Nickel-Catalyzed Cross-Electrophile Coupling of 1,2,3-Benzotriazin-4(3H)-ones with Aryl Bromides.

The nickel-catalyzed cross-electrophile coupling of 1,2,3-benzotriazin-4(3H)-ones with aryl bromides to generate a diverse array of ortho-arylated benzamide derivatives has been developed. The reaction displayed good functional group tolerance with Zn as the reductant. The key to this transformation is the ring opening of benzotriazinones, which undergo a denitrogenative process to obtain various benzamide derivatives (29 examples, 42-93% yield). The scalability of this transformation was demonstrated.

Nickel-Catalyzed Reductive Coupling of γ-Metalated Ketones with Unactivated Alkyl Bromides.

A nickel-catalyzed reductive cross-coupling reaction of aryl cyclopropyl ketones with easily accessible unactivated alkyl bromides to access aryl alkyl ketones has been developed. This strategy facilitates access to various of γ-alkyl-substituted ketones via ring opening of cyclopropyl ketones (26 examples, 50-90% yield). Initial mechanistic studies revealed that the reaction proceeds via radical cleavage of the alkyl bromide.

Catalytic enantioselective reductive domino alkyl arylation of acrylates via nickel/photoredox catalysis.

Nonsteroidal anti-inflammatory drug derivatives (NSAIDs) are an important class of medications. Here we show a visible-light-promoted photoredox/nickel catalyzed approach to construct enantioenriched NSAIDs via a three-component alkyl arylation of acrylates. This reductive cross-electrophile coupling avoids preformed organometallic reagents and replaces stoichiometric metal reductants by an organic reductant (Hantzsch ester). A broad range of functional groups are well-tolerated under mild conditions with high enantioselectivities (up to 93% ee) and good yields (up to 90%). A study of the reaction mechanism, as well as literature precedence, enabled a working reaction mechanism to be presented. Key steps include a reduction of the alkyl bromide to the radical, Giese addition of the alkyl radical to the acrylate and capture of the α-carbonyl radical by the enantioenriched nickel catalyst. Reductive elimination from the proposed Ni(III) intermediate generates the product and forms Ni(I).

Nickel-catalyzed enantioselective vinylation of aryl 2-azaallyl anions.

A unique enantioselective nickel-catalyzed vinylation of 2-azaallyl anions is advanced for the first time. This method affords diverse vinyl aryl methyl amines with high enantioselectivities, which are frequently occurring scaffolds in natural products and medications. This C-H functionalization method can also be extended to the synthesis of enantioenriched 1,3-diamine derivatives by employing suitably elaborated vinyl bromides. Key to the success of this process is the identification of a Ni/chiraphos catalyst system and a less reducing 2-azaallyl anion, all of which favor an anionic vinylation route over a background radical reaction. A telescoped gram scale synthesis and a product derivatization study confirmed the scalability and synthetic potential of this method.

Iron-Catalyzed Tertiary Alkylation of Terminal Alkynes with 1,3-Diesters via a Functionalized Alkyl Radical.

Direct oxidative C(sp)-H/C(sp3 )-H cross-coupling offers an ideal and environmentally benign protocol for C(sp)-C(sp3 ) bond formations. As such, reactivity and site-selectivity with respect to C(sp3 )-H bond cleavage have remained a persistent challenge. Herein is reported a simple method for iron-catalyzed/silver-mediated tertiary alkylation of terminal alkynes with readily available and versatile 1,3-dicarbonyl compounds. The reaction is suitable for an array of substrates and proceeds in a highly selective manner even employing alkanes containing other tertiary, benzylic, and C(sp3 )-H bonds alpha to heteroatoms. Elaboration of the products enables the synthesis of a series of versatile building blocks. Control experiments implicate the in situ generation of a tertiary carbon-centered radical species.

Nickel-catalyzed reductive coupling of homoenolates and their higher homologues with unactivated alkyl bromides.

The catalytic generation of homoenolates and their higher homologues has been a long-standing challenge. Like the generation of transition metal enolates, which have been used to great affect in synthesis and medicinal chemistries, homoenolates and their higher homologues have much potential, albeit largely unrealized. Herein, a nickel-catalyzed generation of homoenolates, and their higher homologues, via decarbonylation of readily available cyclic anhydrides has been developed. The utility of nickel-bound homoenolates and their higher homologues is demonstrated by cross-coupling with unactivated alkyl bromides, generating a diverse array of aliphatic acids. A broad range of functional groups is tolerated. Preliminary mechanistic studies demonstrate that: (1) oxidative addition of anhydrides by the catalyst is faster than oxidative addition of alkyl bromides; (2) nickel bound metallocycles are involved in this transformation and (3) the catalyst undergoes a single electron transfer (SET) process with the alkyl bromide.

Nickel/Photoredox-Catalyzed Asymmetric Reductive Cross-Coupling of Racemic α-Chloro Esters with Aryl Iodides.

A unique nickel/organic photoredox co-catalyzed asymmetric reductive cross-coupling between α-chloro esters and aryl iodides is developed. This cross-electrophile coupling reaction employs an organic reductant (Hantzsch ester), whereas most reductive cross-coupling reactions use stoichiometric metals. A diverse array of valuable α-aryl esters is formed under these conditions with high enantioselectivities (up to 94 %) and good yields (up to 88 %). α-Aryl esters represent an important family of nonsteroidal anti-inflammatory drugs. This novel synergistic strategy expands the scope of Ni-catalyzed reductive asymmetric cross-coupling reactions.

Nickel-Catalyzed C(sp3)-H Arylation of Diarylmethane Derivatives with Aryl Fluorides.

A novel nickel-catalyzed C(sp3)-H arylation with nonactivated aryl fluorides is reported. The use of 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (IMes) as a ligand was found to be critical to the success of the reaction. This new method enables the synthesis of a wide range of triarylmethane derivatives.

Nickel-Catalyzed Desymmetrizing Cross-Electrophile Coupling of Cyclic Meso-Anhydrides.

A Ni-catalyzed desymmetrizing cross-electrophile coupling of cyclic meso-anhydrides with aryl triflates has been successfully demonstrated. This is the only example using cyclic meso-anhydrides in cross-electrophile coupling reactions. A diverse array of valuable γ-keto acid building blocks can be generated under these conditions with excellent functional group tolerance and stereochemical fidelity.

Nickel-Catalyzed Arylation of Heteroaryl-containing Diarylmethanes: Exceptional Reactivity of the Ni(NIXANTPHOS)-based Catalyst.

Nickel(0)-catalyzed cross-coupling of heteroaryl-containing diarylmethanes with both aryl bromides and chlorides has been achieved. The success of this reaction relies on the introduction of a unique nickel/NIXANTPHOS-based catalyst system, which provides a direct route to triarylmethanes from heteroaryl-containing diarylmethanes. Reactivity studies indicate the Ni(NIXANTPHOS)-based catalyst exhibits enhanced reactivity over XANTPHOS derivatives and other Ni(phosphine)-based catalysts in the reactions examined.

Nickel-Catalyzed Allylic Alkylation with Diarylmethane Pronucleophiles: Reaction Development and Mechanistic Insights.

Palladium-catalyzed allylic substitution reactions are among the most efficient methods to construct C-C bonds between sp(3)-hybridized carbon atoms. In contrast, much less work has been done with nickel catalysts, perhaps because of the different mechanisms of the allylic substitution reactions. Palladium catalysts generally undergo substitution by a "soft"-nucleophile pathway, wherein the nucleophile attacks the allyl group externally. Nickel catalysts are usually paired with "hard" nucleophiles, which attack the metal before C-C bond formation. Introduced herein is a rare nickel-based catalyst which promotes substitution with diarylmethane pronucleophiles by the soft-nucleophile pathway. Preliminary studies on the asymmetric allylic alkylation are promising.

Tandem C(sp3)-H Arylation/Oxidation and Arylation/Allylic Substitution of Isoindolinones.

Isoindolinones comprise an important class of medicinally active compounds. Herein we report a straightforward functionalization of the isoindolinones with aryl bromides (22 examples) using a Pd(OAc)2/NIXANTPHOS-based catalyst system. Additionally 3-aryl 3-hydroxy isoindolinone derivatives, which exhibit anti-tumor activity, can be accessed via a tandem reaction. Thus, when the arylation product is exposed to air under basic conditions, in situ oxidation takes place to install the 3-hydroxyl group. Furthermore, a tandem arylation/allylic substitution reaction is advanced in which both the arylation and allylic substitution are catalyzed by the same palladium catalyst. Finally, a tandem arylation/alkylation procedure is presented. These tandem reactions enable the synthesis of a variety of structurally diverse isoindolinone derivatives from common starting materials.

Effects of short-term hyper- and hypo-osmotic exposure on the osmoregulatory strategy of unfed North Pacific spiny dogfish (Squalus suckleyi).

The North Pacific spiny dogfish (Squalus suckleyi) is a partially euryhaline species of elasmobranch that often enter estuaries where they experience relatively large fluctuations in environmental salinity that can affect plasma osmolality. Previous studies have investigated the effects of altered salinity on elasmobranchs over the long term, but fewer studies have conducted time courses to investigate how rapidly they can adapt to such changes. In this study, we exposed unfed (no exogenous source of nitrogen or TMAO) spiny dogfish to hyper- and hypo-osmotic conditions and measured plasma and tissue osmolytes, nitrogen excretion, and changes in enzyme activity and mRNA levels in the rectal gland over 24h. It was shown that plasma osmolality changes to approximately match the ambient seawater within 18-24h. In the hypersaline environment, significant increases in urea, sodium, and chloride were observed, whereas in the hyposaline environment, only significant decreases in TMAO and sodium were observed. Both urea and ammonia excretion increased at low salinities suggesting a reduction in urea retention and possibly urea production. qPCR and enzyme activity data for Na(+)/K(+)-ATPase did not support the idea of rectal gland activation following exposure to increased salinities. Therefore, we suggest that the rectal gland may not be a quantitatively important aspect of the dogfish osmoregulatory strategy during changes in environmental salinity, or it may be active only in the very early stages (i.e., less than 6h) of responses to altered salinity.

Physiological and molecular responses of the spiny dogfish shark (Squalus acanthias) to high environmental ammonia: scavenging for nitrogen.

In teleosts, a branchial metabolon links ammonia excretion to Na(+) uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol(-1) NH4HCO3) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was re-established. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24-48 h. Control ΔP(NH3) (the partial pressure gradient of NH3) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH4(+) electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na(+)/K(+)-ATPase (NKA) increased at 24 h and that of H(+)-ATPase decreased at 48 h, while expression of the potential metabolon components Na(+)/H(+) exchanger2 (NHE2) and carbonic anhydrase IV (CA-IV) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.

Physiological and biochemical strategies for withstanding emersion in two galaxiid fishes.

The galaxiid fishes of the Southern hemisphere display variable tolerance to aerial exposure. Brown mudfish (Neochanna apoda), for example, pseudoaestivate, inhabiting moist soil for months at a time, whereas inanga (Galaxias maculatus) emerse under unfavourable water conditions, but only for periods of a few hours. This study sought to identify the physiological and biochemical strategies that determine emersion tolerance in these species. Nitrogenous waste excretion was measured before and after an experimental emersion period (14 days for mudfish, 6 h for inanga). Both species showed significantly elevated ammonia "washout" upon return to water, but no increase in plasma or muscle ammonia. Post-emersion urea levels were elevated in plasma and muscle in both fish, however the extent of the accumulation did not indicate significant de novo urea production. This was supported by the lack of carbamoyl phosphate synthetase activity in tissues. Consequently, mudfish metabolism was examined to determine whether changes in parameters such as oxygen consumption, carbon dioxide excretion, and/or altered metabolic costs (represented by the key ionoregulatory enzyme Na(+), K(+)-ATPase; NKA) could explain emersion tolerance. Oxygen consumption rates, already very low in immersed mudfish, were largely maintained over the course of emersion. Carbon dioxide excretion decreased during emersion, and a small, but significant, decrease in NKA was noted. These data suggest that the extended emersion capacity of mudfish may result from a generally low metabolic rate that is maintained throughout aerial exposure via cutaneous gas exchange, and which limits the production of potentially toxic nitrogenous waste.

Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus).

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique "pulsing" nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na(+)-K(+)-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.

Zinc hyperaccumulation in squirrelfish (Holocentrus adscenscionis) and its role in embryo viability.

Female squirrelfish (Fam. Holocentridae) can accumulate and temporarily sequester copious amounts of zinc (Zn) in their livers. There, it is initially compartmentalized before a subsequent, estrogen-triggered redistribution to the ovaries. Here we show that cellular uptake of Zn is also influenced by estrogen signaling, and that estrogen increases concentrations of the plasma Zn-binding protein vitellogenin (VTG). However, estrogen-mediated increases in VTG are not sufficient to accommodate the magnitude of hepato-ovarian Zn transfer in female squirrelfish (Holocentrus adscensionis). These findings suggest that holocentrids have acquired the ability to use hormonal cues to drive hepatic uptake and storage of Zn, signal for its physiological redistribution, and influence the capacity for systemic transport of Zn beyond the mediation of increased plasma VTG concentrations. Such specific adaptations suggest an advantage for the oocyte, which is corroborated in further studies where we determined that oocyte Zn concentrations are positively correlated with egg viability in captive-spawned squirrelfish. The novel nature of these findings underlies the importance of Zn in squirrelfish reproductive biology.

Cortisol-sensitive urea transport across the gill basolateral membrane of the gulf toadfish (Opsanus beta).

Gulf toadfish (Opsanus beta) use a unique pulsatile urea excretion mechanism that allows urea to be voided in large pulses via the periodic insertion or activation of a branchial urea transporter. The precise cellular and subcellular location of the facilitated diffusion mechanism(s) remains unclear. An in vitro basolateral membrane vesicle (BLMV) preparation was used to test the hypothesis that urea movement across the gill basolateral membrane occurs through a cortisol-sensitive carrier-mediated mechanism. Toadfish BLMVs demonstrated two components of urea uptake: a linear element at high external urea concentrations, and a phloretin-sensitive saturable constituent (K(m) = 0.24 mmol/l; V(max) = 6.95 micromol x mg protein(-1) x h(-1)) at low urea concentrations (<1 mmol/l). BLMV urea transport in toadfish was unaffected by in vitro treatment with ouabain, N-ethylmaleimide, or the absence of sodium, conditions that are known to inhibit sodium-coupled and proton-coupled urea transport in vertebrates. Transport kinetics were temperature sensitive with a Q(10) > 2, further suggestive of carrier-mediated processes. Our data provide evidence that a basolateral urea facilitated transporter accelerates the movement of urea between the plasma and gills to enable the pulsatile excretion of urea. Furthermore, in vivo infusion of cortisol caused a significant 4.3-fold reduction in BLMV urea transport capacity in lab-crowded fish, suggesting that cortisol inhibits the recruitment of urea transporters to the basolateral membrane, which may ultimately affect the size of the urea pulse event in gulf toadfish.

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.

Sequence, expression and evolutionary relationships of carbamoyl phosphate synthetase I in the toad Xenopus laevis.

The sequence of carbamoyl phosphate synthetase I (CPSase I) cDNA and expression of the enzyme in liver of the toad Xenopus laevis are reported. CPSase I mRNA increases 6-fold when toads are exposed to high salinity for extended periods of time. The deduced 1,494-amino acid sequence of the CPSase I is homologous to other CPSases and reveals a domain structure and conserved amino acids common to other CPSases. A serine residue (S287) is present where there is a cysteine residue required for glutamine-dependent activity in CPSase Types III and II (Type I CPSases utilize only ammonia as nitrogen-donating substrate). A sequence of DNA 964 bases upstream from the ATG start codon for the CPSase I gene is also reported. Phylogenetic analysis for 30 CPSase isoforms, including X. laevis CPSase I, across a wide spectrum of phyla is reported and discussed. The results are consistent with the views that eukaryotic CPSase II as a multifunctional complex evolved from prokaryotic CPSase II and that CPSase I in terrestrial vertebrates and CPSase III in fishes arose from eukaryotic CPSase II by independent events after the divergence of plants in eukaryotic evolution.