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2011/2012 The project deals with the design and development of artificial receptors for monitoring of drugs used for the HIV “Anti-Retroviral Therapy” (ART). Efavirenz, a RT-inhibitor, and its analogues were synthesised to screen with several techniques (surface plasmon resonance, NMR, fluorescence and UV spectroscopy) the new receptors obtained from different approaches. One exploits computational tools to model peptides with high affinity for small molecules. These designed peptides will then be incorporated in polymeric matrix for biosensor purposes. Molecularly imprinted polymers with efavirenz-binding properties were performed during my experience at Queen Mary University of London. Another approach is based on miniaturized receptors obtained from the reduction of the known sequence of a natural receptor as human serum albumin. XXV Ciclo 1984

Multidrug efflux pump inhibitors have a great potential as pharmacological agents that increase the drug susceptibility of bacterial pathogens. Our study was focused on the synthesis and evaluation of the efficiency of resistance-nodulation-division (RND) family efflux pump inhibitors. The efficiency of these inhibitors was investigated on Salmonella enterica ser. typhimurium cells using tetraphenylphosphonium (TPP(+) ) and ethidium cations as the efflux pump substrates. Results of our study indicated that efficiency of the inhibitors depends on the cell outer membrane permeability and method of the assay used. Temperature of the incubation medium and a solvent of the inhibitor used have only minor effect on results of the assay.

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 μg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.

We have identified a 101-amino-acid polypeptide derived from the sequence surrounding the IIA binding site of human albumin. The polypeptide contains residues that make contact with ligands as warfarin in the parent protein, and eight cysteine residues to form disulfide bridges, which stabilize the polypeptide structure. Seventy-four amino acids are located in six [alpha]-helical regions, with the remaining amino acids forming six connecting coil/loop regions. Codon usage optimization was used to express a GST fusion protein in E. coli in yields as high as 4 mg/l. This fusion protein retains its structural integrity and aldolase activity, the ability to direct the stereochemical outcome of a diketone reduction, and its binding capacity to warfarin and efavirenz. Notably, this newly cloned polypeptide represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.

Marine toxins appear to be increasing in many areas of the world. An emerging problem in the Mediterranean Sea is represented by palytoxin (PlTX), one of the most potent marine toxins, frequently detected in seafood. Due to the high potential for human toxicity of PlTX, there is a strong and urgent need for sensitive methods toward its detection and quantification. We have developed an ultrasensitive electrochemiluminescence-based sensor for the detection of PlTX, taking advantage of the specificity provided by anti-PlTX antibodies, the good conductive properties of carbon nanotubes, and the excellent sensitivity achieved by a luminescence-based transducer. The sensor was able to produce a concentration-dependent light signal, allowing PlTX quantification in mussels, with a limit of quantification (LOQ = 2.2 μg/kg of mussel meat) more than 2 orders of magnitude more sensitive than that of the commonly used detection techniques, such as LC-MS/MS. journal article research support, non-u.s. gov't 2012 Sep 25 2012 08 30 imported

2010/2011 Protein scaffolds are stable structures capable to recognize and bind, in different conditions, small guest molecules. They are proteins with known conformation which can be used and modified for the construction of variants. The goal of this work is the identification of peptide-based artificial receptors or catalysts. To this purpose, we have considered two different protein motifs to generate new scaffolds: a synthetic E/K coiled-coil domain, and one of the binding sites of the natural protein Human serum albumin. In order to generate stable peptide hosts, we developed peptide libraries to be selected for both properties, adopting two different approaches: RANDOM mutagenesis and SITE-DIRECTED mutagenesis. XXIV Ciclo 1985

Synthesis and biological evaluation of a new series of potential HIV-1 protease inhibitors incorporating different heterocycles are described. The variation of heteroatom in such molecules has displayed totally different biological activities and a benzothiophene containing inhibitor has shown high potency against wild type HIV-1 protease with IC50 = 60 nM, thanks to the lower desolvation penalty to be payed by such hydrophobic moiety.

The reduction of 1,3-diketones and beta-hydroxyketones with NaBH4 in aqueous acetonitrile is highly stereoselective in the presence of stoichiometric amounts of bovine or human albumin, giving anti 1,3-diols with d.e. up to 96%. The same reaction, without albumin, gives syn and anti 1,3-diols in approximately 1 : 1 ratios. The presence of an aromatic carbonyl group is essential for diastereoselectivity in the NaBH4/albumin reduction of both 1,3-diketones and beta-hydroxyketones. Thus, 3-hydroxy-1-(p-tolyl)-1-butanone is stereoselectively reduced in the presence of albumin, while reduction of its isomer 4-(p-tolyl)-4-hydroxy-2-butanone is not stereoselective. The albumin-controlled reduction is not stereospecific as both enantiomers of 1-aryl-3-hydroxy-1-butanones are reduced to diols with identical stereoselectivities. Circular dichroism of the bound substrates confirms that aromatic ketones are recognized by the protein’s IIA binding site. Binding studies also suggest that 1,3-diketones are recognized in their enol form. From the effect of pH on binding of a diketone it is concluded that, in the complex with the substrate, ionizable residues His242 and Lys199 are in the neutral and protonated forms, respectively. A homology model of BSA was obtained and docking of model substrates confirms the preference of the protein for aromatic ketones. Modelling of the complexes with the substrates also allows to propose a mechanism for the reduction of 1,3-diketones in which the chemoselective reduction of the first (aliphatic) carbonyl is followed by the diastereoselective reduction of the second (aromatic) carbonyl. The role of albumin is thus a combination of chemo- and stereocontrol.

We have synthesized and evaluated a small set of dual action HIV-Protease inhibitors, consisting of a catalytic site reversible inhibitor conjugated with a dimerization peptidic inhibitor, linked via a suitable selective end-modified poly(ethylene glycol), in an effort to obtain a synergistic effect with improved biological behaviour and more powerful pharmacological features, such as water solubility and cell permeability. Low nanomolar activity and synergism were obtained by coupling a Phenylalanine – Proline pseudopeptide catalytic site inhibitor and the Val-Val-Phe tripeptide dimerization inhibitor.

The anthranilic acid diamides represent the most recent class of nonpeptide CCK(1) receptor (CCK(1)-R) antagonists. Herein we describe the second phase of the anthranilic acid C-terminal optimization using nonproteinogenic amino acids containing a phenyl ring in their side chain. The Homo-Phe derivative 2 (VL-0797) enhanced 12-fold the affinity for the rat CCK(1)-R affinity and 15-fold for the human CCK(1)-R relative to the reference compound 12 (VL-0395). The eutomer of 2 (6) exhibited a nanomolar range affinity toward the human CCK(1)-R and was at least 400-fold selective for the CCK(1)-R over the CCK(2)-R. Molecular docking in the modeled CCK(1)-R and its validation by site-directed mutagenesis experiments showed that the 6 binding site overlaps that occupied by the C-terminal bioactive region of the natural agonist CCK. Owing to their interesting properties, new compounds provided by this study represent a solid basis for further advances aimed at synthesis of clinically valuable CCK(1)-R antagonists.

2008/2009 La presente tesi di Dottorato riporta i risultati di ricerche svolte nell’ambito di alcune collaborazioni tra Università ed industria nel campo della progettazione e sviluppo di librerie di trasduttori primari per biosensori. In particolare l’attenzione si è rivolta da un lato a piccoli peptidi come recettori di xanthine (caffeina e teofillina), dall’altro ad una molecola di grandi dimensioni, la palitossina, verso la quale al momento sono stati preparati anticorpi con tecniche più convenzionali. Il contributo di un chimico in questo tipo di progetti è analogo, e consiste in un’attività di sintesi di derivati modificati delle molecole di interesse, nella loro bioconiugazione a proteine ed enzimi, nella sintesi di peptidi, nello studio dell’interazione ligando-recettore attraverso misure di attività, analisi conformazionale e strutturale, modellizzazione. L’attività svolta è brevemente descritta nei punti seguenti. 1) I coiled-coil sono motivi strutturali presenti in natura dati dall’unione di due o più -eliche avvolte su loro stesse al fine di generare un’unica super-elica. L’analisi di tali strutture rivela spesso la presenza di sequenze ripetute di 7 amminoacidi ripetuti (g-a-b-c-d-e-f) con precise caratteristiche: la posizioni a e d per esempio sono occupate sempre da amminoacidi con caratteristiche idrofobiche, atti a generare una interfaccia idrofobica interna alla super-elica molto stabile e non esposta al solvente esterno. Parallelamente, le posizioni e e g sono invece occupate da amminoacidi polari carichi che si trovano sulla superficie esterna della super-elica e possono iteragire tra di loro rinforzando l’interazione e provocando effetti di orientamento. La nostra attenzione si è focalizzata sul coiled-coil denominato EK, sviluppato agli inizi degli anni ’90 da Hodges e colleghi. In questa struttura due catene peptidiche denominate appunto E e K si avvolgono su loro stesse al fine di generare una super-elica anfipatica di notevole stabilità.Partendo da tale struttura e sfruttando la tecnica biomolecolare del phage display che permette l’esposizione di strutture a carattere proteico sulla superficie di un batteriofago, sono state create librerie di EK mutati con l’obiettivo di generare all’interno della super-elica siti recettoriali eventualmente utilizzabili all’interno di biosensori come trasduttori primari per caffeina e teofillina, due piccole molecole organiche altamente correlate e ampiamente presenti nella vita quotidiana. E’ stata prodotta una prima libreria fagica di dimensioni notevoli (108 elementi), all’interno della quale sono stai isolati due cloni specifici per le xanthine; i due cloni sono stati denominati E1 ed E8. La caratterizzazione del binding è avvenuta sia con tecniche biomolecolari che prevedono l’immobilizzazione dell’analita su superficie solida (test ELISA e dot-blot), sia con teniche chimiche con analiti liberi e privi di modificazioni strutturali. Al fine di ridurre il più possibile le dimensioni dello scaffold di partenza è stata sviluppata una seconda libreria fagica in cui è stata randomizzata soltanto la catena K dell’originario EK proposto da Hodges. La libreria finale presenta una dimensione di 108 elementi, tra i quali è stato isolato e caratterizzato attraverso test ELISA il clone denominato KE1. Le ridotte dimensioni di tale peptide (35 amminoacidi) hanno permesso la sua sintesi chimica su una buona scala (40 mg), una cartterizzazione strutturale attraverso spettroscopia di dicroismo circolare, un’analisi dell’interazione in soluzione con la teofillina sfruttando sempre la tecnica CD e quella calorimetrica ed infine un’analisi cristallografica attraverso luce di sincrotrone. 2) Una seconda parte del lavoro è stata finalizzata, invece, allo sviluppo di pool anticorpali mono- e policlonali quali trasduttori primari in biosensori per l’individuazione della palitossina. Tale tossina algale con elevato indice di pericolosità (LD50=0.45 g/kg) negli ultimi anni ha causato notevoli disagi in campo ambientale, inquinando flora e fauna acquatica di diverse zone marittime, compreso il Mediterraneo e l’alto Adriatico. Per lo sviluppo di anticorpi specifici contro tale tossina, sono state condotte immunizzazioni su coniglio e su topo con palitossina coniugata a due tipi di proteine carrier (BSA e KLH) al fine di ridurre la sua tossicità e indurre una risposta immunitaria nell’animale. Il pool anticorpi policolnali di siero di coniglio è stato isolato e caratterizzato attraverso sandwich ELISA contro palitossina libera e palitossina chimicamente modificata allo scopo di ottimizzare tale tipo di test e utilizzarlo come possibile dosatore nei confronti dell’analita presente in campioni di acqua oppure in sistemi più complessi quali mitili o ulteriore fauna marina. This PhD thesis reports the results of research activities carried out at the Dept. of Chemistry of the University of Trieste, in collaboration with several industries, in the field of design and development of libraries of primary transducers for biosensors. We have focused our attention on one side on short peptides as xanthine (caffeine and theophylline) receptors, and on the other side on a very large molecule, palytoxin, which has been considered to raise antibodies by more conventionals techniques. The chemist’s contribution to such projects is similar, consisting in the synthesis of modified target molecules, in their bioconjugation to protein and enzymes, in peptide synthesis, in studying the receptors-ligand interactions by affinity measurements, conformational and structural analysis, modelling. The activity is briefly described in the following points. 1) Coiled-coil are natural motifs made by bundles of two or more -helix that generate a super-helix. Structural analysis of these motifs reveals often the presence of a repeated sequence 7 aminoacids along the helix (g-a-b-c-d-e-f). a and d are hydrophobic aminoacids able to generate an internal hydrophobic core most important for the overall stabilization of the bundle, e and g are polar or charged aminoacids that can contribute to the final stability of the coil via electrostatic interchains interactions. Starting from the EK coiled-coil developed by Hodges et al., made by two different peptide chains (E:EVSALEK; K:KVSALKE) and exploiting phage display techniques, two different phage libraries of E and K mutants were generated, in order to obtain receptors molecules suitable as primary transducer in biosensors for caffeine and theophylline. In the first library, both E and K were randomized, obtaining 108 differents peptide clones; two specific clones was isolated against immobilized caffeine and theophylline, their characterization was carried out both by biomolecular experiments (ELISA test; dot-blot) and by chemical techniques. In a second library, we have further reduced the scaffold size, using only a single K chain. A new library of 108 elements was obtained and characterized. The isolation of KE1 clone as the best xanthine binder, allowed chemical synthesis on a good scale (40 mg); structural characterization and binding analysis of its interaction with theophylline in water was performed by circular dichroism and calorimtrey; the X-ray structure of the peptide was obtained exploiting sincrothrone light. 2) In a second part of this work, the development of mono- and polyclonal antibodies towards palytoxin was stated. Palytoxin is one of the most dangerous algal toxin in nature (LD50=0.4 g/kg in mice) and its presence in the last years in Mediterranean sea, both in the water sea and in fish or mussels, constitues a real problem for tourism economy and for the fish market. In order to obtain specific antibodies as bioreceptors towards palytoxin, a series of immunizations on rabbits were carried out with palytoxin-BSA and palytoxin.KLH conjuagtes in order to generate immune response in the animals; the pool of high reactivity bleeds of rabbits, were isolated and usedto set up ELISA sandwich system towards palytoxin. XXII Ciclo 1979

The cis - and trans -4-benzylparaconic acids and their ethyl esters were synthesized with high enantiomeric excess by hydrolysis of the corresponding diastereomeric lactonic esters using α-chymotrypsin. Thus, at low conversion values, cis - and trans -4-benzyl-5-oxo-3-tetrahydrofurancarboxylic acids were separately isolated with 99% ee and 92% ee, respectively. Both ethyl ester diastereomers were also obtained in enantiopure form. The absolute configuration of the trans -lactonic acid was assigned by 1 H NMR analysis of its ester derivatives with both enantiomers of 1-(9-anthryl)-2,2,2-trifluoroethanol, while that of the cis -lactonic acid was assigned by means of X-ray analysis of a crystalline derivative. The circular dichroism curves of the products obtained are also reported.

A method for the determination of zeranol and its metabolite beta-zearalanol in bovine urine is described. It has been applied to samples from calves given multiple subcutaneous doses of zeranol. Samples were extracted with immunoaffinity columns containing antibodies raised against zeranol and were analysed by gas chromatography-mass spectrometry. The immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was carboxybutylzeranol coupled to bovine serum albumin. Gas chromatography-mass spectrometry was performed in the negative-ion chemical ionization mode, after derivatization of the compounds to their pentafluorobenzyl ethers, and allowed detection of analytes with a sensitivity of 0.01 ppb in spiked urine. The derivatization method and the gas chromatographic determination were also applied to the similar compounds zearalanone, zearalenone and beta-zearalenol. A synthesis of dideuterated zeranol and beta-zearalanol by isotopic exchange is described. These deuterated analogues had an isotopic purity of more than 99% and were used for quantitation of zeranol and beta-zearalanol by isotope dilution mass spectrometry. The recoveries of zeranol and beta-zearalanol, using the immunoaffinity columns, were determined after extraction from spiked urine and were 84 and 64%, respectively. The urines of treated calves were collected for several days after treatments and were analysed after hydrolysis with beta-glucuronidase and arylsulphatase. The samples showed variable but generally decreasing concentrations of zeranol and beta-zearalanol. The levels of beta-zearalanol ranged from less than 0.01 to 98 ppb and were 1.2-3.2 times higher than those of zeranol.

A systematic study of the molecular and electronic structure of the eight possible members in the trans -matrine series and of two dehydro-derivatives, sophocarpine and sophoramine, has been performed. According to density functional theory (DFT) calculations these alkaloids exhibit a variety of form and junction of the four six-membered rings and all but sophocarpine have a strong preference for one conformation. Sophocarpine is predicted to have a marked conformational flexibility at the lactamic nitrogen and to exist as a mixture of two nearly isoenergetic conformers ( C / D - trans and - cis ) in the gas phase or solution. The theoretical predictions are consistent with the available X-ray experimental results as well as IR and NMR evidence. The absolute configuration of the preferred conformer of each compound has been established theoretically and corroborated with the specific optical rotation calculated at the sodium D line. The conformational equilibrium of sophocarpine has also been supported by this physical property. The computed gas-phase proton affinity of matrines indicates a basicity comparable to that of other polycyclic proton sponges. The lowest-energy electronic transitions have been characterized by time-dependent DFT calculations as mainly due to excitations spanning the frontier orbitals π(NCO), n(O), n(N aminic ), and π ∗ (CO). The electronic structures have also been studied by measuring and calculating significant features of the NMR and photoelectron spectra. In particular, a representative set of NMR chemical shifts and nuclear spin–spin coupling constants, obtained with DFT formalisms, compares favourably with experiment. Notably, the stereoelectronic hyperconjugative effects on Δ δ (H eq /H ax ) and Δ 1 J (CH eq /CH ax ) of the >N–CO– groups is correctly accounted for by the theoretical results. Based on ab initio outer valence Green’s function calculations, a reliable description of the uppermost bands in the photoelectron spectra has been advanced. The splitting and sequence of the ionization energies reflect a complex interaction of the n and π chromophores.

Discovery by co-crystallization: High-resolution electron-density maps of HIV-1 aspartyl protease complexed with a potent Phe–Pro isostere-based inhibitor (shown) allowed the determination of the stereochemistry of the inhibitor and an initial assessment of the inhibition properties of this compound without its purification from the epimeric mixture.

An efficient method has been developed for the synthesis of a versatile intermediate bearing azido, hydroxyl and ester functions, a useful precursor for peptidomimetic compounds. The two main features for this synthesis were the use of the Sharpless asymmetric dihydroxylation on thiophene acrylate and the subsequent regioselective ring opening by sodium azide of the cyclic sulfite. Highly chemoselective reduction of the azido alcohol led to a key compound which was utilized for the synthesis of two analogues of commercial anti HIV PR such as nelfinavir and saquinavir. The biological activity and molecular modelling study on these two new potential drugs have been evaluated.

Lipase PS acetylation of diastereomeric dihydro-4-hydroxymethyl-2(3H)-furanones bearing a methyl or a pentyl group at C-5 have been studied. Higher enantioselectivities were found for the cis-isomers with respect to the trans-isomers. They were also higher for the systems bearing the longer alkyl chain. Lipase PS-catalyzed hydrolyses of racemic acetates were found to follow the same trend, although the efficiency of the enzyme was lower than in the acetylation reactions. These results were supported by molecular modelling studies that correctly predicted the maximum stereoselectivity for the cis-isomer of 5-pentyl substituted lactones both in acetylation and in hydrolysis reactions.

Here we report the results of a docking analysis carried out on the homology model of the variable fragment (Fv) region of antibody 312d6. A novel catalytic motif based on an arginine dyad, unprecedented in antibody hydrolases, is identified by this study.

The equilibrium structures of the 11 possible oxo-derivatives of sparteine were investigated with the density functional theory, using the B3LYP functional. The conformational preferences of the seven amino-ketonic sparteines are consonant with those of the parent free base, whose lowest energy conformer consists of a chair–chair quinolizidine A/B-trans system and a boat–chair quinolizidine C/D-trans system. A similar situation also occurs for lactams 2-oxo- (lupanine), 15-oxo-, and 17-oxo-sparteine, whose ketonized ring has a half-chair/sofa, half-chair, and sofa shape, respectively. Unlike the other isomers, the most stable conformer of lactam aphylline (10-oxo-sparteine) adopts the chair–sofa A/B-transoid, chair–chair C/D-cisoid disposition of the two quinolizidine moieties. These theoretical predictions are consistent with the available X-ray experimental results. The electronic structure of the oxo-sparteines was examined by measuring and calculating relevant parameters of their NMR and photoelectron spectra. In particular, a representative set of NMR chemical shifts and nuclear spin–spin coupling constants, calculated by means of DFT formalisms, correlate well with observation. Notably, the effective manifestation of stereoelectronic hyperconjugative effects on Δδ(Heq/Hax) and Δ1J(CHeq/CHax) of the lactams is correctly accounted for by the DFT results. The ab initio outer valence Green's function calculations yielded a consistent description of the main features in the photoelectron spectra, i.e., location, splitting, and sequence of the n(N) and n(O) ionization energies, which also reflect the competing through-bond and through-space interactions within the sparteine framework.

The synthesis of a novel hapten containing a 1,4-diamino-2,3-diolbutane core unit is described. The molecule contains four stereogenic centres and has been synthesised via a stereoselective route. The absolute stereochemistry of each stereogenic carbon atom has been assigned by nuclear Overhauser effect experiments carried out on a number of derivatives. (C) 2004 Elsevier Ltd. All rights reserved.

[reaction: see text] The stereoselective synthesis of a novel proline-containing dipeptide isostere is described. Starting from l-valine, three new contiguous stereocenters are generated by asymmetric induction and epoxide chemistry, while the pyrrolidine ring of proline is introduced in the final step via intramolecular ring opening of the amino acid derived epoxyamine. Proline-containing peptidomimetics are potentially attractive as selective inhibitors of proline-specific enzymes, such as PPIases and retroviral proteases, and as analogues of bioactive peptides.

Recently we described an innovative class of non-peptide CCK 1 antagonists keeping appropriate pharmacophoric groups on the anthranilic acid employed as a molecular scaffold. The lead compound obtained, VL-0395 , characterized by the presence of Phe and the 2-indole moiety at the C- and N-termini of anthranilic acid, respectively, is endowed with submicromolar affinity towards CCK 1 receptors. Thus, we have prepared and tested on CCK receptors a library of VL-0395 analogues in order to investigate the precise topological and essential key interactions of the 2-indole group of the lead with the CCK 1 receptor. The obtained results confirm that this group establishes very specific interactions with this receptor sub-site and may be viewed as a “needle” group.

The equilibrium structures of the lupine alkaloid sparteine, its diastereoisomers (alpha-sparteine and beta-sparteine), and two related tricyclic diamines were investigated with the density functional theory ansatz B3LYP and HF-MP2 method. With the exception of beta-sparteine, these free molecules are structurally simple, since one conformer predominates both in the gas phase and in solution. The mono-protonated forms of all these molecules are characterized by intramolecular asymmetric hydrogen bonds. The di-protonated species have the same structural preferences as the parent free bases. All the theoretical structural predictions are consistent with the available experimental results. The NMR chemical shifts, calculated by the continuous set of gauge transformations formalism with the DFT/B3LYP method, are fairly consistent with NMR observation. The He(I) photoelectron spectra were measured and assigned by means of ab initio outer valence Green's function calculations. The overwhelming predominance of the lowest-energy conformer of all compounds is confirmed by various spectroscopic results. However, the structure of beta-sparteine is very special. The C-13 NMR spectra in solution at room temperature are indicative of an effective C-2 molecular symmetry. From the analysis of the NMR spectra from +20 to -85degreesC it is inferred that the most stable C-1 conformer of beta-sparteine is almost exclusively present in solution. At room temperature, it rapidly interconverts between two enantiomeric forms to produce the NMR spectrum of a time-averaged C-2 conformer. (C) 2003 Elsevier B.V. All rights reserved.

Having successfully obtained new CCK 1 ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK 1 receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N -substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK 1 receptor binding affinity (compound 1 : IC 50 =197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound ( 1 ) (coded VL-0395 ) a receptor binding hypothesis has been provided.

4-Acyl-1H-1,2,3-triazoles are formed from diethylaluminum azide and α′-(N,N-dibenzylamino)-α,β-unsaturated ketones by [3+2] cycloaddition of azide, followed by 1,5 hydride transfer to the β carbon of the triazoline side chain and fragmentation of the tertiary amino group promoted by coordination of the latter to the Lewis acid. The structure of a triazole product is confirmed by X-ray crystallography.

We have extended a highly flexible method for rapidly assembling aspartic protease inhibitors to produce symmetric and asymmetric monohydroxyethylene peptidomimetics. This method is based on the prior synthesis of the central non-cleavable peptide-bond isostere [NH(2)-P(1)psiP1'-NH(2); psi=hydroxyethylene isostere, HNCH(Bz)CHOHCH(2)CH(Bz)NH], with the possibility of accurately controlling its stereochemistry (S,S,S or S,R,S), and subsequently adding appropriate flanking units, chosen from commercially available amino acids, aromatic carboxylic acids, or phenoxyacetic acid (Poa) derivatives. The method was used to make asymmetric inhibitors of general formula Kyn-Xaa-PhepsiPhe-dmPoa, (Kyn=kynurenic acid, Xaa=Val, Thr or D-thienylglycine, M(r)=716-754) and symmetric inhibitors of formula xPoa-PhepsiPhe-xPoa (xPoa=Poa or dimethyl-, hydroxy-, formyl- or acetyl-Poa, M(r)=553-609), with logP(o/w) values ranging from 4.1 to 7.6. Inhibition of HIV-PR did not depend on the stereochemistry of the hydroxyl group, while it depended markedly on the substituents present on the Poa residues, with dmPoa being preferred over Poa or its more hydrophilic derivatives. Conversely, inhibition of Candida albicans Sap2 was higher for the S,S,S epimers, and Poa or its hydrophilic derivatives were preferred over dmPoa.

Amidase antibody 312d6, obtained against the sulfonamide hapten 4 a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1 a-3 a by a factor of 10(3) at pH 8. The mechanisms of both the uncatalyzed and antibody-catalyzed reactions were studied. Between pH 8 and 12 the uncatalyzed hydrolysis of N-toluoylindoles 1 a and 3 a shows a simple first-order dependence on [OH(-)], while hydrolysis of 3 a is zeroth-order in [OH(-)] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2 a is more complex due to the dissociation of the zwitterion into an anion with pK(a) 9.74; hydrolysis of the zwitterionic and the anionic form of 2 a both show simple first-order dependence on [OH(-)]. Absence of (18)O exchange between H(2) (18)O/(18)OH(-) and the substrate, a normal SKIE for both 1 a (k(H)/k(D)=1.12) and 3 a (k(H)/k(D)=1.24) and the value of the Hammett constant rho for hydrolysis of p-substituted amides 3 a-e are consistent with an ester-like mechanism in which formation of the tetrahedral intermediate is rate-determining and the amine departs as anion. The 312d6-catalyzed hydrolysis of 3 a was studied between pH 7.5 and 9, and its independence of pH in this range indicates that water is the reacting nucleophile. Hydrolysis of 3 a is only partially inhibited by the sulfonamide hapten, and this indicates that non-specific catalysis by the protein accompanies the specific process. Only the nonspecific process is observed in the hydrolysis of amides 3 with para substituents other than methyl. Binding studies on the corresponding series of p-substituted sulfonamides 5 a-e confirm the high specificity of antibody 312d6 for p-methyl substituted substrates.

A stereoselective synthesis of hydroxyethylene dipeptide isosteres based on the 1,4-diamino-2-hydroxybutane structure is described. Horner-Emmons olefination of phosphonates derived from alpha-amino acids, stereoselective reduction of the resulting enones to allylic alcohols, and syn epoxidation of the latter lead to enantiomerically pure 1-amino-2-hydroxy-3,4-epoxybutanes, key intermediates in the synthesis. Reductive cleavage of the epoxy alcohols with Red-Al proceeds in a highly regioselective way, giving 1-amino-2,4-dihydroxybutanes, from which diamino alcohol hydroxyethylene isosteres are obtained by selective protection of the secondary 2-hydroxy group, via cyclization to 1,3-oxazolidinone, and further elaboration of the 4-hydroxy. Both C-2 epimers of 1,4-diamino-2-hydroxybutanes are accessible by appropriate choice of the conditions for cyclization. The approach is demonstrated by the synthesis of a series of six hydroxyethylene dipeptide isosteres, including the diamino alcohol core of potent HIV-protease inhibitor ritonavir 18 and its C-2 epimer 11a.

An unprecedented combination of high chemo- and stereoselectivity in the NaBH4 reduction of 1+1 complexes between albumin and aromatic 1,3-diketones results in the formation of anti 1,3-diols with de up to 96%.

A series of new N-substituted anthranilic acid dimer derivatives having a C-terminal Phe residue was synthesized and evaluated for their affinity for CCK receptors. These compounds resulted from a blended approach based firstly on the use of an alternative substructure embedded within asperlicin and secondly on the derivatization of this template with substituents chosen considering the C-terminal primary structure of the endogenous ligand. Although these compounds exhibited a regnylogical-type organization similar to that of CCK-4, they are characterized by about 1000-fold greater affinity for CCK-A receptor than the C-terminal tetrapeptide.

Diethylaluminium cyanide is a highly selective reagent for the ring opening of 2,3-epoxyalcohols under mild conditions; the reaction takes place at C-3, with inversion of configuration, to give 1-cyano-2,3-diols.

2,3-Epoxyalcohols react with diethylaluminium azide under mild conditions to give 3-azido-1,2-diols resulting from the regio- and stereoselective attack of the nucleophile at the epoxide C-3. The high regioselectivity (>25:1) observed with both cis and trans substituted epoxides is not affected by bulky substituents at C-3. The method has been successfully applied also to the synthesis of diaminodiol dipeptide isosteres.

the effects of water and hydrophobic solvents on the stability of the open and closed forms of R. miehei lipase were evaluated with different solvent models. Desolvation of arginine 86 at the water-lipid interface plays a key role in the activation process, while the contribution from hydrophobic stabilization of the open form is less important.

A quantitative study on the effect of ring size in the intramolecular ring opening of epoxides by carbanions is described. Two series of substrates were examined a,a-bis-sulfonyl w-epoxides 1 and a-cyano-a-sulfonyl w-epoxides 2; in each series the carbanion is tethered to the epoxide by a chain of variable length from one to four methylene groups. The nucleophile can attack either electrophilic position of the oxirane ring, or both; exo ring opening of cyano sulfonyl epoxides 2 is followed by a second cyclization leading eventually to bicyclic, fused y-lactones. Both series of epoxides show the same trend of reactivity as a function of ring size, in the formation of three- to seven-membered rings, with reactivity maxima corresponding to the formation of cyclopropane and cyclopentane derivatives. Unlike 8 N 2 ring closure of w-halogeno carbanions, cyclization to a five-membered ring is the fastest process in this case. The ratio kdk5 between formation of three- and five-membered rings drops from over 100, in the SN2 cyclization of w-iodo bis-sulfones, to less than 0.5, in the cyclization of w-epoxy bis-sulfones 1. The difference is discussed in terms of trajectory of approach of the carbanion to the nucleophilic center. Cyclization of cyano sulfonyl epoxide 2a, in which the nucleophilic center and the epoxide are spaced by a single methylene group, is diastereoselective and leads to a bicyclic product with a cis fusion between the y-lactone and the cyclopropane ring.

Bovine and human serum albumins catalyze the aldol reaction of aromatic aldehyedes and acetone, with saturation kinetics and moderate and opposite enantioselectivity. The reaction occurs at the binding site in domain IIa, and is inhibited by warfarin. Kinetic data are consistent with an enamine mechanism. The activity is conserved in a 103 aminoacid peptide derived from the albumin sequence.

A sensitive competitive solid‐phase enzyme immunoassay for the detection of tert‐butylic beta‐agonist drugs was developed. An antiserum was raised in rabbits against the diazo‐conjugate of clenbuterol and the human serum albumin. In the assay, the anticlenbuterol antibody was incubated with the salbutamol‐enzyme conjugate and unlabelled standard or samples in microtitre wells precoated with affinity‐purified antirabbit IgG. The absolute detection limit for clenbuterol and mabuterol was 0.3 ng/ml (15 pg/well) and 50% relative binding at 1.0 ng/ml (50 pg/well). The absolute detection limits for salbutamol and terbutaline were 0.9 and 1.3 ng/ml, respectively. The cross‐reactivity of iso‐propylic parent compounds was quite low (

A quantitative study on the effects of methyl and gem-dimethyl groups in the intramolecular ring opening of epoxides by bis-sulfonyl carbanions is reported for the formation of cyclopropanes. Reaction rates are increased by methyl groups on the chain connecting the nucleophile with the oxirane and depressed by methyl substitution in the epoxide ring. When gem-dimethyl groups are present on the epoxide, ring opening is apparently inhibited. It will be shown that in this case the reaction is reversible and the apparent stabilization of the gem-dimethyl- substituted oxirane is actually due to a combination of effects on both the forward and the reverse reactions. On the basis of current theories on intramolecular reactions, it is suggested that release of ground-state strain and van der Waals repulsions in the transition state can account for the observed reactivity.

The synthesis of four isomers of p -coumaroylquinic acids was performed by esterification of p -acetylcoumaroylchloride with a suitably protected (−)-quinic acid. All isomers have been characterized by means of NMR spectroscopy and circular dichroism. Acyl migration was observed in the synthesis of 3- O - p -coumaroylquinic acid and 4- O - p -coumaroylquinic acid. Calculations on the most stable conformations of all isomers have also been performed to explain the acyl migration observed during the synthesis procedure.

The hydrolysis of N-p-toluoylindole and its 3-formyl derivative, at pH 8.0, are accelerated by a factor of nearly 103 by an antibody obtained against a KLH conjugate of N-p-toluensulfonylindole.

Esterase antibody 522c2, the first example of a catalytic antibody specifically programmed to control the reactivity of functional groups on the side chain of tyrosine, accelerates the hydrolysis of benzoate esters of L-tyrosine and tyrosine-containing dipeptides by a factor of 104 and is moderately active against other benzoate esters.

Two peptidomimetic inhibitors based on a novel epoxyalcohol core were designed to target the epoxide ring at the catalytic aspartates of HIV-protease for irreversible inhibition of the enzyme. The inhibitors were synthesized with a multi-step approach which includes Horner-Emmons olefination of a phenylalanine-derived phosphono ketone, stereoselective reduction of the resulting trans-enones to allylic alcohols and syn-epoxidation of the latters. The epoxyalcohols thus obtained were assayed for their ability to inhibit HIV-PR and were shown to inhibit the protease with IC50 values of 39 and 150 µM, respectively. This confirms that the designed epoxides are recognised with fairly good affinity by the enzyme’s active site, a pre-requisite for selective irreversible inhibition.

We present a method for designing artificial receptors capable of binding with high affinity to a chosen target organic molecule. The primary sequence of the peptide is optimized to maximize its binding affinity. Our algorithm builds on a combination of molecular dynamics, semiflexible docking, and replica exchange Monte Carlo and performs simultaneous sampling in sequence and conformational spaces carefully selecting the degree of flexibility in the mutated peptides. The approach is used to design a decapeptide able to bind efavirenz. The calculated binding energy of the designed peptide (approximately -12 kcal/mol) was confirmed experimentally by fluorescence measurements. NMR spectroscopy confirmed the interactions between the peptide and the efavirenz molecule predicted by the algorithm.