J . Am. Chem. SOC.1980, 102,1635-1631
1635
Carbon- 13 Nuclear Magnetic Resonance Chemical Shifts and Polypeptide Structure Alan E. Tonelli Contribution from Bell Laboratories, Murray Hill, New Jersey 07974. Received May 27, 1980
Abstract: I3CNMR chemical shifts of backbone carbonyl and side chain @ carbons in polypeptides provide information about their structure. Utilization of substituent effects on "C chemical shifts, as applied successfully to synthetic organic polymers, makes it possible to rationalize the relative observed I3C NMR chemical shifts of the backbone carbonyl and side chain @ carbons which depend on polypeptide sequence and conformation. As examples, in the polypeptide sequence -Gly-X-L-Alathe carbonyl carbon of residue X resonates at increasingly higher fields in the series X = L-Ala, L-Pro, Gly. Also the carbon of residue X in the sequence -Gly-X-Gly- resonates downfield from its position in -Gly-X-L-Ala-. These and other sequence-dependent I3C chemical shifts can be understood based on substituent effects (@and y effects). Furthermore, in homopolypeptidesthe downfield shift of the side chain fi carbon resonance observed when passing through the a-helix to the random-coil conformational transition is consistent with the relative "C chemical shifts estimated via the y effect method for the fi carbon in the a-helical and the random-coil conformations.
13CN M R chemical shifts observed for the carbon atoms in synthetic organic polymers can be understood in terms of the polymer chain microstructure.' The effects on 13Cchemical shifts of monomer sequence, configuration, conformation, and defect structures can all be explained based on the substituent effects first deduced for paraffinic hydrocarbon^.^-^ Relative to an unsubstituted carbon, each carbon substituent in the a or 0position produces a downfield shift (deshielding effect) of ca. +10 ppm. On the other hand, a y carbon substituent shields the observed carbon, resulting in an upfield shift of -2 to -3 ppm. This latter shielding effect (y effect) has been shown',5 to not only require a y substituent, but the observed and y carbons must be in a gauche (g) arrangement (see Figure 1). Clearly the y effect on I3C chemical shifts is sensitive to polymer chain conformation. The conformational sensitivity of the y substituent effect has been exploited to understand the 13C N M R chemical shifts and the underlying microstructures of synthetic organic homo- and copolymers.' In this report we attempt the same approach to learn something about the microstructure and conformation of polypeptides. Horsely et aL6 have derived substituent effects for the 13CN M R chemical shifts observed in amino acids (see ref 7 for a refined version). Agreement between observed and estimated 13Cchemical shifts was not found to be as good as that achieved for the paraffinic hydrocarbons or their singly substituted derivatives. These authors suggested that polypeptide sequence effects would be less than a few parts per million based on the relatively small 6 and c shift parameters they derived for the 13Cchemical shifts in amino acids. However, they did not foresee the possibility that the I3C N M R chemical shifts observed in polypeptides might be sensitive to their conformations via y substituent effects as described here. In Figure 2 we present a schematic representation of a polypeptide chain illustrating the backbone and side chain torsional angles which determine its conformation. Among the carbon atoms in each peptide residue only the backbone carbonyl and side chain P carbons have y substituents whose arrangements depend upon the conformation ($,$ rotations) about the backbone (1) A. E. Tonelli and F. C. Schilling, Acc. Chem. Res., in press. (2) H. Spiesecke and W. G. Schneider, J . Chem. Phys., 35, 722-730 (1961). (3) D. M. Grant and E. G. Paul, J. Am. Chem. Soc., 86, 2984-2990 (1964). (4) L. P. Lindeman and J. Q.Adams, Anal. Chem., 43, 1245-1252 (1971). (5) F. A. Bovey, "Proceedings of the International Symposium on Macromolecules", Rio de Janeiro, July 26-31, 1974, E. B. Mano, Ed., Elsevier, Amsterdam, 1975, pp 169-182. (6) W. Horsely, H. Sternlicht, and J. S. Cohen, J . Am. Chem. Soc., 92, 680-686 (1970). (7) J. B. Stoihers, "Carbon-13 NMR Spectroscopy", Academic Press,New York, 1972, pp 478-482. (8) J. J. Edsall, P. J. Flory, J. C. Kendrew, A. M. Liquori, G. Nemethy, G. Ramachandran, and H. A. Scheraga, Biopolymers, 4, 121-130 (1966).
N-C" and P - C ' bonds. Since the y effect on the 13Cchemical shifts in synthetic organic polymers has been shown' to depend upon the gauche arrangements of y substituents with respect to the observed carbon atom, we will focus our attention exclusively on the 13Cchemical shifts of the backbone carbonyl and side chain 0carbon atoms in an attempt to learn something about the polypeptide backbone microstructure, Le., sequence of residues and backbone conformations. Conformational Effects on Polypeptide 13C N M R Chemical Shifts The backbone C-C bonds in synthetic organic polymers are usually constrained9 by 3 kcal/mol inherent rotational barriers to adopt one of the three staggered rotational states t,g' (4 = 0, f120°) depicted in Figure 1. In a polypeptide chain, on the other hand, the intrinsic barriers to rotation about the N-C" and C"-C' 1.O kcal/mol) and nonstaggered backbone bonds are lower (I rotational states are more p r e ~ a l e n t .However, ~ conformational energy maps of the various residues in randomly coiling polypeptides'O do indicate that the most probable backbone conformations still occur in the vicinity of 4 or J, = 0, f120°, though deviations from these three rotational states can be rather large. Consequently, in our treatment of the y effect involving backbone carbonyl and side chain B carbons we will estimate the probabilities" that 4 and rotations adopt values which result in gauche arrangements of C' and C@with other atoms even if 4 and J, # 0, f120°. In Figure 3 we present a portion of a polypeptide chain containing the sequence -Glyl-Glyi+l- or -Glyl-L-Alai+l- in the planar zigzag (4 = J, = 0') Conformation. (Ali peptide bonds are assumed to be planar and trans.) The carbonyl carbon of Gly, is y to the preceding and succeeding carbonyl carbons and to the P-CH3 carbon of the L-Alai+l residue in the -Glyl-L-Alai+l- sequence. The arrangements of these y substituents with C'G!y, depend on the rotations 4i and +i+l about the N-C" bonds in residues i and i 1.
-
+
+
(9) P. J. Flory, "Statistical Mechanics of Chain Molecules", Wiley-Interscience, New York, 1969, Chapters 111, V, and VII. (10) Chapter VI1 in ref 9. (1 1) When estimating the probability that 4 or $ rotations adopt certain p(4' or leading to y gauche interactions involving C' and values 4' or Cfl, we take the following approach
v,
v),
as the expression used, for example, to estimate the probability that the N-C:" bond is in rotational state 4'. Permitting 4 = 4' & 30' in the numerator takes some a c c o ~ n t ~of. ' ~the contribution made by the conformational entropy
0002-7863/80/1502-7635$01 .OO/O 0 1980 American Chemical Society
1636 J . Am. Chem. Soc., Vol. 102, No. 26, 1980
Tonelli
Table 1. Comparison of Predicted and Observed ’C NMR Chemical Shifts of Carbonyl Carbons in Randomly Coiling Polypeptides 3C chemical shift of C’X polypeptide sequence -GlyGly-X-L-Ala-
residue X L-Ala L-Pro Gb
preda obsdb solvent, ref L-Ala 175.8 (171.8)‘ D,O Cpd= 7.0), 13 L-Pro 174.1 D,O (pd = 6.8), 13 Gb 171.8 (168.7)‘ D,O (Pd= 6.0),13 GlyGly-XGlyClyL-Ala L-Ala 176.3 H,O,14,15 L-RO L-Pro 175.8 H,O,14,15 Gb Gly 172.8 H,O,14,15 tert-butoxycarbonyl-X-L-Pro ala L-Ala 172.6 CDCl,, 17 Gly Gb 168.0 CDCI,, 17 ~o~Y(L-P~o,-L-P~o,G~Y,) L-Pro, 175.2 H,O,18 L-Pro, 173.4 H,O,18 GlY3 169.2 H,O,18 polY(W G ~,-Pro Y ,G~Y 1 Pro3 176.2 0.15 M sodium acetate (ph = ,8), 6 Gly,, Gly, 173.0 0.15 M sodium acetate (Ph = 4.8), 6 Gly,,Gly, 173.0 0.15 M sodium acetate (Ph = 4.8), 16 Gly , 170.5 0.15 M sodium acetate (Ph= 4.8),16 Top to bottom corresponds to increasingly upfield chemical shifts. In ppm downfield from tetramethylsilane (Me,Si). ‘In ppm downfield from Me,Si as reported by Grathwohl and Wuthrichlg for Me,SOd, solutions. (a I
(bi 0
0
Figure 3. A portion of a polypeptide chain containing the sequence -Glyl-Glyi+,-L-Ala,+z-or -Gly,-L-Ala,+l-L-Ala,+z-.
lHz%: H H NO 7 - EFFECT ( +z= t
I
J-EFFECT
(+,:g)
Figure 1. (a) Portion of a paraffinic hydrocarbon chain in the all-trans,
planar zigzag conformation. (b) Newman projection along bond 2 in (a) illustrating the y effect.
i
x2&
.c
i
/
Figure 2. Schematic representation of a polypeptide chain in the planar zigzag conformation where all (&J) = Oo,Oo. We adopt the 1966 def-
inition* for polypeptide rotation angles, because it is consistent with the rotation angle convention used for synthetic polymers (see Figure 1 and ref 9).
In a random coil polypeptide containing the -Cly,&lyi+l- and -Gly,-L-Alai+,- sequences, the conformation ($i rotation) about the N-C* bond of the Gly, residue is independentlo of whether or not residue i 1 is Gly or L-Ala. However, rotation $i+l about the N-C” bond of residue i + 1 clearly is sensitivelo to the nature of residue i 1, Gly,, or L-Alaj+*. From the conformational energy maps1° appropriate to Gly and L-Ala residues in randomly coiling polypeptide chains, we estimate” that C’i in -GlyI-L-Alai+,is gauche to either Cli+,or CP,, more than C’i is gauche to C’i+I in -Glyl-Glyi+,-. Assuming that the y gauche effects (upfield shift) of carbonyl and methyl carbons i + 1 upon carbonyl carbon i are comparable,7*12then we would expect the C’i carbon in -GlyrL-Alai+,to resonate upfield from the C’i carbonyl carbon in -ClyI-Glyi+,-.
+
+
(12) This prediction also holds if the y effect of C@,+, on C’i is larger than the y effect of C’i+l on C’i.
This prediction is borne out by the I3C N M R observations performed on the oligomeric peptides Gly-Gly-X-Ala and Gly-GlyX-Gly-Gly by Richarz and WUthrich13 and Curd et al.I4*I5 As another example, compare the 13Cchemical shifts expected for C’, in -Gly1-L-Ala,+,- and Cli+, in -L-Alai+l-L-Alai+2-. Conformational differences between this pair of sequences are the same as in the pair discussed previously. On the basis of y gauche effects, we might expect Cli+,in L-Alai+l to resonate upfield from C’,in Gly,. The opposite is observed by Richarz and WiithrichI3 and this is a consequence of the j3-CH3 carbon in the L-Alai+l residue. B-CH3,+,and C’i+l are j3 to each other and should result in a substantial downfield shift (deshielding j3 effect)7 of C’i+l in L-Alai+, relative to C’i in Gly,. Apparently this downfield B substituent overwhelms the more prevalent upfield y gauche interactions resulting in the carbonyl carbon C’i+l of +Alai+,+ Alai+~-resonating downfield from the Gly, carbonyl in -Gly+ Ala,+,-. Among the residues in the regularly repeatng polypeptide poly(Glyl-Gly2-Pro3-Gly,), Glyl and Gly, are both succeeded by glycine residues. Consequently, in the random coil conformation the probabilities that their carbonyl carbons are y gauche to the carbonyl carbons of the preceding and succeeding residues are the same leading to the prediction of identical VC,for Gly, and GlY4. Gly, is succeeded by Pro3 whose cyclic side group restricts 4% to ca. 120°, resulting in a fixed y gauche arrangement between C‘Gl,,l and C’& We therefore expect vcw to be upfield (shielded) relative to V C ~ ~ , The deshielding effect of the B-CH2 carbon in the Pro3 side groups should move vCM downfield from the other carbonyl carbon resonances. With increasing field we would expect to find the following order of carbonyl carbon chemical shifts: Y C ~V ,C ~ ~ , , ,
,,,.
(13) R. Richarz and K. Wiithrich, Biopolymers, 17,2133-2141 (1978). (14) P. Keim, R. A. Vigna, R. C. Marshall, and F. R. N. Gurd, J . Biol. Chem., 248, 6104-4113 (1973). (15) P. Keim, R. A. Vigna, A. M. Nigen, J. S.Morrow, and F. R. N. Gurd, J . Biol. Chem., 249, 4149-4156 (1974).
J . Am. Chem. SOC.1980, 102, 7637-7643 V C : ~ , Torchia ~~. and Lyerla16 do observe this order in the random coil polypeptide poly(Gly,-Gly,-Pro3-Gly,) (see Table I). Peptide residues with y substituents in their side chain, such as Val, Leu, Asp, Glu, Met, etc., will generally have carbonyl I3C chemical shifts upfield from the C' resonances in Ala due to the additional y effects incurred through x1rotations (see Figure 2) about their C"-C@ side chain bonds.l3-I5 Several more examples of the residue and sequence dependencies of the carbonyl 13C chemical shifts in random coil polypeptides are presented in Table
I. Aside from the possibility of intra-side-chain y interactions in those residues with long side chains (His, Tyr, Glu, Met, Arg, Orn, Lys, etc.), the side chain /3 carbon is involved in y interactions with the C', 0,and N atoms of its own and the succeeding peptide bond. The backbone rotations 4 and $ (see Figure 2) determine whether or not the C@carbon is gauche to the atoms of these peptide bonds. When 4i = 0, 240' Coi is y gauche to C'i-l; when Jli = 0, 120' Cpi is y gauche to Ni+l; and when $i = 180, 300" CBiis y gauche to Ob It is evident that the I3C chemical shift of a residue's side chain @ carbon should reflect the residue's backbone conformation ($,IC.) via the y gauche effect. As an example, in the conformational transition of a polypeptide from the a-helical to the random-coil state, ($,$) change from = l 2Oo,12O0 (right-handed a-helix) to all the low-energy values encompassed by the usual (4,$) conformational energy maplo of a randomly coiling peptide residue. From the probabilities" that $ = 0, 120, 180, 300' obtained from the conformational energy map appropriate to a randomly coiling peptide residue with a /3-CH2 group in its side chain,1° it is possible to estimate20 that the expected difference in the chemical shift of the side chain /3 carbon in the a-helix and the random-coil states is Auce(a-helix - random coil) = 0 . 6 ( y , ~ , ~ -~ ~ e , ~ ) . From I3C N M R studies of carbon- and nitrogen-substituted alkanes, as described by st other^,^ it is apparent that 7 c . N is significantly larger than yc,o. Clearly then we would expect a (16) (17) (18) (19) (1974). (20)
D. A. Torchia and J. R. Lyerla, Jr., Biopolymers, 13,97-114 (1974). W. Voelter and 0. Oster, Org. Mugn. Reson., 5, 547-548 (1973). R. Di Blasi and A. S . Verdini, Biopolymers, 18, 735-738 (1979). C. Grathwohl and K. Wiithrich, J . Mugn. Reson., 13, 217-225
This estimate of the difference in the chemical shift of C8 in the a-helix and the random-coil conformation ignores t h e y effect of C'i+l on CS, when 6,= 0 or 240°, because the probabilities for these two rotational states are relatively small and because' y p , e y$,o we expect va-& in -L-Ala-L-Pro- to come upfield from the resonance position in -L-Ala-X-. This expectation is confirmed by the 13C @ ,, chemical shifts reported in ppm downfield from Me4% for C in tert-butoxycarbonyl-~-Ala-~-Pro,'~ 16.95 (Me2SO) and 15.55 (DzO), and in -Gly-Gly-~-Ala-~-Ala,'~,~~ 18.2 (Me2SO) and 17.7 (D20). Based on the examples discussed in this report, it seems reasonable to conclude that substituent effects (principally y substituents) on the I3C N M R chemical shifts of the backbone carbonyl and side chain /3 carbons can be utilized to understand the microstructure of polypeptides. Even though the quantitative details remain to be established, the relative I3C chemical shifts of backbone carbonyl and side chain @ carbons already provide us with a means to determine the residue sequence and conformations of polypeptide chains. (21) F. A. Bovey, J . Polym. Sci., Mucro. Rev., 9, 1-81 (1975), and references cited therein. (22) H. J. Lader, R. A. Komoroski, and L. Mandelkern, Biopolymers, 16, 895-905 (1977). (23) H. Saito, T. Ohki, M. Kodama, and C. Nagata, Biopolymers, 17, 2587-2599 (1978). (24) We are assuming that the L-Ala-L-Pro and L-Ala-X- peptide bonds
are both trans in this discussion.
Determination of the Absolute Rates of Decay of Arylcarbenes in Various Low Temperature Matrices by Electron Spin Resonance Spectroscopy V. P. Senthilnathan' and Matthew S. Platz* Contribution from the Department of Chemistry, The Ohio State University, Columbus, Ohio 4321 0. Received April 1 1 , 1980
Abstract: The absolute decay rates of diphenylcarbeneand fluorenylidene have been measured by ESR. The decay is pseudo first order and arises from reaction of the carbene with the glassy or crystalline host. The kinetics are sensitive to the chemical nature of the matrix, the viscosity of the matrix, the concentration of the diazo precursor, and the history of the sample with respect to photolysis. The signal decay is nonexponential due to site problems in the matrix. The decay can be fitted to either a t l / * or 11/3 vs. log I dependence. The predominant carbene decay pathway is by hydrogen atom tunneling through a small barrier. This is indicated by very low Arrhenius parameters and anomalous isotope effects. The kinetic study explains the predominance of hydrogen atom abstraction-recombination products observed by other workers.
The chemistry and spectroscopy of arylcarbenes have been exhaustively studied., The solution chemistry of these species
is best interpreted by two reactive states, a very reactive stereoselective singlet and the less reactive, less stereoselective t r i ~ l e t . ~
0002-7863/80/ 1502-7637$01 .OO/O 0 1980 American Chemical Society
