Homobifunctional Crosslinkers

The particular crosslinkers discussed in this section are the types most often referred to in the literature or are commercially available. Many other forms of homobifunctional reagents containing almost every conceivable chain length and reactivity can be found mentioned in the scientific literature. 

Carboxylate groups activated with NHS esters are highly reactive toward amine nucleophiles. In the mid-1970s, NHS esters were introduced as reactive ends of homobifunctional crosslinkers (Bragg and Hou, 1975 Fomant and Fairbanks, 1976). Their excellent reactivity at physiological pH quickly established NHS esters as viable alternatives to the imidoesters predominating at the time (Section 2, this chapter). 

To maximize the modification of amines and minimize the effects of hydrolysis, maintain a high concentration of protein or other target molecule. By adjusting the molar ratio of crosslinker to target molecule(s), the level of modification and conjugation may be controlled to create an optimal product. 

DTSSP reportedly has been used to crosslink the extracytoplasmic domain of the anion exchange channel in human erythrocytes (Staros and Kakkad, 1983), for the characterization 

Reported applications of DSS include crosslinking the A and B subunits of ricin (Montesano et al., 1982), studying human somatotropin and the components of the lactogenic-binding sites of rat liver (Caamano et al., 1983), crosslinking CSF-1 to its cell-surface receptor (Morgan 

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Figure 1.24 A two-step protocol using a homobifunctional crosslinking agent offers more control than single-step methods, but still may result in oligomer formation.

Fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinking agents (Chapter 4, Section 4). Their reaction with amines involves nucleophilic displacement of the fluorine atom with the amine derivative, creating a substituted aryl amine bond (Reaction 9). Detection reagents incorporating reactive aryl chemistry include 2,4-dinitrofluorobenzene and trinitrobenzenesulfonate (Eisen et al., 1953). These compounds form... 

Figure 4.1 The general design of a homobifunctional crosslinking agent. The two reactive groups are identical and typically are located at the ends of an organic spacer arm. The length of the spacer may be designed to accommodate the optimal distance between two molecules to be conjugated.

Figure 4.2 Homobifunctional crosslinkers may be used in a two-step process to conjugate two proteins or other molecules. In the first step, one of the two proteins is reacted with the crosslinker in excess to create an active intermediate. After removal of remaining crosslinker, a second protein is added to effect the final conjugate. Two-step reaction schemes somewhat limit the degree of polymerization obtained when using homobifunctional reagents, but can t entirely prevent it.

Dimethyl pimelimidate (DMP) is a homobifunctional crosslinking agent that has imidoester groups on either end (Thermo Fisher). The imidoesters are amine reactive to give stable amidine linkages with target molecules. The 7-atom bridge created by DMP crosslinks is non-cleavable and... 

Dimethyl suberimidate, DMS, is a homobifunctional crosslinking agent-containing amine-reactive imidoester groups on both ends. The compound is reactive toward the s-amine groups... 

Difluorobenzene derivatives are small homobifunctional crosslinkers that react with amine groups. Conjugation using these compounds results in bridges of only about 3 A in length, potentially providing information concerning very close interactions between macromolecules. 

Glutaraldehyde is the most popular b/s-aldchydc homobifunctional crosslinker in use today. Flowever, a glance at glutaraldehyde s structure is not indicative of the complexity of its possible reaction mechanisms. Reactions with proteins and other amine-containing molecules would be expected to proceed through the formation of Schiff bases. Subsequent reduction with sodium cyanoborohydride or another suitable reductant would yield stable secondary amine... 

Unlike the use of homobifunctional crosslinkers, heterobifunctional compounds usually don t have to be used in large excess with amine particles to prevent aggregation. This is due to the fact that only one of the ends of the crosslinker can react with the amines on the particles. 

Homobifunctional crosslinkers containing thiol-reactive maleimides on each end of a PEG spacer are available in several sizes. These compounds are hydrophilic and react with sulfhydr-yls to produce thioether linkages, which are stable under most conditions. The following compounds can be obtained from Thermo Fisher or Quanta BioDesign. 

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