Synthesis of a new NIR fluorescent Nd complex labeling agent

Fluorescent analysis has been widely used in biological, chemical and analytical research. A useful fluorescent labeling agent should include NIR emission, a large Stoke's shift, and good labeling ability without interfering with the pharmacological profile of the labeled compound. Thus, we planned to develop an M-AMF-DOTA(Nd) derivative composed of an NIR fluorescent moiety and a maleimide conjugating moiety as a new NIR fluorescent labeling agent which fulfills these requirements. M-AMF-DOTA(Nd) was synthesized from 4-amino-fluorescein and was conjugated with an avidin molecule (Avidin-AMF-DOTA(Nd)) through Lys-side chains by reaction with 2-iminothiolane. The fluorescent features of M-AMF-DOTA(Nd) and Avidin-AMF-DOTA(Nd) were comparatively evaluated. A binding assay of Avidin-AMF-DOTA(Nd) with D-biotin and a tumor cell-uptake study were performed to estimate the effects of conjugation on the biological and physicochemical features of the protein. M-AMF-DOTA(Nd) was obtained in 22% overall yield. M-AMF-DOTA(Nd) had a typical NIR fluorescence from the Nd ion (880 nm and 900 nm from 488 nm excitation). Avidin-AMF-DOTA(Nd) was easily synthesized and also had typical NIR fluorescence from the Nd ion without loss of fluorescent intensity. The binding affinity of Avidin-AMF-DOTA(Nd) to D-biotin was equivalent to naive avidin. Avidin-AMF-DOTA(Nd) was taken up by tumor cells in the same manner as avidin conjugated with fluorescein isothiocyanate, an established, widely used fluorescent avidin. Results from this study indicate that M-AMF-DOTA(Nd) is a potential labeling agent for routine NIR fluorescent analysis.