- Microbial reduction and in situ product crystallization coupled with biocatalyst cultivation during the synthesis of 6R-dihydrooxoisophorone
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An in situ product crystallization procedure was developed for a crystalline product formed during microbial reduction coupled with cell cultivation. The model reaction was the asymmetric reduction of 4-oxoisophorone (OIP) by baker's yeast (Saccharomyces cerevisiae). Yeast cells were cultivated fed-batch to reach a maximum concentration of 30 gdw·L-1. The desired product, 6R-dihydrooxoisophorone (DOIP), may be further reduced by baker's yeast to an unwanted by-product; thus, DOIP was removed immediately from the fermenter via an external crystallization loop in this procedure. The OIP reduction rate was five times higher (?0.33 mmol·gdw -1·h-1) as compared to the reduction rate with resting cells. OIP reduction was started when the optimum cell concentration had already been reached in the reactor because the substrate (OIP) at ≥55 mM concentration inhibited cell growth. An appropriate supply of glucose as carbon and energy source was necessary to support the coupled reactions involving cell growth and maintenance and product formation while avoiding formation of metabolic by-products. Final DOIP yield and selectivity were 85% and 99%, respectively, while over 100 g·L-1 of product was obtained in the crystallizer. The product crystals with favorable properties were readily recovered from the crystallizer. These results indicate that product crystallization is not impaired by the solutes present in the fermentation medium.
- Buque-Taboada, Evelyn M.,Straathof, Adrie J. J.,Heijnen, Joseph J.,Van Der Wielen, Luuk A. M.
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- On the bi-enzymatic behaviour of Saccharomyces cerevisiae-mediated stereoselective biotransformation of 2,6,6-trimethylcyclohex-2-ene-1,4-dione
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Baker's yeast has been well-known to have the ability to reduce a variety of substrates into many optically active compounds. One of the important chemicals is (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone or in short, (4R,6R)-actinol, a product formed f
- Uzir, Mohamad Hekarl,Najimudin, Nazalan
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- An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-actinol in a one-pot reduction system
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(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.
- Horita, Shoichiro,Kataoka, Michihiko,Kitamura, Nahoko,Nakagawa, Takuya,Miyakawa, Takuya,Ohtsuka, Jun,Nagata, Koji,Shimizu, Sakayu,Tanokura, Masaru
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p. 440 - 445
(2015/03/05)
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- Synthesis of biotinylated retinoids for cross-linking and isolation of retinol binding proteins
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The synthesis of (3R)-3-[Boc-Lys(biotinyl)-O]-11-cis-retinol bromoacetate and 3-[Boc-Lys(biotinyl)-O]-all trans-retinol chloroacetate is described. These biotinylated retinoids are instrumental in labeling the retinol binding proteins (RBPs) via a nucleophilic displacement of the haloacetate by a residue in the binding site of the protein. The covalently linked biotin will allow for a facile isolation and purification of the protein on a streptavidin column thus rendering the protein ready for a tryptic digest followed by mass spectrometric analysis. The 11-cis retinoid was synthesized via metal reduction of an alkyne intermediate generated from a Horner-Wadsworth-Emmons (HWE) reaction whereas the all-trans was synthesized via two consecutive HWE couplings.
- Nesnas, Nasri,Rando, Robert R,Nakanishi, Koji
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p. 6577 - 6584
(2007/10/03)
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- Reduction of aliphatic and aromatic cyclic ketones to sec-alcohols by aqueous titanium trichloride/ammonia system. Steric course and mechanistic implications
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In contrast to the dissolved metal and metal hydride reductions, the reduction of cyclic ketones by the aqueous TiCl3/NH3 system favours the formation of the less thermodynamically stable axial alcohol. The ammonium ion formed in situ is essential for the reduction to proceed because it behaves as a mild Br?nsted acid in basic medium and favours the protonation of the intermediate ketyl. The corresponding α-hydroxy radical is then rapidly reduced under conditions where the first electron transfer to the substrate takes place. We suggest that the stereoselectivity is determined by the second reduction step, which occurs through the less hindered transition state, regardless of whether the radical to be reduced is thermodynamically favoured or not.
- Clerici, Angelo,Pastori, Nadia,Porta, Ombretta
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p. 2235 - 2243
(2007/10/03)
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- Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13.
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The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides correspo
- Yoshisumi,Wada,Takagi,Shimizu,Nakamori
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p. 830 - 836
(2007/10/03)
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- Total synthesis and biological activities of (+)- and (-)-boscialin and their 1'-epimers
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Natural (-)-boscialin [(-)-1] has recently been described as one of the constituents of various medicinal plants. To obtain more material for investigations of its biological activities, we carried out the synthesis of (-)-1 and its isomers. Starting from the chiral building block 2, the key steps of the synthesis involved a regioselective reduction and a nucleophilic addition. The enantiomer of the natural product, (+)-boscialin [(+)-1], could be obtained via acid-catalyzed epimerization of hydroxyketone 4 to (+)-3. Starting the synthesis with (-)-3 led to (-)-boscialin [(-)-1] with the natural absolute configuration. In addition to (+)- and (-)-boscialin, the corresponding 1'-epimers (+)- and (-)-epiboscialin were also obtained. In vitro assays with (-)-boscialin [(-)-1] and its three stereoisomers were carried out to test for activity against microbes, parasites, and human fibroblasts. The investigations revealed activity against various microbes and against Trypanosoma brucei rhodesiense and also revealed cytotoxicity against human cancer cells.
- Busch, Joachim,Grether, Yvonne,Ochs, Dietmar,Sequin, Urs
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p. 591 - 597
(2007/10/03)
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- Experimental and Computational Studies of Nucleophilic Additions of Metal Hydrides and Organometallics to Hindered Cyclohexanones
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The stereochemistries of nucleophilic additions of several hydride, methyl, and acetylenic Grignard and lithium reagents to hindered cyclohexanones were investigated experimentally. Acetylenic ' reagents attack hindered cyclohexanones from the axial direction, in contrast to the result with other reagents. Ab initio calculations and a modified MM2 transition-state force field were used to study the origins of stereoselectivity.
- Ando, Kaori,Houk,Busch, Joachim,Menasse, Alexander,Sequin, Urs
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p. 1761 - 1766
(2007/10/03)
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- Total synthesis of (±)-phaseic acid
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A highly stereoselective synthesis of (±)-phaseic acid is described. Photochemical reaction of the nitrite derived from trans-4-hydroxy-2,2,6-trimethylcyclohexanone functionalizes the methyl group of the geminal pair cis to the hydroxyl group of the start
- Abrams, Garth D.,Abrams, Suzanne R.,Nelson, Lloyd A. K.,Gusta, Lawrence V.
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p. 5543 - 5554
(2007/10/02)
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- Synthesis of optically active cyclohexanone analogs of the plant hormone abscisic acid
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The abscisic acid analogs (-)-(4R,5R)-, (+)-(4S,5S)-, and (-)-(4S,5R)-4(1E,3Z)-4-(4-carboxy-3-methyl-1,3-butadienyl)-4-hydroxy-3,3,5-trimethylcyclohexanones (dihydroabscisic acids), and (-)-(4S,5R)-, (+)-(4R,5S)-, and (-)-(4R,5R)-4(Z)-4-hydroxy-4-(5-hydroxy-3-methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclohexanones were synthesized from a common precursor, (-)-(6R)-2,2,6-trimethylcyclohexan-1,4-dione, which was readily prepared by the fermentation of oxoisophorone with bakers' yeast.
- Lamb, Nancy,Abrams, Suzanne R.
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p. 1151 - 1162
(2007/10/02)
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- Microbial Conversion of 4-Oxoisophorone by Thermophile, Thermomonospora curvata
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Thermomonospora curvata IFO 12384 was selected from many thermophiles tested as a strain capable of converting 2,6,6-trimethyl-2-cyclohexene-1,4-dione (4-oxoisophorone) into (6R)-2,2,6-trimethyl-1,4-cyclohexadione ((3R)-dihydro-4-oxoisophorone) efficiently.The other conversion products were also isolated and identified by spectrometry.Based on the results of experiments with a degradation sequence, a conversion pathway for 4-oxoisophorone by this thermophile is proposed.The effects of nutritional conditions, temperature, initial pH and the concentration of the substrate on the conversion were examined.Under the optimal conditions, the conversion ratio of 4-oxoisophorone to (3R)-dihydro-4-oxoisophorone was over 95percent.The production rate of (3R)-dihydro-4-oxoisophorone was 86 mg per 1 g of dry cells per hr.
- Hori, Nobuaki,Hieda, Tadaharu,Mikami, Yoichi
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p. 123 - 130
(2007/10/02)
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- Optically active cyclohexane derivatives
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A process for fermentatively hydrogenating and reducing ketoisophorone to produce optically active [4R,6R]-4-hydroxy-2,6,6-trimethyl-cyclohexanone useful as an intermediate in the production of optically active carotenoids and intermediates in the product
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