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(15) J. D. Payzant, A . J. Cunningham, and P. Kebarle, Can. J . Chem., 3242 (19731. (16) M S. '8. Munson, J . Am. Chem. SOC.,87, 2332 (1965). (17) P. Kebarle, R. Yamdagni, K. Hiraoka, and T. E . McMahom, Int. J . Mass Spectrom. Ion Phys., 19, 71 (1977). (18) S. H. Kim and F. W.Karasek, "The Study on the Mobilities of Positive Reactant Ions using N, Carrier Gas", in preparation (1977). (19) I. Dzidic, D. I. Carrol, R. N. Stillwell, and E. C. Horning, Anal. Chem., 48, 1763 (1976)
(5) M. S. Wilson, I. Dzidic, and J. McClosky, Siochim. Siophys. Acta, 240, 623 (1971). (6) J. I. Brauman. J. M. Riveros, and L. K. Blair, J . Am. Chem. SOC.,93, 3914 (1971). 94, 8333 (1972). (7) I. Dzidic, J . Am. Chem. SOC., (8) R. Yamadagni and P. Kebarle, J . Am. Chem. SOC., 95, 3504 (1975). (9) I. Dzidic and J. A. McClosky, Org. Mass Spectrom., 6, 939 (1972). (10) F. W.Karasek and D. M. Kane, Anal. Chem., 45, 576 (1973). (11) E. C. Horning, M. G. Horning, D. I. Carrol, I . Dzidic, and R. N. Stillwell, Anal. Chem., 45, 936 (1973). (12) F. W.Karasek and D. W. Denney, Anal. Chem., 46, 633 (1974). (13) A. Good, D. A. Durden, and P. Kebarle, J Chem. Phys., 5 2 , 212 (1970). (14) P. Kebarle, Adv. Chem. Ser. 72, 24-47 (1968).
RECEIVED for review June 21, 1977. Accepted October 25, 1977.
Test for Racemization in Model Peptide Synthesis by Direct Chromatographic Separation of Diastereomers of the Tetrapeptide Leucylalanylglycylvaline S. B. H. Kent,* A. R. Mitchell,' G. Barany, and R. B. Merrifield The Rockefeller University, New York, New York
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model system that provides a stringent test for racemization in peptide synthesis and have used this to investigate the occurrence of racemization in stepwise solid phase peptide synthesis. The peptide Leu-Ala-Gly-Val has been used as a model for synthetic methods (11-18). Standard analytical conditions on the automatic amino acid analyzer (19) exist for the separation of most of the known peptide by-products (20). As a result of extensive work with the chromatographic separation of diastereomeric dipeptides according to Manning and Moore (21),it seemed likely to us that it would be possible to separate the diastereomers of the tetrapeptide Leu-Ala-Gly-Val. A preliminary investigation with L-Leu-D-Ala-Gly-L-Val showed t h a t it was possible to achieve a large separation of diastereomeric tetrapeptides under standard chromatographic conditions. We therefore prepared diastereomeric tetrapeptide standards and studied their separation as a function of p H to determine the optimal separation. The color yields of the individual peptides were determined and the sensitivity and precision of the determination of D-amino acid-containing peptides were evaluated. Use of the method was illustrated by analyses of stepwise solid phase syntheses of the tetrapeptide.
The peptide Leu-Ala-Gly-Val has been developed as a test for racemization in model peptide synthesis. The single D-amino acid diastereomers, L-Leu-D-Ala-Gly-L-Val and D-Leu-L-AlaGly-L-Val, were separated from one another and from the all L-amino acid tetrapeptide on the standard amino acid analyzer 0.9 X 58 cm column of sulfonated polystyrene resin with 0.2 N sodium citrate buffers. Chromatography at pH 3.49 was chosen as the standard condition for separating the diastereomers. The color yields of the diastereomers were similar to that of valine. The determination of the D-amino acidcontaining diastereomers was accurate above 0.1 % for a standard load of 4 pmol of the tetrapeptide. The limit of detection was less than 0.01% for a 12-pmol load. The analysis was applied to the crude products of stepwise solid phase syntheses; no D-amino acid-containing diastereomers were detected (
