The Merger of Benzophenone HAT Photocatalysis and Silyl Radical-Induced XAT Enables Both Nickel-Catalyzed Cross-Electrophile Coupling and 1,2-Dicarbofunctionalization of Olefins.

A strategy for both cross-electrophile coupling and 1,2-dicarbofunctionalization of olefins has been developed. Carbon-centered radicals are generated from alkyl bromides by merging benzophenone hydrogen atom transfer (HAT) photocatalysis and silyl radical-induced halogen atom transfer (XAT) and are subsequently intercepted by a nickel catalyst to forge the targeted C(sp3)-C(sp2) and C(sp3)-C(sp3) bonds. The mild protocol is fast and scalable using flow technology, displays broad functional group tolerance, and is amenable to a wide variety of medicinally relevant moieties. Mechanistic investigations reveal that the ketone catalyst, upon photoexcitation, is responsible for the direct activation of the silicon-based XAT reagent (HAT-mediated XAT) that furnishes the targeted alkyl radical and is ultimately involved in the turnover of the nickel catalytic cycle.

Accelerated and Scalable C(sp3)-H Amination via Decatungstate Photocatalysis Using a Flow Photoreactor Equipped with High-Intensity LEDs.

Carbon-nitrogen bonds are ubiquitous in biologically active compounds, prompting synthetic chemists to design various methodologies for their preparation. Arguably, the ideal synthetic approach is to be able to directly convert omnipresent C-H bonds in organic molecules, enabling even late-stage functionalization of complex organic scaffolds. While this approach has been thoroughly investigated for C(sp2)-H bonds, only few examples have been reported for the direct amination of aliphatic C(sp3)-H bonds. Herein, we report the use of a newly developed flow photoreactor equipped with high intensity chip-on-board LED technology (144 W optical power) to trigger the regioselective and scalable C(sp3)-H amination via decatungstate photocatalysis. This high-intensity reactor platform enables simultaneously fast results gathering and scalability in a single device, thus bridging the gap between academic discovery (mmol scale) and industrial production (>2 kg/day productivity). The photocatalytic transformation is amenable to the conversion of both activated and nonactivated hydrocarbons, leading to protected hydrazine products by reaction with azodicarboxylates. We further validated the robustness of our manifold by designing telescoped flow approaches for the synthesis of pyrazoles, phthalazinones and free amines.

Decatungstate-Mediated C(sp3 )-H Heteroarylation via Radical-Polar Crossover in Batch and Flow.

Photocatalytic hydrogen atom transfer is a very powerful strategy for the regioselective C(sp3 )-H functionalization of organic molecules. Herein, we report on the unprecedented combination of decatungstate hydrogen atom transfer photocatalysis with the oxidative radical-polar crossover concept to access the direct net-oxidative C(sp3 )-H heteroarylation. The present methodology demonstrates a high functional group tolerance (40 examples) and is scalable when using continuous-flow reactor technology. The developed protocol is also amenable to the late-stage functionalization of biologically relevant molecules such as stanozolol, (-)-ambroxide, podophyllotoxin, and dideoxyribose.

Dual electrocatalysis enables enantioselective hydrocyanation of conjugated alkenes.

Chiral nitriles and their derivatives are prevalent in pharmaceuticals and bioactive compounds. Enantioselective alkene hydrocyanation represents a convenient and efficient approach for synthesizing these molecules. However, a generally applicable method featuring a broad substrate scope and high functional group tolerance remains elusive. Here, we address this long-standing synthetic problem using dual electrocatalysis. Using this strategy, we leverage electrochemistry to seamlessly combine two canonical radical reactions-cobalt-mediated hydrogen-atom transfer and copper-promoted radical cyanation-to accomplish highly enantioselective hydrocyanation without the need for stoichiometric oxidants. We also harness electrochemistry's unique feature of precise potential control to optimize the chemoselectivity of challenging substrates. Computational analysis uncovers the origin of enantio-induction, for which the chiral catalyst imparts a combination of attractive and repulsive non-covalent interactions to direct the enantio-determining C-CN bond formation. This work demonstrates the power of electrochemistry in accessing new chemical space and providing solutions to pertinent challenges in synthetic chemistry.

In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in many shellfish species. Azaspiracid poisoning caused by AZA-contaminated seafood consumption is primarily manifested by diarrhea in humans. To protect human health, AZA-1, AZA-2 and AZA-3 content in seafood has been regulated by food safety authorities in many countries. Recently AZAs have been reported as a low/moderate hERG channel blockers. Furthermore AZA-2 has been related to arrhythmia appearance in rats, suggesting potential heart toxicity. In this study AZA-2 in vitro effects on hERG channel after chronic exposure are analyzed to further explore potential cardiotoxicity. The amount of hERG channel in the plasma membrane, hERG channel trafficking and hERG currents were evaluated up to 12 h of toxin exposure. In these conditions AZA-2 caused an increase of hERG levels in the plasma membrane, probably related to hERG retrograde trafficking impairment. Although this alteration did not translate into an increase of hERG channel-related current, more studies will be necessary to understand its mechanism and to know what consequences could have in vivo. These findings suggest that azaspiracids might have chronic cardiotoxicity related to hERG channel trafficking and they should not be overlooked when evaluating the threat to human health.

In vivo arrhythmogenicity of the marine biotoxin azaspiracid-2 in rats.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in several shellfish species. Azaspiracid poisoning episodes have been described in humans due to ingestion of AZA-contaminated seafood. Therefore, the contents of AZA-1, AZA-2 and AZA-3, the best-known analogs of the group, in shellfish destined to human consumption have been regulated by food safety authorities of many countries to protect human health. In vivo and in vitro toxicological studies have described effects of AZAs at different cellular levels and on several organs, however, AZA target remains unknown. Very recently, AZAs have been demonstrated to block the hERG cardiac potassium channel. In this study, we explored the potential cardiotoxicity of AZA-2 in vivo. The effects of AZA-2 on rat electrocardiogram (ECG) and cardiac biomarkers were evaluated for cardiotoxicity signs besides corroborating the hERG-blocking activity of AZA-2. Our results demonstrated that AZA-2 does not induce QT interval prolongation on rat ECGs in vivo, in spite of being an in vitro blocker of the hERG cardiac potassium channel. However, AZA-2 alters the heart electrical activity causing prolongation of PR intervals and the appearance of arrhythmias. More studies will be needed to clarify the mechanism by which AZA-2 causes these ECG alterations; however, the potential cardiotoxicity of AZAs demonstrated in this in vivo study should be taken into consideration when evaluating the possible threat that these toxins pose to human health, mainly for individuals with pre-existing cardiovascular disease when regulated toxin limits are exceeded.

Microsphere-based immunoassay for the detection of azaspiracids.

Azaspiracids (AZAs) are a group of lipophilic toxins discovered in mussels from Ireland in 1995 following a human poisoning incident. Nowadays the regulatory limit for AZAs in many countries is set at 160 µg of azaspiracid equivalents per kilogram of shellfish meat. In this work a microsphere-based immunoassay has been developed for the detection of AZAs using a Luminex system. This method is based on the competition between AZA-2 immobilized onto the surface of microspheres and free AZAs for the interaction with a monoclonal anti-azaspiracid antibody (mAb 8F4). In this inhibition immunoassay the amount of mAb 8F4 bound to AZA-2 microspheres was quantified using a phycoerythrin-labeled anti-mouse antibody, and the fluorescence was measured with a Luminex analyzer. Simple acetate/methanol or methanol extractions yielded final extracts with no matrix interferences and adequate recovery rates of 86.5 and 75.8%, respectively. In summary, this work presents a sensitive and easily performed screening method capable of detecting AZAs at concentrations below the range of the European regulatory limit using a microsphere/flow cytometry system.

Rhodium-catalyzed and zinc(II)-triflate-promoted asymmetric hydrogenation of tetrasubstituted α,ß-unsaturated ketones.

The asymmetric hydrogenation of tetrasubstituted α,ß-unsaturated ketones has been accomplished using an in situ formed rhodium-Josiphos catalyst. The reaction is enhanced by addition of catalytic zinc(II) triflate, which significantly improves turnover frequency while suppressing epimerization of the products.

Involvement of caspase activation in azaspiracid-induced neurotoxicity in neocortical neurons.

Azaspiracids (AZAs) are a novel group of marine phycotoxins that have been associated with severe human intoxication. We found that AZA-1 exposure increased lactate dehydrogense (LDH) efflux in murine neocortical neurons. AZA-1 also produced nuclear condensation and stimulated caspase-3 activity with an half maximal effective concentration (EC(50)) value of 25.8 nM. These data indicate that AZA-1 triggers neuronal death in neocortical neurons by both necrotic and apoptotic mechanisms. An evaluation of the structure-activity relationships of AZA analogs on LDH efflux and caspase-3 activation demonstrated that the full structure of AZAs was required to produce necrotic or apoptotic cell death. The similar potencies of AZA-1 to stimulate LDH efflux and caspase-3 activation and the parallel structure-activity relationships of azaspiracid analogs in the two assays are consistent with a common molecular target for both responses. To explore the molecular mechanism for AZA-1-induced neurotoxicity, we assessed the influence of AZA-1 on Ca(2+) homeostasis. AZA-1 suppressed spontaneous Ca(2+) oscillations (EC(50) = 445 nM) in neocortical neurons. A distinct structure-activity profile was found for inhibition of Ca(2+) oscillations where both the full structure as well as analogs containing only the FGHI domain attached to a phenyl glycine methyl ester moiety were potent inhibitors. The molecular targets for inhibition of spontaneous Ca(2+) oscillations and neurotoxicity may therefore differ. The caspase protease inhibitor Z-VAD-FMK produced a complete elimination of AZA-1-induced LDH efflux and nuclear condensation in neocortical neurons. Although the molecular target for AZA-induced neurotoxicity remains to be established, these results demonstrate that the observed neurotoxicity is dependent on a caspase signaling pathway.

Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-Jun-N-terminal kinase activation in cultured neurons.

Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro; however, it did not modify membrane potential in mature neurons. Furthermore, in immature cells, AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50mM K(+). Preincubation of the neurons with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride, or ouabain before addition of AZA-1 decreased the AZA-1-induced neurotoxicity and the increase in phosphorylated c-Jun-N-terminal kinase (JNK) caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.

Monoclonal antibodies with orthogonal azaspiracid epitopes.

Azaspiracid antibodies: Immunization of azaspiracid immunoconjugates has elicited monoclonal antibodies with distinct epitopes on the marine toxin; this will open the way toward azaspiracid diagnostics and the detection of contaminated shellfish before they can enter the food supply.

The continuing saga of the marine polyether biotoxins.

The unprecedented structure of the marine natural product brevetoxin B was elucidated by the research group of Nakanishi and Clardy in 1981. The ladderlike molecular architecture of this fused polyether molecule, its potent toxicity, and fascinating voltage-sensitive sodium channel based mechanism of action immediately captured the imagination of synthetic chemists. Synthetic endeavors resulted in numerous new methods and strategies for the construction of cyclic ethers, and culminated in several impressive total syntheses of this molecule and some of its equally challenging siblings. Of the marine polyethers, maitotoxin is not only the most complex and most toxic of the class, but is also the largest nonpolymeric natural product known to date. This Review begins with a brief history of the isolation of these biotoxins and highlights their biological properties and mechanism of action. Chemical syntheses are then described, with particular emphasis on new methods developed and applied to the total syntheses. The Review ends with a discussion of the, as yet unfinished, story of maitotoxin, and projects into the future of this area of research.

Azaspiracid substituent at C1 is relevant to in vitro toxicity.

The azaspiracids are a group of marine toxins recently described that currently includes 20 analogues. Not much is known about their mechanism of action, although effects on some cellular functions have been found in vitro. We used the reported effects on cell viability, actin cytoskeleton, and caspase activation to study the structure-activity relationship of AZA-1 and AZA-2 and the role of the carboxylic acid moiety in toxicity. AZA-1, AZA-2, and the synthetic AZA-2-methyl ester (AZA-2-ME), where the C1 carboxylic acid moiety of AZA-2 was esterified to the corresponding methyl ester moiety, induced a reduction of cell viability in neuroblastoma and hepatocyte cell lines with similar potency and kinetics. Interestingly, the mast cell line HMC-1 was resistant to AZA-induced cytotoxicity. Actin cytoskeleton alterations and caspase activation appeared after treatment with AZA-1, AZA-2, AZA-2-ME, and biotin-AZA-2 (AZA-2 labeled with biotin at C1) in neuroblastoma cells with similar qualitative, quantitative, and kinetics characteristics. Irreversibility of AZA effects on the actin cytoskeleton and cell morphology after short incubations with the toxin were common to AZA-1, AZA-2, and AZA-2-ME; however, 10-fold higher concentrations of biotin-AZA-2 were needed for irreversible effects. AZA-2-ME was rapidly metabolized in the cell to AZA-2, while transformation of biotin-AZA-2 into AZA-2 was less efficient, which explains the different potency in short exposure times. The moiety present at C1 is related to AZA toxicity in vitro. However, the presence of a methyl moiety at C8 is irrelevant to AZA toxicity since AZA-1 and AZA-2 were equipotent regardless of the readout effect.

Cytotoxic effect of azaspiracid-2 and azaspiracid-2-methyl ester in cultured neurons: involvement of the c-Jun N-terminal kinase.

Human poisoning by azaspiracids (AZAs) has emerged as an increasing problem in Europe in recent years. Azaspiracid-2 (AZA-2) is one of the most abundant azaspiracids in nature. Although AZA-2 was recently involved in several toxic episodes leading to human intoxications, there is no information available about its mechanism of action or its cytotoxic effect in cellular models. This paper reports on the neurotoxic effect of azaspiracid-2 and its potential cellular targets. We explore the cellular and cytotoxic effects of AZA-2 and AZA-2-methyl ester (where the carboxylic acid moiety of AZA-2 was converted to the corresponding methyl ester) in cerebellar neurons. Pharmacological tools were used to analyze the role of different signal transduction pathways in the toxicity of AZA-2. The neurotoxicity of AZA-2 and AZA-2-methyl ester was developmentally regulated, exhibiting a higher cytotoxicity in younger cells (2-3 div). After excluding several signal transduction pathways, we found that inhibition of the mitogen-activated protein kinase JNK completely prevented the cytotoxic effect of AZA-2 in neurons. Furthermore, neuronal exposure to AZA-2 or AZA-2-methyl ester caused an increase in the amount of total and phosphorylated JNK and produced nuclear accumulation of the protein. The results presented here point to a common target for AZA-1 and AZA-2 and constitute the first experimental approach to investigate the cytotoxicity of AZA-2 in vitro, establishing an initial approach to probe the mechanism of action of these group of natural toxins.

Chemical Synthesis of the GHIJKLMNO Ring System of Maitotoxin.

As the largest secondary metabolite to be discovered as of yet, the polyether marine neurotoxin maitotoxin constitutes a major structural and synthetic challenge. After its originally proposed structure ( 1) had been questioned on the basis of biosynthetic considerations, we provided computational and experimental support for structure 1. In an effort to provide stronger experimental evidence of the molecular architecture of maitotoxin, its GHIJKLMNO ring system 3 was synthesized. The (13)C NMR chemical shifts of synthetic 3 matched closely those corresponding to the same domain of the natural product providing strong evidence for the correctness of the originally proposed structure of maitotoxin ( 1).

The c-Jun-N-terminal kinase is involved in the neurotoxic effect of azaspiracid-1.

AIMS: Azaspiracids (AZAs) are marine phycotoxins with an unknown mechanism of action, recently implicated in human intoxications. The predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system and exhibits high neurotoxicity in vitro. METHODS: We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Immunocytochemical techniques in combination with confocal microscopy were employed to examine the cellular mechanisms involved in the neurotoxic effect of AZA-1. RESULTS: Several targets for azaspiracid-induced neurotoxicity, specifically the cAMP pathway, or protein kinase C and phosphatidylinositol 3-kinase activation were excluded. Interestingly, the specific c-Jun-N-terminal protein kinase (JNK) inhibitor SP 600125 protected cultured neurons against AZA-induced cytotoxicity. Immunocytochemistry experiments showed that AZA-1 increased the amount of phosphorylated JNK and caused nuclear translocation of the active protein that was prevented by SP 600125. CONCLUSION: Our data constitute the relationship between azaspiracid-induced cytotoxicity and specific modifications in cellular transduction signals, specifically we found that JNK activation is associated with the cytotoxic effect of the toxin. These results should provide the basis to identify the mechanism of action of this group of toxins.

Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells.

Azaspiracid-1 (AZA-1) is a marine toxin discovered in 1995. Besides damage to several tissues in vivo, AZA-1 has been shown to cause cytotoxicity in a number of cell lines and alterations in actin cytoskeleton and cell morphology. We studied the reversibility of AZA-1-induced morphological changes in human neuroblastoma cells and their dependence on caspases and signaling pathways involved in cytoskeleton regulation. Morphological/cytoskeletal changes were clearly observed by confocal microscopy 24h after the addition of toxin, without recovery upon toxin removal. Interestingly, 2min of incubation with AZA-1 was enough for the cytoskeleton to be altered 24-48h later. The activation of caspases by AZA-1 was studied next using a fluorescent caspase inhibitor. A cell population with activated caspases was observed after 48h of exposure to the toxin, but not at 24h. Two fragments and a stereoisomer of AZA-1 were tested to analyze structure-activity relationship. Only ABCD-epi-AZA-1 was active with a similar effect to AZA-1. Additionally, regarding the involvement of apoptosis/cytoskeleton signaling in AZA-1-induced morphological effects, inhibition of caspases with Z-VAD-FMK did not affect AZA-1-induced cytoskeletal changes, suggesting, together with the activation kinetics, that caspases are not responsible for AZA-1-elicited morphological changes. Modulation of PKA, PKC, PI3K, Erk, p38MAPK, glutathione and microtubules with inhibitors/activators did not inhibit AZA-1-induced actin cytoskeleton rearrangement. The JNK inhibitor SP600125 seemed to slightly diminish AZA-1 effects, however due to the effects of the drug by itself the involvement of JNK in AZA-1 toxicity needs further investigation. The results suggest that AZA-1 binds irreversibly to its cellular target, needing moieties located in the ABCDE and FGHI rings of the molecule. Cytotoxicity of AZA-1 has been previously described without reference to the type of cell death, we report that AZA-1 induces the activation of caspases, commonly used as an early marker of apoptosis, and that these proteases are not responsible for AZA-1-induced cytoskeleton disarragement in human neuroblastoma cells.

Effects of azaspiracid-1, a potent cytotoxic agent, on primary neuronal cultures. A structure-activity relationship study.

Azaspiracids (AZAs) are marine phycotoxins with an unknown mechanism of action, implicated in human intoxications. We investigated the effect of azaspiracid-1 (AZA-1) on the cytosolic calcium concentration ([Ca2+]c), intracellular pH (pHi), and neuron viability in neuronal cultures. AZA-1 increased [Ca2+]c and decreased neuronal viability. The effects of several fragments of the AZA-1 molecule (13 different chemical structures) were examined. The ent-ABCD-azaspiracid-1 (2) showed similar potency to AZA-1 (1) in increasing [Ca2+]c but higher cytotoxity than AZA-1. The chemical structures containing only the ABCD or the ABCDE ring domains (3-8) caused a [Ca2+]c increase but did not alter cell viability. The compounds containing only the FGHI ring domain of AZA-1 (9-14) did not modify the [Ca2+]c or the cell viability. Therefore, the effect of AZA-1 on [Ca2+]c depends on the presence of the ABCD or the ABCDE-ring structure, but the complete chemical structure is needed to produce neurotoxic effects.