JP2009060799A - Method for producing (-)-ambroxan (r) - Google Patents
Method for producing (-)-ambroxan (r) Download PDFInfo
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- JP2009060799A JP2009060799A JP2007228877A JP2007228877A JP2009060799A JP 2009060799 A JP2009060799 A JP 2009060799A JP 2007228877 A JP2007228877 A JP 2007228877A JP 2007228877 A JP2007228877 A JP 2007228877A JP 2009060799 A JP2009060799 A JP 2009060799A
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- Prior art keywords
- shc
- ambroxan
- homofarnesol
- reaction
- squalene
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 239000001147 (3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1H-benzo[e][1]benzofuran Substances 0.000 title abstract description 16
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 108091000048 Squalene hopene cyclase Proteins 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 241000640374 Alicyclobacillus acidocaldarius Species 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- KOICZDDAVPYKKX-NCZFFCEISA-N (3e,7e)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CCO KOICZDDAVPYKKX-NCZFFCEISA-N 0.000 claims 1
- KOICZDDAVPYKKX-UHFFFAOYSA-N (3E,7Z)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol Chemical compound CC(=C/CCO)CCC=C(/CCC=C(C)C)C KOICZDDAVPYKKX-UHFFFAOYSA-N 0.000 abstract description 15
- YPZUZOLGGMJZJO-UHFFFAOYSA-N Ambronide Chemical compound C1CC2C(C)(C)CCCC2(C)C2C1(C)OCC2 YPZUZOLGGMJZJO-UHFFFAOYSA-N 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 5
- 239000007979 citrate buffer Substances 0.000 description 5
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 5
- 239000003205 fragrance Substances 0.000 description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229940031439 squalene Drugs 0.000 description 5
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- KIYYUMANFSBVAV-MZIWDXLGSA-N (3r,3as,5ar,5br,7ar,9s,11ar,11br,13ar,13bs)-5a,5b,8,8,11a,13b-hexamethyl-3-propan-2-yl-1,2,3,3a,4,5,6,7,7a,9,10,11,11b,12,13,13a-hexadecahydrocyclopenta[a]chrysen-9-ol Chemical compound C([C@]1(C)[C@H]2CC[C@H]34)C[C@H](O)C(C)(C)[C@@H]1CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@@H]1C(C)C KIYYUMANFSBVAV-MZIWDXLGSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- DLTTWXWDLCGBRD-UHFFFAOYSA-N Hydroxyhopane Natural products CC12CCCC(C)(C)C1CCC1(C)C2CCC2C3(C)CCC(C(C)C)C3(O)CCC21C DLTTWXWDLCGBRD-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- YPZUZOLGGMJZJO-LQKXBSAESA-N ambroxan Chemical compound CC([C@@H]1CC2)(C)CCC[C@]1(C)[C@@H]1[C@]2(C)OCC1 YPZUZOLGGMJZJO-LQKXBSAESA-N 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- HHXYJYBYNZMZKX-PYQRSULMSA-N 22(29)-Hopene Chemical compound C([C@]1(C)[C@H]2CC[C@H]34)CCC(C)(C)[C@@H]1CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@@H]1C(=C)C HHXYJYBYNZMZKX-PYQRSULMSA-N 0.000 description 2
- HHXYJYBYNZMZKX-UHFFFAOYSA-N 3,4:15,16-diepoxy-7-oxo-13(16),14-clerodadien-20,12-olide-(3alpha,4alpha)-form Natural products C12CCC3C4(C)CCCC(C)(C)C4CCC3(C)C1(C)CCC1C2(C)CCC1C(=C)C HHXYJYBYNZMZKX-UHFFFAOYSA-N 0.000 description 2
- 241000589220 Acetobacter Species 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000589174 Bradyrhizobium japonicum Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101710095468 Cyclase Proteins 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- XVULBTBTFGYVRC-UHFFFAOYSA-N Episclareol Natural products CC1(C)CCCC2(C)C(CCC(O)(C)C=C)C(C)(O)CCC21 XVULBTBTFGYVRC-UHFFFAOYSA-N 0.000 description 2
- 229930186351 Hopene Natural products 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000589346 Methylococcus capsulatus Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 241000248418 Tetrahymena pyriformis Species 0.000 description 2
- 241000588902 Zymomonas mobilis Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 150000001923 cyclic compounds Chemical class 0.000 description 2
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 101150012554 shc gene Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- FQTLCLSUCSAZDY-UHFFFAOYSA-N (+) E(S) nerolidol Natural products CC(C)=CCCC(C)=CCCC(C)(O)C=C FQTLCLSUCSAZDY-UHFFFAOYSA-N 0.000 description 1
- CRDAMVZIKSXKFV-FBXUGWQNSA-N (2-cis,6-cis)-farnesol Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C/CO CRDAMVZIKSXKFV-FBXUGWQNSA-N 0.000 description 1
- 239000000260 (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol Substances 0.000 description 1
- XUFAHPAHNJCLLZ-WYEAJDFLSA-N (3as,4s,5as,9as,9br)-4-hydroxy-3a,6,6,9a-tetramethyl-1,4,5,5a,7,8,9,9b-octahydrobenzo[e][1]benzofuran-2-one Chemical compound C([C@@]12C)CCC(C)(C)[C@@H]1C[C@H](O)[C@]1(C)[C@@H]2CC(=O)O1 XUFAHPAHNJCLLZ-WYEAJDFLSA-N 0.000 description 1
- YPZUZOLGGMJZJO-XRGAULLZSA-N (3as,5as,9as,9br)-3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1h-benzo[e][1]benzofuran Chemical compound CC([C@@H]1CC2)(C)CCC[C@]1(C)[C@@H]1[C@@]2(C)OCC1 YPZUZOLGGMJZJO-XRGAULLZSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- FQTLCLSUCSAZDY-ATGUSINASA-N Nerolidol Chemical compound CC(C)=CCC\C(C)=C\CC[C@](C)(O)C=C FQTLCLSUCSAZDY-ATGUSINASA-N 0.000 description 1
- 244000182022 Salvia sclarea Species 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- -1 acetonitrile Chemical compound 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- IMKJGXCIJJXALX-UHFFFAOYSA-N ent-Norambreinolide Natural products C1CC2C(C)(C)CCCC2(C)C2C1(C)OC(=O)C2 IMKJGXCIJJXALX-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930002886 farnesol Natural products 0.000 description 1
- 229940043259 farnesol Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- WASNIKZYIWZQIP-AWEZNQCLSA-N nerolidol Natural products CC(=CCCC(=CCC[C@@H](O)C=C)C)C WASNIKZYIWZQIP-AWEZNQCLSA-N 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 238000006864 oxidative decomposition reaction Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- YMBCJWGVCUEGHA-UHFFFAOYSA-M tetraethylammonium chloride Chemical compound [Cl-].CC[N+](CC)(CC)CC YMBCJWGVCUEGHA-UHFFFAOYSA-M 0.000 description 1
- CRDAMVZIKSXKFV-UHFFFAOYSA-N trans-Farnesol Natural products CC(C)=CCCC(C)=CCCC(C)=CCO CRDAMVZIKSXKFV-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、（−）−３ａ、６、６、９ａ−テトラメチルドデカヒドロナフト［２、１−ｂ］フランの製造方法に関する。 The present invention relates to a process for producing (−)-3a, 6,6,9a-tetramethyldodecahydronaphtho [2,1-b] furan.
３ａ、６、６、９ａ−テトラメチルドデカヒドロナフト［２、１−ｂ］フラン（「アンブロキサン（登録商標）」とも称する）は、香気特性と残香性に優れたアンバー系調合香料に欠かせない化合物である。 3a, 6,6,9a-Tetramethyldodecahydronaphtho [2,1-b] furan (also referred to as “Ambroxan (registered trademark)”) is indispensable for amber-based blended fragrances with excellent aroma characteristics and residual fragrance properties There is no compound.
また、光学異性体を比較すると、（−）−アンブロキサンは典型的なアンバー香気を有し、（＋）−アンブロキサンは弱い木様香気を有するため、香料としての有用性の点から、高い光学純度で（−）−体を得る必要性が高く、（−）−アンブロキサンの不斉合成方法について、現在研究開発が盛んに行われている。 In addition, when the optical isomers are compared, (−)-ambroxan has a typical amber fragrance, and (+)-ambroxan has a weak tree-like fragrance, which is high in terms of usefulness as a fragrance. There is a high need to obtain a (−)-isomer with optical purity, and research and development are actively conducted on asymmetric synthesis methods of (−)-ambroxane.
例えば、下記に示すように、天然植物クラリーセージの抽出物である（−）−スクラレオールを出発原料とし、（＋）−スクラレオライドを経由する製造方法が、（−）−アンブロキサンの好適な工業的製法として知られている（非特許文献１）。
しかしながら、この方法においては、天然原料を用いるために、多段階反応であり操作が迂遠である、供給量と供給安定性が満足のいくものではない、（−）−スクラレオールの酸化分解工程においてクロム酸や過マンガン塩などの酸化剤を用いており環境負荷が大きい、という問題があった。
For example, as shown below, a production method using (−)-sclareol, which is an extract of natural plant clary sage, as a starting material and passing through (+)-sclareolide is a suitable industry for (−)-ambroxan. It is known as a general manufacturing method (Non-patent Document 1).
However, in this method, since natural raw materials are used, the supply amount and the supply stability are not satisfactory because of the multistage reaction and the operation is diverted. In the oxidative decomposition process of (−)-sclareol, There is a problem that an environmental load is large because an oxidizing agent such as acid or permanganate is used.
一方、下記に示すように、スクアレン−ホペン環化酵素（Squalene-Hopene cyclase；以下ＳＨＣともいう）はスクアレンを基質とし、ホペン、ホパノールといった環状化合物を生成する環化酵素であり、Alicyclobacillus acidocaldarius由来、Zymomonas mobilis由来、Bradyrhizobium japonicum由来、 Methylococcus capsulatus由来、Frankiana由来、Acetobacter pasteurianum由来、Tetrahymena pyriformis由来等のものが知られている。中でもAlicyclobacillus acidocaldarius由来酵素は遺伝子配列が解明されており、大腸菌によって組換え酵素を発現させ、安定に機能させる方法が知られている（非特許文献２）。 On the other hand, as shown below, squalene-hopene cyclase (Squalene-Hopene cyclase; hereinafter also referred to as SHC) is a cyclase that produces a cyclic compound such as hopene and hopanol using squalene as a substrate, derived from Alicyclobacillus acidocaldarius , Zymomonas mobilis origin, Bradyrhizobium japonicum origin, Methylococcus capsulatus origin, Frankiana origin, Acetobacter pasteurianum origin, Tetrahymena pyriformis origin, etc. are known. Among them, the gene sequence of the enzyme derived from Alicyclobacillus acidocaldarius has been elucidated, and a method for expressing the recombinant enzyme in Escherichia coli and causing it to function stably is known (Non-patent Document 2).
近年、この酵素を用いて、種々の基質に対する反応性が検討されている。例えば、ファルネソールを基質とした場合、変換率６４％で下記の化合物（ａ），（ｂ），（ｃ），（ｄ）が７：３：４５：９の割合で生成することが報告されている（非特許文献３及び４）。 In recent years, reactivity to various substrates has been studied using this enzyme. For example, when farnesol is used as a substrate, it has been reported that the following compounds (a), (b), (c), and (d) are produced in a ratio of 7: 3: 45: 9 at a conversion rate of 64%. (Non-Patent Documents 3 and 4).
また、ホモファルネソールを基質とした場合、アンブロキサンが生成することが報告されている（非特許文献５）が、変換率は３％と極めて低く、また、（−）−体の光学純度については言及されていないことから、当該文献は、（−）−アンブロキサンの製造方法をなんら開示するものではない。 Moreover, when homofarnesol is used as a substrate, it has been reported that ambroxane is produced (Non-Patent Document 5), but the conversion rate is extremely low at 3%, and the optical purity of the (−)-form is Since it is not mentioned, this document does not disclose any method for producing (−)-ambroxan.
本発明は、スクアレン-ホペン環化酵素を用いた、ホモファルネソールからの（−）−アンブロキサンの製造方法を提供することに関する。 The present invention relates to providing a method for producing (−)-ambroxane from homofarnesol using squalene-hopene cyclase.
本発明者らは、酵素法によるアンブロキサンの製造法を検討したところ、安価に得られるホモファルネソールを基質とし、ｐＨ５．２〜６．６の溶媒中でスクアレン-ホペン環化酵素を作用させることにより、（−）−アンブロキサンが容易に、しかも効率よく製造できることを見出した。 The present inventors examined an enzymatic method for producing ambroxan. As a result, low-cost homofarnesol was used as a substrate, and squalene-hopene cyclizing enzyme was allowed to act in a solvent at pH 5.2 to 6.6. Thus, it was found that (−)-ambroxan can be produced easily and efficiently.
すなわち、本発明は、下記に示すとおり、ｐＨ５．２〜６．６の溶媒中で、ホモファルネソール（化合物（１））に、スクアレン−ホペン環化酵素を作用させることを特徴とする（−）−３ａ、６、６、９ａ−テトラメチルドデカヒドロナフト［２、１−ｂ］フラン（化合物（２））の製造方法に係るものである。 That is, the present invention is characterized in that squalene-hopene cyclizing enzyme is allowed to act on homofarnesol (compound (1)) in a solvent having a pH of 5.2 to 6.6 as described below (-). -3a, 6,6,9a-tetramethyldodecahydronaphtho [2,1-b] furan (compound (2)).
本発明の製造方法によれば、安価な出発原料から容易な方法により、（−）−アンブロキサンを、工業的に有利に得ることができる。 According to the production method of the present invention, (−)-ambroxan can be industrially advantageously obtained from an inexpensive starting material by an easy method.
スクアレン−ホペン環化酵素は、スクアレンを基質とし、ホペン、ホパノールといった環状化合物を生成する環化酵素であり、Alicyclobacillus acidocaldarius由来、Zymomonas mobilis由来、Bradyrhizobium japonicum由来、 Methylococcus capsulatus由来、Frankiana由来、Acetobacter pasteurianum由来、Tetrahymena pyriformis由来等のものが知られている。本発明においては、これらの何れのものも使用できるが、中でもAlicyclobacillus acidocaldarius由来のＳＨＣは、遺伝子配列が解明されており、大腸菌によって組換え酵素を発現させ、安定に機能させる方法が知られていることから（前記非特許文献２）、これを使用するのが好ましい。 Squalene - Hopen cyclization enzyme squalene as a substrate, Hopen, a cyclase to produce cyclic compounds such Hopanoru, from Alicyclobacillus acidocaldarius, from Zymomonas mobilis, from Bradyrhizobium japonicum, from Methylococcus capsulatus, derived Frankiana, from Acetobacter pasteurianum From Tetrahymena pyriformis, etc. are known. In the present invention, any of these can be used. Among them, SHC derived from Alicyclobacillus acidocaldarius has an elucidated gene sequence, and a method of expressing a recombinant enzyme by Escherichia coli and causing it to function stably is known. Therefore (Non-Patent Document 2), it is preferable to use this.
ホモファルネソールに、ＳＨＣを作用させるに当たっては、ＳＨＣであるタンパク質を当該基質に直接的に作用させてもよいし、ＳＨＣを含有する微生物又は当該微生物の処理物、例えば、死菌化細胞、抽出物、粗精製物等の形態で当該基質に作用させてもよい。
ＳＨＣの基質への接触は、ＳＨＣを適当な溶媒、例えば水性溶媒や緩衝液に溶解又は分散させて行うのが好ましく、円滑な反応、操作の容易性などの点から、緩衝液を用いて行うのがさらに好ましい。また、これに有機溶媒を共存させて接触させることもできる。
When SHC is allowed to act on homofarnesol, a protein that is SHC may be directly acted on the substrate, or a microorganism containing SHC or a processed product of the microorganism, for example, a dead cell, an extract, etc. Alternatively, it may be allowed to act on the substrate in the form of a crude product or the like.
The contact of the SHC with the substrate is preferably performed by dissolving or dispersing the SHC in an appropriate solvent, for example, an aqueous solvent or a buffer solution. From the viewpoint of smooth reaction, ease of operation, etc., the buffer is used. Is more preferable. It can also be contacted with an organic solvent.
本発明の反応は、反応収率の点から、溶媒のｐＨを５．２〜６．６として行うのが好ましく、５．２〜６．０として行うのがより好ましい。好適な溶媒としては、ｐＨ調整の点から、例えば、硫酸、塩酸、リン酸等の鉱酸、酢酸、クエン酸などの有機酸、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム等の無機塩基、酢酸ナトリウム、塩化ナトリウム等の塩を添加した水溶液や、クエン酸緩衝液、酢酸緩衝液、燐酸緩衝液等の緩衝液が挙げられ、クエン酸緩衝液がより好ましい。 The reaction of the present invention is preferably carried out at a solvent pH of 5.2 to 6.6, more preferably 5.2 to 6.0 from the viewpoint of reaction yield. Suitable solvents include, for example, mineral acids such as sulfuric acid, hydrochloric acid and phosphoric acid, organic acids such as acetic acid and citric acid, inorganic bases such as sodium hydroxide, potassium hydroxide and sodium carbonate, acetic acid from the viewpoint of pH adjustment. Examples include aqueous solutions to which salts such as sodium and sodium chloride are added, and buffer solutions such as citrate buffer solution, acetate buffer solution, and phosphate buffer solution, and citrate buffer solution is more preferable.
また、有機溶媒としては、例えば、テトラヒドロフラン、ｔ−ブチルメチルエーテル、イソプロピルエーテル等のエーテル類、トルエン、ヘキサン、シクロヘキサン、ヘプタン、イソオクタン、デカン等の炭化水素類、ｔ−ブタノール、メタノール、エタノール、イソプロパノール、ｎ−ブタノール等のアルコール類、ジメチルスルホキサイドなどのスルホキサイド類、アセトン等のケトン類、アセトニトリル等のニトリル類及びこれらの混合物が挙げられる。 Examples of the organic solvent include ethers such as tetrahydrofuran, t-butyl methyl ether, isopropyl ether, hydrocarbons such as toluene, hexane, cyclohexane, heptane, isooctane, decane, t-butanol, methanol, ethanol, isopropanol. , Alcohols such as n-butanol, sulfoxides such as dimethyl sulfoxide, ketones such as acetone, nitriles such as acetonitrile, and mixtures thereof.
当該基質の濃度は特に限定されないが、０．０１〜３％が好ましく、０．０１〜１％がより好ましい。また、ホモファルネソールは、反応系に一括又は連続的に加えることができる。
尚、ホモファルネソールは、ネロリドールを臭素化し、シアノ化し、加水分解することによりホモファルネシル酸とし、さらに還元することにより得ることができる。
The concentration of the substrate is not particularly limited, but is preferably 0.01 to 3%, more preferably 0.01 to 1%. Further, homofarnesol can be added to the reaction system all at once or continuously.
Homofarnesol can be obtained by brominating, cyanating, and hydrolyzing nerolidol to homofarnesyl acid and further reducing it.
反応は、通常３０〜７０℃、好ましくは５０〜６０℃で、通常０．５〜１００時間程度、好ましくは８〜４８時間程度、振とう、撹拌することで行うことができる。 The reaction is usually performed at 30 to 70 ° C., preferably 50 to 60 ° C., usually for about 0.5 to 100 hours, preferably about 8 to 48 hours, with shaking and stirring.
反応系からの化合物（２）の分離回収は、例えば、化合物（２）を含有する反応系に有機溶剤（例えば、ｎ-ヘキサンなど脂肪族炭化水素系溶剤、酢酸エチル、クロロホルムなど非水溶性の有機溶剤、２−プロパノールなどアルコール類等）を一つもしくは複数添加して十分に攪拌した後、水層と有機層に分液させ、化合物（２）を有機層に移行させ、有機層を水層から分離した後に、有機層の溶剤を留去するか、または蒸留、カラムクロマトグラフィーなどにより処理して、化合物（２）を単離精製する方法等が挙げられる。 Separation and recovery of the compound (2) from the reaction system can be achieved, for example, by adding an organic solvent (for example, an aliphatic hydrocarbon solvent such as n-hexane, water-insoluble such as ethyl acetate or chloroform) to the reaction system containing the compound (2). After adding one or more organic solvents, alcohols such as 2-propanol) and stirring sufficiently, the aqueous layer and the organic layer are separated, the compound (2) is transferred to the organic layer, and the organic layer is washed with water. Examples include a method of isolating and purifying the compound (2) by distilling off the solvent of the organic layer after separation from the layer, or treating by distillation, column chromatography or the like.
以下、実験例を示し、本発明をより具体的に説明する。 Hereinafter, experimental examples will be shown to describe the present invention more specifically.
参考例１ ＳＨＣ蛋白質の大腸菌での発現
（１）ＳＨＣ発現ベクターの作製
Alicyclobacillus acidocaldarius NBRC15652株を復元培地（ＮＢＲＣ８６４培地）にて６０℃で培養し、ＭＯ ＢＩＯ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製ＵｌｔｒａＣｌｅａｎTMＭｉｃｒｏｂｉａｌ ＤＮＡ Ｉｓｏｌａｔｉｏｎ ＫｉｔにてゲノムDNAを回収した。回収したゲノムＤＮＡを鋳型とし、Ｐｙｒｏｂｅｓｔポリメラーゼおよびプライマー（プライマー１:5'-GGGGAGGCATATGGCTGAGCAGTTGGTGGAA-3'（配列番号１）、プライマー２:5'-AACGAATTCTCGTCGCCGAGATCGCCACG-3'（配列番号２））を用いてＰＣＲ反応（９８℃１０秒〜５５℃５秒〜７２℃２分 ３０サイクル）によってＳＨＣ遺伝子断片（１.９ｋｂ）を増幅した。
ＳＨＣ遺伝子断片及びｐＥＴ３ａベクターをそれぞれＴａｋａｒａ Ｂｉｏ社製制限酵素ＮｄｅＩ、ＢａｍＨＩによって切断し、両者をＴＯＹＯＢＯ社製Ｌｉｇａｔｉｏｎ Ｈｉｇｈキットを用いて連結させ、プラスミドｐＥＴ３ａ−ＳＨＣを作製した。
Reference Example 1 Expression of SHC protein in E. coli (1) Preparation of SHC expression vector
Alicyclobacillus acidocaldarius NBRC15652 strain was cultured at 60 ° C. in a reconstitution medium (NBRC864 medium), and genomic DNA was collected with UltraClean ™ MicroDNA DNA Isolation Kit manufactured by MO BIO Laboratories, Inc. Using the recovered genomic DNA as a template, PCR reaction using Pyrobest polymerase and primers (Primer 1: 5'-GGGGAGGCATATGGCTGAGCAGTTGGTGGAA-3 '(SEQ ID NO: 1), Primer 2: 5'-AACGAATTCTCGTCGCCGAGATCGCCACG-3' (SEQ ID NO: 2)) The SHC gene fragment (1.9 kb) was amplified by (98 ° C for 10 seconds to 55 ° C for 5 seconds to 72 ° C for 2 minutes 30 cycles).
The SHC gene fragment and the pET3a vector were each cut with restriction enzymes NdeI and BamHI manufactured by Takara Bio, and both were ligated using a Ligation High kit manufactured by TOYOBO to prepare plasmid pET3a-SHC.
ｐＥＴ３ａ−ＳＨＣを大腸菌ＨＢ１０１株に形質転換し、その形質転換体からＲｏｃｈｅ社製Ｈｉｇｈ Ｐｕｒｅ ＰＣＲ Ｐｒｏｄｕｃｔ Ｐｕｒｉｆｉｃａｔｉｏｎ ＫｉｔにてｐＥＴ３ａ−ＳＨＣを抽出、精製した。 pET3a-SHC was transformed into Escherichia coli HB101, and pET3a-SHC was extracted and purified from the transformant using a High Pure PCR Product Purification Kit manufactured by Roche.
＊８６４培地：Solution A(Yeast Extract １ｇ，(NH4)2SO4・7H2O ０．５ｇ，CaCl2・2H2O ０．２５ｇ，KH2PO4 ０．６ｇ， Distilled water ５００ｍＬ，ｐＨ２．５−３．０), Solution B(Glucose １ｇ， Agar(if need) ２０ｇ， Distilled water ５００ｍＬ，ｐＨ５．５−６．０)、Solution AとSolution Bは個々に滅菌処理した後混合した。 * 864 medium: Solution A (Yeast Extract 1 g, (NH 4 ) 2 SO 4 .7H 2 O 0.5 g, CaCl 2 .2H 2 O 0.25 g, KH 2 PO 4 0.6 g, Distilled water 500 mL, pH 2. 5-3.0), Solution B (Glucose 1 g, Agar (if need) 20 g, Distilled water 500 mL, pH 5.5-6.0), Solution A and Solution B were individually sterilized and mixed.
（２）大腸菌におけるＳＨＣの発現およびＳＨＣの抽出
ｐＥＴ３ａ−ＳＨＣを大腸菌ＢＬ２１ＳｔａｒTM（ＤＥ３）株に形質転換し、その形質転換体をＬＢ−Ａｍｐ培地(Bacto Trypton １％，Bact Yeast Extract ０．５％， NaCl １％， Agar(if need) ０．１５％，アンピシリン１００μｇ／ｍＬ（滅菌処理後添加）)にて３０℃で振とう培養した。菌体濁度(ＯＤ６００)が０．４程度まで増加したところで終濃度１００μｇ／ｍＬになるようにイソプロピル-β-D-チオガラクトピラノシド（ＩＰＴＧ）を添加し、ＳＨＣの発現を誘導した。ＩＰＴＧ添加後６〜８時間培養した後、培養液から菌体を遠心分離し、１％ＴｒｉｔｏｎＸ−１００を添加した３００ｍＭクエン酸バッファー（ｐＨ６．０）に再懸濁して超音波処理にて細胞破砕液を調製した。得られた細胞破砕液は遠心分離によって不溶性画分を除去しＳＨＣ抽出液を得た。
(2) Expression of SHC and extraction of SHC in E. coli pET3a-SHC was transformed into E. coli BL21Star ™ (DE3) strain, and the transformant was LB-Amp medium (Bacto Trypton 1%, Bact Yeast Extract 0.5% , NaCl 1%, Agar (if need) 0.15%, ampicillin 100 μg / mL (added after sterilization treatment) at 30 ° C. with shaking. When the bacterial turbidity (OD600) increased to about 0.4, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 100 μg / mL to induce SHC expression. After culturing for 6 to 8 hours after addition of IPTG, the cells are centrifuged from the culture, resuspended in 300 mM citrate buffer (pH 6.0) supplemented with 1% Triton X-100, and disrupted by sonication. A liquid was prepared. From the obtained cell lysate, the insoluble fraction was removed by centrifugation to obtain an SHC extract.
実施例１ ＳＨＣによるホモファルネソールの変換反応
下記表に示すｐＨになるように１００ｍＭクエン酸緩衝液、０．２％ＴｒｉｔｏｎＸ−１００、０．１％ホモファルネソール、及びＳＨＣ抽出液（蛋白質量５ｍｇ）を混合し、６０℃にて１４時間反応させた。ホモファルネソールは、3Ｚ,7Ｚ-体、3Ｅ,7Ｚ-体、3Ｚ,7Ｅ-体、3Ｅ,7Ｅ-体の４つの異性体混合物である。反応液は５分間氷上に静置して反応を停止させ、酢酸エチルを用いて反応物を抽出し、ガスクロマトグラフィーによって分析した。その結果、生成物として(-)-アンブロキサン及び９-エピアンブロキサンが検出された。(-)-アンブロキサンは３Ｅ,７Ｅ-ホモファルネソールから生成したものである。結果を表１に示す。
Example 1 Conversion reaction of homofarnesol by SHC 100 mM citrate buffer solution, 0.2% Triton X-100, 0.1% homofarnesol, and SHC extract (protein mass 5 mg) were adjusted to the pH shown in the following table. The mixture was mixed and reacted at 60 ° C. for 14 hours. Homofarnesol is a mixture of four isomers: 3Z, 7Z-form, 3E, 7Z-form, 3Z, 7E-form, 3E, 7E-form. The reaction solution was left on ice for 5 minutes to stop the reaction, and the reaction product was extracted with ethyl acetate and analyzed by gas chromatography. As a result, (−)-ambroxan and 9-epiambroxan were detected as products. (−)-Ambroxan is produced from 3E, 7E-homofarnesol. The results are shown in Table 1.
ホモファルネソールの変換反応について、非特許文献５のｐＨ５．０における反応（３.０％）と比較して、飛躍的に高収率でアンブロキサンを得ることができた。また、本反応では、(-)-アンブロキサンを不斉的に得ることができた。 Regarding the conversion reaction of homofarnesol, compared with the reaction (3.0%) at pH 5.0 of Non-Patent Document 5, ambroxan was able to be obtained in a significantly high yield. In this reaction, (-)-ambroxan was obtained asymmetrically.
実施例２ ＳＨＣによるホモファルネソールの変換反応２
ｐＨ５．６になるように８０ｍＭクエン酸緩衝液、０．２％ＴｒｉｔｏｎＸ−１００、０.２％ホモファルネソール、及びＳＨＣ抽出液（蛋白質量５ｍｇ）を混合し、６０℃にて６４時間反応させた。実施例１と同様に反応物を分析したところ、生成物として(-)-アンブロキサン及び９-エピアンブロキサンが検出された。３Ｅ,７Ｅ-ホモファルネソールから生成した(-)-アンブロキサンの変換率は６３％であった。
Example 2 Conversion reaction of homofarnesol by SHC 2
80 mM citrate buffer, 0.2% Triton X-100, 0.2% homofarnesol, and SHC extract (protein mass 5 mg) were mixed so that the pH was 5.6, and reacted at 60 ° C. for 64 hours. . When the reaction product was analyzed in the same manner as in Example 1, (-)-ambroxan and 9-epiambroxan were detected as products. The conversion rate of (−)-ambroxan produced from 3E, 7E-homofarnesol was 63%.
参考例２ ＳＨＣによるスクアレンの変換反応
１００ｍＭクエン酸緩衝液（ｐＨ６．０）、０．２％ＴｒｉｔｏｎＸ−１００、０．１％スクアレン、及びＳＨＣ抽出液（蛋白質量５ｍｇ）を混合し、６０℃にて２４時間反応させた。反応液は５分間氷上に静置して反応を停止させ、酢酸エチルを用いて反応物を抽出し、ガスクロマトグラフィーによって分析した。その結果、生成物としてホペン及びホパノールが検出され、その変換率は１００％であった。
Reference Example 2 Conversion reaction of squalene by SHC 100 mM citrate buffer (pH 6.0), 0.2% Triton X-100, 0.1% squalene, and SHC extract (protein mass 5 mg) were mixed and heated to 60 ° C. For 24 hours. The reaction solution was left on ice for 5 minutes to stop the reaction, and the reaction product was extracted with ethyl acetate and analyzed by gas chromatography. As a result, hopper and hopanol were detected as products, and the conversion rate was 100%.
非特許文献５でのＴＬＣ分析結果と同様に、高選択的にホペン及びホパノールを得ることができた。 Similar to the TLC analysis results in Non-Patent Document 5, hopene and hopanol could be obtained with high selectivity.
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