Octavio A. C. Antunes

The High Resolution Crystal Structure of Yeast Hexokinase PII with the Correct Primary Sequence Provides New Insights into Its Mechanism of Action

Hexokinase is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. The crystal structure of yeast hexokinase PII from Saccharomyces cerevisiae without substrate or competitive inhibitor is determined and refined in a tetragonal crystal form at 2.2-Å resolution. The folding of the peptide chain is very similar to that of Schistosoma mansoni and previous yeast hexokinase models despite only 30% sequence identity between them. Distinct differences in conformation are found that account for the absence of glucose in the binding site. Comparison of the current model with S. mansoni and yeast hexokinase PI structures both complexed with glucose shows in atomic detail the rigid body domain closure and specific loop movements as glucose binds. A hydrophobic channel formed by strictly conserved hydrophobic residues in the small domain of the hexokinase is identified. The channels mouth is close to the active site and passes through the small domain to its surface. The possible role of the observed channel in proton transfer is discussed.

The Three‐Component Biginelli Reaction: A Combined Experimental and Theoretical Mechanistic Investigation

Biginelli reactions have been monitored by direct infusion electrospray ionization mass spectrometry (ESI-MS) and key cationic intermediates involved in this three-component reaction have been intercepted and further characterized by tandem MS experiments (ESI-MS/MS). Density functional theory calculations were also used to investigate the feasibility of the major competing mechanisms proposed for the Biginelli reaction. The experimental and theoretical results were found to corroborate the iminium mechanism proposed by Folkers and Johnson, whereas no intermediates directly associated with either the more energy demanding Knoevenagel or enamine mechanisms could be intercepted.

Kinetic Resolution of rac-1-Phenylethanol with Immobilized Lipases: A Critical Comparison of Microwave and Conventional Heating Protocols∥

The lipase-catalyzed kinetic resolution of rac-1-phenylethanol with vinyl acetate as acyl donor and cyclohexane as solvent has been investigated applying both microwave dielectric heating and conventional thermal heating in order to probe the existence of nonthermal microwave effects. All transformations were conducted at 40 degrees C in a dedicated reactor setup that allowed accurate internal reaction temperature measurements with use of fiber-optic probes. Quartz reaction vessels that allow higher levels of microwave power to be administered to the reaction mixture were used for all experiments. For all five studied immobilized lipases, the observed reactivities and enantioselectivities in microwave and oil bath experiments were identical and thus not related to the presence of the microwave field. The effect of magnetic stirring proved critical as too rapid stirring in some instances destroyed the enzyme support structure and led to altered reactivities and selectivities.

Low Accumulation of L90M in Protease from Subtype F HIV-1 with Resistance to Protease Inhibitors Is Caused by the L89M Polymorphism

BACKGROUND This work evaluates the role of subtype F human immunodeficiency virus type 1 (HIV-1) protease (PR) substitutions L89M and L90M in viral replication and resistance to PR inhibitors (PIs). METHODS Subtype B and F PR genes were subjected to site-directed mutagenesis, to create and reverse the methionine at positions 89 and 90. Viruses were re-created in cell culture, and their replicative capacity was assessed by fitness assay. Generated viruses were also phenotyped for PI resistance. RESULTS The subtype F clone (89M90L) showed a replicative capacity comparable to that of the PI-susceptible subtype B clone (89L90L) and was more fit than the L89M mutated subtype B clone (89M90L). Both 89M90M subtype B and F clones presented the lowest fitness s values. The L89M mutation impacted phenotypic resistance to all PIs in half of the subtype F isolates but not in the subtype B isolates. Subtype F isolates presented a phenotypic profile similar to that of subtype B isolates when the M89L mutation was introduced. CONCLUSION The L89M mutation in subtype F viruses is a high genetic barrier to the accumulation of the L90M resistance mutation and can function as a resistance mutation, depending on the presence of other polymorphisms in the subtype F PR backbone.

Production of aromas and fragrances through microbial oxidation of monoterpenes

Aromas and fragrances can be obtained through the microbial oxidation of monoterpenes. Many microorganisms can be used to carry out extremely specific conversions using substrates of low commercial value. However, for many species, these substrates are highly toxic, consequently inhibiting their metabolism. In this work, the conversion ability of Aspergillus niger IOC-3913 for terpenic compounds was examined. This species was preselected because of its high resistance to toxic monoterpenic substrates. Though it has been grown in media containing R-limonene (one of the cheapest monoterpenic hydrocarbons, which is widely available on the market), the species has not shown the ability to metabolize it, since biotransformation products were not detected in high resolution gas chromatography analyses. For this reason, other monoterpenes (alpha-pinene, beta-pinene and camphor) were used as substrates. These compounds were shown to be metabolized by the selected strain, producing oxidized compounds. Four reaction systems were used: a) biotransformation in a liquid medium with cells in growth b) with pre-grown cultures c) with cells immobilized in a synthetic polymer network and d) in a solid medium to which the substrate was added via the gas phase. The main biotransformation products were found in all the reaction systems, although the adoption of previously cultivated cells seemed to favor biotransformation. Cell immobilization seemed to be a feasible strategy for alleviating the toxic effect of the substrate. Through mass spectrometry it was possible to identify verbenone and alpha-terpineol as the biotransformation products of alpha-pinene and beta-pinene, respectively. The structures of the other oxidation products are described.

Terpenoids from Copaiba Cearensis

Abstract Crude oil of Copaifera cearensis was subjected to flash chromatography on KOH-impregnated silica gel from which three fractions were obtained: hexane, dichloromethane and methanol fractions. From the MeOH fraction a new sesquiterpenoid acid was identified.

A new oxo-vanadium complex employing an imidazole-rich tripodal ligand: a bioinspired bromide and hydrocarbon oxidation catalyst.

A vanadyl complex with the ligand (bis(1-methylimidazol-2-yl)methyl)(2-(pyridyl-2-yl)ethyl)amine was synthesized and fully characterized by X-ray crystallography, elemental analyses, cyclic voltammetry and infrared, electronic and electron paramagnetic resonance spectroscopies. This compound was designed under the so called hybrid concept. It shows to be able to promiscuously use hydrogen peroxide to oxidize bromide and to catalyze the oxidation of benzene and cyclohexane with very good selectivities.

3D-QSAR CoMFA/CoMSIA models based on theoretical active conformers of HOE/BAY-793 analogs derived from HIV-1 protease inhibitor complexes

The three-dimensional quantitative structure-activity relationships (3D-QSAR) of a series of HOE/BAY-793 analogs (C(2)-symmetric diol peptidomimetics), developed by Budt and co-workers [Bioorg. Med. Chem. 3 (1995) 559] as inhibitors of HIV-1 protease (HIV-PR), were studied using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA). Theoretical active conformers for these peptidomimetics were generated, derived from modeled protease inhibitor complexes, in order to orient the compounds superposition and to afford a consistent alignment. The best CoMFA model (N=27, q(2)=0.637, R(2)=0.991) showed contributions of the steric (45.7%) and electrostatic (54.3%) fields to the activity, while the best CoMSIA model (N=27, q(2)=0.511, R(2)=0.987) showed contributions of the electrostatic (68.5%) and hydrogen bond donor (37.5%) fields. The models were also external validated using four compounds (test set) not included in the model generation process. The statistical parameters from both models indicate that the data are well fitted and have high predictive ability. Moreover, the resulting 3D CoMFA/CoMSIA contour maps provide useful guidance for designing highly active ligands. The CoMFA/CoMSIA models were also compared with previous 4D-QSAR models [E.F.F. da Cunha, M.G. Albuquerque, O.A.C. Antunes, R.B. de Alencastro, QSAR Comb. Sci. 24 (2005), 240-253.].

Estratégias farmacológicas para a terapia anti-AIDS

The replicative cycle of HIV presents several events. The proteins involved in these events can be anticipated as pharmacological targets, aiming to the development of anti viral agents. Presently, there are fifteen commercially available anti-HIV drugs, which act at substrate binding site of reverse transcriptase (zidovudine, didanosine, zalcitabine, stavudine, lamivudine and abacavir), at a non-substrate binding site of reverse transcriptase (nevirapine, delavirdine and efavirenz), or by inhibiting HIV protease activity (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir). The present review focus both on these established classes of drugs and on new classes of compounds acting on other virus specific steps.

Asymmetric Michael addition of chiral imines to phenylvinylsulfone : preparation of key chiral building blocks for the synthesis of Aspidosperma and Hunteria alkaloids

Abstract Addition of chiral imine 8 to phenylvinylsulfone 9 led, after hydrolytic work-up to adduct 11 (91 % stereoselectivity). This derivative has been converted into target cyclopentanones 14 and 16 .