Cross-bridge conjugates

Figure 4.4 The reaction of DSP with amine-containing molecules yields amide bond crosslinks. The conjugates may be cleaved by reduction of the disulfide bond in the cross-bridge with DTT.

Figure 4.9 EGS reacts with amine-containing molecules to form amide linked conjugates. The ester groups within its cross-bridge are cleavable under alkaline conditions using hydroxylamine.

Figure 5.1 The general design of a heterobifunctional crosslinking agent includes two different reactive groups at either end and an organic cross-bridge of various length and composition. The cross-bridge may be constructed of chemically cleavable components for selective disruption of conjugates.

SMPB contains a hydrophobic cross-bridge and relatively nonpolar ends, which allows the reagent to permeate membrane structures. Due to its water-insolubility, it must be dissolved in an organic solvent prior to adding an aliquot to a reaction mixture. The solvents DMF and DMSO work well for this purpose. A concentrated stock solution prepared in these solvents allows for easy addition of a small amount to a conjugation reaction. Long-term storage in these solvents is not recommended due to slow water pickup and possible hydrolysis of the NHS ester end. 

Thermo Fisher). The small cross-bridge, built from a benzoic acid group, provides crosslinking ability at short intermolecular distances. Reaction of one protein via the NHS ester end of the crosslinker provides a stable derivative that can be incubated with a target molecule and then photolyzed to effect the final conjugation (Figure 5.20). 

Figure 5.23 SAND can be used to modify amine-containing molecules, and then photo-initiate crosslinking to another amine-containing molecule via a ring-expansion process. The conjugates may be disrupted by reduction of the cross-bridge disulfide with DTT.

LC-SPDP (Chapter 5, Section 1.1) is an analog of SPDP containing a hexanoate spacer arm within its internal cross-bridge. The increased length of the extended crosslinker is important in some conjugations to avoid steric problems associated with closely linked macromolecules. However, for the preparation of immunotoxins, no advantages were observed for LC-SPDP over SPDP (Singh et al., 1993). 

Succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), is a heterobifunctional analog of MBS containing an extended cross-bridge (Chapter 5, Section 1.6). The crosslinker has an amine-reactive NHS ester on one end and a sulfhydryl-reactive maleimide group on the other. Conjugates formed using SMPB thus are linked by stable amide and thioether bonds. 

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