128.5, 128.4, 128.3, 128.1, 127.9, 112.5, 99.0, 97.3, 75.8, 74.4, 73.7,

73.4, 73.0, 72.9, 72.8, 71.2, 68.7, 67.7, 62.4, 52.4, 28.1, 26.2, 25.8, 21.6, 18.0,

À4.1, À4.8; FAB MS: m/z: calcd for C₃₈H₅₃N₃NaO₁₂Si: 794.3291; found:

723.3058 [M] .

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-cyclopentylidene-b-l-idopyra-

nosyluronate (50): A mixture of 14 (200 mg, 0.36 mmol) and 37 (95 mg,

0.28 mmol) was coevaporated three times with toluene and dried under

vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(5 mL) and was

stirred for 30 min at room temperature under argon in the presence of

freshly activated powdered 4 ä molecular sieves (150 mg). After cooling

the mixture to À308C, tert-butyldimethylsilyl trifluoromethanesulfonate

(0.1m in dry CH₂Cl₂, 0.4 mL, 0.04 mmol) was added dropwise. The mixture

was warmed to 08C over 1 h. Triethylamine (0.7 mL) was added, the

mixture was filtered through a pad of Celite and the solvent was removed

under reduced pressure. The residue was purified by silica gel column

chromatography (toluene/EtOAc 96:3 ! 90:10) to yield 50 (217 mg,

0.29 mmol, 88%) as a colorless glass. [a]²_D⁴ 75.6 (c 1.00, CH₂Cl₂); IR

794.3298 [MNa] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-l-idopyranosyluronate (53): A

solution of 48 (1.20 g, 1.55 mmol) in dichloroacetic acid (40 mL, 60% aq)

was stirred at room temperature for 3 h, diluted with water and neutralized

with NaHCO₃(24 g). The aqueous phase was extracted three times with

CH₂Cl₂. The combined organic phases were dried over MgSO₄. After

filtration the solvent was removed under reduced pressure to afford 53

(1.05 g, 1.4 mmol, 92%) as an essentially pure white solid. Compound 53

can be further purified by silica gel column chromatography (hexanes/

EtOAc 70:30). FAB MS: m/z: calcd for C₃₅H₄₉N₃O₁₂Si: 731.3086; found:

(thin film on NaCl): nÄ 2955, 2858, 2109, 1744, 1455 cm^À1

;

¹H NMR

731.3002 [M] .

(400 MHz, CDCl₃): d 7.38 7.30 (m, 5H), 5.34 5.29 (m, 2H), 4.68 (t, J

12.0 Hz, 2H), 4.51 (m, 1H), 4.46 (d, J 1.5 Hz 1H), 4.28 (dd, J 2.1, 2.9 Hz,

1H), 4.13 (t, 1H), 4.07 4.02 (m, 2H), 3.87 (t, 1H), 3.80 3.74 (m, 1H), 3.77

(s, 3H), 3.07 (dd, J 3.4, 10.6 Hz, 1H), 2.13 (s, 3H), 2.06 (s, 3H), 2.09 2.04

(m, 2H), 1.78 1.63 (m, 6H), 0.82 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H);

¹³C NMR (125 MHz, CDCl₃): d 170.6, 169.8, 169.1, 137.3, 128.7, 128.3,

121.7, 98.9, 96.9, 76.9, 75.8, 74.4, 74.1, 73.0, 72.7, 71.1, 71.0, 68.9, 62.3, 62.1,

52.6, 37.6, 36.9, 25.7, 23.5, 21.5, 21.0, 18.0, À3.9, À4.9; FAB MS: m/z: calcd

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 3-O-benzyl-l-idopyranosyluronate (54)

From 49: A solution of 49 (770 mg, 1.06 mmol) in dichloroacetic acid

(10 mL, 60% aq) was stirred at room temperature for 3 h, diluted with

water and neutralized with NaHCO₃(7 g). The aqueous phase was

extracted three times with CH₂Cl₂and the combined organic phases were

dried over MgSO₄. After filtration the solvent was removed under reduced

pressure to afford 54 (647 mg, 0.95 mmol, 89%) as

a white solid.

for C₃₅H₅₁N₃O₁₃Si: 749.3191; found: 749.3197 [M] .

Compound 54 can be further purified by silica gel column chromatography

(hexanes/EtOAc 70:30). FAB MS: m/z: calcd for C₃₀H₄₅N₃O₁₃Si: 683.2722;

3,6-Di-O-benzyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-b-l-idopyra-

nosyluronate (51): A mixture of 16 (50 mg, 0.08 mmol) and 35 (25 mg,

0.074 mmol) was coevaporated three times with toluene and dried under

vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(1 mL) and was

stirred for 30 min at room temperature under argon in the presence of

freshly activated powdered 4 ä molecular sieves (20 mg). After cooling the

mixture to À308C, tert-butyldimethylsilyl trifluoromethanesulfonate (0.1m

in CH₂Cl₂, 80 mL, 0.08 mmol) was added dropwise. The mixture was

warmed to room temperature over 1 h. Triethylamine was added, the

mixture was filtered through a pad of Celite and the solvent was removed

under reduce pressure. The residue was purified by silica gel column

chromatography (toluene/EtOAc 95:5 ! 92:8) to yield 51 (55 mg,

0.067 mmol, 92%) as a colorless glass. [a]²_D⁴ 91 (c 1, CHCl₃); IR (thin

found: 683.2743 [M] .

From 50: A solution of 50 (200 mg, 0.27 mmol) in dichloroacetic acid

(3 mL, 60% aq) was stirred at room temperature for 3 h, diluted with water

and neutralized with NaHCO₃(7 g). The aqueous phase was washed three

times with CH₂Cl₂. The combined organic phases were dried over MgSO₄.

After filtration the solvent was removed under reduced pressure to afford

54 (160 mg, 0.23 mmol, 88%) as an essentially pure white solid.

6-O-Acetyl-2-azido 3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl-3-O-benzyl-1,2-O-monochloroacetyl-a/b-

d-glucopyranosyluronate (55): Pyridine (2.1 mL, 24 mmol), monochloro-

acetic anhydride (3.0 g, 9.00 mmol) and DMAP (19 mg, 0.157 mmol) were

added to a solution of 41 (1.3 g, 1.57 mmol) in CH₂Cl₂(21 mL). The solution

was stirred at room temperature for 6 h, water was added and the mixture

was stirred for one additional hour. The organic phase was washed with sat.

NaHCO₃, water and aqueous HCl (10%), dried over MgSO₄and filtered.

The solvent was removed under reduced pressure and the residue was

purified by silica gel column chromatography (hexanes/EtOAc 90:20) to

yield 55 (1.5 g, 1.7 mmol, 96%) as a colorless syrup. FAB MS: m/z: calcd for

film on NaCl): nÄ 2935, 2110, 1766, 1017 cm^À1

;

¹H NMR (500 MHz,

CDCl₃): d 7.39 7.28 (m, 15H), 5.35 (d, J 2.4 Hz, 1H), 4.98 (d, J

3.4 Hz, 1H), 4.84 (d, J 11.3 Hz, 1H), 4.73 (d, J 11.3 Hz, 1H), 4.68 (s,

2H), 4.59 (d, J 11.9 Hz, 1H), 4.46 (d, J 11.6 Hz, 1H), 4.45 (d, J 1.5 Hz,

1H), 4.27 (t, J 2.4 Hz, 1H), 4.11 (s, 1H), 3.98 (s, 1H), 3.80 3.73 (m, 2H),

3.71 (s, 3H), 3.80 3.63 (m, 3H), 3.39 (dd, J 10.4, 3.4 Hz, 1H), 1.65 (s,

3H), 1.41 (s, 3H), 0.84 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H); ¹³C NMR

(125 MHz, CDCl₃) d 169.1, 138.3, 138.2, 137.3, 128.8, 128.5, 128.4, 128.1,

127.9, 127.7, 127.6, 127.5, 112.3, 97.9, 97.1, 80.3, 75.5, 74.9, 73.4, 73.0, 72.9, 72.8,

72.4, 71.3, 70.5, 68.1, 64.4, 52.4, 28.2, 26.3, 26.1, 18.1, À3.6, À4.8; FAB MS:

C₃₉H₅₁Cl₂N₃O₁₄Si: 883.2517; found: 883.2506 [M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl 3-O-benzyl-1,2-O-monochloroacetyl-a/b-d-glucopyranosyluronate

(56): Pyridine (0.65 mL, 7.4 mmol), monochloroacetic anhydride (820 mg,

2.5 mmol) and DMAP (7 mg, 0.06 mmol) were added to a solution of 44

(400 mg, 0.6 mmol) in CH₂Cl₂(6.5 mL). The solution was stirred at room

temperature for 6 h, water was added and the mixture was stirred for one

additional hour. The organic phase was washed with saturated solution of

NaHCO₃, water and 10% HCl, dried over MgSO₄and filtered. The solvent

was removed under reduced pressure and the residue was purified by silica

gel column chromatography (hexanes/EtOAc 90:20) to yield 56 (452 mg,

m/z: calcd for C₃₃H₄₉N₃NaO₁₃Si: 842.3654; found: 842.3696 [MNa] .

6-O-Benzyl-2-azido-3-O-acetyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-l-ido-

pyranosyluronate (52): A mixture of 17 (50 mg, 0.084 mmol) and 35

(25 mg, 0.074 mmol) was coevaporated three times with toluene and dried

under vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(1 mL) and was

stirred for 30 min at room temperature under argon in the presence of

freshly activated powdered 4 ä molecular sieves (20 mg). After cooling the

mixture to À308C, tert-butyldimethylsilyl trifluoromethanesulfonate (0.1m

0.53 mmol 93%) as

C₄₀H₄₃Cl₂N₃O₁₄: 859.2122; found: 859.2113 [M] .

a

colorless syrup. FAB MS: m/z: calcd for

Chem. Eur. J. 2003, 9, No. 1

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157

P. H. Seeberger et al.

FULL PAPER

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

1H), 3.84 3.70 (m, 2H), 3.77 (s, 3H), 3.62 3.49 (m, 2H), 3.32 (dd, J 3.8,

10.4 Hz, 1H), 2.04 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d 170.8, 168.4,

166.5, 160.7, 137.9, 137.8, 137.7, 137.6, 129.0, 128.7, 128.6, 128.5, 128.4, 128.2,

128.1, 128.0, 127.5, 98.4, 92.9, 90.7, 80.1, 79.3, 77.6, 77.5, 75.7, 75.6, 75.2, 75.1,

73.5, 72.6, 70.1, 63.3, 62.3, 53.2, 40.3, 21.0; FAB MS: m/z: calcd for

glucopyranosyl-(1 ! 4)-methyl

3-O-benzyl-2-O-monochloroacetyl-a-d-

glucopyranosyluronate trichloroacetimidate (57)

From 55: Benzylamine (70 mL, 0.63 mmol) was added in three portions,

every 2 h, to a solution of 55 (1 g, 1.2 mmol) Et₂O (40 mL) at 08C and kept

overnight at À208C. The mixture was diluted with CH₂Cl₂, filtered and

washed with aqueous HCl (10%). The organic phase was dried over

MgSO₄, filtered and the solvent was removed under reduced pressure.

Flash chromatography on silica gel (hexanes/EtOAc 90:10 ! 80:20)

afforded 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-de-

oxy-a-d-glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroace-

tyl-d-glucopyranosyluronate (704 mg, 0.87 mmol, 75%) as a white solid.

C₄₀H₄₂Cl₄N₄O₁₃Si: 926.1502; found: 926.1514 [M] .

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldi-

methylsilyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-

levulinoyl-b-d-glucopyranosyluronate (59): Compound 41 (1.12 g,

1.53 mmol) and imidazole (208 mg, 3.05 mmol) were dissolved in CH₂Cl₂

(10 mL) and cooled to À158C. tert-Butyldimethylsilylchloride (253 mg,

1.68 mmol) was added to the mixture and stirring was continued at À158C.

After 5 h tert-butyldimethylsilylchloride (125 mg, 0.83 mmol) was added

and after 6 h imidazole (100 mg, 1.47 mmol) and tert-butyldimethylsilyl-

chloride (253 mg, 1.68 mmol) were added. After 18 h one additional

portion of tert-butyldimethylsilylchloride (70 mg, 0.46 mmol) was added.

After 40 h, water was added and the mixture was warmed to room

temperature. After dilution with EtOAc the mixture was washed with sat.

NaHCO₃and brine. The organic layer was dried over Na₂SO₄and after

filtration the solvent was removed under reduced pressure. Flash chroma-

tography on silica gel (hexanes/EtOAc 9:1 ! 8:2) afforded tert-butyldime-

thylsilyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-de-

oxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-b-d-glucopyranosy-

luronate (1.08 g, 1.28 mmol, 84%) as a colorless foam. [a]²_D⁴ 65.9 (c

1.55, CH₂Cl₂); IR (thin film on NaCl): nÄ 3475, 3031, 2953, 2857, 2106, 1747,

1472, 1254 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.41 7.27 (m, 10H), 5.64

(d, J 3.7 Hz, 1H), 5.07 (d, J 11.0 Hz, 1H), 4.89 (d, J 11.0 Hz, 1H), 4.83

(d, J 11.0 Hz, 1H), 4.81 (d, J 11.0 Hz, 1H), 4.59 (d, J 7.3 Hz, 1H), 4.35

(dd, J 2.1, 11.9 Hz, 1H), 4.14 (dd, J 8.8, 9.5 Hz, 1H), 4.07 (dd, J 3.7,

12.2 Hz, 1H), 3.99 (d, J 9.8 Hz, 1H), 3.79 (s, 3H), 3.76 (at, J 8.8 Hz,

1H), 3.69 3.63 (m, 2H), 3.57 (ddd, J 9.5, 9.2, 2.1 Hz, 1H), 3.52 3.49 (m,

1H), 3.28 3.22 (m, 1H), 2.31 (d, J 2.1 Hz, 1H), 2.10 (s, 3H), 0.92 (s, 9H),

0.89 (s, 9H), 0.15 (s, 6H), 0.00 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): d

171.0, 168.8, 138.5, 138.1, 128.6, 128.5, 127.9, 127.8, 127.7, 127.5, 98.0, 97.7,

83.9, 80.2, 76.6, 75.2, 74.9, 74.8, 74.7, 71.0, 70.8, 63.8, 62.6, 52.8, 26.0, 25.9,

21.1, 18.2, 18.1, À3.5, À4.1, À4.9, À5.0; FAB MS: m/z: calcd for

FAB MS : m/z: calcd for C₃₇H₅₀ClN₃O₁₃Si: 807.2801; found: 807.2796 [M] .

From 61: A mixture of tetrabutylammonium fluoride (1.0m in THF,

0.6 mL) and glacial acetic acid (55 mL, 0.9 mmol) was added dropwise to a

solution of 61 (539 mg, 0.58 mmol) in THF (6 mL) under nitrogen at 08C.

The reaction mixture was warmed to room temperature, stirred for

1.5 h and quenched with brine. After dilution with CH₂Cl₂, the two

phases were separated. The organic phase was dried over MgSO₄, filtered

and the solvent was removed under reduced pressure. The crude

material was purified by silica gel column chromatography (hexane/EtOAc

90:10 ! 80:20) to yield 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldime-

thylsilyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-methyl

3-O-benzyl-2-O-

monochloroacetyl-a/b-d-glucopyranosyluronate (198 mg, 43%) as a white

solid.

A solution of 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

deoxy-a-d-glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroa-

cetyl-d-glucopyranosyluronate (540 mg, 0.7 mmol) and trichloroacetoni-

trile (2 mL, 19.2 mmol) in CH₂Cl₂(14 mL) containing freshly activated 4 ä

molecular sieves (100 mg), was stirred 30 minutes at room temperature.

After cooling to 08C, DBU (45 mL, 0.3 mmol) was added. The mixture was

allowed to reach room temperature after 1 h the mixture was filtered

through a pad of Celite and concentrated. The residue was purified by silica

gel column chromatography (hexanes/EtOAc 85:15) to yield 57 (546 mg,

0.57 mmol, 85%) as a white solid. [a]²_D⁴ 81.5 (c 1.00, CHCl₃); IR (thin

film on NaCl): nÄ 2930, 2106, 1745, 1678, 1252, 1029 cm^À1

;

¹H NMR

C₄₁H₆₃N₃O₁₂Si₂C₄₁H₆₃N₃O₁₂Si₂: 845.3950; found: 845.3925 [M] .

(300 MHz, CDCl₃): d 8.69 (s, 1H), 7.36 7.26 (m, 10H), 6.56 (d, J

3.2 Hz), 5.60 (d, J 3.6 Hz, 1H), 5.14 5.19 (m, 1H), 4.96 4.78 (m, 4H),

4.49 (d, 1H), 4.35 (dd, J 11.8 Hz, 1H), 4.28 4.25 (m, 2H), 4.04 (dd, J

3.8, 12.1 Hz, 1H), 3.99 (d, J 9.5 Hz, 1H), 3.84 3.76 (m, 2H), 3.79 (s, 3H),

3.72 3.61 (m, 2H), 3.50 (m, 1H), 3.26 (dd, J 3.6, 10.2 Hz, 1H), 2.10 (s,

3H), 0.88 (s, 9H), À0.01 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): d 171.0,

168.5, 166.5, 160.8, 138.0, 137.9, 128.8, 128.5, 128.1, 127.8, 127.5, 127.4, 98.2,

92.9, 80.1, 79.6, 75.5, 75.3, 75.0, 74.3, 72.6, 71.3, 70.9, 63.7, 62.5, 53.1, 40.3,

26.0, 21.1, 18.2, À3.5, À4.8; FAB MS: m/z: calcd for C₃₉H₅₀Cl₄N₄O₁₃Si:

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldi-

methylsilyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-b-d-

glucopyranosyluronate (998 mg, 1.18 mmol) and DMAP (432 mg,

3.54 mmol) were dissolved in CH₂Cl₂(10 mL) and a solution of levulinic

anhydride (500 mg, 2.34 mmol) in CH₂Cl₂(5 mL) was added. The reaction

mixture was stirred for 2 h at room temperature and concentrated. Flash

chromatography on silica gel (hexanes/EtOAc 8:2) afforded 59 (1.08 g,

1.14 mmol, 97%) as a colorless foam. ¹H NMR (400 MHz, CDCl₃): d

7.39 7.24 (m, 10H), 5.53 (d, J 3.6 Hz, 1H), 5.04 (dd, J 7.3, 8.8 Hz,

1H), 4.90 4.73 (m, 5H), 4.37 (dd J 1.8, 11.9 Hz, 1H), 4.26 (at, J 9.2,

9.0 Hz, 1H), 4.07 4.00 (m, 2H), 3.86 (dd, J 8.8, 8.7 Hz, 1H), 3.79 (s, 3H),

3.68 3.61 (m, 2H), 3.55 3.52 (m, 1H), 3.25 (dd, J 3.7, 9.9 Hz, 1H), 2.68

2.63 (m, 2H), 2.53 2.48 (m, 2H), 2.12 (s, 3H), 2.08 (s, 3H), 0.89 (s, 9H),

0.87 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.00 (s, 3H), À0.03 (s, 3H); ¹³C NMR

(100 MHz, CDCl₃): d 206.0, 171.2, 170.8, 168.5, 138.0, 137.9, 128.5, 128.4,

127.8, 127.7, 127.6, 127.4, 97.5, 96.1, 82.5, 80.2, 75.2, 75.1, 74.7, 74.5, 74.1, 71.1,

70.8, 63.8, 62.5, 52.7, 37.8, 29.9, 28.0, 26.0, 25.6, 21.0, 18.1, 17.9, À3.6, À4.2,

À5.0, À5.2; FAB MS: m/z: calcd for C₄₆H₆₉N₃O₁₄Si₂: 943.4318; found:

950.1898; found: 950.1892 [M] .

6-O-Acetyl-2-azido 3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl-3-O-benzyl-2-O-monochloroacetyl-a-d-glucopyranosyluronate tri-

chloroacetimidate (58): Benzylamine (70 mL, 0.63 mmol) was added in

three portions, every 2 h, to a solution of 56 (400 mg, 0.46 mmol) in Et₂O

(25 mL) at 08C and kept overnight at À208C. The mixture was diluted with

CH₂Cl₂, filtered and washed with aqueous HCl (10%). The organic phase

was dried over MgSO₄, filtered and the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 90:10 !

80:20) afforded 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyr-

anosyl-(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroacetyl-d-glucopyrano-

syluronate (277 mg, 0.35 mmol, 76%) as a white solid. FAB MS: m/z:

943.4332 [M] .

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldi-

methylsilyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 2-O-allyloxycar-

bonyl-3-O-benzyl-b-d-glucopyranosyluronate (60): tert-Butyldimethylsilyl

(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

calcd for C₃₈H₄₂ClN₃O₁₃Si: 783.2406; found: 783.2400 [M] .

A solution of 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyra-

nosyl-(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroacetyl-d-glucopyranosy-

luronate (226 mg, 0.292 mmol) and trichloroacetonitrile (0.790 mL,

7.6 mmol) in CH₂Cl₂(7 mL) containing freshly activated 4 ä molecular

sieves (100 mg), was stirred 30 minutes at room temperature. After cooling

to 08C, DBU (5 mL, 0.03 mmol) was added. The mixture was allowed to

reach room temperature and after 1 h it was filtered through a pad of Celite

and concentrated. The residue was purified by silica gel column chroma-

tography (hexanes/EtOAc 85:15) to yield 58 (240 mg, 0.26 mmol, 90%) as

a white solid. ¹H NMR (500 MHz, CDCl₃): d 8.70 (s, 1H), 7.38 7.27 (m,

15H), 6.56 (d, J 3.4 Hz, 1H), 5.53 (d, J 3.7 Hz, 1H), 5.17 5.14 (m, 1H),

4.99 4.78 (m, 5H), 4.46 4.42 (m, 2H), 4.28 4.19 (m, 4H), 3.97 3.90 (m,

glucopyranosyl)-(1 ! 4)-methyl

3-O-benzyl-b-d-glucopyranosyluronate

(for the synthesis see the procedure to prepare compound 59) (350 mg,

0.414 mmol) and DMAP (1.01 g, 8.28 mmol) were dissolved in CH₂Cl₂

(10 mL) and cooled to À708C. Allyloxycarbonylchloride (800 mL,

7.54 mmol) was added in three equal portions every 2 h. After the addition

was complete the mixture was warmed to room temperature and stirred

overnight. The mixture was poured into EtOAc and washed with 1n HCl,

brine and sat. NaHCO₃. The organic layer was dried over Na₂SO₄, filtered

and the solvent was removed under reduced pressure. Flash chromatog-

raphy on silica gel (hexanes/EtOAc 9:1 ! 85:15) afforded 60 (347 mg,

0.373 mmol, 90%) as a colorless oil. [a]²_D⁴ 82.6 (c 1.04, CH₂Cl₂); IR

158

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

(thin film on NaCl): nÄ 3037, 2929, 2857, 2106, 1758, 1454, 1252 cm^À1

;

CH₂Cl₂(5 mL) and a solution of levulinic anhydride (390 mg, 1.82 mmol) in

CH₂Cl₂(5 mL) was added. The reaction mixture was stirred for 2 h at room

temperature and concentrated. Flash chromatography on silica gel

¹H NMR (500 MHz, CDCl₃): d 7.39 7.26 (m, 10H), 5.91 5.82 (m, 1H),

5.55 (d, J 3.7 Hz, 1H), 5.35 5.30 (m, 1H), 5.25 5.22 (m, 1H), 4.89 4.71

(m, 6H), 4.62 4.53 (m, 2H), 4.34 (dd, J 2.1, 11.9 Hz, 1H), 4.22 (dd, J

9.5, 9.2 Hz, 1H), 4.05 (dd, J 3.7, 12.2 Hz, 1H), 3.99 (d, J 9.8 Hz, 1H),

3.85 (d, J 9.2 Hz, 1H), 3.80 (s, 3H), 3.68 3.61 (m, 2H), 3.52 3.49 (m,

1H), 3.29 3.25 (m, 1H), 2.10 (s, 3H), 0.89 (s, 9H), 0.87 (s, 9H), 0.12 (s,

3H), 0.10 (s, 3H), 0.00 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): d 171.0,

168.5, 154.1, 138.0, 137.8, 131.4, 128.6, 128.5, 127.9, 127.8, 127.7, 127.5, 119.6,

97.8, 96.1, 82.6, 80.2, 79.1, 75.3, 75.0, 74.8, 74.6, 71.1, 70.8, 69.0, 63.9, 62.5,

52.9, 26.0, 25.6, 21.1, 18.1, 18.0, À3.5, À4.1, À4.9, À5.3; FAB MS: m/z: calcd

(hexanes/EtOAc 8:2) afforded 62 (834 mg, 0.91 mmol, quant.) as

a

colorless foam. [a]²_D⁴ 18.9 (c 1.01, CH₂Cl₂); IR (thin film on NaCl):

nÄ 3036, 2929, 2857, 2108, 1747, 1720, 1454, 1362 cm^À1; ¹H NMR (500 MHz,

CDCl₃): d 7.40 7.26 (m, 15H), 5.50 (d, J 3.7 Hz, 1H), 5.03 (dd, J 7.3,

8.8 Hz, 1H), 4.89 (s, 2H), 4.83 (d, J 10.7 Hz, 2H), 4.74 (d, J 11.3 Hz,

1H), 4.73 (d, J 7.3 Hz, 1H), 4.56 (d, J 11.0 Hz, 1H), 4.27 4.23 (m, 3H),

3.97 (d, J 9.5 Hz, 1H), 3.89 (dd, J 8.8, 10.1 Hz, 1H), 3.82 (dd, J 8.9,

8.5 Hz, 1H), 3.77 (s, 3H), 3.66 3.62 (m, 1H), 3.53 (dd, J 10.1, 8.8 Hz,

1H), 3.31 (dd, J 3.7, 10.4 Hz, 1H), 2.69 2.65 (m, 2H), 2.54 2.49 (m, 2H),

2.14 (s, 3H), 2.04 (s, 3H), 0.86 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); ¹³C NMR

(125 MHz, CDCl₃): d 206.2, 171.3, 170.8, 168.5, 138.0, 137.7, 137.7, 128.7,

128.5, 128.2, 128.2, 128.2, 128.1, 127.8, 127.7, 97.7, 96.1, 82.6, 80.2, 77.6, 75.7,

75.2, 75.2, 75.2, 74.5, 74.4, 69.8, 63.5, 62.4, 52.9, 37.9, 30.0, 28.1, 25.6, 21.0,

18.0, À4.2, À5.2; FAB MS: m/z: calcd for C₄₇H₆₁N₃O₁₄Si: 919.3923; found:

for C₄₅H₆₇N₃O₁₄Si₂: 929.4162; found: 929.4122 [M] .

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldi-

methylsilyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-

monochloroacetyl-b-d-glucopyranosyluronate (61): Pyridine (0.5 mL,

6 mmol), monochloroacetic anhydride (360 mg, 1.00 mmol) and DMAP

(9 mg, 0.05 mmol) were added to a solution of tert-butyldimethylsilyl (6-O-

acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl)-(1 ! 4)-methyl-3-O-benzyl-b-d-glucopyranosyluronate (see the

procedure to prepare 59) (439 mg, 0.52 mmol) in CH₂Cl₂(5 mL). The

solution was stirred at room temperature for 3 h, water was added and the

mixture was stirred for one additional hour. The organic phase was washed

with sat. NaHCO₃, water and aqueous HCl (10%), dried over MgSO₄and

filtered. The solvent was removed under reduced pressure and the residue

was purified by silica gel column chromatography (hexanes/EtOAc 90:20)

to yield 61 (470 mg, 0.51 mmol, 98%) as a colorless syrup. [a]²_D⁴ 30.1

(c 1.00, CHCl₃); IR (thin film on NaCl): nÄ 2932, 2107, 1745, 1254, 1029,

839 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.37 7.28 (m, 10H), 5.50 (d, J

3.4 Hz, 1H), 5.05 (dd, J 7.9, 9.1 Hz), 4.74 (d, J 7.3 Hz), 4.86 (d, J

11.0 Hz, 2H), 4.79 (d, J 11.3 Hz, 1H), 4.68 (d, J 11.3 Hz, 1H), 4.35

(dd, J 1.5, 11.9 Hz, 1H), 4.29 4.23 (m, 1H), 4.04 (dd, J 3.7, 12.2 Hz,

1H), 3.99 (d, J 9.5 Hz, 1H), 3.89 3.83 (m, 2H), 3.79 (s, 3H), 3.78 (d, J

14.9 Hz, 1H), 3.60 3.67 (m, 2H), 3.53-3.51 (m, 1H), 3.28 (dd, J 3.0,

9.2 Hz, 1H), 2.10 (s, 3H), 0.86 (s, 9H), 0.85 (s, 9H), 0.10 (s, 3H), 0.82 (s,

3H), À0.01 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): d 171.0, 168.4, 165.8,

138.0, 137.8, 128.7, 128.5, 128.0, 127.8, 127.7, 127.5, 97.8, 96.0, 82.6, 80.2, 76.4,

75.3, 75.2, 74.7, 74.6, 71.3, 70.8, 63.9, 62.5, 52.9, 26.1, 25.6, 21.1, 18.2, 17.9,

À3.5, À4.1, À4.9, À5.1; FAB MS: m/z: calcd for C₄₃H₆₄ClN₃O₁₃Si:

919.3914 [M] .

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-

glucopyranosyl)-(1 ! 4)-methyl 2-O-allyloxycarbonyl-3-O-benzyl-b-d-glu-

copyranosyluronate (63): tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3,4-

di-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl-3-O-benzyl-b-

d-glucopyranosyluronate (see the procedure to prepare 62), (190 mg,

0.231 mmol) and DMAP (780 mg, 6.38 mmol) were dissolved in CH₂Cl₂

(5 mL) and cooled to À708C. Allyloxycarbonylchloride (600 mL,

5.65 mmol) was added in three equal portions every 2 h. After the addition

was complete the mixture was warmed to room temperature and stirred

overnight. The mixture was poured into EtOAc and washed with 1n HCl,

brine and sat. NaHCO₃. The organic layer was dried over Na₂SO₄, filtered

and the solvent was removed under reduced pressure. Flash chromatog-

raphy on silica gel (hexanes/EtOAc 9:1 ! 85:15) afforded 63 (190 mg,

0.21 mmol, 91%) as a colorless oil. [a]²_D⁴ 21.9 (c 1.25, CH₂Cl₂); IR

(thin film on NaCl): nÄ 3026, 2929, 2858, 2108, 1756, 1252 cm^À1; ¹H NMR

(500 MHz, CDCl₃): d 7.41 7.26 (m, 15H), 5.92 5.84 (m, 1H), 5.52 (d,

J 3.7 Hz, 1H), 5.36 5.32 (m, 1H), 5.26 5.23 (m, 1H), 4.92 4.81 (m,

5H), 4.79 4.73 (m, 2H), 4.63 4.54 (m, 3H), 4.29 4.26 (m, 2H), 4.22 (dd,

J 9.5, 8.9 Hz, 1H), 3.97 (d, J 9.5 Hz, 1H), 3.90 (dd, J 8.8, 10.4 Hz, 1H),

3.84 (dd, J 9.2, 8.8 Hz, 1H), 3.78 (s, 3H), 3.65 3.61 (m, 1H), 3.54 (dd, J

8.9, 10.1 Hz, 1H), 3.32 (dd, J 3.7, 10.4 Hz, 1H), 2.05 (s, 3H), 0.88 (s, 9H),

0.13 (s, 3H), 0.12 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.8, 168.3,

154.0, 137.9, 137.7, 137.6, 131.4, 128.7, 128.7, 128.5, 128.3, 128.2, 128.2, 128.1,

127.9, 127.7, 119.6, 97.8, 96.1, 82.5, 80.2, 79.1, 77.6, 75.7, 75.4, 75.2, 74.9, 74.5,

69.8, 69.0, 63.5, 62.4, 52.9, 25.6, 21.0, 18.0, À4.1, À5.4; FAB MS: m/z: calcd

921.3666; found: 921.366 [M] .

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-

glucopyranosyl)-(1 ! 4)-methyl-3-O-benzyl-2-O-levulinyl-b-d-glucopyra-

nosyluronate (62): Compound 44 (813 mg, 1.15 mmol) and imidazole

(310 mg, 4.55 mmol) were dissolved in CH₂Cl₂(10 mL) and cooled to

À158C. To this mixture tert-butyldimethylsilylchloride (241 mg,

1.60 mmol) was added and stirring was continued at À158C. After 5 h

tert-butyldimethylsilylchloride (50 mg, 0.33 mmol) was added followed

after 16 h by another portion of tert-butyldimethylsilylchloride (100 mg,

0.66 mmol). After 40 h, water was added and the mixture was warmed to

room temperature. After dilution with EtOAc the mixture was washed with

sat. NaHCO₃and brine. The organic layer was dried over Na₂SO₄and after

filtration the solvent was removed under reduced pressure. Flash chroma-

tography on silica gel (hexanes/EtOAc 9:1 ! 8:2) afforded tert-butyldi-

methylsilyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyrano-

for C₄₆H₅₉N₃O₁₄Si: 905.3766; found: 905.3751 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 2-O-levulinyl-3-O-benzyl-a-d-glucopyra-

nosyluronate trichloroacetimidate (64): Compound 59 (1.06 g, 1.12 mmol)

was dissolved in THF (10 mL) and cooled to 08C. Glacial acetic acid

(90 mL, 1.57 mmol) and TBAF (1m in THF, 1.30 mL, 1.30 mmol) were

added in sequence. After 30 min the mixture was poured into Et₂O

(100 mL) and washed three times with brine. The organic layer was dried

over Na₂SO₄, filtered and the solvent was removed under reduced pressure.

The residue was dissolved in CH₂Cl₂(20 mL) and cooled to 08C.

Trichloroacetonitrile (1.7 mL, 17.0 mmol) and DBU (15 mL, 0.1 mmol)

were added and the mixture was stirred at 08C for 1 h and at room

temperature for 3 h. After removal of the solvent under reduced pressure,

flash chromatography on silica gel (hexanes/EtOAc 85:15 ! 70:30) afford-

ed 64 (1.0 g, 1.03 mmol, 92%) as a colorless foam. [a]²_D⁴ 108.9 (c 1.69,

CH₂Cl₂); IR (thin film on NaCl): nÄ 3337, 3031, 2954, 2929, 2857, 2105,

1746, 1720, 1678, 1454 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 8.71 (s, 1H),

7.26 7.40 (m, 10H), 6.54 (d, J 3.7 Hz, 1H), 5.63 (d, J 3.7 Hz, 1H), 5.12

5.16 (m, 1H), 4.80 4.92 (m, 4H), 4.48 (d, J 9.5 Hz, 1H), 4.35 (dd, J 2.1,

11.9 Hz, 1H), 4.21 4.29 (m, 2H), 4.04 (dd, J 4.0, 12.2 Hz, 1H), 3.79 (s,

3H), 3.69 (dd, J 8.5, 10.1 Hz, 1H), 3.64 (dd, J 9.5, 8.5 Hz, 1H), 3.45

3.49 (m, 1H), 3.23 (dd, J 4.0, 10.1 Hz, 1H), 2.61 2.71 (m, 2H), 2.35 2.50

(m, 2H), 2.14 (s, 3H), 2.10 (s, 3H), 0.88 (s, 9H), 0.00 (s, 3H), À0.01 (s, 3H);

¹³C NMR (125 MHz, CDCl₃): d 206.1, 172.0, 171.0, 168.7, 160.7, 138.1,

137.9, 128.7, 128.5, 128.0, 127.7, 127.6, 127.4, 98.1, 93.3, 90.9, 80.1, 79.7, 75.3,

75.3, 74.1, 72.5, 72.4, 71.2, 70.9, 63.7, 62.6, 53.1, 37.8, 30.0, 27.7, 26.0, 21.1, 18.1,

À3.5, À4.9; FAB MS: m/z: calcd for C₄₂H₅₅Cl₃N₄O₁₄Si: 972.2550; found:

syl)-(1 ! 4)-methyl

3-O-benzyl-b-d-glucopyranosyluronate

(752 mg,

0.92 mmol, 80%) as a colorless foam. [a]²_D⁴ 26.2 (c 1.02, CH₂Cl₂);

IR (thin film on NaCl): nÄ 3376, 3049, 2919, 2861, 2107, 1744, 1454, 1362,

1

1252 cm^À1; H NMR (500 MHz, CDCl₃): d 7.42 7.26 (m, 15H), 5.60 (d,

J 4.0 Hz, 1H), 5.08 (d, J 11.0 Hz, 1H), 4.92 4.89 (m, 2H), 4.86 (d, J

10.7 Hz, 1H), 4.84 (d, J 11.0 Hz, 1H), 4.58 (d, J 7.3 Hz, 1H), 4.57 (d, J

10.7 Hz, 1H), 4.27 4.25 (m, 2H), 4.14 (dd, J 8.9, 9.5 Hz, 1H), 3.96 (d, J

9.8 Hz, 1H), 3.91 (dd, J 8.9, 10.4 Hz, 1H), 3.77 (s, 3H), 3.73 (dd, J 9.2,

8.9 Hz, 1H), 3.65 3.61 (m, 1H), 3.59 3.52 (m, 2H), 3.30 (dd, J 4.0,

10.4 Hz, 1H), 2.33 (d, J 2.4 Hz, 1H), 2.04 (s, 3H), 0.93 (s, 9H), 0.16 (s,

3H), 0.15 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.9, 168.6, 138.6,

137.8, 137.7, 128.7, 128.6, 128.3, 128.2, 128.2, 128.1, 127.9, 127.8, 98.0, 97.8,

83.9, 80.2, 77.6, 76.5, 75.6, 75.3, 75.1, 74.9, 74.7, 69.7, 63.5, 62.4, 52.8, 25.8,

21.0, 18.2, À4.1, À5.0; FAB MS: m/z: calcd for C₄₂H₅₅N₃O₁₂Si: 821.3555;

found: 821.3549 [M] .

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-

glucopyranosyl)-(1 ! 4)-methyl

3-O-benzyl-b-d-glucopyranosyluronate

(748 mg, 0.41 mmol) and DMAP (334 mg, 2.73 mmol) were dissolved in

972.2579 [M] .

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0159 $ 20.00+.50/0

159

P. H. Seeberger et al.

FULL PAPER

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 2-O-allyloxycarbonyl-3-O-benzyl-a-d-glu-

copyranosyluronate trichloroacetimidate (65): Compound 60 (271 mg,

0.291 mmol) was dissolved in anhydrous THF (5 mL) and cooled to 08C.

Glacial acetic acid (21 mL, 0.367 mmol) and TBAF (1.0m in THF, 320 mL,

0.32 mmol) were added in sequence. After 30 min the mixture was poured

into Et₂O (50 mL) and washed three times with brine. The organic layer

was dried over Na₂SO₄, filtered and the solvent was removed under

reduced pressure. The residue was dissolved in CH₂Cl₂(11 mL) and cooled

to 08C. Trichloroacetonitrile (450 mL, 4.49 mmol) and DBU (5 mL,

0.033 mmol) were added and the mixture was stirred overnight at room

temperature. After removal of the solvent under reduced pressure, flash

chromatography on silica gel (hexanes/EtOAc 85:15) afforded 65 (190 mg,

0.198 mmol, 68%) as a colorless foam. [a]²_D⁴ 114.0 (c 1.37, CH₂Cl₂);

IR (thin film on NaCl): nÄ 3340, 3026, 2953, 2857, 2105, 1754, 1679, 1454,

ature for 1 h, water was added and the mixture was stirred for one

additional hour. The organic phase was washed with sat. NaHCO₃, water,

and aqueous HCl (10%), dried over MgSO₄and filtered. The solvent was

removed under reduced pressure and the residue was purified by silica gel

column chromatography (hexane/EtOAc 90:20) to yield 68 (707 mg,

0.92 mmol, 95%) as

C₃₄H₄₉N₃O₁₅Si: 767.2933; found: 767.2951 [M] .

a

colorless syrup. FAB MS: m/z: calcd for

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 1,2-O-acetyl-3-O-benzyl-l-idopyranosylur-

onate (69): Pyridine (1.5 mL, 18 mmol), acetic anhydride (1 mL, 11 mmol)

and DMAP (10 mg, 0.08 mmol) were added to a solution of 54 (560 mg,

0.77 mmol) in CH₂Cl₂(10 mL). The solution was stirred at room temper-

ature for 1 h, water was added and the mixture was stirred for one

additional hour. The organic phase was washed with sat. NaHCO₃, water

and aqueous HCl (10%), dried over MgSO₄and filtered. The solvent was

removed under reduced pressure and the residue was purified by silica gel

column chromatography (hexanes/EtOAc 90:20) to yield 69 (619 mg,

1367 cm^À1

;

¹H NMR (500 MHz, CDCl₃): d 8.74 (s, 1H), 7.40 7.26 (m,

10H), 6.63 (d, J 3.4 Hz, 1H), 5.91 5.82 (m, 1H), 5.65 (d, J 3.7 Hz, 1H),

5.34 5.30 (m, 1H), 5.27 5.23 (m, 1H), 5.01 (dd, J 3.4, 9.5 Hz, 1H),

4.93 4.85 (m, 3H), 4.82 (d, J 11.0 Hz, 1H), 4.65 4.57 (m, 2H), 4.50 (d,

J 9.5 Hz, 1H), 4.37 (dd, J 1.8, 12.2 Hz, 1H), 4.29 4.22 (m, 2H), 4.05

(dd, J 4.0, 12.2 Hz, 1H), 3.80 (s, 3H), 3.71 (dd, J 10.1, 8.5 Hz, 1H), 3.65

(dd, J 9.2, 8.5 Hz, 1H), 3.50 3.46 (m, 1H), 3.23 (dd, J 3.7, 10.1 Hz, 1H),

2.11 (s, 3H), 0.89 (s, 9H), 0.00 (s, 3H), À0.01 (s, 3H); ¹³C NMR (125 MHz,

CDCl₃) d 171.0, 168.6, 160.7, 154.2, 138.0, 137.6, 131.2, 128.6, 128.4, 128.0,

127.8, 127.7, 127.4, 119.4, 98.2, 93.2, 90.8, 80.0, 79.4, 75.8, 75.4, 75.2, 74.1, 72.4,

71.2, 70.9, 69.2, 63.6, 62.5, 53.0, 26.0, 21.0, 18.1, À3.6, À4.9; FAB MS: m/z:

0.76 mmol, 99%) as

C₃₉H₅₃N₃O₁₄Si: 815.3297; found: 815.3270 [M] .

a

colorless syrup. FAB MS: m/z: calcd for

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 2-O-acetyl-3-O-benzyl-b-l-idopyranosyluronate

trichloroacetimidate (70): Benzylamine (2.0 mL, 18.3 mmol) was added to

a solution of 68 (650 mg, 0.85 mmol) in Et₂O (50 mL) at 08C. After stirring

at 08C for 4 h the mixture was diluted with CH₂Cl₂, filtered and washed

with HCl (10%). The organic phase was dried over Na₂SO₄, filtered and

the solvent was removed under reduced pressure. Flash chromatography on

silica gel (hexanes/EtOAc 90:10 ! 80:20) afforded 3,6-di-O-acetyl-2-azido-

4-O-tert-butyldimethylsilyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-methyl

calcd for C₄₁H₅₃Cl₃N₄O₁₄Si: 958.2393; found: 958.2392 [M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl 3-O-benzyl-2-O-levulinoyl-a-d-glucopyranosyluronate trichloroa-

cetimidate (66): Compound 62 (345 mg, 0.376 mmol) was dissolved in THF

(10 mL) and cooled to 08C. Glacial acetic acid (27 mL, 0.472 mmol) and

tetrabutylammoniumfluoride (1.0m in THF, 415 mL, 0.415 mmol) were

added sequentially. After 40 min this mixture was poured into EtOAc

(50 mL) and washed with sat. NaHCO₃. The organic layer was dried over

Na₂SO₄, filtered and the solvent was removed under reduced pressure. The

residue was dissolved in CH₂Cl₂(10 mL) and cooled to 08C. Trichloroa-

cetonitrile (750 mL, 7.48 mmol) and DBU (30 mL) were added and the

mixture was stirred for 2 h. After removal of the solvent under reduced

pressure, flash chromatography on silica gel (hexanes/EtOAc 8:2 ! 6:4)

afforded 66 as a colorless foam (302 mg, 0.32 mmol, 85%). [a]²_D⁴ 79.8

(c 1.72, CH₂Cl₂); IR (thin film on NaCl): nÄ 3337, 3063, 3030, 2953, 2108,

1746, 1719, 1677, 1497, 1363 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 8.71 (s,

1H), 7.40 7.25 (m, 15H), 6.53 (d, J 3.4 Hz, 1H), 5.55 (d, J 3.7 Hz, 1H),

5.14 5.11 (m, 1H), 4.95 4.82 (m, 5H), 4.57 (d, J 11.0 Hz, 1H), 4.46 4.42

(m, 1H), 4.28 4.20 (m, 4H), 3.94 (dd, J 8.5, 10.4 Hz, 1H), 3.77 (s, 3H),

3.62 3.58 (m, 1H), 3.51 (dd, J 8.8, 10.1 Hz, 1H), 3.30 (dd, J 3.7, 10.4 Hz,

1H), 2.70 2.60 (m, 2H), 2.51 2.36 (m, 2H), 2.14 (s, 3H), 2.04 (s, 3H);

¹³C NMR (125 MHz, CDCl₃): d 206.1, 172.0, 170.9, 168.6, 160.7, 138.0,

137.8, 137.7, 128.7, 128.7, 128.7, 128.2, 128.1, 127.9, 127.6, 98.3, 93.2, 90.9, 80.1,

79.4, 77.6, 75.7, 75.4, 75.2, 75.0, 72.5, 72.4, 70.0, 63.3, 62.4, 53.2, 37.8, 30.0, 27.7,

21.0; FAB MS: m/z: calcd for C₄₃H₄₇Cl₃N₄O₁₄: 948.2154; found: 948.2118

2-O-acetyl-3-O-benzyl-a/b-l-idopyranosyluronate (443 mg, 72%) as

a

white solid. FAB MS: m/z: calcd for C₃₂H₄₇N₃O₁₄Si: 725.2827; found:

725.2811 [M] .

A solution of 3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-

a-d-glucopyranosyl-(1 ! 4)-methyl 2-O-acetyl-3-O-benzyl-a/b-l-idopyra-

nosyluronate (335 mg, 0.46 mmol) and trichloroacetonitrile (1.3 mL,

12.5 mmol) in CH₂Cl₂(10 mL) containing freshly activated powdered 4 ä

molecular sieves (100 mg) was stirred 30 minutes at room temperature.

After cooling the solution to 08C, DBU (30 mL, 0.2 mmol) was added. The

temperature was allowed to rise and after 1 h stirring, the mixture was

filtered through a pad of Celite and concentrated. The residue was purified

by silica gel column chromatography (hexanes/EtOAc 85:15) yielding 70

(373 mg, 0.43 mmol, 93%) as a white solid. [a]²_D⁴ 53.4 (c 1.00, CHCl₃);

IR (thin film on NaCl): nÄ 2935, 2693, 2109, 1744, 1675, 1372 cm^À1

;

¹H NMR (500 MHz, CDCl₃):

d

8.68 (s, 1H), 7.36 7.30 (m, 5H),

6.41 (s, 1H), 5.26 (dd, J 8.5, 10.4 Hz, 1H), 5.17 (s, 1H), 5.00

(d, J 3.4 Hz, 1H), 4.98 (d, J 1.8 Hz, 1H), 4.81 (d, J 11.6 Hz, 1H),

4.65 (d, J 11.6 Hz, 1H), 4.46 (dd, J 2.1, 12.5 Hz, 1H), 4.27 (s, 1H),

4.09 4.06 (m, 2H), 4.00 3.81 (m, 1H), 3.80 (s, 3H), 3.81 3.79 (m, 1H),

3.05 (dd, J 3.3, 10.7 Hz, 1H), 2.20 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H), 0.84

(s, 9H), 0.49 (s, 3H), 0.30 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.7,

170.3, 169.9, 169.1, 160.3, 137.2, 128.6, 128.1, 128.0, 98.3, 95.7, 73.7, 72.8, 72.7,

72.3, 71.1, 69.2, 68.9, 65.4, 62.4, 61.9, 52.8, 25.8, 21.5, 21.0, 20.9, 18.1, À4.0,

À4.8; FAB MS: m/z: calcd for C₃₄H₄₇Cl₃N₄O₁₄Si: 868.1924; found: 868.1938

[M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl 2-O-allyloxycarbonyl-3-O-benzyl-a/b-d-glucopyranosyluronate tri-

chloroacetimidate (67): Compound 63 (74 mg, 0.082 mmol) was dissolved

in THF (2 mL) and cooled to 08C. Glacial acetic acid (6 mL, 0.1 mmol) and

TBAF (1m in THF, 90 mL, 0.09 mmol) were added. After 30 min the

mixture was poured into Et₂O (50 mL) and washed three times with brine.

The organic layer was dried over Na₂SO₄, filtered and the solvent was

removed under reduced pressure. The residue was dissolved in CH₂Cl₂

(2 mL) and cooled to 08C. Trichloroacetonitrile (120 mL, 1.19 mmol) and

DBU (1.2 mL, 0.008 mmol) were added and the mixture was stirred

overnight at room temperature. After removal of the solvent under

reduced pressure, flash chromatography on silica gel (hexanes/EtOAc

85:15) afforded 67 (63 mg, 0.07 mmol, 83%) as a colorless foam. FAB MS:

[M] .

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-

glucopyranosyl-(1 ! 4)-methyl 2-O-acetyl-3-O-benzyl-b-l-idopyranosylur-

onate) trichloroacetimidate (71): Benzylamine (0.6 mL, 5.5 mmol) was

added to a solution of 69 (600 mg, 0.73 mmol) in Et₂O (15 mL) at 08C.

After stirring at 08C for 5 h the mixture was diluted with CH₂Cl₂, filtered

and washed with HCl (10%). The organic phase was dried over Na₂SO₄and

after filtration purified by silica gel column chromatography (hexanes/

AcOEt 90:10 ! 80:20) to yield 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-

butyldimethylsilyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-methyl 2-O-ace-

tyl-3-O-benzyl-a/b-l-idopyranosyluronate (415 mg, 0.54 mmol, 77%) as a

white solid. FAB MS: m/z: calcd for C₃₇H₅₁N₃O₁₃Si: 773.3191; found:

773.3201 [M] .

m/z: calcd for C₄₂H₄₅Cl₃N₄O₁₄: 934.1998; found: 934.1989 [M] .

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-a-d-gluco-

pyranosyl-(1 ! 4)-methyl 1,2-O-acetyl-3-O-benzyl-a/b-l-idopyranosyluro-

nate (68): Pyridine (2.0 mL, 24 mmol), acetic anhydride (1.4 mL, 15 mmol)

and DMAP (12 mg, 0.1 mmol) were added to a solution of 53 (700 mg,

1.02 mmol) in CH₂Cl₂(16 mL). The solution was stirred at room temper-

A solution of 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

deoxy-a-d-glucopyranosyl-(1 ! 4)-methyl 2-O-acetyl-3-O-benzyl-a/b-l-

idopyranosyluronate (90 mg, 0.12 mmol) and trichloroacetonitrile

(0.34 mL, 3.3 mmol) in CH₂Cl₂(3 mL) containing freshly activated

powdered 4 ä molecular sieves (50 mg) was stirred 30 minutes at room

160

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0160 $ 20.00+.50/0

Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

temperature. After cooling the solution to 08C, DBU (2 mL, 0.012 mmol)

was added. The temperature was allowed to rise and after 1 h stirring, the

mixture was filtered through a pad of Celite and concentrated. The residue

was purified by silica gel column chromatography (hexanes/EtOAc 85:15)

yielding 71 (104 mg, 0.113 mmol, 97%) as a white solid. [a]²_D⁴ 70.3 (c

1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃): d 8.70 (s, 1H), 7.36 7.27 (m,

10H), 6.43 (s, 1H), 5.16 (s, 1H), 4.93 (d, J 3.4 Hz, 1H), 5.00 (d, J 2.1 Hz,

1H), 4.84 (d, J 11.0 Hz, 1H), 4.83 (d, J 11.6 Hz, 1H), 4.76 (d, J

11.0 Hz, 1H), 4.67 (d, J 11.6 Hz, 1H), 4.41 (dd, J 2.1, 12.2 Hz), 4.21 (s,

1H), 3.82 (s, 3H), 4.07 4.02 (m, 2H), 3.75 3.70 (m, 1H), 3.68 3.62 (m,

3H), 3.35 (dd, J 3.5, 9.7 Hz, 1H), 2.17 (s, 3H), 2.07 (s, 3H), 0.88 (s, 9H),

0.05 (s, 3H), À0.01 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.9, 170.1,

168.9, 160.3, 137.9, 137.2, 128.6, 128.5, 128.2, 127.9, 127.8, 127.4, 97.1, 95.4,

80.3, 75.2, 72.6, 72.1, 71.3, 70.8, 70.8, 69.3, 65.4, 64.0, 62.7, 52.7, 26.3, 21.1,

21.0, 18.1, À3.5, À4.8; FAB MS: m/z: calcd for C₃₄H₄₇Cl₃N₄O₁₄Si: 916.2287;

and the solvent was removed under reduced pressure. Flash chromatog-

raphy on silica gel (hexanes/EtOAc 85:15) afforded 74 (527 mg, 0.60 mmol,

70%) as a colorless gum. [a]²_D⁴ 45.5 (c 1.00, CHCl₃); IR (thin film on

NaCl): nÄ 2954, 2109, 1745, 1223, 1072 cm^À1;¹H NMR (500 MHz, CDCl₃):

d 7.37 7.28 (m, 10H), 5.79 5.74 (m, 1H), 5.50 (d, J 3.7 Hz, 1H), 5.09

(dd, J 7.6, 8.8 Hz, 1H), 5.07 4.95 (m, 2H), 4.74 (d, J 7.3 Hz, 1H), 4.87

(d, J 11.3 Hz, 2H), 4.86 (d, J 11.0 Hz, 2H), 4.80 (d, J 11.3 Hz, 1H),

4.70 (d, J 11.0 Hz, 1H), 4.48 (d, 1H), 4.35 (dd, J 2.1, 11.9 Hz, 1H), 4.04

(t, 1H), 4.05 4.00 (m, 2H), 3.89 3.80 (m, 4H), 3.79 (s, 3H), 3.65 3.64 (m,

2H), 3.50 3.42 (m, 2H), 3.28 (dd, J 4.0, 10.1 Hz, 1H), 2.09 (s, 3H), 1.07

1.03 (m, 2H), 1.69 1.58 (m, 2H), 0.88 (s, 9H), À0.01 (s, 6H); ¹³C NMR

(125 MHz, CDCl₃): d 171.0, 168.6, 166.0, 137.9, 137.7, 137.4, 129.0, 128.7,

128.5, 128.1, 128.0, 127.9, 127.75, 127.7, 127.4, 115.0, 100.9, 97.7, 82.5, 80.2,

75.3, 74.9, 74.8, 74.6, 74.5, 71.3, 70.8, 69.5, 63.8, 62.5, 52.9, 44.0, 42.8, 40.6,

30.0, 28.6, 26.0, 21.0, 18.1, À3.5, À4.9; FAB MS: m/z: calcd for

found: 916.2246 [M] .

C₄₂H₃₆ClN₃O₁₃Si: 875.3427; found: 875.3432 [M] .

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-levulinoyl-b-

d-glucopyranosyluronate (72): Compound 64 (292 mg, 0.30 mmol) was

coevaporated three times with toluene, dried under vacuum for 1 h,

dissolved in toluene (5 mL) and 4-penten-1-ol (300 mL, 3.00 mmol) was

added. After cooling the mixture to 08C, TMSOTf (0.1m in toluene, 300 mL,

0.03 mmol) was added dropwise. The mixture was warmed to room

temperature and stirred for 2 h. Triethylamine (0.6 mL) was added and the

solvent was removed under reduced pressure. Flash chromatography on

silica gel (hexanes/EtOAc 85:15) afforded 72 (203 mg, 0.60 mmol, 75%) as

a colorless gum. [a]²_D⁴ 53.1 (c 1.18, CH₂Cl₂); IR (thin film on NaCl):

nÄ 2930, 2858, 2106, 1747, 1362, 1237 cm^À1; ¹H NMR (500 MHz, CDCl₃): d

7.25 7.39 (m, 10H), 5.75 5.84 (m, 1H), 5.54 (d, J 3.7 Hz, 1H), 5.07

(dd, J 7.3, 8.5 Hz, 1H), 4.95 5.04 (m, 2H), 4.88 (d, J 11.0 Hz, 1H),

4.74 4.82 (m, 3H), 4.48 (d, J 7.3 Hz, 1H), 4.35 (dd, J 2.1, 11.9 Hz, 1H),

4.25 (at, J 9.2 Hz, 1H), 4.02 4.07 (m, 2H), 3.84 3.89 (m, 2H), 3.79 (s,

3H), 3.61 3.67 (m, 2H), 3.43 3.51 (m, 2H), 3.23 3.26 (m, 1H), 2.67 2.71

(m, 2H), 2.44 2.58 (m, 2H), 2.14 (s, 3H), 2.03 2.12 (m, 5H), 1.60 1.72

(m, 2H), 0.88 (s, 9H), 0.00 (s, 3H), À0.01 (s, 3H); ¹³C NMR (125 MHz,

CDCl₃): d 206.7, 172.0, 171.5, 169.3, 138.7, 138.6, 138.3, 129.1, 129.0, 128.4,

128.3, 128.3, 127.9, 115.7, 101.7, 98.1, 83.1, 80.7, 75.8, 75.0, 75.0, 74.9, 74.1,

71.7, 71.4, 70.0, 64.4, 63.1, 53.4, 38.5, 30.6, 30.5, 29.2, 28.6, 26.6, 21.6, 18.7,

À3.0, À4.4; FAB MS: m/z: calcd for C₄₅H₆₃N₃O₁₄Si: 897.4079; found:

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroacetyl-b-O-pentenyl-d-gluco-

pyranosyluronate (75): Compound 74 (250 mg, 0.285 mmol) was dissolved

in THF (33 mL). Glacial acetic acid (8 mL) and HF/pyridine complex

(4.8 mL) were added and the solution was stirred at room temperature for

4 d. The mixture was poured into Et₂O and washed with brine, sat.

NaHCO₃and dried over Na₂SO₄. After filtration, the solvent was removed

under reduced pressure. Flash chromatography on silica gel (hexanes/

EtOAc 7:3) afforded 75 (186 mg, 0.244 mmol, 85%) as a colorless foam.

[a]²_D⁴ 10.8 (c 1.0, CHCl₃); IR (thin film on NaCl): nÄ 3485, 2925, 2109,

1747, 1454, 1028 cm^À1

;

¹H NMR (500 MHz, CDCl₃): d 7.43 7.27 (m,

10H), 5.98 5.70 (m, 1H), 5.47 (d, J 3.8 Hz), 5.09 (t, 1H), 5.08 4.97 (m,

2H), 4.95 4.93 (m, 3H), 4.69 (d, J 10.7 Hz, 1H), 4.40 4.35 (m, 1H), 4.59

(dd, J 2.7, 12.4 Hz, 1H), 4.48 (d, J 7.7 Hz, 1H), 4.24 (t, 1H), 4.11 (dd,

J 1.9, 12.4 Hz, 1H), 4.0 (d, J 5.5 Hz, 1H), 3.89 3.79 (m, 4H), 3.79 (s,

3H), 3.78 3.70 (m, 1H), 3.50 3.38 (m, 3H), 3.27 (dd, J 3.8, 10.4 Hz,

1H), 2.98 (brs, 1H), 2.03 2.01 (m, 2H), 2.12 (s, 3H), 1.73 1.59 (m, 2H);

¹³C NMR (125 MHz, CDCl₃): d 172.5, 168.6, 165.9, 138.0, 137.9, 137.7,

128.8, 128.7, 128.4, 128.3, 128.1, 127.7, 115.3, 101.0, 98.0, 82.5, 79.0, 75.5, 75.1,

75.0, 74.8, 74.6, 71.1, 70.5, 69.6, 62.9, 62.6, 53.0, 40.6, 30.0, 28.6, 21.0; FAB

MS: m/z: calcd for C₃₆H₄₄ClN₃O₁₃: 761.2563; found: 761.2557 [M] .

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 2-O-allyloxycarbonyl-3-O-ben-

zyl-b-d-glucopyranosyluronate (76): Compound 65 (93 mg, 0.097 mmol)

was coevaporated three times with toluene, dried under vacuum for 1 h,

dissolved in toluene (5 mL) and 4-penten-1-ol (50 mL, 0.484 mmol) was

added. After cooling the mixture to 08C, TMSOTf (0.1m in toluene, 200 mL,

0.019 mmol) was added dropwise. The mixture was warmed to room

temperature and stirred for 30 min. Triethylamine (200 mL) was added and

the solvent was removed under reduced pressure. Flash chromatography on

silica gel (hexanes/EtOAc 85:15) afforded 76 (57 mg, 0.064 mmol, 66%) as

a colorless gum. [a]²_D⁴ 64.1 (c 1.65, CH₂Cl₂); IR (thin film on NaCl):

897.4067 [M] .

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl

3-O-benzyl-2-O-levulinoyl-b-d-glucopyranosyluronate

(73): Compound 72 (674 mg, 0.75 mmol) was dissolved in THF (80 mL).

Glacial acetic acid (20 mL) and HF/pyridine complex (12 mL) were added

and the solution was stirred at room temperature for 93 h. The mixture was

poured into EtOAc and washed with brine, water, sat. NaHCO₃and dried

over Na₂SO₄. After filtration, the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 8:2 ! 6:4)

afforded 73 (500 mg, 0.64 mmol, 85%) as a colorless oil. [a]²_D⁴ 0.3 (c

1.20, CH₂Cl₂); IR (thin film on NaCl): nÄ 3484, 3037, 2924, 2109, 1746,

nÄ 3065, 2953, 2857, 2106, 1755, 1454, 1369 cm^À1

;

¹H NMR (500 MHz,

CDCl₃): d 7.26 7.39 (m, 10H), 5.73 5.92 (m, 2H), 5.55 (d, J 3.7 Hz,

1H), 5.32 5.37 (m, 1H), 5.19 5.25 (m, 1H), 4.96 5.04 (m, 2H), 4.76 4.90

(m, 5H), 4.55 4.64 (m, 2H), 4.49 (d, J 7.6 Hz, 1H), 4.35 (dd, J 2.1,

11.9 Hz, 1H), 4.21 (at, J 9.2 Hz, 1H), 4.05 (dd, J 4.0, 12.2 Hz, 1H), 4.00

(d, J 9.5 Hz, 1H), 3.86 3.95 (m, 2H), 3.80 (s, 3H), 3.62 3.70 (m, 2H),

3.45 3.50 (m, 2H), 3.29 3.24 (m, 1H), 2.00 2.16 (m, 5H), 1.60 1.77 (m,

2H), 0.89 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d

171.0, 168.6, 154.0, 138.1, 138.0, 137.7, 131.4, 128.6, 128.5, 128.0, 127.8,

127.7, 127.4, 119.6, 115.2, 101.2, 97.8, 82.6, 80.2, 77.4, 75.3, 74.9, 74.7, 74.5,

71.2, 70.9, 69.7, 69.1, 63.9, 62.5, 52.9, 30.0, 28.7, 26.0, 21.1, 18.1, À3.5, À4.9;

1719, 1363 cm^À1

;

¹H NMR (500 MHz, CDCl₃): d 7.43 7.25 (m, 10H),

5.84 5.75 (m, 1H), 5.50 (d, J 3.7 Hz, 1H), 5.07 (dd, J 7.3, 8.5 Hz, 1H),

5.04 4.95 (m, 2H), 4.90 (d, J 11.3 Hz, 1H), 4.88 (d, J 11.3 Hz, 1H), 4.81

(d, J 10.7 Hz, 1H), 4.75 (d, J 10.7 Hz, 1H), 4.57 (dd, J 3.1, 12.5 Hz,

1H), 4.48 (d, J 7.3 Hz, 1H), 4.24 (dd, J 9.2, 8.9 Hz, 1H), 4.12 (dd, J 1.8,

12.5 Hz, 1H), 4.01 (d, J 9.5 Hz, 1H), 3.88 3.83 (m, 2H), 3.78 (s, 3H), 3.74

(dd, J 8.8, 10.0 Hz, 1H), 3.50 3.38 (m, 3H), 3.23 (dd, J 3.7, 10.4 Hz,

1H), 3.05 (d, J 3.4 Hz, 1H), 2.71 2.68 (m, 2H), 2.59 2.45 (m, 2H), 2.15

(s, 3H), 2.11 2.04 (m, 5H), 1.72 1.60 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃): d 206.2, 172.5, 171.5, 168.9, 138.1, 138.0, 137.8, 128.8, 128.6, 128.4,

128.3, 127.9, 127.8, 115.2, 101.2, 97.8, 82.5, 79.1, 75.5, 74.6, 74.5, 74.5, 73.7,

70.9, 70.5, 69.5, 62.9, 62.6, 52.9, 38.0, 30.1, 30.0, 28.7, 28.1, 21.0; FAB MS:

FAB MS : m/z: calcd for C₄₄H₆₁N₃O₁₄Si: 883.3923; found: 883.3930 [M] .

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl 2-O-allyloxycarbonyl-3-O-benzyl-b-d-glucopyranosyluro-

nate (77): Compound 76 (55 mg, 0.062 mmol) was dissolved in THF

(7 mL). Glacial acetic acid (1.75 mL) and HF/pyridine complex (1 mL)

were added and the solution was stirred at room temperature for 5 days.

The mixture was poured into EtOAc and washed with brine, water, sat.

NaHCO₃and dried over Na₂SO₄. After filtration, the solvent was removed

under reduced pressure. Flash chromatography on silica gel (hexanes/

EtOAc 7:3) afforded 77 (41 mg, 0.053 mmol, 85%) as a colorless oil.

[a]²_D⁴ 11.3 (c 1.02, CH₂Cl₂); IR (thin film on NaCl): nÄ 3470, 2922,

m/z: calcd for C₃₉H₄₉N₃O₁₄: 783.3215; found: 783.3206 [M] .

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroa-

cetyl-b-O-d-glucopyranosyluronate (74): Compound 57 (816 mg,

0.856 mmol) was coevaporated three times with toluene, dried in vacuo

for 1 h, dissolved in toluene (30 mL) and 4-penten-1-ol (450 mL, 4.36 mmol)

was added. After cooling the mixture to 08C, TMSOTf (0.1m in toluene,

1.72 mL, 0.17 mmol) was added dropwise. The mixture was warmed to

room temperature and stirred for 48 h. Triethylamine (1.7 mL) was added

1

2109, 1752, 1454, 1367, 1255 cm^À1; H NMR (500 MHz, CDCl₃): d 7.43

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0161 $ 20.00+.50/0

161

P. H. Seeberger et al.

FULL PAPER

7.27 (m, 10H), 5.92 5.84 (m, 1H), 5.82 5.74 (m, 1H), 5.51 (d, J 3.7 Hz,

1H), 5.37 5.31 (m, 1H), 5.23 5.26 (m, 1H), 5.04 4.95 (m, 2H), 4.90 4.79

(m, 4H), 4.77 (d, J 10.7 Hz, 1H), 4.65 4.56 (m, 3H), 4.49 (d, J 7.7 Hz,

1H), 4.21 (dd, J 8.9, 9.4 Hz, 1H), 4.11 (dd, J 1.9, 12.6 Hz, 1H), 3.99 (d,

J 9.5 Hz, 1H), 3.92 3.87 (m, 1H), 3.86 (at, J 9.0 Hz, 1H), 3.79 (s, 3H),

3.74 (dd, J 8.6, 10.3 Hz, 1H), 3.51 3.45 (m, 2H), 3.41 (dd, J 10.0,

8.7 Hz, 1H), 3.25 (dd, J 3.7, 10.4 Hz, 1H), 2.98 (brs, 1H), 2.21 2.06 (m,

5H), 1.74 1.60 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): d 172.5, 168.7,

154.0, 138.1, 138.0, 137.7, 131.4, 128.8, 128.6, 128.4, 128.3, 128.0, 127.8, 119.6,

115.2, 101.3, 98.0, 82.6, 79.1, 75.6, 75.1, 74.9, 74.5, 71.0, 70.5, 69.7, 69.1, 62.9,

62.6, 53.0, 30.0, 28.7, 21.0; FAB MS: m/z: calcd for C₃₈H₄₇N₃O₁₄: 769.3058;

d-glucopyranosyl-(1 ! 4)-methyl 3-O-benzyl-2-O-levulinoyl-b-O-d-gluco-

pyranosyluronate (80): Compound 70 (85 mg, 0.09 mmol) and 73 (60 mg,

0.07 mmol) were coevaporated with toluene and dried under vacuum for

1 h. The mixture was dissolved in CH₂Cl₂(2 mL) and after cooling to

À258C, TMSOTf (90 mL, 0.1m in CH₂Cl₂) was added. The mixture was

stirred for 4 h and then diluted with CH₂Cl₂and filtered through a pad of

Celite. The solvent was removed under reduced pressure and the residue

was purified by silica gel column chromatography (toluene/EtOAc

1

90:10 ! 80:20) to yield 80 (107 mg, 86%) as a syrup. H NMR (500 MHz,

CDCl₃) d 7.37 7.25 (m, 20H), 5.82 5.74 (m, 1H), 5.47 (d, J 3.6 Hz,

1H), 5.26 (d, J 4.6 Hz, 1H), 5.0.7 5.02 (m, 3H), 4.99 4.88 (m, 3H),

4.83 4.65 (m, 8H), 4.47 (d, J 7.3 Hz, 1H), 4.35 4.32 (m, 2H), 4.25 4.18

(m, 2H), 4.07 3.95 (m, 4H), 3.87 3.82 (m, 3H), 3.80 3.73 (m, 2H), 3.72

3.64 (m, 4H), 3.61 (s, 3H), 3.59 3.55 (m, 2H), 3.47 3.43 (m, 1H), 3.27 (dd,

J 3.9, 10.3 Hz, 1H), 3.23 (dd, J 3.3, 10.1, 1H), 2.68 (t, J 6.7 Hz, 2H),

2.57 2.44 (m, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.13 2.05 (m, 5H), 2.02 (s,

3H), 1.70 1.61 (m, 2H), 0.89 (m, 9H), À0.01 (s, 3H), À0.02 (s, 3H);

¹³C NMR (125 MHz, CDCl₃) d 206.2, 171.4, 170.9, 170.8, 170.1, 169.6,

168.9, 138.1, 137.9, 137.8, 137.7, 137.5, 128.7, 128.6, 128.4, 128.2, 128.0, 127.9,

127.8, 127.7, 127.4, 115.2, 101.2, 98.2, 97.7, 97.4, 82.6, 80.2, 78.1, 75.9, 75.2, 75.0,

74.8, 74.5, 74.0, 73.6, 72.7, 71.2, 70.8, 70.2, 70.0, 69.7, 69.5, 63.7, 63.2, 62.6,

61.9, 52.9, 52.1, 38.0, 30.1, 30.0, 28.7, 28.0, 26.0, 21.1, 21.0, 18.1, À3.53, À4.87;

found: 769.3051 [M] .

n-Pentenyl (3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-

a-d-glucopyranosyl)-(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-b-l-idopyra-

nosyluronate)-(1 ! 4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-gluco-

pyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-levulinyl-b-d-glucopyranosyl-

uronate (78): Compound 71 (290 mg, 0.33 mmol) and 73 (173 mg,

0.22 mmol) were coevaporated with toluene and dried under vacuum for

1 h. The mixture was dissolved in CH₂Cl₂(3 mL) and after cooling to

À258C, TMSOTf (330 mL, 0.1m in CH₂Cl₂) was added. The mixture was

stirred for 4 h and then diluted with CH₂Cl₂and filtered through a pad of

Celite. The solvent was removed under reduced pressure and the residue

was purified by silica gel column chromatography (toluene/EtOAc

FAB MS : m/z: calcd for C₇₆H₉₈N₆O₂₆Si: 1538.6300; found: 1538.6249 [M] .

1

90:10 ! 80:20) to yield 78 (289 mg, 88%) as a syrup. H NMR (500 MHz,

n-Pentenyl (3,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1 ! 4)-(6-O-

acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl

3-O-benzyl-2-O-levulinyl-b-d-glucopyranosyluronate (81): Compound 78

(170 mg, 0.11 mmol) was dissolved in THF (13 mL). Glacial acetic acid

(3 mL) and HF/pyridine complex (1.88 mL) were added and the solution

was stirred at room temperature for three days. The mixture was poured

into EtOAc and washed with brine, water, sat. NaHCO₃and dried over

Na₂SO₄. After filtration, the solvent was removed under reduced pressure.

Flash chromatography on silica gel (hexanes/EtOAc 5:3) afforded 81

(135 mg, 86%) as a pale yellow oil. ¹H NMR (500 MHz, CDCl₃) d 7.37

7.15 (m, 15H), 5.82 5.74 (m, 1H), 5.47 (d, J 3.6 Hz, 1H), 5.29 (d, J

4.3 Hz, 1H), 5.21 (t, J 10.1 Hz, 1H), 5.07 5.02 (m, 2H), 4.98 4.90 (m,

3H), 4.79 4.68 (m, 5H), 4.61 (d, J 4.3 Hz, 1H), 4.50 4.46 (m, 2H),

4.32 4.30 (m, 1H), 4.25 4.14 (m, 3H), 4.07 (t, J 5.2 Hz, 1H), 3.98 (d, J

9.5 Hz, 1H), 3.95 (t, J 5.5 Hz, 1H), 3.90 3.82 (m, 4H), 3.74 (t, J

10.1 Hz, 1H), 3.67 (s, 3H), 3.66 (s, 3H), 3.56 3.54 (m, 1H), 3.47 3.42

(m, 2H), 3.27 (dd, J 3.6, 10.3 Hz, 1H), 3.17 (dd, J 3.6, 10.7 Hz, 1H), 3.01

(brs, 1H), 2.68 (t, J 6.7 Hz, 2H), 2.57 2.43 (m, 2H), 2.26 2.08 (m, 17H),

1.70 1.60 (m, 2H); FAB MS: m/z: calcd for C₆₅H₈₀N₆NaO₂₇: 1399.4969;

CDCl₃) d 7.37 7.15 (m, 15H), 5.83 5.75 (m, 1H), 5.47 (d, J 4.0 Hz,

1H), 5.35 (d, J 4.9 Hz, 1H), 5.24 (dd, J 8.8, 10.7 Hz, 1H), 5.13 (d, J

3.3 Hz, 1H), 5.08 5.03 (m, 1H), 4.99 4.90 (m, 4H), 4.80 4.67 (m, 5H),

4.58 (d, J 4.9 Hz, 1H), 4.47 (d, J 7.3 Hz, 1H), 4.37 4.31 (m, 2H), 4.25

4.18 (m, 2H), 4.10 (at, J 6.1 Hz, 1H), 4.06 (dd, J 3.7, 12.2 Hz, 1H),

4.02 3.98 (m, 2H), 3.94 3.91 (m, 1H), 3.88 3.80 (m, 3H), 3.79 3.69 (m,

2H), 3.68 (s, 3H), 3.65 (s, 3H), 3.56 3.54 (m, 1H), 3.48 3.43 (m, 1H), 3.28

(dd, J 6.9, 10.3 Hz, 1H), 3.00 (dd, J 3.3, 10.7 Hz, 1H), 2.68 (t, J 6.7 Hz,

1H), 2.57 2.44 (m, 2H), 2.26 2.01 (m, 17H), 1.71 1.61 (m, 2H); ¹³C NMR

(125 MHz, CDCl₃) d 206.2, 171.4, 170.6, 170.2, 170.0, 169.8, 168.9, 138.1,

138.0, 137.8, 137.5, 129.2, 128.7, 128.6, 128.4, 128.2, 128.1, 127.9, 127.7, 125.5,

115.1, 101.2, 98.3, 98.2, 97.3, 82.5, 78.1, 76.2, 76.0, 75.1, 74.5, 74.4, 74.3, 73.5,

73.0, 72.5, 70.9, 70.4, 70.3, 69.6, 69.4, 68.9, 63.2, 62.4, 61.8, 61.5, 52.8, 52.3,

37.9, 30.0, 29.9, 28.7, 28.0, 25.7, 21.6, 21.5, 21.0, 20.9, 18.0, À3.9, À4.9; FAB

MS: m/z: calcd for C₇₁H₉₄N₆NaO₂₇Si: 1513.5834; found: 1513.5793

[MNa] .

n-Pentenyl (3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-

a-d-glucopyranosyl)-(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyra-

nosyluronate)-(1 ! 4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-a-d-gluco-

pyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroacetyl-b-d-gluco-

pyranosyluronate (79): Compound 71 (360 mg, 0.41 mmol) and 75

(186 mg, 0.24 mmol) were coevaporated with toluene and dried under

vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(3 mL) and after

cooling to À258C, TMSOTf (370 mL, 0.1m in CH₂Cl₂) was added. The

mixture was stirred for 4 h and then diluted with CH₂Cl₂and filtered

through a pad of Celite. The solvent was removed under reduced pressure

and the residue was purified by silica gel column chromatography (toluene/

EtOAc 90:10 ! 80:20) to yield 79 (331 mg, 0.22 mmol, 91%) as a syrup.

[a]²_D⁴ 40.0 (c 1.00, CHCl₃); IR (thin film on NaCl): nÄ 2930, 2107,

found: 1399.9923 [MNa] .

n-Pentenyl (3,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1 ! 4)-(6-O-

acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl

3-O-benzyl-2-O-monochloroacetyl-b-d-glucopyranosyluronate (82): Com-

pound 79 (60 mg, 0.21 mmol) was dissolved in THF (26 mL). Glacial acetic

acid (6.4 mL) and HF/pyridine complex (3.8 mL) were added and the

solution was stirred at room temperature for three days. The mixture was

poured into EtOAc and washed with brine, water, sat. NaHCO₃and dried

over Na₂SO₄. After filtration, the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 5:3) afforded

82 (247 mg, 85%). [a]²_D⁴ 20.4 (c 1.00, CHCl₃); IR (thin film on NaCl):

nÄ 3510, 2924, 2109, 1742 cm^À1; ¹H NMR (500 MHz, CDCl₃): d 7.39 7.27

(m, 20H), 5.80 5.75 (m, 1H), 5.44 (d, J 3.7 Hz, 1H), 5.31 (d, J 4.9 Hz,

1H), 5.22 (t, J 10.2 Hz, 1H), 5.16 5.12 (m, 2H), 5.07 4.83 (m, 5H),

4.73 4.64 (m, 5H), 4.57 4.52 (d, 1H), 4.37 (d, 1H), 4.34 4.25 (m, 3H),

4.08 (m, 1H), 4.00 3.95 (m, 3H), 3.90 3.81 (m, 6H), 3.80 3.72 (m, 2H),

3.69 (s, 3H), 3.67 (s, 3H), 3.60 3.53 (m, 1H), 3.51 3.42 (m, 1H), 3.27 (dd,

J 3.7, 10.7 Hz, 1H), 3.17 (dd, J 3.7, 10.7 Hz, 1H), 3.07 (brs, 1H), 2.14 (s,

3H), 2.13 (s, 3H), 2.12 (s, 3H), 2.05 2.01 (m, 2H), 2.08 (s, 3H), 1.74 1.60

(m, 2H); ¹³C NMR (125 MHz, CDCl₃): d 171.9, 171.3, 171.1, 170.3, 169.7,

168.6, 165.9, 138.0, 137.7, 137.4, 115.3, 100.9, 98.6, 98.4, 97.6, 82.5, 78.2, 75.8,

75.7, 75.1, 75.0, 74.9, 74.7, 74.6, 74.0, 73.8, 72.4, 71.4, 70.2, 70.1, 69.8, 69.5,

68.9, 63.3, 62.5, 61.9, 61.1, 52.9, 52.4, 40.6, 30.0, 28.7, 21.0, 21.0, 20.9; FAB

1728, 1538, 1362 cm^À1

;

¹H NMR (500 MHz, CDCl₃): d 7.39 7.27 (m,

20H), 5.77 5.74 (m, 1H), 5.43 (d, J 3.7 Hz, 1H), 5.35 (d, J 4.9 Hz, 1H),

5.23 (t, J 10.4 Hz, 1H), 5.12 (d, J 3.7 Hz, 1H), 5.07 (t, 1H), 5.01 4.84

(m, 6H), 4.73 4.64 (m, 4H), 4.57 (d, J 4.6 Hz, 1H), 4.47 (d, J 7.6 Hz,

1H), 4.34 (m, 2H), 4.24 4.18 (m, 2H), 4.09 (t, 1H), 4.05 (dd, J 3.3,

12.2 Hz, 1H), 4.00 (m, 2H), 3.92 3.81 (m, 6H), 3.79 3.72 (m, 2H), 3.69 (s,

3H), 3.67 (s, 3H), 3.56 3.54 (m, 1H), 3.45 3.42 (m, 1H), 3.31 (dd, J 3.7,

10.7 Hz, 1H), 3.00 (dd, J 3.7, 10.7 Hz, 1H), 2.13 (s, 6H), 2.10 (s, 3H),

2.05 2.01 (m, 2H), 2.03 (s, 3H), 1.69 1.60 (m, 2H), 0.84 (s, 9H), 0.04 (s,

3H), 0.02 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 171.0, 170.7, 170.2,

169.8, 168.6, 165.9, 137.9, 137.7, 137.5, 128.7, 128.4, 128.2, 128.1, 128.0, 127.9,

127.7, 127.3, 115.3, 101.0, 98.2, 97.7, 82.5, 78.2, 76.0, 75.1, 75.0, 74.7, 74.6, 74.4,

73.0, 72.6, 71.0, 69.8, 69.6, 69.0, 63.3, 62.4, 61.6, 52.4, 40.7, 30.0, 28.7, 26.6,

25.7, 21.8, 21.2, 18.0, À3.9, À4.8; FAB MS: m/z: calcd for C₆₈H₈₉ClN₆O₂₆Si:

1468.5284; found: 1468.5361 [M] .

MS: m/z: calcd for C₆₂H₇₅ClN₆O₂₆: 1354.4420; found: 1354.441 [M] .

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1 ! 4)-

(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

deoxy-a-d-glucopyranosyl-(1 ! 4)-(methyl

2-O-acetyl-3-O-benzyl-a-l-

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

162

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Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

methyl 3-O-benzyl-2-O-levulinoyl-b-O-pentenyl-d-glucopyranosyluronate

(83): Compound 80 (70 mg, 0.045 mmol) was dissolved in THF (5.3 mL).

Glacial acetic acid (1.3 mL) and HF/pyridine complex (0.8 mL) were added

and the solution was stirred at room temperature for three days. The

mixture was poured into EtOAc and washed with brine, water, sat.

NaHCO₃and dried over Na₂SO₄. After filtration, the solvent was removed

under reduced pressure. Flash chromatography on silica gel (hexanes/

EtOAc 5:3) afforded 83 (50 mg, 75%). ¹H NMR (500 MHz, CDCl₃) d

7.40 7.25 (m, 20H), 5.82 5.76 (m, 1H), 5.48 (d, J 3.6 Hz, 1H), 5.25 (d,

J 4.6 Hz, 1H), 5.07 4.89 (m, 6H), 4.84 (s, 2H), 4.80 4.68 (m, 5H), 4.63

(d, J 4.3 Hz, 1H), 4.51 (dd, J 3.5, 12.5 Hz, 1H), 4.47 (d, J 7.3 Hz, 1H),

4.34 4.32 (m, 1H), 4.26 4.20 (m, 2H), 4.09 (dd, J 2.1, 12.5 Hz, 1H), 4.04

(t, J 5.5 Hz, 1H), 4.00 (d, J 9.5 Hz, 1H), 3.95 (t, J 5.5 Hz, 1H), 3.88

3.82 (m, 3H), 3.78 3.71 (m, 3H), 3.69 3.64 (m, 4H), 3.57 (s, 3H), 3.48

3.43 (m, 2H), 3.27 (d, J 3.6, 10.1 Hz, 1H), 3.21 (d, J 3.4, 10.1 Hz, 1H),

2.91 (d, J 4.0 Hz, 1H), 2.68 (t, J 6.7 Hz, 2H), 2.56 2.45 (m, 2H), 2.14 (s,

6H), 2.12 2.05 (m, 8H), 1.69 1.62 (m, 2H); ¹³C NMR (125 MHz, CDCl₃)

d 206.2, 172.1, 171.4, 171.0, 170.0, 169.6, 168.9, 138.1, 138.0, 137.9, 137.5,

129.2, 129.0, 128.8, 128.7, 128.6, 128.4, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8,

115.1, 101.2, 98.4, 98.2, 97.4, 82.5, 78.8, 78.2, 77.4, 75.7, 75.3, 75.2, 75.0, 74.5,

74.4, 73.9, 73.6, 73.4, 71.1, 70.5, 70.2, 70.0, 69.7, 69.4, 63.2, 62.8, 62.7, 61.9,

52.8, 52.2, 37.9, 30.0, 28.7, 28.0, 21.1, 20.9; FAB MS: m/z: calcd for

acid (280 mL, 4.79 mmol) and TBAF (1m in THF) (4.4 mL, 4.4 mmol) was

added to the reaction mixture. After 30 min, the mixture was poured into

Et₂O (300 mL) and washed three times with brine. The organic layer was

dried over Na₂SO₄, filtered and the solvents were removed under reduced

pressure. The residue was dissolved in CH₂Cl₂(100 mL) and cooled to 08C.

Trichloroacetonitrile (6 mL, 59.8 mmol) and DBU (60 mL, 0.4 mmol) were

added at 08C. After stirring the mixture for 1 h at 08C it was concentrated

under reduced pressure. Flash chromatography on silica gel (hexanes/

EtOAc 8:2 then 6:4) afforded 90 (2.0 g, 3.45 mmol, 87%) as a mixture of

anomers (27/73) as a colorless oil. Compounds 90a and 90b were separated

by silica gel column chromatography (toluene/EtOAc 99:1 to 90:1).

Compound 90a: [a]²_D⁴ 57 (c 1, CH₂Cl₂); IR (thin film): nÄ 3300, 2112,

1

1744, 1152 cm^À1; H NMR (400 MHz, CDCl₃): d 8.78 (s, 1H), 7.39 7.29

(m, 5H), 6.44 (d, J 1.0 Hz, 1H), 4.72 4.89 (m, 2H), 4.02 4.25 (m, 3H),

3.77 (dd, J 3.5, 11.0 Hz, 1H), 2.69 2.74 (m, 2H), 2.39 2.58 (m, 2H), 2.17

(s, 3H), 2.05 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) d 206.3, 171.7, 170.9,

161.2, 137.6, 128.7, 128.3, 128.2, 96.7, 80.3, 75.3, 73.2, 69.7, 68.6, 68.0, 38.0,

28.0, 21.0; FAB MS: m/z: calcd for C₂₂H₂₅Cl₃N₄O₈: 601.0636; found:

601.0629. Compound 90b: [a]_D²⁴ À31 (c 1.00, CH₂Cl₂); IR (thin film):

nÄ 3329, 2111, 1744, 1718, 1676 cm^À1; ¹H NMR (400 MHz, CDCl₃): d 8.77

(s, 1H), 7.39 7.28 (m, 5H), 5.64 (d, J 8.0 Hz, 1H), 5.14 (t, J 10.0 Hz,

1H), 4.84 (d, J 11.0 Hz, 1H), 4.73 (d, J 11.0 Hz 1H), 4.26 (dd,

J 5.0, 8.0 Hz, 1H), 4.12 (dd, J 2.0, 10.0 Hz, 2H), 3.77 3.70 (m, 2H),

3.57 (t, J 9.0 Hz, 1H), 2.77 2.63 (m, 2H), 2.54 2.33 (m, 2H), 2.17 (s,

3H), 2.05 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) d 206.3, 171.7, 170.9,

161.2, 137.6, 128.7, 128.3, 128.2, 96.7, 80.3, 75.3, 73.2, 69.7, 65.6, 68.0, 38.0,

28.0, 21.0; FAB MS: m/z: calcd for C₂₂H₂₅Cl₃N₄O₈: 601.0636; found:

C₇₀H₈₄N₆NaO₂₆: 1447.5327; found: 1447.5378 [MNa] .

n-Pentenyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyrano-

syl)-(1 ! 4)-(methyl 3-O-benzyl-a-d-glucopyranosyluronate 1,2)-(3,6-di-

O-acetyl-2-azido-2-deoxy-a-d-glucopyranos-4-yl-monochloro

orthoace-

tate)-(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-

(1 ! 4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl 3-O-benzyl-2-O-monochloroacetyl-b-d-glucopyranosyluro-

nate (88): Compounds 58 (82 mg, 0.09 mmol) and 82 (70 mg, 0.05 mmol)

were coevaporated with toluene and dried under vacuum for 1 h. The

mixture was dissolved in CH₂Cl₂(1 mL) and after cooling to À258C,

TMSOTf (20 mL, 0.1m in CH₂Cl₂) was added. The mixture was stirred for

2 h and then diluted with CH₂Cl₂and filtered through a pad of Celite. The

solvent was removed under reduced pressure and the residue was purified

by silica gel column chromatography (toluene/EtOAc 95:5 ! 80:20) to

601.0626 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyrano-

syl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-b-l-idopyranosyluro-

nate (91): Compound 90 (666 mg, 1.14 mmol) and 35 (300 mg, 0.90 mmol)

were coevaporated three times with toluene and dissolved in anhydrous

CH₂Cl₂(30 mL). Freshly activated molecular sieves 4 ä (600 mg) were

added and the mixture was stirred at room temperature for 1 h. The

mixture was cooled to À788C and tert-butyldimethylsilyl trifluorometha-

nesulfonate 0.1m in CH₂Cl₂(1.2 mL, 0.12 mmol) was added dropwise. This

mixture was stirred for 4 h, while it was allowed to warm to room

temperature. Triethylamine (3 mL) was added and the mixture was filtered

through Celite and evaporated. Flash chromatography (hexanes/EtOAc

70:30) afforded 91 (558 g, 0.72 mmol, 83%) as a colorless foam. [a]²_D⁴ 34

(c 1.00, CH₂Cl₂); IR (thin film): nÄ 2109, 1744, 1720, 1235 cm^À1; ¹H NMR

(400 MHz, CDCl₃): d 7.40 7.27 (m, 10H), 5.36 (d, J 2.4 Hz, 1H), 5.10

(t, 1H), 4.73 (d, J 11.0 Hz, 1H), 4.44 (d, J 11.0 Hz, 1H), 4.23 (t, J

3.0 Hz, 1H), 4.15 (m, 2H), 4.18 4.09 (m, 2H), 3.78 (s, 3H), 3.48 (dd, J

3.0, 10.0 Hz, 1H), 2.67 (t, 2H), 3.34 3.48 (m, 2H), 2.16 (s, 3H), 2.06 (s,

3H), 1.64 (s, 3H), 1.43 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d 206.3,

171.6, 170.9, 169.1, 137.7, 137.1, 128.6, 128.5, 128.4, 128.1, 98.7, 97.2, 75.4, 75.0,

74.4, 73.1, 70.0, 69.4, 64.0, 62.0, 52.6, 37.9, 30.0, 28.3, 28.0, 26.3, 21.0; FAB

1

yield 88 (33 mg, 30%) as a syrup. H NMR (500 MHz, CDCl₃): d 7.39

7.16 (m, 30H), 6.43 (d, J 3.7 Hz, 1H), 6.10 (d, J 3.4 Hz, 1H), 5.73 5.82

(m, 1H), 5.34 (d, J 4.6 Hz, 1H), 5.30 (t, 1H), 5.09 5.01 (m, 1H), 4.98

4.82 (m, 8H), 4.71 4.56 (m, 8H), 4.47 (dd, J 0.9, 7.3 Hz, 1H), 4.40 (dd,

1H), 4.39 4.14 (m, 8H), 4.07 (t, 1H), 3.99 3.89 (m, 6H), 3.88 3.82 (m,

6H), 3.79 3.63 (m, 5H), 3.69 (s, 3H), 3.66 (s, 3H), 3.57 3.44 (m, 3H), 3.39

(dd, J 2.7, 9.5 Hz, 1H), 3.30 (dd, J 3.0, 10.3 Hz, 1H), 3.21 (dd, 1H), 2.36

(s, 3H), 2.14 (s, 3H), 2.10 (s, 3H), 2.06 2.02 (m, 2H), 2.01 (s, 3H), 1.92 (s,

3H), 1.66 1.61 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): d 170.6, 170.9,

169.8, 169.1, 138.5, 138.4, 138.1, 138.0, 136.9, 129.4, 129.3, 129.2, 129.0, 128.8,

128.7, 128.6, 128.5, 128.4, 128.2, 127.9, 122.5, 115.8, 101.5, 99.9, 83.0, 80.6,

78.4, 78.3, 77.9, 76.3, 75.8, 75.6, 75.4, 75.1, 73.4, 71.1, 70.3, 70.1, 63.9, 63.3,

53.4, 53.2, 53.2, 53.0, 41.2, 30.5, 29.2, 21.6, 21.5, 21.4; ES MS: m/z: calcd for

MS: m/z: calcd for C₃₇H₄₅N₃O₁₄: 778.2794; found: 778.2765 [M] .

C₁₀₀H₁₁₅Cl₂N₉O₃₈: 1082.9085; found: 1082.9109 [M2H]²

.

6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyrano-

syl-(1 ! 4)-methyl 3-O-benzyl-l-idopyranosyluronate (92): A solution of

91 (500 mg, 0.66 mmol) in dichloroacetic acid (15 mL, 60% aq) was stirred

for 3 h at room temperature. After dilution with water, the solution was

cooled to 08C and neutralized with NaHCO₃(9 g). The mixture was diluted

with CH₂Cl₂and the two phases were separated. The organic phase was

washed with sat. NaHCO₃and dried over MgSO₄. After filtration the

solvent was removed under reduced pressure to afford 92 (424 mg, 91%) as

a white solid. Compound 92 can be further purified by silica gel column

chromatography (hexane/EtOAc 6:5). FAB MS: m/z: calcd for

tert-Butyldimethylsilyl 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levuli-

noyl-b-d-glucopyranoside (89): Levulinic anhydride (1.86 g, 8.71 mmol)

was added to a solution of 6 (1.97 g, 4.35 mmol) and 4-(dimethylamino)-

pyridine (1.59 g, 13.05 mmol) in CH₂Cl₂(30 mL). After stirring at room

temperature for 5 h, the mixture was poured into EtOAc and extracted

with 1n HCl, brine and sat. NaHCO₃. The organic phase was dried over

Na₂SO₄, filtered and the solvents were removed under reduced pressure.

The crude product was purified by silica gel column chromatography

(hexanes/EtOAc 99:1 ! 90:10) to afford 89 (2.3 g, 4.18 mmol, 96%) as a

colorless oil. [a]²_D⁴ À58 (c 3.00, CH₂Cl₂); IR (thin film): nÄ 2110, 1746,

C₃₄H₄₁N₃O₁₄Si: 715.2589; found: 715.2593 [M] .

1719, 1363 cm^À1

;

¹H NMR (400 MHz, CDCl₃): d 7.20 7.10 (m, 5H),

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glu-

copyranosyl)-(1 ! 4)-methyl-3-O-benzyl-2-O-acetyl-b-d-idopyranosyluro-

nate (93): A solution of 92 (883 mg, 1.23 mmol) and imidazole (336 mg,

4.94 mmol) in CH₂Cl₂(10 mL) was cooled to À158C. tert-Butyldimethylsi-

lylchloride (279 mg, 1.60 mmol) was added and the reaction mixture was

kept at À158C. After 5 h tert-butyldimethylsilylchloride (186 mg,

1.23 mmol) was added. After 40 h water was added and the reaction

mixture was warmed to room temperature. After dilution with EtOAc the

mixture was washed with brine. The organic layer was dried over Na₂SO₄

and the solvent was removed under reduced pressure. Flash chromatog-

raphy on silica gel (hexanes/EtOAc 85:5 ! 70:30) afforded tert-butyldime-

4.82 4.80 (m, 1H), 4.66 4.48 (dd, J 11.5, 50 Hz, 2H), 4.39 (t, J 4 Hz,

1H), 4.01 3.91 (m, 2H), 3.42 3.38 (m, 1H), 3.26 3.22 (m, 2H), 2.59 2.46

(m, 2H), 2.19 2.31 (m, 2H), 1.99 (s, 3H), 1.89 (s,9H) (s, 6H); ¹³C NMR

(100 MHz, CDCl₃): d 206.5, 172.0, 171.0, 138.2, 128.8, 128.7, 128.3, 128.2,

127.4, 127.2, 97.6, 80.2, 76.0, 75.2, 72.5, 71.4, 70.4, 68.6, 67.3, 63.0, 38.2, 37.9,

30.1, 28.2, 25.9, 21.2, 18.4, À4.0, À4.9; FAB MS: m/z: calcd for

C₂₆H₃₉N₃O₈Si: 572.2398; found: 572.2391 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a/b-d-glucopyra-

nosyl trichloroacetimidate (90): A solution of 89 (2.18 g, 3.97 mmol) was

dissolved in THF (30 mL) and cooled to 08C. A solution of glacial acetic

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0163 $ 20.00+.50/0

163

P. H. Seeberger et al.

FULL PAPER

thylsilyl (6-O-acetyl-2-azido-3-benzyl-4-O-levulinoyl-2-deoxy-a-d-gluco-

pyranosyl)-(1 ! 4)-methyl-3-O-benzyl-b-d-idopyranosyluronate (752 mg,

0.92 mmol, 80%) as a colorless foam.

Benzylamine (250 mL, 2.25 mmol) was added in three portions, every 2 h, to

a solution of 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-

glucopyranosyl-(1 ! 4)-methyl 1,2-di-O-acetyl-3-O-benzyl-a/b-l-idopyra-

nosyluronate (300 mg, 0.37 mmol) in Et₂O (10 mL) at 08C and kept

overnight at À208C. The mixture was diluted with CH₂Cl₂, filtered and

washed with aqueous HCl (10%) The organic phase was dried over MgSO₄

and purified by silica gel column chromatography (hexanes/EtOAc

90:10 ! 80:20) to yield 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levu-

Pyridine (1.70 mL, 20.4 mmol), acetic anhydride (1.17 mL, 12.5 mmol) and

DMAP (7 mg, 0.06 mmol) were added to a solution of tert-butyldimethyl-

silyl (6-O-acetyl-2-azido-3-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

methyl-3-O-benzyl-b-d-idopyranosyluronate (520 mg, 0.63 mmol) in

CH₂Cl₂(12 mL). After 6 h at room temperature, water was added and

the mixture was stirred for 1 h. The organic phase was washed with sat.

NaHCO₃, water, and HCl (10% aqueous) and dried over MgSO₄. After

filtration, the solvent was removed under reduced pressure and the crude

was purified by silica gel column chromatography (hexanes/EtOAc 90:20)

to yield tert-butyldimethylsilyl (6-O-acetyl-2-azido-3-benzyl-2-deoxy-4-O-

levulinoyl-a-d-glucopyranosyl)-(1 ! 4)-methyl-2-O-acetyl-3-O-benzyl-b-

d-idopyranosyluronate (532 mg, 97%) as a colorless syrup. Hydrazine

hydrate (83 mL, 1.2 mmol) was added at 08C to a solution of tert-

butyldimethylsilyl (6-O-acetyl-2-azido-3-benzyl-2-deoxy-4-O-levulinoyl-a-

d-glucopyranosyl)-(1 ! 4)-methyl-2-O-acetyl-3-O-benzyl-b-d-idopyrano-

syluronate (200 mg, 0.23 mmol) in pyridine/acetic acid 3:2 (4 mL). After

15 min, acetone (0.5 mL) was added, and the mixture was stirred for 15 min

at room temperature. After evaporation the crude product was purified by

flash chromatography on silica gel (hexanes/EtOAc 85:15) to afford 93

(138 mg, 0.18 mmol, 78%) as a colorless foam.

linoyl-a-d-glucopyranosyl-(1 ! 4)-methyl

2-O-acetyl-3-O-benzyl-a/b-l-

idopyranosyluronate (226 mg, 80%).

A solution of 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-

glucopyranosyl-(1 ! 4)-methyl 2-O-acetyl-3-O-benzyl-a/b-l-idopyranosy-

luronate (400 mg, 0.292 mmol) and trichloroacetonitrile (1.1 mL,

10.4 mmol) in CH₂Cl₂(15 mL) containing freshly activated powdered 4 ä

molecular sieves (100 mg) was stirred 30 minutes at room temperature.

After cooling the solution to 08C DBU (8 mL, 0.052 mmol) was added. The

temperature was allowed to rise and after 1 h stirring, the mixture was

filtered through a pad of Celite and concentrated. The residue was purified

by silica gel column chromatography (hexanes/EtOAc 85:15) to yield 97

(281 mg, 61%) as a white solid. FAB MS: m/z: calcd for C₃₈H₄₃Cl₃N₄O₁₅

923.1682; found: 923.1691 [M] .

:

n-Pentenyl (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyl)-(1 ! 4)-6-O-

acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranoside (98): A mixture

of 97 (77 mg, 0.086 mmol) and 96 (60 mg, 0.066 mmol) was coevaporated

three times with toluene and dried under vacuum for 1 h. The mixture was

dissolved in CH₂Cl₂(1 mL) and was stirred for 30 min at room temperature

under nitrogen. After cooling the mixture to À458C, TMSOTf (86 mL, 0.1m

in CH₂Cl₂) was added dropwise. The mixture was stirred for 1 h and

triethylamine was added. The solvent was removed under reduced pressure

and the residue was purified by silica gel column chromatography

(hexanes/EtOAc 80:20 ! 60:40) to yield n-pentenyl (6-O-acetyl-2-azido-

3-O-benzyl-2-deoxy-4-O-levulinoyl-b-d-glucopyranosyl)-(1 ! 4)-(methyl

2-O-acetyl-3-O-benzyl-a-l-idopyranosyl)-(1 ! 4)-6-O-acetyl-2-azido-3-O-

benzyl-2-deoxy-a-d-glucopyranoside (44 mg, 0.038 mmol, 58%). [a]²_D⁴ 30

n-Pentenyl-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-b-d-

glucopyranoside (95): Compound 90a (380 mg, 0.66 mmol) was dissolved in

CH₂Cl₂(1 mL). Hexanes (10 mL) and 4-penten-1-ol (75 mL, 0.73 mmol)

were added. The reaction mixture was cooled to À108C before BF₃¥ EtO₂

(98.3 mL, 0.066 mmol) was added. The reaction mixture was stirred for

30 min and then triethylamine (1 mL) was added. The solvent was removed

under reduced pressure and the crude was purified by flash chromatog-

raphy on silica gel (hexanes/EtOAc 80:20) to obtain 95 (29 mg, 75%) as a

colorless oil. [a]²_D⁴ À30 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 2111, 1744,

1718 cm^À1; ¹H NMR (400 MHz, CDCl₃): d 7.38 7.28 (m, 5H), 5.88 5.77

(m, 1H), 5.08 4.97 (m, 3H), 4.81 (d, J 11.0 Hz, 1H), 4.66 (d, J 11.0 Hz

1H), 4.29 (d, J 8.0 Hz, 1H), 4.22 (dd, J 5.0, 7.0 Hz, 1H), 4.10 (dd, J 2.0,

10.0 Hz, 1H), 3.96 3.90 (m, 1H), 3.60 3.52 (m, 2H), 3.48 3.38 (m, 2H),

2.76 2.61 (m, 2H), 2.53 2.33 (m, 2H), 2.23 2.17 (m, 3H), 2.16 (s, 3H),

2.07 (s, 3H), 1.81 1.71 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) d 206.4,

171.7, 170.9, 138.1, 137.9, 128.6, 128.2, 128.1, 115.3, 102.4, 80.2, 75.1, 72.2,

70.2, 70.0, 66.2, 62.4, 38.0, 30.2, 30.0, 28.9, 28.0, 21.0; FAB MS: m/z: calcd for

(c 1.00, CH₂Cl₂); IR (thin film): nÄ 3430, 2110, 1742, 1233, 1037 cm^À1

;

¹H NMR (400 MHz, CDCl₃): d 7.41 7.28 (m, 15H), 5.87 5.77 (m, 1H),

5.19 (d, J 3.0 Hz, 1H), 5.10 4.97 (m, 3H), 4.95 (d, J 4.0 Hz, 1H), 4.91

(t, J 3.0 Hz, 1H), 4.79 4.63 (m, 5H), 4.46 (dd, J 2.0, 10.0 Hz, 1H), 4.25

(t, J 1 Hz, 1H), 4.20 (dd, J 2.0, 4.0 Hz, 1H), 4.15 (d, J 4.0 Hz,

1H), 4.07 4.01 (m, 2H), 3.94 3.77 (m, 6H), 3.57 3.53 (m, 1H),

3.51 (s, 3H), 3.47 3.41 (m, 2H), 3.37 3.28 (m, 2H), 2.71 2.67 (m,

2H), 2.52 2.36 (m, 2H), 2.16 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.03

(s, 3H), 1.80 1.68 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d 206.3,

171.6, 170.9, 170.8, 170.1, 169.3, 138.2, 138.1, 137.5, 137.4, 128.7, 128.6, 128.4,

128.3, 128.3, 128.1, 128.0, 127.7, 127.6, 115.3, 102.4, 98.2, 97.4, 81.2, 77.3, 75.2,

74.8, 70.1, 69.0, 68.8, 68.4, 66.4, 62.9, 62.5, 61.7, 52.2, 37.9, 29.9, 28.8, 27.9,

21.2, 20.9 FAB MS: m/z: calcd for C₅₆H₆₈N₆O₂₀: 1167.4380; found: 1167.4415

C₂₅H₃₃N₃O₈: 526.2160; found: 526.2149 [M

n-Pentenyl-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-b-d-glucopyranoside

(96): A solution of hydrazine acetate (8.1 mg, 0.088 mmol) in methanol

(1 mL) was added to a solution of 95 (90 mg, 0.08 mmol) in CH₂Cl₂(8 mL).

After stirring the reaction mixture under nitrogen atmosphere for 1 h,

acetone (1.5 mL) was added and the solvent was removed under reduced

pressure and the crude product was purified by flash chromatography on

silica gel (hexanes/EtOAc 60:40) to afford 96 (29 mg, 0.007 mmol, 91%) as

a colorless oil. [a]²_D⁴ À30 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 3426, 2109,

1740, cm^À1; ¹H NMR (400 MHz, CDCl₃): d 7.41 7.31 (m, 5H), 5.88 5.78

(m, 1H), 5.04 4.97 (m, 3H), 4.94 (d, J 11.0 Hz, 1H), 4.76 (d, J 11.0 Hz

1H), 4.45 (dd, J 4.0, 8.0 Hz, 1H), 4.30 4.23 (m, 2H), 3.97 3.92 (m, 1H),

3.60 3.22 (m, 2H), 3.48 3.38 (m, 2H), 2.63 (s, 1H), 2.21 2.12 (m, 2H),

2.11 (s, 3H), 1.84-.70 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d 129.1,

128.7, 115.5, 102.8, 82.5, 75.7, 74.1, 70.3, 66.3, 63.4, 30.5, 29.1, 21.3; FAB MS:

[M] .

A solution of hydrazine acetate (8.1 mg, 0.088 mmol) in methanol (1 mL)

was added to a solution of n-pentenyl (6-O-acetyl-2-azido-3-O-benzyl-2-

deoxy-4-O-levulinoyl-b-d-glucopyranosyl)-(1 ! 4)-(methyl 2-O-acetyl-3-

O-benzyl-a-l-idopyranosyl)-(1 ! 4)-6-O-acetyl-2-azido-3-O-benzyl-2-de-

oxy-a-d-glucopyranoside (90 mg, 0.08 mmol) in CH₂Cl₂(8 mL). After

stirring the reaction mixture under nitrogen atmosphere for 1 h, acetone

(1.5 mL) was added and the solvent was removed under reduced pressure

and the crude was purified by flash chromatography on silica gel (hexanes/

EtOAc 60:40) to afford 98 (64 mg, 0.062 mmol, 77%) as a colorless foam.

[a]²_D⁴ À5 (c 1.00, CH₂Cl₂); IR (thin film): nÄ 3426, 2109, 1741,

m/z: calcd for C₂₀H₂₇N₃O₆: 428.1702; found: 428.1791 [M] .

6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyrano-

syl-(1 ! 4)-methyl 2-O-acetyl-3-O-benzyl-a/b-l-idopyranosyl) trichloro-

acetimidate (97): Pyridine (0.23 mL, 2.7 mmol), acetic anhydride

(0.16 mL, 1.7 mmol) and DMAP (1 mg, 0.086 mmol) were added to a

solution of 92 (600 mg, 0.86 mmol) in CH₂Cl₂(15 mL). After 6 h at room

temperature, water was added and the mixture was stirred for 1 h. The

organic phase was washed with sat. NaHCO₃, water, and HCl (10%

aqueous) and dried over MgSO₄. After filtration, the solvent was removed

under reduced pressure and the crude product was purified by silica gel

column chromatography (hexanes/EtOAc 90:20) to yield 6-O-acetyl-2-

azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyranosyl-(1 ! 4)-

methyl 1,2-di-O-acetyl-3-O-benzyl-a/b-l-idopyranosyluronate (671 mg,

1235 cm^À1

;

¹H NMR (400 MHz, CDCl₃): d 7.41 7.28 (m, 15H), 5.87

5.77 (m, 1H), 5.20 (d, J 3.0 Hz, 1H), 5.07 4.97 (m, 2H), 4.94 (d, J

4.0 Hz, 1H), 4.91 (t, J 3.0 Hz, 1H), 4.88 4.67 (m, 7H), 4.54 (dd, J 3.0,

11.0 Hz, 1H), 4.44 (dd, J 2.0, 10.0 Hz, 1H), 4.26 4.20 (m, 2H), 4.14 4.04

(m, 2H), 3.95 3.84 (m, 3H), 3.83 3.63 (m, 2H), 3.57 3.53 (m, 2H), 3.52

(s, 3H), 3.47 3.28 (m,4H), 3.21 (dd, J 4.0, 7.0 Hz, 1H), 2.87 (d, J

4.0 Hz, 1H), 2.16 2.13 (m, 1H), 2.09 (s, 3H), 2.08 (s, 6H), 1.78 1.70 (m,

2H); ¹³C NMR (100 MHz, CDCl₃): d 172.3, 170.2, 169.5, 138.2, 138.1,

137.9, 137.5, 128.9, 128.7, 128.5, 128.5, 128.4, 128.3, 128.1, 127.9, 127.7, 115.3,

102.4, 98.3, 98.1, 81.3, 78.9, 75.3, 74.0, 73.3, 73.2, 71.2, 70.6, 69.2, 68.8, 66.4,

63.0, 62.7, 62.5, 52.2, 30.2, 28.9, 21.1, 21.0; FAB MS: m/z: calcd for

95%) as

822.2692; found: 822.2703 [M] .

a

colorless syrup. FAB MS: m/z: calcd for C₃₈H₄₅N₃O₁₆

:

C₅₁H₆₂N₆O₁₈: 1069.4013; found: 1069.4055 [M] .

164

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0164 $ 20.00+.50/0

Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

2-Azido-3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyranosyl tri-

chloroacetimidate (100): Levulinic anhydride (1.4 g, 6.5 mmol) was added

to a solution of 8 (1.4 g, 3.1 mmol) and DMAP (1.2 g, 9.8 mmol) in CH₂Cl₂

(30 mL). After stirring at room temperature for 5 h, the mixture was

poured into EtOAc and extracted with 1n HCl, brine and sat. NaHCO₃.

The organic phase was dried over Na₂SO₄, filtered and the solvents were

removed under reduced pressure. The mixture was purified by silica gel

column chromatography (hexanes/EtOAc 8:2) to afford tert-butyldime-

thylsilyl 2-azido-3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-b-d-glucopyrano-

side (1.81 g, 3.0 mmol, 98%) as a colorless foam. [a]²_D⁴ 22 (c 1.00,

(500 MHz, CDCl₃): d 7.38 7.19 (m, 15H), 5.74 (d, J 4.0 Hz, 1H), 5.11

(d, J 3.6 Hz, 1H), 4.88 (d, J 11.1 Hz, 1H), 4.83 (d, J 11.1 Hz, 1H), 4.67

(s, 2H), 4.61 (d, J 12.0 Hz, 1H), 4.51 (d, J 12.0 Hz, 1H), 4.49 (d, J

6.9 Hz, 1H), 4.48 4.20 (m, 2H), 4.05 (t, J 3.7 Hz, 1H), 3.84 3.71 (m,

4H), 3.65 (s, 3H), 3.63 3.55 (m, 1H), 3.33 (dd, J 3.3, 10.1 Hz, 1H), 2.82

(s, 1H), 1.63 (s, 3H), 1.35 (s, 3H); ¹³C NMR (MHz, CDCl₃) d 170.2, 138.2,

137.8, 137.4, 128.8, 128.7, 128.7, 128.3, 128.2, 128.1, 127.9, 111.0, 98.0, 96.1,

79.3, 77.5, 76.9, 76.5, 75.7, 75.2, 73.9, 73.7, 72.6, 72.2, 71.6, 70.5, 69.7, 62.6,

52.6, 27.4, 25.9; FAB MS: m/z: calcd for C₃₇H₄₃N₃NaO₁₁: 728.2790,; found:

728.2792 [M] .

CH₂Cl₂); IR (thin film): nÄ 2111, 1468, 1365, 1752, cm^À1

;

¹H NMR

2,3,4-O-Benzoyl-b-d-glucopyranosyluronate-(1 ! 4)-O-(2-azido-3,6-di-O-

benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-1,2-O-iso-

propylidene-a-d-glucopyranosyluronate (102): Compounds 94 (30 mg,

0.045 mmol) and 101 (20 mg, 0.03 mmol) were coevaporated three times

with toluene and dried under vacuum for 1 h. The mixture was dissolved in

CH₂Cl₂(1 mL) and was stirred for 30 min at room temperature under

nitrogen. After cooling the mixture to À258C, anhydrous TMSOTf (45 mL,

0.1m in CH₂Cl₂) was added. The mixture was stirred for 2 h at À108C and

overnight at À208C, and then diluted with CH₂Cl₂and filtered. The solvent

was removed under reduced pressure and the residue was purified by silica

gel column chromatography (toluene/EtOAc 95:5 ! 80:20) to yield 102

(300 MHz, CDCl₃): d 7.38 7.27 (m, 10H), 4.80 4.85 (m, 1H), 4.63 (d,

J 11.1 Hz, 1H), 4.46 (d, J 11.1 Hz, 1H), 4.42 4.31 (m, 3H), 3.40 3.35

(m,3H), 3.25 3.20 (m, 2H), 2.51 2.35(m, 2H), 2.22 2.08 (m, 2H), 1.91 (s,

3H), 0.7 (s, 9H), 0.1 (s, 6H); ¹³C NMR (75 MHz, CDCl₃) d 206, 172.0,

138.5, 138.4, 129.0, 128.8, 128.8, 128.7, 128.6, 128.5, 128.3, 128.2, 128.1, 128.0,

97.6, 97.6, 82.7, 80.4, 75.1, 74.1, 73.9, 72.3, 71.5, 70.0, 68.7, 68.5, 66.9, 38.3, 38.1,

30.3, 30.2, 28.4, 28.2, 26.0, 25.9, 18.4, À3.8, À4.8; FAB MS: m/z: calcd for

C₃₁H₄₃N₃NaO₇Si: 620.2762; found: 620.2738 [MNa] .

tert-Butyldimethylsilyl 2-azido-3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-b-

d-glucopyranoside (1.4 mg, 2.4 mmol) was dissolved in THF (25 mL) and

cooled to 08C. TBAF (1.0m in THF, 2.8 mL) and glacial acetic acid (180 mL,

2.5 mmol) were added dropwise simultaneously. The reaction mixture was

warmed to room temperature, stirred for 1.5 h and after dilution with

CH₂Cl₂was washed with sat. NaHCO₃. The organic phase was dried over

MgSO₄and after filtration the solvent was removed under reduced

pressure. The crude material was dried by coevaporation with toluene and

under vacuum. Trichloroacetonitrile (2.6 mL, 25 mmol) and freshly acti-

vated 4 ä powdered molecular sieves (300 mg) were added at a solution of

the crude material in CH₂Cl₂(25 mL) and the mixture was stirred for

30 minutes at room temperature. The reaction mixture was cooled to 08C

and DBU (37 mL, 0.25 mmol) was added. After five minutes the ice bath

was removed and the reaction mixture was stirred for 1 h. After filtration

through a pad of Celite the solvent as removed under reduced pressure.

Flash chromatography on silica gel (hexanes/EtOAc 85:15 with 5%

triethylamine) afforded 100 (1.46 g, 2.34 mmol, 86%). FAB MS: m/z:

(20 mg, 59%) as

a

syrup. [a]²_D⁴ 91 (c 1.00, CH₂Cl₂); ¹H NMR

(400 MHz, CDCl₃): d 7.83 7.80 (m, 9H), 7.54 7.27 (m, 21H), 5.61

5.57 (m, 3H), 5.52 5.47 (m, 1H), 5.27 (d, J 10.9 Hz, 1H), 5.07 (d, J

3.7 Hz, 1H), 4.86 (d, J 5.5 Hz, 1H), 4.75 (d, J 8.0 Hz, 1H), 4.67 (d, J

10.9 Hz, 1H), 4.63 (s, 1H), 4.36 (d, J 12.1 Hz, 1H), 4.30 (d, J 7.7 Hz,

1H), 4.20 4.18 (m, 1H), 4.11 4.01 (m, 1H), 3.99 3.88 (m, 2H), 3.82 3.66

(m, 2H), 3.64 3.60 (m, 1H), 3.51 (s, 3H), 3.32 (m, 3H), 3.21 (s, 3H), 1.43 (s,

3H), 1.31 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.1, 167.0, 165.7,

165.3, 164.8, 137.3, 133.7, 133.6, 133.5, 130.0, 129.9, 129.9, 129.0, 128.9, 128.8,

128.7, 128.5, 128.5, 128.2, 128.1, 127.7, 110.8, 76.9, 74.0, 73.1, 72.2, 71.7, 70.8,

70.7, 70.6, 67.1, 62.6, 52.9, 52.1, 27.1, 25.8; FAB MS: m/z: calcd for

C₆₅H₆₅N₃O₂₀: 1230.4054; found: 1230.4050 [M] .

Methyl 2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-b-l-idopyrano-

syluronate trichloroacetimidate (103): tert-Butyldimethylsilyl trifluorome-

thanesulfonate (448 mL, 1.95 mmol) was added under argon to a solution of

methyl

3-O-benzyl-1,2-O-isopropylidene-a-d-glucofuranosyluronate^[2]

calcd for C₂₇H₂₉Cl₃N₄NaO₇: 649.0994; found: 649.1016 [MNa] .

(600 mg, 1.77 mmol) and 2,6-lutidine (522 mL, 4.48 mmol) in CH₂Cl₂

(4 mL). After stirring for 1 h at room temperature, the reaction mixture

was quenched with the addition of sat. NaHCO₃. The mixture was diluted

with CH₂Cl₂, the two phases were separated and the aqueous phase was

extracted four times with CH₂Cl₂. The combined organic phases were dried

over MgSO₄and after filtration the solvent was removed under reduced

pressure. Flash chromatography on silica gel (hexanes/EtOAc 20:1)

afforded methyl 3-O-benzyl-4-O-tert-butyldimethylsilyl-1,2-O-isopropyli-

dene-b-l-idopyranosyluronate (786 mg, 98%) as a colorless solid. ¹H NMR

(500 MHz, CDCl₃): d 7.39 7.32 (m, 5H), 5.32 (d, J 2.4 Hz, 1H), 4.68 (d,

J 11.9 Hz, 1H), 4.62 (d, J 11.9 Hz, 1H), 4.38 (d, J 1.2 Hz, 1H), 4.06 (m,

1H), 3.94 (brs, 1H), 3.82 (m, 1H), 3.76 (s, 3H), 1.59 (s, 3H), 1.38 (s, 3H),

0.82 (s, 9H), À0.06 (s, 3H), À0.07 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d

169.8, 137.5, 128.8, 128.4, 128.1, 112.3, 96.9, 75.3, 75.1, 72.9, 72.7, 68.0, 52.3,

28.3, 26.6, 25.6, 18.0, À4.4, À5.3; FAB MS: m/z: calcd for C₂₃H₃₆O₇Si:

2-Azido-3,6-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-methyl 3-O-

benzyl-1,2-O-isopropylidene-a-d-glucopyranosyluronate (101): Com-

pound 100 (242 mg, 0.39 mmol) and 31 (100 mg, 0.29 mmol) were

coevaporated three times with toluene and dissolved in CH₂Cl₂(1 mL).

Freshly activated molecular sieves 4 ä (100 mg) were added and the

mixture was stirred at room temperature for 1 h. This mixture was cooled to

À258C and tert-butyldimethylsilyl trifluoromethanesulfonate 0.1m in

anhydrous CH₂Cl₂(39 mL, 0.039 mmol) was added dropwise. This mixture

was stirred for 2.5 h, while it was allowed to warm to room temperature.

Triethylamine (0.5 mL) was added and the mixture was filtered through

Celite and evaporated. Flash chromatography (hexanes/EtOAc 85:15)

afforded 2-azido-3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyra-

nosyl-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glucopyrano-

syluronate (211 mg, 0.26 mmol, 89%) as a colorless foam. [a]²_D⁴ 52

(c 0.8, CH₂Cl₂); IR (thin film on NaCl): nÄ 2955, 2935, 2858, 2110,

1

1753, cm^À1; H NMR (500 MHz, CDCl₃): d 7.38 7.19 (m, 15H), 5.79 (d,

452.2230; found: 452.2211 [M] .

J 3.7 Hz, 1H), 5.22 5.15 (m, 2H), 4.80 (d, J 11.2 Hz, 1H), 4.66 4.58

(m, 4H), 4.48 (d, J 2.6 Hz, 1H), 4.22 4.20 (m, 2H), 4.06 (t, J 3.7 Hz,

1H), 4.00 3.8 (m, 2H), 3.62 (s, 3H), 3.57 3.47 (m, 2H), 3.35 (dd, J 3.4,

10.1 Hz, 1H), 2.63 2.56 (m, 2H), 2.38 2.14 (m, 2H), 2.12 (s, 3H), 1.61 (s,

3H), 1.39 (s, 3H); ¹³C NMR (MHz, CDCl₃): d 206.6, 171.8, 170.3, 138.3,

128.1, 137.5, 128.9, 128.8, 128.7, 128.5, 128.3, 128.3, 128.2, 127.9, 111.3, 98.3,

95.9, 76.0, 75.8, 74.8, 74.2, 74.0, 72.5, 72.2, 71.2, 70.1, 68.8, 63.0, 52.7, 38.1,

30.2. 28.2, 27.8, 26.2; FAB MS: m/z: calcd for C₄₂H₄₉N₃NaO₁₃: 826.3158;

A solution of methyl 3-O-benzyl-4-O-tert-butyldimethylsilyl-1,2-O-isopro-

pylidene-b-l-idopyranosyluronate (800 mg, 1.77 mmol) in dichloroacetic

acid (40 mL, 60% aq) was stirred at room temperature for 3 h, diluted with

water and neutralized with NaHCO₃(24 g). The aqueous phase was washed

three times with CH₂Cl₂and the combined organic phases were dried over

MgSO₄. After filtration, the solvent was removed under reduced pressure

to afford methyl 3-O-benzyl-4-O-tert-butyldimethylsilyl-l-idopyranosylur-

onate (671 mg, 1.62 mmol, 92%) as a white solid. The compound can be

further purified by silica gel column chromatography (hexanes/EtOAc

70:30). FAB MS: m/z: calcd for C₂₀H₃₂O₇Si: 412.1917; found: 412.1896

found: 826.3158 [M] .

Hydrazine hydrate (83 mL, 1.2 mmol) was added at 08C to a solution

2-azido-3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-a-d-glucopyranosyl)-

(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glucopyranosyluro-

nate (200 mg, 0.25 mmol) in pyridine/acetic acid 3:2 (4 mL). After

15 min, acetone (0.5 mL) was added, and the mixture was stirred for

15 min at room temperature. After evaporation the crude product was

purified by flash chromatography (hexanes/EtOAc 85:15) afforded 101

(135 mg, 0.19 mmol, 77%) as a colorless foam. [a]²_D⁴ 61 (c 1, CH₂Cl₂);

[M] .

Pyridine (3.0 mL, 36 mmol), acetic anhydride (2.0 mL, 21.7 mmol) and

DMAP (17 mg, 0.145 mmol) were added to a solution of 3-O-benzyl-4-O-

tert-butyldimethylsilyl-l-idopyranosyluronate (600 mg, 1.45 mmol) in

CH₂Cl₂(20 mL). The solution was stirred at room temperature for 6 h,

water was added and the mixture was stirred for one additional hour. The

organic phase was washed with saturated solution of NaHCO₃, water and

10% HCl, dried over MgSO₄and filtered. The solvent was removed under

IR (thin film on NaCl): nÄ 2953, 2934, 2852, 2110, 1757 cm^À1

;

¹H NMR

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0165 $ 20.00+.50/0

165

P. H. Seeberger et al.

FULL PAPER

reduced pressure and the residue was purified by silica gel column

chromatography (hexanes/EtOAc 90:20) to yield methyl 1,2-di-O-acetyl-3-

O-benzyl-4-O-tert-butyldimethylsilyl-a/b-l-idopyranosyluronate (708 mg,

1.42 mmol, 98%) as a colorless syrup. FAB MS: m/z: calcd for C₂₄H₃₆O₉Si:

(m, 2H), 2.00 (s, 3H), 1.57 1.17 (m, 2H), 0.83 (s, 9H), À0.01 (s, 3H), À0.06

(s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 206.2, 171.4, 170.9, 170.4, 170.1,

168.9, 138.1, 138.2, 138.0, 137.8, 129.2, 128.6, 128.5, 128.4, 128.3, 128.0, 127.9,

127.8, 127.7, 115.1, 101.2, 98.0, 97.3, 82.5, 77.9, 77.4, 77.2, 76.2, 75.1, 74.5, 73.9,

73.5, 73.2, 71.7, 69.8, 69.4, 62.9, 61.7, 52.8, 51.8, 37.9, 30.0, 28.7, 28.0, 25.7, 21.1,

21.0, 27.9, À4.6, À5.1; FAB MS: m/z: calcd for C₆₁H₈₁N₃O₂₁Si: 1219.5132;

496.2129; found: 496.2129 [M] .

Benzylamine (600 mL, 5.4 mmol) was added to a solution of methyl 1,2-di-

O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-a/b-l-idopyranosyluro-

nate (630 mg, 1.27 mmol) in Et₂O (40 mL) at 08C. After 6 h, the mixture

was diluted with CH₂Cl₂, filtered and washed with aqueous HCl (10%).

The organic phase was dried over MgSO₄and after filtration, silica gel

column chromatography (hexanes/EtOAc 90:10 ! 80:20) afforded methyl

2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-l-idopyranosyluronate

(432 mg, 0.95 mmol, 75%) as a white solid. FAB MS: m/z: calcd for

found: 1219.5107 [M] .

n-Pentenyl (methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-

(1 ! 4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl

3-O-benzyl-2-O-levulinoyl-b-d-glucopyranosyluronate

(106): Compound 105 (45 mg, 0.037 mmol) was dissolved in THF (5 mL).

Glacial acetic acid (1.3 mL) and HF/pyridine complex (0.8 mL) were added

and the solution was stirred at room temperature for three days. The

mixture was poured into Et₂O and washed with brine, water, sat. NaHCO₃

and dried over Na₂SO₄. After filtration, the solvent was removed under

reduced pressure. Flash chromatography on silica gel (hexanes/EtOAc 7:3)

afforded 106 (33 mg, 0.03 mmol, 82%) as a colorless foam. ¹H NMR

(500 MHz, CDCl₃): d 7.39 7.27 (m, 15H), 5.49 (d, J 3.9 Hz, 1H), 5.07

4.95 (m, 4H), 4.90 (brs, 1H), 4.85 (brs, 1H), 4.83 4.73 (m, 4H), 4.65 4.62

(m, 4H), 4.47 (d, J 7.0 Hz, 1H), 4.35 (dd, 1H), 4.24 4.19 (m, 2H), 3.99 (d,

J 9.0 Hz, 2H), 3.96 (m, 1H), 3.87 3.81 (m, 3H), 3.74 3.71 (m, 2H), 3.66

(s, 3H), 3.58 (dd, 1H), 3.48 (s, 3H), 3.48 3.43 (m, 1H), 3.28 (dd, J 3.7,

10.4 Hz, 1H), 2.68 (t, 2H), 2.60 2.45 (m, 3H), 2.14 2.07 (m, 11H), 1.68

1.60 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): d 206.2, 171.4, 170.8, 169.7,

169.3, 168.8, 138.1, 137.8, 137.8, 137.3, 129.2, 128.8, 128.6, 128.4, 128.3, 127.9,

127.7, 127.7, 115.1, 101.2, 98.3, 97.6, 82.5, 78.4, 77.4, 75.1, 75.1, 74.8, 74.7, 74.6,

74.4, 73.6, 72.9, 69.8, 69.5, 69.0, 67.9, 67.7, 63.5, 61.9, 52.8, 52.3, 37.9, 30.0,

29.9, 28.7, 28.0, 21.6, 21.1; FAB MS: m/z: calcd for C₅₅H₆₇N₃O₂₁: 1105.4267;

C₂₂H₃₄O₈Si: 454.2023; found: 454.2016 [M] .

A solution of methyl 2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-l-

idopyranosyluronate (500 mg, 1.10 mmol) in CH₂Cl₂(25 mL) was cooled to

08C. Trichloroacetonitrile (1.7 mL, 17.0 mmol) and DBU (25 mL,

0.16 mmol) were added and after stirring the mixture at 08C for 1 h, the

solvent was removed under reduced pressure. Flash chromatography on

silica gel (hexanes/EtOAc 85:15 ! 70:30) afforded 103 (606 mg, 92%) as a

colorless foam. ¹H NMR (500 MHz, CDCl₃) d 8.65 (s, 1H), 7.38 7.31 (m,

5H), 6.41 (s, 1H), 5.11 (m, 1H), 4.90 (d, J 1.8 Hz, 1H), 4.80 (d, J

11.9 Hz, 1H), 4.61 (d, J 11.9 Hz, 1H), 4.12 (m, 1H), 3.78 (s, 3H), 3.66

(brs, 1H), 1.59 (s, 3H), 1.38 (s, 3H), 2.08 (s, 3H), 0.83 (s, 9H), À0.05 (s, 3H),

À0.13 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d 170.7, 170.0, 160.8, 138.0,

129.7, 129.1, 128.9, 128.7, 96.1, 74.2, 72.6, 71.3, 68.8, 66.1, 53.0, 26.2, 26.1, 21.7,

18.5, À3.9, À4.9; FAB MS: m/z: calcd for C₂₄H₃₄Cl₃NO₈Si: 597.119; found:

597.1143 [M] .

found: 1105.4252 [M] .

Methyl 2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-b-l-idopyrano-

syluronate-(1 ! 4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-a-d-glucopyrano-

syl)-(1 ! 4)-methyl 3-O-benzyl-1,2-O-isopropylidene-a-d-glucopyranosyl-

uronate (104): Compounds 103 (27 mg, 0.045 mmol) and 101 (20 mg,

0.03 mmol) were coevaporated three times with toluene and dried under

vacuum for 1 h. The mixture was dissolved in CH₂Cl₂(1 mL) and was

stirred for 30 min at room temperature under nitrogen. After cooling the

mixture to À258C, TMSOTf (45 mL, 0.1m in CH₂Cl₂) was added, The

mixture was stirred for 2 h at À108C and overnight at À208C, and then

diluted with CH₂Cl₂and filtered. The solvent was removed under reduced

pressure and the residue was purified by silica gel column chromatography

(toluene/EtOAc 95:5 ! 80:20) to yield 104 (23 mg, 71%) as a syrup.

[a]²_D⁴ 82 (c 1.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): d 7.40 7.19

(m, 20H), 5.73 (d, J 4.0 Hz, 1H), 5.36 (d, J 4.9 Hz, 1H), 5.01 (d, J

3.6 Hz, 1H), 4.92 4.86 (m, 2H), 4.68 (s, 2H), 4.65 (s, 2H), 4.60 4.52 (m,

4H), 4.21 4.16 (m, 2H), 4.04 3.94 (m, 3H), 3.84 3.68(m, 5H), 3.63 3.60

(m, 3H), 3.57 (s, 3H), 3.47 (s, 1H), 3.30 (dd, J 3.1, 10.3 Hz, 1H), 1.82 (s,

1H), 1.56 (s, 3H), 1.35 (s, 3H), 0.77 (s, 6H), 0.1 (s, 3H), À0.1 (s, 3H);

¹³C NMR (125 MHz, CDCl₃): d 170.4, 170.3, 170.2, 138.5, 138.3, 138.2,

137.5, 128.9, 128.8, 128.7, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 111.1, 98.0,

96.2, 78.1, 76.5, 75.9, 75.8, 73.8, 72.4, 71.8, 71.0, 70.0, 63.0, 52.7, 52.0, 27.7,

26.1, 25.9, 21.3, 18.2, À4.3, À4.9; FAB MS: m/z: calcd for C₅₉H₇₅N₃NaO₁₈Si:

tert-Butyldimethylsilyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-

glucopyranosyl)-(1! 4)-(methyl 3-O-benzyl-2-O-levulinoyl-b-d-glucopyra-

nosyl)-(1 ! 4)-3,6-di-O-acetyl-2-azido-2-deoxy-b-d-glucopyranoside (107):

Compound 66 (365 mg, 0.38 mmol) and 7 (105 mg, 0.26 mmol) were

coevaporated with toluene and dried under vacuum for 1 h. The mixture

was dissolved in CH₂Cl₂(3 mL) and after cooling to À258C, TMSOTf

(20 mL, 0.1m in CH₂Cl₂) was added. The mixture was stirred for 4 h and then

diluted with CH₂Cl₂and filtered through a pad of Celite. The solvent was

removed under reduced pressure and the residue was purified by silica gel

column chromatography (toluene/EtOAc 95:5 ! 80:20) to yield 107

(195 mg, 0.16 mmol, 63%) as a syrup. ¹H NMR (500 MHz, CDCl₃): d

7.37 7.24 (m, 15H), 5.40 (d, J 3.7 Hz, 1H), 4.99 (t, 1H), 4.92 4.87 (m,

3H), 4.84 4.70 (m, 3H), 4.67 (d, J 10.6 Hz, 1H), 4.60 (d, J 7.6 Hz, 1H),

4.57 (d, J 10.9 Hz, 1H), 4.50 4.47 (d, J 10.4 Hz, 1H), 4.42 (d, J 7.6 Hz,

1H), 4.27 4.12 (m, 4H), 3.90 (d, J 9.5 Hz, 1H), 3.85 (dd, J 8.5, 10.4 Hz,

1H), 3.78 3.75 (m, 1H), 3.76 (s, 1H), 3.73 3.60 (m, 2H), 3.55 3.50 (m,

2H), 3.32 3.27 (m, 2H), 2.74 2.63 (m, 2H), 2.59 2.54 (m, 2H), 2.12 (s,

3H), 2.11 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 0.9 (s 9H), 0.15 (s, 3H), 0.14 (s,

3H); ¹³C NMR (125 MHz, CDCl₃): d 206.1, 171.7, 170.8, 170.7, 169.8,

168.1, 145.7, 141.5, 137.7, 137.7, 137.6, 128.7, 128.7, 128.6, 128.6, 128.3, 128.3,

128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 101.3, 97.6, 97.6, 82.5, 80.3, 76.8, 75.7,

75.2, 74.8, 74.7, 74.6, 73.1, 72.8, 71.8, 69.9, 66.5, 63.4, 62.5, 62.3, 52.9, 37.9, 37.7,

29.9, 27.8, 25.7, 21.1, 21.0, 20.9, 18.2, À4.3, À5.0; ES MS: m/z: calcd for

1164.4707; found: 1164.4749 [MNa] .

n-Pentenyl (methyl 2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-a-

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

a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-levulinoyl-b-d-gluco-

pyranosiduronate (105): Compounds 103 (204 mg, 0.34 mmol) and 73

(206 mg, 0.26 mmol) were coevaporated three times with toluene and

dissolved in anhydrous CH₂Cl₂(3 mL). Freshly activated powdered 4 ä

molecular sieves (200 mg) were added and the mixture was stirred at room

temperature for 1 h. The reaction mixture was cooled to À258C and

TMSOTf (340 mL, 0.1m in CH₂Cl₂) was added dropwise. The mixture was

warmed to room temperature over 4 h. Triethylamine was added, the

mixture was filtered through a pad of Celite and the solvent was removed

under reduced pressure. Flash chromatography on silica gel (toluene/

EtOAc 90:10 ! 80:20) afforded 105 (298 mg, 0.24 mmol, 93%) as a syrup.

¹H NMR (500 MHz, CDCl₃): d 7.39 7.27 (m, 15H), 5.80 5.77 (m, 1H),

5.44 (d, J 3.7 Hz, 1H), 5.32 (d, J 5.8 Hz, 1H), 5.07 5.02 (m, 2H), 4.98

4.95 (m, 2H), 4.90 4.87 (m, 1H), 4.79 (d, J 10.9 Hz, 1H), 4.73 4.66 (m,

4H), 4.49 (d, J 5.2, 1H), 4.46 (d, J 7.3 Hz, 1H), 4.38 (dd, 1H), 4.21 4.12

(m, 2H), 3.99 3.91 (m, 2H), 3.89 3.81 (m, 3H), 3.75 (m, 2H), 3.71 (s, 3H),

3.68 (m, 1H), 3.58 (s, 3H), 3.47 3.42 (m, 1H), 3.29 (dd, J 3.3, 10.1 Hz,

1H), 2.68 (m, 2H), 2.56 2.43 (m, 2H), 2.14 (s, 3H), 2.12 (s, 3H), 2.07 2.04

C₅₇H₇₄N₆O₂₀Si: 1190.4727; found: 1190.4735 [M] .

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl-(1 ! 4)-

methyl 3-O-benzyl-2-O-levulinoyl-b-d-glucopyranosyl-(1 ! 4)-(3,6-di-O-

acetyl-2-azido-2-deoxy-a/b-d-glucopyranosyl) trichloroacetimidate (108):

A solution of 107 (80 mg, 0.07 mmol) in THF (1 mL) was cooled to 08C.

Glacial acetic acid (10 mL, 0.17 mmol) and TBAF (1m in THF, 110 mL,

0.11 mmol) were added sequentially. After 30 min the mixture was poured

into Et₂O (100 mL) and washed three times with brine. The organic layer

was dried over Na₂SO₄, filtered and the solvent was removed under

reduced pressure. The residue was dissolved in CH₂Cl₂(20 mL) and the

solution was cooled to 08C. Trichloroacetonitrile (190 mL, 1.9 mmol) and

DBU (5 mL, 0.03 mmol) were added and the mixture was stirred at 08C for

1 h and at room temperature for 3 h. After concentration, flash chroma-

tography on silica gel (hexanes/EtOAc 85:15 ! 70:30) afforded 108 (82 mg,

0.07 mmol, 87%) as

C₅₃H₆₀Cl₃N₇O₂₀: 1219.2959; found: 1219.2963 [M] .

a

colorless foam. FAB MS: m/z: calcd for

n-Pentenyl (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-glucopyrano-

syl)-(1 ! 4)-(methyl 3-O-benzyl-2-O-levulinoyl-b-d-glucopyranosyl)-(1 !

166

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0166 $ 20.00+.50/0

Chem. Eur. J. 2003, 9, No. 1

Heparin Oligosaccharides

140 169

4)-(3,6-di-O-acetyl-2-azido-2-deoxy-a/b-d-glucopyranosyl)-(1 ! 4)-(meth-

yl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1 ! 4)-(6-O-acetyl-2-

azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-ben-

zyl-2-O-levulinoyl-b-d-glucopyranosyluronate (109): Compounds 108

(50 mg, 0.04 mmol) and 106 (30 mg, 0.03 mmol) were coevaporated with

toluene and dried under vacuum for 1 h. The mixture was dissolved in

toluene (1 mL) and after cooling to À258C, TBSOTf (20 mL, 0.1m in

CH₂Cl₂) was added. The mixture was stirred for 2 h and then diluted with

CH₂Cl₂and filtered through a pad of Celite. The solvent was removed

under reduced pressure and the residue was purified by silica gel column

chromatography (toluene/EtOAc 95:5 ! 80:20) to yield 109 (36 mg,

0.02 mmol, 62%) as a syrup. [a]²_D⁴ 35 (c 0.70, CH₂Cl₂); ¹H NMR

(500 MHz, CDCl₃): d 7.36 7.17 (m, 30H), 5.77 5.83 (m, 1H), 5.48 5.45

(m, 2H), 5.41 (d, J 3.7 Hz, 1H), 5.33 (t, 1H), 5.29 (d, J 3.7 Hz, 1H),

5.17 4.90 (m, 7H), 4.87 4.80 (m, 6H), 4.78 4.61 (m, 7H), 4.57 4.53 (m,

2H), 4.45 (d, J 7.9 Hz, 1H), 4.39 (d, J 7.9 Hz, 1H), 4.38 4.22 (m, 2H),

4.20 4.10 (m, 2H), 3.96 (d, J 9.4 Hz, 1H), 3.95 3.84 (m, 2H), 3.82 3.66

(m, 6H), 3.75 (s, 3H), 3.71 (s, 3H), 3.67 (s, 3H), 3.54 3.49 (m, 3H), 3.48

3.43 (m, 1H), 3.30 (dd, J 3.9, 10.4 Hz, 1H), 3.16 (dd, J 3.3, 10.1 Hz, 1H),

3.11 (dd, J 3.0, 10.4, Hz, 1H), 2.69 2.63 (m, 4H), 2.58 2.48 (m, 4H), 2.16

(s, 3H), 2.14 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 2.09 2.02 (m, 2H), 2.02 (s,

3H), 1.87 (s, 3H), 1.66 1.60 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): d

207.2, 206.2, 171.4, 171.2, 171.0, 170.8, 170.7, 170.6, 170.1, 170.0, 169.0,

168.1, 138.2, 137.8, 137.7, 137.6, 129.2, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3,

128.1, 127.9, 127.8, 127.5, 127.3, 125.5, 115.2, 101.5, 101.2, 99.6, 98.1, 97.7, 97.4,

82.9, 82.6, 80.4, 78.0, 77.4, 76.9, 75.8, 75.6, 75.2, 74.9, 74.8, 74.6, 74.4, 74.3,

73.6, 73.1, 71.5, 71.0, 70.0, 69.5, 69.3, 63.5, 63.3, 62.3, 61.9, 61.7, 61.0, 52.9,

52.8, 52.5, 37.9, 37.5, 31.2, 30.1, 30.0, 29.2, 28.7, 28.1, 27.7, 21.7, 21.2, 21.0, 20.9,

20.8; ES MS: m/z: calcd for C₁₀₆H₁₂₅N₉NaO₄₀: 2186.7916; found: 2186.7984

2924, 2108, 1743, 1496, 1453; ¹H NMR (500 MHz, CDCl₃): d 7.36 7.25

(m, 20H), 5.84 5.76 (m, 1H), 5.54 (d, J 4.0 Hz, 1H), 5.28 (d, J 4.0 Hz,

1H), 5.21 (t, J 9.5 Hz, 1H), 5.07 (d, J 3.5 Hz, 1H), 5.03 4.90 (m, 6H),

4.82 4.67 (m, 5H), 4.62 (d, J 4.0 Hz, 1H), 4.50 4.45 (m, 2H), 4.31 4.29

(A part of ABX system, J 12.0, 1.0 Hz, 1H), 4.26 4.23 (B part of ABX

system, J 12.0, 3.5 Hz, 1H), 4.17 4.14 (B part of ABX system, J 12.5,

2.5 Hz, 1H), 4.09 4.05 (m, 2H), 3.96 3.89 (m, 4H), 3.86 (t, J 9.5 Hz,

1H), 3.76 3.73 (m, 2H), 3.67 (s, 3H), 3.65 (s, 3H), 3.57 3.49 (m, 3H), 3.44

(td, J 10.5, 3.5 Hz, 1H), 3.24 (dd, J 4.0, 10.0 Hz, 1H), 3.17 (dd, J 3.5,

10.5 Hz, 1H), 3.02 (d, J 5.0 Hz, 1H), 2.16 2.04 (m, 14H), 1.77 1.70 (m,

2H); ¹³C NMR (125 MHz, CDCl₃): d 172.0, 171.3, 171.1, 170.3, 169.7,

169.1, 138.3, 138.2, 138.1, 137.4, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0,

127.8, 127.7, 115.3, 103.9, 98.6, 98.4, 97.4, 84.1, 81.9, 78.1, 75.75, 75.74, 75.4,

75.0, 74.9, 74.8, 74.4, 74.0, 73.8, 72.4, 71.4, 70.1, 70.0, 69.9, 69.5, 68.9, 63.2,

62.6, 62.0, 61.1, 52.8, 52.4, 30.3, 29.0, 21.0, 21.1, 20.9; ES MS: m/z: calcd for

C₆₇H₈₀N₆NaO₂₅: 1391.5070; found: 1391.5107 [MNa] .

n-Pentenyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-a-d-glucopyranosyl)-(1 !

4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1 ! 4)-(6-

O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl

3-O-benzyl-b-d-glucopyranosyluronate (111): A solution of 82 (50 mg,

0.04 mmol), pyridine (6.3 mL, 0.08 mmol), acetic anhydride (8 mL,

0.08 mmol) and catalytic DMAP was stirred at room temperature for 6 h.

After removal of the solvent under reduced pressure, flash chromatography

on silica gel (hexanes/EtOAc 1.5:1) afforded n-pentenyl (3,4,6-tri-O-acetyl-

2-azido-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-(methyl

2-O-acetyl-3-O-

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

2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl 3-O-benzyl-2-O-levulinyl-b-

d-glucopyranosyluronate (57 mg, 0.08 mmol, quantitative) as a yellow oil.

[a]²_D³ 165 (c 0.10, CHCl₃); ¹H NMR (500 MHz, CDCl₃): d 7.38 7.24

(m, 15H), 5.82 5.74 (m, 1H), 5.48 (d, J 4.0 Hz, 1H), 5.34 (t, J 9.5 Hz,

1H), 5.27 (d, J 4.0 Hz, 1H), 5.08 (d, J 4.0 Hz, 1H), 5.06 4.88 (m, 6H),

4.79 4.66 (m, 6H), 4.47 (d, J 7.5 Hz, 1H), 4.32 4.23 (m, 2H), 4.22 4.18

(m, 2H), 4.14 4.04 (m, 3H), 3.99 (d, J 9.5 Hz, 1H), 3.94 (t, J 5.0 Hz,

1H), 3.87 3.82 (m, 3H), 3.74 (t, J 10.0 Hz, 1H), 3.67 (s, 3H), 3.64 (s, 3H),

3.57 3.55 (m, 1H), 3.47 3.43 (m, 1H), 3.29 3.26 (m, 2H), 2.68 (t, J

6.5 Hz, 2H), 2.57 2.44 (m, 2H), 2.14 2.01 (m, 20H), 1.72 1.59 (m, 2H,

[MNa] .

n-Pentenyl (3,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1 ! 4)-(6-O-

acetyl-3-O-benzyl-2-azido-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-methyl

2,3-di-O-benzyl-b-d-glucopyranosyluronate (110): A solution of 81 (93 mg,

0.07 mmol) and thiourea (230 mg, 3.02 mmol) in DMF/pyridine (10/1,

2 mL) was stirred for 24 h. After removal of the solvent under reduced

pressure, the residue was dissolved in CHCl₃and filtered. The solvent was

removed under reduced pressure and flash chromatography on silica gel

afforded pent-4-enyl (3,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyrano-

pent-CH₂); ¹³C NMR (125 MHz, CDCl₃):

d

206.2, 171.4, 171.0,

170.7, 170.2, 170.1, 169.7, 169.6, 168.8, 138.1, 138.0, 137.8, 137.3, 128.7,

128.6, 128.4, 128.3, 128.2, 127.9, 127.8, 115.1, 101.2, 98.4, 98.3, 97.4, 82.5, 78.2,

75.6, 75.4, 75.0, 74.5, 74.4, 73.9, 73.6, 70.3, 69.8, 69.6, 69.4, 68.6, 68.2, 63.3,

61.9, 61.5, 61.1, 52.8, 52.4, 37.9, 30.0, 28.7, 28.0, 21.0, 20.9, 20.87, 20.83, 20.7;

ES MS: m/z: calcd for C₆₇H₈₂N₆NaO₂₈: 1441.5069; found: 1441.5098

syl)-(1 ! 4)-(methyl

2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-

(1 ! 4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl 3-O-benzyl-b-d-glucopyranosyluronate (79 mg, 0.06 mmol,

90%). [a]²_D³ 12.5 (c 0.80, CHCl₃); IR (thin film on NaCl): nÄ 2914,

2364, 2108, 1743, 1371; ¹H NMR (500 MHz, CDCl₃): d 7.38 7.28 (m,

15H), 5.85 5.77 (m, 1H), 5.57 (d, J 3.5 Hz, 1H), 5.29 (d, J 4.5 Hz, 1H),

5.22 (dd, J 10.5, 9.0 Hz, 1H), 5.07 (d, J 3.0 Hz, 1H), 5.02 4.97 (m, 3H),

4.93 4.90 (m, 2H), 4.82 4.80 (A part of AB system, J_AB 11.0 Hz, 1H),

4.76 4.68 (m, 3H), 4.63 (d, J 4.5 Hz, 1H), 4.49 (A part of ABX system,

J 12.5, 3.5 Hz, 1H), 4.31 (d, J 7.5 Hz, 1H), 4.29 4.24 (m, 2H), 4.16 (B

part of ABX system, J 12.5, 2.0 Hz, 1H), 4.09 4.05 (m, 2H), 4.00 3.84

(m, 5H), 3.77 3.71 (m, 2H), 3.67 (s, 3H), 3.66 (s, 3H), 3.62(dt, J 7.5,

2.0 Hz, 1H), 3.54 3.50 (m, 2H), 3.44(dt, J 9.5, 5.0 Hz, 1H), 3.27 (dd, J

10.5, 4.0 Hz, 1H), 3.17 (dd, J 10.5, 3.5 Hz, 1H), 3.03 (d, J 5.0 Hz, 1H),

2.34 (d, J 2.0 Hz, 1H), 2.20 2.05 (m, 14H), 1.77 1.68(m, 2H); ¹³C NMR

(125 MHz, CDCl₃): d 172.0, 171.3, 171.1, 170.3, 169.8, 169.0, 138.4, 138.2,

138.1, 137.4, 128.7, 128.6, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 115.3, 103.1,

98.6, 98.4, 97.5, 83.9, 78.2, 75.8, 75.0, 74.9, 74.8, 74.7, 74.0, 73.9, 72.4, 71.4,

70.1, 70.0, 69.9, 69.6, 69.0, 63.3, 62.6, 62.0, 61.1, 52.8, 52.4, 30.3, 28.8, 21.1,

21.0, 20.9; ES MS: m/z: calcd for C₆₀H₇₄N₆NaO₂₅: 1301.4601; found:

[MNa] .

A solution of n-pentenyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-a-d-glucopyr-

anosyl)-(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-

(1 ! 4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl

3-O-benzyl-2-O-levulinyl-b-d-glucopyranosyluronate

(42 mg, 0.03 mmol) in pyridine/AcOH (3/2 0.3 mL) was cooled to 08C

and hydrazine hydrate (7.4 mg, 0.15 mmol) was added. After 20 minutes

acetone (2 mL) was added and the ice bath was removed. After stirring the

mixture at room temperature for 30 minutes, the solvent was removed

under reduced pressure and the residue was purified by silica gel column

chromatography (hexanes/EtOAc 1:1) to afford 111 (35 mg, 0.03 mmol,

90%) as a pale yellow oil. [a]²_D³ 138 (c 3.6, CHCl₃); ¹H NMR

(500 MHz, CDCl₃): d 7.38 7.26 (m, 15H), 5.84 5.76 (m, 1H), 5.58 (d,

J 3.5 Hz, 1H), 5.34 (dd, J 10.5, 9.5 Hz, 1H), 5.27 (d, J 4.0 Hz, 1H),

5.08 (d, J 3.5 Hz, 1H), 5.05 4.97 (m, 4H), 4.92 (t, J 4.5 Hz, 1H), 4.89

4.87 (A part of AB system, J_AB 10.5 Hz, 1H), 4.82 4.79 (B part of AB

system, J_AB 10.5 Hz, 1H), 4.76 4.67 (m, 4H), 4.32 4.19 (m, 4H), 4.11

4.04 (m, 4H), 3.97 3.90 (m, 3H), 3.85 (t, J 9.5 Hz, 1H), 3.77 3.71 (m,

2H), 3.67 3.60 (m, 7H), 3.55 3.49 (m, 2H), 3.29 3.25 (m, 2H), 2.40

(brs, 1H), 2.14 2.02 (m, 17H), 1.76 1.69 (m, 2H); ¹³C NMR (500 MHz,

CDCl₃): d 171.0, 170.7, 170.3, 170.1, 169.8, 169.6, 168.9, 138.3, 138.1,

138.0, 137.3, 128.7, 128.6, 128.4, 128.3, 128.2, 127.9, 127.8, 115.3, 103.1, 98.4,

97.5, 83.9, 78.2, 75.6, 75.3, 74.9, 74.8, 74.7, 73.9, 70.3, 70.0, 69.5, 69.3, 68.6,

68.2, 63.3, 61.9, 61.4, 61.0, 52.8, 52.4, 30.3, 28.7, 21.1, 20.90, 20.88, 20.86,

20.79; ES MS: m/z: calcd for C₆₂H₇₆N₆NaO₂₆: 1338.5152; found: 1338.4932

1301.4596 [MNa] .

A solution of n-pentenyl (3,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyra-

nosyl)-(1 ! 4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-

(1 ! 4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-a-d-glucopyranosyl)-

(1 ! 4)-methyl 3-O-benzyl-b-d-glucopyranosyluronate (62 mg, 0.05 mmol)

in CH₂Cl₂(2.0 mL) was added to freshly activated powdered 4 ä molecular

sieves (60 mg). Benzyl bromide (29 mL, 0.24 mmol) was added and the

mixture was stirred at room temperature. After 30 minutes, Ag₂O (67 mg,

0.29 mmol) was added and the mixture was stirred overnight. The mixture

was filtered and the solvent was removed under reduced pressure and flash

chromatography on silica gel afforded 110 (50 mg, 0.04 mmol, 76%) as a

pale yellow oil. [a]²_D⁴ 23.9 (c 1.80, CHCl₃); IR (thin film on NaCl): nÄ

[MNa] .

n-Pentenyl (2-deoxy-2-sodium sulfonatamido-3,4,6-tri-O-sodium sulfona-

to-a-D glucopyranosyl)-(1 ! 4)-(sodium 2-O-sodium sulfonato-a-d-ido-

pyranosyluronate)-(1 ! 4)-(2-deoxy-2-sodium sulfonatamido-6-O-sodium

Chem. Eur. J. 2003, 9, No. 1

¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0947-6539/03/0901-0167 $ 20.00+.50/0

167

P. H. Seeberger et al.

FULL PAPER

sulfonato-a-d-glucopyranosyl)-(1 ! 4)-sodium 2-O-sodium sulfonato-b-d-

glucopyranosyluronate (112): A solution of 111 (44 mg, 0.03 mmol) in THF

(3.0 mL) was cooled to À138C and 50% H₂O₂(1.0 mL) and 0.7m aq. LiOH

(0.8 mL) were added dropwise. The mixture was warmed to 08C over one

hour and at room temperature overnight. Sodium hydroxide solution (4m,

0.8 mL) was added and the mixture was stirred overnight. After acid-

ification to pH 6.0 with 3m HCl in MeOH, the solvent was partially

removed under vacuum. The solution was diluted with EtOAc and the two

phases were separated. The organic phase was washed twice with acidified

aqueous sulfite (pH 3.5) and dried over Na₂SO₄. After filtration, the

solvent was removed under reduced pressure and the residue was purified

on Sephadex LH20 (CH₂Cl₂/EtOH 1:1) affording n-pentenyl (2-azido-2-

deoxy-a-d-glucopyranosyl)-(1 ! 4)-3-O-benzyl-a-l-idopyranosyluronic

acid)-(1 ! 4)-(2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-

3-O-benzyl-b-d-glucopyranosyluronic acid (27 mg, 0.024 mmol, 82%) as a

colorless oil. ES MS: m/z: calcd for C₅₀H₆₂N₆O₂₁: 1105.3865; found:

Acknowledgements

Financial support from the National Institutes of Health (HL-64799 and

HL-62598), the Research Corporation (Research Innovation Award to

P.H.S.), CaP CURE (Research Awards to P.H.S.); CNRI(Italian National

Research Center, Postdoctoral Fellowship for A.B.); FOMEC (Postdoc-

toral Fellowship for H.A.O.), the DAAD (German Academic Exchange

Service, Postdoctoral Fellowship for P.S.); Merck and Boehringer-Ingel-

heim (Predoctoral Fellowships for E.R.P.) is gratefully acknowledged.

Funding for the MIT-DCIF Varian 500 MHz NMR was provided by NSF

(Award CHE-9808061). Funding for the MIT-DCIF Bruker 400 MHz

NMR was provided by NIH (Award 1S10RR13886-01). Funding for the

MIT-DCIF Varian 300 MHz was provided by NSF (Award DBI-9729592).

We thank Dr. Susanne Roehrig for helpful discussions. We thank Prof.

Joseph Zaia (Boston University School of Medicine Mass Spectrometry

Resource funded by NIH/NCRR (Award P41RR10888)) for recording the

mass spectrum of 112.

1105.3806 [MNa] .

A solution of n-pentenyl (2-azido-2-deoxy-a-d-glucopyranosyl)-(1 ! 4)-3-

O-benzyl-a-l-idopyranosyluronic acid)-(1 ! 4)-(2-azido-3-O-benzyl-2-de-

oxy-a-d-glucopyranosyl)-(1 ! 4)-3-O-benzyl-b-d-glucopyranosyluronic

acid (35 mg, 0.03 mmol) and sulfur trioxide/triethylamine complex (88 mg,

0.48 mmol) in DMF (1.5 mmol) was stirred under nitrogen at 508C for 20 h.

Aqueous NaHCO₃(10%, 3 mL) was added at room temperature and the

mixture was stirred for 3.5 h. The reaction mixture was concentrated,

dissolved in MeOH and filtered through a pad of Celite. After removal of

the solvent under reduced pressure, the residue was purified through a

Sephadex G-25 column eluted with 0.2n NaCl. After concentration and

desalting through a Sephadex G-25 column eluted with water, n-pentenyl

(2-azido-2-deoxy-3,4,6-tri-O-sodium sulfonato-a-d-glucopyranosyl)-(1 !

4)-(sodium 3-O-benzyl-2-O-sodium sulfonato-a-d-idopyranosyluronate)-

(1 ! 4)-(2-azido-3-O-benzyl-2-deoxy-6-O-sodium sulfonato-a-d-glucopyr-

anosyl)-(1 ! 4)-sodium 3-O-benzyl-2-O-sodium sulfonato-b-d-glucopyra-

nosyluronate (25 mg, 0.015 mmol, 50%) was obtained as a colorless solid.

¹H NMR (500 MHz, D₂O): d 7.39 7.15 (m, 15H), 5.82 5.74 (m, 1H),

5.25 (d, J 4.0 Hz, 1H), 5.17 (brs, 1H), 5.07 (d, J 3.5 Hz, 1H), 4.99 4.87

(m, 2H), 4.72 4.66 (m, 7H), 4.42 4.38 (m, 2H), 4.32 4.28 (m, 2H), 4.20

4.10 (m, 7H), 3.96 3.69 (m, 8H), 3.55 3.50 (m, 1H), 3.43 (dd, J 10.5,

3.5 Hz, 1H), 3.34 (dd, J 10.0, 4.0 Hz, 1H), 2.06 2.02 (m, 2H), 1.61 1.55

(m, 2H); ¹³C NMR (125 MHz, D₂O): d 139.7, 137.9, 137.4, 137.3, 129.9,

129.4, 129.2, 129.2, 128.9, 128.8, 115.4, 101.4, 98.0, 93.6, 82.8, 80.6, 78.6, 77.3,

76.5, 76.0, 75.3, 75.6, 75.3, 74.6, 73.0, 72.7, 71.4, 70.7, 70.4, 69.3, 68.6, 68.4,

67.4, 66.9, 63.6, 62.2, 29.9, 28.6; ES MS: m/z: calcd for C₅₀H₆₂N₆O₃₉S₆:

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A solution of n-pentenyl (2-azido-2-deoxy-3,4,6-tri-O-sodium sulfonato-a-

d-glucopyranosyl)-(1 ! 4)-(sodium 3-O-benzyl-2-O-sodium sulfonato-a-

d-idopyranosyluronate)-(1 ! 4)-(2-azido-3-O-benzyl-2-deoxy-6-O-sodium

sulfonato-a-d-glucopyranosyl)-(1 ! 4)-sodium

3-O-benzyl-2-O-sodium

sulfonato-b-d-glucopyranosyluronate (25 mg, 0.02 mmol) in EtOH/water

(2/1, 6.0 mL) was treated with a stream of hydrogen in the presence of Pd/C

catalyst (10%, 40 mg) for three days. After filtration on a pad of Celite, the

solvent was removed under reduced pressure. ¹H NMR (500 MHz, D₂O): d

5.41 (brs, 1H), 5.29 (brs, 1H), 5.10 (brs, 1H), 4.78 (brs, 1H), 4.60 4.28

(m, 2H), 4.24 3.57 (m, 17H), 3.55 3.41 (m, 2H), 3.13 (brs, 1H), 1.46 1.41

(m, 2H), 1.18 1.12 (m, 4H), 0.70 (t, J 7.0 Hz, 3H); ES MS: m/z: calcd for

C₂₉H₅₀N₂O₃₉S₆: 620.0085; found: 620.0040 [M À 2H]^2À

.

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NaOH. After 3.5 h, the reaction mixture was concentrated and purified

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Received: May 21, 2002 [F4107]

Chem. Eur. J. 2003, 9, No. 1

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