Hydrocarbon Synthesis via Photoenzymatic Decarboxylation of Carboxylic Acids

A recently discovered photodecarboxylase from Chlorella variabilis NC64A (CvFAP) bears the promise for the efficient and selective synthesis of hydrocarbons from carboxylic acids. CvFAP, however, exhibits a clear preference for long-chain fatty acids thereby limiting its broad applicability. In this contribution, we demonstrate that the decoy molecule approach enables conversion of a broad range of carboxylic acids by filling up the vacant substrate access channel of the photodecarboxylase. These results not only demonstrate a practical application of a unique, photoactivated enzyme but also pave the way to selective production of short-chain alkanes from waste carboxylic acids under mild reaction conditions.


Photoenzymatic synthesis of methane and other light hydrocarbons
The photoenzymatic reactions using CvFAP were performed at 30 °C in total volume of 1.0 mL Tris-HCl buffer (pH 8.5, 100 mM) containing 20% cosolvent (e.g. ethanol or dimethyl sulfoxide (DMSO)). Unless mentioned otherwise, 200 μL of DMSO solution containing 75 mM of tridecane, 300 μL of acetic acid stock solution (500 mM in Tris-HCl buffer, pH 8.5) and 400 μL of Tris-HCl buffer (pH 8.5, 100 mM) were added to a transparent glass vial (total volume 5.0 mL). After the mixture was homogenized under magnetic stirring, 100 μL of CvFAP stock solution (60 μM cell extract in Tris-HCl buffer) was added. The vial was sealed tightly and exposed to blue LED light at 30 °C under gentle magnetic stirring. The homemade setup is shown in Figure S5 Figure S4. After 3 hours, 1.5 mL of the gas in the headspace of the reaction vial (4.0 mL headspace + 1.0 ml liquid) was extracted by a syringe and analysed by Gas Chromatography. The photobiocatalytic experiments were carried out in duplicates from two distinct samples unless indicated otherwise.

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The gas-phase products in the glass vials were extracted with a syringe, injected into the gas-sampling loop of a gas chromatograph (CompactGC 4.0, interscience) equipped with a flame ionization detector (FID, connected to two Rtx-1 capillary columns). Helium was used as carrier gas for the quantification of the hydrocarbons. A typical chromatogram of hydrocarbons detected with the FID is shown in Figure S3, which shows that methane (CH 4 ), ethane (C 2 H 6 ), ethylene (C 2 H 4 ), propane (C 3 H 8 ), isobutane (i-C 4 H 10 ), butane (C 4 H 10 ), isopentane (i-C 5 H 12 ) and pentane (C 5 H 12 ) could be identified at distinct retention times. The peak area for the hydrocarbon products were compared to standards (calibration gases) to obtain the corresponding concentration. Most photobiocatalytic experiments were carried out in duplicates from two distinct samples. Please note that due to the lack of standard gas of propylene and acetylene, the concentration was estimated based on their response factors (0.652 for propylene and 1.07 for acetylene) and single peak observed in GC. 1 The dilution factor of the sampling was determined. The hydrocarbons produced by the photoenzyme ended up in the headspace (total of 4 mL) of the air-tight glass vial. For the GC measurements, 1.5 mL of the headspace was extracted by a syringe. This extraction procedure caused a slight underpressure inside the syringe and the vial, compared to ambient pressure. Therefore, air flowed into the syringe when the syringe was taken out of the gastight vial, diluting the concentration of products in the syringe. Furthermore, the injection process into the GC may further dilute the sample. In order to correct for these dilutions, the dilution factor (DF) of the concentration of products resulting from the extraction and injection procedure was determined. To do so, the vial was filled with standard gases with discrete known CH 4 concentration and the exact sampling procedures were repeated. As presented in Table S1, a DF of 2 was determined independently of the initial CH 4 concentration. Therefore, the actual concentration of product in the headspace the vial is 2-fold higher than that detected via GC.
The total amount of hydrocarbons (mole, N) in the headspace of the vial produced during the reaction was calculated by equation (1): where, C is the concentration of hydrocarbons detected by GC, and 2 is the DF. V and V m are the vial's headspace volume (0.004 L) and the molar volume of ideal gas at room temperature and atmospheric pressure, respectively.

DFT and TD-DFT methodology description
All molecular DFT calculations were carried using the hybrid PBE0 (PBE1PBE) exchange-correlation functional 3 as implemented in Gaussian 09 D.01 program. 4 The all electron 6-311+G(d,p) basis set was used for all atoms.
The polarized continuum model (PCM) with standard parameters for water solvent was used to account for bulk solvent effects during geometry optimization, frequency analysis and excited state calculations. The excited state calculations and simulation of UV-Vis spectra were carried out within the time-dependent DFT formalism. 5 Previous studies confirmed the good accuracy of this methodology for the description of excited-state reactivity and photoexcitation processes. [6][7][8][9] S 23