Multi-mannosides based on a carbohydrate scaffold: Synthesis, force field development, molecular dynamics studies, and binding affinities for lectin con A

Article abstract of DOI:10.1021/jo071248a

(Chemical Equation Presented) A short and efficient strategy for the synthesis of multi-valent mannosides based on a selectively functionalized carbohydrate scaffold was reported involving (i) direct regioselective azidation of unprotected commercial saccharides, (ii) acetylation, (iii) grafting of the mannosyl ligands by click chemistry, and (iv) deacetylation. New glycoclusters with a valency ranging from 1 to 4 and different spatial arrangements of the epitopes were obtained. Binding affinities of the new glycoclusters toward concanavalin A (Con A) lectin were investigated by an enzyme-linked lectin essay (ELLA). The synthetic multi-valent compounds exhibited a remarkable cluster effect with a relative potency per mannoside residue ranging from 8.1 to 9.1 depending on the structures. ELLA experiments were in agreement with the establishment of favorable interactions between triazole ring and Con A, increasing the binding affinity. A new force field topology database was developed in agreement with the GLYCAM 2004 force field. Molecular dynamics performed on representative glyco-conjugates revealed interesting structural features such as rigidity of the scaffold for a well-defined presentation of the ligands and highly flexible mannose counterparts. The new glycoconjugates reported may be promising tools as probes or effectors of biological processes involving lectins.

Full text of DOI:10.1021/jo071248a

Multi-Mannosides Based on a Carbohydrate Scaffold: Synthesis,
Force Field Development, Molecular Dynamics Studies, and Binding
Affinities for Lectin Con A
Se´bastien G. Gouin,*,† Enguerran Vanquelef,‡ Jose´ Manuel Garc´ıa Ferna´ndez,§
Carmen Ortiz Mellet,*,| Franc¸ois-Yves Dupradeau,*,‡ and Jose´ Kovensky*,†
Laboratoire des Glucides UMR CNRS 6219, Faculte´ des Sciences, UniVersite´ de Picardie Jules Verne,
33 Rue Saint-Leu, 80039 Amiens Cedex 1, France, DMAG, EA 3901, Faculte´ de Pharmacie et de
Me´decine, UniVersite´ de Picardie Jules Verne, 1-3 Rue des LouVels, 80037 Amiens Cedex 1, France,
Instituto de InVestigaciones Qu´ımicas, CSIC, Ame´rico Vespucio 49, Isla de la Cartuja, 41092 SeVilla,
Spain, and Departamento de Qu´ımica Orga´nica, Facultad de Qu´ımica, UniVersidad de SeVilla,
Apartado 553, 41071 SeVilla, Spain
jose.koVensky@u-picardie.fr
ReceiVed June 12, 2007
A short and efficient strategy for the synthesis of multi-valent mannosides based on a selectively functionalized
carbohydrate scaffold was reported involving (i) direct regioselective azidation of unprotected commercial
saccharides, (ii) acetylation, (iii) grafting of the mannosyl ligands by click chemistry, and (iv) deacetylation.
New glycoclusters with a valency ranging from 1 to 4 and different spatial arrangements of the epitopes were
obtained. Binding affinities of the new glycoclusters toward concanavalin A (Con A) lectin were investigated
by an enzyme-linked lectin essay (ELLA). The synthetic multi-valent compounds exhibited a remarkable cluster
effect with a relative potency per mannoside residue ranging from 8.1 to 9.1 depending on the structures.
ELLA experiments were in agreement with the establishment of favorable interactions between triazole ring
and Con A, increasing the binding affinity. A new force field topology database was developed in agreement
with the GLYCAM 2004 force field. Molecular dynamics performed on representative glyco-conjugates revealed
interesting structural features such as rigidity of the scaffold for a well-defined presentation of the ligands and
highly flexible mannose counterparts. The new glycoconjugates reported may be promising tools as probes or
effectors of biological processes involving lectins.
Introduction
apoptosis, tumor metastasis, or viral and bacterial infections.2,3
The generally low affinity of monovalent ligands for their
putative lectins can be overcome by using polyfunctional
scaffolds displaying structurally well-defined saccharide units.
Carbohydrate-lectin interactions play a pivotal role in many
biological events including inflammation,1 immune response,
* Corresponding authors. (S.G.G. and J.K.) Fax: (+33) 3 22827568. (C.O.M.)
Fax: (+34) 954-624-960. (F.-Y.D.) Fax: (+33) 3 22827469.
† Faculte´ des Sciences, Universite´ de Picardie Jules Verne.
‡ Faculte´ de Pharmacie et de Me´decine, Universite´ de Picardie Jules Verne.
§ Instituto de Investigaciones Qu´ımicas.
(1) (a) Hanessian, S.; Mascitti, V.; Rogel, O. J. Org. Chem. 2002, 67,
3346-3354. (b) Murphy, P. V.; Hubbard, R. E.; Manallack, D. T.; Wills,
R. E.; Montana, J. G.; Taylor, R. J. K. Bioorg. Med. Chem. 1998, 6, 2421-
2439.
| Universidad de Sevilla.
10.1021/jo071248a CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/03/2007
9032
J. Org. Chem. 2007, 72, 9032-9045

Multi-Mannosides Based on a Carbohydrate Scaffold
The affinity enhancements reached by using this strategy can
be very impressive.4,5 A remarkable example is the decavalent
ligand designed by Bundle and co-workers (STARFISH), which
inhibited Shiga-like toxins in ELISA assays with a 106-fold
enhancement over the monomeric species.6
Carbohydrates have not been investigated much as scaffolds
as compared to dendrimers or polymers.8,17 Most of the
examples on record correspond to glycosylamide scaffolds and
cyclomaltooligosaccharide (cyclodextrin)-centered glycoclus-
ters.18,19 The use of nonreducing mono- or disaccharide tem-
plates where all hydroxyl groups have been elaborated to graft
sugar epitopes has also been explored.20 For in vivo applications,
however, multi-valent ligands based on carbohydrate scaffolds
keeping some free hydroxyl groups may be more appropriate.
Improved hydrophilicity and pharmacokinetics may be expected,
for instance, as compared to peptidic, aromatic, or polymeric
scaffolds.13a,21 Following our ongoing interest in the use of click
chemistry for the synthesis of neoglycoconjugates,22 we present
herein the preparation of new multi-mannosides from regiose-
lectively modified mono-, di-, and trisaccharide templates. The
binding affinities of these glycoconjugates toward the mannose-
specific lectin concanavalin A (Con A) were evaluated by
enzyme-linked lectin assay (ELLA).23 To rationalize the ob-
served affinities, molecular dynamics (MD) calculations were
performed on four representative structures.
Depending both on the nature of the receptor and on the
valence and geometry of the ligand, this gain in stability, the
so-called multi-valent or cluster effect,7 can be explained by
the interplay of different mechanisms, including a chelation
binding mode, clustering of receptors, or statistical rebinding.8
Hundreds of synthetic multi-valent glycomimetics with diverse
spatial arrangements, number of epitopes, and degrees of
freedom have been synthesized. Such glycoconjugates have been
classified as glycodendrimers, glycoclusters, glycopolymers, and
glycoproteins depending on the nature of their scaffold. Different
reactions have been used for grafting the sugar epitopes (e.g.,
glycosilation,9 peptidic coupling, thiourea and oxime bond
formation,10,11 Sonogashira coupling,12 photochemically pro-
moted radical addition of thiols to double bonds,13 and the
Huisgen 1,3-dipolar cycloaddition).14 The last reaction, also
referred to as “click chemistry”, owes its usefulness in part to
its high compatibility with a broad range of functional groups
(e.g., alcohols, carboxylic acids, and amines) in different solvent
systems, including water.15 Furthermore, the generated triazole
ring is stable to hydrolytic cleavage and virtually inert toward
oxidation or reduction.16
Results and Discussion
Synthesis of Multi-Mannosides on Carbohydrate Scaffold.
Commercially available maltose and maltotriose were considered
as polyfunctional cores to generate multi-valency. Regioselective
polyazidation of the fully unprotected sugars was performed in
one pot by using triphenylphosphine/carbon tetrabromide/sodium
azide in dry DMF as the azidation system.24 The number and
the position of the azido groups in the final compound can be
modulated by varying the relative proportions of reagents
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Schmidtke, R.; Weber, W.; Goodman, S. L.; Kessler, H.; Schwaiger, M. J.
Nucl. Med. 2001, 42, 326-336. (b) Beer, A. J.; Haubner, R.; Goebel, M.;
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J. Org. Chem, Vol. 72, No. 24, 2007 9033

Gouin et al.
SCHEME 1. Direct Azidation of Maltose
CHART 1. Structure of Azido Compounds 1-7
SCHEME 2. Synthesis of Cycloadduct 9 by Click Chemistry
(Scheme 1). The crude reaction mixtures were per-O-acetylated
with acetic anhydride-pyridine prior to chromatographic puri-
fication. Despite moderate yields, the direct azidation-acety-
lation methodology is of practical value as compared to
alternative multi-step syntheses employed for accessing polyazi-
dosugars.
The ensemble of azidosugars synthesized by this procedure
is presented in Chart 1. Glucopyranosyl azide 125 and azidosug-
ars 2 and 5 were obtained from D-glucose, maltose, and
maltotriose in 31, 49, and 41% yields, respectively, by using a
2:2:10 PPh3/CBr4/NaN3 molar proportion per monosaccharide
unit, followed by acetylation. When the proportion of the
azidation reagent was increased to 4:4:20, the same protocol
allowed isolation of the anomeric mixture 3(R)/4(â) from
maltose and 6(R)/7(â) from maltotriose in 39 and 26% yields,
repectively. In both cases, the per-O-acetylated anomers could
be separated by flash chromatography.
noside 8 was considered as a suitable partner. Compound 8 was
synthesized from the corresponding peracetylated monosaccha-
ride by treatment with but-3-yn-1-ol in the presence of boron
trifluoride etherate.26 The first cycloaddition trial was performed
with 8 and acetylated glucopyranosyl azide 1 (Scheme 2).
Cycloadduct 9 could be obtained using sodium ascorbate and
copper sulfate as a catalyst in a 4:1 mixture of DMF and water.22
Using DMF instead of the commonly used alcohols, such as
t-butanol, was found to be generally advantageous in terms of
better solubilization of the substrates and faster reaction times.
Water has a positive effect on the reaction as previously
reported.27 Indeed, no cycloaddition product was formed if the
reaction was conducted in dry DMF using the same proportions
of reagents and reactants. Final deacetylation was carried out
with sodium methanolate in methanol using standard Zemple`n
conditions, affording the monovalent ligand 10 in 87% yield.
To check the suitability of the prepared (poly)azidosugars
for click chemistry glycocoating strategies, the alkynyl man-
(26) Mereyela, H. B.; Gurrala, S. R. Carbohydr. Res. 1998, 307, 351-
354.
(27) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
(25) Sabesan, S.; Neira, S. Carbohydr. Res. 1992, 223, 169-185.
9034 J. Org. Chem., Vol. 72, No. 24, 2007

Multi-Mannosides Based on a Carbohydrate Scaffold
The same protocol was sucessfully repeated with polyazi-
dosugars 2-7. Yields ranged from 58 to 76% for the dipolar
cycloaddition reaction ( f 11, 13, 15, 17, 19, and 21) and from
56 to 89% for the Zemple`n deprotection step ( f 12, 14, 16,
18, 20, and 22). The coupling reaction was clean, and total
consumption of the starting substrates was observed in all cases,
with formation of the expected cycloadducts as the only reaction
products (Chart 2). The somewhat moderate isolation yields
were probably due to partial protonation of the triazole ring
over silica gel during purification. Furthermore, the reaction was
found to be highly regioselective, yielding 1,4-disubstituted
1,2,3-triazole-containing pseudo-oligosaccharides. The olefinic
proton associated with the triazole ring was identified as a singlet
at δ ) 7.5-8 ppm. The large ∆(δC-4 - δC-5) values observed
for the different triazoles, ranging from 19 to 25 ppm, cor-
roborated the 1,4-disubstituted structure since much smaller
values would be expected for 1,5-disubstituted regioisomers.28
for aryl mannopyranosides.30 On the basis of molecular model-
ing, NMR, and X-ray evidence, this effect has been ascribed to
stabilizing interactions between aromatic aglycon and protein
amino acids nearby once the mannopyranosyl residue is located
in the binding site of Con A.31 A similar scenario probably
applies in the case of the aglyconic triazole ring.
The divalent derivative 12 exhibited a remarkable cluster
effect. The binding efficiency was 6-fold higher as compared
to monovalent 10, which means 3-fold on a mannose molar
basis. No precipitation was observed during the lectin binding
assay at any concentration, supporting an aggregation indepen-
dent mechanism.32,33
Previous results have shown that the presence of the peroxi-
dase label used in ELLA actually promotes 1:1 sugar ligand/
lectin stoichiometries.34 Since the short distance between the
binding epitopes, which cannot expand the distance between
two recognition sites in Con A (about 65 Å), prevents a chelate
binding mode, the important affinity enhancement observed must
be ascribed to either the existence of an extended binding site
in the lectin that can establish favorable interactions with both
mannosyl residues simultaneously35 or to a sliding mechanism
between the two mannopyranosyl subunits in the maltosyl
scaffold at the primary monosaccharide binding site in Con A.36
The new glycococlusters prepared in this study display
structural differences that are governed by the sugar scaffold
substitution pattern as well as configurational and conforma-
tional biases, offering a good opportunity to test the influence
of such factors on carbohydrate-protein recognition. The whole
ligand set includes R-D-mannopyranosyl conjugates with va-
lencies ranging from 1 to 4. Compounds exhibiting the same
valency, such as 14, 16 and 18, or 20 and 22, differ by the
relative position of the mannosyl residues, one of them being
attached at either a primary C-6 position or the anomeric
position, in that case, with either an R- or â-configuration. As
a suitable protein receptor model, the leguminous tetrameric
lectin Con A, which specifically recognizes R-D-mannopyra-
nosyl residues,29 has been considered.
Going from divalent to trivalent arrangements (14, 16, and
18) led to higher affinities (IC50 ) 33 ( 3, 31 ( 3, and 33 (
4 µM, respectively), although the per mannose binding efficien-
cies were slightly poorer (2.7-2.9). For the tetravalent glyco-
clusters 20 and 22, we observed a similar trend, with binding
affinities of 25 ( 3 and 23 ( 2 µM and valency-corrected
affinities of 2.7 and 2.9. The incorporation of additional
mannosyl units led, therefore, to merely statistical binding
affinity enhancements. This result supports the occurrence of a
sliding process that is optimal for the dimeric compound and
suffers from enthalpy-entropy compensation for higher valent
compounds. The existence of an extended binding site would
Affinity for Con A. The affinities of the prepared glyco-
conjugates toward Con A were evaluated by the ELISA-type
protocol ELLA. This experiment measures the capacity of a
soluble ligand to inhibit the lectin binding to a polymeric ligand
that is used as a coating material on the microtiter well. In the
present case, competitive experiments using horseradish per-
oxidase labeled Con A (HRP-Con A) as the lectin and yeast
mannan as the microplate fixed ligand were carried out in
triplicate. Methyl R-D-mannopyranoside was included in the tests
as a reference compound. Up to eight different concentrations
of each sample were considered, and the percentage of the
inhibition of HRP-Con A-yeast mannan association was
determined spectrophotometrically. The IC50 values (mean of
three measurements), defined as the concentration of synthetic
compound to achieve 50% inhibition of this association, were
determined from the corresponding inhibition curves. IC50 values
are assumed to be inversely proportional to the corresponding
free energy of binding.
(30) (a) Iyer, R. N.; Goldstein, I. J. Immunochemistry 1973, 10, 313. (b)
Van Wauve, J.-P.; Loontiens, F. G.; Carchon, H. A.; De Bruyne, C. K.
Carbohydr. Res. 1973, 30, 249. (c) Loontiens, F. G.; Van Wauve, J.-P.; De
Gussem, R.; De Bruyne, C. K. Carbohydr. Res. 1973, 30, 51. (d) Van
Wauve, J.-P.; Loontiens, F. G.; De Bruyne, C. K. Biochim. Biophys. Acta
1975, 379, 456. (e) Page´, D.; Zanini, D.; Roy, R. Bioorg. Med. Chem. 1996,
4, 1944-1961.
(31) (a) Hamodrakas, S. J.; Alexandraki, E.; Troganis, A.; Stassinopoulou,
C. I. Int. J. Biol. Macromol. 1989, 11, 17-22. (b) Kanellopoulos, P.; Pavlou,
K.; Perrakis, A.; Agianian, B.; Vorgias, C. E.; Mavrommatis, C.; Soufi,
M.; Tucker, P. A.; Hamodrakas, S. J. Struct. Biol. 1996, 116, 345-355.
(32) Corbell, J. B.; Lundquist, J. J.; Toone, E. J. Tetrahedron: Asymmetry
2000, 11, 95-111.
(33) Formation of soluble aggregates between Con A and rigid oligo-
mannosides has been observed. However, in solution, lectin recruitement
should be reflected in activation, rather than inhibition, of the association
of the lectin to the reference mannan fixed in the microplate well surface
upon addition of the multi-valent ligands, resulting in abnormal inhibition
curves, which was not the present case. See: (a) Burke, S. D.; Zhao, Q.;
Schuster, C.; Kiessling, L. L. J. Am. Chem. Soc. 2000, 122, 4518-4519.
(b) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling,
L. L. J. Am. Chem. Soc. 2002, 124, 14922-14933.
(34) Go´mez-Garc´ıa, M.; Benito, J. M.; Rodr´ıguez-Lucena, D.; Yu, J.-
X.; Chmurski, K.; Ortiz Mellet, C.; Gutie´rrez Gallego, R.; Maestre, A.;
Defaye, J.; Garc´ıa Ferna´ndez, J. M. J. Am. Chem. Soc. 2005, 127, 7970-
7971.
Binding affinities of the reference and synthetic glycocon-
jugates for Con A are summarized in Figure 1. The valency-
corrected relative binding potencies expressed per mol of
mannopyranosyl residue relative to the monovalent compound
10 (IC50 ) 267 ( 28 µM) are presented in Figure 2. The
experimental data indicate that 10 is a 3-fold more efficient
ligand for Con A as compared to methyl R-D-mannopyranoside.
Similar affinity enhancements have been previously reported
(35) Con A is known to possess an extended binding site for the Manp-
a(1 f 3)-[Manp-a(1 f 6)]-Man trisaccharide core. See: (a) Mandal, D.
K.; Battachariyya, L.; Koening, S. H.; Brown, R. D., III; Oscarson, S.;
Brewer, C. F. Biochemistry 1994, 33, 1157. (b) Weatherman, R. V.; Mortell,
K. H.; Chervenak, M.; Kiessling, L. L.; Toone, E. J. Biochemistry 1996,
35, 3619-3624.
(28) Rodios, N. A. J. Heterocycl. Chem. 1984, 21, 1169-1173.
(29) (a) Bouckaert, J.; Hamelryck, T.; Wyns, L.; Loris, R. Curr. Opin.
Struct. Biol. 2001, 11, 635-643. (b) Vijayan, M.; Chandra, N. Curr. Opin.
Struct. Biol. 1999, 9, 707-714.
(36) Dam, T. K.; Brewer, C. F. Chem. ReV. 2002, 102, 387-429.
J. Org. Chem, Vol. 72, No. 24, 2007 9035

Gouin et al.
CHART 2. Structure (Isolated Yield) of Glycoclusters 11-22
be expected to be translated into a significant decrease in relative
binding affinity with valency unless the critical fragment is
repeated.
No significant differences were observed for trivalent and
tetravalent ligands with regards to the spatial arrangement of
the epitopes. For trimannosides (14, 16, and 18), the binding
9036 J. Org. Chem., Vol. 72, No. 24, 2007

Multi-Mannosides Based on a Carbohydrate Scaffold
FIGURE 1. Binding affinities of methyl mannoside (ref) and synthetic glycoclusters for Con A.
FIGURE 2. Valency-corrected relative binding potency (per mol of mannopyranoside residue).
affinity remains virtually unchanged when a mannopyranosyl
substituent is switched from the C-6 to the anomeric position,
irrespective of the R- or â-anomeric configuration. Analogously,
the R- and â-tetravalent glycolusters 20 and 22 exhibited
comparable binding affinities.
trans (gt) conformations were selected for the glucose and
mannose derivatives since these conformations are the most
commonly observed in solution for these two monosaccha-
rides.39 In the absence of experimental data for 1-N-methyl-4-
(2-hydroxyethyl)-1,2,3-triazole, the two lowest minima observed
after geometry optimization were chosen. Optimized geometries
presenting intra-molecular hydrogen bonds were not used in the
charge derivation to avoid an over-polarization effect. This
approach is compatible with the use of implicit polarization in
AMBER and GLYCAM force fields.38a,40 Four molecular
orientations for each optimized geometry were generated before
molecular electrostatic potential (MEP) computation and were
involved in the charge fitting procedure to yield reproducible
atom charge values.41 Inter-molecular charge constraints between
methyl and hydroxyl groups were used in charge fitting,
allowing the definition of the required molecular fragments
(Scheme 3A,B). The charge value of each hydrogen bound to
a carbon presenting sp3 hybridization was set to a constrained
value of zero to ensure a compatibility between this work and
the GLYCAM 2004 force field.42
Force Field Development and Atom Charge Value Deri-
vation. Molecular simulations were carried out using the
Glycam 2004 force field.37 However, in the absence of force
field topological fragments and RESP atom charge values for
triazole derivatives in this force field,38 a new force field
topology database (FFTPDB) compatible with glycoclusters 10,
12, 14, 16, 18, 20, and 22, and more generally with any Glc-
R-(1 f 4)-based polymer, was developed. Multiple molecules,
multiple conformations, and multiple molecular orientations
were used in charge derivation. A detailed description of this
procedure is reported in the Experimental Section. Five mol-
ecules, namely, R- and â-D-glucopyranose, methyl R-D-glu-
copyranoside, methyl R-D-mannopyranoside, and 1-N-methyl-
4-(2-hydroxyethyl)-1,2,3-triazole, were considered (Scheme 3A).
Two conformations with the ω dihedral angle about the C5′-
C6′ bond corresponding to gauche, gauche (gg) and gauche,
(39) (a) Marchessault, R. H.; Perez, S. Biopolymers 1979, 18, 2369-
2374. (b) Bock, K.; Duus, J. O. J. Carb. Chem. 1994, 13, 513-543.
(40) Cornell, W. D.; Cieplak, P.; Kollman, P. A. J. Am. Chem. Soc. 1993,
115, 9620-9631.
(41) Pigache, A.; Cieplak, P.; Dupradeau, F.-Y. Automatic and Highly
Reproducible RESP and ESP Charge DeriVation: Application to the
DeVelopment of Programs RED and X RED, 227th ACS National Meeting,
Anaheim, CA, March 28 to April 1, 2004; ACS: Washington, DC, 2004.
(37) (a) Kirschner, K. N.; Woods, R. J. J. Phys. Chem. A 2001, 105,
4150-4155. (b) Basma, M.; Sundara, S.; Calgan, D.; Varnali, T.; Woods,
R. J. J. Comput. Chem. 2001, 22, 1125-1137. (c) Kirschner, K. N.; Woods,
R. J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10541-10545.
(38) (a) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. J. Chem.
Phys. 1993, 97, 10269-10280. (b) Woods, R. J.; Chappelle, R. J. Mol.
Struct. 2000, 527, 149-156.
J. Org. Chem, Vol. 72, No. 24, 2007 9037

Gouin et al.
SCHEME 3. FFTPDB Constructed Using r- and â-D-Glucopyranose, Methyl r-D-Glucopyranoside, Methyl
r-D-Mannopyranoside, and 1-N-Methyl-4-(2-hydroxyethyl)-1,2,3-triazolea
a (A) Inter-molecular (represented by a dashed line) and intra-molecular (represented by a fine dashed line) charge constraints between methyl (belonging
to methyl R-D-glucopyranoside, methyl R-D-mannopyranoside, and 1-N-methyl-4-(2-hydroxyethyl)-1,2,3-triazole) and hydroxyl boxed groups (belonging to
methyl R-D-glucopyranoside, methyl R-D-mannopyranoside, and 1-N-methyl-4-(2-hydroxyethyl)-1,2,3-triazole) were used in charge fitting. (B) Glycoclusters
10, 12, 14, 16, 18, 20, and 22 and polymeric Glc-R-(1 f 4)-based oligosaccharides were built using this new FFTPDB.
Validation of the FFTPDB. This work represents the first
description of the FFTPDB reported. To check the validity of
this FFTPDB and in the absence of experimental data for the
ligands studied, and of triazol derivative units in the Glycam
2004 force field, a comparative study involving the glucose
scaffold [disaccharide Glc-R-(1 f 4) R-Glc] was carried out
using the molecular fragments and charge values taken from
the Glycam 2004 force field and the FFTPDB described in this
work. MD simulations were carried out for 10 ns, in presence
of explicit water molecules, and MD snapshots were generated
every 2 ns. Comparison of these MD snapshots reveals highly
similar three-dimensional structural features (Figure S1). Heavy
atom rmsd between the 10 MD snapshots selected and the
average structure of these snapshots present a maximum rmsd
values of 1.0 Å. This shows the excellent agreement of the
structures obtained using the two approaches and the establish-
ment of a well-defined family of three-dimensional structures
for the glucose-based disaccharide. Indeed, in both computa-
tional conditions, two persistent inter-unit hydrogen bonds (H-
bonds) were observed during MD between the hydroxyl 3 of
the reductive unit and the hydroxyl 2 of the second glucose
unit, each oxygen atom taking successively the role of the
acceptor or donor of the H-bond. These inter-glucose H-bonds
participate in the formation of stable structures for the glucose
scaffold. To further validate our FFTPDB, the evolution of the
ω dihedral angle values for the two glucose units was followed
during MD simulations. Oscillating values corresponding to gt
and gg rotamers (Figures S2A-D) were observed in both
computational conditions, which is consistent with experimental
data.39 Thus taken all together, the results obtained in this
comparative study unambiguously demonstrate the validity of
the FFTPDB reported.
MD Simulation. Structures of glycoclusters 12â, 12R, 16,
and 18 were studied during 10 ns of MD simulation in the
presence of explicit water molecules. Highly similar structural
features were observed for the different glycoclusters studied.
As an illustrative example, the survey for glycocluster 18 is
discussed herein. The presence of H-bonds, which reflects the
formation of a putative stable structure during the simulation,
was first checked. The 10 most frequent H-bonds observed are
reported in Table 1. A single stable H-bond between the HO3′-
O3′ donor of glucose unit 2 and the O2′ acceptor of glucose
unit 1 was detected, demonstrating the absence of a well-defined
three-dimensional structure for glycocluster 18. It is worth noting
that this H-bond was already observed in the disaccharide Glc-
R-(1 f 4) R-Glc simulation. This demonstrates the reproduc-
ibility of the results obtained using different models and
validates the FFTPDB described. On the contrary, several
transient H-bonds were observed during the simulation defining
different local minima.
(42) RRMS (relative root mean square value between MEP calculated
by quantum mechanics and that generated using the derived charge values)
of 0.126 was obtained for the fit. Intra-molecular charge constraints, inter-
molecular charge constraints, charge value equivalencing between the
different molecular orientations and conformations, and the hyperbolic
constraint of the restrained fit are accountable for an increase of 0.005,
0.003, 0.010, and 0.006 of the RRMS value, respectively. These values
rigorously demonstrate the strength and weakness of the approach followed.
The RESP charge derivation procedure was automatically carried out using
the RESP ESP charge Derive (R.E.D.) program (http://q4md-forcefield-
tools.org/RED/).41 The RESP atom charge values and FFTPDB were
submitted in the RESP ESP charge DDataBase (R.E.DD.B.) (http://q4md-
forcefieldtools.org/REDDB/) and are available as the “F-71” R.E.DD.B.
project code as a suite of files in the Tripos mol2 file format considered as
forcefieldlibraryprecursors(http://www.tripos.com/index.php?family)modules,-
SimplePage,,,&page)sup_mol2&s)0).
The Φ (O5′-C1′-O1′-C4′), ψ (C1′-O1′-C4′-C3′), and
ω dihedral angles of the glucose and mannose units of
glycocluster 18 were followed during MD simulation. The
values of those dihedral angles are reported in Figure S3 (see
Supporting Information). The ω dihedral angle about the C5′-
C6′ bond of the glucose scaffold units exhibited striking constant
values corresponding to the gt population (Figure S3A,B). This
result is explained by the establishment of specific stabilizing
interactions within the glycocluster. Indeed, nonclassical H-bond
9038 J. Org. Chem., Vol. 72, No. 24, 2007

Multi-Mannosides Based on a Carbohydrate Scaffold
TABLE 1. Ten Most Frequent H-Bonds Detected during 10 ns
MD Simulation for Glycocluster 18a
triazole derivative units display strikingly different features.
Similar dihedral profiles were observed during the simulation
for triazole units 4, 6, and 8 during the simulation. Consequently,
only the study of the dihedral angle values for unit 4 is presented
in Figure S5A-D as a demonstration. The C5′-C6′-N6-N5
dihedral angles between units 1 and 4 display similar values
during the simulation (Figure S5A). This provides new evidence
for the inter-unit interaction previously described involving the
H7 hydrogen and O5′ endocyclic oxygen of triazole derivative
units 4 and glucose 1, respectively. On the contrary, the three
other dihedral angles studied involving the triazole derivative
units 4 and mannose 3 (C1′-O1′-C1-C2, O1′-C1-C2-C3,
and C1-C2-C3-N4) display highly variable values (Figure
S5B-D). This observation is in agreement with the presence
of different local minima reflecting an important flexibility in
this region of glycocluster 18. The repetition of such dihedral
profiles for the three triazole derivative units led to the formation
of disorganized three-dimensional structures for glycocluster 18
allowing each mannose unit to adopt different orientations. To
further characterize this result, MD snapshots were selected
every nanosecond and superimposed over the last structure using
the pyranose ring atoms belonging to the glucose scaffold.
Figure 3 presents three-dimensional structures of glycoclusters
12â, 12r, 16, and 18 generated using this approach. This clearly
demonstrates the definition of regions with different dynamic
behaviors for each glycocluster. Indeed, independently of the
glycocluster studied, a rigid and well-defined domain for the
glucose scaffold and highly flexible counterparts composed of
the mannose units are observed. Two stabilizing forces were
clearly identified within the glycoclusters that participate in the
definition of the characteristic glucose scaffold structure. The
first one is a H-bond between hydroxyl 3 of the glucose unit of
the reductive side and hydroxyl 2 belonging to the other glucose
unit. The second one is a nonbonding interaction between the
endocyclic O5′ oxygen of glucose unit 1 or 2 and the C7-H7
hydrogen belonging to the triazole ring of unit 4 or 6. Moreover,
the role of the ethylene segments connecting the mannopyra-
nosyl substituents and the triazole rings is central to the high
flexibility observed. It is particularly striking for glycoclusters
16 and 18. Indeed, although the spatial arrangements for R- and
â-anomers are different, each ligand being located on opposite
faces of the scaffold plan, similar ELLA data are obtained for
these two glycoclusters. This clearly shows the importance of
the ethylene groups in the glycocluster building strategy, and
their central role in the flexibility and ELLA results is observed.
On the other hand, the MD simulations performed for 16 and
18 indicated that the rigidity of the maltosyl scaffold virtually
prevented conformations from having simultaneously the three
mannose residues in close spatial proximity, while conforma-
tions having two of them in the same region are frequent. This
dynamic behavior probably explains the high cluster effect
observed for divalent compounds. While sliding of the lectin
between two vicinal mannnosyl ligands is strongly favored,
moving along the maltooligosaccharide scaffold to the next
mannosyl pair has a penalty associated with the adoption of
transient unfavorable conformations. Thus, the results are
compatible with the existence of a sliding mechanism that would
be optimal for the divalent compound that undergoes enthalpy-
entropy compensation for the tri- and tetravalent derivatives at
the origin depending on the structures. The loss of entropy when
the mannose binds to the lectin is compensated by a gain of
a Loose H-bond criteria [distance (acceptor-donor) ) 3.2 Å (3.0 is the
canonical value) and angle (acceptor-hydrogen-donor) ) 120° (180 is
the canonical value)] were selected for H-bond printing ensuring the
detection of both weak and strong H-bonds. Notation of H-bond acceptors
and donors corresponds to “unit number:atom name”. For the schematic
representation of the percentage of occupancy observed during the simula-
tion, the following convention has been used: “blank”, >0-5%; “-”,
5-20%; “+”, 20-40%; and “o”, 40-60%. For notation of the triazole
atom numbers, see the Supporting Information.
interactions were detected between the endocyclic O5′ oxygen
of glucose unit 1 or 2 and the H7 proton at the triazole unit 4
or 6 (Figure S4A-C). Although weak, the nonbonding interac-
tion involving the H7 atom of unit triazole derivative 6 is
unequivocal. Indeed, this atom unambiguously interacts with
the O5′ acceptor of units glucose 1 or 2, after rotation around
the O5′-C5′-C6′-N6 dihedral angle (Figure S4B,C). To
further substantiate this result, model structures were optimized
in the gas phase by ab initio calculations using the B3LYP/6-
311+G** theory level,43 and an optimal distance of 2.75 Å was
found between the H7 and the O5′ atoms for the lowest
minimum. On the contrary, ω dihedral angles for the three
mannose units oscillated between values corresponding to the
expected gt and gg rotamers (Figure S3C-E), which is
consistent with experimental data.39 This observation is another
argument in favor of the validity of the FFTPDB reported. It is
important to emphasize that the HO6′ hydroxyl of mannose units
3, 5, and 7 remained solvated during most of the simulation
time, preventing the formation of stable intra-glycocluster
interactions involving this primary hydroxyl group. Finally, the
Φ and ψ dihedral angles displayed similar values during MD
simulation (Figure S3F-K), corresponding to gauche popula-
tions. This result is a strong argument in favor of the adoption
of a stable structure for the glucose scaffold. In particular, the
dihedral angle ψ observed in glycocluster 18 within the glucose
scaffold was stabilized by the H-bond interaction previously
reported between the HO3′-O3′ donor of glucose unit 2 and
the O2′ acceptors of glucose unit 1 (Table 1).
As compared to the Φ, ψ, and ω dihedral angles of the
glucose and mannose units, the dihedral angles involving the
(43) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio
Molecular Orbital Theory; John Wiley and Sons: New York, 1986.
J. Org. Chem, Vol. 72, No. 24, 2007 9039

Gouin et al.
FIGURE 3. Three-dimensional structures of glycoclusters 12â, 12r, 16, and 18 observed during 10 ns MD simulation. For each glycocluster, MD
snapshots were selected every nanosecond, superimposed over the last structure using the pyranose ring atoms of the glucose scaffold, and displayed
(hydrogen atoms are not represented for clarity). (A) Structures of glycocluster 12â; (B) structures of glycocluster 12r; (C) structures of glycocluster
16; and (D) structures of glycocluster 18.
enthalpy resulting from the establishment of specific electrostatic
interactions between the ligand and the protein within the
binding site.
triphenylphosphine-carbon tetrabromide-sodium azide allows the
requested polyazido templates to be accessed in one pot. In
addition, we have shown that the degree of azidation can be
modulated by acting on the reagent ratio. Further coating with
a model alkynyl mannoside by click chemistry proceeded
cleanly to give the target polyconjugates. Evaluation of the
binding affinity against Con A lectin by ELLA indicated the
existence of stabilizing interactions between the generated 1,2,3-
triazole ring and the protein for a monovalent derivative.
Interestingly, higher valent compounds exhibited strong and
comparable cluster effects (molar relative potencies ranging from
8.1 to 9.1), irrespective of valence (2-4) or spatial arrangement.
The results are compatible with the existence of a sliding
mechanism that would be optimal for the divalent compound
and undergoes enthalpy-entropy compensation for the tri- and
tetravalent derivatives. MD calculations performed on repre-
sentative glycoconjugates indicated that these compounds
combine a remarkable rigidity of the oligosaccharide scaffold
and a relatively high flexibility of the mannosyl branches that
is consistent with the ELLA observations. The possibility of
modulating the orientational properties and dynamic behavior
in multi-valent glycoligands based on regioselectively func-
It is noteworthy that previous work using mannosyl coated
flexible dendritic cores had shown optimal affinity enhancements
for R-D-mannopyranosyl triads, while divalent compounds
exhibited relative binding affinities lower than the monovalent
counterparts.44 The present results underline the importance of
the scaffold architecture on the lectin binding affinity properties
of multi-valent glycoligands. A fine, mechanism-based tuning
of carbohydrate-lectin interactions can be achieved by control-
ling the distance between the binding epitopes, their relative
spatial orientation, and the dynamic behavior of the whole
system.
Conclusion
In summary, we described in this paper a short and efficient
strategy for the synthesis of carbohydrate-centered multi-
mannosides using click chemistry. Regioselective azidation of
unprotected commercial mono- and oligosaccharides with
(44) Baussanne, I.; Benito, J. M.; Ortiz Mellet, C.; Garc´ıa Ferna´ndez, J.
M.; Law, H.; Defaye, J. Chem. Commun. 2000, 1489-1490.
9040 J. Org. Chem., Vol. 72, No. 24, 2007

Multi-Mannosides Based on a Carbohydrate Scaffold
H-4′), 4.83 (1H, dd, J2,3 10.0 Hz, H-2), 4.83 (1H, dd, J1′,2′ 3.9 Hz,
J2′,3′ 10.4 Hz, H-2′), 4.16-4.05 (2H, m, H-4, H-5), 3.84 (1H, m,
H-5′), 3.70 (1H, dd, J5,6a 2.3 Hz, J6a,6b 11.7 Hz, H-6a), 3.52-3.46
(2H, m, H-6b, H-6′a), 3.33 (1H, dd, J5,6′b 5.0 Hz, J6′a,6′b 13.5 Hz,
H-6′b), 2.06, 2.03, 1.99 (15H, 3s, 5 × CH3CO); 13C NMR (75 MHz,
CDCl3): δ ) 170.5, 170.0, 169.9, 169.5 (CO), 95.2 (C-1′R), 86.2
(C-1R), 71.7, 71.4 (C-3, C-4, C-5), 70.6, 70.0(C-2, C-2′), 69.6 (C-
4′), 69.2, 69.0 (C-3′, C-5′), 50.9 (C-6, C-6′), 20.9, 20.6, 20.5 (CH3).
HRMS (ES+): Found 650.1753 C22H29N9O13Na requires 650.1783
[M + Na+].
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(1 f
4)-2,3-di-O-acetyl-6-azido-6-deoxy-â-D-glucopyranosyl Azide (4).
[R]20D +79 (c 0.1, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ ) 5.40
(1H, d, J1′,2′ 3.9 Hz, H-1′), 5.30 (1H, t, J2,3 ) J3,4 9.8 Hz, H-3),
5.26 (1H, t, J2′,3′ ) J3′,4′ 9.6 Hz, H-3′), 4.97 (1H, t, J3′,4′ ) J4′,5′ 9.6
Hz, H-4′), 4.84-4.76 (2H, m, H-2′, H-2), 4.66 (1H, d, J1,2 8.8 Hz,
H-1), 4.07 (1H, t, J′,4 ) J4,5 9.8 Hz, H-4), 3.81-3.69 (3H, m, H-5,
H-5′, H-6′a), 3.50-3.41 (2H, m, H-6a, H-6′b), 3.33 (1H, dd, J5,6b
5.0 Hz, J6a,6b 13.5 Hz, H-6b), 2.03, 2.02, 1.98 (15H, 3s, 5 × CH3-
CO); 13C NMR (75 MHz, CDCl3): δ ) 170.4, 170.2, 170.0, 169.5,
169.4 (CO), 95.2 (C-1′), 87.5 (C-1), 76.2, 74.6, 71.5, 71.4, 70.1,
69.6, 69.1, 68.9 (C-2 to C-5, C-2′ to C-5′), 50.9 (C-6, C-6′), 20.8,
20.7, 20.6, 20.5 (CH3CO). HRMS (ES+): Found 650.1813
C22H29N9O13Na requires 650.1783 [M + Na+].
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-r-D-glucopyranosyl-(1 f
4)-2,3-di-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(1 f 4)-
1,2,3-tri-O-acetyl-6-azido-6-deoxy-D-glucopyranose (5). 1H NMR
(300 MHz, CDCl3) δ ) 6.23 (1H, d, J1R,2R 3.7 Hz, H-1R), 5.68
(1H, d, J1,2 8.1 Hz, H-1â), 5.50-5.23 (10H, m, H-3Râ, H-1′Râ,
H-3′Râ, H-1′′Râ, H-3′′Râ), 4.95-4.90 (4H, m, H-2Râ, H-4′′Râ),
4.77-4.65 (4H, m H-2′Râ, H-2′′Râ), 4.08-3.90 (6H, m, H-4Râ,
H-5Râ, H-4′Râ), 3.80-3.60 (8H, m, H-6aRâ, H-5′Râ, H-6′aRâ,
H-5′′Râ), 3.45-3.4 (6H, m, H-6bRâ, H-6′bRâ, H-6′′aRâ), 3.25-
3.22 (m, 2H, H-6′′bRâ), 1.99, 1.98, 1.97, 1.95, 1.94, 1.93, 1.92
(48H, 7s, 16 × CH3CO); 13C NMR (75 MHz, CDCl3): δ ) 170.5,
170.1, 169.8, 169.7, 169.4, 168.9 (CO), 95.4, 95.3 (C-1′R, C-1′′R,
C-1′â, C-1′′â), 91.2 (C-1â), 88.8 (C-1R), 75.0, 74.0, 72.0, 71.6,
71.4, 71.2, 70.9, 70.5, 70.3, 70.0, 69.7, 69.4, 69.1 (C-2 to C-5Râ,
C-2′ to C-5′Râ, C-2′′ to C-5′′Râ), 50.8 (C-6, C-6′, C-6′′Râ), 20.9,
20.8, 20.6, 20.5 (CH3CO); HRMS (ES+): Found 938.2662
C34H45N9O21Na requires 938.2628 [M + Na+].
tionalized carbohydrate scaffolds makes these conjugates prom-
ising tools as probes or effectors of biological processes.
Experimental Section
General Procedure for the Synthesis of Azido Compounds.
Carbohydrate (1.38 mmol of monosaccharide unit) and sodium azide
(0.903 or 1.801 g, 13.9 or 27.7 mmol) were dissolved in anhydrous
DMF (8 mL) at room temperature (rt). PPh3 (0.726 or 1.456 g,
2.77 or 5.55 mmol) was added to the mixture, and after 30 s of
stirring, CBr4 (0.919 or 1.841 g, 2.77- or 5.55 mmol) dissolved in
anhydrous DMF (2 or 4 mL) was added dropwise (slight exotherm
was observed). The resulting mixture was stirred at rt for 60 h under
nitrogen. Methanol (5 mL) was added, and the solution was filtered.
After evaporation under reduced pressure, water (20 mL) and
toluene (20 mL) were added. The mixture was vigorously stirred,
and ethyl acetate was added dropwise until the system became clear.
The organic layer was extracted with water (3 × 20 mL). Aqueous
layers were combined, and the solution was evaporated under
vacuum. The dry residue was dissolved in 1:1 Pyr/Ac2O (50 mL).
After 12 h at rt, the solvent was removed under reduced pressure,
and the residue was dissolved in ethyl acetate (20 mL). The organic
layer was washed with water (20 mL), dried over Na2SO4, filtered,
and evaporated to dryness. The residue was purified by flash
chromatography (FC) (cyclohexane/ethyl acetate or toluene/diethyl
ether) leading to azido compounds.
General Procedure for the Click Reaction. Compounds 9, 11,
13, 15, 17, 19, and 21 were synthesized using this procedure.
Alkynyl-saccharide 8 (152 mg, 0.38 mmol) and azido-saccharide
4 (80 mg, 0.13 mmol) were dissolved in a DMF/H2O mixture (8:2
mL). Copper sulfate (0.19 mmol) and sodium ascorbate (0.38 mmol)
were added, and the mixture was stirred at rt until MS indicated
the disappearance of starting materials and intermediates (10-16
h). The mixture was poured into a 1:1 H2O/NH4Cl saturated solution
(60 mL) and extracted with EtOAc (4 × 30 mL). The organic layer
was dried over Na2SO4 and filtered, and the solvent was removed
under reduced pressure. The residue was purified by flash chro-
matography to give 15 (177 mg, 76% yield).
General Procedure for Deacetylation. Compounds 10, 12, 14,
16, 18, 20, and 22 were synthesized using this procedure. The
cycloadduct (70 mg) was dissolved in dry MeOH (4 mL). A solution
of sodium methanolate (1 M) in methanol (133 µL) was added,
and the mixture was stirred under N2 at rt until MS indicated the
total disappearance of starting materials and intermediates (3 h).
In some cases, precipitation of the product occurred. If so, water
was added until complete dissolution of the precipitate. Amberlyst
IR120(H+) was added, and the mixture was stirred until the pH
reached 5. The resin was filtered off, and the solution was
evaporated to dryness leading to the pure unprotected product.
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(1 f
4)-1,2,3-tri-O-acetyl-6-azido-6-deoxy-D-GLUCOPYRANOSE (2). 1H
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-r-D-glucopyranosyl-(1 f
4)-2,3-di-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(11 f 4)-
2,3-di-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl Azide (6).
1
[R]20 +155 (c 0.7, CH2Cl2); H NMR (300 MHz, CDCl3) δ )
D
5.56 (1H, d, J1,2 4.2 Hz, H-1), 5.43-5.29 (5H, m, H-3, H-1′, H-3′,
H-1′′, H-3′′), 5.00 (1H, t, J3′′,4′′ ) J4′′,5′′ 9.4 Hz, H-4′′), 4.84 (1H,
dd, J1,2 4.2 Hz, J2,3 10.0 Hz, H-2), 4.82 (1H, dd, J1′′,2′′ 4.4 Hz, J2′′,3′′
10.0 Hz, H-2′′), 4.73 (1H, dd, J1′,2′ 4.4 Hz, J2′,3′ 10.1 Hz, H-2′),
4.17-4.15 (1H, m, H-5), 4.09 (1H, t, J3,4 ) J4,5 9.0 Hz, H-4), 4.07
(1H, t, J3′,4′ ) J4′,5′ 10.4 Hz, H-4′), 3.90-3.70 (5H, m, H-5, H-6a,
H-5′, H-6′a, H-5′′), 3.55-3.40 (3H, m, H-6b, H-6′b, H-6′′a), 3.34
(1H, dd, J5′′,6′′b 4.7 Hz, J6′′a,6′′b 13.5 Hz, H-6′′b), 2.10, 2.03, 2.02
(21H, 3s, 7 × CH3CO); 13C NMR (75 MHz, CDCl3): δ ) 170.7,
170.2, 170.1, 170.0, 169.6 (CO), 95.4, 95.3 (C-1′, C-1′′), 86.4 (C-
1), 71.9, 71.8, 71.5, 71.4, 70.8, 70.7, 70.5, 70.2, 69.6, 69.4, 69.3
(C-2 to C-5, C-2′ to C-5′, C-2′′ to C-5′′), 51.2, 51.0 (C-6, C-6′,
C-6′′), 21.1, 20.8, 20.7, 20.6 (CH3CO); HRMS (ES+): Found
921.2601 C32H42N12O19Na requires 921.2587 [M + Na+].
NMR (300 MHZ, CDCL ) ∆ ) 6.26 (1H, D, J1,2 3.8 HZ, H-1Α),
3
5.72 (1H, D, J1,2 8.1 HZ, H-1Β), 5.50-5.28 (6H, M, H-3ΑΒ, H-1′ΑΒ,
H-3′ΑΒ), 5.01 (4 H, M, H-2ΑΒ, H-4′ΑΒ), 4.82 (2H, M, H-2′ΑΒ),
4.12 (4H, M, H-4ΑΒ, H-5ΑΒ), 3.81 (2H, M, H-5′ΑΒ), 3.67 (2H, M,
H-6AΑΒ), 3.42 (5H, M, H-6BΑΒ, H-6′AΑΒ, H-6′BΒ), 3.29 (1H, DD,
J5′,6′B 5.0 HZ, J6′A,6′B 13.4 HZ, H-6′BΑ), 2.18, 2.03, 2.02, 2.01, 1.98,
1.96, 1.95 (36H, 7S, 12 × CH3CO); 13C NMR (75 MHZ, CDCL ):
3
∆ ) 170.5, 170.2, 170.1, 169.8, 169.5, 169.0 (CO), 95.4 (C-1′Α),
95.2 (C-1′Β), 91.4 (C-1Β), 88.9 (C-1Α), 76.7, 75.1, 74.3, 73.9, 72.1,
71.7, 71.5, 71.0, 69.8, 69.7, 69.3, 69.1 (C-2 TO C-5ΑΒ, C-2′ TO
C-5′ΑΒ,), 51.0, 50.8, 50.7 (C-6ΑΒ, C-6′ΑΒ), 21.0, 20.8, 20.7, 20.6,
20.5 (CH3CO); HRMS (ES+): FOUND 667.1834 C24H32N6O15NA
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(1 f
4)-2,3-di-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(1 f 4)-
2,3-di-O-acetyl-6-azido-6-deoxy-â-D-glucopyranosyl Azide (7).
1
[R]20 +108 (c 0.2, CH2Cl2); H NMR (300 MHz, CDCl3) δ )
D
+
REQUIRES 667.1823 [M + NA ].
5.39-5.25 (5H, m, H-1′, H-1′′, H-3, H-3′, H-3′′), 4.97 (1H, t, J3′′,4′′
) J4′′,5′′ 9.0 Hz, H-4′′), 4.81-4.63 (3H, m, H-2, H-2′, H-2′′), 4.65
(1H, d, J1,2 8.8 Hz, H-1), 4.13 (1H, t, J3,4 ) J4,5 9.3 Hz, H-4), 4.00
(1H, t, J3′,4′ ) J4′,5′ 9.4 Hz, H-4′), 3.90-3.70 (5H, m, H-5, H-6a,
H-5′, H-6′a, H-5′′), 3.55-3.40 (3H, m, H-6b, H-6′b H-6′′a,), 3.32
(1H, dd, J5′′,6′′b 4.7 Hz, J6′′a,6′′b 13.5 Hz, H-6′′b), 2.03, 2.02, 2.01,
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl-(1 f
4)-2,3-di-O-acetyl-6-azido-6-deoxy-R-D-glucopyranosyl Azide (3).
1
[R]20 +162 (c 0.1, CH2Cl2); H NMR (300 MHz, CDCl3) δ )
D
5.56 (1H, d, J1,2 4.2 Hz, H-1), 5.46-5.40 (2H, m, H-3, H-1′), 5.31
(1H, t, J2′,3′ ) J3′,4′ 10.0 Hz, H-3′), 5.01 (1H, t, J3′,4′ ) J4′,5′ 9.6 Hz,
J. Org. Chem, Vol. 72, No. 24, 2007 9041

Gouin et al.
1.99, 1.98 (21H, 5s, 7 × CH3CO); 13C NMR (75 MHz, CDCl3): δ
) 170.5, 170.2, 169.9, 169.5 (CO), 95.4, 95.2 (C-1′, C-1′′), 87.5
(C-1), 76.1, 74.9, 71.6, 71.4, 70.6, 70.5, 70.1, 69.5, 69.2 (C-2 to
C-5, C-2′ to C-5′, C-2′′ to C-5′′), 51.0, 50.9 (C-6, C-6′, C-6′′), 21.0,
20.7, 20.6 (CH3CO); HRMS (ES+): Found 921.2590 C32H42N12O19-
Na requires 921.2587 [M + Na+].
1-[2,3,4,6-Tetra-O-acetyl-â-D-glucopyranos-1-yl]-4-[2,3,4,6-
tetra-O-acetyl-R-D-mannopyranosyloxyethyl]-[1,2,3]-triazol (9).
[R]20D +12 (c 0.1, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ ) 7.76
(1H, s, CHN), 5.80 (1H, d, J1,2 8.7 Hz, H-1), 5.45-5.25 (6H, m,
H-2, H-3, H-4, H-2M, H-3M, H-4M), 4.79 (1H, s, H-1M), 4.23-
3.95 (7H, m, H-5, H-6a, H-6b, H-5M, H-6aM, H-6bM, OCHaCH2),
3.64-3.60 (1H, m, OCHbCH2), 2.99 (2H, m, OCH2CH2), 2.09,
2.04, 1.98, 1.97, 1.95 (24H, 5s, 8 × CH3CO); 13C NMR (75 MHz,
CDCl3): δ ) 170.7, 170.5, 170.3, 170.0, 169.7, 169.4, 168.7, (CO),
145.1 (NCdCH), 121.0 (NCdCH), 97.9 (C-1M), 85.8 (C-1), 75.0,
72.6, 70.6, 69.5, 69.2, 69.0, 67.9, 66.0 (C-2 to C-5, C-2M to C-5M),
67.2 (OCH2CH2), 62.5, 61.9 (C-6, C-6M), 26.3 (OCH2CH2), 20.9,
20.8, 20.7, 20.6, 20.1 (CH3CO); HRMS (ES+): Found 796.2395
C32H43N3O19Na requires 796.2388 [M + Na+].
1-[â-D-Glucopyranos-1-yl]-4-[R-D-mannopyranosyloxyethyl]-
[1,2,3]-triazol (10). [R]20D +29 (c 0.4, H2O); 1H NMR (300 MHz,
D2O) δ ) 8.04 (1H, s, CHN), 5.66 (1H, d, J1,2 8.7 Hz, H-1), 5.45-
5.25 (6H, m, H-2, H-3, H-4, H-2M, H-3M, H-4M), 4.81 (1H, s,
H-1M), 4.20-3.90 (13H, m, H-2 to H-5, H-6a, H-6b, H-2M to
H-5M, H-6aM, H-6bM, OCHaCH2), 3.65-3.60 (1H, m, OCH-
bCH2), 3.02 (2H, m, OCH2CH2); 13C NMR (75 MHz, D2O): δ )
145.9 (NCdCH), 123.5 (NCdCH), 97.7 (C-1M), 87.7 (C-1), 79.2,
76.3, 73.1, 72.7, 70.8, 70.4, 69.3, 66.9 (C-2 to C-5, C-2M to C-5M),
66.5 (OCH2CH2), 61.1, 60.8 (C-6, C-6M), 25.5 (OCH2CH2); HRMS
(ES+): Found 460.1536 C16H27N3O11Na requires 460.1543 [M +
Na+].
2,3,4-Tri-O-acetyl-6-deoxy-6-[4-(2,3,4,6-tetra-O-acetyl-r-D-
mannopyranosyloxyethyl)triazol-1-yl]-R-D-glucopyranosyl-(1 f
4)-1,2,3-tri-O-acetyl-6-deoxy-6-[4-(2,3,4,6-tetra-O-acetyl-R-D-
mannopyranosyloxyethyl)triazol-1-yl]-D-glucopyranose (11). 1H
NMR (300 MHz, CDCl3) δ ) 7.57, 7.53 (4H, 2s, 2 × CHNRâ),
6.18 (1H, d, J1,2 3.7 Hz, H-1R), 5.67 (1H, d, J1,2 8.1 Hz, H-1â),
5.47-5.18 (18H, m, H-3, H-1′, H-3, H-3′, 2 × H-2M, 2 × H-3M,
2 × H-4MRâ), 4.81-4.45 (18H, m, H-2, H-6a, H-6b, H-2′, H-4′,
H-6′a, H-6′b, 2 × H-1MRâ), 4.30-3.80 (20H, m, H-4, H-5, 2 ×
H-5M, 2 × H-6aM, 2 × H-6bM, 2 × OCHaCH2Râ), 3.75-3.62
(6H, m, H-5′, 2 × OCHbCH2Râ), 3.16-2.85 (8H, m, 2 ×
OCH2CH2Râ), 2.11, 2.07, 2.06, 2.04, 2.02, 2.01, 1.98, 1.97, 1.96
(84H, 9s, 14 × CH3CORâ); 13C NMR (75 MHz, CDCl3): δ )
170.8, 170.5, 170.1, 170.0, 169.9, 169.8, 169.6, 169.0, 168.8, 162.6,
(CO), 144.4 (NCdCH), 124.5, 124.0 (NCdCH), 97.9, 97.7, 97.5
(C-1M), 95.7, 95.6 (C-1′Râ), 91.2 (C-1R), 88.6 (C-1â), 71.9, 72.0,
70.2, 69.9, 69.5, 69.4, 69.3, 69.2, 69.1, 68.7, 66.4 (C-2 to C-5,
C-2′ to C-5′, C-2M to C-5M), 67.4, 67.2, 67.0 (OCH2CH2), 62.4
(C-6M), 50.0, 49.9, 49.7 (C-6, C-6′), 26.3 (OCH2CH2), 21.0, 20.8,
20.7, 20.5 (CH3CO); HRMS (ES+): Found 1467.4607 C60H80N6O35-
Na requires 1467.4562 [M + Na+].
2,3,4-Tri-O-acetyl-6-deoxy-6-[4-(2,3,4,6-tetra-O-acetyl-R-D-
mannopyranosyloxyethyl)triazol-1-yl]-R-D-glucopyranosyl-(1 f
4)-2,3-di-O-acetyl-6-deoxy-6-[4-(2,3,4,6-tetra-O-acetyl-R-D-man-
nopyranosyloxyethyl)triazol-1-yl]-R-D-glucopyranosyl-(1 f 4)-
1,2,3-tri-O-acetyl-6-deoxy-6-[4-(2,3,4,6-tetra-O-acetyl-R-D-man-
nopyranosyloxyethyl)triazol-1-yl]-D-glucopyranose (13). 1H NMR
(300 MHz, CDCl3) δ ) 7.68, 7.66, 7.56, 7.54 (6H, 4s, 3 ×
CHNRâ), 6.22 (1H, d, J1,2 3.5 Hz, H-1R), 5.70 (1H, d, J1,2 8.0 Hz,
H-1â), 5.50-5.15 (28H, m, H-3, H-1′, H-3′, H-1′′, H-3′′, 3 × H-2M,
3 × H-3M, 3 × H-4MRâ), 4.84-4.50 (28H, m, H-2, H-6a, H-6b,
H-2′, H-4′, H-6′a, H-6′b, H-2′′, H-4′′, H-6′′a, H-6′′b, 3 × H-1MRâ),
4.45-4.20 (8H, m, H-5, 3 × H-6aMRâ), 4.10-3.82 (22H, m, H-4,
H-5′, 3 × H-5MRâ, 3 × H-6bMRâ, 3 × OCHaCH2Râ), 3.80-
3.65 (8H, H-5′′, 3 × OCHbCH2Râ), 3.17-2.87 (12H, m, 3 ×
OCH2CH2Râ), 2.11, 2.07, 2.06, 2.01, 2.00, 1.97, 1.95, 1.93 (120H,
8s, 20 × CH3CORâ); 13C NMR (75 MHz, CDCl3): δ ) 170.7,
170.5, 170.4, 170.1, 170.0, 169.8, 169.6, 169.4, 169.1 (CO), 144.4,
144.3, 144.0, 143.9 (NCdCH), 124.9, 124.7, 124.0 (NCdCH), 97.8,
97.6 (C-1M), 95.9, 95.3 (C-1′), 91.3 (C-1R), 88.7 (C-1â), 74.5,
74.1, 73.4, 73.3, 72.3, 72.2, 71.8, 70.5, 70.3, 69.8, 69.5, 69.2, 68.6,
66.1 (C-2 to C-5, C-2′ to C-5′, C-2′′ to C-5′′, C-2M to C-5M),
67.3, 67.1 (OCH2CH2), 62.4 (C-6M), 50.0, 49.7, 49.2 (C-6, C-6′,
C-6′′), 26.1 (OCH2CH2), 21.1, 21.0, 20.8, 20.7, 20.5 (CH3CO);
HRMS (ES+): Found 2138.6636 C88H117N9O51Na requires
2138.6736 [M + Na+].
6-Deoxy-6-(4-(mannopyranosyloxyethyl)triazol-1-yl))-R-D-glu-
copyranosyl-(1 f 4)-6-deoxy-6-(4-(mannopyranosyloxyethyl)-
triazol-1-yl))-R-D-glucopyranosyl-(1 f 4)-6-deoxy-6-(4-(man-
nopyranosyloxyethyl)triazol-1-yl))-D-glucopyranose (14). 1H NMR
(300 MHz, D2O) δ ) 7.77, 7.65, 7.61, 7.57 (6H, 4s, 3 × CHNRâ),
5.27 (2H, d, J1′′,2′′ 3.8 Hz, H-1′′Râ) (assignments may be inter-
changed), 5.16 (2H, d, J1′,2′ 3.8 Hz, H-1′Râ)′′Râ) (assignments may
be interchanged), 4.95 (1H, d, J1,2 3.6 Hz, H-1R), 4.82-4.70 (8H,
m, H-6a, 3 × H-1MRâ), 4.45-4.36 (2H, m, H-6bRâ), 4.38 (1H,
d, J1,2 7.8 Hz, H-1â), 4.10-3.00 (68H, m, H-2 to H-5, H-2′ to
H-5′, H-6′a, H-6′b, H-2′′ to H-5′′, H-6′′a, H-6′′b, 3 × H-2M to
H-5M, 3 × H-6aM, 3 × H-6bMRâ), 3.00-2.95 (12H, m, 3 ×
OCH2CH2), 2.70-2.40 (m, 3 × OCH2CH2). 13C NMR (75 MHz,
D2O): δ ) 145.5, 145.2 (NCdCH), 125.3, 125.1, 124.9 (NCd
CH), 101.5 (C-1′′)′′Râ) (assignments may be interchanged), 101.0
(C-1′)′′Râ) (assignments may be interchanged), 100.1 (C-1M), 96.0
(C-1â), 92.0 (C-1R), 81.7, 81.4, 81.3, 75.9, 73.7, 73.1, 72.8, 72.6,
71.9, 71.3, 70.9, 70.4, 67.1, 66.9 (C-2 to C-5, C-2′ to C-5′, C-2′′ to
C-5′′, C-2M to C-5M), 66.6, 66.4, 66.2 (OCH2CH2), 61.2 (C-6M),
51.7, 51.0, 50.3 (C-6, C-6′, C-6′′), 25.5, 25.2 (OCH2CH2); HRMS
(ES+): Found 1298.4572 C48H77N9O31Na requires 1298.4623 [M
+ Na+].
1-{2,3,4-Tri-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-R-D-glucopyranosyl-
(1 f 4)-2,3-di-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-â-D-glucopyranos-1-yl}-
4-[2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyloxyethyl]-[1,2,3]-
1
triazol (15). [R]20 +43 (c 0.1, CH2Cl2); H NMR (300 MHz,
D
CDCl3) δ ) 7.59, 7.54, 7.41 (3H, 3s, 3 × CHN), 5.83 (1H, d, J1,2
9.1 Hz, H-1), 5.50-5.10 (12H, m, H-3, H-1′, H-3′, 3 × H-2M, 3
× H-3M, 3 × H-4M), 4.82-4.50 (10H, m, H-2, H-6a, H-6b, H-2′,
H-4′, H-6′a, H-6′b, 3 × H-1M), 4.25-3.50 (18H, m, H-4, H-5,
H-5′, 3 × H-5M, 3 × H-6aM, 3 × H-6bM, 3 × OCH2CH2), 3.12-
2.80 (6H, m, 3 × OCH2CH2), 2.09, 2.07, 2.05, 2.04, 2.03, 2.02,
2.00, 1.99, 1.97, 1.95 (51H, 10s 17 × CH3CO); 13C NMR (75 MHz,
CDCl3): δ ) 170.7, 170.4, 170.2, 170.1, 169.9, 169.7, 169.6, 168.9
(CO), 144.9, 144.3 (NCdCH), 123.9, 123.8, 121.2 (NCdCH), 97.9,
97.8, 97.5 (C-1M), 96.0 (C-1′), 85.0 (C-1), 75.4, 74.5, 73.5, 70.9,
70.0, 69.5, 69.3, 69.1, 66.0 (C-2 to C-5, C-2′ to C-5′, C-2M to
C-5M), 67.2, 67.1, 66.9 (OCH2CH2), 62.5 (C-6M), 50.3, 50.0 (C-
6, C-6′), 26.1 (OCH2CH2), 20.9, 20.8 (CH3CO); HRMS (ES+):
Found 1850.5839 C76H101N9O43Na requires 1850.5891 [M + Na+].
1-{6-Deoxy-6-[1-(R-D-mannopyranosyloxyethyl)triazol-4-yl]-
R-D-glucopyranosyl-(1 f 4)-6-deoxy-6-[1-(ꢀπ-mannopyranosy-
6-Deoxy-6-[4-(R-D-mannopyranosyloxyethyl)triazol-1-yl]-R-
D-glucopyranosyl-(1 f 4)-6-deoxy-6-[4-(R-D-mannopyranosy-
loxyethyl)triazol-1-yl]-D-glucopyranose (12). 1H NMR (300 MHz,
D2O) δ ) 7.78, 7.63, 7.60 (4H, 3s, 2 × CHNRâ), 5.23 (2H, d,
J1′,2′ 3.3 Hz, H-1′Râ), 4.97 (1H, d, J1,2 3.7 Hz, H-1R), 4.79-4.25
(6H, m, H-6a, 2 × H-1MRâ), 4.39 (1H, d, J1,2 8.2 Hz, H-1â), 4.00-
3.18 (48H, m, H-2 to H-5, H-6b, H-2′ to H-5′, H-6′a, H-6′b, 2 ×
H-2M to H-5M, 2 × H-6aM, 2 × H-6bMRâ), 3.15-3.00 (8H, m,
2 × OCH2CH2Râ), 2.75-2.50 (8H, m, 2 × OCH2CH2Râ); 13C
NMR (75 MHz, D2O): δ ) 145.5, 145.3 (NCdCH), 125.4, 125.1,
124.9 (NCdCH), 101.4 (C-1′), 100.1, 99.9 (C-1M), 96.0 (C-1â),
92.1 (C-1R), 75.9, 73.7, 73.2, 72.8, 72.5, 72.4, 72.0, 71.9, 71.4,
71.1, 70.9, 70.4, 68.1 (C-2 to C-5, C-2′ to C-5′, C-2M to C-5M),
66.9, 66.6, 66.4 (OCH2CH2), 61.2 (C-6M), 51.7, 50.5 (C-6, C-6′),
25.5,25.2(OCH2CH2).HRMS(ES+): Found879.3080C32H52N6O21-
Na requires 879.3083 [M + Na+].
9042 J. Org. Chem., Vol. 72, No. 24, 2007

Multi-Mannosides Based on a Carbohydrate Scaffold
loxyethyl)triazol-4-yl]-â-D-glucopyranos-1-yl}-4-[R-D-mannopy-
ranosyloxyethyl]-[1,2,3]-triazol (16). [R]20D +57 (c 0.1, H2O); 1H
NMR (300 MHz, D2O) δ ) 7.89, 7.83, 7.55 (3H, 3s, 3 × CHN),
5.55 (1H, d, J1,2 8.6 Hz, H-1), 5.34 (1H, d, J1′,2′ 3.7 Hz, H-1′),
4.75-4.65 (4H, m, H-6a, 3 × H-1M), 4.55 (1H, dd, J5,6b 9.3 Hz,
δ ) 170.7, 170.3, 170.2, 170.1, 169.8, 169.7, 169.4 (CO), 144.9,
144.2, 144.0 (NCdCH), 124.7, 123.9, 121.3 (NCdCH), 97.8, 97.7,
97.6, 97.5 (C-1M), 95.8, 95.2 (C-1′, C-1′′), 84.9 (C-1), 74.0, 70.4,
69.8, 69.5, 69.4, 69.3, 69.1, 68.8, 68.7, 68.6, 66.0, 65.9 (C-2 to
C-5, C-2′ to C-5′, C-2′′ to C-5′′, C-2M to C-5M), 67.2, 67.0 (OCH2-
CH2), 62.3 (C-6M), 49.9, 49.4 (C-6, C-6′, C-6′′), 26.0 (OCH2CH2),
20.9, 20.7, 20.1 (CH3CO); HRMS (ES+): Found 2521.8158
J6a,6b 14.7 Hz, H-6b), 4.30-3.25 (34H, m, H-2 to H-5, H-2′ to H-5′,
H-6′a, H-6′b, 3 × H-2M to H-5M, 3 × H-6aM, 3 × H-6bM, 3 ×
OCH2CH2), 3.12-2.85 (4H, m, 2 × OCH2CH2), 2.72-2.50 (2H,
m, OCH2CH2); 13C NMR (75 MHz, D2O): δ ) 145.8, 145.4, 145.2
(NCdCH), 125.5, 125.0, 123.2 (NCdCH), 101.5 (C-1′), 99.9 (C-
1M), 87.1 (C-1), 80.4, 76.1, 75.0, 73.1, 72.7, 72.4, 71.9, 71.3, 70.9,
70.8, 70.3, 66.9 (C-2 to C-5, C-2′ to C-5′, C-2M to C-5M), 66.4,
66.3 (OCH2CH2), 61.1 (C-6M), 51.7, 50.2 (C-6, C-6′), 25.4, 25.2
(OCH2CH2); HRMS (ES+): Found 1136.4042 C42H67N9O26Na
requires 1136.4095 [M + Na+].
C
104H138N12O59Na requires 2521.8065 [M + Na+].
1-{6-Deoxy-6-[1-(R-D-mannopyranosyloxyethyl)triazol-4-yl]-
R-D-glucopyranosyl-(1 f 4)-6-deoxy-6-[1-(R-D-mannopyrano-
syloxyethyl)triazol-4-yl]-R-D-glucopyranosyl-(1 f 4)-6-deoxy-
6-[1-(R-D-mannpyranosyloxyethyl)triazol-4-yl]-â-D-glucopyranos-
1-yl}-4-[R-D-mannopyranosyloxyethyl]-[1,2,3]-triazol (20). [R]20
D
+57 (c 0.1, H2O); 1H NMR (300 MHz, D2O) δ ) 7.86, 7.81, 7.69,
7.52 (4H, 4s, 4 × CHN), 5.52 (1H, d, J1,2 8.5 Hz, H-1), 5.32 (1H,
d, J1′′,2′′ 3.7 Hz, H-1′′)′′Râ) (assignments may be interchanged),
5.26 (1H, d, J1′,2′ 3.9 Hz, H-1′)′′Râ) (assignments may be
interchanged), 4.90-4.70 (5H, m, H-6a, 4 × H-1M), 4.60-4.40
(1H, dd, J5,6b 9.3 Hz, J6a,6b 14.7 Hz, H-6b), 4.20-3.20 (48H, m,
H-2 to H-5, H-2′ to H-5′, H-6′a, H-6′b, H-2′′ to H-5′′, H-6′′a, H-6′′b,
4 × H-2M to H-5M, 4 × H-6aM, 4 × H-6bM, 4 × OCH2CH2),
3.10-2.80 (4H, m, 2 × OCH2CH2), 2.72-2.45 (4H, m, 2 ×
OCH2CH2); 13C NMR (75 MHz, D2O): δ ) 145.8, 145.4, 145.2
(NCdCH), 125.3, 125.0, 123.2 (NCdCH), 101.5, 101.2 (C-1′,
C-1′′), 100.0, 99.9 (C-1M), 87.0 (C-1), 81.4, 80.5, 75.9, 74.8, 73.2,
72.7, 72.5, 71.8, 70.9, 70.8, 70.7, 70.3, 67.0, 66.9 (C-2 to C-5,
C-2′ to C-5′, C-2′′ to C-5′′, C-2M to C-5M), 66.3, 66.1 (OCH2-
CH2), 61.1 (C-6M), 51.6, 50.9, 49.9 (C-6, C-6′, C-6′′), 25.4, 25.1
(OCH2CH2); HRMS (ES+): Found 1555.5693 C58H92N12O36Na
requires 1555.5635 [M + Na+].
1-{2,3,4-Tri-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-R-D-glucopyranosyl-
(1 f 4)-2,3-di-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-R-D-glucopyranos-1-
yl}-4-[2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyloxyethyl]-[1,2,3]-
1
triazol (17). [R]20 +58 (c 0.4, CH2Cl2); H NMR (300 MHz,
D
CDCl3) δ ) 7.59, 7.57, 7.55 (3H, 3s, 3 × CHN), 6.30 (1H, d, J1,2
5.6 Hz, H-1), 6.00 (1H, t, J2,3 ) J3,4 7.4 Hz, H-3), 5.48 (1H, d,
J1′,2′ 3.8 Hz, H-1′), 5.45-5.15 (11H, m, H-2, H-3′, 3 × H-2M, 3
× H-3M, 3 × H-4M), 4.80-4.62 (9H, m, H-6a, H-6b, H-2′, H-4′,
H-6′a, H-6′b, 3 × H-1M), 4.50-3.60 (18H, m, H-4, H-5, H-5′, 3
× H-5M, 3 × H-6aM, 3 × H-6bM, 3 × OCH2CH2), 3.12-2.82
(6H, m, 3 × OCH2CH2), 2.09, 2.08, 2.06, 2.05, 2.02, 1.98, 1.95
(51H, 7s, 17 × CH3CO); 13C NMR (75 MHz, CDCl3): δ ) 170.7,
170.5, 170.3, 170.1, 170.0, 169.8, 169.7, 169.5 (CO), 144.5, 144.1,
143.8 (NCdCH), 124.4, 123.9, 123.6 (NCdCH), 97.6, 97.5 (C-
1M), 95.5 (C-1′), 81.1 (C-1), 72.8, 72.0, 71.4, 69.5, 69.4, 69.1,
68.8, 68.6, 66.0, 65.9, 65.8 (C-2 to C-5, C-2′ to C-5′, C-2M to
C-5M), 67.0, 66.8 (OCH2CH2), 62.4 (C-6M), 50.1, 49.5 (C-6, C-6′),
26.0 (OCH2CH2), 20.9, 20.8, 20.7, 20.3 (CH3CO); HRMS (ES+):
Found 1850.5936 C76H101N9O43Na requires 1850.5891 [M + Na+].
1-{6-Deoxy-6-[1-(R-D-mannopyranosyloxyethyl)triazol-4-yl]-
R-D-glucopyranosyl-(1 f 4)-6-deoxy-6-[1-(R-D-mannopyrano-
syloxyethyl)triazol-4-yl]-R-D-glucopyranos-1-yl}-4-[R-D-man-
nopyranosyloxyethyl]-[1,2,3]-triazol (18). [R]20D +47 (c 0.1, H2O);
1H NMR (300 MHz, D2O) δ ) 7.78, 7.71, 7.54 (3H, 3s, 3 × CHN),
6.08 (1H, d, J1,2 5.9 Hz, H-1), 5.30 (1H, d, J1′,2′ 3.7 Hz, H-1′),
4.92-4.65 (4H, m, H-6a, 3 × H-1M), 4.50-4.32 (2H, m, H-3,
H-6b), 4.10-3.20 (33H, m, H-2, H-4, H-5, H-2′ to H-5′, H-6′a,
H-6′b, 3 × H-2M to H-5M, 3 × H-6′a, 3 × H-6′b, 3 × OCH2-
CH2), 3.00-2.80 (4H, m, 2 × OCH2CH2), 2.62-2.50 (2H, m,
OCH2CH2); 13C NMR (75 MHz, D2O): δ ) 147.8, 147.6 (NCd
CH), 128.1, 128.0, 127.5 (NCdCH), 103.8 (C-1′), 102.6, 102.5
(C-1M), 87.0 (C-1), 83.1, 76.1, 75.7, 75.6, 75.3, 74.9, 74.5, 74.2,
73.9, 73.5, 73.4, 72.8, 72.4 (C-2 to C-5, C-2′ to C-5′, C-2M to
C-5M), 69.5, 69.4 (OCH2CH2), 69.1, 68.9 (C-6M), 54.2, 52.6 (C-
6, C-6′), 28.0, 27.8, 27.7 (OCH2CH2); HRMS (ES+): Found
1136.4055 C42H67N9O26Na requires 1136.4095 [M + Na+].
1-{2,3,4-Tri-O-acetyl-6-[1-(2,3,4,6-tetra-O-acetyl-R-D-man-
nopyranosyloxyethyl)triazol-4-yl]-6-deoxy-R-D-glucopyranosyl-
(1 f 4)-2,3-di-O-acetyl-6-[1-(2,3,4,6-tetra-O-acetyl-R-D-manno-
pyranosyloxyethyl)triazol-4-yl]-6-deoxy-R-D-glucopyranosyl--
(1 f 4)-2,3-di-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-â-D-glucopyranos-1-yl}-
1-{2,3,4-Tri-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-R-D-glucopyranosyl-
(1 f 4)-2,3-di-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-R-D-glucopyranosyl-
(1 f 4)-2,3-di-O-acetyl-6-deoxy-6-[1-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxyethyl)triazol-4-yl]-R-D-glucopyranos-1-
yl}-4-[2,3,4,6-tetra-O-acetyl-â-D-mannopyranosyloxyethyl]-[1,2,3]-
1
triazol (21). [R]20 +64 (c 0.1, CH2Cl2); H NMR (300 MHz,
D
CDCl3) δ ) 7.72, 7.66, 7.59 (4H, 3s, 4 × CHN), 6.30 (1H, d, J1,2
5.6 Hz, H-1), 6.03 (1H, t, J2,3 ) J3,4 7.4 Hz, H-3), 5.47 (1H, d,
J1′′,2′′ 3.8 Hz, H-1′′), 5.46-5.05 (17H, m, H-2, H-1′, H-2′, H-3′,
H-3′′, 4 × H-2M, 4 × H-3M, 4 × H-4M), 5.00-4.35 (14H, m,
H-4, H-6a, H-6b, H-4′, H-6′a, H-6′b, H-2′′, H-4′′, H-6′′a, H-6′′b, 4
× H-1M), 4.32-3.65 (23H, m, H-5, H-5′, H-5′′, 4 × H-5M, 4 ×
H-6aM, 4 × H-6bM, 4 × OCH2CH2), 3.20-2.85 (8H, m, 4 ×
OCH2CH2), 2.12, 2.10, 2.09, 2.07, 2.05, 2.00, 1.99, 1.98, 1.95, 1.93
(69H, 10s, 23 × CH3CO); 13C NMR (75 MHz, CDCl3): δ ) 170.7,
170.4, 170.2, 169.8, 169.7, 169.6, 169.4 (CO), 144.5, 143.9 (NCd
CH), 124.9, 124.6, 123.9, 123.7 (NCdCH), 97.6, 97.5 (C-1M), 95.6,
95.3 (C-1′, C-1′′), 81.2 (C-1), 72.1, 70.0, 69.5, 68.8, 68.7, 68.6,
66.0, 65.9, 65.8 (C-2 to C-5, C-2′ to C-5′, C-2′′ to C-5′′, C-2M to
C-5M), 67.2, 67.1, 66.9 (OCH2CH2), 62.4 (C-6M), 49.9, 49.6, 49.2
(C-6, C-6′, C-6′′), 26.1 (OCH2CH2), 20.9, 20.8, 20.4 (CH3CO);
HRMS (ES+): Found 2521.8137 C104H138N12O59Na requires
2521.8065 [M + Na+].
1-{6-Deoxy-6-[1-(R-D-mannopyranosyloxyethyl)triazol-4-yl]-
R-D-glucopyranosyl-(1 f 4)-6-[1-(R-D-mannopyranosyloxyeth-
yl)triazol-4-yl]-6-deoxy-R-D-glucopyranosyl-(1 f 4)-6-(R-D-
mannopyranosyloxyethyl)triazol-4-yl]-6-deoxy-â-D-glucopyranos-
[1,2,3]-4-[2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyloxyethyl-
1-yl}-4-[R-D-mannopyranosyloxyethyl]-[1,2,3]-triazol (22). [R]20
D
1
]triazol (19). [R]20 +46 (c 0.2, CH2Cl2); H NMR (300 MHz,
+56 (c 0.1, H2O); 1H NMR (300 MHz, D2O) δ ) 7.79, 7.74, 7.67,
7.55 (4H, 4s, 4 × CHN), 6.08 (1H, d, J1,2 5.9 Hz, H-1), 5.31 (1H,
d, J1′′,2′′ 3.6 Hz, H-1′′)′′Râ) (assignments may be interchanged),
5.26 (1H, d, J1′,2′ 3.7 Hz, H-1′)′′Râ) (assignments may be
interchanged), 4.90-4.68 (5H, m, H-6a, 4 × H-1M), 4.50-4.35
(2H, m, H-3, H-6b), 4.22-3.20 (47H, m, H-2, H-4, H-5, H-2′ to
H-5′, H-6′a, H-6′b, H-2′′ to H-5′′, H-6′′a, H-6′′b, 4 × H-2M to
H-5M, 4 × H-6aM, 4 × H-6bM, 4 × OCH2CH2), 3.05-2.80 (4H,
m, 2 × OCH2CH2), 2.60-2.45 (4H, m, 2 × OCH2CH2); 13C NMR
D
CDCl3) δ ) 7.63, 7.59, 7.52, 7.42, (4H, 4s, 4 × CHN), 5.85 (1H,
d, J1,2 9.1 Hz, H-1), 5.50-5.05 (17H, m, H-3, H-1′, H-3′, H-1′′,
H-3′′, 4 × H-2M, 4 × H-3M, 4 × H-4M), 4.83-4.35 (14H, m,
H-2, H-6a, H-6b, H-2′, H-6′a, H-6′b, H-2′′, H-4′′, H-6′′a, H-6′′b, 4
× H-1M), 4.22-3.50 (25H, m, H-4, H-5, H-4′, H-5′, H-5′′, 4 ×
H-5M, 4 × H-6aM, 4 × H6bM, 4 × OCH2CH2), 3.10-2.80 (8H,
m, 4 × OCH2CH2), 2.08, 2.05, 2.03, 2.01, 1.99, 1.98, 1.96, 1.94,
1.92, 1.91 (69H, 10s, 23 × CH3CO); 13C NMR (75 MHz, CDCl3):
J. Org. Chem, Vol. 72, No. 24, 2007 9043

Gouin et al.
(75 MHz, D2O): δ ) 145.3, 145.1, 145.0 (NCdCH), 125.5, 125.3,
125.0 (NCdCH), 101.5, 100.9 (C-1′, C-1′′), 100.1, 99.9 (C-1M),
84.4 (C-1), 81.3, 80.6, 73.2, 73.0, 72.7, 72.5, 71.8, 71.5, 71.3, 70.9,
70.8, 70.3, 69.8, 67.0, 66.9 (C-2 to C-5, C-2′ to C-5′, C-2′′ to C-5′′,
C-2M to C-5M), 66.5, 66.3, 66.1 (OCH2CH2), 61.2, 61.1 (C-6M),
51.6, 50.9, 49.8 (C-6, C-6′, C-6′′), 25.4, 25.1 (OCH2CH2); HRMS
(ES+): Found 1555.5637 C58H92N12O36Na requires 1555.5635 [M
+ Na+].
AMBER OFF force field libraries. A LEaP script is available in
the “F-71” R.E.DD.B. project for this purpose.47
To validate the glycocluster FFTPDB reported, a comparative
study involving the glucose scaffold [disaccharide Glc-R-(1 f 4)
R-Glc] was carried out using the molecular fragments and RESP
charge values taken from the Glycam 2004 force field and the
FFTPDB described in this work. However, in the absence of the
hemiacetal hydroxyl fragment and the corresponding charge values
in the Glycam 2004 force field version available in the AMBER 8
distribution, the charge values were derived anew for this missing
fragment. Charge values were obtained following standard proce-
dures implemented in the R.E.D. program as previously described.41
This was carried out using methanol and an intra-molecular charge
constraint for the methyl group set to the sum of charge values
found in the 1GA and 4GA glucose fragments during the fit. Charge
values of -0.6131 and 0.4141 were found for the oxygen and
hydrogen atoms, respectively, and used to build a LEaP library for
this additional fragment leading to the HO-4GA-1GA tri-unit-based
disaccharides.
Atom Charge Value Derivation, Building, and Validation of
a New FFTPDB. â-D-Glucose, R-D-glucose, methyl R-D-glucopy-
ranoside, methyl R-D-mannopyranoside, and 1-N-methyl-4-(2-
hydroxyethyl)-1,2,3-triazole were involved in the charge derivation
procedure. The ω dihedral angle conformations gg and gt were
chosen for the glucose and mannose derivatives,39 while the two
lowest minima for 1-N-methyl-4-(2-hydroxyethyl)-1,2,3-triazole
found after geometry search were selected. The geometries of the
glucose and mannose derivatives were optimized using dihedral
angle constraints. The HO4′-O4′-C4′-H4′ dihedral angle of the
monosaccharides was constrained to a value of 180°. A second
constraint for the HO2′-O2′-C2′-H2′ dihedral angle of â-glucose
and for the HO3′-O3′-C3′-H3′ dihedral angle of methyl R-man-
nopyranoside was used and set to values of 180 and 60°,
respectively. Frequencies were calculated for all the molecules.
Geometry optimization, frequency calculation, and MEP computa-
tion were carried out using the Gaussian 98 program in the gas
phase,45 while charge fitting was performed using the RESP
program.38a The HF/6-31G** theory level was used in geometry
optimization and frequency calculation.43 The MEP computation
was carried out using the HF/6-31G* theory level43 and the
CHELPG algorithm.46 The molecular orientation of the optimized
geometries was controlled using the rigid-body reorientation
algorithm implemented in the R.E.D. program before MEP com-
putation,41 and four orientations (based on the C1′ C3′ C5′, C5′
C3′ C1′, C2′ C4′ O5′, and O5′ C4′ C2′ atom names for glucose
and mannose derivatives and based on the OH C1 C2, C2 C1 OH,
C2 C3 N4, and N4 C3 C2 atom names for the triazole derivative)
were generated for each optimized geometry. Charge fitting was
performed using a single RESP stage and a hyperbolic constraint
value of 0.01.37,38b Inter-molecular charge constraints between the
methyl group of methyl R-D-glucopyranoside, methyl R-D-man-
nopyranoside, and 1-N-methyl-4-(2-hydroxyethyl)-1,2,3-triazole, the
anomeric 4- or 6-hydroxyl group of methyl R-D-glucopyranoside,
â-D-glucose, and R-D-glucose, or the hydroxyl group of 1-N-methyl-
4-(2-hydroxyethyl)-1,2,3-triazole were set to a target value of zero
in the charge fitting procedure. Intra-molecular charge constraints
between the methyl and the 4-hydroxyl groups of methyl R-D-
glucopyranoside, and for each hydrogen bound to a sp3 carbon,
were also set to a value of zero. The charge derivation procedure
was automatically carried out using a version IV â of the R.E.D.
program.41 The RESP atom charge values and the FFTPDB were
submitted in R.E.DD.B. and are available as the “F-71” R.E.DD.B.
project code. The FFTPDB is available as a suite of molecular
fragments in the Tripos mol2 file format and is used to build
Molecular Dynamics Simulation. Three-dimensional structures
and dynamics of the glucose scaffold [disaccharide Glc-R-(1 f 4)
R-Glc] and glycoclusters 12â, 12R, 16, and 18 were studied using
the AMBER 8 program.48 Each structure was solvated in a truncated
octahedron box of TIP3P explicit water49 and subjected to 10 ns
constant pressure productive MD at 300 K after a 225 ps period of
constant volume equilibration.50 3 ns constant pressure productive
MD was also performed at 320 and 340 K for checking putative
trapping of simulation in local minima. Geometry optimization and
unrestrained MD simulations were carried out using force field
parameters taken from the Glycam 2004 force field,37 and the
FFTPDB was reported. Missing force field parameters for the
triazole and hemiacetal derivatives were obtained from the General
AMBER force field.51 A cutoff value of 9.0 Å and a time step
value of 0.002 ps with the SHAKE algorithm for constraining bonds
involving hydrogens were used during the simulations.52 The 1-4
van der Waals and 1-4 electrostatics interactions were divided by
a scaling factor value of 1.0 as suggested by the Glycam 2004 force
field authors.37 MD was carried out on a PC workstation with an
Intel Core 2 Duo E6400 CPU under a Fedora Core 6 Linux
distribution (http://fedoraproject.org/). Finally, the geometry of
model compounds for each minimum observed during MD simula-
tion was further characterized and optimized by ab initio in the
gas phase using the B3LYP/6-311+G** theory level.43 Molecular
visualization, structure comparison, and structural properties of the
PDB structures and MD snapshots were analyzed using the ptraj
module of the AMBER 8, VMD,53 and InsightII 2000 (Accelrys
Inc., San Diego, CA) programs.
(47) Schafmeister, C. E. A. F.; Ross, W. S.; Romanovski, V. LEaP;
University of California, San Francisco: San Francisco, 1995.
(48) (a) Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.;
Cheatham, T. E., III; DeBolt, S.; Ferguson, D.; Seibel, G.; Kollman, P. A.
Comput. Phys. Commun. 1995, 91, 1-41. (b) Case, D. A.; Darden, T. A.;
Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.;
Merz, K. M.; Wang, B.; Pearlman, D. A.; Crowley, M.; Brozell, S.; Tsui,
V.; Gohlke, H.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Schafmeister,
C.; Caldwell, J. W.; Ross, W. S.; Kollman, P. A. AMBER 8; University of
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Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A.
D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi,
M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.;
Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick,
D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.;
Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,
P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-
Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.;
Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian
98, revision A.11; Gaussian, Inc.: Pittsburgh, PA, 1998.
(49) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J.; Klein, M. L. J.
Chem. Phys. 1983, 79, 926-935.
(50) Dupradeau, F.-Y.; Case, D. A.; Yu, C.; Jimenez, R.; Romesberg,
F. E. J. Am. Chem. Soc. 2005, 127, 15612-15617.
(51) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D.
A. J. Comput. Chem. 2004, 25, 1157-1174.
(52) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys.
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9044 J. Org. Chem., Vol. 72, No. 24, 2007

Multi-Mannosides Based on a Carbohydrate Scaffold
Figures S1-S5 from molecular dynamics simulations, force field
parameters for triazole and hemiacetal derivatives, and FFTPDB
reported with force field atom types, atom names, and RESP atomic
charge values for each molecular fragment. This material is available
free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This work was carried out with financial
support from the Centre National de la Recherche Scientifique
and the Ministe`re De´le´gue´ a` l’Enseignement Supe´rieur et a` la
Recherche.
Supporting Information Available: 1H and 13C NMR spectra
of 2-7 and 9-22, experimental details for ELLA inhibition tests,
JO071248A
J. Org. Chem, Vol. 72, No. 24, 2007 9045