Biotin sulfone as a new tool for synthetic oligosaccharide immobilization: Application to multiple analysis profiling and surface plasmonic analysis of anti-Candida albicans antibody reactivity against α and β (1→2) oligomannosides

Article abstract of DOI:10.1021/jm800099g

As a part of our glycoantigen synthetic program for diagnosis and basic analysis of yeast-related pathogenic mechanisms, a library of 1→2 oligomannosides suitable for immunoanalysis was prepared. The use of biotin sulfone, an oxidized form of biotin, offe

Full text of DOI:10.1021/jm800099g

J. Med. Chem. 2008, 51, 6201–6210
6201
Biotin Sulfone as a New Tool for Synthetic Oligosaccharide Immobilization: Application to
Multiple Analysis Profiling and Surface Plasmonic Analysis of Anti-Candida albicans Antibody
Reactivity against r and ꢀ (1f2) Oligomannosides†
Mayeul Collot,‡ Boualem Sendid,§ Aure´lie Fievez,§ Camille Savaux,§ Annie Standaert-Vitse,§ Marc Tabouret,|
Anne Sophie Drucbert, Pierre Marie Danze´, Daniel Poulain,§ and Jean-Maurice Mallet*,‡
Ecole Normale Supe´rieure, De´partement de Chimie, UMR CNRS 8642, 24 rue Lhomond, 75005 Paris, France, Unite´ Inserm 799,
Physiopathologie des Candidoses, Faculte´ de Me´decine, Poˆle Recherche, CHRU, Place de Verdun, 59045 Lille Cedex, France, Bio-Rad,
Route de Cassel, 59114 SteenVoorde, France, Plateforme d’Etude des Interactions Mole´culaires, IMPRT, IFR114, Faculte´ de Me´decine,
Poˆle Recherche, CHRU, Place de Verdun, 59045 Lille Cedex, France
ReceiVed January 31, 2008
As a part of our glycoantigen synthetic program for diagnosis and basic analysis of yeast-related pathogenic
mechanisms, a library of 1f2 oligomannosides suitable for immunoanalysis was prepared. The use of biotin
sulfone, an oxidized form of biotin, offers a convenient solution for both oligosaccharide synthesis and
immobilization on microspheres and surface plasmon resonance sensors. The application of this new strategy
for the analysis of anti-Candida albicans antibody response through multiple-analyte profiling technology
(Luminex) and with surface plasmonic analysis using biotin tagged synthetic oligosaccharides on avidin
coated surfaces was validated using monoclonal antibodies.
It is preferable to add the lipophilic biotin at an earlier stage of
synthesis than it is to couple it to a lipophilic protected sugar
in an organic solvent compatible with both reactants. Unfortu-
nately, the sulfur present in biotin interferes with a large number
of reactions, in particular with the widely used hydrogenolysis
of the benzyl group.
1. Introduction
Oligosaccharides are important bacterial and fungal antigens.
Being conjugated to proteins or lipids, oligosaccharides elicit
strong antibody responses as a result of infections or im-
munological disorders.1-8 Antioligosaccharide antibodies are
widely used as biomarkers of these different human pathological
conditions, whereas polyclonal or monoclonal antioligosaccha-
ride antibodies raised in animals are used to decipher microbial
biosynthetic and expression pathways in relation to patho-
genesis.9,10 Both basic and clinical applications are based on
the assessment of antioligosaccharide antibody specificity, which
is conferred by the nature of constitutive sugar units, the type
of linkage, and the length of the oligosaccharide chain.11 The
antigenic oligosaccharidic fragments used in these assays
are usually extracted from microbial cell walls by moderate
degradation of polysaccharides or glycoproteins. These hydro-
philic and water-soluble mixtures of antigens need to be
immobilized to a surface, which is not an easy task. This
problem is nicely solved with synthetic oligosaccharides; these
can be prepared in a pure form and with a handle bearing an
anchoring group. Probably the more convenient and universal
of these anchoring groups is biotin. It allows a very strong and
selective binding to a large number of surfaces: arrays, microtiter
plates, microspheres. The binding to surfaces through avidin
allows a precise coating with a controlled density. Biotin can
be added to an unprotected synthetic oligosaccharide, but this
solution does not show a distinct advantage over the use of
natural saccharide. The coupling reaction between a hydrophilic
oligosaccharide and hydrophobic biotin is not straightforward.
Biotin sulfone was first isolated as a natural metabolite of
biotin.12 Similar to biotin, it is strongly recognized by avidin
(binding ratio to avidin vs biotin: 0.33).13 Biotin sulfone has a
reduced nucleophilicity and was expected to be compatible with
benzyl hydrogenolysis. It is readily prepared from biotin by
peracid oxidation.14 Solange Lavielle’s group used this tag,
easily deuterated due to the presence of a sulfone group, for
mass spectroscopy identification of tagged molecules.15 How-
ever, it was never used for immunoassays.
The surface of the pathogenic yeast Candida albicans is
covered by a phosphopepdidomannan (usually designated as
mannan), which is composed of a complex repertoire of
oligomannose sequences11 that vary according to the growth
conditions including in ViVo in the host.16-18 Immunochemical
studies on mannan-derived oligomannosides as well as pioneer-
ing studies involving synthetic oligomannosides have established
that members of this oligomannoside repertoire act as specific
adhesins and/or epitopes.19 Among these epitopes some detect
antibodies associated with inflammatory disorders,20 some
support a protective antibody response,21 and others do not.22
In this context, it is essential for both basic research and
diagnosis to dispose of a convenient and versatile tool for large
scale analysis of the nature of the antibody response against
individual oligomannoses in relation to yeast triggered patho-
genic mechanisms.
†
Dedicated to Professor Marc Julia on the occasion of his 85th birthday.
* To whom correspondence should be addressed. Phone: 33 (0) 1 44 32
33 90. Fax: 33 (0) 1 44 32 33 97. E-mail: Jean-Maurice.Mallet@ens.fr
Website: http://www.chimie.ens.fr/Glycoscience/.
For the above reasons, we envisaged that oligosaccharide
construction from a biotin sulfone could be adapted to such an
objective.
‡
Ecole Normale Supe´rieure, De´partement de Chimie, UMR CNRS 8642.
Unite´ Inserm 799, Physiopathologie des Candidoses, Faculte´ de
§
Me´decine, Poˆle Recherche, CHRU.
|
a Abbreviations: BSTO, biotin sulfone tagged oligosaccharides; EDC,
(1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide hydrochloride; NIS, N-
iodosuccinimide; SPR, surface plasmon resonance; TfOH, triflic acid.
Bio-Rad.
Plateforme d’Etude des Interactions Mole´culaires, IMPRT, IFR114,
Faculte´ de Me´decine, Poˆle Recherche, CHRU.
10.1021/jm800099g CCC: $40.75
2008 American Chemical Society
Published on Web 09/13/2008

6202 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Collot et al.
Figure 1. Synthesized biotin sulfone tagged oligosaccharides (BSTO).
Scheme 1. Iterative Synthesis of R (1f2) Oligomannosides
Repeated until the Tetrasaccharide 20 is Obtaineda
Scheme 2. Iterative Synthesis of ꢀ (1f2) Oligomannosides
Repeated until the Tetrasaccharide 29 is Obtaineda
a Reagents and conditions: (a) 5, NIS (N-iodosuccinimide), TfOH, 4 Å
molecular sieves, CH2Cl2, rt; (b) MeONa, MeOH.
a Reagents and conditions: (a) 6, NIS, TfOH, 4 Å molecular sieves,
CH2Cl2, rt; (b) MeONa, MeOH; (c) Swern oxidation; (d) NaBH4, MeOH.
We therefore constructed a small library of biotin sulfone
tagged oligosaccharides (BSTOa ) (Figure 1) with the aim of
assessing the feasibility and specificity of this strategy in
differentiating anti-C. albicans ꢀ 1f2 oligomannoses, which
are protective in a systemic model of candidiasis, and anti-R
1f2 oligomannoses, which are not. As a reference, we used
monoclonal antibodies whose specificity against these oligom-
annose sequences has been established previously. Considering
present technological evolution in immunoanalysis, the assays
were performed using either multiple analyte profiling technol-
ogy (Luminex) or surface plasmon resonance analysis (BIA-
core). The combination of both methods, which required the
coupling of BSTOs to fluorescent microspheres and SPR
surfaces, was intended to explore the versatility of the same
probes for implementing snapshots and kinetic analysis of
antibody reactivities.
Scheme 3a
a Reagents and conditions: (a) naphthalene-2-thiol, HgBr2, (0.1 equiv),
CH3CN, 60 °C, 24 h, 69%; (b) MeONa, MeOH, rt, 24 h, 80%; (c) BzCl,
NEt3, DMAP, CH2Cl2, rt, 17 h, 90%; (d) naphthalene-2-thiol, HgBr2, (0.1
equiv), CH3CN, 60 °C, 24 h, 63%; (e) MeONa, MeOH, rt, 18 h, 71%; (f)
BzCl, NEt3, DMAP, CH2Cl2, rt, 36 h, 91%; (g) 5, NIS, TfOH, 4 Å molecular
sieves, CH2Cl2, rt, 0.5 h, 64%; (h) MeONa, MeOH, rt, 3 h, 91%.
2. Glycoconjugate Synthesis
Synthesis of the Oligosaccharide Part. Preparation of the
glycoconjugates 1-4 is divided into two stages. The first
involves the synthesis of protected precursors 16, 20, 23, and
29, while the second involves the introduction of biotin followed
by final deprotection, to give, respectively, compounds 1, 2, 3,
4. Synthesis of the precursors is shown in Schemes 1 and 2
and relies on two elongating blocks 5 and 6 (Scheme 3).
Compound 6 is a thionaphth-2-yl mannoside bearing a partici-
pating group (benzoate) in position two. It is a convenient
mannosyl donor for R (1f2) mannoside iterative synthesis
through a two-step sequence: glycosylation-saponification
(Schemes 1, 4).
Compound 6 is the analogous compound in the ꢀ-gluco series.
It allows the construction of ꢀ-mannosides in a four step
sequence: glycosylation-saponification-oxidation-reduction
(Schemes 2, 5).
Although recent advances have been made in ꢀ (1f2)-
mannoside synthesis by the Crich,23 Bundle,24 and Fraser Reid25
groups since our first synthesis published in 1994,26a,b this
method27 is reliable and well adapted for 1f2 mannoside
preparation. The reduction step provides a free alcohol ready
for glycosylation. The hydride reduction occurs from the less
hindered R face of the ketone to give the manno isomer. It was
also very convenient for large scale preparation. In particular,
this procedure avoids sensitive and expensive reagents and the
usually difficult separation of an R/ꢀ mixture: indeed, for both
linkages, the stereoselectivity relies on a participating group in
position 2. The glycosyl donors 5 and 6 were prepared in a
similar way28 from the respective orthoester 729 or 830 and
naphthyl-2-thiol. The acetate in position 2 was replaced by a
benzoate in two steps to give 5 and 6. Compound 14 was
obtained in two steps from 5 and ethyl 6-hydroxy hexanoate.
The naphthalene-2-thiol is a commercially available aromatic
thiol, it is crystalline, almost odorless and less toxic than thio-
phenol, previously used for mannoside preparation. Furthermore,
we recently proposed 2-methyl-5-tert-butylthiophenol as an
improved thiophenol surrogate.31
The iterative strategy for the R mannoside is depicted in
Scheme 4. The synthesis was uneventful and the overall yield
for the introduction of an R manno unit was good (60-78%).

Biotin Sulfone for Synthetic Oligosaccharide Immobilization
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6203
Scheme 4a
a Reagents and conditions: (a) 5, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, rt, 79%; (b) MeONa, MeOH, 77%; (c) 5, NIS, TfOH, 4 Å molecular sieves,
CH2Cl2, 77%; (d) MeONa, MeOH, 87%; (e) 5, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 95%; (f) MeONa, MeOH, 82%.
Scheme 5a
a Reagents and conditions: (a) 6, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 87%; (b) MeONa, MeOH, 90%; (c) oxalyl chloride, DMSO, NEt3, CH2Cl2,
then MeOH, NaBH4, 79%; (d) 6, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 78%; (e) MeONa, MeOH, 91%; (f) oxalyl chloride, DMSO, NEt3, CH2Cl2, then
NaBH4, MeOH (15% Manno + 70% Gluco 25); (g) 6, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 87%; (h) MeONa, MeOH, 86%; (i) oxalyl chloride,
DMSO, NEt3, CH2Cl2, then NaBH4, MeOH (15% Manno + 63% Gluco 28).
The iterative strategy for the ꢀ-mannoside is depicted in
Scheme 5. The selectivity of the reduction step is rather
surprising, although the oxidation-reduction steps gave the
dimannoside with a total manno selectivity, this sequence
led predominantly the gluco isomer on the tri and tetrasac-
charide level. This is in contrast with our previous work26a,b
but using a different reducing agent, superhydride, different
temperature, and aglycon. This length-dependent behavior
is probably due to the helical structure of the ꢀ (1f2)
mannoside (the step is ca. four mannose units24c) as the
reduction of the disaccharide is completely selective. At this
stage, recycling the gluco compounds allowed enough
compound to be available for the subsequent biotinylation
step. The use of an alternative reducing agent for this
transformation is underway.
base of 31 was obtained from the trifluoroacetate salt upon
treatment with an ion-exchange resin.
The methyl esters 16, 20, 23, and 29 were saponified with
sodium hydroxide in aqueous THF (Scheme 7). The acids
32-35 were then condensed with the amine 31 in DMF in
the presence of EDC (1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride) to give the protected biotinylated
compounds (36-39). Oxidation (m-chloroperbenzoic acid)
of these biotin conjugates followed by hydrogenolysis of the
benzyl group (H2, 1.3 atm) allowed access to the final
products (1-4) without any difficulties owing to the inertness
of the biotin sulfone group. The yields are summarized in
Table 1.
Alternatively, in a more direct strategy, we prepared and tried
to use compound 45, the sulfone form of 31 (Scheme 8).
However, this compound was insoluble in most of the solvents
and the coupling reaction failed. It was, therefore, more
Biotinylation. The protected oligomannosides 16, 20, 23, and
29 were then coupled to the amine 31. The amine 3132 was
first prepared in two steps from biotin (Scheme 6). The free

6204 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Collot et al.
Scheme 6a
a Reagents and conditions: (a) EDC, pyridine, 60 °C, 91%; (b) CF3COOH, ultrasonic irradiation, then Amberlite IRA-67, 100%.
Scheme 7a
a Reagents and conditions: (a) NaOH, THF, H2O; (b) 31, EDC, DMAP, DMF; (c) mCPBA, CH2Cl2; (d) H2, Pd/C, MeOH/H2O.
Table 1. Yields of Final Steps
conferred by the anomeric configuration. In the vein, Mab
EBCA1, described as reacting with an R-mannopentaoside,
bound to some extent to the R-mannotetraoside, which was in
our series of BSTOs the closest epitope. Importantly, no
reactivity with members of the ꢀ family was observed.
(a)
(b)
(c) sulfur
(d) benzyl
saponification biotinylation oxidation hydrogenolyse
1 Di R
16 f 32 (96%) f 36 (78%) f 40 (82%) f 1 (89%)
2 Tetra R 20 f 33 (82%) f 37 (68%) f 41 (84%) f 2 (90%)
3 Di ꢀ
23 f 34 (98%) f 38 (79%) f 42 (81%) f 3 (90%)
Surface Plasmon Resonance Analysis of Biotin Sulfone
Tagged Oligomannoside (BSTO) Reactivity with Anticar-
bohydrate Monoclonal Antibodies. The typical results for the
interaction between synthetic oligomannosides and antibodies
are shown in Figure 3. No binding was observed for di R
mannoside 1. The variations of about 5 RU for each Mab are
negligible, and modifications of sensorgramm profiles can be
attributed to composition buffer variations. The presence of
ligand (i.e., 1) was confirmed by the binding of Concanavalin
A (250 nM) on this support (data not shown). The EB CA1
Mab weakly interacted with tetra R mannoside 2 (about 60 RU)
and presented a typical profile of a sensorgramm with associa-
tion and dissociation curves (beginning respectively after 60
and 180 s). EB A2, B6.1, and 5B2 antibodies did not show any
interactions with 2, which is not surprising considering the
specificity of each antibody.
Interactions of antibodies with ꢀ-oligomannosides 3 and 4
were more informative. 5B2 interacted with di-ꢀ mannoside 3
(about 600 RU) and the tetra-ꢀ mannoside 4. B6.1 showed
strong binding only to the tetra-ꢀ mannoside 4. The absence of
a response for EB A2 injections shows the specificity of the
binding.
These experiments are in complete agreement with the known
specificity of the anti-C. albicans Mabs and are consistent with
the microsphere measurements obtained by multiple-analyte
profiling technology (Luminex).
4 Tetra ꢀ 29 f 35 (95%) f 39 (71%) f 43 (94%) f 4 (90%)
preferable to perform the coupling reaction using the more
lipophilic sulfide form.
3. Immunological Evaluation of the Conjugates
Monoclonal Antibodies (Mabs). Synthetic oligomannosides
were tested with a panel of anti-C. albicans glycan Mabs, whose
specificity was established previously. Mab anti-ꢀ (1f3) glucan
was used as a control. Mabs EB CA1, a rat IgM, was described
as reacting with an R (1f2) mannopentaose as a minimal
epitope.33 Mab 5B2, a rat-mouse hybrid IgM, was described
as reacting with ꢀ (1f2) mannosides with a mannobiose as a
minimal epitope.34,35 Mab B6.1, a mouse IgM, has been
described as specific for a ꢀ (1f2) mannotriose.22 Mab EBA2,
reacting with a galactofuranose epitope, was used as a control.
Multiplex Quantification of Biotin Sulfone Tagged Oli-
gomannoside (BSTO) Reactivity with Anticarbohydrate
Monoclonal Antibodies. A total of four BSTO epitopes were
individually coupled to different streptavidin coated microsphere
beads and tested with a panel of anti-C. albicans Mabs directed
against mannoglycoconjugates (EB CA1, 5B2, and B6.1) or ꢀ
(1f3) glucans (Biosupplies). Figure 2 represents the reactivity
of individual Mabs with the panel of BSTO in multiplex assay.
The antigalactofuran Mab did not generate any signal with any
of the mannose residues presented as BSTOs. As expected, Mab
5B2 bound to the members of the ꢀ mannose family but did
not bind to any R mannose. As also expected, its reactivity with
this family started with the mannobiose. As also expected, Mab
B6.1 did not react with ꢀ-mannobiose 3 although it did react
with ꢀ-mannotetraose 4. As for 5B2, no reactivity was observed
against R-mannosides 1 or 2, confirming the strong specificity
4. Conclusions
Extensive studies on the interaction between C. albicans and
its hosts have suggested that the spacial conformation of
ꢀ-mannoses versus R-mannoses represents the support for
differential recognition of C. albicans mannoglycoconjugates

Biotin Sulfone for Synthetic Oligosaccharide Immobilization
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6205
Scheme 8a
a Reagents and conditions: (a) EDC, pyridine, 60 °C, 15 h, 91%; (b) CF3COOH, 5 min, rt, 100%.
Figure 2. Multiplex quantification of biotin sulfone tagged oligomannoside (BSTO) reactivity with anticarbohydrate monoclonal antibodies.
tubes in a Bu¨chi 510 apparatus and are uncorrected. Optical rotations
were measured with a Perkin-Elmer model 241 digital polarimeter
at 22 ( 3 °C. Compound purity was checked by TLC on Silica gel
60 F254 (E. Merck) with detection by charring with sulfuric acid.
Column chromatography was performed on Silica gel 60 (E.
Merck). 1H NMR spectra were recorded with Bru¨ker AM 250, AM
400 instruments. Chemical ionization and FAB mass spectrometry
were recorded with Jeol MS700: CI (gas: ammonia); FAB (matrix:
NBA, NaI).
by the mammalian immune system. This has been observed for
lectins of innate immunity where ꢀ-mannosides have been
shown to react with galectin-3, whereas R-mannosides react with
a large number of C-lectins.15,23-25 Interaction with these
receptors directs the balance between pro- and anti-inflammatory
responses, conditioning the damage to the host.15 This strong
duality in reactivity is also found for adaptative immunity
because the so-called antimannan antibodies generated after C.
albicans infection or immunization distribute between anti-ꢀ-
mannosides, which are “protective” for the host, and anti-R-
mannosides, which are not.17 To make progress in the under-
standing of this complex interplay, it is essential to dispose of
reliable and easy tools for large scale dissection of the antibody
response. In the present study, we used representative members
of anti-R man and anti-ꢀ man antibodies to validate the new
technology we have developed. This development was intended
to present molecules to any kind of support that could be
sensitized with avidin. Using versatile multiplex technology,
as well as the more analytical BIAcore technology, we found
that the BTSO reproduced and refined in simple runs data hardly
accumulated in the literature by EIA and Western blot testing
of yeasts derived oligomannosides preparations.
Typical Hydrogenolysis Procedure. Conjugate 1. To a solution
of 40 (275 mg, 0.201 mmol) in anhydrous methanol (15 mL) was
added Pd/C (10%) (137 mg, 0.5 g/g of substrate). Vacuum and H2
were alternated, and then the mixture was stirred at room temper-
ature under H2 overnight. The mixture was filtered through celite
and concentrated. The residue was dissolved in water (HPLC grade)
and washed with CH2Cl2 (three times). The aqueous layer was
filtered over PTFE 0.45 µm filter and lyophilized to give 1 (56
Experimental Section
mg, 82%) as a white solid. [R]25 +48 (c 1.0, MeOH/H2O (1/1)).
D
General Procedures. All compounds were homogeneous by
TLC analysis and had spectral properties consistent with their
assigned structures. Melting points were determined in capillary
Anal. (C34H60O16N4S) C, H.
Conjugate 2. White solid. Yield: 90%. [R]25 +44 (c 0.4,
D
MeOH/H2O (1/1)). Anal. (C46H80N4O26S) C, H.

6206 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Collot et al.
Figure 3. Surface plasmon resonance analysis.
Conjugate 3. Debenzylation of 42. White solid. Yield: 90%.
D
equiv). The mixture was stirred under argon for 17 h and then
washed with HCl (1 M). The organic layer was dried over MgSO4,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (cyclohexane/EtOAc: 9/1) to give 5
(0.376 g, 90%) as a syrup. Rf: 0.70 (cyclohexane/EtOAc: 8/2).
[R]25 +10 (c 0.5, MeOH/H2O (1/1)). Anal. (C34H60O16N4S) C,
H.
Conjugate 4. Debenzylation of 43. White solid. Yield: 90%.
[R]25 -4.8 (c 1.2, MeOH/H2O (1/1)). Anal. (C46H80N4O26S) C,
D
H.
[R]25 +10 (c 1.0, CHCl3).
D
2-Naphthyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-1-thio-r-D-man-
nopyranoside (5). To a mixture of 10 (0.356 g, 0.600 mmol) and
DMAP (16.2 mg, 0.133 mmol, 0.2 equiv) in anhydrous CH2Cl2 (5
mL) were added at 0 °C, under argon: triethylamine (0.59 mL, 4.20
mmol, 7.0 equiv) and benzoyl chloride (90.5 µL, 0.780 mmol, 1.3
2-Naphthyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-1-thio-ꢀ-D-glu-
copyranoside (6). To a mixture of 12 (6.26 g, 10.57 mmol) and
DMAP (0.64 g, 5.28 mmol, 0.5 equiv) in anhydrous CH2Cl2 (80
mL) were added at 0 °C under argon: triethylamine (7.35 mL, 26.43
mmol, 5.0 equiv) and benzoyl chloride (6.13 mL, 26.43 mmol, 5.0

Biotin Sulfone for Synthetic Oligosaccharide Immobilization
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6207
equiv). The mixture was stirred under argon for 36 h and then
washed with HCl (1 M). The organic layer was dried over MgSO4,
filtered, and concentrated. The residue was dissolved in CH2Cl2,
and the product was precipitated by addition of cyclohexane to give
6 (6.380 g, 91%) as a white solid. Rf: 0.54 (cyclohexane/EtOAc:
TfOH (0.008 mL, 0.088 mmol, 0.5 equiv). After 30 min, the
precipitate was filtered off, and the filtrate was successively washed
with saturated aqueous Na2S2O3, NaHCO3, and brine. The organic
layer was dried over MgSO4, filtered, and concentrated. The residue
was purified by column chromatography on silica gel (cyclohexane/
EtOAc: 9/1 to 8/2) to give 15 (0.156 g; 79%) as a syrup. Rf: 0.35
2/1); mp: 112 °C. [R]25 +29 (c 1.0, CHCl3).
D
(cyclohexane/EtOAc: 8/2). [R]25 +1 (c 1.0, CHCl3).
2-Naphthyl 2-O-Acetyl-3,4,6-tri-O-benzyl-1-thio-r-D-man-
nopyranoside (9). To a mixture of orthoester 7 (3.32 g, 6.56 mmol)
and naphthalene-2-thiol (3.48 g, 21.7 mmol, 3.3 equiv) in anhydrous
CH3CN (10 mL) was added mercury bromide (0.296 g, 0.82 mmol,
0.1 equiv) under argon. The mixture was stirred at 60 °C for 27 h.
The mixture was allowed to cool to room temperature, diluted with
EtOAc, and washed with NaOH (5%) and water. The organic layer
was dried over MgSO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel (cyclohexane/
EtOAc: 9/1) to give 9 (2.85 g, 69%) as a white solid. Rf: 0.50
(cyclohexane/EtOAc: 8/2); mp: 82 °C. [R]25D +14 (c 1.0, CHCl3).
2-Naphthyl 3,4,6-Tri-O-benzyl-1-thio-r-D-mannopyranoside
(10). To a solution of 9 (2.30 g, 3.63 mmol) in methanol (50 mL),
was added sodium (1.6 g, 72.5 mmol, 20.0 equiv). The mixture
was stirred for 22 h and then neutralized (IR-120 H+ resin), filtered,
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (cyclohexane/EtOAc: 8/2) to give 10 (1.070 g,
D
5-Methoxycarbonylpentyl 2-O-(3,4,6-tri-O-benzyl-r-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranoside (16). De-
benzoylation of 15. Column chromatography cyclohexane/EtOAc:
8/2 to 7/3. Syrup. Yield: 97%. Rf: 0.65 (cyclohexane/EtOAc: 1/1).
[R]25 +34 (c 0.5, CHCl3).
D
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-Benzoyl-3,4,6-tri-
O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranoside (17). Gly-
cosylation of 16 with 5. Column chromatography (cyclohexane/
EtOAc: 9/1 to 8/2). Syrup. Yield: 77%. Rf: 0.35 (cyclohexane/
EtOAc: 8/2). [R]25 +12 (c 1.0, CHCl3).
D
5-Methoxycarbonylpentyl 2-O-(2-O-(3,4,6-Tri-O-benzyl-r-D-
mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-
tri-O-benzyl-r-D-mannopyranoside (18). Debenzoylation of 17.
Column chromatography (cyclohexane/EtOAc: 8/2). Syrup. Yield
77%. Rf: 0.15 (cyclohexane/EtOAc: 8/2). [R]25D +32 (c 0.6, CHCl3).
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-(2-O-Benzoyl-3,4,6-
tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-r-D-mannopyranoside (19). Glycosylation of 18 with
5. Column chromatography (cyclohexane/EtOAc: 8/2). Syrup.
Yield: 95%. Rf: 0.30 (cyclohexane/EtOAc: 8/2). [R]25D +20 (c 0.4,
CHCl3).
50%) as a syrup. Rf: 0.20 (cyclohexane/EtOAc: 8/2). [R]25 +20
D
(c 1.0, CHCl3).
2-Naphthyl 2-O-Acetyl-3,4,6-tri-O-benzyl-1-thio-ꢀ-D-glucopy-
ranoside (11). To a mixture of orthoester 8 (10.1 g, 12.6 mmol)
and naphthalene-2-thiol (6.68 g, 41.7 mmol, 3.3 equiv) in anhydrous
CH3CN (32 mL), was added mercury bromide (0.227 g, 0.630
mmol, 0.05 equiv) under argon. The mixture was stirred at 60 °C
for 23 h. After the mixture was cooled to room temperature, it was
diluted with EtOAc and washed with NaOH (5%) and water. The
organic layer was dried over MgSO4, filtered, and concentrated.
The residue was purified by column chromatography on silica gel
(cyclohexane/EtOAc: 9/1) to give 11 (5.06 g, 63%) as a white solid.
Rf: 0.5 (cyclohexane/EtOAc: 3/1); mp: 125 °C. [R]25D +10 (c 1.0,
CHCl3).
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-
r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-
3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-
mannopyranoside (20). Debenzoylation of 19. Column chromato-
graphy (cyclohexane/EtOAc: 7/3). Syrup. Yield: 87%. Rf: 0.20
(cyclohexane/EtOAc: 9/1). [R]25 +38 (c 0.4, CHCl3).
D
5-Methoxycarbonylpentyl 2-O-(2-O-Benzoyl-3,4,6-tri-O-ben-
zyl-ꢀ-D-glucopyranosyl)-3,4,6-tri-O-benzyl-1-O-r-D-mannopyra-
noside (21). Glycosylation of 14 and 6. Column chromatography
(cyclohexane/EtOAc: 8/2). Syrup. Yield: 87%. Rf: 0.44 (cyclohex-
2-Naphthyl 3,4,6-tri-O-benzyl-1-thio-ꢀ-D-glucopyranoside (12).
To a solution of 11 (0.185 g, 0.291 mmol) in methanol (15 mL)
was added sodium (0.324 g, 14.1 mmol, 48 equiv). The mixture
was stirred for 18 h and then neutralized (IR-120 H+ resin), filtered
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (cyclohexane/EtOAc: 8/2) to give 12 (0.122 g,
71%) as a white solid. Rf: 0.41 (cyclohexane/EtOAc: 3/1); mp: 94
ane/EtOAc: 2/1). [R]25 +5 (c 0.5, CHCl3).
D
5-Methoxycarbonylpentyl 2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-glu-
copyranosyl)-3,4,6-tri-O-benzyl-r-d-mannopyranoside (22). De-
benzoylation of 21. Column chromatography (cyclohexane/EtOAc:
°C. [R]25 -6 (c 1.0, CHCl3).
D
8/2. Syrup. Yield: 90%). Rf: 0.35 (cyclohexane/EtOAc: 2/1). [R]25
+9 (c 1.0, CHCl3).
D
5-Ethoxycarboxypentyl 2-O-benzoyl-3,4,6-tri-O-benzyl-r-D-
mannopyranoside (13). To a mixture of 4 (3.000 g, 4.31 mmol),
molecular sieves 4Å (3 g) in anhydrous CH2Cl2 were added
successively under argon: ethyl 6-hydroxy-hexanoate (1.050 mL,
6.460 mmol, 1.5 equiv), NIS (2.420 g, 10.770 mmol, 2.5 equiv),
and TfOH (0.191 mL, 2.150 mmol, 0.5 equiv). After 30 min, the
precipitate was filtered off, and the filtrate was successively washed
with saturated aqueous Na2S2O3, NaHCO3, and brine. The organic
layer was dried over MgSO4, filtered, and concentrated. The residue
was purified by column chromatography on silica gel (cyclohexane/
EtOAc: 9/1 to 8/2) to give 13 (1.910 g, 64%) as a syrup. Rf: 0.45
Typical Swern Oxidation/Reduction Procedure. 5-Methoxy-
carbonylpentyl 2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-mannopyranosyl)-
3,4,6-tri-O-benzyl-r-D-mannopyranoside (23). To a solution of
oxalyl chloride (1.497 mL, 17.424 mmol, 3 equiv) in anhydrous
CH2Cl2 (10 mL) cooled at -78 °C was added dropwise DMSO
(2.474 mL, 34.848 mmol, 6 equiv). After 15 min, a solution of 22
(5.972 g, 5.808 mmol, 1 equiv) in CH2Cl2 (20 mL) was added
dropwise and the mixture was stirred for a further 45 min at -78
°C. Triethylamine (4.888 mL, 34.848 mmol, 6 equiv) was then
added dropwise, and the mixture was allowed to warm up to room
temperature. After 30 min, the mixture was extracted with CH2Cl2
and washed with water. The organic layer was dried over MgSO4,
filtered, and diluted with an equivalent volume of methanol. NaBH4
(0.658 g, 17.424 mmol, 3 equiv) was then added, and the solution
was degassed. The mixture was stirred overnight at room temper-
ature and concentrated. The residue was purified by column
chromatography on silica gel (cyclohexane/EtOAc: 8/2 to 7/3) to
give 23 (4.681 g, 79%) as a syrup. Chromatography (cyclohexane/
EtOAc: 7/3). Syrup. Yield: 79%. Rf: 0.32 (cyclohexane/EtOAc: 2/1)
(cyclohexane/EtOAc: 8/2). [R]25 -7 (c 0.7, CHCl3).
D
Typical Debenzoylation Procedure. 5-Methoxycarbonylpen-
tyl 3,4,6-Tri-O-benzyl-r-D-mannopyranoside (14). To a solution
of 13 (1.910 g, 2.744 mmol) in methanol (30 mL) was added sodium
(0.012 g, 0.548 mmol, 0.2 equiv). The mixture was stirred for 3 h
and then neutralized (IR-120 H+ resin), filtered, and concentrated.
The residue was purified by column chromatography on silica gel
(cyclohexane/EtOAc: 7/3) to give 14 (1.480 g, 91%) as a syrup.
Rf: 0.60 (cyclohexane/EtOAc: 1/1). [R]25 +36 (c 0.8, CHCl3).
D
Typical Glycosylation Procedure. 5-Methoxycarbonylpen-
tyl 2-O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-
3,4,6-tri-O-benzyl-r-D-mannopyranoside (15). To a mixture of
14 (0.102 g, 0.176 mmol) and 5 (0.184 g, 0.264 mmol, 1.5 equiv)
and 4Å molecular sieves (0.100 g) in anhydrous CH2Cl2 (5 mL),
were added under argon NIS (0.099 g, 0.440 mmol, 2.5 equiv) and
[R]25 +0.6 (c 5.0, CHCl3).
D
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-Benzoyl-3,4,6-tri-
O-benzyl-ꢀ-D-glucopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopy-
ranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranoside (24). Glyco-
sylation of 23 with 6. Column chromatography (cyclohexane/

6208 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Collot et al.
EtOAc: 8/2). Syrup. Yield: 78%. Rf: 0.47 (cyclohexane/EtOAc: 8/2).
EtOAc). Syrup; yield: 98%. Rf: 0.38 (cyclohexane/EtOAc: 1/1).
[R]25 -15 (c 1.7, CHCl3).
[R]25 -3 (c 0.9, CHCl3).
D
D
5-Carboxypentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopy-
ranoside (35). Saponification of 29. Column chromatography
(cyclohexane/EtOAc: 5/5, 100% acetone). Syrup; yield: 95%. Rf:
5-Methoxycarbonylpentyl 2-O-(2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-
glucopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-
tri-O-benzyl-r-D-mannopyranoside (25). Debenzoylation of 24.
Column chromatography (cyclohexane/EtOAc: 8/2 to 7/3). Syrup.
Yield: 91%. Rf: 0.41 (cyclohexane/EtOAc: 7/3). [R]25D -34 (c 3.6,
CHCl3).
0.55 (cyclohexane/EtOAc: 5/5). [R]25 -43.4 (c 1.7, CHCl3).
D
.
5-Methoxycarbonylpentyl 2-O-(2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-
mannopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-
tri-O-benzyl-r-D-mannopyranoside (26). Oxidation/reduction of
25. Column chromatography (cyclohexane/EtOAc: 8/2 to 7/3).
Syrup. Yield: 15%; 70% starting material 25 recovered. Rf: 0.32
Typical Biotin Coupling Procedure. 5-Carboxypentyl 2-O-
(3,4,6-Tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-
D-mannopyranoside Biotin Conjugate (36). To a mixture of 32
(500 mg, 0.501 mmol) and 31 (1.5 equiv) in anhydrous DMF (10
mL), were added DMAP (73 mg, 0.601 mmol, 1.2 equiv) and EDC
(143 mg, 0.752 mmol, 1.5 equiv). The mixture was stirred at 60
°C overnight. EDC (95 mg, 1 equiv) was then added to complete
the reaction. The solution was concentrated, and the residue was
extracted with CH2Cl2, washed with water, dried over MgSO4,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH: 9/1) to give 36 (521
mg, 78%) as a syrup. Rf: 0.5 (CH2Cl2/MeOH: 9/1). [R]25D +32 (c
1.6, CHCl3).
(cyclohexane/EtOAc: 7/3). [R]25 -34 (c 1.0, CHCl3).
D
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-(2-O-Benzoyl-3,4,6-
tri-O-benzyl-ꢀ-D-glucopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-r-D-mannopyranoside (27). Glycosylation of 26 with
6. Column chromatography (cyclohexane/EtOAc: 8/2). Syrup.
Yield: 87%. Rf: 0.47 (cyclohexane/EtOAc: 7/3). [R]25D -46 (c 1.4,
CHCl3).
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-
ꢀ-D-glucopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-
3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-
mannopyranoside (28). Debenzoylation of 27. The mixture was
refluxed for 2 h. Column chromatography (cyclohexane/EtOAc:
5-Carboxypentyl 2-O-(2-O-(2-O-(3,4,6-tri-O-benzyl-r-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-man-
nopyranoside Biotin Conjugate (37). Column chromatography
(CH2Cl2/MeOH: 10/1 f 9/1). Syrup; yield: 68%. Rf: 0.53 (CH2Cl2/
7/3). Syrup. Yield: 86%. Rf: 0.41 (cyclohexane/EtOAc: 7/3). [R]25
-42.5 (c 1.2, CHCl3).
D
MeOH: 9/1). [R]25 +25 (c 1.7, CHCl3).
D
5-Carboxypentyl 2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-mannopyrano-
syl)-3,4,6-tri-O-benzyl-r-D-mannopyranoside Biotin Conjugate
(38). Coupling of 34 with 31. Column chromatography (CH2Cl2/
MeOH: 9/1). Syrup; yield: 79%. Rf: 0.51 (CH2Cl2/MeOH: 9/1).
5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-
ꢀ-D-mannopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-
3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-
mannopyranoside (29). Oxidation/reduction of 28. Column
chromatography (cyclohexane/EtOAc: 8/2 to 7/3). Syrup. Yield:
15%; 63% starting material recovered. Rf: 0.37 (cyclohexane/
[R]25 +6 (c 2.8, CHCl3).
D
5-Carboxypentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopy-
ranoside Biotin Conjugate (39). Coupling of 35 with 31. Column
chromatography (EtOAc/acetone/MeOH: 6/3/1). Syrup; yield: 71%.
EtOAc: 7/3). [R]25 -43.2 (c 1.3, CHCl3).
D
(3aS,4S,6aR)-N-[6-[(1,1-Dimethylethoxy)carbonyl]amino-
hexyl]hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentana-
mide (30). To a mixture of D-(+) biotin (0.900 g, 3.68 mmol) and
N-Boc-1,6-diaminohexane hydrochloride (0.929 g, 3.68 mmol, 1
equiv) in anhydrous pyridine (30 mL) was added, under argon, EDC
(0.843 g, 4.41 mmol, 1.2 equiv). The mixture was stirred overnight
and concentrated, the crude product was filtered and washed with
NaOH (1 M) and ether. The product was dried under vacuum to
give 30 (1.448 g, 91%) as a white powder. Rf: 0.44 (CH2Cl2/MeOH:
Rf: 0.47 (EtOAc/Acetone/MeOH: 6/3/1). [R]25 -30.5 (c 1.1,
D
CHCl3).
Typical Biotin Oxidation Procedure. 5-Carboxypentyl 2-O-
(3,4,6-Tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-
D-mannopyranoside Oxidized Biotin Conjugate (40). To a
solution of 36 (455 mg, 0.344 mmol) in anhydrous CH2Cl2 (15
mL) was added m-chloroperbenzoic acid (178 mg, 1.034 mmol, 3
equiv) under argon. The mixture was stirred at room temperature
overnight, washed with a saturated aq NaHCO3, dried over MgSO4,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH: 9/1) to give 40 (384
9/1); mp: 173.7 °C. [R]25 +38 (c 1.0, MeOH).
D
(3aS,4S,6aR)-N-(6-Aminohexyl)hexahydro-2-oxo-1H-thieno[3,4-
d]imidazole-4-pentanamide (31). 30 (0.614 g, 1.390 mmol) was
dissolved in trifluoroacetic acid (5 mL) and sonicated until total
dissolution. After 5 min, the TFAH was evaporated. The residue
was dissolved in methanol, neutralized (Amberlite IRA-67), and
filtered. The methanol was evaporated to give 31 (0.476 g, 100%)
as a syrup.
mg, 82%) as a syrup. Rf: 0.60 (CH2Cl2/MeOH: 9/1). [R]25 +24
D
(c 1.2, CHCl3).
5-Carboxypentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-r-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-man-
nopyranoside Oxidized Biotin Conjugate (41). Column chromato-
graphy (CH2Cl2/MeOH: 9/1). Syrup; yield: 84%. Rf: 0.44 (CH2Cl2/
Typical Saponification Procedure. 5-Carboxypentyl 2-O-
(3,4,6-Tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-
D-mannopyranoside (32). To a solution of 16 (551 mg, 0.544
mmol) in THF (10 mL) was added NaOH (1 M, 3.26 mL). The
mixture was stirred at 60 °C for 24 h, neutralized (IR-120 H+ resin),
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (cyclohexane/EtOAc: 5/5, facetone)
to give 32 (520 mg, 96%) as a syrup. Rf: 0.38 (cyclohexane/EtOAc:
MeOH: 9/1). [R]25 +24 (c 1.2, CHCl3).
D
5-Carboxypentyl 2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-mannopyrano-
syl)-3,4,6-tri-O-benzyl-r-D-mannopyranoside Oxidized Biotin
Conjugate (42). Oxidation of 38. Column chromatography CH2Cl2/
MeOH: 9/1. Syrup; yield: 81%. Rf: 0.42 (CH2Cl2/MeOH: 9/1).
5/5). [R]25 +36 (c 0.5, CHCl3).
[R]25 +5 (c 1.7, CHCl3).
D
D
5-Carboxypentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-r-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-r-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-man-
nopyranoside (33). Column chromatography (cyclohexane/EtOAc:
5/5 f 100% EtOAc). Syrup; yield: 82%. Rf: 0.58 (cyclohexane/
EtOAc: 6/4).
5-Carboxypentyl 2-O-(2-O-(2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-man-
nopyranosyl)-3,4,6-tri-O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-
O-benzyl-ꢀ-D-mannopyranosyl)-3,4,6-tri-O-benzyl-r-D-mannopy-
ranoside Oxidized Biotin Conjugate (43). Oxidation of 39.
Column chromatography (CH2Cl2/MeOH: 9/1). Syrup; yield: 94%.
Rf: 0.39 (CH2Cl2/MeOH: 9/1). [R]25 -23.7 (c 1.3, CHCl3).
D
5-Carboxypentyl 2-O-(3,4,6-Tri-O-benzyl-ꢀ-D-mannopyrano-
syl)-3,4,6-tri-O-benzyl-r-D-mannopyranoside (34). Saponification
of 23. Column chromatography (cyclohexane/EtOAc: 5/5 to 100%
Compound (44). To a mixture of biotin sulfone (0.478 g, 1.731
mmol) and N-Boc-1,6-diaminohexane hydrochloride (0.436 g, 1.731
mmol, 1 equiv) in anhydrous pyridine (18 mL) was added, under

Biotin Sulfone for Synthetic Oligosaccharide Immobilization
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6209
argon, EDC (0.792 g, 4.154 mmol, 2.4 equiv). The mixture was
allowed to stir overnight at 60 °C. The solvent was evaporated,
and the crude product was filtered and washed with CH2Cl2, HCl
(1 M), and acetone. The product was dried under vacuum to give
0.748 g (91%) as a white powder. Rf: 0.55 (CHCl3/MeOH: 5/5);
mp: 237 °C.
Supporting Information Available: NMR and mass spectros-
copy characterization data for the synthesis of compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
Compound (45). 44 (0.100 g, 0.211 mmol) was dissolved in
trifluoroacetic acid (2 mL) and sonicated until total dissolution. After
5 min, the TFA was evaporated. The residue was dissolved in
methanol, neutralized (Amberlite IRA-67), and filtered. The
methanol was evaporated to give 0.080 g (100%) as a syrup.
Monoclonal Antibodies (Mabs). Synthetic oligomannosides
were tested with a panel of anti-C. albicans glycan Mabs, whose
specificity was established previously. Mab anti-ꢀ (1f3) glucan
(Biosupplies, Australia) was used as a control. Mabs EB CA1, a
rat IgM (Bio-Rad Laboratories, France), was described as reacting
with an R (1f2) mannopentaose as a minimal epitope.33 Mab 5B2,
a rat-mouse hybrid IgM, was described as reacting with ꢀ (1f2-
mannosides with a mannobiose as a minimal epitope.34,35 Mab B6.1,
a mouse IgM, has been described as specific for a ꢀ (1f2)
mannotriose.22 Mab EBA2, reacting with a galactofuranose epitope,
was used as a control (Bio-Rad).
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prepared via a four-step procedure. (i) Carboxyl groups on the
surface of the beads were activated in the presence of N-
hydroxysuccinimide and EDC (Pierce Chemicals Co. Rockford, Ill,
USA) to form activated esters. (ii) After three washes in 50 mM
phosphate buffered saline (PBS), pH 7.4, these esters were reacted
for 3 h at room temperature with avidin (Sigma, Saint Quentin
Fallavier, France). (iii) The beads were blocked for 30 min with
50 mM PBS containing 250 mM NH2OH. (iv) After three washes
in PBS, the beads were coupled with biotin sulfone tagged
oligomannosides for 1 h at room temperature and blocked overnight
with 50 mM PBS containing 10% bovine serum albumin. After
washing, the beads were stored at 4 °C.
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Acknowledgment. We thank IMPRT (IFR114) for access
to the BIAcore 3000 instrumentation, and C. Lefevre for
technical assistance. We also thank Me´lanie Jourdain and
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