CN1926241A - Enzymatic preparation of an enantiomerically enriched beta-2-amino acids - Google Patents

Enzymatic preparation of an enantiomerically enriched beta-2-amino acids Download PDF

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CN1926241A
CN1926241A CNA2005800063122A CN200580006312A CN1926241A CN 1926241 A CN1926241 A CN 1926241A CN A2005800063122 A CNA2005800063122 A CN A2005800063122A CN 200580006312 A CN200580006312 A CN 200580006312A CN 1926241 A CN1926241 A CN 1926241A
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伯纳德斯·卡普特因
奎林纳斯·伯纳德斯·布罗克斯特曼
乔纳斯·格拉尔杜斯·西奥多瑞斯·基尔科斯
塞利·密尔森特
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture

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Abstract

The invention relates to a process for the preparation of a enantiomerically enriched beta<2>-amino acid comprising the steps of reacting a stereoselective hydrolytic enzyme with a mixture of enantiomers of a beta< 2>-amino acid ester and collecting the resulting enantiomerically enriched beta< 2>-amino acid. The invention also relates to a process for the preparation of an enantiomerically enriched beta< 2>-amino acid ester comprising the steps of reacting a stereoselective hydrolytic, enzyme with a mixture of enantiomers of a beta< 2>-amino acid ester and collecting remaining enantiomerically enriched beta< 2>-amino acid ester.

Description

The amino acid whose method of β-2-for preparing the enantiomer enrichment with enzyme
The present invention relates to prepare the β of enantiomer enrichment with enzyme 2The method of-amino acid (ester).
The β for preparing the enantiomer enrichment with enzyme 2The method of-amino acid (ester) is not known.At Rossi D.et al. (1977) vol.33, no.12, pp 1557-1559, open among the XP 009030088 BaselISSN 0014-4754: amino acid whose N-phenylacetyl derivative can be hydrolyzed, and produces to have optically active beta-amino acids.
The method of the unprotected beta-amino acids of N-of preparation enantiomer enrichment is disclosed among the EP-A-1 367 129; described method realizes by with lytic enzyme the enantiomeric mixture of the unprotected beta-amino acid esters of N-being carried out enzymic hydrolysis; wherein; hydrolysis is carried out in the two-phase system of water and organic solvent, and described organic solvent can form two-phase system with water under given reaction conditions.
Salamonczyk; G.M.et al.J.Org.Chem.; (1996); vol.61; 6893-6900 discloses a kind of method of involved enzyme a little; it relates to a kind of different compound, wherein, and by reacting the 3-amino-2-methyl-propionic acid for preparing the enantiomer enrichment with unprotected 3-amino-2-methyl-methyl propionate of racemic N-and fixed Candida Antarctica lipase.But this method relates to the β of N-protected 2The β of-amino acid and N-protected 2-amino acid ester.In addition, the shortcoming of this method is that it must use the rapid kinetic resolution of multistep, so that obtaining that enantiomer is excessive can received 3-amino-2-methyl-propionic acid.
The purpose of this invention is to provide a kind of simple enzyme method, be used to prepare the β of enantiomer enrichment 2-amino acid (ester), that is, and need not the rapid kinetic resolution of multistep and/or to β 2-amino acid (ester) carries out the method for N-protected.
This purpose can be reached according to following method according to the present invention, and described method comprises: with the β with stereoselective hydrolytic action enzyme and structural formula 2 representatives 2The enantiomer of-amino acid ester is mixed
The compound reaction, wherein, R 1Represent optional substituted alkyl, and wherein, R 2, R 3And R 4Each represents H, optional substituted (heterocycle) aryl, optional substituted alkyl, OR independently 5, CO 2R 6, C (O) R 7, SR 8, NR 9R 10, OC (O) R 11, wherein, R 5, R 6, R 7, R 8, R 9, R 10And R 11Each represents H, optional substituted alkyl or optional substituted (heterocycle) aryl independently, and, R wherein 2And R 3, R 2And R 4Or R 3And R 4Can form ring with the carbon atom that they connected; And the β that collects the structural formula 1 of the enantiomer enrichment that obtains 2-amino acid
Figure A20058000631200071
Wherein, R 2, R 3And R 4As hereinbefore defined, perhaps collect the β of the structural formula 2 of remaining enantiomer enrichment 2-amino acid ester.
Surprisingly, method of the present invention is simple method, and it does not need the rapid kinetic resolution of multistep and/or to β 2-amino acid (ester) carries out N-protected, and described method can produce that have can the excessive β of received enantiomer 2-amino acid (ester).In addition, method of the present invention is convergent-divergent well, can be used for plant-scale production.
β 2-amino acid (ester) refers to 2 (alpha-positions) upward substituted beta-amino acids (ester).To β 2Sewald is also seen in the definition of-amino acid (ester), Angew.Chem.Int.Ed. (2003), 42,5794-5795.
The β of structural formula 2 2-amino acid (ester) has chiral centre at least on alpha-position; But, the β of structural formula 2 2-amino acid (ester) can also have other chiral centre, for example, and R 2, R 3And R 4The carbon atom that is connected can be a chirality; Work as R 2, R 3And R 4In the time of different, this carbon atom is exactly a chirality.
" mixture of enantiomer " refer to, (R) and (S)-and the random mixture of enantiomer.Typically, the β of utilization structure formula 2 2The racemic mixture of-amino acid ester (that is, and (R): (S)=1: 1), still, certainly, for further enrichment enantiomer, method of the present invention can also carried out on the enantiomeric mixture through the enantiomer enrichment.
Preferably, R 1Represent the alkyl of optional substituted 1-20 C atom, more preferably, (the comprising substituent C atom) of 1-12 carbon atom.For example, R 1Can represent C 1-C 4Alkyl, for example, methyl, ethyl, normal-butyl; Substituted methyl, for example phenmethyl; Perhaps substituted ethyl, for example, three chloroethyls, methoxy ethyl or trifluoroethyl.
Preferably, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R 9, R 10And R 11Each represents the alkyl of H, an optional substituted 1-12 C-atom independently, more preferably, (the comprising substituent C atom) of 1-8 C-atom, perhaps, represents (heterocycle) aryl (comprising substituent C atom) of 2-10 C-atom.Preferably, in the heterocyclic aryl, heteroatoms is selected from the group of N, O and S.
R 2And R 3, R 2And R 4Or R 3And R 4Can form the ring that is preferably 3-6 C-atom.The ring that they form can for example be (mixing) cycloalkyl or (heterocycle) aryl, and wherein each ring can be optional substituted.
Substituent example comprises alkyl, (heterocycle) aryl, alkylsulfonyl, carbalkoxy, aminocarboxyl, nitrile, hydroxyl, alkoxyl group, aryloxy, alkylthio, sulfydryl, amino and fluorine.
Within the scope of the invention, term " the enantiomer enrichment " refers to " (R) of compound-or (S)-enantiomer has enantiomer excessive (e.e.) ".Preferably, enantiomer is excessive to be＞80%, more preferably, and＞85%, further more preferably,＞90%, particularly＞95%, more especially＞97%, further more especially＞98%, the most particularly＞99%.
Collection comprises: for example separate by traditional method, for example, ultrafiltration, concentrate, column chromatography, extraction or crystallization, and with the product (β of enantiomer enrichment of acquisition 2The β of-amino acid or enantiomer enrichment 2-amino acid ester) further reacts.
" hydrolytic action enzyme " (hydrolytic enzyme) also is called as " lytic enzyme " (hydrolase).Lytic enzyme is the enzyme of energy catalytic hydrolysis reaction.Therefore, lytic enzyme interleaves into water molecules at this key by the covalent linkage of cutting compound, destroys compound (that is substrate) and plays a role.Common hydrolase family comprises those enzymes that ester bond, carbon-nitrogen bond, peptide bond and acid anhydrides etc. are played a role.
" hydrolytic action enzyme " refers to have the enzyme of hydrolysising carboxy acid ester's key with the ability of formation corresponding carboxylic acid group.
" stereoselectivity " of hydrolytic action enzyme refers to, the preferential catalysis β of hydrolytic action enzyme 2Wherein a kind of hydrolysis of the enantiomer of-amino acid ester.The stereoselectivity of enzyme can be represented as E-ratio, and this is the ratio of two kinds of enantiomer specificity constant Vmax/Km, as C-S.Chen, and Y Fujimoto, G.Girdaukas, C.J.Sih., J.Am.Chem.Soc.1982,104,7294-7299 is described.Preferably, E-ratio＞5 that the hydrolytic action enzyme has, more preferably, E-ratio＞10, further more preferably, E-ratio＞50, most preferably, E-ratio＞100.
Be suitable for of the present inventionly having stereoselective hydrolytic action enzyme and can for example in a kind of common family of hydrolytic action enzyme, finding; for example in the group of esterase, lipase, proteolytic enzyme, peptase or acyltransferase; preferably, in the group of esterase or lipase.The hydrolytic action enzyme can obtain from eucaryon and prokaryotic cell prokaryocyte, and it includes but not limited to: from those of following Mammals source: pork liver, pig pancreas (for example commercial obtainable II type porcine pancreatic lipase (L-3126, Sigma)); Ren sus domestica and ox pancreas; From plant origin, those of wheatgerm for example; From those of following mould Pseudomonas: Absidia, Aspergillus, Fusarium, Gibberella, Mucor, Neurospora, Trichoderma, Rhizopus, Rhizomucor (for example Rhizomucor miehei), Thermomyces (for example Thermomyces lanugenousus); Those that belong to from following bacterium: Achromobacter, Alcaligenes, Bacillus (for example Bacillus licheniformis), Brevibacterium, Corynebacterium, Providencia, Pseudomonas (for example Pseudomonas fluorescens, Pseudomonas cepase or Pseucomonasalcaligenes), Serratia, Rhodococcus; From those of following yeast belong: Candida (for example Candida rugose or Candida antarctica) and from those of actinomyces Nocardia.
Preferably, have stereoselective hydrolytic action enzyme and can or (for example be classified as peptase in the group of the enzyme that is classified as carboxylic ester hydrolase (EC 3.1.1), EC 3.4.1, EC 3.4.11, EC3.4.21 (more preferably, EC 3.4.21.62), EC 3.4.22 or EC 3.4.23) the group of enzyme in find.
Having stereoselective hydrolytic action enzyme can find in the group of the hydrolytic action enzyme that can obtain by commercial sources.The example of the hydrolytic action enzyme that can obtain by commercial sources is: the enzyme that is provided by Fluka: Candida cylindracea lipase, pig pancreas lipase, Pseudomonasfluorescens lipase, Aspergillus oryzae lipase, Rhizopus niveus lipase, Rhizomucor miehei lipase, Candida Antarctica lipase, Mucor javanicus lipase, Rhizopus arrhizus lipase, Penicillium roqueforti lipase, Candidalipolytica lipase, Pseudomonas sp. lipoprotein lipase, Type B, the Pseudomonascepacia lipoprotein lipase, Chromobacterium viscosum lipoprotein lipase, the Bacillusstearothermophilus esterase, Bacillus thermoglucosidasius esterase, Mucor miehei esterase, Pig Liver Esterase; The enzyme that provides by Altus: Candida rugosa lipase, Mucormiehei lipase, Candida Antarctica B lipase, Candida Antarctica A lipase, Chiro-CLEC-CR, Chiro-CLEC-CR (slurry), Pig Liver Esterase, penioillin acylase, the Carlsberg subtilisin, Chiro-CLEC-BL (slurry), Chiro-CLEC-PC (slurry), Chiro-CLEC-EC (slurry), Aspergillus oryzae proteolytic enzyme, PeptiCLEC-TR (slurry); The enzyme that provides by Recombinant Biocatalysis: ESL-001-07, ESL001-01, ESL-001-01, the ESL-001-02 of band stablizer, ESL-001-03, ESL-001-05; The enzyme that provides by Boehringer-Mannheim: Chirazyme L4 (Pseudomonas sp.), Chirazyme L5 (Candida antarctica segment A), Chirazyme L1 (Burkholderia), Chirazyme L6 (pig pancreas), ChirazymeL7, Chirazyme L8; The enzyme that provides by DSM (Gist-Brocades in the past): Naproxen esterase, Lipomax, Genzyme, Lipoprotein lipase; The enzyme that provides by Novo: Novozyme868, Novozyme435, fixed Candida Antarctica lipase, Nagase enzyme, lipase A-10FG (Rhizopus javanicus); The enzyme that provides by Amano: AmanoAYS, Amano PS, Amano PSD, Amano AKD11, Amano AKD111.
Most preferred have stereoselective hydrolytic action enzyme and be: subtilisin, alcalase2.5L, Bacillus licheniformis proteolytic enzyme, Candida Antarctica lipase A, II type pig pancreas lipase.
Having stereoselective hydrolytic action enzyme can use in any form.For example, the hydrolytic action enzyme can use with following form, for example, dispersion system, solution or the form that is fixed-as rough enzyme, as the enzyme that can obtain by commercial sources, as enzyme from being further purified out by the preparation that commercial sources obtains, as the enzyme of the combination of passing through known purification methods from its source acquisition, whole (alternatively, infiltration and/or fixed) in the cell, described cell has had the stereoselective hydrolytic action enzymic activity that has that needs natively or by genetic modification, perhaps in having this type of active product of cell lysis.
Those of ordinary skills should be understood that, in the method for the invention, that can also use naturally occurring (wild-type) enzyme has an active mutant of hydrolytic action.The mutant of wild-type enzyme can obtain by following method, for example, by modifying with the DNA of induced-mutation technique well known by persons skilled in the art (random mutagenesis, site-directed mutagenesis, orthogenesis (directed evolution), gene swapping (geneshuffling) etc.) to the encoding wild type enzyme, thereby make dna encoding and wild-type enzyme have the different enzyme of at least one amino acid, and by the expression of adorned DNA in suitable (host) cell thus influenced.Aspect (solid) selectivity and/or activity and/or stability and/or solvent resistance and/or pH situation and/or temperature conditions, the mutant with stereoselective hydrolysising protease can have improved character.
For example, can be by at existence with stereoselective hydrolytic action enzymic activity, the some kinds of enzymes or the host cell of expressing enzyme coding gene are screened, select and be used for the stereoselective hydrolytic action enzyme of having of method of the present invention.Usually.Those skilled in the art will know that how to screen and have the active enzyme of wanting.Usually, for selecting suitable enzyme, substrate (for example, the compound of structural formula 2) and the contacted condition of enzyme are chosen to be: make enzyme, substrate and reaction product on the one hand stability and reach good compromise between the speed of reaction (it is increasing under higher temperature usually) on the other hand.Screening to enzyme can be carried out with any scale.With regard to putting into practice the aspect,, use the reaction volume between the 0.15ml to 10ml if screen a large amount of enzymes.
For example, can be by the operational analysis method, for example TLC, HPLC or GC, hydrolytic process to the compound of structural formula 2 in the aqueous solution under the situation that has enzyme is monitored, and filters out the compound that is suitable for structural formula 2 and carries out the stereoselective hydrolytic action enzyme of having of stereo selective hydrolysis (" hydrolysis screening ").
The example of the aqueous solution is water and the water with cosolvent, for example, with the miscible organic solvent of water or with the immiscible solvent of water.The representative examples of organic miscible with water comprises methyl alcohol, ethanol, acetone, dioxane, acetonitrile, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) and dimethyl formamide.Comprise methyl tertiary butyl ether, hexone, toluene, hexane, dimethylbenzene and octane-iso with the immiscible representative examples of organic of water.The amount of cosolvent is unimportant in principle, and it is selected usually to be between 5 to 25%v/v.Substrate is under the situation of liquid, and it can former state be present in the water.Substrate is under the solid situation, also exists cosolvent to be good.
Unimportant on enzyme in " hydrolysis screening "/substrate proportionality principle, it is selected usually to be between 1/20 to 2/1.The amount of used substrate is also inessential in principle, and it can be for example between 5mM to 1.5M.
The pH of " hydrolysis screening " is unimportant in principle, and it can for example selectedly be between 5 to 10, preferably, between 6 to 8, can use aqueous buffer solution, with for example buffer concentration between the 10mM to 500mM, it is remained constant.Perhaps, can will screen the pH that reacts with automatic pH stable system remains constant.The temperature of " hydrolysis screening " is unimportant in principle, and it can selectedly be between 20 to 40 ℃.Perhaps, if used the enzyme that at high temperature to operate, can select higher temperature for use.
Selection to the hydrolytic action enzyme is depended in the selection of the reaction conditions of method of the present invention.Usually, the temperature of present method is selected to be between 0 to 90 ℃, especially, and between 10 to 40 ℃; Usually, the pH of present method is selected to be between 4 to 10, and, usually water, aqueous solvent (for example water/trimethyl carbinol or water/dioxane) or water and with the two-phase system of the immiscible solvent of water (for example, toluene, hexane, heptane, methyl tertiary butyl ether, hexone) as solvent.
Can isolate the method for acid from water or aqueous layer with known being used for, isolate the β of the structural formula 1 of the enantiomer enrichment that obtains by method of the present invention 2-amino acid.Can be with known method itself, for example by extract with organic solvent, crystallization or (ion-exchange) chromatogram, to the β of the structural formula 2 of the enantiomer enrichment of being left that obtains by method of the present invention 2-amino acid ester separates.
Alternatively, to collect the β of structural formula 2 of remaining enantiomer enrichment 2-amino acid ester carries out further hydrolysis, forms the β of corresponding enantiomer enrichment 2-amino acid.It is well known by persons skilled in the art being used for esterolytic method, for example, and can be by using mineral acid (as HCl or H 2SO 4) aqueous solution carries out acid hydrolysis, by carrying out saponification, or come ester is hydrolyzed by enzymic hydrolysis with aqueous sodium hydroxide solution.
Structural formula 2 (wherein, R 1, R 2, R 3And R 4β as hereinbefore defined) 2The mixture of the enantiomer of-amino acid ester can prepare by methods known in the art.For example, the β of structural formula 2 2-
Figure A20058000631200121
Amino acid ester can alternatively, under the situation that appropriate catalyst exists, be gone back original preparation to the corresponding nitrile of structural formula 3 by using appropriate reductant
Wherein, R 1, R 2, R 3And R 4As hereinbefore defined.
R wherein 1, R 2And R 3As hereinbefore defined and R 4Represent the nitrile of the structural formula 3 of H can be by use appropriate reductant, alternatively, under the situation that appropriate catalyst exists, two keys of the corresponding nitrile of structural formula 4 were gone back originally prepare
Figure A20058000631200131
Wherein, R 1, R 2And R 3As hereinbefore defined.
In one embodiment of the invention, can be by use appropriate reductant, alternatively, under the situation that appropriate catalyst exists, the corresponding nitrile of structural formula 4 is carried out a cooking-pot type reduce (that is: the nitrile that does not separate intermediate product structural formula 3), prepare wherein R 1, R 2And R 3As hereinbefore defined and R 4Represent the β of the structural formula 2 of H 2The enantiomer of-amino acid ester mixture.In this reduction, the two keys and the nitrile group of the compound of structural formula 3 all are reduced.
Appropriate reductant (and catalyst combination) is well known by persons skilled in the art, and it comprises, for example, has the H of Pd/ C catalyst 2, have a H of Raney nickel 2, have a H of Pt/ carbon 2, have a H of Ru/ carbon 2Deng.For example, R wherein 1, R 2, R 3And R 4The nitrile of structural formula 3 as hereinbefore defined can be with known method itself, by carrying out nucleophilic 1 with suitable nucleophilic reagent, 4-addition introducing R 4, prepare from the corresponding nitrile of structural formula 4
Figure A20058000631200132
Wherein, R 1, R 2And R 3As hereinbefore defined.Those skilled in the art will know which kind of nucleophilic reagent can be used to introduce the R that wants 4The example of suitable nucleophilic reagent comprises, lithium alkylide, for example lithium methide; Grignard reagent; Alkoxide, for example, the salt of benzylated polyol, for example sylvite; Thio-alcohol, for example thioacetic acid or benzyl sulfhydrate, the two can use with the form of salt; Nitrogen nucleophile, for example amine, for example azanol; Trinitride or imines, for example phthalyl imonium potassium (potassiumphtalimide) or dicarboximide potassium (potassium bisformimide).
Aldehydes or ketones that can also be by structural formula 6 (R wherein 2And R 3As hereinbefore defined)
Figure A20058000631200141
With the nitrile of structural formula 7 (R wherein 1As hereinbefore defined)
Figure A20058000631200142
Under the situation of suitable alkali or dehydrated reagent existence, carry out condensation, prepare wherein R 1, R 2And R 3The nitrile of structural formula 4 as hereinbefore defined.Suitable alkali and dehydrated reagent are well known in the art.Suitable alkali comprises, for example, and piperidines, triethylamine, OH -, the diazabicyclo hendecene (diazabicycloundecene, DBU) and diazabicyclo-nonene (diazabicyclononene, DBN).The example of dehydrated reagent is an ammonium acetate.
Structural formula 2 (wherein, R 1, R 2, R 3And R 4β as hereinbefore defined) 2The mixture of the enantiomer of-amino acid ester can also be by using NH 3Or NH 3The acrylate reactions that the 2-of analogue and structural formula 5 replaces makes
Figure A20058000631200143
Wherein, R 1, R 2, R 3And R 4As hereinbefore defined.
NH 3Analogue refers to, will provide the compound (alternatively, after removing blocking group) of the unhindered amina in the compound of structural formula 2.NH 3The example of analogue comprises benzylamine, dibenzylamine, trinitride, azanol, phthalyl imonium potassium and dicarboximide potassium.
The β of enantiomer enrichment 2The β of-amino acid and enantiomer enrichment 2-amino acid ester can be used as the basic module (building block) of preparation medicine and agrochemistry product.For example, β 2-homo-Threonine can be used to prepare beta-lactam antibiotics active constituents of medicine (EP290385, EP742223, EP371875, EP774463, C Fuganti et al.Org.Bio-Org.Chem (1993), 2247-2249).β 2-amino acid also can for example be used to prepare the active constituents of medicine of thrombocyte coagulation inhibitor, and (US 5,344,957, WO93/07867) or the active constituents of medicine of preparation enkephalinase inhibitor (substituting morphine, treatment diarrhoea, hypertension, cardiac insufficiency and be used as pain killer; EP 634396).In addition, β 2-amino acid/ester also can be used for medicine (Drugs Fut. (2000), 25 (12), active constituents of medicine p1308) that preparation can be used as microbiotic (WO02/41886) or can be used as anti-constipation.
Therefore the present invention also relates to a kind of method, and wherein, available known method own is with the β of enantiomer enrichment prepared in accordance with the present invention 2-amino acid (ester) further is converted into active constituents of medicine.
In addition, the invention still further relates to a kind of method, wherein, can be configured to pharmaceutical composition, wherein comprise described active constituents of medicine and vehicle according to the active constituents of medicine that above obtains.
Be suitable for of the present inventionly having stereoselective hydrolytic action enzyme and also can be used for: from the β of the structural formula (8) of enantiomer enrichment 2-amino acid ester (wherein, R 12Representative and R 1Different optional substituted alkyl)
Figure A20058000631200151
And from R 1Pure R as hereinbefore defined 1OH prepares the β of the structural formula (2) of enantiomer enrichment 2-amino acid ester, wherein, R 1, R 2, R 3And R 4Decide as mentioned.
Preferably, R 1 'Represent the alkyl of optional substituted 1-20 C atom, more preferably, (the comprising substituent C atom) of 1-12 carbon atom.For example, R 1 'Can represent C 1-C 4Alkyl, for example, methyl, ethyl, normal-butyl; Substituted methyl, for example phenmethyl; Perhaps substituted ethyl, for example, three chloroethyls, methoxy ethyl or trifluoroethyl.
Preferably, pure R 1The molar weight of OH is with respect to ratio＞5 of the amount of the amino acid ester of structural formula 8, more preferably, and＞10.
To set forth the present invention by embodiment below, but following embodiment acts on without limits to the present invention.
Embodiment
Embodiment 1 preparation (±)-3-amino-2-phenmethyl-propionic acid methyl ester spirit of salt
Methyl cyanoacetate in dioxane (150mL) (50g, 0.5mol) and phenyl aldehyde (53.5g, add in mixture 0.5mol) acetate (2.9mL, 0.05mol), piperidines (2.0mL, 0.02mol) and 5wt% carbon on palladium (2g).Under hydrogen (1-2bar), mixture was stirred 16 hours in room temperature.Go up at diatomite (celite) then reaction mixture is filtered, filtrate is evaporated to dried.Resistates is dissolved in the ethyl acetate (200mL) again water and salt washing.Dry organic layer on sodium sulfate filters, and is evaporated to driedly, obtains xanchromatic oil (96g, quantitative yield).(50g, 0.25mol) solution in methyl alcohol (300mL) adds on the 5wt% carbon palladium (1g) and 37wt%HCl (33mL 0.4mol), is stirred reaction mixture under the hydrogen of 10bar to this oil.After 17 hours the stirring, filtering solution on diatomite is evaporated to it dried, and resistates is deposited in the ether, obtains (±)-3-amino-the 2-phenmethyl-white powder of propionic acid methyl ester spirit of salt, and productive rate is 61%.
1H-NMR(400MHz，DMSO)δ8.49(s，3H)，7.19-7.30(m，5H)，3.58(s，3H)，3.11-3.21(m，1H)，2.81-3.05(m，4H)。
Embodiment 2 preparation (±)-2-(amino methyl)-4-methyl-valeric acid methyl ester spirit of salt
Methyl cyanoacetate in dioxane (150mL) (50g, 0.5mol) and isobutyric aldehyde (36g, add in mixture 0.5mol) acetate (2.9mL, 0.05mol), piperidines (2.0mL, 0.02mol) and 5wt% carbon on palladium (2g).Under hydrogen (1-2bar), mixture was stirred 18 hours in room temperature.On diatomite, reaction mixture is filtered then, filtrate is evaporated to dried.Resistates is dissolved in the ethyl acetate (200mL) again water and salt washing.Dry organic layer on sodium sulfate filters, and is evaporated to driedly, obtains xanchromatic oil (78g, quantitative yield).(40g, 0.26mol) solution in methyl alcohol (300mL) adds on the 5wt% carbon palladium (1g) and 37wt%HCl (32mL 0.38mol), is stirred reaction mixture under the hydrogen of 10bar to this oil.After 17 hours the stirring, filtering solution on diatomite is evaporated to it dried, and resistates is deposited in the ether, obtains (±)-2-the white powder of (amino methyl)-4-methyl-valeric acid methyl ester spirit of salt, and productive rate is 57%.
1H-NMR(400MHz，DMSO)δ8.24(s，3H)，3.67(s，3H)，2.81-3.09(m，3H)，1.25-1.58(m，3H)，0.98(d，6H)。
Embodiment 3 preparation (±)-2-(amino methyl)-3-methyl-butyric acid methyl ester spirit of salt
Methyl cyanoacetate in ethanol (20mL) (10g, 0.1mol) and acetone (6.38g, add in mixture 0.11mol) acetate (1.2g, 0.02mol), ammonium acetate (0.78g, 0.01mol) and 5wt% carbon on palladium (0.4g).Under hydrogen (1-2bar), mixture was stirred 18 hours in room temperature.After filtering on diatomite, evaporation leaches thing, resistates is dissolved in the ethyl acetate water and salt washing.Dry organic layer on sodium sulfate concentrates under vacuum then, obtains xanchromatic oil (30g).Palladium (1g) and 37wt%HCl on the solution adding 5wt% carbon of this xanchromatic oil in methyl alcohol (200mL) (10.5mL, 0.15mol).After stirring 15 hours under the hydrogen of 10bar, filter reaction mixture on diatomite, concentrated filtrate under vacuum.Carry out post precipitation in acetone, obtained 2-(amino methyl)-3-methyl-butyric acid methyl ester spirit of salt, productive rate is 43%.
1H-NMR(400MHz，DMSO)δ8.18(s，3H)，3.67(s，3H)，3.00(m，2H)，2.65(m，1H)，1.97(m，1H)，0.90(m，6H)。
Embodiment 4 preparation (±)-2-(amino methyl)-butyric acid methyl ester spirit of salt
Methyl cyanoacetate in acetate (16mL) (10g, 0.1mol), acetaldehyde (6.76mL, 0.12mol) and slowly add piperidines (0.34g, acetic acid solution 0.04mol) (4mL) on the 5wt% carbon in the mixture of palladium (0.4g).Under hydrogen (1-2bar), mixture was stirred 17 hours, on diatomite, filter then, concentrate and obtain xanchromatic oil (12g) in room temperature.In this oily methyl alcohol (150mL) solution, add palladium (1.2g) and 37wt%HCl on the 5wt% carbon (12mL, 0.15mol).In room temperature reaction mixture was stirred 15 hours under hydrogen (10bar), filter, concentrate under vacuum, carry out post precipitation in ether, obtained 2-(amino methyl)-butyric acid methyl ester spirit of salt, productive rate is 47%.
1H-NMR(400MHz，DMSO)δ8.24(s，3H)，3.67(s，3H)，2.81-3.09(m，2H)，2.58-2.71(m，1H)，1.61(q，2H)，0.98(t，3H)。
Embodiment 5 preparation (±)-3-amino-2-methyl-propionic acid methyl ester acetates
In methyl alcohol (100mL) to methyl methacrylate (10g, 0.1mol) and benzylamine (10.7g, solution 0.1mol) carries out 4 days backflow, evaporating solvent obtains 3-benzyl ammonia-2-methyl-propionic acid methyl ester, productive rate 100% then.To this ester (4g, add in methyl alcohol 19.3mmol) (70mL) solution palladium (0.4g) and acetate on the 5wt% carbon (2.2mL, 38mmol).Solution carried out 5 hours stirring under the hydrogen of 1-2bar, filter on diatomite then, concentrate under the vacuum, obtain 3-amino-2-methyl-propionic acid methyl ester acetate, productive rate is 85%.
1H-NMR(400MHz，DMSO)δ8.72(s，3H)，3.64(s，3H)，3.09(m，1H)，2.81(m，2H)，2.31(s，3H)，1.15(d，3H)。
Embodiment 6 preparation (S)-3-amino-2-benzyl-propionic acid methyl ester and preparation (R)-3-amino-2-benzyl-
Propionic acid
(10g 44mmol) adds Alcalase Type B 2.5 enzymes (1.25g) to 3-amino-2-benzyl-propionic acid methyl ester spirit of salt in 100mL potassium phosphate buffer (25mM, pH 7.0) in the solution.At room temperature stir the mixture that obtains, during reaction pH is adjusted to pH 7.0 with the 2N NaOH aqueous solution.Stopped reaction during 50% transformation efficiency after 2 hours.Filter reaction mixture is used solid K 2CO 3PH is transferred to pH 9, remove remaining unreacted ester by extract (3 times) with methylene dichloride.Dry organic layer on sodium sulfate filters, and concentrates under vacuum, obtains (the S)-3-amino-2-benzyl-propionic acid methyl ester (ee＞99%) of corresponding isomer enrichment.With phosphoric acid water layer is carried out slow acidifying, up to precipitating by (the R)-3-amino-2-benzyl-propionic acid (ee＞99%) that filters the corresponding isomer enrichment that obtains.
Embodiment 7-14 prepares corresponding isomer richness with the different stereoselective hydrolytic action enzymes that has
The β of collection 2-amino acid
In the solution in the 10mL sodium phosphate buffer (25mM, pH 7.0), add the 200mg enzyme to substrate (200mg), at room temperature stir the mixture that obtains.Reaction in about 50% transformation efficiency, is measured the excessive situation of enantiomer of acid by chirality HPLC monitoring.Calculate corresponding E-ratio, experimental result is listed in the table below in 1.
Table 1. prepares the β of corresponding isomer enrichment with the different stereoselective hydrolytic action enzymes that has 2-amino acid

Claims (13)

1. the β for preparing the structural formula 1 of enantiomer enrichment 2-amino acid whose method
Figure A2005800063120002C1
Wherein, R 2, R 3And R 4Each represents H, optional substituted (heterocycle) aryl, optional substituted alkyl, OR independently 5, CO 2R 6, C (O) R 7, SR 8, NR 9R 10, OC (O) R 11, wherein, R 5, R 6, R 7, R 8, R 9, R1 0And R 11Each represents H, optional substituted alkyl or optional substituted (heterocycle) aryl independently, and, R wherein 2And R 3, R 2And R 4Or R 3And R 4Can form ring with the carbon atom that they connected, described method comprises the steps: to have the β of stereoselective hydrolytic action enzyme and structural formula 2 2The mixture reaction of the enantiomer of-amino acid ester,
Wherein, R 1Represent optional substituted alkyl, R 2, R 3And R 4As hereinbefore defined; And the β that collects the structural formula 1 of the enantiomer enrichment that obtains 2-amino acid.
2. the β for preparing the structural formula 2 of enantiomer enrichment 2The method of-amino acid ester
Wherein, R 1Represent optional substituted alkyl, and, wherein, R 2, R 3And R 4Each represents H, optional substituted (heterocycle) aryl, optional substituted alkyl, OR independently 5, CO 2R 6, C (O) R 7, SR 8, NR 9R 10, OC (O) R 11, wherein, R 5, R 6, R 7, R 8, R 9, R 10And R 11Each represents H, optional substituted alkyl or optional substituted (heterocycle) aryl independently, and, R wherein 2And R 3, R 2And R 4Or R 3And R 4Can form ring with the carbon atom that they connected, described method comprises the steps: to have the β of stereoselective hydrolytic action enzyme and structural formula 2 2The mixture reaction of the enantiomer of-amino acid ester, wherein, R 1, R 2, R 3And R 4As hereinbefore defined; And the β that collects the structural formula 2 of remaining enantiomer enrichment 2-amino acid ester.
3. method as claimed in claim 1 or 2, wherein, described to have stereoselective hydrolytic action enzyme be enzyme from enzyme classification group EC 3.1.1,3.4.21,3.4.22 or 3.4.23.
4. as any described method among the claim 1-3, wherein, described have E-ratio＞5 that stereoselective hydrolytic action enzyme has.
5. as any described method among the claim 2-4, wherein, with known method β itself to the remaining enantiomer enrichment of collecting 2-amino acid ester carries out further hydrolysis.
6. as any described method among the claim 1-5, wherein, the β of structural formula 2 2-amino acid ester is by using appropriate reductant, alternatively, under the situation that appropriate catalyst exists, the corresponding nitrile of structural formula 3 go back prepared originally
Figure A2005800063120003C1
Wherein, R 1, R 2, R 3And R 4As hereinbefore defined.
7. method as claimed in claim 6, wherein, R wherein 1, R 2And R 3As hereinbefore defined, R 4The nitrile of representing the structural formula 3 of H is by using appropriate reductant, alternatively, under the situation that appropriate catalyst exists, the corresponding nitrile of structural formula 4 go back prepared originally
Figure A2005800063120004C1
Wherein, R 1, R 2And R 3As hereinbefore defined.
8. as any described method among the claim 1-5, wherein, R 4Represent H and R 1, R 2And R 3The β of structural formula 2 as hereinbefore defined 2-amino acid ester is by using appropriate reductant, alternatively, and under the situation that appropriate catalyst exists, to R 1, R 2And R 3The corresponding nitrile of structural formula 4 is as hereinbefore defined gone back original preparation
9. method as claimed in claim 6, wherein, R 1, R 2, R 3And R 4As the nitrile of the structural formula 3 of definition in the claim 6, be by carrying out nucleophilic 1 with suitable nucleophilic reagent, 4-addition introducing R 4, from R 1, R 2And R 3The corresponding nitrile of structural formula 4 as hereinbefore defined prepares.
10. as any described method among the claim 7-9, wherein, R 1, R 2And R 3The nitrile of structural formula 4 as hereinbefore defined is the aldehydes or ketones by structural formula 6
R wherein 2And R 3As hereinbefore defined, with the nitrile of structural formula 7
Figure A2005800063120004C3
Wherein, R 1As hereinbefore defined,
Under the situation of suitable alkali or dehydrated reagent existence, carry out condensation, prepare.
11. as claim 1Any described method in-5, wherein, R 1, R 2, R 3And R 4β as any defined structural formula 2 among the claim 1-5 2-amino acid ester is by using NH 3Or NH 3The acrylate reactions that the 2-of analogue and structural formula 5 replaces makes
Figure A2005800063120005C1
Wherein, R 1, R 2, R 3And R 4As hereinbefore defined.
12. as any described method among the claim 1-11, wherein, will be according to the β of the enantiomer enrichment of any described method preparation among the claim 1-11 2-amino acid (ester) further is converted into active constituents of medicine.
13. method as claimed in claim 12 wherein becomes pharmaceutical composition with described active constituents of medicine preparation, described composition comprises described active constituents of medicine and vehicle.
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