Fluorescence spectroscopic profiling of compound libraries.
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 1,536-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.
Simeonov
Anton
A
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.
Jadhav
Ajit
A
Thomas
Craig J
CJ
Wang
Yuhong
Y
Huang
Ruili
R
Southall
Noel T
NT
Shinn
Paul
P
Smith
Jeremy
J
Austin
Christopher P
CP
Auld
Douglas S
DS
Inglese
James
J
D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D015335:Molecular Probes; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D015335:Molecular Probes