Crystal-state 3D-structural characterization of novel, Aib-based, turn and helical peptides.

The crystal-state conformations of the hexapeptide amide Pht-(Aib)(6)-NH-C(CH(3))(2)-O-OtBu (7), the hexapeptide Ac-L-aIle-(Aib)(5)-OtBu (6), the pentapeptide Z-(Aib)(3)-L-Glu(OtBu)-Aib-O-(CH(2))(2)-(1)Nap (5), the tetrapeptides Z-(Aib)(2)-L-His(N(tau)-Trt)-Aib-OMe (4 I) and Z-(Aib)(2)-L-Nva-Aib-OtBu (4 II), the tripeptide Pyr-(Aib)(3)-OtBu (3 I), the dipeptide amides Pyr-(Aib)(2)-(4)NH-TEMPO (3 II) and Piv-(Aib)(2)-NH-C(CH(3))(2)-O-OtBu (3 III), and the dipeptides Pht-Aib-betaAc(6)c-OtBu (2 I), Pht-Aib-NH-C(CH(3))(2)-O-OtBu (2 II) and Boc-gGly-mAib-OH (2 III) have been determined by X-ray diffraction analyses. All peptides investigated are characterized by one or more turn/helix forming Aib residues. Except the three short dipeptides, all are folded into C==O...H--N intramolecularly H-bonded 3(10)-helices, or into various types of beta-turns. In the structure of 6, two independent molecules of opposite screw sense were observed in the asymmetric unit, generating diastereomeric 3(10)-helices.     

Linear oligopeptides: peptaibol antibiotics — preferred conformation of the 2–9 segment of emerimicins III and IV and all related short sequences

With the aim of obtaining information on the effect induced by main-chain length and amino acid sequence on the type of helical structure adopted by naturally-occurring peptides rich in C^α,α-dialkylated residues, an infrared absorption and ¹H nuclear magnetic resonance analysis of chloroform solutions of the protected 2–9 segment of the peptaibol antibiotics emerimicins III and IV,-(Aib)₃-l-Val-Gly-l-Leu-(Aib)₂-, and all related short sequences starting from both the N- and C-termini was performed. The results are consistent with the presence of folded structures of the β-bend type (in the shorter peptides) or 3₁₀-helices (in the longer peptides). Extent of formation and stability of the inter- and intramolecular H-bonds have been assessed as a function of concentration, temperature, addition of dimethylsulphoxide (DMSO) and the free radical 2,2,6,6-tetramethy-1-piperidinyloxy (TEMPO). At high peptide concentration both folded and helical structures tend to self-associate extensively. In the self-association process the N(1)H and N(2)H groups are those acting as H-bonding donors. These results agree well with those obtained in the solid state by X-ray diffraction on the octapeptide itself and selected short sequences.     

Prevention of peptide fibril formation in an aqueous environment by mutation of a single residue to Aib

The behavior of a number of 16 residue polypeptides with a sequence Acetyl-EACARXZAACEAAARQ-amide, where X = V or A and Z = A or Aib, is studied under aqueous conditions. It is shown that the substitution of a single alanine residue by alpha-aminoisobutyric acid (Aib) completely alters both the conformation and the aggregation properties of the peptides. The Ala-Ala (X,Z = A,A) peptide is shown by circular dichroism and FTIR methods to adopt a predominately beta-sheet conformation. Furthermore, the peptide has limited solubility and is shown to form fibrils by electron microscopy and thioflavin T binding assays. In contrast, a single substitution at the center of peptide of alanine to Aib (X,Z = A,Aib) completely abolishes fibril formation and alters the conformation to a mixture of random coil and alpha-helix. The results show that Aib is a strong beta-sheet disrupter that is also able to adopt a helical conformation. This is linked to its role in peptaibol antibiotics. Aib provides an attractive alternative to proline and other substitutions in producing peptide variants with a lower tendency to produce fibril aggregates.     

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.     

Peptoid residues and beta-turn formation.

A set of terminally protected tripeptoids containing a residue of either N-methylglycine or N-isobutylglycine in position i + 1/i + 2 were synthesized and tested for intramolecularly H-bonded beta-turn formation. By exploiting FT-IR absorption and 1H NMR techniques, their folding tendencies were compared with those of a variety of reference peptides. The amount of beta-turn induction and the relative extent of the various types of intramolecularly H-bonded beta-turn conformers were determined in chloroform solution.     

Constrained phenylalanine analogues. Preferred conformation of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue.

Three Tic-containing (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) model peptides were synthesized to assess the tendency of this constrained Phe analogue to fold into a beta-bend and a helical structure, and to adopt a preferred side-chain disposition. The results of the solution conformational analysis, performed by using Fourier transform infrared absorption and 1H nuclear magnetic resonance, indicate that in chloroform the -Aib-D-Tic-Aib-, -(Aib)2-D-Tic-(Aib)2-, and -L-Pro-D-Tic- sequences fold into intramolecularly H-bonded forms to a great extent. An X-ray diffraction analysis on p-BrBz-(Aib)2-DL-Tic-(Aib)2-OMe monohydrate and p-BrBz-L-Pro-D-Tic-NHMe allows us to conclude that, while the pentapeptide methylester forms an incipient (distorted) 3(10)-helix, the dipeptide methylamide adopts a type-II beta-bend conformation. In both cases, the D-Tic side-chain conformation is D, gauche(-). The implications for the use of the Tic residue in designing conformationally restricted analogues of bioactive peptides are briefly discussed.     

Crystallographic structure of a multiple β-turn containing,glycine-rich heptapeptide: A synthetic precursor of the lipopeptaibol antibiotic Trichodecenin I

In continuation of our studies on the structure and function of peptaibol antibiotics, the conformational properties of a sequence analogous to that of Trichodecenin I (Z-Gly-Gly-D -Leu-Aib-Gly-D -Ile-D -Leu-OMe, where Z = benzyloxycarbonyl, Aib = α-aminoisobutyric acid, and OMe = methyl ester) have been investigated crystallographically. This sequence is the mirror image of the naturally occurring molecule and also of the C-terminal heptapeptide of the related lipo-peptaibol Trichogin A IV (where, however, the Leu-OMe residue has replaced the original Leuol residue). The molecule crystallized in the monoclinic system, space group P2₁, Z = 4, and cell parameters a = 11.610(5), b = 33.342(8), c = 11.735(4) Å, β = 110.42(1)*, V = 4257(3) ų. The crystallographic refinement converges at residual values of R = 0.047 and wR₂ = 0.134 on F². In the 1–5 segment the molecular conformation is virtually identical to that one reported from solution nmr studies of a similarly protected sequence [Biopolymers (1995), Vol. 35, pp. 21–29)] and is characterized by β-turns of type I at Gly1-Gly2, II′ at Leu3-Aib4, and I at Aib4-Gly5. In the crystal structure, a β-sheet-like arrangement is seen at the C-terminus. © 1996 John Wiley & Sons, Inc.     

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.     

The crystal structure of Afc-containing peptides

A systematic structural analysis of Afc (9-amino-fluorene-9-carboxylic acid) containing peptides is here reported. The crystal structures of four fully protected tripeptides containing the Afc residue in position 2: Z-X(1)-Afc(2)-Y(3)-OMe (peptide a: X = Y = Gly; peptide b: X = Aib, C(alpha, alpha)-dimethylglycine, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib) have been solved by x-ray crystallography. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in three peptides examined in this paper (peptides a-c). In contrast, Afc was found in a folded conformation, in the 3(10)-helical region, only in the peptide d, in which it is both preceded and followed by the strong helix promoting Aib.     

Synthesis of the protected 6-16 segment of zervamicin 11-2, an application of the azirine/oxazolone method.

The protected 11 amino acid segment (6-16) of the peptaibol zervamicin II-2 was synthesized by using the 'azirine/oxazolone method' for the introduction of all Aib residues. Whereas a 2,2-dimethyl-2H-azirin-3-amine was used as the building block for Aib(7), methyl 2,2-dimethyl-2H-azirine-3-prolinate and -3-(3-hydroxyprolinate) proved to be ideally suited as dipeptide synthons for the introduction of Aib-Pro and Aib-Hyp, respectively. The coupling of Z-protected amino acids or peptide acids with the 2H-azirin-3-amines were performed in 75% to quantitative yield.     

Translocation of an Aib-containing peptide through cell membranes

The biophysical characteristics and channel-forming activity of peptaibols inserted into artificial membranes have been studied over the last 30 years. However, to our knowledge, no studies have addressed directly their behavior in living cells. In this work, a novel strategy has been employed to precisely assess the living cell membrane-penetrating activity of a fluorescein-labeled Aib (α-aminoisobutyric acid)-containing peptide derived from a peptaibol, trichorovin-XIIa (TV-XIIa). We have demonstrated for the first time that the peptide containing an unusual amino acid residue, Aib, is taken up by cells via a non endocytic pathway. The replacement of Aib in the TV-XIIa sequence with Ala inhibits the cellular uptake.     

N-methylation of N(alpha)-acylated, fully C(alpha)-methylated, linear, folded peptides: synthetic and conformational aspects

Peptides characterized by single or multiple N-methylated, C(alpha)-trisubstituted (e.g., protein) amino acids are of great interest in medicinal chemistry. Several naturally occurring peptides, remarkably stable to enzymatic attacks, are based on N-methylated residues. The classical conditions (CH(3)I/Ag(2)O in DMF, 24 h, room temperature) for N-methylation of the peptide function are useful tools for distinguishing solvent exposed from intramolecularly H-bonded -CO-NH- groups in peptides. In this work we have extended this reaction to N(alpha)-acylated, linear peptides based exclusively on helicogenic C(alpha)-tetrasubstituted alpha-amino acids, e.g., Aib (alpha-aminoisobutyric acid) or (alphaMe)Nva (C(alpha)-methyl norvaline) residues. Under the experimental conditions used, only amide monomethylation (on the N-terminal, acylated, residue) takes place. Methylation of internal peptide groups linking two C(alpha)-tetrasubstituted residues was not observed. Our FT-IR absorption, NMR, and X-ray diffraction investigations support the view that the beta-turn and 3(10)-helical conformations preferred by the original peptides are not dramatically perturbed in the derivatives monomethylated at position 1. In particular, the tertiary amide bonds are trans. Conversely, the packing modes in the crystals are strongly influenced by the reduction of the number of H-bonding donors. The MeXxx-Xxx peptide bond is readily disrupted under mild acidic conditions.     

Effect of lengthening of peptide backbone by insertion of chiral beta-homo amino acid residues: conformational behavior of linear peptides containing alternating L-leucine and beta-homo L-leucine residues

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

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 beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.     

Peptides from chiral C alpha,alpha-disubstituted glycines. Crystallographic characterization of conformation of C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] in simple derivatives and model peptides

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral C alpha,alpha-disubstituted glycine C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] have been determined by X-ray diffraction. The derivative is mClAc-L-(alpha Me)Val-OH, and the peptides are Z-L-(alpha Me)Val-(L-Ala)2-OMe monohydrate, Z-Aib-L-(alpha Me)Val-(Aib)2-OtBu, and Ac-(Aib)2-L-(alpha Me)Val-(Aib)2OtBu acetonitrile solvate. The tripeptide adopts a type-I beta-turn conformation stabilized by a 1----4N--H...O = C intramolecular H-bond. The tetra- and pentapeptides are folded in regular right-handed 3(10)-helices. All four L-(alpha Me)Val residues prefer phi, psi angles in the right-handed helical region of the conformational map. The results indicate that: (i) the (alpha Me)Val residue is a strong type-I/III beta-turn and helix former, and (ii) the relationship between (alpha Me)Val chirality and helix screw sense is the same as that of C alpha-monosubstituted protein amino-acids. The implications for the use of the (alpha Me)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.     

Synthetic formyl-methionyl chemoattractants: A conformation-activity study of oxidized tripeptides

The two diastereomeric sulphoxides and the sulphone derived from the formyl-methionyl tripeptide chemoattractant CHO-L-Met-L-Leu-L-Phe-OMe have been synthesized and fully characterized. The diastereomeric sulphoxide tripeptides have been separated at the stage of their N-tert-butyloxycarbonyl synthetic precursors. All of the oxidized sulphur derivatives induce secretion of granule enzymes with ED₅₀s from 1–2×10⁻⁹ M with no significant differences in activity among them. They are also active to the same relative extent in inducing chemotaxis. In parallel, a solution conformational analysis has been performed in solvents of widely different polarities and capabilities of H-bond formation using circular dichroism, infrared absorption and ¹H nuclear magnetic resonance. No significant propensity for formation of intramolecularly (C=O…H-N) H-bonded folded forms has been detected in any of the four tripeptides. Intermolecular S=O…H-N interactions are postulated to tentatively explain the ¹H nuclear magnetic resonance behavior of the Met and, particularly, Leu NH resonances of the two sulphoxide tripeptides in CDCl₃ solution. The biological and conformational data agree with the recently proposed model of the chemotactic peptide receptor of rabbit neurotrophils, which involves the extended backbone of the integrity of the Met side-chain sulphide sulphur atom as a corollary point of ligand interaction.     

Mixed conformation in Cα,α-disubstituted tripeptides: X-ray crystal structures of Z-Aib-Dph-Gly-Ome and Bz-Dph-Dph-Gly-Ome

We report here the synthesis and molecular structure in the solid state of fully protected tripeptides containing C^α,α-diphenylglycine (Dph), namely Z-Aib-Dph-Gly-OMe (Aib: C^α,α-dimethylgrycine) and Bz-Dph-Dph-Gly-OMe. The molecular conformation around the Dph residue, containing two bulky substituents, is fully extended, while the Aib residue, containing two smaller groups on the C^α atom, adopts the typical 3₁₀/α-helical conformation. Gly residues, without substituents on the C^α atom, show different conformational preferences. Each residue seems to behave, from a conformational point of view, independently from the presence of the other residues, and thus mixed local conformations (folded and extended) are present in the crystals. The nonconventional peptide synthesis, using the Ugi reaction, is also reported. © 1994 John Wiley & Sons, Inc.     

Incorporation of azaglutamine residues into peptides synthesised by the ultra-high load solid (gel)-phase technique.

The solid (gel)-phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono-, di- and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N-Activation of the amino-terminus of a resin-bound peptide with bis-(2,4-dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin-bound protected peptides containing the aza-residue. By contrast, coupling of activated amino-acid derivates to the free amino-group of a resin-bound peptide with an aza-residue at the N-terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino-acyl carbazate to a resin-bound isocyanate-activated peptide.     

The crystal structures of the synthetic C‐terminal octa‐ and dodecapeptides of trichovirin

The structures of two synthetic peptides with sequences corresponding to the C‐terminal region of the naturally occurring 14‐residue peptaibol trichovirin have been determined. The crystal structures of 8‐ and 12‐residue segments are presented and are compared with the structures of the tetrapeptide and of the 9‐residue segment, which have been reported earlier. A comparison between these segments leads to the hypothesis that the three‐dimensional structure of trichovirin is to a large extent determined by the properties of a periodically repeating ‐Aib‐Pro‐ pattern in the sequence of the peptide. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

X-Pro peptides: Solution and solid-state conformation of benzyloxycarbonyl-(Aib-Pro)2methyl ester,a type I β-turn

The synthesis of the tetrapeptide benzyloxycarbonyl(α-aminoisobutyryl-L -prolyl)₂-methyl ester (Z-(Aib-Pro)₂-OMe) and an analysis of its conformation in solution and the solid state are reported. Stepwise synthesis using dicyclohexylcarbodiimide leads to racemization at Pro(2). Evidence for the presence of diastereomeric tetrapeptides is obtained from 270-MHz¹H-nmr and 67.89-MHz ¹³C-nmr. The all-L tetrapeptide is obtained by fractional crystallization from ethyl acetate. The NH of Aib(3) is shown to be involved in an intramo-lecular hydrogen bond by variable-temperature ¹H-nmr and the solvent dependence of NH chemical shifts. The results are consistent with a β-turn conformation with Aib(1) and Pro(2) at the corners stabilized by a 4 → 1 hydrogen bond. The molecule crystallizes in the space group P2₁2₁2₁, with a = 8.839, b = 14.938, and c = 22.015 Å. The structure has been refined to an R value of 0.051. The peptide backbone is all-trans, and a 4 → 1 hydrogen bond, between the CO group of the urethane moiety and Aib(3) NH, is observed. Aib(1) and Pro(2) occupy the corner positions of a type I β-turn with ? = ?55.4°, Ψ = ?31.3° for Aib(1) and ? = ?71.6°, Ψ = ?38° for Pro(2). The tertiary amide unit linking Pro(2) and Aib(3) is significantly distorted from planarity (Δω = 14.3°).