α-O-linked glycopeptide mimetics: Synthesis, conformation analysis, and interactions with viscumin, a galactoside-binding model lectin

Article abstract of DOI:10.1002/chem.200901077

Efficient cycloaddition of a silylidene-protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide-mimetic scaffold. Its carbohydrate part was further extended by βl-3-linked galactosylation. T

Full text of DOI:10.1002/chem.200901077

FULL PAPER
DOI: 10.1002/chem.200901077
a-O-Linked Glycopeptide Mimetics: Synthesis, Conformation Analysis,
Interactions with Viscumin, a Galactoside-Binding Model Lectin
ACHTUNGTRENNUNGand
AHCTUNGTRENNUNG
Jesffls JimØnez-Barbero,*[a] Elisa Dragoni,[b, c] Chiara Venturi,[a, b, c] Federico Nannucci,[b]
Ana Ardµ,[a] Marco Fontanella,[a] Sabine AndrØ,[d] Francisco Javier CaÇada,[a]
Hans-Joachim Gabius,[d] and Cristina Nativi*[b, c]
Abstract: Efficient cycloaddition of a
silylidene-protected galactal with
suitable heterodiene yielded the basis
for a facile diastereoselective route to
observed for the disaccharide. The ex-
pected bioactivity was ascertained by
model lectin. The experimental part
was complemented by molecular dock-
ing. The described synthetic route and
the strategic combination of computa-
tional and experimental techniques to
reveal conformational properties and
bioactivity establish the prepared a-O-
linked glycopeptide mimetics as prom-
ising candidates for further exploitation
of this scaffold to give O-glycans for
lectin blocking and vaccination.
a
saturation-transfer-difference
spectroscopy by using the galactoside-
specific plant toxin viscumin as a
NMR
a
glycopeptide-mimetic scaffold. Its
carbohydrate part was further extended
by b1–3-linked galactosylation. The
pyranose rings retain their 4C1 chair
conformation, as shown by molecular
modeling and NMR spectroscopy, and
the typical exo-anomeric geometry was
Keywords: carbohydrates
·
drug
design · glycoproteins · lectins ·
molecular modeling · NMR spec-
troscopy
Introduction
rameters, such as solubility and protection from proteolytic
degradation, to adding versatile docking sites for the recep-
tors that read the sugar-encoded information.[1] A character-
istic of secretory or polarized epithelia, and also encoun-
tered in different cell-surface glycoproteins, is the presence
of mucin-type O-glycosylated positions linked to serine/
Protein glycosylation, which rivals phosphorylation in its fre-
quency, imparts a series of distinct properties to a carrier
backbone. They range from changes in physicochemical pa-
threonine residues via an a-N-d-acetylgalactosACHTUNGTRENNUNGamine
(GalNAc) moiety that are often subject to intimate structur-
al control.[2] When clustered together and arranged in re-
peats, the resulting glycoproteins are referred to as mucins.
They can form a highly stable viscoelastic gel over epithelial
cell surfaces, thus providing a protective barrier against ex-
ternal agents. Equally importantly, their glycan part can me-
diate cell adhesion and attachment processes that initiate
bacterial infection or leukocyte infiltration into inflamed
tissue, here recruiting the sialomucins CD34 or GLYCAM-1
and PSGL-1 as ligands for the C-type lectins of the selectin
group.[3] Since these recognition processes with O-glycans
extend to other lectin classes, such as adhesion/growth-regu-
latory galectins and to clinically relevant events in the meta-
static cascade, it is a challenge to develop bioactive mimetics
that would also be useful as vaccines against mucin-based
tumor-associated antigens.[4] Towards this end, we directed
our efforts to the diastereoselective synthesis of mucin-like
scaffolds, which maintain the key structural features of such
natural glycoproteins and present suitable functional groups
[a] Prof. Dr. J. Jimꢀnez-Barbero, Dr. C. Venturi, Dr. A. Ardꢁ,
Dr. M. Fontanella, Prof. Dr. F. J. CaÇada
Chemical and Physical Biology
Centro de Investigaciones Biolꢂgicas, CSIC
Ramiro de Maeztu 9, 28040 Madrid (Spain)
Fax : (+34)915-360-432
E-mail: jjbarbero@cib.csic.es
[b] Dr. E. Dragoni, Dr. C. Venturi, Dr. F. Nannucci, Prof. Dr. C. Nativi
Dipartimento di Chimica Organica
via della Lastruccia, 13, 50019 Sesto F.no, Firenze (Italy)
Fax : (+39)055-4573570
E-mail: cristina.nativi@unifi.it
[c] Dr. E. Dragoni, Dr. C. Venturi, Prof. Dr. C. Nativi
CERM, University of Florence
via Sacconi, 6, 50019 Sesto F.no, Firenze (Italy)
[d] Dr. S. Andrꢀ, Prof. Dr. H.-J. Gabius
Tierꢃrztliche Fakultꢃt, Lehrstuhl fꢄr Physiologische Chemie
Ludwig-Maximilians-Universitaet
Veterinaerstrasse 13, 80539 Mꢄnchen (Germany)
Fax : (+49)89-2180-2290
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901077.
Chem. Eur. J. 2009, 15, 10423 – 10431
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for the insertion of a saccharidic moiety and one or two pep-
tidic chains or a peptidic chain and a carrier, as required for
generating synthetic vaccines against tumor-associated gly-
copeptide antigens.[5] Since the synthesis of naturally occur-
ring glycopeptides is tedious, often ending up in diastereo-
meric mixtures, one focus in this area is glycopeptide mimet-
ics.[6] As an added advantage, the latter can have improved
stability against chemical and enzymatic hydrolysis and, de-
pending on the type of substitution, improved bioavailability
compared to natural materials.
In the course of the rational design of glycopeptidomimet-
ics, it is mandatory for these neoglyco derivatives to retain
bioactivity, that is, the capacity to serve as ligands for specif-
ic receptors. Thus, synthetic products need to be rigorously
controlled by respective assays, in our case tested against a
plant lectin with affinity to galactosides including the Thom-
sen–Friedenreich antigen, which is often present in mucins,
or its Galb1–3Gal derivative as a model.[7] Viscumin, also re-
ferred to as Viscum album agglutinin (VAA), is a potent
biohazard akin to ricin, whose toxicity can be neutralized by
suitable inhibitors competing with the cognate cell-surface
glycans.[8] This perspective has made the toxin a model for
drug design, for example, applying library approaches in
search of new binding partners.[9] In a more general context,
the strategy of combining preparative synthesis of glycopep-
tide mimetics with affinity testing will not only explore this
substrate class as potential source for new pharmaceuticals
but also enable us to gain insights into structural aspects of
the recognition process, which have relevance for under-
standing and exploiting lectin–carbohydrate interactions.[10]
Scheme 1. Synthesis of a-O-galactosyl scaffold 5. a) pyridine, CHCl3,
458C, 24 h, 70%;[11] b) CF3COOH, CH2Cl2, RT; c) triethylamine, CHCl3,
408C, 40 h, 65% (over two steps).
Scheme 2. Synthesis of deprotected scaffolds 6 and 7. a) HF/pyridine
(70%), CH2Cl2, 08C, 1 h, 90%; b) HF/pyridine (70%), CH2Cl2/MeOH,
RT, 4.5 h, 87%.
Results and Discussion
Synthesis: The entirely diastereoselective synthesis of glyco-
peptide scaffolds that preserve the natural a-O-glycosyl link-
age is based on an efficient cycloaddition between galactal
1, as electron-rich dienophile, and homoglutamate 2, as elec-
tron-poor diene[11] (Scheme 1).
Under these reaction conditions, first the removal of silyli-
dene occurred, followed by nucleophilic cleavage of the
benzyl ester. In fact, if the deprotection reaction is stopped
after one hour instead of four, the benzyl derivative 6 was
isolated in 90% yield. Focusing our attention on the devel-
opment of a synthetically convenient compound for tests as
a bioactive ligand, with perspectives for application as a vac-
cine against a-Tn and a-TF antigens, we started with scaf-
fold 5 to prepare the glycopeptide mimetic 8, its sugar part
then extended to the disaccharide 9 (Scheme 3).
After selective removal of benzyl ester under standard
conditions (90%), scaffold 5 was transformed into derivative
10 (Scheme 4). It presents a free carboxylic residue that is
suitable for the introduction of a serine moiety. Indeed, the
serine methyl ester was treated with 10 in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HATU) and N-methylmorpholine
(NMM) at room temperature and in dimethylformamide as
solvent to give the glycopeptide mimetic 11 in 93% yield.
Removal of the silylidene protecting group, as reported
above, followed by deprotection of the serine carboxylic res-
idue, with lithium hydroxide/water, in tetrahydrofuran/water
The attack of the heterodiene 2 exclusively on the bottom
(a) face of the dienophile 1 resulted in the a-O-glycosyl de-
rivative 3 as the single diastereomer, in a 70% yield. Treat-
ment of 3 with trifluoroacetic acid enabled removal of the
tert-butoxycarbonyl group without affecting the silylidene
protecting group on the galactose moiety, leading to the
amino derivative 4. Under basic conditions, compound 4 un-
derwent cyclization to give the tricyclic lactam 5 in a 65%
yield (calculated over two steps). Galactosyl amino acid 5 is
a versatile scaffold that presents an a-glycosyl linkage and
decorated with three hydroxyls and one carboxylic group.
Treatment of scaffold 5 with hydrogen fluoride/pyridine
(70%) in dichloromethane and methanol at room tempera-
ture facilitated the removal of the silylidene and benzyl
ester protecting groups to produce unprotected scaffold 7 as
a diastereometrically pure compound, in one step and in
87% (Scheme 2).
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a-O-Linked Glycopeptide Mimetics
FULL PAPER
acetylation under standard conditions, in methanol as sol-
vent, resulted in the disaccharide 14 as a methyl ester
(60%). The latter was then treated with HF/pyridine and
lithium hydroxide to obtain the completely unprotected di-
ACHUTNGERNsNUG acAHCTUNGTRNENcUGN haride 9 in 56% yield (calculated over two steps). This
product was further studied to evaluate its conformational
properties.
Conformational analysis in solution by NMR spectroscopy
and molecular modeling: Analysis of the vicinal proton–
proton coupling constants (Table 1) for the pyranose rings
4
of 7 and 8 indicates the exclusive presence of the C1 chair
conformation, as for natural galactosides.[13] Indeed, most of
the NMR parameters regarding the shape of the bicyclic
moieties of these compounds were very similar, thus indicat-
ing no influence from the presence of the Ser moiety in 8.
Results obtained from MM3*-based molecular mechanics
4
calculations[14] also support these observations, with the C1
chair form being more than 3 kJmolÀ1 more stable than the
1S3 skew-boat conformation and much more stable than the
1
alternative C4 chair conformer. The major difference con-
Scheme 3. Structure of a-Tn, a-TF, and scaffolds 8 and 9.
À
cerns the orientation around the C5 C6 torsion angle. For 7,
the observed JH5,H6a and JH5,H6b values (4.6 and 10.1 Hz) are
in agreement with a major conformation of this torsion, in
contrast to the equilibrium usually observed for galactoside
derivatives.[15] On the other hand, the observed intermediate
values (4.5 and 7.9 Hz) for 8 are accounted for by the ex-
pected gt:tg conformational distribution. For 8, the Jab and
Jab’ values are also on an intermediate level (4.0, 5.0 Hz);
this indicates the existence of an equilibrium around the
À
Ca Cb lateral chain. Molecular dynamics simulations (5 ns
with the MM3* force field) were performed (Figures 1 and 2
and Supporting Information). The results disclose that the
chair conformer is very stable, with basically no fluctuations.
Moreover, motion around the pseudoglycosidic F (H1’-C1’-
O-C2) and Y (C1’-O-C2-C3) angles is also rather restricted
for both molecules, with values centered around +808 and
1808. However, fluctuations take place around the region
containing the nitrogen atom, defined by torsions f1 and
f2, giving rise to two geometries (Figure 2).
Scheme 4. Synthesis of scaffold 8. a) Pd(OH)2/C (20%), H2, EtOAc/
CH2Cl2, RT, 2 h, 90%; b) SerACTHUNRGTNE(NUG OMe), N,N-dimethylformamide, NMM,
HATU, RT, 2 h, 93%; c) HF/pyridine (70%), CH2Cl2, RT, 2 h; d) LiOH/
H2O (0.04m), THF/H2O, 0 8C, 1.5 h, 39% (over two steps).
as solvent at 08C (39%, calculated over two steps), afforded
the fully unprotected building block 8.
Glycosylation, under Schmidtꢆs conditions, of scaffold 5
with the glycosyl donor 12[12] then gave the disaccharide 13,
as single b-diastereoisomer in 75% yield (Scheme 5). De-
The first conformer (A) is defined by f1 (defined as
C1-C2-C3-N4) ꢀ+488 and f2 (defined as C2-C3-N4-C5)
ꢀÀ358. The second one (B) is defined by f1, f2 angles
around À458 and +358, respectively (Figures 1 and 2). Com-
parison of the expected couplings with those experimentally
Scheme 5. Synthesis of scaffold 9. a) CH2Cl2 at À308C then TMSOTf at RT, 2.5 h; Et3N (Ph 7), 75%; b) CH3ONa, CH3OH, 08C, 1 h, 60%; c) HF/Py
(70%), RT, 2h, d) LiOH, H2O (Ph 10), RT, 2 h then H3PO4 (Ph 7), 56% (over two steps).
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J. Jimꢀnez-Barbero, C. Nativi et al.
Table 1. Chemical shifts and coupling constants of compounds 7 and 8, measured in D2O (500 MHz, 298 K, phosphate buffer: 20 mm, pH 5.8). The theo-
retical values (Jtheor) were obtained by applying the generalized Karplus equation proposed by Altona to the geometries calculated by the MM3*-based
À
calculations. The gt conformer around the C5 C6 torsion was employed.
Compound 7
Compound 8
J [Hz]
Atom
d (ppm) D2O
J [Hz] (exp)
J [Hz]
J [Hz]
d (ppm)
J [Hz]
J [Hz]
theor (Conf A)
theor (Conf B)
D2O
(exp)
theor (Conf A)
theor (Conf B)
H1’
H2’
H3’
H4’
H5’
H6’a
H6’b
H2a
5.74
3.42
3.75
3.96
4.12
3.70
3.65
2.87
2.67
4.05
1’/2’ 3.0
2’/3’ 11.3
3’/4’ 3.1
4’/5’ <1
5’/6’a 4.6
5’/6’b 10.1
2.6
11.4
2.8
0.8
2.0
2.6
11.4
2.8
0.8
2.3
5.80
3.48
3.77
4.02
4.15
3.73
3.72
3.11
2.9
11.3
2.7
<1
4.5
7.9
1.4
10.9
2.2
0.2
4.7
1.5
10.9
2.3
0.3
1.6
9.7
10.4
10.5
9.8
2a/3 5.3
2b/3 7.7
5.0
11.0
5.1
1.8
5.5
6.5
4.0
11.3
5.0
1.7
H2b
H3
2.82
4.39
H
Ha
b,bꢆ Ser
3.89
4.43
a,b 5.0
a,b’ 4.0
1.0
2.4
10.7
4.5
HN
HN-Ser
7.21 (H2O)
7.67 (H2O)
8.22
Figure 2. Plot of the f1/f2 values sampled during the 5 ns MD simula-
tions performed for 8 with the MM3* force field and the GB/SA solvent
model. The data for 7 are basically identical.
Next, the conformational behavior of the Galb1–3Gal an-
alogue 9 was investigated. In this case, a mixture of stereo-
isomers at the a-carbon of the amino acid was obtained.
Nonetheless, its presence does not preclude testing the bio-
activity, so a qualitative conformational analysis of both iso-
mers was performed. The coupling constant data (see
Table 2) revealed that both galactose residues adopt the
Figure 1. A) The two major conformers of 7 (A and B) according to the
experimental NMR data and MM3*-based molecular mechanics and dy-
namics calculations. This orientation of the lactam defines the two ge
ACHTUNGERTNoNUNG m-
ACHTUNGTRENNUNGetries by the MD. B) Superimposition of the two major conformers of 8
according to the experimental NMR data and MM3*-based molecular
mechanics and dynamics calculations. This orientation of the lactam de-
fines the two geometries, which show rather different spatial orientations
of the Ser moiety. The additional mobility at the Ser moiety is not de-
fined here, although it is manifested in the NMR data.
4
usual C1 chair geometry. The existence of a inter-residual
Gal H1’–Gal H3 NOE indicated the presence of a typical
exo-anomeric geometry[18] around the glycosidic linkage.
Chemical shift and coupling constant data, as deduced from
analysis of the 2D 1H NMR spectra, are given in Table 2.
With this information, molecular-mechanics calculations
were performed for both diastereomers, and the expected
coupling constants were compared to those experimentally
observed. Since the two Ha protons for both diastereomers
À
observed for the C2 C3 fragment enabled us to deduce that
both conformers are present in solution to a significant
extent, with an approximate A/B ratio of 40:60 for 7 and
50:50 for 8. Overall, this slight 10% shift of the conforma-
tional equilibrium is almost negligible considering the DG
parameter. Calculation of the expected J values for the en-
semble deduced from the MD simulations enabled us to
quantitatively explain the experimental observations
(Table 1) and NOEs (Supporting Information).[16,17]
1
can be distinguished in the H NMR spectrum, the couplings
to the corresponding Hb could easily be measured. The
values range between 5.1 and 7.9 Hz (Table 2), thus indicat-
ing that a conformational equilibrium, similar to that de-
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a-O-Linked Glycopeptide Mimetics
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Table 2. Chemical shifts and coupling constants of compound 9, mea-
sured in D2O (500 MHz, 298 K, phosphate buffer: 20 mm, pH 5.8).
Proton
d
J [Hz]
Proton d (ppm)
J [Hz]
(ppm)
H-1
5.74
J
1,2 =2.7
H-1’
4.48, 4.45
for both
isomers
3.55
3.61
3.82
J1’2’ =7.6 and
7.9, for both
isomers
–
–
J3ꢆ,4ꢆ =3.5
–
–
H-2
H-3
H-4
H-5
CH2-6 3.75
H-a 4.05,
(R, S) 3.96
3.57
3.95
4.16
4.14
J
J
–
–
–
J
J
J
J
2,3 =11.1
3,4 =3.2
H-2’
H-3’
H-4’
H-5’
CH2-6’ 3.65
H-b,
4.16
a,b1 =6.7,
2.92–2.65
overlapping
a,b2 =7.9
a,b1’ =5.1,
a,b2’ =6.9
H-b’
(R, S)
Figure 4. Superimposition of the major conformer of 8 with that of Gal-
NAcaOSer. The positions of chair constituents perfectly match each
other. Different orientations of the polar groups of both compounds are
evident, although the existing flexibility might let them occupy similar re-
gions of the conformational space.
scribed above for 7 and 8, is also present. A view of the
major MM3*-based conformers, which are in agreement
with the experimental data, is given in Figure 3.
involved the galactose moiety. Moreover, competitive STD
experiments with lactose, a common ligand for VAA known
to enter the lectin sites, were performed. It was shown that
both 7 and 8 competed with lactose for the contact site; this
underscored their bioactivity as lectin ligands.
Thus, in principle, and according to their conformational
behavior in solution, compounds 7–9 can act as mimetics of
the natural glycopeptides, with exo-anomeric orientation for
the corresponding F glycosidic linkage of 9. Indeed, a super-
imposition of compound 8 with GalNAcaOSer (Figure 4) il-
lustrates a remarkable match of the chairs and the positions
of the polar groups of both compounds. To test whether this
similarity will in fact translate into bioactivity, we proceeded
to perform interaction studies with the plant-toxin-reactive
b1–3-linked galactosides.[7,19]
Further STD NMR assays[21] on 9 in the presence of viscu-
min were also performed (Figure 6). As above for the inter-
actions between 9 and this receptor, but now with ligand/re-
ceptor molar ratios between 30:1 and 200:1, major satura-
tion transfer to the protons in both galactopyranose rings
was observed, especially for the nonreducing end. No trans-
fer to the amino acid protons was evidenced even after long
saturation times; this indicates intimate contact of the lectin
to the sugar moiety of 9. Again, competition experiments
with lactose revealed that both molecules target the same
binding site in the lectin.
Interaction with viscumin: Saturation transfer difference
(STD) NMR spectra[20] of 7 and 8 in the presence of the ga-
ACHTUNGTRENNUNGlacACHTUNGTRENNUNGtoside-specific viscumin (VAA) were recorded (Figure 5).
Two molar ratios for the 7/VAA or 8/VAA mixture were
tested, that is, 20:1 and 50:1. Major saturation transfer to
the protons in the pyranose ring was observed, followed by
minor transfer to H3 at the lactam ring, with basically no
transfer to H2 at this fragment. Thus, the recognition mainly
To rationalize the interaction on the molecular level, the
low-energy conformers of 7–9, as deduced from NMR ex-
periments, were docked into the lectin sites by the program
Autodock. Two sites are known in the lectin dimer, charac-
Figure 3. A) The major conformers of 9 (R configuration for Ca) according to the experimental NMR data and MM3*-based molecular mechanics and
dynamics calculations. The orientation around the glycosidic linkage is defined by the exo-anomeric effect, while, according to MD simulations, mobility
is allowed for Y angle, with a concomitant effect on the lactam ring. A superimposition of the two conformers shown left and right is given in the central.
This moiety, in turn, may also adopt the two geometries shown in Figure 1, for 7 and 8. B) The major conformer of 9 (S configuration for Ca) according
to the experimental NMR data and MM3*-based molecular mechanics and dynamics calculations.
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J. Jimꢀnez-Barbero, C. Nativi et al.
of the carboxy group with the
protein backboneꢆs side chains.
For geometry A, the two meth-
ylene H2a and H2b protons
were oriented outside of the
protein, thus precluding their
contact, as inferred from the
STD experiments. Therefore,
there is a perfect match be-
tween the molecular modeling
results and the experimental
data for bound 8. A similar
conclusion was deduced for the
docking of 9, although in this
case the extension of the addi-
tional galactose moiety permit-
ted docking for any conformer
at the lactam ring as well as re-
taining conformational flexibili-
ty at the interglycosidic torsion
angles (Figure 8). Indeed, the
docked structure perfectly ex-
Figure 5. A) Compound 7 interacts with viscumin, inhibitable by lactose, via the galactose epitope and with
similar affinity, that is in the millimolar range. The concentration of the protein was 25 mm. The bottom part
shows the 500 MHz 1H NMR spectrum of 7. At the top is the STD spectrum of 7 (2 s saturation time) in the
presence of VAA. The 7/VAA molar ratio is 50:1. The off-resonance frequency was set at 50 ppm, while the
on-resonance frequency was established at À0.5 ppm. B) The STD percentages assigned to the particular hy-
drogens. The 78% value below H3’ Gal corresponds to the addition of H3’ and both H6’ protons.
terized by the central presence of Trp38 in the 1a subdo-
main and by Tyr249 in the 2g subdomain.[22] With emerging
insights into pH-dependent changes when comparing the
crystal structure obtained at acidic pH with experimental
binding data under physiological condition, a slight modifi-
cation of the architecture of the site in the 2g subdomain
plains the experimental STD data for the galactose hydro-
gens (with the obvious limitations arising from overlapping)
and the lack of STD effect for protons at the lactam
moiety). These computational data thus establish a structur-
al concept for the experimental data obtained by STD
NMR experiments.
4
was inferred.[22,23] The C1 conformer fitted very well, espe-
cially in the Trp binding site when geometry A was em-
ployed for the lactam ring. The superimposition of the Gal
rings of 8 and 9 of the Autodock solution with that of lac-
tose in the X-ray structure was excellent. In contrast, Auto-
dock did not find a lactose-type structure for the other site
in viscumin for 8, although manual docking could be accept-
able. Conformer B of 8, on the other hand, could not be ac-
commodated at either site (Figure 7) due to steric repulsion
Conclusion
The presented facile synthetic route not only afforded dia-
stereoselectivity, but also a product that maintains the bio-
mimetic conformational properties of the resulting scaffold,
as documented by combined molecular modeling and NMR
spectroscopic analyses. The sugar part retains bioactivity for
Figure 6. Compound 9 interacts with viscumin, mainly through the nonreducing galactose moiety, as can be observed in the STD spectra, and in the mo-
lecular scheme, where the key STD percentages are given. The concentration of the protein was ca. 15 mm. The experiments were performed at 298 K, in
20 mm phosphate buffer at pH 5.8. A) An expanded view of the sugar region with a superimposition of the regular 500 MHz 1H NMR spectrum of 9
(top) onto two STD spectra recorded with 100:1 and 50:1 molar ratios of ligand/VAA, which yielded analogous results. The off-resonance frequency was
set to 50 ppm, while the on-resonance frequency was established at À0.5 ppm. B) The STD percentages for 9. The 76% value corresponds to the addition
of H4 and H5 of the reducing Gal unit, and H5’ of the nonreducing Gal. Negligible STD effects on the protons of the lactam moiety are observed, inde-
pendently of the diastereomer at the amino acid chiral center.
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a-O-Linked Glycopeptide Mimetics
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sugar moiety among the adhesion/growth-regulatory galec-
tins, with preference for galectins-3 and -9, encourages fur-
ther testing at this level of structural complexity, at which
formation of a C-glycoside could even afford a nonhydrolyz-
able galectin ligand.[24]
Experimental Section
Molecular modeling: The low-energy conformers were calculated by
using the MM3* force field,[13] in Maestro.[25] The six-membered ring cis-
fused to the pseudogalactose moiety through C1 and C2 is rather rigid.
Therefore, only a low level of flexibility remains for the lactam moiety.
Two torsion angles were evaluated to account for the six-membered
ring’s shape, namely for 8, torsion angle f1 is defined as N4-C3-C2-C1
and f2 as C5-N4-C3-C2. For all the molecules, the molecular-dynamics
simulations were performed by using the MM3* force field. A tempera-
ture of simulation of 300 K was employed with a time step of 1.5 fs and
an equilibration time of 100 ps. The total simulation time for each com-
pound was 5 ns. In all the molecular-mechanics and -dynamics calcula-
tions, the GB/SA solvation model for water was used.[26] For 9, different
initial geometries of the F/Y glycosidic linkages were used, with syn or
anti conformers for both F and Y angles. Also, the two possible epimers
at the chiral centre of the amino acid were used as starting geometries, as
well as the two possible geometries of the lactam ring, previously de-
duced for 7 and 8 (see text).
Figure 7. The putative binding mode of compound 8 in complex with vis-
cumin, according to AUTODOCK calculations. The expansion of the lec-
tinꢆs Trp site shows interaction with 8 in the preferred A-type conforma-
tion. The methylene protons are not in contact with the polypeptide, in
agreement with the STD observations, which mainly indicate transfer to
the Gal protons. The side chains of the key amino acids interacting
through hydrogen bonds or van der Waals contacts with 8 are shown in
ball and stick form.
the tested model lectin, so this synthetic scaffold is suitable
to present functionally potent docking sites for clinically rel-
evant human lectins, such as the selectins mentioned in the
introduction. The selectivity for the prepared Galb1–3Gal
À
For the C5 C6 torsion of the Gal moieties, only the gt geometry (w, de-
fined as C4-C5-C6-O4 ca. 1808) was considered, since it has been demon-
strated to be the major one for galactose derivatives (O4 in axial orienta-
tion).[15,27]
Figure 8. Different possibilities for the binding mode of compound 9 to VAA, according to AUTODOCK calculations. The expansion of the Trp site of
VAA is shown in panels (B)–(E). The Tyr site is outlined in panels (A) and (F), with the two different possible epimers of the lactam ring. There is not
any hindrance for the recognition of any of them. Panel (B) shows a superimposition of two conformers around the glycosidic torsion angles of the disac-
charide, while panels (C) and (D) depict the two geometries separately. Panel (E) presents a superimposition of the pose shown in (C) with the crystallo-
graphic binding mode of lactose, showing a good match. All these structures perfectly match the experimental STD data, which are mainly observed at
the Gal moiety, with no transfer to the lactam hydrogens. Indeed, the methylene protons point outside the polypeptide chain. The side chains of the key
amino acids interacting through hydrogen bonds or van der Waals contacts with 9 are shown as sticks.
Chem. Eur. J. 2009, 15, 10423 – 10431
ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10429

J. Jimꢀnez-Barbero, C. Nativi et al.
NMR experiments with free compound: 1H NMR spectra were recorded
on Bruker Avance 500 spectrometers in D2O with concentrations of
5 mm. Chemical shifts are reported in ppm with external TSP (2,2,3,3-tet-
radeutero-3-trimethylsilylpropionic acid, 0 ppm) as reference. Vicinal
proton–proton coupling constants were estimated from first-order analy-
sis.
Acknowledgements
This work was supported by a grant from the Ministry of Science and In-
novation of Spain (CTQ2006-10874-C02-01/BQU) and by an EC Marie
Curie Research Training Network grant (MCRTN-CT-2005-19561). The
authors thank the Ente Cassa di Risparmio di Firenze for a PhD grant to
E.D.
2D TOCSY experiments (30 and 70 ms mixing times) were performed by
using a data matrix of 256ꢇ2K to digitize a spectral width of 5000 Hz.
Eight scans were used per increment, with a relaxation delay of 2 s.
2D NOESY (600 and 1000 ms) and 2D T-ROESY experiments (400 and
500 ms) used the standard sequences. Distances were estimated from
NOESY/ROESY experimental data by using the isolated spin-pair ap-
proximation[28] for the data with decreased mixing time and averaging.
Estimated errors were below 10%.
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Received: April 23, 2009
Published online: September 11, 2009
Chem. Eur. J. 2009, 15, 10423 – 10431
ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10431