Proton Magnetic Resonance Study of Peptide ... - Europe PMC

Proc. Nat. Accad. Sci. USA Vol. 70, No. 4, pp. 1199-1203, April 1973

Proton Magnetic Resonance Study of Peptide Conformation: Effect of Trifluoroethanol on Oxytocin and 8-Lysine-Vasopressin (neurohypophyseal hormones/solvent effects/hydrogen bonding/three-dimensional structure/f-turn)

RODERICH WALTER AND J. D. GLICKSON* Department of Physiology, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029; Medical Research Center, Brookhaven National Laboratory, Upton, New York, 11973; and *Laboratory of Molecular Biophysics, University of Alabama Medical Center, Birmingham, Ala. 35294

Communicated by Maurice Goidhaber, February 7, 1973 The usefulness of 2,2,2-trifluoroethanol ABSTRACT titration as a means of distinguishing between intramolecular peptide-peptide hydrogen bonding on the one hand and intermolecular peptide-peptide and peptidesolvent hydrogen bonding on the other has been investigated with neurohypophyseal hormones, and the results have been compared with those of other methods. The chemical shifts (220 MHz) of the resonances of amide NH and aromatic CH protons of oxytocin, lysine vasopressin, deamino-lysine vasopressin, and deamino-8-tosyllysine vasopressin were monitored as the solvent composition was progressively varied from 100% dimethylsulfoxide to 100% 2,2,2-trifluoroethanol. The overall backbone conformation of oxytocin appears to be retained, and possibly somewhat stabilized, during the solvent transition, while the backbone, particularly the acyclic component, of lysine vasopressin and its analogs is subject to solvent-induced perturbation.

Conformational analysis of peptides in solution by proton magnetic resonance (PMR) spectroscopy is aided by the unique capacity of this technique to define the hydrogenbonding states of individual exchangeable protons. For reasons of solubility and simulation of biological media, solvents generally used are hydrogen-bond donors and/or acceptors. Consequently, conformational studies by PMR deal in part with the subtle problem of distinguishing between intrapeptide hydrogen bonding on the one hand and interpeptide and peptide-solvent hydrogen bonding on the other. The most common criteria for distinguishing between these types of hydrogen bonds are rates of exchange of labile protons with the solvent [AH+BY=±AY+BH; Y=D (1), H (2), or T (3)] as well as the temperature dependence of chemical shifts of labile proton resonances (4, 5). A rapid rate of proton exchange and a large upfield shift with increasing temperature have been interpreted as indicative of solute-solute or solutesolvent hydrogen bonding. Recently, it has been suggested that the effect of 2,2,2trifluoroethanol (FE) on the position of peptide amide Abbreviations follow the rules of the IUPAC-IUB Commission on Biochemical Nomenclature in Biochem. J. 126, 773-780 (1972). All optically active amino acids are of the L configuration. Me2SO, dimethylsulfoxide; MeOH, methanol; FXE, 2,2,2-trifluoroethanol; [Lys8Jvasopressin, lysine vasopressin; [flSPpl, Lys8]vasopressin, deamino-lysine vasopressin; TosLys8]vasopressin, deamino3--tosyllysine vasopressin; PMR, proton magnetic resonance.

[,6SP1,,

1199

resonances may distinguish between an internal and external hydrogen-bonded state of these protons (6, 7). For gramicidin S, for which a preferred conformation has been proposed (5, 8-10), resonances of peptide (N-H) hydrogens thought to be internally hydrogen bonded in MeOH shifted moderately to lowfield, whereas resonances of peptide hydrogens exposed to MeOH experienced a dramatic shift to highfield as the mole fraction of F3E increased (6). Qualitatively similar results were observed with gramicidin S in the solvent

transition dimethylsulfoxide (Me2SO)/FXE (6). Furthermore, Kopple (11) has reported a correlation, though limited, between the effects of fluorinated alcohols on NH chemical shifts and other criteria used to distinguish internal and solvent-exposed peptide protons. Ideally, an intramolecular amide hydrogen bond would result in a slow rate of proton exchange as well as a temperatureindependent and solvent-independent NH resonance. However, a lack of complete agreement between these three criteria may be anticipated since they are associated with different phenomena. Thus the rate of hydrogen replacement, a criterion sensitive to kinetic parameters, reflects in the exchange process the rate-limiting step, which may be either the unfolding of the peptide or the actual displacement of the exposed peptide hydrogen (2). Moreover, general acidbase catalysis of the protolysis reaction may complicate interpretation of exchange kinetics (12). In contrast to isotopic exchange, temperature and solvent dependence of chemical shifts are sensitive to equilibrium properties of the peptide. The temperature dependence can reflect lability of hydrogen bonds or transitions between various vibrational states of selective parts of the molecule. Solvent perturbations of amide resonances result from changes of the solvation sphere of a peptide amide proton, as well as from the dependence of the molecular conformation on solvent composition. Delineation of the capabilities and limitations of the various methods for probing the preferred solution conformation of peptides is the driving force of much of present-day research in molecular biology. Towards this end, we have studied the effect of F3E titration on spectra of the neurohypophyseal hormones, oxytocin (Fig. 1) and 8-lysine vasopressin ([Lys8Ivasopressin), as well as on two analogs, deamino-lysine vasopressin ([flSPp1,Lys8jvasopressin) and deamino-8-tosyllysine vasopressin ([3SPp',TosLys8]vasopressin). The effects were compared with the results of previous studies of their ex-

1200

Chemistry: Walter and Glickson

Proc. Nat. Acad. Sci. USA 70

(1973)

protons involved in peptide-solvent hydrogen bonds or unstable intrapeptide hydrogen bonds in Me2SO should respond to addition of FE with a large shift upfield. MATERIALS AND METHODS

Oxytocin, [Lys8]vasopressin, [SPp1,Lys8]vasopressin, and [jtSPPl,TosLys8]vasopressin were from the same batch used previously (19, 20, 26). Specifications of the Varian HR-220 NMR spectrometer have also been described (19). F3E (Matheson, Coleman and Bell, Norwood, Ohio) was fractionally distilled and stored under nitrogen before use; Me2SO (Burdick and Jackson Lab., Muskegon, Mich.) was used without purification. The mole fractions of these nondeuterated solvents were determined from the relative integrals of the CH2 resonance of FXE and the CH3 of Me2SO. Tetramethylsilane was used as an internal chemical shift standard. FIG. 1. Preferred conformation of oxytocin in Me2SO as proposed by Urry and Walter (18).

RESULTS AND DISCUSSION

change properties, temperature dependencies of their amide proton resonances, and other conformational features we have previously described (13-21). Independent PMR work on [Lys8]vasopressin is also being done in other laboratories (22-25). The fact that these hormones are hybrids of relatively conformationally restricted cyclic peptides and more flexible linear oligopeptides, renders neurohypophyseal hormones and their analogs a logical choice for further evaluation of F3E titration as a method for differentiating between intramolecular and intermolecular hydrogen bonds. Resonances of protons involved in stable intrapeptide hydrogen bonds in Me2SO should show either no spectral change or a small downfield shift; the latter may be caused by a further stabilization of the intrapeptide hydrogen bond or by vibrational transitions. Resonances originating from

In the present study, the FE titration was done with peptides being initially dissolved in pure Me2SO rather than MeOH used in other studies (6, 7, 11). This procedure was desirable since PMR assignments of oxytocin (13), [Lys8]vasopressin (16, 19, 23), and its analogs (20) had been made in Me2SO and since neurohypophyseal hormones polymerize in MeOH (unpublished). Changes in chemical shifts of the PMR resonances of the amide NH and aromatic CH protons of these hormones were followed as Me2SO was progressively enriched with FXE (Fig. 2). Results of identical studies on two analogs of the antidiuretic hormone, [,6SPp',Lys8]vasopressin and [3SPp',TosLys8]vasopressin (20) appear in Fig. 3. In tracking the course of specific resonances, we were aided by distinguishing characteristics such as the unique triplet structure of the Gly peptide NH resonance, coupling constants of certain peptide "doublets," and slopes of the titra-

0 10 20 30 40 50 60 70 80 90 100 MOLE FRACTION F3E (%)

FIG. 2. Chemical shifts of lowfield proton resonances of oxytocin and lysine vasopressin ([Lys8]vasopressin) as a function of solvent transition from pure Me2SO to pure F3E. Peptide amide proton resonances are identified by NH ( ); those of primary carboxamide protons by CONH2 ( ); and those of aromatic protons by CH (--- -). The concentration of each hormone was maintained at 3% w/v throughout the titration, and the spectra were recorded at 230.


(1978)

Oxytocin and [Lys]Vasopressin in Trifluoroethanol

tion curves (Figs. 2 and 3). Several observations can be made on the basis of these complex plots. The Asn peptide NH resonance of each of these compounds shifts gradually to lowfield as the F3E concentration increases, which suggests that in Me2SO solution the peptide NH of Asn is involved in a stable intramolecular hydrogen bond that is retained in F3E. This finding is consistent with the slow hydrogen-deuterium exchange rate of the Asn peptide proton in neurohypophyseal peptides (15, 20) and the small effect of temperature on the chemical shift of its resonance in Me2SO (19, 20, 23, 27), both of which favor the assignment of a (-turn with a transannular hydrogen bond between the peptide NH of Asn and the CO of Tyr in oxytocin (27), [Lys8]vasopressin (16, 19, 23), and analogs (20). Concomitant with these resonance shifts one observes a significant increase in the aCH-NH coupling constant of Asn. Whereas in Me2SO this parameter exhibits values of 6.1 i 0.2, 7 i 1, and 6.3 i 0.2 Hz for oxytocin, [Lys8]vasopressin, and [#SP,1,Lys8Jvasopressin (19, 20), respectively, the corresponding coupling constants in F3E are 9.0 i 0.3, 9.0 i 0.5, and 8.5 i 1. The Asn peptide resonance of [flSPP , TosLys8]vasopressin was too broad for accurate estimation of its splitting. The increase of the Asn peptide NH coupling constant suggests enlargement of the dihedral angle between the aCH and the peptide NH hydrogens of Asn. Hence it appears that the (3-turn proposed for the cyclic moiety of oxytocin, [Lys8jvasopressin, and its analogs in Me2SO is stabilized in F3E solution. A broad Tyr peptide resonance has been observed in spectra of all the neurohypophyseal hormones, but not of their deamino derivatives (13, 19, 20, 23, 27). This phenomenon has been attributed to an enhancement of the exchange rate of the Tyr NH by the a-amino groups of the vicinal Cys residue in the hormones (13, 25). Addition of F3E to a solution of oxytocin in Me2SO further broadens the Tyr NH resonance until

it can no longer be observed at concentrations between 10 and 80 mol% F3E. At F3E concentrations higher than 80 mol%, the Tyr NH peak reappears somewhat downfield from its position in Me2SO (Fig. 2), which could reflect an increased tendency of the hydrogen bond between the NH of Tyr and peptide CO of Asn to be. present in F3E. The presence of such a hydrogen bond was suggested for deamino-oxytocin, when dissolved in a mixture of Me2SO-MeOH (27). The Tyr NH resonance of [Lys8]vasopressin, as compared to those of its two deamino analogs, is also broad. In the spectra of [Lys8]vasopressin and [(3SP ILys8]vasopressin, this peak was followed throughout the solvent transition and found to move distinctly to high field upon addition of F3E. The different behavior of the Tyr NH resonances of oxytocin and [Lys8]vasopressin during F3E titration is compatible with the idea of a somewhat different conformation of the two hormones in Me2SO or with a greater susceptibility to F3E-induced conformational perturbations of [Lys8]vasopressin. The slow hydrogen-deuterium exchange of the peptide NH of Gly in Me2SO (15) was part of the argument that led to a fl-turn assignment of the -Cys-Pro-Leu-Gly- sequence in oxytocin, with a hydrogen bond between the peptide NH of Gly and the CO of Cys-6 (18). Slow replacement of the proton of the Leu NH by D20 suggested that this moiety stabilizes the ,B-turn by forming a hydrogen bond with the side-chain carbonyl oxygen of Asn. The small highfield shift observed during F3E titration of the peptide NH resonance of Gly in oxytocin is consistent with retention of this proton in a stable hydrogen bridge, but the marked shift upfield of the Leu NH resonance during the titration suggests that this proton was involved in an unstable hydrogen bond. With [(3SP ITosLys8]vasopressin, the only [Lys8]vasopressin analog yielding measurable deuterium exchange kinetics (19), a slow replacement of the peptide NH of Gly = 9.5

I )

[,BSPp', Lys8JVASOPRESSIN

Tyr (OH)

Tyr (OH)

_

200( -

N

(n

1800 I

4

1700

I

~

Tos (CH)

Asn (CONH2)truOs I

_

rn.

\0 =;

& 0

10 20 30 40 50 60 70 80 90 100 MOLE FRACTION F3E (%)

\

I-

8.0 (n

Gly(NH) *

GlyGln

-.Tyr (CH) mnetu-S..so---O. -*-o-*--- --Asn(CONH2) cis -Gin (CONH2) Cis

_ 8.5 PK

I

Asn (NH

Asn(CONH2)truns _ .a..-* @ .-TGly Gin (CONH2) Iruns = \N...a --° _ _f Phe(C6H5) 7$°-

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1400

A

^ .+ Phe(NH) Lys (NH)_-

~~~~r,Gln

Gly(CONH)trons1500

Lys(6-NH) Tos

Cys (NH)

Cys(NH) ( NH) Lys(NH) Phe ( NH)i\_ Gin (NH) A Tyr (NH) i Gly (NH) Asn (NH) he ,-_ o T Lys (NH3+ ~~_ \S

w u 1600

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I

1201

...

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-8 -

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-£

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Gly(CONH2) cS

Tyr(CH)metoa Asn(CONH2) cis Gl (CONH2)czs Ci

*-*---

Tyr(CH)ortho

g:S3

7.0 =

6.5 -

0 10 20 30 40 50 60 70 80 90 100 MOLE FRACTION F3E (%)

FIG. 3. Chemical shifts of lowfield proton resonances of deamino-lysine vasopressin ([,6SP,',Lys8]vasopressin) and deamino-8-tosyllysine vasopressin ([USPp1,TosLys8]vasopressin) as a function of solvent transition from pure Me2SO to pure F3E. Symbols and experimental conditions are those described under Fig. 2.

1202

Chemistry: Walter and Glickson MOLE FRACTION MeSO (%) 60 40 20 0

100 80

875 II

CH, of F _ -1350

850

0

-I

\OH Ol F

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575

550 ,

0

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_1200,

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0

85H2

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t

OS O CH,

20

40

60

._150 80 100

MOLE FRACTION TRIFLUOROETHANOL (%

FIG. 4. Effect of progressive variation of the solvent from

pure Me2SO to FXE on the chemical shifts of the methyl proton resonance of Me2SO (scale on lower left side), the methylene proton resonance of F3E (scale on upper left), and the hydroxyl proton resonance of F3E (scale on right). The spectra, recorded at 23°, were identical in the presence or absence of neurohypophyseal peptides.

was observed in 10% D20/Me2SO, which suggests that at least in this [Lys8"vasopressin derivative the Gly NH proton is intramolecularly hydrogen bonded. In contrast to oxytocin, the significant shift upfield observed for the peptide NH of Gly with [Lys8]vasopressin and its two analogs during F3E titration argues for the weakness of this hydrogen bond in

Me2SO. In addition, changes in the preferred conformation of the

acyclic peptide portion of [Lys8jvasopressin in going from Me2SO to F3E may also be indicated by spectral changes associated with the peptide NH of the Lys residue. This resonance responds to changes in F3E concentrations in a complex manner, shifting first to lowfield and then to highfield, which indicates that rapid exchange between at least three different states is occurring. That these chemical shift changes reflect, at least in part, a change in the local conformation of the acyclic backbone is suggested by the concomitant diminution of the Lys aCH-NH coupling constant from a value of 7 :1: 1 Hz, 5.4 i 0.3 Hz, and 7 + 1 Hz for [Lys8jvasopressin, [#SPp1,Lys8jvasopressin, and [#SP 1,Tos-

Lys8]vasopressin, respectively, in Me2SO solution to less than [Lys8jvasopressin and [jBSPp1,Lys8Jvasopressin in F3E

2 Hz for

solution. The Lys peptide resonance of the second vasopressin analog was not resolved well enough for estimation of its coupling constant. Comparison of the spectral changes of the acyclic portions of the peptides suggests that the tail of oxytocin to a large degree retains its orientation during the solvent transition, while that of [Lys8]vasopressin and its analogs undergoes detectable conformational alterations. Distinct perturbations of aromatic CH absorptions accompany the solvent transition. Resonances of hydrogens ortho and meta to the hydroxyl of Tyr in oxytocin and [Lys8Jvasopressin shift to highfield as the solvent is enriched with F3E. As observed during the water titration of [Lys8jvasopressin (26), the Phe resonance, which consists of a closely-spaced multiplet in Me2SO, separates into two distinct multiplets when the mole fraction of the other solvent component exceeds 50%. While the Phe aromatic CH peaks of the two analogs of [Lys8jvasopressin behave in a manner similar to the parent compound, the Tyr CH absorptions of these derivatives behave differently-the ortho CH moves downfield while the meta CH moves upfield. These changes in the magnetic environment of aromatic CH hydrogens could originate from


(1973)

changes in the orientation of aromatic side-chains and/or from shielding contributions of the solvent, which includes bulk magnetic susceptibility, van der Waal interaction, diamagnetic anisotropy, electric polarization, and polarizability of the solvent (28). The observed changes are hardly surprising in light of the perturbations of the solvent resonances themselves (Fig. 4). The significant alteration in solvent structure suggested by these data could provide the free energy for conformational transitions in solute molecules. A more compatible solvent pair of Lewis acid/Lewis base than FXE/Me2SO could conceivably lessen the difficulties originating from solvent-induced conformational perturbations. Alternately, a reagent capable of producing significant and selective perturbations of resonances originating from solvent-exposed peptide hydrogens at concentrations too low to bring about conformational changes may be better suited for studying the hydrogenbonding state of protons. Along this line, Kopple and Schamper (29) recently reported the differential line broadening of solvent-exposed NH protons by a stable free radical. Development of additional criteria for the delineation of different types of amide hydrogen bonds is essential to PMR studies of peptide conformation. In conclusion, comparison of results from the limited number of studies of peptide conformation by the F;E titration method suggests that this technique is most promising in distinguishing between solvent-shielded and solvent-exposed peptide hydrogens in relatively rigid cyclic peptides (6). The present study of neurohypophyseal hormones, as well as that of stendomycin (7), reveals that the method of solvent titration may be applied to peptides with greater potential for conformational rearrangements-but the results are rather complex due to the conformational changes that may accompany the solvent transitions. The spectral changes occurring upon addition of F3E to solutions of the neurohypophyseal hormones in Me2SO can be interpreted as follows: the backbone of the cyclic moiety of all molecules studied exists in an essentially stable a-turn conformation maintained by a hydrogen bond between the peptide proton of Asn and carbonyl oxygen of Tyr. Some stabilization of this structure appears to accompany the transition to F3E, and there is an indication that the formation of a hydrogen bond between the peptide proton of Tyr and the peptide carbonyl of Asn may further stabilize the ,-turn of the oxytocin ring. The backbone orientations of the acyclic moieties of the hormones are much more sensitive to addition of F3E, but evidence for retention of the hydrogen bond between the Gly peptide NH and a Cys-6 CO indicates a more stable structure for oxytocin than for vasopressin. A considerable degree of side-chain flexibility is indicated by perturbations of specific resonances of all the hormones, including their aromatic CH absorptions. We thank Dr. D. W. Urry for the use of his laboratory facilities, including the 220-MHz spectrometer. We are also indebted for

technical assistance to Mr. W. Cunningham and Ms. H. Shlank and for the thoughtful editorial help of Ms. P. L. Hoffman. We thank Dr. K. D. Kopple for helpful discussion. This work was supported by USPHS Grants AM-13567 and NSF Grant GB31665, the Mental Health Board of the State of Alabama, and the U.S. Atomic Energy Commission. 1. Berger, A., Loewenstein, A. & Meiboom, S. (1959) J. Amer. Chem. Soc. 81, 62-67. 2. Hvidt, A. & Nielsen, S. 0. (1966) Advan. Protein Chem. 21,

287-386.


(1978)

3. Englander, S. W. (1963) Biochemistry 2, 798-807. 4. Kopple, K. D., Ohnishi, M. & Go, A. (1969) J. Amer. Chem. Soc. 91, 4264-4272. 5. Ohnishi, M. & Urry, D. W. (1969) Biochem. Biophys. Res. Commun. 36, 194-202. 6. Pitner, T. P. & Urry, D. W. (1972) J. Amer. Chem. Soc. 94, 1399-1400. 7. Pitner, T. P. & Urry, D. W. (1972) Biochemistry 11, 41324137. 8. Hodgkin, D. C. & Oughton, B. M. (1957) Biochem. J. 65, 752-756. 9. Schwyzer, R. (1958) in Amino Acids and Peptides with Antimetabolic Activity, ed. Wolstenholme, G. E. W. (J. & A. Churchill, Ltd., London), pp. 171-184. 10. Stern, A., Gibbons, W. A. & Craig, L. C. (1968) Proc. Nat. Acad. Sci. USA 61, 734-741. 11. Kopple, K. D. & Schamper, T. J. (1972) in "Chemistry and biology of peptides," Proc. of the Third Amer. Peptide Symp., ed. Meienhofer, J. (Ann Arbor Sci. Publ., Ann Arbor, Mich.), p. 75. 12. Klotz, I. M. & Frank, B. H. (1965), J. Amer. Chem. Soc. 87, 2721-2728. 13. Johnson, L. F., Schwartz, I. L. & Walter, R. (1969) Proc. Nat. Acad. Sci. USA 64, 1269-1275. 14. Urry, D. W., Quadrifoglio, F., Walter, R. & Schwartz, I. L. (1968) Proc. Nat. Acad. Sci. USA 60, 967-974. 15. Walter, R., Havran, R. T., Schwartz, I. L. & Johnson, L. F. (1971) in Proc. Tenth Eur. Peptide Symp., 1969, ed. Scoffone, E. (North-Holland Publ. Co., Amsterdam), pp. 255-265. 16. Walter, R. (1971) in Structure-Activity Relationships of Protein and Polypeptide Hormones, eds. Margoulies, M. & Greenwood, F. C. (Excerpta Medica, Amsterdam), part 1, pp. 181-193.

Oxytocin and [LysiVasopressin in Trifluoroethanol

1203

17. Walter, R., Gordon, W., Schwartz, I. L., Quadrifoglio, F. & Urry, D. W. (1968) in Proc. Ninth Eur. Peptide Symp., ed. Bricas, E. (North-Holland Publ. Co., Amsterdam), pp. 50-55. 18. Urry, D. W. & Walter, R. (1971) Proc. Nat. Acad. Sci. USA 68, 956-958. 19. Walter, R., Glickson, J. D., Schwartz, I. L., Havran, R. T., Meienhofer, J. &. Urry, D. W. (1972) Proc. Nat. Acad. Sci. USA 69, 1920-1924. 20. Glickson, J. D., Urry, D. W., Havran, R. T. & Walter, R. (1972) Proc. Nat. Acad. Sci. USA 69, 2136-2140. 21. Kotelchuck, D., Scheraga, H. A. & Walter, R. (1972) Proc. Nat. Acad. Sci. USA 69, 3629-3633. 22. Deslauriers, R. & Smith, I. C. P. (1970) Biochem. Biophys. Res. Commun. 40, 179-185. 23. Von Dreele, P. H., Brewster, A. I., Scheraga, H. A., Ferger, M. F.-& du Vigneaud, V. (1971) Proc. Nat. Acad. Sci. USA 68, 1028-1031. 24. Von Dreele, P. H., Brewster, A. I., Bovey, F. A., Scheraga, H. A., Ferger, M. F. & du Vigneaud, V. (1971) Proc. Nat. Acad. Sci. USA 68, 3088-3091. 25. Von Dreele, P. H., Brewster, A. I., Dadok, J., Scheraga, H. A., Bovey, F. A., Ferger, M. F. & du Vigneaud, V. (1972) Proc. Nat. Acad. Sci. USA 69, 2169-2173. 26. Glickson, J. D., Urry, D. W. & Walter, R. (1972) Proc. Nat. Acad. Sci. USA 69, 2566-2569. 27. Urry, D. W., Ohnishi, M. & Walter, R. (1970) Proc. Nat. Acad. Sci. USA 66, 111-116. 28. Jackman, L. M. & Sternhell, S. (1969) Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry (Pergamon Press, Braunschweig), 2nd ed., p. 104. 29. Kopple, K. D. & Schamper, T. J. (1972) J. Amer. Chem. Soc. 94, 3644-3646.