Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3064-3067, May 1986 Agreement with the stretching frequency-conformation correlation of Sugeta, Go, and Miyazawa (Raman spectroscopy) HAROLD E. VAN WART* AND HAROLD A. SCHERAGAt *Department of Chemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-3015; and tBaker Laboratory of Chemistry, Cornell University, Ithaca, NY 14853-1301 Contributed by Harold A. Scheraga, December 16, 1985

ABSTRACT Two hypotheses have been advanced to ex- about the two C-S bonds of the CCSSCC moiety were plain the conformational dependence ofthe disulfide stretching assigned S-S stretching frequencies of approximately 510, frequency of the CCSSCC moiety in unstrained . 525, and 540 cm-l, respectively. Such compounds can adopt conformations that exhibit At about the same time, we had been investigating the disulfide stretching bands near 510, 525, and 540 cm-'. Sugeta effects of rotation about the C-S and S-S bonds of the et al. [Sugeta, H., Go, A. & Miyazawa, T. (1973) Bull. Chem. CCSSCC moiety on its S-S stretching frequency by studying Soc. Jpn. 46, 3407-3411] have attributed these three bands to the Raman spectra of a series of crystalline disulfides whose conformations having none, one, and two trans conformations, structures had been determined by x-ray diffraction tech- respectively, about the C-S bonds. In apparent contradiction to niques (7-10). Unstrained disulfides with GG and G'G' this correlation, Van Wart and Scheraga [Van Wart, H. E. & conformations were found to exhibit S-S stretching bands Scheraga, H. A. (1976) J. Phys. Chem. 80, 1812-1823] found near 508 ± 5 cm-' (7), in agreement with the proposal of that dithioglycolic acid crystals, believed at the time to exist Sugeta et al. (3, 4). However, a commercial sample of only in the trans conformation about both C-S bonds, exhibited dithioglycolic acid, believed from the only crystallographic the S-S stretching band near 510 cm-'. On the basis of this data available at the time to have the TT conformation (R. observation, it was suggested instead that the 510, 525, and 540 Parthasarathy, personal communication), also exhibited the cm 1 bands arose from CCSSCC moieties having none, one, S-S stretch at 508 cm-', in apparent contradiction to the and two A conformations (i.e., those with CC-SS dihedral correlation of Sugeta et al. It was subsequently suggested angles close to 30°), respectively, about the C-S bonds. It has from other experimental and theoretical data (11, 12) that a recently been shown by Nash et al. [Nash, C. P., Olmstead, non--like rotamer with a low CC-SS dihedral angle, M. M., Weiss-Lopez, B., Musker, W. K., Ramasubbu, M. & termed the A conformation, rather than the trans conforma- Parthasarathy, R. (1985) J. Am. Chem. Soc. 107, 7194-7195] tion might be the "missing" rotamer responsible for the that dithioglycolic acid can crystallize in two different forms, higher frequency S-S stretching bands. one with trans and the other with gauche conformations about Recently, Nash et al. (18) have discovered that dithiogly- both C-S bonds, and that these have S-S stretching bands at 536 colic acid crystallizes in two distinct forms, one with GG and and 510 cm-', respectively. These results are confirmed here the other with TT conformations about the C-S bonds. These and it is shown that our earlier data were collected on the authors have also found that the TT and GG forms exhibit the all-gauche (rather than the all-trans) form. Thus, the correla- S-S stretch at 536 and 510 cm-', respectively. This has raised tion proposed by Sugeta et al. is correct. the question as to which form was examined by us earlier (7). It is shown here that Raman data collected on crystals known When the first laser-excited Raman spectra of were to have the TT conformation indeed exhibit the S-S stretching obtained approximately 15 years ago (1, 2), disulfide stretch- band near 540 cm-', in agreement with the data ofNash et al. ing bands attributable to side chains were prominent- (18) and the correlation proposed by Sugeta et al. (3, 4). ly observed. These bands exhibited frequencies in the range Apparently, as will be shown here, the commercial sample of of 480 to 540 cm-1 and attention was soon directed-toward dithioglycolic acid that we examined earlier must have had understanding how the conformation4: of the CCSSCC frag- the GG conformation. ment of cystine and structurally related aliphatic disulfides influenced the observed frequencies. Sugeta, Go, and 4:The conformation of the CCSSCC unit is described by the CS-SC Miyazawa (3, 4) reported that methyl t-butyl disulfide and and the two CC-SS dihedral angles. Rotation about the S-S bond is di-t-butyl disulfide exhibit single S-S stretching bands near hindered, and unstrained disulfides invariably have CS-SC dihedral 525 and 540 cm-1, respectively. They concluded that the angles with absolute values of 85 ± 200. Because of this hindrance and the C2 symmetry of the CSSC group, all unstrained disulfides presence of a trans to the distal S about exist as enantiomers with CS-SC dihedral angles of +85° and -85°. each C-S bond raises the S-S stretching frequency by 15 With regard to the rotamers formed on rotation about the C-S bonds cm-. In contrast, unstrained primary aliphatic disulfides of the CCSSCC unit, we define the C (for cis), A, G (for gauche), [i.e., those in which the carbon adjacent to the B, and T (for trans) conformations as those having CC-SS dihedral disulfide bond are primary (-CH2-) carbon atoms] exhibit S-S angles of approximately 00, ±30°, ±600, ±900, and 1800, respective- at 510 and 525 cm-1 due to the ly. It should be noted that, for a given screw sense of the disulfide stretching bands both bond, conformations of the CCSSCC unit with equal and opposite coexistence of rotational isomers about the C-S bonds (3-7). values of the CC-SS dihedral angle are nonequivalent and not Assuming that rotation about the C-S bond produced only necessarily isoenergetic. For example, when the CS-SC dihedral gauche (G) and trans (T) rotamers, Sugeta et al. (3, 4) angle is +85°, the A, G, and B rotamers having negative CC-SS proposed a frequency-conformation correlation for primary dihedral angles may have higher energies than those having positive disulfides in which the GG, GT or TG, and TT conformations values because of steric repulsions between CH groups on opposite sides ofthe S-S bond. The higher energy rotamers will be designated as the A', G', and B' rotamers. Thus, the G and G' rotamers have The publication costs of this article were defrayed in part by page charge CC-SS dihedral angles of +600 and -60°, respectively, when the payment. This article must therefore be hereby marked "advertisement" CS-SC dihedral angle is +85°, and CC-SS dihedral angles of -60° in accordance with 18 U.S.C. §1734 solely to indicate this fact. and +600, respectively, when the CS-SC dihedral angle is -85°. 3064 Downloaded by guest on September 27, 2021 Chemistry: Van Wart and Scheraga Proc. Natl. Acad. Sci. USA 83 (1986) 3065 MATERIALS AND METHODS Instead of using a commercial sample, as we did previously (7), we have obtained Raman spectra of crystals of dithioglycolic acid kindly provided by Dr. Parthasarathy of the Roswell Park Memorial Institute. These are from the same batch on which the crystallographic structure determi- nation was carried out and are known to have the TT conformation. Raman spectra were recorded for both finely ground crystalline and solution-phase samples held in capil- lary tubes. A Spectra-Physics model 171-18 argon laser was used for excitation and a Spex model 1403 spectrometer was used to obtain the spectra. Data were stored in digital form using a Spex Datamate and the spectra were smoothed by the method of Savitzsky and Golay (13). RESULTS To resolve the question as to which form of dithioglycolic acid had been examined earlier (7), the Raman spectrum of crystals from the batch used for the structural determination ofthe TT form was obtained. The spectrum ofpolycrystalline TT dithioglycolic acid exhibits a clearly defined S-S stretch- ing band at 533 cm-1 (Fig. 1, spectrum A), in reasonable agreement with the frequency of 540 cm-' predicted by the correlation of Sugeta et al. (3, 4) and the value of 536 cm-' found by Nash et al. (18). A single C-S stretching band is C~~~~~~~~ observed at 650 cm-' and an unidentified band is observed at 560 cm-'. On dissolution in either 1 M HCl or 1 M NaOH, 0)~ ~ ~ 6 there is a strong S-S stretching band at 506 cm-' with a shoulder at about 517 cm-' (Fig. 1, spectrum B). When 533 dithioglycolic acid is recovered from HCl solution by rapid rotary evaporation, the resulting polycrystalline material 506 exhibits S-S stretching bands at 506 and 533 cm-', C-S stretching bands at 649 and 661 cm-', and also bands at 560 and 578 cm-' (Fig. 1, spectrum C). Finally, if the crystals of the TT form of dithioglycolic acid are dissolved in methanol and recovered by rotary evaporation, the material obtained exhibits single S-S and C-S stretching bands at 506 cm-' and 662 cm-', respectively, and a band of unknown origin at 579 cm-' (Fig. 1, spectrum D). Several conclusions can be drawn from the data in Fig. 1. D First, the TT conformation of dithioglycolic acid exhibits the S-S stretching band at 533 cm-' (spectrum A). Thus, the 662 sample examined earlier (7) having the S-S stretching band at 579 506 cm-' apparently had the GG conformation. At that time, only the TT conformation was known (R. Parthasarathy, personal communication) and the commercial sample exam- ined was erroneously assumed to have the TT conformation. Raman shift, cm-' Second, the GG form is the most stable in aqueous solution with only a small amount of the GT or TG conformations FIG. 1. Raman spectra: A, polycrystalline TT dithioglycolic acid; indicated by the shoulder near 517 cm-' (spectrum B). Last, B, a solution of the sample from A in either 1 M NaOH or 1 M HCl the conformation of dithioglycolic acid that is adopted in the (the spectrum is the same for both); C, the product recovered by crystalline state is on manner rotary evaporation of a solution of the "A" sample in 1 M HCl; D, highly dependent the in which the product recovered by rotary evaporation ofa solution ofthe "A" the crystals are prepared; spectrum C is apparently that of a sample in methanol. All spectra were recorded using 50 mW of mixture of GG and TT conformations and spectrum D is that 514.5-nm laser light for excitation at an instrumental resolution of 4 of material that is all GG. cm-l and a scan speed of 5 sec/cm-'. DISCUSSION was suggested to be vibrational coupling between the S-C-C bending mode of the trans methyl group and the S-S stretch- The frequency-conformation correlation for cystine residues ing mode. This proposal was supported by normal mode in proteins proposed by Sugeta et al. (3, 4) was derived from calculations (14). data on primary, secondary, and tertiary disulfides. Shortly thereafter, we carried out a systematic study ofthe Dimethyl disulfide, methyl t-butyl disulfide, and di-t-butyl Raman spectra of a series of crystalline strained primary and disulfide exhibit single S-S stretching bands near 510, 525, secondary disulfides of known structure, with strain about and 540 cm-', respectively. Since the latter two compounds the S-S bond (9). We confirmed that, in secondary disulfides, contain one and two methyl groups, respectively, trans to the the presence of a carbon atom trans to the distal S atom distal S atom about the C-S bond(s), Sugeta et al. proposed across each C-S bond of the CCSSCC group raises the S-S that each trans methyl group raised the S-S stretching stretching frequency by approximately 12 cm-1, in good frequency by 15 cm-'. The source of this frequency increase agreement with the proposal of Sugeta et al. (3, 4). It was Downloaded by guest on September 27, 2021 3066 Chemistry: Van Wart and Scheraga Proc. Natl. Acad. Sci. USA 83 (1986) found that, when the effects ofthese trans carbon atoms were It is now possible to present a unified picture of the stable dissected out, lowering the CS-SC dihedral angle below about conformations ofprimary aliphatic disulfides and the manner 50-60° lowered the S-S stretching frequency (9). This trend in which rotation about their C-S and S-S bonds influences was supported by CNDO/2 calculations (15). the S-S stretching frequency. Unstrained disulfides adopt All primary disulfides with alkyl groups larger than a conformations having CS-SC dihedral angles of ± 85 ± 200 methyl group exhibit S-S stretching bands at approximately which, in the present terminology, are called B conforma- 510 and 525 cm-1, respectively. This multiplicity of S-S tions. A survey of the frequency of occurrence of CC-SS stretching bands was attributed to the coexistence of rota- dihedral angles in unstrained primary aliphatic disulfides of tional isomers about the C-S bonds ofthese compounds (3-6) known structure (for data, see refs. 7 and 17) reveals that and is consistent with the observation that only a single band almost all are in the B region with a few in the T region. When persists on freezing and annealing (4). Extrapolating from the a similar survey is taken of the CC-SS dihedral angles of results discussed above, Sugeta et al. assumed that the value cystine residues in proteins, it is found that most occurrences of the S-S stretching frequency for primary, secondary, and are in the B region, some are in the T region, and a few are tertiary disulfides was determined solely by the number of in the B' region (17). It is also seen that the distribution about carbon atoms trans to the distal S atom. Thus, they proposed these conformations is broad with numerous occurrences of that the GG and the TG or GT conformations were respon- CC-SS dihedral angles below 600 and between 1200 and 1500. sible for the S-S stretching bands at 510 and 525 cm-', Collectively, these observations establish that the potential respectively. Put in other words, they assumed that the function for rotation about the C-S bond of primary aliphatic substitution of the trans H atom of each methyl group in disulfides differs appreciably from that of ethane and 1,2- dimethyl disulfide by a methyl group would have the same disubstituted . The rotamers about the C-S bonds are effect on the S-S stretching frequency as the rotation of the not true alkane-like G, G', and T rotamers but rather are the methyl groups of diethyl disulfide about its C-S bonds from B, B', and T conformations. These are apparently the three the G to the T positions. This correlation was drawn from rotamers detected in our earlier Raman work (6) and the analogy to the Raman data for tertiary disulfides rather than mixture ofthe three is consistent with the electron diffraction from structural data. data on methyl ethyl disulfide (16). While alkane-like rota- Our subsequent systematic study of the Raman spectra of mers (e.g., in butane) have the stabilities T > G > G', those primary unstrained disulfides of known structure and with a for the aliphatic disulfides have the order B > T > B'. Thus, variety of CC-SS dihedral angles established that CCSSCC the B rotamer has the lowest energy, in spite of the fact that moieties with GG or G'G' conformations exhibited S-S it has a closer CHI..S contact than the T rotamer. This is stretching bands near 510 cm-' (7), in agreement with the consistent with the abundance of short CHIMES contacts in proposal of Sugeta et al. It was at that time that the crystals crystals ofother (12) and our earlier of dithioglycolic acid thought to have the TT conformation suggestion that weak attractive interactions might exist were examined and also found to have a S-S stretching band between these groups. The potential function for rotation near 510 cm-. This unexpected result prompted us to search about the C-S bond is apparently rather soft, even for low for the "missing rotamer" responsible for the S-S bands near values of the CC-SS dihedral angle. This accounts for the 525 and 540 cm-'. numerous A rotamers found for cystine residues in proteins; A study of the temperature dependence of the low- i.e., the potential function is sufficiently broad to extend into frequency Raman spectrum ofmethyl ethyl disuffide revealed the A region from its peak in the B region. Normally the short the presence of at least three rotational isomers about the C-S CHI..S contacts in these rotamers would have been expected bond (6). An electron diffraction study of the structure of to render them highly unstable. methyl ethyl disulfide was not able to specify the conforma- On the basis ofthe preceding discussion, the conformation tions of all three rotamers uniquely (16). Therefore, the ofthe CCSSCC fragment ofunstrained primary disLilfides can potential function for rotation about the C-S bonds of model be specified by three letters indicating the conformation disulfides was subsequently calculated using the CNDO/2 about the C-S, S-S, and S-C bonds, respectively. The method and evidence for the existence ofa rotamer with a low possible conformations in order ofstability are BBS > (BBT, CC-SS dihedral angle (0-30°) was found (11, 12). This TBB) > TBT > (BBB', B'BB) > (TBB', B'BT) > B'BB'. rotamer, referred to as the A conformation, was predicted to From the preceding discussion, it can be concluded that these have a close contact between the CH group and distal S atom. conformations have S-S stretching frequencies of approxi- On examination of the crystal structures of a number of mately 510, 525, 540, 510, 525, and 510 cm-1, respectively. organosulfur compounds, a large number of close contacts By inference, it is expected that conformations with CC-SS between CH groups and S atoms was discovered (12), lending dihedral angles between the B and T rotamers would have support to the notion that a rotamer with a low CC-SS intermediate frequencies. dihedral angle could be stabilized by a weak attractive Under certain circumstances, such as in cyclic disulfides, interaction between these atoms. Finally, a plot of the the S-S bond can be strained to adopt CS-SC dihedral angles frequency of occurrence of the CC-SS dihedral angles ob- away from the B conformation. This strain lengthens the S-S served for cystine residues in eight proteins was constructed bond and lowers the S-S stretching frequency (8-10, 15). In and about 20% of all occurrences were found in the region of the 0-60° (absolute value) range of CS-SC dihedral angles, the A conformation (7, 17). the S-S stretching frequency varies approximately linearly Based on these findings, it was suggested that adoption of from about 482 to 508 cm-' (9, 10). Thus, the C, A, G, and an A conformation about each C-S bond raised the S-S B conformations about the S-S bond of primary strained stretching frequency by 15 cm-1. An analysis of the S-S disulfides have S-S stretching frequencies of approximately stretching regions in the available Raman spectra of proteins 482, 495, 508, and 508 cm1, respectively. A decrease in this of known structure (7, 17) showed that this correlation fit the frequency is also expected when the dihedral angle is in- data as well as the correlation of Sugeta et al. (3, 4). The data creased to the 120-180° region, but no experimental data on presented here, however, show that our major motivation for such compounds have yet been obtained. Secondary and developing our correlation was incorrect. The new finding of tertiary strained disulfides will have S-S stretching frequen- Nash et al. (18) that the TT conformation of dithioglycolic cies elevated from those predicted from this correlation by acid does indeed exhibit the S-S stretching frequency near about 12-15 cm-1 for each carbon atom trans to the distal S 540 cm-', which is reproduced here (Fig. 1, spectrum A), atom across the C-S bond (3, 4, 7). These correlations can indicates that the correlation of Sugeta et al. is correct. now be used to provide reliable information about the Downloaded by guest on September 27, 2021 Chemistry: Van Wart and Scheraga Proc. Nati. Acad. Sci. USA 83 (1986) 3067

conformations of primary, secondary, and tertiary disulfides 8. Van Wart, H. E., Lewis, A., Scheraga, H. A. & Saeva, F. D. from their Raman spectra. (1973) Proc. Natl. Acad. Sci. USA 70, 2619-2623. 9. Van Wart, H. E. & Scheraga, H. A. (1976) J. Phys. Chem. 80, We are indebted to Dr. R. Parthasarathy for sending us the sample of 1823-1832. dithioglycolic acid used in the present investigation and to Dr. C. P. 10. Van Wart, H. E., Scheraga, H. A. & Martin, R. B. (1976) J. Nash for the information that dithioglycolic acid crystallizes in different Phys. Chem. 80, 1832. conformations and has a S-S stretching frequency forthe TT form of536 11. Van Wart, H. E., Shipman, L. L. & Scheraga, H. A. (1975) J. cm-1. This work was supported by National Institutes of Health Grant Phys. Chem. 79, 1428-1435. GM-27276 and Research Career Development Award AM-01066 to 12. Van Wart, H. E., Shipman, L. L. & Scheraga, H. A. (1975) J. H.E.V.W. and by National Institutes of Health Grant GM-24893 and Phys. Chem. 79, 1436-1447. National Science Foundation Grant DMB84-01811 to H.A.S. 13. Savitzsky, A. & Golay, M. J. E. (1964) Anal. Chem. 36, 1. Lord, R. C. & Yu, N. (1970) J. Mol. Biol. 50, 509-524. 1627-1639. 2. Lord, R. C. & Yu, N. (1970) J. Mol. Biol. 51, 203-213. 14. Sugeta, H. (1975) Spectrochim. Acta Part A 31, 1729-1737. 3. Sugeta, H., Go, A. & Miyazawa, T. (1972) Chem. Lett., 83-86. 15. Van Wart, H. E., Shipman, L. L. & Scheraga, H. A. (1974) J. 4. Sugeta, H., Go, A. & Miyazawa, T. (1973) Bull. Chem. Soc. Phys. Chem. 78, 1848-1853. Jpn. 46, 3407-3411. 16. Yokozeki, A. & Bauer, S. H. (1976) J. Phys. Chem. 80, 5. Scott, D. W. & El-Sabban, M. Z. (1969) J. Mol. Spectrosc. 31, 618-625. 362-367. 17. Van Wart, H. E. & Scheraga, H. A. (1977) Proc. Natl. Acad. 6. Van Wart, H. E., Cardinaux, F. & Scheraga, H. A. (1976) J. Sci. USA 74, 13-17. Phys. Chem. 80, 625-630. 18. Nash, C. P., Olmstead, M. M., Weiss-Lopez, B., Musker, 7. Van Wart, H. E. & Scheraga, H. A. (1976) J. Phys. Chem. 80, W. K., Ramasubbu, M. & Parthasarathy, R. (1985) J. Am. 1812-1823. Chem. Soc. 107, 7194-7195. Downloaded by guest on September 27, 2021