Effect of aromatic amino acid substitutions in the 3-position of cyclic β-casomorphin analogues on μ-opioid agonist/δ-opioid antagonist properties*

The β-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed μ agonist/δ antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37 , 1136-1144]. The 2-Na1³ residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bz1) (4), Tyr(Bz1) (5), 4′-benzoylphenylalanine (Bpa) (6), 4′-benzylphenylalanine (Bzp) (7), thyrnine (Thy) (8), thyroxine (Thx) (9), 4′-biphenylalanine (Bip) (10), 4′-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in μ and δ receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the μ receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the δ receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective δ antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by δ receptor activation. The Bzp³- and Bip³-containing peptides 7 and 10 turned out to be μ antagonists against the μ selective agonist H-Tyr-D-Ala-Phe-Phe-NH₂, in the GPI assay. The other analogues were weak partial μ agonists which displayed remarkably decreased μ receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be δ antagonists in the MVD assay. Analogues 4 and 9 exhibited δ antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher δ antagonist potency against DPDPE and deltorphin I and, in most cases, increased δ receptor affinity. These results indicate that the & delta; receptor tolerates bulky aromatic side chains in the 3-position of cyclic β-casomorphin analogs with either δ agonist or δ antagonist properties. However, these compounds displayed drastically reduced μ receptor affinity in nearly all cases. © Munksgaard 1996.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing α-aminoisobutyric acid

The conformation in aqueous solution of several α-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and ¹H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro² and/or Pro³ in BK stabilizes a degree of β-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB⁷]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a β-turn involving their common Arg¹-Pro²-Pro³-Gly⁴ moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis¹ Pro and the propensity for a β-turn at the N-terminus of the peptide.     

The receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related δ-opioid antagonists contains all trans peptide bonds

Two different models for the receptor-bound conformation of δ-opioid peptide antagonists containing the N-terminal dipeptide segment H-Tyr-Tic (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic² aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ-antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic² residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H-Tyrψ[CH₂NH]. Tic-Phe-Phe-OH and H-Tyrψ[CH₂NH]. MeTic-Phe-Phe-OH (MeTic = 3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ-opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low-energy conformations in which the torsional angle (“ω₁”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low-energy conformations in which “ω₁” had a value of 0°(cis conformation). Conformers with that same bond in the gauche- conformation (“ω₁”= -60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor-bound conformation of δ-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.     

Distinct conformational preferences of three cyclic β-casomorphin-5 analogs determined using NMR spectroscopy and theoretical analysis

The conformational properties of three cyclic β-casomorphin analogs based on the general formula H-Tyr-c[-D-Orn-2-Nal-D-Pro-Xaa-] (2-Nal = 2-naphthylalanine; Xaa = D-Ala, Sar or NMe-Ala) in DMSO solution were investigated using NMR spectroscopy in conjunction with molecular modeling techniques. The D-Ala⁵- and Sar⁵-analogs (compounds 1 and 2, respectively) are potent mixed μ-agonist/§-antagonists with high μ- and §-opioid receptor affinities, whereas the NMe-Ala⁵-analog (compound 3) is a potent μ-agonist and a weak partial §-agonist. Distinct conformational differences emerged for the three compounds studied. Flexibility in the bare ring structures was found to increase in the order 3<2<1. The increased structural rigidity of 3 may be responsible for its low §-receptor affinity as compared to the two other analogs. A low fractional population of conformers containing two cis peptide bonds was found for compound 2 but not for analog 1 or 3. Initial evidence for this observation was obtained from NMR differential line-broadening experiments and later confirmed by molecular mechanics simulations. Comparison of the temperature dependence of amide proton chemical shifts acquired for the three cyclic analogs indicate a large degree of intramolecular hydrogen bonding for 1 but not for the other two peptides. © Munksgaard 1996.     

Testing for cis‘ proline with α-aminoisobutyric acid substitution

Substitution of Pro residues with AIB (α-aminoisobutyric acid) residues in peptides provides a means of evaluating the presence of cis' proline conformations both in solution and, using bioassay data, in a receptor complex. ¹ H n.m.r. has been used to probe the DMSO solution conformation of all seven of the possible AIB/Pro isomers of bradykinin. AIB substitution for Pro² and/or Pro³ appears to stabilize a type III β-turn involving the N-terminal residues, but not an incipient 3₁₀ helix suggested by model peptides. These substitutions are correlated with low biological potencies, suggesting that such conformational features may be incompatible with receptor complexation. Alternatively, AIB⁷ -bradykinin analogs exhibit a variety of long range shift perturbations relative to bradykinin. The data suggests that bradykinin can adopt several folded conformations, including β-turns involving both Ser⁶-Pro⁷-Phe⁸-Arg⁹ and Phe⁵-Ser⁶-Pro⁷-Phe⁸. The relatively high biological activities of the AIB⁷-BK suggest that the complexed form of the peptide is characterized by a cis' Pro⁷ conformation.     

Conformational mimicry II. An obligatory cis amide bond in a small linear pep tide

The structure of Z-Proψ[CN₄]-Ala-OBzl has been determined by X-ray crystallographic techniques. The structure crystallizes in space group P2₁ with cell constants a = 22.176(3) Å, b = 6.141(1)Å, c = 8.275(1) Å, β= 98.31(1), and Z = 2. The structure has been refined to a residual of 0.038 for 2538 independent data. The amide bond between the prolyl and alanyl residues is cis, a result of the presence of the tetrazole ring system, as is the urethane bond linking the benzyloxycarbonyl and the prolyl groups. A comparison of the structures in this study to other structures containing cis amide bonds shows that the tetrazole ring system, when incorporated into peptides, mimics a cis amide bond. Changes in the distance between the α-carbons adjacent to the tetrazole rings in the linear peptide as compared with the bicyclic diketopiperazine required a reassessment of the conformational mimicry with the cis amide bond.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.     

SPECTROSCOPIC STUDY OF THE CONFORMATIONS OF PROLINE-CONTAINING OLIGOPEPTIDES IN THE CRYSTALLINE STATE AND IN SOLUTION

A conformational study has been carried out on a series of linear proline-containing oligopeptides (ZGP, ZGPL, ZGPLG and ZGPLGP) in both the crystalline state and in DMSO-d₆, solution, using Raman and n.m.r. spectroscopy. The amide I and HI bands in the Raman spectra of the crystalline forms indicate the presence of a type I β-bend conformation in ZGPLG and ZGPLGP, but not in ZGP and ZGPL. This result is in agreement with X-ray data on these mole cules. The Raman spectra of these peptides in solution indicate that more than one conformation is present, i.e. that the β-bend structure of the solid form of ZGPLG and ZGPLGP is destabilized by DMSO-d₆. ¹³C and ¹H n.m.r. data also demonstrate the presence of more than one conformation in ZGP, ZGPL, ZGPLG and ZGPLGP in DMSO-d₆ solution. These isomers differ in their con formation (cis and trans) about their Gly-Pro peptide bonds and possibly about the Cα-C' bond of the C-terminal proline in ZGPLGP. For ZGP, ZGPL, ZGPLG and ZGPLGP, the ensemble of conformations in DMSO-d₆ includes C₅ and C₇ hydrogen-bonded rings; in addition, ZGPLGP may contain a small percentage of a β-bend conformation (at Pro₂-Leuj₃) with trans peptides in both Gly-Pro moieties.     

Conformations and conformational interconversions of diastereomeric cyclic tetraprolines

Cyclic tetrapeptides exclusively composed of L- and D-Pro have been studied by theoretical means (conformational searches and molecular mechanics calculations using the CHARMM program) supported by ¹H-NMR spectroscopy, X-ray analysis and chiroptical measurements. We explored the entire conformational space of the diastereomers cyclo(LLLL-Pro₄) (I), cyc1O(LDLD-Pro₄) (II) and CYClo(LLDD-Pro₄) (III) including the low-energy conformations and the related interconversion paths. The conformational interconversions were found to be restricted to cis/trans isomerisations of the amide bonds. Owing to the polycyclic nature of cyclo(Pro₄) most of the cis/trans transitions are hindered by energy barriers higher than 30 kcal/mol (up to 150–200 kcal/mol). A few transitions are characterized by computed energy barriers comparable to those found in linear -Xxx-Pro- sequences (～ 18 kcal/mol), and are therefore experimentally significant. Experimental evidence has been obtained in the case Of CyClo(LDLD-Pro₄), where two enantiomers are interconverted by a series of 4 cis/trans isomerisations ctct→cttt→tttt→tctt→tctc. The Eyring activation parameters of this reaction were determined in H₂O and in DMF by chiroptical measurements (ΔH^#= 44 and 28 kcal/mol; ΔS^#=59 and 22 cal K ^?1 mol^?1, respectively), and correlated with the calculated barriers. In I and III comparable series of four cis/trans isomerisations relate two main conformations with the peptide bond sequences ctct and tctc. In compound I pseudorotational images are interconverted via ctct→ccct→cctt→cctc→tctc. The pathway ctct→ccct→cctt→cctc→tctc. that relates diastereomeric main conformations of III involves exclusively low-energy intermediates; however, the transitions leading to the all-cis conformation are energetically unfavourable, and the conformational space is divided in three insulated domains.     

The pseudo-βI-turn

Synthesis and conformational analysis of three cyclic hexapeptides cyclo(-Gly¹-Pro²-Phe³-Val⁴-Xra⁵-Phe⁶), Xaa= Phe (I), D-Phe (II) and D-Pro (III), were carried out to examine the influence of proline on the formation of reverse turns and the dynamics of hydrophobic peptide regions. Assignment of all ¹H and ¹³C resonances was achieved by homo- and heteronuclear 2D-NMR techniques (TOCSY, ROESY, HMQC, HMQC-TOCSY and HMBCS-270). The conformational analysis is based on interproton distances derived from ROESY spectra and homo- and heteronuclear coupling constants (E.COSY, HETLOC and HMBCS-270). For structural refinements restrained molecular dynamics (MD) simulations in vacuo and in DMSO were performed. Each peptide exhibits two conformations in DMSO solution due to cis-trans isomerism about the Gly-Pro peptide bond. Surprisingly the cis-Gly-Pro segment in the minor isomers is not involved in a βVI-turn, but forms a turn structure with cis-Gly-Pro in the i and i+ 1 positions. Although no stabilizing hydrogen bond is found in this turn, the φ and ψ-angles closely correspond to a βI-turn [Pro²:φ(i+ 1) -60°, ψ(i+ 1) -30° Phe³: φ(i+ 2) -100°, ψ(i+ 2) -50°]. Hence we call this structural element a pseudo-βI-turn. As expected, in the dominating all-trans isomers proline occupies the i+ 1 position of a standard βI-turn. Therefore, cis-trans isomerization of the Gly¹-Pro² amide bond only induces a local conformational rearrangement, with minor structural changes in other parts of the molecule. However, the geometry of the other regions is affected by the chirality of the i+ 1 amino acid for both isomers (βI for Phe⁵, βII′ for D-Phe⁵ or D-Prp⁵).     

Survey of conformational role of ester bonds in a cyclic depsipeptide

The effect of ester bond on the conformation of peptide molecule was studied by designing and synthesizing a model tetradepsipeptide cyclo(-l -Ala-l -Hmb-)z and by analyzing the conformation both theoretically and experimentally. Theoretical analysis showed that both ester and peptide bonds in the calculated low-energy conformations within 3 kcal/mol of the global minimum take a trans but distorted configuration. The distortion is larger in ester bonds than in peptide bonds. Further, the four carbonyls project from one side of the plane of the cyclic backbone, whereas the side chains project from the other side. These results are consistent with the experimental results obtained by NMR measurement; first, the coupling constant deduced from ¹H-NMR species in DMSO-d₆ is consistent with the dihedral angles of the calculated low-energy conformations; second, results of NOE measurement can reproduce the calculated configuration of the carbonyls and side chains. From the consistency between theoretical and experimental results, it is concluded that this model tetradepsipeptide takes an all-trans backbone conformation in solution and this backbone conformation is stabilized by large distortion in the ester bond, which compensates the strain resulted from the 12-membered cyclic backbone structure consisting only of L-residues.     

Combined solid‐phase/solution synthesis of large ribonuclease A C‐terminal peptides containing a non‐natural proline analog*

Abstract: Three large peptides corresponding to the 65–124 (60‐mer), 72–124 (53‐mer), and 77–124 (48‐mer) sequence of bovine pancreatic ribonuclease A (RNase A) were assembled from either two or three shorter protected peptide fragments by chemical coupling in solution. The fragments were synthesized manually by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide chemistry in plastic syringes, and subsequently purified by normal‐phase high‐performance liquid chromatography on a silica gel column. The main aim of this work was to incorporate sterically hindered l ‐5,5‐dimethylproline (dmP) as a substitute for Pro⁹³ into the sequence of RNase A in order to constrain the –Tyr⁹²‐Pro⁹³– peptide group to a single cis‐conformation.     

Probing opioid receptor–ligand interactions by employment of indolizidin‐9‐one amino acid as a constrained Gly2‐Gly3 surrogate in a leucine‐enkephalin mimic

Abstract: The relationship between the conformation and biological activity of Leu‐enkephalin was studied using (2S,6R,8S)‐9‐oxo‐8‐N‐(Boc)amino‐1‐azabicyclo[4.3.0]nonane‐2‐carboxylic acid [(2S,6R,8S)‐ 1 , I⁹AA] as a constrained Gly²‐Gly³ dipeptide surrogate. [I⁹AA]^2,3‐Leu‐enkephalin 12 was assembled using solid‐phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I⁹AA]^2,3‐Leu‐enkephalin 12 exhibited affinities for the µ‐ and δ‐opioid receptors that were three orders of magnitude lower than that of Leu‐enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and ³J_NH‐CαH coupling constants for 12 could not support a hydrogen‐bonded β‐turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu‐enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.     

5,5-Dimethylthiazolidine-4-carboxylic acid (DTC) as a proline analog with restricted conformation

Spectroscopic evidence is presented for the lack of intramolecular hydrogen bonding in a simple peptide derivative of 5,5-dimethylthiazolidine-4-carboxylic acid (Dtc). The infrared spectrum of Boc-Pro-Ile-OMe 1 in nonpolar solvents displays two N-H stretching bands at 3419 and 3330 cm^-1 in CCl₄ and one at 3417 and 3328cin^-1 in CHCl₃. The low frequency band at 3328–3330cm^-1 may be assigned to conformations with an intramolecular hydrogen bond between the Ile N-H and Boc C=O. The band at 3417-3419 cm^-1 is the normal Ile N-H stretch. In the polar solvent CH₃ CN only one NH stretching band at 3365 cm^-1 is observed. The IR spectrum of Boc-Dtc-Ile-OMe 2, on the other hand, displays one N-H stretching band at 3423cm^-1 in CCI, and one at 3418cm^-1 in CHCI₃. The IR spectrum of 2 does not display the N-H stretching band that would arise from intramolecular hydrogen bonding between the Boc C=O and Ile N-H. The lack of intramolecular hydrogen bonding for Boc-Dtc-Ile-OMe 2 was evident also in the NMR spectra in nonpolar solvents. The ¹H-NMR spectrum of the Pro dipeptide 1 in 50% CDCl₃/C₆D₆ at 20° displayed two Ile-NH signals at 6.58 and 7.74 ppm. The latter signal corresponds to the intramolecularly hydrogen bonded Ile-NH in the trans-Boc isomer of 1 (60% of the total population), while the former signal corresponds to the nonhydrogen bonded Ile-NH in the cis-Boc isomer. The ¹H-NMR spectrum of the Dtc dipeptide 2 displayed two slowly exchanging cis- and trans-Boc amide isomers as well, but both amide proton resonances were observed upfield at 6.67 and 6.74 ppm, which correspond only to a nonhydrogen bonded Ile N-H. The X-ray crystal structure of Boc-Dtc displays only a cis-Boc-Dtc urethane amide group and two conformations for the Dtc ring, one in which the beta carbon atom is anti to the carboxyl group and the other in which the gamma sulfur atom is anti to the carboxyl group. Conformational analysis of Ac-Dtc-NHMe suggests that in the hydrogen bonded C7 conformation steric interaction between the syn-beta methyl group and carbonyl group of Dtc adds nonbonded and angle strain energies to counteract the stabilizing coulombic interaction between the Boc C=O and terminal amide N-H. Whereas the C7 conformation is a prominent conformation for peptide derivatives of proline, other conformations are favored in peptide derivatives of Dtc (ψ -ñ 110-150° or ñ 320-360°). These results suggest that, in peptides where substitution of Pro appears to maintain or enhance biological activity, the substitution of Dtc for Pro may test the functional importance of the C7 conformation in that position of the peptide sequence.     

Solution conformations of oligomers of α -aminoisobutyric acid°

The N-acetyl(Aib)nN' -methylamides (with n= 1, 2 and 3) and the N-acetyl-(Aib)3 methyl ester have been synthesized using an oxazolone procedure. An experimental conformational analysis of this series of oligomers has been carried out in water, DMSO-d6 and CDCI3 using n.m.r. techniques, and in chloroform using i.r. spectroscopy. Deuterium exchange rates of amide protons in DMSO-d6 and the rates of change of these proton chemical shifts with temperature in water, DMSO-d6 and CDCI3 indicate that the oligomeric N'-methylamides adopt conformations that have no hydrogen bonds when n = 1, one hydrogen bond when n = 2, and two hydrogen bonds when n = 3, and that Ac(Aib)3OMe has a conformation with one hydrogen bond. An analysis of the N-H stretching region of the i.r. spectra of these compounds in CHCI3 also suggests the existence of these conformational states. These data imply that the peptides adopt the 310-helical and not the α-helical conformation in solution. This conclusion supports the hypothesis that the Aib residue has asymmetric geometry at the C^α atom in solution, similar to that reported in the literature for the crystalline state.     

Cyclic Opioid Peptide Agonists and Antagonists Obtained Via Ring‐Closing Metathesis

The opioid peptide H‐Tyr‐c[D‐Cys‐Phe‐Phe‐Cys]NH₂ cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′‐dimethyltyrosine (Dmt), 3‐[2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid (Dhp) or (2S)‐2‐methyl‐3‐(2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid [(2S)‐Mdp] in the 1‐position were prepared. The peptides were synthesized on solid‐phase by substituting d ‐allylglycine and (2S)‐2‐amino‐5‐hexenoic acid in position 2 and 5, respectively, followed by ring‐closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated –CH₂–CH₂– bridged peptides. All six Tyr¹‐ and Dmt¹‐dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp¹‐ and (2S)‐Mdp¹‐dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15‐membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe³ and Phe⁴ side chains.     

Dipole interaction model predicted π−π* circular dichroism of cyclo(l‐Pro)3 using structures created by semi‐empirical,ab initio,and molecular mechanics methods

Abstract: Cyclo(l ‐Pro)₃ (CP3) is a synthetic peptide created to model cis and torsionally strained peptide bonds that also exhibits a strong distinctive UV circular dichroic (CD) spectrum. Circular dichroic spectra were computed for the amide π ? π* transition using the dipole interaction model for various conformations of the peptide. Conformations of CP3 were created initially from crystal data, and followed by energy minimizations via molecular mechanics using the cvff force field; the effects of additional geometric optimizations by semi‐empirical and ab initio quantum mechanics were investigated. The CD spectra for each conformation were calculated using a variety of different parameters, and each result was compared with the published experimental spectrum [Deber, C.M., Scatturin, A., Vaidya, V.M. & Blout, E.R. (1970) Small cyclic proline peptides: UV absorption and CD. In: Peptides: Chemistry and Biochemistry, Proceedings of the First American Peptide Symposium (Weinstein, B., ed.), Marcel Dekker, New York pp. 163–173]. Herein, two distinct conformations, a C₃ symmetric and an asymmetric form, gave CD predictions that separately did not resemble the experimental spectrum. Energy differences were predicted at various theoretical levels, including MP2 and density functional theory. When the predicted CD spectra for each conformation were multiplied by Boltzmann weighting factors created using heats of formation determined by the AM1 optimizations, the weighted composite CD spectrum created did resemble experiment for the π ? π* transition indicating that both conformations may exist simultaneously in solution.     

Theoretical conformational analysis and synthesis of analogues of the heptapeptide antibiotic K-582 A

A detailed theoretical conformational analysis of the linear heptapeptide antibiotic [Arg²]K-582 A (Arg-Arg-D-Orn-Thr-D-Om-Lys-D-Tyr) was carried out. The results of the computer simulation suggest that the linear peptide has a high propensity to fold in solution into a quasi-cyclic conformation in equilibrium with π(L-D) helices. The synthesis of two inactive analogues with an L-Lys in place of D-Orn³ or D-Orn⁵ confirms the importance of the proposed folding pattern for the occurence of the antimicrobial activity of K-582 A.     

PREFERRED CONFORMATION OF THE tert-BUTOXYCARBONYLAMINO GROUP IN PEPTIDES

Structural parameters, derived from X-ray crystallographic data, have been compiled for amino acid and linear peptide derivatives which contain the N-terminal tert-butoxycarbonyl (Boc) group or its next higher homolog, the tert-amyloxycarbonyl group. The comparison of the geometry of the urethane group in Boc-derivatives with that of the peptide group shows small differences in bond angles about the trigonal carbon, because of altered interactions when a CαH group of a peptide unit is replaced by an ester oxygen. In contrast to the strong preference of the peptide bond for the trans form (except when it precedes proline), the urethane amide bond adopts both the cis and trans conformations in crystals. The cis urethane conformation is preferred in crystals of compounds with a tertiary nitrogen (such as Boc-Pro) or in structures stabilized by strong intermolecular interactions. Conformational energy computations on Boc-amino acid N'-methylamides indicate that the trans and cis conformations of the urethane amide bond have nearly equal energies (even for amino acids other than proline), in contrast to the peptide bond, for which the trans conformation has a much lower energy. The computed increase of the cis content in Boc-amino acid derivatives (as compared with the corresponding N-acetyl derivatives) is consistent with the observed distributions of conformations in crystal structures and with n.m.r. studies in solution. Usually, the substitution of a Boc for an N-acetyl end group does not alter the conformational preferences (as indicated by φ, Ψ values and relative energies) of the amino acid residue which follows the end group when the amide bond is trans. Particular conformations, however, can be stabilized by strong attractive interactions between some side chains (e.g. that of phenylalanine) and the bulky Boc end group.     

Design of peptides with αβ-dehydro residues: Synthesis,crystal structure and molecular conformation of N-Boc-L-Ile-ΔPhe-L-Trp-OCH3

The dehydro-peptide Boc-L-Ile-ΔPhe-L-Trp-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide was crystallized from methanol in an orthorhombic space group P2₁2₁2₁ with a = 10.777(2), b= 11.224(2), c= 26.627(10) Å. The structure was determined by direct methods and refined to an R value of 0.069 for 3093 observed reflections [l≥ 2σ(l)].The peptide failed to adopt a folded conformation with backbone torsion angles: φ₁, = 90.8(8)°, ψ₁= -151.6(6)°, φ₂= 89.0(8)°, ψ₂= 15.9(9)°, φ₃= 165.7(7)°, ψ^T₃= -166.0(7)°. A general rule derived from earlier studies indicates that a three-peptide unit sequence with a ΔPhe at the (i+ 2) position adopts a β-turn II conformation. Because the branched β-carbon residues such as valine and isoleucine have strong conformational preferences, they combine with the ΔPhe residue differently to generate a unique set of conformations in such peptides. The presence of β-branched residues simultaneously at both (i+ 1) and (i+ 3) positions induces unfolded conformations in tetrapeptides, but a β-branched residue substituted only at (i+ 3) positron can not prevent the formation of a folded β-turn II conformation. On the other hand, the present structure shows that a β-branched residue substituted at the (i+ 1) position prevents the formation of a β-turn II conformation. These observations indicate that a β-branched residue at the (i+ 1) position prevents a folded conformation whereas it cannot generate the same degree of effect from the (i+ 3) position. This may be because of the trans disposition of the planar ΔPhe side-chain with respect to the C=O group in the residue. The molecules are packed in an anti-parallel manner to generate N₂-H₂…O₂ (-x,y-1/2, -z+ 3/2) and N^ε1₃-H^ε1₃…O₁(-x,y -1/2, -z+ 3/2) hydrogen bonds.