- Engineering Bacillus subtilis Isoleucine Dioxygenase for Efficient Synthesis of (2 S,3 R,4 S)-4-Hydroxyisoleucine
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Isoleucine dioxygenase (IDO)-catalyzed hydroxylation of isoleucine is a promising method for the synthesis of the diabetic drug (2S,3R,4S)-4-hydroxyisoleucine [(2S,3R,4S)-4-HIL]. However, the low activity of IDO significantly limits its practical application. In this work, a high-throughput screening method was developed and directed evolution was performed on the IDO from Bacillus subtilis, resulting in a double mutant with improvements in specific activity, protein expression level, and fermentation titer of 3.2-, 2.8-, and 9.4-fold, respectively. l-Isoleucine (228 mM) was completely converted to (2S,3R,4S)-4-HIL by the best variant with a space-time yield of up to 80.8 g L-1 d-1, which is the highest record reported so far. With a further increase of the substrate loading to 1 M, a high conversion of 91% could also be achieved. At last, enzymatic synthesis of (2S,3R,4S)-4-HIL was successfully carried out on a 3 L scale, indicating tremendous potential of the IDO variant I162T/T182N for green and efficient production of (2S,3R,4S)-4-HIL.
- Du, Ping,Pan, Jiang,Qian, Xiao-Long,Xu, Jian-He,Yan, Shuai,Yu, Hui-Lei,Zhang, Zhi-Jun
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p. 14555 - 14563
(2020/12/22)
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- Repurposing Nonheme Iron Hydroxylases to Enable Catalytic Nitrile Installation through an Azido Group Assistance
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Three mononuclear nonheme iron and 2-oxoglutarate dependent enzymes, l-Ile 4-hydroxylase, l-Leu 5-hydroxylase and polyoxin dihydroxylase, are previously reported to catalyze the hydroxylation of l-isoleucine, l-leucine, and l-α-amino-δ-carbamoylhydroxyvaleric acid (ACV). In this study, we showed that these enzymes can accommodate leucine isomers and catalyze regiospecific hydroxylation. On the basis of these results, as a proof-of-concept, we demonstrated that the outcome of the reaction can be redirected by installation of an assisting group within the substrate. Specifically, instead of canonical hydroxylation, these enzymes can catalyze non-native nitrile group installation when an azido group is introduced. The reaction is likely to proceed through C - H bond activation by an Fe(IV)-oxo species, followed by azido-directed C-N bond formation. These results offer a unique opportunity to investigate and expand the reaction repertoire of Fe/2OG enzymes.
- Davidson, Madison,McNamee, Meredith,Fan, Ruixi,Guo, Yisong,Chang, Wei-Chen
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supporting information
p. 3419 - 3423
(2019/03/07)
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- Attempt to simultaneously generate three chiral centers in 4-hydroxyisoleucine with microbial carbonyl reductases
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A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5 mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.
- Hibi, Makoto,Takahashi, Koji,Kako, Junko,Wakita, Yuuta,Kodera, Tomohiro,Shimizu, Sakayu,Yokozeki, Kenzo,Ogawa, Jun
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p. 1327 - 1332
(2017/10/05)
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- A practical and efficient total synthesis of potent insulinotropic (2S,3R,4S)-4-Hydroxyisoleucine through a chiral N-protected γ-keto- α-aminoester
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(2S,3R,4S)-4-Hydroxyisoleucine, which exhibits remarkable insulinotropic activity, is expected to be a potent drug to treat type II diabetes. We propose herein a four-step synthesis of the enantiopure natural product on the basis of successive Mannich condensation, catalytic epimerization, N-paramethoxyphenyl deprotection, and diastereoselective reduction. This compact economical and scalable sequence enables to perfectly control three contiguous chiral centers. It does not involve any chromatographic purification, and the desired compound is obtained in >99 % de, >99 % ee, and 22 % overall yield under our optimized conditions.
- De Lamo Marin, Sandra,Catala, Cedric,Kumar, Sreekantha Ratna,Valleix, Alain,Wagner, Alain,Mioskowski, Charles
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experimental part
p. 3985 - 3989
(2010/09/18)
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- An organocatalyzed enantioselective synthesis of (2S,3R,4S)-4- hydroxyisoleucine and its stereoisomers
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A concise enantioselective total synthesis of (2S,3R,4S)-4- hydroxyisoleucine and its stereoisomers is described. A key feature of this protocol is a catalytic enantioselective mannich reaction that is either anti- or syn-selective as genesis of chirality.
- Kumaraswamy, Gullapalli,Jayaprakash, Neerasa,Sridhar, Balasubramanian
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supporting information; experimental part
p. 2745 - 2747
(2010/07/17)
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- METHOD FOR PURIFYING 4-HYDROXYISOLEUCINE
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The present invention aims to provide a method of conveniently isolating and purifying as well as separating and removing (2S,3R,4S)-4-hydroxyisoleucine at a high purity and in a high yield. Specifically, the present invention discloses a purification method of (2S,3R,4S)-4-hydroxyisoleucine or a chemically acceptable salt thereof, which includes the following steps (a), (b) and (c): (a) a step of reacting (2S,3R,4S)-4-hydroxyisoleucine or a chemically acceptable salt thereof in a mixture with an aldehyde compound represented by the formula: Q-CHO wherein Q is an aryl group having a carbon number of 6 to 14, an alkyl group having a carbon number of 1 to 10, a cycloalkyl group having a carbon number of 3 to 10 or a 5- to 10-membered heterocyclic group, each of which optionally has substituent(s), or an equivalent form thereof to give a compound represented by the formula (1) wherein Q is as defined above, (b) a step of extracting the compound represented by the formula (1) with an organic solvent, and (c) a step of converting the compound represented by the formula (1) to (2S,3R,4S)-4-hydroxyisoleucine or a chemically acceptable salt thereof.
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Page/Page column 15
(2009/12/07)
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- Synthesis of hydantoin analogues of (2S,3R,4S)-4-hydroxyisoleucine with insulinotropic properties
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The first synthesis of an optically pure (2R,3R,4S)-hydantoin 2, analogue of (2S,3R,4S)-4-hydroxyisoleucine, was achieved in two steps in un-optimized 35% overall yield from previously reported aldehyde synthon 1. (2R,3R,4S)-Hydantoin is stable at acidic pH. This solves the major drawback of (2S,3R,4S)-4-hydroxyisoleucine that easily cyclizes into inactive lactone. Furthermore, (2R,3R,4S)-hydantoin stimulates the insulin secretion by 150% at 25 μM compared with 4-hydroxyisoleucine and insulin secretagogue drug repaglinide. In view of its stability and biological activity, (2R,3R,4S)-hydantoin represents a good candidate for type-2 diabetes management and control.
- Sergent, Didier,Wang, Qian,Sasaki, N. Andre,Ouazzani, Jamal
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experimental part
p. 4332 - 4335
(2009/04/06)
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- METHODS FOR THE SYNTHESIS OF 4-HYDROXYISOLEUCINE, STEREOISOMERS AND ANALOGS THEREOF
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A method for synthesizing 4-hydroxyisoleucine, stereoisomers and analogs thereof having a general formula I is disclosed herein. The method comprises reacting an alkyl isocyanoacetate of formula III with an acetoin or an acetoin analog of formula II to give an unsaturated N-formyl lactone of formula IV, hydrogenating the unsaturated N-formyl lactone to give N-formyl lactone of formula V and hydrolysing said lactone to yield the desired product of formula I.
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Page/Page column 29
(2009/01/24)
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- Method for the synthesis of 4-hydroxyisoleucine and the derivatives thereof
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The invention relates to a method for the synthesis of two isomers, at function OH, alone or in mixtures, of amino acids α or the derivatives thereof, having general formula (B), wherein: linkage C—O of the 4-position carbon (represented by symbol) denotes one or other of isomers III or IV, or mixtures thereof. Moreover, R1 and R2 represent: a hydrogen atom; or either R1 or R2 represents a hydrogen atom and the other substituent is a radical Ra, an acyl group —CORa, such as acetyl, or a functional group —COORa, —SO2Ra, —N (Ra, Rb), Ra and Rb, which are identical or different, representing a C1-C12 linear or branched alkyl radical, optionally substituted, an aryl group with one or more aromatic rings and heterocycles, comprising between 5 and 8C, optionally substituted, or aralkyl, the alkyl substituent and the aryl group being as defined above; or R1 and R2 both represent a substituent as defined above. R3 represents a hydrogen atom or Ra and R4 has the significance of Ra. The invention is characterised in that it comprises: the isomerisation of a compound having formula (I), wherein R1, R2, Ra, R3 and R4 are as defined above, such as to produce a compound having formula (II); and the reduction of the carbonyl function thereof which, depending on the catalytic system employed and the formula (I) compound used, produces one of the isomers having general formula (III) or (IV) or a mixture thereof having formula (B). The invention can be used for the synthesis of (2S, 3R, 4S)-4-hydroxyisoleucine.
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Page/Page column 10
(2008/06/13)
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- Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626
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Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG-1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM α-ketobutyrate, and 100mM L-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl2·4H2O at 28°C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplex AKU 626. Mn2+ and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.
- Ogawa, Jun,Yamanaka, Hiroyuki,Mano, Junichi,Doi, Yuko,Horinouchi, Nobuyuki,Kodera, Tomohiro,Nio, Noriki,Smirnov, Sergey V.,Samsonova, Natalya N.,Kozlov, Yury I.,Shimizu, Sakayu
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p. 1607 - 1615
(2008/02/09)
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- Compounds and compositions for use in the prevention and treatment of obesity and related syndromes
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The invention relates to 4-hydroxyisoleucine, isomers, analogs, lactones, salts, and prodrugs thereof, to processes for their preparation, and to pharmaceutical compositions comprising the same. More particularly, the invention relates to the use of those compounds in the prevention and treatment of obesity and related syndromes.
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Page/Page column 44
(2010/11/24)
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- DIASTEREOISOMERS OF 4-HYDROXYISOLEUCINE AND USES THEREOF
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The invention relates to configurational isomers 4-hydroxyisoleucine, and to lactones, pharmaceutically acceptable salts, and prodrugs thereof, to processes for their preparation, and to pharmaceutical compositions comprising the same. The isomers of the invention exhibit insulinotropic activity and thus may be useful for the prevention and treatment of disorders of carbohydrate or lipid metabolism, including diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes, and Metabolic Syndrome.
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Page/Page column 27; 1/3
(2008/06/13)
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- Method for preparing diastereoisomers of 4-hydroxy isoleucine
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The invention relates to a method for preparing diastereoisomers of 4-hydroxy isoleucine of formula A ????????CO2H- CH(NH2)- CH(CH3)-CH(OH)-CH3?????(A) which comprises the following steps: deprotection of amine of Formula B reduction and subsequent lactonisation of an intermediate amine derivative of formula C whose substituents at positions 2 and 3 are either R, S or S, S ; or S, R or R, R,hydrolysis of the resulting lactone of formula D where in substituents at positions 2, 3 and 4 are S,R,S or S,R,R when using a protected amine S,RS,S,S or S,S,R when using a protected amine S,SR,S,R or R,S,S, when using a protected amine R,SR,R,R or R,R,S when using a protected amine R,R under conditions to obtain the desired isomer of 4-hydroxy isoleucine. Application for preparing the 8 isomeric forms of 4-hydroxy isoleucine.
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Page/Page column 10; 11
(2008/06/13)
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- Chemoenzymatic synthesis of enantiomerically pure (2S,3R,4S)-4- hydroxyisoleucine, an insulinotropic amino acid isolated from fenugreek seeds
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A short six-step synthesis of (2S,3R,4S)-4-hydroxyisoleucine (1a) with total control of stereochemistry is reported, the last step being the enzymatic resolution by hydrolysis of a N-phenylacetyl lactone derivative using the commercially available penicillin acylase G immobilized on Eupergit C (E-PAC). Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.
- Rolland-Fulcrand, Valerie,Rolland, Marc,Roumestant, Marie-Louise,Martinez, Jean
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p. 873 - 877
(2007/10/03)
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- A practical synthesis of (2S,3R,4S)-4-hydroxyisoleucine, a potent insulinotropic α-amino acid from fenugreek
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An efficient eight-step synthesis of optically pure (2S,3R,4S)-4-hydroxyisoleucine (1), a potent insulinotropic α-amino acid found in the seeds of fenugreek (Trigonella foenum-graecum L.), is achieved in 39% overall yield. The method is suitable for large-scale production of the title compound. The key steps involve the biotransformation of ethyl 2-methylace-toacetate to ethyl (2S,3S)-2-methyl-3-hydroxybutanoate (2) with Geotrichum candidum and an asymmetric Strecker synthesis. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.
- Wang, Qian,Ouazzani, Jamal,Andre Sasaki,Potier, Pierre
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p. 834 - 839
(2007/10/03)
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- Formation of 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) from 4-hydroxy-L-isoleucine and 3-amino-4,5-dimethyl-3,4-dihydro-2(5H)-furanone
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The proposed formation of 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) from 4-hydroxy-L-isoleucine (1) and the corresponding lactone 3-amino-4,5-dimethyl-3,4-dihydro-2(5H)-furanone (2) by thermally induced oxidative deamination was corroborated. The formation of sotolone was studied in model systems by reacting 1 or 2 with different carbonyl compounds in a phosphate buffer at pH 5 at 100 °C for 1 h. The amount of sotolone was quantified by stable isotope dilution assays using 13C2-labeled sotolone as internal standard and GC-MS operating in the selected ion monitoring mode. In general, a-ketoaldehydes were found to be more reactive than a-diketones. Methylglyoxal gave rise to about 64 μg sotolone per mg 1 (7.4 mol %) compared to less than 1μg (0.1 mol %) when reacted with 2,3-pentanedione. Using 2 as the starting material, the yields were increased to 274 μg (35.9 mol %) and 5.4 μg (0.7 mol %), respectively. The optimum pH of the reaction with HIL was 5, representing the best compromise between the lactonization step and the amino-carbonyl reaction. Significant amounts of sotolone were generated only at temperatures higher than 70 °C. The yield increased over a period of 10 h to about 210 μg/mg 1 (23.8 mol %). The Strecker degradation of 1, resulting in 3-hydroxy-2-methylbutanal, was a competitive reaction to the formation of sotolone.
- Blank, Imre,Lin, Jianming,Fumeaux, René,Welti, Dieter H.,Fay, Laurent B.
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p. 1851 - 1856
(2007/10/03)
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