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Biocatalysis 101 - A Chemist's Guide to Starting Biocatalysis

Pablo Díaz-Kruik, David Lim, and Francesca Paradisi

Department of Chemistry, Biochemistry and Pharmaceutical Sciences, Bern, Switzerland

Glossary

API

Active pharmaceutical ingredient

BRENDA

A comprehensive enzyme information system

CALB

Lipase B from Candida antarctica

Cofactor

A non-protein chemical compound or metallic ion that is required for an enzyme's role as a catalyst

DKR

Dynamic kinetic resolution

IRED

Imine reductase

NAD+/NADH

Nicotinamide adenine dinucleotide

Protein expression

Biological process where the protein is synthesized inside a cell

Recombinant DNA

DNA scaffold that contains the protein sequence of interest

TRIS

Tris(hydroxymethyl)aminomethane

1.1 Introduction

1.1.1 Enzymes - the Green and Sustainable Way of the Future

Recent efforts by chemists to actively reduce toxic waste production and minimize costs have led to the discovery of many green and sustainable technologies. Not surprisingly, the use of enzymes, Nature's catalysts, has seen a major resurgence in academic and industrial interest over the past decade - not only for their sustainability and natural activities but for engineering them to perform novel transformations beyond capabilities observed in a synthetic organic lab [1, 2].

The attractiveness of using enzymes for transformations stems from their exquisite regio- and stereoselectivities - something that traditional chemists still struggle to achieve in the lab - that enzymes often execute effortlessly. Moreover, we have seen the emergence of multienzyme cascades for the synthesis of active pharmaceutical ingredients (APIs). A recent landmark example involves the synthesis of molnupiravir (MK-4482), an orally dosed ribonucleoside analogue and inhibitor of influenza viruses, which has demonstrated activity against COVID-19 when administered in animal models [3, 4]. In this work, McIntosh et al. developed a scalable three-step route toward MK-4482 [5]. Using a cascade of five enzymes, MK-4482 could be accessed from 5-isobutyrylribose (Figure 1.1).

To the uninitiated, entering the world of enzyme-catalyzed chemical transformations can be incredibly daunting, especially when one is not equipped with a foundational understanding of what an enzyme is and how these macromolecules work. However, you may be surprised to hear that enzymology and chemistry are not too different from each other at all! With an undergraduate chemistry background, a chemist can easily harness the power of enzymes to perform desired transformations - a fact that we aim to convince you of over the next few pages.

However, while this chapter aims to illustrate the power of enzymes for novel and sustainable transformations, we do not want to inadvertently imply the use of these macromolecules is the be-all-end-all solution - sometimes the use of traditional organic synthesis to access target molecules is the more logical solution. Therefore, when an enzyme might be used is a weighted question often involving the combination of various intricate factors, including efficiency and cost.

Over the following sections, we will do our best to educate you on these factors so that you can begin making an informed decision on this matter. We also aim to convince the reader that the use of enzymes is not limited to biologists and biochemists but also readily available for use by synthetic chemists. With the following breakdown of important considerations to make when using an enzyme, we hope to instill confidence in the reader that a biological catalyst is not too dissimilar to a chemical catalyst and can be readily obtainable from common suppliers.

Figure 1.1 A combined enzymatic cascade/hydroxylamination for the synthesis of molnupiravir (MK-4482).

We will also dispel common misconceptions and myths surrounding the use of enzymes and then give an overview of several classes of reactions that can be performed with enzymes, including recent developments into more exotic transformations such as photobiocatalysis.

This chapter will then conclude with a snippet into recent trends and technologies that have harnessed the use of enzymes in novel ways. We hope that the information gained from reading this chapter will provide a strong foundation for the reader to develop confidence in the use of enzymes and begin their venture into the world of biocatalysis.

1.1.2 Enzymatic and Organic Catalysis Are Not too Different from Each Other

A seasoned chemist may be quite familiar with several stereoselective reactions whereby stereocontrol is dictated by the chiral environment of the reaction. For example, one model for the Corey-Bakshi-Shibata (CBS) reduction involves coordination of the respective carbonyl to the CBS catalyst in a specific spatial orientation, leading to stereoselective reduction of the carbonyl to the corresponding alcohol (Figure 1.2) [6]. Enzymes utilize a very similar concept to this reaction - the catalyst (enzyme) places a reactant (substrate) in a chiral environment (the active site), whereby stereoselectivity is dictated by the local reactive environment, leading to a selective reaction outcome. In the next subsection, we will look at how an enzyme achieves these feats.

Figure 1.2 The CBS reaction has been used in undergraduate texts as a classic example of where an achiral reactant is stereoselectively transformed in a chiral environment to the corresponding product in high enantiomeric excesses.

1.1.3 Enzymes 101

Enzymes are known to accelerate reactions by more than 1017-fold [7]. How an enzyme achieves these colossal rate increases under aqueous conditions requires an understanding of the active site architecture in great molecular detail.

There are 20 essential amino acids found in nature. Enzymes are formed by cellular machinery, which stitch together combinations of these amino acids in a genetically pre-defined sequence, making one very long polymer. This polymer is folded to give a precise three-dimensional structure (Figure 1.3). The active site is defined as the region of the enzyme where substrates bind and undergo catalysis. The catalytic cycle begins with the binding of the substrate in the active site. This process precisely positions all molecules involved in the catalysis (metals, solvents, cofactors, etc.) in their respective orientations ready to achieve regio- and stereoselectivity. Subsequent activation of the substrate initiates the reaction, generating a transition state, which is stabilized by interactions with the active site residues of the enzyme. Following effective conversion of the substrate, the product is then released from the active site of the enzyme, completing one turnover and returning the catalyst back to its original state.

1.2 When Should I Choose an Enzyme over a Chemical Catalyst?

The choice of using a chemical catalyst over a biochemical solution needs to be assessed on a case-by-case basis, often involving a detailed cost-benefit analysis. For example, chemical asymmetric imine reduction often requires the use of expensive precious metals, such as Ir, Rh, Ru, and Pd (Figure 1.4) [8]. While recent methods have moved toward Earth-abundant solutions, such as employing iron or nickel, all these still require decoration with expensive chiral ligands that cannot be recycled [8], making the overall synthesis very environmentally and economically demanding.

In contrast, imine reductases (IREDs) can perform stereoselective reductions without the use of expensive metals and can be performed under aqueous conditions mitigating the need for organic solvents. Since the initial report of IREDs in 2010 [9], many advancements have been made to use these enzymes for novel synthetic transformations [9, 10]. In fact, Matzel et al. published an elegant procedure for performing biocatalytic dynamic kinetic resolutions (DKRs) of aldehydes using IREDs (Figure 1.5). This method exploits the stereo-preference of the enzyme for either the R- or S-chiral center [11].

The use of enzymes in this case showcases the re-opening of the chemical window, enabling unprecedented reaction conditions, merging asymmetric reduction and water media. This would be near impossible to achieve with classical reducing agents, such as NaBH4 or Na(CH3COO)3BH.

Figure 1.3 Proteins are formed by intracellular machinery that uses genetic information (DNA) to form a polymeric amino acid chain, which is then precisely folded to give a protein.

Figure 1.4 Asymmetric reduction of imines to amines in the presence of a chiral catalyst.

Figure 1.5...