3-Chlorobenzoic Acid → (R)-3-hydroxyoctanoyl-CoA

HRID 895801

반응식

HRID 895801 반응식
[P:1]([O:13][CH2:14][C@H:15]1[O:19][C@@H:18]([N:20]2[C:29]3[N:28]=[CH:27][N:26]=[C:24]([NH2:25])[C:23]=3[N:22]=[CH:21]2)[C@H:17]([OH:30])[C@@H:16]1[OH:31])([O:4][P:5]([O:8]P(O)(O)=O)([OH:7])=[O:6])(=[O:3])[OH:2].[Mg+2].[Cl-].[Cl-].[CH3:35][C:36]([C@@H:70]([OH:82])[C:71]([NH:73][CH2:74][CH2:75][C:76]([NH:78][CH2:79][CH2:80][SH:81])=[O:77])=[O:72])([CH2:38]OP(OP(OC[C@H]1O[C@@H](N2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1OP(O)(O)=O)(O)=O)(O)=O)[CH3:37].[OH:83][C@H:84]([CH2:89][CH2:90][CH2:91][CH2:92][CH3:93])[CH2:85][C:86]([O-])=[O:87].S(=O)(=O)(O)O.O[C@H](CCC1C=CC=CC=1)CC(SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)C[O:116][P:117](O)(=[O:145])[O:118]P(O)(=O)OC[C@H]1O[C@@H](N2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1OP(O)(O)=O)=O.O[C@H](CCC1C=CC(F)=CC=1)CC(SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)COP(O)(=O)OP(O)(=O)OC[C@H]1O[C@@H](N2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1OP(O)(O)=O)=O.O[C@H](CCC[C@@H]1OC1)CC(SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)COP(O)(=O)OP(O)(=O)OC[C@H]1O[C@@H](N2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1OP(O)(O)=O)=O.O[C@H](CCC[C@@H]1OC1)CC(O)=O.OC(CCCC=C)CC(O)=O.ClC1C=C(C=CC=1)C(O)=O>>[OH:83][C@H:84]([CH2:89][CH2:90][CH2:91][CH2:92][CH3:93])[CH2:85][C:86]([S:81][CH2:80][CH2:79][NH:78][C:76](=[O:77])[CH2:75][CH2:74][NH:73][C:71](=[O:72])[C@H:70]([OH:82])[C:36]([CH3:35])([CH3:37])[CH2:38][O:8][P:5]([OH:7])(=[O:6])[O:4][P:1]([OH:2])(=[O:3])[O:13][CH2:14][C@H:15]1[O:19][C@@H:18]([N:20]2[C:29]3[N:28]=[CH:27][N:26]=[C:24]([NH2:25])[C:23]=3[N:22]=[CH:21]2)[C@H:17]([OH:30])[C@@H:16]1[O:31][P:117]([OH:145])([OH:118])=[O:116])=[O:87]

참가 물질

12

반응물 11

HCID 4473-Chlorobenzoic AcidHCID 1118Sulfuric AcidHCID 59575'-AtpHCID 87642Coenzyme AHCID 1292433-Hydroxy-7-octenoic acidHCID 5360315Magnesium ChlorideHCID 86308678(R)-3-hydroxyoctanoateHCID 86588296(R)-3-Hydroxy-5-phenylvaleryl-CoAHCID 86588297(R)-3-Hydroxy-5-(4-fluorophenyl)valeryl-CoAHCID 86590092(R,S)-3-Hydroxy-7,8-epoxyoctanoyl-CoA2회HCID 86590093(R,S)-3-hydroxy-7,8-epoxyoctanoic acid

생성물 1

HCID 11966193(R)-3-hydroxyoctanoyl-CoA

실험 절차

(R)-3-hydroxyoctanoyl-CoA was synthesized in accordance with the following procedure, based on the method of Rehm BHA, Kruger N, Steinbuchel A (1998) Journal of Biological Chemistry 273 pp 24044-24051, with the method slightly modified. Acyl-CoA synthetase (manufactured by Sigma Co., Ltd.) was dissolved in a tris hydrochloric buffer solution (50 mM, pH 7.5) containing 2 mM ATP, 5 mM MgCl2, 2 mM CoA and 2 mM (R)-3-hydroxyoctanoate so that the concentration was 0.1 milliunit per microliter. The solution was stored in a hot bat at 37° C., and was sampled at appropriate times to analyze the progress of the reaction by HPLC. Sulfuric acid was added in the sampled reaction solution to make a concentration 0.02 N to stop the enzyme reaction, and thereafter (R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane and removed. For the analysis by HPLC, using a RP18 column (nucleosil C18, 7 μm, Knauser), elution was conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3) as a mobile phase, and absorption spectra of 200 to 500 nm were monitored by a diode array detector, thereby detecting a thioester compound produced through the enzyme reaction. In a similar way, (R)-3-hydroxy-5-phenylvaleryl CoA, (R)-3-hydroxy-5-(4-fluorophenyl) valeryl CoA, (R,S)-3-hydroxy-5-phenoxyvaleryl CoA and (R,S)-3-hydroxy-7,8-epoxyoctanoyl CoA were prepared. Furthermore, (R,S)-3-hydroxy-7,8-epoxyoctanoic acid for use in preparation of (R,S)-3-hydroxy-7,8-epoxyoctanoyl CoA was prepared by epoxidizing unsaturated parts of 3-hydroxy-7-octenoic acid synthesized by the method described in Int. J. Biol. Macromol., 12, 85-91 (1990) with 3-chlorobenzoic acid.

후처리

  1. concentrationso that the concentration
  2. customwas stored in a hot bat at 37° C.
  3. aliquotwas sampled at appropriate times
  4. customthe progress of the reaction by HPLC
  5. customthe enzyme reaction
  6. extraction(R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane
  7. customremoved
  8. washa RP18 column (nucleosil C18, 7 μm, Knauser), elution
  9. concentrationwas conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3)
  10. customas a mobile phase, and absorption spectra of 200 to 500 nm
  11. customproduced through the enzyme reaction
  12. customwas prepared
  13. customsynthesized by the method

출처 및 근거

ORD 기록
ord-c73e303e646b4271b73b699634af9a5f
출처 데이터셋
uspto-grants-2005_07
특허 번호
US06916861B2

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