\makeatletter
\@ifundefined{HCode}
{\documentclass[screen, CRCHIM, Unicode, Thematic, published, HideArticleType]{cedram}
\newenvironment{noXML}{}{}
\def\tsup#1{$^{{#1}}$}
\def\tsub#1{$_{{#1}}$}
\def\thead{\noalign{\relax}\hline}
\def\tbody{\noalign{\relax}}
\def\endthead{\noalign{\relax}\hline}
\def\xsection#1{}
\def\tabnote#1{\vskip4pt\parbox{.83\linewidth}{#1}}
\def\rmeta{\upeta}
\def\rmmu{\upmu}
\def\rmdelta{\updelta}
\def\rmpi{\uppi}
\usepackage{amssymb}
\RequirePackage{etoolbox}
\usepackage{upgreek}
\def\jobid{crchim20230360}
%\graphicspath{{/tmp/\jobid_figs/web/}}
\graphicspath{{./figures/}}
\newcounter{runlevel}
\let\MakeYrStrItalic\relax
\csdef{Seqnsplit}{\\}
\def\refinput#1{}
\newenvironment{xxcenter}{\begin{center}}{\end{center}}
\def\xfigure#1#2{\begin{xxcenter}\includegraphics{#1}\unskip\break#2\end{xxcenter}}
\def\back#1{}
\def\botline{\\\hline}
\usepackage{multirow} 
\def\nrow#1{\@tempcnta #1\relax%
\advance\@tempcnta by 1\relax%
\xdef\lenrow{\the\@tempcnta}}
\def\morerows#1#2{\nrow{#1}\multirow{\lenrow}{*}{#2}}
\def\xmorerows#1#2{#2}
\makeatletter
\g@addto@macro{\UrlBreaks}{\UrlOrds}
\gappto{\UrlBreaks}{\UrlOrds}
\makeatother
\DOI{10.5802/crchim.254}
\datereceived{2023-04-07}
\daterevised{2023-06-25}
\dateaccepted{2023-07-26}
\ItHasTeXPublished
}
{\documentclass[crchim]{article}
\def\CDRdoi{10.5802/crchim.254}
\makeatletter
\def\CDRsupplementaryTwotypes#1#2{}
\let\newline\break
}
\makeatother

\usepackage{upgreek}

\dateposted{2023-12-20}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Chemical Biology}
%%\def\cdr@specialtitle@french{Ch\'emobiologie}
%\makeatother


\title{Synthesis and kinetic evaluation of~analogs of 
(\protect\textit{E})-4-amino-3-methylbut-2-en-1-yl diphosphate, 
a~potent inhibitor of the IspH metalloenzyme}

\alttitle{Synth\`{e}se et \'{e}tude cin\'{e}tique d'analogues du
diphosphate de (\textit{E})-4-amino-3-m\'{e}thylbut-2-\'{e}nyle, un
inhibiteur puissant de la m\'{e}talloenzyme IspH}

\author{\firstname{Beno\^{i}t Eric} \lastname{Petit}}
\address{Equipe Chimie Biologique et Applications Th\'{e}rapeutiques, Institut de Chimie de Strasbourg, UMR 7177, Universit\'{e} de Strasbourg/CNRS, 4, rue Blaise Pascal, 67070 Strasbourg, France}

\author{\firstname{Hannah} \lastname{Jobelius}\CDRorcid{0000-0003-0715-8751}}
\addressSameAs{1}{Equipe Chimie Biologique et Applications Th\'{e}rapeutiques, Institut de Chimie de Strasbourg, UMR 7177, Universit\'{e} de Strasbourg/CNRS, 4, rue Blaise Pascal, 67070 Strasbourg, France}

\author{\firstname{Gabriella Ines} \lastname{Bianchino}\CDRorcid{0000-0002-6553-6063}}
\addressSameAs{1}{Equipe Chimie Biologique et Applications Th\'{e}rapeutiques, Institut de Chimie de Strasbourg, UMR 7177, Universit\'{e} de Strasbourg/CNRS, 4, rue Blaise Pascal, 67070 Strasbourg, France}

\author{\firstname{M\'{e}lina} \lastname{Gu\'{e}rin}\CDRorcid{0009-0006-5986-5828}}
\addressSameAs{1}{Equipe Chimie Biologique et Applications Th\'{e}rapeutiques, Institut de Chimie de Strasbourg, UMR 7177, Universit\'{e} de Strasbourg/CNRS, 4, rue Blaise Pascal, 67070 Strasbourg, France}

\author{\firstname{Franck} \lastname{Borel}\CDRorcid{0000-0002-8905-5336}}
\address{Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000 Grenoble, France}

\author{\firstname{Philippe} \lastname{Chaignon}\CDRorcid{0009-0002-1970-5716}\IsCorresp}
\addressSameAs{1}{Equipe Chimie Biologique et Applications Th\'{e}rapeutiques, Institut de Chimie de Strasbourg, UMR 7177, Universit\'{e} de Strasbourg/CNRS, 4, rue Blaise Pascal, 67070 Strasbourg, France}
\email[P. Chaignon]{p.chaignon@unistra.fr}

\author{\firstname{Myriam} \lastname{Seemann}\CDRorcid{0000-0002-2615-1574}\IsCorresp}
\addressSameAs{1}{Equipe Chimie Biologique et Applications Th\'{e}rapeutiques, Institut de Chimie de Strasbourg, UMR 7177, Universit\'{e} de Strasbourg/CNRS, 4, rue Blaise Pascal, 67070 Strasbourg, France}
\email[M. Seemann]{mseemann@unistra.fr}

\shortrunauthors

\keywords{\kwd{MEP pathway}
\kwd{IspH}
\kwd{LytB}
\kwd{Iron--sulfur clusters} 
\kwd{Inhibitor}
\kwd{(\textit{E})-4-amino-3-methylbut-2-en-1-yl diphosphate} 
\kwd{Anti-infectives}}

\altkeywords{\kwd{Voie du MEP}
\kwd{IspH}
\kwd{LytB}
\kwd{Centre fer-soufre}
\kwd{Inhibiteur}
\kwd{Diphosphate de (\textit{E})-4-amino-3-m\'{e}thylbut-2-\'{e}nyle}
\kwd{Anti infectieux}}

\thanks{Fondation Jean-Marie Lehn, the European Union's Horizon 2020
research and innovation program under the Marie Sklodowska-Curie (Grant
agreement no. 860816),  Universit\'{e} franco-allemande,  IdEx Unistra
(ANR-10-IDEX-0002),  SFRI-STRAT'US project (ANR-20-SFRI-0012).}

\begin{abstract}
Our previous research revealed that
(\textit{E})-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) is one
of the best inhibitors of IspH, a [4Fe--4S]-dependent enzyme involved
in the methylerythritol phosphate pathway that is a valuable target for
the discovery of new antibacterial and antiparasitic drugs as it is
absent in humans. AMBPP has substantial limitations for drug
development due to its poor metabolic stability. Here, we investigate
the replacement of the diphosphate moiety of AMBPP by more stable
mimics: sulfonate, phosphonate or phosphinophosphonate. After synthesis
of the derivatives, enzymatic assays demonstrated that none of these
AMBPP analogs is an efficient IspH inhibitor.
\end{abstract}

\begin{altabstract}
Nos travaux pr\'{e}c\'{e}dents avaient r\'{e}v\'{e}l\'{e} que le
diphosphate de (\textit{E})-4-amino-3-m\'{e}thylbut-2-\'{e}nyle
\'{e}tait un des meilleurs inhibiteurs d'IspH, une enzyme \`{a} centre
[4Fe--4S] impliqu\'{e}e dans la voie du m\'{e}thyl\'{e}rythritol
phosphate, cible pour la d\'{e}couverte de nouveaux agents
antibact\'{e}riens et antiparasitaires. L'AMBPP n'est pas un candidat
optimal pour le d\'{e}veloppement de m\'{e}dicaments d\^{u} \`{a} sa
faible stabilit\'{e} m\'{e}tabolique. Nous pr\'{e}sentons ici, la
premi\`{e}re \'{e}tude des relations structure-activit\'{e} bas\'{e}e
sur la fonction diphosphate de l'AMBPP en \'{e}valuant son remplacement
par un sulfonate, un phosphonate ou un phosphinophosphate. Apr\`{e}s
synth\`{e}se des d\'{e}riv\'{e}s, les tests enzymatiques montrent
qu'aucun de ces analogues de l'AMBPP n'est efficace pour inhiber IspH.
\end{altabstract}

\maketitle

\vspace*{6pt}

\twocolumngrid

\end{noXML}

\section{Introduction}

Drug resistance is a constantly growing issue that poses a major
challenge for the development of new drugs. In the context of  
antibiotic discovery, the  situation is alarming, with antimicrobial
resistant bacteria emerging and spreading all around the world \cite{1}.
Our ability to treat common infectious diseases is now compromised as
some infections are already impossible to treat with the existing
therapeutic repertoire. Antibiotic resistance not only increases
mortality but also has a severe impact on medical expenses and
hospitalization  time. 

\begin{scheme*}[t!]
\includegraphics{sc01}
\vspace*{10pt}
\caption{The MEP pathway.\label{sch1}}
\vspace*{-4pt}
\end{scheme*}


The World Health Organization (WHO) published in 2017 a list of
prioritization of bacteria to guide research and development of novel
antibiotics \cite{2}. In~the latest WHO report dated December 9, 2022
\cite{3}, Dr.~Tedros Adhanom Ghebreyesus, WHO Director-General, stated
that ``Antimicrobial resistance undermines modern medicine and puts
millions of lives at risk''. Given the severe menace caused by
antibiotic-resistant bacteria, it is more than urgent to discover new
antibacterial agents with new modes of action. In this context, the
methylerythritol phosphate pathway (MEP, Scheme~\ref{sch1}),
responsible for the biosynthesis of the universal precursors of
terpenoids in most bacteria, has emerged as an attractive target for
drug development \cite{4,5,6,7}.





Terpenoids, also known as isoprenoids, represent the most diverse
family of natural products, with over 55,000 known compounds. They are
present in all living organisms and are involved in many important
biological processes such as electron transport, cell wall
biosynthesis, and protein prenylation \cite{8,9}. Terpenoids are
biosynthesized by the addition of one or more molecules of isopentenyl
diphosphate (IPP, 1) to its isomer dimethylallyl diphosphate (DMAPP, 2,
Scheme~\ref{sch1}) \cite{8}. Most pathogenic bacteria, including almost
all that were prioritized by the WHO \cite{2}, use the MEP pathway
(Scheme~\ref{sch1}) for the production of IPP and DMAPP, whereas the
biosynthesis of these building blocks relies exclusively on the
mevalonate pathway in humans and animals \cite{10}. This metabolic
difference makes enzymes of the MEP pathway interesting targets for new
antibacterial drug development that are expected to have no or reduced
side effects in humans. Despite the hope sparked by the discovery of
the MEP pathway, only fosmidomycin, an inhibitor of
1-deoxyxylulose-5-phosphate reductoisomerase (DXR), the second enzyme
of the MEP pathway (Scheme~\ref{sch1}), reached clinical trials as an
antimalarial agent (\textit{P. falciparum} is dependent on the MEP
pathway) in combination with clindamycin and piperaquine \cite{11}.
This finding validates the MEP pathway as an innovative target for the
development of new drugs \cite{12}. However, new molecules need to
enter the therapeutic pipeline to combat deadly bacterial
infections.

Here, we focus our efforts on discovering new inhibitors of IspH, also
called LytB, the last enzyme of the MEP pathway.

\begin{scheme*}[t!]
\includegraphics{sc02}
\vspace*{8pt}
\caption{IspH-catalyzed reaction and structures of potent
inhibitors.\label{sch2}}
\vspace*{-.8pc}
\end{scheme*}

IspH contains an oxygen-sensitive [4Fe--4S]\tsup{2+} center that is
essential for catalysis and converts
(\textit{E})-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP,
\textbf{3}) into a mixture of IPP and DMAPP (Scheme~\ref{sch2}).
M\"{o}ssbauer spectroscopy \cite{13,14} and Nuclear Resonance
Vibrational Spectroscopy studies highlighted that the
[4Fe--4S]\tsup{2+} cluster of substrate-free IspH is particular, as one
of its four iron sites is an Fe(II) atom in an octahedral coordination
geometry, linked to three inorganic sulfur atoms of the iron--sulfur
\mbox{cluster} and three water molecules (Scheme~\ref{sch2}) \cite{15}. This
unusual Fe(II) coordination with three labile ligands is at the origin
of the instability of the [4Fe--4S]\tsup{2+} cluster of IspH in the
presence of oxygen. Indeed, the oxidation of this Fe(II) may trigger
the decomposition of the prosthetic group. Consequently, IspH is only
stable under anaerobic conditions, making this enzyme difficult to
study and hence an underexplored target. No crystal structure of
substrate-free IspH in its [4Fe--4S]\tsup{2+} form has been reported as
the apical Fe(II) might dissociate during the crystallization process.
The first X-ray structure of IspH described with an intact [4Fe--4S]
cluster was obtained for the \textit{E. coli} homolog in complex with
HMBPP (Scheme~\ref{sch2}) \cite{16}. Since then, several other IspH
structures harboring the [4Fe--4S] center in complex with ligands have
been published (for a review see \cite{17}).



The IspH mechanism is peculiar and involves bioinorganic and
bioorganometallic intermediates (for reviews see \cite{17,18,19}). It
formally involves removal of the hydroxyl group, transfer of two
electrons from the [4Fe--4S] cluster, and protonation of an
intermediate allylic anion (Scheme~\ref{sch2}). The binding of the OH
group of HMBPP to the unique fourth iron site of the [4Fe--4S]\tsup{2+}
cluster, leading to the change in coordination geometry of this iron
from octahedral to tetrahedral upon binding of the substrate
(Scheme~\ref{sch2}), was shown using M\"{o}ssbauer spectroscopy
\cite{13} and is illustrated in the X-ray structure of the \textit{E.
coli} IspH--HMBPP complex \cite{16}. After reduction of the first
bioinorganic complex (Scheme~\ref{sch2}), the OH group of the substrate
undergoes a rotation to interact with E126, leading to a $\rmpi$-complex
\cite{20,21,22,23}. EPR/ENDOR investigations led to the
characterization of a $\rmeta^{3}$-allyl ($\rmpi$) complex that forms
after water elimination \cite{24,25}. Further reduction of the
paramagnetic $\rmeta^{3}$-allyl ($\rmpi$) complex followed by protonation
at the \textit{si} face of C-2 yields IPP, while protonation at C-4
yields DMAPP \cite{26}.

We and others further exploited the acquired knowledge of the IspH
mechanism to design inhibitors \cite{17}. In this context, we have
already reported two molecular tools that were HMBPP analogs
(Scheme~\ref{sch2}) in which the OH group of HMBPP was replaced by an
amino (AMBPP, \textbf{4}) or a thiol group (TMBPP, \textbf{5}). We had
expected AMBPP and TMBPP to tightly bind to the IspH [4Fe--4S] cluster
but not being capable of undergoing the elimination step.  Enzymatic
studies have revealed that these molecules are very potent inhibitors
of IspH with $K_{i}$ values in the nanomolar range: TMBPP is a
tight-binding inhibitor (IC\tsub{50} $=$ 210~nM, $K_{i}= 20$~nM,
\textit{E. coli} IspH) and AMBPP is a slow-binding inhibitor
(IC\tsub{50} $=$ 150~nM, $K_{i}= 54$~nM, \textit{E. coli} IspH)
\cite{27}. These molecules remain the best IspH inhibitors known to
date \cite{17}. The mode of binding of these inhibitors to the apical
iron of IspH [4Fe--4S] via the thiol or the amino function has been
further confirmed \cite{28,29,30}. In addition to these two inhibitors,
two other potent inhibitors of \textit{A. aeolicus} IspH were reported
(Scheme~\ref{sch2}): a propargyl diphosphate  (IC\tsub{50} $=$
6.7~$\rmmu\mathrm{M}$) and a pyridine diphosphate (IC\tsub{50} $=$
9.1~$\rmmu\mathrm{M}$) \cite{18}. However, all these inhibitors are
diphosphate derivatives and therefore have substantial liabilities with
respect to poor transport across bacterial membranes and inactivation
upon hydrolysis by secreted phosphatases. Barbituric acid analogs, very
different in structure compared to the substrate, were recently
developed. They were found to be less potent IspH inhibitors
(\textbf{B1}: IC\tsub{50} $=$ 22~$\rmmu\mathrm{M}$, \textit{P.
aeruginosa} IspH; \textbf{B2}: IC\tsub{50} $=$ 23~$\rmmu\mathrm{M}$,
\textit{E. coli} IspH, Scheme~\ref{sch2})~\cite{31}. 

All these results comforted us in our approach to optimize these
substrate-based inhibitors. In this context, we report here the
synthesis of analogs of AMBPP \textbf{4}, in which the diphosphate
moiety was replaced by simple mimics, and the results of their
biological evaluation on IspH. 

\section{Results and discussion}

When preparing molecules harboring a diphosphate, this functional group
is usually introduced at the end of the synthesis as its presence leads
to a molecule almost insoluble in most common organic solvents, which
limits further chemical transformations \cite{27,28,29,30,31,32a,32b,32c}.
Moreover, the purification of diphosphorylated molecules is often
tedious. Diphosphate entities are also prone to hydrolysis catalyzed by
phosphatases excreted by bacteria, which would result in the
inactivation of the diphosphate-containing inhibitors. 


To avoid these issues, we investigated the replacement of the
diphosphate moiety of the AMBPP inhibitor with more stable mimics. In
this context, sulfonate, phosphonate or phosphinophosphonate were
chosen. Sulfonate is used as isostere of phosphate as it has a
tetrahedral shape similar to that of the phosphate group but is more
acidic. The methylene phosphonate moiety is an isostere of phosphate
with its phosphorus--carbon bond more stable towards hydrolysis
compared to the phosphorus--oxygen bond of phosphate. These two
moieties are shorter than diphosphate \cite{33}. Finally, the
phosphinophosphonate group is an isostere of the diphosphate group but
less sensitive to hydrolysis. The structures of the corresponding AMBPP
analogs that we investigated are displayed in Figure~\ref{fig1}. 

\begin{figure*}[t!]
\includegraphics{fig01}
\caption{AMBPP and its phosphonate, sulfonate, and phosphinophosphonate
analogs.\label{fig1}}
\end{figure*}

\subsection{Preliminary docking experiments}

Preliminary in silico docking and scoring experiments were performed
using phosphonate \textbf{6}, sulfonate \textbf{7}, and
phosphinophosphonate \textbf{8}. Due to the difference in $\mathrm{pK}_{a}$
values of these different entities and the fact that the activity of
IspH is determined at pH $=$ 8, docking experiments were performed at pH
$=$ 7 and at pH $=$ 8. 


Experiments were carried out starting from the X-ray structure of IspH
in complex with AMBPP (PDB: 3ZGL) \cite{30}. Interestingly, the docking
scores of the sulfonate analog \textbf{7} (D.S.~$=$~${-}$6.93; ${-}$6.92,
Figure~\ref{fig2}) are slightly better than the docking scores of the
parent molecule AMBPP (D.S.~$=$~${-}$6.75; ${-}$5.33, Figure~\ref{fig2}).
Replacement of the diphosphate by a simple phosphonate or a
phosphinophosphonate leads to still acceptable docking scores
(Figure~\ref{fig2}). The corresponding docking poses revealed that
compounds \textbf{6}, \textbf{7}, and \textbf{8} docked in the active
site and placed their sulfonate, phosphonate or phosphinophosphonate
group in the diphosphate binding pocket of AMBPP (Figure~\ref{fig2}).
The amino groups in these AMBPP analogs were also found close to the
apical iron. 

\begin{figure*}[t!]
\includegraphics{fig02}
\caption{Docking experiments were performed using the X-ray structure
of the \textit{E. coli} IspH:AMBPP complex (PDB: 3ZGL). Upper
panel: structural formula and docking score (D.S) at pH $=$ 7 (left) and pH
$=$ 8 (right). Lower panel: docking poses of the compound in the IspH
active site. Docked molecules are depicted with green carbon atoms.
Docked AMBPP is superposed onto its corresponding crystallographic
structure (gray carbon atoms). Iron and sulfur atoms are represented in
orange and yellow, respectively.\label{fig2}}
\vspace*{5pt}
\end{figure*}

Encouraged by these indicators, compounds \textbf{6}, \textbf{7}, and
\textbf{8} were prepared.


\subsection{Chemistry}


Target compounds were synthesized starting from dimethylallyl bromide
\textbf{9}. The syntheses of \textbf{6} and \textbf{8} are
outlined in Scheme~\ref{sch3}. 

\begin{scheme*}[t!]
\includegraphics{sc03}
\caption{Synthesis of phosphonate \textbf{6}  and phosphinophosphonate  \textbf{8} analogs.\label{sch3}}
\vspace*{-8pt}
\end{scheme*}

\subsubsection{Synthesis of (E)-(5-amino-4-methylpent-3-en-1-yl) phosphonate
\textbf{6}}
Dimethylphosphonate \textbf{10}  was synthesized by nucleophilic
substitution of the bromine atom in dimethylallyl bromide \textbf{9}
with deprotonated dimethyl methylphosphonate following the procedure
reported by Wiemer \cite{34}. Phosphonate \textbf{10} was selectively
oxidized with selenium dioxide to yield the corresponding
\textit{E}-configurated aldehyde, which was subsequently reduced by
NaBH\tsub{4} to alcohol \textbf{11}~\cite{35,36}. Displacement of the
hydroxyl group in \textbf{11} with phthalimide under Mitsunobu
conditions yielded \textbf{12}. Methyl phosphoesters and phthalimide
were deprotected by treatment with bromotrimethylsilane and aqueous
ammonia, respectively, to provide the corresponding amine \textbf{6}. 

\subsubsection{Synthesis of (E)-(5-amino-4-methylpent-3-en-1-yl)
phosphinophosphonate \textbf{8}}
Phosphinophosphonate \textbf{16} could not be obtained by sequential
activation of phosphonate \textbf{12} by oxalyl chloride to the
corresponding phosphonic acid chloride and treatment of the latter by
deprotonated dimethyl methylphosphonate. As an alternative, we used the
procedure published by Wiemer and coworkers for the formation of
\textbf{14}~\cite{37}. Subsequent allylic oxidation with selenium dioxide
yielded \textbf{15}. Phosphinophosphonate \textbf{8} was then obtained
following the same strategy used for phosphonate \textbf{6}.


The synthesis of sulfonate \textbf{7} is outlined in Scheme~\ref{sch4}. 

\begin{figure*}[t!]
\vspace*{-2pt}
\includegraphics{fig03}
\vspace*{-2pt}
\caption{IspH enzymatic assays. Decrease in the absorbance of NADPH at
340~nm in the presence or absence of \textbf{6}, \textbf{7},
\textbf{8}, or AMBPP. Conditions: NADPH (2.2~mM), FldA 
($30~\rmmu\mathrm{M}$), FpR1 ($17~\rmmu\mathrm{M}$), IspH 
($0.5~\rmmu\mathrm{M}$) in 50~mM Tris HCl buffer pH $=$ 8 at
37~\textdegree C; HMBPP alone (dark blue) or in the presence of 1~mM of
\textbf{6} (yellow), \textbf{7} (green), \textbf{8} (blue), or 
$25~\rmmu\mathrm{M}$ AMBPP \textbf{4} (orange) that were preincubated
with IspH for 15~min at 37~\textdegree C before initiating the reaction
by addition of HMBPP ($150~\rmmu\mathrm{M}$). Samples were prepared in
a glove box, and additions were performed using a gastight syringe. A
control sample (light gray) was prepared under the same conditions but
replacing HMBPP by buffer.\label{fig3}}
\vspace*{-1pt}
\end{figure*}



\begin{scheme*}[t!]
\includegraphics{sc04}
\vspace*{4pt}
\caption{Synthesis of sulfonate analog \textbf{7}.\label{sch4}}
\vspace*{-10pt}
\end{scheme*}

\subsubsection{Synthesis of (E)-5-amino-4-methylpent-3-ene-1-sulfonate
\textbf{7}}
The sulfonate analog \textbf{7} was prepared in three steps.
Commercially available alkyl bromide \textbf{18} was selectively
oxidized to alcohol \textbf{19}, as reported by~Gaich and Mulzer
\cite{38}. A Mitsunobu reaction using phthalimide as the nucleophile
allowed the synthesis of compound \textbf{20}. Bromide was substituted
by treatment with sodium sulfite, and hydrolysis of the phthalimide by
ammonia yielded \textbf{7}.


Biological experiments were further carried out to test the ability of
\textbf{6}, \textbf{7}, \textbf{8} to act as IspH inhibitors.

\subsection{Biological evaluation}

\textit{E. coli}  IspH contains an oxygen-sensitive [4Fe--4S]\tsup{2+} 
cluster that is essential for catalysis and therefore needs to be
handled in a glove box under a strictly inert (N\tsub{2}) atmosphere.
IspH converts HMBPP into a mixture of IPP and DMAPP in the presence of
an external reduction system. In \textit{E. coli}, the natural
flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH system plays this
role \cite{39,27}. Enzyme activity was therefore determined by
monitoring NADPH consumption. The progress curve (Figure~\ref{fig3},
navy blue) showed a sharp drop in the NADPH concentration in the first
7~min that was due to IspH catalysis under multiple turnover
conditions. The resulting  \textit{E. coli} IspH activity was
$990~\mathrm{nmol}{\cdot}\mathrm{min}^{-1}{\cdot}\mathrm{mg}^{-1}$, in
{agreement} with previous reports \cite{13,23,27}. It should be noticed
that the low-slope region observed after 7~min is due to spontaneous
NADPH degradation at pH $=$ 8. 

We previously reported that AMBPP \textbf{4} is a slow-binding
inhibitor of IspH under the conditions used in this assay and
hypothesized that the slow-binding step might be due to the formation
of the nonprotonated amine required for binding to the apical iron of
the [4Fe--4S]\tsup{2+} cluster \cite{27}. As \textbf{6}, \textbf{7},
and \textbf{8} also contain the amine function, this slow-binding
behavior would also be expected for these compounds. As a consequence,
we tested the ability of \textbf{6}, \textbf{7}, and \textbf{8} to
promote IspH inhibition by first preincubating IspH with each of these
compounds for 15~min, in order to favor the formation of the
enzyme--inhibitor complex, and then initiate the IspH-catalyzed
reaction by the addition of the HMBPP substrate. Progress curves
recorded using \textbf{6}, \textbf{7}, or \textbf{8} at a concentration
of 1 mM displayed the same initial slope as the curve recorded for IspH
with HMBPP alone, indicating the same steady-state rates
(Figure~\ref{fig3}, yellow, green, blue). In contrast, the progress
curve recorded under the same conditions for AMBPP at a concentration
as low as $25~\rmmu\mathrm{M}$ showed a drastic decrease in the IspH
reaction rate, indicative of an \textit{E. coli} IspH activity of
$263~\mathrm{nmol}{\cdot}\mathrm{min}^{-1}{\cdot}\mathrm{mg}^{-1}$
that corresponds to 73\% enzyme inhibition (Figure~\ref{fig3}, orange).
Together, these results reveal that replacement of the diphosphate in
AMBPP with a phosphonate, a sulfonate, or a phosphinophosphonate,
compromise the inhibition potential of the resulting derivatives
\textbf{6}, \textbf{7}, and \textbf{8} towards IspH.\looseness=-1



\section{Conclusion}

Three novel analogs of AMBPP, in which the diphosphate group was
replaced by a sulfonate or a methylene phosphonate or a
phosphinophosphonate, were synthesized and characterized by NMR
spectroscopy and mass spectrometry. In contrast to the parent molecule
AMBPP, which is one of the best two inhibitors known to date for
\textit{E. coli} IspH, a metalloenzyme containing an oxygen sensitive
[4Fe--4S] cluster involved in the MEP pathway, these new molecules
did not affect IspH activity. These results illustrate the essentiality
of the diphosphate group of AMBPP, so far. The lack of inhibition
potential of the sulfonate or methylene phosphonate analogs is most
probably due to the size of these phosphate mimics that are shorter
than diphosphate and might not completely fill the diphosphate binding
pocket of IspH. In contrast, phosphinophosphonate has the same size as
AMBPP and could undergo the same interactions as AMBPP with the
surrounding amino acids of IspH. However, phosphinophosphonates are
known to be less acidic than diphosphates. As a consequence, the
phosphinophosphonate might retain a proton that might weaken some
interactions within the active site. \looseness=-1

Based on the knowledge gained from this study, new inhibitors derived
from AMBPP or other promising IspH inhibitors need to be elaborated.
Such optimization could consist in the use of other diphosphate
isosteres such as difluoromethylphosphonates,
difluoromethanediphosphonates or via structure-based fragment selection
to find new scaffolds binding to the diphosphate pocket of IspH that do
not rely on phosphate chemistry.


\section{Experimental section}
\subsection{Molecular docking}


In silico docking experiments were carried out using the
Schr\"{o}dinger suite 2020-4 (Schr\"{o}dinger LLC, New York, NY, USA).
The X-ray structure of IspH in complex with AMBPP (PDB: 3ZGL) was used
for the studies. The protein structure was processed as previously
described \cite{30}. To generate the docking grid, we used AMBPP as the
reference ligand and the protein model without water molecules. The
binding region was defined by a square box centered on the inhibitor.
Sizes that largely exceeded the volume of the binding site were used
for both the enclosing (10~\AA{} $\times$ 10~\AA{} $\times$ 10~\AA{})
and bounding box (20~\AA{} $\times$ 20~\AA{} $\times$ 20~\AA{}).
LigPrep was used for energy minimization, to generate the 3D structures
of the compounds, and to produce the tautomers and the ionization
states at pH $=$ 7 and pH $=$ 8. The docking study was performed using
Glide's extra precision mode \cite{40,41}. No constraints (such as
hydrogen bond or atom position) were applied to guide the binding.
Results of the in silico docking experiments were sorted according to
the Glide docking score.


\subsection{Syntheses}


All reactions in nonaqueous solvents were conducted under an argon
atmosphere with a magnetic stir bar. All reagents and solvents were
purchased from commercial sources and used without further
purification. Anhydrous CH\tsub{2}Cl\tsub{2} and tetrahydrofuran
(THF) were purchased (99.85\%, water $<$ 50~ppm). All other
solvents were of HPLC grade. Reactions were monitored by thin layer
chromatography (TLC) with silica gel 60-F254 plates. Flash column
chromatography was performed using silica gel (0.04--0.063~mm,
230--400 mesh) under pressure. Yields refer to chromatographically
and spectroscopically pure compounds. 


NMR spectra were recorded on a 300- or 500-MHz spectrometer. All NMR
spectra were measured in CDCl\tsub{3} or D\tsub{2}O solutions and
referenced, respectively, to the residual CHCl\tsub{3} signal ($^{1}$H,
$\rmdelta = 7.26$~ppm; $^{13}$C, $\rmdelta = 77.16$~ppm) or H\tsub{2}O
($\rmdelta = 4.79$~ppm). For $^{31}$P NMR spectroscopy, 85\% phosphoric acid
in D\tsub{2}O was used as external reference 
($\rmdelta = {-}0.85$~ppm). Chemical
shifts and coupling constants are reported in ppm and Hz, respectively.
High-resolution mass spectra were obtained using ESI-TOF.


\subsubsection{Synthesis of (\textit{E})-(5-amino-4-methylpent-3-en-1-yl) 
phosphonate \textbf{6}} 

\noindent
$\bullet$ Synthesis of dimethyl (4-methylpent-3-en-1-yl) phosphonate
\textbf{10}

\xfigure{fx01}{}

Dimethyl methylphosphonate (2.1~mL,~19.4~mmol, 1~eq.) was~added
dropwise at ${-}$78~\textdegree C to a stirred solution of LDA (2~M in
THF, 10~mL, 20~mmol, 1~eq.) in dry THF (80~mL). The reaction mixture
was stirred at ${-}$78~\textdegree C for 15~min before
1-bromo-3-methylbut-2-ene \textbf{9} (2.3~mL, 19.9~mmol, 1~eq.) was
added dropwise. The reaction mixture was stirred at ${-}$78~\textdegree C
for 30 min and then left to stand at 20~\textdegree C overnight. A
saturated aqueous solution of NH\tsub{4}Cl was then added, and the
different layers were separated. The aqueous layer was extracted using
diethyl ether. The combined organic layers were dried over
Na\tsub{2}SO\tsub{4}, filtered, and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography on silica gel (ethyl acetate/petroleum ether, 8:2)
yielding dimethyl (4-methylpent-3-en-1-yl) phosphonate \textbf{10} as an
oil (2.26~g, 11.8~mmol, 61\%, Rf (ethyl acetate/cyclohexane, 9:1) $=$
0.25). \tsup{\textbf{1}}\textbf{H NMR} (500~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 1.61 (3H, s, H-6), 1.68 (3H, s, H-5), 1.70--1.82
(2H, m, H-1), 2.21--2.33 (2H, m, H-2), 3.73 (6H, d, $J = 12.0$~Hz, H-7
$+$ H-8), 5.10 (1H, tq, $J = 7.2$~Hz, $J = 1.5$~Hz, H-3).
\tsup{\textbf{13}}\textbf{C NMR} (125~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 17.8 (C-6), 21.1 (d, $J = 5.0$~Hz, C-2), 25.1 (d, $J
= 137.0$~Hz, C-1), 25.8 (C-5), 52.4 (d, $J = 6.6$~Hz, C-7 $+$ C-8),
123.1 (d, $J = 17.5$~Hz, C-3), 133.1 (d, $J = 2.5$~Hz, C-4).
\tsup{\textbf{31}}\textbf{P NMR} (121~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 34.5.

\medskip\noindent
$\bullet$ Synthesis of dimethyl (\textit{E})-(5-hydroxy-4-\newline
methylpent-3-en-1-yl) phosphonate \textbf{11}

\xfigure{fx02}{}

\vspace*{-10pt}

Dimethyl (4-methylpent-3-en-1-yl) phosphonate \textbf{10} (500~mg, 
2.60~mmol, 1~eq.), SeO\tsub{2} (220~mg, 1.95~mmol, 0.75~eq.) and
tert-butyl hydroperoxide (70\% in water, 1.4~mL, 10.4~mmol, 4~eq.) were
dissolved in CH\tsub{2}Cl\tsub{2} (15~mL). The reaction mixture was
stirred at 20~\textdegree C for 16~h before the reaction was quenched
by the addition of a saturated aqueous solution of NaCl. The different
layers were separated, and the aqueous layer was extracted with
CH\tsub{2}Cl\tsub{2}. The combined organic layers were washed with an
aqueous solution of Na\tsub{2}S\tsub{2}O\tsub{3}, dried over
Na\tsub{2}SO\tsub{4}, filtered, and the solvent was removed under
reduced pressure. The resulting crude product was dissolved in methanol
(7.5~mL), and NaBH\tsub{4} (200~mg, 5.2~mmol,  2~eq.) was added
portionwise. The reaction mixture was stirred at 20~\textdegree C for
2~h before being quenched by the addition of a saturated aqueous
solution of NH\tsub{4}Cl. The resulting mixture was extracted with
diethyl ether, and the combined organic layers were dried over
Na\tsub{2}SO\tsub{4}, filtered, and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography on silica gel (MeOH/DCM, 4:96) 
yielding~dimethyl 
(\textit{E})-(5-hydroxy-4-methylpent-3-en-1-yl) phosphonate \textbf{11}
as an oil (298~mg, 1.43~mmol, 55\%, Rf (ethyl acetate/cyclohexane, 9:1)
$=$ 0.11). \tsup{\textbf{1}}{\textbf{H NMR}} (300~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 1.68 (3H, s, H-6), 1.77--1.84 (2H, m, H-1),
2.31--2.38 (2H, m, H-2), 3.74 (6H, d, $J = 10.0$~Hz, H-7 $+$ H-8), 4.00
(2H, s, H-5), 5.42 (1H, tq, $J = 7.0$~Hz, $J = 1.5$~Hz, H-3).
\tsup{\textbf{13}}\textbf{C NMR} (125~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 13.8 (C-6), 20.8 (d, $J = 5.0$~Hz, C-2), 24.8 (s, $J
= 139.0$~Hz, C-1), 52.5 (d, $J = 6.28$~Hz, C-7 $+$ C-8), 68.6 (C-5),
124.2 (d, $J = 16.25$~Hz, C-3), 136.4 (d, $J = 1.25$~Hz, C-4).
\tsup{\textbf{31}}\textbf{P NMR} (121~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 34.29.

\medskip\noindent
$\bullet$ Synthesis of dimethyl (\textit{E})-(5-(1,3-\newline
dioxoisoindolin-2-yl)-4-methylpent-3-en-1-yl) phosphonate \textbf{12}

\xfigure{fx03}{}

Dimethyl (\textit{E})-(5-hydroxy-4-methylpent-3-en-1-yl) phosphonate
\textbf{11} (398~mg, 1.9~mmol, 1.1~eq.), PPh\tsub{3} (515~mg, 2.0~mmol,
1.1~eq.) and phthalimide (250~mg, 1.7~mmol, 1~eq.) were dissolved in
dry THF (12~mL). DIAD ($430~\rmmu\mathrm{L}$, 2.0~mmol, 1.1~eq.) was
added dropwise at 0~\textdegree C, and the reaction mixture was stirred
at 0~\textdegree C for 30~min and then left to stand at 
20~\textdegree C overnight. The reaction was quenched by the addition
of MeOH (0.5~mL), and the solvent was evaporated under vacuum. The
crude product was purified by column chromatography on silica gel
(ethyl acetate) yielding \textbf{12} as a white solid (260~mg,
0.77~mmol, 45\%, Rf (MeOH/DCM, 5:95) $=$ 0.54).
\tsup{\textbf{1}}\textbf{H NMR} (300~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 1.66 (3H, s, H-6), 1.70--1.81 (2H, m, H-1),
2.24--2.36 (2H, m, H-2), 3.70 (6H, d $J = 9.0$~Hz, H-7 $+$ H-8), 4.18
(2H, s, H-5), 5.31 (1H, tq, $J = 8.7$~Hz, $J = 1.5$~Hz, H-3),
7.70--7.74 (2H, m, H-11 or H-12), 7.81--7.86 (2H, m, H-11 or H-12).
\tsup{\textbf{13}}\textbf{C NMR} (125~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 14.8 (C-6), 20.9 (d, $J = 4.5$~Hz, C-2), 24.5 (d,
$J = 139.0$~Hz, C-1), 44.8 (C-5), 52.4 (d, $J = 6.5$~Hz, C-7 $+$ C-8),
123.5 (C-10), 125.7 (d, $J = 16.9$~Hz, C-3), 130.9 (d, $J = 1.6$~Hz,
C-4), 132.1 (C-11 or C-12), 134.2 (C-11 or C-12), 168.3 (C-9).
\tsup{\textbf{31}}\textbf{P NMR} (121~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$~33.94.

\medskip\noindent
$\bullet$ Synthesis of (\textit{E})-(5-amino-4-methylpent-3-en-1-yl)
phosphonate \textbf{6}


\xfigure{fx04}{}


Me\tsub{3}SiBr ($180~\rmmu\mathrm{L}$, 1.68~mmol, 9~eq.) was added
dropwise to a stirred solution of dimethyl
(\textit{E})-(5-(1,3-dioxoisoindolin-2-yl)-4-methylpent-3-en-1-yl)
phosphonate \textbf{12} (60~mg, 0.178~mmol, 1~eq.) in dry
CH\tsub{2}Cl\tsub{2} (1.5~mL) at 0~\textdegree C. The reaction mixture
was stirred at 0~\textdegree C for 30~min and at 20~\textdegree C for
1~h. Methanol (1~mL) was added, and the reaction was further stirred
for 1~h. The solvents were then evaporated under reduced pressure, and
the resulting oil was dissolved in THF (4~mL). NH\tsub{4}OH (1~mL) was
added, and the reaction mixture was stirred overnight. Solvents were
evaporated under reduced pressure, and the resulting mixture was
purified by column chromatography on silica gel
(Isopropanol/H\tsub{2}O/NH\tsub{4}OH, 7:1:2 to 5:3:2) yielding
\textbf{6} as a white solid, which was dissolved in water, lyophilized,
and further dried under high vacuum (13~mg, 0.074~mmol, 42\%, Rf
(Isopropanol/H\tsub{2}O/NH\tsub{4}OH, 6:2:2) $=$ 0.20).
\tsup{\textbf{1}}\textbf{H NMR} (300~MHz, D\tsub{2}O): $\rmdelta$(ppm)
$=$ 1.61--1.72 (2H, m, H-1), 1.75 (3H, s, H-6), 2.26--2.38 (2H, m,
H-2), 3.53 (2H, s, H-5), 5.62 (1H, tq, $J = 7.5$~Hz, $J = 1.5~$Hz,
H-3). \tsup{\textbf{13}}\textbf{C~NMR} (125~MHz, D\tsub{2}O):
$\rmdelta$(ppm) $=$ 13.5 (C-6), 21.6 (d, $J = 3.75$~Hz, C-2), 27.2 (d,
$J = 132.5$~Hz, C-1), 46.3 (C-5), 127.5 (C-4), 130.9 (d, $J = 15.0$~Hz,
C-3). \tsup{\textbf{31}}\textbf{P NMR} (121~MHz, D\tsub{2}O):
$\rmdelta$(ppm) $=$ 25.32. High-resolution MS (ES-) \textit{m/z}:
[M--H]\tsup{-} (C\tsub{6}H\tsub{13}NO\tsub{3}P) calculated 178.0638,
found 178.0650.

\subsubsection{Synthesis of
(\textit{E})-(((5-amino-4-methylpent-3-en-1-yl) oxidophosphoryl)
methyl) phosphonate \textbf{8}}

\noindent
$\bullet$ Synthesis of dimethyl ((methoxy(4-methylpent-3-en-1-yl)
phosphoryl) methyl) phosphonate \textbf{14}

\vspace*{1pc}

\xfigure{fx05}{}

Oxalyl chloride (3~mL, 35~mmol, 3~eq.) was~added dropwise to a stirred
solution of dimethyl (4-methylpent-3-en-1-yl) phosphonate \textbf{10}
(2.05~g, 11.5~mmol, 1~eq.) and dry DMF (0.10~mL, cat.) in dry
CH\tsub{2}Cl\tsub{2} (60~mL) at 0~\textdegree C. The solution was
stirred overnight at 20~\textdegree C. Solvents were then removed under
reduced pressure, yielding a chlorinated compound, which was used
without further purification. Dimethyl methylphosphonate (3.5~mL,
33~mmol, 2.9~eq.) was added dropwise to a stirred solution of BuLi 
(2.5~M in hexane, 12.5~mL, 31~mmol, 2.7~eq.) at ${-}$78~\textdegree C.
The mixture was further stirred for 30~min before a solution of the
chlorinated compound in CH\tsub{2}Cl\tsub{2} (10~mL) was added
dropwise. The reaction mixture was allowed to reach 20~\textdegree C
while being stirred overnight. A saturated aqueous solution of
NH\tsub{4}Cl was then added, and the different layers were separated.
The aqueous layer was extracted using CH\tsub{2}Cl\tsub{2}. The
combined organic layers were dried over Na\tsub{2}SO\tsub{4}, filtered,
and the solvent was removed under reduced pressure. The resulting
mixture was purified by column chromatography on silica gel (EtOH/PE,
2:8) yielding \textbf{14} (1.50~g, 5.28~mmol, 46\%, Rf (MeOH/DCM, 5:95)
$=$ 0.17) as yellowish oil. \tsup{\textbf{1}}\textbf{H NMR} (300~MHz,
CDCl\tsub{3}): $\rmdelta$(ppm) $=$ 1.63 (3H, s, H-5), 1.68 (3H, s,
H-6), 1.92--2.02 (2H, m, H-1), 2.25--2.35 (2H, m, H-8), 2.33--2.46 (2H,
m, H-2), 3.76 (3H, d, $J = 11.1$~Hz, H-7), 3.81 (6H, d, $J = 11.4$~Hz,
H-9 $+$ H-10), 5.13 (1H, tq, $J = 7.2$~Hz, $J = 1.5$~Hz, H-3).
\tsup{\textbf{13}}\textbf{C NMR} (125, CDCl\tsub{3}): $\rmdelta$(ppm)
$=$ 17.8 (C-6), 20.4 (d,~$J = 4.12$~Hz, C-2), 25.8 (C-5), 26.4 (dd, $J
= 134.2$~Hz, $J = 75.5$~Hz, C-8), 29.5 (d, $J = 96.6$~Hz, C-1), 51.6
(d, $J = 6.9$~Hz, C-7), 53.2 (t, $J = 6.7$~Hz, C-9 $+$ C-10), 122.9 (d,
$J = 15.6$~Hz, C-3) and 133.4 (d, $J = 1.4$~Hz, C-4).
\tsup{\textbf{31}}\textbf{P NMR} (121~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 22.76 (d, $J = 4.4$~Hz), 48.34 (d, $J = 4.5$~Hz).


\medskip\noindent
$\bullet$~Synthesis of dimethyl (\textit{E})-(((5-hydroxy-4-\newline
methylpent-3-en-1-yl) (methoxy)phosphoryl)\newline methyl) phosphonate
\textbf{15}

\xfigure{fx06}{}

Dimethyl ((methoxy(4-methylpent-3-en-1-yl) phosphoryl) methyl)
phosphonate \textbf{14} (911~mg, 3.2~mmol, 1~eq), SeO\tsub{2} (215~mg,
1.9~mmol, 0.6~eq.) and tert-butyl hydroperoxide (70\% in water,
2.15~mL, 15.7~mmol, 5~eq.) were dissolved in CH\tsub{2}Cl\tsub{2}
(20~mL). The reaction mixture was stirred at 20~\textdegree C for 16~h
before being quenched by the addition of a saturated aqueous solution
of NaCl. The different layers were separated, and the aqueous layer was
extracted with CH\tsub{2}Cl\tsub{2}. The combined organic layers were
washed with an aqueous solution of Na\tsub{2}S\tsub{2}O\tsub{3}, dried
over Na\tsub{2}SO\tsub{4}, filtered, and the solvent was removed under
reduced pressure. The resulting crude product was dissolved in
methanol, and NaBH\tsub{4} (150~mg, 3.9~mmol, 1.2~eq.) was added at
0~\textdegree C. The reaction mixture was stirred at room temperature
for 2~h before being quenched by the addition of a saturated aqueous
solution of NH\tsub{4}Cl (2~mL). The solvents were removed under
reduced pressure, and the resulting mixture was purified over column
chromatography on silica gel (MeOH/CH\tsub{2}Cl\tsub{2}, 8:92) yielding
\textbf{15} as an oil (170~mg, 0.56~mmol, 18\%, Rf (MeOH/DCM, 8:92) $=$
0.16). \tsup{\textbf{1}}\textbf{H NMR}  (500~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 1.69 (3H, s, H-6), 1.73 (bs, 1H, OH), 2.00--2.06
(2H, m, H-1), 2.35--2.45 (4H, m, H-2 $+$ H-8), 3.77 (3H, d, $J =$
11.0~Hz, H-7), 3.81 (6H, dd, $J = 11.0$~Hz, $J = 2.0$~Hz, H-9 $+$
H-10), 4.00 (2H, s, H-5), 5.45 (1H, tq, $J = 7.5$~Hz, $J = 1.5$~Hz,
H-3). \tsup{\textbf{13}}\textbf{C NMR} (125, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 13.7 (C-6), 19.9 (d, $J = 4.2$~Hz, C-2), 26.4 (dd,
$J = 134.5$~Hz, $J = 75.8$~Hz, C-8), 29.0 (d, $J = 97.6$~Hz, C-1), 51.5
(d, $J = 6.7$~Hz, C-7), 53.1 (d, $J = 6.5$~Hz, C-9 $+$ C-10), 68.4
(C-5), 123.8 (d, $J = 14.1$~Hz, C-3), 136.6 (d, $J = 1.0$~Hz, C-4).
\tsup{\textbf{31}}\textbf{P NMR} (121~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$~23.07 (d, $J = 4.2$~Hz), 48.26 (d, $J = 4.0$~Hz).


\medskip\noindent
$\bullet$ Synthesis of dimethyl (\textit{E})-(((5-(1,3-\newline
dioxoisoindolin-2-yl)-4-methylpent-3-en-1-yl) (methoxy)phosphoryl)
methyl) phosphonate \textbf{16}

\xfigure{fx07}{}

Dimethyl (\textit{E})-(((5-hydroxy-4-methylpent-3-en-1-yl)
(methoxy)phosphoryl)methyl)phosphonate \textbf{15} (170~mg, 0.57~mmol,
1.2~eq.), PPh\tsub{3} (155~mg, 0.59~mmol, 1.2~eq.) and phthalimide
(70~mg, 0.48~mmol, 1~eq.) were dissolved in dry THF. DIAD
($130~\rmmu\mathrm{L}$, 0.64~mmol, 1.3~eq.) was added dropwise at
0~\textdegree C, and the reaction mixture was stirred at 
0~\textdegree C for 30~min and left to stand at 20~\textdegree C for 
2~h. The reaction was quenched by the addition of MeOH 
$(500~\rmmu\mathrm{L}$), and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography on
silica gel (MeOH/CH\tsub{2}Cl\tsub{2}, 8:92). A second column
chromatography on silica gel (ethyl acetate) yielded \textbf{16} as a
white oil (162~mg, 0.38~mmol, 79\%, Rf (ethyl acetate) $=$ 0.43).
\tsup{\textbf{1}}\textbf{H NMR} (500~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 1.67 (3H, s, H-6), 1.93--2.00 (2H, m, H-1),
2.30--2.42 (4H, m, H-2 $+$ H-8), 3.73 (3H, d, $J = 11$~Hz, H-7), 3.78
(6H, d, $J = 11$~Hz, H-9 $+$ H-10), 4.17 (2H, s, H-5), 5.35 (1H, tq, $J
= 7.5$~Hz, $J = 1.5$~Hz, H-3), 7.70--7.72 (2H, m, H-13), 7.83--7.84
(2H, m, H-14). \tsup{\textbf{13}}\textbf{C NMR} (125~MHz,
CDCl\tsub{3}): $\rmdelta$(ppm) $=$ 14.8 (C-6), 20.1 (d, $J = 16$~Hz,
C-2), 26.4 (dd, $J = 134.1$~Hz, $J = 76.1$~Hz, C-8), 29.0 (d, $J =
97.4$, C-1), 44.7 (C-5), 51.6 (d, $J = 6.3$~Hz, C-7), 53.2 (dd, $J =
6.5$~Hz, $J = 3.1$~Hz, C-9 $+$ C-10), 123.4 (C-13), 125.5 (d, $J =
15.5$~Hz, C-3), 131.2 (d, $J = 1.4$~Hz, C-4), 132.1 (C-14), 134.1
(C-12), 168.3 (C-11). \tsup{\textbf{31}}\textbf{P NMR}~(121~MHz,
CDCl\tsub{3}): $\rmdelta$(ppm) $=$~22.96 (d, $J = 4.0$~Hz), 47.97 (d,
$J = 4.0$~Hz).

\medskip\noindent
$\bullet$ (\textit{E})-(((5-amino-4-methylpent-3-en-1-yl)\newline
oxidophosphoryl) methyl) phosphonate \textbf{8}


\xfigure{fx08}{}


Bromotrimethylsilane ($340~\rmmu\mathrm{L}$, 3.2~mmol, 9.4~eq.) was
added dropwise to a solution of dimethyl  
(\textit{E})-(((5-(1,3-dioxoisoindolin-2-yl)-4-methylpent-3-en-1-yl)
(methoxy)phosphoryl) methyl) phosphonate \textbf{16} (146~mg,
0.34~mmol, 1~eq.) in dry CH\tsub{2}Cl\tsub{2} (3~mL) while being
stirred at 0~\textdegree C. The reaction mixture was further stirred at
0~\textdegree C for 30~min and then at 20~\textdegree C for 1~h.
Methanol was added, and the mixture was further stirred for 1~h.
Solvents were then removed under reduced pressure, and the resulting
oil was dissolved in acetone. A mixture of isopropanol, water and
ammonia (6/2/2) was added, and the mixture was stirred overnight. After
evaporation of the solvents, the crude product was purified by column
chromatography on silica gel (IPA/H\tsub{2}O/NH\tsub{4}OH, 6:2:2 to
5:3:2) yielding \textbf{8} as a~white solid, which was dissolved in
water, lyophilized, and further dried under high vacuum yielding
\textbf{17} (12~mg, 0.046~mmol, 14\%, Rf (IPA/H\tsub{2}O/NH\tsub{4}OH,
6:4:2) $=$ 0.25). \tsup{\textbf{1}}\textbf{H NMR} (300~MHz,
D\tsub{2}O): $\rmdelta$(ppm) $=$ 1.69 (3H, s, H-6), 1.70--1.76 (2H, m,
H-1), 1.98--2.06 (2H, m, H-7), 2.23--2.30 (2H, m, H-2), 3.46 (2H, s,
H-5), 5.54 (1H, t, $J = 7.0$~Hz, H-3). \tsup{\textbf{13}}\textbf{C NMR}
(125~MHz, D\tsub{2}O): $\rmdelta$(ppm) $=$ 13.6 (C-6), 20.4 (d, $J =$
3.6~Hz, C-2), 29.9 (d, $J = 95.0$~Hz, C-1), 30.7 (dd, $J = 119.8$~Hz, $J
= 75.9$~Hz, C-7), 46.2 (C-5), 127.5 (C-4), 130.8 (d, $J = 14.4$~Hz,
C-3). \tsup{\textbf{31}}\textbf{P NMR} (121~MHz, D\tsub{2}O):
$\rmdelta$(ppm) $=$~15.40 (d, $J = 5.5$~Hz), 38.13 (d, $J = 5.5$~Hz).
High-resolution MS (ES-) \textit{m/z}: [M--H]\tsup{-}
(C\tsub{7}H\tsub{16}NO\tsub{5}P\tsub{2}) calculated 256.0509, found
256.0526.


\subsubsection{Synthesis of
(\textit{E})-5-amino-4-methylpent-3-ene-1-sulfonate \textbf{7}}

\noindent
$\bullet$ (\textit{E})-5-bromo-2-methylpent-2-en-1-ol \textbf{18}

\xfigure{fx09}{}

Tert-butyl hydroperoxide (70\% in water, 1.7~mL, 12.6~mmol, 2~eq.)
was added to a suspension of SeO\tsub{2} (340~mg, 3.01~mmol, 0.5~eq.)
stirred at 0~\textdegree C. The reaction mixture was stirred at
0~\textdegree C for 5~min and at 20~\textdegree C for 30~min.
5-Bromo-2-methylpent-2-ene \textbf{17} ($820~\rmmu\mathrm{L}$, 6.13~mmol, 
1~eq.) was added dropwise at 0~\textdegree C. The reaction mixture
was stirred overnight while slowly being allowed to warm up to
20~\textdegree C. It was then diluted with diethyl ether and washed
twice with an aqueous solution of KOH (1M) then brine. The organic
layer was dried over Na\tsub{2}SO\tsub{4}, filtered, and the solvent
was removed under reduced pressure. The crude product was purified by
column chromatography on silica gel (ethyl acetate/petroleum ether,
2:8) yielding \textbf{18} (660~mg, 3.69~mmol, 60\%).
\tsup{\textbf{1}}\textbf{H NMR} (500~MHz, CDCl\tsub{3}):
$\rmdelta$(ppm) $=$ 1.69 (3H, s, H-6), 2.63 (2H, dt, $J = 7.0$~Hz,
6.5~Hz, H-4), 3.39 (2H, t, $J = 7.0$~Hz, H-5), 4.03 (2H, s, H-1), 5.44
(1H, m, H-3). \tsup{\textbf{13}}\textbf{C NMR} (125~MHz,
CDCl\tsub{3}): $\rmdelta$(ppm) $=$ 14.0 (C-6), 31.3 (C-4), 32.6 (C-5),
68.5 (C-1), 122.1 (C-3), 138.1 (C-2).

\medskip\noindent
$\bullet$ (\textit{E})-2-(5-bromo-2-methylpent-2-en-1-yl)\newline
isoindoline-1,3-dione \textbf{19}


\xfigure{fx10}{}

DIAD $(400~\rmmu\mathrm{L}$, 2.0~mmol, 1.2~eq.) was added to a solution
of phthalimide (300~mg, 2.0~mmol, 1.2~eq.), PPh\tsub{3} (530~mg,
2.0~mmol, 1.2~eq.) and \textbf{18} (300~mg, 1.68~mmol, 1~eq.) in dry
THF while stirring at 0~\textdegree C. The reaction mixture was stirred
for 3~h and simultaneously allowed to warm up to 20~\textdegree C. The
reaction was quenched by the addition of MeOH (1~mL), and the solvent
was removed under reduced pressure. The crude product was purified by
column chromatography on silica gel (ethyl acetate/petroleum ether 1:9)
yielding \textbf{19} as an oil (415~mg, 1.35~mmol, 80\%, Rf (ethyl
acetate/cyclohexane, 20:80) $=$ 0.60). \tsup{\textbf{1}}\textbf{H NMR}
(300~MHz, CDCl\tsub{3}): $\rmdelta$(ppm) $=$ 1.68 (3H, s, H-6), 2.59
(2H, dt, $J = 7.2$~Hz, 6.3~Hz, H-4), 3.33 (2H, t, $J = 7.2$~Hz, H-5),
4.22 (2H, s, H-1), 5.37 (1H, m, H-3), 7.69--7.76 (2H, m, H-9),
7.83--7.89 (2H, m, H-10). \tsup{\textbf{13}}\textbf{C NMR} (125~MHz,
CDCl\tsub{3}): $\rmdelta$(ppm) $=$ 15.0 (C-6), 31.5 (C-4), 32.0 (C-5),
44.8 (C-1), 123.5 (C-9), 124.1 (C-3), 132.1 (C-8) 132.7 (C-2), 134.2
(C-10), 168.3 (C-7). 

\medskip\noindent
$\bullet$ (\textit{E})-5-amino-4-methylpent-3-ene-1-sulfonate \textbf{7}

\xfigure{fx11}{}

A solution of sodium sulfite (360~mg, 2.86~mmol, 2.2~eq.) in water
(5~mL) was added to a stirred\unskip\break solution of \textbf{19} (400~mg,
1.30~mmol, 1~eq.) in EtOH (5~mL). The reaction mixture was further
stirred under reflux overnight. The suspension was filtered, and the
filtrate was evaporated under reduced pressure. Nonpolar products were
discarded by filtration on silica gel
(MeOH/CH\tsub{2}Cl\tsub{2}/NEt\tsub{3}, 2/8/0.2), and the most polar
product was recovered and then dissolved in MeOH (2~mL) without further
purification. NH\tsub{4}OH (2~mL) was added, and the reaction mixture
was stirred overnight. Solvents were removed under reduced pressure.
Consecutive column chromatography (isopropanol/H\tsub{2}O/NH\tsub{4}OH,
8:0.8:2 then isopropanol/H\tsub{2}O, 9:1) yielded \textbf{7} as a white
solid, which was dissolved in water, lyophilized, and further dried
under high vacuum (10~mg, 0.056~mmol, 4\%, Rf (IPA/H\tsub{2}O, 8:2) $=$
0.27). \tsup{\textbf{1}}\textbf{H NMR} (300~MHz, D\tsub{2}O):
$\rmdelta$(ppm) $=$ 1.76 (3H, s, H-6), 2.54 (2H, dt, $J = 7.2$~Hz, $J
= 6.2$~Hz, H-2), 2.98 (2H, t, $J = 7.8$~Hz, H-1), 3.55 (2H, s, H-5),
5.60 (1H, m, H-3). \tsup{\textbf{13}}\textbf{C NMR} (125~MHz,
D\tsub{2}O): $\rmdelta$(ppm) $=$ 14.3 (C-6), 23.5 (C-2), 46.8 (C-1),
50.6 (C-5), 128.4 (C-3), 129.9 (C-4). High-resolution MS (ES-)
\textit{m/z}: [M--H]\tsup{-} (C\tsub{6}H\tsub{12}NO\tsub{3}S) calculated
178.0543, found 178.0541.

{\vspace*{-2pt}}

\subsection{Biological experiments}

{\vspace*{-2pt}}

\subsubsection{IspH production}

{\vspace*{-2pt}}

Production and purification of \textit{E. coli} IspH were performed as
previously described \cite{30}. Protein concentration was measured
using the Bradford method with bovine serum albumin as a standard
\cite{42}. Iron was quantified according to Fish \cite{43} and sulfide
as described by Beinert \cite{44}. UV/visible spectrum, iron and sulfur
content of \textit{E. coli} IspH were similar to those previously
reported \cite{13}, in accordance with the presence of
[4Fe--4S]\tsup{2+}  cluster. 

{\vspace*{-2pt}}

\subsubsection{Enzymatic assays}

{\vspace*{-2pt}}

Enzyme activity was determined by monitoring NADPH consumption in the
presence of optimized concentrations of the reducing system under
anaerobic conditions. A HMBPP solution (final concentration 
$150~\rmmu\mathrm{M}$) was added through a gas-tight syringe to a
0.1~cm light path cuvette prepared in an anaerobic glove box and
containing NADPH (2.2~mM), FldA ($30~\rmmu\mathrm{M}$), FpR1 
$(17~\rmmu\mathrm{M})$, IspH ($0.5~\rmmu\mathrm{M}$), and either
\textbf{6} or \textbf{7} or \textbf{8} or AMBPP in 50~mM Tris--HCl pH
$=$ 8 that had previously been incubated for 15~min at 37~\textdegree C. The
reaction was monitored spectrophotometrically at 340~nm with a Cary
100~UV/visible spectrophotometer (Varian) maintained at 
37~\textdegree C using a thermostat equipped with a Peltier element.

{\vspace*{-2pt}}

\section*{Declaration of interests}

{\vspace*{-2pt}}

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.

{\vspace*{-2pt}}

\section*{Funding}

{\vspace*{-2pt}}

This work was funded by the Fondation Jean-Marie Lehn, the European
Union's Horizon 2020 research and innovation program under the Marie
Sklodowska-Curie grant agreement No. 860816 and the ``Universit\'{e}
franco-allemande''. This work of the Interdisciplinary Thematic
Institute InnoVec, as part of the ITI program of the University of
Strasbourg, CNRS and Inserm, was supported by IdEx Unistra
(ANR-10-IDEX-0002) and by SFRI-STRAT'US project (ANR-20-SFRI-0012)
under the framework of the French Investments for the Future Program.

\CDRGrant[EU]{860816}
\CDRGrant[ANR]{ANR-10-IDEX-0002}
\CDRGrant[ANR]{ANR-20-SFRI-0012}

{\vspace*{-2pt}}

\section*{Acknowledgments}

{\vspace*{-2pt}}

We appreciate the help from the staff of the computing facility
provided by the Commissariat \`{a} l'Energie Atomique et aux
\'{e}nergies renouvelables (CEA/DSV/GIPSI), Saclay, France, and the
Centre de Calcul Recherche et Technologie (CEA/CCRT),
Bruy\`{e}res-le-Ch\^{a}tel, France.

\back{}

{\vspace*{-2pt}}

\section*{Supplementary data}

{\vspace*{-2pt}}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-254-suppl.pdf}}

{\vspace*{-2pt}}

\bibliographystyle{crchim}
\bibliography{crchim20230360}
\refinput{crchim20230360-reference.tex}

\end{document}
