Design, Synthesis And Characterization Of FRET Probes Of Albumin

A new homogeneous competitive assay of concentration of protein was proposed based on quenching the fluorescence of tryptophan/tyrosine residues in a protein via Forster-resonance-energy-transfer using a fluorescent probe as the acceptor. A QSAR model was established in order to predict the dissociation constants of FRET probes, and then those compounds with high affinity were designed, synthesized and characterized.The FRET probe is composed of a fluorophore and a protein-specific ligand normally. According with the requirements of FRET probe, three fluorophores were selected. There were naphthylamine, coumarin and dansylamine. Naphthylamine was an ideal fluorophore from evaluating the fluorophore stability, the effects of different solvents and the overlap spectra of fluorophore's excitation spectra and tryptophan's emission spectra. Fatty acid chain were choosed as a specific ligand for it could bind to all domains of human serum albumin.A QSAR model was established in order to screen a FRET probe with high affinity. The dissociation constant of HSA ligands were form -7.63 to-2.49. 20 descriptors of HSA ligands such as logP,MR,CON,and HOMOt were calculated by Chemoffice after geometry optimizing by MM2. The stepwise multiple linear regression were used to obtain a predictive model. Six models were obtained in multiple regression analysis. According to criteria for selecting models, model 9 was the best model with the highest predictive. The importance of independent variables shows by the best model 9:ClogP> DIPY> G> HF> H> Vc. FRET probes were designed to link theα-naphthylamine and a fatty acid by using serine, ethanolamine or ethylene diamine respectively . The model 9 predicted all the designed compounds which showed that the longer of the fatty acid chain, the higher affinity; and three connections had obvious difference. In the article, FRET probes were synthesized withα-naphthylamine and fatty acid chain likes butyric acid, caprylic acid and dodecanoic acid by linking with serine, ethanolamine and ethylene diamine respectively. There were more than 10 compounds to synthesis in order to find the highest affinity one. A Shimadzu RF5301PC fluorospectrometer was used to record the spectra of all the synthesized compounds.NAAC-SER-CA is the only one which could quench the HSA or BSA fluorescence at 340nm and the characteristic fluorescence at 430nm will increase. NAAC-SER and NAAC-EA-CA could be quenching the fluorescence of albumin, but the fluorescence intensity in its specific emission wavelength couldn't be increased. That is to say, fatty acid chain is an essential part of FRET probes.Compared with the NAAC-SER-CA, the complexes of NAAC-Su or NAAC-EDA-Su and albumin had no significant changes in fluorescence spectrum, which indicated that the distance between the carboxyl and the fluorophore was an important effector.The complex of NAAC-SER-DDA and albumin didn't qunchehing the HSA or BSA fluorescence at 340nm compared to NAAC-SER-CA, which suggested that the length of their fatty acid chains have a certain impact.