Transition Metal−Carbonyl Labeling of Biotin and Avidin for Use in Solid-Phase Carbonyl Metallo Immunoassay (CMIA)

Abstract

The preparation of several transition metal-carbonyl tracers of biotin and avidin is described. Multiple labeling of avidin was achieved by acylation of some of its amine-bearing residues with N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl). By varying the initial amount of this complex, protein conjugates with the extent of derivatization of up to 13 were obtained. Biotin was labeled with one (eta5-cyclopentadienyl)manganese tricarbonyl moiety by reaction of biotin hydrazide, whereas multiple labeling was reached by successive conjugation of biotin and N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl) or (eta5-cyclopentadienyl)iron dicarbonyl (eta1-N-maleimidato) to poly-L-lysine or fourth generation Starburst dendrimer. All the conjugates displayed a good to excellent bioaffinity toward their respective counterparts, as measured by competitive enzymatic assays.

Microwave-assisted solid-phase synthesis, cellular uptake, and cytotoxicity studies of cymantrene–peptide bioconjugates

“…In addition, most of these functionalities were introduced by manual coupling. We thus became interested in cymantrene (CpMn(CO) 3 ) as an easy-to-functionalize and robust IR spectroscopic marker [25][26][27][28][29][30][31][32][33] and show here that microwave-assisted solid-phase synthesis allows one to prepare different cymantrene-peptide bioconjugates of increasing complexity in good yield and purity. [Leu 5 ]-enkephalin (ENK) was chosen as a simple pentapeptide with few charged amino acid residues to start with and validate the solid-phase peptide synthesis (SPPS) method.…”

Synthesis, characterization, X-ray crystallography, and cytotoxicity of a cymantrene keto carboxylic acid for IR labelling of bioactive peptides on a solid support

“…Especially metallocenes have been at the focus of intense research efforts, since, for example, the Fe II/III redox couple as in ferrocene allows for an easy electrochemical quantification of the labelled peptide [1][2][3][4][5][6][7][8][9]. Substantially less work has however been carried out based on other properties of organometallic compounds [10][11][12][13][14][15][16][17][18][19][20][21][22]. We are particularly interested in the attachment of IR spectroscopic labels to peptides for biological imaging applications with scanning near-field IR microscopy (SNIM) [23].…”

The Use of Glycidol to Introduce Aldehyde Functions Into Proteins – Application to the Fluorescent Labelling of Bovine Serum Albumin and Avidin

“…We had previously experienced precipitation of avidin when conjugated to more than 10 dicobalt hexacarbonyl IR probes and had attributed it to the additional presence of the hydrophobic Co 2 (CO) 6 moieties on the protein surface. [17] We inferred that such a phenomenon could also be occurring for the AV-LyCH conjugates, as LyCH features a hydrophobic core despite the presence of two sulfonate groups. Thus, to reduce the number of reactive aldehyde functions on AV-diol and AV and therefore to keep unreacted more diol groups, oxidation was carried out at pH 4 for 15 min (instead of 1 h) and coupling of LyCH was performed as above.…”