Synthesis and characterization of D-Alanyl-D-Alanine-Agarose

A new affinity adsorbent, using D-alanyl-D-alanine as ligand, has been prepared. The dipeptide immobilized on Activated CH-Sepharose 4B (D-Ala-D-Ala-AGA) bioselectively binds the glycopeptide antibiotics teicoplanin, vancomycin, ristocetin A (vancomycinlike group of antibiotics) while it does not bind other antibiotics equally active on cell wall biosynthesis but with different target sites, such as penicillin G, cephalosporin C, gardimycin, and bacitracin. Teicoplanin, vancomycin, and ristocetin A have similar binding characteristics for the immobilized dipeptide, as indicated by equilibrium binding experiments. The affinity constants of the three antibiotics for D-Ala-D-Ala-AGA is of the same order of magnitude (10⁵ L mol^-1) and the number of effective binding sites is similar for each antibiotic (6–7 μEq/mL of gel). The adsorption is biospecific as no binding has been observed to immobilized L-alanyl-L -alanine. D-Ala-D-Ala-AGA has been successfully used to purify teicoplanin from mixtures of different complexity and for concomitant extraction and purification from fermentation liquors by both batch adsorption and column chromatography. The antibiotic can be recovered from the resin in high yields by elution at pH 11.