Fluorescent dyes that operate in the long visible / near infra-red (NIR) region of the electromagnetic spectrum are excellent reporter components of molecular probes, fluorescent ligands, enzyme substrates, imaging contrast agents and general tracers. In existing applications of commonly used short and medium wavelength dyes such as coumarins, fluoresceins and rhodamines, assay or tissue components can severely interfere with the fluorescence properties of tracers containing these dyes. For example, in cytochrome P450 (CYP) assays, the enzyme itself can completely quench the reporting signal of such dyes. Longer wavelength / NIR dyes are in very short supply at the present time and those that are commercially available are very expensive or are only available under restrictive license agreements. The main focus of this work is to discover novel, long wavelength near-infrared dyes that are designed for use in high throughput screening (HTS) fluorescent polarization (FP) and fluorescence resonance energy transfer (FRET) biological assays for new drug and agrochemical discovery research. The dyes should also find uses in biomedical in vivo imaging, immunofluorescence microscopy, and fluorescence activated cell sorting and counting. Novel synthetic chemistry pathways will be developed that will allow multi-gram scale synthesis of the dyes in high purity that will result in new products that will be more widely available to the research and scientific community at a lower cost than existing dyes which generally have poorly designed chemical synthetic pathways. There are five specific aims in phase I: 1. Design and Synthesis of a Solution Phase Compound Library of 72 Novel Extended Sulforhodamine Dyes 2. Parallel Purification by Thin Layer Chromatography and Characterization of the Novel Dyes 3. Photophysical Characterization of the New Dyes 4. Assessment of the Performance of the New Dyes in a Model Fluorescence Polarization (FP) Study 5. Assessment of the Performance of the New Dyes in a Fluorescence Resonance Energy Transfer (FRET) Study. A successful phase I study will be followed by a phase II study that involves scale up syntheses and purification of dyes with commercial potential. The dyes will also be tested as components for in vivo optical imaging and a variety of FP and FRET HTS assay kits will be assembled containing the new dyes.
