Folding of peptides characterized by c3Val,a highly constrained analogue of valine

Using a combined chemical/chiral chromatographic approach we synthesized an N-protected derivative of (R)-c(3)Val, a severely conformationally restricted C(alpha)-tetrasubstituted alpha-amino acid characterized by a C(beta,beta)-dimethylated cyclopropane system. A set of terminally protected derivatives and model peptides (to the heptamer level), containing one or two (R)-c(3)Val residues in combination with either Aib or Gly residues, was prepared by solution methods. A detailed solution and crystal-state conformational investigation, based on Fourier transform infrared (FTIR) absorption, (1)H-NMR, and x-ray diffraction techniques, performed in comparison with a similar study on related derivatives and peptides rich in (alphaMe)Val, the prototype of C(alpha)-tetrasubstituted alpha-amino acids of this subfamily, allowed us to conclude the following: (a) c(3)Val is a good beta-bend and helix former, although less efficient than (alphaMe)Val. (b) The relationship between alpha-carbon chirality and screw sense of the folded structure formed is the same as that of (alphaMe)Val, i.e., the (R)-enantiomer has a strong left-handed bias. (c) c(3)Val seems more prone than (alphaMe)Val to fold into a gamma-bend conformation. The conformational propensities of C(beta,beta)-disubstituted Ac(3)c residues are also discussed in comparison with those of the parent cyclopropane residue.     

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.     

Synthesis of chiral alpha-amino aldehydes linked by their amine function to solid support.

The anchoring of an alpha-amino-acid derivative by its amine function on to a solid support allows some chemical reactions starting from the carboxylic acid function. This paper describes the preparation of alpha-amino aldehydes linked to the support by their amine function. This was performed by reduction with LiAlH4 of the corresponding Weinreb amide linked to the resin. The aldehydes obtained were then involved in Wittig or reductive amination reactions. In addition, the linked Weinreb amide was reacted with methylmagnesium bromide to yield the corresponding ketone. After cleavage from the support, the compounds were obtained in good to excellent yields and characterized.     

Enantiomeric recognition of chiral 3,3-bridged-1,1'-binaphthol dimer toward alpha-phenylethylamine and alpha-amino acid ester

The 1,1'-binaphthol-based dimers with p-phenylenebis(2-ethynyl) spacer, (+)-6 and (+)-2, were synthesized as chiral host compounds. (1)H NMR, UV-vis, and fluorescent titration were used to evaluate the enantiomeric recognition abilities of the chiral host dimers toward the guest amine 7 and alpha-amino acid ester 8. The chiral BINOL-based dimers were found to have good enantiomeric recognition ability. The computer simulation of the host-guest complex molecules was carried out to describe the conformational changes of both naphthyl ring in the molecule of chiral host dimer after complexation with the guest molecule.     

The complete chirospectroscopic signature of the peptide 3(10)-helix in aqueous solution

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.     

Stereoselective acylation of a racemic amine with C(alpha)-methyl phenylglycine-based dipeptide 5(4H)-oxazolones

Reactions of a racemic amine with chiral, N(alpha)-acetylated, C(alpha)-methyl l-phenylglycine-based dipeptide 5(4H)-oxazolones proceed diastereoselectively to give predominantly dipeptide alkylamides comprising d-alpha-phenylethylamine. Diastereoselectivity is remarkably sensitive to solvent polarity and reaction temperature but not significantly to the nature of the C(alpha)-tetrasubstituted alpha-amino acid at position 1 of the dipeptide. The beta-turn 3D structures of the aminolysis products were established in CDCl(3) solution by FT-IR absorption and in one case in the crystal state by X-ray diffraction as well.     

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.     

Efficient protocols for the synthesis of enantiopure gamma-amino acids with proteinogenic side chains.

The synthesis of enantiopure gamma-substituted gamma-amino acids with proteinogenic side chains, starting from the corresponding natural alpha-amino acids, was studied. N-Protected amino aldehydes containing various protective groups were prepared from the corresponding amino alcohols by oxidation with NaOCl in the presence of AcNH-TEMPO and directly reacted with methyl, benzyl and tert-butyl phosphoranylidene acetate to produce alpha,beta-unsaturated gamma-amino esters. Simultaneous hydrogenation of the double bond and removal of either the benzyl or benzyloxycarbonyl group led to N- or C-protected gamma-amino acids in high yield. The enantiomeric purity was studied by 1H NMR analysis of Mosher amides and chiral HPLC analysis.     

Computational study of the conformational preferences of the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide.

alpha-Amino acids are important building blocks for the synthesis of a large number of bioactive compounds and pharmaceutical drugs. However, a literature survey revealed that no theoretical conformational study of alpha-amino acids with cage carbon frameworks has been performed to date. This paper reports the results of a conformational study on the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide (cage monopeptide), using molecular mechanics and ab initio methods. The in vacuo Ramachandran maps computed using the different parameterizations of the AMBER force field show the C7eq structure as the most favourable conformation, in contrast to the C7ax structure, that is the lowest energy conformation at the ab initio level. Analysis of these maps reveals the helical preference for the monopeptide and provides the potential for the cage residue to be incorporated into constrained peptide analogues.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Unique reactivity of alpha,beta-unsaturated carboxylic acid imidazolides: catalytic asymmetric synthesis of alpha,beta-epoxy esters and alpha,beta-epoxy carboxylic acid derivatives

Catalytic asymmetric synthesis of alpha,beta-epoxy esters and alpha,beta-epoxy carboxylic acid derivatives is described. Catalytic asymmetric epoxidation of alpha,beta-unsaturated carboxylic acid imidazolides using La-BINOL-Ph(3)As=O complex gave the corresponding alpha,beta-epoxy peroxy tert-butyl esters, which were directly converted to the alpha,beta-epoxy methyl esters by adding methanol to the reaction. This catalytic system had broad generality for epoxidation of various substrates. With the use of 5-10 mol% of the catalyst, both beta-aryl and beta-alkyl-substituted-alpha,beta-epoxy methyl esters were obtained in up to 91% yield and in up to 93% enantiomeric excess. In addition, efficient transformations of alpha,beta-epoxy peroxy tert-butyl esters into the alpha,beta-epoxy amides, alpha,beta-epoxy aldehydes, and gamma,delta-epoxy beta-keto esters are also reported.     

Peroxisome proliferator-activated receptor gamma agonists inhibit adipocyte expression of alpha1-acid glycoprotein

alpha1-Acid glycoprotein (alpha1-AGP) is an acute phase protein that can potentiate cytokine secretion by mononuclear cells and may induce thrombosis by stabilizing the inhibitory activity of plasminogen activator inhibitor-1. Thus, alpha1-AGP may promote pathobiologies associated with type 2 diabetes mellitus (T2DM) including insulin resistance and cardiovascular disease. Here, we demonstrate that antidiabetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists inhibited expression of 3T3-L1 adipocyte alpha1-AGP in a concentration- and time-dependent manner via an apparent PPARgamma-mediated mechanism. As a result, synthesis and secretion of the glycoprotein was reduced. While PPARgamma agonist regulation of genes with functional peroxisome proliferator response elements in their promoter such as phosphoenolpyruvate carboxykinase were unaffected when cellular protein synthesis was inhibited, downregulation of alpha1-AGP mRNA was ablated thereby supporting the proposition that PPARgamma activation inhibits alpha1-AGP expression indirectly. These results suggest a potential novel adipocytic mechanism by which PPARgamma agonists may ameliorate T2DM-associated insulin resistance and cardiovascular disease.     

Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes

In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes.     

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.     

Synthesis of glyoxylyl peptides using an Fmoc-protected alpha,alpha'-diaminoacetic acid derivative.

The synthesis of glyoxylyl peptides by coupling the masked glyoxylic acid derivative (FmocNH)(2)CHCO(2)H, 1, to a peptidyl resin assembled using Fmoc/tert-butyl chemistry has been described recently. Deprotection and cleavage of the peptide from the solid support using TFA was followed by unmasking of the glyoxylyl group in solution in the presence of DBU. [] The glyoxylyl peptide was thus generated using non-oxidizing conditions by comparison with the method based on the periodic oxidation of a seryl-precursor. However, base treatment of the (FmocNH)(2)CHCO(2)-peptide led to the formation of a byproduct besides the desired glyoxylyl peptide. This paper describes an optimized procedure for unmasking the Fmoc-protected alpha,alpha'-diaminoacetic acid moiety in solution which suppressed byproduct formation. Also presented is a series of experiments that permitted a structure and a mechanism of formation for the byproduct to be suggested.     

Metal ion-binding ability of tetrapeptides containing alpha-aminoisobutyric acid.

alpha-Aminoisobutyric acid (Aib), one of the Calpha,alpha-disubstituted glycines, is a sterically hindered amino acid that acts as a conformational constraint in peptides. However, studies for the application of the ability of Aib to control conformation are quite few. The paper focuses on the molecular recognition ability of acyclic oligopeptides containing Aib. Liquid-liquid extraction of nine kinds of metal ions from aqueous layers to nonpolar organic layers with acyclic tetrapeptides, X-Trp-Xaa2-Gly-Xaa4-NH-Ar (X = H or C6H5CH2OCO (Z), Xaa2 = Aib or Gly, Xaa4 = Leu or Ala, Ar = phenyl or 3,5-dimethylphenyl) was examined using picrate as the anion of ion pairs. The extraction behaviour of the metal ions with the tetrapeptides was investigated in the pH range from 3 to 9. In the case of basic pH regions, Cu(II) and Ag(I) were effectively extracted with Trp-Aib-Gly-Leu-NH-Ar. Pd(II) was specifically extracted with Trp-Aib-Gly-Leu-NH-Ar in acidic pH regions. The extraction percent (%E) of the peptide host, which has a 3,5-dimethylphenyl group, was even larger than that of the host, which has a phenyl group. Moreover, Pd(II) was extracted with a peptide host which has Leu and a 3,5-dimethylphenyl group in the absence of picrate as the anion of ion pairs. The free alpha-amino group, the turn conformation and the hydrophobicity of peptide molecules were important factors for the extraction of the metals.     

Crystal-state conformation of Calpha,alpha-dialkylated peptides containing chiral beta-homo-residues.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.     

Bicyclic glutamic acid derivatives

For the second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids via Strecker reaction of chiral ketimines, the cyanide addition as the key stereodifferentiating step produces mixtures of diastereomeric alpha-amino nitrile esters the composition of which is independent of the reaction temperature and the type of the solvent, respectively. The subsequent hydrolysis is exclusively achieved with concentrated H(2)SO(4) yielding diastereomeric mixtures of three secondary alpha-amino alpha-carbamoyl-gamma-esters and two diastereomeric cis-fused angular alpha-carbamoyl gamma-lactams as bicyclic glutamic acid derivatives, gained from in situ stereomer differentiating cyclisation of the secondary cis-alpha-amino alpha-carbamoyl-gamma-esters. Separation was achieved by CC. The pure secondary trans-alpha-amino alpha-carbamoyl-gamma-esters cyclise on heating and treatment with concentrated H(2)SO(4), respectively, to diastereomeric cis-fused angular secondary alpha-amino imides. Their hydrogenolysis led to the enantiomeric cis-fused angular primary alpha-amino imides. The configuration of all compounds was completely established by NMR methods, CD-spectra, and by X-ray analyses of the (alphaR,1R,5R)-1-carbamoyl-2-(1-phenylethyl)-2-azabicyclo[3.3.0]octan-3-one and of the trans-alphaS,1S,2R-2-ethoxycarbonylmethyl-1-(1-phenylethylamino)cyclopentanecarboxamide.     

Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels.

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels.     

Activating mutations in the NH2- and COOH-terminal moieties of the Gs alpha subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation.

The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase.