Highly effective receptors showing di- vs. monosaccharide preference

Article abstract of DOI:10.1039/b719212f

Receptors 1 and 2, incorporating two heterocyclic recognition units as well as oxime- or hydroxymethyl-based hydrogen-bonding sites, were prepared, and their binding properties toward neutral sugars were determined. The design of these receptors was inspi

Full text of DOI:10.1039/b719212f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Highly effective receptors showing di- vs. monosaccharide preference†
Monika Mazik* and Arno C. Buthe
Received 12th December 2007, Accepted 14th February 2008
First published as an Advance Article on the web 14th March 2008
DOI: 10.1039/b719212f
Receptors 1 and 2, incorporating two heterocyclic recognition units as well as oxime- or
hydroxymethyl-based hydrogen-bonding sites, were prepared, and their binding properties toward
neutral sugars were determined. The design of these receptors was inspired by the binding motifs
observed in the crystal structure of protein–carbohydrate complexes. The receptors 1 and 2 are able to
recognize both mono- and disaccharides, with a strong preference for the disaccharides. Both
hydrogen-bonding and interactions of the sugar CH’s with the phenyl rings of the receptor contribute
to the stabilisation of the receptor–sugar complexes. Molecular modeling calculations, synthesis and
binding studies are described.
in Fig. 1. As in natural complexes, the participation of different
Introduction
types of hydrogen-bonding groups in the recognition process was
The interactions observed in the crystal structures of protein–
expected to be favorable for reaching high binding affinity and
carbohydrate complexes1 (for examples, see Fig. 1) inspire the
selectivity of the receptor. It should also be noted that the natural
development of different artificial receptor structures for the
recognition unit consisting of main chain amide of Ser or Thr
and side chain hydroxyl of the same amino acid (see Fig. 1b) has
that acyclic receptors containing two to four recognition units
been successfully mimicked with an aromatic analogue, which was
recognition of carbohydrates.2–5 Our previous studies showed
interconnected by a phenyl, biphenyl or diphenylmethane spacer
used for the construction of receptors showing selectivity for N-
perform effective recognition of carbohydrates through multiple
acetylneuraminic acid over glucuronic acid in competitive media
interactions.5 Depending on the nature and number of recogni-
tion units and connecting bridges used as the building blocks (see
Fig. 2), a variety of structures with different binding properties
could be obtained.
like DMSO or water–DMSO.5e
Recently, we have shown that three-armed oxime-based re-
ceptors are able to bind neutral sugar molecules in chloroform
and water-containing chloroform solutions with high affinity.5c
The using of the oxime groups as hydrogen bonding sites for
carbohydrates was inspired by the interactions involving pairs
of OH · · · N hydrogen bonds, which were observed between
oxime functionalities in the crystal structures of different oxime
molecules.8 Molecular modeling calculations indicated that com-
bining oxime- and aminopyridine-based recognition units, as in
the case of the compound 2, should cause further improvement of
the binding affinity of the new receptor.
As in previously described artificial systems,5 the participation
of the phenyl rings of the receptors 1 and 2 in the interactions
with sugar CH’s was expected to provide additional stabilization
of the receptor–sugar complexes. The character of carbohydrate–
aromatic interactions is still a subject of controversy;9,10 thus, the
studies with suitable model systems provide important insights on
the origin of the carbohydrate–aromatic interactions.
Fig. 1 Examples of hydrogen bonds in the complexes of (a) Galan-
thus nivalis lectin with mannose1f ,1a and (b) concanavalin A with
Mana6(Mana3)Man.1e,1g
To compare the binding properties of receptors 1 and 2 with
the properties of previously published receptors (for example,
receptors 7 and 8, see Fig. 5), the dodecyl b-D-maltoside (3),
dodecyl a-D-maltoside (4), octyl b-D-glucopyranoside (5) and
octyl a-D-glucopyranoside (6) were selected as substrates. The
interactions of the receptors and carbohydrates were investigated
by 1H NMR and fluorescence spectroscopy in organic media.11–13
The 1H NMR binding titration data were analyzed using the
Hostest 5.6 program.14 The fluorescence binding titration data
were analyzed using the Hyperquad 2006 program.15 Stoichiome-
try of the receptor–sugar complexes was determined by mole ratio
plots and by the curve-fitting analysis of the titration data.
In this study, we focused on interactions of receptors 1
and 2 (see Fig. 3 and 4) with neutral sugar molecules. The
design of the receptor 1, containing suitably positioned amine
and hydroxymethyl units, as well as 2-aminopyridine groups6
(as heterocyclic analogues of the asparagine/glutamine primary
amide side chains7), was inspired by the binding motifs shown
Institut fu¨r Organische Chemie der Technischen Universita¨t Braunschweig,
Hagenring 30, 38106, Braunschweig, Germany. E-mail: m.mazik@tu-bs.de;
Fax: +49-531-391-8185; Tel: +49-531-391-5266
† Electronic supplementary information (ESI) available: Description of
the 1H NMR and fluorescence titration experiments (Tables S1–S11). See
DOI: 10.1039/b719212f
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Fig. 2 Examples of spacer and recognition units used by our group for the construction of acyclic carbohydrate receptors.5
Fig. 3 Structures of receptors and sugars investigated in this study.
Fig. 4 Energy-minimized structure of the receptor 1 (a) and 2 (b).
MacroModel V.8.5, OPLS-AA force field, MCMM, 25 000 steps. Color
code: C, blue; N, green; O, red.
Results and discussion
Synthesis of the receptors
benzophenone oxime (14) provided the compounds 1 and 2,
respectively (see Scheme 1).
The base for the synthesis of compounds 1 and 2 was the
compound 11, which was prepared via a reaction of 1,3,5-
tris(bromomethyl)-2,4,6-triethyl-benzene16 (9) with 2 equivalents
of 2-amino-4,6-dimethylpyridine (10).5a The reaction of 1-bromo-
methyl-3,5-bis[(4,6-dimethylpyridin-2-yl)aminomethyl]-2,4,6-tri-
ethylbenzene (11) with 3-aminobenzylalcohol (12) or 3-amino-
Binding studies with disaccharides 3 and 4
b- and a-maltoside, 3 and 4, are poorly soluble in CDCl3, but
could be solubilized in this solvent in the presence of the receptor
1 or 2, indicating favourable interactions between the binding
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Scheme 1 Reaction conditions: (a) 2 equiv. of 10, CH3CN–THF, K2CO3, 48 h (30%); (b) CH3CN–THF, K2CO3, 72 h (73%); (c) CH3CN–THF, K2CO3,
72 h (61%); (d) NH2OH × HCl, NaOH, CH3CH2OH–H2O.
During the titrations of 1 with 3 or 4 the signal due to the
amine NHA of 1 moved downfield by about 0.60 and 0.90 ppm,
respectively; the addition of 0.5 equiv of sugar 3 or 4 led to
practically complete complexation of 1. The NHD signal shifted
downfield with strong broadening (by about 0.4 and 0.8 ppm,
respectively); this signal was overlapping during the titration with
3 or 4, and could not be used for the determination of the binding
1
constants. Furthermore, the H NMR spectra showed changes
in the chemical shifts of the CH3, CH2 and CH resonances of
1 (up- and downfield shifts in the range of 0.03–0.10 ppm; see
Fig. 6 and Table 1). The shifts of the NHA, CH2, CH3 and
aromatic CH protons of 1 were monitored as a function of sugar
concentration; typical titration plots are shown in Fig. 8. The
mole ratio plots indicated the formation of complexes with 2 : 1
receptor–sugar binding stoichiometry. The best fit of the titration
data for 1•3 and 1•4 was obtained with the 2 : 1 receptor–
sugar binding model;14,17 however, the binding constants were
Fig. 5 Structures of the previously studied symmetrical receptors 7 and
8.5c,5k
partners (similar solubility behaviour of the disaccharides 3 and 4
was observed in the presence of the previously described three-
armed oxime-based receptors5c). Thus, the receptor in CDCl3
was titrated with a solution of maltoside dissolved in the same
receptor solution. The complexation between 1 or 2 and both
disaccharides was evidenced by several changes in the NMR
spectra (for examples, see Fig. 6 and 7).
1
too large to be accurately determined by H NMR titrations18
(see Table 1).
Table 1 Association constantsa–d for receptors 1 and 2 and carbohydrates 3–6
Host–guest complex
K
11/M−1
K21 or K12 f/M−1
>100 000b,e
>100 000b,e
180b,f
b21 = K11
K
21 or b12 = K11
K
12/M−2
Ddobsg/ppm
e
1•3
1•4
1•5
2•3
2•4
2•5
2•6
NHA: 0.60; CH2B: −0.09; NHD: 0.80;
CHE: 0.09; CHH: −0.08; CH3 : −0.03
J
NHA: 0.90; CH2B: −0.07; NHD: 0.40;
CHE: 0.09; CHH: −0.07; CH3 : −0.03
J
1830b
3.29 × 105
2.95 × 109
1.31 × 109
1.48 × 106
2.13 × 105
NHA: 1.31; CH2B: −0.15; CHE: 0.14;
CHF: 0.11; CH3 : −0.06
J
371 200c
187 930c
2050b
7950c,e
NOH: −1.70; CHE: 0.16; CHH: −0.10;
CHF: 0.08; CH3 : −0.05
J
7010c,e
NOH: −1.44; CHE: 0.20; CHH: −0.08;
CHF: 0.10; CH3 : −0.06
J
720b,f
NOH: −2.36; NHA: 0.89; CH2B: −0.10;
CH2C: −0.16; CHE: 0.26; CHF: 0.15
790c
270c,f
a Average Ka values from multiple titrations. b Determined on the base of 1H NMR spectroscopic titrations in CDCl3. c Determined on the base of
e
f
fluorescence titrations in CHCl3. d Errors in Ka are less than 10%. K21 corresponds to a 2 : 1 receptor–sugar association constant. K12 corresponds
to a 1 : 2 receptor–sugar association constant. g Largest change in chemical shift observed during the H NMR titrations for the receptor signals (the
1
concentration of receptor was kept constant and that of sugar varied); down- and upfield (−) shifts.
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Fig. 6 (a–c) Partial 1H NMR spectra (400 MHz, CDCl3) of 1 after
addition of (from bottom to top) 0.00–3.13 equiv of b-maltoside 3 ([1] =
1.02 mM). Shown are chemical shifts of the pyridine CHL resonances of
Fig. 7 (a, b) Partial 1H NMR spectra (400 MHz, CDCl3) of 2 after
addition of (from bottom to top) 0.00–3.06 equiv of b-maltoside 3 ([2] =
1.02 mM). Shown are chemical shifts of the CHE,H,F and CH3 resonances
1
1 (for labeling, see formula 1). (b, c) Partial H NMR spectra of 1 after
1
of 2 (for labeling, see formula 2). (c) Partial H NMR spectra of 2 after
addition of (from bottom to top) 0.00–1.95 equiv of a-maltoside 4 ([1] =
1.01 mM). Shown are chemical shifts of the phenyl CH and CH2 resonances
of 1 (protons E, H, F, and B; for labeling, see formula 1).
addition of (from bottom to top) 0.00–3.09 equiv of a-maltoside 4 ([2] =
1.03 mM). Shown are chemical shifts of the CHE,H,F resonances of 2.
The 1H NMR spectra obtained during the titrations of 2
with the disaccharide 3 or 4 showed large shifting of the OH
and NHA resonances; however, the strong broadening of these
resonances prevented their use in the estimation of the binding
constants. The signal due to the NHD of 2 was unobservable
after the addition of only 0.1 equiv of the disaccharide 3 or 4.
The signal due to the oxime OH of 2 moved upfield with strong
broadening by 1.70 and 1.45 ppm after the addition of 3 and 4,
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respectively. This signal was almost unobservable after the
addition of about 0.1 equiv of the corresponding disaccharide,
and became distinct near the saturation, that occurred after the
addition of about 0.8 equiv of the disaccharide 3 or 4. In the case
of the receptor 2, the molecular modeling calculations indicated
the formation of an intramolecular hydrogen bond between the
oxime OH and the pyridine nitrogen of 2, as shown in Fig. 4a. The
existence of the intramolecular hydrogen bond was confirmed
by NMR spectroscopy. The resonance for the oxime OH proton
of 2 in CDCl3 solution was independent of its concentration
and occurred at ∼11.5 ppm (the resonance for the oxime OH
protons of the previously described three-armed oxime-based
receptors5c occurred at 7.5 ppm; 1 mM CDCl3 solution).19 The
observed complexation-induced shifts of the OH and NH signals
indicated important contribution of the OH and NH groups of 2
to the complex formation through formation of intermolecular
hydrogen bonds with the disaccharide 3 or 4.
The complexation between receptor 2 and the both disaccha-
rides was further evidenced by up- and downfield chemical shifts
of the CH2, CH3, pyridine CH and phenyl CH protons (in the
range of 0.03–0.20 ppm; see Table 1 and Fig. 7). The fit of the
NMR shift changes of these resonances agreed with a “mixed”
1 : 1 and 2 : 1 receptor–sugar binding model. The results of the
1H NMR titrations indicated the formation of very strong 1 : 1
complexes (K11 > 100 000 M−1) and weaker complexes with 2 :
1 receptor–sugar stoichiometry (the binding constants were too
large to be accurately determined by 1H NMR titrations).
The formation of strong complexes between the receptor 2
and the disaccharide 3 or 4 was confirmed by fluorescence
spectroscopy20 (the binding properties of the receptor 1 could
not be analysed on the base of fluorescence spectroscopy). The
fluorescence titration experiments were carried out by adding
increasing amounts of the sugar 3 or 4 (both disaccharides
are soluble in CHCl3 in the concentration range required for
fluorescence titrations) to a CHCl3 solution of the receptor 2 (for
example, see Fig. 9a). The best fit of the titration data (at 406
nm) was obtained with a “mixed” 1 : 1 and 2 : 1 receptor–sugar
binding model; this binding model was further supported by the
mole ratio plots. The binding constants for 2•3 were found to be
371 200 (K11) and 7950 (K21) M−1 (b21 = 2.95 × 109
M
−2), whereas
those for 2•4 amounted to 187 930 (K11) and 7010 (K21) M−1 (b21
=
1.31 × 109 M−2).17c Thus, the receptor 2 exhibits about 2-fold higher
binding affinity toward the b-maltoside 3.
According to molecular modeling calculations (see Fig. 10) the
receptor–maltoside complexes are stabilized by hydrogen bonds
between the OH groups as well as the ring-O of the sugar and the
NHA, NHD, pyridine-N, CH2OH (in the case of 1) and oxime-
OH/oxime-N (in the case of 2) of the receptor 1 or 2 (the
participation of the NH and OH groups of the receptors in the
formation of the intermolecular hydrogen bonds with the sugar
1
was confirmed by H NMR spectroscopy; see above). The sugar
OH groups are involved in cooperative and bidentate hydrogen
bonds, similar to interactions in protein–carbohydrate complexes.
Furthermore, CH · · · O/N hydrogen bonds and interactions of
sugar CHs with the phenyl groups of the receptor molecule
provide an additional stabilisation of the receptor–sugar complex.
Examples of noncovalent interactions indicated by molecular
modeling calculations for the complexes formed between receptor
1 or 2 and the disaccharide 3 are given in Table 2.
J
Fig. 8 (a and b) Plot of the observed chemical shifts of the NHA and CH3
resonances of 1 as a function of added b-maltoside 3 (a) or a-maltoside 4
(b). (c) Plot of the observed chemical shifts of the phenyl CH resonances
of 2 as a function of added b-maltoside 3. The [receptor] : [sugar] ratio is
marked.
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Table 2 Examples of noncovalent interactions indicated by molecular modeling calculationsa for the complexes formed between receptor 1 or 2 and
sugar 3
Receptor–substrate complex
Noncovalent interactionsb,c
1•3
(I) pyridine-N · · · HO-3 (g1); (I) NHA · · · OH-2 (g1); (I) CH2OH · · · OH-6 (g1);
2 : 1 receptor–sugar complexb (II) pyridine-N · · · HO-6 (g1); (II) NHA · · · O-ring (g1); (II) NHA · · · OH-3 (g1);
(II) NHD · · · OH-2 (g1); (II) pyridine-N · · · HO-6 (g2); (I) NHD · · · OH-2 (g2)
(I) phenyl (central ring) · · · HC-1 (g1); (I) phenyl (central ring) · · · HC-3 (g1)
(I) phenyl (central ring) · · · HC-5 (g1); (II) phenyl (central ring) · · · HC-2 (g1)
(II) phenyl (central ring) · · · H2C-6 (g1);
(I) phenyl (hydroxymethyl-substituted) · · · HC-3 (g2)
(II) CH2OH · · · NHA (I); (II) pyridine-CH3 · · · N-pyridine (I)
2•3
NHA · · · O-ring (g1); NHA · · · OR; NHD · · · OH-3 (g1); pyridine-N · · · HO-2 (g1)
I
1 : 1 receptor–sugar complex pyridine-N · · · HO-6 (g1); CH3 · · · OH-2 (g2); phenyl-CH · · · OH-3 (g1)
phenyl (oxime-substituted) · · · HO-6 (g2); phenyl (oxime-substituted) · · · HC-1 (g2);
phenyl (central ring) · · · HC-2 (g1); phenyl (central ring) · · · HC-4 (g1)
2•3
(I) NHA · · · O-ring (g1); (I) NHA · · · OH-2 (g1); (I) NHD · · · OH-3 (g1);
2 : 1 receptor–sugar complexb (I) pyridine-N · · · HO-2 (g1); (I) pyridine-N · · · HO-6 (g1); (I) phenyl-CH · · · OH-3 (g1)
(II) NHA · · · OH-2 (g2); (II) NHA · · · OH-3 (g2); (II) NOH · · · OH-6 (g2);
(II) pyridine-N · · · HO-4 (g2);
(I) phenyl (central ring) · · · HC-2 (g1); (I) phenyl (central ring) · · · HC-4 (g1)
(II) phenyl (oxime-substituted) · · · HC-1 (g1); (II) phenyl (oxime-substituted) · · · HC-3 (g1); (II) oxime-N · · · HC-5 (g1)
a MacroModel V.8.5, OPLS-AA force field, MCMM, 50 000 steps. b I and II: two receptors in the 2 : 1 receptor–sugar complex. c g1 and g2: the glucose units
of 3 (for labeling see Fig. 3).
Binding studies with monosaccharides 5 and 6
The 1H NMR titration experiments with b-glucopyranoside 5 were
carried out by adding increasing amounts of the sugar to a CDCl3
solution of the receptor 1 or 2. Similar to the binding studies
with disaccharide 3 or 4, the complexation between 1 or 2 and
glucopyranoside 5 was evidenced by several changes in the NMR
spectra (for examples, see Fig. 11). However, whereas after the
addition of about 0.5 equiv (in the case of receptor 1) or 1 equiv
(in the case of receptor 2) of the disaccharide 3 or 4 almost no more
change was observed in the chemical shift of the receptor signals,
with the monosaccharide 5, chemical shift changes continued to
higher [sugar] : [receptor] ratios. During the titrations of 1 or 2
with 5 the signal due to the amine NHA of 1 moved downfield by
about 1.3 ppm (after the addition of 8 equiv of sugar), whereas
the NHA of 2 shifted downfield by 0.9 ppm. The signal due to the
oxime OH of 2 shifted significantly upfield by about 2.3 ppm with
broadening. Furthermore, the 1H NMR spectra showed changes in
the chemical shifts of the CH3, CH2, and phenyl CH’s protons (up-
or downfield shifts in the range of 0.04–0.26 ppm; see Table 1),
as illustrated in Fig. 11. The curve fitting of the titration data
suggested the existence of 1 : 1 and 1 : 2 receptor–monosaccharide
complexes in the chloroform solution (typical titration curves are
shown in Fig. 12), with a stronger association constant for the 1 :
1 binding and a weaker association constant for the 1 : 2 receptor–
sugar complex. The binding constants for 1•5 were found to be
1830 (K11) and 180 (K12) M−1 (b12 = 3.29 × 105
M
−2), whereas those
for 2•5 amounted to 2050 (K11) and 720 (K12) M−1 (b12 = 1.48 ×
106
M
−2).17c
Fig. 9 Fluorescence titration of receptor 2 with b-maltoside 3 (a) and
b-glucopyranoside 5 (b) in CHCl3; [2] = 0.17 mM; Equiv of 3 = 0.00, 0.07,
0.15, 0.23, 0.31, 0.46, 0.62, 0.77, 0.93, 1.08, 1.24, 1.40, 1.55, 1.71, 1.86,
2.02, 2.17, 2.33, 2.48, and 2.64; Equiv of 5 = 0.00, 0.29, 0.58, 0.88, 1.17,
1.76, 2.35, 2.93, 3.52, 4.11, 4.70, 5.29, 5.87, 6.46, 7.05, 7.64, 8.23, 8.81,
9.40, and 9.99. Excitation wavelength 336 nm.
Interactions between receptor 2 and b-glucopyranoside 5 could
also be detected by fluorescence; however, the spectral changes
observed during the fluorescence titrations with glucopyranoside
5 were less substantial than those observed during the titrations
with disaccharides 3 and 4 (for example, see Fig. 9b). The analysis
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Table 3 Association constantsa,b for receptors 7 and 8 and carbohydrates
3–5
Host–guest
complex
b21 = K11K21 or
b12 = K11
12/M−2
e
K
11/M−1
K21 or K12 f/M−1
K
7•3
7•4
100 500c,g
98 900d,g
65 300c,g
62 000d,g
170c,g
7•5
8•3
8•4
8•5
1730c,f
42 300d,e
16 350d,e
1320c,f
2.94 × 105
5.52 × 109
1.29 × 109
6.42 × 107
8.07 × 107
130 700d
79 100d
48 630c,h
54 920d
1470d,f
a Average Ka values from multiple titrations. b Errors in Ka are less than
10%. c Determined on the base of 1H NMR spectroscopic titrations in
CDCl3. d Determined on the base of fluorescence titrations in CHCl3.
f
e K21 corresponds to a 2 : 1 receptor–sugar association constant. K12
corresponds to a 1 : 2 receptor–sugar association constant. g Results from
ref. 5c. h Results from ref. 5k.
with those determined on the base of the NMR spectroscopic
titrations in CDCl3.
Fluorescence titrations of the receptor 2 with a-glucopyranoside
6 (fluorescence intensity increased with increasing monosaccha-
ride concentration) indicated also the formation of complexes with
1 : 1 and 1 : 2 receptor–monosaccharide binding stoichiometry. The
binding constants for 2•6 were found to be 790 (K11) and 270 (K12)
M−1 (b12 = 2.13 × 105
M
−2). Thus, the complexes formed between
the receptor 1 or 2 and the monosaccharides 5 and 6 are much less
stable than those formed with the disaccharides 3 and 4.
Both the 1H NMR and the fluorescence spectroscopic titrations
clearly show the di- vs. monosaccharide preference of the receptors
1 and 2. It should be noted that oligosaccharides have received
far less attention in the artificial receptor chemistry than the
monosaccharides,21–24 and the selective recognition of oligosac-
charides by receptors using noncovalent interactions is still rare.21
The comparison of the binding properties of the
aminopyridine/oxime-based receptor 2 and the symmetrical
oxime-based receptor 7 (see Table 1 and 3) shows that combining
oxime- and aminopyridine-based recognition units significantly
affect the binding properties. Both receptors, 2 and 7, show strong
di- vs. monosaccharide preference; however, receptor 2 exhibits
higher affinity toward the tested disaccharides. In the case of
receptor 7, both glucose units of the disaccharide 3 or 4 have the
possibility to interact with four phenyl rings of the receptor 7;
these interactions seem to be responsible for the 1 : 1 binding
stoichiometry, similar to the complex between maltose binding
protein (MBP) and maltose.1h Quiocho et al. pointed out that
“the maltose is wedged between four aromatic side chains and the
resulting stacking of these aromatic residues on the faces of the
glucosyl units provides a majority of the van der Waals contacts
in the complex.”1h
Fig. 10 (a) Energy-minimized structure of the 2 : 1 receptor–sugar
complex formed between receptor 1 and b-maltoside 3. (b and c) Ener-
gy-minimized structure of the 2 : 1 and 1 : 1 receptor–sugar complex formed
between receptor 2 and b-maltoside 3. MacroModel V.8.5, OPLS-AA force
field, MCMM, 50 000 steps. Color code: receptor C, blue; receptor N,
green; receptor O, red; the sugar molecule is highlighted in orange.
The symmetrical pyridine-based receptor 8 has been established
as highly effective receptor for both b-glucopyranoside 5 and
maltosides 3/4(see Table 3 and Fig. 13). The preference for the
disaccharides is still observable, but is not so strong as in the case
of 1, 2 or 7 (for comparison, see Table 1 and 3).
of the titration data confirmed the “mixed” 1 : 1 and 1 : 2 receptor–
glucopyranoside binding model; the binding constants determined
on the base of fluorescence titrations in CHCl3 were comparable
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Fig. 11 (a–d) Partial 1H NMR spectra (400 MHz, CDCl3) of 1 after addition of (from bottom to top) 0.00–8.02 equiv of 5 ([1] = 1.02 mM); for labeling,
see formula 1. (e, f) Partial 1H NMR spectra of 2 after addition of (from bottom to top) 0.00–8.03 equiv of 5 ([2] = 1.02 mM); for labeling, see formula 2.
stable than those formed with the monosaccharides 5 and 6
(see Table 1).
Conclusion
Acyclic receptors 1 and 2 containing neutral hydrogen bonding
sites, such as amine, pyridine, hydroxymethyl or oxime groups,
were prepared, and their binding properties towards neutral sugar
molecules studied. The two compounds have been established
as highly effective receptors for b- and a-maltoside, 3 and 4;
the complexes formed with these disaccharides are much more
The formation of receptor–sugar complexes has been charac-
terized by H NMR spectroscopy and confirmed by a second,
independent technique, namely fluorescence spectroscopy (in the
case of the receptor 2). Receptor 1 has the tendency to form
strong 2 : 1 receptor–sugar complexes with b- and a-maltoside
(see Table 1). Both hydrogen-bonding and interactions of the
1
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Fig. 12 Plot of the observed (+) and calculated (—) chemical shifts of the
B
NHA (a), CH2 (b), and CHH (c) resonances of 1 (1.02 mM) as a function
Fig. 13 Fluorescence titration of receptor 8 with b-maltoside 3 (a) and
b-glucopyranoside 5 (b) in CHCl3; [8] = 0.23 mM; Equiv of 3 = 0.00, 0.07,
0.16, 0.25, 0.33, 0.42, 0.50, 0.59, 0.67, 0.75, 0.84, 1.01, 1.18, 1.34, 1.51,
1.68, 2.02, 2.36, 2.69, and 2.86; Equiv of 5 = 0.00, 0.29, 0.58, 0.88, 1.17,
1.76, 2.05, 2.35, 2.64, 2.93, 3.52, 4.11, 4.70, 5.28, 5.87, 7.05, 8.22, 9.40, and
9.89. Excitation wavelength 326 nm.
of added b-glucopyranoside 5. The [receptor] : [sugar] ratio is marked.
sugar CH’s with the phenyl rings of the receptor contribute to
the stabilisation of the receptor–sugar complexes (see Fig. 10a
and Table 2). According to molecular modeling calculations,
the disaccharide 3 is encapsulated in the cavity between the
two receptor molecules in a similar way as in the protein–
sugar complexes. Both glucose units of the disaccharide have the
possibility to interact with four phenyl rings of the two receptor
molecules (two central phenyl rings and two hydroxymethyl-
subsituted phenyl rings; see Fig. 10a), similar to the complex
between maltose binding protein and maltose.
The analysis of the titration data obtained on the base of
1H NMR and fluorescence titrations of the receptor 2 with the
disaccharide 3 or 4 indicated the existence of 1 : 1 and 2 : 1
receptor–maltoside complexes in the chloroform solutions, with a
very high binding constant for the 1 : 1 complexes (see Table 1).
Examples of the energy-minimized structure of the 1 : 1 and 2 : 1
receptor–maltoside complexes are illustrated in Fig. 10c and 10b,
respectively. According to the molecular modeling calculations,
the hydrogen bonding interactions are complemented by the CH-
p interactions between the sugar CH’s and the phenyl rings of the
receptor 2. The phenyl rings provides additional apolar contacts to
a saccharide, similar to sugar-binding proteins, which commonly
place aromatic surfaces against patches of sugar CH groups. The
comparison of the binding properties of the receptor 2 and the
previously described three-armed oxime-based receptors5c shows
that combining oxime- and aminopyridine-based recognition units
significantly affect the binding properties. As in natural complexes,
the participation of different types of hydrogen-bonding groups
in the recognition process is favorable for reaching high binding
selectivity of the receptor.
The binding studies between both receptors and the monosac-
charide 5 or 6 indicated the formation of complexes with 1 : 1
and 1 : 2 receptor–monosaccharide binding stoichiometry (with a
higher binding constant for the 1 : 1 complex; see Table 1). The
results of the NMR and fluorescence titrations clearly showed
that the receptors 1 and 2 are able to recognize both mono- and
disaccharides, with a strong preference for the disaccharides.
Synthetic receptors using noncovalent interactions for sugar
binding provide valuable model systems to study the basic
molecular features of carbohydrate recognition. In this context,
the acyclic receptors represent particularly interesting objects for
systematic studies toward recognition motifs for carbohydrates.
Experimental
Analytical TLC was carried out on silica gel 60 F254 plates.
Melting points are uncorrected. Dodecyl b-D-maltoside (3),
dodecyl a-D-maltoside (4), octyl b-D-glucopyranoside (5), 3-
aminobenzylalcohol (12), and 3-aminobenzophenone (13) are
commercially available.
1-[(3-Hydroxymethyl-phenyl)aminomethyl]-3,5-bis-[(4,6-
dimethylpyridin-2-yl)amino-methyl]-2,4,6-triethylbenzene (1)
A
mixture of 1-bromomethyl-3,5-bis[(4,6-dimethylpyridin-2-
yl)aminomethyl]-2,4,6-triethylbenzene (11) (0.50 g, 0.96 mmol),
3-aminobenzyl alcohol (12) (0.153 g, 1.24 mmol) and K2CO3
1566 | Org. Biomol. Chem., 2008, 6, 1558–1568
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(1 g, 7 mmol) in THF–CH3CN (160 mL, 1 : 1 v/v) was stirred
at room temperature for 72 h (the solution was monitored by
TLC). After filtration and evaporation of solvents, the crude
product was purified by column chromatography (aluminium
oxide; chloroform–diethyl ether, 1 : 6 v/v). Yield 73%. M.p. 74–
75 C. H-NMR (400 MHz, CDCl3): d = 1.24 (m, 9 H), 2.24 (s,
6 H), 2.34 (s, 6 H), 2.75 (m, 6 H), 3.51 (br. s, 1 H), 4.14 (br. s,
2 H), 4.21 (d, J = 4.20 Hz, 2 H), 4.37 (d, J = 4.20 Hz, 4 H), 4.65
(s, 2 H), 6.09 (s, 2 H), 6.35 (s, 2 H), 6.60 (m, 1 H), 6.71 (m, 2 H),
7.19 (t, J = 7.80 Hz, 1 H). 13C-NMR: d = 16.85, 16.90, 21.11,
22.84, 22.91, 24.15, 40.68, 42.22, 65.53, 103.34, 110.63, 111.98,
113.98, 115.89, 129.51, 133.07, 133.20, 142.31, 143.60, 143.68,
148.46, 148.88, 156.74, 158.22. HR-MS (EI) calcd for C36H47N5O:
565.3780; found: 565.3781. Rf = 0.54 (methanol–chloroform 1 : 7,
v/v).
3 For reviews on boronic acid based receptors, using covalent interactions
for sugar binding, see: (a) T. D. James and S. Shinkai, Top. Curr. Chem.,
2002, 218, 159–200; (b) T. D. James, K. R. A. S. Sandanayake and S.
Shinkai, Angew. Chem., 1996, 108, 2038–2050, (Angew. Chem., Int. Ed.,
1996, 35, 1910–1922).
4 For some recent examples of carbohydrate receptors operating through
noncovalent interactions (earlier examples are given in ref. 2a and b),
see ref. 5a–l and: (a) R. Welti, Y. Abel, V. Gramlich and F. Diederich,
Helv. Chim. Acta, 2003, 86, 548–562; (b) R. Welti and F. Diederich,
Helv. Chim. Acta, 2003, 86, 494–503; (c) K. Wada, T. Mizutani and
S. Kitagawa, J. Org. Chem., 2003, 68, 5123–5131; (d) M. Segura, B.
Bricoli, A. Casnati, E. M. Mun˜oz, F. Sansone, R. Ungaro and C.
◦
1
ˇ
Vicent, J. Org. Chem., 2003, 68, 6296–6303; (e) M. Dukh, D. Saman,
ˇ
K. Lang, V. Pouzar, I. Cerny, P. Drasˆar and V. Kra´l, Org. Biomol.
Chem., 2003, 1, 3458–3463; (f) T. Ishi-I, M. A. Mateos-Timoneda,
P. Timmerman, M. Crego-Calama, D. N. Reinhoudt and S. Shinkai,
Angew. Chem., Int. Ed., 2003, 42, 2300–2305; (g) J. M. Benito and M.
Meldal, QASR Comb. Sci., 2004, 23, 117–129; (h) G. Gupta and C. R.
Lowe, J. Mol. Recognit., 2004, 17, 218–235; (i) T. Velasco, G. Lecollinet,
T. Ryan and A. P. Davis, Org. Biomol. Chem., 2004, 2, 645–647; (j) J.-
M. Fang, S. Selvi, J.-H. Liao, Z. Slanina, C.-T. Chen and P.-T. Chou,
J. Am. Chem. Soc., 2004, 126, 645–647; (k) A. Vacca, C. Nativi, M.
Cacciarini, R. Pergoli and S. Roelens, J. Am. Chem. Soc., 2004, 126,
16456–16465; (l) H. Abe, Y. Aoyagi and M. Inouye, Org. Lett., 2005,
7, 59–61; (m) E. Klein, M. P. Crump and A. P. Davis, Angew. Chem.,
Int. Ed., 2005, 44, 298–302; (n) M. G. J. Ten Cate, D. N. Reinhoudt and
M. Crego-Calama, J. Org. Chem., 2005, 70, 8443–8453; (o) H. Abe,
N. Masuda, M. Waki and M. Inouye, J. Am. Chem. Soc., 2005, 127,
16189–16196; (p) H.-P. Yi, X.-B. Shao, J.-L. Hou, C. Li, X.-K. Jiang
and Z.-T. Li, New J. Chem., 2005, 29, 1213–1218; (q) O. Francesconi,
A. Ienco, G. Moneti, C. Nativi and S. Roelens, Angew. Chem., Int. Ed.,
2006, 45, 6693–6696; (r) H. Takeharu, M. Nakamura and Y. Fukazawa,
Heterocycles, 2006, 68, 2477–2482; (s) M. Waki, H. Abe and M. Inouye,
Chem.–Eur. J., 2006, 12, 7839–7847; (t) C. Schmuck and M. Heller, Org.
Biomol. Chem., 2007, 5, 787–791; (u) E. Klein, Y. Ferrand, E. K. Auty
and A. P. Davis, Chem. Commun., 2007, 2390–2392; (v) Y. Ferrand,
M. P. Crump and A. P. Davis, Science, 2007, 318, 619–622.
1-[(3-Acetyl-phenyl)aminomethyl]-3,5-bis-[(4,6-dimethylpyridin-2-
yl)aminomethyl]-2,4,6-triethylbenzene oxime (2)
A
mixture of 1-bromomethyl-3,5-bis[(4,6-dimethylpyridin-2-
yl)aminomethyl]-2,4,6-triethylbenzene (11) (0.42 g, 0.80 mmol), 3-
aminobenzophenone oxime (14) (0.157 g, 1.05 mmol) and K2CO3
(1 g, 7 mmol) in CH3CN–THF (80 mL, 1 : 1, v/v) was stirred
at room temperature for 72 h (the solution was monitored by
TLC). After filtration and evaporation of solvents, the crude
product was purified by column chromatography (aluminium
oxide; chloroform–diethyl ether, 4 : 6 v/v). Yield 61%. M.p. 71–
◦
1
72 C. H-NMR (400 MHz, CDCl3): d = 1.18 (t, J = 7.6 Hz,
6 H), 1.24 (t, J = 7.6 Hz, 3 H), 2.24 (s, 3 H), 2.25 (s, 6 H), 2.36
(s, 6 H), 2.73 (q, J = 7.6 Hz, 2 H), 2.90 (q, J = 7.6 Hz, 4 H), 3.87
(br. s, 1H), 4.32 (d, J = 4.1 Hz, 4 H), 4.39 (s, 2 H), 4.52 (br. s,
2H), 6.11 (s, 2 H), 6.34 (s, 2 H), 6.60 (m, 1 H), 6.79 (m, 1 H),
6.87 (m, 1H), 7.13 (t, J = 7.8 Hz, 1 H), 11.48 (s, 1 H). 13C-NMR:
d = 12.60, 16.56, 16.74, 21.25, 23.01, 23.68, 40.80, 42.90, 103.13,
110.17, 113.88, 115.09, 116.21, 128.90, 132.73, 133.43, 138.20,
143.21, 143.66, 148.24, 149.37, 155.61, 156.37, 158.00. HR-MS
calcd for C37H49N6O (ESI): 593.3974; found: 593.3974. Rf = 0.32
(chloroform–diethyl ether, 4 : 6).
5 (a) M. Mazik and M. Kuschel, Chem.–Eur. J., 2008, 14, 2405–2419;
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Acknowledgements
6 Our previous binding studies showed that 2-aminopyridines provide an
excellent structural motif for binding carbohydrates, associated with
the ability to form cooperative and bidentate hydrogen bonds with the
sugar OH groups, see ref. 5g,j–o.
7 Anslyn and co-workers have exploited the 2-aminopyridine unit for
binding of cyclohexane diols and triols. See: C. Y. Huang, L. A. Cabell
and E. V. Anslyn, J. Am. Chem. Soc., 1994, 116, 2778–2792.
This work was supported by the Deutsche Forschungsgemein-
schaft. We thank Prof. C. S. Wilcox for giving access to the
HOSTEST program.
8 Oximes have received far less attention in supramolecular chemistry
than other compounds such as carboxylic acids and amides. For
some examples, see: (a) M. Mazik, D. Bla¨ser and R. Boese, J. Org.
Chem., 2005, 70, 9115–9122; (b) M. Mazik, D. Bla¨ser and R. Boese,
Tetrahedron, 1999, 55, 7835–7840; (c) M. Mazik, D. Bla¨ser and R.
Boese, Tetrahedron Lett., 1999, 40, 4783–4786; (d) M. Mazik, D. Bla¨ser
and R. Boese, Chem.–Eur. J., 2000, 6, 2865–2873; (e) C. B. Aakero¨y,
A. M. Beatty and D. S. Leinen, CrystEngComm, 2000, 27, 1–6; (f) E. A.
Bruton, L. Brammer, F. C. Pigge, C. B. Aakero¨y and D. S. Leinen,
New J. Chem., 2003, 1084–1094; (g) A. W. Marsman, E. D. Leussink,
J. W. Zwikker, L. W. Jenneskens, W. J. J. Smeets, N. Veldman and A. L.
Spek, Chem. Mater., 1999, 11, 1484; (h) A. W. Marsman, C. A. van
Walree, R. W. A. Havenith, L. W. Jenneskens, M. Lutz, A. L. Spek,
E. T. G. Lutz and J. H. van der Maas, J. Chem. Soc., Perkin Trans. 2,
2000, 501.
References and notes
1 (a) H. Lis and N. Sharon, Lectins, Kluwer Academic Publishers,
Dordrecht, The Netherlands, 2003; (b) F. A. Quiocho, Pure Appl.
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F. A. Quiocho, J. Biol. Chem., 1991, 266, 5202–5219.
2 For reviews, see: (a) A. P. Davis and T. D. James, in Functional Synthetic
Receptors, ed. T. Schrader and A. D. Hamilton, Wiley-VCH, Weinheim,
Germany, 2005, pp. 45–109; (b) A. P. Davis and R. S. Wareham, Angew.
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9 For recent discussions on the importance of carbohydrate–aromatic
interactions, see: (a) G. Terraneo, D. Potenza, A. Canales, J. Jime´nez-
Barbero, K. K. Baldridge and A. Bernardi, J. Am. Chem. Soc.,
2007, 129, 2890–2900; (b) M. I. Cha´vez, C. Andreu, P. Vidal, N.
Aboitiz, F. Freire, P. Groves, J. L. Asensio, G. Asensio, M. Muraki,
F. J. Canˇada and J. Jime´nez-Barbero, Chem.–Eur. J., 2005, 11, 7060–
7074.
10 For examples of CH-p interactions in the crystal structures of the
complexes formed between artificial receptors and carbohydrates, see
ref. 5j.
11 Quiocho has shown that the hydrogen bonds between sugar-binding
proteins and essential recognition determinants on sugars are shielded
from bulk solvent, meaning that they exist in a lower dielectric
environment.1b Thus, investigations with synthetic receptors in organic
media (see also ref. 12) can make an important contribution to
our understanding of the complex carbohydrate binding processes in
nature.
12 Many biological interactions occur in enzyme pockets or in membranes,
meaning that they occur in an environment with a lower dielectric
constant relative to the bulk solvent. For this reason, many theoretical
studies on different enzyme model systems have been performed in a
medium with a lower dielectric constant (mostly e = 5.7, corresponding
to the chlorobenzene). See, for example: (a) K.-B. Cho, Y. Moreau, D.
Kumar, D. A. Rock, J. P. Jones and S. Shaik, Chem.–Eur. J., 2007, 13,
4103–4115; (b) S. P. de Visser, S. Shaik, P. K. Sharma, D. Kumar and
W. Thiel, J. Am. Chem. Soc., 2003, 125, 15779–15788; (c) S. P. de Visser,
J. Phys. Chem. A, 2005, 109, 11050–11057.
A. Vacca, Anal. Biochem., 1995, 251, 374–382; (b) P. Gans, A. Sabatini
and A. Vacca, Talanta, 1996, 43, 1739–1753.
16 K. J. Wallace, R. Hanes, E. Anslyn, J. Morey, K. V. Kilway and J. Siegel,
Synthesis, 2005, 2080–2083.
17 (a) The binding constants were determined in chloroform at 25 ◦C
by titration experiments. Dilution experiments show that receptors
do not self-aggregate in the used concentration range (see ESI†). For
each system at least three 1H NMR titrations were carried out; for
each titration 15–20 samples were prepared; (b) Error in a single Ka
estimation was <10%; (c) K11 corresponds to the 1 : 1 association
constant; K21 corresponds to the 2 : 1 receptor–sugar association
constant; K12 corresponds to the 1 : 2 receptor–sugar association
constant; b21 = K11 × K21; b12 = K11 × K12
.
18 For a review discussing the limitations of the NMR method, see: L.
Fielding, Tetrahedron, 2000, 56, 6151–6170.
19 Intramolecular hydrogen bonds may drastically reduce the binding
affinity of receptors; for some examples, see ref. 5k and: M. Inouye,
T. Miyake, M. Furusyo and H. Nakazumi, J. Am. Chem. Soc., 1995,
117, 12416–12425.
20 (a) For each system at least two fluorescence titrations were carried out;
for each titration 20 samples were prepared; (b) Error in a single Ka
estimation was < 10%.
21 For examples of selective disaccharide binding by macrocyclic receptors
using noncovalent interactions, see ref. 4u and: U. Neidlein and F.
Diederich, Chem. Commun., 1996, 1493–1494.
22 For examples of boronic acid-based receptors for oligosaccharides, see
ref. 3a and reviews: (a) S. Striegler, Curr. Org. Chem., 2003, 7, 81–102;
(b) J. H. Hartley, T. D. James and C. J. Ward, J. Chem. Soc., Perkin
Trans. 1, 2000, 3155–3184.
23 A number of studies have demonstrated that artificial multivalent
carbohydrate ligands possess high affinities for specific carbohydrate-
binding proteins. For examples of such oligosaccharide-based ligands,
see: (a) T. K. Dam and C. F. Brewer, Chem. Rev., 2002, 102, 387–429;
(b) T. K. Lindhorst, Top. Curr. Chem., 2002, 218, 201–235.
24 For examples of oligosaccharide-based model systems for studying
carbohydrate–carbohydrate interactions, see: J. Rojo, J. C. Morales and
S. Penade´s, Top. Curr. Chem., 2002, 218, 45–92.
13 Recognition of neutral sugars in aqueous solution through noncovalent
interactions remains an important challenge in artificial receptor
chemistry; for some examples, see ref. 4m, 4v, 5h and: (a) V. Kra´l,
O. Rusin and F. P. Schmidtchen, Org. Lett., 2001, 3, 873–876; (b) R. D.
Hubbard, S. R. Horner and B. L. Miller, J. Am. Chem. Soc., 2001, 123,
5810–5811; (c) R. Yanagihara and Y. Aoyama, Tetrahedron Lett., 1994,
35, 9725–9728.
14 C. S. Wilcox and N. M. Glagovich, Program HOSTEST 5.6, University
of Pittsburgh, Pittsburgh, PA, 1994.
15 (a) C. Frassineti, S. Ghelli, P. Gans, A. Sabatini, M. S. Moruzzi and
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