Aza-peptides II. X-Ray structures of aza-alanine and aza-asparagine-containing peptides

In order to determine the structural consequences of the N^x/C^xH exchange in aza-peptides, we have solved the crystal molecular structures of some derivatives containing the aza-analogue of asparagine [Z-AzAsn(Me)-NMe₂ (1), Z-AzAsn(Me)-Pro-NHiPr (2) and Piv-Pro-AzAsn(Me)-NHiPr (5), aspartic acid [Z-AzAsp(OEt)-Pro-NHiPr (3) and alanine (Boc-AzAla-Pro-NHiPr (4), by using X-ray diffraction. They reveal that the α-nitrogen accommodates a pyramidal (1–4) or planar (5) structure depending on the sequence. When pyramidal, the α-nitrogen assumes the R (D-like) chiralily. All of the derivatives but 1 adopt either a β₁-folded (2–4) or β_II-folded (5) structure in which the (AzAsn)N^δH bond is intramolecularly hydrogen-bonded to the α-nitrogen. © Munksgaard 1997.     

X-ray structures of aza-proline-containing peptides

The aza-analogue of proline (AzPro) contains a nitrogen atom in place of the CH^α of the cognate residue. The resolution of the crystal structures of seven AzPro-containing peptides, presenting a set of ten AzPro motifs, reveals the structural properties of this particular aza-residue. Because of sterical hindrances, both nitrogen atoms are out of planarity, and the reduced electronic conjugation in the two AzPro-adjacent amide groups probably explains the longer amide bond distances and the weak proton-accepting character of the two pyrazolidine nitrogens. The absolute configuration of both AzPro nitrogens depends on the chemical nature of the sequence. In all cases, the AzPro residue assumes the same intrinsic three-dimensional structure and presents folding tendencies opposed to those induced by proline.     

Molecular design of peptides

The peptide N-Boc-l -Phe-dehydro-Abu-NH-CH₃ was synthesized by the usual workup procedure. The crystals grown from methanol at 4°C belong to the space group P2₁2₁2₁ with a= 7.589(2), b= 13.690(4), c= 21.897(6) Å, Z= 4 and d_c= 1.149(5) g cm^?3 for C₁₉H₂₉N₃O₅·CH₃OH. The peptide crystals were highly sensitive to radiation. The final agreement factor R was 0.055 for 1109 observed reflections (I > 2σ) with data extending to a 2θ value of 103°. The methanol oxygen atom is split into two occupancies. Both sites are involved in identical hydrogen bonding. As a result of substitution of a dehydro-Abu residue at the (i+ 2) position the peptide adopts an ideal β-turn II′ conformation with torsion angles of corner residues as φ₁=63(1)°, ψ₁= - 127(1)°, φ₂= -66(1)° and ψ₂= - 10(1)°, and an intramolecular hydrogen bond N—H ? O of length 3.01(1) Å. This shows that the conformational constraints produced by dehydro-Abu are similar in nature to but different in magnitude than those produced by dehydro-Phe and dehydro-Leu. The methanol–peptide interactions show characteristic features of multiple hydrogen-bond formations involving polar sites of participating peptide and methanol molecules. The packing of the molecules in the unit cell is stabilized by interactions through methanol molecules with the help of several hydrogen bonds.     

Theoretical conformational analysis of brain peptides

Using a semi-empirical method, an a priori conformational analysis of the octadecapeptide β-melanocyte-stimulating hormone (β-MSH) was carried out. The spatial structure of β-MSH can be described by eight low-energy conformations, yielded by combinations of the most stable states of the respective free fragments. Calculations produced the values of all dihedral angles of the backbones and side chains of these forms as well as intra- and inter-residue interaction energies.     

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.

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.     

[1-Desaminopenicillamine, 8-α-hydroxyisocaproic acid] oxytocin. A selective inhibitor in rats of the uterine response to oxytocin

[1-Desaminopenicillamine, 8-α-hydroxyisocaproic acid] oxytocin was synthesized by a 6 + 3 fragment condensation from precursors which had been formed by solution methods. This analog inhibited uterine responses to oxytocin (pA₂ 7.37, 7.9, 6.17; uterus in vitro without Mg++, in vitro with Mg++, and in vivo, respectively) and showed little or no activity in other bioassays.     

Vibrational analysis of peptides,polypeptides, and proteins

The normal modes have been calculated for three kinds of low energy γ-turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a γ-turn, a mirror-related γ-turn, and an inverse γ-turn of CH₃-CO-(L-Ala)_n-NH-CH₃, with n = 3 and n = 5, and for certain ¹⁴C and ¹⁵N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and γ-turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.     

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.     

Vibrational analysis of peptides,polypeptides and proteins

Normal mode calculations have been carried out on three low-energy structures of gramicidin S obtained from conformational energy calculations. When the results on the amide modes are compared with observed bands in the infrared and Raman spectra of crystalline gramicidin S and its N-deuterated derivative, one of the structures is clearly disfavored. Of the other two, one is slightly favored, and it corresponds to the lowest-energy structure obtained from the energy calculations. Spectra from solutions in DMSO and CH₃ OH suggest that the molecular conformation is essentially retained in these solvents.     

Structure,solubility and reactivity of peptides

A conformational study of two protected peptide segments, (1–10 and 11–28), spanning the entire sequence of thymosin α₁, in solvents of different polarity and capability of forming hydrogen bonds, is reported. By using infrared absorption and circular dichroism techniques the occurrence of the random coil conformation, the self-associated β-structure, and the α-helix (the latter adopted only by the longer peptide) was established. The self-associated species of the two peptide segments were disrupted either by adding increasing amounts of hexamethylphosphoramide or by dilution. This structural transition was monitored by the disappearance of the amide-I C=O stretching band of strongly intermolecularly hydrogen-bonded molecules (near 1630 cm^-1) in the infrared absorption spectra. The tendency of these peptides to aggregate is paralleled by a decrease in their solubility. The conformational findings are discussed in terms of the solvent-dependent product yields obtained in the reaction of segment (1–10) with the N^α-deprotected (11–28) segment to give the fully protected thymosin α₁.     

Conformations of peptides containing 1-aminocyclohexanecarboxylic acid (Acc6). Crystal structures of two model peptides

The crystal structures of two peptides containing 1-aminocyclohexanecarboxylic acid (Acc⁶) are described. Boc-Aib-Acc⁶-NHMe · H₂O adopts a β-turn conformation in the solid state, stabilized by an intramolecular 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The backbone conformational angles (φ_Aib = – 50.3°, ψ_Aib = – 45.8°; φ_Acc6 = – 68.4°, ψ_Acc6 = – 15°) lie in between the values expected for ideal Type I or III β-turns. In Boc-Aib-Acc⁶-OMe, the Aib residue adopts a partially extended conformation (φ_Aib = – 62.2°, ψ_Aib = 143°) while the Acc⁶residue maintains a helical conformation (φ_Acc6 = 48°, ψ_Acc⁶= 42.6°). ¹H n.m.r. studies in CDCl₃ and (CD₃)₂SO suggest that Boc-Aib-Acc⁶-NHMe maintains the β-turn conformation in solution.     

Reactions of C-terminal ω-amino acid residues in liquid hydrogen fluoride

The benzyl-ester bond linking the C-terminal δ-aminovaleric acid residue of a peptide to a polymeric support was cleaved with liquid hydrogen fluoride in the presence of anisole, added as scavenger. Instead of the expected peptide with a free carboxyl group at the C-terminus, a peptide terminating in a ketone derivative was obtained. The unusual extent of this known side-reaction was attributed to the effect of the distance between the amino group and the carboxyl group in the C-terminal residue. The results of model experiments corroborated this view.     

Circular dichroic and immunological properties of human choriogonadotropin-β carboxyl terminal peptides

Circular dichroic spectra have been obtained in aqueous solution and in trifluoroethanol for several synthetic (non-glycosylated) human choriogonadotropin carboxyl terminal peptides of the β-subunit ranging in size from 10 residues to 40 residues. There was no evidence for formation of α-helicity or β-structure, but the spectra in 90% (v/v) trifluoroethanol were consistent with the occurrence of β-turns. The Chou-Fasman predictive rules also suggest a high probability of β-turns in these peptides which could result in the occurrence of repeating kinks. Disulfide-linked dimers were also investigated by circular dichroism, and there was evidence of stabilization of particular skewness of the disulfide dihedral angle depending upon the location of the disulfide bond. The single phenylalanyl residue at position 115 in the β-subunit also contributed to the circular dichroic spectra above 250 nm. Antibodies raised to a peptide consisting of residues 111–145 have been shown to contain two immunological determinants, but the sum of antibodies raised to separate determinant sequences do not equal those raised to the full length peptide. These data could reflect the existence of a conformation-related determinant on the 111–145 peptide or stearic hindrance of immunoglobulin binding of two antibodies to the same peptide.     

Synthesis,crystal structure and molecular conformation of the tBuCO-D,L-Ala-Δz-Phe-NhiPr α,β-unsaturated dipeptide

The crystal structure of the tBuCO-d,l -Ala-Δ^z-Phe-NHiPr dipeptide has been solved by X-ray diffraction. The peptide crystallizes in monoclinic space group P2JC with a = 13.445 (3) Å, b = 35.088 (4) Å, c = 14.755(3) Å, β= 116.73(1)°, Z = 12 and d_c= 1.151 g.cm^?3. The three independent molecules per asymmetric unit accommodate a βII-folded conformation, but only one of them contains the typical i + 3 → i interaction characterizing a β-turn. In the other two molecules, the N…O distance exceeds 3.2 Å, a value generally considered the upper limit for hydrogen bonds in peptides. In solution, the βII-turn conformation is largely predominant.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω 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 α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

Synthetic and conformational studies on dehydrophenylalanine containing model peptides

Four model dipeptides containing a Z-dehydrophenylalanine residue (Δ^ZPhe) at the C-terminal, Boc-X-Δ^Z 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.     

The crystal structure of Boc-Pro-Val-Gly-NH2, with a remark on the conformation of the valyl residues in peptides

The crystal structure of Boc-Pro-Val-Gly-NH₂ has been determined: monoclinic; P2₁; a = 9.331 (3) Å, b = 9.532 (4), c = 23.080 (9), β= 91.33 (3)R, Z = 4; R = 0.053 for 3400 reflections with ˙Fo˙,>α(Fo). There are two independent but very similar molecules in the crystal. The peptide main chains are in an extended form, and packed in two kinds of antiparallel β sheets, the (φ, Φ) angles of the central Val residues are (-156°, 146°) and (-139°, 155°), and the mean length of the N- H . 0 hydrogen bonds in the sheets is 2.965 Å. A detailed study of the conformations of the Val residues in oligopeptide crystals shows that the preferred conformation of Val in peptides is: the (φ, Φ) angles close to those of the antiparallel β sheet, and C^γ1 and C^γ2, against N with respect to the C^α– C^β bond, at either (trans, gauche) or (-gauche, gauche). The mean π(NC^αC') angle of such Val residues is 107.9(9)°. A twisting in the β sheets is also discussed.     

Effect of oxidation of β-amyloid precursor protein on its β-secretase cleavage. A model study with synthetic peptides and candidate β-secretases

As the amyloidogenic processing of β-amyloid precursor protein (βAPP) proceeds under conditions of oxidative stress, the methionine-596 residue at the β-secretase cleavage point is likely in an oxidized state. In the present work, possible consequences of the oxidation of Met-596 for the generation of the N-terminus of amyloid p protein were modeled using synthetic peptide substrates, matching 587-606 sequence fragment of βAPP and containing either intact methionine or methionine sulfoxide. Patterns and rates for the cleavage of these substrates by purified mast cell chymase, cathepsin G, cathepsin D, matrix metallopro-teinase-3 and neutrophil elastase, were compared. Only the three first proteases, all previously suggested as candidate β-secretases, preferentially cleaved the “intact” substrate after Met-596. For chymase and cathepsin G, the specificity of this cleavage increased upon a shift from optimal alkaline pH to acidic pH, which is also more compatible with the plausible intracellular localization of amyloidogenic βAPP processing. The substitution of methionine sulfoxide for methionine in the substrate slowed down the cleavage rate for all the enzymes tested, by a factor of 6-15. This was associated with shifts of cleavage preferences to points of minor importance for the “intact” peptide, suggesting a specific resistance of the peptide bond after MetSO-596 against proteolysis.