Bernd A. Nebel

New generation of biocatalysts for organic synthesis.

The use of enzymes as catalysts for the preparation of novel compounds has received steadily increasing attention over the past few years. High demands are placed on the identification of new biocatalysts for organic synthesis. The catalysis of more ambitious reactions reflects the high expectations of this field of research. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the pharmaceutical and chemical industry, and new enzymatic technologies and processes have been established. Enzymes are an important part of the spectrum of catalysts available for synthetic chemistry. The advantages and applications of the most recent and attractive biocatalysts--reductases, transaminases, ammonia lyases, epoxide hydrolases, and dehalogenases--will be discussed herein and exemplified by the syntheses of interesting compounds.

Recent progress in industrial biocatalysis

In recent years, several procedures have been reported for the development of biocatalytic processes. This review focuses on selected examples integrating biocatalysts into a variety of industrially interesting processes ranging from the manufacture of smaller, chiral speciality chemicals to the synthesis of more complex pharmaceutical intermediates. The use of rational protein design, multistep processes and de novo design of enzyme catalysts for the stereocontrolled preparation of important target structures is discussed.

Homogeneous vinyl ester-based synthesis of different cellulose derivatives in 1-ethyl-3-methyl-imidazolium acetate

A homogeneous acylation of cellulose with different vinyl esters in the biodegradable and less toxic ionic liquid 1-ethyl-3-methyl-imidazolium acetate ([EMIM]OAc) is described for the first time. The reaction proceeds in the absence of any additional catalyst and glucose- and cellulose-esters with chain lengths of C8 to C16 are accessible by using equimolar amounts of acyl donor. Cellulose esters with a degree of substitution (DS) in the range of 0.9–3.0 were synthesised successfully. Different reaction parameters like reaction time, temperature and amount of substrate were systematically changed and analysed by NMR, IR and HPLC-GPC. The highest DS was achieved at 80 °C and a reaction time of 2 hours. Taking into consideration the literature, the DS and degree of polymerisation (DP) of fatty acid chloride and vinyl ester-based synthesis routes were compared. Similar DS-values were obtained, but the DP was significantly reduced during the synthesis using fatty acid chlorides in [BMIM]Cl. As an undesirable side reaction, acetates from [EMIM]OAc are bound to the cellulose backbone. The quantity of bound acetate groups during vinyl ester-based synthesis rose with decreasing polarity of the substrates but overall proved to be much lower compared to the literature described anhydride or fatty acid chloride based synthesis routes in [EMIM]OAc. This novel process was extended by using further acyl donors like vinyl benzoate, pivalate and 2-ethylhexanoate to demonstrate the applicability of the vinyl ester-based cellulose modification in [EMIM]OAc. [EMIM]OAc was recycled with an efficiency of ∼90% and reused for subsequent syntheses.

Physicochemical aspects of lipase B from Candida antarctica in bicontinuous microemulsions.

Biotechnology involves applying enzymes in organic synthesis to convert non-natural substrates into enantiomerically pure products under mild reaction conditions. Non-natural substrates are often lipophilic molecules that can hardly be accessed and converted by enzymes in their natural aqueous environment. Bicontinuous microemulsions provide a spongelike nanostructure with a large interfacial area between aqueous and oil domains, which makes them valuable alternative reaction media. In the present study, we introduced lipase B from Candida antarctica into a bicontinuous microemulsion of composition H2O/NaCl-n-octane-pentaethylene glycol monodecylether (C10E5). Phase behavior, partitioning studies, and pulsed-field-gradient NMR measurements revealed that the lipase is mostly adsorbed at the microemulsions interface. Phase diagrams showed a maximum in efficiency with increasing amount of lipase added to the water phase of the microemulsion. It was observed that the ratio between the mass of lipase that is introduced into the system and the mass of lipase that is located at the interface stays constant. Self-diffusion coefficients of all components showed that the presence of the lipase is not influencing the bicontinuity of the microemulsion.

Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure.

In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.

Synthesis of oxyfunctionalized NSAID metabolites by microbial biocatalysts

The synthesis of valuable metabolites and degradation intermediates of drugs, like non-steroidal anti-inflammatory drugs (NSAIDs), are substantially for toxicological and environmental studies, but efficient synthesis strategies and the metabolite availability are still challenging aspects. To overcome these bottlenecks filamentous fungi as microbial biocatalysts were applied. Different NSAIDs like diclofenac, ibuprofen, naproxen and mefenamic acid could be oxyfunctionalized to produce human metabolites in isolated yields of up to 99% using 1 g L−1 of substrate. Thereby the biotransformations using Beauveria bassiana, Clitocybe nebularis or Mucor hiemalis surpass previous reported chemical, microbial and P450-based routes in terms of efficiency. In addition to different hydroxylated compounds of diclofenac, a novel metabolite, 3’,4’-dihydroxydiclofenac, has been catalyzed by B. bassiana and the responsible P450s were identified by proteome analysis. The applied filamentous fungi present an interesting alternative, microbial biocatalysts platform for the production of valuable oxyfunctionalized drug metabolites. Importance The occurrence of pharmaceutically active compounds, such as diclofenac and its metabolites, in the environment, in particular in aquatic systems, is of increasing concern because of the increased application of drugs. Standards of putative metabolites are therefore necessary for environmental studies. Moreover, pharmaceutical research and development requires assessment of the bioavailability, toxicity and metabolic fate of potential new drugs to ensure its safety for users and the environment. Since most of the reactions in the early pharmacokinetics of drugs are oxyfunctionalizations catalysed by P450s, oxyfunctionalized metabolites are of major interest. However, to assess these metabolites chemical synthesis often suffer from multistep reactions, toxic substances, polluting conditions and achieve only low regioselectivity. Biocatalysis can contribute to this by using microbial cell factories. The significance of our research is to complement or even exceed synthetic methods for the production of oxyfunctionalized drug metabolites.

Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197-209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.