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In this chapter, researches focusing on the synthesis, structural characterization and activity of different classes of biomolecules are reported. The covered topics range from the synthesis of new amino acids and foldamers to the structural and functional analysis of proteins, the preparation and characterization of nanosystems for drug delivery applications, the molecular bases of antibiotic resistance spread in Gram-positive bacteria and the study of topology and microstructure of tissues and cells. To this goal, different experimental and computational techniques are exploited, such as biomolecular assays, molecular dynamics, X-ray scattering and IR spectroscopy.

Pursuing the search for a new class of structurally simple mimics of antimicrobial peptides, we optimized a short, cheap and high-yielding synthesis of mono-charged amphiphilic α-hydrazido acid derivatives. The most active derivatives furnished MICs that are among the best values reported in literature for synthetic amphiphilic membranolytic compounds. They exhibited a broad-spectrum in vitro activity against a variety of Gram-positive and Gram-negative bacteria, including two multidrug-resistant strains. In spite of the minimal cationic charge, the best compounds demonstrated to be selective toward bacterial cell membranes over mammalian cell membranes. The relationship between either the antibacterial or the hemolytic activity and the overall lipophilicity furnished an easy way to individuate the best dimensional range for the hydrophobic portions. The importance of a non-disrupted amphiphilicity was also demonstrated. Considering the bioactivity profile and the ease of synthesis, these chemically and proteolitically stable hydrochlorides are suitable for development of a new class of wide-spectrum antibiotics.

Porphyrins are functional elements of important biomolecules, whose assemblies play a central role in fundamental processes such as electron transfer, oxygen transport, enzymatic catalysis, and light harvesting. Here we report an approach to formation of porphyrin supermolecules, a particular type of nanoparticles with unusually strong noncovalent intermolecular interactions. Key differences between the supermolecules and noncovalent nanostructures described earlier are as follows. (1) Supermolecules consist of molecules of the same type without side groups promoting the self-assembly and without any spacers; no surfactant or catalyst to assist the process is needed. (2) They exhibit unusual photophysical properties and remain stable even in organic solvents. Their formation occurs under specially selected conditions at the air-water interface at room temperature. Following this route, we have formed supermolecules of magnesium porphine, a functional element of chlorophyll. The properties of these supermolecules are markedly different from those of the constituent molecules. For example, in contrast to the pink color of the monomer solution, solutions of supermolecules are transparent for visible light and absorb in the ultraviolet and near-infrared regions. We also present atomic force microscopy visualization of the porphyrin two-dimensional nanoaggregates forming at the air-water interface that were predicted in our previous works. This approach offers a guideline for the discovery of new supermolecules, including complex biological ones, and the formation of supermolecular materials with novel properties.

Novel nucleoside analogs were synthesized, in which (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol bears the nucleobase exploiting a 1-oxoethane-1,2-diyl group. Within this synthetic approach, cleavage of the N-phenylethyl group in the starting compound and introduction of the nucleobase-bearing amidic chain were accomplished with a one-pot procedure, simply using bromoacetyl bromide. Moreover, the benzyl carbonate protecting group was used to obtain easy to handle compounds and avoid deprotection of nucleobases occurring under basic conditions. Eventually, directed towards the preparation of short oligonucleotide sequences, both hydroxy functionalities of the iminosugar were orthogonally protected as benzyl carbonate and dimethoxytrityl ether, respectively. Then, exploiting selective orthogonal deprotections and subsequent use of the phosphoramidite method, a dimer phosphite was synthesized, verifying the possibility of building oligomeric structures displaying these novel nucleoside analogs.

Novel analogues of nucleosides tethered on a chiral pyrrolidin-2-one were prepared, but the imide functionality was unstable under basic conditions. Thus, the carbonyl group was removed from pyrrolidin-2-one, and nucleoside analogues bearing a pyrrolidine ring were synthesized, which were unaffected under basic conditions. A nucleoside dimer was also obtained, bearing a carbamate linkage between two units. Graphics for use in the Table of Contents

Starting from a chiral 4-hydroxymethyl pyrrolidin-2-one, an isostere of α-methyl homoserine tethered on a γ-lactam ring was prepared exploiting a stereoselective acylation-methylation sequence, followed by Curtius rearrangement, and structural assignment was confirmed by n.O.e. experiments. By reverting the sequence, the 3-carboxy-3-methyl derivative having the opposite configuration at C-3 was obtained with total stereoselection, but Curtius rearrangement invariably afforded only inseparable mixtures of decomposition products.

Starting from chiral-protected 4-hydroxymethyl pyrrolidin-2-ones, the otherwise elusive 3,4-trans-3,3,4-trisubstituted isosteres of α-methyl homoserine, tethered on a γ-lactam ring, were prepared exploiting stereoselective electrophilic aminations. These reactions led to the isolation and characterization of a novel type of atropisomers, exceedingly stable at room temperature, that were directly converted to the desired products by a novel non-reductive N-N bond cleavage reaction.

The stereoselectivity of the alkylation of chiral pyrrolidin-2-ones leading to conformationally restricted analogues of 3-methylaspartic acid was investigated using density functional theory calculations. The overall stereocontrol is regulated by the relative stability of the lithium enolate intermediates which are also involved in the rate-determining step leading to the methylaspartic acid derivative. The computed results explain the observed data and the different diastereoselectivity can be ascribed to the nature of the alkylating group.

Starting from β-isocupreidine (β-ICD), a series of difunctional catalysts were synthesized to ascertain their usefulness in the asymmetric Morita–Baylis–Hillman (MBH) reaction. The trichloroacetylcarbamate derivative was found to give the (R)-MBH adducts in excellent optical purities (90–99 % ee) and moderate to good yields (43–83 %), in some cases, better than β-ICD. A number of acrylates were also tested and 2,6-dimethyl-4-nitrophenyl acrylate was identified as a suitable alternative to the popular hexafluoroisopropyl acrylate, with both the β-ICD and trichloroacetylcarbamate derivatives. The mechanism of the rate- and selectivity-determining step was ascertained by means of experimental observations and a computational investigation aimed at clarifying the transition state structures is reported. These studies have clarified the reasons for the effectiveness of these structurally related catalysts in the asymmetric MBH reaction between acrylates and aldehydes.

3-Substituted (Z)-2-(bromomethyl)propenoates reacted with (S)-1-(4-methoxyphenyl)ethylamine in the presence of astoichiometric amount of quinidine to yield (R)-3-[(tert-butoxycarbonyl)amino]-2-methylenealkanoates (aza-Morita–Baylis–Hillman adducts) in enantiomerically pure form.

The N-tosyl carbamates 4a-e, easily prepared starting from the Baylis-Hillman adducts 3a-e, underwent cyclization carried out with I(2)/NIS in the presence of NaH, to give the corresponding 2-oxo-1,3-oxazolidines 5a-e in good yield and total stereoselection when the substituent at C-5 is Ar. After the removal of tosyl group, followed by the cleavage of the heterocyclic ring, the alpha-methyl-alpha-amino acids 8a,b and 10 were obtained in good yield as hydrochlorides.

Imines obtained from either chiral 3-amino-4-silyloxymethylpyrrolidin-2-one 5a or 5b underwent alkylation to give, in good yield and total stereoselection, the corresponding 3,3,4-trisubstituted pyrrolidin-2-ones 8a – d where both the amino and the silyloxymethyl groups lie cis to each other, as shown by 1 H NMR spectroscopic data and NOE experiments. By removal of both the imino group and the chiral inducer from 8b , the pyrrolidin-2-one 12 , an isostere of ( R )-2-methylhomoserine 2 and the pyrrolidin-2-one 14 , an isostere of ( R )-2-methylaspartic acid 4 were obtained straightforwardly.

An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an alpha-methylene beta-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly(2)-Gly(3) in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.

Starting from the chiral pyrrolidin-2-one 2, the carboxy group at C-4 underwent homologation, and subsequent removal of the 1-(4-methoxyphenyl)ethyl group gave lactam 6. Alkylation of N-1 with benzyl bromoacetate led to 7, a new conformationally restricted analogue of the dipeptide EG (Glu-Gly). The usefulness of 7 was demonstrated by its eventual conversion into 8, an orthogonally protected analogue ofbioactive tripeptide FEG. In order to provide the biological activity of the new mimetic 9, available from 8 after the removal of the protecting groups, the conformational preference of 9 was ascertained by a detailed conformational analysis and a comparison with that of FEG (1). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

In the presence of DABCO, ethyl glyoxalate and (4S,5R)-1,5-dimethyl-3-acryloyl-4-phenylimidazolidin-2-one gave mixtures of the corresponding Baylis-Hillman adducts enriched in either isomer, depending on the absence or the presence of LiClO4 in the reaction mixture. A diastereomeric mixture in 10:90 dr allowed to definitively establish the mechanism of the reaction leading to trichloroacetamides starting from trichloroacetimidates of the Baylis-Hillman adducts.

In order to explain the high stereocontrol occurring in the iodocyclisation of 3-acylamino-2-methylenealkanoates, either the conformational space of the starting products or the cyclisation reaction potential energy surface (PES) was explored at DFT level of theory, the polarised continuum formalism (PCM) for chloroform being used in order to consider the solvent effect. The observed stereoselection was ascribed to both the near attack conformations (NACs) distribution and the energy differences between the two possible and competitive cyclisation pathways leading to cis- and trans-diastereomers.

A Baylis-Hillman adduct of methyl acrylate and ethyl glyoxalate was converted into the trichloroacetimidate that in the presence of DABCO rearranged to the corresponding trichloroacetamide. Eventually, hydrolysis under acidic conditions, led to the hydrochloride of racemic β-methyleneaspartic acid.

Starting from the homochiral 3-t-butyldimethylsilyloxypyrrolidin-2-one 2, a stereodivergent synthetic route was developed leading to both 3,4-trans- and 3,4-cis-3-amino-4-hydroxymethyl pyrrolidin-2-ones, 19 and 28, that are conformationally restricted analogues of β-homoserine 4. In addition, with a large number of 3,4-disubstituted pyrrolidin-2-ones in hand, a trend was observed for both H-3 chemical shifts and J3,4 values, allowing the configuration to be assigned to either 3-hydroxy or 3-amino derivatives.

The O -silyl derivative 4b , prepared starting from the Baylis–Hillman adduct 4a , underwent cyclization on treatment with ( S )-phenylethylamine, to give to an equimolar mixture of the 4,5- cis -disubstituted pyrrolidin-2-ones 9 and 10 , exclusively, which after separation by silica gel chromatography were both converted into the 3-hydroxy-4-hydroxymethylpyrrolidine 1 a glycosidase inhibitor.

Treatment of both primary alcohols 1a , b and secondary amines 1c , d , tethered to a Michael acceptor with ( R )-phenylethyl isocyanate in the presence of DBU gave in good yield and high stereoselection diastereomeric mixtures of oxazolidin-2-ones 2a , b and 3a , b and imidazolidin-2-ones 2c , d and 3c , d , respectively. The cyclisation reaction was studied computationally by ab initio quantum mechanical methods. The observed stereoselectivity was explained on the basis of the different stability of both anions and transition states leading to 2a and 3a , respectively. The usefulness of the method was proven by conversion of 2a into the enantiomerically pure bioactive amino acid 5 .

Reduction of (4 S ,5 S ,6 S )-4-hydroxymethyl-5-iodo-6-methyl-1,3-oxazin-2-one 2a and (4 S ,5 S ,6 R )-4-hydroxymethyl-5-iodo-6-phenyl-1,3-oxazin-2-one 2b with tributyltin hydride in ethanol afforded 1,3-oxazin-2-one 3a and 1,3-oxazolidin-2-one 4b , respectively. Hydrolysis of 3a and 4b under basic conditions led to enantiomerically pure aminodiols 1a and 1b . Reduction of 2b in refluxing toluene led to the unexpected bicyclic tetrahydrofuro[3 a , d ]-1,3-oxazolidin-2-one 5 as the sole product.

The Baylis-Hillman adducts 1 were treated with a large amount of CCl 3 CN in the presence of DBU without solvent to give in good yield the corresponding trichloroacetimidates 5 which by thermal [3.3]sigmatropic rearrangement were converted into the corresponding (E)-2-trichloroacetylaminomethyl-2-propenoates 6, exclusively. On the contrary, when compounds 5 were treated with a catalytic amount of DABCO in dichloromethane, 2-methylene-3-trichloroacetylamino esters 7 were obtained in good yield. Both 5a and 7a underwent iodocyclization, to give a cyclic intermediate precursor of a polyfunctionalized sequence, and the differences in stereoselectivity were in agreement with computational results.

trans -3-Acetyl-4-alkyl pyrrolidin-2-ones 5a , b and 8a , b undergo reductive amination with NaBH 3 (CN) in CH 3 OH in the presence of CH 3 COONH 4 . By acylation of the reaction product the corresponding 3-acylaminoethylpyrrolidin-2-ones 9a – c and 10a , b were obtained with total stereoselection. The configuration of the newly formed stereogenic centre at C-1″ of the acylaminoethyl chain was assigned on the basis of 1 H NMR data supported by both molecular mechanics and quantomechanical calculations.

The 4-benzyloxymethyl pyrrolidin-2-one, 5 , was alkylated leading to 3,4- trans -disubstituted pyrrolidin-2-one 6 in good yield and total diastereoselection, as shown by 1 H NMR data and NOE experiments. After reduction of the carbonyl group to give the trans -3,4-disubstituted pyrrolidine 7 , and removal of the chiral auxiliary, followed by protection of the nitrogen with t -Boc group, the corresponding N -protected pyrrolidine, 8 was obtained. The cleavage of the benzyl ether moiety, followed by oxidation of the hydroxy function, gave in good yield the corresponding pyrrolidine carboxylic acid 2 , a restricted analog of pregabalin.

threo-N-Alkoxycarbonylamino epoxides 5a–d, containing the oxazolidine moiety, were converted into trans-4,5-disubstituted-2-oxazolidin-2-ones 2 with total regio- and stereoselection by means of nucleophilic intramolecular attack of the carbamate moiety to the protonated oxirane ring. Theoretical calculations confirmed both the regioselection and the preference of the cyclocarbamation reaction vs. the intermolecular attack by the solvent, arising from different behaviour in comparison with the analogous iodonium ions.

By treatment with a catalytic amount of DBU in DCM, N-p-toluenesulphonyl carbamates 6a–c , prepared starting from the corresponding Baylis–Hillman adducts, gave (E)-2-(p-toluenesulphonylaminomethyl)propenoates 3a–c , exclusively. On the contrary, changing DABCO for DBU, 2-methylene-3-p-toluenesulfonylamino esters 4a–f were obtained in good yield starting from N-p-toluenesulphonyl carbamates 6a–f . In analogy, N-acyl carbamates 9a–f were treated with DABCO in DCM to give the 2-methylene-3-acylamino esters 5a–f .

Iodocyclisation of primary homoallylic alcohols 2a – d , containing either a 2- t -butoxy- or a benzyloxycarbonylamino group, was studied, in order to establish the nucleophilic group involved in cyclofunctionalisation. In fact, the N - t -Boc derivative 2a gave the oxazinone 3 , exclusively, whereas starting from the N -Cbz derivative 2b a diastereomeric mixture of substituted tetrahydrofurans 4 and 5 resulted in ratios depending upon the reaction conditions. These results were rationalised by means of computational methods. On the contrary, migration of both the t -butyl or benzyl group to the hydroxy group was observed when both 2c and 2d underwent cyclisation to give the corresponding 5-alkoxymethyl oxazolidin-2-ones 7a , b in low yield, but with high regio- and stereocontrol.

The iodocyclization of allylic carbamates 3a and 3b proceeded with high regio- and stereoselection to give 2-oxobicyclo[4.3.0]nonane 5a as the major product. Both the regio- and diastereoselection of the reaction were investigated with the help of molecular mechanics and quantomechanical calculations. The energetic difference between the competing transition states TS-5a and TS-5b is in good agreement with the experimental results and from the calculated transition structures it appears that steric factors direct the discrimination.

Intramolecular conjugate addition of amide enolates to α,β-unsaturated esters was found to give either of the diastereomeric trans-3,4-disubstituted pyrrolidin-2-ones 6, 10 or 7, 11 as the major products, by choosing the appropriate reaction conditions. The cyclisation performed with NaH in THF afforded mainly 6 and 10, whereas by using sodium ethoxide in ethanol the major products of the cyclisation were isomers 7 and 11, with the opposite configuration at both C-3 and C-4. This behaviour was explained by thermodynamic vs. kinetic control and supported by molecular mechanics and quantomechanical calculations.

A number of trans-3-acetyl-4-alkylpyrrolidin-2-ones 8a–d and 11a,b were prepared and reduction of the keto group, carried out with KBH4 in CH3OH, led mainly to 3-hydroxyethylpyrrolidin-2-ones 13a–d and 19a,b with high stereoselection. Configuration of the newly formed stereogenic centre on the hydroxyethyl chain was assigned by 1H NMR data supported by molecular mechanic calculations and eventually confirmed by X-ray diffraction analysis of p-iodobenzoate derivative 15.

An in vitro study was carried out to verify whether the chain length of a substituent on an indolinic nitroxide could influence its antioxidant activity in different biological environments subjected to oxidative stress. Three distinct indolinic nitroxides were synthesized and compared with vitamin E and Trolox (a hydrophilic analogue of vitamin E), where the only difference between the nitroxides was the length of the hydrocarbon chain in the 2-position of indole, namely 2 (C2), 10 (C10), and 18 (C18) carbons. All the nitroxides were effective in preventing oxidation of bovine serum albumin, but to different extents, with the longer chain derivatives being more efficient. However, the C2 compound was the most efficient in preventing lipid peroxidation in microsomal membranes. The C2 and C18 compounds, Trolox, and vitamin E protected microsomal protein oxidation to the same extent at the highest concentration used (13 μ M). The nitroxide with a C10 chain was less effective in this system. The influence of these compounds on the enzymatic activity of two mitochondrial proteins subjected to oxidative stress was also studied by means of oxygraph measurements. Mitochondrial rotenone-sensitive NADH oxidase and succinate oxidase responded differently to Bu t OOH-induced radical chemistry, and the compounds under study also protected the activity of the two enzymes but to different extents. The results clearly demonstrate that indolinic nitroxides are very efficient antioxidants, protecting both lipids and proteins from peroxidation. The indole structure influences the antioxidant efficacy in biological systems. Copyright © 1996 Elsevier Science Inc.

The oxidative cyclisation of a series of either (S)-N-(2-alken-1-yl)-N-(1-phenyleth-1-yl)-acetoacetamides5a-d and methoxycarbonylacetamides 6a-b, performed by using Mn(OAc)3 · 2H2) and Cu(OAc)2 · H2O in acetic acid, has been examined. The reaction proceeds regioselectively through a 5-exo-mode, leading to 1,3,4-trisubstituted pyrrolidin-2-ones 7a-d,8a-d and 9a-b,10a-b as diastereomeric mixtures in about 2:1 ratio, which are easily separated by silica gel chromatography. The configuration of the pure diastereomers is assigned from 1H NMR data and confirmed by NOE experiments. The observed asymmetric induction has been explained on the basis of molecular mechanics calculations. This cyclisation constitutes a useful tool for the synthesis of biologically active amino acids containing the pyrrolidine ring in both enantiomerically pure forms, such as (R)- and (S)-3-pyrrolidineacetic acid, 1 and 2.

Chiral imines 1a,b, already synthesized in our laboratory, were converted in good yield by reduction into the corresponding N-benzyl-gamma-lactams 2a,b. Desilylation followed by oxidation of the hydroxymethyl functionality gave the N-benzyl-beta-amino acids 7a,b in good yield and high purity. Starting from compound 7a, the corresponding beta-peptoid dimer 8 was prepared, together with its derivatives 9 and 10, this latter displaying conformational restriction about the peptide bond, as evidenced by 1H NMR data.

By Swern oxidation of the hydroxamic acid 3 , prepared from the homochiral imidazolidin-2-one 1 , the transient C-nitroso derivative 4 is obtained, and its cycloaddition to either cyclohexadiene or cyclopentadiene proceeds with high diastereoselection, owing to the conformational stability of 4 . The stereochemical outcome of the reaction is determined from 1 H NMR data and further confirmed by the specific rotation value of 7 , obtained by cleavage of the major cycloadduct 5a .

(3R,4S,1′S)-4-Amino-1-(1′-(4-methoxyphenyl)ethyl)-3-methyl-5-oxopyrrolidine-3-carboxylic acid (AMMOPC) gives a foldamer with an 8-helix conformation. When it was inserted in the 12-helix foldamer of (3R,4S,1′S)-4-amino-1-(1′-(4-methoxyphenyl)ethyl)-5-oxo-pyrrolidine-3-carboxylic acid (AMOPC), it locally induced formation of an 8-helix conformation

Treatment of (3S)-3-methylpiperazine-2,5-dione 6a with LHDMS followed by alkylation of the corresponding enolate with methyl iodide affords (3S,6S)-3,6-dimethyl derivative 7 in 98% de. The same reaction sequence carried out on (3R)-derivative 6b leads to a 93:7 diastereomeric mixture of (3R,6R)-8a and (3R,6S)-8b. Cleavage of the heterocyclic ring of 7 and 8a with 57% HI leads to (S)- and (R)-alanine, respectively. The configuration at C-3 (of 6a and 6b) and at C-6 (of 7 and 8a) can be assigned on the basis of 1 H NMR spectroscopy and conformational analysis performed by MMPMI