Adverse drug reactions following nonresponse in a depressed patient with CYP2D6 deficiency and low CYP 3A4/5 activity

A 47-year-old male taxi driver experienced multiple adverse drug reactions during therapy with clomipramine (CMI) and quetiapine for major depressive disorder, after having been unsuccessfully treated with adequate doses of mirtazapine and venlafaxine. Drug serum concentrations of CMI and quetiapine were significantly increased and pharmacogenetic testing showed a poor metabolizer status for CYP2D6, low CYP3A4/5 activity and normal CYP2C19 genotype. After reduction of the CMI dose and discontinuation of quetiapine, all ADR subsided except for the increase in liver enzymes. The latter improved but did not normalize completely, even months later, possibly due to concomitant cholelithiasis.

Preparation of benzolactams by Pd(II)-catalyzed carbonylation of N-unprotected arylethylamines

An unprecedented NH2-directed Pd(II)-catalytic carbonylation of quaternary aromatic α -amino esters to yield 6-membered 10 benzolactams has been developed. The reaction shows a strong bias to 6-membered lactams over 5-membered ones. The steric hindrance around the amino group seems to be pivotal for the success of the process.

Effects of Cytochrome P450 Enzyme Inhibitors on the Pharmacokinetics of Nonsteroidal Anti-inflammatory Drugs and Venlafaxine

Cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs. Inhibition of CYP enzymes usually increases the plasma concentrations of their substrate drugs and can thus alter the safety and efficacy of these drugs. The metabolism of many widely used nonsteroidal antiinflammatory drugs (NSAIDs) as well as the metabolism of the antidepressant venlafaxine is nown to be catalyzed by CYP enzymes. In the present studies, the effect of CYP inhibition on the armacokinetics and pharmacodynamics of NSAIDs and venlafaxine was studied in clinical trials with healthy volunteers and with a crossover design, by using different antifungal agents as CYP inhibitors. The results of these studies demonstrate that the inhibition of CYP enzymes leads to increased concentrations of NSAIDs. In most cases, the exposure to ibuprofen, diclofenac, etoricoxib, and meloxicam was increased 1.5to 2 fold when they were used concomitantly with antifungal agents. CYP2D6 inhibitor, terbinafine, substantially increased the concentration of parent venlafaxine, whereas the concentration of active moiety of venlafaxine (parent drug plus active metabolite) was only slightly increased. Voriconazole, an inhibitor of the minor metabolic pathway of venlafaxine, produced only minor changes in the pharmacokinetics of venlafaxine. These studies show that an evident increase in the concentrations of NSAIDs may be expected, if they are used concomitantly with CYP inhibitors. However, as NSAIDs are generally well tolerated, use of single doses of NSAIDs concomitantly with CYP inhibitors is not likely to adversely affect patient safety, whereas clinical relevance of longterm concomitant use of NSAIDs with CYP inhibitors needs further investigation. CYP2D6 inhibitors considerably affect the pharmacokinetics of venlafaxine, but the clinical significance of this interaction remains unclear.

Modificação bioquímica da gordura do leite

Recent advances for improving physicochemical and nutritional properties of lipids are reviewed, with emphasis on products attaining by biochemical processing of natural fats and oils. Enzymatic interesterification provides an important route to modify physical and nutritional properties of milkfat without generating trans isomers. This process makes use of lipases, a versatile class of enzyme that is able to perform efficiently the target modification in both solvent and solvent free systems. The present review covers important features of lipases, lipase-catalyzed interesterification reactions and their effects on the composition and texture of the resulting product.

Glicólise do poli(3-hidroxibutirato) por via enzimática

Poly(3-hydroxybutyrate), PHB, is a polymer with broad potential applications because of its biodegradability and biocompatibility. However, its high crystallinity is a limiting factor for many applications. To overcome this drawback, one strategy currently employed involves the reduction of the molecular weight of PHB with the concomitant formation of end-functionalized chains, such as those obtained via glycolysis. The glycolysis of PHB can be catalyzed by acid, base, or organometallic compounds. However, to our knowledge, there are no reports regarding PHB glycolysis catalyzed enzymatically. Among the major types of enzymes used in biocatalysis, the lipases stand out because they have the ability to catalyze reactions in both aqueous and organic media. Thus, in this study, we performed the enzymatic glycolysis of PHB using the lipase Amano PS (Pseudomonas cepacia) with ethane-1,2-diol (ethylene glycol) as the functionalizing agent. The results indicated that the glycolysis was successful and afforded hydroxyl-terminated oligomeric PHB polyols. Nuclear magnetic resonance spectra of the products showed characteristic signals for the terminal hydroxyl groups of the polyols, while thermogravimetric and differential scanning calorimetry analyses confirmed an increase in the thermal stability and a decrease in the crystallinity of the polyols compared with the starting PHB polymer, which were both attributed to the reduction in the molecular weight due to glycolysis.

Catalyzed and non-catalyzed synthesis of bioactive monastrol

The bioactive 3,4-dihydropyrimidin-2(1H)-thione derivative known as Monastrol was synthesized under catalyzed and non-catalyzed conditions through the Biginelli multicomponent reaction under solvent-free conditions. The use of two Lewis acids (FeCl3 and CuCl2) and two Brønsted acids (HCl and CF3COOH) as catalysts improved the reaction yields of the transformation compared with the non-catalyzed reaction. The experiments investigated catalysis and its role, the importance of multicomponent reactions and their green features, and the application of these concepts to the synthesis of a biologically important structure.

MICROWAVE ACTIVATION OF IMMOBILIZED LIPASE FOR TRANSESTERIFICATION OF VEGETABLE OILS

This work investigated the effect of microwave irradiation (MW) on the ethanolysis rate of soybean and sunflower oils catalyzed by supported Novozyme 435 (Candida antarctica). The effects of tert-butanol, water addition and oil:ethanol molar ratio on transesterification were evaluated under conventional heating (CH), and under optimum reaction conditions (with no added water in the system, 10% tert-butanol and 3:1 ethanol-to-oil molar ratio). The reactions were monitored up to 24 h to determine the conditions of initial reaction velocity. The investigated variables under MW (50 W) were: reaction time (5.0-180 min) and mode of reactor operation (fixed power, dynamic and cycles) in the absence and presence of tert-butanol (10% (w/w). The measured response was the reaction conversion in ethyl esters, which was linked to the enzyme catalytic activity. The results indicated that the use of microwave improved the activity at fixed power mode. A positive effect of the association of tert-butanol and MW irradiation on the catalytic activity was observed. The reaction rate improved in the order of approximately 1.5 fold compared to that under CH with soybean oil. Using soybean oil, the enzymatic transesterification under MW for conversion to FAEE (fatty acid ethyl esters) reached >99% in 3h, while with the use of CH the conversions were about 57% under similar conditions.

Molecular modeling of asymmetric induction in heterogeneously catalyzed hydrogenation of the C=O bond

Modifiering av metallytor med starkt adsorberade kirala organiska molekyler är eventuellt den mest relevanta teknik man vet i dag för att skapa kirala ytor. Den kan utnyttjas i katalytisk produktion av enantiomeriskt rena kirala föreningar som behövs t.ex. som läkemedel och aromkemikalier. Trots många fördelar av asymmetrisk heterogen katalys jämfört med andra sätt för att få kirala föreningar, har den ändå inte blivit ett allmänt verktyg för storskaliga tillämpningar. Detta beror t.ex. på brist på djupare kunskaper i katalytiska reaktionsmekanismer och ursprunget för asymmetrisk induktion. I denna studie användes molekylmodelleringstekniker för att studera asymmetriska, heterogena katalytiska system, speciellt hydrering av prokirala karbonylföreningar till motsvarande kirala alkoholer på cinchona-alkaloidmodifierade Pt-katalysatorer. 1-Fenyl-1,2-propandion (PPD) och några andra föreningar, som innehåller en prokiral C=O-grupp, användes som reaktanter. Konformationer av reaktanter och cinchona-alkaloider (som kallas modifierare) samt vätebundna 1:1-komplex mellan dem studerades i gas- och lösningsfas med metoder som baserar sig på vågfunktionsteori och täthetsfunktionalteori (DFT). För beräkningen av protonaffiniteter användes också högst noggranna kombinationsmetoder såsom G2(MP2). Den relativa populationen av modifierarnas konformationer varierade som funktion av modifieraren, dess protonering och lösningsmedlet. Flera reaktant–modifierareinteraktionsgeometrier beaktades. Slutsatserna på riktning av stereoselektivitet baserade sig på den relativa termodynamiska stabiliteten av de diastereomeriska reaktant–modifierare-komplexen samt energierna hos π- och π*-orbitalerna i den reaktiva karbonylgruppen. Adsorption och reaktioner på Pt(111)-ytan betraktades med DFT. Regioselektivitet i hydreringen av PPD och 2,3-hexandion kunde förklaras med molekyl–yta-interaktioner. Storleken och formen av klustret använt för att beskriva Pt-ytan inverkade inte bara på adsorptionsenergierna utan också på de relativa stabiliteterna av olika adsorptionsstrukturer av en molekyl. Populationerna av modifierarnas konformationer i gas- och lösningsfas korrelerade inte med populationerna på Pt-ytan eller med enantioselektiviteten i hydreringen av PPD på Pt–cinchona-katalysatorer. Vissa modifierares konformationer och reaktant–modifierare-interaktionsgeometrier var stabila bara på metallytan. Teoretiskt beräknade potentialenergiprofiler för hydrering av kirala α-hydroxiketoner på Pt implicerade preferens för parvis additionsmekanism för väte och selektiviteter i harmoni med experimenten. De uppnådda resultaten ökar uppfattningen om kirala heterogena katalytiska system och kunde därför utnyttjas i utvecklingen av nya, mera aktiva och selektiva kirala katalysatorer.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

Regioselective modification of galactose-containing polysaccharides in aqueous media

Biorefining is defined as sustainable conversion of biomass into marketable products and energy. Forests cover almost one third of earth’s land area, and account for approximately 40% of the total annual biomass production. In forest biorefining, the wood components are, in addition to the traditional paper and board products, converted into chemicals and biofuels. The major components in wood are cellulose, hemicelluloses, and lignin. The main hemicellulose in softwoods, which are of interest especially for the Nordic forest industry, is O-acetyl galactoglucomannan (GGM). GGM can be isolated in industrial scale from the waste waters of the mechanical pulping process, but is not yet today industrially utilized. In order to attain desired properties of GGM for specific end-uses, chemical and enzymatic modifications can be performed. Regioselective modifications of GGM, and other galactose-containing polysaccharides were done by oxidations, and by combining oxidations with subsequent derivatizations of the formed carbonyl or carboxyl groups. Two different pathways were investigated: activation of the C-6 positions in different sugar units by TEMPO-mediated oxidation, and activation of C-6 position in only galactose-units by oxidation catalyzed by the enzyme galactose oxidase. The activated sites were further selectively derivatized; TEMPO-oxidized GGM by a carbodiimide-mediated reaction forming amides, and GO-oxidized GGM by indium-mediated allylation introducing double or triple bonds to the molecule. In order to better understand the reaction, and to develop a MALDI-TOF-MS method for characterization of regioselectively allylated GGM, α-D-galactopyranoside and raffinose were used as model compounds. All reactions were done in aqueous media. To investigate the applicability of the modified polysaccharides for, e.g., cellulose surface functionalization, their sorption onto pulp fibres was studied. Carboxylation affects the sorption tendency significantly; a higher degree of oxidation leads to lower sorption. By controlling the degree of oxidation of the polysaccharides and the ionic strength of the sorption media, high degrees of sorption of carboxylated polysaccharides onto cellulose could, however, be obtained. Anionic polysaccharides were used as templates during laccase-catalyzed polymerization of aniline, offering a green, chemo-enzymatic route for synthesis of conducting polyaniline (PANI) composite materials. Different polysaccharide templates, such as, native GGM, TEMPO-oxidized GGM, naturally anionic κ-carrageenan, and nanofibrillated cellulose produced by TEMPO-oxidation, were assessed. The conductivity of the synthesized polysaccharide/PANI biocomposites varies depending on the polysaccharide template; κ-CGN, the anionic polysaccharide with the lowest pKa value, produces the polysaccharide/PANI biocomposites with the highest conductivity. The presented derivatization, sorption, and polymerization procedures open new application windows for polysaccharides, such as spruce GGM. The modified polysaccharides and the conducting biocomposites produced provide potential applications in biosensors, electronic devices, and tissue engineering.

Lipase and omega-transaminase catalysis in preparation of alcohol and amine enantiomers

Enantiopure intermediates are of high value in drug synthesis. Biocatalysis alone or combined with chemical synthesis provides powerful tools to access enantiopure compounds. In biocatalysis, chemo-, regio- and enantioselectivity of enzymes are combined with their inherent environmentally benign nature. Enzymes can be applied in versatile chemical reactions with non-natural substrates under synthesis conditions. Immobilization of an enzyme is a crucial part of an efficient biocatalytic synthesis method. Successful immobilization enhances the catalytic performance of an enzyme and enables its reuse in successive reactions. This thesis demonstrates the feasibility of biocatalysis in the preparation of enantiopure secondary alcohols and primary amines. Viability and synthetic usability of the studied biocatalytic methods have been addressed throughout this thesis. Candida antarctica lipase B (CAL-B) catalyzed enantioselective O-acylation of racemic secondary alcohols was successfully incorporated with in situ racemization in the dynamic kinetic resolution, affording the (R)-esters in high yields and enantiopurities. Side reactions causing decrease in yield and enantiopurity were suppressed. CAL-B was also utilized in the solvent-free kinetic resolution of racemic primary amines. This method produced the enantiomers as (R)-amides and (S)-amines under ambient conditions. An in-house sol-gel entrapment increased the reusability of CAL-B. Arthrobacter sp. omega-transaminase was entrapped in sol-gel matrices to obtain a reusable catalyst for the preparation enantiopure primary amines in an aqueous medium. The obtained heterogeneous omega-transaminase catalyst enabled the enantiomeric enrichment of the racemic amines to their (S)-enantiomers. The synthetic usability of the sol-gel catalyst was demonstrated in five successive preparative kinetic resolutions.

The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids) and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin) that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33%.

Bio and chemocatalysis for stereo and regioselective one-pot reaction applications

The development of cost efficient, selective and sustainable chemical processes for production of chiral building blocks is of great importance in synthetic and industrial organic chemistry. One way to reach these objectives is to carry out several reactions steps in one vessel at one time. Furthermore, when this kind of one-pot multi step reactions are catalyzed by heterogeneous chemo- and bio-catalysts, which can be separated from the reaction products by filtration, practical access to chiral small molecules for further utilization can be obtained. The initial reactions studied in this thesis are the two step dynamic kinetic resolution of rac-2-hydroxy-1-indanone and the regioselective hydrogenation of 1,2-indanedione. These reactions are then combined in a new heterogeneously catalyzed one-pot reaction sequence enabling simple recovery of the catalysts by filtration, facilitating simple reaction product isolation. Conclusively, the readily available 1,2-indanedione is by the presented one-pot sequence, utilizing heterogeneous enzyme and transition metal based catalysts, transferred with high regio- and stereoselectivity to a useful chiral vicinal hydroxyl ketone structure. Additional and complementary investigation of homogeneous half-sandwich ruthenium complexes for catalyzing the epimerization of chiral secondary alcohols of five natural products containing additional non-functionalized stereocenters was conducted. In principle, this kind of epimerization reactions of single stereocenters could be utilized for converting inexpensive starting materials, containing other stereogenic centers, into diastereomeric mixtures from which more valuable compounds can be isolated by traditional isolation techniques.

Monooxygenase Diversity and Oxygen Activation within Polyketide Antibiotic Biosynthesis

Molecular oxygen (O2) is a key component in cellular respiration and aerobic life. Through the redox potential of O2, the amount of free energy available to organisms that utilize it is greatly increased. Yet, due to the nature of the O2 electron configuration, it is non-reactive to most organic molecules in the ground state. For O2 to react with most organic compounds it must be activated. By activating O2, oxygenases can catalyze reactions involving oxygen incorporation into organic compounds. The oxygen activation mechanisms employed by many oxygenases to have been studied, and they often include transition metals and selected organic compounds. Despite the diversity of mechanisms for O2 activation explored in this thesis, all of the monooxygenases studied in the experimental part activate O2 through a transient carbanion intermediate. One of these enzymes is the small cofactorless monooxygenase SnoaB. Cofactorless monooxygenases are unusual oxygenases that require neither transition metals nor cofactors to activate oxygen. Based on our biochemical characterization and the crystal structure of this enzyme, the mechanism most likely employed by SnoaB relies on a carbanion intermediate to activate oxygen, which is consistent with the proposed substrate-assisted mechanism for this family of enzymes. From the studies conducted on the two-component system AlnT and AlnH, both the functions of the NADH-dependent flavin reductase, AlnH, and the reduced flavin dependent monooxygenase, AlnT, were confirmed. The unusual regiochemistry proposed for AlnT was also confirmed on the basis of the structure of a reaction product. The mechanism of AlnT, as with other flavin-dependent monooxygenases, is likely to involve a caged radical pair consisting of a superoxide anion and a neutral flavin radical formed from an initial carbanion intermediate. In the studies concerning the engineering of the S-adenosyl-L-methionine (SAM) dependent 4-O-methylase DnrK and the homologous atypical 10-hydroxylase RdmB, our data suggest that an initial decarboxylation of the substrate is catalyzed by both of these enzymes, which results in the generation of a carbanion intermediate. This intermediate is not essential for the 4-O-methylation reaction, but it is important for the 10-hydroxylation reaction, since it enables substrate-assisted activation of molecular oxygen involving a single electron transfer to O2 from a carbanion intermediate. The only role for SAM in the hydroxylation reaction is likely to be stabilization of the carbanion through the positive charge of the cofactor. Based on the DnrK variant crystal structure and the characterizations of several DnrK variants, the insertion of a single amino acid in DnrK (S297) is sufficient for gaining a hydroxylation function, which is likely caused by carbanion stabilization through active site solvent restriction. Despite large differences in the three-dimensional structures of the oxygenases and the potential for multiple oxygen activation mechanisms, all the enzymes in my studies rely on carbanion intermediates to activate oxygen from either flavins or their substrates. This thesis provides interesting examples of divergent evolution and the prevalence of carbanion intermediates within polyketide biosynthesis. This mechanism appears to be recurrent in aromatic polyketide biosynthesis and may reflect the acidic nature of these compounds, propensity towards hydrogen bonding and their ability to delocalize π-electrons.