Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A

Abstract

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 have comparable activity, the absolute structures of (+)-(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.

Scheme 1

Scheme 2

Figure 1

CD spectra of the enantiomers of 1 in methanol; concentration 4.8 × 10⁻⁵ M.

Figure 2

DFT-optimized structures for the absolute lowest energy (left) and second lowest energy (right) conformers of (R)-1.

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/6-311G++(d,p) in methanol. Red dashed line: experimental spectrum for (+)-1. Frequency corrections of +2500 cm⁻¹ and −2000 cm⁻¹ have been applied to calculated spectra in panels A and B, respectively.

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 Zn²⁺ (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.