ABSTRACT
Automated incorporation of haptens at the 3' or 5' end of oligonucleotides required the preparation of the corresponding hapten phosphoramidites. The requisite 1,3-diol framework was prepared in two steps from a carboxylic acid precursor first by a two-carbon homologation using Meldrum's acid to form the corresponding 3-oxo ester and then subsequent reduction to the diol. The primary alcohol was protected with a dimethoxytrityl group, while the secondary alcohol was converted to the reactive phosphoramidite.
Subject(s)
Haptens , Organophosphorus Compounds/chemical synthesis , Automation , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Oligonucleotides/chemistry , Organophosphorus Compounds/immunologyABSTRACT
Upon base composition analysis, oligonucleotides which are labeled at the 3'-terminus with fluorescein or biotin generate an additional, late eluting peak in the HPLC chromatogram. Investigation of this effect revealed the haptens acted as apurinic sites, and phosphodiesterase cleavage of the phosphate bond between the upstream nucleotide and apurinic site is inhibited. Extension of this work with a base-stable apurinic site inserted into all possible junctures of 5'-TGAC-3' tetramers showed this to be a general effect. As a consequence of this work, acid-catalyzed depurination resulting in apurinic sites can be monitored in oligonucleotide synthesis.
Subject(s)
Apurinic Acid/chemistry , Oligodeoxyribonucleotides/chemistry , Phosphoric Diester Hydrolases/metabolism , Base Composition , Biotin/chemistry , Chromatography, High Pressure Liquid , Fluorescein , Fluoresceins/chemistry , Oligodeoxyribonucleotides/metabolism , Substrate SpecificityABSTRACT
A new class of fluorescein derivatives with chemically reactive amino and N-alkylamino "arms" in the 4'-position were synthesized and their utility in the development of fluorescence polarization immunoassays (FPIA) for cortisol and estriol was evaluated. The positioning of the arm in one of the phenolic rings introduced chirality due to hindered rotation and led to rotational isomers. These were separable when brought into a chiral environment, i.e., conjugated to steroid molecules. In the case of cortisol conjugates, the rotamers had similar properties in the FPIA. In the case of estriol conjugates, however, each rotamer exhibited different immunoassay characteristics. The rotamers interconverted at 80 degrees C, with the rate increasing with temperature. An unusual N-alkylation phenomenon by alkanols in acidic medium was observed. A serum cortisol FPIA, developed using an N-alkylamino fluorescein derivative, showed good correlation with a reference RIA.
Subject(s)
Fluoresceins/chemical synthesis , Alkylation , Estriol/blood , Fluorescence Polarization , Humans , Hydrocortisone/blood , Immunoassay/methods , Radioimmunoassay , Stereoisomerism , TemperatureABSTRACT
With the help of NMR spectroscopy the structure assignment of fluorescein mercuric acetate is corrected to 4',5'-bis(acetoxymercury)fluorescein.
