Fluorescence Spectroscopic Profiling of Compound Libraries

• Published in: Journal of medicinal chemistry Vol. 51; no. 8; pp. 2363 - 2371
• Main Authors: Simeonov, Anton, Jadhav, Ajit, Thomas, Craig J, Wang, Yuhong, Huang, Ruili, Southall, Noel T, Shinn, Paul, Smith, Jeremy, Austin, Christopher P, Auld, Douglas S, Inglese, James
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 24.04.2008
• Subjects: Biological and medical sciences; Chromatography, High Pressure Liquid; Fluorescent Dyes; Medical sciences; Miscellaneous; Molecular Probes; Pharmacology. Drug treatments; Spectrometry, Fluorescence - methods; High throughput screening; Fluorescent compound; Chemical compound library; Fluorescence spectrometry
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm701301m

Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70 000 samples across spectral regions commonly utilized in HTS. The quantitative HTS paradigm was utilized to test each sample at seven or more concentrations over a 4-log range in 1536-well format. Raw fluorescence was compared with fluorophore standards to compute a normalized response as a function of concentration and spectral region. More than 5% of library members were brighter than the equivalent of 10 nM 4-methyl umbelliferone, a common UV-active probe. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, fluorescent impurities, novel fluorescent compounds, and the utilization of fluorescence profiling data are discussed.