BIOPOLYMERS
VOL. 16, 1465-1472 (1977)
Cis-Trans Equilibrium and Kinetic Studies of Acetyl-L-Proline and Glycyl-L-Proline H. N. CHENG and F. A. BOVEY, Bell Laboratories, Murray Hill,N e w Jersey 07974
Synopsis By means of carbon-13 nmr (at 25 MHz) the transleis conformer ratio in glycyl-L-proline has been measured in aqueous (DzO) solution over the temperature range 33-96 "C. I t is found that AHo = -4.2 kJ/mole and ASo = -9.7 J/mole/K. Measurements of the 2'1 values for the proline ring carbons yielded values consistent with a fast puckering process involving both the 0-and y-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L-proline, using complete line-shape analysis for the glycyl a-carbon resonance, gave values for the trans cis isomerization as follows: AHf = 83.5 f 0.2 kJ/mole; A S # = 0.0 f 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of A G # of 82.0 f 0.4 kJ/mole for this process in acetyl-L-proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.
-
INTRODUCTION The proline residue occupies a unique place in polypeptide and protein structure because of the conformational restriction introduced by its ring and because of the small energy difference between the cis and trans conformations of the X-Pro bond, X being any preceding amino-acid residue. The cis-trans equilibrium has received considerable study in N,N-dialkyl amides, and model ~ e p t i d e s , l and - ~ in proline homo and copolymers.4,5 Recently, Brandts et a1.6 have proposed that the cis-trans isomerization of X-Pro peptide bonds is a rate-determining step in the renaturation of proteins. If confirmed, this hypothesis would accentuate interest in this equilibrium and in the rate of the equilibration process. However, kinetic and equilibrium data for proline peptides in aqueous solution are very limited,6 and it was with the object of supplying further information on simple proline derivatives that this study was undertaken. Carbon-13 nmr was preferred because potentially usefully proton resonances are usually partially overlapped by the complex proline ring proton spectrum, preventing accurate measurements. In addition, 13C T1 measurements were carried out as a function of temperature to provide apparent energies of activation for overall tumbling and ring puckering. 1465 Q 1977 by John Wiley & Sons, Inc.
1466
CHENG AND BOVEY
EXPERIMENTAL Materials Acetyl-L-proline and glycyl-L-proline were obtained from Cyclo Chemicals, Los Angeles, Calif. Their 13C spectra contained no extraneous resonances and they were used without further purification. D20 of at least 99% isotopic purity was obtained from Diaprep, Inc., Milwaukee, Wisc. All other materials were of reagent grade.
Methods 13C spectra were run using a Varian XL-LOO spectrometer (25.16 MHz for 13C)modified for Fourier-transform operation and interfaced to a Nicolet 1080 c ~ m p u t e r .ProtonJ3C ~ couplings were removed with a noisemodulated decoupling field. Free induction decays were stored in 16K computer locations using dwell times of 100-500 psec (i.e., spectral windows of 1000-5000 Hz). The pulse was located at the most shielded end of the spectrum at 25.160820 Hz;the pulse width was ca. 32 psec for a 90" pulse. For the kinetic study of glycyl-L-proline, a pulse interval of 10 sec was employed. Sample temperatures were measured using a thermocouple immersed in a tube of ethylene glycol; the accuracy is estimated to be f0.5 "C. T Imeasurements were made by both inversion-recovery and progressive saturation methods; the error of the given values is estimated not to exceed f 10%. Samples were prepared to 10.0% (w/v) concentration in D20 in sealed 12-mm tubes deoxygenated by repeated freeze-pump-thaw cycles; 50 h of dioxane was added as reference. Acetyl-L-prolinewas observed at pH 1.98 f 0.02, glycyl-L-proline a t pH 4.85 f 0.03. The simulated spectra for the kinetic studies were generated on a Honeywell6000 computer using the Binsch DNMR programs obtained from Quantum Chemistry Program Exchange, Bloomington, Ind.
RESULTS AND DISCUSSION Cis-Trans Equilibria in Glycyl- proli line Since the glycyl Ca and prolyl Ca resonances broaden excessively in the temperature range of interest, the C@,Cy, and C6peaks (assignments taken from Ref. 9) were employed for trans/cis ratio measurements in D20 over the temperature range 33-96 "C. From all three peaks the equilibrium constant KO = [trans]/[cis]was found to obey the relationship (Fig. 1) In KO = 496/T(K)
- 1.17
(1)
from which it is found that AHo = -4.2 kJ/mole and ASo = -9.7 J/mole/K; the trans conformer is thus favored by enthalpy but disfavored by entropy,
CIS- TRANS EQUILIBRIUM
0.5
1467
:
I
Fig. 1. Equilibrium constant KO = [trans]/[cis]for Glycyl-L-Proline us inverse temperature.
probably owing to opposed effects of steric hindrance and coulombic attraction in the cis conformer and greater solvent immobilization by the trans conformer. O
C
O
P
I
I o ~ c \ c H 2 N H ~
cis
E3NCH2/c\0
trans
In Table I, these data are compared to the information available in the literature for X-Pro peptide bonds. No obvious rationale can be offered for these results, but it is of interest that Gly-L-Pro and poly(G1y-L-Pro) show equivalent equilibrium behavior within experimental error, suggesting that effects of charges are minimal.
Ring Mobility in Glycyl-L-Proline A number of nmr studies have led to the conclusion that in solutions of proline peptides (other than diketopiperazines) the proline ring puckers at Cy or a t both Cy and C/3.11-13 From T I measurements, Deslauriers et a1.I2 deduced that in Gly-L-Pro (in D20), the ring interconverts rapidly between half-chair conformers puckered at Ca and Cy. We have measured the temperature dependence of the ring T I values in this compound; the results are given in Table 11, together with values of T,, calculated on the
CHENG AND BOVEY
1468
TABLE I Equilibrium Enthalpy and Entropy Values for Cis-Trans Conformations About X-Pro Peptide Bonds Compound
Solvent
AHo (kJ/mole)
Ac-L-Pro-N-N-Diisopropylamide HzO Ac-L-Pro-N-N-Diisopropylamide Dioxane X-Proa CDC13 Dz0 Poly(Gly-L-Pro) Gly-L-Pro DzO
A S o (J/mole/k)
Reference
0
0
2
8.4 0 -5.0 -4.2
20.9 4.6 -5.4 -9.6
2 10 4
This work
cH20-B~~
I
a
7'1
X = C~HSCH~OCO-NHCHCO-
TABLE I1 (sec) and 7 C (psec) Values for Gly C" and Proline Ring Carbons of Gly-L-Pro in DzO
34.0 49.1 64.4 74.0
1.2 2.0 3.0 3.6
20 12 8.2 6.8
0.9 1.3 1.7 1.9
54 38 29 26
1.1 1.6 2.3 2.6
22 15 10.7 9.4
1.1 1.7 2.4 3.0
22 14 10 8
0.8 1.2 1.8 2.1
31 20 13.6 11.7
TABLE I11 Ring Correlation Times, Tc(ring), in psec for Glycyl-L-Proline (Calculated from the Observed Correlation Times by Eq. (2)) Temp ("C)
CP
CY
C6
34" 49O 64" 74"
38 26 17 15
38 24 16 12
71 47 26 21
assumption of isotropic reorientation in the extreme narrowing limit.14 These values represent both the cis and trans conformers, which were the same within experimental error, as has been generally ~bserved.'l-'~ (For N,N-dialkylamides, results are not consistent; different 7'1 values are reported for syn and anti methyl carbons in N,N-dimethylf~rmamide,'~ whereas for N,N-dimethylacetamide the values are the same.16) Adopting the now commonly used approximate treatment of such data, in which the observed motion of each ring C-H vector is taken as the sum
CIS- TRANS EQUILIBRIUM
1469
Fig. 2. Arrhenius plot of proline ring correlation Tc(ring) of Glycyl-L-Proline; data from Table 111.
of the overall molecular tumbling and the internal ring motions, we have 1 / ~(ring) , = 1 / ~(ring, , obs) - 1 / ~( ,2 )
(2)
where T , (ring) is the correlation time of the ring C-H vector apart from the contribution of overall molecular tumbling, T , (ring, obs) is the effective correlation time of the ring C-H vectors, appearing in Table I1 and T , ( Z ) is the correlation time for molecular tumbling, taken as equal to that calculated for Pro Ccrin Table 11. Values thus calculated for ring) are shown in Table 111. From an Arrhenius plot of this data, shown in Figure 2 , we find the following apparent activation energies in kJ/mole: Overall Motion (from Pro-Ca)
Cfl Cr C6
17.1 21.7
25.5 26.3
The value of 17.1 kJ/mole for the tumbling of the molecule is reasonable for motion controlled primarily by rotational diffusion. (For glycine a value of 16.3 kJ/mole has been reported.I7) The values for the other ring carbons are consistent with a coordinated ring puckering process involving all of them, but such data do not really provide a clear-cut indication of the process involved.
CHENG AND BOVEY
1470
-
TABLE IV Kinetic Data for the Trans Cis Isomerization of Glycyl-L-Proline (Measured Using Gly Cn) T (OK)
k t , (sec-')
AGft, (kJ/mole)
350.4 354.3 356.0 359.3 364.9 366.5 367.1 369.3
3.0 3.5 4.5 5.0 8.0 9.5 9.5 10.0
83.0 83.5 83.2 83.7 83.6 83.5 83.6 84.0
Kinetics of Cis-Trans Interconversion The large chemical shift range of 13Cmakes it potentially attractive for kinetic measurements, particularly for fairly fast exchange reactions. (The recent controversy concerning the equivalence of Fourier-transform and continuous wave nmr spectra does not apply to natural abundance C13nmr because the C13 nuclei are for the most part not coupled.18-20) For the present case, involving a relatively slow reaction observed over a limited range of temperatures (the high limit being the boiling point of water), one must choose pairs of conformer resonances which are sufficiently close so that coalescence will occur within this range. One must also of course be sure that pulse intervals are sufficiently long (at least 3T1) so that the relative areas of the conformer resonances are correct (this requirement is less critical if the T I values are the same) and also take adequate account of possible unequal nuclear Overhauser enhancements. 1) Glycyl-L-Proline. The glycyl Ca,which in slow exchange shows a separation of 5.0 Hz for the cis and trans conformers, was selected as most suitable for kinetic studies; 2'1 values were found to be the same for each form within experimental error and NOE should be close to maximum.13 Rate measurements were made by total line-shape fitting (see Experimental section) a t eight temperatures within a 20° range below the coalescence temperature. The relative populations of the cis and trans forms a t each temperature were taken from Figure 1. Initial values of Tz for the computer program were taken from the linewidths of the dioxane reference and the static peak separation Av, was taken as the value a t ambient temperature. These parameters were varied slightly in the subsequent refinement to obtain a best fit of the data. The results are shown in Table IV. Although there is a slight apparent increase in AG Z t c with temperature, this is within the probable experimental error of the measurements. It is thus found that AH#tc is 83.5 f 0.2 kJ/mole and AS#,, is 0.0 f 10 J/ mole/K. (An approximate line-shape analysis of the Pro Ca resonances gave a value of AG Z t c of 88 f 4 kJ/mole.) 2 ) Acetyl-L-Proline. None of the proton-bearing carbons of acetylL-proline shows coalescence below 100 "C. The carbonyl resonances
a
I
CH20-But
I
I
CHz
CfiHs
83.5 82.0 79.4
ca. 82.5
75.7 87.4 ca. 82.8 ca. 96.2 ca. 73
AGf or E, (kJ/mole)
'H dynamic nmr dynamic nmr I3C dynamic nmr dynamic nmr
'H dynamic nmr 'H dynamic nmr PH jump 'H nmr equilibration 'H dynamic nmr
Method
R = CGH&H~OCO-NHCHCO-,C6H&H20CO-NHCHCO-, C~HSCHZOCO-NHCH~CO-NHCH~CO-, and NHzCHzCO-NHCHzCO-.
R = &H&Hz and But.
R-L-Pro-O-But Gly-I>-proline Acetyl-L-proline Acetyl-L-proline
Neat Neat H2O HzO CDCl:, MezSO CDC13 DzO D20 12M NaSCN in DzO
N,N- dimethylacetamide N,N-dimethylformamide L- Ala-L-Pro Poly-(L-proline) (I 11) RO-CO-L-Pro-O-But a
-
Solvent
Compound
TABLE V Activation Energies for Cis-Trans Equilibration in Proline Peptides and Related Compounds
10,24 This work This work This work
21,22 21,22 6 23 10,24
Reference
x
b
1472
CHENG AND BOVEY
showed prohibitively long TI values: a t 32.2 "C ca. 25 sec for the acetyl carbonyl and 18 sec for the carboxyl carbonyl and a t 94.4 "C, 125 and 52 sec for the acetyl and carboxyl carbonyls, respectively. From the acetyl carbonyl coalescence, observed to occur a t 90.5 "C, and the slow exchange peak separation of 7.0 Hz, a AG Z t c of 82.0 f 0.4 kJ/mole was calculated. In 12M NaSCN, the acetyl methyl 13C resonance of acetyl-L-proline, which is a singlet in H20, splits into cis and trans peaks separated by ca. 0.15 ppm (ca. 3.8 Hz). From coalescence measurements on this doublet and the acetyl carbonyl doublet, a AG#tc of 79.4 f 0.4 kJ was calculated. Thus, the presence of this salt lowers the barrier by about 2.6 kJ/mole. The kinetic data are summarized in Table V from this work and from published work on related compounds. The conclusion may be drawn that the cis-trans barrier for X - P r o peptide bonds in aqueous solution is very similar to that observed for simpler N,N-dialkyl amides as neat liquids or in organic solvents and for a substituted seryl-L-prolineester in chloroform. The poly(-L-proline) cis-trans isomerization in D2O appears to be characterized by a somewhat higher barrier, while that of the cis-trans equilibration in an oxycarbonyl (i.e., urethane) link preceding proline is lower.
References 1. Stewart, W. E. & Siddall, T. H., I11 (1970) Chem. Rev. 70,517-519. 2. Madison, V. & Schellman, J. (1970) Biopolymers 9,511-567. 3. Thomas, W. A. & Williams, M. K. (1972) J.C.S. Chem. Commun., 994. 4. Torchia, D. A. (1972) Biochemistry 11,1462-1468. 5. Dorman, D. E., Torchia, D. A. & Bovey, F. A. (1973) Macromolecules 6 , 8 0 4 2 . 6. Brandts, J. F., Halvorson, H. R. & Brennan, M. (1975) Biochemistry 14,4953-4963. 7. Sternlicht, H. & Zuckerman, D. M. (1972) Rev. Sci. Instrum. 43,525-529. 8. Binsch, G. (1969) J. Amer. Chem. Soc. 91,1304-1309. 9. Dorman, D. E. & Bovey, F. A. (1973) J. Org. Chem. 38,2379-2383. 10. Maia, H. L., Orrel, K. 0. & Rydon, H. N. (1971) Chem. Commun., 1209-1210. 11. Fossel, E. T.,Easwaran, K. R. K. & Blout, E. R. (1975) Biopolymers 14,927-935. 12. Deslauriers, R., Smith, I. C. P. & Walter, R. (1974) J. Biol. Chem. 249,4149-4156. 13. Torchia, D. A. & Lyerla, J. R., Jr. (1974) Biopolymers 13,97-114. 14. Lyerla, J. R., Jr. & Levy, G. C. (1974) in Topics in Carbon-13 NMR Spectroscopy, Levy, G. C., Ed., Wiley-Interscience, New York, Vol. 1, pp. 79-143. 15. Levy, G. C. & Nelson, G. L. (1972) J. Amer. Chem. Soc. 94,4897-4901. 16. Giannini, D. D., Armitage, I. M., Pearson, H., Grant, D. M. & Roberts, J. D. (1975) J . Amer. Chem. Soc. 97,3416-3419. 17. Cutnell, J. D., Glasel, J. A. & Hruby, V. J. (1975) Org. Magn. Reson. 7,256. 18. Kaplan, J. I. (1972) J. Chem. Phys. 57,5615-5616; (1973) 59,990. 19. Ernst, R. R. (1973) J. Chem. Phys. 59,989. 20. Ernst, R. R., Aue, W. P., Bartholdi, E,, Hohener, A. & Schaublin, S. (1974) Pure Appl. Chem. 37,47-59. 21. Drakenberg, T., Dahlqvist, K.-I. & ForsBn, S. (1972) J. Phys. Chem. 76,2178-2183. 22. Jackman, L. M. (1975) in Dynamic Nuclear Magnetic Resonance Spectroscopy, Jackman, L. M. & Cotton, F. A., Eds., Academic, New York, pp. 203-252. 23. Torchia, D. A. & Bovey, F. A. (1971) Macromolecules 4,246-251. 24. Maia, H. C., Orrel, K. G. & Rydon, H. N. (1976) J.C.S. Perkin II, 761-763.
Received September 24,1976 Accepted November 8,1976
VOL. 16, 1465-1472 (1977)
Cis-Trans Equilibrium and Kinetic Studies of Acetyl-L-Proline and Glycyl-L-Proline H. N. CHENG and F. A. BOVEY, Bell Laboratories, Murray Hill,N e w Jersey 07974
Synopsis By means of carbon-13 nmr (at 25 MHz) the transleis conformer ratio in glycyl-L-proline has been measured in aqueous (DzO) solution over the temperature range 33-96 "C. I t is found that AHo = -4.2 kJ/mole and ASo = -9.7 J/mole/K. Measurements of the 2'1 values for the proline ring carbons yielded values consistent with a fast puckering process involving both the 0-and y-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L-proline, using complete line-shape analysis for the glycyl a-carbon resonance, gave values for the trans cis isomerization as follows: AHf = 83.5 f 0.2 kJ/mole; A S # = 0.0 f 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of A G # of 82.0 f 0.4 kJ/mole for this process in acetyl-L-proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.
-
INTRODUCTION The proline residue occupies a unique place in polypeptide and protein structure because of the conformational restriction introduced by its ring and because of the small energy difference between the cis and trans conformations of the X-Pro bond, X being any preceding amino-acid residue. The cis-trans equilibrium has received considerable study in N,N-dialkyl amides, and model ~ e p t i d e s , l and - ~ in proline homo and copolymers.4,5 Recently, Brandts et a1.6 have proposed that the cis-trans isomerization of X-Pro peptide bonds is a rate-determining step in the renaturation of proteins. If confirmed, this hypothesis would accentuate interest in this equilibrium and in the rate of the equilibration process. However, kinetic and equilibrium data for proline peptides in aqueous solution are very limited,6 and it was with the object of supplying further information on simple proline derivatives that this study was undertaken. Carbon-13 nmr was preferred because potentially usefully proton resonances are usually partially overlapped by the complex proline ring proton spectrum, preventing accurate measurements. In addition, 13C T1 measurements were carried out as a function of temperature to provide apparent energies of activation for overall tumbling and ring puckering. 1465 Q 1977 by John Wiley & Sons, Inc.
1466
CHENG AND BOVEY
EXPERIMENTAL Materials Acetyl-L-proline and glycyl-L-proline were obtained from Cyclo Chemicals, Los Angeles, Calif. Their 13C spectra contained no extraneous resonances and they were used without further purification. D20 of at least 99% isotopic purity was obtained from Diaprep, Inc., Milwaukee, Wisc. All other materials were of reagent grade.
Methods 13C spectra were run using a Varian XL-LOO spectrometer (25.16 MHz for 13C)modified for Fourier-transform operation and interfaced to a Nicolet 1080 c ~ m p u t e r .ProtonJ3C ~ couplings were removed with a noisemodulated decoupling field. Free induction decays were stored in 16K computer locations using dwell times of 100-500 psec (i.e., spectral windows of 1000-5000 Hz). The pulse was located at the most shielded end of the spectrum at 25.160820 Hz;the pulse width was ca. 32 psec for a 90" pulse. For the kinetic study of glycyl-L-proline, a pulse interval of 10 sec was employed. Sample temperatures were measured using a thermocouple immersed in a tube of ethylene glycol; the accuracy is estimated to be f0.5 "C. T Imeasurements were made by both inversion-recovery and progressive saturation methods; the error of the given values is estimated not to exceed f 10%. Samples were prepared to 10.0% (w/v) concentration in D20 in sealed 12-mm tubes deoxygenated by repeated freeze-pump-thaw cycles; 50 h of dioxane was added as reference. Acetyl-L-prolinewas observed at pH 1.98 f 0.02, glycyl-L-proline a t pH 4.85 f 0.03. The simulated spectra for the kinetic studies were generated on a Honeywell6000 computer using the Binsch DNMR programs obtained from Quantum Chemistry Program Exchange, Bloomington, Ind.
RESULTS AND DISCUSSION Cis-Trans Equilibria in Glycyl- proli line Since the glycyl Ca and prolyl Ca resonances broaden excessively in the temperature range of interest, the C@,Cy, and C6peaks (assignments taken from Ref. 9) were employed for trans/cis ratio measurements in D20 over the temperature range 33-96 "C. From all three peaks the equilibrium constant KO = [trans]/[cis]was found to obey the relationship (Fig. 1) In KO = 496/T(K)
- 1.17
(1)
from which it is found that AHo = -4.2 kJ/mole and ASo = -9.7 J/mole/K; the trans conformer is thus favored by enthalpy but disfavored by entropy,
CIS- TRANS EQUILIBRIUM
0.5
1467
:
I
Fig. 1. Equilibrium constant KO = [trans]/[cis]for Glycyl-L-Proline us inverse temperature.
probably owing to opposed effects of steric hindrance and coulombic attraction in the cis conformer and greater solvent immobilization by the trans conformer. O
C
O
P
I
I o ~ c \ c H 2 N H ~
cis
E3NCH2/c\0
trans
In Table I, these data are compared to the information available in the literature for X-Pro peptide bonds. No obvious rationale can be offered for these results, but it is of interest that Gly-L-Pro and poly(G1y-L-Pro) show equivalent equilibrium behavior within experimental error, suggesting that effects of charges are minimal.
Ring Mobility in Glycyl-L-Proline A number of nmr studies have led to the conclusion that in solutions of proline peptides (other than diketopiperazines) the proline ring puckers at Cy or a t both Cy and C/3.11-13 From T I measurements, Deslauriers et a1.I2 deduced that in Gly-L-Pro (in D20), the ring interconverts rapidly between half-chair conformers puckered at Ca and Cy. We have measured the temperature dependence of the ring T I values in this compound; the results are given in Table 11, together with values of T,, calculated on the
CHENG AND BOVEY
1468
TABLE I Equilibrium Enthalpy and Entropy Values for Cis-Trans Conformations About X-Pro Peptide Bonds Compound
Solvent
AHo (kJ/mole)
Ac-L-Pro-N-N-Diisopropylamide HzO Ac-L-Pro-N-N-Diisopropylamide Dioxane X-Proa CDC13 Dz0 Poly(Gly-L-Pro) Gly-L-Pro DzO
A S o (J/mole/k)
Reference
0
0
2
8.4 0 -5.0 -4.2
20.9 4.6 -5.4 -9.6
2 10 4
This work
cH20-B~~
I
a
7'1
X = C~HSCH~OCO-NHCHCO-
TABLE I1 (sec) and 7 C (psec) Values for Gly C" and Proline Ring Carbons of Gly-L-Pro in DzO
34.0 49.1 64.4 74.0
1.2 2.0 3.0 3.6
20 12 8.2 6.8
0.9 1.3 1.7 1.9
54 38 29 26
1.1 1.6 2.3 2.6
22 15 10.7 9.4
1.1 1.7 2.4 3.0
22 14 10 8
0.8 1.2 1.8 2.1
31 20 13.6 11.7
TABLE I11 Ring Correlation Times, Tc(ring), in psec for Glycyl-L-Proline (Calculated from the Observed Correlation Times by Eq. (2)) Temp ("C)
CP
CY
C6
34" 49O 64" 74"
38 26 17 15
38 24 16 12
71 47 26 21
assumption of isotropic reorientation in the extreme narrowing limit.14 These values represent both the cis and trans conformers, which were the same within experimental error, as has been generally ~bserved.'l-'~ (For N,N-dialkylamides, results are not consistent; different 7'1 values are reported for syn and anti methyl carbons in N,N-dimethylf~rmamide,'~ whereas for N,N-dimethylacetamide the values are the same.16) Adopting the now commonly used approximate treatment of such data, in which the observed motion of each ring C-H vector is taken as the sum
CIS- TRANS EQUILIBRIUM
1469
Fig. 2. Arrhenius plot of proline ring correlation Tc(ring) of Glycyl-L-Proline; data from Table 111.
of the overall molecular tumbling and the internal ring motions, we have 1 / ~(ring) , = 1 / ~(ring, , obs) - 1 / ~( ,2 )
(2)
where T , (ring) is the correlation time of the ring C-H vector apart from the contribution of overall molecular tumbling, T , (ring, obs) is the effective correlation time of the ring C-H vectors, appearing in Table I1 and T , ( Z ) is the correlation time for molecular tumbling, taken as equal to that calculated for Pro Ccrin Table 11. Values thus calculated for ring) are shown in Table 111. From an Arrhenius plot of this data, shown in Figure 2 , we find the following apparent activation energies in kJ/mole: Overall Motion (from Pro-Ca)
Cfl Cr C6
17.1 21.7
25.5 26.3
The value of 17.1 kJ/mole for the tumbling of the molecule is reasonable for motion controlled primarily by rotational diffusion. (For glycine a value of 16.3 kJ/mole has been reported.I7) The values for the other ring carbons are consistent with a coordinated ring puckering process involving all of them, but such data do not really provide a clear-cut indication of the process involved.
CHENG AND BOVEY
1470
-
TABLE IV Kinetic Data for the Trans Cis Isomerization of Glycyl-L-Proline (Measured Using Gly Cn) T (OK)
k t , (sec-')
AGft, (kJ/mole)
350.4 354.3 356.0 359.3 364.9 366.5 367.1 369.3
3.0 3.5 4.5 5.0 8.0 9.5 9.5 10.0
83.0 83.5 83.2 83.7 83.6 83.5 83.6 84.0
Kinetics of Cis-Trans Interconversion The large chemical shift range of 13Cmakes it potentially attractive for kinetic measurements, particularly for fairly fast exchange reactions. (The recent controversy concerning the equivalence of Fourier-transform and continuous wave nmr spectra does not apply to natural abundance C13nmr because the C13 nuclei are for the most part not coupled.18-20) For the present case, involving a relatively slow reaction observed over a limited range of temperatures (the high limit being the boiling point of water), one must choose pairs of conformer resonances which are sufficiently close so that coalescence will occur within this range. One must also of course be sure that pulse intervals are sufficiently long (at least 3T1) so that the relative areas of the conformer resonances are correct (this requirement is less critical if the T I values are the same) and also take adequate account of possible unequal nuclear Overhauser enhancements. 1) Glycyl-L-Proline. The glycyl Ca,which in slow exchange shows a separation of 5.0 Hz for the cis and trans conformers, was selected as most suitable for kinetic studies; 2'1 values were found to be the same for each form within experimental error and NOE should be close to maximum.13 Rate measurements were made by total line-shape fitting (see Experimental section) a t eight temperatures within a 20° range below the coalescence temperature. The relative populations of the cis and trans forms a t each temperature were taken from Figure 1. Initial values of Tz for the computer program were taken from the linewidths of the dioxane reference and the static peak separation Av, was taken as the value a t ambient temperature. These parameters were varied slightly in the subsequent refinement to obtain a best fit of the data. The results are shown in Table IV. Although there is a slight apparent increase in AG Z t c with temperature, this is within the probable experimental error of the measurements. It is thus found that AH#tc is 83.5 f 0.2 kJ/mole and AS#,, is 0.0 f 10 J/ mole/K. (An approximate line-shape analysis of the Pro Ca resonances gave a value of AG Z t c of 88 f 4 kJ/mole.) 2 ) Acetyl-L-Proline. None of the proton-bearing carbons of acetylL-proline shows coalescence below 100 "C. The carbonyl resonances
a
I
CH20-But
I
I
CHz
CfiHs
83.5 82.0 79.4
ca. 82.5
75.7 87.4 ca. 82.8 ca. 96.2 ca. 73
AGf or E, (kJ/mole)
'H dynamic nmr dynamic nmr I3C dynamic nmr dynamic nmr
'H dynamic nmr 'H dynamic nmr PH jump 'H nmr equilibration 'H dynamic nmr
Method
R = CGH&H~OCO-NHCHCO-,C6H&H20CO-NHCHCO-, C~HSCHZOCO-NHCH~CO-NHCH~CO-, and NHzCHzCO-NHCHzCO-.
R = &H&Hz and But.
R-L-Pro-O-But Gly-I>-proline Acetyl-L-proline Acetyl-L-proline
Neat Neat H2O HzO CDCl:, MezSO CDC13 DzO D20 12M NaSCN in DzO
N,N- dimethylacetamide N,N-dimethylformamide L- Ala-L-Pro Poly-(L-proline) (I 11) RO-CO-L-Pro-O-But a
-
Solvent
Compound
TABLE V Activation Energies for Cis-Trans Equilibration in Proline Peptides and Related Compounds
10,24 This work This work This work
21,22 21,22 6 23 10,24
Reference
x
b
1472
CHENG AND BOVEY
showed prohibitively long TI values: a t 32.2 "C ca. 25 sec for the acetyl carbonyl and 18 sec for the carboxyl carbonyl and a t 94.4 "C, 125 and 52 sec for the acetyl and carboxyl carbonyls, respectively. From the acetyl carbonyl coalescence, observed to occur a t 90.5 "C, and the slow exchange peak separation of 7.0 Hz, a AG Z t c of 82.0 f 0.4 kJ/mole was calculated. In 12M NaSCN, the acetyl methyl 13C resonance of acetyl-L-proline, which is a singlet in H20, splits into cis and trans peaks separated by ca. 0.15 ppm (ca. 3.8 Hz). From coalescence measurements on this doublet and the acetyl carbonyl doublet, a AG#tc of 79.4 f 0.4 kJ was calculated. Thus, the presence of this salt lowers the barrier by about 2.6 kJ/mole. The kinetic data are summarized in Table V from this work and from published work on related compounds. The conclusion may be drawn that the cis-trans barrier for X - P r o peptide bonds in aqueous solution is very similar to that observed for simpler N,N-dialkyl amides as neat liquids or in organic solvents and for a substituted seryl-L-prolineester in chloroform. The poly(-L-proline) cis-trans isomerization in D2O appears to be characterized by a somewhat higher barrier, while that of the cis-trans equilibration in an oxycarbonyl (i.e., urethane) link preceding proline is lower.
References 1. Stewart, W. E. & Siddall, T. H., I11 (1970) Chem. Rev. 70,517-519. 2. Madison, V. & Schellman, J. (1970) Biopolymers 9,511-567. 3. Thomas, W. A. & Williams, M. K. (1972) J.C.S. Chem. Commun., 994. 4. Torchia, D. A. (1972) Biochemistry 11,1462-1468. 5. Dorman, D. E., Torchia, D. A. & Bovey, F. A. (1973) Macromolecules 6 , 8 0 4 2 . 6. Brandts, J. F., Halvorson, H. R. & Brennan, M. (1975) Biochemistry 14,4953-4963. 7. Sternlicht, H. & Zuckerman, D. M. (1972) Rev. Sci. Instrum. 43,525-529. 8. Binsch, G. (1969) J. Amer. Chem. Soc. 91,1304-1309. 9. Dorman, D. E. & Bovey, F. A. (1973) J. Org. Chem. 38,2379-2383. 10. Maia, H. L., Orrel, K. 0. & Rydon, H. N. (1971) Chem. Commun., 1209-1210. 11. Fossel, E. T.,Easwaran, K. R. K. & Blout, E. R. (1975) Biopolymers 14,927-935. 12. Deslauriers, R., Smith, I. C. P. & Walter, R. (1974) J. Biol. Chem. 249,4149-4156. 13. Torchia, D. A. & Lyerla, J. R., Jr. (1974) Biopolymers 13,97-114. 14. Lyerla, J. R., Jr. & Levy, G. C. (1974) in Topics in Carbon-13 NMR Spectroscopy, Levy, G. C., Ed., Wiley-Interscience, New York, Vol. 1, pp. 79-143. 15. Levy, G. C. & Nelson, G. L. (1972) J. Amer. Chem. Soc. 94,4897-4901. 16. Giannini, D. D., Armitage, I. M., Pearson, H., Grant, D. M. & Roberts, J. D. (1975) J . Amer. Chem. Soc. 97,3416-3419. 17. Cutnell, J. D., Glasel, J. A. & Hruby, V. J. (1975) Org. Magn. Reson. 7,256. 18. Kaplan, J. I. (1972) J. Chem. Phys. 57,5615-5616; (1973) 59,990. 19. Ernst, R. R. (1973) J. Chem. Phys. 59,989. 20. Ernst, R. R., Aue, W. P., Bartholdi, E,, Hohener, A. & Schaublin, S. (1974) Pure Appl. Chem. 37,47-59. 21. Drakenberg, T., Dahlqvist, K.-I. & ForsBn, S. (1972) J. Phys. Chem. 76,2178-2183. 22. Jackman, L. M. (1975) in Dynamic Nuclear Magnetic Resonance Spectroscopy, Jackman, L. M. & Cotton, F. A., Eds., Academic, New York, pp. 203-252. 23. Torchia, D. A. & Bovey, F. A. (1971) Macromolecules 4,246-251. 24. Maia, H. C., Orrel, K. G. & Rydon, H. N. (1976) J.C.S. Perkin II, 761-763.
Received September 24,1976 Accepted November 8,1976
