Molar Yield Research Articles

A process for p-hydroxystyrene production from glycerol based on cell-free biosynthesis system

As an essential chemical, p-hydroxystyrene has attracted much attention and is widely used in the field of semiconductors. However, the current production methods have some disadvantages, such as low molar conversion yield, p-hydroxystyrene toxicity, and high substrate cost. To overcome these problems, an five enzymes cell-free cascade, consisting of alditol oxidase from Streptomyces coelicolor A3 (ScALDO), dehydratase from Paralcaligenes ureilyticus (PuDHT), tyrosine phenol lyase from Citrobacter freundii (CfTPL), tyrosine ammonia lyase from Rhodotorula glutinis (RgTAL) and phenylacrylate decarboxylase from Enterobacter sp. (EsPAD), was constructed for the de novo biosynthesis of p-hydroxystyrene from glycerol. Through process strategy optimization, a molar conversion yield of more than 84.2% was finally obtained from 10 mM crude glycerol. It is worth mentioning that the reaction system performed an excellent titer using 50 mM crude glycerol, and the p-hydroxystyrene concentration reached 30.32 mM, which exceeded the maximum titer of fermentation. This showed that our system is robust to p-hydroxystyrene and crude glycerol. In addition, a ‘two-step purification process’ was designed to produce pure p-hydroxystyrene. This is the first study focused on the cell-free biosynthesis of p-hydroxystyrene, and the highest conversion yield and titer thus far was obtained for p-hydroxystyrene de novo biosynthesis.

The impact of methanol mass transport on its conversion for the production of hydrogen and oxygenated reactive species in sono-irradiated aqueous solution

This study aims principally to assess numerically the impact of methanol mass transport (i.e., evaporation/condensation across the acoustic bubble wall) on the thermodynamics and chemical effects (methanol conversion, hydrogen and oxygenated reactive species production) of acoustic cavitation in sono-irradiated aqueous solution. This effect was revealed at various ultrasound frequencies (from 213 to 1000 kHz) and acoustic intensities (1 and 2 W/cm2) over a range of methanol concentrations (from 0 to 100%, v/v). It was found that the impact of methanol concentration on the expansion and compression ratios, bubble temperature, CH3OH conversion and the molar productions inside the bubble is frequency dependent (either with or without consideration of methanol mass transport), where this effect is more pronounced when the ultrasound frequency is decreased. Alternatively, the decrease in acoustic intensity decreases clearly the effect of methanol mass transport on the bubble sono-activity. When methanol mass transfer is eliminated, the decrease of the bubble temperature, CH3OH conversion and the molar yield of the bubble with the rise of methanol concentration was found to be more amortized as the wave frequency is reduced from 1 MHz to 213 kHz, compared to the case when the mass transport of methanol is taken into account. Our findings indicate clearly the importance of incorporating the evaporation and condensation mechanisms of methanol throughout the numerical simulations of a single bubble dynamics and chemical activity.

Sulfosuccinic acid-based metal-center catalysts for the synthesis of HMF from carbohydrates

5-Hydroxymethylfurfural (HMF) has been highlighted as a promising platform chemical and a keystone for the implementation of carbohydrate-based biorefineries. However, substrate cost remains a hurdle for the industrialization of HMF. Using Cr, Zr, and Ti precursors, three novel bifunctional catalysts with sulfosuccinic acid as ligand were developed, and their catalytic properties tested on the synthesis of HMF. Cr-SSA was the most effective in producing HMF, reaching 60% yield after 3 h. Similar results were obtained from sucrose, reaching 60% molar yield. Kinetic models were developed to provide a better understanding of the reactions’ pathway, suggesting that glucose-to-fructose isomerization was the preferred pathway for HMF formation when using Cr-SSA.

Improving the production of 9α-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Δ1-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum

Background9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking Δ1‑dehydrogenation and regulating metabolic flux.ResultsFive 3-Ketosteroid-Δ1-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MFΔkstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MFΔkstD could remarkably reduce the contents of 9,21 ‑dihydroxy‑20‑methyl‑pregna‑4‑en‑3‑one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminating the two by-products. Compared with MFΔkstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9‑OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol.ConclusionsKstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.

Experimental Study of Bio-Hydrogen Production by Clostridium beijerinckii from Different Substrates

Glucose, alcohol stillage and glycerol were used as substrates for bio-hydrogen production by the newly isolated strain Clostridium beijerinckii 6A1 under batch conditions. High molar yields of hydrogen from the studied organic substrates were observed. There was a neat difference in the metabolic pathways of substrate digestion when hexose-based substrate or glycerol were used. The products of glycerol digestion showed that a pathway with no formic acid formation as intermediate was probable. In this case, considerable concentrations of acetic and propionic acid (up to 6 g dm−3) and small amounts of butanol were observed after 48 h. When glucose or hexose-based substrates were used, considerable amounts of formic acid (up to 6 g dm−3), i.e., the pathway proposed for Clostridia mixed cultures, were appropriate for the observed process of hydrogen release. For these substrates, considerable amounts of propionic acid in concentrations up to 1 g dm−3 were observed. That is why the pathway proposed for mixed cultures seemed more appropriate for our experiments carried out with hexose-based substrates. When hexoses were used, substrate digestion stopped the formation of acetic acid, propionic acid and ethanol. Probably, these intermediates are inhibitors to the further digestion to other products.

Bioconversion of ferulic acid and vanillin to vanillic acid by cold-adapted Paraburkholderia aromaticivorans AR20-38: impact of culture conditions

PurposeBiovalorization of lignin-derived aromatic monomers such as ferulic acid (FA) has attracted considerable interest. The cold-adapted strain Paraburkholderia aromaticivorans AR20-38 converts FA to the value-added product vanillic acid (VA), without further VA degradation. The efficiency of the bioconversion of FA to VA was optimized by studying culture conditions.MethodsVarious cultivation parameters (agitation, temperature, FA concentration, nutrient supplementation) were assessed to increase biomass production and shorten the cultivation time, while obtaining high VA production yields. The fate of the intermediate vanillin was also studied. Lignin monomers and degradation products (FA, vanillin, VA) were quantified via UV/Vis-HPLC.ResultFull bioconversion of 5 mM FA occurred over a broad temperature range of 5–30 °C. Concentrations up 30 mM FA were utilized as the sole carbon source at 20 °C. Molar VA yields (> 90%) produced from 5 to 12.5 mM FA and from 15 to 17.5 mM FA (82–87%) were not significantly different at 10 °C and 20 °C. The supplementation of the mineral medium with monosaccharides (glucose, fructose, mannose) and/or N-rich complex compounds (yeast extract, casamino acids) resulted in high biomass production, accelerated FA bioconversion, and high molar yields (96–100%). The presence of the N-rich compounds alone or in combination with glucose reduced the incubation time necessary to convert FA to VA. Vanillin, formed as an intermediate during FA degradation, was consumed and converted to VA before FA metabolization, when added in combination with FA. Vanillin bioconversion was significantly accelerated in the presence of glucose.ConclusionThe variation of culture conditions improved the efficiency of the studied strain to convert FA via vanillin to VA and demonstrated remarkable FA bioconversion under varying environmental conditions, especially temperature, substrate concentration, and nutrient availability, which is of importance for potential future application.

The effect of metformin and drinking water quality variation on haloacetamide formation during chlor(am)ination of acetaminophen

Acetaminophen (Apap) is widely used and is known to form toxic haloacetamides (HAcAms) during chlorination. Metformin (Met) is a typical medication with usage much higher than that of Apap and its ubiquitous presence in the environment is known. The objective of this study was to investigate the effects of Met which contains multiple amino groups potentially joining reactions and different chlorination methods on HAcAm formation from Apap. In addition, a major drinking water treatment plant (DWTP) using the largest river in southern Taiwan was sampled to study the influence of Apap in a DWTP on the HAcAm formation. Results showed increasing dichloroacetamide (DCAcAm) molar yields of Apap at a Cl/Apap molar ratio of 5 during chlorination (0.15%) and two-step chlorination (0.03%). HAcAms were formed by the chlorine substitution of hydrogen on the methyl group in Apap followed by the cleavage of the bonding between nitrogen and aromatic. While a high Cl/Apap ratio during chlorination led to reactions between chlorine and HAcAms formed decreasing the HAcAm yields, the two-step chlorination further reduced the HAcAm formation during chlorination by a factor of 1.8–8.2. However, Met which limitedly formed HAcAms increased the DCAcAm yields of Apap by 228% at high chlorine dosages during chlorination and by 244% during two-step chlorination. In the DWTP, trichloroacetamide (TCAcAm) formation was important. The formation was positively correlated with NH4+, dissolved organic carbon (DOC), and specific ultraviolet absorbance (SUVA). DCAcAm dominated in the presence of Apap. The DCAcAm molar yields were 0.17%–0.27% and 0.08%–0.21% in the wet and dry seasons, respectively. The HAcAm yields of Apap in the DWTP were limitedly changed between different locations and seasons. Apap could be one important cause for HAcAm formation in a DWTP, as the presence of other pharmaceuticals such as Met possibly worsens the situation in chlorine applications.

Rational design of a novel halotolerant ATP regeneration system for biocatalytic CTP production

AbstractBACKGROUNDAcetyl phosphate/acetate kinase system is a cost‐saving and efficient ATP regeneration system. However, it suffers from accumulation of salts during catalytic reactions. It inhibits the activity of acetate kinase (ACK), a crucial enzyme of this process, and hence decreases the rate of product accumulation and yield of CTP.RESULTSA new halotolerant ACK enzyme based ATP regeneration platform was established in this research by applying the concept of rational design to optimize its building blocks. Selection of residues for rational design followed several rules. First, the less conserved residue positions on the protein surface were considered. Second, avoiding significant residue interactions, suchas salt bridges. Third, following the feature of nature occurring halotolerant enzymes, the basic and neutral surface residues were replaced with acidic ones. Our mutants represent high catalytic activities and high IC50 value (inhibit activity by 50%) at high salt concentrations. Also, when the halotolerant ACK was applied to catalyze CTP production, the maximum titer was 213.5 ± 1.6 mM (which is 12.3‐fold higher than that of the traditional process), the productivity was 13.3 ± 0.1 mM L−1 h−1, and the molar yield to 5′‐cytidylate monophosphate (CMP) and utilization efficiency of energy were 92.4% and 72.7%, respectively.CONCLUSIONThis strategy can be applied to modify other enzymes in the industry and gives insight into resolving salt accumulation during biocatalytic processes. In addition, the halotolerant Adenosine Triphosphate (ATP) regeneration system built in this work can be applied to the synthesis of several other high energy compounds. © 2023 Society of Chemical Industry (SCI).

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17β-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.

Mechanistic insights allows high yield assembly of giant unilamellar vesicles in solutions of physiological ionic strengths and studies of the effects of intercellular variation on circadian timekeeping.

Giant unilamellar vesicles or GUVs are single-walled closed phospholipid bilayer membranes that resemble minimal biological cells. A general understanding of the mechanism of solvent-free GUV assembly from lamellar lipid films on surfaces is lacking. Our group has developed a quantitative framework using sedimentation and confocal microscopy to reproducibly measure the “molar yield” and distribution of sizes of GUVs. The molar yield provides a quantitative metric to understand the effects of physical variables on the process of assembly. Using this technique, we show that the assembly of GUVs composed of zwitterionic lipids can be explained by the so called budding and merging (BNM) model. The model explains the evolution of yields as a function of i) increasing surface concentration of lipid, ii) increasing ionic strength of the solution, and iii) increasing charge of the membrane. The mechanistic insight that budding is temporally distinct from merging allows, via modulation of ionic strength, the ability to obtain high yields of GUVs in buffers of arbitrary ionic strengths and valencies, including of physiological ionic strengths. The GUV buds remain attached to the substrate until harvested, which allows for diffusive loading of protein cargo into the vesicles. We find that the distribution of protein concentrations in the vesicles mimics the intercellular distribution of protein in cells. The GUVs thus offer a tractable in vitro platform for studying the effects of variability on biophysical phenomena such as circadian timekeeping.

Modeling the mechanism of giant unilamellar vesicle (GUV) assembly.

Giant unilamellar vesicles (GUVs) are cell-mimetic compartments that can be assembled from amphiphilic lipid molecules in aqueous environments. There has been an increasing interest in the use of bio-mimetic compartments for therapeutics and drug delivery which requires the efficient production of large numbers of GUVs. Despite the long history of producing GUVs, the mechanism of their assembly from films of lipids on surfaces is not yet fully understood. The recently proposed budding and merging (BNM) model by the Subramaniam group shows that unlike on flat surfaces such as glass, the formation of GUVs from films of lipids on cylindrical nanofibers can be spontaneous. GUV formation occurs through the formation of nanobuds that merge to form micron sized GUVs. Here, we report quantitative experiments on the effect of changing the surface concentration of zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) on the molar yields and distribution of sizes of GUVs obtained from assembly on cellulose nanofibers. We find that both the yields and the sizes of the GUVs showed a peaked response, increasing to a maximum of 30% yield at an optimal concentration of 17 nmol/cm2 before decreasing for higher surface concentrations of lipids. We conduct simulations using the BNM model as a framework to account for the effects of multiple bilayers on the cylindrical nanofibers. We find that the evolution of the molar yield and distribution of GUV diameters seen in experiments were reproduced in the simulations. By varying simulation parameters, we predict optimal conditions and substrates to obtain high yields of GUVs composed of zwitterionic lipids. For the highest yield and largest sizes, we require monodisperse fibers with small diameters and large aspect ratios. Similar results can also be obtained by increasing the energy of the system available for the budding of bilayers.

Osmotic pressure enables facile high yield assembly of cell-like giant unilamellar vesicles in solutions of physiological ionic strength.

Giant unilamellar vesicles (GUVs) are single-walled, closed phospholipid bilayers that are useful as cellular mimics for synthetic biology and drug delivery. The assembly of GUVs in solutions of ionic concentrations >150 mM Na/KCl (salty solutions) is challenging. Here, we investigate the effects of temperature, chemical identity of small molecule and macromolecular polymers on the molar yield of free-floating GUVs in salty solutions using high resolution confocal microscopy and large dataset image analysis. We find that low gelling temperature agarose is the singular compound that produces free-floating GUVs at molar yields >10 % in salty solutions. All other macromolecular polymers, such as polyvinyl alcohol (PVA), fructose, and ultralow gelling temperature agarose, produced yields of GUVs of <7 %. Surface images show that for these other compounds, the formation of large numbers of lipid-polymer pseudobuds due to polymer dewetting reduces yields of GUVs. We propose a free-energy model of GUV assembly to explain the effects of polymers on the assembly of GUVs. Modulating the adhesion potential by varying ion concentration and valency yielded experimental data that supports our model's prediction - the free-energy for bud formation can be reduced by a contribution to the osmotic pressure of dissolving polymers. These results provide a quantitative comparison between established and novel methodology, as well as experimental and theoretical framework to guide studies for obtaining GUVs in physiological salt solutions.

Exploring Br-'s roles on non-brominated NDMA formation during ozonation: Reactive oxygen species contribution and brominated intermediate path validation.

Bromide ions (Br-) affected non-brominated nitroso-dimethylamine (NDMA) formation during ozonation, but the mechanism is still unclear. 1,1,1',1'-tetramethyl-4,4'-(methylene-di-p-phenylene) di-semicarbazide (TMDS) was chosen to further probe this problem. The results indicated that low levels of Br- (≤20μM) enhanced NDMA from 3.27 to 7.56μg/L, while its amount slightly dropped to 6.22μg/L raising Br- to 100μM. It was experimentally verified that intermediates 1,1-dimethylsemicarbazide (DMSC) and 1,1-dimethylhydrazine (UDMH) played important roles on promoting NDMA generation, whose contribution rates were 40.2% and 32.2%, respectively. The brominated substances with higher NDMA molar yields were detected. ∙OH reduced NDMA formation without Br-, while it played promotion role with Br-; the corresponding contribution rates were -26.9% and 29.2%, respectively. No matter with or without Br-, both ∙O2- and lO2 brought a boost to NDMA formation, their contribution ratios were 34.9% and 58.1% without Br-, while raised significantly to 64.6% and 81.5% when Br- existed. Br- not only facilitated NDMA formation, but also benefited the degradation of TMDS. Based on the calculation results and intermediates detected, the influence mechanisms of Br- were proposed. The results would provide theoretical basis and technical guarantee for treating NDMA precursors and bromide co-existing water in the future.

Efficient synthesis of Ala-Tyr by L-amino acid ligase coupled with ATP regeneration system.

The multi-enzyme coupling reaction system has become a promising biomanufacturing platform for biochemical production. Tyr is an essential amino acid, but the limited solubility restricts its use. Tyrosyl dipeptide has been paid more attention due to its higher solubility. In this study, an efficient enzymatic cascade of Ala-Tyr synthesis was developed by a L-amino acid ligase together with polyphosphate kinase (PPK). Two L-amino acid ligases from Bacillus subtilis and Bacillus pumilus were selected and applied for Ala-Tyr synthesis. The L-amino acid ligase from B. subtilis (Bs) was selected and coupled with the PPK from Sulfurovum lithotrophicum (PPKSL) for regenerating ATP to produce Ala-Tyr in one pot. In the optimization system, 40.1mM Ala-Tyr was produced within 3h due to efficient ATP regeneration with hexametaphosphate (PolyP(6)) as the phosphate donor. The molar yield was 0.89mol/mol based on the substrates added, while the productivity of Ala-Tyr achieved 13.4mM/h, which were the highest yield and productivity ever reported about Ala-Tyr synthesis with L-amino acid ligase.

Improved terephthalic acid production from p-xylene using metabolically engineered Pseudomonas putida

Terephthalic acid (TPA) is an important commodity chemical used as a monomer of polyethylene terephthalate (PET). Since a large quantity of PET is routinely manufactured and consumed worldwide, the development of sustainable biomanufacturing processes for its monomers (i.e. TPA and ethylene glycol) has recently gained much attention. In a previous study, we reported the development of a metabolically engineered Escherichia coli strain producing 6.7 g/L of TPA from p-xylene (pX) with a productivity and molar conversion yield of 0.278 g/L/h and 96.7 mol%, respectively. Here, we report metabolic engineering of Pseudomonas putida KT2440, a microbial chassis particularly suitable for the synthesis of aromatic compounds, for improved biocatalytic conversion of pX to TPA. To develop a plasmid-free, antibiotic-free, and inducer-free biocatalytic process for cost-competitive TPA production, all heterologous genes required for the synthetic pX-to-TPA bioconversion pathway were integrated into the chromosome of P. putida KT2440 by RecET-based markerless recombineering and overexpressed under the control of constitutive promoters. Next, TPA production was enhanced by integrating multiple copies of the heterologous genes to the ribosomal RNA genes through iteration of recombineering-based random integration and subsequent screening of high-performance strains. Finally, fed-batch fermentation process was optimized to further improve the performance of the engineered P. putida strain. As a result, 38.25 ± 0.11 g/L of TPA was produced from pX with a molar conversion yield of 99.6 ± 0.6%, which is equivalent to conversion of 99.3 ± 0.8 g pX to 154.6 ± 0.5 g TPA. This superior pX-to-TPA biotransformation process based on the engineered P. putida strain will pave the way to the commercial biomanufacturing of TPA in an industrial scale.

Resource utilization and catalytic pyrolysis conversion mechanism of polyacrylate solid waste

It is a great challenge for the sustainable production and application of water-based coatings and inks in terms of realizing the green resource utilization of polyacrylate solid waste (PSW) and avoid its secondary pollution. In this paper, the pyrolysis of PSW catalyzed by HZSM-5 in a fixed bed was investigated, and its reaction conditions were optimized by a Box-Behnken design. The results indicated that PSW could be translated into benzene, toluene and xylene (BTX) with a molar carbon yield of up to 51.24 % at the optimized conditions, which is much higher than those of the solid waste catalytic pyrolysis by HZSM-5 reported in existing literatures. The yield and composition of BTX products were significantly influenced by the structural characteristics of PSW. TG-MS indicated that the conversion process of PSW to BTX through monomer cracking, dealkylation of aromatic hydrocarbons, dehydrogenation of alkanes and aromatization of alkynes/alkenes. Among them, the catalytic effect of HZSM-5 on the dealkylation reaction of catalytic pyrolysis of PSW was proposed for the first time. Benzene was mainly derived from dealkylation and aromatization, whereas toluene and xylene were mainly derived from aromatization. The results enriched the existing mechanism of solid-waste catalytic pyrolysis using HZSM-5 to prepare BTX.

An Efficient 4-Step Synthesis of Favipiravir with Industrial Potential

An efficient, economical synthetic route is developed for the antiviral drug favipiravir using a four-steps instead of a six-step protocol. Starting with 6-bromo-3-hydroxypyrazine-2-carboxamide the method offers an overall 65% molar yield. The prime intermediate 3,6-difluoropyrazine-2-carbonitrile was synthesized by reacting 3,6-dichloropyrazine-2-carbonitrile with potassium fluoride using tetrabutyl-ammonium bromide (TBAB) as a phase transfer catalyst in toluene and DMSO medium at reflux temperature (120 ºC). A simple purification method from fluoro intermediate was developed by making dicyclohexylamine salt and a neutral compound using hydrogen peroxide. The prepared pure favipiravir is fully characterized using NMR Mass and IR techniques. Validated analytical HPLC methods assessed the purity of the drug. The quality of the drug synthesized in terms of assay and impurities is well within the ICH and pharmacopeia standards.

High yield production of 1,4-cyclohexanediol and 1,4-cyclohexanediamine from high molecular-weight lignin oil.

The complete utilization of all lignin depolymerization streams obtained from the reductive catalytic fractionation (RCF) of woody biomass into high-value-added compounds is a timely and challenging objective. Here, we present a catalytic methodology to transform beech lignin-derived dimers and oligomers (DO) into well-defined 1,4-cyclohexanediol and 1,4-cyclohexanediamine. The latter two compounds have vast industrial relevance as monomers for polymer synthesis as well as pharmaceutical building blocks. The proposed two-step catalytic sequence involves the use of the commercially available RANEY® Ni catalyst. Therefore, the first step involves the efficient defunctionalization of lignin-derived 2,6-dimethoxybenzoquinone (DMBQ) into 1,4-cyclohexanediol (14CHDO) in 86.5% molar yield, representing a 10.7 wt% yield calculated on a DO weight basis. The second step concerns the highly selective amination of 1,4-cyclohexanediol with ammonia to give 1,4-cyclohexanediamine (14CHDA) in near quantitative yield. The ability to use RANEY® Ni and ammonia in this process holds great potential for future industrial synthesis of 1,4-cyclohexanediamine from renewable resources.

Optimizing lactic acid production for agricultural wastes revalorization via sugars hydrothermal conversion with zirconia-based catalysts: response surface methodology

The hydrothermal conversion of a complex sugars solution comprising glucose, arabinose, and xylose with zirconia-based catalysts to produce lactic acid (LA) was studied. Catalysts were synthesized by a simple template-assisted sol–gel method. Alternative template removal procedures, such as refluxing ethanol extraction, were compared to the conventional direct calcination. The catalyst obtained after ethanol extraction (Z-Ex) reported the highest specific surface area (323 m2 g−1), total pore volume (0.21 cm3 g−1) and density of acid sites (1.111 µmol m−2), with a significant contribution of strong acid sites. On the other hand, template removal by thermal treatment transformed the hydrous zirconia into crystalline tetragonal phase, reducing the specific surface area and removing most of the acid sites. Catalytic tests were carried out in a stirred batch reactor, resulting Z-Ex the most selective towards LA production. A four-factor experimental design was performed for Z-Ex catalyst by varying temperature, reaction time, initial pressure, and catalyst/reactants ratio. Results were analyzed using the response surface methodology (RSM) to maximize the LA production. A molar yield of 61.2% towards LA and complete sugars conversion were reached at optimized operating conditions: 189 °C, 208 min, 0.5 g catalyst g−1 reactants, and 10 bar N2. Additionally, fitting models for main by-products molar yields (furans, glyceraldehyde, glycolic, formic, acetic, and levulinic acids) were obtained with good accuracy prediction. All responses were jointly studied to understand the reaction mechanisms involved in generation of intermediaries and undesired degradation products.Graphical Abstract

Microwave heating for sustainable valorization of almond hull towards high-added-value chemicals

Microwave (MW) treatment promotes homogeneous heating compared to conventional methods, thus increasing the recovery of high-added-value compounds and leading to a considerably lower amount of both by-products and side reactions. Therefore, the main goal of this work is to valorize almond hull (AH) via microwave (MW)-assisted radiation (0–200 W, 0–300 psi, 100–190 °C, 10–40 min). In this context, two different pathways were evaluated. Firstly, the transformation of AH into levulinic acid (LA), one of the major bio-based chemicals obtained from lignocellulosic biomass. The so-called almond hull extractives-free biomass (AH-EFB) led to the best results after using both Lewis (AlCl 3 ⋅6 H 2 O, 1 mol/L, 87 % molar yield) and Brønsted ( p -toluenesulfonic ( p -TsOH), 0.25 mol/L, 91 % molar yield) acids, at 190 °C for 20 min. This latter not only provides a sustainable system in contrast to mineral acids such as H 2 SO 4 or HCl, but also the possibility of being recovered and recycled for further transformations. In a parallel secondary experiment, the recovery of biologically active compounds (BACs) was studied separately. For this purpose, antioxidant assays and phenolic profiling were carried out, which demonstrated that MW was more efficient than traditional methods (i.e. soaking) based on obtained values in terms of scavenging activity and polyphenols. Overall, this valorization approach involves most of the Green Chemistry principles, thus contributing to the development of almond biorefineries. • Bioenergy and biofuels can be obtained from almond by-products. • Almond hull can provide high-added-value chemicals. • MW treatment increased the extraction of polyphenols and antioxidant activity.