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Herein we report the preparation and characterization of the iron and ruthenium organometallic maleimides (η5-Cp*)Fe(CO)2(η1-maleimidato) (Cp* = pentamethyl cyclopentadienyl) and (η5-Cp)Ru(CO)2(η1-maleimidato). The molecular structure of the ruthenium complex was confirmed by X-ray diffraction study. The reactivity of the metallocarbonyl maleimide derivatives toward biologically meaningful thiols, namely cysteine ethyl ester hydrochloride, glutathione (GSH) and human serum albumin (HSA) was investigated. To get better insight into the reaction mechanism, kinetic studies of the reaction with GSH and HSA were performed as well as quantum-chemical calculations and QTAIM analysis. We found out that the reactivity of the organometallic maleimides towards thiols depended on: 1) the metal present in the structure, 2) the nature of the capping aromatic ligand due to differences in electron donating capabilities and steric demands and 3) additional interactions like hydrogen bonding in (η5-Cp*)Fe(CO)2(η1-maleimidato) and carbonyl … carbonyl interactions in (η5-Cp)Ru(CO)2(η1-maleimidato).

International audience; Two half-sandwich d6-rhodium(III) complexes of the general formula [(η5-Cp*)Rh(N^N)Cl]Cl where N^N is a phenanthroline or a bispyridine methane derivative carrying a thiol-targeting maleimide or chloroacetamide function were synthesized and characterized. Both complexes were able to catalyse the transfer hydrogenation of 2,2,2-trifluoroacetophenone in aqueous medium using formate or phosphite as hydrogen donor. Covalent anchoring of these complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins with transfer hydrogenase properties. Under optimized conditions of pH, hydrogen donor concentration and catalyst load, conversion of substrate was nearly quantitative within 24 h at 40 °C and the (S)-enantiomer was obtained preferably albeit with a modest enantiomeric excess of 710%. Covalent docking simulations complemented the experimental findings suggesting a molecular rationale for the observed low enantioselectivity. The harmonious use of experimental and theoretical approaches represents an unprecedented starting point for driving the rational design of artificial metalloenzymes built up from papain with higher catalytic efficiency.

Metallocarbonyl-labeled gold nanoparticles (displaying specific and intense absorption bands assigned to the stretching vibrations of the carbonyl ligands in IR spectroscopy) were derivatized with an anti-mouse IgG antibody to provide IR reporter immunoprobes that were characterized by several analytical techniques like ELISA, IR and transmission electron microscopy (TEM). These immunoprobes were able to detect a mouse monoclonal anti-staphylococcal enterotoxin A (SEA) antibody chemisorbed to a planar gold-coated glass sensor. The formation of the immune complex between metallocarbonyl–gold nanoparticle-antibody bioconjugate and anti-SEA antibody was assessed by mid-IR surface and atomic force microscopy (AFM) analysis.

We describe here the synthesis of the first bioorthogonal cross-linking reagent based on aminocaproic acid core with a hydrazide function at one end to react with glycoproteins and an alkyne group at the other end for Cu(I)-catalyzed click chemistry to azide-derivatized probes. As an application, this cross-linker was used to orthogonally conjugate profluorescent 3-azido-7-hydroxycoumarin to immunoglobulin G (IgG). An immunoassay showed that IgG was mostly not affected by the Cu(I)-catalyzed click chemistry conditions. Successful conjugations and retained immunoreactivity demonstrate the potential of this new bioorthogonal cross-linker in chemoselective ligation.

We designed and synthesized new organometallic Fe complex for application as IR marker of noble metal nanoparticles. The novel metallocarbonyl succinimide complex possesses a disulfide group, which enables its covalent binding to noble metals. The disulfide metallocarbonyl complex was chemisorbed to gold nanoparticles of different sizes and the properties of the AuNp-metallocarbonyl conjugates were determined on the basis of UV–vis and IR spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM) and inductively-coupled plasma mass spectrometry (ICP-MS). A surface enhanced infrared absorption (SEIRA) effect was observed during IR analysis of the AuNp-metallocarbonyl conjugates by IR transmission spectroscopy.

Times Cited: 1 Article English Cited References Count: 32; Two hydrolytic enzymes, namely lysozyme and trypsin, were covalently immobilized onto stainless steel surfaces using wet chemistry processes. The immobilization strategy took advantage of the spontaneous physisorption of the polymer poly(ethylene imine) (PEI) onto stainless steel to yield a firmly attached, thin organic layer containing a high density of primary amine functions. Both enzymes were then covalently grafted to the surface via a glutaraldehyde cross-linker. Alternatively, a thicker underlayer of PEI was chemisorbed by cross-linking two PEI layers by glutaraldehyde. The effective presence of both enzymes on the stainless steel surfaces and their relative amount were assessed by immunochemical assays employing specific anti-enzyme antibodies. Eventually, the hydrolytic activity of the immobilized enzymes was evaluated by local enzymatic tests with suitable substrates. This work demonstrates that, although the amount of enzymes did not vary significantly with the underlayer thickness, their hydrolytic activity could be much improved by increasing the distance from the oxide surface and, likely, by favoring their accessibility. Our data suggest that the immobilization of enzymes on solid oxide surfaces is feasible and efficient, and that the enzymes retain catalytic activity. It may thus provide a promising route towards biofilm-resistant materials. (C) 2009 Published by Elsevier Inc.

Thioredoxin reductase is often over expressed in tumor tissues and consequently, inhibitors of TrxR are actively studied. TrxR is present in different isoforms and the role of the mitochondrial isoform is still not completely elucidated. In tumors, TrxR2 inhibition can address cells to apoptosis by altering mitochondrial permeability. This enzyme, directly or through its natural substrate Trx2, is crucial in the redox regulation of many target proteins, such as Prx3 and Grx2. Another protein redox regulated by the mitochondrial thioredoxin system is Cyclophilin D, a key molecule in the permeability transition process. Many inhibitors, such as metal complexes, are able to interact with the selenol group of TrxR, but their action is often nonselective for TrxR2 isoform. In order to hamper TrxR2, determining a redox imbalance, the inhibitors have to reach the mitochondrial matrix compartment and therefore mitochondriotropic agents could be a useful tool to block TrxR2 bringing to cell death. Interesting chemical features are offered by tamoxifen-like metallocifens, that are able to reach the mitochondrial environment, driven by the negative charge of mitochondrial membrane potential, and to inhibit TrxR determining a large increase of ROS production finalized to apoptosis.

This paper describes the elaboration of a model immunosensor monitored by polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM-D), as well as its reactivity using PM-RAIRS as transduction technique. To prove its relevance, this immunosensor was applied to the detection of benzo[a]pyrene (BaP), a carcinogenic polycylic aromatic hydrocarbon. Very few immunoassays, and even fewer immunosensors, have been described for the assay of BaP, making it an interesting target for analytical device development. The PM-RAIRS immunosensor was constructed on planar gold-coated sensors functionalized with cystamine then glutaraldehyde. Antibodies were immobilized through their affinity to protein G, covalently coupled to the aldehyde layer. In a first stage, a model mouse IgG was utilized to optimize the elaboration parameters; then, a monoclonal anti-PAH antibody was used and detection tests were performed, monitored by PM-RAIRS. The successive functionalization steps were monitored by PM-RAIRS and QCM-D. The binding of the proteins to gold surface, their saturation coverages and association constants, as well as their capture efficiencies were discussed. BaP capture by the antibody layer was evidenced by the appearance of a new ν(C–H) band at 3039 cm−1 typical of aromatic C–H bonds. The integrated area of this band varied linearly with the BAP concentration within the range of tested concentrations, with a limit of detection close to 3 μM. This represents the first example of direct, label-free immunodetection of a low molecular weight molecule by PM-RAIRS transduction. The simplicity and the rapid response of this IR sensor make it already very worthwhile to preliminary on-site measurements.

Dialkyl- and diphenyl phosphites react with the (η5-C5H5)M(CO)x(η1-N-maleimidato) (M = Fe, Mo; x = 2 or 3) complexes giving products of the phospha-Michael addition to the η1-N-maleimidato ligand. One of these complexes (M = Fe, x = 2) was characterized by X-ray diffraction. The synthesized metallocarbonyl azaphosphonates and the corresponding iron phosphonic acid act as inhibitors of certain serine hydrolases (AChE and BChE). The kinetic assays were performed and revealed that inhibition mechanism depends strongly on the enzyme and the structure of the inhibitor.

The cationic (η6-arene)ruthenium complexes 6–9 containing a chloroacetamide or a maleimide functional group on the arene ligand were synthesized and successfully used to introduce ruthenium(II) species to the active site of the cysteine endoproteinase papain in a site-directed and covalent fashion as shown by enzymatic and ESI-MS studies.

Binary mixtures of 11-mercaptoundecanoic acid (MUA) and other thiols of various lengths and terminal functions were chemisorbed on gold-coated surfaces via S-Au bonds to form mixed self-assembled monolayers (SAMs). Several values of the inole fraction of MUA in the thiol mixtures were tested and the structure and composition of the resulted thin films were characterized by X-ray photoelectron spectroscopy (XPS) and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The results made it clear that co-adsorption of MUA with thiols of similar chain length led to well-ordered monolayers whereas the co-adsorption of MUA with shorter thiols yielded less crystalline-like thin films, but with more reactive carboxylic acid terminal groups. This criterion appeared decisive for efficient covalent binding of Stapkylococcus aureus Protein A (PrA), a protein that displays high affinity for the constant fragment (Fc) of antibodies of the IgG type from various mammal species. The ability of immobilized Protein A to recognize and bind a model IgG appeared to be optimal for the mixed SAM of MUA and the short-chain, omega-hydroxythiot 6-mercaptohexanol in the proportion 1-3. (c) 2006 Elsevier B.V. All rights reserved.

Syntheses of Fischer carbene complexes with various hydrophilic tethers (4, 6a, 6b, 6c, 8a, 8b, 14a, 14b, 15 and 19) are described. The water-soluble carbene complex, 14b, was used to label and pegylate bovine serum albumin (BSA) without affecting its conformation. The red complex, 19, underwent a sharp color change to yellow on reaction with BSA, a feature that is potentially useful for developing assay methods.

Tricarbonyl cyclohexa-1,3-diene iron(0) derivatives of two biologically active molecules, N-acetyl cysteine and N-acetyl histamine and of a primary amine, n-butylamine, have been prepared by reaction of [tricarbonyl (1-4-η-5-N-pyridiniocyclohexa-1,3-diene) iron] tetrafluoroborate, a precursor of the highly reactive cation [Fe(CO)3(1-5-η-C6H7)](+), and characterized spectroscopically. Kinetic studies revealed that the reaction of N-acetyl histamine and n-butylamine was much faster than the reaction of N-acetyl cysteine. Acid/base titration of metal-carbonyl labelled histamine was achieved by IR spectroscopy and revealed that the imidazole ring substituted by the metal-carbonyl unit was slightly more basic than the parent compound. To cite this article: M. Mokhtari et al., C. R. Chimie 8 (2005).

The first poly(amidoamine) (PAMAM) dendrimers tethered with both (g 5 -cyclopentadienyl) rhenium tricarbonyl (CpRe(CO)3) units and polyethylene glycol (PEG) chains were prepared and characterized by combining NMR spectroscopy and Fourier-transform IR spectroscopy. Grafting of CpRe(CO)3 units was achieved by reductive amination of formyl-CpRe(CO)3 with the peripheral amines of generation 3 and 4 PAMAMs to yield dendrimers labeled with a variable number of CpRe(CO)3 units, ranging from 8 to 14 for PAMAM-G3 and 17–30 for PAMAM-G4. PEG chains of different lengths were then attached to some of the remaining peripheral amines, and their respective ability to improve the solubility of the metallodendrimers in aqueous buffered media was evaluated. These metallodendrimers represent new infrared probes designed to be coupled to immunological reagents for the amplification of the IR signal in carbonyl metallo immunoassay (CMIA). � 2004 Elsevier B.V. All rights reserved.

The synthesis, through a cross-metathesis reaction, of the first chiral peptide nucleic acid (PNA) monomer labelled with a Fischer-type carbene complex of chromium is reported. IR analysis of the new bioconjugate shows that the Cr(CO) 4 moiety represents a suitable spectroscopic probe for diagnostic purposes.

This article reviews the current methods available to covalently label peptides and proteins with transition organometallic entities in a side-chain-selective and covalent manner. Almost all these methods take advantage of the reactivity of the nucleophilic groups borne by most proteins, namely the ϵ-amino function of lysine residues and the thiol function of cysteine residues. In this way, the unusual physical and/or spectroscopic properties of transition metals and their organo-complexes may be exploited in different areas of biology and medicine, including analytical biochemistry, protein three-dimensional structure analysis and radio-pharmaceuticals development. To cite this article: M. Salmain, G. Jaouen, C. R. Chimie 6 (2003).

The immobilisation of biomolecules on gold surfaces has attracted a lot of attention due to the various ways of transducing biorecognition events at such surfaces and of course to its broad field of applications. The current challenge is two-fold: first, to produce a functionalised surface able to bind a given biomolecule in a complex environment, but also resistant to nonspecific adsorption; second, to assess the protein–ligand interaction by a suitable physical method. In our attempt to conceive a new type of biosensor, the biotin/avidin system was chosen as a model of biological (ligand/receptor) interaction. The first task was achieved by developing a two-step procedure, briefly consisting in the chemisorption of the short diamine disulfide cystamine to a gold substrate followed by chemical coupling with the N -succinimidyl ester of biotin. The presence of biotin molecules both specifically and unspecifically bound to the gold surface was assessed by Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) and XPS. Undesired nonspecific binding of biotin was minimised by increasing the chain length of the chemisorbed amine to which was coupled the N -succinimidyl ester of biotin. Chemical characterisation of the adsorbed layer was performed after each step by FT-IRRAS. For the second task, the protein avidin was labelled with an alkyne Co 2 (CO) 6 probe, a transition metal carbonyl entity that yields three characteristic ν CO signals far from the peptide region of absorption, before its interaction with the modified surface. Molecular recognition was checked and quantified by FT-IRRAS. The occurrence of nonspecific adsorption of avidin was measured by exposing the biotinylated substrates to a solution of labelled avidin pre-saturated by biotin. Methods to reduce nonspecific binding of avidin were also shown.

Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) was successively used to monitor the covalent immobilization of biotin molecules onto a planar gold substrate covered with a self-assembled monolayer of cystamine and to transduce the molecular recognition of avidin and biotin. This detection was greatly facilitated and made selective by the labeling of avidin and of biotin with various transition metal carbonyl probes. The binding of avidin to the surface was optimized by blocking the nonspecific binding sites by adsorption of an unrelated protein, bovine serum albumin. This work exemplifies the feasibility of detecting biomolecular associations involving molecules of any size at a liquid/solid interface by using a simple and accessible surface analysis technique.

High purity gaseous CO stored in a steel tank contains a volatile iron carbonyl compound that easily contaminates electrochemical samples saturated with CO. The impurity yields a voltammetric reduction peak at −2.0 V in non-aqueous solvents (acetonitrile, acetone and tetrahydrofurane) and IR absorption at 2024 and 2001 cm −1 characteristic of metal carbonyls. This impurity is Fe(CO)5 and can be removed efficiently by bubbling CO through aqueous NaOH solution. This purification procedure is adequate for the electrochemical studies of metal carbonyls under CO atmosphere. Failure to remove this volatile impurity may lead to misinterpretation of electrocatalytic and ligand substitution reactions.

The electrophilic property of metallocarbenes of Fischer-type was used to label amino acid derivatives and a model protein in a regio-specific manner with a transition heavy metal complex. Hence, the reaction of (CO) 5 WC(OMe)Me ( 1 ) was shown to involve exclusively the amino group of a series of α-amino esters whether this function was carried by the α-carbon or by the side chain, leading to stable aminocarbenes. With a model protein, namely bovine serum albumin, labelling also occurred involving solely the amine function of some of its lysine residues. Coupling yield was shown to depend both on the pH where the reaction was carried out, and on the quantity of carbene. This work could find an application in the field of protein X-ray crystallography.

Pyrylium ions are useful reagents to selectively modify the amino group of protein lysine residues. This property was exploited here to label proteins with chromium tricarbonyl complexes in the form of 4-benchrotrenyl pyridinium ions. Kinetic studies of the reaction of a series of benchrotrenyl pyrylium salts with water and n-butylamine were performed and revealed that the overall reactivity of these compounds was highly dependent on their substitution pattern. These compounds could find application in protein X-ray crystallography.

We describe here the development of a new, non-isotopic immunological assay termed CMIA (carbonyl metallo immunoassay) that uses metal carbonyl complexes as tracers and Fourier transform infrared spectroscopy (FT-IR) as the detection method. This assay is based on the particular spectral features of these complexes, which show very strong absorption bands in the 1800–2200 cm −1 spectral range where proteins and organic molecules do not absorb. In Section 1 , the optimisation of the quantitative detection of these tracers is detailed. In Section 2 , the implementation of mono-CMIA is described, including the CMIA assays of three antiepileptic drugs (carbamazepine, phenobarbital, phenytoin). Finally, extension to the simultaneous double- and triple-CMIA of these drugs is reported.

The first example of simultaneous triple carbonylmetalloimmunoassay (CMIA), a non-isotopic immunological method, is presented herein. This method, originally developed for mono-assays, uses various transition metal carbonyl complexes as labels and sensitive quantitation of these tracers by Fourier transform infrared (FT-IR) spectroscopy, thanks to the characteristic νCO bands appearing in the 1800–2200 cm−1 region. We show here that this method offers an advance in the challenging field of simultaneous multi-immunoassay, with the example of the assay of three antiepileptic drugs (carbamazepine, phenobarbital and diphenylhydantoin).

The bioorganometallic complex (S)-[3-CpFe(CO)2(η1-N-succinimidato)]glutathione 1 resulting from the reaction of glutathione with (η5-Cp)Fe(CO)2(η1-N-maleimidato) is shown to inhibit in a competitive manner the enzymatic activity of glutathione (S)-transferase. Moreover, when the Fp fragment is removed by photochemical means, the resulting compound 2 loses this property, which thus appears to be due to the presence of the organometallic moiety.

The first transition metal-carbonyl water-soluble imidoester has been prepared in several steps and characterised by classical spectroscopic methods. It is aimed at the selective conjugation of amine-containing biomolecules such as proteins in aqueous medium, leading to conjugates that retain their net charge. A preliminary reactivity study with bovine serum albumin (BSA) is reported.

Site-specific labelling of the cysteine-containing peptide glutathione with a ferrocene group was achieved by reaction with ferrocenylmethanol in aqueous acidic medium. The resulting peptide was shown to be a potent competitive inhibitor of the biologically important enzyme glutathione-(S)-transferase. This approach may prove general for the labelling of proteins with ferrocene.

Resume Nous avons recemment mis au point un nouvel immunodosage non isotopique, appele carbonyl metallo immunoassay (CMIA) qui utilise comme traceur des complexes organometalliques et comme methode de detection l'infrarouge a transformee de Fourier (IR-TF). Cet immunodosage a deja ete decrit pour un medicament antiepileptique, le phenobarbital et une hormone steroide endogene, le cortisol. Nous decrivons ici l'extension de ce dosage CMIA au cas de deux autres medicaments antiepileptiques, la diphenylhydantoine (DPH) et la carbamazepine (CBZ). Pour ces deux medicaments, nous avons tout d'abord synthetise des immunogenes specifiques qui nous ont permis d'obtenir des anticorps polyclonaux anti-DPH et anti-CBZ qui possedent un titre eleve et une bonne specificite. Nous avons ensuite prepare, pour ces deux molecules, des traceurs organometalliques. Pour la DPH, nous avons choisi un complexe du chrome tricarbonyle [Cr(CO) 3 -DPH] et pour la CBZ un complexe du dicobalt hexacarbonyle [Co 2 (CO) 6 -CBZ]. Ces complexes ont ete selectionnes car ils fournissent en infrarouge, dans la region spectrale etudiee (1850–2150 cm −1 ), des signaux caracteristiques et intenses qui n'ont aucune zone de recouvrement. Ainsi, la simple mesure de la valeur d'absorbance d'une des bandes caracteristiques de chaque complexe permet l'analyse quantitative simultanee du melange des deux traceurs. Nous avons utilise cette propriete unique pour realiser le premier multidosage CMIA simultane de ces deux medicaments.

For the development and applications of organometallic moieties as potential tracers for biological assays, several methods are explored for the labelling of a polyfunctional and nearly insoluble biomolecule with a dicobalt hexacarbonyl marker. This can be done successfully using an organometallic-like Bolton-Hunter reagent. The metal-carbonyl isomeric adducts are obtained in the expected proportions and are characterized by high performance liquid chromatography, IR spectroscopy and fast atom bombardment/mass spectroscopy.

Resume Nous decrivons ici un nouvel immunodosage non isotopique qui utilise comme traceurs des complexes organometalliques du cobalt ou du manganese carbonyle et comme methode de detection l'infrarouge a transformee de Fourier. Le principe de ce dosage est base sur les proprietes spectrales particulieres de ces complexes qui ont de tres fortes bandes d'absorption dans la region 1800–2200 cm−1, une region dans laquelle les proteines et les molecules organiques n'absorbent pas. Pour demontrer la faisabilite de ce dosage nous sommes interesses au cas de deux analytes : le phenobarbital et le cortisol. Dans les deux cas nous avons effectue une caracterisation complete d'anticorps polyclonaux specifiques de ces haptenes par la methode CMIA (valeur du titre des anticorps, courbes d'etalonnage, etude de la specificite). Lors des etudes de reproductibilite nous avons obtenu des valeurs de coefficient de variation de 5%. Ces resultats montrent que les dosages effectues par CMIA sont fiables et reproductibles.

A new non-radioisotopic immunoassay procedure, which we have termed carbonylmetalloimmunoassay (CMIA), is described. The tracers used in this approach are organometallic carbonyl complexes that can be detected at femtomole levels (300-700 fmol) by Fourier transform infrared (FT-IR) spectroscopy. The validity of the technique has been tested in a phenobarbital assay using as the marker a cyclopentadienylmanganese (I) tricarbonyl (cymantrene) moiety, ethyl acetate extraction to separate the free and bound organometallic fractions, and FT-IR spectroscopy to detect the CO stretching modes of the organometallic label. Typical dilution and standard curves obtained with this CMIA procedure are presented. The method was of comparable sensitivity to a [14C] radioimmunoassay (RIA) for the detection of phenobarbital. A comparison of the results for phenobarbital assays by both CMIA and RIA showed that higher titres were obtained using the CMIA method. The standard curves suggest that CMIA is a reliable and reproducible immunoassay procedure for phenobarbital.

The new compounds [η5-C5H4COOH)M(CO)n,Me] [M  Mo, n = 3 (2); M  Fe, n = 2 (5)] were synthesized in 61% and 63% yields, respectively, and treated with N-hydroxysuccinimide to yield the corresponding activated ester derivatives [(η5-C5H4COONS)-M(CO)nMe] [M  Mo, n = 3 (3); M  Fe, n = 2, (6). The metal-activated ester complex, [(η(su5)-C5H5Mo(CO)3(CH2COONS)] (8) was obtained similarly, the ester unit being bonded directly to the metal rather than to the π-bonded cyclopentadienyl. The reactivity and potential of the above species as labelling agents for amino-acids is discussed.

Resume Nous presentons dans cet article les bases d'un nouveau concept en immunoanalyse : l'IRIA ( Infrared Immunoassay ). Il s'agit d'un dosage competitif en phase heterogene qui s'appuie sur trois criteres : 1. l'utilisation de marqueurs organometalliques ou de clusters; 2. la separation des fractions libre et liee par un solvant organique; 3. la detection du traceur par spectroscopie infrarouge transformee de Fourier (IR-TF).