Alessandro Volonterio

The trifluoroethylamine function as peptide bond replacement.

Peptides play a fundamental role in a number of physiological processes, and can be used as drugs in various therapeutic classes. However, a major drawback of peptides is represented by their low bioavailability, consequently they have to be injected or administered in the form of expensive formulations. For this reason, the design and synthesis of metabolically stable peptide analogues that can either mimic or block the bioactivity of natural peptides or enzymes is an important issue in bioorganic and medicinal chemistry research. The replacement of a scissile peptide bond represents a viable and popular approach in the rational design of peptidomimetics. Peptidomimetics find applications as drugs, in protein engineering and so on. This is evident from the wealth of therapeutically useful peptidomimetic leads incorporating any of the peptide bond surrogates that are currently available. Therefore, within the realm of peptide mimics, the replacement of amide bonds with appropriate functionalities is a classic approach in medicinal chemistry and drug discovery. Some peptide bond surrogates, such as the alkene ( CR=CH ) or fluoroalkene ( CF=CH ) functions, are purely geometric replacements preserving little or none of the capacity of the native peptide bond to undertake electrostatic interactions or hydrogen-bonding with the receptor. Other amide replacements are known which retain the geometry of the amide bond or maintain the hydrogen bond-accepting properties of the amide, such as the ketomethylene ( COCH2 ) or depsipeptide ( CO2 ) functions. However, there are a few functional groups, which are capable of preserving the hydrogen bond-donating properties of the amide. Among them, one can mention sulfonamides ( SO2 NH ), anilines, secondary alcohols, hydrazines, and certain heterocycles. How to minimize the basicity of an NH donor, so that a NH2 + moiety is not formed at physiological pH, is the main issue for identifying a truly effective NH amide replacement. In fact, ammonium ions are poorly tolerated deep in the active site of a protein where binding interactions cannot compensate for the energetic cost of desolvation. This is a serious drawback of popular peptide bond surrogates, such as the methyleneamino ( CH2 NH-) function. Currently, a powerful new strategy is emerging for replacing the peptide bond with a very effective surrogate: the stereogenic trifluoroethylamine function. Indeed, a trifluoroethyl group can replace the carbonyl of an amide and generate a metabolically stable, poorly basic amine that maintains the excellent hydrogen bond of an amide. This strategy was first proposed by our research group, and was used to replace both a glycine amide bond and a malonamide of a partially modified retro (PMR) peptide. The main properties featured by the trifluoroethylamino group are: 1) very low NH basicity, 2) a CHACHTUNGTRENNUNG(CF3)NHCH backbone angle close to 1208, 3) a C CF3 bond substantially isopolar with the C=O, and 4) structural analogy with the tetrahedral proteolytic transition state. Furthermore, the sp hybridization of all the atoms forming the stereogenic trifluoroethylamine moiety is expected to allow a better orientation of the atoms in the receptors’ active sites, thus optimizing the energetically favorable interactions (hydrogen-bonds, van der Waals, hydrophobic, etc.). With this in mind, in 2000 we published the first examples of peptidomimetic structures incorporating a trifluoroethylamine unit replacing the retropeptide bond. In that first work, we presented a number of PMR and partially modified retro-inverso (PMRI) peptides (Figure 1) in

Ψ[CH(CF3)NH]Gly-peptides: synthesis and conformation analysis

Psi[CH(CF(3))NH]Gly peptides, a conceptually new class of peptidomimetics having a stereogenic trifluoroethylamine group as a natural peptide-bond surrogate, have been synthesized by stereoselective addition of alpha-amino acid esters to trans-3,3,3-trifluoro-1-nitropropene. Long range nuclear Overhauser effects, detected via ROESY experiments, provided evidence that model Psi[CH(CF(3))NH]Gly-tetrapeptides incorporating a trifluoroethylamine mimic of the dipeptide loop Pro-Gly can be represented by an ensemble of unfolded and folded conformations. The latter are driven by the formation of a hydrogen bond between a carbonyl group and the aminic proton of the trifluoroethylamine unit. MD calculations indicate a population of conformers which adopt folded beta turn structures for all the L-peptides; on the other hand, a D-stereochemistry at the Pro residue induces a natural folding towards a beta hairpin conformation driven by the formation of a second hydrogen bond, regardless of the stereochemistry of the stereogenic peptide bond surrogate.

Chitosan-Graft-Branched Polyethylenimine Copolymers: Influence of Degree of Grafting on Transfection Behavior

Background Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEIx) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. Methodology/Principal Findings With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEIx derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEIx series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEIx copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI2.7% was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. Conclusions/Significance This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEIx copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.

Solution/solid-phase synthesis of partially modified retro-ψ[NHCH(CF3)]-peptidyl hydroxamates

Abstract The synthesis of a novel family of partially-modified (PM) retropeptidyl hydroxamates incorporating a [CH(CF 3 )CH 2 CO] unit as a surrogate of the conventional malonyl group, has been accomplished both in solution and in solid-phase. The key step is the Michael-type N -addition of free or polymer bound α-amino hydroxamates to 3-( E -enoyl)-1,3-oxazolidin-2-ones, which takes place very effectively, although with low stereocontrol. A number of tri- and tetra-peptidyl hydroxamates were obtained either in diastereomerically pure form (by solution-phase synthesis, after chromatographic purification), or as mixtures of two epimers in very good chemical purity (by solid-phase, after release from the resin), demonstrating that this method is suitable for preparing combinatorial libraries of PM retro- ψ [NHCH(CF 3 )]-peptidyl hydroxamates for screening as metalloprotease inhibitors.

Synthesis of Multifunctional PAMAM−Aminoglycoside Conjugates with Enhanced Transfection Efficiency

The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4-neamine, -paromomycin, and -neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.

Enantiomerically pure α-fluoroalkyl-α-amino acids: Synthesis of (R)-α-difluoromethyl-alanine and (S)-α-difluoromethyl-serine

Abstract Hydrocyanation of enantiomerically pure N-Cbz α-fluoroalkyl β-sulfinylenamines 1 occurs smoothly by treatment with KCN or by addition of trimethylsilycyanide or diethyl phosphorocyanidate to preformed sodium derivatives of 1. The diastereoisomeric difluoro α-aminonitriles 2b have been transformed in the unnatural amino acids (R)-α-difluoromethyl-alanine and (S)-α-difluoromethyl-serine.

Stereoselective Total Synthesis of Enantiomerically Pure 1-Trifluoromethyl Tetrahydroisoquinoline Alkaloids

Enantiomerically pure 1-trifluoromethyl-tetrahydroisoquinoline alkaloid analogues, in which C-1 is a quaternary stereogenic centre, have been synthesized by stereoselective intramolecular Pictet-Spengler reaction of the N-arylethyl γ-trifluoro-β-iminosulfoxide (R)-3, and subsequent elaborations of the sulfinyl auxiliary. The absolute stereochemistry of the stereogenic centre was determined by X-ray diffraction on the α-phenylpropionic ester (1R,2′S)-10.

Novel pyrazole derivatives as neutral CB1 antagonists with significant activity towards food intake

In spite of rimonabants withdrawal from the European market due to its adverse effects, interest in the development of drugs based on CB1 antagonists is revamping on the basis of the peculiar properties of this class of compounds. In particular, new strategies have been proposed for the treatment of obesity and/or related risk factors through CB1 antagonists, i.e. by the development of selectively peripherally acting agents or by the identification of neutral CB1 antagonists. New compounds based on the lead CB1 antagonist/inverse agonist rimonabant have been synthesized with focus on obtaining neutral CB1 antagonists. Amongst the new derivatives described in this paper, the mixture of the two enantiomers (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-hydroxyethyl)-4-methyl-1H-pyrazole ((±)-5), and compound 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl]-4-methyl-1H-pyrazole ((Z)-6), showed interesting pharmacological profiles. According to the preliminary pharmacological evaluation, these novel pyrazole derivatives showed in fact both neutral CB1 antagonism behaviour and significant in vivo activity towards food intake.

The “Non-Oxidative” Chloro-Pummerer Reaction: Novel Stereospecific Entry to Vicinal Chloroamines and Aziridines

This article describes a new, useful synthetic tool, the “Non-Oxidative” Chloro-Pummerer Reaction (NOCPR), which allows for the use of enantiomerically pure α-Li alkylsulfoxides as chiral α-chloroalkyl carbanions with N-protected imines. In this reaction the sulfinyl group of N-alkoxycarbonyl-β-sulfinylamines derived from aryl-, fluoroalkyl- and alkylimines is displaced by a chlorine atom in a one-pot reaction with clean stereoinversion at carbon. Several 1,2-chloroamines produced via NOCPR were transformed into the corresponding aziridines. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Solution/solid-phase synthesis of partially modified retro- and retro-inverso-ψ[NHCH(CF3)]-peptidyl hydroxamates and their evaluation as MMP-9 inhibitors

The synthesis of a novel family of partially modified (PM) retro- and retro-inverso-peptidyl hydroxamates, each incorporating a [CH(CF3)CH2CO] unit as a surrogate for the conventional malonyl group, has been accomplished both in solution and in solid phase. The key step is the Michael-type N-addition of free or polymer-bound α-amino hydroxamates to 3-[(E)-enoyl]-1,3-oxazolidin-2-ones, which takes place in high yields, although with low stereocontrol. This method is suitable for the preparation of combinatorial libraries of PM retro-ψ[NHCH(CF3)]-peptidyl hydroxamates for screening as metalloprotease inhibitors. A number of tri- and tetrapeptidyl hydroxamates were indeed obtained either in diastereomerically pure form by solution-phase synthesis followed by chromatographic purification, or as mixtures of two epimers by solid-phase synthesis and release from the resin. X-ray diffraction of a Tfm-retropeptidyl hydroxamate showed an interesting turn-like conformation with an intramolecularly hydrogen-bonded nine-membered ring, and a nearly planar geometry of the NH group bound to the CH(CF3) group. Three retro-peptidyl hydroxamates were submitted to bioassays, and displayed the capacity to reduce MMP-9 (Gelatinase B) gelatinolytic activity.