Peptide-based carbohydrate receptors

Article abstract of DOI:10.1002/chem.201303777

A broad spectrum of physiological processes is mediated by highly specific noncovalent interactions of carbohydrates and proteins. In a recent communication we identified several cyclic hexapeptides in a dynamic combinatorial library that interact selectively with carbohydrates with high binding constants in water. Herein, we report a detailed investigation of the noncovalent interaction of two cyclic hexapeptides (Cys-His-Cys (which we call HisHis) and Cys-Tyr-Cys (which we call TyrTyr)) with a selection of monosaccharides and disaccharides in aqueous solution. The parallel and antiparallel isomers of HisHis or TyrTyr were synthesized separately, and their interaction with monosaccharides and disaccharides in aqueous solution was studied by isothermal titration calorimetry, NMR spectroscopic titrations, and circular dichroism spectroscopy. From these measurements, we identified particularly stable complexes (K_a>1000 M^-1) of the parallel isomer of HisHis with N-acetylneuraminic acid and with methyl-α-D-galactopyranoside as well as of both isomers of TyrTyr with trehalose. To gain further insight into the structure of the peptide-carbohydrate complexes, structure prediction was performed using quantum chemical methods. The calculations confirm the selectivity observed in the experiments and indicate the formation of multiple intermolecular hydrogen bonds in the most stable complexes. Peptide talk: Cyclic peptides (see figure) are potent and selective receptors for carbohydrates in aqueous solution. A detailed investigation of the noncovalent interaction of two cyclic hexapeptides with a selection of monosaccharides and disaccharides in aqueous solution is described.

Full text of DOI:10.1002/chem.201303777

DOI: 10.1002/chem.201303777
Full Paper
&
Peptides
Peptide-Based Carbohydrate Receptors
Melanie Rauschenberg, Sateesh Bandaru, Mark P. Waller, and Bart Jan Ravoo*[a]
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Abstract: A broad spectrum of physiological processes is
mediated by highly specific noncovalent interactions of car-
bohydrates and proteins. In a recent communication we
identified several cyclic hexapeptides in a dynamic combina-
torial library that interact selectively with carbohydrates with
high binding constants in water. Herein, we report a detailed
investigation of the noncovalent interaction of two cyclic
hexapeptides (Cys-His-Cys (which we call HisHis) and Cys-
Tyr-Cys (which we call TyrTyr)) with a selection of monosac-
charides and disaccharides in aqueous solution. The parallel
and antiparallel isomers of HisHis or TyrTyr were synthesized
separately, and their interaction with monosaccharides and
disaccharides in aqueous solution was studied by isothermal
titration calorimetry, NMR spectroscopic titrations, and circu-
lar dichroism spectroscopy. From these measurements, we
identified particularly stable complexes (Ka >1000mÀ1) of the
parallel isomer of HisHis with N-acetylneuraminic acid and
with methyl-a-d-galactopyranoside as well as of both iso-
mers of TyrTyr with trehalose. To gain further insight into the
structure of the peptide–carbohydrate complexes, structure
prediction was performed using quantum chemical meth-
ods. The calculations confirm the selectivity observed in the
experiments and indicate the formation of multiple intermo-
lecular hydrogen bonds in the most stable complexes.
Introduction
they all posses a similar size and similar functional groups but
only differ in their stereochemistry. Recent papers on synthetic
lectins exploit hydrogen bonding and CH–p interactions[4] or
(non-biomimetic) boronic acids[5] to obtain highly selective and
potent carbohydrate receptors that are also able to operate
under aqueous conditions. Artificial receptors based on 8-hy-
droxyquinoline were recently shown to display binding prefer-
ence for glycopyranosides.[6] Another useful type of host–guest
complex includes the acetylene-linked pyridine/pyridone mac-
rocycles that bind to mannosides.[7] Arguably the most spec-
tacular advances in the area of synthetic lectins come from
Davis and co-workers, who recently synthesized a cagelike re-
ceptor that binds glucose with excellent selectivity versus
other simple carbohydrates (for example, approximately 50:1
versus galactose) and sufficient affinity for glucose sensing in
blood.[8]
In biology, proteins that bind to oligosaccharides are called lec-
tins.[1] Lectins play important roles in cell–cell recognition pro-
cesses, inflammation, and infection of cells by viruses and bac-
teria. Lectins are an attractive yet difficult target for drug dis-
covery and diagnostics. Carbohydrates also play a prominent
role in cell–cell recognition.[2] Embedded in cell membranes in
the form of glycolipids or glycoproteins, they are often found
in the plasma membranes of eukaryotic cells. Oligosaccharides
enhance the hydrophilic properties of the lipids and proteins
and stabilize the conformation of membrane proteins. The
human blood-group antigens are a well-known example of
specific protein–carbohydrate interaction at the cell surface:
the A, B, and 0 antigens are closely related oligosaccharides
that are linked to lipids, and the ability of antibodies to differ-
entiate between these glycolipids with only slightly different
structures leads to specific immune reactions. Similarly, bacteri-
al proteins can also selectively bind to oligosaccharides on the
cell surfaces.
In 2010, we described the identification of carbohydrate re-
ceptors from tripeptides using dynamic combinatorial chemis-
try and the thiol–disulfide exchange reaction under thermody-
namic equilibrium.[9] We showed that the N- and C-terminal
cysteine residues of tripeptides Cys-X-Cys form disulfide bonds
with a second Cys-X-Cys tripeptide to yield exclusively cyclic
hexapeptides (Scheme 1). The cyclic hexapeptides obtained
from cysteine and histidine, Cys-His-Cys (which we call HisHis),
and cysteine and tyrosine, Cys-Tyr-Cys (which we call TyrTyr),
showed strong and selective interactions (Ka >1000mÀ1 in
aqueous solution at neutral pH) with N-acetyl neuraminic acid
(NANA) and trehalose (Tre), respectively.
Owing to the complexity and stereochemical diversity of car-
bohydrates, the identification of “synthetic lectins” poses a phe-
nomenal challenge to supramolecular chemists.[3] Binding of
lectins to carbohydrates is mediated by hydrogen bonding,
CH–p interactions, and/or metal-ion complexation. The large
number of hydroxyl groups on the carbohydrate is similar to
the surrounding biological solvent (i.e., water), which is there-
fore the main competitor for the binding site. To bind, any
given carbohydrate receptor must be able to discriminate be-
tween water and the carbohydrate so that the latter is prefer-
entially bound. The discrimination between substrate and
water is far from trivial, since carbohydrates are complex and
“camouflaged” by hydroxyl groups.[3] Furthermore, the differen-
ces between individual carbohydrates are very small because
It should be emphasized that during the thiol–disulfide ex-
change reaction in the dynamic combinatorial library of Cys-X-
Cys peptides, two constitutional isomers (parallel and antipar-
allel Cys-X-Cys; see Scheme 1) were formed. In our previous
work, only the affinity and selectivity of the mixture of isomers
was investigated. Herein, we report the synthesis and charac-
terization of the parallel and antiparallel isomers by using
solid-phase peptide chemistry and an orthogonal protection
scheme. The affinity and selectivity of each isomer for a range
of carbohydrates were studied by isothermal titration calorime-
try (ITC), NMR spectroscopy, and circular dichroism (CD) titra-
tions.
[a] Dr. M. Rauschenberg, Dr. S. Bandaru, Dr. M. P. Waller, Prof. Dr. B. J. Ravoo
Organic Chemistry Institute
Westfꢀlische Wilhelms-Universitꢀt Mꢁnster
Corrensstrasse 40, 48149 Mꢁnster (Germany)
E-mail: b.j.ravoo@uni-muenster.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303777.
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triphenylmethyl
(trityl,
Trt)
groups were selected in view of
the possibility of selectively
cleaving the Acm group and si-
multaneously forming the de-
sired disulfide bond. The Trt
group is readily deprotected
when cleaving the peptide of
the resin. In the case of the par-
allel isomers of HisHis and TyrTyr,
the free thiol function is used to
perform the disulfide exchange
reaction, which leads to the for-
mation of the first disulfide
bridge. Upon cleavage of the
Acm group with iodine under
acidic conditions, the second di-
sulfide bridge was obtained in
reasonable yield.[19]
Scheme 1. Parallel and antiparallel isomers of HisHis and TyrTyr and their most important carbohydrate targets.
Furthermore, quantum chemical calculations were per-
formed to provide structural insight into this class of peptide–
carbohydrate complexes. A balanced description of the various
noncovalent intermolecular interactions within the complex is
the key to model realistic structures. For large systems (>100
atoms), density functional theory (DFT) has gained popularity
owing to good accuracy combined with favorable scaling.[10]
The deficiencies of traditional Kohn–Sham DFT toward long-
range dispersion interactions[11–13] can be corrected using an
additional dispersion correction term (DFT-D).[14] DFT has been
extensively validated across an extremely large number of
benchmark studies of weakly interacting dimers,[15–17] and
therefore is a good choice for novel structure prediction of
these peptide-based carbohydrate receptors.
In the case of the antiparallel isomers of HisHis and TyrTyr,
the free thiol function was derivatized with a reactive disulfide
by using dithiodipyridine to prevent formation of the unde-
sired linear homodimer (Scheme 3).[20] Afterwards, the inverse
Acm-protected tripeptide was coupled using the thiol–disul-
fide exchange reaction to yield the desired (inverse) linear
hexapeptide. Upon cleavage of the Acm group under the
same conditions as mentioned above, the antiparallel cyclic
dimer was obtained in good yield.
Details of the synthesis of the parallel and antiparallel cyclic
hexapeptides can be found in the Supporting Information. For
the purpose of comparison, the mixture of isomers was also
synthesized simply by stirring the tripeptides (Cys-His-Cys or
Cys-Tyr-Cys) with two free thiol functions under basic condi-
Results and Discussion
Synthesis and characterization
of cyclic peptides
Initial attempts to synthesize the
parallel and antiparallel isomers
of HisHis and TyrTyr on 4-meth-
ylbenzhydrylamine (MBHA) resin
by using solid-phase peptide
chemistry and orthogonal pro-
tection groups failed.[18] No
product could be isolated after
cleavage from the resin. This
might be due to the formation
of mixed disulfides on the
beads, which leads to cross-link-
ing of the peptide backbone.
Therefore, orthogonal protection
Scheme 2. Synthesis of the parallel isomers of HisHis (3a) and TyrTyr (3b): i) 20% piperidine in DMF, 20 min, RT/
Fmoc-His(Trt)ÀOH (Fmoc=fluorenylmethyloxycarbonyl) or Fmoc-Tyr(tBu)OH, N,N’-diisopropylcarbodiimide
(DIPCDI), Oxyma pure, DMF, 2 h, RT/20% piperidine in DMF, 20 min, RT/Fmoc-Cys(Acm)ÀOH, DIPCDI, Oxyma pure,
DMF, 2 h, RT; ii) 94% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS), 2.5% H2O, and 2.5% 1,2-ethanedithiol
groups on the Cys residues and
disulfide formation in solution
were applied (see Scheme 2).
The acetamidomethyl (Acm) and (EDT), 6 h, RT; iii) Milli-Q, aqueous NaOH, 4 d, RT; iv) 118 mm I2, 334 mm HCl, AcOH, 2 h, RT.
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(3a) and antiparallel HisHis (7a)
with NANA, it is evident that
both isomers interact with NANA
in a 1:2 complex, but a signifi-
cantly stronger binding of the
parallel isomer is observed (see
Table 1; Figure S3 in the Sup-
porting Information). Neverthe-
less, both HisHis isomers are
potent cooperative receptors for
the binding of two equivalents
of NANA in aqueous solution.
The binding constants (Ka) are
higher, the binding enthalpy
(DH) is more negative, and the
binding entropy (DS) is more
positive for the parallel HisHis
than the antiparallel HisHis.
These findings are in agreement
with HPLC data reported previ-
ously for a dynamic combinatori-
al library of peptides that con-
tain Cys-His-Cys, which displayed
a higher amplification of the par-
Scheme 3. Synthesis of the antiparallel isomers of HisHis (7a) and TyrTyr (7b): i) 20% piperidine in DMF, 20 min,
RT/Fmoc-His(Trt)ÀOH or Fmoc-Tyr(tBu)OH, DIPCDI, Oxyma pure, DMF, 2 h, RT/20% piperidine in DMF, 20 min, RT/
Fmoc-Cys(Trt)ÀOH, DIPCDI, Oxyma pure, DMF, 2 h, RT; ii) 94% TFA, 1% TIS, 2.5% H2O, and 2.5% EDT, 6 h, RT; iii) di-
thiodipyridine (1 equiv), 2m HOAc/MeOH=10:1, 24 h, RT; iv) compound 1a or 1b, 0.1% HOAc/10 mm NH4OAc
(pH 6.0), 24 h, RT; v) 118 mm I2, 334 mm HCl, AcOH, 2 h, RT.
tions for 2 days. The purity of the isomers as well as the com-
position of the mixture was determined with HPLC by using
hydrophilic interaction liquid chromatography (HILIC). The
HPLC traces for both isomers of HisHis and TyrTyr as well as
the mixture of isomers are shown in Figures S1 and S2 of the
Supporting Information.
allel isomer of HisHis relative to the antiparallel isomer of
HisHis upon addition of NANA.[9] It can be seen in Table 1 that
for each isomer of HisHis, the binding of the second molecule
of NANA is considerably more favorable than the binding of
the first molecule of NANA: K2 is more than one order of mag-
nitude larger than K1 in each case. Binding of the second
NANA is driven by a substantial increase in entropy, which
likely originates from significant dehydration of both HisHis
and NANA upon complexation. This clearly suggests that coop-
erativity is the result of receptor preorganization by the first
NANA, which leads to an entropic penalty for the binding of
the first NANA relative to the second NANA. The higher affinity
of the parallel HisHis than the antiparallel HisHis should be
a consequence of the structural difference and the relative sta-
bility of each isomer, which will be discussed on the basis of
the DFT results (see below).
Isothermal titration calorimetry
The selectivity of the interaction of the HisHis and TyrTyr iso-
mers with different carbohydrates was studied using ITC. In
our previous work we had established that the mixture of
HisHis isomers binds NANA in a cooperative 1:2 complex (1
molecule of HisHis binds 2 molecules of NANA) with rather
strong binding constants K1 =70mÀ1 and K2 =7.76ꢁ103 mÀ1 at
neutral pH in water.[9] From the ITC titration of parallel HisHis
Table 1. Thermodynamic data for the interaction of HisHis and TyrTyr isomers (parallel, antiparallel, and a mixture of both) with selected carbohydrates
measured by isothermal titration calorimetry.
Peptide
Carbohydrate
NANA
n
Ka [mÀ1
]
DG [kJmolÀ1
]
DH [kJmolÀ1
]
DS [JKÀ1 molÀ1
]
K1 =72.7
K2 =7.76ꢁ103
K1 =143
K2 =5.08ꢁ103
K1 =94
K2 =990
9.28ꢁ103
7.96ꢁ103
n.a.[a]
DG1 =À10.6
DG2 =À22.2
DG1 =À12.6
DG2 =À21.2
DG1 =À11.3
DG2 =À17.1
À10.6
DH1 =À6.27
DH2 =À1.54
DS1 =14.6
DS2 =69.4
DS1 =3.0
DS2 =61.8
DS1 =19.5
DS2 =48.6
78.7
75.4
n.a.[a]
56.5
42.3
mixed HisHis
parallel HisHis
antiparallel HisHis
1:2
1:2
1:2
DH1 =À11.40
DH2 =À2.73
DH1 =À5.46
DH2 =À2.61
À0.39
NANA
NANA
mixed HisHis
MeGal
MeGal
MeGal
Tre
Tre
Tre
1:1
1:1
n.a.[a]
1:1
1:1
1:1
parallel HisHis
antiparallel HisHis
mixed TyrTyr
parallel TyrTyr
antiparallel TyrTyr
À22.7
À0.22
n.a.[a]
n.a.[a]
2.85ꢁ103
930
1.19ꢁ103
À9.7
À2.89
À16.7
À4.07
À18.1
À5.73
41.5
[a] Not applicable.
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Unexpectedly, investigation of the interaction of parallel
HisHis and antiparallel HisHis with methyl-a-d-galactopyrano-
side (MeGal) by ITC revealed a weakly endothermic but highly
entropically favored binding of the parallel HisHis isomer exclu-
sively (see Figure S4 in the Supporting Information). The raw
data obtained from this titration could be readily fitted to
a 1:1 model and gave a binding constant of Ka =5.12ꢁ103 mÀ1.
In the case of antiparallel HisHis, only a very small heat of dilu-
tion was observed in the ITC measurement (see Figure S4 in
the Supporting Information). Thus, in contrast to the interac-
tion of HisHis with NANA, the interaction of HisHis with MeGal
is 1:1 (not 1:2), endothermic (not exothermic), and highly selec-
tive toward the parallel isomer. Evidently, the selective interac-
tion of the parallel isomer of HisHis with NANA can only be ex-
plained from the structure and the relative stability of the par-
allel and antiparallel isomers, which will be discussed on the
basis of the DFT results (see below).
1
Figure 1. Aromatic region of the H NMR spectra of parallel HisHis, NANA,
and their 1:1, 1:2, and 1:5 mixtures.
It should be emphasized that the monosaccharides methyl
a-d-glucopyranoside (MeGlc), methyl a-d-mannopyranoside
(MeMan), and methyl a-d-fucopyranoside (MeFuc) as well as
the disaccharides sucrose (Suc) and trehalose (Tre) showed no
significant interaction with any of the HisHis isomers (see Fig-
ure S5 in the Supporting Information). These control experi-
ments confirm that parallel HisHis is a selective receptor for
NANA and MeGal that does not bind to several very similar
carbohydrates.
pression of the water signal by using an excitation sculpting
pulse sequence identifies NH and OH protons that are not ex-
changing or slowly exchanging with the aqueous solvent.[21]
The aromatic region of the NMR spectra obtained for parallel
HisHis (2 mm), NANA (2 mm), their 1:1 mixture (2 mm each),
their 1:2 mixture (2 mm parallel HisHis and 4 mm NANA), and
their 1:5 mixture (2 mm parallel HisHis and 10 mm NANA) are
shown in Figure 1. Upon addition of one equivalent of NANA
to parallel HisHis, the imidazole protons are shifted downfield
(Figure 1: marked by a blue diamond and orange circle are
shifted by d=0.041 and 0.10 ppm, respectively) owing to hy-
drogen-bond formation. This is confirmed by the simultaneous
downfield shift of the imidazole NH protons (Figure 1; marked
by a green square and shifted by d=0.058 ppm). Furthermore,
amide protons of the peptide backbone of HisHis (red penta-
gon) are also shifted downfield by d=0.086 ppm. At the same
time, the amide proton of NANA (black triangle) does not
show any shift, which indicates that it is not involved in the in-
teraction with parallel HisHis. Addition of two equivalents of
NANA leads to further downfield shifts of the imidazole reso-
nances (Figure 1: marked by a blue diamond and orange circle
are shifted by d=0.018 and 0.042 ppm, respectively), the imi-
dazole NH protons (marked by a green square and shifted by
d=0.026 ppm), and the amide protons in the backbone
(marked by a red pentagon and shifted by d=0.036 ppm). The
observed shifts are only half of what was found for the 1:1
mixture. Further addition of NANA up to five equivalents did
not result in significant shifts of any proton. The NMR spectro-
scopic experiments indicate that binding of parallel HisHis and
NANA involves the formation of hydrogen bonds by the imida-
zole residues of HisHis as well as the peptide backbone of
HisHis. The amide group of NANA does not participate in the
binding.
The selectivity of the TyrTyr isomers with different carbohy-
drates was also studied using ITC (see Table 1; Figure S6 in the
Supporting Information). In our previous work we had estab-
lished that the mixture of TyrTyr isomers binds with Tre in
a 1:1 complex with a rather strong binding constant Ka =2.85ꢁ
103 mÀ1 at neutral pH in water.[9] It was found that both isomers
of TyrTyr bind with Tre with nearly the same affinity. The inter-
action is exothermic and entropically driven. The raw data
could be readily fitted with a 1:1 model (see Figure S6 in the
Supporting Information) to give the binding constants shown
in Table 1. It should be noted that the quality of the ITC data
was affected by the low solubility of TyrTyr in the phosphate
buffer at the concentrations necessary for ITC. The observation
that both isomers of TyrTyr have the same affinity for Tre is in
agreement with HPLC data reported previously for a dynamic
combinatorial library of peptides that contain Cys-Tyr-Cys,
which displayed an equal amplification of each isomer upon
addition of Tre.[9] We note that the monosaccharides NANA,
MeGal, MeGlu, MeMan, and MeFuc as well as the disaccharide
Suc showed no interaction with either of the TyrTyr isomers
(see Figure S7 in the Supporting Information). These control
experiments confirm that TyrTyr is a selective receptor for Tre
relative to several very similar carbohydrates.
NMR spectroscopic titrations
1
The formation of hydrogen bonds in the peptide–carbohydrate
From H NMR spectroscopic water suppression experiments
1
complexes was studied by using H NMR spectroscopic meas-
with antiparallel HisHis (2 mm) and NANA (0–10 mm), the spec-
tra shown in Figure 2 were obtained. In this case, the down-
field shift observed for the imidazole protons (Figure 2:
marked by a blue diamond and orange circle are shifted by
urements of the parallel and antiparallel isomers of HisHis iso-
mers as well as of the 1:1 and 1:2 complex of each HisHis
isomer with NANA in 100 mm phosphate buffer at pH 7.4. Sup-
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1
Figure 2. Aromatic region of the H NMR spectra of antiparallel HisHis,
NANA, and their 1:1, 1:2, and 1:5 mixtures.
d=0.020 and 0.044 ppm, respectively) and the amide protons
in the peptide backbone (marked by a red pentagon and shift-
ed by d=0.043 ppm) in the presence of one equivalent of
NANA is only half of the shift that was found for the parallel
HisHis isomer. A shift of the imidazole NH protons (green
square) cannot be determined owing to overlapping with the
amide signal of NANA. No significant shift was detected for the
amide proton of NANA (black triangle). Addition of two equiv-
alents NANA results in further downfield shifts of the imidazole
protons (Figure 2: marked by a blue diamond and orange
circle are shifted by d=0.026 and 0.057 ppm, respectively) and
the amide protons in the backbone (marked by a red penta-
gon and shifted by d=0.058 ppm). Relative to the parallel
isomer of HisHis, for which the second equivalent of NANA re-
sulted in smaller shifts, the shifts observed for the antiparallel
isomer are slightly larger than the ones found for the 1:1 com-
plex. When the amount of NANA was increased to five equiva-
lents, no further shifts were detected.
1H NMR spectroscopic water suppression experiments of the
parallel isomer of HisHis (2 mm) and d-galactopyranose (Gal;
0–10 mm) in 100 mm phosphate buffer at pH 7.4 showed
a downfield shift of the imidazole protons (Figure 3a: marked
by an orange circle and blue diamond and are shifted by d=
0.10 and 0.038 ppm, respectively) of HisHis in the presence of
one equivalent of Gal, thus indicating hydrogen-bond forma-
tion. Additionally, the imidazole NH protons (Figure 3a, marked
by a green square and shifted by d=0.059 ppm) shifted down-
field. In contrast, two CH protons (Figure 3b, black and red
squares) of Gal are shifted upfield by d=0.025 and 0.023 ppm,
respectively, which also indicates hydrogen-bond formation of
the corresponding OH functions. Thus, the NMR spectroscopic
experiments indicate that binding of parallel HisHis and Gal in-
volves the formation of hydrogen bonds by the imidazole resi-
dues of HisHis with OH groups of Gal. Addition of up to five
equivalents of Gal did not result in any changes relative to the
spectra when one equivalent of Gal was added. Experiments
with antiparallel HisHis and Gal resulted in no significant shifts
of the imidazole moieties or of the carbohydrate protons (Fig-
Figure 3. 1H NMR spectra of A) the aromatic region and B) the carbohydrate
region of parallel HisHis (3a), Gal, their 1:1 mixture, and their 1:5 mixture.
ure S7 in the Supporting Information), thus indicating no sig-
nificant interaction, which is in good agreement with the re-
sults obtained by ITC titration.
In summary, the results obtained by 1H NMR spectroscopy
and ITC for the complexes of the isomers of HisHis with NANA
and MeGal, respectively, are fully consistent, but the basis for
the observed selectivity remains uncertain. One explanation
might be the different conformations of the parallel and anti-
parallel HisHis isomers. Nuclear Overhauser effect (NOE) meas-
urements of either of the isomers in the presence of NANA
and Gal were inconclusive owing to the high flexibility of the
receptors, which gives rise to a large number of different con-
formations present in the solution. In case of the TyrTyr and
Tre, no significant shifts can be detected upon addition of Tre
(see Figures S9 and S10 in the Supporting Information). Also in
this case, NOE measurements were inconclusive owing to the
large number of possible conformations present in solution.
CD measurements
The carbohydrate complexes of HisHis and TyrTyr were also in-
vestigated by CD measurements. It was found that the isomers
of HisHis did not display significant changes in their CD spec-
trum in the presence of NANA so that no meaningful informa-
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Figure 5. A) CD spectra upon titration of 80 mm trehalose into a 2 mm solu-
tion of the antiparallel isomer of TyrTyr in phosphate buffer (100mm,
pH 7.4). B) Fit of the titration data.
give a binding constant Ka =802mÀ1 that corresponds to a free
energy of binding DG=À16.7 kJmolÀ1 (Figure 4c). These find-
ings are entirely consistent with the ITC data reported in
Table 1. In the case of the antiparallel isomer of TyrTyr, a small
yet significant decrease in the CD signal at 230 nm[22] occurred
upon addition of Tre (Figure 5a). The data from this titration
could also be readily fitted to a 1:1 model (Figure 5b), thus
giving a slightly higher binding constant Ka =1.21ꢁ103 mÀ1
Figure 4. A) CD spectra upon titration of 80 mm trehalose to a 2 mm solu-
tion of the parallel isomer of TyrTyr in phosphate buffer (100mm, pH 7.4).
B) Job plot of the titration data. C) Fit of the titration data.
that corresponds to
a free energy of binding DG=
À17.7 kJmolÀ1, which again is in good agreement with the
data obtained from ITC titrations. Thus, CD measurements indi-
cate that parallel and antiparallel TyrTyr receptors have differ-
ent conformations, which each undergo a significant rear-
rangement upon binding of the carbohydrate Tre.
tion could be obtained for this complex by using this method.
However, the tyrosine moieties in TyrTyr show significant UV
absorption and Cotton bands at higher wavelength. The differ-
ent conformations of the parallel and antiparallel isomers are
evident from the different CD signals for each isomer (see Fig-
ures 4a and 5a). The CD effect observed for the tyrosine moiet-
ies upon addition of Tre is rather small but the disulfide bonds
show a significant CD effect at 230 nm for both the parallel
and the antiparallel isomer.[22] A small but significant increase
in this CD band upon addition of Tre was observed for the par-
allel isomer of TyrTyr (Figure 4a), thus indicating that the con-
formation of the disulfide bonds of the cyclic peptide changes
upon binding of the carbohydrate. The CD effect was plotted
in a Job plot, which has a pronounced maximum at 0.5, thus
indicating a 1:1 stoichiometry of the complex (Figure 4b).
Indeed, the CD data could be readily fitted with a 1:1 model to
Quantum chemical calculations
Quantum chemical calculations were performed for the com-
plexes of the parallel isomer of HisHis with NANA and MeGal.
For the sake of comparison, calculations were also performed
for the nonbinding combinations of the parallel isomer of
HisHis with MeMan as well as the antiparallel isomer of HisHis
with MeGal. Furthermore, the complexes of the parallel and
antiparallel complexes of TyrTyr with Tre were calculated. It is
reasonable to assume that the carboxylic acid group of NANA
is deprotonated at neutral pH (NANAÀ1). The pKa values of the
amino acids (pKa,COOH Cys=1.7 and pKa,NH Cys=10.4) suggest
2
that the peptides should exist in a zwitterionic form at neutral
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pH in an aqueous environment. However, the neutral form is
more stable in the gas phase owing to the lack of solva-
tion,[23,24] and therefore the density functional geometry opti-
mizations were performed on the neutral form of the peptides.
To test the effect of the protonation state in the gas phase, we
calculated the mono-, di-, and triprotonated HisHis binding en-
ergies with NANAÀ1, which resulted in values of À202, À405,
and À685 kJmolÀ1, respectively, at the BLYP-D3/def2-TZVP
level of theory. This is to be expected as the Coulombic inter-
action increases in the absence of the solvent environment. To
investigate the effect of an implicit solvation model, conduc-
tor-like screening model (COSMO)-corrected (e=78.25) binding
energies of all complexes are reported in Table 2. A set of di-
Figure 6. A) Parallel HisHis and B) antiparallel HisHis optimized at the BLYP-
D3/def2-TZVP level of theory. Only the hydrogen atoms on the imidazoles
are shown. All others are removed to increase the clarity of the diagram.
Table 2. Average electronic binding energy (BE) of the three lowest con-
formations at the BLYP-D3/def2-TZVP level of theory.[a]
[a]
Complex
BEgas [kJmolÀ1
]
BECOSMO [kJmolÀ1
]
The 1:1 complex of parallel HisHis with NANA
paraHisHis@NANAÀ1
paraHisHis@(NANAÀ1
paraHisHis@MeGal
paraHisHis@MeMan
antiHisHis@MeGal
paraTyrTyr@Tre
À202.1
À211.2
À60.02
À36.33
À28.97
À102.8
À111.3
À47.81
À87.27
À24.87
À8.99
)
2
A preliminary geometry optimization resulted in a lower-
energy conformation of the parallel isomer of HisHis bound to
NANA in a hypothetical 1:1 complex versus the antiparallel
isomer of HisHis bound to NANA in a 1:1 complex. The energy
difference was around 20 kJmolÀ1 at the PM3 level of theory,
and the coordinates are provided in the Supporting Informa-
tion. The preference for the complex with the parallel isomer
was also observed in the experimental data. Therefore, we pre-
dominately concentrate on the parallel isomer hereafter.
+6.79
À33.50
À37.81
antiTyrTyr@Tre
[a] Solvent binding energies were obtained using COSMO e=78.25.
verse starting orientations were created for each combination
of peptide and carbohydrate. We optimized 64 separate con-
formations for each complex, and we observed a large variety
of binding energies (see the Supporting Information). The aver-
age binding energies of the three lowest-energy conforma-
tions (BLYP-D3/def2-TZVP) are tabulated in Table 2.
Among the best three gas-phase DFT-optimized structures
calculated, the one that shows the highest accordance with
1
the H NMR spectroscopic results was selected (Figure 7). This
structure shows linear hydrogen bonds (O-H-O angle 1768) of
the carboxylate at the C terminus of the peptide with the car-
boxylic acid and a hydroxyl of NANA. In addition, the structure
features a hydrogen bond between the two opposing imida-
zole rings (N-H-N angle 1718), whereas the second NH is clearly
oriented towards the NANA, which suggests another potential
hydrogen bond. Thus, the calculated structure shows that
NANA binds on the outside of the peptide (exo receptor)
rather than forming an inclusion complex (endo receptor), as
Carbohydrate complexes of HisHis
The parallel and antiparallel isomers of HisHis were each opti-
mized in the gas phase by using DFT. The parallel and the anti-
parallel alignment of the tripeptides in the macrocycle lead to
very different conformations. The gas-phase conformation of
the parallel isomer of HisHis is best described as an “open”
form, whereas the conformation of the antiparallel isomer ap-
pears much more compact and resembles a helical motif (see
Figure 6). The antiparallel isomer of HisHis was calculated to be
around 10 kJmolÀ1 more stable than the parallel isomer in the
gas phase in the absence of any carbohydrate. The antiparallel
isomer is possibly stabilized owing to the favorable dipole–
dipole interaction of the two constituting tripeptides, which
are essentially oriented in an antiparallel orientation. These ini-
tial findings suggest that the open conformation of parallel
HisHis offers easy access to the imidazole moieties and there-
fore to strong interaction in the case of NANA. In contrast, the
compact and helical form of the antiparallel HisHis might
hinder carbohydrate binding.
Figure 7. Parallel HisHis bound to NANA (paraHisHis@NANAÀ1) optimized at
the BLYP-D3/def2-TZVP level of theory correlating best to the data from
1H NMR spectra. Hydrogen atoms that participate in hydrogen bonding are
shown. All others are removed to increase the clarity of the diagram.
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might be intuitively expected from a cyclic peptide. The struc-
ture also shows that parallel HisHis undergoes a substantial
conformational change to optimize hydrogen bonding with
NANA, and the interaction involves multiple hydrogen bonds
with the peptide backbone as well as the imidazole residues.
We emphasize that in the NMR spectra of a 1:1 mixture of par-
allel HisHis and NANA a pronounced downfield shift of the imi-
dazole protons was observed (see above). Thus, the calcula-
tions provide a reasonable explanation for the shifts in the
NMR spectroscopic titration. The paraHisHis@NANAÀ1 complex
was further modeled in an aqueous medium, that is, an implic-
it COSMO-type solvation model was used. As can be seen from
Table 2, a significant reduction in the binding energy is ob-
served in solution relative to the gas phase. This reduction is
to be expected as the charges are effectively screened by the
aqueous environment.
on the outside of the cyclic peptide. Upon comparison of the
structures of paraHisHis@NANAÀ1 and paraHisHis@(NANAÀ1)2 it
is evident that the peptide unfolds upon binding the second
molecule of NANA and that each imidazole ring is now form-
ing hydrogen bonds (O-H-N angle 173 and 1728, and N-H-O
angle 1778) with NANA. Furthermore, NANA is also bound by
an amide proton in the backbone by a hydrogen bond (N-H-O
angle 1788). We note that the hydrogen bonds in the calculat-
1
ed structures correlate with the shifts observed in the H NMR
spectra (see above). The smaller shift of the imidazole protons
upon addition of the second equivalent of NANA relative to
the addition of the first one is due to the imidazole ring that
serves simultaneously as a hydrogen-bond acceptor and
donor. This rearrangement might also explain the large entro-
py change detected on the second binding event with ITC be-
cause water molecules are expelled to the bulk solvent during
the second binding of the NANA.
Interestingly, the gas-phase binding energy of the 1:2 com-
plex (À211 kJmolÀ1) is only marginally higher than calculated
for the 1:1 complex (À202 kJmolÀ1), see Table 2, which indi-
cates anticooperative binding in the gas phase. This suggests
that solvation plays a substantial role in these receptors, as co-
operative binding was observed for the ITC measurements in
an aqueous environment. Indeed, the COSMO implicit solva-
tion model calculations show additivity (but not cooperativity)
in the 1:2 complex, with approximately twofold binding
energy for the 1:2 complex relative to the 1:1 complex (see
Table 2).
The 1:2 complex of parallel HisHis with NANA
In the complex of the parallel isomer of HisHis bound to two
molecules of NANA (paraHisHis@(NANAÀ1)2), two possible bind-
ing processes were investigated with DFT. A stepwise-like path-
way was compared to a concerted case. In the stepwise case,
the first NANA initially binds to HisHis, and then a subsequent
binding of the second NANA molecule completes the 1:2 com-
plex by using a two-step geometry optimization procedure.
Concerted addition occurs when the two NANA molecules si-
multaneously bind with the HisHis as a 1:2 complex therefore
using a one-step geometry optimization procedure. The corre-
sponding binding energies of the 1:2 paraHisHis@(NANAÀ1
)
2
stepwise complex are given in the Supporting Information,
and the best conformation is presented in Figure 8. The opti-
The 1:1 complexes of parallel and antiparallel HisHis with
MeGal
Interestingly, only the parallel isomer of HisHis binds to MeGal
with a binding constant that is on the same order of magni-
tude as for NANA (Table 1). In Figure 9, the gas-phase DFT-opti-
mized structure for the complex paraHisHis@MeGal is shown.
The binding energy of this complex in the gas phase is
À60 kJmolÀ1. It is evident from Figures 7–9 that the structures
of the complexes of MeGal and NANA with parallel HisHis
show a totally different binding mode. The parallel HisHis exo
Figure 8. Parallel HisHis bound to two molecules of NANA (para@(NANAÀ1)2)
optimized at the BLYP-D3/def2-TZVP level of theory correlating best to the
1
data from H NMR spectra. Hydrogen atoms that participate in hydrogen
bonding are shown. All others are removed to increase the clarity of the dia-
gram.
mization procedures failed to converge for the concerted path-
way, thus suggesting that the simultaneous binding of two
anionic ligands to the HisHis is not energetically favorable. The
fact that the stepwise pathway is more favorable in the com-
putations is in accord with the ITC data, which clearly indicate
a cooperative binding of NANA.
Figure 9. Parallel HisHis bound to MeGal at the BLYP-D3/def2-TZVP level of
theory correlating best with data from the H NMR spectra. Hydrogen atoms
1
Also in this case, the parallel isomer of HisHis acts as an exo
receptor because both NANA molecules appear to be bound
that participate in hydrogen bonding are shown. All others are removed to
increase the clarity of the diagram.
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receptor binds MeGal through a strong hydrogen bond with
the imidazole moieties and the peptide backbone. One of the
imidazole moieties forms a hydrogen bond (N-H-O angle 1778)
with the MeGal, whereas the second one forms an additional
hydrogen bond (N-H-O angle 1718) with the backbone of the
peptide (Figure 9). This might be the reason for the observed
1
downfield shift in the H NMR spectra for the imidazole pro-
tons (Figure 3). On the other hand, protons from Gal show
a upfield shift, which indicates a (His)N-H-O(MeGal) interaction
because the environment around the aromatic protons is more
electron rich in the complex. The reason for the upfield shift of
the CH protons of Gal upon binding to parallel HisHis might
also be explained by the formation of hydrogen bonds (N-H-O
angle 1788) between the receptor and the OH function on C2
and C3 of the carbohydrate. A significant binding energy of
À24.9 kJmolÀ1 is also observed when using a solvation model
for the complex between parallel HisHis and MeGal (Table 2).
This binding energy is substantially lower than for the com-
plexes of HisHis with NANA (see Table 2). This reduction in
binding energy is presumably related to the absence of formal
charge on MeGal so that it may be assumed that electrostatic
interactions significantly stabilize the gas-phase complex of
parallel HisHis and NANA.
Figure 10. A) Parallel TyrTyr and B) antiparallel TyrTyr at the BLYP-D3/def2-
TZVP level of theory. Hydrogen atoms that participate in hydrogen bonding
are shown. All others are removed to increase the clarity of the diagram.
In contrast, although a weak binding is observed in the gas-
phase calculation for the complex of antiparallel HisHis and
MeGal (see Figure S11 in the Supporting Information), no bind-
ing is found when a solvation model is employed (Table 2). To
verify whether the selectivity observed by experiment is repro-
duced by the DFT calculations, another negative control calcu-
lation was carried out: the hypothetical complex of parallel
HisHis and MeMan was also calculated (see Figure S12 in the
Supporting Information). Although significant interaction is cal-
culated for the gas phase, only a very small binding energy
(À9 kJmolÀ1) is observed when using a solvation model. Thus,
the results from the calculations are entirely consistent with
the experimental data.
The 1:1 complexes of parallel and antiparallel TyrTyr with Tre
In contrast to the parallel and antiparallel HisHis receptors de-
scribed above, the parallel and antiparallel TyrTyr show nearly
identical binding constants for the interaction with Tre in the
ITC and CD experiments. The DFT-optimized paraTyrTyr@Tre
and antiTyrTyr@Tre complexes are presented in Figure 11. It
can be observed that the structure of paraTyrTyr@Tre exhibits
hydrogen bonding to the carboxylate terminus of the peptide
to form a rather “open” complex. Tre is bound by two hydro-
gen bonds (O-H-O angle 1778) to the C=O and NÀH functions
of two adjacent amide bonds of parallel TyrTyr. Furthermore,
a nonlinear hydrogen bond (O-H-O angle 1688) is formed be-
tween the carboxylate terminus of the receptor and an OH
function of Tre. Thus, the complex features three strong hydro-
gen bonds between the peptide and the carbohydrate, and
the cyclic peptide appears to function as an exo receptor. In
contrast, the antiTyrTyr@Tre complex features a hydrogen-
bond interaction of the OH at the C6 of Tre with the Tyr side
group (O-H-O angle 1788) and moreover additional hydrogen
bonds (O-H-O angle 1718) between the C and N terminus of
the backbone and the OH at C3 and C4 of Tre. Thus, the calcu-
lations suggest that Tre is bound exclusively through hydrogen
bonding with the peptide backbone of parallel TyrTyr, whereas
it forms hydrogen bonds with the tyrosine moiety as well as
the peptide backbone of antiparallel TyrTyr. Finally, the opti-
mized complexes paraTyrTyr@Tre and antiTyrTyr@Tre suggest
that Tre binds slightly more strongly to the antiparallel isomer
than to the corresponding parallel isomer of TyrTyr. This differ-
ence at the DFT level of theory in the gas phase is around
10 kJmolÀ1 and reduces to around 4 kJmolÀ1 with the inclusion
of solvation in the COSMO model. This small energetic differ-
ence between the isomers is at least qualitatively in accord
Carbohydrate complexes of TyrTyr
The relative stabilities of the parallel and antiparallel isomers of
TyrTyr in the gas phase were computed with DFT calculations
(see Figure 10). The antiparallel isomer was found to be
around 13 kJmolÀ1 more stable than the parallel isomer. This is
presumably owing to the intramolecular hydrogen bonding of
the peptide backbones in the antiparallel isomer. The gas-
phase DFT-optimized structure of the parallel isomer displays
no such intramolecular hydrogen-bonding interaction. In addi-
tion, the antiparallel isomer is possibly stabilized owing to the
favorable dipole–dipole interaction of the constituting tripep-
tides, which are essentially oriented in an antiparallel orienta-
tion (as described above for HisHis). Also in this case, the paral-
lel isomer appears to have a rather “open” conformation,
whereas the antiparallel is more compact and “closed”. We
note that the differing conformations of the parallel and anti-
parallel isomers are also clearly observed in the CD spectra
(see Figures 4 and 5)
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mations of the each isomer. Whereas the parallel isomer of
HisHis has an open structure by which the imidazole moieties
can be easily accessed, the antiparallel isomer of HisHis has
a closed structure that might hamper the interaction between
the imidazoles and the carbohydrate. According to the calcula-
tions, both HisHis and TyrTyr are exo receptors for their carbo-
hydrate targets, which are not included in the macrocycle but
bind edge-on to the peptides. The calculations confirm the se-
lectivity observed in the experiments and indicate the forma-
tion of multiple intermolecular hydrogen bonds in the most
stable complexes. These findings have significantly increased
our understanding of a new class of biomimetic carbohydrate
receptors that had previously been identified using a dynamic
combinatorial library of peptides. It is hoped that one day
these receptors may find application in biomedicine and bio-
technology.
Experimental Section
HPLC
The parallel and antiparallel isomers as well as the mixture of iso-
mers were analyzed with a LC setup that comprised a Shimadzu
LC20 prominence HPLC system. For controlling the LC, LCsolutions
(version 1.0.0.1, Shimadzu) was used. The separation was carried
out using a ZORBAX HILIC Plus, 2.1ꢁ100 mm, 3.5 mm. The flow
rate was 0.10 mLminÀ1, the oven was maintained at 258C, and the
injection volume was 5 mL. Acetonitrile (90%) and NH4OAc (10%,
10 mm, pH 6.8) were used as the mobile phase. The complete run
time was set to 55 min. Samples were prepared by dissolving
a minimum amount of the solid peptide in 1 mL ACN/NH4OAc
(90:10).
Figure 11. Complexes of A) parallel and B) antiparallel TyrTyr with trehalose
at the BLYP-D3/def2-TZVP level of theory. Structures that correlate best with
1
the data from the H NMR spectra are shown. Hydrogen atoms that partici-
pate in hydrogen bonding are shown. All others are removed to increase
the clarity of the diagram.
with our reported ITC and CD data, which shows very similar
binding constants for both isomers of TyrTyr with Tre.
Conclusion
Isothermal titration calorimetry
We have investigated two biomimetic carbohydrate receptors,
HisHis and TyrTyr, which are composed of two tripeptides, Cys-
His-Cys and Cys-Tyr-Cys, linked by two disulfides. The parallel
and antiparallel isomer of each cyclic peptide was synthesized
separately. It was found that the parallel isomer of HisHis binds
NANA with a significantly higher affinity than the correspond-
ing antiparallel isomer. Nevertheless, both isomers are high-af-
finity receptors for NANA, and they display a cooperative bind-
ing of two molecules of NANA in aqueous solution at neutral
pH. In contrast, although the parallel HisHis isomer binds
strongly to MeGal, no interaction was found for the antiparallel
HisHis and MeGal by ITC and NMR spectroscopy. In the case of
the TyrTyr isomers and Tre, the antiparallel isomer has a slightly
higher affinity towards the carbohydrate target than the paral-
lel isomer. Four other very similar carbohydrates showed no
significant interaction with any isomer of the two peptides.
Quantum chemical calculations were used to model these pep-
ITC measurements were performed using a NanoITC system (Calo-
rimetry Sciences Cooperation, USA) and ITC Run software. Sample
solutions were prepared by dissolving the appropriate amount of
peptide in phosphate buffer (100 mm, pH 7.4). Before filling the
cell, the solution was degassed for 30 min. The peptide—2 mm for
parallel HisHis and TyrTyr, or 1 mm for antiparallel HisHis and
TyrTyr—was titrated with a degassed solution (30 min) of NANA
(40 mm), MeGal (25 mm), or Tre (12 mm) in phosphate buffer. All
measurements were performed at 238C using a stirring rate of
300 rpm and a 400 s interval between each injection. To determine
the heat of dilution, a carbohydrate solution was titrated into
phosphate buffer (100 mm, pH 7.4). The heat of dilution was sub-
tracted from the raw heat data. The data was fitted to a 1:1 or 1:2
model by using a spreadsheet method.[25]
1H NMR spectroscopic water suppression experiments
The NMR spectroscopic samples were prepared by dissolving the
appropriate amount of peptide and carbohydrate in phosphate
buffer (0.8 mL, 100 mm, pH 7.4). Typically, peptide (2 mm) was
mixed with carbohydrate (0, 2, 4, or 10 mm). After mixing, the sam-
ples were measured using a Varian Inova 500 NMR at 300 K using
the 1D wet sequence to suppress the water signal. Chemical shifts
are reported as relative values versus tetramethylsilane and were
calculated using MestReNova (version 6.0.3–5604, MestRelab Re-
search S.L.).
1
tide–carbohydrate complexes. With reference to H NMR spec-
troscopy water suppression experiments, a possible complex
structure was selected from the three best DFT structures. Fur-
thermore, the inclusion of implicit solvation models was found
to be crucial to understand the relative difference between the
complexes in experiments. Differences in the affinities for each
carbohydrate may be explained by the totally different confor-
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CD titrations
Acknowledgements
CD titrations were performed using a J815 CD spectrometer
(JASCO). The data were analyzed using OriginPro 8.0 (OrginLab Co-
operation, Northhampton, USA, version 8.0724(B724)). As back-
ground, the used phosphate buffer was measured and subtracted
after measuring each sample. A 1.0 mm solution of parallel TyrTyr
or a 2.0 mm solution of antiparallel TyrTyr in phosphate buffer was
put into a quartz glass cuvette (104F-QS, 10.00 mm, Brand), mea-
sured from 180 to 300 nm nine times, and afterwards accumulated
using the Spectra Manager Version 2 software (version 2.08.04,
JASCO). An 80 mm Tre solution in buffer was added in 1.7 mL steps.
A blank measurement was performed by titrating the correspond-
ing aliquots of buffer into a 1.0 or 2.0 mm peptide solution. All
measurements were performed after mixing the solutions and
waiting for 60 s before starting the measurements. The data were
fitted to a 1:1 model.
The Deutsche Forschungsgemeinschaft (DFG) is gratefully ac-
knowledged for financial support (IRTG 1143 and SFB 858). This
work was supported by COST CM1005 “Supramolecular
Chemistry in Water”.
Keywords: carbohydrates · lectins · molecular recognition ·
peptides · receptors
[1] H. Lis, N. Sharon, Chem. Rev. 1998, 98, 637–674.
[2] a) A. Varki, Glycobiology 1993, 3, 97–130; b) R. A. Laine, Glycobiology
1994, 4, 759–767; c) B. T. Houseman, M. Mrksich, Top. Curr. Chem. 2002,
218, 1–44; d) T. K. Lindhorst, Essentials of Carbohydrate Chemistry and
Biochemistry; Wiley-VCH Weinheim, 2007.
[3] a) A. P. Davis, R. S. Wareham, Angew. Chem. 1999, 111, 3160–3179;
Angew. Chem. Int. Ed. 1999, 38, 2978–2996; b) S. Kubik, Angew. Chem.
2009, 121, 1750–1753; Angew. Chem. Int. Ed. 2009, 48, 1722–1725.
[4] a) E. Klein, M. P. Crump, A. P. Davis, Angew. Chem. 2005, 117, 302–306;
Angew. Chem. Int. Ed. 2005, 44, 298–302; b) Y. Ferrand, M. P. Crump,
A. P. Davis, Science 2007, 318, 619–622; c) K. Ramꢂrez-Gualito, R. Alonso-
Rios, B. Quiroz-Garcia, A. Rojas-Aguilar, D. Diaz, J. Jimenez-Barbero, G.
Cuevas, J. Am. Chem. Soc. 2009, 131, 18119–18128; d) M. Mazik, Chem.
Soc. Rev. 2009, 38, 935–956; e) Y. Ferrand, E. Klein, N. P. Barwell, M. P.
Crump, J. Jimꢃnez-Barbero, C. Vicent, G.-J. Boons, S. Ingale, A. P. Davis,
Angew. Chem. 2009, 121, 1807–1811; Angew. Chem. Int. Ed. 2009, 48,
1775–1779; f) N. P. Barwell, M. P. Crump, A. P. Davis, Angew. Chem.
2009, 121, 7809–7812; Angew. Chem. Int. Ed. 2009, 48, 7673–7676;
g) N. P. Barwell, A. P. Davis, J. Org. Chem. 2011, 76, 6548–6857.
[5] a) G. Wulff, Angew. Chem. 1995, 107, 1958–1979; Angew. Chem. Int. Ed.
Engl. 1995, 34, 1812–1832; b) S. Striegler, Curr. Org. Chem. 2003, 7, 81–
102; c) G. Heinrichs, M. Schellentrager, S. Kubik, Eur. J. Org. Chem. 2006,
4177–4186; d) A. Pal, M. Bꢃrubꢃ, D. Hall, Angew. Chem. 2010, 122,
1534–1537; Angew. Chem. Int. Ed. 2010, 49, 1492–1495.
Computational methods
Herein we applied a multi-tiered meta-optimization strategy:
1) The metaheuristic particle swarm optimization (PSO) technique
was employed to iteratively improve on a candidate solution with
regards to maximizing the binding energy. The particle swarm opti-
mization calculations were carried out using an in-house code that
utilizes the JSwarm API.[26] The dimensions of acceptable search
space were determined by trial and error. In total twelve degrees
of freedom were used as the search space (x, y, z, a, b, g), which
are simply a Cartesian framework with corresponding Euler angles
for rigid rotation. The internal degrees of freedom for the mono-
mers were frozen.
2) A simulated annealing (SA)-based approach was used, whereby
a single carbohydrate molecule binds to the HisHis (1:1) or a dimer
(1:2) is constructed in either a concerted or stepwise fashion. Ten
diverse structures were heated to 600 K, simulated for 300 ps, then
cooled to 300 K, and simulated for a further 300 ps. The atom-cen-
tered density matrix propagation (ADMP) method implemented
within Gaussian 09[27] was used for the molecular dynamics trajec-
tories. The PM3 Hamiltonian was used for the ADMP trajectories. A
total of ten high/low cycles were performed, and a single random
snapshot was extracted from each of the cooling cycles, therefore
we generated 640 conformations for each complex. We have ran-
domly taken 64 conformers to be subsequently optimized with
DFT in the subsequent step.
[6] M. Mazik, C. Gefferet, Org. Biomol. Chem. 2011, 9, 2319–2326.
[7] H. Abe, Y. Chida, H. Kurokawa, M. Inouye, J. Org. Chem. 2011, 76, 3366–
3371.
[8] C. Ke, H. Destecroix, M. P. Crump, A. P. Davis, Nat. Chem. 2012, 4, 718–
723.
[9] M. Rauschenberg, S. Bomke, U. Karst, B. J. Ravoo, Angew. Chem. 2010,
122, 7498–7503; Angew. Chem. Int. Ed. 2010, 49, 7340–7345.
[10] M. P. Waller, H. Kruse, C. Mꢄck-Lichtenfeld, S. Grimme, Chem. Soc. Rev.
2012, 41, 3119–3128.
[11] P. Hobza, J. Sponer, T. Reschel, J. Comput. Chem. 1995, 16, 1315–1325.
ˇ
[12] J. Cerny, P. Hobza, Phys. Chem. Chem. Phys. 2005, 7, 1624–1626.
[13] E. R. Johnson, R. A. Wolkow, G. A. DiLabio, Chem. Phys. Lett. 2004, 394,
334–338.
[14] a) S. Grimme, J. Chem. Phys. 2006, 124, 034108; b) S. Grimme, J. Antony,
S. Ehrlich, H. Krieg, J. Chem. Phys. 2010, 132, 154104.
3) For the DFT calculations, TURBOMOLE was used with the BLYP
functional combined with Grimme’s third-generation dispersion
correction, in conjunction with the def2-TZVP[28] basis set. The reso-
lution-of-the-identity approximations together with suitable auxili-
ary fitting functions from the TURBOMOLE library were applied
throughout. The corresponding binding energies are shown in the
above (see Table 2).
4) COSMO was invoked within TURBOMOLE 6.3[29] to model solva-
tion effects. COSMO is a well-known continuum solvation model,
whereby a cavity within a dielectric continuum of permittivity (e) is
created by the solute molecule. The dielectric constant of e=78.35
was chosen to model the aqueous environment of the experiment.
COSMO single-point energies were computed at the RI-BLYP/def2-
TZVP level of theory (see Table 2).
ˇ
[15] P. Jurecka, J. Sponer, J. Cerny, P. Hobza, Phys. Chem. Chem. Phys. 2006,
8, 1985–1993.
ˇ
ˇ
ˇ
[16] L. Grꢅfovꢅ, M. Pitonꢅk, J. Rezꢅc, P. Hobza, J. Chem. Theory Comput. 2010,
6, 2365–2376.
[17] S. T. Schneebeli, A. D. Bochevarov, R. A. Friesner, J. Chem. Theory
Comput. 2011, 7, 658–668.
[18] a) R. Matsueda, S. Higashida, F. Alberico, D. Andreu, Peptide Res. 1992, 5,
262–264; b) A. K. Galande, A. F. Spatola, Org. Lett. 2003, 5, 3431–3434.
[19] S. Zhang, F. Lin, M. A. Hossain, F. Shabanpoor, G. W. Tregear, J. D. Wade,
Int. J. Pept. Res. Ther. 2008, 14, 301–305.
[20] a) D. Andreu, F. Alberico, N. A. Sole, M. C. Munson, M. Ferrer, G. Barany
in Methods in Molecular Biology (Eds: M. W. Pennington, B. M. Dunn),
Humana Press: Totowa, 1994, pp. 91–160; b) M. S. Bernatowicz, R. Mat-
sueda, G. R. Matsueda, Int. J. Pept. Protein Res. 1986, 28, 107–112; c) A.
Schulz, K. Adermann, M. Eulitz, S. M. Feller, C. Kardinal, Tetrahedron
2000, 56, 3889–3891.
[21] a) T. L. Hwang, A. J. Shaka, J. Magn. Reson. Ser. A 1995, 112, 275–279;
b) P. Besenius, K. P. van den Hout, H. M. H. G. Albers, T. F. A. de Greef,
L. L. C. Olijve, T. M. Hermans, B. F. M. de Waal, P. H. H. Bomans, N. A. J. M.
Chem. Eur. J. 2014, 20, 2770 – 2782
www.chemeurj.org
2781
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Sommerdijk, G. Portale, A. R. A. Palmans, M. H. P. van Genderen,
J. A. J. M. Vekemans, E. W. Meijer, Chem. Eur. J. 2011, 17, 5193–5203.
[22] G. Pescitelli, L. Di Bari, N. Berova, Chem. Soc. Rev. 2011, 40, 4603–4625.
[23] K. ꢆsapaz, W. S. Young, D. Bashford, C. L. Brooks, D. A. Case, J. Phys.
Chem. 1996, 100, 2698–2705.
[24] M. Knapp-Mohammadya, K. J. Jalkanena, F. Nardib, R. C. Wadeb, S.
Suhaia, Chem. Phys. 1999, 240, 63–77.
[25] J. Huskens, H. van Bekkum, J. A. Peters, Comput. Chem. 1995, 19, 409–
416.
M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Ko-
bayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyen-
gar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B.
Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,
S. Dapprich, A. D. Daniels, ꢆ. Farkas, J. B. Foresman, J. V. Ortiz, J.
Cioslowski, D. J. Fox, Gaussian, Inc., Wallingford CT, 2009.
[28] A. Schꢇfer, C. Huber, R. Ahlrichs, J. Chem. Phys. 1994, 100, 5829–5835.
[29] TURBOMOLE V6.3 2011, a development of University of Karlsruhe and
Forschungszentrum Karlsruhe GmbH, 1989–2007, TURBOMOLE GmbH,
since 2007; available from http://www.turbomole.com.
[26] P. Cingolani, http://jswarm-pso.sourceforge.net/.
[27] Gaussian 09, Revision A.1, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.
Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Men-
nucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian,
A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro,
Received: September 26, 2013
Published online on February 13, 2014
Chem. Eur. J. 2014, 20, 2770 – 2782
www.chemeurj.org
2782
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim