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\DOI{10.5802/crchim.408}
\datereceived{2024-12-12}
\daterevised{2025-04-01}
\dateaccepted{2025-05-23}
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\dateposted{2025-07-18}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}


\title{Practical examples of biocatalysis in industry}

\alttitle{Exemples pratiques de biocatalyse dans l'industrie}
         
\author{\firstname{Amelia K.} \lastname{Gilio}\CDRorcid{0000-0003-2834-035X}}
\address{Pfizer Global Research and Development, Groton, CT, 06340, USA}

\author{\firstname{Miguel A.} \lastname{Abdo}}
\addressSameAs{1}{Pfizer Global Research and Development, Groton, CT, 06340, USA}

\author{\firstname{Carlos A.} \lastname{Martinez}}
\addressSameAs{1}{Pfizer Global Research and Development, Groton, CT, 06340, USA}

\author{\firstname{Andrew T.} \lastname{Palaia}}
\addressSameAs{1}{Pfizer Global Research and Development, Groton, CT, 06340, USA}

\author{\firstname{Jovan}\nobreakauthor\lastname{Livada}\IsCorresp}
\addressSameAs{1}{Pfizer Global Research and Development, Groton, CT, 06340, USA}
\email[J. Livada]{jovan.livada@pfizer.com}

\shortrunauthors

\keywords{\kwd{Enzyme}
\kwd{Biocatalysis}
\kwd{Hydrolase}
\kwd{Ketoreductase}
\kwd{Imine-reductase}
\kwd{Ene-reductase}
\kwd{Transaminase}}

\altkeywords{\kwd{Enzyme}
\kwd{Biocatalyse}
\kwd{Hydrolase}
\kwd{C\'{e}to-r\'{e}ductase}
\kwd{Imine-r\'{e}ductase}
\kwd{Ene-r\'{e}ductase}
\kwd{Transaminase}}

\begin{abstract}
Biocatalysis has been used
in industrial settings, especially in the
pharmaceutical industry, for decades. In this review, we showcase the
six most prominent enzyme classes in industrial biocatalysis:
keto-reductases, ene-reductases, imine-reductases, transaminases,
oxygenases, and hydrolases. For each class, several examples are
presented that highlight the substrate scope and enantioselectivity of
these biotransformations from milligram to multikilogram scale.
\end{abstract}

\begin{altabstract}
La biocatalyse est utilis\'{e}e depuis des d\'{e}cennies dans les
milieux industriels, en particulier dans l'industrie pharmaceutique.
Dans cette revue, nous pr\'{e}sentons les six classes d'enzymes les
plus importantes en biocatalyse industrielle : les
c\'{e}to-r\'{e}ductases, les ene-r\'{e}ductases, les
imine-r\'{e}ductases, les transaminases, les oxyg\'{e}nases et les
hydrolases. Pour chaque classe, nous pr\'{e}sentons plusieurs exemples
qui mettent en \'{e}vidence l'\'{e}tendue des substrats et
l'\'{e}nantios\'{e}lectivit\'{e} de ces biotransformations, de
l'\'{e}chelle du milligramme \`{a} celle du kilogramme.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

Biocatalytic reactions are characterized by exceptional chemo-, regio-,
and enantioselectivity, 
making biocatalysis a key enabling technology
in asymmetric synthesis. In this review, we highlight the most
prominent enzyme classes, and more importantly, dozens of successfully
implemented examples. We provide relevant conditions for successful
biocatalytic reaction execution, including buffers, pH, temperature,
and cofactor recycling systems when applicable. A significant number of
enzymes are readily accessible through commercial suppliers or through
preparation via fermentation of recombinant microorganisms. Crude
lyophilized enzyme formulations have facilitated widespread
distribution of biocatalysts, while genetic engineering has advanced
contemporary enzymatic processes by improving reaction selectivity and
productivity. These processes are performed under a diverse array of
conditions and in many cases at concentrations comparable to those
achieved in traditional synthetic chemistry methods~\cite{1}.

\section{Ketoreductases (KREDs)}\label{sec2}

Enzymatic reduction of ketones and aldehydes are well-established
methods for the synthesis of chiral alcohols. These transformations are
catalyzed by a diverse array of enzyme families, including alcohol
dehydrogenases (ADHs) and aldo-keto reductases (AKRs), collectively
referred to as ketoreductases (KREDs) or carbonyl reductases (CREDs).
In their active sites, these enzymes bind NAD(P)H, which delivers a
hydride to the carbonyl of interest, producing the chiral
alcohol~\cite{2}.  

Most examples utilize a second enzyme for NAD(P)H cofactor recycling.
Others employ a single enzyme for both ketone reduction and cosubstrate
oxidation of propan-2-ol to acetone~\cite{3}. Generally, KREDs exhibit
broad substrate specificity, although the reduction of ketones with
bulky substituents is lacking. For instance, an intermediate for an
investigational $\upgamma$-secretase inhibitor for Alzheimer's
Disease treatment (Scheme~\ref{sch1}), 1,1-dimethylethyl
(${R}$)-2-hydroxypentanoate \textbf{2}, was synthesized via enzymatic
reduction of a ketoester~\cite{4}. The reaction, conducted with 35~kg
of ketoester in phosphate buffer and glycerol, utilized low enzyme
loadings of ADH-108L and glucose dehydrogenase (GDH), with efficient
NAD$^{+}$ recycling by GDH and glucose.

\begin{scheme*}
\vspace*{5pt}
\includegraphics{sc01}
\vspace*{8pt}
\caption{\label{sch1}KRED-catalyzed reduction toward a
$\upgamma$-secretase inhibitor.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc02}
\vspace*{5pt}
\caption{\label{sch2}KRED-catalyzed reduction toward montelukast.}
\vspace*{-6pt}
\end{scheme*}

\begin{scheme*}
\vspace*{2pt}
\includegraphics{sc03}
\vspace*{5pt}
\caption{\label{sch3}KRED-catalyzed reduction toward
($S$)-licarbazepine.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc04}
\vspace*{4pt}
\caption{\label{sch4}KRED-catalyzed reduction toward ticagrelor.}
\vspace*{-5pt}
\end{scheme*}

Another example employed an engineered KRED, CDX-026, in the synthesis
of an intermediate for montelukast, the active ingredient in
Singulair{\textregistered} from Merck (Scheme~\ref{sch2})~\cite{1}. The
reaction, performed on 230~kg of ketone \textbf{3} in a mixture of
propan-2-ol, toluene, and triethanolamine buffer, achieved a 97\%
yield. Crystallization of the alcohol as monohydrate drove the reaction
to high conversion without acetone removal, with CDX-026 recycling
NADP$^{+}$ by oxidizing propan-2-ol.


Additionally, an engineered KRED facilitated the scalable synthesis of
(${S}$)-licarbazepine, an anticonvulsant~\cite{5}. After four rounds of
engineering and a design of experiments (DoE) made up of 32
experiments, the process was conducted on a 500 mL scale, 50 g of
ketone, and 1\% w/w of KRED CDX-021. This produced
(${S}$)-licarbazepine \textbf{6} in 96\% yield after distillation and
filtration (Scheme~\ref{sch3}). KRED CDX-021 recycled NADP$^{+}$ by
oxidizing propan-2-ol, with a nitrogen sweep removing acetone to drive
the reaction forward.

\begin{scheme*}
\vspace*{-1pt}
\includegraphics{sc05}
\vspace*{4pt}
\caption{\label{sch5}KRED-catalyzed reduction that produces
dehydroepiandrosterone.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc06}
\vspace*{2pt}
\caption{\label{sch6}KRED-catalyzed dynamic kinetic resolution towards
a GPR40 partial agonist.
\vspace*{-4pt}}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc07}
\vspace*{6pt}
\caption{\label{sch7}KRED-catalyzed reduction toward ipatasertib.}
\vspace*{-6pt}
\end{scheme*}

A wild-type KRED from \textit{Leifsonia} sp.\ S749 was used to
synthesize (${S}$)-2-chloro-1-(3,4-difluorophenyl)ethanol \textbf{8},
an intermediate for {ticagrelor}, a treatment for acute coronary
{syndromes}~\cite{6}. The enzyme, overexpressed in recombinant
\textit{E.~coli}, was used in concentrated conditions with propan-2-ol
and phosphate buffer, recycling NAD$^{+}$ by oxidizing isopropanol
(Scheme~\ref{sch4}). The reaction was optimized to substrate loadings
of 500~g/L and resulted in space time yields of 145.8~mmol/L/h. The
same enzyme also converted a different substrate,
(${R}$)-1-(3,5-bis(trifluoromethyl)-phenyl)ethanol, also at 500~g/L
substrate loading with high yield and high  stereoselectivity.

\begin{scheme*}
\vspace*{3pt}
\includegraphics{sc08}
\vspace*{7pt}
\caption{\label{sch8}KRED-catalyzed reduction toward navoximod.}
\vspace*{-6pt}
\end{scheme*}

An example of a bulky substrate involved a wild-type KRED from
\textit{Sphingomonas wittichii} for the reduction of
5-androstene-3,17-dione \textbf{9} to dehydroepiandrosterone (DHEA)
\textbf{10}, a precursor for steroidal drugs
(Scheme~\ref{sch5})~\cite{7}. This KRED was identified by a
colorimetric screen in the reverse oxidative direction, to streamline
the identification of enzymes which acted on the desired regio- and
stereoisomer. The resulting highly stereo- and regioselective reaction
produced DHEA with minimal by-products and up to 150~g/L substrate
loadings under biphasic reaction conditions.

Merck reported a KRED process that involved a dynamic kinetic
resolution for the synthesis of an intermediate for a GPR40 partial
agonist~\cite{8}. Following assessments of the kinetic and free energy
profiles of the KRED reaction, and by performing six rounds of enzyme
engineering, KRED-264 provided the desired \textit{trans} diastereomer
in high yield and diastereomeric ratio (Scheme~\ref{sch6}).


Diastereoselectivity was also a beneficial characteristic of the KRED
employed in Roche's \mbox{synthesis} of investigational cancer therapeutic
ipatasertib. The process offered higher yields and diastereomeric
ratios than those obtained by the ruthenium- or palladium-catalyzed
processes explored in parallel. A commercially available KRED was
therefore applied to this route and achieved highly diastereoselective
reduction and NADPH regeneration via propan-2-ol oxidation. The overall
chemoenzymatic synthesis of ipatasertib has been performed on a
hundred-kilogram scale (Scheme~\ref{sch7})~\cite{9}.

Another report from Roche detailed the synthesis of an intermediate for
the indoleamine 2,3-dioxygenase inhibitor navoximod. Both ketone
moieties of the diketone intermediate were initially targeted for
biocatalytic reductions with a screen of 500 KREDs performed; however,
a metal hydride reduction was ultimately favored for the central
ketone. Their KRED panel was successfully screened resulting in a
process scaled up to 50~g scale, achieving high yield and selectivity
with low enzyme loading (Scheme~\ref{sch8})~\cite{10}.



\section{Ene-reductases (EREDs)}\label{sec3}

Ene-reductases (EREDs), a subset of the oxidoreductase enzyme family,
are known for their ability to facilitate the asymmetric reduction of
electron-deficient alkenes~\cite{11}. Distinct from KREDs and
imine-reductases (IREDs), which directly utilize NAD(P)H, EREDs operate
through a unique mechanism involving hydride transfer from a
non-covalently associated, reduced flavin mononucleotide (FMNH$_{2}$)
cofactor to the substrate alkene~\cite{12,13}. The subsequent reduction
process of FMN is mediated by NAD(P)H~\cite{14}.


The classification of ene-reductases encompasses five distinct classes,
with the Old Yellow Enzyme representing the most extensively researched
category~\cite{15}. The initial demonstration of ene-reductase activity
dates back to 1932, as documented by Warburg and Christian~\cite{16}. It
was not until 1995 that EREDs were employed in the synthesis of chiral
intermediates. After this pivotal development, numerous instances of
ERED applications in biocatalysis have been documented.

A recent scholarly article detailed the characterization of a novel
ERED derived from \textit{Pichia angusta} (\textit{Pa}ER), which
exhibited remarkable stereoselectivity in the reduction of
ketoisophorone \textbf{17} to (${R}$)-levodione \textbf{18} as
depicted in Scheme~\ref{sch9}~\cite{17}. The reaction was conducted with
an impressive substrate concentration of 154~g/L, utilizing whole
\textit{E.~coli} cells that housed both the ERED and the GDH cofactor
recycling enzyme.

\begin{scheme*}
\vspace*{4pt}
\includegraphics{sc09}
\vspace*{6pt}
\caption{\label{sch9}Preparative-scale reduction of ketoisophorone.}
\end{scheme*}

Another example characterized by almost perfect stereoselectivity
involves an ERED isolated from~\textit{Lactobacillus casei}
(\textit{Lac}ER). This ene-reductase was used to catalyze the reduction
of (5${R}$)-carvone \textbf{19}, yielding (2${R}$,
5${R}$)-dihydrocarvone \textbf{20} as illustrated in
Scheme~\ref{sch10}~\cite{18}. The enzyme facilitated the production of
this compound with a diastereomeric excess (de) of 99\%. Additionally,
this ERED demonstrated versatility by functioning in a one-pot \mbox{cascade}
\mbox{reaction} alongside a KRED, culminating in the synthesis of
(1${S}$, 2${R}$, 5${R}$)-dihydrocarveol.

\begin{scheme*}
\includegraphics{sc10}
\vspace*{6pt}
\caption{\label{sch10}Analytical-scale reduction of (5$R$)-carvone.}
\end{scheme*}

In instances where the catalytic characteristics of ERED catalysts are
deemed suboptimal, enzyme-engineering techniques can be harnessed to
enhance the enzymatic properties. A 2015 study demonstrated that enzyme
engineering could modify the enantioselectivity of OYE 1 from
\textit{S.~pastorianus} through the introduction of a singular W116V
mutation~\cite{19}. In a separate investigation, a consensus mutagenesis
approach was applied to the 12-oxophytodienoate reductase from
\textit{L.~esculentum} (tomato) applied to cyclohex-2-en-1-one-based
substrates, resulting in an improved enantioselectivity exceeding 99\%
ee~\cite{20}. Furthermore, a rational design strategy was employed to
truncate the flexible loops of the nicotinamide-dependent cyclohexenone
reductase (NCR), thereby augmenting both the thermal stability and
solvent resistance of the ERED~\cite{21}.

The NCR ERED has also been documented to function effectively at
elevated substrate concentrations. For instance, a study focusing on
the reduction of 1-(cyclohex-1-en-1-yl)ethan-1-one \textbf{21} to
1-cyclohexylethan-1-one \textbf{22} reported a substrate loading
capacity of 100~g/L, achieving a 99\% conversion over a span of 110~h
(Scheme~\ref{sch11})~\cite{22}.

\begin{scheme*}
\includegraphics{sc11}
\vspace*{6pt}
\caption{\label{sch11}Reduction of 1-(cyclohex-1-en-1-yl)ethanone via NCR.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc12}
\vspace*{6pt}
\caption{\label{sch12}Analytical-scale reduction of a potential
intermediate in pregabalin synthesis.}
\vspace*{-4pt}
\end{scheme*}

EREDs have been employed in the synthesis of pharmaceutical
intermediates, notably in the \mbox{production} of pregabalin, a medication
with anticonvulsant, analgesic, and anxiolytic properties for treating
neurological conditions. Ethyl (${E}$)-3-cyano-5-methylhex-2-enoate
\textbf{23} underwent reduction by 12-oxophytodienoate reductase (OPR1)
to yield ethyl (${S}$)-3-cyano-5-methylhexanoate \textbf{24}, as
depicted in Scheme~\ref{sch12}~\cite{23}.

\begin{scheme*}
\includegraphics{sc13}
\vspace*{5pt}
\caption{\label{sch13}Analytical-scale reduction of a potential
intermediate in tesaglitazar synthesis.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc14}
\vspace*{5pt}
\caption{\label{sch14}Demonstrated ERED reduction of a carboxylic acid substrate.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc15}
\vspace*{5pt}
\caption{\label{sch15}ERED reduction of C${=}$N bonds in oximes.}
\vspace*{-5pt}
\end{scheme*}

EREDs have also been utilized in the synthesis of intermediates for
pharmaceuticals, exemplified by tesaglitazar, a drug under
investigation for the treatment of type 2 diabetes~\cite{24}. The
specific \mbox{reaction} involves the reduction of
(\textit{Z})-2-ethoxy-3-(4-methoxyphenyl)acrylaldehyde \textbf{25} to
(${S}$)-2-ethoxy-3-(4-methoxyphenyl)propanoic acid \textbf{26},
executed with high efficiency and selectivity (100\% conversion; 98\%
ee), employing Old Yellow Enzyme 3 (OYE3) as the biocatalyst
(Scheme~\ref{sch13}).


Ene-reductases have also been shown to catalyze reactions that are not
limited only to activated olefins. Two recent reports have shown the
expansion of the ERED substrate scope. The first report demonstrated
that EREDs can reduce several \mbox{carboxylic} acid substrates~\cite{25}, of
which the most successful example involved the stereoselective
reduction of 2-benzylacrylic acid \textbf{27} (Scheme~\ref{sch14}). 


The second report showed a novel feature of EREDs, the reduction of
C${=}$N in oximes (Scheme~\ref{sch15})~\cite{26}. Several enzymes
exhibited this activity with the most notable members being OYEs 1, 2
and 3. In addition, for seven out of the eight oxime substrates tested,
EREDs have demonstrated catalysis characterized by an isolated yield
greater than 50\%, at 10~mM substrate loading.


Therefore, in recent years, the type of activities that EREDs exhibit
have been significantly expanded. We do not see this trend slowing down
soon. 

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc16}
\vspace*{4pt}
\caption{\label{sch16}Telescoped condensation and reduction toward the
synthesis of  ($S$,$S$)-sertraline.}
\vspace*{-6pt}
\end{scheme*}

\section{Imine-reductases (IREDs)}\label{sec4}

Imine reductases (IREDs) are a class of enzymes commonly used for the
formation of secondary and tertiary amines, and less frequently primary
amines. As the name suggests, these enzymes catalyze the \mbox{reduction} of
imine C${=}$N bonds with a NADPH cofactor. This activity was first shown
by Nagasawa and coworkers in 2009 with two enzymes from strains of
\textit{Streptomyces} reducing 2-methyl-1-pyrroline to (${R}$)- or
(${S}$)-2-methylpyrrolidine~\cite{27}. Further research expanded the
panel of known IREDs and showed that these enzymes work well with a
range of ketones and imines including cyclic and acyclic, 
\mbox{aromatic,} and
aliphatic ketones~\cite{28}. For the amine {partner}, IREDs prefer smaller
amines such as methyl, allyl, and cyclopropyl amines. Couplings with
ammonia, however, can be difficult. Regardless, larger amines like
benzylamine and isoindoline~\cite{29} have been reported to be accepted.
A recent report even showed a successful coupling of an aldehyde with
${\upalpha}$-(1-aminoethyl)naphthalene~\cite{30}.


Being NADPH-dependent, IREDs are typically used in tandem with an NADPH
recycling system. Many of these are the same systems used in KRED
reactions. The most used system is the
glucose-dehydrogenase (GDH)-catalyzed oxidation of glucose to gluconic
acid. As the glucose oxidation step is non-reversible, this imparts a
driving force on the imine reduction step. Other recycling systems have
been reported such as alcohol dehydrogenase (ADH) oxidation of alcohols
to carbonyls (typically propan-2-ol to acetone)~\cite{31,32}, soluble
hydrogenase (SH) reduction of NADP$^{+}$ with hydrogen gas~\cite{33},
phosphite dehydrogenase (PTDH) oxidation of phosphite to
phosphate~\cite{34}, and formate dehydrogenase (FDH) oxidation of
formic acid to carbon dioxide~\cite{35}. While these systems are very
attractive from an atom economy standpoint, glucose oxidation remains
the most commonly used in industry. 

In 2017, Pfizer gave a brief report of an IRED-catalyzed reduction to
synthesize (${S}$,${S}$)-sertraline \cite{36}. Since the aryl ketimine
\textbf{32} did not form spontaneously, the team synthesized the imine
by chemical means and fed it into the IRED reduction. Via this route,
IR064 from \textit{Myxococcus fulvus} was identified as a hit for the
reduction with the desired selectivity at both chiral centers and 95\%
ee (Scheme~\ref{sch16}). Enzyme engineering efforts focused on IR064
allowed the team to identify variants with improved activity and
selectivity. One variant with 5 mutations gave a 2.3-fold improvement
in activity with 95\% ee~\cite{36}.

In the same year, Aleku and coworkers reported a new IRED from
\textit{Aspergillus oryzae} which, in \mbox{addition} to reducing imines, also
catalyzed the condensation of a ketone and amine to form the imine for
reduction~\cite{37}. This subset of IREDs has been dubbed reductive
aminases or RedAms, and following their discovery, a mechanism of
enzymatic reductive amination was proposed~\cite{38}. Since then,
several articles reporting the use of RedAms to produce various amines
have been published, and additional IREDs that show this activity have
been discovered. \looseness=1

\begin{scheme*}
\includegraphics{sc17}
\vspace*{5pt}
\caption{\label{sch17}Pilot-scale reductive amination toward the
synthesis of GSK2879552.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc18}
\vspace*{5pt}
\caption{\label{sch18}Preparative-scale reductive amination toward the
synthesis of abrocitinib.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc19}
\vspace*{5.5pt}
\caption{\label{sch19}Preparative-scale reductive amination with IR007-143.}
\vspace*{-6pt}
\end{scheme*}

The first of these reports came in 2019 from GSK showing the use of an
IRED from \textit{Saccharothrix espanaensis} (IR-46) to resolve
\textit{trans}-phenylcyclopropylamine \textbf{35} and use it in the
reductive amination of an aldehyde (Scheme~\ref{sch17})~\cite{39}.
After three rounds of enzyme engineering, Schober and coworkers
reported that they were able to run the reaction at 20.1~g/L aldehyde
\textbf{35} with 1.2~wt\% enzyme loading to reach 84\% isolated yield
of the desired product amine obtained with 99.7\% ee. They noted that,
although IREDs are typically most active at neutral to slightly basic
pH, a slightly acidic pH was needed for product and substrate
stability, and product solubility. 

A few years later, Pfizer reported using an IRED from
\textit{Streptomyces purpureus} (\textit{Sp}RedAm) to couple isopropyl
3-ketocyclobutylcarboxylate \textbf{38} with methylamine for the
production of an intermediate in the synthesis of abrocitinib~\cite{40}.
The wild-type enzyme (40~wt\%) was able to convert 20~g/L of ketone
with 2 equiv of methylamine to give the desired product in 27\%
isolated yield. The engineered enzyme (\textit{Sp}RedAm-R3-V6) was used
in this reaction at 230~kg scale with 125~g/L ketone and 1.5~wt\%
enzyme. Reaction performance under these conditions improved to 92\%
assay yield with 73\% isolated yield and 99.5:0.5 dr
(Scheme~\ref{sch18}).


\begin{scheme*}
\includegraphics{sc20}
\vspace*{6pt}
\caption{\label{sch20}Analytical-scale screening for Ene-IRED reactivity.}
\end{scheme*}

Another report from Pfizer showed the use of a larger amine in a
resolution and reductive amination of hydroxyketone
\textbf{40}~\cite{41}. As part of a synthesis toward an investigatory
CDK2/4/6 inhibitor, the team identified an IRED from
\textit{Amycolatopsis azurea}, dubbed IR007, which resolved the racemic
hydroxyketone via reductive amination with benzylamine as the donor to
produce the desired amine as a single stereoisomer. After engineering
IR007 for improved activity and stability, variant IR007-143 was
identified, which resolved the ketone with only 2.5~wt\% catalyst
loading to give 35\% isolated yield and 98.4\% ee after 48~h
(Scheme~\ref{sch19}). This process was developed as an alternative to a
transaminase-catalyzed reaction discussed in a later section of this
review. With~higher chiral purity and easier product isolation, this
IRED approach was selected to move \mbox{forward}.

\begin{scheme*}
\includegraphics{sc21}
\vspace*{6pt}
\caption{\label{sch21}Analytical-scale screening for Ene-IRED reactivity.}
\vspace*{-6pt}
\end{scheme*}

Most recently, Thorpe and coworkers have reported a new type of
activity from IREDs. In 2022, the group showed the use of an IRED with
${\upalpha},{\upbeta}$-unsaturated imines to reduce not only the
C${=}$N bond but also the C${=}$C bond~\cite{42}. From a screen of 389 IREDs,
44 enzymes were identified which reduced the ene--imine to the fully
saturated piperidine \textbf{43} (Scheme~\ref{sch20}). 

An additional 12 IREDs were identified that gave the reduction of only
the C${=}$C double bond resulting in \textbf{45}. The enzyme with the
highest activity for the full reduction was dubbed EneIRED and was
selected for further study. After reaction optimization, EneIRED was
able to catalyze the condensation and reduction of methylcyclohexenone
\textbf{46} and cyclopropylamine to 61\% conversion with only 1.1~equiv
of amine (Scheme~\ref{sch21}). When the reaction was performed with 
20~equiv, the final product was isolated as the chlorhydrate salt in 69\%
yield. To test the scalability of the new reaction, the
condensation/reduction of 3-methylcyclohex-2-en-1-one and
cyclopropylamine was run at mmol scale with 50~mM enone and 5 equiv of
amine. The desired product of this reaction was isolated in 77\% yield.

\section{Transaminases}\label{sec5}

Another class of enzymes for amination are transaminases. These enzymes
are used to synthesize primary amines from a ketone or aldehyde using a
pyridoxal phosphate (PLP) cofactor and a sacrificial amine donor such
as isopropylamine. This is achieved via a two-step mechanism where the
amine donor is deaminated by the PLP cofactor to form a ketone and
pyridoxamine (PMP). An amination step then transfers the amine group on
to the target carbonyl, reforming PLP in the process. During this
reaction, the enzyme can preferentially form one epimer of the amino
compound. In cases where the starting carbonyl compound has additional
stereogenic centers, the transaminase may also show preference for one
of the starting material stereoisomers and set both chiral centers of
the product in a single reaction. ${\upalpha}$-Transaminases are
limited to the formation of ${\upalpha}$-amino and ${\upalpha}$-keto
acids whilst \unskip\break ${\upomega}$-transaminases exhibit a much
wider substrate scope and are employed much more widely for
biocatalysis.

\begin{scheme*}
\includegraphics{sc22}
\vspace*{6pt}
\caption{\label{sch22}Preparative-scale synthesis of a chiral building block.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc23}
\vspace*{6pt}
\caption{\label{sch23}Pilot-scale synthesis of chiral amine.}
\vspace*{-6pt}
\end{scheme*}

In 2019, the biocatalysis team at Merck reported the use of a
transaminase for the synthesis of an intermediate for a CGRP receptor
antagonist, {4-amino-\textit{N}-Boc-3-fluoropiperidine
\textbf{49}}~\cite{43}. Starting from the racemic fluoroketone
\textbf{48} and screening at pH 10.5 to promote epimerization of the
chiral fluoride center, the team identified ATA-303 from Codexis as a
hit for the desired product stereoisomer. Under initial screening
conditions, the ATA-303 gave the desired isomer with 79\% ee and 16:1
dr (Scheme~\ref{sch22}). \mbox{Without} any enzyme engineering, the process was
intensified to 100~g/L ketone and 2~wt\% enzyme. This process resulted
in 66\% isolated yield, with 94\% ee and 15:1~dr.

\begin{scheme*}
\includegraphics{sc24}
\vspace*{5pt}
\caption{\label{sch24}Preparative-scale transamination with ATA-71 and
($R$)-MBA.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc25}
\vspace*{5pt}
\caption{\label{sch25}Industrial-scale transamination with ATA-47.}
\vspace*{-4pt}
\end{scheme*}

One obstacle commonly encountered with transaminases is the fact that
these reactions are controlled by the reaction equilibrium and require
a large excess of amine donor to push the reaction \mbox{forward}.
Fortunately, several methods have been developed to introduce a
thermodynamic or entropic driving force into the reaction. With
isopropylamine as the amine donor, the reaction can be pushed to
completion by removing the acetone by-product with a constant nitrogen
sweep, or by applying a slight vacuum. A report from Pfizer in 2021
showed the use of another commercial transaminase (ATA-200) in the
synthesis of 3-amino-4$'$-Boc-spiro[oxolane-2,4$'$-piperidine]
\textbf{51}~\cite{44}. In this process, the spiroketone was converted to
the amine using 2.5 equiv of IPA and applying a constant nitrogen sweep
with periodic vacuum at 220~torr. As in the previous example, no enzyme
engineering was needed, and the process was successfully implemented at
50~g/L ketone and 8~wt\% enzyme. The optimized conditions resulted in
the reaction where the desired amine was isolated with 82\% yield and
98\% ee (Scheme~\ref{sch23}). \mbox{Additionally,} the enzymatic synthesis
enabled the formation of \textbf{51} as a single enantiomer, avoiding
the use of \mbox{hazardous} reagents such as bromine, sodium azide, and
triphenylphosphine.

Alternatively, when methylbenzylamine (MBA) is used as the amine donor,
evaporation of the by-product can be replaced by removal extraction. In
2022~\cite{41}, a report showcased this in a transamination/resolution
of hydroxyketone \textbf{52} (Scheme~\ref{sch24}). In this report, the
authors explain that while isopropylamine was initially used for
development, it led to an unfavorable reaction equilibrium causing the
reaction to stall at low conversion. Applying the nitrogen sweep helped
increase conversion but also caused significant solvent loss and
deactivation of the enzyme. To remedy this, the team used (${R}$)-MBA
instead, as using this donor imparted a more favorable equilibrium. One
caveat to this is that the formation of acetophenone can be detrimental
to the enzyme at higher concentrations. To account for this phenomenon,
the reaction was run as a biphasic mixture with 25~vol\% toluene to
remove the acetophenone as it is formed. These conditions allowed the
reaction of 50~g/L ketone and 16~wt\% enzyme load to reach 40\% yield
over 3 steps with a very favorable 97\% ee.

It should be noted that when using a chiral amine donor (e.g.,
methylbenzylamine, alanine, etc.) it is important to use the correct
enantiomer. Typically, this is the isomer with the same chirality as
the desired product (e.g., (${R}$)-MBA was used in this example to
generate the (${R}$)-amine).

A less common method of driving the transaminase equilibrium forward is
by removing the desired product from the reaction mixture as it is
formed. A 2017 report showcases the synthesis of
2-amino-6,8-difluorotetralin \textbf{56}~\cite{45}. The Pfizer
biocatalysis group showed that using ATA-47 from c-LEcta GmbH \mbox{enabled}
the conversion of 6,8-difluoro-beta-tetralone \textbf{55} to the
desired (${S}$)-amine in 94\% yield (Scheme~\ref{sch25}). More
importantly, they reported that due to low solubility of starting
material and product, the product precipitated in the form of
phosphoric acid salt as it was formed.

Other transaminase reactions involve the removal of pyruvate (formed
from alanine) that can be achieved using lactate dehydrogenase (LDH) or
pyruvate decarboxylase (PDC). A downside to using LDH is that it
requires a NADPH recycling system to continue removing pyruvate. On the
other hand, PDC yields acetaldehyde and carbon dioxide that can be
removed by vacuum or a nitrogen stream due to their
volatility~\cite{46}.

Another obstacle with transaminase processes is that they are typically
limited to aldehydes or small ketones (e.g., methylo, cyclic ketones).
However, a large area of transaminase research has focused on expanding
the binding pocket of transaminases to accommodate larger substrates.
Several groups have reported successfully expanding the active site of
a transaminase to accommodate carbonyls as large as phenyl tert-butyl
ketone~\cite{47}. In fact, in 2010, Codexis and Merck reported a
transaminase that after only one round of engineering, was able to
transaminate pro-sitagliptin ketone \textbf{57}
(Scheme~\ref{sch26})~\cite{48}. This variant achieved less than 1\%
conversion with high enzyme load; however, this was a huge improvement
considering the lack of any hits in the initial screen. After several
additional rounds of enzyme and process engineering, the team was able
to develop an enzyme which converted the target ketone in 88\% assay
yield at 134~g/L with only 4~wt\% enzyme.


\begin{scheme*}
\includegraphics{sc26}
\vspace*{5pt}
\caption{\label{sch26}Transamination of the bulky pro-sitagliptin ketone.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc27}
\vspace*{5pt}
\caption{\label{sch27}100 kg scale hydroxylation of a-isophorone by P450-BM3.}
\vspace*{-6pt}
\end{scheme*}

\section{Cytochrome P450}\label{sec6}

Cytochrome P450 monooxygenases (P450s or CYPs) are heme enzymes which
perform oxidations with molecular oxygen and often with the aid of
NADPH cofactor and a redox partner protein~\cite{49,50}. P450s perform
a diverse range of functionalizations from oxidation of heteroatoms, to
C--C bond formation or cleavage, to aromatic hydroxylation of aromatic
compounds. However, they are most often used to achieve the
hydroxylation of sp$^{3}$ C--H bonds. The~ability to stereo- and
regioselectively activate a previously inactivated C--H has been
captivating due to the ability of P450s to eliminate the need for
multistep synthetic processes. P450s do come with many limitations such
as low stability, limited substrate scope and low activity, which have
prevented them from replacing these synthetic steps on a larger scale.
Engineering has been applied to combat these limitations, often with
P450-BM3 from \textit{Bacillus megaterium} as the starting point for
these enzyme-modifying methods~\cite{51}.

An example of P450-BM3 reaching a kilogram scale production was
demonstrated by Kaluzna et~al.~\cite{52} where they synthesized
4-hydroxy-${\upalpha}$-isophorone \textbf{60} from ${\upalpha}$-isophorone
\textbf{59}. At a 1 L scale, Kaluzna et~al.\ were able to obtain
unprecedented product concentration of 10~g/L and a space time yield of
1.5~g/L/h (Scheme~\ref{sch27}). Upon extensive reaction engineering, the
process was scaled up to a 100~L scale, however the authors noted that
at this scale, the reaction was restricted by the oxygen transfer rate
(OTR) and by self-deactivation of the enzyme.

To overcome some of the restrictions of P450s, fusion enzymes have been
designed. Many \mbox{families} of P450s require a reductase partner enzyme for
NADPH binding. An electron is transferred from the reductase partner to
the P450 heme domain, allowing the desired oxidation reaction to
proceed with the target substrate. In a so-called self-sufficient P450,
the reductase domain is already fused to the heme domain, however
nonnatural self-sufficient P450s have also been developed to improve
the catalytic efficiency~\cite{53}. 

\begin{scheme*}
\includegraphics{sc28}
\vspace*{6pt}
\caption{\label{sch28}Two-step oxidation of (${+}$)-valencene to (${+}$)-nootkatone.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc29}
\vspace*{6pt}
\caption{\label{sch29}Tertiary hydroxylation of benzylic CH by P450-BM3
variant P450$_{\mathrm{PL}2}$.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc30}
\vspace*{6pt}
\caption{\label{sch30}P450-catalyzed biaryl coupling of arylomycin.}
\vspace*{-6pt}
\end{scheme*}

This has also been taken a step further, by Kokorin et~al.~\cite{54},
where a dehydrogenase enzyme has been fused to a self-sufficient P450
to eliminate the need for a separate cofactor recycling enzyme,
resulting in an increase of activity and catalytic efficiency. Kokorin
and Urlacher~\cite{55} then expanded on this work by applying a fusion
enzyme to the two-step oxidation of the insecticide and food additive
(${+}$)-nootkatone \textbf{62} from (${+}$)-valencene \textbf{61} with 
1.5~times increase in activity compared to the individual enzymes
(Scheme~\ref{sch28}).

Although P450s are often considered to have narrow substrate scopes,
Zhang et~al.~\cite{56} have demonstrated that hydroxylation at a
tertiary carbon position is possible with P450s. Tertiary hydroxylation
is synthetically often difficult to achieve on inactivated C--H bonds
and even more difficult to achieve in high enantiomeric excess. Zhang
et~al.\ engineered P450-BM3 variant P450$_{\mathrm{PL}2}$ to reach up to
99\% ee and 32\% yield upon ${\upalpha}$-hydroxylation of a benzylic C--H
bond with a range of substituted substrates\break (Scheme~\ref{sch29}).


Aside from hydroxylations, alternative functionalizations by P450s are
being explored on process scales. A recent report by Genentech has
demonstrated that engineered P450s are a viable replacement for
Suzuki--Miyaura couplings on a gram scale by performing an oxidative
biaryl macrocyclization to produce an arylomycin antibiotic core
\textbf{66}. \mbox{Molinaro} et~al.~\cite{57} obtained 1.3~g of product
in 64\% yield from a 3 L fermentation process after \mbox{purification}
(Scheme~\ref{sch30}).


Bond connections that do not occur in natural biological processes have
also been performed by P450s. Inspired by P450's non-natural carbene
transfers with N--H and S--H insertions to form C--N or C--S
bonds~\cite{58,59}, Kan et~al.~\cite{60} proposed that a {similar} method
could be achieved with Si--H insertions to form carbon--silicon bonds.
As C--Si bonds are present in a diversity of compounds which include
materials and pharmaceutical compounds, upon engineering cytochrome c
from \textit{Rhodothermus marinus} (\textit{Rma} cyt c), Kan et~al.\ were
able to stereoselectively insert a C--Si bond into
4-(dimethylsilyl)aniline \textbf{67}. Moreover, \textit{Rma} cyt c was
engineered to chemoselectively form the C--Si bond despite the presence
of a competing nitrogen atom in the molecule. Preparative-scale
reactions with whole cells resulted in over 95\% conversion and a 70\%
isolated yield with 98\% ee and a total turnover number (TTN) of 3410
(Scheme~\ref{sch31}).\looseness=-1

\begin{scheme*}
\includegraphics{sc31}
\vspace*{5pt}
\caption{\label{sch31}Chemo- and stereoselective carbene Si--H insertion
of to 4-(dimethylsilyl)aniline.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc32}
\vspace*{6pt}
\caption{\label{sch32}Hydroxylation by GriE of an intermediate in the
chemoenzymatic synthesis of \mbox{manzacidin-C.}}
\vspace*{-6pt}
\end{scheme*}

\section{Iron/${\upalpha}$-ketoglutarate-dependent
{\unskip\break}oxygenases}\label{sec7}

Like P450s, iron/${\upalpha}$-ketoglutarate-dependent oxygenases or
iron/2-oxoglutarate-dependent oxygenases (Fe/${\upalpha}$KGs or 2ODs) are
iron-coordinating enzymes which perform oxidations with molecular
oxygen. However, unlike P450s, Fe/${\upalpha}$KGs are non-heme
iron-binding enzymes and instead recruit an ${\upalpha}$-ketoglutarate
(${\upalpha}$KG) cofactor which coordinates to the iron via a catalytic
triad and assists in electron transfer to the substrate of interest.
Fe/${\upalpha}$KGs, first discovered in the 1960s~\cite{61}, have many
important roles in biological processes, both in humans and many other
organisms~\cite{62}. As such, Fe/${\upalpha}$KGs can perform a diverse
range of C--H functionalizations, but biocatalytic applications are
often focused on their halogenation or hydroxylation capabilities.

One such enzyme, is the putative leucine hydroxylase GriE, which
performs sequential oxidations at the delta position of L-leucine,
forming 4-methylproline by cyclization via a spontaneous reductive
amination in the biosynthesis of griselimycin~\cite{63}. Zwick and
Renata~\cite{64,65}, report a proof of concept where they employ GriE
as the first Fe/${\upalpha}$KG to be used in a natural product synthesis.
They share that GriE can produce an array of \mbox{substituted} pyrrolidine
building blocks and also demonstrate the utility of the enzymes in
natural product synthesis by performing the chemoenzymatic preparation
of manzacidin-C (Scheme~\ref{sch32}), a bromopyrrole alkaloid first
isolated from sea sponge \textit{Hymeniacidon} sp.~\cite{66} Zwick and
Renata reported a photocatalytic azidation to L-leucine which is
followed by a single ${\Delta}$ hydroxylation by GriE, creating the
hydroxylated product intermediate with a yield of 95\% on a 130~mg
scale.\looseness=-1

Most recently, the industrial potential of Fe/${\upalpha}$KG hydroxylases
has been used by scientists at Merck who have incorporated an
Fe/${\upalpha}$KG into the synthesis of the oncology treatment
belzutifan, by \mbox{performing} a stereoselective hydroxylation on a
non-native fluoroindanone intermediate \textbf{72}~\cite{67,68,69}. This
{biocatalytic} step replaced five chemical synthesis steps reducing the
process mass intensity (PMI) by 43\%, again illustrating the
environmental advantages to employing biocatalysts in industry.
Cheung-Lee et~al.~\cite{67} reported that, despite a P450 reaching
higher conversions in their initial screens, a Fe/${\upalpha}$KG
hydroxylase, FoPip4H from \textit{Fusarium oxysporum}, was selected for
further development in this process due to the resulting economic and
time benefits. After 15 rounds of directed evolution, FoPip4H Rd16BB
showed a 3000-fold improvement in catalytic efficiency over the wild
type. Furthermore, DiRocco et~al.~\cite{68} stated that an overall
improvement of more than 400,000-fold was observed in comparison to the
wild type when enzyme loading, reaction concentration, yield, and
enantioselectivity were achieved. At kilogram scale, the final variant
exhibited yields and ee of more than 90\% and than 99\%, respectively
(Scheme~\ref{sch33}).\looseness=-1

\begin{scheme*}
\vspace*{-1pt}
\includegraphics{sc33}
\vspace*{5pt}
\caption{\label{sch33}Hydroxylation of a
fluoroindanone intermediate in the synthesis of belzutifan.}
\vspace*{-2pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc34}
\vspace*{5pt}
\caption{\label{sch34}Preparative-scale hydroxylation of lysine by K3H-1.}
\vspace*{-7pt}
\end{scheme*}

Another scaled up application of Fe/${\upalpha}$KGs was demonstrated by
Hara et~al.~\cite{70}. They reported the preparative-scale synthesis of
(2${S}$,3${S}$)-3-hydroxylysine \textbf{75}, among other
hydroxylysines, which can be an intermediate in the synthesis of
(${-}$)-balanol, a protein kinase C inhibitor. The lysine hydroxylases
tested in this study were identified by sequence analysis of the
clavaminic acid synthase superfamily with K3H-1 showing the greatest
productivity. High substrate loadings were achieved of up to 600~mM
lysine, and after 52 h, a 40 mL reaction yielded 531~mM (86.1~g/L) of
(2${S}$,3${S}$)-3-hydroxylysine, with 89\% molar conversion from whole
cells expressing K3H-1 (Scheme~\ref{sch34}). The authors did not report
the diastereoselectivity of the enzyme. \looseness=-1

\begin{scheme*}
\includegraphics{sc35}
\vspace*{6pt}
\caption{\label{sch35}Halogenation of a martinelline core analog.}
\end{scheme*}

Aside from hydroxylations, halogenations with Fe/${\upalpha}$KG enzymes
are being reported more frequently in the literature. In 2005 the first
Fe/${\upalpha}$KG halogenase, SyrB2, was discovered to perform
monochlorination of a L-threonine substrate covalently tethered to
carrier protein, SyrB1~\cite{71}. The requirement of a carrier protein
limited the biocatalytic applicability of these enzymes. However, in
2014, WelO5, isolated from \textit{Fischerella ambigua} and
\textit{Hapalosiphon welwitschia}, was identified as the first
Fe/${\upalpha}$KG halogenase to work on a freestanding small molecule.
WelO5 performed a stereoselective monochlorination of an aliphatic
carbon in the native substrates 12-\textit{epi}-fischerindole U and
12-\textit{epi}-hapalindole C~\cite{72}, and has subsequently been
engineered for biocatalytic applications. Mutagenesis studies on these
halogenases were performed by Novartis and the Buller team in
Switzerland~\cite{73,74}, using a homolog of WelO5 called WelO5$^*$ with
95\% sequence identity from \textit{Hapalosiphon welwitschia}. This was
the first study to evolve a Fe/${\upalpha}$KG halogenase for a substrate
other than the native indole-alkaloids. Hayashi et~al.\ were able to
evolve WelO5$^*$ to halogenate fragments of a substrate analog of
martinelline \textbf{76}, a bradykinin receptor agonist. After two
rounds of mutagenesis, the final variant was able to perform both
chlorination and bromination to the martinelline core analog reaching
95\% conversion at 10~mM substrate \mbox{loadings}
(Scheme~\ref{sch35}). It was noted that this mutant reduced the
formation of unwanted side  \mbox{products}, exhibiting 290- and
400-fold increases in TTN and catalytic efficiency, respectively, over
the wild-type enzyme. No diastereoselectivity of the catalyst was
reported in the work of Hayashi et~al. 

\begin{scheme*}
\includegraphics{sc36}
\vspace*{6pt}
\caption{\label{sch36}Halogenation of a dGMP.}
\vspace*{-6pt}
\end{scheme*}


The Fe/${\upalpha}$KG halogenases act on natural products. However,
Fe/${\upalpha}$KG amino acid halogenases and Fe/${\upalpha}$KG nucleotide
halogenases have also recently been discovered. BesD catalyzes the
chlorination, bromination and azidation of aliphatic carbons in amino
acids~\cite{75}. In 2020, an Fe/${\upalpha}$KG {nucleotide} halogenase
called AdeV was the first Fe/${\upalpha}$KG to perform halogenations of
nucleotides by carrying out a C2$'$-chlorination to
2$^{\prime}$-deoxyadenosine-5$^{\prime}$-monophosphate (dAMP)
\textbf{78} (Scheme~\ref{sch36})~\cite{76}. Despite the narrow substrate scope of AdeV, these
nucleotide halogenases have the potential to \mbox{catalyze} the halogenation
of nucleotide analogs for therapeutic purposes such as anticancer
medicine like clofaribine or antiviral drugs such as uprifosbuvir.
Recent developments in nucleotide halogenases have been made by 
Ni et~al.~\cite{77} who have identified enzymes VaNTH and CtNTH which can
carry out chlorinations, brominations and azidations at the C2$'$
position of 2$'$-deoxyguanosine monophosphate (dGMP) and exhibit a more
than 43-fold improvement in catalytic efficiency when compared with
AdeV acting on dAMP. Furthermore Ni et~al.\ demonstrated that the
activity and nucleotide specificity can be modulated with engineering,
demonstrating the potential that these enzymes hold for nucleotide
modifications.\looseness=-1

\section{Monoamine oxidases}\label{sec8}

Monoamine Oxidases (MAOs) selectively oxidize amines to form imines
with the aid of molecular oxygen and a flavin adenine dinucleotide
(FAD) cofactor, producing hydrogen peroxide as a byproduct. A monoamine
oxidase from \textit{Aspergillus niger} (MAO-N) was first discovered by
Schilling and Lerch~\cite{78} and exhibits high ${S}$-stereoselectivity
for amines at the ${\upalpha}$-carbon position. This can be exploited in
kinetic resolutions and deracemizations to access a single
(${R}$)-enantiomer of an amine starting material when combined with a
chemical reducing agent such as BH$_{3}$NH$_{3}$. Wild type MAO-N
exhibits a narrow substrate scope of alkylamines but this has been
\mbox{substantially} broadened by engineering, with three key variants D5, D9
and D11 often employed~\cite{79}.

The pharmaceutical applicability of these MAO-N variants was
demonstrated by the analytical-scale syntheses of solifenacin and
levocetirizine. Their utility outside of deracemizations was also
demonstrated by two approaches with the MAO-N D9 variant via the
synthesis of (${R}$)-harmicine \textbf{81}, an alkaloid which shows
anti-leishmania activity~\cite{79}. The first approach to synthesizing
this active pharmaceutical ingredient (API) involved a deracemization
step to afford the final product in more than 99\% ee and 95\% yield.
The second approach reduced the overall syntheses to two synthetic
steps by using an oxidative asymmetric Pictet--Spengler reaction
resulting in more than 99\% ee and 83\% conversion
(Scheme~\ref{sch37}).

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc37}
\vspace*{5pt}
\caption{\label{sch37}Two routes to ($R$)-harmicine with MAO-N D9.}
\vspace*{-2pt}
\end{scheme*}


\begin{scheme*}
\includegraphics{sc38}
\vspace*{6pt}
\caption{\label{sch38}Synthesis of a quinoline heterocycle by MAO-N D11
variant.}
\vspace*{-7pt}
\end{scheme*}

Recently, it has been demonstrated that MAOs can oxidize
1,2,3,4-tetrahydroquinolines (THQs) into quinolines and
2-quinolones~\cite{80} which are prevalent scaffolds in many APIs.
Biotransformations were successful with unsubstituted or
electron-donating substituents to THQ, with the ethoxy-substituted THQ
affording 84\% \mbox{conversion} (Scheme~\ref{sch38}). 
\mbox{However,} bulkier or
electron-withdrawing substituents performed poorly. In-silico studies
did note that steric and binding constraints may be hindering the
activity with bulkier substituted substrates. To overcome this, Xiang
et~al.\ also employed a horseradish peroxidase.\looseness=-1

\begin{scheme*}
\includegraphics{sc39}
\vspace*{6pt}
\caption{\label{sch39}Enzymatic cascade using MAO-N D11 and an R-IRED
for the allylation of tetrahydroisoquinolines.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc40}
\vspace*{6pt}
\caption{\label{sch40}MAO synthetic step in the synthesis of the P2
moiety of boceprivir.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc41}
\vspace*{6pt}
\caption{\label{sch41}Enzymatic cascade using 6-HDNO and an eneIRED for
the synthesis of a Niraparib intermediate.}
\vspace*{-7pt}
\end{scheme*}

This focus on THQ-like molecules was taken even further by the Turner
group. They introduced a chemoenzymatic cascade where they employed
MAO-N and R-IRED enzymes alongside a chemical allylation resulting in
C--C bond formation at the C1 position of tetrathydroisoquinoline
(THIQ) \textbf{86}~\cite{81}. Sangster et~al.\ noted that there have
been few reports of enantioselective allylation of cyclic imines
despite their prevalence in many pharmaceutical scaffolds. Here they
demonstrated that the scaffolds can be synthesized on a preparative
scale at high enantioselectivity of up to 98\% ee and purified yields
of 64\% (Scheme~\ref{sch39}). Furthermore, on analytical scales, a range
of substituents both on the aryl ring and boryl--allyl partner were
accepted with good conversions, albeit lower ee values, demonstrating
the broad substrate scope that can be reached by this cascade.
\looseness=-1

However, MAOs have not only been used to synthesize APIs on an
analytical scale. Merck and Codexis employed a MAO for the synthesis of
protease inhibitor boceprevir, used in the treatment of chronic
hepatitis C infections~\cite{82}. MAO-N was evolved through 4
engineering rounds into MAON401, which replaced a synthetic resolution
step in the synthesis of the P2 moiety of boceprevir \textbf{90}. By
replacing this synthetic step, Merck increased the product yield by
150\% and reduced overall process waste (E-factor) by 63.1\%,
demonstrating the environmental advantages of employing biocatalytic
steps in industrial processes (Scheme~\ref{sch40}). The
stereoselectivity of this MAO-N-catalyzed step was not reported. 
\looseness=-1

Aside from MAO-N, other ${S}$-selective FAD-dependent monoamine
oxidases have been identified including human monoamine oxidases
MAO-A~\cite{83}, MAO-B~\cite{84}, and cyclohexylamine oxidase from
\textit{Brevibacterium oxydans}~\cite{85,86}. Only one ${R}$-selective
MAO is reported in the literature by Heath  et~al.~\cite{87}
6-hydroxy-D-nicotine oxidase (6-HDNO) has been shown to isolate
secondary and tertiary ${S}$-amines upon enzyme engineering. 6-HDNO has
been included in cascades alongside IREDS~\cite{88}. Such a cascade
reported by Harawa et~al.~\cite{89} employs 6-HDNO and an EneIRED for
the synthesis of 3-substituted and 3,4-disubstituted piperidines as
intermediates in the synthesis of ovarian cancer drug Niraparib. 6-HDNO
oxidizes a tetrahydropyridinium (THP) to form a dihydropyridinium (DHP)
which is then enantioselectively reduced by an EneIRED to form the
3-substituted piperidine, which was obtained in 68\% yield
(Scheme~\ref{sch41}).

\begin{scheme*}
\includegraphics{sc42}
\vspace*{7pt}
\caption{\label{sch42}Esterase desymmetrization in the synthetic
process of CGRP receptor antagonist (VIII).}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc43}
\vspace*{7pt}
\caption{\label{sch43}Lipase and nitrilase approaches to synthesize
chiral 3-substituted cyclohexanone building blocks.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc44}
\vspace*{7pt}
\caption{\label{sch44}Epoxide hydrolase resolution involved in the
synthetic process of an acid transport (iBAT) inhibitor GSK2330672.}
\vspace*{-6pt}
\end{scheme*}

\section{Hydrolases}\label{sec9}

Hydrolases are a well-established enzymatic class in biocatalysis,
represented by a broad range of enzymes, operating on esters and amides
(hydrolysis catalyzed by proteases, esterases, and lipases); alcohols
and amines (in lipase-catalyzed acylations); and nitriles and epoxides
(hydrolysis catalyzed by nitrilases and epoxide hydrolases,
respectively). Their mechanisms involve a catalytic triad to enable
catalysis~\cite{90}. Lipases are hydrolases that can operate in the
presence of organic solvents, including biphasic systems and without a
strict immobilization requirement~\cite{91}. The following examples,
highlight various substrates and typical conditions employed for
preparative-scale applications.

A biocatalytic desymmetrization was employed in the synthesis of the
chiral lactam motif of the CGRP receptor antagonist (VIII). Researchers
at Merck developed an esterase-catalyzed hydrolysis of the symmetrical
diester \textbf{93} to achieve the chiral monoacid \textbf{94} in high
yield (96\%) (Scheme~\ref{sch42})~\cite{43}. Commercially available pig
liver esterase was used for this transformation which was successfully
scaled to multikilogram scale at high substrate loading (100~g/L). Only
a modest ee of 89\% was obtained from this enzymatic process, which
could however be efficiently increased to 99\% by a chiral salt
resolution in a subsequent synthetic step. 

Researchers at GSK developed hydrolase resolutions to the
enantiocomplementary compounds \textbf{97} and \textbf{100}
(Scheme~\ref{sch43})~\cite{92}. A commercial lipase and a wild-type
nitrilase were selected from screening for rapid process enablement to
deliver multigram quantities of each material. 

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc45}
\vspace*{5pt}
\caption{\label{sch45}Nitrilase resolution process to access an
enantiopure nitrile building block for ipatasertib.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc46}
\vspace*{5pt}
\caption{\label{sch46}A hydrolase resolution used to synthesize a key
chiral intermediate in the synthetic process of a glucagon-like
peptide-1 receptor agonist (GLP-1-RA).}
\vspace*{-8pt}
\end{scheme*}

An epoxide hydrolase (EH) process was developed by GSK to resolve
racemic epoxide \textbf{101}, a key intermediate used to access the
ileal bile acid transport (iBAT) inhibitor GSK2330672 (XVII) for type 2
diabetes and cholestatic pruritus (Scheme~\ref{sch44})~\cite{93}. The
wild-type enzyme used for this reaction was identified from a panel
comprised of literature and metagenomically sourced biocatalysts. This
enzyme had sufficient activity and substrate tolerance to enable a 22 g
scale-up with very high \mbox{substrate} loading (338~g/L). It was noted that
enzyme engineering may be able to improve the selectivity of the enzyme
to increase the yield of \textbf{103} while maintaining high
enantioselectivity. 

Genentech and Roche utilized a nitrilase to access an enantiopure
nitrile building block for ipatasertib (Scheme~\ref{sch45})~\cite{94}. As
part of their efforts to develop a long-term manufacturing synthesis of
this API, a nitrile resolution process was developed in which the
(${R}$)-enantiomer of racemic nitrile \textbf{104} was selectively
hydrolyzed to the corresponding acid \textbf{105}, leaving the desired
(${S}$)-enantiomer \textbf{106} unreacted. A commercial enzyme from
c-LEcta GmbH was employed in this transformation as a liquid
preparation. It was found that high concentrations of sulfate and
phosphate ions enhanced the enzyme activity and polar organic
cosolvents had a detrimental effect on the enzyme performance. This led
to a process being implemented on 200 g scale at very high \mbox{substrate}
loading (235~g/L) without organic cosolvents (Scheme~\ref{sch45}). 

Another example of a successfully implemented hydrolase into a
biocatalytic process involved an \mbox{engineered} variant of MC064,
MC064-0797. This hydrolase was responsible for resolving a key racemic
amide \textbf{107} in the synthetic process of an oral late-stage
glucagon-like peptide-1 receptor agonist \mbox{(GLP-1-RA)} used in the
treatment of type 2 diabetes mellitus (T2DM) and for weight loss~\cite{95,96}.
Two rounds of enzyme engineering improved the wild-type
enzyme to support a 16 g reaction at low enzyme loads of 2.5~wt\%
(Scheme~\ref{sch46}). The reaction yielded a close to perfect resolution
of 48\% conversion and \textbf{108} with an ee of 91\%. The ee of the
resulting product could be improved by using a chiral salt
crystallization as a subsequent step. 

A nitrilase was also employed by Gilead and Almac for the
enantioselective, dynamic kinetic resolution of
(${R}$)-2-methoxymandelic acid \textbf{111}~\cite{97}. A one-pot
process was devised to exploit the reversible nature of the cyanohydrin
formation step by performing a dynamic kinetic resolution with
nitrilase BCJ2315 isolated from \textit{Burkholderia cenocepacia}
J2315. A multipronged approach was developed to improve the reaction
performance and reduce enzyme deactivation by carrying out process
development alongside in-silico-guided enzyme engineering. A scaled-up
process at 10~g/L substrate loadings with the M100 mutant resulted in
70\% isolated yield and 97\% ee\break in \textbf{111} (Scheme~\ref{sch47}).

\begin{scheme*}
\includegraphics{sc47}
\vspace*{5pt}
\caption{\label{sch47}One-pot preparation of ($R$)-2-methoxymandelic acid.}
\vspace*{-3pt}
\end{scheme*}

Ghosh and Nyalapatla presented a total synthesis of (${+}$)-amphirionin-4
which exhibits proliferation-promoting activity in ST-2 cells~\cite{98}. 
This synthetic route includes a lipase resolution step to \mbox{isolate} an
acetate intermediate \textbf{113} with readily available
\textit{Burkholderia cepacia} lipase (Amano PS-30). Using lipases in
biocatalytic reactions is beneficial in this process since it requires
a high solvent content. The resilience of this lipase to nonaqueous
environments is demonstrated in the following example where
\mbox{approximately} 50\% of the reaction solution was THF
(Scheme~\ref{sch48}).

\begin{scheme*}
\includegraphics{sc48}
\vspace*{5pt}
\caption{\label{sch48}Lipase PS-30 catalyzes lactone resolution.}
\vspace*{-6pt}
\end{scheme*}

\section{Enzyme and process engineering considerations}\label{sec10}

Enzyme engineering has proven to be a key enabler to developing
biocatalytic processes in the pharmaceutical sector~\cite{99,100}.
However, the rapid engineering of enzymes for complex and insoluble
substrates remains a bottleneck. The use of computational tools to
design and accelerate enzyme engineering is \mbox{proving} to be of vital
importance in the development of robust enzyme
catalysts~\cite{101,102,103}. Among those, machine-learning-assisted
enzyme engineering and ancestral sequence reconstruction (ASR) have the
potential to accelerate enzyme
engineering~\cite{104,105,106,107,108,109}. High-throughput platforms
include microfluidics with enhanced capabilities to acquire enzyme
kinetic data. These novel methods have the potential to make game
changing leaps in how biocatalysis development is performed,
particularly by enabling the screening of more complex multisite
recombination libraries~\cite{110,111,112,113}.

From a process chemistry implementation perspective, the solubility and
complexity of substrates, and the need to run processes at high
substrate loadings, often lead to processes operating as slurries.
Efficient protein removal remains a challenge which is especially
heightened in cascade processes when there are many enzymes present in
the system. Ultimately, enzymes engineered for high specific activity
and stability, enabling high utilization factors (ratio of substrate
load to enzyme load), are required to minimize challenges in downstream
processing. A strong control strategy demands fate and purge studies of
enzymes, and monitoring using gel electrophoresis and MS techniques has
become essential. Moreover, well-defined process acceptable ranges
(PARs), ensuring robustness and easily defined normal operating ranges
(NORs) for enzyme steps, minimizes failures at manufacturing scale.
Although there is great promise for the successful combination of flow
chemistry and biocatalysis, the vast majority of biocatalysis processes
are still run in batch mode, mainly due to substrate insolubility in
aqueous solutions. The scientific literature is dominated by examples
of immobilized enzymes operated in packed bed reactors, however, there
is huge potential for growth in the flow biocatalysis area on
{alternative} systems~\cite{114,115,116}. Photobiocatalysis is an
emerging area in the field of biocatalysis; during the past few years,
it has been used to generate new reaction pathways for asymmetric
synthesis, resulting in new-to-nature transformations {enabled} by
photo-bio-redox systems, in situ production of hydrogen peroxide, as
well as cascade reactions~\cite{117,118}.

\section{Final remarks}\label{sec11}

As demonstrated by the examples mentioned in this review, biocatalysis
continues to be highly impactful for key synthetic challenges in the
pharmaceutical industry. Well-established enzyme classes such as
ketoreductases and transaminases have become the go-to methods for the
synthesis of chiral alcohols or amines, respectively, as well as
hydrolases for desymmetrizations or resolution processes. In addition,
imine-reductase, reductive aminases and ene-reductases have
increasingly joined the top tier of enzymes enabling highly feasible
transformations~\cite{92}.

Looking ahead to the continued growth of biocatalysis implementation in
the pharmaceutical industry, certain gaps in the biocatalytic toolbox
remain. Amide bond synthesis, present in many pharmaceutical drugs is
still a challenge, despite advances being made in the use of ligases
for these transformations~\cite{119,120,121}. Functional group
interconversions starting from carboxylic acids are still
underexploited, and carboxylic acid reductases (CARs) offer great
potential to bridge the gap and enable amine and alcohol synthesis from
acid derivatives~\cite{122}. Continued development that involves
engineering and efficient enzyme expression of these complex
ATP-dependent reactions is anticipated in the coming years. Enzymatic
halogenations have great potential for pharmaceutical synthesis due to
the ubiquitous presence of halogens in pharmaceutical route scouting.
Implementable enzymatic solutions \mbox{remain} elusive primarily due to low
catalytic activities and very limited substrate specificity~\cite{123,124}.
Applications of underutilized biocatalysts for the synthesis of
chiral amines, sulfoxides, carboxylic acids, and amino acid analogs is
mainly associated to low activities and limited substrate
scope~\cite{125}. Finally, there are still limited options for general
C--C bond forming in biocatalytic transformations; despite
lyase-catalyzed C--C bond formation being represented in scattered
examples over the past two decades, there is a need for the discovery
of novel and broad scope enzymes to perform enzymatic
carboligations~\cite{126,127}. The repertoire of reactions available to
synthetic chemists is rapidly growing, aided by the emergence of
sophisticated computational tools to sort through ever-expanding public
and \mbox{private} enzyme databases~\cite{128,129,130}.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that  could benefit from this article, and have
declared no affiliations other than their research organizations.

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