ENZYMATIC RESOLUTION OF ANTIDEPRESSANT DRUG PRECURSORS IN AN UNDERGRADUATE LABORATORY

The use of biocatalysts in synthetic chemistry is a conventional methodology for preparing enantiomerically enriched compounds. Despite this fact, the number of experiments in chemical teaching laboratories that demonstrate the potential of enzymes in synthetic organic chemistry is limited. We describe a laboratory experiment in which students synthesized a chiral secondary alcohol that can be used in the preparation of antidepressant drugs. This experiment was conducted by individual students as part of a Drug Synthesis course held at the Pharmacy Faculty, Lisbon University. This laboratory experiment requires six laboratory periods, each lasting four hours. During the first four laboratory periods, students synthesized and characterized a racemic ester using nuclear magnetic resonance spectroscopy and gas chromatography. During the last two laboratory periods, they performed enzymatic hydrolysis resolution of the racemic ester using Candida antarctica lipase B to yield enantiomerically enriched secondary alcohol. Students successfully prepared the racemic ester with a 70%-81% overall yield in three steps. The enzymatic hydrolysis afforded (R)- secondary alcohol with good enantioselectivity (90%-95%) and reasonable yields (10%-19%). In these experiments, students were exposed to theoretical and practical concepts of aromatic acylation, ketone reduction, esterification, and enzymatic hydrolysis.

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.

ESTUDO DA IMOBILIZAÇÃO DE LIPASE EM SÍLICA OBTIDA PELA TÉCNICA SOL-GEL

The objective of this work was the immobilization of the enzyme Candida antarctica lipase B (CAL B) using the sol-gel method of immobilization and three different initiators of the polymerization reaction: one acid (HCl), one basic (NH4OH) and the other nucleophilic (HBr). Tetraethylorthosilicate was used as the silica precursor. The influence of the additive PEG 1500 on immobilization was assessed. The efficiency of the process was evaluated considering the esterification activity of the xerogels. The immobilization process provided enhanced thermal stability, storage and operational aspects relative to the free enzyme. Storage temperature proved one of the main variables to be considered in the process, with the xerogels stored under refrigeration showing better results in terms of residual activity (nearly 200 days with ≥ 90% residual activity of basic and nucleophilic xerogels) when compared with storage at ambient temperature (nearly 40 days). The results demonstrated the possibility of reuse of derivatives and a greater number of cycles (nine), considering a residual activity of 50%.

Lipase-catalyzed Approaches towards Secondary Alcohols: Intermediates for Enantiopure Drugs

The use of enantiopure intermediates for drug synthesis is a trend in pharmaceutical industry. Different physiological effects are associated with the enantiomers of chiral molecules. Thus, the safety profile of a drug based on an enantiopure active pharmaceutical ingredient is more reliable. Biocatalysis is an important tool to access enantiopure molecules. In biocatalysis, the advantage of selectivity (chemo-, regio- and stereoselectivity) is combined with the benefits of a green synthesis strategy. Chemoenzymatic syntheses of drug molecules, obtained by combining biocatalysis with modern chemical synthesis steps usually consists of fewer reaction steps, reduced waste production and improved overall synthetic efficiency both in yields and enantio- and/or diastereoselectivities compared with classical chemical synthesis. The experimental work together with the literature review clearly indicates that lipase catalysis is highly applicable in the synthesis of enantiopure intermediates of drug molecules as the basis to infer the correct stereochemistry. By lipase catalysis, enantiopure secondary alcohols used as intermediates in the synthesis of Dorzolamide, an antiglaucoma drug, were obtained. Enantiopure _-hydroxy nitriles as potential intermediates for the synthesis of antidepressant drugs with 1-aryl-3- methylaminopropan-1-ol structure were also obtained with lipases. Kinetic resolution of racemates was the main biocatalytic approach applied. Candida Antarctica lipase B, Burkholderia cepacia lipase and Thermomyces lanuginosus lipase were applied for the acylation of alcohols and the alcoholysis of their esters in organic solvents, such as in diisopropyl ether and tert-butyl methyl ether. Candida Antarctica lipase B was used under solvent free conditions for the acylation of ethyl 3-hydroxybutanoate.

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.

ORGANOSILICON BIOTECHNOLOGY: A BIO-INSPIRED APPROACH TO THE HYDROLYSIS OF ALKOXYSILANES and THE LIPASE-CATALYZED SYNTHESIS OF SILOXANE-CONTAINING POLYESTERS AND POLYAMIDES

The first part of this thesis studied the capacity of amino acids and enzymes to catalyze the hydrolysis and condensation of tetraethoxysilane and phenyltrimethoxysilane. Selected amino acids were shown to accelerate the hydrolysis and condensation of tetraethoxysilane under ambient temperature, pressure and at neutral pH (pH 7±0.02). The nature of the side chain of the amino acid was important in promoting hydrolysis and condensation. Several proteases were shown to have a capacity to hydrolyze tri- and tet-ra- alkoxysilanes under the same mild reaction conditions. The second part of this thesis employed an immobilized Candida antarctica lipase B (Novozym-435, N435) to produce siloxane-containing polyesters, polyamides, and polyester amides under solvent-free conditions. Enzymatic activity was shown to be temperature dependent, increasing until enzyme denaturation became the dominant pro-cess, which typically occurred between 120-130ᵒC. The residual activity of N435 was, on average, greater than 90%, when used in the synthesis of disiloxane-containing polyesters, regardless of the polymerization temperature except at the very highest temperatures, 140-150ᵒC. A study of the thermal tolerance of N435 determined that, over ten reaction cycles, there was a decrease in the initial rate of polymerization with each consecutive use of the catalyst. No change in the degree of monomer conversion after a 24 hour reaction cycle was found.

Lipase-Mediated Enzymatic Catalysis For The Synthesis of New Chiral Polymers

Immobilized lipase B from Candida antarctica (N435) was investigated as a potential biocatalyst to generate silicone-based chiral polymers from monomers derived from the enzymatic dihydroxylation of bromobenzene. Several conditions and parameters have been investigated for this purpose and lipase transesterification preference to each of the free secondary alcohols in the chiral monomers was documented. The N435 was challenged with a series of substrates where the free alcohol moieties were systematically protected in order to study the substrate preference(s) for the transesterification reactions.

Cyclotetrasiloxane Frameworks for the Chemoenzymatic Synthesis of Oligoesters

Immobilized lipase B from Candida antarctica (Novozym® 435, N435) was utilized as part of a chemoenzymatic strategy for the synthesis of branched polyesters based on a cyclotetrasiloxane core in the absence of solvent. Nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were utilized to monitor the reactions between tetraester cyclotetrasiloxanes and aliphatic diols. The enzyme-mediated esterification reactions can achieve 65– 80% consumption of starting materials in 24–48 h. Longer reaction times, 72–96 h, resulted in the formation of cross-linked gel-like networks. Gel permeation chromatography of the polymers indicated that the masses were Mw ¼ 11 400, 13 100, and 19 400 g mol 1 for the substrate pairs of C7D4 ester/ octane-1,8-diol, C10D4 ester/pentane-1,5-diol and C10D4 ester/octane-1,8-diol respectively, after 48 h. Extending the polymerization for an additional 24 h with the C10D4 ester/octane-1,8-diol pair gave Mw ¼ 86 800 g mol 1. To the best of our knowledge this represents the first report using lipase catalysis to produce branched polymers that are built from a cyclotetrasiloxane core.

Chemoenzymatic synthesis of boron-containing chiral amines and amides

The first application of lipases as catalysts to obtain optically active boron-containing amines and amides is described. We studied several reaction conditions to achieve the kinetic resolution of boron-containing amines via enantioselective acylation mediated by Candida antarctica lipase B (CAL-B). Excellent enantioselectivity (E>200) and high enantiomeric excess (up to >99%) of both the remaining amines and amides were obtained. (C) 2010 Elsevier Ltd. All rights reserved.

First stereocontrolled acetylation of a hydroxypropargylpiperidone by lipase CALB

Hydroxypropargylpiperidones rac-1-3 were efficiently obtained by a one-pot three-component coupling reaction; enantioenriched propargylpiperidones were then obtained by a kinetic resolution process using the lipase from Candida antarctica. Lipase CALB has been shown to efficiently catalyse the stereocontrolled acetylation of hydroxypropargylpiperidones rac-3 by promoting stereodiscrimination at the carbinolic centre. The enzymatic catalytic processes allow the separation of the (S,R)- and (S,S)-3 diastereoisomers into the corresponding acetates produced as a (R,S)- and (R,R)-6 diastereoisomeric pair. The CALB was able to discriminate the stereogenic centre of the secondary (R)-enantiomer of rac-3 according to the Kaslauzkas rule. The remote stereogenic centre was not discriminated by the lipase. The functionalised enantioenriched diastereoisomers obtained are important building blocks in organic synthesis. (C) 2010 Elsevier Ltd. All rights reserved.

Isosorbide Polyesters from Enzymatic Catalysis

The synthesis of isosorbide aliphatic polyesters is demonstrated by the use of Novozym 435, a catalyst consisting of Candida antarctica lipase B immobilized on a macroporous support Several experimental procedures were tested and azeotropic distillation was most effective in removing low mass byproduct Furthermore, the use of diethyl ester derivatives of diacid comonomers gave isosorbide copolyesters with highest Isolated yield and molecular weights The length of the diacid aliphatic chain was less restrictive, but with a clear preference for longer aliphatic chains The molecular mass values of the obtained products were equivalent or higher than those obtained by nonenzymatic polymerizations, a clear illustration of the potential of enzymatic over conventional catalysis The ability of Novozym 435 to catalyze the synthesis of isosorbide polyester with weight-average molecular weights in excess of 40000 Da was unexpected given that isosorbide has two chemically distinct secondary hydroxyl groups This is the first example in which isosorbide polyesters were synthesized by enzyme catalysis, opening a large array of possibilities for this important class of biomass-derived building blocks Because these polymers are potential biomaterials the total absence of conventional Lewis acid catalyst residues represents a major Improvement in the toxicity of the material

Biosurfactants production by yeasts using soybean oil and glycerol as low cost substrate

Biosurfactants are bioactive agents that can be produced by many different microorganisms. Among those, special attention is given to yeasts, since they can produce many types of biosurfactants in large scale, using several kinds of substrates, justifying its use for industrial production of those products. For this production to be economically viable, the use of residual carbon sources is recommended. The present study isolated yeasts from soil contaminated with petroleum oil hydrocarbons and assessed their capacity for producing biosurfactants in low cost substrates. From a microbial consortium enriched, seven yeasts were isolated, all showing potential for producing biosurfactants in soybean oil. The isolate LBPF 3, characterized as Candida antarctica, obtained the highest levels of production - with a final production of 13.86 g/L. The isolate LBPF 9, using glycerol carbon source, obtained the highest reduction in surface tension in the growth medium: approximately 43% of reduction after 24 hours of incubation. The products obtained by the isolates presented surfactant activity, which reduced water surface tension to values that varied from 34 mN/m, obtained from the product of isolates LBPF 3 and 16 LBPF 7 (respectively characterized as Candida antarctica and Candida albicans) to 43 mN/m from the isolate LPPF 9, using glycerol as substrate. The assessed isolates all showed potential for the production of biosurfactants in conventional sources of carbon as well as in agroindustrial residue, especially in glycerol.

Síntese enzimática de biosurfactante e sua aplicabilidade na indústria do petróleo

Enzymes have been widely used in biosynthesis/transformation of organic compounds in substitution the classic synthetic methods. This work is the first writing in literature of enzymatic synthesis for attainment the biossurfactants, the use glucose sucrose, ricinoleic acid e castor oil as substratum, and as biocatalyst, used immobilized lipase Thermomyces lanuginose, Rhizomucor miehei and the Candida antarctica lipase B; alkaline protease and neutral protease from Bacillus subtillis and yeast Saccharomyces cerevisiaeI. The analysis of HPLC (high performance liquid chromatography) showed that highest conversions were reached of used the alkaline protease from Bacillus subtillis. Laboratory tests, to evaluate the applicability, indicated that the produced biosurfactantes had good stability in presence of salts (NaCl) and temperature (55 e 25°C), they are effective in the reduction of the superficial tension and contac angle, but they have little foaming capacity, when compared with traditional detergents. These results suggest that the prepared surfactants have potential application as wetting agent and perforation fluid stabilizer

Isolamento de leveduras de um consórcio especializado e avaliação de seu potencial na produção de biossurfactantes em fontes alternativas de carbono

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Imobilização de lipases por adsorção e ligação covalente em derivados de agarose e quitosana e sua aplicação em biocatálise

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