Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid.

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

The condensation of 1,3-diketones with hydrazine to access 4-pyrazoles is a well-established synthetic route that travels through a 4-pyrazol-1-ium intermediate. In the route to a 3,5-diphenyl-4-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[]pyrazole. Replacing the 3,5-diphenyl groups with 2-furanyl groups decreases the calculated rate of the 1,5-sigmatropic shift by 6.

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

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

Bioorthogonal 4-pyrazole "click" reagents.

4-Pyrazoles are emerging as useful click reagents. Fluorinating the saturated center enables 4-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 4-pyrazoles for bioorthogonal chemistry applications, we investigated the Diels-Alder reactivity and biological stability of three 4-oxo-substituted 4-pyrazoles.

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

Geminal Repulsion Disrupts Diels-Alder Reactions of Geminally Substituted Cyclopentadienes and 4-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-4-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 -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 -dimethyl group in the transition state.

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.

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.

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

4-Pyrazoles are emerging scaffolds for "click" chemistry. Late-stage fluorination with Selectfluor is found to provide a reliable route to 4-fluoro-4-methyl-4-pyrazoles. 4-Fluoro-4-methyl-3,5-diphenyl-4-pyrazole (MFP) manifested 7-fold lower Diels-Alder reactivity than did 4,4-difluoro-3,5-diphenyl-4-pyrazole (DFP), but higher stability in the presence of biological nucleophiles.

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.

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.

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

The Diels-Alder reactivity of 4,4-difluoro-3,5-diphenyl-4-pyrazole was investigated experimentally and computationally with -bicyclo[6.1.0]non-4-yne.

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.