Complexation of acetyl-D-alanyl-D-alanine by antibiotic A35512B.

Antibiotic A3 5512B1.2) is a member of the glycopeptide family of antibiotics; complete structures have been reported for two members of this group, vancomycin3' and ristocetin A4' a) . Both vancomycin and ristocetin have been shown to form complexes with mucopeptides containing the terminal dipeptide D-alanyl-D-alaninee) ; this interaction within the bacterial cell inhibits crosslinking of the cell wall and leads to eventual lysis of the organism. Chemical attack on A35512B2) produces degradation products which are closely related to those obtained from ristocetin A7), suggesting structural similarities between the two antibiotics. Thus, A35512B might also be expected to form complexes with mucopeptide analogs. This paper reports the observation of such a complex with acetyl-D-alanyl-D-alanine (Ac-D-Ala-D-Ala). The interactions of glycopeptide antibiotics with acetyl-D-alanyl-D-alanine and related molecules have been examined by NMR methods (both vancomycin8-11) and ristocetinl2'18)) and by UV difference spectroscopy (chiefly vancomycin6'14-18)). The UV difference method was used to examine complex formation between Ac-D-Ala-D-Ala and A35512B in the work reported here. In order to compare the binding results for A35512B with those for a more thoroughly characterized glycopeptide, an identical experiment was conducted using vancomycin. A tandem-cell arrangementa.la) was used for titration of Ac-D-Ala-D-Ala into a solution of the antibiotic (A35512B or vancomycin). Initial concentrations were 2.00x10-1m Ac-D-Ala-DAla (0.404 mg/ml), 1.60 x 10-4 M A3 5512B (0.313 mg/ml), and 1.72 x 10-4 M vancomycin hydrochloride (0.256 mg/ml). The solvent for all solutions was 0.02m sodium citrate buffer, pH 5.115). The total lightpath of each cell was 2 cm, and the initial volume of antibiotic solution was 0.8 ml in each case. Examples of the UV difference spectra produced by the addition of Ac-DAla-n-Ala to A35512B or to vancomycin are shown in Fig. 1. Fig. 1. UV difference spectra induced by Ac-D-AlaD-Ala by complex formation with A35512B or with vancomycin. For A35512B, Ao'=1.33 x 10-4 M; [peptide]/ [antibiotic] =2.50. For vancomycin, Ao' =1.43 x 10-4M; [peptide]/[antibiotic] =2.33.