The utility of alkyl amine modified fluorescent dyes is described in the Molecular Probes Handbook (9th Edition, Section 3.3 Derivitization Reagents for Carboxylic Acids and Glutamine). They offer a limited number of dyes for sale and the invention described here has the same inherent advantages. The novel molecular linker structures described here simplify single isomer synthesis and provide dye analogs with increased fluorescent brightness. Certain analogs have improved dye properties due to lower pKa and large Stokes shift.
The carboxylic acids of water-soluble biopolymers such as proteins can be coupled to hydrazines, hydroxylamines and amines in aqueous solution using water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). Including N-hydroxysulfosuccinimide in the reaction mixture has been shown to improve the coupling efficiency of EDAC-mediated protein-carboxylic acid conjugations. To reduce intra- and inter-protein coupling to lysine residues which is a common side reaction, carbodiimide-mediated coupling should be performed in a concentrated protein solution at a low pH, using a large excess of the nucleophile.
Peptide synthesis research has led to the development of numerous methods for coupling carboxylic acids to amines in organic solution. One such method involves the conversion of carboxylic acids to succinimidyl esters or mixed anhydrides. Dicyclohexylcarbodiimide and diisopropylcarbodiimide are widely used to promote amide formation in organic solution. Another recommended derivatization method for coupling organic solvent-soluble carboxylic acids, including peptides, to aliphatic amines without racemization is the combination of 2,2′-dipyridyldisulfide and triphenylphosphine.
Molecular Probes (Invitrogen) provides a wide selection of carboxylic acid-reactive reagents including several different Dapoxyl, ALEXA FLUOR, BODIPY, fluorescein, OREGON GREEN, rhodamine, TEXAS RED and QSY Hydrazine Derivatives, Hydroxylamine Derivatives and Amine Derivatives, all of which are particularly useful for synthesizing drug analogs and as probes for fluorescence polarization immunoassays. These probes all require a coupling agent such as a carbodiimide to react with carboxylic acids; they do not spontaneously react with carboxylic acids in solution. They do, however, react spontaneously with the common amine-reactive functional groups including succinimidyl esters and isothiocyanates.
A transglutaminase-catalyzed transamidation reaction of glutamine residues in some proteins and peptides enables their selective modification by amine-containing probes. This unique method for selective protein modification requires formation of a complex consisting of the glutamine residue, the aliphatic amine probe and the enzyme. It has been found that a short aliphatic spacer in the amine probe enhances the reaction. Although dansyl cadaverine D113 has been probably the most widely used reagent, ALEXA FLUOR cadaverines, OREGON GREEN 488 cadaverine, fluorescein cadaverine, tetramethylrhodamine cadaverine, TEXAS RED cadaverine and BODIPY TR cadaverine are among the most fluorescent transglutaminase substrates available. The intrinsic transglutaminase activity in sea urchin eggs has been used to covalently incorporate dansyl cadaverine during embryonic development.
Transamidation of cell-surface glutamine residues by the combination of a transglutaminase enzyme and a fluorescent or biotinylated aliphatic amine can form stable amides. Impermeability of the enzyme restricts this reaction to a limited number of proteins on the cell surface. This technique was used to selectively label erythrocyte band 3 protein with dansyl cadaverine and proteins of the extracellular matrix with fluorescein cadaverine.
Transglutaminase-mediated labeling of a protein using dansyl cadaverine.