Monofunctional and Zero -Length Cross -Linking Reagents
From a logical point of view, cross-linking reagents would contain two functional groups, which react with target components, thus linking them together. The two functional groups in the crosslinker could be identical as in homobifunctional reagents (Chapter 5), or different as in heterobifunctional reagents (Chapter 6). However, there are some compounds that do not belong to either of these two classes, but are still able to effect cross-linking of two proteins or other macromolecules. These compounds are generally simple and readily available and may be classiŠed into two types: the monofunctional cross-linkers and the zero-length cross-linkers. Although monofunctional cross-linkers have one reactive group, part of the molecule is incorporated in the Šnal cross-linked product. On the other hand, zero-length reagents induce direct joining of two intrinsic chemical groups of proteins or other molecules without introduction of any extrinsic material. During the cross-linking reaction, atoms are eliminated from the reactants, thus shortening the distance between the two linked moieties. This approach contrasts with other cross-linking reactions in which a spacer is always incorporated between the two cross-linked groups. For this reason, intramolecular cross-linking can only occur between very closely juxtaposed residues in a macromolecular complex. Reagents that catalyze the formation of disulŠde bonds, for example, are zerolength cross-linkers. Other reagents condense carboxyl and primary amino groups to form amide bonds, hydroxyl and carboxyl groups to form esters, thiol and carboxyl groups to form thioesters, and so on. Many of these reagents simply act as activating agents converting one of the components, for example, carboxyl groups, into a reactive species. Examples of these reactions are multitudinous in organic synthesis wherein two molecules are condensed to form a new compound. Carboxylic acids, for instance, are activated to acyl chlorides for the synthesis of esters or amides. In the synthesis of polypeptides, the carboxyl group is activated by carbodiimide to a reactive intermediate that the amino group attacks. However, many of the activation reactions are too harsh to be used with native proteins. They either disrupt the three-dimensional structure of the macromolecule or cause extensive modiŠcation of the biologically active component. Only mild reagents that do not cause denaturation are useful for the cross-linking. This chapter will focus on these compounds. Other reagents that are not suitable for cross-linking proteins, but which have been used to couple macromolecules to solid supports, will be discussed in a later chapter.