Computational and experimental investigations of mono-septanoside binding by Concanavalin A: Correlation of ligand stereochemistry to enthalpies of binding

Article abstract of DOI:10.1039/c0ob00425a

Structure-energy relationships for a small group of pyranose and septanose mono-saccharide ligands are developed for binding to Concanavalin A (ConA). The affinity of ConA for methyl "manno" β-septanoside 7 was found to be higher than any of the previousl

Full text of DOI:10.1039/c0ob00425a

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PAPER
Computational and experimental investigations of mono-septanoside binding
by Concanavalin A: correlation of ligand stereochemistry to enthalpies of
binding†
Michael R. Duff Jr., W. Sean Fyvie, Shankar D. Markad, Alexandra E. Frankel, Challa V. Kumar,*
Jose´ A. Gasco´n* and Mark W. Peczuh*
Received 15th July 2010, Accepted 7th October 2010
DOI: 10.1039/c0ob00425a
Structure-energy relationships for a small group of pyranose and septanose mono-saccharide ligands
are developed for binding to Concanavalin A (ConA). The affinity of ConA for methyl “manno”
b-septanoside 7 was found to be higher than any of the previously reported mono-septanoside ligands.
Isothermal titration calorimetry (ITC) in conjunction with docking simulations and quantum
mechanics/molecular mechanics (QM/MM) modeling established the specific role of binding enthalpy
in the structure–energy relations of ConA bound to natural mono-saccharides and unnatural
mono-septanosides. An important aspect in the differential binding among ligands is the deformation
energy required to reorganize internal hydroxyl groups upon binding of the ligand to ConA.
shown to contribute to protein–carbohydrate associations.13–16
Introduction
These forces bear a connection to the dual nature of carbohydrates
The integral role that protein–carbohydrate interactions play in a
themselves. The hydroxyl groups on a given sugar residue are
variety of biological processes is now well established.1–5 Exam-
critical for H-bonding with its protein partner while also being
ples include protein-folding, viral attachment to host cells, im-
important for solvation. Hydroxyl groups form a H-bonding
munomodulation, enzymes involved in carbohydrate metabolism,
“ribbon” around the perimeter of the monosaccharide residue.
and cell attachment to the extra-cellular matrix, among others.
Alternatively, the C–H bonds collectively define apolar patches
Because of the potential benefits, inhibiting certain protein–
carbohydrate interactions that lead to cellular pathologies can
that can associate with the p-system of aromatic amino acid
residues. Solvation and desolvation of apolar patches is distinct
provide a valuable entry into new therapeutics. A soluble polysac-
from solvation of the hydrogen bonding array. Specific arrange-
charide ligand of galectin-3 (gal-3), for instance, was recently
ments of H-bonding arrays and apolar patches as defined by
approved for clinical trials to treat multiple cancers.6,7 The release
specific sugar residues alone or as part of an oligosaccharide then
of new influenza viruses from a host cell involves breaking up
define how a protein binds to a carbohydrate.
protein–carbohydrate interactions via hydrolysis of the polysac-
Synthetic analogs of pyranose sugars (e.g., oseltamivir) have
R
charide. Oseltamivir (Tamifluꢀ) is a >$1b/year carbohydrate
been extensively studied. In general, such analogs should find
analog8 that works by inhibiting the key glycosidase in the process,
a neuraminidase.9 The relevance of protein–carbohydrate interac-
tions in biology, therefore, motivates fundamental investigations
on the parameters that govern their association and also on the
development of new carbohydrate analogs as potential competing
ligands.
use in glycobiology and as therapeutic leads.17 The literature
contains only a few reports, however, of septanoses being used
as analogs of natural pyranose sugars to investigate protein–
carbohydrate interactions.18–20 Tauss et al.20 reported on the ability
of p-nitrophenyl L-idoseptanosides 1 and 2 to serve as substrates
of exo-glycosidases. Both a- and b- anomers of the septanoside
were prepared for the investigation. The L-idoseptanosides (1 and
2) were chosen because they share the same stereochemistry as D-
glucose from C1–C4 (Fig. 1). Molecular modeling studies showed
that minimized structures of 1 and 2 positioned their aglycones
and C2–C4 hydroxyl groups in similar orientations relative to 3
and 4, respectively.20 As measured by the overall efficiency of the
enzymatic reaction using a-glucosidase, 1 was a poor substrate
(kcat/KM = 24 s-1 M-1) when compared to the pyranose substrate 3
(kcat/KM = 2.4 ¥ 104 s-1 M-1). The data also showed a factor of 10
A number of weak non-covalent forces, working in concert,
underpin the binding of carbohydrates by lectins and glyco-
enzymes.10–12 Hydrogen bonding, van der Waals interactions, and
desolvation of the ligand and binding site residues have all been
Department of Chemistry, The University of Connecticut, 55 North Eagleville
Road, Storrs, CT, 06269, USA. E-mail: mark.peczuh@uconn.edu
† Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for all newly reported compounds. Computed QM/MM
structures not shown in the main text are provided. Further details on the
thermodynamic cycle used in our calculations and approximations are also
provided. See DOI: 10.1039/c0ob00425a
difference in the relative binding affinities of 1 versus 3 (KM
=
2.9 and 0.27 mM, respectively). Similar results were obtained
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We endeavored, therefore, to understand how septanosides
interacted with ConA more precisely and how this association
corresponded to the way pyranosides are bound. In the same way
that the initial mono-septanoside ligand of ConA could loosely
be considered a ring expanded analog of D-glucose (Fig. 1), we
expected ring expanded analogs of D-mannose and D-galactose
to behave like their pyranoside counterparts. Reported here is
the calorimetric determination of association constants for ConA
binding to several new seven membered ring sugar analogs. We
further show the origin of the relevant protein–ligand interactions
as they pertain to enthalpic contributions in detail. This is
accomplished by correlating the binding data with structural
information from docking and Quantum Mechanical/Molecular
Mechanical (QM/MM) calculations. We show that there is a very
good qualitative correlation between the gas-phase QM/MM
binding energy and the experimental enthalpy of binding. This
allows us to make a structure–energy correlation. Further, the
implications of the results in terms of utilizing septanoses as
analogs of pyranoses are discussed.
Methyl glycoside ligands
Methyl glycosides used in this investigation are shown in Fig. 2.
The absolute affinities of ConA for monosaccharides are generally
low; however, the literature has several examples of approaches
for increasing this affinity via polyvalent presentation of the
ligand.27–31 Moreover, ConA also has a higher affinity for the
Man a-1-3,1-6 trisaccharide, which is the biologically relevant
ligand.32–34 We chose to analyze binding between a series of
monosaccharides because access to the septanoside ligands was
straightforward and structure–energy relationships could be de-
veloped. Ring expanded analogs 6–8 were chosen for comparison
to each other and also to their pyranose counterparts 9–11. Fig. 2
organizes the ligands based on whether the ligand is a septanoside
or pyranoside (columns) and also on ring stereochemistry (rows).
Each series reflects the stereochemical configuration of C2–
C5 (pyranose numbering) of glucose, mannose, and galactose,
respectively.
Methyl b-septanoside 6 of the gluco- series serves as a starting
point for comparison of the monosaccharides in this study. As
previously mentioned, 6 was the ring-expanded ligand with the
highest affinity for ConA in our earlier investigation. The new
methyl b-septanosides 7 and 8 vary one stereocenter on the
ring relative to 6; in manno- analog 7, C3 has been inverted
(septanose numbering) whereas in galacto- analog 8 C5 was
inverted. Stereocenters C3–C6 in 6–8 correspond to the C2–
C5 stereocenters of 9–11. Synthesis of methyl b-septanosides
6–8 was accomplished by the epoxidation and methanolysis
of carbohydrate based oxepines35 followed by removal of the
protecting groups on the C3–C7 hydroxyls.36,37
Fig. 1 Septanose and pyranose glycosides: L-idoseptanosides (1, 2),
D-glucosides (3, 4), D-glycero-D-septanosides (5, 6).
when comparing the b-glycosides 2 and 4 using b-glucosidase.
Albeit with low affinity, even in comparison to low affinities of the
“native” substrates, the results of this investigation showed that
natural glycosidases could, in fact, bind septanosides and achieve
enzymatic turn-over in the hydrolysis of these substrates.
The binding of methyl b-septanosides such as 6 to the plant
lectin Concanavalin A was previously reported by this laboratory.
Structural information from X-ray crystallographic investiga-
tions of Concanavalin A (ConA) complexed with a-pyranose
monosaccharides21–26 showed that the primary contacts between
the ligand and the protein were along C3–C6. Our design of
septanose ligands therefore included an exocyclic hydroxymethyl
group (e.g., C7 in 5 and 6). Association constants obtained by
Isothermal Titration Calorimetry (ITC) showed that methyl b-
septanoside 6 was a weak ligand of ConA (Ka = 450 M-1). For
comparison, methyl a-glucoside 9 and methyl a-mannoside 10
(their six-member ring counterparts; Fig. 2) gave affinity constants
of 2000 and 7900 M-1, respectively. 1H NMR Saturation Transfer
Difference (STD) competition experiments confirmed that binding
to 6 was at the pyranoside binding site. The investigation showed
that b-septanosides were bound by ConA but a-septanosides
such as 5 were not. Remarkably, this selectivity for anomeric
configuration is opposite to that observed for ConA binding to
pyranosides. That is, a-pyranosides are ligands of ConA while b-
pyranosides are not.19 An important conclusion that was drawn
from these preliminary studies of protein–septanose interactions
was that, based on a given protein, septanoses may be bound
with low affinity. In both the glycosidase and the lectin examples,
presentation of specific hydroxyl groups by the septanosides to
mimic the “target” pyranosides was shown to be important for
binding. It remained unclear exactly how the changes to individual
stereocenters of the ligands correlated with their ability to be
bound by ConA.
Additional molecules were prepared in an effort to obtain
further details regarding the structure–energy relationships that
had been observed (Fig. 3). The first pair, oxepanes 12 and 13,
were prepared by hydrogenation of their oxepine precursors along
with concomitant hydrogenolysis of the benzyl ether protecting
groups (Scheme 1). Methyl a-septanoside 14 and methyl 2-O-
methyl b-septanoside 15 were also targeted. Preparation of 14
from a mannose-derived oxepine had been described earlier.37
Methyl 2-O-methyl septanoside 15 was prepared by epoxidation
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Fig. 2 Methyl b-septanosides 6–8 and methyl a-pyranosides 9–11 used as ligands of ConA. Each row is organized by related ring stereochemistries:
“gluco”, “manno” and “galacto”. Changes in stereochemistry are denoted by yellow/blue highlights.
Scheme 1 Preparation of oxepanes 12 and 13.
and methanolysis of the glucose-derived oxepine followed by
methylation of the C2 hydroxyl group and subsequent debenzyla-
tion (Scheme 2) (The Experimental Section details the synthesis
of newly reported compounds 12, 13, and 15.). We had originally
planned to synthesize a family of C2-acylated septanosides by
this route where acylation at C2 would have substituted for
the alkylation step. However, acyl migration proved to be a
complicating side-reaction during the hydrogenolysis of the benzyl
protecting groups and so attempts at preparing 2-O-acyl analogs
of 6 were abandoned.
charide ligands that were studied by ITC. We were especially
interested in discerning the differences in affinity between the
septanoside ligands and their pyranoside counterparts. Modeling
of all protein–ligand complexes was based on the X-ray structure
of Concanavalin A (ConA) with 9 (PDB access code: 1GIC chain
A).23 We analyzed the binding of seven ligands (5–10, 15), for
which thermodynamic data had been obtained (Table 1). These
complexes span the entire range of binding affinities observed,
and can help understand the origin of relevant interactions. To take
advantage of the steric constraints imposed by the protein cavity
we used the co-crystal (ConA bound to 9) to create the different
stereoisomers by modifying the existing ligand in situ. After
in situ modification with the program Maestro38 and removal of
all crystallographic waters except Wat335 (see discussion below),
Computational approach
We turned to a computational approach to gain greater insight into
the atomic level interactions between ConA and the monosac-
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Scheme 2 Preparation of methyl 2-O-methyl-b-septanoside 15.
treated at the quantum mechanical (QM) level and the rest of the
protein at the MM level. Cuts between the QM and MM region
were treated with the frozen-orbital method as implemented in
Qsite. The MM region was treated with the OPLSAA force field,41
and the QM region with Density Functional Theory (DFT). The
functional B3LYP and basis set 6-31g* were used in all QM
calculations.
We considered two sets of calculations, one in which the protein
remained frozen in its crystallographic position while only the
ligand was relaxed, and another where the ligand and the specific
residues Asn14, Leu99, Tyr100, Asp208, and Arg228 were relaxed.
It will be shown below that both sets of calculations produce
similar results and both lead to the same analysis regarding the
differential binding among the ligands.
The gas phase binding energy (Eb) is defined as:
Eb = EQM/MM - E◦
- EQM ,
(1)
QM/MM
Fig. 3 Additional ligands of ConA used in the investigation: Oxepanes
12 and 13 derived from D-glucose and D-mannose, respectively; methyl
“manno” a-septanoside 14 and methyl 2-O-methyl “gluco” b-septanoside
15.
where EQM/MM is the QM/MM energy of the protein–ligand
complex, E◦
is the QM/MM energy of the protein alone
QM/MM
including Asp208 in the QM region, and EQM is the QM energy
of the ligand alone. For the latter calculations we optimized the
structure in vacuum using the program Jaguar.42
each ligand (including 9) was re-docked with the program Glide,39
always maintaining the structure of the protein frozen. The
docking algorithm in Glide allows for flexibility and bond rotation
and uses scoring functions predominantly based on a molecular
mechanics (MM) force field.
Conformational search. As mentioned above, a conforma-
tional search of internal degrees of freedom inside the protein
cavity was carried out by Glide. For the ligand in the gas phase,
the starting geometry was that obtained by the highest scored
conformation using Glide. In addition, we considered other con-
formations with different OH rotamers and ring conformations.
Docking computations were followed by QM/MM energy
minimizations using Qsite,40 where the ligand and Asp208 were
Table 1 Thermodynamics of bindinga for ConA with ligands
Ligand
Ka/M-1
N
DG/kcal mol-1
DH/kcal mol-1
TDS/kcal mol-1
5b
6b
6b
7
NB
—
—
—
—
5.2 0.3 ¥ 102
4.5 0.7 ¥ 102
8.4 0.4 ¥ 102
NB
3.2 0.9
-3.7 0.1
-3.6 0.09
-4.0 0.03
—
-0.83 0.1
-2.7 0.2
-5.5 0.6
—
2.9 0.2
0.95 0.3
-1.5 0.6
—
1c
0.9 0.1
—
8
9b
2.0 0.2 ¥ 103
7.9 1.0 ¥ 103
NB
0.87 0.03
0.83 0.18
—
—
-4.5 0.03
-5.5 0.1
—
-3.5 0.9
-4.5 0.03
—
0.98 0.2
1.1 0.2
—
—
3.4 0.2
1.7 0.3
10b
11d
14
15
15
NB
—
—
4.3 1.0 ¥ 102
1.9 0.3 ¥ 102
9.1 3.1
-3.6 0.1
-3.1 0.1
-0.13 0.05
-1.4 0.2
1c
a Titrations were run at 298 K. [ConA] was between 0.200–0.300 mM and [5–15] was between 15–30 mM. ConA was dimeric under the experimental
conditions. Buffer: 50 mM 3,3-dimethylglutarate pH 5.2, 250 mM NaCl, 1 mM CaCl2, 1 mM MnCl2. b Data from ref. 19 c Data fit with the ConA : ligand
stoichiometry manually fixed at 1 : 1; therefore N = 1. d Data from ref. 46. NB = no binding was detected under these conditions.
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Selection of these rotamers was guided by a previous study in
which a rigorous conformational analysis of septanosides 5 and 6
was done by Monte Carlo (MC) simulations.36 In fact, our mini-
mum energy structures of 5 and 6 were the same as those obtained
in these previous MC simulations. From the study of DeMatteo
et al.36 it was concluded that the septanosides studied were rigid
enough to prefer one conformation at room temperature. Thus,
the sole use of the lowest energy configurations, found in both
the Glide/QM/MM and gas phase QM calculations, to correlate
binding energies with experimental enthalpies appears to be a
reasonable assumption.
DDH = DDH0 + (DDHcomplex - DDHlig).
(3)
We now argue that, due to the similar nature of the ligands
considered, DDHcomplex - DDHligand is negligible. This would not
be the case for structurally dissimilar ligands. Our assumption is
supported by studies carried out by Toone’s group on protein–
carbohydrate binding.43 In that work, a direct measure of the
solvent reorganization contribution to enthalpy was obtained by
measuring the binding enthalpies in D2O relative to H2O. Key
to our study was that the difference in the enthalpy of solva-
tion (DDHlig) across several monosaccharides (including methyl
pyranosides 9 and 10, for example) was of the order of 0.2 kcal
mol-1.44,45 The other contributor to the solvent reorganization is
the solvation of the protein–ligand complex (DHcomplex). Although
no experimental results for this contribution to the protein–
ligand complexes have been determined, it is logical to assume
that, since all ligands utilize a similar set of interactions when
binding to the protein (see results below), DDHcomplex should be
of a similar magnitude as DDHlig but with the opposite sign.
This assumption is also supported a posteriori by the good
qualitative and quantitative correlation between the gas-phase
QM/MM calculations and the experimental enthalpies of binding
(see Table 2). Although inclusion of solvent effects, via implicit
or explicit solvent treatments would be certainly desirable and
would likely yield better results, the computational cost of such
a treatment would be significantly higher at the present level of
theory (i.e. DFT-QM/MM). Thus, assuming a constant effect of
the solvent throughout the various ligands as argued above, the
absolute binding energies were no longer meaningful. Instead, our
interest was focused on relative binding energies (DEb), rather than
absolute binding energies, which we assume, as argued above, to
be a good approximation of DDH0. Again, we emphasize that
for general ligands this may not be the case, but because of the
incremental difference between any two monosaccharides, this
approximation appears well founded.
Structural waters and solvation. Solvent reorganization is
an important contributor to the binding process of all these
carbohydrates.43 The change in enthalpy of the binding event
(DH), which takes place in solution, can be constructed via a
thermodynamic cycle (Fig. 4) by adding up the following terms: 1)
individual desolvation enthalpies of the apo-protein and ligand,
-DHlig and -DHprot, 2) binding of ligand to protein in the gas phase,
DH0, and 3) solvation of the protein complex, DHcomplex. Thus, the
total change in enthalpy is written as:
DH = DH0 + (DHcomplex - DHlig - DHprot).
(2)
It is also important to mention recent work from the Woods lab-
oratory on the involvement of structural waters in the binding of
ConA to the natural mannose trisaccharide and a related analog.15
That work addressed the role of a conserved bound water molecule
which makes hydrogen bonds to Asn14, Arg228, and Asp16 and
remains bound upon ligand binding. This water becomes relevant
as the central mannosyl residue of the trisaccharide possesses a
hydroxyl group very close to that water. It was shown that the
replacement of this group by a hydroxyethyl side chain resulted in
a more strained conformation of this water molecule with respect
Fig. 4 Thermodynamic cycle describing the separation of the observed
binding enthalpy in solution into the gas phase binding enthalpy and
solvent reorganization enthalpy.
In the present work, we have focused on the relative enthalpy of
binding, under the assumption of a rigid cavity (DDHprot = 0):
Table 2 Comparison between the calculated binding energy with the experimental binding enthalpies. Values in parentheses correspond to a QM/MM
calculation in which residues Asn14, Leu99, Tyr100, Asp208, and Arg228 are relaxed
Ligand
Ed /kcal mol-1
Erb/kcal mol-1
Eb/kcal mol-1
DEb/kcal mol-1
DDHa (exp.)/kcal mol-1
5
6
7
8
9
10
15
23.1
18.6
14.4
24.3
13.7
14.3
17.1
-87.1
-86.2
-87.2
-80.2
-84.8
-86.1
-85.6
-64.0 (-58.3)
-67.6 (-61.8)
-72.8 (-67.3)
-55.9 (-51.9)
-71.1 (-66.2)
-71.8 (-66.3)
-68.5 (-62.6)
8.8 (9.0)
5.2 (5.5)
0.0 (0.0)
16.9 (15.4)
1.7 (1.1)
1.0 (1.0)
4.3 (4.7)
no binding
4.67/2.8
0.0
no binding
2.0
1.0
5.37/4.1
a DDH = DH(ConA·ligand) - DH(ConA·7).
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to its cavity affecting the enthalpy of binding. All other waters in
the binding site appeared to be displaced upon binding. Therefore,
considering this important observation we included this water
molecule (Wat335 in 1GIC.pdb) in all QM/MM calculations.
Hydrogen atoms were added to this molecule and their orientation
optimized to form hydrogen bonds to Asn14, Arg228, and Asp16
following the findings of Kadirvelraj et al.15
ligands (by factors of 5–10) compared to the pyranoside binders.
One objective of the computational work presented here was to
develop a way to rationalize this observation.
To our surprise, 12 and 13 were very poor ligands for ConA.
Weak exothermic binding was observed in the corrected ITC
traces, but curve fitting of the data gave poor fits and highly
variable association constants and stoichiometries. We interpreted
this to mean that 12 and 13 were binding, but with Ka values
that were significantly lower than the ones reported for the other
ligands in Table 1. The oxepanes were intended to measure if
the aglycone moiety contributed to binding. Preliminary manual
overlays of 5 and 6 in the binding pocket of the 9–ConA co-
complex19 suggested that the methyl aglycone of 5 was making
unfavorable van der Waals contacts with the protein that were
likely responsible for the lack of binding between ConA and 5.
Such an unfavorable interaction was largely, but not completely,
eliminated in 6. We reasoned that removing the aglycone entirely
(and also the C2 stereocenter) could maintain the important
contacts with ConA at C3–C7 while removing the unfavorable
aglycone–protein interaction. We also anticipated that the absence
of an anomeric center could influence the conformational behavior
of the ring. Therefore, comparison of the binding data of 12 and
13 with those of 6 and 7 suggested that the anomeric centers of 6
and 7 were, in fact, crucial for binding to ConA.
Results and discussion
ITC Studies
The binding interactions between ConA and specific monosac-
charides have been quantified by calorimetric titrations where the
heat produced or absorbed during the titration has been detected.
The ITC data for one of the substrates (7) are shown in Fig. 5.
Each addition of the ligand solution to the solution of ConA
resulted in the release of heat, and a concomitant peak is observed
in the data trace (Fig. 5, left). As the titration progressed, the heat
released diminished progressively due to the gradual saturation
of the available binding sites. The area data are then integrated,
analyzed, and binding isotherms constructed (Fig. 5, right).
Titration of ConA with methyl “manno” a-septanoside 14 was
to confirm that, even in the higher affinity series (i.e. methyl
“manno” b-septanoside 7), stereochemistry at the anomeric center
was in fact critical for binding. Lack of any discernable binding of
14 supported this assumption. Methyl b-2-O-methyl-septanoside
15 was used to gauge whether addition of specific functional
groups at the C2 position could increase affinity for ConA. A
preliminary manual overlay of 15 at the binding site on ConA
suggested that methyl b-septanoside 6 was “de-phased” relative
to its pyranose counterpart 9;19 that is, C2 of the septanose
partially overlapped with the anomeric carbon (C1) of 9 in the
binding pocket. Acylation/alkylation at C2 of the septanose was
therefore intended to mimic the aglycone of the pyranose. Binding
by ConA to 15 was of a similar magnitude to that observed
for the related methyl “gluco” b-septanoside 6; it seemed that
functionalization at the C2 hydroxyl group increased the affinity
for ConA only marginally. Based on the modest binding behavior,
the complications with acylation at C2 and anticipated difficulties
of alkylation with other groups at this position, more detailed
structure affinity relationships at this position were not pursued.
Overall, the experimental binding data provided a model for the
interaction where affinity depended on individual stereocenters of
the monosaccharide (both pyranose and septanose) ligands and
also the presence of an anomeric center, presumably as a rigidifying
feature.
Fig. 5 ITC of methyl “manno” b-septanoside 7 into ConA. Titration
conditions are given in Table 1. (Left) Raw injection traces from titration
of 7 into ConA (red) and dilution of 7 into buffer (black). (Right) Titration
curve generated from raw data.
Analysis of the data with specific binding models that gave best
fits to the data were used to obtain the binding enthalpy (DH),
binding entropy (DS), binding free energy (DG), affinity constant
(Ka), and the number of binding sites per ConA (N) extracted
(Table 1). Such analysis of the ITC data with other substrates
has been carried out and resulting thermodynamic parameters
collected in Table 1, which are discussed below.
Binding of the pyranoside ligands 9–10 and septanosides 6,
7, and 15 to ConA was exothermic. A notable parallel between
the trend in binding affinities for methyl b-septanosides 6–8 and
methyl a-pyranosides 9–11 became apparent. The rank ordering
of ConA affinities for the pyranosides is methyl a-mannoside 10
(7900 M-1) followed by methyl a-glucoside 9 (2000 M-1). Methyl
a-galactoside 11 is not bound by ConA.46 The same ordering of
affinities for methyl b-septanosides held true. Manno- analog 7
(850 M-1) was bound more tightly than gluco- analog 6 (520 M-1)
and the galacto- analog 8 showed no binding in our assay. The data
clearly suggested that the methyl b-septanosides bind in a manner
analogous to methyl a-pyranosides based on this parallel. A key
point is that the septanoside binders were consistently poorer
It should be noted that the concentration of ConA used in the
above titrations, when combined with low affinities of ligands 6
and 15, resulted in c parameters of 0.10 to 0.16.47 These values
are outside the optimal experimental window (c = 10–500). A
consequence of the low c values is that the binding stoichiometry,
expressed as parameter N in Table 1, deviated from the expected
value of 1.48 As was done previously, the data for 15 were fit
where the N value was manually set at 1. This was done to
approximate the DH value in such a scenario. The magnitude of
DH can be underestimated when the c value is low. As presented
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below, the experimental DH values where N was either allowed
to float or was fixed were correlated to the calculated DH values
separately.
frozen cavity while the open triangle corresponds to the case with
a relaxed cavity. The experimental DH values used in this plot
are those in which N was allowed to float. These two datasets
gave correlation coefficients of 0.968 and 0.965, respectively. Both
coefficients are of sufficient magnitude to indicate that both models
captured the most important aspects of the interaction. If the same
correlation is carried out with the DDH values for ligands 6 and
15 where N was fixed at 1, a correlation of 0.885 and 0.865 is
obtained, respectively, suggesting that fixing the N values may, in
fact, overestimate the experimental DH values.
Binding energy contributions
In order to analyze the origin of the different binding energies
amongst the ligands, we separated the binding energy into two
contributions. First, we determined a deformation energy (Ed ),
defined as the energy required to deform the gas-phased optimized
structure into the conformation adopted by the ligand inside the
protein. Second, we computed the rigid-binding energy (Erb), using
the deformed ligand for the gas phase calculations in eqn (1),
instead of the relaxed one. Thus, the binding energy can also be
expressed as:
Structure–energy relationships
The correlation between the computational binding energies and
the experimentally determined DDH values gave us confidence
to use the computed structures to piece together the protein–
ligand interactions contributing to the range of binding energies
observed. Fig. 7 shows the Glide-QM/MM structure of ConA
bound to methyl a-glucoside 9 according to the frozen cavity
QM/MM model. The calculated complex reproduces the co-
crystal structure almost exactly, resulting in a root mean square
(4)
Eb = Ed + Erb.
Table 2 shows the computed relative binding energies for ConA
bound to 5–10 and 15 (DEb) along with their decomposition in
the various terms. Since we were particularly interested in the
deformation effect of the ligand alone, we made the separation of
rigid binding and deformation using the computational model
which assumes a frozen protein cavity. Thus, for the set of
calculations with a relaxed cavity, we only report the relative
binding energies. The reference system was chosen to be the
complex between ConA and methyl “manno” b-septanoside 7
because it had the greatest enthalpic contribution to binding of
the ligands investigated (Table 2). The binding energies show a
very good qualitative agreement with the experimental enthalpy
values (DDH) for all ligands and for both sets of calculation (frozen
protein and relaxed cavity). In both experiment and computation,
the complex of ConA with 7 displayed the largest enthalpy of
binding. The computed values of +17 kcal mol-1 for ConA·8
and +9 kcal mol-1 for ConA·5 were also consistent with the lack
of binding observed by ITC (Table 1). Complexes that showed
intermediate DEb and DDH values for ConA bound to 6, 9, 10, and
15 also correlated well (Fig. 6). The two plots in Fig. 6 demonstrate
the correlation. The solid squares correspond to the case with a
˚
deviation (RMSD) of 0.3 A. One immediate observation is that
Asp208 is the acceptor of two hydrogen-bonds from the glycoside,
a feature common to all the ligands considered. In this regard,
it should be expected that elimination of Asp208 would cause a
substantial decrease in binding affinity.
Fig. 7 QM/MM structure of the ConA·9 complex. All immediate
residues form hydrogen-bonds with the methyl a-D-glucoside are displayed
as thin green lines.
The enthalpic contribution to the binding affinity in this
complex primarily arises from the seven hydrogen-bonds which
connect it to a number of residues in the ConA binding pocket
(see Fig. 7). In particular, 9 makes two hydrogen bonds to the side
chain of Asp208, two to the backbone of Leu99, one to each of
the backbones of Arg228 and Tyr100, and one to the side chain of
Asn14. The side chain of Leu99 is positioned so that a-pyranosides
such as 9 matched the binding pocket, and escape unfavorable van
der Waals contacts between the aglycone and Leu99. The aglycone
of a b-pyranosides, on the other hand, would need to occupy
the same space as Leu99 and are therefore not bound by ConA.
Fig. 6 Correlation between the computed binding energy and the
experimental enthalpy of binding. Solid line represents a linear regression
(correlation coeff. = 0.968) using the solid square data. Dashed line
represents a linear regression (correlation coeff. = 0.962, see text for
discussion) for open triangles data.
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Fig. 8 QM/MM structures of complex ConA with ligands 6, 7, 8, and 10 showing the main interactions with the protein.
We also investigated one other complex containing a pyranoside
ring, methyl a-mannoside 10. According to our calculations there
is a small increase in the difference in binding energies (DEb)
between 10 and 9 (0.7 kcal mol-1 and 0.1 kcal mol-1 for the two
computational models considered) in qualitative agreement with
the experimental increase in the enthalpy of binding.
binding. Therefore, the origin of the more favorable affinity of
ConA for the pyranoside must be dominated by entropy. Indeed,
due to the additional stabilization of ConA·7 versus ConA·10
coming from more favorable van der Waals contacts (DH(ConA·7)
- DH(ConA·10) < 0), 7 is more tightly bound than 10. Thus,
it is expected that 10, in the ConA·10 complex, should have a
larger conformational entropy than 7 in the ConA·7 complex
(TDS(ConA·7) - TDS(ConA·10) < 0), which is in agreement
with the ITC data. On the other hand, a similar comparison of
gluco-configured methyl glycosides 6 and 9 revealed that 9 takes
considerably less energy to adopt the bound conformation than
does 6. Such an enthalpic difference is what finally dominates,
resulting in larger binding affinities for the complex ConA·9 with
respect to ConA·6, which is also in agreement with the ITC data.
We next sought to determine the origin of the different affinities
between the two methyl b-septanosides. The difference of ~ 5 kcal
mol-1 in the calculated binding energy between methyl “manno”
b-septanoside 7 and methyl “gluco” b-septanoside 6 comes mostly
from differences in the deformation energy. It takes about 4 kcal
mol-1 more energy to reconfigure ligand 6 into a conformation
appropriate for binding relative to 7.
Fig. 8A shows that 10 is bound by ConA in a very sim-
ilar way as 9, maintaining the same number and type of
hydrogen-bonds. Both methyl “manno” b-septanoside 7 (Fig. 8B)
and methyl “gluco” b-septanoside 6 (Fig. 8C) are the seven-
membered ring homologs of methyl a-mannoside 10 and methyl a-
glucoside 9, respectively. The seven hydrogen bonds that are critical
to binding pyranosides were conserved for both of the methyl b-
septanoside ligands. Inspection of the components that contribute
to the binding energies of manno-configured methyl glycosides 7
and 10 showed that the energy required to deform the molecule to
fit in the ConA active site was the same for both molecules (Table
2). The main difference, therefore, came from different interactions
with the protein. Considering the van der Waals contribution to
the protein–ligand interaction energy, which is readily available in
QM/MM calculations, revealed that the complex ConA·7 is about
1.5 kcal mol-1 more stable than its six-member ring counterpart
(ConA·10), despite having an overall less favorable free energy of
As noted previously, the conformation of unbound 6 is the same
as we had determined in our earlier conformational analysis.36
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Fig. 9 Changes in the number of internal OH–O interactions upon deformation to fit the active site.
We argue that, based on this correspondence, the conformation
of free ligand 7, following the present minimization protocols, is
also accurate. Inspection of the pairs of structures (unbound and
bound for both 6 and 7) shows that the main difference between
them comes from the reorientation of the ring hydroxyl groups in
going from the free to the bound state (Fig. 9). Reorientation of
the ring hydroxyls is governed by the need to make the C5 and C7
hydroxyl groups available to donate hydrogen-bonds to Asp208.
One way to describe these changes is to count the number of
electrostatic OH ◊ ◊ ◊ O interactions before and after binding. In
methyl “manno” b-septanoside 7, this number changes from three
to two (see arrows in Fig. 9), while in methyl “gluco” b-septanoside
6 it changes from five to three, accounting for most of the difference
in binding energy between these two ligands. A similar analysis can
be invoked for ligand 15 (figure shown in Supporting Information),
which has the same stereochemistry as 6, but contains a methyl
ether rather than a hydroxyl group at C2.
C6 (septanoside numbering) for productive protein–septanoside
interactions. A similar argument can be applied to the ConA·5
complex. The QM/MM structure of this complex reveals the seven
hydrogen bonds between ligand and protein are conserved (figure
and analysis shown in Supporting Information). Thus, the only
contributor to destabilization comes from a large deformation
energy upon binding titrations of ConA with methyl “manno” a-
septanoside 14 also did not show evidence of binding. Whereas the
stereochemistry at C5 in 8 prevented binding, it was the anomeric
(C1) configuration of 14 that obstructed protein association.
Methyl septanosides 7 and 14 share the same stereochemistry
around the ring from C2–C6; the only difference between them is
that 7 is a methyl b-septanoside and 14 is a methyl a-septanoside.
Results obtained were consistent with our earlier observation
that a-septanosides were not ligands of ConA. This reversal of
selectivity (b-anomers bound in preference to a-anomers) for
septanoside binding relative to pyranosides (a-anomers bound
in preference to b-anomers) was attributed to a combination of
septanoside conformation in the bound state and the interaction
between the aglycone methyl group and the Leu99 side chain.
The configuration of stereocenters in oxepanes 12 and 13
is consistent with that of glucose and mannose, respectively.
Titration data for oxepanes 12 and 13 suggested that they may
be ligands of ConA but, if so, their affinities were below those
reported for the weakest binders (6 and 15) in Table 1. These
results reinforced the fact that the anomeric center was a necessary
feature for binding and that it likely aided in rigidifying the
structure of the septanoside. We propose that these oxepanes are
in fact bound by ConA, but with very low affinity. Glycerol, for
example, makes contacts to ConA in the monosaccharide binding
pocket, as evidenced in X-ray structures.49 Overall, the ligands
that demonstrated low or no affinity for ConA were essential to
the development of a general model of how structural features
affected affinity Table 2.
Non-binding ligands
A few of the ligands were either not bound by ConA (i.e. 8,
14) or were only very weakly bound (i.e. 12, 13) as measured
by ITC. The low stability of the ConA·8 complex (Fig. 8D)
clearly comes from the combined effect of breaking the hydrogen
bonds between the ligand and Asn14 and Leu99 and the excessive
energy required to induce fitting. As in the deformation of 6, two
favorable electrostatic OH ◊ ◊ ◊ O hydrogen-bonding interactions in
8 are lost in going from the unbound to the bound conformation.
Additionally, an unfavorable dipole–dipole interaction is induced
by the two hydroxyl groups at C2 and C4 (Fig. 8D) which
are pointing in different directions. The inability of methyl
“galacto” b-septanoside 8 to be bound by ConA parallels the
behavior of methyl a-galactoside 11. This observation reinforced
the importance of the stereochemistry of the ring from C3–
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Hz, 1H), 3.56–3.51 (m, 1H), 3.41 (dd, J = 11.6, 7.0 Hz, 1H), 3.24
(ddd, J = 9.3, 6.8, 2.8 Hz, 1H), 3.18–3.10 (m, 2H), 1.82–1.78 (m,
2H); 13C NMR (CD3OD) 75 MHz d 82.3, 79.3, 74.9, 71.2, 66.4,
64.4, 34.3; HRMS [M + Na]+ m/z calcd for C7H14O5Na 201.0733,
found 201.0727.
Conclusions
The observation and analysis of ConA binding to monosac-
charides reported here leads to three major conclusions: first,
methyl b-septanosides are bound by ConA in a manner that is
analogous to methyl a-pyranosides. Second, the greater binding
affinity of pyranosides versus septanosides has a different origin
for glucosides and mannosides; in glucosides, the lower affinity of
the septanosides is dominated by an unfavorable enthalpy contri-
bution, while in mannosides, the lower affinity of the septanoside is
dominated by an unfavorable conformational entropy. Third, the
hypothesis of a constant solvent reorganization enthalpy amongst
the ligands considered has been confirmed.
An important contributor to the differential binding enthalpy
among natural and unnatural monosaccharides is the energy
required to reorient the hydroxyl groups of a given ligand. This
is mainly the result of the loss of two internal contacts involving
the hydroxyls, which are always needed to bind Asp208. Since
the stereochemistry is different among many of these ligands, this
reorientation is different among them. The two non-binding events
found experimentally and computationally were rationalized in
terms of an unfavorable enthalpic contribution due to both the
deformation energy and the loss of intermolecular hydrogen bonds
with the protein when complexed.
1,6-Anhydro-2-deoxy-D-glycero-D-mannoseptitol (13)
20% Pd(OH)2/C (0.157 g) was added to a solution of oxepine 17
(0.100 g, 0.187 mmol) in MeOH (10 mL). The reaction vessel was
purged with N2, then H2 was introduced via a balloon attachment.
The mixture was allowed to stir overnight (15 h) at rt. The vessel
was purged with N2 before being exposed to the atmosphere.
The mixture was then filtered through a short pad of celite.
The celite was washed with MeOH (4 ¥ 5 mL) and the solvent
was removed from the combined filtrates by rotary evaporation
under reduced pressure to give 1,6-anhydro-2-deoxy-D-glycero-D-
mannoseptitol (13) (0.028 g, 93%). [a]D -2.05 (c 1.00, CH3OH);
1H NMR (CD3OD) 300 MHz d 4.10 (dddd, J = 9.0, 1.7, 1.6, 1.5
Hz, 1H), 4.02 (ddd, J = 12.4, 5.2, 5.0 Hz, 1H), 3.74–3.69 (m, 2H),
3.60–3.47 (m, 3H), 3.35–3.28 (m, 1H), 2.13 (dddd, J = 18.1, 9.1,
5.3, 5.3 Hz, 1H), 18.0–1.71 (m, 1H); 13C NMR (CD3OD) 100 MHz
d 86.0, 80.2, 72.2, 71.1, 68.1, 64.9, 34.2; HRMS [M + Na]+ m/z
calcd for C7H14O5Na 201.0733, found 201.0728.
Methyl 3,4,6-tetra-O-benzyl-2-O-methyl-b-D-glycero-D-
gulosepanoside (19)
Experimental section
Provided here are schemes and procedures for the preparation
of molecules 12, 13, and 15. Characterization data used to
confirm structures of intermediates and products is provided in
the supporting information.
Unless other conditions were specified, reactions were con-
ducted at room temperature under nitrogen atmosphere and all
the solvents and reagents were purchased commercially and used
without further purification. Reactions were monitored by TLC
To an ice cold solution of methyl 3,4,5,7-tetra-O-benzyl-b-D-
glycero-D-guloseptanoside 18 (0.077 g 0.132 mmol) in THF (5
mL) was added sodium hydride 60% oil dispersion (0.006 g, 0.145
mmol) followed by methyl iodide (0.024 mL, 0.296 mmol) in two
portions. The reaction was allowed to warm to rt and stirred for
18 h. The reaction was then quenched with the addition of 5 mL
EtOAc–H2O (1 : 1). The mixture was concentrated and extracted
with DCM (3 ¥ 20 mL). The combined extract was dried with
Na2SO4 and the solvent was removed under reduced pressure. The
resulting residue was purified via column chromatography using
7 : 3 hexanes : EtOAc as eluent to give methyl 3,4,6-tetra-O-benzyl-
2-O-methyl-b-D-glycero-D-gulosepanoside (19) as a colorless oil
(0.063 g, 80%). [a]D +14.79 (c 3.21, CDCl3); IR (KBr) cm-1
3087.66, 3062.98, 3030.11, 2930.80, 2862.78, 1960.11, 1872.13,
1811.78, 1722.81, 1604.74, 1496.60, 1453.47, 1387.69, 1361.08,
1229.92, 1276.26, 1206.95, 1110.10, 1074.43, 1028.00, 987.35,
˚
(silica gel, 60 A, F254, 250 nm) with visualization conducted either
under UV light or by charring with 2.5% p-anisaldehyde in H2SO4,
acetic acid, and ethanol solution. Flash column chromatography
˚
was conducted on silica gel (60 A, 32–63 mm). Optical rotations
were measured at 22 2 ◦C. 1H NMR spectra were recorded at 300,
400 and 500 MHz with chemical shifts referenced to the residual
signal in CDCl3 (dH 7.27 ppm) or CD3OD (dH 3.31 ppm). 13C
spectra were collected at 75, 100 and 125 MHz and referenced to
the residual signal in CDCl3 (dC 77.2) or CD3OD (dC 49.0 ppm).
1
910.51, 736.29, 697.40, 600.75, 484.17; H NMR (CDCl3) 300
MHz d 7.40–7.20 (m, 20H), 4.72–4.54 (m, 8H), 4.40 (d, J = 11.25
Hz, 1H), 4.07 (ddd, J = 9.7, 6.4, 2.3 Hz, 1H), 4.00–3.95 (m, 2H),
3.72 (dd, J = 10.2, 2.0 Hz, 1H), 3.67–3.57 (m, 3H), 3.55 (s, 3H),
3.46 (s, 3H); 13C NMR (CDCl3) 75 MHz d 138.7, 138.5, 138.4,
128.6, 128.5(2), 128.3, 128.1, 128.0, 127.9, 127.8, 127.6(2), 106.0,
83.0, 81.4, 79.2, 77.9, 75.8, 73.6, 73.5, 73.4, 73.2, 71.7, 59.2 56.4;
FAB-MS [M + Na]+ m/z calcd for C37H42O7Na+ 621.2823, found
621.2801.
1,6-Anhydro-2-deoxy-D-glycero-D-glucoseptitol (12)
20% Pd(OH)2/C (0.106 g) was added to a solution of oxepine 16
(0.112 g, 0.209 mmol) in MeOH (7 mL). The reaction vessel was
purged with N2, then H2 was introduced via a balloon attachment.
The mixture was allowed to stir overnight (15 h) at rt. The vessel
was purged with N2 before being exposed to the atmosphere.
The mixture was then filtered through a short pad of celite.
The celite was washed with MeOH (4 ¥ 5 mL) and the solvent
was removed from the combined filtrates by rotary evaporation
under reduced pressure to give 1,6-anhydro-2-deoxy-D-glycero-D-
glucoseptitol (12) as a beige solid (0.035 g, 99%). [a]D -12.62 (c
Methyl 2-O-methyl-b-D-glycero-D-gulosepanoside (15)
20% Pd–OH/C (0.053 g) was added to a solution of 19 (0.062 g,
0.103 mmol) in MeOH (5 mL). The reaction vessel was purged with
N2, then H2 and was kept under an H2 atmosphere via a balloon
attachment. The mixture was allowed to stir 15 h at rt. The vessel
1
1.96, CH3OH); H NMR (CD3OD) 500 MHz d 3.77–3.71 (m,
1H), 3.69 (dd, J = 11.0, 2.8 Hz, 1H), 3.61 (ddd, J = 12.1, 5.2, 4.6
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was then evacuated of H2 and purged with N2 before being exposed
to the atmosphere. The mixture was filtered through a short pad
of celite and washed with CH3OH (4 ¥ 5 mL). The solvent was
removed from the combined filtrates by rotary evaporation under
reduced pressure to give 15 as an off-white solid (0.024 g, 99%);
[a]D +6.34 (c 1.30, CH3OH); 1H NMR (CD3OD) 300 MHz d 4.48
(d, J = 3.6 Hz, 1H), 3.87 (d, J = 11.3, 2.3 Hz, 1H), 3.78–3.70
(m, 2H), 3.62–3.49 (m, 3H), 3.48 (s, 3H), 3.47 (s, 3H); 13C NMR
(CD3OD) 75 MHz d 108.0, 86.8, 79.8, 75.4, 74.8, 71.6, 64.4, 60.0,
56.6; FAB-MS [M + Na]+ m/z calcd for C9H18O7Na+ 261.0945,
found 261.0941.
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Acknowledgements
Bikash Surana (U. of Connecticut) helped with the synthesis and
characterization of compound 12. Martha Morton and Srikanth
Rapole (U. of Connecticut) are thanked for technical assistance
with NMR and mass spectrometry, respectively. M.W.P. thanks
the National Science Foundation (NSF) for a CAREER Award
(CHE-0546311). J.A.G thanks financial support from the Camille
and Henry Dreyfus foundation and NSF for a CAREER Award
(CHE-0847340). C. V. K. and M. R. D. thank the NSF Division
of Materials Research (DMR-0604815, and DMR-1005609) for
partial financial support of this work.
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