Absolute Configuration, Optical Activity and Raman Microscopy of L and D- Glutamic Acid Leonardo Y. Fox-Uribe 1 , Yedith Soberanes 1 , Valeria Guzman-Luna 2 , Gloria Saab-Rincon 2 , Javier Hernández-Paredes 3 and Rogerio R. Sotelo-Mundo 1 1. Laboratorio de Estructura Biomolecular, Centro de Investigacion en Alimentacion, y Desarrollo, A.C., Carretera a Ejido La Victoria km 0.6 s/n, Hermosillo, Sonora, C.P. 83304, Mexico. 2. Departamento de Ingenieria Celular y Biocatalisis. Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico. 3. Posgrado en Nanotecnología, Departamento de Fisica, Universidad de Sonora (UNISON), Edificio 3R, Blvd. Luis Encinas J. y Rosales s/n Col. Centro, Hermosillo, Sonora, C.P. 83000, Mexico. Amino acids are the building blocks of proteins. Among this diverse group of molecules, glutamic acid (Glu - C5H9NO4) plays a key role as a component of enzymes, either with a structural role or as part of the catalysis in the active site to perform biological function [1]. Charged transfer RNAs are loaded with L stereoisomers, so proteins synthesized by the ribosome are exclusively comprised of L-amino acid residues. However, D-Glu is used in formation and development of bacterial peptidoglycan [2]. The determination of the absolute configuration of chiral compounds is critical in many fields of science and technology. In this work, we carried out single-crystal X-ray diffraction (SCXRD) experiments with Cu Kα radiation to determine the absolute configuration of glutamic acid in both enantiomeric forms ( L/D). L and D-Glu isomers were also analyzed to determine their optical activity and structural information. The amino acids were purchased from Sigma-Aldrich and used without further recrystallization. The crystallization experiments were: 1) 50 mmol of L-glutamic acid (≥ 99% pure); m.p.: 206-208 °C. 1 H NMR (D2O, DSS, 400 MHz): δ = 2.14 (m, J = 7.2 Hz, 2H, Ha), δ = 2.55 (m, J = 8,8 Hz, 2H, Hb), δ = 3.80 (t, J = 6.8 Hz, 1H, Hc) was dissolved in 1 mL of ultrapure water (Milli-Q water, Merck Millipore); 2) 50 mmol of D-glutamic acid (≥ 99% pure); m.p.: 210-211°C. 1 H RMN NMR (D2O, DSS, 400 MHz): δ = 2.14 (m, J =7.2 Hz, 2H, Ha), δ = 2.55 (m, J = 8 Hz, 2H, Hb), δ = 3.80 (t, J =6,4 Hz, 1H, Hc) was dissolved in 1 mL of ultrapure water. SCXRD experiments of L-Glu and D-Glu were collected on a Bruker D8-QUEST diffractometer with a Cu Kα microfocus source (λ=1.5418 Å) equipped with a CMOS detector (300 (2) K). Optical activity of L-Glu and D-Glu was done on a Jasco 710 spectropolarimetrer (Jasco Inc., Easton MD, USA) on a 0.2 cm 3 quartz with 1 mm path-length containing 20 mM Tris-HCl pH 8.0, 20 mM NaF and 6 mM of each L-Glu/D-Glu. A separate experiment was done with a mixture of 3 mM DL-Glu each. Data was reported as positive or negative ellipticity vs. wavelength. Solid-state Raman spectra were taken in a Horiba Jobin-Yvon LabRam HR high-resolution Raman microscope, equipped with a charge-coupled device detector and an excitation laser source with a wavelength of 632.8 nm. After refinement of the Flack parameter [3], the absolute configuration was determined. L-Glu had an absolute configuration “sinister - S” with a Flack parameter value 0.03 (3), whereas D-Glu had an absolute configuration as “rectum - R” and Flack parameter of 0.09 (4) (Figure 1). The optical activity of each enantiomer was evaluated using circular dichroism. An opposite optical activity was found for the two enantiomers (Figure 2a). As expected, an equimolar mixture of L-Glu and D-Glu gave a near zero ellipticity for the 200-260 nm range. Therefore, L-Glu is classified as levorotatory (-) and D-Glu as dextrorotatory (+). Raman spectra showed the main structural characteristics of the compounds, from 1382 doi:10.1017/S1431927617007577 Microsc. Microanal. 23 (Suppl 1), 2017 © Microscopy Society of America 2017 https://doi.org/10.1017/S1431927617007577 Downloaded from https://www.cambridge.org/core. IP address: 54.196.56.151, on 29 Oct 2021 at 11:21:48, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.