WO2007039079A1 - Process for the synthesis of beta amino acids - Google Patents
Process for the synthesis of beta amino acids Download PDFInfo
- Publication number
- WO2007039079A1 WO2007039079A1 PCT/EP2006/009050 EP2006009050W WO2007039079A1 WO 2007039079 A1 WO2007039079 A1 WO 2007039079A1 EP 2006009050 W EP2006009050 W EP 2006009050W WO 2007039079 A1 WO2007039079 A1 WO 2007039079A1
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- WO
- WIPO (PCT)
- Prior art keywords
- biocatalyst
- formula
- rhodococcus
- dsm
- amino acids
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 150000001576 beta-amino acids Chemical class 0.000 title claims abstract description 14
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 8
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 7
- -1 N-acetyl methyl ester Chemical class 0.000 claims abstract description 39
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 239000011942 biocatalyst Substances 0.000 claims abstract description 23
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 10
- 230000007062 hydrolysis Effects 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims description 18
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 10
- 108090000531 Amidohydrolases Proteins 0.000 claims description 9
- 102000004092 Amidohydrolases Human genes 0.000 claims description 9
- 125000003118 aryl group Chemical group 0.000 claims description 9
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 9
- 241001464989 Rhodococcus globerulus Species 0.000 claims description 8
- 241000158504 Rhodococcus hoagii Species 0.000 claims description 8
- 230000002255 enzymatic effect Effects 0.000 claims description 8
- 125000005843 halogen group Chemical group 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 230000003301 hydrolyzing effect Effects 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 229910052760 oxygen Chemical group 0.000 claims description 5
- 239000001301 oxygen Chemical group 0.000 claims description 5
- 229920001184 polypeptide Polymers 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
- 150000001412 amines Chemical class 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- PQNFLJBBNBOBRQ-UHFFFAOYSA-N indane Chemical compound C1=CC=C2CCCC2=C1 PQNFLJBBNBOBRQ-UHFFFAOYSA-N 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- 239000011593 sulfur Chemical group 0.000 claims description 4
- CXWXQJXEFPUFDZ-UHFFFAOYSA-N tetralin Chemical compound C1=CC=C2CCCCC2=C1 CXWXQJXEFPUFDZ-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 125000005842 heteroatom Chemical group 0.000 claims description 3
- 125000002853 C1-C4 hydroxyalkyl group Chemical group 0.000 claims description 2
- 108090000371 Esterases Proteins 0.000 claims description 2
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 2
- VZWXIQHBIQLMPN-UHFFFAOYSA-N chromane Chemical compound C1=CC=C2CCCOC2=C1 VZWXIQHBIQLMPN-UHFFFAOYSA-N 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 2
- 239000012429 reaction media Substances 0.000 claims description 2
- ASFAFOSQXBRFMV-LJQANCHMSA-N 3-n-(2-benzyl-1,3-dihydroxypropan-2-yl)-1-n-[(1r)-1-(4-fluorophenyl)ethyl]-5-[methyl(methylsulfonyl)amino]benzene-1,3-dicarboxamide Chemical compound N([C@H](C)C=1C=CC(F)=CC=1)C(=O)C(C=1)=CC(N(C)S(C)(=O)=O)=CC=1C(=O)NC(CO)(CO)CC1=CC=CC=C1 ASFAFOSQXBRFMV-LJQANCHMSA-N 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 description 12
- 239000001257 hydrogen Substances 0.000 description 12
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 7
- 150000002431 hydrogen Chemical class 0.000 description 7
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 6
- 229910052801 chlorine Inorganic materials 0.000 description 6
- 239000000460 chlorine Substances 0.000 description 6
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 229910052731 fluorine Inorganic materials 0.000 description 5
- 239000011737 fluorine Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 4
- 229910052794 bromium Inorganic materials 0.000 description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 description 4
- 239000011707 mineral Substances 0.000 description 4
- 235000010755 mineral Nutrition 0.000 description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000000707 stereoselective effect Effects 0.000 description 4
- 125000001424 substituent group Chemical group 0.000 description 4
- 239000011573 trace mineral Substances 0.000 description 4
- 235000013619 trace mineral Nutrition 0.000 description 4
- 125000004765 (C1-C4) haloalkyl group Chemical group 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical group C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 0 CCC(C(C**(C)C)C(*)=O)N*(*)=O Chemical compound CCC(C(C**(C)C)C(*)=O)N*(*)=O 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 125000003545 alkoxy group Chemical group 0.000 description 3
- 125000003710 aryl alkyl group Chemical group 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 125000000714 pyrimidinyl group Chemical group 0.000 description 3
- YQTCQNIPQMJNTI-UHFFFAOYSA-N 2,2-dimethylpropan-1-one Chemical group CC(C)(C)[C]=O YQTCQNIPQMJNTI-UHFFFAOYSA-N 0.000 description 2
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 2
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 2
- 125000003282 alkyl amino group Chemical group 0.000 description 2
- 125000002947 alkylene group Chemical group 0.000 description 2
- 125000005428 anthryl group Chemical group [H]C1=C([H])C([H])=C2C([H])=C3C(*)=C([H])C([H])=C([H])C3=C([H])C2=C1[H] 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 125000003983 fluorenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3CC12)* 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 2
- 125000005928 isopropyloxycarbonyl group Chemical group [H]C([H])([H])C([H])(OC(*)=O)C([H])([H])[H] 0.000 description 2
- TZIHFWKZFHZASV-UHFFFAOYSA-N methyl formate Chemical group COC=O TZIHFWKZFHZASV-UHFFFAOYSA-N 0.000 description 2
- 125000004043 oxo group Chemical group O=* 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 125000006413 ring segment Chemical group 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 125000005930 sec-butyloxycarbonyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(OC(*)=O)C([H])([H])[H] 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 125000000229 (C1-C4)alkoxy group Chemical group 0.000 description 1
- 125000001637 1-naphthyl group Chemical group [H]C1=C([H])C([H])=C2C(*)=C([H])C([H])=C([H])C2=C1[H] 0.000 description 1
- 125000001622 2-naphthyl group Chemical group [H]C1=C([H])C([H])=C2C([H])=C(*)C([H])=C([H])C2=C1[H] 0.000 description 1
- MGWGWNFMUOTEHG-UHFFFAOYSA-N 4-(3,5-dimethylphenyl)-1,3-thiazol-2-amine Chemical group CC1=CC(C)=CC(C=2N=C(N)SC=2)=C1 MGWGWNFMUOTEHG-UHFFFAOYSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical group C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- PAVMRYVMZLANOQ-UHFFFAOYSA-N N-(1-phenylethyl)acetamide Chemical compound CC(=O)NC(C)C1=CC=CC=C1 PAVMRYVMZLANOQ-UHFFFAOYSA-N 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- 108010073038 Penicillin Amidase Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 125000001589 carboacyl group Chemical group 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004296 chiral HPLC Methods 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 125000003016 chromanyl group Chemical group O1C(CCC2=CC=CC=C12)* 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 125000002541 furyl group Chemical group 0.000 description 1
- 125000001188 haloalkyl group Chemical group 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000004030 hiv protease inhibitor Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 125000003387 indolinyl group Chemical group N1(CCC2=CC=CC=C12)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 125000004594 isoindolinyl group Chemical group C1(NCC2=CC=CC=C12)* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000005956 isoquinolyl group Chemical group 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- JCXJVPUVTGWSNB-UHFFFAOYSA-N nitrogen dioxide Inorganic materials O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000005936 piperidyl group Chemical group 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000001422 pyrrolinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 125000005493 quinolyl group Chemical group 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000004460 silage Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 125000006318 tert-butyl amino group Chemical group [H]N(*)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P13/04—Alpha- or beta- amino acids
Definitions
- the present invention relates to a process for the synthesis of beta-amino acids using a microorganism as a biocatalyst and to the biocatalyst catalyzing this process.
- Beta-amino acids are used in medicinal chemistry and show biological activity. Beta-amino acids may be found in e.g. beta-lactam antibiotics, HIV-protease inhibitors and further enzyme inhibitors. Classical organic synthesis or enzyme-catalyzed resolution may be used to synthesise beta-amino acids. Enzymes commonly used to resolve derivatives of alpha- amino acids have narrow substrate tolerance towards beta-amino acids. Due to the narrow substrate tolerance the biocatalytic approach is less used. Some examples using lipases or penicillin G acylase as catalysts are described.
- the present invention therefore provides a process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives, characterized in that a biocatalyst is used.
- the present invention provides a process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives using a biocatalyst characterized in that the biocatalyst is stereoselective ⁇ hydrolyzing the N-acetyl methyl ester derivatives.
- the advantages of the process of the invention are that whole cells as biocatalysts as well as isolated enzymes may be used to catalyze the process and the reaction medium may be distilled water.
- the product may easily be separated by simple extraction with organic solvents under acidic conditions without pH control or pH adjustment during the hydrolysis.
- biocatalyst means a purified enzyme, from original or recombinant source, a partially purified enzyme, a crude cell extract, enzymes immobilized or caught in partially broken cells or cell debris, a protein with enzymatic activity, a polypeptide with enzymatic activity, a living microorganism or dead microorganism containing a protein with enzymatic activity or a cell extract of such a microorganism.
- the biocatalyst may be used in solution, suspension or in immobilized form according to routine processes, e.g. batch or continuous, known in the art.
- a preferred embodiment is a biocatalyst, the substrate of which may be hydrolysed to beta- amino acids.
- the biocatalyst according to the invention is preferably selected from the group of (a) a protein with said enzymatic activity and (b) a living microorganism or dead microorganism containing a protein with said enzymatic activity or a cell extract of such a microorganism.
- a dead microorganism in context with the present invention is e.g. a microorganism in a disintegrated form in which the cell wall and/or cell membrane is mechanically or chemically disrupted or removed.
- the biocatalyst of the invention may be an amido hydrolase or enzyme. If not stated otherwise, all these terms include not only the naturally occurring, authentic sequence of the protein of the enzyme, which are preferred embodiments of the invention, but also all mutants, variants and fragments thereof which exhibit amido hydrolase activity, preferably the same stereoselective activity as the natural enzyme.
- the biocatalyst may be obtainable from a microorganism selected from the group of Rhodococcus globerulus and Rhodococcus equi.
- the biocatalyst is selected from the group consisting of Rhodococcus globerulus K1/1, DSM 10337, and Rhodococcus equi Ac6, DSM 10278 as disclosed in the International Application WO 97/41214 which is incorporated by reference.
- Isolation of a microorganism naturally expressing amido-hydrolase activity of the invention may be achieved by a selection process comprising inoculating a selection medium with natural samples such as soil, water, or plant silage, said selection medium comprising N- acetyl methyl ester derivatives as sole carbon source.
- a selection process comprising inoculating a selection medium with natural samples such as soil, water, or plant silage, said selection medium comprising N- acetyl methyl ester derivatives as sole carbon source.
- the racemic substrates described above in the context with the hydrolysis reaction are used for the selection.
- the selection medium also contains all essential ingredients necessary for allowing growth of microorganisms, such as mineral salts, N-sources and trace elements.
- a suitable selection medium used for performing the invention comprises 3 g racemic N- acetyl-1-phenylethylamine, 3 ml Trace element solution SL-6, and 1 I mineral salt solution ML1 (pH 7.0), whereby the composition of the mineral salt solution ML1 is 5 g K2HPO4, 0.2 g MgS ⁇ 4 * 7H2 ⁇ , 20 mg CaCt ⁇ , 20 mg FeS ⁇ 4 * 7H2 ⁇ , 1.5 g (NH4)2SO4 i and 1 I Aqua deion (pH 7.0); and the composition of the trace element solution SL-6 is 20 mg NiCl2 * 6H2 ⁇ , 200 mg CoCl2 * 6H2 ⁇ , 30 mg MnCl2*4H2 ⁇ , 10 mg CuCl2 * 2H2 ⁇ , 300 mg H3BO3, 30 mg Na2Mo ⁇ 4 * 2H2 ⁇ , 100 mg ZnS ⁇ 4 * 7H2 ⁇ , and 1 I Aqua deion.
- This recipe may be varied as long as sufficient trace elements,
- the enzyme of the invention which may be a polypeptide may be used in enriched or, preferably, purified form.
- an enzyme of the invention is particularly capable of catalyzing the following stereoselective reaction (A)
- racemic acylamide of the formula (1) is stereoselective ⁇ hydrolyzed with an amido hydrolase of the invention, which is a polypeptide with amido hydrolase enzymatic activity but without lipase- or esterase-activity, and which is capable of stereoselective ⁇ hydrolysing the racemic acylamide of formula (1 ), and wherein said hydrolysis results in the R- (or S-) amine of the formula (2) and the acid of formula (3) and the S- (or R-) amide of formula (4), wherein in formula (1) to (4) .
- an amido hydrolase of the invention which is a polypeptide with amido hydrolase enzymatic activity but without lipase- or esterase-activity, and which is capable of stereoselective ⁇ hydrolysing the racemic acylamide of formula (1 ), and wherein said hydrolysis results in the R- (or S-) amine of the formula (2) and the acid of formula (3) and the S- (or R-) amide of formula (4), where
- R 1 is NH 2 or O-alkyl
- R 2 is aryl or d-C 4 aryl; unsubstituted or substituted by C 1 -C 4 alkyl, C 1 -C 4 BIkOXy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 haloalkyl, hydroxy, amino, halogeno, nitro, sulfo or cyano; or wherein the rings may contain one or two heteroatoms selected from nitrogen, sulfur and oxygen; preferably indane, tetraline or chromane, unsubstituted or substituted with methoxy, ethoxy, halogeno, methyl, ethyl, nitro, cyano, amino, hydroxy, trifluoromethyl; and
- R 3 is H or an aliphatic acyl residue; preferably C 1 -C 4 alkyl; and more preferably methyl.
- Aryl is, for example a homo- or heterocyclyl.
- a suitable ring system is, for example, a single or double ring system having from 3 to 10 ring atoms, is bonded via a carbon atom or via a nitrogen atom and contains up to 4 hetero atoms selected from oxygen, nitrogen, sulfur, and sulfur linked to 1 or 2 oxygen atoms; which in addition may also be fused with 1 or 2 phenyl radicals or with 1 or 2 cycloalkyl radicals, cycloalkyl preferably having from 5 to 7 ring atoms; and which may be unsaturated or partially or fully saturated.
- Examples for aryl are phenyl, napthyl, biphenylyl, anthryl, fluorenyl, thienyl.
- phenyl-lower alkyl e.g. benzyl, hydroxy-lower alkyl, e.g. hydroxymethyl or 2-hydroxyethyl, hydroxy, lower alkoxy, e.g. methoxy or ethoxy, amino, lower alkylamino, e.g. methyl-, ethyl- or tertbutyl- amino, di-iower alkylamino, e.g.
- aryl is unsubstituted or substituted by one or more substituents selected from methyl, hydroxymethyl, 2-hydroxyethyl, hydroxy, methoxy; ethoxy, amino, methyl-amino, ethyl-amino, tert-butyl-amino, dimethylamino, diethyl-amino, carboxy, methoxy-carbonyl, isopropoxy-carbonyl, sec-butoxy-carbonyl, tert-butoxy-carbonyl, fluorine, chlorine, bromine, acetyl or pivaloyl, nitro, oxo and/or by cyano.
- a racemic acylamide is hydrolyzed wherein R 3 is H or Ci-C 3 alkyl, more preferably Cialkyl.
- R 3 is most preferably d-Caalkyl.
- the term 'substituted' means that the moiety in question can be substituted by one to three identical or different substituents, preferably one or two identical or different, most preferably by one substituent selected from the group consisting of Ci-C 8 alkyl (preferably methyl), haloalkyl (preferably trifluoromethyl), halogen (preferably fluorine or chlorine), amino, nitro, and Ci-C 8 alkoxy (preferably methoxy).
- aryl standing alone or being member or an aralkyl moiety is a carbocyclic radical in which at least one ring is in the form of a 6-membered aromatic ring (i.e. a benzene ring).
- a 6-membered aromatic ring i.e. a benzene ring.
- Preferred are phenyl, naphthyl, such as 1- or 2- naphthyl, biphenylyl, such as, especially, 4-biphenylyl, anthryl, and fluorenyl, and also such ring systems having one or more fused saturated rings.
- aralkyl stands for an aliphatic radical substituted by an aryl moiety, wherein the aliphatic radical is an unbranched or branched Ci-C 8 alkylene (preferably Ci-C 3 alkylene, most preferably methylene or ethylene) and the aryl moiety is a carbocyclic radical as defined above.
- aralkyl stands for a radical of the formula
- R4 and R5 are each independent of each other hydrogen, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, halogeno, amino, cyano, nitro or C 1 -C 4 alkoxy; preferably hydrogen, methyl, ethyl, trifluoromethyl, fluorine, chlorine, bromine, iodine, amino, nitro, cyano or methoxy; more preferably hydrogen, methyl, fluorine, chlorine, cyano, nitro or methoxy.
- n is an integral number selected from 0, 1 and 2; R4 and R5 are each independent of each other hydrogen or cyano.
- X is oxygen, carbon or nitrogen; preferably oxygen and carbon; and R4 and R5 are each independent of each other hydrogen, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, . halogeno, amino, cyano, nitro or C.-C4 alkoxy; preferably hydrogen, methyl, ethyl, iodo, bromo, trifluoromethyl, chloro, fluoro, amino, cyano, nitro, methoxy or ethoxy.
- the present invention provides the hydrolysis of trans- and cis-N- acetyl-2-aminocyclohexane carboxylic acid methyl ester:
- N-acetyl methyl ester derivative of the formula 3a-15a is stereoselective ⁇ hydrolised with an amido hydrolase of the invention, which is a polypeptide with amine hydrolase enzymatic activity and which is capable of stereoselective ⁇ hydrolysing the racemic substrate of formula 3a to 15a, wherein 3a to 10a is and R1 is
- R1 is hydrogen
- R1 is methyl formate
- R1 is tert-butyl
- R 1 is methoxy
- R1 is fluoro
- R1 is chloro
- R1 is phenyl
- R1 is nitrogen dioxide
- R1 is hydrogen, R2 is methoxy and R3 is hydrogen 12a: R1 is methoxy, R2 is methoxy and R3 is hydrogen 13a: R1 is R2 is ethylene oxide, R3 is hydrogen 14a: R1 is hydrogen, R2 is methoxy, R3 is methoxy
- the biocatalyst may be obtainable from a microorganism selected from the group of Rhodococcus globerulus K1/1 , DSM 10337, and Rhodococcus equi Ac6, DSM 10278.
- each racemic substrate ( ⁇ )-3a-15a was dissolved in 5 ml of methanol and the solutions were added to 50 ml of distilled water.
- 50ml cell suspension of Rhodococcus equi DSM 10278 or Rhodococcus globerulus DSM 10337 obtained by addition of 8.5g wet cells in 900 ml phosphate buffer pH7.0, was added and the mixtures were shaken at 200 rpm and 30°C. After 64 hours the pH was adjusted to 2 by adding cone. HCI and extracted 3 times with CH 2 CI 2 .
- the organic phases were dried (MgSO 4 ) and the solvent removed under vacuum. The obtained residues were analyzed by chiral HPLC.
- the pH of the aqueous phases containing the amines 3-15 was adjusted to 8 and acetic acid anhydride was added. Usual workup gave the enantiomeric N-acetyl derivatives which were also submitted for HPLC-analysis.
- Rhodococcus globerulus K1/1 Rhodococcus 10337 (deposited September 23, 1995) Rhodococcus equiAc ⁇ , DSM 10278 (deposited September 23, 1995)
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Abstract
A process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives using a biocatalyst.
Description
Process for the synthesis of beta amino acids
The present invention relates to a process for the synthesis of beta-amino acids using a microorganism as a biocatalyst and to the biocatalyst catalyzing this process.
Beta-amino acids are used in medicinal chemistry and show biological activity. Beta-amino acids may be found in e.g. beta-lactam antibiotics, HIV-protease inhibitors and further enzyme inhibitors. Classical organic synthesis or enzyme-catalyzed resolution may be used to synthesise beta-amino acids. Enzymes commonly used to resolve derivatives of alpha- amino acids have narrow substrate tolerance towards beta-amino acids. Due to the narrow substrate tolerance the biocatalytic approach is less used. Some examples using lipases or penicillin G acylase as catalysts are described.
There is a need to provide improved processes to synthesize beta-amino acids using a biocatalyst.
The present invention therefore provides a process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives, characterized in that a biocatalyst is used.
In another aspect the present invention provides a process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives using a biocatalyst characterized in that the biocatalyst is stereoselective^ hydrolyzing the N-acetyl methyl ester derivatives.
The advantages of the process of the invention are that whole cells as biocatalysts as well as isolated enzymes may be used to catalyze the process and the reaction medium may be distilled water. The product may easily be separated by simple extraction with organic solvents under acidic conditions without pH control or pH adjustment during the hydrolysis.
The stereoselective hydrolysis of N-acetyl methyl ester derivatives provides easy separation of the enantiomers. With an entiomerically pure pharmaceutical or agrochemical drug side effects e.g. triggered by the therapeutically ineffective enantiomer can be avoided.
As used herein, the term "biocatalyst" means a purified enzyme, from original or recombinant source, a partially purified enzyme, a crude cell extract, enzymes immobilized or caught in partially broken cells or cell debris, a protein with enzymatic activity, a polypeptide with enzymatic activity, a living microorganism or dead microorganism containing a protein with enzymatic activity or a cell extract of such a microorganism.
The biocatalyst may be used in solution, suspension or in immobilized form according to routine processes, e.g. batch or continuous, known in the art.
A preferred embodiment is a biocatalyst, the substrate of which may be hydrolysed to beta- amino acids. The biocatalyst according to the invention is preferably selected from the group of (a) a protein with said enzymatic activity and (b) a living microorganism or dead microorganism containing a protein with said enzymatic activity or a cell extract of such a microorganism. A dead microorganism in context with the present invention is e.g. a microorganism in a disintegrated form in which the cell wall and/or cell membrane is mechanically or chemically disrupted or removed.
The biocatalyst of the invention may be an amido hydrolase or enzyme. If not stated otherwise, all these terms include not only the naturally occurring, authentic sequence of the protein of the enzyme, which are preferred embodiments of the invention, but also all mutants, variants and fragments thereof which exhibit amido hydrolase activity, preferably the same stereoselective activity as the natural enzyme.
The biocatalyst may be obtainable from a microorganism selected from the group of Rhodococcus globerulus and Rhodococcus equi. Preferably the biocatalyst is selected from the group consisting of Rhodococcus globerulus K1/1, DSM 10337, and Rhodococcus equi Ac6, DSM 10278 as disclosed in the International Application WO 97/41214 which is incorporated by reference.
Isolation of a microorganism naturally expressing amido-hydrolase activity of the invention may be achieved by a selection process comprising inoculating a selection medium with natural samples such as soil, water, or plant silage, said selection medium comprising N- acetyl methyl ester derivatives as sole carbon source. In the preferred embodiments of the
selection process, the racemic substrates described above in the context with the hydrolysis reaction are used for the selection.
Apart from the carbon source, the selection medium also contains all essential ingredients necessary for allowing growth of microorganisms, such as mineral salts, N-sources and trace elements.
A suitable selection medium used for performing the invention comprises 3 g racemic N- acetyl-1-phenylethylamine, 3 ml Trace element solution SL-6, and 1 I mineral salt solution ML1 (pH 7.0), whereby the composition of the mineral salt solution ML1 is 5 g K2HPO4, 0.2 g MgSθ4*7H2θ, 20 mg CaCtø, 20 mg FeSθ4*7H2θ, 1.5 g (NH4)2SO4i and 1 I Aqua deion (pH 7.0); and the composition of the trace element solution SL-6 is 20 mg NiCl2*6H2θ, 200 mg CoCl2*6H2θ, 30 mg MnCl2*4H2θ, 10 mg CuCl2*2H2θ, 300 mg H3BO3, 30 mg Na2Moθ4*2H2θ, 100 mg ZnSθ4*7H2θ, and 1 I Aqua deion. This recipe may be varied as long as sufficient trace elements, mineral salts, and N-source are provided.
The enzyme of the invention which may be a polypeptide may be used in enriched or, preferably, purified form. In one embodiment of the invention an enzyme of the invention is particularly capable of catalyzing the following stereoselective reaction (A)
S- (or R) amine acid R- (or S) amide
1 2 3 4 or
R1 is NH2 or O-alkyl
R2 is aryl or d-C4aryl; unsubstituted or substituted by C1-C4 alkyl, C1-C4BIkOXy, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 haloalkyl, hydroxy, amino, halogeno, nitro, sulfo or cyano; or wherein the rings may contain one or two heteroatoms selected from nitrogen, sulfur and oxygen; preferably indane, tetraline or chromane, unsubstituted or substituted with methoxy, ethoxy, halogeno, methyl, ethyl, nitro, cyano, amino, hydroxy, trifluoromethyl; and
R3 is H or an aliphatic acyl residue; preferably C1-C4 alkyl; and more preferably methyl.
Aryl is, for example a homo- or heterocyclyl. A suitable ring system is, for example, a single or double ring system having from 3 to 10 ring atoms, is bonded via a carbon atom or via a nitrogen atom and contains up to 4 hetero atoms selected from oxygen, nitrogen, sulfur, and sulfur linked to 1 or 2 oxygen atoms; which in addition may also be fused with 1 or 2 phenyl radicals or with 1 or 2 cycloalkyl radicals, cycloalkyl preferably having from 5 to 7 ring atoms; and which may be unsaturated or partially or fully saturated. Examples for aryl are phenyl, napthyl, biphenylyl, anthryl, fluorenyl, thienyl. furyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, benzimidazolyl, quinolyl, isoquinolyl, 3,1-benzofuranyl, chromanyl, cyclohexa- [b]pyrrolyl, cyclohexa[b]pyridyl, [b]pyrimidinyl, pyrrolidinyl, pyrrolinyl, cyclohexa[b]pyrazinyl, cyclohexa[b]pyrimidinyl, imidazolidyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, S,S-dioxo-thiomorpholinyl, indolinyl, isoindolinyl, 4,5,6,7-tetrahydro indolyl, 1 ,2,3,4- tetrahydroquinolyl or 1 ,2,3,4-tetrahydroisoquinolyl, for example one of the last-mentioned radicals, being unsubstituted or substituted by one or more substituents selected from lower alkyl, e.g. methyl, phenyl, 1- or 2-naphthyf, phenyl-lower alkyl, e.g. benzyl, hydroxy-lower alkyl, e.g. hydroxymethyl or 2-hydroxyethyl, hydroxy, lower alkoxy, e.g. methoxy or ethoxy, amino, lower alkylamino, e.g. methyl-, ethyl- or tertbutyl-
amino, di-iower alkylamino, e.g. dimethyl- or diethyl-amino, carboxy, lower alkoxycarbonyl, for example methoxy-, isopropoxy-, sec-butoxy- or tert-butoxy-carbonyl, phenyl- or naphthyl-lower alkoxycarbonyl, e.g. benzyloxycarbonyl, halogen, for e fluorine, chlorine, bromine or iodine, especially chlorine or bromine, lower alkanoyl, e.g. acetyl or pivaloyl, nitro, oxo and/or by cyano.
In a preferred embodiment aryl is unsubstituted or substituted by one or more substituents selected from methyl, hydroxymethyl, 2-hydroxyethyl, hydroxy, methoxy; ethoxy, amino, methyl-amino, ethyl-amino, tert-butyl-amino, dimethylamino, diethyl-amino, carboxy, methoxy-carbonyl, isopropoxy-carbonyl, sec-butoxy-carbonyl, tert-butoxy-carbonyl, fluorine, chlorine, bromine, acetyl or pivaloyl, nitro, oxo and/or by cyano.
In a preferred embodiment of the above reaction A, a racemic acylamide is hydrolyzed wherein R3 is H or Ci-C3 alkyl, more preferably Cialkyl. In particular, if an hydrolase obtainable from Rhodococcus globerulus or Rhodococcus equi is used, R3 is most preferably d-Caalkyl.
The term 'substituted' means that the moiety in question can be substituted by one to three identical or different substituents, preferably one or two identical or different, most preferably by one substituent selected from the group consisting of Ci-C8 alkyl (preferably methyl), haloalkyl (preferably trifluoromethyl), halogen (preferably fluorine or chlorine), amino, nitro, and Ci-C8 alkoxy (preferably methoxy).
In accordance with the present invention aryl standing alone or being member or an aralkyl moiety is a carbocyclic radical in which at least one ring is in the form of a 6-membered aromatic ring (i.e. a benzene ring). Preferred are phenyl, naphthyl, such as 1- or 2- naphthyl, biphenylyl, such as, especially, 4-biphenylyl, anthryl, and fluorenyl, and also such ring systems having one or more fused saturated rings.
In a preferred embodiment aralkyl stands for an aliphatic radical substituted by an aryl moiety, wherein the aliphatic radical is an unbranched or branched Ci-C8 alkylene (preferably Ci-C3 alkylene, most preferably methylene or ethylene) and the aryl moiety is a carbocyclic radical as defined above. In a more preferred embodiment aralkyl stands for a radical of the formula
wherein
R4 and R5 are each independent of each other hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, halogeno, amino, cyano, nitro or C1-C4 alkoxy; preferably hydrogen, methyl, ethyl, trifluoromethyl, fluorine, chlorine, bromine, iodine, amino, nitro, cyano or methoxy; more preferably hydrogen, methyl, fluorine, chlorine, cyano, nitro or methoxy.
Further preferred compounds are
wherein n is an integral number selected from 0, 1 and 2; R4 and R5 are each independent of each other hydrogen or cyano.
Further preferred compounds are of formulae
wherein X is oxygen, carbon or nitrogen; preferably oxygen and carbon; and R4 and R5 are each independent of each other hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, . halogeno, amino, cyano, nitro or C.-C4 alkoxy; preferably hydrogen, methyl, ethyl, iodo, bromo, trifluoromethyl, chloro, fluoro, amino, cyano, nitro, methoxy or ethoxy.
In a preferred embodiment the present invention provides the hydrolysis of trans- and cis-N- acetyl-2-aminocyclohexane carboxylic acid methyl ester:
(±)-trans-fa 1 S,2S-1 1R,2R-1a
(±)-cis-2a 1S.2S-2 1R,2R-2a
In another preferred embodiment of the invention an enzyme of the invention is capable of catalyzing the following stereoselective reaction:
(±)-3a-"/5a (R)-3a-15a (S)-3-15
wherein a N-acetyl methyl ester derivative of the formula 3a-15a is stereoselective^ hydrolised with an amido hydrolase of the invention, which is a polypeptide with amine hydrolase enzymatic activity and which is capable of stereoselective^ hydrolysing the racemic substrate of formula 3a to 15a, wherein 3a to 10a is
and R1 is
3a: R1 is hydrogen
4a: R1 is methyl formate
5a: R1 is tert-butyl
6a R 1 is methoxy
7a: R1 is fluoro
8a: R1 is chloro
9a: R1 is phenyl
10a: R1 is nitrogen dioxide
and wherein 11a to 14a is
11a: R1 is hydrogen, R2 is methoxy and R3 is hydrogen 12a: R1 is methoxy, R2 is methoxy and R3 is hydrogen 13a: R1 is R2 is ethylene oxide, R3 is hydrogen 14a: R1 is hydrogen, R2 is methoxy, R3 is methoxy
and wherein 15a is
Example
400mg of each racemic substrate (±)-3a-15a was dissolved in 5 ml of methanol and the solutions were added to 50 ml of distilled water. Finally 50ml cell suspension of Rhodococcus equi DSM 10278 or Rhodococcus globerulus DSM 10337, obtained by addition of 8.5g wet cells in 900 ml phosphate buffer pH7.0, was added and the mixtures were shaken at 200 rpm and 30°C. After 64 hours the pH was adjusted to 2 by adding cone. HCI and extracted 3 times with CH2CI2. The organic phases were dried (MgSO4) and the solvent removed under vacuum. The obtained residues were analyzed by chiral HPLC. The pH of the aqueous phases containing the amines 3-15 was adjusted to 8 and acetic acid anhydride was added. Usual workup gave the enantiomeric N-acetyl derivatives which were also submitted for HPLC-analysis.
The selectivities (E) for each substrate are summarized in table 1 :
Table 1
The following microorganisms are deposited according to the Budapest Treaty with the DSM - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. The deposition has been done as disclosed in the filing of the published International Application WO 97/41214.
Rhodococcus globerulus K1/1 , DSM 10337 (deposited September 23, 1995) Rhodococcus equiAcβ, DSM 10278 (deposited September 23, 1995)
Claims
1. A process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives, characterized in that a biocatalyst is used.
2. The process of claim 1 wherein the biocatalyst is obtainable from a microorganism selected from the group of Rhodococcus globerulus and Rhodococcus equi.
3. The process of claim 1 or 2 wherein the biocatalyst is obtainable from a microorganism selected from the group of Rhodococcus globerulus, DSM 10337, and Rhodococcus equi Ac6, DSM 10278.
4. The process of any preceding claim wherein the biocatalyst is stereoselective^ hydrolyzing the N-acetyl methyl ester derivatives.
5. The process according to any preceding claim, which has the following reaction scheme,
R- (or S) amide 
4 or
S-) amine of the formula (2) and the acid of formula (3) and the S- (or R-) amide of formula (4), wherein in formula (1 ) to (4) .
R1 is NH2 or lower alkyl
R2 is aryl or Ci-C4aryl; unsubstituted or substituted by Ci-C4alkyl, Ci-C4alkoxy, C1-
C4hydroxyalkyl, C1-C4aminoalkyl, Ci-C4haloalkyl, hydroxy, amino, halogeno, nitro, sulfo or cyano; or wherein the rings contain one or two heteroatoms selected from nitrogen, sulfur and oxygen; preferably indane, tetraline or chromane, unsubstituted or substituted with methoxy, ethoxy, halogeno, methyl, ethyl, nitro, cyano, amino, hydroxy, trifluoromethyl; and
R3 is H or an aliphatic acyl residue; preferably d-C4alkyl; and more preferably methyl.
6. The process of claim 1 to 5 wherein the reaction medium is distilled water.
7. A biocatalyst catalyzing the process of claims 1 to 6.
8. The biocatalyst according to claim 7 being an amido hydrolase.
9. The biocatalyst of claim 7 or 8 obtainable from a microorganism selected from the group consisting of Rhodococcus globerulυs K1/1 , DSM 10337, and Rhodococcus equi Ac6, DSM 10278.
10. The beta amino acids obtained by the process of claim 5.
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| GB0519200A GB0519200D0 (en) | 2005-09-20 | 2005-09-20 | Organic compounds |
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| JP2013505710A (en) * | 2009-09-25 | 2013-02-21 | ビーエーエスエフ ソシエタス・ヨーロピア | Amidase and its use to produce 3-aminocarboxylic esters |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5518903A (en) * | 1993-08-13 | 1996-05-21 | Chisso Corporation | Process for producing optically active 3-aminobutanoic acid and the ester intermediates |
| WO1997041214A1 (en) * | 1996-04-25 | 1997-11-06 | Novartis Ag | Biocatalysts with amine acylase activity |
| WO1998050575A1 (en) * | 1997-05-01 | 1998-11-12 | G.D. Searle & Co. | Method and apparatus for preparation of chiral beta amino acids |
-
2005
- 2005-09-20 GB GB0519200A patent/GB0519200D0/en not_active Ceased
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5518903A (en) * | 1993-08-13 | 1996-05-21 | Chisso Corporation | Process for producing optically active 3-aminobutanoic acid and the ester intermediates |
| WO1997041214A1 (en) * | 1996-04-25 | 1997-11-06 | Novartis Ag | Biocatalysts with amine acylase activity |
| WO1998050575A1 (en) * | 1997-05-01 | 1998-11-12 | G.D. Searle & Co. | Method and apparatus for preparation of chiral beta amino acids |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013505710A (en) * | 2009-09-25 | 2013-02-21 | ビーエーエスエフ ソシエタス・ヨーロピア | Amidase and its use to produce 3-aminocarboxylic esters |
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