Biomedical subjects

G Jaouen

Publications and source records attributed to G Jaouen.

27 records · Page 2Linked to original sources

PubMedDate not supplied

(N-succinimidyl 4-pentynoate)hexacarbonyldicobalt: a transition-metal carbonyl complex having similar uses to the Bolton-Hunter reagent.

The synthesis of the transition-metal carbonyl complex (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt [[(C4H4O2N)O(CO)CH2CH2C identical to CH]Co2(CO)6] is described. This cobalt carbonyl complex is structurally similar to the Bolton-Hunter conjugation reagent and has been successfully employed as a nonradioactive tracer for labeling the drug carbamazepine. The metal carbonyl tracer can be detected at extremely low concentrations (ca. 1 pmol) by FT-IR spectroscopy in the v(CO) region (2150-1800 cm-1). The cobalt carbonyl labeled carbamazepine retains good recognition for anti-carbamazepine antibodies. This novel labeling procedure, which can be broadly termed carbonylmetalloimmunoassay (CMIA), has considerable potential for assaying a wide range of biological materials.

Cobalt↗

PubMedDate not supplied

Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy.

Labeling of bovine serum albumin (BSA) and anti-human thyroid stimulating hormone (hTSH) monoclonal antibodies (mAbs) was performed using (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt (NSCo2(CO)6). Conditions of coupling were different depending on the protein to be labeled, denaturation of the mAbs occuring with high percentages of organic solvent in the reaction mixture. The influence of reaction time and initial concentration of NSCo2(CO)6 was examined. They were both shown to affect the final coupling rate of the metal carbonyl probe. Preservation of the immunoreactivity toward 125I-hTSH was observed for five conjugates having different NSCo2(CO)6: mAb molar ratios when compared to unmodified and peroxidase-labeled mAbs. Finally, a preliminary study of the quantitative detection of the metal carbonyl mAbs on microtiter wells was achieved using Fourier transform infrared spectroscopy.

Animals↗

PubMedDate not supplied

Synthesis of cobalt carbonyl complexes of cortisol and testosterone. Study of their recognition by specific polyclonal antibodies.

We have synthesized organometallic complexes of steroids (cortisol, testosterone, dihydrotestosterone) for potential use as tracers in nonisotopic carbonyl-metal immunoassays (CMIA). An ethynyl/CO2(CO)6 fragment at the end of a five-atom spacer was coupled to position 3 of the steroid skeleton. In the case of cortisol, we exploited the difference in reactivity of the ketone and enone functions toward amines in order to form an enamine which was then made to react with carboxymethylamine to yield 3-[(carboxymethyl)oxime] steroid. Activation of the carboxylic acid function with N,N'-dicyclohexylcarbodiimide in the presence of propargylamine introduced an acetylenic function at the end of the spacer. The triple bond was then complexed by CO2(CO)8 to form complexes 5a-c. Complexes for use in CMIA should be stable in biologic media and effectively recognize specific antibodies. Complexes 5a-c were stable in the buffers we use in biochemical tests. Their cross reactivities for anti-cortisol and anti-testosterone antibodies ranged from 50 to 110% according to batch, indicating, first, that the addition of an organometallic complex in position 3 of the steroid skeleton does not hinder recognition between the organometallic steroid and antibody and, second, that their individual behavior differs substantially according to antibody batch. Although all of these complexes could be used as tracers in CMIA, it is necessary, in each case, to establish which tracer-antibody duo gives rise to the most sensitive immunoassay.

Animals↗

PubMedDate not supplied

Labeling of proteins by organometallic complexes of rhenium. (I). Synthesis and biological activity of the conjugates.

We describe herein a totally new pathway for the introduction of rhenium in the form of low oxidation state organometallic complexes covalently attached to various proteins. The synthesis of several rhenium conjugates takes advantage of the specificity of N-succinimidyl esters for amino residues. Conjugation experiments were carried out under various conditions, and analysis of the conjugates was performed by FT-IR spectroscopy. Yields were optimized and reached 50%. Furthermore, the conjugate resulting from the coupling of N-succinimidyl 4-[eta 5-cyclopentadienylrhenium tricarbonyl] 4-oxobutanoate to an anti-hTSH monoclonal antibody retained a satisfactory immunoreactivity. Finally, IR detection of conjugates adsorbed onto nitrocellulose membranes was achieved and response was found to be related to the coupling extent of the conjugate.

Amino Acids↗

PubMedDate not supplied

[eta 5-Cyclopentadienyl]metal tricarbonyl pyrylium salts: novel reagents for the specific conjugation of proteins with transition organometallic labels.

New specific reagents for the conjugation of organo transition metal species to proteins are described. These reagents are pyrylium salts bearing a (eta 5-C5H4)M(CO)3 (M = Mn and Re) at position 4. They couple with simple amines (n-butylamine and tert-butylamine) and to lysine side chains of proteins (bovine serum albumin and lysozyme) with varying yields. In almost all cases, the final conjugated species is a pyridinium salt, with the exception of lysozyme, for which the reaction ends at the divinylogous amide form. Differences in reactivity for bovine serum albumin and lysozyme can be explained in terms of differences of isoelectric point and steric local environment around the reactive lysine residue.

Acetonitriles↗

PubMedDate not supplied

Cyclopentadienyl iron dicarbonyl (eta 1-N-phthalimidato) complexes containing an isothiocyanate function: synthesis and application to protein side-chain selective labeling.

The two first transition metal carbonyl isothiocyanates were prepared in high yield within two steps from photolysis of CpFe(CO)2I and 3- or 4-aminophthalimide in the presence of diisopropylamine followed by reaction with thiophosgene/triethylamine. Their reaction with a model amino acid, i.e. beta-alanine, was performed and led to the expected thioureas. When reacted with bovine serum albumin in aqueous medium, conjugates bearing 6-10 iron-carbonyl fragments were obtained and characterized by Fourier transform infrared spectroscopy, thus demonstrating the usefulness of these reagents for the selective and covalent labeling of proteins.

Iron Compounds↗

PubMedDate not supplied

Synthesis of CpFe(CO)(L) complexes of hydantoin anions (Cp = eta5-C5H5, L = CO, PPh3), and the use of the 5,5-diphenylhydantoin anion complexes as tracers in the nonisotopic immunoassay CMIA of this antiepileptic drug.

As part of our ongoing development of the CMIA nonisotopic immunoassay method, in which the tracers are metal carbonyl complexes and detection is by Fourier transform infrared spectroscopy, we examined the potential use as tracers of the complexes CpFe(CO)2(5,5-diphenylhydantoin) 2d and CpFe(CO)(PPh3)(5, 5-diphenylhydantoin) 3. The present study involved the synthesis of a series of hydantoin complexes (2a-2d), in particular that of the derivative of 5,5-diphenylhydantoin 2d. The structure of 2d was confirmed by X-ray crystallography. The infrared analysis, establishing the position and intensity of the characteristic metal-carbonyl peaks of complexes 2d and 3 in the 1850-2200 cm-1 region, shows that measurement of the absorbance values of these characteristic peaks will permit quantitative analysis in the picomole range, the norm for routine use in immunoassay and thus suitable for use as CMIA tracers. Cross-reaction rates of these tracers with anti-DPH specific antibodies show that 2d and 3 are both recognized by anti-DPH antibodies (cross-reaction rates 43 and 20%, respectively). In developing a CMIA of DPH with these tracers, it was found that 3, with a single, intense band at 1977 cm-1, had very promising IR characteristics for use in multiassay CMIA, but probably owing to its relatively weak affinity for the antibodies, it was not possible to develop a CMIA for DPH using this tracer. Complex 2d, however, showed better recognition by the antibodies, and using this complex as a tracer, it was possible to develop a particularly sensitive monoassay of DPH by the CMIA method.

Anions↗

PubMedDate not supplied

Use of heavy-metal clusters in the design of N-succinimidyl ester acylation reagents for side-chain-specific labeling of proteins.

New heavy transition metal carbonyl markers for protein labeling, containing an "Mn(CO)11" (M = Ru, Os, n = 3; M = Ir, n = 4) moiety, were prepared by reaction of "lightly stabilized" clusters with an N-succinimidyl ester functionalized phosphine, namely N-succinimidyl 3-diphenylphosphine-propionate (DPPS). The reaction of Os3(CO)11(DPPS) with the model amino acid beta-alanine was performed and led to the expected amide. From the reaction of Mn(CO)11(DPPS) with bovine serum albumin (BSA) in mixed organic/aqueous medium, conjugates bearing a fairly high number of metal carbonyl fragments were obtained, thus demonstrating the usefulness of this class of reagents for the selective and covalent graft of heavy metal clusters to side chain of proteins.

Acylation↗

PubMedDate not supplied

[Contribution of simultaneous multiple immunoassays to biology].

During the last years, research in the area of immunoanalysis has been oriented towards the marketing of more reliable, faster and low cost kits. In this field, simultaneous multiple immunoanalysis that offer long-time ignored original and specific advantages, has undergone tremendous improvements triggered by striking technological innovations and the advent of lanthanide chelate based markers. Simultaneous multiple immunoassays have turned from semi-quantitative methods restricted to pharmacology to quantitative methods for medical biology. We chose first to describe several commercial systems among the most representative then we stressed on the main emerging systems based on optical or electrochemical detection as well as some immunosensor devices. The array of simultaneous multiple immunoassays currently available appears attractive enough so as to take a significant area within the analytical arsenal at the biologist disposal.

Biology↗