Vibrational Circular Dichroism Spectra Using the FVS-6000

Download PDF January 22, 2020

Introduction

It is generally understood that chiral compounds have different bioactivities depending upon the absolute configuration of each compound. Some familiar examples include glutamic acid and thalidomide. L-glutamic acid demonstrates the “Umami” taste*1, while D-glutamic acid has a bitter taste, similarly, the R- form of thalidomide is a sedative, but the S- form has teratogenic activity. Thus, the separation and study of chiral compounds is critical for many reasons.

The functionalities of chiral compounds have been studied for the development of advanced molecules for many applications. The study of chiral compounds has spread to several fields such as natural products, pharmaceuticals and other functional molecules, and it can be pointed out that among those studies, the structural analysis of chiral compounds is a very important topic. X-ray Diffraction (XRD), Nuclear Magnetic Resonance (NMR) and Electronic Circular Dichroism (ECD) using UV/Vis light are employed as primary methods for the structural analysis of chiral compounds. In this paper, the measurement of chiral compounds by Vibrational Circular Dichroism (VCD) using infrared light will be outlined.

VCD is a method to measure the difference of absorbance intensity between left-hand and right-hand circularly polarized light as shown in Figure 1. It is an advantage of VCD that this method can be applied to almost all organic compounds in the same way as infrared (IR) spectroscopy. In addition, by comparing the measurement results with calculated results by ab-initio molecular orbital calculations, the absolute configuration of the sample can be determined. However, since the peak intensity of VCD spectra are 1,000 to 10,000 times weaker than that of standard IR spectra, therefore spectroscopic instruments with high sensitivity and stability with very small baseline fluctuations are required. The FVS-6000 VCD system includes the highest sensitivity narrow band detectors, coupled with optical filter technology and a thermostatted Photo Elastic Modulator (PEM) to accurately measure the weak VCD peaks. The measurement results of several typical chiral compounds and hemoglobin as a model protein using the FVS-6000 are reported here.

*1 Umami is considered to be the fifth taste sensation in addition to sweet, acid, salty and bitter tastes.

Figure 1: Principles of VCD spectroscopy
Figure 2: External view of the FVS-6000

Keywords

vibrational circular dichroism, VCD, alpha pinene, absolute stereochemistry, IR spectroscopy, protein secondary structure, alpha helix, beta sheet, ab-initio structural calculation

Results

Measurement results

Figures 3 through 6 illustrate the measurement results for alpha-pinene; 1,1-Bi-2-naphthol; proline and hemoglobin, respectively. Both IR and VCD spectra can be obtained by the FVS-6000. The analysis of molecular structure and absolute configuration of chiral compounds can be determined from both the IR and the VCD spectra.

Figure 3 demonstrates the IR and VCD spectra of alpha-pinene which is a typical standard molecule used to validate a VCD instrument. IR spectra of the R- and S- forms of alpha-pinene are completely overlapped, while their VCD spectra are symmetric, clearly identifying each alpha-pinene enantiomer. Since the measured VCD spectra correlate accurately with typical alpha-pinene spectra, it is confirmed that high quality VCD spectra can be obtained using the FVS-6000.

Figure 4 outlines measurement results for 1,1-Bi-2-naphthol which is used as a ligand for transition-metal catalyzed asymmetric synthesis and is a precursor for chiral ligands such as BINAP. The small peaks due to the anisotropy factor g value (VCD peak/IR peak) around 1600 and 1500 cm-1 attributed to the benzene ring are clearly shown. These results demonstrate that the FVS-6000 is a very effective system for evaluation of chiral compounds which have similar structures.

Figure 5 illustrates the measurement results of the amino acid proline. Good, symmetrical VCD spectra were obtained for both the D- and L- forms. Since amino acids demonstrate different bioactivities between the D- and L- forms, studies regarding structure and bioactivity are increasingly popular. Liquid samples can be easily measured by VCD so the structural analysis of amino acids can be performed in typical physiological conditions.

Figure 6 contains the measurement results for hemoglobin. Hemoglobin is a spherical protein rich in alpha-helix structures, its VCD spectrum is very different from that of concanavalin A, which is rich in beta-sheet structures.

The correlation of VCD spectral results with information obtained from ECD and IR can provide much more accurate secondary structure analysis of proteins in solvents. We also believe that VCD can also be a powerful tool for the analysis of other chiral macro molecules such as DNA and chiral polymers.

In this paper, the standard performance and measurement results of typical chiral compounds using the FVS-6000 were reported and we believe the FVS-6000 can be an essential and indispensable tool for analysis of chiral compounds.

Measurement condition

Model name:FVS-6000
Resolution:4 cm-1
Detector:MCT-V
Accumulation:1000 (alpha-pinene and 1,1?-Bi-2-naphthol), 2500 (proline), 2000 (hemoglobin)
Figure 3: Spectra of alpha-pinene
(neat, 50 um pathlength BaF2 liquid cell)
Figure 4: Spectra of 1,1-Bi-2-naphthol
(solvent: CHCl3, concentration: 0.162 M, 50 um pathlength BaF2 liquid cell)
Figure 5: Spectra of proline
(solvent: D2O, concentration: 0.9 M, 25 um pathlength CaF2 liquid cell)
Figure 6: Spectra of hemoglobin
(solvent: D2O, concentration: 50 mg/mL,
25 um pathlength CaF2 liquid cell)

About the Author

Dr. Carlos Morillo received his Post Doc at Advanced Industrial Science & Technology in Fukuoka and was a Research Scientist at Kyushu University in Japan where he lived for several years. Carlos received his Doctor of Engineering from Kyushu University and his Masters and BS from Simon Bolivar University in Caracas Venezuela. He is an Applications Scientist at JASCO.