Modifications of solid phase peptide synthesis to obtain homogeneous oligoprolines in high yield.
When oligoprolines containing a [3H]proline in the second residue from the carboxyl terminus are synthesized by the standard solid phase method using stepwise addition of prolines there appears, in addition to the oligoprolines, a steady increase of ninhydrin-negative, but radioactive, impurities. These impurities were found to be N-trifluoroacetyl oligoprolines, and a substantial amount of diketopiperazine was present at the diproline stage. The trifluoroacetyl blockage of the N-termini was almost complete by the time 14 residues were added. To eliminate these side reactions, a block of Boc[3H]Pro2 was coupled to prolyl-resin in order to avoid the diprolyl-resin stage, and 25% CF3CO2H in CHCl3 was replaced by 4N HCl in dioxane as the deprotecting reagent. These changes eliminated the formation of diketopiperazine and trifluoroacetyl impurities, but still a steady increase of ninhydrin-negative, but radioactive, impurity was seen, reaching 22% after three couplings. These side reactions were traced to the neutralization step, and in order to avoid them it became necessary to eliminate the neutralization step, and replace it by incremental additions of Et3N during the coupling step. As a result of these modifications, we now obtain homogeneous length oligoprolines upon cleavage from the resin, with no further purification, when analyzed by reversed-phase liquid chromatography.
D002845:Chromatography; D004588:Electrophoresis, Cellulose Acetate; D007202:Indicators and Reagents; D009842:Oligopeptides / Q000032:analysis / Q000138:chemical synthesis*; D010446:Peptide Fragments; D010879:Piperazines; D011392:Proline / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D013057:Spectrum Analysis
D007202:Indicators and Reagents; D009842:Oligopeptides; D010446:Peptide Fragments; D010879:Piperazines; D011392:Proline