Separation and determination of α-amino acids by boroxazolidone formation

Abstract

Reaction of an α-amino acid (α-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2–5 in which both the α-amino and the α-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the > N:B bond is coordinate, involving the free electron pair of nitrogen, thereby satisfying the octet rule for the second electron shell of boron (Group IIIA). Consequently, the α-amino function of the boroxazolidone can be primary, secondary, or tertiary, as demonstrated by boroxazolidone formation with glycine, N-methylglycine, and N,N-dimethylglycine. On reaction with DPBA, the α-AA moiety of N-terminal γ-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of α-AA may be separated by reversed-phase (RP)-HPLC (AA-DPBA/RP-HPLC) enabling the derivatization procedure to be used as a precolumn reaction for α-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the ϵ-amino group of lysine. Furthermore, it does not complex with amide bonds of the peptide backbone or to any side chains of the common amino acids. Reaction of an α-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze α-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from α-AA and from those peptides which contain an α-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted α-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of α-amino acids that are derived from post-translational modification of protein side chains.