Reactive carbodiimide linkage

Carbodiimides are used to mediate the formation of amide or phosphoramidate linkages between a carboxylate and an amine or a phosphate and an amine, respectively (Hoare and Koshland, 1966 Chu et al., 1986 Ghosh et al., 1990). Regardless of the type of carbodiimide, the reaction proceeds by the formation of an intermediate o-acylisourea that is highly reactive and short-lived in aqueous environments. The attack of an amine nucleophile on the carbonyl group of this ester results in the loss an isourea derivative and formation of an amide bond (see Reactions 11 and 12). The major competing reaction in water is hydrolysis. 

Three main forms of amine-reactive AMCA probes are commonly available. One of them is simply the free acid form of AMCA, which can be used to couple to amine-containing molecules using the carbodiimide reaction (Chapter 3, Section 1.1). The other two are active-ester derivatives of AMCA—the water-insoluble NHS ester and the water-soluble sulfo-NHS ester forms—both of which spontaneously react with amines to create stable amide linkages. All of them react under mild conditions with primary amines in proteins and other molecules to form highly fluorescent derivatives. 

A number of BODIPY derivatives that contain reactive groups able to couple with amine-containing molecules are commonly available. The derivatives either contain a carboxy-late group, which can be reacted with an amine in the presence of a carbodiimide to create an amide bond, or an NHS ester derivative of the carboxylate, which can react directly with amines to form amide linkages. The three discussed in this section are representative of this amine-reactive BODIPY family. The two NHS ester derivatives react under alkaline conditions with primary amines in molecular targets to form stable, highly fluorescent derivatives. The carboxylate derivative can be coupled to an amine using the EDC/sulfo-NHS reaction discussed in Chapter 3, Section 1.2. 

Figure 19.15 The carbodiimide EDC can be used in the presence of sulfo-NHS to create reactive sulfo-NHS ester groups on a carrier protein. Subsequent coupling with an amine-containing hapten can be done to create amide bond linkages.

N-substituted carbodiimides can react with carboxylic acids to form highly reactive, O-acylisourea derivatives that are extremely short-lived (Reaction 11). This active species then can react with a nucleophile such as a primary amine to form an amide bond (Reaction 12) (Williams and Ibrahim, 1981). Other nucleophiles are also reactive. Sulfhydryl groups may attack the active species and form thioester linkages, although these are not as stable as the bond formed with an amine. 

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). 

Chu et al. (1983, 1986) and Ghosh et al. (1990) describe modified carbodiimide protocols using the water-soluble reagent EDC instead of DCC. They also incorporate a second reactive intermediate, a phosphorimidazolide, created from the reaction of the phosphomonoester at the 5 -terminus of DNA with EDC in the presence of imidazole. A reactive phosphorimidazolide will rapidly couple to amine-containing molecules to form a phosphoramidate linkage (Fig. 398). The chemical reaction had been used previously to effect the formation of phosphodiester linkages between short DNA strands (Shabarova et al., 1983). 

Modified polyisocyanates are prepared by incorporating at least one linkage into monomeric polyisocyanates. Such linkages include urethane, carbodiimide, allophanate, biuret, amide, imide, isocyanurate, and oxazolidone. These modifications provide some advantages, e.g., lower vapor pressure, increased viscosity, and controlled reactivity. 

However, more frequently employed has been the commercial polystyrene-supported carbodiimide 5 (0.9-1.4mmolg 1), another bound variant of DCC, although showing similar reactivity. In this supported reagent the tether to the polystyrene backbone has greater activity than the N-methylene linkage present in reagent 2 (R = Cy). 

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