br Activatable bioorthogonal reactions reagents br Cycloprop
Activatable bioorthogonal reactions/reagents
Cyclopropenes as bioorthogonal reagents for IEDDA substrates Cyclopropenes have become popular bioorthogonal reagents due to their small size, inertness to biological nucleophiles, ability to be genetically encoded (Yu et al., 2012), and participation in cycloaddition reactions with 1,3-dipoles and IEDDA substrates such as s-tetrazines (Ravasco, Monteiro, & Trindade, 2017), 1,2,4-triazines (Kamber et al., 2015), and o-quinones (Gahtory et al., 2018). Cyclopropenes are also useful synthetic targets due to their importance as synthetic intermediates (Edwards, Rubina, & Rubin, 2016; Rubin, Rubina, & Gevorgyan, 2006; Zhu, Wei, & Shi, 2011), e.g., for polymerization (Elling, Su, & Xia, 2016) or unnatural Firefly Luciferase synthesis (Zhang & Fox, 2006). Over the last decade, cyclopropenes have been appended to various biomolecules like glycans (Späte et al., 2014), lipids (Kaur et al., 2018; Yang, Šečkutė, Cole, & Devaraj, 2012), proteins (Li et al., 2014; Patterson, Jones, & Prescher, 2014), and nucleic acids (Seckute, Yang, & Devaraj, 2013) to reveal their function in cells and whole organisms. Of these biological applications, majority of them utilize tetrazine ligation where cyclopropenes act as dienophiles. Historically, tetrazine ligation was first reported by Carboni and Lindsey. They observed that the characteristic red-violet color of the tetrazine disappears upon heating with olefins or acetylenic compounds (Carboni & Lindsey, 1959) with concomitant evolution of nitrogen gas. Building upon this seminal study, Sauer and coworkers conducted thorough spectrometric studies to measure kinetic rate constants of the tetrazine ligation for a series of dienophiles with different electronic densities, steric constraints, and ring strains. This seminal study provided experimental values of the quantitative observations made by Carboni and Lindsey; it has set the basis for now routinely applied bioorthogonal tetrazine ligation (Thalhammer, Wallfahrer, & Sauer, 1990). Both these studies found the rate of tetrazine ligation to increase with increasing electron density of the alkene dienophiles or with decreasing electron density of the tetrazines. Increasing the ring size of the cyclic dienophiles from three to eight, thereby decreasing the ring strain, decreases the rate of adduct formation. Interestingly, the trans form of the eight-member ring, trans-cyclooctene (fastest dienophile tested) and cyclooctyne do not follow this general trend. Cyclopropene, the most strained ring, is only second to trans-cyclooctene. However, disubstituted 3,3-dimethyl cyclopropene display very diminished reactivity compared to their monosubstituted analog. This decrease in the reactivity is due to steric blocking of the incoming tetrazine. The IEDDA inactive 3,3-disubstituted cyclopropenes are active toward the 1,3-dipole cycloaddition, for example, in photoclick chemistry. Prescher and coworkers exploited this significant difference in reactivity between C3 mono- and di-substituted cyclopropenes for orthogonal labeling of proteins in vitro using a tetrazine and tetrazole respectively (Kamber et al., 2013), and with azides for SPAAC (Patterson, Nazarova, Xie, Kamber, & Prescher, 2012).
Caged cyclopropenes: Unreactive to tetrazines, activated by light or enzymes
Protocols for HPLC assays and syntheses of caged cyclopropenes
The ubiquitin–proteasome system (UPS) is important for biological processes such as protein degradation and protein function regulation. In general, poly-ubiquitinated proteins are degraded by proteasomes, and some proteins acquire new features following ubiquitination. Therefore, ubiquitination is essential for protein homeostasis. However, aberrations in protein ubiquitination are associated with several diseases, including cancer, neurodegenerative disorders, and autoimmune diseases., , , Therefore, small molecule modulators that can control the UPS are potential therapeutic agents for the treatment of such diseases.