Reaction #1573411

ord-44857b5b9e014069a38d644e4a6daf46

Reaction equation

NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADH
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
O=S(=O)([O-])[O-].[Mg+2]
MgSO4
O=C(CCCC(=O)N1C(=O)OC[C@@H]1c1ccccc1)c1ccc(F)cc1
(S)-1-(4-Fluoro-phenyl)-5-(2-oxo-4-phenyl-oxazolidin-3-yl)-pentane-1,5-dione
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
CC(C[*:1])[*:2].[Al]
aluminum polypropylene
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
→
O=C(CCCC(=O)N1COC[C@@H]1C1C=CC=CC1=O)c1ccc(F)cc1
5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione

Reactants

NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADH
—
organic layer
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
O=S(=O)([O-])[O-].[Mg+2]
MgSO4
O=C(CCCC(=O)N1C(=O)OC[C@@H]1c1ccccc1)c1ccc(F)cc1
(S)-1-(4-Fluoro-phenyl)-5-(2-oxo-4-phenyl-oxazolidin-3-yl)-pentane-1,5-dione
—
Na NADP
—
IA isopropyl alcohol
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
CC(C[*:1])[*:2].[Al]
aluminum polypropylene
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH

Reagents

None

Solvents

CC(C)O
isopropylalcohol
—
triethanolamine(chloride)
COC(C)(C)C
MTBE
CC#N
acetonitrile

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITION100 μl of cell lysate was added to a deep well plate (Costar #3960)
  2. 2
    Temperatureheat
  3. 3
    Otherseal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), reactions
  4. 4
    workup.WAITwere run for at least 16 hrs at ambient temperature
  5. 5
    workup.ADDITIONwas added per well
  6. 6
    Othercentrifuged (4000 rpm, 10 min, 4° C.)

Procedure

KREDs described in Table 5 of Example 1 are screened using NADH and NADPH as cofactors and glucose dehydrogenase/glucose or isopropylalcohol (“IPA”) as co-factor regeneration system. 100 μl of cell lysate was added to a deep well plate (Costar #3960) containing 25 μl 5 mg/ml Na-NADP (Oriental Yeast) and 2 mM MgSO4 in 100 mM triethanolamine(chloride) (pH7.0), and 125 IA isopropyl alcohol containing 2 g/L (S)-1-(4-Fluoro-phenyl)-5-(2-oxo-4-phenyl-oxazolidin-3-yl)-pentane-1,5-dione. After sealing the plates with aluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), reactions were run for at least 16 hrs at ambient temperature. At the end of the reaction 1 ml acetonitrile (for reversed phase HPLC) or MTBE (for normal phase HPLC) was added per well. Plates were resealed, shaken for 20 minutes, and centrifuged (4000 rpm, 10 min, 4° C.). 200 μl of the organic layer was transferred into a new shallow-well microtiter plate for analysis.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US09422530B2uspto-grants-2016_08