Coupling nucleophiles, carboxylic groups

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. 

Biotin-hydrazide also may be used to couple with carboxylate-containing molecules. Hydrazidcs can be coupled with carboxylic acid groups by using the carbodiimide reaction (Chapter 3, Section 1.1). The carbodiimide activates a carboxylate to an o-acylisourea intermediate. Biotin-hydrazide can react with this intermediate via nucleophilic addition to form a stable covalent bond. 

A coupling reaction, broadly defined, joins two fragments with the accompanying loss of a small molecule (or ion). In one well-known coupling reaction, the nucleophilic amino group of one amino acid reacts with the electrophilic carboxyl carbon of a second amino acid to form an amide bond with the loss... 

The carboxylate group of a glycine residue is activated by phosphorylation and then coupled to the amino group of phosphoribosylamine. A new amide bond is formed while the amino group of glycine is free to act as a nucleophile in the next step. 

The two reactions most commonly employed for the modification of protein carboxyl groups have been acid-catalyzed esterification (Wilcox 1967), and coupling with nucleophiles mediated by a water-soluble carbodiimide. The latter reaction has been exploited widely in the determination of the carboxyl group content of proteins as well as in studies of carboxyl group function, and would at present appear to be the reaction of choice for these purposes. 

Additional reactions may occur in constitutionally favourable cases Cdomino reactions ), for example, 2-quinolones result from an ordinary Heck coupling, followed by a cyclization step due to nucleophilic attack of the amine at a carboxylic group (eq. (14)) [14e]. Bicyclic nitrogen heterocycles were reported by Buchwald [45]. For a comprehensive review of applications, the reader is referred to the article by de Meijere and Meyer [16 a]. 

These potential limitations, however, have been largely eliminated. Early attempts describe modifications of the metal surface with thin insoluble layers, such as silica with subsequent functionalization via silane compounds [16]. The SAM approach created a tool for convenient introduction of various surface functionalities that can be used for immobilization [6j. Examples include SAMs that are cw-terminated with hydroxyl or carboxyl groups, which can be activated for covalent coupling via nucleophilic reactions. In addition, this modification with epoxy groups leads to activated surfaces that can be directly used for nucleophilic linkage. 

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