Specific and quantitative labeling of biomolecules using click chemistry

Abstract

Specific and highly efficient fluorescent labeling techniques for biomolecules, especially for proteins, are required for the quantitative analyses of bio-phenomena and for subsequent systems biology. Although expression of exogenous proteins fused with fluorescent tags, such as green fluorescent protein, is the most widely used method for quantitative bio-analysis, the following problems need to be considered carefully: (1) precise stoichiometric control in living cells is difficult, and (2) the bulkiness of the fluorescent tags restricts analysis of the inherent physical and biological properties of the proteins. Therefore, novel techniques to specifically and stoichiometrically label intrinsic proteins or other biomolecules in living cells should be developed. Click chemistry reactions (e.g., Huisgen cycloaddition and Staudinger ligation) are the most promising approaches for this purpose, because these chemical reactions have following advantages: (1) bioorthogonal reactions; (2) mild reaction conditions suitable for fragile biomolecules, cells, and tissues; (3) extremely high reaction ratio; (4) small size of the functional groups for the cross-coupling reactions; (5) stable covalent bonding; and (6) simple metabolic labeling procedures in living cells, using various biomolecular analogs. Diverse quantitative biological studies have been carried out using this technology (e.g., quantification of novel synthesized proteins and observation of post-translational modifications). In this review, I explain the basics of chemical probing with click chemistry, and discuss its recent applications in the field of quantitative biology. Furthermore, I discuss the capability, significance, and future of the chemical probing of proteins, with an emphasis on the use of click chemistry in the field of the quantitative biology.

Development of Activity-Based Chemical Probes for Human Sirtuins

“…39 This reaction can tolerate a broad array of functional groups and has been widely used in interrogating biological events. 40,41 Conjugation of the probe–enzyme complex with azido-dye or azido-biotin would enable the subsequent visualization or enrichment. The general labeling strategy is shown in Scheme 2.…”

Customised nucleic acid libraries for enhanced aptamer selection and performance

“…In the last decade, Cu(I)-catalysed alkyne-azide cycloaddition (CuAAC) (click chemistry) became a widely used technique to functionalise biomolecules with various modifications [31]. The click reaction can be driven in a quantitative manner and is biorthogonal, as the DNA damaging properties of the Cu(I) catalyst have been overcome by the addition of Cu(I)-chelating agents, for example tris(benzyltriazolylmethyl)amine (TBTA) [32].…”

Reactivity of 7‐Azanorbornenes in Bioorthogonal Inverse Electron‐Demand Diels–Alder Reactions

“…[8][9][10] Despite the widespread use of the CuAAC reaction, the toxicity of copper that the reaction rates correlate well with an increase in sp 2 character of the 7-nitrogen atoms: The ease of conversion of the more stable exo conformer into the more reactive endo conformer may lower the activation energy of the first ratedetermining hetero-Diels-Alder step. [8][9][10] Despite the widespread use of the CuAAC reaction, the toxicity of copper that the reaction rates correlate well with an increase in sp 2 character of the 7-nitrogen atoms: The ease of conversion of the more stable exo conformer into the more reactive endo conformer may lower the activation energy of the first ratedetermining hetero-Diels-Alder step.…”