Syntheses And Characterizations Of Bifunctional Tripyridinery Chelate Intermediates For Time-Resolved Fluoroimmunoassay

Time-resolved fluoroimmunoassay is a new immunoassay technology established in 1980s. It using lanthanide chelates as the fluorescence probes is an established immunoassay method. The most important advantage of TRFIA is that it allows easy distinction of the specific fluorescence signal of long-lived lanthanide fluorescence probe from the short-lived background signal present in most biological samples, and also obviates the problems associated with scattering light of the optical components, since the fluorescence of lanthanide fluorescence probe is long-lived with large Stokes shift and sharp emission band. Therefore the sensitivity of TRFIA is higher than those of other immunoassay methods.The synthesis of bifunctional chelator was the key of time-resolved fluorescence immunoassay. The so-called bifunctional chelator could be coordinated with rare earth ion; on the other hand, it could also contain appropriate functional to be covalently attached to biomolecules. Tripyridine multiacid derivatives are the famous bifunctional chelatores for their use in DSLFIA. The syntheses of heterocycle molecule, functions about protein connection and rare earth chelating are the key to realize the bifunction.In this paper, tetra(ethyl) N,N,N',N'-[2,6-bis(3-aminomethyl-pyrazol-1-yl)-4-(4-nitro phenylethynyl)pyridine] tetrakis (acetate) and several important intermediates were designed, prepared and characterized according to correlative literature with the aim to satisfied the need of bifuncional chelatores for direct solid lanthanide time-resolved fluorescence immunoassay (DSLTRFIA). These intermediates were prepared from 2, 6-dibromopyridine by oxidation, nitration, reduction, nucleophilic substitution, diazotization- Sandmeyer and NBS reactions and so on. During the experimentation, the structure of the products were determined by DSC, IR, 1H-NMR, MS, element analysis and so on to prove the reliability of these structures and synthetic methods. Otherwise, the reaction conditions and mechanism of several steps were also studied.The nitrogen and oxygen atoms in the molecule of tetra (ethyl) N, N, N', N'-[2, 6-bis (3-aminomethyl -pyrazol-1–yl)-4-bromopyridine] tetrakis (acetate) could form strong complexes with lanthanide metals in an aperture figure. The bromine atom in this molecule could be substituted by other functionals in order to attach to protein.The other intermediates such as 2,6-bis (3-methyl-pyrazol-1-yl)-4-bromopyridine and 2,6-bis (3-bromomethyl-pyrazol-1-yl)-4-bromopyridine and so on in our experiment not only provide methods for itselves, but also give elicitation for syntheses of other tribipyridineryptates.