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Carmofur, a 5-fluorouracil (5-FU) derivative, was initially developed as an antineoplastic agent to treat colorectal cancer. Through drug repurposing efforts, it has been identified as a potent covalent inhibitor of the main protease of SARS-CoV-2 (Mpro), making it a promising therapeutic agent against COVID-19. However, previous synthetic procedures suffer from low yields or long reaction times. In this study, benchtop 19F nuclear magnetic resonance (NMR) spectroscopy enables the real-time quantitative monitoring and characterization of the synthesis of carmofur by providing kinetic insight. Furthermore, its proton lock capabilities no longer require the use of deuterated solvents and have enabled convenient and rapidly scalable synthesis of our compound. Here, we present the application of benchtop 19F NMR as an efficient method for optimizing the synthesis of carmofur and its future application in the synthesis of related 5-FU analogs.

Benchtop nuclear magnetic resonance (NMR) spectroscopy has enabled the monitoring and optimization of chemical transformations while simultaneously providing kinetic, mechanistic, and structural insight into reaction pathways with quantitative precision. Moreover, benchtop NMR proton lock capabilities further allow for rapid and convenient monitoring of various organic reactions in real-time, as the use of deuterated solvents is not required. The complementary role of 19F NMR-based kinetic monitoring in the fluorination of bioactive compounds serves many benefits in the drug discovery process, since fluorinated motifs additionally improve drug pharmacology. In this study, 19F NMR spectroscopy was utilized to monitor the synthesis of novel trifluorinated analogs of monastrol, a small molecule dihydropyrimidine kinesin-Eg5 inhibitor, and to probe the mechanism of the Biginelli cyclocondensation, a multicomponent reaction used to synthesize dihydropyrimidine and tetrahydropyrimidines through a Bronsted- or Lewis-acid catalyzed cyclocondensation between ethyl acetoacetate, thiourea, and an aryl aldehyde. In the present study, a trifluorinated ketoester serves a dual purpose as being the source of the trifluoromethyl group in our fluorinated dihydropyrimidines and as a spectroscopic handle for real time reaction monitoring and tracking of reactive intermediates by 19F NMR. Further, upon extending this workflow to a diverse array 3- and 4-substituted aryl aldehydes, we were able to derive Hammett linear free energy relationships (LFER) to determine stereoelectronic effects of para- and meta- substituted aryl aldehydes to corresponding reaction rates and mechanistic routes. In addition, we used density functional theory (DFT) calculations to corroborate our experimental results through the thermodynamic values of key intermediates in each mechanism. Finally, these studies cumulate in the synthesis of a novel trifluorinated analog of monastrol and its subsequent biological evaluation in vitro. More broadly, we show an application of benchtop 19F NMR spectroscopy as an analytical tool in the real-time investigation of a mechanistically and chemically complex multicomponent reaction mixture.

Carmofur, a 5-fluorouracil derivative, was initially developed as an antineoplastic agent to treat colorectal cancer. Through drug repurposing efforts, it has been identified as a potent covalent inhibitor of the main protease of SARS-CoV-2 (Mpro), making it a promising therapeutic agent against COVID-19. However, previous synthetic procedures suffer from low yields, or long reaction times. In this study, benchtop 19F nuclear magnetic resonance spectroscopy (NMR) enables the real-time quantitative monitoring and characterization of the synthesis of carmofur by providing kinetic insight. Furthermore, its proton lock capabilities no longer require the use of deuterated solvents, and has enabled convenient, and rapidly scalable synthesis of our compound. Here, we present the application of benchtop 19F NMR as an efficient method for optimizing the synthesis of carmofur and its future application in the synthesis of related 5-FU analogs.

Tigilanol tiglate is a natural product diterpenoid in clinical trials for the treatment of a broad range of cancers. Its unprecedented protein kinase C isoform selectivity make it and its analogues exceptional leads for PKC-related clinical indications, which include human immunodeficiency virus and AIDS eradication, antigen-enhanced cancer immunotherapy, Alzheimer’s disease and multiple sclerosis. Currently, the only source of tigilanol tiglate is a rain forest tree, Fontainea picrosperma, whose limited number and restricted distribution (northeastern Australia) has prompted consideration of designed tree plantations to address supply needs. Here we report a practical laboratory synthesis of tigilanol tiglate that proceeds in 12 steps (12% overall yield, >80% average yield per step) and can be used to sustainably supply tigilanol tiglate and its analogues, the latter otherwise inaccessible from the natural source. The success of this synthesis is based on a unique strategy for the installation of an oxidation pattern common to many biologically active tiglianes, daphnanes and their analogues.

In December of 2019, a novel coronavirus was first identified in Wuhan, China, and has since spread around the world, leaving a largely unsolved biomedical problem in its wake. Upon entry into host cells, the main protease is essential for the replication of viral RNA, which is what allows the virus to replicate inside humans. Inhibition of the main protease has been investigated as a potential strategy for inhibition of the viral replication cycle. Here, we designed a combinatorial library of small molecules and performed high-throughput virtual screening to identify a series of hit compounds that may serve as potential inhibitors of the main protease. In our design of covalent inhibitors of the coronavirus protease, we modeled a library of 361 peptidomimetic Michael acceptor small molecules, which are designed to engage the nucleophilic cysteine residue in the active site of the protease in an irreversible 1,4-conjugate addition. We then employed a variety of computational tools to determine the binding affinity of our designed compounds when bound to the protease active site, where we determined that cationic side chains are potentially beneficial for inhibition of SARS-CoV-2.

The aggregation of the 37-amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β-islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on-path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full-length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full-length IAPP.

Organisms inhabiting the rocky intertidal zone must tolerate both aquatic and terrestrial stresses encountered over the tidal cycle, particularly those that occur during emersion such as desiccation, hypoxia, and thermal stress. We tested whether episodic exposure to elevated body temperatures that are possible in future climate scenarios leads to up-regulation of the antioxidant defense system or, alternatively, to accumulation of oxidative damage. We quantified antioxidant capacities against peroxyl and hydroxyl radicals, catalase enzymatic activity, and lipid peroxidation—a form of oxidative damage—in gill, mantle, and adductor muscle from mussels (Mytilus californianus) exposed to three treatments for 4 weeks: immersed continuously in seawater (15 ± 1 °C), exposed to moderate temperature air (21.8 ± 0.1 °C) for 4 h per day, or exposed to warm air (30 ± 0.1 °C) for 4 h per day. We quantified antioxidants and oxidative damage under both baseline conditions and following a common, acute heat stress challenge. Gill tissue accumulated lipid peroxidation damage during the acute thermal challenge independent of acclimation history, and this tissue expressed the lowest baseline levels of defense against peroxyl radicals that promote lipid peroxidation. There was no up-regulation of measured components of the antioxidant system in any tissue following the acclimation regimes. There was also no evidence for chronic accumulation of lipid oxidative damage. Based on these patterns, we hypothesize that the costs of repairing lipid peroxidation are less than the costs of mounting a constitutive, baseline antioxidant defense against extreme events that tend to be rare and relatively unpredictable in nature.