Coupling reactions using carbodiimides

CAUTION Take care when handling DCCI use rubber gloves, protective eye wear and a good fume hood. 

About 10 /imol of the acid or acids in 25 p of the alcohol and 5 fil of pyridine are treated with a slight molar excess of DCCI and shaken gently at intervals over 30-120 minutes at room temperature. N,AT-Dicyclohexylurea precipitates out and is spun down. The supernatant can then be analysed directly by gas chromatography. With solid alcohols, more pyridine and heating at 40-80 °C may be necessary [116]. 

An extract of plasma acids was made by acid extraction with heptane-isopropyl alcohol (3 7). The residue from this extraction, in a reaction vial, was dissolved in 0.1 ml of 0.325 M CDI in freshly distilled ethanol or hydrocarbon-stabilized chloroform. 10% Triethylamine in dry methanol (1 ml) was added. After one minute or more, 3 ml of 1M NaOH saturated with carbon tetrachloride was added, with vortex mixing for 2 minutes. After releasing the pressure in the vial by puncturing the septum, the vial was centrifuged to separate the layers and the lower (chloroform) layer was sampled with a 

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General Procedures for Coupling Reaction Using Carbodiimides... 

Trichloroethyl esters 16 2.6 Coupling reactions using carbodiimides 22... 

Hydrazide-containing compounds also can be coupled to carboxylate groups using a carbodiimide-mediated reaction. Using bifunctional hydrazide reagents, carboxylates can be modified to possess terminal hydrazide groups able to conjugate with other carbonyl compounds (Chapter 4, Section 8). 

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. 

The more activated the ester, the less stable is the compound. All the esters mentioned above can be used as shelf-stable reagents except benzotriazolyl esters, which decompose too readily. In addition to their use as activated forms of the A - a I ko x y ca r bo n y I am i n o acids, the esters derived from hydroxamic acids are implicated as intermediates in coupling reactions in which the A-hydroxy compounds have been added to promote efficient coupling between an acid and a primary or secondary amine (see Section 2.10). It is pertinent to mention that the O-acylisourea generated from carbodiimides (see Section 2.02) is an activated ester but one of nature different than those alluded to above. 

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... 

Amine-reactive biotinylation reagents contain functional groups off biotin s valeric acid side chain that are able to form covalent bonds with primary amines in proteins and other molecules. Two basic types are commonly available NHS esters and car-boxylates. NHS esters spontaneously react with amines to form amide linkages (Chapter 2, Section 1.4). Carboxylate-containing biotin compounds can be coupled to amines via a carbodiimide-mediated reaction using EDC (Chapter 3, Section 1.1). 

Moreover, Voinea et al. attached antibodies against vascular cell adhesion molecule-1 (VCAM-1) overexpressed on activated human endothelial cells on liposomes with the intention of using them as drug carriers [162], A-gluraryl-PE was used as membrane anchor for the antibody coupling via its free amino groups after its activation with carbodiimide. There is no necessity of antibody modification before the coupling reaction. 

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