Imidazole → Diethylstilbestrol

HRID 1487812Notes

Reaction equation

HRID 1487812 reaction equation
CC1(C)S[C@@H]2[C@H](N[C:11]([C@H:13](N)[C:14]3[CH:15]=[CH:16][CH:17]=[CH:18][CH:19]=3)=O)C(=O)N2[C@H]1C(O)=O.CC(S[C@@H:29]1O[C@H:33]([CH2:35]O)[C@H:32](O)[C@H:31]([OH:38])[C@H:30]1O)C.C(O)[C:41](N)([CH2:44]O)[CH2:42][OH:43].[Na+].[Cl-].N1C=CN=[CH:51]1>OCC(CO)O>[CH3:17][CH2:16]/[C:15](/[C:35]1[CH:33]=[CH:32][C:31]([OH:38])=[CH:30][CH:29]=1)=[C:14](\[C:19]1[CH:18]=[CH:51][C:42]([OH:43])=[CH:41][CH:44]=1)/[CH2:13][CH3:11]

Participants

7

Reactant 5

HCID 795ImidazoleHCID 5234Sodium ChlorideHCID 6249AmpicillinHCID 6503Tris(hydroxymethyl)aminomethaneHCID 656894isopropyl beta-D-thiogalactopyranoside

Product 1

HCID 448537Diethylstilbestrol

Reagents, catalysts & solvents 1

HCID 753GlycerolSolvent

Procedure

Cells of E. coli 7Δ harboring pELAC-MBP-DES-HT was grown in LB medium containing 100 μg/mL ampicillin in a test tube at 37° C. up to OD610nm˜2. 2 mL of the resulting culture was inoculated into 100 mL of LB medium supplemented with IPTG (final concentration of 0.1 mmol/L) in a 500 ml Sakaguchi flask at 30° C., for 8 hours. Induced cells were harvested from 1.6 L of cultivation broth, re-suspended in 200-240 mL of HT-II buffer (50 mM Tris-HCL, pH 8.0, 0.3 M NaCl, 10 mM imidazole, 15% glycerol), and sonicated using sonicator (INSONATOR 201M, KUBOTA). The debris was removed by centrifugation at 4° C. and 14,000 rpm for 15 minutes followed by filtration through 0.45 mm filter (Millipore). A solution of crude proteins was loaded onto HisTALON Superflow cartridge, 5 ml (Clontech) using AKTA avant 25 (GE Healthcare) in accordance with the manufacturer's recommendations. Fractions containing MBP fused His6-tagged DES were combined, and desalted using PD-10 columns (GE Healthcare) equilibrated with SC-buffer (50 mM Tris-HCl, pH8.0, 0.3 M NaCl, 15% glycerol).

Workup

  1. customsonicated
  2. customThe debris was removed by centrifugation at 4° C.
  3. filtration14,000 rpm for 15 minutes followed by filtration through 0.45 mm
  4. filtrationfilter (Millipore)
  5. additionFractions containing MBP

Sources & evidence

ORD record
ord-c3c83c6ec1d242dab994a04706ea86ce
Source dataset
uspto-grants-2016_08
Patent number
US09428783B2

Reaction notes

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