Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions

DOI: 10.1002/chem.200601103

Preparation and Use of Microarrays Containing Synthetic Heparin

Oligosaccharides for the Rapid Analysis of Heparin–Protein Interactions

Christian Noti,^{[a, b]}Jose L. de Paz,^{[a, b]}Laura Polito,^[a]and Peter H. Seeberger*^[a]

Abstract: Heparin is a highly sulfated,

linear polymer that participates in a

plethora of biological processes by in-

teraction with many proteins. The

chemical complexity and heterogeneity

of this polysaccharide can explain the

fact that, despite its widespread medi-

cal use as an anticoagulant drug, the

structure–function relationship of de-

fined heparin sequences is still poorly

understood. Here, we present the

chemical synthesis of a library contain-

ing heparin oligosaccharides ranging

from di- to hexamers of different se-

quences and sulfation patterns. An

amine-terminated linker was placed at

the reducing end of the synthetic struc-

tures to allow for immobilization onto

N-hydroxysuccinimide activated glass

slides and creation of heparin microar-

rays. Key features of this modular syn-

thesis, such as the influence of the

amine linker on the glycosidation effi-

ciency, the use of 2-azidoglucose as gly-

cosylating agents for oligosaccharide

assembly, and the compatibility of the

protecting group strategy with the sul-

fation-deprotection steps, are discussed.

Heparin microarrays containing this

oligosaccharide library were construct-

ed using a robotic printer and em-

ployed to characterize the carbohy-

drate binding affinities of three hepa-

rin-binding growth factors. FGF-1,

FGF-2 and FGF-4 that are implicated

in angiogenesis, cell growth and differ-

entiation were studied. These heparin

chips aided in the discovery of novel,

sulfated sequences that bind FGF, and

in the determination of the structural

requirements needed for recognition

by using picomoles of protein on a

single slide. The results presented here

highlight the potential of combining

oligosaccharide synthesis and carbohy-

drate microarray technology to estab-

lish a structure–activity relationship in

biological processes.

Keywords:

glycomics

carbohydrates

· glycosylation

·

heparin · microarrays

Introduction

cludes chondroitin sulfate, keratan sulfate, and dermatan

sulfate. HLGAGs serve important biological functions by

binding to different growth factors, enzymes, morphogens,

cell adhesion molecules, and cytokines.^[1–3]

Proteoglycans are major components of the extracellular

matrix that surround all mammalian cells. Different core

proteins anchor glycosaminoglycan polysaccharides in the

outside of the lipid bilayer. Heparin and heparan sulfate

(heparin-like glycosaminoglycans, HLGAG) are the most

complex members of this family of molecules that also in-

Heparin^[4]is a linear, unbranched, highly sulfated polysac-

charide composed of disaccharide units consisting of an

uronic acid 1,4-linked to a d-glucosamine unit. The uronic

acid residues are more often l-iduronic acid (90%) than its

C5 epimer d-glucuronic acid (10%). Aprototypical heparin

disaccharide unit contains three sulfate groups. These sulfate

groups render heparin one of the most acidic macromole-

cules in nature. O-Sulfation normally occurs at C2 of the

uronic acids and at C3 and/or C6 of the glucosamine. In ad-

dition, the glucosamine nitrogen may be sulfated, acetylated

or, less frequently, may remain unmodified, thus resulting in

48 possible disaccharides that make up heparin.^[5]

[a] C. Noti, Dr. J. L. de Paz, Dr. L. Polito, Prof. Dr. P. H. Seeberger

Laboratory for Organic Chemistry

Swiss Federal Institute of Technology (ETH) Zürich

Wolfgang-Pauli-Strasse 10, HCI F315, 8093 Zürich (Switzerland)

Fax : (+41)44-633-1235

E-mail: seeberger@org.chem.ethz.ch

[b] C. Noti, Dr. J. L. de Paz

These authors contributed equally to this work.

The structural variability is responsible for the interaction

of heparin with a wide variety of proteins.^[6]The chemical

complexity explains the fact that the structure–activity rela-

tionship of heparin is still poorly understood despite its

widespread medical use. The most thoroughly studied hepa-

Supporting information for this article is available on the WWW

under http://www.chemeurj.org/ or from the author: Spectral copies

of new compounds, image of an array after incubation with FGF-1,

and binding curves for compounds 32, 23, 51, and 77 after incubation

with FGF-1 and FGF-4).

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rin binding protein is the serine protease inhibitor anti-

thrombin III (AT III) that interacts with thrombin and factor

Xa in the blood-coagulation cascade.^[7,8]The heparin–AT III

interaction is responsible for the anticoagulant activity of

heparin and is the only system where the exact sequence of

heparin that associates with the protein has been identified.

Many growth factors, including the fibroblast growth fac-

tors^[9](FGFs), bind to the extracellular matrix of target tis-

sues by interacting with HLGAGs. The FGF family of pro-

teins contains 23 different members and is involved in devel-

opmental and physiological processes including cell prolifer-

ation, differentiation, morphogenesis, and angiogenesis.^[1–3]

The most thoroughly studied members of the FGF family

are FGF-1 (acidic FGF) and FGF-2 (basic FGF). FGF sig-

naling involves binding to specific cell-surface tyrosine

kinase receptors (FGFRs) and is tightly regulated by

HLGAGs that facilitate the formation of FGF-FGFR com-

plexes. Therefore, the various FGFs require heparin for bio-

logical activity, presumably to generate and stabilize ternary

signaling complexes with FGFRs. High-resolution X-ray

crystal structures of FGF-FGFR-heparin complexes^[10–13]

have been determined to provide insights into structural as-

pects of this physiologically relevant interaction. These stud-

ies demonstrate the complexity of the molecular mechanism

involved in heparin-mediated FGF signaling.

The minimum heparin sequences required for FGF-1 and

FGF-2 binding have been determined by using oligosacchar-

ides derived by depolymerization of isolated heparin.^[14–18]

Usually, tetra- and hexasaccharides are sufficient to bind

FGFs with high affinity. The iduronate 2-O-sulfate and the

N-sulfate of glucosamine are critical modifications that are

required for both FGF-1 and FGF-2 signaling. The 6-O-sul-

fation has been shown to be critical for FGF-1 signaling but

it is not essential for FGF-2. However, these structure–func-

tion studies are limited by the structural modifications intro-

duced in heparin sequences using both enzymatic and chem-

ical depolymerization methods.^[19]

involve carbohydrates since the multivalent display of li-

gands on a surface (cluster effect^[25]) overcomes the relative

weakness of these interactions by mimicking cell–cell inter-

faces. Moreover, carbohydrate microarrays possess a ple-

thora of potential applications in glycomics including the

rapid determination of the binding profile of carbohydrate-

binding proteins,^[26–32]the detection of specific antibodies for

the diagnosis of diseases,^[33]the characterization of carbohy-

drate-cell recognition events^[34,35]and the high-throughput

screening of inhibitors to prevent carbohydrate–protein in-

teractions.^[36]

Here, we report the preparation and use of microarrays

containing synthetic heparin oligosaccharides.^[37]For that

purpose, a library of heparin oligosaccharides ranging from

di- to hexamers with different sequences and sulfate group

distributions was synthesized. An amine-terminated linker

at the reducing end of all synthetic structures allows for im-

mobilization onto glass slides and creation of heparin arrays.

Additionally, this linker strategy is compatible with auto-

mated solid phase approaches.^[37]The heparin chips were

employed for the rapid analysis of heparin–FGF interac-

tions, by determining the carbohydrate-binding affinities of

three heparin-binding growth factors, FGF-1, FGF-2 and

FGF-4 using picomoles of protein on a single slide. The re-

sults presented here demonstrate the potential of synthetic

heparin oligosaccharide microarrays to elucidate the role of

defined heparin sequences in biological processes, creating

an opportunity for the discovery of novel therapeutic inter-

ventions for a variety of disease states.

Results and Discussion

Synthetic strategy: Over the last two decades, synthetic ap-

proaches aiming at the preparation of a broad range of

HLGAG fragments have been developed.^[38–47]The prepara-

tion of sufficient quantities of fully differentiated building

blocks incorporating appropriate protecting groups is partic-

ularly important for the synthesis of complex oligosacchar-

ides such as heparin.^[48]Ahighly convergent, modular syn-

thetic approach for the assembly of defined libraries of

HLGAGs is of great interest and requires careful consider-

ation of the many synthetic challenges presented by the

great diversity of native structures. The placement of specif-

ic temporary protecting groups at the 4-hydroxyl group of

each building block is needed to allow for ready deprotec-

tion in anticipation of chain elongation. The hydroxyl

groups to be O-sulfated and those that have to remain free

have to be protected differentially. Commonly, acyl groups

serve to mark the hydroxyl groups to be sulfated, while

benzyl ethers mask hydroxyl groups that will not be modi-

fied. The amine group of d-glucosamine requires the place-

ment of different protecting groups such as azides.

Fractionated heparin is derived from mammalian organs.

This source implies a potential risk of contamination with

pathogens such as viruses or prions. Thus, there is significant

interest in identifying alternatives to isolated material. The

chemical synthesis of defined heparin oligosaccharides is of

utmost importance in order to establish more-detailed struc-

ture–activity relationships and correlate specific sequences

and sulfation patterns with protein binding and biological

activity.^[20,21]

Carbohydrate microarrays,^[22–24]carrying tens or hundreds

of different sugars that are bound covalently in small spots

on solid surfaces, are becoming a standard tool for glycobi-

ologists to screen large numbers of sugars and to elucidate

the role of carbohydrates in biological systems. The minia-

turized array methodology is particularly well suited for in-

vestigations in the field of glycomics, because only tiny

amounts of both analyte and ligand are required for one ex-

periment and several thousand binding events can be

screened in parallel on a single glass slide. In addition, car-

bohydrate microarrays are ideal to detect interactions that

We envisioned a modular strategy for the synthesis of a li-

brary of amine-functionalized heparin oligosaccharides

(Scheme 1). Oligosaccharide I displays the general structure

of the target molecules that contain the GlcN-IdoArepeat-

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P. H. Seeberger et al.

ing unit of the major sequence of heparin and an amine-ter-

minated linker at the anomeric position of the reducing end

for immobilization purposes. Different sulfation patterns, in-

volving position 2 of the iduronic acid units and positions 2

and 6 of the glucosamine units, can be obtained by appropri-

ate combination of deprotection and sulfation steps. These

final structures are derived from fully protected oligosac-

charides II. The location of the sulfate groups at the desired

positions is anticipated by the protecting groups. Either n-

pentenyl glycosides or amine-protected pentyl glycosides

were used for the protection of the reducing end. When n-

pentenyl glycosides were employed, radical addition of 2-

(benzyloxycarbonylamino)-1-ethanethiol to the pentenyl

moiety was performed before sulfated oligosaccharides were

generated and printed onto glass slides. Notably, the n-pen-

tenyl group^[49]mimics the octanediol linker we developed

for automated solid-phase oligosaccharide synthesis.^[50]The

fully protected oligosaccharides II were built up using three

different building blocks: iduronic acid monosaccharide 1

that constitutes the reducing end of the chain,^[51]disacchar-

ide repeating unit 2 and glucosamine 3 for capping.^[42]Akey

feature of this modular assem-

side. The first target molecule was disaccharide 10 with the

sulfation pattern of the major region of heparin. The union

of glycosyl acceptor 5 and trichloroacetimidate 3 followed

by radical elongation of the pentenyl moiety using 2-(benzyl-

oxycarbonylamino)-1-ethanethiol^[55]furnished disaccharide 6

in good yield. Treatment of 6 with lithium hydroperoxide

and then with an aqueous solution of KOH afforded 7 in

73% yield. Under these saponification conditions, we ob-

served the complete conversion of sulfide to sulfone without

detectable sulfoxide intermediates. Reduction of the azido

group using PMe₃in THF^[56]gave disaccharide 8 in 89%

yield. This transformation required the addition of an aque-

ous solution of NaOH to avoid the formation of the corre-

sponding stable betaine where the phosphazene intermedi-

ate deprotonated the carboxylic acid.^[57]The sulfation of

both amino and hydroxyl groups was carried out by treat-

ment with SO₃·Py in pyridine to give 9. Global deprotection

by hydrogenolysis afforded the sulfated disaccharide 10.

Next, the preparation of longer oligosaccharide sequences

was performed by combining disaccharide building block 2

with the reducing end module 5. Coupling of 11^[51]with or-

bly is the combined use of 2-

azidoglucopyranose trichloroa-

cetimidates as glycosyl donors

and iduronic acid units as glyco-

syl acceptors for the stereose-

lective preparation of 1,2-cis

glycosidic linkages. In accord-

ance with previous reports, the

conformation of the iduronic

acid directs the stereochemical

outcome of these glycosyla-

tions.^[52]This approach is con-

venient for solid phase oligosac-

charide synthesis since 2-azido-

glucopyranose trichloroacetimi-

dates are better glycosylating

agents than iduronic acids.^[53,54]

Oligosaccharide assembly and Scheme 1. Retrosynthetic analysis for the preparation of amine-functionalized heparin oligosaccharides.

preparation of amine-function-

alized heparin oligosaccharides:

We initiated oligosaccharide as-

sembly by preparing the reduc-

ing end. Trichloroacetimidate 1

was converted to n-pentenyl

glycoside 5 by coupling with n-

pentenyl alcohol followed by

removal of the levulinoyl pro-

tecting group from the C4 hy-

droxyl (Scheme 2). The pres-

ence of

a participating acyl

group at position C2 of the

iduronic acid allowed for the

completely selective prepara-

Scheme 2. Synthesis of disaccharide 10. a) 4-Penten-1-ol, TMSOTf, CH₂Cl₂, ꢀ258C, 81%; b) H₂NNH₂·H₂O,

Py/AcOH, CH₂Cl₂, 91%; c) 3, TMSOTf, CH₂Cl₂, ꢀ258C, 81%; HS(CH₂)₂NHZ, AIBN, THF, 758C, 78%;

AHCTREUNG

tion of the desired trans glyco- d) LiOH, H₂O₂; KOH, MeOH, 73%; e) PMe₃, THF, NaOH, 89%; f) SO₃·Py, Py, 87%; g) H₂, Pd/C, quant.

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Heparin Oligosaccharides

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thogonally protected 2-azido-2-deoxyglucose 12^[42]furnished

disaccharide 13 in excellent yield after treatment with

TMSOTf at ꢀ258C (Scheme 3). Disaccharide 13 was con-

forded a mixture of partially sulfated tetrasaccharides. On

the basis of this finding, the deprotection–sulfation sequence

was altered. O-Sulfation of 19 was achieved by treatment

with SO₃·Et₃N in DMF at 558C.

Tetrasaccharide 20 was ob-

tained in 80% and unambigu-

ously characterized by ESI-MS

1

analyses and H NMR spectros-

copy. Staudinger reduction of

20 to form 21 followed by N-

sulfation using SO₃·Py in a mix-

ture triethylamine/pyridine af-

forded 22 in good yield. Finally,

hydrogenolytic cleavage of the

benzyl and benzyloxycarbonyl

Scheme 3. Synthesis of disaccharide repeating unit 2. a) TMSOTf (cat.), CH₂Cl₂, ꢀ258C, 97%; b) HOAc,

TBAF (1m in THF), THF, 98%; c) DBU, Cl₃CCN, CH₂Cl₂, 08C, 98%.

verted to glycosylating agent 2 by cleavage of the silyl ether

at the anomeric position and reaction with trichloroacetoni-

trile and DBU. The condensation of glycosyl acceptor 5 and

trichloroacetimidate 2, followed by removal of the levulinoyl

ester from the C4 hydroxyl group, produced trisaccharide

acceptor 16, that served as key intermediate for further

elongation reactions (Scheme 4). Trisaccharide 16 was

capped with building block 3 to afford tetrasaccharide 17

(88% yield), that was submitted to the deprotection–sulfa-

tion sequence. Tetrasaccharide 17 was treated with 2-(benzyl-

oxycarbonylamino)-1-ethanethiol and AIBN to give 18 in

61% yield. Aconsiderable amount (24%) of unreacted 17

was also isolated from the reaction mixture. Longer reaction

times or the use of more AIBN led to the detection of ther-

mal decomposition products. The hydrolysis of ester groups

was performed with lithium hydroperoxide and followed by

KOH to give 19 (88%). The reduction of the azide protect-

ing groups followed by simultaneous O- and N-sulfation af-

groups furnished 23 in excellent yield.

Glycosylation of 16 with disaccharide repeating unit 2 af-

forded pentasaccharide 24 in 74% yield (Scheme 5). Dele-

vulinoylation at C4 to yield 25 followed by capping with

building block 3 yielded hexasaccharide 26 in 82% over two

steps. Radical elongation of the pentenyl moiety using 2-

(benzyloxycarbonylamino)-1-ethanethiol and AIBN trans-

formed 26 to 27 (59%). Aconsiderable amount (19%) of

unreacted starting material was again isolated from the reac-

tion mixture. Treatment of 27 with lithium hydroperoxide

and then KOH to furnish 28 followed by O-sulfation using

SO₃·Et₃N in DMF gave 29 in excellent yield.^[58]Reduction

of the azido group using Staudinger conditions to give 30

followed by N-sulfation using SO₃·Py afforded 31 that was

submitted to hydrogenolysis to yield sulfated hexasaccharide

32. These amine-functionalized heparin oligosaccharides 10,

23 and 32 were ready for immobilization on chip surfaces

and microarray experiments.

Scheme 4. Assembly of tetrasaccharide 23. a) 2, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 4 MS, 91%; b) H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, 86%; c) 3,

TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 4 MS, 88%; d) HS-CH₂-CH₂-NHZ, AIBN, THF, 758C, 61%; e) LiOH, H₂O₂; KOH, MeOH, 88%; f) SO₃·Et₃N, DMF,

558C, 80%; g) PMe₃, THF, NaOH, quant.; h) SO₃·Py, Py, TEA, 78%; i) H₂, Pd/C, quant.

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Scheme 5. Synthesis of hexasaccharide 32. a) 2, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 4 MS, 74%; b) H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, 96%; c) 3, TMSOTf

(cat.), CH₂Cl₂, ꢀ208C, 4 MS, 85% (26); d) HS-CH₂-CH₂-NHZ, AIBN, THF, 758C, 59%; e) LiOH, H₂O₂; KOH, MeOH, 92%; f) SO₃·Et₃N, DMF,

558C, 97%; g) PMe₃, THF, NaOH, 85%; h) SO₃·Py, Py, TEA, 77%; i) H₂, Pd/C, quant.

Oligosaccharide assembly using an amine-protected pentyl

linker and preparation of heparin oligosaccharides with dif-

ferent sulfation patterns: To avoid elaborate linker modifi-

cations at the late stage of oligosaccharide synthesis and to

improve upon the moderate yields observed during the radi-

cal elongation of the pentenyl moiety of tetra- and hexasac-

charides, we decided to explore the use of an amine-protect-

ed pentyl linker that was installed at the reducing end prior

to oligosaccharide construction. Coupling of building block

1 with n-benzyloxycarbonyl-5-aminopentane-1-ol^[59]afforded

the desired reducing end building block 33 that was readily

converted to glycosyl acceptor 34 (Scheme 6). Coupling of

disaccharide 2 with acceptor 34 afforded trisaccharide 35 in

a mere 41% yield after purification by flash column chro-

matography. Aconsiderable amount of unreacted starting

material (21%) and silylated acceptor (35%) was also re-

covered from the reaction mixture. After cleavage of the

levulinic acid protecting group at C4 using hydrazine mono-

Scheme 6. Assembly of hexasaccharides 39 and 45. a) 1, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 45% (33); b) 1, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, then

H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, 56% (40); c) H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, 95%; d) 2, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 4 MS, 41% (35), 70%

(41); e) H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, 86% (36), 89% (42); f) 2, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 4 MS, 34% (37), 82% (43); g) H₂NNH₂·H₂O,

Py/AcOH, CH₂Cl₂, 98% (38), 86% (44); h) 3, TMSOTf (cat.), CH₂Cl₂, ꢀ208C, 4 MS, 62% (39), 80% (45).

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Heparin Oligosaccharides

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hydrate in a pyridine/acetic acid solution, acceptor 36 was

glycosylated with disaccharide 2. Pentasaccharide 37 was ob-

tained in only 34% yield accompanied by recovered accept-

or (19%) and silylated acceptor (33%). Removal of the

protecting group at C4 to furnish 38 and capping with glu-

cosamine 3 afforded 62% of hexasaccharide 39.

Since all glycosylations with the benzyloxycarbonyl-5-

amino linker were low yielding, we considered the use of a

(benzyl)benzyloxycarbonyl-amino linker that had been suc-

cessfully used^[60,61]in oligosaccharide synthesis. This linker

avoids the undesired deactivating effect of the -NH group^[62]

and was prepared from n-benzyloxycarbonyl-5-aminopen-

tane-1-ol by benzylation of the amine group via an O-trity-

lated intermediate (see Experimental Section). Glycosida-

ly, saponification followed by Staudinger reduction, N-acety-

lation and hydrogenolysis yielded non-sulfated hexasacchar-

ide 52. Starting from compound 45, hydrolysis of the acyl

groups followed by azido reduction, selective N-sulfation

using SO₃·Py in a mixture of triethylamine/MeOH/NaOH

and global deprotection by hydrogenolysis afforded oligo-

saccharide 56 that contains sulfate groups at position 2 of

glucosamine.

Preparation of heparin oligosaccharides containing the alter-

native sequence IdoA-GlcN: To access further distributions

of sulfate groups we adapted our synthetic approach to the

synthesis of heparin oligosaccharides that contain the alter-

native IdoA-GlcN sequence. Permanent benzyl protecting

groups are placed at position 6 of the glucosamine units.

Three different disaccharide building blocks had to be pre-

pared to achieve this goal (Scheme 8). Glycosylation of the

n-benzyloxycarbonyl-5-aminopentane-1-ol linker with tri-

chloroacetimidate 57^[38]followed by regioselective opening

of the benzylidene acetal afforded 58. Union of 58 and tri-

chloroacetimidate 11 gave disaccharide 59 in 60% yield.

The reducing end unit 60 was obtained after removal of the

temporary levulinoyl protecting group at C4 by treatment

with hydrazine monohydrate. Elongation disaccharide 64

was constructed by condensation of 11 with glucosamine ac-

ceptor 61^[42]to give 62. Removal of the silyl protecting

group was followed by conversion to the glycosylating

agent. Building block 66 was synthesized as precursor to

capping disaccharide 69. Starting from the 1,2-locked idur-

onic acid 65,^[51]hydrolysis of the isopropylidene acetal fol-

lowed by selective silylation of the anomeric position, acety-

lation, desilylation and trichloroacetimidate activation af-

forded 66 (Scheme 8). Combination of 66 with glucosamine

acceptor 61 afforded, after removal of the silyl group and in-

stallation of the imidate, the desired capping disaccharide

tion of iduronic acid 1 with n-(benzyl)-benzyloxycarbonyl-5-

A

aminopentane-1-ol followed by cleavage of the levulinoyl

group at C4 gave alcohol 40 (Scheme 6). This new reducing

end building block was elongated via intermediates 41 and

43 to trisaccharide 42 and pentasaccharide 44 using iterative

TMSOTf-catalyzed glycosylation with 2. Cleavage of the

levulinoyl protecting group was achieved with hydrazine

monohydrate in the presence of pyridine/acetic acid solu-

tion. The use of the (benzyl)benzyloxycarbonyl-amino linker

increased the efficiency of the glycosidation reactions (70

and 82% yield, respectively) as planned. Subsequently, cou-

pling with capping building block 3 afforded the desired

hexasaccharide 45 in 80% yield after chromatography.

With the fully protected oligosaccharides 39 and 45 at

hand, we planned the deprotection–sulfation sequence in

order to prepare amine-functionalized sugars with different

sulfation patterns (Scheme 7). Commencing with compound

39, saponification followed by O-sulfation, Staudinger re-

duction, selective N-acetylation and hydrogenolysis afforded

hexasaccharide 51 with sulfate groups at position 6 of the

glucosamine and position 2 of the iduronic acid. Alternative-

Scheme 7. Preparation of heparin oligosaccharides with different sulfation patterns. a) LiOH, H₂O₂; KOH, MeOH, 71%; b) SO₃·Et₃N, DMF, 558C, 88%;

c) PMe₃, THF, NaOH; Ac₂O, TEA, MeOH, 67%; d) H₂, Pd/C, 88%; e) PMe₃, THF, NaOH, 77%; f) Ac₂O, TEA, MeOH, quant.; g) H₂, Pd/C, quant.;

h) LiOH, H₂O₂; KOH, MeOH, 63%; i) PMe₃, THF, NaOH, 83%; j) SO₃·Py, TEA, MeOH, NaOH, quant.; k) H₂, Pd/C, 97%.

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Scheme 8. Synthesis of disaccharide building blocks 60, 64, and 69. a) n-benzyloxycarbonyl-5-aminopentane-1-ol, TMSOTf (cat.), CH₂Cl₂, ꢀ258C, 92%;

b) SiH(Et)₃, CH₂Cl₂, 08C, TFA, 60%; c) 11, TMSOTf (cat.), CH₂Cl₂, ꢀ258C, 60%; d) H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, quant.; e) 11, TMSOTf (cat.),

CH₂Cl₂, ꢀ258C, 86%; f) HOAc, TBAF (1m in THF), THF, 90%; g) DBU, Cl₃CCN, CH₂Cl₂, 08C, 92%; h) TFA/H₂O, CH₂Cl₂, then imidazole, TDSCl,

CH₂Cl₂, ꢀ288C, 77%, two steps; i) Py, Ac₂O, DMAP (cat.), 96%; j) HF·Py (70%), THF, then DBU, Cl₃CCN, CH₂Cl₂, 08C, 98%, two steps; k) 61,

TMSOTf (cat.), CH₂Cl₂, ꢀ258C, 80%; l) HOAc, TBAF (1m in THF), THF, 80%; m) DBU, Cl₃CCN, CH₂Cl₂, 08C, 96%.

69. With the three disaccharide units 60, 64 and 69 in hand,

assembly of hexasaccharide 72 was accomplished

(Scheme 9). Coupling of 60 with elongation disaccharide 64

afforded the fully protected tetrasaccharide 70 in 75% yield.

Removal of the levulinoyl group at C4 and coupling with

capping disaccharide 69 resulted in hexasaccharide 72.

Next, deprotection and sulfation of the structures contain-

ing the IdoA-GlcN sequence allowed for the preparation of

further heparin oligosaccharides with different sulfate group

patterns (Scheme 10). Commencing with 72, saponification,

O-sulfation, Staudinger reduction, N-sulfation and hydroge-

nolysis afforded hexasaccharide 77 with sulfate groups at po-

sition 2 of both glucosamine and iduronic acid. Alternative-

ly, saponification followed by O-sulfation, Staudinger reduc-

tion, N-acetylation and hydrogenolysis yielded hexasacchar-

ide 79 with sulfate groups at position 2 of iduronic acid.

From compound 71, hydrolysis of the acyl groups followed

by azide reduction, selective N-sulfation and global depro-

tection by hydrogenolysis gave tetrasaccharide 83 that con-

tains sulfate groups at position 2 of the glucosamine.

Carbohydrate microarray analysis: The library of synthetic

heparin oligosaccharides (Figure 1) was used in microarray

experiments to evaluate FGF binding to defined sequences

and to determine the influence of length and sulfation pat-

tern on carbohydrate recognition. The amine-terminated

linker was employed to immobilize the sugars onto N-hy-

droxysuccinimide (NHS) activated glass slides. The covalent

attachment strategy is convenient due to the reproducibility

of amide bond formation.^[63]Disulfated monosaccharides

IdoA(2,4-di-OSO₃) 84 and GlcNSO₃

(6-OSO₃) 85^[37]

A

(Figure 1) were also included in the microarray analysis as

well as non-sulfated IdoAmonosaccharide 86 (negative con-

trol) and an amine-functionalized heparin sample^[64](87,

average molecular weight 5 kDa). The spotting process was

miniaturized by using a non-contact robotic printer to gener-

ate an array of 832 spots (Figure 2). No anti-evaporation

agent, such as DMSO or glyc-

erol, was added in the printing

solution, as recommended by

the manufacturer, because

these additives decrease bind-

ing and/or destroy spot mor-

phology. The nature of the Co-

deLink slides does not require

that the printed spots remain

solubilized for effective immo-

bilization. The binding assay in-

volved initial hybridization

with the FGF protein, followed

by incubation with anti FGF-

polyclonal antibodies. Finally,

Scheme 9. Assembly of hexasaccharide 72. a) 64, TMSOTf (cat.), CH₂Cl₂, ꢀ258C, 4 MS, 75%;

b) H₂NNH₂·H₂O, Py/AcOH, CH₂Cl₂, 90%; c) 69, TMSOTf (cat.), CH₂Cl₂, ꢀ258C, 4 MS, 59%.

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Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

printed oligosaccharide spots

was directly observed. No bind-

ing of the antibodies without

FGFs to spots was observed.

The fluorescence signals were

quantified by using the appro-

priate software.

Incubation of FGF-2 with the

microarray revealed binding at

spots corresponding to hexasac-

charides 32, 51 and 77, tetrasac-

charide 23, and heparin 87

along with less binding to disac-

charide 10 and iduronic acid

monosaccharide 84. No binding

was detected for hexasacchar-

ides 52, 56 and 79 indicating

that at least two sulfate groups

per disaccharide are needed for

FGF-2 recognition. The obser-

vation that tetrasaccharide 23,

Scheme 10. Preparation of oligosaccharides 77, 79 and 83. a) LiOH, H₂O₂; KOH, MeOH, 86% (73);

b) SO₃·Et₃N, DMF, 558C, 86 % (74); c) PMe₃, THF, NaOH, 69% (75); d) SO₃·Py, Py, TEA, 92% (76); e) H₂,

Pd/C, 95% (77); f) Ac₂O, TEA, MeOH, 94% (78); g) H₂, Pd/C, 90% (79); h) LiOH, H₂O₂; KOH, MeOH,

82% (80); i) PMe₃, THF, NaOH, quant. (81); j) SO₃·Py, TEA, MeOH, NaOH, 82% (82); k) H₂, Pd/C, 85%

(83).

containing the GlcNSO

A

OSO₃)-IdoA(2-OSO₃) repeat-

AHCTREUNG

Figure 1. Amine-functionalized heparin oligosaccharides employed in the microarray experiments.

incubation with fluorescently labelled secondary antibody

detected any bound protein. Scanning the slide for fluores-

cence produced images (Figure 2), where FGF binding to

ing unit, is bound to FGF-2 agrees with previously reported

data.^[3]These earlier experiments typically used heparin

fragments derived by depolymerization of isolated heparin.

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8671

P. H. Seeberger et al.

the sulfate group at position 6

of the glucosamine unit likely is

not necessary for FGF-2 bind-

ing. This observation is again in

agreement with published re-

ports.^[65]

Microarray incubation with

FGF-1 revealed overall de-

creased affinity with hexasac-

charide 32 and heparin sample

87 as the best binders, followed

by

monosaccharide

84

(Figure 4). The presence of a

2,4-O-sulfation pattern, not

found in heparin, may explain

the high affinity of 84. Indeed,

compounds such as myo-inosi-

tol hexasulfate and sucrose oc-

tasulfate have been shown to

bind FGF-1 with high affini-

ty.^[66,67]The discovery of small

sulfated molecules, such as

monosaccharide 84, that strong-

ly bind FGF-1 has important

consequences for the design of

novel angiogenic inhibitors with

anticancer properties.^[68,69]The

fact that hexasaccharides 51

and 77 bound FGF-1 with

lower affinity than FGF-2 sug-

gests that higher negative

charge density, with three sul-

fate groups per disaccharide, is

essential to enhance FGF-1

binding. These results were con-

firmed by the construction of

Figure 2. Top: Image of a microarray after incubation with FGF-2. Bottom: Fluorescence signal observed for

each arrayed carbohydrate binding to FGF-2. Four different concentrations of sugar solutions, ranging from

16 mm to 2 mm from left to right were employed. All samples were printed in replicates of sixteen.

the

corresponding

binding

curves (see Supporting Infor-

mation). Interestingly, we also

Interestingly, the observed binding of FGF-2 to oligosac-

charides as short as disaccharide 10 may be explained by an

increased affinity of these compounds due to the multivalent

display on the glass surfaces.^[32]Additionally, by comparing

the fluorescence signals for monosaccharides 84 and 85, a

simple non-specific interaction between a cluster of negative

charges displayed on the glass surface and the basic amino

acids of the heparin binding site of FGF-2 can be ruled out.

After this initial assay, a second microarray experiment

was designed to study compounds that possess a similar

binding affinity to FGF-2 such as hexasaccharides 32, 51 and

77 and tetrasaccharide 23 (Figure 3). Sugars were spotted at

12 different concentrations ranging from 5 mm to 28 nm.

Plotting the fluorescence intensity against the concentration

of printed carbohydrates generates binding curves. These

binding curves indicated that FGF-2 binds the oligosacchar-

ides in the following order: 32>77>23>51. Since the bind-

ing curves for compounds 32 and 77 are almost identical,

found that non-sulfated hexasaccharide 52 bound FGF-1 at

high concentration. This result may confirm an earlier

report^[70]showing that nonsulfated heparin di- and trisac-

charides were able to activate several FGFs. These findings

suggest that FGF can specifically recognize structural fea-

tures of the non-sulfated carbohydrate backbone of heparin,

independent of ionic interactions with sulfate groups.

Heparin glass slides were also incubated with FGF-4.^[17,18]

Initial visual analysis indicated FGF-4 binding to hexasac-

charide 32 and heparin 87 along with less binding to com-

pounds 23, 51 and 77 (Figure 5). These results were con-

firmed by analysis of the corresponding binding curves (see

Supporting Information). The difference in binding between

hexa- 32 and tetrasaccharide 23 suggests the importance of

an additional disaccharide repeating unit for FGF-4 recogni-

tion. Also, these results indicate the significant effect of sul-

fate group distribution on FGF-4 binding at the hexasac-

charide level.

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Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

mal structural requirements for carbohydrate recognition is

extracted. The chemical synthesis of amine-functionalized

heparin sequences allowed for their immobilization onto

glass surfaces and the creation of heparin microarrays for

the rapid analysis of carbohydrate–protein interactions. We

employed a modular strategy for the assembly of oligosac-

charides with differential sulfation. Key features of this ap-

proach, such as the influence of the linker on the assembly

efficiency, and the compatibility of the protecting group

strategy with the sulfation–deprotection steps, have been

discussed in detail. These heparin chips have been employed

to characterize the carbohydrate binding affinities of three

heparin-binding growth factors, FGF-1, FGF-2 and FGF-4

that are implicated in angiogenesis, cell growth and differen-

tiation. In addition, the discovery of novel small sulfated

molecules, such as monosaccharide 84, that bind FGF has

important consequences for the design of novel angiogenic

inhibitors with anticancer properties.

The miniaturized chip format offers important advantages

over classical methods, such as the ability to screen several

thousand binding events on a single glass slide and the min-

iscule amounts of both analyte and ligand (ꢁpicomoles) re-

quired for one experiment. Therefore, heparin arrays are ex-

pected to fundamentally assist in the establishment of struc-

ture–activity relationships for heparin sequences. However,

the limitation of preparing hundreds or thousands of hepa-

rin oligosaccharides needs to be overcome for wider applica-

tions of heparin chips. Despite the impressive advances in

the automated assembly of oligosaccharides during the last

decade, the solid-phase synthesis of HLGAGs is still a diffi-

cult goal to achieve. In this context, our group is currently

working on the automated solid phase synthesis of these

heparin oligosaccharides in order to expand the complexity

and utility of heparin arrays. We

Figure 3. Binding of FGF-2 to compounds 32, 23, 51, and 77. Top: Slide

image obtained from a fluorescence scan following FGF-2 incubation.

Each grid has increasing concentrations of carbohydrate from 28 nm to

5 mm from left to right with sixteen replicates per sample. Bottom: Bind-

ing curves obtained by plotting fluorescence signal against concentration

of carbohydrate delivered to surface.

envision that the combination of

the automated oligosaccharide

synthesis and the carbohydrate

microarray

technology

will

become essential to better un-

derstand the role of heparin in

biological processes.

Experimental Section

General information: All chemicals

used were reagent grade and used as

supplied except where noted. Dichloro-

methane (CH₂Cl₂) and tetrahydrofuran

(THF) were purified by a Cycle-Tainer

solvent delivery system. Pyridine and

triethylamine were distilled over CaH₂

prior to use. Analytical thin layer chro-

matography (TLC) was performed on

Figure 4. Fluorescence signal observed for each carbohydrate binding to FGF-1. Four different concentrations

of sugar solutions, ranging from 2 mm to 16 mm, were employed.

Conclusion

Merck silica gel 60 F₂₅₄plates (0.25 mm). Compounds were visualized by

UV irradiation or dipping the plate in an anisaldehyde solution followed

by heating. Flash column chromatography was carried out using forced

flow of the indicated solvent on Fluka Kieselgel 60 (230–400 mesh). Gel

In summary, these studies using microarrays of synthetic

heparin oligosaccharides expand our understanding of FGF–

heparin interactions. Precious information about the mini-

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8673

P. H. Seeberger et al.

Heparin array fabrication: Heparin

oligosaccharides were spatially arrayed

onto NHS-activated CodeLink slides

by use of an automated arraying robot

in sodium phosphate buffer (pH 9.0,

50 mm). Slides were printed in 50%

relative humidity at 228C, followed by

incubation overnight in

a saturated

NaCl chamber that provides a 75%

relative humidity environment. The

robot delivered 1 nL of sugar solutions

at four different concentrations (2 mm,

400 mm, 80 mm, and 16 mm) and the re-

sulting spots had an average diameter

of 200 mm with a distance of 500 mm

between the centers of adjacent spots.

All samples were printed in replicates

of sixteen. Slides were then washed

with water (3) to remove the un-

bound carbohydrates from the surface.

Remaining succinimidyl groups were

quenched by placing slides in a solu-

tion preheated to 508C that contained

100 mm ethanolamine in sodium phos-

phate buffer (pH 9.0, 50 mm) for 1 h.

Slides were rinsed several times with

distilled water, dried by centrifugation

and stored in a desiccator prior to use.

Binding assay: Solutions used for chip

hybridizations were sterile filtered

through a 0.2 mm syringe filter prior to

use. The FGF hybridization solutions

were prepared by diluting the stock

solutions to

a concentration of 4–

20 mgmL^ꢀ1with PBS buffer (pH 7.5,

10 mm) containing BSA(1%). Array

incubations were performed as fol-

lows: 100 mL of FGF solution were

placed between array slides and plain

coverslips and incubated for 1 h at

room temperature. The arrays were

Figure 5. Top: Image of an array after incubation with FGF-4. Bottom: Fluorescence signal observed for each

arrayed carbohydrate binding to FGF-4. Four different concentrations of sugar solutions, ranging from 16 mm

to 2 mm from left to right were employed. All samples were printed in sixteen replicates.

filtration chromatography was carried out using Sephadex LH-20 from

Amersham Biosciences.

washed with PBS (pH 7.5, 10 mm) containing 1% Tween 20 and 0.1%

BSA, twice with water, and then centrifuged for 5 min to ensure dryness.

For detection of bound FGF, arrays were incubated with anti-human

FGF polyclonal antibody (4–20 mgmL^ꢀ1) and then washed as above. Fi-

nally, AlexaFluor-546-labelled anti-rabbit IgG (20 mgmL^ꢀ1) was used as

secondary antibody and again washed as above.

¹H and ¹³C NMR spectra were recorded on

a Varian VXR-300

(300 MHz) or Bruker DRX500 (500 MHz) spectrometer. When NMR

spectra of sulfated compounds were recorded, triethylamine was usually

added to improve the resolution of the iduronic acid signals. High-resolu-

tion mass spectra (HR MALDI MS) were performed by the MS service

at the Laboratory for Organic Chemistry (ETH Zürich). ES-MS were

run on an Agilent 1100 Series LC/MSD instrument. IR spectra were re-

corded on a Perkin–Elmer 1600 FTIR spectrometer. Optical rotations

were measured using a Perkin–Elmer 241 polarimeter.

Image acquisition and signal processing: Heparin arrays were scanned by

using a LS400 scanner and fluorescence intensities from these scans were

integrated on Scan Array Express and Gen Spotter software. Signal to

background was typically ꢂ 50:1. The local background was subtracted

from the hybridization signal of each separate spot and the mean intensi-

ty of each spot was used for data analysis. Spot finding was automatically

performed, followed by manual fitting to correct spot deviations. Data

presented are the average of 16 spots on the same array; errors are the

standard deviations for each measurement.

Microarray analysis: All aqueous solutions were made from nanopure

water. Solutions used for chip hybridizations were sterile filtered through

a 0.2 mm syringe filter prior to use. Recombinant human basic Fibroblast

Growth Factor (FGF-2), anti-human FGF-2, recombinant human acidic

Fibroblast Growth Factor (FGF-1), recombinant human FGF-4, and anti-

human FGF-4 were purchased from PeproTech EC (London, UK). Anti-

human FGF-1 was purchased from Santa Cruz Biotechnology Inc (CA).

The primary anti-FGF antibodies were rabbit polyclonal antibodies, de-

tected by using goat anti-rabbit IgG labelled with Alexa Fluor 546 dye

(Molecular Probes). CodeLink slides were purchased from Amersham

Biosciences. Microarrays were constructed using a Perkin–Elmer noncon-

tact printer. For all incubations, 100 mL of protein solution were applied

to the slide by using HybriSlip Hybridization Covers from Grace Bio-

Labs (Bend, OR). Slides were scanned using a LS400 scanner from

Tecan (Männedorf, Switzerland) and quantified using Scan Array Ex-

press (Perkin Elmer) and Gene Spotter (MicroDiscovery GmbH, Berlin,

Germany) software.

n-Pentenyl (methyl 2-O-pivaloyl-3-O-benzyl-4-O-levulinoyl)-a-l-idopyra-

nosyluronate (4): Glycosyl trichloroacetimidate 1 (480 mg, 0.77 mmol)

was dissolved in CH₂Cl₂. 4-Penten-1-ol (100 mg, 0.84 mmol) was added

and the reaction was cooled to ꢀ208C, before TMSOTf (20 mL,

0.08 mmol) was added. The reaction mixture was allowed to warm to

ꢀ108C over 30 min. The reaction was quenched with triethylamine at

ꢀ258C and allowed to warm to room temperature. Purification by flash

chromatography (hexanes/EtOAc 4:1) yielded in the desired compound 4

(350 mg, 83%). [a]^R_D^T=ꢀ53.9 (c=1, CHCl₃); ¹H NMR (500 MHz,

CDCl₃): d = 7.38–7.28 (m, 5H), 5.81–5.76 (m, 1H), 5.23 (t, J=4.5 Hz,

1H), 5.00 (dd, J=1.5, 3.5 Hz, 1H), 4.97–4.93 (m, 2H), 4.92–4.91 (m, 1H),

4.89 (d, J=2.0 Hz, 1H), 4.80 (d, J=11.5 Hz, 1H), 4.69 (d, J=11.5 Hz,

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Heparin Oligosaccharides

FULL PAPER

1H), 3.77–3.74 (m, 1H), 3.71 (dd, J=2.0, 4.0 Hz, 1H), 3.52–3.49 (m, 1H),

2.77–2.69 (m, 2H), 2.55–2.52 (m, 2H), 2.17 (s, 3H), 2.16–2.11 (m, 2H),

1.73–1.67 (m, 2H), 1.20 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): d = 206.2,

177.5, 171.9, 169.3, 138.2, 137.8, 128.5–127.7, 115.1, 98.7, 77.5, 77.2, 77.0,

73.3, 72.3, 68.3, 68.2, 66.7, 66.1, 52.7, 38.9, 37.9, 30.4, 29.9, 28.7, 28.1, 27.2;

IR (thin film on NaCl): n˜ =3065, 2957, 2933, 2874, 1741, 1538, 1496, 1438,

PMe₃in THF (42 mL of a 1m solution) was added and the reaction was

allowed to stir for 2 h. The reaction mixture was neutralized with a 0.1m

solution of HCl, concentrated and the residue was eluted from a Sepha-

dex LH-20 chromatography column with MeOH/CH₂Cl₂1:1 to afford 8

(17 mg, 89%). ¹H NMR (300 MHz, CD₃OD): d = 7.40–7.20 (m, 20H),

5.30 (d, J=3.6 Hz, 1H), 5.07 (s, 2H), 4.99 (d, J=11.7 Hz, 1H), 4.83–4.59

(m, 7H), 4.31 (brs, 1H), 4.09–3.43 (m, 12H), 3.22–3.18 (m, 2H), 2.96–

2.88 (m, 2H), 1.75–1.41 (m, 6H); ES-MS: m/z: calcd for C₄₈H₆₁O₁₅N₂S:

937.4; found: 937.2 [M+H]⁺.

1398, 1306, 1277, 913 cm^ꢀ1

; HR MALDI MS: m/z: calcd for

C₂₉H₄₀O₁₀Na: 571.2519; found: 571.2501 [M+Na]⁺.

n-Pentenyl (methyl 2-O-pivaloyl-3-O-benzyl)-a-l-idopyranosyluronate

(5): Compound 4 (0.35 g, 0.64 mmol) was dissolved in CH₂Cl₂. Pyridine

(1.5 mL) and acetic acid (1.0 mL) were added, followed by the addition

of hydrazine monohydrate (45 mL, 1.27 mmol). The reaction mixture was

stirred at room temperature for 90 min, diluted with CH₂Cl₂, quenched

with acetone and evaporated to dryness. Product 5 was isolated as foam

after flash column chromatography on silica gel (hexanes/EtOAc 9:1)

SO₃·Py (43 mg, 0.27 mmol) was added to a solution of 8 (17 mg, 18 mmol)

in anhydrous pyridine (2 mL). After stirring for 4 h at room temperature

under an argon atmosphere, the reaction mixture was quenched with trie-

thylamine (0.2 mL) and diluted with MeOH (1 mL) and CH₂Cl₂(1 mL).

The solution was layered on the top of a Sephadex LH-20 chromatogra-

phy column that was eluted with MeOH/CH₂Cl₂1:1. The fractions con-

taining the sulfated disaccharide were pooled and evaporated to dryness.

The residue was converted into the sodium salt by elution from a column

of Dowex 50WX4-Na⁺with MeOH/H₂O 9:1 to give 9 (20 mg, 87%).

(261 mg, 91%). [a]_D^RT

=

ꢀ36.2 (c=1, CHCl₃); ¹H NMR (500 MHz,

CDCl₃): d = 7.37–7.24 (m, 5H), 5.84–5.71 (m, 1H), 5.01–4.99 (m, 1H),

4.97–4.93 (m, 2H), 4.87 (d, J=1.8 Hz, 1H), 4.81 (d, J=11.7 Hz, 1H), 4.61

(d, J=11.7 Hz, 1H), 4.05 (d, J=10.2 Hz, 1H), 3.82 (s, 3H), 3.81–3.74 (m,

1H), 3.70–3.68 (m, 1H), 3.52–3.49 (dt, J=7.8, 9.6 Hz, 1H), 2.67 (d, J=

12.0 Hz, 1H), 2.13 (dd, J=6.9, 13.5 Hz, 2H), 1.77–1.67 (m, 2H), 1.23 (s,

9H); ¹³C NMR (125 MHz, CDCl₃): d = 177.4, 170.7, 138.6, 138.3, 129.1–

128.3, 115.7, 99.5, 74.9, 72.4, 68.9, 68.8, 68.2, 67.1, 53.1, 39.5, 30.9, 29.3,

27.8, 1.7; IR (thin film on NaCl): n˜ =3582, 2918, 1739, 1496, 1455, 1398,

1368, 1276, 1141, 912 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₂₄H₃₄O₈Na:

473.2151; found: 473.2146 [M+Na]⁺.

¹H NMR (300 MHz, CD₃OD): d

= 7.46–7.20 (m, 20H), 5.39 (d, J=

3.3 Hz, 1H), 5.27 (brs, 1H), 5.17 (d, J=11.1 Hz, 1H), 5.07 (s, 2H), 4.80–

4.68 (m, 6H), 4.53 (brs, 1H), 4.43–4.35 (m, 2H), 4.24 (m, 1H), 4.16 (brs,

1H), 3.94 (m, 1H), 3.79–3.48 (m, 7H), 3.22–3.18 (m, 2H), 2.92–2.87 (m,

2H), 1.74–1.42 (m, 6H); ES-MS: m/z: calcd for C₄₈H₅₉O₂₄N₂S₄: 1175.2;

found: 1175.0 [M+2H]^ꢀ; calcd for C₄₈H₅₈O₂₄N₂S₄: 587.5; found: 587.0.

Asolution of 9 (20 mg, 16 mmol) in MeOH/H₂O (2 mL/1 mL) was hydro-

genated in the presence of 10% Pd/C. After 24 h, the suspension was fil-

tered and concentrated to give 10 (13 mg, quantitative). [a]²_D⁴=ꢀ3.9 (c=

0.38, H₂O); ¹H NMR (300 MHz, D₂O): d = 5.35 (s, 1H), 5.15 (s, 1H),

4.50 (s, 1H), 4.37 (m, 1H), 4.23–4.18 (m, 3H), 4.05–3.96 (m, 2H), 3.75–

3.06 (m, 11H), 1.91–1.54 (m, 6H); ¹³C NMR (75 MHz, D₂O): d = 177.5,

163.5, 101.3, 99.8, 78.4, 78.3, 73.6, 72.6, 71.9, 70.8, 69.1, 60.7, 55.3, 51.6,

30.5, 26.9, 23.5; ES-MS: m/z: calcd for C₁₉H₃₄O₂₂N₂S₄Na: 793.0; found:

792.8 [M+Na+H]^ꢀ.

5-[2-(Benzyloxycarbonylamino)-1-thioethyl] pentyl (6-O-acetyl-2-azido-

3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1!4)-methyl 3-O-benzyl-

2-O-pivaloyl-a-l-idopyranosiduronate (6): TMSOTf (40 mL of a 0.45m so-

lution in anhydrous CH₂Cl₂) was added at ꢀ258C, under argon, to a solu-

tion of 3 (102 mg, 0.18 mmol) and 5 (67 mg, 0.15 mmol) in anhydrous

CH₂Cl₂(1 mL). After 30 min, the reaction mixture was quenched with

triethylamine, concentrated and the residue was purified by flash chroma-

tography (toluene/EtOAc 16:1s) to yield the desired pentenyl glycoside

(104 mg, 81%). ES-MS: m/z: calcd for C₄₆H₆₁O₁₃N₄: 877.0; found: 877.2

[M+NH₄]⁺.

tert-Butyldimethylsilyl (methyl 2-O-acetyl-3-O-benzyl-4-O-levulinoyl-a-l-

idopyranosyluronate)-(1!4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-b-d-

glucopyranoside (13): Glucosamine acceptor 12 (0.85 g, 1.87 mmol) and

iduronic acid trichloroacetimidate 11 (1.31 g, 2.25 mmol) were combined

in a flask, coevaporated with toluene (5) and dried under vacuum. The

starting materials were dissolved in CH₂Cl₂(27 mL) and cooled to

ꢀ208C. TMSOTf (41 mL, 0.23 mmol) was added and the reaction was al-

lowed to warm to ꢀ108C over 30 min. The reaction was quenched at

ꢀ208C with triethylamine and the solvent was removed under reduced

pressure. Purification by flash chromatography (hexanes/EtOAc 1:1)

yielded disaccharide 13 (1.59 g, 97%). [a]^R_D^T=ꢀ39.6 (c=1, CHCl₃);

¹H NMR (500 MHz, CDCl₃): d = 7.39–7.21 (m, 10H), 5.10 (s, 1H), 5.08

(t, J=3.0 Hz, 1H), 4.99 (d, J=2.5 Hz, 1H), 4.85 (s, 1H), 4.74 (d, J=

12.0 Hz, 1H), 4.71 (d, J=11.1 Hz, 1H), 4.70 (d, J=11.0 Hz, 1H), 4.60 (d,

J=11.0 Hz, 1H), 4.53 (d, J=8.1 Hz, 1H), 4.52 (dd, J=2.5, 12.0 Hz, 1H),

4.14 (dd, J=5.5, 11.5 Hz, 1H), 3.85 (t, J=9.5 Hz, 1H), 3.80 (t, J=3.0 Hz,

1H), 3.50–3.47 (m, 1H), 3.44 (s, 3H), 3.36 (dd, J=7.5, 10.0 Hz, 1H), 3.23

(t, J=9.5 Hz, 1H), 2.81–2.74 (m, 1H), 2.67–2.61 (m, 1H), 2.58–2.52 (m,

1H), 2.49–2.43 (m, 1H), 2.17 (s, 3H), 2.08 (s, 3H), 2.7 (s, 3H), 0.93 (s,

9H), 0.15 (s, 3H), 0.14 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d = 206.2,

171.9, 170.8, 170.7, 170.1, 168.6, 138.1, 137.5, 128.6–127.6 (CH-Ar), 97.7,

97.4, 81.0, 74.8, 74.7, 73.6, 72.8, 72.6, 68.9, 68.1, 67.3, 66.8, 62.5, 52.3, 37.7,

30.0, 28.0, 25.8, 21.1, 21.0, 18.2. ꢀ4.2, ꢀ5.0; IR (thin film on NaCl): n˜ =

3032, 2959, 2929, 2857, 2111, 1743, 1497, 1407, 1367, 1260, 1158, 1107,

908 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₄₂H₅₇N₃O₁₆SiNa: 894.3457;

found: 894.3462 [M+Na]⁺.

Asolution of this disaccharide (64 mg, 74 mmol), AIBN (4.9 mg, 30 mmol)

and 2-(benzyloxycarbonylamino)-1-ethanethiol (236 mg, 1.12 mmol) in

THF (6 mL) was stirred at 758C under an argon atmosphere. After 12 h,

the reaction mixture was concentrated and the residue was subjected to

flash chromatography (hexanes/EtOAc 4:1!2:1) to afford 6 (62 mg,

78%). [a]²_D⁴= +6.0 (c=0.5, CHCl₃); ¹H NMR (300 MHz, CDCl₃): d =

7.38–7.25 (m, 20H), 5.19 (brs, 1H), 5.10 (s, 2H), 5.05–5.04 (m, 2H), 4.96

(dd, J=3.9, 4.8 Hz, 1H), 4.87–4.69 (m, 6H), 4.59 (d, J=11.1 Hz, 1H),

4.33 (dd, J=2.1, 12.3 Hz, 1H), 4.22–4.13 (m, 2H), 3.99–3.71 (m, 7H),

3.56–3.26 (m, 5H), 2.66–2.62 (m, 2H), 2.49–2.45 (m, 2H), 2.01 (s, 3H),

1.61–1.41 (m, 6H), 1.23 (s, 9H); ¹³C NMR (75 MHz, CDCl₃): d = 177.2,

170.3, 169.6, 137.5, 137.4, 137.3, 128–127.4, 98.8, 98.2, 79.9, 77.6, 75.5,

75.4, 74.7, 74.0, 73.0, 69.9, 69.7, 69.4, 68.8, 66.7, 63.3, 62.5, 52.3, 40.1, 38.9,

38.2, 32.2, 31.7, 29.4, 29.1, 27.2, 25.4, 20.9; IR (thin film on NaCl): n˜ =

3446, 3005, 2923, 2102, 1728, 1512, 1144 cm^ꢀ1; ES-MS: m/z: calcd for

C₅₆H₇₄O₁₅N₅S: 1088.5; found: 1088.4 [M+NH₄]⁺; HR MALDI MS: m/z:

calcd for C₅₆H₇₀N₄O₁₅SNa: 1093.4451; found: 1093.4430 [M+Na]⁺.

Disaccharide 10: H₂O₂(30%, 0.52 mL) and a solution of LiOH (1 N,

0.88 mL) were added at 08C to a solution of 6 (49 mg, 46 mmol) in THF

(1.6 mL). After stirring for 16 h at room temperature, the mixture was

cooled to 08C and MeOH (2.7 mL) and a solution of KOH (3m, 1.6 mL)

were added. After stirring for 16 h at room temperature, the reaction

mixture was neutralized with IR-120-H⁺Amberlite resin and then fil-

tered and concentrated. The residue was dissolved in CH₂Cl₂, washed

with H₂O, dried (MgSO₄), concentrated in vacuo and purified by flash

Methyl 2-O-acetyl-3-O-benzyl-4-O-levulinoyl-a-l-idopyranosiduronate-

(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a/b-d-glucopyranosyl) tri-

chloroacetimidate (2): Disaccharide 13 (1.02 g, 1.14 mmol) was dissolved

in anhydrous THF (16 mL). Glacial acetic acid (145 mL, 2.52 mmol) and

TBAF (1m in THF, 2.30 mL, 2.28 mmol) were added and the mixture

was stirred at room temperature for 4 h. The mixture was diluted with

EtOAc, extracted with NaHCO₃and with brine. The organic layer was

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

1

chromatography (CH₂Cl₂/MeOH 8:1) to afford 7 (32 mg, 73%). H NMR

(300 MHz, CD₃OD): d = 7.39–7.18 (m, 20H), 5.25 (s, 1H), 5.07 (m, 3H),

4.84–4.59 (m, 6H), 4.49 (d, J=11.1 Hz, 1H), 4.28 (brs, 1H), 3.97–3.41

(m, 12H), 3.21–3.17 (m, 2H), 2.90–2.84 (m, 2H), 1.68–1.37 (m, 6H); ES-

MS: m/z: calcd for C₄₈H₆₁O₁₅N₅S: 979.4; found: 980.0 [M+NH₄]⁺.

Compound 7 (20 mg, 21 mmol) was dissolved in THF (3.5 mL) and treat-

ed with a 0.1m aqueous solution of NaOH (0.4 mL). Then, a solution of

Chem. Eur. J. 2006, 12, 8664 – 8686

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

8675

P. H. Seeberger et al.

rification by flash chromatography on silica gel (hexanes/EtOAc 7:3!

1:1) afforded 14 as a white solid (870 mg, 98%).

(500 MHz, CDCl₃): d = 7.40–7.27 (m, 25H), 5.77–5.68 (m, 1H), 5.24 (d,

J=4.6 Hz, 1H), 4.98–4.84 (m, 8H), 4.79–4.58 (m, 10H), 4.53 (d, J=

11.1 Hz, 1H), 4.38 (dd, J=1.9, 12.5 Hz, 1H), 4.21–4.15 (2dd, J=2.2, 2.3,

12.1, 13.7 Hz, 2H), 4.12 (dd, J=3.7, 12.2 Hz, 1H), 4.07–4.05 (m, 1H),

3.99–3.97 (m, 1H), 3.89–3.87 (m, 3H), 3.83–3.68 (m, 7H), 3.53 (s, 3H),

3.52–3.42 (m, 3H), 3.25 (dd, J=3.6, 10.3 Hz, 2H), 2.05 (m, 2H), 2.04 (s,

3H), 2.0 (s, 3H), 1.91 (s, 3H), 1.66–1.59 (m, 2H), 1.21 (s, 9H); ¹³C NMR

(125 MHz, CDCl₃): d = 177.7, 170.8, 170.7, 170.0, 169.9, 169.6, 138.2,

137.9, 137.8, 137.8, 137.7, 137.6, 128.8–127.7, 115.1, 99.2–98.1 (4C_anomeric),

80.1, 78.3, 77.7, 75.9, 75.6, 75.5, 75.4, 75.1, 75.0, 74.4, 74.3, 74.1, 73.3, 73.2,

70.4, 70.3, 70.1, 70.0, 69.8, 69.6, 68.6, 63.5, 63.4, 62.5, 62.1, 60.6, 52.4, 52.2,

39.0, 30.3, 30.2, 29.9, 28.9, 27.5, 27.4, 27.3, 21.0, 20.9, 20.9, 14.4; IR (thin

film on NaCl): n˜ =3026, 2943, 2861, 2102, 1738, 1497, 1369, 1143,

1025 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₇₇H₉₂N₆O₂₅Na: 1523.6010;

found: 1523.6012 [M+Na]⁺.

Compound 14 (860 mg, 1.13 mmol) was dissolved in anhydrous CH₂Cl₂

(12 mL) and cooled to 08C. Trichloroacetonitrile (1.14 mL, 11.34 mmol)

and a catalytic amount of DBU were added. After 30 min, the solvents

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

EtOAc 1:1) afforded a mixture (1:1.5) of 2a and 2b as a white solid

(1.01 mg, 98%). ¹H NMR (300 MHz, CDCl₃): d = 8.76 (brs, NH, 1H),

7.38–7.22 (m, 10H), 6.41 (d, J=3.4 Hz, 0.4H), 5.63 (d, J=8.4 Hz, 0.6H),

5.12–5.07 (m, 2H), 4.96–4.86 (m, 2H), 4.72–4.62 (m, 4H), 4.47–4.45 (m,

1H), 4.23–4.21 (m, 1H), 4.05–4.01 (m, 2H), 3.83–3.67 (m, 4H), 3.47–3.38

(m, 3H), 2.74–2.48 (m, 4H), 2.16 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H);

¹³C NMR (125 MHz, CDCl₃): d = 206.9, 172.4, 171.2, 171.1, 170.7, 170.6,

169.23, 169.19, 161.6, 161.2, 138.2, 138.1, 137.9, 137.8, 129.2–128.2, 98.3,

98.2, 97.4, 95.1, 81.8, 79.2, 75.7, 75.6, 74.8, 74.7, 74.5, 73.5, 73.4, 73.3, 73.2,

72.8, 68.73, 68.68, 67.9, 67.7, 67.6, 67.5, 66.4, 63.9, 62.5, 62.4, 52.9, 38.24,

38.22, 30.5, 28.47, 28.45, 21.61, 21.59, 21.57; HR MALDI MS: m/z: calcd

for C₃₈H₄₃Cl₃N₄O₁₅Na: 923.1688; found: 923.1691 [M+Na]⁺.

5-[2-(Benzyloxycarbonylamino)-1-thioethyl] pentyl (6-O-acetyl-2-azido-

3,4-di-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)-methyl 3-O-benzyl-2-O-piva-

loyl-a-l-idopyranosiduronate (18): Asolution of 17 (29 mg, 19 mmol),

AIBN (1.3 mg, 7.7 mmol) and 2-(benzyloxycarbonylamino)-1-ethanethiol

(61 mg, 0.29 mmol) in THF (3 mL) was stirred at 758C under an argon

atmosphere. After 7 h, the reaction mixture was concentrated and the

residue was subjected to flash chromatography (hexanes/EtOAc 4:1!

2:1!1:1) to afford 18 (20 mg, 61%) and unreacted starting material

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-pivaloyl-a-l-idopyranosiduronate (16): Disacchar-

ide 2 (165 mg, 0.17 mmol) and reducing end building block 5 (65 mg,

0.14 mmol) were combined in a flask, coevaporated with toluene (5)

and dried under vacuum. The starting materials were dissolved in CH₂Cl₂

(1.6 mL) and freshly activated 4 molecular sieves (350 mg) were

added. This mixture was stirred for 30 min at room temperature under an

argon atmosphere. After cooling the mixture to ꢀ208C, TMSOTf (3 mL,

0.07 mmol) was added and the reaction was allowed to warm to ꢀ108C

over 30 min. The reaction was quenched at ꢀ208C with triethylamine

and filtrated over Celite. Removal of the solvent under reduced pressure

and purification by flash chromatography (hexanes/EtOAc 1:1) and size-

exclusion chromatography (Sephadex LH-20 MeOH/CH₂Cl₂1:1) yielded

15 (156 mg, 91%) and rearranged disaccharide.

1

(7 mg, 24%). [a]²_D⁴=+13.6 (c=0.8, CHCl₃); H NMR (500 MHz, CDCl₃):

d = 7.30–7.18 (m, 30H), 5.23 (d, J=4.7 Hz, 1H), 5.10 (brs, 1H), 5.05 (s,

2H), 4.97 (d, J=3.7 Hz, 1H), 4.95 (d, J=3.6 Hz, 1H), 4.93 (d, J=3.6 Hz,

1H), 4.90–4.84 (m, 3H), 4.79–4.76 (m, 3H), 4.73–4.65 (m, 5H), 4.63–4.57

(m, 2H), 4.52 (d, J=11.2 Hz, 1H), 4.37 (m, 1H), 4.22–4.15 (m, 2H), 4.10

(dd, J=3.7, 12.4 Hz, 1H), 4.06 (m, 1H), 3.98 (dd, J=4.9, 5.4 Hz, 1H),

3.90–3.85 (m, 3H), 3.83–3.80 (m, 2H), 3.75 (dd, J=8.9, 10.0 Hz, 1H),

3.69–3.66 (m, 5H), 3.53 (s, 3H), 3.51–3.41 (m, 2H), 3.32–3.31 (m, 2H),

3.23 (dd, J=3.6, 10.2 Hz, 1H), 3.19 (dd, J=3.4, 10.2 Hz, 1H), 2.58 (m,

2H), 2.41 (m, 2H), 2.05 (s, 3H), 2.00 (s, 3H), 1.91 (s, 3H), 1.53–1.50 (m,

4H), 1.37–1.36 (m, 2H), 1.15 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): d =

177.7, 170.8, 170.7, 170.0, 169.9, 169.6, 156.5, 137.9, 137.83, 137.80, 137.67,

137.61, 136.7, 129.2–127.7, 99.1, 98.4, 98.3, 98.1, 80.1, 78.3, 77.8, 75.9,

75.63, 75.58, 75.4, 75.1, 75.0, 74.4, 74.1, 73.3, 73.2, 70.4, 70.3, 70.1, 70.0,

69.8, 69.6, 69.0, 67.0, 63.45, 63.37, 62.5, 62.1, 60.6, 52.5, 52.2, 40.4, 39.0,

32.4, 31.9, 29.9, 29.6, 29.3, 27.5, 27.3, 25.5, 21.2, 21.02, 20.97, 20.96, 14.4,

Trisaccharide 15 (145 mg, 0.13 mmol) was dissolved in CH₂Cl₂. Pyridine

(320 mL) and acetic acid (210 mL) were added, followed by the addition

of hydrazine monohydrate (13 mL, 0.26 mmol). The reaction mixture was

stirred at room temperature for 90 min, diluted with CH₂Cl₂, quenched

with acetone and evaporated to dryness. Trisaccharide acceptor 16 was

isolated (114 mg, 86%) after purification by flash chromatography on

silica gel (hexanes/EtOAc 1:1). ¹H NMR (500 MHz, CDCl₃): d = 7.33–

7.20 (m, 15H), 5.77–5.69 (m, 1H), 5.01–4.88 (m, 6H), 4.79–4.57 (m, 8H),

4.4 (dd, J=2.2, 12.5 Hz, 1H), 4.19 (dd, J=3.6, 12.6 Hz, 1H), 4.07 (t, J=

4.5 Hz, 1H), 3.91–3.87 (m, 3H), 3.81 (t, J=9.4 Hz, 1H), 3.73–3.63 (m,

6H), 3.47–3.42 (m, 4H), 3.26 (dd, J=3.7, 10.2 Hz, 1H), 2.53 (d, J=

10.7 Hz, 1H), 2.07–2.02 (m, 2H), 2.02 (s, 3H), 2.0 (s, 3H), 1.66–1.60 (m,

2H), 1.15 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): d = 177.6, 170.7, 169.9,

169.7, 169.3, 138.2, 137.8, 137.7, 137.4, 128.8–127.6, 115.1, 99.2, 98.5, 98.4,

78.6, 75.6, 75.3, 75.1, 74.4, 73.2, 72.9, 70.1, 69.9, 69.5, 69.2, 68.5, 68.1, 67.9,

63.7, 62.2, 52.4, 52.3, 38.9, 33.9, 32.1, 30.3, 29.9, 29.6, 28.9, 27.3, 22.9, 21.1,

21.0, 14.3; IR (thin film on NaCl): n˜ =3025, 2944, 2861, 1741, 1497, 1439,

14.3; IR (thin film on NaCl): n˜ =3011, 2930, 2110, 1739, 1369, 1030 cm^ꢀ1

;

HR MALDI MS: m/z: calcd for C₈₇H₁₀₅N₇O₂₇SNa: 1734.6671; found:

1734.6640 [M+Na]⁺.

Tetrasaccharide 23: H₂O₂(30%, 0.29 mL) and a solution of LiOH (1m,

0.49 mL) were added at 08C to a solution of 18 (27 mg, 16 mmol) in THF

(0.9 mL). After stirring for 16 h at room temperature, the mixture was

cooled to 08C and MeOH (1.5 mL) and a solution of KOH (3m, 0.9 mL)

were added. After stirring for 16 h at room temperature, the reaction

mixture was neutralized with IR-120-H⁺Amberlite resin and then fil-

tered and concentrated. The residue was eluted from a Sephadex LH-20

chromatography column with MeOH/CH₂Cl₂1:1 to afford 19 (21 mg,

88%). ¹H NMR (300 MHz, CD₃OD): d = 7.45–7.18 (m, 30H), 5.29 (d,

J=3.0 Hz, 1H), 5.15–4.96 (m, 5H), 4.84–4.61 (m, 11H), 4.42 (d, J=

10.8 Hz, 1H), 4.21 (brs, 1H), 4.07 (brs, 1H), 3.98–3.50 (m, 20H), 3.24–

3.19 (m, 2H), 2.99–2.85 (m, 2H), 1.78–1.48 (m, 6H); ES-MS: m/z: calcd

for C₇₄H₉₀O₂₅N₈S: 1522.5; found: 1523.2 [M+NH₄]⁺.

1365, 1143, 1027, 908 cm^ꢀ1

; HR MALDI MS: m/z: calcd for

C₅₅H₆₉N₃O₂₀Na: 1114.4372; found: 1114.4376 [M+Na]⁺.

n-Pentenyl (6-O-acetyl-2-azido-3,4-O-dibenzyl-2-deoxy-a-d-glucopyrano-

syl)-(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-pivaloyl-a-l-idopyranosiduronate (17): Trisac-

charide 16 (138 mg, 0.12 mmol) and cap building block

3 (102 mg,

SO₃·NEt₃(55 mg, 0.31 mmol) was added to a solution of 19 (23 mg,

15 mmol) in anhydrous DMF (1.5 mL). After stirring for 24 h at 558C

under an argon atmosphere, the reaction mixture was quenched with trie-

thylamine (0.2 mL) and diluted with MeOH (1 mL) and CH₂Cl₂(1 mL).

The solution was layered on the top of a Sephadex LH-20 chromatogra-

phy column that was eluted with MeOH/CH₂Cl₂1:1. The fractions that

contained the sulfated tetrasaccharide were pooled and evaporated to

dryness to give 20 (24 mg, 80%). ¹H NMR (300 MHz, CD₃OD): d =

7.49–7.15 (m, 30H), 5.40 (brs, 1H), 5.20 (brs, 1H), 5.14 (d, J=3.9 Hz,

1H), 5.08 (s, 2H), 5.00 (d, J=3.3 Hz, 1H), 4.93–4.63 (m, 8H), 4.58 (brs,

1H), 4.51 (brs, 1H), 4.42–4.06 (m, 8H), 3.98–3.95 (m, 3H), 3.92–3.53 (m,

0.18 mmol) were combined in a flask, coevaporated with toluene (5)

and dried under vacuum. The starting materials were dissolved in CH₂Cl₂

(1.6 mL) and freshly activated 4 molecular sieves (300 mg) were

added. This mixture was stirred for 30 min at room temperature under an

argon atmosphere. After cooling the mixture to ꢀ208C, TMSOTf (4 mL,

0.02 mmol) was added and the reaction was allowed to warm to ꢀ108C

over 30 min. The reaction was quenched at ꢀ208C with triethylamine

and filtrated over Celite. Removing of the solvent under reduced pres-

sure and purification by flash chromatography (hexanes/EtOAc 1:1) and

size exclusion chromatography (Sephadex LH-20 MeOH/CH₂Cl₂1:1) af-

forded 17 (167 mg, 88%). [a]^R_D^T=+13.8 (c=1, CHCl₃); ¹H NMR

8676

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

9H), 3.42 (d, J=3.3, 10.2 Hz, 1H), 3.31–3.12 (m, 5H), 3.07–2.92 (m, 2H),

1.79–1.46 (m, 6H); ES-MS: m/z: calcd for C₇₄H₈₅O₃₇N₇S₅: 911.5; found:

11H), 4.41–4.34 (m, 2H), 4.18–4.10 (m, 2H), 4.06 (t, J=4.4 Hz, 1H), 3.95

(t, J=5.3 Hz, 1H), 3.89–3.66 (m, 13H), 3.58–3.50 (m, 1H), 3.46–3.42 (m,

7H), 3.25–3.22 (m, 2H), 2.71–2.56 (m, 2H), 2.49–2.37 (m, 2H), 2.11 (s,

3H), 2.06–2.02 (m, 2H), 2.4 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.97 (s,

3H), 1.65–1.59 (m, 2H), 1.16 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): d =

206.1, 177.7, 171.9, 170.8, 170.7, 170.1, 169.9, 169.6, 168.9, 138.2, 137.9,

137.8, 137.7, 137.6, 137.5, 128.8–127.8, 115.1, 99.2–97.8 (5C_anomeric), 78.3,

78.2, 75.9, 75.6, 75.1, 74.9, 74.8, 74.4, 74.1, 73.8, 73.5, 73.2, 73.1, 70.5, 70.3,

70.1, 70.0, 69.8, 69.6, 68.7, 68.6, 68.5, 67.7, 63.4, 63.3, 62.1, 61.9, 52.4, 52.3,

52.0, 39.0, 37.7, 30.3, 29.9, 28.9, 27.9, 27.5, 27.3, 21.0, 21.0, 20.9; IR (thin

film on NaCl): n˜ =3022, 2945, 2872, 1729, 1497, 1438, 1369, 1150, 1072,

1031 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₉₁H₁₁₀N₆O₃₄Na: 1853.6961;

found: 1853.6965 [M+Na]⁺.

911.6 [M+2H]^2ꢀ

.

Compound 20 (24 mg, 12 mmol) was dissolved in THF (3 mL) and treated

with a 0.1m aqueous solution of NaOH (0.7 mL). Then, a solution of

PMe₃in THF (98 mL of a 1m solution) was added and the reaction was

allowed to stir for 6 h. The reaction mixture was neutralized with a 0.1m

solution of HCl, concentrated and the residue was eluted from a Sepha-

dex LH-20 chromatography column with MeOH/CH₂Cl₂1:1 to afford 21

(23 mg, quantitative). ¹H NMR (300 MHz, CD₃OD): d = 7.41–7.10 (m,

30H), 5.47 (d, J=2.1 Hz, 1H), 5.33 (d, J=3.9 Hz, 1H), 5.27 (d, J=

3.9 Hz, 1H), 5.19 (brs, 1H), 5.08 (s, 2H), 5.01–4.12 (m, 22H), 3.98–3.32

(m, 12H), 3.24 (m, 2H), 2.97 (m, 2H), 1.78–1.48 (m, 6H); ES-MS: m/z:

calcd for C₇₄H₈₇O₃₇N₃S₅: 442.2; found: 442.4 [M]^4ꢀ

C₇₄H₈₈O₃₇N₃S₅: 590.0; found: 590.2 [M+H]^3ꢀ; calcd for C₇₄H₈₉O₃₇N₃S₅:

885.5; found: 885.8 [M+2H]^2ꢀ

;

calcd for

n-Pentenyl (6-O-acetyl-2-azido-3,4-O-dibenzyl-2-deoxy-a-d-glucopyrano-

syl)-(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

2-O-acetyl-3-O-benzyl-a-l-idopyranosyluronate)-(1!4)-(6-O-

Triethylamine (0.2 mL) and then SO₃·Py (18 mg, 0.11 mmol) were added

to a solution of 21 (21 mg, 11 mmol) in anhydrous pyridine (1 mL). After

stirring for 2 h at room temperature under an argon atmosphere, the re-

action mixture was diluted with MeOH (1 mL) and CH₂Cl₂(1 mL). The

solution was layered on the top of a Sephadex LH-20 chromatography

column that was eluted with MeOH/CH₂Cl₂1:1. The fractions that con-

tained the sulfated tetrasaccharide were pooled and evaporated to dry-

ness. The residue was converted into the sodium salt by elution from a

column of Dowex 50WX4-Na⁺with MeOH/H₂O 9:1 to give 22 (18 mg,

acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1!4)-methyl 3-

O-benzyl-2-O-pivaloyl-a-l-idopyranosiduronate (26): Fully protected

pentasaccharide 24 (258 mg, 0.14 mmol) was dissolved in CH₂Cl₂. Pyri-

dine (0.33 mL) and acetic acid (0.22 mL) were added, followed by the ad-

dition of hydrazine monohydrate (14 mL, 0.28 mmol). The reaction mix-

ture was stirred at room temperature for 90 min, diluted with CH₂Cl₂,

quenched with acetone and evaporated to dryness. Pentasaccharide ac-

ceptor 25 was isolated after purification (234 mg, 96%) by column chro-

matography on silica gel (hexanes/EtOAc 7:3). Acceptor 25 (110 mg,

64.4 mmol) and cap building block 3 (76 mg, 0.13 mmol) were combined

in a flask, coevaporated with toluene (5) and dried under vacuum. The

starting materials were dissolved in CH₂Cl₂(1.6 mL) and freshly activat-

ed 4 molecular sieves (220 mg) were added. This mixture was stirred

for 30 min at room temperature under an argon atmosphere. After cool-

ing the mixture to ꢀ208C, TMSOTf (1.55 mL, 7.6 mmol) was added and

the reaction was allowed to warm to ꢀ108C over 30 min. The reaction

was quenched at ꢀ208C with triethylamine and filtered through Celite.

Removing of the solvent under reduced pressure and purification by

flash chromatography (hexanes/EtOAc 3:2) and size exclusion chroma-

tography (Sephadex LH-20 MeOH/CH₂Cl₂1:1) afforded hexasaccharide

26 (116 mg, 85%). [a]^R_D^T=+14.3 (c=1, CHCl₃); ¹H NMR (500 MHz,

CDCl₃): d = 7.40–7.19 (m, 35H), 5.78–5.70 (m, 1H), 5.25 (d, J=4.9 Hz,

1H), 5.23 (d, J=3.3 Hz, 1H), 5.05–4.51 (m, 26H), 4.39 (dd, J=1.55,

10.8 Hz, 1H), 4.32 (dd, J=1.6, 12.5 Hz, 1H), 4.21–4.04 (m, 5H), 3.99 (t,

J=5.9 Hz, 1H), 3.95 (t, J=5.9 Hz, 1H), 3.91–3.65 (m, 10H), 3.59–3.42

(m, 10H), 3.26–3.19 (m, 3H), 2.06–2.02 (m, 8H), 1.99–1.98 (3s, 9H), 1.90

(s, 3H), 1.64–1.61 (m, 2H), 1.16 (s, 9H); ¹³C NMR (125 MHz, CDCl₃):

d = 177.7, 170.8, 170.8, 170.7, 170.0, 169.9, 169.8, 169.7, 169.6, 138.2,

138.0, 137.9, 137.8, 137.8, 137.7, 137.6, 137.6, 128.8–127.8, 115.1, 99.2–97.9

(6C_anomeric), 80.0, 78.3, 78.0, 77.8, 75.9, 75.8, 75.6, 75.5, 75.3, 75.1, 75.0,

74.4, 74.3, 74.2, 74.0, 73.3, 73.2, 70.6, 70.5, 70.41, 70.39, 70.1, 70.0, 69.9,

69.8, 69.6, 68.6, 63.5, 63.4, 63.1, 62.6, 62.1, 61.9, 60.6, 53.6, 52.5, 52.3, 52.2,

52.1, 51.1, 39.0, 30.3, 30.2, 29.9, 28.9, 28.8, 27.5, 27.4, 27.3, 21.2, 21.0, 21.0,

20.9, 20.9, 20.9, 14.4, 14.3; IR (thin film on NaCl): n˜ =3025, 2943, 2871,

1738, 1497, 1439, 1154, 1072, 1031 cm^ꢀ1; HR MALDI MS: m/z: calcd for

C₁₀₈H₁₂₇N₉O₃₇Na: 2164.823; found: 2164.829 [M+Na]⁺.

1

78%). H NMR (300 MHz, CD₃OD): d = 7.52–7.06 (m, 30H), 5.54 (brs,

1H), 5.38–5.33 (m, 3H), 5.17 (d, J=11.1 Hz, 1H), 5.08 (s, 2H), 5.03 (d,

J=1.2 Hz, 1H), 4.94–4.64 (m, 9H), 4.56 (brs, 1H), 4.47–4.44 (m, 2H),

4.40–4.11 (m, 6H), 4.01–3.94 (m, 3H), 3.79–3.47 (m, 11H), 3.24–3.19 (m,

2H), 2.94–2.89 (m, 2H), 1.73–1.44 (m, 6H); ES-MS: m/z: calcd for

C₇₄H₈₈O₄₃N₃S₇: 643.3; found: 643.3 [M+3H]^3ꢀ; cald for C₇₄H₈₇O₄₃N₃S₇Na:

650.7; found: 650.8 [M+Na+2H]^3ꢀ; calcd for C₇₄H₈₈O₄₃N₃S₇Na: 976.5;

found: [M+Na+3H]^2ꢀ

.

Asolution of 22 (15 mg, 7 mmol) in MeOH/H₂O (2 mL/1 mL) was hydro-

genated in the presence of 10% Pd/C. After 24 h, the suspension was fil-

tered, concentrated and eluted from a column of Dowex 50WX4-Na⁺

with MeOH/H₂O 1:9 to give 23 (11 mg, quantitative). [a]²_D⁴=+16.4 (c=

1

0.25, H₂O); H NMR (500 MHz, D₂O): d = 5.28 (d, J=3.2 Hz, 1H), 5.25

(d, J=2.7 Hz, 1H), 5.10 (brs, 1H), 4.99 (brs, 1H), 4.70 (brs, 1H), 4.38

(brs, 1H), 4.29–3.85 (m, 11H), 3.60–3.10 (m, 15H), 1.74–1.40 (m, 6H);

ES-MS: m/z: calcd for C₃₁H₅₂O₄₁N₃S₇Na: 684.5; found: 684.5

[M+Na+3H]^2ꢀ

; calcd for C₃₁H₅₁O₄₁N₃S₇Na₂: 695.5; found: 695.5

[M+2Na+2H]^2ꢀ. NMR data for calcium salt (after addition of CaCl₂;

20 mL of a 0.5m solution in D₂O): ¹H NMR (500 MHz, D₂O): d = 5.27

(brs, 1H), 5.24 (d, 1H), 5.19 (d, 1H), 5.08 (brs, 1H), 4.80 (brs, 1H), 4.42

(brs, 1H), 4.26–4.21 (m, 3H), 4.14–4.08 (m, 6H), 3.98–3.85 (m, 3H),

3.73–3.62 (m, 3H), 3.54–3.44 (m, 5H), 3.35 (m, 2H), 3.20–3.12 (m, 4H),

1.74–1.40 (m, 6H); HSQC anomeric cross peaks (500 MHz, D₂O): d=

(5.2799.0), (5.2497.4), (5.1997.0), (5.0898.6).

n-Pentenyl (methyl 2-O-acetyl-3-O-benzyl-4-O-levulinoyl-a-l-idopyrano-

syluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-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-pivaloyl-a-l-idopyranosiduronate (24): Trisac-

charide 16 (224 mg, 0.21 mmol) and disaccharide 2 (240 mg, 0.27 mmol)

were combined in a flask, coevaporated with toluene (5) and dried

under vacuum. The starting materials were dissolved in CH₂Cl₂(4.5 mL)

and freshly activated 4 molecular sieves (730 mg) were added. This

mixture was stirred for 30 min at room temperature under an argon at-

mosphere. After cooling the mixture to ꢀ208C, TMSOTf (5 mL;

25.6 mmol) was added and the reaction was allowed to warm to ꢀ108C

over 30 min. The reaction was quenched at ꢀ208C with triethylamine

and filtrated over Celite. Removing of the solvent under reduced pres-

sure and purification by flash chromatography (hexanes/EtOAc 1:1) and

size exclusion chromatography (Sephadex LH-20 MeOH/CH₂Cl₂1:1) af-

forded pentasaccharide 24 (278 mg, 74%). ¹H NMR (500 MHz, CDCl₃):

d = 7.40–7.27 (m, 25H), 5.77–5.68 (m, 1H), 5.77–5.68 (m, 1H), 5.24 (d,

J=4.7 Hz, 1H), 5.10 (d, J=3.0 Hz, 1H), 4.98–4.79 (m, 9H), 4.73–4.55 (m,

5-[2-(Benzyloxycarbonylamino)-1-thioethyl] pentyl (6-O-acetyl-2-azido-

3,4-di-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)-(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-pivaloyl-a-l-

idopyranosiduronate (27): Asolution of 26 (113 mg, 53 mmol), AIBN

(3.5 mg,

21 mmol)

and

2-(benzyloxycarbonylamino)-1-ethanethiol

(167 mg, 0.79 mmol) in degassed THF (4 mL) was stirred at 758C under

an argon atmosphere. After 9 h, the reaction was quenched with cyclo-

hexene (200 mL) and the mixture was concentrated and purified by flash

chromatography (hexanes/EtOAc 4:1!2:1!3:2) to afford 27 (73 mg,

59%) and unreacted starting material (22 mg, 19%). [a]²_D⁴=+11.2 (c=1,

CHCl₃); ¹H NMR (500 MHz, CDCl₃): d = 7.31–7.19 (m, 40H), 5.24 (d,

J=4.5 Hz, 1H), 5.23 (d, J=5.0 Hz, 1H), 5.09 (brs, 1H), 5.05 (s, 2H), 4.97

(m, 2H), 4.94 (d, J=4.0 Hz, 1H), 4.91–4.77 (m, 9H), 4.73–4.51 (m, 12H),

Chem. Eur. J. 2006, 12, 8664 – 8686

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

8677

P. H. Seeberger et al.

4.37 (m, 1H), 4.30 (m, 1H), 4.20 (dd, J=2.1, 12.0 Hz, 1H), 4.17–4.04 (m,

4H), 3.98 (m, 1H), 3.94 (m, 1H), 3.91–3.66 (m, 11H), 3.69 (s, 3H), 3.58

(m, 1H), 3.53 (s, 3H), 3.48 (s, 3H), 3.45–3.41 (m, 2H), 3.32–3.30 (m, 2H),

3.25–3.22 (m, 3H), 2.58 (m, 2H), 2.41 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H),

1.98 (s, 3H), 1.97 (s, 3H), 1.91 (s, 3H), 1.54–1.51 (m, 4H), 1.37–1.36 (m,

3.36 (m, 38H), 1.98 (m, 2H), 1.81 (m, 2H), 1.70 (m, 2H); ES-MS: m/z:

calcd for C₄₃H₇₀O₆₀N₄S₁₀Na₂: 984.0; found: 984.0 [M+2Na+5H]^2ꢀ; calcd

for C₄₃H₆₈O₆₀N₄S₁₀Na₄1006.0; found: 1006.0 [M+4Na+3H]^2ꢀ; calcd for

C₄₃H₇₀O₆₀N₄S₁₀Na: 648.3; found: 648.4 [M+Na+5H]^3ꢀ

.

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-pivaloyl-3-O-benzyl-4-

O-levulinoyl-a-l-idopyranosyluronate (33): Trichloroacetimidate

2H), 1.15 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): d

= 177.7, 170.82,

1

170.80, 170.7, 170.0, 169.90, 169.88, 169.7, 169.6, 137.93, 137.86, 137.81,

137.66, 137.64, 137.60, 136.7, 129.9- 127.7, 99.1, 98.4, 98.24, 98.19, 97.9,

80.1, 78.3, 78.1, 77.8, 75.9, 75.8, 75.63, 75.59, 75.55, 75.3, 75.12, 75.09, 75.0,

74.4, 74.1, 73.3, 73.2, 70.6, 70.5, 70.45, 70.40, 70.1, 70.0, 69.9, 69.8, 69.6,

69.0, 66.9, 63.5, 63.3, 63.1, 62.6, 62.1, 61.9, 60.6, 52.5, 52.19, 52.15, 40.4,

39.0, 32.4, 31.9, 29.9, 29.6, 29.3, 27.3, 25.5, 22.9, 21.2, 21.00, 20.98, 20.94,

20.93, 14.4, 14.3; IR (thin film on NaCl): n˜ =2982, 2103, 1739, 1600, 1369,

(420 mg, 0.67 mmol) and N-benzyloxycarbonyl-5-aminopentan-1-ol

(480 mg, 2.0 mmol) were combined in a flask, coevaporated with toluene

(5) and dried under vacuum overnight. Afterwards, the starting materi-

als were dissolved in CH₂Cl₂under an argon atmosphere. The reaction

was cooled to ꢀ208C, TMSOTf (37 mL, 0.2 mmol) was added and the re-

action was allowed to warm to ꢀ108C over 30 min. The reaction was

quenched with triethylamine at ꢀ258C and allowed to warm to room

temperature. Flash chromatography (hexanes/EtOAc 9:1) afforded the

desired compound 33 (212 mg, 45%). [a]^R_D^T=ꢀ35.1 (c=1, CHCl₃);

¹H NMR (300 MHz, CDCl₃): d = 7.35–7.25 (m, 10H), 5.23 (t, J=2.5 Hz,

1H), 5.07 (s, 2H), 4.93 (s, 1H), 4.89–4.87 (m, 1H), 4.85 (d, J=2.2 Hz,

1H), 4.78 (dd, J=11.5, 14.6 Hz, 1H), 4.69 (dd, J=11.5, 14.9 Hz, 1H),

3.78–3.69 (m, 5H), 3.49–3.46 (m, 1H), 3.14–3.07 (m, 2H), 2.76–2.68 (m,

2H), 2.55–2.50 (m, 2H), 2.17 (s, 3H), 1.59–1.33 (m, 6H), 1.19 (s, 9H);

¹³C NMR (75 MHz, CDCl₃): d = 205.9, 177.4, 171.7, 169.1, 156.4, 137.6,

136.7, 128.5–127.5, 98.5, 73.2, 72.2, 68.5, 68.1, 66.8, 66.6, 66.1, 52.5, 40.9,

38.9, 38.7, 29.7, 29.7, 29.6, 28.9, 27.9, 27.1, 27.1, 23.4; IR (thin film on

NaCl): n˜ =3450, 3015, 2933, 2872, 1721, 1513, 1436, 1364, 1149, 1097,

1056 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₃₇H₄₉NO₁₂Na: 722.3152;

found: 722.3134 [M+Na]⁺.

1026 cm^ꢀ1

; HR MALDI MS: m/z: calcd for C₁₁₈H₁₄₀N₁₀O₃₉SNa:

2375.8970; found: 2375.8892 [M+Na]⁺.

Hexasaccharide 32: H₂O₂(30%, 0.47 mL) and a solution of LiOH (1m,

0.8 mL) were added at 08C to a solution of 27 (44 mg, 19 mmol) in THF

(1.5 mL). After stirring for 16 h at room temperature, the mixture was

cooled to 08C and MeOH (2.5 mL) and a solution of KOH (3m, 1.5 mL)

were added. After stirring for 16 h at room temperature, the reaction

mixture was neutralized with IR-120-H⁺Amberlite resin, then filtered

and concentrated. The residue was eluted from a Sephadex LH-20 chro-

matography column with MeOH/CH₂Cl₂1:1 to afford 28 (35 mg, 92%).

¹H NMR (300 MHz, CD₃OD): d = 7.40–7.20 (m, 40H), 5.27 (m, 2H),

5.12–5.08 (m, 4H), 4.83–4.45 (m, 20H), 4.28 (brs, 1H), 4.15 (m, 1H),

3.96–3.47 (m, 28H), 3.34 (m, 2H), 2.93–2.92 (m, 2H), 1.75–1.44 (m, 6H);

ES-MS: m/z: calcd for C₁₀₀H₁₁₆O₃₅N₁₀SNa: 2071.7; found: 2072.2

[M+Na]⁺.

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-pivaloyl-3-O-benzyl-a-

l-idopyranosyluronate (34): Compound 33 (75 mg, 0.11 mmol) was dis-

solved in CH₂Cl₂. Pyridine (0.25 mL) and acetic acid (0.17 mL) were

added, followed by the addition of hydrazine monohydrate (11 mL,

0.21 mmol). The reaction mixture was stirred at room temperature for

90 min, diluted with CH₂Cl₂, quenched with acetone and evaporated to

dryness. Product 34 was isolated after purification (61 mg, 95%) by

column chromatography on silica gel (hexanes/EtOAc 4:1). [a]^R_D^T=ꢀ28.5

(c=1, CHCl₃); ¹H NMR (300 MHz, CDCl₃): d = 7.34–7.25 (m, 10H),

5.04 (s, 2H), 4.97 (t, J=1.1 Hz, 1H), 4.91 (s, 1H), 4.83 (d, J=11.5 Hz,

1H), 4.79–4.69 (m, 2H), 4.60 (d, J=11.5 Hz, 1H), 4.07 (d, J=10.9 Hz,

1H), 3.79–3.67 (m, 5H), 3.50–3.44 (m, 1H), 3.15–3.09 (m, 2H), 2.69 (d,

J=12.1 Hz, 1H), 1.63–1.33 (m, 6H), 1.21 (s, 9H); ¹³C NMR (75 MHz,

CDCl₃): d = 176.6, 169.9, 169.4, 156.4, 137.6, 136.7, 128.6–127.6, 98.8,

74.3, 71.8, 68.6, 68.2, 67.6, 66.6, 52.4, 40.9, 38.9, 29.7, 29.1, 23.4; IR (thin

film on NaCl): n˜ =3574, 3453, 3008, 2939, 2872, 1725, 1516, 1497, 1455,

1302, 1141, 1050 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₃₂H₄₃NO₁₀Na:

624.2785; found: 624.2788 [M+Na]⁺.

SO₃·NEt₃(58 mg, 0.32 mmol) was added to a solution of 28 (22 mg,

11 mmol) in anhydrous DMF (1 mL). After stirring for 16 h at 558C

under an argon atmosphere, the reaction mixture was quenched with trie-

thylamine (0.2 mL) and diluted with MeOH (1 mL) and CH₂Cl₂(1 mL).

The solution was layered on the top of a Sephadex LH-20 chromatogra-

phy column that was eluted with MeOH/CH₂Cl₂1:1. The fractions that

contained the sulfated hexasaccharide were pooled and evaporated to

dryness to give 29 (28 mg, 97%). ¹H NMR (300 MHz, CD₃OD): d =

7.42–7.18 (m, 40H), 5.21–5.16 (m, 2H), 5.09–5.08 (m, 6H), 4.96–2.89 (m,

52H), 1.75–1.44 (m, 6H); ES-MS: m/z: calcd for C₁₀₀H₁₁₃O₅₃N₁₀S₇: 841.7;

found: 842.4 [M+3H]^3ꢀ

.

Compound 29 (28 mg, 10 mmol) was dissolved in THF (3.5 mL) and treat-

ed with a 0.1m aqueous solution of NaOH (0.8 mL). Then, a solution of

PMe₃in THF (124 mL of a 1m solution) was added and the reaction was

allowed to stir for 8 h. The reaction mixture was neutralized with a 0.1m

solution of HCl, concentrated and the residue was eluted from a Sepha-

dex LH-20 chromatography column with MeOH/CH₂Cl₂1:1 to afford 30

(23 mg, 85%). ES-MS: m/z: calcd for C₁₀₀H₁₁₉O₅₃N₄S₇: 815.7; found:

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3-O-benzyl-4-O-

levulinoyl-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-piva-

loyl-a-l-idopyranosiduronate (35): Reducing end building block 34

(200 mg, 0.33 mmol) and disaccharide 2 (360 mg, 0.40 mmol) were com-

815.6 [M+3H]^3ꢀ

.

Triethylamine (0.2 mL) and then SO₃·Py (21 mg, 0.13 mmol) were added

to a solution of 30 (23 mg, 9 mmol) in anhydrous pyridine (1 mL). After

stirring for 2 h at room temperature under an argon atmosphere, the re-

action mixture was diluted with MeOH (1 mL) and CH₂Cl₂(1 mL). The

solution was layered on the top of a Sephadex LH-20 chromatography

column that was eluted with MeOH/CH₂Cl₂1:1. The fractions that con-

tained the sulfated hexasaccharide were pooled and evaporated to dry-

ness. The residue was converted into the sodium salt by elution from a

column of Dowex 50WX4-Na⁺with MeOH/H₂O 9:1 to give 31 (20 mg,

bined in

a flask, coevaporated with toluene (5) and dried under

vacuum. The starting materials were dissolved in CH₂Cl₂(5 mL) and

freshly activated 4 molecular sieves (550 mg) were added. This mixture

was stirred for 30 min at room temperature under an argon atmosphere.

After cooling the mixture to ꢀ208C, TMSOTf (7 mL, 0.04 mmol) was

added and the reaction was allowed to warm to ꢀ108C over 30 min. The

reaction was quenched at ꢀ208C with triethylamine and filtrated over

Celite. Removing of the solvent under reduced pressure and purification

by flash chromatography (hexanes/EtOAc 1:1) afforded 35 (183 mg,

41%). [a]^R_D^T=ꢀ16.7 (c=1, CHCl₃); ¹H NMR (300 MHz, CDCl₃): d =

7.38–7.25 (m, 20H), 5.15 (d, J=3.0 Hz, 1H), 5.11–5.08 (m, 3H), 5.03 (d,

J=3.3 Hz, 2H), 4.97 (t, J=4.3 Hz, 1H), 4.88–4.86 (m, 2H), 4.82 (d, J=

10.7 Hz, 2H), 4.75–4.71 (m, 4H), 4.67 (d, J=10.8 Hz, 2H), 4.49 (d, J=

11.6, 1H), 4.24 (d, J=11.6, 1H), 4.13–4.09 (m, 1H), 3.95–3.91 (m, 3H),

3.81–3.72 (m, 6H), 3.50–3.45 (m, 4H), 3.34 (dd, J=3.6, 10.2 Hz, 1H),

3.19–3.12 (m, 2H), 2.74–2.66 (m, 2H), 2.52–2.46 (m, 2H), 2.16 (s, 3H),

2.09 (s, 3H), 2.06 (s, 3H), 1.62–1.33 (m, 6H), 1.19 (s, 9H); ¹³C NMR

(75 MHz, CDCl₃): d = 205.9, 177.5, 171.7, 170.6, 169.9, 169.7, 168.71,

156.1, 137.6, 137.4, 136.7, 128.5–127.6, 98.9, 98.1, 97.8, 78.2, 77.2, 75.4,

1

77%). H NMR (500 MHz, CD₃OD): d = 7.38–7.01 (m, 40H), 5.56 (brs,

1H), 5.47 (brs, 1H), 5.33 (d, J=3.0 Hz, 1H), 5.31 (brs, 1H), 5.25 (m,

2H), 5.11 (d, J=11.0 Hz, 1H), 5.02 (s, 2H), 5.00 (brs, 1H), 4.97 (brs,

1H), 4.74–3.44 (m, 45H), 3.17–3.14 (m, 2H), 2.88–2.85 (m, 2H), 1.69–

1.42 (m, 6H); ES-MS: m/z: calcd for C₁₀₀H₁₁₇O₆₂N₄S₁₀: 671.5; found:

671.6 [M+4H]^4ꢀ

.

Asolution of 31 (8 mg, 2.7 mmol) in MeOH/H₂O (2 mL/1 mL) was hydro-

genated in the presence of 10% Pd/C. After 24 h, the suspension was fil-

tered, concentrated and eluted from a column of Dowex 50WX4-Na⁺

with MeOH/H₂O 1:9 to give 32 (6 mg, quantitative). [a]²_D⁴=+30.4 (c=

0.25, H₂O); ¹H NMR (500 MHz, D₂O): d = 5.52 (brs, 1H), 5.46 (brs,

1H), 5.34 (brs, 1H), 5.29 (brs, 1H), 5.05 (brs, 1H), 4.98 (brs, 1H), 4.79–

8678

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

74.9, 74.7, 74.0, 73.7, 73.2, 73.0, 70.0, 70.0, 69.6, 68.9, 68.6, 68.3, 67.6, 66.6,

63.3, 61.9, 52.2, 52.1, 40.9, 38.8, 37.6, 29.8, 29.7, 29.1, 27.9, 27.1, 23.3, 20.9,

20.9; IR (thin film on NaCl): n˜ =3446, 3015, 2944, 2872, 2103, 1739, 1605,

1513, 1492, 1451, 1308, 1149, 1103, 1031 cm^ꢀ1; HR MALDI MS: m/z:

calcd for C₆₈H₈₄N₄O₂₄Na: 1363.537; found: 1363.534 [M+Na]⁺.

asaccharide 39 (51 mg, 62%). [a]^R_D^T=+16.1 (c=1, CHCl₃); ¹H NMR

(500 MHz, CDCl₃): d = 7.34–7.23 (m, 40H), 5.29 (d, J=4.7 Hz, 1H),

5.27 (d, J=4.6 Hz, 1H), 5.06 (brs, 2H), 5.01 (d, J=3.8 Hz, 2H), 4.98 (d,

J=3.6 Hz, 1H), 4.95 (d, J=3.7 Hz, 1H), 4.92–4.81 (m, 9H), 4.76–4.55 (m,

12H), 4.38–4.36 (m, 1H), 4.31–4.29 (m, 1H), 4.22–4.03 (m, 5H), 3.99–

3.96 (m, 1H), 3.95–3.92 (m, 1H), 3.91–3.57 (m, 15H), 3.54–3.40 (m, 8H),

3.25–3.19 (m, 3H), 3.11 (q, J=6.7 Hz, 2H), 2.10 (s, 3H), 2.09 (s, 3H),

2.04–2.03 (2s,s 6H), 1.96 (s, 3H), 1.51–1.47 (m, 4H), 1.37–1.34 (m, 2H),

1.20 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): d = 177.5, 170.6, 170.5, 169.8,

169.7, 169.5, 169.4, 156.4, 137.7, 137.66, 137.62, 137.59, 137.46, 137.42,

136.7, 128.6–127.6, 98.9, 98.2, 98.0, 97.7, 79.9, 78.1, 77.9, 77.3, 75.7, 75.6,

75.40, 75.39, 75.1, 74.9, 74.8, 74.2, 74.0, 73.1, 73.0, 70.4, 70.3, 70.25, 70.18,

69.9, 69.8, 69.7, 69.5, 68.9, 66.6, 63.3, 63.2, 62.9, 62.4, 61.8, 61.7, 52.3,

52.01, 51.96, 41.0, 38.8, 29.70, 29.66, 29.1, 27.1, 23.3, 20.82, 20.76, 20.7; IR

(thin film on NaCl): n˜ =3682, 3446, 3025, 2944, 2872, 2113, 1739, 1600,

1513, 1436, 1148, 1108, 1031 cm^ꢀ1; HR MALDI MS: m/z: calcd for

C₁₁₆H₁₃₆N₁₀O₃₉Na: 2315.886; found: 2315.891 [M+Na]⁺.

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3-O-benzyl-4-O-

levulinoyl-a-l-idopyranosyluronate)-(1!4)-(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)-methyl 3-O-benzyl-2-O-pivaloyl-a-l-

idopyranosiduronate (37): Pyridine (320 mL) and AcOH (210 mL) were

added to a solution of 35 (176 mg, 0.13 mmol) in CH₂Cl₂(1.4 mL) fol-

lowed by the addition of hydrazine monohydrate (13 mL, 0.26 mmol).

After stirring the reaction mixture for 1 h under an argon atmosphere,

acetone (1 mL) was added and the solvents were removed under reduced

pressure. The crude product was purified by flash chromatography on

silica gel (hexanes/EtOAc 1:1). This deprotected trisaccharide 36

(135 mg, 0.11 mmol) and disaccharide 2 (127 mg, 0.14 mmol) were com-

bined in a flask, coevaporated with toluene(5) and dried under vacuum.

The mixture was dissolved in CH₂Cl₂(2.4 mL), freshly activated 4 mo-

lecular sieves (390 mg) were added and the mixture was stirred for

30 min at room temperature under argon. After cooling to ꢀ258C,

TMSOTf (3 mL, 14.3 mmol) was added and the reaction was allowed to

warm to ꢀ108C over 30 min. The reaction mixture was cooled back to

ꢀ258C, quenched with triethylamine and filtrated over Celite. After re-

moval of the solvent under reduced pressure, purification by flash chro-

matography (hexanes/EtOAc 7:3) and size-exclusion chromatography

(Sephadex LH-20 MeOH/CH₂Cl₂1:1) afforded pentasaccharide 37

(68 mg, 34%). [a]^R_D^T=ꢀ6.2 (c=1, CHCl₃); ¹H NMR (300 MHz, CDCl₃):

d = 7.37–7.23 (m, 30H), 5.30 (d, J=4.7 Hz, 1H), 5.15 (d, J=2.8 Hz,

1H), 5.08 (brs, 3H), 5.03 (d, J=3.7 Hz, 1H), 4.99–4.85 (m, 7H), 4.79–

4.60 (m, 12H), 4.45–4.39 (m, 2H), 4.24–4.17 (m, 2H), 4.12 (t, J=4.6 Hz,

1H), 4.02–3.98 (m, 1H), 3.94–3.70 (m, 12H), 3.66–3.51 (m, 2H), 3.49–

3.48 (2s, 6H), 3.32–3.27 (m, 2H), 3.18–3.12 (m, 2H), 2.76–2.44 (m, 4H),

2.17 (s, 3H), 2.10–2.04 (4s, 12H), 1.64–1.36 (m, 6H), 1.21 (s, 9H);

¹³C NMR (75 MHz, CDCl₃): d = 205.6, 177.5, 171.7, 170.6, 170.5, 169.8,

169.7, 169.4, 168.7, 156.4, 137.7, 137.6, 137.5, 137.4, 137.3, 136.7, 128.6–

127.6, 98.9, 98.2, 98.1, 97.8, 78.1, 78.0, 77.3, 75.7, 75.4, 74.9, 74.7, 74.6,

74.2, 73.9, 73.6, 73.3, 73.0, 70.2, 70.0, 69.9, 69.9, 69.8, 69.5, 68.9, 68.5, 68.3,

67.5, 66.6, 63.1, 61.9, 61.7, 52.3, 52.2, 51.9, 40.9, 38.8, 37.6, 29.8, 29.7, 29.0,

27.8, 27.3, 27.1, 20.8, 20.7; IR (thin film on NaCl): n˜ =3446, 3026, 2954,

2879, 2103, 1739, 1605, 1513, 1492, 1369, 1308, 1103, 1072, 1031 cm^ꢀ1; HR

MALDI MS: m/z: calcd for C₉₉H₁₁₉N₇O₃₆Na: 2004.759; found: 2004.765

[M+Na]⁺.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentan-1-ol: N-Benzyloxycarbon-

yl-5-aminopentan-1-ol (3.1 g, 12.7 mmol) was dissolved in pyridine

(12 mL) and cooled to 08C. Tritylchloride (3.9 g, 13.9 mmol) was added

and the reaction mixture was stirred overnight at room temperature. Ex-

traction with water, saturated aqueous NaHCO₃and brine, drying over

MgSO₄and removing of the solvent afforded after purification by flash

column chromatography (hexanes/EtOAc 9:1) the tritylated alcohol

(5.6 g, 92%).

The obtained product (4.0 g, 8.34 mmol) was dissolved in DMF and

cooled to 08C. NaH (0.4 g, 10.0 mmol) was added and the reaction mix-

ture was stirred for 10 min at 08C before BnBr (1 mL, 9.17 mmol) and

catalytic amount of TBAI were added. The solution was stirred for

90 min at room temperature and afterwards extracted wit EtOAc. Drying

over MgSO₄and removing of the solvent yielded after purification by

flash column chromatography (hexanes/EtOAc 40:1) in the fully protect-

ed linker (4.45 g, 94%).

This linker (3.1 g, 5.4 mmol) was dissolved in CH₂Cl₂(35 mL) and treated

with a TFA/H₂O solution (1 mL, 3%). Stirring for 3 h at room tempera-

ture, neutralization with NaHCO₃and washing with brine yielded after

purification by flash column chromatography (hexanes/EtOAc 2:1!1:3)

in N-(benzyl)-benzyloxycarbonyl-5-aminopentan-1-ol (1.63 g, 92%).

¹H NMR (300 MHz, CDCl₃): d = 7.37–7.19 (m, 10H), 5.19 (d, J=5.4 Hz,

2H), 4.51 (s, 2H), 3.59–3.53 (m, 2H), 3.29–3.22 (m, 2H), 2.13 (s, 1H),

1.55–1.29 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): mixture of rotamers: d

= 156.8/156.4, 137.9, 136.8/136.7, 128.6–127.2, 67.3, 62.5, 50.6/50.3, 47.1/

46.3, 32.3, 27.9/27.5, 22.9; IR (thin film on NaCl): n˜ =3620, 3466, 2995,

2933, 2861, 1687, 1605, 1584, 1492, 1471, 1421, 1364, 1123, 1046,

1005 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₂₀H₂₅NO₃Na: 350.1732;

found: 350.1722 [M+Na]⁺.

N-Benzyloxycarbonyl-5-aminopentyl (6-O-acetyl-2-azido-3,4-O-dibenzyl-

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 2-O-acetyl-3-O-benzyl-a-l-idopyrano-

syluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyr-

anosyl)-(1!4)-methyl 3-O-benzyl-2-O-pivaloyl-a-l-idopyranosiduronate

(39): Pentasaccharide 37 (72 mg, 36.2 mmol) was dissolved in CH₂Cl₂. Pyr-

idine (90 mL) and acetic acid (60 mL) were added, followed by the addi-

tion of hydrazine monohydrate (4 mL, 72.6 mmol). The reaction mixture

was stirred at room temperature for 90 min, diluted with CH₂Cl₂,

quenched with acetone and evaporated to dryness. Product 38 was isolat-

ed after purification (68 mg, 98%) by column chromatography on silica

gel (hexanes/EtOAc 7:3).

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl (methyl 2-O-pivaloyl-3-O-

benzyl-a-l-idopyranosyluronate (40): Trichloroacetimidate

1 (440 mg,

0.70 mmol) and N-(benzyl)-benzyloxycarbonyl-5-aminopentan-1-ol

AHCTREUNG

(690 mg, 2.1 mmol) were combined in a flask, coevaporated with toluene

(5) and dried under vacuum. The starting materials were dissolved in

CH₂Cl₂and cooled to ꢀ208C, then TMSOTf (25 mL, 0.14 mmol) was

added and the reaction was allowed to warm to ꢀ108C over 30 min.

Quenching with triethylamine at ꢀ258C and removing of the solvent

yielded after purification by flash chromatography (hexanes/EtOAc 9:1)

in the desired product (340 mg, 61%).

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-pivaloyl-3-O-benzyl-4-

O-levulinoyl-a-l-idopyranosyluronate (260 mg, 0.33 mmol) was dissolved

in CH₂Cl₂. Addition of pyridine (0.78 mL) and acetic acid (0.52 mL) were

added, followed by the addition of hydrazine monohydrate (32 mL,

0.66 mmol). The reaction mixture was stirred for 90 min at room temper-

ature under an argon atmosphere and afterwards diluted with CH₂Cl₂,

quenched with acetone and evaporated to dryness. Product 40 was isolat-

ed after purification (207 mg, 91%) by column chromatography on silica

gel (hexanes/EtOAc 4:1). [a]^R_D^T=ꢀ28.5 (c=1, CHCl₃); ¹H NMR

(300 MHz, CDCl₃): d = 7.33–7.15 (m, 15H), 5.16 (d, J=4.8 Hz, 2H),

4.96 (s, 1H), 4.89 (s, 1H), 4.82 (s, 1H), 4.78 (dd, J=11.7, 57.6 Hz, 1H),

4.59 (dd, J=11.7, 57.6 Hz, 1H), 4.45 (brs, 2H), 4.05–4.01 (m, 1H), 3.79–

Pentasaccharide acceptor 38 (68 mg, 36.1 mmol) and cap building block 3

(42 mg, 72.2 mmol) were combined in a flask, coevaporated with toluene

(5) and dried under vacuum. The mixture was dissolved in CH₂Cl₂

(1 mL), freshly activated 4 molecular sieves (130 mg) were added and

the mixture was stirred for 30 min at room temperature under argon.

After cooling to ꢀ258C, TMSOTf (1.3 mL, 7.2 mmol) was added and the

reaction was allowed to warm to ꢀ108C over 30 min. The reaction mix-

ture was cooled down to ꢀ258C, quenched with triethylamine and filtrat-

ed over Celite. After removal of the solvent under reduced pressure, pu-

rification by flash chromatography (hexanes/EtOAc 7:3) and size-exclu-

sion chromatography (Sephadex LH-20 MeOH/CH₂Cl₂1:1) afforded hex-

Chem. Eur. J. 2006, 12, 8664 – 8686

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

8679

P. H. Seeberger et al.

3.65 (m, 5H), 3.43–3.38 (m, 1H), 3.21–3.13 (m, 2H), 2.67 (d, J=12.1 Hz,

1H), 1.63–1.19 (m, 15H); ¹³C NMR (75 MHz, CDCl₃): mixture of rotam-

ers: d = 176.9, 170.2, 156.9/156.4, 138.1, 137.8/137.0, 128.7–127.4 (CH-

Ar), 99.0, 74.5, 71.9, 68.9, 68.4, 67.8, 67.4, 66.8, 52.6, 50.7/50.4, 47.3/46.4,

39.1, 29.4, 28.2/27.7, 27.3, 23.6; IR (thin film on NaCl): n˜ =3569, 3005,

2933, 2871, 1739, 1692, 1497, 1471, 1456, 1426, 1302, 1097, 1051 cm^ꢀ1; HR

MALDI MS: m/z: calcd for C₃₉H₄₉NO₁₀Na: 714.3254; found: 714.3236

[M+Na]⁺.

rotamers: d = 205.8, 177.4, 171.7, 170.6, 170.5, 169.9, 169.7, 169.4, 168.7,

156.7/156.2, 138.0, 137.7, 137.6, 137.5, 137.4, 137.3, 136.9/136.8, 128.6–

127.6 (CH-Ar), 98.9, 98.2, 98.1, 97.7, 78.1, 78.0, 77.3, 75.7, 75.4, 74.9, 74.7,

74.6, 74.2, 73.9, 73.6, 73.3, 73.0, 70.2, 70.0, 69.92, 69.86, 69.7, 69.5, 69.0,

68.5, 68.3, 67.5, 67.2, 63.1, 61.9, 61.7, 52.3, 52.2, 51.9, 50.5/50.3, 47.1/46.2,

38.8, 37.6, 29.8, 29.2, 27.8, 27.6, 27.2, 23.3, 20.8, 20.7; IR (thin film on

NaCl): n˜ =3015, 2944, 2872, 2103, 1739, 1692, 1492, 1456, 1369, 1149,

1072, 1031 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₁₀₆H₁₂₅N₇O₃₆Na:

2094.806; found: 2094.801 [M+Na]⁺.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3-O-

benzyl-4-O-levulinoyl-a-l-idopyranosyluronate)-(1!4)-(6-O-acetyl-2-

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl (6-O-acetyl-2-azido-3,4-O-

azido-3-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1!4)-methyl

3-O-

dibenzyl-2-deoxy-a-d-glucopyranosyl)-(1!4)-(methyl

2-O-acetyl-3-O-

benzyl-2-O-pivaloyl-a-l-idopyranosiduronate (41): Reducing end build-

ing block 40 (130 mg, 0.19 mmol) and disaccharide 2 (210 mg, 0.23 mmol)

were combined in a flask, coevaporated with toluene (5) and dried

under vacuum. The starting materials were dissolved in CH₂Cl₂(4.2 mL)

and freshly activated 4 molecular sieves (660 mg) were added. This

mixture was stirred for 30 min at room temperature under an argon at-

mosphere. After cooling the mixture to ꢀ208C, TMSOTf (4 mL,

22.5 mmol) was added and the reaction was allowed to warm to ꢀ108C

over 30 min. The reaction was quenched at ꢀ208C with triethylamine

and filtrated over Celite. Removing of the solvent under reduced pres-

sure and purification by flash chromatography (hexanes/EtOAc 1:1)

yielded in 41 (188 mg, 70%). [a]^R_D^T=ꢀ14.4 (c=1, CHCl₃); ¹H NMR

(300 MHz, CDCl₃): d = 7.36–7.22 (m, 25H), 5.17–5.15 (m, 3H), 5.11 (t,

J=4.1 Hz, 1H), 5.03–5.00 (m, 2H), 4.95 (t, J=4.3 Hz, 1H), 4.88–4.85 (m,

2H), 4.81 (dd, J=10.8, 45.5 Hz, 1H), 4.74–4.70 (m, 5H), 4.67 (dd, J=

10.8, 45.5 Hz, 1H), 4.48–4.44 (m, 3H), 4.23–4.19 (m, 1H), 4.11 (t, J=

4.4 Hz, 1H), 3.94–3.90 (m, 3H), 3.87–3.67 (m, 6H), 3.50–3.41 (m, 4H),

3.34 (dd, J=3.7, 10.3 Hz, 1H), 3.22–3.12 (m, 2H), 2.73–2.65 (m, 2H),

2.52–2.46 (m, 2H), 2.16 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.62–1.21 (m,

6H), 1.19 (s, 9H); ¹³C NMR (75 MHz, CDCl₃): mixture of rotamers: d =

205.6, 177.2, 171.5, 170.4, 169.62, 169.54, 168.5, 156.6/156.0, 137.8, 137.42,

137.39, 137.2, 136.8/136.5, 128.4–127.2, 98.8, 98.0, 97.7, 78.2, 77.3, 76.7,

75.4, 74.8, 74.74, 74.66, 74.1, 73.6, 73.2, 73.0, 70.0, 69.7, 69.5, 68.96, 68.95,

68.5, 68.3, 67.5, 67.2, 63.3, 61.9, 52.3, 52.2, 50.6/50.3, 47.2/46.2, 38.9, 37.6,

29.9/29.8, 29.3, 28.1, 27.9, 27.6, 27.3/27.2, 23.4, 21.03, 20.99; IR (thin film

on NaCl): n˜ =3015, 2933, 2103, 1739, 1692, 1497, 1451, 1436, 1421, 1369,

1149, 1067, 1031 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₇₅H₉₀N₄O₂₄Na:

1453.584; found: 1453.581 [M+Na]⁺.

benzyl-a-l-idopyranosyluronate)-(1!4)-(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)-methyl 3-O-benzyl-2-O-pivaloyl-a-l-idopyrano-

siduronate (45): Pentasaccharide 43 (134 mg, 64.6 mmol) was dissolved in

CH₂Cl₂(1 mL). Pyridine (150 mL) and acetic acid (100 mL) were added,

followed by the addition of hydrazine monohydrate (6 mL, 129 mmol).

The reaction mixture was stirred 90 min at room temperature, diluted

with CH₂Cl₂, quenched with acetone (1 mL) and evaporated to dryness.

Product 44 was isolated after purification (110 mg, 86%) by flash column

chromatography on silica gel (hexanes/EtOAc 1:1). Pentasaccharide ac-

ceptor 44 (110 mg, 55.7 mmol) and cap building block 3 (64 mg, 112 mmol)

were combined in a flask, coevaporated with toluene (5) and dried

under vacuum. The mixture was dissolved in CH₂Cl₂(1.5 mL), freshly ac-

tivated 4 molecular sieves (200 mg) were added and the mixture was

stirred for 30 min at room temperature under argon. After cooling to

ꢀ258C, TMSOTf (1.5 mL, 6.2 mmol) was added and the reaction was al-

lowed to warm to ꢀ108C over 30 min. The reaction mixture was cooled

back to ꢀ258C, quenched with triethylamine and filtrated over Celite.

After removal of the solvent under reduced pressure, purification by

flash chromatography (hexanes/EtOAc 3:2) and size-exclusion chroma-

tography (Sephadex LH-20 MeOH/CH₂Cl₂1:1) afforded hexasaccharide

45 (106 mg, 80%). [a]^R_D^T=+14.6 (c=1, CHCl₃); ¹H NMR (500 MHz,

CDCl₃): d = 7.36–7.22 (m, 45H), 5.30 (d, J=4.7 Hz, 1H), 5.28 (d, J=

4.8 Hz, 1H), 5.17–5.14 (m, 2H), 5.02–5.00 (m, 2H), 4.98 (d, J=3.5 Hz,

1H), 4.96 (d, J=3.7 Hz, 1H), 4.94–4.90 (m, 4H), 4.87–4.81 (m, 5H),

4.76–4.74 (m, 3H), 4.71–4.64 (m, 6H), 4.63–4.60 (m, 2H), 4.58–4.56 (m,

2H), 4.48–4.45 (m, 2H), 4.43–4.40 (m, 1H), 4.36–4.34 (m, 1H), 4.26–4.14

(m, 4H), 4.09 (t, J=4.3 Hz, 1H), 4.04 (t, J=4.9 Hz, 1H), 4.00 (t, J=

5.1 Hz, 1H), 3.95–3.89 (m, 4H), 3.87–3.77 (m, 4H), 3.74–3.70 (m, 4H),

3.65–3.61 (m, 1H), 3.59–3.55 (m, 3H), 3.53–3.49 (m, 4H), 3.45–3.42 (m,

1H), 3.29–3.15 (m, 5H), 2.08–1.96 (5s, 15H), 1.59–1.46 (m, 4H), 1.32–

1.24 (m, 2H), 1.19 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): mixture of ro-

tamers: d = 177.6, 170.8, 170.7, 170.0, 169.95, 169.90, 169.88, 169.7, 169.7,

156.9/156.4, 138.2, 137.93, 137.88, 137.8, 137.72, 137.67, 137.65, 137.6,

137.1, 128.8–127.5, 99.1, 98.4, 98.2 (double intensity), 98.1, 97.2, 80.1,

78.3, 78.1, 78.0, 77.8, 77.5, 76.0, 75.9, 75.8, 75.63, 75.59, 75.6, 75.3, 75.12,

75.10, 75.0, 74.4, 74.19, 74.17, 73.3, 73.2, 70.6, 70.53, 70.46, 70.4, 70.1, 70.0,

69.91, 69.89, 69.7, 69.1, 67.3, 63.5, 63.3, 63.1, 62.6, 62.1, 61.9, 52.6, 52.20,

52.17, 50.7/50.5, 47.3/46.4, 39.0, 29.4, 28.2/27.7, 27.4, 27.3, 27.2, 23.5, 21.01,

20.98, 20.95, 20.93, 20.92; IR (thin film on NaCl): n˜ =3025, 2952, 2872,

2109, 1740, 1692, 1495, 1435, 1369, 1103, 1073, 1029 cm^ꢀ1; HR MALDI

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3-O-

benzyl-4-O-levulinoyl-a-l-idopyranosyluronate)-(1!4)-(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)-methyl 3-O-benzyl-2-O-

pivaloyl-a-l-idopyranosiduronate (43): Pyridine (320 mL) and AcOH

(210 mL) were added to a solution of 41 (188 mg, 0.13 mmol) in CH₂Cl₂

(1.6 mL) followed by the addition of hydrazine monohydrate (13 mL,

0.26 mmol). After stirring the reaction mixture for 1 h under an argon at-

mosphere, acetone (1 mL) was added and the solvents were removed

under reduced pressure. The crude product 42 was purified by flash chro-

matography on silica gel (hexanes/EtOAc 1:1). Deprotected trisaccharide

42 (122 mg, 91.5 mmol) and disaccharide 2 (121 mg, 109.8 mmol) were

combined in a flask, coevaporated with toluene (5) and dried under

vacuum. The mixture was dissolved in CH₂Cl₂(2.4 mL), freshly activated

4 molecular sieves (390 mg) were added and the mixture was stirred

for 30 min at room temperature under argon. After cooling to ꢀ258C,

TMSOTf (3 mL, 11.2 mmol) was added and the reaction was allowed to

warm to ꢀ108C over 30 min. The reaction mixture was cooled back to

ꢀ258C, quenched with triethylamine and filtrated over Celite. After re-

moval of the solvent under reduced pressure, purification by flash chro-

matography (hexanes/EtOAc 7:3) and size exclusion chromatography

(Sephadex LH-20 MeOH/CH₂Cl₂1:1) afforded the desired pentasacchar-

ide 43 (156 mg, 82%). [a]_D^RT=ꢀ7.1 (c=1, CHCl₃); ¹H NMR (300 MHz,

CDCl₃): d = 7.40–7.23 (m, 35H), 5.30 (d, J=4.6 Hz, 1H), 5.16–5.14 (m,

3H), 5.10 (t, J=3.8 Hz, 1H), 5.03–4.85 (m, 8H), 4.79–4.61 (m, 11H),

4.48–4.40 (m, 4H), 4.24–4.16 (m, 2H), 4.11 (t, J=4.4 Hz, 1H), 4.02–3.98

(m, 1H), 3.95–3.60 (m, 14H), 3.50 ꢀ3.49 (2s, 6H), 3.32–3.20 (m, 4H),

2.71–2.42 (m, 4H), 2.15 (s, 3H), 2.09–2.02 (4s, 12H), 1.54–1.46 (m, 4H),

1.27–1.20 (m, 2H), 1.19 (s, 9H); ¹³C NMR (75 MHz, CDCl₃): mixture of

MS: m/z: calcd for

[M+Na]⁺.

C₁₂₃H₁₄₂N₁₀O₃₉Na: 2405.933; found: 2405.930

Hexasaccharide 51: H₂O₂(30%, 0.50 mL) and a solution of LiOH (1m,

0.8 mL) were added at 08C to a solution of 39 (45 mg, 20 mmol) in THF

(1.5 mL). After stirring for 16 h at room temperature, the mixture was

cooled to 08C and MeOH (2.5 mL) and a solution of KOH (3m, 1.5 mL)

was added. After stirring for 16 h at room temperature, the reaction mix-

ture was neutralized with IR-120-H⁺Amberlite resin, filtered and con-

centrated. The residue was purified by Sephadex LH-20 chromatography

(MeOH/CH₂Cl₂1:1) to afford 46 (27 mg, 71%). ¹H NMR (300 MHz,

CD₃OD): d = 7.43–7.17 (m, 40H), 5.30 (m, 3H), 5.13–5.09 (m, 3H), 5.03

(s, 2H), 4.95–4.44 (m, 17H), 4.24 (m, 1H), 4.13–3.02 (m, 30H), 1.60–1.38

(m, 6H); ES-MS: m/z: calcd for C₉₈H₁₁₀O₃₃N₁₀: 997.4; found: 977.2

[Mꢀ2H]^2ꢀ

.

8680

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

SO₃·NEt₃(36 mg, 0.20 mmol) was added to a solution of 46 (13 mg,

6.6 mmol) in anhydrous DMF (1.5 mL). After stirring for 16 h at 558C

under an argon atmosphere, the reaction mixture was quenched with trie-

thylamine (0.3 mL) and diluted with MeOH (1 mL) and CH₂Cl₂(1 mL).

The solution was purified by Sephadex LH-20 chromatography (MeOH/

CH₂Cl₂1:1). The fractions that contained the sulfated hexasaccharide

were pooled and evaporated to dryness to give 47 (15 mg, 88%). ES-MS:

m/z: calcd for C₉₈H₁₀₉O₅₁N₁₀S₆: 811.1; found: 811.2 [M+3H]^3ꢀ; calcd for

Hexasaccharide 56: H₂O₂(30%, 0.31 mL) and a solution of LiOH (1m,

0.50 mL) were added at 08C to a solution of 45 (28 mg, 12 mmol) in THF

(1 mL). After stirring for 16 h at room temperature, the mixture was

cooled to 08C and MeOH (1.5 mL) and

a solution of KOH (3m,

0.94 mL) was added. After stirring for 16 h at room temperature, the re-

action mixture was neutralized with IR-120-H⁺Amberlite resin and then

filtered and concentrated. The residue was purified by Sephadex LH-20

chromatography (MeOH/CH₂Cl₂1:1) to afford 53 (15 mg, 63%).

¹H NMR (300 MHz, CD₃OD): d = 7.40–7.19 (m, 45H), 5.33 (brs, 2H),

5.14–5.06 (m, 5H), 4.99–4.56 (m, 18H), 4.44 (s, 2H), 4.28 (brs, 1H), 4.17–

C₉₈H₁₀₈O₅₁N₁₀S₆: 608.1; found: 608.3 [M+2H]^4ꢀ

.

Compound 47 (15 mg, 5.7 mmol) was dissolved in THF (2 mL) and treat-

ed with a 0.1m aqueous solution of NaOH (1.3 mL). Then a solution of

PMe₃in THF (204 mL of a 1m solution) was added and the reaction was

stirred for 8 h. The reaction mixture was neutralized with a 0.1m solution

of HCl and concentrated. ES-MS: m/z: calcd for C₉₈H₁₁₅O₅₁N₄S₆: 785.1;

3.47 (m, 30H), 1.47–1.23 (m, 6H); ES-MS: m/z: calcd for C₁₀₅H₁₁₆O₃₃N₁₀

:

1022.4; found: 1022.4 [Mꢀ2H]^2ꢀ

.

Compound 53 (15 mg, 7.3 mmol) was dissolved in THF (2.4 mL) and

treated with a 0.1m aqueous solution of NaOH (0.29 mL). Then a solu-

tion of PMe₃in THF (44 mL of a 1m solution) was added and the reaction

was stirred for 3 h. The reaction mixture was neutralized with a 0.1m so-

lution of HCl, concentrated and the residue was purified by Sephadex

LH-20 chromatography (MeOH/CH₂Cl₂1:1) to afford 54 (12 mg, 83%).

found: 785.4 [M+3H]^3ꢀ

.

The residue was dissolved in MeOH (1 mL), and then triethylamine

(10 mL) and acetic anhydride (5 mL) were added. After stirring for 1 h at

room temperature, the reaction mixture was purified by Sephadex LH-20

chromatography (MeOH/CH₂Cl₂1:1). The fractions that contained the

sulfated hexasaccharide were pooled and evaporated to dryness. The resi-

due was converted into the sodium salt by elution from a column of

Dowex 50WX4-Na⁺with MeOH/H₂O 9:1 to give 48 (10 mg, 67%).

¹H NMR (500 MHz, CD₃OD): d = 7.38–7.06 (m, 40H), 6.03 (brs, 1H),

5.99 (brs, 1H), 5.10 (brs, 1H), 5.05–3.06 (m, 53H), 2.05 (s, 3H), 2.02 (s,

3H), 2.01 (s, 3H), 1.60–1.39 (m, 6H); HSQC anomeric cross peaks

(500 MHz, CD₃OD): d = (6.0393.7), (5.9992.8), (5.1098.9), (4.63

ES-MS: m/z: calcd for C₁₀₅H₁₂₆O₃₃N₄: 985.4; found: 985.2 [M+2H]²⁺

.

An aqueous solution of NaOH (0.1m, 0.3 mL), and then SO₃·Py (15 mg,

92 mmol) were added to a solution of 54 (12 mg, 6.1 mmol) in MeOH

(1 mL), triethylamine (300 mL). After stirring for 2 h at room tempera-

ture, the reaction mixture was neutralized with a 0.1m solution of HCl

and purified by Sephadex LH-20 chromatography (MeOH/CH₂Cl₂1:1).

The fractions that contained the hexasaccharide were pooled and evapo-

rated to dryness. The residue was converted into the sodium salt by elu-

tion from a column of Dowex 50WX4-Na⁺with MeOH/H₂O 9:1 to give

55 (14 mg, quantitative). ¹H NMR (500 MHz, CD₃OD): d = 7.34–7.06

(m, 45H), 5.34 (d, J=3.5 Hz, 1H), 5.31 (d, J=3.4 Hz, 1H), 5.29 (d, J=

3.8 Hz, 1H), 5.18 (brs, 1H), 5.13 (brs, 1H), 5.08–5.01 (m, 3H), 4.90 (brs,

1H), 4.87 (brs, 1H), 4.73–4.46 (m, 15H), 4.38 (brs, 2H), 4.11–3.39 (m,

31H), 1.44–1.21 (m, 6H); HSQC anomeric cross peaks (500 MHz,

CD₃OD): d = (5.3496.3), (5.3196.3), (5.2996.3), (5.1899.4), (5.13

99.4), (4.87101.1); ES-MS: m/z: calcd for C₁₀₅H₁₂₂O₄₂N₄S₃: 1103.3;

found: 1103.5 [M+H]^2ꢀ; calcd for C₁₀₅H₁₂₁O₄₂N₄S₃: 735.2; found: 735.3

94.8),

(4.6195.1),

(4.5497.0);

ES-MS:

m/z:

.

calcd

for

C₁₀₄H₁₂₀O₅₄N₄S₆Na: 834.5; found: 834.8 [M+Na+2H]^3ꢀ

Asolution of 48 (7.0 mg, 2.6 mmol) in MeOH/H₂O (2 mL/0.5 mL) was hy-

drogenated in the presence of 10% Pd/C. After 24 h, the suspension was

filtered over Celite and concentrated to give 51 (4.3 mg, 88%). ¹H NMR

(500 MHz, D₂O): d = 5.06 (m, 2H), 5.02 (d, J=3.7 Hz, 1H), 5.01 (d, J=

3.9 Hz, 1H), 4.99 (d, J=3.7 Hz, 1H), 4.96 (brs, 1H), 4.79–4.78 (m, 2H),

4.43 (d, J=2.1 Hz, 1H), 4.21–3.51 (m, 28H), 3.42 (dd, J=9.7, 9.8 Hz,

1H), 2.87 (m, 2H), 1.93 (s, 3H), 1.92 (s, 3H), 1.92 (s, 3H), 1.55–1.31 (m,

6H); HSQC anomeric cross peaks (500 MHz, D₂O): d = (5.0699.4),

(5.0699.4), (5.0294.0), (5.0194.0), (4.9993.5), (4.9698.8); ES-MS:

[M]^3ꢀ

.

Asolution of 55 (14.0 mg, 6.0 mmol) in MeOH/H₂O (2 mL/1 mL) was hy-

drogenated in the presence of 10% Pd/C. After 24 h, the suspension was

filtered over Celite and concentrated to give 56 (8.5 mg, 97%). ¹H NMR

(500 MHz, D₂O): d = 5.23 (d, J=3.2 Hz, 1H), 5.21 (d, J=3.2 Hz, 1H),

5.19 (d, J=3.2 Hz, 1H), 4.86 (brs, 1H), 4.83 (brs, 1H), 4.73 (brs, 1H),

4.67 (m, 2H), 4.35 (brs, 1H), 3.96–3.46 (m, 25H), 3.32 (dd, J=9.5,

9.7 Hz, 1H), 3.09–3.04 (m, 3H), 2.89–2.82 (m, 2H), 1.56–1.42 (m, 4H),

1.32–1.28 (m, 2H); HSQC anomeric cross peaks (500 MHz, D₂O): d =

(5.2395.4), (5.2195.5), (5.1995.5), (4.86101.5), (4.83101.6),

(4.73100.6); ES-MS: m/z: calcd for C₄₁H₆₈O₄₀N₄S₃: 676.1; found: 675.7

m/z: calcd for C₄₇H₇₁O₅₂N₄S₆Na₃: 892.0; found: 892.7 [M+3Na+H]^2ꢀ

.

Hexasaccharide 52: Compound 46 (14 mg, 7.2 mmol) was dissolved in

THF (2.2 mL) and treated with a 0.1m aqueous solution of NaOH

(0.27 mL). Then a solution of PMe₃in THF (43 mL of a 1m solution) was

added and the reaction was stirred for 3 h. The reaction mixture was neu-

tralized with a 0.1m solution of HCl, concentrated and the residue was

purified by Sephadex LH-20 chromatography (MeOH/CH₂Cl₂1:1) to

afford 49 (10 mg, 77%). ES-MS: m/z: calcd for C₉₈H₁₁₆O₃₃N₄: 938.4;

found: 938.7 [Mꢀ2H]^2ꢀ

.

[M+H]^2ꢀ; calcd for C₄₁H₆₇O₄₀N₄S₃Na: 687.1; found: 686.8 [M+Na]^2ꢀ

.

Triethylamine (10 mL) and acetic anhydride (5 mL) were added to a solu-

tion of 49 (10 mg, 5.3 mmol) in MeOH (1 mL). After stirring for 1 h at

room temperature, the reaction mixture was purified by Sephadex LH-20

chromatography (MeOH/CH₂Cl₂1:1). The fractions that contained the

hexasaccharide were pooled and evaporated to dryness. The residue was

converted into the sodium salt by elution from a column of Dowex

50WX4-Na⁺with MeOH/H₂O 9:1 to give 50 (11 mg, quantitative).

¹H NMR (300 MHz, CD₃OD): d = 7.36–7.15 (m, 40H), 5.23 (m, 2H),

5.03 (s, 2H), 4.94–4.42 (m, 21H), 4.18–3.46 (m, 29H), 3.07–3.03 (m, 2H),

1.82–1.68 (3s, 9H), 1.61–1.39 (m, 6H); ES-MS: m/z: calcd for

N-Benzyloxycarbonyl-5-aminopentyl-2-azido-3,6-di-O-benzyl-2-deoxy-b-

d-glucopyranoside (58): Trichloroacetimidate 57 (487 mg, 0.92 mmol) and

N-benzyloxycarbonyl-5-aminopentan-1-ol (660 mg, 2.77 mmol) were co-

evaporated with toluene (3), dried under vacuum overnight and dis-

solved in anhydrous CH₂Cl₂(5 mL). The reaction mixture was cooled to

ꢀ258C and TMSOTf (53 mL, 0.27 mmol) was added dropwise and the so-

lution was allowed to warm to ꢀ108C. After 1 h the reaction was

quenched with triethylamine and the solvents were evaporated. The

crude product was purified by silica gel chromatography (toluene/EtOAc

9:1) affording N-benzyloxycarbonyl-5-aminopentyl 2-azido-4,6-O-benzyli-

dene-3-O-benzyl-2-deoxy-b-d-glucopyranoside (512 mg, 92%). ¹H NMR

(300 MHz, CDCl₃): d = 7.49–7.25 (m, 15H), 5.59 (brs, 0.5H), 5.56 (brs,

0.5H), 5.09 (s, 2H), 4.90 (d, J=11.1 Hz, 1H), 4.78 (d, J=11.4 Hz, 1H),

4.36–4.30 (m, 2H), 3.92–3.86 (m, 1H), 3.82–3.66 (m, 2H), 3.60–3.32 (m,

4H), 3.21–3.19 (m, 2H), 1.71–1.40 (m, 6H).

C

₁₀₄H₁₂₂O₃₆N₄: 1001.4; found: 1001.6 [Mꢀ2H]^2ꢀ

.

Asolution of 50 (10 mg, 5.0 mmol) in MeOH/H₂O (2 mL/0.5 mL) was hy-

drogenated in the presence of 10% Pd/C. After 24 h, the suspension was

filtered over Celite and concentrated to give 52 (6.2 mg, quantitative).

¹H NMR (500 MHz, D₂O): d = 5.05 (d, J=3.5 Hz, 1H), 5.04 (d, J=

3.5 Hz, 1H), 5.02 (d, J=3.5 Hz, 1H), 4.77 (m, 2H), 4.72 (brs, 1H), 4.65–

4.60 (2br s, 2H), 4.36 (brs, 1H), 3.94–3.89 (m, 3H), 3.80–3.46 (m, 25H),

3.33 (m, 1H), 2.85–2.82 (m, 2H), 1.89–1.88 (3s, 9H), 1.52 (m, 4H), 1.32

The above-mentioned compound (512 mg, 0.85 mmol) and triethylsilane

(0.81 mL, 5.10 mmol) were dissolved in anhydrous CH₂Cl₂(15 mL) under

(m, 2H); HSQC anomeric cross peaks (500 MHz, D₂O): d

=

(5.05

a nitrogen atmosphere at 08C and trifluoroacetic acid (0.38 mL,

94.5), (5.0494.5), (5.0294.5), (4.77101.7), (4.77101.7), (4.74

5.10 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 CH₂Cl₂and phase separation, the

100.7); ES-MS: m/z: calcd for C₄₇H₇₈O₃₄N₄: 621.2; found: 621.0

[M+2H]²⁺

.

Chem. Eur. J. 2006, 12, 8664 – 8686

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

8681

P. H. Seeberger et al.

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 (toluene/acetone 9:1) afforded prod-

uct 58 (308 mg, 60%) as a white solid. [a]²_D⁴=ꢀ7.7 (c=1.08, CHCl₃);

¹H NMR (300 MHz, CDCl₃): d = 7.38–7.30 (m, 15H), 5.09 (s, 2H), 4.91

(d, J=11.4 Hz, 1H), 4.76 (d, J=11.4 Hz, 1H), 4.57 (2d, J=11.9 Hz, 2H),

4.27 (d, J=7.8 Hz, 1H), 3.94–3.86 (m, 1H), 3.72 (d, J=5.1 Hz, 2H), 3.61

(dt, J=1.5, 5.8 Hz, 1H), 3.56–3.48 (m, 1H), 3.43–3.34 (m, 2H), 3.26–3.16

(m, 3H), 2.61 (d, J=2.5 Hz, 1H), 1.69–1.40 (m, 6H); ¹³C NMR (75 MHz,

CDCl₃): d = 128.5–127.6, 102.1, 82.4, 75.1, 73.8, 73.7, 72.0, 70.2, 69.9,

66.7, 65.7; IR (thin film on NaCl): n˜ =3518, 3404, 2112, 1728, 1512, 1353,

1077 cm^ꢀ1; HR MALDI MS: m/z: calcd for C₃₃H₄₀N₄O₇Na: 627.2789;

found: 627.2795 [M+Na]⁺.

3.75 (brs, 1H), 3.67 (dd, J=2.2, 4.5 Hz, 1H), 3.61 (br d, 1H), 3.33–3.30

(m, 5H), 3.15 (t, J=5.7 Hz, 1H), 2.74–2.64 (m, 1H), 2.60–2.44 (m, 2H),

2.41–2.36 (m, 1H), 2.10 (s, 3H), 2.00 (s, 3H), 0.87 (s, 9H), 0.09 (s, 3H),

0.08 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d = 206.2, 172.0, 170.1, 168.7,

138.4, 138.1, 137.7, 128.6–127.4, 97.6, 97.5, 81.2, 75.5, 74.3, 74.2, 73.4,

72.72, 72.70, 69.0, 68.33, 68.26, 67.1, 66.4, ꢀ4.0, ꢀ4.1; IR (thin film on

NaCl): n˜ =2849, 2112, 1743, 1369, 1159, 1107, 1066, 836 cm^ꢀ1

; HR

MALDI MS: m/z: calcd for C₄₇H₅₁N₃O₁₄SiNa: 942.3815; found: 942.3798

[M+Na]⁺.

Methyl 2-O-acetyl-3-O-benzyl-4-O-levulinoyl-a-l-idopyranosiduronate-

(1!4)-(2-azido-3,6-di-O-benzyl-2-deoxy-a/b-d-glucopyranosyl) trichloro-

acetimidate (64): Disaccharide 62 (160 mg, 0.17 mmol) was dissolved in

anhydrous THF (3 mL). Glacial acetic acid (21 mL, 0.37 mmol) and

TBAF (1m in THF, 0.34 mL, 0.34 mmol) were added and the reaction

mixture was stirred at room temperature for 4 h. The mixture was diluted

with EtOAc and extracted with NaHCO₃and brine. The organic layer

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

Flash chromatography on silica gel (hexanes/EtOAc 3:2!1:1) afforded

product 63 as a white solid (119 mg, 90%). Compound 63 (119 mg,

0.15 mmol) was dissolved in anhydrous CH₂Cl₂(2 mL) and cooled to

08C. Trichloroacetonitrile (0.15 mL, 1.48 mmol) and catalytic amount of

DBU were added. After 30 min, the solvents were removed in vacuo.

Flash chromatography on silica gel (hexanes/EtOAc 1:1) afforded a mix-

ture (1:1.8) of 64a and 64b as a white solid (0.12 mg, 92%). ¹H NMR

(300 MHz, CDCl₃): d = 8.72 (brs, NH, 1H), 7.40–7.17 (m, 15H), 6.44 (d,

J=3.3 Hz, 0.3H), 5.64 (d, J=8.1 Hz, 0.6H), 5.25 (d, J=5.4 Hz, 0.6H),

5.07–4.89 (m, 3H), 4.72–4.47 (m, 6H), 4.24–4.12 (m, 1H), 4.05–3.94 (m,

0.3H), 3.85–3.52 (m, 5.7H), 3.40–3.36 (2s, 3H), 2.84–2.70 (m, 1H), 2.66–

2.38 (m, 3H), 2.17 (s, 3H), 2.06 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d

N-(Benzyloxycarbonyl)-5-aminopentyl (methyl 2-O-acetyl-3-O-benzyl-4-

O-levulinoyl-a-l-idopyranosiduronate)-(1!4)-2-azido-3,6-di-O-benzyl-2-

deoxy-b-d-glucopyranoside (59): Trichloroacetimidate 11 (185 mg,

0.32 mmol) and acceptor 58 (160 mg, 0.26 mmol) were coevaporated with

toluene (3), dried under vacuum overnight and dissolved in anhydrous

CH₂Cl₂(3 mL). The reaction mixture was cooled to ꢀ258C and TMSOTf

(6 mL, 0.03 mmol) was added dropwise. The solution was warmed to

ꢀ108C over 1 h. Triethylamine was added to quench the reaction and the

solvents were evaporated. The crude product was purified by silica gel

chromatography (hexanes/EtOAc 3:2!1:1) affording disaccharide 59

(160 mg, 60%). [a]²_D⁴=ꢀ14.4 (c=3.6, CHCl₃); ¹H NMR (500 MHz,

CDCl₃): d = 7.35–7.12 (m, 20H), 5.13 (brs, 1H), 5.03 (brs, 2H), 5.00

(brs, 1H), 4.94 (d, J=2.1 Hz, 1H), 4.82 (brs, 1H), 4.70–4.44 (m, 6H),

4.18 (d, J=7.6 Hz, 1H), 3.90 (t, J=9.8 Hz, 1H), 3.85–3.81 (m, 1H), 3.75–

3.72 (m, 1H), 3.66 (m, 2H), 3.47–3.43 (m, 1H), 3.38–3.29 (m, 5H), 3.17–

3.11 (m, 3H), 2.74–2.68 (m, 1H), 2.58–2.46 (m, 2H), 2.41–2.35 (m, 1H),

2.10 (s, 3H), 1.98 (s, 3H), 1.60–1.56 (m, 2H), 1.50–1.44 (m, 2H), 1.40–

=

206.2, 172.98, 171.96, 170.2, 170.1, 168.6, 161.3, 160.9, 138.3, 138.1,

1.34 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): d

= 206.2, 171.9, 170.1,

137.9, 137.8, 137.6, 137.5, 128.7–127.4, 97.5, 97.4, 97.1, 95.0, 75.0, 74.7,

74.2, 73.8, 73.6, 73.5, 73.3, 72.8, 72.74, 72.68, 72.5, 68.31, 68.26, 67.9, 67.8,

67.1, 66.9, 66.6, 66.5, 66.0, 63.6, 52.3, 37.8, 30.0, 28.1, 21.1; IR (thin film

on NaCl): n˜ =2954, 2113, 1728, 1374, 1272, 1046 cm^ꢀ1; HR MALDI MS:

m/z: calcd for C₄₃H₄₇Cl₃N₄O₁₄Na: 971.2047; found: 971.2030 [M+Na]⁺.

168.7, 156.6, 138.3, 138.2, 138.1, 137.6, 136.9, 128.7–127.5, 102.5, 97.5,

81.3, 75.5, 74.7, 74.3, 73.4, 72.8, 72.7, 69.9, 68.4, 68.2, 67.1, 66.8, 66.7, 66.6,

66.5, 52.2, 41.2, 37.8, 29.9, 29.8, 29.3, 28.1, 23.4, 21.1; IR (thin film on

NaCl): n˜ =3456, 3025, 2943, 2112, 1738, 1718, 1518, 1456, 1369, 1159,

1107, 1046 cm^ꢀ1

1047.4210; found: 1047.423 [M+Na]⁺.

; HR MALDI MS: m/z: calcd for C₅₄H₅₄N₄O₁₅:

Methyl 2-O-acetyl-3,4-O-benzyl-a/b-l-idopyranosiduronate trichloroacet-

imidate (66): Compound 65 (235 mg, 0.55 mmol) was dissolved in CH₂Cl₂

(10 mL) and a solution of trifluroacetic acid (407 mL, 5.48 mmol) in H₂O

(98 mL, 5.48 mmol) was added. The reaction was stirred for 4 h at room

temperature and then quenched with a saturated Na₂CO₃solution. The

mixture was washed with water and extracted with CH₂Cl₂(300 mL). The

organic phase was dried over MgSO₄, filtered and the solvents were

evaporated and dried under vacuum, affording the diol as pale yellow oil

(193 mg, 91%).

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3-O-benzyl-a-l-

idopyranosiduronate)-(1!4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-d-gluco-

pyranoside (60): Compound 59 (198 mg, 0.19 mmol) was dissolved in

CH₂Cl₂. Pyridine (0.41 mL) and acetic acid (0.8 mL) were added, fol-

lowed by the addition of hydrazine (19 mL, 0.39 mmol). The reaction mix-

ture was stirred at room temperature for 90 min, diluted with CH₂Cl₂,

quenched with acetone and evaporated to dryness. The crude product

was purified by silica gel chromatography (hexanes/EtOAc 3:2!1:2) af-

fording acceptor 60 in quantitative yield (181 mg). [a]²_D⁴=ꢀ6.6 (c=1.2,

The resulting crude product was dissolved in CH₂Cl₂(2 mL) and imida-

zole (135 mg, 2.00 mmol) was added. The solution was cooled to ꢀ288C

and thexyldimethylsilyl chloride (0.13 mL, 0.65 mmol) was added. The re-

action was stirred at ꢀ258C for 40 h, quenched afterwards with saturated

aqueous NaHCO₃(1 mL) and allowed to warm to room temperature.

The reaction was diluted with CH₂C1₂and washed with brine. The aque-

ous layer was extracted with CH₂C1₂and the combined organic phases

were dried over MgSO₄, filtered, concentrated under reduced pressure,

and purified with flash silica column chromatography (hexanes/EtOAc

4:1) to afford methyl (thexyldimethylsilyl 3,4-O-benzyl-a/b-l-idopyrano-

sid)uronate as an a/b mixture (203 mg, 77%). ¹H NMR (300 MHz,

CDC1₃): major anomer: d = 7.38–7.23 (m, 10H), 5.01 (d, J=0.9 Hz,

1H), 4.57 (d, J=11.7 Hz, 1H), 4.53 (s, 1H), 4.50 (d, J=1.8 Hz, 2H), 4.43

(d, J=11.7 Hz, 1H), 3.90 (t, J=3.9, 1H), 3.81–3.78 (m, 1H), 3.71–3.70

(m, 4H), 3.64–3.59 (m, 1H), 1.70–1.60 (m, 1H), 0.91–0.88 (m, 12H), 0.24

(s, 3H), 0.18 (s, 3H); ¹³C NMR (125 MHz, CDC1₃): d = 169.1, 137.3,

136.8, 128.5–127.7, 74.1, 73.8, 73.5, 72.6, 72.3, 68.7, 20.2, 19.9, ꢀ1.9, ꢀ3.5;

HR MALDI MS: m/z: calcd for C₂₉H₄₂O₇SiNa: 553.2592; found:

553.2582 [M+Na]⁺.

1

CHCl₃); H NMR (300 MHz, CDCl₃): d = 7.40–7.23 (m, 20H), 5.14 (brs,

1H), 5.09 (s, 2H), 4.96 (d, J=1.8 Hz, 1H), 4.95–4.92 (m, 1H), 4.74–4.55

(m, 6H), 4.24 (d, J=7.5 Hz, 1H), 3.98–3.86 (m, 3H), 3.71 (d, J=3.0 Hz,

2H), 3.52–3.32 (m, 7H), 3.26–3.18 (m, 3H), 2.03 (s, 3H), 1.69–1.40 (m,

6H); ¹³C DEPT NMR (75 MHz, CDCl3): d = 128.4–127.2, 102.2, 97.6,

80.9, 75.0, 74.3, 74.2, 73.1, 72.0, 69.6, 67.9, 67.7, 66.9, 66.5, 66.3, 51.9, 40.9,

29.5, 29.0, 23.1, 20.9; IR (thin film on NaCl): n˜ =3017, 2941, 2117, 1744,

1722, 1516, 1451, 1370, 1100 cm^ꢀ1; HR MALDI MS: m/z: calcd for

C₄₉H₅₈N₄O₁₄Na: 949.3842; found: 949.3845 [M+Na]⁺.

tert-Butyldimethylsilyl (methyl 2-O-acetyl-3-O-benzyl-4-O-levulinoyl-a-l-

idopyranosiduronate)-(1!4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-d-gluco-

pyranoside (62): Trichloroacetimidate 11 (845 mg, 1.45 mmol) and accept-

or 61 (604 mg, 1.21 mmol) were coevaporated with toluene (3), dried

under vacuum overnight, and dissolved in anhydrous CH₂Cl₂(5 mL). The

reaction mixture was cooled to ꢀ258C and TMSOTf (26 mL, 0.15 mmol)

was added dropwise. The solution was warmed to ꢀ108C over 1.5 h. Trie-

thylamine was added to quench the reaction and the solvents were

evaporated to dryness. The crude product was purified by silica gel chro-

matography (hexanes/EtOAc 4:1!3:2) affording disaccharide 62

(970 mg, 86%). [a]²_D⁴=ꢀ20.4 (c=1.04, CHCl₃); ¹H NMR (500 MHz,

CDCl₃): d = 7.34–7.12 (m, 15H), 5.15 (brs, 1H), 5.07 (brs, 1H), 5.00

(brs, 1H), 4.84 (brs, 1H), 4.66–4.44 (m, 7H), 3.94 (t, J=5.7 Hz, 1H),

This compound (203 mg, 0.38 mmol) was dissolved in pyridine (3 mL)

and acetic anhydride (77 mL, 0.76 mmol) and DMAP (77 mg, 0.06 mmol)

were added. The reaction was stirred at room temperature overnight.

Quenching with MeOH (2 mL) and evaporation of the solvents under re-

duced pressure afforded after purification with flash silica gel column

8682

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

chromatography (hexanes/EtOAc 9:1) methyl (thexyldimethylsilyl 2-O-

acetyl-3,4-O-benzyl-a/b-l-idopyranosid)uronate (210 mg, 96%) as a clear

oil (a/b 1:2). ¹H NMR (300 MHz, CDCl₃): d = 7.38–7.17 (m, 10H), 5.07

(d, J=1.8, 1H), 4.90 (m, 1H), 4.64 (d, J=12.0 Hz, 1H), 4.54 (d, J=

11.7 Hz, 1H), 4.49 (d, J=2.1 Hz, 1H), 4.41 (d, J=3.9 Hz, 2H), 3.82 (t,

J=3.0 Hz, 1H), 3.72–3.70 (m, 4H), 2.05 (s, 3H), 1.66–1.58 (m, 1H), 0.88–

0.84 (m, 12H), 0.24 (s, 3H), 0.15 (s, 3H); ¹³C NMR (75 MHz, CDCl₃):

d = 170.9, 168.9, 137.4, 137.2, 128.5–127.8, 92.8, 74.2, 73.1, 72.6, 72.5,

72.41, 72.38, 67.9, 52.97, 51.94, 51.92, 33.9, 24.7, 20.98, 20.96, 20.2, 19.7,

18.5, 18.2, ꢀ1.9, ꢀ3.9; HR MALDI MS: m/z: calcd for C₃₁H₄₄O₈SiNa:

595.2698; found: 595.2687 [M+Na]⁺.

(t, J=3.9 Hz, 1H), 4.85–4.44 (m, 9H), 4.19 (t, J=9.3 Hz, 1H), 3.93–3.87

(m, 2H), 3.85–3.76 (m, 2H), 3.74–3.71 (m, 1H), 3.68–3.60 (m, 2H), 3.45

(s, 3H), 1.94 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): d = 170.2, 169.8,

161.0, 138.1, 137.9, 137.7, 128.7–127.6, 98.1, 95.0, 75.4, 74.96, 74.91, 74.2,

74.1, 73.6, 73.2, 73.0, 70.2, 69.8, 67.8, 63.2, 52.0, 21.2; IR (thin film on

NaCl): n˜ =3005, 2913, 2103, 1739, 1672, 1369, 1287, 1072, 1046 cm^ꢀ1; HR

MALDI MS: m/z: calcd for C₄₅H₄₇Cl₃N₄O₁₂Na: 963.2148; found:

963.2131 [M+Na]⁺.

N-Benzyloxycarbonyl-5-aminopentenyl (methyl 2-O-acetyl-3-O-benzyl-4-

O-levulinoyl-a-l-idopyranosiduronate)-(1!4)-(2-azido-3,6-di-O-benzyl-2-

deoxy-a-d-glucopyranosyl)-(1!4)-(methyl

2-O-acetyl-3-O-benzyl-a-l-

The obtained compound (210 mg, 0.37 mmol) was dissolved THF (4 mL),

and HF·pyridine (70% solution, 240 mL) was added. The reaction was

stirred at room temperature for 28 h, then diluted with water, extracted

with CH₂Cl₂(250 mL), dried over MgSO₄, filtered, and concentrated

under reduced pressure. Purification with flash silica column chromatog-

raphy (hexanes/EtOAc 3:2!1:1) provided the desired hemiacetal

(139 mg, 88%). HR MALDI MS: m/z: calcd for C₂₃H₂₆O₈Na: 453.1520;

found 453.1512 [M+Na]⁺.

idopyranosiduronate)-(1!4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-d-gluco-

pyranoside (70): Trichloroacetimidate 64 (120 mg, 0.13 mmol) and accept-

or 60 (106 mg, 0.12 mmol) were coevaporated with toluene (3), dried

under vacuum overnight and then dissolved in anhydrous CH₂Cl₂(2 mL).

Freshly activated molecular sieves 4 (450 mg) were added and the mix-

ture was stirred at room temperature for 1 h. The reaction mixture was

cooled to ꢀ258C and TMSOTf (3 mL, 0.02 mmol) was added. The solu-

tion was warmed to ꢀ108C over 45 min. Triethylamine was added to

quench the reaction and the solvents were evaporated. The crude product

was purified by silica gel chromatography (toluene/acetone 7:3) affording

tetrasaccharide 70 as a white foam (148 mg, 75%). [a]²_D⁴=+40.3 (c=0.7,

CHCl₃); ¹H NMR (500 MHz, CDCl₃): d = 7.38–7.19 (m, 35H), 5.26 (d,

J=2.9 Hz, 1H), 5.21 (brs, 1H), 5.09 (brs, 2H), 5.05 (t, J=2.9 Hz, 1H),

4.92 (d, J=3.9 Hz, 1H), 4.91–4.89 (m, 2H), 4.86 (t, J=2.7 Hz, 1H), 4.80

(d, J=3.0 Hz, 1H), 4.73–4.67 (m, 8H), 4.61 (brs, 1H), 4.58–4.55 (m, 3H),

4.48 (d, J=12.3 Hz, 1H), 4.22 (d, J=8.3 Hz, 1H), 3.99 (t, J=4.0 Hz, 1H),

3.94–3.87 (m, 4H), 3.81 (t, J=3.7 Hz, 1H), 3.76–3.70 (m, 4H), 3.65 (t, J=

9.5 Hz, 1H), 3.59 (d, J=10.1 Hz, 1H), 3.54–3.49 (m, 1H), 3.41–3.73 (m,

3H), 3.34 (dd, J=4.0, 10.9 Hz, 1H), 3.32 (s, 3H), 3.26 (t, J=9.7 Hz, 1H),

3.22–3.18 (m, 2H), 2.79–2.72 (m, 1H), 2.65–2.59 (m, 1H), 2.56–2.50 (m,

1H), 2.47–2.41 (m, 1H), 2.16 (s, 3H), 2.03 (s, 3H), 1.95 (s, 3H), 1.66–1.64

(m, 2H), 1.55–1.51 (m, 2H), 1.47–1.41 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃): d = 206.2, 171.9, 170.2, 170.0, 169.3, 168.7, 156.6, 138.4, 138.3,

138.1, 137.9, 137.6, 137.5, 136.9, 128.8–127.4, 102.4, 98.0, 97.7, 97.5, 81.3,

78.4, 75.4, 75.2, 74.7, 74.6, 74.34, 74.27, 73.6, 73.5, 73.4, 73.24, 73.20, 73.1,

73.0, 71.7, 69.9, 68.9, 68.7, 68.6, 68.4, 68.0, 67.7, 66.9, 66.8, 66.7, 66.5, 63.6,

60.6, 52.20, 51.93, 41.2, 37.8, 30.0, 29.3, 28.0, 23.4, 21.2, 21.1, 14.4; IR

(thin film on NaCl): n˜ =2923, 2113, 1738, 1451, 1369, 1046 cm^ꢀ1; HR

MALDI MS: m/z: calcd for C₉₀H₁₀₃N₇O₂₇Na: 1736.679; found:1736.677

[M+Na]⁺.

This hemiacetal (139 mg, 0.32 mmol) was dissolved in CH₂Cl₂(5 mL), tri-

chloroacetonitrile (0.32 mL, 3.23 mmol) was added and the solution was

cooled to 08C. DBU (2 mL) was added to the cooled reaction mixture,

and the reaction was allowed to warm to room temperature over 30 min.

The reaction was concentrated under reduced pressure and purified with

flash silica gel column chromatography (hexanes/EtOAc 7:3) to afford 66

(a/b mixture 1:7) (182 mg, 98%) as a white foam. ¹H NMR (300 MHz,

CDCl₃): d = 8.61 (s, 1H), 7.36–7.15 (m, 10H), 6.43 (brs, 1H), 5.12 (m,

1H), 4.96 (d, J=2.1 Hz, 1H), 4.75 (d, J=12.3 Hz, 1H), 4.58 (d, J=

12.0 Hz, 1H), 4.51 (d, J=11.7 Hz, 1H), 4.43 (d, J=12.0 Hz, 2H), 3.88 (m,

1H), 3.82 (m, 1H), 2.05 (s, 3H); HR MALDI MS: m/z: calcd for

C₂₅H₂₆Cl₃NO₈Na: 596.0616; found: 596.0605 [M+Na]⁺.

tert-Butyldimethylsilyl (methyl 2-O-acetyl-3,4-O-benzyl-a-l-idopyranosi-

duronate)-(1!4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-d-glucopyranoside

(67): Trichloroacetimidate 66 (182 mg, 0.32 mmol) and acceptor 61

(131 mg, 0.26 mmol) were coevaporated with toluene (3), dried under

vacuum, and dissolved in anhydrous CH₂Cl₂(2 mL). The reaction mix-

ture was cooled to ꢀ258C and TMSOTf (6 mL, 0.03 mmol) was added

dropwise. The solution was warmed to ꢀ108C over 1.5 h. Triethylamine

was added to quench the reaction and the solvents were evaporated. The

crude product was purified by silica gel chromatography (hexanes/EtOAc

4:1) affording disaccharide 67 (192 mg, 80%). [a]²_D⁴=ꢀ31.4 (c=1.19,

CHCl₃); ¹H NMR (300 MHz, CDCl₃): d = 7.34–7.26 (m, 20H), 5.30 (d,

J=1.1 Hz, 1H), 4.88 (m, 1H), 4.80 (d, J=3.0 Hz, 1H), 4.76–4.43 (m,

9H), 3.98 (t, J=8.7 Hz, 1H), 3.81–3.80 (m, 2H), 3.68–3.67 (m, 2H), 3.44

(s, 3H), 3.41–3.22 (m, 3H), 1.95 (s, 3H), 0.93 (s, 9H), 0.15 (s, 3H), 0.13

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3-O-benzyl-a-l-

idopyranosiduronate)-(1!4)-(2-azido-3,6-di-O-benzyl-2-deoxy-a-d-gluco-

pyranosyl)-(1!4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosiduro-

nate)-(1!4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-d-glucopyranoside (71):

Tetrasaccharide 70 (60 mg, 0.03 mmol) was dissolved in CH₂Cl₂(1 mL).

Pyridine (70 mL) and acetic acid (40 mL) were added, followed by the ad-

dition of hydrazine (4 mL, 0.07 mmol). The reaction mixture was stirred

at room temperature for 90 min. The reaction was then diluted with

CH₂Cl₂, quenched with acetone and evaporated to dryness. The crude

product was purified by silica gel chromatography (hexanes/EtOAc 3:1!

1:1) to afford acceptor 71 (49 mg, 90%). [a]²_D⁴=+0.4 (c=0.9, CHCl₃);

(s, 3H); ¹³C NMR (125 MHz, CDCl₃): d

= 206.2, 172.0, 170.1, 168.7,

138.4, 138.1, 137.7, 128.6–127.4, 97.6, 97.5, 81.2, 75.5, 74.3, 74.2, 73.4,

72.72, 72.70, 69.0, 68.31, 68.26, 67.1, 66.4, ꢀ4.02, ꢀ4.10; IR (thin film on

NaCl): n˜ =3007, 2930, 2859, 2112, 179, 1454, 1370, 1062 cm^ꢀ1

; HR

MALDI MS: m/z: calcd for C₄₉H₆₂N₃O₁₂SiNa: 934.3917; found: 934.3897

[M+Na]⁺.

Methyl

2-O-acetyl-3,4-O-benzyl-a-l-idopyranosiduronate-(1!4)-(2-

trichloroacetimi-

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

¹H NMR (500 MHz, CDCl₃): d

= 7.40–7. 21 (m, 35H), 5.26 (d, J=

date (69): Disaccharide 67 (192 mg, 0.21 mmol) was dissolved in anhy-

drous THF (3 mL). Glacial acetic acid (26 mL, 0.46 mmol) and TBAF

(1m in THF, 0.42 mL, 0.42 mmol) were added and the mixture stirred at

room temperature for 4 h. The mixture was diluted with EtOAc and ex-

tracted with NaHCO₃and brine. The organic layer was dried over

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

tography on silica gel (hexanes/EtOAc 3:2!1:1) afforded 68 as a white

solid (134 mg, 80%).

2.5 Hz, 1H), 5.13 (brs, 1H), 5.09 (brs, 2H), 4.94–4.89 (m, 3H), 4.85 (d,

J=2.0 Hz, 1H), 4.82 (d, J=3.0 Hz, 1H), 4.75–4.54 (m, 11H), 4.48 (d, J=

12.5 Hz, 1H), 4.23 (d, J=8.0 Hz, 1H), 4.04 (d, J=9.5 Hz, 1H), 3.99 (t,

J=3.0 Hz, 1H), 3.96–3.88 (m, 4H), 3.76–3.65 (m, 6H), 3.60–3.49 (m,

2H), 3.41–3.33 (m, 6H), 3.31 (s, 3H), 3.27 (t, J=10.0 Hz, 1H), 3.22–3.18

(m, 2H), 2.65 (d, J=11.5 Hz, 1H), 2.01 (s, 3H), 1.96 (s, 3H), 1.67–1.50

(m, 3H), 1.46–1.40 (m, 3H); ¹³C NMR (125 MHz, CDCl₃): d = 170.2,

169.8, 169.34, 169.31, 156.6, 138.4, 138.3. 138.0, 137.99, 137.6, 137.5, 136.9,

128.8–127.3, 102.5, 102.4, 98.1, 98.0, 97.9, 97.8, 81.4, 78.4, 75.4, 75.1, 74.7,

74.6, 74.2, 73.6, 73.5, 73.2, 72.7, 71.7, 70.0, 68.9, 68.7, 68.61, 68.55, 68.1,

68.0, 67.8, 66.8, 66.5, 63.6, 52.2, 51.9, 41.2, 29.8, 29.3, 23.4, 21.1, 20.9; IR

Product 68 (134 mg, 0.17 mmol) was dissolved in anhydrous CH₂Cl₂

(2 mL) and cooled to 08C. Trichloroacetonitrile (0.17 mL, 1.68 mmol)

and catalytic amount of DBU were added. After 30 min, the solvents

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

EtOAc 7:3) afforded a mixture (1:2.5) of 69a and 69b as a white solid

(152 mg, 96%). ¹H NMR (300 MHz, CDCl₃): d = 8.68 (brs, NH, 1H),

7.35–7.19 (m, 20H), 6.39 (d, J=3.6 Hz, 1H), 5.36 (d, J=4.2 Hz, 1H), 4.92

(thin film on NaCl): n˜ =2933, 2112, 1743, 1456, 1369, 1102, 1041 cm^ꢀ1

;

HR MALDI MS: m/z: calcd for C₈₅H₉₇N₇O₂₅Na: 1638.6426;

found:1638.642 [M+Na]⁺.

Chem. Eur. J. 2006, 12, 8664 – 8686

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

8683

P. H. Seeberger et al.

N-Benzyloxycarbonyl-5-aminopentyl (methyl 2-O-acetyl-3,4-di-O-benzyl-

a-l-idopyranosiduronate)-(1!4)-(2-azido-3,6-di-O-benzyl-2-deoxy-a-d-

glucopyranosyl)-(1!4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosi-

duronate)-(1!4)-(2-azido-3,6-di-O-benzyl-2-deoxy-a-d-glucopyranosyl)-

(1!4)-(methyl 2-O-acetyl-3-O-benzyl-a-l-idopyranosiduronate)-(1!4)-

2-azido-3,6-di-O-benzyl-2-deoxy-b-d-glucopyranoside (72): Disaccharide

donor 69 (48 mg, 0.05 mmol) and tetrasaccharide acceptor 71 (69 mg,

0.04 mmol) were dried under vacuum overnight and dissolved in anhy-

drous CH₂Cl₂(1 mL). The reaction mixture was cooled to ꢀ258C and

TMSOTf (40 mL of a 64 mm solution in anhydrous CH₂Cl₂) was added

dropwise. The solution was warmed to ꢀ108C and stirred for 1 h. Trie-

thylamine was added, the reaction mixture was concentrated and the resi-

due was purified by flash chromatography (toluene/EtOAc 6:1, then hex-

anes/EtOAc 2:1!3:2) to give 72 (60 mg, 59%). [a]²_D⁴=ꢀ9.7 (c=1,

CHCl₃); ¹H NMR (500 MHz, CDCl₃): d = 7.34–7.08 (m, 55H), 5.30 (d,

J=5.2 Hz, 1H), 5.28 (d, J=4.9 Hz, 1H), 5.20 (d, J=3.0 Hz, 1H), 5.03 (s,

2H), 4.92 (d, J=3.6 Hz, 1H), 4.85–4.69 (m, 8H), 4.65–4.43 (m, 19H),

4.15 (d, J=8.0 Hz, 1H), 4.00–3.80 (m, 10H), 3.74 (dd, J=5.2, 5.8 Hz,

1H), 3.68–3.43 (m, 11H), 3.42 (s, 3H), 3.33 (s, 3H), 3.31 (s, 3H), 3.29–

3.18 (m, 4H), 3.15–3.11 (m, 2H), 1.91 (s, 3H), 1.86 (s, 3H), 1.85 (s, 3H),

1.58–1.57 (m, 2H), 1.50–1.44 (m, 2H), 1.39–1.34 (m, 2H); ¹³C NMR

(125 MHz, CDCl₃): d = 170.2, 169.89, 169.88, 169.47, 169.43, 156.6, 138.4,

138.3, 138.19, 138.15, 138.11, 138.10, 138.07, 137.8, 137.7, 137.6, 136.9,

129.2–127.5, 102.4, 98.4, 98.02, 97.99, 97.90, 97.7, 81.3, 78.22, 78.16, 76.1,

75.6, 75.5, 75.4, 75.2, 74.79, 74.76, 74.6, 74.1, 73.9, 73.83, 73.76, 73.65,

73.63, 73.57, 73.3, 73.21, 73.16, 71.7, 71.6, 71.02, 70.96, 70.54, 70.49, 69.9,

69.2, 68.9, 68.5, 67.5, 66.8, 66.5, 63.2, 63.1, 52.05, 52.00, 51.96, 41.2, 31.1,

29.9, 29.8, 29.3, 23.4, 21.1, 21.0, 20.9; HSQC anomeric cross peaks

Triethylamine (0.3 mL) and then SO₃·Py (20 mg, 0.13 mmol) were added

to a solution of 75 (11 mg, 4.4 mmol) in anhydrous pyridine (1.5 mL).

After stirring for 3 h at room temperature under an argon atmosphere,

the reaction mixture was diluted with MeOH (1 mL) and CH₂Cl₂(1 mL).

The solution was purified by Sephadex LH-20 chromatography (MeOH/

CH₂Cl₂1:1s). The fractions that contained the sulfated hexasaccharide

were pooled and evaporated to dryness. The residue was converted into

the sodium salt by elution from a column of Dowex 50WX4-Na⁺with

MeOH/H₂O 9:1 to give 76 (11 mg, 92%). ¹H NMR (500 MHz, CD₃OD):

d = 7.40–7.01 (m, 55H), 5.98 (brs, 1H), 5.73 (brs, 1H), 5.45 (brs, 1H),

5.27–5.23 (m, 3H), 5.06 (d, J=2.8 Hz, 1H), 4.97 (s, 2H), 4.89–4.81 (m,

4H), 4.72–4.70 (m, 2H), 4.67–4.56 (m, 6H), 4.52–4.37 (m, 10H), 4.31–

3.43 (m, 29H), 3.03–3.00 (m, 2H), 1.54–1.32 (m, 6H); HSQC anomeric

cross peaks (500 MHz, CD₃OD): d = (5.9893.4), (5.7393.8), (5.46

97.6), (5.2796.4), (5.0695.9), (4.71101.8); ES-MS: m/z: calcd for

C

₁₁₉H₁₃₃O₅₁N₄S₆: 875.2; found: 875.5 [M+3H]^3ꢀ

C₁₁₉H₁₃₂O₅₁N₄S₆: 656.1; found: 656.5 [M+2H]^4ꢀ

;

calcd for

.

Asolution of 76 (10 mg, 3.5 mmol) in MeOH/H₂O (2 mL/0.5 mL) was hy-

drogenated in the presence of 10% Pd/C. After 24 h, the suspension was

filtered and concentrated to give 77 (6 mg, 95%). ¹H NMR (500 MHz,

D₂O): d = 5.23 (d, J=3.6 Hz, 1H), 5.17 (d, J=2.9 Hz, 1H), 5.14 (brs,

1H), 5.08 (brs, 1H), 5.05 (brs, 1H), 4.73 (brs, 1H), 4.71 (d, J=1.7 Hz,

1H), 4.65 (brs, 1H), 4.40 (d, J=8.4 Hz, 1H), 4.24–3.42 (m, 26H), 3.12–

3.09 (m, 2H), 2.90–2.84 (m, 3H), 1.60–1.51 (m, 4H), 1.40–1.33 (m, 2H);

HSQC anomeric cross peaks (500 MHz, D₂O): d = (5.2296.8), (5.17

97.3), (5.1499.2), (5.0899.0), (5.0599.1), (4.41101.5); ES-MS: m/z:

calcd for C₄₁H₆₈O₄₉N₄S₆: 796.0; found: 796.4 [M+4H]^2ꢀ

.

Hexasaccharide 79: Triethylamine (10 mL) and acetic anhydride (10 mL)

were added to a solution of 75 (18 mg, 7 mmol) in MeOH (1 mL). After

stirring for 1 h at room temperature, the reaction mixture was purified by

Sephadex LH-20 chromatography (MeOH/CH₂Cl₂1:1). The fractions

that contained the sulfated hexasaccharide were pooled and evaporated

to dryness. The residue was converted into the sodium salt by elution

from a column of Dowex 50WX4-Na⁺with MeOH/H₂O 9:1 to give 78

(17 mg, 94%). ¹H NMR (500 MHz, CD₃OD): d = 7.34–7.07 (m, 55H),

5.82 (brs, 1H), 5.63 (brs, 1H), 5.44 (brs, 1H), 5.01–4.95 (m, 6H), 4.69–

4.46 (m, 21H), 4.41–3.33 (m, 30H), 3.01–2.98 (m, 2H), 1.95 (s, 3H), 1.94

(s, 3H), 1.92 (s, 3H), 1.50–1.23 (m, 6H); HSQC anomeric cross peaks

(500 MHz, CD₃OD): d = (5.8294.7), (5.6394.9), (5.4499.0), (4.66

95.7), (4.6295.8), (4.32101.0); ES-MS: m/z: calcd for C₁₂₅H₁₃₉O₄₅N₄S₃:

837.2; found: 837.6 [M]^3ꢀ; calcd for C₁₂₅H₁₃₉O₄₅N₄S₃Na: 1267.4; found:

(500 MHz, CDCl₃):

d = (5.3097.90), (5.2897.99), (5.2098.02),

(4.9298.4), (4.8597.7), (4.15102.4); IR (thin film on NaCl): n˜ =3036,

2954, 2113, 1739, 1369, 1108, 1046 cm^ꢀ1; HR MALDI MS: m/z: calcd for

C

₁₂₈H₁₄₂N₁₀O₃₆Na: 2417.9480; found: 2417.9560 [M+Na]⁺.

Hexasaccharide 77: H₂O₂(30%, 0.33 mL) and a solution of LiOH (1m,

0.55 mL) were added at 08C to a solution of 72 (31 mg, 13 mmol) in THF/

MeOH (4 mL/2 mL). After stirring for 16 h at room temperature, the

mixture was cooled to 08C and MeOH (6.8 mL) and a solution of KOH

(3m, 1 mL) were added. After stirring for 16 h at room temperature, the

reaction mixture was neutralized with IR-120-H⁺Amberlite resin, fil-

tered and concentrated. The residue was purified by Sephadex LH-20

chromatography (MeOH/CH₂Cl₂1:1) to afford 73 (25 mg, 86%).

¹H NMR (300 MHz, CD₃OD): d = 7.46–7.17 (m, 55H), 5.61 (brs, 1H),

5.55 (brs, 1H), 5.32 (brs, 1H), 5.11 (brs, 1H), 5.02–3.06 (m, 58H), 1.61–

1.42 (m, 6H); ES-MS: m/z: calcd for C₁₁₉H₁₂₈O₃₃N₁₀: 1112.5; found:

1267.4 [M+Na]^2ꢀ

.

Asolution of 78 (17 mg, 6.4 mmol) in MeOH/H₂O (2 mL/0.5 mL) was hy-

drogenated in the presence of 10% Pd/C. After 24 h, the suspension was

filtered over Celite and concentrated to give 79 (9.0 mg, 90%). ¹H NMR

(500 MHz, D₂O): d = 5.01 (m, 3H), 4.94 (m, 2H), 4.73 (brs, 1H), 4.67

(brs, 1H), 4.66 (brs, 1H), 4.35 (d, J=7.8 Hz, 1H), 4.16 (m, 2H), 4.10 (m,

3H), 3.92–3.40 (m, 24H), 2.84–2.81 (m, 2H), 1.95–1.90 (3s, 9H), 1.53–

1.43 (m, 4H), 1.28–1.25 (m, 2H); ES-MS: m/z: calcd for C₄₇H₇₄O₄₃N₄S₃:

739.1; found: 739.0 [M+H]^2ꢀ; calcd for C₄₇H₇₃O₄₃N₄S₃Na: 750.1; found:

1112.1 [Mꢀ2H]^2ꢀ

.

SO₃·NEt₃(37 mg, 0.20 mmol) was added to a solution of 73 (30 mg,

13 mmol) in anhydrous DMF (1.5 mL). After stirring for 16 h at 558C

under an argon atmosphere, the reaction mixture was quenched with trie-

thylamine (0.3 mL) and diluted with MeOH (1 mL) and CH₂Cl₂(1 mL).

The solution was purified by Sephadex LH-20 chromatography (MeOH/

CH₂Cl₂1:1). The fractions that contained the sulfated hexasaccharide

were pooled and evaporated to dryness to give 74 (30 mg, 86%).

¹H NMR (300 MHz, CD₃OD): d = 7.42–7.12 (m, 55H), 5.44 (brs, 1H),

5.42 (brs, 1H), 5.38 (brs, 1H), 5.18 (d, J=3.6 Hz, 1H), 5.15 (d, J=

4.2 Hz, 1H), 5.04 (s, 2H), 4.96 (d, J=1.8 Hz, 1H), 4.83–4.42 (m, 22H),

4.34–3.04 (m, 32H), 1.63–1.38 (m, 6H); ES-MS: m/z: calcd for

750.2 [M+Na]^2ꢀ

.

Tetrasaccharide 83: H₂O₂(30%, 0.57 mL) and a solution of LiOH (1m,

0.95 mL) were added at 08C to a solution of 71 (53 mg, 33 mmol) in THF/

MeOH (6.8 mL/3.4 mL). After stirring for 16 h at room temperature, the

mixture was cooled to 08C and MeOH (11.5 mL) and a solution of KOH

(3m, 1.7 mL) was added. After stirring for 16 h at room temperature, the

reaction mixture was neutralized with IR-120-H⁺amberlite resin and

then was filtered and concentrated. The residue was purified by Sepha-

dex LH-20 chromatography (MeOH/CH₂Cl₂1:1) to afford 80 (40 mg,

82%). ¹H NMR (300 MHz, CD₃OD): d = 7.43–7.16 (m, 35H), 5.24 (d,

J=2.7 Hz, 1H), 5.17 (brs, 1H), 5.12 (d, J=3.6 Hz, 1H), 5.04 (s, 2H),

4.86–4.52 (m, 13H), 4.40 (d, J=11.4 Hz, 1H), 4.32 (d, J=7.5 Hz, 1H),

4.14 (m, 1H), 4.02–3.64 (m, 16H), 3.57–3.53 (m, 2H), 3.45 (m, 1H), 3.13–

3.07 (m, 2H), 1.64–1.42 (m, 6H); ES-MS: m/z: calcd for C₇₉H₈₈O₂₃N₇:

1502.6; found: 1502.0 [MꢀH]^ꢀ.

C

₁₁₉H₁₂₇O₄₂N₁₀S₃: 821.2; found: 821.2 [M]^3ꢀ

.

Compound 74 (30 mg, 11 mmol) was dissolved in THF (4 mL) and treated

with a 0.1m aqueous solution of NaOH (1.7 mL). Then, a solution of

PMe₃in THF (276 mL of a 1m solution) was added and the reaction was

allowed to stir for 8 h. The reaction mixture was neutralized with a 0.1m

solution of HCl, concentrated and the residue was purified by flash chro-

matography on silica gel (EtOAc/Py/H₂O/AcOH 25:5:3:1) and Sephadex

LH-20 chromatography (MeOH/CH₂Cl₂1:1) to afford 75 (20 mg, 69%).

¹H NMR (300 MHz, CD₃OD): d = 7.46–7.13 (m, 55H), 5.53 (brs, 1H),

5.47 (brs, 1H), 5.44 (brs, 1H), 5.27–5.24 (m, 2H), 5.06–4.50 (m, 25H),

4.42–3.52 (m, 30H), 3.13–3.03 (m, 2H), 1.70–1.40 (m, 6H); ES-MS: m/z:

calcd for C₁₁₉H₁₃₄O₄₂N₄S₃: 1193.4; found: 1193.8 [M+H]^2ꢀ; calcd for

Compound 80 (25 mg, 17 mmol) was dissolved in THF (4 mL) and treated

with a 0.1m aqueous solution of NaOH (1.0 mL). Then, a solution of

PMe₃in THF (132 mL of a 1m solution) was added and the reaction was

C

₁₁₉H₁₃₃O₄₂N₄S₃: 795.2; found: 795.5 [M]^3ꢀ

.

8684

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 8664 – 8686

Heparin Oligosaccharides

FULL PAPER

stirred for 5 h. The reaction mixture was neutralized with a 0.1m solution

of HCl, concentrated and the residue was purified by Sephadex LH-20

chromatography (MeOH/CH₂Cl₂1:1) to afford 81 (24 mg, quantitative).

ES-MS: m/z: calcd for C₇₉H₉₂O₂₃N₃: 1450.6; found: 1450.0 [MꢀH]^ꢀ.

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An aqueous solution of NaOH (0.1m, 0.3 mL), and then SO₃·Py (14 mg,

88 mmol) were added to a solution of 81 (11 mg, 7.6 mmol) in MeOH

(1 mL), triethylamine (300 mL). After stirring for 3 h at room tempera-

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and purified by Sephadex LH-20 chromatography (MeOH/CH₂Cl₂1:1).

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4.49 (m, 14H), 4.20–3.64 (m, 17H), 3.60 (m, 1H), 3.54–3.46 (m, 3H),

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Asolution of 82 (12.0 mg, 7.1 mmol) in MeOH/H₂O (2 mL/0.5 mL) was

hydrogenated in the presence of 10% Pd/C. After 24 h, the suspension

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Acknowledgements

This research was supported by ETH Zürich and the European Commis-

sion (Marie Curie Fellowship for J.L.P.). We thank Dr. Jens Sobek (Func-

tional Genomics Center, Zürich) for assistance in the construction of mi-

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Received: July 29, 2006

Published online: October 25, 2006

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Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions

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