Chemoselective Cyclopropanation over Carbene Y-H Insertion Catalyzed by an Engineered Carbene Transferase.

Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene transfer reactions including cyclopropanation and Y-H insertion (Y = N, S, Si, B). For these and synthetic carbene transfer catalysts alike, achieving high chemoselectivity toward cyclopropanation in olefin substrates bearing unprotected Y-H groups has proven remarkably challenging due to competition from the more facile carbene Y-H insertion reaction. In this report, we describe the development of a novel artificial metalloenzyme based on an engineered myoglobin incorporating a serine-ligated Co-porphyrin cofactor that is capable of offering high selectivity toward olefin cyclopropanation over N-H and Si-H insertion. Intramolecular competition experiments revealed a distinct and dramatically altered chemoselectivity of the Mb(H64V,V68A,H93S)[Co(ppIX)] variant in carbene transfer reactions compared to myoglobin-based variants containing the native histidine-ligated heme cofactor or other metal/proximal ligand substitutions. These studies highlight the functional plasticity of myoglobin as a "carbene transferase" and illustrate how modulation of the cofactor environment within this metalloprotein scaffold represents a valuable strategy for accessing carbene transfer reactivity not exhibited by naturally occurring hemoproteins or transition metal catalysts.

Mini Heme-Proteins: Designability of Structure and Diversity of Functions.

Natural heme proteins may have heme bound to poly-peptide chain as a cofactor via noncovalent forces or heme as a prosthetic group may be covalently bound to the proteins. Nature has used porphyrins in diverse functions like electron transfer, oxidation, reduction, ligand binding, photosynthesis, signaling, etc. by modulating its properties through diverse protein matrices. Synthetic chemists have tried to utilize these molecules in equally diverse industrial and medical applications due to their versatile electro-chemical and optical properties. The heme iron has catalytic activity which can be modulated and enhanced for specific applications by protein matrix around it. Heme proteins can be designed into novel enzymes for sterio specific catalysis ranging from oxidation to reduction. These designed heme-proteins can have applications in industrial catalysis and biosensing. A peptide folds around heme easily due to hydrophobic effect of the large aromatic ring of heme. The directional property of co-ordinate bonding between peptide and metal ion in heme further specifies the structure. Therefore heme proteins can be easily designed for targeted structure and catalytic activity. The central aromatic chemical entity in heme viz. porphyrin is a very ancient molecule. Its presence in the prebiotic soup and in all forms of life suggests that it has played a vital role in the origin and progressive evolution of living organisms. Porphyrin macrocycles are highly conjugated systems composed of four modified pyrrole subunits interconnected at their α -carbon atoms via methine (=CH-) bridges. Initial minimalist models of hemoproteins focused on effect of heme-ligand co-ordinate bonding on chemical reactivity, spectroscopy, electrochemistry and magnetic properties of heme. The great sensitivity of these spectroscopic features of heme to its surrounding makes them extremely useful in structural elucidation of designed heme-peptide complexes. Therefore heme proteins are easier to work on for designing novel proteins for industrial and medical applications.

Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids.

An important strategy used in engineering of hemoproteins to generate artificial enzymes involves replacement of heme with an artificial cofactor after removal of the native heme cofactor under acidic conditions. Replacement of heme in an enzyme with a nonnatural metalloporphyrinoid can significantly alter the reactivity of the enzyme. This chapter describes the design and synthesis of three types of artificial metalloporphyrinoid cofactors consisting of mono-, di-, and tri-anionic ligands (tetradehydrocorrin, porphycene, and corrole, respectively). In addition, practical procedures for the preparation of apo-hemoproteins, incorporation of artificial cofactors, and characterization techniques are presented. Furthermore, the representative catalytic activities of artificial enzymes generated by reconstitution of hemoproteins are summarized.

Current status of synthetic hemozoin adjuvant: A preliminary safety evaluation.

Although adjuvants are a "must-have" component of successful vaccines, there are very few adjuvants licensed for use in humans, there is therefore an urgent need to develop new and safer adjuvants. Synthetic hemozoin (sHZ), a chemical analog of hemozoin which is produced by the malaria parasite, exhibits a potent adjuvant effect which enhances antigen-specific immune responses to vaccines. The potency of sHZ adjuvanticity is not limited to malaria specific vaccines, it has also been demonstrated to be effective in influenza and dog allergy models. While the synthesis of uniformly sized sHZ with consistent characteristics has proven difficult, we have recently successfully optimized the manufacture of sHZ product with an optimal adjuvant effect. Here, we summarize recent developments on the adjuvant properties of optimized sHZ adjuvant, including its good laboratory practice (GLP) non-clinical safety profile in animals. These studies ensure the safety of optimized sHZ product to be readily used as vaccine adjuvant beforehand in veterinary medicine.

Chemical Synthesis of the 20†kDa Heme Protein Nitrophorin†4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation.

The chemical synthesis of the 184-residue ferric heme-binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α-ketoacid-hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild-type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well-suited for the preparation of hydrophobic protein targets.

Mimicking hemoproteins: a new synthetic metalloenzyme based on a Fe(III)-mesoporphyrin functionalized by two helical decapeptides.

A new metalloenzyme formed by a Fe(III)-mesoporphyrin IX functionalized by two helical decapeptides was synthesized to mimic function and structural features of a hemoprotein active site. Each decapeptide comprises six 2-aminoisobutyric acid residues, which constrain the peptide to attain a helical conformation, and three glutamic residues for improving the solubility of the catalyst in aqueous solutions. The new compound shows a marked amphiphilic character, featuring a polar outer surface and a hydrophobic inner cavity that hosts the reactants in a restrained environment where catalysis may occur. The catalytic activity of this synthetic mini-protein was tested with respect to the oxidation of L- and D-Dopa by hydrogen peroxide, showing moderate stereoselectivity. Structural information on the new catalyst and its adduct with the L- or D-Dopa substrate were obtained by the combined use of spectroscopic techniques and molecular mechanics calculations.

The single crystal X-ray structure of ß-hematin DMSO solvate grown in the presence of chloroquine, a ß-hematin growth-rate inhibitor.

Single crystals of solvated ß-hematin were grown from a DMSO solution containing the antimalarial drug chloroquine, a known inhibitor of ß-hematin formation. In addition, a kinetics study employing biomimetic lipid-water emulsion conditions was undertaken to further investigate the effect of chloroquine and quinidine on the formation of ß-hematin. Scanning electron microscopy shows that the external morphology of the ß-hematin DMSO solvate crystals is almost indistinguishable from that of malaria pigment (hemozoin), and single crystal X-ray diffraction confirms the presence of µ-propionato coordination dimers of iron(III) protoporphyrin IX. The free propionic acid functional groups of adjacent dimers hydrogen bond to included DMSO molecules, rather than forming carboxylic acid dimers. The observed exponential kinetics were modeled using the Avrami equation, with an Avrami constant equal to 1. The decreased rate of ß-hematin formation observed at low concentrations of both drugs could be accounted for by assuming a mechanism of drug adsorption to sites on the fastest growing face of ß-hematin. This behavior was modeled using the Langmuir isotherm. Higher concentrations of drug resulted in decreased final yields of ß-hematin, and an irreversible drug-induced precipitation of iron(III) protoporphyrin IX was postulated to account for this. The model permits determination of the equilibrium adsorption constant (K(ads)). The values for chloroquine (log K(ads) = 5.55 ± 0.03) and quinidine (log K(ads) = 4.92 ± 0.01) suggest that the approach may be useful as a relative probe of the mechanism of action of novel antimalarial compounds.

De novo design, synthesis and characterisation of MP3, a new catalytic four-helix bundle hemeprotein.

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four-helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix-loop-helix/heme/helix-loop-helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP-like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix-forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe(III)-MP3 possesses peroxidase-like activity comparable to R38A-hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.

Manipulating cofactor binding thermodynamics in an artificial oxygen transport protein.

We report the mutational analysis of an artificial oxygen transport protein, HP7, which operates via a mechanism akin to that of human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of 13. Paradoxically, it also decreases heme binding affinity by a factor of 5 in the reduced state and 60 in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates for that. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins.

Innate inflammatory response to the malarial pigment hemozoin.

Malaria is an infectious disease caused by parasites of the genus Plasmodium. This intraerythrocytic protozoan produces hemozoin (HZ), an insoluble crystalline metabolite resulting from the heme detoxification mechanism. This review will focus on HZ biosynthesis and synthetic preparation, but in particular on its effect on host's innate inflammatory responses.

Effects of axial pyridine coordination on a saddle-distorted porphyrin macrocycle: stabilization of hexa-coordinated high-spin Fe(III) and air-stable low-spin iron(II) porphyrinates.

We have reported here the effect of axial ligand L (L: pyridine/substituted pyridine) on Fe(III)(tn-OEP)Cl/Fe(III)(tn-OEP)ClO(4) that first form high-spin Fe(III)(tn-OEP)(L)(2).X (X: Cl, ClO(4)) which, on longer exposure, spontaneously auto reduce to a series of air stable Fe(II)(tn-OEP)(L)(2) complexes. The introduction of four nitro groups into the meso-positions of octaethyl porphyrin (tn-OEP), severely distorts the porphyrin macrocycle which enables the facile isolation of a rare family of high-spin Fe(III)(tn-OEP)(L)(2)(+) in a saddle distorted macrocyclic environment. The synthesis and characterization of high-spin Fe(III)(tn-OEP)(L)(2).X and low-spin Fe(II)(tn-OEP)(L)(2) are reported. The X-ray structures of Fe(II)(tn-OEP)(py)(2), Fe(II)(tn-OEP)(4-CNpy)(2) and Fe(II)(tn-OEP)(3-Clpy)(2) have been determined in which the axial ligands are orientated nearly perpendicular to each other. Electrochemical data obtained from cyclic voltammetric study for Fe(II)(tn-OEP)(L)(2) reveals the one electron oxidations at very high positive potentials which readily explains why the complexes are so stable in air. However, spectroscopic characterizations such as magnetic and EPR measurements in both solid and solution, and (1)H NMR in solution demonstrates the high-spin nature of Fe(III)(tn-OEP)(L)(2).X. Molecular orbital calculations using DFT for five coordinate Fe(III)(tn-OEP)Cl shows a(2u)-like HOMO that is expected for a saddle distorted porphyrin but for six coordinate Fe(III)(tn-OEP)(L)(2).X results in switch of the HOMO from a(2u) to a(1u). However, metal d(x(2)-y(2)) and porphyrin a(1u) bonding interaction is symmetrically unfavorable and thus responsible for high-spin nature of the complexes reported here. The porphyrin cores (tn-OEP) are found to be least distorted in Fe(III)(tn-OEP)(H(2)O)(2).ClO(4) with a core size of 2.061 A while, for Fe(II)(tn-OEP)(py)(2), the macrocycle is distorted most with lowest core size of 1.961 A; thus shows a significant and unprecedented core expansion of 0.1 A in the series.

Autofluorescence of condensed heme aggregates in malaria pigment and its synthetic equivalent hematin anhydride (beta-hematin).

The condensed crystalline phase of iron(III) protoporphyrin IX either isolated from parasite culture as malaria pigment (hemozoin) or synthetic equivalent hematin anhydride exhibits a solid-state autofluorescence characterized by an excitation maximum of 555 nm and an emission maximum of 577 nm. The excitation spectrum maximum at 555 nm corresponds to the Q(0,0) band in the absorption spectrum which represents the lowest singlet of the material. This suggests that the fluorescent emission is due to the heme condensed phase. The photoluminescence lifetime of tau(f) = 2.7 +/- 0.8 ns as measured at four wavelengths between 550 and 600 nm is in the range of Frankel exciton in porphyrinic condensed phases. The material is shown to have an optical band gap of 2.04 eV characteristic of a semiconductor. Luminescence is markedly dependent upon the degree of hydration and the emission does not seem to be caused by presence of zinc(II) protoporphyrin IX or free-base protoporphyrin IX in the lattice. The autofluorescence can be used for in vivo tracking of hemozoin, for determination of parasitemia levels, and for infection monitoring and possibly for drug screening studies.

Various strategies for obtaining artificial hemoproteins: from "hemoabzymes" to "hemozymes".

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.

Probing hydrogen bonding to bound dioxygen in synthetic models for heme proteins: the importance of precise geometry.

Distal hydrogen bonding in natural dioxygen binding proteins is crucial for the discrimination between different potential ligands such as O(2) or CO. In the present study, we probe the chemical requirements for proper distal hydrogen bonding in a series of synthetic model compounds for dioxygen-binding heme proteins. The model compounds 1-Co to 7-Co bear different distal residues. The hydrogen bonding in their corresponding dioxygen adducts is directly measured by pulse EPR spectroscopy. The geometrical requirements for this interaction to take place were found to be narrow and very specific. Only two model complexes, 1-Co and 7-Co, form a hydrogen bond to bound dioxygen, which was characterized in terms of geometry and nature of the bond. The geometry and dipolar nature of this interaction in 1-Co-O(2) is more similar to the one in natural cobalt myoglobin (Co-Mb), making 1-Co the best model compound in the entire series.

Recent advances in understanding the mechanism of hemozoin (malaria pigment) formation.

The recent literature on hemozoin/beta-hematin formation is reviewed, with an emphasis on the mechanism of its formation. Recent findings from unrelated organisms that produce hemozoin, namely the malaria parasite Plasmodium falciparum, the worm Schistosoma mansoni and the kissing bug Rhodnius prolixus all of which consume human hemoglobin show that the formation of this crystalline substance occurs within or at the surface of lipids. Biomimetic experimental models of the lipid-water interface as well as computational studies indicate that these lipid environments are probably extraordinarily efficient at producing hemozoin. A rethink is now needed, with a new emphasis on Fe(III)PPIX in non-aqueous environments that mimic lipids and indeed within the lipid environment itself. These findings are explored and discussed in the context of earlier studies on beta-hematin formation.

Artificial hemoprotein nanotubes.

Artificial hemoprotein nanotubes have been prepared by a layer-by-layer deposition technique with human serum albumin (HSA) incorporating the synthetic heme (FeP) [HSA-FeP] using an anodic porous alumina template; each of the liberated tubules has a very uniform outer/inner diameter and can reversibly bind dioxygen (O(2)) at 25 degrees C.

Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins.

Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.

Kinetics of beta-haematin formation from suspensions of haematin in aqueous benzoic acid.

Kinetics of beta-haematin (synthetic malaria pigment) formation from haematin have been studied in the presence of aqueous benzoic acid and derivatives of benzoic acid. Formation of the beta-haematin product is demonstrated by X-ray diffraction and IR spectroscopy. Reactions were followed by determining the fraction of unreacted haematin at various time points during the process via reaction of extracted aliquots with pyridine. The kinetics can be fitted to the Avrami equation, indicating that the process involves nucleation and growth. Reaction kinetics in stirred benzoic acid are similar to those previously observed in acetic acid, except that benzoic acid is far more active in promoting the reaction than acetic acid. The reaction reaches completion within 2 h in the presence of 0.050 M benzoic acid (pH 4.5, 60 degrees C). This compares with 1 h in the presence of 4.5 M acetic acid and 4 h in the presence of 2 M acetic acid. The reaction rate in benzoic acid is not affected if the stirring rate is decreased to zero, but very vigorous stirring appears to disrupt nucleation. The rate constant for beta-haematin formation in benzoic acid has a linear dependence on benzoic acid concentration and follows Arrhenius behaviour with temperature. There is a bell-shaped dependence on pH. This suggests that the haematin species in which one propionate group is protonated and the other is deprotonated is optimal for beta-haematin formation. When the reaction is conducted in para-substituted benzoic acid derivatives, the log of the rate constant increases linearly with the Hammett constant. These findings suggest that the role of the carboxylic acid may be to disrupt hydrogen bonding and pi-stacking in haematin, facilitating conversion to beta-haematin. The large activation energy for conversion of precipitated haematin to beta-haematin suggests that the reaction in vivo most likely involves direct nucleation from solution and probably does not occur in aqueous medium.

Probing the role of the covalent linkage of ferrocene into a chloroquine template.

A new therapeutic approach to malaria led to the discovery of ferroquine (FQ, SR97276). To assess the importance of the linkage of the ferrocenyl group to a 4-aminoquinoline scaffold, two series of 4-aminoquinolines, structurally related to FQ, were synthesized. Evaluation of antimalarial activity, physicochemical parameters, and the beta-hematin inhibition property indicate that the ferrocene moiety has to be covalently flanked by a 4-aminoquinoline and an alkylamine. Current data reinforced our choice of FQ as a drug candidate.

Human serum albumin hybrid incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer as an O2-binding site.

We have found that recombinant human serum albumin (HSA) incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer can reversibly bind and release O2 under physiological conditions (pH 7.3, 37 degrees C) like hemoglobin and myoglobin. beta-2-Methylimidazolyl-tailed porphyrinatoirons (6a, 6b) are synthesized via four steps from the atropisomers of tetrakis(o-aminophenyl)porphyrin. The stereochemistry of the alpha,alpha,alpha,beta-conformer has been determined by NMR spectroscopy. 6a and 6b form stable O2-adduct complexes in toluene solution at room temperature. The association rate constants of O2 are 3.1- and 1.9-fold lower than those of the corresponding alpha,alpha,alpha,alpha-conformers (1a, 1b), indicating that the three substituents (cyclohexanamide or pivalamide groups) are close to each other on the porphyrin platform and construct a narrow encumbrance around the O2-coordination site. Although 6a and 6b are incorporated into the hydrophobic domains of HSA to produce the albumin-heme hybrid, only HSA-6a can bind O2 in aqueous medium. The cyclohexanamide fences are necessary for the tailed porphyrinatoiron to form a stable O2-adduct complex under physiological conditions. The O2-binding affinity (P(1/2)) of HSA-6a is 45 Torr (37 degrees C), and the O2 transporting efficiency between lungs and muscle tissues in the human body is estimated to be identical to that of human red blood cells. The HSA-6a solution will become one of the most promising materials for red blood cell substitutes, which can be manufactured on an industrial scale.