Synthesis of glycosaminoglycan oligosaccharides - An unexpected inhibitory effect of a remote N-acetyl group upon trichloroacetimidate-mediated couplings

Article abstract of DOI:10.1002/ejoc.200300791

In order to prepare biologically relevant heparan sulfate (HS) tetrasaccharide fragments containing an N-acetylated glucosamine at the reducing end, we studied the glycosylation reaction between the 2-azidoglucose trichloroacetimidate disaccharide donor 1 and a range of 4′-OH-uronyl disaccharide acceptors with an N-acetylglucosamine at the reducing terminus. Although we tried several condensation conditions, no tetrasaccharide was formed. We show that the failure of these reactions is due to the presence of the N-acetyl group, which inhibits the trichloroacetimidate-mediated glycosylation, since the analogous reaction proceeds smoothly once the N-acetyl group has been replaced by an azide. In the latter case, we show that the careful optimisation of the solvent system is a powerful way to obtain high yields and α-stereoselectivity in coupling reactions of 1 with the 4-OH of a GlcUA acceptor. Thus, in a THF/Et₂O (9:1) system, we obtained the GlcUA-β-(1→4)-GlcN₃-α-(1→4)-GlcUA-β- (1→4)-GlcN₃ tetrasaccharide 16α/β in 90% isolated yield and 92:8 α/β ratio, as compared to 57% yield and 70:30 α/β ratio when CH₂Cl₂ was used. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300791

FULL PAPER
Synthesis of Glycosaminoglycan Oligosaccharides ؊ An Unexpected Inhibitory
Effect of a Remote N-Acetyl Group upon Trichloroacetimidate-Mediated
Couplings
[a]
[a]
[a]
[a]
´
´
Ricardo Lucas, Daniel Hamza, Andre Lubineau, and David Bonnaffe*
Keywords: Oligosaccharides / Combinatorial chemistry / Glycosylation
In order to prepare biologically relevant heparan sulfate (HS)
tetrasaccharide fragments containing an N-acetylated gluco-
samine at the reducing end, we studied the glycosylation re-
action between the 2-azidoglucose trichloroacetimidate dis-
the N-acetyl group has been replaced by an azide. In the
latter case, we show that the careful optimisation of the
solvent system is a powerful way to obtain high yields and
α-stereoselectivity in coupling reactions of 1 with the 4-OH
accharide donor 1 and a range of 4Ј-OH-uronyl disaccharide of a GlcUA acceptor. Thus, in a THF/Et2O (9:1) system, we
acceptors with an N-acetylglucosamine at the reducing ter-
minus. Although we tried several condensation conditions,
no tetrasaccharide was formed. We show that the failure of
these reactions is due to the presence of the N-acetyl group,
obtained
the
GlcUA-β-(1Ǟ4)-GlcN3-α-(1Ǟ4)-GlcUA-β-
(1Ǟ4)-GlcN3 tetrasaccharide 16α/β in 90% isolated yield
and 92:8 α/β ratio, as compared to 57% yield and 70:30 α/β
ratio when CH2Cl2 was used.
which inhibits the trichloroacetimidate-mediated glycosyl- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
ation, since the analogous reaction proceeds smoothly once
Germany, 2004)
Introduction
polymerisation with heparin lyase III, and its structure was
determined, by MS analyses, to be a non-sulfated tetrasac-
charide containing an inner N-unsubstituted glucosamine
and an N-acetyl glucosamine at the reducing end.[7] In
addition, there is strong evidence that the terminal uronic
acid is GlcUA and not IdoUA.[7] The two tetra-
saccharides GlcUAϪGlcNH2ϪGlcUAϪGlcNAc (A) and
GlcUAϪGlcNH2ϪIdoUAϪGlcNAc (B) are thus candi-
dates as the minimum structure recognized by the 10E4
antibody. We felt that as part of an ongoing program aimed
at the preparation of libraries of HS fragments,[8] tetrasac-
charides A and B could be interesting biological targets in
the development of a strategy for the synthesis of HS frag-
ments containing an N-acetylated glucosamine at the reduc-
ing end. For the synthesis of compound A, our strategy
relied upon a Schmidt trichloroacetimidate-mediated coup-
ling between the 2-azido disaccharide donor 1 and the N-
acetyl glucosamine-containing disaccharide acceptor 2
(Figure 1).
Heparan sulfate (HS) is a member of the glycosaminogly-
can (GAG) family. This linear sulfated polysaccharide com-
prises the repetition of a basic disaccharide in which a
uronic acid is 1,4-linked to a 2-deoxy-2-aminoglucose. HS
is one of the most heterogeneous biopolymers since various
epimerisation and sulfation patterns (sulfoforms) may occur
along the chain.[1Ϫ3] The uronic acid may be either -glucu-
ronic (GlcUA) or -iduronic (IdoUA), while O-sulfation
may occur at the 2-position of the uronic acid and the 3-
and/or 6-positions of the amino sugar. The glucosamine ni-
trogen may be sulfated, acetylated or, less frequently, un-
modified. There is growing evidence that the formation of
different HS structures is tightly controlled during bio-
synthesis, with the presumed goal of generating sequences
with biological specificity.[4] In fact, HS chains, either at
the cell surface or in the extracellular matrix, interact and
regulate the activity of numerous proteins such as growth
factors, cytokines, chemokines, viral proteins and coagu-
The key point in this approach is to obtain good α-
lation factors.[5,6] Recently, it has been shown that a he- stereoselectivity in the glycosylation reaction between ac-
paran sulfate antigen, recognized by the monoclonal anti- ceptor 2 and donor 1. The non-participating azide at the 2-
body 10E4, co-distributes precisely with the prion lesions position of the donor should allow for a 1,2-cis glycosyl-
in the brain of scrapie-infected mice. An antigen-positive ation. It has been recently shown that total α-selectivity un-
fragment from heparan sulfate was isolated after partial de- der such coupling conditions may be obtained when the
glucuronyl acceptor is conformationally constrained by a
1,2-O-isopropylidene derivative.[9,10] However, this strategy
is not applicable when the GlcUA acceptor is already linked
to another sugar, as in compound 2. A quick survey of the
literature revealed that, although reactions between donors
[a]
Laboratoire de Chimie Organique Multifunctionnelle, UMR
´
8614 ‘‘Glycochimie Moleculaire’’,
´
Bat. 420, Universite Paris Sud, 91405 Orsay Cedex, France
Fax: (internat.) ϩ33-1-69154715
E-mail: david.bonnaffe@icmo.u-psud.fr
Eur. J. Org. Chem. 2004, 2107Ϫ2117
DOI: 10.1002/ejoc.200300791
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R. Lucas, D. Hamza, A. Lubineau, D Bonnaffe
FULL PAPER
Figure 1. Retrosynthetic strategy for the preparation of GlcUA-
containing HS fragments with an N-acetyl glucosamine at the re-
ducing end
˚
Scheme 1. Reagents and conditions: a) i. PhBCl2, Et3SiH, 4 A MS,
Et2O, Ϫ40 °C, 97%; ii. (COCl)2, DMSO, CH2Cl2, Ϫ78 °C and then
Et3N; iii. Na2HPO4, NaClO2, tBuOH/H2O, β-isoamylene, Ϫ10 °C
to r.t; iv. Cs2CO3, MeI, DMF, 76%; b) i. C8H14(Me2PhP)2IrIPF6,
H2,THF, r.t; ii. HgO/HgCl2, acetone/H2O, 9:1, 83% two steps; c)
Cl3CCN, DBU, CH2Cl2, 15 min 88%; d) PPh3, THF/H2O, 9:1, 16 h,
r.t; ii. Ac2O, Pyridine, 83%; e) TFA, CH2Cl2, 15 min, 94%
containing a non-participating azido[11Ϫ13] or O-benzyl
group[14Ϫ16] and non-conformationally-locked glucuronyl
acceptors may indeed lead to some unwanted β-anomer,
this is not the general trend. Indeed, in many cases, the
α-anomer was reported to be formed as the sole isolated
stereoisomer.[14,15,17Ϫ23] We thus felt rather confident that
we would be able to find highly α-diastereoselective glycos-
ylation conditions between 1 and 2. These two compounds
will be prepared from the key disaccharide 3, for which we
have recently described an efficient multigram synthesis.[8]
This disaccharide building block is central to our strategy
for the preparation of GlcUA-containing GAG fragments.
The tetrasaccharide formed will comprise the same protect-
ing group pattern at the anomeric and 4ЈЈЈ-positions, al-
lowing us to perform further iterative chain elongation
using sequential addition of disaccharides.
comprising allyl to vinyl isomerisation with an
[Ir(C8H14)(PMe2Ph)2][PF6] catalyst activated by hydrogen,
followed by cleavage of the resulting enol ether using HgO/
HgCl2 in a water-acetone mixture.[26] The activation of the
anomeric position of 5, as a trichloroacetimidate, was car-
ried out under standard conditions to give a 3:10 α/β mix-
ture of the required donor 1 in excellent yield.
Ester 3 was also used as a precursor to the disaccharide
acceptor 2. The azido group was readily transformed in
83% yield to the N-acetyl derivative 6 by Staudinger[27] re-
duction and conventional acetylation (Scheme 1). The re-
moval of Ph3PO, after these two steps, was performed using
LH-20 size exclusion chromatography, this greatly facili-
tated the purification of compound 6, which was otherwise
difficult to perform by silica gel chromatography. The cleav-
age of the p-methoxybenzyl group was then achieved with
10% TFA in dichloromethane (DCM)[28] to give the desired
disaccharide acceptor 2 in excellent yield (Scheme 1). We
found that this method for the cleavage of the p-meth-
oxybenzyl group gave superior yields to the use of DDQ.
Results and Discussion
Preparation of Disaccharide Donor 1 and Disaccharide
Acceptor 2
The glucuronic building block 3 was prepared from di-
saccharide 4 using a slight modification of a protocol that
we have recently described (Scheme 1).[8] First, a PhBCl2/
Et3SiH-mediated reductive opening of the p-methoxy-
benzylidene moiety of compound 4 was followed by Swern
oxidation as described.[8] We then performed the oxidation
Glycosylation Reactions with Disaccharide Acceptors
Containing N-Acetyl Glucosamine at the Reducing End
We initially investigated the glycosylation reaction be-
of the resulting aldehyde to ester 3 using first NaO2Cl-me- tween donor 1 and acceptor 2 under standard conditions,
diated oxidation to the carboxylic acid,[24,25] followed by using CH2Cl2 as solvent and TMSOTf as the promoter at
methylation using methyl iodide and Cs2CO3 in DMF. We Ϫ20 °C or room temperature, but the reactions were slug-
found this oxidation method more reliable than alkaline gish with only trace amounts of the desired tetrasaccharide
iodine in methanol, especially when performing the reaction 13α/β isolated, as evidenced by mass spectrometry. The typi-
on a larger scale (Ͼ1 g). We thus obtained the glucuronic cal course of the reaction involved the slow hydrolysis of
building block 3 from the disaccharide 4 in a good and donor 1 over several hours. Addition of further quantities
reproducible overall yield (76%).
of promoter — up to one equivalent — served only to speed
From disaccharide 3, the glucuronic donor 1 was pre- up this hydrolysis. Thus, after treatment of the reaction mix-
pared in a few high-yielding steps (Scheme 1). First the an- ture, the acceptor could be partly recovered in addition to
omeric alcohol 5 was obtained using a two-step deallylation compound 5, resulting from donor hydrolysis, and the am-
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Synthesis of Glycosaminoglycan Oligosaccharides
FULL PAPER
ide 12, resulting from the rearrangement of trichloroacetim- merisation of an -iduronyl-containing disaccharide into
idate 1. We then looked at the effects of changing the pro- tetra-, hexa- and octasaccharides in high yields.[29] Thus, in
moter to BF3·Et2O, TBDMSOTf or Sn(OTf)2 as well as the order to compare the reactivity of -glucuronyl acceptor 2
presence or absence of molecular sieves; in all cases the re- with a similar -iduronyl acceptor, we prepared compound
actions were unsuccessful.
10 from the known disaccharide 9,[8] using a Staudinger
We reasoned that, in compound 2, the nucleophilicity of azide reduction and selective N-acetylation with acetic an-
the 4Ј-OH may be reduced by the proximity of the carboxy- hydride in MeOH (Scheme 2). In addition, the coupling re-
methyl group that would thus be the cause of the failure of action of 1 and 10 would also provide easy access to the
the glycosylation reaction. In order to test this hypothesis, biologically important tetrasaccharide B. However, when
we prepared acceptor 8, in three steps and 73% overall the IdoUA acceptor 10 was treated with the donor 1 and
yield, from the non-oxidised precursor 4 (Scheme 2). Unfor- TMSOTf, the desired tetrasaccharide 15 was not obtained
tunately, the glycosylation reactions between this new ac- (Scheme 3).
ceptor 8 and donor 1 also failed to generate the desired
tetrasaccharide. Irrespective of the promoter and reaction
conditions used, only hydrolysis and rearrangement of do-
nor 1 were observed (Scheme 3).
The 4-position of -iduronyl derivatives has been de-
scribed as being more reactive than that of their -glucu-
ronyl counterparts,[9] and we have performed the oligo-
The above experiments showed unambiguously that the
failure of the trichloroacetimidate-mediated coupling of do-
nor 1 with acceptor 2 was not linked to the low reactivity
of the 4Ј-hydroxyl of the glucuronyl moiety. However, since
the three disaccharide acceptors 2, 8 and 10 contain an N-
acetylated glucosamine at their reducing end, we began to
suspect that this acetamido group may exert an inhibitory
effect in the trichloroacetimidate-mediated glycosylation,
even remote from the reacting centre. A quick survey of
the literature shows that numerous successful glycosylation
reactions have been performed on N-acetyl-containing
acceptors.[30Ϫ32] In the GAG field, we have already per-
formed the coupling of a trichloroacetimidate with the 3-
OH of a 2-acetamidoglucosamine acceptor,[33] while others
have successfully performed glycosylation at the 4-OH of a
terminal N-acetylglucosamine in a tetrasaccharide ac-
ceptor,[34] or used a trisaccharide donor containing an N-
acetylglucosamine at the non-reducing end.[20] Nevertheless,
the low reactivity of the 4-OH group in N-acetylglucosam-
ine derivatives in some glycosylation reactions has long
been known.[35] It has been shown that 4-OH nucleophilic-
ity is variable and linked to the protecting group used for
3-OH. In the case of glycosylation reactions with bromide
donors, an acetyl group at the 3-position gives low glycosyl-
ation yields, while allyl or benzyl groups allow efficient
Scheme 2. Synthesis of acceptors 7, 8, 10 and 11; reagents and
conditions: a) i. PPTS, MeOH, reflux, 10 min, 86%; ii. AcCl, Py,
Ϫ20 °C, 90%; b) i. PPh3, THF/H2O, 9:1, 16 h. r.t; ii. Ac2O, MeOH,
94%.c) i. PPh3, THF/H2O, 9:1, 16 h. r.t; ii. Ac2O, MeOH, 97%; d) couplings.[30] More recently, it has been shown that in sulf-
TFA, CH2Cl2, 15 min, 96%
Scheme 3. Couplings reactions of donor 1 with acceptors 2, 7, 8, 10 and 11; for reagents, conditions and α/β ratio see Table 1
Eur. J. Org. Chem. 2004, 2107Ϫ2117
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FULL PAPER
Table 1. Coupling optimisation studies with donor 1 and acceptors 2, 8, 9 and 11 using standard conditions (1.3 equiv. donor, 0.12 ,
Ϫ30° to Ϫ20 °C, 0.1 equiv. promoter and without molecular sieves)
Entry
Acceptor
Solvent
Ratio
Promotor
Tetrasaccharide
Yield (%)
α/β[a]
1
2
3
4
5
6
7
8
9
11
11
11
11
11
11
11
11
11
11
7
CH2Cl2[c]
Ϫ
Ϫ
Ϫ
Ϫ
9:1
8:2
5:5
9:1
3:1
9:1
9:1
9:1
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TBDMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
16
16
16
16
16
16
16
16
16
16
17
13
57[b]
70:30
70:30
68:32
93:7
CH2Cl2
55[b]
Et2O
THF
74
66
90
81
60
55
45
57
THF/Et2O
THF/CH2Cl2
THF/CH2Cl2
THF/Et2O
Dioxane/toluene[d]
THF/toluene
CH2Cl2
92:8
88:12
87:13
90:10
82:18
84:16
50:50[e]
10
11
12
42
2
THF/Et2O
No desired reaction
under standard conditions
13
14
8
10
THF/Et2O
THF/Et2O
9:1
9:1
TMSOTf
TMSOTf
14
15
[a]
α/β ratio was determined by HPLC after isolation of the tetrasaccharide fraction by LH-20 chromatography (see Exp. Sect. for
conditions). [b] Amide yield was determined with respect to donor: 32% and 17% for entries 1 and 2 respectively. [c] Glycosylation reaction
performed by inverse procedure. Reaction at 0 °C to avoid solvent freezing. Based on isolated yields after column chromatography.
[d]
[e]
[36]
oxide-
or trichloroacetimidate-mediated[37] glycosyla- leading thus to the hydrolysis products 2 and 5 after pro-
tions, the reactivity of glucosaminyl 4-OH acceptors, bear- longed exposure to a Lewis acid, when spotted on a TLC
ing the same group in the 3-position, is markedly influenced plate or upon quenching the reaction. We were unable to
by the protecting group of the amino function. Indeed, an isolate this imidate, but we cannot exclude that the product
acetamido-containing acceptor was shown to be ten times of the condensation of 1 and 2, characterised by MS analy-
less reactive than its azido counterpart and three times less ses as the awaited tetrasaccharide 13, was in fact an imidate.
than the corresponding N-phthalimido acceptor. An expla-
In order to ascertain this unforeseen and, to the best of
nation for this low reactivity has been proposed by Crich our knowledge, previously unreported remote inhibitory ef-
and Dudkin, who demonstrated the occurrence of intermo- fect of the NHAc group upon these trichloroacetimidate-
lecular hydrogen bonds involving the glucosamine amide based glycosylations, we decided to study glycosylation re-
group. They showed that these interactions are involved in actions on disaccharide acceptors 7 and 11 in which the
the low reactivity of N-acetyl acceptors since the introduc- NHAc group has been replaced by an azide.
tion of a protecting group favouring an intramolecular hy-
Glycosylation Reactions with Disaccharide Acceptors
Containing 2-Azidoglucose at the Reducing End
drogen bond enhanced the reactivity by inhibiting the inter-
molecular one.[36] Although the reacting OH in acceptor 2
is far from the NHAc group, we wondered whether such
Acceptor 11 was prepared in excellent yield by TFA
intermolecular bonds could also explain the absence of re- cleavage of the p-methoxybenzyl group of the key inter-
activity of acceptor 2. We thus performed glycosylation re- mediate 3. We then investigated the coupling reactions of
actions between donor 1 and acceptor 2 in a THF/Et2O donor 1 and acceptor 11 and we were pleased to find that,
mixture that should be able to break intermolecular H- in CH2Cl2 and with TMSOTf as promoter, the desired
bonds, but, once again, no tetrasaccharide was formed tetrasaccharide 16 was formed, albeit in moderate yield
(Table 1, entry 12). It appears therefore that intermolecular (57%) and an α/β ratio of 70:30 (Scheme 3, Table 1, entry
hydrogen bonding is not responsible for the lack of reactiv- 1).[38] In this reaction, all the donor 1 was consumed after
ity of compound 2.
15 min at Ϫ30 °C and no further evolution of the reaction
More recently, Liao and Auzanneau have proposed an was observed by TLC analysis. Along with tetrasaccharide
alternative explanation for the low reactivity of the 4-OH 16, an α/β mixture of amide 12 (32%) and traces of hydroly-
in N-acetamido-containing acceptors which does not ex- sis product 5 were formed. We first attributed the moderate
clude that reported previously. They found that the oxygen yield of this reaction to the low reactivity of the acceptor
of the acetamido group may act as a competitive nucleo- that would allow the rearrangement of trichloroacetimidate
phile in glycosylation reactions, leading to a kinetically fav- 1 into the trichloroacetamide by-product 12 to compete
oured imidate.[37] This compound may go on to rearrange with the tetrasaccharide formation. The ‘‘inverse pro-
to the thermodynamically more stable glycoside, albeit in cedure’’ has been reported to give better yields with unreac-
only 50% yield, thus lowering the overall yield of the glycos- tive acceptors.[39] We thus performed a reaction adding first
ylation reaction. In our case, it is possible that the reaction TMSOTf to the acceptor 11 and then the donor over 5 min.
of the donor 1 with acceptor 2 may also lead to a similar However, no improvements in the yield nor in the α/β ratio
imidate, that would be unable to rearrange to the glycoside were obtained this way (Table 1, entry 2).
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Synthesis of Glycosaminoglycan Oligosaccharides
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As in the case of the NHAc acceptor 2, we thought that tained with the latter. However, when we diluted the THF
the reactivity of the 4Ј-OH in 11 could be lowered by the with 50% CH2Cl2, the yield dropped to 60% (Table 1, entry
proximity of the C-5 carboxymethyl group. We thus used 7). Toluene has been found to be the best solvent in some
compound 7 as acceptor in a glycosylation reaction with condensations between a 2-O-benzylated glucosyl donor
donor 1 in the same conditions as for acceptor 11 and we and 4-OH uronic oligosaccharide containing ac-
obtained the tetrasaccharide 17 in a 42% yield and an α/β ceptors,[14,15] while dioxane/toluene (3:1) mixtures have
ratio of 50:50 (Scheme 3, Table 1, entry 11). This experi- been reported to enhance the α/β ratio in thioglycoside-me-
ment demonstrates clearly that the replacement of the C-5 diated glycosylations.[42] We thus conducted reactions be-
carboxymethyl function by an acetoxymethyl group in such tween 1 and 11 in dioxane or THF with toluene as the co-
4Ј-OH disaccharide acceptors does not have a marked influ- solvent, but this change was not beneficial in our case and
ence on the yield and stereochemical outcome of the reac- only resulted in lower yields similar to that observed with
tion. Similar results have already been observed in the con- the THF/CH2Cl2 (1:1) mixture (Table 1, entries 9 and 10).
densation of a 2-azidoglucose trichloroacetimidate donor We then decided to study the effect of changing the pro-
with a 4Ј-OH glucosyl disaccharide acceptor protected as a moter from TMSOTf to TBDMSOTf, in order to see
6Ј-O-TBDMS. In this case, a mixture of α- and β-anomers whether this could still further enhance the diastereoselec-
was obtained from which a 66% yield of the pure α-anomer tivity of the reaction, but this change only resulted in a
could be isolated.[40] However, no comparison between the lower yield (Table 1, entry 8).
4-OH glucosyl acceptor and its glucuronyl counterpart was
made in this article.
We have thus found that the most efficient promoter and
reaction solvent for the glycosylation of 11 with 1 are
These first experiments showed that in CH2Cl2 we faced TMSOTf and a THF/Et2O (9:1) mixture. With these new,
a dual reactivity and diastereoselectivity problem. It has optimal conditions we decided to return to our original
long been known that the solvent used in a glycosylation couplings of donor 1 with the N-acetyl-containing ac-
has a profound impact on the outcome of the reac- ceptors 2, 8 and 10. Unfortunately the reactions between
tion.[35,39,41,42] Under SN1 conditions the effect of ethers on donor 1 and acceptors 2, 8 or 10 were as unsuccessful under
the oxycarbenium intermediate generally results in the for- these optimised conditions as they had been in pure CH2Cl2
mation of the thermodynamically more stable glycoside an- (Table 1, entries 12, 13 and 14). In order to evaluate if a
omer, due to stabilisation of the epimeric oxonium anomer difference in nucleophilicity between the 4Ј-OH of N-acetyl
by the reverse anomeric effect.[41,43] We began our optimis- and azido containing acceptors could explain the difference
ation studies by repeating the condensation in Et2O as the in reactivity between both groups, we looked at the 1H
solvent and we were pleased to observe a marked increase chemical shifts of their 4Ј-OH (Table 2), but no general
in the yield to 74% (Table 1, entry 3). In accordance with tendency can be drawn from these results. This shows once
the change in solvent polarity,[44] the donor was consumed more that the absence of tetrasaccharide formation with ac-
more slowly in Et2O than in CH2Cl2 (30Ϫ45 min versus ceptors 2, 8 or 10 is linked to the presence of the N-acetyl
less than 15 min), while the formation of the amide 12 was group.
reduced to around 5%. Thus, as expected, the rearrange-
1
Table 2. H chemical shifts of the 4Ј-OH in acceptors 2 and 7Ϫ11
ment of the imidate was probably inhibited by the disrup-
tion of the oxycarbenium/leaving group ion pair by the chel-
Compound
δ (ppm)
Amino function
ating solvent. However, quite surprisingly, the α/β ratio re-
mained unchanged when using Et2O. We thus turned to
THF, a solvent with a higher polarity and donor number
than Et2O[44] that may both favour the SN1 pathway and
the stability of the oxycarbenium-solvent intermediate. In-
deed, the diastereoselectivity of the reaction between donor
1 and acceptor 11 in THF was greatly improved leading to
a 93:7 α/β ratio, but the isolated yield of tetrasaccharide 16
α/β dropped to 66%. However, in contrast to the reaction
2
11
8
2.87
2.96
2.81
2.60
2.87
2.678
NHAc
N3
NHAc
N3
NHAc
N3
7
10
9
in dichloromethane, this lower yield was not linked to the Conclusion
competitive formation of the amide 12, whose proportion
was low in the reaction mixture. We then decided to study
We have demonstrated a complete inhibitory effect of an
the effect of the addition of a co-solvent to the THF, and N-acetyl group, even remote from the 4Ј-OH nucleophilic
conducted the reactions in a THF/Et2O (9:1) or THF/ centre, in the coupling of donor 1 with a range of disacchar-
CH2Cl2 (8:2) mixture. We were pleased to find that, in both ide acceptors containing such an N-acetyl group, since the
cases, the yields were improved with respect to THF alone analogous coupling proceeds smoothly once the N-acetyl
while the α/β ratios remained excellent (Table 1, entry 5 and moiety is replaced by an azide. Our results are compatible
6). When Et2O was used as co-solvent, the results were with the formation of an imidate between the donor and
slightly better than with CH2Cl2, since yields as high as 90% the N-acetyl group of the acceptors as proposed by Liao
with 92:8 α/β selectivity were obtained with the former and Auzanneau.[37] We have shown that a careful optimis-
while only 81% isolated yield and 88:12 selectivity were ob- ation of the solvent system is a powerful way to obtain high
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R. Lucas, D. Hamza, A. Lubineau, D Bonnaffe
FULL PAPER
analysis. The mixture was then re-cooled to 0 °C and a saturated
aq. solution of NaHSO3 (40 mL) was added with stirring. The mix-
ture was allowed to warm to room temp. and after 1 h NaHSO3
(20 mL), brine (20 mL) and EtOAc (100 mL) were added. The or-
ganic phase was separated and the aqueous layer was extracted
with CHCl3 (3 ϫ 50 mL) and EtOAc (3 ϫ 50 mL). The organic
layers were combined, dried over MgSO4 and concentrated under
reduced pressure to yield 1.86 g of the crude acid. TLC (toluene/
acetone, 1:1), plate pre-treated by dipping in 2% Et3N/DCM, then
dried in vacuo prior to applying samples. Rf ϭ 0.29.
yields and α-stereoselectivity in coupling reactions with oli-
gosaccharides containing a 4-OH GlcUA acceptor that can-
not be conformationally constrained. We have thus ob-
tained the tetrasaccharide 16α/β in 90% isolated yield and
92:8 α/β ratio. The transformation of tetrasaccharide 16α
into the biologically relevant tetrasaccharide A is currently
under optimisation in our laboratory and will be reported
in due course.
The crude acid from the previous step and Cs2CO3 (726 mg,
2.23 mmol, 1.1 equiv.) were dissolved in DMF (10 mL) and stirred
for 10 min at room temp. To the resulting suspension was added
MeI (1.72 g, 754 µL, 12.1 mmol, 6 equiv.) and the reaction stirred
for 16 h, after which time no starting material was visible by TLC
analysis. The reaction mixture was diluted with diethyl ether
(200 mL) and washed with water (10 mL), saturated aq. NaHCO3
(15 mL), 10% aq. Na2S2O3 (20 mL) and brine (20 mL). The diethyl
ethereal fraction was dried over MgSO4 and concentrated under
reduced pressure to afford a yellow oil. Purification by flash chro-
matography (petroleum ether/EtOAc, 4:1) afforded 3 as a clear,
colourless oil (1.41 g, 76%). C52H57N3O12: M ϭ 916.0 g/mol.
Experimental Section
Abbreviations: DDQ ϭ dichlorodicyanobenzoquinone, PPTS ϭ
pyridinium para-toluenesulfonate, TMSOTf
triflate, TBDMSOTf ϭ tert-butyldimethylsilyl triflate, TFA ϭ tri-
fluoroacetic acid.
ϭ
trimethylsilyl
General Remarks: All moisture-sensitive reactions were performed
under an argon atmosphere using oven-dried glassware. Evapor-
ation was performed under vacuum with a water bath temperature
below 40 °C. All solvents were dried over standard drying agents
and freshly distilled prior to use. Flash column chromatography
was performed on Silica Gel 60 A.C.C. (6Ϫ35 µm). Reactions were
monitored by TLC on glass Silica Gel 60 F254 plates with detection
by UV at 254 nm and by charring with 5% ethanolic H2SO4. Gel
filtration chromatography were performed on LH-20 (Pharmacia)
eluting with CH2Cl2/MeOH, 1:1. Optical rotations were measured
on a Jasco DIP 370 digital polarimeter. NMR spectra were re-
corded at room temperature with Bruker 400 MHz or AC 250 spec-
trometers. 13C NMR were performed at 100 MHz. Me4Si or solvent
signals were used for δ calibration (CDCl3: 13C δ ϭ 77.0 ppm).
Phase-sensitive COSY were performed by recording 256 or 512
FIDs with 1024 complex data points. Prior to Fourier transform,
the data were zero-filled to 1024 points in the F1 dimension and
multiplied with a π/3-shifted sinebell function in both dimensions.
HSQC were performed by recording 128 FIDs with 1024 complex
data points. Prior to Fourier transform, the data were zero-filled to
1024 points in the F1 dimension and multiplied with a π/3-shifted
squared sinebell function in both dimensions. Elemental analyses
were performed at the CNRS (Gif sur Yvette, France). MS spectra
were recorded in the positive or negative mode on a Finnigan MAT
95 S using electrospray ionisation. For the determination of the α/
β ratio for tetrasaccharide 16, size-exclusion chromatography was
first performed followed by HPLC analyses of the pooled tetrasac-
charide fractions. HPLC was performed using a Nucleosil C18 5µ
200 ϫ 4.6 mm column (Hypersil) and UV detection at 220 and
254 nm. The elution was performed at 0.8 mL/min with a linear
water (5% CH3CN)/CH3CN gradient (10:90 to 5:95 over 10 min)
followed by 17 min isocratic elution (95:5).
[Methyl 2,3-Di-O-benzyl-4-O-(4-methoxybenzyl)-β-D-glucopyranosyl-
uronate]-(1Ǟ4)-2-azido-3,6-di-O-benzyl-2-deoxy-
D
-glucopyranose
(5): A solution of 3 (1.07 g, 1.17 mmol) in THF (5 mL) was de-
gassed under vacuum and (1,5-cyclooctadiene)bis(methyldiphenyl-
phosphane)iridium() hexafluorophosphate catalyst was added
(16 mg), followed by further degassing of the mixture. The suspen-
sion was stirred for 15 min and then the catalyst was activated with
hydrogen at room temperature for 2 min and the solution became
nearly colourless. The reaction mixture was degassed again and
stirred for 1.5 h. The solvent was then evaporated and the residue
was dissolved in acetone/water, 9:1 (20 mL) and HgO (303 mg,
1.41 mmol, 1.2 equiv.) and HgCl2 (349 mg, 1.28 mmol, 1.1 equiv.)
were added. After 1 h, the solution was filtered through a pad of
celite 545 and the solvent was evaporated. The residue was diluted
with EtOAc (150 mL) and washed with H2O (2 ϫ 50 mL) and satu-
rated aq. Na2S2O3 solution (50 mL). The aqueous layer was ex-
tracted with EtOAc (2 ϫ 50 mL) and the organic layers were dried
over MgSO4 and concentrated. The residue was purified by flash
chromatography (petroleum ether/EtOAc, 2:1 to 1:1), to yield 5
(846 mg, 83%) as a 2:1 α/β mixture of anomers along with 32 mg
(3%) of the starting material 3.TLC (petroleum ether/EtOAc, 2:1).
1
Rf ϭ 0.25. H NMR (400 MHz, CDCl3): δ ϭ 7.46Ϫ7.31 (m, 3 H,
Ph α and β), 7.37Ϫ7.20 (m, 27 H, Ph α and β), 7.13 (d, Jortho
ϭ
8.8 Hz, 3 H, MeOϪPh), 6.82 (d, Jortho ϭ 8.8 Hz, 3 H, MeOϪPh),
5.23 (d, J1,2 ϭ 3.2 Hz, 1 H, H-1a α), 5.12 (d, Jgem ϭ 10.4 Hz, 1 H,
CH2Ph α), 5.03 (d, Jgem ϭ 10.4 Hz, 0.5 H, CH2Ph β), 4.88Ϫ4.47
(m, 9 H, CH2Ph α and β), 4.47Ϫ4.40 (m, 2 H, H-1a β, H-1b α and
β), 4.34 (d, Jgem ϭ 13.5 Hz, 1 H, CH2Ph α), 4.33 (d, Jgem ϭ 13.5 Hz,
Allyl [Methyl 2,3-Di-O-benzyl-4-O-(4-methoxybenzyl)-β-D-glucopyr- 0.5 H, CH2Ph β), 4.07Ϫ3.67 (m, 13.5 H, H-4a, H-6a, H-5a, H-4b,
anosyluronate]-(1Ǟ4)-O-2-azido-3,6-di-O-benzyl-2-deoxy-α-
D-gluco- H-3a, H-5b, CH3OϪPh α and β), 3.57 (s, 3 H, COOCH3 β), 3.56
pyranoside (3): Reductive ring opening of 4 and subsequent Swern
(s, 1.5 H, COOCH3 α), 3.52Ϫ3.16 (m, 6 H, H-3b, H-2b, H-2a, OH
α and β) ppm. 13C NMR (100.0 MHz, CDCl3): δ ϭ 168.4 (CϭO),
oxidation gave the corresponding aldehyde as described pre-
viously.[8] A solution of NaH2PO4 (2.42 g, 15.8 mmol, 7.8 equiv.) 159.1 (C-OMe, CH3OϪPh), 138.1Ϫ137.3 (Cquaternary,arom),
in water (4.5 mL) was added to a rapidly stirring solution of this
129.9Ϫ127.3 (Ph, CH3OϪPh), 113.5 (MeOϪPh), 102.6 (C-1b α),
aldehyde (1.80 g, 2.03 mmol) and β-isoamylene (2-methyl-2-butene) 102.5 (C-1b β), 95.8 (C-1a β), 91.6 (C-1a α), 83.6 (C-3b α and β),
(23.5 mL, 191 mmol, 94 equiv.) in tert-butyl alcohol (9.5 mL). The 81.9 (C-2b α and β), 79.0 (C-3a α and β), 77.7 (C-4b α and β), 77.1
resulting biphasic mixture was cooled to Ϫ10 °C and a solution of
NaO2Cl (1.08 g, 9.8 mmol, 4.8 equiv.) in tert-butyl alcohol/H2O,
(C-4 α and β), 75.3, 75.1, 75.0, 74.9, 74.8, 74.7, 74.3; 74.2, 74.1 (C-
5b α and β), 73.1 (CH2Ph), 70.3 (C-5a α and β), 67.3 (C-6a α and
3:1 (19 mL) was added over 45 min whereupon a yellow colour was β), 66.8 (C-2a α and β), 63.0 (C-2a α and β), 55.0 (CH3OϪPh α
observed. The reaction was allowed to warm to room temp. over
16 h after which time no starting material was visible by TLC
and β), 52.1 (COOCH3 α and β) ppm. C49H53N3O12 (876): calcd.
C 67.17, H 6.10, N 4.80; found C 67.11, H 6.25, N 4.79.
2112
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 2107Ϫ2117

Synthesis of Glycosaminoglycan Oligosaccharides
[Methyl 2,3-Di-O-benzyl-4-O-(4-methoxybenzyl)-β-
FULL PAPER
-glucopyranosyl- Ja,b ϭ 5.0, Ja,cc ϭ Ja,ct ϭ 1.5 Hz, 1 H, CH2ϪCHϭCH2), 4.09 (t,
D
uronate]-(1Ǟ4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α,β-
D
-gluco-
J4,3 ϭ J4,5 ϭ 9.6 Hz, 1 H, H-4a), 3.96 (ddt, Jgem ϭ 13.0, Ja,b ϭ 6.0,
pyranosyluronate)trichloroacetimidate (1): CCl3CN (580 µL,
8.8 mmol, 9 equiv.) and DBU (20 µL) were added to a solution of
Ja,cc ϭ Ja,ct ϭ 1.5 Hz, 1 H, CH2ϪCHϭCH2), 3.89 (dd, J6,5 ϭ 3.2,
J6,6Ј ϭ 11.2 Hz, 1 H, H-6a), 3.82Ϫ3.73 (m, 5 H, CH3OϪPh, H-4b,
5 (846 mg, 0.96 mmol) in dry CH2Cl2 (10 mL). After stirring for H-5b), 3.66Ϫ3.57 (m, 5 H, H-3a, H-5a, COOCH3), 3.52Ϫ3.39 (m,
15 min the solvent was evaporated to dryness and the residue was
purified by flash chromatography (petroleum ether/EtOAc, 2:1, 1%
Et3N) to yield 1 (870 mg, 88%) as a 3:10 α/β mixture of anomers.
3 H, H-6Јa, H-2b, H-3b), 1.81 (s, 3 H, CH3CONH) ppm. 13C NMR
(100 MHz, CDCl3): δ ϭ 169.4, 168.6 (CϭO), 159.0 (CϪOMe,
CH3OϪPh), 139.0Ϫ133.7 (Cquaternary,arom.), 133.5 (CH2ϪCHϭ
TLC β/α (petroleum ether/EtOAc, 2:1, 1% Et3N). Rf ϭ 0.73/0.65. CH2), 129.8Ϫ127.2 (Ph, CH3OϪPh), 117.3 (CH2ϪCHϭCH2),
IR (thin film): ν˜ ϭ 3338 (νNϪH), 3090, 3063, 3031, 3000 (νCϪHarom), 113.5 (MeOϪPh), 102.6 (C-1b), 96.4 (C-1a), 83.7 (C-3b), 82.0 (C-
2955, 2910, 2813 (νCϪHaliph), 2113 (νN3), 1750 (νCϭO COOMe), 2b), 79.0 (C-4b), 77.3, 77.1 (C-3a, C-4a), 75.3, 74.9, 74.3 (CH2Ph),
1674 (νCϭNH), 1612, 1514, 1497, 1454, 1361, 1284, 1250, 1143,
74.0 (C-5a), 73.2, 73.0 (CH2Ph), 70.6 (C-5b), 68.0 (CH2ϪCHϭ
CH2), 67.2 (C-6a), 55.0 (CH3OϪPh), 52.0 (COOCH3), 51.6 (C-2a),
1068, 1032. 1H NMR (400 MHz, CDCl3, selected NMR spectro-
scopic data for the β anomer): δ ϭ 8.69 (s, 1 H, NH), 7.45Ϫ7.40 23.1 (CH3CONH). C54H61NO13 (932.1): calcd. C 69.59, H 6.60, N
(m, 2 H, Ph), 7.40Ϫ7.20 (m, 18 H, Ph), 7.14 (d, Jortho ϭ 8.4 Hz, 2 1.50; found 69.59, 6.71, 1.37. ESI-MS calcd. for
C
H
N
H, MeOϪPh), 6.83 (d, Jortho ϭ 8.4 Hz, 2 H, MeOϪPh), 5.56 (d, C54H61NNaO13 [M ϩ Na]: m/z ϭ 954.4; found 954.3.
J1,2 ϭ 8.4 Hz, 1 H, H-1a), 5.06 (d, Jgem ϭ 10.8 Hz, 1 H, CH2Ph),
Allyl (Methyl 2,3-Di-O-benzyl-β-
acetamido-3,6-di-O-benzyl-2-deoxy-α-
D
-glucopyranosyluronate)-(1Ǟ4)-2-
4.85 (d, Jgem ϭ 10.8 Hz, 1 H, CH2Ph), 4.80 (d, Jgem ϭ 10.8 Hz, 1
H, CH2Ph), 4.76 (d, Jgem ϭ 10.8 Hz, 1 H, CH2Ph), 4.72 (d, Jgem
D-glucopyranoside (2):
ϭ
CF3COOH (150 µL) was added to a solution of 6 (145 mg,
0.15 mmol) in CH2Cl2 (1 mL) at room temperature. After stirring
for 15 min, the reaction mixture, which had become purple, was
neutralised with Et3N (300 µL) at 0 °C and concentrated. The resi-
due was purified by flash chromatography ( toluene/acetone, 4:1 to
1:1) to yield 2 (114 mg, 94%) as a colourless gum. TLC (toluene/
acetone, 3:1): Rf ϭ 0.29. [α]3D0 ϭ ϩ43 (CHCl3, c ϭ 1.1). IR (thin
film): ν˜ ϭ 3275 (3550Ϫ3150, νOϪH and νNϪH), 3088, 3063, 3030,
3000 (νCϪHarom), 2958, 2923, 2851 (νCϪHaliph), 1753 (νCϭO
COOMe), 1658 (νCϭO NHAc), 1550, 1528, 1454, 1438, 1378, 1360,
1313, 1286, 1265, 1214, 1168, 1121, 1068, 1028. 1H NMR
(400 MHz, CDCl3): δ ϭ 7.42Ϫ7.20 (m, 20 H, Ph), 5.88 (dddd, J ϭ
11.0 Hz, 1 H, CH2Ph), 4.69 (d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.68
(d, Jgem ϭ 10.5 Hz, 1 H, CH2Ph), 4.60 (d, Jgem ϭ 10.8 Hz, 1 H,
CH2Ph), 4.64 (d, Jgem ϭ 12.4 Hz, 1 H, CH2Ph), 4.56 (d, J1,2
ϭ
8.0 Hz, 1 H, H-1b), 4.53 (d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 4.42 (d,
Jgem ϭ 12.4 Hz, 1 H, CH2Ph), 4.13 (t, J4,3 ϭ J4,5 ϭ 9.2 Hz, 1 H,
H-4a), 3.87 (dd, J6,5 ϭ 3.2, J6,6Ј ϭ 11.6 Hz, 1 H, H-6a), 3.82 (dd,
J4,5 ϭ 10.0, J4,3 ϭ 9.2 Hz, 1 H, H-4b), 3.79Ϫ3.73 (m, 4 H,
PhϪOCH3, H-5b), 3.67Ϫ3.56 (m, 5 H, H-2a, H-6Јa, COOCH3),
3.50 (t, J3,2 ϭ J3,4 ϭ 9.2 Hz, 1 H, H-3b), 3.48Ϫ3.38 (m, 3 H, H-
3a, H-5a, H-2b) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 168.7
(CϭO), 161.2 (CϭNH), 159.3 (CϪOMe, CH3OϪPh), 138.3Ϫ137.8
(Cquaternary,arom.), 130.0Ϫ127.6 (Ph, CH3OϪPh), 113.8 (MeOϪPh),
102.6 (C-1b), 96.7 (C-1a), 84.0 (C-3b), 82.1 (C-2b), 81.0 (C-3a),
79.2 (C-4b), 76.1 (C-4a), 76.0, 75.7 (CH2Ph), 75.3 (C-5a), 75.2, 74.6
(CH2Ph), 74.4 (C-5b), 73.3 (CH2Ph), 67.0 (C-6a), 65.2 (C-2a), 55.3
17.0, 10.5, 6.0, 5.0 Hz, 1 H, CH2ϪCHϭCH2), 5.26 (br.d, JNH,2
ϭ
8.8 Hz, 1 H, NH), 5.25 (dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭ
CH2), 5.20 (dq, J ϭ 10.5, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 4.91 (d,
J1,2 ϭ 4.5 Hz, 0.4 H, H-1a), 4.89 (d, J1,2 ϭ 3.5 Hz, 0.6 H, H-1a),
4.84 (s, 2 H, CH2Ph), 4.77 (s, 2 H, CH2Ph), 4.64 (d, Jgem ϭ 12.0 Hz,
1 H, CH2Ph), 4.57 (2 d separated by 0.005 ppm, Jgem ϭ 12.0 Hz, 1
H, CH2Ph of two NHAc rotamers), 4.42 (d, J1,2 ϭ 7.4 Hz, 0.4 H,
H-1b of one NHAc rotamers), 4.41 (d, J1,2 ϭ 7.4 Hz, 0.6 H, H-
1b), 4.35 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.29Ϫ4.21 (m, 1 H, H-
(CH3OϪPh), 52.4 (COOCH3) ppm. C51H53Cl3N4O12:
M ϭ
1020.3 g/mol.
Allyl [Methyl 2,3-Di-O-benzyl-4-O-(4-methoxybenzyl)-β-
D-glucopyr-
anosyluronate]-(1Ǟ4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-α-
D
-
glucopyranoside (6): A solution of 3 (180 mg, 0.19 mmol) and PPh3
(62 mg, 0.236 mmol) in THF/H2O, 9:1 (1 mL) was stirred for 16 h
at room temperature. After concentrating to dryness, the residue
was dissolved in pyridine (1 mL) and Ac2O (0.5 mL) was added.
The reaction mixture was stirred overnight, the solvent was re-
moved and the residue was purified by gel-filtration chromatogra-
phy to remove Ph3PO. Fractions containing the product were con-
centrated and purified by flash chromatography (toluene/acetone,
3:1) to yield 6 (145 mg, 83%). TLC (toluene/acetone, 3:1). Rf ϭ
0.38. [α]3D0 ϭ ϩ37 (CHCl3, c ϭ 1.0). IR (thin film): ν˜ ϭ 3275
(3550Ϫ3150, νOϪH and νNϪH), 3088, 3063, 3030, 3000 (νCϪHarom),
2958, 2923, 2851 (νCϪH aliph), 1753 (νCϭO COOMe), 1658 (νCϭO
NHAc), 1550, 1528, 1454, 1438, 1378, 1360, 1313, 1286, 1265,
1214, 1168, 1121, 1068, 1028. 1H NMR (400 MHz, CDCl3): δ ϭ
7.38Ϫ7.25 (m, 20 H, Ph), 7.16 (d, Jortho ϭ 8.8 Hz, 2 H, MeOϪPh),
6.82 (d, Jortho ϭ 8.8 Hz, 2 H, MeOϪPh), 5.88 (dddd, J ϭ 17.0,
10.5, 6.0, 5.0 Hz, 1 H, CH2ϪCHϭCH2), 5.26 (dq, J ϭ 17.0, 1.5 Hz,
2a), 4.16Ϫ4.08 (m, 2 H, H-4a, CH2ϪCHϭCH2), 3.94 (ddt, Jgem
13.0, Ja,b ϭ 6.0, Ja,cc ϭ Ja,ct ϭ 1.5 Hz, 1 H, CH2ϪCHϭCH2), 3.89
(dd, J6.5 ϭ 3.5, J6,6Ј ϭ 11.0 Hz, 1 H, H-6a), 3.80 (t, J4,3 ϭ J4,5
ϭ
ϭ
9.2 Hz, 1 H, H-4b), 3.68Ϫ3.47 (m, 7 H, H-3b, H-5a, H-5b, CO-
OCH3, H-6Јa), 3.39Ϫ3.28 (m, 2 H, H-2b, H-3b); 2.96 (br. s, 1 H,
OH); 1.81 (s, 3 H, CH3CONH) ppm. 13C NMR (100 MHz,
CDCl3): δ ϭ 169.6, 169.5 (CϭO), 139.0Ϫ137.8 (Cquaternary,arom.),
133.5 (CH2ϪCHϭCH2), 128.4Ϫ127.0 (Ph, CH3OϪPh), 117.3
(CH2ϪCHϭCH2), 102.5 (C-1b), 96.4 (C-1a), 83.1 (C-3b), 81.3 (C-
2b), 77.4 (C-3a), 76.7 (C-4a), 75.1, 74.9, 73.6 (CH2Ph), 73.0 (C-5b),
72.6 (CH2Ph), 71.9 (C-4b), 70.6 (C-5a), 68.0 (CH2ϪCHϭCH2),
67.3 (C-6a), 52.2 (COOCH3), 51.5 (C-2a), 23.1 (CH3CONH).
C46H53NO12 (811.9): calcd. C 68.05, H 6.58, N 1.73, O 23.65; found
C
67.71,
H 6.63, N 1.66, O 23.73. ESI-MS calcd. for
C46H53NNaO12 [M ϩ Na]: m/z ϭ 834.4; found 834.3.
1 H, CH2ϪCHϭCH2), 5.24 (d, JNH,2 ϭ 9.2 Hz, 1 H, NH), 5.21 (dq, Allyl (6-O-Acetyl-2,3-di-O-benzyl-β-
J ϭ 10.5, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 4.93 (d, Jgem ϭ 12.0 Hz, 1 ido-3,6-di-O-benzyl-2-deoxy-α- -glucopyranoside (7): A solution of
H, CH2Ph), 4.89 (d, J1,2 ϭ 4.0 Hz, 1 H, H-1a), 4.86Ϫ4.76 (m, 4 H, 4 (390 mg, 0.44 mmol) and PPTS (25 mg, 0.10 mmol) in MeOH
CH2Ph), 4.68 (d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 4.63 (d, Jgem (10 mL) was heated for 10 min at reflux. The temperature was then
12.0 Hz, 1 H, CH2Ph), 4.58 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.52 decreased to room temp. and the solution was neutralised with
D-glucopyranosyl)-(1Ǟ4)-2-az-
D
ϭ
(d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 4.46 (d, J1,2 ϭ 7.6 Hz, 1 H, H-
1b), 4.42 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.22 (ddd, J2,3 ϭ 10.8,
Et3N (0.1 mL) and concentrated. The residue was purified by flash
chromatography (petroleum ether/EtOAc, 4:1 to 1:1) to yield the
J2,NH ϭ 9.2, J2,1 ϭ 4.0 Hz, 1 H, H-2a), 4.13 (ddt, Jgem ϭ 13.0, corresponding diol (290 mg, 86%). Acetyl chloride (19 µL,
Eur. J. Org. Chem. 2004, 2107Ϫ2117
www.eurjoc.org  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2113

´
R. Lucas, D. Hamza, A. Lubineau, D Bonnaffe
FULL PAPER
0.28 mmol, 1.1 equiv.) was added to a cooled (Ϫ20 °C) solution of
J5,4 ϭ 9.0 Hz, 1 H, H-5b), 2.81 (br. s, 1 H, OH), 1.86 (s, 3 H,
diol (200 mg, 0.25 mmol) in pyridine (2.0 mL). After 2.5 h, EtOH OCOCH3), 1.75 (s, 3 H, CH3CONH) ppm. 13C NMR (100 MHz,
(1 mL) was added and the mixture was warmed to room tempera-
ture. The solvents were then evaporated and the residue was puri-
fied by flash chromatography (petroleum ether/EtOAc, 4:1 to 1:1)
CDCl3): δ ϭ 171.6, 169.7 (CϭO), 139.1Ϫ137.5 (Cquaternary,arom.),
133.6 (CH2ϪCHϭCH2), 128.4Ϫ127.3 (Ph, CH3OϪPh), 117.3
(CH2ϪCHϭCH2), 102.3 (C-1b), 96.5 (C-1a), 83.8 (C-3b), 82.1 (C-
to afford 7 (188 mg, 90%). TLC (petroleum ether/EtOAc, 3:2): Rf ϭ 2b), 77.3 (C-3a), 76.5 (C-4a), 75.3, 74.8, 73.3 (CH2Ph), 73.2 (C-5b),
0.50. [α]3D0 ϭ ϩ19 (CHCl3, c ϭ 1.1). IR (thin film): ν˜ ϭ 3450
72.6 (CH2Ph), 70.8 (C-5a), 70.1 (C-4b), 68.1 (CH2ϪCHϭCH2),
(3550Ϫ3150, νOϪH), 3089, 3063, 3030, 3010 (νCϪHarom), 2959, 2924, 67.6 (C-6a), 63.0 (C-6b), 51.7 (C-2a), 23.2 (CH3CONH), 20.5 (OC-
2866, 2852 (νCϪHaliph), 2109 (νN3), 1742 (νCϭO OAc), 1497, 1454, OCH3). C47H55NO12 (825.9): calcd. C 68.35, H 6.71, N 1.70, O
1
1364, 1242, 1138, 1119, 1053, 1028. H NMR (400 MHz, CDCl3): 23.25; found C 67.98, H 6.49, N 1.62, O 23.15. ESI-MS calcd. for
δ ϭ 7.38Ϫ7.18 (m, 20 H, Ph), 5.88 (dddd, J ϭ 17.0, 10.5, 6.0, C47H55NNaO12 [M ϩ Na]: m/z ϭ 848.4; found 848.3.
5.0 Hz, 1 H, CH2ϪCHϭCH2), 5.29 (dq, J ϭ 17.0, 1.5 Hz, 1 H,
CH2ϪCHϭCH2), 5.18 (dq, J ϭ 10.5, 1.5 Hz, 1 H, CH2ϪCHϭ
CH2), 5.06 (d, Jgem ϭ 10.8 Hz, 1 H, CH2Ph), 4.86 (d, J1,2 ϭ 3.6 Hz,
Allyl (Methyl 2-O-Acetyl-3-O-benzyl-α-
(1Ǟ4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-α-
L
-idopyranosyluronate)-
D
-glucopyranoside
(10): Compound 9[8] (136 mg, 0.18 mmol) was treated as described
for compound 6, giving, after flash chromatography (toluene/ace-
tone, 3:1), 128 mg of 10 (97%). TLC (toluene/acetone, 3:1): Rf ϭ
0.25. [α]3D0 ϭ ϩ15 (CHCl3, c ϭ 1.0). IR (thin film): ν˜ ϭ 3275
(3550Ϫ3150, νOϪH and νNϪH), 3095, 3063, 3030, 3004 (νCϪHarom),
2955, 2922, 2864, 2851 (νCϪHaliph), 1739 (νCϭO OAc), 1660 (νCϭO
NHAc), 1604, 1550, 1536, 1498, 1454, 1446, 1373, 1316, 1235,
1211, 1169, 1156, 1102, 1041. 1H NMR (400 MHz, CDCl3): δ ϭ
7.42Ϫ7.12 (m, 15 H, Ph), 5.87 (dddd, J ϭ 17.0, 10.5, 6.0, 5.0 Hz,
1 H, CH2ϪCHϭCH2), 5.35 and 5.34 (2 d, JNH,2 ϭ 10 Hz, 1 H,
NH, 2 rotamers), 5.25 (dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭ
CH2), 5.20 (br. d, J ϭ 10.5 Hz, 1 H, CH2ϪCHϭCH2), 5.16 (br. s,
1 H, H-1b), 4.99 (d, J5,4 ϭ 2.0 Hz, 1 H, H-5b), 4.96 (br. s, 1 H, H-
2b), 4.82 (d, J1,2 ϭ 3.6 Hz, 1 H, H-1a), 4.72 (d, Jgem ϭ 11.2 Hz, 1
1 H, H-1a), 4.81 (d, Jgem ϭ 11.2 Hz, 1 H, CH2Ph), 4.74 (d, Jgem
ϭ
11.2 Hz, 1 H, CH2Ph), 4.71 (d, Jgem ϭ 11.2 Hz, 1 H, CH2Ph), 4.70
(d, Jgem ϭ 11.2 Hz, 1 H, CH2Ph), 4.58 (d, Jgem ϭ 12.0 Hz, 1 H,
CH2Ph), 4.56 (d, Jgem ϭ 10.8 Hz, 1 H, CH2Ph), 4.33Ϫ4.37 (m, 3
H, H-1b, CH2Ph, H-6b), 4.12 (ddt, Jgem ϭ 13.0, Ja,b ϭ 5.0, Ja,cc
Ja,ct ϭ 1.5 Hz, 1 H, CH2ϪCHϭCH2), 4.05 (dd, J6,5 ϭ 2.0, J6,6Ј
12.5 Hz, 1 H, H-6Јb), 4.01 (t, J4,3 ϭ J4,5 ϭ 10.0 Hz, 1 H, H-4a),
3.98 (ddt, Jgem ϭ 13.0, Ja,b ϭ 6.0, Ja,cc ϭ Ja,ct ϭ 1.5 Hz, 1 H,
CH2ϪCHϭCH2), 3.84 (dd, J6,5 ϭ 2.0, J6,6Ј ϭ 10.8 Hz, 1 H, H-6a),
3.82 (dd, J3,2 ϭ 8.8, J3,4 ϭ 10.0 Hz, 1 H, H-3a), 3.61 (dt, J5,6
J5,6 ϭ 2.0, J5,4 ϭ 10.0 Hz, 1 H, H-5a), 3.41 (dd, J6,5 ϭ 1.6, J6,6Ј
ϭ
ϭ
ϭ
ϭ
10.8 Hz, 1 H, H-6Јa), 3.35Ϫ3.29 (m, 2 H, H-4b, H-2a), 3.24 (dd,
2,3 ϭ 8.8, J2,1 ϭ 7.6 Hz, 1 H, H-2b), 3.18 (dd, J3,4 ϭ J3,2 ϭ 8.8 Hz,
J
1 H, H-3b), 3.09Ϫ3.05 (ddd, J5,6 ϭ 2.0, J5,6Ј ϭ 4.0, J5,4 ϭ 9.6 Hz,
1 H, H-5b), 2.60 (br. s, 1 H, OH), 1.90 (s, 3 H, OCOCH3) ppm.
13C NMR (100 MHz, CDCl3): δ ϭ 169.0 (CϭO), 138.0Ϫ137.5
(Cquaternary,arom.), 133.1 (CH2ϪCHϭCH2), 128.2Ϫ127.3 (Ph,
CH3OϪPh), 117.7 (CH2ϪCHϭCH2), 102.3 (C-1b), 96.5 (C-1a),
83.5 (C-3b), 81.9 (C-2b), 77.9 (C-3a), 76.2 (C-4a), 75.2, 74.7, 74.6,
73.3 (CH2Ph), 73.1 (C-5b), 70.3 (C-5a), 69.8 (C-4b), 68.3
(CH2ϪCHϭCH2), 67.2 (C-6a), 62.9 (C-6b), 62.4 (C-2a), 20.5 (OC-
OCH3) ppm. C45H51N3O11 (809.9): calcd. C 66.73, H 6.35, N 5.17,
O 21.73; found C 66.49, H 6.44, N 4.87, O 21.73. ESI-MS calcd.
for C45H51N3NaO11 [M ϩ Na]: m/z ϭ 832.3; found. 832.5.
H, CH2Ph), 4.67 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.65 (d, Jgem
11.2 Hz, 1 H, CH2Ph), 4.52 (s, 2 H, CH2Ph), 4.45 (d, Jgem
ϭ
ϭ
12.0 Hz, 1 H, CH2Ph), 4.33 (td, J2,1 ϭ 3.6, J2NH ϭ J2,3 ϭ 10.0 Hz,
1 H, H-2a), 4.14 (br. dd, Jgem ϭ 13.0, Ja,b ϭ 5.0 Hz, 1 H,
CH2ϪCHϭCH2), 4.06 (t, J4,3 ϭ J4,5 ϭ 9.6 Hz, 1 H, H-4a), 3.96
(br. s, 1 H, H-4b), 3.94 (br. dd, Jgem ϭ 13.0, Ja,b ϭ 6.0 Hz, 1 H,
CH2ϪCHϭCH2), 3.79Ϫ3.75 (m, 2 H, H-5a, H-6a), 3.72 (t, J3,2
ഠ
J3,4 ϭ 2.8 Hz, 1 H, H-3b), 3.67Ϫ3.62 (m, 2 H, H-3a, H-6Јa), 3.45
(s, 3 H, COOCH3), 2.87 (br. s, 1 H, OH), 2.05 (s, 3 H, OCOCH3),
1.75 (s, 3 H, CH3CONH) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ
169.4, 169.3 169.0 (CϭO), 138.0Ϫ137.2 (Cquaternary,arom.), 133.4
(CH2ϪCHϭCH2), 128.2Ϫ127.0 (Ph, CH3OϪPh), 117.3
(CH2ϪCHϭCH2), 97.6 (C-1b), 96.5 (C-1a), 78.1 (C-3a), 74.9 (C-
4a), 74.5 (C-3b), 73.0, 72.0, (CH2Ph), 71.0 (C-5b), 68.2 (C-4b), 68.1
(CH2ϪCHϭCH2), 68.0 (C-6a), 67.7 (C-5b), 67.1 (C-2b), 51.8 (C-
2a), 51.7 (COOCH3), 23.0 (OCOCH3), 20.7 (CH3CONH) ppm.
C41H49NO13 (763.8): calcd. C 64.47, H 6.47, N 1.83; found C 64.19,
H 6.44, N 1.73. ESI-MS calcd. for C41H49NNaO13 [M ϩ Na]:
m/z ϭ 786.3; found 786.3.
Allyl
(6-O-Acetyl-2,3-di-O-benzyl-β-
D-glucopyranosyl)-(1Ǟ4)-2-
acetamido-3,6-di-O-benzyl-2-deoxy-α-
D-glucopyranoside (8): Com-
pound 7 (200 mg, 0.246 mmol) was treated as described for com-
pound 6, giving, after flash chromatography (toluene/acetone, 3:1),
184 mg of 8 (94%). TLC (toluene/acetone, 2:1): Rf ϭ 0.45. [α]3D0
ϭ
ϩ31 (CHCl3, c ϭ 1.0). IR (thin film): ν˜ ϭ 3260 (3550Ϫ3150, νOϪH
and νNϪH), 3088, 3061, 3029, 3009 (νCϪHarom), 2938, 2918, 2899,
2859 (νCϪHaliph), 1741 (νCϭO OAc), 1655 (νCϭO NHAc), 1576,
1562, 1536, 1496, 1453, 1364, 1313, 1278, 1259, 1239, 1207, 1169,
1143, 1117, 1091, 1052, 1028. 1H NMR (400 MHz, CDCl3): δ ϭ Allyl (Methyl 2,3-Di-O-benzyl-β-
D-glucopyranosyluronate)-(1Ǟ4)-
7.28Ϫ7.16 (m, 20 H, Ph), 5.82 (dddd, J ϭ 17.0, 10.5, 6.0, 5.0 Hz,
2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside (11): Com-
1 H, CH2ϪCHϭCH2), 5.22 (d, JNH,2 ϭ 7.2 Hz, 1 H, NH), 5.20 pound 3 (285 mg, 0.31 mmol) was treated as described for the prep-
(dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 5.14 (dq, J ϭ 10.5, aration of compound 2. Flash chromatography (petroleum ether/
1.5 Hz, 1 H, CH2ϪCHϭCH2), 4.84 (d, J1,2 ϭ 3.6 Hz, 1 H, H-1a),
EtOAc, 4:1 to 1:1 ) gave 11 (235 mg, 96%) as a colourless oil. TLC
4.82 (d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.80 (d, Jgem ϭ 10.0 Hz, 1 (petroleum ether/EtOAc, 2:1): Rf ϭ 0.4. [α]3D0 ϭ ϩ45 (CHCl3, c ϭ
H, CH2Ph), 4.77Ϫ4.68 (m, 3 H, CH2Ph), 4.59 (d, Jgem ϭ 12.0 Hz, 1.0). IR (thin film): ν˜ ϭ 3466 (3550Ϫ3150, νOϪH), 3086, 3063, 3030,
1 H, CH2Ph), 4.53 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.37Ϫ4.33 3010 (νCϪHarom), 2955, 2922, 2851 (νCϪHaliph), 2108 (νN3), 1751
(m, 2 H, CH2Ph, H-1b), 4.27 (dd, J6,5 ϭ 4.4, J6,6Ј ϭ 12.0 Hz, 1 H,
H-6b), 4.18 (ddd, J2,1 ϭ 3.6, J2,NH ϭ 7.2, J2,3 ϭ 9.0 Hz, 1 H, H-
(νCϭO COOMe), 1607, 1584, 1497, 1453, 1360, 1261, 1245, 1210,
1161, 1118, 1066, 1027. 1H NMR (400 MHz, CDCl3): δ ϭ
2a), 4.10Ϫ4.02 (m, 3 H, CH2ϪCHϭCH2, H-6Јb, H-4a), 3.89 (ddt, 7.45Ϫ7.20 (m, 20 H, Ph), 5.98 (dddd, J ϭ 17.0, 10.5, 6.0, 5.0 Hz,
Jgem ϭ 13.0, Ja, b ϭ 6.0, Ja,cc ϭ Ja,ct ϭ 1.5 Hz, 1 H, CH2ϪCHϭ 1 H, CH2ϪCHϭCH2), 5.40 (dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭ
CH2), 3.84 (dd, J6,5 ϭ 3.2, J6,6Ј ϭ 10.8 Hz, 1 H, H-6a), 3.61 (br. d, CH2), 5.28 (dq, J ϭ 10.5, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 5.13 (d,
J5,4 ϭ 10.0, 1 H, H-5a), 3.57 (dd, J3,4 ϭ 10.8, J3,2 ϭ 9.0 Hz, 1 H, Jgem ϭ 10.5 Hz, 1 H, CH2Ph), 4.96 (d, J1,2 ϭ 3.6 Hz, 1 H, H-1a),
H-3a), 3.48 (dd, J6,5 ϭ 1.6, J6,6Ј ϭ 10.8 Hz, 1 H, H-6Јa), 3.36Ϫ3.20
4.88 (s, 2 H, CH2Ph), 4.69 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.68
(m, 2 H, H-4b, H-3b, H-2b), 3.13 (ddd, J5,6Ј ϭ 2.2, J5,6 ϭ 4.4, (d, Jgem ϭ 10.5 Hz, 1 H, CH2Ph), 4.69 (m, 2 H, CH2Ph), 4.42 (d,
2114
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 2107Ϫ2117

Synthesis of Glycosaminoglycan Oligosaccharides
FULL PAPER
J1,2 ϭ 7.6 Hz, 1 H, H-1b), 4.37 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), Data for 16β: TLC (toluene/EtOAc, 6:1): Rf β 0.34. [α]3D0 ϭ ϩ3
4.24Ϫ4.06 (m, 3 H, H-4a, CH2ϪCHϭCH2), 3.94Ϫ3.83 (m, 3 H, (CHCl3, c ϭ 1.0). 1H NMR (400 MHz, CDCl3): δ ϭ 7.41Ϫ7.15
H-6a, H-3a, H-4b), 3.70 (dd, J5,6 ϭ J5,6 ϭ 1.5, J5,4 ϭ 9.6 Hz, 1 H,
H-5a), 3.59 (s, 3 H, COOCH3), 3.57 (d, J5,4 ϭ 8.5 Hz, 1 H, H-5b), 8.8 Hz, 2 H, CH3OϪPh), 5.89 (dddd, J ϭ 17.0, 10.5, 6.0, 5.0 Hz,
3.50 (dd, J6Ј,5 ϭ 1.5, J6,6Ј ϭ 10.8 Hz, 1 H, H-6Јa), 3.42 (dd, J2,1 1 H, CH2ϪCHϭCH2), 5.31 (dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭ
3.6, J2,3 ϭ 10.4 Hz, 1 H, H-2a), 3.37 (dd, J2,1 ϭ 7.6, J2,3 ϭ 9.6 Hz, CH2), 5.19 (dq, J ϭ 10.5, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 5.02 (d,
1 H, H-2b), 3.32 (t, J3,4 ϭ J3,2 ϭ 9.6 Hz, 1 H, H-3b), 2.90 (br. s, 1 Jgem ϭ 11.5 Hz, 1 H, CH2Ph), 5.01 (d, Jgem ϭ 10.0 Hz, 1 H,
H, OH) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 169.4 (CϭO), CH2Ph), 4.99 (d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.87 (d, J1,2
138.3Ϫ137.5 (Cquaternary,arom.), 133.1 (CH2ϪCHϭCH2), 3.6 Hz, 1 H, H-1a), 4.79 (d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.73 (d,
(m, 36 H, Ph), 7.15Ϫ7.06 (m, 6 H, Ph, CH3OϪPh), 6.78 (d, Jortho ϭ
ϭ
ϭ
128.3Ϫ127.1 (Ph, CH3OϪPh), 117.7 (CH2ϪCHϭCH2), 102.4 (C- Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.68Ϫ4.58 (m, 6 H, CH2Ph), 4.57
1b), 96.6 (C-1a), 83.1 (C-3b), 81.4 (C-2b), 77.9 (C-3a), 76.8 (C-5a), (d, Jgem ϭ 10.5 Hz, 1 H, CH2Ph), 4.53 (d, Jgem ϭ 10.5 Hz, 1 H,
75.0, 74.9, 74.7 (CH2Ph), 73.7 (C-5b), 73.1 (CH2Ph), 71.4 (C-4b),
CH2Ph), 4.42 (d, J1,2 ϭ 7.6 Hz, 1 H, H-1c), 4.35Ϫ4.23 (m, 4 H,
70.3 (C-4a), 68.4 (CH2ϪCHϭCH2), 67.2 (C-6a), 62.4 (C-2a), 52.2 CH2Ph, H-1d, H-1b), 4.12 (br. dd, Jgem ϭ 13.0, Ja,b ϭ 5.0 Hz, 1 H,
(COOCH3) ppm. C44H49N3O11 (795.9): calcd. C 66.39, H 6.21, N CH2ϪCHϭCH2), 4.08 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.08Ϫ3.95
5.28, O 22.11; found C 66.51, H 6.29, N 5.06, O 21.88.
(m, 4 H, CH2ϪCHϭCH2, H-4a, H-4c, H-4b), 3.83 (dd, J6,5 ϭ 2.5,
J6,6Ј ϭ 11.0 Hz, 1 H, H-6a), 3.79 (dd, J ϭ 9.0, J6,5 ϭ 9.0 Hz, 1 H,
H-3a), 3.84Ϫ3.73 (m, 5 H, H-3c, H-4d, PhϪOCH3), 3.67 (dd,
J6,5 ϭ 3.0, J6,6Ј ϭ 10.5 Hz, 1 H, H-6c), 3.649 (s, 3 H, COOCH3),
3.64 (d, J5,4 ϭ 3.5 Hz, 1 H, H-5d), 3.61 (d, J5,4 ϭ 3.5 Hz, 1 H, H-
5d), 3.56 (br. d, J5,4 ϭ 9.0 Hz, 1 H, H-5a), 3.53 (s, 3 H, COOCH3),
Allyl [Methyl 2,3-di-O-benzyl-4-O-(4-methoxyphenyl)-β-
D
-gluco-
pyranosyluronate]-(1Ǟ4)-(2-azido-3,6-di-O-benzyl-2-deoxy-
pyranosyl)-(1Ǟ4)-O-(methyl 2,3-di-O-benzyl-β-
ate)-(1Ǟ4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-
D-gluco-
D-glucopyranosyluron
D
-gluco-
pyranoside (16): A solution of TMSOTf (10%, 76 µL of a 0.55 m
in CH2Cl2) was added to a cooled solution (Ϫ30 °C) of 11 (240 mg,
0.301 mmol) and 1 (431 mg, 0.422 mmol, 1.4 equiv.) in THF/Et2O,
9:1 (500 µL). The solution was stirred for 2 h at this temperature,
and then neutralised with Et3N (100 µL). The solvent was evapo-
rated and the residue was purified by flash chromatography (tolu-
ene/EtOAc, 20:1 to 6:1) to give 413 mg 16α (83% isolated yield)
and 36 mg 16β (7% isolated yield). Data for 16 α: TLC (toluene/
EtOAc, 6:1 ): Rf α 0.42. [α]3D0 ϭ ϩ39 (CHCl3, c ϭ 1.0). IR (thin
film): ν˜ ϭ 3084, 3063, 3030, 3004 (νCϪHarom), 2954, 2923, 2865,
2852 (νCϪHaliph), 2109 (νN3), 1750 (νCϭO COOMe), 1610, 1585,
1513, 1497, 1453, 1438, 1361, 1249, 1216, 1141, 1090, 1064, 1027.
1H NMR (400 MHz, CDCl3): δ ϭ 7.50Ϫ7.21 (m, 40 H, Ph), 7.19
(d, Jortho ϭ 8.4 Hz, 2 H, CH3OϪPh), 6.88 (d, Jortho ϭ 8.4 Hz, 2 H,
CH3OϪPh), 5.97 (dddd, J ϭ 17.0, 10.5, 6.0, 5.0 Hz, 1 H,
CH2ϪCHϭCH2), 5.57 (d, J1,2 ϭ 4.0 Hz, 1 H, H-1c), 5.39 (dq, J ϭ
3.45 (br. d, J6,6Ј ϭ 10.5 Hz, 1 H, H-6Јc), 3.38 (t, J3,2 ϭ J3,4
ϭ
9.0 Hz, 1 H, H-3d), 3.34Ϫ3.21 (m, 6 H, H-6Јa, H-2a, H-2c, H-3b,
H-2d, H-2b), 3.16 (br. d, J5,4 ϭ 9.0 Hz, 1 H, H-5c) ppm. 13C NMR
(100 MHz, CDCl3): δ ϭ 168.7, 167.4 (CϭO), 159.1 (CϪOCH3,
MeOϪPh), 138.8Ϫ137.3 (Cquaternary,arom.), 133.0 (CH2ϪCHϭCH2),
129.8Ϫ127.0 (Ph, CH3OϪPh), 117.8 (CH2ϪCHϭCH2), 113.5
(CH3OϪPh), 102.4 (C-1d), 102.3 (C-1b), 101.7 (C-1c), 96.5 (C-1a),
83.6 (C-3d, C-3b), 81.9 (C-2d), 81.7 (C-2b), 80.8, 79.2, 78.9, 77.9,
76.1, 75.4, 75.1, 74.9, 74.7, 74.4, 74.1; 73.7, 73.2 (C-5b, C-5d), 72.7,
70.3; 68.4 (CH2ϪCHϭCH2), 67.2; 66.9, 66.1 (C-6c, C-6a), 62.3 (C-
2c, C-2a), 55.0 (CH3OϪPh), 52.4, 52.1 (COOCH3) ppm.
C93H100O22N6 (1653.8): calcd. C 67.54, H 6.09, N 5.08; found C
67.48, H 6.21, N 4.85.
[Methyl 2,3-di-O-benzyl-4-O-(4-methoxybenzyl)-β-
D
-glucopyranos-
-glucopyranose
trichloroacetamide (12): TLC β anomer (toluene/acetone, 7:1): Rf ϭ
17.0, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 5.28 (dq, J ϭ 10.5, 1.5 Hz, 1 yluronate]-(1Ǟ4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-
H, CH2ϪCHϭCH2), 5.19 (d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 5.12
D
(d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 4.84 (d, J1,2 ϭ 4.0 Hz, 1 H, H- 0.33. IR (thin film): (ν˜ ϭ 3300 (3450Ϫ3250, νNϪH), 3084, 3064,
1a), 4.81 (d, Jgem ϭ 11.5 Hz, 1 H, CH2Ph), 4.76Ϫ4.37 (m, 11 H, 3031, 3004 (νCϪHarom), 2957, 2924, 2865, 2852 (νCϪHaliph), 2114
CH2Ph), 4.52 (d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.46 (d, Jgem
ϭ
(νN3), 1753 (νCϭO COOMe), 1728 (νCϭO CONHCCl3), 1612, 1585,
11.0 Hz, 1 H, CH2Ph), 4.41 (d, J1,2 ϭ 7.5 Hz, 1 H, H-1d), 4.35 (d,
1514, 1496, 1454, 1439, 1360, 1282, 1249, 1216, 1176, 1138, 1089,
Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.27 (d, Jgem ϭ 12.0 Hz, 1 H, 1063, 1030. 1H NMR (400 MHz, CDCl3) (β anomer): δ ϭ
CH2Ph), 4.25 (d, J1,2 ϭ 8.0 Hz, 1 H, H-1b), 4.21 (br. dd, Jgem
13.0, Ja,b ϭ 5.0 Hz, 1 H, CH2ϪCHϭCH2), 4.14Ϫ4.01 (m, 4 H, 8.8 Hz, 2 H, CH3OϪPh), 7.04 (d, J1,NH ϭ 9.5 Hz, 1 H, NH), 6.77
CH2ϪCHϭCH2, H-4a, H-4c, H-4b), 3.98 (dd, J6,5 ϭ 1.6, J6,6Ј ϭ (d, Jortho ϭ 8.8 Hz, 2 H, CH3OϪPh), 5.04 (d, Jgem ϭ 10.4 Hz, 1 H,
11.2 Hz, 1 H, H-6a), 3.90Ϫ3.80 (m, 7 H, H-6c, H-4d, H-3a, CH2Ph), 4.84 (t, J1,NH ϭ J1,2 ϭ 9.5 Hz, H-1a), 4.79 (d, Jgem
ϭ
7.34Ϫ7.40 (m, 2 H, Ph), 7.38Ϫ7.15 (m, 18 H, Ph), 7.09 (d, Jortho ϭ
ϭ
PhϪOCH3, H-3c), 3.77 (d, J5,4 ϭ 9.6 Hz, 1 H, H-5b), 3.73 (d,
J5,4 ϭ 10.0 Hz, 1 H, H-5d), 3.65 (br. d, J5,4 ϭ 10.5 Hz, 1 H, H-
11.0 Hz, 1 H, CH2Ph), 4.75 (d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.70
(d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.65 (d, Jgem ϭ 11.0 Hz, 1 H,
5a), 3.61 (s, 3 H, COOCH3), 3.58Ϫ3.38 (m, 7 H, H-6Јa, H-5c, H- CH2Ph), 4.63 (d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 4.59 (d, Jgem
3b, H-3d, H-6Јc, H-2b, H-2d), 3.38 (dd, J2,1 ϭ 4.0, J2,3 ϭ 10.4 Hz, 10.4 Hz, 1 H, CH2Ph), 4.52 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.46
1 H, H-2a), 3.37 (dd, J2,1 ϭ 4.0, J2,3 ϭ 10.4 Hz, 1 H, H-2c), 3.18 (d, Jgem ϭ 10.4 Hz, 1 H, CH2Ph), 4.38 (d, J1,2 ϭ 7.2 Hz, 1 H, H-
(s, 3 H, COOCH3) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 168.4, 1b), 4.31 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.04 (t, J4,3 ϭ J4,5
167.9 (CϭO), 159.1 (CϪOCH3, MeOϪPh), 138.1Ϫ137.3 (Cquaterna- 9.5 Hz, 1 H, H-4a), 3.79 (dd, J6,5 ϭ 2.4, J6,6Ј ϭ 11.2 Hz, 1 H, H-
ry,arom.), 133.1 (CH2ϪCHϭCH2), 129.9Ϫ126.9 (Ph, CH3OϪPh), 6a), 3.74 (t, J4,3 ϭ J4,5 ϭ 10.0 Hz, 1 H, H-4b), 3.73 (s, 3 H,
117.7 (CH2ϪCHϭCH2), 113.54 (CH3OϪPh); 102.5 (C-1d); 102.3 PhϪOCH3), 3.66 (d, J5,4 ϭ 10.0 Hz, 1 H, H-5b), 3.54 (s, 3 H, CO-
ϭ
ϭ
(C-1b); 97.2 (C-1c), 96.6 (C-1a), 83.8, 83.7 (C-3d, C-3b), 82.2 (C- OCH3), 3.51Ϫ3.44 (m, 2 H, H-6Јa, H-3a), 3.39Ϫ3.30 (m, 3 H, H-
2d), 81.7 (C-2b), 79.0 (C-4d), 77.9 (C-3a), 77.0 (C-3c), 76.9, 76.5 3b, H-2a, H-2b), 3.26 (br. d, J5,4 ϭ 9.5 Hz, 1 H, H-5a) ppm. 13C
(C-4a, C-4c), 75.3, 75.0, 74.9, 74.7, 74.6, 74.5, 74.3, 74.2 (CH2Ph),
74.1 (C-4b), 73.4, 73.3 (C-5b, C-5d), 70.6 (C-5c), 70.3 (C-5a), 68.4
(CH2ϪCHϭCH2), 66.9, 66.8 (C-6c, C-6a), 62.4 (C-2c), 62.3 (C-
2a), 55.0 (CH3O-Ph), 52.0, 51.7 (COOCH3) ppm. C93H100O22N6
(1653.8): calcd. C 67.54, H 6.09, N 5.08; found C 67.39, H 6.12,
N, 4.95.
NMR (100 MHz, CDCl3): δ ϭ 168.4 (CϭO), 161.7 (NHϪCϭO),
159.0 (CϪOMe, MeOPh), 138.0Ϫ137.1 (Cquaternary,arom.),
129.8Ϫ127.4 (Ph, CH3OϪPh), 113.8 (MeOϪPh), 102.3 (C-1b),
92.0 (CCl3), 83.6, 81.8, 81.6 (C-3a), 75.7 (C-4b), 75.3, 75.0, 74.4,
74.2, 73.1, 67.0 (C-6a), 65.2 (C-2a), 55.0 (CH3OϪPh), 52.1 (CO-
OCH3) ppm. ESI-MS calcd. for C51H53N4O12Cl3 [M ϩ Na]: m/z ϭ
Eur. J. Org. Chem. 2004, 2107Ϫ2117
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2115

´
R. Lucas, D. Hamza, A. Lubineau, D Bonnaffe
FULL PAPER
1041.3/1403.3; found 1041.3/1043.3. C51H53Cl3N4O12:
1020.3 g/mol.
M
ϭ
H, CH2Ph), 4.42 (d, J1,2 ϭ 8.0 Hz, 1 H, H-1c), 4.30 (d, Jgem ϭ
12.0 Hz, 1 H, CH2Ph), 4.29 (d, Jgem ϭ 12.0 Hz, 1 H, CH2Ph), 4.28
(d, Jgem ϭ 11.0 Hz, 1 H, CH2Ph), 4.25 (d, J1,2 ϭ 7.5 Hz, 1 H, H-
1d), 4.19Ϫ4.13 (m, 3 H, H-1b, 2CH2Ph), 4.11 (ddt, Jgem ϭ 13.0,
Ja,b ϭ 5.0, Ja,b ϭ 1.5 Hz, 1 H, CH2ϪCHϭCH2), 4.01Ϫ3.93 (m, 3
Allyl [Methyl 2,3-di-O-benzyl-4-O-(4-methoxyphenyl)-β-D-gluco-
pyranosyluronate]-(1Ǟ4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α,β-
glucopyranosyl)-(1Ǟ4)-O-(6-O-acetyl-2,3-di-O-benzyl-β-
D
-
-
D
H, H-4b, H-4a, CH2ϪCHϭCH2), 3.82 (dd, J6,6Ј ϭ 3.0, J6,5
ϭ
glucopyranosyl)-(1Ǟ4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-gluco-
12.8 Hz, 1 H, H-6a), 3.79 (dd, J3,4 ϭ 8.5, J3,4 ϭ 10.0 Hz, 1 H, H-
3a), 3.75 (dd, J6,6Ј ϭ 3.5, J6,5 ϭ 11.0 Hz, 1 H, H-6c), 3.73Ϫ3.71
(m, 5 H, H-3c, H-4d, Ph-OCH3), 3.67 (dd, J4,5 ϭ 9.6, J4,3 ϭ 8.8 Hz,
1 H, H-4c), 3.62 (d, J5,4 ϭ 9.6 Hz, 1 H, H-5d), 3.59Ϫ3.56 (m, 2 H,
H-5a, H-6a), 3.51 (s, 3 H, COOCH3), 3.40Ϫ3.36 (m, 3 H, H-6Јa,
H-6Јb, H-6Јc), 3.32Ϫ3.28 (m, 5 H, H-2c, H-2a, H-5c, H-3b, H-2b),
3.26Ϫ3.22 (m, 1 H, H-5c), 3.22 (dd, J2,1 ϭ 7.5, J2,3 ϭ 9.0 Hz, 1 H,
H-2d), 3.03 (ddd, J5,6 ϭ 1.5, J5,6Ј ϭ 3.0, J5,4 ϭ 10.0, 1 H, H-5b),
1.85 (s, 3 H, OCOCH3) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ
170.3, 168.4 (CϭO), 159.0 (CϪOCH3, MeOϪPh), 138.4Ϫ137.0
(Cquaternary,arom.), 133.1 (CH2ϪCHϭCH2), 129.3Ϫ126.8 (Ph,
CH3OϪPh), 117.7 (CH2ϪCHϭCH2), 113.5 (CH3OϪPh), 102.4 (C-
1d), 102.0 (C-1b), 101.3 (C-1c), 96.5 (C-1a), 83.6 (C-3b), 82.7 (C-
3d), 81.9, 81.8 (C-2b, C-2d), 841.2, 78.9, 77.7, 77.0, 76.2, 75.3, 75.0,
74.8, 74.3, 73.2, 72.8, 72.3, 70.3; 68.3 (CH2ϪCHϭCH2), 67.0 (C-
6c, C-6a), 66.2 (C-6b), 62.3 (C-2a, C-2c), 55.0 (CH3OϪPh), 52.1
(COOCH3), 20.4 (OCOCH3) ppm. C94H102N6 O22 (1667.8): calcd.
C 67.69, H 6.16, N 5.04; found, C 67.67, H 6.51, N 4.42. ESI-MS
calcd. for C94H102N6NaO22 [M ϩ Na]: m/z ϭ 1689.7 (94%), 1690.7
(100%); found 1689.7 (93%), 1690.7 (100%).
pyranoside (17): A solution of TMSOTf (6%, 10 µL of a 0.55 m
solution in CH2Cl2) was added to a cooled solution (Ϫ30 °C) of 7
(65 mg, 0.082 mmol) and 1 (100 mg, 0.098 mmol, 1.2 equiv.) in
CH2Cl2 (200 µL). The solution was stirred for 2 h at this tempera-
ture, and then neutralised with Et3N. The solvent was evaporated
and the crude reaction mixture was passed through a gel-filtration
chromatography column (LH-20) with CH2Cl2/MeOH, 1:1 as elu-
ent to afford a 50:50 α/β mixture of the corresponding tetrasacchar-
ides. Flash column chromatography (toluene/EtOAc, 9:1 to 6:1) af-
forded 28 mg 17α and 28 mg 17β (42% overall). Data for 17α: TLC
1
(toluene/EtOAc, 6:1): Rf α 0.66. [α]3D0 ϭ ϩ26 (CHCl3, c ϭ 1.0). H
NMR (400 MHz, CDCl3): δ ϭ 7.40Ϫ7.35(m, 2 H, Ph), 7.34Ϫ7.12
(m, 38 H, Ph), 7.07 (d, Jortho ϭ 8.4 Hz, 2 H, CH3OϪPh), 6.76 (d,
Jortho ϭ 8.4 Hz, 2 H, CH3OϪPh), 5.86 (dddd, J ϭ 17.0, 10.5, 6.0,
5.0 Hz, 1 H, CH2ϪCHϭCH2), 5.53 (d, J1,2 ϭ 4.0 Hz, 1 H, H-1c),
5.28 (dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 5.17 (br. d, J ϭ
10.5 Hz, 1 H, CH2ϪCHϭCH2), 5.08 (d, Jgem ϭ 10.8 Hz, 1 H,
CH2Ph), 5.01 (d, Jgem ϭ 10.8 Hz, 1 H, CH2Ph), 4.92 (d, Jgem
ϭ
10.8 Hz, 1 H, CH2Ph), 4.86 (d, J1,2 ϭ 3.5 Hz, 1 H, H-1a),
4.79Ϫ4.71 (m, 6 H, CH2Ph), 4.70Ϫ4.53 (m, 7 H, CH2Ph), 4.48 (d,
Jgem ϭ 12.5 Hz, 1 H, CH2Ph), 4.45 (d, Jgem ϭ 12.5 Hz, 1 H,
CH2Ph), 4.39 (d, J1,2 ϭ 7.2 Hz, 1 H, H-1d), 4.35 (d, Jgem ϭ 12.8 Hz,
1 H, CH2Ph), 4.32 (d, Jgem ϭ 12.8 Hz, 1 H, CH2Ph), 4.29 (d, J1,2 ϭ
7.2 Hz, 1 H, H-1b), 4.20 (dd, J6,5 ϭ 1.6 Hz J6,6Ј ϭ 10.0 Hz, 1 H,
H-6b), 4.10 (br. dd, Jgem ϭ 13.0, Ja,b ϭ 5.0 Hz, 1 H, CH2ϪCHϭ
Acknowledgments
We thank Pr. T. Feizi for stimulating discussions on 10E4 epitope,
`
the CNRS, the Ministere de l’Education Nationale et de la Recher-
che, the National Agency for AIDS research (ANRS), Ensemble
Contre le SIDA-Sidaction (R. L.) and the Medical Research Coun-
cil (D. H.) for funding.
CH2), 4.03Ϫ3.94 (m,
3 H, H-4c, H-4a, CH2ϪCHϭCH2),
3.85Ϫ3.74 (m, 6 H, H-6a, H-6Јb, H-6c, H-4d, H-3a, H-3c), 3.72 (s,
3 H, PhϪOCH3), 3.65Ϫ3.54 (m, 4 H, H-5d, H-5c, H-5a, H-4b),
3.50 (s, 3 H, COOCH3), 3.46 (t, J3,2 ϭ J3,4 ϭ 9.0 Hz, 1 H, H-3b),
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J. D. Esko, U. Lindhal, J. Clin. Invest. 2001, 108, 169Ϫ173.
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ϭ
[2]
3.5, J2,3 ϭ 10.0 Hz, 1 H, H-2c), 3.29 (dd, J2,1 ϭ 7.2, J2,3 ϭ 9.0 Hz,
1 H, H-2b), 3.24 (dd, J2,1 ϭ 4.0, J2,3 ϭ 10.5 Hz, 1 H, H-2c),
3.27Ϫ3.21 (m, 1 H, H-5b), 1.63 (s, 3 H, OCOCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ ϭ 170.1, 168.4 (CϭO), 159.0 (CϪOCH3,
MeOϪPh), 138.4Ϫ137.2 (Cquaternary,arom.), 133.0 (CH2ϪCHϭCH2),
129.8Ϫ127.1 (Ph, CH3OϪPh), 117.7 (CH2ϪCHϭCH2), 113.54
(CH3OϪPh), 102.7 (C-1d), 101.8 (C-1b), 97.7 (C-1c), 96.5 (C-1a),
84.2 (C-3b), 83.7 (C-3d), 82.8 (C-2b), 81.6 (C-2d), 78.9 (C-4d), 77.6,
77.5 (C-3a, C-3c), 75.3Ϫ73.0 (CH2Ph, H-4a, H-4c), 71.9 (C-5b),
71.2 (C-4b), 70.2 (C-5a), 68.3 (CH2ϪCHϭCH2), 67.0 (C-6c), 66.8
(C-6a), 63.0 (C-6b), 62.4 (C-2a), 62.2 (C-2c), 55.0 (CH3OϪPh),
52.0 (COOCH3), 20.2 (OCOCH3) ppm. C94H102N6O22 (1667.8):
calcd. C 67.69, H 6.16, N 5.04; found C 67.11, H 6.22, N 4.71;.
ESI-MS calcd. for C94H102N6NaO22 [M ϩ Na]: m/z ϭ 1689.7
(94%), 1690.7 (100%); found 1689.8 (90%), 1690.8 (100%).
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8.8 Hz, 2 H, CH3OϪPh), 6.77 (d, Jortho ϭ 8.8 Hz, 2 H, CH3OϪPh),
5.87 (dddd, J ϭ 17.0, 10.5, 6.0, 5.0 Hz, 1 H, CH2ϪCHϭCH2), 5.29
(dq, J ϭ 17.0, 1.5 Hz, 1 H, CH2ϪCHϭCH2), 5.17 (dq, J ϭ 10.5,
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Jgem ϭ 11.2 Hz, 1 H, CH2Ph), 4.72 (d, Jgem ϭ 11.2 Hz, 1 H,
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2116
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 2107Ϫ2117

Synthesis of Glycosaminoglycan Oligosaccharides
FULL PAPER
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