Taming the 1,5-sigmatropic shift across protonated spirocyclic 4H-pyrazoles.

The condensation of 1,3-diketones with hydrazine to access 4H-pyrazoles is a well-established synthetic route that travels through a 4H-pyrazol-1-ium intermediate. In the route to a 3,5-diphenyl-4H-pyrazole containing a cyclobutane spirocycle, density functional theory calculations predict and experiments show that the protonated intermediate undergoes a rapid 1,5-sigmatropic shift to form a tetrahydrocyclopenta[c]pyrazole. Replacing the 3,5-diphenyl groups with 2-furanyl groups decreases the calculated rate of the 1,5-sigmatropic shift by 6.2 × 105-fold and enables the isolation of new spirocyclic 4H-pyrazoles for click chemistry.

Computational study of an oxetane 4H-pyrazole as a Diels-Alder diene.

We combine the effects of spirocyclization and hyperconjugation to increase the Diels-Alder reactivity of the 4H-pyrazole scaffold. A density functional theory (DFT) investigation predicts that 4H-pyrazoles containing an oxetane functionality at the saturated center are extremely reactive despite having a relatively high-lying lowest unoccupied molecular orbital (LUMO) energy.

Spirocyclization enhances the Diels-Alder reactivities of geminally substituted cyclopentadienes and 4H-pyrazoles.

The Diels-Alder reactivity of 5-membered dienes is tunable through spirocyclization at the saturated center. As the size of the spirocycle decreases, the Diels-Alder reactivity increases with the cyclobutane spirocycle, spiro[3.4]octa-5,7-diene, being the most reactive. Density functional theory calculations suggest that spiro[3.4]octa-5,7-diene dimerizes 220,000-fold faster than 5,5-dimethylcyclopentadiene and undergoes a Diels-Alder reaction with ethylene 1,200-fold faster than 5,5-dimethylcyclopentadiene. These findings show that spirocyclization is an effective way to enhance the Diels-Alder reactivity of geminally substituted 5-membered dienes.

Bioorthogonal 4H-pyrazole "click" reagents.

4H-Pyrazoles are emerging as useful click reagents. Fluorinating the saturated center enables 4H-pyrazoles to react rapidly as Diels-Alder dienes without a catalyst but compromises the stability of these dienes under physiological conditions. To identify more stable 4H-pyrazoles for bioorthogonal chemistry applications, we investigated the Diels-Alder reactivity and biological stability of three 4-oxo-substituted 4H-pyrazoles. We found that these dienes undergo rapid Diels-Alder reactions with endo-bicyclo[6.1.0]non-4-yne (BCN) while being much more stable to biological nucleophiles than their fluorinated counterparts. We attribute the rapid Diels-Alder reactivity of the optimal oxygen-substituted pyrazole to a combination of antiaromaticity, predistortion, and spirocyclization. Their reactivity and stability suggest that 4-oxo-4H-pyrazoles can be useful bioorthogonal reagents.

Geminal Repulsion Disrupts Diels-Alder Reactions of Geminally Substituted Cyclopentadienes and 4H-Pyrazoles.

We have experimentally and computationally explored the sluggish Diels-Alder reactivities of the geminally substituted 5,5-dimethylcyclopentadiene and 5,5-dimethyl-2,3-diazacyclopentadiene (4,4-dimethyl-4H-pyrazole) scaffolds. We found that geminal dimethylation of 1,2,3,4-tetramethylcyclopentadiene to 1,2,3,4,5,5-hexamethylcyclopentadiene decreases the Diels-Alder reactivity towards maleimide by 954-fold. Quantum mechanical calculations revealed that the decreased Diels-Alder reactivities of gem-dimethyl substituted cyclopentadienes and 2,3-diazacyclopentadienes are not a consequence of unfavorable steric interactions between the diene and dienophile as reported previously, but a consequence of the increased repulsion within the gem-dimethyl group in the transition state. The findings have implications for the use of cyclopentadienes in "click" chemistry.

Acceleration of 1,3-Dipolar Cycloadditions by Integration of Strain and Electronic Tuning.

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.

Synthesis and Diels-Alder Reactivity of 4-Fluoro-4-Methyl-4H-Pyrazoles.

4H-Pyrazoles are emerging scaffolds for "click" chemistry. Late-stage fluorination with Selectfluor® is found to provide a reliable route to 4-fluoro-4-methyl-4H-pyrazoles. 4-Fluoro-4-methyl-3,5-diphenyl-4H-pyrazole (MFP) manifested 7-fold lower Diels-Alder reactivity than did 4,4-difluoro-3,5-diphenyl-4H-pyrazole (DFP), but higher stability in the presence of biological nucleophiles. Calculations indicate that a large decrease in the hyperconjugative antiaromaticity in MFP relative to DFP does not lead to a large loss in Diels-Alder reactivity because the ground-state structure of MFP avoids hyperconjugative antiaromaticity by distorting into an envelope-like conformation like that in the Diels-Alder transition state. This predistortion enhances the reactivity of MFP and offsets the decrease in reactivity from the diminished hyperconjugative antiaromaticity.

Differential Effects of Nitrogen Substitution in 5- and 6-Membered Aromatic Motifs.

Invited for the cover of this issue is the group of Ronald T. Raines at the Massachusetts Institute of Technology. The image depicts the consequence of replacing carbon with nitrogen in aromatic systems, represented by Kekulé's allegorical snake. Read the full text of the article at 10.1002/chem.202000825.

A Chemical Probe for Dehydrobutyrine.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Differential Effects of Nitrogen Substitution in 5- and 6-Membered Aromatic Motifs.

The replacement of carbon with nitrogen can affect the aromaticity of organic rings. Nucleus-independent 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 1011 -fold increase in reactivity upon nitrogen substitution at the 4- and 5-positions, whereas a 5-membered heteroaromatic diene, furan, experiences a comparatively incidental 102 -fold increase in reactivity upon nitrogen substitution at the 3- and 4-positions.

Hyperconjugative Antiaromaticity Activates 4H-Pyrazoles as Inverse-Electron-Demand Diels-Alder Dienes.

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

Selectivity within a Family of Bacterial Phosphothreonine Lyases.

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.