An Organic Chemist's Guide to Mediated Laccase Oxidation

Abstract Laccases are oxidases that only require O2 as a terminal oxidant. Thus, they provide an attractive green alternative to established alcohol oxidation protocols. However, laccases typically require catalytic amounts of mediator molecules to serve as electron shuttles between the enzyme and desired substrate. Consequently, laccase‐mediator systems are defined by a multitude of parameters such as, e. g., the choice of laccase and mediator, the respective concentrations, pH, and the oxygen source. This complexity and a perceived lack of comparable data throughout literature represent an entry burden into this field. To provide a solid starting point, particularly for organic chemists, we herein provide a time‐resolved, quantitative laccase and mediator screening based on the oxidation of anis alcohol as model reaction. We measured the redox potentials of mediators under the reaction conditions to relate them to their performance. Lastly, for particularly efficient laccase‐mediator pairs, we screened important reaction parameters, resulting in an optimized setup for mediator‐assisted laccase catalyzed oxidations.


Optical rotation
To measure the optical rotation of the chiral compounds the modular circular polarimeter 500 (MCP 500) from Anton Paar with a 100 mm long cuvette of a 3 mm diameter was used. The measurements took place at 20 °C and a wavelength of 589 nm with c = 1.0 for most substances as indicated at the respective protocols.
UV/VIS measurements UV/VIS measurements were performed on a plate reader Zenyth 3100 from Anthos.

Oxygen-meter
Oxygen saturation measurements were carried out with Firesting-O2. The software for processing was Pyro Workbench.

Nuclear Magnetic Resonance (NMR)
NMR spectra were recorded on a Brucker Avance UltraShield 400 spectrometer (400 MHz machine) or a Brucker Avance III HD 600 spectrometer (600 MHz machine). Spectra were calibrated to the solvent residual signal [1] . Coupling constants (J) are given in Hz and chemical shifts (δ) in ppm. Assignments are based on COSY, HSQC and HMBC spectra and follow IUPAC nomenclature. Determined according to phase-sensitive experiments (HSQC, APT, DEPT or DEPTQ) for 13 C-NMRs virtual coupling patterns are given for (s, d, t, q for CH3, CH2, CH, C).
For in-situ reduction N-oxyl radicals in the NMR tube, addition of 55 mg of phenylhydrazine in 0.6 ml CDCl3 was utilized, following a modified literature protocol. [2] Gas Chromatography (GC) GC analysis of anisaldehyde oxidations was carried out on a Trace Dual GC, equipped with a standard capillary column (BGB5, 30 m x 0.25 mm ID, 0.50 μm film) with an FID detector. Carrier gas: helium, injector: 230 °C; column flow: 2.0 ml/min; method for quantification: 80 °C (0°C/min,1 min) → 80−280 °C (40 °C/min, 7 min). The software for processing used was Chromeleon.

Melting points
Melting points were recorded on a Kofler-type Leica Galen III and are uncorrected

Enzyme activity measurement
The enzyme activity was determined photometrically at 25°C using ABTS as substrate. Into a well plate, 50 µL of a 0.01 M ABTS solution and then, 170 µL of a 0.05 mg/ml solution of laccase was added. The absorbance change was recorded at 405 nm with 5-sec pre-read shake (ε(ABTS)=36.8 lꞏmmol -1 ꞏcm -1 at 420 nm). The activity was determined from the linear range of the curve. Equation 21 describes the slope of the linear section of the curve:

A.2. Control Experiments
The set-up of our model system using anisaldehyde is depicted in Scheme 1 and the control experiments in Table 1: Scheme 1: Reaction conditions of the laccase-mediated oxidation of anis alcohol as the model substrate

A.3. Determination of Influence of Redox-State of Mediators in the Laccase-Mediated Oxidation of Anis Alcohol
Accompanying our mediator screening, we conducted a short study on the influence of oxidation state on an N-oxyl based mediator on its reactivity in the laccase-mediated oxidation of anis alcohol. According to the mechanism of the laccase-mediated oxidation of benzyl alcohol, elucidated by Tromp et al. [3] , this should not lead to differences in mediator performance. In order to conduct this investigation, methoxy-TEMPO (8) was converted into its respective oxidized and reduced forms acordding to literature protocols [4] (Scheme 2). All species (15, 8, and 16) were screened via our anis alcohol system.
Scheme 2: Conversion of methoxy-TEMPO (8) in the reduced form (15) and the oxidized form (16). Table 2, there is no significant difference in mediator performance between the three possible oxidation states. This implies that similar results can be expected regardless of which species is provided. This was reassuring, as we utilized AZADOL (3) instead of even more expensive AZADO (iii) in our following mediator screening. Additionally, this information is beneficial for those who develop synthetic routes towards new N-oxyl mediators, as those can be simplified by targeting the most accessible or stable oxidation state. Spectral data in accordance with the literature. [5] S6 A.4.2. Synthesis of 4-methoxy-2,2,6,6-tetramethyl-1-oxopiperidinium tetrafluoroborate 25

As shown in
Procedure: Oxidation Following a modified literature protocol [4b] , methoxy-TEMPO 11 (2.00 g, 10.7 mmol, 1.00 equiv.) was dispersed in H2O (6.4 ml). At room temperature, HBF4 (48%, 1.96 g, 10.7 mmol, 1.00 equiv.) was slowly added dropwise over a period of 10 min. After the orange slurry turned to a yellow color, NaOCl (2.95 g, 5.37 mmol, 0.50 equiv.) was added over 15 min at 0 °C and stirred for an additional 1 h at 0 °C. The reaction mixture was filtered, and the yellow crystalline precipitate was washed with ice-cold 5% NaHCO3 (4 ml), water (8 ml), and ice-cold Et2O (80 ml) to yield the product as the bright yellow solid. The solid was dried in a desiccator overnight, yielding 1.77 g of a solid. 1 H-NMR indicated that the product was 99% pure. An aliquot of 1.00 g of the crude product was recrystallized in 14 ml H2O yielding 780 mg (78% recovery) of target product 16 in yellow needles. Spectral data in accordance with the literature. [6] *The recrystallization of an already pure product (according to 1 H-NMR) was carried out to determine whether the decomposition at 97°C still takes place for recrystallized product 25, which it did.

A.5. Quantification of Screenings via Quantitative NMR for Anis Aldehyde System
The NMR samples in CDCl3 supplemented with 20 mM 3,4,5-trimethoxy benzaldehyde 17 as internal standard were quantified via equation 1.
mx, mstd…masses of analyte x and internal standard std in g MWx and MWstd…molecular weights in g/mol Px and Pstd…purities (=1) nHx and nHstd…number protons generating the selected signals for integration Ax and Astd…areas for the selected peak Table 3 shows the selected 1 H-NMR signals used for quantification in the anis aldehyde system. The results using equation 1 of those signals were then averaged for each compound to lead to an average mass of analyte x. One example of an 1 H-NMR of an anis alcohol is shown in Figure 1.

A.6. Investigations of the Stoichiometric Oxidant
Another crucial factor determining the efficacy of a laccase-mediator system is the accessibility of the stoichiometric oxidant, i.e. molecular oxygen. According to procedures usually found in the literature, the reaction mixture is saturated via an external oxygen source (usually an oxygen balloon on a syringe that is put into the solution). After saturation, then the oxygen source is connected via the gas phase to the reaction mixture (e.g., the balloon is placed on a septum of a closed vial or flask). We were particularly interested in following the oxygen saturation of the reaction mixture over time using the standard protocol for our anis alcohol oxidation. We utilized an oxygen meter to follow the oxygen concentration during a laccase-TEMPO mediated oxidation of anis alcohol according to our standard screening conditions (Scheme 3), as shown in Figure 2. The diagram in Figure 2 shows the oxygen saturation in the reaction mixture over time. The first drastic increase of oxygen concentration arises from the addition of an oxygen balloon. Regarding the O2-intake, it is very common to saturate reaction mixtures of several milliliters before applying the laccase for extended periods of time, i.e., 30 min in the Arends publication. [7] In our case, however, full saturation (~450 % air sat.) was achieved within only two minutes. This is an important realization for the time-saving performing of reactions.
After full saturation, the oxygen balloon was removed. This caused no significant change to the oxygen saturation in the reaction mixture (without laccase). Upon the addition of the laccase, the oxygen saturation in the reaction mixture decreases almost linearly from around 450% to near 0% air sat. If another oxygen balloon was applied, the re-saturation occurred.