Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders.

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Influencing factors of hospital-acquired COVID-19 prevention and control status among emergency support frontline healthcare workers under closed-loop management: a cross-sectional study.

Background: This study aimed to understand the hospital-acquired COVID-19 infection rate and infection prevention and control status of emergency support frontline healthcare workers (ESFHCWs) under closed-loop management, and to explore the related factors affecting hospital-acquired COVID-19 prevention and control status. Methods: The study site was a provincial-level tertiary hospital in the Xinjiang Uygur Autonomous Region specializing in treating COVID-19 patients. ESFHCWs were assigned from different hospitals in Zhejiang Province to provide emergency medical support in this specialized hospital. All ESFHCWs were managed using a closed loop. A self-designed questionnaire was used to estimate basic information, work experience, and the status of infection prevention and control (SIPC). A total of 269 ESFHCWs responded to the questionnaire. A generalized linear regression model was used to estimate the factors influencing SIPC. Results: There were six hospital-acquired COVID-19 cases, with an infection rate of 2.23%. The independent risk factors influencing COVID-19 prevention and control status were work seniority, anxiety disorder, and consumption of gastrointestinal, anti-inflammatory and anti-asthmatic, and hypnotic sedative drugs. Compared with ESFHCWs with more than 10 years of work seniority, ESFHCWs with less than 5 years of work seniority and 5-10 years of work seniority had lower COVID-19 SIPC scores. Among ESFHCWs with anxiety disorder, the SIPC score was significantly lower than that of ESFHCWs without anxiety disorder. The SIPC scores of ESFHCWs taking other medications (gastrointestinal, anti-inflammatory and anti-asthmatic, and hypnotic sedative drugs) were lower than those of ESFHCWs who did not. Conclusion: The closed-loop management method may be effective in reducing the infection rate of hospital-acquired COVID-19 among ESFHCWs. HCWs with less than 10 years of work seniority, anxiety disorder, and other medications (gastrointestinal, anti-inflammatory and anti-asthmatic, and hypnotic sedative drugs) were probably not suitable for participating in emergency assistant actions because of their poor SIPC scores. Further studies are needed to develop the selection criteria for ESFHCWs.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in rapid coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing C. parvum, a parasite of cattle and humans, and C. tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward- genetic analysis of parasite biology and host specificity.

Diversity-oriented synthesis encoded by deoxyoligonucleotides.

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Nickel-Catalyzed, Enantioselective Hydrofluoromethylation of Olefins: Access to Chiral α-Fluoromethylated Amides and Esters.

We herein report the nickel-catalyzed enantioselective hydrofluoromethylation of enamides and enol esters with CH2FI as the fluoromethyl source to enable the diversity-oriented synthesis (DOS) of chiral α-fluoromethylated amides as well as esters with features of wide functional group compatibility as well as excellent enantioselectivity. The synthetic value of this protocol was demonstrated by transformations of the resulted α-fluoromethylated amides to different scaffolds including amine, oxazoline, thiazoline, and α-fluoromethylated tetrahydroquinoline.

Ligand-Enabled ß-Methylene C(sp3 )-H Arylation of Masked Aliphatic Alcohols.

Despite recent advances, reactivity and site-selectivity remain significant obstacles for the practical application of C(sp3 )-H bond functionalization methods. Here, we describe a system that combines a salicylic-aldehyde-derived L,X-type directing group with an electron-deficient 2-pyridone ligand to enable the ß-methylene C(sp3 )-H arylation of aliphatic alcohols, which has not been possible previously. Notably, this protocol is compatible with heterocycles embedded in both alcohol substrates and aryl coupling partners. A site- and stereo-specific annulation of dihydrocholesterol and the synthesis of a key intermediate of englitazone illustrate the practicality of this method.

δ-C-H Mono- and Dihalogenation of Alcohols.

Alkoxy radicals have long been known to enable remote C-H functionalization via 1,5-hydrogen atom abstraction. However, methods for their generation traditionally have relied upon highly oxidizing metals, ultraviolet radiation, or preformed peroxide intermediates, which has prevented the development of many desirable transformations. Herein we report a new bench-stable precursor that decomposes to free alkoxy radicals via a previously unreported single-electron oxidation pathway. This new precursor enables the fluorination and chlorination of remote C-H bonds under exceptionally mild conditions with exceedingly high monoselectivity. Iterative use of this precursor enables the introduction of a second halogen atom, granting access to remote dihalide motifs, including CF2 and CFCl.

Reversing conventional site-selectivity in C(sp3)-H bond activation.

One of the core barriers to developing C-H activation reactions is the ability to distinguish between multiple C-H bonds that are nearly identical in terms of electronic properties and bond strengths. Through recognition of distance and molecular geometry, remote C(sp2)-H bonds have been selectively activated in the presence of proximate ones. Yet achieving such unconventional site selectivity with C(sp3)-H bonds remains a paramount challenge. Here we report a combination of a simple pyruvic acid-derived directing group and a 2-pyridone ligand that enables the preferential activation of the distal γ-C(sp3)-H bond over the proximate ß-C(sp3)-H bonds for a wide range of alcohol-derived substrates. A competition experiment between the five- and six-membered cyclopalladation step, as well as kinetic experiments, demonstrate the feasibility of using geometric strain to reverse the conventional site selectivity in C(sp3)-H activation.

Ligand-accelerated non-directed C-H functionalization of arenes.

The directed activation of carbon-hydrogen bonds (C-H) is important in the development of synthetically useful reactions, owing to the proximity-induced reactivity and selectivity that is enabled by coordinating functional groups. Palladium-catalysed non-directed C-H activation could potentially enable further useful reactions, because it can reach more distant sites and be applied to substrates that do not contain appropriate directing groups; however, its development has faced substantial challenges associated with the lack of sufficiently active palladium catalysts. Currently used palladium catalysts are reactive only with electron-rich arenes, unless an excess of arene is used, which limits synthetic applications. Here we report a 2-pyridone ligand that binds to palladium and accelerates non-directed C-H functionalization with arene as the limiting reagent. This protocol is compatible with a broad range of aromatic substrates and we demonstrate direct functionalization of advanced synthetic intermediates, drug molecules and natural products that cannot be used in excessive quantities. We also developed C-H olefination and carboxylation protocols, demonstrating the applicability of our methodology to other transformations. The site selectivity in these transformations is governed by a combination of steric and electronic effects, with the pyridone ligand enhancing the influence of sterics on the selectivity, thus providing complementary selectivity to directed C-H functionalization.

Enantioselective amine α-functionalization via palladium-catalysed C-H arylation of thioamides.

Saturated aza-heterocycles are highly privileged building blocks that are commonly encountered in bioactive compounds and approved therapeutic agents. These N-heterocycles are also incorporated as chiral auxiliaries and ligands in asymmetric synthesis. As such, the development of methods to functionalize the α-methylene C-H bonds of these systems enantioselectively is of great importance, especially in drug discovery. Currently, enantioselective lithiation with (-)-sparteine followed by Pd(0) catalysed cross-coupling to prepare α-arylated amines is largely limited to pyrrolidines. Here we report a Pd(II)-catalysed enantioselective α-C-H coupling of a wide range of amines, which include ethyl amines, azetidines, pyrrolidines, piperidines, azepanes, indolines and tetrahydroisoquinolines. Chiral phosphoric acids are demonstrated as effective anionic ligands for the enantioselective coupling of methylene C-H bonds with aryl boronic acids. This catalytic reaction not only affords high enantioselectivities, but also provides exclusive regioselectivity in the presence of two methylene groups in different steric environments.

Efficient synthesis of heterocyle-fused ß-naphthylamines via intramolecular addition to a cyano group initiated by nucleopalladation of alkynes.

A palladium(II)-catalyzed efficient synthesis of heterocycle-fused ß-naphthylamines was accomplished via nucleophilic addition of a carbon-palladium bond to the intramolecular cyano group initiated by nucleopalladation (oxypalladation or aminopalladation) of alkynes.

Pd(II)-catalyzed one-step construction of cycloalkane-fused indoles and its application in formal synthesis of (±)-aspidospermidine.

A highly efficient, redox-free Pd(II)-catalyzed tandem cyclization reaction initiated by intramolecular aminopalladation of alkynes followed by nucleophilic addition to nitriles is developed. This method provides a versatile approach for the synthesis of six- to eight-membered ring-fused indoles in one step and has also shown advantages in the formal synthesis of (±)-aspidospermidine.