Synthesis and antiproliferative activity of a tetrahydrofuran analog of FR901464.

FR901464 is a natural product that exhibits antiproliferative activity at single-digit nanomolar concentrations in cancer cells. Its tetrahydropyran-spiroepoxide covalently binds the spliceosome. Through our medicinal chemistry campaign, we serendipitously discovered that a bromoetherification formed a tetrahydrofuran. The tetrahydrofuran analog was three orders of magnitude less potent than the corresponding tetrahydropyran analogs. This study shows the significance of the tetrahydropyran ring that presents the epoxide toward the spliceosome.

Synthesis and Conformational Analysis of FR901464-Based RNA Splicing Modulators and Their Synergism in Drug-Resistant Cancers.

FR901464 is a cytotoxic natural product that binds splicing factor 3B subunit 1 (SF3B1) and PHD finger protein 5A (PHF5A), the components of the human spliceosome. The amide-containing tetrahydropyran ring binds SF3B1, and it remains unclear how the substituents on the ring contribute to the binding. Here, we synthesized meayamycin D, an analogue of FR901464, and three additional analogues to probe the conformation through methyl scanning. We discovered that the amide-containing tetrahydropyran ring assumes only one of the two possible chair conformations and that methylation of the nitrogen distorts the chair form, dramatically reducing cytotoxicity. Meayamycin D induced alternative splicing of MCL-1, showed strong synergism with venetoclax in drug-resistant lung cancer cells, and was cancer-specific over normal cells. Meayamycin D incorporates an alkyl ether and shows a long half-life in mouse plasma. The characteristics of meayamycin D may provide an approach to designing other bioactive L-shaped molecules.

Pre-mRNA splicing-associated diseases and therapies.

Precursor mRNA (pre-mRNA) splicing is an essential step in human gene expression and is carried out by a large macromolecular machine called the spliceosome. Given the spliceosome's role in shaping the cellular transcriptome, it is not surprising that mutations in the splicing machinery can result in a range of human diseases and disorders (spliceosomopathies). This review serves as an introduction into the main features of the pre-mRNA splicing machinery in humans and how changes in the function of its components can lead to diseases ranging from blindness to cancers. Recently, several drugs have been developed that interact directly with this machinery to change splicing outcomes at either the single gene or transcriptome-scale. We discuss the mechanism of action of several drugs that perturb splicing in unique ways. Finally, we speculate on what the future may hold in the emerging area of spliceosomopathies and spliceosome-targeted treatments.

Recent Progress on One-Pot Multisubstrate Screening.

Traditionally, new synthetic reactions have been developed using a model substrate to screen reaction conditions before testing the optimized conditions with a range of more complex substrates. In 1998, Gao and Kagan pooled multiple substrates in one pot to study the generality of an enantioselective method. Although such one-pot multisubstrate screenings may be powerful, few applications have appeared in the literature. With the advancement of various chromatography techniques, it may be time to revisit this underutilized platform. This review article discusses the applications of one-pot multisubstrate screenings as a method for developing new synthetic methods.

Improved Synthesis of the Amine Fragment of FR901464 and Thailanstatins through the Development of a Convenient N-Detosylation Method.

FR901464 and thailanstatins are potent cytotoxic natural products that share an amine-containing tetrahydropyran ring. We previously reported the synthesis of the tetrahydropyran component. Here, we changed the protecting group for the amine from Boc to tosyl, improving yields and the time economy. A highlight of the revised synthetic scheme is the use of lithium, t-butanol, and ethylenediamine in THF (nontraditional Birch reduction conditions) for the N-detosylation.

Scalable Birch reduction with lithium and ethylenediamine in tetrahydrofuran.

The Birch reduction dearomatizes arenes into 1,4-cyclohexadienes. Despite substantial efforts devoted to avoiding ammonia and cryogenic conditions, the traditional, cumbersome, and dangerous procedure remains the standard. The Benkeser reduction with lithium in ethylenediamine converts arenes to a mixture of cyclohexenes and cyclohexanes; this is operationally easier than the Birch reduction but does not afford 1,4-cyclohexadienes. Here, we report a Birch reduction promoted by lithium and ethylenediamine (or analogs) in tetrahydrofuran at ambient temperature. Our method is easy to set up, inexpensive, scalable, rapid, accessible to any chemical laboratory, and capable of reducing both electron-rich and electron-deficient substrates. Our protocol is also compatible with organocuprate chemistry for further functionalization.

Acetal Addition to Electron-Deficient Alkenes with Hydrogen Atom Transfer as a Radical Chain Propagation Step.

We describe a visible-light-promoted addition of a hydrogen atom and an acetal carbon toward various electron-deficient alkenes. 1,3-Dioxolane is converted to its radical species in the presence of persulfate and an iridium catalyst upon visible light irradiation, which then reacts with electron-deficient alkenes. The reaction operates via a radical chain mechanism, a less commonly observed pathway for this class of transformation. Hydrogen atom transfer from 1,3-dioxolane to α-malonyl radicals is corroborated by experimental and density functional theory studies.

Using Ligand-Accelerated Catalysis to Repurpose Fluorogenic Reactions for Platinum or Copper.

The development of a fluorescent probe for a specific metal has required exquisite design, synthesis, and optimization of fluorogenic molecules endowed with chelating moieties with heteroatoms. These probes are generally chelation- or reactivity-based. Catalysis-based fluorescent probes have the potential to be more sensitive; however, catalytic methods with a biocompatible fluorescence turn-on switch are rare. Here, we have exploited ligand-accelerated metal catalysis to repurpose known fluorescent probes for different metals, a new approach in probe development. We used the cleavage of allylic and propargylic ethers as platforms that were previously designed for palladium. After a single experiment that combinatorially examined >800 reactions with two variables (metal and ligand) for each ether, we discovered a platinum- or copper-selective method with the ligand effect of specific phosphines. Both metal-ligand systems were previously unknown and afforded strong signals owing to catalytic turnover. The fluorometric technologies were applied to geological, pharmaceutical, serum, and live cell samples and were used to discover that platinum accumulates in lysosomes in cisplatin-resistant cells in a manner that appears to be independent of copper distribution. The use of ligand-accelerated catalysis may present a new blueprint for engineering metal selectivity in probe development.

Methyl Scanning and Revised Binding Mode of 2-Pralidoxime, an Antidote for Nerve Agent Poisoning.

Organophosphorus nerve agents (OPNAs) inhibit acetylcholinesterase (AChE) and, despite the Chemical Weapons Convention arms control treaty, continue to represent a threat to both military personnel and civilians. 2-Pralidoxime (2-PAM) is currently the only therapeutic countermeasure approved by the United States Food and Drug Administration for treating OPNA poisoning. However, 2-PAM is not centrally active due to its hydrophilicity and resulting poor blood-brain barrier permeability; hence, these deficiencies warrant the development of more hydrophobic analogs. Specifically, gaps exist in previously published structure activity relationship (SAR) studies for 2-PAM, thereby making it difficult to rationally design novel analogs that are concomitantly more permeable and more efficacious. In this study, we methodically performed a methyl scan on the core pyridinium of 2-PAM to identify ring positions that could tolerate both additional steric bulk and hydrophobicity. Subsequently, SAR-guided molecular docking was used to rationalize hydropathically feasible binding modes for 2-PAM and the reported derivatives. Overall, the data presented herein provide new insights that may facilitate the rational design of more efficacious 2-PAM analogs.

Fluorogenic Probe Using a Mislow-Evans Rearrangement for Real-Time Imaging of Hydrogen Peroxide.

Hydrogen peroxide (H2 O2 ) mediates the biology of wound healing, apoptosis, inflammation, etc. H2 O2 has been fluorometrically imaged with protein- or small-molecule-based probes. However, only protein-based probes have afforded temporal insights within seconds. Small-molecule-based electrophilic probes for H2 O2 require many minutes for a sufficient response in biological systems. Here, we report a fluorogenic probe that selectively undergoes a [2,3]-sigmatropic rearrangement (seleno-Mislow-Evans rearrangement) with H2 O2 , followed by acetal hydrolysis, to produce a green fluorescent molecule in seconds. Unlike other electrophilic probes, the current probe acts as a nucleophile. The fast kinetics enabled real-time imaging of H2 O2 produced in endothelial cells in 8 seconds (much earlier than previously shown) and H2 O2 in a zebrafish wound healing model. This work may provide a platform for endogenous H2 O2 detection in real time with chemical probes.

Total Synthesis of Meayamycin B.

Meayamycin B is currently the most potent modulator of the splicing factor 3b subunit 1 and used by dozens of research groups. However, current supply for this natural product analogue is limited because of the lengthy synthetic scheme. Here, we report a more concise, more cost-effective, and greener synthesis of this compound by developing and employing a novel asymmetric reduction of a prochiral enone to afford an allylic alcohol with high enantioselectivity. In addition to this reaction, this synthesis highlights a scalable Mukaiyama aldol reaction, Nicolaou-type epoxide opening reaction, stereoselective Corey-Chaykovsky-type reaction, and a modified Horner-Wadsworth-Emmons Z-selective olefination. We also discuss a Z-E isomerization during the α,ß-unsaturated amide formation. The new synthesis of meayamycin B consists of 11 steps in the longest linear sequence and 24 total steps.

Total Synthesis of the Reported Structure of Stresgenin B Enabled by the Diastereoselective Cyanation of an Oxocarbenium.

We report the first total synthesis of the reported structure of the heat shock protein expression inhibitor stresgenin B. The synthesis features (1) diastereoselective cyanation of an oxocarbenium intermediate en route to the synthetically challenging α-amido dioxolane, (2) Pd-catalyzed hydration of an unstable nitrile, and (3) late-stage Au-catalyzed Meyer-Schuster rearrangement or Ce-mediated Peterson olefination to furnish the exocyclic α,ß-unsaturated ester. Our synthetic endeavors allowed us to conclude that the structure of stresgenin B requires revision.

Noneffervescent Method for Catalysis-Based Palladium Detection with Color or Fluorescence.

Palladium is a highly valuable metal in automobile, chemical, and pharmaceutical industries. The metal is generally quantified by atomic absorption spectrometry or inductively coupled plasma mass spectrometry. These techniques are tedious and require expensive instruments that are operated mostly off site. As cost-effective and user-friendly alternatives to these techniques, we previously reported two practical fluorometric or colorimetric methods to quantify palladium. Both methods rely on the use of NaBH4, which cannot be stored in solution for more than 10 days. Commercially available solutions of NaBH4 are partially or fully degraded to di- or triborohydride species and cannot be used for palladium(0)-catalyzed allylic C-O bond cleavage for quantification purposes. Here, we report a new method that replaces NaBH4 with NH2NH2 for the palladium-catalyzed deallylation of fluorogenic and colorimetric chemodosimeter resorufin allyl ether. This method is slower but as sensitive as the most recent method from our laboratory. The method is selective for palladium and depends on the presence of tri(2-furyl)phosphine as a palladium ligand and NH2NH2 as a palladium-reducing reagent.

Synthesis of Violaceic Acid and Related Compounds through Aryl Triazene.

MPC1001 is a potent anticancer natural product that contains a violaceic acid moiety. Herein we report the total synthesis of the natural product violaceic acid and its derivative. In this approach, a triazene-directed Ullman coupling proved to be highly effective. We converted the triazene to a hydroxy group by means of a palladium-catalyzed reaction. Treatment of the triazene with trifluoroacetic acid generated an arenediazonium ion that produced an aryl radical, leading to the protodediazoniation and a tricyclic product.

Online sensing of palladium in flowing streams.

Rapid palladium (Pd) catalyzed deallylation of an uncoloured reagent within a flowing stream affords a dose dependent colour formation that can be used for convenient online analysis of trace levels of Pd contamination using a modified HPLC instrument. An application to the online sensing of Pd breakthrough from a flow through Pd adsorption cartridge is described. An alternative configuration of the instrumentation allows the rapid (<1 min) and accurate measurement of Pd levels within samples injected via a conventional HPLC autosampler.

Influenza virus mRNA trafficking through host nuclear speckles.

Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

A competitive and reversible deactivation approach to catalysis-based quantitative assays.

Catalysis-based signal amplification makes optical assays highly sensitive and widely useful in chemical and biochemical research. However, assays must be fine-tuned to avoid signal saturation, substrate depletion and nonlinear performance. Furthermore, once stopped, such assays cannot be restarted, limiting the dynamic range to two orders of magnitude with respect to analyte concentrations. In addition, abundant analytes are difficult to quantify under catalytic conditions due to rapid signal saturation. Herein, we report an approach in which a catalytic reaction competes with a concomitant inactivation of the catalyst or consumption of a reagent required for signal generation. As such, signal generation proceeds for a limited time, then autonomously and reversibly stalls. In two catalysis-based assays, we demonstrate restarting autonomously stalled reactions, enabling accurate measurement over five orders of magnitude, including analyte levels above substrate concentration. This indicates that the dynamic range of catalysis-based assays can be significantly broadened through competitive and reversible deactivation.

Comment on "Virtues and limitations of Pittsburgh green for ozone detection" by C. C. Beltrán, E. A. Palmer, B. R. Buckley and F. Iza, Chem. Commun., 2015, 51, 1579.

We previously developed Pittsburgh Green homoallyl ether to quantify trace ozone. Independently, problems were reported when the method was used for excess ozone. Here, we discuss the origin of the reported problems and demonstrate that when this method is used according to our previous report, no problems occur.

Discoveries, target identifications, and biological applications of natural products that inhibit splicing factor 3B subunit 1.

Covering: 1992 to 2015The natural products FR901464, pladienolide, and herboxidiene were discovered as activators of reporter gene systems. Unexpectedly, these compounds target neither transcription nor translation; rather, they target splicing factor 3B subunit 1 of the spliceosome, causing changes in splicing patterns. All of them showed anticancer activity in a low nanomolar range. Since their discovery, these molecules have been used in a variety of biological applications.