1. GC-MS Studies on the Conversion and Derivatization of γ-Glutamyl Peptides to Pyroglutamate (5-Oxo-Proline) Methyl Ester Pentafluoropropione Amide Derivatives
Alexander Bollenbach, Dimitrios Tsikas Molecules. 2022 Sep 15;27(18):6020. doi: 10.3390/molecules27186020.
Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, γ-Glu-Cys-Gly) is the most abundant intra-cellular dicarboxylic tripeptide with multiple physiological roles. In biological samples, glutathione exists in its reduced form GSH and in two stable oxidized forms, i.e., in its symmetric disulfide form GSSG and as S-glutathionyl residue in proteins. S-Glutathionylation is a post-translational modification, which is involved in several pathophysiological processes, including oxidative stress. The GSH-to-GSSG molar ratio is widely used as a measure of oxidative stress. γ-Glutamyl is the most characteristic structural moiety of GSH. We performed gas chromatography-mass spectrometry (GC-MS) studies for the development of a highly specific qualitative and quantitative method for γ-glutamyl peptides. We discovered intra-molecular conversion of GSH, GSSG, γ-Glu-Cys and of ophthalmic acid (OPH; γ-glutamyl-α-amino-n-butyryl-glycine) to pyroglutamate (pGlu; 5-oxo-proline, also known as pidolic acid) during their derivatization with 2 M HCl/CH3OH (60 min, 80 °C). For GC-MS analysis, the methyl esters (Me) were further derivatized with pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v; 30 min, 65 °C) to their PFP derivatives. At longer reaction times, pGlu is hydrolyzed to Glu. Internal standards were prepared by derivatizing GSH, GSSG, γ-Glu-Cys and OPH in 2 M HCl/CD3OD. Quantification of the Me-PFP derivative of pGlu was performed in the electron-capture negative-ion chemical ionization (ECNICI) mode by selected-ion monitoring (SIM) of the mass-to-charge (m/z) ions 269 for unlabeled pGlu (d0Me-PFP-pGlu) and m/z 272 for the in situ prepared deuterium-labeled pGlu (d3Me-PFP-pGlu). Although not inherent to the analysis of small peptides, the present GC-MS method is useful to study several biochemical aspects of GSH. Using pentafluorobenzyl bromide (PFB-Br) as the derivatization reagent, we found that synthetic pGlu is converted in aqueous acetone (60 min, 50 °C) into its pentafluorobenzyl (PFB) ester (PFB-pGlu). This derivatization procedure is useful for the GC-MS analysis of free pGlu in the ECNICI mode. Quantitative analysis of PFB-pGlu by GC-MS requires the use of stable-isotope labeled analogs of pGlu as an internal standard.
2. Structure-CaSR-Activity Relation of Kokumi γ-Glutamyl Peptides
Yusuke Amino, Masakazu Nakazawa, Megumi Kaneko, Takashi Miyaki, Naohiro Miyamura, Yutaka Maruyama, Yuzuru Eto Chem Pharm Bull (Tokyo). 2016;64(8):1181-9. doi: 10.1248/cpb.c16-00293.
Modulation of the calcium sensing receptor (CaSR) is one of the physiological activities of γ-glutamyl peptides such as glutathione (γ-glutamylcysteinylglycine). γ-Glutamyl peptides also possess a flavoring effect, i.e., sensory activity of kokumi substances, which modifies the five basic tastes when added to food. These activities have been shown to be positively correlated, suggesting that kokumi γ-glutamyl peptides are perceived through CaSRs in humans. Our research is based on the hypothesis that the discovery of highly active CaSR agonist peptides will lead to the creation of practical kokumi peptides. Through continuous study of the structure-CaSR-activity relation of a large number of γ-glutamyl peptides, we have determined that the structural requirements for intense CaSR activity of γ-glutamyl peptides are as follows: existence of an N-terminal γ-L-glutamyl residue; existence of a moderately sized, aliphatic, neutral substituent at the second residue in an L-configuration; and existence of a C-terminal carboxylic acid, preferably with the existence of glycine as the third constituent. By the sensory analysis of γ-glutamyl peptides selected by screening using the CaSR activity assay, γ-glutamylvalylglycine was found to be a potent kokumi peptide. Furthermore, norvaline-containing γ-glutamyl peptides, i.e., γ-glutamylnorvalylglycine and γ-glutamylnorvaline, possessed excellent sensory activity of kokumi substances. A novel, practical industrial synthesis of regiospecific γ-glutamyl peptides is also required for their commercialization, which was achieved through the ring opening reaction of N-α-carbobenzoxy-L-glutamic anhydride and amino acids or peptides in the presence of N-hydroxysuccinimide.
3. Gamma-L-glutamyl-L-glutamate is an endogenous dipeptide in the rat olfactory bulb which activates N-methyl-D-aspartate receptors
X Li, O Orwar, J Persson, M Sandberg, I Jacobson Neurosci Lett. 1993 May 28;155(1):42-6. doi: 10.1016/0304-3940(93)90669-c.
The concentration of gamma-L-glutamyl-L-glutamate (gamma-glu-glu), a potent displacer of excitatory amino acid receptor binding, and other structurally related dipeptides were determined in extracts of the rat olfactory bulb by a novel liquid chromatographic method. Whole-cell patch clamp recordings of currents from freshly isolated neurons showed that gamma-glu-glu produced inward currents at negative holding potentials, provided microM concentrations of glycine were added and no Mg(2+)-ions were present, indicating activation of N-methyl-D-aspartate receptors. Consistently, geometry optimization of gamma-glu-glu using molecular mechanics calculations, suggested a bent conformer with structural features supporting this view.
