You are here : Home > BioCE team > Research topics of the team > Artificial metalloenzymes or the tomorrow synthetic chemistry

Artificial metalloenzymes or the tomorrow synthetic chemistry

Published on 24 January 2019

Dr Caroline Marchi Delapierre
Maître de Conférences Université Grenoble Alpes
Laboratoire Chimie et Biologie des Métaux
17 avenue des Martyrs
38 054 Grenoble Cedex 09
Phone: 33 (0)4 38 78 91 04
Fax: 33 (0)4 38 78 91 24

Scientific project

The goal of our project is to develop hybrid active catalysts under asymmetric oxidations by favoring oxygen transfer reactions. The approach we develop involves the development of artificial metalloenzymes: bioinspired inorganic complexes are introduced in hydrophobic cavities of proteins whose scaffolding is chiral.

Artificial metalloenzyme: An achiral complex in a chiral peptide environment. A new hybrid biocatalyst

Homogeneous catalysis is the best method to introduce stereochemistry into pharmaceutically active molecules but the industrial constraints (economy, freedom to operate, process robustness) remains a significant issue today. Biocatalysis, on the other hand, could be an alternative but the restrain scope of substrates for example has slowed down its application.
Then, there is a need for a new generation of catalysts combining clean processes and controlled asymmetry.

One original solution deals with the combination of homogeneous catalysis and biocatalysis via the design of artificial metalloenzymes. This new field, still in its infancy, is very promising. The original hybrids, formed by the embedment of an inorganic complex (playing the role of active site) into a protein (playing the role of the selective or chiral auxiliary) should gain new properties taking advantages of the two catalytic fields:

• broad substrate scope. The inorganic catalyst will be able to drive several types of catalysis and substrate;
• ease for recycling. The hybrid system will be in aqueous media. Product extraction is often sufficient;
• access to both enantiomers via transformation of the inorganic catalyst;
aqueous media. Insertion of the catalyst in a polypeptide will lead to its solubilization in water;
gain in stability. Protein scaffold should prevent inter-molecular undesired reactions such as catalyst destruction;
• great tool for improvement of enantioselectivity by site directed mutagenesis and chemical modifications of the inorganic complex (active site).

Our project deals with the design of artificial metalloenzymes in the field of oxidation catalysis. Two general directions are under exploration:

• the development of new cleaner processes for enantioselective oxidations,
• the development of new cleaner processes for aromatic transformations via oxidation (remediation) with O2.

The fundamental question that could be answered via these strategies relies on the control of the second coordination sphere of the catalyst.

Our aim is to use this strategy to provide enantioselective oxidation of alkanes, alkenes and sulfides via an oxygen transfer reaction. The insertion of an oxygen atom into a prochiral molecule is an option far more convenient than reduction of a ketone for example. Moreover, the previous reported oxygenase activities are still under industrial standard. In particular, we focus on asymmetric catalysts for hydroxylation, for which solutions are still to be discovered. Depending upon the quality of our designed biocatalyst, the use of drugs precursors is planned. A special care is afforded to aromatic hydroxylation, in order to search for clean degradation processes. This is based on the fact that the transformation should solubilize molecules such as HAP.
Our strategy is not only to characterize the catalytic properties of our hybrids but to fully characterize them spectroscopically too. For this purpose, various spectroscopies are available in the laboratory: UV-visible, EPR, Mössbauer or in collaboration: Raman, X-rays, crystallography.

Two recent studies:

The proof of concept: Synthesis of new “NikA-FeL” hybrids and their catalytic properties towards thioether oxidation.

Figure 1: Selection of metal complexes candidates for their insertion into NikA.

Because it has been demonstrated that NikA, a periplasmic nickel-binding protein involved in nickel uptake in Escherichia coli, is able to bind inorganic compounds like Fe(III)-EDTA, (Journal of the American Chemical Society, 2005) we have considered it as a suitable target for the design of new artificial metalloenzymes. Based on our previous studies, we have designed EDTA-like mononuclear iron complexes mimicking active sites of non-heme iron oxygenases. For that, we have varied the number and the position of the carboxylate groups as well as the number and the nature of aromatic or cyclic groups (Figure 1). The different inorganic complexes and the corresponding NikA/FeL hybrids were characterized in solution and by X-ray crystallography (Journal of Biological Inorganic Chemistry, 2012).
The crystal structures of hybrids were solved at 2.0 Å resolution (
Figure 2).

Figure 2: Crystal structures of 5NikA (top) and 6NikA hybrids (bottom).

These hybrids were tested for their catalytic efficiency towards thioethers oxidation. When the reference substrate for sulfoxidation, thioanisole, was used, high reactivity of this sulfide led to an unexpected side oxidation reaction with the protein itself. This competitive side reaction does not allow us to determine the best catalyst and the selectivity of the reaction catalyzed by the hybrid was quite poor. So, we turned to molecular docking to ensure a large screening of sulfides presenting the thioanisole motif in their backbone (Angewandte Chemie International Edition, 2013). Through the hundreds of molecules giving a positive hit, we selected a family easy to synthesize and sharing some features with the omeprazole skeleton. A series of substrates were synthesized (Figure 3).

Figure 3: Molecular docking of a new family of substrate member inside 4
NikA (left) and selected synthesized members (right).

All the hybrids were tested as catalysts for the oxidation of the new substrates. The results show clearly that only hybrids containing a non fully coordinated iron metal center are able to oxidize this type of substrate. More importantly, we tested analogs of 1a substrate and found a direct correlation between the selectivity and the binding mode of the susbtrate into the protein that validated our method (Angewandte Chemie International Edition, 2013).

In cristallo hydroxylation of aromatics.

Recently, we used our own technology based on the insertion of inorganic catalysts into NikA, to decipher an oxidation mechanism (Nature Chemistry, 2010). We combined model synthetic chemistry and protein X-ray crystallography to unravel the catalytic cycle of an aromatic dihydroxylation reaction catalyzed by a complex mimicking the active site of iron monooxygenases. The target arene was incorporated into the iron complex that was bound to the crystallized NikA. In this manner, the hydroxylated species was generated in an intramolecular reaction. The protein-bound arene-containing iron complex was able to activate dioxygen in the presence of a reductant, which led to the formation of a catechol as a sole product. Structure determination and Resonance Raman spectroscopic measurements performed on flash-cooled crystals were used to characterize four intermediates as well as the end product (Figure 4). This study revealed that the hydroxylation is carried out by an iron peroxo-generated hydroxyl radical species.

Figure 4: Crystal structures of NikA-bound iron complex (NikA/1) at different aromatic hydroxylation stages.
1: Complex 1 in its binding site.
2: Reduced NikA/1 hybrid.
3: Diatomic oxo intermediate prior hydroxylation.
4: Doubly hydroxylated iron complex-NikA species.

Our in situ determination of a catalytic cycle illustrates that a protein scaffold can control reactions smoothly, allowing the structural observation of transient species, without interfering with the nature of the reaction. Solution studies are unlikely to provide such a degree of detail.
Consequently, our approach may have a general application in the study of challenging chemical reaction intermediates using protein crystallography. This outcome is the basis for the design of the new catalysts for O
2-mediated aromatics degradation that are currently under investigation in our laboratory.


Lopez S, Rondot L, Cavazza C, Iannello M, Boeri-Erba E, Burzlaff N, Strinitz F, Jorge-Robin A, Marchi-Delapierre C and Ménage S
Efficient conversion of alkenes to chlorohydrins by a Ru-based artificial enzyme.
Chemical Communications, 2017, 53(25): 3579-3582

Lopez S, Rondot L, Leprêtre C, Marchi-Delapierre C, Ménage S and Cavazza C
Cross-linked artificial enzyme crystals as heterogeneous catalysts for oxidation reactions.
Journal of the American Chemical Society, 2017, 139(49): 17994-18002

Rondot L, Girgenti E, Oddon F, Marchi-Delapierre C, Jorge-Robin A and Ménage S
Catalysis without a headache: Modification of ibuprofen for the design of artificial metalloenzyme for sulfide oxidation.
Journal of Molecular Catalysis A: Chemical, 2016, 416: 20-28

Marchi-Delapierre C, Rondot L, Cavazza C and Ménage S
Oxidation catalysis by rationally designed artificial metalloenzymes.
Israel Journal of Chemistry, 2015, 55(1): 61-75

Rull J, Nonglaton G, Costa G, Fontelaye C, Marchi-Delapierre C, Ménage S and Marchand G
Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide.
Applied Surface Science, 2015, 354: 285-297

Esmieu C, Cherrier MV, Amara P, Girgenti E, Marchi-Delapierre C, Oddon F, Iannello M, Jorge-Robin A, Cavazza C and Ménage S
Oxygenase built from scratch: Substrate binding site identified using a docking approach.
Angewandte Chemie International Edition, 2013, 52(14): 3922-3925

Hall N, Orio M, Jorge-Robin A, Gennaro B, Marchi-Delapierre C and Duboc C
Vanadium thiolate complexes for efficient and selective sulfoxidation catalysis: A mechanistic investigation.
Inorganic Chemistry, 2013, 52(23): 13424-13431

Cherrier MV, Girgenti E, Amara P, Iannello M, Marchi-Delapierre C, Fontecilla-Camps JC, Ménage S and Cavazza C
The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design.
Journal of Biological Inorganic Chemistry, 2012, 17(5): 817-829

Oddon F, Girgenti E, Lebrun C, Marchi-Delapierre C, Pécaut J and Ménage S
Iron coordination chemistry of N
2Py2 ligands substituted by carboxylic moieties and their impact on alkene oxidation catalysis.
European Journal of Inorganic Chemistry, 2012, 2012(1): 85–96

Cavazza C, Bochot C, Rousselot-Pailley P, Carpentier P, Cherrier MV, Martin L, Marchi-Delapierre C, Fontecilla-Camps JC and Menage S
Crystallographic snapshots of the reaction of aromatic C-H with O2 catalysed by a protein-bound iron complex.
Nature Chemistry, 2010, 2(12): 1069-1076

Hazimeh H, Mattalia JM, Marchi-Delapierre C, Kanoufi F, Combellas C and Chanon M
Structural effects in radical clocks and mechanisms of grignard reagent formation: Special effect of a phenyl substituent in a radical clock when the crossroads of selectivity is at a metal/solution interface.
European Journal of Organic Chemistry, 2009, 2009(17): 2775-2787

Rousselot-Pailley P, Bochot C, Marchi-Delapierre C, Jorge-Robin A, Martin L, Fontecilla-Camps JC, Cavazza C and Ménage S
The protein environment drives selectivity for sulfide oxidation by an artificial metalloenzyme.
Chembiochem, 2009, 10(3): 545-552

Marchi-Delapierre C, Jorge-Robin A, Thibon A and Ménage S
A new chiral diiron catalyst for enantioselective epoxidation.
Chemical Communications, 2007, 11: 1166-1168

Mattalia JM, Marchi-Delapierre C, Hazimeh H and Chanon M
The reductive decyanation reaction: Chemical methods and synthetic applications.
Arkivoc, 2006, 0000

Gimbert C, Lumbierres M, Marchi C, Moreno-Manas M, Sebastian RM and Vallribera A
Michael additions catalyzed by phosphanes.
Tetrahedron, 2005, 61: 8598-8605

Hazimeh H, Mattalia JM, Marchi-Delapierre C, Barone R, Nudelman NS and Chanon M
Radical clocks and electron transfer. Compared crown ether effects in the reactivity of potassium and magnesium towards 1-bromo-2-but-3-énylbenzene. The incidence of homogeneous versus heterogeneous electron transfer on selectivity.
Journal of Physical Organic Chemistry, 2005, 18: 1145-1160