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The 1,3-dipolar cycloaddition between azides and alkynes provides new means to probe and control biological processes. A major challenge is to achieve high reaction rates with stable reagents. The optimization of alkynyl reagents has relied on two strategies: increasing strain and tuning electronics. We report on the integration of these strategies. A computational analysis suggested that a CH → N aryl substitution in dibenzocyclooctyne (DIBO) could be beneficial. In transition states, the nitrogen of 2-azabenzo-benzocyclooctyne (ABC) engages in an n→π* interaction with the C=O of α-azidoacetamides and forms a hydrogen bond with the N–H of α-diazoacetamides. These dipole-specific interactions act cooperatively with electronic activation of the strained π-bond to increase reactivity. We found that ABC does indeed react more quickly with α-azidoacetamides and α-diazoacetamides than its constitutional isomer, dibenzoazacyclooctyne (DIBAC). ABC and DIBAC have comparable chemical stability in a biomimetic solution. Both ABC and DIBO are accessible in three steps by the alkylidene carbene-mediated ring expansion of commercial cycloheptanones. Our findings enhance the accessibility and utility of 1,3-dipolar cycloadditions and encourage further innovation.

The Diels-Alder reactivity of 4,4-difluoro-3,5-diphenyl-4H-pyrazole (DFP) was investigated experimentally and computationally with endo-bicyclo[6.1.0]non-4-yne (BCN). The computationally predicted rate enhancement from hyperconjugative antiaromaticity induced by fluorination of cyclopentadienes at the 5-position extends to 5-membered heterocyclic dienes containing a saturated center. 4,4-Difluoro-4H-pyrazoles are new electron-deficient dienes with rapid reactivities towards strained alkynes.

Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.

The replacement of carbon with nitrogen can affect the aromaticity of organic rings. Nucleusindependent chemical shift (NICS) calculations at the center of the aromatic π-systems reveal that incorporating nitrogen into 5-membered heteroaromatic dienes has only a small influence on aromaticity. In contrast, each nitrogen incorporated into benzene results in a sequential and substantial loss of aromaticity. The contrasting effects of nitrogen-substitution in 5-membered dienes and benzene are reflected in their Diels-Alder reactivities as dienes. 1,2-Diazine experiences a 99 billion-fold increase in reactivity upon nitrogen-substitution at the 4-and 5positions, whereas a 5-membered heteroaromatic diene, furan, experiences a comparatively incidental 62-fold increase in reactivity upon nitrogen-substitution at the 3-and 4-positions.