Proline Peptide Bonds Research Articles

Role of peptidyl-prolyl-cis/trans-isomerases in pathologic processes

Enzymes grouped into a superfamily of peptidyl-prolyl-cis/trans-isomerases (PPIases) possess chaperone activity and fold proteins into active configuration by catalyzing slow cis/trans isomerization on proline-peptide bonds. PPIases are conservative and abundant among Pro-and Eukaryotes. Many proteins involved in disease processes require modification; thus, PPIases can play an important role in pathogenesis. Analysis of the experimental data published so far allows us to classify the involvement of PPIases in pathologic process as follows: (1) Modification of outer membrane porines and channels, components of secretion systems, and secreted proteins by FkpA-like and SurA-like PPIases. Such modification helps to avoid or suppress host cell immune response; (2) Use of host PPIases (like ROC1 of Arabidopsis thaliana) for modification of effector proteins necessary for pathogenesis. Bacterial protein surA participates in suppression of host immune reaction and can be recognized as elicitor of immune response in other host. SurA is essential for formation of bacterial flagella and pile. We suggest that SurA protein can be involved in activation of some effector proteins, while other effectors, for their activation in host cell, may need PPIases of ROC1-type. Viruses and some other obligate parasites use host PPIases for their own protein modification as an essential step for the pathogen multiplication. In this case modified host PPIases are capable to suppress the pathogenesis. That makes the PPIases potential targets for gene therapy.

Proline Conformation-Dependent Antimicrobial Activity of a Proline-Rich Histone H1 N-Terminal Peptide Fragment Isolated from the Skin Mucus of Atlantic Salmon

A 30-residue N-terminally acetylated peptide derived from the N-terminal part of histone H1 was identified as the dominant antimicrobial peptide in skin mucus from Atlantic salmon (Salmo salar). The peptide (termed salmon antimicrobial peptide [SAMP H1]) was purified to homogeneity by a combination of reversed-phase and cation-exchange chromatographies. By Edman degradation of the deacetylated peptide and by sequencing of the PCR-amplified DNA that encodes the peptide, the complete amino acid sequence was determined to be AEVAPAPAAAAPAKAPKKKAAAKPKKAGPS. The theoretical molecular weight of N-terminally acetylated SAMP H1 was calculated to be 2,836, which is the same as that determined by matrix-assisted laser desorption ionization mass spectrometry. The peptide was active against both gram-negative and -positive bacteria. The N-terminal acetyl group was not necessary for activity since deacetylation did not reduce the activity. A synthetic peptide whose sequence was identical to that of the isolated fragment was initially inactive but could be activated by binding it to a cation-exchange column. Treatment of the synthetic peptide when it was bound to the exchange column with peptidylproline cis-trans-isomerase increased the amount of active peptide, indicating that isomerization of the proline peptide bond(s) was necessary for activation of the synthetic peptide. Comparison of the active and inactive forms by circular dichroism and chromatographic analyses suggests that the active form, both the natural and the synthetic forms, is more structured, condensed, and rigid than the inactive form, which has a more nonstructured conformation. This work shows for the first time the importance of proline isomers in the activity of an antimicrobial peptide.

Proline Isomerization in Unfolded Ribonuclease A

Unfolded ribonuclease A (RNaseA) consists of a mixture of fast refolding (UF) and slow-refolding (Us) species. The slow UF in equilibrium Us equilibration reaction is rate-limited by proline peptide bond isomerization. Investigations of the dependence on temperature of the UF in equilibrium Us equilibrium have led to conflicting results and different molecular interpretations. Here the dependence on temperature of the UF:US ratio was reinvestigated by using a new assay for the fast-folding molecules UF. Between 0 degrees C and 60 degrees C the proportion of UF present in unfolded RNase A at 6 M guanidine . HCl was found to be independent of temperature. Consequently, no conclusions can be drawn regarding the role and importance of particular prolines in the UF in equilibrium US transition solely from these results.

Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A

The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 micros to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-micros dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of approximately 80 micros is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.

Conformationally tailored N-[(2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)carbonyl]proline templates as molecular tools for the design of peptidomimetics. Design and synthesis of fibrinogen receptor antagonists.

The proline peptide bond was shown by 2D proton NMR studies to exist exclusively in the trans conformation in benzyl (2S)-1-[[(2S)-2-methyl-6-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl]-2-pyrrolidinecarboxylate [(S,S)-11], benzyl (2S)-1-[[(2S)-2-methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl]-2-pyrrolidinecarboxylate [(S,S)-9], and in the corresponding 6-amino and 7-amino carboxylic acids (S,S)-3 and (S,S)-4. On the other hand, the diastereomers (R,S)-11 and (R,S)-9 containing an (R)[2-methyl-6/7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl moiety, and the diastereoisomers (R,S)-3 and (R,S)-4 incorporating an (R)[6/7-amino-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl moiety were found to exist as equilibria of trans(63-83%) and cis(17-37%) isomers. These conformationally defined templates were applied in the construction of RGD mimetics possessing antagonistic activity at the platelet fibrinogen receptor.

Analysis of the isomeric composition of the proline peptide bond in an angiotensin-converting enzyme inhibitor using capillary electrophoresis

The cis and trans isomeric composition of a proline peptide bond can be determined by routine free-solution capillary electrophoresis measurements provided that one isomeric form is preferentially stabilized by a dissociable ionic group. This capability is illustrated using the angiotensin converting enzyme (ACE) inhibitor (S)-1- N-[1-(ethoxycarbonyl)-3-phenylpropyl]- l-ala- l-pro, which has the trade name enalapril. Electropherograms indicate that the two isomeric forms of enalapril can be separated with baseline resolution at 15 °C using capillary buffers having pH values in the dissociation ranges of the enalapril carboxyl group, p K cis and p K trans of 2.6 and 3.1, and of the enalapril amine group, p K cis and p K trans of 5.9 and 5.6. Such baseline resolution indicates that the isomeric composition does not change during analysis, facilitating measurement of the isomer composition of a sample prior to its injection into the capillary. Thus the effect of pH, ionic strength, or an aprotic solvent on the isomeric composition of enalapril can be measured under uniform analytical conditions. The trans isomer composition changes from 68% in the cationic form, pH <2, to 50% in the isoelectric form, pH ∼4.5, to 60% in the anionic form, pH >7. Addition of salt to the isoelectric form or addition of an aprotic solvent to any form prior to analysis increases the trans isomer composition. Similar analyses can be made using the alternative ACE inhibitors captopril and enalaprilat.

Solid-state NMR investigation of the buried X-proline peptide bonds of bacteriorhodopsin.

The role of proline residues in the photocycle of bacteriorhodopsin (bR) is addressed using solid-state NMR. (13)C and (15)N chemical shifts from X-Pro peptide bonds in bR are assigned from REDOR difference spectra of pairwise labeled samples, and correlations of chemical shifts with structure are explored in a series of X-Pro model compounds. Results for the three membrane-embedded X-Pro bonds of bR indicate only slight changes in the transition from the resting state of the protein to either the early or late M state of the protonmotive photocycle. These results suggest that the buried prolines serve a principally structural role in bR.

1H-NMR parameters of common amino acid residues measured in aqueous solutions of the linear tetrapeptides Gly-Gly-X-Ala at pressures between 0.1 and 200 MPa

For the interpretation of chemical shift changes induced by pressure in proteins, a comparison with random-coil data is important. For providing such a data basis, the pressure dependence of the 1H-NMR chemical shifts of the amino acids X in the random-coil model peptides Gly-Gly- X-Ala were studied for the 20 common amino acids at two pH values (pH 5.0 and 5.4) at 305 K, in the pressure range from 0.1 to 200 MPa. The largest shift changes Δδ with pressure p can be observed for the backbone amide protons. The average linear pressure coefficient δ Δp is 0.38 ppm GPa −1, with a root mean square deviation of 0.2 ppm GPa −1. In contrast to the downfield shift typical for amide protons, the H α-resonances typically shift upfield, with a pressure coefficient of −0.025 ppm GPa −1 and a root mean square deviation of 0.05 ppm GPa −1. The side chain resonances are only weakly influenced by pressure, on average they are shifted by 0.014 ppm GPa −1 , with a root mean square deviation of 0.14 ppm GPa −1. The exceptions are the side chain amide protons of asparagine and glutamine. Here, values of 0.214 (Asn H δ21), 0.417 (Asn H δ22), 0.260 (Gln H ε21) and 0.395 (Gln H ε22) ppm GPa −1 can be observed. In both cases, the pressure dependent shift is larger for the pro-E proton than for the pro-Z proton. Within the limits of error the equilibrium constant for the trans- and cis-conformers at the proline peptide bond is independent of pressure in the pressure range studied.

Preferred proline puckerings in cis and trans peptide groups: implications for collagen stability.

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.

Influence of chirality of the preceding acyl moiety on the cis/trans ratio of the proline peptide bond.

We report that the cis/trans ratio of the proline peptide bond can be strongly influenced by the chirality of the acyl residue preceding proline. Acyl moieties derived from (2S)-2,6-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid (8) and (2R)-3-methoxy-2-methyl-2-(4-methyl-2-nitrophenoxy)-3-oxopropanoic acid (5) in acyl-Pro molecules influence isomerization of the proline peptide bond constraining the omega dihedral angle to the trans orientation. Structures of benzyl (2S)-1-([(2S)-2,6-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl)-2-pyrrolidinecarboxylate (3) derived from 2D (1)H NMR conformational analysis and crystallographic data exhibit only the trans conformation of proline peptide bond. On the other hand the diastereomer 4, which contains an (R) acyl moiety, exhibits two sets of signals in (1)H NMR spectra. The signals were assigned to trans (72%) and cis (28%) conformers. Crystallographic analysis of 4 showed that only the cis conformation is present in the crystalline state. The (1)H NMR chemical shift pattern of three sets of signals observed in 2 was observed also in benzyl (2S)-1-[(2R/S)-3-methoxy-2-methyl-2-(4-methyl-2-nitrophenoxy)-3-oxopropanoyl]-2-pyrrolidinecarboxylate. (R)-Carboxylic acid 5, after coupling with (S)-ProOBn, yielded benzyl (2S)-1-[(2R)-3-methoxy-2-methyl-2-(4-methyl-2-nitrophenoxy)-3-oxopropanoyl]-2-pyrrolidinecarboxylate (6), which in DMSO-d(6) exhibited only the trans conformation of the proline peptide bond. These results suggest that in these particular cases acyl-Pro peptide bond isomerization is strongly influenced by the stereochemistry of the acyl residue preceding proline. (2S)-2,6-Dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid (8) and (2R)-3-methoxy-2-methyl-2-(4-methyl-2-nitrophenoxy)-3-oxopropanoic acid (5) are promising chiral peptidomimetic building blocks that can be used as acyl moieties to force the proline peptide bond into the trans conformation in a variety of acyl-Pro molecules.

Synthesis of optically pure N-Boc-protected (2 R,3 R)- and (2 R,3 S)-3-fluoroprolines

Non-protein amino acids (2 R,3 R)- and (2 R,3 S)-3-fluoroprolines were synthesized as novel probes for studying the cis/ trans isomerization of the amino acylproline peptide bond. The N-Boc-protected target compounds were obtained as optically pure material starting from (2 S,3 S)-3-hydroxyproline.

Intramolecular Catalysis of the Cis−Trans Isomerization of Proline Peptide Bonds in Cyclic Disulfide-Containing Peptides

Intramolecular Catalysis of the Cis−Trans Isomerization of Proline Peptide Bonds in Cyclic Disulfide-Containing Peptides

Hydrolysis Mechanism of the Phenylalanine−Proline Peptide Bond Specific to HIV-1 Protease: Investigation by the ab Initio Molecular Orbital Method

The protein hydrolysis mechanism by the HIV-1 protease (HIV-1 PR) was studied using ab initio molecular orbital calculations with a model compound. The initial model compound was constructed based on the result of a 100 ps molecular dynamics (MD) simulation of the enzyme−substrate (ES) complex under physiologic conditions, and consists of an acetate/acetic acid pair for the two catalytic-site Asp residues, a water molecule acting on the hydrolysis, and a dimethylacetamide for the substrate. This study suggests that the hydrolysis mechanism of the Phe-Pro peptide bond specific to the HIV-1 PR consists of three elementary reactions: first, the reaction of the formation of the amide hydrate intermediate; second, the reaction of the protonation of the proline nitrogen of the substrate; and third, the reaction of the C−N bond cleavage of the substrate. The rate-determining step is the protonation of the substrate proline nitrogen, and its activation energy is 23.95 kcal/mol. This result strongly suggests that protein hydrolysis by the HIV-1 PR occurs in vivo.

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed α-β topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three β-stands and to the first α-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 °C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.

Structural characterization and optimization of antibody-selected phage library mimotopes of an antigen associated with autoimmune recurrent thrombosis.

The presence of high titers of anti-cardiolipin antibodies (ACA's) of autoimmune origin, which are known to bind to plasma beta2-glycoprotein I (aka apolipoprotein H), correlates clinically with autoimmune recurrent thrombosis. Soluble beta2-glycoprotein I binds to solid-phase ACA (immobilized on a surface plasmon resonance chip) with a Kd of 1.4 microM, but if the reactants are reversed and beta2-glycoprotein I is on the solid-phase support, then the Kd is 52 nM. This 27-fold difference in affinity reflects the avidity/entropic advantage obtained for an antibody binding to an antigen that is made multivalent because it is attached to a solid phase. A mimotope of this antigen, selected from a phage display peptide library screen with an ACA, has been shown to bind to solid-phase ACA as a phage, using surface plasmon resonance. This peptide is representative of the motif from 37 peptides obtained in a previously reported phage library screen with this ACA (1). A synthetic version of this peptide, referred to as P4, has the sequence: A1G2P3C4I5L6L7A8R9D10R11C12P13G14, and binds to its selecting antibody with a Kd of 42 nM. NMR data indicate that proline-13 is present in both cis and trans configurations, and that these two geometries dramatically affect the overall tertiary structure of the molecule. The peptide lacking this proline binds severalfold better to the ACA, consistent with at least one of these structures having low affinity for binding ACA. Replacement of the arginine-9 position with a proline decreases binding affinity to ACA 10-fold. Another phage library-selected peptide has a proline in position 9, but also has a leucine in position 5, instead of isoleucine. Since its affinity for ACA is nearly as good as that for peptide P4, the phage library screening must have selected for a non-beta-branched amino acid in this position to compensate for the adverse effects of the arginine-9 to proline-9 substitution. The solution structure of a modified version of the antibody-selected phage peptide P4 with the central proline was determined. This peptide has one turn comprised of Ala-Pro-Asp-Arg, with the proline peptide bond in the cis configuration, and another turn that contains the disulfide and adjacent residues. If the disulfide is replaced by a thioether, and the central proline by an alpha-methyl proline, in an attempt to make the peptide more biologically stable, there is little adverse effect on affinity for ACA. The thioether bond/turn is fairly well defined with a Calpha to Calpha separation of 4.9 +/- 0.8 A. The alpha-methyl proline adopts the trans configuration, and this central Ala-(alpha-methyl-Pro)-Asp-Arg turn adopts a distorted type I turn conformation with a probable i to i+3 hydrogen bond. Modeling studies suggest that the proline peptide bond configuration switched from cis to trans in the presence of the alpha-methyl group on proline because of steric hindrance with the beta-carbon of the preceding residue. Overall, this peptidomimetic molecule is structurally very similar to the peptide with natural amino acids, with an rmsd difference of only 1.37 A, when comparing backbone atoms.

Local control of peptide conformation: stabilization of cis proline peptide bonds by aromatic proline interactions.

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constant, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database.

Effect of prolyl isomerase on the folding reactions of staphylococcal nuclease.

The low-temperature fluorescence-detected refolding of staphylococcal nuclease (SNase) can be described by three slow kinetic phases. The slowest phase is absent in the P117G mutant of SNase. Peptidyl prolyl cis-trans isomerase (cyclophilin), which has been shown to catalyze the slow folding reactions of some proteins, was employed to determine which of the refolding reactions of SNase and P117G SNase involve proline isomerization. We report here that all three folding phases of the wild type and the slower phase of P117G SNase are catalyzed by prolyl isomerase, indicating that proline isomerization is involved in these fluorescence-detected phases in the refolding of SNase. Since the rates of these phases are denaturant-dependent, we conclude that the slow folding steps involve isomerization of non-native cis proline peptide bonds and are tightly coupled to denaturant-sensitive structural changes.

Remarkably slow folding of a small protein

Equilibrium denaturation of the 72 amino acid α/β-protein MerP, by acid, guanidine hydrochloride, or temperature, is fully reversible and follows a two-state model in which only the native and unfolded states are populated. A cis- trans equilibrium around a proline peptide bond causes a heterogeneity of the unfolded state and gives rise to a slow- and a fast folding population. With a rate constant of 1.2 s −1 for the major fast folding population, which has none of the common intrinsically slow steps, MerP is the slowest folding protein of this small size yet reported.

UV resonance Raman spectroscopy and hydrogen bonding of the proline peptide bond

UV resonance Raman (UVRR) spectroscopy with 210 nm laser excitation enhances the ≈ 1460 cm −1 CN stretching vibration of the X-Pro peptide bond, which is designated as amide IIp. Studies of dipeptides reveal that the amide IIp frequency is insensitive to proline cis/trans isomerization and to pyrrolidine ring puckering, but is sensitive to hydrogen bonding of the X-Pro carbonyl group. A 9 cm −1 downshift in the amide IIp frequency of Gly-Pro when the pH is raised from 6.5 to 10.5 is shown to result from the loss of an intramolecular hydrogen bond from the terminal amino group, since this shift is absent when the N-terminus is blocked by a t-BOC group. Studies of solvent hydrogen bonding to N-acetylpyrrolidine (NAP), a convenient model of the X-Pro bond, showed a linear increase with solvent acceptor number for amide IIp and a concomitant decrease in the CO stretching vibration (amide Ip). The amide IIp band profile is reported for native and unfolded states of cytochrome c, myoglobin and lysozyme. Comparison with X-ray crystallographic coordinates indicates that the amide IIp frequency is ≈ 1460 cm −1 for strongly hydrogen bonded X-Pro carbonyl groups, but decreases to ≈ 1445 cm −1 in the absence of hydrogen bonding. The observed frequency is ≈ 1460 cm −1 for the unfolded proteins, reflecting strong hydrogen bonding from solvent molecules.

Conformationally Restricted TRH Analogs: The Compatibility of a 6,5-Bicyclic Lactam-Based Mimetic with Binding to TRH-R

A pair of conformationally restricted TRH analogs have been synthesized. Both analogs have the central histidine of TRH replaced by a cyclohexylalanine unit, and both analogs contain a 6,5-bicyclic lactam ring fusing the proline peptide bond into a trans conformation. The analogs differ at the bridgehead stereocenter. The synthesis of the analogs utilized an anodic amide oxidation reaction to selectively functionalize a proline derivative and a titanium tetrachloride-initiated cyclization−rearrangement sequence to assemble the desired bicyclic ring skeleton. The analogs were compared with the unrestricted cyclohexyalanine-containing TRH analog (cyclohexyl-Ala2-TRH) in order to determine how the added lactam ring affected the affinity and the potency of the analog for TRH-R. Both the affinity and potency of the restricted analogs were found to be critically dependent on the bridgehead stereochemistry of the bicyclic ring. The analog having R stereochemistry at the bridgehead was 478 times more potent than ...