COMMUNICATIONS TO THE EDITOR
Oct. 20, 1962
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WATER-SOLUBLE HELICAL POLYPEPTIDES’,*
Sir: We report here the synthesis and some physicalchemical properties of high molecular weight watersoluble polypeptides capable of assuming either .O0I random or helical conformations in aqueous s o h tions. The new polypeptides are derivatives of poly-a,L-glutamic acid and certain copolymers thereof where the 7-carboxyl group has reacted with hydrophilic amino compounds to form y amido derivatives. The synthesis makes use of amide-ester i n t e r ~ h a n g e . ~This reaction has been applied t o polypeptide^^^ and some rotatory data have been reported recently.4b A typical preparation of such a water-soluble polypeptide is given. Poly-y-benzyl-a,L-glutamate ( ( ~ ~ ~ =1 ~2.0, ) c = 0.2% in dichloroacetic acid; MW, 350,000) 4.38 g. (0.02 mole) was dissolved in 10.4 g. (0.08 mole) of 2-(N-morpholinyl) -ethylamhe and heated in an oil bath a t 70’ for 70 hr. The solution a t this state was completely miscible with water and showed no ester absorption in its infrared spectrum. The solution was dissolved in 50 ml. of3water, dialyzed for 48 hr. with four changes of water, 20 40 60 80 )O lyophilized and dried in vacuo a t 50’; yield 3.6 g. (75%) Calcd. for (CllH19N308)n. C, 54.74; H, V O L U M E 7- M E T H A N O L 7.95; N, 17.40. Found: C, 55.05; H, 7.65; N, Fig. 1.-The dependence of bo (calculated from the 17.01. The poly-y-N- [2-morpholinylethyl]-a-~glutamamide, I, had an intrinsic viscosity of 0.82 in Moffitt equations over the wave length range 313 to 578 mp using XO = 212 mh) upon the solvent composition for water0.2 M aqueous NaCl. If the amide-ester inter- methanol solutions of poly-7-5-[2-morpholinylethyl]-CU,Lchange is run at higher temperatures or in a sealed glutamamide. tube, lower molecular weight products are obtained. Similar reactions were employed to prepare the undergoes a transformation from a random form in copolypeptides described below. It has been water solution to a helical conformation in water found that, using the above procedure, poly-y- solutions containing more than about 50% of methyl-a,L-glutamate also undergoes aminolysis to methanol. When I was dissolved in a water soluyield I. I is completely soluble in water from pH tion containing 2 NaC1, i t showed a bo of -lSO 1.5 to 13 and is also soluble in various ratios of indicating a helix content of -307,. Measurement methyl alcohol or dioxane and water. Lower mo- of the short wave length optical rotatory dispersion lecular weight preparations of I are soluble in of I in 95% methanolic solution showed the negamethyl alcohol alone. tive 225 mp Cotton effects and the positive 190 Measurements of the rotatory dispersion of I in mM Cotton effectg characteristic of the a-helical various methanol-water solutions have been made conformation. The infrared spectra of oriented over the wave length region 313-578 m,u and the films of I, obtained from either water or niethanoldata calculated using the Moffitt equation,h from water solutions, showed infratred dichroism charwhich bo values have been obtained. A plot of acteristic of the a-helical conformation. l o these bo values as a function of solvent composition Since, until this time, there has been no reported is shown in Fig. 1. Assuming that the magnitude of preparation of a synthetic high molecular weight bo in the range 0 to 630 indicates the percentage non-ionic water-soluble polypeptide containing only helix present in s0lution,6~~ it is apparent that I one optical isomer and having an a-helical conformation in water, i t was of some interest to see whether ( 1 ) This is Polypeptides. XXXIX. For the preceding paper helix formation in water solution could be obtained in this series see ref. 9. (2) This work was supported in part by the Office of the Surgeon through the preparation of suitable copolypeptides General, Department of the Army. of I. Copolypeptides of y-benzyl-L-glutamate (3) R. Baltzly, 1. X.Berger and A. A. Rothstein, J . A m . Chem. with L-alanine, L-leucine and L-methionine in variSoc., 73,4149 (1950). ous mole ratios were prepared and treated in the (4) (a) N. Lupu, A. Yaron, M. Sela and A. Berger, Bull. Res. Council Israel, lOA, 47 (1961). (b) A. Yaron, S . Lupu, A. Berger and M. manner described above to replace the y-ester group Sela, ibid., l l A , 87 (1962). We thank the referee for bringing t o our by 2-morpholinylethylamine. Those containing up attention this note which had not been available t o us. to 10 mole per cent. leucine, or up to 40 mole per (5) W. Moffitt and J. T. Yang, Proc. Null. A c a d . Sci., U . S., 43,
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596 (19.56). (6) C. Cohen and A . G. Szent-Gyorgyi, J . A m . Chem. Soc., 79, 248
(1957). (7) For recent reviews on the use of bo see E. R. Blout, Chapter 17 in “Optical Rotatory Dispersion,” C. Djerassi, Editor, McGraw-Hill Book Company, &-ern York, S . Y . . 1960; P. Urnes and P. Doty in “.4dvancer. in Protein Chemistry,” Vol. 16, C. B. Anfinsen, h-,L. Anson, K. Bailey and J. T. Edsall, Editors, Academic Press, Inc., New York, N. Y.. le)t31, p. 401.
( 8 ) N. S. Simmons, C. Cohen, A. G. Szent-Gyorgyi, D. B. Wetlaufer and E. R. Blout, J . A m . Chem. Soc., 83,4766 (1961); Beychok and E. R. Blout, J . Mol. B i d . , 3, 769 (1961). (9) E. R. Blout, 1. Scbmier a n d N. S. Simmons, J . A m . Chem. S o c . , 84, 3193 (1962). (10) See for example C. H. Bamford, A. Elliott and W. E. Hanby in ”Synthetic Polypeptides,” Academic Press, Inc., S e w York, X. Y., 19.56, p. 147, and T . Miyazawa and E. K. Blout, J . A m . Chem. Soc., 83, 712 (1961).
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COMMUNICATIONS TO THE EDITOR 70C
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tion than L-alanine residues. Also, it is clear that polypeptide helix formation in water solution depends not only on the formation of intramolecular peptide hydrogen bonds but also is aided by “hydrophobic bond” and dispersion force stabilizations’l provided by residues such as methionine and alanine. Further, these new water-soluble polypeptides having high helix contents, can be transformed by reagents known to denature proteins into completely random conformations.
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(11) For a discussion see W. Kauzmann in “Advances in Protein Chemistry,” Vol. 14, C. B. Anfinsen, Jr., M. L. Anson, K. Bailey and J. T. Edaall, Editors, Academic Press, Inc., New York, N. Y..1959.
-bo
CHILDREN’S CANCER RESEARCH FOUNDATION AND DEPARTMENT OF BIOLOGICAL CHEMISTRY R. K. KULKARNI HARVARD MEDICAL SCHOOL E. R. BLOUT 15, MASSACHUSETTS BOSTON RECEIVED AUGUST17, 1962
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FLUORINE N.M.R. SPECTROSCOPY. XII. PROOF OF OPPOSITE SIGNS FOR THE “DIRECT” CARBON13 COUPLING CONSTANTS TO HYDROGEN AND T O FLUORINE
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Sir :
The use of “spin decoupling” (double resonance) for the determination of relative signs of coupling MOLE % constants in fluorocarbon derivative^^-^ and in C O - A M I N O ACID RESIDUE proton corn pound^^^^ has become important as Fig. 2 -The dependence of bo (calculated from the Moffitt an experimental check on theoretical predictions. equation6 over the wave length range 313 to 578 mp using The work of Lauterbur and Kurland6 was particXO = 212 mp) upon amino acid composition for a series of ularly significant, since i t was a test of the prohigh molecular weight copolypeptides derived from ?-IT- posa17 that “direct” couplings of C13 to protons 12 - morpholinylethyl] - LY,L- glutamamide and L - alanine, have the same sign as vicinal proton-proton 0-0-0: poly - - N - [2-morpholinylethyl] -cu,Lglutamamide couplings. and L-methionine, A-A-A. The solvent in all cases w a s water The predictions of signs and magnitudes of containing 0.2 M NaCl. All measurements were performed couplings involving fluorine, though less certain at 25 f 1’ except those on the copolypeptides containing than those for protons, nevertheless represent a 30, 35 and 40q/,L-methionine with which compounds the further development of the theory. I t is of prime measurements were made at 5 =IC1”. iniportance to test the suppositioii that the “dicent. alanine or methionine were water-soluble. rect” couplings, J(C13F) and J(Cl3H), have the Some rotatory dispersion data of the water-soluble same sign. This has now been done for the particcopolypeptides are shown in Fig. 2, where bo is ularly simple case of CHC12F, dichlorofluoroplotted as a function of mole per cent. coamino acid methane. Experimental.-The basic n.m.r. spectrometer residue content. The solubility of the copolypeptides containing 30 or more per cent. L-methionine and techniques were as previously described.R,9 is increased a t lower temperatures so the dispersion For the double resonance work a Model SD-60 measurements on these compounds were made a t Spin Decoupler’O was employed. It was equipped 5’. The data reveal that, by incorporation of with modules designed for strong irradiation of -40% L-methionine in I, it is possible to obtain an fluorine nuclei a t 37.65 mc./sec., while observing essentially completely helical water-soluble syn- protons a t 40.000 mc./sec. (symbolized as H { F ] decoupling’) and for F{H}decoupling with irradiathetic polypeptide, Other experiment were performed with the tion a t 42.50 mc./sec. With this equipment some copolypeptide containing 30 mole percent L-alanine ten watts of radiofrequency power is available t o which, in 0.2 M NaC1, had a bo value of -331 in- the probe; however, far less than this is required dicating slightly more than 50’% helix content. since the F-H coupling constant in CHCllF is (1) J. D. Baldeschwieler, C k e m . Reus., 63, i n press (1963). When this copolypeptide’s rotatory dispersion was (2) D. P. Evans, Mol. P h y s . , 5, 183 (1962). measured in 8 M urea or in 8.6 M lithium bromide (3) S. L. Manatt and D. D. Elleman, J . A m . Chefn. Soc., 84, 1305 solution, the bo value was found to be zero. (1962). From the experiments and the data reported (4) D. D. Ellernan and S. I,. Manatt, J . C h e m . P h r s . , 36, 1945 here we can conclude the following. The helical (1962). (5) S. I,, ivlnnrtt and D. D. Elleman, J . A m . Chent. SOL.,84, 1579 form of poly-7-N- [2-morpholinylethyl]-cr,~-glu(1962). tamamide is not stable in water solution but this (6) P. C. Lauterbur and R. 1. Kurland, ibid., 84,3405 (1962). polypeptide may be converted to a helical conforma(7) M.Karplus, ibid.,84, 2458 (1962). (8) G. Filipovich and G. V. D. Tiers, J . Pkrs. Chem., 8S, 761 tion by changing to less polar solvents such as methanol or dioxane. In certain water soluble (1959). (9) G. V. D. Tiers, J. Chem. P h y s . , 56, 2263 (1962). polypeptides L-methionine residues are more effec( 101 Manufactured by t h e Nuclear Magnetic Resonance Specialties tive in rxomoting helix formation in aqueous solu- Co., Inc., Box 145, Grcensburg Road, New Kensington, Pa. 20
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