Generation of enantiomeric amino acids during acid hydrolysis of peptides detected by the liquid chromatography/tandem mass spectroscopy.

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Chemistry & Biodiversity (Impact Factor: 1.81). 06/2010; 7(6):1644-50. DOI: 10.1002/cbdv.200900309
Source: PubMed

ABSTRACT The number of reports indicating the occurrence of D-amino acids in various proteins and natural peptides is increasing. For a usual detection of peptidyl D-amino acids, proteins or peptides are subjected to acid hydrolysis, and the products obtained are analyzed after cancellation of the effect of amino acid racemization during the hydrolysis. However, this method does not seem reliable enough to determine the absence or presence of a small amount of innate D-amino acids. We introduce a modification of an alternative way to distinguish true innate D-amino acids from those artificially generated during hydrolysis incubation. When model peptides (L-Ala)(3), D-Ala-(L-Ala)(2) are hydrolyzed in deuterated hydrochloric acid (DCl), only newly generated D-amino acids are deuterated at the alpha-H-atom. Both innate D-amino acids and artificially generated ones are identified by the combination of high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry equipped with a chiral column. When a peptide containing D-Phe residues was analyzed by this method, the hydrolysis-induced conversion to L-Phe was similarly identified.

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    ABSTRACT: Amino acids are essential for life, and have many functions in metabolism. One particularly important function is to serve as the building blocks of peptides and proteins, giving rise complex three dimensional structures through disulfide bonds or crosslinked amino acids. Peptides are frequently cyclic and contain proteinogenic as well as nonproteinogenic amino acids in many instances. Since most of the proteinogenic α-amino acids contain at least one stereogenic center (with the exception of glycine), the stereoisomers of all these amino acids and the peptides in which they are to be found may possess differences in biological activity in living systems. The impetus for advances in chiral separation has been highest in the past 25 years and this still continues to be an area of high focus. The important analytical task of the separation of isomers is achieved mainly by chromatographic and electrophoretic methods. This paper reviews indirect separation approaches, i.e. derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers of unusual amino acids and related compounds, with emphasis on the literature published from 1980s. The main aspects of the chiral derivatization of amino acids are discussed, i.e. derivatization on the amino group, transforming the molecules into covalently bonded diastereomeric derivatives through the use of homochiral derivatizing agents. The diastereomers formed (amides, urethanes, urea and thiourea derivatives, etc.) can be separated on achiral stationary phases. The applications are considered, and in some cases different derivatizing agents for the resolution of complex mixtures of proteinogenic d,l-amino acids, non-proteinogenic amino acids and peptides/amino acids from peptide syntheses or microorganisms are compared.
    Journal of Chromatography A 04/2013; · 4.61 Impact Factor