Получение и изучение медико-биологических свойств меченного технецием-99м противомикробного препарата норфлоксацина гидрохлорида

Проведены исследования по созданию стандартного реагента для получения меченного 99mТс норфлоксацина гидрохлорида (НФГ). Оценку влияния компонентов реакционной смеси на радиохимическую чистоту получаемого препарата проводили методом тонкослойной хроматографии. На экспериментальных животных (кроликах) с моделью воспаления различной локализации показана функциональная пригодность меченого антибиотика для диагностики воспалительных процессов

Reaction and protein engineering employing a carbonyl reductase from candida parapsilosis

Biocatalytic manufacturing of chiral alcohols as building blocks for the synthesis of pharmaceuticals and fine chemicals is of growing relevance in the chemical industry. The biocatalytic route to provide these compounds by asymmetric reduction of cheap ketones constitutes a competitive synthesis route with respect to selectivity, productivity, cost-effectiveness and sustainability. Major challenges to establish economically feasible biocatalytic processes are addressed; on the one hand by reaction engineering facilitating efficient use of the resources and high space-time yields and on the other hand by protein engineering to supply appropriate enzymes to meet the process benchmarks. In this thesis, the characterization of a promising carbonyl reductase from the yeast Candida parapsilosis (CPCR2), which is able to selectively reduce various ketones to the corresponding alcohols, is presented. Molecular modeling permitted the prediction of indicator substrates and experimental verification enabled the assignment of sequence to function of two CPCR isoenzymes (CPCR1 & CPCR2). Applying CPCR2, a novel concept for producing chiral alcohols in molar amounts via an easy operation mode and straightforward work-up is introduced. Throughout the development of this so-called “neat substrate system”, which is composed of pure substrates and the whole cell catalyst, the key reaction parameters were elucidated and optimized. Moreover, the concept was further expanded towards different substrates, other ketone reducing enzymes and alternative operation modes. The “neat substrate system” demonstrates the first example for efficient biocatalytic alcohol production in a reaction medium lacking any bulk solvent. The versatility and efficiency of this system, developed here, might leverage this technique to become widely applicable. Protein engineering has become a powerful tool for tailoring enzymes to meet certain requirements like increased activity, stability or selectivity. In this thesis, CPCR2 was subjected to semi-rational protein engineering for the first time. The establishment of an appropriate protein expression procedure and a NADH-depletion activity assay in microliter formate enabled screening of CPCR2 variant libraries. The main engineering goals were the enlargement of the substrate spectrum and stabilization of the enzyme. A semi-rational approach led to substantial activity increase towards cyclohexanone substrates by exchange of leucine to methionine located in the substrate binding pocket (CPCR2-L119M). In particular, kcat for the reduction of 2-methyl cyclohexanone was increased more than 7-fold. The effect was explained on the molecular level by in silico substrate docking. The overall findings led to the assumption that more conservative amino acid substitutions might be more appropriate for altering the substrate scope of CPCR2. This may guide future strategies to modify the substrate acceptance of this enzyme class. Former studies on CPCR2 revealed severe inactivation at water-organic interfaces, which limits the application of the enzyme in biphasic systems. Previous attempts to stabilize the enzyme by rational protein engineering led to the double mutant CPCR2-(A275N, L276Q) showed increased stability as well as activity. This variant was adopted as a starting point for semi-rational optimization. Simultaneous site saturation and screening of these two positions revealed variants with improved activity and stability superior to the previous variant. The best variant found was CPCR2-(A275S, L276Q) exhibiting 1.4-fold higher activity, a deltaT50 of +5.2 °C in thermoresistance and 1.6-fold increased interfacial stability. Analysis of the single mutations suggested cooperativity of the amino acids at position 275 and 276, which are located at the dimer interface and close to the binding pocket. Experimental data as well as computational analysis indicated a contribution to stability by position 275 and an impact on activity by position 276. Structural investigation of the model predicted the establishment of an inter-subunit hydrogen bond by threonine at position 275 being responsible for stabilization and direct interactions of residue 276 with the substrate modulating activity. Taken together, a sequence to function assignment was achieved for identification of CPCR1 and CPCR2. A novel concept for biocatalytic alcohol manufacture, the “neat substrate system”, was developed and expanded to possible broad applications. CPCR2 was subjected to protein engineering yielding new variants with enlarged substrate scope, increased activity and stability

Reaction and protein engineering employing a carbonyl reductase from candida parapsilosis

Biocatalytic manufacturing of chiral alcohols as building blocks for the synthesis of pharmaceuticals and fine chemicals is of growing relevance in the chemical industry. The biocatalytic route to provide these compounds by asymmetric reduction of cheap ketones constitutes a competitive synthesis route with respect to selectivity, productivity, cost-effectiveness and sustainability. Major challenges to establish economically feasible biocatalytic processes are addressed; on the one hand by reaction engineering facilitating efficient use of the resources and high space-time yields and on the other hand by protein engineering to supply appropriate enzymes to meet the process benchmarks. In this thesis, the characterization of a promising carbonyl reductase from the yeast Candida parapsilosis (CPCR2), which is able to selectively reduce various ketones to the corresponding alcohols, is presented. Molecular modeling permitted the prediction of indicator substrates and experimental verification enabled the assignment of sequence to function of two CPCR isoenzymes (CPCR1 & CPCR2). Applying CPCR2, a novel concept for producing chiral alcohols in molar amounts via an easy operation mode and straightforward work-up is introduced. Throughout the development of this so-called “neat substrate system”, which is composed of pure substrates and the whole cell catalyst, the key reaction parameters were elucidated and optimized. Moreover, the concept was further expanded towards different substrates, other ketone reducing enzymes and alternative operation modes. The “neat substrate system” demonstrates the first example for efficient biocatalytic alcohol production in a reaction medium lacking any bulk solvent. The versatility and efficiency of this system, developed here, might leverage this technique to become widely applicable. Protein engineering has become a powerful tool for tailoring enzymes to meet certain requirements like increased activity, stability or selectivity. In this thesis, CPCR2 was subjected to semi-rational protein engineering for the first time. The establishment of an appropriate protein expression procedure and a NADH-depletion activity assay in microliter formate enabled screening of CPCR2 variant libraries. The main engineering goals were the enlargement of the substrate spectrum and stabilization of the enzyme. A semi-rational approach led to substantial activity increase towards cyclohexanone substrates by exchange of leucine to methionine located in the substrate binding pocket (CPCR2-L119M). In particular, kcat for the reduction of 2-methyl cyclohexanone was increased more than 7-fold. The effect was explained on the molecular level by in silico substrate docking. The overall findings led to the assumption that more conservative amino acid substitutions might be more appropriate for altering the substrate scope of CPCR2. This may guide future strategies to modify the substrate acceptance of this enzyme class. Former studies on CPCR2 revealed severe inactivation at water-organic interfaces, which limits the application of the enzyme in biphasic systems. Previous attempts to stabilize the enzyme by rational protein engineering led to the double mutant CPCR2-(A275N, L276Q) showed increased stability as well as activity. This variant was adopted as a starting point for semi-rational optimization. Simultaneous site saturation and screening of these two positions revealed variants with improved activity and stability superior to the previous variant. The best variant found was CPCR2-(A275S, L276Q) exhibiting 1.4-fold higher activity, a deltaT50 of +5.2 °C in thermoresistance and 1.6-fold increased interfacial stability. Analysis of the single mutations suggested cooperativity of the amino acids at position 275 and 276, which are located at the dimer interface and close to the binding pocket. Experimental data as well as computational analysis indicated a contribution to stability by position 275 and an impact on activity by position 276. Structural investigation of the model predicted the establishment of an inter-subunit hydrogen bond by threonine at position 275 being responsible for stabilization and direct interactions of residue 276 with the substrate modulating activity. Taken together, a sequence to function assignment was achieved for identification of CPCR1 and CPCR2. A novel concept for biocatalytic alcohol manufacture, the “neat substrate system”, was developed and expanded to possible broad applications. CPCR2 was subjected to protein engineering yielding new variants with enlarged substrate scope, increased activity and stability

A two-step biocatalytic cascade in micro-aqueous medium: using whole cells to obtain high concentrations of a vicinal diol

Although single- and multi-step biocatalytic approaches show persuasive advantages for the synthesis of especially chiral compounds (e.g. high chemo- and stereoselectivity), their application often suffers from low substrate loads and hence low space-time-yields. We herein present a synthetic cascade approach in which lyophilised, recombinant whole cells are applied in micro-aqueous reaction systems yielding extremely high space-time-yields. As an example we investigated the two-step synthesis of 1-phenylpropane-1,2-diol starting from cheap aldehydes and achieved high selectivities (ee/de > 99%) and high product concentrations. The new concept of running biocatalytic cascades in a mixture of high substrate loads and organic solvents under addition of small amounts of highly concentrated buffer is not only very easy-to-apply, but also exhibits several economic and ecologic advantages. On the one hand the usage of whole, lyophilised cells circumvents time-consuming enzyme purification as well as addition of expensive cofactors (here ThDP and NADPH). Additionally, catalyst and product workup is facilitated by the application of organic solvents (here MTBE). On the other hand, the employment of whole cells very effectively circumvents stability problems of biocatalysts in unconventional media enabling the addition of extremely high substrate loads (up to 500 mM in our example) and is therefore an easy and effective approach for multi-step biocatalysis. After optimisation, the combination of a carboligation step followed by a second oxidoreduction step with whole cell catalysts afforded an efficient two-step cascade for the production of 1-phenylpropane-1,2-diol with space-time yields up to 327 g L−1 d−1 and an E-factor of 21.3 kgwaste kgproduct−1

Stereoselective Two-Step Biocatalysis in Organic Solvent: Toward All Stereoisomers of a 1,2-Diol at High Product Concentrations

Biotransformations on larger scale are mostly limited to cases in which alternative chemical routes lack sufficient chemo-, regio-, or stereoselectivity. Here, we expand the applicability of biocatalysis by combining cheap whole cell catalysts with a microaqueous solvent system. Compared to aqueous systems, this permits manifoldly higher concentrations of hydrophobic substrates while maintaining stereoselectivity. We apply these methods to four different two-step reactions of carboligation and oxidoreduction to obtain 1-phenylpropane-1,2-diol (PPD), a versatile building block for pharmaceuticals, starting from inexpensive aldehyde substrates. By a modular combination of two carboligases and two alcohol dehydrogenases, all four stereoisomers of PPD can be produced in a flexible way. After thorough optimization of each two-step reaction, the resulting processes enabled up to 63 g L–1 product concentration (98% yield), space-time-yields up to 144 g L–1 d–1, and a target isomer content of at least 95%. Despite the use of whole cell catalysts, we did not observe any side product formation of note. In addition, we prove that, by using 1,5-pentandiol as a smart cosubstrate, a very advantageous cofactor regeneration system could be applied