O-Acylisourea intermediates

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. 

The reactions involved in an EDC-mediated conjugation are discussed in Chapter 3, Section 1.1 (Note EDC is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride MW 191.7 and is sometimes referred to as EDAC). The carbodiimide first reacts with available carboxylic groups on either the carrier or hapten to form a highly reactive o-acylisourea intermediate. The activated carboxylic group then can react with a primary amine to form an amide bond, with release of the EDC mediator as a soluble isourea derivative. The reaction is quite efficient with no more than 2 hours required for it to go to completion and form a conjugated immunogen. 

DCC (dicyclohexylcarbodiimide) and the carboxylic acid are able to form an O-acylisourea intermediate, which offers reactivity similar to the corresponding carboxylic acid anhydride ... 

Other effective ways of carboxyl activation involves the use of carboxylphos-phonium [56] and O-acylisourea intermediates [57]. 

EDC-mediated coupling This step is crucial for the success of the immobilization procedure. EDC reacts with the carboxyl groups to form an amine-reactive O-acylisourea intermediate. If this intermediate does not encounter an amine, it will hydrolyze and regenerate the carboxyl group. In the presence of N-hydroxysulfosuccinimide (Sulfo-NHS), efficiency of EDC coupling is increased by the formation of an amine-reactive Sulfo-NHS ester intermediate, which has... 

Carbodiimide method, a procedure for peptide bond formation using carbodi-imides, R-N=C=N-R, such as dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC) and water-soluble carbodi-imides. The carbodiimide reacts in a one-pot procedure with the carboxylate anion of the carboxy component to form a highly reactive O-acylisourea intermediate. The former reacts immediately with the amino function of the amino component to yield the desired peptide derivative and the urea byproduct. Indeed, a more complex mechanism must be taken into consideration. Unwanted side reactions are racemization via the oxazolone mechanism and formation of the unreactive N-acylurea by base-catalyzed acyl migration from the isourea oxygen to nitrogen. The side reactions can be diminished by preparing the O-acylisourea at 0 °C... 

The mechanisms described in the preceding paragraphs are the ones generally proposed for the explanation of racemization, but it is far from certain that other pathways are not involved. For instance it seems to be possible that the repeatedly observed loss of chiral integrity of the activated residue in coupling of peptides with the aid of dicyclohexylcarbodiimide is due to intramolecular proton abstraction by the basic center in the reactive O-acylisourea intermediate ... 

Attack of the carboxyl-component on the O-acylisourea intermediate is facile, unimpeded by the polymer. In fact, the symmetrical anhydride can first be generated in solution and if desired DCU removed prior to the addition of the acylating mixture to the resin-bound amine-component. In this mode of coupling the lifetime of the overly reactive O-acylisourea is significantly reduced with the simultaneous reduction in the extent of side reactions such as N-acyl-urea formation. The symmetrical anhydride approach, considered earlier as too expensive, became with the decrease in price of Boc-amino acids quite popular in recent years. 

Fig. 2.4 Reaction pathways in caibodiimide mediated polyesterification. 1 O-acylisourea intermediate, 2 urea, 3 acid anhydride, 4 N-acylpyridinium intermediate, and 5 TV-acyl urea side product Adapted from Ref. [18]...

Activation of the carboxylic acid moiety of the amino acid is required to be able to react with the Mx-amino group of the growing peptide chain. The first step is the reaction with an electrophile, in some cases in the presence of a base. Carbodiimide-based coupling reagents, such as DCC or DIG (A,A"-diisopropylcarbodiimide), have been used for decades (Figs. 6 and 7). Potential side reactions with carbodiimide-based reagents include the O-to-N rearrangement of the O-acylisourea intermediate and overactivation by formation of the symmetrical anhydride, which can lead to epimer-ization (Fig. 5). These side reactions can be prevented by the addition of auxiliary nucleophiles such as 1-hydroxybenzotriazole... 

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