Preparation of synthetic glycoconjugates as potential vaccines against Shigella flexneri serotype 2a disease

Article abstract of DOI:10.1039/b400986j

The synthesis of three neoglycopeptides incorporating carbohydrate haptens, differing in length, covalently linked to a non natural universal T helper peptide is disclosed. They were synthesized according to a blockwise strategy based on the condensation

Full text of DOI:10.1039/b400986j

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Preparation of synthetic glycoconjugates as potential vaccines
against Shigella flexneri serotype 2a disease†‡
Karen Wright, Catherine Guerreiro, Isabelle Laurent, Françoise Baleux and
Laurence A. Mulard*
Unité de Chimie Organique, URA CNRS 2128, Institut Pasteur, 28 rue du Dr Roux, 75 724,
Paris, Cedex 15, France. E-mail: lmulard@pasteur.fr
Received 21st January 2004, Accepted 26th March 2004
First published as an Advance Article on the web 26th April 2004
The synthesis of three neoglycopeptides incorporating carbohydrate haptens, differing in length, covalently linked to
a non natural universal T helper peptide is disclosed. They were synthesized according to a blockwise strategy based
on the condensation of appropriate di-, tri-, and tetrasaccharide trichloroacetimidate donors onto an azidoethyl
2-acetamido-2-deoxy-β--glucopyranoside acceptor. Use of thiol–maleimide coupling chemistry allowed
site-selective efficient conjugation.
onstrate the efficacy of a vaccine made of the corresponding
Introduction
detoxified LPS covalently linked to the recombinant exoprotein
Since the discovery of Shigella dysenteriae type 1 (Shiga’s
A of Pseudomonas aeruginosa.20 Conversion of polysaccharide
bacillus) more than a century ago,2 shigellosis or bacillary
T-independent antigens to T-dependent ones through their
dysentery has been known as a serious infectious disease,
covalent attachment to a carrier protein has had a tremendous
occurring only in humans.3 In a recent survey of the literature
impact in the field of bacterial vaccines. Several such neoglyco-
published between 1966 and 1997,4 the number of episodes
conjugate vaccines are currently in use against Haemophilus
of shigellosis occurring annually throughout the world was
estimated to be 164.7 million, of which 163.2 million were in
influenzae b,21 Neisseria meningitidis,22 and Streptococcus
pneumoniae.23 These polysaccharide–protein conjugate vaccines
developing countries. Up to 1.1 million annual deaths were
are highly complex structures, whose immunogenicity depends
associated with shigellosis during the same period. Occurrence
on several parameters amongst which are the length and nature
of the disease is seen as a correlate of sanitary conditions, and
of the saccharide component as well as its loading on the
those are not likely to improve rapidly in areas at risk. The
protein. It is reasonably admitted that control of these param-
financial status of the populations in which shigellosis exists
eters is somewhat difficult when dealing with polysaccharides
in its endemic or epidemic forms, as well as the emerging
purified from bacterial cell cultures. As recent progress in
resistance to antimicrobial drugs,5–9 limit the impact of such
carbohydrate synthesis allows access to complex saccharides,
drugs. Of the four species of Shigellae, Shigella flexneri is
it has been suggested that the use of well-defined synthetic
the major species responsible for the endemic form of the
oligosaccharides may allow better control, and consequently
disease, with serotype 2a being the most prevalent. The critical
the optimisation, of these parameters. Indeed, available data
importance of the development of a vaccine against Shigellae
on S. dysenteriae type 1 indicate that neoglycoconjugates
infections was first outlined in 1987.10 Due to increasing
incorporating di-, tri- or tetramers of the O-SP repeating unit
resistance of all groups of Shigellae to antibiotics,7 it remained
were more immunogenic than a detoxified LPS–human serum
a high priority as stated by the World Health Organization ten
albumin reference conjugate.24 Besides, it was reported that
years later.11 In the meantime, several experimental vaccines
short oligosaccharides comprising one repeating unit may be
have gone through field evaluation,12–14 but there are as yet no
immunogenic in animal models.25,26 Another critical parameter
licensed vaccines for shigellosis.
Shigella’s lipopolysaccharide (LPS) is a major surface anti-
gen of the bacterium. The corresponding O-specific poly-
saccharide domain (O-SP) is both an essential virulence factor
in the design of neoglycoconjugate vaccines is the carrier
protein. As potential applications for these vaccines are expand-
ing, the need for new carrier proteins licensed for human use
is growing.27 It has been suggested,28 and later on demon-
and the target of the infected host’s protective immune
strated,29,30 that synthetic peptides representing immunodo-
response.15,16 Indeed, using the pulmonary murine model for
minant T-cell epitopes could act as carriers in polysaccharide
shigellosis, it was recently demonstrated that the presence
locally, preliminary to infection, of a secretory antibody of iso-
type A, specific for an epitope located on the O-SP moiety of
the LPS of Shigella flexneri 5a, prevented any host homologous
and oligosaccharide conjugates. Besides, the use of T-cell
epitopes offers several advantages, including potential access to
well-defined conjugates with no risk of epitopic suppression, as
this latter phenomenon appeared to be a major drawback of
infection.17 Based on the former hypothesis that serum IgG
protein carriers.31–34 Polypeptides containing multiple T-cell
anti-LPS antibodies may confer specific protection against
epitopes have been generated in order to address the extensive
shigellosis,18 several polysaccharide–protein conjugates, target-
polymorphism of HLA molecules.35 In other strategies,
ing either Shigella sonnei, S. dysenteriae 1 or S. flexneri serotype
universal T-helper epitopes compatible with human use have
2a, were evaluated in humans.14,19 In the case of S. sonnei,
been characterized, for example from tetanus toxoid,36 or
recent field trials allowed J. B. Robbins and co-workers to dem-
engineered such as the pan HLA DR-binding epitope
(PADRE).37 Recently, covalent attachment of the human milk
oligosaccharide, lacto-N-fucopentose II, to PADRE resulted
† See ref. 1.
in a linear glycopeptide of comparable immunogenicity to
‡ Electronic supplementary information (ESI) available: experimental
details for compounds 5, 8, 15–17, 21–24 and 38–40. See http://
www.rsc.org/suppdata/ob/b4/b400986j/
that of a glycoconjugate employing HSA as the carrier.38Along
the same lines, a PADRE glycoconjugate was recently shown
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Scheme 1 Retrosynthetic analysis of the target neoglycopeptides 1, 2 and 3. (a, X and Z stand for -Ala, cyclohexyl Ala, and ε-aminohexanoic acid,
respectively).
to induce
a
strong T-cell dependent antibody response
conjugates. Three fully synthetic linear neoglycopeptides 1,2
and 3, corresponding to haptens ECD, B(E)CD, and AB(E)CD,
respectively, were synthesized according to a strategy built
on the concept of chemoselective ligation which allows the
selective one-point attachment of the free B and T epitopes in
aqueous media. All conjugates involve the peptide PADRE as
the universal T-cell epitope.
specific for the Tn antigen in both outbred and HLA transgenic
mice.39
Based on these converging data, we focused on the develop-
ment of well-defined neoglycopeptides as an alternative to
polysaccharide–protein conjugate vaccines targeting infections
caused by S. flexneri 2a. The target neoglycopeptides were
constructed by covalently linking a short peptide, serving as a
T-helper epitope, to appropriate carbohydrate haptens, serving
as B epitopes mimicking the S. flexneri 2a O-SP. We have
employed a rational approach involving a preliminary study of
the interaction between the bacterial O-SP and homologous
protective monoclonal antibodies, which helped to define the
carbohydrate haptens.
Retrosynthetic analysis of the saccharidic haptens (Scheme 1)
Analysis of S. flexneri 2a O-SP suggests that, due to the 1,2-
cis glycosidic linkage involved, construction of the EC di-
saccharide is probably the most demanding. Besides, prior work
in this laboratory has shown that the C–D glycosidic linkage
was an appropriate disconnection site when dealing with the
blockwise synthesis of oligosaccharide fragments of S. flexneri
2a O-SP.45,46,48 These observations supported the design of a
synthetic strategy common to all three targets. Basically, it relies
on (i) the condensation of an EC (4),44 B(E)C (5)48 or AB(E)C
(6) donor to a D acceptor (7), functionalized at the anomeric
position with an azidoethyl spacer; and (ii) elongation of the
spacer with introduction of a masked thiol group to allow its
coupling onto a PADRE peptide derivatized by a maleimido
group on a C-terminal lysine (8). The carbohydrate synthesis
relies on the trichloroacetimidate (TCA) methodology51 and
the use of known building blocks whenever possible. It is
worthy of note that the strategy disclosed herein differs from
that developed for the preparation of the corresponding methyl
glycosides.45,47
Results and discussion
The O-SP of S. flexneri 2a is a heteropolysaccharide defined
by the pentasaccharide repeating unit I.40,41 It features a linear
tetrasaccharide backbone, which is common to all S. flexneri
O-antigens, except serotype 6, and comprises an N-acetyl
glucosamine (D) and three rhamnose residues (A, B and C).
The serotype specificity is associated with the presence of an
α--glucopyranose residue linked at C-4 of rhamnose C. Besides
the known methyl glycoside of the EC disaccharide,42,43 a set
of di- to pentasaccharides corresponding to frame-shifted
fragments of the repeating unit I,44–47 an octasaccharide,48 and
more recently a decasaccharide,49 representative of fragments
of S. flexneri 2a O-SP have been synthesized in this laboratory.
Based on the use of these compounds as molecular probes
for mapping at the molecular level the binding characteristics
of a set of protective monoclonal antibodies against S. flexneri
2a infection,50 fragments ECD, B(E)CD and AB(E)CD
were selected as haptens that could act as B epitopes in the
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
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Scheme 2 (a) see ref. 46; (b) see ref. 45; (c) see ref. 55; (d) cat. MeONa, MeOH, rt, 2 h; (e) Me2C(OMe)2, cat. CSA, DMF, rt, 2 h; (f) cat. TfOH,
4 Å-MS, CH2Cl2, Ϫ40 ЊC rt, 3 h; (g) 95% aq. TFA, CH2Cl2, 0 ЊC; 15 min; (h) 10% Pd/C, 1 M aq. HCl, EtOH/EtOAc, rt, 24 h.
Synthesis of the aminoethyl ECD building block 18 (Scheme 2)
terminator, was chosen as the precursor to residue B. Benzoyl-
ation of diol 11 to give 1946 was performed by regioselective
opening of the cyclic orthoester intermediate as described.46
Glycosylation of the latter by donor 20, with activation by
a catalytic amount of TMSOTf, proceeded smoothly in Et2O
to yield the fully protected trisaccharide 21 (89%), which was
de-O-allylated into the hemiacetal 22 (80%) following a two
step process involving (i) iridium()-catalysed isomerisation
of the allyl glycoside to the prop-1-enyl glycoside62 and (ii)
subsequent hydrolysis.54,63 The selected trichloroacetimidate
leaving group was introduced by treatment of 22 with tri-
chloroacetonitrile in the presence of a catalytic amount
of DBU, which resulted in the formation of 5 (99%). Conden-
sation of the latter with acceptor 7 was performed in CH2Cl2 in
the presence of a catalytic amount of trifluoromethanesulfonic
acid (TfOH) to give the required tetrasaccharide 23 (76%).
Acidic hydrolysis of the latter using 95% aq. TFA gave the
intermediate diol 24 in 95% yield. Deacylation of 24 followed
by debenzylation and concomitant conversion of the azide
into the corresponding amine with hydrogen in the presence
of Pd/C under acidic conditions gave the key aminoethyl
tetrasaccharide 25 in a yield of 77% after gel filtration. Again,
compound 25 was purified by RP-HLPC before elongation of
the spacer for conjugation.
The now easily accessible disaccharide donor 4,46 with a
benzoyl participating group at C-2C, was used as the precursor
to the EC moiety in the construction of 1. It was prepared,
as described,44 in 5 steps and 45% overall yield from 2,3,4,6-
tetra-O-benzyl-β--glucopyranosyl trichloroacetimidate (9)52,53
and allyl 2,3-O-isopropylidene-α--rhamnopyranoside (10)54
through the key intermediate diol 1145 (69% from 10). Introduc-
tion of the azidoethyl spacer to a glucosaminyl intermediate
was performed according to a known procedure55 by coupling
of azidoethanol56 onto the oxazoline57 12 to give the triacetate
13.55 We have shown on several occasions in the S. flexneri
series, that protection of the 4- and 6-OH groups of precursors
to residue D with an isopropylidene acetal was appropriate,
especially when such precursors are involved in a blockwise
synthesis based on the disconnection at the C–D linkage.46,48
Thus, Zemplén deacetylation of 13 gave the triol 14 which was
converted to the key acceptor 7 (81% from 13) upon reaction
with 2,2-dimethoxypropane under acid catalysis. When the
latter was glycosylated with the donor 4 in the presence of
BF3.OEt2 in CH2Cl2, the fully protected trisaccharide 15 was
isolated in 58% yield together with the diol 16 (30%), resulting
from partial loss of the isopropylidene acetal. When 4 and 7
were glycosylated in the presence of a catalytic amount of
TMSOTf, no side reaction was observed, and the condensation
product 15 was obtained in 86% yield. Conversion of 15 into 16
(87%) was more conveniently achieved by acidic hydrolysis of
the former with 95% aq. TFA. Debenzoylation of 16 gave the
tetraol 17 (94%) which was subsequently transformed into the
aminoethyl trisaccharide 18 (69%) by hydrogenation in the
presence of palladium-on-charcoal (Pd/C) and 1 M aq. HCl to
convert the formed amine to its hydrochloride salt as others
have pointed out that hydrogenolysis using Pd/C in the presence
of a free amine was sluggish and low-yielding.58–60 In order to
prevent any side-reaction at a latter stage of the synthesis, 18 was
subsequently submitted to reverse-phase HPLC (RP-HPLC).
Synthesis of the aminoethyl AB(E)CD building block 37
(Scheme 4)
The synthesis of 37 is based on the condensation of acceptor 7
and donor 6, which resulted from the selective deallylation and
anomeric activation of the key intermediate tetrasaccharide 33.
The latter was obtained by two routes following either a block
strategy (route 1) based on the condensation of an AB di-
saccharide donor (30) and the EC disaccharide acceptor 19, or
a linear strategy (route 2) involving the stepwise elongation of
19. The construction of the donor 30 was based on the use of
the known allyl rhamnopyranoside 26,64 having permanent pro-
tecting groups at C-3 and C-4, as the precursor to residue B,
and the trichloroacetimidate chain terminator 27,65 acting as a
precursor to residue A. Condensation of the two entities in the
presence of a catalytic amount of TMSOTf resulted in the fully
Synthesis of the aminoethyl B(E)CD building block 25 (Scheme 3)
The known rhamnopyranosyl trichloroacetimidate 20,61
acetylated at its 2-, 3-, and 4-OH groups thus acting as a chain
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
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Scheme 3 (a) see ref. 46; (b) cat. TMSOTf, Et2O, Ϫ50 ЊC rt, 2 h; (c) i. cat. [Ir(COD){PCH3(C6H5)2}2]ϩPF6Ϫ, THF, rt, 20 h, ii. HgO, HgCl2,
acetone/water, rt, 2 h; (d) CCl3CN, DBU, CH2Cl2, rt, 30 min; (e) cat. TfOH, 4 Å-MS, 1,2-DCE, 65 ЊC, 1 h; (f) 50% aq. TFA, CH2Cl2, 0 ЊC, 1 h;
(g) i. cat. MeONa, MeOH, rt, 18 h; ii. 10% Pd/C, 1 M aq. HCl, EtOH/EtOAc, rt, 72 h.
Scheme 4 (a) cat. TMSOTf, Et2O, Ϫ70 ЊC rt, 8 h; (b) i. cat. [Ir(COD){PCH3(C6H5)2}2]ϩPF6Ϫ, THF, rt, 16 h, ii. HgO, HgBr2, acetone/water, rt,
Ϫ
1 h; (c) CCl3CN, DBU, CH2Cl2, rt, 2 h; (d) cat. TMSOTf, Et2O, Ϫ60 ЊC Ϫ30 ЊC, 2 h; (e) see ref. 49; (f) i. cat. [Ir(COD){PCH3(C6H5)2}2]ϩPF6
,
THF, rt, 16 h, ii. HgO, HgCl2, acetone/water, rt, 1 h; (g) cat. TMSOTf, 4 Å-MS, CH2Cl2, rt, 3 h; (h) i. 50% aq. TFA, CH2Cl2, 0 ЊC; 2 h; ii. cat.
MeONa, MeOH, 55 ЊC, 2 h; (i) 10% Pd/C, 1 M aq. HCl, EtOH/EtOAc, rt, 2 h.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
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Scheme 5 (a) SAMA-Pfp, 0.1 M phosphate buffer (pH 7.4), rt, 45 min; (b) i. maleimide butyric acid, DCC, CH2Cl2, 10 min; ii. TFA : TIS : H2O
(95 : 2.5 : 2.5); (c) HONH2.HCl, H2O, CH3CN, 0.5 M phosphate buffer (pH 6.0), rt, 1 h.
protected 28 (96%), which was selectively de-O-allylated into
29 (84%) according to the protocol described above for the
preparation of 22. Subsequent treatment of 29 with trichloro-
acetonitrile and a catalytic amount of DBU gave the required
30 (96%). Glycosylation of 19 with the latter under TMSOTf
promotion afforded the fully protected tetrasaccharide 33 in
55% yield. No β-anomer was detected. The stereochemical
outcome of this glycosylation step involving a rhamnosyl donor
glycosylated at C-2, thus lacking any participating group at this
position is not without precedent. Related examples involving
rhamnopyranosyl donors may be found in the synthesis of
oligosaccharides representative of the capsular polysaccharide
of the β-hemolytic Streptococcus Group A,66 or of the O-Ag of
Serratia marcescens O1867 as well as in our own work on S.
flexneri serotype 2a.49 Route 1 was considered initially in order
to prevent extensive consumption of the EC disaccharide 11.
Given the relatively low yield of coupling of 19 and 30, route 2
was considered as well. Of all precursors to 34, only that to
residue B, namely the donor and potential acceptor 31,68
differed from those used in route 1. Conventional glycosylation
of disaccharide 19 and 31 and subsequent selective deacetyl-
ation using methanolic HBF4, gave the acceptor 32 in 70% yield
from 19.49 The trisaccharide 32 was glycosylated with trichloro-
acetimidate 27 in an analogous fashion to glycosylation of 19
with 30, yielding 33 (92%). Anomeric de-O-allylation of this
key intermediate, as described above for the preparation of 22,
gave the corresponding hemiacetal 34 (90%) which was con-
verted into the required trichloroacetimidate 6 (88%) upon
treatment with trichloroacetonitrile and DBU. Condensation
of donor 6 with the glucosaminyl acceptor 7 was performed
under promotion by TfOH or TMSOTf, which resulted in the
fully protected pentasaccharide 35 in 62% and 80% yield,
respectively. Following the process described for the prepar-
ation of 25, compound 35 was submitted to acid hydrolysis
(97%) and subsequent deacylation to give the partially
deblocked 36 (87%), which was next converted to the amino-
ethyl pentasaccharide 37 upon treatment with hydrogen in the
presence of Pd/C. Final RP-HPLC purification resulted in
the isolation of 37 in 53% yield.
Synthesis of the target neoglycopeptides 1-3 (Scheme 5)
In all cases, chemoselective ligation of the B and T epitopes was
achieved through coupling of the carbohydrate haptens
pre-functionalized with a thiol function and a maleimide group
introduced at the C terminus of the T helper peptide. Such a
strategy was chosen in order to exploit the high reactivity and
specificity of thiol groups towards the maleimide functional-
ity,69 which allows specific and high-yielding modification of
the former in the presence of other nucleophiles.70 It was used
previously under various forms in the coupling of carbohydrate
haptens to either proteins71,72 or peptides.30,72 To our know-
ledge, in all the reported cases the maleimide functionality was
introduced onto the carbohydrate hapten. On the contrary,
our strategy relies on the introduction of this activating group
to the T helper peptide. The immunogenicity of various
maleimide-derived coupling reagents was evaluated in a model
system.73 Based on the reported data,73 4-(N-maleimido)-n-
butanoyl was selected as the linker, and incorporated by
covalent linkage to the side chain amino group of a lysine
residue added to the C-terminus of the PADRE sequence
(PADRE-Lys). It is worth mentioning that the strategy
described herein differs somewhat from that described by others
when demonstrating the usefulness of PADRE in the construc-
tion of immunogenic neoglycopeptides.38
The lysine-modified PADRE (PADRE-Lys) was assembled
using standard Fmoc chemistry solid-phase peptide synthesis.74
Standard side chain protecting groups were used, except for
that of the C-terminal lysine side chain which was protected
by the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methyl-
butyl (ivDde) group.75 Indeed, this orthogonal protecting group
strategy allows specific introduction of the maleimide group to
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
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the C-terminal lysine, upon selective cleavage of the ivDde
group by hydrazine. The thiol functionality was introduced
onto the carbohydrate haptens as a masked thiol function
(acetylthioester), which is easily generated in situ during the
conjugation process. Thus, reaction of 18, 25 and 37, with
S-acetylthioglycolic acid pentafluorophenyl ester (SAMA-Pfp)
resulted in the site-selective elongation of their aminoethyl
spacer with a thioacetyl acetamido linker. Derivatization could
be monitored by RP-HPLC with detection at 215 nm. Under
these conditions, the required thioacetyl-armed intermediates,
38, 39 and 40 were isolated in 53%, 74%, and 75% yield, respect-
ively. Their structure was confirmed based on MS and NMR
analysis. Conjugation of the carbohydrate haptens to the male-
imido activated PADRE-Lys (8) was performed in phosphate
buffer at pH 6.0 in the presence of hydroxylamine76 and
monitored by RP-HPLC. Lastly, RP-HPLC purification gave
the target neoglycopeptides 1, 2 and 3 as single products, whose
identity was assessed by MS analysis, in yields of 58%, 48% and
46%, respectively.
gas, by electrospray mass spectrometry (ES-MS), or by fast
atom bombardment mass spectrometry (FAB-MS) in the
positive-ion mode using dithioerythritol/dithio--threitol (4 : 1,
magic bullet) as the matrix, in the presence of NaI, and xenon
as the gas. High-resolution mass spectra were obtained by
Matrix Assisted Laser Desorption Ionisation mass spectro-
metry (MALDI-MS). Anhydrous dichloromethane (CH2Cl2),
1,2-dichloroethane (1,2-DCE) and Et2O, sold on molecular
sieves were used as such. 4 Å powder molecular sieves were kept
at 100 ЊC and activated before use by heating at 250 ЊC under
vacuum. Solid phase peptide synthesis was performed using
standard Fmoc chemistry protocols on a Pioneer peptide
synthesiser
(AppliedBiosystem).
Fmoc-Lys(iv-Dde)-OH,
Fmoc-Cha-OH, Fmoc--Ala-OH, Fmoc-ε-Ahx-OH and Boc-
-Ala-OH were purchased from NovaBiochem. All others
reagents and amino acids were purchased from Applied
Biosystem.
2-Azidoethyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-ꢀ-D-
glucopyranoside (7)
1 M methanolic sodium methoxide (1 mL) was added to a
solution of 1355 (3.6 g, 8.65 mmol) in MeOH (20 mL) and the
mixture was stirred at rt for 2 h. The mixture was neutralised
with Amberlite IR-120 (Hϩ) resin and filtered. The filtrate was
concentrated to give crude 14 (2.6 g). Camphorsulfonic acid
(200 mg, 0.9 mmol) was added to a solution of triol 14 (2.3 g) in
a mixture of DMF (4 mL) and 2,2-dimethoxypropane (4 mL).
After 3 h at rt, low boiling point solvents were evaporated under
reduced pressure and more 2,2-dimethoxypropane (2 mL, 15.8
mmol) was added. The mixture was stirred for 2 h at rt, Et3N
was added, and the mixture was concentrated. The crude
product was purified by column chromatography (solvent A,
19 : 1) to give 7 as a white solid (1.97 g, 78% from 13),
[α]D Ϫ91.1 (c 1.0); 1H NMR: δ 6.15 (d, 1H, JNH,2 = 5.9 Hz, NH),
4.70 (d, 1H, J1,2 = 8.3 Hz, H-1), 4.05 (m, 1H, OCH2), 3.97–3.89
(m, 2H, H-6a, 3), 3.79 (pt, 1H, J5,6b = J6a,6b = 10.5 Hz, H-6b),
3.70 (m, 1H, OCH2), 3.62–3.46 (m, 3H, H-2, 4, OCH2), 3.35–
3.26 (m, 3H, H-5, CH2N3), 2.05 (s, 3H, CH3CO), 1.52 (s, 3H,
Conclusion
The synthesis of three fully synthetic glycopeptides incorporat-
ing tri-, tetra-, and pentasaccharide haptens representative of
fragments of the O-SP of S. flexneri serotype 2a covalently
linked to the PADRE-sequence, which acts as a universal T cell
epitope compatible with human use is reported. The carbo-
hydrate haptens were selected based on a preliminary study of
the recognition of synthetic oligosaccharides with homologous
protective antibodies. They were synthesized following a
common block strategy, in a form allowing their coupling
by chemical ligation onto a maleimido-activated PADRE.
Evaluation of the immunogenicity of the conjugates in mice is
ongoing.
Experimental
General methods
C(CH3)2), 1.44 (s, 3H, C(CH3)2); 13C NMR: δ 172.4 (C᎐O),
᎐
Optical rotations were measured for CHCl3 solutions at 25 ЊC,
except where indicated otherwise, with a Perkin-Elmer auto-
matic polarimeter, Model 241 MC. TLC on precoated slides of
Silica Gel 60 F254 (Merck) was performed with solvent mixtures
of appropriately adjusted polarity consisting of A, CH2Cl2/
methanol; B, cyclohexane/ethyl acetate, C, cyclohexane/acetone
and D, toluene/ethyl acetate. Detection was effected when
applicable, with UV light, and/or by charring with orcinol
(35 mM) in 4 N aq. H2SO4. Preparative chromatography was
performed by elution from columns of Silica Gel 60 (particle
size 60–43 µm). RP-HPLC (215 nm) used a Kromasil 5 µm C18
100 Å 4.6 × 250 mm analytical column (1 mL minϪ1). NMR
spectra were recorded at 20 ЊC for solutions in CDCl3 unless
stated otherwise, on a Bruker Advance 400 spectrometer
(400 MHz for 1H, 100 MHz for 13C). External references:
100.9 (C-1), 100.0 (C(CH3)2), 74.3 (C-4), 71.8 (C-3), 68.6
(OCH2), 67.3 (C-5), 62.0 (C-6), 58.7 (C-2), 50.7 (CH2N3), 29.0
(C(CH3)2), 23.6 (CH3CO), 19.1 (C(CH3)2). CI-MS for
C13H22N4O6 (M, 330) m/z 331 [M ϩ H]ϩ. Anal. Calcd. for
C13H22N4O6 0.5H2O: C, 46.01; H, 6.83; N, 16.51%. Found: C,
46.37; H, 6.69; N, 16.46%.
.
2-Aminoethyl ꢁ-D-glucopyranosyl-(1 4)-ꢁ-L-rhamnopyranosyl-
(1 3)-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (18)
The trisaccharide 17 (368 mg, 0.38 mmol) was dissolved in a
mixture of EtOH (10 mL) and EtOAc (1 mL). A 1 M solution
of aq. HCl (0.77 mL) was added. The mixture was stirred under
hydrogen in the presence of 10% Pd/C (400 mg) for 24 h. The
mixture was diluted with water and filtered. The filtrate was
concentrated, then freeze-dried. The residue was dissolved in a
solution of NaHCO3 (75 mg) in water (1 mL) and purified by
passing first through a column of C18 silica gel (eluting with
water), then through a column of Sephadex G10 (eluting with
water) to give, after lyophilization, 18 (151 mg, 69%). Starting
from 75 mg of the latter, further RP-HPLC purification gave
46 mg of RP-HPLC pure 18. HPLC (215 nm): Rt 4.09 min
(Kromasil 5 µm C18 100 Å 4.6 × 250 mm analytical column,
using a 0–20% linear gradient over 20 min of CH3CN in 0.01 M
1
for solutions in CDCl3, TMS (0.00 ppm for both H and 13C);
for solutions in D2O, dioxane (67.4 ppm for 13C) and trimethyl-
1
silyl-3-propionic acid sodium salt (0.00 ppm for H). Proton
signal assignments were made by first-order analysis of the
spectra as well as analysis of two-dimensional 1H-1H corre-
lation maps (COSY) and selective TOCSY experiments. In
addition to s, d, t, and q, multiplicity is given as pt for pseudo t,
and br s for broad s. Of the two magnetically non-equivalent
geminal protons at C-6, the one resonating at lower field
is denoted H-6a and the one at higher field is denoted H-6b.
Interchangeable assignments in the 13C NMR spectra are
marked with an asterisk. Sugar residues in oligosaccharides are
serially lettered according to the lettering of the repeating unit
of the O-SP and identified by a subscript in listing of signal
assignments. Low-resolution mass spectra were obtained by
either chemical ionisation (CI-MS) using NH3 as the ionising
1
aq. TFA at 1 mL minϪ1 flow rate). H NMR (D2O): δ 4.97 (d,
1H, J1,2 = 3.8 Hz, H-1E), 4.78 (d, 1H, J1,2 = 1.2 Hz, H-1C), 4.54
(d, 1H, J1,2 = 8.6 Hz, H-1D), 4.02 (m, 1H, H-5C), 4.00–3.90 (m,
3H, H-5E, 6aD, CH2O), 3.88–3.67 (m, 6H, H-2C, 2D, 3C, 6aE,
6bE, 6bD, CH2O), 3.61 (dd, 1H, J = 9.8, J = 9.1 Hz, H-3E), 3.60–
3.42 (m, 5H, H-2E, 4C, 4D, 4E, 5D), 3.54 (m, 1H, H-3D), 3.03 (m,
2H, CH2NH2), 2.00 (s, 3H, CH3CO), 1.25 (d, 3H, J5,6 = 6.3 Hz,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
1523

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H-6C); 13C NMR (D O): δ 175.2 (C᎐O), 101.6 (C-1 ), 100.7
9.7 Hz, H-5A), 4.21 (m, 1H, OCH2), 4.10 (dd, 1H, H-2B), 4.02
(m, 1H, OCH2), 3.97 (dd, 1H, J2,3 = 3.0, J3,4 = 9.2 Hz, H-3B),
3.82 (dq, 1H, J4,5 = 9.4 Hz, H-5B), 3.71 (dd, 1H, H-4B), 1.43
᎐
2
C
(C-1D), 100.0 (C-1E), 82.1 (C-3D), 81.4 (C-4C), 76.3 (C-2E), 73.1
(C-3E), 72.2 (C-5E), 71.9 (C-4D), 71.3 (C-2C), 69.7 (C-4E), 69.3
(C-3C), 68.8 (C-5D), 68.5 (C-5C), 66.0 (CH2O), 60.9 (C-6D), 60.5
(C-6E), 55.5 (C-2D), 39.8 (CH2NH2), 22.5 (CH3CO), 17.1
(C-6C). ES-MS for C22H40N2O15 (M, 572) m/z 573 [M ϩ H]ϩ.
HRMS (MALDI) Calcd for C22H40N2O15Na: 595.2326. Found:
595.2341.
(d, 3H, J5,6 = 6.1 Hz, H-6B), 1.37 (d, 3H, J5,6 = 6.2 Hz, H-6A); 13
C
NMR: δ 166.3, 165.9, 165.7 (C᎐O), 139.0–127.9 (CH᎐, Ph),
᎐
᎐
117.8 (CH ᎐), 99.9 (C-1 ), 98.3 (C-1 ), 80.6 (C-4 ), 80.2 (C-3 ),
᎐
2
A
B
B
B
76.5 (C-2B), 76.0, 72.9 (2C, CH2Ph), 72.3 (C-4A), 71.0 (C-2A*),
70.4 (C-3A*), 68.7 (C-5B), 68.1 (OCH2), 67.5 (C-5A), 18.4 (C-6B),
18.1 (C-6A). FAB-MS for C50H50O12 (M, 842.3) m/z 865.1 [M ϩ
Na]ϩ. Anal. Calcd. for C50H50O12: C, 71.24; H, 5.98%. Found:
C, 71.21; H, 5.99%.
2-Aminoethyl ꢁ-L-rhamnopyranosyl-(1 3)-[ꢁ-D-glucopyrano-
syl-(1 4)]-ꢁ-L-rhamnopyranosyl-(1 3)-2-acetamido-2-deoxy-
ꢀ-D-glucopyranoside (25)
(2,3,4-Tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-(1 2)-3,4-di-O-
benzyl-ꢁ/ꢀ-L-rhamnopyranose (29)
An ice cold solution of 95% aq. TFA (2.4 mL) in CH2Cl2 (21.6
mL) was added to the tetrasaccharide 23 (1.93 g, 1.40 mmol).
The mixture was kept at 0 ЊC for 5 min, then diluted with
toluene and concentrated. Toluene was co-evaporated from the
residue. The residue was dissolved in MeOH (65 mL), and a
1 M solution of sodium methoxide in MeOH (3 mL) was
added. The mixture was left to stand at rt for 18 h, then neutral-
ised with Amberlite IR-120 (Hϩ) resin, and filtered. The filtrate
was concentrated, and the residue was purified by column
chromatography (solvent B, 9 : 1) to give 24 (1.38 g, 89%) as a
colourless foam. The tetrasaccharide 24 (1.38 g, 1.25 mmol)
was dissolved in a mixture of EtOH (35 mL) and EtOAc (3.5
mL). A 1 M solution of aq. HCl (2.5 mL) was added. The
mixture was stirred under hydrogen in the presence of 10% Pd/
C (1.5 g) for 72 h, then diluted with water and filtered. The
filtrate was concentrated, then freeze-dried. The residue was
dissolved in a solution of 5% aq. NaHCO3 and purified by
passing first through a column of C18 silica (eluting with water),
then through a column of Sephadex G10 (eluting with water) to
give, after lyophilization, 25 (693 mg, 77%). Further HPLC
purification of 373 mg of the latter gave 351 mg of RP-HPLC
pure 25. HPLC (215 nm): Rt 4.78 min (Kromasil 5 µm C18 100
Å 4.6 × 250 mm analytical column, using a 0–20% linear gradi-
ent over 20 min of CH3CN in 0.01 M aq. TFA at 1 mL minϪ1
flow rate). 1H NMR (D2O): δ 5.10 (d, 1H, J1,2 = 3.7 Hz, H-1E),
4.89 (d, 1H, J1,2 = 1.1 Hz, H-1B), 4.73 (d, 1H, J1,2 = 1.0 Hz,
H-1C), 4.50 (d, 1H, J1,2 = 8.6 Hz, H-1D), 4.08 (dq, 1H, J4,5 = 9.3
Hz, H-5C), 3.96 (m, 2H, H-2B, CH2O), 3.88–3.64 (m, 12H,
H-2C, 2D, 3B, 3C, 4C, 5B, 5E, 6aD, 6bD, 6aE, 6bE, CH2O), 3.59 (pt,
1H, J2,3 = J3,4 = 9.4 Hz, H-3E), 3.52 (dd, 1H, J2,3 = 8.6, J3,4 = 9.7
Hz, H-3D), 3.48–3.33 (m, 5H, H-2E, 4B, 4D, 4E, 5D), 3.15 (m, 2H,
CH2NH2), 1.98 (s, 3H, CH3CO), 1.27 (d, 3H, J5,6 = 6.3 Hz,
H-6C), 1.20 (d, 3H, J5,6 = 6.3 Hz, H-6B); 13C NMR (D2O): δ 174.8
1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium
hexafluorophosphate (25 mg) was dissolved in THF (10 mL)
and the resulting red solution was degassed in an argon stream.
Hydrogen was then bubbled through the solution, until the
colour had changed to yellow. The solution was then degassed
again in an argon stream. A solution of 28 (4.71 g, 5.59 mmol)
in THF (40 mL) was degassed and added. The mixture was
stirred at rt overnight, then concentrated. The residue was taken
up in acetone (350 mL) and water (82 mL). Mercuric bromide
(3.23 g, 8.96 mmol) and mercuric oxide (2.64 g, 12.2 mmol)
were added to the mixture, which was protected from light. The
suspension was stirred at rt for 1 h, then concentrated. The
residue was taken up in CH2Cl2 and washed three times with
sat. aq. KI, then with brine. The organic phase was dried and
concentrated. The residue was purified by column chromato-
graphy (solvent B, 3 : 1) to give 29 (3.87 g, 84%) as a colourless
foam. 1H NMR (α anomer): δ 8.15–7.12 (m, 25H, Ph),
5.94–5.88 (m, 3H, H-2 , 3 , CH᎐), 5.70 (pt, 1H, J = 9.7 Hz,
᎐
A
A
3,4
H-4A), 5.31 (dd, 1H, J1,OH = 3.0 Hz, H-1B), 5.28 (br s, 1H, H-1A),
4.98 (d, 1H, J = 11.0 Hz, CH2Ph), 4.82–4.68 (m, 3H, CH2Ph),
4.31 (dq, 1H, J4,5 = 9.8 Hz, H-5A), 4.13 (dd, 1H, J1,2 = 2.1 Hz,
H-2B), 4.06–3.99 (m, 2H, H-3B, 5B), 3.72 (pt, 1H, J3,4 = J4,5 = 9.4
Hz, H-4B), 2.79 (br s, 1H, OH-1B), 1.41 (d, 3H, J5,6 = 6.2 Hz,
H-6B), 1.37 (d, 3H, J5,6 = 6.3 Hz, H-6A); 13C NMR (α anomer):
δ 166.2, 165.9, 165.7 (C᎐O), 138.9–127.9 (Ph), 99.7 (C-1 ), 94.2
᎐
A
(C-1B), 80.5 (C-4B), 79.6 (C-3B), 77.6 (C-2B), 76.5, 72.5 (2C,
CH2Ph), 72.3 (C-4A), 71.0 (C-2A*), 70.4 (C-3A*), 68.8 (C-5B),
67.6 (C-5A), 18.5 (C-6B*), 18.1 (C-6A*). FAB-MS for C47H46O12
(M, 802.3) m/z 825.1 [M ϩ Na]ϩ. Anal. Calcd. for C47H46O12.0.5
H2O: C, 69.53; H, 5.84%. Found: C, 69.55; H, 5.76%.
(C᎐O), 103.2 (br s, C-1 ), 101.4 (C-1 ), 100.9 (C-1 ), 98.6 (C-1 ),
(2,3,4-Tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-(1 2)-3,4-di-O-
benzyl-ꢁ/ꢀ-L-rhamnopyranosyl trichloroacetimidate (30)
᎐
B
C
D
E
81.9 (C-3D), 79.0 (br s, C-3C), 76.6 (br s, C-4C), 76.3 (C-2E), 72.9
(C-3E), 72.3 (2C, C-5E, 4B), 71.8 (C-4D), 71.1 (br s, C-2C), 70.5
(C-2B, 3B), 69.7, 69.5 (2C, C-5B, 4E), 69.2, 68.8 (C-5D, 5C), 67.9
(CH2O), 61.0 (C-6D), 60.8 (C-6E), 55.5 (C-2D), 40.0 (CH2NH2),
22.6 (CH3CO), 18.0 (C-6C), 17.0 (C-6B). FAB-MS for C28H50-
N2O19 (M, 718.3) m/z 741 [M ϩ Na]ϩ. HRMS (MALDI) Calcd
for C28H50N2O19Na: 741.2905. Found: 741.2939.
The hemiacetal 29 (3.77 g, 4.71 mmol) was dissolved in CH2Cl2
(15 mL) and the solution was cooled to 0 ЊC. Trichloro-
acetonitrile (2.5 mL) was added, then DBU (200 µL). The
mixture was stirred at rt for 2 h. Toluene was added, and
co-evaporated twice from the residue. The crude material was
purified by flash chromatography (solvent B, 4 : 1 ϩ 0.1% Et3N)
to give 30 as a white foam (4.29 g, 96%). Some hydrolyzed
material 29 (121 mg, 3%) was eluted next. The trichloro-
acetimidate 30, isolated as an α/β mixture had 1H NMR
(α anomer): δ 8.62 (s, 1H, NH), 8.20–7.18 (m, 25H, Ph), 6.31
(s, 1H, H-1B), 5.94 (dd, 1H, J1,2 = 1.6 Hz, H-2A), 5.89 (dd, 1H,
J2,3 = 3.4, J3,4 = 9.9 Hz, H-3A), 5.71 (pt, 1H, H-4A), 5.27 (br s,
1H, H-1A), 5.02 (d, 1H, J = 10.8 Hz, CH2Ph), 4.84 (d, 1H,
J = 11.9 Hz, CH2Ph), 4.79 (d, 1H, CH2Ph), 4.72 (d, 1H,
CH2Ph), 4.36 (dq, 1H, J4,5 = 9.8 Hz, H-5A), 4.13 (dd, 1H, H-2B),
4.03–3.97 (m, 2H, H-3B, 5B), 3.80 (pt, 1H, J3,4 = 9.5 Hz, H-4B),
1.45 (d, 3H, J5,6 = 6.1 Hz, H-6B), 1.40 (d, 3H, J5,6 = 6.2 Hz,
Allyl (2,3,4-tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-(1 2)-3,4-di-
O-benzyl-ꢁ-L-rhamnopyranoside (28)
TMSOTf (11 µL, 59 µmol) was added to a solution of the
rhamnoside 26 (2.26 g, 5.88 mmol) and the trichloroacetimidate
27 (4.23 g, 6.82 mmol) in anhydrous Et2O (60 mL) at Ϫ70 ЊC.
The reaction mixture was stirred for 8 h while the cooling bath
was slowly coming back to rt. Et3N (100 µL) was added, and the
mixture was stirred at rt for 15 min. Solvents were evaporated,
and the crude material was purified by column chromatography
(solvent B, 49 : 1 9 : 1), to give 28 as a white foam (4.78 g,
1
96%). [α]D ϩ81.7 (c 1.0); H NMR: δ 8.17–7.12 (m, 25H, Ph),
H-6A); 13C NMR (α anomer): δ 166.2, 165.9, 165.7 (3C, C᎐O),
᎐
5.97–5.85 (m, 3H, H-2 , 3 , CH᎐), 5.67 (pt, 1H, J = 9.6 Hz,
160.8 (C᎐NH), 138.6–128.2 (Ph), 99.9 (C-1 ), 97.2 (C-1 ),
᎐
A B
᎐
A
A
3,4
H-4 ), 5.34–5.19 (m, 3H, H-1 , CH ᎐), 5.01 (d, 1H, J = 9.0 Hz,
91.4 (CCl3), 79.9 (C-4B), 79.1 (C-3B), 76.2 (CH2Ph), 74.9 (C-2B),
73.3 (CH2Ph), 72.1 (C-4B), 71.7 (C-5B), 71.0 (C-2A), 70.2 (C-3A),
67.8 (C-5A), 18.4 (C-6B), 18.0 (C-6A). Anal. Calcd. for
᎐
2
A
A
CH2Ph), 4.92 (d, 1H, J1,2 = 1.3 Hz, H-1B), 4.82–4.74 (m, 2H,
CH2Ph), 4.71 (d, 1H, J = 11.8 Hz, CH2Ph), 4.31 (dq, 1H, J4,5
=
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
1524

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C49H46Cl3NO12: C, 62.13; H, 4.89; N, 1.48%. Found C, 61.81; H,
4.86, N, 1.36%.
4.61 (br s, 1H, H-2B), 4.58–4.47 (m, 5H, H-5A, CH2Ph), 4.38 (d,
1H, J = 12.0 Hz, CH2Ph), 4.24 (br d, 1H, J3,4 = 8.5 Hz, H-3C),
4.09–3.99 (m, 3H, H-3E, 5C, 5E), 3.86 (pt, 1H, J3,4 = J4,5 = 8.9 Hz,
H-4C), 3.80–3.60 (m, 6H, H-3B, 4E, 4B, 5B, 6aE, 6bE), 3.54 (dd,
1H, H-2E), 3.17 (d, 1H, OH), 1.46 (d, 3H, J5,6 = 6.0 Hz, H-6A),
1.42 (d, 3H, J5,6 = 6.0 Hz, H-6C), 1.16 (d, 3H, J5,6 = 5.7 Hz,
Allyl (2,3,4-tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-(1 2)-(3,4-di-
O-benzyl-ꢁ-L-rhamnopyranosyl)-(1 3)-[(2,3,4,6-tetra-O-
benzyl-ꢁ-D-glucopyranosyl)-(1 4)]-2-O-benzoyl-ꢁ-L-rhamno-
pyranoside (33)
H-6B); 13C NMR (α anomer): δ 166.0, 165.6, 165.2 (C᎐O),
᎐
138.9–127.2 (Ph), 101.1 (br s, C-1B), 99.7 (C-1A), 98.1 (C-1E),
91.6 (C-1C), 81.9 (C-3E), 81.1 (C-2E), 79.9 (C-4B), 79.4 (br s,
C-3C), 78.9 (C-3B), 78.3 (br s, C-4C), 77.6 (C-4E), 76.1 (C-2B),
75.8, 75.3, 75.1, 74.0, 73.1 (5C, CH2Ph), 72.7 (C-2C), 72.1
(C-4A), 71.4 (C-5E), 71.1 (CH2Ph), 70.8 (C-2A), 70.2 (C-3A), 69.4
(C-5B), 68.3 (C-6E), 67.7 (C-5C), 67.3 (C-5A), 19.0 (C-6A), 18.2
(C-6C), 17.9 (C-6B). FAB-MS for C94H94O22 (M, 1574) m/z 1597
[M ϩ Na]ϩ. Anal. Calcd. for C94H94O22: C, 71.65; H, 6.01%.
Found: C, 71.48; H, 6.17%.
(a) The acceptor 19 (465 mg, 0.56 mmol) was dissolved in Et2O
(3 mL). The solution was cooled to Ϫ60 ЊC and TMSOTf
(65 µL, 0.36 mmol) was added. The donor 30 (690 mg, 0.73
mmol) was dissolved Et2O (6 mL) and added to the acceptor
solution in two portions with an interval of 30 min. The
mixture was stirred at Ϫ60 ЊC to Ϫ30 ЊC over 2 h. Et3N (100
µL) was added. The mixture was concentrated and the residue
was purified by column chromatography (solvent B, 7 : 1) to
give 33 (501 mg, 55%).
(b) A solution of the donor 27 (1.41 g, 2.25 mmol) and the
acceptor 3249 (1.07 g, 1.79 mmol) in anhydrous Et2O (88 mL)
was cooled to Ϫ60 ЊC. TMSOTf (63 µL) was added, and the
mixture was stirred at Ϫ60 ЊC to Ϫ20 ЊC over 2.5 h. Et3N was
added (100 µL). The mixture was concentrated and the residue
was purified by column chromatography (solvent D, 49 : 1) to
give 33 (2.66 g, 92%); [α]D ϩ74.1 (c 0.5); 1H NMR: δ 8.11–7.06
(2,3,4-Tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-(1 2)-(3,4-di-O-
benzyl-ꢁ-L-rhamnopyranosyl)-(1 3)-[(2,3,4,6-tetra-O-benzyl-
ꢁ-D-glucopyranosyl)-(1 4)]-2-O-benzoyl-ꢁ/ꢀ-L-rhamno-
pyranosyl trichloroacetimidate (6)
The hemiacetal 34 (412 mg, 0.26 mmol) was dissolved in
CH2Cl2 (5 mL) and the solution was cooled to 0 ЊC. Trichloro-
acetonitrile (0.26 mL) was added, then DBU (4 µL). The
mixture was stirred at 0 ЊC for 1 h. The mixture was concen-
trated and toluene was co-evaporated from the residue. The
residue was purified by flash chromatography (solvent B, 4 : 1 ϩ
(m, 50H, Ph), 6.00–5.87 (m, 3H, H-2 , 3 , CH᎐), 5.72 (pt, 1H,
᎐
A
A
J3,4 = J4,5 = 9.9 Hz, H-4A), 5.49 (dd, 1H, J1,2 = 2.0, J2,3 = 3.0 Hz,
H-2 ), 5.35–5.22 (m, 3H, H-1 , CH ᎐), 5.12 (d, 1H, J = 1.6
᎐
C
A
2
1,2
Hz, H-1B), 5.08 (d, 1H, J1,2 = 3.2 Hz, H-1E), 4.96 (br s, 1H,
H-1C), 4.98–4.62 (m, 7H, CH2Ph), 4.57 (br s, 1H, H-2B), 4.54–
4.31 (m, 6H, H-5A, CH2Ph), 4.21–4.16 (m, 2H, H-3C, OCH2),
4.09–3.99 (m, 3H, H-3E, 5E, OCH2), 3.84 (m, 2H, H-4C, 5C),
1
0.1% Et3N) to give 6 (393 mg, 88%). H NMR (α anomer):
δ 8.75 (s, 1H, NH), 8.10–7.03 (m, 50H, Ph), 6.42 (d, 1H, J1,2
2.0 Hz, H-1C), 5.90 (m, 2H, H-2A, 3A), 5.74 (pt, 1H, J3,4 = J4,5
=
=
3.77–3.54 (m, 6H, H-3B, 4B, 4E, 5B, 6aE, 6bE), 3.49 (dd, 1H, J2,3
=
9.8 Hz, H-4A), 5.59 (br s, 1H, H-2C), 5.32 (br s, 1H, H-1A), 5.15
(br s, 1H, H-1B), 5.09 (d, 1H, J1,2 = 2.7 Hz, H-1E), 5.04–4.79 (m,
6H, CH2Ph), 4.70–4.42 (m, 7H, H-2B, 5A, CH2Ph), 4.32 (d, 1H,
J = 12.0 Hz, CH2Ph), 4.24 (m, 1H, H-3C), 4.09–3.96 (m, 3H,
H-3E, 5B, 5E), 3.91 (m, 1H, H-4C), 3.74–3.57 (m, 6H, H-3B, 4B,
4E, 5C, 6aE, 6bE), 3.52 (dd, 1H, J1,2 = 3.4, J2,3 = 9.8 Hz, H-2E),
1.42 (d, 3H, J5,6 = 6.0 Hz, H-6C 1.39 (d, 3H, J5,6 = 6.2 Hz, H-6A),
1.10 (d, 3H, J5,6 = 4.3 Hz, H-6B); 13C NMR (α anomer): δ 166.3,
9.7 Hz, H-2E), 1.42 (d, 3H, J5,6 = 6.2 Hz, H-6A), 1.37 (d, 3H, J5,6
= 5.5 Hz, H-6C), 1.11 (d, 3H, J5,6 = 5.9 Hz, H-6B); 13C NMR:
δ 166.0, 165.9, 165.4, 165.1 (C᎐O), 138.7–127.1 (CH᎐, Ph),
᎐
᎐
117.8 (CH ᎐), 101.3 (br s, C-1 ), 99.6 (C-1 ), 97.9 (C-1 ), 96.1
᎐
2
B
A
E
(C-1C), 81.9 (C-3E), 81.0 (C-2E), 80.1 (br s, C-3C), 79.8 (C-4B),
78.9 (C-3B), 77.9 (br s, C-4C), 77.4 (C-4E), 75.9 (C-2B), 75.6,
75.0, 74.9, 73.9, 72.9 (CH2Ph), 72.4 (C-2C), 71.9 (C-4A), 71.2
(C-5E), 70.9 (CH2Ph), 70.7 (C-2A), 70.0 (C-3A), 69.2 (C-5B), 68.5
(OCH2), 68.1 (C-6E), 67.6 (C-5C), 67.2 (C-5A), 18.8 (C-6A), 18.1
(C-6C), 17.8 (C-6B). FAB-MS for C97H98O22 (1614) m/z 1637
[M ϩ Na]ϩ. Anal. Calcd. for C97H98O22.H2O: C, 71.31; H, 6.17%.
Found: C, 71.35; H, 6.21%.
165.9, 165.8, 165.5 (C᎐O), 160.5 (C᎐NH), 138.7–127.2 (Ph),
᎐
᎐
101.2 (C-1B), 99.7 (C-1A), 98.3 (C-1E), 94.3 (C-1C), 90.9 (CCl3),
82.2 (C-3E), 81.3 (br s, C-2E), 80.0 (2C, C-3C, 4B), 79.0 (C-3B),
77.9 (C-4E), 77.5 (br s, C-4C), 76.4 (br s, C-2B), 76.0, 75.5, 75.4,
74.4, 73.3 (5C, CH2Ph), 72.2 (C-4A), 71.7 (C-5E), 71.3 (CH2Ph),
71.1 (C-2A*), 71.0 (C-2C*), 70.7 (br s, C-5C*), 70.4 (C-3A), 69.8
(br s, C-5B*), 68.4 (C-6E), 67.6 (C-5A), 19.1 (br s, C-6C), 18.2
(C-6A), 18.1 (C-6B). Anal. Calcd. for C96H94Cl3NO22.H2O: C,
66.34; H, 5.57; N, 0.81%. Found: C, 66.26; H, 5.72; N, 0.94%.
(2,3,4-Tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-(1 2)-(3,4-di-O-
benzyl-ꢁ-L-rhamnopyranosyl)-(1 3)-[(2,3,4,6-tetra-O-benzyl-
ꢁ-D-glucopyranosyl)-(1 4)]-2-O-benzoyl-ꢁ/ꢀ-L-rhamno-
pyranose (34)
2-Azidoethyl (2,3,4-tri-O-benzoyl-ꢁ-L-rhamnopyranosyl)-
(1 2)-(3,4-di-O-benzyl-ꢁ-L-rhamnopyranosyl)-(1 3)-[(2,3,4,6-
tetra-O-benzyl-ꢁ-D-glucopyranosyl)-(1 4)]-(2-O-benzoyl-ꢁ-L-
rhamnopyranosyl)-(1 3)-2-acetamido-2-deoxy-4,6-O-isopropyl-
idene-ꢀ-D-glucopyranoside (35)
1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium
hexafluorophosphate (12.5 mg) was dissolved in THF (5 mL)
and the resulting red solution was degassed in an argon stream.
Hydrogen was then bubbled through the solution, causing the
colour to change to yellow. The solution was then degassed
again in an argon stream. A solution of 33 (1.14 g, 0.70 mmol)
in THF (15 mL) was degassed and added. The mixture was
stirred at rt overnight. The mixture was concentrated. The
residue was taken up in acetone (7 mL) and water (0.7 mL).
Mercuric chloride (285 mg, 1.05 mmol) and mercuric oxide (303
mg, 1.4 mmol) were added to the mixture, which was protected
from light. The mixture was stirred at rt for 1 h, then concen-
trated. The residue was taken up in CH2Cl2 and washed three
times with sat. aq. KI, then with brine. The organic phase was
dried and concentrated. The residue was purified by column
chromatography (solvent B, 7 : 3) to give 34 (992 mg, 90%) as a
(a) The tetrasaccharide donor 6 (500 mg, 0.29 mmol) and the
acceptor 7 (140 mg, 0.42 mmol) were dissolved in 1,2-DCE
(5 mL) and 4 Å-MS (400 mg) were added. The mixture was
stirred at rt for 2 h. The mixture was cooled to 0 ЊC and TfOH
(7 µL, 72 µmol) was added. The mixture was stirred at 0 ЊC to rt
over 1 h 30 min. The mixture was then heated at 65 ЊC for 1 h 30
min. The mixture was allowed to cool, Et3N (0.5 mL) was
added, and the mixture was stirred at rt for 20 min. The mixture
was diluted with CH2Cl2 and filtered through a pad of Celite.
The filtrate was concentrated and purified by column
chromatography (solvent B, 4 : 3) to give 35 (340 mg, 62%).
(b) The tetrasaccharide donor 6 (250 mg, 145 µmol) and the
acceptor 7 (67 mg, 204 µmol) were dissolved in CH2Cl2 (1.5 mL)
and 4 Å-MS (200 mg) were added. The mixture was stirred at
Ϫ40 ЊC for 30 min and TMSOTf (5 µL) was added. The
mixture was stirred at rt over 3 h, triethylamine was added, and
1
colourless foam. H NMR (α anomer): δ 8.16–7.05 (m, 50H,
Ph), 5.97–5.88 (m, 2H, H-2A, 3A), 5.74 (pt, 1H, J3,4 = J4,5 = 9.8
Hz, H-4A), 5.56 (br s, 1H, H-2C), 5.35 (br s, 1H, H-1A), 5.29 (d,
1H, J1,2 = 1.7 Hz, H-1C), 5.18 (br s, 1H, H-1B), 5.01 (d, 1H, J1,2
=
3.1 Hz, H-1E), 4.99–4.78 (m, 6H, CH2Ph), 4.68 (d, 1H, CH2Ph),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
1525

View Article Online
the mixture was stirred at rt for 15 min. The mixture was diluted
with CH2Cl2 and filtered through a pad of Celite. The filtrate
was concentrated and purified by column chromatography
(s, 3H, CH3CO), 1.28 (d, 3H, H-6C), 1.22 (m, 6H, H-6A, 6B); 13
NMR (D O): δ 175.3 (C᎐O), 102.9 (C-1 ), 101.7 (br s, C-1 ),
101.4 (C-1C), 100.9 (C-1D), 97.9 (br s, C-1E), 81.8 (C-3D), 79.8
(C-2B), 79.4 (br s, C-3C), 76.3 (C-2E), 75.1 (br s, C-4C), 72.9,
72.4, 72.4, 72.2, 71.7, 71.2 (br s), 70.5 (2C), 70.4, 70.1, 70.0,
69.7, 69.6, 69.4, 68.7, 66.7 (CH2O), 61.0 (2C, C-6D, 6E), 55.5
C
᎐
2
A
B
(solvent B, 9 : 1 1 : 1) to give 35 (219 mg, 80%). [α]D ϩ64.0
1
(c 1); H NMR: δ 8.14–7.10 (m, 50H, Ph), 6.31 (d, 1H, JNH,2
=
7.6 Hz, NH), 6.01 (dd, 1H, J1,2 = 1.6 Hz, H-2A), 5.98 (dd, 1H,
J2,3 = 3.3 Hz, H-3A), 5.79 (pt, 1H, J3,4 = J4,5 = 9.9 Hz, H-4A), 5.47
(dd, 1H, J1,2 = 1.6, J2,3 = 2.0 Hz, H-2C), 5.41 (br s, 1H, H-1A),
5.24 (m, 1H, H-1B), 5.21 (d, 1H, J1,2 = 3.2 Hz, H-1E), 5.10
(d, 1H, J1,2 = 8.3 Hz, H-1D), 5.05 (br s, 1H, H-1C), 5.04–4.81 (m,
4H, CH2Ph), 4.73–4.68 (m, 2H, CH2Ph), 4.65 (br s, 1H, H-2B),
4.62–4.42 (m, 8H, CH2Ph), 4.55 (m, 1H, H-5A), 4.39 (pt, 1H,
J2,3 = J3,4 = 9.5 Hz, H-3D), 4.23–4.12 (m, 3H, H-3C, 5C, 5E), 4.08
(pt, 1H, J3,4 = J4,5 = 9.3 Hz, H-3E), 4.02–3.97 (m, 2H, H-6aD,
CH2O), 3.91 (pt, 1H, J3,4 = J4,5 = 9.3 Hz, H-4C), 3.85–3.66
(m, 8H, H-3B, 4B, 4E, 5B, 6bD, 6aE, 6bE, CH2O), 3.63 (pt, 1H,
J4,5 = 9.3 Hz, H-4D), 3.59 (dd, 1H, J2,3 = 9.7 Hz, H-2E), 3.47 (m,
1H, H-5D), 3.41–3.33 (m, 2H, H-2D, CH2N3), 3.16 (m, 1H,
CH2N3), 2.21 (s, 3H, CH3CO), 1.53–1.47 (m, 9H, H-6A,
(CH3)2C), 1.39 (d, 3H, J5,6 = 6.2 Hz, H-6C), 1.19 (d, 3H,
J5,6 = 5.Hz, H-6B); 13C NMR: δ 172.3, 166.4, 166.3, 165.9, 165.5
(C-2 ), 39.9 (CH NH ), 22.6 (CH C᎐O), 18.2 (C-6 ), 17.2
᎐
D
2
2
3
C
(C-6A), 17.0 (C-6B). HRMS (MALDI) Calcd for C34H60N2O23
ϩ H: 865.3665. Found: 865.3499.
General procedure for the preparation of targets 1, 2 and 3
The oligosaccharide bearing a masked thiol group (5.8 µmol)
was dissolved in water (500 µL) and added to a solution of 8 (13
mg, 7.4 µmol) in a mixture of water (1 mL), CH3CN (200 µL)
and 0.5 M phosphate buffer (pH 5.7, 1.2 mL). 117 µL of a
solution of hydroxylamine hydrochloride (139 mg mLϪ1) in
0.5 M phosphate buffer (pH 5.7) was added, and the mixture
was stirred for 1 h. RP-HPLC purification gave the pure
neoglycopeptide
PADRE-lys-(thiomethyl)carbonylaminoethyl ꢁ-D-glucopyrano-
syl-(1 4)-ꢁ-L-rhamnopyranosyl-(1 3)-2-acetamido-2-deoxy-
ꢀ-D-glucopyranoside (1)
(C᎐O), 139.3–127.5 (Ph), 101.8 (br s, C-1 ), 100.3 (C-1 ), 100.0
᎐
B
D
(C-1A), 99.9 (C(CH3)2), 98.0 (2C, C-1C, 1E), 82.1 (C-3E), 81.5
(C-2E), 80.4 (br s, C-3C), 80.2 (C-4E*), 79.4 (C-4B*), 78.0 (C-3B),
77.9 (br s, C-4C), 76.6 (C-3D), 76.4 (C-2B), 76.0, 75.4, 75.3, 74.2,
73.5 (5C, CH2Ph), 73.4 (C-4D*), 73.2 (C-2C), 72.3 (C-4A), 71.8
(C-2A), 71.5 (CH2Ph), 71.2 (C-5E), 70.5 (C-3A), 69.7 (C-5B), 69.1
(OCH2), 68.8 (C-6E), 67.9 (C-5C), 67.7 (C-5A), 67.6 (C-5D), 62.7
(C-6D), 59.2 (C-2D), 51.0 (CH2N3), 29.5 (C(CH3)2), 24.0
(CH3CO), 19.2 (C(CH3)2), 19.1 (C-6A), 18.5 (C-6C), 18.2 (C-6B).
FAB-MS for C107H114N4O27 (M, 1886) m/z 1909 [M ϩ Na]ϩ.
Anal. Calcd. for C107H114N4O27: C, 68.07, H, 6.09; N, 2.97%.
Found: C, 68.18, H, 6.07; N, 2.79%.
62% from 38. HPLC (230 nm): Rt 10.40 min (100% pure,
Kromasil 5 µm C18 100 Å 4.6 × 250 mm analytical column,
using a 0–20% linear gradient over 20 min of CH3CN in
0.01 M aq. TFA at 1 mL minϪ1 flow rate). ES-MS Calcd for
C109H181N23O35S: 2405.85. Found: 2405.52.
PADRE-lys-(thiomethyl)carbonylaminoethyl ꢁ-L-rhamno-
pyranosyl-(1 3)-[ꢁ-D-glucopyranosyl)-(1 4)]-ꢁ-L-rhamno-
pyranosyl-(1 3)-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (2)
48% from 39. HPLC (230 nm): Rt 11.60 min (100% pure,
Kromasil 5 µm C18 100 Å 4.6 × 250 mm analytical column,
using a 20–50% linear gradient over 20 min of CH3CN in
0.01 M aq. TFA at 1 mL minϪ1 flow rate). ES-MS Calcd for
C125H191N23O39S: 2552.99. Found: 2551.90.
2-Aminoethyl ꢁ-L-rhamnopyranosyl-(1 2)-ꢁ-L-rhamnopyrano-
syl-(1 3)-[ꢁ-D-glucopyranosyl)-(1 4)]-ꢁ-L-rhamnopyranosyl-
(1 3)-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (37)
An ice cold solution of 50% aq. TFA (2.1 mL) in CH2Cl2 (8 mL)
was added to the pentasaccharide 35 (283 mg, 0.15 mmol). The
mixture was kept at 0 ЊC for 2 h, then diluted with toluene and
concentrated. Toluene was co-evaporated from the residue.
Chromatography of the residue (solvent B, 7 : 3 1 : 1) gave the
intermediate diol (265 mg, 96%). The latter (265 mg) was
dissolved in MeOH (6 mL), and a 1% solution of methanolic
sodium methoxide (4.0 mL) was added. The mixture was stirred
at 55 ЊC for 2 h, then neutralised with Dowex X8 (Hϩ) resin,
and filtered. The filtrate was concentrated. The mixture was
purified by column chromatography (solvent A, 100 : 0 1 95 :
5) to give 36 (195 mg, 87%) as a colourless foam, whose
structure was confirmed from mass spectrometry analysis
(FAB-MS for C76H94N4O23 (M, 1430) m/z 1453 [M ϩ Na]ϩ).
Pentasaccharide 36 (171 mg, 0.11 mmol) was dissolved in EtOH
(18 mL). A 1 M solution of aq. HCl (210 µL) was added. The
mixture was stirred under hydrogen in the presence of 10% Pd/
C (96 mg) for 2 h. The mixture was diluted with EtOH and
water, then filtered through a pad of Celite. The filtrate was
concentrated and preliminarily purified by passing through
a column of C18 silica (eluting with water). The residue was
purified by RP-HPLC to give, after lyophilization, 37 (50 mg,
53%). HPLC (215 nm): Rt 5.87 min (Kromasil 5 µm C18 100 Å
4.6 × 250 mm analytical column, using a 0–20% linear gradient
over 20 min of CH3CN in 0.01 M aq. TFA at 1 mL minϪ1 flow
rate). 1H NMR (D2O): δ 5.15 (d, 1H, J1,2 = 3.7 Hz, H-1E), 5.00
(br s, 1H, H-1A), 4.92 (d, 1H, J1,2 = 1.1 Hz, H-1B), 4.76 (br s, 1H,
H-1C), 4.53 (d, 1H, J1,2 = 8.6 Hz, H-1D), 4.10 (m, 1H, H-5C),
4.03 (m, 2H, H-2A, 2B), 4.01 (m, 3H, H-4A, 4B, CH2O), 3.88–
3.38 (m, 7H, H-2C, 2D, 3A, 6aD, 6bD, 6aE, CH2O), 3.69–3.76
(m, 7H, H-3B, 3C, 3E, 4C, 5A, 5B, 6bE), 3.52 (pt, 1H, H-3D), 3.33–
3.54 (m, 5H, H-2E, 4D, 4E, 5D, 5E), 3.09 (m, 2H, CH2NH2), 1.98
PADRE-lys-(thiomethyl)carbonylaminoethyl ꢁ-L-rhamnopyr-
anosyl-(1 2)-ꢁ-L-rhamnopyranosyl-(1 3)-[ꢁ-D-glucopyrano-
syl)-(1 4)]-ꢁ-L-rhamnopyranosyl-(1 3)-2-acetamido-2-deoxy-
ꢀ-D-glucopyranoside (3)
46% from 40. HPLC (230 nm): Rt 10.33 min (100% pure,
Kromasil 5 µm C18 100 Å 4.6 × 250 mm analytical column,
using a 20–50% linear gradient over 20 min of CH3CN in 0.01
M aq. TFA at 1 mL minϪ1 flow rate). ES-MS Calcd for
C121H201N23O43S: 2698.14. Found: 2698.09.
Acknowledgements
The authors are grateful to J. Ughetto-Monfrin (Unité de
Chimie Organique, Institut Pasteur) for recording all the NMR
spectra. The authors thank the Bourses Mrs Frank Howard
Foundation for the postdoctoral fellowship awarded to K. W.,
and the Institut Pasteur for its financial support (grant no. PTR
99).
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 8 – 1 5 2 7
1527