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A link has been established between energy metabolism and sleep homeostasis. The ketone bodies acetoacetate and β-hydroxybutyrate, generated from the breakdown of fatty acids, are major metabolic fuels for the brain under conditions of low glucose availability. Ketogenesis is modulated by the activity of peroxisome proliferator-activated receptor alpha (PPARα), and treatment with a PPAR activator has been shown to induce a marked increase in plasma acetoacetate and decreased β-hydroxybutyrate in mice, accompanied by increased slow-wave activity during non-rapid eye movement (NREM) sleep. The present study investigated the role of ketone bodies in sleep regulation. Six-hour sleep deprivation increased plasma ketone bodies and their ratio (acetoacetate/β-hydroxybutyrate) in 10-week-old male mice. Moreover, sleep deprivation increased mRNA expression of ketogenic genes such as PPARα and 3-hydroxy-3-methylglutarate-CoA synthase 2 in the brain and decreased ketolytic enzymes such as succinyl-CoA: 3-oxoacid CoA transferase. In addition, central injection of acetoacetate, but not β-hydroxybutyrate, markedly increased slow-wave activity during NREM sleep and suppressed glutamate release. Central metabolism of ketone bodies, especially acetoacetate, appears to play a role in the regulation of sleep homeostasis.

A library of 16 2-substituted methyl acetoacetates containing ferrocenyl or phenyl units was designed to disclose differences in the antimicrobial activity of ferrocene-containing compounds and their phenyl analogs. Two methyl acetoacetates, whose structures do not contain an aromatic nucleus, were also included in order to probe the inherent activity of the scaffold itself. The acetoacetates were synthesized (low-to-good yields) and fully characterized by spectral (MS, IR, UV–Vis, 1D and 2D NMR) and electrochemical (cyclic voltammetry) techniques. Single-crystal X-ray analysis has been performed for methyl 2-acetyl-2-(ferrocenylmethyl)-5-methylhex-4-enoate. All compounds have demonstrated in vitro antimicrobial activity against six bacterial (three Gram-positive and three Gram-negative) and two fungal strains with minimal inhibitory concentration values of 0.0050–20.6 \(\upmu \hbox {mol mL}^{-1}\). The most active compound was 2-acetyl-2-(ferrocenylmethyl)-4-methylpent-4-enoate whose activity was comparable to that of nystatin against the yeast Candida albicans. Agglomerative hierarchical clustering statistical analysis of the antimicrobial assay data demonstrated that ferrocene-containing compounds have statistically different and greater antimicrobial activity when compared to their phenyl analogs.

The purpose of this study was to assess the metabolic profile of plasma samples from cows with clinical and subclinical ketosis. According to clinical signs and 3-hydroxybutyrate plasma levels, 81 multiparous Holstein cows were selected from a dairy farm 7 to 21 d after calving. The cows were divided into 3 groups: cows with clinical ketosis, cows with subclinical ketosis, and healthy control cows. (1)H-Nuclear magnetic resonance-based metabolomics was used to assess the plasma metabolic profiles of the 3 groups. The data were analyzed by principal component analysis, partial least squares discriminant analysis, and orthogonal partial least-squares discriminant analysis. The differences in metabolites among the 3 groups were assessed. The orthogonal partial least-squares discriminant analysis model differentiated the 3 groups of plasma samples. The model predicted clinical ketosis with a sensitivity of 100% and a specificity of 100%. In the case of subclinical ketosis, the model had a sensitivity of 97.0% and specificity of 95.7%. Twenty-five metabolites, including acetoacetate, acetone, lactate, glucose, choline, glutamic acid, and glutamine, were different among the 3 groups. Among the 25 metabolites, 4 were upregulated, 7 were downregulated, and 14 were both upregulated and downregulated. The results indicated that plasma (1)H-nuclear magnetic resonance-based metabolomics, coupled with pattern recognition analytical methods, not only has the sensitivity and specificity to distinguish cows with clinical and subclinical ketosis from healthy controls, but also has the potential to be developed into a clinically useful diagnostic tool that could contribute to a further understanding of the disease mechanisms.

The catalytic application of a novel manganese-containing periodic mesoporous organosilica with ionic-liquid framework (Mn@PMO-IL) in the Biginelli reaction was investigated. First, the Mn@PMO-IL nanocatalyst was prepared and characterized by TEM, SEM, X-ray photoelectron spectroscopy, and nitrogen-sorption analysis. The catalyst was then used in the one-pot Biginelli condensation of various aldehydes with urea and alkyl acetoacetates under solvent-free conditions. The corresponding dihydropyrimidone products were obtained in high to excellent yields and selectivities at short reaction times. Moreover, the catalyst was recovered and successfully reused many times with no notable decrease in activity and selectivity.

Reactions of alkyl acetoacetates, aromatic aldehydes, ammonium acetate, and 1,3-cyclohexanedione afford alkyl 4-aryl-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylates. The structure of compounds obtained was established with the help of IR, 1H NMR, and mass spectra and by X-ray diffraction analysis.

The reaction of 2,5-dimethyl- or 1,2,5-trimethyl-6-aminoindoles with β-ketoesters (methyl and ethyl acetoacetates, diethyl oxaloacetate) was found to proceed chemoselectively via the interaction between an amino group of indole and the carbonyl function of ketoester, leading to the formation of enamines. In contrast to enamines synthesized from similar aminoindoles and β-diketones, enamines obtained from β-ketoesters can easily undergo thermal cyclization, furnishing pyrrolo[3,2-f]quinolines with an angular catenation of rings.

In this article, we report the first examples of carbofluorination of unactivated alkenes. Under catalysis by AgNO3, the reactions of unactivated alkenes with Selectfluor reagent and active methylene compounds such as acetoacetates or 1,3-dicarbonyls in CH2Cl2–H2O–HOAc solution afforded the corresponding three-component condensation products under mild conditions. Furthermore, with the promotion of NaOAc, the AgOAc-catalyzed carbofluorination of various unactivated alkenes with Selectfluor and acetone proceeded smoothly in aqueous solution at 50 °C. The carbofluorination was efficient and highly regioselective, and enjoyed a broad substrate scope and wide functional group compatibility. These formal fluorine atom transfer radical addition reactions provide a convenient entry to structurally divergent, polyfunctional organofluorine compounds as versatile synthetic intermediates.

INTRODUCTION: While the KD holds promise as a therapeutic option for brain cancer patients, stringency of the diet impacts compliance. We have previously demonstrated that a high fat/low carbohydrate diet, similar to the Ketogenic Diet [KD], can reduce tumor progression and enhance survival in an orthotopic xenograft model. However, while this diet is less restrictive than the classic KD it still involves significant changes to a patient’s diet. Two of the primary physiological changes that occur when on the KD are a reduction in glucose and an increase in ketone bodies. These physiological changes are mimicked by providing ketone esters [KE] in the diet, and we [AMP, DPD], have recently shown that ketone esters can reduce glucose, elevate ketone bodies and enhance survival in a metastatic cancer model. HYPOTHESIS: We hypothesize that KE [1,3 butanediol acetoacetate diester] will reduce glucose, elevate ketones, reduce tumor progression and enhance survival in an orthotopic xenograft GBM model using a PDX model. APPROACH: NON/SCID animals implanted with patient-derived GBM cells were fed a standard diet [SD], or SD + KE [20%] till they reached endpoint. Body weight, plasma glucose, and ketones were measured weekly and overall survival assessed. RESULTS: While the KE is bitter and can have poor compliance, we found that supplementing with 1% Stevia increased palatability based on food consumption and body weight. Comparing to SD, KE supplemented diet reduced plasma glucose (145.5 ± 5.3 vs 121.7 ± 5.7), increased ketone bodies [ß-hydroxybutarate, 0.7 ± 0.15 vs.1.3 ± 0.1] and enhanced median survival [47 ± 6.2 vs 60.8 ± 1.9, days]. CONCLUSION: Ketone esters can be effectively delivered orally together with a standard diet, and produce similar physiological changes [reduction in glucose and elevation in ketones] and enhance survival as the more restrictive KD in NON/SCID animals.

Functionalized diazo acetoacetates are prepared by an efficient Mukaiyama aldol reaction between 3-TBSO-2-diazo-3-butenoate with aldehydes and acetals under mild reaction conditions. A variety of substituted aldehydes and the corresponding acetals are both accessible in good to excellent yields through this methodology. MgI2 etherate (MgI2·(OEt2)n) is the preferred catalyst and, the addition proceeds without decomposition of the diazo moiety. In addition, this MgI2·(OEt2)n-catalyzed Mukaiyama aldol reaction shows unique chemoselectivity towards aldehydes and acetals.

An ultra-high-throughput screening system for NADPH-dependent enzymes, such as stereospecific alcohol dehydrogenases, was established. It is based on the [2Fe-2S] cluster-containing transcriptional regulator SoxR of Escherichia coli that activates expression of soxS in the oxidized but not in the reduced state of the cluster. As SoxR is kept in its reduced state by NADPH-dependent reductases, an increased NADPH demand of the cell counteracts SoxR reduction and increases soxS expression. We have taken advantage of these properties by placing the eyfp gene under the control of the soxS promoter and analyzed the response of E. coli cells expressing an NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (LbAdh), which reduces methyl acetoacetate to (R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlated with the substrate concentration added and with LbAdh enzyme activity, supporting the NADPH responsiveness of the sensor. These properties enabled sorting of single cells harboring wild-type LbAdh from those with lowered or without LbAdh activity by fluorescence-activated cell sorting (FACS). In a proof-of-principle application, the system was used successfully to screen a mutant LbAdh library for variants showing improved activity with the substrate 4-methyl-2-pentanone.

Ketosis occurs as a metabolic consequence of negative energy balance in post-calving lactating dairy cows. Metabolism of free fatty acids, released from adipose tissue, generates excessive amounts of acetoacetate (AcAc), β-hydroxybutyrate (BHB), and acetone (Ac) in the liver, which are released into the blood. The effects of ketone bodies on endothelial cells include increased rates of portal vein and liver blood flow and decreased cytokine secretion in response to both bacterial and viral infections. The aim of the current study was to understand the effects of AcAc, BHB and Ac, on expression of connexin 43 (Cx43) and gap junctional intercellular coupling (GJIC) in bovine aortic endothelial cells (BAECs). Confocal microscopy, Western blotting, and real-time quantitative RT-PCR indicated that Cx43 mRNA and protein expression increased after endothelial cell exposure to ketone bodies and that this was accompanied by upregulation of GJIC and cell migration. These effects were most obvious when BAECs were treated with a combination of the three ketones. Ketone bodies were shown to activate ERK and p38 MAPK as early as 3h after treatment and an ERK inhibitor (PD98059) or p38 MAPK inhibitor (SB203580) were found to antagonise the ketone-induced increase in Cx43 protein expression. Thus, ketone bodies up-regulate Cx43 expression and GJIC in BAECs via activation of ERK and p38 MAPK.

Reactive oxygen species scavenging effect of Pleioblastus kongosanensis f. aureostriatus leaf extract against O2(-), OH and H2O2 were investigated by chemiluminescence methods in vitro. Bamboo grass leaves were extracted with 70% ethanol solution and sequentially partitioned with solvents in an order of increasing polarity. Among fractions of different polarity, BuOH and EtOAc fractions showed powerful scavenging activities than others, and showed better scavenging ability on OH than that of O2(-)and H2O2, with IC50 of 0.55 μg/mL and 0.60 μg/mL, respectively. Both OH-induced DNA damage model by chemiluminescence assay and plasmid pUC18 double-strand break model by agarose gel electrophoresis showed that BuOH and EtOAc fractions had remarkable concentration-dependent prevention effect on the OH-induced damage of DNA attribute to their good scavenging effects on ROS. Results from the compositional analysis of different fractions indicate that the flavonoids in the Pleioblastus kongosanensis f. aureostriatus leaf may be responsible for its ROS scavenging activity and DNA damage prevention ability.

Diabetes and alcohol abuse may cause severe metabolic disturbances that can be fatal. These may be difficult to diagnose in autopsies based solely on macroscopical and histological findings. In such cases, metabolic markers, such as postmortem glucose and ketone levels, can provide supporting information. Glucose or combined glucose and lactate, the Traub value, is often used to indicate hyperglycemia. The use of the Traub value, however, has been questioned by some, because the lactate levels are known to elevate in postmortem samples also due to other reasons than glycolysis of glucose molecules. Ketoacidosis can be detected by analyzing ketone body levels, especially beta-hydroxybutyric acid (BHB). Acetone is also elevated in severe cases of ketoacidosis. Here, we have evaluated the value of these biomarkers for postmortem determination of the metabolic disturbances. Retrospective data of 980 medico-legal autopsies performed in Finland, where glucose, lactate and ketone bodies were analyzed, was collected. Our findings show that the Traub value indicates hyperglycemia, even when glucose levels are low. For diagnosis, evaluation of complementing markers, e.g. ketone bodies and glycated hemoglobin is needed. Our results show that BHB can be used for screening and diagnosis of ketoacidosis. Acetone alone is not sufficient, since it is elevated only in the most severe cases. We also found that alcohol abuse rarely causes severe ketoacidosis. However, sporadic cases do exist where ketone body levels are extremely high. Despite this, alcoholic ketoacidosis is very rarely diagnosed as the cause of death.

We investigated the association of the levels of ketone bodies (KBs) with hyperglycemia and with 62 genetic risk variants regulating glucose levels or type 2 diabetes in the population-based Metabolic Syndrome in Men (METSIM) study, including 9,398 Finnish men without diabetes or newly diagnosed type 2 diabetes. Increasing fasting and 2-h plasma glucose levels were associated with elevated levels of acetoacetate (AcAc) and β-hydroxybutyrate (BHB). AcAc and BHB predicted an increase in the glucose area under the curve in an oral glucose tolerance test, and AcAc predicted the conversion to type 2 diabetes in a 5-year follow-up of the METSIM cohort. Impaired insulin secretion, but not insulin resistance, explained these findings. Of the 62 single nucleotide polymorphisms associated with the risk of type 2 diabetes or hyperglycemia, the glucose-increasing C allele of GCKR significantly associated with elevated levels of fasting BHB levels. Adipose tissue mRNA expression levels of genes involved in ketolysis were significantly associated with insulin sensitivity (Matsuda index). In conclusion, high levels of KBs predicted subsequent worsening of hyperglycemia, and a common variant of GCKR was significantly associated with BHB levels.

carnitine, and the (S)-enantiomer is an importantstarting material for hydroxymethylglutaryl-CoA reductaseinhibitors [11, 13]. Enzymatic reduction of ethyl-4-chloro-3-oxobutanoate (ECOB) by some reductase enzymes is auseful process in the asymmetric synthesis of chiral ECHB[1, 17, 19].These enzymatic reduction reactions require a largeamount of the expensive cofactor NADPH; hence, practicalapplications require efficient recycling of the cofactor [5,16]. Generally, cofactors can be regenerated through“coupled-substrate” [14, 21] and “coupled-enzyme” [5, 11,15] approaches. The “coupled-enzyme” method is the moregeneral approach and utilizes the first enzyme for thedesired biotransformation and the second to regenerate thecofactor. Until now, some X-ray crystal structures of carbonylreductase enzymes have been elucidated and their ternarycomplexes with substrate and cofactor have been disclosed[4, 8, 10]. Based on these early X-ray structures, manyhomology and docking models for other reductases havebeen suggested [8]. Detailed structural information aboutsubstrate- and cofactor-binding sites is very important tounderstand their reaction mechanisms. In addition, newmutant enzymes can be constructed through rational designand site-directed mutagenesis to change their cofactorpreference or enhance enantioselectivity [2, 12, 23].In a previous study, we generated a variety of microbialreductases in E. coli cells [3]. Among these, the yeastreductase YOR120W converted ECOB exclusively into(R)-ECHB. In this study, we purified and characterizedYOR120W and conducted a coupling reaction with Bacillus

An efficient synthetic procedure for the functionalized spiro[furan-3,3′-indoline] derivatives was successfully developed by domino reactions of N-phenacylpyridinium bromides or N-ethoxycarbonylmethylenepyridinium bromide with isatinylidene acetoacetate in the presence of triethylamine in ethanol at room temperature. The mechanism included sequential Michael addition of the in situ generated pyridinium ylide and intramolecular substitution of enolate.

The Acylase "Amano" (AA)-catalyzed synthesis of valuable pyridin-2-ones via domino Knoevenagel condensation-Michael addition-intramolecular cyclization-oxidization reaction between aldehyde, cyanoacetamide and ethyl acetoacetate or cyclohexyl acetoacetate was developed in the sense of a one-pot strategy. Various aliphatic, aromatic and hetero-aromatic pyridin-2-ones could also be produced in the reaction. The mechanism was illustrated according to the controlled reaction, pyridin-2-one was formed via the oxidization by oxygen at the final step. This simple and efficient enzymatic domino reaction not only widens its application of AA to organic synthesis, but is also an attractive way for the synthesis of heterocyclic compounds.

AbstactAim Diabetic ketoacidosis is a life-threatening complication of Type 1 diabetes. Blood β-hydroxybutyrate testing is now widely available as an alternative to urine acetoacetate testing for detecting ketosis. The aim of this study was to review the effectiveness of capillary or serum β-hydroxybutyrate compared with urine acetoacetate testing in prevention and management of diabetic ketoacidosis. Methods MEDLINE, EMBASE, EBM Reviews, The Cochrane Library and CINAHL (until April 2012, no language restrictions, studies in humans) were searched for experimental and observational studies comparing the effectiveness of blood β-hydroxybutyrate and urine acetoacetate testing. Outcomes examined were prevention of diabetic ketoacidosis, time to recovery from diabetic ketoacidosis, healthcare costs and patient or caregiver satisfaction. Additional sources included reference lists, conference proceedings and contact with experts in the field. Results Four studies (two randomized controlled trials and two cohort studies) met eligibility criteria, including 299 participants across 11 centres. Risk of bias was low to moderate. Blood ketone testing compared with urine testing was associated with reduced frequency of hospitalization (one study), reduced time to recovery from diabetic ketoacidosis (three studies), cost benefits (one study) and greater satisfaction (one study, intervention group only). No study assessed prevention of diabetic ketoacidosis. Meta-analysis could not be performed because of heterogeneity in study design and published data. Conclusions There is evidence suggesting that blood β-hydroxybutyrate testing is more effective than urine acetoacetate testing in reducing emergency department assessment, hospitalization and time to recovery from diabetic ketoacidosis, as well as potentially lowering healthcare expenditure. Further research in both young people and adults is needed.

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB + Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB + E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ∆atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ∆atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ∆atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ∆atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB + E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.

Treatment of [Re2(CO)8(CH3CN)2] with 2,7-dipyridinyl-1,8-naphthyridine (bpnp) thermally provided mononuclear [(N,N′-bpnp)Re(CO)3Cl] (1). Further reaction of 1 with rhenium carbonyls yielded ortho-metallation dinuclear rhenium complex 2. Both complexes were characterized by spectroscopic analyses and single-crystal X-ray determination. Complex 1 also reacted with [PdMeCl(COD)] and [Ir(COD)Cl]2 by means of ortho-metallation to yield the corresponding heterodinuclear species. Complex 2 appears to be an excellent precatalyst to promote the insertion of terminal alkynes into acetoacetates, which subsequently undergo cyclization to form 2-pyranones.

The biocatalytic anti-Prelog stereoselective reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate {(R)-EHB} was successfully conducted in the aqueous system using immobilized Acetobacter sp. CCTCC M209061 cells. Among various microorganisms tested, Acetobacter sp. CCTCC M209061 gave the best performance and showed great potential for the bioreduction of EAA to (R)-EHB. Acetobacter sp. CCTCC M209061 cells were immobilized on calcium alginate and then coated with chitosan, and the immobilized cells clearly surpassed the free cells in terms of better thermal stability, storability and recyclability. The optimal co-substrate for the reaction was found to be glucose. The optimal substrate concentration, buffer pH, glucose concentration, reaction temperature, biocatalyst concentration and shaking speed were 35.0 mmol/L, 5.5, 80.0 mmol/L, 35 °C, 0.45 g/mL and 200 rpm, respectively. Under the optimized conditions, the initial reaction rate, the yield and the enantiomeric excess (e.e.) of the product were 1.28 μmol/min, 82.6% and above 99.0%, respectively, which were much higher than those reported previously. The efficient whole-cell biocatalytic process was feasible on a 500-mL preparative scale, and the immobilized cells showed excellent operational stability and could be re-used for at least 10 batches. Additionally, the product e.e. constantly remained above 99.0% regardless of re-use cycles of the biocatalyst.

Mice with humanized chimeric liver are promising in vivo tools to evaluate the efficacy of novel compounds or vaccine induced antibodies directed against pathogens that infect the human liver. In addition they can be used to study the human-type metabolism of medicinal compounds and hepatotoxicity.

Objective Acute brain injury (ABI) is a catastrophic event, leading to disruption of the normal cerebral metabolic pathways and a subsequent cerebral energy deficit. Ketones (beta-hydroxybutyrate (BHB) and acetoacetate) may represent an alternative metabolic substrate with the potential to improve cerebral energy supply and decrease injury. The purpose of this study was to evaluate baseline ketone concentrations in the ABI population. Methods Thirty-eight patients with ABI were enrolled into the study and followed for up to 7 days. We collected arterial blood samples immediately after admission and daily to measure the levels of BHB and acetoacetate. Where possible, matching cerebrospinal fluid (CSF) specimens were also collected. Results During the study period, plasma BHB levels were increased initially but normalized by day 3 while acetoacetate levels remained within the normal range. The change in BHB was significant. There were 30 observations in 10 patients where BHB could be measured in both blood and CSF. When the data were averaged over patients there was a weak correlation between blood and CSF BHB (Spearman's ρ = 0.62, p = 0.054). Conclusion Blood ketone concentrations remain low within the ABI population. An external source of ketones will be required to increase blood concentrations to clinically relevant levels.

Dairy cows with ketosis are characterized by oxidative stress, hepatic damage, and hyperketonemia. Acetoacetate (AA) is the main component of ketone bodies in ketotic cows, and is associated with the above pathological process. However, the potential mechanism was not illuminated. Therefore, the aim of this study was to investigate the mechanism of AA-induced hepatic oxidative damage in ketotic cows. Compared with healthy cows, ketotic cows exhibited severe oxidative stress and hepatic damage. Moreover, the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of bovine hepatocytes. In this process, AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit nuclear factor E2-related factor 2 (Nrf2) expression, nuclear localization, and DNA-binding affinity, thereby inducing the overexpression of pro-apoptotic molecules Bax, Caspase 3, Caspase 9, PARP and inhibition of anti-apoptotic molecule Bcl-2. Antioxidant N-acetylcysteine (NAC) treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, these results indicate that AA triggers hepatocytes apoptosis via the ROS-mediated MAPKs pathway in ketotic cows.

A carbonyl reductase ( cr ) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363 mM with highest turnover number (112.77 ± 3.95 s −1 ). Solitary recombineering of such catalytic cell reproduced CHBE 161.04 g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp ( crp *, F136I) into this heterologous E. coli host yielded CHBE 477.54 g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88 g/L/gDCW. The CHBE production has thus been upscaled to 8–12 times than those reported so far. qRT-PCR studies revealed that both membrane efflux channels such as acrAB as well as ROS scavenger genes such as ahpCF have been activated by engineering crp . Moreover, membrane protecting genes such as manXYZ together with solvent extrusion associated genes such as glpC have been upregulated inside mutant host. Although numerous proteins have been investigated to convert COBE to CHBE; this is the first approach to use engineering triad involving crp engineering, recombinant DNA engineering and transporter engineering together for improving cell performance during two-phase biocatalysis.

A series of novel alkyl 5-hydroxy-7-aryl-5-methyl-1,3-dioxo-2-phenyl hexahydropyrazolo[1,2-a][1,2,4]triazole-6-carboxylates and alkyl 5-aryl-7-methyl-1,3-dioxo-2-phenyl-1,2,3,5-tetra hydropyrazolo[1,2-a][1,2,4]triazole-6-carboxylates have been synthesized. The synthesis of hydroxy pyrazolo[1,2-a][1,2,4]triazoles has been achieved by DBU mediated one pot reaction of aromatic aldehydes, alkyl acetoacetates and 4-phenylurazole at 35 °C in ethanol–water (1:1). Dehydration of hydroxy pyrazolo[1,2-a][1,2,4]triazoles has been achieved using conc. H2SO4 to afford dehydrated pyrazolo[1,2-a][1,2,4]triazoles in high yields. The structural assignments have been made by spectral data and unambiguously confirmed by X-ray analysis. Good yields, operational simplicity and moderate conditions are the advantages of the reactions.

An unprecedented Bronsted base ionic liquid tuned selective aminolysis of ester carbonyl of acetoacetates is demonstrated to achieve acetoacetamide derivatives. Other imidazolium ionic liquid performs an efficient cyclization catalysis involving acetoacetate-carbonyl groups and o-phenylenediamine at elevated temperature to produce benzimidazoles via C–C bond cleavage of intermediate 1,5-benzodiazepinones under solvent-free conditions.

A complication of diabetes is diabetic ketoacidosis (DKA), which if left untreated is a life threatening condition. Prompt and accurate diagnosis of DKA is required for the commencement of life saving interventions. Measurements of ketone bodies in DKA have usually been through nitroprusside urine acetoacetate testing. The aim of this systematic review was to examine whether capillary β-hydroxybutyrate (β-OHB) testing is more accurate compared to other diagnostic methods of DKA. The following electronic databases were searched: EBSCO Host, MEDLINE, PSYCHInfo, CINAHL and Science Direct for publications from 1 January 2005 and up to and including 1 January 2016. Inclusion criteria were: Adults 18 years and over and known type 1 or type 2 diabetes. Retrospective and prospective observation studies were included. A total of nine studies met the inclusion criteria. Capillary β-OHB was found to have high sensitivity, specificity, positive predictive value and negative predictive value in identifying DKA compared to urinary ketone testing.

The concept of noncovalent organocatalysis by means of networks of cooperative hydrogen bonds (NCHB organocatalysis) has been explored. Arylideneureas were chosen as ideal substrates because of their powerful donor-acceptor properties. We have examined their uncatalyzed, direct Mannich reaction with acetoacetates in comparison with that catalyzed by a number of salan derivatives capable of providing a network of cooperative hydrogen bonds. Catalyst D [(R,R)-N,N'-bis(salicyl)cyclohexane-1,2-diamine] was found to drive the above direct Mannich reaction in an enantioselective manner, thereby allowing the synthesis of several Biginelli dihydropyrimidinones with high enantioselectivity. DFT calculations (B3LYP-D-PCM/6-31+G*//B3LYP/6-31+G*) revealed that the NCHB organocatalyst lowers the energy barrier of the reaction. The NCHB organocatalysts appear to function as biomimetic catalysts.

The first asymmetric, Bronsted acid catalyzed Biginelli-like reaction of a ketone has been developed, employing N-substituted isatins as carbonyl substrates, and urea and alkyl acetoacetates as further components. BINOL-derived phosphoric acid catalysts have been used to achieve the synthesis of a small library of chiral, enantioenriched spiro(indoline-pyrimidine)-diones derivatives. The absolute configuration of the new spiro stereocenter was assessed on diastereoisomeric derivatives through computer-assisted NMR spectroscopy. X-ray diffractometry allowed the disclosure of the overall molecular conformation in the solid state and the characterization of the crystal packing of a Br-substituted Biginelli-like derivative, while computational studies on the reaction transition state allowed us to rationalize the stereochemical outcome.

To determine whether basal lipid vacuolization characteristic of ketoacidosis could be induced with short-term hypertriglyceridemia, adult Sprague Dawley rat kidneys were perfused in an isolated perfused kidney model with, and without, 11.3 mM (10 g/L) of triglycerides in Krebs-Henseleit buffer, for 1 and 2 h (n = 5/group). Additional treatments included perfusion with triglycerides with 20 mM of β-hydroxybutyrate and 2 mM of acetoacetate (n = 5) and perfusion with triglycerides with 70 mM of glucose (n = 1). Basal vacuolization was produced in all groups, but differed in morphology to that reported in postmortem studies. There was no further increase in vacuolization after 2 h of perfusion compared to 1 h (p = 0.24), and the addition of ketones did not alter the morphology or extent of vacuolization. This study using an ex vivo model has confirmed that isolated hypertriglyceridemia is sufficient to cause basal lipid vacuolization in renal tubular epithelial cells, but with different morphology to vacuoles observed in lethal ketoacidosis at autopsy.

A novel one pot synthesis of pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine-diones, via a five-component reaction, involving, hydrazine hydrate, ethyl acetoacetate, and 1,3-dimethyl barbituric acid, an appropriate aryl aldehydes and ammonium acetate catalyzed via both of heterogeneous and homogeneous catalysis in water, is reported.

It was the first report that the concentrated hydrolyzates from the enzymatic hydrolysis of dilute NaOH (3 wt%)-soaking rice straw at 30 °C was used to form [Bmim]PF 6 -hydrolyzate (50:50, v/v) media for bioconverting ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl ( R )-4-chloro-3-hydroxybutanoate [( R )-CHBE] (>99% e.e. ) with recombinant E. coli CCZU-A13. Compared with pure glucose, the hydrolyzates could promote both initial reaction rate and the intracellular NADH content. Furthermore, emulsifier OP-10 (20 mM) was employed to improve the reductase activity. Moreover, Hp-β-cyclodextrin (0.01 mol Hp-β-cyclodextrin/mol COBE) was also added into this bioreaction system for enhancing the biosynthesis of ( R )-CHBE from COBE by E. coli CCZU-A13 whole-cells. The yield of ( R )-CHBE (>99% e.e. ) from 800 mM COBE was obtained at 100% in the [Bmim]PF 6 -hydrolyzate (50:50, v/v) media by supplementation of OP-10 (20 mM) and Hp-β-CD (8 mM). In conclusion, an effective strategy for the biosynthesis of ( R )-CHBE was successfully demonstrated.

Elevated levels of blood interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor–α (TNF–α) increase insulin resistance and result in inflammation. It is not clear whether elevated blood level of acetoacetate (ACAC) and decreased blood level of glucose, which are the predominant characteristics of clinical biochemistry in ketotic dairy cows, increase proinflammatory cytokines and subsequent inflammation. The objective of this study was to test the hypothesis that ACAC and glucose activate the NF-κB signalling pathway to regulate cytokines expression in bovine hepatocytes. Bovine hepatocytes were cultured with ACAC (0–4·8 mm) and glucose (0–5·55 mm) with or without NF-κB inhibitor PDTC for 24 h. The secretion and mRNA levels of cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The NF-κB signalling pathway activation was evaluated by western blotting. Results showed that the secretion and expression of IL-1β, IL-6 and TNF-α increased in an ACAC dose-dependent manner. Additionally, there was an increase in the secretion and mRNA expression of these three cytokines in glucose treatment group, which increased significantly when the glucose concentrations exceed 3·33 mm. Furthermore, both ACAC and glucose upregulated NF-κB p65 protein expression and IκBα phosphorylation levels. However, these effects were reduced by PDTC. These results demonstrate that elevated levels of ACAC and glucose increase the synthesis and expression of proinflammatory factors by activating NF-κB signalling pathway in hepatocytes, which may contribute to inflammation injury in ketotic dairy cows.

Cyclocondensation of salicylaldehydes with alkyl acetoacetates and 2-aminobenzothiazoles or 2-amino-thiadiazole/thiazoles under l -proline catalysis gives 4-hetarylamino substituted chromanecarboxylate derivatives. The mechanism involving the Mannich/hemiketalization cascade reaction and the observed stereoselectivity of the three component process are discussed.

A series of novel pyrazole amide derivatives were designed and synthesized by multi-step reactions from phenylhydrazine and ethyl 3-oxobutanoate as starting materials, and their structures were characterized by NMR, MS and elemental analysis. The antifungal activity of the title compounds was determined. The results indicated that some of title compounds exhibited moderate antifungal activity. Furthermore, DFT calculations were used to study the structure-activity relationships (SAR).

Background Many tissues play an important role in metabolic homeostasis and the development of diabetes and obesity. We hypothesized that the circulating redox metabolome is a master metabolic regulatory system that impacts all organs and modulates reactive oxygen species (ROS) production, lipid peroxidation, energy production and changes in lipid turnover in many cells including adipocytes. Methods Differentiated human preadipocytes were exposed to the redox couples, lactate (L) and pyruvate (P), β–hydroxybutyrate (βOHB) and acetoacetate (Acoc), and the thiol-disulfides cysteine/ cystine (Cys/CySS) and GSH/GSSG for 1.5–4 hours. ROS measurements were done with CM-H2DCFDA. Lipid peroxidation (LPO) was assessed by a modification of the thiobarbituric acid method. Lipolysis was measured as glycerol release. Lipid synthesis was measured as 14C-glucose incorporated into lipid. Respiration was assessed using the SeaHorse XF24 analyzer and the proton leak was determined from the difference in respiration with oligomycin and antimycin A. Results Metabolites with increasing oxidation potentials (GSSG, CySS, Acoc) increased adipocyte ROS. In contrast, P caused a decrease in ROS compared with L. Acoc also induced a significant increase in both LPO and lipid synthesis. L and Acoc increased lipolysis. βOHB increased respiration, mainly due to an increased proton leak. GSSG, when present throughout 14 days of differentiation significantly increased fat accumulation, but not when added later. Conclusions We demonstrated that in human adipocytes changes in the external redox state impacted ROS production, LPO, energy efficiency, lipid handling, and differentiation. A more oxidized state generally led to increased ROS, LPO and lipid turnover and more reduction led to increased respiration and a proton leak. However, not all of the redox couples were the same suggesting compartmentalization. These data are consistent with the concept of the circulating redox metabolome as a master metabolic regulatory system.

The effect of three-day fasting on cardiac ischemic tolerance was investigated in adult male Wistar rats. Anesthetized open-chest animals (pentobarbitone 60 mg/kg, i.p.) were subjected to 20-min left anterior descending coronary artery occlusion and 3-h reperfusion for infarct size determination. Ventricular arrhythmias were monitored during ischemia and at the beginning (3 min) of reperfusion. Myocardial concentrations of beta-hydroxybutyrate and acetoacetate were measured to assess mitochondrial redox state. Short-term fasting limited the infarct size (48.5±3.3 % of the area at risk) compared to controls (74.3±2.2 %) and reduced the total number of premature ventricular complexes (12.5±5.8) compared to controls (194.9±21.9) as well as the duration of ventricular tachycardia (0.6±0.4 s vs. 18.8±2.5 s) occurring at early reperfusion. Additionally, fasting increased the concentration of beta-hydroxybutyrate and beta-hydroxybutyrate/acetoacetate ratio (87.8±27.0) compared to controls (7.9±1.7), reflecting altered mitochondrial redox state. It is concluded that three-day fasting effectively protected rat hearts against major endpoints of acute I/R injury. Further studies are needed to find out whether these beneficial effects can be linked to altered mitochondrial redox state resulting from increased ketogenesis.

Type 1 diabetic patients have hyperketonemia, elevated levels of pro-inflammatory and oxidative stress markers, and a higher incidence of vascular disease. This study examines the hypothesis that hyperketonemia increases reactive oxygen species (ROS) and is in part responsible for increased expression of adhesion molecules in monocytes. THP-1 monocytes were treated with acetoacetate (AA) or β-hydroxybutyrate (BHB) (0–10 mmol/L) for 24 h. Results show that AA, but not BHB, increases ROS production in monocytes. Pretreatment of monocytes with N-acetylcysteine (NAC) inhibited AA-induced ROS production. AA treatment induced upregulation of LFA-1 and pretreatment of monocytes with NAC or an inhibitor to p38 MAPK inhibited this upregulation in monocytes. This suggests that physiological concentrations of AA can contribute to increased ROS and activation of p38 MAPK, which may be responsible for AA-induced upregulation of LFA-1 in monocytes. Thus, hyperketonemia contributes to the risk for cardiovascular disease in type 1 diabetes.

Hereditary tyrosinemia type I (HT1) is an autosomal recessive disease caused by a deficiency in human fumarylacetoacetate (FAA) hydrolase (FAH), which is the last enzyme in the catabolic pathway of tyrosine. Several reports suggest that intracellular accumulation of intermediates of tyrosine catabolism, such as FAA and succinylacetone (SA) is important for the pathogenesis in liver and kidney of HT1 patients. In this work, we examined the effect of FAA and SA on DNA glycosylases initiating base excision repair (BER), which is the most important pathway for removing mutagenic DNA base lesions. In vitro assays monitoring DNA glycosylase activities demonstrated that FAA but not SA inhibited base removal. In particular, the Neil1 and Neil2 DNA glycosylases were strongly inhibited, whereas inhibition of Nth1 and Ogg1 were less efficient. These DNA glycosylases initiate excision of a broad range of mutagenic oxidative base lesions. Further, FAA showed a modest inhibitory effect on the activity of the alkylbase DNA glycosylase Aag and no significant inhibition of the uracil DNA glycosylase Ung2. These data indicate that FAA inhibition of DNA glycosylases removing oxidative base lesions in HT1 patients may increase mutagenesis, suggesting an important mechanism for development of hepatocarcinoma and somatic mosaicism.

Microwave-assisted synthesis of 3,4-dihydropyrimidin-2-thiones via Biginelli reaction from aldehydes, acetoacetates, and thiourea in the presence of iodine under solvent-free conditions has been accomplished in good yields and purity without chromatographic separation.

Nitric oxide adds to methyl acetoacetate in the presence of KOH in methanol at room temperature to form potassium acetylsydnonate N-oxide (K1) with an (E)-diazeniumdiolation and potassium acetate diazenium diolate (K(2)2) from a (Z)-diazeniumdiolation. A study of the reaction with LiOH, NaOH, and NMe(4)OH and with ethyl acetate substrate reveals that the temperature of the reaction greatly influences the nitric oxide reactivity. At 23 °C, nitric oxide adds to give both E and Z products, whereas at -5 °C the gas reacts almost exclusively to give Z addition. The (Z)-diazeniumdiolation products, namely, the alkali metal and NMe(4)(+) salts of methyl and ethylbutenoate-2-diazeniumdiolate-3-hydroxylate (3(2-) and 4(2-)), are isolated in good yields. The alkali metal salts are not amenable for recrystallization because of their ready decomposition in aqueous solutions. However, [NMe(4)](2)[MeC(O)C(N(2)O(2))CO(2)Me] is readily recrystallized from a methanol/acetonitrile solvent mixture. The crystals are unambiguously characterized by X-ray crystallography. NMR spectra for all of the 3(2-) and 4(2-) salts reveal the presence of two isomers in aq solutions. But the structure of the NMe(4)(+) salt contains only one of the isomers. Our attempts to cyclize the isolated and purified butenoatediazeniumdiolates from the (Z)-diazeniumdiolation to the E-containing sydnonate products were unsuccessful. TGA/DSC data for all of the products demonstrate the thermal instability of the salts at high temperatures. The salts decompose exothermally possibly with the release of N(2)O among other gases.

Verapamil, diltiazem and nifedipine are Ca(2+)-channel blockers used in cardiovascular diseases. We report here that the Escherichia coli AtoSC signaling is inhibited by those blockers. AtoSC two-component system plays a pivotal role in sophisticated signaling networks in E. coli regulating processes implicated in bacterial homeostasis and pathogenicity. The Ca(2+)-channel blockers abrogated the in vitro full-length AtoS kinase autophosphorylation. However, they demonstrated no effect on the AtoS cytoplasmic form autophosphorylation. AtoC protected AtoS from verapamil or diltiazem but not from nifedipine, when the two constituents formed complex. The blockers did not affect the AtoS≈P to AtoC phosphotransfer. The blockers-mediated AtoSC inhibition was verified in vivo on the atoDAEB expression, which was inhibited only in AtoSC-expressing bacteria upon acetoacetate. The AtoS and AtoC protein or their genes transcription levels were unaffected by the blockers. Blockers demonstrated differential effects in the regulation of both the cytosolic- and most potently the membrane-bound-cPHB. Extracellular Ca(2+) counteracted the verapamil-mediated effect on cPHB only in atoSC(+) cells. Extracellular Ca(2+) reversed the diltiazem-mediated cPHB decreases in cells of both genetic backgrounds, yet a Ca(2+)-concentration dependent reversion was observed only in the AtoSC-regulated cPHB. Nifedipine caused a more pronounced cPHB down-regulation that was not reversed by extracellular Ca(2+). The AtoSC signaling inhibition by Ca(2+)-channel blockers used for human treatment, and their differential effects on cPHB-formed Ca(2+)-channels, signify their implications in bacterial-host interactions through the two-component signaling and could stimulate the design of Ca(2+)-channels blockers derivatives acting as inhibitors of two-component systems.

AtoSC two-component system plays a pivotal role in many regulatory indispensable Escherichia coli processes. AtoSCDAEB regulon, comprising the AtoSC system and the atoDAEB operon, regulates the short-chain fatty acids catabolism. We report here, that AtoSC up-regulates the high-molecular weight PHB biosynthesis, in recombinant phaCAB + E. coli , with the Cupriavidus necator phaCAB operon. PHB accumulation was maximized upon the acetoacetate-mediated induction of AtoSC, under glucose 1% w/v, resulting in a yield of 1.73 g/l with a biopolymer content of 64.5% w/w. The deletion of the atoSC locus, in the Δ atoSC strains, resulted in a 5 fold reduction of PHB accumulation, which was restored by the extrachromosomal introduction of the AtoSC system. The deletion of the atoDAEB operon triggered a significant decrease in PHB synthesis in Δ atoDAEB strains. However, the acetoacetate-induced AtoSC system in those strains increased PHB to 1.55 g/l, while AtoC expression increased PHB to 1.4 g/l upon acetoacetate. The complementation of the Δ atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. The individual inhibition of β-oxidation and mainly fatty-acid biosynthesis pathways by acrylic acid or cerulenin respectively, reduced PHB biosynthesis. Under those conditions the introduction of the atoSC locus or the atoSCDAEB regulon was capable to up-regulate the biopolymer accumulation. The concurrent inhibition of both the fatty acids metabolic pathways eliminated PHB production. PHB up-regulation in phaCAB + E. coli , by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay, provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards the biotechnologically improved polyhydroxyalkanoates biosynthesis.

Microalgal photo-biocatalysis is a green technique for asymmetric synthesis. Asymmetric reduction of nonnatural prochiral ketones to produce chiral alcohols by microalgal photo-biocatalysis was studied in this work. Acetophenone (ACP) and ethyl acetoacetate (EAA) were chosen as model substrates for aromatic ketones and β-ketoesters, respectively. Two prokaryotic cyanophyta and two eukaryotic chlorophyta were selected as photo-biocatalysts. The results proved that nonnatural prochiral ketones can be reduced by microalgal photo-biocatalysis with high enantioselectivity. Illumination is indispensable to the photo-biocatalysis. For aromatic ketone, cyanophyta are eligible biocatalysts. For ACP asymmetric reduction reaction, about 45% yield and 97% e.e. can be achieved by the photo-biocatalysis reaction with Spirulina platensis as biocatalyst. On the contrary, chlorophyta are efficient biocatalysts for β-ketoester asymmetric reduction reaction among the four tested algae. For EAA asymmetric reduction reaction, about 70% yield and 90% e.e. can be achieved with Scenedesmus obliquus as biocatalyst. The microalgae used in this study outperformed other characterized biocatalysts such as microbial and plant cells.

Acridinediones were synthesized by the one-pot Hantzsch condensation of an aromatic aldehyde, 5,5-dimethyl-1,3-cyclohexanedione, and aniline/4-methylaniline in refluxing water. This method has then been extended to the four-component reaction of an aromatic aldehyde, 5,5-dimethyl-1,3-cyclohexanedione, ethyl acetoacetate and ammonium acetate for the synthesis of polyhydroquinoline derivatives. This is an environmentally friendly and efficient procedure providing good to excellent yields.

Human cysticercosis caused by Taenia crassiceps is rare however it is considered of zoonotic risk. The treatment of the infected patients was successful when using albendazole or praziquantel. The active forms of albendazole inhibit the glucose uptake and the active forms of praziquantel alter glycogen levels and nutrients absorption. The aim of this study was to analyze the production of organic acids that indicate the oxidation of fatty acids and the use of alternative energy sources from T. crassiceps cysticerci removed from the peritoneal cavity of mice treated with low dosages of albendazole (5.75 and 11.5mg/kg) or praziquantel (3.83 and 7.67mg/kg). The beta-hydroxibutyrate production was higher by the larval stage cysticerci in all treated groups and the propionate production was higher in final stage cysticerci treated with 11.5mg/kg of albendazole when compared to the control group. The larval stages of cysticerci from the groups treated with 5.75mg/kg of albendazole and 3.83mg/kg of praziquantel produced more urea than the initial and final stages which indicate amino acids breakdown. We conclude that it was possible to detect the fatty acid oxidation and amino acids breakdown which indicate the use of alternative energy production sources as the used dosages only cause a partial blockage of the glucose uptake and leads to metabolic alterations in the cysticerci. The metabolic behavior observed after host treatment was different from former descriptions of the in vitro one which indicates great host-parasite interaction.

Sucrose esters consisting of acetoacetates and fatty acids were invented as novel biobased amine-cured and autoxidation dual-cure coating systems, in the presence of diamine crosslinker and metal salt driers. Acetoacetylated sucrose soyate (ASS) was an intramolecular system containing both acetoacetates and fatty acids in the same molecule. Miscible bicomponent blends, made of ASS and partially enamine alkyl substituted acetoacetylated sucrose (p-EAS), was an intermolecular curing system. Compatibility between the two components was achieved by reacting acetoacetylated sucrose (AS) with alkyl amines, in which the compatibility could be adjusted by either the alkyl chain length or the degree of enamine alkyl substitution, DES. Sucrose esters were characterized by gel permeation chromatography (GPC), Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and differential scanning calorimetry (DSC). The properties and drying times of coatings cured through autoxidation, amine-acetoacetate reactions, and by dual-curing were studied. It was found that acetoacetate chemistry improved the attributes of sucrose ester of fatty acids in ambient curing conditions, as well as providing dual-cure coatings having a good balance of properties.

The axial-equatorial conformational isomer distribution of the reactant diazoacetoacetate or its metal carbene intermediate is reflected in Rh(II) catalyzed oxonium ylide forming reactions of 3-(trans-2-arylvinyl)tetrahydropyranone- 5-diazoacetoacetates that afford diastereoisomeric products for both the symmetry-allowed [2,3]- and the formally symmetry-forbidden [1,2]-oxonium ylide rearrangements.