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customer's publication Biocatalysis: From academic curiosity to manufacturing mainstream – the emergence of enzymes as catalysts in API manufacture

Chimica Oggi - Chemistry Today

The modern era of pharmaceuticals, based on synthetic and the types of reactions accessible were limited almost production of target enzymes. Piramal Pharma Solutions range of different enzymes. For example the ever expanding compounds rather than natural extracts, emerged around the entirely to resolution of racemic mixtures by ester hydrolysis or applied this approach to the ketosulfone reductase so that the range of genome sequences (currently >100,000) are a middle of the 20th century. Subsequently, the vast majority of transesterification using lipase enzymes, developed for the enzyme could be expressed in the bacterial host Escherichia source of information enabling the rapid identification of pharmaceutical active ingredients have been synthesised food processing and detergent industries rather than for coli at levels around one hundred times greater than was potential target genes. In addition, more than 120,000 protein using reactive stoichiometric reagents or simple catalysts such pharmaceutical synthesis. Access to a wider range of possible in the native Neurospora crassa. The isolated enzyme structures are listed in the RCSB protein database which, as metals or strong acids. Although the role of enzymes as reactions and enzyme selectivities required skills in has the advantage that, unlike live cells, it can be stored until when combined with a knowledge of the catalytic catalysts in biological systems has been known since the microbiology, genetic engineering and enzymology, required and the reduction reaction can be carried out in a mechanism, allows targeted modification of an enzyme’s second half of the 19th century, until quite recently their disciplines not historically associated with synthetic organic conventional chemical reactor rather than requiring a amino acid sequence to improve enzyme performance. Even application in pharmaceutical synthesis had been restricted to chemistry. Within Piramal Pharma Solutions the interaction of fermenter, this provides additional flexibility for process location when the reaction mechanism and structure of an enzyme a few niche applications such as the hydroxylation of steroids. these disciplines has been nurtured to form global and scheduling. The ketoreductase enzyme has been are not known its activity, selectivity, stability and other multidisciplinary teams that work together to solve complex characterised and belongs to the short chain dehydrogenase/ characteristics can be optimised through a process of There are multiple reasons synthetic problems. reductase family, structurally it is comprised of a dimer of two random mutation and selection of improved variants, a why enzymes were identical subunits each of 28,000 Daltons (Figure 2). The role of procedure often described as directed evolution. previously considered The synthesis of an anti- the enzyme in Neurospora crassa is currently unknown. unsuitable for glaucoma drug, Over the last 25 years enormous advances have been made pharmaceutical synthesis, dorzolamide, serves as a As is often the case, extreme specialisation is a double-edged in biotechnology and this understanding has provided the the primary ones being good example of how the sword and enzymes often suffer from this; for example an tools necessary to make enzyme catalysis the first choice for multidisciplinary approach enzyme that is an excellent catalyst for reducing a particular the synthesis of many complex pharmaceuticals. The close • that the availability of has been applied. The ketone may be completely unreactive with even close cooperation of biologists, chemists and process engineers has enzymes was limited synthesis of dorzolamide analogues. Fortunately, the various tools of modern genetic been critical in the application of these tools to industrial • enzymology was not Figure 1. Ketosulfone reductase goes via a ketosulfone and protein engineering enable access to an almost infinite scale synthesis. Despite the huge advances in biotechnology catalysed reduction yields part of an organic hydroxysulfone with which must be reduced the story is not over yet - current research into the de novo chemists training diastereomeric excess greater stereoselectively to design of totally novel protein structures offers the potential of than 98% • enzymes were provide the required enzyme activities not found in Nature. Watch this space! perceived to be diastereoisomer. Reduction incompatible with the with hydride reagents organic solvents typically used in chemical synthesis resulted in the wrong diastereoisomer (~90% selectivity) as a • why would anyone want to use a complex, water soluble result of induction from the chiral methyl substituent. polypeptide (typically in excess of 30,000 Daltons) to Consequently the potential for enzymatic reduction to solve this catalyse a reaction when zinc dust or sulfuric acid could problem was investigated (Figure 1). Screening of commercially be used instead? available ketoreductases failed to identify an enzyme with suitable selectivity so a range of microorganisms were screened Things however, began to change during the late 1980s when to identify potential biocatalysts. Several candidate tighter regulations were imposed by the regulatory authorities microorganisms were identified, the fungus Neurospora crassa on the control of stereochemistry in pharmaceuticals. It was was selected for process development and the reaction was known that enzymes exhibit extreme levels of selectivity as a scaled to hundreds of kilograms batch size. For several years the Contacts consequence of their complex 3-dimensional structure and process was operated in a fermentation mode using live cells. Robert A. Holt ([email protected]) diastereomeric purity, igniting renewed interest in enzymes as Figure 2. Structural model of ketosulfone reductase dimer Andre Jakoblinnert ([email protected]) catalysts for organic chemistry. One of the most significant scientific advances underpinning with bound NADPH cofactor. The left subunit is depicted as the protein surface whilst the www.piramalpharmasolutions.com As mentioned above, during the 1980s and 1990s the range of the success of large scale biocatalysis is the development of right subunit is depicted showing the secondary structure enzymes available for process scale chemistry was very small gene cloning and over-expression, enabling the cost-efficient

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