Molecular recognition of N-acetylneuraminic acid with acyclic benzimidazolium- and aminopyridine/guanidinium-based receptors

Article abstract of DOI:10.1021/jo061901e

(Chemical Equation Presented) Acyclic receptors incorporating neutral and cationic recognition sites show effective binding of N-acetylneuraminic acid (Neu5Ac), the most naturally abundant sialic acid, in highly competitive solvents such as dimethyl sulfo

Full text of DOI:10.1021/jo061901e

Molecular Recognition of N-Acetylneuraminic Acid with Acyclic
Benzimidazolium- and Aminopyridine/guanidinium-Based Receptors
Monika Mazik* and Hu¨seyin Cavga
Institut fu¨r Organische Chemie der Technischen UniVersita¨t Braunschweig, Hagenring 30,
38106 Braunschweig, Germany
m.mazik@tu-bs.de
ReceiVed September 14, 2006
Acyclic receptors incorporating neutral and cationic recognition sites show effective binding of
N-acetylneuraminic acid (Neu5Ac), the most naturally abundant sialic acid, in highly competitive solvents
such as dimethyl sulfoxide (DMSO) and water/DMSO. Receptors 6b and 7b are able to form neutral/
charge-reinforced hydrogen bonds and ion pairs with Neu5Ac, similar to sialic acid-binding proteins.
Syntheses and binding properties of the artificial receptors are discussed.
Introduction
that possess a relatively simple, acyclic structure and that are
expected to form complexes with carbohydrates through neutral
and charge-reinforced hydrogen bonds in combination with
interactions between the faces of the sugar and the aromatic
rings of the receptor (similar to interactions found in the crystal
structures of sugar-binding proteins3). The acyclic scaffold
provides simplicity in the synthetic plan for many modifications
of the receptor structure, supplying a base for systematic studies
toward recognition motifs for carbohydrates.
In this study, we focused on the interactions of acyclic
benzimidazolium- and aminopyridine/guanidinium-based recep-
tors (7b and 6b, respectively) with N-acetylneuraminic acid (for
R- and â-anomers, see formulas 1R and 1â), which is the most
common occurring sialic acid, in competitive media like
dimethyl sulfoxide (DMSO) or water/DMSO. N-Acetylneuramin-
ic acid (Neu5Ac) and Neu5Ac-containing ligands play a key
The molecular recognition of carbohydrates by artificial
receptors remains an important challenge in artificial receptor
chemistry (for reviews on carbohydrate recognition with arti-
ficial receptors, see ref 1). As part of our program aimed at the
development of receptor molecules for neutral and ionic sugars,
we have already reported a series of receptors for neutral
carbohydrates,2 which are particularly challenging substrates to
recognize. Our interest in this area concentrates on receptors
* To whom correspondence should be addressed: phone +49-531-391-5266;
fax +49-531-391-8185.
(1) (a) Davis, A. P.; James, T. D. In Functional Synthetic Receptors;
Schrader, T., Hamilton, A. D., Eds.; Wiley-VCH: Weinheim, Germany,
2005; pp 45-109. (b) Davis, A. P.; Wareham, R. S. Angew. Chem., Int.
Ed. 1999, 38, 2979-2996. (c) Armitage, B. A. Bioinspired organic
chemistry. Annu. Rep. Prog. Chem., Sect. B: Org. Chem. 2002, 98, 503-
541. (d) For a review on boronic acid-based receptors, see James, T. D.;
Shinkai, S. Top. Curr. Chem. 2002, 218, 159-200. (e) Striegler, S. Curr.
Org. Chem. 2003, 7, 81-102.
(3) (a) Lis, H.; Sharon, N. Lectins; Kluwer Academic Publishers:
Dordrecht, The Netherlands, 2003. (b) Weiss, W. I.; Drickamer, K. Annu.
ReV. Biochem. 1996, 65, 441-473. (c) Lis, H.; Sharon, N. Chem. ReV. 1998,
98, 637-674. (d) Quiocho, F. A. Pure. Appl. Chem. 1989, 61, 1293-1306.
(e) Lemieux, R. U. Chem. Soc. ReV. 1989, 18, 347-374. (f) Quiocho,
F. A.; Wilson, D. K. Nature 1989, 340, 404-408. (g) Vyas, N. K.; Vyas,
M. N.; Quiocho, F. A. Science 1988, 242, 1290-1295. (h) Ravishankar,
R.; Ravindran, N.; Suguna, A.; Surolia, A.; Vijayan, M. Curr. Sci. 1997,
72, 855. (i) Spurlino, S., P.; Lu, G.-Y.; Quiocho, F. A. J. Biol. Chem. 1991,
266, 5202-5219.
(2) (a) Mazik, M.; Cavga, H. J. Org. Chem. 2006, 71, 2957-2963.
(b) Mazik, M.; Kuschel, M.; Sicking, W. Org. Lett. 2006, 8, 855-858.
(c) Mazik, M.; Cavga, H.; Jones, P. G. J. Am. Chem. Soc. 2005, 127, 9045-
9052. (d) Mazik, M;, Radunz, W.; Boese, R. J. Org. Chem. 2004, 69, 7448-
7462. (e) Mazik, M., Sicking, W. Tetrahedron Lett. 2004, 45, 3117-3121.
(f) Mazik, M;, Bandmann, H.; Sicking, W. Angew. Chem., Int. Ed. 2000,
39, 551-554. (g) Mazik, M.; Sicking, W. Chem. Eur. J. 2001, 7, 664-
670. (h) Mazik, M.; Radunz, W.; Sicking, W. Org. Lett. 2002, 4, 4579-
4582. (i) Mazik, M.; Ko¨nig, A. J. Org. Chem. 2006, 71, 7854-7857.
10.1021/jo061901e CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/09/2007
J. Org. Chem. 2007, 72, 831-838
831

Mazik and Cavga
FIGURE 1. Examples of neutral/charge-reinforced hydrogen bonds
and ion pairing in the crystal structures formed between Neu5Ac-
containing ligands and different proteins: (a, b) methyl R-sialoside/
rhesus rotavirus hemagglutinin;3a,5c (c, d) NeuAc(R2-3)Galâ4Glc/
polyoma virus;3c,5b (e) NeuAc/influenza neuraminidase complex.5f,g
In the case of receptors 6b and 7b, both the neutral and ionic
hydrogen bonds, as well as ion pairing, were expected to
stabilize the complexes with Neu5Ac, similar to sugar-binding
proteins. Furthermore, the participation of the central phenyl
ring of 6b and 7b in CH···π interactions with sugar CHs was
expected to provide additional stabilization of the receptor-
sugar complexes. The formation of the above-mentioned
interactions was also indicated by molecular modeling studies
(Figures 2-5); examples of binding motifs found by molecular
modeling are shown in Figures 3 and 5.
In the design of artificial receptors for anions, the guanidinium
and amidinium groups have proved to be very popular motifs;
both groups are strongly basic and remain protonated over a
wide pH range (pKa values typically range between 11 and 13).
Several excellent reviews exist in the literature covering the
use of artificial guanidinium- and amidinium-containing receptor
systems in supramolecular chemistry.6 In the area of sugar
recognition, these units have mostly been incorporated into
different boronic acid-based receptors.1d,7
role in a variety of biological processes,3a,4 including different
cellular recognition processes. N-Acetylneuraminic acid is
known to be overexpressed on the cell surface of tumor cells;
furthermore, sialic acids are frequently used as a recognition
unit in influenza viruses,3a including H5N1 influenza A viruses.4b
The biological recognition processes involving sialoglycocon-
jugates use both neutral and charge-reinforced hydrogen bonds,
as well as ion pairing for sugar binding; some examples of these
interactions are shown in Figure 1.
The analyses of the crystal structures of the complexes formed
between the Neu5Ac-containing ligands and sialic acid-binding
lectins showed that, with the exception of polyoma virus (see
Figure 1c), the carboxylate moiety of Neu5Ac interacts with
the main-chain amide groups, polar side chains (especially
serine, see Figure 1a), and ordered water molecules rather than
fully charged side chains.3a,b,5a-c In contrast, formation of ion
pairs with the Neu5Ac carboxylate (Figure 1e) appears to be a
common feature of neuraminidases.3b,5d-f In the complexes of
sialic acid-binding lectins, the glycerol side chain of Neu5Ac
is often involved in charge-reinforced hydrogen bonds with
positively charged amino acids, such as an arginine side chain,
as shown in Figure 1b. The acetamido moiety of Neu5Ac is
also frequently a significant recognition determinant (for
example, see Figure 1d).
(6) (a) Hartley, J. H.; James, T. D.; Ward, C. J. J. Chem. Soc., Perkin
Trans. 1 2000, 3155-3184. (b) Houk, R. J. T.; Tobey, S. L.; Anslyn, E. V.
Top. Curr. Chem. 2005, 255, 199-229. (c) Schmidtchen, F. P., Berger, M.
Chem. ReV. 1997, 97, 1609-1646. (d) Fitzmaurice, R. J.; Kyne, G. M.;
Douheret, D.; Kilburn, J. D. J. Chem. Soc., Perkin Trans. 1 2002, 841-
864. (e) Kubik, S.; Reyheller, C.; Stu¨we, S. J. Inclusion Phenom.
Macrocyclic Chem. 2005, 52, 137-187. (f) Yoon, J.; Kim, S. K.; Singh,
N. J.; Kim, K. S. Chem. Soc. ReV. 2006, 35, 355-360. (g) Best, M. D.;
Tobey, S. L.; Anslyn, E. V. Coord. Chem. ReV. 2003, 240, 3-15.
(7) (a) Wiskur, S. L.; Lavigne, J. J.; Metzger, A.; Tobey, S. L.; Lynch,
V.; Anslyn, E. V. Chem.sEur. J. 2004, 10, 3792-3804. (b) Segura, M.;
Alcazar, V.; Prados, P.; de Mendoza, J. Tetrahedron 1997, 53, 13119-
13128. (c) Yang, W.; Yan, J.; Fang, H.; Wang, B. Chem. Commun. 2003,
792-793. (d) For a receptor operating through noncovalent interactions,
see Schmuck, C.; Schwegmann, M. Org. Lett. 2006, 8, 1649-1652.
(8) (a) Otsuka, H.; Uchimura, E.; Koshino, H.; Okano, T.; Kataoka, K.
J. Am. Chem. Soc. 2003, 125, 3493-3502. (b) Djanashvili, K.; Frullano,
L.; Peters, J. A. Chem.sEur. J. 2005, 11, 4010-4018. (c) Yamamoto, M.;
Takeuchi, M.; Shinkai, S. Tetrahedron 1998, 54, 3125-3140. (d) He, M.;
Johnson, R. J.; Escobedo, J. O.; Beck, P. A.; Kim, K. K.; St. Luce, N. N.;
Davis, C. J.; Lewis, P. T.; Fronczek, F. R.; Melancon, B. J.; Mrse, A. A.;
Treleaven, W. D.; Strongin, R. M. J. Am. Chem. Soc. 2002, 124, 5000-
5009. (e) Zhang, T.; Anslyn, E. V. Org. Lett. 2006, 8, 1649-1652.
(4) (a) Schauer, R.; Schukla, A. K.; Schroder, C.; Muller, E. Pure. Appl.
Chem. 1984, 56, 907-921. (b) Shinya, K.; Ebina, M.; Yamada, S.; Ono,
M.; Kasai, N.; Kawaoka, Y. Nature 2006, 440, 435-436.
(5) (a) Watowich, S. J.; Skehel, J. J.; Wiley, D. C. Structure 1994, 2,
719-731. (b) Stehle, T.; Yan, Y.; Benjamin, T. L.; Harrison, S. C. Nature
1994, 369, 160-163. (c) Dormitzer, P. R.; Sun, Z. Y.; Wagner, G.; Harrison,
S. C. EMBO J. 2002, 21, 885-897. (d) Varshese, J. N.; Laver, W. G.;
Colman, P. M. Nature 1983, 303, 35-44. (e) Crennell, S.; Garman, E.;
Laver, G.; Vimr, E.; Taylor, G. Structure 1994, 2, 535-544. (f) Varghese,
J. N.; McKimm-Breschkin, J.; Caldwell, J. B.; Kortt, A. A.; Colman, P. M.
Proteins: Struct., Funct., Genet. 1992, 14, 327-332. (g) Sears, P.; Wong,
C.-H. Angew. Chem., Int. Ed. 1999, 38, 2300-2324.
832 J. Org. Chem., Vol. 72, No. 3, 2007

Recognition of Neu5Ac by Acyclic Receptors
FIGURE 2. Energy-minimized structures of the 1:1 and 1:2 complexes formed between receptor 6b and Neu5Ac 1â (MacroModel v. 6.5, Amber*
force field, Monte Carlo conformational searches, 50 000 steps). (a) Two different representations of the 1:1 receptor/sugar complex. (b) Space-
filling representations of the 1:2 receptor/sugar complex. Color code: receptor C, blue; receptor N, green; the sugar molecules are highlighted in
yellow or orange.
Some representatives of boronic acid-based receptors, using
covalent interactions for sugar binding, were developed for the
recognition of N-acetylneuraminic acid.8 Kataoka and co-
workers8a have studied the anomalous binding profile of
3-(propionamido)phenylboronic acid with Neu5Ac in aqueous
solution with varying pH. The molecular recognition of sialic
acid end groups by phenylboronates has been reported by Peters
and co-workers.8b Shinkai and co-workers8c have described a
FIGURE 3. Examples of neutral/charge-reinforced hydrogen bonds
and ion pairing found by molecular modeling studies of the 1:1 complex
between receptor 6b and Neu5Ac 1â (MacroModel v. 6.5, Amber*
receptor system that features two-point interactions of boronic
acid-diol complexation and Zn(II)-carboxylate coordination.
The interactions between Neu5Ac and xanthene dyes containing
force field, Monte Carlo conformational searches, 50 000 steps).
well-positioned boronic acid have been studied by Strongin and
co-workers.8d Zhang and Anslyn8e have explored the binding
properties of a cadmium-centered tris-boronic acid receptor
toward various anionic sugars, including N-acetylneuraminic
acid.
6-methylpyridine. The reaction of 3 with aqueous ammonia gave
the amino derivative 4, which was transformed into compound
5 by reaction with di-Boc-protected S-methylisothiourea in the
presence of triethylamine (see Scheme 1). The crystal structure
of 5 is shown in Figure 6; it should be noted that the three
arms of 5 point to the same face of the central phenyl ring,
while the ethyl groups point in the opposite direction. The
protective groups of 5 were removed with trifluoroacetic acid,
and the obtained salt was transformed into 6a, which was further
converted into the hydrochloride 6b. The trihydrochloride 7b
was prepared from 7a, which was synthesized by reaction of
benzene-1,3,5-tricarbonyl chloride with 2-aminomethylbenz-
imidazole in the presence of triethylamine.
Results and Discussion
Synthesis of the Receptors. The synthesis of 6b started from
1,3,5-tris(bromomethyl)-2,4,6-triethylbenzene,9 which was con-
verted into compound 3 via a reaction with 2 equiv of 2-amino-
(9) For examples of other systems based on triethylbenzene frame, see
(a) Stack, T. D. P.; Hou, Z.; Raymond, K. N. J. Am. Chem. Soc. 1993, 115,
6466. (b) Metzger, A.; Lynch, V. M.; Anslyn, E. V. Angew. Chem., Int.
Ed. 1997, 36, 862-865. (c) Niikura, K.; Metzger, A.; Anslyn, E. V. J. Am.
Chem. Soc. 1998, 120, 8533-8534. (d) Cabell, L. A.; Best, M. D.; Lavigne,
J. J.; Schneider, S. E.; Perreault, D. M.; Monahan, M.-K.; Anslyn, E. V.
J. Chem. Soc., Perkin Trans. 2 2001, 315-323. (e) Rekharsky, M.; Inoue,
Y.; Tobey, S.; Metzger, A.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 120,
8533-8534. (f) Wiskur, S. L.; Lavigne, J. J.; Metzger, A.; Tobey, S. L.;
Lynch, V.; Anslyn, E. V. Chem.sEur. J. 2004, 10, 3792-3804. (g) Kim,
S.-G.; Ahn, K. H. Chem.sEur. J. 2000, 6, 3399-3403. (h) Turner, D. R.;
Peterson, M. J.; Steed, J. W. J. Org. Chem. 2005, 71, 1598-1608. (i) Vacca,
A.; Nativi, C.; Cacciarini, M.; Pergoli, R.; Roelens, S. J. Am. Chem. Soc.
2005, 126, 16456-16465.
Binding Studies. The interactions of the receptors 6b and
7b, operating through noncovalent interactions, with Neu5Ac
were investigated by 1H NMR spectroscopy and microcalorim-
etry (isothermal titration calorimetry, ITC).
1H NMR Titrations. The 1H NMR binding titration data were
analyzed by use of the Hostest 5.610 and the HypNMR 2006
(10) Wilcox, C. S.; Glagovich, N. M. Program HOSTEST 5.6; University
of Pittsburgh: Pittsburgh, PA, 1994.
J. Org. Chem, Vol. 72, No. 3, 2007 833

Mazik and Cavga
FIGURE 4. Energy-minimized structure of the 1:3 complex (different representations) formed between receptor 7b and Neu5Ac 1â (MacroModel
v. 6.5, Amber* force field, Monte Carlo conformational searches, 50 000 steps). Color code: receptor C, blue; O, red; N, green; the sugar molecules
are highlighted in yellow and orange.
FIGURE 5. Examples of neutral/charge-reinforced hydrogen bonds and ion pairing found by molecular modeling studies of the 1:3 complex
between receptor 7b and Neu5Ac 1â (MacroModel v. 6.5, Amber* force field, Monte Carlo conformational searches, 50 000 steps).
by adding increasing amounts of the tetramethylammonium salt
of Neu5Ac (2)12 to a solution of the host 6b or 7b. In addition,
inverse titrations were performed in which the concentration
of 2 was held constant and that of receptor was varied. Neu5Ac
in solution is in an equilibrium between R- and â-pyranose forms
(structures 2R and 2â in the case of the tetramethylammonium
salt of Neu5Ac; for studies on mutarotation of Neu5Ac, see ref
13). The 1H NMR spectra14 showed that the complexes formed
with 6b and 7b include Neu5Ac predominantly in the â-form.
(12) Tetralkylammonium ions are commonly used as countercations in
FIGURE 6. Crystal structure of 5 (hydrogen-bonded ethanol molecule
is shown).
the binding studies of anions. For a recent discussion of the solvent and
countercation effects, see Sessler, J. L.; Gross, D. E.; Cho, W.-S.; Lynch,
V. M.; Schmidtchen, F. P.; Bates, G. W.; Light, M. E.; Gale, P. A. J. Am.
Chem. Soc. 2006, 128, 12281-12288.
(13) The anomeric configuration of Neu5Ac as a â-anomer is more stable
in solution, as demonstrated by the rapid mutarotation undergoing from
the R- to the â-anomer. For studies in water, see Friebolin, H.; Kunzelman,
P.; Supp, M.; Brossmer, R.; Keilich, G.; Ziegler, D. Tetrahedron Lett. 1981,
22, 1383-1386.
programs.11 The stoichiometry of the receptor-sugar complexes
was determined by mole ratio plots and by curve-fitting analysis
of the titration data. The titration experiments were carried out
(11) Gans, P. University of Leeds, Leeds, U.K., 2006.
834 J. Org. Chem., Vol. 72, No. 3, 2007

Recognition of Neu5Ac by Acyclic Receptors
1
FIGURE 7. Partial H NMR spectra (400 MHz; D2O/DMSO-d6, 1:9 v/v) of receptor 6b after addition of (from bottom to top) 0.00, 0.20, 0.40,
A,B
061, 0.82, 1.02, 1.33, 1.64, 1.95, 2.26, 2.56, 2.87, 3.28, 3.70, and 4.10 equiv of 2 ([6b] ) 0.90 mM). Shown are chemical shifts of the (a) CH2
,
(b) CH3C, and (c) pyridine CHD,E resonances (for labeling, see formula 6b).
SCHEME 1a
FIGURE 8. (a) Chemical shift changes observed for pyridine CHF of
6b during the titration of 6b with 2 in D2O/DMSO-d6 (1:9 v/v). The
[receptor]:[sugar] ratio is marked. (b) Chemical shift changes observed
for CH3 protons of 2 during the titration of 2 with 6b (inverse titration).
The [sugar]:[receptor] ratio is marked.
Complexation between receptor 6b and the tetramethylam-
monium salt of Neu5Ac (2) in DMSO-d6 and D2O/DMSO-d6
(1:9 v/v) was evidenced by several changes in the NMR spectra
D2O/DMSO-d6 mixtures (1:9 v/v) the association constants were
still at the upper limit of values, for which the 1H NMR
technique is meaningful (the binding constants were found to
be Ka1 ) 5.3 × 105 and Ka2 ) 3.7 × 104 M-1).15
(see, for example, Figure 7).15a,b The upfield shifts of the CH2 ,
B
CH3C (for labeling, see structure of 6b) and pyridine CH protons
of 6b were monitored as a function of sugar concentration
(typical titration curves are shown in Figures 8a and S1-1,
Supporting Information). The signals for the pyridine CH
protons moved in the range of 0.50-0.67 ppm, whereas those
for the CH2 and CH3 protons shifted in the range of 0.12-0.20
ppm. The pyridine CHs broaden during the titration and became
distinct near saturation, which occurred after the addition of 2
equiv of 2. The best fit of the titration data was obtained with
the mixed 1:1 and 1:2 receptor-sugar binding model; this model
was further supported by the mole ratio plots (see Figure S2a,
Supporting Information). Binding of 2 in DMSO proved to be
too strong to be followed quantitatively (Ka > 106 M-1).16 In
The 1H NMR spectra obtained during the titrations at constant
sugar concentration (inverse titrations) in DMSO-d6 showed
large shifting of the sugar hydroxyl resonances; however, the
strong broadening of these resonances prevented their use in
the estimation of the binding constants. Consequently, chemical
shift changes of the amide NH (see Figure S1-2a, Supporting
Information) and CH3 protons of 2 were monitored (downfield
shifts by 0.10 and 0.03 ppm, respectively; such shifts are
consistent with the binding motifs shown in Figure 3c). After
the addition of 0.5 equiv of 6b, almost no change was observed
in the NMR spectra (see Figure 8b). The best fit of the titration
data was obtained with the pure 1:2 receptor-sugar binding
model; however, the binding constant was too large to be
(14) For 1H and 13C NMR studies on Neu5Ac in D2O and DMSO-d6,
see ref 8a and Gervay, J.; Batta, G. Tetrahedron Lett. 1994, 35, 3009-
3012.
(15) (a) Dilution experiments show that receptor does not self-aggregate
in the used concentration range. (b) For each system at least four titrations
were carried out (for each titration 10-21 samples were prepared). (c) The
reproducibility of the Ka values was (10-25%. (d) Ka1 corresponds to the
1:1 association constant, Ka2 corresponds to the 1:2 receptor/sugar association
constant, and Ka3 corresponds to the 1:3 receptor/sugar association constant.
1
accurately determined by H NMR titrations. In the first part
of the inverse titration the sugar is the excess component and
the receptor is the minor component, thus, the 1:1 complex is
(16) For a review discussing the limitations of the NMR method, see
Fielding, L. Tetrahedron 2000, 56, 6151-6170.
J. Org. Chem, Vol. 72, No. 3, 2007 835

Mazik and Cavga
FIGURE 9. Partial 1H NMR spectra (400 MHz, DMSO-d6) of receptor 7b after addition of (from bottom to top) 0.00, 0.15, 0.31, 0.47, 0.63, 0.79,
0.95, 1.11, 1.59, 1.83, 2.07, 2.39, 2.79, and 3.10 equiv of 2 ([7b] ) 0.95 mM). Shown are chemical shifts of the (a) CH2, (b) benzimidazole CH,
(c) phenyl CH, and (d) amide NH resonances.
formed followed by the immediate formation of a 1:2 receptor-
sugar complex.
Complexation between receptor 7b and sugar 2 in DMSO-
d6 or water containing DMSO-d6 ([receptor]:[water] ) 1:1300;
H2O/DMSO 1:40 v/v) was evidenced by the upfield shift of
the receptor amide protons (0.50 ppm) as well as changes of
the chemical shifts of the CH2 and phenyl and benzimidazole
CH resonances (see Figure 9). The CH2 and phenyl CH signals
moved upfield by 0.25 and 0.17 ppm, respectively, whereas
those for benzimidazole CH protons shifted in the range of
0.25-0.35 ppm. After the addition of 3 equiv of 2, almost no
change was observed in the NMR spectra (see Figures 10a and
FIGURE 10. (a) Plot of the chemical shifts of the amide NH
S1-3, Supporting Information). The chemical shift changes
indicated that three binding processes occurred during the
titration. The best fit of the titration data was obtained with a
mixed 1:1, 1:2, and 1:3 receptor-sugar binding model (incor-
porated in the HypNMR 2006 program), indicating a very strong
association constant for 1:1 binding and weaker association
constants for 1:2 and 1:3 receptor-sugar complexes. However,
the binding constant for the first Neu5Ac molecule (Ka1) derived
from the model incorporating a final receptor-sugar stoichi-
ometry of 1:3 was too large (Ka1 > 106 M-1) to be accurately
resonances of 7b as a function of added 2 in DMSO-d6. The [receptor]:
[sugar] ratio is marked. (b) Plot of the chemical shifts of the CH3
resonances of 2 as a function of added 7b (inverse titration). The [sugar]:
[receptor]ratio is marked.
In the case of the inverse titrations at constant sugar
concentration, the addition of only 0.3 equiv of 7b led to
practically complete complexation of 2, indicating the formation
of 1:3 receptor-sugar complexes (the amide NH and CH3
signals of 2 were monitored; see Figures 10b and S1-2b,
Supporting Information). The 1:3 receptor-sugar binding stoi-
1
determined by H NMR titrations.
836 J. Org. Chem., Vol. 72, No. 3, 2007

Recognition of Neu5Ac by Acyclic Receptors
FIGURE 11. ITC titration of receptors 6b and 7b with Neu5Ac 2. (a) Isotherm for titration of 1.00 mM 6b with 10 µL aliquots of 6.18 mM 2 in
H2O/DMSO (1:9 v/v) at 25 °C. (b) Isotherm for titration of 0.70 mM 7b with 10 µL aliquots of 7.98 mM 2 in H2O/DMSO (1:9 v/v) at 25 °C. The
molar ratio of the sugar to the receptor is given (for thermodynamic parameters, see Tables 1 and 2).
TABLE 1. Results of ITC Titrations of 6b and 7b with 2 in H2O/DMSO (1:9 v/v) at 25 °Ca
â1
â2
â3
∆H1
∆H2
∆H3
T∆S1
T∆S2
T∆S3
6b‚2
7b‚2
1.5 × 105
4.6 × 109
-15.7
-12.6
-11.8
-18.7
13.8
17.7
43.4
39.0
2.0 × 105
11.9 × 109
13.4 × 1013
-22.6
58.5
a In the ligand binding program of Digitam, the equilibrium constants (âi) and the reaction enthalpies for the overall reaction are determined. â1 ) Ka1
(M-1); â2 ) Ka1Ka2 (M-2); â3 ) Ka1Ka2Ka3 (M-3). ∆Hi and T∆Si are given in kilojoules per mole. Errors in ∆H are less than 5%.
TABLE 2. Thermodynamic Parameters Evaluated by ITC Studies for 6b‚2 and 7b‚2 in H2O/DMSO (1:9 v/v) at 25 °Ca
Ka1b (∆G)
Ka2b (∆G)
Ka3b (∆G)
∆H1s
∆H2s
∆H3s
T∆S1s
T∆S2s
T∆S3s
6b‚2
7b‚2
1.5 × 105 (-29.5)
3.2 × 104 (-25.7)
-15.7
-12.6
3.9
-6.1
13.8
17.7
29.6
21.2
2.0 × 105 (-30.2)
6.1 × 104 (-27.3)
1.2 × 104 (-23.1)
-3.9
19.2
a ∆His, reaction enthalpies for the stepwise reaction. Ka is given in liters per mole; ∆G, ∆H, and T∆S are given in kilojoules per mole. Errors in Ka range
from 9% to 15%; Errors in ∆H are less than 5%. b See ref 15d.
chiometry was also indicated by mole ratio plots (see Figure
S2b, Supporting Information). A satisfactory fit of the titration
data to the 1:3 receptor-sugar binding model yielded Ka value
The microcalorimetric data showed that the binding processes
are exothermic and entropically favored (see Tables 1 and 2).
>106 M-1
.
Conclusions
Typical binding motifs found by molecular modeling studies
in the 1:3 complex between receptor 7b and Neu5Ac 1â are
shown in Figure 5, showing interactions between receptor and
sugar molecules (Figure 5a-c), as well as between the bound
sugars (for example, see Figure 5d).
Microcalorimetric Titrations. The microcalorimetric titra-
tions were carried out in a mixture of H2O/DMSO (1:9 v/v) at
298 K by use of a Thermometric titration calorimetric system
(Thermometric, Sweden). The reproducibility of the calorimeter
was checked with the complexation of Ba2+ by 18-crown-6. A
solution of Neu5Ac 2 (6-8 mM) was titrated into a 0.7-1 mM
solution of receptor 6b or 7b (see, for example, the caption for
Figure 11). The data obtained were analyzed by use of the
Digitam 4.1 software provided by Thermometric (heat of
dilution was corrected).
Receptors 6b and 7b, including neutral and cationic binding
sites, proved to be very effective receptors for N-acetyl-
neuraminic acid, the most naturally abundant sialic acid. As in
natural complexes, the charge-reinforced hydrogen bonds and
ion pairs seem to be the major driving force for receptor-
Neu5Ac association in highly competitive media.
According to molecular modeling studies, the neutral and
ionic recognition sites of 6b and 7b should be able to participate
in the following interactions: (i) ion pairing and ionic hydrogen-
bonding between the guanidinium/benzimidazolium residue of
the receptor and the carboxylate group of Neu5Ac (for example,
see Figures 3a, 5a and 5c); (ii) charge-reinforced hydrogen
bonds17 between the guanidinium/benzimidazolium residue of
the receptor and the acetamido/hydroxy groups of Neu5Ac (see
Figures 3a and 5a); (iii) neutral hydrogen bonds (including also
CH‚‚‚O/N hydrogen bonds) between the neutral recognition sites
of the receptor and the acetamido/hydroxy groups of Neu5Ac
In the case of 6b, the best fit of the titration data was obtained
with the mixed 1:1 and 1:2 receptor-sugar binding model. The
binding constants for 6b‚2 were found to be 1.5 × 105 M-1
(Ka1) and 3.2 × 104 (Ka2)15d M-1 and are of the same magnitude
as those determined by NMR spectroscopy in D2O/DMSO-d6
(1:9 v/v).
(17) The studies in the area of drug design found that a neutral-neutral
hydrogen bond is worth up to about 1.5 kcal mol-1, which is equivalent to
a maximum 15-fold increase in binding, whereas a hydrogen bond between
a charged and a neutral component can contribute up to 3000-fold to the
binding of a substrate (up to 4.7 kcal mol-1). See (a) Davis, A. M.; Teague,
S. J. Angew. Chem., Int. Ed. 1999, 38, 736-749. (b) Fersht, A. R.; Shi,
J.-P.; Knill-Jones, J.; Lowe, D. M.; Wilkinson, A. J.; Blow, D. M.; Brick,
P.; Carter, P.; Waye, M. M. Y.; Winter, G. Nature 1985, 314, 845-851.
(c) Williams, D. H.; Searle, M. S.; Mackay, J. P.; Gerhard, U.; Maplestone,
R. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1172-1178.
In the case of 7b, the microcalorimetric data indicated that
three binding processes occurred during the titration (similar
to NMR titrations in water containing DMSO-d6). The binding
constants derived from the model incorporating a final receptor-
sugar stoichiometry of 1:3 were found to be Ka1 ) 2.0 × 105
M-1, Ka2 ) 6.1 × 104 M-1, and Ka3 ) 1.2 × 104 M-1
.
J. Org. Chem, Vol. 72, No. 3, 2007 837

Mazik and Cavga
(see Figure 3b,c; the neutral binding sites of 7b consist of amide
groups as used by nature, whereas those of 6b comprise
aminopyridine moieties as heterocyclic analogues of the aspar-
agine/glutamine primary amide side chains); and (iv) CH···π
interactions between the CHs of Neu5Ac and the aromatic
groups of the receptor (similar to sugar-binding proteins, which
commonly place aromatic surfaces against patches of sugar CH
groups).
receptor-sugar complexes at higher Neu5Ac concentrations.
The mixed 1:1, 1:2, and 1:3 receptor-sugar binding model was
further supported by microcalorimetric titrations in H2O/DMSO
(1:9 v/v). The binding constants were found to be Ka1 ) 2.0 ×
105 M-1, Ka2 ) 6.1 × 104 M-1, and Ka3 ) 1.2 × 104 M-1 (see
Table 2). The microcalorimetric titrations revealed that the
enthalpy of binding is negative (apart from ∆H2s for 6b‚2; see
Table 2) and the binding processes are entropically favored (see
Tables 1 and 2).18 The 1H NMR spectra showed that the
complexes include Neu5Ac predominantly in the â-form. The
receptors 6b and 7b combine ease of synthesis and the ability
to form strong complexes with Neu5Ac in highly competitive
solvents.
1H NMR titrations at constant concentration of 6b suggested
the formation of complexes with 1:1 and 1:2 receptor/sugar
binding stoichiometry. Complexation between 6b and 2 in
DMSO-d6 proved to be too strong to be accurately determined
1
by H NMR titrations (Ka1 > 106 M-1). In a mixture of D2O/
DMSO-d6 (1:9 v/v), the binding constants Ka1 and Ka215d were
found to be in the range of 105 M-1 and 104 M-1, respectively.
The microcalorimetric titrations carried out in a mixture of H2O/
DMSO (1:9 v/v) verified the mixed 1:1 and 1:2 receptor/sugar
binding model. The values of the binding constants determined
by microcalorimetry (Ka1 ) 1.5 × 105 M-1 and Ka2 ) 3.2 ×
104 M-1; see Table 2) and the NMR spectroscopy are of the
same magnitude.
Acknowledgment. This work was supported by the Deutsche
Forschungsgemeinschaft. We thank Professor Dr. R. Boese and
Dipl.-Ing. D. Bla¨ser (Institut fu¨r Anorganische Chemie der
Universita¨t Duisburg-Essen) for performing the X-ray measure-
ments.
Supporting Information Available: Syntheses of compounds
1
3-7; H and 13C NMR spectra of compounds 3-5, 6b, and 7b;
examples of inverse titrations (1H NMR titration of 2 with receptors
6b and 7b); 1H NMR titrations of 6b or 7b with 2 (typical titration
curves); representative mole ratio plots; and X-ray data for
compound 5. This material is available free of charge via the
Internet at http://pubs.acs.org.
In the case of receptor 7b, the 1H NMR titrations in DMSO-
d6 indicated that very strong 1:1 complexes are formed (Ka1
>
106 M-1), followed by the formation of weaker 1:2 and 1:3
(18) For a discussion of the energetics of protein-carbohydrate interac-
tions, see Toone, E. J. Curr. Opin. Struct. Biol. 1994, 4, 719-728.
JO061901E
838 J. Org. Chem., Vol. 72, No. 3, 2007