On the preparation of carbohydrate-protein conjugates using the traceless Staudinger ligation

Article abstract of DOI:10.1021/jo0505472

The nature of a linker used for preparing glycoconjugate vaccines is of utmost importance as it may lead to immunogenic biomolecules. We report the conjugation of carbohydrate haptens to protein carriers leading to potential vaccines using the traceless Staudinger ligation. The ligation relies on the selective transfer of a phosphane substituent to an azide to form a native amide bond in the final product upon release of an oxidized phosphane byproduct. We designed new phosphinofunctionalized cross-linkers suitable for protein carrier derivatization. We evaluated their utility in preparing conjugates using both synthetic and purified bacterial carbohydrates. The use of a borane-protected phosphane which is deprotected at the time of the ligation reaction led to the best results observed thus far in terms of stability toward oxidation and reactivity.

Full text of DOI:10.1021/jo0505472

On the Preparation of Carbohydrate-Protein Conjugates Using
the Traceless Staudinger Ligation
Cyrille Grandjean,*,† Alain Boutonnier,‡ Catherine Guerreiro,† Jean-Michel Fournier,‡ and
Laurence A. Mulard*,†
Unite´ de Chimie Organique, URA CNRS 2128 and Unite´ du Chole´ra et des Vibrions, Centre National de
Re´fe´rence des Vibrions et du Chole´ra, Institut Pasteur, 25-28 rue du Docteur Roux,
75724 Paris Cedex 15, France
grandjean.cyr@wanadoo.fr; lmulard@pasteur.fr
Received March 17, 2005
The nature of a linker used for preparing glycoconjugate vaccines is of utmost importance as it
may lead to immunogenic biomolecules. We report the conjugation of carbohydrate haptens to protein
carriers leading to potential vaccines using the traceless Staudinger ligation. The ligation relies
on the selective transfer of a phosphane substituent to an azide to form a native amide bond in the
final product upon release of an oxidized phosphane byproduct. We designed new phosphino-
functionalized cross-linkers suitable for protein carrier derivatization. We evaluated their utility
in preparing conjugates using both synthetic and purified bacterial carbohydrates. The use of a
borane-protected phosphane which is deprotected at the time of the ligation reaction led to the
best results observed thus far in terms of stability toward oxidation and reactivity.
Introduction
pling reaction should be site-selective and efficient. This
avoids forming ill-defined glycoconjugates and the result-
ant loss of costly starting materials. The choice of a
method is often restricted by the solubility of starting
materials and products in organic solvents as well as by
the pH and temperature stability of these materials. The
availability of functional groups and the stability and
biocompatibility, in particular the absence of immuno-
genicity of the resulting covalent bond, are also impor-
tant. Attachment via an amide bond is attractive since
this type of bond is ubiquitous in nature. However, the
basic amine/carboxylic acid condensation reaction is not
often convenient. Indeed, yields are usually low as the
activated species are hydrolyzed in a competing side-
reaction.4
It has long been known that conjugating carbohydrate
haptens to appropriate protein carriers often results in
biomolecules capable of inducing anti-carbohydrate an-
tibodies.1 This has stimulated the development of glyco-
conjugates as potential anti-infectious and anti-cancer
vaccines. Some of these are currently licensed as phar-
maceuticals.2
Numerous methods have been developed to link oli-
gosaccharides covalently to proteins.3 Ideally, the cou-
* To whom correspondence should be adressed. Fax: +33 (0)-
145688404.
† Unite´ de Chimie Organique.
‡ Unite´ du Chole´ra et des Vibrions, Centre National de Re´fe´rence
des Vibrions et du Chole´ra.
(1) Goebel, W. F.; Avery, O. T. J. Exp. Med. 1931, 54, 431-436.
(2) Robbins, J. B.; Schneerson, R.; Anderson, P.; Smith, D. H. J. Am.
Med. Assoc. 1996, 9, 1181-1185.
(3) (a) Dick, W., Jr.; Beurret, M. In Conjugate Vaccines: Contribu-
tions to Microbiology and Immunology; Cruse, J. M., Lewis, R. E.,
Eds.: Karger: Basel, 1989; Vol. 10, pp 48-114. (b) Jennings, H. J.;
Sood, R. K. In Neoglycoconjugates. Preparation and Applications; Lee,
Y., Lee, R., Eds.; Academic Press: New York, 1994; Chapter 10, pp
325-371. (c) Pozsgay, V. Adv. Carbohydr. Chem. Biochem. 2003, 56,
153-199.
These limitations may be circumvented if the amide
link is created by a chemoselective ligation reaction. This
is the coupling of two mutually and uniquely reactive
(4) (a) Staros, J. V.; Wright, R. W.; Swingle, D. M. Anal. Biochem.
1986, 156, 220-222. (b) Bioconjugate Techniques; Hermanson, G. T.,
Ed.; Academic Press: San Diego, 1995. (c) Wu, X.; Ling, C.-C., Bundle,
D. R. Org. Lett. 2004, 6, 4407-4410.
10.1021/jo0505472 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/11/2005
J. Org. Chem. 2005, 70, 7123-7132
7123

Grandjean et al.
SCHEME 1. Principle of the Staudinger Reaction and of Its Chemoselective Ligation Variant
functional groups on two biomolecules in an aqueous
environment.5 The direct coupling of an azide with a thio
acid to give the corresponding amide, in the absence of
protecting groups, has been recently reported.6 However,
transition metals, trace amounts of which are difficult
to remove from the product, are sometimes required to
promote the reaction. Also, the utility of this azide/thio
acid condensation for the preparation of biomolecules has
not yet been demonstrated.
aqueous medium and results in amide-linked products
(Scheme 1). One of the three aryl groups on the phos-
phorus atom is functionalized by an ester, such as a
methyl ester, at the position ortho to the phosphorus. The
aza-ylide now reacts preferentially via a nucleophilic
attack of the nitrogen atom on the ester, eliminating a
methoxide, rather than being hydrolyzed. This transfor-
mation was so efficient that it was executed at the surface
of living cells.7a
The Staudinger ligation, which couples two biomol-
ecules bearing an azide and an engineered trivalent
phosphine, respectively, has been used successfully for
cell surface engineering, for probing post-translational
modification, for coating microarrays, and for the block-
wise synthesis of peptides and proteins.7-9 The Staudinger
reaction10 consists of the nucleophilic attack of a phos-
phine on an azide to give a phosphazide, which through
the loss of nitrogen gives a reactive intermediate aza-
ylide, which is further hydrolyzed upon addition of water
to give a phosphine oxide and an amine.11 This reaction
tolerates a wide range of reactants. It is mainly limited
by the sensitivity of the phosphine to oxidation. Careful
design of a triarylphosphine allowed Saxon and Bertozzi
to trap the aza-ylide intermediate intramolecularly. This
avoids shortcut hydrolysis of the aza-ylide in a truly
However, any bioconjugate produced by using this
method contains a triarylphosphine oxide residue. The
presence of this phosphine oxide may not be suitable for
preparing glycoconjugate vaccines, and it is highly desir-
able to remove it from the actual glycoconjugates. Indeed,
this side product possesses all characteristics of a hap-
ten,1 although it has not yet been tested. Therefore, the
desired immune response may be partly diverted or even
absent, preventing extensive use of this method in the
development of immunogens. For instance, partial diver-
sion of the immune response has been occasionally
documented for maleimide spacers:12 when observed in
the series, their immunogenicity was shown to increase
from the aliphatic members to the alicyclic ones, with
the aromatic linkers being the most immunogenic.
Saxon and Bertozzi13 and Nilsson et al.8a have inde-
pendently prepared a new generation of phosphines
which are released from the conjugated product, in a so-
called traceless Staudinger ligation, to give a true amide
link, thus allowing broader use of the reaction. In this
new generation, the phosphine is incorporated in the
leaving group and not in the transferred acyl group
(Chart 1).
(5) Lemieux, G. A.; Bertozzi, C. R. TIBTECH 1998, 16, 506-513.
Peri, F.; Nicotra, F. Chem. Commun. 2004, 623-627.
(6) Shangguan, N.; Katukojvala, S.; Greenberg, R., Williams, L. J.
J. Am. Chem. Soc. 2003, 125, 7754-7755. Fazio, F.; Wong, C.-H.
Tetrahedron Lett. 2003, 44, 9083-9085.
(7) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010. (b)
Vocadlo, D. J.; Hang, H. C.; Hanover, J. A.; Bertozzi, C. R. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 9116-9121. (c) Kiick, K. L.; Saxon, E.,
Tirell, D. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
19-24. (d) Ko¨hn, M.; Wacker, R.; Peters, C.; Schro¨der, H.; Soule`re, L.;
Breinbauer, R.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int.
Ed. 2003, 42, 5830-5834.
Previous studies have shown the difficulty in tuning
the structure of the phosphine to avoid the two main
limitations: premature oxidation of the phosphorus atom,
(8) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001,
3, 9-12. (b) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Org. Chem.
2002, 67, 4993-4996. (c) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.;
Raines, R. T. J. Am. Chem. Soc. 2003, 125, 5268-5269.
(9) For a review, see: Ko¨hn, M.; Breinbauer, R. Angew. Chem., Int.
Ed. 2004, 43, 3106-3116.
(12) (a) Peeters, J. M.; Hazendonk, T. G.; Beuvery, E. C.; Tesser, G.
I. J. Immunol. Methods 1989, 120, 133-143. (b) Boeckler, C.; Frisch,
B.; Muller, S.; Schuber, F. J. Immunol. Methods 1996, 191, 1-10. (c)
Buskas, T.; Li, Y.; Boons, G.-J. Chem. Eur. J. 2004, 10, 3517-3524.
(13) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2,
2141-2143.
(10) Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635-646.
(11) Tin, W. Q.; Wang, Y. A. J. Org. Chem. 2004, 69, 4299-4308.
7124 J. Org. Chem., Vol. 70, No. 18, 2005

Preparation of Carbohydrate-Protein Conjugates
CHART 1. Known Phosphines Designed To Test
the Traceless Staudinger Ligation
expected acid 12 in an acceptable 60% yield. Subsequent
deprotection with DABCO gave 10a in a quantitative
yield. However, although the phosphorus atom was
stabilized by two phenyl groups, this compound was
highly susceptible to deleterious oxidation. Isolation of
the phosphino-thioester was extremely difficult, with the
purified product being partially oxidized to 10b during
analysis.15
To avoid a premature oxidation, we used the inductive
effect of p-chloro substituents on the phenyl groups to
stabilize the phosphine. The halogen atoms could also
favor intramolecular acylation by increasing the nucleo-
philic character of the nitrogen atom of the aza-ylide
intermediate. Thus, chloromethylphosphonic dichloride
was reacted with commercially available (4-chlorophe-
nyl)magnesium bromide as described by Herd et al.16 to
give intermediate 13, which was then treated with
potassium thioacetate to give 14 in 54% overall yield.
Potassium thioacetate proved better for the nucleophilic
substitution than the previously used thioacetic acid/
triethylamine mixture.8a Thioester 14 was then deacety-
lated with sodium methoxide to give an intermediate
thiol, which was trapped with glutaric anhydride to afford
acid 15 in 53% yield. Reduction of 15 with excess
trichlorosilane gave air-stable phosphino-thioester 16.
We then investigated whether these phosphino-func-
tionalized cross-linkers were suitable for a Staudinger
ligation process. In preliminary experiments, we used the
known 2-azidoethyl 2-acetamido-2-deoxy-â-D-glucopyra-
noside 1717 as a model azide (Scheme 3).
We obtained the required amide 18 in 30% yield on
reaction of phosphine 8, which was shown to be inert
toward oxidation, with azide 17 in CH3CN/H2O. As for
compound 2,13 the reaction proceeded very slowly and
reached completion after 96 h. However, unlike for
compound 2, there was predominant hydrolysis of the
aza-ylide intermediate resulting in amine 19 being
formed in 70% yield (Table 1, entry a). This may be due
to increased steric hindrance in the intermediates when
replacing the acetyl in 2 by the longer acyl chain in 8.
Merkx et al.18a and Bianchi et al.18b have recently
reported similar results when attempting to synthesize
peptides or glycopeptides using closely related C-terminal
peptide or amino acid o-(diphenylphosphine)phenyl es-
ters.
as in dicyclohexylphosphine 5, and competitive hydrolysis
of the iminophosphorane intermediate, as for 1 and 3.
The use of compound 413 overcame these problems but
needed prolonged reaction times, whereas phosphines 213
and 68a did not need such long reaction times. Therefore,
we designed new phosphino-functionalized cross-linkers
derived from these two reagents but adapted them to
allow construction of potential glycoconjugate vaccines.
In this paper, we report the synthesis of such cross-
linkers. We assessed their stability toward oxidation
when stored either neat or in aqueous solution We
evaluated their reactivity in model traceless Staudinger
ligations. The most promising reagents were activated
as a succinimidyl ester and coupled to tetanus toxoid (TT)
or bovine serum albumin (BSA). The resulting phosphine-
derivatized carriers were further conjugated to either
synthetic bacterial carbohydrates or purified carbohy-
drates functionalized with a pendent azido group. Finally,
we assessed the efficiency of the ligation by determining
the carbohydrate/protein ratio and evaluating the anti-
genicity of the glycoconjugates.
Results and Discussion
Synthesis and Evaluation of Phosphino-Func-
tionalized Cross-Linkers. The synthesis of phosphane
cross-linkers bearing a carboxylic function was easily
devised as the derivatization of proteins using activated
ester reagents is an extremely convenient and well-
known technique.14 Thus, o-(diphenylphosphino)phenol
7, previously used by Saxon et al. as a precursor of
compound 2,13 was reacted gently with glutaric anhydride
to give the carboxy-functionalized phosphine 8 in about
74% yield (Scheme 2). Next, we attempted to prepare the
phosphino-thioester 10a from the known thioacetate 9.8b
According to a one-pot, two-step procedure, thioacetate
9 was treated with sodium methoxide and glutaric
anhydride, yielding several products. Since a side product
resulting from the premature oxidation of the phosphorus
atom was isolated, we turned to the borane protected
phosphine 11,8 a synthetic precursor of 9. The reaction
of 11 under the previously mentioned conditions gave the
When the condensation was carried out with the air-
stable phosphino-thioester 16, amide 18 was formed as
the major product with only traces of the hydrolyzed
product 19. However, the reaction stopped at 40%
conversion, using a 2:1 molar ratio of 16 versus 17, with
the remaining phosphane 16 being completely oxidized
(Table 1, entry c). TLC of pure phosphine 16, in aceto-
nitrile, confirmed its susceptibility toward oxidation.
Therefore, we developed a one-pot deprotection and
(15) The propensity of the phosphino-thioesters to be oxidized has
also been noticed by Professor R. T. Raines who, now, deprotects these
reagents immediately prior to their use for conjugation. Personal
communication.
(16) Herd, O.; Hessler, A., Hingst, M.; Machnitzki, P.; Tepper, M.;
Stelzer, O. Catal. Today 1998, 42, 413-420.
(17) Hasegawa, A.; Terada, T.; Ogawa, H.; Kiso, M. J. Carbohydr.
Chem. 1992, 11, 319-331.
(18) (a) Merkx, R.; Rijkers, D. T. S.; Kemmink, J.; Liskamp, R. M.
J. Tetrahedron Lett. 2003, 44, 4515-4548. (b) Bianchi, A.; Russo, A.;
Bernardi, A. Tetrahedron: Asymmetry 2005, 16, 381-386.
(14) Bioconjugate Techniques; Hermanson, G. T., Ed.; Acadamic
Press: San Diego, 1995; pp 417-671.
J. Org. Chem, Vol. 70, No. 18, 2005 7125

Grandjean et al.
SCHEME 2. Synthesis of Phosphino-Functionalized Crosslinkersa
a Reagents and conditions: (a) NaOMe (1.1 equiv), then glutaric anhydride (0.9 equiv), DMF, 0 °C to rt, 30 min; (b) DABCO (1 equiv),
toluene, 40 °C, 4 h, quantitative; (c) ClPhMgBr (2 equiv), THF, reflux, 24 h; (d) KSAc (1.1 equiv), THF, reflux, overnight; (e) HSiCl3 (15
equiv), ClCH2CH2Cl, reflux, 72 h.
SCHEME 3. Model Staudinger Ligation
Staudinger ligation procedure using synthetic intermedi-
ate 12, whose phosphorus atom is protected against
oxidation. This method has recently been used to prepare
lactams. In the lactam synthesis, the phosphine was kept
protected as the borane complex until the cyclization step
to avoid a spontaneous, premature, Staudinger ligation.19
As expected, when 17 and 12 were heated in the presence
of a slight excess of a strong base, such as DABCO, amide
18 was formed almost quantitatively (Table 1, entry d).
Moreover, RP-HPLC showed that phosphine 12 was not
significantly degraded over a 1 month period when kept
at 4 °C in various aqueous solutions ranging from pH
5.5 to 8.3. Stability in aqueous solution is important as
common protein carriers such as TT are kept in aqueous
buffers once derivatized. Finally, unreacted phosphane
was removed from the carrier by hydrolysis of 12 and 8
with an excess of hydroxylamine (Table 1, last column),
to yield the corresponding glutaryl-modified protein.
We selected compounds 12 and 8 for the preparation
of glycoconjugates. They were activated into their corre-
sponding succinimide ester 20 and 21 in 90% and 63%
yield, respectively (Scheme 4).
Preparation of Carbohydrate-Protein Conju-
gates. A large excess of heterobifunctional cross-linker
20 was used to derivatize ꢀ-amino groups of TT lysines
(19) David, O.; Meester, W. J. N.; Biera¨ugel, H.; Schoemaker, H.
E.; Hiemstra, H.; van Maarseveen, J. H. Angew. Chem., Int. Ed. 2003,
42, 4373-4375.
7126 J. Org. Chem., Vol. 70, No. 18, 2005

Preparation of Carbohydrate-Protein Conjugates
TABLE 1. Properties of the Phosphino-functionalized Crosslinkers
yields of productsa (%)
experimental
conditions
entry
a
phosphine
stability
azide 17
amide 18
amine 19
hydrolysisb
8
stable upon storage
neat or in solution
30
70
rt, 96 h
200 equiv of NH2OH
52% after 1 h
95% after 4 h
b
c
10a
16
rapidly oxidizedc
stable (stored neat)
rapidly oxidized
in solution
stable (stored neat
or in solution)
61
37
2
rt, 96 h
d
12
>95
<5
DABCO (4 equiv),
40 °C, 4 h
50 equiv of NH2OH,
>95% after 1 h
a Yield determined by RP-HPLC. b Reaction performed in potassium phosphate buffer 0.2 M, pH 8.3. c Too unstable to be determined.
SCHEME 4. Activation of
SCHEME 5. Derivatization and Conjugation of
the Protein Carriersa
Phosphino-functionalized Crosslinkersa
a Reagents and conditions: (a) HOSu (1 equiv), EDC (1 equiv),
CH2Cl2, overnight.
to give the phosphino-functionalized carrier 22. Analo-
gously, reaction of cross-linker 21 with TT or BSA gave
the corresponding protein carriers 23 and 24, respectively
(Scheme 5). Due to its low solubility, we used a somewhat
smaller but still large excess of borane-phosphine com-
plex 21.
We compared the SELDI-TOF (surface enhance laser
desorption ionization-time of flight) mass spectra of these
different preparations with those of the parent protein
to determine the extent of derivatization (Figure 1). The
degrees of derivatization were in the range of 10-20 mol
of phosphane per mole of protein depending on the assay
and on the protein, with a higher substitution being
observed for BSA. These rates remained inferior to those
we obtained when using either highly hydrophilic or
small cross-linkers, such as maleimide-functionalized or
bromoacetylated succinimidyl esters. For these cross-
linkers, 30-40 linker-to-protein molar ratios were typi-
cally observed.20 After derivatization, 24 was reacted with
an excess of hydroxylamine at pH 8.3 and the resulting
protein analyzed by SELDI-TOF mass spectroscopy
(Figure 1e). This confirmed that the phosphane residues
could be completely removed from the protein, resulting
in a modified protein bearing only glutaryl chains.
We next prepared carbohydrate haptens bearing a
complementary azide group. As a representative ex-
ample, we selected a synthetic pentasaccharide 34,
corresponding to the biological repeating unit of the O-SP
of Shigella flexneri 2a LPS (Chart 2).21
a Reagent and conditions: (a) Potassium phosphate buffer 0.1
M, pH 7.4, 47 °C, 6 h; (b) DABCO (40 + 40 equiv), DMF/NaCl
0.05 M 5:1, 40-45 °C, 4 h. m and p are average values. For further
details, see Table 2.
We also used the polysaccharide moiety (pmLPS) of the
lipopolysaccharide (LPS) of Vibrio cholerae O1 serotype
Inaba, a causative agent of cholera. This surface polysac-
charide is the major target of the human’s protective
immune response against this disease.22 Thus, LPS,
purified from bacterial culture, was treated with mild
acid treatment to remove the lipid A, giving the corre-
sponding pmLPS 35 in about 25% yield (Chart 2).20 Both
compounds 34 and 35 possess a unique amine functional-
ity which can be used for derivatization. Amines can be
(20) Grandjean, C.; Boutonnier, A.; Dassy, B.; Mulard, L. A.;
Fournier, J.-M. Unpublished results.
(21) Wright, K.; Guerreiro, C.; Laurent, I.; Baleux, F.; Mulard, L.
A. Org. Biomol. Chem. 2004, 2, 1518-1527.
(22) Robbins, J. B.; Scheerson, R.; Szu, S. C. In New generation of
Vaccines. Second Edition, Revised and Expanded; Levine, M. M.,
Woodrow, G. G., Kapper, J. B., Cobon, G. S., Eds.; Marcel Dekker: New
York, 1997; Chapter 52, pp 803-815.
J. Org. Chem, Vol. 70, No. 18, 2005 7127

Grandjean et al.
FIGURE 1. SELDI-TOF-MS analysis of TT, BSA, and their phosphino-functionalized derivatives. The average content of phosphine
residues was determined on the basis of a 374 or 342 Da mass increment per incorporated molecule of 20 or 21, respectively: (a)
TT; (b) 22, average ratio phosphine/protein: 9.5; (c) 23, average ratio phosphine/protein: 12.4; (d) BSA; (e) 24, average ratio
phosphine/protein: 17.4; (f) 24 after NH2OH treatment for 1 h, theoretical average molecular mass: 68,562 Da. For further
details, see the Supporting Information.
SCHEME 6. Synthesis of Azido-Functionalized
Linkersa
We then coupled 34 with 37 to give the azido-function-
alized pentasaccharide (penta-C3N3, 25) in 70% yield.
Similarly, we coupled 39 and 41 with pmLPS 35 in
PBS (pH 7.3) to give the azido-containing derivatives
[pmLPSC6N3, 26 and pmLPSC12N3, 27] in 38 and 58%
yield, respectively. We recovered 20-25% of the starting
polysaccharide after RP-HPLC purification. We charac-
terized 26 and 27 by MALDI-TOF-MS analysis. Com-
parison of their MS spectra with that of the parent
compound 35 showed an increase of 140 or 253 Da,
respectively, for all signals from the polymeric mixture.
These were consistent with the introduction of a single
azido-linker on 35.
For the traceless Staudinger ligation we found we could
not use a strong base such as DABCO under the previ-
ously mentioned conditions to couple 26 or 27 with the
derivatized proteins 23 or 24. We found predominant,
premature cleavage of the thioester bond under these
conditions. In aqueous buffers, the pH was either too
acidic or too basic for the decomplexation to occur. Under
basic conditions, hydrolysis was strongly favored com-
pared to the decomplexation-iminophosphorane forma-
tion/acyl transfer sequence. However, we found accept-
able levels of Staudinger products in the organic medium
where the ion pairs are only slightly dissociated and the
pH effect is minimized. Activated carriers 23 and 24
tended to precipitate in acetonitrile, which had been used
in our initial assays. Therefore, 5/1 (v/v) DMF/aq NaCl
0.05 M was typically used for the conjugation. Thus,
haptens 25, 26 and 27 were successfully coupled to TT
derivative 23 under these conditions to give conjugates
28, 29 and 30, referred to as TT-C3Penta, TT-C6pmLPSI
and TT-C12pmLPSI, respectively. We also conjugated 26
to BSA-derivative 24 to give 31, referred to as BSA-C6-
pmLPS (Scheme 5 and Table 2). Similarly, phosphino-
functionalized TT 22 was reacted with 26 or 27 according
to the optimized procedure developed by Kiick et al.7c to
give conjugates 32 and 33, referred as TT-C6pmLPSII and
TT-C12pmLPSII, respectively (Scheme 5 and Table 2).
a Reagents and conditions: (a) NaN3 (1.5 equiv), CH3CN, reflux,
3 h, 51%; (b) HOSu (1 equiv), EDC (1 equiv), CH2Cl2, rt, overnight;
(c) NaN3 (2 equiv), DMF, 85 °C, 3 h, 82%; (d) 6-aminocaproic acid
(1 equiv), Et3N (2 equiv), DMF, rt, 4 h, quantitative.
selectively converted into azides by a metal-catalyzed
diazo transfer reaction24 or by coupling to azido-func-
tionalized linkers.25 Using the latter approach, we reacted
3-bromopropionic acid or 6-bromocaproic acid with so-
dium azide to give intermediates 36 and 38 in 51% and
82% yield, respectively (Scheme 6). Treatment of these
derivatives with N-hydroxysuccinimide gave the corre-
sponding activated esters 37 and 39 in 51% and 80%
yield, respectively. Partial water solubility of compounds
36 and 37 resulted in a loss of product during workup,
explaining the observed modest yields. We then reacted
compound 39 with 6-aminocaproic acid to afford deriva-
tive 40, which was transformed into the long N-hydrox-
ysuccinimidyl ester spacer 41 in 87% overall yield.
(23) Vinogradov, E. V.; Bock, K.; Holst, O.; Brade, H. Eur. J.
Biochem. 1995, 233, 152-158.
(24) (a) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett.
1996, 37, 6029-6032. (b) Nyffeler, P. T.; Liang, C.-H.; Koeller, K. M.;
Wong, C.-H. J. Am. Chem. Soc. 2002, 124, 10773-10778.
(25) Wang, C. C.-Y.; Seo, T. S.; Li, Z.; Ruparel, H.; Ju, J. Bioconjugate
Chem. 2003, 14, 697-701.
All conjugates were isolated between 45% and 80%
yields (Table 2). The carbohydrate contents were esti-
mated using a colorimetric method, based on the an-
7128 J. Org. Chem., Vol. 70, No. 18, 2005

Preparation of Carbohydrate-Protein Conjugates
CHART 2. Structures of the Model Carbohydrate Haptens and of Their Azido-Functionalized Derivativesa
a The pmLPS structure is according to Vinogradov et al.23 The point of attachment (dotted line) between the specific polysaccharide
(O-SP) and the core has not yet been clearly established.
throne reaction.27 Carbohydrate/protein molar ratios were
in the range of 2.7-7.0. These values can be easily
explained by the low degree of derivatization of the
protein carriers and the small excess of carbohydrate
haptens used for the ligation reactions (12-20 equiv/
protein). Competitive hydrolysis of the aza-ylide inter-
mediates when using carrier 22 or of the thioester bonds
when using carriers 23 or 24 may also account for the
overall moderate sugar/protein ratios.
We assessed the antigenicity of derivatives 29-33,
obtained from Inaba pmLPS, by ELISA inhibition assays,
to test whether the functionality of the carbohydrate
haptens was altered upon conjugation (Table 3). All
conjugates inhibited the interaction between V. cholerae
O1 serotype Inaba LPS and the monoclonal antibody
I-24-2. This is an IgG3 known to possess a protective
activity against V. cholerae O1 oral challenge in the
suckling mouse model.28 We could not strictly compare
the different conjugates because of the differences in their
extent of derivatization. Antigenicity of glycoconjugates
29 and 30 was somewhat lower than antigenicity of Inaba
LPS used as a reference compound. This apparent loss
may be caused by the use of an organic solvent, namely
DMF, and/or heating to perform the conjugation. How-
ever, we found that 31, obtained using the same protocol
from BSA instead of TT, as well as 32 and 33 were almost
as potent as Inaba LPS. These latter results suggested
that, for these glycoconjugates, neither pmLPS deriva-
tization nor the ligation reaction conditions strongly
altered the unique antigenic determinant recognized by
mIgG I-24-2. This determinant is known to involve
residues from both the core and the O-SP.29
(26) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J.
Biol. Chem. 1951, 193, 265-275.
(27) Herbert, D.; Phipps, P. J.; Strange, R. E. Methods Microbiol.
1971, 5B, 209-344.
(28) Bougoudogo, F.; Ve´ly, F.; Nato, F.; Boutonnier, A.; Gounon, P.;
Mazie, J.-C.; Fournier, J.-M. Bull. Inst. Pasteur 1995, 93, 273-283.
(29) Villeneuve, S.; Boutonnier, A.; Mulard, L. A.; Fournier, J.-M.
Microbiology 1999, 145, 2477-2484.
J. Org. Chem, Vol. 70, No. 18, 2005 7129

Grandjean et al.
TABLE 2. Characterization of the Conjugates
carbohydrate
derivatized
carriera
sugar/protein
ratio (mol/mol)
entry
a
conjugate
hapten
methodb
A
yieldc (%)
50
28
25
23
12.4
23
12.4
23
12.4
24
17
22
5.1
7.3
6.5
7.0
2.7
3
pentaC3N3
26
b
c
d
e
f
29
30
31
32
33
A
A
A
B
B
70
60
52
59
83
pmLPSC6N3
27
pmLPSC12N3
26
pmLPSC6N3
26
pmLPSC6N3
27
pmLPSC12N3
9.5
22
9.5
a Compound number followed by the extent of derivatization. b Method A: DABCO (40 + 40 equiv), DMF/aq NaCl 0.05 M 5:1, 40-45
°C, 4 h. Method B: Potassium phosphate buffer 0.1 M, pH 7.4, 47 °C, 6 h. c Yields were calculated on the basis of the weight of protein
in the conjugates compared with the amount of starting protein estimated by the Lowry method using BSA as the standard.26
TABLE 3. Inhibition of the Interaction between Inaba
Succinimidyl 2-(Diphenylphosphanyl)phenyl Glut-
arate 20. To a stirred solution of 8 (418 mg, 1.07 mmol) and
N-hydroxysuccinimide (122 mg, 1.07 mmol) in CH2Cl2 (4 mL)
was added EDC (205 mg, 1.07 mmol), and the mixture was
stirred at rt overnight. The crude reaction mixture was diluted
with CH2Cl2, and this solution washed with 0.1 N aq HCl and
then brine. The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure.
The crude residue was purified by chromatography on silica
gel (eluent: CH2Cl2/EtOAc 96:4) to give the succinimidyl ester
20 (470 mg, 90%) as a colorless oil: Rf ) 0.71 (EtOAc/CH2Cl2
4:96); 1H NMR (400 MHz, CDCl3) δ 7.41-7.32 (m, 11 H), 7.17
(t, 1 H, J ) 3.8 Hz), 7.15 (t, 1 H, J ) 7.1 Hz), 6.88-6.85 (m, 1
H), 2.74 (br s, 4 H), 2.59 (t, 2 H, J ) 7.3 Hz), 2.43 (t, 2 H, J )
7.2 Hz), 1.93 (q, 2 H, J ) 7.2 Hz; 13C NMR (100 MHz, CDCl3)
δ 170.8, 169.6 (2 C), 168.5, 153.0 (d, 1 C, J ) 17.3 Hz), 135.8
(d, 2 C, J ) 10.0 Hz), 134.4 (d, 4 C, J ) 20.6 Hz), 134.1 (d, 1
C, J ) 0.9 Hz), 130.7 (d, 1 C, J ) 14.7 Hz), 130.4, 129.6 (2 C),
129.1 (d, 4 C, J ) 7.3 Hz), 126.7, 123.0, 32.8, 30.3, 26.0 (2 C),
19.8; 31P NMR (162 MHz, CDCl3) δ 15.0; positive FAB-MS m/z
522.2 [M + Na]+, 490.2 [M + H]+. Anal. Calcd. for C27H24NO6P‚
1/3H2O: C, 65.45; H, 5.02; N, 2.83; O, 20.45. Found: C, 65.54;
H, 4.87; N, 2.73; O, 20.19.
Borane-{4-Carboxythiobutyric acid S-[(diphenylphos-
phanyl)methyl] ester} Complex 12. Compound 11 (520 mg,
1.80 mmol) was dissolved in DMF (2.8 mL) and degassed under
vacuum. Argon was then bubbled through the solution cooled
at 0 °C for 45 min. Sodium methoxide (108 mg, 2.0 mmol) was
added in several portions at 0 °C. The mixture was stirred at
0 °C for 30 min, and then glutaric anhydride (186 mg, 1.62
mmol) was added. The reaction mixture was stirred for another
20 min at 0 °C, poured over an ice-water mixture, and
extracted with CH2Cl2. The organic layer was washed with
0.1 M aq citrate buffer at pH 3, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude residue
was purified by flash chromatography on silica gel (eluent:
cyclohexane/EtOAc 6:4) to give the acid 12 (390 mg, 60%) as
an oil: Rf ) 0.18 (EtOAc/cyclohexane 3:7); 1H NMR (400 MHz,
CDCl3) δ 10.18 (br s, 1 H), 7.68-7.54 (m, 4 H), 7.48-7.30 (m,
6 H), 3.73 (d, 2 H, J ) 6.7 Hz), 2.55 (t, 2 H, J ) 7.3 Hz), 2.25
(t, 2 H, J ) 7.3 Hz), 1.84 (q, 2 H, J ) 7.3 Hz), 1.51-0.52 (br
m, 3 H; 13C NMR (100 MHz, CDCl3) δ 196.5, 178.7, 133.0 (d,
4 C, J ) 9.4 Hz), 132.1 (d, 2 C, J ) 2.3 Hz), 129.3 (d, 4 C, J )
10.1 Hz), 127.9 (d, J ) 55.5 Hz), 42.7, 32.9, 23.8 (d, J ) 35.2
Hz), 20; 31P NMR (162 MHz, CDCl3) δ 20.6 (d, J ) 41.5 Hz);
positive CI-MS m/z 378 [M + NH4]+, 362 [M + H]+, 347 [M +
BH3]+; HRMS (FAB) calcd for [M + H - H2]+ 359.1046, found
359.1042.
LPS and MAb I-24-2 by LPS or Conjugates 29-33
a
entry
inhibitor
IC50 (ng‚mL-1
)
a
b
c
d
e
f
V. cholerae O1 Inaba LPS
TT-C6pmLPSI
45
820
410
100
110
100
29
30
31
32
33
TT-C12pmLPSI
BSA-C6pmLPS
TT-C6pmLPSII
TT-C12pmLPSII
a The amount of inhibitor is calculated from its estimated sugar
content.
Conclusion
The traceless Staudinger ligation reaction has been
successfully applied to the conjugation of carbohydrate
haptens to immunogenic protein carriers, giving a fully
stable and biocompatible amide link between the haptens
and protein carriers. Further work should aim at in-
creasing the water solubility of the phosphino-function-
alized linker. This should give greater derivatization of
the carrier and, consequently, contribute to an improved
sugar/protein ratio. A phosphine that is totally stable
toward oxidation, which would allow intramolecular
acylation in the absence of an organic cosolvent, is still
in need.
Experimental Section
2-(Diphenylphosphanyl)phenyl Glutarate 8. Diphenyl-
(2-hydroxyphenyl)phosphine 7 (758 mg, 2.72 mmol) was dis-
solved in dry DMF (3 mL) under argon at 0 °C, and sodium
hydride (60% dispersion in oil, 120 mg, 3 mmol, 1.1 equiv) was
added in several portions. The reaction mixture was stirred
at 0 °C for 20 min, and then solid glutaric anhydride (311 mg,
2.73 mmol) was added. After 30 min at 0 °C, the reaction
mixture was poured over 0.1 N HCl and extracted with CH2-
Cl2. The organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude residue was
purified by flash chromatography on silica gel (cyclohexane/
EtOAc 8:2) to give acid 8 (786 mg, 74% for the two steps) as a
colorless oil: Rf ) 0.46 (EtOAc/cyclohexane 3:7); 1H NMR (400
MHz, CDCl3) δ 10.81 (br s, 1 H), 7.83-7.74, 7.68-7.58, 7.55-
7.49, 7.48-7.36, 7.27-7.13 and 7.94-7.90 (m, 14 H), 2.48-
2.40 (m, 4 H), 1.92 (q, 2 H, J ) 7.2 Hz); 13C NMR (100 MHz,
CDCl3) δ 179.4, 171.3, 153.1 (d, J ) 17.1 Hz), 136.0-129.1 (12
C), 126.7, 123.1, 33.3 (2 C), 19.8; 31P NMR (162 MHz, CDCl3)
δ 14.8; positive CI-MS m/z 393 [M + NH4]+; HR-MS (FAB)
m/z 393.1276 (calcd for [M + H]+ C23H22BO4P 393.1256).
Preparation of Succinimidyl Derivative 21. To a solu-
tion of acid 12 (500 mg, 1.39 mmol) and N-hydroxysuccinimide
(160 mg, 1.39 mmol) in CH2Cl2 (4 mL) was added EDC (267
mg, 1.39 mmol), and the mixture stirred at rt overnight. The
7130 J. Org. Chem., Vol. 70, No. 18, 2005

Preparation of Carbohydrate-Protein Conjugates
crude reaction mixture was diluted with CH2Cl2 and washed
with 0.1 N aq HCl and then brine. The organic layer was dried
over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude residue was purified by chroma-
tography on silica gel (cyclohexane/EtOAc 7:3) to give the
succinimidyl ester 21 (400 mg, 63%) as an oil: Rf ) 0.32
(EtOAc/cyclohexane 4:6); 1H NMR (400 MHz, CDCl3) δ 7.76-
7.70 (m, 4 H), 7.58-7.45 (m, 6 H), 3.74 (d, 2 H, J ) 6.6 Hz),
2.86 (br s, 4 H, 2 CH2), 2.62 (t, 2 H, J ) 7.2 Hz), 2.50 (t, 2 H,
J ) 7.4 Hz), 1.95 (q, 2 H, J ) 7.3 Hz), 1.51-0.52 (br m, 3 H);
13C NMR (100 MHz, CDCl3) δ 196.0, 169.3 (2 C), 168.1, 133.0
(d, 4 C, J ) 9.4 Hz), 132.2 (d, 2 C, J ) 2.3 Hz), 129.2 (d, 4 C,
J ) 10.1 Hz), 127.8 (d, J ) 55.4 Hz), 42.1, 30.0, 26.0 (2 C),
23.8 (d, J ) 35.2 Hz), 20.7; 31P NMR (162 MHz, CDCl3) δ 20.73
(d, J ) 64.3 Hz); positive CI-MS m/z 475 [M + NH4]+, 444 [M
+ BH2]+; HRMS (FAB) calcd for [M + H - H2]+ 456.1210,
found 456.1200.
buffer saline, pH 7.3 (0.9-1.5 µL), was added the succinimidyl
ester 39, or 41 [3 × 10 equiv dissolved in CH3CN (50 µL)], in
three portions every 2 h. Following an additional reaction
period of 2 h, the crude reaction mixture was purified by RP-
HPLC (gradient: 0% B for 5 min then 0-30% B over 60 min).
The collected fractions were diluted with H2O, frozen, and
freeze-dried to give the corresponding activated-pmLPS 26, or
27, as a white foam. pmLPSC6N3 26 (8.5 mg, 38%) was
obtained as well as recovered starting material 35 (5.2 mg,
24%): (negative MALDI-TOF-MS) m/z 7197, 6951, 6702, 6455,
6207, 5960, 5713, 5466, 5219, 4970 (M - H)-, 7169, 6922, 6675,
6429, 6181, 5933, 5686, 5438, 5192 (M - N2 - H)- (22-mer to
14-mer). pmLPSC12N3 27 (7.3 mg, 58%) was obtained as well
as recovered starting material 35 (2.0 mg, 20%): (negative
MALDI-TOF-MS) m/z 7312, 7066, 6814, 6572, 6321, 6070 (M
- H)- (22-mer to 17-mer).
Derivatization of the Protein Carriers. In a typical
experiment, a stock solution of TT (18 mg, 820 µL, 0.12 µmol)
was diluted with 0.2 M potassium phosphate buffer saline, pH
7.3 (340 µL). To this solution was added succinimidyl 2-(diphe-
nylphosphanyl)phenyl glutarate 20 (3 × 2.61 mg dissolved in
50 µL of CH3CN) or the borane-phosphine complex 21 (3 ×
1.63 mg dissolved in 100 µL of CH3CN) in three portions every
2 h. Following an additional reaction period of 4 h, the crude
reaction mixtures were dialyzed against 0.1 M potassium
phosphate buffer, pH 6 (3 × 2 L over 2 days), at 4 °C to remove
excess reagent. Phosphino-functionalized TT 22 and 23 were
used without further purification.
Similarly, BSA (5 mg, 0.08 µmol) was diluted with 0.2 M
potassium phosphate buffer saline, pH 7.3 (340 µL). To this
solution was added the borane-phosphine complex 21 (3 ×
1.02 mg dissolved in 100 µL of CH3CN, 3 × 30 equiv), in three
portions every 2 h. Following an additional reaction period of
4 h, the crude reaction mixture was dialyzed against 0.1 M
potassium phosphate buffer, pH 6 (3 × 2 L over 2 days) at 4
°C to remove excess reagent. Phosphino-functionalized BSA
24 was used without further purification. The extent of
derivatization of these protein carriers was determined by
comparison of the SELDI-TOF mass spectra of compounds 22-
24 with those of their parent protein.
6-Azidohexanoic Acid 38. To a stirred solution of 6-bro-
mohexanoic acid (1.5 g, 7.7 mmol) in DMF (5 mL) was added
sodium azide (1.0 g, 15.4 mmol), and the mixture was heated
at 85 °C for 3 h. The crude reaction mixture was diluted in
CH2Cl2, and this solution washed with 0.1 N aq HCl. The
organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to give 38 (1.0 g, 82%)
as an oil, which was used without further purification: Rf )
1
0.63 (EtOAc/CH2Cl2 3:7); H NMR (400 MHz, CDCl3) δ 11.00
(br s, 1 H), 3.29 (t, 2 H, J ) 6.9 Hz), 2.39 (t, 2 H, J ) 7.4 Hz),
1.73-1.58 (m, 4 H), 1.49-1.40 (m, 2 H); 13C NMR (100 MHz,
CDCl3) δ 180.3, 51.6, 34.2, 28.9, 26.5, 24.5; CI-MS m/z 175 [M
+ NH4]+.
6-Azidohexanoic Acid Succinimidyl Ester 39. To a
stirred solution of 6-azidohexanoic acid 38 (590 mg, 3.76 mmol,
1 equiv) and N-hydroxysuccinimide (432 mg, 3.76 mmol) in
CHCl3/DMF 9:1 (1 mL) was added EDC (720 mg, 3.76 mmol),
and the mixture stirred at rt overnight. The crude reaction
mixture was diluted with CH2Cl2 and washed with 1 N aq HCl,
5% aq NaHCO3, and then brine. The organic layer was dried
over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to give the succinimidyl ester 39 (760 mg,
80%) as an oil which was used without further purification:
1
Rf ) 0.76 (EtOAc/CH2Cl2 3:7); H NMR (400 MHz, CDCl3) δ
3.29 (t, 2 H, J ) 6.8 Hz), 2.82 (br s, 4 H), 2.62 (t, 2 H, J ) 7.4
Hz), 1.77 (q, 2 H, J ) 7.4 Hz), 1.68-1.56 (m, 2 H), 1.54-1.44
(m, 2 H); 13C NMR (100 MHz, CDCl3) δ 169.6 (2 C), 168.8,
51.5, 31.1, 28.8, 26.2, 26.0 (2 C), 24.5; CI-MS m/z 272 [M +
NH4]+. Anal. Calcd for C10H14N4O4: C, 47.24; H, 5.55; N, 22.04.
Found: C, 47.11; H, 5.55; N, 22.05.
Staudinger Ligation Procedures. Method A. Azido-
activated carbohydrate hapten 25, 26, or 27 was added in one
portion to the corresponding modified TT or BSA, 23 or 24,
respectively, in solution in DMF/aq NaCl 0.05 M 5/1, at a 20:1
molar ratio. DABCO (40 equiv) (at a 60 mg‚mL-1 solution in
H2O) was added to the reaction mixture and further heated
at 45 °C. Another 40 equiv of DABCO was added 1 h later.
The reaction mixture was further heated for another 3 h. Then,
2 M NH2OH,HCl in 0.1 M phosphate buffer, pH 6 (20-40 µL),
was added to the mixture, and the reaction was kept at room
temperature for a further 2 h. Purification and analyses of the
conjugates 28-31 were performed as for the derivatization
procedure.
Method B. Azide-containing oligosaccharides 26 and 27
were mixed with the phosphino-functionalized TT 22 in a 0.2
M phosphate buffer saline solution at a 12:1 molar ratio.
Reaction mixtures were heated at 47 °C for 6 h. The crude
reaction mixtures were then dialyzed against 0.1 M potassium
phosphate buffer saline, pH 7.4 (3 × 2 L over 2 days) at 4 °C,
and further purified by gel permeation chromatography on a
Sepharose CL-6B column (1 m × 160 mm) (Pharmacia Bio-
tech), using 0.1 M potassium phosphate buffer saline, pH 7.4
as eluent, at a flow rate of 0.2 mL.min-1, with optical density
at 280 nm and refractive index detection. The fractions
containing the conjugates were pooled and concentrated using
Vivaspin 15R centrifugal concentrators (Vivascience), having
a membrane cutoff of 10 000 Da, and at a centrifugal force of
5000g. The conjugates 32 and 33 were stored at 4 °C in the
presence of thimerosal (0.1 mg.mL-1) and assessed for total
carbohydrate27 and protein content.26
Derivatization of the Carbohydrate Haptens: Prepa-
ration of 25 and Derivatization of Pentasaccharide 3421
with N-Hydroxysuccinimidyl Ester 37. To a solution of 34
(10.5 mg, 13 µmol) in 0.2 M potassium phosphate buffer saline,
pH 7.3 (1 mL), was added 37 dissolved in CH3CN (50 µL), (3
× 26.9 mg, 3 × 130 µmol) in three portions at rt every 2 h.
After an additional period of 2 h, the crude reaction mixture
was concentrated under reduced pressure and the residue
purified by RP-HPLC (gradient: 0-5% B over 15 min). The
collected fractions were diluted with H2O, frozen, and freeze-
dried to give the corresponding activated pentasaccharide 25
as a white foam (8.5 mg, 70%): 1H NMR (400 MHz, D2O)
(selected data) δ 5.13 (d, 1 H, J ) 2.7 Hz), 5.00 (br s, 1 H),
4.90 (br s, 1 H), 4.73 (br s, 1 H), 4.48 (d, 1 H, J ) 8.6 Hz), 2.46
(t, 2 H, J ) 6.0 Hz), 1.92 (s, 3 H), 1.27 (d, 3 H, J ) 6.2 Hz),
1.24 (d, 3 H, J ) 6.0 Hz), 1.22 (d, 3 H, J ) 5.9 Hz); 13C NMR
(100 MHz, D2O) (selected data) δ 102.9, 101.5, 101.4, 101.0,
97.9, 81.8, 79.7, 79.4, 76.3, 72.9, 72.4, 72.3, 72.2, 71.7, 71.1,
70.4, 70.4, 70.1, 70.0, 69.7, 69.6, 69.3, 68.8, 68.5, 61.1, 61.0,
55.6, 47.6, 39.7, 35.4, 22.6, 18.2, 17.2, 17.0; positive FAB-MS
m/z 984 [M + Na]+; HRMS (FAB) calcd for [M + Na]+
984.3761, found 984.3777.
Preparation of cOmpounds 26 and 27: Derivatization
of pmLPS 35 with Succinimidyl Esters 39 and 41. To a
solution of 35 (1.5 to 3.6 µmol) in 0.2 M potassium phosphate
J. Org. Chem, Vol. 70, No. 18, 2005 7131

Grandjean et al.
Acknowledgment. We are grateful to Prof. Ronald
T. Raines for advice. We thank Bernadette Coddeville
(Universite´ des Sciences et Technologies de Lille, France)
and Jacques d’Alayer (Institut Pasteur) for the MALDI-
TOF and SELDI-TOF mass spectroscopy experiments,
respectively. We also thank Joe¨l Ughetto-Monfrin (Unite´
de Chimie Organique, Institut Pasteur) for running the
NMR experiments. We are grateful to the Institut
Pasteur for financial support (Programmes Transver-
saux de Recherche).
Supporting Information Available: General procedures,
experimental procedures and analyses for compounds 10a,b,
13-16, 18, 25-27, and 33-41. This material is available free
of charge via the Internet at http://pubs.acs.org.
JO0505472
7132 J. Org. Chem., Vol. 70, No. 18, 2005