13c and 1h nmr studies of helix-coil transition of poly(β-benzyl-l-aspartate) and...

BIOPOLYMERS VOL. 16, 2521-2532 (1977)

13C and lH NMR Studies of Helix-Coil Transition of Poly(@-Benzyl-L-Aspartate) and Poly(y-Benzyl-

L-Glutamate): Behavior in Nonprotonating Solvent Mixtures, and Origin of Solvent -Induced

Chemical Shift

YASUYUKI SUZUKI, YOSHIO INOUE, and RIICHIRO CHflJO, Department of Polymer Chemistry, Tokyo Institute of Technology,

Ookayama 2-12-2, Meguro-ku, Tokyo, Japan

Synopsis

From the results of 13C-nmr measurement of poly(P-benzyl-L-aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(0-benzyl-L-aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the a-helix. The chemical shift differences in the benzyl group carbons of poly(y-benzyl-L-glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (0, C') originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(P-benzyl-L-aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.

INTRODUCTION

Helix-coil transition of synthetic polypeptides in solution using organic acids as coiled solvents has been extensively studied by 1H-nmr.1-2 There are two possible mechanisms for the breakdown of the helix by the acids: formation of the hydrogen bondings between the acids and amide groups of polypeptide^,^-^ and protonation to the amide groups by the acid^.^-^ Recently, E. M. Bradbury et aL9 reported helix-coil transition of some poly- and copolypeptides in nonprotonating solvent mixtures [dimethyl sulphoxide(Me2SO)/deuterated chloroform(CDCl3)]. They found that the a-CH proton behaved in a similar manner as when in trifluoroacetic acid

In our previous papers, we reported some regularity of the chemical shifts (F~AcOH).

2521

0 1977 by John Wiley & Sons, Inc.

2522 SUZUKI, INOUE, AND C H ~ J ~

TABLE I Chemical Shift Differences Ahelidcoil in FaAcOH/CDC13 Mixtures

Ahelidcoil (PPm)"

Poly(Glu( OBzl)) Poly(Asp(OBz1))

c=oamide -2.7 0 Ce -3.3 -1.2 C@ 2.0 0.9 C7 -0.5 -

c=o,ter 1.7 1.9 0 ? ? Cbenzyl 1.4 1.2 c1 phenyl -0.4 -1.3 c2-6 phenyl 0.5 0.3

" AheIix/.mil = &oil - &helix.

in poly(y-benzyl-L-glutamate) [p~ly(Glu(OBzl))]~~ and poly(0-benzyl- a asp art ate) [poly(Asp(OBzl))] l1 accompanied by the helix-coil transition (shown in Table I): (1) for poly(Glu(OBzl)), the sign of the chemical shift differences alternate along the side chain if the sign of the chemical shift difference of oxygen atom is considered to be negative; (2) for poly- (Asp(OBzl)), the behavior of the chemical shift differences can be divided

and the alternation of the sign along the side chain is also observed in each group; and (3) the carbons that have the same suffix have the same sign of the chemical shift differences between these two polymers.

Morishima et a1.12 reported the I3C chemical shifts induced by the protonation in the trifluoroacetic acid solution for various aliphatic amines and N-heterocyclic six-membered ring compounds. They found that the protonation-induced I3C shift depends on the orientation of the nitrogen lone-pair electrons and on the conformation of the intervening carbon skeleton. Further, they found that there is an alternation effect in the N-protonation 13C shifts for n-butylamine and so on; protonation shifts are in the order of Ca < CD, C@ > Cr, Cr < C6. They also suggested that the 0-protonation-induced I3C shift decreases along the a-chain (I. Morishima, private communication).

In this paper, we investigate poly(Asp(OBz1)) in MezSO/CDCl3 mixtures primarily by 13C-nmr in order to obtain the information about the backbone and the side-chain conformations and the relation between the polypeptide and the solvent used. Further, we show the chemical shifts of model compounds induced by the solvents, especially coiled solvents, in com- parison with the chemical shifts of poly(Glu(OBz1)) and poly(Asp(OBz1)) accompanied by the helix-coil transition.

into two groups, Ca and cs, and C=Oester, Cbenzylt c1 phenyl and c2-6 phenyl

NMR STUDIES OF HELIX-COIL TRANSITION 2523

EXPERIMENTAL

The samples of poly(Glu(OBz1)) (DP ca. 820) and poly(Asp(OBz1)) (DP ca. 70) were purchased from Kyowa Hakko Company, Ltd. Model compounds such as N,N-diethyl acetamide, benzylbutyrate, and benzylpropionate were purchased from Tokyo Kasei Company, Ltd. Deuterated chloroform and dimethyl sulphoxide-ds, isotopically 99.8%, were purchased from Commissariat B 1’Energie. Polypeptide solutions were of concen- tration 15% (w/v) for F3AcOH/CDCl3 solutions and 5% (w/v) for Me2SO/ CDC13 solutions. The concentrations of model compounds were the same as the concentrations of the corresponding polypeptides.

lH-nmr spectra were obtained on JEOL PS-100 spectrometer operating at 100 NHz and tetramethyl silane (Me4Si) was used for lock signal in order to obtain the chemical shift of the sample. l3C-nmr spectra were obtained on the same instrument equipped with the PFT-100 Fourier transform system.

RESULTS AND DISCUSSION

All measurements for poly(Asp(OBz1)) were performed at 82°C. The dependence of the chemical shifts of all of protons of poly(Asp(OBz1)) in Me2SO/CDCl3 mixtures were investigated in order to ensure the behavior of the chemical shift change of the polypeptide used. P-CHz, benzyl-CHa, and phenyl H are almost insensitive to the solvent composition. The chemical shift values of a-CH are 4.29 and 4.63 ppm (6) in the helix and the coil, respectively. The shift difference Ahelix/coil between the helix and the coil is thus 0.34 ppm. It is the same as the value reported by Bradbury et

The chemical shift values of NH are 8.70 and 7.86 ppm ( 6 ) and the shift difference Ahelix/coil is 0.84 ppm. Bradbury et aL9 reported 8.75 and 8.15 ppm (6); we have no explanation for this discrepancy. From the change of these chemical shifts, the helix-coil transition region is found around 30-50% (v/v) Me2SO.

Figure 1 shows the dependence of the chemical shifts of all of carbon atoms on the Me2SO% (v/v) in the Me2SO/CDC13 mixtures. In Table I1 are summarized the helix-coil transition midpoints and the shift differences Ahelix/coil for each of these carbon atoms. The plateau values were used to decide these values. From these results, the following is found:

1. All carbons show upfield shifts in the region of 10-20% (v/v) Me2SO

2. Only C=Oamide, Ca, and Ca show large shifts in the transition re-

3. The transition region of the side-chain carbon, Cfl, appears a t lower

with the increase of the proportion of Me2SO.

gion.

Me,SO% (v/v) than that of each of backbone carbons.

Model compounds were used in order to estimate the contribution induced by the solvents used in the chemical shifts of polypeptides [poly-

2524 SUZUKI, INOUE, AND C H ~ J ~

TABLE I1 Chemical Shift Differences &,elix/coil and the Transition Midpoints of Poly(Asp(OBz1)) in

MeZSO/CDC13 Mixtures

Transition Ahelix/coila Midpoint

( P P d (% MeZSO)

c=oamide -1.6 47

C=O,ter 0 -

c1 phenyl 0 -

cZ-6 phenyl -0.1 -

C- -1.5 46 C@ 1.9 42

Cbenzyt -0.3 -

a Ahelix/coil = &oil - dhelix.

172

169

135

129 1 d

E 65

51

49 50 f :: 34


(Glu(OBzl)), poly(Asp(OBzl))]. In the helix-coil transition of poly- (Glu( OBzl)) and poly(Asp(OBzl)), solvent molecules, especially coiled solvent molecules (F3AcOH or MezSO), can interact with two sites: the amide group of the backbone and the ester group of the side chain. So the influence of this interaction on the chemical shifts must be taken into account to interpret the chemical shifts accompanied by the helix-coil transition. In order to separate these effects, two kinds of model compounds were used: one simulates the amide group of the backbone, and the other, the ester group of the side chain. N,N-diethyl acetamide was used as a model of the former, because it would be less likely to self-associate than N-ethyl acetamide.13 Benzylbutyrate and benzylpropionate were used as models corresponding to the side chain of poly(Glu(OBz1)) and poly(Asp( OBzl)), respectively. There are two carbons corresponding to the backbone a-carbon in the case of N,N-diethyl acetamide; one is C-methyl carbon and the other is methylene carbon. Methyl carbon corresponds to the @-carbon of the side chain.

Figure 2 shows the dependence of the chemical shifts of the carbons of N,N-diethyl acetamide on the F,AcOH% (v/v) in the F3AcOH/CDCl3 mixtures. Downfield shifts are induced by F3AcOH at the carbonyl and methylene carbons, while upfield shifts are induced at C-methyl and methyl carbons. Alternation of the sign of the induced chemical shifts are observed for two carbons bonded by the common cr-bond each other; methyl and methylene carbons of N-ethyl group and carbonyl and C-methyl carbons of acyl group. The sign of the induced shift of C-methyl carbon, which corresponds to the a-carbon, is opposite to that of methylene carbon which is also corresponds to the a-carbon. The a-carbon of the polypeptide is placed between amide group on the C-terminal side and that on the N- terminal side. Therefore, if the results of N,N-diethyl acetamide were simply applied to the interpretation of the behavior in polypeptide, both positive and negative chemical shifts can be induced at the a-carbon by the interaction between amide group of the polypeptide and F~AcOH, furthermore even canceling could occur.

Figure 3 also shows the dependence of the chemical shifts of the carbons of N,N-diethyl acetamide on the percent Me2SO (v/v) in the Me2SO/CDCl3 mixtures. Two methylene carbons and methyl carbons (i.e., anti and syn carbons) are coalesced with each other in CDCl3 because of higher temperature (42OC). Two characteristic patterns of the chemical shifts are found in contrast with the case of F3AcOH/CDC13 solvent mixtures. All carbons show upfield shifts with the increase of the ratio of MezSO and the magnitude of the chemical shifts induced by Me2SO is much smaller than those by F3AcOH.

Figure 4 shows the dependence of the chemical shifts of benzylbutyrate on the percent F3AcOH (v/v) in the F3AcOH/CDCl3 mixtures. C1 phenyl shows an upfield shift by F3AcOH and the remaining carbons show downfield shifts by F3AcOH. The magnitude of the chemical shift of each carbon induced by F3AcOH gradually decreases along bonds from carbonyl

2526 SUZUKI, INOUE, AND C H ~ J J ~

E 170 169 40

12 11

0 10 20 30 40 50 60 70 0 10 20 30 LO 50 60 70 FIAcOH '1.

Fig. 2. Chemical shifts of all of carbons of N,N-diethyl acetamide in ppm downfield from Me& vs the volume ratio of F&OH in the F3AcOH/CDC13 mixtures.

L3 ._ 42

LO E 39 - 38 E

4 41

8 22 21

t COCH,

I I e--_.-, 1 *-.-.-

0 20 LO 60 80 100 Me,SO %

Fig. 3. Chemical shifts of all of carbons of N,N-diethyl acetamide in ppm downfield from Me.& vs the volume ratio of MezSO in the Me2SO/CDCl3 mixtures: (0) measured at 42OC, (x) measured at room temperature.


E 2 E a

c

a

70 69 68 67 6 6

38 37 36

20 19 18

13 12

etc . - t

0 10 20 30 LO 50 60

181 180 179 178 177 176 175 17L 173

136 135

130 129 128

0 10 20 30 LO 50 60 F,AcOH '1.

Fig. 4. Chemical shifts of all of carbons of benzylbutyrate in ppm downfield from internal Me& vs the volume ratio of F3AcOH in the F3AcOHKDC13 mixtures.

172

137

E a 6L a

29 E CHZ I 26

28 .-.---.---s,-, '"i , , l-j -.-.--.--.- 27 I

8

0 20 LO 60 80 100 Me2S0 '1.

Fig. 5. Chemical shifts of all of carbons of benzylpropionate in ppm downfield from internal Me& vs the volume ratio of MezSO in the Me2SOKDC13 mixtures.

2528 SUZUKI, INOUE. AND C H ~ J J ~

carbon to methyl carbon on the side of the alkyl group, while on the side of the benzyl group it also decreases but being accompanied with the alternation of the sign if the induced shift of oxygen atom is considered to be negative. For benzylpropionate, similar dependence of the chemical shifts on the solvent composition is observed.

Figure 5 shows the dependence of the chemical shifts of benzylpropionate on the percent MeZSO (v/v) in the Me2SO/CDC13 mixtures. All carbons show upfield shifts with the increase of the ratio of MeZSO, similar to the case of N,N-diethyl acetamide in the same solvent mixtures. The magnitude of the chemical shifts induced by Me2SO gradually decreases in proportion to the distance from carbonyl carbon along bond.

In order to compare clearly the chemical shifts of polypeptides accompanied by the helix-coil transition with those of the model compounds induced by the solvent used, the latter is plotted against the former. These correlations are shown in Figs. 6-9. Figure 6 shows the correlation described above of the case of poly(Glu(OBz1)) in F3AcOHKDC13. There is a negative correlation between the induced chemical shifts in the backbone model (i.e., N,N-diethyl acetamide) and the chemical shifts of the backbone accompanied by the helix-coil transition. For the side chain, the carbons can be divided into two groups: (1) C=Owkr, cbwi, CI phenyl, and C2-6 phenyl and (2) Ca, Cfl, and Cr. There is a positive correlation between the induced chemical shifts in the side-chain model (i.e., benzylbutyrate) and the chemical shifts of the side-chain carbons (1) accompanied by the helix-coil transition, while there is no correlation with respect to the side-chain carbons (2).

In the case of poly(Glu(OBz1)) in F3AcOH/CDC13, the chemical shifts of the side-chain carbons (1) can be roughly interpreted only by the chemical shifts induced by the interaction between the ester group and

Fig. 6. Correlation between the chemical shifts (abscissa) in poly(Glu(OBz1)) accompanied by the helix-coil transition and the chemical shifts (ordinate) in the model compounds induced by F3AcOH. 6 - ( v / v ) ~ d a ~ - 6 c ~ c l ~ was used as the chemical shift difference in model compound induced by FdcOH: (0) estimated from N,N-diethyl acetamide, (0) estimated from benzylbutyrate.

2529 NMR STUDIES OF HELIX-COIL TRANSITION

Fig. 7. Correlation between the chemical shifts (abscissa) in poly(Asp(OBz1)) accompanied by the helix-coil transition and the chemical shifts (ordinate) in the model compounds induced by F3AcOH. 640%(v/v)F&OH - ~ C D C ~ ~ was used as the chemical shift difference in model compound induced by F3AcOH: (0) estimated from N,N-diethyl acetamide, (0 ) estimated from benzylpropionate.

Fig. 8. Correlation between the chemical shifts (abscissa) in poly(Asp(OBz1)) accompanied by the helix-coil transition and the chemical shifts (ordinate) in the model compound induced by F3AcOH; benzylbutyrate was used as the side-chain model.

FAcOH. On the other hand, the shifts of the side-chain carbons (2) cannot be explained by the same reason as the side-chain carbons (1). They are also considered to be affected by the backbone which undergoes helix-coil transition, including the breakdown of the intrahydrogen bondings and whose peptide interacts with F3AcOH. In addition to these influences, it can be considered that the conformational change of the side chain affects the chemical shifts of the side chain carbons. Ca, which is derived from methyl carbon of benzylbutyrate, is not affected by the inductive effect due to the interaction between the ester group and F3AcOH. The plot of Cr

2530 SUZUKI, INOUE, AND C H ~ J 6

is situated in the second quadrant, i.e., the sign of the chemical shift of Cr induced by F3AcOH is opposite to that accompanied by the helix-coil transition. So the chemical shift difference of Cr originates not only from the interaction between the ester group of the side chain and F~AcOH, but also from some other unknown factors.

Figures 7 and 8 show the correlation described above in the case of poly(Asp(OBz1)) in F3AcOH/CDC13. In Fig. 8, benzylpropionate is used as the side-chain model and in Fig. 9, benzylbutyrate is used. Induced chemical shift of methyl carbon of benzylpropionate, corresponding to the a-carbon of the backbone, is 0 ppm, while that of methylene carbon of benzylbutyrate that corresponds to the same carbon is 0.6 ppm. There may be some peculiarity about the methyl carbon at the end of the chain, if it is close to the site of interaction. So benzylbutyrate is more favorable than benzylpropionate as the side-chain model in the case of poly(Asp(OBz1)). It is used in the following discussion.

The correlation between the chemical shifts of the backbone of poly(Asp(OBz1)) accompanied by the helix-coil transition and those of N,N-diethyl acetamide induced by F3AcOH is negative as in the case of poly(Glu(OBz1)). The correlation about the side chain is positive except for the a-carbon. In this case, the chemical shift induced by the interaction between the ester group of the side chain and F3AcOH affects the a-carbon. The correlation about the side chain of poly(Asp(OBz1)) is better than that of poly(Glu(OBz1)). From these results, the chemical shifts of the side chain of poly(Asp( OBzl)) accompanied by the helix-coil transition can be roughly explained if only the interaction between the ester group of the side chain and F3AcOH was taken into account.

Figure 9 shows the correlation between the chemical shifts in poly- (Asp(OBz1)) in Me2SO/CDC13 accompanied by the helix-coil transition and those in the model compounds induced by the solvent. There is no correlation and all plots fall on the third and the fourth quadrants. Consid- ering these results, in addition to the results of the model compounds (Figs. 3 and 5), the findings 1 and 2 for poly(Asp(OBz1)) (designated in an earlier part of this paper) indicate the solvation of MezSO to the side chain in the

benz I ester 0 -1 0

amide -2

ppm

Fig. 9. Correlation between the chemical shifts (abscissa) in poly(Asp(0Bzl)) accompanied by the helix-coil transition and the chemical shifts (ordinate) in the model compounds induced by Me2SO (0) estimated from N,N-diethyl acetamide, (0 ) estimated from benzylpro- pion ate .


region more than MezSO 20% (v/v), where the backbone keeps the a- helix.

For the correlation between the chemical shifts of poly(Asp(OBz1)) and poly(Glu(OBz1)) accompanied by the helix-coil transition and those of model compounds induced by F~AcOH, there is a positive correlation in a part of the side chain [which is designated as (1) in this paper]. These results mean that the relation between the chemical shift differences of the part of the side-chain carbons can be roughly interpreted only by the chemical shifts induced by the interaction between the ester group and F3AcOH. There is poor [poly(Asp(OBzl))] or no [poly(Glu(OBzl))] correlation with respect to Ca, CS, and Cr [Cr: only in the case of poly- (Glu(OBzl))]. There may be two or more origins of the chemical shift differences of these carbons as described in an earlier part of this paper. So these chemical shift differences originate not only from the interaction between the ester group of the side chain and F~AcOH, but also from other factors described in an earlier part. In the case of Ca, the chemical shift difference is affected by the inductive effect due to the interaction between the ester group and F3AcOH if Ca is close to the ester group [poly- (Asp(OBzl))] and not affected if it is away from the ester group [poly- (Glu(OBzl))].

CONCLUSION

In contrast to the dependence of the chemical shifts of the side-chain carbons of poly(Asp(OBz1)) on the percent F3AcOH (v/v) in the F3AcOH/ CDC13 mixtures,ll the chemical shifts of the side-chain carbons of poly(Asp(OBzl)), except C@, are almost independent of the solvent composition around the helix-coil transition region in solution of MezSO/CDCl3 mixtures. The side-chain carbons show upfield shifts in the region of 10-20% (v/v) MezSO with the increase of the proportion of MeZSO. For the model compounds, all carbons also show upfield shifts with the increase of the proportion of Me2SO in the MeZSOJCDC13 mixtures. Therefore, the upfield shifts observed in the region of 10-20% (v/v) Me2SO with the increase of the proportion of MezSO are considered to be induced by the solvation of MezSO to the side chain, so the side chains of poly(Asp(OBz1)) in solutions of MezSO/CDC13 mixtures are solvated by MezSO in the region more than Me2SO 20% (v/v), where the backbone keeps the a-helix.

N,N-Diethyl acetamide, which corresponds to a model of the backbone of the polypeptide, protonates on the carbonyl oxygen in strong acids.14 Simply applying the results of N,N-diethyl acetamide in solution of F3AcOH/CDC13 mixtures to the interpretation of the behavior in polypeptide, both positive and negative chemical shifts can be induced at a- carbon by 0-protonation-furthermore, even canceling could occur.

From the result of the correlation between the chemical shifts in poly- (Glu(OBz1)) in F3AcOH/CDC13 accompanied by the helix-coil transition and those in model compounds induced by F~AcOH, the chemical shifts

2532 SUZUKI, INOUE, AND CHoJ6

of a part of the side chain [designated as (l)] can be roughly interpreted only by the chemical shifts induced by the interaction between the ester group and FAcOH, and the remaining side-chain carbons (2) cannot be explained by the same reason as the side-chain carbons (1). There may be some other unknown factors for the origin of the chemical shift differences of the side-chain carbons (2). Ca is not affected by the inductive effect due to the interaction between the ester group of the side chain and F3AcOH.

According to the results of the correlation between the chemical shift differences in poly(Asp(OBz1)) in F3AcOH/CDC13 accompanied by the helix-coil transition and those in model compounds induced by FsAcOH, the chemical shift differences of the side-chain carbons can be roughly explained if only the interaction between the ester group of the side chain and F3AcOH is taken into account. The chemical shifts induced by the interaction between the ester group of the side chain and F3AcOH affect the chemical shift difference of Ca.

The authors wish to express their sincere thanks to Professor Morishima for his helpful discussion on inductive effect.

References

1. Bovey, F. A. (1974) Mucromol. Rev. 9 , 1 4 1 . 2. Bradbury, E. M., Cary, P. D., Crane-Robinson, C. & Hartman, P. G. (1973) Pure Appl.

3. Ferretti, J. A. & Ninham, B. W. (1970) Macromolecules 3,30-34. 4. Bovey, F. A. (1968) Pure Appl. Chem. 16,417-432. 5. Stewart, W. E., Mandelkern, L. & Glick, R. E. (1967) Biochemistry 6,143-149. 6. Nagayama, K. (1972) Tumpakushitsu Kukusan Koso 17,653-668. 7. Hanlon, S. (1966) Biochemistry 5,2049-2061. 8. Hanlon, S. & Klotz, I. M. (1965) Biochemistry 4,3748. 9. Bradbury, E. M., Crane-Robinson, C., Paolillo, L. & Temussi, P. (1973) Polymer 14,

Chem. 36,53-92.

303-308. 10. Suzuki, Y., Inoue, Y. & ChtijB, R. (1975) Biopolyrners 14,1223-1230. 11. Sase, S., Suzuki, Y., Inoue, Y. & ChtijjB, R. (1977) Biopolymers 16,95107. 12. Morishima, I., Yoshikawa, K., Okada, K., Yonezawa, T. & Goto, K. (1973) J. Am. Chem.

13. Hinton, J. F. & Lander, K. H. (1972) J. Mugn. Reson. 6,586-599. 14. Gillespe, R. J. & Birchall, T. (1963) Can. J. Chem. 41,148-155.

SOC. 95,165-171.

Received December 30,1976 Accepted March 24,1977

13c and 1h nmr studies of helix-coil transition of poly(β-benzyl-l-aspartate) and...

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