BIOPOLY MERS VOL. 14, 1223-1230 (1975)

Carbon-13 and Proton NMR Studies of Helix-Coil Transition of Poly(7-benzyl-L-glutamate)

YASUYUIiI SUZUBI, YOSHIO INOUE, and RIICHIRO CHfJJ6*, Department of Polymer Chemistry, Tokyo Institute of Technology, aokayama,

Meyuro-ku, Tokyo, J a p a n

Synopsis

The helix-coil conformational transition undergone by poly (7-benayl-Lglutamate) in solutions of trifluoroacetic acid and deuterated chloroform was studied by proton and carbon-13 nmr. The results indicate that in the case of the solventrinduced helix-coil transition, the side chain assumes a helical conformation before the backbone. I n the thermally induced helix-coil transition, the results indicate the existence of an inter- mediate state, which is between the a-helix and random coil and is free from intramolecu- lar hydrogen bonding.

INTRODUCTION

Helix-coil transitions of synthetic polypeptides in solution have been extensively studied by many analytical means, namely, ir, CD, ORD, and nmr spectroscopy. For proton nmr, Bovey et al.' obtained the first spectrum of a polypeptide a t 40 MHz in 1959. After this, kinetic, equi- librium, double peaks of the a-CH, and many other mechanisms or phe- nomena have been studied by proton Proton nmr spectra of polypeptides in solution at 100 MHz are rather broad because of their limited molecular mobility and only the a-CH and NH signals, when ob- servable, shift significantly in the transition region. On the other hand, carbon-13 nmr has a wide range of chemical shifts and does not have com- plex spin-spin coupling in decoupling experiments. For these reasons, carbon-13 nmr has recently been used for studies of polypeptides.15-19 In the case of poly (ybenzyl-L-glutamate), Rradbury and c o - ~ o r k e r s ~ ~ obtained spectra in the helical state, transition region, coiled state, and the spin lattice relaxation times T I in the helical state. Allerhand and Oldfield16 also investigated T I and the nuclear Overhauser effect (NOE) in the helical and coiled states.

As carbon-13 nmr has a wide range of chemical shifts, i t can give informa- tion on each carbon in a residue, i.e., both backbone and side chain. In this paper, we show, in comparison with proton nmr, the results of a carbon- 13 nmr study of solvent [deuterated chloroform(CDCl3)/trifluoroacetic

* TO whom correspondence should be addressed. 1223

0 1975 by John Wiley & Sons, Inc.

1224 SUZUKI, INOUE, AND CHUJO

2 -

acid (TFA) ] induced and thermally induced helix-coil transitions of poly- ( ybenzyl-L-glut amate) [poly (Glu (OBzl)) 1.

/--- 0- NH

I 1

EXPERIMENTAL

The sample of poly(Glu(OBz1)) was purchased from Iiyowa Hakko Company, Ltd. and had a molecular weight of about 1S0,OOO. Solution coricentrations were 1.5% (w/v) for each solvent. Proton nmr spectra were obtained on a JEOL 1's-100 spectrometer operating a t 100 MHx and tetramethylsilane (TAIS) was used for lock signal in all cases. Carbon-13 nmr spectra were obtained on a ,JEOL 1%-100 spectrometer equipped with the PFT-100 I'ourier transform system operating at 25.15 3IHz and having a field-frequency control system based on the deuterium resoiiancc signal derived from the CDCl, contained in thc solvent mixture. The sample was contained in 5-mm-0.d. tubes for proton nmr and S-mm-0.d. tubes for carbon-13 nmr. Internal TMS was used as reference. Trifluoroacetic acid (TFA) was purchased from Tokyo Iiasci Company, Ltd. Deuterated chloroform (CDCIJ), isotopically 99.7%, was purchased from 1Ierek.

RESULTS AND DISCUSSION

Solvent-Induced Helix-Coil Transition

All measurements were performed at 25.0"C. Figure 1 shows the de- pendence of chemical shifts of various protons of poly(Glu(OBe1)) in CDC13-TFA mixtures on the volume ratio of TFA in mixed solvent. Side- chain protons are almost insensitive to the volume ratio. Backbone pro- tons show sharp shifts in the transition region. NH proton peaks could

' t a-C H

a 5 -Lo-o-o-o-ooL~p.o benzyl-CH2 a

TFA volume Fig. 1. Chemical shifts of various protons of poly(Glu(OBz1)) in ( 7 ) vs. the volume ratio

of TFA in the mixed solvent CDC13-TFA mixtures at 25'C.

POLY ( ~-BENZYL-L-GLUTA~\IATE) 1225

E a a

5 0 0 10 20 30

TFA volume "lo

Fig. 2. Chemical shift of wCH proton vs. the voluine ratio of TFA in the mixed solvent CDCl,-TFA mixtures a t 25°C.

150 100 50 ppm from TMS

0

Fig. 3. Carbon-I3 nmr spectra of poly(Clu(OBz1)) in the coiled region [30y0 (v/v)- Unlabeled quartets are as- TFA].

signed to the T F A carbonyl and CF, carbons, and triplet is the CDC13 carbon. Assignments are labeled on corresponding peaks.

not be observed in the helical region because of line broadening and coales- cence with the wing of the phenyl peak. 1:igurc. 2 shows the chemical shift of the a-CH proton. The sample of poly(Glu(OBz1)) used was of such high molecular weight that the a-CH proton peak behaved as a single shift- ing peak over the helix-coil transition r e g i o ~ i . ~ ~ * ~ ~ This peak is observed at 6.05, for the samples in the helical state and a t 5.387 for those in the coiled state. Thc shift difference between the helical and the coiled states is thus 0.67 ppm. From this figure, the helix-coil transition midpoint is found at 14.5y0 (v/v) TFA.

Figure 3 shows a carbon-13 nmr spectrum of poly(Glu(OBz1)) in the coiled region [30% (v/v)TFR]. The assignmcnts are those of Bradbury

It is the same ah the value reported in other papcrs.g,19

1226 SUZUKI, INOUE, AND CHUJO

180

178

176

138

137

I32

130

71

rr, 69

E 58 2 E 56 a

L

a 5L

32

30

28

26

5 10 15 20 25 30 TFA%(v/v)

Fig. 4. Chemical shifts of various carbons of poly(Glu(OBz1)) in ppm down-field from internal TMS vs. the volume ratio of TFA in the mixed solvent CDC1,-TFA mixtures a t 25°C.

and coworker^'^ and are labeled on each peak in the figure. In Figure 4, the chemical shifts of the various carbon nuclei (which were measured with respect to the TATS carbon signal) are plotted against the TFA% (v/v) in the CDCL-TFA mixture. The backbone carbons, C, and C=Oamlde show large down-field shifts in the coil-helix transition. The side-chain carbons, c,, Cbenzyl, C=Oester, and CZ--6 phenyl show up-field shifts and other carbons show down-field shifts in the transition. The transition region of the backbone carbons appcars a t lowcr TFA% (v/v) than that of the side-chain carbons. Table I summarizes the helix-coil transition midpoints and the shift differences Aco,l,helix observed for each of these carbon peaks.

From these results, chemical groups containing a carbon nucleus can be divided into two classes according to their chemical shifts and helix-coil transition midpoints (transition region) : (1) the backbone carbons, C=Oamlde and C,, which show large down-field shifts of 2.7 and 3.3 ppm, respectively, in the coil-to-helix transition and have helix-coil transition midpoints of 14.5-15.070 (v/v)TFA; ( 2 ) the sidc-chain carbons, which mainly show up-field shifts in the transition and have midpoints of 17.0-18.070 (v/v)TFA.

POLY ( 7-BENZYGL-GLUTAMATE) 1227

TABLE I Solvent-Induced Helix-Coil Transition Midpoints and Shift Differences Between the

Helix and the Coil of Various Carbons

Transition Midpoint (TFAY0)* Aooilihelix (pprn)b

15.0 14.5 18.0

17.5 17.0

-

-2 .7 - 3 . 3

2.0 -0 .5

1 . 7 1.4

-0.4 0 .

Experimental error is +0.5y0 (v/v)TFA. Minus sign indicates the down-field shift in coil-to-helix transition.

The magnitudes of the shift differences of the backbone carbons are larger than those of the side-chain carbons. The large shift difference of C=Oester is perhaps due to the interaction with the solvent, especially TFA. Since CB is directly bonded to the backbone carbon, C,, the shift difference may be expected to be considerable. The helix-coil transition midpoints of the side-chain carbons are found a t 17.0-18.070 (v/v)TFA, a higher concentration than that of the backbone carbons. It is considered that the solvation, particularly with TFA, is one of the driving forces of the helix-coil transition. Side-chain chemical shifts are considered to be sensi- tive to the solvation. The transition region of side-chain carbons is a t a higher concentration of TFA than that of the backbone carbons. From this result, we have considered that the transition of coil-helix of backbone is induced by liberation from the solvation to the side chain. So the side chain assumes a different conformation from the solvent coiled state before the backbone takes a helical conformation in the coil-to-helix transition.

For the side-chain orientation, two models have been considered : inter-side-chain and intra-side-chain orientations. Silverman and ScheragaZ0 have proposed a model of stacking of the phenyl group side chain for poly(L-phenylalanine), while Yan et a1.21 have proposed the model in which the side chain takes a helical conformation (which does not mean helical backbone but helical side chain). If the side-chain orientation is due to the former mechanism, the backbone must be in an a-helical state simultaneously with the orientation in the side chain. The transition mid- point of the backbone, C,, must be as large as, or larger than, that of the side chain in vol yo TFA. The latter model is not inconsistent with the experimental results obtained. We can say that in the case of poly(G1u- (OBzl)), the intra-side-chain orientation model appears more probable.

Thermally Induced Helix-Coil Transition

All measurements were performed a t 23.3% (v/v) TFA. Figure 5 shows the temperature dependence of the chemical shifts of various protons in a res- idue. Coil-to-helix transition cannot be performed completely as yet, even a t the highest temperature observed. For the solvent used, the boiling

1228

Q Q

5 - - w

4 -

3 -

2 -

1

SUZUKI, INOUE, AND CHUJO

-0- _ _ -*-*__o- - d . ' - . -- benzyl-

CH2

_e__D __o__d- phenyl H - -0 NH

I I I I

point of TFA is (312°C and that of CDCl, is 71.S"C. So the experiment at higher temperatures cannot be carried out. Thus, the value of chemical shift in helical state must be determined by anothrr way. This value is obtained as 5.967 for a-CH proton in the other solvent mixtures [leyo (v/v)TFA]. The helix-coil transition midpoint is cstin1atc.d ac; 60.0"C using this valuc..

Figure 6 shows the temperature dependence of thc chemical shifts of the various carbons. As does the solvent-induced helix-coil transition (Figure 4), thc backbone carbons shou a large do\\ n-field shift in the coil-to- hclix transition. But the backbone carbons C, and C=OamlCle bchavc in a diffcrcnt manner in the transition rcygion. Tablc I1 summarizes thc hclix- coil transition midpoints and thcl shift diffcrcnccs Acolllhellx in thc thermally induced transition. Thc shift diffcrmccs of thc hackboni~ carbons are the same as in the solwnt-induced hc.lix-coi1 transition. Thc, side-chain shift diffcrcnces arc smaller than thosc found in thcl solvent-induccld transition. This means that the backbone orientation occurs in the sanw n a y in both cases, but that the side-chain orientation doc5 not.

Carbon-13 hclix-coil transition midpoints, i n particular C,, arc diffcrcnt from thosc obscmwd in proton nmr (a-CH GO.0"C). Thc possible origin of thc a-CH proton shift has bccn considcrcd to 1 x 1 1) the diffcrenccx in local field induccd by th(, shi~lding cffcct of the barl,bonc and the side chain between thc hclix arid thc coil, or 2 ) the diffcrcwci. corresponding to the prcscncc or absence of wlvation. If thr origin of t h r a-CH proton shift is mainly ducl to the latter, the a-CH transition midpolnt ha. to bc identical with the value obicrvcd in C=-Oalnldr (50.0"C). Discrcyxincics bet\\ ('en thcxc result.; are not yet adequately intcrprc.tcd.

POLY (-,-BENZY L-L-GLUTAM ATE ) 1229

54

32

30

TABLE I1 Thermally Induced Helix-Coil Transition Midpoints and Shift Differences

Transition Midpoint ("C)* AeotliheIir (pprn)

C=Oarndie 50.5 -2.8 C, 46.0 - 3 . 2

(1 .2) -1 .1 c, 49 ..5

C=O,,ter 48.5 0 . 7 Cbenzyl - 0.4

- CB

c1 p h e w 1 47 .o -0.8 0 - c2--6 phem 1

* Experimental error is + 1 "C.

-

-

-

132

I I I

25 35 4 5 55 65 75 ec

Fig. 6. Chemical shifts of various carbons of poly(Glu(OBz1)) in ppm down-field from TRIS in 23.3YG (v/v)TFA vs. temperature.

The helix-coil transition midpoint of C, (46.0"C) is different from that of C=OaInlde (50.5OC). The C, transition is mainly derived from the conformational change of the backbone while the C=Oamlde transition is derived from the formation of intramolecular hydrogen bonding. This difference suggests the existence of an intermediate state, which lies bc-

1230 SUZUKI, INOUE, AN11 CHUJO

tween the a-helical and randomly coiled states. This state is probably free from the intramolecular hydrogen bonding, but is in the same confor- mation as that of the helix. If this is the case, the two kinds of observed midpoints (4G.O"C and 50.5"C) may correspond to the tempcrature of the formation of the same conformation as helix and that of the formation of hydrogen bonding, respectively.

Refer en ce s 1. Bovey, F. A,, Tiers, G. V. D. & Filipovich, G. (1959) J . Polym. Sci. 38,73-90. 2. Goodman, M. & Masuda, Y. (1964) Biopolymers 2, 107-112. 3. Markeley, J . L., Meadow, I). H. & Jardetzky, 0. (1967) J . Mol. Biol. 2'7, 25-40, 4. Ferretti, J . A. (1967) J . Chem. SOC., Chem. Commun., 1030-1032. 5. Bovey, F. A. (1968) Pure Appl. Chem. 16, 417432. 6. Bradbury, J . H. & Fenn, M. D. (1969) Aust. J . Chem. 22, 357-371. 7. Joube-t, E. J., Lotan, N. & Scheraga, H. A. (1970) Biochemistry 9, 2197-2211. 8. Nagayama, K. & Wada, A. (1972) Chem. Phys. Lett. 16, 50-51. 9. Nagayama, K. (1972) Tampakushitsu Kakusan Koso 17, 653-668.

10. Bradbury, E. RI., Crane-Robinson, C. & Rattle, H. W. E. (1970) Polymer 11,

11. Ferretti, J . A., Jernigan, R. L. & Weiss, G. H. (1973) J . Polym. Sci. Symp. 42,

12. Jernigan, R . L., Ferretti, J . A. & Weiss, G. H. (1973) Macromolecules 6, 684-687. 13. Ferretti, J . A. & Jernigan, R. L. (1973) Macromolecules 6, 687-692. 14. hfilstien, J. B. & Ferretti, J. A. (1973) Biopclymers 12, 2335-2349. 15. Paolillo, L., Tancredi, T., Temussi, P. A., Trivellone, E., Bradbury, E. M. &

16. Allerhand, A. & Oldfield, E. (1973) Biochemistry 12, 3428-3433. 17. Boccalon, G., Verdini, A. S. & Giacometti, G. (1972) J . Amer. Chem. Soc. 94,

18. Dorman, I). E., Torchia, D. A. & Bovey, F. A. (1973) Macromolecules 6, 80-82. 19. Bradbury, E. M., Cary, P. D., Crane-Robinson, C. & Hartman, P. G. (1973)

20. Silverman, D. N. & Scheraga, H. A. (1971) Biochemistry 10, 1340-1347. 21. Yan, J. F., Vanderkooi, G. & Scheraga, H. A. (1968) J . Chem. Phys. 49, 2713-

277-288.

1051-1059.

Crane-Robinson, C. (1972) J . Chem. SOC. Chem. Commun. 335-336.

3639-3641.

Pure Appl. Chem. 36, 53-92.

2726.

Received August 21, 1974 Accepted January 31, 1975