戊二醛化学交联注意事项.doc

Glutaraldehyde Glutaraldehyde is the most popular bis-aldehyde homobifunctional crosslinker in use today. However, a glance at glutaraldehydes 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 linkages (Chapter3, Sections 4.4 and 5.3). This reaction sequence certainly is possible, but other crosslinking reactions also occur. Glutaraldehyde in aqueous solutions can form polymers containing points of unsaturation due to aldol formation (Figure 5.25) (Chapter 15, Section 2.1) (Hardy etal., 1969, 1976;Monsan et al., 1975). Such ,-unsaturated glutaraldehyde polymers are highlyreactive toward nucleophiles, especially primary amines. Reaction with a protein results in alkylation of available amines, forming stable secondary amine link-ages. These glutaraldehyde modified proteins still may react with other amine-containing molecules either through the Schiff base pathway or through addition at other points of unsaturation (Figure 5.26). The proposed reaction mechanism of conjugation using these polymer conjugates may explain the stability of proteins crosslinked by glutaraldehyde that has not been reduced. Schiff base formation alone would not yield stable crosslinked products without reduction. In addition, a number of other potential reactions of glutaraldehyde in aqueous solution also can contribute to its stable crosslinking ability. These include reactions involving hemiacetal rings sometimes combined with aldol formation products, which can couple to amine groups without the formation of Schiff base linkages (see Chapter15, Section 2.1, for an in-depth discussion of these reactions).FIGURE 5.24 Two amine-containing molecules can be crosslinked by formaldehyde through formation of a quaternary ammonium salt with subsequent dehydration to an immonium cation intermediate. This active species then can react with a second amine compound to form stable secondary amine bonds.Crosslinking using glutaraldehyde polymers can be difficult to reproduce and scale up. Since the exact glutaraldehyde state in solution, including its potential polymer size and structure, is difficult to determine, the exact nature of the conjugates formed by this method may be indeterminable as well. The age of a glutaraldehyde solution is another variable, because the older the solution the more polymer could be formed. Fresh glutaraldehyde often will not yield the same results as aged solutions. Some methods to control the glutaraldehyde activation and coupling process have been successfully carried out for the immobilization of affinity ligands (Chapter15, Section 2.1); therefore, it also may be possible to use such methods to better control conjugate formation in solution.A third method of using glutaraldehyde in conjugation reactions is through its ability to react rapidly with hydrazide groups. A molecule containing hydrazide functionalities or modified to contain them (Chapter2,Section 4.5) can be conjugated with another molecule containing either amines or hydrazides. Glutaraldehyde will react with the hydrazide groups to form hydrazone linkages. When two macromolecules are crosslinked in solutions that contain multiple sites of conjugation, the multivalent hydrazone bonds will be strong enough to create a stable conjugate. If a small molecule is involved,however, reduction of the hydrazone with sodium cyanoborohydride is recommended to produce a leak-resistant bond.Glutaraldehyde has been used extensively as a homobifunctional crosslinking reagent, especially for antibodyenzyme conjugations (Avrameas, 1969; Avrameas and Ternynck, 1971) and to produce vaccine immunogens (De Filette etal., 2011; Chapter19, Section 7). To help overcome its tendency to form large-molecular-weight polymers upon crosslinking two proteins, a two-step protocol often is employed. In this regard, one protein first is reacted with glutaraldehyde and purified away from excess reagent. The second protein then is added to effect the conjugate formation. See the introduction to this chapter and Chapter2, Section 1.2 as well as Chapter20, Section 1.2 for additional information on the use of glutaraldehyde and two-step crosslinking procedures.FIGURE 5.25 Glutaraldehyde in aqueous solution ma