Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

Effective microbiological testing during biopharmaceutical drug development and manufacturing is crucial for ensuring sterility, determining antimicrobial effectiveness, detecting microbial contamination or bioburden levels, analyzing endotoxins, and implementing environmental monitoring programs.

	Growing pressures to increase productivity, flexibility, and cost-effectiveness and the unique properties of many next-generation therapies are challenging today’s microbiologists.

Articles

Pressures to accelerate current and next-gen therapies are challenging traditional microbiological testing models.

Microbiological Testing: Time is of the Essence

A small-molecule APIs become increasingly complex, designing concise, practical, and economical synthetic routes for their commercial production becomes increasingly challenging. There is, consequently, growing demand for cost-effective, highly efficient catalysts that mediate atom-economical, environmentally friendly transformations that can replace traditional, non-catalytic processes that require multiple steps. A summary of selected new catalysts that show promise for pharmaceutical intermediate and API synthesis is presented in the following.

	Silicon-containing compounds are of interest as potential drug candidates because silicon can impart greater stability and lead to compounds with improved solubilities and pharmacokinetic properties. There are two common routes to heteroarylsilanes: stoichiometric reaction of a silicon electrophile with a heteroaromatic organometallic intermediate (prepared via a Grignard reaction) or rhodium- or iridium-catalyzed C-H silylation in the presence of an excess of a hydrogen acceptor. Neither approach is practical on an industrial scale, and both suffer from limited functional group tolerance.

	Researchers in the Grubbs and Stoltz groups at the California Institute of Technology fortuitously discovered an attractive alternative method based on the inexpensive base potassium tert-butoxide (KOtBu) (1). When investigating a reaction to convert biomass to chemicals via the breakage of C-O bonds using an iron catalyst, KOtBu, and a hydrosilane as a hydride equivalent, they observed that silylated heteroaromatics were formed in minor quantities in a control reaction using only KOtBu and the hydrosilane. In addition, they noted that the yield of silylated products increased as the reaction temperature decreased.

	The cross-dehydrogenative heteroaromatic C-H silylation reaction with hydrosilanes was found to proceed best using a catalytic amount of KOtBu (1-20 mol%) in the absence of an acceptor, with only hydrogen gas as the byproduct. The fact that a common base catalyzes this important reaction was quite surprising, and thus the researchers went to great lengths to confirm that the KOtBu was indeed the catalyst and not some unknown impurity.

	The reaction is believed to proceed via a radical mechanism that is completely different from the mechanisms observed with transition-metal catalysts. Its scope is also very broad. Indoles with a variety of substituents on the nitrogen and various positions on the arene ring and many different electron-neutral and electron-rich N-, O-, and S-containing heterocyclic compounds are suitable substrates. Carbonyl groups are generally not tolerated except when protected as acetals. Some groups (bromide, iodide, cyano, and nitro substituents) do hinder the reaction. Interestingly, fluoride, chloride, trifluoromethyl, epoxide, N-alkyl aziridine, pyridine, and tertiary amine and phosphine groups do not.

	Notably, even simple arenes serve as good substrates. Substitution affects the regioselectivity; however, ortho-substitution occurs with anisole, while directing-group-free C(sp3)-H silylation leads to silylated benzyl derivatives with toluene and similar compounds. The researchers are also exploring the reaction of non-aromatic compounds (e.g., aliphatic compounds, alkenes, and alkynes).

	The scientists have also shown that the cross-dehydrogenative heteroaromatic C-H silylation reaction is scalable. When run neat using N-methyl indole and 1.5 equivalents of triethylsilane at 45 °C on a 100-gm scale, desired C2 silylated product was obtained in 76% yield with a greater than 20:1 regioselectivity after simple filtration and distillation.

	The researchers also demonstrated the utility of the reaction by silylating the antihistamine thenalidine and the antiplatelet drug ticlopidine in 58-68% yield with high chemo- and regioselectivity, indicating the applicability of the transformation for late-stage modification of pharmaceutically relevant compounds.

	Sites of unsaturation (alkenes in particular) are widely present in pharmaceutical compounds and their introduction can be achieved using a variety of methods, such as the elimination of alcohols, halides or other leaving groups, the reduction of acetylenes, and Wittig and Diels-Alder reactions. Dehydrogenation of alkanes is highly attractive because it allows for the flexible placement of alkenes. In addition, when performed under acceptor-less or terminal oxidant-free conditions, only hydrogen is generated as the byproduct. It is, however, a highly endothermic reaction traditionally mediated by expensive noble-metal catalysts at high temperature. Enzyme-based systems do proceed at room temperature, but require the use of a full equivalent of a terminal oxidant, and thus hydrogen recovery is not possible.

	Scientists at Princeton University have overcome these issues with the development of a reaction that is noble-metal-free, does not require the use of a terminal oxidant (and thus generates hydrogen), and proceeds at mild temperatures (2). Their approach is based on key aspects of the iron-based desaturase system, which catalyzes dehydrogenation reactions via step-wise hydrogen atom removal. The first hydrogen atom transfer (HAT) occurs from the alkane substrate to a high-valent oxo species, which is much more difficult than the second, which can occur via HAT or electron transfer followed by proton transfer.

	In their new approach, the researchers use cooperative catalysis with a dual-catalyst system to achieve the two steps. The polyoxometalate tetra-n-butylammonium decatungstate (TBADT) is easy to synthesize and has been shown to mediate HAT reactions of unactivated alkanes under near-UV light irradiation. For the second catalyst, the researchers were interested in cobaloximes because they are known to catalyze radical reactions including the reversible formation of alkenes and cobalt hydrides and have been shown to mediate water reduction under photo-irradiation conditions. Cobaloxime pyridine chloride (COPC) was selected because it is also readily synthesized.

	When used together (1.5 mol% TBADT and 0.75 mol% COPC) under irradiation at 323 nm, which is the absorbance maximum of TBADT, cyclooctane was converted to cyclooctene in 23% yield. The researchers also showed that cyclopentane was converted to cyclopentene and cyclopentadiene, while cyclohexane was dehydrogenated to cyclohexane rather than benzene, which commonly occurs under noble-metal catalysis.

	The reactivity of ethyl isovalerate was also interesting. Rather than produce the thermodynamically most stable product, which is generally the case with noble-metal catalysts, the dehydrogenation proceeded to form the skipped-enone product. The researchers are also exploring the tolerance for functional groups other than esters.

	While the initial results of the cooperative catalysis approach with simple alkanes are not as efficient as reactions with noble-metal catalysts, the researchers believe there is potential to improve the reaction. In fact, when secondary alcohols are used as substrates, the yields increase (i.e., 83% for 1-phenylethanol).

	Researchers at the Scripps Research Institute (TSRI) developed a catalyst that mediates C-H activation in the synthesis of heterocycles (3). Using an N-methoxy amide group as both a directing group and an anionic ligand, the PdX2 (X = ArCONOMe) catalyst is generated in situ from a Pd(0) source using air as the oxidant. With the PdX2 species localized near the target C-H bond, the natural preference of the metal to coordinate to the heteroatom is avoided. The catalyst has been used to synthesize a number of different compound types, including furans, benzofurans, benzothiophenes, indoles, pyrroles, thiazoles, pyrazoles, imidazoles, pyridines, quinolines, pyrazines, pyrimidines, pyrazoles, and thiazoles.

	Catalysts that mediate other types of C-H activation reactions have also been developed by TSRI researchers. In one case, sequential diarylation is achieved to afford non-natural β -Ar-β-Ar’-β-α-amino acids with diastereomeric ratios of greater than 20:1 (4). These compounds are building blocks for peptide drugs with structural diversity that may make them resistant to enzyme degradation. In the second reaction, palladium-catalyzed enantioselective C-H iodination is used to achieve the kinetic resolution of chiral amines (5). Notably, the reaction proceeds at room temperature and involves only inexpensive and commercially available reagents.

	Coupling of heteroatom-substituted olefins

	Carbon-carbon coupling reactions have become, in recent years, fundamental transformations for the commercial production of small-molecule APIs. Researchers at TSRI recently developed a new coupling reaction for the preparation of highly substituted, uniquely functionalized compounds catalyzed by an inexpensive iron complex (6). The reaction is also attractive because it is performed open to the air at room temperature.

	While typically run in ethanol, the researchers showed that reactions run in vodka, gin, whiskey, tequila, beer, and wine also afforded the desired coupling products. In addition, they demonstrated the broad scope of the reaction by synthesizing more than 60 different compounds, most of which were new chemical entities not previously reported.

, 518, 80-84 (2015).
	2. J. G. West, D. Huang, and E.J. Sorensen, 

, 515, 389-393 (2014).
	4. J-Q Yu, et al., 

, 343 (6176), 1216-1220 (2014).
	5. J-Q. Yu, et al., 

, 346 (6208), 451-455 (2014).
	6. P.S. Baran, et al., 

New catalysts show promise for pharmaceutical intermediate and API synthesis.

Cost-Effective Catalysts Could Replace Traditional Multi-Step Processes

Although pharmaceutical manufacturers prefer to formulate APIs in solid-dosage form, many drug candidates do not readily form stable crystalline solids. In addition, a growing percentage of APIs in development suffer from poor bioavailability and/or other undesirable properties. There are many approaches for addressing these issues, such as the formation of amorphous dispersions, salts, and cocrystals.

	Cocrystals are attractive because, like solvates and hydrates, they allow the modification of an API’s properties without altering its chemical structure. They also provide an option for APIs that lack ionic functionality and thus cannot form salts. The synthesis of cocrystals may also be advantageous from a cost standpoint (1).

	There has been, however, significant hesitation in the pharmaceutical industry regarding the use of cocrystals since the issuance of FDA’s initial guidance on cocrystals in April 2013 (2).

	In that guidance, FDA defined cocrystals as “drug product intermediates (DPIs)” rather than APIs. This decision raised questions about expiration dates, intellectual property protections, and the consistency of the US approach with those adopted by other regulatory bodies (1).

	A new draft guidance issued by FDA in August 2016 (3) redefines cocrystals as APIs. Classified as a special case of solvates and hydrates in which the coformer is nonvolatile, cocrystals are now to have a regulatory classification similar to that of polymorphs of APIs.

	Cocrystals have crystalline lattice structures that are different from the pure API and consequently often have different physical and chemical properties. The formation of cocrystals can reduce hygroscopicity and change the particle size and shape and melting point to help overcome compaction problems for improved manufacturability. The stability of an API can also be enhanced. As importantly, dissolution properties in the body can be significantly modified. In addition, because cocrystals are neutral combinations of APIs with conformers, they can be prepared for compounds without acid or base functionality, which is required for salt formation.

	The most common property targeted for improvement via cocrystal formation is an improved aqueous dissolution rate compared to an API free acid or base, according to G. Patrick Stahly, chief operating officer at Triclinic Labs. “Cocrystals are like amorphous materials in that they provide a supersaturated concentration of an API in the body for a time sufficient for absorption to occur (a supersaturating drug delivery system),” he explains. He adds that it needs to be realized, however, that a cocrystal typically does not affect the equilibrium solubility of an API; cocrystals usually dissociate in solution because they are held together by relatively weak chemical bonds.

	Cocrystals also may offer benefits from an intellectual property perspective, according to Stahly. “At the current level of the science, it is not possible to predict cocrystal properties based on the molecular structures of the components. Solid-form screening, including cocrystal screening, is an empirical process. Since the formation of cocrystals is currently unpredictable, cocrystals that are discovered and found to have advantageous properties can be patented,” he notes.

	It should be recognized, however, that cocrystals are still not regarded as new APIs. “Drug products that are designed to contain a new cocrystal are considered analogous to a new polymorph of the API,” according to the 2016 FDA draft guidance.

	The redefinition of cocrystals as APIs is important for several reasons, according to Stahly. First, it allows the use of existing FDA guidance to provide information necessary for development. Second, existing manufacturing facilities and supply chains that make and distribute APIs can be used, and there is no need to add cocrystal manufacturing capabilities to existing drug-product manufacturing facilities. Third, API expiry dating begins at the time of cocrystal manufacture. Fourth, there is now consistency among FDA guidance, international guidance, and scientific reality.

	“After the first FDA guidance was issued, there was confusion within the pharmaceutical industry about the viability of cocrystals. My personal experiences led me to believe that scientists (myself included) were unhappy because they felt the guidance was wrong from a scientific standpoint. I believe the guidance presented unnecessary obstacles to development of cocrystals, which would discourage some companies from utilizing them. Those dealing with the facilities and regulatory issues seemed to be more pessimistic. There was talk that cocrystals were ‘dead’ in the pharmaceutical industry,” comments Stahly.

	Triclinic Labs did cocrystal screening and other development activities for clients in the period between issuance of the old and new guidance, indicating that not everyone felt that the original guidance eliminated the use of cocrystals.

	With the new guidance, however, cocrystals were, if ‘dead’ before, now ‘reborn’, according to Stahly. “The development pathway is clear and cocrystals should now be considered another tool in the box of tools used to improve solid-state properties,” he says.

	An issue addressed by the new FDA guidance is the nature of the interaction of components in a cocrystal where one component is acidic and the second component is basic. The drug manufacturer must demonstrate that there are no ionic interactions in such a cocrystal, which can be done either using the acid dissociation constants of the API and the coformer or via spectroscopic analysis.

	It is important, according to Stahly, to realize that there are various analytical techniques that can be used to probe the extent of proton transfer. “A common method is single-crystal x-ray structure determination, which is an excellent technique only if used properly. It is easy for a novice to believe protons have been actually located when often they are simply left to ride on the atom to which they are bonded; the protons are in assumed instead of refined positions,” he notes. A single-crystal study is not always necessary, however, and other techniques such as infrared (IR) spectroscopy can often be used.

	What drove the agency to make this significant change to its guidance on cocrystals? Stahly believes the strong response from industry was the major driving force. In 2012 at an Indo-US Bilateral Meeting held from Feb. 2-4, 2012, 46 attendees co-authored a publication presenting their consensus “… on the need to define cocrystals more broadly and to classify them like salts” and their recommendation “… that the classification of cocrystals in pharmaceutical science should be consistent with current scientific thought” (4).

	Also in 2012, a working group of the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ Consortium) submitted a comment paper to FDA recommending that “FDA considers classification of co-crystals within existing guidance[s], which provides discrete, industry understood points where quality attributes are measured and controlled to ensure product quality and patient safety (i.e., the drug substance and drug product)” (5).

	“The primary messages from those groups were that the original guidance was inconsistent with both current scientific understanding of the nature of cocrystals and existing FDA guidance relating to drug development. In addition, the guidance would lead to negative regulatory and manufacturing consequences and be inconsistent with the European Medicines Agency provisional guidance outlining submission requirements for cocrystals. It appears that FDA seriously considered industry’s comments and revised the guidance,” Stahly observes.

Guidance for Industry: Regulatory Classification of Pharmaceutical Co-crystals

 (Silver Spring, MD, April 2013).
	3. FDA, 

Draft Guidance for Industry: Regulatory Classification of Pharmaceutical Co-crystals

 (Silver Spring, MD, Aug. 2016).
	4. S. Aitipamula, et al., 

.12, 2147-2152 (2012).
	5. IQ Consortium, “IQ Comments on FDA Draft Guidance Regulatory Classification of Pharmaceutical Co-Crystals,” March 1, 2012.

	When referring to this article, please cite it as C. Challener, “’Rebirth’ of Cocrystals?" 

FDA redefines cocrystals as APIs and not drug-product intermediates.

“Rebirth” of Cocrystals?

The incorporation of fluorine into APIs continues to be a key approach to improving important properties of candidate drug substances. While there are numerous methods for introducing trifluoromethyl groups, effective routes for selectively incorporating a single fluorine atom at a chiral center remain limited. Expensive, specialized fluorinating reagents and/or precious metal catalysts are often required to achieve the latter transformations. Researchers at GlaxoSmithKline (GSK) reported a simpler, cost-effective method for the preparation of fluorolactams via copper-catalyzed selective, direct fluorination using fluorine (F2) gas. This article summarizes the GSK research efforts (1).

	Although incorporation of fluorine atoms into APIs is known to increase their lipophilicity and impart other beneficial pharmacokinetic properties, there remain few commercial drugs with single fluorine atoms introduced at chiral quaternary carbons due to the difficulty in achieving this chemistry at scale. Numerous methods have been introduced in recent years for the enantioselective synthesis of these types of compounds, but most suffer from high cost.

	Specialized reagents based on Selectfluor (chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate), transition-metal catalyzed reactions, and routes mediated by organocatalysts have all been reported. Unfortunately, all of these methods rely on reagents and/or ligands that often require multiple steps to synthesize and are typically too expensive to allow their use at commercial scale. In addition, most of these processes result in the production of large volumes of waste, which further reduces their practicality for large-scale manufacturing.

	As part of its environmental strategy, GSK aims to make its operations carbon neutral by 2050 (2). The company uses methods based on both qualitative and quantitative criteria to evaluate the atom economy and level of waste generation for proposed synthetic strategies. The obtained process mass intensity (PMI) values for different routes are compared to identify the most effective synthetic strategy.

	Scaling up a chiral fluorolactam building block

	Recently GSK researchers needed to scale up a process for the synthesis of chiral fluorolactam building blocks (enantiopure 2-fluoro-1,3-amidoesters) for the preparation of a series of spleen tyrosine kinase inhibitors as pre-clinical candidates. Three possible existing methods were considered: enantioselective fluorination of malonate esters using N-fluorobenzenesulfonimide (NFSI) and a Zn(OAc)

/DBFOX-Ph (4,6-dibenzofurandiyl-2,2’-bis(4-phenyloxazoline)) catalyst followed by formation of the amide, chiral alkylation of fluoromalonate derivatives followed by formation of the amide, or palladium-catalyzed fluorination using NFSI.

	Initially, the palladium-catalyzed route was selected for production of 100 g of the first chiral fluorolactam. The researchers found, however, that preparation of the catalyst involved a complex, multistep synthesis with low selectivity at large scale, leading to the need for extensive purification. A green metric analysis of the route identified issues with the enantioselective fluorination step due to the fact that NFSI is prepared using fluorine gas and the loss of material resulting from the low selectivity during catalyst preparation.

	As a second attempt, the GSK researchers considered the use of a fluoromalonate derivative. In this approach, a fluoromalonate ester would be converted to the desired fluorolactams via simple chemical transformations, which would then be subjected to enzyme-catalyzed resolution.

	The desired fluoromalonate was prepared via direct fluorination of the corresponding dimethyl malonate ester using fluorine gas. The reaction was catalyzed by copper nitrate and performed in acetonitrile, affording the desired product in high yield, even at the 50-g scale. Notably, the mass intensity was quite low.

	This fluoromalonate was then converted to the racemic fluorolactam derivative in three steps: Michael addition of acrylonitrile to form the nitrile, hydrogenation of the nitrile over palladium, and base-catalyzed ring closure. Hydrolase-catalyzed resolution of the nitrile, salt, and fluorolactam was then attempted.

	Ultimately after screening numerous enzymes, the GSK researchers found an effective method for the hydrolase-promoted amidation of the reduced salt to the fluorolactam in phosphate buffer with high enantioselectivity. The inexpensive enzyme CAL-B 10 000, a recombinant Candida Antartica Lipase B that is commercially available in multi-kilogram and tonne quantities, was selected. The enzyme was shown to be quantitatively recoverable and reusable three times without loss of activity.

	The researchers then further optimized each step in the conversion of the fluoromalonate ester to the reduced salt. The resulting three-step synthesis of the fluorolactam was found to have a PMI of 201, which was more than four times lower than that of the initial route based on palladium-catalyzed fluorination using NFSI (925).

	The optimized alternative route based on direct fluorination using fluorine gas was found to be highly competitive from a PMI perspective compared to the approach based on methods previously reported in the literature. A large percentage of the PMI value for the optimized synthesis is due to the final resolution step, which leads to loss of 50% of the product (as is the case in all resolutions).

	Despite this loss, the cost of the fluorination-amidase process was also found to be significantly lower than that for syntheses based on specialized (and expensive) electrophilic fluorinating agents prepared from fluorine gas and palladium catalysis. The researchers attributed this result to the facile recycling of the enzyme catalyst and solvents and the use of an inexpensive copper catalyst for the fluorination step. They also noted that the low cost and straightforward synthesis make this strategy suitable for scale up.

. 18, 1313-1318 (2016).
	2. GSK, Green Chemistry, www.gsk.com/en-gb/behind-the-science/how-we-do-business/green-chemistry/ accessed Aug. 12, 2016.

A simpler, cost-effective method for the preparation of fluorolactams may be suitable for scale-up.

Researchers Identify a Greener Fluorination Reaction

	When referring to this article, please cite it as C. Challener, "The Search for Practical and Economical Catalysts,” 

Researchers develop catalysts that mediate complex transformations under conditions appropriate for commercial manufacture.

The Search for Practical and Economical Catalysts

Pressures on drug manufacturers to reduce development timelines and costs while also providing safer and more effective medications are driving the pharmaceutical industry to seek more effective, alternative manufacturing solutions. In addition to continuous processes, both branded drug companies and contract development and manufacturing organizations are seeking greener routes to the production of complex APIs--routes that consume fewer resources and produce less waste. Replacement of more traditional reactions mediated by transition-metal catalysts with those facilitated by enzymes, or biocatalysts, is one option receiving growing attention. Jim Lalonde, senior vice-president of R&D with enzyme engineering company Codexis, shares his perspective on the benefits of biocatalysis for API synthesis.

: Why is biocatalysis considered to be greener than chemo (transition metal) catalysis?

: Biocatalysis is considered to be greener than transition metal catalysis in some key regards. Using the comparison of Merck’s second-generation process for sitagliptin (Rh/Josiphos asymmetric hydrogenation) with its third-generation biocatalytic transaminase process is illustrative, as it meets the green chemistry principles of safety, atom economy, waste prevention, and energy efficiency. Firstly, we eliminated the use of rhodium, which is considered toxic and had to be removed with an absorbent (which also generated a solid waste). Secondly, we eliminated the need for high-pressure, high-temperature hydrogenation and replaced it with a process that runs at ambient pressure and near ambient temperature. This change makes the biocatalytic process inherently safer, less energy-consumptive, and less expensive. The hydrogenation had to be performed in an expensive, custom high-pressure reaction vessel, while the biocatalytic transformation is conducted in standard reactors. The use of the transaminase catalyst also eliminated several synthetic steps, gave a higher overall yield, reduced the solvent demand, and resulted in a high purity product. The latter is often a feature of biocatalytic processes with engineered enzymes, as we can ‘evolve’ the enzyme until we have one that meets or exceeds product purity requirements. This high degree of customization is not typically possible with transition metal catalysts.

: What are the key changes that have led to growing interest in the use of biocatalysis in the pharmaceutical industry?

: The key advancement that has led to a large increase in the application of biocatalysis in the pharmaceutical industry has been the availability of enzyme engineering technologies. Previously, enzyme development took too long to allow the wide application of biocatalysts in commercial manufacturing, except for later-generation processes.

	Advances in genetics (genomic databases and next-gen sequencing), high-throughput biochemistry and robotics, data analysis, and computational modeling have enabled the rapid development of novel, customized biocatalysts that are optimized for API manufacture with improved efficiencies and reduced costs.

: When in the drug development cycle have biocatalysts most likely been used and why? Is this changing? If so, what is driving the change?

: Biocatalysts are used throughout the drug development process, from discovery through clinical Phases I, II, and III, to commercialization and even for generic drugs. The biggest change, however, is that biocatalysis is now becoming a first approach, rather than one of last resort. GlaxoSmithKline and Merck, through licensing our technology, now have the ability to rapidly demonstrate proof of concept with custom biocatalysts during the development of first scale-up routes to produce kilogram quantities. This milestone typically occurs in Phase I or II. With the in-house capability to then optimize an enzyme in months, they can meet their clinical development demands with enzyme-based routes. The second major change, which is just beginning to take hold, is that enzymes can enable the rapid synthesis of complex drug molecules that are not easily made using traditional organic chemistry. Consequently, many more candidates can be synthesized early in discovery programs.

: What are the most recent enzymes introduced to the market for API synthesis on a large scale? What might be expected in the next several years?

: Examples of new enzymes recently made available for API manufacture include an acylase specific for simvastatin manufacture that can regiospecifically acylate monocolin J, eliminating the need for protection and deprotection steps; imine reductases that reductively couple ketones with amines for the convergent synthesis of secondary and tertiary chiral amine drug targets; large libraries of transaminases and ketone reductases capable of enantiospecific synthesis of primary amines and secondary alcohols; and oxidases for the manufacture of asymmetric sulfoxides like esomeprazole and armodafinil.

	Of these transformations, currently there are only conventional catalysts for asymmetric oxidation, reduction of ketones, and asymmetric reductive amination. The enzymes are superior to conventional catalysts in that an enzyme with the targeted enantioselectivity can be developed for almost any substrate and is tolerant for virtually all functionality in the molecule.

	In development, there are enzymes for direct hydroxylation of unactivated C-H bonds, enzymes for direct synthesis of amides (no need for protection, activation, or deprotection steps), enzymes for regiospecific glycosylation of hydroxyl groups, and enzymes for the site-specific conjugation of proteins. There are no conventional transition metal catalysts for any of these transformations.

: What are the key advances in protein engineering and related technologies that have had the greatest impact on biocatalyst development in recent years? What might be expected in the next several years?
	

: Current engineering advances are such that wild-type enzymes are no longer used, and virtually all biocatalysts are engineered. Computational advances, metagenomics, gene synthesis, and sequencing advances have had the largest impact. Computational advances include molecular modeling in which docking of a target substrate into enzyme models can be performed 

 with sufficient accuracy to dramatically accelerate development of suitable biocatalysts. Data mining and machine learning algorithms that can correlate the performance data for a biocatalyst with the structure of the enzyme also guide the design of more optimal enzymes.

	Genomic databases, which contain sequence information on a vast and growing number of organisms, provide access to diverse enzyme starting points for engineering programs. The cost to synthesize and sequence genes has dropped exponentially, so we now typically sequence thousands of variants per week per development program. This information is used in the data mining algorithms. The combined effect of all of these advances is such that an enzyme development program that took several man-years a decade ago now can happen in a few months.

	Current molecular models of enzymes are static and based on X-ray crystallography, which is only a snap shot of a dynamic structure. As computational models improve with increased computational speed, we can expect to develop predictive models for protein structure that do not require crystallographic structure determination and molecular dynamics that will better model the kinetics of catalysis. Using these predictive models, one should need to only make and test a small number of variants to come up with a commercial biocatalyst. We should also be able to expand the types of enzymes to ones that catalyze non-natural transformations that are desirable to organic chemists.

: What about whole cell production of pharmaceutical intermediates and APIs? What is the state of this technology?

: Whole cell production of pharmaceuticals has been established for decades for the manufacture of natural product drugs (e.g., antibiotics such as streptomycin and the immunosuppressant Tacrolimus). Semisynthetics, or drugs that are made from fermentation followed by chemical modification, often have enhanced efficacy, however. Recently, routes to natural and semisynthetic drugs that are rare in nature (e.g., artemisinin from the sweet wormwood plant and opiods from poppies) have been produced using synthetic biology; the complete biosynthetic pathway for these drugs has been installed in an easily fermentable yeast, vastly lowering the cost of production and simplifying the supply chain for these valuable drugs. In addition, we can now create enzymes that act on non-natural molecules and even ones that catalyze non-natural reactions, which will allow the direct fermentation of a much wider range of drug molecules.

: Is there anything else about biocatalysis as a green alternative that is worth noting?
	

: We continue to be surprised by the breadth of chemistries possible using biocatalysis. Pharma companies estimate that anywhere from 25-75% of their pipeline can benefit from biocatalytic methods. In the area of drug discovery, there is a trend toward molecules of higher complexity with higher specificity and thus both lower dosage requirements and fewer side effects. Enzymes that have been designed for selective transformations allow the rapid creation of libraries of these complex molecules.

Drug companies and CDMOs seeking greener routes to the production of complex APIs may look to biocatalysis.