Mimicking the binding motifs found in the crystal structures of protein-carbohydrate complexes: An aromatic analogue of serine or threonine side chain hydroxyl/main chain amide

Article abstract of DOI:10.1002/ejoc.200700295

An aromatic analogue of the side chain hydroxyl/main chain amide of Ser or Thr was used for the construction of artificial receptors for N-acetylneuraminic acid (Neu5Ac), the most common occurring sialic acid. The acyclic receptors, incorporating only neu

Full text of DOI:10.1002/ejoc.200700295

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200700295
Mimicking the Binding Motifs Found in the Crystal Structures of Protein–
Carbohydrate Complexes: An Aromatic Analogue of Serine or Threonine Side
Chain Hydroxyl/Main Chain Amide
Monika Mazik*[a] and Alexander König[a]
Keywords: Molecular Recognition / Receptors / Carbohydrates / Hydrogen Bonds / Supramolecular Chemistry
An aromatic analogue of the side chain hydroxyl/main chain
amide of Ser or Thr was used for the construction of artificial
receptors for N-acetylneuraminic acid (Neu5Ac), the most
common occurring sialic acid. The acyclic receptors, incorpo-
tive solvent, such as water-containing [D6]DMSO. Both re-
ceptors show remarkable selectivity for Neu5Ac over
glucuronic acid (GlcA).
D-
rating only neutral recognition sites, are able to bind Neu5Ac
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
–2
with an overall binding constant β2 of 105
M
in a competi-
idine- and pyrimidine-based receptors.[3f] In this study we
focused on mimicking the binding motifs shown in Fig-
ure 1a, b.
Introduction
The binding motifs observed in the crystal structures of
protein–carbohydrate complexes[1] provide much of the in-
spiration for the development of artificial carbohydrate re-
ceptors.[2] The protein–carbohydrate interactions involve
hydrogen bonding, van der Waals forces, interactions of
sugar CHs with aromatic residues of the protein, and metal
coordination. Quiocho et al. pointed out that “hydrogen
bonds are the main factors in conferring specificity and af-
finity to protein–carbohydrate interactions”.[1a] The hydro-
gen bonds have both neutral and ionic character, and are
both direct and water-mediated. Ion pairing and ionic hy-
drogen bonding are frequently observed in the complex-
ation of proteins with charged sugars, such as N-acetylneur-
aminic acid (3), which is the most commonly occurring
sialic acid. It should be noted that the design of both selec-
tive and effective carbohydrate receptors operating through
noncovalent interactions in competitive media still repre-
sents a significant challenge.
Figure 1. (a) Examples of charge-reinforced hydrogen bonds in the
complex between 2-α-O-methyl N-acetylneuraminic acid and rhe-
sus rotavirus hemagglutinin.[1e,4a] (b) Neutral hydrogen bonds in
the complex between Manα6(Manα3)Man and concanavaline
A.[1c,4b] (c) Aromatic analogue of the side chain hydroxyl/main
chain amide groups of Ser or Thr.
The analyses of the crystal structures of sialic acid bind-
ing lectins with bound N-acetylneuraminic acid (Neu5Ac)
or Neu5Ac-containing ligands showed that, with the excep-
tion of the polyoma virus, the carboxylate moiety of Ne-
u5Ac 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[1c,1d]
(in contrast, formation of ion pairs with the Neu5Ac car-
boxylate appears to be a common feature of neuraminid-
ases[1d]).
Our interest in the area of sugar recognition with artifi-
cial receptors concentrates on receptors that possess a rela-
tively simple, acyclic structure and that are expected to
complex uncharged carbohydrates through neutral and
charge-reinforced hydrogen bonds in combination with the
CH–π interactions between the sugar CHs and the aromatic
rings of the receptor.[3] Recently, we showed that binding
motifs (including cooperative and bidentate hydrogen
bonds) observed in the complexation of uncharged sugars
with lectins can be successfully mimicked with acyclic pyr-
The binding motif shown in Figure 1a was found, for
example, in the crystal structure of 2-α-O-methyl-N-acetyl-
neuraminic acid with rhesus rotavirus hemagglutinin.[1e,4a]
The noncovalent interactions shown in Figure 1a consist of
two charge-reinforced hydrogen bonds: one from the side
[a] Institut für Organische Chemie der Technischen Universität
Braunschweig,
Hagenring 30, 38106 Braunschweig
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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M. Mazik, A. König
SHORT COMMUNICATION
chain hydroxyl group of Ser190 to the carboxylate oxygen,
and one from the main chain amide functionality of Ser190
to the sugar carboxylate. Neutral hydrogen bonds formed
between sugar hydroxyl groups and the side chain hydroxyl
moiety of Ser or Thr, as well as the main chain amide func-
tionality of the same amino acid, could also be observed in
the crystal structures of protein–carbohydrate complexes. In
the crystal structure of concanavaline A with Manα6-
(Manα3)Man (see Figure 1b), for example, the α1-3-linked
mannose contributes to the binding of the trisaccharide by
hydrogen bonds between the 3- and 4-OH groups and the
hydroxyl moiety of Thr15, as well as between the 3-OH and
the main chain NH group of the same amino acid.[1c,4b]
The binding motifs shown in Figure 1a, b inspired the
design of an aromatic analogue of the side chain hydroxyl/
main chain amide groups of Ser or Thr, as shown in Fig-
ure 1c (recognition unit I). Artificial receptors consisting of
more than one recognition unit I were expected to complex
Neu5Ac (3) through multiple interactions, incorporating
charge-reinforced hydrogen bonds with the Neu5Ac carbox-
ylate and neutral hydrogen bonds with the sugar hydroxy
groups (2-OH, 4-OH, and the OHs of the glyceryl side
chain). Furthermore, interactions with the acetamido moi-
ety and the CH units of Neu5Ac were expected to provide
further stabilization of the receptor–sugar complexes. In the
complexes of sialic acid binding lectins, the glyceryl side
chain of Neu5Ac is often involved in hydrogen bonds; the
acetamido moiety of Neu5Ac is frequently a significant re-
cognition determinant.
Neu5Ac and Neu5Ac-containing ligands play a key role
in a variety of biological processes,[1e,4a,5] including different
cellular recognition processes. Specificity for Neu5Ac-con-
taining ligands is expressed by the hemagglutinins of nu-
merous viruses, notably of influenza (including H5N1 influ-
enza A viruses[5]), as well as several others, such as Sendai,
Newcastle disease, and polyoma viruses.[1e] Furthermore,
Neu5Ac is known to be overexpressed on the cell surface
of tumor cells. Thus, the design of artificial receptors for
the recognition of Neu5Ac may serve as a basis for the de-
velopment of new therapeutics or sensors.[6]
Figure 2. Energy-minimized structure of the (a) 1:1 complex be-
tween receptor 1 and Neu5Ac 3β (two different representations),
(b) 1:2 receptor/sugar complex between 1 and 3β (two different
representation), (c) 1:1 (left) and 1:2 (right) receptor/sugar complex
between 2 and 3β. (d) Examples of charge-reinforced hydrogen
bonds indicated by molecular modeling (MacroModel V.8.5,
OPLS-AA force field, MCMM, 50000 steps). Color code: receptor
C, blue; receptor N, green; receptor O, red; the sugar molecule is
highlighted in yellow or orange.
The interactions of receptors 1 and 2[7] with Neu5Ac
1
were investigated by H NMR spectroscopy in water-con-
taining [D6]DMSO ([receptor]/[H2O] ≈ 1:100; the solubility
behavior of 1 and 2 prevents the binding studies in both
pure water and nonpolar solvents, such as chloroform). The
binding properties of 1 and 2 towards Neu5Ac were com-
pared with those towards glucuronic acid (GlcA, 5), an an-
Results and Discussion
Molecular modeling calculations indicated that
a
3,3Ј,5,5Ј-tetrasubstituted diphenylmethane or a 3,3Ј,5,5Ј- ionic sugar actively participating in biotransformations and
tetrasubstituted biphenyl scaffold (see formula 1 and 2, detoxification processes of a number of endogenous com-
respectively) provides a cavity of the correct shape and size pounds, through hepatic glucuronidation.[8] The titration
for Neu5Ac encapsulation. According to the molecular experiments were carried out by adding increasing amounts
modeling studies, receptors 1 and 2, including four recogni- of the tetramethylammonium salt of Neu5Ac (for α- and β-
tion units I, should be able to form both 1:1 and 1:2 recep- anomer, see formulas 4α and 4β) or tetramethylammonium
tor/sugar complexes with Neu5Ac (see Figure 2).
-glucuronate (6) to a solution of receptor 1 or 2.[9] The 1H
Receptors 1 and 2 were prepared by the reaction of 2- NMR spectroscopic binding titration data were analyzed
amino-4-methylphenol (15) with 3,3Ј,5,5Ј-diphenylmeth- by using the Hostest 5.6 program.[10] Additionally, compar-
anetetracarbonyl (14) or 3,3Ј,5,5Ј-biphenyltetracarbonyl tet- ative binding studies were carried out with neutral sugars,
rachloride (22) (see Schemes 1 and 2, and the Supporting such as methyl β--glucopyranoside (7), methyl β--galac-
Information).
topyranoside (8), -maltose (9), and -lactose (10).
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Mimicking the Binding Motifs in Protein–Carbohydrate Complexes
SHORT COMMUNICATION
Scheme 1. a) 20% Oleum; b) CH3OH, HCl; c) 10% NaOH; d) 50% H2SO4; e) SOCl2, THF; f) 15 (9 equiv.), THF.
Scheme 2. a) NaNO2, 20% HCl, KI; b) PdCl2(dppf), KOAc/DMF, 80 °C, 2 h; c) 17 (2 equiv.), PdCl2(dppf), CsF, 80 °C, 12 h; d) 10%
NaOH; e) 50% H2SO4; f) SOCl2, THF; g) 15 (10 equiv.), THF.
During the titration of 1[11] with 4 (used as an anomeric plexation between 1 and 4 was further evidenced by chemi-
mixture), the signals due to the NH and OH protons of 1 cal shift changes of the CH units of 1 (in the range of 0.03–
shifted downfield with strong broadening of the peaks and 0.07 ppm; Figure 3a, b), which were monitored as a func-
they were almost unobservable after the addition of about tion of sugar concentration.[12] The best fit of the titration
1 equiv. of 4 (see Figure 3c), which indicates the important data was obtained with the mixed 1:1 and 1:2 receptor/
contribution of the OH and NH groups of 1 to the forma- sugar binding model (for comparison of the plots obtained
tion of the complex in the competitive solvent. The com- for the different binding models, see Figures S1a and S1b,
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M. Mazik, A. König
SHORT COMMUNICATION
Supporting Information); the binding constants were were found to be Ka1 = 930 –1 and Ka2 = 120 –1 (β2 =
found to be Ka1 = 140 –1 and Ka2 = 1450 –1 (β2
=
1.11ϫ105 –2).[11d]
2.03ϫ105 –2). Typical titration curves are shown in Fig-
The comparison of the overall binding constants β2 indi-
cates that diphenylmethane-based receptor 1 exhibits about
a twofold higher affinity for sugar 4 than biphenyl-based
receptor 2 in the competitive solvent (Figure 3).
ure 3d, e.
1
The H NMR binding studies between 1 or 2 and sugar
6 indicated that the interactions with GlcA are less favor-
able than those with Neu5Ac. The chemical shifts of the
OH, NH, and CH signals (see Figure 4) were affected
weakly (some CH signals almost did not move, ∆δ Ͻ
0.01 ppm) during the titrations with 6. The NH and OH
signals of 1 or 2 broaden during the titrations, but in con-
trast to the titrations with 4, were still observable, even after
the addition of 20 equiv. of 6. Among the CH signals, the
signal of the CH proton in the neighboring position to the
OH group of 1 or 2 shows the largest shift (∆δmax
=
0.018 ppm in the case of 2; in the case of 1 the changes
were smaller). However, the binding constants could not be
accurately determined on the basis of the small chemical
shift changes; the Hostest program indicated Ka
≈
100 –1 for 2·6 (the titration curve is shown in Figure S1c).
Thus, the 1H NMR spectroscopic binding studies suggested
high selectivity for Neu5Ac over GlcA (of a factor of about
1000 in the case of 2), which indicates an important contri-
bution of the glyceryl side chain and the acetamido moiety
of Neu5Ac to the formation of the complex, in line with
the observation in natural complexes.
Figure 3. (a–c) Partial 1H NMR spectra (400 MHz; water-contain-
ing [D6]DMSO) of receptor 1 after the addition of (from bottom
to top) 0.0–19 equiv. of 4 ([1] = 0.51 m). Shown are chemical
shifts of the phenyl CH (a, b), OH and NH (c) resonances of 1.
(d,e) Plots of the chemical shifts of the phenyl CHs of 1 as a func-
Figure 4. (a) Partial 1H NMR spectra (400 MHz; water-containing
[D6]DMSO) of receptor 1 after the addition of 0.0–19 equiv. of 6
([1] = 0.45 m). Shown are the phenyl CH signals of 1. (b,c) Partial
1H NMR spectra of receptor 2 after the addition of 0.0–19 equiv.
of 6 ([2] = 0.59 m). Shown are the phenyl CHs (b), NH and OH
signals (c) of 2.
1
tion of added 4. (f–h) Partial H NMR spectra of receptor 2 after
the addition of 0.0–21 equiv. of 4 ([2] = 0.50 m). Shown are chem-
ical shifts of the phenyl CH (f), biphenyl CH (g), OH and NH (h)
resonances of 2.
The comparative binding studies with neutral sugars 7–
Similar to 1, the NH and OH signals of receptor 2 10 showed that the interactions between the neutral binding
broaden during the titration with 4, and were almost unob- partners are unable to effectively compete with the interac-
servable after the addition of about 1 equiv. of 4 (see Fig- tions with the solvent molecules. The complexation-induced
1
ure 3h). Furthermore, the H NMR spectra showed small shifts observed after the addition of 20 equiv. of 7–10 into
changes in the chemical shifts of the CH protons of 2 (in the solution of 1 or 2 in water-containing [D6]DMSO were
the range of 0.02–0.05 ppm; Figure 3f, g). The spectro- very small (largest changes ≈ 0.015 ppm for NH/OH signals,
scopic changes indicated stronger 1:1 binding followed by ≈ 0.005 for CH signals of 1 or 2). It should be noted that
weaker association of the second sugar molecule. The best the spectral changes observed after the addition of disac-
fit of the titration data was obtained with the mixed 1:1 and charides 9 and 10 were more substantial than those ob-
1:2 receptor-sugar binding model; the binding constants served in the presence of monosaccharides 7 and 8. These
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Mimicking the Binding Motifs in Protein–Carbohydrate Complexes
SHORT COMMUNICATION
results indicate that neutral sugars need receptors contain-
ing both neutral and ionic recognition sites for effective
complexation in competitive media (see also ref.[3h]).
[1]
[2]
a) F. A. Quiocho, Pure Appl. Chem. 1989, 61, 1293–1306; b)
R. U. Lemieux, Chem. Soc. Rev. 1989, 18, 347–374; c) H. Lis,
N. Sharon, Chem. Rev. 1998, 98, 637–674; d) W. I. Weiss, K.
Drickamer, Annu. Rev. Biochem. 1996, 65, 441–473; e) H. Lis,
N. Sharon, Lectins, Kluwer Academic Publishers, Dordrecht,
The Netherlands, 2003.
For reviews on carbohydrate recognition with artificial recep-
tors, see: a) A. P. Davis, R. S. Wareham, Angew. Chem. 1999,
111, 3161–3179; Angew. Chem. Int. Ed. 1999, 38, 2979–2996;
b) A. P. Davis, T. D. James in Functional Synthetic Receptors
(T. Schrader, A. D. Hamilton, Eds.), Wiley-VCH, Weinheim,
Germany, 2005, p. 45–109; c) For a review on boronic acid
based receptors, see: T. D. James, S. Shinkai, Top. Curr. Chem.
2002, 218, 159–200.
a) M. Mazik, H. Bandmann, W. Sicking, Angew. Chem. 2000,
112, 562–565; Angew. Chem. Int. Ed. 2000, 39, 551–554; b) M.
Mazik, W. Sicking, Chem. Eur. J. 2001, 7, 664–670; c) M. Ma-
zik, W. Radunz, W. Sicking, Org. Lett. 2002, 4, 4579–4582; d)
M. Mazik, W. Radunz, R. Boese, J. Org. Chem. 2004, 69, 7448–
7462; e) M. Mazik, W. Sicking, Tetrahedron Lett. 2004, 45,
3117–3121; f) M. Mazik, H. Cavga, P. G. Jones, J. Am. Chem.
Soc. 2005, 127, 9045–9052; g) M. Mazik, A. König, J. Org.
Chem. 2006, 71, 7854–7857; h) M. Mazik, H. Cavga, J. Org.
Chem. 2006, 71, 2957–2963; i) M. Mazik, M. Kuschel, W. Sick-
ing, Org. Lett. 2006, 8, 855–858; j) M. Mazik, H. Cavga, J.
Org. Chem. 2007, 72, 831–838.
a) P. R. Dormitzer, Z. Y. Sun, G. Wagner, S. C. Harrison,
EMBO J. 2002, 21, 885–897; b) J. H. Naismith, R. A. Field, J.
Biol. Chem. 1996, 271, 972–976.
K. Shinya, M. Ebina, S. Yamada, M. Ono, N. Kasai, Y. Ka-
waoka, Nature 2006, 440, 435–436.
For examples of boronic acid based receptors for Neu5Ac
(using covalent interactions for sugar binding), see: a) H. Ot-
suka, E. Uchimura, H. Koshino, T. Okano, K. Kataoka, J.
Am. Chem. Soc. 2003, 125, 3493–3502; b) K. Djanashvili, L.
Frullano, J. A. Peters, Chem. Eur. J. 2005, 11, 4010–4018; c)
M. Yamamoto, M. Takeuchi, S. Shinkai, Tetrahedron 1998, 54,
3125–3140; d) M. He, R. J. Johnson, J. O. Escobedo, P. A.
Beck, K. K. Kim, N. N. S. t. Luce, C. J. Davis, P. T. Lewis, F. R.
Fronczek, B. J. Melancon, A. A. Mrse, W. D. Treleaven, R. M.
Strongin, J. Am. Chem. Soc. 2002, 124, 5000–5009; e) T. Zhang,
E. V. Anslyn, Org. Lett. 2006, 8, 1649–1652.
Conclusions
The analysis of the binding motifs in the protein–carbo-
hydrate complexes has inspired the design of an aromatic
analogue of the side chain hydroxyl/main chain amide
groups of Ser or Thr. This recognition unit (Figure 1c) was
used for the construction of artificial receptors for the com-
plexation of Neu5Ac, the most naturally abundant sialic
acid. Receptors 1 and 2 are able to form 1:2 receptor/sugar
complexes with Neu5Ac with the overall binding constant
β2 of 105 –2 in water-containing [D6]DMSO ([receptor]:
[water] ≈ 1:100). The affinity of 1 is about twofold higher
than that of 2. Binding of Neu5Ac is achieved through a
combination of neutral and charge-reinforced hydrogen
bonds as well as CH–π interactions.
Receptors 1 and 2 are able to distinguish between two
anionic sugars, Neu5Ac and GlcA, with high selectivity for
Neu5Ac over GlcA (of a factor of about 1000 in the case
of 2). Thus, acyclic receptors 1 and 2, which contain only
neutral recognition sites and operate through noncovalent
interactions, exhibit remarkable binding selectivity in the
competitive solvent.
[3]
[4]
[5]
[6]
Further improvement of the binding affinity is expected
with receptors constructed on the basis of recognition unit
II as the intermolecular interactions formed through such
receptors should compete more effectively with the intra-
molecular hydrogen bonds (see Ia, Ib, and II).
[7]
For examples of other carbohydrate receptors incorporating
phenolic hydroxy groups, see: a) H. Abe, Y. Aoyagi, M. Inouye,
Org. Lett. 2005, 7, 59–61; b) S. Anderson, U. Neidlein, V.
Gramlich, F. Diederich, Angew. Chem. 1995, 107, 1722–1726;
Angew. Chem. Int. Ed. Engl. 1995, 34, 1596–1600; c) A. Bähr,
A. S. Droz, M. Püntener, U. Neidlein, S. Anderson, P. Seiler,
F. Diederich, Helv. Chim. Acta 1998, 81, 1931–1963; d) T. Mi-
zutani, T. Kurahashi, T. Murakami, N. Matsumi, H. Ogoshi,
J. Am. Chem. Soc. 1997, 119, 8991–9001; e) Y. Aoyama, Y.
Tanaka, S. Sugahara, J. Am. Chem. Soc. 1989, 111, 5397–5404.
The synthesis of new receptors of this type and complex-
ation studies with different sugar molecules, including 2-α-
O-methyl Neu5Ac, which serves as a model for Neu5Ac
bound to a neighboring unit in glycoproteins,[6b,13] are in
progress.
The acyclic scaffold of the receptors provides simplicity
in the synthetic plan for many modifications to the struc-
ture of the receptor, which provides a basis for systematic
studies directed towards recognition motifs of carbo-
hydrates.
[8]
[9]
M. Segura, V. Alcazar, P. Prados, J. de Mendoza, Tetrahedron
1997, 53, 13119–13128, and references therein.
Tetralkylammonium ions are commonly used as countercations
in the binding studies of anions. For a recent discussion of the
solvent and countercation effects, see: J. L. Sessler, D. E. Gross,
W.-S. Cho, V. M. Lynch, F. P. Schmidtchen, G. W. Bates, M. E.
Light, P. A. Gale, J. Am. Chem. Soc. 2006, 128, 12281–12288.
´
[10]
[11]
C. S. Wilcox, N. M. Glagovich, Program HOSTEST 5.6; Uni-
versity of Pittsburgh, Pittsburgh, PA, 1994.
Supporting information (see footnote on the first page of this arti-
cle): Syntheses of 1 and 2; additional titration curves.
a) Dilution experiments show that receptors 1 and 2 do not
self-aggregate in the used concentration range; b) For each sys-
tem, at least four titrations were carried out; for each titration
15–21 samples were prepared; c) Errors in Ka are Ͻ15%; d)
The binding constants were determined at 25 °C. Ka1 corre-
sponds to the 1:1 association constant. Ka2 corresponds to the
1:2 receptor/sugar association constant.
Acknowledgments
Financial Support by the Deutsche Forschungsgemeinschaft is
gratefully acknowledged.
Eur. J. Org. Chem. 2007, 3271–3276
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M. Mazik, A. König
SHORT COMMUNICATION
[12] As noted by Schneider and Yatsimirsky, under properly chosen
conditions reliable association constants can be obtained even
if the maximally observed shift changes are Ͻ0.03 ppm, see:
H.-J. Schneider, A. Yatsimirsky, Principles and Methods in
Supramolecular Chemistry, John Wiley & Sons, Chichester,
2000.
charides: Their Synthesis and Biological Roles, Oxford Univer-
sity Press, New York, 2000; b) T. K. Lindhorst, Essentials of
Carbohydrate Chemistry and Biochemistry, Wiley-VCH,
Weinheim, Germany, 2000; c) R. Schauer, Angew. Chem. 1973,
85, 128–140; Angew. Chem. Int. Ed. Engl. 1973, 12, 127–138.
Received: April 3, 2007
[13] The naturally occurring oligosaccharides contain α-linked N-
acetylneuraminic acid, see: a) H. Osborn, T. Khan, Oligosac-
Published Online: May 29, 2007
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