Selective detection of the carbohydrate-bound state of concanvalin A at the single molecule level

The labeling of molecules with charge-transfer dyes, such as 5-(dimethylamino)-1-napthalenesulfonyl (dansyl) chloride, is a powerful tool for examining the solvent shell of attached substances. This investigation describes the synthesis and application of a new charge transfer label, based on trans-1-[p-(N,N-dimethylamino)phenyl]-trans- 4-(p-nitrophenyl)-1,3-butadiene (NND). Unlike many commonly used fluorophores, the quantum yield of NND decreases over 4 orders of magnitude upon changing from nonpolar to polar environments. In addition, several derivatives of NND undergo little photodecomposition and can be detected at the picomolar level in a confocal fluorescence correlation spectrometer. In conjunction with recent detection of single molecules in solution, this paper describes a method to discriminate between single free and carbohydrate-bound aggregates of the Jack Bean lectin, concanavalin A (Con A). To this end, two derivatives of NND were constructed possessing an additional functional handle. One derivative, alkenyne 4, was efficiently attached to the beta-anomeric position of glucopyranosides. Transients from single aggregates of this fluorophore were detected in solutions which contained both Con A and maltoside 1, and not the corresponding glucoside 2. This result is in agreement with the known affinity of Con A for alpha-glucopyranosides and not beta-glucopyranosides. A full description of the synthesis of these dyes, their solvochromatic properties, and the method used for single aggregate detection is provided herein.