Proc. Nat. Acad. Sci. USAVol. 69, No. 7, pp. 1825-1829, July 1972

Conformational Interconversions of the Cyclic Hexapeptide Cyclo(Pro-Gly)3* t(peptides/NMR/alkali metal complexes)

CHARLES M. DEBER, DENNIS A. TORCHIAT, SIMON C. K. WONG, AND ELKAN R. BLOUT

Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115; and tBell Laboratories, Murray Hill,New Jersey 07974

Contributed by Elkan R. Blout, April 24, 1972

ABSTRACT A cyclic hexapeptide, cyclo(Pro-Gly-Pro-Gly-Pro-Gly), hasbeen synthesized; its solution conforma-tions were examined by 220-MHz nuclear magnetic reso-nance spectroscopy. The solution structures have beendeduced, and shown to vary as a function of solvent polar-ity. In addition, it has been found that this cyclic peptidebinds alkali metal cations. While the predominant con-formation of this cyclic peptide is 3-fold symmetric in theapolar solvent methylene chloride, an asymmetric struc-ture is preferred in some polar solvents (water, dimethyl-sulfoxide). However, addition of alkali metal salts, such assodium thiocyanate, to dimethylsulfoxide solutions ofthe peptide shifts the conformational equilibrium infavor of a second type of C3-symmetric structure, presum-ably the result of the formation of a stable peptide-metalion complex. Nuclear magnetic resonance data suggestthat the peptide in methylene chloride solution takes up aconformation containing three cis' ProCaC=O bonds andthree cis Gly-Pro peptide bonds; that water and dimethyl-sulfoxide stabilize a conformer in which one (or two)sets of such bonds of a given Pro-Gly unit have undergoneinterconversion to trans'/trans forms; and that alkalimetal cations complex the cyclic peptide in a Cs-symmetricall-trans'/trans structure.

The biological activity and structural simplicity of manycyclic peptides have generated considerable interest in thedetermination of their conformations, with the ultimate goalof ascertaining structure-function relationships. High-resolution nuclear magnetic resonance (NMR) spectroscopy,useful for the study of solution conformations of cyclic pep-tides, can provide information for structural determinations(1,2).Many naturally-occurring cyclic peptides contain proline,

and it is believed that investigations of synthetic proline-containing cyclic peptides composed of repeating oligopeptideunits (3-5) may clarify the structural role of the prolylresidue in biologically active cyclic peptides. An additionaladvantage of the investigation of proline peptides is that thenumber of possible conformers of the peptide backbone islimited by the restrictions to rotation about the N-CCa bondof the prolyl residues. Another consideration is the recentdemonstration of the presence of cis- and trane-X-Pro peptidebonds (where X is any amino-acid residue preceeding a prolineresidue), in both linear (6-8) and cyclic (4, 5) proline-contain-ing peptides. Since the barrier to rotation about the peptide

* Dedicated to our late colleague, Dr. Simon C. K. Wong, whowas a gentleman, a scholar, and a scientist.t This is the fourth paper in the series: "Cyclic Peptides." Thepreceding paper is ref. 5.$ Present address: Polymer Division, National Bureau of Stan-dards, Washington, D.C. 20234.

1825

bond is estimated to be at least 15-20 Cal (9), different cyclicpeptide conformations resulting from the presence of cis- andtrans-X-Pro peptide bonds yield separate NMR spectra, thuspermitting direct determination of the populations of theconformers present in solution under different conditions (e.g.,various solvents or temperatures).

In NMR analyses of cyclo(Pro-Ser-Gly-Pro-Ser-Gly) [c-(Pro-Ser-Gly)2] and its retro-isomer cyclo(Ser-Pro-Gly-Ser-Pro-Gly) [c-(Ser-Pro-Gly)2], it was shown (4, 5) that in bothcompounds C2-symmetric intramolecularly hydrogen-bondedconformations having all peptide bonds trans were in equilib-rium with conformations [both asymmetric (4) and symmetric(5) ] containing cis-X-Pro peptide bonds. The present reportdescribes the synthesis of, and NMR studies on, cyclo(L-Pro-Gly-L-Pro-Gly-L-Pro-Gly) [designated c-(Pro-Gly)3], inwhich the presence of three prolyl residues in alternating posi-tions excludes intramolecularly hydrogen-bonded C2-symmet-ric conformations similar to those found for c-(Pro-Ser-Gly)2and c-(Ser-Pro-Gly)2. In the apolar solvent [2H]methylenechloride (CD2CI2), a 3-fold symmetric conformation is deducedfor c-(Pro-Gly)3 in which Pro Ca C=O bonds are cis' andGly-Pro peptide bonds are cis. In the polar solvent [2H]-dimethylsulfoxide ([U-2H]Me2SO)§, a single asymmetric con-former of c-(Pro-Gly)3 is predominant; mixed ([U-2H]Me2SO-CD2Cl2 solvents are used to study the equilibrium between thetwo conformers. The preferred conformation of c-(Pro-Gly)3 inwater is also asymmetric. Lastly, we report that alkali metalcations complex c-(Pro-Gly)3 in [U-2H]Me2SO solution in asymmetric trans'/trans conformation.

RESULTS AND DISCUSSION

Conformation of Cyclo(Pro-Gly)3 in Methylene Chloride.When c-(Pro-Gly)3 (depicted schematically in Fig. 1) isdissolved in CD2Cl2, the 220-MHz NMR spectrum in Fig. 2ais obtained (NH portion not shown). The solubility of thiscyclic peptide in CD2Cl2 afforded an opportunity for the studyof its conformation(s) unperturbed by interaction with oxy-gen-containing solvents. Most of the assignments indicated forvarious resonances are readily made by inspection (3, 4), withprolyl ,B- and y-proton assignments confirmed by spin de-coupling. Noteworthy in Fig. 2a is the magnetic equivalence ofthe three Pro-Gly units, indicating overall Crsymmetry forthe predominant conformation in CD2C12. In attempts todeduce the specific nature of this Crsymmetric conformer, it isnecessary to consider first the most likely isomeric structures

§ [U-2H]Me2SO is also called dimethylsulfoxide-do by spectros-copists.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 2,

202

0

Proc. Nat. Acad. Sci. USA 69 (1972)

FIG. 1. Diagrammatic representation of cyclo(Pro-Gly)3. Pro(41', ipl, wi) and Gly (0)2 4)2, w2 ) residue rotation angles are in-dicated.

of c-(Pro-Gly),, namely, those arising from the cis (Gly =1800) and trans (Gly co = 00) ¶ rotational isomers of the(planar) Gly-Pro peptide bonds (6). Four distinct conformersof this kind are possible: cis-cis-cis, cis-cis-trans, cis-trans-trans, and trans-trans-trans. Furthermore, the rotationalpotential about the Pro Ca C=O bond is characterized(10) by two regions of low conformational energy, withminima at cis' (Pro 4 - 125°) and trans' (Pro 4 - 3000).If rotational isomers about the Pro Ca C=O bond areincludedll, the number of possible conformers increases to 16,of which only four have C3-symmetry: those with three cis'bonds and either three cis or three trans Gly-Pro peptidebonds; and those with three trans' bonds, and either three cisor three trans Gly-Pro peptide bonds. The NMR spectrumobserved in CD2Cl2 can be assigned with reasonable certaintyto a single C3-symmetric conformer on the basis of the follow-ing considerations.

First, the two relatively small and nearly equal JNa, (GlyH-N-C,-H2) constants (about 2.5 and 4.0 Hz) (Fig. 2a)require (11) that in each of the three Pro-Gly units, the di-

¶ For explanations of conventions used in dihedral angle nomen-clature, see Edsall, J. T., Flory, P. J., Kendrew, J. C., Liquori,A. M., Nemethy, G., Ramachandran, G. N. & Scheraga, H. A.(1966) J. Biol. Chem. 241, 1004. For convenience, this former"standard" convention is used herein.

11 While the barrier to interconversion between cis' and trans'forms of Pro Ca-C=O bonds is not believed to be high enoughat room temperature to give separate NMR spectra for eachform, considerations based upon molecular geometry of c-(Pro-Gly)3, and upon experimental data gathered from spectrathroughout this study, suggest that in any given c-(Pro-Gly)3conformer one form only (either cis' or trans') will be significantlypopulated once other conformational degrees of freedom havebeen specified. However, the possibility cannot be rigorouslyexcluded that the observed resonances result from rapid averagingof the conformations proposed with those containing mixedcis'/trans' Pro Ca-C==O bonds.

hedral angles formed by the Gly NH proton and the two GlyCaH's are both approximately 1200, or, in terms of peptidebackbone rotational angles, the three Gly 4 angles 00.

Second, the appearance of the Pro CaH as a doublet (at 5.25r) in c-(Pro-Gly)3 recalls a similar doublet (at 4.95 r in CD2-Cl2) in the NMR spectrum of cyclo(L-prolyl)3 (3). The con-formations of the pyrrolidine rings in both cyclic peptides may,therefore, be similar, and possibly related to a commonorientation of neighboring peptide bonds, i.e., cis in both in-stances [Pro-Pro in cyclo(L-prolyl)3, Gly-Proinc-(Pro-Gly),3].

Third, inspection of Corey-Pauling-Koltun (CPK) mo-lecular models indicates that (a) the sequences trans'-cis andcis'-trans in a given Pro-Gly unit have unfavorably closeatomic contacts, especially between neighboring carbonyloxygen atoms; (b) both in these "mixed" Pro-Gly units and intrans'/trans Pro-Gly units, the sum of the two Gly JNacoupling constants is 10-12 Hz, in disagreement with theexperimentally-observed sum of JNa values (about 6.5 Hz);and (c) three peptide carbonyl oxygens (the three Pro and/orthe three Gly carbonyl oxygens) in the all-trans'-all-transstructures are oriented toward each other and in close proxim-ity, resulting in strong repulsive electrostatic interactions inthe relatively nonpolar CD2Cl2 solvent.The data therefore indicate that cyclo(Pro-Gly), exists as a

Cr-symmetric conformer in CD2CI2, designated [S], that con-tains three cis' ProCa-C==O, and three cis Gly-Pro peptidebonds. This conformer is illustrated schematically in Fig. 1.Once these conformational features have been specified, pep-tide bond planarity, cyclic geometry, and steric requirementsbring Gly '/ angles to about 00. The backbone conformationof [S] can thus be described approximately by three sets ofPro (4), /', co) angles - (1200, 1250, 00), and three sets of Gly(4), /', w) angles - (0°, 00, 1800).

Effects of Polar Solvents on Conformation. In our studies ofcyclo(Pro-Gly)3 in methylene chloride, it quickly becameevident that small amounts of water strongly influenced thepopulations of c-(Pro-Gly)3 conformations, as well as thechemical shifts of Gly NH protons. Therefore, we examinedthe NH region of the NMR spectrum of a dry sample ofc-(Pro-Gly)3 in CD2Cl2, the water content of which was<0.01% as determined by the absence of an H20 resonance.

T

FIG. 2. Portion of the 220-MHz NMR spectra of cyclo(Pro-Gly)3 in (a) CD2C12, and (b) CD2Cl2 plus about 0.2% H20. Temp-erature = 23°C. Concentration: 20 mg/md (a), 15 mg/ml (b).Chemical shifts (r scale) given in ppm downfield from internalMe4Si.

1826 Chemistry: Deber et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 2,

202

0

Conformational Interconversions of Cyclo(Pro-Gly)3 1827

In the resulting spectrum (Fig. 3a), minor NH resonancesA1, A2, and A3 were visible at 1.9, 3.3, and 3.5 T, respectively,and their area was found (with a computer of average tran-sients) to contain 10-15% of the total NH area**. Theseresonances became more pronounced when approximately0.2% water (less than 1 mol percent) was added (Fig. 3b).Note that the upfield portion of the spectrum run in this samesolvent (Fig. 2b) also reveals the growth of minor resonances(visible between 5.4 and 5.8 T, but only barely evident in 2a).The presence of 0.2% water leads also to sharper resonances(compare 2b with 2a) and to a perceptible increase in thevalues of the Gly JNa (from about 2.5 and 4.0 Hz in dry CD2C12to about 3.0 and 4.5 Hz in 0.2% H20).A clear indication that the minor resonances correspond to an

asymmetric conformation of cyclo(Pro-Gly)3 stabilized bywater is seen in Fig. 3 a-c, which shows that the single NHresonance due to the three Gly NH's of predominant sym-metric conformation [S] in dry CD2Cl2 (3a) is replaced by aset of three NH resonances having equal areas. This set ofresonances is visible in both 3a and b, and is fully developed inpure water (0.5% acetic acid added to retard exchange)(Fig. 3c). Hence, in water a single asymmetric structure (hav-ing three magnetically nonequivalent Gly NH's) is thepredominant conformation.

Since the polar solvent dimethylsulfoxide, unlike water, ismiscible with CD2Cl2, it was possible to monitor the conforma-tional transitions just described over the entire range ofsolvent compositions. Thus, when aliquots of [U-2H]Me2SOare added to a solution of c-(Pro-Gly)3 in CD2Cl2 (Fig. 4),again three "minor" resonances of equal area are seen to growin size (Al, A2, A3) at the expense of S, until they are approxi-mately each the same size as S (4c), and finally predominate(4d)tt. The polar solvent [U-2H]Me2SO therefore stabilizespredominantly a single asymmetric conformation of c-(Pro-Gly)3 in which the three Gly NH protons are nonequivalentand appear as three separate multiplets of equal area, similarto those observed in water.A sample of c-(Pro-Gly)3, dried from [U-2H]Mle2SO and

redissolved in CD2CI2, had the symmetric [S] conformation,confirming that the asymmetric conformer is in dynamicequilibrium with the symmetric conformer [S]. It is sug-gested from symmetry considerations and CPK model studiesthat these conformational transformations involve rotations ofapproximately 1800 about both the Pro ;/ angle and the Gly w

angle in one (or two) Pro-Gly units, i.e., a "flip" of the entireH 0

1 1sequence of atoms -C-N-CH2-C- of a given Pro-Glyub

0unit. (Note that the Pro-Gly peptide bond remains trans.)

** In one experiment, a particularly dry CD2Cl2 solution was

obtained, as judged from (a) the upfield-shifted positions of theminor NH resonances (2.00, 3.35, and 3.55 r) and their relativelydecreased population (5-10% of the total NH area), and (b)the rather poorly resolved resonances in the upfield region of thespectrum, possibly due to a tendency of c-(Pro-Gly)3 to "aggre-gate."tt In pure [U-2H]Me2SO (4e), [S] still gives a small resonancenear 1.8 'r, combined with another conformation (the symmetrictrans'/trans conformer [S']?), which gives a small resonance atabout 1.9 r (better resolved in 4d).

1.0T

FIG. 3. Peptide N-H region of 220 MHz spectra of cyclo(Pro-Gly)3 in (a) CD2C12; (b) CD2C12 plus about 0.2% H20; and (c)H20 (containing 0.5% acetic acid, V/V). The resonance due to Alis obscured by S in (b). Temperature = 230C. Concentrations:20 mg/ml (a); 15 mg/ml (b); 7 mg/mI (c). The signal-to-noiseratio in (c) was improved by 64 scans in a computer of averagetransients. Chemical shifts in (a) and (b) (T scale) given in ppmdownfield from Me4Si; in (c), from internal t-butyl alcohol takenat 8.77 r.

While it is certain that the predominant conformer in [U-2H]-Me2SO is asymmetric, a clear choice is not possible betweenconformer [A] (one "unit flip"), and conformer [A'] (two"unit flips").

Interaction of Cyclo(Pro-Gly)s with Alkali Thiocyanate Salts.Evidence for specific interaction between sodium thiocyanate(NaSCN) and cyclo(Pro-Gly)3 is seen in Fig. 5, which showsthe changes in the NH resonances resulting from addition ofNaSCN to a solution of c-(Pro-Gly)3 in [U-2H]Me2SO. As thesalt concentration increases, the population of the asymmetricconformation decreases, with the parallel increase of thepopulation of a new symmetric conformation, signaled by thegrowth of the resonance designated S*. It is clear from Fig.5e that about 90% of the c-(Pro-Gly)3 molecules become sym-metric when the molar ratio NaSCN/c-(Pro-Gly)3 = 7. Theupfield portion of spectrum 5e, appearing in Fig. 6, should becompared with Fig. 2a, where prominent differences areapparent in the chemical shifts, shapes, and coupling constantsof most resonances, e.g., the Pro CaH resonance is now atriplet, and the Gly JNa are now increased.Because the conformation corresponding to S* resonances is

C3-symmetric, Gly-Pro peptide bonds must be all-cis or all-trans (see above). Since in the all-cis structure (i.e., conforma-tion [S]), the three Pro and three Gly carbonyl groups arerigidly fixed with C=0 axes approximately parallel and oxy-gen-oxygen distances - 4-5 iA, the C=0 groups are thus notwell-disposed to bind a sodium cation (with radius about 1 A).However, when the three Gly-Pro peptide bonds are trans andthe Pro Ca C=0 bonds are trans', structures can readily bemade with either the three Gly C=O's or the three Pro C=O'sdirected inward toward the center of the molecule in closeproximity (as previously noted) and well-positioned to bind asodium cation. The flexibility required to build these twotypes of trans'/trans structures is provided by the broadregion of low energy about the trans' minimum in the potentialwell of the isolated Pro residue (10). Conformations of c-(Pro-

Proc. Nat. Acad. Sci. USA 69 (1972)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 2,

202

0

Proc. Nat. Acad. Sci. USA 69 (1972)

FIG. 4. Peptide N-H regions of 220-MHz NMR spectra ofcyclo(Pro-Gly)3 (a) in CD2Cl2; (b), (c), and (d) in CD2C12-[U-2H]Me2SO mixtures; and (e) in [U-2H]Me2SO. In (a)-(e), themole fractions Mf of [U-2H]Me2SO are 0.0, 0.1, 0.25, 0.5, and 1.0,respectively. Temperature = 230C. Concentrations: 20 mg/ml(a); 12 mg/ml (b), (c), and (d); 15 mg/ml (e). In (b)-(e), thesignal-to-noise ratio was improved by 4-16 scans in a computer.Chemical shifts (T scale) given in ppm downfield from internalMe4Si.

Gly)3, having their sets of Gly or Pro carbonyl oxygens proxi-mal and best oriented to bind Na+, have Pro angles cor-

responding to the opposing limits of this broad well; when theGly carbonyls are proximal (at Pro 41 e 280°), the conforma-tion is designated [SG*], and when Pro carbonyls are proximal(at Pro 4

- 340°), the conformation is designated [Sp*]. Both[SG*] and [Sp*] appear favorable for complexing Na+, and thesodium cation may alternately (and rapidly on the NMR timescale) be bound and unbound to either the set of three Glycarbonyls [SG*] or the set of three Pro carbonyls [Sp*]. TheS* resonances in Fig. 5 (and Fig. 6) are taken, therefore, torepresent averaged resonances corresponding to these twotypes of conformers. The experimental values of JNa obtainedfrom Fig. 6 (5.0 and 5.5 Hz) are in good agreement with theaverage JNa predicted when Karplus-type equations (11-14) are

applied to these two equally populated conformations in rapidequilibrium, i.e., when the three Gly angles are alternately3000 [SG*I and 240° [Sp*] as determined from the models, thecalculated average JNa, values are in the range 4.5-5.3 Hz and6.0-6.5 Hz. Backbone rotational angles approximately de-scribing conformation [SG*] are: three sets each of Pro

T

FIG. 5. 220 MHz spectra of peptide N-H region of cyclo(Pro-Gly)3 (a) in [U-2H]Me2SO; (b)-(e), in [U-2H]Me2SO withNaSCNadded. In (b)-(e), the molar ratios of Na+ to c-(Pro-Gly)3 are 0.5,1, 2, and 7, respectively. Temperature = 230C. Concentrations ofc-(Pro-Gly)3: 15 mg/ml. In (a), (b), and (c), 8 scans were accu-mulated in a computer to improve the signal. Chemical shifts(r scale) in ppm downfield from Me4Si.

(4, 4', W) angles - (1200, 2800, 00) and Gly ( w,4, c) angles~(3000, 3300, 00). Those approximately describing [Sp*] are:Pro (4), 4, w) - (1200, 340°, 00) and Gly (4, A, w) ~ (2400,300, 00).An alternative conformation [S*], midway between [SG*]

and [Sp*] having Pro (4, 4', w) - (1200, 300°, 0°) and Gly(4), 4', a)~ (2700, 00, 00), may be considered in Which bothsets of carbonyls of a given c-(Pro-Gly)3 molecule would besimultaneously available for complexation with sodium ionson either (and/or both) faces of the cyclic peptide. Such aconformer seems less likely, since (a) sets of [S*] carbonyloxygen atoms are significantly less proximal than the Gly's in[SG*] or the Pro's in [Sp*], and (b) the experimentally-observed JNa do not compare as well to the calculated Gly JNa,(5.0-6.5 and 7.0-7.5) corresponding to such a structure.The stoichiometry of the c-(Pro-Gly)3-salt complex remains

to be determined. Since three peptide carbonyls may not besufficient to complex the cation effectively, the c-(Pro-Gly)3-NaSCN system may be a "sandwich" complex, with thesodium ion associated on the average with two [SG*] con-formers, two [SP*] conformers, or with one of each, but at anymoment bound to a total of six carbonyl groups.While sodium was found to be the most effective ion, experi-

ments with other alkali metal thiocyanates demonstratedtheir ability to complex c-(Pro-Gly)3 in a symmetric con-

1828 Chemistry: Deber et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 2,

202

0

Conformational Interconversions of Cyclo(Pro-Gly)3 1829

formation. Thus, in [U-2H]Me2SO solutions at a molar ratio ofMSCN/c-(Pro-Gly)3 = 8, where M = K+, Li+, Rb+, andCs+, the following fractions of symmetric c-(Pro-Gly)3 con-former were observed: K+, -0.75; Li+ - Rb+, -0.50; andCs+, -0.25 (compared with Na+ 0.90 under similar condi-tions). The uncertainty in these measurements is estimated tobe i=10%.The results reported above are of interest in relation to the

ion-transport properties of naturally-occurring cyclic pep-tides. The data indicate that c-(Pro-Gly)3 is a synthetic cyclichexapeptide that complexes alkali metal cations; similarinteractions of metal cations with a synthetic bicyclopenta-peptide have been reported (15). The preference for sodiumshown by c-(Pro-Gly)3 parallels the recent finding (16) thatthe naturally-occurring cyclic decapeptide, antamanide, alsocomplexes sodium more effectively than it does potassium.The fact that the striking conformational transitions ofcyclo(Pro-Gly)3 (as a function both of solvent polarity andalkali metal ion concentration) are directly observable byNMR illustrates the potential of this cyclic peptide as amodel for the study of peptide-solvent interactions.

MATERIALS AND METHODS

Synthesis. t-Butyloxycarbonyl-Gly-Pro-OH was convertedto its mixed anhydride with isobutylchloroformate and N-methylmorpholine, and treated with Gly-Pro-benzyl esterhydrochloride to give the tetrapeptide, t-Boc-Gly-Pro-Gly-Pro-OBz. After hydrogenation of this latter material int-BuOH with 10% Pd-C, the acid obtained was converted to asimilar mixed anhydride, and reacted with Gly-Pro-benzylester hydrochloride to produce the hexapeptide t-Boc-Gly-Pro-Gly-Pro-Gly-Pro-OBz. This ester was then hydro-genated to yield the hexapeptide acid, treatment of which withp-nitrophenol and dicyclohexylcarbodiimide gave the activeester t-Boc-Gly-Pro-Gly-Pro-Gly-Pro-ONp; its t-Boc groupwas removed with HCl-ethyl acetate. The resulting hydro-chloride was cyclized in pyridine; when the acetone-insolublefraction of the crude reaction product was dissolved in di-methylformamide, a 38% yield was obtained of cyclo(Gly-Pro-Gly-Pro-Gly-Pro) [=cyclo(Pro-Gly-Pro-Gly-Pro-Gly) ]T as a dimethylformamide complex. Crystallizationfrom methanol-ether gave the free cyclic peptide, mp 205-2100 C, with a portion of the crystals persisting in the melt untilits decomposition at 310-3150C. Infrared spectra, mass spectra(molecular ion peak 462), and elemental analysis, in additionto the NMR spectra presented herein, confirmed the identityof this compound. Details of the synthesis and characteriza-tion of c-(Pro-Gly)3 will be published elsewhere.

NMR Spectra. NMR spectra were obtained with the HR-220 spectrometer at Bell Laboratories. Homonuclear spindecoupling was accomplished by a General Radio 1107-Aaudio oscillator. In some instances, a Nicolet time averagingcomputer with 1024 channels was used in conjunction with

FIG. 6. The upfield portion of the 220 MHz spectrum of cyclo-(Pro-Gly)3 in [U-2H]Me2SO containing NaSCN. The molar ratioNaSCN/c-(Pro-Gly)3: 7. Temperature = 23°C. Concentrationof c-(Pro-Gly)3 is 15 mg/ml.

the HR-220 instrument to improve the signal-to-noise ratioof the spectra. In nonaqueous solvents, tetramethylsilanewas used as an internal reference, while in aqueous solution,the t-butyl resonance at 8.77 r (relative to sodium 2,2-di-methyl-2-silapentane-5-sulfonate) of [2H ]tert-butyl alcohol wasused as internal reference.

The work at Harvard was supported, in part, by U.S. PublicHealth Service Grants AM-07300 and AM-10794.

1. Hassall, C. H. & Thomas, W. A. (1971) Chem. Brit. 7, 145-153, and references therein.

2. For a review, see Bovey, F. A., Brewster, A. I., Patel,D. J., Tonelli, A. E. & Torchia, D. A. (1972) Accounts Chem.Res., in press.

3. Deber, C. M., Torchia, D. A. & Blout, E. R. (1971) J.Amer. Chem. Soc. 93, 4893-4897.

4. Torchia, D. A., di Corato, A., Wong, S. C. K., Deber, C. M.,& Blout, E. R. (1972) J. Amer. Chem. Soc. 94, 609-615.

5. Torchia, D. A., Wong,. S. C. K., Deber, C. M. & Blout,E. R. (1972) J. Amer. Chem. Soc. 94, 616-620.

6. Deber, C. M., Bovey, F. A., Carver, J. P. & Blout, E. R.(1970) J. Amer. Chem. Soc. 92, 6191-6198.

7. Madison, V. & Schellman, J. A. (1970) Biopolymers 9,511-567.

8. Torchia, D. A. (1972) Biochemistry 11 2 1462-1468.9. For a review, see Stewart, W. E. & Siddall, T. H., III

(1970) Chem. Rev. 70, 517-551.10. Schimmel, P. R. & Flory, P. J. (1969) J. Mol. Biol. 34,

105-120.11. Barfield, M. & Karplus, M. (1969) J. Amer. Chem. Soc. 91,

1-10, and references therein.12. Tonelli, A. E. & Bovey, F. A. (1970) Macromolecules 3,

410-411.13. Bystrov, V. F., Portnova, S. L., Tsetlin, V. I., Ivanov,

V. T. & Ovchinnikov, Yu. A. (1969) Tetrahedron 25, 493-515.14. Ramachandran, G. N., Chandrasekaran, R. & Kopple,

K. D. (1971) Biopolymers 10, 2113-2131.15. Schwyzer, R., Tun-Kyi, A., Caviezel, M. & Moser, P.

(1970) Helv. Chim. Acta 53, 15-27.16. Ivanov, V. T., Miroshnikov, A. I., Abdullaev, N. D.,

Senyavina, L. B., Arkhipova, S. F., Uvanova, N. N.,Khalilulina, K. Kh., Bystrov, V. F. & Ovehinnikov, Yu. A.(1971) Biochem. Biophys. Res. Commun. 42, 654-663.

tt The latter designation is used in the text with residues re-versed from the order used in the synthesis for clarification inpresentation of data.

Proc. Nat. Acad. Sci. USA 69 (1972)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 2,

202

0