Federico Berti

Short peptides as biosensor transducers

This review deals with short peptides (up to 50 amino acids) as biomimetic active recognition elements in sensing systems. Peptide-based sensors have been developed in recent years according to different strategies. Synthetic peptides have been designed on the basis of known interactions between single or a few amino acids and targets, with attention being paid to the presence of peptide motifs known to allow intermolecular self-organization of the sensing peptides over the sensor surface. Sensitive and sophisticated sensors have been obtained in this way, but the use of designed peptides is limited by severe difficulties in their in silico design. Short peptides from random phage display have been selected in a random way from large, unfocussed, and often preexisting and commercially available phage display libraries, with no design elements. Such peptides often perform better than antibodies, but they are difficult to select when the target is a small molecule because of the need to immobilize it with considerable modifications of its structure. Artificial, miniaturized receptors have been obtained from the reduction of the known sequence of a natural receptor down to a synthesizable and yet stable one. Alternatively, binding sites have been created over a designed, stable peptide scaffold. Short peptides have also been used as active elements for the detection of their own natural receptors: pathogenic bacteria have been detected with antimicrobial and cell-penetrating peptides, but key challenges such as detection of bacteria in real samples, improved sensitivity, and improved selectivity have to be faced. Peptide substrates have been conjugated to fluorescent quantum dots to obtain disposable sensors for protease activity with high sensitivity. Ferrocene–peptide conjugates have been used for electrochemical sensing of protease activity.

Regio- and stereoselective ring opening of 2,3-epoxyalcohols with diethylaluminium azide

Abstract 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.

Interaction of chlorogenic acids and quinides from coffee with human serum albumin.

Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-γ-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide), finding dissociation constants of the albumin-chlorogenic acids and albumin-quinides complexes in the micromolar range, between 2 and 30μM. Such values are comparable with those of the most powerful binders of albumin, and more favourable than the values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5-γ-quinide, we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dissociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by the quinide has also been demonstrated.

Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies.

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.

Effect of Size and N-Terminal Residue Characteristics on Bacterial Cell Penetration and Antibacterial Activity of the Proline-Rich Peptide Bac7

Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo.

Sandwich ELISA assay for the quantitation of palytoxin and its analogs in natural samples

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.

Determination of zeranol and β-zearalanol in calf urine by immunoaffinity extraction and gas chromatography—mass spectrometry after repeated administration of zeranol

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.

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists.

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.

Ring-opening of epoxyalcohols by diethylaluminium cyanide. Regio- and stereoselective synthesis of 1-cyano-2,3-diols

Abstract 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.

One-step stereospecific synthesis of α,β-dehydroamino acids and dehydropeptides.

Abstract Dehydroamino acids and dehydropeptides were prepared by a one-pot reaction employing diethyl chloroposphate in the presence of sodium hydride. The reaction is stereospecific and proceeds without racemization.