for better polymerization methods led to the use of of hydrogen chloride, and the resulting tripeptide active trichlorophenyl and later of pentachlorophenyl active ester hydrochloride was polymerized in dimethylesters. formamide (50 % solution) in the presence of triethylamine to poly-(y-t-butyl-L-glutamyl-L-alanyl-y-l-butylN-Carbobenzoxy-a-benzyl-L-asparticacid pentaL-glutamic acid) (VI) in 42% yield. The t-butyl ester chlorophenyl ester (I), m.p. 151-152", [ c x ] ~ ~20.0" D groups from polymer VI were removed by 90% tri(c 2.36, chloroform), was obtained in 95 % yield from fluoracetic acid and the water-soluble poly-a-L-gluN-carbobenzoxy-a-benzyl-L-aspartate and pentachlorotamyl-L-alanyl-L-glutamic acid (VII) was obtained in phenol using the DCCI m e t h ~ d . ~Anal. Calcd. for 94% yield. Further purification was achieved by Cz5H1806NC15:C, 49.57; H , 3.00; N, 2.31. Found: dialysis l6 for the removal of low molecular weight C, 49.57; H, 3.24; N, 2.35. Hydrogen bromide polypeptides and possible cyclic peptides. Anal. cleavage of I [IO g. of I in 140 ml. of 8.517, solution of Calcd. for (C13H19N307.0.5HZO)o;:C, 46.1 ; H, 5.94; hydrogen bromide in acetic acid-dichloroacetic acid N, 12.4; equiv. wt., 169. Found: C, 46.34; H, 6.41; (1 : 6)] gave after two recrystallizations from absolute N, 12.10; equiv. wt., 173.5; intrinsic viscosity in dialcohol 84 % a-benzyl-P-pentachlorophenyl-L-aspartate chloroacetic acid, 0.12 dl./g. Molecular weight as hydrobromide (11), m.p. 201-202°, [ c x ] ~ ~ 7.12" D (c 3.38, determined by D N P method was 20,000. Retention dimethylformamide). Anal. Calcd. for C17H1304Nof optical purity was 98 f 2%.16 C15Br: C, 36.96; H, 2.37; N, 2.54. Found: C, Similarly N-carbobenzoxyglycylglycyl-L-phenyl- 37.04; H, 2.46; N , 2.87. Active ester I1 polymerized alanine pentachlorophenyl ester was polymerized to readily in dimethylformamide in the presence of a poly-(glycylglycyl-L-phenylalanine)in 58 % yield. tertiary amine to poly-0-(a-benzyl)-L-aspartic acid (111) Polypeptides with known repeating sequence of in 95 % yield of which 62 % was a polypeptide with an amino acids containing other than a-peptide bonds, average molecular weight of 10,000, as determined by e.g., y-glutamyl residues, are also important. l7 Prepaend-group analysis. (During the polymerization of ration of poly-(7-L-glutamyl-y-aminobutyric acid) 11, the higher molecular weight polypeptide precipitated (VIII) from N-carbobenzoxy-a-t-butyl-L-glutamyl-y- from the dimethylformamide solution (62 %) and the aminobutyric acid pentachlorophenyl ester (IX), m.p. lower molecular weight fraction was precipitated by 175-176", [ a I z 6-3" ~ (c 2.0, chloroform), was achieved ether.) The intrinsic viscosity of I11 in dichloroacetic in 49 % over-all yield after dialysis; intrinsic viscosity acid is 0.09 dl./g. Anal. Calcd. for ( C I ~ H ~ ~ O ~ N ) ~ : 0.135 dl./g. Anal. Calcd. for(C9Hl4N2O4),:C , 50.51 ; C, 64.38; H, 5.40; N, 6.88. Found: C, 63.90; H, 6.55; N , 13.10. Found: C, 51.03; H, 7.08; H, 5.41 ; N , 6.76. Catalytic hydrogenation of I11 gave N, 12.6. optically pure water-soluble poly-@+aspartic acid (IV) Other polypeptides containing different amino acid in 99% yield. After dialysis polypeptide IV was residues were also prepared by this general procedure. obtained in 50% yield; intrinsic viscosity in dichloroacetic acid 0.11 dl./g.; molecular weight, as deAcknowledgment. This work was supported by termined by the D N P method, 19,000; equivalent Research Grants G M 06579 and 08795 from the Naweight 125.1, calcd. 124. Anal. Calcd. for ( C 4 H 5 0 3 tional Institutes of Health, Public Health Service. N.0.5 H 3 0 ) m ; C, 38.79; H, 4.80; N, 11.21. Found: (15) During the extensive dialysis the loss for various batches was C, 39.01; H, 4.55; N, 11.42. between 38 and 6 2 Z . (16) The retention.of the optical purity was established by determinThe optical purity of the dialyzed sample was esing the specific rotation of the total hydrolysate of the polypeptides and tablished by determining the optical purity of the comparing that with the specific rotation of the corresponding amino acids treated under the same conditions. aspartic acid after total hydrolysis; 58.6 mg. of sample (17) J. Kovacs, U. R. Ghatak, G . N. Schmit, F. Koide, J. W. Goodwas refluxed for 24 hr. in 3 ml. of 12 N hydrochloric man, and D. E. Nitecki, International Symposium o n the Chemistry of acid, the solution was evaporated to dryness, the resiNatural Products, Kyoto, Japan, April 1964; Abstracts of Papers, pp. 175-1 78. due was dried for 24 hr. over sodium hydroxide and phosphorus pentoxide and then dissolved in 5 ml. of J. Kovacs, A. Kapoor water, and the specific rotation was determined with a Department of Chemistry Sf. John's University, New York, New York Rudolph Model 200AS-80Q spectropolarimeter; [ a ] 2 6 ~ Received September 3, 1964 19.65" (c 1.25, water). As a control, 68.3 mg. of aspartic acid was treated in the same manner; [ a I z 6 ~ 20.03 " (c 1.35, water), which indicated at least 98.1 optical purity. The exclusive presence of P-peptide Poly-P-L-aspartic Acid. Synthesis through linkages was determined by Hofmann degradation.5 Pentachlorophenyl Active Ester Poly-@-L-asparticacid was also prepared through the and Conformational Studies a-t-butyl ester derivative. N-Carbobenzoxy-a-t-butylSir : L-aspartic acid pentachlorophenyl ester (V), m.p. Utilization of the pentachlorophenyl active ester 121-123", [ c Y ] ~37.5" ~ D (c 2, chloroform) [Anal. Calcd. method for the synthesis of poly-@-aspartic acid has for CzzH2~O8NClj: C, 46.20; H, 3.54; N, 2.45. Found: resulted in optically pure high molecular weight poly(2) A few trichlorophenyl and pentachlorophenyl esters were reported peptide, which was needed for biological and physical first by G . Kupryszewski and M. Kaczmarek, Rocrniki Chem., 35, 935, 1533 (1961); K . Stich and H. A. Lehmann [Helv. Chim. Acta, 46, chemical investigations. The importance of optically 1887 (1963)], studied the kinetics of aminolysis of different chlorophenyl pure poly-0-aspartic acids for immunochemical studies esters. was emphasized in our earlier paper, where a synthesis (3) D. F. Elliot and D. W. Russell, Biochem. J . , 66,49P (1957). (4) M. Sela and A . Berger, J . Am. Chem. Soc., 77, 1893 (1955). through p-nitrophenyl ester was also reported. Search (1) J . Kovacs, R. Ballina, and R. Rodin, Chem. Ind. (London), 1955 (1963).
( 5 ) J. Kovacs and I. Konyves. Naturwiss., 41, 333 (1954); J . Kovacs, H. Nagy-Kovacs, I. Konyves, J. Csiszbr, T. Vajda, and H. Mix, J . Org. Chem., 26, 1084 (1961).
Communications to the Editor
119
hump in the titration curve near p H 3 is most likely associated with this structural change, since similar anomalies have been observed for PGA.lnsll This conclusion is supported by two additional lines 4.64 of evidence: (i) The rate of deuterium exchange with amide hydrogen in D 2 0 solution was followed by the ~ infrared absorption method of Blout, et ~ 1 . ' The exchange a t room temperature was complete within 10 min. at p D 7.5, but required 94 min. for half completion at p D 2.8. As in the case of PGA,12 the decrease in 3.8 exchange rate at low p D may result from the participa14 I I I I 0 I 2 3 4 5 6 tion of amide groups in hydrogen bonds among each PH other. (ii) Heating a solution at p H 2.34 produced a Figure 1. Optical rotation, ultraviolet absorption, a n d titration gradual change in optical rotation in the negative direcof poly-P-~-aspartic acid, mol. wt. 19,000, in 0.2 M N a C l : (0) tion, suggesting a gradual breaking up of a secondary residue-molar extinction coefficient at 200 mp, concentration structure whose stability is roughly comparable to that 0.034 g./dl. ; ( 0 ) specific rotation at 546 mp, concentration 0.62 of PGA.13 g./dl. Titration a t concentration = 0.0276 g./dl. Ordinate scales The details of the secondary structure cannot be are adjusted to make the ranges of the various measurements approximately coincide. stated with certainty a t present. However, a onestranded helical structure is more appealing than an intermolecular aggregate, in view of the fact that the C, 45.85; H , 3.48; N, 2.361 gave after catalytic analogous structures in poly-a-L-lysine are distinguished hydrogenation and polymerization poly-P-(a-t-butyl)by the appearance of hypochromism in the helix and L-aspartic acid (VI), which on treatment with 90% hyperchromism in the aggregate.I4 A number of trifluoroacetic acid yielded polypeptide IV ; intrinsic poly-P-amino acid helices have been constructed with viscosity in dichloroacetic acid 0.125 dl./g. ; molecular Courtauld stereomodels.'j One helix that appears to weight, as determined by the DNP method, 24,000. be particularly favorable on the basis of the criteria The physical properties of poly-P-L-aspartic acid laid down by Pauling, Corey, and Branson16 is formed solutions have been examined for the purpose of learnby hydrogen bonding each carbonyl to the third amide ing about possible secondary structure in this material. group removed toward the N-terminus. This model Evidence for such secondary structure was reported has 3.4 residues/turn and a n axial translation of 1.58 A.,/ previously6 from a study of low molecular weight polyresidue. It is necessarily right-handed for @-amino 6-L-aspartic acid prepared via the N-carboxyanhydride. acids with the L-configuration at the a-carbon (aThe higher molecular weight samples prepared by the amino acid convention). present method seemed more suitable for this purpose, It is interesting to note that a homologous compound, since the stability conferred on an ordered structure as poly-y-D-glutamic acid, has also been found to show a result of cooperative effects increases with the molecuchanges in optical rotation with degree of dissocialar weight. In view of the current interest in these tion.17,I8 Edelhoch and Lippoldt l7 interpreted their structures, a brief report of results is appropriate at data t o mean that the un-ionized form is hypercoiled this time. as a result of random intramolecular hydrogen bonds, In Figure 1 are shown the dependence upon p H of the while Rydon l8 has suggested that a helical structure specific rotation [a'326546 and the residue-molar extinction would account for the observed effects. coefficient a t 200 mp, which is within the amide Acknowledgments. This investigation was supported absorption band centered at 190 mp. The degree of by Public Health Service research grants (GM-10882, dissociation of carboxyl groups shown was determined GM-06579, GM-08795) from the Division of General by potentiometric titration with 1 M NaOH. The Medical Sciences, Public Health Service. changes in optical properties with p H are strikingly similar to those observed for poly-a-L-glutamic acid (IO) A. Wada, Mol. Phys., 3, 409 (1960). (PGA)7-g and ascribed in that case to a transition (11) M . Nagasawa and A . Holtzer, J . A m . Chem. Soc., 86, 538 ( 1964). from helix to random coil as the molecule becomes (12) E. R . Blout, C. de Loze, and A. Asadourian, ibid., 83, 1895 charged. The change in rotation a t 546 m p and the (1961). hypochromic effect at 200 m p are smaller for poly-P-L(13) G. D . Fasman, D . Lindblow, and E. Bodenheimer, ibid., 84, 4977 (1962). aspartic acid than for PGA, but are in the same direc(14) K. Rosenheck and P. Doty, Proc. Natl. Acad. Sci. U.S., 47, tion. Furthermore, neither effect can be simply a 1775 (1961). measure of the degree of dissociation of carboxyl (15) J. Applequist, unpublished observations. (16) L. Pauling, R . B. Corey, and H. R . Branson, Proc. N a f l . Acad. groups, since most of the change occurs when the Sci. U . S., 37, 205 (1951). molecules are less than 5 0 z dissociated. It is there(17) H. Edelhoch and R. E. Lippoldt, Biochim. Biophys. Acta, 45, 205 (1960). fore concluded that the observed changes arise from an (18) H. N. Rydon, J . Chem. Soc., 1328 (1964). ordered secondary structure which is stable only at very low degrees of electostatic charge. The anomalous J. Kovacs, R. Ballina, R. L. Rodin Department of Chemistry St. John's University, N e w Y o r k , N e w Y o r k
(6) D . Balasubramanian and J . Applequist, Abstracts, 145th National Meeting of the American Chemical Society, Sept. 1963, New York, N. Y . . p. 67C. (7) M. Idelson and E. R . Blout, J . A m . Chem. Soc., 80, 4631 (1958). (8) I
