Ampicillin → (R)-4-cyano-3-hydroxybutyric acid

HRID 414886Notes

Reaction equation

HRID 414886 reaction equation
[CH3:1][C:2]1(C)S[C@@H]2[C@H](NC([C@H](N)C3C=CC=CC=3)=O)C(=O)N2[C@H:3]1[C:21]([OH:23])=[O:22].CC(S[C@@H]1[O:34][C@H](CO)[C@H](O)[C@H](O)[C@H]1O)C.[Si]([CH:44]=[N+:45]=[N-])(C)(C)C>CCOC(C)=O>[OH:34][C@H:2]([CH2:1][C:44]#[N:45])[CH2:3][C:21]([OH:23])=[O:22]

Participants

5

Reactant 3

HCID 6249AmpicillinHCID 167693TrimethylsilyldiazomethaneHCID 656894isopropyl beta-D-thiogalactopyranoside

Product 1

HCID 10236005(R)-4-cyano-3-hydroxybutyric acid

Reagents, catalysts & solvents 1

HCID 8857Ethyl AcetateSolvent

Procedure

Putative nitrilase up-mutants were assayed in triplicate. Each transformant was grown in 5 mL LB (100 μg/mL ampicillin), at 37° C., 220 rpm for 18 h. The overnight culture was diluted 2-fold and nitrilase expression induced at 37° C., 220 rpm with 0.1 mM IPTG for 6 h. Cells were harvested by centrifugation, washed in 100 mM pH 7 sodium phosphate buffer and then re-suspended in 1 mL of 100 mM HGN in 100 mM pH 7 sodium phosphate buffer. Reactions were allowed to proceed for at least 36 h at 22° C. with gentle agitation. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.5, nitrile; Rf=0.0, acid). Cells and other debris were removed by centrifugation and the treated with one volume methanol prior to lyophilization. The lyophilizate was re-suspended in methanol and treated with TMS-diazomethane (10 equivalents, 2 M solution in hexanes) until gas evolution ceased and yellow color persisted in order to prepare the methyl ester for GC analysis. Selected nitrilase variants producing (R)-(−)-3-hydroxy-4-cyanobutyric acid of 95% ee or greater were then evaluated for performance at 2.25 M HGN.

Workup

  1. additionThe overnight culture was diluted 2-fold
  2. custominduced at 37° C.
  3. washwashed in 100 mM pH 7 sodium phosphate buffer
  4. waitto proceed for at least 36 h at 22° C. with gentle agitation
  5. customReaction progress
  6. customCells and other debris were removed by centrifugation
  7. additionthe treated with one volume methanol

Sources & evidence

ORD record
ord-546e0220487c4ba2b06ab44e1d21749e
Source dataset
uspto-grants-2012_01
Patent number
US08088613B2

Reaction notes

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