- Highly Focused Library-Based Engineering of Candida antarctica Lipase B with (S)-Selectivity Towards sec-Alcohols
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Candida antarctica lipase B (CALB) is one of the most extensively used biocatalysts in both academia and industry and exhibits remarkable (R)-enantioselectivity for various chiral sec-alcohols. Considering the significance of tailor-made stereoselectivity in organic synthesis, a discovery of enantiocomplementary lipase mutants with high (R)- and (S)-selectivity is valuable and highly desired. Herein, we report a highly efficient directed evolution strategy, using only 4 representative amino acids, namely, alanine (A), leucine (L), lysine (K), tryptophan (W) at each mutated site to create an extremely small library of CALB variants requiring notably less screening. The obtained best mutant with three mutations W104V/A281L/A282K displayed highly reversed (S)-selectivity towards a series of sec-alcohol with E values up to 115 (conv. 50%, ee 94%). Compared with the previously reported (S)-selective CALB variant, W104A, a single mutation provided less selectivity, while the synergistic effects of three mutations in the best variant endow better (S)-selectivity and a broader substrate scope than the W104A variant. Structural analysis and molecular dynamics simulation unveiled the source of reversed enantioselectivity. (Figure presented.).
- Cen, Yixin,Li, Danyang,Xu, Jian,Wu, Qiongsi,Wu, Qi,Lin, Xianfu
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p. 126 - 134
(2018/12/05)
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- LipG9-mediated enzymatic kinetic resolution of racemates: Expanding the substrate-scope for a metagenomic lipase
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Enzymes are the main biocatalysts of biological systems and nowadays they play an important role in asymmetric organic synthesis. Microorganisms are the main source for enzymes, however, just a very small portion of them are culturable at lab conditions and, as an alternative, metagenomics approaches allow new enzymes to be accessed from so-called “non-culturable” microorganisms. Several classes of metagenomic enzymes have been described in literature. Nevertheless, studies about their potential for asymmetric biotransformation are underexploited. Therefore, we present our recent efforts to establish the substrate-scope of LipG9, a metagenomic lipase, in enzymatic kinetic resolution (EKR) of chiral substances. LipG9 was previously isolated, immobilized and successfully applied in EKR of aliphatic alcohols. In this study, a series of resolvable chiral substances were assayed with LipG9, and secondary benzyl alcohols/esters were preferentially resolved in a much superior enantioselectivity (E > 200) than those described for aliphatic alcohols (E from 4 to 63). In an opposite way, Im-LipG9 did not present activity for tertiary alcohols, amines and lactones. When compared to commercial lipases, Im-LipG9 enantioselectivity was superior to Candida rugosa lipase and equivalent to Candida antarctica lipase B. Thus, the chemo and enantioselectivity of LipG9 in EKR reactions were identified and its potential for asymmetric synthetic approaches was demonstrated.
- Thomas, Juliana Christina,Alnoch, Robson Carlos,Costa, Allen Carolina dos Santos,Bandeira, Pamela Taisline,Burich, Martha Daniela,Campos, Suelem Kluconski,de Oliveira, Alfredo Ricardo Marques,de Souza, Emanuel Maltempi,Pedrosa, Fabio de Oliveira,Krieger, Nadia,Piovan, Leandro
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- Base-catalyzed selective esterification of alcohols with unactivated esters
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A practical and efficient base-catalyzed esterification has been developed for the facile synthesis of a broad range of esters from simple alcohols with unactivated tert-butyl esters. This protocol could be conducted at mild conditions, providing esters in high to excellent yields with good functional tolerance. Mechanistic studies provided evidence of an exchange of the tert-butyl alkoxide metal with the alcohol, producing a new alkoxide to participate in the transesterification reaction.
- Zhang, Chunyan,Zhang, Guoying,Luo, Shizhong,Wang, Chunfu,Li, Huiping
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supporting information
p. 8467 - 8471
(2018/12/01)
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- Expanding substrate scope of lipase-catalyzed transesterification by the utilization of liquid carbon dioxide
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Secondary alcohols having bulky substituents on both sides of the chiral center are often poor substrates for most lipases. Here we reported that substrate scopes of two of the most used lipases, Candida antarctica lipase B and Burkholderia cepacia lipase, were found to be expanded toward more bulky secondary alcohols such as 1-phenyl-1-dodecanol and 2-methyl-1-phenyl-1-propanol by simply using them in liquid carbon dioxide as a solvent. The effects of solvents, reaction pressure, and pre-treatment of the enzyme with liquid CO2on this acceleration phenomenon were also studied.
- Hoang, Hai Nam,Matsuda, Tomoko
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p. 7229 - 7234
(2016/10/26)
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- Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulfolobus tokodaii by site-saturation mutagenesis
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An archaeon GGG(A)X-type esterase (ST0071) can catalyze the hydrolysis of various acetates of secondary alcohols, but shows low enantioselectivity. Using structure-guided site-saturation mutagenesis, we successfully identified a G274W variant that has excellent selectivity compared with that of wild-type ST0071.
- Ozaki, Masanaru,Kawakami, Norifumi,Ohta, Hiromichi,Miyamoto, Kenji
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p. 249 - 252
(2016/12/16)
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- Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: Manipulation of the transition state
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Secondary alcohols having bulky substituents on both sides of the hydroxy group are inherently poor substrates for most lipases. In view of this weakness, we redesigned a Burkholderia cepacia lipase to create a variant with improved enzymatic characteristics. The I287F/I290A double mutant showed a high conversion and a high E value (>200) for a poor substrate for which the wild-type enzyme showed a low conversion and a low E value (5). This enhancement of catalytic activity and enantioselectivity of the variant resulted from the cooperative action of two mutations: Phe287 contributed to both enhancement of the (R)-enantiomer reactivity and suppression of the (S)-enantiomer reactivity, while Ala290 created a space to facilitate the acylation of the (R)-enantiomer. The kinetic constants indicated that the mutations effectively altered the transition state. Substrate mapping analysis strongly suggested that the CH/π interaction partly enhanced the (R)-enantiomer reactivity, the estimated energy of the CH/π interaction being -0.4 kcal mol-1. The substrate scope of the I287F/I290A double mutant was broad. This biocatalyst was useful for the dynamic kinetic resolution of a variety of bulky secondary alcohols for which the wild-type enzyme shows little or no activity. The Royal Society of Chemistry 2012.
- Ema, Tadashi,Nakano, Yasuko,Yoshida, Daiki,Kamata, Shusuke,Sakai, Takashi
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supporting information; experimental part
p. 6299 - 6308
(2012/09/05)
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- Mutant lipase-catalyzed kinetic resolution of bulky phenyl alkyl sec-alcohols: A thermodynamic analysis of enantioselectivity
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The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.
- Vallin, Michaela,Syren, Per-Olof,Hult, Karl
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experimental part
p. 411 - 416
(2010/12/19)
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- Asymmetric transfer hydrogenation of ketones in aqueous solution catalyzed by rhodium(III) complexes with C2-symmetric fluorene-ligands containing chiral (1R,2R)-cyclohexane-1,2-diamine
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Two C2-symmetric bis(sulfonamide) ligands containing fluorene-chiral (1R,2R)-cyclohexane- 1,2-diamine were complexed to Rh III(Cp*) and used as catalyst to reduce aromatic ketones. The corresponding chiral secondary alcohols were obtained in 87-100percent ee and 85-99percent yield, under asymmetric transfer hydrogenation (ATH) conditions using aqueous sodium formate as the hydride source. With acetophenone, 94percent ee and 86-97percent yield was achieved with substrate/catalyst (S/C) ratio of 10,000.
- Montalvo-Gonza?lez, Rube?n,Cha?vez, Daniel,Aguirre, Gerardo,Parra-Hakea, Miguel,Somanathan, Ratnasamy
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experimental part
p. 431 - 435
(2010/07/16)
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- One-pot sequence for reductive-acetylation of carbonyl compounds with (N-methylimidazole)(tetrahydroborato)zinc complex
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Reductive-acetylation of variety of aliphatic and aromatic aldehydes and ketones, α,β-unsaturated carbonyl compounds are examined efficiently with (N-methylimidazole)(tetrahydroborato)zinc complex, [Zn(BH4) 2(nmi)], under mild condition in THF at room temperature or reflux conditions. The corresponding acetates were obtained in excellent yields (90-98 %).
- Setamdideh, Davood,Khezri, Behrooz
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experimental part
p. 5766 - 5772
(2012/07/28)
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- Baeyer-Villiger monooxygenase-catalyzed kinetic resolution of racemic α-alkyl benzyl ketones: enzymatic synthesis of α-alkyl benzylketones and α-alkyl benzylesters
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The application of three BVMOs for the enantioselective oxidation of 3-phenylbutan-2-ones with different substituents in the aromatic moiety is described. By choosing the appropriate biocatalyst and substrate combination, chiral ketones and esters can be obtained with excellent enantiopurities. This methodology could also be applied to the resolution of racemic α-alkyl benzylketones with longer alkyl chains as well as with two substituted α-substituted benzylacetones. A kinetic analysis revealed that the BVMOs studied effectively convert all tested compounds showing that the enzymes are tolerant towards the substrate structure while being highly enantioselective. These properties render BVMOs as valuable biocatalysts for the preparation of compounds with high interest in organic synthesis.
- Rodriguez, Cristina,Gonzalo, Gonzalo de,Torres Pazmino, Daniel E.,Fraaije, Marco W.,Gotor, Vicente
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experimental part
p. 1168 - 1173
(2009/10/02)
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- Empirical method for predicting enantioselectivity in catalytic reactions: demonstration with lipase and oxazaborolidine
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We derived a novel equation capable of predicting the degree of enantioselectivity in a catalytic reaction without any knowledge of the reaction mechanism and/or the transition-state structure, and tested the validity of this equation by changing substrates systematically in the lipase or oxazaborolidine-catalyzed reactions. A good correlation was observed between the predicted and observed E values, and the stereochemistry of the products could be predicted correctly in most cases (28 out of 30).
- Ema, Tadashi,Ura, Norichika,Yoshii, Masataka,Korenaga, Toshinobu,Sakai, Takashi
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supporting information; experimental part
p. 9583 - 9591
(2010/01/06)
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- Immobilized Manihot esculenta preparation as a novel biocatalyst in the enantioselective acetylation of racemic alcohols
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The enzymatic preparation obtained from a discard of Manihot esculenta roots has been successfully immobilized on calcium alginate hydrogels. This preparation has been tested as a chiral biocatalyst in the enzymatic acylation of a set of racemic aromatic alcohols. Depending on the reaction conditions, excellent enantioselectivities can be achieved. Some parameters that can alter the biocatalytic properties of the enzyme, such as solvent, temperature, acyl donor and substrate structure have been studied exhaustively in order to establish a deeper knowledge of this novel biocatalyst.
- Machado, Luciana L.,Lemos, Telma L.G.,de Mattos, Marcos Carlos,de Oliveira, Maria da Conceicao F.,de Gonzalo, Gonzalo,Gotor-Fernandez, Vicente,Gotor, Vicente
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p. 1418 - 1423
(2008/12/20)
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- DDQ catalyzed benzylic acetoxylation of arylalkanes: a case of exquisitely controlled oxidation under sonochemical activation
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Acetoxylated arylalkanes are selectively obtained via sonochemical activation of DDQ catalyzed benzylic oxidation of arylalkanes in the presence of anhydrous acetic acid. The method gives an exquisite control of benzylic acetoxylation under ultrasound, in contrast to the uncontrolled oxidation observed under conventional heating or microwave activation. In addition, the developed method could be?a useful strategy for the synthesis of industrially important enantiopure benzyl alcohols due to the easy amenability of obtained acetoxylated products toward chiral resolution.
- Kumar, Vinod,Sharma, Abhishek,Sharma, Meenakshi,Sharma, Upendra K.,Sinha, Arun K.
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p. 9718 - 9723
(2008/02/12)
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- Microscale determination of the absolute configuration of α-aryl- substituted alcohols by the CD exciton chirality method
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The absolute configurations of a broad spectrum of aryl alcohols 1 have been determined for the first time by the CD exciton chirality method. The configurational assignment is additionally verified by computer modeling and lipase-catalyzed acetylation of the racemic alcohols. The CD-spectroscopic data have revealed that the S enantiomers of the benzoate derivatives 2 display a positive first Cotton effect and the R enantiomers a negative one at around 228 nm. Thus, the sense of the first Cotton effect of the benzoate derivative 2 allows a reliable assignment of the absolute configuration of the corresponding alcohol 1.
- Adam, Waldemar,Lukacs, Zoltan,Viebach, Kirsten,Humpf, Hans-Ulrich,Saha-Moeller, Chantu R.,Schreier, Peter
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p. 186 - 190
(2007/10/03)
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- Benzotriazole: A novel synthetic auxiliary
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Benzotriazole and aldehydes react reversibly to give addition products: in the presence of amines and other NH-compounds water can be eliminated to form products of type Bt-CHR-NR′R″. The latter are versatile intermediates for the preparation of primary, secondary, and tertiary amines and in the alkylation of hydroxylamines, hydrazines, amides, thioamides, and sulfonamides. Polyfunctional amines and other polyfunctional compounds can also be prepared, and they enable significant extending of Mannich reaction. Similar oxygen compounds Bt-CRR′-OR″ enable new syntheses of ethers and esters. Reactions in which benzotriazole is eliminated rather than substituted open up new pathways to enamines, enol ethers, and nitrones. The methodology is capable of extension to a variety of vinylogous systems including benzenoid and heteroaromatic derivatives. In addition to acting as a versatile leaving group, benzotriazolyl residues activate neighboring CH bonds to proton loss and a variety of such applications is described.
- Katritzky, Alan R.,Rachwal, Stanislaw,Hitchings, Gregory J.
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p. 2683 - 2732
(2007/10/02)
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- A novel synthesis of esters via substitution of the benzotriazolyl group in 1-(benzotriazol-1-yl)alkyl esters with organozinc reagents
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Aldehydes are converted by thionyl chloride and benzotriazole into 1-(1-chloroalyl)benzotriazoles which react with sodium carboxylates to give 1-(benzotriazol-1-yl)alkyl esters. 3. In the alternative route, 3 are prepared by substitution of one of the acetoxy groups in acylals with benzotriazole. The benzotriazolyl moiety in 3 is substituted by an alkyl, an aryl or an alkynyl group upon treatment with an organic reagent in a new versatile synthesis of esters 4.
- Katritzky,Rachwal,Rachwal
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- ASYMMETRIC ACYLATION OF ALCOHOLS IN THE PRESENCE OF CHIRAL TERTIARY AMINES
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The asymmetric acylation of secondary alcohols with acetic anhydride in the presence of chiral tertiary amines, synthesized from S-(-)-α-phenylethylamine, was investigated.The optical yield of the obtained alcohols was not greater than 2.4percent.It was established that in the presence of S-(-)-N,N-dimethyl-α-phenylethylamine the unreacted alcohol is rich in the S isomer.On the basis of this relationship the S configuration was proposed for (-)-4-iodo-, (-)-4-chloro-, and (-)-3-nitrophenylehanols.
- Potapov, V. M.,Dem'yanovich, V. M.,Khlebnikov, V. A.,Koval'skaya, S. S.
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p. 1602 - 1606
(2007/10/02)
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- Photolysis and Thermolysis of 3-n-Butyl-3-phenyldiazirine
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The photochemical and thermal decompositions of 3 butyl-3-phenyldiazirine have been investigated in acetic acid and acetic acid, and in other solvents with and without m-chloroperbenzoic acid.The results are consistent with the fact that the oxidation products are derived mainly from the reactions of 1-diazo-1-phenylpentane.
- Liu, Michael T. H.,Palmer, Glenn E.,Chishti, Najmul H.
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