Determination of the absolute configuration of resolved enantiomers
Bo Wang is a research scientist, BioTools, and Laurence A. Nafie is chief technology officer, BioTools and distinguished professor
emeritus, Syracuse University. Elena Eksteen is senior manager, business development and planning, Chiral Technologies
During the small-molecule drug development process, chromatographic resolution is an effective way for separating racemic
compounds into single enantiomers for use in bioassays and toxicology studies. Chromatography also is a tool that can be used
to establish pharmacokinetic and toxicological properties of candidate drug compounds (1). Vibrational circular dichroism
(VCD) technology is an important tool for determining the absolute configuration (AC) of enantiomers and is used by pharmaceutical
companies for molecular structure characterization of chiral molecules. Knowledge of the AC is vital in drug discovery, development,
and the regulatory requirements for investigational new drug submissions (2).
VCD is the small difference in the IR absorbance of a chiral molecule for left versus right circularly polarized light. The
determination of AC using VCD is a method for assigning the absolute stereochemistry of chiral molecules. It supplements,
or in some cases, replaces the previous gold-standard method of anomalous X-ray diffraction that requires a pure single crystal
of one enantiomer of the chiral molecule. VCD requires no crystallization and no chemical modification or derivatization of
the chiral molecule. Assignments of AC are made by comparing the solution-state VCD and IR absorbance spectra to the corresponding
quantum chemical calculations of the same spectra. When the VCD spectra agree, the AC chosen for the calculations is the same
as that of the sample. If the signs of the calculated VCD are opposite to that of the measured VCD, the sample has the opposite
AC. In addition, valuable information about the conformation or conformations of the chiral molecule in solution also may
be obtained. Instrumentation for the measurement of VCD (Chiral IR-2X, BioTools) and software (Gaussian 09) for the calculation
of VCD are commercially available as are services for performing AC determinations using VCD.
Figure 1 (Chiral): Separation of a racemic mixture. (FIGURES 1–3 (CHIRAL) ARE COURTESY OF THE AUTHORS)
A client requested enantiomer separation of a racemic mixture to obtain pure enantiomers needed for toxicology studies. In
preparation for clinical trials, the client also requested AC measurements for an IND submission. The racemic compound was
screened against several chiral stationary phases to identify a method appropriate for the separation of 50 to 100 g of the
mixture. Figure 1 (Chiral) shows the separation of the racemic mixture using a Chiralpak IA column (Chiral Technologies).
Figure 2 (Chiral): IR absorbance (bottom), vibrational circular dichroism VCD (middle) and VCD noise spectra (top) of the
two enantiomers before baseline subtraction. The two enantiomers show identical IR spectra, as expected, but opposite-signed
VCD spectra for every IR band.
Using the conditions of the separation method for scale-up, 80 g were separated, and pure enantiomers were isolated for toxicology
studies and for VCD-based AC measurements of the pure enantiomers. VCD spectra of the resolved enantiomers were measured using
a Chiral IR-2x instrument (BioTool) The IR and VCD spectra are shown in Figure 2 (Chiral). Figure 3 (Chiral) demonstrates
the comparison of experimental to theoretical spectra.
Figure 3 (Chiral): Comparison between observed vibrational circular dichroism (VCD) for one enantiomer and the corresponding
calculated VCD for the S,S-configuration of this enantiomer. The agreement in signs between the two spectra yields an absolute
configuration assignment of S,S for the enantiomer.
1. K. Valko, Eur. Pharm. Rev. Issue 5, 40 (2010).
2. H. Yanan et al., Appl. Spectrosc. 65 (7), 699–723 (2011).