Figure 4.

Effects of varying the dose (1, 3.3, 10, 33, and 100 μm) of ring D-modified GA derivatives on the production of GA₁ from substrate GA₂₀ in the presence of AtGA3ox1. The same amount of enzyme was added to each reaction mixture. The initial GA₂₀ concentration was 10 μm. Cofactors added included 0.1 mm Fe²⁺, 5 mm ascorbate, 5 mm 2-oxoglutarate, 2 mm NADPH, and 2 mg BSA mL⁻¹. The control is 10 μm GA₂₀ alone, plus cofactors (white circle, upper left). A, endo-dihydro GA₅ (r² = 0.98); B, exo-dihydro GA₅ (r² = 0.82); C, endo-dihydro GA₂₀ (r² = 0.95); D, exo-dihydro GA₂₀ (r² = 0.89). The reaction period was 10 min. Quantification of GA₁ was accomplished by GC-MS-SIM using a known amount of [²H₂]GA₁ which was added after stopping the reaction mixture, just before extraction with EtOAc.