Synthesis of Conjugates for a Barbiturate Screening Assay

Abstract

Novel derivatives of barbiturates functionalized with free carboxylic acids were designed and synthesized. Coupling of 5-cyclopentyl-5-carboxycrotylbarbituric acid via its active ester to an aminofluorescein derivative produced a fluorescent tracer. Conjugation of the 5-cyclopentenyl-5-carboxyethylbarbituric acid via its mixed anhydride to thyroglobulin allowed for subsequent development of a polyclonal antibody which was evaluated for binding in a fluorescence polarization immunoassay format with various barbiturates. The binding studies showed good cross-reactivity of a variety of barbiturates containing both aromatic and aliphatic 5-substituents with the tested antisera. The relationship between the immunogen architecture, the chemical structure of the binding analytes, and the characteristics of the antisera is also presented.

Chemoenzymatic Synthesis of 3‘-O-(Carboxyalkyl)fluorescein Labels

“…These derivatives allow for conjugation of ligands through the 5-or 6-position of the phenyl ring of fluorescein. Additionally, 4′-(aminomethyl)fluorescein has been utilized for the preparation of tracers used in commercially available immunoassays (10,11). This fluorescein derivative allows for conjugation through the xanthinyl ring of fluorescein.…”

Optimization and Immunological Characterization of a Photochemically Coupled Lysergic Acid Diethylamide (LSD) Immunogen

“…Depending on the size of the protein and the distance between it and the hapten, areas surrounding the point of attachment may be obscured from the immune system. This is of particular importance for small molecules such as drugs of abuse, for which immunogen design plays a fundamental role in determining the overall antibody specificity ( − ). As a result, cross-reactivity studies can provide valuable insights into possible structural attachments which have taken place in the immunogen.…”