Separation and identification of the DL-forms of short-chain peptides using a new chiral resolution labeling reagent.

Abstract

There are several reports of D-amino acids being the causative molecules of serious diseases, resulting in the formation of, for example, prion protein and amyloid β. D-Amino acids in peptides and proteins are typically identified by sequencing each residue by Edman degradation or by hydrolysis with hydrochloric acid for amino acid analysis. However, these approaches can result in racemization of the L-form to the D-form by hydrolysis and long pre-treatment for hydrolysis. To address these problems, we aimed to identify the DL-forms of amino acids in peptides without hydrolysis. Here, we showed that the DL-forms in peptides which are difficult to separate on a chiral column can be precisely separated by labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamine-amide (D-FDLDA). Additionally, the peptides could be quantitatively analyzed using the same labeling method as for amino acids. Furthermore, the detection sensitivity of a sample labeled with D-FDLDA was higher than that of the conventional reagents Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (L-FDAA) and Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA) used in Marfey's method. The proposed method for identifying DL-forms of amino acids in peptides is a powerful tool for use in organic chemistry, biochemistry, and medical science.