Total synthesis of anticoagulant pentasaccharide fondaparinux

Article abstract of DOI:10.1002/cmdc.201400019

The anticoagulant pentasaccharide fondaparinux was synthesized using an improved and optimized synthetic strategy including a convergent [3+2] coupling approach, orthogonal protecting groups and various glycosyl donors. The new methods of glycosylation were also used for controlling the stereochemical configuration and improving the yield of the glycosylation. In addition, HPLC and NMR methods to monitor the process of total synthesis of fondaparinux were employed. This work provides a comprehensive elaboration for the synthesis and analysis of fondaparinux based on related literature, as well as abundant information for the synthesis of heparin-like oligosaccharides. A matter of protection! The anticoagulant pentasaccharide fondaparinux was synthesized using an improved and optimized synthetic strategy. The process of total synthesis was monitored by HPLC and NMR. This work will contribute to continued improvement of the multistep production of fondaparinux and provide abundant information for the synthesis of heparin-like oligosaccharides.

Full text of DOI:10.1002/cmdc.201400019

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DOI: 10.1002/cmdc.201400019
Total Synthesis of Anticoagulant Pentasaccharide
Fondaparinux
Tiehai Li, Hui Ye, Xuefeng Cao, Jiajia Wang, Yonghui Liu, Lifei Zhou, Qiang Liu,
Wenjun Wang, Jie Shen,* Wei Zhao,* and Peng Wang*[a]
The anticoagulant pentasaccharide fondaparinux was synthe-
sized using an improved and optimized synthetic strategy in-
cluding a convergent [3+2] coupling approach, orthogonal
protecting groups and various glycosyl donors. The new meth-
ods of glycosylation were also used for controlling the stereo-
chemical configuration and improving the yield of the glycosy-
lation. In addition, HPLC and NMR methods to monitor the
process of total synthesis of fondaparinux were employed. This
work provides a comprehensive elaboration for the synthesis
and analysis of fondaparinux based on related literature, as
well as abundant information for the synthesis of heparin-like
oligosaccharides.
Introduction
Heparin and heparan sulfate, members of the glycosaminogly-
cans (GAGs) family, are structurally related linear polyanionic
polysaccharides, consisting of a-1,4-linked glucosamine and
uronic acid (either d-glucuronic or l-iduronic) disaccharide re-
peating units, which are heavily O- and N-sulfated.[1] They are
present on the surface of most animal cells as well as in the
basement membranes and extracellular matrices.[2] It is well es-
tablished that heparin and heparan sulfate play significant
roles in various biological processes such as blood coagulation,
bacterial and viral infection, inflammation, growth factor regu-
lation, cell adhesion, cell growth, tumor metastasis, lipid me-
tabolism and diseases of the nervous system.[3]
fested with the case of worldwide distribution of contaminated
animal-sourced heparin in 2007, which caused hundreds of pa-
tient deaths in the United States, and raised the concerns over
the reliability and safety of animal-sourced heparins and LMW
heparins.[6]
A unique pentasaccharide domain in some heparin chains
was discovered that blocks factor Xa in the coagulation cas-
cade. In light of the discovery, a homogeneous chemically syn-
thesized this pentasaccharide was patented by Choay[7a] and
Sanofi,[7b] and later led to the debut of antithrombotic drug
fondaparinux (Arixtaꢀ, Figure 1) on the market by GlaxoSmith-
Kline in the USA in 2002.[7] Fondaparinux has been demonstrat-
Heparin and heparan sulfate are widely used as anticoagu-
lant drugs for major cardiovascular and orthopedic surgeries
such as hip fractures, knee surgery or hip replacement, pre-
venting the occurrence of venous thrombosis.[4] Current hepa-
rin-based treatment in the clinic mainly relies on the heteroge-
neous unfractionated (UF, molecular weight average ~14000)
or low-molecular-weight (LMW, molecular weight average
~6000) heparins that are either extracted from natural animal
sources (porcine intestine or bovine lung) or degraded forms
of such extractions.[5] The problems associated with UF and
LMW heparin therapies are the challenges in product quality
control in the process of its preparation owing to the non-uni-
formity of the enormous bulk of sources. The problem mani-
Figure 1. Structure of fondaparinux (Arixtraꢀ).
ed to display superior antithrombotic activity and brings about
antithrombin-mediated activity against factor Xa exclusively,
not against thrombin.[8] As a pure synthetic pentasaccharide, it
is much easier to control product quality in terms of reproduc-
tion reliability and purity. Furthermore, the synthetic homoge-
nous pentasaccharide has multiple advantages over the afore-
mentioned two forms in terms of pharmacokinetics (such as
longer circulation half-life and low IC50 value) and product
quality control.[9] Therefore, it is very significant and urgent for
clinical applications to synthesize the homogeneous pentasac-
charide with a highly efficient approach. In 2011, Dr. Reddy’s
Laboratories Ltd marketed fondaparinux in the USA, a bioequi-
valent generic version of Arixtraꢀ using a different patented
[a] Dr. T. Li,+ H. Ye,+ X. Cao, J. Wang, Y. Liu, L. Zhou, Q. Liu, W. Wang,
Dr. J. Shen, Dr. W. Zhao, Prof. P. Wang
State Key Laboratory of Medicinal Chemical Biology
and College of Pharmacy, Nankai University
Synergetic Innovation Center of Chemical Science and Engineering (Tianjin)
94 Weijin Road, Tianjin 300071 (P.R. China)
E-mail: jieshen@nankai.edu.cn
wzhao@nankai.edu.cn
pwang@nankai.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400019.
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Results and Discussion
synthetic process developed by Alchemia Ltd.[7c] In addition,
a variety of synthetic methods for the preparation of heparin-
like oligosaccharides have been developed over the past two
decades.[7f–h,10] Various analogues of the antithrombin III bind-
ing pentasaccharide domain of heparin were synthesized by
the van Boeckel and Petitou labs, and structure–activity rela-
tionship (SAR) studies revealed that essential sulfate and car-
boxylate substituents were located at opposite sides of the
pentasaccharide molecule.[10b] Using a straightforward synthesis
of the l-idopyranosyl building block and a-glycosylation by
glucosamine-derived donors for the total synthesis of heparin
oligosaccharides was accomplished by Hung and co-work-
ers.[10j,k] A modular strategy toward the synthesis of heparin-
like oligosaccharides in a sequen-
Synthetic strategy for fondaparinux
Fondaparinux consists of a-1,4-linked glucosamine with uronic
acids (d-glucuronic acid and l-iduronic acid), as well as b-1,4-
linked d-glucuronic and a-1,4-linked l-iduronic acid with d-glu-
cosamine. The positions of O-sulfations are C-6 of glucosamine,
C-2 of iduronic acid, and rarely C-3 of glucosamine. C-2 amino
groups of glucosamine are all N-sulfated (Figure 1).
As illustrated in Scheme 1, we envisaged the preparation of
fully protected pentasaccharide to be prepared by a conver-
gent and stereocontrolled [3+2] approach. The protecting
tial glycosylation protocol using
sulfonium triflate activator sys-
tems was developed in the
van der Marel lab.[10a] Boons and
co-workers developed a modular
approach for the parallel combi-
natorial synthesis of well-defined
heparin oligosaccharide library
for SAR studies by utilizing a rela-
tively small number of selectively
protected and flexible disacchar-
ide building blocks.[10i] A highly
efficient one-pot methodology
for the synthesis of heparin-like
oligosaccharides utilizing thio-
glycosides with well-defined re-
activity as building blocks was
developed by Wong’s lab.[10h]
Moreover, Huang’s lab also de-
veloped the preactivation-based
one-pot approach combinatorial
synthesis of heparin-like oligo-
saccharides for the analysis of
heparin–protein interactions.[10f]
Based on these synthetic strat-
egies of heparin-like oligosac-
charides, we established an im-
proved and optimized synthetic
strategy that exploited a set of
closely related building blocks
Scheme 1. Synthetic strategy of fondaparinux.
and new methods of glycosyla-
tion to assemble fondaparinux
pentasaccharide. We also ap-
plied HPLC and NMR methods to monitor the process of total
synthesis of fondaparinux. Furthermore, the glycosylation of
uronic acid building blocks with low reactivity imposed by the
C-5 ester was investigated.
group strategy was established for O-sulfation and selective N-
sulfation, as well as stereoselectivity of glycosylation. Acetyl
and benzoyl protecting hydroxy groups intended to be O-sul-
fated, benzyl ethers were used as permanent protection
groups for other free hydroxy groups. Azido groups were used
as amino functionalities for subsequent N-sulfation, as well as
performing non-neighboring group participation for the intro-
duction of 1,2-cis-glycosidic linkages. The benzoyl protecting
C-2 hydroxy of iduronic acid (building block 5) allowed the ste-
reoselective introduction of 1,2-trans-glycosidic linkages by
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neighboring group participation. The 1-thio iduronic acid
building block 5 displayed a highly disarmed class of glycosyl
donor, it was activated by 1-benzene sulfinyl piperidine/triflic
anhydride (BSP/Tf2O) to couple with building block 6.[10a,11] It is
worth mentioning that levulinoyl (Lev) protecting the C-2 hy-
droxy group of glucuronic acid, which can be selectively re-
moved and transferred to other protecting hydroxy groups,
was envisaged to construct b-1,4-linked d-glucuronic with glu-
cosamine.[10i] However, due to the inherent low reactivity im-
posed by the C-5 ester and the disarmed Lev protecting
group, coupling this lowly active glucuronic acid donor with
glucosamine acceptor led to unsuccessful glycosylation. There-
fore, using the armed benzyl ether (Bn) protecting C-2 hydroxy
group of glucuronic acid (building block 3) to enhance the ac-
tivity as the glycosyl donor, a Ag2CO3-mediated coupling of
glycosyl bromide (building block 3) with building block 4
under mild condition slowly led to the desired disaccharide.[10e]
In addition, the 9-fluorenylmethyl carbonate (Fmoc) protecting
the C-4 hydroxy group of building block 5 and the chloracetyl
group protecting building block 3 can be easily removed with
triethylamine and thiourea, respectively, to give the corre-
sponding glycosyl acceptors for glycosylation without affecting
other protecting groups.[10e,i]
glucosamine hydrochloride with imidazole-1-sulfonyl azide as
a diazotransfer reagent gave 2-azido-2-deoxy-d-glucopyranose
8 in 85% yield using our previously reported methods.[12]
Bringing a solution of 8 in methanol with 10% HCl to reflux af-
forded methyl glycoside 9 as a mixture of anomers, which
were treated with PhCH(OMe)2 and camphorsulfonic acid (CSA)
in acetonitrile to give 4,6-O-benzylidence 10.[10d] Benzylation of
the remaining 3-hydroxy group with BnBr and NaH in THF
gave the corresponding a/b anomers. The desired a anomer
11 was separated by crystallization from petroleum ether/ethyl
acetate mixtures in 49% yield. Hydrolysis of the 4,6-O-benzyli-
dene acetal gave diol 12 using 80% AcOH. Selective protection
of the 6-hydroxy group with benzoate ester gave the desired
building block 6 in 69% yield over two steps.
Glucosazide building blocks 2 and 4 were synthesized using
the common intermediate 1,6-anhydro-2-azido-2-deoxy-b-d-
glucopyranose 14 (Scheme 3), which can be easily prepared
from readily available d-glucal 13 based on previously reported
methods.[13] Benzylation of intermediate 14 using benzyl bro-
mide and NaH in N,N-dimethylformamide (DMF) gave the
benzyl ether 15 in 92% yield. 1,6-anhydride 15 was subjected
to acetolysis in the presence of catalytic amount of tert-butyl-
dimethylsilyl trifluoromethanesulfonate (TBSOTf) to afford 1,6-
diacetate 16 in 92% yield. Selective removal of the anomeric
acetyl group using NH3 in the presence of THF/MeOH (7:3)
gave lactol 17 in 85% yield; the resulting lactol was converted
into desired trichloroacetimidate 2 using trichloroacetonitrile
and 1,8-diazazdicycloundec-7-ene (DBU) in 93% yield.[10d] In ad-
dition, selective silylation of the C-4 hydroxy of intermediate
14 gave silyl ether 18 using tert-butyldimethylsilyl chloride
(TBDSCl) and imidazole in 90% yield.[13b] The remaining C-3 hy-
droxy group of 18 was protected
Preparation of monomeric building blocks
Based on the synthetic strategy for fondaparinux, five strategi-
cally chosen monosaccharide building blocks 2, 3, 4, 5 and 6
were initially synthesized. The glucosazide building block 6
was synthesized starting from commercially available d-glucos-
amine hydrochloride 7 in six steps (Scheme 2). Treatment of d-
with acetyl ester giving 19,
which was then subjected to de-
silylation with TBAF and AcOH
to provide desired building
block 4 in 94% yield.[13b,14]
The l-iduronic acid building
block 5 was efficiently synthe-
sized as illustrated in Scheme 4.
The commercially available diac-
Scheme 2. Reagents and conditions: a) Imidazole-1-sulfonyl azide, K2CO3, CuSO4, MeOH, RT, overnight, 85%;
b) 10% HCl/MeOH, reflux, 908C, 24 h; c) CSA, PhCH(OMe)2, CH3CN, 508C, 5 h, 65% (two steps); d) NaH, BnBr, THF,
08C to RT, overnight, 49%; e) 80% HOAc, 608C, 6 h; f) BzCl, pyridine,CH2Cl2, ꢀ208C, 3 h, 69% (two steps).
etone a-d-glucose 20 was con-
verted into 1,6-anhydro-b-d-ido-
Scheme 3. Reagents and conditions: a) NaH, BnBr, DMF, 08C to RT, overnight, 92%; b) TBSOTf, Ac2O, 08C, 20 min, 92%; c) NH3, THF/MeOH (7:3), 08C, 85%;
d) CCl3CN, DBU, CH2Cl2, RT, 3 h, 93%; e) TBDSCl, imidazole, DMF, 08C to RT, overnight, 90%; f) Ac2O, pyridine, CH2Cl2, 08C to RT, overnight, 89%; g) TBAF,
AcOH, THF, RT, overnight, 94%.
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nylsulfoxide/triflic
anhydride
(Ph2SO/Tf2O) as a powerful pro-
moter system can activate dis-
armed 1-thio uronic acid build-
ing blocks 37, facilitating its con-
densation
with
glucosazide
building block 4.[10a,18] Unfortu-
nately, the strategy of glycosyla-
tion could not give the desired
product, but substantial decom-
position of the glycosyl donor
and acceptor were observed by
TLC. It was considered that this
glucuronic acid donor was too
inactive due to the inherent low
.
Scheme 4. Reagents and conditions: a) TMSOTf, Ac2O, 08C to RT, 2 h, 81%; b) BF3 Et2O, PhSH, CH2Cl2, 08C to RT, 4 h,
82%; c) 1. NaOCH3, CH3OH, RT, 12 h; 2. PhCH(OCH3)2, CSA, DMF, RT, 6 h; d) BzCl, pyridine, CH2Cl2, 08C to RT, over-
night, 77% (three steps); e) 80% AcOH, 808C, 2 h, 72%; f) 1. TEMPO, BAIB, CH2Cl2/H2O, RT, 4 h; 2. CH3I, KHCO3,
DMF, 08C to RT, 4 h, 50% (two steps); g) FmocCl, pyridine, DMAP, CH2Cl2, 08C to RT, 3 h, 78%.
pyranose derivative 21 by the methods of Hung and Fꢂge-
Table 1. Glycosylation of glucouronic acid derivates with acceptor 4.
di.[10j,15] Treatment of 21 with Ac2O and catalytic amounts of tri-
methylsilyl trifluoromethanesulfonate (TMSOTf) gave tetra-
acetate 22 in 81% yield, which was readily converted into thio-
glycoside 23 in the presence of thiophenol and BF3·Et2O in
82% yield.[10d] Using standard methods, the acetate esters of
23 were removed with catalytic amounts of NaOCH3. Then,
4,6-O-benzylidene acetal 24 was formed using PhCH(OCH3)2
and CSA in DMF. The remaining hydroxy group of 24 was ben-
zoylated to give 25 in 77% yield over three steps. Removal of
the 4,6-O-benzylidene group of 25 using 80% AcOH gave diol
26 in 72% yield, which was subjected to selective oxidation of
the resulting primary hydroxy group with 2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPO) in the presence of iodobenzene diace-
tate (BAIB) as co-oxidant.[16] The resulting carboxyl groups were
then esterified in the presence of KHCO3 and MeI to give de-
sired 27 in 50% yield over two steps.[10h] Blocking the remain-
ing 4-hydroxy group with Fmoc in the presence of FmocCl,
4-dimethylaminopyridine (DMAP) and pyridine gave l-iduronic
acid building block 5 in 78% yield.[17]
Donors
Activators
Yield [%] (a/b)[c]
37
33
3
Ph2SO/Tf2O/TTBP[a]
Ph2SO/Tf2O/TTBP[a]
ND[d]
58 (4.6:1)
72 (1:7)
[b]
Ag2CO3
[a] Conditions: 4 ꢃ MS, CH2Cl2, ꢀ608C, then ꢀ408C to RT. [b] Conditions:
4 ꢃ MS, CH2Cl2, RT. [c] Anomeric ratio determined from anomeric mixture
by 1H NMR. [d] ND: no desired product.
reactivity imposed by the C-5 ester and the disarmed Lev pro-
tecting group at the C-2 position to lead to unsuccessful glyco-
sylation.[19] In order to improve the reactivity of the glucouron-
ic acid building block, disarmed levulinoyl (Lev) protecting C-2
hydroxy group was changed to armed benzyl ether giving 33.
Coupling 33 with 4 gave the corresponding disaccharide 40 as
a mixture of anomers (a/b=4.6:1) using the same glycosyla-
tion conditions; however, the ratio of desired b anomer was
too low. It was observed that Ag2CO3-mediated coupling of
glycosyl bromide 3 with 4 under mild condition can slowly
form disaccharide 40 as a mixture of anomers (a/b=1:7).[7c,10e]
The glucouronic acid building blocks 3, 33 and 37 were syn-
thesized as described in Scheme 5. The treatment of commer-
It was initially envisaged to synthesize glucouronic acid
building block 37 with levulinoyl (Lev) protecting the C-2 hy-
droxy group for the construct b-1,4-linked d-glucuronic acid
with 4 due to neighboring group participation (Table 1). Diphe-
Scheme 5. Reagents and conditions: a) BF3·Et2O, TolSH, CH2Cl2, 08C to RT, 4 h, 86%; b) 1. NaOCH3, CH3OH, RT, 2 h; 2. PhCH(OCH3)2, CSA, DMF, RT, 6 h, 83%
(two steps); c) NaH, BnBr, DMF, 08C to RT, overnight, 85%; d) 1. CH2Cl2/TFA/H2O, 10:1:0.1, RT, 20 min; 2. TEMPO, BAIB, CH2Cl2/H2O, RT, 5 h; 3. CH3I, KHCO3,
DMF, 08C to RT, 4 h, 42% for 32, 40% for 36 (three steps); e) (ClCH2CO)2O, pyridine, CH2Cl2, 08C to RT, 6 h, 86% for 33, 82% for 37; f) IBr, CH2Cl2, RT, 4 h, 72%;
g) 1. (Bu2Sn)O, toluene, reflux, 6 h; 2. BnBr, CsF, DMF, RT, overnight, 63% (two steps); h) LevOH, DCC, DMAP, CH2Cl2, 08C to RT, overnight, 83%.
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cially available b-peracetylated glucose 28 with para-toluene-
thiol (TolSH) in the presence of BF3·Et2O afforded 29 in 86%
yield. Using standard methods, the acetate esters of 29 were
removed with catalytic amounts of NaOCH3, and then 4,6-O-
benzylidene acetal 30 was generated using PhCH(OCH3)2 and
CSA in DMF. Benzylation of 30 using benzyl bromide and NaH
in DMF gave the benzyl ether 31 in 85% yield. The benzyli-
dene acetal of 31 was removed by TFA in CH2Cl2 and water,
followed by selective oxidation
the activation of BSP,[10a] a powerful promoter system devel-
oped by the Crich laboratory.[11] Activation of the anomeric thi-
ophenyl group of idouronate 5 with the BSP/Tf2O reagent
system proceeded efficiently for condensation of the glucosa-
zide building block 6 to afford the desired disaccharide 38 in
56% yield. Subsequently, the Fmoc group can be readily re-
moved with triethylamine to give 39 as an acceptor for the
next glycosylation[10i] (Scheme 6).
of the resulting primary hydroxy
group with TEMPO in the pres-
ence of BAIB as co-oxidant.[10i]
The resulting carboxyl groups
were then esterified in the pres-
ence of KHCO3 and MeI to afford
desired 32 in 42% yield over
Scheme 6. Reagents and conditions: a) BSP, Tf2O, 4 ꢃ MS, CH2Cl2, ꢀ608C, then ꢀ408C to RT, 4 h, 56%; b) Et3N,
three steps.[10h] The free C-4 hy-
droxy group of this glucouronic
acid building block was protect-
CH2Cl2, RT, 2 h, 88%.
ed with a chloracetyl group in the presence of chloroacetic
acid anhydride and pyridine. The chloracetyl group can be
readily removed with thiourea as an acceptor for glycosyla-
tion.[7c] Thioglycoside 33 was converted into the corresponding
glycosyl bromide 3 with IBr in 72% yield.[20] In addition, regio-
selective benzylation of the C-3 hydroxy of 30 by treatment
with dibutyltin oxide, followed by reaction with benzyl bro-
mide in the presence of CsF in DMF afforded 34 in 63% yield.
The C-2 hydroxy of 34 was protected with Lev to give 35 by
reaction with levulinic acid (LevOH) in the presence of N,N’-di-
cyclohexylcarbodiimide (DCC) and DMAP.[10d] Subsequently, glu-
couronic acid building block 37 can be readily obtained using
the same methods of synthesis of 33 from 31.
Condensation of glucouronic acid building block 3 with glu-
cosazide building block 4 slowly formed disaccharide 40 as
a mixture of anomers (a/b=1:7) in the presence of silver car-
bonate[7c,10e] (Scheme 7). The resulting mixture of anomers was
subjected to removal of the chloracetyl group using thiourea
to give the corresponding disaccharide anomers, which can be
separated by silica column chromatography to give desired
b-1,4-linked disaccharide 41 as an acceptor for next coupling
in 55% yield over two steps. A TBSOTf-mediated coupling of
the trichloroacetimidate donor 2 with acceptor 41 afforded de-
sired trisaccharide 42 in 78% yield. Opening of the anhydro
bridge of 42 and subsequent deacetylation of the anomeric
center gave lactol 43 in 64% yield over two steps.[10k] The re-
sulting lactol was converted into desired trichloroacetimidate
44 using trichloroacetonitrile and K2CO3 in 81% yield. A con-
vergent [3+2] coupling of this trichloroacetimidate donor 44
and acceptor 39 in the presence of TMSOTf afforded the fully
protected pentasaccharide 45 in 68% yield. The convergent
[3+2] coupling approach improved the utilization of difficultly
Glycosylation and assembly of fully protected pentasacchar-
ide
Thioglycosides of the idouronate were reported by van der
Marel and co-workers to be effective glycosyl donors under
Scheme 7. Reagents and conditions: a) Ag2CO3, 4 ꢃ MS, CH2Cl2, RT, 4 days; b) thiourea, CHCl3, 608C, 24 h, 55% (two steps); c) TBSOTf, 4 ꢃ MS, toluene, ꢀ208C
to RT, 2 h, 78%; d) 1. TBSOTf, Ac2O, 08C, 20 min; 2. NH3, THF/MeOH (7:3), 08C, 5–10 min, 64% (two steps); e) CCl3CN, K2CO3, CH2Cl2, 4 h, 81%; f) TMSOTf, 4 ꢃ
MS, CH2Cl2, ꢀ208C to RT, 2 h, 68%.
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prepared l-iduronic acid building block due to the use of dis-
accharide 39 as an acceptor and efficient synthesis of 39 by
the BSP/Tf2O-mediated promoter system.
Conclusions
In summary, the total synthesis of anticoagulant pentasacchar-
ide fondaparinux has been improved and optimized. We estab-
lished an effective protecting group strategy for O-sulfation
and selective N-sulfation, as well as, stereoselective glycosyla-
tion. The BSP/Tf2O-mediated promoter system contributed to
coupling disarmed thioglycosides of idouronate with appropri-
ate glucosazide building blocks. The convergent [3+2] cou-
pling approach also improved the utilization of difficultly pre-
pared l-iduronic acid building block. In addition, the HPLC and
NMR methods were used to monitor the process of total syn-
thesis of fondaparinux. Overall, we report a comprehensive
elaboration for the synthesis and analysis of fondaparinux. We
believe that the continued improvement of the multistep pro-
duction of fondaparinux will be a critical task for medicinal and
process chemistry worldwide.
Preparation of fondaparinux
With the fully protected pentasaccharide 45 in hand, the
acetyl, benzoyl and methyl esters of 45 were saponified by
a two-step procedure using LiOH and H2O2 in THF, and then
NaOH in methanol to afford partially deprotected 46 (Sche-
me 8).[10i] O-sulfation of the resulting hydroxy groups of 46 was
achieved with sulfur trioxide trimethylamine complex in DMF.
The crude of O-sulfated product was purified to give 47 by
Dowex 50WX4 Na+-form and Sephadex LH-20 columns, re-
spectively. Removal of the benzyl ethers and reducing azido of
47 gave amine 48 by catalytical hydrogenation in the presence
of Pd/C, followed by selective N-sulfation of 48 employing pyr-
idinium sulfur trioxide by maintaining pH 9.5 with 2m NaOH.
Finally, the crude N-sulfated product was purified to give fon-
daparinux (1) by Dowex 50WX4 Na+-form and Sephadex G-25
columns, respectively.[10e,h]
Experimental Section
General procedures: All reagents were purchased from commer-
cially sources and used without further purification. All solvents
were available commercially dried or freshly dried and distilled
prior to use. Reactions were monitored by thin-layer chromatogra-
phy (TLC) using silica gel GF254 plates with detection using short-
wave UV light (l=254 nm) and staining with 10% phosphomolyb-
dic acid in EtOH or a p-anisaldehyde solution (EtOH/p-anisalde-
hyde/AcOH/H2SO4, 135:5:4:1.5), followed by heating on a hot
plate. Column chromatography was conducted using silica gel
(200–300 mesh) with EtOAc and petroleum ether or EtOAc (or
CH2Cl2) and MeOH as eluent. H NMR and 13C NMR were recorded
1
with a Bruker AV 400 spectrometer at 400 MHz and 100 MHz, re-
spectively, using CDCl3, CD3OD and D2O as solvents. Chemical
shifts (d) are reported in ppm from CDCl3 (d=7.26 ppm for
1
1H NMR, d=77.00 ppm for 13C NMR), CD3OD (3.31 ppm for H NMR,
d=49.00 ppm for 13C NMR). Coupling constants are reported in
Hertz. High-resolution mass spectra (HRMS) were obtained on
a Varian QFT-ESI mass spectrometer. Compound purity was deter-
mined by HPLC analysis on a Waters e2695 system with a Waters
Atlantis T3 column (4.6 mmꢄ150 mm, 5 mm) and Dinoex Carbopac
PA column (14 mmꢄ250 mm, 5 mm) at 1.0 mLminꢀ1 using a Waters
2998 photodiode array detector at 220 nm.
Methyl O-(methyl 2-O-benzoyl-3-O-benzyl-4-O-(4-(((9H-fluoren-9-
yl)methoxy)carbonyloxy)-a-l-idopyranosyluronate)-(1!4)-2-
azido-6-O-benzoyl-3-benzyl-2-deoxy-a-d-glucopyranoside (38): A
solution of 5 (430 mg, 0.6 mmol), 1-benzene sulfinyl piperidine
(BSP; 176 mg, 0.9 mmol) and 4 ꢃ molecular sieves (500 mg) in
anhyd CH2Cl2 (10 mL) was stirred at RT under Ar atmosphere for
30 min. The reaction mixture was cooled ꢀ608C, and Tf2O (139 mL,
0.84 mmol) was added. The temperature was slowly raised to
ꢀ408C and stirred at this temperature for 15 min. Then a solution
of 6 (372 mg, 0.9 mmol) in CH2Cl2 (2.0 mL) was added, and the re-
action mixture was allowed to slowly warm to RT. TLC analysis
showed complete conversion of starting material to a major prod-
uct (petroleum ether/EtOAc 8:1, Rf =0.22). The reaction was
quenched by the addition of Et3N (234 mL, 1.68 mmol) and filtered.
The filtrate was concentrated in vacuo and purified by silica gel
column chromatography (petroleum ether/EtOAc, 10:1) to afford
Scheme 8. Reagents and conditions: a) 1. LiOH, H2O2, THF, ꢀ58C to RT, 16 h;
2. 4m NaOH, MeOH, RT, 24 h, then 358C, 12 h; b) SO3·NMe3, DMF, 508C,
24 h, Dowex 50WX4 Na+-form and Sephadex LH-20, 66% (two steps); b) H2,
Pd/C, tBuOH/H2O, 4 days; (d) SO3·Pry, 2m NaOH, 4 h, Dowex 50WX4 Na+-
form and Sephadex G-25, 62% (two steps).
1
compound 38 as a white solid (343 mg, 56%): H NMR (400 MHz,
CDCl3): d=3.47 (s, 6H), 3.51 (dd, J=3.6 Hz, J=10.0 Hz, 1H), 3.92 (t,
J=9.6 Hz, 1H), 4.02–4.12 (m, 3H), 4.18 (t, J=7.6 Hz, 1H), 4.31 (dd,
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J=8.4, 10.0 Hz, 1H), 4.38 (dd, J=8.0, 10.0 Hz, 1H), 4.52 (dd, J=4.0,
12.0 Hz, 1H), 4.74–4.87 (m, 6H), 5.08 (d, J=2.4 Hz, 1H), 5.11 (d, J=
3.6 Hz, 1H), 5.30 (s, 1H), 5.50 (s, 1H), 7.24–7.36 (m, 14H), 7.41–7.47
(m, 6H), 7.55–7.61 (m, 2H), 7.79 (d, J=7.2 Hz, 2H), 8.08 ppm (d, J=
7.6 Hz, 4H); 13C NMR (100 MHz, CDCl3): d=46.58, 52.19, 55.43,
62.61, 63.78, 67.18, 67.78, 69.34, 70.32, 71.50, 73.09, 73.15, 74.97,
75.37, 78.70, 97.75, 98.43, 120.08, 125.07, 125.20, 127.22, 127.52,
127.71, 127.96, 128.11, 128.17, 128.26, 128.38, 128.50, 129.04,
129.76, 129.84, 130.03, 133.06, 133.48, 137.06, 137.63, 141.20,
141.24, 142.92, 143.22, 154.30, 165.32, 166.06, 168.42 ppm; HRMS
(ESI): m/z [M+NH4]+ calcd for C57H57N4O15: 1307.3815, found:
1307.3823; Anal. RP-HPLC: tR =21.13 min (MeOH/H2O, 78:22;
purity: 95.52%).
conversion of starting material to a major product (CH2Cl2/CH3OH,
50:1, Rf =0.49). The reaction mixture was concentrated in vacuo,
and after the addition of CH2Cl2 (20 mL), washed with saturated
NaHCO3 solution (12 mL). The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The crude product was
purified by silica gel column chromatography (petroleum ether/
EtOAc/CH2Cl2, 3:1:1) to yield 41 as a white solid (310 mg, 83%):
1H NMR (400 MHz, CDCl3): d=2.03 (s, 3H), 3.13 (s, 1H), 3.43–3.52
(m, 2H), 3.58 (s, 1H), 3.69 (dd, J=1.6, 7.6 Hz, 1H), 3.72 (s, 3H), 3.76
(d, J=9.6 Hz, 1H), 3.84 (dd, J=8.4, 9.2 Hz, 1H), 3.95 (d, J=7.6 Hz,
1H), 4.50 (d, J=5.6 Hz, 1H), 4.57 (d, J=7.2 Hz, 1H), 4.70 (d, J=
10.8 Hz, 1H), 4.75–4.80 (m, 2H), 4.91 (d, J=10.8 Hz, 1H), 5.22 (s,
1H), 5.41 (s, 1H), 7.18–7.30 ppm (m, 10H); 13C NMR (100 MHz,
CDCl3): d=20.97, 52.69, 58.83, 64.87, 70.69, 71.54, 74.01, 74.09,
75.12, 75.34, 76.21, 80.86, 82.98, 100.20, 103.66, 127.72, 127.75,
127.88, 128.12, 128.37, 128.43, 138.31, 138.41, 169.10, 169.51 ppm;
HRMS (ESI): m/z [M+NH4]+ calcd for C29H37N4O11: 617.2453, found:
617.2447; Anal. RP-HPLC: tR =9.27 min (CH3CN/H2O, 45:55; purity:
99.09%).
Methyl O-(methyl 2-O-benzoyl-3-O-benzyl-a-l-idopyranosyluro-
nate)-(1!4)-2-azido-6-O-benzoyl-3-benzyl-2-deoxy-a-d-gluco-
pyranoside (39): Et3N (2.1 mL, 15 mmol) was added to a solution
of compound 38 in CH2Cl2 (8 mL). The mixture was stirred at RT for
2 h. TLC analysis showed complete conversion of starting material
to a major product (petroleum ether/EtOAc, 4:1, Rf =0.21). The
mixture was concentrated in vacuo, and the crude product was pu-
rified by silica gel column chromatography (petroleum ether/
EtOAc, 4:1) to yield 39 as a white solid (211 mg, 88%): 1H NMR
(400 MHz, CDCl3): d=3.45–3.47 (m, 7H), 3.86–3.91 (m, 2H), 4.01–
4.04 (m, 3H), 4.48 (dd, J=3.2, 12.0 Hz, 1H), 4.68–4.4.72 (m, 2H),
4.77–4.84 (m, 4H), 4.97 (s, 1H), 5.21 (s, 1H), 5.40 (s, 1H), 7.26–7.43
(m, 14H), 7.51–7.57 (m, 2H), 7.91 (d, J=7.6 Hz, 2H), 8.03 ppm (d,
J=7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=52.08, 55.46, 62.74,
63.86, 67.97, 68.14, 68.84, 69.30, 72.70, 74.88, 75.03, 75.41, 78.77,
98.10, 98.49, 127.46, 127.66, 128.14, 128.21, 128.44, 128.56, 128.58,
129.81, 133.13, 133.71, 137.24, 137.75, 165.06, 166.07, 169.52 ppm;
HRMS (ESI): m/z [M+NH4]+ calcd for C42H47N4O13: 815.3134, found:
815.3137; Anal. RP-HPLC: tR =34.52 min (MeOH/H2O, 85:15; purity:
92.06%).
O-(6-O-Acetyl-2-azide-3,4-di-O-benzyl-2-deoxy-a-d-glucopyrano-
syl)-(1!4)-O-(Methyl
2,3-di-O-benzyl-b-d-glucopyranosyluro-
nate)-(1!4)-2-O-acetyl-1,6-anhydro-2-azido-2-deoxy-b-d-gluco-
pyranose (42): A solution of 2 (881 mg, 1.54 mmol), compound 41
(0.72 g, 1.28 mmol) and 4 ꢃ molecular sieves (500 mg) in anhyd tol-
uene (15 mL) was stirred at RT under Ar atmosphere for 30 min.
The reaction mixture was cooled to ꢀ208C, TBSOTf (0.136 mL,
0.593 mmol) was added, and the reaction mixture was allowed to
slowly warm to RT within 2 h. TLC analysis showed complete con-
version of starting material to a major product (CH2Cl2/acetone,
50:1, Rf =0.44). The reaction was quenched by the addition of Et3N
and filtered. The filtrate was concentrated in vacuo and purified by
silica gel column chromatography (petroleum ether/EtOAc/CH2Cl2
5:1:1!CH2Cl2/acetone=300:1) to yield 42 as a white solid (1.01 g,
1
O-(Methyl 2,3-di-O-benzyl-4-O-chloroacetyl-b-d-glucopyranosyl-
uronate)-(1!4)-2-O-acetyl-1,6-anhydro-2-azido-2-deoxy-b-d-glu-
copyranose (40): A solution of 39 (620 mg, 1.18 mmol), compound
4 (809 g, 3.54 mmol) and 4 ꢃ molecular sieves (800 mg) in anhyd
CH2Cl2 (15 mL) was stirred at RT under Ar atmosphere for 30 min,
then Ag2CO3 (650 mg, 2.36 mmol) was added. The mixture was
stirred at RT under Ar atmosphere for 4 days. TLC analysis showed
complete conversion of starting material to a major product
(CH2Cl2/CH3OH, 50:1, Rf =0.61). The reaction mixture was filtered
through Celite. The filtrate was concentrated in vacuo and purified
by silica gel column chromatography (petroleum ether/EtOAc, 4:1
and then CH2Cl2/CH3OH 100:1!50:1) to yield a mixed product
with a/b anomers as a white solid (523 mg, 66%): b anomer:
1H NMR (400 MHz, CDCl3): d=2.13 (s, 3H), 3.26 (s, 1H), 3.67–3.71
(m, 4H), 3.75 (s, 3H), 3.80–3.87 (m, 2H), 3.98 (d, J=14.0 Hz, 1H),
4.03 (d, J=7.6 Hz, 1H), 4.60 (d, J=5.2 Hz), 4.65 (d, J=11.6 Hz), 4.70
(d, J=6.8 Hz, 1H), 4.81 (d, J=10.8 Hz), 4.87 (d, J=11.6 Hz, 1H),
5.03 (d, J=11.2 Hz), 5.21 (dd, J=10.0, 9.2 Hz, 1H), 5.30 (s, 1H), 5.53
(s, 1H), 7.26–7.41 ppm (m, 10H); 13C NMR (100 MHz, CDCl3): d=20.
01, 40.40, 52.87, 58.95, 64.96, 70.70, 72.06, 72.40, 73.81, 75.25,
75.30, 76.12, 80.81, 81.21, 100.29, 102.10, 128.00, 128.24, 128.47,
138.04, 138.08, 166.00, 167.28, 169.25 ppm; HRMS (ESI): m/z [M+
NH4]+ calcd for C31H38ClN4O12: 693.2169, found: 693.2169; Anal. RP-
HPLC: tR =23.82 min (MeOH/H2O, 70:30; purity: 97.84%).
78%): H NMR (400 MHz, CDCl3): d=2.03 (s, 3H), 2.09 (s, 3H), 3.21
(s, 1H), 3.26 (dd, J=4.0, 10.0 Hz, 1H), 3.51 (t, J=9.6 Hz, 1H), 3.59–
3.66 (m, 3H), 3.75–3.79 (m, 5H), 3.85–3.91 (m, 1H), 3.96 (d, J=
9.6 Hz, 1H), 4.01 (d, J=7.2 Hz, 1H), 4.15 (t, J=9.6 Hz, 1H), 4.23–
4.24 (m, 2H), 4.53–4.56 (m, 2H, H-5), 4.67 (d, J=7.6 Hz, 1H), 4.73
(d, J=10.4 Hz, 1H), 4.79–4.86 (m, 4H), 5.01–5.04 (m, 2H), 5.21 (s,
1H), 5.47 (s, 1H), 5.53 (d, J=4.0 Hz, 1H), 7.24–7.35 ppm (m, 20H);
13C NMR (100 MHz, CDCl3): d=20.82, 20.98, 52.75, 58.80, 62.22,
63.24, 64.90, 69.59, 70.49, 73.71, 74.46, 74.88, 74.93, 75.07, 75.09,
75.45, 75.85, 77.43, 79.99, 81.40, 83.72, 97.72, 100.20, 103.13,
127.38, 127.55, 127.77, 127.90, 127.97, 128.03, 128.13, 128.35,
128.43, 128.49, 137.51, 137.59, 138.05, 138.14, 168.15, 169.11,
170.64 ppm; HRMS (ESI): m/z [M+NH4]+ calcd for C51H60N7O16:
1026.4091, found: 1026.4090; Anal. RP-HPLC: tR =27.55 min
(MeOH/H2O, 82:18; purity: 84.57%).
O-(6-O-Acetyl-2-azide-3,4-di-O-benzyl-2-deoxy-2-a-d-glucopyra-
nosyl)-(1!4)-O-(Methyl 2,3-di-O-benzyl-b-d-glucopyranosyluro-
nate)-(1!4)-3,6-di-O-acetyl-2-azido-2-deoxy-a/b-d-glucopyra-
nose (43): TBSOTf (21.4 mL, 0.1 mmol) was added to a solution of
compound 42 (1.01 g, 1.0 mmol) in Ac2O (10 mL) under Ar atmos-
phere in an ice bath for 20 min. TLC analysis showed complete
conversion of starting material to a major product (petroleum
ether/EtOAc/CH2Cl2, 3:1:1, Rf =0.56). The reaction was quenched by
the addition of Et3N (0.1 mL). The mixture was concentrated in
vacuo with an oil pump. The crude product was purified by silica
gel column chromatography (petroleum ether/EtOAc/CH2Cl2, 6:1:1)
to yield a white solid (966 mg, 87%): HRMS (ESI): m/z [M+NH4]+
calcd for C55H66N7O19: 1128.4408, found: 1128.4409.
O-(Methyl 2,3-di-O-benzyl-b-d-glucopyranosyluronate)-(1!4)-2-
O-acetyl-1,6-anhydro-2-azido-2-deoxy-b-d-glucopyranose
(41):
Thiourea (189 mg, 2.48 mmol) was added to the solution of com-
pound 40 (419 mg, 0.62 mmol) in MeOH/CHCl3 (1:1, 10 mL). The
mixture was stirred at 608C for 24 h. TLC analysis showed complete
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A solution of the above white solid (888 mg, 0.8 mmol) in THF/
MeOH (v/v 7:3, 30 mL) was stirred under NH3 in an ice bath for
about 5–10 min. TLC analysis showed complete conversion of start-
ing material to a major product (petroleum ether/EtOAc/CH2Cl2,
3:1:1, Rf =0.18). The mixture was concentrated in vacuo, and the
crude product was purified by silica gel column chromatography
(petroleum ether/EtOAc/CH2Cl2, 6:1:1!3:1:1) to yield 43 as a white
solid (735 mg, 86%): HRMS (ESI): m/z [M+NH4]+ calcd for
C53H64N7O18: 1086.4302, found: 1086.4312; Anal. RP-HPLC: tR =
35.02 and 37.09 min (CH3CN/H2O, 70:30; purity: 97.15%).
7.49–7.53 (m, 2H), 8.00 (d, J=7.6 Hz, 2H), 8.10 ppm (d, J=7.2 Hz,
2H); 13C NMR (100 MHz, CDCl3): d=20.65, 20.83, 52.25, 52.66,
55.41, 60.71, 61.35, 62.22, 62.37, 63.28, 63.37, 69.15, 69.30, 69.72,
71.52, 72.17, 73.05, 74.42, 74.47, 74.99, 75.08, 75.21, 75.44, 75.52,
75.73, 76.57, 76.87, 77.26, 78.50, 80.13, 81.72, 83.73, 97.57, 97.63,
98.27, 98.43, 103.23, 127.23, 127.56, 127.72, 127.88, 127.96, 128.07,
128.27, 128.36, 128.54, 128.57, 128.67, 129.07, 129.85, 129.92,
133.00, 133.46, 137.32, 137.36, 137.56, 137.97, 138.05, 165.35,
166.07, 168.35, 168.35, 169.72, 170.00, 170.10, 170.63 ppm; HRMS
(ESI): m/z [M+NH4]+ calcd for C95H105N10O30: 1866.7032, found:
1866.7004; Anal. RP-HPLC: tR =24.17 min (CH3CN/H2O, 90:10;
purity: 98.95%).
O-(6-O-Acetyl-2-azide-3,4-di-O-benzyl-2-deoxy-a-d-glucopyrano-
syl)-(1!4)-O-(Methyl
2,3-di-O-benzyl-b-d-glucopyranosyluro-
nate)-(1!4)-3,6-di-O-acetyl-2-azido-2-deoxy-a/b-d-glucopyrano-
syl trichloroacetimidate (44): Cl3CCN (1.0 mL) was added to a solu-
tion of compound 43 (532 mg, 0.5 mmol) and K2CO3 (138 mg,
1.0 mmol) in anhyd CH2Cl2 (10 mL) under Ar atmosphere at RT. The
mixture was stirred at RT for 2 h. TLC analysis showed complete
conversion of starting material to a major product (petroleum
ether/EtOAc/CH2Cl2, 3:1:1, Rf =0.69). The reaction mixture was con-
centrated in vacuo, and the crude product was purified by silica
gel column chromatography (petroleum ether/EtOAc/CH2Cl2, 6:1:1)
to yield 44 with a/b anomers as a white solid (490 mg, 81%):
Methyl O-(2-azide-3,4-di-O-benzyl-2-deoxy-a-d-glucopyranosyl)-
(1!4)-O-(2,3-di-O-benzyl-b-d-glucopyranosyluronic acid)-(1!4)-
O-(2-azido-2-deoxy-a-d-glucopyranosyl)-(1!4)-O-(3-O-benzyl-a-
l-idopyranosyluronic acid)-(1!4)-2-azido-3-benzyl-2-deoxy-a-d-
glucopyranoside (46): A solution of H2O2 (30%, 5.1 mL) was added
to a cooled (ꢀ58C) solution of compound 45 (185 mg, 0.1 mmol)
in THF (10 mL), and LiOH(aq) (1.25m, 2.4 mL) was added dropwise.
After 16 h at RT, MeOH (6 mL) and NaOH (4m, 3 mL) were added,
and the reaction mixture stirred for 24 h at RT. In the case that the
reaction had not gone to completion, stirring was continued at
358C for an additional 12 h. The reaction mixture was acidified (6m
HCl) and diluted with H2O. The compound was extracted with
CH2Cl2, washed with 10% Na2SO3(aq) and H2O. The aqueous layer
was extracted with CH2Cl2 (2ꢄ). The organic layers were combined,
dried (Na2SO4) and filtered. The filtrate was concentrated in vacuo
to give 46 as white powder (135 mg, 91%): 1H NMR (400 MHz,
CD3OD): d=3.28 (dd, J=3.2, 10.4 Hz, 1H), 3.36 (dd, J=3.6, 10.0 Hz,
1H), 3.41 (s, 3H), 3.50 (t, J=8.0 Hz, 1H), 3.64–3.72 (m, 6H), 3.74–
3.82 (m, 6H), 3.88–3.97 (m, 7H), 3.99–4.08 (m, 4H), 4.55–4.61 (m,
2H), 4.67–4.70 (m, 2H), 4.72 (d, J=8.0 Hz, 1H), 4.77–4.79 (m, 3H),
4.84–4.68 (m, 2H), 4.86–4.91 (m, 3H), 5.00 (d, J=10.8 Hz, 1H), 5.13
(d, J=3.2 Hz, 1H), 5.30 (d, J=4.4 Hz, 1H), 5.54 (d, J=3.6 Hz, 1H),
7.19–7.36 (m, 28H), 7.44 ppm (d, J=7.2 Hz, 2H); 13C NMR
(100 MHz, CD3OD): d=55.57, 60.61, 61.12, 61.61, 64.53, 64.61,
64.73, 71.31, 71.94, 72.43, 72.76, 73.18, 73.52, 74.88, 75.40, 75.55,
75.73, 75.93, 76.18, 76.25, 76.29, 77.41, 78.49, 79.12, 79.61, 79.72,
80.94, 83.17, 85.29, 98.95, 99.18, 100.05, 102.32, 103.99, 128.48,
128.52, 128.62, 128.83, 128.93, 129.03, 129.18, 129.36, 129.40,
129.48, 139.38, 139.53, 139.63, 139.69, 139.73, 172.57 ppm; HRMS
(ESI): m/z [M+NH4]+ calcd for C73H87N10O25: 1503.5838, found:
1503.5833; Anal. RP-HPLC: tR =31.40 min (Eluent A: CH3CN,
Eluent B: 10 mm NH4HCO3 buffer; gradient: 0–10 min 10% A, 10–
35 min 10–80% A, 35–38 min 80% A; purity: 91.99%).
1
b anomer: H NMR (400 MHz, CDCl3): d=2.03 (s, 3H), 2.05 (s, 3H),
3.27 (dd, J=3.6 Hz, J=10.0 Hz, 1H), 3.42 (t, J=8.8 Hz, 1H), 3.50–
3.51 (m, 2H), 3.58–3.60 (m, 1H), 3.69–3.75 (m, 2H), 3.77 (s, 3H),
3.80–3.87 (m, 3H), 4.05 (t, J=9.2 Hz, 1H), 4.11 (dd, J=7.2, 14.4 Hz,
1H), 4.18–4.21 (m, 2H), 4.26 (d, J=11.6 Hz, 1H), 4.33 (d, J=8.0 Hz,
1H), 4.41 (d, J=12.0 Hz, 1H), 4.55 (d, J=11.6 Hz, 1H), 4.68 (d, J=
11.6 Hz, 1H), 4.76 (d, J=11.2 Hz, 1H), 4.80–4.86 (m, 3H), 4.96 (d, J=
10.8 Hz, 1H), 5.09 (t, J=9.6 Hz, 1H), 5.50 (d, J=3.2 Hz, 1H), 5.66 (d,
J=8.4 Hz, 1H),7.22–7.35 (m, 20H), 8.77 ppm (brs, 1H, NH);
13C NMR (100 MHz, CDCl3): d=20.68, 20.85, 52.72, 60.41, 61.40,
62.22, 63.24, 63.30, 69.71, 71.94, 73.73, 74.39, 74.98, 75.03, 75.27,
75.53, 80.13, 81.92, 83.82, 90.26, 96.35, 97.58, 103.13, 127.30,
127.72, 127.82, 127.90, 128.05, 128.08, 128.39, 128.48, 128.54,
137.56, 138.03, 160.62, 168.31, 169.97, 170.13, 170.67 ppm; HRMS
(ESI): m/z [M+NH4]+ calcd for C55H64Cl3N8O18: 1229.3399, found:
1229.3393.
Methyl O-(6-O-Acetyl-2-azide-3,4-di-O-benzyl-2-deoxy-a-d-gluco-
pyranosyl)-(1!4)-O-(Methyl 2,3-di-O-benzyl-b-d-glucopyranosy-
luronate)-(1!4)-O-(3,6-di-O-acetyl-2-azido-2-deoxy-a-d-gluco-
pyranosyl)-(1!4)-O-(Methyl 2-O-benzoyl-3-O-benzyl-a-L-idopyr-
anosyluronate)-(1!4)-2-azido-6-O-benzoyl-3-benzyl-2-deo-xy-a-
d-glucopyranoside (45):
A solution of donor 44 (310 mg,
0.25 mmol), disaccharide acceptor 39 (168 mg, 0.21 mmol) and 4 ꢃ
molecular sieves (0.5 g) was stirred in anhyd CH2Cl2 (8 mL) under
Ar atmosphere for 30 min. Then reaction mixture was cooled to
ꢀ208C, and after the addition of TMSOTf (3.8 mL, 0.021 mmol), was
allowed to slowly warm to RT within 2 h. TLC analysis showed
complete conversion of starting material to a major product (pe-
troleum ether/EtOAc/CH2Cl2, 3:1:1, Rf =0.55). The reaction was
quenched by the addition of Et3N (0.1 mL) and filtered. The filtrate
was concentrated in vacuo and purified by silica gel column chro-
matography (petroleum ether/EtOAc/CH2Cl2 7:1:1!CH2Cl2/acetone
Methyl O-(2-azide-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-a-d-gluco-
pyranosyl)-(1!4)-O-(2,3-di-O-benzyl-b-d-glucopyranosyluronic
acid)-(1!4)-O-(2-azido-2-de-oxy-3,6-di-O-sulfo-a-d-glucopyrano-
syl)-(1!4)-O-(3-O-benzyl-2-O-sulfo-a-l-ido-pyranosyluronic
acid)-(1!4)-2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-a-d-glucopyr-
anoside heptasodium salt (47):
A solution of 46 (45 mg,
0.03 mmol) and trimethylamine sulphur trioxide complex (209 mg,
0.15 mmol) in N,N-dimethylformamide (DMF; 3 mL) was stirred for
24 h at 508C. The reaction mixture was cooled to RT and then con-
centrated in vacuo. The resulting residue was passed through
a column of Dowexꢀ50WX4 Na+-form using CH3OH as eluent. The
fractions containing the product were concentrated in vacuo, and
the residue was passed through a column of Sephadex LH-20
using MeOH/CHCl3 (1:1) as eluent to afford 47 as a white powder
(24 mg, 72%): 1H NMR (400 MHz, CD3OD): d=3.34–3.86 (m, 2H),
3.45 (s, 3H), 3.50 (dd, J=3.6, 10.4 Hz, 1H), 3.58–3.64 (m, 2H), 3.72
(t, J=9.6 Hz, 1H), 3.83–3.95 (m, 6H), 3.98–4.06 (m, 3H), 4.11–4.19
1
80:1) to yield 45 as a white solid (262 mg, 68%): H NMR (400 MHz,
CDCl3): d=2.02–2.23 (3 s, 9H), 3.21 (dd, J=3.2, 10.8 Hz, 1H), 3.26
(dd, J=3.6, 10.0 Hz, 1H,), 3.35 (s, 3H), 3.38–3.43 (m, 2H), 3.53 (s,
3H), 3.66–3.72 (m, 2H), 3.74 (s, 3H), 3.82–3.92 (m, 4H), 3.97–4.06
(m, 3H), 4.10–4.21 (m, 4H), 4.26 (d, J=12.0 Hz, 1H), 4.34 (d, J=
8.0 Hz, 1H), 4.42–4.47 (m, 2H), 4.53–4.65 (m, 4H), 4.72–4.86 (m,
9H), 4.95–4.97 (m, 2H), 5.10 (d, J=3.2 Hz, 1H), 5.25–5.34 (m, 2H),
5.49 (d, J=3.6 Hz), 5.72 (d, J=6.0 Hz, 1H), 7.20–7.44 (m, 34H),
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(m, 3H), 4.26–4.33 (m, 4H), 4.39 (d, J=11.2 Hz, 1H), 4.50 (d, J=
10.4 Hz), 4.59–4.61 (m, 2H), 4.63 (d, J=4.0 Hz, 1H), 4.66–4.78 (m,
4H), 4.81–4.84 (m, 4H), 4.93–5.00 (m, 5H), 5.20 (d, J=3.6 Hz, 1H),
5.42 (s, 1H), 5.52 (d, J=4.0 Hz, 1H), 7.13–7.37 (m, 28H), 7.47 ppm
(d, J=7.2 Hz, 2H); 13C NMR (100 MHz, CD3OD): d=55.76, 64.40,
64.76, 65.03, 66.15, 67.00, 67.48, 67.87, 71.01, 71.15, 71.36, 71.41,
71.56, 71.85, 73.57, 74.08, 74.75, 75.24, 75.92, 76.06, 76.17, 76.21,
76.25, 77.72, 79.32, 79.91, 81.21, 83.21, 85.29, 95.92, 98.91, 99.21,
99.99, 103.40, 128.19, 128.27, 128.44, 128.46, 128.65, 128.74,
128.94, 129.17, 129.23, 129.30, 129.33, 129.34, 129.43, 129.55,
129.95, 138.95, 139.05, 139.54, 140.01, 140.12, 172.58 ppm; HRMS
(ESI): m/z calcd for C73H82N9O40S5 [MꢀH]ꢀ: 1884.3268, found:
1884.3083, calcd for C73H85N10O40S5 [Mꢀ2H+NH4]ꢀ: 1901.3534,
found: 1901.3312, calcd for C73H81N9O40S5 [(Mꢀ2H)/2]ꢀ: 941.6592,
found: 941.6575; Anal. RP-HPLC: tR =20.33 min (Eluent A: CH3CN,
Eluent B: 10 mm Na2HPO4 buffer; gradient: 0–30 min 10–70% A,
35–40 min 70% A; purity: 88.50%).
calcd for C31H43N3Na8O49S8 [(M+8Naꢀ10H)/2]ꢀ 840.3962, found:
840.4034; Anal. RP-HPLC: tR =16.18 min (Eluent A: 0.001% DMSO
in H2O, Eluent B: 11.7% NaCl in H2O; gradient: 0–5 min 50% B, 5–
25 min 50–90% B, 25–30 min 90% B; purity: 95.50%).
Acknowledgements
This work was supported by the National Basic Research Pro-
gram of China (973 Program, No. 2010CB529106, 2012CB910300)
and the Natural Science Foundation of China (No. 21332006,
21372130). The authors would like to thank Jeffrey Meisner
(Emory University, Atlanta, GA, USA) for proof reading.
Keywords: fondaparinux
·
glycosylation
·
heparin
·
pentasaccharides
Methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-a-d-glucopyranosyl)-
(1!4)-O-(b-d-glucopyranosyluronic
acid)-(1!4)-O-(2-deoxy-2-
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sulfamido-3,6-di-O-sulfo-a-d-glucopyranosyl)-(1!4)-O-(2-O-
sulfo-a-l-idopyranosyluronic acid)-(1!4)-2-de-oxy-2-sulfamido-
6-O-sulfo-a-d-glucopyranoside decasodium salt (1): A solution of
compound 47 in MeOH/tBuOH/H2O (2:1:1, 4 mL) was hydrogenat-
ed in the presence of 10% Pd/C (40 mg) and 8 atm H2 at RT for
1
4 days. A H NMR spectrum showed no signals of aryl groups. The
mixture was filtered through Celite, and the filtrate was concentrat-
ed in vacuo to give 48 as a gray-green solid (16 mg, 100%): HRMS
(ESI): m/z calcd for C31H51N3O40S5 [(Mꢀ2H)/2]ꢀ: 632.5326, found:
632.5329, calcd for C31H50N3NaO40S5 [(Mꢀ3H+Na)/2]ꢀ: 643.5236,
found: 643.5239, calcd for C31H49N3Na2O40S5 [(Mꢀ4H+2Na)/2]ꢀ:
654.5151, found: 654.5152, calcd for C31H48N3Na3O40S5 [(Mꢀ5H+
3Na)/2]ꢀ: 665.5061, found: 654.5058.
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3118–3133.
The above amino alcohol 48 was dissolved in H2O (2 mL), and the
solution was adjusted to pH 9.5 through addition of 2m NaOH(aq)
.
Sulfur trioxide pyridine complex (32 mg, 0.2 mmol) was added in
five equal portions at half-hour intervals at RT, and the solution
was maintained at pH 9.5 via calibration with 2m NaOH(aq). After
stirring for 4 h, the solution was adjusted to pH 7–8 using 0.1m
HCl, and passed through a column of Dowex 50WX4 Na+-form
using H2O as eluent. The fractions containing the product were
lyophilized, and the residue was passed through a column of Se-
phadex G-25 using 0.2m NaCl(aq) as an eluent. The fractions con-
taining product were lyophilized, and the crude product was sub-
jected to desalting through a Sephadex G-25 column eluted with
H2O to give compound 1 as a white solid (12 mg, 62%, over two
1
steps): H NMR (400 MHz, D2O): d=3.22–3.28 (m, 2H), 3.40–3.45 (m,
5H), 3.53–3.66 (m, 3H), 3.73–3.86 (m, 5H), 3.93–3.97 (m, 2H), 4.10–
4.17 (m, 4H), 4.23–4.40 (m, 6H), 4.46 (d, J=10.8 Hz, 1H), 4.61 (d,
J=7.6 Hz, 1H), 4.79 (m, 1H), 5.00 (d, J=3.2 Hz, 1H), 5.17 (d, J=
2.8 Hz, 1H), 5.48 (d, J=2.8 Hz, 1H), 5.60 ppm (d, J=3.6 Hz, 1H);
13C NMR (100 MHz, D2O): d=58.15, 59.36, 60.42, 60.64, 68.69, 69.04,
69.42, 71.24, 71.72, 72.31, 72.53, 72.63, 72.78, 73.83, 75.45, 75.80,
78.73, 78.77, 78.89, 79.08, 79.41, 79.65, 99.02, 100.39, 100.99,
102.24, 103.88 ppm; HRMS (ESI): m/z calcd for C31H50N3NaO49S8
[(M+Naꢀ3H)/2]ꢀ 763.4594, found: 763.4598; calcd for
C31H49N3Na2O49S8 [(M+2Naꢀ4H)/2]ꢀ 774.4504, found: 774.4500;
calcd for C31H48N3Na3O49S8 [(M+3Naꢀ5H)/2]ꢀ 785.4413, found:
785.4414; calcd for C31H47N3Na4O49S8 [(M+4Naꢀ6H)/2]ꢀ 796.4323,
found: 796.4318; calcd for C31H46N3Na5O49S8 [(M+5Naꢀ7H)/2]ꢀ
807.4233, found: 807.4249; calcd for C31H45N3Na6O49S8 [(M+
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6Naꢀ8H)/2]ꢀ
818.4142,
found:
818.4142;
calcd
for
C31H44N3Na7O49S8 [(M+7Naꢀ9H)/2]ꢀ 829.4070, found: 829.4053;
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1071 – 1080 1079

CHEMMEDCHEM
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Published online on April 11, 2014
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