Characterization in aqueous medium of an FMN semiquinone radical stabilized by the enzyme-like microenvironment of a modified polyethyleneimine.

The elusive flavin semiquinone intermediate found in flavoproteins such as cryptochromes has been obtained in aqueous solution by single electron reduction of the natural FMN cofactor using sodium ascorbate. This species was formed in the local hydrophobic microenvironment of a modified polyethyleneimine and characterized by UV-Visible, fluorescence and EPR spectroscopies.

Artificial iron hydrogenase made by covalent grafting of Knölker's complex into xylanase: Application in asymmetric hydrogenation of an aryl ketone in water.

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.

Selective Formation of an FeIV O or an FeIII OOH Intermediate From Iron(II) and H2 O2 : Controlled Heterolytic versus Homolytic Oxygen-Oxygen Bond Cleavage by the Second Coordination Sphere.

We demonstrate that the devised incorporation of an alkylamine group into the second coordination sphere of an FeII complex allows to switch its reactivity with H2 O2 from the usual formation of FeIII species towards the selective generation of an FeIV -oxo intermediate. The FeIV -oxo species was characterized by UV/Vis absorption and Mössbauer spectroscopy. Variable-temperature kinetic analyses point towards a mechanism in which the heterolytic cleavage of the O-O bond is triggered by a proton transfer from the proximal to the distal oxygen atom in the FeII -H2 O2 complex with the assistance of the pendant amine. DFT studies reveal that this heterolytic cleavage is actually initiated by an homolytic O-O cleavage immediately followed by a proton-coupled electron transfer (PCET) that leads to the formation of the FeIV -oxo and release of water through a concerted mechanism.

Aerobic Baeyer-Villiger Oxidation Catalyzed by a Flavin-Containing Enzyme Mimic in Water.

Direct incorporation of molecular oxygen into small organic molecules has attracted much attention for the development of new environmentally friendly oxidation processes. In line with this approach, bioinspired systems mimicking enzyme activities are of particular interest since they may perform catalysis in aqueous media. Demonstrated herein is the incorporation of a natural flavin cofactor (FMN) into the specific microenvironment of a water-soluble polymer which allows the efficient reduction of the FMN by NADH in aqueous solution. Once reduced, this artificial flavoenzyme can then activate molecular dioxygen under aerobic conditions and result in the Baeyer-Villiger reaction at room temperature in water.

Redox Self-Adaptation of a Nitrene Transfer Catalyst to the Substrate Needs.

The development of iron catalysts for carbon-heteroatom bond formation, which has attracted strong interest in the context of green chemistry and nitrene transfer, has emerged as the most promising way to versatile amine synthetic processes. A diiron system was previously developed that proved efficient in catalytic sulfimidations and aziridinations thanks to an FeIII FeIV active species. To deal with more demanding benzylic and aliphatic substrates, the catalyst was found to activate itself to a FeIII FeIV L. active species able to catalyze aliphatic amination. Extensive DFT calculations show that this activation event drastically enhances the electron affinity of the active species to match the substrates requirements. Overall this process consists in a redox self-adaptation of the catalyst to the substrate needs.

Neocarzinostatin-based hybrid biocatalysts with a RNase like activity.

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=13.6M(-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.

A diiron(III,IV) imido species very active in nitrene-transfer reactions.

Metal-catalyzed nitrene transfer reactions arouse intense interest as clean and efficient procedures for amine synthesis. Efficient Rh- and Ru-based catalysts exist but Fe alternatives are actively pursued. However, reactive iron imido species can be very short-lived and getting evidence of their occurrence in efficient nitrene-transfer reactions is an important challenge. We recently reported that a diiron(III,II) complex is a very efficient nitrene-transfer catalyst to various substrates. We describe herein how, by combining desorption electrospray ionization mass spectrometry, quantitative chemical quench experiments, and DFT calculations, we obtained conclusive evidence for the occurrence of an {Fe(III) Fe(IV) NTosyl} intermediate that is very active in H-abstraction and nitrene-transfer reactions. DFT calculations revealed a strong radical character of the tosyl nitrogen atom in very low-lying electronic configurations of the Fe(IV) ion which are likely to confer its high reactivity.

Interplay of chloride levels and palladium(ii)-catalyzed O-deallenylation bioorthogonal uncaging reactions.

The palladium-mediated uncaging reaction of allene substrates remains a promising yet often overlooked strategy in the realm of bioorthogonal chemistry. This method exhibits high kinetic rates, rivaling those of the widely employed allylic and propargylic protecting groups. In this study, we investigate into the mechanistic aspects of the C-O bond-cleavage deallenylation reaction, examining how chloride levels influence the kinetics when triggered by Pd(ii) complexes. Focusing on the deallenylation of 1,2-allenyl protected 4-methylumbelliferone promoted by Allyl2Pd2Cl2, our findings reveal that reaction rates are higher in environments with lower chloride concentrations, mirroring intracellular conditions, compared to elevated chloride concentrations typical of extracellular conditions. Through kinetic and spectroscopic experiments, combined with DFT calculations, we uncover a detailed mechanism that identifies AllylPd(H2O)2 as the predominant active species. These insights provide the basis for the design of π-allylpalladium catalysts suited for selective uncaging within specific cellular environments, potentially enhancing targeted therapeutic applications.

Unprotected amine transfer performed by non-heme iron(II) complexes.

Direct amination of C-H or CC bonds using unprotected amino groups is very challenging, especially with earth abundant metal ions. Here we show that a bioinspired iron(II) complex catalyses the double amination of its dangling benzyl branch in the presence of hydroxylamine derivatives as the unprotected amine donor and that the replacement of the benzyl branch by a methyl group also allows the aziridination of styrene.

Bioinspired symmetrical and unsymmetrical diiron complexes for selective oxidation catalysis with hydrogen peroxide.

Two new symmetrical and unsymmetrical diiron(iii) complexes were synthesized and characterized by X-ray diffraction analysis, mass spectrometry, UV-visible and Mössbauer spectroscopies. They proved to be good catalysts for alkene and alkane oxidation reactions by H2O2 in acetonitrile solution, and interesting effects of both the nature and the symmetry of the complexes were observed on catalysis in the presence of water.

Combining medium effects and cofactor catalysis: metal-coordinated synzymes accelerate phosphate transfer by 10(8).

The systematic exploration of the modification of polyethylene imine with guanidinium and octyl groups has led to the identification of a catalyst, CD6, which accelerates the phosphate transfer reaction of HPNP (2-hydroxypropyl-4-nitrophenyl phosphate) in the presence of divalent metals such as Zn(2+), Co(2+), Mg(2+) or Ni(2+). CD6 exhibits saturation kinetics that are described by Michaelis-Menten parameters K(m) ranging from 2.5-8 mM and k(cat) ranging from 0.0014-0.09 s(-1). For Zn(II)-CD6 this corresponds to an overall acceleration k(cat)/k(uncat) of 3.8x10(5) and a catalytic proficiency (k(cat)/K(m))/k(uncat) of 1.5x10(8). Catalysis by Zn(II)-CD6 is specifically inhibited by inorganic phosphate, allowing turnover regulation by product inhibition. This effect stands in contrast to Zn(II)-catalysed transesterification of HPNP in water or by the synzymes Co(II)-CD6 and Ni(II)-CD6, with which no such interference by product is observed. These characteristics render synzyme Zn(II)-CD6 an efficient enzyme model that reflects enzyme-like properties in a wide range of features.

Relating chemical and biological diversity space: a tunable system for efficient gene transfection.

Polyethyleneimine (PEI), a well-established nonviral transfection reagent, was combinatorially modified with varying proportions of methyl, benzyl, and n-dodecyl groups to create a library of 435 derivatized polymers. Screening of this library for transfection, DNA binding, and toxicity allows systematic correlation of the biological properties of our polymers to their derivatizations. Combinations of derivatizations bring about a 100-fold variation in transfection efficiency between library members. The best PEI derivatives exhibit increases in transfection efficiency of more than 80-fold over unmodified PEI (up to 28+/-7 % of cells transfected) and rival commercial reagents such as Lipofectamine 2000 (21+/-10 %) and JetPEI (32+/-5.0 %). In addition, we can identify compounds that are specifically tuned for efficient transfection in CHO-K1 over Ishikawa cells and vice versa, demonstrating that the approach can lead to cell-type selectivity of at least one order of magnitude. This work demonstrates that multivalent derivatization of a polymeric framework can create functional diversity substantially greater than the structural diversity of the derivatization building blocks and suggests an approach to a better understanding of the molecular underpinnings of transfection as well as their exploitation.

Elucidating light-induced charge accumulation in an artificial analogue of methane monooxygenase enzymes using time-resolved X-ray absorption spectroscopy.

We report the use of time-resolved X-ray absorption spectroscopy in the ns-µs time scale to track the light induced two electron transfer processes in a multi-component photocatalytic system, consisting of [Ru(bpy)3]2+/ a diiron(iii,iii) model/triethylamine. EXAFS analysis with DFT calculations confirms the structural configurations of the diiron(iii,iii) and reduced diiron(ii,ii) states.

A diiron complex mediates an intramolecular aliphatic hydroxylation by various oxygen donors.

In the presence of hydrogen peroxide, m-chloroperbenzoic acid or an iodosyl arene, the tert-butyl group of the ligand H(L-t-Bu) in the complex [Fe2(L-t-Bu)(mpdp)]2+ is quantitatively hydroxylated to a butanolate terminally bound to one iron in [Fe2(L-t-Bu - H + O)(mpdp)]2+, and mass spectrometry experiments indicate that the reaction proceeds according to different mechanisms.

Bio-inspired electron-delivering system for reductive activation of dioxygen at metal centres towards artificial flavoenzymes.

Development of artificial systems, capable of delivering electrons to metal-based catalysts for the reductive activation of dioxygen, has been proven very difficult for decades, constituting a major scientific lock for the elaboration of environmentally friendly oxidation processes. Here we demonstrate that the incorporation of a flavin mononucleotide (FMN) in a water-soluble polymer, bearing a locally hydrophobic microenvironment, allows the efficient reduction of the FMN by NADH. This supramolecular entity is then capable of catalysing a very fast single-electron reduction of manganese(III) porphyrin by splitting the electron pair issued from NADH. This is fully reminiscent of the activity of natural reductases such as the cytochrome P450 reductases with kinetic parameters, which are three orders of magnitude faster compared with other artificial systems. Finally, we show as a proof of concept that the reduced manganese porphyrin activates dioxygen and catalyses the oxidation of organic substrates in water.

Catalytic aziridination of olefins and amidation of thioanisole by a non-heme iron complex.

A non-heme iron complex catalyses the aziridination of various olefins and the amidation of thioanisole in good yields at the expense of an aryl iodinane.

Polyethylene imine derivatives ('synzymes') accelerate phosphate transfer in the absence of metal.

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.

Spectroscopic and electrochemical characterization of an aqua ligand exchange and oxidatively induced deprotonation in diiron complexes.

Reaction of the unsymmetrical phenol ligand 2-((bis(2-pyridylmethyl)amino)methyl)-6-(((2-pyridylmethyl)benzylamino)methyl)-4-methylphenol (HL-Bn) or its 2,6-dichlorobenzyl analogue (HL-BnCl(2)) with Fe(H(2)O)(6)(ClO(4))(2) in the presence of disodium m-phenylenedipropionate (Na(2)(mpdp)) followed by exposure to atmosphere affords the diiron(II,III) complexes [Fe(2)(L-Bn)(mpdp)(H(2)O)](ClO(4))(2) and [Fe(2)(L-BnCl(2))(mpdp)(CH(3)OH)](ClO(4))(2), respectively. The latter complex has been characterized by X-ray crystallography. It crystallizes in the monoclinic system, space group P2(1)/n, with a = 13.3095(14) A, b = 20.1073(19) A, c = 19.4997(19) A, alpha = 90 degrees, beta = 94.471(2) degrees, gamma = 90 degrees, V = 5202.6(9) A(3), and Z = 4. The structure of the compound is very similar to that of [Fe(2)(L-Bn)(mpdp)(H(2)O)](BPh(4))(2) determined earlier, except for the replacement of a water by a methanol on the ferrous site. Magnetic measurements of [Fe(2)(L-Bn)(mpdp)(H(2)O)](BPh(4))(2) reveal that the two high-spin Fe ions are moderately antiferromagnetically coupled (J = -3.2(2) cm(-)(1)). Upon dissolution in acetonitrile the terminal ligand on the ferrous site is replaced by a solvent molecule. The acetonitrile-water exchange has been investigated by various spectroscopic techniques (UV-visible, NMR, Mössbauer) and electrochemistry. The substitution of acetonitrile by water is clearly evidenced by Mössbauer spectroscopy by a reduction of the quadrupole splitting value from 3.14 to 2.41 mm/s. In addition, it causes a 210 mV downshift of the oxidation potential of the ferrous site and a similar reduction of the stability domain of the mixed-valence state. Exhaustive electrolysis of a solution of [Fe(2)(L-Bn)(mpdp)(H(2)O)](2+) shows that the aqua diferric species is not stable and undergoes a chemical reaction which can be partly reversed by reduction to the mixed-valent state. This and other electrochemical observations suggest that upon oxidation of the diiron center to the diferric state the aqua ligand is deprotonated to a hydroxo. This hypothesis is supported by Mössbauer spectroscopy. Indeed, this species possesses a large quadrupole splitting value (DeltaE(Q) >or= 1.0 mm.s(-)(1)) similar to that of analogous complexes with a terminal phenolate ligand. This study illustrates the drastic effects of aqua ligand exchange and deprotonation on the electronic structure and redox potentials of diiron centers.