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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Importance of ligand exchanges in Pd(II)-Brønsted acid cooperative catalytic approach to spirocyclic rings.

Increasing number of reports in the most recent literature convey the use of palladium and Brønsted acids as cooperative catalytic partners. However, the mechanistic understanding of several such cooperative catalytic reactions and the origin of cooperativity continue to remain limited. In transition metal catalysis, it is typically assumed that the native ligands, such as the acetates in palladium acetate, are retained throughout the catalytic cycle. Herein, we convey the significance of invoking ligand exchanges in transition metal catalysis by using the mechanism of a representative cooperative dual-catalytic reaction. Density functional theory (M06 and B3LYP) computations have been employed to decipher the mechanism of Pd(II)-Brønsted acid catalyzed migratory ring expansion reaction of an indenyl cyclobutanol to a spirocyclic indene bearing a quaternary carbon. The molecular role of water, benzoquinone and phosphoric acid has been probed by computing the energetics using several combinations of all these as ligands on palladium. Of the two key mechanistic possibilities examined, a Wacker-type pathway (involving a semipinacol ring expansion of cyclobutanol followed by a reductive elimination) is found to be energetically more preferred over an allylic pathway wherein the ring expansion in a Pd-π-allyl intermediate occurs subsequent to the initial allylic C-H activation. The Gibbs free energies of the transition states with the native palladium acetate are much higher than a Pd-bis-phosphate species generated through ligand exchanges.

Mechanistic insights on cooperative asymmetric multicatalysis using chiral counterions.

Cooperative multicatalytic methods are steadily gaining popularity in asymmetric catalysis. The use of chiral Brønsted acids such as phosphoric acids in conjunction with a range of transition metals has been proven to be effective in asymmetric synthesis. However, the lack of molecular-level understanding and the accompanying ambiguity on the role of the chiral species in stereoinduction continues to remain an unresolved puzzle. Herein, we intend to disclose some novel transition state models obtained through DFT(B3LYP and M06) computations for a quintessential reaction in this family, namely, palladium-catalyzed asymmetric Tsuji-Trost allylation of aldehydes. The aldehyde is activated as an enamine by the action of a secondary amine (organocatalysis), which then adds to an activated Pd-allylic species (transition metal catalysis) generated through the protonation of allyic alcohol by chiral BINOL-phosphoric acid (Brønsted acid catalysis). We aim to decipher the nature of chiral BINOL-phosphates and their role in creating a quaternary chiral carbon atom in this triple catalytic system. The study reports the first transition state model capable of rationalizing chiral counterion-induced enantioselectivity. It is found that the chiral phosphate acts as a counterion in the stereocontrolling event rather than the conventional ligand mode.

Rational design of catalysts for asymmetric diamination reaction using transition state modeling.

The stereoselective synthesis of 1,2-diamines has remained a formidable challenge. A recent palladium-catalyzed asymmetric diamination of conjugated double bonds using di-tert-butyldiaziridinone appears promising. The axially chiral binol phosphoramidite ligands are successful in offering high enantioselectivity. The density functional theory investigations revealed that the energies of the stereocontrolling transition states for the C-N bond formation depend on a number of weak non-covalent interactions such as C-H···π, C-H···O and anagostic interactions. We envisaged that the modulation in these interactions in the transition states, through subtle changes in chiral phosphoramidite substituents, could be exploited toward steering the stereoselectivity. The effect of systematic modifications on both 3,3' positions of the binol as well as on the amido nitrogen on the stereochemical outcome is predicted. It is identified that high enantioselectivity requires a balance between the nature of the substituents on binol and amido groups. The reduced size of the amido substituents demands increased bulk on the binol whereas lowering the size on the binol demands increased bulk on the amido for higher stereoselectivity. The substituent at the α-position of the amido group is found to be vital and appears to be a hot spot for modifications. These insights derived from studies on the stereocontrolling transition states could help improve the catalytic efficacies in palladium-catalyzed asymmetric diamination reactions.

Axially chiral imidodiphosphoric Acid catalyst for asymmetric sulfoxidation reaction: insights on asymmetric induction.

Insights into chiral induction for an asymmetric sulfoxidation reaction involving a single oxygen atom transfer are gained through analyzing the stereocontrolling transition states. The fitting of the substrate into the chiral cavity of a new class of imidodiphosphoric Brønsted acids, as well as weak CH⋅⋅⋅π and CH⋅⋅⋅O noncovalent interactions, are identified as responsible for the observed chiral induction.

Unveiling the importance of catalyst framework and non covalent interactions in an asymmetric Fe-catalyzed O-H insertion: insights from computational tools.

Fe-based catalysts as well as enzymes typically yield low stereoselectivity for carbene insertion into X-H bonds. Here, we have utilized DFT methods to understand the mechanism and unusually high enantioselectivity in an Fe-spiroBox catalyzed carbene insertion reaction into the O-H bond of aliphatic alcohols. Our transition state model shows a unique binding of the reaction intermediates to the chiral catalyst enabled by weak non covalent interactions that is absent in other X-H insertion reactions.

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.

Developmental malformation of primary and permanent dentition: a rare sequel of trauma.

Dentofacial injuries that occur prior to the eruption of primary teeth can result in developmental disturbances not only in the primary but also in permanent dentition. Here we report a rare case of long term sequelae of trauma in a female child of 4 to 5 months of age which resulted in dilaceration and impaction of maxillary primary central incisors and subsequent enamel hypoplasia of the permanent maxillary central incisors.