Reevaluation of the reaction of formaldehyde at low concentration with amino acids

Many studies have been reported on the reaction of formaldehyde (FA) with amino acids or proteins, and FA is assumed to react with the alpha-amino group as well as some of the side chain groups. In most of these investigations a large excess of FA relative to amino acids or proteins was employed. In the present study, however, we carried out the reaction with a smaller excess of FA in order to clarify the reactivity, firstly with the alpha-amino groups, and secondly with specific side chain groups. No evidence for so-called Schiff base (-N = CH2) formation was obtained in the reaction with the alpha-amino groups, but the formation of an acid-labile N-hydroxymethyl compound as a major product was suggested by NMR and IR at greater than pH 9.2. There was no indication, however, of the presence of such a product below pH 9.2, and the amount of N-hydroxymethyl product increased in parallel with the reaction pH. The higher the reaction pH (greater than or equal to 9.2), the greater the consumption of FA, up to 2 mol/mol Ala. In addition, the larger the excess of FA, the smaller the amount of free amino group remaining (at pH 9.7). In the assignment of IR spectra, discrete absorption bands of the alpha-carboxyl group of Ala were observed, which reflected ionization states of the alpha-amino group, and these were utilized for analysis of the reaction mechanism. Furthermore, among amino acids with side chain groups, His, Trp, and Arg showed high reactivity and Asn showed moderate reactivity. The products were relatively stable and were purified and subjected to instrumental analyses. Sixteen other amino acids including Tyr and Lys did not yield stable products. The products from Arg were unique because of the non-involvement of the amino group, and were reversibly converted to the original Arg upon acid hydrolysis. The products from His, Trp and Asn all involved amino or amide nitrogen forming cyclic ring structures with methylene derived from FA. The chemical structures of these products were determined on the basis of elemental analyses, MS and NMR.