- Iterative Alanine Scanning Mutagenesis Confers Aromatic Ketone Specificity and Activity of L-Amine Dehydrogenases
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Direct reductive amination of prochiral ketones catalyzed by amine dehydrogenases is attractive in the synthesis of active pharmaceutical ingredients. Here, we report the protein engineering of L-Bacillus cereus amine dehydrogenase to allow reactivity on synthetically useful aromatic ketone substrates using an iterative, multiple-site alanine scanning mutagenesis approach. Mutagenesis libraries based on molecular docking, iterative alanine scanning, and double-proximity filter approach significantly expand the scope of active pharmaceutical ingredients relevant building blocks. The eventual quintuple mutant (A115G/T136A/L42A/V296A/V293A) showed reactivity toward aromatic ketones 12 a (5-phenyl-pentan-2-one) and 13 a (6-phenyl-hexan-2-one), which have not been reported to serve as targets of reductive amination by currently available amine dehydrogenases. Docking simulation and tunnel analysis provided valuable insights into the source of the acquired specificity and activity.
- Mu, Xiaoqing,Wu, Tao,Mao, Yong,Zhao, Yilei,Xu, Yan,Nie, Yao
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p. 5243 - 5253
(2021/11/16)
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- Enzymatic Primary Amination of Benzylic and Allylic C(sp3)-H Bonds
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Aliphatic primary amines are prevalent in natural products, pharmaceuticals, and functional materials. While a plethora of processes are reported for their synthesis, methods that directly install a free amine group into C(sp3)-H bonds remain unprecedented. Here, we report a set of new-to-nature enzymes that catalyze the direct primary amination of C(sp3)-H bonds with excellent chemo-, regio-, and enantioselectivity, using a readily available hydroxylamine derivative as the nitrogen source. Directed evolution of genetically encoded cytochrome P411 enzymes (P450s whose Cys axial ligand to the heme iron has been replaced with Ser) generated variants that selectively functionalize benzylic and allylic C-H bonds, affording a broad scope of enantioenriched primary amines. This biocatalytic process is efficient and selective (up to 3930 TTN and 96percent ee), and can be performed on preparative scale.
- Jia, Zhi-Jun,Gao, Shilong,Arnold, Frances H.
-
supporting information
p. 10279 - 10283
(2020/07/27)
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- Enantioselective Bioamination of Aromatic Alkanes Using Ammonia: A Multienzymatic Cascade Approach
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Chiral amines are common drug building blocks and important active pharmaceutical ingredients. Preparing these functionalized compounds from simple materials, such as alkanes, is of great interest. We recently developed an artificial bioamination cascade for the C?H amination of cyclic alkanes by combining P450 monooxygenase, alcohol dehydrogenase, and amine dehydrogenase. Herein, this system has been extended to the synthesis of chiral aromatic amines. In the first hydroxylation step, process optimization increased the conversion to 77 %. Two stereoselectively complementary alcohol dehydrogenases and an amine dehydrogenase were selected for the bioconversion of aromatic hydrocarbons to amines. The amination reaction was optimized with respect to cofactor addition and enzyme dosage. Isopropanol was added to decrease ketone intermediate accumulation in the amination step, which further enhanced the overall conversion. This cascade system converted a panel of hydrocarbon substrates into the corresponding amines with excellent optical purity (>99 % ee) and moderate conversion ratios (13–53 %).
- Chen, Fei-Fei,Wang, Hui,Xu, Jian-He,Yu, Hui-Lei,Zheng, Yu-Cong
-
-
- Deracemization of Racemic Amines to Enantiopure (R)- and (S)-amines by Biocatalytic Cascade Employing ω-Transaminase and Amine Dehydrogenase
-
A one-pot deracemization strategy for α-chiral amines is reported involving an enantioselective deamination to the corresponding ketone followed by a stereoselective amination by enantiocomplementary biocatalysts. Notably, this cascade employing a ω-transaminase and amine dehydrogenase enabled the access to both (R)-and (S)-amine products, just by controlling the directions of the reactions catalyzed by them. A wide range of (R)-and (S)-amines was obtained with excellent conversions (>80 %) and enantiomeric excess (>99 % ee). Finally, preparative scale syntheses led to obtain enantiopure (R)- and (S)-13 with the isolated yields of 53 and 75 %, respectively.
- Yoon, Sanghan,Patil, Mahesh D.,Sarak, Sharad,Jeon, Hyunwoo,Kim, Geon-Hee,Khobragade, Taresh P.,Sung, Sihyong,Yun, Hyungdon
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p. 1898 - 1902
(2019/02/27)
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- Generation of amine dehydrogenases with increased catalytic performance and substrate scope from ε-deaminating L-Lysine dehydrogenase
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Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Guided by structural information from computational models, we create AmDHs that can convert pharmaceutically relevant aromatic ketones with conversions up to quantitative and perfect chemical and optical purities. These AmDHs are created from an unconventional enzyme scaffold that apparently does not operate any asymmetric transformation in its natural reaction. Additionally, the best variant (LE-AmDH-v1) displays a unique substrate-dependent switch of enantioselectivity, affording S- or R-configured amine products with up to >99.9% enantiomeric excess. These findings are explained by in silico studies. LE-AmDH-v1 is highly thermostable (Tm of 69 °C), retains almost entirely its catalytic activity upon incubation up to 50 °C for several days, and operates preferentially at 50 °C and pH 9.0. This study also demonstrates that product inhibition can be a critical factor in AmDH-catalysed reductive amination.
- Tseliou, Vasilis,Knaus, Tanja,Masman, Marcelo F.,Corrado, Maria L.,Mutti, Francesco G.
-
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- Mapping the substrate scope of monoamine oxidase (MAO-N) as a synthetic tool for the enantioselective synthesis of chiral amines
-
A library of 132 racemic chiral amines (α-substituted methylbenzylamines, benzhydrylamines, 1,2,3,4-tetrahydronaphthylamines (THNs), indanylamines, allylic and homoallylic amines, propargyl amines) was screened against the most versatile monoamine oxidase (MAO-N) variants D5, D9 and D11. MAO-N D9 exhibited the highest activity for most substrates and was applied to the deracemisation of a comprehensive set of selected primary amines. In all cases, excellent enantioselectivity was achieved (e.e. >99%) with moderate to good yields (55–80%). Conditions for the deracemisation of primary amines using a MAO-N/borane system were further optimised using THN as a template addressing substrate load, nature of the enzyme preparation, buffer systems, borane sources, and organic co-solvents.
- Herter, Susanne,Medina, Florian,Wagschal, Simon,Benha?m, Cyril,Leipold, Friedemann,Turner, Nicholas J.
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p. 1338 - 1346
(2017/10/06)
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- Asymmetric Synthesis of Chiral Primary Amines by Ruthenium-Catalyzed Direct Reductive Amination of Alkyl Aryl Ketones with Ammonium Salts and Molecular H2
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A ruthenium/C3-TunePhos catalytic system has been identified for highly efficient direct reductive amination of simple ketones. The strategy makes use of ammonium acetate as the amine source and H2 as the reductant and is a user-friendly and operatively simple access to industrially relevant primary amines. Excellent enantiocontrol (>90% ee for most cases) was achieved with a wide range of alkyl aryl ketones. The practicability of this methodology has been highlighted by scalable synthesis of key intermediates of three drug molecules. Moreover, an improved synthetic route to the optimal diphosphine ligand C3-TunePhos is also presented.
- Tan, Xuefeng,Gao, Shuang,Zeng, Weijun,Xin, Shan,Yin, Qin,Zhang, Xumu
-
supporting information
p. 2024 - 2027
(2018/02/19)
-
- In vitro biocatalytic pathway design: Orthogonal network for the quantitative and stereospecific amination of alcohols
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The direct and efficient conversion of alcohols into amines is a pivotal transformation in chemistry. Here, we present an artificial, oxidation-reduction, biocatalytic network that employs five enzymes (alcohol dehydrogenase, NADP-oxidase, catalase, amine dehydrogenase and formate dehydrogenase) in two concurrent and orthogonal cycles. The NADP-dependent oxidative cycle converts a diverse range of aromatic and aliphatic alcohol substrates to the carbonyl compound intermediates, whereas the NAD-dependent reductive aminating cycle generates the related amine products with >99% enantiomeric excess (R) and up to >99% conversion. The elevated conversions stem from the favorable thermodynamic equilibrium (K′eq = 1.88 × 1042 and 1.48 × 1041 for the amination of primary and secondary alcohols, respectively). This biocatalytic network possesses elevated atom efficiency, since the reaction buffer (ammonium formate) is both the aminating agent and the source of reducing equivalents. Additionally, only dioxygen is needed, whereas water and carbonate are the by-products. For the oxidative step, we have employed three variants of the NADP-dependent alcohol dehydrogenase from Thermoanaerobacter ethanolicus and we have elucidated the origin of the stereoselective properties of these variants with the aid of in silico computational models.
- Knaus, Tanja,Cariati, Luca,Masman, Marcelo F,Mutti, Francesco G.
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p. 8313 - 8325
(2017/10/19)
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- Biocatalytic transamination with near-stoichiometric inexpensive amine donors mediated by bifunctional mono- and di-amine transaminases
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The discovery and characterisation of enzymes with both monoamine and diamine transaminase activity is reported, allowing conversion of a wide range of target ketone substrates with just a small excess of amine donor. The diamine co-substrates (putrescine, cadaverine or spermidine) are bio-derived and the enzyme system results in very little waste, making it a greener strategy for the production of valuable amine fine chemicals and pharmaceuticals.
- Galman, James L.,Slabu, Iustina,Weise, Nicholas J.,Iglesias, Cesar,Parmeggiani, Fabio,Lloyd, Richard C.,Turner, Nicholas J.
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supporting information
p. 361 - 366
(2017/08/14)
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- Amine dehydrogenases: Efficient biocatalysts for the reductive amination of carbonyl compounds
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Amines constitute the major targets for the production of a plethora of chemical compounds that have applications in the pharmaceutical, agrochemical and bulk chemical industries. However, the asymmetric synthesis of α-chiral amines with elevated catalytic efficiency and atom economy is still a very challenging synthetic problem. Here, we investigated the biocatalytic reductive amination of carbonyl compounds employing a rising class of enzymes for amine synthesis: amine dehydrogenases (AmDHs). The three AmDHs from this study-operating in tandem with a formate dehydrogenase from Candida boidinii (Cb-FDH) for the recycling of the nicotinamide coenzyme-performed the efficient amination of a range of diverse aromatic and aliphatic ketones and aldehydes with up to quantitative conversion and elevated turnover numbers (TONs). Moreover, the reductive amination of prochiral ketones proceeded with perfect stereoselectivity, always affording the (R)-configured amines with more than 99% enantiomeric excess. The most suitable amine dehydrogenase, the optimised catalyst loading and the required reaction time were determined for each substrate. The biocatalytic reductive amination with this dual-enzyme system (AmDH-Cb-FDH) possesses elevated atom efficiency as it utilizes the ammonium formate buffer as the source of both nitrogen and reducing equivalents. Inorganic carbonate is the sole by-product.
- Knaus, Tanja,B?hmer, Wesley,Mutti, Francesco G.
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p. 453 - 463
(2017/08/14)
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- Whole-Cell Biocatalysts for Stereoselective C-H Amination Reactions
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Enantiomerically pure chiral amines are ubiquitous chemical building blocks in bioactive pharmaceutical products and their synthesis from simple starting materials is of great interest. One of the most attractive strategies is the stereoselective installation of a chiral amine through C-H amination, which is a challenging chemical transformation. Herein we report the application of a multienzyme cascade, generated in a single bacterial whole-cell system, which is able to catalyze stereoselective benzylic aminations with ee values of 97.5 %. The cascade uses four heterologously expressed recombinant enzymes with cofactors provided by the host cell and isopropyl amine added as the amine donor. The cascade presents the first example of the successful de novo design of a single whole-cell biocatalyst for formal stereoselective C-H amination.
- Both, Peter,Busch, Hanna,Kelly, Paul P.,Mutti, Francesco G.,Turner, Nicholas J.,Flitsch, Sabine L.
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p. 1511 - 1513
(2016/02/14)
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- Identification of novel thermostable ω-transaminase and its application for enzymatic synthesis of chiral amines at high temperature
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A novel thermostable ω-transaminase from Thermomicrobium roseum which showed broad substrate specificity and high enantioselectivity was identified, expressed and biochemically characterized. The advantage of this enzyme to remove volatile inhibitory by-products was demonstrated by performing asymmetric synthesis and kinetic resolution at high temperature.
- Mathew, Sam,Deepankumar, Kanagavel,Shin, Giyoung,Hong, Eun Young,Kim, Byung-Gee,Chung, Taeowan,Yun, Hyungdon
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p. 69257 - 69260
(2016/08/05)
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- From racemic alcohols to enantiopure amines: Ru-catalyzed diastereoselective amination
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A commercially available ruthenium(II) PNP-type pincer catalyst (Ru-Macho) promotes the formation of α-chiral tert-butanesulfinylamines from racemic secondary alcohols and Ellmans chiral tert-butanesulfinamide via a hydrogen borrowing strategy. The formation of α-chiral tert-butanesulfinylamines occurs in yields ranging from 31% to 89% with most examples giving >95:5 dr.
- Oldenhuis, Nathan J.,Dong, Vy M.,Guan, Zhibin
-
supporting information
p. 12548 - 12551
(2014/12/10)
-
- Enantioselective synthesis of (R)-2-arylpropanenitriles catalysed by ene-reductases in aqueous media and in biphasic ionic liquid-water systems
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The enantioselective reduction of α-methylene nitrile derivatives catalysed by ene-reductases affords the corresponding (R)-2-arylpropanenitriles with high conversion values. The reaction is investigated either in aqueous medium (with an organic cosolvent or by loading the substrate onto hydrophobic resins) or in a biphasic ionic liquid-water system. The use of ionic liquids, herein with isolated ene-reductases, is found to improve the work-up and the substrate recovery method. The synthetic manipulation of the final chiral nitrile derivatives indicates how this biocatalysed method can be exploited for the preparation of a wide range of chiral compounds.
- Brenna, Elisabetta,Crotti, Michele,Gatti, Francesco G.,Manfredi, Alessia,Monti, Daniela,Parmeggiani, Fabio,Santangelo, Sara,Zampieri, Davila
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p. 2425 - 2431
(2014/08/18)
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- Transaminases applied to the synthesis of high added-value enantiopure amines
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Critical parameters affecting the stereoselective amination of (hetero)aromatic ketones using transaminases have been studied, such as temperature, pH, substrate concentration, cosolvent, and source and percentage of amino donor, to further optimize the production of enantiopure amines using both (S)- and (R)-selective biocatalysts from commercial suppliers. Interesting enantiopure amino building blocks have been obtained, overcoming some limitations of traditional chemical synthetic methods. Representative processes were scaled up, affording halogenated and heteroaromatic amines in enantiomerically pure form and good isolated yields.
- Paul, Caroline E.,Rodriguez-Mata, Maria,Busto, Eduardo,Lavandera, Ivan,Gotor-Fernandez, Vicente,Gotor, Vicente,Garcia-Cerrada, Susana,Mendiola, Javier,De Frutos, Oscar,Collado, Ivan
-
supporting information
p. 788 - 792
(2014/07/08)
-
- Modification of supported Pd catalysts by alkalic salts in the selective racemization and dynamic kinetic resolution of primary amines
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Supported Pd nanoparticles (PdNPs) were modified with various alkalic salts by incipient wetness impregnation method. The effect of the salts on the catalytic activity and selectivity behavior of Pd catalysts for the racemization of (S)-1-phenylethylamine was investigated. The presence of alkalic salts can greatly enhance the selectivity of Pd catalysts, without significantly decreasing the catalytic activity. This modification method is suitable for PdNPs supported on various supports, such as micro/mesoporous silica and activated carbon. Combined with the immobilized lipases (Novozyme 435), this catalyst system can efficiently catalyze the dynamic kinetic resolution (DKR) of 1-phenylethylamine with yield and enantioselectivity up to 97% and 99%, respectively. The Royal Society of Chemistry.
- Jin, Qianru,Jia, Guoqing,Zhang, Yanmei,Li, Can
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p. 464 - 471
(2014/02/14)
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- One-pot one-step deracemization of amines using ω-transaminases
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In this study, we developed a one-pot one-step deracemization method for the production of various enantiomerically pure amines using two opposite enantioselective ω-TAs. Using this method, various aromatic amines were successfully converted to their (R)-forms (>99%) with good conversion.
- Shin, Giyoung,Mathew, Sam,Shon, Minsu,Kim, Byung-Gee,Yun, Hyungdon
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p. 8629 - 8631
(2013/09/23)
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- Nickel nanoparticles as racemization catalysts for primary amines
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By combining bases that are known to racemize benzylic amines with a nickel(II) salt, active nickel nanoparticles were obtained that can be used as catalysts in the racemization of both aliphatic and benzylic primary amines. The nanoparticles are stable in the ionic liquid tetrabutylammonium bromide and can complete most racemizations within a few hours with excellent selectivity. The problem of the incompatibility of the strongly reducing racemization catalyst and the enzymatic amine resolution catalyst was overcome by using a two-pot system with a biphasic racemization step. Consecutive contact of a nonane layer that contained the amine with the acylating enzyme and with the racemizing Ni nanoparticles in the ionic liquid allowed the 50 % amide yield limit of a kinetic resolution to be successfully surpassed. Copyright
- Geukens, Inge,Plessers, Eva,Seo, Jin Won,De Vos, Dirk E.
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p. 2623 - 2628
(2013/07/11)
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- Enantioselective reduction of ketoxime ethers with borane-oxazaborolidines and synthesis of the key intermediate leading to (S)-rivastigmine
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The reduction of representative alkyl aryl (E)-ketoxime O-benzyl ethers with borane catalyzed by terpene oxazaborolidines, derived from (1R)-nopinone and (1R)-camphor, gave the corresponding amines with 82-99% ee. Oxazaborolidines derived from (1S)-2-carene and (1S)-3-carene were less selective. (S)-1-(3-Methoxyphenyl)ethanamine (94% ee) the key intermediate in the synthesis of (S)-rivastigmine, was obtained by the reduction of (E)-1-(3-methoxyphenyl) ethanone O-benzyl oxime with borane/oxazaborolidine generated from (S)-valinol.
- Pakulski, Marcin M.,Mahato, Sanjit K.,Bosiak, Mariusz J.,Krzeminski, Marek P.,Zaidlewicz, Marek
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p. 716 - 721
(2012/09/21)
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- ω-Transaminase-catalyzed kinetic resolution of chiral amines using l-threonine as an amino acceptor precursor
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Kinetic resolution of chiral amines using l-threonine as a cosubstrate was demonstrated by a biocatalytic strategy in which (S)-selective ω-transaminase (ω-TA) was coupled with threonine deaminase (TD), eliminating the need to use an expensive keto acid as an amino acceptor. The coupled enzyme reaction enabled simultaneous production of enantiopure (R)-amine and l-homoalanine which are pharmaceutically important building blocks. To extend the versatility of this strategy to production of both enantiomers of chiral amines, (R)-selective ω-TA coupled with TD was employed to produce (S)-amine.
- Malik, M. Shaheer,Park, Eul-Soo,Shin, Jong-Shik
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supporting information; experimental part
p. 2137 - 2140
(2012/09/25)
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- Cyclohexylamine oxidase as a useful biocatalyst for the kinetic resolution and dereacemization of amines
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The biocatalytic performance of a cloned cyclohexylamine oxidase derived from Brevibacterium oxydans IH-35A towards structurally different amines was investigated. Cycloalkyl primary amines, alkyl aryl amines, and α-carbon-substituted aliphatic amines were identified as suitable substrates for the biocatalyst based on an activity assay. Kinetic resolutions of several amines by either recombinant whole cells or crude enzyme extracts prepared therefrom gave enantiomerically pure (R)-amines besides the corresponding ketones. When cyclohexylamine oxidase in combination with a borane-ammonia complex as reducing agent was applied to the deracemization of several substrates, excellent enantiomeric ratios (>99:1) and good isolated yields (62%-75%) of the corresponding (R)-amines were obtained.
- Leisch, Hannes,Grosse, Stephan,Iwaki, Hiroaki,Hasegawa, Yoshie,Lau, Peter C.K.
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experimental part
p. 39 - 45
(2012/03/07)
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- Halogen-bonding interaction stabilizing cluster-type diastereomeric salt crystals
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O-Ethyl 4-chlorophenylphosphonothioic acid (1) was newly synthesized and applied as a chiral selector for the enantioseparation of racemic l-(4-halophenyl)ethylamines (halo = F, Cl, Br, I; 2a-d) through diastereomeric salt formation. The phosphonothioic acid 1 showed an excellent chirality-recognition ability for the fluorinated and iodinated amines 2a and 2d with the dramatic switch of the absolute configuration of the enantio-enriched isomers in the deposited salts from R for the amine 2a to S for the amine 2d. The X-ray crystallographic analyses of the four pairs of diastereomeric salts revealed that halogen-bonding interaction in the salt crystals plays a very important role for the switch.
- Kobayashi, Yuka,Maeda, Jin,Ando, Tetsuo,Saigo, Kazuhiko
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experimental part
p. 685 - 690
(2011/10/09)
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- COMPOSITIONS AND METHODS FOR CYCLOFRUCTANS AS SEPARATION AGENTS
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The present invention relates to derivatized cyclofructan compounds, compositions comprising derivatized cyclofructan compounds, and methods of using compositions comprising derivatized cyclofructan compounds for chromatographic separations of chemical species, including enantiomers. Said compositions may comprise a solid support and/or polymers comprising derivatized cyclofructan compounds.
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Page/Page column 45-49; 62
(2010/12/31)
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- Optical resolution reagent and manufacturing method of optically active amines that uses it
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PROBLEM TO BE SOLVEDTo provide an effective reagent for optical resolution which produce an optically active amines by resolving the (+/-)-amines and the method for producing the optically active amines characterized by using the same reagent. SOLUTION The O-alkylthiophosphoric acid represented as the following formula (1) is effective for the optical resolution of various amines.
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Page/Page column 19-20
(2008/06/13)
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- A new hydrogen-bonding motif for chiral recognition in the diastereomeric salts of racemic 1-phenylethylamine derivatives with enantiopure O-ethyl phenylphosphonothioic acid
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(Chemical Equation Presented) An enantiopure phosphonothioic acid showed a unique and superior chiral recognition ability, arising from its P-stereogenicity, for racemic 1-phenylethylamine derivatives through diastereomeric crystallization. Spherical molecular clusters, associated by hydrogen bonds and CH/π interactions, aggregated with high symmetry in the less-soluble diastereomeric salts.
- Kobayashi, Yuka,Morisawa, Fumi,Saigo, Kazuhiko
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p. 4227 - 4230
(2007/10/03)
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- Ruthenium-catalysed asymmetric hydrosilylation of ketoximes using chiral oxazolinylferrocenylphosphines
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Chiral ruthenium(II) complexes, RuCl2(PPh3)(oxazolinylferrocenylphosphine), have been found to be effective catalysts for asymmetric hydrosilylation of ketoximes to give the corresponding primary amines in good yields with high enant
- Takei,Nishibayashi,Ishii,Mizobe,Uemura,Hidai
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p. 2360 - 2361
(2007/10/03)
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- A high-performance, tailor-made resolving agent: Remarkable enhancement of resolution ability by introducing a naphthyl group into the fundamental skeleton1
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A novel resolving agent, 2-naphthylglycolic acid (2-NGA), was designed for p-substituted 1-arylethylamines on the basis of the consideration that a rigid and large naphthyl group would be favorable for the close packing of supramolecular hydrogen-bond sheets formed between the carboxy groups of 2-NGA and the amino groups of p-substituted 1-arylethylamines. Racemic 2-NGA was readily available from commercially available raw materials, and both enantiopure forms could be obtained by simple diastereomeric resolution with enantiopure 1-phenyl-ethylamine. Thus-prepared enantiopure 2-NGA was found to have an excellent resolution ability not only for p-substituted 1-arylethylamines, but also for a wide variety of chiral primary amines. X-Ray crystallographic analyses of the less- and more-soluble diastereomeric salts revealed that this excellent resolution ability of 2-NGA arose from the formation of a supramolecular hydrogen-bond sheet with the primary amine, as we had expected, and also from the possible achievement of an infinite chain of CH... π interaction between its naphthyl group and the aromatic group of the amine, which was formed in the hydrophobic region of the supramolecular hydrogen-bond sheet.
- Kinbara, Kazushi,Harada, Yoshiko,Saigo, Kazuhiko
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p. 1339 - 1347
(2007/10/03)
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