Novel 7-(dimethylamino)fluorene-based fluorescent probes and their binding to human serum albumin.
A novel solvatochromic fluorescent molecule, 9,9-dibutyl-7-(dimethylamino)-2-fluorenesulfonate 2 was synthesized from 2-nitrofluorene in moderate yield. The fluorescence spectra of 2 and 7-(dimethylamino)-2-fluorenesulfonate 1 shift to shorter wavelengths as the polarity of the medium decreases. Both 1 and 2 bind to hydrophobic sites of human serum albumin (HSA). The apparent binding constants were determined by fluorescence titration to be 0.37 x 10(6) M(-1) for 1 and 2.2 x 10(6) M(-1) for 2. The energy of the Trp-214 fluorescence of HSA is transferred to the HSA-bound fluorophores with near 100% efficiency. The covalent bonding of acrylodan (AC) to Cys-34 has little effect on the binding affinity of 2 to HSA or fluorescent behavior of HSA-bound 2. Bound 2 also has little effect on the fluorescence of AC, but 2-->AC and Trp-214-->2-->AC resonance energy transfers were observed. Competitive binding between the fluorene compounds and other ligands such as 1-anilino-8-naphthalenesulfonate, aspirin, S-(+)-ibuprofen and phenylbutazone were also studied fluorometrically. The results indicated that the primary binding site of 2 to HSA is site II in domain IIIA, whereas 1 binds to site I in domain IIA, but a different region from the phenylbutazone binding site. Because of its large molar absorptivity, strong fluorescence, sensitivity to its environment, and high binding constant to HSA, 2 can be used successfully in the study of proteins and their binding properties.
Department of Chemistry, Chungnam National University, Daejeon, 305-764, Korea. khkoh@cnu.ac.kr
Kwanghee Koh
KK
Park
Joon Woo
JW
Park
Andrew D
AD
Hamilton
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000042:Absorption; D001667:Binding, Competitive; D032701:Biomimetics; D005449:Fluorenes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014867:Water / Q000737:chemistry
D005449:Fluorenes; D005456:Fluorescent Dyes; D008024:Ligands; D012709:Serum Albumin; D014867:Water; C038518:acrylodan; D015081:2-Naphthylamine