Carbohydrate-π interactions: What are they worth?

Article abstract of DOI:10.1021/ja803960x

Protein-carbohydrate interactions play an important role in many biologically important processes. The recognition is mediated by a number of noncovalent interactions, including an interaction between the α-face of the carbohydrate and the aromatic side chain of the protein. To elucidate this interaction, it has been studied in the context of a β-hairpin in aqueous solution, in which the interaction can be investigated in the absence of other cooperative noncovalent interactions. In this β-hairpin system, both the aromatic side chain and the carbohydrate were varied in an effort to gain greater insight into the driving force and magnitude of the carbohydrate-π interaction. The magnitude of the interaction was found to vary from -0.5 to -0.8 kcal/mol, depending on the nature of the aromatic ring and the carbohydrate. Replacement of the aromatic ring with an aliphatic group resulted in a decrease in interaction energy to -0.1 kcal/mol, providing evidence for the contribution of CH-π interactions to the driving force. These findings demonstrate the significance of carbohydrate-π interactions within biological systems and also their utility as a molecular recognition element in designed systems.

Full text of DOI:10.1021/ja803960x

Published on Web 10/10/2008
Carbohydrate-π Interactions: What Are They Worth?
Zachary R. Laughrey, Sarah E. Kiehna,‡ Alex J. Riemen, and Marcey L. Waters*
Department of Chemistry, CB 3290, UniVersity of North Carolina,
Chapel Hill, North Carolina 27599
Received June 3, 2008; E-mail: mlwaters@email.unc.edu
Abstract: Protein-carbohydrate interactions play an important role in many biologically important processes.
The recognition is mediated by a number of noncovalent interactions, including an interaction between the
R-face of the carbohydrate and the aromatic side chain of the protein. To elucidate this interaction, it has
been studied in the context of a ꢀ-hairpin in aqueous solution, in which the interaction can be investigated
in the absence of other cooperative noncovalent interactions. In this ꢀ-hairpin system, both the aromatic
side chain and the carbohydrate were varied in an effort to gain greater insight into the driving force and
magnitude of the carbohydrate-π interaction. The magnitude of the interaction was found to vary from
-0.5 to -0.8 kcal/mol, depending on the nature of the aromatic ring and the carbohydrate. Replacement
of the aromatic ring with an aliphatic group resulted in a decrease in interaction energy to -0.1 kcal/mol,
providing evidence for the contribution of CH-π interactions to the driving force. These findings demonstrate
the significance of carbohydrate-π interactions within biological systems and also their utility as a molecular
recognition element in designed systems.
Introduction
Many biological processes, including bacterial cell wall
recognition, viral and bacterial infections, and fertilization, rely
on carbohydrate-protein interactions.1,2 Additionally, glyco-
sylation as a post-translational modification affects the hydration
and conformation of a protein.3,4 Because of its significance in
biology, understanding the driving force for binding of carbo-
hydrates in water is an active area of research.5-7 In addition
to hydrogen bonding, a common feature of carbohydrate-binding
proteins is the interaction of the R-face of the carbohydrate with
the face of an aromatic side chain (Figure 1).8,9 Carbohydrate-π
interactions have been investigated through a variety of analyti-
cal techniques, including NMR, IR, molecular modeling, and
X-ray analysis.10-16 The Simons group utilized IR and molec-
Figure 1. (a) Interaction between glucose and Trp 183 in the Escherichia
coli chemoreceptor protein (PDB entry 2GBP).9 (b) Interaction geometry
for Trp and Ac4Glc in the context of a ꢀ-hairpin peptide.21
ular modeling to examine the interaction.17 Jimenez-Barbero
et al.18-20 have used NMR and molecular modeling to examine
the driving force for binding of oligosaccharides to the hevein
domain and variations thereof. These studies indicate that
‡ Current address: Nanotope, Inc., 8025 Lamon Ave., Suite 450, Skokie,
IL 60077.
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2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 14625–14633 14625

A R T I C L E S
Laughrey et al.
Figure 2. Structures of the ꢀ-hairpin and the X and Z side chains.
Results and Discussion
carbohydrate-π interactions are important for the recognition
of carbohydrates and that these interactions are dependent on
the electronic nature of the aromatic group. However, there is
a limited amount of experimental data investigating the
favorable contribution of carbohydrate-π interactions in
isolation.15,16 Given the importance of carbohydrate recogni-
tion in biology, a better understanding of the role of
carbohydrate-π interactions is warranted.
We previously reported an attractive interaction between a
tryptophan and a diagonally cross-strand tetraacetylglucoserine
[Ser(Ac4Glc)] that stabilized the folding of a ꢀ-hairpin.21 An
examination of the proton NMR shifts of the carbohydrate
protons demonstrated that the interaction was primarily through
the R-face of the carbohydrate and the face of the Trp side chain,
suggesting a carbohydrate-π interaction. Upfield shifting and
NOE data were consistent with the geometry shown in Figure
1b. The magnitude of the interaction was found to be -0.8 kcal/
mol, which is greater than the magnitude of a Lys-Trp
cation-π interaction measured in the same model system.22
However, when the acetyl groups on the glucose were removed,
the interaction between the carbohydrate and tryptophan was
lost. The reduction was attributed to the increased desolvation
cost of the unprotected glucose.
Design. The 12-residue sequence used in this study was based
on previously described peptides in which a stabilizing carbo-
hydrate-π interaction between a Trp at position 2 and tetraa-
cylated glucose at position 9 was explored.21 Several key
features were maintained, including a +3 charge to provide
solubility and discourage aggregation, an Asn-Gly turn nuclea-
tor sequence, and hydrophobic clusters on both the HB face
(Val 3, Val 5, and Ile 10) and the NHB face (X 2 and Leu 11)
of the hairpin. Aromatic amino acids and carbohydrates were
placed in positions 2 (X) and 9 (Z), respectively. These positions
have been shown to allow diagonal cross-strand interactions
and provide ample room for the bulky side chains.22-25 The
glycosylated series were synthesized via literature methods
and introduced into the peptide chain as Fmoc-protected
amino acids (see the Supporting Information).21,26-31
Characterization of Structure. ꢀ-Hairpin structure charac-
terization was accomplished by a number of NMR measure-
ments, including carbohydrate chemical shifts, R-hydrogen (HR)
chemical shifts, glycine splitting, and cross-strand nuclear
(23) Hughes, R. M.; Waters, M. L. J. Am. Chem. Soc. 2005, 127, 6518–
6519.
To further investigate the efficacy of an isolated carbohy-
drate-π interaction in aqueous solution, two series of peptides
were synthesized and studied. In the first series, aromatic or
aliphatic residues (X) were incorporated in close proximity to
a Ser(Ac4Glc) on the face of a ꢀ-hairpin (Figure 2). In the second
series, Trp was used as the aromatic residue at position X and
the nature of the carbohydrate (Z) in Ser(Z) was varied.
(24) Hughes, R. M.; Waters, M. L. J. Am. Chem. Soc. 2006, 128, 13586–
13591.
(25) Tatko, C. D.; Waters, M. L. Protein Sci. 2003, 12, 2443–2452.
(26) Salvador, L. A.; Elofsson, M.; Kihlberg, J. Tetrahedron 1995, 51,
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2002, 3, 775–782.
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36, 2953–2956.
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4026–4028.
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2001, 66, 2327–2342.
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9
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Carbohydrate-π Interactions: What Are They Worth?
A R T I C L E S
Figure 3. Electrostatic potential maps of the side chains at position X in the ꢀ-hairpin: (a) indole; (b) naphthalene; (c) benzene; (d) cyclohexane. The maps
were generated using MacSpartan at the HF/6-31G* level, with an isodensity value of 0.02 and a range of -25 kcal/mol (red, electron-rich) to 25 kcal/mol
(blue, electron-poor).
Table 1. Fraction Folded and ∆G°(folding) at 298 K for ꢀ-Hairpinsa
∆δGly
fraction folded
(Gly splitting)c
fraction folded
(HR)d
∆G°(folding)
(kcal mol-1 e
X
Z
(ppm)b
)
Trpf
1-Nal
2-Nal
Phe
Ac4Glc
Ac4Glc
Ac4Glc
Ac4Glc
Ac4Glc
0.484
0.484
0.471
0.325
0.242
0.85
0.86
0.83
0.57
0.45
0.83 (0.02)
0.83 (0.03)
0.81 (0.04)
0.51 (0.13)
0.42 (0.12)
-1.03
-1.08
-0.94
-0.17
0.12
Cha
a Conditions: 50 mM sodium acetate-d4, pH 4.0 (uncorrected) at 298
K, referenced to DSS. b Error is (0.005 ppm. c Error is (0.01. d The
HR fraction folded was determined from the average of the values for
Val 3, Val 5, Lys 8, and Ile 10. Standard deviations are shown in
parentheses. e ∆G° was determined using glycine-splitting values; the
error is (0.05 kcal/mol. f Previously reported in ref 21.
Figure 4. Fraction folded as determined from HR chemical shifts. Values
for WS(Ac4Glc) were originally reported in ref 21.
Overhauser effects (NOEs), as described below. NMR spec-
troscopy provides insight into the geometry of the interaction,
as the carbohydrate protons are shifted upfield when in close
proximity to the face of the aromatic side chains because of
ring-current effects.32 The extent of downfield shifting of HR
relative to the random coil is an indicator of the extent of ꢀ-sheet
conformation at each position along the strand. Downfield
shifting of HR by >0.1 ppm is indicative of ꢀ-hairpin forma-
tion.33 The fraction folded at each residue can be determined
by comparing the observed HR chemical shifts to those in the
unfolded and fully folded states (as obtained from an unfolded
control peptide and a cyclic control peptide, respectively; see
the Experimental Section).56 Alternatively, glycine splitting,
when compared to a cyclic control, acts as a global indicator of
ꢀ-hairpin conformation.34 Fraction-folded values determined
from HR shifting and Gly splitting were generally in good
agreement. Finally, long-distance cross-strand NOEs between
side chains are consistent with ꢀ-hairpin structure and were
observed for all peptides.
Figure 5. (a) Upfield shifting of carbohydrate protons in peptides WS(Ac4-
Glc), 1-NalS(Ac4Glc), 2-NalS(Ac4Glc), PheS(Ac4Glc), and ChaS(Ac4Glc).
Conditions: 50 mM sodium acetate-d4, pH 4.0 (uncorrected) at 298 K,
referenced to DSS. Values for WS(Ac4Glc) were originally reported in
ref 21.
Variation of the Aromatic Side Chain (X). To examine the
role of the aromatic side chain, X, a series of peptides were
synthesized in which Trp was replaced with other aromatic or
hydrophobic side chains, including 1-Nal, 2-Nal, Phe, and Cha
(Figure 2), while the carbohydrate Z was maintained as Ac4Glc.
1-Nal was substituted for Trp to investigate the significance
of the NH group in Trp. In addition, 1-Nal has a greater surface
area than Trp (161 Å2 for 1-Nal compared with 147 Å2 for Trp),
and the electron density on the face of the ring is not as great
(Figure 3). This substitution results in a well-folded peptide that
is folded to the same extent as WS(Ac4Glc) within experimental
error (Table 1 and Figure 4). The protons on the R-face (H1,
H3, and H5) are all shifted upfield, indicating that the R-face
packs against the aromatic face of 1-Nal (Figure 5), similar to
the shifting observed for WS(Ac4Glc). Protons H6 and H6′ were
also found to be shifted to a lesser extent, indicating that the
exocyclic CH2 interacts with the aromatic face. This interaction
and geometry has precedent in galactose-binding lectins. While
the acetyl groups could not be assigned, the maximum upfield
shift was e0.07 ppm (assuming that the methyl group peak that
was farthest downfield in the control peptide was the farthest
upfield in the ꢀ-hairpin). This is significantly less than the shifts
of the protons of the R-face of the carbohydrate, which ranged
from -0.6 to -1.2 ppm, and indicates that the acetyl groups
play little or no direct role in the stabilizing interaction. NOESY
NMR displayed long-distance cross-strand interactions in the
peptide, indicating that the peptide is properly folded in a
ꢀ-hairpin structure. Strong NOEs were also observed between
the carbohydrate and 1-Nal (Figure 6), although not as
extensively as seen with Trp.
Because the ꢀ-sheet propensity of each amino acid influences
the stability of the folded state, one cannot directly compare
the extents of folding of two peptides in which X has been varied
and attribute differences exclusively to side chain-side chain
interactions. To determine the energetic contribution of the
(32) Pople, J. A. J. Chem. Phys. 1956, 24, 1111.
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9
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A R T I C L E S
Laughrey et al.
Figure 6. Unambiguous NOEs (red arrows) observed between X (Trp, 1-Nal, 2-Nal, Phe, or Cha) and the carbohydrate side chain. Unambiguous NOEs are
defined as ones that can be definitively assigned to a particular set of protons.
diagonal side chain-side chain interaction alone, a double
mutant cycle was completed.33-39 A double mutant cycle
replaces two interacting side chains with two noninteracting
ones. A single mutation disrupts the interaction of interest
but could additionally cause other changes to stability (i.e.,
ꢀ-sheet propensity, hydrogen bonding, etc.). The double
mutant corrects for these unintentional changes, leaving only
the noncovalent interaction of interest. In this study, X and
Ser(Z) were exchanged for Leu and Ser, respectively. Leu
was chosen because it has a high ꢀ-sheet propensity that
Table 2. Diagonal Interaction Energies between Residues 2 and 9
As Determined by Double Mutant Cycles
X
Z
∆∆G (kcal mol-1 a
)
Trpb
1-Nal
2-Nal
Phe
Ac4Glc
Ac4Glc
Ac4Glc
Ac4Glc
Ac4Glc
-0.8
-0.7
-0.7
-0.5
-0.1
Cha
a Error is (0.1 kcal mol-1
.
b Previously reported in ref 21.
minimizes the net loss of ꢀ-hairpin stability. Ser was chosen
because it has a small polar side chain that does not interact
diagonally. The double-mutant cycle revealed that the
interaction of 1-Nal with Ser(Ac4Glc) has a magnitude similar
to that of the Trp-Ser(Ac4Glc) interaction (Table 2).
2-Nal was substituted at the X position to determine the
influence of the orientation difference between 1-Nal and 2-Nal
on the carbohydrate-π interaction. The results were similar to
(35) Serrano, L.; Bycroft, M.; Fersht, A. R. J. Mol. Biol. 1991, 218, 465–
475.
(36) Schreiber, G.; Fersht, A. R. J. Mol. Biol. 1995, 248, 478–486.
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9
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Carbohydrate-π Interactions: What Are They Worth?
A R T I C L E S
Table 3. Thermodynamic Parameters for Folding at 298 K
Obtained from Thermal Denaturation of the Peptides
peptide
∆H° (kcal mol-1
)
∆S° (cal mol-1 K-1
)
∆Cp (cal mol-1 K-1
)
WS(Ac4Glc)a
FS(Ac4Glc)
ChaS(Ac4Glc)
-5.9
-4.23
-2.96
-16.4
-13.77
-10.32
-112
-77
-88
a Previously reported in ref 21.
those for both 1-Nal and Trp, indicating that the carbohydrate
interacts in a favorable manner with 2-Nal via stacking with
the aromatic side chain despite the differences in orientation of
the two side chains (Table 1 and Figure 4). The upfield shifting
of the peptide HR protons demonstrates that the ꢀ-hairpin is
well-formed throughout the peptide with a stability similar to
those of WS(Ac4Glc) and 1-NalS(Ac4Glc). Additionally, the
carbohydrate shifts are similar in magnitude to those for the
other two peptides with large aromatic groups (Figure 5): the
protons of the carbohydrate’s R-face are shifted significantly
upfield, indicating an interaction with the aromatic face. Long-
distance NOEs between the carbohydrate’s R-face and 2-Nal
also support a stacking geometry (Figure 6). The magnitude of
the interaction of 2-Nal with Ac4Glc also was found to be similar
to those involving Trp and 1-Nal, as determined from the double
mutant cycle (Table 2).
The situation changed when the smaller Phe group was placed
in the sequence at position 2. The fraction folded decreases from
0.85 to 0.57 (Figure 4 and Table 1). Phe is known to have a
lower ꢀ-sheet propensity than Trp,40,57 but the double mutant
cycle indicates that the loss in ꢀ-hairpin stability is due in part
to a weakening of the carbohydrate-π interaction (Table 2).
Additionally, the carbohydrate’s R-face protons are not shifted
upfield nearly as much as with the larger aromatic X groups.
There are significantly fewer unambiguous NOEs between the
Phe side chain and the face of the carbohydrate than between
the larger aromatic side chains and the carbohydrate. The smaller
changes in the chemical shifts of both the HR and the
carbohydrate protons indicate a less folded hairpin and a less
favorable carbohydrate-π interaction.
When the aromatic nature of the X side chain was removed
by replacing Phe with Cha, the stability of the hairpin was
further reduced (Table 1), despite the fact that Cha has been
shown to have a higher ꢀ-sheet propensity than Phe.41 A double
mutant cycle indicates that the interaction of Cha with Ac4Glc
is weaker than that of Phe with Ac4Glc (Table 2), despite the
similar facial surface area. In contrast to the results for the
aromatic peptides in this series, there are no unambiguous NOEs
between the cyclohexane side chain and the carbohydrate (Figure
6). Since Cha is not aromatic, no shifting of the carbohydrate
protons is observed.
To provide additional insight into the effect of the X group
on the interaction with Ac4Glc, we performed thermal dena-
turations on WS(Ac4Glc), FS(Ac4Glc), and ChaS(Ac4Glc) using
variable-temperature NMR.33 Fitting of the data provided ∆H°,
∆S°, and ∆Cp values for folding (Table 3). Since the only
change in the peptide sequence is the X group at position 2,
changes in the driving force for folding can be attributed to the
role of that residue in stabilizing the folded state. One can see
that for Trp and Phe, folding is enthalpically more favorable
than for Cha, which does not significantly interact with Ac4Glc.
Figure 7. Trp-Gal interaction in the binding pocket of galectin.43
Table 4. Fraction Folded and ∆G°(folding) for ꢀ-Hairpins at 298 Ka
∆δGly
fraction folded
(Gly splitting)c
fraction folded
(HR)d
∆G°(folding)
X
Z
(ppm)b
(kcal/mol)e
Trpf Ac4Glc
0.484
0.466
0.85
0.82
0.73
0.70
0.88
0.65
0.64
0.83 (0.02)
0.77 (0.04)
0.66 (0.09)
0.64 (0.02)
0.81 (0.01)
0.63 (0.02)
0.60 (0.01)
-1.03
-0.90
-0.59
-0.50
-1.18
-0.37
-0.34
Trp
Trp
Trp
Trp
Ac4Gal
Ac3GlcNAc 0.421
GlcNAc
Me4Glc
0.392
0.50
0.383
0.375
Trpf Glc
Trp
OH
a Conditions: 50 mM sodium acetate-d4, pH 4.0 (uncorrected) at 298
K, referenced to DSS. b Error is (0.005 ppm. c Error is (0.01. d The
HR fraction folded was determined from the average of the values for
Val 3, Val 5, Lys 8, and Ile 10. Standard deviations are shown in
parentheses. e ∆G° was determined using glycine-splitting values; the
error is (0.05 kcal/mol. f Previously reported in ref 21.
This is consistent with an enthalpic driving force for the
interaction of the carbohydrate with the aromatic ring, as has
been observed in other systems,5,42 and is suggestive of
CH(δ+)-π and dispersion forces as major contributors to the
interaction.
Variation of the Carbohydrate. The carbohydrate was also
varied while Trp was used as X, and the resulting impact on
the side chain-side chain interaction was explored. Previous
studies comparing Ac4Glc and Glc suggested that Ac4Glc
formed a favorable carbohydrate-π interaction, but the desol-
vation cost appeared to be too high for Glc to interact favorably
with Trp.21 In this work, two other acetylated carbohydrates,
Ac4Gal and Ac3GlcNAc (Figure 2) were substituted for Ac4Glc.
Ac4Gal was used to examine the effect of the stereochemistry
at C4 on the carbohydrate-π interaction, and Ac3GlcNAc was
chosen to investigate the replacement of oxygen with nitrogen
at C2. We also studied the deprotected counterpart, GlcNAc,
in which only the nitrogen at C2 is acetylated. Lastly, we
investigated Me4Glc to further explore the role of desolva-
tion and determine the role (if any) of the acetyl groups.
The only difference between Ac4Glc and Ac4Gal is the
orientation of the alcohol at C4 (equatorial vs axial, repectively)
(Figure 2). The binding sites of many galactose-binding proteins
(galectins) contain an aromatic residue that interacts with the
“hydrophobic cluster” made up of C4, C5, and C6 (Figure 7).43
The upfield shift of the 6/6′ protons of Ac4Glc in WS(Ac4Glc)
suggests that such an interaction at C4, C5, and C6 may be
feasible in the ꢀ-hairpin. Thus, we replaced Ac4Glc with Ac4Gal
and investigated its interaction with Trp. There is only a small
change in the fraction folded for WS(Ac4Gal) relative to
WS(Ac4Glc), as measured by glycine splitting and HR shifts
(Table 4 and Figure 8). NOEs between the sugar and Trp
indicate that the interaction occurs on the R-face of the sugar,
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Acharya, K. R. Biochemistry 1998, 37, 13930–13940.
9
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A R T I C L E S
Laughrey et al.
relative to WS(Ac4Glc): the fraction folded decreases from 0.85
to 0.73 (Figure 8 and Table 4), and the double mutant cycle
demonstrates that the interaction energy is reduced by 0.2 kcal
mol-1 (Table 5). However, the protons of the Ac3GlcNAc R-face
are not nearly as upfield-shifted as those of WS(Ac4Glc) (Figure
9). The proton at C1 is the only one that was shifted significantly,
suggesting a change in geometry due to the amide at C2. This in
turn suggests that the energetic term from the double mutant cycle
arises from a favorable interaction other than the carbohydrate-π
interaction. Indeed, the downfield shifting of the NH group of
Ac3GlcNAc (9.34 ppm) suggests that it may participate in a
hydrogen bond. The Trp NH is not significantly shifted, however
[10.16 ppm in WS(Ac3GlcNAc) vs 10.11 ppm in an unfolded
control peptide].
When the acetyl groups were removed from the Ac3GlcNAc,
the fraction folded decreased only slightly (0.73 for Ac3GlcNAc
vs 0.70 for GlcNAc). However, the carbohydrate protons are
significantly less shifted than those of the other carbohydrates,
with the greatest shift occurring at C6 (Figure 9). In fact, the
chemical shifts of GlcNAc are similar to those of Glc, which
do not display any significant interaction with Trp.21 There are
several weak unambiguous NOEs between Trp and the R-
face of the carbohydrate (Figure 10). The double mutant cycle
indicates that the interaction energy for GlcNAc is comparable
to that for Ac3GlcNAc. Thus, it appears that some sort of
favorable interaction is present, but it occurs via a different
geometry than seen with other carbohydrates. However, NMR
data provides no evidence of hydrogen bonding involving either
the GlcNAc NH (7.82 ppm in this peptide vs 7.88 ppm in the
unfolded control peptide) or the Trp NH (10.17 ppm in this
peptide vs 10.11 ppm in the unfolded control peptide).
Figure 8. Fraction folded as determined from HR chemical shifts. Values
for WS(Ac4Glc) were originally reported in ref 21.
Figure 9. Upfield shifting of carbohydrate protons in peptides. Values for
WS(Ac4Glc) were originally reported in ref 21.
We also investigated the peptide in which the acetyl protecting
groups of Ac4Glc were replaced with methyl groups to probe
the role of desolvation and determine if there is a specific
influence of the acetyl groups. The peptide WS(Me4Glc) is
equally well folded as WS(Ac4Glc) (Table 4, Figure 8) and
exhibits numerous NOEs between the Trp residue and the R-face
of the sugar (Figure 10), indicating that Me4Glc also forms a
favorable interaction with Trp. Double mutant cycles indicate
that the magnitudes of the interactions for Me4Glc and Ac4Glc
are within the experimental error of each other. This appears to
suggest that protection of the hydroxyl groups, and hence
reduction of the desolvation cost, is indeed the primary reason
for the difference between the carbohydrate-π interactions for
peptides WS(Ac4Glc) and WS(Me4Glc) and that for WS(Glc)
containing the unprotected glucose. However, Ac4Glc and
Me4Glc do not behave identically: peptide WS(Me4Glc) does
not demonstrate the same extent of upfield shifting of the
carbohydrate protons at positions C1, C3, and C5 as does peptide
WS(Ac4Glc) [0.4-0.6 ppm for WS(Me4Glc) versus 0.6-1.35
ppm for WS(Ac4Glc)], despite the similar stability of the
ꢀ-hairpins. This may be due to competition of the methyl groups
for interaction with the Trp, as the methyl groups were also
shifted upfield by as much as 0.25 ppm. Indeed, the magnitude
of the upfield shift of the methyl groups was very similar to
that observed by Cuevas and co-workers44 in their study of
carbohydrate-π interactions with Me5Glc. Thus, it appears that
while Me4Glc forms a favorable interaction with Trp, the
interaction of the R-face of the sugar is not the only contributor
to the interaction.
Table 5. Diagonal Interaction Energies As Determined by Double
Mutant Cycles
X
Y
∆∆G (kcal mol-1 a
)
Trpb
Trp
Trp
Trp
Trp
Ac4Glc
Ac4Gal
Ac3GlcNAc
GlcNAc
Me4Glc
-0.8
-0.7
-0.6c
-0.5c
-0.8c
a Error is (0.1 kcal mol-1
.
b Previously reported in ref 21. c Al-
though the interaction energy for this mutant is similar to that of the
Trp-Ac4Glc interaction, the NMR data suggest that an interaction other
than the carbohydrate-π interaction is contributing. See text for details.
as was seen for Ac4Glc. Inspection of the carbohydrate chemical
shifts reveals that the protons of the R-face (C1, C3, and C5)
are shifted by the greatest amount relative to a random coil and
that C4 is not shifted significantly (Figure 9), indicating the
same geometry as for WS(Ac4Glc) rather than that seen in
galectins. This may be due to conformational restrictions of
the system rather than a specific preference for one geometry
over the other. The extent of shifting at positions 1, 3, and
5 of Ac4Gal is similar to that observed in Ac4Glc, suggesting
that the interaction with the R-face is equally as favorable.
This is consistent with the interaction energy determined from
double mutant cycles (Table 5), which is within the experi-
mental error of that measured for Ac4Glc.
Another common carbohydrate found in nature is GlcNAc. This
carbohydrate has two distinctive features: nitrogen replaces oxygen
at C2, and this nitrogen is acetylated. Both of these differences
affect how the carbohydrate interacts with the face of Trp. The
tetraacetylated sugar Ac3GlcNAc was used for direct comparison
with Ac4Glc. The presence of the amide reduces the interaction
(44) Bautista-Iba´n˜ez, L.; Ram´ırez-Gualito, K.; Quiroz-Garc´ıa, B.; Rojas-
Aguilar, A.; Cuevas, G. J. Org. Chem. 2008, 73, 849–857.
9
14630 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Carbohydrate-π Interactions: What Are They Worth?
A R T I C L E S
Figure 10. Unambiguous NOEs (red arrows) observed between the carbohydrate side chain (Ac4Gal, Ac3GlcNAc, GlcNAc, or Me4Glc) and Trp. Unambiguous
NOEs are defined as ones that can be definitively assigned to a particular set of protons.
Discussion
which mutation of an aromatic residue in the binding pocket
abolishes binding of the carbohydrate.20,47 Because naturally
occurring aliphatic residues have different sizes and shapes than
aromatic residues, the results from protein mutation studies have
been difficult to attribute solely to the loss of aromaticity. Since
Phe and Cha have the same facial surface area, the comparison
within the ꢀ-hairpin model system is more direct, and it clearly
indicates that aromaticity influences the interaction energy. The
preference for interaction of Ac4Glc with Phe over Cha and
the greater enthalpic driving force for folding of FS(Ac4Glc)
relative to ChaS(Ac4Glc) point to CH(δ+)-π interactions as a
significant contributor to the driving force of the interaction:
since cyclohexane is more polarizable than benzene, dispersion
forces would be expected to be stronger for Cha than for Phe.48
Moreover, Cha is also more hydrophobic than Phe, arguing
against the hydrophobic effect as the primary driving force for
the interaction. This is consistent with the finding of Jime´nez-
Barbero et al.20 that variation of the electronic structure of the
aromatic ring influences carbohydrate binding in the hevein
domain.
The system described here has made possible the systematic
investigation of carbohydrate-π interactions in the absence of
other cooperative noncovalent interactions, such as hydrogen
bonds, thus allowing for the quantification of the binding energy
and an exploration of the factors that contribute to these
interactions. Variation of the X side chain provides significant
insight into the nature and driving force of the carbohydrate-π
interaction. The similar interactions of Ac4Glc with Trp, 1-Nal,
and 2-Nal confirm that Ac4Glc interacts primarily with the face
of the aromatic ring and that any hydrogen bonding to the NH
of Trp is at best a minor contributor to the interaction (e0.1
kcal/mol). The fact that 1-Nal and 2-Nal interact similarly
indicates that this model system has enough flexibility to
optimize the interaction when the orientation of the aromatic
ring is varied. Comparison of Trp to Phe indicates that the
surface area of the aromatic ring impacts the magnitude of the
interaction (-0.8 kcal/mol for Trp vs -0.5 kcal/mol for Phe).
The interaction between carbohydrates and aromatic groups
has been variously described in terms of the hydrophobic effect,
dispersion forces, and CH-π interactions.20,45,46 Comparison
of Phe versus Cha at position X indicates that the carbohy-
drate-π interaction is more favorable than an equivalent
hydrophobic interaction between Ac4Glc and an aliphatic side
chain (-0.5 kcal/mol for Phe vs -0.1 kcal/mol for Cha). This
is similar to what has been seen in protein mutation studies, in
Variation of the carbohydrate provides insight into the balance
of features that influence this interaction. Within the ꢀ-hairpin
model system, it appears that interaction on the R-face of the
carbohydrate is most favorable, even when another “hydropho-
bic” surface is present, as in Ac4Gal. For Ac3GlcNAc and
GlcNAc, the interaction energy decreased and the geometry of
(47) Flint, J.; Bolam, D. N.; Nurizzo, D.; Taylor, E. J.; Williamson, M. P.;
Walters, C.; Davies, G. J.; Gilbert, H. J. J. Biol. Chem. 2005, 280,
23718–23726.
(45) Muraki, M.; Morii, H.; Harata, K. Protein Pept. Lett. 1998, 5, 193–
198.
(46) Muraki, M.; Morii, H.; Harata, K. Protein Eng. 2000, 13, 385–389.
(48) Ma, J. C.; Dougherty, D. A. Chem. ReV. 1997, 97, 1303–1324.
9
J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008 14631

A R T I C L E S
Laughrey et al.
is considerable. In contrast, the interaction of Cha with Ac4Glc
has the same magnitude as its interaction with Lys (-0.1 kcal/
mol).22
Lastly, these studies also provide evidence for a novel method
of influencing protein structure. In structural studies of glyco-
sylated proteins and peptides, it has generally been found that
stabilization of the folded state occurs because glycosylation
rigidifies the peptide backbone, thereby destabilizing the
unfolded state.49-55 In contrast, in the system reported here,
incorporation of a carbohydrate-π interaction results in en-
thalpic stabilization of the folded structure through a specific
interaction.
Figure 11. Electrostatic potential maps of the side chains at position Z in
the ꢀ-hairpin: (a) Ac4Glc; (b) Glc; (c) Me4Glc. The maps were generated
using MacSpartan at the HF/6-31G* level, with an isodensity value of 0.02
and a range of -25 kcal/mol (red, electron-rich) to 25 kcal/mol (blue,
electron-poor).
Conclusion
This study provides insight into the role of carbohydrate-π
interactions in carbohydrate recognition by proteins. The
energetic contribution of the carbohydrate-π interaction be-
tween the R-face of the pyranose ring and the face of an aromatic
ring was found to range from -0.5 to -0.8 kcal mol-1 and is
dependent on the nature of both the aromatic ring and the
carbohydrate. Of significance is the fact that a favorable
interaction was only observed when the hydroxyl groups of the
carbohydrate were protected using either acetyl groups or methyl
groups. This implies a significant cost for desolvation of the
sugar. However, the NMR data suggests that the interaction with
Ac4Glc is more favorable than that with Me4Glc, which may
imply an electronic tuning of the interaction. Moreover, the
preferential interaction of the pyranose ring with the face of a
phenyl group as opposed to a cyclohexyl ring suggests that
CH(δ+)-π interactions play a measurable role in the interac-
tion. These studies provide a better physical understanding of
the driving force behind carbohydrate-π interactions as well
as insight into their magnitudes and significance relative to other
noncovalent interactions that have been measured for the same
model system. In addition to providing insight into the recogni-
tion of carbohydrates by proteins, we expect that the findings
of this study will be useful in the development of new and
improved receptors for carbohydrate recognition.
the interaction changed, likely because of the presence of the
amide at C2, which is expected to have stronger interactions
with the solvent than the corresponding ester does.
A comparison of Ac4Glc to Me4Glc was made to address
the roles of desolvation and electrostatics in the interaction. We
have previously shown that Ac4Glc interacts favorably with Trp,
with an interaction energy of approximately -0.8 kcal/mol, and
that removal of the acetyl groups results in the loss of the
favorable interaction. We attributed this to differences in
desolvation cost, although the electron-withdrawing nature of
the acetyl groups also results in differences in the partial charges
on the R-faces of Ac4Glc and Glc, as indicated by electrostatic
potential maps (Figure 11). Thus, we investigated the interaction
of Me4Glc with Trp because it has an electrostatic potential map
similar to that of Glc but its desolvation cost is significantly
reduced. The observed stabilizing interaction (∆∆G ) -0.8
kcal/mol) indicates that paying the desolvation cost is indeed
enough to allow for a favorable carbohydrate-π interaction and
that there is nothing unique about the acetylated glucose.
However, the NMR shifts of Me4Glc indicate that interaction
of Trp with the R-face is reduced and that direct interaction
with the polarized methyl groups also occurs. Thus, a direct
comparison of the role of electrostatics in the interactions of
Trp with Ac4Glc and Me4Glc is not possible, as there are other
contributors to the interaction energy. Nonetheless, the NMR
data suggest that the weaker polarization of the R-face of the
sugar may reduce the carbohydrate-π interaction and that the
interaction between the methyl groups and Trp provides a
compensating interaction.
Since the carbohydrate-π interaction is only observed in this
system when the hydroxyl groups are protected, the question
arises as to whether this interaction is significant in carbohydrate-
binding proteins, where the carbohydrate is unprotected. We
have shown that the role of the protecting groups is to desolvate
the sugar to allow for interaction with the aromatic ring. Within
a carbohydrate-binding protein, hydrogen-bonding groups are
preorganized for the same task. Thus, it appears that nature uses
cooperative interactions between the aromatic ring and hydrogen-
bonding groups to desolvate and bind the carbohydrate.42 Indeed,
obtaining this sort of cooperative binding may be the primary
challenge in designing synthetic receptors for carbohydrates in
water.7
Experimental Section
Peptide Synthesis and Purification. All of the peptides were
synthesized on Fmoc-PAL-PEG-PS amide resin using standard
solid-phase protocols on a continuous flow Pioneer Peptide
Synthesizer (Applied Biosystems). Fmoc-amino acids (4-6 equiv)
were activated and coupled with 0.45 M HBTU/HOBt in dimeth-
ylformamide (DMF). The following protecting groups were used:
Arg(Pbf), Asn(Trt), Cys(Trt), Gln(Trt), Lys(Boc), Ser(tBu), Thr(t-
Bu), and Trp(Boc). Deprotection of the Fmoc groups was achieved
with 2% piperidine/2% 1,8-diazabicyclo[5.4.0]undec-7-ene in DMF.
(49) Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1993, 115, 3352–
3353.
(50) Wyss, D. F.; Choi, J. S.; Li, J.; Knoppers, M. H.; Willis, K. J.;
Arulanandam, A. R. N.; Smolyar, A.; Reinherz, E. L.; Wagner, G.
Science 1995, 269, 1273–1278.
(51) Live, D. H.; Kumar, R. A.; Beebe, X.; Danishefsky, S. J. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 12759–12761.
(52) O’Connor, S. E.; Imperiali, B. J. Am. Chem. Soc. 1997, 119, 2295–
2296.
We have measured a wide range of noncovalent interactions
within the same peptide model system, so a direct comparison
can be made between them. Surprisingly, the energy of the
interaction between Ac4Glc and Trp is larger than that of the
cation-π interaction between Lys and Trp (-0.4 kcal/mol)22,25
but similar in magnitude to that of the interaction between KMe3
and Trp (-1.0 kcal/mol).23 Thus, the carbohydrate-π interaction
(53) Imperiali, B.; O’Connor, S. E. Chem. Biol. 1998, 5, 427–437.
(54) Imperiali, B.; O’Connor, S. E. Curr. Opin. Chem. Biol. 1999, 3, 643–
649.
(55) Bann, J. G.; Peyton, D. H.; Ba¨chinger, H. P. FEBS Lett. 2000, 473,
237–240.
(56) Syud, F. A.; Espinosa, J. F.; Gellman, S. H. J. Am. Chem. Soc. 1999,
121, 11577–11578.
(57) Smith, C. K.; Regan, L. Acc. Chem. Res. 1997, 30, 153–161.
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14632 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Carbohydrate-π Interactions: What Are They Worth?
A R T I C L E S
All of the peptides were acylated at the N-terminus using a 5%
acetic anhydride/6% lutidine/DMF solution and amidated at the
C-terminus. Peptide resin cleavage and deprotection were performed
simultaneously by treatment with 95% trifluoroacetic acid (TFA)/
2.5% triisopropylsilane/2.5% H2O for 2-3 h under nitrogen. The
TFA was removed by distillation under vacuum. The crude peptides
were precipitated with cold ether, extracted into water, lyophilized,
and then dissolved and purified by reverse-phase (RP) HPLC using
a Vydac C18 semipreparative column. The peptides were eluted with
a linear gradient of solvent A (95% H2O/5% acetonitrile with 0.1%
TFA) and solvent B (95% acetonitrile/5% water with 0.1% TFA)
from 0 to 30% B and detected by monitoring at 220 and 280 nm.
Molecular weights were determined using electrospray-ionization
mass spectrometry. Disulfide bonds were formed by dimethyl
sulfoxide oxidation of purified peptides in phosphate-buffered saline
(pH 7.4). The peptides were then repurified by RP HPLC.
NMR Spectroscopy. Solutions for NMR spectroscopy with
concentrations of 1-3 mM were analyzed on a Varian Inova 600
MHz instrument. Samples were dissolved in D2O buffered with 10
mM acetate-d3 (pH 4.2) and referenced to DSS. NMR spectra were
collected with 8-64 scans using a 1.5 s presaturation. All of the
2D NMR experiments used pulse sequences from the Chempack
software, including TOCSY, gCOSY, and ROESY. TOCSY and
gCOSY experiments were performed with 4-8 scans in the first
dimension and 256 scans in the second dimension. ROESY
experiments were performed with 32 scans in the first dimension
and 256-512 scans in the second dimension. All of the spectra
were analyzed using standard window functions (sinebell and
Gaussian with shifting). Assignments were made using standard
methods. Thermal denaturations were performed in duplicate in
5-10 °C increments. The temperature was calibrated with methanol
and ethylene glycol standards using Varian macros.
Determination of Fraction Folded. To determine the chemical
shifts of the fully folded state, 14-residue disulfide-linked ana-
logues of the peptides were synthesized with the sequence Ac-
CRXVTVNGKS(Z)ILQC-NH2, where X ) Trp, 1-Nal, 2-Nal,
Cha, or Phe and S(Z) ) Ser(Ac4Glc), Ser(Ac4Gal), Ser(Ac3-
GlcNAc), Ser(GlcNAc), or Ser(Me4Glc), and characterized by
NMR. To determine the chemical shifts of the unfolded state, a
series of seven-residue peptides were synthesized and character-
ized. The sequences of these peptides were Ac-RXVTVNG-
NH2 (X ) 1-Nal, 2-Nal, Cha, or Phe) and Ac-NGKZILQ-NH2
[Z ) Ser(Ac4Gal), Ser(Ac3GlcNAc), Ser(GlcNAc), or Ser(Me4-
Glc)]. The 7-mers with either Trp or Ser(Ac4Glc) had previously
been described.21 The fraction folded based on HR chemical
shifts was determined using eq 1:
control peptide. The fraction folded based on glycine splitting was
determined with the eq 2:
(∆δGly
)
hairpin
fraction folded )
(2)
(∆δGly cycle
)
Double Mutant Cycles. Double-mutant cycles were performed
in order to quantify the interaction between the series of carbohy-
drates and the side chain X. Single mutant peptides in which
Ser(Ac4Glc), Ser(Ac4Gal), Ser(Ac3GlcNAc), Ser(GlcNAc), or
Ser(Me4Glc) was replaced by Ser or 1-Nal, 2-Nal, Phe, or Cha was
replaced by Leu were prepared. The double mutant contained
both substitutions. The singly-mutated peptides RWVTVNGK-
SILQ and RLVTVNGKS(Ac4Glc)ILQ as well as the double
mutant RLVTVNGKSILQ were previously reported.21 Difficul-
ties arose in the synthesis of the cyclic RLVTVNGKS(Me4-
Glc)ILQ control, so the glycine-splitting value for the cyclic
RLVTVNGKS(Ac4Glc)ILQ control was used instead. The energy
of folding for each peptide was determined from the difference
in the chemical shifts of the glycine hydrogens. The side chain
interaction energy was then determined using eq 3:
∆∆G(X-Z) ) ∆GXZ - ∆GXS - ∆GLZ + ∆GLS
(3)
Thermal Denaturation. Variable-temperature NMR was used
to perform the thermal denaturation experiments. A temperature
range of 275-330 K was explored in 5 K increments. The
temperature was calibrated using methanol and ethylene glycol
standards. The change in glycine chemical-shift difference was used
to determine the fraction folded at each temperature. The fraction
folded was then plotted against temperature and the data fitted with
eq 4:
x
RT
exp
(
)
fraction folded )
(4)
x
1 + exp
(
)
RT
where
T
x ) T ∆S298° + ∆Cp° ln
- ∆H298° + ∆Cp°(T - 298 K)
[
(
)]
298 K
(5)
Acknowledgment. This work was supported by funding from
the National Institutes of Health, Institute of General Medical
Sciences (GM072691 and GM071589).
Supporting Information Available: Synthetic procedures for
glycosylated amino acids, NMR assignments, data from double
mutant cycles, and thermodynamic data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
δobs - δ0
δ100 - δ0
fraction folded )
(1)
where δobs is the observed chemical shift, δ100 is the chemical shift
for the cyclic peptide, and δ0 is the chemical shift for the unfolded
JA803960X
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J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008 14633