INBRE

a. Objectives: The goal of this project is to use combinatorial synthesis to develop fluorescent chemosensors for the marine toxin saxitoxin (STX) which causes paralytic shellfish poisoning (PSP).

b. Approach: Saxitoxin binds specifically the orphan site 1 on voltage-gated sodium channels with high affinity. The receptor site has been characterized, and includes two characteristic features. One is an array of amino acid side chains that complement structural features of the toxin. These structural features of the side chains facilitate and strengthen binding of the toxin to the receptor site. A second feature is a solvent-excluded pocket in which the amino acid side chains are arrayed. This preorganized feature of toxin receptor sites will be mimicked by design of synthetic receptors at the nanoscale (nanosensors). To optimize the sensitivity and the selectivity of the nanosensor, we will employ combinatorial synthesis techniques to optimize binding in libraries of peptidic host molecules immobilized on solid support (Argogel® solid-phase synthesis beads). By not limiting ourselves to natural amino acids, we will be able to produce short peptide sequences that wrap around toxins and bind them by providing an array of side chains similar to the native receptor. To mimic the solvent-excluded pocket of protein receptor sites, we will incorporate the combinatorially-designed peptide at the core of a dendritic polymer, still on a polystyrene synthesis bead.

The marriage of combinatorial design and dendrimer synthesis on solid support will provide large libraries (up to 100,000 members) of polypeptide hosts inside dendritic polymers, with each individual host molecule attached uniquely to a polystyrene bead. To our knowledge, this is the first time combinatorially designed peptidic hosts have been incorporated into a dendrimer. Qualitative evaluation of toxin binding can be done simply with a fluorescence microscope. Quantitative analysis will be done with a specific host after it has been synthesized in bulk.

c. Expected Results: At present, environmental monitoring for aquatic toxins is most commonly done by mouse bioassay. Alternative methods, such as liquid chromatography coupled with mass spectroscopy (LC-MS) are extraordinarily expensive and not suitable for high-throughput analysis. In order to move away from mouse bioassay, an inexpensive, fast method is needed. We anticipate that this project will identify nanoscale sensors attached to polystyrene beads that can detect toxins using only a hand-held UV lamp and a magnifying glass.

The science behind the design of toxin sensors will lead to further developments as well. These synthetic receptors could also be used to immobilize the toxins. Although beyond the scope of the present work, the same design features we will use for mimicking toxin receptor sites can also be used to mimic enzyme receptor sites. Thus, by using models of enzyme active sites, we anticipate being able to use this methodology to mimic enzyme reactions to produce solid phase catalysts.s.s.

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Updated 10/31/2005

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and the National Center for Research Resources (P20 RR-16460).

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