Pyrrolic tripodal receptors for the molecular recognition of carbohydrates: Ditopic receptors for dimannosides

Article abstract of DOI:10.1002/chem.201204298

Synthetic ditopic receptors, designed for the molecular recognition of dimannosides, have been prepared by bridging two monotopic units effectively recognizing mannosides with linkers of the appropriate size and flexibility, endowed with hydrogen-bonding groups. Affinities toward the α and β glycosides of the biologically relevant Manα(1-2)Man disaccharide were measured by NMR spectroscopy and isothermal titration calorimetry (ITC) in polar organic media (30-40 % DMF in chloroform). Significant selectivities and affinities in the micromolar range were observed in most cases, with two newly designed receptors being the most effective receptors of the set, together with a distinct preference of the dimannosides for the (S) enantiomer of the receptor in all cases. A 3D view of the recognition mode was elucidated by a combined NMR spectroscopic/molecular modeling approach, showing the dimannoside included in the cleft of the receptor. Compared to the monotopic precursors, the ditopic receptors showed markedly improved recognition properties, proving the efficacy of the modular receptor design for the recognition of disaccharides. Pac-man loves sugar: Unprecedented recognition of the biologically relevant Manα(1-2)Man disaccharidic epitope has been achieved by bridging two mannoside binding units into a ditopic biomimetic receptor through hydrogen-bonding linkers of appropriate length and flexibility (see figure). Copyright

Full text of DOI:10.1002/chem.201204298

DOI: 10.1002/chem.201204298
Pyrrolic Tripodal Receptors for the Molecular Recognition of Carbohydrates:
Ditopic Receptors for Dimannosides
Oscar Francesconi,[b] Cristina Nativi,[b] Gabriele Gabrielli,[b] Matteo Gentili,[b]
Marco Palchetti,[b] Beatrice Bonora,[b] and Stefano Roelens*[a]
Abstract: Synthetic ditopic receptors,
designed for the molecular recognition
of dimannosides, have been prepared
by bridging two monotopic units effec-
tively recognizing mannosides with
linkers of the appropriate size and flex-
ibility, endowed with hydrogen-bonding
groups. Affinities toward the a and b
glycosides of the biologically relevant
in polar organic media (30–40% DMF
in chloroform). Significant selectivities
and affinities in the micromolar range
were observed in most cases, with two
newly designed receptors being the
most effective receptors of the set, to-
gether with a distinct preference of the
dimannosides for the (S) enantiomer of
the receptor in all cases. A 3D view of
the recognition mode was elucidated
by a combined NMR spectroscopic/mo-
lecular modeling approach, showing
the dimannoside included in the cleft
of the receptor. Compared to the mon-
otopic precursors, the ditopic receptors
showed markedly improved recognition
properties, proving the efficacy of the
modular receptor design for the recog-
nition of disaccharides.
Keywords: binding · carbohydrates ·
mannosides · molecular recognition ·
synthetic receptors
ManaACHTUNGTRENNUNG(1–2)Man disaccharide were
measured by NMR spectroscopy and
isothermal titration calorimetry (ITC)
Introduction
nition processes are usually the terminal mono- or oligosac-
charides, among which mannosides are frequent and play a
central role in both pathological and therapeutic processes.
Indeed, mannosides are recognized by a variety of lectins[3]
and are involved in infections caused by several high-risk
pathogens, like pathogenic yeasts (Candida),[4] bacteria (mi-
cobacterium tubercolosis),[5] and viruses (HIV and Hepatitis
HCV).[6] On the other hand, antibodies, such as 2G12[7] or
PGT128,[8] lectins, such as cyanovirin-N,[9] microvirin,[10] or
actinohivin,[11] and other natural non-peptidic molecules,
such as benanomicin A and pradimicin A,[12] which show
potent antiviral activity, owe their antiviral properties to the
specific recognition of terminal oligomannosides on the
pathogen surface.
Molecular recognition of carbohydrates has become a topic
of high interest in the past decade. The impetus for research
in this field is largely provided by the prominence of carbo-
hydrate recognition events in biological processes,[1] which
stimulated the development of artificial biomimetic recep-
tors,[2] not only to mimic the natural receptors and to under-
stand recognition mechanisms in biological systems, but also
to develop new therapeutic and diagnostic tools for medi-
cine and technology.
In biological systems, carbohydrate recognition is exerted
by dedicated classes of biomolecules, such as lectins and an-
tibodies, which govern the interaction between living cells
and other cells or pathogens through the specific recognition
of the saccharidic chains of glycoconjugates exposed on cell
or pathogen surfaces.[1c] The epitopes involved in the recog-
A particularly relevant example of pathological conse-
quences of mannose recognition is the binding of the DC-
SIGN lectin of the immune system dendritic cells to the
viral glycoprotein gp120 on the HIV envelope, which trig-
gers the infection of human cells by HIV.[13] In this binding
event, the ManaACHTUNTRGNEUNG(1–2)Man dimannoside motif is the smallest
[a] Dr. S. Roelens
Istituto di Metodologie Chimiche (IMC)
Consiglio Nazionale delle Ricerche (CNR)
Dipartimento di Chimica, Universitꢀ di Firenze
Polo Scientifico e Tecnologico, Via della Lastruccia, 13
50019 Sesto Fiorentino, Firenze (Italy)
Fax : (+39)055-457-3570
fragment of the Man9 epitope of the gp120 glycans required
for recognition by the DC-SIGN lectin.[14]
It thus appears that oligomannosides are targets of high
interest in the design of artificial receptors, especially in the
search for effective carbohydrate binding agents (CBAs) for
therapeutic purposes.[13] However, the endeavor dedicated
to the design of structures for the recognition of carbohy-
drates has been so far focused mainly on monosaccharidic
targets, to an extent that examples of biomimetic receptors
selectively recognizing oligosaccharides through noncovalent
interactions are still rare.[15] Most remarkably, although re-
ceptors showing preference for the most commonly availa-
E-mail: stefano.roelens@unifi.it
[b] Dr. O. Francesconi, Prof. C. Nativi, Dr. G. Gabrielli, M. Gentili,
M. Palchetti, B. Bonora
Dipartimento di Chimica, Universitꢀ di Firenze
Polo Scientifico e Tecnologico
50019 Sesto Fiorentino, Firenze (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204298.
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FULL PAPER
ble cellobiose, maltose, lactose, and sucrose saccharides
have been reported, displaying excellent recognition proper-
ties in some cases,[15a] to the best of our knowledge, we are
not aware of any report on the recognition of dimannosides
or oligomannosides through synthetic receptors.
In the course of our studies on the design of biomimetic
receptors for carbohydrates, we have described a family of
aminopyrrolic tripodal structures showing the highest affini-
ties and selectivities for a- and b-mannosides reported to
date.[16] Due to the biological significance of oligomanno-
sides, the results obtained encouraged us to further explore
the recognition of dimannosidic targets by adjusting the ar-
chitecture of our mannose-selective receptors.
We would like to describe here the successful design of a
new generation of ditopic diaminopyrrolic structures featur-
ing unprecedented recognition properties toward the glyco-
sides of the ManaACHTUNGTRENNUNG(1–2)Man dimannoside. The results assess
the new structures as the first receptors reported in the
chemical literature capable of effectively recognizing diman-
nosides in polar organic media. Although admittedly recog-
nition in water could not be evaluated, due to the insolubili-
ty of the receptors, we believe that the described structures
represent a significant leap in the recognition of biologically
relevant oligomannosides.
Scheme 1. Synthesis of n-octyl(a-d-mannopyranosyl)-(1!2)-a-d-manno-
pyranoside (OctACTHUNTGRNEUNG(aMan)aMan). Reagents and conditions: a) n-octanol,
trimethylsilyl trifluoromethanesulfonate (TMSOTf), CH2Cl2, 08C, 15 min,
61%; b) CH3ONa 1m solution in CH3OH, RT, 45 min, 95%; c) com-
pound 2, TMSOTf, CH2Cl2, 08C, 50 min, 60%; d) CH3ONa 1m solution
in CH3OH, RT, 1.5 h, then H2, [Pd(OH)2]/C, CH2Cl2/CH3OH 1:1, RT,
16 h, 84%. Bn=benzyl.
Results and Discussion
Design: Having available a family of structures effectively
recognizing monomannosides, the logical evolution of our
studies involved choosing biologically relevant dimannosidic
targets and modifying the architecture of the receptors to
optimize the affinities and the selectivities toward the
chosen dimannosides. Because the Mana
encountered in important recognition processes, the a- and
b-octyl glycosides of the Mana(1–2)Man dimannoside (Oct-
(aMan)aMan and Oct(aMan)bMan) were chosen to test
ACHTUNGTREN(NGNU 1–2)Man motif is
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
the binding abilities of the modified structures, whereas the
diaminopyrrolic receptor 1, which was prepared in both
enantiomerically pure forms[17] and gave best recognition re-
sults with the related monomannosides OctaMan and Octb
Man,[16] was selected as the appropriate building block to
construct modified architectures.
The design of the receptors was based on the idea of
bridging two units of 1 by an appropriate linker to build a
ditopic structure capable of recognizing the two mannose
units of the dimannoside through a tweezer-like architec-
ture. Because markedly different recognition properties
were exhibited by the two enantiomers of 1 toward Octb
Man, the derived ditopic receptors were also expected to
show selectivity in the recognition of the dimannosides and
were thus prepared in both enantiomerically pure forms for
recognition studies.
The octyl chain was necessary to ensure the solubility in
polar organic solvents required for binding measurements.
The synthesis of the a-octyl glycoside was achieved by a
standard glycosidation procedure. The trichloroacetimidate
2[18] was glycosidated with n-octanol, deacetylated, and gly-
cosylated on the free hydroxyl group with a second equiva-
lent of compound 2. The resulting a-dimannoside was de-
protected to the desired OctACTHNUGRTNEUNG(aMan)aMan in good yields
(see Scheme 1 for yields, reagents, and conditions).
Synthesis of glycosides: Because Oct
(aMan)bMan were not commercially available, the target
glycosides were prepared as described in Schemes 1 and 2.
(aMan)aMan and Oct-
For the synthesis of the b-octyl glycoside, because of the
well-known difficulty of obtaining b-mannosides, Oct-
AHCTUNGTREG(NNNU aMan)bMan was prepared through the intramolecular agly-
ACHTUNGTRENNUNG
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S. Roelens et al.
Scheme 3). The gem-dimethyl group in the linker induces a
convenient twisting of the two pyrrolic moieties of the dia-
ldehyde 10,[22] and presumably of the two halves of the di-
topic structure as well, which may appropriately match the
twist of the two mannose units of the dimannoside.[23] On
the other hand, the single carbon hinge may impose confor-
mational restrictions to the structure, which may hinder
ligand binding.
The more flexible CH2NHCH2 bridging moiety was thus
implemented in the structure of compound 12, which was
prepared by condensation of compound 11 with the corre-
sponding dialdehyde 13, followed by reduction of the Schiff
base (Scheme 3). In addition to providing a more flexible
linker, the hydrogen-bonding ability of the aminic group
may result beneficial to the recognition properties of the
structure. The dialdehyde 13 was prepared by condensing.
the mono-protected pyrroledialdeyde 14[24] with ammonia,
reducing the Schiff base, and subsequently deprotecting the
formyl groups (Scheme 4).
The rigid, non-hydrogen bonding, p-xylyl moiety imple-
mented in the structure of compound 15 served as a refer-
ence to evaluate the impact of the hydrogen-bonding groups
in the linker of the previous structures and the role of the
size and the rigidity of the bridging moiety. Receptor 15 was
prepared from terephthalaldehyde following the general
procedure shown in Scheme 3.
Scheme 2. Synthesis of n-octyl(a-d-mannopyranosyl)-(1!2)-b-d-manno-
pyranoside (Oct(aMan)bMan). Reagents and conditions: a) n-octanol,
ACHTUNGTRENNUNG
2,3-dichloro-5,6,-dicyano-1,4-benzoquinone (DDQ), CH2Cl2, RT, 1 h;
b) 2,6-di(tert-butyl)-4-methylpyridine (DTBMP), AgOTf (Tf=triflate),
SnCl2, CH2Cl2, RT, 3.5 h, 72%; c) compound 2, TMSOTf, CH2Cl2, 08C,
1 h, 63%; d) K2CO3, CH3OH, RT, 3 h, then H2, [Pd(OH)2]/C, CH2Cl2/
CH3OH 1:1, RT, 16 h, 71%.
cone delivery (IAD) proce-
dure.[19] The mannosyl fluoride
6[20] was reacted with n-octanol
under oxidative conditions to
give the benzylic octylacetal in-
termediate. The latter trans-
posed under Lewis acidic catal-
ysis to deliver the octyl agly-
cone to the anomeric position
from the same face, stereoselec-
tively giving the b-glycoside 7
in good overall yields. Com-
pound 7 was subsequently gly-
cosylated on the free hydroxyl
group with compound 2 and de-
protected to OctACHTUNGTRENNUNG(aMan)bMan
through the same procedure de-
scribed for the corresponding a
anomer (see Scheme 2 for
yields, reagents and conditions).
Synthesis of the receptors: The
ditopic receptor 9, featuring a
single carbon linker, could be
prepared by condensing the di-
pyrrylmethane dialdehyde 10[21]
with the aminic precursor 11[16a]
of the monotopic receptor 1,
and reducing the resulting
Scheme 3. Synthesis of receptors 9, 12, and 15 from enantiomerically pure (R)- and (S)-11. Reagents and con-
ditions: a) 5,5’-(propane-2,2-diyl)bis(1H-pyrrole-2-carbaldehyde), CHCl3, 708C, 3 h, then NaBH4 suspension in
CH3OH, RT, 3 h, 46%; b) compound 13, CHCl3, RT, 18 h, then NaBH4 suspension in CH3OH, RT, 3 h, 40%;
Schiff base with NaBH4 (see c) terephthalaldehyde, CHCl3, RT, 18 h, then NaBH4 suspension in CH3OH, RT, 4 h, 58%.
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Pyrrolic Receptors for the Molecular Recognition of Carbohydrates
FULL PAPER
to the tetramine 19. The latter was condensed with 2,5-di-
formyl pyrrole and reduced to the tetraminopyrrolic inter-
mediate 20, featuring the cyclic unit of the receptor. Stau-
dinger reduction of the azide, followed by further condensa-
tion with 2,5-diformyl pyrrole gave, after reduction of the
Schiff base, the ditopic receptor 16.
To obtain enantiopure receptors of opposite chirality, all
the described syntheses were performed starting from the
enantiopure diaminocyclohexane reagents of (R,R) and
(S,S) configuration, providing structures of all-R and all-S
configuration, respectively.
It should be noted that the designed structures are confor-
mationally flexible, thus, not preorganized in a tweezer-like
conformation. This conformation may be achieved upon
complexation if the correct binding geometry would provide
the required energy gain. Improved binding capabilities with
respect to the monotopic receptors would therefore suggest
a convergent arrangement of the two units into a tweezer-
like conformation and would support the efficacy of the
modular design.
Scheme 4. Synthesis of linker 13. Reagents and conditions: a) NH3,
CH3OH, 608C, 3 h, then NaBH4, RT, 2 h, then compound 14; CH3OH,
RT, 3 h, then NaBH4, RT, 2 h, 80%; b) HCl 1m, H2O, RT, 2.5 h, 72%.
Eventually, a receptor featuring the narrowest cleft of the
set was obtained by replacing the diaminocyclohexane frag-
ment of the pyrrolic bridge with a simple amine moiety in
structure 16. The latter would provide information on the
cleft size required for the binding to a disaccharidic ligand.
Receptor 16 was prepared starting from the dibromo-mono-
azido reactant 17[25] as described in Scheme 5. Compound 17
was reacted with sodium acetate, and the resulting diacetate
was hydrolyzed to the diol and oxidized to the dialdehyde
18 by pyridinium chlorochromate (PCC). The dialdehyde
was condensed with the mono-BOC-protected diaminocy-
clohexane and the Schiff base was reduced and deprotected
Recognition studies: To assess the improvement achieved
through the described structure modifications, the affinities
of the ditopic receptors for Oct
ACHTUNGTREN(NUGN aMan)aMan and Oct-
AHCTUNGTRENNUNG
1
Association constants were measured by H NMR spectro-
scopic titrations according to a previously described proto-
col,[26] which consists in the simultaneous fit of the complex-
ation induced shifts[27] of all the available signals from both
the receptor and the glycoside to the appropriate association
model by nonlinear regression analysis.
The main issue to be addressed in this study was the selec-
tion of an appropriate medium that would provide the solu-
bility of both reactants required for binding measurements,
as the dimannosides were soluble in strongly polar solvents,
whereas the receptors were soluble in solvents of low per-
mittivity, but not in water. A mixture of CDCl3 and DMF
turned out to be the medium of choice, ensuring an overlap
of the solubility ranges by allowing the appropriate tuning
of the polarity by simply changing their mole ratio. We
found that 30% of DMF in CDCl3 (v/v) was an appropriate
polar medium to run the whole set of measurements. We
also found that binding constants measured in this medium
were comparable to those measured in acetonitrile, when
using the values obtained in the latter solvent for the bind-
ing of 1 to monomannosides as reference,[16a] giving an indi-
cation of the competitivity of the medium, which can be
useful to compare results with previous data. The results ob-
tained for receptors 9, 12, 15, and 16 with Oct
ACHTUNGTRENUN(NG aMan)aMan
and Oct(aMan)bMan are reported in Table 1, together with
AHCTUNGTRENNUNG
Scheme 5. Synthesis of receptor 16. Reagents and conditions: a) AcONa,
DMF, 1008C, 2 h, >99%; b) K2CO3, CH3OH, RT, 70 h, >99%; c) pyridi-
nium chlorochromate, CH2Cl2, RT, 2 h, 95%; d) N-tert-butoxycarbonyl-
1,2-diaminocyclohexane, CHCl3, 808C, 15 h, then NaBH4, CH3OH, RT,
3 h, >99%; e) trifluoroacetic acid (TFA), CH2Cl2, RT, 3 h, 63%; f) pyr-
role-2,5-dicarbaldehyde, CHCl3, RT, 15 h, then NaBH4, CH3OH, RT,
1.5 h, 96%; g) PhP3, H2O, THF, RT, 15 h, 87%; h) pyrrole-2,5-dicarbalde-
hyde, CHCl3, RT, 15 h, then NaBH4, CH3OH, RT, 1 h, 70%. BOC=tert-
butoyloxycarbonyl.
those obtained for receptor 1 and with OctaMan and Octb
Man for a direct comparison of the binding properties of
mono- versus ditopic receptors, and of mono- versus diman-
nosides in the same medium.
Because multiple complex species were found in most
cases, affinities were assessed from the measured binding
constants through the BC050 parameter, a binding descriptor
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S. Roelens et al.
Table 1. Cumulative formation constants (logb)[a] and intrinsic median binding concentration (BC500)[b] for receptor to glycoside (R/G) complexes of
octyl mannosides and octyl dimannosides in CDCl3/DMF 70:30.
Receptor[c]
OctaMan
OctbMan
Oct
logb (R/G)
N
OctACHTGNUTRENNUNG
logb (R/G)
logb (R/G)
BC050 [mm]
(352ꢀ18)
logb (R/G)
BC050 [mm]
BC050 [mm]
(R)-1
(3.41ꢀ0.02) (1:1)
(5.83ꢀ0.06) (2:1)
(4.96ꢀ0.17) (1:2)
(3.59ꢀ0.02) (1:1)
(6.13ꢀ0.05) (2:1)
(4.93ꢀ0.21) (1:2)
(2.380ꢀ0.003) (1:1)
G
(2.66ꢀ0.07) (1:1)
(4.70ꢀ0.08) (2:1)
(4.80ꢀ0.06) (1:2)
(3.56ꢀ0.08) (1:1)
(6.51ꢀ0.09) (2:1)
(1453ꢀ128) (2.76ꢀ0.04) (1:1)
(1908ꢀ166)
(4.51ꢀ0.09) (2:1)
(5.04ꢀ0.02) (2:1)
(S)-1
A
A
(3.15ꢀ0.05) (1:1)
(5.84ꢀ0.07) (2:1)
(3.00ꢀ0.07) (1:1)
(5.16ꢀ0.12) (2:1)
(R)-9
(4173ꢀ28) (2.39ꢀ0.03) (1:1)
E
(3.03ꢀ0.01) (1:1)
(2.88ꢀ0.01) (1:1)
(4.95ꢀ0.04) (2:1)
(S)-9
(2.82ꢀ0.03) (1:1)
(4.71ꢀ0.12) (1:2)
(3.24ꢀ0.04) (1:1)
(5.41ꢀ0.07) (1:2)
(3.56ꢀ0.07 (1:1)
(6.05ꢀ0.10) (1:2)
(2.228ꢀ0.002) (1:1)
(1289ꢀ82) (3.00ꢀ0.08) (1:1)
T
(3.49ꢀ0.08) (1:1)
(5.56ꢀ0.50) (2:1)
(3.11ꢀ0.14) (1:1)
(6.42ꢀ0.11) (2:1)
(3.63ꢀ0.06) (1:1)
(7.75ꢀ0.08) (2:1)
(2.75ꢀ0.03) (1:1)
(5.35ꢀ0.10) (1:2)
(3.15ꢀ0.08) (1:1)
(5.21ꢀ0.32) (2:1)
(3.16ꢀ0.04) (1:1)
(5.22ꢀ0.15) (1:2)
(3.40ꢀ0.05) (1:1)
(4.80ꢀ0.10) (1:1)
(8.28ꢀ0.17) (2:1)
(3.50ꢀ0.06) (1:1)
(5.73ꢀ0.28) (2:1)
(4.19ꢀ0.04) (1:1)
(10.39ꢀ0.16) (2:2)
(2.524ꢀ0.009) (1:1)
(5.29ꢀ0.15) (1:2)
(3.32ꢀ0.07) (1:1)
(5.86ꢀ0.09) (1:2)
(3.66ꢀ0.08) (1:1)
(6.12ꢀ0.17) (1:2)
(R)-12
(S)-12
(R)-15
(S)-15
(R)-16
(S)-16
R
E
N
E
(5913ꢀ33) (2.020ꢀ0.002) (1:1)
T
(2.76ꢀ0.01) (1:1)
(3.99ꢀ0.28) (1:2)
(2.97ꢀ0.06) (1:1)
(5.27ꢀ0.11) (1:2)
(3.30ꢀ0.04) (1:1)
(5.81ꢀ0.19) (1:2)
(1659ꢀ73) (2.554ꢀ0.001) (1:1)
E
(2.81ꢀ0.01) (1:1)
(4.66ꢀ0.03) (1:2)
(3.30ꢀ0.02) (1:1)
(5.64ꢀ0.10) (1:2)
(3.45ꢀ0.04) (1:1)
(6.43ꢀ0.11) (1:2)
(828ꢀ86 ) (2.94ꢀ0.02) (1:1)
R
(5.88ꢀ0.05) (2:1)
(3.12ꢀ0.03) (1:1)
(5.04ꢀ0.18) (1:2)
A
A
(5.75ꢀ0.1)6 (2:1)
[a] Measured by 1H NMR spectroscopy (400–900 MHz) from titration experiments at T=298 K. Formation constants were obtained by nonlinear least-
square regression analysis of the NMR data by simultaneous fit of all available signals from both reagents. [b] Calculated from the logb values by using
the “BC50 Calculator” program (see ref. [28]). [c] See reference [17].
that quantitatively defines the overall affinity of a receptor
for a ligand.[29] The BC500 values calculated for the investigat-
ed systems through the “BC50 Calculator”[28] are also report-
ed in Table 1. Gratifyingly, it can be appreciated that all the
ditopic receptors consistently bound the dimannosides more
effectively than the monomannosides, whereas the opposite
was true for the monotopic receptor 1.
the adaptivity of the receptor at the expenses of selectivity.
In agreement with these conclusions, receptor 16 behaves
much like receptor 12, although exhibiting weaker affinities:
most likely, the smaller size of the flexible, monopyrrolic
bridge does not affect the binding capabilities of 16, but the
lack of one of the two pyrrolic rings results in increased
BC050 values. Altogether, bridging monotopic binding units
with a linker of appropriate size and flexibility and endowed
with effective binding groups proved to be a successful strat-
egy.
To confirm the interesting results obtained, we challenged
the binding abilities of the best performing receptors 9 and
12 toward OctACTHNUTRGENNG(U aMan)aMan and OctACHUTNGTRENN(GUN aMan)bMan in a more
competitive medium. To this end, the amount of DMF in
the mixture was raised to 40% and the extent of the weak-
ening effect of the increased polar solvent on the affinities
was assessed by replicating the NMR spectroscopic titra-
tions. A typical example is depicted in Figure 1, in which a
marked shift of the signals of both reactants is evident,
Although the design of the ditopic receptors appeared
well suited for the recognition of the disaccharides, the re-
sults clearly showed that the bridging arm played a crucial
role. Receptor 15, featuring the p-xylyl moiety in the linker,
gave the worst results of the set showing, with a single ex-
ception, affinities in the millimolar range toward both
mono- and dimannosides. On the contrary receptor 9, fea-
turing a dipyrrolic bridge, gave the best affinity of the whole
set, showing toward the b-dimannoside a BC050 value in the
low micromolar range. Even more interestingly, the b-di-
mannoside was recognized by the chiral receptor with an
outstanding enantioselectivity, the (S) enantiomer being
more effective than the (R) enantiomer by nearly two
orders of magnitude. On the other hand, the best affinity for
the a-dimannoside was shown by receptor 12; the latter
gave excellent BC050 values for both dimannosides, but
showed rather shallow enantioselectivity and a/b discrimina-
tion.
showing that binding of (S)-9 to OctACTHNUTRGNEUNG(aMan)bMan is still
substantial in the more competitive medium. Because multi-
ple complex species were still detectable, to avoid ambigui-
ties in the definition of the equilibrium model, two inde-
pendent titrations at different concentrations of the reac-
tants were performed for each receptor/ligand system inves-
tigated, and the two set of data were simultaneously fitted
including all available signals from both the receptor and
the dimannoside. This approach drastically reduced the cor-
relation of the data, providing well-defined models and reli-
able binding constants. The results obtained are shown in
The above-discussed results indicate a substantial contri-
bution to the recognition from the pyrrolic groups of the
linker, where the twisted bridge of receptor 9 appears to be
determinant for the matching of the enantiomers to the
ligand, whereas the more flexible linker of 12 seems to favor
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Pyrrolic Receptors for the Molecular Recognition of Carbohydrates
FULL PAPER
finities are still monitored in
most cases, indicating that rec-
ognition properties tolerate
well the increasing competition
from the medium.
The binding affinities ob-
tained by NMR spectroscopy
were further confirmed by iso-
thermal titration calorimetry
(ITC), as an independent tech-
nique, in 40% DMF in CHCl3.
The issue of the presence of
multiple complex species was
addressed through the same ap-
proach followed for the NMR
data, by combining independent
titrations run at different reac-
tant concentrations into a si-
multaneous fit of all data, when
necessary to remove ambigui-
ties in the definition of the
model. The ITC results of (S)-9
with OctACTHUNRTGNEUNG(aMan)bMan are re-
ported in Figure 2 as an illustra-
tive case, to be compared to the
corresponding NMR spectro-
scopic titration and data treat-
ment displayed in Figure 1. The
binding constants measured by
ITC and the BC050 values calcu-
lated for receptors 9 and 12
with Oct
(aMan)bMan are reported in
Table 3.
ACHTUNGTREN(NUGN aMan)aMan and Oct-
AHCTUNGTRENNUNG
Figure 1. 1H NMR spectroscopic titrations (400 MHz, CDCl3/DMF 60:40, 298 K) of Oct
ACTHNUGTRNEN(UG aMan)bMan
(12.7 mm, set 1; and 6.03 mm, set 2) with (S)-9 (12.5 mm). a) Selected spectral regions from the 1H NMR spec-
Comparison of the observed
affinities with those obtained
by NMR spectroscopy shows a
generally good agreement, al-
though not excellent in all
cases. The discrepancy is clearly
apparent in the models detect-
ed from the two techniques, as
up to four binding constants
tra of a single titration (set 1) showing the signal shifts observed for an increasing receptor to glycoside (R/G)
~
mole ratio. Representative signals of the receptor (left) and of the glycoside (right) are reported: NH-Pyrr’
&
^
*
(endocyclic pyrrole), NH-Pyrr (exocyclic pyrrole), CH-1a (aMan anomeric proton), CH-1 (octyl bMan
anomeric proton). b) Plot of the complexation-induced shifts of the corresponding receptor (left) and glycoside
(right) proton signals along the titrations. Symbols are experimental data points; lines (c set 1, a set 2)
are best fit curves obtained through nonlinear regression by simultaneous fit of all available signals from the
two independent titrations at different reactant concentrations.
Table 2, where the measured binding constants and the cal-
culated BC050 values for receptors 9 and 12 toward the two
dimannosides are reported. Comparison of the results with
the corresponding data from Table 1 shows, apart from the
expected affinity decrease, that the binding abilities follow
essentially the same trend in the two different media, featur-
ing the (S) enantiomers performing better than the (R)
enantiomers for both receptors, although exhibiting better
selectivities for receptor 9 and higher affinities for receptor
12. What appears to suffers the most from the more compet-
itive medium is the selectivity; indeed, the main difference
appears to be the shallower enantiomeric as well as anome-
ric discrimination, the larger affinities being affected more
heavily than the smaller ones. Nevertheless, micromolar af-
were measured by NMR spectroscopy, whereas no more
than two could be obtained by ITC. Such a discrepancy can
be ascribed to the higher definition of the equilibrium sys-
tems that can be obtained by NMR spectroscopy with re-
spect to ITC, because the larger number of data points
(many shift data for several signals of both reactants) and
the higher sensitivity of the shift modulation to the presence
of different complex species, allow for a much finer decon-
volution of the binding isotherm by the former technique.
As a result, the number of species (and therefore of binding
constants) that can be appreciated by ITC is limited by the
intrinsic resolution of the technique.[30]
However, taking into account the formation constants for
the 1:1 complexes, which give the most relevant contribution
Chem. Eur. J. 2013, 19, 11742 – 11752
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11747

S. Roelens et al.
Table 2. Cumulative formation constants[a] and intrinsic median binding
concentration[b] for receptor to glycoside (R/G) complexes of octyl di-
mannosides in CDCl3/DMF 60:40 from NMR spectroscopic data.
Receptor
Oct
logb (R/G)
(aMan)aMan
OctACTHNGUTERN(UNG aMan)bMan
logb (R/G)
BC050 [mm]
BC050 [mm]
(R)-9
(2.31ꢀ0.04) (1:1)
(3.61ꢀ0.18) (1:2)
(6.34ꢀ0.24) (2:2)
(2.92ꢀ0.03) (1:1)
(4.79ꢀ0.07) (2:1)
(3.74ꢀ0.34) (1:2)
(3.30ꢀ0.10) (1:1)
(5.95ꢀ0.18) (2:1)
(5.32ꢀ0.28) (1:2)
(8.73ꢀ0.35) (2:2)
(3.48ꢀ0.08) (1:1)
(5.38ꢀ0.27) (2:1)
(5.33ꢀ0.14) (1:2)
(9.15ꢀ0.17) (2:2)
1
G
ACHTUNGTRENNUNG
4.31ꢀ0.02) (1:2)
(S)-9
A
(R)-12
R
(S)-12
G
[a] Measured by H NMR spectroscopy (400–900 MHz) from titration ex-
periments at T=298 K. Formation constants were obtained by nonlinear
least-square regression analysis of NMR data by simultaneous fit of all
available signals from both reagents. [b] Calculated from the logb values
by using the “BC50 Calculator” program (see ref. [28]).
to the affinity, it can be clearly seen that the agreement be-
tween the NMR spectroscopic and the ITC values is very
good indeed, the data for (S)-12 showing the only significant
deviation. Thus, within the discussed limits, the overall affin-
ity pattern and the measured binding constants are essential-
ly confirmed by the two independent techniques.
Structural studies: A description of the binding mode char-
acterizing the investigated receptor–carbohydrate complexes
in solution was attempted by combining NMR techniques
with molecular modeling calculations. This approach was
complicated by some basic issues affecting these systems:
indeed, the occurrence of multiple complex species of com-
parable stability at room temperature prevented the identifi-
cation of suitable conditions for an NMR spectroscopic in-
vestigation, which required the 1:1 complex to be the preva-
lent species; on the other hand, the structures of the recep-
tors were too flexible to obtain well-defined energy minima
in the conformational space of the investigated complexes.
Fortunately, we found that titration data obtained at 508C
could be adequately fitted to the 1:1 model, thus allowing
the selection of conditions under which useful amounts of
the desired complex could be formed in solution, despite
the lower affinity of the receptor for the disaccharide at that
temperature.
Figure 2. ITC results of Oct
60:40 at T=298 K. a) Titration of (S)-9 (0.48 mm) with Oct
(7.71 mm). b) Titration of Oct(aMan)bMan (0.27 mm) with (S)-9
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
&
(19.2 mm). c) Plot of the experimental data points for titrations a) ( )
*
and b) ( ). Crosshair symbols (+) are calculated points obtained by si-
multaneous fit of all data through nonlinear regression.
The complex of receptor (S)-9 with OctACTHNUGRTENUNG(aMan)bMan was
selected as a representative system and its structural fea-
tures were studied at 508C in CDCl3/[D7]DMF 60:40 by
NMR techniques. Under these conditions, a logb value of
(2.51ꢀ0.04) was measured for the 1:1 complex from a titra-
tion of the glycoside with (S)-9, which would cause nearly
80% of complex to be formed at a concentration of diman-
noside and receptor of 5 and 15 mm, respectively, the free
reactants accounting for the rest of the species in solution.
A further issue was the severe overlap of the saccharide
signals with those of the receptor. This issue was addressed
by following the proton resonances along the titration
through HSQC 2D spectra, which deconvoluted the overlap-
ped signals over the 13C dimension and provided chemical
shift variations for all dimannoside signals. Signal assign-
ments were unambiguously obtained through HSQC, DQ-
COSY, and TOCSY 2D spectra. The chemical shifts of the
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Pyrrolic Receptors for the Molecular Recognition of Carbohydrates
FULL PAPER
Table 3. Cumulative formation constants[a] and intrinsic median binding concentra-
tion[b] for receptor to glycoside (R/G) complexes of octyl dimannosides in CDCl3/
DMF 60:40 obtained from the ITC data.
straints. The protocol returned a few families of
structures, only one of which was in agreement with
the observed chemical shift data (Figure S1 in the
Supporting Information). The lowest-energy
member of the family (eighteen structures were
found within an energy window of 6.1 kJmolꢁ1) was
let to relax in a single-point energy minimization
removing all constraints, resulting in the structure
reported in Figure 4 as the global minimum.
Several intermolecular hydrogen bonds between
pyrrolic/aminic NH groups of the receptor and OH
groups of the disaccharide could be found in the
structure of the complex, showing that both the
monosaccharidic units are interacting with the mon-
otopic subunits of the ditopic receptor. This feature
Receptor
Oct
N
OctACHTGNUTRENNUNG
logb (R/G)
logb (R/G)
(R)-9
(S)-9
(2.66ꢀ0.01) (1:1)
(4.88ꢀ0.08) (2:1)
(2.812ꢀ0.003) (1:1)
U
(2.253ꢀ0.004) (1:1)
ACHTUNGTRENNUNG
(3.426ꢀ0.007) (1:1)
(5.98ꢀ0.02) (2:1)
(3.20ꢀ0.01) (1:1)
(6.18ꢀ0.03) (2:1)
(4.036ꢀ0.008) (1:1)
(6.06ꢀ0.04) (2:1)
ACHTUNGTRENNUNG
(R)-12
(S)-12
(3.16ꢀ0.01) (1:1)
(5.82ꢀ0.02) (2:1)
(3.90ꢀ0.02) (1:1)
(7.12ꢀ0.09) (2:1)
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
[a] Measured by ITC titration experiments at T=298 K. Formation constants were ob-
tained by nonlinear least-square regression analysis of calorimetric data by simultane-
ous fit of combined titrations. [b] Calculated from the log b values using the “BC50
Calculator” program (see ref. [28]).
Table 4. Chemical shifts and chemical shift differences of the relevant
protons of Oct
ACHTUNGTRENNUNG(aMan)bMan free and OctHCATUNGTRNE(NUGN aMan)bMan bound to (S)-9 in
the 1:1 complex in CDCl3/[D7]DMF 60:40 at T=323 K.[a]
Nucleus[b]
d [ppm]
CSD [ppm]
free
bound
H-1a
H-3a
H-4a
H-5a
H-1
H-2
H-4
H-7’
H-7’
4.6719
3.1989
3.1796
3.5150
3.8998
3.4532
3.0821
3.2481
2.8692
4.1694
2.6143
1.4186
3.0325
3.5482
3.1216
2.5815
2.7940
2.3697
0.5025
0.5846
1.7610
0.4825
0.3516
0.3316
0.5006
0.4541
0.4995
Figure 3. Plot of the chemical shift differences between free Oct-
ACHUTNGREN(NUG aMan)bMan and OctCAHTUNGTRENN(UGN aMan)bMan bound to (S)-9 in the 1:1 complex in
CDCl3/[D7]DMF 60:40 at T=323 K.
[a] Chemical shifts were obtained by nonlinear regression of the experi-
mental data from titration of Oct(aMan)bMan with (S)-9. [b] Nuclei la-
beled with lower-case “a” belong to the aMan fragment of Oct-
ACHTUNGTRENNUNG(aMan)bMan.
AHCTUNGTRENNUNG
not only accounts for the observed high affinity of (S)-9 for
OctACTHUNRTGNEUNG(aMan)bMan, but also explains the preference for di-
mannosides shown by the ditopic receptors with respect to
their monotopic counterparts, therefore validating the mod-
ular design of our architectures.
An independent support to the calculated structure was
obtained by NOESY spectra, that were run under the
free and bound dimannoside obtained from the titration are
reported in Table 4; the corresponding chemical shift differ-
ences (CSD) are depicted in Figure 3.
It can be immediately seen that all protons experience an
upfield shift caused by the aromatic shielding effect, indicat-
ing the inclusion of the dimannoside in the cleft of the re-
ceptor; most remarkably, the H-4a proton, showing an out-
standing shift of nearly 2 ppm, which demonstrated its loca-
tion in close proximity to the aromatic moiety of one of the
two subunits of the receptor. This feature is analogous to
that observed for the (S)-1/OctaMan complex, the binding
mode of which was elucidated in a previous paper.[16b] This
evidence suggested that modeling calculations may take ad-
vantage of the latter structure as a starting point for a con-
above-described conditions (OctACHTGNUTERNNU(G aMan)bMan, 5 mm; (S)-9,
15 mm; CDCl3/[D7]DMF 60:40; T=323 K) (Figure 5). Inter-
molecular NOE cross peaks between the pyrrolic NH
groups of the receptor and the CH protons of the sacchari-
dic backbone were unambiguously detected in the 2D
NOESY map, demonstrating the inclusion of the disacchar-
ide in the receptor cleft. The interatomic distances calculat-
ed from the global minimum structure for the experimental-
ly observed NOE contacts reported in Table 5 are in good
agreement with a disposition of the a-mannosyl residue with
formational search on the (S)-9/OctACTHNUTRGNEUNG(aMan)bMan complex.
Table 5. Observed intermolecular NOE contacts and corresponding in-
teratomic distances calculated from the global minimum structure of the
The two dihedral angles about the a-glycosidic linkage, de-
termining the relative orientation of the two monosacchar-
ides, were also included among the constraints for the con-
formational search, by using the value reported in a study
on the dimannoside conformation.[23a] The conformational
search was then performed following a well-established mo-
lecular mechanics protocol (see the Supporting Informa-
tion),[31] imposing, in a first approximation, the above con-
complex of (S)-9 with Oct
ACHTUNGTRENNUNG
(S)-9
AHCTUNGTRENN(GUN aMan)bMan (interatomic distance [ꢂ])
NH-A
NH-B
NH-B
H-3a (3.80)
H-1a (3.02)
H-2a (3.54)
[a] From the 500 MHz NOESY spectrum of a 3:1 receptor-to-glycoside
mixture in CDCl3/[D7]DMF 60:40 at T=323 K.
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11749

S. Roelens et al.
Figure 4. Global minimum structure obtained from a search of the con-
formational space for the complex between (S)-9 and Oct(aMan)bMan.
AHCTUNGTRENNUNG
Hydrogen bonds involving pyrrolic and aminic NH groups are depicted
ꢁ
as dashed lines. Selected hydrogen bond length [ꢂ]: N H(B)···OH-2a
ꢁ
ꢁ
ꢁ
ꢁ
2.48, O H(2a)···NH 2.27, O H(3a)···NH 2.30, N H···OH-4a 2.27, N
ꢁ
ꢁ
ꢁ
H(A)···OH-3 2.15, N H···OH-3 2.37, N H(B)···OH-6 2.69, O H(6)···NH
2.74. Nuclei labeled with lower-case “a” indicate the aMan fragment of
OctACHTUNGTRENNUNG(aMan)bMan; NH-A/NH-B indicate the NH protons of the exocyclic
and endocyclic pyrroles, respectively; NH indicate the aminic NH pro-
tons (see Figure 5).
Figure 5. 500 MHz NOESY spectrum of a (S)-9 to OctACTHNURTGNEU(GN aMan)bMan 3:1
mixture in CDCl3/[D7]DMF 60:40 at T=323 K. Intermolecular NOEs be-
tween the NH-A and NH-B protons (the NH protons of the exocyclic
and endocyclic pyrroles, respectively) of (S)-9, and the H-1a, H-2a, and
H-3a protons of the dimannoside are indicated by solid squares. Intramo-
lecular NOEs of the receptor are indicated by dotted circles. A schematic
representation of the intermolecular NOEs found between (S)-9 and Oct-
its b face over the aromatic scaffold, which causes the ob-
served large shift of the H-4a proton, and confirms the con-
sistency between the calculated structure and the combined
NMR experimental data. The NOE cross peaks between the
AHCTUNGTRENN(GUN aMan)bMan is depicted on the bottom.
to new ditopic structures capable of recognizing biologically
relevant dimannosides with improved affinities with respect
to their monotopic counterparts. Among the structures pre-
pared, receptors 9 and 12 gave the most interesting results,
the (S) enantiomer of the former giving the highest selectivi-
bMan fragment of OctACTHNUGRTENUNG(aMan)bMan and the other subunit
of the receptor, which could be expected from the global
minimum structure, could not be identified unambiguously
because of the severe overlap of inter- and intramolecular
cross peaks from the reacting partners.
ty toward OctACTHUNTGRNEUNG(aMan)bMan, the latter showing affinities
Finally, further support to the calculated structure is pro-
vided by the NOE contact observed between the H-1a and
the H-2 protons of the dimannoside (Figure S2 in the Sup-
porting Information), which is in agreement with the dihe-
dral angles about the glycosidic linkage between the two
mannose units obtained from the global minimum structure.
We believe that the calculated structure gives a close de-
scription of the actual binding mode characterizing the mo-
lecular recognition of the dimannosides by ditopic amino-
pyrrolic receptors.
toward both dimannosides in the micromolar range even in
a very competitive polar solvent. In all cases, the (S) enan-
tiomer proved to be more effective than the (R) enantiomer,
demonstrating a distinct enantiodiscrimination of the diman-
nosides toward the ditopic structures.
A combined approach through NMR techniques and mo-
lecular modeling calculations to the complex between Oct-
AHCTUNGTREG(NNNU aMan)bMan and (S)-9 provided a description of the bind-
ing mode characterizing the recognition of the investigated
dimannosides by the newly designed ditopic receptors and
also provided a rationale for the observed preference for di-
mannosides exhibited by the ditopic receptors compared to
their monotopic counterparts.
Conclusion
Although highly polar and very competitive, the medium
used in this study (30–40% DMF in chloroform) is still
quite far from an aqueous medium, thus, admittedly, the ob-
served recognition properties were not detected under bio-
The results described in this paper have clearly shown that
bridging two units of an effective receptor for mannosides
through an appropriately designed linker does indeed lead
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Pyrrolic Receptors for the Molecular Recognition of Carbohydrates
FULL PAPER
[12] Y. Nakagawa, Y. Masuda, K. Yamada, T. Doi, K. Takegoshi, Y. Igar-
ashi, Y. Ito, Angew. Chem. 2011, 123, 6208–6212; Angew. Chem. Int.
Ed. 2011, 50, 6084–6088.
[13] a) J. Balzarini, Nat. Rev. Microbiol. 2007, 5, 583–597; b) C. N. Scan-
lan, J. Offer, N. Zitzmann, R. A. Dwek, Nature 2007, 446, 1038–
1045.
[14] H.-K. Lee, C. N. Scanlan, C.-Y. Huang, A. Y. Chang, D. A. Calarese,
R. A. Dwek, P. M. Rudd, D. R. Burton, I. A. Wilson, C.-H. Wong,
Angew. Chem. 2004, 116, 1018–1021; Angew. Chem. Int. Ed. 2004,
43, 1000–1003.
mimetic conditions. Yet, the binding abilities exhibited by
the ditopic receptors appear very promising for the future
development of biomimetic recognition in a physiological
medium. Considering that recognition of oligomannosides
by artificial receptors is not documented in the chemical lit-
erature, the structures reported in this work enriched the
carbohydrate recognition toolbox of hitherto unprecedented
biomimetic receptors for dimannosides.
[15] a) B. Sookcharoenpinyo, E. Klein, Y. Ferrand, D. B. Walker, P. R.
Brotherhood, C. Ke, M. P. Crump, A. P. Davis, Angew. Chem. Int.
Ed. 2012, 51, 4586–4590; b) Y. Jiao, J. Zhang, L. Zhang, Z. Lin, C.
He, C. Duan, Chem. Commun. 2012, 48, 6022–6024; c) H. Abe, Y.
Chida, H. Kurokawa, M. Inouye, J. Org. Chem. 2011, 76, 3366–
3371; d) M. Mazik, A. C. Buthe, Org. Biomol. Chem. 2009, 7, 2063–
2071; e) M. Mazik, A. C. Buthe, Org. Biomol. Chem. 2008, 6, 1558–
1568; f) Y. Ferrand, M. P. Crump, A. P. Davis, Science 2007, 318,
619–622; g) T. Reenberg, N. Nyberg, J. Ø. Duus, J. L. J. van Dongen,
M. Meldal, Eur. J. Org. Chem. 2007, 5003–5009; h) O. Rusin, K.
Lang, V. Krꢄl, Chem. Eur. J. 2002, 8, 655–663; i) A. S. Droz, U. Nei-
dlein, S. Anderson, P. Seiler, F. Diederich, Helv. Chim. Acta 2001,
84, 2243–2289.
[16] a) C. Nativi, O. Francesconi, G. Gabrielli, A. Vacca, S. Roelens,
Chem. Eur. J. 2011, 17, 4814–4820; b) A. Ardꢄ, F. J. CaÇada, C.
Nativi, O. Francesconi, G. Gabrielli, A. Ienco, J. Jimꢅnez-Barbero, S.
Roelens, Chem. Eur. J. 2011, 17, 4821–4829.
[17] In the present work, the R and S descriptors are used to designate
the receptors of all-R and all-S absolute configuration of the six ster-
eocenters, respectively.
Acknowledgements
High-field NMR spectra were acquired at the Magnetic Resonance
Center (CERM) facilities of the Universitꢀ di Firenze. We thank M.
Lucci for technical assistance. Ente Cassa di Risparmio di Firenze (Italy)
is acknowledged for granting an ITC nano calorimeter (grant no.
2009.0576).
[1] a) The Sugar Code: Fundamentals of Glycosciences (Ed.: H.-J.
Gabius), Wiley-VCH, Weinheim, 2009; b) T. K. Lindhorst, Essentials
of Carbohydrate Chemistry and Biochemistry, 3rd ed., Wiley-VCH,
Weinheim, 2007; c) Carbohydrates in Chemistry and Biology, Part I,
Vol. 2 (Eds.: B. Ernst, G. W. Hart, P. Sinaꢃ), Wiley-VCH, Weinheim,
2000; d) Carbohydrates in Chemistry and Biology, Part II, Vol. 4
(Eds.: B. Ernst, G. W. Hart, P. Sinaꢃ), Wiley-VCH, Weinheim, 2000.
[2] a) M. Mazik, RSC Adv. 2012, 2, 2630–2642; b) S. Jin, Y. Cheng, S.
Reid, M. Li, B. Wang, Med. Res. Rev. 2010, 30, 171–257; c) D. B.
Walker, G. Joshi, A. P. Davis, Cell. Mol. Life Sci. 2009, 66, 3177–
3191; d) A. P. Davis, Org. Biomol. Chem. 2009, 7, 3629–3638; e) M.
Mazik, Chem. Soc. Rev. 2009, 38, 935–956; f) S. Kubik, Angew.
Chem. 2009, 121, 1750–1753; Angew. Chem. Int. Ed. 2009, 48, 1722–
1725; g) A. P. Davis T. D. James in Functional Synthetic Receptors
(Eds.: T. Schrader, A. D. Hamilton), Wiley-VCH, Weinheim, 2005,
pp. 45–109.
[3] Carbohydrates in Chemistry and Biology, Part II, Vol. 4 (Eds.: B.
Ernst, G. W. Hart, P. Sinaꢃ), Wiley-VCH, Weinheim, 2000, pp. 533–
609.
[4] S.-C. Cheng, L. A. B. Joosten, B.-J. Kullberg, M. G. Netea, Infect.
Immun. 2012, 80, 1304–1313.
[5] T. B. Geijtenbeek, S. J. Van Vliet, E. A. Koppel, M. Sanchez-Her-
nandez, C. M. Vandenbroucke-Grauls, B. Appelmelk, Y. Van Kooyk,
J. Exp. Med. 2003, 197, 7–17.
[6] a) T. B. Geijtenbeek, D. S. Kwon, R. Torensma, S. J. Van Vliet, G. C.
van Duijnhoven, J. Middel, I. L. Cornelissen, H. S. Nottet, V. N. Ke-
wal Ramani, D. R. Littman, C. G. Figdor, Y. Van Kooyk, Cell 2000,
100, 587–597; b) P. Y. Lozach, A. Amara, B. Bartosch, J. L. Virelizi-
er, F. Arenzana-Seisdedos, F. L. Cosset, R. Altmeyer, J. Biol. Chem.
2004, 279, 32035–32045.
[7] S.-K. Wang, P.-H. Liang, R. D. Astronomo, T.-L. Hsu, S.-L. Hsieh,
D. R. Burton, C.-H. Wong, Proc. Natl. Acad. Sci. USA 2008, 105,
3690–3695.
[8] R. Pejchal, K. J. Doores, L. M. Walker, R. Khayat, P.-S. Huang, S.-K.
Wang, R. L. Stanfield, J.-P. Julien, A. Ramos, M. Crispin, R. Depet-
ris, U. Katpally, A. Marozsan, A. Cupo, S. Maloveste, Y. Liu, R.
McBride, Y. Ito, R. W. Sanders, C. Ogohara, J. C. Paulson, T. Feizi,
C. N. Scanlan, C.-H. Wong, J. P. Moore, W. C. Olson, A. B. Ward, P.
Poignard, W. R. Schief, D. R. Burton, I. A. Wilson, Science 2011,
334, 1097–1103.
[9] C. A. Bewley, Structure 2001, 9, 931–940.
[10] S. Shahzad-ul-Hussan, E. Gustchina, R. Ghirlando, G. Marius Clore,
C. A. Bewley, J. Biol. Chem. 2011, 286, 20788–20796.
[11] H. Tanaka, H. Chiba, J. Inokoshi, A. Kuno, T. Sugai, A. Takahashi,
Y. Ito, M. Tsunoda, K. Suzuki, A. Takenaka, T. Sekiguchi, H.
Umeyama, J. Hirabayashi, S. Omura, Proc. Natl. Acad. Sci. USA
2009, 106, 15633–15638.
[18] B. Richichi, C. Nativi, L. Luzzatto, R. Notaro, G. LaMarca, Bioorg.
Chem. 2011, 39, 88–93.
[19] Y. Ito, T. Ogawa, Angew. Chem. 1994, 106, 1843; Angew. Chem. Int.
Ed. Engl. 1994, 33, 1765.
[20] I. Cumpstey, A. J. Fairbanks, A. J. Redgrave, Org. Lett. 2001, 3,
2371–2374.
[21] J. B. Love, J. A. Blake, C. Wilson, S. D. Reid, A. Novak, P. B. Hitch-
cock, Chem. Commun. 2003, 1682–1683.
[22] S. P. Mahanta, B. S. Kumar, S. Baskaran, C. Sivasankar, P. K. Panda,
Org. Lett. 2012, 14, 548–551.
[23] a) D. Chen, Y. Yao, Z. Wei, S. Zhang, P. Tu, B. Liu, M. Dong,
Comput. Theor. Chem. 2013, 1010, 45–52; b) T. Srikrishnan, M. S.
Chowdhary, K. L. Matta, Carbohydr. Res. 1989, 186, 167–175.
[24] C. Nativi, M. Cacciarini, O. Francesconi, G. Moneti, S. Roelens, Org.
Lett. 2007, 9, 4685–4688.
[25] K. Niikura, E. V. Anslyn, J. Chem. Soc. Perkin Trans. 2 1999, 2769–
2776.
[26] A. Vacca, C. Nativi, M. Cacciarini, R. Pergoli, S. Roelens, J. Am.
Chem. Soc. 2004, 126, 16456–16465.
[27] All investigated systems showed time-averaged signals for the free
and complexed species, due to fast exchange on the NMR spectro-
scopic time scale.
[28] BC50 Calculator is a computer program developed for PC platforms
and is available for free upon request from the corresponding
author. For a detailed description of the equations used by the pro-
gram for computing BC50 and BC050, see: A. Vacca, O. Francesconi,
S. Roelens, Chem. Rec., 2012, 12, 544–566.
[29] The BC050 (intrinsic median binding concentration) descriptor is de-
fined as the total concentration of receptor necessary for binding
50% of the ligand when the fraction of bound receptor is zero, that
is, when forming the first complex molecule. This value coincides
with the dissociation constant Kd for 1:1 complexes, whereas it can
be viewed as a “global” dissociation constant when more than one
complex is present in solution. For a comprehensive account of the
descriptor, see: A. Vacca, O. Francesconi, S. Roelens, Chem. Rec.
2012, 12, 544–566.
[30] It should be emphasized that thermodynamic parameters are even
more severely affected than binding constants by
a
simplified
Chem. Eur. J. 2013, 19, 11742 – 11752
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S. Roelens et al.
model, and should therefore be taken with great care (see the Sup-
porting Information, Table S1).
Mari, F. J. CaÇada, J. Jimꢅnez-Barbero, A. Bernardi, G. Marcou, I.
Motto, I. Velter, F. Nicotra, B. La Ferla, Eur. J. Org. Chem. 2006,
2925–2933; d) S. Mari, H. Posteri, G. Marcou, D. Potenza, F. Mi-
cheli, F. J. CaÇada, J. Jimꢅnez-Barbero, A. Bernardi, Eur. J. Org.
Chem. 2004, 5119–5225.
[31] For examples of application of NMR techniques assisted by molecu-
lar modeling, see: a) J. Jimꢅnez-Barbero, E. Dragoni, C. Venturi, F.
Nannucci, A. Ardꢄ, M. Fontanella, S. Andrꢅ, F. J. CaÇada, H. J.
Gabius, C. Nativi, Chem. Eur. J. 2009, 15, 10423–10431; b) M. Ko-
lympadi, M. Fontanella, C. Venturi, S. Andrꢅ, H. J. Gabius, J. Jimꢅ-
nez-Barbero, P. Vogel, Chem. Eur. J. 2009, 15, 2861–2873; c) S.
Received: December 3, 2012
Published online: July 9, 2013
11752
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 11742 – 11752