Probing the heparin - Antithrombin III interaction using synthetic pentasaccharides bearing positively charged groups

Article abstract of DOI:10.1002/1099-0690(200212)2002:23<3954::AID-EJOC3954>3.0.CO;2-2

Four heparin pentasaccharides bearing either one (i.e. 3b and 4b) or two (i.e. 3c and 4c) positively charged amino groups at the reducing end have been synthesized and evaluated for their antithrombin III mediated anti-Xa activity. The positively charged groups were introduced to interact specifically with the negatively charged amino acid residues Glu113 and Asp117 of antithrombin III, which are located in the heparin binding site in close proximity to the reducing end of the saccharide. It turned out that the target compounds 3b,c and 4b,c exhibited relatively low anti-Xa activities, indicating unfavorable interactions between the new pentasaccharides and antithrombin III rather than the anticipated enhancement of association. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200212)2002:23<3954::AID-EJOC3954>3.0.CO;2-2

FULL PAPER
Probing the Heparin؊Antithrombin III Interaction Using Synthetic
Pentasaccharides Bearing Positively Charged Groups
[a]
[a]
[b]
´
Jeroen D. C. Codee, Gijsbert A. van der Marel, Constant A. A. van Boeckel,* and
Jacques H. van Boom*[a]
Keywords: Antithrombotics / Heparin / C-Glycosides / Inhibitors / Oligosaccharides / Carbohydrates
Four heparin pentasaccharides bearing either one (i.e. 3b
and 4b) or two (i.e. 3c and 4c) positively charged amino
groups at the reducing end have been synthesized and
evaluated for their antithrombin III mediated anti-Xa activity.
The positively charged groups were introduced to interact
specifically with the negatively charged amino acid residues
Glu113 and Asp117 of antithrombin III, which are located in
the heparin binding site in close proximity to the reducing
end of the saccharide. It turned out that the target com-
pounds 3b,c and 4b,c exhibited relatively low anti-Xa activit-
ies, indicating unfavorable interactions between the new
pentasaccharides and antithrombin III rather than the an-
ticipated enhancement of association.
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
essential (marked with an exclamation mark ‘‘!’’ in Fig-
ure 1), contributing (marked with an asterisk ‘‘*’’) or nones-
Pentasaccharide 1a (see Figure 1) constitutes the sential. The latter insight led inter alia to the design and
antithrombin III (AT III) binding domain of heparin, an synthesis of the highly potent pentamer 2a (SanOrg 34006
anticoagulant polysulfated glycosaminoglycan.[1,2] AT III or Idraparinux Sodium).[9]
requires activation by heparin to exert its activity as a cru-
cial inhibitor of coagulation factors Xa and thrombin (IIa).
The action of the heterogeneous drug heparin is mainly
governed by a unique pentasaccharide domain, also known
as fragment DEFGH, in some of the polysaccharide chains.
The pentasaccharide moiety has a distinct mode of action
in that it solely activates AT III towards inhibition of factor
Xa, but not of thrombin. On the basis of this knowledge,
pentamer 1b (Org 31540/SR 90107A or Fondaparinux So-
dium), the α-O-methyl glycoside of 1a, has been developed
as a new antithrombotic drug (Arixtra).[3] In addition, a
vast array of analogs[4] such as desulfated,[5] decarb-
oxylated,[4] epimeric,[4] flexible,[6] conformationally con-
strained[7] and non-glycosaminoglycan[8] derivatives of the
pentasaccharide have been synthesized and tested for their
biological activity. In this respect it is noteworthy that of all
derivatives the binding to AT III (Kd) is proportional to
the anti-factor Xa activity measured. The outcome of the
structure-activity relationship (SAR) studies revealed
whether the mode of interactions of the individual carb-
oxylate and sulfate groups in 1 and 2 with AT III are either
Figure 1. Pentasaccharides 1a,b and 2a,b; groups highlighted with
‘‘!’’ are essential for AT III activation, whilst the groups with ‘‘*’’
increase the biological activity
[a]
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden
University
Recently, the crystal structure[10] of AT III in complex
with pentasaccharide 2b nicely corroborated the interac-
tions of the pentasaccharide with the antithrombin binding
site. Thus, all essential sulfate and carboxylate groups inter-
P. O. Box 9502, 2300 RA Leiden, The Netherlands
Fax: (internat.) ϩ 31-71/527-4307
E-mail: boom j@chem.leidenuniv.nl
[b]
Lead Discovery Unit, N. V. Organon
P. O. Box 20, 5340 BH Oss, The Netherlands
3954
 2002 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/02/1223Ϫ3954 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 3954Ϫ3965

Probing the HeparinϪAntithrombin III Interaction
FULL PAPER
act directly with various positively charged Lys and Arg
residues of AT III. However, the crystal structure does not
explain why several analogs, in which particular sulfates are
replaced by phosphates or carboxylates, are hardly active.[4]
Furthermore, close inspection of the binding site not only
revealed the presence of positively charged amino acids, but
also negatively charged amino acid residues in the proxim-
ity of the anomeric center of unit H. As shown in Figure 2,
the carboxylate groups of Glu113 and Asp117 are separated
from the α-O-methyl group and the anomeric center in 2b
˚
by approximately 6Ϫ8 A.
Figure 3. Structure of pentasaccharides 3aϪe and 4aϪe
ible by condensation of 3b and 4b, with a diaminopropionic
acid derivative.
At this stage it is noteworthy that the target pentamers
3b,c and 4b,c have an unprecedented sulfation pattern,
which prevents the use of the parent pentasaccharide 2a as
a reference in assessing the contribution of the positively
charged groups of the pentasaccharides 3b,c and 4b,c in
their interaction with AT III. This drawback has been over-
come by using the easily accessible N-benzyloxycarbonyl-
ated (Z) counterparts 3d,e and 4d,e as reference com-
pounds.
Figure 2. Part of the AT IIIϪpentasaccharide binding site showing
Glu113 and Asp117 in proximity of the anomeric region of 2b in
the crystal structure of the AT IIIϪpentasaccharide complex
We here report the synthesis and anti-Xa activity of the
four pentasaccharides 3b,c and 4b,c, as well as their refer-
ence compounds 3d,e and 4d,e.
Based on the above observation, it was envisaged that
replacement of the α-O-methyl group in 2a by an α-C-glyco-
sidic ethylamine tether, as in compound 3a (see Figure 3),
may result in additional binding interactions with the nega-
tively charged amino acid residues Glu113 and Asp117 in
AT III. The crystal structure also shows the possibility of
accommodating the corresponding β-C-anomer, implying
that 4a may give rise to similar binding interactions with
the same amino acids.
The presence, however, of the terminal amino group in
both 3a and 4a may also lead to unwanted intramolecular
salt bridge formation with proximal sulfate groups. In order
to reduce such undesired intramolecular interactions, the
proximal R1 sulfate in 3a, as well as both sulfates R1 and
R2 in 4a, were replaced by methyl groups resulting in new
pentasaccharide derivatives 3b and 4b, respectively.
Results and Discussion
It was expected, based on the well-established synthetic
route[11] of the pentasaccharide 2a, that the assembly of the
target compounds 3b,c as well as 4b,c could be accomp-
lished using the appropriately protected and functionalized
monomeric H-building blocks 5 and 6 (see Figure 4), re-
spectively. Retrosynthetic analysis revealed that the pre-
paration of both the α- and the β-C-glycoside could be ef-
fected by a stereoselective transformation of a 1,2-anhydro
Apart from this, it is evident that the ethylamino tether
in 3b and 4b also opens the way of introducing additional
positive charges through further functionalization. For in-
stance, pentasaccharide analogs bearing two positively
charged amino groups (i.e. pentamers 3c and 4c) are access- Figure 4. H-building blocks 5 and 6
Eur. J. Org. Chem. 2002, 3954Ϫ3965
3955

´
J. D. C. Codee, G. A. van der Marel, C. A. A. van Boeckel, J. H. van Boom
FULL PAPER
Scheme 1. a) TBSCl, imidazole, DMF, 60 °C, 16 h, 8: 75%; b) DMDO, acetone, CH2Cl2, 0 °C, 5 min; c) NaCCH, ZnCl2, THF, 0 °C to
room temp., 1 h, 10: 75% in 2 steps; d) MeI, NaH, DMF, 0 °C, 1 h, 11: 93%, 18: 93%; e) i. BH3·THF, 2-methyl-2-butene, THF, 0 °C,
1 h; ii. H2O2, NaOH, H2O, 1 h; iii. LiAlH4, THF, 0 °C, 5 min, 12: 81% in 3 steps; f) i. MsCl, pyridine, 16 h; ii. NaN3, DMF, 60 °C, 3 h,
˚
13: 87%, 20: 85% in 2 steps; g) TBAF, THF, 3 h, 5: 99% 6: 86%; h) allylMgCl, THF, mol. sieves 4 A, 30 min, 16: 93% in 2 steps; i) TFA,
pyrrole, CH2Cl2, 1 min, 17: 90%; j) i. O3, CH2Cl2, MeOH, Ϫ70 °C, 2 h; ii. DMS, Ϫ50 °C to room temp., 16 h; iii. LiAlH4, 30 min, 19:
72% in 3 steps
sugar into either the α-ethynyl or the β-allyl-C-glycoside fol- menced, as outlined in Scheme 2, with the synthesis of the
lowing the protocol developed by Leeuwenburgh et. fully protected α(1Ǟ4)-linked dimer 22. Thus, glycosylation
al.[12a,12b]
of 5 at low temperature (Ϫ20 °C) with known[16] ethyl 2,4,6-
Accordingly, the known[13] dibenzylated -glucal 7 was tri-O-acetyl-3-O-methyl-1-thio-α/β--idopyranose (21) us-
converted into the required H-α unit 5 by the sequence of ing N-iodosuccinimide (NIS) and a catalytic amount of
events depicted in Scheme 1. Thus, silylation of 7, followed triflic acid (TfOH), proceeded with a high degree of stereo-
by epoxidation of fully protected 8 with dimethyldioxirane selectivity to give the α-linked dimer 22, as gauged by NMR
(DMDO),[14] afforded the key 1,2-anhydro derivative 9 in spectroscopy, in an excellent yield. Zemplen deacetylation
´
an excellent yield. Ring opening of the epoxide with sodium of 22 and transient protection of the 4Ј,6Ј-diol in the re-
acetylide in the presence of zinc chloride[12a,12b] gave, after sulting triol derivative with an acetonide group was fol-
methylation of the newly introduced hydroxy group in 10, lowed by methylation and then unmasking of the acetal
the α-ethynyl-C-glucoside 11. Hydroboration of 11 with di- functionality. Subsequent chemoselective TEMPO oxida-
siamylborane, and subsequent in situ oxidation of the inter- tion[17] of the primary alcohol function gave, after ester-
mediate vinylborane with hydrogen peroxide, was followed ification, the benzyl ester derivative 23 in an overall yield
by reduction of the resulting crude aldehyde leading to of 49% based on 22. The crucial introduction of the α-gly-
isolation of alcohol 12 in a yield of 81% over the three steps. cosidic bond between the iduronic acid acceptor 23 and the
Reaction of the mesyl derivative of 12 with sodium azide known[11] trimeric α/β-imidate donor 24 in the presence of
afforded, after desilylation, the H-α unit 5 in an overall the activating agent trimethylsilyl triflate (TMSOTf) pro-
yield of 36% based on 7.
ceeded uneventfully to give the expected[11] fully protected
In a similar fashion, the H-β unit 6 was prepared (see pentamer 25 in a yield of 77% based on 24. Unmasking of
Scheme 1) from the known fully protected glucal 14.[12b] the benzyl protecting groups by hydrogenation and con-
The sequence of events commenced by ring-opening of ep- comitant reduction of the azide function in 25 gave, after
oxide 15 with allylmagnesium chloride[12] to afford the β- deacetylation, pentamer 26 which, in turn, could be readily
allyl-C-glucoside 16 as the sole product in an excellent transformed into the required reference pentamers 3d,e, as
yield. Acidolysis[15] of the trityl protecting group in 16 and well as the target compounds 3b,c. Thus, masking of the
subsequent bis-methylation of 17 gave the fully protected amino function in 26 with the benzyloxycarbonyl (Z) group
alkene derivative 18 in 84% yield over the two steps. Ozonol- was followed by sulfation of the six hydroxy groups (i.e.
ysis of the terminal alkene moiety and reduction of the both three primary and secondary OH groups in 27) led to
resulting aldehyde led to the primary alcohol 19, which was the isolation of 3d. Removal of the temporary Z protecting
transformed, by the same sequence of reactions employed group in the latter pentamer completed the synthesis of the
earlier in the transformation of 12 into 5, into building first target compound 3b. Condensation of 26 with the O-
block 6 in an overall yield of 41% (based on 14).
succinimidyl ester of 2,3-bis(Z-amino)propionic acid[18]
Having the terminal β-C-glycoside building unit 5 in gave pentamer 28 in a yield of 50%. Subjection of 28 to
hand, the construction of the pentasaccharides 3b,c com- the same sulfation conditions as mentioned above gave the
3956
Eur. J. Org. Chem. 2002, 3954Ϫ3965

Probing the HeparinϪAntithrombin III Interaction
FULL PAPER
˚
Scheme 2. a) NIS, TfOH, mol. sieves 4 A, toluene, Ϫ20 °C, 30 min, 22 96%, 29 88%; b) i. KOtBu, MeOH, 2 h; ii. dimethoxypropane,
pTsOH, DMF, 1 h; iii. MeI, NaH, DMF, 0 °C, 30 min; iv. HOAc, H2O, 60 °C, 1 h; v. TEMPO, NaOCl, NaHCO3, TBACl, NaCl, CH2Cl2/
˚
H2O, 0 °C, 3 h; vi. BnBr, KHCO3, DMF, 2 h, 23: 49% 30: 50% in 6 steps; c) TMSOTf, mol. sieves 4 A, CH2Cl2, Ϫ20 °C, 1 h, 25: 77%,
31: 75%; d) i. H2. Pd/C, tBuOH/H2O, 16 h; ii. NaOH, MeOH/H2O, 16 h, 26: 77%, 32: 80%; e) ZϪOSu, N-methylmorpholine, DMF/
H2O, 30 min, 27: 50%, 33: 50%; f) SO3.Et3N, DMF, 55 °C, 16 h, 3d: 100%, 3e: 100%, 4d: 100%; g) H2, Pd/C, H2O, 4 h, 3b: 100%, 3c:
100%, 4b: 100%, 4c: 100%; h) 2,3-bis(Z-amino)propionic acid succinimidyl ester, N-methylmorpholine, DMF, 30 min, 28: 55%, 34: 60%;
i) 0.2  HCl, 4 °C, 16 h, 4d: 90% in 2 steps
Eur. J. Org. Chem. 2002, 3954Ϫ3965
3957

´
J. D. C. Codee, G. A. van der Marel, C. A. A. van Boeckel, J. H. van Boom
FULL PAPER
additional reference compound 3e. Moreover, removal of of the amino group in 3b, 4b with the residues Glu113 and
both Z protecting groups from 3e resulted in the isolation Asp117, but would also hinder the 3-O-sulfate binding[10]
of the second target pentamer 3d containing two, instead of with Arg46 and Arg47 of AT III.[21] The 6-O-sulfate in 3b
one, terminal amino groups.
can likewise form an intramolecular salt bridge with the
The corresponding β-pentasaccharides 4b,c and their proximal amino function, which may explain the lower ac-
benzyloxycarbonylated counterparts were synthesized in an tivity of compound 4b as compared to 3b. In addition, anal-
analogous manner starting from the β-C-glycoside 6 ogs, 3c and 4c, comprising two positively charged groups
(Scheme 2). Thus, glycosylation of 6 with the -idose donor have lost almost all anti-Xa activity, probably due to sim-
21[16] furnished the α-linked disaccharide 29, which was ilar reasons.
converted into the iduronic acid acceptor 30 and sub-
sequently coupled with imidate donor 24 to provide pen-
Conclusion
tamer 31. Complete deprotection led to pentasaccharide 32,
of which the amine function was capped with a Z group.
Interestingly, sulfation of the hydroxy groups in 3 under the
same conditions used in the sulfation of 27 was accompan-
ied, as evidenced by NMR spectroscopy, by unwanted par-
tial N-sulfation to give 34. Fortunately, the N-sulfate group
could be removed selectively under mild acidic condi-
tions[19] to yield the reference pentasaccharide 4d. Reduc-
tion of the benzyloxycarbonyl function provided the target
pentamer 4b. To complete the set of target compounds, pen-
tamer 32 was transformed into compounds 4c,d as de-
scribed for the conversion of 26 into 3c,d.
This paper describes the synthesis and biological evalu-
ation of four heparin pentasaccharide analogs 3b,c and 4b,c,
containing positively charged groups tethered to their redu-
cing end and designed to interact specifically with the am-
ino acid residues Glu113 and Asp117 in the AT III heparin
binding site. The biological activities of our pentasacchar-
ides reveal that these specific amino acid residues are not
readily available for additional binding interactions as was
anticipated on basis of the crystal structure of pentamer 2b
in complex with AT III. It is assumed that the formation of
intramolecular salt bridges between the newly introduced
amine functions and the sulfate groups of the pentasacchar-
ide is more favorable than the formation of intermolecular
salt bridges with the amino groups with Glu113 and Asp117.
Inhibition of Factor Xa
The AT III mediated anti-factor Xa activities of the pen- It is tempting to speculate that the incorporation of a rigid
tasaccharides 3bϪe and 4bϪd and of pentamers 1b and 2a spacer between the positively charged amino groups and
are recorded in Table 1. It is evident that saccharides 3bϪe the pentamer can lead to the required additional interac-
and 4bϪe display a lower activity than the parent pentasac- tions with the target amino acids. On the other hand, previ-
charide 2a and natural 1b. Furthermore, it can be seen that ous studies[4Ϫ9] show that the interaction of pentasacchar-
the activity of compounds 3b and 4b, having one positively ides with AT III is highly specific in nature and not merely
charged amino function, is lower than their benzyloxycar- based on straightforward electrostatic interactions. In con-
bonylated counterparts 3d and 4d. For this reason it is un- clusion, even with a very detailed crystal structure of the
likely that the incorporated amino function interacts favor- AT III pentasaccharide complex at our disposal it remains
ably with the negatively charged target amino acids Glu113 a fascinating game of trial and error to construct more po-
and Asp117. Possibly, the positively charged Arg24, posi- tent and simplified analogs of the unique heparin pentasac-
tioned between Glu113 and Asp117, prevents these residues charide domain.
from interaction with the pentamer, whilst being essential
for the integrity of the AT III conformation.[20] Notwith-
Experimental Section
standing our intentions, it cannot be excluded that the am-
ino function in both pentamers 3b and 4b, forms an intra-
molecular salt bridge with the O-sulfate at the 3-position.
This phenomenon would not only prevent the interaction
General Methods: Dichloromethane and toluene were refluxed with
P2O5 for 3 h and then distilled. THF and diethyl ether were re-
fluxed with LiAlH4 and distilled directly before use. All anhydrous
˚
solvents were stored over molecular sieves (3 or 4 A). All chemicals
Table 1. Anti-Xa activities of compounds 3 and 4
(Fluka, Acros, Merck) were used as received. Reactions were per-
formed under an inert gas under strictly anhydrous conditions at
ambient temperature. Traces of water in the reagents were removed
by coevaporation with toluene or DMF. 1H and 13C NMR spectra
were recorded with a Jeol JNM FX 200 (200 and 50.1 MHz, re-
spectively), a Bruker DPX 300 (300 and 75.1 MHz) or a Bruker
Pentamer
(α anomers)
Anti-Xa activity[a] Pentamer
(β anomers) Activity [U/Mg]
Anti-Xa
[U/Mg]
3b
77 Ϯ 4
3d (reference) 105 Ϯ 14
4b
4c (reference) 183 Ϯ 23
4d 18 Ϯ 1
4e (reference) 49 Ϯ 1
2a 938 Ϯ 18
21 Ϯ 4
1
DMX 600 (600 MHz). H NMR chemical shifts (δ) in CDCl3 are
reported relative to tetramethylsilane. For proton spectra in aque-
ous solutions (D2O) the residual HDO peak was set at δ ϭ
4.80 ppm. Mass spectra were recorded with a PE/SCIEX API 165
equipped with an Electrospray Interface (PerkinϪElmer). Column
chromatography was performed on Merck silica gel 60
(0.040Ϫ0.063 mm). TLC analysis was conducted on DC-Fertigfol-
3c
18 Ϯ 2
28 Ϯ 4
440
3e (reference)
1b
[a] AT III mediated anti-Xa activities were determined by an amido-
lytic method adapted from Teien and Lie.[22]
3958
Eur. J. Org. Chem. 2002, 3954Ϫ3965

Probing the HeparinϪAntithrombin III Interaction
ien (Schleicher & Schuell, F1500, LS254) or HPTLC aluminum CϵC) 67.5, 70.0, 71.0, 75.3, 82.9 (C-3,4,5,6,7), 127.3Ϫ128.1
FULL PAPER
sheets (Merck, silica gel 60, F254). Compounds were visualized by
UV absorption (254 nm), and by spraying with 20% H2SO4 in eth-
anol or with a solution of (NH4)6Mo7O24·4H2O (25 g/L) and
(NH4)4Ce(SO4)4·2H2O (10 g/L) in water or 3% ninhydrin in ethanol
followed by charring at 140 °C. Gel permeation chromatography
was accomplished on Toyopearl HW40S. Preparative HPLC was
performed with a Biocad Vision (Applied Biosystems, Inc.) using
an Alltima C-18 5µ encapped semi-preparative column. Gradient
elution was performed at 20 °C by building up a gradient of aceton-
itrile in 0.1% aqueous TFA at a flow rate of 5 mL/min.
(CHarom), 137.9, 138.4 (Cq,arom) ppm.
α-Ethynyl-C-glucoside 11: Alcohol 10 (3.33 g, 6.91 mmol) and
methyl iodide (0.94 mL, 13.8 mmol) were dissolved in DMF
(35 mL) and cooled in an ice bath. Sodium hydride (0.331 g,
7.60 mmol) was added and stirring was continued for 1 h. The mix-
ture was diluted with diethyl ether (100 mL) and washed once with
saturated aqueous NH4Cl and water. The aqeous layers were com-
bined and extracted three times with diethyl ether. The combined
organic layers were dried (MgSO4) and the solvents evaporated to
dryness. Silica gel column chromatography (0Ϫ10% EtOAc in light
petroleum ether) gave pure 11 (3.18 g, 6.40 mmol, 93%). Rf ϭ 0.80
(light petroleum ether/EtOAc, 4:1, v/v). 1H NMR (CDCl3): δ ϭ
0.039 (s, 6 H, CH3 TBS), 0.84 (s, 9 H, tBu), 2.65 (d, J ϭ 2.2 Hz, 1
H, 1-H), 3.43 (s, 3 H, OMe), 3.43 (m, 1 H), 3.65 (m, 4 H) 3.93 (m,
Glucal 8: tert-Butyldimethylsilyl chloride (5.21 g, 34.6 mmol) and
imidazole (4.71 g, 69.2 mmol) were added to a solution of 3,6-O-
dibenzylglucal[13] (5.64 g, 17.3 mmol) in DMF (80 mL). The reac-
tion mixture was brought to 60 °C and stirred for 16 h, after which
methanol (5 mL) was added and the mixture was concentrated. The
residue was dissolved in diethyl ether and washed with water, after
1 H, 4, 5, 6, 7, 8, 8Ј-H), 4.62 (AB, 2 H, CH2Bn), 4.93 (dd, J3,4
5.9, J ϭ 2.2 Hz, 1 H, 3-H), 5.02 (AB, 2 H, CH2Bn), 7.36 (m, 10
ϭ
which the aqueous layer was extracted three times with diethyl H, CH Ph) ppm. 13C{1H} NMR (CDCl3): δ ϭ Ϫ5.11, Ϫ3.87 (CH3,
ether. The organic layers were dried (MgSO4) and the solvents
evaporated in vacuo. The crude product was purified by silica gel
column chromatography (0 to 2% EtOAc in toluene) to give the
TBS), 17.94 (Cq tBu), 25.88 (CH3 tBu), 58.18 (OMe), 68.88, 73.10,
74.52 (CH2 Bn, C-8), 77.46 (Cq CϵC), 65.42, 70.31, 74.92, 81.59,
82.41 (C-3,4,5,6,7), 126.95Ϫ128.14 (CHarom), 138.02, 139.08
fully protected glucal 8 (5.71 g, 13.0 mmol) in 75% yield. Rf ϭ 0.90 (Cq,arom) ppm.
(light petroleum ether/EtOAc, 3:1, v/v). 1H NMR (CDCl3): δ ϭ
α-Hydroxyethyl-C-glycoside 12: Freshly distilled 2-methyl-2-butene
0.055 (s, 3 H, CH3 TBS), 0.073 (s, 3 H, CH3 TBS), 0.85 (s, 9 H,
tBu), 3.76, 3.95 (m, 5 H, 3,4,5,6,6Ј-H), 4.58 (AB, 2 H, CH2Bn),
4.59 (AB, 2 H, CH2Bn), 4.86 (dd, J2,1 ϭ 5.9, J2,3 ϭ 2.2 Hz, 1 H,
2-H), 6.44 (d, J1,2 ϭ 5.9 Hz, 1 H, 1-H), 7.27 (m, 10 H, CH Ph)
ppm. 13C{1H} NMR (CDCl3): δ ϭ Ϫ5.0, Ϫ4.0 (CH3, TBS), 18.2
(Cq tBu), 26.0 (CH3 tBu), 68.9, 70.0, 73.4 (CH2 Bn, C-6), 68.7,
77.2, 78.5 (C-3,4,5), 99.4 (C-2), 127.5Ϫ129.1 (CHarom), 138.3, 138.5
(Cq,arom), 144.8 (C-1) ppm.
(3.4 mL, 32.0 mmol) was added to a chilled borane solution in
THF (1.0 , 12.8 mL, 12.8 mmol) and the mixture was stirred for
1 h, after which a solution of alkyne 11 (3.178 g, 6.40 mmol) in
THF (6 mL) was added and stirring was continued for 30 min, after
which TLC analysis showed complete conversion of the starting
material. Aqueous sodium hydroxide (3 , 12.8 mL, 38.4 mmol)
was added dropwise, immediately followed by a solution of hydro-
gen peroxide (35% in water, 4.0 mL, 46.12 mmol). The mixture was
stirred for 1 h after which it was diluted with diethyl ether, washed
with water and brine. The aqueous layers were collected, extracted
1,2-Anhydro-α-D-glucopyranose 9: Glucal 8 (4.50 g, 10.2 mmol) was
dissolved in dichloromethane (30 mL) and the solution was cooled
to 0 °C. A solution of DMDO in acetone[16] (ca. 0.08 , 150 mL, twice with EtOAc and the combined organic layers were dried with
12 mmol) was added and after stirring for 5 min the reaction mix- MgSO4, filtered and concentrated. The resulting oil was coevapor-
ture was concentrated to provide epoxide 9 as a yellow oil (4.65 g, ated thrice with toluene, dissolved in THF (20 mL) and cooled to
10.2 mmol, 100%). 1H NMR (CDCl3): δ ϭ 0.037 (s, 3 H, CH3 0 °C. Lithium aluminum hydride (267 mg, 7.04 mmol) was added
TBS), 0.039 (s, 3 H, CH3 TBS), 0.83 (s, 9 H, tBu), 3.00 (d, J2,1
ϭ
and the mixture was stirred for 5 min. The reaction was quenched
by dropwise addition of aqueous sodium hydroxide (3 ) until gas
evolution had ceased. Dilution of the mixture with EtOAc was fol-
2.2 Hz, 1 H, 2-H), 3.70 (m, 5 H, 3,4,5,6,6Ј-H), 4.64 (AB, 2 H,
CH2Bn), 4.65 (AB, 2 H, CH2Bn), 4.97 (d, J1,2 ϭ 2.9 Hz, 1 H, 1-
H), 7.31 (m, 10 H, CH Ph) ppm. 13C{1H} NMR (CDCl3): δ ϭ lowed by the addition of MgSO4, the mixture was stirred for 10 min
Ϫ5.3, Ϫ4.2 (CH3, TBS), 18.0 (Cq tBu), 25.8 (CH3 tBu), 51.9 (C-2), and filtered through Celite. The filter cake was rinsed thoroughly
68.4, 71.6, 73.2 (CH2 Bn, C-6), 67.8, 70.2 (C-3,4,5), 79.8 (C-1),
127.4Ϫ128.3 (CHarom), 137.5, 138.0 (Cq,arom) ppm.
with EtOAc and the solution was concentrated in vacuo. The crude
product was purified by silica gel column chromatography
(10Ϫ50% EtOAc in light petroleum ether) to give pure alcohol 12
(2.66 g, 5.15 mmol) in 81% yield. Rf ϭ 0.20 (light petroleum ether/
EtOAc, 3:1, v/v). 1H NMR (CDCl3): δ ϭ Ϫ0.03 (s, 3 H, CH3 TBS),
Ϫ0.01 (s, 3 H, CH3 TBS), 0.85 (s, 9 H, tBu), 1.82 (m, 1 H, 2-H),
2.15 (m, 1 H, 2Ј-H) 3.40 (s, 3 H, OMe), 3.44 (m, 4 H), 3.79 (m, 4
H), 4.34 (m, 1 H, 1,1Ј,3,4,5,6,7,8,8Ј-H), 4.62 (AB, 2 H, CH2Bn),
4.93 (AB, 2 H, CH2Bn), 7.36 (m, 10 H, CH Ph) ppm. 13C{1H}
NMR (CDCl3): δ ϭ Ϫ5.0, Ϫ3.8 (CH3, TBS), 17.9 (Cq tBu), 25.8
(CH3 tBu), 27.7 (C-2), 58.32 (OMe), 61.4 (C-1), 70.0, 73.3, 74.6
(CH2 Bn, C-8), 71.5, 72.6, 73.5, 81.7, 82.4 (C-3,4,5,6,7),
127.1Ϫ128.3 (CHarom), 137.8, 139.0 (Cq,arom) ppm.
α-Ethynyl-C-glucoside 10: A solution of ZnCl2 in THF (2 , 26 mL,
52 mmol) was added to a suspension of sodium acetylide (18 wt%
in xylenes/mineral oil, 16 mL, 51 mmol) in THF (50 mL) at 0 °C,
after which the mixture was stirred for 10 min. The resulting solu-
tion was added at 0 °C to a mixture of the crude 1,2-anhydroglu-
cose 9 (10.2 mmol) in THF (50 mL) and the reaction mixture was
stirred for 1 h. The mixture was poured into saturated aqueous
NH4Cl, washed with water, dried with MgSO4 and concentrated.
Silica gel column chromatography (10Ϫ20% EtOAc in light petro-
leum ether) gave the pure α-ethynyl adduct (3.69 g, 7.65 mmol,
1
75%). Rf ϭ 0.60 (light petroleum ether/EtOAc, 2:1, v/v). H NMR
(CDCl3): δ ϭ 0.011 (s, 3 H, CH3 TBS), 0.048 (s, 3 H, CH3 TBS), Azide 13: Alcohol 12 (0.880 g, 1.71 mmol) was dissolved in dichlor-
0.86 (s, 9 H, tBu), 2.16 (d, J ϭ 7.3 Hz, 1 H, OH), 2.63 (d, J ϭ omethane (10 mL). Mesyl chloride (0.34 mL, 3.41 mmol) and pyr-
2.2 Hz, 1 H, 1-H), 3.67 (m, 5 H), 3.95 (m, 1 H, 3, 4,5,6,7,8,8Ј-H), idine (0.68 mL, 8.53 mmol) were added and the reaction mixture
4.58 (AB, 2 H, CH2Bn), 4.77 (dd, J3,4 ϭ 5.1, J ϭ 2.2 Hz, 1 H,
was stirred for 16 h, after which TLC analysis showed clean conver-
3-H), 4.80 (AB, 2 H, CH2Bn), 7.32 (m, 10 H, CH Ph) ppm.
sion into a faster running product; Rf ϭ 0.50 (light petroleum ether/
13C{1H} NMR (CDCl3): δ ϭ Ϫ5.1, Ϫ4.1 (CH3, TBS), 17.8 (Cq EtOAc, 3:1, v/v). The mixture was diluted with diethyl ether and
tBu), 25.7 (CH3 tBu), 68.6, 73.0, 74.4 (CH2 Bn, C-8), 77.5 (Cq
washed with water. After the organic phase was dried (MgSO4) and
Eur. J. Org. Chem. 2002, 3954Ϫ3965
3959

´
J. D. C. Codee, G. A. van der Marel, C. A. A. van Boeckel, J. H. van Boom
FULL PAPER
concentrated, the crude mesylate was coevaporated three times with
toluene and taken up in DMF (5 mL). Sodium azide (290 mg,
β-Hydroxyethyl-C-glucoside 19: Alkene 18 (1.31 g, 3.02 mmol)
was dissolved in dichloromethane/methanol (7:1, 30 mL) and co-
4.46 mmol) was added and the mixture was heated for 3 h at 60 oled to Ϫ70 °C. An ozone/oxygen mixture was bubbled through
°C. The solution was allowed to cool to room temperature, diluted
with diethyl ether and washed with water, after which the aqueous
layer was extracted thrice with diethyl ether. The combined organic
phases were dried (MgSO4) and concentrated. The azide was puri-
fied by silica gel column chromatography (10% EtOAc in light pet-
the solution until a blue color persisted. Then oxygen was passed
through for 30 min, after which the mixture was brought to Ϫ50
°C, dimethyl sulfide (3 mL) was added and the mixture was allowed
to reach room temperature overnight. The mixture was concen-
trated, coevaporated three times with toluene, dissolved in THF
roleum ether) and obtained as a yellow oil (0.805 g, 1.49 mmol) in and cooled in an ice bath. Lithium aluminum hydride (126 mg,
87% yield. Rf ϭ 0.80 (light petroleum ether/EtOAc, 3:1, v/v). 1H
3.32 mmol) was added and the mixture was stirred for 30 min, after
NMR (CDCl3): δ ϭ Ϫ0.020 (s, 3 H, CH3 TBS), Ϫ0.015 (s, 3 H, which the reaction was quenched by dropwise addition of aqueous
CH3 TBS), 0.82 (s, 9 H, tBu), 1.93 (m, 2 H, 2,2Ј-H), 3.36 (s, 3 H, sodium hydroxide (3 , 1.5 mL). The mixture was diluted with
OMe), 3.53 (m, 8 H), 4.24 (m, 1 H, 1,1Ј,3,4,5,6,7,8,8Ј-H), 4.57 (AB,
2 H, CH2Bn), 4.79 (AB, 2 H, CH2Bn), 7.31 (m, 10 H, CH Ph) after which the mixture was filtered through Celite. The filter cake
ppm. 13C{1H} NMR (CDCl3): δ ϭ Ϫ5.0, Ϫ3.8 (CH3, TBS), 24.5
was rinsed thoroughly with EtOAc and the solution was concen-
EtOAc, MgSO4 was added and stirring was continued for 10 min,
(Cq tBu), 25.9 (CH3 tBu), 29.6 (C-2), 47.8 (C-1), 58.2 (OMe), 69.7, tarted in vacuo. The crude alcohol was purified by silica gel column
73.3, 74.5 (CH2 Bn, C-8), 70.2, 71.0, 73.0, 81.7, 82.2 (C-3,4,5,6,7), chromatography (10Ϫ50% EtOAc in light petroleum ether) to give
127.1Ϫ128.7 (CHarom), 139.0 (Cq,arom) ppm.
19 (0.952 g, 2.16 mmol) in 72% yield. Rf ϭ 0.20 (light petroleum
ether/EtOAc, 3:1, v/v). H NMR (CDCl3): δ ϭ 0.055 (s, 6 H, CH3
1
H-α Building Block 5: A solution of tetrabutylammonium fluoride
in THF (1 , 1.8 mL, 1.8 mmol) was added to Azide 13 (0.805 g,
1.49 mmol) in THF (10 mL). The mixture was stirred for 3 h after
which it was washed twice with water. The organic layer was dried
(MgSO4), concentrated and the crude product was purified by silica
gel column chromatography (10Ϫ50% EtOAc in light petroleum
ether) to give the pure 5 in 99% yield (0.630 g, 1.48 mmol). Rf ϭ
0.35 (light petroleum ether/EtOAc, 3:1, v/v). 1H NMR (CDCl3):
δ ϭ 1.90 (m, 2 H, 2,2Ј-H), 2.76 (br. s, 1 H, OH), 3.50 (s, 3 H,
OMe), 3.53 (m, 8 H), 4.18 (m, 1 H, 1,1Ј,3,4,5,6,7,8,8Ј-H), 4.56 (s,
2 H, CH2Bn), 4.77 (AB, 2 H, CH2Bn), 7.31 (m, 10 H, CH Ph)
ppm. 13C{1H} NMR (CDCl3): δ ϭ 35.0 (C-2), 47.5 (C-1), 58.1
(OMe), 69.3, 73.1, 73.9 (CH2 Bn, C-8), 69.3, 69.7, 72.4, 79.3, 80.5
(C-3,4,5,6,7), 127.2Ϫ128.1 (CHarom), 137.8, 138.3 (Cq,arom) ppm.
TBS), 0.89 (s, 9 H, tBu), 1.76 (m, 1 H), 2.01 (m, 1 H, 2,2Ј-H), 2.72
(br. s, 1 H, OH), 2.95 (t, J ϭ 9.5 Hz, 1 H), 3.12Ϫ3.56 (m, 4 H),
3.80 (m, 4 H, 1,1Ј,3,4,5,6,7,8,8Ј-H), 3.56 (s, 6 H, OMe), 4.85 (AB,
2 H, CH2Bn), 7.37 (m, 5 H, CH Ph) ppm. 13C{1H} NMR (CDCl3):
δ ϭ Ϫ5.5, Ϫ5.4 (CH3, TBS), 18.1 (Cq tBu), 25.7 (CH3 tBu), 33.8
(C-2), 60.1, 60.5 (OMe), 61.1, 62.2 (C-1,8), 75.4 (CH2 Bn), 79.1,
79.6, 83.8, 86.6(C-3,4,5,6,7), 127.6, 127.9, 128.3 (CHarom), 138.5
(Cq,arom) ppm.
Azide 20: Azide 20 was synthesized from alcohol 19 as was de-
scribed for 13 in 85%: Rf ϭ 0.75 (light petroleum ether/EtOAc, 3:1,
v/v).1H NMR (CDCl3): δ ϭ 0.051 (s, 6 H, CH3 TBS), 0.89 (s, 9 H,
tBu), 1.65 (m, 1 H), 2.10 (m, 1 H, 2,2Ј-H), 2.86 (t, J ϭ 9.5 Hz, 1
H), 3.07Ϫ3.57 (m, 5 H), 3.81 (m, 3 H, 1,1Ј,3,4,5,6,7,8,8Ј-H), 3.57
(s, 6 H, OMe), 4.85 (AB, 2 H, CH2Bn), 7.32 (m, 5 H, CH Ph) ppm.
13C{1H} NMR (CDCl3): δ ϭ Ϫ5.6, Ϫ5.2 (CH3, TBS), 18.1 (Cq
tBu), 25.7 (CH3 tBu), 31.3 (C-2), 47.7 (C-1), 60.3, 60.7 (OMe), 61.9
(C-8), 75.2 (CH2 Bn), 75.5, 79.3, 79.4, 84.0, 86.6 (C-3,4,5,6,7),
127.5, 127.9, 128.2 (CHarom), 138.5 (Cq,arom) ppm.
β-Allyl-C-glucoside 17: β-Allyl-C-glycoside 16[12b] (7.15 g,
11.0 mmol) and pyrrole (4.58 mL, 66.0 mmol) were dissolved in
dichloromethane (250 mL) and trifluoroacetic acid (1.67 mL,
22.0 mmol) was added dropwise to the resulting solution. After
1 min, saturated aqueous NaHCO3 (100 mL) was added. The or-
ganic layer was extracted and washed with water, dried with
MgSO4 and the solvents were evaporated. Silica gel column chro-
matography (0Ϫ15% EtOAc in light petroleum ether) yielded pure
17 (4.039 g, 9.90 mmol, 90%). Rf ϭ 0.50 (light petroleum ether/
H-β Building Block 6: Desilylation of 20 was performed as de-
scribed for 5 to give 6 in 86% yield: Rf ϭ 0.25 (light petroleum
ether/EtOAc, 3:1, v/v). 1H NMR (CDCl3): δ ϭ 1.70 (m, 1 H, 2-H),
2.14 (m, 1 H, 2Ј-H), 1.87 (br. t, J ϭ 4.9 Hz, 1 H, OH), 2.90 (t, J ϭ
8.8 Hz, 1 H), 3.17Ϫ3.72 (m, 7 H), 3.87 (m, 1 H, 1,1Ј,3,4,5,6,7,8,8Ј-
H), 3.56 (s, 6 H, OMe), 4.85 (s, 2 H, CH2Bn), 7.38 (m, 5 H, CH
Ph) ppm. 13C{1H} NMR (CDCl3): δ ϭ 31.1 (C-2), 47.8 (C-1), 60.5,
60.8 (OMe), 61.7 (C-8), 75.0 (CH2Bn), 76.0, 78.9, 79.8, 83.9, 86.4
(C-3,4,5,6,7), 127.5, 127.8, 128.2 (CHarom), 138.4 (Cq,arom) ppm.
1
EtOAc, 3:1, v/v). H NMR (CDCl3): δ ϭ 0.10 (s, 3 H, CH3 TBS),
0.12 (s, 3 H, CH3 TBS), 0.92 (s, 9 H, tBu), 2.20 (m, 1 H, 3-H), 2.51
(m, 1 H, 3Ј-H), 3.28 (m, 4 H), 3.56 (m, 2 H) 3.80 (m, 1 H,
4,5,6,7,8,9,9Ј-H), 4.79 (AB, 2 H, CH2Bn), 5.08 (m, 2 H, 1, 1Ј-H),
5.83 (m, 1 H, 2-H), 7.36 (m, 10 H, CH Ph) ppm. 13C{1H} NMR
(CDCl3): δ ϭ Ϫ4.7, Ϫ3.9 (CH3, TBS), 17.9 (Cq tBu), 25.9 (CH3
tBu), 37.0 (C-3), 62.2 (C-9), 75.2 (CH2 Bn) 71.1, 73.6, 78.4, 80.1,
87.2 (C-4,5,6,7,8), 117.1 (C-1), 127.3, 127.6, 128.5 (CHarom), 134.3
(C-2), 138.7 (Cq,arom) ppm.
Disaccharide 22: C-Glycoside 5 (0.371 g, 0.869 mmol) and idose
donor 21[12] (0.411 g, 1.13 mmol) were dissolved in toluene (10 mL)
˚
and molecular sieves (4 A) were added. The suspension was cooled
to Ϫ20 °C and stirred for 30 min. A solution of N-iodosuccinimide
(0.391 g, 1.74 mmol) and TfOH (11µL, 0.130 mmol) in dioxane/
dichloromethane (1:1, 10 mL) was added dropwise. After 30 min,
β-Allyl-C-glucoside 18: β-Allyl-C-glucoside 17 was methylated as
described for 11. Yield: 93%. Rf ϭ 0.85 (light petroleum ether/ the reaction mixture was filtered through Celite into an aqueous
1
EtOAc, 4:1, v/v). H NMR (CDCl3): δ ϭ 0.00 (s, 3 H, CH3 TBS), NaHCO3/Na2S2O3 solution, diluted, extracted and washed with
0.051 (s, 3 H, CH3 TBS), 0.88 (s, 9 H, tBu), 2.26 (m, 1 H, 3-H), water. The organic layer was dried with MgSO4, filtered and con-
2.52 (m, 1 H, 3Ј-H), 2.91 (m, 1 H), 3.00Ϫ3.56 (m, 3 H), 3.81 (m, 3
centrated in vacuo to give the crude disaccharide, which was puri-
H, 4,5,6,7,8,9,9Ј-H), 3.56 (s, 6 H, OMe), 4.85 (AB, 2 H, CH2Bn), fied by silica gel column chromatography (10Ϫ20% EtOAc in light
5.07 (m, 2 H, 1,1Ј-H), 5.90 (m, 1 H, 2-H), 7.40 (m, 10 H, CH Ph) petroleum ether) to give pure 22 (0.608 g, 0.834 mmol, 96%): Rf ϭ
ppm. 13C{1H} NMR (CDCl3): δ ϭ Ϫ5.4, Ϫ5.1 (CH3, TBS), 18.3
0.60 (toluene/EtOAc, 1:1, v/v),1H NMR (CDCl3, HH-COSY): δ ϭ
(Cq tBu), 25.8 (CH3 tBu), 36.1 (C-3), 60.4, 60.7 (OMe), 62.1 (C-9), 1.89 (m, 2 H, 2H,2ЈH-H), 1.89 (s, 3 H, CH3 Ac), 2.00 (s, 3 H, CH3
75.4 (CH2Bn), 78.3, 78.8, 83.3, 86.9 (C-4,5,6,7,8), 116.8 (C-1), Ac), 2.07 (s, 3 H, CH3 Ac), 3.39 (m, 2 H, 1H,1ЈH-H), 3.40 (s, 3 H,
127.5, 128.0, 128.3 (CHarom), 134.7 (C-2), 138.8 (Cq,arom) ppm.
OMe), 3.46 (m, 1 H, 3G-H), 3.47 (m, 1 H, 4H-H), 3.49 (s, 3 H,
3960
Eur. J. Org. Chem. 2002, 3954Ϫ3965

Probing the HeparinϪAntithrombin III Interaction
FULL PAPER
OMe), 3.60 (m, 2 H), 3.69 (m, 2 H, 5H,6H,7H,8H-H), 3.90 (m, 3 H,
Pentasaccharide 25: GH acceptor 23 (87 mg, 0.12 mmol) and DEF
8ЈH,6G,6ЈG-H), 4.21 (m, 1 H, 3H-H), 4.54 (AB, 2 H, CH2 Bn), 4.59 donor 24[11] (166 mg, 0.16 mmol) were dissolved in dichlorome-
˚
(m, 1 H, 5G-H), 4.74 (m, 1 H, 4G-H), 4.80 (m, 1 H, 2G-H), 4.80 thane (2 mL) and molecular sieves (4 A) were added. The suspen-
(AB, 2 H, CH2 Bn), 4.98 (br. s, 1 H, 1G-H), 7.27Ϫ7.36 (m, 10 H,
CH Ph) ppm. 13C{1H} NMR (CDCl3): δ ϭ 20.5Ϫ20.7 (CH3 Ac),
24.6 (C-2H), 48.5 (C-1H), 58.2, 58.5 (CH3 OMe), 61.9, 68.7, 73.1,
sion was cooled to Ϫ20 °C and stirred for 30 min. A solution of
trimethylsilyl trifluoromethanesulfonate (3.3 µL, 18 µmol) in
dichloromethane (0.1 mL) was added dropwise. After 30 min, the
74.6 (CH2 Bn, C-6G,8H), 63.1, 65.9, 67.1, 70.3, 71.6, 73.4, 74.5, reaction mixture was filtered through Celite into an aqueous
79.5, 81.7 (C-3H,4H,5H,6H,7H,2G,3G,4G,5G), 96.6 (C-1G), 127.3, NaHCO3 solution, diluted, extracted and washed with water. The
128.1 (CHarom), 137.9, 138.6 (Cq,arom), 169.5, 170.1 (CϭO Ac) ppm. organic layer was dried with MgSO4, filtered and concentrated in
vacuo to give the crude pentasaccharide, which was purified by
silica gel column chromatography (0Ϫ10% acetone in dichlorome-
Disaccharide 23: Disaccharide 22 (0.250 g, 0.343 mmol) was dis-
solved in methanol/dioxane (1:1, 4 mL) and a catalytic amount of
KOtBu (4 mg) was added. The mixture was stirred for 3 h, neutral-
ized with Dowex-Hϩ, filtered and concentrated. The triol was co-
evaporated three times with toluene and dissolved in DMF/dime-
thoxypropane (5:1, 5 mL). A catalytic amount of p-toluenesulfonic
acid was added and the mixture was stirred for 1 h and sub-
sequently diluted with diethyl ether (20 mL), washed with saturated
aqueous NaHCO3 and water, dried (MgSO4), filtered and concen-
trated to give the crude acetonide [Rf ϭ 0.60 (toluene/EtOAc, 3:1,
v/v)], which was coevaporated three times with toluene, dissolved
in DMF (4 mL) and methylated with iodomethane (43 µL,
0.68 mmol) and sodium hydride (15 mg, 0.38 mmol) at 0 °C. The
mixture was diluted with diethyl ether (20 mL) and washed with
saturated aqueous NH4Cl (10 mL). The aqueous layer was ex-
tracted three times with diethyl ether and the organic phases were
combined, dried (MgSO4), filtered and concentrated. The residual
oil was dissolved in 80% aqueous acetic acid (4 mL), heated at 60
°C for 1 h and coevaporated with toluene (4 ϫ 5 mL). Silica gel
column chromatography (50Ϫ100% EtOAc in light petroleum
ether) yielded the pure diol [Rf ϭ 0.10 (toluene/EtOAc, 3:1, v/v)].
The diol was dissolved in dichloromethane (2.5 mL) and a catalytic
amount (1 mg) of 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)
was added, followed by a solution of potassium bromide (8 mg)
and tetrabutylammonium chloride (10 mg) in saturated aqueous
NaHCO3 (1.5 mL). The mixture was brought to 0 °C and a mixture
of 13% aqueous NaOCl (1.3 mL), saturated aqueous NaHCO3
thane) followed by passage through a column of Sephadex LH-20,
which was eluted with dichloromethane/methanol (1:1). The appro-
priate fractions were pooled to give pure 25 (148 mg, 0.093 mmol,
77%). Rf ϭ 0.40 (dichloromethane/acetone, 9:1, v/v). 1H NMR
(CDCl3, HH-COSY, HH-TOCSY): D: δ ϭ 3.05 (t, J4,3 ϭ J4,5
ϭ
9.1 Hz, 1 H, 4-H), 3.13 (dd, J2,1 ϭ 3.7, J2,3 ϭ 9.8 Hz, 1 H, 2-H),
3.33 (t, J3,2 ϭ J3,4 ϭ 9.4 Hz, 1 H, 3-H), 3.43 (m, 1 H, 5-H), 4.24
(m, 1 H, 6-H), 4.31 (m, 1 H, 6Ј-H), 5.54 (d, J1,2 ϭ 3.7 Hz, 1-H)
ppm; E: 2.88 (t, J2,1 ϭ J2,3 ϭ 8.1 Hz, 1 H, 2-H), 3.30 (t, J3,2
ϭ
J3,4 ϭ 9.0 Hz, 1 H, 3-H), 3.84 (d, J5,4 ϭ 9.8 Hz, 1 H, 5-H), 3.93 (t,
J4,3 ϭ J4,5 ϭ 9.2 Hz, 1 H, 4-H), 4.10 (d, J1,2 ϭ 7.9 Hz, 1 H, 1-H)
ppm; F: 3.41 (m, 1 H, 2-H), 3.56 (m, 1 H, 4-H), 4.02 (m, 1 H, 5-
H), 4.21 (m, 1 H, 6-H), 4.45 (m, 1 H, 6Ј-H), 5.17 (d, J1,2 ϭ 4.0 Hz,
1 H, 1-H), 5.38 (t, J3,2 ϭ J3,4 ϭ 9.6 Hz, 1 H, 3-H) ppm; G: 2.97 (t,
J2,1 ϭ J2,3 ϭ 6.8 Hz, 1 H, 2-H), 3.66 (t, J3,2 ϭ J3,4 ϭ 8.0 Hz, 1 H,
3-H), 3.87 (dd, J4,3 ϭ 8.1, J4,5 ϭ 6.1 Hz, 1 H, 4-H), 4.47 (d, J5,4
ϭ
6.0 Hz, 1 H, 5-H), 5.26 (d, J1,2 ϭ 6.0 Hz, 1 H, 1-H) ppm; H: 1.83
(m, 1 H, 2-H), 1.93 (m, 1 H, 2Ј-H), 3.37 (m, 3 H, 1, 1Ј, 4Ј-H), 3.61
(m, 2 H, 5, 7-H), 3.70 (m, 2 H, 8, 8Ј-H), 3.78 (t, J6,5 ϭ J6,7
ϭ
8.4 Hz, 1 H, 6-H), 4.16 (m, 1 H, 3-H) ppm; OMe: 3.21, 3.38, 3.44,
3.45, 3.49, 3.52, 3.53, 3.55 (8 ϫ s, 3 H); Ac: 1.90, 2.01, 2.12 (3 ϫ
s, 3 H) ppm; CH2 Bn: 4.57, 4.60, 4.72, 5.11, 5.14 (5 ϫ AB, 2 H)
ppm; CH Ph: δ ϭ 7.73 (m, 25 H) ppm. ES-MS: m/z ϭ 1621.0 [M
ϩ Na]ϩ.
Pentasaccharide 26: Fully protected pentamer 25 (89 mg,
(0.76 mL) and saturated aqueous NaCl (1.5 mL) was added drop- 0.056 mmol) was dissolved in tert-butyl alcohol/water (2:1, 3 mL)
wise over a period of 2 h after which the mixture was stirred for
another hour. The layers were separated, and the dichloromethane
fraction was extracted three times with water. The aqueous phases
were combined, brought to pH ϭ 1 with 1  HCl and extracted
five times with EtOAc. The organic phases were dried (MgSO4)
and two drops of acetic acid and 10% palladium on carbon
(100 mg) were added. The heterogeneous mixture was stirred under
hydrogen for 16 h, after which the catalyst was filtered off and the
solvents were evaporated. The resulting oil was taken up in a mix-
ture of 0.4  NaOH (2.6 mL) and methanol (0.9 mL) and stirred
and concentrated to give the crude acid, which was transformed overnight. The reaction mixture was neutralized with 1  HCl and
into its benzyl ester by treatment with benzyl bromide (0.12 mL, concentrated to 1 mL. The solution was passed through a Toyope-
1.01 mmol) and KHCO3 (69 mg, 0.69 mmol) in DMF (5 mL) for arl HW40S column, which was eluted with acetonitrile/water (1:9).
2 h. The reaction mixture was diluted with EtOAc (20 mL), washed
with water, dried (MgSO4), filtered and concentrated to dryness.
Silica gel column chromatography (50Ϫ100% EtOAc in light petro-
leum ether) yielded pure disaccharide 23 (0.121 mg, 0.168 mmol,
The appropriate fractions were pooled and lyophilized to give the
fully deprotected pentamer 26 as a fluffy white solid (43 mg,
0.043 mmol, 77%). Rf ϭ 0.20 (EtOAc/pyridine/acetic acid/water,
8:7:1.6:4 v/v/v/v). 1H NMR (D2O, HH-COSY, HH-TOCSY): D:
49%): Rf ϭ 0.85 (EtOAc).1H NMR (CDCl3, HH-COSY): δ ϭ 1.65 δ ϭ 3.27 (m, 1 H, 4-H), 3.29 (m, 1 H, 2-H), 3.51 (m, 1 H, 3-H),
(br. s, 1 H, OH), 1.83 (m, 1 H, 2H-H), 1.92 (m, 1 H, 2ЈH-H), 3.23 3.67 (m, 1 H, 5-H), 3.72 (m, 2 H, 6, 6Ј-H), 5.44 (d, J1,2 ϭ 3.8 Hz,
(m, 1 H, 2G-H), 3.24 (s, 3 H, OMe),3.35 (s, 3 H, OMe), 3.39 (m, 3
1-H) ppm; E: 3.23 (m, 1 H, 2-H), 3.51 (m, 1 H, 3-H), 3.73 (m, 1
H, 1H,1ЈH,4H-H), 3.41 (s, 3 H, OMe), 3.52 (m, 1 H, 3G-H), 3.65 (m, H, 5-H), 3.81 (m, 1 H, 4-H), 4.53 (d, J1,2 ϭ 7.8 Hz, 1-H) ppm; F:
4 H), 3.87 (m, 1 H, 5H,6H,7H,8H,8ЈH-H), 3.99 (br. d, J ϭ 10.1 Hz, 1 3.47 (m, 1 H, 2-H), 3.56 (m, 1 H, 4-H), 3.74 (m, 1 H, 3-H), 3.78
H, 4G-H), 4.15 (m, 1 H, 3H-H), 4.57 (s, 2 H, CH2 Bn), 4.73 (AB, 2 (m, 1 H, 6-H), 3.84 (m, 2 H, 5, 6Ј-H), 5.10 (d, J1,2 ϭ 3.9 Hz, 1-H);
H, CH2 Bn), 4.87 (br. s, 1 H, 5G-H), 5.01 (AB, 2 H, CH2 Bn), 5.16 G: 3.43 (m, 1 H, 2-H), 3.72 (m, 1 H, 3-H), 4.17 (m, 1 H, 4-H), 4.62
(br. s, 1 H, 1G-H), 7.26Ϫ7.34 (m, 15 H, CH Ph) ppm. 13C{1H}
(d, J5,4 ϭ 2.4 Hz, 1 H, 5-H), 5.01 (d, J1,2 ϭ 2.1 Hz, 1 H, 1-H) ppm;
NMR (CDCl3): δ ϭ 25.1 (C-2H), 47.9 (C-1H), 58.1, 58.2, 58.4 H: 1.89 (m, 1 H, 2-H), 2.10 (m, 1 H, 2Ј-H), 3.09 (m, 2 H, 1, 1Ј-H),
(OMe), 66.3, 69.1, 73.5, 74.0 (CH2 Bn, C-8H), 67.1, 68.4, 69.9, 71.8, 3.40 (m, 1 H, 4-H), 3.59 (m, 1 H, 6-H), 3.62 (m, 1 H, 7-H), 3.75
74.5, 75.6, 76.0, 78.8, 81.2 (C-3H,4H,5H,6H,7H,2G,3G,4G,5G), 97.2 (m, 2 H, 5, 8-H), 3.81(m, 1 H, 8Ј-H), 4.32 (m, 1 H, 3-H) ppm;
(C-1G), 127.2Ϫ128.3 (CHarom), 137.8, 138.5 (Cq,arom), 169.4 (Cϭ OMe: 3.44, 3.47, 3.50, 3.51, 3.54, 3.60, 3.61, 3.62 (8 ϫ s, 3 H) ppm.
O) ppm.
ES-MS: m/z ϭ 996.3 [M ϩ H]ϩ.
Eur. J. Org. Chem. 2002, 3954Ϫ3965
3961

´
J. D. C. Codee, G. A. van der Marel, C. A. A. van Boeckel, J. H. van Boom
FULL PAPER
Pentasaccharide 27: Pentamer 26 (46 mg, 0.046 mmol) was dis-
solved in DMF/water (1:4, 1.25 mL). N-(Benzyloxycarbonyloxy)s-
in 2 mL of acetonitrile/water (1:9) and passed through a column of
Toyopearl HW40S, which was eluted with acetonitrile/water (1:9).
uccinimide (17 mg, 0.069) and N-methylmorpholine (17 µL, The product fractions were pooled, concentrated and passed
0.138 mmol) were added and the mixture was stirred for 30 min,
concentrated to dryness and taken up in 2 mL of acetonitrile/water
(1:9). The resulting mixture was chromatographed on a Toyopearl
HW40S column, which was eluted with acetonitrile/water (1:9).
The product fractions were pooled and lyophilized. The resulting
pentamer (47 mg) was purified by preparative HPLC (elution was
effected by applying a gradient of 25Ϫ30% acetonitrile) to give pure
27 (26 mg, 0.023 mmol, 50%). Rf ϭ 0.40 (EtOAc/pyridine/acetic
through a column of Dowex 50 WX-4 (Naϩ form). The eluate was
lyophilized to give target pentasaccharide 3b (3.3 mg, 2.0 µmol,
100%). 1H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.28 (m,
2 H, 2, 4-H), 3.50 (m, 1 H, 3-H), 3.83 (m, 1 H, 5-H), 4.05 (m, 1 H,
6-H), 4.24 (m, 1 H, 6Ј-H), 5.42 (d, J1,2 ϭ 3.7 Hz, 1-H); E: 3.20 (t,
J2,1 ϭ J2,3 ϭ 8.0 Hz, 1 H, 2-H), 3.48 (m, 1 H, 3-H), 3.67 (d, J5,4
ϭ
9.8 Hz, 1 H, 5-H), 3.84 (m, 1 H, 4-H), 4.61 (d, J1,2 ϭ 7.8 Hz, 1 H,
1-H); F: 3.92 (t, J4,3 ϭ J4,5 ϭ 9.6 Hz, 1 H, 4-H), 4.12 (m, 1 H, 5-
acid/water 8:7:1.6:4 v/v/v/v). 1H NMR (D2O, HH-COSY, HH- H), 4.24 (m, 1 H, 6-H), 4.25 (m, 1 H, 2-H), 4.36 (m, 1 H, 6Ј-H),
TOCSY): D: δ ϭ 3.26 (t, J4,3 ϭ J4,5 ϭ 9.8 Hz, 1 H, 4-H), 3.30 (dd, 4.51 (t, J3,2 ϭ J3,4 ϭ 9.6 Hz, 1 H, 3-H), 5.34 (d, J1,2 ϭ 3.6 Hz, 1
J2,1 ϭ 3.8, J2,3 ϭ 10.0 Hz, 1 H, 2-H), 3.43 (m, 2 H, 3, 5-H), 3.69
H, 1-H); G: 3.39 (br. s, 1 H, 2-H), 3.80 (br. s, 1 H, 3-H), 4.12 (m,
1 H, 4-H), 4.48 (br. s, 1 H, 5-H), 5.08 (br. s, 1 H, 1-H); H: 1.86 (m,
1 H, 2-H), 2.06 (m, 1 H, 2Ј-H), 3.16 (m, 2 H, 1, 1Ј-H), 3.39 (m, 1
H, 4-H), 3.88 (m, 1 H, 6-H), 4.04 (m, 1 H, 8-H), 4.07 (m, 1 H, 3-
(m, 2 H, 6, 6Ј-H), 5.47 (d, J1,2 ϭ 3.7 Hz, 1-H); E: 3.23 (dd, J2,1
7.9, J2,3 ϭ 9.1 Hz, 1 H, 2-H), 3.55 (m, 1 H, 3-H), 3.87 (t, J4,3
ϭ
ϭ
J4,5 ϭ 9.0 Hz, 1 H, 5-H), 4.04 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 4.59
(d, J1,2 ϭ 7.8 Hz, 1-H); F: 3.48 (m, 1 H, 2-H), 3.58 (m, 1 H, 4-H), H), 4.33 (m, 1 H, 7-H), 4.58 (m, 1 H, 8Ј-H), 4.75 (m, 1 H, 5-H);
3.65 (m, 1 H, 5-H), 3.70 (m, 1 H, 3-H), 3.80 (m, 2 H, 6, 6Ј-H), 5.13 OMe: 3.48, 3.48, 3.51, 3.53, 3.57, 3.58, 3.58, 3.59 (8 ϫ s, 3 H). ESI-
(d, J1,2 ϭ 3.8 Hz, 1-H); G: 3.44 (m, 1 H, 2-H), 3.73 (m, 1 H, 3-H),
4.18 (m, 1 H, 4-H), 5.02 (br. s, 1 H, 5-H), 5.03 (br. s, 1 H, 1-H);
H: 1.62 (m, 1 H, 2-H), 1.84 (m, 1 H, 2Ј-H), 3.18 (m, 2 H, 1, 1Ј-H),
3.33 (m, 1 H, 4-H), 3.58 (m, 2 H, 6, 7-H), 3.64 (br. s, 1 H, 8-H),
3.66 (m, 1 H, 5-H), 3.76 (m, 1 H, 8Ј-H), 4.22 (m, 1 H, 3-H); OMe:
3.34, 3.44, 3.47, 3.52, 3.52, 3.58, 3.58, 3.61 (8 ϫ s, 3 H); CH2 Bn:
5.07 (m, 2 H); CH Ph: 7.39 (m, 5 H); ES-MS: m/z ϭ 1167.0 [M
ϩ Na]ϩ.
MS: m/z ϭ 738.0 [M Ϫ 2 H]2Ϫ, 491.0 [M Ϫ 3 H]3Ϫ
.
Pentasaccharide 28: Pentamer 26 (24 mg, 24 µmol) and 2,3-bis(Z-
amino)propionic acid succinimidyl ester (70 mg, 145 µmol) were
dissolved in DMF (1 mL) and N-methylmorpholine (9.2 µL, 72
µmol) was added. The mixture was stirred for 30 min, concentrated
to dryness, taken up in 2 mL of acetonitrile/water (9:1) and applied
to a Toyopearl HW40S column, which was eluted with the same
solvent system. The appropriate fractions were collected and lyo-
philized. The resulting pentamer (24 mg) was purified by preparat-
ive HPLC (elution was effected applying a gradient of 30Ϫ35%
acetonitrile) to give pentasaccharide 28 as a white fluffy solid
Pentasaccharide 3d: Pentamer 27 (8.4 mg, 7.4 µmol) was dissolved
in water (1 mL) and passed through a column of Dowex 50 WX-
4Hϩ. The eluate was concentrated, lyophilized, coevaporated thrice
with DMF and dissolved in DMF (1 mL). Triethylamine sulfur tri-
(12 mg, 50%). Rf
ϭ 0.40 (EtOAc/pyridine/acetic acid/water,
oxide complex (5 equiv. for each hydroxy function, 40 mg, 8:7:1.6:4 v/v/v/v). 1H NMR (D2O, H-HCOSY, HH-TOCSY): D:
0.022 mmol) was added under nitrogen. The flask was sealed and
heated at 55 °C overnight. A solution of NaHCO3 (4 equiv. for
each triethylamineϪsulfur trioxide complex, 75 mg, 0.089 mmol) in
1.5 mL of water was added at 0 °C, and the resulting mixture was
δ ϭ 3.27 (t, J3,2 ϭ J3,4 ϭ 9.7 Hz, 1 H, 4-H), 3.30 (m, 1 H, 2-H),
3.49 (m, 2 H, 3, 5-H), 3.72 (m, 2 H, 6, 6Ј-H), 5.46 (d, J1,2 ϭ 3.7 Hz,
1-H); E: 3.24 (m, 1 H, 2-H), 3.55 (m, 1 H, 3-H), 3.87 (t, J4,3
ϭ
J4,5 ϭ 9.1 Hz, 1 H, 4-H), 3.96 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 4.59
stirred for 1 h, after which it was concentrated to a small volume (d, J1,2 ϭ 7.8); F: 3.49 (m, 1 H, 2-H), 3.60 (m, 1 H, 4-H), 3.72 (m,
and applied to a Toyopearl HW40S column, which was eluted with 2 H, 3, 5-H), 3.82 (m, 2 H, 6, 6Ј-H), 5.14 (d, J1,2 ϭ 3.8 Hz, 1-H);
acetonitrile/water (1:9). The product fractions were pooled, concen- G: 3.44 (br. s, 1 H, 2-H), 3.72 (m, 1 H, 3-H), 4.19 (m, 1 H, 4-H),
trated and passed through a column of Dowex 50 WX-4 (Naϩ
4.98 (br. s, 1 H, 5-H), 5.03 (br. s, 1-H); H: 1.67 (m, 1 H, 2-H), 1.79
(m, 1 H, 2Ј-H), 3.16 (m, 1 H, 1-H), 3.28 (m, 1 H, 1Ј-H), 3.32 (m,
form). The eluate was lyophilized to give target pentasaccharide 3d
(13.2 mg, 100%). 1H NMR (D2O, HH-COSY, HH-TOCSY): D: 1 H, 4-H), 3.58 (m, 2 H, 6, 7-H), 3.65 (m, 1 H, 5-H), 3.72 (m, 1 H,
δ ϭ 3.32 (m, 2 H, 2, 4-H), 3.51 (m, 1 H, 3-H), 3.86 (m, 1 H, 5-H), 8-H), 3.82 (m, 1 H, 8Ј-H), 4.19 (m, 1 H, 3); OMe: 3.33, 3.46, 3.47,
4.10 (m, 1 H, 6-H), 4.27 (m, 1 H, 6Ј-H), 5.45 (d, J1,2 ϭ 3.7 Hz, 1-
3.53, 3.54, 3.60, 3.60, 3.62 (8 ϫ s, 3 H), CH2 Bn: 5.07 (m, 4 H);
CH Ph: 7.42 (m, 10 H), H-α: 4.19 (m, 1 H); H-β: 3.36 (m, 1 H);
H); E: 3.26 (m, 1 H, 2-H), 3.51 (m, 1 H, 3-H), 3.71 (d, J5,4
ϭ
9.8 Hz, 1 H, 5-H), 3.87 (m, 1 H, 4-H), 4.65 (d, J1,2 ϭ 7.9 Hz, 1 H, H-βЈ: 3.49 (m, 1 H). ES-MS: m/z ϭ 1350.7 [M ϩ H]ϩ.
1-H); F: 3.98 (m, 1 H, 4-H), 4.18 (m, 1 H, 5-H), 4.26 (m, 1 H, 6-
Pentasaccharide 3e: Pentamer 28 was sulfated as described for 27.
1H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.30 (m, 2 H,
2, 4-H), 3.50 (m, 1 H, 3-H), 3.85 (m, 1 H, 5-H), 4.07 (m, 1 H, 6-
H), 4.24 (m, 1 H, 6Ј-H), 5.43 (d, J1,2 ϭ 3.7 Hz, 1-H); E: 3.22 (m,
1 H, 2-H), 3.49 (m, 1 H, 3-H), 3.69 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-H),
3.85 (m, 1 H, 4-H), 4.62 (d, J1,2 ϭ 7.9 Hz, 1 H, 1-H); F: 3.94 (t,
J4,3 ϭ J4,5 ϭ 9.6 Hz, 1 H, 4-H), 4.14 (m, 1 H, 5-H), 4.25 (m, 1 H,
6-H), 4.28 (dd, J2,1 ϭ 3.5, J2,3 ϭ 10.1 Hz, 1 H, 2-H), 4.38 (m, 1 H,
6Ј-H), 4.51 (m, 1 H, 3-H), 5.36 (d, J1,2 ϭ 3.3 Hz, 1 H, 1); G: 3.30
(m, 1 H, 2-H), 3.75 (br. s, 1 H, 3-H), 4.14 (m, 1 H, 4-H), 4.51 (m,
1 H, 5-H), 5.03 (br. s, 1 H, 1-H); H: 1.66 (m, 1 H, 2-H), 1.80 (m,
1 H, 2Ј-H), 3.16 (m, 1 H, 1-H), 3.30 (m, 1 H, 4-H), 3.34 (m, 1 H,
1Ј-H), 3.85 (m, 2 H, 6-H), 3.92 (m, 1 H, 3-H), 4.09 (m, 1 H, 8-H),
4.24 (m, 1 H, 7-H), 4.51 (m, 1 H, 8Ј-H), 4.71 (br. s, 1 H, 5-H);
OMe: 3.38, 3.51, 3.52, 3.54, 3.56, 3.60, 3.61, 3.62 (8 ϫ s, 3 H); CH2
Bn: 5.06 (m, 4 H); CH Ph: 7.37 (m, 10 H); H-α: 4.21 (m, 1 H); H-
H), 4.31 (dd, J2,1 ϭ 3.6, J2,3 ϭ 10.1 Hz, 1 H, 2-H), 4.39 (m, 1 H,
6Ј-H), 4.54 (m, 1 H, 3-H), 5.40 (d, J1,2 ϭ 3.6 Hz, 1 H, 1-H); G:
3.44 (br. s, 1 H, 2-H), 3.79 (t, J3,2 ϭ J3,4 ϭ 3.5 Hz, 1 H, 3-H), 4.18
(m, 1 H, 4-H), 4.54 (br. s, 1 H, 5-H), 5.09 (br. s, 1 H, 1-H); H: 1.73
(m, 1 H, 2-H), 1.82 (m, 1 H, 2Ј-H), 3.23 (m, 2 H, 1, 1Ј-H), 3.32
(m, 1 H, 4-H), 3.93 (m, 1 H, 6-H), 3.98 (m, 1 H, 3-H), 4.10 (m, 1
H, 8-H), 4.26 (m, 1 H, 7-H), 4.54 (m, 1 H, 8Ј-H), 4.75 (m, 1 H, 5-
H); OMe: 3.40, 3.51, 3.52, 3.54, 3.56, 3.60, 3.61, 3.62 (8 ϫ s, 3 H);
CH2 Bn: 5.10 (AB, 2 H); CH Ph: 7.41 (m, 5 H). ESI-MS: m/z ϭ
803.8 [M Ϫ 2 H]2Ϫ, 535.8 [M Ϫ 3 H]3Ϫ, 402.0 [M Ϫ 4 H]4Ϫ
.
Pentasaccharide 3b: Pentamer 3d (3.6 mg, 2.0 µmol) was dissolved
in 1 mL of water and one drop of acetic acid and 10% palladium
on carbon (5 mg) were added. The heterogeneous mixture was
stirred under hydrogen for 5 h, after which the catalyst was filtered
off and the solvents were evaporated. The pentamer was taken up
3962
Eur. J. Org. Chem. 2002, 3954Ϫ3965

Probing the HeparinϪAntithrombin III Interaction
FULL PAPER
β: 3.38 (m, 1 H); H-βЈ: 3.52 (m, 1 H). ESI-MS: m/z ϭ 914.4 [M Ϫ
(t, J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H), 4.08 (m, 1 H, 5-H), 4.22 (m, 1
H, 6-H), 4.53 (m, 1 H, 6Ј-H), 5.12 (1 H, J1,2 ϭ 3.7 Hz, 1 H, 1-H),
5.39 (t, J3,2 ϭ J3,4 ϭ 9.7 Hz, 1 H, 3-H); G: 3.11 (dd, J2,1 ϭ 5.7,
J2,3 ϭ 6.9 Hz, 1 H, 2-H), 3.68 (t, J3,2 ϭ J3,4 ϭ 6.9 Hz, 1 H, 3-H),
3.96 (dd, J4,3 ϭ 6.9, J4,5 ϭ 5.2 Hz, 1 H, 4-H), 4.64 (d, J5,4 ϭ 5.2 Hz,
1 H, 5-H), 5.10 (d, J1,2 ϭ 5.7 Hz, 1 H, 1-H); H: 1.60 (m, 1 H, 2-
H), 2.00 (m, 1 H, 2Ј-H), 2.85 (t, J4,3 ϭ J4,5 ϭ 9.2 Hz, 1 H, 4-H),
3.10 (t, J6,5 ϭ J6,7 ϭ 9.2 Hz, 1 H, 6-H), 3.17 (dt, J3,4 ϭ 9.2 Hz J3,2/
2 H]2Ϫ, 609.2 [M Ϫ 3 H]3Ϫ
.
Pentasaccharide 3c: Pentamer 3e was deprotected as described for
1
3d: H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.32 (m, 2
H, 2, 4-H), 3.51 (m, 1 H, 3-H), 3.83 (m, 1 H, 5-H), 4.06 (m, 1 H,
6-H), 4.25 (m, 1 H, 6Ј-H), 5.44 (d, J1,2 ϭ 3.3 Hz, 1-H); E: 3.22 (t,
J2,1 ϭ J2,3 ϭ 8.1 Hz, 1 H, 2-H), 3.50 (m, 1 H, 3-H), 3.69 (d, J5,4
ϭ
9.8 Hz, 1 H, 5-H), 3.85 (m, 1 H, 4-H), 4.63 (d, J1,2 ϭ 7.8 Hz, 1 H,
1); F: 3.95 (t, J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H), 4.15 (m, 1 H, 5-H),
4.25 (m, 1 H, 6-H), 4.30 (m, 1 H, 2-H), 4.38 (m, 1 H, 6Ј-H), 4.52
(m, 1 H, 3-H), 5.37 (d, J1,2 ϭ 3.6 Hz, 1 H, 1-H); G: 3.42 (m, 1 H,
2-H), 3.80 (m, 1 H, 3-H), 4.15 (br. s, 1 H, 4-H), 4.52 (br. s, 1 H, 5-
H), 5.10 (br. s, 1 H, 1-H); H: 1.76 (m, 1 H, 2-H), 1.88 (m, 1 H, 2Ј-
H), 3.34 (m, 1 H, 1-H), 3.37 (m, 2 H, 1Ј, 4-H), 3.90 (m, 1 H, 6-H),
4.00 (m, 1 H, 3-H), 4.06 (m, 1 H, 8-H), 4.30 (m, 1 H, 7-H), 4.56
(m, 1 H, 8Ј-H), 4.74 (m, 1 H, 5-H); OMe: 3.42, 3.49, 3.49, 3.52,
3.54, 3.58, 3.60, 3.60 (8 ϫ s, 3 H); H-α: 3.53 (m, 1 H); H-β: 2.85
(m, 1 H); H-βЈ: 2.97 (m, 1 H). ESI-MS: m/z ϭ 1560.2 [M Ϫ H]Ϫ.
ϭ 2.4 Hz, 1 H, 3-H), 3.26 (ddd, J7,6 ϭ 10.0, J7,8 ϭ 2.0, J7,8Ј
5.7 Hz, 1 H, 7-H), 3.36 (m, 3 H, 1, 1Ј, 4-H), 3.44 (t, J5,4 ϭ J5,6
9.2 Hz, 1 H, 5-H), 3.57 (m, 1 H, 8-H), 3.96 (m, 1 H, 8Ј-H); OMe:
3.36, 3.42, 3.48, 3.49, 3.50, 3.52, 3.54, 3.55 (9 ϫ s, 3 H); Ac: 1.91,
2.08, 2.11 (3 ϫ s, 3 H); CH2 Bn: 4.61, 4.83, 5.12, 5.29 (5 ϫ AB, 2
H); CH Ph: 7.40 (m, 20 H); ES-MS: m/z ϭ 1546.0 [M ϩ Na]ϩ.
ϭ
2Ј
ϭ
Pentasaccharide 32: Pentamer 32 was obtained from 31 in 80%
yield. Rf ϭ 0.20 (EtOAc/pyridine/acetic acid/water, 8:7:1.6:4 v/v/v/
1
v). H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.26 (m, 2
H, 2, 4-H), 3.52 (m, 1 H, 3-H), 3.64 (m, 1 H, 5-H), 3.71 (m, 2 H,
6, 6Ј-H), 5.44 (d, J1,2 ϭ 3.8 Hz, 1-H); E: 3.21(m, 1 H, 2-H), 3.52
(m, 1 H, 3-H), 3.81 (m, 2 H, 4, 5-H), 4.55 (d, J1,2 ϭ 8.1 Hz, 1-H);
F: 3.50 (m, 1 H, 2-H), 3.57 (m, 1 H, 4-H), 3.72 (m, 1 H, 3-H), 3.81
(m, 3 H, 5, 6, 6Ј-H), 5.12 (d, J1,2 ϭ 3.8 Hz, 1-H); G: 3.45 (m, 1 H,
2-H), 3.71 (m, 1 H, 3-H), 4.14 (br. s, 1 H, 4-H), 4.54 (br. s, 1 H, 5-
H), 5.00 (d, J1,2 ϭ 3.8 Hz, 1-H); H: 1.77 (m, 1 H, 2-H), 2.11 (m, 1
H, 2Ј-H), 2.94 (t, J4,3 ϭ J4,5 ϭ 9.4 Hz, 1 H, 4-H), 3.11 (m, 2 H, 1,
1Ј-H), 3.26 (m, 1 H, 6-H), 3.36 (m, 2 H, 3, 7-H), 3.53 (m, 1 H, 5-
H), 3.90 (m, 2 H, 8,8Ј-H); OMe: 3.45, 3.50, 3.51, 3.53, 3.53, 3.55,
3.59, 3.60, 3.61 (9 ϫ s, 3 H); ES-MS: m/z ϭ 1011.0 [M ϩ H]ϩ.
Disaccharide 29: The H-β anomer 29 was synthesized from 6 and
21 as described for 22 (88% yield): Rf ϭ 0.60 (toluene/EtOAc, 1:1
v/v). 1H NMR (CDCl3, HH-COSY): δ ϭ 1.65 (m, 1 H, 2H-H), 2.07
(s, 3 H, CH3 Ac), 2.08 (m, 1 H, 2ЈH-H), 2.09 (s, 3 H, CH3 Ac), 2.12
(s, 3 H, CH3 Ac), 2.88 (t, J4,3 ϭ J4,5 ϭ 9.2 Hz, 1 H, 4H-H), 3.10 (t,
J6,5 ϭ J6,7 ϭ 8.6 Hz, 1 H, 6H-H), 3.24 (dt, J3,2:2Ј ϭ 2.5, J3,4
ϭ
9.6 Hz, 3H-H), 3.39 (m, 3 H, 1H,1ЈH,7H-H), 3.47 (m, 1 H, 5H-H),
3.49 (s, 3 H, OMe), 3.53 (s, 3 H, OMe), 3.56 (s, 3 H, OMe), 3.65
(dd, J8,7 ϭ 6.1, J8,8Ј ϭ 11.2 Hz, 1 H, 8H-H), 3.91 (dd, J8Ј,7 ϭ 2.0,
J8Ј,8 ϭ 11.2 Hz, 1 H, 8ЈH-H), 4.19 (dd, J6,6Ј ϭ 2.5, J6/6Ј,5 ϭ 6.7 Hz,
2 H, 6G,6ЈG-H), 4.45 (dt, J5,4 ϭ 1.8, J5,6/6Ј ϭ 6.5 Hz, 5G-H), 4.84
(m, 1 H, 4G-H), 4.84 (s, 2 H, CH2 Bn), 4.89 (m, 1 H, 2G-H), 4.90
(m, 1 H, 1G-H), 7.27Ϫ7.41 (m, 5 H, CH Ph) ppm. 13C{1H} NMR
(CDCl3): δ ϭ 20.32 (CH3 Ac), 30.75 (C-2H), 47.22 (C-1H), 57.62,
60.05, 60.38 (CH3 OMe), 62.08, 66.81, 74.81 (CH2 Bn, C-6G,8H),
63.05, 66.26, 74.15, 75.33, 77.87, 80.15, 83.64, 86.16 (C-
3H,4H,5H,6H,7H,2G,3G,4G,5G), 97.80 (C-1G), 127.19, 127.49, 127.92
(CHarom), 138.17 (Cq,arom), 168.91, 168.92, 169.52 (CϭO Ac).
Pentasaccharide 33: Pentasaccharide 33 was prepared as described
for 27 and purified by HPLC analysis (elution was effected apply-
ing a gradient of 25Ϫ30% acetonitrile). Rf ϭ 0.40 (EtOAc/pyridine/
acetic acid/water, 8:7:1.6:4 v/v/v/v). 1H NMR (D2O, HH-COSY,
HH-TOCSY): D: δ ϭ 3.27 (m, 1 H, 4-H), 3.31 (dd, J2,1 ϭ 3.8,
J2,3 ϭ 10.0 Hz, 1 H, 2-H), 3.46 (m, 2 H, 3, 5-H), 3.71 (m, 2 H, 6,
6Ј-H), 5.47 (d, J1,2 ϭ 3.8 Hz, 1-H); E: 3.25 (m, 1 H, 2-H), 3.56 (m,
1 H, 3-H), 3.88 (m, 1 H, 4-H), 4.06 (d, J5,4 ϭ 9.2 Hz, 1 H, 5-H),
4.61 (d, J1,2 ϭ 7.8 Hz, 1 H, 1-H); F: 3.48 (m, 1 H, 2-H), 3.58 (m,
1 H, 4-H), 3.67 (m, 1 H, 5-H), 3.73 (m, 1 H, 3-H), 3.79 (m, 2 H,
6, 6Ј-H), 5.13 (1 H, J1,2 ϭ 3.8 Hz, 1 H, 1); G: 3.46 (m, 1 H, 2-H),
3.73 (m, 1 H, 3-H), 4.14 (br. s, 1 H, 4-H), 4.81 (br. s, 1 H, 5-H),
5.05 (br. s, 1 H, 1-H); H: 1.53 (m, 1 H, 2-H), 1.92 (m, 1 H, 2Ј-H),
2.91 (t, J4,3 ϭ J4,5 ϭ 9.3 Hz, 1 H, 4-H), 3.20 (m, 4 H, 1, 1Ј, 3, 6-
H), 3.27 (m, 1 H, 7-H), 3.53 (m, 1 H, 5-H), 3.73 (m, 2 H, 8,8Ј-H);
OMe: 3.42, 3.46, 3.48, 3.52, 3.52, 3.53, 3.58, 3.59, 3.61 (9 ϫ s, 3
H); CH2 Bn: 5.08 (AB, 2 H); CH Ph: 7.40 (m, 5 H); ES-MS:
m/z ϭ 1144.8 [M ϩ H]ϩ.
Disaccharide 30: Disaccharide 30 was obtained from 29 (50%
yield): Rf ϭ 0.85 (EtOAc). 1H NMR (CDCl3, H-HCOSY): δ ϭ
1.51 (m, 1 H, 2H-H), 1.97 (m, 1 H, 2ЈH-H), 2.81 (t, J4,3 ϭ J4,5
ϭ
9.2 Hz, 1 H, 4H-H), 3.13 (m, 2 H, 3H,6H-H), 3.29 (m, 3 H, 1H,
1ЈH,7H-H), 3.38 (m, 1 H, 2G-H), 3.44 (s, 3 H, OMe), 3.44 (m, 1 H,
5H-H), 3.46 (s, 3 H, OMe), 3.50 (s, 3 H, OMe), 3.52 (s, 3 H, OMe),
3.58 (m, 1 H, 3G-H), 3.75 (dd, J8,7 ϭ 5.4, J8,8Ј ϭ 11.4 Hz, 1 H, 8H-
H), 3.94 (dd, J8Ј,7 ϭ 1.8, J8Ј, 8 ϭ 11.4 Hz, 1 H, 8ЈH-H), 4.03 (m, 1
H, 4G-H), 4.80 (d, J5,4 ϭ 1.6 Hz, 1 H, 5G-H), 4.83 (s, 2 H, CH2
Bn), 5.09 (br. s, 1 H, 1G-H), 5.26 (AB, 2 H, CH2 Bn), 7.26Ϫ7.34
(m, 10 H, CH Ph) ppm. 13C{1H} NMR (CDCl3): δ ϭ 31.22 (C-
2H), 47.57 (C-1H), 57.80, 58.36, 60.47, 60.86 (OMe), 66.65, 67.51,
75.30 (CH2 Bn, C-8H), 67.85, 68.08, 74.98, 75.67, 75.72, 78.29,
80.30, 84.02, 86.26 (C-3H,4H,5H,6H,7H,2G,3G,4G,5G), 98.02 (C-1G),
127.62Ϫ128.44 (CHarom), 135.44, 138.44 (Cq,arom), 169.69 (CϭO).
Pentasaccharide 4d: Pentamer 33 was sulfated as described for 3d,
followed by treatment with 0.2  HCl at 4 °C for 16 h, after which
the mixture was concentrated to a small volume and passed
through a Toyopearl HW40S column, eluted with acetonitrile/water
(1:9). The product fractions were pooled, concentrated and passed
through a column of Dowex 50 WX-4 (Naϩ form). The eluate was
lyophilized to give target pentasaccharide 4d: 1H NMR (D2O3,
Pentasaccharide 31: Pentasaccharide 31 was obtained from 30 and
1
24 in 75% yield: Rf ϭ 0.55 (dichloromethane/acetone, 9:1 v/v). H HH-COSY, HH-TOCSY): D: δ ϭ 3.29 (m, 2 H, 2, 4-H), 3.51 (m,
NMR (CDCl3, HH-COSY, HH-TOCSY): D: δ ϭ 3.05 (t, J4,3
ϭ
1 H, 3-H), 3.85 (m, 1 H, 5-H), 4.09 (m, 1 H, 6-H), 4.25 (m, 1 H,
J4,5 ϭ 9.7 Hz, 1 H, 4-H), 3.11 (dd, J2,1 ϭ 3.8, J2,3 ϭ 9.7 Hz, 1 H,
6Ј-H), 5.45 (d, J1,2 ϭ 3.7 Hz, 1-H); E: 3.24 (m, 1 H, 2-H), 3.50 (m,
2-H), 3.32 (t, J3,2 ϭ J3,4 ϭ 9.7 Hz, 1 H, 3-H), 3.44 (m, 1 H, 5-H), 1 H, 3-H), 3.70 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 3.87 (t, J4,3 ϭ J4,5
ϭ
4.22 (m, 1 H, 6-H), 4.30 (m, 1 H, 6Ј-H), 5.53 (d, J1,2 ϭ 3.8 Hz, 1-
H); E: 2.94 (dd, J2,1 ϭ 7.9, J2,3 ϭ 9.0 Hz, 1 H, 2-H), 3.32 (t, J3,2 ϭ
9.5 Hz, 1 H, 4-H), 4.65 (d, J1,2 ϭ 7.9 Hz, 1 H, 1-H); F: 3.95 (t,
J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H), 4.14 (m, 1 H, 5-H), 4.26 (m, 1 H,
J3,4 ϭ 9.0 Hz, 1 H, 3-H), 3.85 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 3.92 6-H), 4.29 (m, 1 H, 2-H), 4.39 (m, 1 H, 6Ј-H), 4.55 (t, J3,2 ϭ J3,4 ϭ
(dd, J4,3 ϭ 9.0, J4,5 ϭ 9.7 Hz, 1 H, 4-H), 4.13 (d, J1,2 ϭ 7.9 Hz, 1 9.7 Hz, 1 H, 3-H), 5.35 (d, J1,2 ϭ 3.7 Hz, 1 H, 1-H); G: 3.36 (br. s,
H, 1-H); F: 3.44 (dd, J2,1 ϭ 3.7 Hz J2,3 ϭ 9.7 Hz, 1 H, 2-H), 3.58 1 H, 2-H), 3.81 (br. s, 1 H, 3-H), 4.11 (m, 1 H, 4-H), 4.42 (br. s, 1
Eur. J. Org. Chem. 2002, 3954Ϫ3965
3963

´
J. D. C. Codee, G. A. van der Marel, C. A. A. van Boeckel, J. H. van Boom
FULL PAPER
H, 5-H), 4.95 (br. s, 1 H, 1-H); H: 1.57 (m, 1 H, 2-H), 1.69 (m, 1 H-βЈ: 3.50 (m, 1 H) ppm. ESI-MS: m/z ϭ 880.8 [M Ϫ 2 H]2Ϫ, 587.0
H, 2Ј-H), 3.24 (m, 1 H, 3-H), 3.25 (m, 2 H, 1, 1Ј-H), 3.09 (t, J4,3 ϭ
J4,5 ϭ 9.0 Hz, 1 H, 4-H), 3.32 (m, 2 H, 6-H), 3.35 (m, 1 H, 7-H),
[M Ϫ 3 H]3Ϫ
.
Pentasaccharide 4c: Pentamer 4e was deprotected as described for
3.69 (m, 1 H, 8-H), 3.95 (m, 1 H, 8Ј-H), 4.31 (t, J5,4 ϭ J5,6
ϭ
1
3b: H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.36 (m, 2
9.0 Hz, 1 H, 5-H); OMe: 3.47, 3.47, 3.48, 3.51, 3.53, 3.55, 3.59,
H, 2, 4-H), 3.54 (m, 1 H, 3-H), 3.88 (m, 1 H, 5-H), 4.16 (m, 1 H,
6-H), 4.30 (m, 1 H, 6Ј-H), 5.49 (d, J1,2 ϭ 3.5 Hz, 1-H) ppm; E:
3.28 (t, J2,1 ϭ J2,3 ϭ 8.7 Hz, 1 H, 2-H), 3.54 (m, 1 H, 3-H), 3.71
3.60, 3.61 (9 ϫ s, 3 H); CH2 Bn: 5.10 (AB, 2 H); CH Ph: 7.40 (m,
5 H). ESI-MS: m/z ϭ 770.8 [M Ϫ 2 H]2Ϫ, 513.6 [M Ϫ 3 H]3Ϫ
.
Pentasaccharide 4b: Pentamer 4d was deprotected as described for
3b. H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.31 (m, 2
(d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 3.90 (m, 1 H, 4-H), 4.67 (d, J1,2
7.8 Hz, 1 H, 1-H) ppm; F: 3.99 (t, J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H),
ϭ
1
H, 2, 4-H), 3.50 (m, 1 H, 3-H), 3.82 (m, 1 H, 5-H), 4.11 (m, 1 H, 4.16 (m, 1 H, 5-H), 4.30 (m, 2 H, 2,6-H), 4.41 (m, 1 H, 6Ј-H), 4.56
6-H), 4.25 (m, 1 H, 6Ј-H), 5.44 (d, J1,2 ϭ 3.6 Hz, 1-H) ppm; E: (t, J3,2 ϭ J3,4 ϭ 9.7 Hz, 1 H, 3-H), 5.41 (d, J1,2 ϭ 3.5 Hz, 1 H, 1-
3.23 (t, J2,1 ϭ J2,3 ϭ 8.0 Hz, 1 H, 2-H), 3.50 (m, 1 H, 3-H), 3.71 H) ppm; G: 3.49 (br. s, 1 H, 2-H), 3.85 (m, 1 H, 3-H), 4.16 (br. s,
(d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 3.85 (m, 1 H, 4-H), 4.63 (d, J1,2 1 H, 4-H), 4.51 (br. s, 1 H, 5-H), 5.05 (br. s, 1 H, 1-H) ppm; H:
8.0 Hz, 1 H, 1-H) ppm; F: 3.95 (t, J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H), 1.69 (m, 1 H, 2-H), 2.09 (m, 1 H, 2Ј-H), 3.16 (t, J4,3 ϭ J4,5
4.09 (m, 1 H, 5-H), 4.25 (m, 1 H, 6-H), 4.29 (m, 1 H, 2-H), 4.36 9.0 Hz, 1 H, 4-H), 3.37 (m, 4 H, 1, 1Ј,3,6-H), 3.51 (m, 1 H, 7-H),
(m, 1 H, 6Ј-H), 4.53 (t, J3,2 ϭ J3,4 ϭ 9.7 Hz, 1 H, 3-H), 5.36 (d, 3.85 (m, 1 H, 8-H), 3.98 (m, 1 H, 8Ј-H), 4.37 (t, J5,4 ϭ J5,6
J1,2 ϭ 3.7 Hz, 1 H, 1-H) ppm; G: 3.39 (br. s, 1 H, 2-H), 3.81 (br. 9.0 Hz, 1 H, 5-H); OMe: 3.54, 3.54, 3.56, 3.57, 3.57, 3.59, 3.63,
ϭ
ϭ
ϭ
s, 1 H, 3-H), 4.13 (br. s, 1 H, 4-H), 4.50 (br. s, 1 H, 5-H), 4.97 (br.
3.65, 3.65 (9 ϫ s, 3 H) ppm; H-α: 3.63 (m, 1 H) ppm; H-β: 3.02
(dd, Jβ,α/βЈ ϭ 7.1, Jβ,βЈ/α ϭ 13.1 Hz, 1 H) ppm; H-βЈ: 3.19 (m, 1 H)
s, 1 H, 1-H) ppm; H: 1.79 (m, 1 H, 2-H), 2.13 (m, 1 H, 2Ј-H), 3.12
(m, 1 H, 4-H), 3.13 (m, 2 H, 1, 1Ј-H), 3.32 (m, 1 H 3-H), 3.39 (m, ppm. ESI-MS: m/z ϭ 747.0 [M Ϫ2 H]2Ϫ, 497.2 [M Ϫ 3 H]3Ϫ
2 H, 6, 7-H), 3.91 (m, 2 H, 8,8Ј-H) 4.32 (t, J5,4 ϭ J5,6 ϭ 8.9 Hz, 1
.
H, 5-H); OMe: 3.50, 3.50, 3.53, 3.53, 3.53, 3.55, 3.59, 3.60, 3.61 (9
ϫ s, 3 H) ppm. ESI-MS: m/z ϭ 703.8 [M Ϫ 2 H]2Ϫ, 469.0 [M Ϫ
Acknowledgments
3 H]3Ϫ
.
This work was financially supported by the Council for Chemical
Sciences of the Netherlands Organization for Scientific Research
(CW-NWO), the Netherlands Technology Foundation (STW) and
N. V. Organon. We are grateful to F. Lefeber and K. Erkelens (Lei-
den University) for recording NMR spectra, H. van den Elst (Lei-
den University) and W. Verweij (N. V. Organon) for recording mass
spectra and T. G. van Dinther and J. G. H. W. BoveeϪvan de Pasch
(N. V. Organon) for measuring anti-Xa activity.
Pentasaccharide 35: Pentamer 35 was prepared as described for 28
and purified by HPLC (elution was effected applying a gradient of
25Ϫ45% acetonitrile). Rf ϭ 0.45 (EtOAc/pyridine/acetic acid/water,
8:7:1.6:4 v/v/v/v). 1H NMR (D2O, HH-COSY, HH-TOCSY): D:
δ ϭ 3.27 (m, 1 H, 4-H), 3.30 (dd, J2,1 ϭ 3.7, J2,3 ϭ 9.4 Hz, 1 H, 2-
H), 3.46 (m, 2 H, 3, 5-H), 3.71 (m, 2 H, 6,6Ј-H), 5.46 (d, J1,2
3.7 Hz, 1-H) ppm; E: 3.17 (m, 1 H, 2-H), 3.46 (m, 1 H, 3-H), 3.85
(m, 1 H, 4-H), 3.99 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-H), 4.81 (d, J1,2
ϭ
ϭ
[1]
J. Choay, J. C. Lormeau, M. Petitou, P. Sinay¨, J. Fareed, Ann.
7.8 Hz, 1 H, 1-H) ppm; F: 3.46 (m, 1 H, 2-H), 3.58 (m, 1 H, 4-H),
3.67 (m, 1 H, 5-H), 3.73 (m, 1 H, 3-H), 3.79 (m, 2 H, 6, 6Ј-H), 5.11
(1 H, J1,2 ϭ 3.4 Hz, 1 H, 1-H) ppm; G: 3.45 (m, 1 H, 2-H), 3.71
(m, 1 H, 3-H), 4.14 (br. s, 1 H, 4-H), 4.78 (br. s, 1 H, 5-H), 4.98
(br. s, 1 H, 1-H) ppm; H: 1.45 (m, 1 H, 2-H), 1.91 (m, 1 H, 2Ј-H),
2.83 (t, J4,3 ϭ J4,5 ϭ 9.3 Hz, 1 H, 4-H), 3.17 (m, 4 H, 1,1Ј,3,6-H),
3.27 (m, 1 H, 7-H), 3.55 (m, 1 H, 5-H), 3.59 (m, 1 H, 8-H), 3.71
(m, 1 H, 8Ј-H) ppm; OMe: 3.40, 3.45, 3.46, 3.50, 3.51, 3.52, 3.55,
3.58, 3.59 (9 ϫ s, 3 H); CH2 Bn: 5.08 (m, 4 H) ppm; CH Ph: 7.48
(m, 10 H); H-α: 4.21 (m, 1 H); H-β: 3.41 (m, 1 H) ppm; H-βЈ: 3.51
(m, 1 H) ppm. ES-MS: m/z ϭ 1431 [M ϩ 3 Na Ϫ 2 H]ϩ, 727 [M
N. Y. Acad. Sci. 1981, 370, 644Ϫ649.
[2]
L. Tunberg, G. Bäckström, U. Lindahl, Carbohydr. Res. 1982,
100, 393Ϫ410.
[3]
K. A. Bauer, D. W. Hawkins, P. C. Peters, M. Petitou, J.-M.
Herbert, C. A. A. van Boeckel, D. G. Meuleman, Cardiovasc.
Drug Rev. 2002, 20, 37Ϫ52.
C. A. A. van Boeckel, M. Petitou, Angew. Chem. Int. Ed. Engl.
[4]
1993, 32, 1671Ϫ1818.
M. Petitou, P. Duchaussoy, L. Lederman, J. Chaoy, P. Sinay,
[5]
Carbohydr. Res. 1988, 179, 163Ϫ172.
C. A. A. van Boeckel, J. E. M. Basten, H. Lucas, S. F. van
[6]
Aelst, Angew. Chem. Int. Ed. Engl. 1988, 27, 1177Ϫ1178.
ϩ 4 Na Ϫ 2 H]2ϩ
.
[7] [7a]
N. Sakairi, J. E. M. Basten, G. A. van der Marel, C. A. A.
van Boeckel, J. H. van Boom, Chem. Eur. J. 1996, 2,
Pentasaccharide 4e: Pentamer 35 was sulfated as described for 3d.
1H NMR (D2O, HH-COSY, HH-TOCSY): D: δ ϭ 3.30 (m, 2 H,
2, 4-H), 3.51 (m, 1 H, 3-H), 3.87 (m, 1 H, 5-H), 4.10 (m, 1 H, 6-
H), 4.27 (m, 1 H, 6Ј-H), 5.45 (d, J1,2 ϭ 3.7 Hz, 1-H) ppm; E: 3.24
(m, 1 H, 2-H), 3.51 (m, 1 H, 3-H), 3.71 (d, J5,4 ϭ 9.7 Hz, 1 H, 5-
H), 3.87 (t, J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H), 4.65 (d, J1,2 ϭ 7.8 Hz,
1 H, 1-H) ppm; F: 3.96 (t, J4,3 ϭ J4,5 ϭ 9.7 Hz, 1 H, 4-H), 4.12
(m, 1 H, 5-H), 4.27 (m, 1 H, 6-H), 4.28 (m, 1 H, 2-H), 4.39 (m, 1
1007Ϫ1012. [7b] S. K. Das, J.-M. Mallet, J. Esnault, P.-A. Drig-
´
uez, P. Duchaussoy, P. Sizun, J.-P. Herault, J.-M. Herbert, M.
Petitou, P. Sinay¨, Angew. Chem. Int. Ed. 2001, 40, 1670Ϫ1673.
[7c]
S. K. Das, J.-M. Mallet, J. Esnault, P.-A. Driguez, P. Du-
´
chaussoy, P. Sizun, J.-P. Herault, J.-M. Herbert, M. Petitou, P.
Sinay¨, Chem. Eur. J. 2001, 7, 4821Ϫ4834.
[8]
[9]
J. E. M. Basten, G. Jaurant, B. Olde-Hanter, P. Duchaussoy,
M. Petitou, C. A. A. van Boeckel, Bioorg. Med. Chem. Lett.
1992, 2, 905Ϫ908.
H, 6Ј-H), 4.56 (t, J3,2 ϭ J3,4 ϭ 9.7 Hz, 1 H, 3-H), 5.35 (d, J1,2
ϭ
P. Westerduin, C. A. A. van Boeckel, J. E. M. Basten, M. A.
Broekhoven, H. Lucas, A. Rood, H. van der Heijden, R. G.
M. van Amsterdam, T. G. van Dinther, D. G. Meuleman, A.
Visser, G. M. T. Vogel, J. B. L. Damm, G. T. Overklift, Bioorg.
Med. Chem. 1994, 2, 1267Ϫ1280.
L. Jin, J.-P. Abrahams, R. Skinner, M. Petitou, R. N. Pike, R.
W. Carrell, Proc. Nat. Acad. Sci. USA 1997, 94, 14683Ϫ14688.
H. van der Heijden, T. Geertsen, M. Pennekamp, R. Willems,
D. J. Vermaas, P. Westerduin, Abstr. Pap. 9th Europ. Carbohydr.
Symp. (Utrecht) 1997, 154; see also ref.[7]
3.5 Hz, 1 H, 1-H) ppm; G: 3.34 (br. s, 1 H, 2-H), 3.81 (br. s, 1 H,
3-H), 4.12 (br. s, 1 H, 4-H), 4.42 (br. s, 1 H, 5-H), 4.92 (br. s, 1 H,
1-H) ppm; H: 1.55 (m, 1 H, 2-H), 1.94 (m, 1 H, 2Ј-H), 3.02 (t,
J4,3 ϭ J4,5 ϭ 9.0 Hz, 1 H, 5-H), 3.24 (m, 3 H, 1,1Ј,3-H), 3.30 (m,
2 H, 6-H), 3.34 (m, 1 H, 7-H), 3.67 (m, 1 H, 8-H), 3.93 (m, 1 H,
8Ј-H), 4.31 (t, J5,4 ϭ J5,6 ϭ 9.0 Hz, 1 H, 5-H) ppm; OMe: 3.47,
3.48, 3.51, 3.53, 3.56, 3.59, 3.61, 3.61, 3.61 (9 ϫ s, 3 H) ppm; CH2
Bn: 5.09 (m, 4 H) ppm; CH Ph: 7.41 (m, 10 H) ppm, H-α: 4.20
(dd, Jα,β ϭ 5.5, Jα,βЈ ϭ 7.1 Hz, 1 H) ppm; H-β: 3.45 (m, 1 H) ppm;
[10]
[11]
3964
Eur. J. Org. Chem. 2002, 3954Ϫ3965

Probing the HeparinϪAntithrombin III Interaction
FULL PAPER
P. Westerduin, J. E. M. Basten, M. A. Broekhoven, V. de
Kimpe, W. H. A. Kuijpers, C. A. A. van Boeckel, Angew. Chem.
Int. Ed. Engl. 1996, 35, 331Ϫ333.
In this respect it is of interest to note that the crystal structure
of the latent form of antithrombin in complex with pentasacch-
aride 2b[10] shows that Arg24 is directed away from the sacchar-
ide, and that the Glu113 and Asp117 residues are positioned
[12] [12a]
[19]
[20]
M. A. Leeuwenburgh, C. M. Timmers, G. A. van der Ma-
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