AFFINITY PURIFICATION METHOD USING A REVERSIBLE BIOTINYLATING REAGENT FOR PEPTIDES SYNTHESIZED BY THE SOLID-PHASE TECHNIQUE

The specific interaction between biotin and avidin was explored as the basis of an affinity purification procedure for peptides synthesized by the solid-phase technique. In this affinity purification procedure, a reversible biotinylating reagent, 2-[(N-biotinyl)-aminoethylsulphonyllethyl p-nitrophenyl carbonate (BASEC), was synthesized. Using this reagent, the procedure involves the following sequence of four reactions: (i) attachment of the biotin to the N-terminus of the peptide-resin through a base-labile sulphonylethoxycarbonyl linkage at the final step of solid-phase peptide synthesis; (ii) acid treatment to remove side-chain protecting groups and cleave the biotin-modified peptide from the resin; (iii) affinity purification of the biotinyl-peptide on an avidin-agarose column; and (iv) base (5% ammonia solution) treatment to remove the biotin moiety from the peptide. To facilitate this purification procedure, unreacted amino groups were acetylated in each step during solid-phase synthesis. The usefulness of this method was demonstrated by the purification of several peptides synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase technique.