Peptide mimics for structural features in proteins. Crystal structures of three heptapeptide helices with a C-terminal 6-->1 hydrogen bond.

The crystal structure determination of three heptapeptides containing alpha-aminoisobutyryl (Aib) residues as a means of helix stabilization provides a high-resolution characterization of 6-->1 hydrogen-bonded conformations, reminiscent of helix-terminating structural features in proteins. The crystal parameters for the three peptides, Boc-Val-Aib-X-Aib-Ala-Aib-Y-OMe, where X and Y are Phe, Leu (I), Leu, Phe (II) and Leu, Leu (III) are: (I) space group P1, Z = 1, a = 9.903 A, b = 10.709 A, c = 11.969 A, alpha = 102.94 degrees, beta = 103.41 degrees, gamma = 92.72 degrees, R = 4.55%; (II) space group P21, Z = 2, a = 10.052 A, b = 17.653 A, c = 13.510 A, beta = 108.45 degrees, R = 4.49%; (III) space group P1, Z = 2 (two independent molecules IIIa and IIIb in the asymmetric unit), a = 10.833 A, b = 13.850 A, c = 16.928 A, alpha = 99.77 degrees, beta = 105.90 degrees, gamma = 90.64 degrees, R = 8.54%. In all cases the helices form 3(10)/alpha-helical (or 3(10)helical) structures, with helical columns formed by head-to-tail hydrogen bonding. The helices assemble in an all-parallel motif in crystals I and III and in an antiparallel motif in II. In the four crystallographically characterized molecules, I, II, IIIa and IIIb, Aib(6) adopts a left-handed helical (hL) conformation with positive phi, psi values, resulting in 6-->1 hydrogen-bond formation between Aib(2) CO and Leu(7)/Phe(7) NH groups. In addition a 4-->1 hydrogen bond is seen between Aib(3) CO and Aib(6) NH groups. This pattern of hydrogen bonding is often observed at the C-terminus of helices proteins, with the terminal pi-type turn being formed by four residues adopting the hRhRhRhL conformation.

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

Two crystals structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue alpha, alpha-di-n-butylglycyl, reveal a mixed 3(10)/alpha-helical conformation. Residues 1-7 adopt phi, psi values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has phi, psi values of -40, -37 degrees and -46, -40 degrees in 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 C50H91N9O12, space group P2(1), with a = 9.789(1) Angstrom, b = 20.240(2) Angstrom, c = 15.998(3) Angstrom, beta = 103.27(1); Z = 2, R = 10.3% for 1945 data observed >3 sigma(F) and C50H91N9O12. 3H(2)O, space group P2(1), with a = 9.747(3) Angstrom, b = 21.002(8) Angstrom, c = 15.885(6) Angstrom, beta = 102.22(3)degrees, Z = 2, R = 13.6% for 2535 data observed >3 sigma(F). The observation of a helical conformation at Dbg suggests that the higher homologs in the alpha, alpha-dialkylated glycine series also have a tendency to stabilize peptide helices. (C) Munksgaard 1996.

Polypeptide Helices in Hybrid Peptide Sequences

A new class of polypeptide helices in hybrid sequences containing alpha-, beta-, and gamma-residues is described. The molecular conformations in crystals determined for the synthetic peptides Boc-Leu-Phe-Val-Aib-beta Phe-Leu-Phe-Val-OMe 1 (beta Phe: (S)-beta(3)-homophenylalanine) and Boc-Aib-Gpn-AibGpn-OM2(Gpn:1-(aminomethyl)cycl hexaneacetic acid) reveal expanded helical turns in the hybrid sequences (alpha alpha beta)(n) and (ay), In 1, a repetitive helical structure composed Of C-14 hydrogen-bonded units is observed, whereas 2 provides an example of a repetitive C-12 hydrogen-bonded structure. Using experimentally determined backbone torsion angles for the hydrogen-bonded units formed by hybrid sequences, we have generated energetically favorable hybrid helices. Conformational parameters are provided for C-11, C-12, C-13, C-14, and C-15 helices in hybrid sequences.

Characterization of Alkali Induced Formation of Lanthionine, Trisulfides, and Tetrasulfides from Peptide Disulfides using Negative Ion Mass Spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfied linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide (Boc - Cys - Pro - Leu - Cys - NHMe) and an acyclic peptide, oxidized glutathione, bis ((gamma)Glu Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of (CH)-H-alpha or (CH)-H-beta protons of Cys residues, with Subsequent elimination of H2S or H2S2. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S-2. (J Am Soc Mass Spectrom 2009, 20, 783-791) (C) 2009 American Society for Mass Spectrometry.

Characterization of Alkali Induced Formation of Lanthionine, Trisulfides, and Tetrasulfides from Peptide Disulfides using Negative Ion Mass Spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfied linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide (Boc - Cys - Pro - Leu - Cys - NHMe) and an acyclic peptide, oxidized glutathione, bis ((gamma)Glu Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of (CH)-H-alpha or (CH)-H-beta protons of Cys residues, with Subsequent elimination of H2S or H2S2. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S-2. (J Am Soc Mass Spectrom 2009, 20, 783-791) (C) 2009 American Society for Mass Spectrometry.

Helical Conformations of Hexapeptides Containing N-Terminus Diproline Segments

The role of N-terminus diproline segments in facilitating helical folding in short peptides has been investigated in a set of model hexapeptides of the type Piv-Xxx-Yyy-Aib-Leu-Aib-Phe-OMe (Piv, pivaloyl). Nine sequences have been investigated with the following N-terminus dipeptide segments: (D)Pro-Ala (4) and Pro-Psi Pro (5, Psi, pseudoproline), Ala-Ala (6), Ala-Pro (7), Pro-Ala (8), Aib-Ala (9), Ala-Aib (10). The analog sequences Piv-Pro-Pro-Ala-Leu-Aib-Phe-OMe (2) and Piv-Pro-Pro-Ala-Aib-Ala-Aib-OMe (3) have also been studied. Solid state conformations have been determined by X-ray crystallography for peptides 4, 6, and 8 and compared with the previously determined crystal structure of peptide 1 (Boc-Pro-Pro-Aib-Leu-Aib-Val-OMe); (Rai et al., JACS 2006, 128, 7916-7928). Peptides 1 and 6 adopt almost identical helical conformations with unfolding of the helix at the N-terminus Pro (1) residue. Peptide 4 reveals the anticipated (D)Pro-Ala type II' beta-turn, followed by a stretch of 3(10)-helix. Peptide 8 adopts a folded conformation stabilized by four successive 4 -> 1 intramolecular hydrogen bonds. Ala (2) adopts an alpha(L) conformation, resulting in a type II beta-turn conformation followed by a stretch of 3(10)-helix. Conformational properties in solution were probed using solvent perturbation of NH chemical shifts which permit delineation of hydrogen bonded NH groups and nuclear Overhauser effects (NOEs) between backbone protons, which are diagnostic of local residue conformations. The results suggest that continuous helical conformations are indeed significantly populated for peptides 2 and 3. Comparison of the results for peptides 1 and 2, suggest that there is a significant influence of the residue that follows diproline segments in influencing backbone folding. (C) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 360-370, 2010.

Structural studies of model peptides containing beta-, gamma- and delta-amino acids

The crystal structures of five model peptides Piv-Pro-Gly-NHMe (1), Piv-Pro-beta Gly-NHMe (2), Piv-Pro-beta Gly-OMe (3), Piv-Pro-delta Ava-OMe (4) and Boc-Pro-gamma Abu-OH (5) are described (Piv:pivaloyl; NHMe: N-methylamide; beta Gly:beta-glycine; OMe:O-methyl ester; delta Ava:delta-aminovaleric acid; gamma Abu:gamma-aminobutyric acid). A comparison of the structures of peptides 1 and 2 illustrates the dramatic consequences upon backbone homologation in short sequences. 1 adopts a type II beta-turn conformation in the solid state, while in 2, the molecule adopts an open conformation with the beta-residue being fully extended. Piv-Pro-beta Gly-OMe (3), which differs from 2 by replacement of the C-terminal NH group by an O-atom, adopts an almost identical molecular conformation and packing arrangement in the solid state. In peptide 4, the observed conformation resembles that determined for 2 and 3, with the delta Ava residue being fully extended. In peptide 5, the molecule undergoes a chain reversal, revealing a beta-turn mimetic structure stabilized by a C-H center dot center dot center dot O hydrogen bond.

An unusual C-H center dot center dot center dot O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix

An unusual C-terminal conformation has been detected in a synthetic decapeptide designed to analyze the stereochemistry of helix termination in polypeptides. The crystal structure of the decapeptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe reveals a helical segment spanning residues 1-7 and helix termination by formation of a Schellman motif, generated by (D)Ala(8) adopting the left-handed helical (alpha(L)) conformation. The extended conformation at (D)Leu(9) results in a compact folded structure, stabilized by a potentially strong C-H ... O hydrogen bond between Ala(4) (CH)-H-alpha and (D)Leu(9)CO. The parameters for C-H ... O interaction are Ala(4) (CH)-H-alpha .. O=C (D)Leu(9) distance 3.27 Angstrom C-alpha-H .. O angle 176 degrees, and O .. H-alpha distance 2.29 Angstrom. This structure suggests that insertion of contiguous D-residues may provide a handle for the generation of designed structures containing more than one helical segment folded in a compact manner. (C) 2000 Academic Press.

Conformations of dehydrophenylalanine containing peptides: nmr studies of an acyclic hexapeptide with two delta Z-Phe residues

The conformation of an acyclic dehydrophenylalanine (delta Z-Phe) containing hexapeptide, Boc-Phe-delta Z-Phe-Val-Phe-delta Z-Phe-Val-OMe, has been investigated in CDCl3 and (CD3)2SO by 270-MHz 1H-nmr. Studies of NH group solvent accessibility and observation of interresidue nuclear Overhauser effects (NOEs) suggest a significant solvent-dependent conformational variability. In CDCl3, a population of folded helical conformations is supported by the inaccessibility to solvent of the NH groups of residues 3-6 and the detection of several NiH----Ni + 1H NOEs. Evidence is also obtained for conformational heterogeneity from the detection of some Ci alpha H----Ni + 1H NOEs characteristic of extended strands. In (CD3)2SO, the peptide largely favors an extended conformation, characterized by five solvent-exposed NH groups and successive Ci alpha H----Ni + 1H NOEs for the L-residues and Ci beta H----Ni + 1H NOEs for the delta Z-Phe residues. The results suggest that delta Z-Phe residues do not provide compelling conformational constraints.

Synthetic and conformational studies on dehydrophenylalanine containing model peptides

Four model dipeptides containing a Z-dehydrophenylalanine residue (ΔZPhe) at the C-terminal, Boc-X-ΔZ Phe-NHMe (X = Ala (1), Gly (2), Pro (3), and Val (4)), have been synthesised and their solution conformations investigated by 270 MHz 1H n.m.r. and i.r. spectroscopy. N.m.r. studies on these peptides clearly show the presence of intramolecularly hydrogen bonded structures in CHCl3 solutions while such structures appear to be absent in the corresponding saturated peptides. This conclusion is also supported by i.r. studies. Studies of the nuclear Overhauser effect provided evidence for the occurrence of a significant population of β-turn structures in solvents like CDCl3 and (CD3)2SO. The observed NOES are consistent with a major contribution from Type II β-turn structure in CDCl3, while in (CD3)2SO solutions there is evidence of a partially extended structure also.

Stereochemically constrained peptides. Theoretical and experimental studies on the conformations of peptides containing 1-aminocyclohexane carboxylic acid

Conformational energy calculations on the model system N-acetyl- 1 -aminocyclohexanecarboxylic acid N'methylamide (Ac-Acc6-NHMe), using an average geometry derived from 13 crystallographic observations, establish that the Acc6 residue is constrained to adopt conformations in the helical regions of In contrast, the a,a-dialkylated residue with linear hydrocarbon side chains, a,a-di-n-propylglycine favors fully extended backbone structures (6 1= $ = 180'). The crystal structures of two model peptides, Boc-(Acc6),-OMe (type 111 @-turn at -Acc6(1)-Acc6(2)-) and Boc-Pro-Acc6-Ala-OMe (type I1 P-turn at -Pro-Acc6-), establish that Acc6 residues can occupy either position of type 111 P-turns and the i + 2 position of type I1 @-turns. The stereochemical rigidity of these peptides is demonstrated in solution by NMR studies, which establish the presence of one intramolecular hydrogen bond in each peptide in CDCI, and (CDJ2S0. Nuclear Overhauser effects permit characterization of the @-turn conformations in solution and establish their similarity to the solid-state structures. The implications for the use of Acc6 residues in conformational design are considered.

Conformations of dehydrophenylalanine containing peptides. NMR studies on three tripeptides with a central dehydrophenylalanyl residue.

Three tripeptides containing a central Z-dehydrophenylalanine residue (Δz-Phe), Boc-L-Phe-Δz-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 Δz-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 Δz-Phe residue.

Conformations of dehydrophenylalanyl containing peptides. Nuclear Overhauser effect study of two acyclic

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (Δz-Phe) at position 2 or 3, Boc-Leu-Ala-Δz-Phe-Leu-OMe (1) and Boc-Leu-Δz-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 (CαjH ⇆ Ni+1H and NiH ⇆ Ni+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-Δz-Phe- Type II β-turn structure and a second species with Δz-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-Δz-Phe-Ala- segment and an almost completely extended conformation.

Probing the role of the C-H center dot center dot center dot O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides

The structural characterization in crystals of three designed decapeptides containing a double D-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val- (D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all L analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed a-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-(HO)-O-... hydrogen bond between residue 4 (CH)-H-alpha and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C-alpha atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt at conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-(HO)-O-... hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.

Conformational properties of hybrid peptides containing alpha and omega-amino acids

This review briefly surveys the conformational properties of guest omega-amino acid residues when incorporated into host alpha-peptide sequences. The results presented focus primarily on the use of beta- and gamma-residues in alphaomega sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between alpha-peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and beta-hairpin conformations permits the characterization of backbone conformational parameters for beta- and gamma-residues inserted into regular alpha-polypeptide structures. Substituted beta- and gamma-residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral beta,beta-disubstituted gamma-amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C-beta-C-gamma (theta(1)) and C-alpha-C-beta (theta(2)) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.