- Engineering the large pocket of an (S)-selective transaminase for asymmetric synthesis of (S)-1-amino-1-phenylpropane
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Amine transaminases offer an environmentally benign chiral amine asymmetric synthesis route. However, their catalytic efficiency towards bulky chiral amine asymmetric synthesis is limited by the natural geometric structure of the small pocket, representing a great challenge for industrial applications. Here, we rationally engineered the large binding pocket of an (S)-selective ?-transaminase BPTA fromParaburkholderia phymatumto relieve the inherent restriction caused by the small pocket and efficiently transform the prochiral aryl alkyl ketone 1-propiophenone with a small substituent larger than the methyl group. Based on combined molecular docking and dynamic simulation analyses, we identified a non-classical substrate conformation, located in the active site with steric hindrance and undesired interactions, to be responsible for the low catalytic efficiency. By relieving the steric barrier with W82A, we improved the specific activity by 14-times compared to WT. A p-p stacking interaction was then introduced by M78F and I284F to strengthen the binding affinity with a large binding pocket to balance the undesired interactions generated by F44. T440Q further enhanced the substrate affinity by providing a more hydrophobic and flexible environment close to the active site entry. Finally, we constructed a quadruple variant M78F/W82A/I284F/T440Q to generate the most productive substrate conformation. The 1-propiophenone catalytic efficiency of the mutant was enhanced by more than 470-times in terms ofkcat/KM, and the conversion increased from 1.3 to 94.4% compared with that of WT, without any stereoselectivity loss (ee > 99.9%). Meanwhile, the obtained mutant also showed significant activity improvements towards various aryl alkyl ketones with a small substituent larger than the methyl group ranging between 104- and 230-fold, demonstrating great potential for the efficient synthesis of enantiopure aryl alkyl amines with steric hindrance in the small binding pocket.
- Liu, He,Wang, Hualei,Wei, Dongzhi,Xie, Youyu,Xu, Feng,Xu, Xiangyang,Yang, Lin
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p. 2461 - 2470
(2021/04/22)
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- A Simple Biosystem for the High-Yielding Cascade Conversion of Racemic Alcohols to Enantiopure Amines
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The amination of racemic alcohols to produce enantiopure amines is an important green chemistry reaction for pharmaceutical manufacturing, requiring simple and efficient solutions. Herein, we report the development of a cascade biotransformation to aminate racemic alcohols. This cascade utilizes an ambidextrous alcohol dehydrogenase (ADH) to oxidize a racemic alcohol, an enantioselective transaminase (TA) to convert the ketone intermediate to chiral amine, and isopropylamine to recycle PMP and NAD+ cofactors via the reversed cascade reactions. The concept was proven by using an ambidextrous CpSADH-W286A engineered from (S)-enantioselective CpSADH as the first example of evolving ambidextrous ADHs, an enantioselective BmTA, and isopropylamine. A biosystem containing isopropylamine and E. coli (CpSADH-W286A/BmTA) expressing the two enzymes was developed for the amination of racemic alcohols to produce eight useful and high-value (S)-amines in 72–99 % yield and 98–99 % ee, providing with a simple and practical solution to this type of reaction.
- Li, Zhi,Tian, Kaiyuan
-
supporting information
p. 21745 - 21751
(2020/09/21)
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- Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone
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ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., R-analysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the R-analysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2×104-fold activity increase for 1-phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with >99% ee of a resulting (S)-amine. (Figure presented.).
- Kim, Hong-Gon,Han, Sang-Woo,Shin, Jong-Shik
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p. 2594 - 2606
(2019/05/15)
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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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- Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope, co-solvent tolerance and biocatalyst immobilization
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In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50 mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.
- L?we, Jana,Ingram, Aaron A.,Gr?ger, Harald
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p. 1387 - 1392
(2018/03/21)
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- Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines
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The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.
- Pavlidis, Ioannis V.,Wei?, Martin S.,Genz, Maika,Spurr, Paul,Hanlon, Steven P.,Wirz, Beat,Iding, Hans,Bornscheuer, Uwe T.
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p. 1076 - 1082
(2016/11/02)
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- Expanding Substrate Specificity of ω-Transaminase by Rational Remodeling of a Large Substrate-Binding Pocket
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Production of structurally diverse chiral amines via biocatalytic transamination is challenged by severe steric interference in a small active site pocket of ω-transaminase (ω-TA). Herein, we demonstrated that structure-guided remodeling of a large pocket by a single point mutation, instead of excavating the small pocket, afforded desirable alleviation of the steric constraint without deteriorating parental activities toward native substrates. Molecular modeling suggested that the L57 residue of the ω-TA from Ochrobactrum anthropi acted as a latch that forced bulky substrates to undergo steric interference with the small pocket. Removal of the latch by a L57A substitution allowed relocation of the small pocket and dramatically improved activities toward various arylalkylamines and alkylamines (e.g., 1100-fold increase in kcat/KM for α-propylbenzylamine). This approach may provide a facile strategy to broaden the substrate specificity of ω-TAs.
- Han, Sang-Woo,Park, Eul-Soo,Dong, Joo-Young,Shin, Jong-Shik
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p. 2712 - 2720
(2015/09/01)
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- Engineering the active site of the amine transaminase from vibrio fluvialis for the asymmetric synthesis of aryl-alkyl amines and amino alcohols
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Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of α-hydroxy ketones and aryl-alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30-fold increased activity in the conversion of (S)-phenylbutylamine and (R)-phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98 ee. Excavating the active site: The constrained active site of the amine-transaminase from Vibrio fluvialis was engineered in order to achieve the conversion of bulky ketones. This led to the discovery of mutants with a 30-fold increase in activity which enabled the asymmetric synthesis of (S)-phenylbutylamine and (R)-phenylglycinol
- Nobili, Alberto,Steffen-Munsberg, Fabian,Kohls, Hannes,Trentin, Ivan,Schulzke, Carola,H?hne, Matthias,Bornscheuer, Uwe T.
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p. 757 - 760
(2015/03/14)
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- Mechanism-Guided Engineering of ω-Transaminase to Accelerate Reductive Amination of Ketones
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Asymmetric reductive amination of ketones using ω-transaminases (ω-TAs) offers a promising alternative to the chemocatalytic synthesis of chiral amines. One fundamental challenge to the biocatalytic strategy is the very low enzyme activities for most ketones compared with native substrates (i.e., cat/KM for acetophenone). The W58L mutant afforded an efficient synthesis of enantiopure amines (i.e., >99% ee) using isopropylamine as an amino donor.
- Han, Sang-Woo,Park, Eul-Soo,Dong, Joo-Young,Shin, Jong-Shik
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p. 1732 - 1740
(2015/06/02)
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- Microwave-Enhanced Asymmetric Transfer Hydrogenation of N-(tert-Butylsulfinyl)imines
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Microwave irradiation has considerably enhanced the efficiency of the asymmetric transfer hydrogenation of N-(tert-butylsulfinyl)imines in isopropyl alcohol catalyzed by a ruthenium complex bearing the achiral ligand 2-amino-2-methylpropan-1-ol. In addition to shortening reaction times for the transfer hydrogenation processes to only 30 min, the amounts of ruthenium catalyst and isopropyl alcohol can be considerably reduced in comparison with our previous procedure assisted by conventional heating, which diminishes the environmental impact of this new protocol. This methodology can be applied to aromatic, heteroaromatic and aliphatic N-(tert-butylsulfinyl)ketimines, leading, after desulfinylation, to the expected primary amines in excellent yields and with enantiomeric excesses of up to 96 %. Microwave irradiation promotes the asymmetric transfer hydrogenation of N-(tert-butylsulfinyl)imines in 2-propanol catalysed by a ruthenium complex bearing an achiral β-amino alcohol as ligand. After desulfinylation, α-branched primary amines containing aromatic, heteroaromatic and aliphatic substituents are obtained in excellent yields and with enantiomeric excesses of up to 96 %.
- Pablo, Oscar,Guijarro, David,Yus, Miguel
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p. 7034 - 7038
(2016/02/19)
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- Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4- phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors
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We report synthesis and optimization of a series of (3S,5R)-5-(2,2- dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of P1, P2 and P 3 portions led to a promising 3
- Mori, Yutaka,Ogawa, Yasuyuki,Mochizuki, Akiyoshi,Nakamura, Yuji,Fujimoto, Teppei,Sugita, Chie,Miyazaki, Shojiro,Tamaki, Kazuhiko,Nagayama, Takahiro,Nagai, Yoko,Inoue, Shin-Ichi,Chiba, Katsuyoshi,Nishi, Takahide
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p. 5907 - 5922
(2013/09/12)
-
- One-pot synthesis of chiral nonracemic amines
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One-pot five-component reactions of oxathiazolidine-S-oxides with mesitylmagnesium bromide, lithium bis(trimethylsilyl)amide, aldehydes and Grignard reagents afford chiral nonracemic amines or sulfinamides in good yields and high stereoselectivities.
- Roe, Caroline,Hobbs, Heather,Stockman, Robert A.
-
experimental part
p. 9452 - 9459
(2012/01/06)
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- Asymmetric synthesis of chiral primary amines by transfer hydrogenation of N -(tert -Butanesulfinyl)ketimines
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(Figure presented) The diastereoselective reduction of (R)-N-(tert- butanesulfinyl)ketimines by a ruthenium-catalyzed asymmetric transfer hydrogenation process in isopropyl alcohol, followed by desulfinylation of the nitrogen atom, is an excellent method to prepare highly enantiomerically enriched α-branched primary amines (up to >99% ee) in short reaction times (1-4 h). (1S,2R)-1-Amino-2-indanol has been shown to be a very efficient ligand to perform this transformation. Ketimines bearing either an aryl or a heteroaryl group and an alkyl group as substituents of the iminic carbon atom are very good substrates for this process. The reduction of a dialkyl ketimine could also be achieved, affording the expected amine with moderate optical purity (69% ee). Some amines which are precursors of very interesting biologically and pharmacologically active compounds have been prepared in excellent yields and enantiomeric excesses.
- Guijarro, David,Pablo, Oscar,Yus, Miguel
-
supporting information; experimental part
p. 5265 - 5270
(2010/10/21)
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- Efficient kinetic resolution of racemic amines using a transaminase in combination with an amino acid oxidase
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A range of enantiomerically pure (R)- and (S)-configured chiral amines has been prepared in excellent e.e. (99%) by combining a transaminase enzyme with an amino acid oxidase and catalytic quantities of pyruvate.
- Truppo, Matthew D.,Turner, Nicholas J.,Rozzell, J. David
-
supporting information; experimental part
p. 2127 - 2129
(2009/09/06)
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- Asymmetric synthesis of primary amines via the spiroborate-catalyzed borane reduction of oxime ethers
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The enantioselective borane reduction of O-benzyloxime ethers to primary amines was studied under catalytic conditions using the spiroborate esters 5-10 derived from nonracemic 1,2-amino alcohols and ethylene glycol. Effective catalytic conditions were achieved using only 10% of catalyst 5 derived from diphenylvalinol in dioxane at 0°C resulting in complete conversion to the corresponding primary amine in up to 99% ee.
- Huang, Xiaogen,Ortiz-Marciales, Margarita,Huang, Kun,Stepanenko, Viatcheslav,Merced, Francisco G.,Ayala, Angel M.,Correa, Wildeliz,De Jesus, Melvin
-
p. 1793 - 1795
(2008/02/02)
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- Rational design, synthesis, and structure-Activity relationships of aryltriazoles as novel corticotropin-releasing factor-1 receptor antagonists
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Following the discovery of the very high binding affinity of 4-anilinopyrimidines against corticotropin-releasing factor receptor-1 (CRF 1) (e.g., 1, Ki = 2 nM), a new series of triazoles bearing different groups has been synthesized
- Lowe, Richard F.,Nelson, Jodene,Dang, Trunghau N.,Crowe, Paul D.,Pahuja, Anil,McCarthy, James R.,Grigoriadis, Dimitri E.,Conlon, Paul,Saunders, John,Chen, Chen,Szabo, Thomas,Chen, Ta Kung,Bozigian, Haig
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p. 1540 - 1549
(2007/10/03)
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- Synthesis of enantiopure 1-aryl-1-butylamines and 1-aryl-3-butenylamines by diastereoselective addition of allylzinc bromide to imines derived from (R)-phenylglycine amide
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The synthesis of enantiopure 1-aryl-1-butylamines via a highly diastereoselective addition of allylzinc bromide to imines derived from (R)-phenylglycine amide is reported. These are synthesised by a three-step procedure, which involves: (a) formation of the chiral imines; (b) asymmetric addition of the allylzinc reagent; (c) removal of the chiral auxiliary by means of a reductive or non-reductive method. The reductive method provides 1-aryl-1-butylamines whereas the non-reductive method preserves the double bond to afford 1-aryl-3-butenylamines. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.
- Dalmolen, Jan,Van Der Sluis, Marcel,Nieuwenhuijzen, Jose W.,Meetsma, Auke,De Lange, Ben,Kaptein, Bernard,Kellogg, Richard M.,Broxterman, Quirinus B.
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p. 1544 - 1557
(2007/10/03)
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- Asymmetric synthesis of allyl- and α-allenylamines from chiral imines and alkynes via (η2-imine)Ti(O-i-Pr)2 complexes
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(Matrix presented) The reaction of a divalent titanium reagent Ti(O-i-Pr)4/2i-PrMgX with optically active arylaldimines derived from arylaldehydes and O-methylphenylglycinol provided, in a highly diastereoselective manner, chiral (η2-imine)Ti(O-i-Pr) 2 complexes, which in turn reacted with 1-alkynes or propargyl compounds to give optically active allyl- and α-allenylamines, respectively.
- Fukuhara, Kohki,Okamoto, Sentaro,Sato, Fumie
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p. 2145 - 2148
(2007/10/03)
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- Synthesis of optically-active benzylic amines; asymmetric reduction of ketoxime ethers with chiral oxazaborolidines
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The preparation of novel optically active benzylic amines by the enantioselective reduction of phenone oximes using chiral oxazaborolidine is described. The choice of the chiral 1,2-amino alcohol (S)-diphenylvalinol as chiral inducer and that of the benzy
- Fontaine, Evelyne,Namane, Claudie,Meneyrol, Jerome,Geslin, Michel,Serva, Laurence,Roussey, Eliane,Tissandie, Stephanie,Maftouh, Mohamed,Roger, Pierre
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p. 2185 - 2189
(2007/10/03)
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- Diastereoselective addition of allylzinc bromide to imines derived from (R)-phenylglycine amide
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equation presented The highly diastereoselective addition of allylzinc bromide to imines derived from (R)-phenylglycine amide is reported. Homoallylamines with high enantiomeric purity are obtained from the adducts in three steps on removal of the chiral
- Van Der Sluis, Marcel,Dalmolen, Jan,De Lange, Ben,Kaptein, Bernard,Kellogg, Richard M.,Broxterman, Quirinus B.
-
p. 3943 - 3946
(2007/10/03)
-
- Enantioselective syntheses of α-phenylalkanamines via intermediate addition of Grignard reagents to chiral hydrazones derived from (R)-(-)-2- aminobutan-1-ol
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The hydrazine (R)-(-)-28 was obtained in four steps from 2-aminobutan- 1-ol (R)-(-)-11, and reacted with benzaldehyde to give the hydrazone (R)-(- )-29. Nucleophilic addition of various alkyl Grignard reagents to the latter yielded the corresponding trisubstituted hydrazines (R,R)-30a-g in 70-89% yields and having d.e.s=100% (1H and 13C NMR). Catalytic hydrogenolysis of these hydrazines afforded the corresponding (R)(+)-α-phenylalkanamines (R)- (+)-31a-g having e.e.s=90-92% (chiral GPC).
- Bataille, Patricia,Paterne, Michel,Brown, Eric
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p. 2181 - 2192
(2007/10/03)
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- Chiral Oximes in Asymmetric Synthesis. Addition of Organometallic Reagents to O-(1-Phenylethyl) Aldoximes
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Addition of Grignard and organolithium reagents to O-(1-phenylethyl) aldoximes in the presence of boron trifluoride etherate gives secondary hydroxylamines in 21-84percent yield with up to 95percent diastereomeric excess.
- Brown, David S.,Gallagher, Peter T.,Lightfoot, Andrew P.,Moody, Christopher J.,Slawin, Alexandra M. Z.,Swann, Elizabeth
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p. 11473 - 11488
(2007/10/02)
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- Asymmetric Reduction of Ketoxime O-Alkyl Ethers with Chirally Modified NaBH4-ZrCl4
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Reducing agents prepared from sodium borohydride (NaBH4), zirconium tetrachloride (ZrCl4), and chiral amino alcohols have been successfully applied to the enantioselective reduction of oxime ethers.Optically active primary amines were obtained in high enantiomeric excess ( : : : .
- Itsuno, Shinichi,Sakurai, Yoshiki,Shimizu, Koji,Ito, Koichi
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p. 1859 - 1863
(2007/10/02)
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- Asymmetric Reduction of Ketoxime O-Alkyl Ethers with Sodium Borohydride-Lewis Acid
-
Novel hydride agents formed by combining Lewis acids with sodium borohydride (NaBH4) reduce quantitatively ketoxime O-alkyl ethers to the corresponding optically active primary amines with high enantioselectivities (up to 95percent e.e.) in the presence of chiral amino alcohols.
- Itsuno, Shinichi,Sakurai, Yoshiki,Shimizu, Koji,Ito, Koichi
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p. 1548 - 1549
(2007/10/02)
-
