Homobifunctional cross-linkers

Carboxylate groups activated with N-hydroxysuccinimide (NHS) esters are highly reactive toward amine nucleophiles. In the mid-1970s, NHS esters were introduced as reactive ends of homobifunctional cross-linkers (Bragg and Hou, 1975 Lomant and... 

Figure 121 Homobifunctional cross-linkers 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 cross-linker in excess to create an active intermediate. After removal of remaining cross-linker, 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.

NHS- or sulfo-NHS ester-containing homobifunctional cross-linkers react with... 

Difluorobenzene derivatives are small homobifunctional cross-linkers 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 bis-aldehyde homobifunctional cross-linker in use today. However, 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 re-ductant would yield stable secondary amine linkages (Chapter 2, Section 5.3, and Chapter 3, Section 4). This reaction sequence certainly is possible, but other cross-linking reactions also are feasible. 

Glutaraldehyde is among the earliest homobifunctional cross-linkers employed for protein conjugation (Chapter 4, Section 6.2). It reacts with amine groups through several routes, including the formation of Schiff base linkages, which can be reduced... 

Figure 353 The homobifunctional cross-linker DMS may be used to conjugate PE-containing liposomes with proteins via amidine bond formation.

Glutaraldehyde is a homobifunctional cross-linker containing an aldehyde residue at both ends of a 5 carbon chain. Its primary reactivity is toward amine groups, but the reaction may occur by more than one mechanism. As discussed in Chapter 4, Section... 

DSS is a homobifunctional cross-linker containing an amine-reactive NHS ester at both ends (Chapter 4, Section 1.2). Reaction of the reagent in excess with diamine-... 

Figure 408 The homobifunctional cross-linker DSS may be used to conjugate an enzyme to a 5 -diamine modified oligonucleotide. The NHS ester groups on DSS reart with the amines to form amide bonds.

Figure 14.6 Homobifunctional cross-linkers to connect identical functional groups to surfaces.

The noncovalent adsorption of proteins by p.CP is experimentally simple, but suffers from the disadvantage that the attachment can be reversible by rinsing the pattern with certain buffers and detergents or replacement by other proteins in solution. Moreover, the orientation of the deposited protein is not controlled. Delamarche et al. proposed the use of stamps modified with poly(ethylene oxide) silanes.100 The modification was conducted by oxidation of the PDMS stamp and reaction with APTES to yield an amino-functionalized surface. The next step was the reaction with homobifunctional cross-linker BS3 to bind surface amino groups with poly(ethylene glycol) (PEG) chains (Fig. 14.10). 

---