Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation

MOPS acts as a Good buffer and electron donor and prevents the degradation of catalysts by reactive species in aerated photobiocatalysis.


General considerations
All chemicals were obtained from Sigma Aldrich (highest purity available) and used without further purification unless otherwise stated.

Enzyme Activity and Stability Measurements
Enzyme activities were measured by monitoring the substrate-dependent decrease in NADPH absorbance at 340 nm (ε 340 = 6.22 mM -1 cm -1 ) in 100 mM Tris-HCl or 100 mM MOPS (pH 7.5).
Standard assays for the activity measurement of CHMO Acineto contained 0.05 μM enzyme, 100 μM NADPH and 0.5 mM cyclohexanone. Measurements were done according to a previously published procedure 1 . Oxidation of NADPH was followed for 120 s at 30 ºC in a Shimadzu spectrophotometer (UV-1800) featuring a thermo-controlled 6-cell positioner (CPS-240A). All kinetic measurements were performed in triplicate unless otherwise stated. Enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute under the specified conditions. Specific activities were calculated from the observed rate constants (k obs ), which were obtained by fitting the initial rate of the absorbance changes to a linear regression (Origin 2017 for Windows). Stability measurements were performed by incubating 10 μM enzyme at 30 ºC in 100 mM Tris-HCl or 100 mM MOPS, pH 7.5, containing 100 μM FAD (CHMO Acineto ). Aliquots were taken at different time points and added to a cuvette containing 0.1 mM NADPH and 0.5 mM substrate to test for catalytic activity. Experimental data were fitted to an exponential decay equation using the Origin Pro software (Origin 8.5 for Windows). Data are reported as ± 1 SD ̅ (n=3) unless otherwise stated.

Light Source
For the daylight lamp experiments, the lamp was placed at 20 cm distance from the 96 well-plate.
The light intensity on the plate level was measured with an Ocean Optics Spectrophotometer (USB2000+) at 450 nm for the daylight lamp (28 μW/cm 2 ). The region where the light intensity remained the same was determined and the experiments performed in the area where the amount of energy was constant.

Gas Chromatography (GC) Analysis
For GC analysis, 50 μL of the reaction mixture were added to 1.5 mL Eppendorf tubes containing 150 μL ethyl acetate supplemented with 1 mM methyl benzoate as internal standard. Samples were vortexed at maximum speed (IKA Vortex 4 basic) for 30 s and centrifuged for 1 min (VWR Silverstar bench top centrifuge). The organic layer was transferred into a new 1.5 mL Eppendorf tube and dried over Na 2 SO 4 . After centrifugation, the supernatant was transferred to a 1.5 mL GC glass vial equipped with a 0.1 mL micro-insert and subjected to GC analysis. For that, 50 μL aliquots were taken over time and added to 2 mM ABTS and 5.8 U/mL HRP to a final volume of 500 μL. The absorption spectrum was recorded for each sample and the amount of H 2 O 2 quantified after calibration curve with commercial H 2 O 2 at 734 nm (intercept = 0.067; slope = 0.02702 ± 0.0008, Adj-R 2 > 0.99).

Synthesis of 2,2,6-trimethyl-1,4-cyclohexanedione (levodione)
Ketoisophorone (1a) (500 mg, 3.3 mmol, 1 eq.) was placed in a 3-neck-round bottomed flask and ethyl acetate (5 mL) was added. 75 mg of Palladium on charcoal (75 mg, 10% w/w were added to the mixture and the mixture was evacuated and purged with argon three times. After the last purging step, the evacuated state was maintained and the Schlenk-line tube was exchanged with a hydrogen balloon. The apparatus was flushed with H 2 and the mixture was stirred overnight. Upon reaction completion (monitored by GC/MS analysis), the reaction mixture was filtered over Celite and the solvent was removed under reduced pressure (428 mg crude yield). The crude product (100 mg) was mixed with Dess-Martin-Periodinane (DMP, 300 mg, 0.7 mmol, 1.06 eq. vs 1c) in CH 2 Cl 2 and stirred for 15 minutes at room temperature. After completion (monitored by TLC), the reaction was extracted with a 10% w/v Na 2 S 2 O 3 solution followed by extraction with a saturated solution of NH 4 Cl. The aqueous layers were re-extracted with CH 2 Cl 2 and the resulting organic layer was dried over Na 2 SO 4 and filtered through silica. The solvent was removed under reduced pressure resulting in a beige solid (1b, 42 mg).