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.
Aita
Kazuki
K
Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Temma
Takashi
T
Shimizu
Yoichi
Y
Kuge
Yuji
Y
Seki
Koh-Ichi
K
Saji
Hideo
H
D000818:Animals; D001360:Avidin / Q000378:metabolism; D001692:Biological Transport; D001710:Biotin / Q000378:metabolism; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D007259:Infrared Rays; D008301:Maleimides / Q000737:chemistry; D009354:Neodymium / Q000737:chemistry*; D009942:Organometallic Compounds / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006573:Heterocyclic Compounds, 1-Ring; D008301:Maleimides; D009942:Organometallic Compounds; D001360:Avidin; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; C043592:maleimide; D009354:Neodymium; D001710:Biotin