Nucleophilic fluorescent derivatizing

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group results in the formation of an amide bond (Figure 9.22). Derivatization of AMCA off its carboxylate group causes no major effects on its fluorescent properties. Thus, proteins and other macromolecules may be labeled with this intensely blue probe and easily detected by fluorescence microscopy and other techniques. 

Extending the utility of fluorescence to various nonfluorophores is achieved via chemical derivatization methods, also termed labeling or tagging methods (Reaction 11.2). Numerous commercial fluorescent tags are available with disparate reactive functional groups. For example, derivatives of fluorescein, fluorescein isothiocyanate (FITC) are reactive toward nucleophiles such as amines and sulfhydryl groups. 

Similarly, the wild type of CCMV could be chemically derivatized using amine-reactive fluorescent anhydrides or via activation of surface-exposed carboxylates and subsequent reaction with fluorophores bearing nucleophilic amine groups. In addition, genetically engineered CCMV with surface-exposed cysteine residues could be used for additional thiol selective attachment of fluorophores and peptides. 

---