Novel 7-(dimethylamino)fluorene-based fluorescent probes and their binding to human serum albumin

Abstract

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.

Synthesis and Structure of Benzotriazolyl Fluorenes

“…The compound 1b was prepared by reacting 1a with n-butyl bromide in DMF using cesium carbonate as a base as described previously. 13 Reduction of nitro group of 1a-b with atmospheric hydrogen gas in the presence of 5% Pd/C at room temperature provided the corresponding 2-aminofluorenes 2a-b in 85 ~ 96% yields. Nucleophilic aromatic substitution of 2,4-dinitrofluorobenzene with 2-aminofluorenes 2a-b in DMSO at room temperature yielded 2-(N-2,4-dinitrophenyl)aminofluorene compounds 3a-b in 90 ~ 97% yields.…”

“…Sulfonation of 5a-b with concentrated sulfuric acid yielded 7-benzotriazol-1-ylfluorene-2-sulfonic acids 6a-b. Their structures were characterized by 1 H NMR, 13 C NMR, and elemental analysis, and the structures of 5b and 6b were further confirmed by single crystal X-ray analysis. Fluorene derivatives and benzotriazole compounds are known to have various useful photophysical and biological properties, respectively, and thus the fluorene derivatives having benzotriazole substituent prepared in this study are expected to exhibit interesting characteristics.…”

“…Alkylation at the 9-position of the fluorene ring can be easily carried out utilizing the acidity of the hydrogens at the 9-position, 5,6,9,[13][14][15][16]21 and introduction of substituents at 2-or 7-position was also effectively accomplished by electrophillic substitution of the fluorene ring. 5,9,[12][13][14][15]21 Further manipulation at 2-or 7-position has been carried out by standard procedures starting from the appropriate fluorene compounds having diversely transformable substituents such as nitro and/or halogen group.…”

“…Alkylation at the 9-position of the fluorene ring can be easily carried out utilizing the acidity of the hydrogens at the 9-position, 5,6,9,[13][14][15][16]21 and introduction of substituents at 2-or 7-position was also effectively accomplished by electrophillic substitution of the fluorene ring. 5,9,[12][13][14][15]21 Further manipulation at 2-or 7-position has been carried out by standard procedures starting from the appropriate fluorene compounds having diversely transformable substituents such as nitro and/or halogen group. In particular, various metal-catalyzed coupling reactions of bromo-or iodo-fluorenes with stannyl compounds (Stille reaction), 1,9 alkenes (Heck reaction), [1][2][3]5,6,15,16 arylboronic acid (Suzuki reaction), 10,18,21 terminal alkynes (Sonogashira reaction), 10 or amines (Buchwald-Hartwig or Ullmann reaction) 5,9,16 provided a variety of alkenyl-, aryl-, alkynyl-, or amino-substituted fluorene derivatives.…”

“…The synthesis and characterization of fluorene derivatives have attracted much attention in recent years because of their interesting properties and potential applications in various fields, such as two-photon absorption dyes, [1][2][3][4][5][6][7][8][9] fluorescent probes, [10][11][12][13][14][15][16][17] and light-emitting materials. [18][19][20][21] It has also been shown that fluorene derivatives exhibit high binding affinity to human serum albumin, 13 β2-integrins, 22 and β-amyloid.…”