Bioconjugation groups

Figure 7 Luminescent lanthanides. (Modified from Reference 3.) (a) Structure of the LanthaScreen Tb probe from Invitrogen. The bioconjugation group is typically a NHS ester, isothiocyanate, or maleimide group, (b) The unique sharp emission profile of the LanthaScreen Tb probe Xex = 343 nm.

Nagasaki Y, Iijima M, Kato M, Kataoka K (1995) Primary amino-terminal heterobifunctional poly(ethylene oxide). Facile synthesis of poly(ethylene oxide) with a primary amino group at one end and a hydroxyl group at the other end. Bioconjug Chem 6 702-704... 

Figure 1.6 The ionizable amino acids possess some of the most important side-chain functional groups for bioconjugate applications. The C- and N-terminal of each polypeptide chain also is included in this group.

Figure 1.22 The solvent accessibility of lysine residues in the Fc region of an antibody is illustrated by highlighting the lysine groups in solid gray. Some lysine e-amine groups are extremely accessible to conjugation, while others are only partially exposed, making them difficult to modify in bioconjugation reactions.

Acylation reactions can be done at the nucleophilic sites on pyrimidines using activated forms of carboxylic acids. Acylation of functional groups in nucleotides typically is used for protection during synthesis (Reese, 1973). However, for bioconjugate applications, the reactivity of native groups on pyrimidines is not as great as that obtained using an amine-terminal spacer derivative, such as those described in Chapter 27, Section 2.1. Yields and reaction rates are typically low for direct acylation or alkylation of pyrimidine bases, especially in aqueous environments. 

Figure 2.1 Three types of fluorophenyl esters have been used for coupling to amine-containing molecules. The PFP and TFP esters are relatively hydrophobic and typically have better stability toward hydrolysis in aqueous solution than NHS esters. The STP ester is water-soluble due to the negatively charged sulfonate group, and it provides better solubility to associated crosslinkers or bioconjugation reagents similar to that of a sulfo-NHS ester group.

Chemical groups that specifically react with carboxylic acids are limited in variety. In aqueous solutions, the carboxylate functionality displays rather low nucleophilicity. For this reason, it is unreactive with the great majority of bioconjugate reagents which couple through a nucleophilic addition process. 

The following sections briefly describe three cycloaddition reactions that can be used to form bioconjugates. These reactions represent highly specific reactant pairs that have a chemoselec-tive nature, meaning they mainly react with each other and not other functional groups, such as those found on biomolecules. For a complete discussion of chemoselective ligation reactions, see Chapter 17. 

Typically, the boronic acid group is part of an aminophenyl boronic acid derivative, and this group has been used for bioconjugation and affinity chromatography purposes (Burnett et al., 1980 O Shannessy and Quarles, 1987). A common partner for a phenyl boronic acid group in bioconjugation is the salicylhydroxamic acid (SHA) group (Chapter 17, Section 3) (Reaction 59). 

Click chemistry refers to the reaction between an azido functional group and an alkyne to form a [3 + 2] cycloaddition product, a 5-membered triazole ring. This reaction has been used for many years in organic synthesis to form heterocyclic rings. Normally, the click reaction requires high temperatures, and this was the main reason that it was not used as a bioconjugation tool. However, it was discovered that in aqueous solutions and in the presence of Cu(I), the reaction kinetics are dramatically accelerated to provide high yields even at room temperature and ambient pressures (Rostovtsev et al., 2002 Tornoe et al., 2002 Sharpless et al., 2005). 

The advantage of the click reaction for bioconjugation is that the reactant pair is not reactive with any other functional group encountered in biological systems. This property of... 

Figure 7.8 The pendent groups available on the surface of dendrimer molecules are highly varied. Some groups provide functional or reactive groups for bioconjugation, while other groups create unique solubility characteristics for the dendrimer.

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