Synthesis of polysubstituted azepanes by dearomative ring expansion of nitroarenes.

The synthesis of functionalized nitrogen heterocycles is integral to discovering, manufacturing and evolving high-value materials. The availability of effective strategies for heterocycle synthesis often biases the frequency of specific ring systems over others in the core structures of bioactive leads. For example, while the six- and five-membered piperidine and pyrrolidine are widespread in medicinal chemistry libraries, the seven-membered azepane is essentially absent and this leaves open a substantial area of three-dimensional chemical space. Here we report a strategy to prepare complex azepanes from simple nitroarenes by photochemical dearomative ring expansion centred on the conversion of the nitro group into a singlet nitrene. This process is mediated by blue light, occurs at room temperature and transforms the six-membered benzenoid framework into a seven-membered ring system. A following hydrogenolysis provides the azepanes in just two steps. We have demonstrated the utility of the strategy with the synthesis of several azepane analogues of piperidine drugs.

Structural basis for the simultaneous recognition of NEMO and acceptor ubiquitin by the HOIP NZF1 domain.

Ubiquitination of NEMO by the linear ubiquitin chain assembly complex (LUBAC) is essential for activating the canonical NF-κB signaling pathway. While the NZF1 domain of the HOIP subunit of LUBAC recognizes the NEMO substrate, it is unclear how it cooperates with the catalytic domains in the ubiquitination process. Here, we report a crystal structure of NEMO in complex with HOIP NZF1 and linear diubiquitin chains, in which the two proteins bind to distinct sites on NEMO. Moreover, the NZF1 domain simultaneously interacts with NEMO and Ile44 surface of a proximal ubiquitin from a linear diubiquitin chain, where the C-term tail of the ubiquitin is in the proximity of the NEMO ubiquitination site (Lys285). We further propose a model for the linear ubiquitination of NEMO by HOIP. In the model, NZF1 binds the monoubiquitinated NEMO and recruits the catalytic domains to the ubiquitination site, thereby ensuring site-specific ubiquitination of NEMO.

The Nucleoside/Nucleotide Analogs Tenofovir and Emtricitabine Are Inactive against SARS-CoV-2.

The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19.

Synthesis of Bis-heteroaryls Using Grignard Reagents and Pyridylsulfonium Salts.

Herein are reported ligand-coupling reactions of Grignard reagents with pyridylsulfonium salts. The method has wide functional group tolerance and enables the formation of bis-heterocycle linkages, including 2,4'-, 2,3'-, and 2,2'-bipyridines, as well as pyridines linked to pyrimidines, pyrazines, isoxazoles, and benzothiophenes. The methodology was successfully applied to the synthesis of the natural products caerulomycin A and E.

Don't Ditch the Laptop Just Yet: A Direct Replication of Mueller and Oppenheimer's (2014) Study 1 Plus Mini Meta-Analyses Across Similar Studies.

In this direct replication of Mueller and Oppenheimer's (2014) Study 1, participants watched a lecture while taking notes with a laptop (n = 74) or longhand (n = 68). After a brief distraction and without the opportunity to study, they took a quiz. As in the original study, laptop participants took notes containing more words spoken verbatim by the lecturer and more words overall than did longhand participants. However, laptop participants did not perform better than longhand participants on the quiz. Exploratory meta-analyses of eight similar studies echoed this pattern. In addition, in both the original study and our replication, higher word count was associated with better quiz performance, and higher verbatim overlap was associated with worse quiz performance, but the latter finding was not robust in our replication. Overall, results do not support the idea that longhand note taking improves immediate learning via better encoding of information.

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila.

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents >95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

Synthesis of Pyridylsulfonium Salts and Their Application in the Formation of Functionalized Bipyridines.

An S-selective arylation of pyridylsulfides with good functional group tolerance was developed. To demonstrate synthetic utility, the resulting pyridylsulfonium salts were used in a scalable transition-metal-free coupling protocol, yielding functionalized bipyridines with extensive functional group tolerance. This modular methodology permits selective introduction of functional groups from commercially available pyridyl halides, furnishing symmetrical and unsymmetrical 2,2'- and 2,3'-bipyridines. Iterative application of the methodology enabled the synthesis of a functionalized terpyridine with three different pyridine components.

Structural investigation of 2-naphthyl phenyl ether inhibitors bound to WT and Y181C reverse transcriptase highlights key features of the NNRTI binding site.

Human immunodeficiency virus (HIV)-1 remains as a global health issue that is primarily treated with highly active antiretroviral therapy, a combination of drugs that target the viral life cycle. One class of these drugs are non-nucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral reverse transcriptase (RT). First generation NNRTIs were troubled with poor pharmacological properties and drug resistance, incentivizing the development of improved compounds. One class of developed compounds are the 2-naphthyl phenyl ethers, showing promising efficacy against the Y181C RT mutation. Further biochemical and structural work demonstrated differences in potency against the Y181C mutation and binding mode of the compounds. This work aims to understand the relationship between the binding mode and ability to overcome drug resistance using macromolecular x-ray crystallography. Comparison of 2-naphthyl phenyl ethers bound to Y181C RT reveal that compounds that interact with the invariant W229 are more capable of retaining efficacy against the resistance mutation. Additional modifications to these compounds at the 4-position, computationally designed to compensate for the Y181C mutation, do not demonstrate improved potency. Ultimately, we highlight important considerations for the development of future HIV-1 drugs that are able to combat drug resistance.

Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+ patient.

A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.

Sleep restores place learning to the adenylyl cyclase mutant rutabaga.

Sleep plays an important role in regulating plasticity. In Drosophila, the relationship between sleep and learning and memory has primarily focused on mushroom body dependent operant-learning assays such as aversive phototaxic suppression and courtship conditioning. In this study, sleep was increased in the classic mutant rutabaga (rut2080) and dunce (dnc1) by feeding them the GABA-A agonist gaboxadol (Gab). Performance was evaluated in each mutant in response to social enrichment and place learning, tasks that do not require the mushroom body. Gab-induced sleep did not restore behavioral plasticity to either rut2080 or dnc1 mutants following social enrichment. However, increased sleep restored place learning to rut2080 mutants. These data extend the positive effects of enhanced sleep to place learning and highlight the utility of Gab for elucidating the beneficial effects of sleep on brain functioning.

Decarboxylative Conjunctive Cross-coupling of Vinyl Boronic Esters using Metallaphotoredox Catalysis.

The synthesis of complex alkyl boronic esters through conjunctive cross-coupling of vinyl boronic esters with carboxylic acids and aryl iodides is described. The reaction proceeds under mild metallaphotoredox conditions and involves an unprecedented decarboxylative radical addition/cross-coupling cascade of vinyl boronic esters. Excellent functional-group tolerance is displayed, and application of a range of carboxylic acids, including secondary α-amino acids, and aryl iodides provides efficient access to highly functionalized alkyl boronic esters. The decarboxylative conjunctive cross-coupling was also applied to the synthesis of sedum alkaloids.

Ultrafast core-excited electron dynamics in model crystalline organic semiconductors.

Electron transfer is key to the operation of devices based on molecular (organic) semiconductors. Others have shown that electron transfer in the solid state often proceeds on sub-50 fs timescales, the details of which can be difficult to temporally resolve using pump-probe spectroscopy. A popular technique to measure average time scales for such rapid electron-transfer events is the core-hole clock implementation of resonant Auger electron spectroscopy at a single X-ray absorption edge. This is often done on relatively small molecules with core-excited states that are highly localized. We have used resonant Auger spectroscopy to probe sub-50 fs electron dynamics of two relatively large model organic semiconductors: Cu phthalocyanine (CuPc) along with its fluorinated analog, F16CuPc. We have interrogated electron dynamics simultaneously at N and C K-edges, along with calculations of initial and final states participating in the core-excited states. Our measurements show that the electron dynamics differ substantially across the two absorption edges for a given molecule, and that there are significant differences at a given edge between the two derivatives. X-ray spectroscopy calculations suggest that the extension of π-electron density onto peripheral F atoms in F16CuPc is implicated in the large change in ultrafast electron dynamics upon fluorination. We believe our results have important implications for analysis of core-hole clock measurements on relatively large organic semiconductors.

Influence of conjugation length on ultrafast electronic tunneling in organic semiconductor thin films.

Electron delocalization in conjugated organic molecules is a rate-limiting step in maximizing the charge generation efficiency of next generation photovoltaics and molecular electronics. In particular, ultrafast (<50 fs) delocalization is an important aspect that has been beyond the scope of traditional optical experiments. In this work, we use resonant photoemission spectroscopy to probe electron delocalization timescales as a function of conjugation length by examining an oligothiophene chemical series containing 4-, 5- and 6-mers. We find that above a certain photon energy threshold, the 5-mer, quinquenthiophene, displays the largest ultrafast tunneling rates, roughly three times faster than the 6-mer, sexithiophene. We argue that differences in thin-film molecular packing cannot satisfactorily explain our results, and we speculate that the differences in ultrafast electron dynamics may be a manifestation of the odd/even effect.

Activity and fidelity of human DNA polymerase α depend on primer structure.

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

Few-layer, large-area, 2D covalent organic framework semiconductor thin films.

In this work, we synthesize large-area thin films of a conjugated, imine-based, two-dimensional covalent organic framework at the solution/air interface. Thicknesses between ∼2-200 nm are achieved. Films can be transferred to any desired substrate by lifting from underneath, enabling their use as the semiconducting active layer in field-effect transistors.

Pneumatic switched angle spinning NMR probe with capacitively coupled double saddle coil.

Switched angle spinning (SAS) experiments can be used for generating isotropic-anisotropic correlations in oriented samples in a single experiment. In order for these methods to become widespread, specialized hardware is required. Here we describe the electronic and mechanical design and performance of a double-resonance SAS probe. Unlike many previous SAS probe implementations, the focus here is on systems where the dipolar couplings are partially averaged by molecular motion. This probe has a moving double saddle coil capacitively coupled to the stationary circuit. Angle switching is accomplished by a steam engine-type pneumatic mechanism. The speed and stability of the switching hardware for SAS experiments are demonstrated using spectra of model compounds.