PURIFICATION OF SYNTHETIC PEPTIDES WITH THE AID OF REVERSIBLE CHROMATOGRAPHIC PROBES

The application of reversible chromatographic probes for the purification of synthetic peptides by reversed-phase (RP) and affinity chromatography is described. In previous work the concept of a one-step purification procedure based on the acid-stable fluorenylmethoxycarbonyl (Fmoc) group was considered. Similar probes were used to purify peptides from 17 to 104 residues in length, using ion-exchange and RP media. The RP probes described here represent simplified versions of those reported previously. Further, the application of a new probe containing biotin, for affinity chromatography, is demonstrated. Stepwise solid-phase peptide synthesis was performed using both Boc and Fmoc chemistries and N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping reagent. The chromatographic probes were incorporated on to the ''target'' peptide as active succinimidyl carbonate derivatives. After cleavage using either hydrogen fluoride or trifluoroacetic acid, purification was performed on either RP media or immobilized avidin. Treatment with organic base yielded the free purified product. The efficiency of the new probe molecules and the capping procedure was demonstrated by the purification of two polypeptides, 46 and 101 residues in length.