Efficient selective preparation of methyl-1,2,4-tri-O-acetyl-3-O-benzyl-β-L-idopyranuronate from methyl 3-O-benzyl-L-iduronate

Article abstract of DOI:10.1016/S0008-6215(02)00525-6

Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-L-idopyranuronate 6β/6α, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key synthon in heparin/heparan sulfate synthesis. The ¹H and ¹³C NMR spectra of the furanose-pyranose mixture of 4, a

Full text of DOI:10.1016/S0008-6215(02)00525-6

Carbohydrate Research 338 (2003) 681–686
www.elsevier.com/locate/carres
Note
Efficient selective preparation of
methyl-1,2,4-tri-O-acetyl-3-O-benzyl-b-
L
-idopyranuronate from
methyl 3-O-benzyl- -iduronate
L
Anna Dilhas, David Bonnaffe´*
Laboratoire de Chimie Organique Multifonctionnelle, UMR 8614 ‘Molecular Glycochemistry’, Bat. 420 Uni6ersite´ Paris Sud,
F-91405 Orsay, France
Received 4 November 2002; accepted 27 November 2002
Abstract
Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-
L
-idopyranuronate 6b/6a, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key
synthon in heparin/heparan sulfate synthesis. The 1H and 13C NMR spectra of the furanose–pyranose mixture of 4, after
dissolution and equilibration in d4-methanol, were fully assigned allowing to expect that 4 could crystallise in the b-pyranose form.
New acetylation conditions able to trap this form were subsequently devised, allowing the isolation of 83% of pure 6b by simple
crystallisation, along with 9% of the 6b/6a mixture. This represents a major advantage over the previously published procedure,
especially on multigram scales. © 2003 Elsevier Science Ltd. All rights reserved.
Keywords: L-Idose; L-Iduronic acid; Pyranose form equilibration; Acetylation; Heparin; Heparan sulfate
1. Introduction
responsible for the selective binding of a protein. In the
total synthesis of HS/HP fragments, activated uronic
L
-Iduronic acid (
L
-IdoUA) is a key component of der-
acids have been shown to be efficient donors in glycosy-
lation reactions.12,13 However, efficient access to
L-
matan sulfate (DS), heparin (HP) and heparan sulfate
(HS). These linear sulfated oligosaccharides are mem-
bers of the glycosaminoglycan family and play a crucial
role in all organisms, not only through their structural
functions but also by selectively binding and regulating
the activity of numerous proteins. This is especially true
for HS1,2 which is present at the cell surface, as proteo-
glycan,3 where it may interact with growth factors, such
as FGFs,4 interleukins and interferons,5 chemokines,6
viral proteins,7 prion proteins8 and adhesion
molecules.9 HS thus plays a key role in many biological
processes such as development,10 tumour growth and
metastasis.11 Among the biopolymer family, HS shows
one of the highest level of molecular diversity. A cur-
rent challenge in glycochemistry is thus the preparation
of HS fragment libraries in order to find sequences
IdoUA synthons suitable for activation as
donors is often a serious challenge. The a/b mixture of
methyl 1,2,4-tri-O-acetyl-3-O-benzyl- -idopyranuro-
L-iduronyl
L
nate 6a/6b, that may easily be converted into a bromide
donor, has been a key synthon in earlier heparin frag-
ment syntheses.13 However, its preparation by acetyla-
tion of methyl 3-O-benzyl- -iduronate 4 (Scheme 1)
L
with Ac2O in pyridine13a leads to 40% of furanose
derivatives 5a/5b that have to be separated from 6a/6b
by chromatography. This is probably one of the rea-
sons for the use of
L-idose synthons that are more
prone to cyclise in the pyranose form. However, in
these cases C-6 oxidation has to be performed at a later
stage on elaborated material.13b,14 We have recently
shown that the addition of tris(phenylthio)methyl-
lithium on 3-O-benzyl-1,2-O-isopropylidene-a-
D-xylo-
dialdose (1) is a totally stereoselective and high yielding
route to the -IdoUA synthon 2 easily scalable up to
L
* Corresponding author. Tel.: +33-1-69154719; fax: +33-
1-69154715
E-mail address: david@icmo.psud.fr (D. Bonnaffe´).
100 g.15 This reaction is now a key step for the prepara-
tion of HS/HP fragments that we are currently synthe-
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(02)00525-6

682
A. Dilhas, D. Bonnaffe´ / Carbohydrate Research 338 (2003) 681–686
sising in our laboratory. In order to avoid tedious
chromatography resulting from unselective acetylation
then recorded which allowed the identification of the
1H spin system (H-1 to H-5) of each compound, while
of methyl 3-O-benzyl-
L-iduronate 4 in large-scale syn-
a HSQC experiment allowed total attribution of the 13
C
thesis of 6a/6b from 1, we undertook a study of this
step in order to reduce the amount of furanose deriva-
tives 5a/5b.
spectrum of the sugar carbon atom ring. A HMBC map
was used to determine whether a component was in the
furanose or pyranose form: for the pyranose com-
pounds, cross peaks were found between H-1 and C-5
(4.97 and 75.5 ppm for 4-b-Pyr; 5.17 and 70.6 ppm for
4-a-Pyr) and H-5 and C-1 (4.54 and 94.4 ppm for
4-b-Pyr; 4.80 and 96.7 for 4-a-Pyr). On the other hand,
for the furanose component there are cross peaks be-
tween H-1 and C-4 (5.28 and 79.0 ppm for 4-b-Fur; 5.08
and 82.2 ppm for 4-a-Fur) and H-4 and C-1 (4.52 and
104.2 ppm for 4-a-Fur). For the pyranose derivatives,
attribution of the anomeric configuration was based on
the existence of J4 W coupling constants of 1.0 Hz
between H-3 and H-1 and H-2 and H-4 in the spectrum
of the pyranose compound with H-1 chemical shift at
2. Results and discussion
Methyl 3-O-benzyl- -iduronate (4) is a crystalline com-
L
pound.13a,15 We reasoned that if 4 could crystallise in a
pyranose form and if acetylation could be performed in
a solvent in which the crystals would be only sparingly
soluble, we could trap this pyranose form and thus
enhance the proportion of 6a and/or 6b. In order to
address the first question, we dissolved crystals of 4 in
1
d4-methanol and followed the evolution of its H NMR
1
5.17 ppm. This is only possible for 4-a-Pyr in a C4
spectrum over time. The first spectrum, taken 3 min
after dissolution, shows the presence of a major com-
pound with H-1 at 4.97 ppm. This compound has been
previously shown to be in the a-pyranose form, based
on the multiplicity of the anomeric proton (broad sin-
glet).13a We decided to test this hypothesis by fully
conformation. On the contrary, there is only one J4 W
coupling constant between H-2 and H-4 in the spec-
trum of the other pyranose component (l H-1 4.97
1
ppm) as expected for 4-b-Pyr in a C4 conformation.
This later assignment was confirmed by the measure-
ment in the HMBC map of a H-1 C-1 coupling con-
stant of 162 Hz, which is characteristic of the presence
of an axial hydrogen at the anomeric position of a
pyranose compound.16 For the furanose derivatives,
attribution of the anomeric configuration was based on
the chemical shift of the anomeric carbon, since for
1
attributing the H and 13C spectra of the pyranose–
furanose mixture once the equilibrium had been
reached. After 120 min at room temperature, a mixture
of four compounds with anomeric proton chemical
shifts at 4.97, 5.08, 5.17 and 5.28 ppm was formed in
the proportions 41:17:18:24. A COSY spectrum was
Scheme 1.

A. Dilhas, D. Bonnaffe´ / Carbohydrate Research 338 (2003) 681–686
683
Table 1
1H and 13C NMR data for compounds 4-b-Pyr, 4-a-Pyr, 4-b-Fur and 4-a-Fur
Compound % a H/C
1
2
3
4
5
CH2(Bn)
CꢀO
Me
3.75
1
4-b-Pyr 41%
4-a-Pyr 18%
l H b
4.97
J1,2 1.0
3.69
3.84
J3,4 3.5
3.98
J4,5 1.5
4.54
4.67; 4.64
Jgem 12.0
J (Hz)
J2,4 1.0
J2,3 3.5
69.7
l
13C b
94.4
77.8
68.9
75.5
4.80
73.3
171.5
52.5
3.75
1
l H b
5.17
3.63
3.73
4.00
4.74; 4.69
J (Hz)
J1,2 3.0
J2,4 1.0
J2,3 4.5
70.0
J1,3 1.0
J3,4 4.5
78.4
J4,5 2.5
Jgem 12.0
l
13C b
96.7
69.9
70.6
4.30
73.7
172.4
174.3
174.1
52.5
3.66
1
4-b-Fur 24%
4-a-Fur 17%
l H b
5.28
J1,2 4.5
96.9
4.31
J2,3 6.5
76.5
4.23
J3,4 6.5
84.0
4.50
J4,5 3.5
79.0
4.79; 4.59
Jgem 11.5
73.9
J (Hz)
l
13C b
71.2
4.36
52.5
3.65
1
l H b
5.08
J1,2 2.5
104.2
4.21
J2,3 4.5
81.1
4.10
J3,4 7.0
85.3
4.52
J4,5 4.0
82.2
4.72; 4.52
Jgem 12.0
73.7
J (Hz)
l
13C b
71.4
52.5
a Proportion of each compound after equilibration for 120 min after dissolution of 4 in d4-methanol.
1
b l are in ppm relative to the d4-methanol signal at 3.31 for H and 49.0 for 13C.
Fig. 1. Evolution of the composition in 4-b-Pyr, 4-a-Pyr, 4-b-Fur and 4-a-Fur after dissolution of crystalline 4 in d4-methanol,
based upon the integration of the anomeric proton signals.
and carbons, carbonyls and methoxy groups were per-
formed using HMBC and HSQC maps, allowing com-
furanoses in which the substituents at C-1 and C-2 are
trans oriented, the signals of the anomeric carbon
atoms are always found at a lower field as compared to
those of the corresponding cis isomer.17 Thus the fura-
nose anomeric 13C atom with chemical shift at 104.2
ppm was assigned to 4-a-Fur and that at l 96.9 ppm to
4-b-Fur. Further attributions of the benzylic protons
1
plete assignment of the H and 13C spectra (Table 1).
In order to establish the nature of the crystalline
1
form of 4, we followed the evolution of its H NMR
spectrum after dissolution in d4-methanol. Fig. 1 show
the course of the integration of each anomeric proton

684
A. Dilhas, D. Bonnaffe´ / Carbohydrate Research 338 (2003) 681–686
over time. The major compound present 3 min after
dissolution is 4-b-Pyr (83%). Fig. 1 shows that its
concentration decreases steadily over time, while the
a-pyranose anomer and the furanose derivatives are
formed. These results show unambiguously that the
crystalline form of 4 is the b-pyranose form.
tivity for the formation of the pyranose compound 6b,
along with 3–4% of 6a and only 2–3% of the 6b
furanose derivative (Entries 4 and 5). In order to deter-
mine the influence of the temperature on this excellent
selectivity, we dissolved crystalline 4-b-Pyr in pyridine
and performed the reactions at −40 °C either in the
classical conditions with Ac2O13a or under our condi-
tions with AcCl, DMAP after dilution with CH2Cl2.
The results are similar for both experiments (Entries 2
and 6) resulting in an acetylated furanose–pyranose
ratio similar to that observed at room temperature and
a slight increase of 5a at the detriment of 5b. We have
thus shown that the dramatic increase of acetylated
derivative 6b-pyranose in our conditions was linked to
the trapping of the b-pyranose crystalline form and not
to a temperature or solvent effect. These results also
rationalise a similar selectivity observed in the anomeric
silylation of 4-b-Pyr.18 Once the reaction conditions
have been optimised, the reaction was tested on multi-
gram scales and allowed to reproductively isolate, after
extraction and crystallisation from Et2O, 83% of pure
6b without the need for chromatography. From the
mother liquors, a further amount of 6a–6b mixture
could be isolated by chromatography giving a total
isolated yield in pyranose derivatives of 92%. More-
over, we have also shown that a careful crystallisation
of 4 is not essential for the selectivity. The crude
crystals obtained after the evaporation of the trifl-
uoroacetic–water mixture used for the removal of the
1,2-isopropylidene group in 213a may be used in the
acetylation step without any loss of selectivity (Entry
7).
We began our study by investigating the reaction
conditions required to trap the b-pyranose form of the
crystals of 4 by acetylation. As a reference, a reaction
with acetic anhydride in pyridine at room temperature
was performed as described.13a After the reaction had
reached completion, an aqueous work-up was carried
out and the crude reaction mixture was analysed by 13C
NMR spectroscopy in order to determine the relative
proportion of 5a- and 5b-furanose and 6a- and 6b-py-
ranose derivatives. 1H NMR spectroscopy was also
used as control, but could not be used to determine
precisely the relative proportions of 5a and 6b due to
signal overlapping (see Table 2 for chemical shifts). We
found a 8:29:18:45 ratio for 5a–5b–6a–6b (Entry 1,
Table 2) similar to that reported previously.13a We then
suspended the crystalline 4b-Pyr compound in methyl-
ene chloride, and added pyridine, 2,4-dimethylaminopy-
ridine and acetyl chloride after lowering the
temperature to −20 °C. As expected, we observed a
dramatic increase in the pyranose forms and obtained
80% of 6b (Entry 3). Since we were working in a 4:1
CH2Cl2–pyridine mixture, we considered that dissolu-
tion and equilibration may occur at −20 °C, which
would allow the formation of the acetylated furanose
derivatives. We thus lowered the temperature to −40
and −50 °C and obtained, in both cases, a 94% selec-
Table 2
a
Entry
Conditions
Temperature (°C)
Compounds (%) b
Isolated yields (%) c
5a
5b
6a
6b
Crist. 6b
6a+6b
1
2
3
4
5
6
7
A
A
B
B
B
20
−40
−20
−40
−50
−40
−40
8
18
8
29
22
6
2
3
15
20
6
4
3
48
40
80
94
94
49
94
nd.
nd.
nd.
83
nd.
nd.
60
nd.
nd.
nd.
92
nd.
nd.
70
C
15
18
2
18
4
B d
a A: acetylation using Ac2O in pyridine as described.13a B: acetylation in CH2Cl2 with 6.0 equiv AcCl, 10 equiv pyridine and
0.1 equiv DMAP. C: crystalline 4b-Pyr was first dissolved in 10 equiv pyridine, then CH2Cl2 was added and the rest of the
reaction was performed as for B.
1
b Product ratio was determined by NMR: 13C: l (ppm): 98.7 (C-1 5a); 92.7 (C-1 5b); 91.2 (C-1 6a); 89.8 (C-1 6b) and H: l
(ppm): 6.11 (d, J 3.0 Hz, H-1 5a); 6.43 (d, J 4.5 Hz, H-1 5b), 6.24 (t, J 1.5 Hz, H-1 6a), 6.09 (d, J 1.5 Hz, H-1 6b).
c Crystalline 6b was obtained after extraction and crystallisation from diethyl ether–petroleum ether; remaining 6a+6b mixture
was obtained after flash chromatography of the mother liquor.
d Conditions B were used on the crude crystals obtained after removal of the 1,2-O-isopropylidene group of 3.

A. Dilhas, D. Bonnaffe´ / Carbohydrate Research 338 (2003) 681–686
685
3. Conclusion
We have thus shown that methyl 3-O-benzyl-
4.3. Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-b-L-idopyra-
nuronate (6b)
L-
iduronate 4 crystallises in the b-pyranose form by care-
ful assignment of its 1H and 13C NMR spectra in
d4-methanol after equilibration into a- and b-pyranose
and furanose forms. This allowed us to define reaction
conditions in which the acetylated pyranose compound
6b could be isolated in high yield by simple crystalliza-
tion, a major improvement over the previously de-
scribed conditions.13a This strategy for the enhancement
of the pyranose content, after derivatisation of the
anomeric position, should be applicable to reactions
other than acylation or silylation as long as it is possi-
ble to find reactions conditions where 4b-Pyr could be
only sparingly soluble.
Crystalline methyl 3-O-benzyl-
-iduronate 413a (4.2 g,
L
14.1 mmol) was suspended in anhyd CH2Cl2 (70 mL) at
0 °C. The mixture was brought to −40 °C and 2,4-
dimethylaminopyridine (172 mg, 1.4 mmol, 0.1 equiv),
Py (11.3 mL, 141 mmol, 10 equiv) and acetyl chloride
(6.0 mL, 84.6 mmol, 6 equiv) were added. After stirring
at this temperature for 10 h, the mixture was diluted
with CH2Cl2 (200 mL) and the resulting organic phase
was washed with satd NaHCO3 solution (3×50 mL),
water (2×50 mL), 1 M H2SO4 (3×50 mL) and water
(3×50 mL). The organic layer was passed through a
phase separator filter paper and concentrated under
diminished pressure. The residue was crystallised from
Et2O giving 5.0 g 6b (11.7 mmol, 83%). Evaporation of
the mother liquor followed by flash chromatography
gave an additional 0.5 g amount of a 6a–6b (1/2)
mixture (combined yield of 6a/6b: 92%).
4. Experimental
4.1. General methods
Acknowledgements
All moisture-sensitive reactions were performed under
an Ar atmosphere using oven-dried glassware. All sol-
vents were dried over standard drying agents and
freshly distilled prior to use. Flash column chromatog-
raphy was performed on silica gel 60 A C.C. (6–35 mm,
SDS). Reactions were monitored by TLC on silica gel
60 F254 with detection by charring with H2SO4. Melting
points were determined with a capillary apparatus and
This work was supported by the ANRS (Agence
Nationale pour la Recherche sur le SIDA), Sidaction
and the CNRS ‘Physique Chimie du Vivant’ program.
We thank Professor A. Lubineau for helpful
discussions.
are uncorrected. All products have characteristics simi-
13a
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