Carboxyl group conjugation amines

Figure 1.88 The maleimide group of BMPA reacts with a thiol-containing molecule to result in a modification having a terminal carboxylate group. Amine-containing molecules then can be conjugated to the carboxylate using a carbodiimide reaction with EDC.

Figure 9.59 One of the first methods of preparing water-soluble QDs was to use thioacetic acid modification of the nanocrystal surface. This resulted in a negative charge on the surface of each dot that provides like charge repulsion of particles suspended in aqueous solution. The carboxylate group also could be used for conjugation with amine-containing molecules.

Biocytin should not be used in a carbodiimide reaction to modify proteins or other molecules, since it contains both a carboxylate and an amine group. A carbodiimide-mediated reaction, as suggested for D-biotin previously, would cause self-conjugation and polymerization of this reagent. 

Figure 18.25 Amino-PEG -carboxylate compounds contain a primary amine on one end and a carboxylate group on the other end. They can be used to add water-soluble spacer arms to molecules or surfaces. Using an amine-reactive group, the amino-PEG -carboxylate compound can be coupled via an amide bond, thus leaving the carboxylate end free for further conjugation reactions. Avoid the use of single-step EDC conjugation reactions, as this will polymerize the amino-PEGK-carboxylate by reacting with both ends.

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. 

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