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\DOI{10.5802/crchim.281}
\datereceived{2023-10-15}
\daterevised{2023-11-10}
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\dateposted{2024-02-12}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{French Women in Chemistry in 2024}
%%\def\cdr@specialtitle@french{Les chimistes fran\c{c}aises en 2024}
%\makeatother

\title{The need for Open Labs for fostering interdisciplinarities in
Modern Chemistry. Biocatalysis: a necessary tool for synthetic
chemists}

\alttitle{Plus de synergies entre les laboratoires pour favoriser
l'interdisciplinarit\'{e} en chimie moderne. Biocatalyse~: un outil
n\'{e}cessaire pour les chimistes de synth\`{e}se}

\author{\firstname{Juliette} \lastname{Martin}\CDRorcid{0000-0003-1756-848X}}
\address{70 All\'{e}e Graham Bell, 30035 N\^{i}mes -- SEQENS, France}
\email{juliette.martin@seqens.com}

\keywords{\kwd{Biocatalysis}\kwd{Enzymes}\kwd{Catalysis}\kwd{Asymmetric
synthesis}\kwd{Sustainability}\kwd{Green chemistry}}

\altkeywords{\kwd{Biocatalyse}\kwd{Enzymes}\kwd{Catalyse}
\kwd{Synth\`{e}se asym\'{e}trique}\kwd{Durabilit\'{e}}\kwd{Chimie verte}}

\begin{abstract}
Among the different fields of chemical approaches available to the
synthetic chemists, biocatalysis is only being slowly adopted,
especially due to a lack of know-how and practical experience. However,
enzymatic catalysis is a mature technology, and this should encourage
more chemists to take further ownership and expand applications in
modern chemistry.

In the industrial sector, some pharmaceutical companies have been
pioneers in the use and acceptance of biocatalysis for the unique
properties that enzymes can deliver. In this article, the key concepts
for biocatalysis applications will be addressed including some
recommendations to new practitioners. Some industrial examples will be
presented, such as polycyclization, regioselective acylation, alkene
asymmetric bioreduction, \textit{meso} desymmetrization, kinetic
resolution and aldolization. Biocatalysis provides economic and
environmental benefits, its awareness should be enhanced, especially in
a \textit{momentum} of conducting hybrid approaches and multicatalysis.
Open your Labs!
{\vspace*{-2pt}}
\end{abstract}

\begin{altabstract}
Parmi les diff\'{e}rents domaines d'approches en chimie dont disposent
les chimistes organiciens, la biocatalyse n'est que lentement
adopt\'{e}e, notamment en raison d'un manque de connaissances et
d'exp\'{e}rience pratique. Cependant, la catalyse enzymatique est une
technologie mature, ce qui devrait encourager davantage les chimistes
\`{a} s'approprier davantage et \`{a} \'{e}tendre le champ
d'applications en chimie de synth\`{e}se.

Dans le secteur industriel, certaines soci\'{e}t\'{e}s pharmaceutiques
ont \'{e}t\'{e} pionni\`{e}res dans l'utilisation et l'acceptation de
la biocatalyse pour les propri\'{e}t\'{e}s uniques que peuvent offrir
les enzymes. Dans cet article, les concepts cl\'{e}s pour les
applications de la biocatalyse seront abord\'{e}s, y compris quelques
recommandations destin\'{e}es aux nouveaux utilisateurs. Quelques
exemples industriels seront pr\'{e}sent\'{e}s, tels que la
polycyclisation, l'acylation r\'{e}gios\'{e}lective, la
bior\'{e}duction asym\'{e}trique des alc\`{e}nes, la
d\'{e}sym\'{e}trisation de compos\'{e}s \textit{meso}, la
r\'{e}solution cin\'{e}tique et l'aldolisation. La biocatalyse apporte
des avantages \'{e}conomiques et environnementaux. Une plus grande
sensibilisation vis-\`{a}-vis de la biocatalyse m\'{e}rite d'\^{e}tre
prise en compte, d'autant plus les approches hybrides et de
multicatalyse sont en train de prendre de l'ampleur. \^{O}tez les
fronti\`{e}res entre les laboratoires~!
\end{altabstract}

\maketitle

{\vspace*{8pt}}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

For the past decade, the need to produce compounds with more complexity
in terms of group functionality and chemical structure has expanded
tremendously, including drug discovery in the pharmaceutical field and
materials science. Moreover, the increasing pressure to reduce energy
consumption, protect the environment and conserve natural resources
challenge synthetic methods.

In response to this demand, the execution of complex chemical syntheses
requires expert knowledge, usually acquired over many years of study
and hands-on laboratory practice. However, when cross-fertilization of
multi-competencies is achieved, the outcome may lead to incredible
avenue of innovation and enlarge the vision on synthetic methods and
strategies. Among the different tools available to synthetic chemists,
enzymatic catalysis remains underused. Indeed, there is a lack of
knowledge and training in this area which contribute mainly to the slow
progress.

Considering the significant advances in biotechnologies over the past
two decades, the emergence of plethora new enzymes has occurred.
Contemporary tools are facilitating the access to new ways of
conducting chemical transformations and should complement chemical
intuition.

By examining the current key concepts, this article aims to demystify
biocatalysis for bench chemists so that they may embrace it as a tool
rather than fear it as a too complex technology. Spur the curiosity of
synthetic chemists who are willing to fulfill the gaps in knowledge and
increase the number of successful stories in biocatalysis, especially
within the trends of building multicatalysis approaches.

To familiarize yourself with biocatalysis, books by Grogan~\cite{1} 
and Faber~\cite{2}, and comprehensive contemporary review which have
been compiled by Faber \etal~\cite{3} and Whittall \etal~\cite{4},  are
worth reading. The recent review by Lin \etal~\cite{5} provides a
convenient correlation of enzyme catalyzed reactions with named organic
chemical reactions that organic chemists are familiar with.

\section{Biocatalysis is in its Golden Age}\label{sec2}

The emergence of catalysis in the 18{th} century has had a
transformative impact over the decades in many fields, including the
production of chemicals, healthcare, materials, agriculture and in the
environmental sector. A majority of all commercial chemicals are
produced by methods involving at least one catalytic step. Still,
research in catalysis remains one of the most dynamic areas of chemical
synthesis research, and in addition, the quest for more sustainable
methods encourages synthetic chemists to consider catalysis as a
central science and as a key contributor. 

Traditionally, catalysis has been subdivided into three fields:
heterogeneous catalysis, homogeneous catalysis and biocatalysis. It can
also be classified in terms of thermo-, electro-, and photocatalytic
processes within the three fields. These disciplines have been
developed mostly independently to address specific scientific and
distinct catalytic challenges. Since the late 1990s, a series of
fundamental studies on metal-free chiral catalysts, such as ketones,
thioureas, amines and amino-acids for asymmetric catalysis have sparked
a new craze for organocatalysis~\cite{6}. It is a promising
alternative, generally exhibiting better moisture and air tolerance, as
well as excellent compatibility with various functional groups.
However, their main limitation is their low efficiency. In many cases,
it is necessary to load a high quantity of catalyst and the selectivity
is difficult to control. Besides, in some cases the molecular weight of
the catalyst is superior to the weight of the desired product. The need
to develop highly chemoselective and efficient catalysts remains a
challenge in synthetic chemistry both in organocatalysis and in
organometallic catalysis.

In recent years, cross-fertilization between these distinct fields and
complementary hybrid approaches to catalysts design have begun to
address some of the inherent limitations of the traditional fields.
These efforts have enabled combining some of the most attractive
features of the disciplines, such as chemocatalysis. 

In this article, enzymatic chemistry will be the main focus,
highlighting the considerable progress that has been made over the past
two decades leading to a mature technology in organic synthesis. Major
advances in enzyme discovery and engineering have brought with them a
surge in the development and implementation of biocatalytic reactions
in organic syntheses executed in both academic and industrial settings.
Moreover, they offer retrosynthetic disconnections that can be quite
distinct from typical chemical small-molecule-mediated transformations.

\subsection{State-of-the-art}\label{ssec21}

The amazing efficiency with which living organisms build complex
molecules from abundant starting materials has inspired chemists for
centuries. To produce natural compounds, there are necessarily
individual enzymes that have specific tasks for specific compounds.
Enzymes excel at catalyzing chemical transformations in complex
cellular media and, despite their selectivity, some of them are even
evolving \textit{in~vivo} to specifically handle non-natural
chemicals~\cite{8}.

Innovation inspired by nature has led to recent significant
achievements in the field of biocatalysis, enabling scientists to mimic
this approach by using evolutionary strategies to create and tune
enzymes for a broad and diversified applications~\cite{9a,9b,9c,9d}.
One of the major
advances has been the development of directed evolution of enzymes
which has enabled the adaptation of enzymes and their optimization to
the needs of catalysis. The work on directed evolution has been
rewarded with the Nobel Prize in Chemistry in 2018 to Professor Frances
Arnold\footnote{Nobel Price received with Gregory Winter and Georges P.
Smith, for their work on directed evolution of peptides and antibodies
via phages.}.

Enzymes have a promiscuous behavior which is useful for exploring other
types of reactions that have not yet been discovered. Indeed, catalytic
promiscuity~\cite{11} is defined as the ability of an enzyme to
catalyze secondary reactions. Interestingly, promiscuous enzymes
provide the opportunity for new reaction applications through molecular
evolution in the laboratory, through protein engineering techniques and
reaction engineering.

Another major breakthrough for the discovery of new enzymes has been
the extraordinary advances in high-throughput DNA sequencing and also
the development of computer-assisted tools for protein modeling
combined with protein engineering techniques. All these technical
advances have made it possible to enlarge the application of
biocatalysis in multiple synthetic routes~\cite{12,13}. Today, the
number of reactions accessible by a biocatalytic approach has
considerably expanded~\cite{14}. However, for neophytes, its use seems
restricted to certain experts, whereas it should be more widespread
among synthetic chemists.

For the synthetic chemists, biocatalysis offers a number of advantages:
\begin{enumerate}[(1)]
\item enzymes as catalysts can typically deliver superior regio-,
stereo-, and enantioselectivity,
\item biocatalysis can significantly shorten multi-step synthesis
routes by allowing reactions and reaction sequences, which cannot be
carried out with classical chemical means,
\item biocatalysis is generally associated with mild reaction
conditions, {e.g.}, avoiding the use of toxic reagents and high
temperature or pressure.
\end{enumerate}
However, chemists have preconceived ideas on biocatalysis such as:
\begin{enumerate}[(1)]
\item enzymatic process is necessarily carried out in very dilute
conditions and can only work in water,
\item the enzymes are not stable,
\item high-cost contribution of enzymes in the process,
\item the reaction does not work for compounds that are not soluble in
water,
\item or native enzymes will not be active on substrates that are
chemically different from the natural substrate. 
\end{enumerate}
These considerations are not anymore valid. Biocatalysis in organic
synthesis has faced many challenges related to stability, substrate
scope, and restricted reaction conditions. Enzymes can be easy to
handle, and it should be acknowledged that enzymatic reactions can be
carried out in combination of water with organic solvents (even fully
excluding the presence of water) and, in addition the stability and
productivity of biocatalysts for commercial applications are now widely
demonstrated~\cite{15}.
Cost contribution of the biocatalyst into the process is
an important milestone. General rules for cost analysis have been
established for biocatalyst production based on biocatalyst format
(whole cells, soluble or immobilized enzyme), product cost, market size
and value, and the requirements for the implementation of biocatalytic
processes~\cite{16} have been carefully analyzed. Enzymes still are the
exception rather than the rule in the synthesis of fine chemicals and
pharmaceuticals; biocatalysis is often regarded as a second generation
process. Although it holds great promise as a powerful tool for
application in process chemistry, the number of applications at
commercial scale is still low. By means of enzyme and/or reaction
engineering, cost-effective process is achievable. For illustration, in
one of the following case study referring to enantioselective
aldolization to produce D-serine at manufacturing scale, the
biocatalyst cost contribution is less than~5\%.

\subsection{The experimental method for implementing biocatalysis} 
\label{ssec22}

When designing the retrosynthesis pathway to produce the targeted
molecule, the integration of the enzymatic approach should be
considered as it may enlarge the route alternatives and/or potential
short-cuts. To select the enzymatic step, the disconnection approach
through functional group consideration, will help to determine the
appropriate enzyme family. To plan the required tests to be carried
out, the following features have to be considered.

\subsubsection{Access to enzymes}\label{sssec221}

Enzyme providers, such as Prot\'{e}us by Seqens, offer enzyme
collections in single vials and/or microtiterplate format for rapid
screening (Seqenzym$^{\text{\textregistered}}$\ kits) and will ensure
availability at larger scale.

\xfigure{fx01}{Customized Seqenzym$^{\text{\textregistered}}$ kit.}

Cofactor-independent enzymes are usually preferred for organic
synthesis because they don't require cofactor recycling. Nevertheless,
there are known methods to recycle cofactor-dependent enzymes:
\begin{itemize}
\item NADH can be recycled by adding \textit{iso}propanol or using
Formate DeHydrogenase (FDH) with formic acid.
\item NADPH can be recycled using Glucose DeHydrogenase (GDH) and
glucose or NADPH-dependent FDH variants.
\item Pyridoxal-5$'$-phosphate (PLP)-dependent enzymes usually do not
need cofactor recycling, but adding a small amount of PLP maybe
recommended for stability.
\item Thiamine diphosphate in lyases is sufficiently accessible and
does not require recycling.
\item Efficient ADP recycling systems for ATP-dependent enzymes are
currently lacking, so whole cells with glucose supplementation are
used.
\end{itemize}

\subsubsection{Screening conditions}\label{sssec222}

The enzyme screening approach needs to be combined with adequate
reaction conditions to evaluate the technical feasibility and to
determine the best enzyme candidates. Once the size of the enzyme
library is addressed, the implementation of the most appropriate
analytical method is required for activity measurement. Regarding the
reaction conditions, the next features should be taken into account:

Most enzymes can tolerate the presence of organic solvents. Typically,
the concentration of water-miscible organic solvents (like DMSO, DMF,
lower molecular weight alcohols) should not exceed 10--20\% (v/v).
Enzymes may tolerate water-non-miscible solvents (toluene, ethers),
leading to biphasic systems that can ease product isolation and
potentially favor product formation. Certain enzymes, especially
lipases, can be highly active in pure organic solvents. Noteworthy,
maintaining appropriate pH and temperature is crucial for enzyme
activity.

When working with enzymes, it is important to evaluate the risk of
substrate and product inhibition.

According to the activity results, further confirmation and statement
on enzymes performance will need to be carried out independently, in
order to select the best enzyme candidate in respect of the targeted
criteria, such as stability and robustness under the chosen reaction
conditions. At this point, acceptable specifications can be reached
within the selected enzyme. For industrial applications, the choice of
criteria acceptance and relevance at the screening stage is very
important prior to further development studies.

Stability, selectivity and activity, are the prominent criteria for all
types of catalysis, homogeneous, heterogeneous and biocatalysis, but
they may be evaluated in a very different way. Enzymes are proteins and
they may be typically stable in the range of moderate temperatures and
moderate pH values, unless using extremozymes~\cite{17}. Under these
conditions they can be remarkably active. Several enzymes may be
limited only by diffusion of the starting material to the active site,
allowing however extremely high turnover frequencies once the medium
composition is fulfilled. Air is often not a problem for enzymes, while
many homogenous and heterogeneous catalysts will be oxidized and
deactivated by air, even at room temperature. 

Choosing the best route depends on factors like enzyme availability,
starting material cost, solubility, product isolation, and
productivity. Careful evaluation of the entire process is necessary
before making a decision.

\subsubsection{Enzyme properties improvement}\label{sssec223}

To achieve both an excellent selectivity and a broad substrate scope,
modern biocatalysis relies on libraries of enzymes, similar to the
catalyst libraries used in chemoselective metal-based catalysis.
Noteworthy, compared to other catalyst-based chemical transformations,
when observing initial poor performance with an enzyme, such as very
low conversion, it remains yet a good starting point for further
remarkable improvement, as recognition (or affinity) between the enzyme
and the substrate remains the key feature. In the case where the
diversity of available libraries does not provide the \mbox{desired}
\mbox{specifications,} nor within studied reaction conditions, it may be
required to engineer an existing biocatalyst for a specific substrate
shape or even identify/evolve a new biocatalyst for a substrate for
which no catalyst is available at all. 

Moreover, enzymes have a chiral 3D structure that interacts at multiple
contact points with the substrate, thus explaining activity and
selectivity. As a result, through protein engineering, modifications of
protein sequences and therefore of their 3D structures, will alter the
properties of a biocatalyst indefinitely until reaching the targeted
specifications. Conversely, the selectivity and reactivity of typical
chemical catalysts are more limited to the available structures of the
catalysts themselves or of their ligands.

\subsection{In the field of pharmaceuticals manufacturing}
\label{ssec23}

The pharmaceutical industry has already adopted biocatalysis as an
essential tool~\cite{18} for providing efficient and sustainable
catalytic routes toward APIs. The synthesis of active pharmaceutical
ingredients (APIs) can be particularly challenging as they become more
complex molecules. Chemists recognize the value of enzymes related to
exceptional selectivity, in terms of stereo-, regio-, and
chemoselectivity in many biotransformations. This field has gained
impressive developments offering robust, scalable processes along with
a very diversified panel of enzymes that are required.

Today, the pharmaceutical industry is facing significant challenges
associated with environmental regulations, including productivity and
costs~\cite{19}, which provides the opportunity to expand synthetic
methods within green chemistry technologies. Consequently, the
identification of innovative, cost-effective and environmentally
responsible synthetic methods becomes a ``Must-Have''. 

\subsection{How sustainable is biocatalysis?}\label{ssec24}

Enzyme catalysis is a clean, green, and sustainable alternative to
traditional synthetic catalysts, as enzymes exhibit an exquisite
selectivity and efficiency, and work under mild reaction conditions
(significantly less energy consumption), while eliminating the use of
hazardous solvents and reagents (less waste), making it a green
alternative to classical strategies~\cite{20}. The conciseness,
environmental friendliness, and atom economy of synthetic methods have
become important aspects of synthetic chemistry (Figure~\ref{fig1}).

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Biocatalysis is aligned with the green chemistry
principles.}
\end{figure*}

In terms of process-based chemistry, biocatalysis is aligned with all
the principles of green chemistry. Alignment with five of the
principles is a consequence of enzymes generally exhibiting high
\mbox{chemo-}, regio-, and stereoselectivities that are in general difficult
to achieve with traditional catalytic methods. In particular, the
(near) perfect enantioselectivities observed with highly engineered
enzymes are practically inimitable. 

The use of enzymes in organic synthesis has clear economic and
environmental benefits. Enzymes are produced from readily available and
inexpensive renewable resources and the cost of production is,
therefore, essentially stable and predictable. This contrasts with the
serious environmental costs of precious metals and the disruptive price
fluctuations caused by competing demand from other
industries~\cite{21}. Moreover, the significant costs associated with
removing traces of noble metals from end products to the very low
levels that are demanded by pharmaceutical regulatory authorities, are
prohibitive. \looseness=-1

Regarding the production of enzymes, you need to know how they are
produced, but a synthetic chemist should focus on their use within the
awareness of mechanistic insights in the chemical transformation.

\subsection{How are enzymes produced?}\label{ssec25}

An enzyme is a polypeptide chain that includes on average 200--600
amino-acids, hence it is necessarily produced by fermentation. At
industrial scale, depending on the production host---for example:
bacteria or yeast, the cultivation times are around 24~h--72~h.

In most enzyme-based catalysis, the term biocatalyst refers typically
to a crude cell extract from the production strain ({e.g.}, 
\textit{E.~coli}), which may contain many other enzymes that, in
general, do not interfere with the intended reaction. The whole cells
containing the non-natural expressed enzyme may also be used as
catalyst preparation, thanks to the recombinant DNA technology. Enzyme
purification by chromatography is prohibitively expensive on a large
scale, explaining the prevalence of crude \mbox{preparations} for biocatalysis
applications. Certain production strains ({e.g.}, \textit{Yarrowia
lipolytica}, \textit{Pichia pastoris}) allow the secretion of enzymes
into the aqueous medium in which the production strain grows, making
separation straightforward and leading directly to a rather pure enzyme
preparation. 

In the following case studies, the use of either a single enzyme
(provided as crude preparation) or in combination of different enzymes
in one-pot have been implemented. For illustration, below are cited
some of our work, where biocatalysis was applied successfully with key
considerations for limiting the number of chemical steps, such as
protection and deprotection steps. But also, for replacing metal-based
catalytic process by an enzyme in an asymmetric reduction of alkenes,
desymmetrization of prochiral compounds, and in kinetic resolution
steps to avoid separative chromatography.

\section{Applications}\label{sec3}

\subsection{Biocatalytic synthesis of (${-}$)-Ambrox}\label{ssec31}

The terpenoid molecule: (${-}$)-8,12-epoxy-13,14,15,{\ubreak}16-tetranorlabdane,
so-called (${-}$)-Ambrox, is a valuable ingredient related to
Ambergris, exhibiting amber and woody scent. It is widely used in the
fragrance industry. For a longtime, it has been produced starting from
the diterpenic alcohol, sclareol~\cite{22a,22b}. The latter is readily
extracted in sufficient quantities from clary sage (\textit{Salvia
sclarea} L.). The historical and chemical synthesis consists of seven
steps involving long reaction times and hazardous reagents such as
peracetic acid, lithium aluminum hydride, and butyllithium, a
stoichiometric oxidation with sodium \mbox{periodate,} and the generation of
enormous amounts of waste in addition to the 76\% global yield of the
desired product (Scheme~\ref{sch1}).

\begin{scheme*}
\includegraphics{sc01}
{\vspace*{6pt}}
\caption{\label{sch1}Chemical route to (${-}$)-Ambrox starting from
sclareol~\cite{1}.}
\end{scheme*}

Later, a green two-step process has been reported~\cite{23},
which involves the
conversion of sclareol to ambradiol, catalyzed by whole cells of
\textit{Hyphozyma roseoniger}, followed by cyclization to
(${-}$)-Ambrox over a Ca--Y zeolite at ambient temperature, both steps
proceeding in 98\% yield (Scheme~\ref{sch2}).

\begin{scheme*}
\includegraphics{sc02}
{\vspace*{6pt}}
\caption{\label{sch2}Chemo-enzymatic route to (${-}$)-Ambrox starting
from sclareol.}
\end{scheme*}

More recently, Eichhorn and Schroeder described a new industrial method
for producing (${-}$)-Ambrox starting from ($E$,$E$)-homofarnesol, which
includes a biocatalytic step using a Squalene Hopene Cyclase (SHC)
enzyme (Scheme~\ref{sch3})~\cite{24}.

\begin{scheme*}
\includegraphics{sc03}
{\vspace*{6pt}}
\caption{\label{sch3}SHC-mediated (${-}$)-Ambrox synthesis from
$\upbeta$-farnesene via ($E$,$E$)-homofarnesol.}
\vspace*{7pt}
\end{scheme*}

Squalene-Hopene Cyclase (SHC) enzymes are remarkable because they
transform the C30 terpene squalene into the pentacyclic product hopene
through a unique cationic polycyclization process involving multiple
C--C bond formations. This transformation is initiated by the
protonation of the C${=}$C double bond of the terminal isoprene unit. The
properties of the SHC enzyme were improved by evolving the wild type
enzyme to produce a biocatalyst suited for application at industrial
scale (Scheme~\ref{sch4})~\cite{25}.

\begin{scheme*}
\includegraphics{sc04}
{\vspace*{6pt}}
\caption{\label{sch4}Biocatalytic reaction with evolved SHC enzyme:
work with Prot\'{e}us by Seqens~\cite{4}.}
\end{scheme*}

The engineering strategy generated a significant range of new variants
that were then tested experimentally in the reaction~\cite{25}. As a
result, the best variants resulted in the following: 
\begin{itemize}
\item Improved ($E$,$E$)-homofarnesol conversion properties. With one
round of random mutagenesis, 1.5- to 10-fold compared to the wild type
enzyme.
\item Conversion of 125~g/L ($E$,$E$)-homofarnesol. The maximal
productivity observed was about 10~g/L/h, with approximately a 10-fold
improvement with the best variant compared to the wild type enzyme.
\item Further optimization of the reaction conditions was carried out
by DoE (Design of Experiments) with the best performing variant.
\end{itemize}

\subsection{Regioselective acylation}\label{ssec32}

Simvastatin is a lipolipidemic compound that lowers the cholesterol and
triglycerides circulating in the blood, this drug is marketed by Merck
as Zocor. Historically, the manufacturing chemical route synthesis,
involved six chemical steps starting from lovastatin, including
protection and deprotection steps (Scheme~\ref{sch5}).

\begin{scheme*}
\vspace*{-1.5pt}
\includegraphics{sc05}
{\vspace*{5pt}}
\caption{\label{sch5}Chemical route to simvastatin starting from
lovastatin.}
\end{scheme*}

Following the hydrolysis of the natural product lovastatin to give
monacolin J, this latter was then converted to simvastatin by
lactonization. Subsequent protection of the hydroxyl group followed by
acylation to introduce the dimethylbutyryl side chain yields the
protected form of simvastatin, which is then deprotected to yield
simvastatin. The overall process needs six steps which are technically
and economically demanding.

By integrating an enzymatic approach, the overall chemical steps could
be reduced to only two steps (Scheme~\ref{sch6}), thanks to a
regioselective acylation using an acyl transferase with a cheap acyl
donor, e.g. dimethylbutyryl phosphonate~\cite{26}, and starting from
the lactone-hydrolyzed form of lovastatin. The benefits of using
acyl-phoshonates are: 
\begin{itemize}
\item low cost acyl donors easy to produce,
\item acyl transferases accommodate acyl-phosphonates donors,
\item non reactive leaving groups.
\end{itemize}

\begin{scheme*}
\includegraphics{sc06}
{\vspace*{4pt}}
\caption{\label{sch6}Biocatalytic route to simvastatin starting from
lovastatin.}
\end{scheme*}

\vspace*{-2pt}

\subsection{Alkene asymmetric bioreduction}\label{ssec33}

\vspace*{-1pt}

Danoprevir is a protease inhibitor used for the treatment of hepatitis
C virus (HCV). During the Covid-19 pandemic crisis, some antiviral
drugs were evaluated to repositioning on Covid-19 treatment. This
product was discovered by Array BioPharma (now Pfizer) and first
licensed to Roche for development and commercialization. The
modified-peptidic type macrocyclic structure of Danoprevir is built
according to a 20 steps convergent synthesis~\cite{27}. To build
Danoprevir, the retrosynthetic pathway considers three main
disconnections, that lead to three fragments: an hydroxy
proline~\textbf{1}, a protected amino ester~\textbf{2}, and a
substituted cyclopropane~\textbf{3}. The key steps will be two peptide
couplings and a ring closing metathesis (Scheme~\ref{sch7}).

\begin{scheme*}
\includegraphics{sc07}
{\vspace*{6pt}}
\caption{\label{sch7}Retrosynthetic pathway of danoprevir compound.}
\vspace*{1pt}
\end{scheme*}

Regarding the synthesis of~\textbf{2}, the unnatural $\upalpha$-amino
acid derivative possessing a terminal alkene, was produced by a
rhodium-catalyzed asymmetric hydrogenation of the dehydroamino
ester~\textbf{4}~\cite{28} (Scheme~\ref{sch8}).

\begin{scheme*}
\includegraphics{sc08}
{\vspace*{6pt}}
\caption{\label{sch8}Asymmetric hydrogenation with a rhodium-based
chiral catalyst.}
\vspace*{1pt}
\end{scheme*}

Despite a perfect enantio- and chemo-selectivity for the conjugated
alkene, the use of this transition metal catalyst suffers from a
tedious preparation of the chiral diphosphine ligand and from the
actual price of rhodium, which has a commercial price that has
increased by a factor of 8--13 between 2015--2020.

A cheaper alternative was envisaged by using a metal-free catalytic
step, such as the reduction of the enoate analog~\textbf{4} by an ene
reductase (ERED) (Scheme~\ref{sch9})~\cite{29}.

\begin{scheme*}
\includegraphics{sc09}
{\vspace*{6pt}}
\caption{\label{sch9}Enzymatic reduction of a dehydro-amino
intermediate.}
\end{scheme*}

This NAD(P)-dependent ERED family of enzymes can reduce selectively
substituted alkenes conjugated to an electron-withdrawing group meaning
that chemo-selectivity between the two alkenes in~\textbf{4} could be
achieved. Generally, the ERED-catalyzed reactions proceed with high
enantio-selectivity in mild conditions without hydrogen gas. An
efficient system for the regeneration of the NAD(P)H cofactor could be
easily implemented to afford a greener\break process.

At Prot\'{e}us by Seqens, the asymmetric bioreduction of a
dehydro-amino ester analog has been carried out (Scheme~\ref{sch10}).
The EREDs tested have allowed to selectively reduce only one of the
alkene\break functions.

\begin{scheme*}
\includegraphics{sc10}
{\vspace*{6pt}}
\caption{\label{sch10}Bioreduction of dehydro-amino acid derivative
with ERED Seqenzym$^{\text{\textregistered}}$.}
\end{scheme*}

A panel of twenty ene reductases (EREDs)
Seqenzym$^{\text{\textregistered}}$\ have been tested. Within the
exclusion of two enzyme candidates, all exhibited activity and the
conversion rate during this first series of screening was moderate
(around 30\%) but still high degree of enantioselectivity were
detected. Optimization of the reaction conditions is also on its\break way.

Depending on the scale of production, replacing a reaction catalyzed by
a transition metal by an enzymatic reaction can result in a competitive
\mbox{process.} This is explained by the volatility of the price of transition
metals compared to the stable prices of raw materials used in the
enzyme production (glucose, mineral salts, etc.). In addition, there is
a strong scale effect with a cost reduction during the production of a
biocatalyst making the latter competitive for the intermediate
production on a large scale. Furthermore, when evaluating the
manufacturing cost, the overall process must be considered, including
potential purification steps to remove some\break impurities.

\subsection{Desymmetrization strategy}\label{ssec34}

Understanding that desymmetrization is the transformation of a
substrate that results in the loss of a symmetry element, that
precludes chirality (plane of symmetry, center of symmetry), this
concept is one the most efficient strategy for obtaining
enantioenriched molecules, within 100\% attainable yield. The exquisite
advantage of enzymes being highly specific, makes enzymatic
desymmetrization of prochiral molecules an obvious and efficient method
for obtaining enantiomerically pure compounds.

To desymmetrize 2-Me-propanedi-acetate and a cyclopentan-diester
analog, a screening test of 80 enzymes, such as lipases, were realized
and implemented at large scale. In both examples, we reached high
enantioselectivity (ee {${>}$} 98\%) (Scheme~\ref{sch11}).

\begin{scheme*}
{\vspace*{-2pt}}
\includegraphics{sc11}
{\vspace*{6pt}}
\caption{\label{sch11}Enzymatic desymmetrization with lipases starting
from 2-Me-propanedi-acetate and cyclopentan-diester analog.}
\end{scheme*}

Lipases are astonishing catalysts with a vast range of applications
including the synthesis of esters/acids, and polymers. The broad
specificity of the substrates, as well as their regio-, stereo-, and
enantioselectivity, are the differentiating factors of these enzymes.
They are also attractive biocatalysts in the kinetic resolution of
racemic mixtures and they are highly robust in organic solvents.

\subsection{Kinetic resolution}\label{ssec35}

For the substituted aminoester~\textbf{5}, a lipase was identified in
the collection of Seqenzym$^{\text{\textregistered}}$\ for the kinetic
resolution of racemic 4-amino-butanoate derivative, with high
enantioselectivity (Scheme~\ref{sch12}).

\begin{scheme*}
\includegraphics{sc12}
{\vspace*{6pt}}
\caption{\label{sch12}Resolution of $\upbeta$-aminoester derivative.}
{\vspace*{-4pt}}
\end{scheme*}

However, the lipase candidate suffered from thermal stability issue
during the course of the reaction. Thus, it was decided to run an
experimental statistical strategy with DoE approach (Design of
Experiments), to immobilize the enzyme by absorption on the most
efficient resin support. Ten different resin carriers were tested. This
work resulted in implementing a new biocatalyst. As shown in
Figure~\ref{fig2}, the new immobilized enzyme, allowed running the
resolution process without the presence of the undesired enantiomer,
even at prolonged time. The resulting biocatalyst had exclusive
stereopreference in favor of the ($R$)-configuration. Figure~\ref{fig2}
exhibits the results obtained with the corresponding immobilized
enzyme.\looseness=-1

\begin{figure}
\includegraphics{fig02}
\caption{\label{fig2}Conversion and enantioselectivity profiles during
kinetic resolution with the corresponding immobilized enzyme.}
\end{figure}

We were able to measure the enantiomeric ratio $E =
(k_{\mathrm{cat}}^{R}/K_{m}^{R})/(k_{\mathrm{cat}}^{S}/K_{m}^{S})$,
which is a measure of the intrinsic selectivity of the catalyst and
therefore gives a concise representation of the enantioselective
properties of an enzyme in reactions involving chiral
compounds~\cite{30}. As such, we obtained a value of $E>150$ with the
new immobilized enzyme, to be compared with the initial value of 
$E=60$. Consequently, we were able to improve the productivity by a
factor of~44. By reaching such high enantiomeric ratio, this leads to
the optimum process for ensuring full chirality control, without the
presence of the undesired enantiomer at manufacturing scale and in
multi-batch campaigns. 

In the second example represented in Scheme~\ref{sch13}, the kinetic
resolution was carried out on a racemic compound: $\upalpha$-methyl
$\upalpha$-alkyl aldehyde derivative using a ketoreductase (KRED) from
Seqenzym$^{\text{\textregistered}}$, leading to the desired
enantioenriched $\upbeta$-alcohol product ($\mathrm{ee} > 98\%$). By working on
the process conditions, we were able to manufacture at large scale
starting with 400~g/L of substrate, the cofactor system NAD$^{+}$/NADH
was regenerated by using \textit{iso}propanol (IPA) as co-substrate.
Conversion was completed within 8~h, and the desired product was
isolated within 47\% yield, after trapping the remaining
enantioenriched aldehyde intermediate with bisulfite salt reagent to
make the isolation process easier.

\begin{scheme*}
{\vspace*{1pt}}
\includegraphics{sc13}
{\vspace*{6pt}}
\caption{\label{sch13}Kinetic resolution of (${\pm}$)-$\upalpha$-methyl
$\upalpha$-alkyl-aldehyde with KRED Seqenzym$^{\text{\textregistered}}$.}
{\vspace*{2pt}}
\end{scheme*}

The scope with other aldehyde substrates was investigated with KRED
Seqenzym$^{\text{\textregistered}}$\ family leading to preferential
($R$)- or ($S$)-enantiomers as presented below (Scheme~\ref{sch14}).

\begin{scheme*}
\includegraphics{sc14}
{\vspace*{6pt}}
\caption{\label{sch14}Scope of products formed with KRED
Seqenzym$^{\text{\textregistered}}$\ by kinetic resolution of racemic
aldehydes.}
{\vspace*{1pt}}
\end{scheme*}

\subsection{Enantioselective aldolization}\label{ssec36}

In this work, an enzymatic process, for producing non-natural
amino-acid e.g. D-serine, has been developed and implemented, which is
a key building block for the synthesis of several APIs. The enzymatic
aldolization step is carried out on glycine as starting material
(Scheme~\ref{sch15}). This approach is an alternative strategy to
access D-serine and to avoid conventional chromatographic separation
process.

\begin{scheme*}
\includegraphics{sc15}
{\vspace*{6pt}}
\caption{\label{sch15}Enzymatic aldolization of glycine substrate.}
\end{scheme*}

It is important to mention that this process leading to very high
enantioselectivity is also carried out at high substrate concentration
(${>}$300~g/L), and that the cost impact of the biocatalyst added in
the process contributes to less than 5\% of the 
\mbox{overall} \mbox{chemical}
production cost. Noteworthy, enzyme residues in the final product
isolation are below detection limit. Besides, the enzymatic
aldolization conditions were implemented into a cascade reaction for
producing ($S$)-ketose~\cite{31} (Scheme~\ref{sch16}). 

\begin{scheme*}
{\vspace*{2pt}}
\includegraphics{sc16}
{\vspace*{10pt}}
\caption{\label{sch16}Enzymatic cascade reaction from glycine to
(3S)-ketose derivative. Step 2 and step 3 are simultaneous.}
\end{scheme*}

\section{The emergence of multi-catalysis systems}\label{sec4}

Driven by the demand for more efficient and sustainable chemical
processes, and bringing solutions for more complex molecules, the field
of catalysis \mbox{continues} to evolve rapidly. With the aim of minimizing
isolation and purification steps during multistep syntheses, concurrent
catalytic cascades have attracted increasing attention and, recently,
there has been a growing interest in developing multi-catalytic
systems. 

The biocatalytic route developed by Merck \& Co. for the manufacture of
the anti-HIV drug islatravir illustrates how efficient cascades
reactions are, along with protein engineering for fine tuning enzyme
performances (Scheme~\ref{sch17})~\cite{32}.

\begin{scheme*}
\includegraphics{sc17}
{\vspace*{6pt}}
\caption{\label{sch17}Chemical structure of islatravir.}
\end{scheme*}

Multi-catalysis emerges not only to enlarge the avenue of new methods
but also to offer potential new valuable short-cuts to the synthetic
practitioner. Within multi-catalytic processes, either multiple
catalysts execute single reactions, or precise sequences of multiple
catalytic reactions occur in a one-pot fashion. Cascade reactions can
achieve more than the separated individual steps. This is particularly
the case when an equilibrium reaction is coupled with an irreversible
catalytic step.

The combination of several catalysts from different fields, such as
chemocatalysis and biocatalysis, has been developed. For example,
Bornscheuer and coworkers developed a method for producing chiral
biaryl amines by combining a biocatalytic method, with an amino
transfer using an optimized transaminase enzyme, with a chemocatalysis
Suzuki-Miyaura Coupling (SMC) reaction~\cite{33}  (Scheme~\ref{sch18}).

\begin{scheme*}
\includegraphics{sc18}
{\vspace*{6pt}}
\caption{\label{sch18}Combination of the Suzuki--Miyaura cross-coupling
reaction with engineered transaminases.}
\end{scheme*}

The ability of such species to work under ``enzyme-compatible''
conditions continues to trigger the development of linear cascades.

\section{Perspectives}\label{sec5}

Remarkably, multi-catalytic sequences can undergo through otherwise
unfeasible pathways thanks to, for instance, prospectively unstable
intermediates being converted as soon as formed. Complex molecules need
to be produced, especially in the pharmaceutical industry and the
design of methods to access these compounds in a minimum of steps is
highly desirable. For illustration, the synthesis of Eribulin
(Figure~\ref{fig3}), targeting anticancer treatment, is a highly
complex molecule possessing 19 stereogenic centers. It was 
designed~\cite{34} thanks to synthetic chemist's skills. Still, among
the first process generation, a 62-step sequence was required for the
preparation of this drug which obviously leaves room for further
improved productivity and route design, potentially by using some
biocatalytic steps.

\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}Eribulin structure.}
\end{figure}

\section{The awareness of biocatalysis for synthetic chemists must be
strengthen}\label{sec6}

Some synthetic chemists might be hesitant to adopt biocatalysis due to
features like unfamiliarity with the techniques, limited access to
biocatalysts, concerns about scalability, and perception that
biocatalysis could be more complex than traditional chemical methods.
However, as biocatalysis continues to progress, its benefits such as
high selectivity (chemo- and enantioselectivity) and environmentally
friendly conditions, begin to be recognized and more chemists are going
to be motivated to explore its potential. However, most of synthetic
chemists are not familiar with the principles, techniques, and
applications of biocatalysis. Chemists who are more accustomed to
traditional chemical methods might not have the necessary knowledge or
experience to effectively use biocatalysis in their work. This lack of
``familiarity'' could lead to a reluctance to adopt biocatalysis as a
viable option for their synthetic processes.

To address the unawareness of synthetic chemists with biocatalysis,
several features should be considered:
\begin{itemize}
\item \textit{Education and collaborations}. Some
academic\footnote{ESAB: European Society of Applied Biocatalysis.}
and industrial organizations\footnote{Such as SEQENS.}
can offer workshops, seminars,
and training sessions focused on biocatalysis. This would help
synthetic chemists understand the principles, advantages, and
techniques involved, making them more comfortable with incorporating
biocatalysis into their work.

\item \textit{Access to biocatalysts}. An increasing number of
commercially available enzymes (see footnote 3)---ready to be used are
available. Nevertheless, the need to establish repositories or networks
that provide synthetic chemists with easy access to enzymes and
biocatalyst-related tools.

\item \textit{Practical experience}. Training opportunities where
synthetic chemists can work directly with biocatalysts under the
guidance of experts. Practical experience will demystify the process
and boost confidence in using biocatalysis.
\end{itemize}
By addressing the unfamiliarity through education, cooperation, and
practical experience, the barriers preventing synthetic chemists from
using biocatalysis can be gradually overcome. Communicating on success
stories with synthetic chemists can inspire others and demonstrate the
feasibility and benefits of adopting biocatalytic approaches.

\section{Concluding remarks}\label{sec7}

Current biotechnology offers a comprehensive arsenal of powerful
approaches to discover, improve and apply enzymes to produce
value-added compounds. Enzyme-catalyzed reactions have already expanded
the landscape of attractive retrosynthetic disconnections to
selectively build new bonds and design fundamentally new syntheses of
complex molecular structures.

Despite the growing impact of biocatalysis in industrial chemistry, the
full potential of this technology is yet to be unlocked, especially for
reactions that are highly underexplored in enzyme catalysis or not yet
known to the enzyme universe. We hope to bring this knowledge to the
attention of chemists to bridge the educational gap between organic
chemistry and biocatalysis~\cite{37,38a,38b}.

Within industrial organizations, manufacturing active ingredients is
paving the way by taking the best from different fields of expertise,
including synthetic chemists, process engineers and technobiologists. 

This leads to unique opportunity to collaborate across each competency
by sharing their knowledge, know-how and perspectives for allowing the
discovery of new strategic paths which will enable considerable
synthetic economies. Interdisciplinarity is also highly associated with
innovation. 

Innovative organizations with integrated competencies and capacities,
such as high throughput screening platform, can offer these services,
such as Seqens.

\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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