24 publications

24 publications

Active Site Topology of Artificial Peroxidase-like Hemoproteins Based on Antibodies Constructed from a Specifically Designed Ortho-carboxy-substituted Tetraarylporphyrin

Mahy, J.-P.

Eur. J. Biochem. 1998, 257, 121-130, 10.1046/j.1432-1327.1998.2570121.x

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin {α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin]}, and which exhibit in the presence of this α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra‐aryl‐substituted porphyrin has shown that : (a) the central iron(III) atom of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] is not recognized by either of the two antibodies; and (b) the ortho‐carboxylate substituents of the meso‐phenyl rings of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o‐COOHPh)4‐porphyrinH2 as well as mono‐ and di‐ortho‐carboxyphenyl‐substituted porphyrins suggests that the three carboxylates in the α, α, β position are recognized by both 13G10 and 14H7 with the two in the α, β positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two‐thirds of the α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O‐O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.


Metal: Fe
Ligand type: ---
Host protein: Antibody 13G10 / 14H7
Anchoring strategy: Antibody
Optimization: Chemical & genetic
Reaction: Peroxidation
Max TON: ---
ee: ---
PDB: ---
Notes: ---

An Artificial Enzyme Made by Covalent Grafting of an FeII Complex into β-Lactoglobulin: Molecular Chemistry, Oxidation Catalysis, and Reaction-Intermediate Monitoring in a Protein

Banse, F.; Mahy, J.-P.

Chem. - Eur. J. 2015, 21, 12188-12193, 10.1002/chem.201501755

An artificial metalloenzyme based on the covalent grafting of a nonheme FeII polyazadentate complex into bovine β‐lactoglobulin has been prepared and characterized by using various spectroscopic techniques. Attachment of the FeII catalyst to the protein scaffold is shown to occur specifically at Cys121. In addition, spectrophotometric titration with cyanide ions based on the spin‐state conversion of the initial high spin (S=2) FeII complex into a low spin (S=0) one allows qualitative and quantitative characterization of the metal center’s first coordination sphere. This biohybrid catalyst activates hydrogen peroxide to oxidize thioanisole into phenylmethylsulfoxide as the sole product with an enantiomeric excess of up to 20 %. Investigation of the reaction between the biohybrid system and H2O2 reveals the generation of a high spin (S=5/2) FeIII(η2‐O2) intermediate, which is proposed to be responsible for the catalytic sulfoxidation of the substrate.


Metal: Fe
Ligand type: Poly-pyridine
Host protein: ß-lactoglobulin
Anchoring strategy: Covalent
Optimization: ---
Reaction: Sulfoxidation
Max TON: 5.6
ee: 20
PDB: ---
Notes: ---

Artificial Metalloenzymes with the Neocarzinostatin Scaffold: Toward a Biocatalyst for the Diels–Alder Reaction

Mahy, J.-P.; Ricoux, R.

ChemBioChem 2016, 17, 433-440, 10.1002/cbic.201500445

A new artificial enzyme formed by associating NCS‐3.24 with a copper complex catalyzed the Diels–Alder cyclization of cyclopentadiene with 2‐azachalcone and led to an increase in the formation of the exo‐products. Molecular modeling proposed the preferred relative positioning of both the Trojan horse complex and the two substrates.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 33
ee: ---
PDB: ---
Notes: Up to endo/exo ratio 62:38

Artificial Peroxidase-Like Hemoproteins Based on Antibodies Constructed from a Specifically Designed Ortho-Carboxy Substituted Tetraarylporphyrin Hapten and Exhibiting a High Affinity for Iron-Porphyrins

Mahy, J.-P.

FEBS Lett. 1996, 395, 73-76, 10.1016/0014-5793(96)01006-X

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)α,α,α,β‐mesotetrakis‐orthocarboxyphenyl‐porphyrin (Fe(ToCPP))‐KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG, and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True K d values of 2.9 × 10−9 M and 5.5 × 10−9 M have been determined for the two IgG1‐Fe(ToCPP) complexes. Those values are the best ones ever reported for iron‐porphyrin‐antibody complexes. UV‐vis. studies have shown that the two IgG1‐Fe(ToCPP) complexes were highspin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2α‐Fe(ToCPP) complex was a low‐spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1 ‐Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2′‐azinobis (3ethylbenzothiazoline‐6‐sulfonic acid (ABTS) 5‐fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron‐porphyrin had no effect on its catalytic activity. k cat values of 100 min−1 and 63 min−1 and k cat/K m. values of 105 M−1 s−1 and 119 M−1 s−1 have been found respectively for the two IgG1‐Fe(ToCPP) complexes.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 13G10
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 105 M-1 * s-1

Coordination Chemistry of Iron(III)-Porphyrin-Antibody Complexes Influence on the Peroxidase Activity of the Axial Coordination of an Imidazole on the Iron Atom

Mahy, J.-P.

Eur. J. Biochem. 2002, 269, 470-480, 10.1046/j.0014-2956.2001.02670.x

An artificial peroxidase‐like hemoprotein has been obtained by associating a monoclonal antibody, 13G10, and its iron(III)–α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin [Fe(ToCPP)] hapten. In this antibody, about two‐thirds of the porphyrin moiety is inserted in the binding site, its ortho‐COOH substituents being recognized by amino‐acids of the protein, and a carboxylic acid side chain of the protein acts as a general acid base catalyst in the heterolytic cleavage of the O–O bond of H2O2, but no amino‐acid residue is acting as an axial ligand of the iron. We here show that the iron of 13G10–Fe(ToCPP) is able to bind, like that of free Fe(ToCPP), two small ligands such as CN–, but only one imidazole ligand, in contrast to to the iron(III) of␣Fe(ToCPP) that binds two. This phenomenon is general for a series of monosubstituted imidazoles, the 2‐ and 4‐alkyl‐substituted imidazoles being the best ligands, in agreement with the hydrophobic character of the antibody binding site. Complexes of antibody 13G10 with less hindered iron(III)–tetraarylporphyrins bearing only one [Fe(MoCPP)] or two meso‐[ortho‐carboxyphenyl] substituents [Fe(DoCPP)] also bind only one imidazole. Finally, peroxidase activity studies show that imidazole inhibits the peroxidase activity of 13G10–Fe(ToCPP) whereas it increases that of 13G10–Fe(DoCPP). This could be interpreted by the binding of the imidazole ligand on the iron atom which probably occurs in the case of 13G10–Fe(ToCPP) on the less hindered face of the porphyrin, close to the catalytic COOH residue, whereas in the case of 13G10–Fe(DoCPP) it can occur on the other face of the porphyrin. The 13G10–Fe(DoCPP)–imidazole complex thus constitutes a nice artificial peroxidase‐like hemoprotein, with the axial imidazole ligand of the iron mimicking the proximal histidine of peroxidases and a COOH side chain of the antibody acting as a general acid‐base catalyst like the distal histidine of peroxidases does.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 13G10
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 15200 M-1 * s-1

Coordination Chemistry Studies and Peroxidase Activity of a New Artificial Metalloenzyme Built by the “Trojan Horse” Strategy

Mahy, J.-P.

J. Mol. Catal. A: Chem. 2010, 317, 19-26, 10.1016/j.molcata.2009.10.016

In the general context of green chemistry, a considerable research effort is devoted to the elaboration of new artificial metalloproteins that catalyze, under mild conditions, the oxidation of a wide range of organic compounds, using cheap and environmentally friendly oxidants. A new artificial hemoprotein was obtained by the so-called “Trojan horse” strategy involving the non-covalent insertion of a cationic iron–porphyrin–estradiol cofactor into an anti-estradiol antibody. UV–vis titrations showed the formation of a 1/2 antibody/cofactor complex with a dissociation constant KD = 4.10−7 M. UV–vis determination of the Fe-imidazole binding constants showed that the protein provided a weak steric hindrance around the iron–porphyrin cofactor. The antibody–estradiol–iron–porphyrin complex displayed a peroxidase activity and catalyzed the oxidation of ABTS by H2O2 with about double the efficiency of the iron–porphyrin–estradiol alone. Kinetic studies revealed that this was due to a faster formation of the intermediate high valent iron–oxo species in the presence of the antibody protein. Consequently, the association of an anti-estradiol antibody with an iron–porphyrin–estradiol cofactor leads to a new artificial hemoprotein with an interesting peroxidase activity and the “Trojan horse” strategy appears as a valuable method to generate artificial metalloenzymes that could act as biocatalysts for selective oxidations.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 7A3
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: k1 = 574 M-1 * min-1

From "Hemoabzymes" to "Hemozymes": Towards new Biocatalysts for Selective Oxidations

Review

Mahy, J.-P.

Chem. Commun. 2015, 51, 2476-2494, 10.1039/c4cc08169b

The design of artificial hemoproteins that could catalyze selective oxidations using clean oxidants such as O2 or H2O2 under ecocompatible conditions constitutes a real challenge for a wide range of industrial applications. In vivo, such reactions are performed by heme-thiolate proteins, cytochromes P450, which catalyze the oxidation of substrates by dioxygen in the presence of electrons delivered from NADPH by cytochrome P450 reductase. Several strategies were used to design new artificial hemoproteins that mimic these enzymes. The first one involved the non-covalent association of synthetic hemes with monoclonal antibodies raised against these cofactors. This led to the first generation of artificial hemoproteins or “hemoabzymes” that displayed a peroxidase activity, and in some cases catalyzed the regioselective nitration of phenols by H2O2/NO2 and the stereoselective oxidation of sulfides by H2O2. The second one involved the non-covalent association of easily affordable non-relevant proteins with metalloporphyrin derivatives, using either the “Trojan Horse strategy” or the “host–guest” strategy. This led to a second generation of artificial hemoproteins or “hemozymes”, some of which were found able to catalyze the stereoselective oxidation of organic compounds such as sulfides and alkenes by H2O2 and KHSO5.


Notes: ---

Hemoabzymes - Different Strategies for Obtaining Artificial Hemoproteins based on Antibodies

Review

Mahy, J.-P.

Appl. Biochem. Biotechnol. 1998, 75, 103-127, 10.1007/Bf02787712

Besides existing models of chemical or biotechnological origin for hemoproteins like peroxidases and cytochromes P450, catalytic antibod ies (Abs) with a metalloporphyrin cofactor represent a promising alter native route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that, until now, the first strategy for obtaining such artificial hemoproteins has been to produce antipor phyrin Abs, raised against various free-base, N-substituted, Sn-,Pd-,or Fe-porphyrins. Four of them exhibited, in the presence of the corre sponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/Km values of 102 to 5 × 103/M/s. This value remained low when com pared to that of peroxidases, probably because neither a proximal ligand of the Fe, nor amino acid residues participating in the catalysis of the heterolytic cleavage of the O—O bond of H2O2, have been induced in those Abs. This strategy has been shown to be insufficient for the elabo ration of effective models of cytochromes P450, because only one set of Abs, raised againstmeso-tetrakis(para-carboxyvinylphenyl)porphyrin, was reported to catalyze the nonstereoselective oxidation of styrene by iodosyl benzene using a Mn-porphyrin cofactor, and attempts to gener ate Abs having binding sites for both the substrate and the metallopor phyrin cofactor, using as a hapten a porphyrin covalently linked to the substrate, were not successful. A second strategy is then proposed, which involves the chemical labeling of antisubstrate Abs with a metallopor phyrin. As an example, preliminary results are presented on the covalent linkage of an Fe-porphyrin to an antiestradiol Ab, in order to obtain semisynthetic catalytic Abs able to catalyze the selective oxidation of steroids.


Notes: ---

Hemoabzymes: Towards New Biocatalysts for Selective Oxidations

Mahy, J.-P.

J. Immunol. Methods 2002, 269, 39-57, 10.1016/S0022-1759(02)00223-5

Catalytic antibodies with a metalloporphyrin cofactor or «hemoabzymes», used as models for hemoproteins like peroxidases and cytochrome P450, represent a promising route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin antibodies, raised against various free-base, N-substituted Sn-, Pd- or Fe-porphyrins. Five of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/Km values of 3.7×103–2.9×105 M−1 min−1. This value remained, however, low when compared to that of peroxidases. This strategy has also led to a few models of cytochrome P450. The best of them, raised against a water-soluble tin(IV) porphyrin containing an axial α-naphtoxy ligand, was reported to catalyze the stereoselective oxidation of aromatic sulfides by iodosyl benzene using a Ru(II)-porphyrin cofactor. The relatively low efficiency of the porphyrin–antibody complexes is probably due, at least in part, to the fact that no proximal ligand of Fe has been induced in those antibodies. We then proposed to use, as a hapten, microperoxidase 8 (MP8), a heme octapeptide in which the imidazole side chain of histidine 18 acts as a proximal ligand of the iron atom. This led to the production of seven antibodies recognizing MP8, the best of them, 3A3, binding it with an apparent binding constant of 10−7 M. The corresponding 3A3–MP8 complex was found to have a good peroxidase activity characterized by a kcat/Km value of 2×106 M−1 min−1, which constitutes the best one ever reported for an antibody–porphyrin complex. Active site topology studies suggest that the binding of MP8 occurs through interactions of its carboxylate substituents with amino acids of the antibody and that the protein brings a partial steric hindrance of the distal face of the heme of MP8. Consequently, the use of the 3A3–MP8 complexes for the selective oxidation of substrates, such as sulfides, alkanes and alkenes will be undertaken in the future.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 3A3
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 33000 M-1 * s-1

Hemozymes Peroxidase Activity Of Artificial Hemoproteins Constructed From the Streptomyces Lividans Xylanase A and Iron(III)-Carboxy-Substituted Porphyrins

Mahy, J.-P.

Bioconjug. Chem. 2008, 19, 899-910, 10.1021/bc700435a

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-α4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 µM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV–visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.


Metal: Fe
Ligand type: Porphyrin
Host protein: Xylanase A (XynA)
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 1083 M-1 * s-1

Incorporation of Manganese Complexes into Xylanase: New Artificial Metalloenzymes for Enantioselective Epoxidation

Mahy, J.-P.; Ricoux, R.

ChemBioChem 2012, 13, 240-251, 10.1002/cbic.201100659

Enantioselective epoxidation: An artificial metalloenzyme obtained by noncovalent insertion of MnIII‐meso‐tetrakis(para‐carboxyphenyl)porphyrin Mn(TpCPP) into xylanase 10A from Streptomyces lividans as a host protein was able to catalyse the oxidation of para‐methoxystyrene by KHSO5 with a 16 % yield and the best enantioselectivity (80 % in favour of the R isomer) ever reported for an artificial metalloenzyme.


Metal: Mn
Ligand type: Porphyrin
Host protein: Xylanase A (XynA)
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Epoxidation
Max TON: 21
ee: 80
PDB: ---
Notes: ---

Neocarzinostatin-Based Hybrid Biocatalysts for Oxidation Reactions

Mahy, J.-P.; Ricoux, R.

Dalton Trans. 2014, 43, 8344-8354, 10.1039/c4dt00151f

An anionic iron(III)-porphyrin–testosterone conjugate 1-Fe has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called ‘Trojan Horse’ strategy. This new 1-Fe-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the chemoselective and slightly enantioselective (ee = 13%) sulfoxidation of thioanisole by H2O2. Molecular modelling studies show that a synergy between the binding of the steroid moiety and that of the porphyrin macrocycle into the protein binding site can explain the experimental results, indicating a better affinity of 1-Fe for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the Fe-porphyrin complex is sandwiched between the two subdomains of the protein providing with good complementarities. However, the artificial cofactor entirely fills the cavity and its metal ion remains widely exposed to the solvent which explains the moderate enantioselectivity observed. Some possible improvements in the “Trojan Horse” strategy for obtaining better catalysts of selective oxidations are presented.


Metal: Fe
Ligand type: Porphyrin
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 6
ee: 13
PDB: ---
Notes: ---

Neocarzinostatin-Based Hybrid Biocatalysts with a RNase like Activity

Mahy, J.-P.; Ricoux, R.

Bioorg. Med. Chem. 2014, 22, 5678-5686, 10.1016/j.bmc.2014.05.063

A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N′(17′-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called ‘Trojan horse’ strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (kcat/KM = 13.6 M−1 s−1 at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the ‘Trojan horse’ strategy for obtaining better catalysts of selective reactions will be further studied.


Metal: Zn
Ligand type: Poly-pyridine
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 13.6 M-1 * s-1

New Activities of a Catalytic Antibody with a Peroxidase Activity: Formation of Fe(II)–RNO Complexes and Stereoselective Oxidation of Sulfides

Mahy, J.-P.

Eur. J. Biochem. 2004, 271, 1277-1283, 10.1111/j.1432-1033.2004.04032.x

In order to estimate the size of the cavity remaining around the heme of the 3A3–microperoxidase 8 (MP8) hemoabzyme, the formation of 3A3–MP8–Fe(II)‐nitrosoalkane complexes upon oxidation of N‐monosubstituted hydroxylamines was examined. This constituted a new reaction for hemoabzymes and is the first example of fully characterized Fe(II)–metabolite complexes of antibody–porphyrin. Also, via a comparison of the reactions with N‐substituted hydroxylamines of various size and hydrophobicity, antibody 3A3 was confirmed to bring about a partial steric hindrance on the distal face of MP8. Subsequently, the influence of the antibody on the stereoselectivity of the S‐oxidation of sulfides was examined. Our results showed that MP8 alone and the antibody–MP8 complex catalyze the oxidation of thioanisole by H2O2 and tert‐butyl hydroperoxide, following a peroxidase‐like two‐step oxygen‐transfer mechanism involving a radical–cation intermediate. The best system, associating H2O2 as oxidant and 3A3–MP8 as a catalyst, in the presence of 5% tert‐butyl alcohol, led to the stereoselective S‐oxidation of thioanisole with a 45% enantiomeric excess in favour of the R isomer. This constitutes the highest enantiomeric excess reported to date for the oxidation of sulfides catalyzed by hemoabzymes.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 3A3
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 82
ee: 45
PDB: ---
Notes: ---

New Biocatalysts Mimicking Oxidative Hemoproteins: Hemoabzymes

Review

Mahy, J.-P.

C. R. Chim. 2007, 10, 684-702, 10.1016/j.crci.2006.12.014

Catalytic antibodies with a metalloporphyrin cofactor or “hemoabzymes”, used as models for hemoproteins like peroxidases and cytochrome P450s, represent a promising route to catalysts tailored for selective oxidation reactions. The first strategy has been to produce anti-porphyrin antibodies, raised against various N-substituted- and meso-carboxyaryl-porphyrins, which led to monoclonal antibodies exhibiting, in the presence of the corresponding iron-porphyrin cofactor, a significant peroxidase activity. We ourselves obtained an artificial hemoprotein by associating a monoclonal antibody, 13G10, and its iron(III)-α,α,α,β-meso-tetrakis(ortho-carboxyphenyl)porphyrin (Fe(ToCPP)) hapten, which exhibited a significant peroxidase activity. Biological studies suggested that in this antibody, a carboxylic acid side chain of the protein participated in the catalysis, but no amino acid residue acting as an axial ligand of the iron was detected. Therefore, to provide the iron atom with an axial ligand, we raised antibodies against microperoxidase 8, a heme octapeptide containing a histidine bound to the iron atom. This strategy was successful, as an antibody–microperoxidase 8 complex (3A3–MP8) led to the best kcat/Km ever reported for antibody–porphyrin complexes. The ability of the 3A3–MP8 complex to catalyze the selective oxidation of substrates was studied and it was found able to catalyze the regioselective nitration of aromatics by NO2−/H2O2 as well as the stereoselective oxidation of sulfides like thioanisole by H2O2. Other strategies based on antibodies have to be developed to obtain more efficient biomimetic systems for cytochrome P450s. A first one could involve the modification of anti-substrate antibodies by covalent linkage of an iron(III)-porphyrin close to the binding site of the substrate, to obtain an artificial hemoprotein able to catalyze its regioselective oxidation.


Notes: ---

Oxidation Catalysis via Visible-Light Water Activation of a [Ru(bpy)3]2+ Chromophore BSA–Metallocorrole Couple

Gross, Z.; Mahy, J.-P.

Dalton Trans. 2016, 45, 706-710, 10.1039/c5dt04158a

Light induced enantioselective oxidation of an organic molecule with water as the oxygen atom source is demonstrated in a system where chirality is induced by a protein, oxygen atom transfer by a manganese corrole, and photocatalysis by ruthenium complexes.


Metal: Mn
Ligand type: Corrole
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 21
ee: 16
PDB: ---
Notes: Water as oxygen source

Oxidation of Organic Molecules in Homogeneous Aqueous Solution Catalyzed by Hybrid Biocatalysts (Based on the Trojan Horse Strategy)

Mahy, J.-P.

Tetrahedron: Asymmetry 2010, 21, 1593-1600, 10.1016/j.tetasy.2010.03.050

New anionic metalloporphyrin–estradiol conjugates have been synthesized and fully characterized, and have been further associated to a monoclonal anti-estradiol antibody 7A3, to generate new artificial metalloenzymes following the so-called ‘Trojan Horse’ strategy. The spectroscopic characteristics and dissociation constants of these complexes were similar to those obtained for the artificial metalloproteins obtained by association of cationic metalloporphyrin–estradiol conjugates to 7A3. This demonstrates that the nature of the porphyrin substituents, anionic or cationic, had little influence on the association with the antibody that is mainly driven by the tight association of the estradiol anchor with the binding pocket of the antibody. These new biocatalysts appeared to have an interesting catalytic activity in oxidation reactions. The iron(III)–anionic-porphyrin–estradiol-antibody complexes were found able to catalyze the chemoselective and slightly enantioselective (ee = 10%) sulfoxidation of sulfides by H2O2. The Mn(III)–porphyrin–estradiol-antibody complexes were found to catalyze the oxidation of styrene by KHSO5, the Mn(III)–cationic-porphyrin–estradiol-antibody complexes even showing the highest yields so far reported for the oxidation of styrene catalyzed by artificial metalloproteins. However, a lack of chemoselectivity and enantioselectivity was observed, which was probably due to a weak interaction of the metalloporphyrin cofactor with the binding pocket of antibody 7A3, as suggested by the similar UV–visible characteristics and catalytic activities obtained with both anionic and cationic porphyrins.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 7A3
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 9
ee: 10
PDB: ---
Notes: ---

Metal: Mn
Ligand type: Porphyrin
Host protein: Antibody 7A3
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Epoxidation
Max TON: 105
ee: ---
PDB: ---
Notes: Imidazole as co-catalyst

Receptor-Based Artificial Metalloenzymes on Living Human Cells

Ghattas, W.; Mahy, J.-P.

J. Am. Chem. Soc. 2018, 140, 8756-8762, 10.1021/jacs.8b04326

Artificial metalloenzymes are known to be promising tools for biocatalysis, but their recent compartmentalization has led to compatibly with cell components thus shedding light on possible therapeutic applications. We prepared and characterized artificial metalloenzymes based on the A2A adenosine receptor embedded in the cytoplasmic membranes of living human cells. The wild type receptor was chemically engineered into metalloenzymes by its association with strong antagonists that were covalently bound to copper(II) catalysts. The resulting cells enantioselectively catalyzed the abiotic Diels–Alder cycloaddition reaction of cyclopentadiene and azachalcone. The prospects of this strategy lie in the organ-confined in vivo preparation of receptor-based artificial metalloenzymes for the catalysis of reactions exogenous to the human metabolism. These could be used for the targeted synthesis of either drugs or deficient metabolites and for the activation of prodrugs, leading to therapeutic tools with unforeseen applications.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 24
ee: 35
PDB: ---
Notes: ---

Regioselective Nitration of Phenol Induced by Catalytic Antibodies

Mahy, J.-P.

J. Protein Chem. 2002, 21, 473-477, 10.1023/A:1021351120772

Catalytic antibodies with a metalloporphyrin cofactor represent a new generation of biocatalysts tailored for selective oxidations. Thus monoclonal antibodies, 3A3, were raised against microperoxidase 8 (MP8), and the corresponding 3A3-MP8 complexes were shown previously to have a high peroxidase activity. This paper shows that those complexes also catalyzed efficiently the nitration of phenol into 2- and 4-nitrophenol by NO2 − in the presence of H2O2. pH dependence studies suggested that no amino acid from the antibody protein participated in the heterolytic cleavage of the O-O bond of H2O2. The inhibition of the reaction by cyanide and radical scavengers suggested a MP8-mediated peroxidase-like mechanism, involving the reduction of high-valent iron-oxo species by NO2 − and phenol producing, respectively, NO2 · and phenoxy radicals, which then reacted to give nitrophenols. Finally, the antibody protein appears to have two major roles: (i) it protects MP8 toward oxidative degradations and (ii) it induces a regioselectivity of the reaction toward the formation of 2-nitrophenol.


Metal: Fe
Ligand type: Amino acid; Porphyrin
Host protein: Antibody 3A3
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: C-H oxidation
Max TON: 36
ee: ---
PDB: ---
Notes: Nitration of phenol

Selective Oxidation of Aromatic Sulfide Catalyzed by an Artificial Metalloenzyme: New Activity of Hemozymes

Mahy, J.-P.

Org. Biomol. Chem. 2009, 7, 3208, 10.1039/b907534h

Two new artificial hemoproteins or “hemozymes”, obtained by non covalent insertion of Fe(III)-meso-tetra-p-carboxy- and -p-sulfonato-phenylporphyrin into xylanase A from Streptomyces lividans, were characterized by UV-visible spectroscopy and molecular modeling studies, and were found to catalyze the chemo- and stereoselective oxidation of thioanisole into the S sulfoxide, the best yield (85 ± 4%) and enantiomeric excess (40% ± 3%) being obtained with Fe(III)-meso-tetra-p-carboxyphenylporphyrin-Xln10A as catalyst in the presence of imidazole as co-catalyst.


Metal: Fe
Ligand type: Porphyrin
Host protein: Xylanase A (XynA)
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 145
ee: 40
PDB: ---
Notes: ---

Studies of the Reactivity of Artificial Peroxidase-Like Hemoproteins Based on Antibodies Elicited Against a Specifically Designed ortho-Carboxy Substituted Tetraarylporphyrin

Mahy, J.-P.

FEBS Lett. 1999, 443, 229-234, 10.1016/S0014-5793(98)01703-7

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10‐Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80°C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2–6‐fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H2O2 of a variety of reducing cosubstrates such as 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), o‐phenylenediamine (OPD), 3,3′,5,5′‐tetramethylbenzidine (TMB) and 3,3′‐dimethoxybenzidine 3–8‐fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10‐Fe(ToCPP) or Fe(ToCPP) shows that K m(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k cat values. With Fe(ToCPP) as catalyst the k cat value remains constant around 100 min−1 whereas with the 13G10‐Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min−1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid‐base catalyst, to the heterolytic cleavage of the O‐O bond of H2O2 leading to the highly reactive iron(V)‐oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10‐Fe(ToCPP) complex.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 13G10
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: TOF = 4.7 min-1

Synthesis of a New Estradiol–Iron Metalloporphyrin Conjugate Used to Build up a New Hybrid Biocatalyst for Selective Oxidations by the ‘Trojan Horse’ Strategy

Mahy, J.-P.

Tetrahedron Lett. 2008, 49, 1865-1869, 10.1016/j.tetlet.2008.01.022

The synthesis of a new cationic iron metalloporphyrin–estradiol conjugate is reported. After a study of its association with the anti-estradiol antibody 7A3 by UV–visible spectroscopy, the influence of the antibody on the sulfoxidation of thioanisole by H2O2 catalyzed by the iron–metalloporphyrin has been investigated.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 7A3
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 12
ee: 8
PDB: ---
Notes: ---

Various Strategies for Obtaining Artificial Hemoproteins: From "Hemoabzymes" to "Hemozymes"

Mahy, J.-P.

Biochimie 2009, 91, 1321-1323, 10.1016/j.biochi.2009.03.002

The design of artificial hemoproteins that could lead to new biocatalysts for selective oxidation reactions of organic compounds presents a huge interest especially in pharmacology, both for a better understanding of the metabolic profile of drugs and for the synthesis of enantiomerically pure molecules that could be involved in the design of drugs. The present results show that the so-called “host-guest strategy” that involves the non-covalent incorporation of anionic water-soluble iron-porphyrins into xylanase A from Streptomyces lividans, a low cost protein, leads to such an artificial hemoprotein that is able to perform the stereoselective oxidation of sulfides.


Metal: Fe
Ligand type: Porphyrin
Host protein: Xylanase A (XynA)
Anchoring strategy: Supramolecular
Optimization: Chemical
Reaction: Sulfoxidation
Max TON: ---
ee: 36
PDB: ---
Notes: ---

Various Strategies for Obtaining Oxidative Artificial Hemoproteins with a Catalytic Oxidative Activity: From "Hemoabzymes" to "Hemozymes"?

Review

Mahy, J.-P.

J. Porphyr. Phthalocyanines 2014, 18, 1063-1092, 10.1142/S1088424614500813

The design of artificial hemoproteins that could lead to new biocatalysts for selective oxidation reactions using clean oxidants such as O 2 or H 2 O 2 under ecocompatible conditions constitutes a really promising challenge for a wide range of industrial applications. In vivo, such reactions are performed by heme-thiolate proteins, cytochromes P450, that catalyze the oxidation of drugs by dioxygen in the presence of electrons delivered from NADPH by cytochrome P450 reductase. Several strategies were used to design new artificial hemoproteins to mimic these enzymes, that associate synthetic metalloporphyrin derivatives to a protein that is supposed to induce a selectivity in the catalyzed reaction. A first generation of artificial hemoproteins or "hemoabzymes" was obtained by the non-covalent association of synthetic hemes such as N-methyl-mesoporphyrin IX, Fe(III) -α3β-tetra-o-carboxyphenylporphyrin or microperoxidase 8 with monoclonal antibodies raised against these cofactors. The obtained antibody-metalloporphyrin complexes displayed a peroxidase activity and some of them catalyzed the regio-selective nitration of phenols by H 2 O 2/ NO 2 and the stereo-selective oxidation of sulphides by H 2 O 2. A second generation of artificial hemoproteins or "hemozymes", was obtained by the non-covalent association of non-relevant proteins with metalloporphyrin derivatives. Several strategies were used, the most successful of which, named "host-guest" strategy involved the non-covalent incorporation of metalloporphyrin derivatives into easily affordable proteins. The artificial hemoproteins obtained were found to be able to perform efficiently the stereoselective oxidation of organic compounds such as sulphides and alkenes by H 2 O 2 and KHSO 5.


Notes: ---