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The novel antibacterial peptide mimetic alaphosphin (L-alanyl-L-1-aminoethylphosphonic acid) selectively inhibited peptidoglycan biosynthesis in both gram-negative and gram-positive bacteria. It induced accumulation of uridine diphosphate-N-acetyl-muramyl-tripeptide in gram-positive organisms and significantly reduced the intracellular pool levels of D-alarnie. Alaphosphin was actively transported into bacterial cells by stereospecific peptide permeases and was subsequently hydrolyzed by intracellular aminopeptidases to yield L-1-aminoethylphosphonic acid. This alanine mimetic rapidly accumulated inside susceptible cells to yield a concentration which was 100-to 1,000-fold in excess of that of the precursor peptide in the surrounding medium. In the case of susceptible gram-negative organisms, it was shown that 1-aminoethylphosphonic acid was incorporated into a metabolite which was tentatively identified as uridine diphosphate-N-acetylmuramyl-aminoethylphosphonate. The Alaphosphin is representative of a novel series of antibacterial phosphonopeptides which was designed to mimnic the terminal dipeptide moiety (D-Ala-D-Ala) of bacterial cell wall peptidoglycan (2). Other papers (3, 5) have described the antibacterial properties of selected phosphonopeptides and have given some indication of structure-activity relationships in this series. Several points of evidence have suggested that transport into the bacterial cell is an important factor for activity. In this paper we describe investigations which confirm this and elucidate later stages in the mechanism of action of alaphosphin on typical bacteria. MATERIALS AND METHODSChemicals. Alaphosphin [Ala-Ala(P)] and L-and D-1-aminoethylphosphonic acids [Ala(P)] were prepared by the methods of Atherton et al. (5). '4C-labeled alaphosphin was prepared by R. J. Francis by using similar methods. All other radiochemicals were purchased from the Radiochemical Centre, Amersham, England. Chemicals were of analytical grade or equivalent and were purchased from either BDH

Peptide mimetics with C-terminal residues simulating natural amino acids have been designed as inhibitors of bacterial cell wall biosynthesis. The phosphonopeptide series consisting of various l and d residues of natural amino acids combined with 1-aminoalkyl (and aryl-alkyl-) phosphonic acid residues had the most interesting antibacterial properties when the C-terminal residue was l -1-aminoethylphosphonic acid. The in vitro antibacterial activities of representative phosphonodi- to phosphonohexapeptides were investigated. The antibacterial action of the active compounds has been explained in terms of transport into the bacterial cell and intracellular release of the alanine mimetic, which interferes with the biosynthesis of the peptidoglycan of the bacterial cell wall.

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