- Rapid and Quantitative Profiling of Substrate Specificity of ω-Transaminases for Ketones
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ω-Transaminases (ω-TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high-throughput activity assay of ω-TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω-TA activities toward ketones in a 96-well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω-TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild-type ω-TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω-TA activity was also demonstrated by supplementing the assay mixture with color-developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω-TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH-based assay is expected to be promising for high-throughput screening of enzyme collections and mutant libraries to fish out the best ω-TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.
- Han, Sang-Woo,Shin, Jong-Shik
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p. 3287 - 3295
(2019/06/21)
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- Ring-opening reaction of Bus- and SES-protected aziridines using lithiated dithianes
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The scope and limitation of the ring-opening reaction of sulfonyl-activated aziridines using lithiated dithianes was investigated. Nucleophilic attack of lithiated dithianes on aziridines containing tert-butylsulfonyl (Bus) and 2-(trimethylsilyl)ethylsulf
- Sakakibara, Ken,Nozaki, Kyoko
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experimental part
p. 502 - 507
(2009/07/18)
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- An ammonia equivalent for the dimethyltitanocene-catalyzed intermolecular hydroamination of alkynes
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(Equation presented) Commercially available α-aminodiphenylmethane 1 (benzhydrylamine) serves as a convenient ammonia equivalent in the dimethyltitanocene-catalyzed intermolecular hydroamination of alkynes. The primary formed imines can be hydrogenated and cleaved directly to the corresponding primary amines by catalytic hydrogenation using Pd/C as catalyst.
- Haak, Edgar,Siebeneicher, Holger,Doye, Sven
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p. 1935 - 1937
(2007/10/03)
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- Convenient Access to Primary Amines by Employing the Barbier-Type Reaction of N-(Trimethylsilyl)imines Derived from Aromatic and Aliphatic Aldehydes
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A new versatile preparation of primary amines via benzylation of aromatic and aliphatic aldimines is described. Sonochemical and traditional methods for generation of the reactive intermediates are compared and contrasted. Competitive reactions were analyzed via free energy relationships to support the proposed alkylative mechanism.
- Gyenes, Ferenc,Bergmann, Kathryn E.,Welch, John T.
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p. 2824 - 2828
(2007/10/03)
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- ALKYLATION OF 2-AZAALLYL ANIONS; A VERSATILE PRIMARY AMINE SYNTHESIS
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Imines from primary amines and mesityl 2-pyridyl ketone react with LDA followed by treatment with an alkyl halide and hydrolysis to give the original amine alkylated at the alpha-position.
- Hornback, Joseph M.,Murugaverl, Balasingam
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p. 5853 - 5856
(2007/10/02)
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- Synthesis and Evaluation of 3-Substituted Analogues of 1,2,3,4-Tetrahydroisoquinoline as Inhibitors of Phenylethanolamine N-Methyltransferase
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1,2,3,4-Tetrahydroisoquinoline (THIQ) and aryl-substituted derivatives of THIQ are potent inhibitors of the enzyme that catalyzes the formation of epinephrine - phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28).In previous studies, we found that substitution of the 3-position of THIQ with a methyl group resulted in enhanced activity as an inhibitor for 3-methyl-THIQ (8) with respect to THIQ itself.To more fully delineate this region of the PNMT active site, we have synthesized and evaluated other 3-substituted THIQ analogues that vary in both steric and electronic character.Extension of the methyl side chain in 8 by a single methylene unit results in diminished potency for 3-ethyl-THIQ (13), suggesting that this zone of the active site is spatially compact; furthermore, the region of steric intolerance may be located principally on only "one side" of the 3-position of bound THIQs, since the carbonyl containing (bent) analogues 3-(methoxycarbonyl)-THIQ (10) and 3-(aminocarbonyl)-THIQ (12) are much less capable of forming a strong enzyme-inhibitor dissociable complex compared to straight-chain derivatives possessing a similar steric component.The good activity of 3-(hydroxymethyl)-THIQ (11) as a PNMT inhibitor cannot be explained solely by steric tolerance for this side chain.We believe that an active-site amino acid residue capable of specific (i.e., hydrogen bond) interactions is located in close proximity to the 3-position of bound THIQs and that association of the OH functionality with this active-site residue results in the enhanced in vitro potency of this analogue (Ki = 2.4 μM) compared to that of THIQ (Ki = 10.3 μM).Incorporation of a hydroxymethyl substituent onto the 3-position of the potent PNMT inhibitor 7,8-dichloro-THIQ (SKF 64139, Ki = 0.24 μM) did not result in the same enhancement in inhibitor potency for 17 (Ki = 0.38 μM).This result suggests that simultaneous binding in an optional orientation of the aromatic halogens, secondary amine, and side-chain hydroxyl functionalities to the PNMT active site is not allowed in this analogue.
- Grunewald, Gary L.,Sall, Daniel J.,Monn, James A.
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p. 824 - 830
(2007/10/02)
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- SYNTHESIS OF N-(1-PHENYL-2-PROPYL)-2,5-DIPHENYLPENTYLAMINE AND SOME RELATED COMPOUNDS AS POTENTIAL NEUROTROPIC AND CARDIOVASCULAR DRUGS
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Heating of 2,5-diphenylvaleric acid with 2-phenylethylamine, 1-phenyl-2-propylamine, 1-phenyl-2-butylamine (IX), 1-(4-methoxyphenyl)-2-propylamine, 1-(4-methoxyphenyl)-2-butylamine (X) and 1-(4-dimethylaminophenyl)-2-propylamine to 200-210 deg C resulted
- Vejdelek, Zdenek,Nemec, Jiri,Protiva, Miroslav
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p. 1487 - 1493
(2007/10/02)
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- Probes of the Active Site of Norepinephrine N-Methyltransferase: Effect of Hydrophobic and Hydrophilic Interactions on Side-Chain Binding of Amphetamine and α-Methylbenzylamine
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A series of ω-substituted analogues of amphetamine and α-methylbenzylamine were prepared and evaluated as inhibitors of norepinephrine N-methyltransferase (NMT).These included several alkyl side chain extended analogues (1-5), as well as the terminally hydroxylated derivatives phenylalanol (6a) and Phenylglycinol (7a).None of the alkyl-substituted derivatives displayed appreciable activity as inhibitors; however, the hydroxylated analogues were up to twofold more potent than the parent compounds.The positive contribution of the side-chain hydroxy suggests that theterminal methyl group of the lead compounds is situated close to a hydrophilic area or hydrogen bonding functional group within the active site.
- Grunewald, Gary L.,Monn, James A.,Rafferty, Michael F,,Borchardt, Ronald T.,Krass, Polina
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p. 1248 - 1250
(2007/10/02)
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