was hydrogenated in the usual manner over palladium +12.8 (c 2.0, water). Anal. Calcd. for ClzHleNz03: N, 11.9. Found: N, 12.1. Paper chromatography in methanol (20 ml.) to yield 0.13 g. (867J of the di+13.2' (c 2.1, water); lit.zz [ a J Z o ~showed one spot, Rr 0.62, with I-butanol-acetic acidpeptide: [alZ5~ (22) w. Grassmann, E. Wiinsch, and A. Riedel, Ber., 91,455 (1958). water, and one spot, Rf0.80, with phenol-water.
A Nuclear Magnetic Resonance Study of the Structures of L- and meso-Cystine in Aqueous Solutions' Jay A. Glasel Contributionf r o m Columbia University, Department of Biochemistry, College of Physicians and Surgeons, New York, New York. Received April 26,1965 The n.m.r. spectra of L-cystine, meso-cystine, and their dimethyl esters in acidic and basic solutions are analyzed. The results indicate that the possible conjigurations f o r L-cystine are stablized b y intramolecular interactions between the two moieties. This is in contrast to mesocystine where no stabilization is observed.
R' x-$ ;i.
I
There are two cases which must be considered in a discussion of the cystines in solution. The first is when R = R'. This is either all L- or all D-cystine. The Introduction two structures 1and I1 do not necessarily have the same There have been several analyses of the n.m.r. spectra energy. This is apparent from the use of space fillof the L-amino acids, including L-cystine. Taddei ing models and is, of course, theoretically valid. Thus, and Pratt2 have observed the chemical shift changes of the possibility of one of the structures (I or 11) being the various protons as a function of pH. P a ~ h l e r , ~ ?predominant ~ in solution obtains. Fujiwara and Arata,5*6and Martin and Mathur' have The second cases arises in a cystine molecule where analyzed the high-resolution spectra of these acids in a one moiety is of the L optical isomer, and the other is fashion which can be interpreted to give the relative of the D variety. This is defined as meso, or internally populations of the three classical rotamers derived compensated, cystine. For this case, by the same argufrom considering the a-amino acids as derivatives of ment as above, both structures I and I1 have the same ethane. energy. The case of the cystines is, however, more compliThere have been speculations" concerning the possicated than these studies would indicate, and some of bility of an endocyclic configuration of L-cystine brought the peculiarities of their spectra may throw some light about by binding between the opposing amino and caron the interpretation of the spectra of the other amino boxyl groups of the two moieties. The presence of acids. this structure would fit in with the observation that The structure of L-cystine in solution has been concystine has a strong temperature coefficient of molecsidered by others&'O theoretically, in connection with ular optical rotation, indicating an equilibrium bethe very large optical activity exhibited by the moletween structural forms. cule. This is a factor of 10 greater than any other All previous analyses of the n.m.r. spectra of the amino acid. The disulfide ultraviolet absorption band simple amino acids in solution are based on the classical is optically active,lO"and studies of this phenomenon in rotamer structures which neglect intramolecular intersolution indicate that there is only very highly hindered actions other than steric hindrance in the trans and rotation about the disulfide bond. In fact it would gauche configurations. Absence of intramolecular appear that the disulfide portion of the structure of the interactions leads to the conclusion that both L- and cystines is represented by structures I and 11, in which meso-cystine should have the same n.m.r. spectra. the dihedral angle is 90". It was of some interest to determine whether or not this is true. (1) This work supported by Grant GB-1788 from the National Science Foundation. (2) F. Taddei and L. Pratt, J. Chem. SOC.,1553 (1964). ( 3 ) K. G. R. Pachler, Spectrochim. Acta, 19, 2085 (1963). (4) K. G. R. Pachler, ibid., 20, 281 (1964). ( 5 ) S. Fujiwara and Y.Arata, Bull. Chem. SOC.Japan, 36, 578 (1963). (6) S. Fujiwara and Y. Arata, ibid., 37, 344 (1964). (7) R. B. Martin and R. Mathur, J. Am. Chem. Soc., 87, 1065 (1965). (8) M. Calvin, U. S. Atomic Energy Commission, UCRL-2438, 1954. (9) A. Fredga, Acta Chem. Scand., 4, 1307 (1950). (10) C. Djeressi, "Optical Rotatory Dispersion," McGraw Hill Book Co., Inc., New York, N. Y.,1960. (loa) NOTEADDED IN PROOF. A recent investigation of this point has been made: S. Beychok, Proc. Natl. Acad. Sci. U.S., 53,999 (1965).
5412
Journal of the American Chemical Society / 87:23
Experimental Section The n.m.r. spectra were taken with a DA-60 Varian n.m.r. spectrometer at an in-probe temperature of 26 + I " as measured by the ethylene glycol method. An internal standard of TSS (3-trimethylsilyl-1-propanesulfonic acid sodium salt) was used. pH measurements were made with a Beckman one-drop electrode useful in the range 0-11 pH units, and no attempt was (11) L. Fieser, Rec. Tray. Chim., 69, 410 (1950).
December 5, I965
Table I.
Comparison of Computed N.m.r. Constants for L-Cystine, meso-Cystine, and L-Cysteinea Computation scheme
6ax(o)
90 %) whose spectrum is discussed below, and a small amount of L-cystine ( 11) displayed the spectrum disGussed below for the meso-cystine commercial preparation. However, when the solution was titrated again to pH
