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C Toniolo

Publications and source records attributed to C Toniolo.

At least 37 records · Page 2Linked to original sources
PubMed2001

Solid-phase synthesis of peptides containing the spin-labeled 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC).

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino4-carboxylic acid (TOAC) is a nitroxide spin-labeled, achiral Calpha-tetrasubstituted amino acid recently shown to be not only an effective beta-turn and 3(10)/alpha-helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid-phase synthesis in an automated apparatus.

Amino Acids↗
PubMed2001

Ac10c: a medium-ring, cycloaliphatic Calpha,alpha-disubstituted glycine. Incorporation into model peptides and preferred conformation.

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cyclodecane-1-carboxylic acid (Ac10c), an alpha-amino acid conformationally constrained through a medium-ring Calphai <--> Calphai cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac10c monomer and homo-dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT-IR absorption and 1H NMR techniques. Furthermore, the molecular structures of two derivatives (Z-Ac10c-OH and Fmoc-Ac10c-OH) and two peptides (the dipeptide ester Z-Ac10c-L-Phe-OMe and the tripeptide ester Z-Aib-Ac10c-Aib-OtBu) were determined in the crystal state using X-ray diffraction. The experimental results support the view that beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac10c, the third largest cycloaliphatic C(alpha,alpha)-disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1-amino-cycloalkane-1-carboxylic acid (Ac(n)c, with n = 3-12) series, which represents the prerequisite for our recent proposal of the 'Ac(n)c scan' concept.

Crystallography, X-Ray↗
PubMed2001

Analogs of the antimicrobial peptide trichogin having opposite membrane properties.

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.

Amino Acid Sequence↗
PubMed2000

The first water-soluble 3(10)-helical peptides.

Two water-soluble 3(10)-helical peptides are synthesized and fully characterized for the first time. The sequence of these terminally blocked heptamers comprises two residues of the Calpha-trisubstituted alpha-amino acid 2-amino-3-[1-(1,4,7-triazacyclononyl)]propanoic acid and five residues of a Calpha-tetrasubstituted alpha-amino acid (either alpha-aminoisobutyric acid or isovaline). Using CD and NMR techniques we were able to show that both heptapeptides are well structured in water, and that the type of conformation adopted is indeed the ternary 3(10)-helix.

Journal Article↗
PubMed2000

CIDEP effects of intramolecular quenching of singlet and triplet excited states by nitroxide radicals in oligopeptides: a potentially useful new method for investigating peptide secondary structures in solution.

Two hexapeptides, each bearing one photoactive alpha-amino acid (Bin or Bpa) and one nitroxide-containing TOAC residue, have been synthesized and fully characterized. FT-IR absorption measurements indicate that a 3(10)-helical conformation is adopted by these peptides in solution. As two amino acid units separate the photoactive residue from TOAC in the peptide sequences, the two moieties face each other at a distance of about 6 A after one complete turn of the ternary helix. Irradiation by a light pulse from an excimer laser populates the excited states localized on the chromophores. An intramolecular interaction between the singlet (Bin) or triplet (Bin and Bpa) excited states and the doublet state of the TOAC nitroxide makes a spin-selective decay pathway possible, that produces transient spin polarization. In addition, in order to determine whether the intramolecular exchange interaction occurs through-bond or through-space, we have prepared linear and cyclic TOAC-Bin dipeptide units. A CIDEP study revealed that a through-space intramolecular interaction is operative. The observation of spin polarization makes the two helical hexapeptides suitable models to test the possibility of application of this novel technique to conformational studies of peptides in solution.

Cyclic N-Oxides↗
PubMed2000

A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes.

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-¿Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]¿ [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.

Aminoisobutyric Acids↗
PubMed2000

Conformational restriction through C alpha i <--> C alpha i cyclization: Ac12c, the largest cycloaliphatic C alpha,alpha- disubstituted glycine known.

Two complete series of N-protected, monodispersed oligopeptide esters to the pentamer level from 1-aminocyclododecane-1-carboxylic acid (Ac(12)c), an alpha-amino acid conformationally constrained through C(alpha)(i) <--> C(alpha)(i) cyclization, and either L-Ala or Aib residues, along with the N-protected Ac(12)c homopeptide alkylamide series from monomer to trimer, have been synthesized by solution methods and fully characterized. The solution-preferred conformations of these peptides have been assessed by Fourier transform ir absorption and (1)H-nmr techniques. Moreover, the molecular structures of one derivative (Z-Ac(12)c-OH) and three peptides [the tripeptide ester Z-L-Ala-Ac(12)c-L-Ala-OMe, the tripeptide alkylamide Z-(Ac(12)c)(3)-NHiPr, and the tetrapeptide ester Z-(Aib)(2)-Ac(12)c-Aib-OtBu (Aib, alpha-aminoisobutyric acid)] have been determined in the crystal state by x-ray diffraction. The results obtained point to the conclusion that beta-bends and 3(10)-helices are preferentially adopted by peptides based on Ac(12)c, the largest cycloaliphatic C-disubstituted glycine known. A comparison with the structural tendencies extracted from published works on peptides from Aib, the prototype of C-disubstituted glycines, and the other extensively studied members of the class of 1-aminocycloalkane-1-carboxylic acids (Ac(n) c, with n = 3-9), is made and the implications for the use of the Ac(12)c residue in the Ac(n) c scan approach of conformationally restricted analogues of bioactive peptides are briefly discussed.

Alanine↗
PubMed2000

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 Förster 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.

Circular Dichroism↗
PubMed2000

Electron spin resonance of TOAC labeled peptides: folding transitions and high frequency spectroscopy.

The unnatural, conformationally constrained nitroxide amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) stabilizes helical structure and provides a means for studying rigidly spin labeled peptides by electron spin resonance (ESR). Two new directions in TOAC research are described. The first investigates intermediates formed during alpha-helix unfolding. Double TOAC labeled alpha-helical peptides were unfolded at low temperature in aqueous solution with increasing concentrations of guanidine hydrochloride. Comparison of ESR spectra from two doubly labeled peptides suggests that 3(10)-helix emerges as an intermediate. The second research direction involves the use of high frequency ESR (140 GHz) at low temperature to assess dipolar couplings and, hence, distances between TOAC pairs in a series of 3(10)-helical peptides. Preliminary simulations suggest that high frequency ESR is able to extract correct distances between 6 and 11 A. In addition, the spectra appear to be very sensitive to the relative orientation of the TOAC labels.

Cyclic N-Oxides↗
PubMed2000

Interaction between TOAC free radical and photoexcited triplet chromophores linked to peptide templates.

The intramolecular quenching of photoexcited triplet states by free radicals linked to peptide templates was studied by time-resolved electron paramagnetic resonance (EPR) with pulsed laser excitation. The systems investigated are 3(10)-helix forming peptides, having in the amino acid sequence the free radical 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and a triplet precursor, such as Bin, Bpa, or Trp, incorporated at different relative positions. Upon interaction with the excited triplet the TOAC radical spin sublevel populations assume values that differ from the Boltzmann equilibrium values. This spin polarization effect produces EPR lines in emission whose time evolution reflects the triplet quenching rate. In particular, in a series of peptides labeled with Bpa and TOAC at successive positions in the 3(10)-helix, radical-triplet interaction was observed in all cases. However, for the peptide where Bpa and TOAC are at positions 2 and 4 the rate of triplet quenching is lower than for the other peptides in the series. In addition, the radical-excited triplet complex in the quartet spin state was observed in a peptide containing fullerene (C(60)) as a triplet precursor and TOAC.

Carbon↗
PubMed2000

An azacrown-functionalized peptide as a metal ion based catalyst for the cleavage of a RNA-model substrate.

The previously synthesized, terminally blocked heptapeptide Ac-Aib-ATANP-Aib-Aib-ATANP-Aib-Aib-OMe (1a), where ATANP is (S)-2-amino-3-[1-(1,4,7-triazacyclononane)]propanoic acid and Aib is alpha-aminoisobutyric acid, which is soluble in neutral water where it largely adopts a 3(10)-helical conformation, has been studied, as bimetallic complex [metal ions: Cu(II), Ni(II), Zn(II)], for the transphosphorylation catalysis of the RNA-model substrate 2-(hydroxypropyl)-p-nitrophenyl phosphate (HPNP). A detailed analysis was carried out with the Zn(II) dinuclear complex. Comparison with the mononuclear Zn(II) complex with 1,4,7-triazacyclononane (3) points to cooperativity between the two Zn(II) ions in the process catalyzed by 1a-2Zn(II). On the contrary, the dinuclear Zn(II) complex of dipeptide Ac-(ATANP)(2)-OMe (2), lacking any ordered conformation, is less active than 3-Zn(II). The kinetic analysis suggests the following: (a) the peptide is conformationally very robust and does not loose activity up to 50 degrees C; (b) the substrate binds to the peptide-Zn(II) complex, although not all modes of complexation allow us to take advantage of the cooperativity between the two metal centers. The maximum rate acceleration estimated at pH 7 for the fully bound substrate is ca. 200-fold compared with the uncatalyzed process.

Alanine↗
PubMed2000

Preferred conformation of peptides based on cycloaliphatic C(alpha,alpha)-disubstituted glycines: 1-amino-cycloundecane-1-carboxylic acid (Ac11c).

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cycloundecane-1-carboxylic acid (Ac11c), an alpha-amino acid conformationally constrained through a medium-ring C(i)alpha<-->C(i)alpha cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac11c monomer and homo-dimer alkylamides, have been synthesized by solution methods and fully characterized. The preferred conformation of these model peptides has been assessed in deuterochloroform solution by FT-IR absorption and 1H-NMR techniques. Furthermore, the molecular structures of one derivative (Z-Ac11c-OH) and two peptides (the tripeptide ester Z-Aib-Ac11c-Aib-OtBu and the pentapeptide ester Z-Ac11c-(Aib)2-Ac11c-Aib-OtBu) have been determined in the crystal state by X-ray diffraction. The experimental results support the view that beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac11c, the second largest cycloaliphatic C(alpha,alpha)-disubstituted glycine known. This investigation has allowed the authors to approach the completion of a detailed conformational analysis of the whole 1-amino-cycloalkane-1-carboxylic acid (Ac(n)c, with n = 3-12) series, which represents the prerequisite for their recent proposal of the 'Ac(n)c scan' concept.

Acids, Carbocyclic↗
PubMed2000

(alphaMe)Aun: a highly lipophilic, chiral, Calpha-tetrasubstituted alpha-amino acid. Incorporation into model peptides and preferred conformation.

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.

Amino Acids↗
PubMed2000

(alphaMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides.

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.

Crystallization↗
PubMed2000

Structural features of linear, homo-Aib-based peptides in solution: a spectroscopic and molecular mechanics investigation.

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 properties of a series of linear, homo-Aib peptides in methanol (a structure-supporting solvent) were investigated. These compounds have the general formula P(Aib)nN, where Aib is alpha-aminoisobutyric acid, N is naphthalene and P is the monomethylated protoporphyrin IX, the two latter chromophores being covalently attached to the peptide C- and N-termini, respectively, while n=3, 6 and 9. According to 1H NMR and IR spectra, the peptides investigated largely populate a 3(10)-helical structure in CDCl3, which is also a structure-supporting solvent. Both steady-state and time-resolved fluorescence measurements show a strong quenching of the N emission that parallels an increase of the P fluorescence intensity, suggesting the occurrence of long-range energy transfer from 1N* to ground-state P. Comparison of quenching efficiencies and lifetime pre-exponents with those obtained theoretically from the deepest energy minimum conformers is very satisfactory. The computed structures, built up by partially taking into account the solvent medium, exhibit a rigid, highly compact arrangement, owing to both the 3(10)-helix conformation of the backbone chain and the very few peptide-to-chromophore covalent linkages. As a result, only one or two stable conformations for each peptide were theoretically found, 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, which implies that interconversion among conformational substates of the N linkages is slower than 10 ns, corresponding to the upper limit of the energy transfer characteristic time.

Aminoisobutyric Acids↗
PubMed1999

A new class of pseudopeptide antagonists of the kinin B1 receptor containing alkyl spacers.

Four previously reported kinin receptor peptide antagonists, including the B1 receptor-selective peptides desArg10-HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg10-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-Arg-Arg-NH-(CH2)10-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA2 = 7.1) as a kinin B1 receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B1 receptor (rat ileum) and antagonist activity at the kinin B2 receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg10-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B1 receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on Calpha-tetrasubstituted alpha-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc).

Amino Acid Sequence↗
PubMed1999

Orientation and immersion depth of a helical lipopeptaibol in membranes using TOAC as an ESR probe.

Trichogin GA IV is a lipopeptaibol antibiotic characterized by the sequence nOct-Aib1-Gly-Leu-Aib4-Gly-Gly-Leu-Aib8-Gly-Ile- Lol (nOct: n-octanoyl; Aib: alpha-aminoisobutyric acid; Lol, leucinol), which exhibits membrane-modifying properties. We synthesized step-by-step by solution methods three trichogin analogues, each with a single Aib --> 2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid (TOAC) substitution. The similarity in the conformational propensities of the Calpha-tetrasubstituted alpha-amino acids Aib and TOAC allowed us to exploit these analogues to investigate the orientation and therefore the mechanism of action of trichogin in the membranes by the electron spin resonance (ESR) technique. A conformational analysis by Fourier transform ir absorption and CD in different organic solvents and in a membrane-mimetic environment indicated that the conformation of the natural lipopeptaibol remains almost unchanged in the three analogues. Moreover, for all of the analogues permeability measurements revealed membrane-modifying properties comparable to those of trichogin. Our ESR investigation demonstrated that, in liposomes based on phosphatidylcholine, trichogin lays parallel to the membrane surface with its hydrophobic face oriented toward the membrane interior. These results suggest that trichogin might modify membrane permeability via a carpet-like mechanism, at least in liposomes and in the absence of a transmembrane potential.

Anti-Bacterial Agents↗