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2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C(alpha)-tetrasubstituted alpha-amino acid. Here, we describe the molecular and crystal structures, as determined by X-ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)(2).1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solvate, and five TOAC-containing, terminally protected, linear peptides ranging in length from tetra- to hepta-peptides. Incipient and fully developed, regular or distorted 3(10)-helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X-ray diffraction structure of an intramolecularly cyclized side product resulting from a C-activated TOAC residue has also been determined.

Out of all nitrophenyl esters of N(alpha)-protected alpha-amino acids Z-Trp-ONP(o) is the only one which is deep orange colored in the crystalline state. Any change in N(alpha)-protection, nature of amino acid, spatial separation between Trp and the ester-group or position of the nitro-substitutent in the aromatic ring of the ester function results in a loss of this characteristic property. We solved the molecular and crystal structure of Z-l-Trp-ONP(o) by X-ray diffraction analysis and investigated its color changes and visible (vis) and infrared (IR) absorption spectra in the solid state as a function of the amino acid derivative/KBr (w/w) ratio in the pellets. This investigation was extended to toluene solutions of different Z-Trp-ONP(o) concentrations by use of vis absorption and proton magnetic resonance spectroscopic techniques. The onset of the orange color correlates closely with the appearance of a concentration-dependent absorption band near 500 nm and concentration-dependent shifts of the urethane and indole NH proton resonances. Our observations can be explained by the formation of an intermolecular charge transfer complex involving the Trp indole and the -ONP(o) nitrophenyl as the donor and the acceptor moieties, respectively.

The novel Calpha-tetrasubstituted alpha-amino acid Calpha-methyl, Calpha-cyclohexylglycine was prepared by hydrogenation of its Calpha-methyl, Calpha-phenylglycine precursor. Terminally protected homodi-, homotri-, and homotetrapeptides from Calpha-methyl, Calpha-cyclohexylglycine and co-oligopeptides to the pentamer level in combination with Gly or alpha-aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT-IR) spectrophotometer absorption, 1H NMR, and X-ray diffraction techniques, support the contention that this Calpha-methylated, Cbeta-trisubstituted aliphatic alpha-amino acid is an effective beta-turn and 3(10)-helix inducer in tri- and longer peptides as its Calpha-methyl valine parent compound, but partially divergent from the corresponding aromatic Calpha-methyl, Calpha-diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.

A conformationally restricted analog of the N-terminal 12-residue peptide segment of the HA2 subunit of the PPV/34, PR/8/34, and Jap/57 strains of influenza virus hemagglutinin was synthesized containing three residues of Calpha-methyl-valine. This peptide has a higher content of helical structure in the presence of 50% trifluoroethanol or when interacting with liposomes of egg phosphatidylcholine compared with the conformationally more flexible control peptide with the native sequence. The control and analog peptides had opposite effects on membrane curvature as measured by shifts in the bilayer-to-hexagonal phase transition temperature of a synthetic phosphatidylethanolamine derivative. The control peptide promoted more negative curvature, particularly at acidic pH and was also more potent than the analog in promoting lipid mixing. The results indicate that the ability of the peptide to sample a broader range of conformations is required for the influenza fusion peptide to destabilize membranes and that a rigid helical structure is less fusogenic. The difference between the two peptides and between pH 7.4 and pH 5.0 show a correlation between the ability to promote negative curvature and to accelerate lipid mixing.

We synthesized using solution-phase methods three analogs of [l-Leu11-OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N-terminal n-octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic Calpha,alpha-disubstituted glycine l-(alphaMe)Aun, respectively [partial (alphaMe)Aun scan]. FT-IR absorption and CD analyses unequivocally show that the main three-dimensional structural features of [l-Leu11-OMe] trichogin GA IV are preserved in the analogs. Also, [l-Leu11-OMe] trichogin GA IV and the three Nalpha-acetylated l-(alphaMe)Aun analogs exhibit strictly comparable membrane-modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the Nalpha-blocking group to the side-chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l-Leu11-OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.

Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, Calpha-methylated alpha-amino acid L-(alphaMe)Nva with a short, linear side-chain. A set of terminally protected model peptides to the pentamer level containing either (alphaMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (alphaMe)Nva peptides were also synthesized using side-chain hydrogenation of the corresponding Calpha-methyl, Calpha-allylglycine (Mag) peptides. A detailed solution and crystal-state conformational analysis based on FT-IR absorption, 1H NMR and X-ray diffraction techniques allowed us to define that: (i) (alphaMe)Nva is an effective beta-turn and 3(10)-helix former; and (ii) the relationship between (alphaMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. L-(alphaMe)Nva induces the preferential formation of right-handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on alpha-amino acids with a Calpha-methyl substituent and a Calpha-linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.

Using a chemo-enzymatic approach we prepared the highly lipophilic, chiral, Calpha-methylated alpha-amino acid (alphaMe)Aun. Two series of terminally protected model peptides containing either D-(alphaMe)Aun in combination with Aib or L-(alphaMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT-IR absorption, 1H NMR and CD techniques, allowed us to determine the preferred conformation of this 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 D-(alphaMe)Aun favors the formation of the left-handed 3(10)-helical conformation.