Nature stays natural: two novel chemo-enzymatic one-pot cascades for the synthesis of fragrance and flavor aldehydes

Novel synthetic strategies for the production of high-value chemicals based on the 12 principles of green chemistry are highly desired. Herein, we present a proof of concept for two novel chemo-enzymatic one-pot cascades allowing for the production of valuable fragrance and flavor aldehydes. We utilized renewable phenylpropenes, such as eugenol from cloves or estragole from estragon, as starting materials. For the first strategy, Pd-catalyzed isomerization of the allylic double bond and subsequent enzyme-mediated (aromatic dioxygenase, ADO) alkene cleavage were performed to obtain the desired aldehydes. In the second route, the double bond was oxidized to the corresponding ketone via a copper-free Wacker oxidation protocol followed by enzymatic Baeyer–Villiger oxidation (phenylacetone monooxygenase from Thermobifida fusca), esterase-mediated (esterase from Pseudomonas fluorescens, PfeI) hydrolysis and subsequent oxidation of the primary alcohol (alcohol dehydrogenase from Pseudomonas putida, AlkJ) to the respective aldehyde products. Eight different phenylpropene derivatives were subjected to these reaction sequences, allowing for the synthesis of seven aldehydes in up to 55% yield after 4 reaction steps (86% for each step).


General remarks
If not mentioned otherwise, all glassware and media used for the cultivation of E. coli were sterilized via autoclavation prior use.All stock solutions of substrates used for the biotransformation were prepared in ethanol (EtOH) or Acetonitrile (ACN) (usually 0.5 M) and stored in the freezer at -20 °C for a limited amount of time.All stock solutions used for the cultivation of Escherichia coli (E.coli) and the induction of protein production (e.g., antibiotics, inducer) were prepared in water or EtOH and sterilized via sterile filtration through the use of a syringe filter.All substances used for the biotransformation or cultivation of E. coli and synthetic procedures were purchased from Sigma Aldrich or a comparable vendor for chemicals.The following practices were carried out in all synthetic procedures unless stated otherwise.All glassware was flame dried before use, and to guarantee water and oxygen exclusion for sensitive reactions, Schlenk techniques were employed.In general, reactions were carried out under slight argon pressure and stirred magnetically.Liquid reagents were added by syringe through a rubber septum, and solid reagents were added in a slight argon countercurrent.NMR-analysis ( 1 H-NMR) was performed on the Bruker Avance 400 at 400 MHz.Depending on the substance, measurements were performed in chloroform-d6.
Anisaldehyde, basic KMnO 4, and phosphomolybdic acid/cerium sulfate solution were used as dip reagents to visualize the compounds.
The following solvents were used for synthesis or purification:  ACN and DMSO were obtained in p.a. grade and used as received.
 H 2 O was obtained from an ultrapure filtration system.
 PE and EtOAc used for column chromatography were obtained in technical grade.

Sample preparation for GC analysis
The aqueous sample taken from the biotransformation was transferred into a 1.5 ml Eppendorf vessel charged with 300 µl of ethyl acetate containing 1 mM of methyl benzoate as internal standard.The tube was shaken and centrifuged for 1 min at 14000 rpm.The organic phase was then transferred into a second Eppendorf vessel charged with Na 2 SO 4 .After a second centrifugation step for 1 min at 14000 rpm the supernatant was transferred into a GC vial and measured immediately or stored in the freezer at -30 °C for later measurement.

Sample preparation for HPLC analysis
The aqueous sample taken from the biotransformation was diluted with 100 µl of ACN in a 1.5 ml Eppendorf vessel.The mixture was centrifuged for 1 min at 14000 rpm and then transferred into an HPLC vial via a syringe, passing the solution through a filter.Samples were then directly measured or stored in the freezer at -30 °C for later measurements.

GC-Analysis
GC analysis for the extracted samples, as described in chapter 2, was performed on a Thermo Fisher Scientific Trace 1310 Dual GC composed of an FID detector and two Rxi-5Sil MS columns (length 15, ID 0.25 mm, film thickness 1.0 µm).GC quantification was performed with internal referencing employing 1 mM of methyl benzoate as an internal standard.

Methods:
For all compounds in the same substrate class 1-8 (ketone, alcohol, aldehyde, and acid) the same analytical method was utilized.

Methods:
For all compounds in the same substrate class 1-8 (ketone, alcohol, aldehyde, and acid), the same analytical method was utilized.

General procedure for protein production and biotransformation with whole-cell biocatalysts
A pre-culture containing the expression plasmid for the desired enzyme was grown in a 15 ml falcon tube charged with 5 ml LB-Miller medium, supplemented with the appropriate antibiotic for 19 hours at 37 °C and 220 rpm.The following day, a 20-500 ml flask was charged with TB-Medium (1/5 of the flask volume) and the appropriate antibiotic and inoculated with 1 % (v/v) of the pre-culture.The flask was then shaken at (37 °C, 150 -170 rpm) until the culture reached an OD 590 of 0.6.Subsequently, the appropriate inducer (and auxiliary salts) was added to the suspension.After shaking the culture for 22 hours at 20-25 °C at 150 rpm, the cells were harvested by centrifugation (6000 rcf, 10 min).To prepare the biocatalyst, the supernatant was removed, and the cell pellet was resuspended in sodium phosphate buffer (50 mM, pH 7.4).After a second centrifugation and removal of the supernatant, the cell pellet was resuspended in sodium phosphate buffer (volume determined by the desired OD) to obtain the washed biocatalyst.Biotransformation was performed in 8 ml screw cap vials.For this, 1 ml of the whole-cell biocatalyst was combined with the desired substrate dissolved in a co-solvent.
Biotransformation was then performed at 30-50 °C for 24 hours.Samples (150 µl or 100 µl) were taken after 0, 1, 5, and 24 hours and analyzed via GC or (U)HPLC.A pre-culture of the respective E. coli strains containing the expression plasmid for the Baeyer-Villigermonooxygenase (PAMO or TmCHMO), and the alcohol dehydrogenase (AlkJ) were grown in a 15 ml flacon tube filled with 5 ml of LB-Miller medium, supplemented with the appropriate antibiotic (1 mg / ml chloramphenicol for AlkJ and 1 mg / ml Ampicillin for PAMO) for 19 hours at 220 rpm.

Gene expression and protein production conditions for ADO, PAMO, TmCHMO, and AlkJ
Subsequently, a 250 ml flask charged with TB medium and the appropriate antibiotic was inoculated with the respective pre-culture (one for the BVMO and one for the ADH).The cultures were grown until an OD 590 of 0.5-0.6 was reached at 37 °C and 150-170 rpm.A stock solution of an appropriate inducer was then added to reach a final concentration of 0.2 vol% of 20 % L-Arabinose for PAMO or TmCHMO and 1 mM IMPT for AlkJ.The flasks were shaken at 150 rpm at 25 °C for 22 hours to produce sufficient protein.The next day the cells were harvested by centrifugation (6000 rfc, 10 min, 4 °C), washed with sodium phosphate buffer, and centrifuged again (6000 rcf, 10 min, 4 °C).Lastly, the cell pellet was resuspended with an appropriate volume of sodium phosphate buffer (50 mM, pH 7.4) to obtain a final OD of 40.The two cell suspensions were mixed in a 1:1 ratio to generate the mixed culture biocatalyst with a final relative OD of 20 for each component.The biotransformation was performed in 8 ml screw cap glass vials, with 1 ml of the mixed culture biocatalyst and 100 U (µmol/min for ~3 mg of powder) of PfeI added as a lyophilized powder.The substrate was added last from a stock solution to obtain a final concentration of 5 mM.The biotransformation was then performed at 30 or 33 °C.Samples were taken after 0, 1, 5, and 24 hours.Analysis was performed with GC or HPLC.

Chemo-enzymatic one-pot reaction using a whole-cell mixed culture biocatalyst with PAMO /TmCHMO, AlkJ, and PfeI
The mixed culture biocatalyst was prepared according to SI 5.3.An appropriate volume of sodium phosphate buffer (100 mM, pH 7.4) was added for the final resuspension to obtain a final OD 590 of 80.
The cell suspensions were then mixed in a 1:1 ratio to obtain the mixed culture biocatalyst with a relative OD of 40 for each component.For the chemo-enzymatic one-pot reaction, 0.2 ml of the reaction mixture obtained after the Wacker oxidation was diluted with 0.2 ml of H 2 O.The mixture's pH was then adjusted by adding 2 N NaOH (usually 10-20 µl) to 7 -8.Subsequently, water was added to a final volume of 0.5 ml.To this, 0.5 ml of the mixed culture biocatalyst was added together with PfeI 100 U (µmol/min for ~3 mg of powder), as a lyophilized powder.Biotransformation was then performed at 30 or 33 °C (for TmCHMO or PAMO, respectively).Samples were taken after 0, 1, 5, and 24 hours.Analysis was performed with GC or HPLC.

5.5
Optimized procedure for the chemo-enzymatic one-pot reaction using a whole-cell mixed culture biocatalyst with PAMO /TmCHMO, AlkJ, and PfeI The mixed culture biocatalyst was prepared according to SI 5.3.An appropriate volume of sodium phosphate buffer (200 mM, pH 7.4) was added for the final resuspension to obtain a final OD 590 of 80.
The cell suspensions were then mixed in a 1:1 ratio to obtain the mixed culture biocatalyst with a relative OD of 40 for each component.For the chemo-enzymatic one-pot reaction, 0.2 ml of the reaction mixture obtained after the Wacker oxidation was diluted with 0.25 ml of PB buffer (200 mM, pH 7.4), and 25 µl EtOH and H 2 O were then added.To this mixture, 0.5 ml of the mixed culture biocatalyst was added together with 100 U PfeI (µmol/min for ~3 mg of powder) as a lyophilized powder.Biotransformation was then performed at 30 or 33 °C (for TmCHMO or PAMO, respectively).
Samples were taken after 0, 1, 5, and 24 hours.Analysis was performed with GC or HPLC.

Preparation of lyophilized cells containing PfeI
A pre-culture of an E. coli strain containing a pGaston::PfeI vector was cultivated in a 15 ml flacon tube charged with 5 ml LB-Miller medium and supplemented with Ampicillin (5 µl, 100 mg/ml).The culture was grown for 19 hours at 190 rpm.Subsequently, a 1000 ml baffled flask charged with 200 ml TBmedium and supplemented with Ampicillin (200 µl, 100 mg/ml) was inoculated with 1 % (v/v) of the pre-culture.The culture was grown at 37 °C at 170 rpm until an OD 590 of 0.29 was reached.Protein production was induced by the addition of 20 % L-Rhamnose (0.2 % v/v).The cells were then incubated for 3 hours at 37 °C, followed by harvesting through centrifugation (6000 rcf. 10 min.).The cell pellet was resuspended in sterile water (10 ml) in a 15 ml Falcon tube.After snap-freezing the suspension in liquid nitrogen, the cells were then lyophilized for 24 hours.The resulting off-white powder was stored at -80 °C.The esterase activity was determined photometrically via a p-nitrophenol acetate assay 1 .

ADO chemo-enzymatic one-pot biotransformation and substrate scope
The whole-cell biocatalyst was prepared according to the general procedure for protein production and biotransformation.For the chemo-enzymatic one-pot reaction, the product (1b-8b) obtained after the isomerization was dissolved in EtOH to get a solution of 0.5 M and added to 1 ml of the whole cell suspension (OD 590 30) to obtain a final concertation of 5 -8 mM.The biotransformation was then performed at 50 °C, 220 rpm.Samples were taken after 0, 1, 5, and 24 hours and analyzed via GC (Figure 3).ADO only showed activity toward substrate 7b (and 8b after hydrolysis of the acetate) The desired aldehyde vanillin (7f) was formed with a yield of 50 % (GC) after 5 hours.For 8b, solubility issues of the substrate in the aqueous medium led to recovery issues in the 0-and 1-hour samples, as seen in the GC trace (Figure 1 b).Vanillin 7f was obtained with a yield of 61 % (GC) after 24 hours.

ADO biotransformation with 4-(prop-2-en-1-yl)phenol
To test the hypothesis that the activity and substrate acceptance of ADO strongly depends on the stabilizing effect of hydrogen bonding with the para hydroxyl group of the substrate anethol 4b was demethylated and subjected to ADO in a preparative scale experiment (30 ml OD 590 , 30, 5 mM).The biotransformation was performed at 50 °C with 220 rpm.After 24 hours, the product was extracted with ethyl acetate, and the solvent was dried over Na 2 SO 4 and filtered.After removing the solvent under reduced pressure via rotary evaporation, the residue was analyzed via 1 H-NMR (Figure 2).The obtained NMR spectrum (red) is shown overlayed with the spectrum of the starting material (cyan).
The characteristic aldehydic proton signal at 9.85 ppm is clearly visible, and the aromatic signals at 7.80 and 6.95 ppm indicate a partial oxidation to the corresponding aldehyde.

AlkJ, PAMO, and TmCHMO substrate scope
To test for the substrate scope of the Baeyer-Villiger-monooxygenases PAMO and TmCHMO and the alcohol dehydrogenase AlkJ, small-scale biotransformations were performed with the purified substrate.The whole-cell biocatalyst was prepared according to the general procedure for protein production and biotransformation.For this, 1 ml of the whole-cell suspension was filled into an 8 ml screw-capped glass vial, followed by the addition of a solution of the respective substrate (10 µl, 0.5 M in EtOH) to obtain a final concentration of 5 mM.The biotransformation was then performed at 30 °C for TmCHMO and AlkJ and 37 °C for PAMO at 220 rpm.Samples were taken after 0, 1, 5, and 24 hours and analyzed via GC (Figure 3-5).

PAMO, PfeI and AlkJ enzymatic cascade with purified substrate -GC analysis
Figure 6: Results of the biotransformation using a whole-cell mixed culture biocatalyst (PAMO, AlkJ and, PfeI) with substrates 1c-8c (5 mM).The substrate dissolved in EtOH (0.5 M) was added to the suspension.Samples were taken after 0, 1, 5, and 24 hours and analyzed via GC.Overoxidation of the formed aldehyde was observed (not quantified with GC).

PAMO, PfeI and AlkJ chemo-enzymatic cascade -GC analysis
Figure 7 Results of the chemo-enzymatic one-pot reaction using a whole-cell mixed culture biocatalyst (PAMO, AlkJ and, PfeI) with substrates 1c-8c (max.~1.5-4 mM).The substrate was added as a solution in the form of the reaction mixture obtained after the Wacker oxidation.Samples were taken after 0, 1, 5, and 24 hours and analyzed via GC.Overoxidation of the formed aldehyde was observed (not quantified with GC).

TmCHMO, PfeI and AlkJ chemo-enzymatic cascade -GC analysis
Figure 8: Results of the chemo-enzymatic sequential one-pot reaction using a whole-cell mixed culture biocatalyst (TmCHMO, AlkJ, and PfeI) with substrates 1c-6c (~1.5-4 mM).The substrate was added as a solution in the form of the reaction mixture obtained after the Wacker oxidation (1a-6a).Samples were taken after 0, 1, 5, and 24 hours and analyzed via GC.Overoxidation of the formed aldehyde was observed (not quantified with GC).

Optimization of the chemo-enzymatic sequential reaction cascade -(U)HPLC analysis
The Insufficient mass recovery encountered in GC analysis (as seen in 5.10 -5.12) suggested the formation of a side product that was not identifiable with the analytical method deployed.
Exchanging the analytical system from GC to HPLC analysis revealed the formation of major amounts of the corresponding carboxylic acid during the enzymatic cascade, making the optimization of the enzymatic cascade necessary.We discovered that increasing the buffer concentration and adding 2.5 vol% EtOH into the biotransformation mixture could almost entirely suppress the formation of undesired carboxylic acid (Figure 9).The phenotype of the colony picked for protein expression also seemed to have a noticeable impact on the system's efficiency.As an undesired side effect, the addition of EtOH led to a slight decrease in the transformation speed (Figure 9) The optimized protocol for the biotransformation can be found in chapter 5.5.

PAMO, PfeI, and AlkJ chemo-enzymatic cascade -(U)HPLC analysis
Figure 10 Results of the optimized chemo-enzymatic sequential one-pot reaction using a whole-cell mixed culture biocatalyst (PAMO, AlkJ, and PfeI) with substrates 1c-8c (~1.3-4 mM).The substrate was added as a solution in the form of the reaction mixture obtained after the Wacker oxidation (1a-8a).Samples were taken after 0, 1, 5, and 24 hours and analyzed via (U)HPLC.Overoxidation of the formed aldehyde was observed in small amounts.

Large-scale biotransformation using 4a
For the preparative-scale biotransformation, the Wacker oxidation was performed with allyl anisole 4a (37 mg) under standard conditions with 9.5 ml of H 2 O and 0.5 ml ACN as co-solvent.After the complete consumption of the starting material was confirmed, the pH of the solution was adjusted to 7.4 through the addition of 2 N NaOH.Removal of the precipitates via centrifugation led to a yellow solution.This solution was added to 50 ml of the mixed culture biocatalyst (OD 20 TmCHMO, OD 20 AlkJ) together with 100 U (µmol/min for ~3 mg of powder) of PfeI in a 250 ml flask equipped with a rubber septum to prevent the evaporation of the reaction components.The biotransformation was conducted at 30 °C and 220 rpm.Samples were taken after 0, 1, 5, and 19 hours.At the 0 h mark of the biotransformation the yield of the Wacker-oxidation was determined to be 71 %.After GC analysis (Figure 11) confirmed complete consumption of the starting material, the suspension was centrifuged, the supernatant transferred into a separating funnel, and extracted with EtOAc (3 x 30 ml).Removal of the solvent resulted in a brown residue (31 mg) which was filtered through a pad of silica. 1 H-NMR analysis of the residue revealed a mixture of the desired aldehyde (34 % after four steps) and the corresponding carboxylic acid obtained after overoxidation (Figure 12).

5.16
General procedure for the Wacker-oxidation of phenylpropenes 2 A reaction vessel equipped with a septum and magnetic stirring was charged with Pd(TFA) 2 , NaTFA (0.2 equiv.),FeCl 3 (Fe 2 (SO 4 ) 3 .The vessel was then evacuated and flushed with Argon utilizing standard Schlenk techniques.Degassed water (three freeze-thaw cycles) was then added to the solids resulting in a yellow solution.This was followed by the slow addition of 1a-8a in a solution of ACN via a syringe.
The mixture was then stirred vigorously in the dark for 24 hours.After full consumption of the starting material was confirmed via TLC, the reaction mixture was used directly for the subsequent biotransformation or stored in the freezer for later use.For the isolation of the product, the mixture was extracted with EtOAc instead.The combined organics were washed once with saturated NaHCO 3 solution and brine.After drying the solution over Na 2 SO 4, and filtration, the removal of the solvent via rotary evaporation resulted in a brown oil.The crude product was further purified by column chromatography.

General procedure for the isomerization of phenylpropenes
A brown glass reaction vessel equipped with magnetic stirring was filled with 1a-8a.To this, PdCl 2 was then added, and the reaction was stirred for 24 hours.The conversion was monitored via 1 H-NMR.The mixture was then used directly for the subsequent biotransformation or stored in the freezer for later use.For the catalyst recovery, the mixture was diluted with EtO 2 and centrifuged (5 min, 14000 rpm).
The supernatant was then removed.The residue (PdCl 2 ) was then rewashed with a fresh portion of solvent, and the procedure was repeated.The wash fractions were combined, and the solvent was removed under reduced pressure, leaving a brown oil, which was further purified via flash chromatography if necessary.The catalyst residue could be directly reused for a new batch without further treatment.

Isomerization and catalyst recovery
To demonstrate the possibility of catalyst recovery and reusability of said catalyst, the standard isomerization procedure was followed (chapter 5.17), emploing 7a as the model substrate (1 g, 6.09 mmol) together with PdCl2 (54 mg, 5 mol%).After complete conversion was achieved, the catalyst was recovered as mentioned above, and the isomerization restarted with the recovered PdCl 2 .This process was repeated two more times.The amount of catalyst recovered after each run can be found in table table

Synthesis of 4-(prop-2-en-1-yl)phenol 3
A flame-dried three-necked round bottom flask equipped with a condenser and a dropping funnel was charged with Al (S) (0.26 g, 9.2 mmol, 2.8 equiv.)and I 2(S) .(1.41 g, 11.0 mmol, 3.3 equiv.)The atmosphere of the reaction vessel was then replaced by Argon, utilizing standard Schlenk techniques.ACN (25 ml) and DMSO (0.65 g, 8.2 mmol, 2.5 equiv.)were then added, followed by heating to 80 °C for 30 minutes under vigorous stirring.To this mixture, a solution of anethol (0.50 g, 3.3 mmol, 1.0 equiv. in 2 ml ACN) was added dropwise via the dropping funnel over a period of 30 minutes.The reaction was then stirred for 18 h at 80 °C.After full consumption of the starting material was confirmed via TLC, the heating was removed, allowing the mixture to cool down to room temperature.The reaction was then quenched via the addition of 2 N HCl, and the product was extracted with EtOAc (4 x 10 ml).The combined organic phases were then washed with aq.Na 2 S 2 SO 3 and brine.The solution was then dried over Na 2 SO 4 , filtered, and the solvent removed under reduced pressure.The resulting crude was then purified via flash chromatography resulting in a colorless oil, which solidified in the freezer.

Figure 1
Figure 1 Chemo-enzymatic biotransformation with 7b and 8b and ADO.Biotransformation with 7b was performed with 8 mM of the substrate and 5 mM for substrate 8b.

Figure 2 :
Figure 2: 1 H-NMR of the product of the biotransformation of 4-(prop-2-en-1yl)phenol with ADO (red) in comparison with the 1 H-NMR of the starting material (cyan).

Figure 3 :Figure 4 :
Figure 3: Conversions of the substrates 1c -8c after 1h and 24 h with PAMO.All substrates were successfully oxidized to the desired products.

Figure 5 :
Figure 5: Conversions of the substrates 1e -8e after 1h and 24 h with AlkJ.All substrates were successfully oxidized to the desired products.

Figure 9 :
Figure 9: Difference in the product distribution obtained after the chemo-enzymatic reaction sequence with PAMO/AlkJ and PfeI.A) The standard conditions (see 5.4) led to the exclusive formation of the corresponding carboxylic acid after 24 hours.B) The optimized conditions (see 5.5) led to a sharp decrease in acid formation.

Table 1 :
Typical conditions for gene expression and protein production for the enzymes used in this work.