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.     

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.     

Enantio- and diastereoselective syntheses of cyclic Calpha-tetrasubstituted alpha-amino acids and their use to induce stable conformations in short peptides

C(alpha)-tetrasubstituted alpha-amino acids are widely used to design and prepare peptides and peptide mimics with constrained conformations. Subcategories of these compounds are cyclic C(alpha)-tetrasubstituted alpha-amino acids, in which both alpha-substituents are covalently connected. This survey presents recent advances in the synthesis and application of cyclic C(alpha)-tetrasubstituted alpha-amino acids in a systematic order beginning with cyclopropane amino acids, continuing with four, five, six membered rings, and ring structures larger than six-membered. We discuss synthetic routes to the cyclic C(alpha)-tetrasubstituted alpha-amino acids and their use as conformation determining elements in peptides.     

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.     

C(alpha)-methyl proline: a unique example of split personality

Methylation at the C(alpha)-position of a Pro residue was expected to lock the preceding tertiary amide (omega) torsion angle of the resulting (alphaMe)Pro to the trans disposition and to restrict the phi,psi surface to the single region where the 3(10)/alpha-helices are found (in this five-membered ring residue phi is severely constrained to about +/-65 degrees by its cyclic nature). The results of the present X-ray diffraction work on a selected set of four N(alpha)-blocked, (alphaMe)Pro-containing, dipeptide N'-alkylamides clearly show that, although the region of the conformational map largely preferred by (alphaMe)Pro would indeed be that typical of 3(10)/alpha-helices, the semi-extended [type-II poly(Pro)(n) helix] region can also be explored by this extremely sterically demanding C(alpha)-tetrasubstituted alpha-amino acid. In addition, the known high propensity for beta-turn formation of the Pro residue is further enhanced in peptides based on its C(alpha)-methylated derivative.     

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.     

Chirality transferring [3,3] sigmatropic rearrangement of (1-Acyloxy-2-alkenyl)trianlkylsilane synthesis of optically active vinylsilane-containing alpha-amino acid

A vinylsilane-containing alpha-amino acid and alpha,alpha-disubstituted alpha-amino acid 2 having two contiguous asymmetric carbon centers at their alpha and beta positions were synthesized in an optically active form by ester-enolate Claisen rearrangement of the alpha-acyloxysilane 1 as the key step, where the chirality of an alpha-acyloxy-TBDMS group was completely transferred to the rearranged product.     

Crystal-state 3D-structural characterization of novel 3(10)-helical peptides.

The crystal-state conformations of two octapeptides, pBrBz-(D-Iva)8-OtBu (8I) and Ac-[L-(alphaMe)Val]8-OH (8II), the heptapeptide Z-[L-(alphaMe)Val]7-OH (7), the hexapeptide Z-[L-(alphaMe)Leu]6-OtBu (6) and the tetrapeptide alkylamide Z-(Aib)2-L-Glu(OMe)-L-Ala-L-Lol (5) were assessed by x-ray diffraction analyses. Two independent molecules are observed in the asymmetric unit of each L-(alphaMe)Val homo-peptide. All four homo-peptides are folded in a regular 3(10)-helical structure (only the C-terminal H-bonded conformation of the D-Iva octapeptide is distorted to a type-I beta-turn). The hydroxyl groups of the C-terminal carboxyl moieties of the two L-(alphaMe)Val homo-peptides participate in an oxy-analogue of the type-III beta-turn conformation. While the two L-(alphaMe)Val 3(10)-helices are right-handed, the D-Iva and L-(alphaMe)Leu helices are left-handed. The tetrapeptide alkylamide is 3(10)-helical at the N-terminus, but it is mixed 3(10)/alpha-helical at the C-terminus.     

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.     

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.     

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.     

Structural features of linear (alphaMe)Val-based peptides in solution by photophysical and theoretical conformational studies

In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time-resolved fluorescence data and molecular mechanics results, the conformational features of a series of linear, L-(alphaMe)Val-based peptides have been investigated in methanol. These foldamers have the general formula F[(alphaMe)Val](r)-T-[(alphaMe)Val](2)NHtBu, where (alphaMe)Val = C(alpha)-methylvaline and r = 0-3, while F [= fluoren-9-ylmethoxycarbonyl (Fmoc)] and T [= 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-carboxylic (Toac)] are a fluorophoric N(alpha)-protecting group and a nitroxide-based alpha-amino acid quencher, respectively. According to ir and CD spectra, the longest term of the series (r = 3) attains a 3(10)-helical structure, while the other peptides populate an intramolecularly H-bonded, 3(10)-helix-like conformation affected by dynamic helical distortions, which are enhanced by the shortness of the backbone chain. Such distortions are reflected in both the energy of the stretching mode and the molar extinction coefficient of the H-bonded N-H groups, the former being higher and the latter smaller than those of a stable 3(10)-helix. Steady-state and time-resolved fluorescence measurements in methanol show a strong quenching of Fmoc by the Toac residue, located at different helix positions, depending on the r value. Comparison of quenching efficiencies and lifetime preexponents with those theoretically obtained from the deepest energy minimum conformers, assuming a F?rster mechanism, is satisfactory. The computed structures exhibit a rather compact arrangement, which accounts for the few sterically favored conformations for each peptide, in full agreement with the time-resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, implying that interconversion among conformational substates involving the probes is slower than the energy transfer rate.     

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.     

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.     

Aib residues in peptaibiotics and synthetic sequences: analysis of nonhelical conformations

The alpha-aminoisobutyric (Aib) residue has generally been considered to be a strongly helicogenic residue as evidenced by its ability to promote helical folding in synthetic and natural sequences. Crystal structures of several peptide natural products, peptaibols, have revealed predominantly helical conformations, despite the presence of multiple helix-breaking Pro or Hyp residues. Survey of synthetic Aib-containing peptides shows a preponderance of 3(10)-, alpha-, and mixed 3(10)/alpha-helical structures. This review highlights the examples of Aib residues observed in nonhelical conformations, which fall 'primarily' into the polyproline II (P(II)) and fully extended regions of conformational space. The achiral Aib residue can adopt both left (alpha(L))- and right (alpha(R))-handed helical conformations. In sequences containing chiral amino acids, helix termination can occur by means of chiral reversal at an Aib residue, resulting in formation of a Schellman motif. Examples of Aib residues in unusual conformations are illustrated by surveying a database of Aib-containing crystal structures.     

Gαs protein C‐terminal α‐helix at the interface: does the plasma membrane play a critical role in the Gαs protein functionality?

The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, Galphabetagamma) mediate the signalling process of a large number of receptors, known as G protein-coupled receptors. The C-terminal domain of the heterotrimeric G protein alpha-subunit plays a key role in the selective activation of G proteins by their cognate receptors. The interaction of this domain can take place at the end of a cascade including several successive conformational modifications. Galpha(s)(350-394) is the 45-mer peptide corresponding to the C-terminal region of the Galpha(s) subunit. In the crystal structure of the Galpha(s) subunit it encompasses the alpha4/beta6 loop, the beta6 beta-sheet segment and the alpha5 helix region. Following a previous study based on the synthesis, biological activity and conformational analysis of shorter peptides belonging to the same Galpha(s) region, Galpha(s)(350-394) was synthesized and investigated. The present study outlines the central role played by the residues involved in the alpha4/beta6 loop and beta6/alpha5 loops in the stabilization of the C-terminal Galpha(s)alpha-helix. H(2)O/(2)H(2)O exchange experiments, and NMR diffusion experiments show interesting evidence concerning the interaction between the SDS micelles and the polypeptide. These data prompt intriguing speculations on the role of the intracellular environment/cellular membrane interface in the stabilization and functionality of the C-terminal Galpha(s) region.     

Preferred solution conformation of peptides rich in the lipophilic, chiral, C(alpha)-methylated alpha-amino acid (alpha Me)Aoc.

The lipophilic, chiral, C(alpha)-methylated alpha-amino acid L-(alphaMe)Aoc (2-methyl-2-amino-octanoic acid) was prepared using a chemo-enzymatic approach. Two series of terminally protected model peptides, from dimer through to hexamer, containing L-(alphaMe)Aoc in combination with either Gly or Aib, were synthesized by solution methods and were fully characterized. A solution conformational analysis, based on FT-IR absorption, 1H-NMR and circular dichroism (CD) techniques, was performed with the aim at determining the preferred conformation of this novel amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that L-(alphaMe)Aoc favours the formation of the right-handed 3(10)-helical conformation.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Peptides from chiral C alpha,alpha-disubstituted glycines. Synthesis and characterization, conformational energy computations and solution conformational analysis of C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] derivatives and model peptides.

Conformational energy computations on Ac-L-(alpha Me)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral C alpha-methyl, C alpha-alkylglycyl residue. We have synthesized and characterized a variety of L-(alpha Me)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, 1H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the L-(alpha Me)Val residue turns out to be a strong beta-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other C alpha-methyl, C alpha-alkylglycyl residues.     

Nuclear magnetic resonance analyses of side chain conformations of histidine and aromatic amino acid derivatives. Solvent and pH dependence

Stereoselectively beta-deuterated species were synthesized of Ac-His-NHMe, Ac-His-OEt, Ac-His-OH and H-His-NHMe, which are useful as models of histidine residues in peptides. From the spectral comparison of 1H n.m.r., the beta-proton resonances of the normal species were unambiguously assigned. In (C2H3)2SO, C2(2)H5O2H, C2H3O2H, and C5(2)H5N solution and in aqueous solution, the lower-field and higher-field components of beta-proton resonances of the four histidine derivatives are assigned to the pro-R and pro-S protons, respectively. The alternative assignments apply for Ac-His-NHMe, Ac-His-OEt and Ac-His-OH in non-polar solvents such as C2HCl3. Vicinal coupling constants 3J alpha beta S and 3J alpha beta R were obtained for calculating the fractional populations of rotamers about the C alpha-C beta bond. The rotamer populations depend little on the ionization states of the alpha-amino and carboxyl groups or the imidazole ring. The rotamer populations depend significantly on the solvent polarity, similar to those of Phe, Tyr and Trp derivatives. For the two beta-proton resonances of His, Phe, Tyr, and Trp derivatives in a variety of solvents, linear relationships are found between the differences in chemical shifts and the differences in vicinal coupling constants.