774 J . Org, Chem., Vol. 38, No.
4,1973
HANCOCX, PRESCOTT, VAGELOS,AND MARSHALL
Solvation of the Polymer Matrix. Source of Truncated and Deletion Sequences in Solid Phase Synthesis' WILLIAXS.HANCOCK,~ DAVIDJ. PRESCOTT, P. R O Y V A G E L O S , AND GARLAND R. l I A R S H A L L * 3 8 4 Departments of Physiology and Biophysics and of Bzological Chemistry, Washington University Medical School, St. Louis, Missouri 635110 Received November 9,l97a An automated procedure utilizing hydrogen chloride-36 for monitoring the free amine in automated solid phase synthesis was developed. Discrepancies were found between the values determined by this procedure and those from amino acid analysis in the synthesis of a peptide, residues 63-74, of acyl carrier protein. These results led to a hypothesis of dynamic solvation changes of the polymer matrix as synthesis proceeds. The effects of chain termination by acetylation were also in agreement with the hypothesis. Dynamic solvation changes of the polymer matrix leads to the sequence-dependent problems of solid phase synthesis, both truncated and deletion sequences. It may also be responsible for difficultiesencountered with monitoring procedures and with attempts to terminate unreacted peptide chains. Based on these observations, a modified procedure of solid phase peptide synthesis was developed which significantly improved the synthesis of residues 63-74 of acyl carrier protein.
Since the introduction of the solid phase method for peptide synthesis by llerrifield in 1962,6 an enormous number of peptides have been prepared.6 A more striking achievement, however, has been the synthesis by the solid phase procedure of several large proteins with high biological activity, e.g., ribonuclease A,? fragment Pz of Staphylococcus aureus nuclease T,s soybean trypsin i n h i b i t ~ rand , ~ acyl carrier protein.'O Despite these impressive achievements, many peptides have not been prepared in an adequate yield and the cause of such failures is still not clear despite extensive studies of the sequence-dependent" problems of solid phase synthesis.6$12-16l I o s t failures in the method have been attributed to incomplete coupling and deprotection steps.6j16-21 Fortunately, a wide variety of methods has been established for the formation of a peptide bond,18 and recently several new resin sup(1) This work was supported by Grants No. HE-10406 and AM-13025 from the C S P H S and G r a n t GB-5142X from the Kational Science Foundation. (2) Recipient, George Murray Scholarship (University of Sdelaide, Australia). (3) To whom correspondence should be addressed. (4) Established Investigator, American 'Heart Association. (5) R. B. Merrifield, F e d . Proc., 21, 412 (1962). ( 6 ) (a) G. R. Marshall and R . E. Merrifield, "Biochemical ilspects of Reactions on Solid Supports," Academic Press, New York, N . Y . , 1971; (b) A . Marglin and R. B.Merrifield, Ann. Rev. Biochem., 39, 841 (1970). (7) B. G u t t e and R . B. Merrifield, J . B i d . Chem., 246, 1922 (1971). (8) D . A. Ontjes and C . E. hnfinsen, Proc. N a t . Acad. Sci. U . S.,64, 428 (1969). (9) K. Noda, J. S. Terada, N. Mitsuyasu, &I. Waki, T. K a t o , and N. Izumiya, Naturwissenschaften, 58, 147 (1971). (10) (a) W. S.Hancock, D. J. Prescott, W.L. Nulty, J. Weintraub, P . R. Vagelos, and G. R . Marshall, J . Amer. Chem. Soc., 93, 1799 (1971); (b) W .S. Hancock, D . J. Prescott, G. R . Marshall, and P . R . Vagelos, J . B i d . Chem., 247, 6224 (1972). (11) I t has been established that, these failures are not necessarily associated with a particular amino acid residue, and therefore are described as sequence-dependent problems. (12) E. Bayel., II. Eckstein, IC. Hagele, W, A. Konig, W. Bruning, H . Hagemaier, and W. P a r r , J . Amer. Chem. Soc., 92, 1735 (1970). (13) E. Bayer, H. Hagenmaier, G. Jung, W.P a r r , H. Eokstein, P . Hunaiker, and R. E . Sievers, "Peptides 1971," North-Holland Publishing Co., New York, N. Y., 1971, pp 65-73. (14) H. Hagenmaier, y'etrahedrom Lett., 283 (1970). (15) R . C. Sheppard, "Peptides 1971," North-Holland Publishing Co., New York, N. Y , , 1971. (16) F. C . Testa11 and A. B. Robinson, J . 010. Chem., 35, 2842 (1970). (17) F. Chuen-Heh Chou, R . I
