RESUMO
A series of novel p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-thiazoles derivatives (2a-f, 3a-f and 5-8) were synthesized by initial condensation of p-toluenesulfonylthiosemicarbazide 1 with a series of α-halogenocarbonyls in acetone or dimethylformamide (DMF)/acetone, mixture. All our synthesized compounds were submitted for further acylation reaction in the presence of acetic anhydride. The structures of newly synthesized derivatives 2a-f, 3a-f and 5-8 were confirmed by IR, (1)H-NMR, EIMS spectral data and elemental analysis. Compounds 2a, 2c, 2d, 2e and 3a showed significant anticancer activities (IC(50)<10 µM) on both prostate DU-145 and hepatocarcinoma Hep-G2 cancer cell lines.
Assuntos
Antineoplásicos/síntese química , Hidrazinas/síntese química , Tiazóis/síntese química , Compostos de Tosil/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Hidrazinas/farmacologia , Espectroscopia de Ressonância Magnética , Tiazóis/farmacologia , Compostos de Tosil/farmacologiaRESUMO
Our group has developed a stereospecific enzymatic method, which is very efficient for the in vitro synthesis of l-[(15)N]serine, l-[(15)N]methionine and l-[(15)N]glutamic acid. These amino acids were prepared from the corresponding alpha -ketoacids in the suitable enzymatic systems. The bacterial NAD-dependent amino acid dehydrogenases alanin dehydrogenase, leucin dehydrogenase and glutamate dehydrogenase were used as catalysts. Glucose dehydrogenase was used for the regeneration of NADH and (15)NH(4)Cl as isotopically labelled material at 99 at.% (15)N. All reactions are inexpensive and easy to perform on a synthetically useful scale (1-10g) giving high yields of l-amino acids. The (15)N isotope content was determined by mass spectrometry.
Assuntos
Aminoácidos/biossíntese , Aminoácidos/química , Enzimas/metabolismo , Isótopos de Nitrogênio/química , Alanina Desidrogenase/metabolismo , Biocatálise , Glucose 1-Desidrogenase/metabolismo , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/biossíntese , Ácido Glutâmico/química , Marcação por Isótopo , Cetoácidos/metabolismo , Leucina Desidrogenase/metabolismo , Espectrometria de Massas , Metionina/biossíntese , Metionina/química , NAD/metabolismo , Serina/biossíntese , Serina/químicaRESUMO
Anatoxin-a (ANTX) and homoanatoxin-a (HANTX) are low molecular weight neurotoxic secondary amines of 165 and 179 Da, respectively. We applied matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the detection of ANTX and HANTX directly on lyophilized and fresh filaments of axenic strains of the genus Oscillatoria, using 2,5-dihydroxybenzoic acid as matrix and purified ANTX and HANTX as references. To counteract the span of low molecular mass ions (< m/z 1000) generated by the matrix, we induced the matrix-suppression effect to obtain high quality ANTX/HANTX MALDI signals. MALDI desorption/ionization of the matrix-ANTX and the matrix-HANTX generated protonated molecules [M+H](+) at m/z 166.12322 and 180.1372, respectively. The masses obtained from the analysis of lyophilized filaments of the ANTX-producer Oscillatoria sp. strain PCC 9240 (m/z 166.15) and of fresh filaments of the HANTX-producers Oscillatoria sp. strains PCC 6506 (m/z 180.1375), PCC 9029 (m/z 180.1334) and PCC 10111 (m/z 180.13996) corresponded to the protonated molecular ions of ANTX and HANTX, respectively. Therefore, the application of MALDI-TOF-MS for the detection of cyanobacterial anatoxins in clonal and axenic strains of the cyanobacterial culture collections worldwide may help to assess ANTX/HANTX incidence among cyanobacteria.
Assuntos
Anabaena , Toxinas Bacterianas/análise , Compostos Bicíclicos Heterocíclicos com Pontes/análise , Neurotoxinas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tropanos/análise , Anabaena/química , Anabaena/metabolismo , Animais , Toxinas Bacterianas/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Toxinas de Cianobactérias , Cromatografia Gasosa-Espectrometria de Massas/métodos , Peso Molecular , Neurotoxinas/química , Tropanos/químicaRESUMO
In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.
Assuntos
Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/enzimologia , Núcleosídeo-Fosfato Quinase/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Ativação Enzimática , Guanosina Trifosfato/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Núcleosídeo-Fosfato Quinase/química , Uridina Monofosfato/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Two neurotoxic alkaloids, anatoxin-a and its homologue homoanatoxin-a, were purified from the filamentous cyanobacteria Oscillatoria sp. strain 193 (PCC 9240) and Oscillatoria formosa NIVA CYA-92 (PCC 10111), respectively, and characterized by mass spectrometry. Biological activity was determined by examining the capacity of the toxins to competitively inhibit the binding of radiolabelled bungarotoxin to acetylcholine receptors, using post-synaptic membrane fractions of Torpedo electric tissue. Inhibition was concentration dependent, with a K(i) of 5.4+/-1.1x10(-8) M for anatoxin-a and 7.4+/-0.9x10(-8) M for homoanatoxin-a. Their high affinities for the nicotinic cholinergic receptors were exploited to adapt the radioligand-binding assay for routine detection of this class of neurotoxins directly in low-molecular-mass cell extracts of cyanobacteria. Confirmation of the results and toxin identification were achieved by coupled gas chromatography-mass spectrometry (GC/MS). Seventy-six axenic strains, representative of 13 genera, were analysed. Five strains of the genus Oscillatoria, hitherto unknown for their toxicity, inhibited bungarotoxin binding. GC/MS revealed that Oscillatoria sp. strains PCC 6407, PCC 6412 and PCC 9107 synthesized exclusively anatoxin-a, whereas both anatoxin-a and homoanatoxin-a were produced by strain PCC 9029. Oscillatoria sp. strain PCC 6506, an isolate co-identic with strain PCC 9029, also produced both neurotoxins, but their respective presence depended upon growth conditions. The latter results suggest that regulatory differences in at least some of the cyanobacterial strains may account for the preferential synthesis of only one of the two neurotoxins or for their simultaneous occurrence.