Crosslinkers cleavable

Figure 4.6 DSS reacts with two amine-containing molecules to form amide bond crosslinks. The cross-bridge is non-cleavable.

Figure 4.7 DST may be used to crosslink amine-containing molecules, forming amide bond linkages. The central diol of the cross-bridge is cleavable by treatment with sodium periodate.

Figure 4.8 BSOCOES reacts with amine-containing molecules to create amide bond crosslinks. The internal sulfone group is cleavable under alkaline conditions.

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... 

The differences within these families of reagents generally relate to the length of the spacer or bridging portion of the molecule. Occasionally, the bridging portion itself is designed to be cleavable by one of a number of methods (Chapter 8). The great majority of homobifunctional, sulfhydryl-reactive crosslinkers mentioned in the literature are not readily available from commercial sources and would have to be synthesized to make use of them. The ones listed in this section are obtainable from Thermo Fisher. 

Reported structures for homobifunctional aryl azides include a biphenyl derivative and a naphthalene derivative (Mikkelsen and Wallach, 1976), a biphenyl derivative containing a central, cleavable disulfide group (Guire, 1976), and a compound containing a central l,3-diamino-2-propanol bridge between phenyl azide rings that are nitrated (Guire, 1976). The only commercially available homobifunctional photoreactive crosslinker is BASED. 

Figure 4.21 BASED can react with molecules after photoactivation to form crosslinks with nucleophilic groups, primarily amines. Exposure of its phenyl azide groups to UV light causes nitrene formation and ring expansion to the dehydroazepine intermediate. This group is highly reactive with amines. The cross-bridge of BASED is cleavable using a disulfide reducing agent.

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.

SADP, N-succinimidyl-(4-azidophenyl)l,3 -dithiopropionate, is a photoreactive heterobifunctional crosslinker that is cleavable by treatment with a disulfide reducing agent (Thermo Fisher). The crosslinker contains an amine-reactive NHS ester and a photoactivatable phenyl azide group, providing specific, directed coupling at one end and nonselective insertion capability at the other end. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

The use of periodate as a cleavage agent does have advantages, however. Unlike the use of cleavable crosslinkers that contain disulfide bonds which require a reductant to break the conjugate, cleavage of diol-containing crosslinks with periodate typically preserves the indigenous disulfide bonds and tertiary structure of proteins and other molecules. As a result, with most proteins bioactivity usually remains unaffected after mild periodate treatment. 

Finally, the small amine-reactive and photoreactive crosslinker, NHS-ASA (Chapter 5, Section 3.1), can be iodinated to provide a non-cleavable radioactive conjugate. 

Other investigators, however, have demonstrated that conjugations of antibody with intact, two-subunit toxins can be done using non-cleavable crosslinkers such as NHS ester-maleimide heterobifunctionals (Chapter 5, Section 1) (Myers et al., 1989). Presumably, the toxin is still able to release the A chain after the antibody has bound to the cell, since the conjugation process does not permanently attach the two toxin subunits together—only the toxin to the antibody. 

A-chain immunotoxins, however, may not be quite as cytotoxic as conjugates formed from intact toxin molecules (Manske et al., 1989). In an alternative approach to A chain use, the intact toxin of two-subunit proteins is directly conjugated to a monoclonal without isolation of the A chain. Conjugation of an antibody with intact A-B chain toxins can be done without a cleavable linker, as long as the A chain can still separate from the B chain once it is internalized. Therefore, it is important to avoid intramolecular crosslinking during the conjugation process which can prevent release of the A-B complex. In addition, since the B chain... 

However, since SMCC forms nonreversible thioether linkages with sulfhydryl groups, it only can be used in the preparation of immunotoxins if intact A-B toxins are employed in the conjugate. In such conjugates, the A chain still have the potential for reductive release from the B-chain subunit after cellular docking and internalization. Immunotoxins prepared with A-chain or single-subunit toxins will not display cytotoxicity if crosslinked with SMCC, since the crosslinker does not create cleavable disulfide bonds upon conjugation. 

The method for the preparation of immunotoxins with SMPB is identical to that used for MBS (above). Since the thioether bonds formed with sulfhydryl-containing molecules are non-cleavable, A-chain isolates or single-chain toxin molecules can not be conjugated with antibodies with retention of cytotoxicity. Only intact A-B toxin molecules may be used with this crosslinker, since the A chain still is capable of being reductively released from the complex. 

Figure 28.5 This PIR compound contains NHS esters at both ends to capture interacting proteins through amide bond formation. It also contains MS cleavable bonds that release a central reporter group, which can be used to identify crosslinked peptides by mass spec.

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