Synthesis and biological evaluation of a heparin-like hexasaccharide with the structural motifs for binding to FGF and FGFR

Article abstract of DOI:10.1002/ejoc.200400799

A heparin-like hexasaccharide structure (2) containing the two trisaccharide sequences that interact with acidic fibro-blast growth factor (FGF-1) and with its receptor (FGFR) has been designed on the basis of cristallographic data. This hexasaccharide has been effectively synthesized by a completely stereoselective modular approach and biologically evaluated by determination of its capacity to stimulate FGF-1-induced mitogenic activity. It was found that this molecule did not show an appreciable FGF-1 activating effect. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005.

Full text of DOI:10.1002/ejoc.200400799

FULL PAPER
Synthesis and Biological Evaluation of a Heparin-Like Hexasaccharide with
the Structural Motifs for Binding to FGF and FGFR
José Luis de Paz[a] and Manuel Martín-Lomas*[a]
Keywords: Growth factor activation / Heparin / Synthesis design / Oligosaccharides / Carbohydrates
A heparin-like hexasaccharide structure (2) containing the
two trisaccharide sequences that interact with acidic fibro-
blast growth factor (FGF-1) and with its receptor (FGFR) has
been designed on the basis of crystallographic data. This
hexasaccharide has been effectively synthesized by a com-
pletely stereoselective modular approach and biologically
evaluated by determination of its capacity to stimulate FGF-
1-induced mitogenic activity. It was found that this molecule
did not show an appreciable FGF-1 activating effect.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
acceptor-bound strategy,[7,8] allowed for the preparation of
several hexa- and octasaccharides with different charge dis-
tributions and orientations.[9–11] In terms of overall confor-
mation, these synthetic compounds, like heparin itself,[12]
presented well defined three-dimensional helical structures
that determine the spatial orientation of the sulfate
groups.[9–11,13]
Among these synthesized hexa- and octasaccharides,
compound 1 (Figure 1) was able to induce the mitogenic
activity of acidic FGF (FGF-1), the first member of the
FGF family, showing a maximum activating effect of the
same order as heparin.[13] The three-dimensional structure
of hexasaccharide 1 displays all the sulfate groups on only
one side of the right-handed helix, in contrast to naturally
occurring heparin oligosacharides, in which the sulfate
groups are distributed on both sides of the helix. These were
unexpected biological results and we speculated that this
particular arrangement of negative charges may influence
the formation and the geometry of the FGF oligomers that
had been proposed to participate in FGF activation.[10] In
any case, they confirmed the importance of negative charge
distribution in the activation and seem to strongly support
the specificity of the process.[13]
The role of the sulfate groups in the interactions between
heparin and this family of proteins was analysed by Pelleg-
rini,[14] who overlaid the available crystallographic struc-
tures of FGF–heparin[15,16] and FGF–FGFR–heparin com-
plexes,[17,18] examining the protein–heparin contacts. This
study revealed that the interaction between heparin and
FGFR involves a single disaccharide containing three sul-
fate groups at the nonreducing end of the molecule, as indi-
cated in Figure 2. For the interaction between heparin and
FGF this author proposed a trisaccharide binding sequence
at the reducing end, with three sulfate groups distributed
on the same side of the helical structure as in the biolo-
gically active hexasaccharide 1.
Introduction
Fibroblast growth factors (FGFs) constitute a family of
signalling polypeptides involved in a variety of biological
processes through stimulation of key cellular functions after
binding to specific receptors at the cell surface (FGFRs).[1]
The biological activity of FGFs is tightly regulated by hepa-
rin or heparan sulfate (HS) glycosaminoglycans (GAGs),
which interact both with the FGFs and the FGFRs in the
stimulation process.[2–4] The precise structural requirements
for the GAG chains to regulate FGF activation in this pro-
cess are not yet well understood at the molecular level.[2]
HS-GAGs are linear polysaccharides composed of alternat-
ing units of variously sulfated d-glucosamine (GlcN) and
l-iduronic (IdoA) or d-glucuronic (GlcA) acid units,[5] and
a main difficulty in the study of FGF activation is the in-
herent heterogeneity of the GAGs in terms of sequence, size
and sulfation pattern. The availability of homogeneous oli-
gosaccharides with precisely defined molecular structures
would most probably represent a major contribution to the
elucidation of the molecular mechanism of FGF acti-
vation.[2]
In the context of a programme involving the molecular
basis of FGF activation we have developed a modular ap-
proach for a completely stereoselective synthesis of oligo-
saccharides containing the GlcN–IdoA repeating unit of
the major sequence of heparin.[6] This approach, which has
been successfully extended to the solid phase through an
[a] Grupo de Carbohidratos, Instituto de Investigaciones Quím-
icas, CSIC,
Country Américo Vespucio, s/n, Isla de La Cartuja, 41092 Sev-
illa, Spain
Fax: +34-95-4460565
E-mail: manuel.martin-lomas@iiq.csic.es
Supporting information for this article is available on the
WWW under http://www.eurjoc.com or from the author.
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DOI: 10.1002/ejoc.200400799
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J. L. de Paz, M. Martín-Lomas
FULL PAPER
Figure 1. Structure and schematic representation of hexasaccharide 1.
Figure 2. Proposed heparan sulfate sequence binding FGF and FGFR.[14]
Extension of the analysis to include interactions with
Results and Discussion
both FGF and FGFR suggests an oligosaccharide sequence
with a pattern of sulfate groups to link FGF and FGFR in
a 1:1:1 complex (Figure 2).[14]
As indicated in Scheme 1, the synthesis of 2 was envis-
aged as progressing from the fully protected hexasaccharide
On the basis of this working hypothesis we have adapted 3, which could be prepared from building blocks 4, 5 and
our synthetic strategy to the preparation of hexasaccharide 6. These compounds have been prepared in a multigram
2, which contains the proposed FGF-activating heparin se- scale from the known iduronic acid 7,[19] disaccharide 8[6,9]
quence (Figure 3), in order to evaluate its biological behav- and N-acetylglucosamine derivative 9.[20]
iour. The expected spatial orientation of the negative
Scheme 2 summarizes the synthesis of these building
charges of 2 is similar to that in hexasaccharide 1, with five blocks. The nonreducing end 4 was prepared from 7 in four
sulfate groups displayed on one side of the helical structure, steps. Benzoylation[21] at position 2, followed by benzylation
but compound 2 contains the alternative monosaccharide under neutral conditions,[22] gave 13 in moderate yield. Al-
sequence IdoA-GlcN, with the l-iduronic acid unit at the though no regioselectivity was observed in the benzoylation
nonreducing end and the d-glucosamine residue at the re- of diol 7 with 1 equiv. of BzCN, this synthetic route was
ducing end.
chosen because it allows for rapid access to the conveniently
Figure 3. Structure and schematic representation of hexasaccharide 2.
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Synthesis and Biological Evaluation of a Heparin-Like Hexasaccharide
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Scheme 1. Retrosynthetic analysis. TDS = dimethylthexylsilyl.
Scheme 2. Synthesis of building blocks. Reagents and conditions: a) BzCN, Et3N (cat.), MeCN, –40 °C, 42% 11 + 40% 12; b) Ag2O,
DMF, BnBr, 62%; c) (HF)n·Py, THF, –15 °CǞ0 °C, 81%; d) Cl3CCN, K2CO3, 86%; e) (HF)n·Py, THF, –15 °CǞ0 °C, 82%; f) Cl3CCN,
K2CO3, 87%; g) TMSOTf, CH2Cl2, 76%; h) NaBH3CN, HCl/Et2O, THF, 90%. TMSOTf = trimethylsilyl trifluoromethanesulfonate.
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protected iduronate unit 12, and the also obtained re- detection of small amounts of the corresponding orthoester
gioisomer 11 can be recycled or used to prepare other hepa- intermediate.
rin-like oligosaccharides with different sulfation patterns
Removal of the levulinic ester in 18 with hydrazine ace-
needed for different synthetic purposes. Compound 13 was tate[27] proceeded in 93% yield to give 19. This product was
then desilylated[23] (Ǟ14, 81%) and activated as a trichlo- converted into its benzyl ether 20 in 75% yield by use of
roacetimidate[24] 4 (86%).
BnBr and silver oxide[22] (Ag2O) in order to introduce a
Building block 5 was obtained from known disaccharide required permanent protecting group at position 2 of the
8[6,9] by conventional levulinoylation[25] (Ǟ15, 83%), desi- D unit. Removal of the benzylidene acetal[28] (Ǟ21, 84%)
lylation (Ǟ16, 82%) and anomeric activation (Ǟ5, 87%).
and selective benzoylation of the resulting diol afforded ac-
Trisaccharide acceptor 6 was prepared by regioselective ceptor 22 (99%). Glycosylation of 22 with donor 4 at room
reductive opening[26] of the benzylidene acetal group in 17, temperature with 0.15 equiv. of TMSOTf gave hexasaccha-
which had been effectively synthesized in our laboratory by ride 3 in 52% yield. A considerable amount (44%) of un-
stereoselective glycosylation of 9[20] with trichloroacetimid- changed 22 was also isolated from the reaction mixture.
ate 10.[9] Compound 6 was to constitute the reducing end
Hexasaccharide 3 was then subjected to the deprotection/
of the hexasaccharide sequence (2) and, as established in sulfation sequence. The removal of methoxycarbonyl and
our previous studies,[6] was provided with an α-isopropyl acyl groups was performed with lithium hydroperoxide and
group at the anomeric position.
then aqueous/alcoholic potassium hydroxide in order to
The coupling of 6 with donor 5 afforded pentasaccharide minimise elimination.[29,30] The resulting partially protected
18 in 59% yield (Scheme 3). A considerable amount (18%) hexasaccharide 23 was sulfated by treatment with SO3·Py
of unchanged 6 was also isolated from the reaction mixture. complex in pyridine. Close monitoring by TLC revealed
We found that the glycosylation was more efficient when that a mixture of partially O-sulfated products was ob-
performed with 0.15 equiv. of TMSOTf for at least 3 h. The tained after stirring for 24 h. It was necessary to purify that
use of less promoter or shorter reaction times led to the mixture by Sephadex LH-20 chromatography and to treat
Scheme 3. Synthesis of hexasaccharide 2. Reagents and conditions: a) TMSOTf, CH2Cl2, 59% + 18% of recovered 6; b) hydrazine acetate,
CH2Cl2, 93%; c) Ag2O, DMF, BnBr, 75% + 15% of recovered 19; d) EtSH, pTsOH (cat.), 84%; e) BzCN, Et3N (cat.), MeCN, –40 °C,
99%; f) TMSOTf, CH2Cl2, 52% + 44% of recovered 22; g) H2O2, LiOH aq, THF; KOH aq, MeOH, 96%; h) SO3·Py, Py, 50%; i) H2,
Pd/C, MeOH, H2O; SO3·Py, H2O, pH = 9.5, 93%.
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Synthesis and Biological Evaluation of a Heparin-Like Hexasaccharide
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the resulting residue with fresh SO3·Py complex in order charide structure presents a pattern of sulfate groups, as
to complete O-sulfation and to isolate pure 24 (50%). The shown in Figure 2, to bind both FGF-1 and FGFR in a
extremely low reaction rate of this step could be explained 1:1:1 complex. The three-dimensional shape of this hexa-
by the presence of the deactivating acetamido group.[31] saccharide structure would display five sulfate groups on
Similar results have been obtained by us in the O-sulfation one side of the helical structure. This structural feature, but
of trisaccharides containing acetamido groups.[20]
with the alternative monosaccharide sequence, is present in
Finally, hydrogenolytic cleavage of the benzyl groups and hexasaccharide 1, previously synthesized by us,[10] which
simultaneous reduction of the azido groups in 24, followed was able to stimulate FGF-1-induced mitogenic activity
by selective N-sulfation, yielded compound 2 (93%), which with a maximum activating effect of the same order as hep-
was purified by gel permeation chromatography by the pro- arin.[13] We therefore synthesized hexasaccharide 2 by a
tocol previously reported for heparin-like oligosaccha- completely stereoselective modular approach developed by
1
rides.[30] The H and 13C NMR spectra of hexasaccharide us[6] that has proven to be effective to construct a variety of
2 were fully assigned by conventional 1D and 2D spec- heparin-like oligosaccharides of different size and negative
troscopy (Table 1). These assignments were carried out by charge distribution both in solution[9–11] and in solid
identification of the spin systems of the residues and further phase,[7,8] and tested its capacity to stimulate FGF-1-in-
connections based on interresidue NOE. The chemical shift duced mitogenesis. Hexasaccharide 2 did not show an ap-
values were in agreement with those expected according to preciable activating effect. This result clearly shows that the
reported data for chemically modified heparin.[32,33]
presence of the recognition sites in the oligosaccharide con-
The induction of the mitogenic activity of FGF-1 by struct alone did not suffice to trigger the biological process,
hexasaccharide 2 was tested by a reported procedure.[34] It and that many other factors have to be considered to ac-
was found that this molecule did not show an appreciable count for the stimulation capacity of these synthetic oligo-
activating effect in spite of its containing the minimal bind- saccharides.
ing sequences found by Pellegrini to be involved in the in-
teractions of FGF-1 and of FGFR with heparin on the ba-
Experimental Section
sis of existing X-ray crystallographic data. This is an inter-
esting result, particularly in relation to those previously ob-
tained by us with synthetic hexasaccharide 1 and several
other synthetic oligosaccharides.[13] It further confirms the
complexity of the FGF activation, in which small variations
in the oligosaccharide sequence, size or sulfation pattern
may have a dramatic effect on the capacity of the oligosac-
charide to induce mitogenic activity of the protein.
General Remarks: Thin layer chromatography (TLC) analyses were
performed on silica gel 60 F254 precoated on aluminium plates
(Merck) and the compounds were detected by staining with sulfuric
acid/ethanol (1:9) or with anisaldehyde solution [anisaldehyde
(25 mL) with sulfuric acid (25 mL), ethanol (450 mL) and acetic
acid (1 mL)], followed by heating at over 200 °C. Column
chromatography was carried out on silica gel 60 (0.2–0.5 mm, 0.2–
0.063 mm or 0.040–0.015 mm; Merck). Optical rotations were de-
termined with a Perkin–Elmer 341 polarimeter. H and 13C NMR
1
spectra were acquired on Bruker DPX 300, DRX 400 and
DRX 500 spectrometers, and chemical shifts are given in ppm (δ)
relative to tetramethylsilane as an internal reference or relative to
D2O. Elemental analyses were performed with a Leco CHNS-932
apparatus, after drying of analytical samples over phosphorus pent-
oxide for 24 h. The FAB mass spectrum of compound 23 was mea-
sured by the Mass Spectrometry Service, Facultad Química, Sevilla,
with a Kratos MS-80 RFA spectrometer. The ES mass spectrum of
compound 24 was measured by the Laboratorio de Aplicaciones,
Conclusions
The analysis of the interactions between heparin and the
proteins of the FGF family carried out by Pellegrini[14] by
overlaying the available crystallographic structures of FGF–
heparin[15,16] and FGF–FGFR–heparin[17,18] complexes
permits one to design a hexasaccharide structure containing
FGF and FGFR trisaccharide binding sequences at the re-
ducing and the nonreducing end respectively. This hexasac- Thermo, Instituto Química-Física Rocasolano, Madrid, with a
Table 1. Observed proton and carbon chemical shifts (in ppm) for hexasaccharide 2.
A
B
C
D
E
F
1-H
2-H
3-H
4-H
5-H
6-H/6Ј-H
C-1
C-2
C-3
C-4
C-5
4.95
3.87
3.79
3.70
4.09
4.32/4.32
95.19
54.21
70.08
77.69
69.23
67.38
5.18
4.28
4.16
4.06
4.68
5.40
3.22
3.61
3.67
3.86
3.86/3.78
96.79
58.40
70.00
77.63
71.50
60.17
4.90
3.69
4.05
4.05
4.74
5.34
3.22
3.66
3.73
3.97
4.28/4.21
95.79
58.36
70.16
76.94
69.51
66.71
5.14
4.27
4.07
3.95
4.81
99.83
76.96
69.96
76.25
70.06
101.99
69.70
69.21
75.62
69.76
99.46
74.50
69.25
69.53
69.22
C-6
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LCQ Deca ThermoFinnigan spectrometer. The ES mass spectrum
of compound 2 was measured by the Laboratorio Espectrometría
chromatography (3:2 hexane/AcOEt) to yield 14 (213 mg, 81%) as
an α/β mixture: TLC (3:2 hexane/AcOEt), Rf = 0.36. 1H NMR
Masas, SidI, Universidad Autónoma Madrid, with a HP1100 MSD (500 MHz, CDCl3) (0.6:0.4 α/β): δ = 8.03–6.98 (m, 15 H, Ph), 5.48
spectrometer.
(br. s, 0.6 H, 1α-H), 5.26 (br. s, 0.4 H, 1β-H), 5.18 (br. s, 0.4 H, 2β-
H), 5.13 (br. s, 0.6 H, 2α-H), 4.97 (d, J4,5 = 2.5 Hz, 0.6 H, 5α-H),
4.81–4.26 (m, 4.4 H, 5β-H, CH2Ph), 4.57 (m, 1 H, OHα), 4.06 (m,
0.4 H, 3β-H), 4.02 (m, 0.6 H, 3α-H), 3.92 (m, 0.6 H, 4α-H), 3.80
(m, 0.4 H, 4β-H), 3.73 (2s, 3 H, COOCH3 α and β) ppm. 13C NMR
(125 MHz, CDCl3): δ = 170.1, 169.4, 166.4, 165.8, 137.3–127.7
(Ph), 93.2, 92.1, 74.3, 74.0, 73.8, 73.1, 72.79, 72.75, 72.71, 72.2,
72.0, 68.4, 67.9, 67.8, 52.34, 52.27 ppm. C28H28O8 (492.5): C 68.28,
H 5.73; found C 67.88, H 5.55.
Methyl (Dimethylthexylsilyl 2-O-Benzoyl-3-O-benzyl-β-L-idopyr-
anoside) Uronate (12): BzCN (887 mg, 6.77 mmol) and catalytic
Et3N were added to a cooled (–40 °C) solution of 7 (2.84 g,
6.44 mmol) in dry CH3CN (30 mL). After 1 h, MeOH was added
and the mixture was allowed to reach room temperature. The sol-
vent was evaporated and the residue was dissolved in MeOH and
concentrated to dryness. The purification was carried out by flash
chromatography (8:1Ǟ5:1 hexane/AcOEt) to afford 12 (1.41 g,
40%) and regioisomer 11 (1.48 g, 42%). [α]2D0 = +41.1 (c = 2.1,
CHCl3); TLC (3:1 hexane/AcOEt), Rf = 0.54. 1H NMR (500 MHz,
CDCl3): δ = 7.99–7.24 (m, 10 H, Ph), 5.27 (br. s, 1 H, 2-H), 5.22
(br. s, 1 H, 1-H), 4.76–4.70 (2d, Jgem = 12.0 Hz, 2 H, CH2Ph), 4.60
O-(Methyl 2-O-Benzoyl-3,4-di-O-benzyl-α,β-L-idopyranosyluronate)
Trichloroacetimidate (4): Cl3CCN (0.49 mL, 4.9 mmol) and K2CO3
(67 mg, 0.49 mmol) were added to a solution of 14 (200 mg,
0.41 mmol) in dry CH2Cl2 (3 mL). After stirring at room tempera-
ture for 5 h, the mixture was then filtered off and concentrated in
vacuo, and the residue was purified by chromatography over a short
silica gel column (2:1 hexane/AcOEt + 1% triethylamine) to yield
4 (222 mg, 86%) as an α/β mixture. TLC (2:1 hexane/AcOEt), Rf
= 0.57. 1H NMR (500 MHz, CDCl3) (0.55:0.45 α/β): δ = 8.71–8.66
(2s, 1 H, NH β and α), 8.05–7.10 (m, 15 H, Ph), 6.57 (s, 0.45 H,
1β-H), 6.35 (s, 0.55 H, 1α-H), 5.57 (br. s, 0.55 H, 2α-H), 5.43 (br. s,
0.45 H, 2β-H), 5.05 (br. s, 0.45 H, 5β-H), 4.87–4.38 (m, 4.55 H, 5α-
H, CH2Ph), 4.17 (m, 0.55 H, 3α-H), 4.02 (m, 0.9 H, 3β-H, 4β-H),
(d, J4,5 = 1.0 Hz, 1 H, 5-H), 4.03 (br. d, 1 H, 4-H), 3.96 (dd, J2,3
J3,4 = 3.0 Hz, 1 H, 3-H), 3.80 (s, 3 H, COOCH3), 3.03 (d, J4,OH
=
=
12.0 Hz, 1 H, OH), 1.52 (m, 1 H, CH(CH3)2), 0.76–0.72 (4s, 12 H,
C(CH3)2 and CH(CH3)2), 0.20–0.12 (2s, 6 H, Si(CH3)2) ppm. 13C
NMR (125 MHz, CDCl3): δ = 169.0, 165.4, 137.2–127.9 (Ph), 93.2,
76.1, 74.3, 72.7, 69.4, 67.8, 52.2, 33.9, 24.8, 20.0, 19.8, 18.5, 18.3,
–2.0, –3.5 ppm. C29H40O8Si·1/2H2O (553.7): C 62.90, H 7.46; found
C 62.94, H 7.50.
Data for Regioisomer 11: [α]2D0 = +16.2 (c = 1.3, CHCl3); TLC (3:1
hexane/AcOEt), Rf = 0.69. 1H NMR (500 MHz, CDCl3): δ = 8.04–
7.31 (m, 10 H, Ph), 5.43 (br. s, 1 H, 4-H), 5.16 (br. s, 1 H, 1-H),
4.87–4.70 (2d, Jgem = 12.0 Hz, 2 H, CH2Ph), 4.74 (d, J4,5 = 2.0 Hz,
1 H, 5-H), 4.10 (dd, J2,3 = J3,4 = 2.5 Hz, 1 H, 3-H), 3.68 (s, 3 H,
COOCH3), 3.66 (m, 1 H, 2-H), 2.52 (d, J2,OH = 4.5 Hz, 1 H, OH),
1.70 (m, 1 H, CH(CH3)2), 0.94–0.91 (4s, 12 H, C(CH3)2 and
3.91 (m, 0.55 H, 4α-H), 3.76 (2s, 3 H, COOCH3 α and β) ppm. 13
C
NMR (125 MHz, CDCl3): δ = 169.2, 168.3, 166.0, 165.5, 160.5,
160.2, 137.3–127.9 (Ph), 95.5, 94.9, 91.0, 90.6, 75.0, 74.2, 73.9, 73.0,
72.9, 72.7, 72.6, 72.3, 70.8, 69.7, 66.1, 65.5, 52.38, 52.35 ppm.
Methyl 4-O-(2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
D-
glucopyranosyl)-3-O-benzyl-2-O-levulinoyl-α,β- -idopyranosuronate
L
CH(CH3)2), 0.31–0.24 (2s,
6
H, Si(CH3)2) ppm. 13C NMR
(16): An excess of (HF)n·Py complex (13.1 mL) was added to a
cooled (–15 °C) solution of 15 (2.39 g, 2.65 mmol) in dry THF
(63 mL). The reaction mixture was then warmed up to 0 °C and
stirred under argon. After 24 h, CH2Cl2 (300 mL) was added and
the mixture was washed with H2O (2 × 150 mL) and saturated
NaHCO3 solution (100 mL) until neutral pH. The organic layer
was dried (MgSO4) and concentrated in vacuo. The residue was
purified by flash chromatography (1:1 hexane/AcOEt) to yield 16
(1.65 g, 82%) as an α/β mixture: TLC (1:1 hexane/AcOEt), Rf =
0.46. 1H NMR (500 MHz, CDCl3) (0.55:0.45 α/β): δ = 7.45–7.24
(m, 15 H, Ph), 5.53 (2s, 1 H, PhCHO α and β), 5.36 (br. d, J1α,OH
= 6.5 Hz, 0.55 H, 1α-H), 5.11 (br. d, J1β,OH = 7.5 Hz, 0.45 H,1β-
H), 4.98–4.65 (m, 6.5 H, 2-H, 5α-H, 1Ј-H, CH2Ph), 4.59 (m, 1 H,
5β-H, OHβ), 4.51!(d, 0.55 H, OHα), 4.28 (m, 1 H, 6Јa-H), 4.11–
3.98 (m, 2 H, 3-H, 4-H), 3.92 (m, 1 H, 3Ј-H), 3.85–3.66 (m, 3 H,
4Ј-H, 5Ј-H, 6Јb-H), 3.78–3.75 (2s, 3 H, COOCH3 α and β), 3.41
(dd, J1Ј,2Ј = 3.5, J2Ј,3Ј = 10.0 Hz, 0.45 H, 2Јβ-H), 3.35 (dd,
J1Ј,2Ј = 3.0, J2Ј,3Ј = 9.5 Hz, 0.55 H, 2Јα-H), 2.73–2.55 (m, 4 H,
OCO(CH2)2), 2.09–2.08 (2s, 3 H, COCH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 207.5, 206.5, 172.8, 172.3, 169.7, 168.9,
137.9–126.1 (Ph), 101.4, 97.9, 97.7, 93.3, 92.4, 82.41, 82.38, 76.5,
76.1, 74.89, 74.84, 73.8, 73.3, 72.9, 72.8, 72.7, 72.5, 68.6, 68.5, 68.3,
68.1, 67.6, 63.31, 63.28, 63.21, 63.0, 52.5, 52.4, 38.2, 37.8, 29.7,
28.1, 27.9 ppm. C39H43N3O13 (761.8): C 61.49, H 5.69, N 5.52;
found C 61.21, H 5.72, N 5.47.
(125 MHz, CDCl3): δ = 168.1, 165.7, 137.5–126.0 (Ph), 94.3, 75.4,
72.9, 68.7, 67.8, 52.3, 34.1, 25.1, 20.4, 20.1, 18.7, 18.5, –1.84,
–3.31 ppm. C29H40O8Si·1/2H2O (553.7): C 62.90, H 7.46; found C
63.12, H 7.38.
Methyl (Dimethylthexylsilyl 2-O-Benzoyl-3,4-di-O-benzyl-β-L-ido-
pyranoside) Uronate (13): Benzyl bromide (295 μL, 2.48 mmol) was
added to a cooled (0 °C) solution of 12 (135 mg, 0.25 mmol) and
freshly prepared Ag2O (132 mg, 0.57 mmol) in dry DMF (0.8 mL).
After stirring for 8 h at room temperature, the solution was filtered
through celite and concentrated. The residue was purified by flash
chromatography (20:1 hexane/AcOEt) to afford 13 (98 mg, 62%).
[α]2D0 = +21.9 (c = 0.5, CHCl3); TLC (4:1 hexane/AcOEt), Rf =
1
0.46. H NMR (500 MHz, CDCl3): δ = 8.06–7.06 (m, 15 H, Ph),
5.19 (m, 2 H, 1-H, 2-H), 4.74–4.60 (2d, Jgem = 12.0 Hz, 2 H,
CH2Ph), 4.56 (d, J4,5 = 2.0 Hz, 1 H, 5-H), 4.41–4.32 (2d, Jgem
=
11.5 Hz, 2 H, CH2Ph), 3.96 (dd, J2,3 = J3,4 = 2.5 Hz, 1 H, 3-H),
3.79 (br. s, 1 H, 4-H), 3.74 (s, 3 H, COOCH3), 1.53 (m, 1 H,
CH(CH3)2), 0.76–0.72 (4s, 12 H, C(CH3)2 and CH(CH3)2), 0.19–
0.13 (2s, 6 H, Si(CH3)2) ppm. 13C NMR (125 MHz, CDCl3): δ =
169.2, 166.3, 137.5–127.7 (Ph), 93.2, 74.1, 73.7, 73.1, 72.7, 72.6,
68.0, 52.0, 34.0, 24.8, 20.2, 19.8, 18.5, 18.3, –1.9, –3.3 ppm.
C36H46O8Si (634.9): C 68.11, H 7.30; found C 67.77, H 7.16.
Methyl 2-O-Benzoyl-3,4-di-O-benzyl-α,β-
An excess of (HF)n·Py complex (2 mL) was added to a cooled
(–15 °C) solution of 13 (340 mg, 0.54 mmol) in dry THF (10 mL). O-(Methyl 4-O-(2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
L-idopyranosuronate (14):
The reaction mixture was then warmed up to 0 °C and stirred un-
der argon. After 24 h, CH2Cl2 (75 mL) was added and the mixture
was washed with H2O (2×40 mL) and saturated NaHCO3 solution
(40 mL) until neutral pH. The organic layer was dried (MgSO4)
and concentrated in vacuo. The residue was purified by flash
D-glucopyranosyl)-3-O-benzyl-2-O-levulinoyl-α,β-L-idopyranosyluron-
ate) Trichloroacetimidate (5): Cl3CCN (1.7 mL, 17.4 mmol) and
K2CO3 (220 mg, 1.60 mmol) were added to a solution of 16
(1.102 g, 1.45 mmol) in dry CH2Cl2 (10 mL). After stirring at room
temperature for 6 h, the mixture was then filtered off and concen-
1854
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 1849–1858

Synthesis and Biological Evaluation of a Heparin-Like Hexasaccharide
FULL PAPER
trated in vacuo, and the residue was purified by chromatography
4.96–4.87 (m, 4 H, 1A-H, 2B-H, CH2Ph), 4.84–4.56 (m, 14 H, 5B-
over a short silica gel column (3:2 hexane/AcOEt + 1% triethyl- H, 5D-H, 6A-H, 6ЈA-H, CH2Ph), 4.23 (m, 2 H), 4.14–3.98 (m, 7
amine) to yield 5 (1.142 g, 87%) as an α/β mixture. TLC (3:2 hex- H), 3.93–3.82 (m, 4 H), 3.77–3.62 (m, 6 H), 3.47–3.37 (2s, 6 H,
ane/AcOEt), Rf = 0.45. H NMR (500 MHz, CDCl3) (0.6:0.4 α/β): COOCH3), 3.32 (m, 2 H, 2C-H, 2E-H), 2.61–2.35 (m, 4 H, OC-
1
δ = 8.73 (2s, 1 H, NH β and α), 7.48–7.24 (m, 15 H, Ph), 6.44 (br. s,
0.4 H, 1β-H), 6.25 (br. s, 0.6 H, 1α-H), 5.53 (2s, 1 H, PhCHO α
and β), 5.31 (br. s, 0.6 H, 2α-H), 5.19 (br. s, 0.4 H, 2β-H), 5.04–
O(CH2)2), 2.08 (s, 3 H, COCH3), 1.84 (s, 3 H, NHCOCH3), 1.20
(s, 9 H, OCOC(CH3)3), 1.22–1.10 (2d, 6 H, J = 6.0 Hz, CH-
(CH3)2) ppm. 13C NMR (125 MHz, CDCl3): δ = 205.8, 177.4,
4.65 (m, 6 H, 5-H, 1Ј-H, CH2Ph), 4.31–3.63 (m, 7 H, 3-H, 4-H, 3Ј- 171.8, 169.75, 169.67, 169.1, 166.2, 138.8–127.7 (Ph), 101.5, 98.7,
H, 4Ј-H, 5Ј-H, 6Јa-H, 6Јb-H), 3.79–3.77 (2s, 3 H, COOCH3 α and 98.1, 97.9, 97.7, 95.8, 82.5, 78.5, 78.2, 76.5, 75.8, 75.6, 75.2, 75.1,
β), 3.41 (m, 1 H, 2Ј-H), 2.78–2.60 (m, 4 H, OCO(CH2)2), 2.12–2.06 74.8, 74.6, 74.5, 73.93, 73.90, 73.83, 73.51, 73.47, 71.6, 71.4, 71.0,
(2s, 3 H, COCH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 206.2,
70.5, 70.4, 70.0, 69.5, 68.6, 67.6, 63.3, 63.1, 62.91, 62.84, 52.3, 51.9,
206.0, 172.4, 172.0, 168.6, 167.9, 160.4, 160.0, 138.0–126.0 (Ph), 51.7, 38.9, 37.7, 29.6, 27.8, 27.1, 23.4, 23.3, 21.7 ppm.
101.5, 101.4, 98.2, 97.5, 95.5, 94.6, 90.9, 90.6, 82.4, 76.6, 76.2, 75.0, 103H117N7O30 (1933.1): C 64.00, H 6.10, N 5.07; found C 64.00,
C
74.9, 74.2, 73.5, 73.3, 72.5, 72.1, 70.9, 69.0, 68.53, 68.47, 66.2, 65.5, H 6.04, N 4.99.
63.4, 63.3, 63.2, 63.0, 52.52, 52.48, 37.87, 37.81, 29.7, 28.0,
27.8 ppm.
Isopropyl O-(2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
glucopyranosyl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-α- -idopyranosyl-
uronate)-(1Ǟ4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α- -glucopyr-
-idopyranosyluron- anosyl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α- -idopyranos-
ate)-(1Ǟ4)-2-acetamido-6-O-benzoyl-3-O-benzyl-2-deoxy-α- -glu- yluronate)-(1Ǟ4)-2-acetamido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-
-glucopyranoside (19): A solution of hydrazine acetate (35 mg,
D-
L
Isopropyl O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α-
D-glucopyranosyl)-
D
(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α-
L
L
D
copyranoside (6): Sodium cyanoborohydride (10.5 mL of a 1 m solu-
tion in dry THF) was added to a solution of 17 (829 mg,
0.70 mmol) in dry THF (18 mL). The reaction mixture was stirred
for 10 min and then a solution of HCl in Et2O (1 m) was added
dropwise until the mixture became acidic. The mixture was neutral-
ized with saturated NaHCO3 solution (5 mL), diluted with Et2O
(125 mL) and washed with saturated NaHCO3 solution (75 mL)
and H2O (75 mL). The organic layer was dried (MgSO4) and con-
centrated in vacuo. The residue was purified by flash chromatog-
D
0.382 mmol) in methanol (1 mL) was added to a solution of 18
(568 mg, 0.294 mmol) in CH2Cl2 (15 mL). After the reaction mix-
ture had been stirred under argon for 3 h, acetone (9 mL) was
added and the solvent was removed under reduced pressure and
the crude product was purified by flash chromatography on silica
gel (1:1 hexane/AcOEt) to afford 19 (500 mg, 93%). [α]2D0 = +11.5
(c = 0.9, CHCl3); TLC (1:1 hexane/AcOEt), Rf = 0.25. H NMR
(500 MHz, CDCl3): δ = 8.11–7.27 (m, 40 H, Ph), 5.50 (s, 1 H,
PhCHO), 5.36 (m, 2 H, 1B-H, NHCOCH3), 5.25 (br. s, 1 H, 1D-
1
raphy (75:1 CH2Cl2/MeOH) to yield 6 (747 mg, 90%): [α]2D0 = +34.0
1
(c = 0.8, CHCl3); TLC (25:1 CH2Cl2/MeOH), Rf = 0.50. H NMR H), 5.04 (m, 2 H, 2B-H, 1C-H or E), 4.97 (m, 1 H, 1C-H or E),
(500 MHz, CDCl3): δ = 8.09–7.19 (m, 25 H, Ph), 5.40 (d, J1,2
5.0 Hz, 1 H, 1B-H), 5.30 (d, JNH,2 = 9.5 Hz, 1 H, NHCOCH3),
5.04–5.00 (m, 2 H, 2B-H, 1C-H), 4.87–4.85 (m, 2 H, 1A-H,
=
4.90–4.85 (m, 4 H, 1A-H, 5B-H or 5D-H, CH2Ph), 4.76–4.72 (m,
6 H, 5B-H or 5D-H, CH2Ph), 4.68–4.54 (m, 7 H, 6A-H, 6ЈA-H,
CH2Ph), 4.27 (m, 2 H, 2A-H), 4.19 (m, 1 H), 4.15–3.99 (m, 6 H),
CH2Ph), 4.80–4.65 (m, 6 H, 5B-H, CH2Ph), 4.57–4.47 (m, 4 H, 6A- 3.95–3.79 (m, 4 H), 3.68–3.55 (m, 9 H), 3.43–3.30 (2s, 6 H, CO-
H, 6ЈA-H, CH2Ph), 4.23 (ddd, J1,2 = 4.0 Hz, 1 H, 2A-H), 4.10 (m, OCH3), 3.35 (m, 1 H, 2C-H or E), 1.82 (s, 3 H, NHCOCH3), 1.18
2 H, 4A-H, 4B-H), 4.00 (m, 2 H, 5A-H, 3B-H), 3.85–3.56 (m, 7 H,
3A-H, 3C-H, 4C-H, 5C-H, 6C-H, 6ЈC-H, CH(CH3)2), 3.38 (s, 3 H, (CH3)2) ppm. 13C NMR (125 MHz, CDCl3): δ = 177.5, 169.8,
COOCH3), 3.20 (dd, J1,2 = 3.5, J2,3 = 10.0 Hz, 1 H, 2C-H), 2.88 169.3, 166.2, 138.7–126.1 (Ph), 101.5, 101.2, 98.3, 97.9, 96.0, 95.9,
(br. s, 1 H, OH), 1.80 (s, 3 H, NHCOCH3), 1.18 (s, 9 H, OC- 82.1, 78.5, 78.3, 77.4, 76.0, 75.4, 75.2, 74.9, 74.4, 73.8, 73.7, 73.6,
(s, 9 H, OCOC(CH3)3), 1.22–1.09 (2d, 6 H, J = 6.0 Hz, CH-
OC(CH3)3), 1.20–1.07 (2d, 6 H, J = 6.0 Hz, CH(CH3)2) ppm. 13C
NMR (125 MHz, CDCl3): δ = 177.5, 169.8, 169.4, 166.2, 138.7–
127.5 (Ph), 98.5, 97.7, 95.8, 79.3, 78.5, 76.1, 75.4, 74.9, 74.5, 73.7,
73.6, 72.1, 72.0, 70.9, 70.8, 70.4, 69.5, 69.4, 62.9, 62.7, 52.3, 51.8,
38.9, 27.1, 23.33, 23.25, 21.7 ppm. C64H76N4O18·H2O (1207.4): C
63.67, H 6.51, N 4.64; found C 63.87, H 6.31, N 4.67.
73.3, 72.7, 72.2, 71.8, 70.8, 70.5, 70.4, 69.5, 68.4, 63.3, 63.2, 63.1,
63.0, 52.3, 52.2, 51.7, 38.8, 27.1, 23.4, 23.3, 21.7 ppm.
C98H111N7O28·H2O (1853.0): C 63.52, H 6.15, N 5.29; found C
63.32, H 6.14, N 5.28.
Isopropyl O-(2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
glucopyranosyl)-(1Ǟ4)-O-(Methyl 2,3-Di-O-benzyl-α- -idopyranos-
-gluco-
-idopyr-
D-
L
Isopropyl O-(2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
D-
yluronate)-(1Ǟ4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α-
pyranosyl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α-
D
glucopyranosyl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-levulinoyl-α-
L-
L
idopyranosyluronate)-(1Ǟ4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-
α- -glucopyranosyl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α-
-idopyranosyluronate)-(1Ǟ4)-2-acetamido-6-O-benzoyl-3-O-ben-
zyl-2-deoxy-α- -glucopyranoside (18): TMSOTf (300 μL of a 0.38 m
anosyluronate)-(1Ǟ4)-2-acetamido-6-O-benzoyl-3-O-benzyl-2-de-
oxy-α- -glucopyranoside (20): Benzyl bromide (219 μL, 1.84 mmol)
D
D
L
was added to a cooled (0 °C) solution of 19 (338 mg, 0.184 mmol)
and freshly prepared Ag2O (85 mg, 0.368 mmol) in dry DMF
(0.7 mL). After stirring for 12 h at room temperature, the solution
was filtered through celite and concentrated. The residue was puri-
fied by flash chromatography (3:1 toluene/AcOEt) to afford 20
(264 mg, 75%) and starting material (50 mg, 15%). [α]2D0 = +23.2
D
solution in dry CH2Cl2) was added at room temperature, under
argon, to a solution of 6 (596 mg, 501 μmol) and 5 (681 mg,
0.752 mmol) in dry CH2Cl2 (12 mL). After 3 h, saturated NaHCO3
solution (4 mL) and CH2Cl2 (200 mL) were added and the mixture
was washed with H2O (100 mL). The organic layer was dried
(MgSO4) and concentrated in vacuo, and the residue was purified
by flash column chromatography (4:1 Ǟ2:1 toluene/AcOEt) to
yield 18 (568 mg, 59%) and unreacted acceptor 6 (108 mg, 18%).
1
(c = 0.5, CHCl3); TLC (3:1 toluene/AcOEt), Rf = 0.24. H NMR
(500 MHz, CDCl3): δ = 8.12–7.17 (m, 45 H, Ph), 5.58 (s, 1 H,
PhCHO), 5.50 (d, J1,2 = 6.4 Hz, 1 H, 1D-H), 5.43 (d, J1,2 = 5.2 Hz,
1 H, 1B-H), 5.31 (d, JNH,2 = 9.3 Hz, 1 H, NHCOCH3), 5.22 (d,
J1,2 = 3.8 Hz, 1 H, 1E-H), 5.08 (dd, J2,3 = 5.5 Hz, 1 H, 2B-H), 5.04
[α]2D0 = +16.9 (c = 0.9, CHCl3); TLC (1:1 hexane/AcOEt), Rf =
1
0.21. H NMR (500 MHz, CDCl3): δ = 8.13–7.16 (m, 40 H, Ph), (d, J1,2 = 3.5 Hz, 1 H, 1C-H), 4.97–4.84 (m, 7 H, 1A-H, CH2Ph),
5.55 (s, 1 H, PhCHO), 5.47 (d, J1,2 = 5.0 Hz, 1 H, 1D-H), 5.34 (m,
2 H, 1B-H, NHCOCH3), 5.07–5.04 (m, 3 H, 2D-H, 1C-H, 1E-H),
4.80–4.68 (m, 7 H, 5B-H, 6A-H, CH2Ph), 4.63–4.56 (m, 3 H, 6ЈA-
H, CH2Ph), 4.51 (d, Jgem = 12.0 Hz, 1 H, CH2Ph), 4.45 (d, J4,5
=
Eur. J. Org. Chem. 2005, 1849–1858
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1855

J. L. de Paz, M. Martín-Lomas
FULL PAPER
6.0 Hz, 1 H, 5D-H), 4.25 (m, 2 H, 2A-H, 6E-H), 4.18–3.99 (m, 7
H, 4A-H, 5A-H, 3B-H, 4B-H, 4C-H, 3D-H, 4D-H), 3.96–3.85 (m,
5D-H, 6ЈA-H, CH2Ph), 4.39 (m, 1 H, 6ЈE-H), 4.24 (ddd, J1,2
3.5 Hz, 1 H, 2A-H), 4.14–3.95 (m, 8 H, 4A-H, 5A-H, 3B-H, 4B-H,
=
4 H, CH(CH3)2, 5C-H, 3E-H, 4E-H), 3.80–3.65 (m, 5 H, 3A-H, 4C-H, 3D-H, 4D-H, 5E-H), 3.87–3.83 (m, 2 H, CH(CH3)2, 5C-H),
3C-H, 6C-H, 5E-H, 6ЈE-H), 3.55 (s, 3 H, COOCH3), 3.54 (m, 1 H, 3.77 (m, 1 H, 6C-H), 3.72–3.57 (m, 4 H, 3A-H, 3C-H, 3E-H, 4E-
6ЈC-H), 3.43 (dd, 1 H, 2D-H), 3.36 (m, 2 H, 2C-H, 2E-H), 3.30 (s, H), 3.56 (s, 3 H, COOCH3), 3.52 (m, 1 H, 6ЈC-H), 3.41 (dd, 1 H,
3 H, COOCH3), 1.84 (s, 3 H, NHCOCH3), 1.24 (s, 9 H, OC- 2D-H), 3.35 (dd, J1,2 = 3.8, J2,3 = 10.5 Hz, 1 H, 2C-H), 3.27 (s, 3
OC(CH3)3), 1.24–1.12 (2d, 6 H, J = 6.0 Hz, CH(CH3)2) ppm. 13C
NMR (125 MHz, CDCl3): δ = 177.4, 169.7, 169.5, 169.4, 166.2,
138.8–126.2 (Ph), 101.5, 100.1, 99.7, 98.3, 97.8, 95.9, 82.6, 81.5,
H, COOCH3), 3.23 (dd, J1,2 = 3.3, J2,3 = 9.8 Hz, 1 H, 2E-H), 1.82
(s, 3 H, NHCOCH3), 1.21 (s, 9 H, OCOC(CH3)3), 1.21–1.09 (2d, J
= 6.0 Hz, 6 H, CH(CH3)2) ppm. 13C NMR (125 MHz, CDCl3): δ
79.0, 78.5, 78.2, 76.2, 76.1, 75.6, 75.48, 75.46, 75.1, 74.8, 74.7, 74.6, = 177.4, 169.8, 169.51, 169.46, 167.2, 166.2, 138.7–127.8 (Ph),
73.9, 73.8, 73.5, 72.3, 71.6, 71.2, 71.0, 70.4, 69.5, 68.6, 67.3, 63.4,
62.9, 62.8, 52.2, 51.9, 51.7, 38.9, 27.2, 23.4, 23.3, 21.7, 21.5 ppm.
100.1, 99.1, 98.3, 97.7, 95.8, 81.2, 78.7, 78.5, 78.0, 76.24, 76.18,
75.6, 75.2, 74.9, 74.8, 74.61, 74.58, 73.91, 73.85, 73.5, 72.2, 71.6,
C105H117N7O28 (1925.1): C 65.51, H 6.13, N 5.09; found C 65.39, 71.2, 71.0, 70.6, 70.4, 69.5, 67.3, 63.0, 62.8, 52.2, 51.9, 51.7, 38.9,
H 5.93, N 5.13.
27.1, 23.4, 23.3, 21.7 ppm. C105H117N7O29·H2O (1959.2): C 64.37,
H 6.12, N 5.00; found C 64.29, H 6.13, N 5.01.
Isopropyl
O-(2-Azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1Ǟ4)-O-(Methyl
(1Ǟ4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α-
(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α-
2,3-Di-O-benzyl-α-
L
-idopyranosyluronate)- Isopropyl O-(Methyl 2-O-Benzoyl-3,4-di-O-benzyl-α-
-glucopyranosyl)- uronate)-(1Ǟ4)-O-(2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-
-idopyranosyluron- glucopyranosyl)-(1Ǟ4)-O-(Methyl 2,3-Di-O-benzyl-α- -idopyranos-
ate)-(1Ǟ4)-2-acetamido-6-O-benzoyl-3-O-benzyl-2-deoxy-α- -glu- yluronate)-(1Ǟ4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α- -gluco-
pyranosyl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α- -idopyr-
anosyluronate)-(1 Ǟ4)-2-acetamido-6-O-benzoyl-3-O-benzyl-2-de-
oxy-α- -glucopyranoside (3): TMSOTf (150 μL of a 0.17 m solution
L-idopyranosyl-
D
D-
L
L
D
D
copyranoside (21): EtSH (48 μL, 0.65 mmol) and catalytic pTsOH
were added to a solution of 20 (250 mg, 0.13 mmol) in dry CH2Cl2
(4 mL). After stirring for 2 h under argon, the mixture was neutral-
ized with solid NaHCO3, diluted with CH2Cl2 (75 mL), washed
with H2O (50 mL), dried (MgSO4) and concentrated to dryness.
The purification of the residue was carried out by flash chromatog-
raphy (1:2 hexane/AcOEt) to yield 21 (201 mg, 84%). [α]2D0 = +35.0
(c = 0.5, CHCl3); TLC (1:2 hexane/AcOEt), Rf = 0.51. H NMR
(500 MHz, CDCl3): δ = 8.07–7.17 (m, 40 H, Ph), 5.46 (d, J1,2
6.5 Hz, 1 H, 1D-H), 5.39 (d, J1,2 = 5.0 Hz, 1 H, 1B-H), 5.27 (d,
L
D
in dry CH2Cl2) was added at room temperature, under argon, to a
solution of 22 (178 mg, 92 μmol) and 4 (105 mg, 0.166 mmol) in
dry CH2Cl2 (2 mL). After 3 h, saturated NaHCO3 solution (2 mL)
and CH2Cl2 (50 mL) were added and the mixture was washed with
H2O (35 mL). The organic layer was dried (MgSO4) and concen-
trated in vacuo, and the residue was purified by flash column
chromatography (3:1 toluene/AcOEt) to yield 3 (115 mg, 52%) and
1
=
JNH,2 = 9.0 Hz, 1 H, NHCOCH3), 5.19 (d, 1 H, 1E-H), 5.02 (m, 2 unreacted acceptor (79 mg, 44%). [α]2D0 = +24.7 (c = 0.7, CHCl3);
H, 2B-H, 1C-H), 4.92–4.43 (m, 19 H, 1A-H, 5B-H, 5D-H, 6A-H, TLC (3:1 toluene/AcOEt), Rf = = 0.35. 1H NMR (500 MHz,
6ЈA-H, CH2Ph), 4.21 (m, 1 H, 2A-H), 4.10–3.98 (m, 7 H), 3.81– CDCl3): δ = 8.06–7.09 (m, 60 H, Ph), 5.58 (d, J1,2 = 6.0 Hz, 1 H,
3.62 (m, 10 H), 3.50 (s, 3 H, COOCH3), 3.49 (m, 1 H), 3.38 (dd, 1
H, 2D-H), 3.32 (m,1 H, 2C-H), 3.26 (s, 3 H, COOCH3), 3.16 (m,
1 H, 2E-H), 3.00 (br. s, 1 H, OH), 2.31 (br. s, 1 H, OH), 1.80 (s, 3
1F-H), 5.40 (d, J1,2 = 7.0 Hz, 1 H, 1D-H), 5.38 (d, J1,2 = 5.5 Hz,
1 H, 1B-H), 5.27–5.20 (m, 2 H, NHCOCH3, 2F-H), 5.12 (d, J1,2
3.5 Hz, 1 H, 1E-H), 5.03–5.00 (m, 2 H, 2B-H, 1C-H), 4.96–4.41
=
H, NHCOCH3), 1.19 (s, 9 H, OCOC(CH3)3), 1.19–1.07 (2d, 6 H, (m, 25 H, 1A-H, 6A-H, 6ЈA-H, 5B-H, 5F-H, 6E-H, 6ЈE-H,
J = 6.0 Hz, CH(CH3)2) ppm. 13C NMR (125 MHz, CDCl3): δ = CH2Ph), 4.21–4.18 (m, 2 H, 5D-H, 2A-H), 4.14 (dd, J3,4 = J4,5
177.4, 169.9, 169.6, 169.4, 166.2, 138.7–127.7 (Ph), 100.0, 99.1, 9.2 Hz, 1 H, 4E-H), 4.09–3.93 (m, 7 H, 4A-H, 5A-H, 3B-H, 4B-H,
98.2, 97.7, 95.7, 81.2, 79.1, 78.9, 78.4, 76.2, 76.1, 75.6, 75.0, 74.9, 4C-H, 3D-H, 3F-H), 3.90–3.77 (m, 5 H, CH(CH3)2, 5C-H, 4D-H,
74.8, 74.61, 74.57, 73.9, 73.5, 72.3, 72.2, 71.6, 71.2, 71.0, 70.9, 70.4, 5E-H, 4F-H), 3.73–3.59 (m, 4 H, 3A-H, 3C-H, 3E-H, 6C-H), 3.46
=
62.8, 62.7, 61.8, 52.2, 51.9, 51.7, 38.9, 27.1, 23.3, 23.2, 21.6 ppm.
C98H113N7O28·H2O (1855.0): C 63.45, H 6.25, N 5.29; found C
63.52, H 6.31, N 5.28.
(m, 1 H, 6ЈC-H), 3.41–3.40 (2s, 6 H, COOCH3), 3.36–3.29 (m, 3
H, 2C-H, 2D-H, 2E-H), 3.24 (s, 3 H, COOCH3), 1.80 (s, 3 H,
NHCOCH3), 1.18 (s, 9 H, OCOC(CH3)3), 1.18–1.07 (2d, J =
6.0 Hz, 6 H, CH(CH3)2) ppm. 13C NMR (125 MHz, CDCl3): δ =
178.0, 170.4, 170.3, 170.06, 170.03, 166.77, 166.68, 165.9, 139.3–
128.5 (Ph), 100.6, 99.5, 98.8, 98.7, 98.3, 96.4, 82.2, 79.6, 79.0, 78.6,
78.5, 76.92, 76.89, 76.5, 76.44, 76.37, 76.2, 76.1, 75.73, 75.68, 75.4,
75.2, 74.5, 74.3, 74.0, 73.6, 73.0, 72.4, 72.20, 72.16, 71.9, 71.7, 71.0,
70.6, 70.1, 67.8, 63.5, 63.4, 62.5, 52.8, 52.27, 52.23, 39.5, 30.3, 27.7,
24.0, 23.8, 22.2 ppm. C133H143N7O36 (2415.7): C 66.13, H 5.97, N
4.06; found C 66.06, H 5.88, N 4.34.
Isopropyl O-(2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-
ranosyl)-(1Ǟ4)-O-(Methyl 2,3-Di-O-benzyl-α- -idopyranosyluron-
ate)-(1Ǟ4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α- -glucopyranos-
yl)-(1Ǟ4)-O-(Methyl 3-O-Benzyl-2-O-pivaloyl-α- -idopyranosyl-
uronate)-(1Ǟ4)-2-acetamido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-
D-glucopy-
L
D
L
D-
glucopyranoside (22): BzCN (100 μL of a 1.1 m solution in dry
CH3CN) and catalytic Et3N were added to a cooled (–40 °C) solu-
tion of 21 (190 mg, 103 μmol) in dry CH3CN (3 mL). After 30 min,
additional BzCN was added (20 μL of a 1.1 m solution in dry
CH3CN) until starting material disappeared. After 1 h, MeOH was
added and the mixture was allowed to reach room temperature.
The solvent was evaporated and the residue was dissolved in
MeOH and concentrated to dryness. The purification was carried
out by flash chromatography (75:1 CH2Cl2/MeOH) to afford 22
(198 mg, 99%). [α]2D0 = +33.4 (c = 0.6, CHCl3); TLC (75:1 CH2Cl2/
Isopropyl O-(3,4-Di-O-benzyl-α-
O-(2-Azido-3-O-benzyl-2-deoxy-α-
(2,3-Di-O-benzyl-α- -idopyranosyluronic Acid)-(1Ǟ4)-O-(2-Azido-
3,6-di-O-benzyl-2-deoxy-α- -glucopyranosyl)-(1Ǟ4)-O-(3-O-Ben-
zyl-α- -idopyranosyluronic Acid)-(1Ǟ4)-2-acetamido-3-O-benzyl-2-
deoxy-α- -glucopyranoside (23): H2O2 (30%, 0.55 mL) and LiOH
solution (1 n, 0.9 mL) were added to a solution of 3 (33 mg,
L-idopyranosyluronic Acid)-(1Ǟ4)-
D
-glucopyranosyl)-(1 Ǟ 4)-O-
L
D
L
D
MeOH), Rf = 0.28. 1H NMR (500 MHz, CDCl3): δ = 8.10–7.17 14 μmol) in THF (1.6 mL) at –5 °C. After stirring for 24 h at room
(m, 45 H, Ph), 5.48 (d, J1,2 = 6.4 Hz, 1 H, 1D-H), 5.41 (d, J1,2
=
temperature, the mixture was cooled to 0 °C and MeOH (2.8 mL)
5.3 Hz, 1 H, 1B-H), 5.30 (d, JNH,2 = 9.1 Hz, 1 H, NHCOCH3),
and KOH solution (3 n, 1.6 mL) were added. After stirring for 24 h
5.24 (d, 1 H, 1E-H), 5.07–5.03 (m, 2 H, 2B-H, 1C-H), 4.93–4.65 at room temperature, the reaction mixture was neutralized with IR-
(m, 15 H, 1A-H, 6A-H, 5B-H, 6E-H, CH2Ph), 4.61–4.46 (m, 5 H,
120-H+ Amberlite resin and was then filtered and concentrated.
1856
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 1849–1858

Synthesis and Biological Evaluation of a Heparin-Like Hexasaccharide
FULL PAPER
The residue was eluted from a Sephadex L20-H chromatography
of HCl (0.1 n) and was then subjected to chromatography over a
Sephadex G-25 column with a 0.9% solution of NaCl. The appro-
priate fractions were pooled and passed through a column of
Dowex 50WX4-Na+ with a solution of NaCl (0.5 m) as the eluent
and then through a column of Sephadex G-25 with H2O/MeOH
(9:1) as the eluent. The fractions containing the final hexasaccha-
ride were lyophilized to give 2 (5.4 mg, 93%). Before NMR spectro-
scopic studies, it was useful to make a last elution over a column
of Dowex 50WX4-Na+ (H2O/MeOH, 9:1) in order to avoid the
formation of calcium salts instead of sodium salts and to obtain
column with MeOH/CH2Cl2 (1:1) to afford 23 (26 mg, 96%). TLC
1
(12:1 CH2Cl2/MeOH), Rf = 0.39. H NMR (500 MHz, MeOD): δ
= 7.46–7.10 (m, 45 H, Ph), 5.37–5.14 (m, 5 H, 1B-H, 1C-H, 1D-
H, 1E-H, 1F-H), 3.59–3.48 (m, 3 H, 2C-H, 2D-H, 2E-H), 1.83 (s,
3 H, NHCOCH3), 1.28–1.16 (2d, J = 6.1 Hz, 6 H, CH(CH3)2) ppm.
FAB MS: m/z 1998 [M + Na]+.
Isopropyl
Acid)-(1Ǟ4)-O-(2-Azido-3-O-benzyl-2-deoxy-6-O-sulfo-α-
pyranosyl)-(1 Ǟ 4)-O-(2,3-Di-O-benzyl-α- -idopyranosyluronic
Acid)-(1 Ǟ 4)-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-α- -gluco-
pyranosyl)-(1 Ǟ 4)-O-(3-O-Benzyl-2-O-sulfo-α- -idopyranosyl-
uronic Acid)-(1 Ǟ 4)-2-acetamido-3-O-benzyl-2-deoxy-6-O-
sulfo-α- -glucopyranoside Heptasodium Salt (24): Sulfur triox-
O-(3,4-Di-O-benzyl-2-O-sulfo-α-
L-idopyranosyluronic
D-gluco-
L
1
better resolution in the spectra. H NMR (500 MHz, D2O, 25 °C):
D
δ = 3.89 (m, 1 H, CH(CH3)2), 1.99 (s, 3 H, NHCOCH3), 1.19–
1.09 (2d, J = 6.2 Hz, 6 H, CH(CH3)2) ppm. (The complete NMR
spectrum assignment of the sugar protons appears in Table 1). 13C
NMR (125 MHz, D2O, 25 °C): δ = 71.56 (CH(CH3)2), 22.2
(NHCOCH3), 22.7 and 20.9 (CH(CH3)2) ppm. (The complete
NMR spectrum assignment of the sugar carbons appears in
Table 1.) ES MS: m/z 872.3 (M – 2Na)–2.
L
D
ide–pyridine complex (40 mg, 0.253 mmol) was added to a solution
of 23 (25 mg, 13 μmol) in dry Py (1.5 mL). (This reactive complex
had been previously washed with H2O, EtOH and CH2Cl2 and
dried, because this kind of sulfating material usually contains a lot
of acid). After stirring for 20 h at room temperature under argon,
the mixture was diluted with MeOH (1 mL) and CH2Cl2 (1 mL)
and the solution was layered on the top of a Sephadex L20-H
chromatography column, which was eluted with MeOH/CH2Cl2
(1:1). The fractions containing sulfated products were pooled and
the solvents were evaporated to dryness. The residue was subjected
to the same process (sulfation and purification by Sephadex L20-
H) until no evolution was observed by TLC (8:5:3:1 ethyl acetate/
Py/H2O/AcOH) and was then converted into the corresponding so-
dium salt by elution from a column of Dowex 50WX4-Na+ with
MeOH/H2O (9:1) to give 24 (15 mg, 50%). TLC (8:5:3:1 ethyl ace-
Acknowledgments
We thank Dr. P. M. Nieto for helpful discussions, Ms, Sara López
Galán for technical assistance and Mr. Jesús Quintanilla (Labora-
torio Aplicaciones Thermo, Instituto Química-Física Rocasolano,
Madrid) for recording the mass spectrum of 24.
1
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7.36–7.11 (m, 45 H, Ph), 5.63–5.43 (2 br. s, 2 H, 1F-H, 1B-H), 5.34
(br. s, 1 H, 1D-H), 5.22 (d, J1,2 = 4.0 Hz, 1 H, 1E-H), 5.08 (d, Jgem
= 11.0 Hz, 1 H, CH2Ph), 4.99–4.93 (m, 3 H, 1C-H, 5-H B or 5F-
H, CH2Ph), 3.56–3.49 (m, 3 H, 2C-H, 2D-H, 2E-H), 1.59 (s, 3 H,
NHCOCH3), 1.22–1.08 (2d, J = 6.0 Hz, 6 H, CH(CH3)2). ES MS:
m/z 2382 [M –3Na + 2H]–.
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Isopropyl O-(2-O-Sulfo-α-
L
-idopyranosyluronic Acid)-(1Ǟ4)-O-(2-
-glucopyranosyl)-(1Ǟ4)-O-(α-
Acid)-(1Ǟ4)-O-(2-Deoxy-2-sulfamido-α-
Deoxy-2-sulfamido-6-O-sulfo-α-
Idopyranosyluronic
D
L-
D-
glucopyranosyl)-(1Ǟ4)-O-(2-O-Sulfo-α-
L-idopyranosyluronic Acid)-
(1Ǟ4)-2-Acetamido-2-deoxy-6-O-sulfo-α-
D-glucopyranoside Nona-
sodium Salt (2): A solution of 24 (8.0 mg, 3.3 μmol) in MeOH/H2O
(1.8 mL/0.4 mL) was hydrogenated in the presence of 10% Pd/C.
After 24 h the suspension was filtered and concentrated to give the
desired product, which was homogeneous by TLC analysis with
ethyl acetate/Py/H2O/AcOH (3:5:3:1) as the eluent (Rf, 0.24). No
aromatic signal was detected by NMR spectroscopy: 1H NMR
(500 MHz, D2O): δ = 5.27–5.26 (2 br. s, 2 H, 1F-H, 1B-H), 5.23–
5.17 (2 d, J1,2 = 3.5 Hz, 2 H, 1C-H, 1E-H), 4.98 (d, J1,2 = 3.5 Hz,
1 H, 1A-H), 4.94 (d, J1,2 = 4.0 Hz, 1 H, 1D-H), 4.90–4.75 (3d, 3
H, 5B-H, 5D-H, 5F-H), 4.39–4.24 (m, 7 H), 4.18–4.02 (m, 6 H),
3.97–3.72 (m, 13 H), 2.93–2.82 (2 dd, 2 H, 2C-H, 2E-H), 2.05 (s, 3
H, NHCOCH3), 1.24–1.14 (2d, 6 H, J = 6.0 Hz, CH(CH3)2) ppm.
This compound was directly used for the N-sulfation.
The hydrogenated hexasaccharide (7 mg) was dissolved in H2O
(1 mL) and the pH value of the solution was adjusted to 9.5 with
a solution of NaOH (1 n). A pyridine–sulfur trioxide complex
(5 mg, 5 equivalents for each amine group) was added and the pH
value was maintained at 9.5 by subsequent addition of a solution
of NaOH (1 n). Second, third and fourth additions of the pyridine–
sulfur trioxide complex were made after stirring for 2, 4 and 6 h,
respectively. After 24 h the mixture was neutralised with a solution
Eur. J. Org. Chem. 2005, 1849–1858
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1857

J. L. de Paz, M. Martín-Lomas
FULL PAPER
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Received: November 08, 2004
1858
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 1849–1858