NADPH → (4aR,4bS,6aS,7S,9aS,9bS,11aR)-N-(tert-butyl)-4a,6a-dimethyl-2-oxohexadecahydro-1H-indeno(5,4-f)quinoline-7-carboxamide

HRID 2256813

반응식

HRID 2256813 반응식
[CH3:1][C@@:2]12[C@@H:10]([C:11]([NH:13][C:14]([CH3:17])([CH3:16])[CH3:15])=[O:12])[CH2:9][CH2:8][C@H:7]1[C@@H:6]1[CH2:18][CH2:19][C@H:20]3[NH:26][C:24](=[O:25])[CH:23]=[CH:22][C@:21]3([CH3:27])[C@H:5]1[CH2:4][CH2:3]2.C1N=C(N)C2N=CN([C@@H]3O[C@H](COP(OP(OC[C@H]4O[C@@H](N5C=C(C(N)=O)CC=C5)[C@H](O)[C@@H]4O)(O)=O)(O)=O)[C@@H](O)[C@H]3OP(O)(O)=O)C=2N=1.C1N(CCCS(O)(=O)=O)CCOC1.C(O)[C@H]1O[C@H](O[C@]2(CO)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O.C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O>>[CH3:1][C@@:2]12[C@@H:10]([C:11]([NH:13][C:14]([CH3:15])([CH3:16])[CH3:17])=[O:12])[CH2:9][CH2:8][C@H:7]1[C@@H:6]1[CH2:18][CH2:19][C@H:20]3[NH:26][C:24](=[O:25])[CH2:23][CH2:22][C@:21]3([CH3:27])[C@H:5]1[CH2:4][CH2:3]2

참가 물질

6

반응물 5

HCID 5884NADPHHCID 5988SucroseHCID 6049Ethylenediaminetetraacetic AcidHCID 57363FinasterideHCID 70807Mops

생성물 1

HCID 9864387(4aR,4bS,6aS,7S,9aS,9bS,11aR)-N-(tert-butyl)-4a,6a-dimethyl-2-oxohexadecahydro-1H-indeno(5,4-f)quinoline-7-carboxamide

실험 절차

To obtain sufficient transformed inhibitor for mass spectral analysis, a preparation of baculovirus-expressed enzyme was employed that consisted of all protein sedimenting between 1,000 to 100,000×g. The enzyme-[3H]finasteride complex was formed in a solution containing 1.39 μM enzyme (41,000 units, 141 mg protein), 1.14 μM unlabeled finasteride (15.0 nmol), 0.0113 uM tracer [3H]finasteride (1.56 μCi), and 573 μM NADPH, in a buffer composed of 0.1M MOPS, 1M sucrose, and 10 mM EDTA at pH 7.20 in a total volume of 13.1 mL. The enzyme was dispersed in the solution with a dounce and stirred for 45 min as it warmed to room temperature, which produced 73% inhibition. The solution was then dialyzed overnight at 4° C. in 14,000 Da cutoff dialysis tubing (SPECTRAPOR 2) against 2 changes of 4L water containing 1 mM MOPS and 1 mM EDTA at pH 7.20. This resulted in 88% retention of the radioactivity. (Analysis of the radioactivity lost to dialysis indicated 62% chromatographed as [3H]dihydrofinasteride, and the remainder as [3H]finasteride. This corresponds to loss of 8% of the total starting radioactivity as [3H]dihydrofinasteride, implying a partition ratio of 1.08 in this instance.)

후처리

  1. customTo obtain sufficient
  2. customa preparation of baculovirus-expressed enzyme
  3. customThe enzyme-[3H]finasteride complex was formed in a solution
  4. additionThe enzyme was dispersed in the solution with a dounce
  5. customwhich produced 73% inhibition
  6. customThe solution was then dialyzed overnight at 4° C. in 14,000 Da cutoff dialysis tubing (SPECTRAPOR 2) against 2 changes of 4L water containing 1 mM MOPS and 1 mM EDTA at pH 7.20
  7. customThis resulted in 88% retention of the radioactivity
  8. custom(Analysis of the radioactivity lost to dialysis
  9. customchromatographed as [3H]dihydrofinasteride

출처 및 근거

ORD 기록
ord-d573dc965959415a872967e2c02b2cc3
출처 데이터셋
uspto-grants-1997_08
특허 번호
US05656613

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