Fluorescence Properties of Benz[f]indole, a Wavelength and Quenching Selective Tryptophan Analog

Abstract

Tryptophan analogues with unique spectral and photophysical properties offer intrinsic fluorescent probes for studying peptide-protein and protein-protein interactions. Two benzannulated indole derivatives, benz-[f]indole and 3-methylbenz[f]indole, were synthesized and their fluorescence was characterized. The absorption and fluorescence emission spectra are red shifted about 75 nm to the red of the spectra of indole and 3-methylindole. INDO/S-CIS computations indicate two nearly degenerate lowest excited singlet states, analogous to the 1 L a and 1 L b transitions of indoles but with almost collinear transition moments. The limiting excitation anisotropy spectra increase and the emission anisotropy spectra decrease with increasing wavelength, probably due to vibronic coupling of the 1 L a state with higher energy B states. Solvent and temperature effects on the wavelength of maximum emission argue that emission occurs from 1 L a in polar environments. The fluorescence quantum yields and lifetimes are high and essentially independent of temperature and solvent isotope. Quantum yields are 0.80 for both compounds. Lifetimes are 19 ns for benz[f]indole and 25 ns for 3-methylbenz[f]indole. The fluorescence is not quenched by most protein functional groups in Stern-Volmer experiments, though it is quenched weakly by strong electron acceptors, such as histidine and cysteine. These results indicate that two nonradiative processes of indoles, solvent quenching and excited-state proton transfer, are not important decay pathways of benzannulated indoles. A third nonradiative process, excited-state electron transfer, occurs in benzannulated indoles under more limited conditions than in indoles.

Two-Step FRET as a Structural Tool

“…Fluorescence decays were measured by time-correlated single photon counting using picosecond laser excitation. A DCM dye laser system was used for 330 nm excitation. , A Ti-sapphire laser system was used for 465 nm excitation with the following modifications . The doubled output was passed through a beam splitter, focused by a concave lens, and used to excite the sample.…”

Attenuation of DNA Replication by HIV-1 Reverse Transcriptase near the Central Termination Sequence

“…The fluorescent guanine analogue 6-MI was substituted for guanosine at position 26 on the template strand of the 25/36-mer across from the incoming nucleotide. A fluorescence quantum yield of 0.10 was measured for 25/36-mer 6-MI in RT reaction buffer at 350 nm excitation wavelength, 25 °C, relative to quinine sulfate ( , ). The relative quantum yield of 2.5 μM 25/36-mer 6-MI at 37 °C decreased ∼20% after overnight preincubation with 5 μM total RT at 4 °C.…”

RNA Polymerase Alters the Mobility of an A-Residue Crucial to Polymerase-Induced Melting of Promoter DNA

“…The (320) ) 4.48 × 10 3 M -1 cm -1 for 2AP in 2AP Fork was calculated from A(320) using the measured DNA concentration. Steady-state fluorescence was measured in the ratio mode on a SLM 8000C photon counting spectrofluorometer as described elsewhere (27). An excitation wavelength of 320 nm was used to avoid exciting the tryptophans in RNAP.…”

“…Fluorescence decays were measured by time-correlated single photon counting using a picosecond DCM dye laser system as described elsewhere (27,29). Decay curves were acquired at 370 nm emission wavelength (16-nm band pass) in 1024 channels of 13-52 ps/channel using a PCA3 multichannel analyzer (Oxford Instruments).…”