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\DOI{10.5802/crchim.395}
\datereceived{2024-11-27}
\daterevised{2025-03-13}
\dateaccepted{2025-03-20}
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\dateposted{2025-05-16}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{clarification}

\title{Accelerating the implementation of biocatalysis in
pharmaceutical research and development portfolio}

\alttitle{Acc\'{e}l\'{e}ration de l'impl\'{e}mentation de la
biocatalyse dans le portefeuille de recherche et d\'{e}veloppement
pharmaceutique}

\author{\firstname{Alain} \lastname{Rabion}\IsCorresp}
\address{Sanofi's  Chemistry, Manufacturing and Control, 1 impasse
des ateliers, 94400 Vitry-sur-Seine, France}
\email[A. Rabion]{alain.rabion@sanofi.com}
                 
\author{\firstname{Davide} \lastname{Panigada}\CDRorcid{0000-0003-0287-8426}}
\addressSameAs{1}{Sanofi's  Chemistry, Manufacturing and Control, 1
impasse des ateliers, 94400 Vitry-sur-Seine, France}
\email[D. Panigada]{davide.panigada@sanofi.com}

\author{\firstname{Sebastien} \lastname{Roy}}
\addressSameAs{1}{Sanofi's  Chemistry, Manufacturing and Control, 1
impasse des ateliers, 94400 Vitry-sur-Seine, France}
\email[S. Roy]{sebastien.roy@sanofi.com}

\author{\firstname{Antony} \lastname{Bigot}\CDRorcid{0000-0003-3320-3755}}
\addressSameAs{1}{Sanofi's  Chemistry, Manufacturing and Control, 1
impasse des ateliers, 94400 Vitry-sur-Seine, France}
\email[A. Bigot]{antony.bigot@sanofi.com}

\author{\firstname{Jason\nobreakauthor S.}\nobreakauthor\lastname{Tedrow}\CDRorcid{0000-0002-5663-2360}}
\address{Sanofi's Chemistry, Manufacturing and Control, 350 Water St,
Cambridge (U.S.) Massachusetts 02141, USA}
\email[J. S. Tedrow]{jason.tedrow@sanofi.com}

\def\thanksname{Note}

\thanks{This article is submitted by invitation of the editorial
committee. All authors are employees of Sanofi and may hold stock
and/or stock options.}

\shortrunauthors

\keywords{\kwd{Biocatalysis}
\kwd{Transaminase}
\kwd{Ketoreductase}
\kwd{Tetrahydrofuran derivative}
\kwd{Chirality}
\kwd{Green chemistry}
\kwd{Enzyme evolution}}

\altkeywords{\kwd{Biocatalyse}
\kwd{Transaminase}
\kwd{C\'{e}toreductase}
\kwd{D\'{e}riv\'{e} tetrahydrofuranique}
\kwd{Chiralit\'{e}}
\kwd{Chimie verte}
\kwd{\'{E}volution enzymatique}}


\begin{abstract}
Biocatalysis is widely regarded as a sustainable technology to innovate
Active Pharmaceutical Ingredient (API) synthesis. Biotransformation is
considered a unique technology in the drug manufacturing world to
unlock synthetic routes to complex chiral motifs. Sanofi is
accelerating the development of strong mindset and efficient
capabilities in biocatalysis to foster API delivery and promote greener
solutions for its synthetic processes.

In this article, we will describe how Sanofi is building its internal
biocatalytic capabilities in order to address new challenges related to
sustainability and the growing complexity of its portfolio. The
outcomes of recent studies will be presented demonstrating the
successful implementation of the biocatalysis technology across early
and late-stage development of our pipeline assets.
\end{abstract}

\begin{altabstract}
La biocatalyse est largement pr\'{e}sent\'{e}e comme une technologie
durable permettant d'innover dans la synth\`{e}se des principes actifs
pharmaceutiques (API). La biotransformation est consid\'{e}r\'{e}e
comme une technologie unique dans le monde de la synth\`{e}se de
m\'{e}dicaments car elle permet, \textit{via} des voies
synth\'{e}tiques originales et souvent fortement \'{e}nantio- et/ou
diast\'{e}r\'{e}os\'{e}lectives, l'acc\`{e}s \`{a} des structures
chirales complexes. Sanofi acc\'{e}l\`{e}re le d\'{e}veloppement d'un
fort \'{e}tat d'esprit et des capacit\'{e}s n\'{e}cessaires
vis-\`{a}-vis de la biocatalyse afin d'acc\'{e}l\'{e}rer la fabrication
d'API et de promouvoir une solution plus \'{e}cologique pour ses
proc\'{e}d\'{e}s de synth\`{e}se.

Dans cet article, nous d\'{e}crirons comment Sanofi d\'{e}veloppe des
capacit\'{e}s en biocatalyse en interne afin de relever les nouveaux
d\'{e}fis li\'{e}s au d\'{e}veloppement durable et \`{a} la
complexit\'{e} croissante de son portefeuille de mol\'{e}cules
chimiques. Les r\'{e}sultats d'\'{e}tudes r\'{e}centes seront
pr\'{e}sent\'{e}s, d\'{e}montrant la r\'{e}ussite de la mise en
\oe{}uvre de la technologie de biocatalyse dans les phases pr\'{e}coces
et avanc\'{e}es de d\'{e}veloppement de nos principes actifs.
\end{altabstract}

\shortrunauthors

\maketitle

\vspace*{11pt}

\twocolumngrid

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\section{Introduction}\label{sec1}

In the past decade, advances in protein engineering, genomic database
mining, and computational methods associated with high-throughput
screening tools and DNA library synthesis have enabled a step change in
biocatalysis. As a consequence, the nature of biocatalytic
transformations can be nearly infinitely tunable and optimizable with
billions of combinations of alterations to tailor specific reactivity
and selectivity~\cite{1}. Due to modern directed evolution
techniques~\cite{2a,2b}, advanced artificial intelligence (AI)~\cite{3},
machine learning (ML) techniques~\cite{4a,4b}, the introduction of
non-natural amino acids and new reactivity~\cite{1}, the potential of
biocatalysis to transform modern drug manufacturing has been expanded. 
The field has moved beyond traditional like-for-like enzyme
replacements to wholly new routes designed around system
biocatalysis~\cite{5a,5b}. Sanofi, among others, has identified
biotransformations as one of the most transformative technologies to
date in the synthesis of today's complex new drug candidates.  In this
article, we describe how Sanofi is accelerating the implementation of
biocatalysis in its \mbox{pharmaceutical} research and development portfolio.
For details, original publications and patents are referenced. 

\begin{scheme*}
\vspace*{2pt}
\includegraphics{sc01}
\vspace*{8pt}
\caption{\label{sch1}Examples of biocatalytic transformation at
Sanofi~\cite{10}.}
\vspace*{-2pt}
\end{scheme*}

\section{History of Sanofi use and production of enzymes}\label{sec2}

Sanofi has extensive experience in the synthesis of molecules of
interest, including small molecules and recombinant enzymes, through
fermentation processes. 

Sanofi also used enzyme technologies to produce small molecules at
various stages of their development. Discovery capabilities were
developed to produce drug metabolites for quantification and profiling
of in-vivo generated compounds. The CYP450 enzymes of microorganisms
are well described for their metabolizing ability, as ``the microbial
model for drug metabolism''. This type of technology was developed
internally, and the use of such enzymes has been proven to be an
efficient way to produce either Phase I or Phase II
metabolites~\cite{6} in their labeled~\cite{7a,7b} or non-labeled form.
Another class of enzyme used for the synthesis of
Carbon-14-radiolabeled \mbox{metabolite} is nitrilase. We showed that
radiolabeled nitrile derivatives can be hydrolyzed to get the
corresponding radiolabeled acid, using mild enzymatic
conditions~\cite{8,9}. In addition to the use of biocatalysis for
small-scale pharmaceutical metabolite synthesis, implementation of
enzyme technologies for the scalable synthesis of pharmaceutical
advanced intermediates was also developed. Castro et~al.~\cite{10}
identified commercial lipase for the enantioselective hydrolysis of
racemic carboxylic ester and for the desymmetrization of a symmetrical
diester to achieve a chiral monoacid ester product 
(Scheme~\ref{sch1}).

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig01}
\vspace*{-4pt}
\caption{\label{fig1}Sanofi charter on Planet Care environmental
sustainability program.}
\vspace*{-2pt}
\end{figure*}

By taking advantage of internal history in using enzymes for
synthesizing optically molecules, and in fermentation processes, Sanofi
decided in early 2020, to build a fully integrated end-to-end
biocatalysis platform within the Process Chemistry department belonging
to the R\&amp;D organization.  This platform aims to provide chemists with
efficient tools for improving the environmental impact of API synthesis
and addressing the challenges associated with the growing complexity of
molecules entering \mbox{development}.

\section{Biosynthesis technology to address new challenges from the
R\&amp;D portfolio}\label{sec3}

Sanofi has a strong ambition to build a sustainable environment with a
global approach to minimizing impacts. Three main axes have been
defined and are described in Figure~\ref{fig1}.  A key pillar toward achieving
these environmental commitments revolves around reducing the
environmental footprint of manufacturing processes through an ecodesign
approach. This includes reduced water and solvent use, eliminating
toxic metals, reagents and solvents, and lowering energy requirements. 
To meet our long-term environmental sustainability commitment,
technological innovations such as biocatalysis, are required.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Evolution of the structural complexity in Sanofi's
small molecule portfolio.}
\end{figure*}

\subsection{Molecular complexity of next generation small molecule APIs}

A general trend at Sanofi, and elsewhere, is that new molecular
entities entering development are increasing in complexity
(Figure~\ref{fig2})~\cite{11}. The trend is moving away from
traditional activators/inhibitors toward a next-generation approach of
stabilizing or disrupting complex protein--protein interactions
(next-generation small molecules) to obtain the desired effects. 
Additionally, the introduction of bispecific small molecule degraders
has also increased the overall complexity and size of the synthetic
targets. With increased complexity comes chemistry manufacturing and
control (CMC) risks to supply, \mbox{resources}, time to clinic and/or cost
manufacture. The utilization of biotransformation is a key lever to
help us manage the increase in synthetic complexity.  


\section{How we proceed to accelerate the implementation of
biocatalysis in the Sanofi Pharmaceutical R\&amp;D Portfolio}\label{sec4}

Sanofi's strategy is based on three pillars. 

\subsection{Development of a route-scouting mindset for an innovative
route design approach}\label{sec41}

We consider that organic chemists and biochemists need to work together
to design routes integrating a biocatalytic retrosynthesis approach,
supported by a strong network of external experts and partners
(Figure~\ref{fig3}). The biosynthetic technologies team is integrated
into the global Process Chemistry organization which favors scientific
exchange through brainstorming sessions on innovative chemical route
design. Furthermore, enzymatic transformations can be considered for
the design of clinical and commercial routes thanks to new enzyme
engineering techniques that can quickly deliver an effective
biocatalyst in a short time.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Route design workflow.}
\end{figure*}

\subsection{Contribution all along value chain
(Figure~\myRef{fig4})}\label{sec42}

At the research stage, the main input from biocatalysis is either to
produce metabolites or to generate diversity through late-stage
functionalization (LSF) for accelerating compound discovery~\cite{12}.
Currently, we are using biotransformation early in discovery as a
diversity engine to aid candidate generation.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Implementation of biocatalysis all along the value
chain.}
\end{figure*}

Starting in preclinical development, a first generation of synthetic
routes is used for delivering preclinical and clinical batches with a
focus on speed to the clinic. A retrosynthetic strategy integrating
biotransformation from the beginning has proven to be of great
interest.  Accelerating pressure on clinical timelines is squeezing
development timelines and shifting our strategy toward fewer cycles of
route selection and development.  In some cases, this can mean keeping
the same disconnection strategy throughout the product's life cycle,
but optimizing, concatenating and replacing less sustainable
technologies with highly selective and efficient technologies.  This
could involve using an off-the-shelf enzyme, readily accessible at
scale, that may not be optimal for the system but can sustain projects
within a short timeframe (see example in Section~\ref{sec5}).  Another
option is to utilize a non-enzymatic strategy that could be replaced by
a more sustainable biocatalyzed sequence later in the development
phase. An illustrative example from our R\&amp;D portfolio of such chemical
transformation is the asymmetric hydrogenation catalyzed by a very
expensive chiral ruthenium catalyst (Scheme~\ref{sch2})~\cite{13}. 

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc02}
\vspace*{10pt}
\caption{\label{sch2}Initial ruthenium-catalyzed
hydrogenation~\cite{13} and KRED-mediated biocatalytic
reaction~\cite{14} affording chiral hydroxy intermediate \textbf{B}.}
\vspace*{-10pt}
\end{scheme*}

In addition to the costly precious ruthenium catalyst, the
diastereoisomeric ratio obtained after the chemo selective reduction
step was moderate (84:16), negatively impacting the process efficiency,
manufacturing costs, and waste generation. To anticipate potential
constraints regarding large-scale drug supply, we initiated a study on
biocatalytic transformation using ketoreductase (KRED)
enzymes~\cite{14}.

One enzyme was found to be highly selective toward the desired
\textit{cis} alcohol product \textbf{B} (\textit{cis}/\textit{trans}\ ${=}$\ 
99:1 and enantiomeric excess ${\sim}$90\%). Our results demonstrate that
the KRED enzyme is an alternative to the ruthenium catalyst for the
reduction of the very bulky ketone substrate \textbf{A} to the chiral
alcohol product~\textbf{B}.

Once the project has moved into late clinical development and, drug
supply has been taken out of the critical path, the time and resources
needed to find the most ideal way to utilize biotransformation was
investigated. At that stage, the chemical route for launch is selected
and we can introduce the fully engineered enzyme as if we can replace
one catalyst by another one. The objective at this point is to meet the
prerequisites of an industrial process, 
including  {productivity}, yield,
turnover number, robustness, cost. Finally, the incorporation of the
biocatalytic strategy early in development, when possible, allows us to
focus our efforts on enzyme engineering and optimization instead of
compressing enzyme development on top of additional route selection
activities. 

\subsection{Building efficient capacity}\label{sec43}

A biosynthetic technologies team was created, designed for end-to-end
biocatalyst development and deployment, able to take in charge the
different studies related to biocatalysis involved at early and late
development phases. 

A global workflow has been established and related capacities and
expertise have been developed internally with the support of key
partners to rapidly implement biocatalytic process: 
\begin{itemize}
\item Molecular biology platform including expertise in enzyme
discovery and in the design of new enzyme through protein engineering
approaches such as directed evolution or more rational engineering.
This is supported by internal protein physical modeling. Regarding the
large computational power needed for the enzyme searches and multimodal
modifications we have implemented strategic partnerships in order to
enhance our in-house capabilities. 
\item Reaction engineering capacities including medium- and
high-throughput equipment and homothetic vessels for the development
and the optimization studies of biocatalytic reaction. 
\item Enzyme production platform including various host fermentation
and formulation techniques for biocatalysts production.
\item Pilot capacities for the upscaling of biocatalytic processes and
the production of enzymes at medium scale by fermentation.
\item Analytical platform capable of developing analytical methods for
reaction monitoring, conducting structural analyses for product and
impurity identification, and characterizing biocatalytic enzyme.
\vspace*{5pt}
\end{itemize}

\section{Recent examples of enzyme use in the manufacture of APIs
across early- and late-stage development}\label{sec5}

One of the first projects, using biocatalysts in manufacturing and
studied in the newly created biosynthesis technology platform, was the
new route toward a potent and selective LRRK2 inhibitor via chiral
intermediate \textbf{1}~\cite{14,15,16a,16b,16c}.
One key intermediate of the
route, for the early preclinical batches, was the chiral 3-hydroxy
4-methyl tetrahydrofuran \textbf{4}, which could be prepared by an
enantio- and diasteroselective reduction of ketone \textbf{5}
(Scheme~\ref{sch3})~\cite{15,17}.

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc03}
{\vspace*{4pt}}
\caption{\label{sch3}Disconnection of \textbf{1} toward simpler
fragments.}
\vspace*{-2pt}
\end{scheme*}

Access to the challenging fragment \textbf{4} for early clinical
deliveries was first achieved through the enabling route presented in
Scheme~\ref{sch4}. 

\begin{scheme*}
\includegraphics{sc04}
{\vspace*{4pt}}
\caption{\label{sch4}Enabling route to chiral tetrahydrofuran
derivative \textbf{4}.}
\vspace*{-8pt}
\end{scheme*}

In this first-generation route, it should be noted that to increase the
enantiomeric excess of \textbf{4}, a lipase-catalyzed kinetic
resolution (esterification) was performed, using Lipase Amano
AK~\cite{18} from \textit{Pseudomonas fluorescens} (commercially
available from Sigma-Aldrich) in the presence of vinyl acetate. This
resulted in an isolated enantio-enriched (ee\ ${=}$\ 94\%)
O-acetylated intermediate \textbf{8} which was then
deprotected to generate enantio-enriched secondary alcohol \textbf{4}
(ee = 94\%). However, this suboptimal enantiomeric excess necessitates
performing a chiral SFC later in the synthesis, as requested by the
project. 


This synthetic route faced CMC risks related to the supply of 50 kg of
intermediate \textbf{4}, resources, time to clinic and cost of
manufacture. Therefore, alternate chemistry was quickly required to
advance the program.

Having solved the challenge associated with the preparation of
\textbf{5} through a safe process~\cite{15}, we then turned our
attention to the challenging task of improving both the yield and the
enantiomeric excess of compound \textbf{4}, while diminishing the
number of steps.  

Starting from methyl ketone \textbf{5}, we explored the synthesis of
\textbf{4} through asymmetric reduction with KRED enzymes
(Scheme~\ref{sch5}) which are known to catalyze the reduction of
sterically hindered ketones into the corresponding chiral
alcohol~\cite{19a,19b}. Due to short project timelines, we went for
commercial or readily available enzymes for first deliveries while
postponing the expensive and time-consuming enzyme evolution studies
until later. Following an internal screening of KRED enzymes from
commercially available kits, a very selective and active enzyme was
found and selected for further process development.

\begin{scheme}
\includegraphics{sc05}
\vspace*{8pt}
\caption{\label{sch5}Synthesis of \textbf{4} via KRED reaction: global
reaction scheme principle.}
\vspace*{-5pt}
\end{scheme}

Interestingly, the complete conversion of the two enantiomers of the
ketone into one main diastereoisomer of the hydroxy methyl
tetrahydofuran, \textbf{4} demonstrated that a dynamic kinetic
resolution is occurring under the reaction conditions
(Scheme~\ref{sch6}).

\begin{scheme*}
\includegraphics{sc06}
\vspace*{5pt}
\caption{\label{sch6}Kinetic resolution (KR) and dynamic reductive
kinetic resolution (DRKD) based on ketoreductase-catalyzed reduction.}
\vspace*{-5pt}
\end{scheme*}

The optimized laboratory process was further refined to fit the scale
and was successfully scaled in the pilot plant to 40 kg of ketone
\textbf{5} per batch (Table~\ref{tab1}). Yield and enantiomeric excess
like those observed at laboratory scale were obtained, demonstrating
the robustness of the biocatalytic process.  

\begin{table*}
\caption{\label{tab1}Scale up results for preparation of alcohol 4}
\begin{tabular}{cccccc}
\thead
Batch type &amp; Starting material &amp; Product &amp; ee (\%)$^{\mathrm{a}}$ &amp;
\textit{cis}/\textit{trans}  Ratio$^{\mathrm{a}}$ &amp; Yield (\%)$^*$\\
\endthead
Demo batch &amp; \01.155 kg &amp; 0.86 kg &amp; ${&gt;}$99.9  &amp; 16.7 &amp; 73.2  \\
Pilot plant \#1 &amp; 39.95 kg\0  &amp; 31.7 kg &amp; ${&gt;}$99.9  &amp; 13.4 &amp; 78.0 \\
Pilot Plant \#2 &amp; 33.75 kg\0  &amp; 25.6 kg &amp; ${&gt;}$99.9  &amp; 14.9 &amp; 75.0
\botline
\end{tabular}
\xxtabnote{$^{*}$Assay
yield. $^{\mathrm{a}}${Calculation: Ratio
{\textit{cis}}{/}\textit{trans}: $R\ {=}\ \sum$[\textit{cis}({\textit{S,S}})\ 
${+}$\ \textit{cis}(\textit{R,R})]/$\sum$[\textit{trans}({\textit{R,S}})\ 
${+}$\ \textit{trans}({\textit{S,R}})]. Enantiomeric excess: {ee} (\%) ${=}$
100\ ${\times}$\ [\textit{cis}({\textit{S,S}})\ ${-}$\ 
\textit{cis}(\textit{R,R})]/$\sum$[\textit{cis}({\textit{S,S}})\ ${+}$\ 
\textit{cis}(\textit{R,R})]}.}
\vspace*{5pt}
\end{table*}

A qualitative comparison between the enabling route (3 steps from
ketone \textbf{5}) and the biocatalytic alternative (1 step from ketone
\textbf{5}) is shown in Table~\ref{tab2}~\cite{15}. 

\begin{table*}
\caption{\label{tab2}Qualitative comparison between enabling and
biochemical processes from ketone 5 to alcohol~4}
\begin{tabular}{ccccccccc}
\thead
{Route} &amp; \parbox[t]{1.2cm}{\centering Number of steps} &amp; 
\parbox[t]{1.6cm}{\centering Work up  Chromato.} &amp;
\parbox[t]{1.5cm}{\centering Operating temp} &amp; 
\parbox[t]{1.9cm}{\centering Solvent with  constraints} &amp;
\parbox[t]{1.4cm}{\centering Isolated yield (\%)}  &amp; 
\parbox[t]{1.2cm}{\centering \textit{cis}/\textit{trans} ratio} 
&amp; {ee (\%)} &amp; \parbox[t]{1cm}{\centering PMI (kg/kg)}\vspace*{2pt} \\
\endthead
Enabling$^{\mathrm{a}}$ &amp; 3 &amp; 1 &amp; ${-}$65~\textdegree C &amp; CH$_{2}$Cl$_{2}$ &amp; \08  &amp; ${&gt;}$30 &amp; {\mn}93.0  &amp; 1142 \\
Enzymatic$^{\mathrm{b}}$ &amp; 1 &amp; 0 &amp; {\mn}30~\textdegree C &amp; None  &amp; 75  &amp; 14.9 &amp; ${&gt;}$99.9 &amp; \0151
\botline
\end{tabular}
\tabnote{$^{\mathrm{a}}$Kilolab campaign, $^{\mathrm{b}}$Pilot plant campaign \#2.}
\vspace*{2pt}
\end{table*}

The one step enzymatic process outperforms the three steps chemical
reactions in terms of yield, waste generated, scalability, and
timelines. Ultimately, the enzymatic route was the only viable
alternative for large scale clinical batch manufacturing to meet
timelines and quality.

After unlocking the production of preclinical and clinical batches with
the implementation of the \mbox{ketoreductase} process to produce the
chiral hydroxy methyl tetrahydrofuran derivative, we looked for an
optimal synthesis route in anticipation of the commercial launch
(Scheme~\ref{sch7})\footnote{A. Rabion, ``Accelerating the
implementation of Biocatalysis in the Sanofi Pharma portfolio:
Synthetic Routes to complex chiral tetrahydrofuran derivatives'', in 
\textit{Oral Communication CBSO24 30th Symposium, October 7, 2024, Cap
Hornu, France}.}.

\begin{scheme*}
\vspace*{4pt}
\includegraphics{sc07}
\vspace*{10pt}
\caption{\label{sch7}Disconnections integrating hydroxy methyl
tetrahydrofuran \textbf{4} and amino methyl tetrahydrofuran
\textbf{9}.}
\vspace*{-2pt}
\end{scheme*}

The preclinical route based on the chiral hydroxy methyl
tetrahydrofuran derivative intermediate \textbf{4} was first
assessed~\cite{15}. The enantiomerically pure compound \textbf{4}
obtained from KRED-mediated reduction was introduced at the next step
of synthesis under Mitsunobu conditions. It quickly became apparent
that this transformation was not efficient enough due to the formation
of by-products such as triphenylphosphine oxide as well as reduced form
of DIAD used in the Mitsunobu reaction. Additionally, an excess of
compound \textbf{4} (3 equiv) was necessary, likely due to
decomposition under the reaction conditions~\cite{21}. 

To install the chiral fragment into the molecule, the
\textit{trans}-(3\textit{R},4\textit{R})-amino methyl tetrahydrofuran
derivative \textbf{9} was identified as a potential intermediate. It
was demonstrated that it could be introduced into the global synthesis
through an efficient nucleophilic substitution reaction without
degrading the diastereoselectivity.  Having solved the challenge
associated with the introduction of the chiral fragment onto the
molecule, we then turned our attention to the challenging task of
preparing the chiral amino methyl tetrahydrofuran derivative
\textbf{9}. 

Two alternative routes have been evaluated in parallel, namely
alternative 1 (chemical pathway) and alternative 2 (enzymatic pathway)
starting from the same methyl ketone intermediate \textbf{5}
(Scheme~\ref{sch8}). The chemical route initially studied produced the
first grams of the chiral (3\textit{R},4\textit{R})-amino methyl
tetrahydrofuran derivative \textbf{9}, which allowed validation of the
end of the synthesis. It should be noted that diastereo- and
enantioselectivity were improved through (\textit{S})-mandelate salt
formation but remained moderate (\textit{trans}/\textit{cis}\ ${=}$\ 88:12
and ee\ ${\sim}$\ 93\%). The overall yield of this four-step synthesis was
low (${\sim}$15\%) and generated high quantities of waste. This chemical
route was not suitable for commercial batch manufacturing, and it was
decided to investigate enzymatic approaches. 

\begin{scheme*}
\includegraphics{sc08}
\vspace*{10pt}
\caption{\label{sch8}Chemical pathway and enzymatic transamination
pathway toward the synthesis of trans amino methyl tetrahydrofuran
\textbf{9}.}
\end{scheme*}

Starting from methyl ketone \textbf{5}, we explored the synthesis of
the amino methyl tetrahydrofuran derivative \textbf{9} through
asymmetric \textit{trans} amination with transaminase
enzymes (see footnote 1)~\cite{22a,22b}.

At first, commercial enzymes were used for the proof-of-concept.
Following an internal screening of 83 transaminase enzymes from
commercially available kits, 19 enzymes demonstrated good biocatalytic
activity (assay yield\ ${&gt;}$\ 50\%). We identified one enzyme
(transaminase ATA-047) that was able to \mbox{deliver} the amino methyl
tetrahydrofuran derivative \textbf{9} with high selectivity toward the
desired \textit{trans} (3\textit{R},4\textit{R})-diastereoisomer (assay
yield\  ${\sim}$\ 99\% after 24 h reaction, ee\ ${&gt;}$\ 99\%,
\textit{trans}/\textit{cis}\ ${&gt;}$\ 30) under screening conditions.
Interestingly, the reaction occurred under dynamic kinetic resolution
and only the \textit{trans} (3\textit{R},4\textit{R})-diastereoisomer
\textbf{9} was produced starting from the racemic ketone \textbf{5}. To
demonstrate the potential of the transaminase approach, a scale trial
in 100~mL thermostated glass reactor with overhead stirring containing
was performed. \mbox{Applying} a low enzyme/substrate ratio (1:10), a high
conversion rate (assay yield \textbf{9}\ ${=}$\ 92.7\%) was observed
after 48 h reaction with excellent stereoselectivity\break
(Figure~\ref{fig5}).

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Time course of transamination reaction 
of \textbf{5} with ATA-047 at 100 mL scale.}
{\vspace*{2pt}}
\end{figure*}


Conditions: transaminase ATA47 1 mg/mL; ketone \textbf{5}\  ${=}$\  100 mM;
isopropylamine\  ${=}$\  0.9 M; PLP\  ${=}$\  10 mM; sodium phosphate buffer
100 mM, pH 9; \unskip\break temperature\  ${=}$\  30~\textdegree C; volume
${=}$\  100 mL. See footnote for calculation\footnote{Calculation : ee\ 
${=}$\  [\textit{trans}(\textit{3}\textit{R},4\textit{R})\ ${-}$\ 
\textit{trans}(3\textit{S},4\textit{S})]/[\textit{trans}(3\textit{R},4\textit{R})\ ${+}$\ 
\textit{trans}(3{\textit{S,}}4\textit{S})]; conversion \textit{cis}
(\%) ${=}$ [\textit{cis}(3\textit{R},4\textit{S})\ ${+}$\ 
\textit{cis}(3\textit{S},4\textit{R})]/{\ubreak}[\textit{cis}(3\textit{R},4\textit{S})\ ${+}$\ 
\textit{cis}(3\textit{S},4\textit{R})\ ${+}$\ 
\textit{t}{\textit{rans}}(3\textit{R},4\textit{R})\ ${+}$\ 
\textit{trans}(3\textit{S},4\textit{S})\ ${+}$\ Ketone]; conversion
\textit{trans} (\%)\ ${=}$\ [\textit{trans}(3\textit{R},4\textit{R})\ 
${+}$\ 
\textit{trans}(3\textit{S}{,}4\textit{S})]/[\textit{cis}(3\textit{R},4\textit{S})\ ${+}$\ 
\textit{cis}(3\textit{S},4\textit{R})\ ${+}$\ 
\textit{trans}(3\textit{R},4\textit{R})\ ${+}$\ 
\textit{trans}(3\textit{S},4\textit{S})\ ${+}$\ Ketone].}.

Although the commercial enzyme performed well, industrial objectives
required a more efficient biocatalyst with better stability and higher
substrate loading compatibility. Thus, in parallel with the testing of
commercial kits, we carried out a genome mining approach through
literature search and database in-silico screening. Fifty wild-type
transaminases were selected for cloning and screening assays. 
\mbox{Two~enzymes} were found to be very active in the {formation} of the amino
methyl tetrahydrofuran derivative \textbf{9} (screening conditions),
e.g., transaminase from \textit{Chromobacterium violaceum} (assay yield
amino methyl tetrahydrofuran\  ${\sim}$\  90\%), providing mainly the
\textit{trans}-(3\textit{R},4\textit{R})-diastereoisomer and
\textit{Arthrobacter sp} (assay yield amino methyl tetrahydrofuran\ 
${\sim}$\  50\%) providing mainly the \textit{cis}-diastereoisomers.
Enantioselectivity could be reversed during enzyme evolution. Thus,
with such results in hand, we considered these two enzymes to be good
hits to start an enzyme evolution campaign to achieve the industrial
goal of an efficient biocatalyst.

The third example deals with the optimization of a sulfotransferase
enzyme for the synthesis of one of our late-stage assets~\cite{23}. The
biocatalytic process used $3'$-phosphoadenosine-$5'$-phosphosulfate (PAPS)
as a donor of the SO$_{3}$ group and was viewed as unsustainable and a
significant cost driver.  As an alternative to reduce the process cost,
a PAPS recycling system was envisaged. We demonstrated how it was
possible to rapidly increase the activity of an enzyme for a
non-natural reaction in a few optimization rounds through enzyme
evolution campaign. In the literature, it is known that the enzyme Aryl
sulfotransferase IV (AST-IV) from rat can produce the PAPS from a
SO$_{3}$ donor much cheaper, the $p$-nitrophenyl sulfate
($p$-NPS) (Scheme~\ref{sch9}). 

\begin{scheme}
\vspace*{4pt}
\includegraphics{sc09}
\vspace*{15pt}
\caption{\label{sch9}PAPS regeneration system. AST-IV catalyzes the
transfer of the sulfo group from the less expensive donor,
$p$-NPS.}
\vspace*{-9pt}
\end{scheme}

However, this is the reverse reaction of this enzyme since its natural
function is the sulfation of $p$-NP starting from PAPS. Therefore, the
challenge was to force an enzyme to be efficient against its natural
reaction equilibrium.

An optimization program was started with a directed evolution approach
by recombination of homologues. This method consists of taking
advantage of some homologues of the starting gene and recombining them
randomly to produce new sequences among which we might find some
improved versions of the enzyme. In this first round, 12,000 clones
were screened, and we achieved a twofold activity increase in PAPS
production. Even though we found more efficient variants, this method
did not lead to an enzyme active enough to reach the goal, and we
decided to use a more structure-guided rational design approach. 

By using computational tools and molecular modeling, it is possible to
predict mutations that might enhance the enzyme performance. Thanks to
the 3D modelling tools, we were able to reconstitute the structure of
the protein and identify the binding region for the substrates and
products (PAP/$p$-NPS and PAPS/$p$-NP) in the catalytic site. The
challenge was then to find how to increase PAPS formation. We decided
to create variants to destabilize the binding between the enzyme and
the $p$-NP in order to favor the binding of $p$-NPS as a substrate. In
this case we found one mutant able to increase the activity of the
enzyme sevenfold (Figure~\ref{fig6}). 

\begin{figure}
\includegraphics{fig06}
\caption{\label{fig6}$p$-NP production achieved by Isoleucine 239
mutant compared to wild type enzyme.}
\end{figure}

After this first positive result we decided to launch a more in-depth
study. We tested a list of other ${\sim}$100 variants targeting both the
binding site (to directly increase the activity) and the entire protein
with the idea of stabilizing the activity of the enzyme during the
reaction. After this additional round, we identified 10 mutations that,
in combination, were able to increase the activity of the protein more
than 10-fold, maintaining a high level of activity throughout the
reaction. 

This final variant of the enzyme can sustain the recycling of PAPS and
allow an efficient sulfonation of the target, reducing the manufacture
cost of the process (Figure~\ref{fig7}).

\begin{figure}
\includegraphics{fig07}
\caption{\label{fig7}Activity of final variant (Var09) of AST-IV.}
\end{figure}

\section{Outlook and opportunities}\label{sec6}

Environmental considerations drive our processes all along development
and industrial phases. This is a key factor in accelerating the
implementation of our internal Biosynthetic Technologies platform. A
key success factor for the implementation of biocatalysis is the
collaboration between organic chemists and biochemists to design new
and more efficient chemical routes enabled by biocatalysis.

The examples described in this article show that the implementation of
enzymatic transformations in the synthesis of complex molecules of our
R\&amp;D portfolio has a positive impact on environmental footprint and can
be in some cases the only viable alternative for large scale clinical
batch manufacturing to meet timelines and quality.

We have built a very efficient toolbox to investigate common
biocatalytic transformations using well-developed classes of enzymes
(such as imine reductase (IRED), transaminase, ketoreductase (KRED),
ene-reductase or nitrilase). However, the molecules entering our R\&amp;D
portfolio are becoming increasingly complex. Innovative retrosynthetic
pathway integrating biocatalytic step will be of great interest to
overcome associated CMC risks. Diversification of our toolbox with less
well-investigated classes is needed to enlarge the investigation in the
repertoire of biocatalytic reaction on non-natural substrates.
Currently, certain gaps in the toolbox remain. The biocatalysis
community (academics and industrials) is addressing these challenges
and new enzymatic platforms for industrial chemistry should arise in
the near future~\cite{1}.

Protein engineering is of great importance for developing enzymes for
biocatalytic transformations at an industrial scale. We decided to
build an in-house molecular biology platform (expertise and
capabilities) that allows us to tailor enzyme performance to meet
demanding industrial requirements. A proof-of-concept was achieved
through the development of an efficient AST-IV enzyme. Looking to the
future, the ability in expanding the field of computational methods,
such as artificial intelligence, machine learning or data management
will support our internal strategy toward designing highly efficient
enzyme for non-natural substrates within reduced cycles time. This will
be possible thanks to: (1) the better identification of suitable enzymes
from protein sequence information accessible in databanks (genomic
database mining); (2) smarter mutant libraries design.  

\section*{Abbreviations}

CMC, chemistry, manufacturing and control; DIAD, diisopropyl
azodicarboxylate; DKR, dynamic kinetic resolution; DNA,
deoxyribonucleic acid; IPA, isopropyl alcohol; IRED, imine reductase;
KRED, ketoreductase; PMI, process mass intensity; PLP: pyridoxal
5$^{\prime}$-phosphate; R\&amp;D, research and development; SFC,
supercritical fluid chromatography.

\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.

\section*{Author contributions}

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

\section*{Acknowledgments}

All authors would like to express their gratitude to all the Sanofi
teams who contributed to the work presented in this manuscript. Their
names can be found in the references. 

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