Optimized aminolysis conditions for cleavage of N-protected hydrophobic peptides from solid-phase resins

Solid-phase synthesis and aminolysis cleavage conditions were optimized to obtain N- and C-terminally protected hydrophobic peptides with both high qu ality and yield. Uncharged 'WALP' peptides, consisting of a central (Leu-Al a), repeating unit (where n=5, 10.5 or 11.5) flanked on both sides by Trp ' anchors', and gramicidin A (gA) were synthesized using 9-fluorenylmethoxyca rbonyl chemistry from either Wang or Merrifield resins. For WALP peptides, the N-terminal amino acid was capped by coupling N-acetyl- or N-formyl-Ala or -Gly to the peptide/resin or by formylation of the completed peptide/res in with para-nitrophenylformate (p-NPF). N-Terminal acetyl- or formyl-Ala r acemized when coupled as an HOBt-ester to the resin-bound peptide, but not when the peptide was formylated with p-NPF. Racemization was avoided at the last step by completing the peptide with acety]- or formyl-Cly. For both W ALP peptides and gA, cleavage conditions using ethanolamine or ethylenediam ine were optimized as functions of solvent, time, temperature and resin typ e. For WALP peptides, maximum yields of highly pure peptide were obtained b y cleavage with 20% ethanolamine or ethylenediamine in 80% dichloromethane for 48 h at 24 degreesC, N-Acetyl-protected WALP peptides consistently gave higher yields than those protected with N-formyl. For gA, cleavage with 20 % ethanolamine or ethylenediamine in 80% dimethylformamide for 48 h at 24 C gave excellent results. For both WALP peptides and gA, decreasing the clea vage time to 4h and increasing the temperature to 40-55 degreesC resulted i n significantly lower yields. The inclusion of hexafluoroisopropanol in the cleavage solvent mixture did not improve yields for either gA or WALP pept ides.