Synthesis and anticoagulant activity of bioisosteric sulfonic-acid analogues of the antithrombin-binding pentasaccharide domain of heparin

Article abstract of DOI:10.1002/chem.201201041

Two pentasaccharide sulfonic acids that were related to the antithrombin-binding domain of heparin were prepared, in which two or three primary sulfate esters were replaced by sodium-sulfonatomethyl moieties. The sulfonic-acid groups were formed on a monosaccharide level and the obtained carbohydrate sulfonic-acid esters were found to be excellent donors and acceptors in the glycosylation reactions. Throughout the synthesis, the hydroxy groups to be methylated were masked in the form of acetates and the hydroxy groups to be sulfated were masked with benzyl groups. The disulfonic-acid analogue was prepared in a [2+3] block synthesis by using a trisaccharide disulfonic acid as an acceptor and a glucuronide disaccharide as a donor. For the synthesis of the pentasaccharide trisulfonic acid, a more-efficient approach, which involved elongation of the trisaccharide acceptor with a non-oxidized precursor of the glucuronic acid followed by post-glycosidation oxidation at the tetrasaccharide level and a subsequent [1+4] coupling reaction, was elaborated. In vitro evaluation of the anticoagulant activity of these new sulfonic-acid derivatives revealed that the disulfonate analogue inhibited the blood-coagulation-proteinase factor Xa with outstanding efficacy; however, the introduction of the third sulfonic-acid moiety resulted in a notable decrease in the anti-Xa activity. The difference in the biological activity of the disulfonic- and trisulfonic-acid counterparts could be explained by the different conformation of their L-iduronic-acid residues. The thin red line: The lower activity of the trisulfonic-acid analogue of the antithrombin-binding domain of heparin revealed that the substitution pattern (or number of sulfonates) affected the inhibitory potency of these derivatives for factor Xa. Copyright

Full text of DOI:10.1002/chem.201201041

FULL PAPER
DOI: 10.1002/chem.201201041
Synthesis and Anticoagulant Activity of Bioisosteric Sulfonic-Acid
Analogues of the Antithrombin-Binding Pentasaccharide Domain of Heparin
Mihꢀly Herczeg,[b] Lꢀszlꢁ Lꢀzꢀr,[b] Zsuzsanna Bereczky,[c] Katalin E. Kçvꢂr,[d]
Istvꢀn Timꢀri,[d] Jꢀnos Kappelmayer,[e] Andrꢀs Liptꢀk,[b] Sꢀndor Antus,[b] and
Anikꢁ Borbꢀs*[a]
Dedicated to Professor Ferenc Fꢀlçp on the occasion of his 60th birthday
Abstract: Two pentasaccharide sulfonic
acids that were related to the antith-
rombin-binding domain of heparin
were prepared, in which two or three
primary sulfate esters were replaced by
sodium-sulfonatomethyl moieties. The
sulfonic-acid groups were formed on
a monosaccharide level and the ob-
disulfonic-acid analogue was prepared
in a [2+3] block synthesis by using a tri-
saccharide disulfonic acid as an accept-
or and a glucuronide disaccharide as
a donor. For the synthesis of the penta-
saccharide trisulfonic acid, a more-effi-
cient approach, which involved elonga-
tion of the trisaccharide acceptor with
a non-oxidized precursor of the glucur-
onic acid followed by post-glycosida-
tion oxidation at the tetrasaccharide
level and a subsequent [1+4] coupling
reaction, was elaborated. In vitro eval-
uation of the anticoagulant activity of
these new sulfonic-acid derivatives re-
vealed that the disulfonate analogue in-
hibited the blood-coagulation-protei-
nase factor Xa with outstanding effica-
cy; however, the introduction of the
third sulfonic-acid moiety resulted in
a notable decrease in the anti-Xa activ-
ity. The difference in the biological ac-
tivity of the disulfonic- and trisulfonic-
acid counterparts could be explained
by the different conformation of their
l-iduronic-acid residues.
tained
carbohydrate
sulfonic-acid
esters were found to be excellent
donors and acceptors in the glycosyla-
tion reactions. Throughout the synthe-
sis, the hydroxy groups to be methylat-
ed were masked in the form of acetates
and the hydroxy groups to be sulfated
were masked with benzyl groups. The
Keywords: anticoagulants · carbo-
hydrates · glycosylation · saccha-
rides · sulfonic acid
Introduction
Heparin polysaccharide and its chemically- or enzymatically
fragmented derivatives (low-molecular-weight heparins,
LMWHs) are widespread anticoagulant agents.[1] Heparin
exerts its therapeutic effect by binding to the endogenous
anticoagulant protein antithrombin III (AT-III), which re-
sults in the deactivation of thrombin and factor Xa, the piv-
otal enzymes in the coagulation cascade.[2] Heparin prepara-
tions that are obtained from animal organs are complex
mixtures of polysaccharides. Their application may be ac-
companied by severe bleeding owing to overdosing, allergic
reactions, and heparin-induced thrombocytopenia (HIT).[3]
Fractionated heparins are more bioavailable, possess
a more-predictable anticoagulant activity, and have fewer
side-effects. However, these derivatives are still heterogene-
ous and polydisperse and the risk of inducing HIT has also
been reported.[4]
[a] Dr. A. Borbꢀs
Department of Pharmaceutical Chemistry
Medical and Health Science Center
University of Debrecen, 4010, Debrecen, P.O. Box 70 (Hungary)
E-mail: borbas.aniko@science.unideb.hu
[b] Dr. M. Herczeg, Dr. L. Lꢀzꢀr, Prof. Dr. A. Liptꢀk, Prof. Dr. S. Antus
Research Group for Carbohydrates of the
Hungarian Academy of Sciences, University of Debrecen
4010, Debrecen, P.O. Box: 94 (Hungary)
[c] Dr. Z. Bereczky
Clinical Research Center, Medical and Health Science Center
University of Debrecen, 4032, Debrecen, 98 Nagyerdei krt (Hungary)
[d] Prof. Dr. K. E. Kçvꢁr, I. Timꢀri
Department of Inorganic and Analytical Chemistry
University of Debrecen, 4010, Debrecen, P.O. Box 21 (Hungary)
[e] Prof. Dr. J. Kappelmayer
The identification of the specific AT-III-binding pentasac-
charide domain of heparin[5] and detailed structure–activity
relationship (SAR) studies by using synthetic oligosacchar-
ides have led to the development of the antithrombotic drug
known as Arixtra (1),[6,7] which is a pentasaccharide that se-
lectively inhibits factor Xa and minimizes the risk of bleed-
ing and many other unfavorable side-effects in anticoagulant
Department of Clinical Biochemistry and Molecular Pathology
Medical and Health Science Center, University of Debrecen
4032, Debrecen, 98 Nagyerdei krt (Hungary)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201041; including detailed
1
experimental procedures, selected H, 13C, and 2D NMR spectra, and
evaluation of the inhibitory activity of compounds 1–4 towards factor
Xa.
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therapy. However, its synthesis represents a real challenge,
owing to the presence of uronic acids and a-d-glucosamine,
most of which are N- and O-sulfated at selected positions.
The observation that the N-sulfate groups can be replaced
by O-sulfates without causing a detrimental effect on their
activity paved the way for the synthesis of second-genera-
tion antithrombotic pentasaccharides with simplified struc-
tures that are much easier to synthesize than the glycosami-
noglycan derivatives.[6] Idraparinux[8] (2), the most-potent
representative of these analogues, was considered as a prom-
ising anticoagulant drug candidate. However, its develop-
ment was terminated in the final phase of clinical trials[9]
owing to its extremely long elimination half-life (about
60 days). Thus, the need for new synthetic anticoagulants
with fewer adverse effects and feasible synthesis persists.
From the SAR studies of synthetic analogues of heparin
pentasaccharides, it is known that the negatively charged
groups are essential for the activation of AT-III and that the
type of charge is also crucial: the carboxylate groups may
not be exchanged for sulfate esters and the essential sulfate
moiety cannot be replaced by a phosphate group without af-
fecting the activity.[6] However, the replacement of the sul-
fate groups with sulfonic-acid moieties has not been investi-
gated until now. Phosphonate derivatives have long been
known to be good bioisosteres of phosphates[10] and it has
recently been shown that the isosteric phosphonate and sul-
fonate analogues[11] of mannose 6-phosphate present higher
affinity to the cation-independ-
compound 2 by systematic replacement of the sulfate esters
with
sodium-sulfonatomethyl moiety.[12–14] Herein, we
report the synthesis and factor Xa inhibitory activity of two
pentasaccharide sulfonic acids that contain the sulfonato-
methyl moieties at positions 6 of the F and H (3) or D, F,
and H units (4, Figure 1).
a
Results and Discussion
The target compounds (3 and 4) were built from 6-C-sulfo-
natomethyl-containing glucosides and uronic acids, which
were bound to each other through alternating 1,2-cis- and
1,2-trans-glycosidic linkages. The main goal of our strategy
was the formation of the carboxylic-acid- and sulfonic-acid
functions in the form of esters at a monosaccharide level,
thereby precluding the need for additional synthetic steps at
the pentasaccharide level. Throughout the synthesis, hy-
droxy groups that were meant to be methylated were
masked in the form of acetates and hydroxy groups that
were meant to be sulfated were masked with benzyl groups.
At the same time, this protecting-group strategy ensures the
stereocontrol in the glycosylation steps. We planned [2+3]
block syntheses in which the common FGH trisaccharide of
the two pentasaccharides can be used as an acceptor.
Of the three building blocks of the FGH trisaccharide
(Scheme 1), the synthesis of methylsulfonatomethyl-contain-
ent mannose-6-phosphate re-
ceptor than to the natural com-
pounds. By analogy, we as-
sumed that exchanging the sul-
fate esters for sulfonate moie-
ACHTUNGTRENNUNGties in heparin pentasaccharide
may result in bioactive mimetic,
which might bind to the antith-
rombin with enhanced affinity.
Moreover, the resistance of the
sulfonate moiety to hydrolases
could also be advantageous.
Therefore, we decided to pre-
pare isosteric sulfonic-acid ana-
logues of the AT-III domain of
heparin to obtain new and se-
lective factor Xa inhibitors. As
a starting point of this investi-
gation, Idraparinux (2) was
chosen as the lead compound
because it possesses very high
anticoagulant
activity
and,
owing to its non-glycosamino-
glycan-type structure, it is much
easier to synthesize compared
to Arixtra. We have previously
reported the preparation of sul-
fonic-acid analogues of the EF,
Figure 1. Structures of the synthetic antithrombotic drug Arixtra (1), its non-glycosaminoglycan analogue Idra-
parinux (2), and the target pentasaccharides that contained two (3) or three sulfonatomethyl moieties (4) as
GH, and DEF fragments of bioisosteric substituents of the sulfate esters.
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Analogues of the Pentasaccharide Domain of Heparin
FULL PAPER
tion of compound 11, followed
by removal of the benzylidene
acetal, afforded 4,6-diol 13,
which was isolated in its pure
b form.
The desired uronic-acid ac-
ceptor could be synthesized di-
rectly from compound 13 in
a selective (2,2,6,6-tetramethyl-
piperidin-1-yl)oxyl (TEMPO)-
based oxidation reaction,[20]
which was performed by using
a catalytic amount of TEMPO
and 2 equivalents of [bis-
AHCTUNGERTG(NNUN acetoxy)iodo]benzene
(BAIB)[21] to give the glucuron-
ic acid, which was subsequently
transformed into methyl ester
14 with diazomethane.
Glycosylation of uronate ac-
ceptor 14 with phenylthiogluco-
side 9 in the presence of N-io-
dosuccinimide (NIS) and TfOH
exclusively gave a-linked disac-
Scheme 1. Sulfonate- and urinate-ester building blocks for the FGH trisaccharide. Reagents and conditions:
a) (COCl)2, DMSO, DIPEA, ꢀ608C, 2 h; b) EtO3SCH2PO(OEt)2, nBuLi, THF, DIPEA, ꢀ688C to RT, 5 h;
G
c) H2/Pd/C, EtOH, Et3N, 48 h; d) 0.1m H2SO4, 608C, 12 h; e) PhCHO, CF3COOH, RT, 4 h; f) Ac2O, py, 24 h;
g) 70% CF3COOH, RT, 3 h; h) TEMPO, BAIB, CH2Cl2/water 2:1; 45 min; i) CH2N2 (Et2O), THF, 08C.
DIPEA=N,N-diisopropylethylamine, py=pyridine.
ing acceptor 5 has been reported recently, which involved
a stereoselective free-radical addition of bisulfite onto an
appropriate exomethylene derivative as the key step.[12] The
other 6-deoxy-6-sulfonatomethyl building block (9) that we
planned to use as a donor was not achievable by using this
transformation because the formation of the reactive sulfate
radical anion from sodium hydrogen sulfite requires a per-
charide 15 in 83% yield (Scheme 2). Common reagents that
are useful for the selective deacetylation of the anomeric
position of compound 15 were excluded owing to the sus-
ceptibility of sulfonate esters toward nitrogen nucleophiles.
Thus, dibutyltin oxide[22] was used to liberate the anomeric
position, thereby affording compound 16, treatment of
which with trichloroacetonitrile and DBU resulted in the
corresponding imidate (17). Glycosylation of 6-deoxy-6-
methylsulfonatomethyl acceptor 5 with disaccharide donor
17, upon activation with trimethylsilyltriflate (TMSOTf), af-
forded trisaccharide 18, which contained the desired b-link-
age between units G and H. The coupling product could be
used directly as an acceptor owing to the auspicious side-re-
action of cleavage of the p-methoxybenzyl protecting group
under the slightly acidic conditions of the reaction.
The synthesis of glucuronide disaccharide imidate 19 has
been recently reported[13] and its glycosylation capability has
been demonstrated for sulfonic-acid-containing monosac-
charide acceptors, thereby affording their related trisacchar-
ides in good yields. In this case, the reaction of donor 19
with trisaccharide acceptor 18 resulted in the desired penta-
saccharide (20), although only in low yield. Nevertheless,
the fully protected pentasaccharide, which contained the re-
ACHTUNGTRENNUNGoxybenzoate catalyst, which is incompatible with the oxidiz-
able thioaglycone.
In this case, the Horner–Wadsworth–Emmons (HWE) re-
action[11c,15,16] was used to introduce the methanesulfonic-
acid moiety into dialdose 7, which itself was obtained from
compound 6[17] by Swern oxidation. The reaction of the sul-
fonyl-stabilized phosphonate anion[15] with compound 7 ste-
reoselectively gave the a,b-unsaturated sulfonate ester (8)
as its E isomer. Catalytic hydrogenation of compound 8 in
the presence of triethylamine gave thioglycoside donor 9,
which contained a selectively removable p-methoxybenzyl
(PMB) group at position 4.
In the synthesis of glycosaminoglycan oligosaccharides,
the preparation of the l-iduronic-acid building blocks is
always laborious and troublesome because neither l-idose
nor l-iduronic acid are commercially available. Our ap-
proach was to use compound 14, the simplest available l-
iduronic-acid building block, which is useful as an acceptor
and is easily transformable into a donor, in two well-estab-
lished procedures, which have been elaborated for the 3-O-
benzyl-counterpart,[5b,18] for the synthesis. l-Idose derivative
10[19] was prepared from d-glucose by using the C-5-epimeri-
zation method.[5b] Acid hydrolysis of epoxide 10 resulted in
3-O-methyl-l-idose, which was treated with benzaldehyde in
the presence of trifluoroacetic acid (TFA)[18] to give com-
pound 11 with an a/b-anomeric ratio of about 1:2. Acetyla-
quired uronic-acid units and two sulfonatomethyl moieACHTUNGTRENNUNGties,
could be isolated in an amount that was sufficient for the re-
maining transformations. First, compound 20 was subjected
to Zemplꢁn deacetylation to liberate the hydroxy groups to
obtain compound 21. Subsequent deprotection of the sulfon-
ic-acid esters by nucleophilic displacement with sodium
iodide in acetone gave the disodium salt (22), whose stan-
dard methylation by using methyl iodide and sodium hy-
dride afforded the desired compound 23, which possessed
all of the required methyl ethers. Deprotection of the car-
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Scheme 2. Synthesis of the common trisaccharide acceptor of the target
pentasaccharides. Reagents and conditions: a) NIS, TfOH, CH2Cl2, THF,
4 ꢄ M.S., ꢀ508C, 1 h; b) Bu2SnO, MeOH, 558C, 2 h; c) CCl3CN, DBU,
CH2Cl2, 08C, 30 min; d) TMSOTf, CH2Cl2, 4 ꢄ M.S., ꢀ208C, 1 h. DBU=
Scheme 3. ACHTUNTRGNEU[GN 3+2] Block synthesis of the pentasaccharide disulfonic acid.
Reagents and conditions: a) TMSOTf, CH2Cl2, THF, 4 ꢄ M.S., 08C to
RT, 5 h; b) MeOH, NaOMe, RT, 6 h; c) NaI, acetone, RT, 24 h; d) NaH,
MeI, DMF, 08C, 2 h; e) 0.2m NaOH, MeOH/THF 1:1, RT, 24 h; f) H2
(10 bar), Pd/C (10%), EtOH/AcOH 29:1, 24 h; g) SO3·Et3N, DMF, then
Dowex (Na+).
1,8-diazabicycloACHTUNGTRENNUNG[5.4.0]undec-7-ene.
boxylic-ester groups by saponification, followed by unmask-
ing of the hydroxy groups that were meant to be sulfated by
catalytic hydrogenolysis, furnished compound 25 in high
yield. O-Sulfation was achieved by using SO3·Et3N to give,
after treatment with Dowex Na+ ion-exchange resin, com-
pound 3 as the first isosteric sulfonatomethyl analogue of
Idraparinux (Scheme 3).
The moderate yield of the [2+3] coupling reaction with
glucuronide disaccharide donor 19 forced us to consider an-
other synthetic route to the pentasaccharide trisulfonic acid.
Our new strategy was based on the stepwise elongation of
trisaccharide 18 and on creation of the glucuronic-acid resi-
due by post-glycosidation oxidation[5,8,23] on a tetrasaccharide
level. Thus, compound 26,[13] which is a non-oxidized precur-
sor of glucuronic acid, was coupled to trisaccharide acceptor
18 to give the fully protected compound 27 in 83% yield.
Removal of the p-methoxybenzylidene group by mild acid
hydrolysis, followed by an advantageous two-step procedure
that involved a TEMPO/BAIB-based selective oxidation
and transformation of the carboxylic acid into the corre-
sponding methyl ester, afforded tetrasaccharide 29, which
was ready for the final chain-elongation step (Scheme 4).
The sulfonatomethyl-containing thioglycoside donor (34)
was prepared from known compound 30,[24] which was
equipped with all of the required methyl-ether groups, in
three steps, as described above for the synthesis of com-
pound 9 (Scheme 5). However, in this case, Swern oxidation
of the primary alcohol group afforded a mixture of the re-
quired dialdose (31) and itꢃs a,b-unsaturated counterpart
(32). Such an elimination reaction is well-known upon oxi-
dation of 4-O-acyl-[25a,b] or 4-O-sulfonyl[25c] derivatives into
dialdoses but was surprising for compound 30, which pos-
sessed a 4-O-alkyl substituent. Oxidation of compound 30
was also carried out by using Dess–Martin periodinane[26] as
a reagent, which again provided the same mixture of com-
pounds 31 and 32. HWE olefination of compound 31 and
subsequent selective hydrogenation of compound 33 pro-
ceeded smoothly, thereby affording the fully methylated sul-
fonatomethyl-containing derivative (34) in high yield.
Coupling of donor 34 to tetrasaccharide acceptor 29 upon
activation with NIS/TfOH furnished pentasaccharide 35
with the desired a-interglycosidic linkage. The fully protect-
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Analogues of the Pentasaccharide Domain of Heparin
FULL PAPER
Scheme 4. Elongation of the FGH trisaccharide with a non-oxidized pre-
cursor of glucuronic acid and formation of the carboxylic function at the
tetrasaccharide level. Reagents and conditions: a) NIS, AgOTf, CH2Cl2,
4 ꢄ M.S., ꢀ208C to RT, 1 h; b) 80% AcOH, RT, 1 h; c) TEMPO, BAIB,
CH2Cl2/water 2:1, 6 h; d) CH2N2 (Et2O), THF, 08C.
Scheme 6. Synthesis of the pentasaccharide trisulfonic acid. Reagents and
conditions: a) NIS, TMSOTf, CH2Cl2, THF, 4 ꢄ M.S., ꢀ508C, 1 h;
b) MeOH, NaOMe, RT, 12 h; c) NaI, acetone, RT, 24 h; d) NaH, MeI,
DMF, 08C, 2 h; e) 0.2m NaOH, MeOH, THF, RT, 24 h; f) H2 (10 bar),
Pd/C (10%), EtOH/AcOH 29:1, 24 h; g) SO3·Et3N, DMF, then Dowex
(Na+).
Next, the inhibitory activities of new sulfonic-acid deriva-
tives 3 and 4 towards factor Xa, as well as of reference com-
pounds 1 and 2, were determined (Table 1). Disulfonate an-
Table 1. Inhibitory activity of compounds 1–4 towards factor Xa.
Compound
Activity [Umgꢀ1
1195
(ꢁ189)
1911
(ꢁ193)
2153
(ꢁ153)
384
(ꢁ139)
]
1
2
3
4
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Scheme 5. Synthesis of sulfonic-acid-containing donor 34. Reagents and
conditions: a) (COCl)2, DMSO, DIPEA, ꢀ608C, 2 h; b) EtO3SCH2PO-
alogue 3 inhibited the blood-coagulation proteinase factor
Xa with higher efficacy than Idraparinux, although the in-
troduction of the third sulfonic-acid moiety (4) resulted in
a notable decrease in anti-Xa activity.
G
EtOH, Et3N, 48 h, 73%.
ed pentasaccharide gave, upon Zemplꢁn deacetylation and
subsequent deprotection of the sulfonic acid esters, trisodi-
um salt 36. Standard methylation of the free hydroxy groups
and subsequent saponification of the carboxylic-ester func-
tions provided pentasodium salt 37. Liberation of the hy-
droxy groups, which were meant to be sulfated, by catalytic
hydrogenolysis afforded compound 38. O-Sulfation of the
tetraol by using SO3·Et3N gave compound 4 as an isosteric
trisulfonic-acid analogue of Idraparinux (Scheme 6).
The difference between the biological activities of com-
pounds 3 and 4 could be explained by a conformational
analysis of their iduronic-acid residues. l-Iduronic acid can
1
2
adopt three conformations (4C1, C4, and SO) in heparin-like
pentasaccharides (Figure 2) and the equilibrium between
the conformers is highly dependent on the sulfation pattern
of the adjacent residues.[6a] The conformational flexibility of
2
the l-iduronic acid (i.e., its ability to switch to SO) is known
to be required for antithrombin activation[27] and this resi-
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A. Borbꢀs et al.
modification of the conformation of the l-iduronic-acid resi-
due.
From a synthetic point of view, the most-remarkable find-
ing was that the uronic-acid residues were tolerant of the
sodium-hydride-mediated methylation reaction (22!23 and
33!34), although b-elimination of the base-sensitive uronic
acids is very common under such conditions. The role of sul-
fonic-acid moieties in stabilizing the uronic-acid units to-
wards base is worthy of further investigation.
Figure 2. The three conformers (4C1, 1C4, and 2SO) of l-iduronic acid that
are similar in energy and may be present in heparin.
due adopts the 2SO conformation in antithrombin-bound syn-
thetic pentasaccharides.[28] The 3J
ACTHNUTRGNE(UNG H,H) coupling constants
Experimental Section
for unit G in compounds 3 and 4 clearly demonstrate that
the introduction of the third sulfonic-acid unit significantly
affected the conformation, thus decreasing the population of
General information: Optical rotations were measured at RT on
a Perkin–Elmer 241 automatic polarimeter. TLC analysis was performed
on Kieselgel 60 F254 (Merck) silica-gel plates with visualization by im-
mersing in a sulfuric-acid solution (5% in EtOH) followed by heating.
Column chromatography was performed on silica gel 60 (Merck 0.063–
0.200 mm), Sephadex G-25, and Sephadex LH-20 (Sigma–Aldrich, bead
size: 25–100 mm). Organic solutions were dried over MgSO4 and concen-
trated under vacuum. 1H and 13C NMR spectroscopy (1H: 200, 360, 400,
and 500 MHz; 13C: 50.3, 90.54, 100.28, and 125.76 MHz) were performed
on Bruker AC-200, Bruker DRX-360, Bruker DRX-400, and Bruker
Avance II 500 spectrometers at 258C. Chemical shifts are referenced to
SiMe4 or sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS, d=
0.00 ppm for 1H nuclei) and to residual solvent signals (CDCl3: d=
77.00 ppm, CD3OD: d=49.15 ppm for 13C nuclei). MS (MALDI-TOF)
analysis was carried out in positive reflectron mode on a BIFLEX III
mass spectrometer (Bruker, Germany) with delayed-ion extraction. The
matrix solution was a saturated solution of 2,4,6-trihydroxy-acetophenone
(THAP) in MeCN. Elemental analysis was performed on an Elementar
Vario MicroCube instrument.
2
the bioactive SO conformer (Table 2).
Table 2. 3J
and in its locked conformation.
ACHTUNGTRENNUNG(H,H) Coupling constants [Hz] for the l-iduronic-acid residue
Locked conformation[a]
Residue
4C1
1C4
2S0
1[b]
2
3
4
3J
3J
3J
N
7.6
8.5
9.5
0.0
3.7
5.5
1.3
1.4
2.7
3.1
7.5
3.6
2.6
5.5
2.2
2.8
5.1
3.1
3.6
8–9
2–3
[a] Literature data for the l-iduronic-acid unit in synthetic pentasacchar-
ides, see ref. [28b]; [b] literature data, see refs. [6a,28c].
Conclusions
(E)-Phenyl-2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethylene-4-O-
(4-methoxybenzyl)-1-thio-b-d-glucopyranoside (8): A solution of ethyl di-
ethylphosphorylmethanesulfonate[15] (2.2 g, 9.6 mmol) in dry THF
(24 mL) was cooled to ꢀ788C and a solution of nBuLi (2.5m, 4 mL,
9.6 mmol) in n-hexane was added slowly. After stirring at ꢀ788C for
20 min, a solution of aldehyde 7 (5.45 g, 9.6 mmol) in dry THF (24 mL)
was added dropwise. Stirring was continued for 45 min at ꢀ788C and
then for a further 4 h at RT. The mixture was diluted with CH2Cl2 and
the organic layer was washed with water, dried, and concentrated. The
crude product was purified by column chromatography on silica gel (n-
hexane/EtOAc, 7:3) to give compound 8 (4.68 g, 72%) as a colorless
syrup. [a]2D3 =+1.4 (c=0.11, CHCl3); Rf =0.47 (n-hexane/EtOAc, 7:3);
1H NMR (360 MHz, CDCl3): d=7.51–6.84 (m, 19H; ArH), 6.99 (dd, 3J-
In conclusion, two isosteric sulfonic-acid analogues of the
synthetic anticoagulant pentasaccharide Idraparinux were
prepared by exchanging two or three primary sulfate esters
for a sodium-sulfonatomethyl moiety. The common trisac-
charide part of the two target compounds was constructed
from l-iduronic acid and two monosaccharide sulfonic acids,
thus demonstrating that sulfonic-ester-containing glycosides
could serve as excellent building blocks in oligosaccharide
synthesis, both as donors or acceptors. Pentasaccharide di-
sulfonic acid 3 was obtained from a [2+3] block synthesis by
utilizing trisaccharide acceptor 18 and a glucuronide disac-
charide donor (19). However, this approach was low-yield-
ing, owing to the low reactivity of the uronic-acid-containing
donor (19). Elongation of trisaccharide acceptor 18 with
a non-oxidized precursor of the glucuronic acid, followed by
post-glycosidation oxidation at a tetrasaccharide level and
a subsequent [1+4] coupling reaction, was a highly efficient
strategy that resulted in trisulfonic acid pentasaccharide 4.
The disulfonate analogue inhibited the blood-coagulation
proteinase factor Xa with outstanding efficacy, thereby pro-
viding the first evidence that biologically essential sulfate
esters can be replaced by a sulfonatomethyl group without
any detriment to the anticoagulant activity. However, the
significantly lower activity of the trisulfonic-acid analogue
revealed that the substitution pattern (or the number of sul-
fonate moieties) heavily influenced the inhibitory potency
of the derivatives towards factor Xa, presumably through
A
(H6,H7)=15.2 Hz, 1H; H-6), 6.53 (dd, 3J
ACHUTGTNRNEUNG ACHTUNGTRENNUNG(H5,H7)=
AHCTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(90 MHz, CDCl3): d=159.4, 137.9, 137.5, 128.8 (5ꢅArCq), 143.1 (C-6),
132.5–113.9 (ArC), 125.6 (C-7), 87.5 (C-1), 86.3, 80.5, 79.8, 76.2 (C-2, C-3,
C-4, C-5), 75.7, 75.4, 74.9 (3ꢅPhCH2), 66.8 (CH2), 55.1 (OCH3), 14.7 ppm
(CH3); MS (MALDI-TOF): m/z calcd for C37H40O8S2Na: 699.21
[M+Na]+; found: 699.14; elemental analysis calcd (%) for C37H40O8S2:
C 65.66, H 5.96, S 9.47; found: C 65.60, H 5.92, S 9.43.
Phenyl-2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-4-O-(4-me-
thoxybenzyl)-1-thio-b-d-glucopyranoside (9): To a solution of compound
8 (6.24 g, 0.016 mol) in EtOH (96%, 30 mL) were added Pd/C (10%,
75 mg) and Et3N (45 mL). The reaction mixture was stirred under a H2 at-
mosphere (1 bar) for 48 h. The catalyst was filtered through a pad of
Celite and the filtrate was concentrated. The crude product was purified
by column chromatography on silica gel (n-hexane/EtOAc, 7:3) to give
compound 9 (4.7 g, 75%) as a colorless syrup. [a]2D3 =+1.3 (c=0.44,
CHCl3); Rf =0.38 (n-hexane/EtOAc, 7:3); 1H NMR (360 MHz, CDCl3):
d=7.50–6.83 (m, 19H; ArH), 4.93–4.54 (m, 7H; H-1, 3ꢅPhCH2), 4.16
&
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Analogues of the Pentasaccharide Domain of Heparin
FULL PAPER
(m, 2H; CH2), 3.77 (s, 3H; OCH3), 3.67 (t, 3J
(t, 3J
(H,H)=9.1 Hz, 1H), 3.27 (m, 2H), 3.02 (m, 2H), 2.32, 1.83 (2ꢅm,
2H; H-6a,b), 1.33 ppm (t, 3J(H,H)=7.1 Hz, 3H; CH3); 13C NMR
(90 MHz, CDCl3): d=159.4, 138.1, 137.7, 132.8, 129.6 (5ꢅArCq), 132.8–
113.8 (ArC), 87.1 (C-1), 86.4, 81.1, 80.2, 76.6 (C-2, C-3, C-4, C-5), 75.7,
75.4, 74.8 (3ꢅPhCH2), 65.9 (CH2), 55.1 (OCH3), 46.3 (C-7), 26.0 (C-6),
15.0 ppm (CH3); MS (MALDI-TOF): m/z calcd for C37H42O8S2Na: 701.22
[M+Na]+; found: 701.17; elemental analysis calcd (%) for C37H42O8S2:
C 65.46, H 6.24, S 9.45; found: C 65.50, H 6.31, S 9.49.
(H,H)=8.6 Hz, 1H), 3.47
83.6, 82.9, 82.3, 81.2, 80.0, 79.6, 79.1, 79.0, 78.6, 78.2, 76.1, 74.5, 74.1, 72.5,
70.7, 68.9, 68.5, 67.9, 67.1 (skeleton carbons), 74.9, 73.6, 73.3 (4ꢅPhCH2),
67.6 (C-6-D), 66.2 (CH2), 60.5, 60.2, 59.4, 59.1, 58.1, 55.4, 55.3, 52.2, 51.8
(9ꢅOCH3), 46.3, 45.8 (2ꢅC-7), 25.8, 25.4 (2ꢅC-6), 20.9, 20.7 (2ꢅ
AcCH3), 15.0 ppm (CH3); MS (MALDI-TOF): m/z calcd for
C82H108O34S2Na: 1723.61 [M+Na]+; found: 1723.79; elemental analysis
calcd (%) for C82H108O34S2: C 57.87, H 6.40, S 3.77; found: C 57.72,
H 6.32, S 3.63.
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
Methyl-(6-O-benzyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1!4)-
[methyl-(3-O-methyl-b-d-glucopyranosyl)-uronate]-(1!4)-(2,3-di-O-
benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-a-d-glucopyranosyl)-(1!4)-
[methyl-(3-O-methyl-a-l-idopyranosyl)-uronate]-(1!4)-2,3-di-O-benzyl-
6-deoxy-6-C-methoxysulfonylmethyl-a-d-glucopyranoside (21): To a solu-
tion of compound 20 (330 mg, 0.20 mmol) in MeOH (5 mL) was added
NaOCH3 (2.2 mg, 0.041 mmol). The reaction mixture was stirred for 6 h
and monitored by TLC. After the complete disappearance of the starting
material, the mixture was neutralized with AcOH and concentrated. The
crude product was purified by column chromatography on silica gel
(CH2Cl2/acetone, 9:1) to give compound 21 (261 mg, 83%) as a colorless
syrup. [a]2D3 =+4.7 (c=0.09, CHCl3); Rf =0.42 (CH2Cl2/acetone, 9:1);
1H NMR (360 MHz, CDCl3): d=7.32–7.27 (m, 25H; ArH), 5.37 (d, 3J-
Methyl-(2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-a-d-glucopyr-
anosyl)-(1!4)-[methyl-(2-O-acetyl-3-O-methyl-a-l-idopyranosyl)-uro-
nate]-(1!4)-2,3-di-O-benzyl-6-deoxy-6-C-methoxysulfonylmethyl-a-d-
glucopyranoside (18): To a solution of compound 17 (1.6 g, 1.64 mmol)
and compound 5[12] (1.07 g, 2.3 mmol) in dry CH2Cl2 (48 mL) was added
4 ꢄ molecular sieves (1.5 g). After 30 min, the mixture was cooled to
ꢀ208C and a solution of TMSOTf (30 mL, 0.164 mmol) in dry CH2Cl2
(1.5 mL) was added. After stirring for 1 h at ꢀ208C, TLC analysis
showed the complete consumption of the donor. The reaction mixture
was diluted with CH2Cl2 (100 mL) and filtered. The filtrate was washed
with a saturated aqueous solution of NaHCO3 (50 mL), water (50 mL),
dried, and concentrated. The crude product was purified by column chro-
matography on silica gel (n-hexane/EtOAc, 1:1) to give compound 18
(1.27 g, 67%) as a syrup. [a]2D3 =+2.0 (c=0.23, CHCl3); Rf =0.38 (n-
AHCTUNGERTG(NNUN H,H)=3.2 Hz, 1H), 5.20 (s, 1H), 5.02–4.49 (m, 14H), 4.30–4.26 (m, 2H;
CH2), 3.99–3.91 (m, 3H), 3.86 (s, 3H; OCH3), 3.84–3.61 (m, 7H), 3.59,
3.53, 3.50, 3.49, 3.47, 3.42, 3.39 (7ꢅs, 24H; 8ꢅOCH3), 3.55–3.05 (m,
17H), 2.47–2.31, 2.06–1.91 (2ꢅm, 4H; H-6a,b-F,H), 1.39 ppm (t, 3J-
1
hexane/EtOAc, 1:1); H NMR (400 MHz, CDCl3): d=7.53–7.44 (m, 20H;
ArH), 5.39 (d, 3J
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(H,H)=7.1 Hz, 3H; CH3); 13C NMR (90 MHz, CDCl3): d=169.6, 168.2
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
(2ꢅCO), 138.4, 138.3, 137.8, 137.6, 136.8 (5ꢅArCq), 128.6–126.8 (ArC),
103.0, 100.4, 97.6, 96.4, 94.3 (5ꢅC-1), 84.9, 82.9, 81.2, 80.2, 79.5, 79.3,
78.9, 78.6, 78.2, 77.5, 74.7, 74.1, 73.3, 70.6, 70.5, 68.8, 67.9, 67.1, 65.2 (skel-
eton carbons), 74.9, 74.8, 73.9, 73.2, 73.1 (5ꢅPhCH2), 67.5 (C-6-D), 66.0
(CH2), 60.5, 60.1, 59.9, 59.0, 57.5, 55.3, 55.0, 52.1 (9ꢅOCH3), 45.9, 45.8
(2ꢅC-7), 26.1, 25.0 (2ꢅC-6), 14.9 ppm (CH3); MS (MALDI-TOF): m/z
calcd for C78H104O32S2Na: 1639.58 [M+Na]+; found: 1640.01; elemental
analysis calcd (%) for C78H104O32S2: C 57.91, H 6.48, S 3.96; found:
C 58.01, H 6.57, S 3.99.
ACHTUNGTRENNUNG
3
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
3.50 (3ꢅs, 9H; 3ꢅOCH3), 3.65–3.64 (m, 1H; H-2), 3.58–3.56 (m, 1H; H-
2”), 3.53–3.49 (m, 1H; H-7a), 3.39–3.29 (m, 4H; H-4“, H-7”a,b, H-7b),
2.60 (s, 1H; H-4“-OH), 2.57–2.54 (m, 1H; H-6a), 2.47–2.41 (m, 1H; H-
6”a), 2.18 (s, 3H; AcCH3), 2.14–2.09 (m, 2H; H-6b, H-6“b), 1.55 ppm (t,
ACHTUNGTRENNUNG
3J(H,H)=7.1 Hz, 3H; CH3); 13C NMR (100 MHz, CDCl3): d=169.9,
169.2 (2ꢅCO), 138.7, 138.3, 137.8, 137.7 (4ꢅArCq), 128.5–127.1 (ArC),
98.5 (C-1”), 97.8 (C-1), 97.6 (C-1’), 80.5 (C-3“), 79.9 (C-2), 79.7 (C-2”),
79.2 (C-4), 76.7 (C-3’), 75.0, 74.9, 73.3, 73.1 (4ꢅPhCH2), 74.3 (C-4’), 73.2
(C-4“), 69.8 (C-5”), 69.1 (C-5’), 68.2 (C-2’), 67.9 (C-5), 66.0 (CH2), 58.3,
55.4, 55.3, 51.7 (4ꢅOCH3), 46.1, 45.8 (C-7, C-7“), 25.7 (C-6, C-6”), 20.8
(AcCH3), 14.9 ppm (CH3); MS (MALDI-TOF): m/z calcd for
C56H72O22S2Na: 1183.38 [M+Na]+; found: 1183.19; elemental analysis
calcd (%) for C56H72O22S2: C 57.92, H 6.25, S 5.52; found: C 58.01,
H 6.41, S 5.61.
Methyl-(6-O-benzyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1!4)-
[methyl-(3-O-methyl-b-d-glucopyranosyl)-uronate]-(1!4)-[sodium (2,3-
di-O-benzyl-6-deoxy-6-C-sulfonatomethyl-a-d-glucopyranosyl)]-(1!4)-
[methyl-(3-O-methyl-a-l-idopyranosyl)-uronate]-(1!4)-[sodium (2,3-di-
O-benzyl-6-deoxy-6-C-sulfonatomethyl-a-d-glucopyranoside)] (22): To
a solution of compound 21 (93 mg, 0.06 mmol) in acetone (3.5 mL) was
added NaI (28 mg, 0.18 mmol). After 24 h, the mixture was concentrated
and the residue was purified by column chromatography on Sephadex
LH-20 (MeOH) to give compound 22 (63 mg, 68%) as a syrup. [a]D23
+5.9 (c=0.05, CHCl3); Rf =0.28 (CH2Cl2/MeOH, 9:1); 1H NMR
(360 MHz, CD3OD): d=7.33–7.21 (m, 25H; ArH), 5.37 (d, 3J
(H,H)=
=
Methyl-(6-O-benzyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1!4)-
[methyl-(2-O-acetyl-3-O-methyl-b-d-glucopyranosyl)-uronate]-(1!4)-
(2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-a-d-glucopyranosyl)-
(1!4)-[methyl-(2-O-acetyl-3-O-methyl-a-l-idopyranosyl)-uronate]-(1!
4)-2,3-di-O-benzyl-6-deoxy-6-C-methoxysulfonylmethyl-a-d-glucopyrano-
side (20): To a solution of compound 18 (300 mg, 0.26 mmol) and com-
pound 19[12] (365 mg, 0.52 mmol) in dry CH2Cl2 (15 mL) was added 4 ꢄ
molecular sieves (1 g). After 30 min, the mixture was cooled to 08C and
a solution of TMSOTf (10 mL in 300 mL dry CH2Cl2, 0.055 mmol) was
added. After stirring for 2 h at 08C and for a further 5 h at RT, TLC anal-
ysis showed the complete consumption of the donor molecule. The reac-
tion mixture was diluted with CH2Cl2 (30 mL) and filtered. The filtrate
was washed with a saturated aqueous solution of NaHCO3 (10 mL) and
water (10 mL), dried, and concentrated. The crude product was purified
by column chromatography on silica gel (CH2Cl2/acetone, 9:1) to give
compound 20 (167 mg, 38%) as a syrup. [a]2D3 =+5.2 (c=0.07, CHCl3);
Rf =0.51 (CH2Cl2/acetone, 9:1); 1H NMR (360 MHz, CDCl3): d=7.33–
ACHTUNGTRENNUNG
3.2 Hz, 1H), 5.16 (s, 1H), 5.04–4.47 (m, 14H), 3.95–3.65 (m, 8H), 3.61,
3.54, 3.46, 3.43, 3.35 (5ꢅs, 24H; 8ꢅOCH3), 3.49–2.85 (m, 17H), 2.49–
2.33, 2.09–1.93 ppm (2ꢅm, 4H; H-6a,b-F,H); 13C NMR (90 MHz,
CD3OD): d=171.7, 170.5 (2ꢅCO), 140.5, 140.1, 139.5, 139.1 (5ꢅArCq),
129.5–128.1 (ArC), 104.9, 102.2, 98.7, 97.6, 96.3 (5ꢅC-1), 87.3, 84.3, 82.8,
82.2, 82.1, 81.2, 80.6, 80.4, 79.2, 76.6, 76.2, 75.7, 73.0, 72.0, 70.7, 70.4, 69.6,
68.1 (skeleton carbons), 76.0, 74.8, 74.5, 73.9 (5ꢅPhCH2), 69.3 (C-6-D),
61.0, 60.7, 59.3, 58.6, 55.7, 53.1, 52.9 (8ꢅOCH3), 28.3, 27.8 ppm (2ꢅC-6);
MS (MALDI-TOF): m/z calcd for C75H96Na3O32S2: 1641.50 [M+Na]+;
found: 1641.57; elemental analysis calcd (%) for C75H96Na2O32S2:
C 55.62, H 5.97, S 3.96; found: C 55.55, H 5.89, S 3.89.
Methyl-(6-O-benzyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1!4)-
[methyl-(2,3-di-O-methyl-b-d-glucopyranosyl)-uronate]-(1!4)-[sodium
(2,3-di-O-benzyl-6-deoxy-6-C-sulfonatomethyl-a-d-glucopyranosyl)]-(1!
4)-[methyl-(2,3-di-O-methyl-a-l-idopyranosyl)-uronate]-(1!4)-[sodium
(2,3-di-O-benzyl-6-deoxy-6-C-sulfonatomethyl-a-d-glucopyranoside)]
(23): To a solution of pentasaccharide diol 22 (48 mg, 0.03 mmol) in dry
DMF (1 mL) was slowly added NaH (3 mg, 0.072 mmol) at 08C. After
stirring for 30 min at 08C, MeI (4 mL, 0.072 mmol) was added. When
complete conversion of the starting material into a main spot had been
observed by TLC analysis (2 h at 08C), CH3OH (1 mL) was added. The
reaction mixture was stirred for 5 min and the solvents were evaporated.
7.18 (m, 25H; ArH), 5.36 (d, 3J
1.6 Hz, 1H), 4.93–4.44 (m, 16H), 4.30–4.24 (m, 2H; CH2), 4.03 (t, 3J-
ACHTUNGTRENNUNG(H,H)=9.1 Hz, 1H), 3.90 (s, 3H; OCH3), 3.84–3.59 (m, 10H), 3.59, 3.48,
(H,H)=3.6 Hz, 1H), 5.17 (d, 3J
ACHUTGTNRNEUNG AHCUTNGTREN(NUGN H,H)=
3.47, 3.42, 3.33, 3.29 (6ꢅs, 24H; 8ꢅOCH3), 3.53–3.25 (m, 6H), 3.25–3.09
(m, 6H), 2.41–2.19 (m, 2H), 2.14, 2.03 (2ꢅs, 6H; 2ꢅAcCH3), 1.94–1.78
ACTHNUTRGNEUNG
(m, 2H), 1.40 ppm (t, 3J(H,H)=7.1 Hz, 3H; CH3); 13C NMR (90 MHz,
CDCl3): d=170.2, 169.3, 169.2, 168.0 (4ꢅCO), 138.9, 138.6, 137.9, 137.7,
137.6 (5ꢅArCq), 128.3–126.9 (ArC), 101.4, 98.2, 97.7, 97.5, 96.8 (5ꢅC-1),
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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A. Borbꢀs et al.
The crude product was purified by column chromatography on Sephadex
LH-20 (MeOH) to give compound 23 (34 mg, 70%) as a syrup. [a]D23
+6.0 (c=0.12, CHCl3); Rf =0.44 (CH2Cl2/MeOH, 9:1); 1H NMR
(360 MHz, CD3OD): d=7.46–7.36 (m, 25H; ArH) 5.50 (d, 3J
(H,H)=
3.5 Hz, 1H), 5.39 (d, 3J(H,H)=3.5 Hz, 1H), 5.17 (d, 3J
(H,H)=3.1 Hz,
1H), 5.10–4.61 (m, 13H), 4.01–3.80 (m, 10H), 3.73, 3.70, 3.69, 3.62, 3.58,
3.56, 3.52, 3.48 (8ꢅs, 30H; 10ꢅOCH3), 3.71–3.46 (m, 5H), 3.44–2.99 (m,
10H), 2.65–2.49, 2.19–2.01 ppm (2ꢅm, 4H; 2ꢅH-6a,b); 13C NMR
(90 MHz, CD3OD): d=171.4, 170.1 (2ꢅCO), 140.2, 139.9, 139.2, 139.1
(4ꢅArCq), 129.2–128.0 (ArC), 104.5, 99.8, 98.5, 97.6, 97.2 (5ꢅC-1), 86.6,
85.1, 84.0, 82.9, 82.5, 81.6, 80.4, 80.0, 79.6, 78.4, 76.4, 75.3, 74.1, 71.8, 71.0,
70.9, 70.1 (skeleton carbons), 75.8, 74.3, 73.9 (5ꢅPhCH2), 69.0 (C-6-D),
61.2, 61.0, 60.8, 59.9, 59.4, 55.6, 52.9, 52.8 (10ꢅOCH3), 27.9, 27.5 ppm (2ꢅ
C-6); MS (MALDI-TOF): m/z calcd for C77H100Na3O32S2: 1669.53
[M+Na]+; found: 1669.57; elemental analysis calcd (%) for
C77H100Na2O32S2: C 56.13, H 6.12, S 3.89; found: C 56.09, H 6.08, S 3.81.
sulfonato-6-C-sulfonatomethyl-a-d-glucopyranoside)] (3): A solution of
compound 25 (60 mg, 0.05 mmol) in dry DMF (2.5 mL) was treated with
SO3·Et3N (227 mg, 1.25 mmol). After stirring for 24 h at 508C, the reac-
=
tion mixture was neutralized with
a saturated aqueous solution of
AHCTUNGTRENNUNG
NaHCO3 (525 mg, 6.25 mmol). The resulting mixture was concentrated.
The crude product was treated with Dowex ion-exchange resin (Na+) and
purified by column chromatography on Sephadex G-25 (water) to give
G
ACHTUNGTRENNUNG
compound
MeOH); Rf =0.28 (CH2Cl2/MeOH/water, 6:7:1); 1H NMR (500 MHz,
D2O): d=5.47 (d, 3J(H1,H2)=3.2 Hz, 1H; H-1-D), 5.41 (d, 3J
(H1,H2)=
2.6 Hz, 1H; H-1-F), 5.26 (d, 3J(H1,H2)=2.3 Hz, 1H; H-1-G), 5.13 (d, 3J-
(H1,H2)=3.3 Hz, 1H; H-1-H), 4.86 (s, 1H; H-5-G), 4.68 (d, J
7.7 Hz, 1H; H-1-E), 4.62 (t, 3J(H2,H3)=9.4 Hz, 1H; H-3-H), 4.56 (t, 3J-
(H2,H3)=9.2 Hz, 1H; H-3-F), 4.37 (dd, 3J(H1,H2)=3.5 Hz, 3J
(H2,H3)=
9.7 Hz, 1H; H-2-H), 4.33–4.27 (m, 2H; H-2-F, H-6a-D), 4.15–4.13 (m,
2H; H-6b-D, H-4-G), 4.03 (t, 3J
(H4,H5)=9.2 Hz, 1H; H-5-F), 3.94–3.88
3 (62 mg, 73%) as a
white foam. [a]2D3 =+5.3 (c=0.11,
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
3
A
ACHTUNGTRENNUNG(H1,H2)=
AHCTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(m, 3H; H-5-D, H-4-E, H-5-H), 3.84–3.75 (m, 4H; H-5-E, H-4-H, H-4-F,
H-3-G), 3.67, 3.64, 3.63, 3.59, 3.57, 3.54 (6ꢅs, 24H; 8ꢅOCH3), 3.61–3.55
(m, 3H; H-2-G, H-3-E, H-3-D), 3.36–3.29 (m, 3H; H-2-E, H-2-D, H-4-
D), 3.02–2.98 (m, 4H; H-7a,b-F, H-7a,b-H), 2.44–2.37 (m, 2H; H-6a-F,
H-6a-H), 2.03–1.96 ppm (m, 2H; H-6b-F, H-6b-H); 13C NMR (125 MHz,
D2O): d=175.2, 175.1 (2ꢅCO), 102.8 (C-1-E), 101.3 (C-1-G), 97.9 (C-1-
H), 96.9 (C-1-D), 94.5 (C-1-F), 86.6 (C-3-E), 83.9 (C-2-E), 82.8 (C-3-D),
81.4 (C-2-D), 79.4, 78.8, 77.7, 76.5 (C-5-E, C-4-H, C-4-F, C-3-G), 78.9 (C-
4-D), 78.4 (C-2-G), 78.2 (C-3-H), 77.1 (C-3-F), 76.7 (C-2-H), 76.0 (C-2-
F), 75.1 (C-4-E), 73.8 (C-4-G), 71.4 (C-5-G), 71.0 (C-5-F), 70.3, 69.6 (C-5-
H, C-5-D), 66.9 (C-6-D), 61.3, 61.1, 60.8, 60.3, 60.2, 59.9, 58.9, 56.2 (8ꢅ
OCH3), 48.2, 48.0 (C-7-F, C-7-H), 27.2, 27.0 ppm (C-6-F, C-6-H); MS
(ESI): m/z: 838.0 [Mꢀ7Na]2ꢀ, 551.0 [Mꢀ6Na]3ꢀ; elemental analysis calcd
(%) for C40H59Na9O47S7: C 27.88, H 3.45, S 13.03; found: C 23.89, H 4.37,
S 10.89.
Methyl-(6-O-benzyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1!4)-
[sodium (2,3-di-O-methyl-b-d-glucopyranosyl)-uronate]-(1!4)-[sodium
(2,3-di-O-benzyl-6-deoxy-6-C-sulfonatomethyl-a-d-glucopyranosyl)]-(1!
4)-[sodium (2,3-di-O-methyl-a-l-idopyranosyl)-uronate]-(1!4)-[sodium
(2,3-di-O-benzyl-6-deoxy-6-C-sulfonatomethyl-a-d-glucopyranoside)]
(24): A solution of pentasaccharide 23 (117 mg, 0.071 mmol) in MeOH
and THF (1:1, 2 mL) was treated with an aqueous solution of NaOH
(1 mL, 0.2m). After stirring for 24 h at RT, TLC analysis showed com-
plete conversion of the carboxylic esters into their corresponding sodium
salts. The mixture was neutralized with acetic acid and concentrated. The
crude product was purified by column chromatography on silica gel
(CH2Cl2/MeOH, 7:3) and then by column chromatography on Sephadex
LH-20 (MeOH) to give compound 24 (98 mg, 83%) as a white foam.
[a]2D3 =+6.6 (c=0.14, MeOH); Rf =0.56 (CH2Cl2/MeOH, 7:3); 1H NMR
(360 MHz, CD3OD): d=7.47–7.37 (m, 25H; ArH), 5.54 (s, 1H), 5.33–
4.62 (m, 15H), 4.19–3.83 (m, 9H), 3.74, 3.69, 3.63, 3.57, 3.48 (5ꢅs, 24H;
8ꢅOCH3), 3.77–3.31 (m, 6H), 3.27–2.68 (m, 10H), 2.75–2.51, 2.17–
2.01 ppm (2ꢅm, 4H; 2ꢅH-6a,b); 13C NMR (90 MHz, CD3OD): d=
140.5, 139.8, 139.6, 139.5, 139.3 (5ꢅArCq), 129.3–128.1 (ArC), 100.0, 98.6,
98.0 (5ꢅC-1), 86.9, 85.5, 84.3, 82.9, 82.6, 81.8, 81.0, 80.4, 80.3, 77.1, 71.9,
70.8, 70.5 (skeleton carbons), 76.3, 75.7, 74.5, 74.3, 73.8 (5ꢅPhCH2), 69.3
(C-6-D), 61.3, 60.9, 60.6, 60.0, 59.5, 58.8, 55.6 (8ꢅOCH3), 28.4, 28.2 ppm
(2ꢅC-6); MS (MALDI-TOF): m/z calcd for C75H94Na5O32S2: 1685.47
[M+Na]+; found: 1685.61; elemental analysis calcd (%) for
C75H94Na4O32S2: C 54.15, H 5.70, S 3.85; found: C 54.01, H 5.63, S 3.76.
Methyl-[2,3-di-O-acetyl-4,6-O-(4-methoxybenzylidene)-b-d-glucopyrano-
syl]-(1!4)-(2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-a-d-glu-
copyranosyl)-(1!4)-[methyl-(2-O-acetyl-3-O-methyl-a-l-idopyranosyl)-
uronate]-(1!4)-2,3-di-O-benzyl-6-deoxy-6-C-methoxysulfonylmethyl-a-
d-glucopyranoside (27): To
a solution of compound 18 (500 mg,
0.43 mmol) and compound 26[13] (368 mg, 0.77 mmol) in dry CH2Cl2
(17 mL) was added 4 ꢄ molecular sieves (1 g). After stirring for 30 min,
the mixture was cooled to ꢀ208C and solutions of NIS (399 mg,
1.78 mmol) in dry THF (500 mL) and AgOTf (72 mg, 0.28 mmol) in dry
toluene (500 mL) were added. After stirring for 1 h at ꢀ208C, TLC analy-
sis (CH2Cl2/acetone, 95:5) showed complete consumption of the donor.
The reaction mixture was neutralized with Et3N (100 mL), diluted with
CH2Cl2 (100 mL), and filtered. The filtrate was washed with an aqueous
solution of Na2S2O3 (10%, 50 mL), a saturated aqueous solution of
NaHCO3 (50 mL), and water (50 mL), dried, and concentrated. The
crude product was purified by column chromatography on silica gel
(CH2Cl2/acetone, 95:5) to give compound 27 (543 mg, 83%) as a syrup.
[a]2D3 =+5.8 (c=0.12, CHCl3); Rf =0.42 (CH2Cl2/acetone, 95:5); 1H NMR
(360 MHz, CDCl3): d=7.33–6.83 (m, 24H; ArH), 5.31–5.18 (m, 3H),
4.94–4.46 (m, 14H), 4.29–4.27 (m, 2H; CH2), 3.95–3.67 (m, 6H), 3.89,
3.76, 3.49, 3.33, 3.31 (5ꢅs, 15H; 5ꢅOCH3), 3.54–3.13 (m, 12H), 2.67–2.29
(m, 4H; 2ꢅH-6a,b), 2.09, 2.04, 2.00 (3ꢅs, 9H; 3ꢅAcCH3), 1.40 ppm (t,
Methyl-(2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1!4)-[sodium (2,3-di-
O-methyl-b-d-glucopyranosyl)-uronate]-(1!4)-[sodium (6-deoxy-6-C-sul-
fonatomethyl-a-d-glucopyranosyl)]-(1!4)-[sodium (2,3-di-O-methyl-a-l-
idopyranosyl)-uronate]-(1!4)-[sodium (6-deoxy-6-C-sulfonatomethyl-a-
d-glucopyranoside)] (25):
A
mixture of compound 24 (95 mg,
0.057 mmol), which was dissolved in EtOH/AcOH (96%, 29:1, 6 mL),
and Pd/C (10%, 60 mg) was stirred in an autoclave under a H2 atmos-
phere (10 bar) for 24 h. The catalyst was filtered through a pad of Celite
and the filtrate was concentrated. The crude product was purified by
column chromatography on Sephadex G-25 (water) to give compound 25
(62 mg, 90%) as a white foam. [a]2D3 =+6.9 (c=0.20, MeOH); Rf =0.19
(CH2Cl2/MeOH/water, 7:6:1); 1H NMR (360 MHz, D2O): d=5.46 (d, 3J-
ACTHNUTRGNEUNG
3J(H,H)=7.1 Hz, 3H; CH3); 13C NMR (90 MHz, CDCl3): d=170.0,
ACHTUNGTRENNUNG AHCTUNGTRNEN(UGN H,H)=
(H,H)=3.1 Hz, 1H), 5.13 (s, 1H), 4.78–4.75 (m, 2H), 4.61 (d, 3J
169.8, 169.6, 169.3 (4ꢅCO), 160.0, 138.7, 138.6, 137.7, 137.6, 129.0 (5ꢅ
ArCq), 128.3–126.5 (ArC), 113.4 (Cac), 101.3, 98.1, 97.7, 97.5 (4ꢅC-1),
82.0, 80.1, 79.4, 79.1, 78.1, 77.9, 76.0, 74.0, 72.8, 71.9, 68.9, 68.5, 67.9, 67.2
(skeleton carbons), 74.9, 74.6, 73.7, 73.3 (4ꢅPhCH2), 68.0 (C-6-E), 66.1
(CH2), 58.1, 55.4, 55.2, 55.1, 51.8 (5ꢅOCH3), 46.3, 45.8 (2ꢅC-7), 25.8,
25.4 (2ꢅC-6), 20.9, 20.6, 20.4 (3ꢅAcCH3), 15.0 ppm (CH3); MS
(MALDI-TOF): m/z calcd for C74H92O30S2Na: 1547.50 [M+Na]+; found:
1547.37; elemental analysis calcd (%) for C74H92O30S2: C 58.26, H 6.08,
S 4.20; found: C 57.88, H 5.91, S 3.98.
7.5 Hz, 1H), 4.12 (s, 1H), 3.84–3.70 (m, 10H), 3.66, 3.65, 3.62, 3.56, 3.54,
3.51, 3.40 (7ꢅs, 24H; 8ꢅOCH3), 3.68–3.49 (m, 4H), 3.28–2.99 (m, 11H),
2.39–2.25, 1.99–1.83 ppm (2ꢅm, 4H; 2ꢅC-6a,b); 13C NMR (90 MHz,
D2O): d=175.5, 175.4 (2ꢅCO), 103.5, 100.5, 99.8, 96.8, 95.8 (5ꢅC-1),
86.6, 84.5, 84.0, 82.7, 82.6, 81.5, 79.2, 77.5, 77.1, 75.5, 74.9, 72.7, 72.6, 72.4,
72.2, 72.0, 71.6, 71.5, 70.2, 69.9 (skeleton carbons), 61.5, 61.1, 60.8, 60.3,
59.9, 59.8, 58.8, 56.0 (8ꢅOCH3), 60.5 (C-6-D), 48.4, 48.2 (2ꢅC-7), 27.2,
26.9 ppm (2ꢅC-6); MS (MALDI-TOF): m/z calcd for: C40H64Na5O32S2
1235.23 [M+Na]+; found: 1235.45; elemental analysis calcd (%) for
C40H64Na4O32S2: C 39.61, H 5.32, S 5.29; found: C 39.53, H 5.24, S 5.19.
Methyl-(2,3-di-O-acetyl-b-d-glucopyranosyl)-(1!4)-(2,3-di-O-benzyl-6-
deoxy-6-C-ethoxysulfonylmethyl-a-d-glucopyranosyl)-(1!4)-[methyl-(2-
O-acetyl-3-O-methyl-a-l-idopyranosyl)-uronate]-(1!4)-2,3-di-O-benzyl-
Nona-sodium [methyl-(2,3,4-tri-O-methyl-6-O-sulfonato-a-d-glucopyra-
nosyl)-(1!4)-(2,3-di-O-methyl-b-d-glucopyranosyl-uronate)-(1!4)-(6-
deoxy-2,3-di-O-sulfonato-6-C-sulfonatomethyl-a-d-glucopyranosyl)]-(1!
4)-(2,3-di-O-methyl-a-l-idopyranosyl-uronate)-(1!4)-(6-deoxy-2,3-di-O-
6-deoxy-6-C-methoxysulfonylmethyl-a-d-glucopyranoside (28):
A solu-
tion of compound 27 (660 mg, 0.43 mmol) in AcOH (80%, 14 mL) was
stirred for 1 h at RT. After the reaction had been completed, the mixture
&
8
&
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Analogues of the Pentasaccharide Domain of Heparin
FULL PAPER
was concentrated. The residue was purified by column chromatography
on silica gel (CH2Cl2/MeOH, 95:5) to give compound 28 (417 mg, 68%)
as a syrup. [a]2D3 =+13.7 (c=0.11, CHCl3); Rf =0.42 (CH2Cl2/MeOH,
1H), 3.88 (s, 3H; OCH3), 3.86–3.65 (m, 8H), 3.54, 3.51, 3.50, 3.47, 3.40,
3.33, 3.30 (7ꢅs, 21H; 7ꢅOCH3), 3.39–3.07 (m, 10H), 2.98 (dd, 3J-
ACHUTNGRENNUG CAHTUNGTRENNUNG
(H1,H2)=2.8 Hz, 3J
1
95:5); H NMR (360 MHz, CDCl3): d=7.32–7.28 (m, 20H; ArH), 5.16 (s,
3
1H), 4.97–4.53 (m, 14H), 4.46 (d, 3J
N
(t, JACTHGNUTERNNUG
(H,H)=7.1 Hz, 6H; 2ꢅCH3); 13C NMR (90 MHz, CDCl3): d=169.8,
2H; CH2), 3.89, 3.47, 3.32, 3.30 (4ꢅs, 12H; 4ꢅOCH3), 3.84–3.50 (m, 9H),
169.3, 169.1, 169.0, 167.4 (5ꢅCO), 138.6, 138.5, 137.5, 137.4 (4ꢅArCq),
128.1–126.3 (ArC), 100.9, 97.9, 97.4, 97.3, 96.7 (5ꢅC-1), 82.7, 82.1, 81.2,
79.9, 79.6, 78.9, 78.8, 77.8, 75.8, 74.9, 74.1, 73.8, 73.3, 71.6, 69.0, 68.6, 68.2,
67.6, 66.8 (skeleton carbons), 74.6, 74.0, 73.4, 72.9 (4ꢅPhCH2), 65.9 (2ꢅ
CH2), 60.3, 60.2, 58.8, 57.8, 55.1, 52.2, 51.5 (8ꢅOCH3), 46.0, 45.5 (3ꢅC-
7), 25.6, 25.4 (3ꢅC-6), 20.6, 20.4, 20.2 (3ꢅAcCH3), 14.7 ppm (CH3); MS
(MALDI-TOF): m/z calcd for C79H108O37S3Na: 1767.56 [M+Na]+; found:
1767.49; elemental analysis calcd (%) for C79H108O37S3: C 54.35, H 6.24,
S 5.51; found: C 54.09, H 5.98, S 5.38.
3.37–3.13 (m, 11H), 2.37–2.28 (m, 2H), 2.07, 2.04, 2.02 (3ꢅs, 9H; 3ꢅ
AcCH3), 1.91–1.86 (m, 2H), 1.39 ppm (t, 3J
ACTHNUTRGNE(UNG H,H)=7.1 Hz, 3H; CH3);
13C NMR (90 MHz, CDCl3): d=171.2, 170.1, 169.5, 169.3 (4ꢅCO), 138.6,
138.5, 137.7, 137.6 (4ꢅArCq), 128.3–126.7 (ArC), 100.5, 98.2, 97.6, 97.4
(4ꢅC-1), 81.3, 80.1, 79.2, 79.1, 78.8, 78.0, 76.0, 75.5, 73.9, 71.7, 69.3, 69.1,
68.5, 67.9, 67.2 (skeleton carbons), 74.9, 74.8, 73.7, 73.3 (4ꢅPhCH2), 66.2
(CH2), 58.1, 55.4, 55.2, 51.7 (4ꢅOCH3), 46.3, 45.7 (2ꢅC-7), 25.7, 25.2 (2ꢅ
C-6), 20.8, 20.6, 20.4 (3ꢅAcCH3), 15.0 ppm (CH3); MS (MALDI-TOF):
m/z calcd for C66H86O29S2Na: 1429.46 [M+Na]+; found: 1429.49; elemen-
tal analysis calcd (%) for C66H86O29S2: C 56.32, H 6.16, S 4.56; found:
C 55.99, H 5.93, S 4.11.
Nona-sodium [methyl-(6-deoxy-2,3,4-tri-O-methyl-6-C-sulfonatomethyl-
a-d-glucopyranosyl)-(1!4)-(2,3-di-O-methyl-b-d-glucopyranosyl-uro-
nate)-(1!4)-(6-deoxy-2,3-di-O-sulfonato-6-C-sulfonatomethyl-a-d-gluco-
pyranosyl)]-(1!4)-(2,3-di-O-methyl-a-l-idopyranosyl-uronate)-(1!4)-(6-
deoxy-2,3-di-O-sulfonato-6-C-sulfonatomethyl-a-d-glucopyranoside)] (4):
Compound 38 (30 mg, 0.023 mmol) was O-sulfated in an analogous
manner to that described for the synthesis of compound 3. The crude
product was treated with Dowex ion-exchange resin (Na+) and then puri-
fied by column chromatography on Sephadex G-25 (water) to give com-
pound 4 (33 mg, 84%). [a]D23 =+5.1 (c=0.07, water); Rf =0.33 (CH2Cl2/
Methyl-[methyl-(2,3-di-O-acetyl-b-d-glucopyranosyl)-uronate]-(1!4)-
(2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-a-d-glucopyranosyl)-
(1!4)-[methyl-(2-O-acetyl-3-O-methyl-a-l-idopyranosyl)-uronate]-(1!
4)-2,3-di-O-benzyl-6-deoxy-6-C-methoxysulfonylmethyl-a-d-glucopyrano-
side (29): To a vigorously stirring solution of compound 28 (400 mg,
0.30 mmol) in CH2Cl2 (6 mL) and water (3 mL) were added TEMPO
(9 mg, 0.06 mmol) and BAIB (193 mg, 0.6 mmol). After stirring for 6 h at
RT, the reaction mixture was quenched by addition of an aqueous solu-
tion of Na2S2O3 (10%, 5 mL). The phases were separated and the aque-
ous layer was extracted with EtOAc (2ꢅ5 mL). The combined organic
layers were dried and concentrated. The crude glucuronic acid was dis-
solved in THF (5 mL) and treated with diazomethane in Et2O at 08C.
When complete conversion of the uronic acid was observed by TLC anal-
ysis, the mixture was concentrated. The crude product was purified by
column chromatography on silica gel (CH2Cl2/acetone, 85:15) to give
compound 29 (359 mg, 88%) as a colorless syrup. [a]2D3 =+11.2 (c=0.12,
CHCl3); Rf =0.56 (CH2Cl2/acetone, 85:15); 1H NMR (360 MHz, CDCl3):
d=7.33–7.19 (m, 20H; ArH), 5.18 (s, 1H), 5.02–4.52 (m, 14H), 4.47 (d,
1
3
MeOH/water, 6:7:1); H NMR (500 MHz, D2O): d=5.51 (d, J
3.3 Hz, 1H; H-1-D), 5.43 (d, 3J(H1,H2)=3.2 Hz, 1H; H-1-F), 5.13 (d, 3J-
(H1,H2)=3.3 Hz, 1H; H-1-H), 5.09 (d, 3J
(H1,H2)=2.6 Hz, 1H; H-1-G),
4.79 (s, 1H; H-5-G), 4.68 (d, 3J(H1,H2)=7.5 Hz, 1H; H-1-E), 4.61 (t, 3J-
(H2,H3)=9.3 Hz, 1H; H-3-H), 4.53 (t, 3J
(H2,H3)=9.4 Hz, 1H; H-3-F),
4.37 (dd, 3J(H1,H2)=3.6 Hz, 3J
(H2,H3)=9.8 Hz, 1H; H-2-H), 4.33 (dd,
3J(H1,H2)=3.2 Hz, 3J
(H2,H3)=10.0 Hz, 1H; H-2-F), 4.16 (s, 1H; H-4-
G), 4.06 (t, J(H4,H5)=9.1 Hz, 1H; H-5-F), 3.95–3.87 (m, 2H; H-4-E, H-
ACHTUNGTRENNUNG(H1,H2)=
AHCTUNGTRENNUNG
G
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
E
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
3
AHCTUNGTRENNUNG
5-H), 3.82–3.74 (m, 5H; H-5-D, H-5-E, H-4-H, H-4-F, H-3-G), 3.66, 3.62,
3.61, 3.58, 3.56, 3.55, 3.45 (7ꢅs, 24H; 8ꢅOCH3), 3.61–3.55 (m, 3H; H-2-
G, H-3-E, H-3-D), 3.33–3.29 (m, 2H; H-2-E, H-2-D), 3.19–2.99 (m, 7H;
H-4-D, H-7a,b-D, H-7a,b-F, H-7a,b-H), 2.46–2.37 (m, 2H; H-6a-F, H-
6a-H), 2.23–2.17 (m, 1H; H-6a-D), 2.04–1.96 (m, 2H; H-6b-F, H-6b-H),
1.92–1.86 ppm (m, 1H; H-6b-D); 13C NMR (125 MHz, D2O): d=175.8,
175.3 (2ꢅCO), 102.9 (C-1-E), 101.3 (C-1-G), 98.0 (C-1-H), 96.0 (C-1-D),
94.6 (C-1-F), 86.9 (C-3-E), 83.7 (C-2-E), 83.3 (C-4-D), 82.1 (C-3-D), 81.6
(C-2-D), 79.3 (C-2-G) 78.9 (C-4-H) 78.5, 78.3, 77.6 (C-5-E, C-4-F, C-3-G),
77.1 (C-3-H), 76.6 (C-3-F), 76.5 (C-2-H), 76.0 (C-2-F), 73.8 (C-4-E, C-4-
G), 71.4 (C-5-G), 71.0 (C-5-F), 70.3 (C-5-H), 69.8 (C-5-D), 61.4, 61.1,
60.4, 60.3, 59.8, 58.9, 56.2 (8ꢅOCH3), 48.2, 48.0 (C-7-D, C-7-F, C-7-H),
3J
ACHTUNGTRENNUNG(H,H)=3.2 Hz, 1H), 4.28–4.22 (m, 2H; CH2), 3.89, 3.55, 3.48, 3.33, 3.31
(5ꢅs, 15H; 5ꢅOCH3), 3.84–3.66 (m, 8H), 3.50–3.10 (m, 9H), 2.38–2.24
(m, 2H), 2.07, 2.05, 2.02 (3ꢅs, 9H; 3ꢅAcCH3), 1.93–1.83 (m, 2H),
ACHTUNGTRENNUNG
1.38 ppm (t, 3J(H,H)=7.1 Hz, 3H; CH3); 13C NMR (90 MHz, CDCl3):
d=170.4, 170.1, 169.4, 169.2, 168.4 (5ꢅCO), 138.8, 138.5, 137.6, 137.5
(ArCq), 128.2–126.2 (ArC), 100.8, 98.0, 97.6, 97.4 (4ꢅC-1), 82.1, 80.0,
79.7, 79.0, 78.0, 76.0, 74.3, 74.1, 74.0, 71.5, 69.7, 68.7, 68.3, 67.7, 67.0 (skel-
eton carbons), 74.8, 74.1, 73.5, 73.1 (4ꢅPhCH2), 66.1 (CH2), 57.9, 55.3,
55.2, 52.3, 51.7 (5ꢅOCH3), 46.2, 45.6 (2ꢅC-7), 25.7, 25.5 (2ꢅC-6), 20.8,
20.4, 20.3 (3ꢅAcCH3), 14.8 ppm (CH3); MS (MALDI-TOF): m/z calcd
for C67H86O30S2Na: 1457.45 [M+Na]+; found: 1457.31; elemental analysis
calcd (%) for C67H86O30S2: C 56.06, H 6.04, S 4.47; found: C 55.72, H 5.81,
S 4.05.
27.2, 27.0 ppm (C-6-D, C-6-F, C-6-H); MS (ESI): m/z: 836.97 [Mꢀ7Na]2ꢀ
,
550.33 [Mꢀ6Na]3ꢀ; elemental analysis calcd (%) for C41H61Na9O46S7:
C 28.61, H 3.57, S 13.04; found: C 26.00, H 4.34, S 11.28.
Methyl-(6-O-deoxy-6-C-ethoxysulfonylmethyl-2,3,4-tri-O-methyl-a-d-glu-
copyranosyl)-(1!4)-[methyl-(2,3-di-O-acetyl-b-d-glucopyranosyl)-uro-
nate]-(1!4)-(2,3-di-O-benzyl-6-deoxy-6-C-ethoxysulfonylmethyl-a-d-glu-
copyranosyl)-(1!4)-[methyl-(2-O-acetyl-3-O-methyl-a-l-idopyranosyl)-
uronate]-(1!4)-2,3-di-O-benzyl-6-deoxy-6-C-methoxysulfonylmethyl-a-
Acknowledgements
The work was supported by the TꢆMOP 4.2.1/B-09/1/KONV-2010–0007
project, which is co-financed by the European Union and the European
Social Fund.
d-glucopyranoside (35): To
a solution of compound 29 (350 mg,
0.244 mmol) and compound 34 (154 mg, 0.366 mmol) in dry CH2Cl2
(10 mL) was added 4 ꢄ molecular sieves (1 g). After 30 min, the stirring
mixture was cooled to ꢀ208C and
a solution of NIS (124 mg,
0.549 mmol) in dry THF (300 mL) and TMSOTf (10 mL, 0.11 mmol) were
added. After stirring for 1 h at ꢀ208C, TLC analysis (CH2Cl2/acetone,
9:1) showed complete conversion of the donor molecule. The reaction
mixture was neutralized with Et3N (20 mL), diluted with CH2Cl2 (50 mL),
and filtered. The filtrate was washed with an aqueous solution of
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Received: March 27, 2012
Published online: && &&, 0000
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Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Analogues of the Pentasaccharide Domain of Heparin
FULL PAPER
Saccharides
M. Herczeg, L. Lꢁzꢁr, Z. Bereczky,
K. E. Kçvꢂr, I. Timꢁri, J. Kappelmayer,
A. Liptꢁk, S. Antus,
A. Borbꢁs* .................... &&&& — &&&&
The thin red line: The lower activity of
the trisulfonic-acid analogue of the
antithrombin-binding domain of hepa-
rin revealed that the substitution pat-
tern (or number of sulfonates) affected
the inhibitory potency of these deriva-
tives for factor Xa.
Synthesis and Anticoagulant Activity
of Bioisosteric Sulfonic-Acid
Analogues of the Antithrombin-
Binding Pentasaccharide Domain of
Heparin
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
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&
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These are not the final page numbers!