Model-assisted DoE software: optimization of growth and biocatalysis in Saccharomyces cerevisiae bioprocesses.

Bioprocess development and optimization are still cost- and time-intensive due to the enormous number of experiments involved. In this study, the recently introduced model-assisted Design of Experiments (mDoE) concept (Möller et al. in Bioproc Biosyst Eng 42(5):867, https://doi.org/10.1007/s00449-019-02089-7 , 2019) was extended and implemented into a software ("mDoE-toolbox") to significantly reduce the number of required cultivations. The application of the toolbox is exemplary shown in two case studies with Saccharomyces cerevisiae. In the first case study, a fed-batch process was optimized with respect to the pH value and linearly rising feeding rates of glucose and nitrogen source. Using the mDoE-toolbox, the biomass concentration was increased by 30% compared to previously performed experiments. The second case study was the whole-cell biocatalysis of ethyl acetoacetate (EAA) to (S)-ethyl-3-hydroxybutyrate (E3HB), for which the feeding rates of glucose, nitrogen source, and EAA were optimized. An increase of 80% compared to a previously performed experiment with similar initial conditions was achieved for the E3HB concentration.

Melanoma-Time to fast or time to feast? An interplay between PPARs, metabolism and immunity.

Development and progression of melanoma can be accelerated by intensification of particular metabolic pathways, such as aerobic glycolysis and avid amino acid catabolism, and is accompanied by aberrant immune responses within the tumor microenvironment. Contrary to other cancer types, melanoma reveals some unique tissue-specific features, such as melanogenesis, which is intertwined with metabolism. Nuclear peroxisome proliferator-activated receptors (PPARs) take part in regulation of systemic and cellular metabolism, inflammation and melanogenesis. They appear as a focal regulatory point for these three distinct processes by occupying the intersection among AMP-dependent protein kinase (AMPK), mammalian target of rapamycin (mTOR) and PPAR gamma coactivator 1-alpha (PGC-1α) signalling pathways. When deregulated, they may accelerate melanoma malignant growth. Presenting the contribution of PPARα and PPARγ in melanoma biology, we attempt to ask how two contrasting metabolic states: obesity and fasting, can change progression of the disease and possible outcome of the treatment. This short essay is aimed to provoke a discussion about some practical implications for melanoma prevention and treatment, especially: how metabolic manipulation may be exploited to overcome immunosuppression and support immune checkpoint blockade efficacy.

Modification of Starch via the Biginelli Multicomponent Reaction.

An efficient and straightforward modification of starch using renewable and commercially available aromatic aldehydes (benzaldehyde, vanillin, and p-anisaldehyde) and urea via the Biginelli multicomponent reaction is reported in this work. First, starch acetoacetate (SAA) with a degree of substitution ranging from 1.4 to 2.5, depending on the reaction time or the molar ratio of reactants, is prepared. SAA is then modified with different aromatic aldehydes and urea via the Biginelli reaction. The modified products are characterized by ATR-IR, NMR, and gel permeation chromatography (GPC). The processability of the products is also investigated using a hot press instrument, revealing that glycerol is a suitable and renewable plasticizer for the Biginelli products.

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental study backed by density functional calculations.

The role of pH-dependent ionic structures of L-amino acids in catalysis has been investigated for the two-component Mannich reactions between dimethyl malonate (DMM)/ethyl acetoacetate (EAA) and imines. As catalysts, L-amino acids performed well, even better than corresponding base catalysts and provided the ß-amino carbonyl compounds in very high yields. Density functional calculations were used to gain the mechanistic insight of the reaction. High catalytic efficiency of amino acids was attributed to the facile formation of carbanion intermediate through barrierless transition state TS1 (- 19.43 kcal/mol) and then its stabilization owing to carbanion interaction with protonated amino acid.

Direct assessment of renal mitochondrial redox state using hyperpolarized 13 C-acetoacetate.

PURPOSE: The purpose of this study was to investigate the hyperpolarized ketone body 13 C-acetoacetate (AcAc) and its conversion to 13 C-ß-hydroxybutyrate (ßOHB) in vivo, catalyzed by ß-hydroxybutyrate dehydrogenase (BDH), as a novel direct marker of mitochondrial redox state. METHODS: [1,3-13 C2 ]AcAc was synthesized by hydrolysis of the ethyl ester, and hyperpolarized via dissolution DNP. Cold storage under basic conditions resulted in sufficient chemical stability for use in hyperpolarized (HP) MRI studies. Polarizations and relaxation times of HP [1,3-13 C2 ]AcAc were measured in a clinical 3T MRI scanner, and 8 rats were scanned by dynamic HP 13 C MR spectroscopy of a slab through the kidneys. Four rats were scanned after acute treatment with high dose metformin (125 mg/kg, intravenous), which is known to modulate mitochondrial redox via inhibition of mitochondrial complex I. An additional metformin-treated rat was scanned by abdominal 2D CSI (8 mm × 8 mm). RESULTS: Polarizations of 7 ± 1% and 7 ± 3%, and T1 relaxation times of 58 ± 5 s and 52 ± 3 s, were attained at the C1 and C3 positions, respectively. Rapid conversion of HP AcAc to ßOHB was detected in rat kidney in vivo, via the C1 label. The product HP ßOHB was resolved from closely resonating acetate. Conversion to ßOHB was also detected via 2D CSI, in both kidney as well as liver regions. Metformin treatment resulted in a significant increase (40%, P = 0.01) of conversion of HP AcAc to ßOHB. CONCLUSION: Rapid conversion of HP AcAc to ßOHB was observed in rat kidney in vivo and is a promising new non-invasive marker of mitochondrial redox state. Magn Reson Med 79:1862-1869, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Current progress in asymmetric Biginelli reaction: an update.

The Biginelli reaction, involving a three-component reaction of an aromatic aldehyde, urea and ethyl acetoacetate, has emerged as an extremely useful synthetic tool to organic chemists for the synthesis of 3,4-dihydropyrimidine-2-(1H)-ones and related heterocyclic compounds. In the past decades, the asymmetric variants of this reaction have been at the forefront of investigations in several research groups. In 2013, we highlighted the developments occurred in the asymmetric version of the Biginelli reaction. This review article focuses on the recent developments of asymmetric Biginelli reaction covers the literature going back to 2012.

Utility of Von Pechman synthesis of coumarin reaction for development of spectrofluorimetric method for quantitation of salmeterol xinafoate in pharmaceutical preparations and human plasma.

Simple, precise and selective spectrofluorimetric technique was evolved for quantitation of selective ß2 agonist drug namely salmeterol xinafoate (SAL). Utilizing its phenolic nature, a method was described based on the reaction of the studied drug with ethyl acetoacetate (EAA) to yield extremely fluorescent coumarin product which can be detected at 480 nm (λex  = 420 nm). The procedure obeys Beer's law with a correlation coefficient of r = 0.9999 in the concentration range between 500 and 5000 ng ml-1 with and 177 ng ml-1 for limit of detection (LOD) and limit of quantification (LOQ), respectively. Diverse reaction variables influencing the firmness and formation of the coumarin product were accurately examined and modified to ensure greatest sensitivity of the procedure. The proposed technique was performed and examined according to the US Food and Drug Administration (FDA) guidelines for bio-analytical methods and was efficiently applied for quantitation of SAL in both pharmaceutical preparations (% recovery = 100.06 ± 1.07) and spiked human plasma (% recovery = 96.64-97.14 ± 1.01-1.52).

Determination of Formaldehyde in Water Samples by High-Performance Liquid Chromatography with Methyl Acetoacetate Derivatization.

A high-performance liquid chromatography method with methyl acetoacetate derivatization via the Hantzsch reaction was developed for the analysis of formaldehyde (HCHO) in several water samples. Under optimized conditions, HCHO was detected within 4 min and was not affected by excessive derivatization reagents. The calibration curve constructed from the peak height of HCHO was linear, with a correlation coefficient of 0.9998. The relative standard deviation of the peak height from ten replicates was 0.29%. The detection and quantitative limits were 0.96 µg/L and 3.16 µg/L, respectively. A recovery test of HCHO was performed to compare the developed method with the official analysis method (DNPH method). The developed method was used to determine the HCHO levels in several water samples (tap water, river water, and waste water).

ReactELISA: Monitoring a Carbon Nucleophilic Metabolite by ELISA-a Study of Lipid Metabolism.

We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.

Inclusion of Ethyl Acetoacetate Bearing 7-Hydroxycoumarin Dye by ß-Cyclodextrin and its Cooperative Assembly with Mercury(II) Ions: Spectroscopic and Molecular Modeling Studies.

The inclusion of the fluorescent organic dye, ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-oxopropanoate (1) by the host ß-cyclodextrin (ß-CD), and its response toward mercuric ions (Hg2+ ), was studied by UV/Vis, fluorescence, and 1 H NMR spectroscopic analyses, mass spectrometry and molecular modeling studies. 1 H NMR measurements together with molecular modeling studies for dye 1 demonstrate that it exhibits two tautomeric forms (keto and enol); however, when the dye is included into the ß-CD cavity, the enol form predominates. Moreover, by using spectroscopic and spectrometry techniques, a 1:1 stoichiometry was determined for the complexes formed between dye 1 (enol form) and ß-CD, with a binding constant (Kb1 =1.8×104 m-1 ) and for the dye 1 (keto form)-Hg2+ (Kb2 =2.3×103 m-1 ). Interestingly, in the presence of 1-ß-CD complex and mercuric ions, a ternary supramolecular system (Hg-1-ß-CD complex) was established, with a 1:1:1 stoichiometry and a Kb3 value of 4.3×103 m-1 , with the keto form of the dye being the only one present in this assembly. The three-component system provides a starting point for the development of novel and directed supramolecular assemblies.

High Glass Transition Temperature Renewable Polymers via Biginelli Multicomponent Polymerization.

A novel and straightforward one-pot multicomponent polycondensation method was established in this work. The Biginelli reaction is a versatile multicomponent reaction of an aldehyde, a ß-ketoester (acetoacetate) and urea, which can all be obtained from renewable resources, yielding diversely substituted 3,4-dihydropyrimidin-2(1H)-ones (DHMPs). In this study, renewable diacetoacetate monomers with different spacer chain lengths (C3, C6, C10, C20) were prepared via simple transesterification of renewable diols and commercial acetoacetates. The diacetoacetate monomers were then reacted with renewable dialdehydes, i.e., terephthalaldehyde and divanillin in a Biginelli type step-growth polymerization. The obtained DHMP polymers (polyDHMPs) displayed high molar masses, high glass transition temperatures (Tg) up to 203 °C and good thermal stability (Td5%) of 280 °C. The Tg of the polyDHMPs could be tuned by variation of the structure of the dialdehyde or the diacetoacetate component.

Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20.

Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate for the synthesis of "blockbuster" drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bio-reductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of ChKRED20 to allow a higher reaction temperature. After one round of error-prone PCR (epPCR) library screening followed by the combination of beneficial mutations, a triple-mutant MC135 was successfully achieved with substantially enhanced thermostablity. The activity of MC135 at 50 °C was similar to the wild type. However, at its temperature optima of 65 °C, the mutant displayed 63 % increase of activity compared to the wild type and remained >95 % activity after being incubated for 15 days, while the wild type had a half-life of 11.9 min at 65 °C. At a substrate/catalyst ratio of 100 (w/w), the mutant catalyzed the complete conversion of 300 g/l substrate within 1 h to yield enantiopure (S)-CHBE with an isolated yield of 95 %, corresponding to a space-time yield of 1824 mM/h.

A general chemical shift decomposition method for hyperpolarized (13) C metabolite magnetic resonance imaging.

Metabolic imaging with hyperpolarized carbon-13 allows sequential steps of metabolism to be detected in vivo. Potential applications in cancer, brain, muscular, myocardial, and hepatic metabolism suggest that clinical applications could be readily developed. A primary concern in imaging hyperpolarized nuclei is the irreversible decay of the enhanced magnetization back to thermal equilibrium. Multiple methods for rapid imaging of hyperpolarized substrates and their products have been proposed with a multi-point Dixon method distinguishing itself as a robust protocol for imaging [1-(13) C]pyruvate. We describe here a generalized chemical shift decomposition method that incorporates a single-shot spiral imaging sequence plus a spectroscopic sequence to retain as much spin polarization as possible while allowing detection of metabolites that have a wide range of chemical shift values. The new method is demonstrated for hyperpolarized [1-(13) C]pyruvate, [1-(13) C]acetoacetate, and [2-(13) C]dihydroxyacetone. Copyright © 2016 John Wiley & Sons, Ltd.

A Novel and Efficient Five-Component Synthesis of Pyrazole Based Pyrido[2,3-d]pyrimidine-diones in Water: A Triply Green Synthesis.

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.

Design, Synthesis, DFT Study and Antifungal Activity of Pyrazolecarboxamide Derivatives.

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).

Development and Application of O-(Trimethylsilyl)aryl Fluorosulfates for the Synthesis of Arynes.

A class of o-(trimethylsilyl)aryl fluorosulfates was synthesized by a concise method and successfully used as aryne precursors for the first time. Different trapping agents such as azides, furans, and acyl acetoacetates could successfully react with the aryne precursors under mild conditions with good to excellent yields.

Metal-free arylation of ethyl acetoacetate with hypervalent diaryliodonium salts: an immediate access to diverse 3-aryl-4(1H)-quinolones.

A clean arylation protocol of ethyl acetoacetate was developed using hypervalent diaryliodonium salts under mild and metal-free conditions. The scope of the reaction, using symmetric and unsymmetric iodonium salts with varying sterics and electronics, was examined. Further, this method has been applied for the synthesis of antimalarial compound ELQ-300, which is currently in preclinical development.

Tandem catalytic oxidative deacetylation of acetoacetic esters and heteroaromatic cyclizations.

One pot syntheses of furan, thiophene, and pyrrole were accomplished by oxidative deacetylation using Mn(III)/Co(II) catalysts and the Paal-Knorr reaction from 1,5-dicarbonyl compounds, which are prepared from the conjugate addition of ethyl acetoacetate to α,ß-unsaturated carbonyl compounds. The oxidative deacetylation and reductive cyclization of ß-ketoesters derived from ethyl acetoacetate and o-nitrobenzyl bromides efficiently produced diversely substituted indoles.

Novel hyperbranched polysiloxanes containing acetoacetyl groups synthesized through transesterification reaction.

Development of an innovative strategy to prepare hyperbranched polysiloxanes (HBPS) is highly desirable due to the significant shortcomings of conventional fabrication approaches: the precursors need pre-synthesis, the hydrosilylation reaction is conducted using costly catalysts, and hydrolysis of organosiloxanes easily results in gelation. Here, novel HBPS containing acetoacetyl groups (HBPS-Ac) are synthesized through a cost-efficient and easily controllable transesterification reaction. It is shown by gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR), (1) H NMR, and gas chromatography (GC) measurements that the polymerization process is a straightforward technique to prepare new HBPS. The polymers are capable to remove formaldehyde due to the highly efficient reaction of the active methylene in the acetoacetyl group with formaldehyde at room temperature. Notably, coatings incorporating 4 wt% of the polymers allow for formaldehyde absorption, while integrated performances are kept almost unaffected. Therefore, HBPS-Ac are promising as scavengers for formaldehyde.

TiCl4-promoted condensation of methyl acetoacetate, isobutyraldehyde, and indole: a theoretical and experimental study.

The mechanism of the TiCl4-promoted condensation of methyl acetoacetate, isobutyraldehyde, and indole was studied by a combination of theoretical and experimental techniques. The energy profile of plausible reaction paths was evaluated by DFT calculations, and various reaction intermediates were isolated or observed in solution by NMR spectroscopy. Theoretical and experimental results indicate that the reaction proceeds in three steps, all promoted by titanium: (1) formation of the enolate ion of methyl acetoacetate, (2) Knoevenagel condensation of the enolate ion and aldehyde, and (3) Michael addition of indole to the Knoevenagel adduct. The study sheds light on the role of titanium in the reaction, providing a mechanistic model for analogous reactions.