Biocatalysis in Medicinal Chemistry: Challenges to Access and Drivers for Adoption

Abstract

The use of biocatalysis in the manufacture of small molecule active pharmaceutical ingredients has seen a marked increase over the past decade. Driven by academic and industrial interest in the application of enzymes as catalysts for transforming chemical routes, the biocatalytic toolbox available to a chemist has continued to expand. Despite this, the application of biocatalysis in early discovery chemistry has trailed in comparison to its use in manufacturing routes. The authors offer their perspective on the adoption of biocatalysis in the early discovery space: highlighting challenges including enzyme supply and the biocatalysis business model, as well as recent trends that could spur more collaboration and access to enzymes for early discovery R&D activities.

As medicinal chemistry finds the need to access more stereochemically dense molecular structures, it is unsurprising that there is an increasing desire for chemical transformations that are more cost-effective, atom economical, robust, and translatable from the small reaction vessel to the pilot plant reactor. Biocatalysis, broadly defined here as the use of microorganisms or enzyme preparations to catalyze chemical transformations, meets these requirements for a growing number of reactions and has been increasingly adopted in late stage route development and active pharmaceutical ingredient (API) manufacture. Technological improvements in directed evolution, molecular biology, gene sequencing, and automation have resulted in a continually improving biocatalytic toolbox. However, the adoption of enzymatic catalysis earlier in the discovery process has lagged in comparison to its use in late stage processes. If biocatalytic reactions are often a preferred option in late stage development allowing chemists to access highly selective transformations so efficiently,¹ why has it not been implemented in earlier stages of the discovery cycle such as lead optimization or early route development? The authors propose some considerations and challenges that may play a role and highlight some emerging developments that may facilitate the use of biocatalysis earlier in pharmaceutical discovery and development.

“Evolution” of Nature’s Toolbox

While there are numerous examples of enzymes being used on an industrial scale to produce valuable molecules across the chemical industry,^2,3 many of these examples have harnessed the natural properties of specific enzymes for their inherent stereo- and chemoselectivity. In order to access the broad chemical space that is demanded by the pharmaceutical industry, enzymes with expanded reactivity were needed. The academic and industrial communities have actively worked over the years to address these limitations through the discovery and characterization of many useful wild-type enzymes mined from nature. Further engineering of wild-type enzymes to optimize chemical processes and discover new chemistry has also been maturing rapidly within this field.⁴ Directed evolution has proven to be a highly effective approach to engineering enzymes for the manufacture of fine chemicals and pharmaceuticals.⁵ Highlighting the broad impact of this technology, Frances H. Arnold was a recipient of the 2018 Nobel Prize in Chemistry for her contributions to the “directed evolution of enzymes.” The revolution that has taken place in the popularity and utility of directed evolution has extended to its development and application in areas including metabolite generation,⁶ chemical diversification,⁷ and the discovery of new enzymatic functions by unlocking non-natural mechanisms.^8,9

Since Louis Pasteur reported the first example of modern biocatalysis in 1858 when he reported the kinetic resolution of racemic tartaric acid using penicillium glaucum,¹⁰ the field has undergone a renaissance beginning in the late 20th century. We will not attempt to summarize the recent progress within this perspective, but instead, we would like to offer a perspective on some of the practical challenges presented when considering the implementation of this technology in discovery chemistry.

First and Foremost, How Can Enzymes Be Obtained?

This is a seemingly simple and reasonable question, but it has the potential to have a complex answer. In the most straightforward case where a biocatalytic reaction employs a well-established class of enzymes such as lipases, there are commercial suppliers from which an enzyme can be ordered and received just like any other chemical reagent. However, as the chemical toolbox of enzymes has evolved, these modified enzymes have often diverged from readily available wild-type enzymes. For a chemist exploring a potential biocatalytic transformation, the question arises, “How can an enzyme be obtained if it is not commercially available?” Publications will frequently cite identifying information including specific DNA sequences, construct details, and expression conditions. Converting a DNA sequence into a lyophilized enzyme powder or whole cell preparation can require numerous steps including, but not limited to polymerase chain reactions (PCR), molecular cloning, bacterial cell culture, cell disruption, and protein purification. These tasks require equipment (PCR machines, shaking incubators, centrifuges, cell disruption apparatus, lyophilizers) and reagents (bacterial cells, growth media and flasks, DNA/protein gels) that are not typically found in a synthetic chemistry lab. If there is access to a department where such expertise and equipment is available, the gene of interest can often be obtained via custom synthesis at select vendors to prepare the enzyme in-house.

Detailed Enzyme Preparation Methods Are Critical

Access to the equipment and expertise necessary to produce your own recombinant enzyme is merely one potential difficulty when attempting to perform a biocatalytic reaction. Lack of reproducibility in preparation and form can affect the stability of the enzyme and thus the overall success of a chemical transformation.¹¹ Optimization of the production of a particular enzyme is a science in and of itself, where procedures need to be established for DNA construct engineering (encoding the enzyme sequence of interest), host-cell growth and expression (getting a cell to produce the enzyme of interest), and downstream processing to obtain the final enzyme form (isolation and formulation of the enzyme of interest). The expression host, promoter choice, DNA sequence (codon-optimization), use of fusion proteins and amino acid tags, growth/fermentation conditions, and downstream processing conditions used to produce the enzyme can all potentially influence enzyme activity and stability. Depending on the enzyme, deviations in these factors have the potential to impact the reaction efficiency and purity profile of a given reaction.

For a chemist, there are many different formulations of an enzyme that can be accessed, each with its own set of advantages and limitations. Understanding the necessary enzyme form can be very important as the stability and activity profile of an enzyme can vary according to the specific combination of preparation conditions. Enzymes can be prepared in a purified form or as a protein mixture. They can also be produced in different physical forms with some of the more common options being stabilized solutions, lyophilized or spray-dried powders, and whole cell pastes. In the biocatalysis field where enzymes are typically used as reagents or catalysts, they are generally used without extensive purification as this can add dramatically to the cost of production. A lyophilized powder of an enriched enzyme mixture with no extensive purification is generally the preferred option. This powdered form is easily weighable and transferable like any other solid reagent, and it can be stored for extended periods of time in a suitable freezer according to the enzyme’s stability profile.

With the appropriate facilities, expertise, and information, producing wild-type enzymes (or those published in the literature) for smaller scale chemistry in the early discovery space is indeed possible. However, for many enzymes that have been specifically engineered to be more robust and to offer a wider scope of reactivity, obtaining this information may be much more difficult.

The Biocatalysis Industry Model: Why Disclosing Detailed Enzyme Information Is Tricky Business

There are two general operating models in place for those wanting to employ biocatalysis to produce small molecule APIs: “buy” or “build”. The “buy” model is restricted to procurement through commercial suppliers, whereas the “build” model relies on the investment in the creation of internal bespoke departments. In the “buy” model, enzymes are purchased from commercial suppliers, often as a panel or kit for a desired transformation. In this model, when larger quantities of the enzyme of interest are desired, they are then ordered from the same vendor. Should optimization be required, there are also selected suppliers that can perform directed evolution services usually at a nontrivial cost. This model is particularly attractive for those who are doing exploratory work with biocatalysis as enzyme suppliers will actively work to make their portfolio of commercial enzymes readily accessible for any interested party to rapidly test. This pathway allows for companies without biocatalysis departments to utilize these chemistries, but they sacrifice control over enzyme supply and freedom to operate should the application of the enzyme move from discovery into the manufacturing space. In the “build” model, biocatalysis and enzyme evolution capabilities are maintained in-house. This model requires a large capital investment into scientific expertise, technology platforms, and infrastructure but benefits the security of supply for enzymes incorporated into future manufacturing routes and allows for increased control over intellectual property of developed processes. In general, for those looking to adopt biocatalysis in chemical manufacture, many will first subscribe to the “buy” model before garnering the necessary buy-in and investment to move to the “build” model, making both operating structures key to the current biocatalysis landscape.

For those companies that subscribe to the “build” model and actively engage in the directed evolution of enzymes, a tangible advantage that arises from the creation of collections of evolved enzymes is the generation of additional diversity in chemical reactivity. These proprietary collections offer a potential competitive advantage, and companies may thus limit disclosure of exhaustive sequence–activity relationships. The same considerations generally apply to those companies that operate as suppliers for the “buy” model. These companies have likely invested a significant amount of R&D effort to develop enzymes that will form the basis of their commercial offering. While the business model for the discovery and development of engineered enzymes remains in the competitive space, the intellectual property relating to these valuable assets will be guarded and protected via patents where possible, making access for the wider chemistry community an issue.

Why Accessibility Matters: The Need for Speed in Medicinal Chemistry

Pharmaceutical innovation thrives on efficiency and speed, driven by competitive landscapes and a strict timeline of deliverables. Unlike commercial manufacture where elongated timelines allow for optimization of a biocatalytic transformation via directed evolution, the utilization of biocatalytic reactions in discovery chemistry is less prevalent, and the pressure for reduction of design-make-test-analyze¹² cycle time will often steer a medicinal chemist to go for the tried-and-true methods over new technologies. The power of enzymatic catalysis in late stage development lies in the ability to take an initial hit and optimize it via directed evolution toward the desired process target, but this process takes time; a typical round of directed evolution takes between 4 and 6 weeks to deliver an improved enzymatic variant. This is an extraordinarily long time to a medicinal chemist who then would likely turn to more traditional approaches, such as transition metal catalysis or organocatalysis to name a few.

For enzymatic transformations to be readily adopted in this space where time is so critical, enzymes need to be (1) readily available, (2) proven fit-for-purpose from the initial screen without extensive optimization, and (3) provide robust and reproducible reactivity with a substrate scope that is broad and/or predictable. Consider a hypothetical situation where enzyme supplies are not rate-determining, either through in-house capability or commercial access: a key pharmacophore within a chemical template is a stereochemically defined α-substituted benzylic amine. Accessing this stereochemical motif could be readily accomplished through a variety of metal-catalyzed options (e.g., imine reduction, C(sp³)–H amination, etc.) or chiral auxiliaries. However, in this situation, biocatalysis with an aminotransaminase could prove a superior method for several reasons: (1) the reactions are run under mild conditions without the need for an inert atmosphere or dry conditions, (2) enzymes offer exceptional chemoselectivity where often only the desired product and starting material are observed, and (3) enzymes are notorious for their exquisite levels of enantiocontrol. The authors would argue that 20% yield/99% ee is more useful than a 60% yield/70% ee to the chemist because difficult chiral preparative chromatographic separations are avoided and recovered starting material could easily be isolated and resubjected to the reaction conditions, if needed.

Future Directions and Potential Opportunities

In the past few decades, the impact of the field of biocatalysis has already been observed in the increasing access to commercial enzymes from selected vendors via the “buy” model. Ketoreductases, lipases, esterases, and aminotransaminases have become “established” biocatalytic enzyme classes with a growing number of commercially available options. Newer classes including imine reductases, reductive aminases, ene reductases, nitrilases, and P450s are becoming more readily available as published reports of reactivity generate more interest among the chemistry community. In addition, many academic research programs continue to focus on the discovery and characterization of new enzyme classes, adding examples of new reactivity and simplified transformations to the chemists’ toolbox.¹³

For those companies, like GSK or most other large pharmaceutical companies, who are fortunate to have invested early in the “build” model, the internal knowledge generated from years of reaction optimization and enzyme engineering becomes a major benefit in this environment where cycle time is paramount. By creating panels of biocatalysts for different chemical transformations and providing the infrastructure necessary to screen and analyze these reactions in high-throughput, a chemist becomes able to perform and analyze an enzyme screen quickly in a time frame of mere days. Furthermore, by investing in data systems to capture and render this screening data searchable in conjunction with other reaction searching tools (i.e., Reaxys or SciFinder), databases can be built that are a more comprehensive source for synthetic planning and retrosynthetic analyses. It would be ideal if, for example, a specific R-selective ketoreductase could not only be selected for an elaborate ketone structure based on information from structurally similar compounds but would then definitively deliver the corresponding alcohol stereoselectively without significant optimization. As the number of practitioners and literature reports increase with the biocatalysis boom (Figure 1), the need for full panel screening for each new substrate would be replaced by confident selection of a specific enzyme to perform a reaction based on literature reports.

Figure 1.

Incidence of literature reports on biocatalysis between the years 1970–2018. Chart details the number of literature reports of a SciFinder search conducted in July 2019 using the search terms “biocatalysis” and “reaction”. Results were restricted to journal articles.

The “build” model also allows for the exploration of chemistries that allow synthetic routes to be transformed rather than simply modified. New enzyme classes and novel reactivity are reported in the literature at a seemingly rapid rate, but the utility of such enzymes might be limited. As detailed in the sections above, producing an enzyme that is ready for use is not trivial without the appropriate infrastructure and expertise. The level of expertise required increases for unknown enzyme classes as there are seldom adequate procedures for their preparation to confirm activity. However, with dedicated biocatalysis experts, chemistry teams have been able to rise to this challenge and have developed new technologies to enable truly transformative enzymatic reactions.¹⁴

Conclusion

As the demand for enzymatic reactions increases, the incentive for commercial suppliers to enter the market and invest in capabilities to increase production of these enzymes will grow. There are still significant challenges surrounding the development and commercial model of biocatalytic enzymes, especially as these relate to the consistency and quality of enzyme preparations, intellectual property, and freedom-to-operate around the development of emerging enzyme classes. The current paradigm does not greatly incentivize broad disclosure of findings, but if the significant research and development costs can be shared among industrial and academic parties in a precompetitive manner, this could pioneer a course to greater access of this chemistry to the entire community. This push toward comprehensive disclosure could dramatically alter the current landscape and potentially allow for a second renaissance in the field of biocatalysis. The early 21st century has been exciting and transformational thus far, providing significant momentum for years to come.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.