Isopropylamino and isobutylamino groups as recognition sites for carbohydrates: Acyclic receptors with enhanced binding affinity toward β-galactosides

Article abstract of DOI:10.1021/jo100982x

Binding motifs observed in the crystal structures of protein-carbohydrate complexes, in particular the participation of the isopropyl/isobutyl side chain of valine/leucine in the formation of van der Waals contacts, have inspired the design of new artificial carbohydrate receptors. The new compounds, containing a trisubstituted triethylbenzene core, were expected to recognize sugar molecules through a combination of NH→O and OH→N hydrogen bonds, CH→π interactions, and numerous van der Waals contacts. ¹H NMR spectroscopic titrations in competitive and noncompetitive media, as well as binding studies in two-phase systems, such as dissolution of solid carbohydrates in apolar media and phase transfer of sugars from aqueous into organic solvents, revealed effective recognition of neutral carbohydrates and β- vs α-anomer binding preferences in the recognition o' glycosides as well as significantly increased binding affinity of the receptors toward β-galactoside in comparison with the previously described receptors.

Full text of DOI:10.1021/jo100982x

pubs.acs.org/joc
Isopropylamino and Isobutylamino Groups as Recognition Sites for
Carbohydrates: Acyclic Receptors with Enhanced Binding Affinity
toward β-Galactosides
Monika Mazik* and Claudia Sonnenberg
€
Institut fu€r Organische Chemie der Technischen Universitat Braunschweig, Hagenring 30, 38106
Braunschweig, Germany
m.mazik@tu-bs.de
Received May 19, 2010
Binding motifs observed in the crystal structures of protein-carbohydrate complexes, in particular
the participation of the isopropyl/isobutyl side chain of valine/leucine in the formation of van der
Waals contacts, have inspired the design of new artificial carbohydrate receptors. The new compounds,
containing a trisubstituted triethylbenzene core, were expected to recognize sugar molecules through
a combination of NH O and OH N hydrogen bonds, CH π interactions, and numerous
3 3 3
3 3 3
3 3 3
van der Waals contacts. 1H NMR spectroscopic titrations in competitive and noncompetitive media,
as well as binding studies in two-phase systems, such as dissolution of solid carbohydrates in apolar
media and phase transfer of sugars from aqueous into organic solvents, revealed effective recognition
of neutral carbohydrates and β- vs R-anomer binding preferences in the recognition of glycosides as
well as significantly increased binding affinity of the receptors toward β-galactoside in comparison
with the previously described receptors.
Introduction
Carbohydrate-protein interactions play a key role in a
wide range of biological processes.1,2a The structural basis for
selective carbohydrate recognition by carbohydrate-binding
proteins has been intensively investigated by X-ray crystallog-
raphy.2 It hasbeenshownthat selectivity is achievedthrougha
combination of hydrogen bonding to the sugar hydroxyl
groups with hydrophobic packing, often including CH-π
interactions between the sugar CH groups and aromatic
FIGURE 1. Examples of hydrogen bonds and van der Waals
contacts in the complex of Galanthus Nivalis Agglutinin with
ManR3(ManR6)Man.2a,f
(1) (a) Osborn, H.; Khan, T. Oligosaccharides: Their Synthesis and
Biological Roles; Oxford University Press: New York, 2000. (b) Lindhorst,
T. K. Essentials of Carbohydrate Chemistry and Biochemistry; Wiley-VCH:
Weinheim, Germany, 2007. (c) Dwek, R. A.; Butters, T. D. Chem. Rev. 2002,
102, 283–284. (d) Gabius, H. J.; Siebert, H. C.; Andre, S.; Jimenez-Barbero,
J.; Rudiger, H. ChemBioChem 2004, 5, 741–764. (e) Bertozzi, C. R.; Kiessling,
L. L. Science 2001, 291, 2357–2364.
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Netherlands, 2003. (b) Lis, H; Sharon, N. Chem. Rev. 1998, 98, 637–674.
(c) Quiocho, F. A. Pure Appl. Chem. 1989, 61, 1293–1306. (d) Weiss, W. I.;
Drickamer, K. Annu. Rev. Biochem. 1996, 65, 441–473. (e) Lemieux, R. U.
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1996, 4, 1339–1352.
amino acid side chains, such as indole of tryptophan and
phenyl group of phenylalanine. Furthermore, numerous van
der Waals contacts, involving all the atoms of the bound
saccharides, were shown to contribute significantly to the
binding affinity and selectivity.2c Examples of hydrogen-
bonding interactions and van der Waals contacts in a protein-
carbohydrate complex are shown in Figure 1. The participation
6416 J. Org. Chem. 2010, 75, 6416–6423
Published on Web 09/09/2010
DOI: 10.1021/jo100982x
r
2010 American Chemical Society

Mazik and Sonnenberg
JOCArticle
FIGURE 2. Structures of receptors 1-5.
FIGURE 3. Structures of sugars investigated in this study.
of the primary amide group of asparagine and the isopropyl
group of valine (see Figure 1) in the formation of hydrogen
bonds and van der Waals contacts, respectively, has inspired the
design of artificial receptor3-5 1 (see Figure 2), which was
expected to be able to recognize a sugar molecule through a
combination of hydrogen bonding, CH-π interactions,6 and
van der Waals contacts. Instead of the primary amide group
shown in Figure 1, we have used the 2-aminopyridine unit, which
can be regarded as a heterocyclic analogue of the asparagine/
glutamine primary amide side chain and was shown to be an
effective recognition group for carbohydrates.7 The binding
properties of 1 toward selected monosaccharides (see Figure 3)
were compared with those of compounds 2-5shown in Figure 2.
1H NMR spectroscopic titrations in competitive and non-
competitive media, as well as binding studies in two-phase
(3) For reviews on carbohydrate recognition with artificial receptors
using noncovalent interactions, see: (a) Davis, A. P.; James, T. D. In
Functional Synthetic Receptors; Schrader, T., Hamilton, A. D., Eds.,
Wiley-VCH: Weinheim, Germany, 2005. (b) Davis, A. P.; Wareham, R. S.
Angew. Chem. 1999, 111, 3161-3179; Angew. Chem., Int. Ed., 1999, 38,
2979-2996. (c) Walker, D. B.; Joshi, G.; Davis, A. P. Cell. Mol. Life Sci.
2009, 66, 3177–3191. (d) Mazik, M. Chem. Soc. Rev. 2009, 38, 935–956.
(e) Jin, S.; Cheng, Y.; Reid, S.; Li, M.; Wang, B. Med. Res. Rev. 2010, 30, 171–257.
(4) For examples of carbohydrate receptors operating through noncova-
lent interactions see refs 3 and 7 and: (a) Ferrand, Y.; Klein, E.; Barwell,
(5) For reviews on boronic acid-based receptors, which use covalent
interactions for sugar binding, see refs 3a,3e and: (a) James, T. D.; Shinkai,
S. Top. Curr. Chem. 2002, 218, 159–200. (b) James, T. D.; Sandanayake, K. R. A.
S.; Shinkai, S. Angew. Chem. 1996, 108, 2038-2050; Angew. Chem., Int. Ed.
1996, 35, 1910-1922.For a recent example of a boronic acid-based receptor, see:
(c) Phillips, M. D.; Fyles, T. M.; Barwell, N. P.; James, T. D. Chem. Commun.
2009, 6557–6559. For a recent discussion on o-hydroxymethyl phenylboronic
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D. G. J. Org. Chem. 2008, 73, 6471–6479.
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(6) For recent discussions on the nature of the CH-π interactions, see:
(a) Nishio, M.; Umezawa, Y.; Honda, K.; Tsuboyama, S.; Suezawa, H.
CrystEngComm 2009, 11, 1757–1788. (b) Takahashi, O; Kohno, Y.; Nishio,
M. Chem. Rev. 2010, DOI: 10.1021/cr100072x.
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Yamamoto, T.; Inouye, M. Chem. Commun. 2009, 2121–2123. (d) Abe, H.;
Horii, A.; Matsumoto, S.; Shiro, M.; Inouye, M. Org. Lett. 2008, 10, 2685–2688.
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9573. (f) Klein, E.; Ferrand, Y.; Auty, E. K.; Davis, A. P. Chem. Commun. 2007,
2390–2392. (g) Nativi, C.; Cacciarini, M.; Francesconi, O.; Moneti, G.; Roelens,
S. Org. Lett. 2007, 9, 4685–4688. (h) Ferrand, Y.; Crump, M. P.; Davis, A. P.
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Mazik and Sonnenberg
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SCHEME 1. Synthesis of Compounds 1-5a
aKey: (a) AlCl3, CH3CH2Br, 0 °C to room temperature, 12 h (85%);9 (b) 33% HBr in CH3COOH, ZnBr2, (CH2O)n, 90 °C, 16 h (94%); (c) 2 equiv of
2-amino-4,6-dimethylpyridine, CH3CN/THF, K2CO3, room temperature, 3 days (20%); (d) 4 equiv of isopropylamine (14), THF/CH3CN, K2CO3,
room temperature, 2 days (75%); (e) 4 equiv of isobutylamine (15), THF/CH3CN, K2CO3, room temperature, 2 days (71%); (f) potassium phthalimide,
dimethyl sulfoxide, 95 °C, 8 h, (57%); (g) hydrazine hydrate, ethanol/toluene, reflux, 19 h, KOH (43%);7b (h) 3.3 equiv of isopropylamine (14), THF/
CH3CN, K2CO3, room temperature, 3 days (97%); (i) 3.3 equiv of isobutylamine (15) THF/CH3CN, K2CO3, room temperature, 3 days (96%).
systems, such as dissolution of solid carbohydrates in apolar
media and phase transfer of sugars from aqueous into organic
solvents, revealed both effective recognition of neutral carbohy-
drates and interesting binding preferences8 of the acyclic recep-
tors 1 and 2.
Binding Studies in Two-Phase Systems: Liquid-Solid and
Liquid-Liquid Extractions. Extractions of methyl pyrano-
sides, such as β-glucoside 6b, R-glucoside 7b, β-galactoside 8b,
and R-galactoside 9, from the solid state into a CDCl3 solution10
of receptor 1 or 2 (1 mM) provided evidence for stronger com-
plexation of the β-anomers 6b and 8b (see Table 1). The pre-
ference of 1 and 2 for β- vs R-glycoside indicated by liquid-solid
Results and Discussion
1
extractions was further confirmed by H NMR spectroscopic
Synthesis of the Receptors. The basis for the synthesis of
compounds 1-3 was 1,3-bis(bromomethyl)-5-[(4,6-dimethyl-
pyridin-2-yl)aminomethyl]-2,4,6-triethylbenzene (13).7b The
reaction of 13 with isopropylamine (14) or isobutylamine
(15) provided compounds 1 and 2, respectively. The treat-
ment of 13 with potassium phthalimide gave compound 16,
which was converted into 1,3-bis(aminomethyl)-5-[(4,6-di-
methylpyridin-2-yl)aminomethyl]-2,4,6-triethylbenzene (3)
via a reaction with hydrazine. Compounds 4 and 5 were
prepared through a reaction of 1,3,5-tris(bromomethyl)-2,4,
6-triethylbenzene with isopropylamine (14) or isobutylamine
(15), respectively. The synthesis of compounds 1-5 is sum-
marized in Scheme 1.
titrations (see below). The extraction experiments indicated that
the isopropylamino-based compound 1 is a more powerful
monosaccharide receptor than 2, containing isobutylamino
groups. In comparison to the receptors 1 and 2, the extraction
experiments indicated a lower level of affinity of compounds
3-5 toward the tested monosaccharides (see Table 1).
(9) Wallace, K. J.; Hanes, R.; Anslyn, E.; Morey, J.; Kilway, K. V.; Siegel,
J. Synthesis 2005, 2080–2083.
(10) The dissolution of solid carbohydrates in apolar media provides
valuable means of studying carbohydrate recognition by organic-soluble
receptors. For examples of receptors which are able to dissolve solid
carbohydrates in apolar media, see refs 3a, 4b, and 7a,7b,7p,7r and:
€
(a) Bahr, A.; Felber, B.; Schneider., K.; Diederich, F. Helv. Chim. Acta
2000, 83, 1346–1376. (b) Inouye, M.; Chiba, J.; Nakazumi, H. J. Org. Chem.
1999, 64, 8170–8176. (c) Inouye, M.; Miyake, T.; Furusyo, M.; Nakazumi, H.
J. Am. Chem. Soc. 1995, 117, 12416.
(8) For a discussion on selectivity in supramolecular host-guest complexes,
see: Schneider, H.-J.; Yatsimirsky, A. Chem. Soc. Rev. 2008, 37, 263–277.
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FIGURE 4. (a, c) Partial 1H NMR spectra (400 MHz; CDCl3) of receptor 1 before (bottom) and after the addition of β-glucoside 6a (a) and
β-galactoside 8a (c): [1] = 0.97 mM, 0.00-4.80 equiv of 6a or 8a. (b) Partial 1H NMR spectra of sugar 6a before (bottom) and after the addition
of receptor 1 (inverse titration): [6a] = 0.78 mM, 0.00-4.99 equiv of 1. (d, e) Partial 1H NMR spectra of receptors 3 (d) and 4 (e) before (bottom)
and after the addition of β-galactoside 8a (b): [3] = 0.96 mM, 0.00-4.85 equiv of 8a; [4] = 0.91 mM, 0.00-4.48 equiv of 8a.
TABLE 1. Solubilization of Sugars in CDCl3 by Receptors 1-5 (1 mM
Solutions)
described by Davis et al.; see refs 11a, 11b and see also ref
7b) of methyl β-D-glucoside (6b) and methyl β-D-galactoside
(8b) from aqueous solution into chloroform revealed that
compound 1 (1 mM chloroform solution) is capable of
extracting about 0.15 equiv of β-glucoside 6b and β-galacto-
side 8b from 1 M aqueous solutions (control experiments
were performed in the absence of the receptor).
Binding Studies in Homogeneous Solution. The interactions
of the receptors and carbohydrates were investigated by 1H
NMR spectroscopic titrations in CDCl3 and DMSO-d6/
CDCl3 mixtures. The stoichiometry of the receptor-sugar
complexes was determined by mole ratio plots12 (for exam-
ples, see the Supporting Information) and by a curve-fitting
analysis of the titration data.13-15
sugar
sugar/1a sugar/2a sugar/3a sugar/4a sugar/5a
β-D-glucoside 6b
R-D-glucoside 7b
β-D-galactoside 8b
R-D-galactoside 9
0.79
0.41
0.84
0.38
0.60
0.21
0.66
0.30
0.34
0.14
0.30
0.23
0.41
0.15
0.43
0.24
0.24
0.10
0.24
0.20
aMolar ratios of sugar to receptor occurring in solution (the 1H NMR
signals of the corresponding sugar were integrated with respect to the
receptor’s signals to provide the sugar to receptor ratio; control experi-
ments were performed in the absence of the receptor).
We were interested to see whether 1 would be able to
extract carbohydrates from aqueous solution into nonpolar
solvent.11 Studies of the extraction (using the procedure
(11) For examples of macrocyclic receptors which are able to extract
sugars from water into nonpolar organic solutions, see: (a) Velasco, T.;
Lecollinet, G.; Ryan, T.; Davis, A. P. Org. Biomol. Chem. 2004, 2, 645–647.
(b) Ryan, T.; Lecollinet, G.; Velasco, T.; Davis, A. P. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 4863–4866. (c) Aoyama, Y.; Tanaka, Y.; Sugahara, S.
J. Am. Chem. Soc. 1989, 111, 5397. (d) Aoyama, Y.; Tanaka, Y.; Toi, H.;
Ogoshi, H. J. Am. Chem. Soc. 1988, 110, 634.
(12) For a description of the mole ratio method, see: (a) Schneider, H.-J.;
Yatsimirsky, A. Principles and Methods in Supramolecular Chemistry; Wiley:
Chichester, U.K., 2000; p 148. (b) Tsukube, H.; Furuta, H.; Odani, A.;
Takeda, Y.; Kudo, Y.; Inoue, Y.; Liu, Y.; Sakamoto, H.; Kimura, K. In
Comprehensive Supramolecular Chemistry; Atwood, J.-L., Davis, J. E. D.,
€
MacNicol, D. D., Vogtle, F., Eds.; Pergamon: Oxford, U.K., 1996; Vol. 8, p
425.
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Mazik and Sonnenberg
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TABLE 2. Association Constantsa,b for Receptors 1-5 and Carbohydrates 6a-8a
host-guest complex
solvent
K11 (M-1
)
K21 or K12 (M-1
)
β
21 = K11K21 or β12 = K11K12 (M-2
)
1 6a
3
CDCl3
28800
2550
4360
44540
3830
12600
1660
19400
4580
5950
4420
5200
2800
530c
190d
210d
1680c
300d
450c
280d
940c
150c
310c
220c
340c
260c
1.52 ꢀ 107
4.85 ꢀ 105
9.15 ꢀ 105
7.48 ꢀ 107
1.15 ꢀ 106
5.67 ꢀ 106
4.64 ꢀ 105
1.82 ꢀ 107
6.87 ꢀ 105
1.84 ꢀ 106
9.72 ꢀ 105
1.76 ꢀ 106
7.28 ꢀ 105
5% DMSO-d6/CDCl3
CDCl3
CDCl3
5% DMSO-d6/CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
1 7a
3
1 8a
3
2 6a
3
2 7a
3
2 8a
3
3 6a
3
3 8a
4 6a
3
3
4 8a
5 6a
CDCl3
CDCl3
3
3
aAverage Ka values from multiple titrations in CDCl3. bErrors in Ka are less than 20%. cK21 corresponds to 2:1 receptor-sugar association constant.
dK12 corresponds to 1:2 receptor-sugar association constant.
Addition of octyl pyranosides 6a-8a to a CDCl3 solution
of 1-5 (the concentration of receptor was kept constant and
that of the corresponding sugar was varied) caused various
changes in the 1H NMR spectrum of the receptor. In partic-
ular, the signal due to the NHA of 1-3 moved significantly
downfield (in the range of 1.2-2.6 ppm, see Table S4 in the
also noted that the symmetrical isopropylamino-based com-
pound 4, possessing only three NH groups as hydrogen
bonding sites, showed a significantly decreased affinity
(about 10 times lower) to β-glucoside 6a but a level of affinity
toward β-galactoside 8a similar to that of the previously
described7p symmetrical aminopyridine-based receptor (K11
=
E
Supporting Information), while those due to CH2B and CH3
5200 M-1 for 4 8a vs K11 = 3070 M-1 for the previously
3
moved upfield (further changes in chemical shift are given in
Table S4; see also Figure 4 and Figure S1 in the Supporting
Information). In some cases splitting of the signals due to the
described receptor7p and 8a).
Since the binding constants for 1 6a and 1 8a in CDCl3
3
3
were determined to be higher than 1 ꢀ 104 M-1, additional
1H NMR titrations in a more polar solvent (DMSO-d6/
CDCl3 mixture) were carried out (for a review discussing
the limitations of the NMR spectroscopy method, see ref 16).
The best fit of the titration data was obtained with the
“mixed” 1:1 and 1:2 receptor-sugar binding model; the
results are given in Table 2. As expected for the recognition
of polar molecules, the affinities decreased as the solvent
polarity increased.17
B
CH2 ,CH2C,orCH3H group was observed, as given in Table S4.
The complexation-induced chemical shifts of the NHA and
CH3E,F protons (for labeling, see Figure 2) were monitored for
the determination of the binding constants, which are summarized
in Table 2. In addition to the 1:1 complexes, binding constants
for complexes of higher stoichiometry (1:2 or 2:1 receptor-
sugar complexes) were measured; the values of K12 or K21 are
considerably smaller than those of K11 (see Table 2). The
binding studies showed that the interactions of 1 and 2 with
β-glycosides 6a and 8a are more favorable than those with
R-glucoside 7a. The interactions of 6a-8a with receptor 1 were
shown to be stronger than those with 2. In addition, the bind-
ing affinities of 1 and 2 toward 6a/8a were shown to be signifi-
cantly higher than those of 3 (see Table 2 and Figure 4d),
indicating an important role of the isopropyl or isobutyl
groups in the complex formation. When the aminopyridine
group in 1/2 was replaced by the isopropylamino (compound
4) or isobutylamino group (compound 5), the expected de-
crease in the binding affinity to the tested glycosides was
observed (see Table 2). Compound 4, which was found to be
a more effective receptor than the isobutylamino-based
compound 5, showed affinities toward β-glucoside 6a and
β-galactoside 8a similar to those of compound 3. It should be
The interactions between the monosaccharides 6a/8a and
the receptors 1/2 were also investigated on the basis of
inverse titrations in which the concentration of the sugar
was held constant and that of the receptor varied. During the
titration of 6a/8a with 1 or 2 in CDCl3, the signals due to the
OH protons of 6a and 8a shifted downfield with strong
broadening and became almost indistinguishable from the
baseline after the addition of only 0.1 equiv of the receptor,
indicating the participation of the sugar OH groups in the
formation of hydrogen bonds. The addition of 1 or 2 to a
CDCl3 solution of 6a or 8a, furthermore, caused significant
upfield shifts of the sugar CH signals (see, for example
Figure 4b), indicating the participation of the sugar CH
units in the formation of the CH π interactions with the
3 3 3
(16) Fielding, L. Tetrahedron 2000, 56, 6151–6170.
(13) (a) Wilcox, C. S.; Glagovich, N. M. Program HOSTEST 5.6;
University of Pittsburgh, Pittsburgh, PA, 1994. (b) Cowart, M. D.; Sucholeiki,
I.; Bukownik, R. R.; Wilcox, C. S. J. Am. Chem. Soc. 1988, 110, 6204–6210.
(c) The HOSTEST program is designed to fit data to different binding
models, which include both “pure” binding models, taking into considera-
tion the formation of only one type of complex in solution (1:1, 1:2, or 2:1
receptor-substrate complex), and “mixed” binding models containing more
than one type of complex in solution (for example, 1:1 and 1:2 or 1:1 and 2:1
receptor-substrate complex).
(17) For a discussion on solvent effects in carbohydrate binding by
synthetic receptors, see: Klein, E.; Ferrand, Y.; Barwell, N. P.; Davis, A. P.
Angew. Chem. 2008, 120, 2733-2736; Angew. Chem., Int. Ed., 2008, 47,
2693-2696.
(18) For discussions on the importance of carbohydrate-aromatic inter-
actions, see: (a) Tsuzuki, S.; Uchimaru, T.; Mikami, M. J. Phys. Chem. B
ꢀ
2009, 113, 5617–5621. (b) Terraneo, G.; Potenza, D.; Canales, A.; Jimenez-
Barbero, J.; Baldridge, K. K.; Bernardi, A. J. Am. Chem. Soc. 2007, 129,
ꢀ
2890–2900. (c) Chavez, M. I.; Andreu, C.; Vidal, P.; Aboitiz, N.; Freire, F.;
ꢁ
ꢀ
(14) Hynes, M. J. J. Chem. Soc., Dalton Trans. 1993, 311–312.
Groves, P.; Asensio, J. L.; Asensio, G.; Muraki, M.; Canada, F. J.; Jimenez-
Barbero, J. Chem. Eur. J. 2005, 11, 7060–7074. (d) Screen, J.; Stanca-
Kaposta, E. C.; Gamblin, D. P.; Liu, B.; Macleod, N. A.; Snoek, L. C.;
Davis, B. G.; Simons, J. P. Angew. Chem., Int. Ed. 2007, 46, 3644–3648.
(e) Kiehna, S. H.; Laughrey, Z. R.; Waters, M. L. Chem. Commun. 2007,
(15) (a) The titration data were analyzed by non-linear regression anal-
ysis, using the program HOSTEST 5.613 and EQNMR.14 (b) The error in Ka
was <20%. (c) K11 corresponds to the 1:1 association constant. K21
corresponds to the 2:1 receptor-sugar association constant. K12 corresponds
ꢀ
4026–4028. (f) Morales, J. C.; Penades, S. Angew. Chem., Int. Ed. 1998, 37,
654–657.
to the 1:2 receptor-sugar association constant. β21 = K11 ꢀ K21, β12 = K11
ꢀ
K12
.
6420 J. Org. Chem. Vol. 75, No. 19, 2010

Mazik and Sonnenberg
JOCArticle
benzene ring of the corresponding receptor (for discussions
on the importance of carbohydrate-aromatic interactions,
see ref 18; for examples of CH-π interactions in the crystal
structures of the complexes formed between artificial re-
ceptors and carbohydrates, see ref 7o). Among the CH
signals, the signal due to the 2-CH proton of 6a and 8a
showed the largest shift (for example, 1.7 and 2.1 ppm for
the titration 6a 1 and 8a 1, respectively). Analysis of the
data supported the “mixed” 1:1 and 1:2 sugar-receptor
binding model and provided binding constants which are
identical, within the limits of uncertainty, with those deter-
mined from titrations where the roles of receptor and
substrate were reversed.
3
3
In comparison to the previously described symmetrical,
three-armed aminopyridine-based receptor,7p which shows a
β-glucoside vs β-galactoside binding preference, compounds
1 and 2 exhibit an inverse preference and a significantly
higher binding affinity toward the β-galactoside 8 (about 10
times higher in the case of 1). It should be also noted that an
enhancement of the binding affinity toward β-galactoside
was recently observed for the imidazole/aminopyridine- and
indole/aminopyridine-based receptors 17 and 18 (see Figure 5).7a
In contrast to these results, the phenanthroline/aminopyridine-
based receptors 19 and 20 show a high binding affinity
toward R-galactoside 9 (K11 > 105 M-1 in CDCl3) as well as
high R- vs β-galactoside preference.7b,d
Molecular Modeling. According to molecular modeling
calculations the 1:1 complex between receptor 1 and β-gala-
ctoside 8b can be stabilized by at least six hydrogen bonds
(NH O and OH N hydrogen bonds, see Table 3) and
3 3 3
3 3 3
interactions of sugar 2-CH with the central phenyl group of
the receptor molecule. Furthermore, CH O and CH
interactions (as indicated by the calculations, some of these
interactions can be of hydrogen bonding type) provide an
additional stabilization of the receptor-sugar complex (see
N
FIGURE 5. Structures of the recently described receptors 17/187a
showing β-galactoside vs β-glucoside as well as β- vs R-anomer
binding preferences and receptors 19/207b,d showing R- vs β-anomer
binding preferences in the recognition of glycosides.
3 3 3
3 3 3
FIGURE 6. Energy-minimized structure of the 1:1 (a) and 2:1 complexes (b) formed between receptor 1 and methyl β-galactoside 8b
(MacroModel V.8.5, OPLS-AA force field, MCMM, 50 000 steps): (gray) receptor C; (blue) receptor N; (yellow) sugar molecule.
J. Org. Chem. Vol. 75, No. 19, 2010 6421

Mazik and Sonnenberg
JOCArticle
FIGURE 7. Examples of noncovalent interactions (hydrogen bonding and van der Waals contacts) indicated by molecular modeling studies in
1:1 complexes of receptor 1 with β-galactoside 8b (a) and β-glucoside 6b (b) as well as in 1:1 complexes of receptor 2 with β-galactoside 8b (c) and
β-glucoside 6b (d) (MacroModel V.8.5, OPLS-AA force field, MCMM, 50 000 steps).
TABLE 3. Examples of Noncovalent Interactions Indicated by Molec-
ular Modeling Calculationsa for the Complexes Formed between Receptor
1 and Sugar 8b
Conclusions
Crystal structures of protein--carbohydrate complexes
revealed the participation of the isopropyl/isobutyl side
chain of valine/leucine in van der Waals contacts with the
carbohydrate substrates. We were interested to see whether
isopropyl and isobutyl groups would be suitable building
blocks for artificial carbohydrate receptors. Compounds 1
and 2, containing a trisubstituted triethylbenzene core, were
expected to recognize sugar molecules through a combina-
1:1 receptor-sugar complex
HND HO-2;
2:1 receptor-sugar complexb
(I) HND HO-2; (I) NHD O-CH3
3 3 3
3 3 3
3 3 3
NHD O-CH3
3 3 3
H
H
CH(CH3)2H
CH(CH3)2
OCH3
OH-2
(I) CH(CH3)2
OH-2
3 3 3
3 3 3
3 3 3
(I) NHD OH-3; (I) HND HO-4
(I) pyridine-N HO-6
3 3 3
3 3 3
HND HO-4
3 3 3
3 3 3
NHD OH-3
CH(CH3)2
(I) NHA O-ring
(I) pyridine-CH3
3 3 3
3 3 3
H
E
OH-3
OH-4
OH-3
OH-6
(I) phenyl HC-6; (I) phenyl HC-2
3 3 3
3 3 3
J
tion of NH O and OH N hydrogen bonds, CH
3 3 3 3 3 3
π
CH3CH2
CH3CH2
3 3 3
L3 3 3
3 3 3
3 3 3
3 3 3
3 3 3
interactions,6 and numerous van der Waals contacts. 1H NMR
spectroscopic titrations and binding studies in two-phase
systems, such as the dissolution of solid carbohydrates in
apolar media, revealed effective recognition of neutral carbo-
hydrates, β- vs R-anomer binding preferences in the recogni-
tion of glycosides, and considerably increased binding affin-
ity toward β-galactoside in comparison with the previously
described symmetrical aminopyridine-based receptor7p and
other acyclic receptors.7f Although 1:1 complexes predomi-
nate in the solution, the presence of 1:2 or 2:1 receptor-sugar
complexes, depending on the titration conditions, was also
detected. Compound 1, containing isopropylamino groups,
was shown to be a more effective carbohydrate receptor than
the isobutylamino-based compound 2. Liquid-liquid extra-
ctions demonstrated ability of 1 to extract monosaccharides
from water into chloroform; such an ability is interesting,
considering that the receptor possesses a very simple, acyclic
structure. In comparison to the previously described recep-
tor, incorporating three aminopyridine-based recognition
units,7p receptor 1 showed significantly increased affinity
to β-galactoside (about 10 times higher affinity) but de-
creased affinity toward β-glucoside (about 2 times lower).
The affinities of 3 for the tested monosaccharides were
shown to be considerably lower than those of 1 and 2. Tighter
binding of monosaccharides by 1/2 compared to 3 has been
attributed to van der Waals contacts between the monosac-
charide substrate and the isopropyl/isobutyl groups, which
are absent in 3. The replacement of the aminopyridine group
(II) NHD OH-6
3 3 3
pyridine-N HO-6
(II) NHA OH-2; (II) HNA HO-3
3 3 3
3 3 3
NHA O-ring
pyridine-N H3CO
(II) (CH3)2CHG OH-6
(II) phenyl HC-1; (II)phenyl HC-3
(II) phenyl HC-5
3 3 3
3 3 3
3 3 E3
3 3 3
3 3 3
pyridine-CH3
phenyl HC-2
3 3 3
OH-6
3 3 3
3 3 3
(II) HND HC-4
3 3 3
aMacroModel V.8.5, OPLS-AA force field, MCMM, 50 000 steps.
bI and II denote two receptors in the 2:1 receptor-sugar complex; for
labeling see Figure 2.
Table 3 and Figure 6a). In the case of 1 8b, the 4-OH group
3
of the β-galactoside 8b seems to be better positioned in the
binding pocket of 1 than the 4-OH of the β-glucoside 6b in
the 1 6b complex (for comparison, see Figure 7a,b).
3
In the case of a 2:1 receptor-sugar complex between 1 and
8b, the two receptor molecules almost completely enclose the
sugar, leading to involvement of all sugar hydroxyl groups in
interactions with the two receptor molecules (see Table 3 and
Figure 6b). The OH groups are involved in the formation of
cooperative hydrogen bonds, which result from the simulta-
neous participation of a sugar OH as donor and acceptor
of hydrogen bonds; the phenyl groups of both receptors
stack on the sugar ring, and both sides of the pyranose
ring are involved in CH π interactions (see Table 3 and
Figure 6b).
3 3 3
The preference of 2 for β-galactoside vs β-glucoside
shown by 1H NMR titrations was also indicated by molec-
ular modeling calculations (for examples of noncovalent
interactions in 2 8b and 2 6b, see Figure 7c,d).
3
3
6422 J. Org. Chem. Vol. 75, No. 19, 2010

Mazik and Sonnenberg
JOCArticle
in 1 and 2 by an isopropylamino or isobutylamino unit,
respectively, results in a decrease in the binding constants.
The affinity of the symmetrical isopropylamino-based re-
ceptor 4 toward the selected β-glycosides was shown to be
similar to that of 3 but higher than that of 5. In comparison to
the previously described symmetrical aminopyridine-based
receptor,7p compound 4, possessing only three NH groups as
hydrogen bonding sites, showed a significantly decreased
affinity (about 10 times lower) to β-glucoside 6a but a similar
affinity toward β-galactoside 8a. Considering the simple
structure of 4,19 the binding affinity toward β-galactoside
is noteworthy.
41 °C. 1H NMR (400 MHz, CDCl3, 0.07 M): δ 1.13 (d, J = 6.3
Hz, 12 H), 1.19-1.27 (m, 9 H), 2.21 (s, 3 H), 2.35 (s, 3 H), 2.76 (q,
J = 7.6 Hz, 4 H), 2.82 (q, J = 7.6 Hz, 2 H), 2.93 (sept, J = 6.3
Hz, 2 H), 3.71 (s, 4 H), 4.28 (br s, 1 H), 4.34 (d, J = 4.0 Hz, 2 H),
6.06 (s, 1 H), 6.32 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ
16.8, 21.1, 22.7, 24.2, 40.6, 45.4, 50.1, 103.6, 113.6, 132.6, 134.3,
142.42, 142.6, 148.6, 156.6, 158.3 ppm. HR-MS (EI): m/z calcd
for C28H46N4 438.372 25, found 438.372 27. Rf = 0.10 (CHCl3/
CH3OH, 7:1 v/v).
1,3-Bis[(isobutylamino)methyl]-5-[(4,6-dimethylpyridin-2-yl)-
aminomethyl]-2,4,6-triethylbenzene (2). Yield: 71%. Mp: 42-
44 °C. 1H NMR (400 MHz, CDCl3, 0.03 M): δ 0.93 (d, J = 6.6
Hz, 12 H), 1.20-1.28 (m, 9 H), 1.77 (sept, J = 6.6 Hz, 2 H), 2.22
(s, 3 H), 2.35 (s, 3 H), 2.54 (d, J = 6.7 Hz, 4 H), 2.76 (q, J = 7.6
Hz, 4 H), 2.83 (q, J = 7.6 Hz, 2 H), 3.68 (s, 4 H), 4.18 (br s, 1 H),
Similar to our previous studies,7a,b the binding studies
with compounds 1-3 demonstrated that, depending on the
nature of the recognition units used as the building blocks for
the acyclic receptor structures, effective carbohydrate recep-
tors with different binding preferences can be generated. The
exact prediction of the binding preference is still further
away, and it is hoped that systematic studies toward recogni-
tion units for carbohydrates will contribute significantly to
the solution of this unsolved problem. In this context, the
acyclic receptors represent particularly interesting objects
for such systematic studies. It should be noted that artificial
carbohydrate receptors using noncovalent interactions for
sugar binding provide valuable model systems to study the
underlying principles of carbohydrate-based molecular re-
cognition processes and may serve as a basis for the devel-
opment of saccharide sensors or new therapeutics.20
4.34 (d, J = 4.20 Hz, 2 H), 6.07 (s, 1 H), 6.33 (s, 1 H) ppm. 13
C
NMR (100 MHz, CDCl3): δ 16.8, 16.9, 20.8, 21.1, 21.4, 22.7,
24.2, 28.4, 40.7, 48.1, 59.2, 103.4, 113.6, 132.5, 134.8, 142.4,
142.7, 148.6, 156.7, 158.4 ppm. HR-MS (EI): m/zcalcd for C30H50N4
466.403 55, found 466.403 57. Rf = 0.15 (CHCl3/CH3OH, 7:1 v/v).
General Procedure for the Synthesis of Compounds 4 and 5. To
a mixture of isopropylamine (14; 0.32 mL, 3.74 mmol) or
isobutylamine (15; 0.36 mL, 3.74 mmol), CH3CN (30 mL),
and K2CO3 (0.5 g) was added dropwise a THF/CH3CN (40 mL,
4:1) solution of 1,3,5-tris(bromomethyl)-2,4,6-triethylbenzene
(12; 0.5 g, 1.13 mmol). After complete addition, the mixture was
stirred at room temperature for 36 h. The solvents were then
removed under vacuum; the crude product was washed several
times with water and dried.
1,3,5-Tris[(isopropylamino)methyl]-2,4,6-triethylbenzene (4).
1
Yield: 97%. Mp: 69-70 °C. H NMR (400 MHz, CDCl3): δ
1.11 (d, J = 6.3 Hz, 18 H), 1.23 (t, J = 7.5 Hz, 9 H), 2.79 (q, J =
7.5 Hz, 6 H), 2.91 (sept, J = 6.3 Hz, 3 H), 3.67 (s, 6H). 13C NMR
(100 MHz, CDCl3): δ 16.9, 22.6, 22.9, 45.7, 49.8, 134.5, 141.7.
HR-MS (EI): m/z calcd for C24H45N3 375.361 34, found
375.361 28. Rf = 0.10 (CHCl3/CH3OH, 5:1 v/v).
Experimental Section
Analytical TLC was carried out on silica gel 60 F254 plates
employing chloroform/methanol mixtures as the mobile phase.
Melting points are uncorrected. Sugars 6-9 are commercially
available. The binding studies are described in the Supporting
Information.
General Procedure for the Synthesis of Compounds 1 and 2. To
a mixture of isopropylamine (14; 0.21 mL, 0.15 g, 2.49 mmol) or
isobutylamine (15; 0.26 mL, 0.18 g, 2.49 mmol), CH3CN
(20 mL), and K2CO3 (0.18 g, 1.29 mmol) was added dropwise
a THF/CH3CN (25 mL, 4:1) solution of 1,3-bis(bromomethyl)-
5-[(4,6-dimethylpyridin-2-yl)aminomethyl]-2,4,6-triethylben-
zene (13; 0.30 g, 0.62 mmol). After complete addition, the
mixture was stirred at room temperature for 24 h. The solvents
were then removed under vacuum; the crude product was puri-
fied by column chromatography (silica gel, CHCl3/CH3OH, 7:1).
1,3-Bis[(isopropylamino)methyl]-5-[(4,6-dimethylpyridin-2-yl)-
aminomethyl]-2,4,6-triethylbenzene (1). Yield: 75%. Mp: 39-
1,3,5-Tris[(isobutylamino)methyl]-2,4,6-triethylbenzene (5).
1
Yield: 96%. Mp: 38-39 °C. H NMR (400 MHz, CDCl3): δ
0.93 (d, J = 6.7 Hz, 18 H), 1.24 (t, J = 7.5 Hz, 9 H), 1.77 (sept,
J = 6.7 Hz, 3 H), 2.54 (d, J = 6.7 Hz, 6H), 2.80 (q, J = 7.5 Hz,
6 H), 3.67 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 16.9, 20.8,
22.6, 28.3, 48.2, 59.2, 134.5, 141.9. HR-MS (ESI): m/z calcd for
C27H52N3 (Mþ þ H) 418.416 12, found 418.416 03. Rf = 0.65
(CHCl3/CH3OH, 5:1 v/v).
Acknowledgment. Financial support by the Deutsche
Forschungsgemeinschaft is gratefully acknowledged.
Supporting Information Available: Copies of the 1H and 13
C
NMR spectra of 1, 2, 4, and 5 (Figures S4-S12), representative
mole ratio plots (Figures S2 and S3), and further examples of 1H
NMR titrations (Figures S1a and S1b), descriptions of the H
1
(19) Artificial carbohydrate receptors very closely related to ours have
been recently presented, although binding constants in CDCl3 were only in
the range of 58-465 M-1; see: Fernandez-Trillo, F.; Fernandez-Megia, E.;
Riguera, R. J. Org. Chem. 2010, 75, 3878–3881.
(20) (a) Davis, A. P. Org. Biomol. Chem. 2009, 7, 3629–3638. (b) Kubik, S.
Angew. Chem. 2009, 121, 1750-1753; Angew. Chem., Int. Ed., 2009, 48,
1722-1725. (c) Mazik, M. ChemBioChem 2008, 9, 1015–1017.
NMR titration experiments (Tables S1-S3), and changes in
chemical shift observed during 1H NMR titrations of com-
pounds 1-3 with sugars 6a-8a in CDCl3 (Table S4). This
material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 75, No. 19, 2010 6423