Coupling with Amines

Figure 3.3 EDC may be used in tandem with sulfo-NHS to create an amine-reactive protein derivative containing active ester groups. The activated protein can couple with amine-containing compounds to form amide bond linkages.

Figure 5.21 The reaction sequence of crosslinking with sulfo-SANPAH involves first derivatizing an amine-containing molecule using its NHS ester end to create an amide bond. Exposure to UV light then causes ring expansion to the dehydroazepine derivative, which can couple with amines to form the final conjugate.

Figure 5.24 SADP reacts with amines via its NHS ester end to produce amide bonds. The modified molecule then may be photoactivated to create a nucleophile-reactive dehydroazepine intermediate able to covalently couple with amine-containing compounds.

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. 

The carboxyl groups on the surface may be activated to an NHS ester for coupling with amine-containing biomolecules according to the following protocol, based on the method of Benters et al., 2002. 

The imidazole carbamate group is more stable to hydrolysis in aqueous buffer than the NHS-carbonate group, which is similar in reactivity to an NHS ester. However, this means that the imidazole carbamate also is slower to react and couple with amines. NHS-carbonate reactions usually go to completion within 1-2 hours at room temperature, whereas imidazole carbamates typically require higher pH conditions and overnight incubations to get maximal yield of ligand coupling. 

Figure 14.20 Particles containing reactive epoxy groups can be coupled with amine-, thiol-, or hydroxyl-containing molecules.

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins, using a carbodiimide reaction with EDC. A similar protocol to that previously described for coupling to carboxylate polymer particles may be used. The following protocol is based on the method of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli 0157. 

The periodate-oxidized liposomes may be used immediately to couple with amine-containing molecules such as proteins (see Section 7.6), or they may be stored in a lyophilized state in the presence of sorbitol (Friede et al., 1993) for latter use. 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

With bifunctional NHS ester reagents, one of three modification products can occur with proteins (1) a dead-end linkage wherein one end of the crosslinker has attached to an amine group within a protein and the other end has hydrolyzed and not formed an attachment, (2) an intra-protein crosslink wherein the PIR reagent has been coupled at both ends to amines within the same protein, or (3) an inter-protein crosslink wherein both ends of the PIR reagent have been coupled with amines on two different protein molecules (Figure 28.7). 

Generally the coupling of diazonium salt is carried out under neutral to slightly alkaline conditions. In coupling with amines or phenols, it has been demonstrated that the active species are the diazonium cation, the free amine,... 

Halofuranes also couple with amines, although usually less efficiently than halothiophenes. 2-, and 3-bromofurane were converted into the... 

Another variant of this reaction has also been described51. In this case, carbonyl compounds were coupled with amines and a,[3-unsaturated nitroalkanes to form 1,3,4,5-alkyl substituted pyrroles on AI2O3 as the solid support, Scheme 5.32. After 15 min irradiation, the products were obtained in 71—86% yield. It was found that substitution at the a-position of the nitroalkane was essential for the reaction. While... 

Sodium periodate can be used to oxidize hydroxyl groups on adjacent carbon atoms, forming reactive aldehyde residues suitable for coupling with amine- or hydrazide-... 

---