Are You a Bio-Catalytic Converter?
Speed and efficiency are everything in pharmaceutical synthesis – and that’s why biocatalysis is an increasingly attractive option
Alice Dunbabin |
The industry continues to face unrelenting demand for faster and more efficient chemical processes. In response, chemists continue to seek new solutions to expand the toolkit. One increasingly important approach in the chemist’s toolkit is biocatalysis. Already a well-established methodology, the profile of biocatalysis was further raised in 2018 when Frances H. Arnold won the Nobel Prize in Chemistry for her group’s work in the area of directed evolution. In short, they developed novel bioengineering methods that harnessed the principles of evolution to access new biocatalysts in the laboratory. Breakthroughs in this area and others have enabled chemocatalysts to be replaced by biocatalysts in many industrial processes – and they have also opened up new avenues for small molecule transformations in the pharmaceutical industry.
Bio versus chemo
Compared with chemocatalysts, biocatalysts offer many advantages. Many catalytic processes use expensive transition metals to mediate transformations; these are unnecessary with cheaper biocatalysts, which also remove the need for high temperatures and pressures. Chemocatalysts are also often moisture and air sensitive, requiring strictly anhydrous conditions. Biocatalysis, on the other hand, is usually conducted in aqueous media. Put simply, the cost of drug development can be reduced.
Biocatalysts also offer exquisite selectivity. With chemocatalysis, transition metals often require ligands to create a 3D structure that influences the selectivity, which further drives up costs. Biocatalysts have chemo-, regio- and stereo-selectivity built in; they only bind specific substrates in certain conformations to the active site. Also, thanks to their high selectivity, biocatalysts often bypass the need for addition and removal of protecting groups.
Ensuring the safety of reactions is of paramount importance to chemists. In addition to cost savings above, the use of ambient temperatures and pressures, aqueous media (rather than flammable solvents) and avoidance of metals with limited availability, makes biocatalysis a much safer option than chemo-catalyzed routes. In addition, biocatalysts provide an environmentally friendly and more sustainable route for small molecule transformations.
For drug developers to benefit from biocatalysis, a screening process is needed to find the most suitable enzyme for a reaction. Screening can be done in-house using commercially available kits or outsourced to a specialist company.
Once an enzyme of interest is found, it may need to be engineered to make it fit for purpose. For example, the chosen enzyme may need to be altered to increase its tolerance to heat or organic solvents. Many factors, including temperature, pH, and substrate concentrations, can affect enzymes and their active sites – and all factors must be considered during the engineering phase. As an enzyme’s shape is directly related to its function, with the active site complementary to the shape of its specific substrate, the structure can also be modified so that different substrates fit the active site. Advances in this field have allowed many unnatural substrates to be converted, further increasing the applicability of biocatalysts.
Cheaper? Check. Safer? Check.
Biocatalysts are also non-toxic and biodegradable – and they can be reused multiple times when immobilized on a support. Alongside waste reduction, fewer toxic solvents are required. Lower environmental footprint? Check.
Given the advantages, why are some chemists reluctant to add biocatalysis to their toolkit? The ubiquity and reliability of transition metals and other chemocatalysis approaches certainly give them a head start. But biocatalysis does have one distinct disadvantage: enzyme screening and engineering are not quick processes – although advances in screening technology are being made (see box: Engineering Advances). One example is the use of simple-to-use colorimetric screening assays that reduce the work required to find successful hits by simply changing color when the desired reaction has occurred. In silico modeling of enzyme active sites has also aided enzyme engineering and mutation. By computationally visualizing the interactions between the substrate and the enzyme active site, changes can be made to avoid unfavorable interactions and increase the likelihood of a successful transformation.
Many enzymes require a cofactor to function, which can also pose challenges. Transaminases, for example, require an enzymatic amount of pyridoxal phosphate to covalently bind the substrate, which is straightforward to implement in a process. But there are times where the addition of cofactors can add extra complications to reactions; keto reductases, for example, need NADPH – a cofactor used in anabolic reactions – to function. As this cofactor can be very expensive, it is usually necessary to recycle these cofactors by adding in another enzyme and cosubstrate to the reaction. Fortunately, as the recyclable cofactors are well known, the reactions can be easily adapted to incorporate them.
There can also be challenges relating to the use of biocatalysis during the process development stage; for example, insolubility of the reaction materials in water. Enzymes typically tolerate 10-20 percent of an organic cosolvent, but if this does not provide enough solubility, it is possible to engineer enzymes with higher tolerance for organic solvents.
Finally, at the end of a reaction, emulsions and foaming can be problematic. After all, enzymes are proteins, so they can naturally denature and unfold, resulting in aggregates. Various solutions have been developed to overcome this issue, including the addition of cosolvents and anti-foaming agents.
Taking into account these limitations, choosing a biocatalyst for a process should still be relatively straightforward. After identifying which class of enzyme is able to perform your transformation, a screening kit of that class can be purchased – or the enzyme screening can be outsourced. Once a hit is identified, optimization can be carried out by looking at parameters (such as temperature, pH, and substrate concentration), before moving on to process development and addressing any issues with cofactor recycling or isolation of the product from the aqueous media. During this process, the need for enzyme engineering could also arise to combat problems with enzyme stability, heat or solvent tolerance.
The Evergrowing Enzyme Inventory
Some enzyme classes have been extensively studied and are widely available, whereas others are relatively new to the field. Enzymes are named based on the reaction they catalyze and most enzymes can perform their reaction in the forward or reverse direction. For example, lipases catalyze the stereoselective formation or hydrolysis of esters, and are commonly used in the kinetic resolution of racemic alcohols, amines and acids. Other examples include transaminases, which catalyze the formation of chiral amines, and ketoreductases, which perform the stereoselective reduction of ketones and the oxidation of alcohols. The list of enzyme-catalyzed reactions continues to extend as more novel enzymes are discovered, with enzymes such as ene reductases, imine reductases, and nitrilases all now being available. Some enzymes even perform named reactions, such as Baeyer-Villager monooxygenases and Pictet-Spenglerases.
To further extend the scope of biocatalysis, scientists are using metagenomics. By analyzing environmental samples, scientists have been able to discover new and exciting enzymes from all over the world; for example, in deep oceans, arctic ice, and even in the sands of Peru.
Biocatalysis is an evolving success story in the industry. In my view, one of the most important accomplishments using biological catalysts was the development of the anti-diabetic drug, sitagliptin (1). Its manufacture originally used a rhodium-catalyzed process, which required hydrogen pressure during the reaction. This catalyst was then replaced with a transaminase biocatalyst, which provided higher stereoselectivity than the chemical process, along with excellent yields. As well as better productivity, waste was reduced, and the cost decreased as the rare metal was no longer required.
Another recent success was the application of a ketoreductase in the synthesis of the asthma drug montelukast (2). A suitable ketoreductase was obtained through a directed evolution approach, and the reaction was higher yielding with an improved enantiomeric excess.
There are still improvements in the field of biocatalysis that can be made, particularly improving the engineering of enzymes and exploring enzyme cascades. Nature could also teach us more; thousands of enzymes are found in nature, with more being uncovered every day, and we can still learn from the many existing enzyme cascades and pathways to design more elegant processes. And, if cost and efficiency aren’t your main drivers, by embracing biocatalysts, we can also shift toward a greener and more sustainable future in drug development.
- CK Saville et al., “Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture,” Science, 329, 305-309 (2010). DOI: 10.1126/science.1188934
- J Liang et al., “Development of a Biocatalytic Process as an Alternative to the (−)-DIP-Cl-Mediated Asymmetric Reduction of a Key Intermediate of Montelukast,” Org. Process Res. Dev., 14, 193–198 (2010). DOI: 10.1021/op900272d