Synthesis of conformationally locked carbohydrates: A skew-boat conformation of L-iduronic acid governs the antithrombotic activity of heparin

Article abstract of DOI:10.1002/1521-3773(20010504)40:9<1670::AID-ANIE16700>3.0.CO;2-Q

Conformational flexibility of L-iduronic acid is a key feature of this typical component of heparin. Three pentasaccharides have now been synthesized, which are analogues of the active site of heparin and in which the single L-iduronic acid is conformatio

Full text of DOI:10.1002/1521-3773(20010504)40:9<1670::AID-ANIE16700>3.0.CO;2-Q

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moleculeº, camphor, where Dn/n ꢀ 10 8 was achieved.[9] Even
if more recent techniques could provide higher resolving
power,[10] a full rotational analysis for the IR spectrum of
camphor would be more difficult than for fluorooxirane, and
the experiment itself does not provide a value for DpvE. The
data calculated here are also sufficient for an estimate of the
equilibrium constant for racemization[28] from Equations (9)
and (10) where x and y explicitly show the deviation from
unity of the prefactor and the exponential factor.
[24] H. Hollenstein, D. Luckhaus, J. Pochert, M. Quack, G. Seyfang,
Angew. Chem. 1997, 109, 136 ± 138; Angew. Chem. Int. Ed. Engl. 1997,
36, 140 ± 143.
[25] H. Gross, G. Grassi, M. Quack, Chem. Eur. J. 1998, 4, 441 ± 448.
[26] H. Sengstschmid, A. Bauder, D. Luckhaus, M. Quack, unpublished
results.
[27] M. Quack, Nova Acta Leopoldina 1999, 81, 137 ± 173.
[28] M. Quack, J. Stohner, Chirality, 2001, in press.
[29] B. Char, K. Geddes, G. Gonnet, B. Leong, M. Monagan, S. Watt,
MAPLE V, Waterloo Maple Software, Waterloo, Ontario, 1990.
q
qvib;Rq
Rexp( DrH00/RT)
ˆ
rot;Rexp( DrH00/RT)
qvib;S qrot;S
(9)
R;S
K
ˆ
ꢀ
eq
qS
(1 ‡ x)(1 ‡ y)
(10)
Within the SHAA ,[20, 21] the equilibrium constant for
R;S
16
racemization at 300 K is K ꢀ 1 ‡ 8.20  10
with x ꢀ
eq
17
1.01 Â 10
and y ꢀ 8.30 Â 10 16. These values have been
obtained with perturbation theory and high precision arith-
metic (MAPLE V).[29] To our knowledge, this is the first time
that these contributions have been explicitly estimated,
although it should be understood that the harmonic approx-
imation is not quite adequate.[28]
Synthesis of Conformationally Locked
Carbohydrates: A Skew-Boat Conformation of
l-Iduronic Acid Governs the Antithrombotic
Activity of Heparin
Sanjoy K. Das, Jean-Maurice Mallet, Jacques Esnault,
Pierre-Alexandre Driguez, Philippe Duchaussoy,
Philippe Sizun, Jean-Pascal Herault, Jean-Marc
Received: October 31, 2000
Revised: February 12, 2001 [Z16028]
Â
Herbert, Maurice Petitou,* and Pierre SinayÈ*
The conformational flexibility[1] of l-iduronic acid, a typical
monosaccharide component of heparin, most probably ex-
plains its remarkable protein adaptability and resulting
diverse biological activities.[2, 3] The analysis of this feature
has been the matter of a long controversy,[4] which not
surprisingly originates from the complexity of the heparin
primary structure. A major breakthrough in heparinology has
been the identification,[5, 6] followed by the total synthesis,[7, 8]
of a well-defined pentasaccharide sequence inserted into the
heparin chain, which specifically binds to antithrombin
(AT)Ða physiological inhibitor of activated blood coagula-
tion factorsÐand amplifies its action. This is the molecular
origin of the anticoagulant and antithrombotic activities of
heparin.
[1] J. H. vanꢁt Hoff in La Chimie dans lꢀEspace (Ed.: P. M. Bazendijk),
Rotterdam, 1887.
[2] W. J. Hehre, L. Radom, P. von R. Schleyer, J. A. Pople, Ab initio
Molecular Orbital Theory, Wiley, New York, 1986.
[3] M. Quack, Angew. Chem. 1989, 101, 588 ± 604; Angew. Chem. Int. Ed.
Engl. 1989, 28, 571 ± 586.
[4] M. Quack, Chem. Phys. Lett. 1986, 132, 147 ± 153.
[5] A. L. Barra, J. B. Robert, L. Wiesenfeld, Europhys. Lett. 1988, 5, 217 ±
222.
[6] A. Bauder, A. Beil, D. Luckhaus, F. Müller, M. Quack, J. Chem. Phys.
1997, 106, 7558 ± 7570.
[7] V. Letokhov, Phys. Lett. A 1975, 53, 275 ± 276.
[8] O. Kompanets, A. Kukudzhanov, V. Letokhov, L. Gervits, Opt.
Commun. 1976, 19, 414 ± 416.
[9] E. Arimondo, P. Glorieux, T. Oka, Opt. Commun. 1977, 23, 369 ± 372.
Â
[10] C. Daussy, T. Marrel, A. Amy-Klein, C. Nguyen, C. Borde, C.
Chardonnet, Phys. Rev. Lett. 1999, 83, 1554 ± 1557.
[11] R. Compton, unpublished work, cited in R. F. Service, Science 1999,
286, 1282 ± 1283.
[12] ªChemical Evolution: Physics of the Origin and Evolution of Lifeº:
A. Bakasov, T. K. Ha, M. Quack in Proc. 4th Trieste Conf. 1995
(Eds.: J. Chela-Flores, F. Raulin), Kluwer, Netherlands, 1996,
pp. 287 ± 296.
[13] A. Bakasov, T. K. Ha, M. Quack, J. Chem. Phys. 1998, 109, 7263 ±
7285.
[14] R. A. Hegstrom, D. W. Rein, P. G. H. Sandars, J. Chem. Phys. 1980, 73,
2329 ± 2341.
[15] S. F. Mason, G. E. Tranter, Mol. Phys. 1984, 53, 1091 ± 1111.
[16] P. Lazzeretti, R. Zanasi, Chem. Phys. Lett. 1997, 279, 349 ± 354.
[17] A. Bakasov, M. Quack, Chem. Phys. Lett. 1999, 303, 547 ± 557.
[18] J. K. Laerdahl, P. Schwerdtfeger, Phys. Rev. A 1999, 60, 4439 ±
4453.
[19] R. Berger, M. Quack, J. Chem. Phys. 2000, 112, 3148 ± 3158.
[20] M. Quack, J. Stohner, Phys. Rev. Lett. 2000, 84, 3807 ± 3810.
[21] M. Quack, J. Stohner, Z. Phys. Chem. 2000, 214, 675 ± 703.
[22] J. Laerdahl, P. Schwerdtfeger, H. Quiney, Phys. Rev. Lett. 2000, 84,
3811 ± 3814.
1
The H NMR spectroscopic data of this original synthetic
pentasaccharide in aqueous solution were easily extracted and
[*] Prof. P. SinayÈ, Dr. S. K. Das, Dr. J.-M. Mallet, Dr. J. Esnault
Â
Â
Departement de Chimie, Associe au CNRS
Â
Ecole Normale Superieure
24 Rue Lhomond, 75231 Paris cedex 05 (France)
Fax : (‡33)1-44-32-33-97
E-mail: pierre.sinay@ens.fr
Â
Dr. M. Petitou, Dr. P.-A. Driguez, Dr. P. Duchaussoy, Dr. J.-P. Herault,
Dr. J.-M. Herbert
Â
Departement Cardiovasculaire/Thrombose
Â
Sanofi-Synthelabo
195 route dꢁEspagne, 31036 Toulouse cedex (France)
Fax : (‡33)5-61-16-22-86
E-mail: maurice.petitou@sanofi-synthelabo.com
Dr. P. Sizun
Â
DARA, Sanofi-Synthelabo
371 rue du professeur Joseph Blayac
34184 Montpellier cedex (France)
[23] A. Beil, D. Luckhaus, R. Marquardt, M. Quack, J. Chem. Soc. Faraday
Discuss. 1994, 99, 49 ± 76.
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were best explained by taking into
account the participation of the
unusual 2S0 skew-boat conformer
4
in addition to the classical C1 and
1C4 chair forms of the single l-
iduronic acid component.[9] This
was confirmed by force field studies
and energy computations.[10] The 2S0
conformer was shown to be the
major contributor to the conforma-
tional equilibrium of l-iduronic acid
in this pentasaccharide.[11]
In order to establish the relation-
ship between conformation and AT
activity, we performed total synthe-
ses of three pentasaccharides in
which the single l-iduronic residue
is conformationally locked, either in
the 1C4, 4C1, or 2S0 form. The
known[12] synthetic methylated pen-
tasaccharide 1 (Scheme 1) was chos-
en as the reference compound for
several reasons. Firstly, it binds
strongly to AT and selectively in-
hibits the blood coagulation factor -
Xa; secondly, the presence both of
methoxy groups in place of free
hydroxyl functions and of O- in
place of N-sulfonates largely sim-
plifies the corresponding synthetic
approach. Lastly, the hydroxyl
group at the C-2 position of l-
iduronic acid unit G in 1 is methy-
lated and not sulfated. Among the
various sulfate groups present in the
original pentasaccharide, the one on
the C-2 atom of l-iduronic acid was
found not to be essential for activity.
This auspicious feature is critical, in-
asmuch as it is a prerequisite for the
Scheme 2. a) The three most energetically stable conformers of the methylated iduronic acid unit G of the
biologically active pentasaccharide 1. b) The three conformationally locked l-iduronic acid mimics, which
have been synthesized and inserted in 1 in place of the unit G. c) The three resulting synthetic analogues of
the biologically active pentasaccharide 1 in which the l-iduronic acid G unit is unambiguously locked in
2
synthesis of a locked S0 skew-boat
conformer.
In the conformational formula of
1 shown in Scheme 1 the l-iduro-
nate unit G is represented exclu-
sively for the sake of simplicity in its 2S0 form. There is
actually an equilibrium in aqueous solution between the three
conformers: 1C4>2S0>4C1 (Scheme 2a). As indicated in
1
2
4
either the C4, S0, or C1 form, as shown in (b).
Scheme 2b, we have locked the l-iduronate ring G in one of
these conformations, and the three corresponding pentasac-
charides 2 ± 4 (Scheme 2c) have been synthesized.
The 1C4 and 4C1 chair forms are
well-defined conformers which are
rather easy to lock. The challenging
chemical question is how to isolate the
2S0 form from the pseudorotational
itinerary of the pyranoid ring.[13] We
reasoned that a covalent connection
between carbon atoms C-2 and C-5
would necessarily confine this itiner-
ary to the section 2S0>2,5B>5S1.
Scheme 1. The synthetic pentasaccharide 1 selected as the reference compound in this work.
Angew. Chem. Int. Ed. 2001, 40, No. 9 ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
More precisely, a one-atom bridge
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would freeze the unwanted 2,5B conformation, whereas a more
flexible two-atom bridge would probably allow the equili-
brium between the three forms, and a shift toward the more
stable skew-boat form. In the case of l-iduronic acid, this
equilibrium would be shifted to the wanted and favorable 2S0,
wherein the substituents are equatorially and isoclinally
oriented (see Scheme 2b).
In order to bridge the C-2 and C-5 positions, we first
synthesized a protected disaccharide 9 (Scheme 3). The easily
available[14] ketone 5 was transformed in three steps into the
tetraacetate 7. The first step, the face-selective addition of
vinyl magnesium bromide, is explained by a chelation of the
magnesium to the ring oxygen atom of 5. The b-acetate 7,
whose structure has been confirmed by NMR spectroscopy, is
well suited[15] for selective 1,2-transglycosylation of the
known[16] alcohol 8. The derivative 9 was then easily trans-
formed (see Scheme 3) into the key alcohol 14, which is a
locked protected equivalent of the G-H section of the
pentasaccharide 1. The known[17] trisaccharidic imidate 15,
which has successfully been used for the synthesis of the
parent active pentasaccharide 1,
was then condensed with the alco-
hol 14 to afford the protected
pentasaccharide 16. A sequence
of well-established reactions[18]
then gave the pentasaccharide 2.
1
Comparison of the H NMR cou-
pling constants of the locked l-
iduronic acid residue in 2 (J1,2
1.3, J2,3 ˆ 1.4, J3,4 ˆ 2.7, J2,4
ˆ
ˆ
0.5 Hz) with those reported by
Köll et al.[19] for methyl-2,6-anhy-
dro-b-d-mannopyranoside deriva-
2
tives that exist in the S0 confor-
mation (J1,2 ˆ 1.2 ± 1.4, J2,3 ˆ 1.2 ±
2.4, J3,4 ˆ 3.0 ± 3.4, J2,4 ˆ 0.7 Hz)
confirms that our derivative
adopts
a similar conformation.
Much to our delight, the 2S0-
locked pentasaccharide 2 was able
to strongly activate AT with re-
spect to factor Xa (Table 1).
As shown schematically in
Scheme 4, a pivotal compound of
type 9 has also been used to
construct 1C4- and 4C1-locked
blocks, which were then trans-
formed into the two pentasacchar-
ides 3 and 4.[20, 21] A pentasacchar-
ide similar to 3 has already been
synthesized,[22] but it bears a sul-
fate group at the C-2 position of l-
iduronic acid so its properties
cannot be directly compared with
those of our locked compounds to
assess the influence of the confor-
mation of l-iduronic acid on the
biological properties. It should be
noted that the 4C1 chair conforma-
tion of the G unit of 4 is ensured
by the methoxymethyl group at-
tached to the C-5 atom, which
replaces the hydrogen atom of l-
iduronic acid (Scheme 2b).
ˆ
Scheme 3. General strategy used to synthesize the pentasaccharide 2. a) CH2 CHMgBr, THF, 08C, 1 h,
70%; b) 1. IR-120 H ion-exchange resin, H2O, 80 8C, 6 h; 2. Ac2O, pyridine, RT, 16 h, 75% (two steps); c) 8,
‡
TMSOTf, CH2Cl2, 788C, 2 h, 85%; d) 1. MeONa, MeOH, 08C then RT, 3 h; 2. (CH3O)2C(CH3)2, p-TsOH,
acetone, RT, 16 h, 70% (two steps); e) 1. (COCl)2, DMSO, CH2Cl2, 788C, 45 min; 2. LiEt3BH, THF,
788C then RT, 1 h, 70% (two steps); f) 1. Ac2O, pyridine, RT, 3 h; 2. AcOH, 608C, 2 h, 70% (two steps);
3. TsCl, pyridine, RT, 3 h, 80%; g) NaOH, EtOH, 708C, 3 h, 70%; h) 1. O3, CH2Cl2, 788C, Me2S, then
2-methyl-2-butene, tBuOH, H2O, NaH2PO4, NaClO2, RT, 16 h; 2. BnBr, Bu4NI, KHCO3, DMF, RT, 5 h,
80% (three steps); i) TBDMSOTf, CH2Cl2, Et2O, 208C, 30 min, 67%; j) 1. H2, Pd/C, AcOH, 408C, 12 h;
2. NaOH, H2O, 55 8C, 3 h, 86% (two steps); 3. Et3N ´ SO3, DMF, 558C, 18.5 h, 85%. Bn ˆ benzyl, DMF ˆ
dimethylformamide, DMSO ˆ dimethyl sulfoxide, TBDMS ˆ tert-butyldimethylsilyl, Tf ˆ triflate ˆ tri-
fluoromethanesulfonyl, TMS ˆ trimethylsilyl, Ts ˆ tosyl ˆ toluene-4-sulfonyl.
As shown in Table 1, the two
pentasaccharides 3 and 4, which
contain the G unit locked in the
4C1 and 1C4 conformations, respec-
tively, only very slightly potentiate
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Table 1. Activity a against factor Xa by the pentasaccharides 1 ± 4 and the
reference compound 17.
changes of the pyranose ring have important significance in
the biology of heparin. Other recent studies have also
emphasized the importance of boat or skew-boat conformers
in biology.[25±28] We believe that the importance of the
conformational flexibility of carbohydrates is now very clear
and is going to emerge as a key feature in this field of science.
1
[c]
Compound
a [umg ]
1[a]
2
3
1208 Æ 63
1073 Æ 61
115 Æ 3
4
43 Æ 3
17[b]
1345 Æ 65
Received: November 23, 2000 [Z16162]
[a] The activity against factor Xa of the genuine nonmethylated synthetic
1
pentasaccharide is 1013 Æ 52 umg
. [b] Compound 17 is a synthetic
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heparin standard, Bz ˆ benzoyl.
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[20] A full account of the syntheses of 3 and 4 will be separately described
elsewhere.
[21] In the case of the pentasaccharide 4, the glucuronic acid unit E has
also been modified, with the H-5 atom being replaced by an ethyl
group. This is a direct consequence of the selected converging strategy
that has been selected for the sake of simplicity. We have, however,
shown that replacement of the H-5 atom by an ethyl group in
compound 1 does not affect its biological activity (see Table 1).
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Scheme 4. Our synthetic strategy is based on the construction of a key
structural element in which the C-5 atom is harnessed with two appendages,
an equatorially oriented protected hydroxymethyl group and an axially
oriented vinyl group. Through classical manipulations, this architecture can
be selectively converted into one of the three conformers.
the inhibition of the blood coagulation protease factor Xa.
This work unambiguously demonstrates for the first time that
2
the AT-bound l-iduronic acid unit G adopts the unusual S0
conformation and clearly explains how the unique conforma-
tional behavior of l-iduronic acid translates in terms of
biological activity.
It is likely that the accessibility of the three conformers is
the basis for the high versatility of glycosaminoglycans
(GAGs) that contain l-iduronic acid for binding to basic sites
of interacting proteins. Although the 2S0 skew-boat conformer
nicely governs the antithrombotic activity of heparin, it could
very well be that the chair forms are critical in other situations,
such as high affinity for fibroblast growth factors.[23] The
availability of the three synthetic locked conformers offers a
unique opportunity to replace any flexible l-iduronic acid
residue in a GAG chain by a conformationally defined
counterpart, thus providing a direct tool for general explora-
tion of the relationship between conformational flexibility
and biological properties of GAGs.
[25] P. E. Marszalek, A. F. Oberhauser, Y.-P. Pang, J. M. Fernandez, Nature
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As laid out by Barton,[24] a conformational analysis of cyclic
molecules is critical for the understanding of their biological
action. We have demonstrated here that the conformational
Angew. Chem. Int. Ed. 2001, 40, No. 9
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