Separation of betti reaction product enantiomers: Absolute configuration and inhibition of botulinum neurotoxin A

Article abstract of DOI:10.1021/ml200028z

The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde, and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to h

Full text of DOI:10.1021/ml200028z

LETTER
pubs.acs.org/acsmedchemlett
Separation of Betti Reaction Product Enantiomers: Absolute
Configuration and Inhibition of Botulinum Neurotoxin A
John H. Cardellina II,† Rebecca C. Vieira,† Vanessa Eccard,† Janet Skerry,† Vicki Montgomery,†
Yvette Campbell,† Virginia Roxas-Duncan,† William Leister,‡ Christopher A. LeClair,‡ David J. Maloney,‡
Daniele Padula,§ Gennaro Pescitelli,§ Ilja Khavrutskii,|| Xin Hu,|| Anders Wallqvist,|| and Leonard A. Smith*,^
†Division of Integrated Toxicology, U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Maryland, United States
‡NIH Chemical Genomics Center, National Human Genome Research Institute, NIH, 9800 Medical Center Drive, MSC 3370,
Bethesda, Maryland, United States
§Department of Chemistry, University of Pisa, Pisa, Italy
Biotechnology High Performance Computer Software Application Institute, Telemedicine and Advanced Technology Research Center,
U.S. Army Medical Research and Materiel Command, Frederick, Maryland, United States
^Office of Chief Scientist, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, United States
S Supporting Information
b
ABSTRACT: The racemic product of the Betti reaction of
5-chloro-8-hydroxyquinoline, benzaldehyde, and 2-aminopyri-
dine was separated by chiral HPLC to determine which
enantiomer inhibited botulinum neurotoxin serotype A. When
the enantiomers unexpectedly proved to have comparable
activity, the absolute structures of (þ)-(R)-1 and (ꢀ)-(S)-1
were determined by comparison of calculated and observed
circular dichroism spectra. Molecular modeling studies were
undertaken in an effort to understand the observed bioactivity
and revealed different ensembles of binding modes, with
roughly equal binding energies, for the two enantiomers.
KEYWORDS: Chiral resolution, Betti reaction products, TDDFT CD calculations, molecular docking, inhibition of botulinum
neurotoxin
e recently reported the identification of 7-substituted
8-hydroxyquinolines (e.g., 1), products of the Betti reac-
recombinant BoNT/A protease light chain (LC).1 To our
surprise, both compounds displayed similar IC50 values, 1.0
and 1.1 μM for (þ)-1 and (ꢀ)-1, respectively. We subsequently
evaluated their potential to inhibit the biological activity of
BoNT/A holotoxin in murine neuroblastoma N2a cells.1 No
difference was observed in percent inhibition (P > 0.05) of
SNAP-25 cleavage for both enantiomers and the racemate (()-1
at the four concentrations tested (60, 45, 30, and 15 μM). We
then examined the efficacy of these compounds in mouse phrenic
nerve hemidiaphragm preparations (MPNHDA).1 Similar to
observations in HPLC and cell-based assays, both (þ)-1 and
(ꢀ)-1 were equipotent (P = 0.94) in the tissue-based assay. At 2
μM concentrations, both enantiomers dramatically delayed (P =
1.58 ꢁ 10ꢀ8 and 2.30 ꢁ 10ꢀ6 for (þ)-1 and (ꢀ)-1, respectively)
the BoNT/A-induced paralytic half-time 3-fold. The compara-
tive testing of (()-1 and the two enantiomers is summarized in
Table 1.
W
tion, as leads to potential chemotherapies for botulinum poison-
ing.1 The Betti reaction2 originated as a condensation of Schiff
bases with 2-naphthol,3,4 but it was later extended to other
nucleophilic aromatic substrates, e.g., 8-hydroxyquinoline.5
While a chiral center is formed in this reaction, the products
are typically racemic mixtures, as the reagents are usually achiral
(Scheme 1). Over the years, there have been several efforts to
resolve Betti product racemates, most of which involve preparing
diastereomeric salts with chiral acids.6ꢀ8
Chiral chromatography seemed an obvious alternative for
resolution of these racemates, but we examined a total of six
chiral columns and 18 different methods before observing
sufficient resolution of the two enantiomers on a Chiralcel OD
column to permit semipreparative purification of adequate
quantities of (þ)-1 and (ꢀ)-1 for evaluation in our bioassays
and assignment of absolute configuration (Figures S1 and S2 of
the Supporting Information).
Initial comparison of the botulinum neurotoxin serotype A
(BoNT/A) inhibitory activity of the (þ) and (ꢀ) enantiomers of
1 was accomplished via an HPLC-based assay using a full-length
Received: February 3, 2011
Accepted: March 6, 2011
Published: March 11, 2011
r
2011 American Chemical Society
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|

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We then turned to assigning the absolute configuration of
(þ)-1 and (ꢀ)-1 via comparison of calculated and experimental
electronic dichroism (CD) spectra. As a prelude, we determined
the 3-dimensional conformation of 1 through a series of NMR
experiments. Proton and carbon resonances were assigned from a
combination of COSY, HSQC, and HMBC experiments (Table
S1, Supporting Information). Numerous NOE interactions were
observed in the NOESY experiment (Scheme 2), with those of
NH, OH, H6, and H9 providing the greatest insight into the
conformation of the compound. While all NMR data were
obtained for the racemic mixture, the (S)-enantiomer is shown
for ease of portraying the structural analysis. The observed NOE
between H6 and H18a,b and lack of NOE between the C8
hydroxyl proton and H18a,b indicate that the phenyl ring is
rotated away from the C8 OH. The NOE between the OH and
the NH, as well as the OH and H9 interaction, supports this
conformation. The phenyl ring assumes a position perpendicular
to the quinoline ring system to minimize steric interactions. This
is supported by NOEs of H18a,b with H6, H9, and the NH.
Interestingly, H15 and H16 both have an NOE with the NH, but
not with H9, suggesting that the pyridine ring is oriented so as to
place the ring nitrogen toward H9, while H15 and H16 are
projected away. Also, this allows the pyridine ring to be ortho-
gonal to the quinoline and pseudoparallel to the phenyl ring,
further minimizing steric interactions. A locked conformation of
the compound due to hydrogen bonding between the lone pair of
the NH and the proton of the C8 hydroxyl is highly probable.
However, the high number of NOEs for the NH (OH, H6, H9,
H18a,b, and H19a,b) implies that the structure may not be
entirely rigid.
(TDDFT) method.11,12 The enantiomers of 1 show almost
mirror image CD spectra in methanol, as expected (Figure 1).
Due to the presence of three different aromatic chromophores,
these spectra feature many bands between 200 and 350 nm. For
the (þ)-enantiomer, the first band appearing in the 280ꢀ340 nm
region is broad and positive, followed by a moderately intense
negative band centered around 260 nm and two stronger bands, a
positive one at 245 nm and a negative one around 220 nm.
Several rotatable bonds in the structure of 1, most directly
affecting the relative orientation of the chromophores, made
obtaining a reliable set of input structures crucial for CD
calculations. A preliminary molecular-mechanics conformational
search was run with the MMFF force field, using a starting
geometry with (R) absolute configuration (see Supporting
Information for details). All low-energy structures obtained were
optimized with the DFT method at the B3LYP/6-31G(d) level,
converging to a set of nine distinct conformers within 10 kJ/mol.
Their energies and populations at 298.15 K were estimated with
B3LYP/6-311þþG(d,p) in methanol (PCM solvent model).13
The low-energy DFT structures were then checked against the
conformational picture provided by NMR experiments, bearing
in mind that the coexistence of several low-energy minima
rendered interpretation of NOEs in terms of a single conforma-
tion questionable. The calculated structures may be divided into
Scheme 2
To assign the absolute configuration of (þ)- and (ꢀ)-1, their
electronic CD spectra9,10 were recorded in solution and com-
pared with those calculated using the time-dependent DFT
Scheme 1
Table 1. Comparative Testing of (()-1, (þ)-1, and (ꢀ)-1a
HPLC assay
N2a cell culture assay
MPNHDAb
% inhibition
5 μM
IC50
% inhibition of SNAP-25 cleavage
minutesc,d
sample
20 μM
(μM)
60 μM
45 μM
30 μM
15 μM
2 μM
(()-1
(þ)-1
(ꢀ)-1
92
94
91
88
90
90
1.5
1.1
1.0
91
96
96
90
94
93
74
89
69
41
56
54
NDe
191f
188f
a Assays conducted as described in ref 1 and the Supporting Information. b Mouse phrenic nerve hemidiaphragm assay. c Average time to 50% loss of
twitch tension (min). d Average value for BoNT/A toxin control was 63 min. e Not determined. f P value < 0.001 (highly significant) for comparison with
values recorded for the BoNT/A control; statistical analyses performed using SigmaPlot 10 (Systat Software, San Jose, CA).
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two subsets according to the rotation around the C7ꢀC9 bond.
In one (major) subset, composed of four lowest-energy con-
formers, H9 is directed toward the OH. Within this subset, two
conformers are especially stable (absolute minimum, 30.8%
population at 298.15 K; second lowest minimum, þ0.37 kJ/
mol, 26.5%; Figure 2); the structures and relative energies for all
minima are reported in the Supporting Information. In the
second (minor) subset, composed of the remaining five con-
formers accounting for 25% overall population, H9 is again in the
plane of the quinoline ring but directed toward C6. These
findings corroborate the observed NOEs that NH and H9 each
have with both the C8 OH and H6.
Taking the lowest-energy structure as a test molecule,
TDDFT calculations were run using different combinations of
hybrid DFT functionals (B3LYP, PBE0, CAM-B3LYP, BH&
HLYP) and basis sets (SVP, TZVP, aug-TZVP),14 in vacuo or in
methanol. The three basis sets led to very similar results (using
B3LYP), except for a small red shift observed for all computed
transitions on increasing the basis size. It appears that the two
“standard” functionals B3LYP and PBE0 led to calculated
transition energies underestimated with respect to the experi-
ment, while the opposite was true for Coulomb-attenuated
B3LYP (CAM-B3LYP) and the half a d-half functional
BH&HLYP. Looking, for example, at the absorption band
measured at 250 nm, the calculated transition wavelength was
270 and 230 nm with B3LYP/aug-TZVP and CAM-B3LYP/
SVP, respectively. Apart from a systematic wavelength shift, the
shape of the calculated CD spectrum was similar in all cases.
Finally, including the solvent model in CAM-B3LYP/SVP
calculations did not appreciably change calculated frequencies
and spectra.
TDDFT calculations were then run on all low-energy struc-
tures using the two combinations B3LYP/aug-TZVP and CAM-
B3LYP/SVP in vacuo. The resulting CD spectra were weighted
with the respective Boltzmann factors (estimated from B3LYP/
6-311þG(d,p) internal energies in methanol) at 298.15 K
and summed to afford weighted average spectra. In all cases,
input structures had (R) absolute configuration. Figure 3 displays
spectra computed with the two methods for the lowest-energy
structure and the weighted averages over nine structures. Apart
from the already discussed wavelength shift (taken into account
by a frequency correction in Figure 3), the overall shapes of the
four spectra are quite similar, especially in the low-energy region
(where TDDFT calculations are intrinsically more accurate).15
CAM-B3LYP/SVP results agree especially well with the experi-
mental spectrum for (þ)-1 in sign, position, and intensity of
bands (Figure 3b). Therefore, the absolute configuration of the
enantiomers of compound 1 may be assigned as (þ)-(R)-1 and
(ꢀ)-(S)-1. It must be noted that the calculated average CD
spectrum is the superposition of very heterogeneous component
spectra; thus, the apparent bands are actually the convolution
from several transitions (Supporting Information).
The unexpected observation of virtually equivalent BoNT/
A-inhibitory activity for the enantiomers in three different
bioassays prompted us to examine the bound state of the ligands
to rationalize the apparent lack of discrimination. Given the
observed pharmacological activity, we hypothesized that both
enantiomers bind BoNT/A, presumably in the Zn2þ-containing
active site. Nevertheless, experimental observations suggest that
binding of a similar ligand is not exclusively due to Zn2þ
chelation.1,16 To test this hypothesis computationally, we as-
sessed relative binding free energies for the enantiomers by
performing Thermodynamic Integration with Molecular Dy-
namics simulations17 (see the Supporting Information for
details). Because the protonation state of the bound ligand is
unknown, we assumed that both enantiomers bind the active site
Zn2þ without ionization of the OH group. Using 10 lowest stable
conformations of each ligand, we generated corresponding
complex conformations by superposing the quinoline moiety
onto a template complex derived with AutoDock4.0.18ꢀ21 The
resulting calculations indicated that the (ꢀ)-S-enantiomer would
Figure 1. CD spectra of the enantiomers of 1 in methanol; concentra-
tion 4.8 ꢁ 10ꢀ5 M.
Figure 2. DFT-optimized structures for the absolute lowest energy (left) and second lowest energy (right) conformers of (R)-1.
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Figure 3. CD spectra calculated for (R)-1 with two TDDFT methods: (A) B3LYP/aug-TZVP; (B) CAM-B3LYP/SVP. Blue lines: spectra calculated
for the lowest-energy DFT structure (divided by 2 for better comparison). Black lines: averages of spectra calculated for nine low-energy DFT structures
weighted with Boltzmann factors at 298.15 K using populations estimated with B3LYP/6-311Gþþ(d,p) in methanol. Red dashed line: experimental
spectrum for (þ)-1. Frequency corrections of þ2500 cmꢀ1 and ꢀ2000 cmꢀ1 have been applied to calculated spectra in panels A and B, respectively.
both (þ)-R-1 and (ꢀ)-S-1 to BoNT/A, along with the corre-
sponding binding features, we performed GROMOS-style
clustering22 of the sparsely saved simulation trajectories in the
physical state for each of the 10 starting configurations of the (R)-
and (S)- complexes. (Table S3 of the Supporting Information
summarizes the 16 most prominent features describing the two
ensembles and comprising H-bonding interactions, coordinating
bonds to the Zn2þ ion and πꢀπ interactions.) The most robust
features include coordinate bonds of atoms O, N1 (quinoline),
and N12 (pyridine) to the Zn2þ ion. Depending on the number
of bonds, the ligand can be monodentate or bidentate. When
both O and N12 chelate the Zn2þ, the N10 (amine) atom is
brought sufficiently close to the Zn2þ that this coordination
mode could be considered as tridentate. The most common
H-bonding interaction is that of the coordinated OH group with
the carboxyl of E224.
Clustering analysis of the trajectories of the proteinꢀligand
complexes captured the diversity of the binding modes that can
be described by a few features. Most of these features are
common to both enantiomers and pertain to specific interactions
of the ligand with the binding site residues. We found that the
(ꢀ)-S-1 ensemble, on average, has more interaction features per
cluster, 3.6 versus 3.0 for that of the (þ)-R-1. In addition, the
(ꢀ)-S-1 ensemble has a larger number of unique interactions
with the active site compared to that of the (þ)-R-1. These
observations are consistent with the computed binding free
energy difference. Figure 4 illustrates examples of the likely
binding mode for each enantiomer.
Only a few interactions exist that can differentiate between the
two enantiomers. In particular, the N10HꢀE224 interaction is
Figure 4. Examples of binding modes for the (R)-enantiomer (A) and
(S)-enantiomer (B). Active site residues (yellow) of the protein (green)
participate in binding of the Zn2þ (magenta) and the ligand (gray). Blue
and red colors correspond to oxygen and nitrogen atoms. Certain active
site residues and hydrogen atoms are omitted for clarity. The red dashed
lines show some coordinating and hydrogen bonds.
realized only by the (R)-enantiomer, whereas the interactions
OH-Y366, N12ꢀF163(NH), and Ar:Phe-Y366 are only realized
by the (S)-enantiomer. Although it is possible that different
realizations of common interaction features by the enantiomers
can further contribute to binding disparity, quantifying those
differences could be daunting.
The fact that our calculations favor BoNT/A binding by the
(ꢀ)-S-1 suggests that, in reality, the ligands might bind differ-
ently. Unlike experimental measurements, our calculated values
provide a more limited assessment of the relative binding
affinities of the two enantiomers under specified assumptions.
It is also possible that the ligand can undergo epimerization,
bind BoNT/A by 3.8 ( 2.4 kcal/mol more favorably than its
(þ)-R-counterpart.
The large standard deviation in the free energy calculations
indicates significant variability in the conformational ensembles
of the bound ligands. To analyze the variety of binding modes of
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LETTER
similar to that of ibuprofen23,24 in the presence of certain
enzymes.25
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In conclusion, we separated the enantiomers of 1 (chiral
HPLC), established their chemical structure (NMR) and abso-
lute configuration (CD, molecular modeling), evaluated their
BoNT/A inhibitory activity, and explored their docking motifs
with BoNT/A LC. To our knowledge, this is the first use of CD
calculations to assign the absolute configuration of Betti reac-
tion product enantiomers. While chiral chromatography has
recently been applied to the separation of naphthol-based Betti
products,26,27 this is the first such separation of Betti products
comprised of 8-hydroxyquinoline, an aryl aldehyde and an
aryl amine.
While, in a vast majority of cases, one enantiomer of a racemic
drug or drug candidate has significantly greater pharmacological
activity than the other, as has recently been demonstrated for one
BoNT/A inhibitor,28 we found essentially equivalent BoNT/
A-inhibitory activity in (þ)-(R)-1 and (ꢀ)-(S)-1. However, this
unexpected finding can be explained by the proposed docking
motifs for the two enantiomers—different ensembles, but nearly
equivalent in energy. We are currently seeking to confirm those
binding models by crystallizing each enantiomer in the active site
of BoNT/A.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental protocols for
b
synthesis, chiral separation, and biological testing of (()-1, 1H
and 13C NMR assignments and NOE interactions, CD calcula-
tions leading to assignment of the absolute configurations of
(þ)-1 and (ꢀ)-1, and molecular modeling and docking of (þ)-
and (ꢀ)- 1 into the BoNT/A light chain. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(15) Casida, M. E.; Jamorski, C.; Casida, K. C.; Salahub, D. R.
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dependent density-functional response theory: characterization and
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Corresponding Author
*E-mail: Leonard.Smith@amedd.army.mil. Telephone: 301-619-
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’ ACKNOWLEDGMENT
This work was supported by a grant to L.A.S. from the Defense
Threat Reduction Agency, JSTO-CBD Project Number
3.10037_07_RD_B. R.C.V. was funded by a National Research
Council Research Associateship Award. This work was spon-
sored by the U.S. Department of Defense High Performance
Computing Modernization Program (HPCMP), under the High
Performance Computing Software Applications Institutes
(HSAI) initiative. We thank James Bougie for help with the
chiral HPLC separation. Opinions or assertions contained herein
are the private views of the authors and are not to be construed as
reflecting the official views of the United States Department of
Defense.
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