Ligation reaction

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. 

Figure 11.22 Azido-sialic acid-containing glycans can be labeled in vivo with biotin-PEG-phosphine using the Staudinger ligation reaction, which forms an amide bond.

Figure 13.4 APTS-modified surfaces may be further derivatized with amine-reactive crosslinkers to create additional surface characteristics and reactivity. Modification with NHS-PEG4-azide forms a hydrophilic PEG spacer terminating in an azido group that can be used in a click chemistry or Staudinger ligation reaction to couple other molecules.

Figure 17.3 Maleimide-modified glass slides (1) can be derivatized using two chemoselective ligation reactions to create biotin modifications. In the first step, alkyne-PEG4-cyclopentadiene linkers (2) are added to the maleimide groups using a Diels-Alder reaction. In the second reaction, an azido-PEG4-biotin compound (3) is reacted with the terminal alkyne on the slide using click chemistry to result in another cycloaddition product, a triazole ring.

Figure 17.12 Azido derivatives of sugars can be used as monomers for glycan and carbohydrate synthesis by cells. Such modifications can be probed using click chemistry or Staudinger ligation reactions.

Figure 17.18 The Staudinger ligation reaction uses a modified phosphine derivative containing an electrophilic group that acts as a trap for the nucleophilic nitrogen in the intermediate aza-ylide. The resultant shift yields an amide bond derivative between the phosphine-containing molecule and the azide-containing molecule.

Figure 17.20 An azido-palmitic acid derivative can be added to cells to obtain palmitoylated proteins that contain an azide group able to participate in the Staudinger ligation reaction. Biotinylation of these post-translationally modified sites then can be done in vivo using a biotin-phosphine reagent.

Another important variation of the Staudinger ligation reaction described above involves the use of cleavable aryl groups on the triphenylphosphine component, which allows for... 

Figure 17.25 The native chemical ligation reaction can be used to form larger peptides from smaller peptides, if one contains a cysteine residue at its N-terminal and the other one contains a thioester on its C-terminal. Reaction of the peptide derivatives gives a native peptide (amide) bond.

Muir et al. (1998) realized that the intein reaction could be used to facilitate a native chemical ligation with a synthetic N-terminal cysteine-containing peptide or cysteine-containing molecule. With the discovery of a mutant intein that could form an intermediate thioester but not go on to complete the splice and ligation reaction (Xu and Perler, 1996 Chong et al.,... 

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

However, if the expressed protein is treated on the affinity support using thiophenol, this also will release the protein and result in a phenylthioester at its C-terminal, which is the reactive intermediate imminendy suitable for native chemical ligation. Treatment of this activated thioester protein with a N-terminal cysteine peptide induces the native chemical ligation reaction and couples the peptide to the expressed protein through an amide bond (Severinov and Muir, 1998) (Figure 17.27). 

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

Figure 18.11 NHS-PEG4-azide can be used to modify an amine-containing molecule to create an amide derivative terminating in azido groups. The azide modifications then can be used in a click chemistry reaction that forms a triazole linkage with an alkyne-containing molecule. Alternatively, the azide derivative can be used in a Staudinger ligation reaction with a phosphine derivative, which results in an amide bond linkage.

Lemieux, G.A., and Bertozzi, C.R. (1998) Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol. 16(12), 506-513. 

A logical extension of the work on phosphoryl cleavage reactions is the study of the reverse, ligation reaction. The principle of microscopic reversibility offers the comforting thought that the requirements for catalysis are basically the same, and Watson-Crick base-pairing provides a simple... 

Templating by oligopeptides is a more complicated proposition than the essentially linear solution available for nucleotide systems. Regular structures can be designed, but conformations typically depend on pH, and template aggregation is potentially more of a problem. Two groups have achieved success in the sort of template-mediated ligation reactions we have discussed for nucleotide systems, and their results illustrate the problems.160,611... 

Ligation Reaction. The nick in the phosphate backbone is repaired by DNA ligase. A similar excision repair mechanism exists in mammalian cells (see, e.g., Cleaver, 1983). 

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