A rational approach to heparin-related fragments - Synthesis of differently sulfated tetrasaccharides as potential ligands for fibroblast growth factors

Article abstract of DOI:10.1002/1099-0690(200107)2001:14<2727::aid-ejoc2727>3.0.co;2-8

Heparin-like tetrasaccharides 1-3, differing in their sulfation pattern at position 6 of the glucosamine units, were synthesised. The three compounds are putative ligands for fibroblast growth factors and have the unusual sequence (GlcN-IdoA). They were o

Full text of DOI:10.1002/1099-0690(200107)2001:14<2727::aid-ejoc2727>3.0.co;2-8

FULL PAPER
A Rational Approach to Heparin-Related Fragments ؊ Synthesis of
Differently Sulfated Tetrasaccharides as Potential Ligands for Fibroblast
Growth Factors
Laura Poletti,[a] Martin Fleischer,[a] Christian Vogel,[a] Marco Guerrini,[b]
Giangiacomo Torri,[b] and Luigi Lay*[a]
Keywords: Carbohydrates / Growth factors / Oligosaccharides
Heparin-like tetrasaccharides 1−3, differing in their sulfation
fibroblast growth factors and have the unusual sequence
pattern at position 6 of the glucosamine units, were syn- (GlcN-IdoA). They were obtained from two common disacch-
thesised. The three compounds are putative ligands for
aride precursors by a versatile synthetic procedure.
Introduction
identification of the minimal saccharide structure required
for binding; and (ii) the role of the sulfation pattern. As a
matter of fact, preliminary binding studies on FGF-2 and
FGF-1 have indicated that 2-O-sulfation of -idopyranosyl-
uronic acid and N-sulfation of -glucosamine moieties are
essential for the interaction with FGFs. In contrast, the role
played by 6-O-sulfation is still unclear.[10] Moreover, the
strict relationship observed between heparin structure and
biological activity of the growth factor suggests that modi-
fication of the sulfation pattern of heparin fragments can
be an important means of regulation of the response of cells
to distinct members of the FGF family.
The synthesis of a number of heparin-related fragments,
different in length, in composition of monosaccharide units
and/or in sulfation pattern, may provide a contribution to
these topics. Moreover, conformational analysis and model-
ling of these fragments should be useful for acquisition of
information on structure-activity relationships.
In order to establish how the conformational and binding
properties are influenced by different sulfation patterns, we
began a project aimed at the exploration of a new series of
heparin-related fragments containing a glucosamine unit at
the nonreducing terminus. These types of oligomers are ra-
ther unusual, since heparin fragments obtained by com-
monly used degradation methods, either enzymatic or
chemical, generally bear a uronic acid at the nonreducing
end. We focussed our attention on the significance of the 6-
O-sulfation of the glucosamine units, which, as mentioned
above, is still unclear. We therefore planned a synthetic ap-
proach[11] suitable for obtaining all possible oligosacchar-
ides of various length, in which the O-6 position of each
glucosamine unit may or may not be sulfated.
Our synthetic strategy was based on the synthesis and
coupling of two versatile disaccharides 4 and 5, bearing a
pattern of orthogonal protective groups. Adoption of such
an approach should make it possible, in principle, to build
up new families of differently sulfated disaccharides, tetra-
saccharides or even longer oligosaccharides. Here we de-
scribe the application of this approach to the synthesis of
Fibroblast growth factors (FGFs) constitute a large fam-
ily of at least seven structurally related, multifunctional pro-
teins with a variety of growth and differentiation activit-
ies.[1,2] They are highly mitogenic for vascular endothelial
cells and are among the most potent inducers of angiogen-
esis, which is involved in several critical physiological
events, including organogenesis, wound healing and solid
tumour growth. These functions are mediated by interac-
tion of the growth factors with high-affinity cell-surface re-
ceptors and subsequent alterations in gene expression
within responsive cells.
FGFs display relatively high binding affinities for
glycosaminoglycans,[3Ϫ7] such as heparan sulfate (HS) and
heparin, this latter being a linear, heterogeneously sulfated,
anionic polysaccharide, composed of alternating -iduronic
and -glucosamine units and almost ubiquitous in animal
tissues.
The most intensely studied and best characterised mem-
bers of the FGFs are FGF-2 (basic FGF) and FGF-1
(acidic FGF). Their interaction with HS proteoglycans at
the cell surface and in the extracellular matrix is thought to
be of functional significance, serving as storage depots for
growth factors and protecting them from various degradat-
ive or inactivation processes. The addition of heparin in-
duces the release of growth factors in active form and their
binding facilitates interaction with high-affinity signalling
receptors on the cell surface. From these findings, it seems
likely that heparin may play a central role in the regulation
of vascular cell growth.[8,9]
As far as the study of the binding between FGFs and
heparin is concerned, two main problems are evident: (i)
[a]
Department of Organic and Industrial Chemistry, University
of Milan,
Via Venezian 21, 20133 Milan, Italy
Fax: (internat.) ϩ 39-02/266-4952
E-mail: llay@mailserver.unimi.it
[b]
Institute of Chemistry and Biochemistry ‘‘G. Ronzoni’’,
Via Colombo 81, 20131 Milan, Italy
Eur. J. Org. Chem. 2001, 2727Ϫ2734
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0714Ϫ2727 $ 17.50ϩ.50/0
2727

L. Poletti, M. Fleischer, C. Vogel, M. Guerrini, G. Torri, L. Lay
FULL PAPER
tetrasaccharides 1؊3 (Figure 1), corresponding to the un- disaccharide 5 was employed for the synthesis of the trichlo-
usual sequence (GlcN-IdoA). In tetrasaccharides 1؊3, in roacetimidate 9 by the same sequence as described for the
which both the idopyranosiduronyl residues are invariably preparation of 8 (59% overall yield, Scheme 2).
2-O-sulfated, the glucosaminyl units contain three possible
permutations of the functionalisation of the primary posi-
tion (6-OH and/or 6-O-sulfate). These fragments fulfil one
of the basic requirements for binding to FGFs (presence
of 2-O-sulfated iduronic acid and N-sulfated glucosaminyl
units) and should allow the importance of 6-O-sulfation to
be evaluated through proper biological assays.
Scheme 1. Synthesis of acceptors 6 and 7 (All ϭ allyl)
Figure 1. Tetrasaccharides 1؊3; Pr ϭ propyl
Results and Discussion
According to our methodology, tetrasaccharides 1؊3
were synthesised by coupling disaccharide building blocks
derived from disaccharides 4 and 5,[11] strategically pro-
tected in order to permit elongation both at the reducing
and at the nonreducing ends. We chose benzyl ethers as
permanent groups and acetates as temporary groups to pro-
tect those hydroxy groups intended to be O-sulfated.
Disaccharide 4 was converted into acceptors 6 and 7 by
hydrolysis of the benzylidene acetal with a 70% aqueous
solution of trifluoroacetic acid, followed by regioselective 6-
O-benzylation (52% overall yield from 4) or 6-O-acetylation
Scheme 2. Synthesis of donors 8 and 9 (All ϭ allyl)
With these versatile building blocks in hand, the synthesis
(85% overall yield from 4), respectively (Scheme 1). Disac- of the tetrasaccharides 1؊3 was carried out in a straightfor-
charide 4 also afforded glycosyl donor 8 in 84% overall ward manner. Thus, coupling between donor 9 and ac-
yield (Scheme 2) by a two-step deallylation [isomerization ceptor 7, performed at Ϫ20 °C in the presence of TMSOTf,
of the allyl group to the propenyl group with (1,5-cyclo- afforded tetrasaccharide 10, containing four O-acetylated
octadiene)bis(methyldiphenylphosphane)iridium hexaflu- hydroxy groups, in 50% yield. Similarly, tetrasaccharides 11
orophosphate catalyst[12] under hydrogen, followed by treat- and 12 were obtained by coupling of acceptor 7 with donor
ment with iodine in moist THF] and final conversion of the 8 (63% yield) and of acceptor 6 with donor 8 (62% yield)
sugar hemiacetal into the corresponding trichloroacetimid- respectively, also at Ϫ20 °C and using TMSOTf as a Lewis
ate[13] (trichloroacetonitrile, cat. DBU). On the other hand, acid catalyst (Scheme 3).
Scheme 3. Synthesis of tetrasaccharides 10؊12
2728
Eur. J. Org. Chem. 2001, 2727Ϫ2734

A Rational Approach to Heparin-Related Fragments
FULL PAPER
1
of a participating group at C-2 of the glycosyl donor, and
this was further confirmed by NMR spectroscopy (Table 1
and Table 2). In the H NMR spectra, the signals corre-
sponding to the anomeric protons appear as doublets in the
δ ϭ 5.00Ϫ5.20 range (Table 1), in full agreement with data
describing heparin-like oligosaccharide synthesis reported
in previous papers.[14,15]
Table 1. H NMR (CD3OD, 500 MHz, 303 K) of tetrasaccharides
10؊12; coupling constants are given in Hz
1
Unit
d
δ
c
δ
b
δ
a
δ
Compd. H-n
(Jn,nϩ1
)
(Jn,nϩ1
)
(Jn,nϩ1
)
(Jn,nϩ1)
10
11
12
1
2
4.96
(3.6)
3.39
5.14
(3.1)
4.91
5.04
(3.4)
3.35
5.02
(2.2)
4.90
(10.3)
3.70
(3.3)[a] (10.3)
(n.d.)
3.97
Table 2. 13C NMR (CD3OD, 125.72 MHz, 303 K) of tetrasacchar-
ides 10؊12
3
4.02
(3.8)[a]
4.08
(n.d.)
4.78
3.81
(8.8)
3.52
(10.1)
3.80
(2.2)
4.33
(12.1)
4.07
(8.4)[a]
3.89
(3.4)[a]
4.10
4
Compd.
Unit
a
b
c
d
(n.d.)
3.86
(n.d.)
4.82
5
10
C-1
C-2
C-3
C-4
C-5
102.0
72.0
77.1
76.0
73.0
167.6
72.9
135.0
117.8
101.1
67.5
82.2
78.9
74.0
66.0
101.9
72.9
76.5
76.9
71.5
168.0
101.0
67.3
83.9
81.8
74.1
66.5
(12.5)
4.46
6
(14.2)[b]
4.22
6Ј
(р 2)[c]
C-6
OAc 2.09/2.08/2.07/1.96
C-1Јall
C-2Јall
C-3Јall
1Ј-Hall
2Ј-Hall
3Ј-Hall
4.23/4.08
5.90
5.31/5.17
11
C-1
C-2
C-3
C-4
C-5
99.0
69.5
73.5
74.0
68.9
166.8
98.2
64.5
79.5
71.1
70.4
64.6
99.2
69.5
74.6
74.1
69.8
167.2
99.5
64.1
77.0
83.4
73.0
69.3
101.9
1
2
3
4
5
6
4.99
(3.4)
3.40
(9.8)
3.87
(n.d.)
3.72
(n.d.)
3.9Ϫ3.7
(n.d.)
4.19
(n.d.)
3.69
5.62
5.04
(2.3)
4.88
(n.d.)
3.99
(n.d.)
4.04
(3.3)
4.75
4.96
(3.5)
3.38
(9.2)
3.69
5.02
(2.2)
4.89
(10.2)
3.96
(n.d.)
4.02
C-6
(n.d.)
3.85
CHPh
C-1Јall
C-2Јall
C-3Јall
69.7
134.9
117.5
(n.d.) (3.2)[a]
4.23
(n.d.)
4.82
12
C-1
C-2
C-3
C-4
C-5
98.9
69.4
73.5
73.7
69.0
167.2
98.3
64.6
79.5
72.7
74.6
64.5
98.9
69.4
75.4
74.6
69.2
168.1
99.7
64.1
76.9
83.5
69.5
69.0
102.6
3.77
6Ј
CHPh
OAc
1Ј-Hall
2Ј-Hall
3Ј-Hall
2.07/2.05/2.01
4.22/4.07
5.91
C-6
CHPh
C-1Јall
C-2Јall
C-3Јall
5.31/5.16
69.8
134.9
117.4
1
2
3
4
5
6
4.98
(3.6)
3.40
(10.0)
3.88
(8.6)
3.73
(n.d.)
4.20
5.21
(2.4)
4.90
(2.9)
4.02
(n.d)
4.02
(n.d.)
4.77
4.94
(3.6)
3.37
(10.5)
3.67
(n.d.)
3.76
(n.d.)
4.00
5.01
(2.2)
4.88
(2.8)
3.95
(3.4)
4.08
(3.2)
4.80
The conversion of the intermediates 10؊12 into the tetra-
saccharides 1؊3 involved O-deacetylation, O-sulfation, re-
moval of the protecting groups and final N-sulfation, and
was accomplished as follows. Tetrasaccharide 10 was O-de-
(n.d.)
3.88
(n.d.)
`
acetylated under Zemplen conditions (quantitative) and
submitted to O-sulfation using sulfur trioxideϪ
trimethylamine complex in dry DMF at 50 °C (Scheme 4).
The 2a,6b,2c,6d-tetra-O-sulfated derivative 13, obtained in
quantitative yield, was purified by filtration through a short
(n.d.)
3.66
5.69
2.08/2.07
4.21/4.06
5.90
3.77
6Ј
CHPh
OAc
1Ј-Hall
2Ј-Hall
3Ј-Hall
1
column of silica gel. The H NMR spectrum of 13 showed
the expected chemical shifts of the signals of protons associ-
ated with sulfuric esters (H-2a: δ ϭ 4.51; H-6b,6Јb: δ ϭ
4.32; H-2c: δ ϭ 4.59; H-6d,6Јd: δ ϭ 4.20) (Table 3, for 13C
NMR see Table 4).
5.29/5.15
[a]
[b]
The signal appears as a triplet with Jn,nϪ1 ϭ Jn,nϩ1. Ϫ Calcu-
lated on H-6Ј signal. Ϫ Value of J6Ј,5
[c]
.
Hydrolysis of the methyl esters was carried out with a
The configuration of the newly formed glycosidic bond 1.25  aqueous solution of LiOH in methanol; the com-
was expected in all cases to be α, because of the presence pound was directly converted into the hexasodium salt by
Eur. J. Org. Chem. 2001, 2727Ϫ2734
2729

L. Poletti, M. Fleischer, C. Vogel, M. Guerrini, G. Torri, L. Lay
FULL PAPER
Scheme 4. Deacetylation and sulfation of tetrasaccharides 10؊12
Scheme 5. Deprotection and N-sulfation of tetrasaccharides 13؊15 afforded target compounds 1؊3
initial elution of the product on Dowex 50 W (Hϩ form)
resin, followed by a second elution on the same resin in
Naϩ form. Hydrogenolysis of benzyl ethers with concomit-
ant reduction of the allyl groups and the azido functions,
and N-sulfation of the amino groups at controlled pH
values (buffered at 9.0 with satd. aq. NaHCO3 solution)
gave tetrasaccharide 1 as a propyl glycoside and octasodium
salt (Scheme 5). Final purification of 1 was achieved by de-
salting on Sephadex GϪ10. The same reaction sequence
meters. In the description of the NMR spectra a, b, c, d refer to
monosaccharide units in the oligosaccharides (a ϭ reducing end);
13C NMR signals corresponding to aromatic carbon atoms are
omitted. Ϫ Melting points were determined with a Büchi apparatus
and are not corrected. Ϫ Optical rotations were measured at room
temperature (23 °C) with a PerkinϪElmer 241 polarimeter. Ϫ TLC
was carried out on Merck 60 F254 silica gel plates (0.25 mm thick-
ness), and spots were viewed by spraying with a solution containing
H2SO4 (31 mL), ammonium molybdate (21 g) and Ce(SO4)2 (1 g)
in 500 mL of water, followed by heating at 110 °C for 5 min. Ϫ
was applied to compounds 11 and 12, which gave pure Column chromatography was performed by the flash procedure,
using Merck 60 silica gel (230Ϫ400 mesh). Ϫ Elemental analyses
were performed using a Carlo Erba elemental analyzer 1108.
tetrasaccharides 2 and 3, respectively (Scheme 5).
Conclusion
Allyl 2-Azido-3,6-di-O-benzyl-2-deoxy-α-
(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosiduronate) (6): Com-
pound 4[11] (882 mg, 1.21 mmol) was dissolved in dichloromethane
D-glucopyranosyl-(1Ǟ4)-
L
We have reported a synthetic approach suitable for pro-
duction of a family of heparin-like oligosaccharides with (10 mL) and cooled to 0 °C. A 70% aq. solution of trifluoroacetic
acid (7 mL) was added and the reaction mixture was stirred at the
same temperature for 2 h. After neutralisation with satd. NaHCO3,
the reaction mixture was extracted with chloroform (5 ϫ 5 mL),
dried (Na2SO4), filtered and concentrated. The residue was dis-
solved in toluene (30 mL), and Bu2SnO (451 mg, 1.81 mmol) was
added. The reaction mixture was refluxed for 3 h in a two-necked
flask, equipped with a DeanϪStark apparatus, and the volume was
then reduced to one third of its original value. After the mixture
had cooled to 60 °C, benzyl bromide (430 µL, 3.63 mmol) and
TBAI (670 mg, 1.81 mmol) were added. The reaction mixture was
then heated at 95Ϫ100 °C for 16 h, and the solvent was evaporated.
The residue was purified by flash chromatography (8:2, hexane/
EtOAc) affording compound 6 (470 mg, 52%) as a colourless oil.
Ϫ [α]2D3 ϭ Ϫ2.5 (c ϭ 1.05, chloroform). ؊ C39H45N3O12 (747.8):
calcd. C 62.64, H 6.07, N 5.62; found C 62.38, H 6.00, N 5.73. Ϫ
the unusual sequence (GlcN-IdoA) and with all possible 6-
O-sulfation permutations in the glucosamine units. This
methodology has been successfully applied to the synthesis
of tetrasaccharides 1؊3 and, in principle, can be extended
to the preparation of larger oligosaccharides. Binding stud-
ies of compounds 1؊3 towards FGF-1, aimed at elucidat-
ing the role of 6-O sulfation on glucosamine units, are un-
derway and will be published elsewhere.
Experimental Section
General: 1H NMR and 13C NMR spectra were recorded with
Bruker AC 300, Bruker AMX 500 and Varian Gemini 200 spectro-
2730
Eur. J. Org. Chem. 2001, 2727Ϫ2734

A Rational Approach to Heparin-Related Fragments
FULL PAPER
1
Table 3. H NMR (CD3OD, 500 MHz, 303 K) of tetrasaccharides Table 4. 13C NMR (CD3OD, 125.72 MHz, 303 K) of tetrasaccha-
13؊15; coupling constants are given in Hz
rides 13؊15
Compd.
13
Unit
H-n
d
δ
c
δ
b
δ
a
δ
Unit
a
b
c
d
Compd.
(Jn,nϩ1
)
(Jn,nϩ1
)
(Jn,nϩ1
)
(Jn,nϩ1)
C-1
C-2
C-3
C-4
C-5
99.8
72.8
72.9
72.3
68.2
170.3
64.4
135.0
117.0
97.1
64.8
79.3
73.9
71.2
67.0
99.1
71.2
73.2
74.7
67.8
170.1
99.2
64.8
81.2
78.8
71.5
66.7
13
1
2
3
4
5
4.88
(3.3)
3.33
5.34
(р 2)
4.59
(n.d.)
4.26
(n.d.)
3.95
5.12
(3.5)
3.31
(n.d.)
3.75
(n.d.)
3.89
(n.d.)
3.87
5.22
(р 2)
4.51
(n.d.)
4.30
(n.d.)
3.90
C-6
C-1Јall
C-2Јall
C-3Јall
(9.1)[a]
3.55
(n.d.)
4.16
(9.9)
3.76
(n.d.)
4.20
(1.8)
4.94
(3.0)[b]
4.81
14
C-1
C-2
C-3
C-4
C-5
100.0
72.7
72.9
72.4
68.3
170.6
97.3
64.9
79.4
73.8
71.4
67.2
99.3
71.1
72.8
73.8
67.8
170.5
98.4
64.6
77.4
83.3
64.2
64.3
100.9
(n.d.)
4.32
6
6Ј
1Ј-Hall
2Ј-Hall
3Ј-Hall
4.24/4.10
5.94
5.31/5.14
C-6
CHPh
C-1Јall
C-2Јall
C-3Јall
69.5
134.8
117.2
14
1
2
3
4
5
4.92
(3.3)
3.40
(9.9)
3.96
(10.0)
3.64
(n.d.)
4.12
5.37
(р 2)
4.60
(n.d.)
4.23
(n.d.)
3.91
(n.d.)
4.93
5.13
(3.2)
3.34
(10.3)
3.77
(9.4)
3.93
(10.0)
3.87
15
C-1
C-2
C-3
C-4
C-5
102.7
76.4
75.0
74.6
71.5
170.1
100.0
67.5
82.4
75.9
72.3
71.9
102.6
76.5
76.8
76.1
71.5
169.8
101.7
67.2
80.3
86.3
72.2
67.4
101.3
C-6
(n.d.)
3.65
(2.7)
4.32
CHPh
C-1Јall
C-2Јall
C-3Јall
6
6Ј
CHPh
1Ј-Hall
2Ј-Hall
3Ј-Hall
72.6
134.7
117.3
5.57
4.23/4.10
5.94
5.32/5.14
11.7 Hz, 1 H, CHHPh), 4.82Ϫ4.83 (m, 3 H, H-5a, CH2Ph), 4.87
(d, 1 H, J1,2 ϭ 3.6 Hz, H-1b), 4.95 (t, 1 H, J2,1 ϭ J2,3 ϭ 3.8 Hz,
H-2a), 5.06 (d, 1 H, J1,2 ϭ 3.8 Hz, H-1a), 5.16Ϫ5.33 (m, 2 H,
CH2CHϭCH2), 5.82Ϫ5.93 (m, 1 H, CH2CHϭCH2), 7.42Ϫ7.22 (m,
15HAr). Ϫ 13C NMR (CDCl3, 75.46 MHz): δ ϭ 20.75 (s, OAc),
52.11 (s, CH3COO), 68.35, 69.58, 72.21, 73.65, 74.79, (5 t, 3
CH2Ph, C-6b, CH2CHϭCH2), 62.67, 67.71, 67.81, 70.37, 72.20,
72.43, 72.56, 79.14, (8 d, C-2a, C-2b, C-3a, C-3b, C-4a, C-4b, C-
5a C-5b), 97.68, 97.22, (2 d, C-1a, C-1b), 117.36 (t, CH2CHϭCH2),
137.42 (d, CH2CHϭCH2), 169.52, 169.99 (2q, CϭO).
15
1
2
3
4
5
6
5.03
(3.3)
3.32
(n.d.)
3.99
(10.1)
3.64
(9.7)
4.15
5.43
(р 2)
4.60
(n.d.)
4.29
5.11
(3.1)
3.28
(n.d.)
3.73
(n.d.)
3.79
5.20
(р 2)
4.49
(р 2)
4.28
(n.d.)
4.14
(n.d.)
4.79
(n.d.)
4.01
(2.0)[c]
4.84
(n.d.)
3.67
(n.d.)
3.70
(4.2)[d]
3.94
Allyl 6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-α-
(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosiduronate) (7):
D-glucopyranosyl-
(11.0)
3.73
L
6Ј
Compound 4[11] (345 mg, 0.47 mmol) was dissolved in dichlorome-
thane (3 mL) and cooled to 0 °C. A 70% aq. solution of trifluo-
roacetic acid (3 mL) was added and the reaction was stirred at the
same temperature for 2 h. The reaction mixture was then neut-
ralised with satd. NaHCO3, extracted with chloroform (5 ϫ 5 mL),
dried (Na2SO4), filtered and concentrated. The residue was dis-
solved in vinyl acetate (5 mL) and Lipase P from Pseudomonas ce-
pacia (303 mg) was added. The reaction mixture was stirred at 37
°C for 36 h, and it was then filtered through a Celite pad and con-
CHPh
1Ј-Hall
2Ј-Hall
3Ј-Hall
5.58
4.22/4.08
5.93
5.31/5.14
[a]
[b]
Calculated on H-4 signal. Ϫ
The signal appears as a triplet
with Jn,nϪ1 ϭ Jn,nϩ1. Ϫ [c] Calculated on H-5 signal. Ϫ [d] Calculated
on H-6 signal.
1H NMR (CDCl3, 300 MHz): δ ϭ 2.12 (s, 3 H, OAc), 2.57 (br. s, centrated. The crude product was purified by flash chromatography
1 H, OH), 3.23 (m, 1 H, H-2b), 3.60 (dd, 1 H, J6Ј,5 ϭ 3.9 Hz, J6Ј,6 ϭ (7:3, hexane/EtOAc), affording compound 7 (280 mg, 85%) as a
10.0 Hz, H-6Јb), 3.67Ϫ3.79 (m, 7 H, H-3b, H-4b, H-5b, H-6b, white solid. Ϫ M.p. 50Ϫ51 °C. Ϫ [α]2D3 ϭ ϩ29.2 (c ϭ 1.02, chloro-
CH3COO), 3.92 (t, 1 H, J2,3 ϭ J3,4 ϭ 3.8 Hz, H-3a), 4.04Ϫ4.10 (m, form). Ϫ C34H41N3O13 (699.7): calcd. C 58.36, H 5.91, N 6.01;
2 H, H-4a, CHHCHϭCH2), 4.25 (m, 1 H, CHHCHϭCH2), 4.52 found C 58.27, H 6.02, N 6.30. Ϫ 1H NMR (CDCl3, 300 MHz):
(d, J ϭ 11.9 Hz, 1 H, CHHPh), 4.59 (d, J ϭ 11.9 Hz, 1 H, δ ϭ 3.23 (dd, 1 H, J2,1 ϭ 3.6, J2,3 ϭ 10.1 Hz, H-2b), 3.44 (dd,
CHHPh), 4.65 (d, J ϭ 11.7 Hz, 1 H, CHHPh), 4.79 (d, J ϭ J3,4 ϭ 8.7, J4,5 ϭ 9.8 Hz, H-4b), 3.73 (dd, 1 H, J3,2 ϭ 10.1, J3,4
ϭ
Eur. J. Org. Chem. 2001, 2727Ϫ2734 2731

L. Poletti, M. Fleischer, C. Vogel, M. Guerrini, G. Torri, L. Lay
FULL PAPER
8.7 Hz, H-3b), 3.78 (s, 3 H, COOCH3), 3.77Ϫ3.84 (m, 1 H, H-5b), 5b, CH3OOC), 4.01 (br. s, 1 H, H-3a), 4.14Ϫ4.20 (m, 2 H, H-4a,
3.93 (t, 1 H, J3,2 ϭ J3,4 ϭ 2.4 Hz, H-3a), 4.04Ϫ4.16 (m, 2 H, H-6Јb), 4.33 (dd, 1 H, J6,6Ј ϭ 12.4, J6,5 ϭ 1.6 Hz, H-6b), 4.58 (d,
CHHCHϭCH2, H-4a), 4.21Ϫ4.33 (m, 2 H, CHHCHϭCH2, H- J ϭ 11.1 Hz, 1 H, CHHPh), 4.66 (d, J ϭ 11.6 Hz, 1 H, CHHPh),
6Јb), 4.56 (dd, 1 H, J6,5 ϭ 3.3 Hz, J6,6Ј ϭ 12.4 Hz, H-6b), 4.67 (d, 4.80Ϫ4.84 (m, 4 H, CH2Ph, 2 CHHPh), 4.89 (d, 1 H, J1,2 ϭ 3.5 Hz,
J ϭ 11.8 Hz, 1 H, CHHPh), 4.83 (d, J ϭ 11.8 Hz, 1 H, CHHPh),
H-1b), 4.98 (d, 1 H, J5,4 ϭ 1.7 Hz, H-5a), 5.14 (br. s, H-2a), 6.41
4.85 (br. s, 3 H, CH2Ph, H-1b), 4.90 (d, 1 H, J5,4 ϭ 3.5 Hz, H-5a), (s, 1 H, H-1a), 7.45Ϫ7.22 (m, 15 H, HAr), 8.68 (s, 1 H, NH),Ϫ 13C
4.98 (t, 1 H, J2,3 ϭ J2,1 ϭ 2.4 Hz, H-2a), 5.06 (br. s, 1 H, H-1a), NMR (CDCl3, 75.46 MHz): δ ϭ 20.79 (q, CH3CϭO), 52.50 (q,
5.17Ϫ5.36 (m, 2 H, CH2CHϭCH2), 5.81Ϫ6.00 (m, 1 H, CH2CHϭ COOCH3), 62.33 (t, C-6b), 72.43, 74.83, 75.44, (3 t, CH2Ph), 63.42,
CH2), 7.24Ϫ7.50 (m, 10 H, HAr).
Ϫ
13C NMR (CDCl3, 65.44, 69.08, 70.07, 72.10, 72.55, 77.58, 80.07, (8 d, C-2a, C-2b, C-
3a, C-3b, C-4a, C-4b, C-5a, C-5b), 95.41, 97.33, (2 d, C-1a, C-1b),
75.46 MHz): δ ϭ 20.74 (OAc), 52.16 (q, COOCH3), 62.72, 69.22,
72.38, 75.04 (4 t, C-6b, CH2CHϭCH2, 2 CH2Ph), 62.96, 67.81, 160.13 (s, CϭNH), 168.70, 169.89, 170.47 (3 s, CϭO).
70.54, 70.94, 72.55, 73.02, 78.96 (7 d, 8C, C-2a, C-2b, C-3a, C-3b,
Allyl 6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-
syl-(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosiduronyl)-
(1Ǟ4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α- -glucopyranosyl-
(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosiduronate)
D-glucopyrano-
C-4a, C-4b, C-5a, C-5b), 97.51, 97.86 (2 d, C-1a, C-1b), 117.38 (t,
CH2CHϭCH2), 133.50 (d, CH2CHϭCH2), 169.60, 169.90, 171.64
(3 s, CϭO).
L
D
L
2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
D-glucopyranosyl-
(10): A solution of trimethylsilyl triflate (0.1  in dry dichlorome-
thane, 276 µL) was added dropwise at Ϫ20 °C to a solution of
donor 9 (92 mg, 0.10 mmol) and acceptor 7 (65 mg, 92.0 µmol) in
dry dichloromethane under argon. After 10 min, the reaction mix-
ture was neutralised with triethylamine, concentrated and purified
by flash chromatography (7:3, hexane/EtOAc), affording tetrasac-
charide 10 as a white glassy solid (65 mg, 50%). Ϫ [α]2D3 ϭ ϩ15.6
(c ϭ 1, chloroform). Ϫ C72H82N6O25 (1431.5): calcd. C 60.41, H
(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosyluronate) Tri-
L
chloroacetimidate (8): Compound 4[11] (729 mg, 2.05 mmol) was
dissolved in dry THF (15 mL). The solution was carefully degassed.
(1,5-Cyclooctadiene)bis(methyldiphenylphosphane)iridium hexa-
fluorophosphate catalyst (2 mg) was added, followed by further de-
gassing of the mixture. The catalyst was activated under hydrogen
for 2 min, and the reaction mixture was then stirred at room tem-
perature for 3 h. The solvent was evaporated, the residue was dis-
solved in a 4:1 THF/H2O mixture (30 mL) and iodine (1 g,
4.10 mmol) was added. After 10 min, the solution was diluted with
water and extracted with chloroform (3 ϫ 10 mL). The organic
layers were washed with a freshly prepared 5% solution of NaHSO3
until decoloured, dried (Na2SO4), filtered and concentrated. A
short filtration through silica gel (6:4, hexane/EtOAc) afforded the
deallylated compound (1 g, 73%) as a mixture of α/β anomers,
which was used in the next step without further characterisation.
The crude compound (1 g, 1.50 mmol) was dissolved in dry dichlo-
romethane (20 mL), followed by addition of trichloroacetonitrile
(1.5 mL, 15 mmol) and a catalytic amount of DBU. After stirring
at room temperature for 45 min, the reaction mixture was concen-
trated and purified by flash chromatography (7:3, hexane/EtOAc
ϩ 1% TEA), affording donor 8 (1.07 g, 84%) as a brown oil. The
compound was obtained as 3:2 α/β mixture, as measured by inte-
gration of the anomeric signals in the 1H NMR spectrum, and was
used directly for the glycosylation step without further characteris-
ation. Ϫ 1H NMR (CDCl3, 300 MHz): δ ϭ 2.05, 2.09 (2 s, 3 H,
OAc αϩβ), 3.35Ϫ3.41 (m, 1 H, H-2b αϩβ), 3.62Ϫ3.69 (m, 2 H, H-
4b, H-6ЈЈb αϩβ), 3.79 (s, 3 H, COOCH3 αϩβ), 3.80Ϫ3.89 (m, 1 H,
H-5a αϩβ), 3.93Ϫ4.01 (m, 2 H, H-3a αϩβ), 4.05Ϫ4.16 (m, 1 H,
H-4a αϩβ), 4.25Ϫ4.32 (m, 1 H, H-6Јb αϩβ), 4.64Ϫ4.94 (m, 6 H,
H-1b αϩβ, 2 CH2Ph, H-5a β), 4.99 (d, 3/5 H, J5,4 ϭ 1.8 Hz, H-5a
α), 5.14 (br. s, 3/5 H, H-2a α), 5.28 (br. t, 2/5 H, H-2a β), 5.51 (s,
1
5.77, N 5.87; found C 60.32, H 6.00, N 5.42. Ϫ H NMR and 13C
NMR: See Table 1 and 2.
Allyl 2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
anosyl-(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosyluro-
nyl)-(1Ǟ4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α- -glucopyra-
nosyl-(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosiduron-
D-glucopyr-
L
D
L
ate) (11): A mixture of donor 8 (486 mg, 0.57 mmol) and acceptor
7 (236 mg, 0.34 mmol) was dissolved in dry dichloromethane
(3 mL) and cooled to Ϫ20 °C under argon. A solution of trimethyl-
silyl triflate (0.1  in dry dichloromethane, 674 µL) was added
dropwise and the solution was stirred for 20 min at the same tem-
perature, and then neutralised with triethylamine. The solvent was
evaporated and the obtained residue was purified by flash chroma-
tography (7:3, hexane/EtOAc), affording compound 11 as a glassy
solid (294 mg, 63%). Ϫ [α]2D3 ϭ ϩ1.3 (c ϭ 1, chloroform). Ϫ
C70H78N6O24 (1387.4): calcd. C 60.60, H 5.67, N 6.06; found C
60.61, H 5.60, N 6.15. Ϫ 1H NMR and 13C NMR: See Table 1
and 2.
Allyl 2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
anosyl-(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosyluro-
nyl)-(1Ǟ4)-2-azido-3,6-di-O-benzyl-2-deoxy-α- -glucopyrano-
syl-(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α- -idopyranosiduronate)
D-glucopyr-
L
D
L
(12): A mixture of donor 8 (448 mg, 0.53 mmol) and acceptor 6
(232 mg, 0.31 mmol) was dissolved in dry dichloromethane
(3.5 mL) and cooled to Ϫ20 °C under argon. A solution of trime-
thylsilyl triflate (0.1  in dry dichloromethane, 930 µL) was added
dropwise and the solution was stirred for 20 min at the same tem-
perature, and then neutralised with triethylamine. The solvent was
evaporated and the obtained residue was purified by flash chroma-
tography (75:20, hexane/EtOAc), affording tetrasaccharide 12 as a
glassy solid (280 mg, 62%). Ϫ [α]2D3 ϭ ϩ1.5 (c ϭ 1, chloroform). Ϫ
C75H82N6O23 (1435.5): calcd., C 62.75, H 5.76, N 5.85; found C
62.45, H 5.76, N 5.84. Ϫ 1H NMR and 13C NMR: See Table 1
and 2.
2/5 H, CHPh β), 5.53 (s, 3/5 H, CHPh α), 4.78 (d, 2/5 H, J1,2
ϭ
1.7 Hz, H-1β), 6.41 (br. s, 3/5 H, H-1a α), 7.26Ϫ7.49 (m, 15 H,
HAr), 8.66, 8.68 (2 s, 2 H, NH αϩβ).
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-
D
-glucopyranosyl-
(1Ǟ4)-(methyl 2-O-acetyl-3-O-benzyl-α-
L
-idopyranosyluronyl) Tri-
chloroacetimidate (9): Compound 5[11] (488 mg, 0.62 mmol) was
submitted to the same procedure as described for the preparation
of compound 8, affording an α/β mixture of donor 9 (328 mg, 59%)
as a yellow oil, the major product being the α anomer.
α Anomer: [α]2D3 ϭ ϩ1.9 (c ϭ 0.98, chloroform). Ϫ C40H43Cl3N4O13
(894.1): calcd. C 53.73, H 4.85, N 6.27; found C 53.72, H 4.90, N Allyl 2-Azido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-α-
D
-glucopyrano-
-idopyranosyluronyl)-
(1Ǟ4)-2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-α- -glucopyranosyl-
-idopyranosiduronate) Tetra-
1
6.25. Ϫ H NMR (CDCl3, 300 MHz): δ ϭ 2.02 (s, 3 H, OAc), 2.18 syl-(1Ǟ4)-(methyl 3-O-benzyl-2-O-sulfo-α-
L
(s, 3 H, OAc), 3.32 (dd, 1 H, J2,3 ϭ 10.2, J2,1 ϭ 3.5 Hz, H-2b), 3.53
D
(t, 1 H, J4,3 ϭ J4,5 ϭ 9.5 Hz, H-4b), 3.77Ϫ3.90 (m, 5 H, H-3b, H- (1Ǟ4)-(methyl 3-O-benzyl-2-O-sulfo-α-
L
2732
Eur. J. Org. Chem. 2001, 2727Ϫ2734

A Rational Approach to Heparin-Related Fragments
FULL PAPER
kis(trimethylammonium) Salt (13): A solution of NaOMe in dry (4 mL), maintaining a pH of ca 9.0 by addition of solid NaHCO3.
methanol (0.9 , 20 µL) was added to a solution, cooled to 0 °C, After 48 h, the solid was filtered off and the solution was concen-
of compound 10 (53 mg, 37.0 µmol) in dry methanol (2 mL). The trated. Elution on Sephadex G10 (eluent 9:1, H2O/EtOH, V0
solution was stirred at the same temperature for 4 h and at room 20 mL, Vt ϭ 50 mL) afforded pure compound 1 (44 mg, 90%) as a
temperature for 3 h; it was then neutralised with IR-120 (Hϩ form) glassy solid. [α]2D3
ϩ26.5 (c 0.29, water).
ϭ
Ϫ
ϭ
ϭ
Ϫ
and the resin was filtered off and the solvent evaporated. The
residue was dissolved in dry DMF (2.5 mL) and sulfur
trioxideϪtrimethylamine complex (70 mg, 0.51 mmol) was added.
The reaction was heated to 50 °C and stirred for 56 h at this tem-
perature; a further portion of sulfur trioxideϪtrimethylamine com-
plex (24 mg, 0.17 mmol) was added, and after a total of 72 h the
reaction was quenched by addition of methanol (2 mL) and stirred
for 1 h. The solvent was evaporated and the residue was purified
by flash chromatography (8:3, chloroform/methanol), affording
compound 13 (67 mg, quant.) as a glassy solid. Ϫ [α]2D3 ϭ Ϫ40.5
(c ϭ 1, methanol). Ϫ C76H110N10O33S4 (1819.9): calcd. C 50.15, H
C27H38N2Na6O39S6 (1344.9): no combustion analysis was per-
1
Table 5. H NMR (D2O, 500 MHz, 298 K) spectra of tetrasaccha-
rides 1؊3; coupling constants are given in Hz
Unit
H-n
d
δ
c
δ
b
δ
a
δ
Compd.
(Jn,nϩ1
)
(Jn,nϩ1
)
(Jn,nϩ1
)
(Jn,nϩ1)
1
1
2
5.38
(3.5)
3.25
5.28
(2.9)
4.36
5.34
(3.5)
3.28
5.14
(2.9)
4.24
1
6.09, N 7.69; found C 50.00, H 6.15, N 7.58. Ϫ H NMR and 13C
(10.3)
3.65
(5.4)
4.23
(10.3)
3.72
(5.3)
4.21
NMR: See Table 3 and 4.
3
(9.7)
3.57
(9.7)
3.99
(n.d.)
4.36
(n.d.)
4.21
(3.8)
4.10
(2.7)
4.85
(9.2)
3.79
(9.2)
4.03
(2.1)
4.39
(11.4)
4.27
(3.9)
4.06
(2.7)
4.52
Allyl 2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
anosyl-(1Ǟ4)-(methyl 3-O-benzyl-2-O-sulfo-α- -idopyranosyluro-
nyl)-(1Ǟ4)-2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-α- -glucopy-
ranosyl-(1Ǟ4)-(methyl 3-O-benzyl-2-O-sulfo-α- -idopyranosiduro-
D-glucopyr-
4
L
D
5
L
nate) Tris(trimethylammonium) Salt (14): Compound 11 (220 mg,
0.16 mmol) was submitted to the same procedure as compound 10,
affording sulfated tetrasaccharide 14 (264 mg, quant.) as a glassy
solid. Ϫ [α]2D3 ϭ Ϫ29.9 (c ϭ 1, methanol). Ϫ C73H99N9O30S3
(1678.8): calcd. C 52.22, H 5.94, N 7.51; found, C 52.28, H 5.88,
6
6Ј
(n.d.)
(n.d.)
1Ј-Hpr 3.65/3.49
1
N 7.50. Ϫ H NMR and 13C NMR: See Table 3 and 4.
2Ј-Hpr
3Ј-Hpr
1.55
0.92
Allyl 2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-
anosyl-(1Ǟ4)-(methyl 3-O-benzyl-2-O-sulfo-α- -idopyranosyluro-
nyl)-(1Ǟ4)-2-azido-3,6-di-O-benzyl-2-deoxy-α- -glucopyranosyl-
(1Ǟ4)-(methyl 3-O-benzyl-2-O-sulfo-α- -idopyranosyduronate) Bis-
D-glucopyr-
L
2
1
2
5.40
(3.5)
3.23
(10.4)
3.66
(n.d.)
3.46
(n.d.)
3.85
(n.d.)
3.87
(n.d.)
3.80
5.25
(2.8)
4.36
(5.6)
4.24
(3.5)
4.11
(2.7)
4.85
5.35
(3.6)
3.29
(10.5)
3.71
(n.d.)
3.80
D
L
(trimethylammonium) Salt (15): Compound 12 (254 mg, 0.18 mmol)
was submitted to the same procedure as compound 10, affording
compound 15 (276 mg, 96%.) as a glassy solid. Ϫ [α]2D3 ϭ Ϫ3.0 (c ϭ
1, methanol). Ϫ C77H96N8O27S2 (1629.7): calcd. C 56.74, H 5.93,
N 6.87; found C 56.77, H 5.90, N 6.81. Ϫ 1H NMR and 13C NMR:
See Table 3 and 4.
3
4
(n.d.)
4.03
5
(2.1)[a]
4.28
6
Propyl 2-Deoxy-2-sulfamino-6-O-sulfo-α-
(2-O-sulfo-α- -idopyranosyluronic acid)-(1Ǟ4)-2-deoxy-2-sulfami-
no-6-O-sulfo-α- -glucopyranosyl-(1Ǟ4)-(2-O-sulfo-α- -idopyrano-
D-glucopyranosyl-(1Ǟ4)-
(11.5)
4.42
L
6Ј
(2.2)[a] [b]
D
L
(n.d.)
syluronic acid) Hexasodium Salt (1). ؊ Saponification: A solution
of LiOH in water (1.25 , 114 µL, 14 equiv.) was added dropwise
to a solution of tetrasaccharide 13 (67 mg, 37.0 µmol), dissolved in
1:1:1 THF/MeOH/H2O (6 mL), and cooled to 0 °C. After 2.5 h,
the reaction mixture was allowed to warm to room temperature
and three further portions of LiOH solution (14, 7 and 7 equiv.,
respectively) were added over the following 7 h. The reaction mix-
ture was then diluted with methanol (1 mL) and eluted through a
column of Dowex 50 X 8 resin (Hϩ form, eluent 9:1, MeOH/H2O).
The eluted solution was concentrated and eluted through a column
of Dowex 50 X 8 (Naϩ form, same eluent) affording the hydrolysed
tetrasaccharide as the hexasodium salt. Ϫ Hydrogenolysis: The
product of the previous reaction was dissolved in 2:1 MeOH/H2O
(6 mL) with a few drops of glacial HOAc. Pd(OH)2/C catalyst
(40 mg, 0.20 equiv.) was added and the reaction mixture was stirred
under hydrogen at room temperature. After 48 h, a further portion
of catalyst (30 mg, 0.15 equiv.) was added and after a further 16 h,
the reaction mixture was filtered through a Celite pad and ly-
ophilised, giving the unprotected tetrasaccharide. Ϫ N-Sulfation:
Sulfur trioxideϪtrimethylamine complex (156 mg, 1.11 mmol) was
added to the hydrogenated product, dissolved in satd. aq NaHCO3
1Ј-Hpr 3.70/3.52
2Ј-Hpr
3Ј-Hpr
1.62
0.88
3
1
2
3
4
5
6
5.31
(3.6)
3.23
(10.4)
3.68
5.26
(1.8)
4.35
(3.9)
4.25
(n.d.)
4.05
(2.3)
4.89
5.36
(3.6)
3.25
(10.3)
3.70
(n.d.)
3.70
(n.d.)
3.89
(n.d.)
3.84
(n.d.)
3.78
5.15
(2.7)
4.24
(4.9)
4.23
(n.d.)
4.05
(2.7)
4.52
(n.d.)
3.46
(9.7)[c]
3.84
(n.d.)
3.88
6Ј
1Ј-Hpr
2Ј-Hpr
3Ј-Hpr
[a]
[b]
[c]
Calculated on H-6 signal. Ϫ
appears as a triplet with Jn,nϪ1 ϭ Jn,nϩ1
Value of J6Ј,5. Ϫ
The signal
2733
.
Eur. J. Org. Chem. 2001, 2727Ϫ2734

L. Poletti, M. Fleischer, C. Vogel, M. Guerrini, G. Torri, L. Lay
FULL PAPER
1
formed on this precious material. Ϫ H NMR and 13C NMR: See Propyl 2-Deoxy-2-sulfamino-α-
D
-glucopyranosyl-(1Ǟ4)-(2-O-sulfo-
-idopyranosyluronic acid)-(1Ǟ4)-2-deoxy-2-sulfamino-6-O-sulfo-
-glucopyranosyl-(1Ǟ4)-(2-O-sulfo-α- -idopyranosyluronic acid)
Table 5 and 6.
α-L
α-
D
L
Tetrasodium Salt (3): Compound 15 (141 mg, 86.0 µmol) was sub-
mitted to the same deprotection procedure as compound 13, af-
Propyl 2-Deoxy-2-sulfamino-α-D-glucopyranosyl-(1Ǟ4)-(2-O-sulfo-
α-L-idopyranosyluronic acid)-(1Ǟ4)-2-deoxy-2-sulfamino-6-O-sulfo-
fording tetrasaccharide 3 (98 mg, 90%) as a glassy solid. Ϫ [α]2D3
ϭ
α-
D
-glucopyranosyl-(1Ǟ4)-(2-O-sulfo-α-L-idopyranosyluronicacid)
ϩ10.9 (c ϭ 0.33, water). Ϫ C27H40N2Na4O33S4 (1140.8): no com-
bustion analysis was performed on this precious material. Ϫ 1H
NMR and 13C NMR: See Table 5 and 6.
Pentasodium Salt (2): Compound 14 (161 mg, 96.0 µmol) was sub-
mitted to the same deprotection procedure as compound 13, afford-
ing tetrasaccharide 2 (116 mg, 93%) as white crystals. Ϫ [α]2D3
ϭ
ϩ13.2 (c ϭ 0.60, water). Ϫ C27H39N2Na5O36S5 (1242.9): no com-
bustion analysis was performed on this precious material. Ϫ 1H
NMR and 13C NMR: See Table 5 and 6.
Acknowledgments
We thank the EU (CARENET-2 project, contract ERB-FMRX-
CT 96Ϫ0025), the MURST (COFIN 2000, prot. MM03155477)
and the CNR (Centro di Studio sulle Sostanze Organiche Naturali)
for financial support.
Table 6. 13C NMR (D2O, 125.72 MHz, 298 K) of tetrasaccharides
1؊3
Compd.
Unit
a
b
c
d
[1]
W. H. Burgess, T. Maciag, Annu. Rev. Biochem. 1989, 58,
575Ϫ606.
[2]
1
C-1
C-2
C-3
C-4
C-5
101.5
78.8
71.4
78.8
71.4
178.2
73.3
24.8
13.1
99.9
60.9
72.6
79.0
72.0
69.2
102.1
78.4
71.5
78.8
72.2
177.9
100.1
60.9
74.0
72.1
72.8
72.2
M. Klagsburn, Curr. Opin. Cell. Biol. 1990, 2, 857Ϫ863.
[3]
L. Kjellen, U. Lindahl, Annu. Rev. Biochem. 1991, 60,
443Ϫ475.
[4]
B. Casu, Adv. Carbohydr. Chem. 1985, 43, 51Ϫ134 and refer-
ences cited therein.
S. Faham, R. E. Hileman, J. R. Fromm, R. J. Linhardt, D. C.
Rees, Science 1996, 271, 1116Ϫ1120.
R. E. Hileman, J. R. Fromm, J. M. Wiler, R. J. Linhardt,
BioEssays 1998, 20, 156Ϫ167.
L. Pellegrini, D. F. Burke, F. Von Delft, B. Mulloy, T. L. Blund-
ell, Nature 2000, 107, 1029Ϫ1034.
H. Mach, D. B. Volkin, C. J. Burke, C. R. Middaugh, R. J.
Linhardt, J. R. Fromm, D. Loganathan, L. Mattsson, Biochem-
istry 1993, 32, 5480Ϫ5489.
S. Guimond, M. Maccarana, B. B. Olwin, U. Lindahl, A. C.
[5]
C-6
C-1Јpr
C-2Јpr
C-3Јpr
[6]
[7]
2
C-1
C-2
C-3
C-4
C-5
101.2
78.2
70.9
78.6
70.8
177.5
73.2
24.8
13.0
99.7
60.7
72.3
78.6
71.9
69.1
101.9
73.4
71.5
78.5
72.0
178.0
99.6
60.8
73.9
72.7
74.4
63.1
[8]
[9]
C-6
Rapraeger, J. Biol. Chem. 1993, 268, 23906Ϫ23914.
[10]
C-1Јpr
C-2Јpr
C-3Јpr
M. Maccarana, B. Casu, U. Lindahl, J. Biol. Chem. 1993,
268, 23898Ϫ23905.
[11]
B. La Ferla, L. Lay, M. Guerrini, L. Poletti, L. Panza, G.
Russo, Tetrahedron 1999, 55, 9867Ϫ9880.
[12]
3
C-1
C-2
C-3
C-4
C-5
101.4
78.7
71.3
78.4
71.1
177.8
73.0
25.1
13.1
101.1
60.9
72.3
80.2
73.9
63.1
101.9
77.3
70.3
78.8
70.9
177.5
99.8
61.0
73.9
72.8
74.5
62.6
J. J. Oltvoort, C. A. A. Van Boeckel, J. H. De Koning, J. H.
Van Boom, Synthesis 1981, 305Ϫ308.
[13]
R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1986, 25, 212Ϫ235.
[14]
J.-C. Jacquinet, M. Petitou, P. Duchassoy, I. Lederman, J.
Choay, G. Torri, P. Sinay¨, Carbohydr. Res. 1984, 130, 221Ϫ241.
M. Nilsson, C.-M. Svahn, J. Westman, Carbohydr. Res. 1993,
[15]
C-6
C-1Јpr
C-2Јpr
C-3Јpr
246, 161Ϫ172.
Received December 22, 2000
[O00655]
2734
Eur. J. Org. Chem. 2001, 2727Ϫ2734