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the filtrate after evaporation upon trituration with Although several model peptides have been coupled ether (17.7 mg, 90%, mp 163-170" dec). successfully to peptide polymers, our preliminary The product was characterized as follows: the experience in coupling sequences of the staphylococcal NH,-terminal residue was determined by the dansyl nuclease indicates that refinement of conditions may be chloride method12 to be glutamic acid, without a trace required when using longer peptides or when coupling of the previous leucine amino terminus; thin layer certain pairs of carboxyl- and amino-terminal residues. chromatography gave a single spot (Rf 0.1),I3 and highFor example, coupling the hydrophobic octapeptide or voltage electrophoresis gave single spots in pyridinium tetrapeptide sequences (both containing COOH-teracetate buffer pH 3.6 ( R f 0.78) and at pH 6.5 (Rf0.61), minal valine) corresponding to residues 32-39 or 36-39 with L-lysine as reference (Rf 1.0); digestion with of staphylococcal nuclease' to the nonapeptide polymer aminopeptidase M I 4 (AP-M) was complete as judged containing residues 40-48, with NHz-terminal aspartic by paper and thin layer chromatography and gave acid, gave only 20-30Z coupling with either NEPIS ratios upon amino acid analysis of Glu 0.96, Lys 1.66, or DCC. The aspartic acid residue may offer special Ser 1.00, Leu 1.0, Pro 0.86, confirmed by acid hydroldifficulty. Thus, the dipeptide Z-L-Phe-L-Tyr-OH was ysis (6 N HC1, sealed evacuated tube, 110", 20 hr) coupled readily with DCC-HOSu to Leu-Pro polymer and consistent with 100% coupling efficiency without (aminopeptidase digestion gave Phe 1.03, Tyr 0.98, racemization a t the carboxyl-terminal serine residue. Leu 1.0, Pro 1.03; dansyl showed all Phe; thin layer Coupling conditions with NEPIS, in which reaction chromatography and electrophoresis systems all gave a time was held to 2 hr with fourfold excess of soluble single spot), but under identical conditions was coupled tetrapeptide and in which only twofold excess was only to the extent of 40% to the /3-benzyl-L-aspartylused for 6- to 48-hr reaction time, gave incomplete terminal nonapeptide polymer. reactions. Nevertheless, the modification of the solid-phase The same peptide coupling reaction was carried out synthetic approach described here offers the advantages with DCC plus hydroxysuccinimide (HOSu) as the of fewer steps in a long sequence and of convenience in coupling agent. Complete coupling without detectsubstituting residues in structural analog studies. able racemization was obtained in 2 hr at room temFurthermore, coupling peptide fragments rather than perature. Dansylation showed NH2-terminal glutamic individual amino acid monomers should give a major acid only and complete digestion with AP-M yielded the advantage in the purification of the polypeptide prodfollowing amino acid ratios: Glu 1.06, Lys 1.99, Ser uct. The peptides with incomplete sequences that 1.01, Leu 1.0, Pro 0.98. Reaction for 4 hr at O", or accumulate from less than quantitative coupling at reaction at room temperature using DCC without any step might be removed if they differ from the HOSu, gave a product in which dansyl analysis demoncompleted peptide by the several residues constituting strated a small proportion (
