Bogna Rudolf

Synthesis of metal-carbonyl-dendrimer-antibody immunoconjugates: Towards a new format for carbonyl metallo immunoassay

We report the preparation of metal‐carbonyl–dendrimer–antibody conjugates. These metal‐carbonyl‐multilabeled antibodies are designed to be used in a new solid‐phase‐format carbonyl metallo immunoassay (CMIA). A fourth‐generation polyamidoamine dendrimer was labeled with 10–25 (η5‐cyclopentadienyl)iron dicarbonyl (η1‐N‐succinimidyl) entities. An antibody was chemically modified at its carbohydrate chains by a site‐directed process used to preserve the antigen–antibody binding site. The antibody was then coupled with the dendrimer labeled with 10 metal carbonyl groups. An average of 1.4 labeled dendrimers were grafted per antibody molecule. These metal‐carbonyl–dendrimer–antibody conjugates were used as new universal detection reagents that recognize their specific antigens. The antigens were spotted onto nitrocellulose membranes and detected by using the conjugates in combination with Fourier transform infrared spectroscopy. A detection level in the range 5–200 pmol per membrane was achieved. This approach opens the way to a new CMIA format.

(η5-cyclopentadienyl)Fe(CO)2-complex of maleimide anion: An organometallic carbonyl probe for biomolecules containing HS groups

Abstract Synthesis of (η5-cyclopentadienyl)Fe(CO)2(η1-N-maleimidato) complex and its reaction with L-cysteine ethyl ester hydrochloride and glutathione are reported. This reaction enables introduction of a metal carbonyl probe into biomolecules containing HS groups.

Cysteine‐Specific, Covalent Anchoring of Transition Organometallic Complexes to the Protein Papain from Carica papaya

Site‐directed and covalent introduction of various transition metal–organic entities to the active site of the cysteine endoproteinase, papain, was achieved by treatment of this enzyme with a series of organometallic maleimide derivatives specially designed for the purpose. Kinetic studies made it clear that time‐dependent irreversible inactivation of papain occurred in the presence of these organometallic maleimides as a result of Michael addition of the sulfhydryl of Cys25. The rate and mechanism of inactivation were highly dependent on the structure of the organometallic entity attached to the maleimide group. Combined ESI‐MS and IR analysis indicated that all the resulting papain adducts contained one organometallic moiety per protein molecule. This confirmed that chemospecific introduction of the metal complexes was indeed achieved. Thus, three novel reagents for heavy‐atom derivatization of protein crystals, which include ruthenium, rhenium and tungsten, are now available for the introduction of electron‐dense scatterers for phasing of X‐ray crystallographic data.

Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay.

We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamidoamine (G4-PAMAM) dendrimers carrying (i) (η(5)-cyclopentadienyl) iron dicarbonyl succinimidato complexes as infrared (IR) probes, (ii) nitroaniline entities as nuclear magnetic resonance (NMR) probes, (iii) acetamide groups for surface neutralization, and (iv) hydrazide-terminated spacer arms for the reaction with aldehyde. To preserve a high binding affinity, the conjugation was performed on the carbohydrate moieties located on the Fc fragment. The resulting conjugates were characterized by Fourier transform-IR, ultraviolet (UV), and high-mass matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. On the basis of relative concentration ratios of IR probes and antibody, an average labeling of 30 IR probes per antibody was reached (i.e., more than twice the value obtained with our previous strategy that generated no spacer arm). Immunoassays revealed that the antibody-dendrimer conjugates retained 55.1% of immunoreactivity on average with respect to underivatized antibody. Finally, the conjugates were used to quantify their antigen by solid-phase carbonyl metallo immunoassay (CMIA). Results showed a significant enhancement of the IR signal, demonstrating the efficiency of the new conjugation strategy and the potential of the new antibody-dendrimer conjugates as universal immunoanalytical reagents.

Metallo-carbonyl complexes based on the CpFe(CO)2(η1-N-imidato) system as protein labelling reagents: reactivity and selectivity studies using bovine serum albumin as a model protein

Transition metallo-carbonyl complexes are useful bioprobes to study molecular recognition processes such as ligand-receptor interactions, owing to their intense absorption bands in the mid-IR spectral range, which enable their detection at the picomole level with FT-IR spectrometers. New chemical methods for the introduction of cyclopentadienyl iron dicarbonyl fragments into proteins have been developed. Synthesis of a maleimide and of an N-succiminidyl ester and their reactivity with a model protein, namely bovine serum albumin (BSA), are described here. Results show that BSA can be labelled by both reagents at neutral to basic pH, and, as for the maleimide, side-chain selectivity can be tuned as a function of experimental coupling conditions.

Addition of imidazoles and aminoacids to the ethylenic bond in (η5-C5H5)Fe(CO)2(η1-N-maleimidato)

Abstract ( η 5 -C 5 H 5 )Fe(CO) 2 ( η 1 -N-maleimidato) 1 reacts in water solution at pH = 7 with imidazole, histidine methyl ester and carnosine to give the products of the addition of the heterocyclic moiety to the ethylenic bond of the maleimidato ligand. Addition of the primary amino group of glycine and β-alanine to 1 takes place at pH = 10 and 60°C. These results are relevant for labelling of peptides and proteins with 1 .

Inhibition and photo-deinhibition of glutathione (S)-transferase activity by an organometallic complex: (S)-[3-CpFe(CO)2(η1-N-succinimidato)]glutathione

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.

Metallo-carbonyl conjugates of biotin and biocytin

Abstract Coupling of biotin with CpFe(CO) 2 (η 1 - N (1)-4-aminosulfonamido) ( 1a ) and CpFe(CO)(P(OMe) 3 ) (η 1 - N (1)-4-aminosulfonamide) ( 1b ) afforded corresponding metallo-carbonyl biotin amides 2a – b . Reaction of N e -biotinyl- l -lysine (biocytin) with CpFe(CO) 2 (η 1 - N -3-isothiocyanatophtalimidato) ( 4a ), CpFe(CO) 2 (η 1 - N -4-isothiocyanatophtalimidato) ( 4b ), CpFe(CO) 2 (η 1 - N (1)-4-isothiocyanatosulfonamido) ( 5 , this compound was prepared from 1a and thiophosgene and its structure confirmed by spectral data and by the X-ray diffraction method) gave bioconjugates 6a – c . The HABA-avidin test revealed high affinities of all synthesised bioconjugates to avidin.

Reactivity of the maleimidato ligand in CpFe(CO)2(η1-N-maleimidato) in Diels-Alder reaction with 1,3-diphenylisobenzofuran. The crystal structure of [CpFe(CO)2(η1-N-1,4-diphenyl-1,4-epoxy-1,4-dihydronaphthalene-2,3-dicarboxyimidato)]

Abstract The title complex reacts with 1,3-diphenylisobenzofuran to afford the Diels-Alder adduct [CpFe(CO) 2 ( η 1 -N-1,4-diphenyl-1,4-epoxy-1,4-dihydronaphthalene-2,3-dicarboxyimidato)] 2 in nearly quantitative yield. The competitive experiment showed that this reaction is around three times slower than the reaction of the parent maleimide with 1,3-diphenylisobenzofuran. The X-ray crystal structure of 2 has been determined. It revealed a scrolled configuration resulting from an endo transition state in Diels-Alder reaction and from various hydrogen bonds and π-π interactions.

η1-N-succinimidato complexes of iron, molybdenum and tungsten as reversible inhibitors of papain.

Recently we have found that the metallocarbonyl complexes (eta(5)-C(5)H(5))M(CO)(x)(eta(1)-N-maleimidato) (M=Fe, Mo, W; x=2 or 3) bearing a maleimide function were irreversible inhibitors of the enzyme papain. To get further insight into the binding mechanism of these compounds we synthesized the related complexes (eta(5)-C(5)H(5))M(CO)(x)(eta(1)-N-succinimidato) (M=Fe, Mo, W; x=2 or 3) that lacked the ethylenic bond responsible for alkylation of the cysteine 25 thiol group in the papains catalytic pocket. We performed kinetic studies of the interaction of the synthesized complexes towards papain. We found that they act as reversible inhibitors of the enzyme with IC(50) values in the range 480-1700microM. Docking experiments confirmed binding of these complexes to the enzymes catalytic pocket.