3. Diversity-oriented enzymatic synthesis of cyclopropane building blocks
3.1 Leveraging biocatalysis in the drug development pipeline
Since the beginning of the second wave of biocatalysis,1 enzymes have been engineered for and applied to the synthesis of myriad molecules of industrial interest, from low-value, high- volume fuels2,3 and specialty chemicals4 to value-added fine chemicals such as flavors and fragrances.5 One application where the advantages of biocatalysis have truly shone is the synthesis of pharmaceutical compounds. The chemo-, regio-, and stereoselectivity required in the synthesis of complex pharmaceuticals can be met by a single enzyme engineered for this specific function. The high value of pharmaceutical compounds offsets the substantial cost of protein engineering and biocatalyst production. It is therefore unsurprising that intensive academic and industrial research and development have gone into the discovery and engineering of enzymes to synthesize pharmaceutical compounds.6–8
The early stages of a drug discovery and development pipeline require the ability to rapidly generate tens to hundreds of grams of a candidate drug for further studies.9 For biocatalysts to outperform the competing chemical processes, the speed at which they can carry a project from initial activity determination to industrial production is crucial.8–10 The field of protein engineering has made great strides toward faster and more efficient biocatalyst development, which has begun to bring biocatalysis into the drug development pipeline, albeit after the drug candidate has been identified and is in clinical trials. Integrating biocatalysis even earlier in the drug development pipeline requires examining and overcoming current limitations of biocatalysts, namely their narrow substrate scope and the time required to engineer them for the desired activity. To determine where biocatalysts can fit earlier in the drug discovery pipeline, we must look at the workflow of the drug discovery pipeline and examine what is needed at each stage (Figure 3-1). The drug discovery development pipeline starts with identifying a target for treatment, typically a key protein for which an inhibitor could be developed. Following this, a screen to assay fitness (e.g. protein inhibition) is developed, and large libraries of small molecules are tested. Molecules identified at this stage (lead fragments) are used as a core motif for further derivatization and optimization. When a molecule has been optimized and has met the features required for its function, it will move into clinical trials for approval by the FDA and other regulatory agencies.
Figure 3-1. Drug development pipeline. Target identification: determination of which biomolecule of interest will be targeted for the therapeutic effect. Compound screening and hit validation: screen small-molecule libraries and determine which have the desired therapeutic effect. Lead identification and optimization: lead fragments are derivatized and decorated to improve their therapeutic effect. Clinical trials: drug candidates are tested for their efficacy and safety to be approved by regulatory bodies (e.g. FDA).
The lead identification and optimization stage of drug discovery, in which lead fragments are diversified to improve their efficacy, is a highly attractive stage for the incorporation of biocatalysts. At this stage, a lead fragment might already require several synthetic steps to produce and generating decorated analogs requires either selective functionalization of the lead fragment or modifying the synthetic route to incorporate diverse functional groups.
Enzymes have been shown to excel at selective late-stage functionalization in natural product and pharmaceutical syntheses, installing functional groups with high selectivity.9,11 Developing a set of enzymes to selectively produce dozens or hundreds of decorated analogs of the lead fragment will take a long time – at a stage of drug development in which speed is a crucial factor. Instead, employing an enzyme to produce a single analog of the complex chiral lead fragment with high selectivity, which is readily derivatized by commonly used methods in diversity-oriented synthesis, would then allow for the use of standard medicinal chemistry diversification libraries and pipelines which have been developed and optimized over decades (Figure 3-2).12
Target identification
Compound screening, hit
validation
Lead identification,
optimization
Clinical trials
Figure 3-2. Expanding cyclopropane diversity through selective installation of derivatizable functional groups. (a) Enzymes can catalyze reactions with high specificity and selectivity, but often suffer from a narrow substrate scope. (b) Combinatorial chemistry approaches can be used to install diverse functional groups, but can lack the stereoselectivity control needed to generate the target compound. (c) Enzymes with high activity and selectivity for a single transformation can be coupled with combinatorial chemistry approaches to generate a diverse array of substituted cyclopropanes.
Combining traditional synthesis and enzymatic synthesis has been a cornerstone of the biocatalysis field for decades; these chemoenzymatic approaches use the specificity and selectivity of enzymes together with well-established synthetic steps, where the optimal catalyst for each step is used. In many cases, a key synthetic step which sets one or more stereocenters is performed enzymatically, preceded or followed by chemical synthetic steps.13–15 Coupling enzymes with combinatorial chemistry has precedent as well;
researchers have shown the use of chemoenzymatic approaches to diversify small molecules, with decarboxylases generating substrates for olefin metathesis,16 halogenases generating substrates for cross-coupling reactions,17,18 and carbene transferases installing nitrile moieties for conversion to a variety of functional groups.19
Substituted cyclopropanes are prevalent in pharmaceutical and agrochemical compounds.20–22 The ability to rapidly produce derivatives of a stereopure cyclopropane could be used in lead fragment optimization to assist future drug discovery and development efforts. Boronate ester moieties are ubiquitous in medicinal chemistry due to their robust activity in the presence of a wide range of functional groups and their efficacy in convergent synthesis of complex molecules.12 Therefore, I envisioned that an enzymatically produced pinacolboronate (Bpin)-substituted chiral cyclopropane could be used as a substrate for Suzuki-Miyaura cross-coupling reactions to generate a diverse array of substituted cyclopropanes. In light of our previous studies that showed that heme proteins can be engineered to generate the cyclopropane-containing pharmaceuticals,23-26 I chose to develop a chemoenzymatic strategy to produce a cyclopropane motif with a functional handle, which could then be derivatized to form substituted chiral cyclopropanes. To realize this approach, I set out to engineer heme proteins to catalyze the stereoselective cyclopropanation via carbene transfer of ethyl diazoacetate (EDA) and vinylboronic acid pinacol ester, generating 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-cyclopropanecarboxylic acid ethyl ester (Figure 3-3).
Figure 3-3. Proposed enzymatic reaction of vinylboronic acid pinacol ester (1) and ethyl diazoacetate (2) to form the cyclopropylboronate ester (3).