Obesity aggravates neuroinflammatory and neurodegenerative effects of bisphenol A in female rats.

Bisphenol A (BPA), a common plasticizer, is categorized as a neurotoxic compound. Its impact on individuals exhibits sex-linked variations. Several biological and environmental factors impact the degree of toxicity. Moreover, nutritional factors have profound influence on toxicity outcome. BPA has been demonstrated to be an obesogen. However, research on the potential role of obesity as a confounding factor in BPA toxicity is lacking. We studied the neurodegenerative effects in high-fat diet (HFD)-induced obese female rats after exposure to BPA (10 mg/L via drinking water for 90 days). Four groups were taken in this study - Control, HFD, HFD + BPA and BPA. Cognitive function was evaluated through novel object recognition (NOR) test. Inflammatory changes in brain, and changes in hormonal level, lipid profile, glucose tolerance, oxidative stress, and antioxidants were also determined. HFD + BPA group rats showed a significant decline in memory function in NOR test. The cerebral cortex (CC) of the brain showed increased neurodegenerative changes as measured by microtubule-associated protein-2 (MAP-2) accompanied by histopathological confirmation. The increased level of neuroinflammation was demonstrated by microglial activation (Iba-1) and protein expression of nuclear factor- kappa B (NF-КB) in the brain. Obesity also caused significant (p < 0.05) increase in lipid peroxidation accompanied by reduced activities of antioxidant enzymes (glutathione S-transferase, catalase and glutathione peroxidase) and decrease in reduced-glutathione (p < 0.05) when compared to non-obese rats with BPA treatment. Overall, study revealed that obesity serves as a risk factor in the toxicity of BPA which may exacerbate the progression of neurological diseases.

Downregulation of ATF-4 Attenuates the Endoplasmic Reticulum Stress-Mediated Neuroinflammation and Cognitive Impairment in Experimentally Induced Alzheimer's Disease Model.

Protein aggregation is invariably associated with the inflammation as a factor in Alzheimer's disease (AD). We investigated the interaction between downstream factors of endoplasmic reticulum (ER) stress pathway and inflammation, with implications in cognitive impairment in AD. Amyloid-ß (Aß)(1-42) was administered by bilateral intracerebroventricular (icv) injection in the brain of adult male Wistar rats to experimentally develop AD. The cognitive impairment was assessed by measuring behavioral parameters such as Morris water maze and novel object recognition tests. Levels of pro-inflammatory cytokines such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α and anti-inflammatory cytokines IL-4 and IL-10 were measured by the enzyme-linked immunosorbent assay (ELISA) in different rat brain regions. Inflammatory marker proteins such as cyclo-oxygenase (COX)-2 and phosphorylation of nuclear factor kappa B (NF-КB) (p65) were measured by the western blotting. Gene expression of ER stress downstream factors such as ATF-4, CHOP, and GADD-34 was analyzed by qRT-PCR. Histological studies were performed to check Aß accumulation and neuronal degeneration. Integrated stress response inhibitor (ISRIB) was used to confirm the specific role of ER stress-mediated inflammation in cognitive impairment. Administration of Aß(1-42) resulted in alteration in levels of inflammatory cytokines, inflammatory proteins, and mRNA levels of ER stress downstream factors. ISRIB treatment resulted in attenuation of Aß(1-42)-induced ER stress, inflammation, neurodegeneration, and cognitive impairment in rats. These results indicate that ER stress-mediated inflammation potentiates the cognitive impairment in AD. An understanding of cascade of events, interaction of ER stress which was a hallmark of the present investigation together with inflammation and modulation of downstream signalling factors could serve as potent biomarkers to study AD progression. Schematic representation of interaction between ER stress and inflammation. Administration of Aß(1-42) resulted in ER stress which caused the activation of factors of PERK pathway, inflammation, neuronal degeneration, and cognitive impairment. ISRIB treatment caused downregulation of ATF-4 and attenuation of inflammation indicating a role of ER stress-mediated inflammation in the cognitive impairment in AD. The site of action of ISRIB is shown in blue color.

Is Enol Always the Culprit? The Curious Case of High Enantioselectivity in a Chiral Rh(II) Complex Catalyzed Carbene Insertion Reaction.

The mechanism of Rh2 (S-NTTL)4 catalyzed carbene insertion into C(3)-H of indole is investigated using DFT methods. Since the commonly accepted enol mechanism cannot account for enantioinduction, a concerted oxocarbenium pathway was proposed in an earlier work using a model catalyst. However, after considering the full catalytic system, this study finds that akin to other reactions, here, too, the enol pathway is of lower energy, which now naturally raises a conundrum regarding the mode of chiral induction. Herein, a new water promoted mechanistic pathway involving a metal-associated enol intermediate hydrogen bonding and stereochemical model are proposed to solve this puzzle. It is shown how the catalyst bowl-shaped structure along with substrate-catalyst binding is crucial for achieving high levels of enantioselectivity. A stereodetermining water-assisted proton transfer is proposed and confirmed through deuterium-labeling experiments. The water molecules are held together by H-bonding interactions with the carboxylate ligands that is reminiscent of enzyme catalysis. Although several previous studies have aimed at understanding the mechanism of metal catalyzed carbene insertion reactions, the origin of high stereoinduction especially with chiral metal complexes remains unclear, and till date there is no transition state model that can explain the high enantioselectivity with such chiral Rh complexes. The metal-associated enol pathway is currently underrepresented in catalytic cycles and may play a crucial role in catalyst design. Since the enol pathway is commonly adopted in other metal-catalyzed X-H insertion reactions involving a diazoester, the presented results are not specific to the current reaction. Therefore, this study could provide the direction for achieving high levels of enantioselectivity which is otherwise difficult to achieve with a single metal catalyst.

Role of mutations in a chemoenzymatic enantiodivergent C(sp3)-H insertion: exploring the mechanism and origin of stereoselectivity.

New-to-nature enzymes have emerged as powerful catalysts in recent years for streamlining various stereoselective organic transformations. While synthetic strategies employing engineered enzymes have witnessed proliferating success, there is limited clarity on the mechanistic front and more so when considering molecular-level insights into the role of selected mutations, dramatically escalating catalytic competency and selectivity. We have investigated the mechanism and correlation between mutations and exquisite stereoselectivity of a lactone carbene insertion into the C(sp3)-H bond of substituted aniline, catalyzed by two mutants of a cytochrome P450 variant, "P411" (engineered through directed evolution) in which the axial cysteine has been mutated to serine, utilizing various computational tools. The pivotal role of S264 and L/R328 mutations in the active site has been delineated computationally using two cluster models, thus rationalizing the enantiodivergence. This report provides much-needed insights into the origin of enantiodivergence, furnishing a mechanistic framework for understanding the anchoring effects of H-bond donor residues with the lactone ring. This study is expected to have important implications in the rational design of stereodivergent enzymes and toward successful in silico enzyme designing.

Investigating the Nature of the Onium Ylide and Michael Acceptor in a Rhodium(I)-Catalyzed Multicomponent Reaction.

We computationally study the mechanistic pathway for the synthetically valuable cascading N-H functionalization followed by the C-C bond-forming reaction. The impetus to study such multicomponent reactions catalyzed by Rh(I) arises from the highly fluxional nature of the onium ylide involved, which is often not amenable to experimental detection. Our results throw light on an interesting mechanistic paradigm where the binding of the ylide to the metal plays a crucial role. The study provides some much-needed insights to expand the scope of these highly valuable methodologies to a broader range of asymmetric reactions.

Oxidized Bridged Carbenoids as Viable Intermediates in a Fe(III) Catalyzed C-H Insertion Reaction.

Fe catalyzed carbene insertion reactions present an efficient route for direct C-H functionalization. The use of Fe(III) in place of the widely used Fe(II) presents several benefits. However, the mechanistic understanding of Fe(III) severely lags behind Fe(II) complexes. One of the major unsolved issues relates to the formation of bridged versus terminal metallocarbenes. Even though the oxidized bridged carbenoid complexes have been isolated and found to be thermodynamically more stable, they are generally considered a dead end for the catalytic cycle. In the current report, the formation and the subsequent reactions of the bridged carbenoid complexes for an Fe(TPP)Cl catalyzed C(sp2 )-H insertion are investigated. Using DFT calculations, it is observed that both mono and bis oxidized bridged carbenoid complexes can participate in the catalytic cycle. Importantly, for the first time, a mechanistic pathway showing that these bridged species are not a dead end in Fe catalysis is presented. Their existence in other reactions might be more prevalent than what is currently believed. The current study will have important implications in utilizing Fe(III) complexes for other insertion reactions, especially for heme containing enzymes which necessarily need to be carried out under anaerobic/reducing conditions.

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Does an Enol Pathway Preclude High Stereoselectivity in Iron-Catalyzed Indole C-H Functionalization via Carbene Insertion?

C-H functionalization of indoles via Fe carbenoids presents an attractive strategy to obtain biologically important structural motifs. However, obtaining good stereoselectivity with Fe has been a significant challenge. It is unclear whether the low selectivity is due to a radical pathway or an ionic mechanism involving metal-free species. We therefore present a density functional theory (DFT) study of indole alkylation with diazoacetates catalyzed by Fe(ClO4)TMEDA/spirobisoxazoline and myoglobin. We explore three mechanistic pathways: nucleophilic, radical, and oxocarbenium routes. The nucleophilic pathway is the most feasible with the formation of an enol species that tautomerizes to furnish the alkylated indole. While this mechanism is routinely proposed, the stereochemical model has been conspicuously absent until now. We show that the conventionally invoked enol pathway is not responsible for the low enantiomeric excess. The enol intermediate can stay coordinated to the catalyst via different binding sites placing the enol in proximity to the chiral environment and affecting the stereoselective proton transfer. Both the binding strength and the chiral environment are crucial for obtaining high selectivity. Our study provides the much needed insights for the modest-low selectivities of Fe systems and could help in expediting the discovery of an efficient catalytic system. These mechanistic underpinnings could also be applicable to other metal (Rh, Pd, Cu, etc.)-catalyzed X-H insertion reactions.

Copper-Catalyzed Aerobic Cross-Dehydrogenative Coupling of ß-Oxime Ether Furan with Indole.

Heterobiaryls serve as relevant structural motifs in many fields of high applicative importance such as drugs, agrochemicals, organic functional materials etc. Cross-dehydrogenative coupling involving direct oxidation of two C-H bonds to construct a C-C bond is actively being pursued as a more benign and 'greener' alternative for synthesizing heterobiaryls. Herein, we report a Cu(I)-catalyzed cross-dehydrogenative coupling of indoles and furans, two of the most important aromatic heterocycles using air as the terminal oxidant. The reaction proceeds with regio- and chemoselectivity to give the cross-coupled products in good to excellent yields generally. A broad substrate scope with respect to both the coupling partners has been demonstrated to prove the generality of this reaction. This represents the hitherto unexplored cross-dehydrogenative coupling methodology to obtain an indole-furan biaryl motif.

Aryne Three-Component Coupling Involving CS2 for the Synthesis of S-Aryl Dithiocarbamates.

A facile synthesis of biologically important S-aryl dithiocarbamates has been demonstrated by the aryne three-component coupling involving CS2 and aliphatic amines. This transition-metal-free and mild reaction is scalable and operates with good functional group compatibility. Preliminary mechanistic experiments, including density functional theory studies, are also provided. Moreover, with 3-triflyloxybenzynes, a unique four-component coupling incorporating tetrahydrofuran was observed.

DFT study on Ir-quinoid catalyzed C-H functionalization: new radical reactivity or direct carbene transfer?

DFT methods are used to probe the mechanism of a newly developed Ir-quinoid catalyzed C(sp3)-H functionalization of 1,4 dienes. The lowest energy pathway proceeds via an old-school concerted C-H insertion as opposed to a unique hydrogen atom transfer process proposed previously. The concertedness of the reaction shows an intriguing dependence on sterics of the diene leading to either inserted or dehydrogenated products. We use these new insights to tune the axial ligand, and design a more efficient catalyst.

A computational approach to understand the role of metals and axial ligands in artificial heme enzyme catalyzed C-H insertion.

Engineered heme enzymes such as myoglobin and cytochrome P450s metalloproteins are gaining widespread importance due to their efficiency in catalyzing non-natural reactions. In a recent strategy, the naturally occurring Fe metal in the heme unit was replaced with non-native metals such as Ir, Rh, Co, Cu, etc., and axial ligands to generate artificial metalloenzymes. Determining the best metal-ligand for a chemical transformation is not a trivial task. Here we demonstrate how computational approaches can be used in deciding the best metal-ligand combination which would be highly beneficial in designing new enzymes as well as small molecule catalysts. We have used Density Functional Theory (DFT) to shed light on the enhanced reactivity of an Ir system with varying axial ligands. We look at the insertion of a carbene group generated from diazo precursors via N2 extrusion into a C-H bond. For both Ir(Me) and Fe systems, the first step, i.e., N2 extrusion is the rate determining step. Strikingly, neither the better ligand overlap with 5d orbitals on Ir nor the electrophilicity on the carbene centre play a significant role. A comparison of Fe and Ir systems reveals that a lower distortion in the Ir(Me)-porphyrin on moving from the reactant to the transition state renders it catalytically more active. We notice that for both metal porphyrins, the free energy barriers are affected by axial ligand substitution. Further, for Fe porphyrin, the axial ligand also changes the preferred spin state. We show that for the carbene insertion into the C-H bond, Fe porphyrin systems undergo a stepwise HAT (hydrogen atom transfer) instead of a concerted hydride transfer process. Importantly, we find that the substitution of the axial Me ligand on Ir to imidazole or chloride, or without an axial substitution changes the rate determining step of the reaction. Therefore, an optimum ligand that can balance the barriers for both steps of the catalytic cycle is essential. We subsequently used the QM cluster approach to delineate the protein environment's role and mutations in improving the catalytic activity of the Ir(Me) system.

NHC-Catalyzed Desymmetrization of N-Aryl Maleimides Leading to the Atroposelective Synthesis of N-Aryl Succinimides.

Although the construction of axially chiral C-C bonds leading to the atroposelective synthesis of biaryls and allied compounds are well-known, the related synthesis of compounds bearing axially chiral C-N bonds are relatively rare. Described herein is the N-heterocyclic carbene-catalyzed atroposelective synthesis of N-aryl succinimides having an axially chiral C-N bond via the desymmetrization of N-aryl maleimides. The NHC involved intermolecular Stetter-aldol cascade of dialdehydes with prochiral N-aryl maleimides followed by oxidation afforded N-aryl succinimides in good yields and ee values. Preliminary studies on rotation barrier for the C-N bond, the temperature dependence, and detailed DFT studies on mechanism are also provided.

Catalytic Enantioselective Desymmetrizing Fischer Indolization through Dynamic Kinetic Resolution.

The first catalytic enantioselective Fischer indolization of prochiral diketones containing enantiotopic carbonyl groups is developed and shown to proceed through dynamic kinetic resolution (DKR). Catalyzed by the combination of a spirocyclic chiral phosphoric acid and ZnCl2 (Lewis acid assisted Brønsted acid), this direct approach combines 2,2-disubstituted cyclopentane-1,3-diones with N-protected phenylhydrazines to furnish cyclopenta[b]indole derivatives containing an all-carbon quaternary stereocenter with good to excellent enantioselectivities.

N-Heterocyclic Carbene-Catalyzed Formal [6+2] Annulation Reaction via Cross-Conjugated Aza-Trienolate Intermediates.

The diverse reactivity of N-heterocyclic carbenes (NHCs) in organocatalysis is due to the possibility of different modes of action. Although NHC-bound enolates and dienolates are known, the related NHC-bound cross-conjugated aza-trienolates remain elusive. Herein, we demonstrate the NHC-catalyzed formal [6+2] annulation of nitrogen-containing heterocyclic aldehydes with α,α,α-trifluoroacetophenones leading to the formation of versatile pyrrolooxazolones (29 examples). The catalytically generated cross-conjugated aza-trienolates (aza-fulvene type) underwent smooth [6+2] annulation with electrophilic ketones to afford the product in moderate to good yields under mild conditions. Preliminary DFT studies on the mechanism are also provided.

Exploring the challenges of computational enzyme design by rebuilding the active site of a dehalogenase.

Rational enzyme design presents a major challenge that has not been overcome by computational approaches. One of the key challenges is the difficulty in assessing the magnitude of the maximum possible catalytic activity. In an attempt to overcome this challenge, we introduce a strategy that takes an active enzyme (assuming that its activity is close to the maximum possible activity), design mutations that reduce the catalytic activity, and then try to restore that catalysis by mutating other residues. Here we take as a test case the enzyme haloalkane dehalogenase (DhlA), with a 1,2-dichloroethane substrate. We start by demonstrating our ability to reproduce the results of single mutations. Next, we design mutations that reduce the enzyme activity and finally design double mutations that are aimed at restoring the activity. Using the computational predictions as a guide, we conduct an experimental study that confirms our prediction in one case and leads to inconclusive results in another case with 1,2-dichloroethane as substrate. Interestingly, one of our predicted double mutants catalyzes dehalogenation of 1,2-dibromoethane more efficiently than the wild-type enzyme.

Postoperative Analgesia with Intrathecal Nalbuphine versus Intrathecal Fentanyl in Cesarean Section: A Double-Blind Randomized Comparative Study.

BACKGROUND: Nalbuphine when used as adjuvant to hyperbaric bupivacaine has improved the quality of perioperative analgesia with fewer side effects. Fentanyl is a lipophilic opioid with a rapid onset following intrathecal injection. It does not cause respiratory depression and improves duration of sensory anesthesia without producing significant side effects. AIM: This study aims to compare the postoperative analgesia of intrathecal nalbuphine and fentanyl as adjuvants to bupivacaine in cesarean section. METHODOLOGY: A prospective, randomized, double-blind, and comparative study was conducted on 150 parturients of American Society of Anesthesiologists (ASA) physical status I and II of age group 20-45 years with normal coagulation profile undergoing cesarean section under spinal anesthesia. These patients were randomized into three groups with fifty patients in each group. Group I received 2 ml of 0.5% hyperbaric bupivacaine (10 mg) plus 0.4 ml nalbuphine (0.8 mg), Group II received 2 ml of 0.5% hyperbaric bupivacaine (10 mg) plus 0.4 ml fentanyl (20 µg), and Group III received 2 ml of 0.5% hyperbaric bupivacaine (10 mg) plus 0.4 ml of normal saline. RESULTS: The mean duration of effective analgesia was 259.20 ± 23.23 min in Group I, 232.70 ± 13.15 min in Group II, and 168.28 ± 7.55 min in Group III. The mean number of rescue analgesics required was significantly lower (P < 0.001) in Group I as compared to Group II and III. CONCLUSION: Both intrathecal nalbuphine 0.8 mg and fentanyl 20 µg are effective adjuvants to 0.5% hyperbaric bupivacaine in subarachnoid block. However, intrathecal nalbuphine prolongs postoperative analgesia maximally and may be used as an alternative to intrathecal fentanyl in cesarean section.

Exploring the Mechanism and Stereoselectivity in Chiral Cinchona-Catalyzed Heterodimerization of Ketenes.

Catalytic heterodimerization of ketenes can lead to important four-membered ß-lactones. A recent asymmetric organocatalytic [2 + 2] cycloaddition between methylketene (MK) and methylphenylketene (MPK) in the presence of pseudoenantiomeric cinchona catalysts (trimethylsilylquinine (TMSQ) or methylquinidine (MeQd)) provided ß-lactones with high enantio- and diastereoselectivities. We employ DFT(M06-2X) computations to understand the mechanism and the origin of stereoselectivity in this ketene heterodimerization. The mechanism is found to involve the formation of an ammonium enolate first, by the action of the quinuclidine tertiary amine of the cinchona catalyst on MK. A stepwise pathway wherein the MK-cinchona enolate (enolate-A) adds to MPK in the selectivity-determining C-C bond formation step leading to the R-Z and S-Z product respectively with TMSQ and MeQd catalysts is predicted. The inclusion of LiClO4 is found to favor the C-C bond formation transition state to the S-E isomer in the case of MeQd and the R-E isomer with TMSQ catalysts. In the most preferred transition states, more effective C-H···π (between the phenyl ring of the EPK and the catalyst) and C-H···O interactions (between the catalyst and LiClO4) are noticed than that in the higher energy analogues, underscoring the importance of noncovalent interactions in enantio- and diastereocontrol.

Exploring the Development of Ground-State Destabilization and Transition-State Stabilization in Two Directed Evolution Paths of Kemp Eliminases.

Computer-aided enzyme design presents a major challenge since in most cases it has not resulted in an impressive catalytic power. The reasons for the problems with computational design include the use of nonquantitative approaches, but they may also reflect other difficulties that are not completely obvious. Thus, it is very useful to try to learn from the trend in directed evolution experiments. Here we explore the nature of the refinement of Kemp eliminases by directed evolution, trying to gain an understanding of related requirements from computational design. The observed trend in the directed evolution refinement of KE07 and HG3 are reproduced, showing that in the case of KE07 the directed evolution leads to ground-state destabilization, whereas in the case of HG3 the directed evolution leads to transition-state stabilization. The nature of the different paths of the directed evolution is examined and discussed. The present study seems to indicate that computer-aided enzyme design may require more than calculations of the effect of single mutations and should be extended to calculations of the effect of simultaneous multiple mutations (that make a few residues preorganized effectively). However, the analysis of two known evolution paths can still be accomplished using the relevant sequences and structures. Thus, by comparing two directed evolution paths of Kemp eliminases we reached the important conclusion that the more effective path leads to transition-state stabilization.

Misunderstanding the preorganization concept can lead to confusions about the origin of enzyme catalysis.

Understanding the origin of the catalytic power of enzymes has both conceptual and practical importance. One of the most important finding from computational studies of enzyme catalysis is that a major part of the catalytic power is due to the preorganization of the enzyme active site. Unfortunately, misunderstanding of the nontrivial preorganization idea lead some to assume that it does not consider the effect of the protein residues. This major confusion reflects a misunderstanding of the statement that the interaction energy of the enzyme group and the transition state (TS) is similar to the corresponding interaction between the water molecules (in the reference system) and the TS, and that the catalysis is due to the reorganization free energy of the water molecules. Obviously, this finding does not mean that we do not consider the enzyme groups. Another problem is the idea that catalysis is due to substrate preorganization. This more traditional idea is based in some cases on inconsistent interpretation of the action of model compounds, which unfortunately, do not reflect the actual situation in the enzyme active site. The present article addresses the above problems, clarifying first the enzyme polar preorganization idea and the current misunderstandings. Next we take a specific model compound that was used to promote the substrate preorganization proposal and establish its irrelevance to enzyme catalysis. Overall, we show that the origin of the catalytic power of enzymes cannot be assessed uniquely without computer simulations, since at present this is the only way of relating structure and energetics.