BIOCATALYSIS


Coupled chemoenzymatic reactions in continuous flow


This three-part article highlights the state of the art in the field of coupled chemoenzymatic reactions in continuous flow. Three different approaches are discussed in terms of their advantages and disadvantages and their future development. Part One describes single-reactor processes in vitro.


chemical engineer. The effectiveness with which nutrients are converted into complex chemical building blocks required for the cell metabolism is still a distant goal for any man-made chemical factory. Metabolic pathways, consisting of enzymatic sequential and coupled reactions, lie at the core of any living system and have been optimised by evolution over billions of years to create the phenomenon of life as we know it. In the field of applied biocatalysis, chemists are constantly trying to recognise the principles responsible for the efficiency of cell metabolism and to exploit them in organic synthesis.1-3 There are three biological principles whose implementation may be regarded as important milestones in this field and which can be used for the classification of existing


F


biotransformations. One of these principles is that a single reaction


or a long time, the living cell has been considered to be a perfect chemical factory, whose organisational principles can inspire every organic chemist and


historically were the first to be applied in the chemical industry and until now remain the most abundant among industrial biotransformations.


“Hybrid systems, in which the


strengths of both chemical and biological


approaches are combined, have proven to be


step of a given metabolic pathway proceeds in a very specific manner due to the intrinsically high chemo-, regio- and stereoselectivity of the enzyme catalysing this step. This principle is the soul of applied biocatalysis and has already been widely exploited in the chemical industry for decades in the production of chemicals by enzymatic processes.4-6 Biotransformations solely based on this principle, ie ‘single-reaction, single-enzyme’ systems, may be classified as first-generation enzymatic processes, which


28 sp2 Inter-Active September/October 2012


powerful tools for organic synthesis”


The second biological principle states that cell metabolism is a continuous process. Every metabolically active living cell is an open system that requires a constant flux of nutrients in order to stay alive. The numerous continuous chemocatalytic processes implemented on laboratory and on industrial scales prove that this principle can be effectively transferred to technical systems as well.7-11 Biotransformations, which combine both of the biological principles mentioned, may be regarded as ‘single-reaction, single- enzyme continuous- flow’ systems and classified as second- generation enzymatic processes. Such biotransformations naturally evolve from the corresponding first- generation ‘single- reaction’ batch processes and are often economically more attractive than their ancestors due to the higher productivity that they afford.


Therefore, it is not surprising that second- generation biotransformations have received much attention in recent years.12 The third biological principle says that cell metabolism is a complex network of reactions coupled through common substrates and products: Multistep syntheses of metabolites are conducted in sequential reactions catalysed by spatially aligned enzymatic complexes, while coupled parallel reactions are used to regenerate costly cofactors or to enable thermodynamically unfavourable steps.


The practical realisation of this principle is a dream for every organic chemist who wishes to perform a multistep organic synthesis of a desired compound in one pot, without isolation of the intermediates. And this dream has recently become reality: In the past few decades the concept of reaction cascades has become increasingly popular and has been proven to be a viable synthetic route to many classes of organic compounds.13-15 Biotransformations consisting of coupled sequential and/or parallel reactions catalysed by one or several enzymes may be regarded as third-generation enzymatic processes. The interest in such systems is prompted by their obvious economical potential: in situ regeneration of expensive cofactors and reduction of downstream processing steps decreases production costs and generation of waste.16,17 Therefore such processes are generally expected to have a higher ‘green index’18 and E-factor (kilograms of total waste produced per kilogram of product).19 One may also consider so-called chemoenzymatic reaction sequences, ie multi-step-reaction systems in which chemical and biocatalytic reaction steps are coupled, as third-generation biotransformations. These hybrid systems, in which the strengths of both chemical and biological approaches are combined, have proven to be powerful tools for organic synthesis and thus have nowadays become a hot topic in applied biocatalysis.20 From a formal point of view, reactions such as the lipase-catalysed hydrolysis of triglycerides to glycerol and fatty acids, or amylase-catalysed hydrolysis of amylose to glucose, should also be classified as multi-step-reaction processes because they proceed stepwise via a sequence of intermediates. However, such transformations differ from third-generation multi-step-reaction enzymatic processes in the sense that every reaction step is formally the same, ie it is catalysed by the same enzyme under the same reaction conditions


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