J. Am. Chem. SOC. zyxwvu 1987, 109, zyxwvu 4473-4477 4473 Conformational Properties of 2,4-Methanoproline zyxwvutsrqponmlkjihgfed (2-Carboxy-2,4-methanopyrrolidine) in Peptides: Evidence for 2,4-Methanopyrrolidine Asymmetry Based on Solid-state X-ray Crystallography, ‘H NMR in Aqueous Solution, and CND0/2 Conformational Energy Calculations zyxwvutsrqponmlkjihgfedcbaZYXW S. Talluri, G. T. Montelione, G. van Duyne, L. Piela,+ J. Clardy, and H. A. Scheraga* Contribution from the Baker Laboratory of Chemistry, Cornel1 University, Ithaca, New York zyxwvutsr 14853-1301. Received November 3, 1986 Abstract: The crystal structure of the terminally blocked amino acid N-acetyl-2,4-methanoproline-N’-methylamide (Ac- 2,4-MePro-NHMe) has been determined by X-ray crystallography zyxwvu (R zyxwvu = 0.05). In the solid state, the Ac-2,CMePro peptide group is trans, and slightly nonplanar (wo = -178.4’). The unit cell was found to contain two conformations of Ac-2,4- MePro-”Me which are characterized by the values of the backbone dihedral angles 9 (i29O) and I ) (=~114.6O), and by distortions of the side-chain 2,4-methanopyrrolidine ring conformation. These two conformations are related to each other as mirror images. ‘H NMR spectroscopy was used to identify the 2,4-methanopyrrolidine asymmetry in Ac-~-Tyr-2,4-Me- Pro-”Me in water. CND0/2 conformational energy calculations on Ac-2,4-MePro-NHMe and related analogues indicate that the preference for asymmetric sidechain conformations and nonzero values of 4 arises primarily from inter-residueinteractions, and particularly from unfavorable interactions between the carbonyl group of the preceding peptide with the carbonyl group of the symmetric 2,4-MePro residue (e.g., unfavorable nonbonded 1-4 C’-C’ interactions). These results indicate that proper modeling of the conformational properties of 2,4-MePro in peptides requires that its backbone (4) and side-chain conformational chirality be taken into account. 1. Introduction Proline and hydroxyproline are unique among the commonly occurring amino acids found in proteins in so far as they are the only ones that form tertiary peptide bonds (rather than secondary peptide bonds) and have cyclic side-chain structures. These structural properties of proline and its derivatives result in unique constraints on the conformational space of sequences containing proline or hydroxyproline.1d For example, L-proline constrains the conformational space of the preceding L-amino acid residue in an amino acid sequence so as to disfavor a-helical backbone conformation^.^^^ On the other hand, X-Pro-Y sequences’ with trans X-Pro peptide bonds and X-Pro sequences with cis X-Pro peptide bonds have a strong tendency to form type-189g and type-VI8 @-bends at Pro-Y3J0 and at X-Pro,6 respectively. These well- understood conformational properties of proline in peptides suggest that chemical analogues of p r ~ l i n e ~ l - ’ ~ may be useful in molecular engineering approaches to designing polypeptides with desired structures and functions. In a recent paper,I5 we described solution N M R studies which demonstrate that replacement of L-proline in small peptides with the bicyclic proline analogue 2,4-methanopr0linel~-~~ results in selective stabilization of the trans tertiary peptide bond confor- mation. These results suggest that 2,4-MePro18 may be a useful L-proline analogue for polypeptide molecular design. For this reason, further studies of the conformational properties of 2,4- MePro in peptides are now in progress in our laboratory. In order to carry out energy calculations to explore the con- formational properties of 2,4-MePro in peptides,19reliable values of the fixed bond lengths and bond angles for 2,4-MePro in a peptide must be obtained from X-ray crystallography. Although a crystal structure for the free amino acid (Le., without peptide bonds) has been reported eIsewhere,l6 these data are inadequate as input for conformational energy calculations since several essential bond lengths and bond angles can be obtained only from the crystal structures of molecules containing peptide bonds (e.g., the C’-N-Ca bond angle for an X-2,CMePro peptide bond cannot be parameterized properly from the structure of the free amino acid). For this reason, the terminally blocked amino acid Ac- 2,4-MePro-NHMeIs was crystallized and its structure was de- + On leave from the Department of Chemistry, University of Warsaw, Warsaw, Poland, 1984-1986. termined by X-ray crystallography. In the course of this analysis, it was observed that the peptide Ac-2,4-MePro-NHMe exists in two conformations. In these conformations, the side chain (2,4- methanopyrrolidine) lacks a plane of symmetry. The two con- formations are mirror images of each other. This has important consequences for the conformational properties of 2,4-MePro in peptides. A method for detecting this conformational distortion in peptides by ‘H N M R is proposed and used to provide evidence for structural asymmetry in Ac-~-Tyr-2,4-MePro-NHMe in aqueous solution. In addition, CNDO/2 calculations are presented and compared with experimental data in order to identify the ~~ (1) Schimmel, P. R.; Flory, P. J. J. Mol. Biol. 1968, 34, 105. (2) Zimmerman, zyxwv S. S.; Pottle, M. S.; Nemethy, G.; Scheraga, H. A. (3) Zimmerman, S. S. Scheraga, H. A. Biopolymers 1977, 16, 811. (4) Nemethy, G.; Scheraga, H. A. Biopolymers 1982, 21, 1535. (5) Visquez, M.; Nemethy, G.; Scheraga, H. A. Macromolecules 1983, 16, 1043. (6) Oka, M.; Montelione, G. T.; Scheraga, H. A. J. Am. Chem. SOC. 1984, 106, 7959. (7) We use the notation X-Pro-Y to designate a tripeptide sequence of L-amino acids, where X always refers to the L-amino acid preceding L-proline, and Y to the L-amino acid following L-proline. (8) The classification of P-bend types used here is described in ref 9. (9) Lewis, P. N.; Momany, F. A,; Scheraga, H. A. Biochim. Biophys. Acfa (10) Chain reversals involving four residues may be characterized by the where i is the index of the (11) Galardy, R. E.; Alger, J. R.; Liakopoulou-Kyriakides, M. Inf. J. (12) Delaney, N. G.; Madison, V. Inf. J. Peptide Protein Res. 1982, 19, (13) Delaney, N. G.; Madison, V. J. Am. Chem. SOC. 1982, 104, 6635. (14) Flippen-Anderson, J. L.; Gilardi, R.; Karle, I. L.; Frey, M. H.; Opella, S. J.; Gierasch, L. M.; Goodman, M.; Madison, V.; Delaney, N. G. J. Am. Chem. SOC. 1983, 105, 6609. (15) Montelione, G. T.; Hughes, P.; Clardy, J.; Scheraga, H. A. J. Am. Chem. SOC. 1986, 108, 6765. (16) Bell, E. A.; Qureshi, M. Y.; Pryce, R. J.; Janzen, D. H.; Lemke, P.; Clardy, J. J. Am. Chem. SOC. 1980, 102, 1409. (17) Hughes, P.; Martin, M.; Clardy, J. Tetrahedron Left. 1980,21,4579. (18) Throughout the text, we have adopted the abbreviation 2,4-MePro for the a-amino acid 2,4-methanop;roline (2-carboxy-methanopyrrolidine), and Ac- and -”Me for terminal acetyl and N-methyl groups, respectively. (19) Piela, L.; NCmethy, G.; Scheraga, H. A. J. Am. Chem. SOC. following paper in this issue. Macromolecules 1977, 10, 1. 1973, 303, 21 1. values of the dihedral angles #i+,, J.i+l, $I,+~. first residue on the amino terminal side of the bend sequence. Peptide Protein Res. 1982, 19, 123. 543. 0002-7863/87/1509-4473$01.50/0 0 1987 American Chemical Society