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The BI-3802 was designed by Boehringer Ingelheim and could be obtained free of charge through the Boehringer Ingelheim open innovation portal opnMe.com, associated with its negative control.
Dextrose, a simple sugar (monosaccharide), is an important carbohydrate in biology.
Synonyms: Glucose; Dextrose; NSC 287045
4.5
*For Research Use Only! Not for Human Use. We Do Not Sell to Patients.
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Jung, Juliano Ten Kathen ; Marques, Luiza Souza ; Felipeto, Victor Machado ; Lima, Gabriel Corrêa ; Nogueira, Cristina Wayne ;
Abstract: Post-traumatic stress disorder (PTSD) often involves disrupted neuroendocrine, inflammatory, and cell death pathways. In this study, we investigated the neuroprotective effects of β-hydroxybutyrate (BHB) on a PTSD model in male and female Wistar rats. Rats were exposed to a PTSD-like model, and twenty-four hours later, they received BHB (250 mg/kg) or glucose (5 %) twice daily, for seven days. Twelve hours after the last injection, the rats were challenged on behavioral tests for locomotor activity, anxiety-like phenotype, and contextual fear-related behaviors. Twenty-four hours after the last behavioral test, samples of serum, amygdala, and hippocampus were collected. Corticosterone, anxiety index, and freezing behavior were increased, and the expression of GRα/β, FKBP51, BAX/Bcl-2, and TNFα in the amygdala and hippocampus were different in the PTSD rats. BHB prevented behavioral deficits, normalized corticosterone and GR, and apoptosis signaling in both sexes, but glucose did not rescue these alterations. In females, inflammatory mediators (TNFα, IBA-1) were enhanced and partially suppressed by BHB. Correlations between anxiety-like phenotype and fear behaviors, and between measures of behaviors and levels of molecular and corticosterone changes, indicate a unified stress response. Our findings suggest that BHB shows promise as a neuroprotective treatment for PTSD in both male and female rats.
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Keywords: Post-traumatic stress disorder ; β-hydroxybutyrate ; Glucocorticoid receptor ; Sex differences ; amygdala ; hippocampus
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Febuxostat Analogs as Anti-Quorum Sensing and Antibacterial Agents
Singh, Praveen Kumar ; Zore, Matej ; San-Martin-Galindo, Paola ; Kornmayer, Moritz M ; Reigada, Inés ; Hanski, Leena , et al.
Abstract: Febuxostat, a marketed anti-gout drug, has been reported to inhibit quorum sensing (QS), biofilm formation, and virulence in Gram-negative bacteria Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum CV026. However, no structure-activity relationship (SAR) studies have been reported to date. To address this gap, we synthesized 27 novel febuxostat analogs and evaluated their QS inhibitory activity using wild-type C. violaceum and the mutant strain CV026. This dual-strain assay enabled differentiation between quorum sensing inhibition and quorum quenching mechanisms, allowing determination of whether QS interference occurred at the level of signal synthesis or signal reception. Here, we demonstrated that febuxostat acts as a QQ at concentrations lower than previously reported, strongly inhibiting violacein production in CV026 without affecting bacterial viability. At 400 µM, several analogs showed comparable or stronger activity than febuxostat in one or both strains. At 40 µM, trifluoromethoxyphenyl analog 49 emerged as the most effective QS inhibitor in the wild-type strain, whereas cyclopentyl analog 24 was most active in CV026, with a few other derivatives also retaining notable activity. We also assessed whether febuxostat and its analogs exhibit antibacterial activity. While febuxostat showed no antibacterial activity, seven analogs (26–30, 33, and 49) exhibited minimum inhibitory concentrations (MICs) ranging from 2.5 to 40 µM against Gram-positive bacteria Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, and E. faecium. On the other hand, no activity was observed against Gram-negative bacteria. In cytotoxicity studies, the two most potent antibacterial compounds 29 (MIC = 5 µM) and 30 (MIC = 10 µM), showed no cytotoxicity against the mammalian cell line A549, while they were less tolerated by THP-1 cells. In summary, our results provide SAR insights into febuxostat analogs and highlight their potential as novel anti-QS and antibacterial agents against multidrug-resistant bacteria.
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Keywords: antibacterial activity ; Chromobacterium violaceum ; drug discovery ; febuxostat ; quorum sensing ; Staphylococcus aureus
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Reynolds, Donald L ; Simpson, E Barry ; Hille, Matthew M ;
Abstract: Enterococcus faecalis (E. faecalis) is a ubiquitous microbe occurring in the environment and in the intestinal tract of poultry. E. faecalis has been identified in cases of egg infertility and/or decreased hatchability and can cause amyloid arthropathy in older laying chickens. E. faecalis produces cytolysin, a bacterial exotoxin that can cause lysis of erythrocytes. It has been difficult to demonstrate this virulence trait using conventional culture methods with sheep blood agar. A 96-well microplate hemolysis assay, along with a culture method incorporating glucose and L-arginine into the culture media, is described that demonstrates the production of cytolysin in E. faecalis isolates of avian origin. Additionally, the results show that horse and sheep erythrocytes were susceptible to lysis by the E. faecalis cytolysin, but cow and chicken erythrocytes were less susceptible.
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Keywords: Enterococcus faecalis ; hemolysis ; cytolysin ; avian
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| CAS No. : | 50-99-7 |
| Formula : | C6H12O6 |
| M.W : | 180.16 |
| SMILES Code : | O=C[C@@H]([C@H]([C@@H]([C@@H](CO)O)O)O)O |
| Synonyms : |
Glucose; Dextrose; NSC 287045
|
| English Name : | (2R,3S,4R,5R)-2,3,4,5,6-Pentahydroxyhexanal |
| MDL No. : | MFCD00148912 |
| InChI Key : | GZCGUPFRVQAUEE-SLPGGIOYSA-N |
| Pubchem ID : | 107526 |
| GHS Pictogram: | |
| Signal Word: | |
| Hazard Statements: | |
| Precautionary Statements: | |
| Class: | |
| UN#: | |
| Packing Group: |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 29.89 %Chromat. | With Aspergillus flavus NFCCI 2364 culture filtrate In aq. buffer at 55℃; for 24 h; Microbiological reaction | General procedure: FOS production was carried out by adding 1ml of enzyme samples collected at various time intervals to 3ml of 50percent (w/v) sucrose dissolved in 0.1M citrate buffer (pH 5.5) for period of 24h at 55°C. The amount of FOS formation in the samples was analyzed by high performance liquid chromatography (HPLC, Waters) with sugar-pak column (6.5×300mm) and refractive index (RI) differential detector (RI 2414). |
| 25.31 %Chromat. | With Aspergillus niger SI 19 culture filtrate In aq. buffer at 55℃; for 24 h; Microbiological reaction | General procedure: FOS production was carried out by adding 1ml of enzyme samples collected at various time intervals to 3ml of 50percent (w/v) sucrose dissolved in 0.1M citrate buffer (pH 5.5) for period of 24h at 55°C. The amount of FOS formation in the samples was analyzed by high performance liquid chromatography (HPLC, Waters) with sugar-pak column (6.5×300mm) and refractive index (RI) differential detector (RI 2414). |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 92.4% | With barium hydroxide octahydrate In lithium hydroxide monohydrate at 25℃; for 48h; Inert atmosphere; | 7 Take 1.5774 g of Ba(0H)2 · 8H20, dissolved in 20 mL of deionized water, and then weighed 0.18 g of glucose. The high purity N2 was passed at a flow rate of 0.6 L / min for 3 min, after which the reactor was quickly sealed and transferred to 25 ° C Constant temperature shaker, the speed of 100r / min, reaction 48h. After the reaction, 5 mL of the reaction solution was taken, diluted twice with 5 mL of 0.5 M H2SO4, and the effect of Ba2 + on the column was removed. Dilution liquid into liquid chromatography to measure substrate conversion and lactic acid yield. The yield of substrate was 98.9%, the yield of lactic acid was 92.4% Lactic acid selectivity was 93.4%. |
| 81% | With Aluminum Chloride; stannous chloride In lithium hydroxide monohydrate at 189.84℃; for 2h; Inert atmosphere; Autoclave; | |
| 76.2% | With erbium(III) chloride In lithium hydroxide monohydrate at 240℃; for 0.5h; Inert atmosphere; Autoclave; | Reaction test and product analysis General procedure: All reactions were carried out in a 35 mL stainless steel autoclaveequipped with a mechanical stirrer. In a typical experiment, 0.1 gof substrate material, 0.05 g of catalyst and 30 mL of water wereadded to the reactor, after which the autoclave was purged threetimes with N2 and then pressurized to 2.0 MPa with N2 at roomtemperature. The reaction mixture was heated to 240 C unless otherwisestated and held at that temperature for 30 min with stirringat 600 rpm. After each reaction the reactor was quickly cooled toroom temperature using an ice/water mixture and then depressurized.The post-reaction sample was diluted with mobile phasesolution prior to analysis.Sample analyses were performed on a Shimadzu LC-20AT HPLCsystem equipped with a RID-10A detector and a Bio-Rad AminexHPX-87H ion exclusion column (300×7.8 mm), using 0.005 MH2SO4 as the mobile phase at a flow rate of 0.5 mL min-1. Thecolumn temperature was 50 C and the detector was set to 45 C.The amount of product was determined using calibration curvesgenerated with standard solutions. |
| 74% | With lanthanum trifluoromethanesulphonate at 250℃; for 1h; | |
| 65.5% | With potassium hydroxide In lithium hydroxide monohydrate at 100℃; for 0.5h; Autoclave; Inert atmosphere; Green chemistry; chemoselective reaction; | |
| 65.2% | With dealuminated Zn-Sn-beta zeolite In lithium hydroxide monohydrate at 190℃; for 8h; Autoclave; | 8 Example 8 (0045) Catalyst preparation: The beta zeolites are first mixed with a concentrated nitric acid solution with a mass ratio of 1:110 and stirred at 100° C. for 20 h. Then the mixture is centrifuged and washed for 8 times and dried at 80° C. for 2 h and 150° C. for 10 h to obtain dealuminated beta zeolites. 1 g of the dealuminated beta zeolites is mixed with 99.46 mg of tin acetate (0.42 mmol Sn) and 92.19 mg of zinc acetate (0.42 mmol Zn) and the mixture is grinded for 20 min and calcined in air at 550° C. for 6 h to obtain dealuminated Zn-Sn-beta zeolite catalyst. (0046) Catalytic reaction: The catalytic reaction is carried out in a Teflon-lined stainless steel autoclave. The autoclave is charged with 225 mg of glucose, 160 mg of dealuminated Zn-Sn-beta zeolite catalyst, and 10 g of deionized water. The autoclave is placed in a rotary oven at 190° C. for 8 h with a rotate rate of 20 r/min. After centrifugation at 5000 r/min for 2 min, the remaining liquid is analyzed with High HPLC. The yield of lactic acid turns out to be 65.2%. |
| 19.6% | With YNbO4 In lithium hydroxide monohydrate at 139.84℃; for 5h; | |
| 5% | With Calcium hydroxide In lithium hydroxide monohydrate at 300 - 400℃; for 0.00833333 - 0.05h; | |
| With potassium hydroxide | ||
| With calcium hydroxide powder at 165 - 235℃; unter Druck; | ||
| Einfluss von Insulin bei der Vergaerung durch Lactobacillus bulgaricus; | ||
| Einfluss von Insulin bei der Vergaerung durch Lactobacillus acidophilus; | ||
| unter der Einw. eines aus dem Pariser Leitungswasser isolierten Bacteriums; hierbei entsteht intermediaer Brenztraubensaeure; | ||
| bei der Vergaerung durch Bact. xylinum; | ||
| bei der Vergaerung durch Azotobacter Chroococcum; | ||
| With potassium hydroxide at 25℃; Einfluss der Alkali-Konzentration; | ||
| With potassium hydroxide at 50℃; Einfluss der Alkali-Konzentration; | ||
| With anhydrous sodium carbonate | ||
| With barium dihydroxide | ||
| With alkali | ||
| With sodium hydroxide | ||
| With diluted alkali | ||
| With sodium hydroxide at 37℃; | ||
| With barium dihydroxide at 60℃; | ||
| bei der Vergaerung durch Bact. coli unter verschiedenen Bedingungen; | ||
| With calcium carbonate at 165 - 235℃; unter Druck; | ||
| With sodium hydroxide | ||
| Einw. von Rhizopus chinensis; | ||
| 75 %Chromat. | Stage #1: D-glucose With aluminum(III) oxide; potassium hydroxide at 180℃; for 0.666667h; Microwave irradiation; Stage #2: With sulfuric acid In lithium hydroxide monohydrate | |
| With oxidovanadium(IV) sulfate In lithium hydroxide monohydrate at 159.84℃; for 1.5h; Autoclave; Inert atmosphere; | ||
| With lithium hydroxide monohydrate; sodium hydroxide at 100℃; for 2h; | 1.i Example 1 - Reaction of saccharide with sodium hydroxide i) Glucose Example 1 - Reaction of saccharide with sodium hydroxide i) Glucose To a 500 mL flask was charged NaOH (15.98 g). Water (22.46 mL) was added to the alkali and the mixture was heated to 100 ±2 °C. An aqueous solution of glucose (1.0 M, 100 mL) was then added to the hot alkali over a period of 120 minutes. After completion of the addition the reaction mixture was cooled to ambient temperature, quenched with HC1 (aq.) and analysed by HPLC for lactic acid selectivity. The % lactic acid selectivity as determined by HPLC was 41.9% (±1%). ii) Xylose To a 100 mL flask was charged NaOH (4 - 5 equivalents based on the amount of xylose). A small amount of water (5 - 15 mL) was added to the alkali and the mixture was heated to 100 ±2 °C. An aqueous solution of xylose (0.2 M to 4 M, 50 mL) was then added to the hot alkali over a period of 25 - 120 minutes. After completion of the addition the reaction mixtures were cooled to ambient temperature, quenched with HCl (aq.) and analysed by HPLC for lactic acid selectivity. | |
| Einw. von verschiedenen Bakterien; | ||
| With Ca-WO3 In lithium hydroxide monohydrate at 160℃; for 4.66667h; Autoclave; Inert atmosphere; | 2 (2) Catalytic conversion of biomass sugars by Ca-WO3 catalyst: 0.1 g of Ca-WO3 catalyst, 5 g of glucose, and 40 ml of water were added to the autoclave.After exhausting the air with nitrogen, the reaction was carried out at 160 ° C for 280 min.A lactic acid yield (relative to the glucose raw material) of 88 mol% was obtained. | |
| 15 %Chromat. | With γ-Al2O3 In lithium hydroxide monohydrate at 170℃; for 4h; Autoclave; | |
| 88.6 %Chromat. | With sodium hydroxide In lithium hydroxide monohydrate for 2h; Inert atmosphere; UV-irradiation; Sealed tube; | |
| 71.0 %Chromat. | With potassium hydroxide In lithium hydroxide monohydrate at 70℃; for 0.5h; Irradiation; |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 95.69% | Stage #1: D-glucose; ethylamine With hydrogen In methanol at 25 - 35℃; for 0.5h; Stage #2: In methanol at 60 - 80℃; for 12h; Sealed tube; Inert atmosphere; | 1-5 Example 2: Put 1000g of methanol into the hydrogenation kettle, add 180g (1mol) of glucose, add 180g (4mol) of monoethylamine under stirring, keep it at 25-35°C for 30 min; add 9g of aluminum nickel iron ternary powder catalyst,Sealed, the system is replaced with nitrogen, the hydrogen pressure is maintained at 1.0~2.0MPa, the system temperature is at 60~80°C, and the reaction is stirred for 12 hours. Cooling, depressurizing, filtering to remove the catalyst, the filtrate is frozen, crystallized, filtered with stirring, and the solid is dried N-ethyl-D-glucamine 200g, yield 95.69%. The titrated content is 99.6%. |
| With nickel kieselguhr; water at 90 - 100℃; Hydrogenation; | ||
| With nickel kieselguhr; water at 90 - 100℃; Hydrogenation; |
[ 50-99-7 ]
[ 16732-66-4 ]

[ 50-99-7 ]
[ 2595-97-3 ]
[ 491-71-4 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 81% | With Montmorillonite K-10 clay at 90℃; for 0.15h; microwave irradiation; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 79% | With cerium(III) sulfate at 90℃; for 0.216667h; microwave irradiation; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| Stage #1: D-glucose In phosphate buffer at 50℃; for 168000h; Stage #2: 2,3-Diaminonaphthalene In water at 50℃; for 1h; Further stages. Further byproducts.; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With 1,1'-carbonyldiimidazole In methanol; dichloromethane | 1 Ethyl 2-ethoxy-3-[4-[2-(4-pyridine-2-ylbenzoylamino)ethoxy]phenyl]propionate (ethyl ester of exemplification No. 1-35 compound) EXAMPLE 1 Ethyl 2-ethoxy-3-[4-[2-(4-pyridine-2-ylbenzoylamino)ethoxy]phenyl]propionate (ethyl ester of exemplification No. 1-35 compound) Hydrazine-hydrate (80%, 0.125 ml) was added to a solution of ethyl 2-ethoxy-3-[4-(2-phthaloyliminoethoxy)phenyl]propionate (760 mg), which is the product of reference example 1, in methanol (5 ml) and the mixture was allowed to stand at room temperature for 1.5 hours. At the end of this time the reaction mixture was concentrated. The residue was partitioned between ethyl acetate and water and the layers were separated. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated by evaporation in vacuum to afford an amino derivative. Separately, carbonyldiimidazole (400 mg) was added to a suspension of 4-pyridine-2-ylbenzoic acid (400 mg) in anhydrous dichloromethane (10 ml) and the mixture was stirred at room temperature for 1.5 hours to give a clear solution. To this clear solution, a solution of the amino derivative produced as described above in dichloromethane (5 ml) was added and the mixture was stirred at room temperature for 30 minutes. To this reaction mixture 4-pyridine-2-ylbenzoic acid (200 mg) and carbonyldiimidazole (170 mg) were added. After the mixture was allowed to stand overnight, the reaction mixture was concentrated by evaporation in vacuum. The residue was partitioned between ethyl acetate and water and the layers were separated. The organic layer was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified via chromatography on a silica gel column using dichloromethane/methanol=20/1 as the eluant to afford the title compound (135 mg) as a gum. 1H-NMR (270 MHz, CDCl3): δ ppm 1.08-1.29 (6H, m), 2.95 (2H, d, J=6.5 Hz), 3.35 (1H, quintuplet, J=7.0 Hz), 3.60 (1H, quintuplet, J=7.0 Hz), 3.83-4.30 (7H, m), 6.72 (1H, t, J=4.5 Hz), 6.86 (1H, t, J=8.5 Hz), 7.17 (2H, d, J=8.5 Hz), 7.25-7.40 (1H, m), 7.72-8.01 (4H, m), 8.07 (2H, d, J=8.5 Hz), 8.70-8.80 (1H,m). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 92.1% | With sodium hydroxide In methanol; water | 16 EXAMPLE 16 EXAMPLE 16 A mixture of methanol 60 ml, water 30 ml, 97% sodium hydroxide 12.4 g and 2-nitro-2'-hydroxy-5'-methylazobenzene 12.9 g was heated and stirred at 45°~50° C. 9-fluorenone 1.0 g and then glucose 5.5 g were added to the resultant mixture over 30 minutes while stirring. The mixture was further stirred at 75° C. (boiling point) for 7 hours. As this result, the azobenzene disappeared to produce 2-(2'-hydroxy-5'-methylphenyl)benzotriazole-N-oxide. Water 50 ml was added to the reaction liquor thus obtained, and the liquor was neutralized with 62% sulfuric acid 19 g to precipitate a crystal. The crystal thus obtained was separated by filtration, and the separated crystal was fully washed with water and further with methanol. The crystal thus washed was dried to produce 11.1 g of 2-(2'-hydroxy-5'-methylphenyl)benzotriazole having a melting point of 138° to 140° C. at the yield of 92.1%. |
| 92.1% | With sodium hydroxide In methanol; water | 16 Example 16 Example 16 A mixture of methanol (60 ml), water (30 ml), 97% sodium hydroxide (12.4 g) and 2-nitro-2'-hydroxy-5'-methyl azobenzene (12.9 g) was heated and stirred at 45~50°C. 9-fluorenone (1.0 g) and then glucose (5.5 g) were added to the resultant mixture over 30 minutes while stirring. The mixture was further stirred at 75°C (boiling point) for 7 hours. As a result, the azobenzene disappeared to produce 2-(2'-hydroxy-5'-methylphenyl)benzotriazole-N-oxide. Water (50 ml) was added to the reaction liquor thus obtained, and the liquor was neutralized with 62 % sulfuric acid (19 g) to precipitate a crystal. The crystal thus obtained was separated by filtration, and the separated crystal was fully washed with water and further with methanol. The crystal thus washed was dried to produce 11.1 g of 2-(2'-hydroxy-5'-methylphenyl)benzotriazole having a melting point of 138 to 140°C at the yield of 92.1 %. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With sodium hydroxide In water at 20℃; for 18h; | 12 EXAMPLE 12 This Example details the synthesis of glucose hydroxypropyl trimethylammonium chloride. A round bottom 250 ml flask was fitted with a mechanical stirrer. Into the flask was charged 1 M sodium hydroxide (55.5 ml, 55.5 mmol), Clearsweet 95 (10 g, 55.5 mmol) and 3-chloro-2-hydroxypropyl trimethylammonium chloride (15 ml, 55.5 mmol). Clearsweet 95 is a trademark of Cargill and contains 95.4% glucose, 2.8% maltose and 1.8% higher sugars. The 3-chloro-2-hydroxypropyl trimethylammonium chloride was sourced from Aldrich Chemical Company as a 60% active material in water. It is also available as Quat 188 from the Dow Chemical Company. The reactants were stirred at room temperature for 18 hours. Water was removed under reduced pressure at 50° C. to give a heterogeneous colorless syrup. Filtration through glass wool afforded glucose hydroxypropyl trimethylammonium chloride as a homogeneous clear and colorless syrup: m/z (ESI; M+ -Cl-) 296; HPLC (Column: YMC-ODS-AQ, S5, 120A, 4.6*250 mm; Flow: 1 ml/min isocratic, 100% water; Detector: RI; Temperature: 35° C.) tr 3.11 min (Clearsweet 95-tr 3.09 min; CHPTMAC-tr 3.39 min). |
[ 1132-61-2 ]
[ 50-99-7 ]
[ CAS Unavailable ]
[ 8013-01-2 ]
[ CAS Unavailable ]
[ 89232-77-9 ]
[ CAS Unavailable ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Amycolatopsis fastidiosa MA7344 In water at 32℃; for 168h; Microbiological reaction; |

[ 67-47-0 ]
[ 57-48-7 ]
[ 59432-60-9 ]
[ 50-99-7 ]
[ 81129-73-9 ]
[ 470-69-9 ]
[ 13133-07-8 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With water;phosphoric acid; at 50 - 95℃; for 0.25 - 0.75h;pH 1.75 - 3.75; | Figure 4, from a second laboratorial work, is a TLC - Thin Layer Chromatography on silicagel 60 (Merck) developed with the mixture isopropanol : ethyl acetate : water 5:1 :2 as mobile phase and with partial runs of 1/3, 2/3, and 3/3 of the front line and developed with orcinol : sulfuric acid : methanol at 1000C for 5 minutes. This figure illustrates the profile of FOS - Fructooligosaccharides when one hydrolyzes purified inulin from dahlia roots (5g%) with phosphoric acid at pH = 2.5 at 850C during 15 minutes (15), 30 minutes (30), and 45 minutes (45), indicating that the modulation of a single kinetic parameter - time of hydrolysis - already allows to govern the quantitative relation of FOS > fructose (cases 15 and 30) or the opposite (FOS < fructose; 45). A similar strategy for FOS > fructose may also be governed by the other parameters (pH itself or temperature of hydrolysis), as shown for hydrolysis of inulin with phosphoric or citric acids at pHs from 1.75 to 3.75 in the range of 5O0C to 95oC (as better explained in Figure 5). In Figure 4, (f) and (g) denotes for free fructose and glucose, respectively. GP refers to the Degree of Polymerization. It is remarkable that phosphoric or citric hydrolyses of inulin may be effectively addressed <n="27"/>to the preferential preparation of FOS - Fructooligosaccharides since when addressed to a higher fructose content (lane 45), some amount of the co-product HMF - hydroxymethylfurfural turns clearly visible in the front zone of the chromatogram. Its companion spot most probably is a DFA (difructose anhydride).; Figure 5 is a bar graphic comparing the effect of the kinetic parameters such as temperature and time of hydrolysis once fixed the hidrogenionic potential at pH = 2.5 and their respective capacities for the modulation on the qualitative nature of the products from the hydrolyses of dahlia inulin with phosphoric or citric acids when the substrate is used at a concentration of 5g%. According to the intended innovation in this patent request - the preferential production of FOS or FrutoOligoSaccharides - is obviously that, for each range of temperature, namely, 750C, 850C ou 950C, either in the phosphoric or in the citric hydrolyses, the formation of FOS is preferential in (8x2 =) 16 assays, except for those two - at 950C and during 25 minutes - where fructose shows predominance with respect to FOS. Incidentally, those two exceptional conditions - which are not the scope of this patent request - also led to the formation of some HMF - hydroxymethylfurfural - undesirable in a inulin hydrolysis, with the attenuating condition that a less expensive practice as activated charcoal is able to remove the contaminant HMF. Concerning the reaction yield reported to the initial inulin input, the diluted phosphoric acid guarantees in the times of 25 min at 850C and of 15 min at 950C percentages of hydrolyses up to 80%, being the most of the products - 76% and 63%, respectively - FOS or frutooligosaccharides and being the remaining fructose since HMF is no longer detected under these conditions of hydrolysis. The same approach is attained with citric acid although with somewhat reduced yields - 64% or 76%, but even so FOS correspond to 78% and 74% of the hydrolysis products.; Figure 6 derives from another practical example of laboratory work, namely a high performance liquid chromatography or HPLC in a column of 10 micra microparticles of silica gel derivatized with amino groups and provided by Spectraphysics. Twenty microliters of a citric hydrolyzate of inulin at 10% obtained at pH 2.5 during 5 or 15 minutes at 850C were applied to the column and elution proceeded with 70% acetonitrile at a 1 mL/min flow rate and the monitoring was carried out with DRI- differential refraction index. It is shown that in both conditions <n="28"/>- 5 and 15 minutes - inulin is converted, by citric acid, into a family of FOS - FructoOligoSaccharides with DP - Degree of Polymerization - 3 to 18 or more (considering the analytical capacity of the referred column), still emphazing that fructose, with respect to FOS concentration, contributes with a maximum of 25% and a minimum of 5%.; Figure 7, resulting from another practical laboratory work, is also - like Figure 6 - a HPLC but carried out with samples arising from phosphoric acid hydrolyses under the same conditions of those described in Figure 6. The qualitative profiles for FOS are quite similar in both figures. In 8 of 9 assays, FOS predominates. In the nineth - 25 minutes of hydrolysis at higher temperature - FOS and fructose contents are more or less equivalent. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Tetradecanoic acid 1-methylethyl ester;artemisinic acid producing yeast strain; In water; for 123.4h;Enzymatic reaction;Product distribution / selectivity; | 5.13 Example 13: Extractive Fermentation of yeast producing artemisinic acid; [00137] An artemisinic acid producing yeast strain was run in a carbon restricted, mixed glucose and ethanol feed, fed-batch fermentation. The starting aqueous medium volume was 690 mL, which consisted of 620 mL batch medium and 70 mL seed culture. The OD600 of the two seed flasks were 6.3. Prior to inoculation, 200 mL of isopropyl myristate (IPM) was added to the reactor. Exponential feed was initiated at 22.5 hours after both the glucose and ethanol in the batch medium was consumed.[00138] The exponential phase of the feed continued until 43.1 hours when it reached a maximum of 10 g glucose & ethanol/hr/L fermentor volume. The feed was then reduced to a linear feed of 5 g glucose & ethanol/hr/L fermentor volume. The aqueous fermentor volume increased to 1.52 L by the end of the run (approximately 122 hr after inoculation) and no volume was removed from the fermentation except for sampling. Because the aqueous volume in the reactor was increasing throughout the run (and sample volume was being removed), the ratio of IPM to the total volume (IPM + aqueous phase) changed from approximately 0.23 at the start of the fermentation to 0.09 at the end of the fermentation.[00139] In the reactor, the IPM and aqueous cell broth forms a well mixed mixture or emulsion. At early times in the process (28 hrs or less), samples of the mixture, taken from the fermentor, will begin to phase separate when left standing for several minutes. At later times (40 hrs or more), the mixture becomes a more stable emulsion as the yeast cell density increases.[00140] At approximately 123.4 hours after inoculation, fermentation was ended and the cell broth and IPM mixture was harvested. The liquid mixture was removed from the reactor vessel by gravity flow (siphoning) and the total volume was measured prior to further processing. The total volume of the cell broth and IPM mixture was approximately 1680 mL. The liquid mixture was transferred to two 1-L centrifuge bottles, and centrifuged for 25 minutes at 24C and 7459 X G to separate the phases. The organic and aqueous phases appeared to separate with little interface emulsion remaining and the cells pelletted firmly to bottom and side of the centrifuge bottle. After centrifugation, the light phase (IPM) was removed from the centrifuge bottles by serological pipette and collected in a graduated cylinder. The remaining aqueous phase and cells were centrifuged again for 40 minutes at <n="34"/>24C and 7459 X G to separate any addition IPM that was missed in the first pass. Approximately 154-165 mL total of IPM was collected.5.14 Example 14: Purification of artemisinic acid from IPM layer[00141] Isopropyl myristate (IPM) was isolated from an artemisinic acid fermentation described in Example 13. IPM ( 100 mis) was mixed with 400 mis of 1 % NaPO4* 12H2O and the pH adjusted to 10.7 by the addition of 5N NaOH. The solution was then stirred at ambient temperature for 60 minutes. After mixing, the solution was allowed to gravity settle in a separatory funnel for 60 minutes at ambient temperature. The bottom aqueous phase was drawn off from the upper IPM phase. The bottom aqueous phase was run through a liquid: liquid annular centrifugal contactor (CINC industries) to ensure complete removal of any residual IPM. The solution was mixed and the pH adjusted to 5.0 with 5N H2SO4. The acidification resulted in the formation of a fine white precipitate which was captured on a 0.45 micron PTFE filter, rinsed with purified water and then dried. Analysis of the dried precipitate by GC-FID gave artemisinic acid purities of 87-90% by area and 82-84% by weight.5.15 Example 15: Purification of artemisinic acid from IPM layer with SDS[00142] Isopropyl myristate (IPM) was isolated from an artemisinic acid fermentation described in Example 13. IPM ( 100 mis) was mixed with 900 mis of 1 % NaPO4* 12H2O and the pH adjusted to 10.7 by the addition of 5N NaOH. The solution was then stirred at ambient temperature for 60 minutes. After mixing, the solution was allowed to gravity settle in a separatory funnel for 60 minutes at ambient temperature. The bottom aqueous phase was drawn off from the upper IPM phase. The bottom aqueous phase was run through a liquid:liquid annular centrifugal contactor (CINC industries) to ensure complete removal of any residual IPM. A 10% w/v sodium dodecyl sulfate (SDS) solution was added to approximately 850 mis of the aqueous phase to bring the final SDS concentration to 0.03%. The solution was mixed and the pH adjusted to 5.0 with 5N H2SO4. The acidification resulted in the formation of a fine white precipitate which was captured on a 0.45 micron PTFE filter, rinsed with purified water and then dried. Analysis of the IPM before and after aqueous extraction showed that 5% of the artemisinic acid remained in the IPM after extraction (-95% yield). Analysis of the filtrate after precipitation showed that 2% of the a... |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With hydrogenchloride; water for 2h; Reflux; | ||
| With water Acidic conditions; | ||
| With sulfuric acid In methanol at 100℃; for 0.333333h; |
| With acetic acid In water at 100℃; for 2h; | Acid hydrolysis General procedure: Each new compounds (2 mg) was refluxed with 2N aq. CH3COOH (5 ml) for 2 h at 100°C. Afterextraction with CHCl3 (3 × 5 ml), the aqueous layer was repeatedly evaporated to dryness with MeOH until neutral, and then analysed by TLC over silica gel (MeCOEt-isoPrOH-Me2CO-H2O 20:10:7:6) by comparison with authentic samples (L-rhamnose Rf 0.65; d-glucose Rf 0.40)(Nguyen et al. 2015; 2016). | |
| With hydrogenchloride In methanol at 100℃; for 2h; | 1.5. Hydrolysis of the isolated compounds General procedure: Each isolated compound (100 μg) was refluxed with 10% HCl in 50% methanol for 2 hrs (100C). Aqueous solution of the hydrolyzate was then extracted with diethyl ether and ethyl acetate. Aglycons were identified using UHPLC-DAD-MS/MS and TLC with authentic samples. Sugars were identified using TLC in the following mobile phases: n-propanol-ethyl acetate-water (7:2:1; v/v); ethyl acetate-acetic acid-water (3:1:3; v/v); chloroform-methanol-acetic acid-water (7:3:1:0.5; v/v). |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 16.5% | Stage #1: D-glucose With pyrrolidine In ethanol at 60℃; for 4h; Stage #2: With acetic acid In ethanol at 75℃; for 40h; | 1; 4 Embodiment 1 Preparation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one: in a 500 mL three-necked round bottom flaskIn the process, add 36g of D-glucose (0.2mol), 28g of tetrahydropyrrole (0.4mol) and 250mL of absolute ethanol.In a blender, stir at 60C for 4h until it is clear. Then dissolve 24g of acetic acid (0.4mol) in 50mL of absolute ethanol and place it in a constant pressure dropping funnel.Slowly dropwise into the three-necked flask, and the temperature was raised to 75 ° C. and the reaction was continued for 40 h. After the reaction, cool to room temperature, 45C, 75Pa conditionsAfter the solvent was distilled off under reduced pressure, a viscous Maillard reaction product was obtained.Using silica gel chromatography column-polyamide resin column-silica gel column chromatography three times for viscous Maillard reaction productsIsolation and purification. First, dissolve the Maillard reaction product with methanol, add silica gel (100 ~ 200 mesh), mix well and reduce the pressureEvaporate the solvent to loose sand, and add it to the first silica gel column (200 ~ 300 mesh silica gel) as a dry sample. DichloromethaneMethane / methanol = 50: 1 mixed solvent is used as the eluent. The eluate is detected by the detector. When it contains 2,3-dihydro-3,5-dihydroxy 6-methyl-4H-pyran-4-one When the crude product flows out, the detector introduces the effluent to the inlet of the transfer pump and passes through the transfer pump.Conveyed to the first-stage concentration device at the upper end of the polyamide resin column. The first stage concentration device receives a certain volume of effluentAfter that, the stirring heating device was started, concentrated to a certain volume, and then flowed into the polyamide resin column. Similarly, the crude product is polyamideAfter the resin column and the second silica gel column, a pure 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one product was obtained.The total yield was 16.54%. After nuclear magnetic resonance and GC-MS detection, it was confirmed to be the target with a purity of more than 98%. (Of which polyamideThe resin column was mixed with petroleum ether / acetone = 7: 1 as the eluent, and the second silica gel column was mixed with dichloromethane / methanol = 50: 1.The combined solvent is the eluent. |
| 16% | With piperidine; acetic acid In ethanol at 65 - 73℃; for 36h; | |
| 7% | Stage #1: D-glucose With piperidine In ethanol at 90℃; for 1.5h; Stage #2: With acetic acid In ethanol for 22h; Reflux; |
| Stage #1: D-glucose With piperidine; acetic acid; triethylamine at 20 - 70℃; Stage #2: With mercaptoacetic acid In ethanol at 75℃; for 20.6667h; | 4 Example 4: Preparation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (synthesis with the use of glucose, piperidine and thioglycolic acid) 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one was prepared by use of glucose, piperidine and thioglycolic acid in accordance with Isabelle Cutzach et al.: J. Agric. Food Chem. 47:1663-67, 1999 (modified). Next the synthesis method reported in this document will be briefly described. To a suspension of D-glucose (600 g, 3.33 mol) and triethylamine (192 mL, 3.33 mol), piperidine (329 mL, 3.33 mol) and acetic acid (192 mL, 3.33 mol) were added under stirring at room temperature. The obtained mixture was stirred under heating at 70°C for 1 hour. After cooling to room temperature by standing, the mixture was allowed to stand in a refrigerator overnight. The solid thus precipitated was washed with 4 L of a solvent mixture of ethanol-acetone (1:1) and collected by filtration. Then, it was heated to 35°C and dried under reduced pressure to give a piperidine derivative. A solution of the obtained piperidine derivative (176 g, 0.711 mol) in ethanol (1.6 L) was stirred under heating at 75°C. A solution of thioglycolic acid (49.6 ml, 0.713 mol) in ethanol (135 mL) was dropped thereinto over 40 minutes and the mixture was heated to 75°C and stirred for additional 20 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to give crude 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one. The crude 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one thus obtained was subjected to column chromatography successively with Daiso SP 120-40/60-ODS-B (manufactured by DAISO) and CHP20P (manufactured by MITSUBISHI CHEMICAL Co.). Thus a purified 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one preparation was obtained. In the above synthesis method, the purified 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one preparation was obtained at a yield of 6.93 g of the purified 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one preparation per 600 g of glucose. |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With recombinant C,N-terminal 6xHis-tagged rabbit cecum umbgl3B β-glucosidase; water at 28℃; for 0.25h; aq. phosphate buffer; | ||
| With C-terminally His6-tagged Oryza sativa L. β-glucosidase OsTAGG2 In aq. acetate buffer at 37℃; for 0.166667h; Enzymatic reaction; | ||
| With rice BGlu1 β-glucosidase, Q178A mutant In aq. acetate buffer at 30℃; Enzymatic reaction; |
| With Cellulosimicrobium cellulans strain 21 GH1 β-glucosidase CcBgl1B, recombinant, molecular weight: 57 kDa; water In aq. phosphate buffer at 30℃; for 2h; Enzymatic reaction; | ||
| Multi-step reaction with 2 steps 1: Cellulosimicrobium cellulans strain 21 GH1 β-glucosidase CcBgl1B, recombinant, molecular weight: 57 kDa; water / aq. phosphate buffer / 0.17 h / 30 °C / pH 6 / Enzymatic reaction 2: Cellulosimicrobium cellulans strain 21 GH1 β-glucosidase CcBgl1B, recombinant, molecular weight: 57 kDa; water / aq. phosphate buffer / 2 h / 30 °C / pH 6 / Enzymatic reaction |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| In methanol for 1h; Reflux; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Thermobifida fusca cellulase Cel9A at 50℃; for 24h; aq. acetate buffer; Enzymatic reaction; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Thermobifida fusca cellulase Cel5A at 50℃; for 24h; aq. acetate buffer; Enzymatic reaction; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 18.4 %Chromat. 2: 9.7 %Chromat. 3: 6.5 %Chromat. 4: 58.4 %Chromat. | Stage #1: D-glucose In water at 28℃; for 168h; Microbiological reaction; Stage #2: methanol With hydrogenchloride |
[ 50-99-7 ]
[ 67-63-0 ]
[ 67-56-1 ]
[ 57-55-6 ]
[ 623-61-0 ]
[ 107-21-1 ]
[ 116-09-6 ]
[ 67-64-1 ]
[ 56-81-5 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| In water at 300 - 375℃; Autoclave; | 3 Example 3; Various batch reactions using the Materials of Example 2 were completed. Table 2 presents the yields of products according to the methods of the invention generated from several simple sugars. In these runs aqueous solutions of the sugar at room temperature were pumped into the hot alcohol under pressure. |
[ 50-99-7 ]
[ 67-63-0 ]
[ 67-56-1 ]
[ 96-26-4 ]
[ 108-21-4 ]
[ 623-61-0 ]
[ 107-21-1 ]
[ CAS Unavailable ]
[ 116-09-6 ]
[ 67-64-1 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| In water at 300℃; for 0.3h; Stainless steel flow reactor; | 4 Example 4; Continuous reactions using the Materials of Example 2 were completed. The yields of products obtained from a continuous run are presented in Table 3. A reaction mixture under high pressure (from approximately 1500 psi to 3500 psi) was pumped sequentially through a high temperature zone (from approximately 200° C. to 325° C.), a heat exchanger, a backpressure regulator, and then into a collection vessel. The reaction mixture was generated by pumping an aqueous solution of glucose at room temperature into a heated source of 2-propanol (temperature from approximately 275° C. to 325° C.) which was being pumped into the high temperature zone. aContinuous reaction pumped through a stainless steel tube having about a 5 min residence time and a length to diameter ratio of about 20. Temperature was 300° C., and the sample was collected over a period of 18 min. Glucose input during this time was 0.64 g and solvent included 32 mL of 2-propanol/5% water. ball yields given in grams per gram starting material cDetected by NMR in the D2O solution as the hydrate but reported as the aldehyde. disopropyl ester tentatively assigned as isopropyl glycolate eisopropyl ester tentatively assigned as isopropyl acetate funidentified products not soluble in alcohol gunidentified products soluble in alcohol htotal identified products derived from carbohydrate material iproduced by the oxidation of 2-propanol |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With ammonium sulfate; potassium dihydrogenphosphate; magnesium sulfate heptahydrate; calcium(II) chloride dihydrate; potassium hydrogenphosphate trihydrate; vitamin B1; biotin In water; glycerol at 37℃; for 126h; Microbiological reaction; | 1.2 1.2 Preparation of a Succinate-Containing Fermentation BrothA 1.0 ml aliquot of the working cell bank of the succinate production strain is removed from the freezer compartment and stored at room temperature for 10 minutes. 100 μl thereof are withdrawn under sterile conditions and distributed under sterile conditions with an inoculating loop on an LB+glucose agar plate. After incubation at 37° C. for 20 hours, the plate of cell material is withdrawn under sterile conditions and used to inoculate the preculture flasks (2000 ml round-bottom flasks with 4 chicanes, which comprise 300 ml of preculture medium). The preculture flasks are incubated at 37° C. and 110 rpm on an incubation shaker (from Infors, Multitron type) for six hours. For the start of the main culture, 3.51 of batch medium are inoculated with 250 ml of the preculture. The cultivation is effected at 37° C. for 120 h in a stirred 5 l bioreactor (from Infors, ISF 100) with a 6-blade disk stirrer and four baffles, pH and pO2 electrodes. Within the first 5.5 h (aerobic growth phase), the cultivation is effected at a constant speed of 900 rpm and sparging with sterile air (3.0 l/min), then switched to 250 rpm and sparging with sterile CO2 gas (0.18 l/min). The pH is controlled to 7.0 at the start with 20% NaOH solution and 20% HCl soluftion. From 5.5 h, instead of NaOH solution, 25% NH4OH solution is used and the pH is regulated to 6.8. The feed medium is metered in according to a fixed metering profile which ensures that the glucose concentration in the fermentation broth is always between 10 and 30 g/l. For the feed metering, a balance (from Satorius, LP6200S), a metering module (from Satorius, YFC02Z-V2) and a pump (from Meredos, HP60) are used. During the cultivation, the course of the optical density is determined at a measurement wavelength of 600 nm (OD600) with a photometer (from Pharmacia Biotech, Ultrospec 2000). The concentrations of glucose and succinate in the cell-free supernatant (filtration of the culture broth with Braun syringes, 2 ml injection and syringe filter attachment from Millipore, Millipore GP; Ø 33 mm; pore width 0.22 μm; PES Express membrane) are quantified by means of HPLC (stationary phase: Aminex HPX-87 H, 300×7.8 mm [from Biorad], mobile phase: 5 mM H2SO4, RI detection). At the end of the cultivation, the fermentation broth is discharged into 3 l Erlenmeyer flasks and sterilized at 121° C. in an autoclave for 20 minutes.Before the further processing, the biomass is preferably removed, for example by means of laboratory centrifuge.The preculture medium comprises the constituents specified in Table 2. To prepare the preculture medium, 22.0 g of glucose monohydrate, 10.0 g of yeast extract, 20.0 g of tryptone, 0.9 g of K2HPO4.3H2O, 1.14 g of KH2PO4, 0.25 g of CaCl2.2H2O, 3.0 g of (NH4)2SO4, 0.5 g of MgSO4.7H2O, 20 ml of a biotin solution (50 mg/l, demineralized water as the solvent) and 1 ml of a thiamine solution (1 g/l, demineralized water as the solvent) were added to 0.95 liter of demineralized water. With stirring, the pH is adjusted to 7.0 with 2N NaOH solution, then the solution is made up to 1.0 liter with demineralized water. The sterilization is effected by sterile filtration through Stericups with Millipore Express PLUS membrane with pore width 0.22 μm (from Millipore). TABLE 2 Preculture medium Medium constituent Concentration Glucose monohydrate 22.0 g/l Yeast extract 10.0 g/l Tryptone 20.0 g/l K2HPO4•3H2O 0.9 g/l KH2PO4 1.14 g/l CaCl2•2H2O 0.25 g/l (NH4)2SO4 3.0 g/l MgSO4•7H2O 0.5 g/l Biotin 1.0 mg/l Thiamine 1.0 mg/l The batch medium for the main culture comprises the constituents specified in Table 3. To prepare the batch medium, 154.0 g of glucose monohydrate, 35.0 g of yeast extract, 70.0 g of tryptone, 3.15 g of K2HPO4.3H2O, 3.99 g of KH2PO4, 088 g of CaCl2. 2H2), 10.5 g of (NH4)2SO4, 1.75 g of MgSO4.7H2O, 70 ml of a biotin solution (50 mg/l, demineralized water as the solvent) and 3.5 ml of a thiamine solution (1 g/l, demineralized water as the solvent) are added to 3.30 liters of demineralized water. With stirring, the pH is adjusted to 7.0 with 2N NaOH solution, then the solution is made up to 3.5 liters with demineralized water, The sterilization is effected by sterile filtration by means of Stericups with Millipore Express PLUS membrane with pore width 0.22 μm (from Millipore). TABLE 3 Batch medium for the main culture Medium constituent Concentration Glucose monohydrate 44.0 g/l Yeast extract 10.0 g/l Tryptone 20.0 g/l K2HPO4•3H2O 0.9 g/l KH2PO4 1.14 g/l CaCl2•2H2O 0.25 g/l (NH4)2SO4 3.0 g/l MgSO4•7H2O 0.5 g/l Biotin 1.0 mg/l Thiamine 1.0 mg/l For the preparation of the feed medium, 550 g of glucose monohydrate are dissolved in 1 liter of demineralized water for the main culture. The sterilization is effected by sterile filtration through Stericups with Millipore Express PLUS membrane with pore width 0.22 μm (from Millipore). | |
| Stage #1: D-glucose In aq. phosphate buffer at 37℃; Microbiological reaction; Stage #2: With ammonium hydroxide; ammonium bicarbonate In aq. phosphate buffer at 37℃; for 48h; Microbiological reaction; | 1 Example 1 Fermentative Production of Succinic Acid Example 1 Fermentative Production of Succinic Acid E. coli strain KJ122 was inoculated in a minimum media consisting of NBS, 100 mM MOPS, 2% glucose, 1 mM MgSO4, trace element and 0.1 mM CaCl2 at 37° C. Once the cell density reaches OD=5, the inoculums were transferred to a fermenter containing initial medium consisting of 25 mM KH2PO4, 3 mM MgSO4, 2 mM betaine, and 8 ppm of antifoam 204. Fermentation was run in a fed-batch mode with glucose as the carbohydrate source. As succinic acid was being produced, a solution of 7M NH4OH and 3M NH4HCO3 was metered into the fermentor to maintain the pH at around 6.5-7 and to provide a source for CO2. After 48 hours, the fermentation was completed. Biomass was removed by a tangential flow microfiltration unit. The filtered broth composition is shown in Table 1. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With hydrogenchloride; triethanolamine; magnesium sulfate In n-heptane; water | 10 7.10 Example 10 Preparative Scale Production of (S)-1-[2',6'-dichloro-3'-fluorophenyl]-ethanol To a 500 mL jacketed three neck round bottom flask equipped with an Ace Glass mechanical stirrer (75 mm diameter teflon stirrer blade), and a pH electrode connected to an automatic titrator for pH-controlled addition of base on-demand via a delivery tube into the vessel, was added water (120 mL), triethanolamine (1.8 g) and then hydrochloric acid to adjust the pH to 7.0. Magnesium sulfate was added as a 1M solution (120 μL, 0.12 mmoles, 14.4 mg of MgSO4). The solution was heated to 30° C. with heating fluid circulating through the flask's jacket. Glucose (20 g) was added followed by Na-NADP (120 mg), GDH (0.50 g) and KRED having SEQ ID No. 38 (0.50 g). The pH stat was set to maintain the pH at 7.0+-0.1 by the addition of 4N NaOH through the delivery tube. 2',6'-dichloro-3'-fluoroacetophenone (50 g) was added to start the reaction. The electrode required periodic rinsing to remove enzyme derived material. Additional glucose was added in portions as the reaction proceeded: 10 g at 104 min (after 17.5 mL of 4 N NaOH had been added), 5 g at 275 min (after 35.2 mL of 4 N NaOH had been added), 5 g at 379 min (after 42 mL of 4 N NaOH had been added), and 8 g at 488 min (after 47 mL of 4 N NaOH had been added). The reaction was stopped after 24 hr. Heptane (150 mL) was then added and the mixture was heated to 40° C. for 45 min. After cooling to 30° C. the resulting mixture was poured into a separatory funnel and the majority of the bottom aqueous layer was drained. The top layer, a heptane emulsion, was filtered (350 mL, 85 mm diameter coarse filter) through a celite pad under vacuum. The filter was washed with heptane (150 mL) and the filtrate was transferred to a separatory funnel and the two phases were separated. The heptane phase was concentrated on a rotary vacuum evaporator (˜50° C., ˜150 mmHg increasing to 40 mmHg) to yield (S)-1-[2',6'-dichloro-3'-fluorophenyl]-ethanol as an oil (47.8 g, 94%) which crystallized upon standing. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 8.25%Chromat.; 29.89%Chromat.; 25.26%Chromat. | With Aspergillus flavus NFCCI 2364 culture filtrate; In aq. buffer; at 55℃; for 24h;pH 5.5;Microbiological reaction; | General procedure: FOS production was carried out by adding 1ml of enzyme samples collected at various time intervals to 3ml of 50% (w/v) sucrose dissolved in 0.1M citrate buffer (pH 5.5) for period of 24h at 55C. The amount of FOS formation in the samples was analyzed by high performance liquid chromatography (HPLC, Waters) with sugar-pak column (6.5×300mm) and refractive index (RI) differential detector (RI 2414). |
| 5.94%Chromat.; 23.74%Chromat.; 25.31%Chromat. | With Aspergillus niger SI 19 culture filtrate; In aq. buffer; at 55℃; for 24h;pH 5.5;Microbiological reaction; | General procedure: FOS production was carried out by adding 1ml of enzyme samples collected at various time intervals to 3ml of 50% (w/v) sucrose dissolved in 0.1M citrate buffer (pH 5.5) for period of 24h at 55C. The amount of FOS formation in the samples was analyzed by high performance liquid chromatography (HPLC, Waters) with sugar-pak column (6.5×300mm) and refractive index (RI) differential detector (RI 2414). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| In ethanol; water Reflux; Inert atmosphere; | ||
| In ethanol; water at 80℃; for 6h; | 14.A; V.B Method A Method A Scheme V, Step B: Charge to a 20 L three neck flask a solution of 4-methylindoline (1000 g; 7.51moles) in ethanol: water (8000 ml: 1000 ml) and D-glucose (1480g; 8.25 moles). Heat the mixture for 6 hours at 80° C. Concentrate the mixture under reduced pressure and dissolve the residue in pyridine : dichloromethane (8000 ml: 8000 ml). Add dimethylaminopyridine (91.79 g; 0.75 moles) and cool the reaction mixture to 10° C. (internal temperature). Add acetic anhydride (9000 ml) dropwise. When the addition is complete stir the reaction mixture for 1 hour at 45° C. and then stir overnight at room temperature. Concentrate the mixture under reduced pressure. Add ethyl acetate (20 L) and water (10 L) to the residue. Separate the organic layer and extract the aqueous layer with ethyl acetate (2*10 L). Combine the organic layers and wash with a saturated solution of citric acid (5 Kg) in water. Dry the organic layer over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Crystallise the residue from ethanol to give the title compound (3205.5 g; 92.11% yield): mass spectrum (m/z): 464.2 (M+1). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With trifluoroacetic acid; In water; at 120℃; for 4h; | Compound 1 (about 4 mg) was added into a solution of water (1 mL) and 2N aqueous CF3COOH(2 mL), heated to 120°C under reflux conditions for 4 h. The mixture was diluted with water (2 mL) and then extracted with EtOAc (3 £2 mL). The combined organic phase was washed with brine and evaporated to dryness to afford the aglycones. The aqueous phase was concentrated. Then dry pyridine (1 mL) and L-cysteine methyl ester hydrochloride (2 mg) were added into the residue. Each mixture was reacted at 60°C for 1 h, and 0.5mL of (trimethylsilyl) imidazole dissolved in H2O was added, followed by heating to dryness at 60°C for 2 h. Each dried reactant was extracted with n-hexane (3 £ 1 mL) and H2O (1 mL, each). The n-hexane fraction was subjected to GC (column: Rtx-1, 0.25mm i.d. 0.25 mm, length 30 m). The conditions of GC were flame ionization detector; column temperature 100?180°C (10°C min?1)and 180?230°C (3°C min21); injector temperature 250°C; detector temperature 300°C and the carrier gas (N2, 0.8mLmin21). Under these conditions, these sugars of each reactants were identified by comparison with authentic samples: tR (min) 7.56 (D-ribose), 7.85 (L-ribose), 10.49 (D-glucose) and 11.10 (L-glucose). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Viscozyme L (-fructosyltransferase) immobilized on chitosan spheres; In aq. acetate buffer; at 50℃;pH 4.5;Flow reactor; | Production of invert sugar was carried using a solution of sucrose150 g/L diluted in 50 mM sodium acetate buffer pH 4.5. The solu-tion was pumped at flow rates of (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.7) mL/min in both fluidized and packed bed reactors. Four bed vol-umes of solution were passed through the column before taking the sample to achieve stationary state. These experiments were carriedout in duplicate, changing the column bed at each experiment |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 92.11% | Stage #1: 4-methyl-2,3-dihydro-1H-indole; D-glucose In ethanol; water at 80℃; for 6h; Large scale; Stage #2: acetic anhydride With pyridine; dmap In dichloromethane at 10 - 45℃; Large scale; | 2.B [(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(4-methylindolin-1-yl)tetrahydropyran-2-yl]methyl acetate Method B [0031] Scheme I, Step A: Charge to a 20 L three neck flask a solution of 4-methylindoline (1000 g; 7.51 moles) in ethanol: water (8000 ml: 1000 ml) and D-glucose (1480 g; 8.25 moles). Heat the mixture for 6 hours at 80° C. Concentrate the mixture under reduced pressure and dissolve the residue in pyridine:dichloromethane (8000 ml:8000 ml). Add dimethylaminopyridine (91.79 g; 0.75 moles) and cool the reaction mixture to 10° C. (internal temperature). Add acetic anhydride (9000 ml) dropwise. When the addition is complete stir the reaction mixture for 1 hour at 45° C. and then stir overnight at room temperature. Concentrate the mixture under reduced pressure. Add ethyl acetate (20 L) and water (10 L) to the residue. Separate the organic layer and extract the aqueous layer with ethyl acetate (2×10 L). Combine the organic layers and wash with a saturated solution of citric acid (5 Kg) in water. Dry the organic layer over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Crystallise the residue from ethanol to give the title compound (3205.5 g; 92.11% yield): mass spectrum (m/z): 464.2 (M+1). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 85% | With acetic acid; In ethanol; for 10.0h;Reflux; | General procedure: To solution of 4 (10 mmol) in absoluteethanol, different sugars (10 mmol) were added and then glacial acetic acid (1 ml) was added to the reaction mixture which was refluxed for 10h (TLC).The solvent was evaporated or concentrated under reduced pressure and the product was filtered off to afford 5(a-d) (83 - 88%) yields. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Cellulosimicrobium cellulans strain 21 GH1 β-glucosidase CcBgl1B, recombinant, molecular weight: 57 kDa; water In aq. phosphate buffer at 30℃; for 0.166667h; Enzymatic reaction; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Cellulosimicrobium cellulans strain 21 GH1 β-glucosidase CcBgl1B, recombinant, molecular weight: 57 kDa; water In aq. phosphate buffer at 30℃; for 0.166667h; Enzymatic reaction; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Cellulosimicrobium cellulans strain 21 GH1 β-glucosidase CcBgl1B, recombinant, molecular weight: 57 kDa; water In aq. phosphate buffer at 30℃; for 0.166667h; Enzymatic reaction; |
[ 50-99-7 ]
[ 79-14-1 ]
[ 80-69-3 ]
[ 2089584-55-2 ]
[ 473-81-4 ]
[ 526-95-4 ]
[ 6556-12-3 ]
[ 1986-15-8 ]
[ 25525-21-7 ]
[ 124-38-9 ]
[ 2382861-64-3 ]
[ 144-62-7 ]
[ 64-19-7 ]
[ 526-83-0 ]
[ 1198-69-2 ]
[ 826-91-5 ]
[ 389-36-6 ]
[ 2782-04-9 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With 5% Pt/titania; water-d2; oxygen at 80℃; for 24h; |
[ 50-99-7 ]
[ 79-14-1 ]
[ 80-69-3 ]
[ 2089584-55-2 ]
[ 473-81-4 ]
[ 526-95-4 ]
[ 6556-12-3 ]
[ 1986-15-8 ]
[ 25525-21-7 ]
[ 124-38-9 ]
[ 2382861-64-3 ]
[ 144-62-7 ]
[ 64-19-7 ]
[ 526-83-0 ]
[ 1198-69-2 ]
[ 389-36-6 ]
[ 2782-04-9 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Pt/SiO2; water-d2; oxygen at 60℃; for 24h; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With hydrogenchloride In water at 60℃; for 1.5h; | 3.5. Acid hydrolysis of 6-8 A solution of 6e8 (1.0 mg) in 6 N HCl was stirred at 60 C for1.5 h. After cooling, the mixtures were extracted with EtOAc. The aqueous layer was concentrated in vacuo followed by TLC examination and optical rotation measurement. The optical rotation of the glucose of 6-8 are as follows: [a]D25 45.0 (c 0.044, H2O) for 6,[a]D25 42.3 (c 0.052, H2O) for 7, and [a]D25 40.9 (c 0.044, H2O) for 8. By comparing optical rotation with D-glucose: [a]D25 42.3 (c0.106, H2O), the glucose in compounds 6-8 were determined to be D-configurations |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With hydrogenchloride In water at 60℃; for 1.5h; | 3.5. Acid hydrolysis of 6-8 A solution of 6e8 (1.0 mg) in 6 N HCl was stirred at 60 C for1.5 h. After cooling, the mixtures were extracted with EtOAc. The aqueous layer was concentrated in vacuo followed by TLC examination and optical rotation measurement. The optical rotation of the glucose of 6-8 are as follows: [a]D25 45.0 (c 0.044, H2O) for 6,[a]D25 42.3 (c 0.052, H2O) for 7, and [a]D25 40.9 (c 0.044, H2O) for 8. By comparing optical rotation with D-glucose: [a]D25 42.3 (c0.106, H2O), the glucose in compounds 6-8 were determined to be D-configurations |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With hydrogenchloride In water at 60℃; for 1.5h; | 3.5. Acid hydrolysis of 6-8 A solution of 6e8 (1.0 mg) in 6 N HCl was stirred at 60 C for1.5 h. After cooling, the mixtures were extracted with EtOAc. The aqueous layer was concentrated in vacuo followed by TLC examination and optical rotation measurement. The optical rotation of the glucose of 6-8 are as follows: [a]D25 45.0 (c 0.044, H2O) for 6,[a]D25 42.3 (c 0.052, H2O) for 7, and [a]D25 40.9 (c 0.044, H2O) for 8. By comparing optical rotation with D-glucose: [a]D25 42.3 (c0.106, H2O), the glucose in compounds 6-8 were determined to be D-configurations |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With Escherichia coli BL21 recombinant α-glucosidase In aq. buffer at 60 - 100℃; | 4.9. Transglycosylation Reaction The reaction mixture, which consisted of 30% maltose and 5 g enzyme in sodium phosphatebuffer, was incubated at 60 °C for 2 h. Reaction mixtures were incubated at 100 °C for 5 min toinactivate the enzyme, and the samples were then centrifuged at 12,000 g for 10 min and analyzedby HPAEC. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 20% 2: 16% 3: 14% 4: 8% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride; lanthanum(lll) triflate at 120℃; for 2h; Sealed tube; | |
| 1: 20% 2: 16% 3: 11% 4: 7% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Sealed tube; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 48% 2: 7% 3: 7% | Stage #1: Eucalyptus cellulose With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Stage #2: With water at 120℃; for 4h; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 50% 2: 8% 3: 7% 4: 6% | Stage #1: Pinus cellulose With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Stage #2: With water at 120℃; for 4h; | |
| 1: 24% 2: 17% 3: 11% 4: 8% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Sealed tube; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 46% 2: 10% 3: 9% 4: 8% | Stage #1: Pinus cellulose, unbleached With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Stage #2: With water at 120℃; for 4h; | |
| 1: 23% 2: 16% 3: 12% 4: 7% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Sealed tube; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 31% 2: 20% 3: 16% 4: 16% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 6h; Sealed tube; | |
| 1: 29% 2: 18% 3: 13% 4: 8% | With choline chloride; silver trifluoromethanesulfonate; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 2h; Sealed tube; | |
| 1: 28% 2: 16% 3: 16% 4: 11% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 12h; Sealed tube; |
| 1: 27% 2: 21% 3: 20% 4: 12% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 8h; Sealed tube; |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 24% 2: 8% 3: 6% | With tin(II) trifluoromethanesulfonate; choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 2h; Sealed tube; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1: 26% 2: 7% | With choline chloride; oxalic acid; 1-butyl-3-methylimidazolium chloride at 120℃; for 16h; Sealed tube; |

[ 3615-41-6 ]
[ 50-99-7 ]
[ 491-71-4 ]| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With sulfuric acid; water; at 80℃; for 2h; | A MeOH solution of 1 (3 mg) was treated with H2SO4 solution (5%), hydrolyzed on a waterbath at 80 C for 2 h, cooled, diluted with H2O, and extracted with EtOAc. The extract was washed with H2O, dried over anhydrous Na2SO4, and evaporated to dryness. The solid was dissolved in MeOH, after which the aglycon crystallized, mp 325-330. UV spectrum (a, lambdamax, nm): 265, 345; +NaOMe - 267, 405; +AlCl3 - 274, 396; + AlCl3/HCl - 275,394; + NaOAc - 274, 355. The aqueous fraction was neutralized by AV-17 anion-exchanger (OH--form). Sugars were identified by comparing paper chromatograms with authentic D-glucose and L-rhamnose using system 3. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 89% | With E. coli whole cells harboring coumarin C-glucosyltransferase from Morus alba (MaCGT) at 30℃; for 24h; Enzymatic reaction; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 62% | With hydrogenchloride In water monomer at 40℃; | 1-A 1.2 mM aminoalbendazole (CAS 80983-36-4) (commercially available: Albendazole amine) and 0.5 M glucose were added to a hydrochloric acid solution (pH 3.45) to prepare a reaction mixture, and reacted at 40±1° C. in a Teflon-coated rubber-stopped glass vial, and diluted with acetate buffer (0.5 M, pH 5.8) to terminate the reaction. The solvent was removed, and purified by column chromatography using dichloromethane and 10% methanol to obtain an albendazole-glucose conjugate compound in a yield of 62%. The product was analyzed by 1H-NMR spectrum to confirm the formation of an albendazole-glucose conjugate compound (see FIG. 5). |
| 62% | With hydrogenchloride In water monomer at 40℃; | 1-A 1.2 mM aminoalbendazole (CAS 80983-36-4) (commercially available: Albendazole amine) and 0.5 M glucose were added to a hydrochloric acid solution (pH 3.45) to prepare a reaction mixture, and reacted at 40±1° C. in a Teflon-coated rubber-stopped glass vial, and diluted with acetate buffer (0.5 M, pH 5.8) to terminate the reaction. The solvent was removed, and purified by column chromatography using dichloromethane and 10% methanol to obtain an albendazole-glucose conjugate compound in a yield of 62%. The product was analyzed by 1H-NMR spectrum to confirm the formation of an albendazole-glucose conjugate compound (see FIG. 5). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With ammonium sulfate; potassium dihydrogenphosphate; magnesium sulfate heptahydrate In water for 48h; Microbiological reaction; | 1 Example 1 - Process for making a first halogenated psilocybin derivative from halogenated tryptophan feedstock. Yeast (Saccharomyces cerevisiae) was genetically engineered to enable bioconversion of commercially obtained, halogenated, simple indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast (Saccharomyces cerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was engineered to include three enzymatic steps through chromosomal homologous recombination of integration cassettes as described previously (Dastmalchi et al., 2019, Nat. Chem Biol.15: 384-390; Chen et al., 2018, Nat. Chem Biol. 14: 738- 743). The three steps were comprised of PsiH (SEQ.ID NO: 4), PsiK (SEQ.ID NO: 6) and PsiM (SEQ.ID NO: 8), encoded by SEQ.ID NO: 3, SEQ.ID NO: 5 and SEQ.ID NO: 7, respectively, with addition of in-frame, C-terminal HA (SEQ.ID NO: 81, SEQ.ID NO: 82), V5 (SEQ.ID NO: 87, SEQ.ID NO: 88), and FLAG (SEQ.ID NO: 85, SEQ.ID NO: 86) epitope tags, respectively. DNA (SEQ.ID NO: 95) encoding a fifth enzyme, PcCPR (SEQ.ID NO: 96) was also integrated following PCR-based, C-terminal, in-frame addition of epitope tag c-MYC (SEQ.ID NO: 83, SEQ.ID NO: 84) to support functionality of PsiH. Integration cassettes were built using yeast promoter sequences amplified from S. cerevisiae genomic DNA as described (Dastmalchi et al. 2019; Chen et al. 2018) enabling constitutive gene expression. Amplified promoters included PGK1 (SEQ.ID NO: 77), TDH3 (SEQ.ID NO: 78), CLN1 (SEQ.ID NO: 79), and UGA1 (SEQ.ID NO: 80). Two integration cassettes were assembled: the first (SEQ.ID NO: 74) harbored tagged PsiH and PcCPR, and the second (SEQ.ID NO: 75) harbored tagged PsiK and PsiM. Successive genomic integration of these cassettes was performed as described previously (Chen et al.2018). Following stable integration of these two cassettes, the strain was further manipulated by transformation with a yeast episomal vector encoding a promiscuous decarboxylase, ClostSporTDC (pMM1- pTDH3-ClostSporTDC-His-tCYC1) (SEQ.ID NO: 76) enabling efficient decarboxylation of halogenated tryptophan intermediates and/or feedstocks. For this Example heterologous expression of a non native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-1. For scaled-up production of halogenated product, culturing was performed as follows. Seed cultures were inoculated in SD-drop-out medium overnight. The overnight culture was then divided into two flasks containing 500 ml each of SD-drop-out medium containing 2% (w/v) glucose, 0.3%(w/v) KH2PO4, 0.05% (w/v) MgSO4.7H2O, 0.5% (w/v) (NH4)2SO4 plus 500 μM 6-fluorotryptophan (ThermoFisher Scientific) for conversion by Sc-1. Yeast cultures were grown for 48h. Cultures were then centrifuged (10,000g x 5 minutes) to remove cellular content, and culture broth containing secreted halogenated product was stored at -80 °C until further processing. To determine successful biosynthesis of 6-fluoro-psilocybin (compound with formula (XI)), culture broth was analyzed using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific), employing a modified version of a method described previously (Chang et al., 2015, Plant Physiol.169: 1127-1140), with the exception that liquid chromatography was carried out using an UltiMate 3000 HPLC (Thermo Fisher Scientific) equipped with a Poroshell 120 SB- C18 column (Agilent Technologies) instead of an Accela HPLC system (Thermo Fisher Scientific) equipped with a Zorbax C18 column (Agilent Technologies). Briefly, 10 microliters of sample was injected at a flow rate of 0.5 mL/min and a gradient of solvent A (water with 0.1 % of formic Acid) and solvent B (ACN with 0.1% formic Acid) as follows: 100% to 0% (v/v) solvent A over 5 min; isocratic at 0% (v/v) for 1 min; 0% to 100% (v/v) over 0.1 min; and isocratic at 100% (v/v) for 1.9 min. Total run time was 8 minutes. Heated ESI source and interface conditions were operated in positive ion mode as follows: vaporizer temperature, 400°C; source voltage, 3 kV; sheath gas, 60 au, auxiliary gas, 20 au; capillary temperature, 380°C; capillary voltage, 6 V; tube lens, 45 V. Instrumentation was performed as a single, HR scan event using Orbitrap detection of m/z in the range of 100-500 m/z. Ion injection time was 300 ms with scan time of 1 s. External and internal calibration procedures ensured <2 ppm error to facilitate elemental formulae predictions. Singly protonated product with exact m/z and expected elemental formula matching 6-fluoro-psilocybin having chemical formula (XI): (XI) eluted at 1.9 minutes (EIC, see: FIG.10). | |
| With ammonium sulfate; potassium dihydrogenphosphate; magnesium sulfate heptahydrate In water for 48h; Microbiological reaction; | 1 Example 1 - Process for making a first halogenated psilocybin derivative from halogenated tryptophan feedstock. Yeast (Saccharomyces cerevisiae) was genetically engineered to enable bioconversion of commercially obtained, halogenated, simple indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast (Saccharomyces cerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was engineered to include three enzymatic steps through chromosomal homologous recombination of integration cassettes as described previously (Dastmalchi et al., 2019, Nat. Chem Biol.15: 384-390; Chen et al., 2018, Nat. Chem Biol. 14: 738- 743). The three steps were comprised of PsiH (SEQ.ID NO: 4), PsiK (SEQ.ID NO: 6) and PsiM (SEQ.ID NO: 8), encoded by SEQ.ID NO: 3, SEQ.ID NO: 5 and SEQ.ID NO: 7, respectively, with addition of in-frame, C-terminal HA (SEQ.ID NO: 81, SEQ.ID NO: 82), V5 (SEQ.ID NO: 87, SEQ.ID NO: 88), and FLAG (SEQ.ID NO: 85, SEQ.ID NO: 86) epitope tags, respectively. DNA (SEQ.ID NO: 95) encoding a fifth enzyme, PcCPR (SEQ.ID NO: 96) was also integrated following PCR-based, C-terminal, in-frame addition of epitope tag c-MYC (SEQ.ID NO: 83, SEQ.ID NO: 84) to support functionality of PsiH. Integration cassettes were built using yeast promoter sequences amplified from S. cerevisiae genomic DNA as described (Dastmalchi et al. 2019; Chen et al. 2018) enabling constitutive gene expression. Amplified promoters included PGK1 (SEQ.ID NO: 77), TDH3 (SEQ.ID NO: 78), CLN1 (SEQ.ID NO: 79), and UGA1 (SEQ.ID NO: 80). Two integration cassettes were assembled: the first (SEQ.ID NO: 74) harbored tagged PsiH and PcCPR, and the second (SEQ.ID NO: 75) harbored tagged PsiK and PsiM. Successive genomic integration of these cassettes was performed as described previously (Chen et al.2018). Following stable integration of these two cassettes, the strain was further manipulated by transformation with a yeast episomal vector encoding a promiscuous decarboxylase, ClostSporTDC (pMM1- pTDH3-ClostSporTDC-His-tCYC1) (SEQ.ID NO: 76) enabling efficient decarboxylation of halogenated tryptophan intermediates and/or feedstocks. For this Example heterologous expression of a non native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-1. For scaled-up production of halogenated product, culturing was performed as follows. Seed cultures were inoculated in SD-drop-out medium overnight. The overnight culture was then divided into two flasks containing 500 ml each of SD-drop-out medium containing 2% (w/v) glucose, 0.3%(w/v) KH2PO4, 0.05% (w/v) MgSO4.7H2O, 0.5% (w/v) (NH4)2SO4 plus 500 μM 6-fluorotryptophan (ThermoFisher Scientific) for conversion by Sc-1. Yeast cultures were grown for 48h. Cultures were then centrifuged (10,000g x 5 minutes) to remove cellular content, and culture broth containing secreted halogenated product was stored at -80 °C until further processing. To determine successful biosynthesis of 6-fluoro-psilocybin (compound with formula (XI)), culture broth was analyzed using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific), employing a modified version of a method described previously (Chang et al., 2015, Plant Physiol.169: 1127-1140), with the exception that liquid chromatography was carried out using an UltiMate 3000 HPLC (Thermo Fisher Scientific) equipped with a Poroshell 120 SB- C18 column (Agilent Technologies) instead of an Accela HPLC system (Thermo Fisher Scientific) equipped with a Zorbax C18 column (Agilent Technologies). Briefly, 10 microliters of sample was injected at a flow rate of 0.5 mL/min and a gradient of solvent A (water with 0.1 % of formic Acid) and solvent B (ACN with 0.1% formic Acid) as follows: 100% to 0% (v/v) solvent A over 5 min; isocratic at 0% (v/v) for 1 min; 0% to 100% (v/v) over 0.1 min; and isocratic at 100% (v/v) for 1.9 min. Total run time was 8 minutes. Heated ESI source and interface conditions were operated in positive ion mode as follows: vaporizer temperature, 400°C; source voltage, 3 kV; sheath gas, 60 au, auxiliary gas, 20 au; capillary temperature, 380°C; capillary voltage, 6 V; tube lens, 45 V. Instrumentation was performed as a single, HR scan event using Orbitrap detection of m/z in the range of 100-500 m/z. Ion injection time was 300 ms with scan time of 1 s. External and internal calibration procedures ensured <2 ppm error to facilitate elemental formulae predictions. Singly protonated product with exact m/z and expected elemental formula matching 6-fluoro-psilocybin having chemical formula (XI): (XI) eluted at 1.9 minutes (EIC, see: FIG.10). |
Tags: D-Glucose | Glucose | D-(+)-Glucose | Dextrose | Endogenous Metabolite | monosaccharide | general metabolism response | hBON | 50-99-7 |
Precautionary Statements-General | |
| Code | Phrase |
| P101 | If medical advice is needed,have product container or label at hand. |
| P102 | Keep out of reach of children. |
| P103 | Read label before use |
Prevention | |
| Code | Phrase |
| P201 | Obtain special instructions before use. |
| P202 | Do not handle until all safety precautions have been read and understood. |
| P210 | Keep away from heat/sparks/open flames/hot surfaces. - No smoking. |
| P211 | Do not spray on an open flame or other ignition source. |
| P220 | Keep/Store away from clothing/combustible materials. |
| P221 | Take any precaution to avoid mixing with combustibles |
| P222 | Do not allow contact with air. |
| P223 | Keep away from any possible contact with water, because of violent reaction and possible flash fire. |
| P230 | Keep wetted |
| P231 | Handle under inert gas. |
| P232 | Protect from moisture. |
| P233 | Keep container tightly closed. |
| P234 | Keep only in original container. |
| P235 | Keep cool |
| P240 | Ground/bond container and receiving equipment. |
| P241 | Use explosion-proof electrical/ventilating/lighting/equipment. |
| P242 | Use only non-sparking tools. |
| P243 | Take precautionary measures against static discharge. |
| P244 | Keep reduction valves free from grease and oil. |
| P250 | Do not subject to grinding/shock/friction. |
| P251 | Pressurized container: Do not pierce or burn, even after use. |
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| P261 | Avoid breathing dust/fume/gas/mist/vapours/spray. |
| P262 | Do not get in eyes, on skin, or on clothing. |
| P263 | Avoid contact during pregnancy/while nursing. |
| P264 | Wash hands thoroughly after handling. |
| P265 | Wash skin thouroughly after handling. |
| P270 | Do not eat, drink or smoke when using this product. |
| P271 | Use only outdoors or in a well-ventilated area. |
| P272 | Contaminated work clothing should not be allowed out of the workplace. |
| P273 | Avoid release to the environment. |
| P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
| P281 | Use personal protective equipment as required. |
| P282 | Wear cold insulating gloves/face shield/eye protection. |
| P283 | Wear fire/flame resistant/retardant clothing. |
| P284 | Wear respiratory protection. |
| P285 | In case of inadequate ventilation wear respiratory protection. |
| P231 + P232 | Handle under inert gas. Protect from moisture. |
| P235 + P410 | Keep cool. Protect from sunlight. |
Response | |
| Code | Phrase |
| P301 | IF SWALLOWED: |
| P304 | IF INHALED: |
| P305 | IF IN EYES: |
| P306 | IF ON CLOTHING: |
| P307 | IF exposed: |
| P308 | IF exposed or concerned: |
| P309 | IF exposed or if you feel unwell: |
| P310 | Immediately call a POISON CENTER or doctor/physician. |
| P311 | Call a POISON CENTER or doctor/physician. |
| P312 | Call a POISON CENTER or doctor/physician if you feel unwell. |
| P313 | Get medical advice/attention. |
| P314 | Get medical advice/attention if you feel unwell. |
| P315 | Get immediate medical advice/attention. |
| P320 | |
| P302 + P352 | IF ON SKIN: wash with plenty of soap and water. |
| P321 | |
| P322 | |
| P330 | Rinse mouth. |
| P331 | Do NOT induce vomiting. |
| P332 | IF SKIN irritation occurs: |
| P333 | If skin irritation or rash occurs: |
| P334 | Immerse in cool water/wrap n wet bandages. |
| P335 | Brush off loose particles from skin. |
| P336 | Thaw frosted parts with lukewarm water. Do not rub affected area. |
| P337 | If eye irritation persists: |
| P338 | Remove contact lenses, if present and easy to do. Continue rinsing. |
| P340 | Remove victim to fresh air and keep at rest in a position comfortable for breathing. |
| P341 | If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
| P342 | If experiencing respiratory symptoms: |
| P350 | Gently wash with plenty of soap and water. |
| P351 | Rinse cautiously with water for several minutes. |
| P352 | Wash with plenty of soap and water. |
| P353 | Rinse skin with water/shower. |
| P360 | Rinse immediately contaminated clothing and skin with plenty of water before removing clothes. |
| P361 | Remove/Take off immediately all contaminated clothing. |
| P362 | Take off contaminated clothing and wash before reuse. |
| P363 | Wash contaminated clothing before reuse. |
| P370 | In case of fire: |
| P371 | In case of major fire and large quantities: |
| P372 | Explosion risk in case of fire. |
| P373 | DO NOT fight fire when fire reaches explosives. |
| P374 | Fight fire with normal precautions from a reasonable distance. |
| P376 | Stop leak if safe to do so. Oxidising gases (section 2.4) 1 |
| P377 | Leaking gas fire: Do not extinguish, unless leak can be stopped safely. |
| P378 | |
| P380 | Evacuate area. |
| P381 | Eliminate all ignition sources if safe to do so. |
| P390 | Absorb spillage to prevent material damage. |
| P391 | Collect spillage. Hazardous to the aquatic environment |
| P301 + P310 | IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician. |
| P301 + P312 | IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell. |
| P301 + P330 + P331 | IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. |
| P302 + P334 | IF ON SKIN: Immerse in cool water/wrap in wet bandages. |
| P302 + P350 | IF ON SKIN: Gently wash with plenty of soap and water. |
| P303 + P361 + P353 | IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower. |
| P304 + P312 | IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell. |
| P304 + P340 | IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing. |
| P304 + P341 | IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
| P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
| P306 + P360 | IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes. |
| P307 + P311 | IF exposed: call a POISON CENTER or doctor/physician. |
| P308 + P313 | IF exposed or concerned: Get medical advice/attention. |
| P309 + P311 | IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician. |
| P332 + P313 | IF SKIN irritation occurs: Get medical advice/attention. |
| P333 + P313 | IF SKIN irritation or rash occurs: Get medical advice/attention. |
| P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
| P337 + P313 | IF eye irritation persists: Get medical advice/attention. |
| P342 + P311 | IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician. |
| P370 + P376 | In case of fire: Stop leak if safe to Do so. |
| P370 + P378 | In case of fire: |
| P370 + P380 | In case of fire: Evacuate area. |
| P370 + P380 + P375 | In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion. |
| P371 + P380 + P375 | In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion. |
Storage | |
| Code | Phrase |
| P401 | |
| P402 | Store in a dry place. |
| P403 | Store in a well-ventilated place. |
| P404 | Store in a closed container. |
| P405 | Store locked up. |
| P406 | Store in corrosive resistant/ container with a resistant inner liner. |
| P407 | Maintain air gap between stacks/pallets. |
| P410 | Protect from sunlight. |
| P411 | |
| P412 | Do not expose to temperatures exceeding 50 oC/ 122 oF. |
| P413 | |
| P420 | Store away from other materials. |
| P422 | |
| P402 + P404 | Store in a dry place. Store in a closed container. |
| P403 + P233 | Store in a well-ventilated place. Keep container tightly closed. |
| P403 + P235 | Store in a well-ventilated place. Keep cool. |
| P410 + P403 | Protect from sunlight. Store in a well-ventilated place. |
| P410 + P412 | Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF. |
| P411 + P235 | Keep cool. |
Disposal | |
| Code | Phrase |
| P501 | Dispose of contents/container to ... |
| P502 | Refer to manufacturer/supplier for information on recovery/recycling |
Physical hazards | |
| Code | Phrase |
| H200 | Unstable explosive |
| H201 | Explosive; mass explosion hazard |
| H202 | Explosive; severe projection hazard |
| H203 | Explosive; fire, blast or projection hazard |
| H204 | Fire or projection hazard |
| H205 | May mass explode in fire |
| H220 | Extremely flammable gas |
| H221 | Flammable gas |
| H222 | Extremely flammable aerosol |
| H223 | Flammable aerosol |
| H224 | Extremely flammable liquid and vapour |
| H225 | Highly flammable liquid and vapour |
| H226 | Flammable liquid and vapour |
| H227 | Combustible liquid |
| H228 | Flammable solid |
| H229 | Pressurized container: may burst if heated |
| H230 | May react explosively even in the absence of air |
| H231 | May react explosively even in the absence of air at elevated pressure and/or temperature |
| H240 | Heating may cause an explosion |
| H241 | Heating may cause a fire or explosion |
| H242 | Heating may cause a fire |
| H250 | Catches fire spontaneously if exposed to air |
| H251 | Self-heating; may catch fire |
| H252 | Self-heating in large quantities; may catch fire |
| H260 | In contact with water releases flammable gases which may ignite spontaneously |
| H261 | In contact with water releases flammable gas |
| H270 | May cause or intensify fire; oxidizer |
| H271 | May cause fire or explosion; strong oxidizer |
| H272 | May intensify fire; oxidizer |
| H280 | Contains gas under pressure; may explode if heated |
| H281 | Contains refrigerated gas; may cause cryogenic burns or injury |
| H290 | May be corrosive to metals |
Health hazards | |
| Code | Phrase |
| H300 | Fatal if swallowed |
| H301 | Toxic if swallowed |
| H302 | Harmful if swallowed |
| H303 | May be harmful if swallowed |
| H304 | May be fatal if swallowed and enters airways |
| H305 | May be harmful if swallowed and enters airways |
| H310 | Fatal in contact with skin |
| H311 | Toxic in contact with skin |
| H312 | Harmful in contact with skin |
| H313 | May be harmful in contact with skin |
| H314 | Causes severe skin burns and eye damage |
| H315 | Causes skin irritation |
| H316 | Causes mild skin irritation |
| H317 | May cause an allergic skin reaction |
| H318 | Causes serious eye damage |
| H319 | Causes serious eye irritation |
| H320 | Causes eye irritation |
| H330 | Fatal if inhaled |
| H331 | Toxic if inhaled |
| H332 | Harmful if inhaled |
| H333 | May be harmful if inhaled |
| H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled |
| H335 | May cause respiratory irritation |
| H336 | May cause drowsiness or dizziness |
| H340 | May cause genetic defects |
| H341 | Suspected of causing genetic defects |
| H350 | May cause cancer |
| H351 | Suspected of causing cancer |
| H360 | May damage fertility or the unborn child |
| H361 | Suspected of damaging fertility or the unborn child |
| H361d | Suspected of damaging the unborn child |
| H362 | May cause harm to breast-fed children |
| H370 | Causes damage to organs |
| H371 | May cause damage to organs |
| H372 | Causes damage to organs through prolonged or repeated exposure |
| H373 | May cause damage to organs through prolonged or repeated exposure |
Environmental hazards | |
| Code | Phrase |
| H400 | Very toxic to aquatic life |
| H401 | Toxic to aquatic life |
| H402 | Harmful to aquatic life |
| H410 | Very toxic to aquatic life with long-lasting effects |
| H411 | Toxic to aquatic life with long-lasting effects |
| H412 | Harmful to aquatic life with long-lasting effects |
| H413 | May cause long-lasting harmful effects to aquatic life |
| H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
Sorry,this product has been discontinued.
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