134003-04-6Relevant articles and documents
Efficient synthesis of the intermediate of abacavir and carbovir using a novel (+)-γ-lactamase as a catalyst
Gao, Shuaihua,Zhu, Shaozhou,Huang, Rong,Lu, Yingxiu,Zheng, Guojun
, p. 3878 - 3881 (2015)
Abstract The enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) are key chiral synthons in the synthesis of antiviral drugs such as carbovir and abacavir. (+)-γ-Lactamase can be used as a catalyst in the enzymatic preparation of optically pure (-)-γ-lactam. Here, a (+)-γ-lactamase discovered from Bradyrhizobium japonicum USDA 6 by sequence-structure guided genome mining was cloned, purified and characterized. The enzyme possesses a significant catalytic activity towards γ-lactam. The active site of the (+)-γ-lactamase was studied by homologous modeling and molecular docking, and the accuracy of the prediction was confirmed by site-specific mutagenesis. The (+)-γ-lactamase reveals the great practical potential as an enzymatic method for the efficient production of carbocyclic nucleosides of pharmaceutical interest.
An efficient route to all eight stereoisomers of a tri-functionalised cyclopentane scaffold for drug discovery
Smith, Mark E.B.,Lloyd, Michael C.,Derrien, Nadine,Lloyd, Richard C.,Taylor, Stephen J.C.,Chaplin, David A.,Casy, Guy,McCague, Raymond
, p. 703 - 705 (2001)
A route to all eight stereoisomers of 3-(tert-butoxycarbonylamino)-4-hydroxycyclopentanecarboxylic acid methyl ester is presented; these products should prove to be valuable scaffolds in pharmaceutical discovery.
Novel screening methods - The key to cloning commercially successful biocatalysts
Taylor, Stephen J. C.,Brown, Rob C.,Keene, Phil A.,Taylor, Ian N.
, p. 2163 - 2168 (1999)
Providing sufficient biocatalyst to support the demands of multi tonne product supply can be problematical. Here we describe how screening for and cloning a γ-lactamase overcame biocatalyst supply issues, and greatly improved the actual biocatalytic process. The isolation of an expressing γ- lactamase clone from a gene library necessitated a combination of classical molecular biology techniques together with innovative screening methods to identify a functional clone. Once isolated the enzyme was characterised with regard to its process performance and proved to be active at 500 g L-1 substrate. Further development of the recombinant fermentation and downstream processing has resulted in the ability to produce sufficient biocatalyst from one 500 l fermentation to resolve 5 metric tonnes of (±)-lactam, whilst simplifying the process chemistry greatly.
Chemical preparation method of 2-azabicyclo [2.2.1] hept-5-ene-3-one with optical activity
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Paragraph 0023; 0029-0031; 0041; 0047-0049; 0059; 0065-0067, (2020/05/08)
The invention discloses a chemical preparation method of 2-azabicyclo [2.2.1] hept-5-ene-3-one with optical activity. The chemical preparation method comprises the following steps: racemic 2-azabicyclo [2.2.1] hept-5-ene-3-one which is easily available on the market is used as a raw material and is subjected to chemical resolution to obtain (+/-) 4-aminocyclopent-2-ene-1-carboxylic acid methyl ester with optical activity, (+/-) 4-aminocyclopent-2-ene-1-carboxylic acid is prepared by hydrolysis, and the (+/-) 4-aminocyclopent-2-ene-1-carboxylic acid is subjected to intramolecular condensation to obtain the 2-azabicyclo [2.2.1] hept-5-ene-3-one with optical activity. The method has the advantages that the use of an enzyme fermentation method is avoided, the repeatability is good, and the yield is high.
Stereo- and regiocontrolled synthesis of highly functionalized cyclopentanes with multiple chiral centers
Nonn, Melinda,Binder, Adrienn,Volk, Balázs,Kiss, Loránd
, p. 1199 - 1209 (2020/03/17)
The synthesis of some highly substituted three-dimensional cyclopentanes with multiple chiral centers and with high regiochemical and stereochemical diversity has been accomplished starting from cyclopentadiene-derived aminocyclopentenecarboxylic acids. The small-molecular design consisted of stereo- and regiocontrolled functionalization of the starting cyclopentene β- and γ-amino acids through oxirane formation/oxirane opening and afforded regio- and diastereoisomers of orthogonally protected aminocyclopentanecarboxylates.
Promiscuous (+)-γ-lactamase activity of an amidase from nitrile hydratase pathway for efficient synthesis of carbocyclic nucleosides intermediate
Li, Hongxia,Zhu, Shaozhou,Zheng, Guojun
supporting information, p. 1071 - 1076 (2018/03/01)
Based on bioinformatics analysis, the promiscuous (+)-γ-lactamase activity of an amidase was identified in Rhodococcus erythropolis PR4 and found to be involved in the nitrile hydratase pathway. The amidase is highly enantioselective and can be used in the kinetic resolution of the Vince lactam. The known structure provides a rare insight into the catalytic mechanism of (+)-γ-lactamase with absolute chiral selectivity. This lactamase was cloned, purified, biochemically characterized, and demonstrated to be an ideal catalyst for the preparation of carbocyclic nucleosides of pharmaceutical interest. The chiral selectivity of this enzyme was investigated by molecular docking and site-specific mutagenesis, which provides a foundation for further engineering of these versatile biocatalysts.
Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases
Devamani, Titu,Rauwerdink, Alissa M.,Lunzer, Mark,Jones, Bryan J.,Mooney, Joanna L.,Tan, Maxilmilien Alaric O.,Zhang, Zhi-Jun,Xu, Jian-He,Dean, Antony M.,Kazlauskas, Romas J.
supporting information, p. 1046 - 1056 (2016/02/05)
Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ～100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.
Enhanced enzymatic synthesis of the enantiopure intermediate for the blockbuster drug intermediate abacavir through a two-step enzymatic cascade reaction
Galla, Zsolt,Forró, Enik?,Fül?p, Ferenc
, p. 729 - 731 (2016/08/01)
A very efficient enzymatic two-step cascade reaction was devised (E?>?200) for the resolution of activated γ-lactams (±)-1 and (±)-2. The N-hydroxymethyl group worked as a traceless activating group, when the reactions were performed with H2O (0.5?equiv) in the presence of benzylamine (1?equiv) in i-Pr2O at 60?°C. The ring-opened enantiomerically pure γ-amino acids (1S,4R)-6 (ee?=?99%, intermediate of abacavir) and (1S,3R)-8 (ee?=?99%) and unreacted lactams (1S,4R)-1 and (1R,4S)-2 (ee???96%) were obtained in good yields (?43%). Treatment of (1S,4R)-1 and (1R,4S)-2 with 18% HCl or NH4OH resulted in (1R,4S)-6·HCl and (1S,3R)-8·HCl or (1S,4R)-3 and (1R,4S)-4 quantitatively, with ee???96%.
Study on immobilization of (+) γ-lactamase using a new type of epoxy graphene oxide carrier
Li, Wei,Wen, Huijiao,Shi, Qianqiao,Zheng, Guojun
, p. 270 - 276 (2016/02/05)
In this article, a new type of graphene oxide material was obtained by modification with epoxy chloropropane, which was used for the (+) γ-lactamase immobilization research. The enzymatic properties of the immobilized (+) γ-lactamase were systematically tested and compared with those of the free enzyme. The free and immobilized enzymes have the same optimum temperature (80 °C) and pH (8.0), the range of pH tolerance increased from pH 8.0-9.0 to pH 4.0-10.0 after immobilization, and the immobilized enzyme is less tolerant to heat than the free enzyme. Kinetic experiments showed that the Km value of the immobilized enzyme was about 2.66 times higher than that of the free enzyme, whereas Vmax decreased 40%. Subsequently, the apparent activation energies (Ea) of the free and immobilized enzymes became 40.03 and 35.62 kJ/mol, respectively. Finally, a reusability assay demonstrated that 70% of the enzyme activity remained after 15 repeated batch experiments.
Method for synthesizing (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride
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Paragraph 0017; 0018, (2017/07/23)
The invention relates to a method for synthesizing (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride. According to the method, the reaction conditions are mild, (1S,4R)-(-)-2-azabicyalo[2,2,1]hepta-5-alkene-3-ketone is directly used, (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride is obtained through the reactions of hydrolysis and reducing, the yield is high, and the optical purity is high.
