Indolizidine 239Q and quinolizidine 275I. Major alkaloids in two Argentinian bufonid toads (Melanophryniscus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ligand Exchange Inducing Efficient Incorporation of CisPt Derivatives into Ureasil-PPO Hybrid and Their Interactions with the Multifunctional Hybrid Network

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Complexation of some trivalent lanthanides, scandium(III) and thorium(IV) by benzylidenepyruvates in aqueous solution

The protonation constants of 4-methylbenzylidenepyruvate (4Me-BP) and 4-isopropylbenzylidenepyruvate (4IP-BP) as well as the stability constants of their binary 1:1 complexes with Cu(II), La(III), Pr(III), Sm(III), Eu(III), Yb(III), Sc(III) and Th(IV) have been determined spectrophotometrically in aqueous solution at 25°C and ionic strength 0.500 M, maintained with sodium perchlorate. For all metal ions considered, the stability changes move in the same direction as the pKa of the ligands. Linear free energy relationships, as applied to oxygen donor substances, suggest the -COCOO- moiety as the metal binding site of the ligands. The results are discussed mainly taking into account that benzylidenepyruvates, besides the α-keto canonical form, may display other forms in aqueous solution with changing pH and the possible occurrence of extra intra-ligand charge polarization, induced by metal ions.

Structure of thrombin complexed with selective non-electrophilic inhibitors having cyclohexyl moieties at P1

The crystal structures of five new non-electrophilic β-strand-templated thrombin active-site inhibitors have been determined bound to the enzyme. Four co-crystallize with hirugen and inhibitor isomorphously to produce thrombin-hirugen crystals (monoclinic, space group C2), while one co-crystallizes in the hexagonal system, space group P65. A 1,4-substituted cyclohexyl moiety is conserved at the P1 position of all the inhibitors, along with a fused hetero-bicyclic five- and six-membered ring that occupies the P2 site. Amino, amidino and aminoimidazole groups are attached to the cyclohexyl ring for recognition at the S1 specificity site, while benzylsulfonyl and diphenyl groups enhance the binding at the S3 subsite. The cyclohexyl groups at the P1 positions of three of the inhibitors appear to be in the energetically favored chair conformation, while the imidazole-substituted cyclohexyl rings are in a boat conformation. Somewhat unexpectedly, the two cyclohexyl-aminoimidazole groups bind differently in the specificity site; the unique binding of one is heretofore unreported. The other inhibitors generally mimic arginyl binding at S1. This group of inhibitors combines the nonelectrophilicity and selectivity of DAPA-like compounds and the more optimal binding features of the S1-S3 sites of thrombin for peptidic molecules, which results in highly potent (binding constants 12 nM-16 pM, one being 1.1 μM) and selective (ranging from 140 to 20 000 times more selective compared with trypsin) inhibitors of thrombin. The binding modes of these novel inhibitors are correlated with their binding constants, as is their selectivity, in order to provide further insight for the design of therapeutic antithrombotic agents that inhibit thrombin directly at the active site.

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: Electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4- carboxylic acid (TOAC). TOAC replaced Asp 1 (TOAC 1-AII) and Val 3 (TOAC 3-AII) in AII and was inserted prior to Arg 1 (TOAC 0-BK) and replacing Pro 3 (TOAC 3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (TC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, τ C increased due to viscosity effects. Calculation of τ Cpeptide/τ CTOAC ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC 1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC 3 derivatives acquired more restricted conformations. Fluorescence spectra of All and its derivatives were especially sensitive to the ionization of Tyr 4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC 3-AII The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation. © 2004 Wiley Periodicals, Inc.

Purificação de lacases PPO-I de Botryosphaeria rhodina

Laccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4°C-18°C over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion-exchange chromatography on DEAE-cellulose. The purified laccase was identified as a glycoprotein, weight molecular 113 kDa, consisting of 40% protein and 60% carbohydrate identified by HPAEC-PAD as fucose, galactose, mannose, glucose and glucosamine.

Biosynthetic origins of the isoprene units of 4-nerolidylcatechol in Potomorphe umbellata

The biosynthetic origins of the isoprene units of 4-nerolidylcatechol (1), the major constituent of Potomorphe umbellata, have been studied through feeding experiments with [14C]- and [13C]-glucose, and with precursors of the mevalonic acid and triose/pyruvate pathways, namely, [2- 14C]-mevalonolactone and [U-14C]-glyceraldehyde-3- phosphate, respectively. The pattern of incorporation of label from [1- 13C]-glucose into 1 was determined by quantitative 13C NMR spectroscopy. The labelling pattern revealed that the additive was specifically incorporated, and that the isoprene units of the sesquiterpenoid moiety of 4-nerolidylcatechol were derived from both the mevalonic acid and the triose/pyruvate pathways. The results indicate that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for the biosynthesis of 1. ©2005 Sociedade Brasileira de Química.

β-lactam antibiotics epitope mapping with STD NMR spectroscopy: A study of drug-human serum albumin interaction

Molecular recognition events are key issues in many biological processes. STD NMR (saturation transfer difference nuclear magnetic resonance spectroscopy) is one of the techniques used to understand such biological interactions. Herein, we have investigated the interactions of four β-lactam antibiotics belonging to two classes (cephalosporins and penicillins) with human serum albumin (HSA) by 1H STD NMR revealing that the interaction between the aromatic moiety and HSA is responsible for the binding efficiency. Thus, the structural differences from the five to six-membered thio ring in penicillins and cephalosporins do not seem to influence antibiotic-albumin interactions. © 2012 Sociedade Brasileira de Química.

The acylpolyamines from spider venoms

Acylpolyamines are low molecular mass toxins occurring exclusively in the venoms from solitary wasps and some groups of spiders. Their chemical structures have been elucidated using hyphenated techniques of mass spectrometry, such as LC-MS and MS/MS, or through direct analysis with different types of NMR analyses. The chemical structures of the acylpolyamine toxins from the venoms of Nephilinae orb-web spiders appear to be organized into four parts based on the combinatorial way that the chemical building blocks are bound to each other. An aromatic moiety (part I) is connected through a linker amino acid (part II) to a polyamine chain (part III), which in turn may be connected to an optional tail (part IV). The polyamine chains were classified into seven subtypes according to the different combinations of chemical building blocks. These polyamine chains, in turn, are connected to one of three chromophore moieties: a 2,4-dihydroxyphenyl acetyl group, a 4-hydroxyindolyl acetyl group, or an indolyl acetyl group. They may be connected through an asparagine residue or sometimes through the dipeptide ornithyl asparagine. Also, nine different types of backbone tails may be attached to the polyamine chains. These toxins are noncompetitive blockers of ionotropic glutamate receptors with neuroprotective action against the neuronal death and antiepileptic effect. Thus, compounds of this class of spider venom toxin seem to represent interesting molecular models for the development of novel neuropharmaceutical drugs. © 2012 Elsevier B.V. All rights reserved.

Light emission from tryptophan oxidation by hypobromous acid

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N-formylkynurenine, kynurenine, 1,2,3,3a,8,8a-hexahydro-3a-hydroxypyrrolo[2,3-b]-indole-2-carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α-amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant.

Experimental NMR and MS study of benzoylguanidines. Investigation of E/Z isomerism

Molecules containing the guanidinic nuclei possess several pharmacological applications, and knowing the preferred isomers of a potential drug is important to understand the way it operates pharmacologically. Benzoylguanidines were synthesized in satisfactory to good yields and characterized by NMR, Electrospray Ionization Mass Spectrometry (ESI-MS) and Fourrier Transform InfraRed Spectroscopy techniques (FTIR). E/Z isomerism of the guanidines was studied and confirmed by NMR analysis in solution (1H-13C Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple-Bond Correlation (HMBC), 1H-15N HMBC, 1H- 1H Correlation Spectroscopy (COSY) and Nuclear Overhauser Effect Spectroscopy (NOESY) experiments) at low temperatures. Compounds with p-Cl and p-Br aniline moiety exist mainly as Z isomer with a small proportion of E isomer, whereas compounds with p-NO2 moiety showed a decrease in proportion of isomer Z. The results are important for the application of these molecules as enzymatic inhibitors. Copyright © 2013 John Wiley & Sons, Ltd.

Nitensidine A, a guanidine alkaloid from Pterogyne nitens, induces osteoclastic cell death

Nitensidine A is a guanidine alkaloid isolated from Pterogyne nitens, a common plant in South America. To gain insight into the biological activity of P. nitens-produced compounds, we examined herein their biological effects on osteoclasts, multinucleated giant cells that regulate bone metabolism by resorbing bone. Among four guanidine alkaloids (i.e., galegine, nitensidine A, pterogynidine, and pterogynine), nitensidine A and pterogynine exhibited anti-osteoclastic effects at 10 μM by reducing the number of osteoclasts on the culture plate whereas galegine and pterogynidine did not. The anti-osteoclastic activities of nitensidine A and pterogynine were exerted in a concentration-dependent manner, whereas nitensidine A exhibited an approximate threefold stronger effect than pterogynine (IC50 values: nitensidine A, 0.93 ± 0.024 μM; pterogynine, 2.7 ± 0.40 μM). In the present study, the anti-osteoclastic effects of two synthetic nitensidine A derivatives (nitensidine AT and AU) were also examined to gain insight into the structural features of nitensidine A that exert an anti-osteoclastic effect. The anti-osteoclastic effect of nitensidine A was greatly reduced by substituting the imino nitrogen atom in nitensidine A with sulfur or oxygen. According to the differences in chemical structures and anti-osteoclastic effects of the four guanidine alkaloids and the two synthetic nitensidine A derivatives, it is suggested that the number, binding site, and polymerization degree of isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their anti-osteoclastic effects. © 2013 Springer Science+Business Media Dordrecht.

Nitensidine A, a guanidine alkaloid from Pterogyne nitens, is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity. © 2013 Elsevier GmbH. All rights reserved.

Estudo da atividade biológica de compostos de paládio (II)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Investigação fitoquímica e avaliação da atividade antioxidante das folhas e frutos de Eugenia jambolana Lam. (Myrtaceae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)