Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c)

The crystal structures of four peptides incorporating l-aminocycloheptane-l-carboxylic acid (Ac₇c) are described. Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe adopt β-turn conformations stabilized by an intramolecular 4 × 1 hydrogen bond, the former folding into a type-I/III β-turn and the latter into a type-II β-turn. In the dipeptide esters, Boc-Aib-Ac₇c-OMe and Boc-Pro-Ac₇c-OMe, the Ac₇c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac₇c-OMe found in the asymmetric unit. The cycloheptane ring of Ac₇c residues adopts a twist-chair conformation in all the peptides studied. ¹H-NMR studies in CDCl₃ and (CD₃)₂SO and IR studies in CDCl₃, suggest that Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe maintain the β-turn conformations in solution.     

Conformations of dehydrophenylalanine containing peptides Nuclear Overhauser effect study of two acyclic tetrapeptides

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (Δ^z-Phe) at position 2 or 3, Boc-Leu-Ala-Δ^z-Phe-Leu-OMe (1) and Boc-Leu-Δ^z-Phe-Ala-Leu-OMe (2), have been synthesized and their solution conformations investigated by 270MHz ¹H n.m.r. spectroscopy. In peptide 1 the Leu(4) NH group appears to be partially shielded from solvent, while in peptide 2 both Ala(3) and Leu(4) NH groups show limited solvent accessibility. Extensive difference nuclear Overhauser effect (n.O.e.) studies establish the occurrence of several diagnostic inter-residue n.O.e.s (Cα_jH ? N_i+1H and N_iH ? N_i+1H) between backbone protons. The simultaneous observation of “mutually exclusive” n.O.e.s suggests the presence of multiple solution conformations for both peptides. In peptide 1 the n.O.e. data are consistent with a dynamic equilibrium between an -Ala-Δ^z-Phe- Type II β-turn structure and a second species with Δ^z-Phe adopting a partially extended conformation with Ψ values of ± 100° to ± 150°. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive β-turn structure for the -Leu-Δ^z-Phe-Ala- segment and an almost completely extended conformation.     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and ¹H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II ß-turn in the solid state with Met and Aib as the corner residues (øMet =- 51.8^o, øMet = 139.5^o, øAib = 58.1^o, øAib = 37.0^o). A single, weak 4 -> 1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N—O 3.25 Å, N-H—O 128.4^o). ¹Hn.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagneti radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCI₃ and (CD₃)₂SO. Nuclear Overhauser effects observed between Met Cα H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II ß-turns in these solvents.     

H N.M.R. STUDIES OF PROTECTED α-AMINOISOBUTYRIC ACID CONTAINING PEPTIDES

The benzylic methylene protons in a large number of benzyloxycarbonyl α-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 → 1 hydrogen bonded β-turn is not possible, the benzylic -CH₂- protons appear as a singlet in CDCl₃ and have a very small chemical shift difference in (CD₃)₂SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro β-turn is retained in large peptide fragments of alamethicin.     

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.     

Acyclic peptides as conformational models

The tripeptide Boc-Aib-Leu-Pro-NHMe crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 9.542, b = 15.200, c = 18.256 Å and Z = 4. Each peptide is associated wth two water molecules in the asymmetric unit of the crystal. The structure has been solved by direct methods and refined to an R-value of 0.069. The peptide adopts a structure without any intramolecular hydrogen bond. The three residues occupy distinctly different regions of the Ramachandran map: Aib in the left-handed 3₁₀-helical region (± = 67°, ± = 23°), Leu in the β-sheet region (± = - 133°, ± = 142°) and Pro in the poly (Pro) II region (± = - 69°, ± = 151°). An interesting observation is that each water molecule participates in four hydrogen bonds with distorted tetrahedral coordination about the oxygen atom.     

Peptide models for β-turns.

The circular dichroism spectra of four β-turn model peptides, Z-Aib-Pro-Aib-Pro-OMe (1), Piv-Pro-Aib-NHMe (2), Piv-Pro-D-Ala-NHMe (3) and Piv-Pro-Val-NHMe (4) have been examined under a wide range of solvent conditions, using methanol, hexafluoroisopropanol and cyclohexane. Type I and Type II β-turns have been observed for peptides 1 and 2 respectively, in the solid state, while the Pro-D-Ala sequence adopts a Type II β-turn in a related peptide crystal structure. A class C spectrum is observed for 1 in various solvents, suggesting a variant of a Type I (III) structure. The Type II β-turn is characterized by a CD spectrum having two positive CD bands at ? 230 nm and ? 202 nm, a feature observed in Piv-Pro-D-Ala-NHMe in cyclohexane and methanol and for Piv-Pro-Aib-NHMe in methanol. Peptide 2 exhibits solvent dependent CD spectra, which may be rationalized by considering Type II, III and V reverse turn structures. Piv-Pro-Val-NHMe adopts non-β-turn structures in polar solvents, but exhibits a class B spectrum in cyclohexane suggesting a population of Type I β-turns.     

Stabilization of β-turn conformations in enkephalins

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α-aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly. ¹H n.m.r. studies of Tyr-Aib-Gly-Phe-Met-NH₂, Tyr-Aib-Aib-Phe-Met-NH₂ and Tyr-Gly-Aib-Phe-Met-NH₂ demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t-butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β-turn centred at positions 2 and 3 is proposed for the Aib²-Gly³analog. In the Gly²-Aib³analog, the β-turn has Aib³-Phe⁴as the corner residues. The Aib²-Aib³analog adopts a consecutive β-turn or 3₁₀ helical conformation. High in vivo biological activity is observed for the Aib²and Aib²-Aib³analogs, while the Aib³peptide is significantly less active.     

Crystal structures of a nonapeptide helix containing α, α-di-n-butylglycine (Dbg), Boc-G1y-Dbg-Ala-Val-Ala-Leu-Aib-Val-Leu-OMe

Two crystal structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue α,α-di-n-butylglycyl, reveal a mixed 3₁₀-α-helical conformation. Residues 1-7 adopt φ, ψ values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has φ, ψ values of -40, -37° and -46, -407° in the two crystals with the two butyl side chains mostly extended in each. Peptide molecules in the crystals pack into helical columns. The crystal parameters are: C₅₀ H₉₁ N₉ O₁₂, space group P2₁, with a= 9.789(1)Å;, b= 20.240(2) Å. c= 15.998(3) Å. β= 103.97(1): Z= 2, R=10.3% for 1945 data observed < 3σ(F) and C₅₀H₉₁N₉O₁₂· 3H₂O, space group P2₁ with a= 9.747(3)Å, b= 21.002(8) Å, c= 15.885(6) Å, β= 102.22(3). Z= 2. R=13.6% for 2535 data observed < 3σ(F) The observation of a helical conformation at Dbg suggests that the higher homologs in the α,α-dialkylated glycine series also have a tendency to stabilize peptide helices. © Munksgaard 1996.     

Conformations of dehydrophenylalanine containing peptides.

Three tripeptides containing a central Z-dehydrophenylalanine residue (Δ^z-Phe), Boc-L-Phe-Δ^z-Phe-X-OMe (X = L-Val 1, L-Leu 2 and X = L-Ala 3) have been synthesized and their solution conformations investigated by 270 MHz ¹H NMR spectroscopy. In all three peptides, conformations involving the X residue NH in an intramolecular hydrogen bond were favoured in CDCl₃ solutions. Studies of the nuclear Overhauser effect (NOE) provided support for a Type II β turn conformation in these peptides with Phe and Δ^z-Phe occupying the i + 1 and i + 2 positions, respectively. Significantly different conformations lacking any intramolecular hydrogen bonds were observed for peptide 1 in (CD₃)₂SO. NOE results were consistent with a significant population of molecules having semi-extended conformations (ø > 100°) at the Δ^z-Phe residue.     

Synthesis and conformational study of two L-prolyl-L-leucyl-glycinamide analogues with a reduced peptide bond

The peptide bond between Pro-Leu or Leu-Gly in Pro-Leu-Gly-NH₂ was replaced by a CH₂-NH function. The ¹H and ¹³C n.m.r. studies demonstrated that HCl·Pro-Leu (CH₂-NH)Gly-NH₂ 10 adopted a conformation in DMSO that is similar to the previously postulated β-turn for the natural hormone. This was not the case for the other analogue. In vivo tests on 10 revealed an activity approximately equal to the natural compound and an increased toxicity.     

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.     

Conformation of model,alanine and proline containing tetrapeptides in water

CD spectra of model alanine and prolyl-alanine tetrapeptides were measured at different _pH values. An analysis of the spectra shows that proline in position 2 or 4 of a tetrapeptide favours folding of the peptide chain, and unfolding when it is in position 3. Changes in CD spectra evidence growing amounts of the β-turn conformation upon increasing ^pH, independent of proline position in the peptide chain.     

Use of 13C-n.m.r. for the characterisation of linkages in acidic peptides.

A method involving the measurement of ¹³C-n.m.r. spectra in the presence and absence of lanthanides has been found to be useful for distinguishing α-linked and β-linked aspartyl residues in peptides. Lanthanides were found to induce shifts of 2–4 p.p.m. in the resonances of β-carbons of α-linked aspartyl residues, or α-carbons of β-linked aspartyl residues, whilst no shifts were observed in the resonances of carbons positioned further from ionisable carboxyl groups. These shifts were more marked than the corresponding shifts induced by pH-ionisation, and thus are more useful for distinguishing α from β peptides linkages. The method has been applied to a comparison between solution and solid-phase synthesis of aspartyl-rich peptides. The two activation peptides of human trypsinogen, Asp-Asp-Asp-Asp-Lys and Ala-Pro-Phe-Asp-Asp-Asp-Asp-Lys were synthesised by solution and solid-phase methods, respectively, the latter employing Fmoc-protection of amino groups during synthesis. The purified products were found to contain aspartyl residues that were exclusively α-linked.     

Peptides from chiral Cα,α-disubstituted glycines

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral C^α,α-disubstituted glycine Cα-methyl, Cα-isopropylglycine [(αMe)Val] have been determined by X-ray diffraction. The derivative is mClAc-l -(α Me)Val-OH, and the peptides are Z-l -(αMe)Val-(l -Ala)₂-OMe monohydrate, Z-Aib-L-(αMe)Val-(Aib)₂-OtBu, and Ac-(Aib)₂-l -(αMe)Val-(Aib)₂OtBu acetonitrile solvate. The tripeptide adopts a type-I β-turn conformation stabilized by a 1 ← 4N-H . O=C intramolecular H-bond. The tetra- and pentapeptides are folded in regular right-handed 3₁₀-helices. All four L-(αMe)Val residues prefer φ, Ψ angles in the right-handed helical region of the conformational map. The results indicate that: (i) the (αMe)Val residue is a strong type-I/III β-turn and helix former, and (ii) the relationship between (αMe)Val chirality and helix screw sense is the same as that of Cα-monosubstituted protein amino-acids. The implications for the use of the (αMe)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.     

Crystal and molecular structure of tert. -butyloxycarbonyl-L-prolyl-α-aminoisobutyryl-L-alanyl-α-aminoisobutyrate methyl ester

Boc-Pro-Aib-Ala-Aib-OMe crystallizes in the orthorhombic space group P2₁2₁2 with cell dimensions a = 17.701 (3)Å, b = 17.476 (4)Å, c = 9.686 (2)Å, V = 2996.3 ų. The first three residues form a single turn of a 3₁₀-helix stabilized by two intramolecular hydrogen bonds. Comparison of the conformation of the methyl ester of the tetrapeptide with that of its benzyl ester shows differences in the individual torsion angles of up to 29°, although the overall conformation is conserved.     

The preferred solid-state conformation of (αMe)Trp peptides

The two Z-l -Ala-d l -(xMe)Trp-NH₂ diastereomeric dipeptides were synthesized from (Z-l -Ala)₂O and H-dl -(xMe)Trp-NH₂. The latter racemate, prepared by phase-transfer catalyzed alkylation of the N^α-benzylidene derivative of alanine amide followed by acidic hydrolysis of the resulting Schiff base, was characterized by X-ray diffraction. The molecular and crystal structure of Z-l -Ala-l -(αMe)Trp-NH², separated from its diastereomer by silica-gel column chromatography, was determined by X-ray diffraction analysis. Both independent molecules in the asymmetric unit of the dipeptide adopt a type-II β-bend conformation. However, only the more regularly folded conformation of molecule B is stabilized by a 1←4 C=O…H—N intramolecular H bond. The present results indicate that: (i) the Cα-methylated (αMe)Trp residue is a strong β-bend and helix former, and (ii) the relationship between (αMe)Trp chirality and helix screw sense tends to be opposite to that of protein amino acids. The implications for the use of the (αMe)Trp residue in designing conformationally restricted analogs of bioactive peptides are briefly discussed. ©Munksgaard 1995.     

Type II‘β-bend conformation of tert.-butyloxycarbonyl-L-amino-succinyl-L-alanyl-glycine methyl ester in the solid state

Boc-L-Asu-L-Ala-Gly-OMe crystallizes in the monoclinic space group P2₁ with cell dimensions a = 14.315(3) Å, b = 9.280(2) Å, c = 14.358(3) Å, β= 103.63 (1) d?, V= 1853.4(9) ų, with two molecules in the asymmetric unit. The conformation of the two molecules is characterized by a type II' β-bend, similar to that predicted earlier by potential energy calculations, stabilized by an intramolecular hydrogen bond. I.r. and ¹H-n.m.r. data show that the folded conformation is also stable in chloroform solution.     

Conformationally restrained peptides: crystal structure of tert-butyloxycarbonyl-L-alanyl-D-alanyl-D-aminosuccinyl-glycyl-L-alanine methyl ester

The solid-state structure of a heterochiral peptide embodying a D-aminosuccinyl peptide (D-ASU) and a D-Ala was studied in order to analyse the effects of Asu and amino acids with inverse chirality on peptide conformation. The crystal structure has been determined by X-ray diffraction techniques and refined to a final R factor of 0.043. The molecule adopts an unusual overall 'S-shape’ conformation due to two consecutive type II β-turns. In this molecule it is possible to compare a type II β-bend conformation (L-Ala¹-D-Ala²) favoured by the presence of a D-residue at second corner to a type II β-turn (D-Asu³-Gly⁴) favoured by the presence of a D-ASU residue at first corner. In agreement with previous studies, this structure confirms that the Asu has a high propensity to adopt a type II or II′β-bend conformation and that it may be used as a strong determinant of these structural motifs. © Munksgaard 1996.     

N-Methyl peptides

The natural occurrence of N-methyl peptides in various plant metabolites has made N-methylation a subtle and attractive possible modification for structure-activity relationship studies of endogeneous peptides. However, little is known about the conformational specificity induced by the N-methylation of a given peptide, and particularly concerning the β-turn conformation. A spectroscopic investigation (i.r., n.m.r., CD) and X-ray diffraction experiments have been carried out on tBuCO-X-Me-Y-NHMe blocked dipeptides (X = Gly, L-Ala, L-Pro, and Y = Gly, and L- or D-Ala, Leu, Phe) with reference to the homologous desmethylated species. The influence of the N-methylation on conformation depends to a large extent on the chirality of the X and Y residues. Homochiral sequences are the most affected, with a strong preference for the βVI-folded conformation containing a middle cis amide bond. Heterochiral sequences are essentially unaffected and retain the βII-folded conformation with a trans middle amide bond. Glycine-containing sequences undergo a more complex perturbation according to the X or Y position of the Gly residue. The available data for larger N-methyl peptides are consistent with our observations, suggesting that these simple dipeptides well reflect the conformational perturbations induced by N-methylation on the β-turn conformation.