Conformational analysis of cyclic tetrapeptides by molecular mechanics calculations

Molecular mechanics calculations of the cyclic tetrapeptide ring system for the cis, trans, cis, trans amide bond sequences for cyclo tetraglycine and cyclo tetraalanine have been carried out. A systematic search of conformational space was carried out by using Still's RINGMAKER in an attempt to find the global minimum for each amide bond sequence. Ring system structures were optimized by using the BAKMOD program. A comparison of 11 experimentally determined cyclic tetrapeptide conformations with the lowest energy calculated conformations showed that only 4 of 11 known cyclic tetrapeptides adopted the lowest energy conformation. However, when the destabilization energy between cyclo tetraalanine and cyclo tetraglycine was calculated, 10 of the 11 experimentally determined conformations for cyclic tetrapeptides with alternating cis, trans, cis, trans amide bond sequences adopted the conformations with the least de-stabilization energy. The relationship between the molecular mechanics calculations and empirical rules for predicting cyclic tetrapeptide conformations is discussed.     

Cyclic peptides

Cyl-2 is a phytotoxic cyclic tetrapeptide with the sequence of cyclo(-D-Tyr(Me)-L-Ile-L-Pip-Aoe-): Aoe, 2-amino-8-oxo-9,10-epoxydecanoic acid. The configuration of Aoe is unknown. Three analogs, cyclo(-D-Tyr(Me)-L-Ile-L-Pip-L(and D)-Leu-) (1 and 2) and cyclo (-D-Tyr(Me)-L-Ile-L-Pro-D-Leu-) (3), were synthesized and spectroscopic features of these analogs were compared. ¹ H-N.m.r. spectra of cyclo(-D-Tyr(Me)-L-Ile-L-Pip-L-Leu-) (1), cyclo(-D-Tyr(Me)-L-Ile-L-Pro-L-Leu-) (4), and Cyl-2 showed a similar pattern. On the other hand, the spectra of analogs 2 and 3 showed a different pattern, the Ile-Pro bond in 3 being in cis conformation.     

A light-activated beta-turn scaffold within a somatostatin analog: NMR structure and biological activity

Somatostatin owes its biological activity to the presence of a well-defined beta-turn centered around the tetrapeptide Phe-Trp-Lys-Thr. We have developed a light-activated beta-turn scaffold, 1, with the ability to template a beta-turn conformation within the somatostatin tetrapeptide only upon photolysis. The three-dimensional structure of the trans cyclic peptide I obtained by NMR revealed no beta-turn conformation; however, when isomerized to the cis form II with light, the solution structure of the resulting cyclic peptide was found to contain a type II' beta-turn within the Phe-Trp-Lys-Thr sequence. Binding assays with the SRIF receptor demonstrated that the cis peptide displayed enhanced affinity for the receptor over the trans form.     

Synthesis and renin inhibitory activity of angiotensinogen analogues having dehydrostatine, Leu psi [CH2S]Val, or Leu psi [CH2SO]Val at the P1-P1' cleavage site

The synthesis and in vitro renin inhibitory potencies of angiotensinogen (ANG) analogues having amide (CONH) bond replacements at P1-P1', the Leu-Val cleavage site, corresponding to Leu psi[CH2SO]Val, and the trans olefinic analogue of statine (Sta), 4(S)-amino-6-methyl-2(E)-heptenoic acid (dehydrostatine, Dhs), are reported. These are compared to P1-P1' Leu psi[CH2NH]Val-, Sta-, or Phe-Phe-substituted analogues of the same template. The Dhs pseudodipeptide was found to be an adequate mimic of a trans CONH bond and gave a peptide, H-Pro-His-Pro-Phe-His-Dhs-Ile-His-D-Lys-OH, approximately equal in potency to a Phe-Phe-containing inhibitor, but 200-fold less potent than its Sta-substituted congener. That the enhanced potency of the Sta-containing peptide most likely depends on hydrogen bonding as well as tetrahedral geometry is indicated by the 50-100-fold lower potency of the tetrahedral Leu psi[CH2S]Val and Leu psi[CH2SO]Val analogues as compared to the Leu psi[CH2NH]Val-containing congener.     

Synthesis, conformational analysis, and cytotoxicity of conformationally constrained aplidine and tamandarin A analogues incorporating a spirolactam beta-turn mimetic

With the aim of studying the contribution of the beta II turn conformation at the side chain of didemnins to the bioactive conformation responsible for their antitumoral activity, conformationally restricted analogues of aplidine and tamandarin A, where the side chain dipeptide Pro8-N-Me-d-Leu7 is replaced with the spirolactam beta II turn mimetic (5R)-7-[(1R)-1-carbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]nonane, were prepared. Additionally, restricted analogues, where the aplidine (pyruvyl9) or tamandarin A [(S)-Lac9] acyl groups are replaced with the isobutyryl, Boc, and 2-methylacryloyl groups, were also prepared. These structural modifications were detrimental to cytotoxic activity, leading to a decrease of 1-2 orders of magnitude with respect to that exhibited by aplidine and tamandarin A. The conformational analysis of one of these spirolactam aplidine analogues, by NMR and molecular modeling methods, showed that the conformational restriction caused by the spirolactam does not produce significant changes in the overall conformation of aplidine, apart from preferentially stabilizing the trans rotamer at the pyruvyl9-spirolactam amide bond, whereas in aplidine both cis and trans rotamers at the pyruvyl9-Pro8 amide bond are more or less equally stabilized. These results seem to indicate a preference for the cis form at that amide bond in the bioactive conformation of aplidine. The significant influence of this cis/trans isomerism upon the cytotoxicity suggests a possible participation of a peptidylprolyl cis/trans isomerase in the mechanism of action of aplidine.     

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.     

Probing the role of proline in peptide hormones. NMR studies of bradykinin and related peptides

The use of NMR methods to study conformational and dynamic aspects of the proline residues in the nonapeptide bradykinin is reviewed. NMR analyses involve considerations of bistable equilibria which include the cis/trans conformational heterogeneity of the imide bond, the cis'/trans' regions of conformational stability which characterize rotation about the C alpha-CO bond (dihedral angle psi), and the interconversion of the pyrrolidine ring of proline between puckered C gamma-endo and C gamma-exo conformations. These conformational features are all characterized by different kinetic behavior, are interdependent with peptide bond conformation, and exhibit sensitivity to amino acid substitutions. Thus, the substitution of Gly6 for Ser6 increases the fractional cis probability of the sixth peptide bond from 0.1 to 0.35. Substitutions of alpha-aminoisobutyric acid (AIB) residues for proline introduce conformational constraints analogous to those in cis' proline. Correlations of pyrrolidine ring conformation and dynamics with the cis/trans ratio of the imide bond have also been observed in model systems. Conformational and activity analyses of [AIB7]-bradykinin provided a stimulus for the development of the first bradykinin antagonist by Stewart and Vavrek (Vavrek RJ and Stewart JM, Competitive antagonists of bradykinin. Peptides 6: 161-164, 1985).     

Synthesis of HC toxin and related cyclopeptides containing the (L-Ala-D-Ala-L-Ada-D-Pro) sequence

In studies leading to HC toxin synthesis, a phytotoxic cyclic tetrapeptide with the sequence cyclo (L-Ala-D-Ala-L-Aoe-D-Pro), we have determined optimal conditions for the cyclization which constitutes one of the most important steps in the synthesis of the toxin. All four possible sequences containing an optically active precursor, i.e. L-Ada = (2 S)-2-amino-9-decenoic acid instead of Aoe, have been prepared and subjected to cyclization. Owing to the differences in racemization risk during activation of the terminal carboxyl aminoacid different cyclization procedures have been applied. Cyclopeptide yields and selectivity between cyclomonomer and dimer both containing the title sequence are mainly controlled by the linear precursor sequence. The cyclic tetrapeptide is only obtained with D-proline in the C-terminal position, the best yield reached by the -ONSu activation method. Starting from the peptide, the (9S, 9R) HC toxin epimer on the epoxidic carbon atom has been further synthesized in two steps.     

Conformational study of cyclosporin A in acetone at low temperature

The conformation of cyclosporin A (CsA), an undecapeptide with seven N-methylated amino acids, was studied in acetone at 193 K. Previous studies of the conformation of CsA in different solvents, in the cyclosporin-cyclophilin complex and in complexes with LiCl showed that the conformation of the free and the bound CsA are different. Differences were observed at the conformation of the MeLeu⁹-MeLeu¹⁰ peptide bond, which is cis in solution and trans in the complex, and in the orientation of the amide protons and the N-Me groups. By using acetone, which is a proton acceptor, we wanted to influence the orientation of the amide protons. In the conditions used in this study a new conformation is found, which differs as well from the one previously observed in solution as from the Conformation observed in the complex. This conformation has a cis peptide bond between MeLeu⁹ and MeLeu¹⁰. The trans conformation of the peptide bond MeLeu⁹-MeLeu¹⁰, which is necessary for biological activity, was not induced. One of the amide protons is involved in an intramolecular H-bridge stabilising a β-turn around Sar³MeLeu⁴, and three of the seven NMe groups are oriented to the centre of the molecule. © Munksgaard 1994.     

Solid-state trans-cis isomerization of captopril determined by thermal Fourier transform infrared (FT-IR) microspectroscopy

Thermal Fourier transform infrared (FT-IR) microspectroscopy was used to investigate the conformational isomerization of captopril in the solid state. The result indicates that the IR peak intensity of captopril for original bands decreased dramatically at 102 degrees C, but for new bands it increased with the rise of temperature. The frequency of C=O stretching mode for carboxylic acid and for amide was located at a higher wavenumber of 1747 cm(-1) and at a lower frequency of 1591 cm(-1) as compared with the general compound, suggesting the existence of trans isomer of captopril in the solid state by intramolecular hydrogen bonding. Beyond 102 degrees C, several new bands at 1720, 1645, and 1610 cm(-1) were observed with the rise of temperature, indicating the coexistence of a cis isomer. However, the cis isomer could transform gradually to the trans isomer after cooling. The thermodynamics of equilibrium mixture of cis/trans isomers were also studied. The trans isomer was more stable than the cis isomer, but the cis isomer was favored at the higher temperature.     

A new class of high affinity ligands for the neurokinin A NK2 receptor: psi (CH2NR) reduced peptide bond analogues of neurokinin A4-10.

Analogues of [Leu10]NKA4-10 were synthesized in which each of the amide bonds was sequentially replaced with the reduced amide psi (CH2NH) bond to determine the effect of this structural modification on the antagonism of NKA binding to the HUB NK2 receptor. [psi (CH2-NH)9,Leu10]NKA4-10 (6) retained significant affinity for the NK2 receptor (IC50 = 115 nM) and showed weak partial stimulation of PI turnover (approximately 10-15% of NKA maximum). 6 behaves as a competitive antagonist of NKA-stimulated PI turnover with a pA2 = 6.7. The secondary amine of the psi (CH2NH) moiety of 6 was converted to a tertiary amine by alkylation. This modification was found to have a small effect upon receptor affinity but did result in attenuation of partial agonist activity. A combination of amino acid substitutions and psi (CH2NH) alkylation yielded [beta Ala8,psi (CH2N(CH2)2CH3)9,Phe10]NKA4-10 (21) which has very high affinity for the HUB NK2 receptor. This compound inhibited [125I]NKA binding with an IC50 = 1 nM which is equal to the receptor affinity of NKA. Compound 21 also shows very weak partial agonism of PI turnover (< or = 5% of NKA maximum) which makes this the most potent member of a new class of NKA ligands: psi(CH2NR)9-NKA4-10 analogues which potently antagonize NKA binding and possess minimal partial agonist activity.     

A catalogue of phi,psi and omega torsion angles for tetrapeptides in proteins

This work examined the degree of consistency, over multiple occurrences in several proteins, of conformations of tetrapeptides. A data base of 114 proteins was taken from the Brookhaven Data Bank. The number of occurrences of every tetrapeptide in these proteins was counted. For each tetrapeptide which occurred more than 6 times all phi, psi and omega angles were calculated and averaged over the occurrences. Due to the limitations of the data base there were only 24 tetrapeptides in this category. This does not provide sufficient information for the extensive prediction of structures which are not homologous to those already in the data base but the effectiveness of structure prediction can be expected to increase with the size of the data base.     

Conformational studies of cyclic tetrapeptides

The conformations of chlamydocin and cyclo (Ala-Aib-Phe-D-Pro)(Ala⁴-chlamy-docin) in chloroform have been investigated by nuclear magnetic resonance, infrared and circular dichroism spectroscopy. The data obtained from these experiments establish an all transoid, bis γ-turn conformation for both compounds in chloroform with the following torsional angles (± 20d?): Ala⁴4-chlamydocin: Aib, Ø+ 60d?, Ψ– 50d?; ω+ 160d?; Phe Ø– 120d?, Ψ+ 120d?, ω– 160d?; D-Pro Ø+ 60d?, Ø– 55d?, ω+ 160d?; Ala Ø– 110d?, Ψ+ 110d?, ω– 160d?. Chlamydocin adopts a closely related conformation in neat chloroform. Nuclear Overhauser Effect (NOE) data are utilized to assign amide bond geometries in the cyclic tetrapeptide ring system.     

Synthesis and conformational properties of the lanthionine-bridged opioid peptide [D-AlaL2,AlaL5]enkephalin as determined by NMR and computer simulations.

We report the synthesis and conformational analysis by means of NMR and computer simulations of a novel opioid peptide with the sequence [formula: see text], which we write as [formula: see text], abbreviated [D-AlaL2,L-AlaL5]EA, where AlaL denotes each of the lanthionine amino acid ends linked by a monosulfide bridge and EA indicates enkephalinamide. Data from 2D NMR (HOHAHA and ROESY) provide short-range NOEs that are used as constraints in molecular modeling; measurement of coupling constants shows that chi 1 (D-AlaL2) is predominantly in either the t or g- conformation, and temperature coefficient data suggest the participation of the AlaL5 amide proton in an intramolecular hydrogen bond. The use of NOE and hydrogen-bond constraints in a distance-geometry program yields a large number of initial conformations compatible with the data. Energy minimization of these structures using CHARMM results in three families of backbone ring conformations, labled A1, A2, and B. The torsion chi 1 in D-AlaL2 remains close to trans for all three conformations. Molecular dynamics in vacuo at 300 K show that these three families of conformers interconvert, with concerted shifts in two of the three torsions psi(Phe), phi(AlaL5), and chi(AlaL5). The [D-AlaL2,L-AlaL5]EA is superactive in the guinea pig ileum (GPI) and mouse vas deferens (MVD) in vitro tests and also in the rat hot plate test in vivo. At the same time, this analog with a constrained 13-membered ring shows virtually no selectivity with a ratio IC50 (MVD)/IC50 (GPI) of 0.882.     

Design,synthesis, and testing of potential antisickling agents. 9. Cyclic tetrapeptide homologs as mimics of the mutation site of hemoglobin S

As part of our continuing search for new agents which might be useful for the treatment of sickle-cell anemia, we have synthesized two cyclic tetrapeptide homologs, cyclo(-Val-Glu[-Thr-Pro-]-OH) (1a) and cyclo(-Phe-Glu[-Thr-Pro-]-OH (1b), and a tetrapeptide lactone homolog cyclo(H-T-hr-Pro-Val-Glu-OH) (2). The intent was that these peptides would mimic a tetrapeptide region around the mutation site of HbS and thus be able to bind at the acceptor site of HbS and thereby inhibit polymerization. The synthesis of the linear peptides was accomplished in solution using both the polymeric reagent (PHBT) and DCC/HOBT methods; cyclization was accomplished by an improved method. ¹³C-n.m.r. studies were performed which allowed us to assign the conformation about the Thr-Pro bond in 1a and 2 as trans. The cyclic peptides were tested for their ability to increase the solubility of HbS under deoxygenating conditions, but only 1a had any antigelling activity, albeit low.     

Analogues of growth hormone-releasing factor (1-29) amide containing the reduced peptide bond isostere in the N-terminal region

Previous peptide structure-activity investigations employing the psi[CH2NH] peptide bond isostere have produced antagonists when inserted into various sequences. These include bombesin, in which the incorporation of Leu13 psi[CH2NH]Leu14 produced a potent antagonist, and tetragastrin, with which Boc-Trp-Leu psi[CH2NH]Asp-Phe-NH2 is an antagonist. In this study, we chose to investigate the effect of this isostere on growth hormone-releasing factor (1-29) amide. Analogues were prepared by solid-phase synthesis and the isosteres incorporated by racemization-free reductive alkylation with a preformed protected amino acid aldehyde in the presence of NaBH3CN. The aldehydes were prepared by the reduction of the protected N,O-dimethyl hydroxamates with LiAlH4 at 0 degrees C. The purified analogues were assayed in a 4-day primary culture of male rat anterior pituitary cells for growth hormone (GH) release. Potential antagonists were retested in the presence of GRF(1-29)NH2. The following results were obtained: At position 5-6, a very weak agonist was produced with much less than 0.01% activity. Incorporation of the isostere in positions 1-2, 2-3, and 6-7 gave weak agonists with approximately 0.1% activity. Agonists with 0.39% and 1.6% activity were produced by incorporation at 10-11 and 3-4, respectively. The analogue [Ser9 psi[CH2NH]Tyr10]GRF(1-29)NH2 was found to be an antagonist in the 10 microM range vs 1 nM GRF and had no agonist activity at doses as high as 0.1 mM.     

Synthesis and pharmacological evaluation of an analogue of the peptide hormone oxytocin that contains a mimetic of an inverse gamma-turn

Oxytocin is a neurohypophyseal peptide hormone that induces labor and lactation in mammals. An inverse gamma-turn mimetic corresponding to the tripeptide Ile-Val-Asn has been synthesized and incorporated instead of residues 3-5 of oxytocin to probe the hypothesis that a gamma-turn involving these residues is found in the receptor bound conformation of oxytocin. In the turn mimetic, residues i and i + 1 are connected by a psi[CH(2)O] isostere while a covalent methylene bridge replaces the hydrogen bond that is often found between residues i and i + 2 in gamma-turns. The turn mimetic was assembled from three types of building blocks: an azido epoxide, an alpha-bromo acid, and a protected beta-amino alcohol. The oxytocin analogue did not induce contractions of the uterus nor did it inhibit oxytocin-induced contractions. It is suggested that the loss of bioactivity is mainly due to the presence of a psi[CH(2)O] isostere instead of an amide bond between residues i and i + 1 in the turn mimetic.     

Vinyl sulfide cyclized analogues of angiotensin II with high affinity and full agonist activity at the AT(1) receptor

Vinyl sulfide cyclized analogues of the octapeptide angiotensin II that are structurally related to the cyclic disulfide agonist c[Hcy(3,5)]Ang II have been prepared. The synthesis relies on the reaction of the mercapto group of a cysteine residue in position 3 with the formyl group of allysine incorporated in position 5 of angiotensin II. A mixture of the cis and the trans isomers was formed, and these were separated and isolated by RP-HPLC. Thus, the three-atom CH(2)[bond]S[bond]S element of the AT(1) receptor agonist c[Hcy(3,5)]Ang II has been displaced by a bioisosteric three-atom S[bond]CH[double bond]CH element. A comparative conformational analysis of the 13-membered ring systems of c[Hcy(3,5)]Ang II and the 13-membered cyclic vinyl sulfides with cis and trans configuration, respectively, suggested that all three systems adopted very similar low-energy conformations. This similarity was also reflected in the bioactivity. Both of the compounds that contained the ring systems encompassing the cis or trans vinyl sulfide elements between positions 3 and 5 exhibited K(i) values less than 2 nM and exerted full agonism at the AT(1) receptor. In contrast, vinyl sulfide cyclization involving the amino acid residues 5 and 7 rendered inactive compounds. The cyclic vinyl sulfides that have agonist activity were both shown to possess low-energy conformers compatible with the previously proposed 3D model for the bioactive conformation of Ang II.     

The cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues

Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) is an endogenous tetrapeptide that combines potency and efficacy with high affinity and selectivity toward the μ opioid receptor, the most responsible for analgesic effects in the central nervous system. The presence of the Pro(2) represents a crucial factor for the ligand structural and conformational properties. Proline is in fact an efficient stereochemical spacer, capable of inducing favorable spatial orientation of aromatic rings, a key factor for ligand recognition and interaction with receptors. Here the Pro(2) has been replaced by 4(S)-NH(2)-2(S)-proline (cAmp), a proline/GABA cis-chimera residue. This bivalent amino acid maintains the capacity to influenc the tetrapeptide conformation and offers the opportunity to generate new linear models and unusually constrained cyclic analogues characterized by an N-terminal Tyr bearing a free α-amino group. The results indicate that the new analogues do not show affinity for both δ and κ opioid receptors and bind only poorly to the μ receptors (for cyclopeptide 9: K(i)(μ) = 660 nM; GPI (IC(50)) = 1.4% at 1 μM; for linear tetrapeptide acid 13: K(i)(μ) = 2000 nM; GPI (IC(50)) = 0% at 1 μM; for linear tetrapeptide amide 15: K(i)(μ) = 310 nM; GPI (IC(50)) = 894 nM).     

Structural features of thePip/AzPip couple in the crystalline state: influence of the relative AzPip location in an azadipeptide sequence upon the induced chirality and conformational characteristics

Abstract: Azapipecolic (AzPip) is a pipecolic (Pip) residue analogue containing a nitrogen atom in place of the C^αH group. AzPip was introduced into two reverse dipeptide sequences,Piv‐AzPip‐l ‐Ala‐NHiPr I and Boc‐l ‐Ala‐AzPip‐NHiPr II in order to evaluate, in the crystalline state, the influence of thel ‐Ala‐induced chirality upon the prochiral AzPip residue, and therefore the resulting conformational characteristics, according to the relative position of the AzPip residue. Piv‐dl ‐Pip‐NHMe III served as a control derivative for comparison between the properties of the two different heterocycles of Pip and AzPip residues. Piperidine and hexahydropyridazine rings have a few characteristics in common: chair conformation, axial disposition of the C‐terminal backbone substituent and the cisoid form of the N‐terminal tertiary amide function. An almost pure sp³ hybridization state is observed for the substituted nitrogen atom N^α, so that l ‐Ala induces an AzPip (R) or (S) chirality when it follows or precedes, respectively, the azaresidue in such a pseudodipeptide sequence. If both I and II compounds present a short NH…N contact between the sp² tertiary amide nitrogen atom and the NH of the next secondary amide function, whatever the chiral nature of the sequence, the heterochiral azadipeptide I adopts a rather totally extended conformation while the homochiral azadipeptide II is folded by a β‐VI turn‐like structure stabilized by a classical 4→1 intramolecular hydrogen bond.