<html><head><meta name="color-scheme" content="light dark"></head><body><pre style="word-wrap: break-word; white-space: pre-wrap;">\makeatletter
\@ifundefined{HCode}
{\documentclass[screen,CRCHIM,Unicode,biblatex,published]{cedram}
\addbibresource{crchim20240991.bib}
\newenvironment{noXML}{}{}
\RequirePackage{etoolbox}
\usepackage{upgreek}
\def\jobid{crchim20240991}
%\graphicspath{{/tmp/\jobid_figs/web/}}
\graphicspath{{./figures/}}
\newcounter{runlevel}
\let\MakeYrStrItalic\relax
\csdef{Seqnsplit}{\\}
\def\refinput#1{}
\def\ubreak{\unskip\break}
\def\back#1{}                                         
\let\rmgamma\upgamma
\let\rmomega\upomega
\usepackage{multirow} 
\DOI{10.5802/crchim.388}
\datereceived{2024-11-28}
\daterevised{2025-02-14}
\dateaccepted{2025-02-21}
\ItHasTeXPublished
}
{\documentclass[crchim]{article}
\usepackage[T1]{fontenc} 
\def\CDRdoi{10.5802/crchim.388}
\let\newline\break
\let\ubreak\relax
\makeatletter
\def\CDRsupplementaryTwotypes#1#2{}
}
\makeatother

\usepackage{upgreek}

\dateposted{2025-03-21}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}


\title{Nucleoside chemistry: a challenge best tackled together} 

\alttitle{Chimie des nucl\'{e}osides : Un d\'{e}fi qu'il vaut mieux relever ensemble}

\author{\firstname{Sarah} \lastname{Westarp}\CDRorcid{0000-0001-6498-8810}}
\address{Chair of Bioprocess Engineering, Institute of Biotechnology,
Faculty III Process Sciences, Technische Universit\"{a}t Berlin,
Ackerstrasse 76, 13355 Berlin, Germany}
\email[S. Westarp]{s.westarp@tu-berlin.de}

\author{\firstname{Peter} \lastname{Neubauer}\CDRorcid{0000-0002-1214-9713}}
\addressSameAs{1}{Chair of Bioprocess Engineering, Institute of Biotechnology,
Faculty III Process Sciences, Technische Universit\"{a}t Berlin,
Ackerstrasse 76, 13355 Berlin, Germany}
\email[P. Neubauer]{peter.neubauer@tu-berlin.de}

\author{\firstname{Anke} \lastname{Kurreck}\CDRorcid{0000-0001-6919-725X}\IsCorresp}
\address{BioNukleo GmbH, Ackerstrasse 76, 13355 Berlin, Germany}
\email[A. Kurreck]{anke.kurreck@bionukleo.com}

\def\thanksname{Note}
\thanks{This article was submitted upon invitation by the guest editor Juliette Martin}

\keywords{\kwd{Nucleoside synthesis}
\kwd{Biocatalysis}
\kwd{Chemoenzymatic synthesis}
\kwd{\textit{N}-glycosylation}
\kwd{Nucleoside analogue}}

\altkeywords{\kwd{Synth\`{e}se de
nucl\'{e}osides}\kwd{Biocatalyse}\kwd{Synth\`{e}se
chimio-enzymatique}\kwd{\textit{N}-glycosylation}\kwd{Analogue de
nucl\'{e}oside}} 

\begin{abstract}
Nucleoside analogues (NAs) are among the most successful small-molecule classes
in the fight against cancer and viral infections. Their importance has
recently been further underlined by the approval of sofosbuvir for the
treatment of hepatitis C virus infections and by developments related
to the corona pandemic. In view of the enormous interest from the
academic world and the pharmaceutical industry, a variety of chemical
and enzymatic synthesis routes have been developed since the first
approval of an NA. The selective chemical synthesis of \tralicstex{Î²}{$\upbeta$}-anomeric NAs through chemical
\textit{N}-glycosylation remains a challenging and, sometimes, a
prohibitively expensive process, thus limiting the available chemical
space. In response, full enzymatic cascades for the synthesis of
NAs have been developed since the discovery of suitable
biocatalysts. Despite significant success, particularly due to advances
in enzyme engineering, constraints persist due to a limited substrate
scope of the available biocatalysts. This mini-review aims to address
both the challenges and potential of chemical and biocatalytic
approaches in NA synthesis. It also illustrates how a
combined strategy could substantially enhance synthesis efficiency as
demonstrated by a few existing examples. Thus the authors hope to
encourage scientists from both domains to join their efforts to drive
innovative solutions in this field.
\end{abstract}

\begin{altabstract}
Les analogues nucl\'{e}osidiques font partie de la classe de petites
mol\'{e}cules les plus efficaces dans la lutte contre le cancer et les
infections virales. Leur importance a r\'{e}cemment \'{e}t\'{e}
soulign\'{e}e par l'approbation du sofosbuvir pour le traitement des
infections par le virus de l'h\'{e}patite C et par les
d\'{e}veloppements li\'{e}s \`{a} la pand\'{e}mie de corona. Compte
tenu de l'int\'{e}r\^{e}t consid\'{e}rable du monde universitaire et de
l'industrie pharmaceutique, diverses voies de synth\`{e}se chimiques et
enzymatiques ont \'{e}t\'{e} mises au point depuis la premi\`{e}re
approbation d'un analogue nucl\'{e}osidique. La synth\`{e}se chimique
s\'{e}lective d'analogues nucl\'{e}osidiques \tralicstex{Î²}{$\upbeta$}-anom\'{e}riques 
par \textit{N}-glycosylation chimique
reste un processus difficile et parfois d'un co\^{u}t prohibitif, ce
qui limite l'espace chimique disponible. En r\'{e}ponse, des cascades
enzymatiques compl\`{e}tes pour la synth\`{e}se d'analogues de
nucl\'{e}osides ont \'{e}t\'{e} d\'{e}velopp\'{e}es depuis la
d\'{e}couverte de biocatalyseurs appropri\'{e}s. Malgr\'{e} des
succ\`{e}s significatifs, notamment gr\^{a}ce aux progr\`{e}s de
l'ing\'{e}nierie enzymatique, des limites persistent en raison de
l'\'{e}tendue limit\'{e}e des substrats des biocatalyseurs disponibles.
Cette mini-revue a pour but d'aborder les d\'{e}fis et le potentiel des
approches chimiques et biocatalytiques dans la synth\`{e}se des
analogues de nucl\'{e}osides. Elle illustre \'{e}galement comment une
strat\'{e}gie combin\'{e}e pourrait am\'{e}liorer consid\'{e}rablement
l'efficacit\'{e} de la synth\`{e}se, comme le d\'{e}montrent quelques
exemples existants. Les auteurs esp\`{e}rent ainsi encourager les
scientifiques des deux domaines \`{a} unir leurs efforts pour trouver
des solutions innovantes dans ce domaine.
\end{altabstract}

\shortrunauthors

\maketitle
{\vspace*{1.8pc}}

\twocolumngrid

\end{noXML}

\section{Introduction}

Nucleoside analogues (NAs) are highly significant pharmaceutical
agents. Their relevance is based on the biological functions of their
natural counterparts (Scheme~\ref{sch1}a) as building blocks of nucleic
acids or as regulators of gene expression, mRNA translation, and cell
signaling. The main fields of application of nucleoside drugs are the
treatment of cancer and viral infections~\cite{bib1,bib2}. The desired
activity is achieved by alteration of the canonical nucleoside scaffold
and its functional groups (Scheme~\ref{sch1}b). The resulting
{${\upbeta}$-NAs} inhibit key enzymes such as
methyltransferases, deaminases, and polymerases, thereby preventing
infection or curing diseases. Additionally, the application of NAs has
been approved for the treatment of the autoimmune disease multiple
sclerosis~\cite{bib3}, and some NAs even exhibit
antibiotic properties \cite{bib4,bib5}. 

\begin{scheme*}
\includegraphics{sc01}
\vspace*{6pt}
\caption{\label{sch1}Nucleoside scaffolds: (a) canonical nucleosides and (b) nucleoside
analogues.  Numbering convention (blue numbers) and reported
modification sites (yellow circles). Yellow circles within nucleobases
signify modifications of the core structure such as heteroatom
position or incomplete{\unskip\break}  rings.}
{\vspace*{-8pt}}
\end{scheme*}

Given the significance of NAs as therapeutic agents,
various chemical, biocatalytic, and {chemoenzymatic} methods have been
developed (Schemes~\ref{sch2} and~\ref{sch3}). In this mini-review,
chemical synthesis refers to processes that do not involve biocatalysts
(enzymes) in contrast to biocatalytic synthesis. Chemoenzymatic
synthesis, on the other hand, denotes methods that combine both
chemical and enzymatic strategies. These terms specifically refer to
the individual reaction steps or corresponding cascade reactions under
consideration. If the synthesis of starting materials were included in
this discussion, the enzymatic production of NAs would
always be classified as a chemoenzymatic approach since the precursor
building blocks are typically derived from synthetic sources.

\begin{scheme*}[t!]
\includegraphics{sc02}
\vspace*{9pt}
\caption{\label{sch2}\textit{N}-glycosylation as a key step in nucleoside
analogue synthesis. Chemical glycosylation reactions are adapted 
from~\cite{bib10}. B$_{1}$---nucleobase 1 and  B$_{2}$---nucleobase 2.
Nucleobases typically have either a purine or a pyrimidine scaffold. NP,
nucleoside phosphorylase; NDT, nucleoside
2$'$-deoxyribosyltransferase.}
{\vspace*{-10pt}}
\end{scheme*}



\begin{scheme*}[t!]
\includegraphics{sc03}
\vspace*{8pt}
\caption{\label{sch3}Chemoenzymatic workflows. (a) Chemoenzymatic
cascade reaction towards molnupuravir~\cite{bib9}. (b) Biocatalytic
base diversification of synthetic 4$'$-thiouridine and subsequent
ethynylation via a Sonogashira cross-coupling reaction \cite{bib20}.
(c) Section of the synthesis route to the cyclic dinucleotide MK-1454
\cite{bib31}. NP,  nucleoside phosphorylase.}
{\vspace*{-7pt}}
\end{scheme*}



Chemical synthesis is the gold standard in NA
production. Recently, this has been impressively demonstrated by Kothapalli
and colleagues in their comprehensive review on synthetic routes for
NAs with antiviral activity~\cite{bib6}. Chemical
synthesis routes were described for all the 14 compounds investigated
while a biocatalytic route was only outlined for one compound, namely
islatravir~\cite{bib7}. This highlights the significant potential of
synthetic chemistry and explains the number of approaches available for
NA synthesis.


Since the discovery of nucleoside phosphorylases~\cite{bib8},
biochemists have focused on enzymatic processes for NA
production to overcome the limitations of chemical methods. The
identification of additional suitable biocatalysts for NA synthesis has
broadened the scope of enzymatic methods, and the production of a wide
range of NAs has been reported. The field has experienced a
revival driven by the demand for sustainable manufacturing processes
and the challenges posed by the COVID-19 pandemic{\unskip\break} \cite{bib7,bib9}.



\textit{N}-Glycosylation is a key step in nucleoside (bio)chemistry,
facilitating the formation of {${\upbeta}$-nucleosides} from a sugar
synthon and a \mbox{nucleobase.} In synthetic chemistry, over time, three core
strategies for the \textit{N}-glycosylation step have been developed.
The Vorbr\"{u}ggen glycosylation is \mbox{frequently} used but is known to
produce nucleosides with poor ${\upalpha}/{\upbeta}$ anomeric purity,
particularly for {2$'$-deoxynucleosides,} and exhibits low
\mbox{regioselectivity} for purine nucleosides
(Scheme~\ref{sch2}, {Equation~(1)}). To address these
challenges, several {alternative synthesis} methods have been introduced
as reviewed by Wang and colleagues~\cite{bib10}. Some of these
approaches, such as gold-catalyzed \textit{N}-glycosylation with ester
or ortho ester ribosyl donors or the use of halide leaving groups,
offer good regio- and stereoselectivity, even for
2$'$-deoxyribosides~\cite{bib10,bib11}
(Scheme~\ref{sch2}, Equations~({2})~and~({3})). To meet the
ongoing demand for novel NAs to be synthesized and
evaluated as potential drugs, numerous specialized synthesis routes
have been developed over time~\cite{bib12,bib13}. For example,
intramolecular ring formation represents an alternative strategy and
was first described by Hager and Liotta in 1991~\cite{bib14}. It
involves the formation of the \textit{N}-glycosidic bond prior to ring
closure. This method provides greater control over the stereochemical
configuration of the \textit{N}-glycosidic bond and offers more
versatility compared to the aforementioned \textit{N}-glycosylation
reactions. More recent efforts have focused on developing 
protecting-group-free synthesis routes~\cite{bib15}.




All reported biocatalytic \textit{N}-glycosylation reactions are either
transglycosylation or direct glycosylation reactions (Scheme~\ref{sch2},
Equations~({4})--({6})). These reactions utilize enzymes
from nucleotide salvage pathways and are anomer-specific. Two
enzyme classes have been identified as efficient biocatalysts for the
synthesis of {${\upbeta}$}-nucleosides: nucleoside phosphorylases and
nucleoside 2$'$-deoxyribosyltransferases~\cite{bib16,bib17,bib18}.
Recent advancements include one-pot nucleobase diversification of
unconventional sugar modifications, such as 4$'$-methylated 
nucleosides~\cite{bib19} and 4$'$-thionucleosides~\cite{bib20}, using nucleoside
phosphorylases as well as a comprehensive characterization of the
substrate scope of \textit{Ll}NDT-2~\cite{bib21}. To enhance the
competitiveness of biocatalysis compared to synthetic chemistry,
several strategies have been explored to (i) improve solubility, (ii)
address thermodynamic limitations, and (iii) broaden the range of
substrates. For instance, employing thermostable enzyme variants
enables reactions at temperatures up to 90~{\textdegree}C and the use
of solvents, which can significantly increase substrate 
concentration~\cite{bib22,bib23,bib24}. Thermodynamic limitations can be overcome
through direct glycosylation~\cite{bib25,bib26} and the removal of
released phosphate using enzymes like sucrose phosphorylase and pyruvate
oxidase~\cite{bib7,bib9}. Additionally, enzyme engineering presents a
promising approach to broaden the substrate range of biocatalysts~\cite{bib7,bib9,bib27}.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Strengths and drawbacks of chemical and
biocatalytic synthesis routes.}
\end{figure*}

This review provides an overview of advantages and drawbacks of purely
chemical and biocatalytic synthesis routes. Regarding chemoenzymatic
approaches, solutions are presented that overcome still existing
challenges of both synthetic chemistry and biocatalysis, showcasing the
advantages of \mbox{combining} both methods. By presenting the key strategies
of NA synthesis, the authors aim to inspire researchers from both
disciplines to collaborate to harness the full potential of this
integrated approach. Hopefully, this will help address the existing
challenges in NA synthesis and pave the way for more
efficient production methods.

\section{Strengths and weaknesses of synthetic chemistry and biocatalysis}

The success of synthetic chemistry in NA synthesis can
be attributed to several key advantages: the wide availability of sugar
synthons, the ability to work with high substrate loads, and the
tolerance for diverse and often extensive modifications (Figure~\ref{fig1}).
Disregarding factors such as yield, efficiency, and sustainability,
synthetic chemistry offers the flexibility to develop a process for
virtually any NA. However, despite its established
dominance, chemical synthesis routes for NAs still
show several drawbacks, including the requirement for harsh reaction
conditions, multiple protection and deprotection steps, and challenges
with regard to regioselectivity and stereoselectivity. These factors
often result in complex multistep procedures, which in sum decrease
overall process efficiency~\cite{bib28}.


In contrast, biocatalytic synthesis routes are highly regio- and
stereoselective, enabling efficient one-pot cascade reactions under
mild reaction conditions. De novo enzyme development and
enzyme engineering now also allow the synthesis of highly modified
compounds. However, despite significant advancements, biocatalytic
production still faces challenges, such as the limited tolerance for
large substituents and certain thermodynamic constraints
(Figure~\ref{fig1}).


\section{Chemoenzymatic approaches for NA synthesis}


In the literature on nucleoside chemistry, only a few chemoenzymatic
synthesis routes have been described so far. Most of the reported
methods primarily focus on biocatalytic endgame reactions, utilizing
either nucleoside phosphorylases or nucleoside
2$'$-deoxyribosyltransferases. This section aims to highlight three
recent cascade synthesis strategies for NAs, each designed to leverage
the complementary strengths of both chemical and enzymatic methods. Two
examples illustrate biocatalytic reactions that have been coupled with
a chemical reaction step, and one example demonstrates a chemical
synthesis that incorporates a biocatalytic step.


One impressive chemoenzymatic cascade reaction is the 
synthesis of molnupuravir, which was reported by McIntosh and
colleagues. After the C5-acylation of ribose by \textit{Candida
antarctica} lipase~B, ribosyl-1-phosphate was formed using 
engineered 5-(\textit{S})-methylthioribose kinase and
\textit{N}-glycosylation was realized by engineered uridine
phosphorylase (Scheme~\ref{sch3}a)~\cite{bib9}. The
reaction was driven by a {sophisticated} ATP regeneration system based on
pyruvate oxidase and acetate kinase. 
The last reaction step required
the interconversion of the amidic carbonyl in the uracil ring to the
corresponding oxime and was performed using synthetic chemistry. It was
necessary to follow this regime as uridine phosphorylase does not
accept cytidine derivatives as substrates. Hence, this approach duly
shows how to overcome substrate limitations of the biocatalyst by
implementing a chemical step.

The second example is work from our own group: starting from commercially
available ribose, where 1$'$-OAc-4$'$-thioribose was initially obtained in
seven synthetic steps. From there, Pummerer-type glycosylation yielded
${\upbeta}$-4$'$-thiouridine with good selectivity~\cite{bib29,bib30}. We
employed thermostable nucleoside phosphorylase to convert
4$'$-thiouridine, for example, to 4$'$-thio-5-iodouridine with 82\% yield. For
this, 4 equiv of base was applied in the transglycosylation reaction.
After purification by semi-preparative HPLC, a Pd-catalyzed Sonogashira
cross-coupling reaction gave 4$'$-thio-5-ethynyluridine in quantitative yields
(Scheme~\ref{sch3}b). A final synthetic step was
necessary since 5-ethynyluridine is thermodynamically unfavored with
respect to most other nucleosides and therefore hard to access by
nucleoside phosphorylase catalyzed transglycosylation. In the same
publication, we demonstrated the versatility of this enzymatic platform
by synthesizing a range of other halogenated purine and pyrimidine
nucleosides~\cite{bib20}.\looseness=1 


          
The third example is the synthesis of nucleotide analogue MK-1454
reported by Benkovic and colleagues. In this process, engineered
${\upalpha}$-ketoreductase is used in between chemical
transformations to ensure the correct anomeric configuration of the
\mbox{2$'$-hydroxy} group from a 2$'$-ketonucleoside intermediate. This
approach exhibited superior diastereoselectivity compared to several
traditional catalysts explored by the authors~\cite{bib31}
(Scheme~\ref{sch3}c).



The limited number of examples of chemoenzymatic synthesis routes can
likely be attributed to the highly specialized environments in which
chemists and biochemists typically work. A 2023 survey by Gallou 
et~al.\ 
examined how decision-makers in the chemical industry perceive
biocatalysis. The findings revealed that they tend to rely more on the
strengths of their own discipline to solve problems rather than
seeking solutions from other fields. Interestingly, however, most
participants in the survey recognized the potential of biocatalysis~\cite{35}.
This raises the question of how to facilitate the development
of new chemoenzymatic synthesis methods. If approached effectively,
nucleoside chemistry could greatly benefit from the complementary
strengths of both fields (Figure~\ref{fig1}). 

\section{Conclusion}

The most recent reviews on nucleoside chemistry and sugar synthesis
primarily focus on the achievements and challenges within synthetic
chemistry \cite{bib12,bib32}. 
Even though Wang et~al.\ and Cosgrove and Miller
acknowledge the potential of biocatalysts and highlight their
respective strengths, they still predominantly emphasize chemical
approaches~\cite{bib10,34}. We believe that integrating both strategies is
key to overcoming the most pressing challenges since biocatalytic
synthesis can address some of the limitations inherent in chemical
synthesis and vice versa.

\section*{CRediT author contributions}

SW, AK: Conceptualization. SW: Methodology,  Formal Analysis, 
Investigation,  Resources,  Data Curation,  Writing---Original
Draft. SW, AK, PN: Writing---Review \&amp; Editing. SW: Visualization.
AK: Supervision. PN: Project Administration, Funding Acquisition.

\section*{Declaration of interest}

Anke Kurreck is CEO of BioNukleo GmbH, Peter Neubauer is a member of
the advisory board. The authors declare no conflict of interest.

\section*{Acknowledgment}

The authors thank Laura Hillebrand for valuable feedback on the
chemical section of the manuscript.

\back{}

\printbibliography
\refinput{crchim20240991-reference.tex}

\end{document}
</pre></body></html>