Multienzyme One‐Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids

Abstract The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S‐Adenosylmethionine (SAM)‐dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi‐enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non‐natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C‐1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

EcMAT and TfNCS (Δ29TfNCS) purification: Cell pellets were lysed as described above. The supernatant was collected by centrifugation at 4 °C, 10000 rpm for 15 min, then filtered through a 0.2 μm cellulose acetate springe filter. An empty cartridge charged with Ni-NTA (8 mL) was washed with 40 mL of MilliQ water, followed by 40 mL of binding buffer (50 mM HEPES, 20 mM imidazole (Sigma-Aldrich), 100 mM NaCl, pH 7.5). The filtered supernatant was then passed through the Ni-NTA column, and the column was washed with wash buffer (40 mL, 50 mM HEPES, 40 mM imidazole, pH 7.5) to remove some background protein. The bound protein was then eluted with elution buffer (50 mM HEPES, 500 mM imidazole, 100 mM NaCl, pH 7.5) until all the protein was collected. The eluent containing pure enzyme was concentrated using a vivaspin (10000 MW) at 4 °C, 8000 rpm for 5 min until 2.5 mL eluent remained. Then the concentrated eluent was desalted into 3.5 mL of 50 mM HEPES (pH 7.5), using a SephadexTM G-25 in PD-10 column (GE Healthcare lifesciences). The concentration of the pure protein was measured by OD280 using a Nanodrop. The protein was aliquoted and stored at -20 °C in 10% glycerol. To check the protein purity, the expression supernatant, and pure protein were examined using an SDS gel ( Figure S3). CjCNMT purification: The purification of CjCNMT was carried out via an Äkta Purifier (GE Healthcare) according to the following protocol. Lysis buffer (40 mM Tris HCl, 100 mM NaCl, 20 mM imidazole, pH 8.0), elution buffer (40 mM Tris HCl, 100 mM NaCl, 500 mM imidazole; pH 8.0) and desalting buffer (100 mM potassium phosphate, pH 7.5) were used. The cell pellet was stored at -20 °C. For lysis it was thawed and resuspended in lysis buffer using 2-5 mL per gram of wet weight. Then lysozyme and DNaseI (each 0.1 mg/mL) were added and incubated on ice for 30 min. Cell disruption was achieved by using a French Press (1,81 kbar at 5 °C). The lysate was cleared by centrifugation at 10,000 x g for 30 min at 4°C and subsequently filtered through a 0.2 µm filter membrane. For purification through IMAC a Ni-NTA column was equilibrated with lysis buffer. Then the cleared sample was loaded onto the column with a volumetric flow rate of 2.0 mL/min. The unbound or unspecific bound protein was washed away with lysis buffer until the absorption at 280 nm reached the baseline. Further, the His6-tagged protein was eluted using a step gradient of lysis buffer and elution buffer (first step: 5% (v/v) elution buffer, second step: 60% (v/v) elution buffer, third step: 100% elution buffer). The target enzyme was found in the fraction using 60% elution buffer. A sample of each fraction was collected and monitored via SDS-PAGE analysis. The fraction containing the enriched protein were desalted using PD10 columns. Freeze-drying was carried out and the protein was stored and -20 °C. Figure S4. SDS-PAGE analysis of purified protein; 1-CjCNMT (41 kDa) sample after desalting, the calculated molecular weight inside the parentheses always includes the His6-tag.

Chemicals, NMR spectroscopy and mass spectrometry
The solvents and chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA) or Alfa Aesar (Thermo Fisher Scientific Inc., MA, USA). All chemicals were purchased in the highest purity available and were used as supplied. Adenosine 5′-triphosphate (ATP) was purchased as a disodium salt hydrate Grade II, ≥98.5% (HPLC), crystalline, from Sigma-Aldrich. S-(5'-Adenosyl)-L-methionine (SAM) was purchased as the p-toluenesulphonate from Sigma-Aldrich. 1 H and 13 C NMR spectra were recorded respectively at 600 MHz and 150 MHz on a Bruker Avance 600 spectrometer or at 700 MHz and 176 MHz on a Bruker Avance 700 spectrometer in the stated solvent. Chemical shifts (in ppm) are quoted relative to tetramethylsilane and referenced to residual protonated solvent. Coupling constants (J) are measured in Hertz (Hz) and multiplicities for 1 H NMR coupling are shown as s (singlet), d (doublet), t (triplet), and m (multiplet). Mass spectra were obtained using either a VG70-SE or MAT 900XP spectrometer at the Department of Chemistry, University College London. All optical rotations were measured on a Bellingham and Stadley ADP 430 polarimeter with a path length of 0.5 cm.

Analytical and preparative HPLC
Achiral analytical HPLC methods were performed with a HPLC system Agilent 1260 Infinity II system equipped with a DAD detector.

Method C2:
The chiral separation of compound (rac)-3 and (S)-3 were achieved with a Supelco Astec ChirobioticTM T column (25 cm × 4.6 mm) and a flow speed of 0.2 mL/min at 30 °C. The injection volumes were 5 μL. Compounds were detected by UV absorbance at 230 nm. An isocratic mobile phase 20 mM NH4OAc pH 4:MeOH (70:30) was used over 120 min.

Method C3:
The chiral separation of compound (rac)-11 and (S)-11 were achieved with an Supelco Astec ChirobioticTM T2 column (25 cm × 4.6 mm) and a flow speed of 1 mL/min at 30 °C. The injection volume was 5 μL. Products were detected via UV absorbance at 230 nm. Methanol (0.2% AcOH, 0.1% TEA) was used as a mobile phase over 40 min.

Single step methylation reactions for preliminary screening
The assay was conducted in a 96-well plate. Each well contained the reaction mixture consisting of 50 mM HEPES pH 7.5, 20 mM MgCl2, 0.5 mM substrate (1-14), 3 mM SAM, and the specific methyltrasferase RnCOMT, MxSafC or Cj-6-OMT as clarified lysate (10% v/v) in a total volume of 100 μL. The assay was performed at 37 °C, 750 rpm for 90 min unless otherwise specified. After this time, the reaction was quenched with 100 μL of MeOH. All the experiments were performed in duplicate and the reaction mixture was prepared freshly for each experiment. The conversion yields reported refer to depletion of starting material and are calculated using calibration curves for each substrate. When no starting material was left at the end of the reaction a quantitative conversion was reported.

Comparison between pure enzymes and clarified lysates for RnCOMT and MxSafC
The assay was conducted in Eppendorf vials. Each vial contained the reaction mixture consisting of 50 mM HEPES pH 7.5, 20 mM MgCl2, 2.5 mM substrate 1-3, 2.5 mM SAM, and the specific methyltrasferase RnCOMT, MxSafC as clarified lysate (10% v/v) or pure enzyme (0.4 mg/mL) in a total volume of 100 μL. The assay was performed at 37 °C, 450 rpm for 4 h. After this time, the reaction was quenched with 100 μL of MeOH and clarified by centrifugation ( Figure S4).

Multi-enzyme methylation reactions with the SAM supply system (scale-up optimization)
Small scale assays were carried out in order to find the optimal conditions for the scale-up reactions. Optimised conditions are reported. The reaction mixture consisted of 50 mM HEPES pH 7.5, 20 mM MgCl2, 200 mM KCl, 5 mM substrate (1-14), ATP (1-4 equiv), Lmethionine (1-4 equiv), in a total volume of 200 μL. The methylation step was started by adding 10% v/v RnCOMT lysate together with EcMAT (0.4 mg/mL) and EcMTAN (0.025 mg/mL). Finally, the pure EcMAT and EcMTAN were replaced by the clarified lysate and 10% v/v EcMAT lysate and 2.5% v/v EcMTAN lysate were added.
The assay was performed at 37 °C, 450 rpm in eppendorf vials for 19 h unless otherwise specified. After this time, the reaction was quenched with 100 μL of MeOH.

Four-enzymes cascade to (S)-1-benzyl-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol (S)-6-OMe-1
Dopamine hydrochloride (9.45 mg, 0.05 mmol), phenylacetaldehyde (8.4 μl in 0.5 mL of CH3CN, 0.075 mmol), sodium ascorbate (9.9 mg, 0.05 mmol), and 10% v/v of TfNCS clarified lysate desalted into 50 mM HEPES (pH 7.5), using a SephadexTM G-25 in PD-10 column (GE Healthcare lifesciences), were combined with HEPES (50 mM pH 7.5) to a total volume of 5 mL. The reaction was stirred at 37 °C for 16 h. Once the reaction had completed, the yellow suspension was centrifuged, and the supernatant used for the next step. ATP (55 mg, 0.10 mmol) and L-methionine (15 mg, 0.10 mmol) were combined in HEPES buffer (50 mM) and the pH was adjusted to 7.5. The ATP/L-methionine mixture was added to the supernatant. The methylation step was initiated upon addition of 10% v/v of EcMAT lysate, 2% v/v of EcMTAN lysate, and 10% v/v of RnCOMT lysate in a total volume of 10 mL containing 20 mM MgCl2 and 200 mM KCl. The reaction was incubated overnight at 37 °C and 180 rpm. After this time an aliquot was taken to calculate the yield by HPLC against the standard product (see calibration curve below). The reaction was centrifuged, the supernatant was basified with NaHCO3 and extracted with EtOAc (3 x 10 mL). The organic fractions were collected, evaporated under vacuum and the residue purified using preparative HPLC (method B). Fractions containing the desired product were freeze-dried to give (S)-1a as white powder (TFA salt, 10 mg, 55% yield, 96% yield by HPLC). Trace impurities (5%) were also identified by NMR spectroscopy corresponding to the regioisomeric product methylated at O-7. 1  AU 280 nm concentration (mM)

Five-Enzymes Cascade to (S)-1-(4-hydroxy-3-methoxybenzyl)-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol (S)-6,4'-(OMe)2-3
Dopamine hydrochloride (19 mg, 0.1 mmol), sodium ascorbate (20 mg, 0.1 mmol), sodium pyruvate (5.5 mg, 0.05 mmol), CvTAm 10% v/v lysate, and 10% v/v of TfNCS lysate desalted into 50 mM HEPES (pH 7.5), using a SephadexTM G-25 in PD-10 column (GE Healthcare lifesciences), were combined with HEPES (50 mM pH 7.5) containing PLP 0.5 mM to a total volume of 5 mL. The reaction was stirred at 37 °C until completion. Once the reaction had completed, the yellow suspension was centrifuged, and the supernatant used for the next step. ATP (55 mg, 0.10 mmol) and L-methionine (15 mg, 0.10 mmol) were combined in HEPES buffer 50 mM and the pH was adjusted to 7.5. The ATP/ L-methionine mixture was added to the supernatant and the methylation step was initiated upon addition of 10% v/v of EcMAT lysate, 2% v/v of EcMTAN lysate, and 10% v/v of Cj-6-OMT lysate in a total volume of 10 mL containing 20 mM MgCl2 and 200 mM KCl. The reaction was incubated overnight at 37 °C and 180 rpm. After this time an aliquot was taken to calculate the yield by HPLC against the product standard (see calibration curve below). The reaction was centrifuged and the supernatant used for the next step. A solution of ATP and L-methionine (0.75 mL, 100 mM in HEPES buffer 50 mM with the pH adjusted to 7.5) was added to the supernatant and the methylation step was initiated upon addition of EcMAT lysate (1 mL), EcMTAN lysate (0.2 mL), and MxSafC lysate (1 mL). The reaction was incubated overnight at 37 °C and 180 rpm. After this time the mixture was centrifuged and the supernatant was basified with NaHCO3 and extracted with EtOAc (3 x 10 mL). The organic fractions were collected, evaporated under vacuum and the residue purified using preparative HPLC (method B). Fractions containing the desired product were freeze-dried to give norreticuline (S)-6,4'-(OMe)2-3 as white powder (TFA salt, 5.6 mg, 27% yield, 70% yield by HPLC). 1

Synthesis of 6'-methoxy-3',4'-dihydro-2'H-spiro[cyclopentane-1,1'-isoquinolin]-7'-ol 6-OMe-10
Compound 10 (TFA salt, 16 mg, 0.05 mmol), ATP (55 mg, 0.1 mmol), and L-methionine (15 mg, 0.1 mmol) were combined in HEPES buffer 50 mM pH 7.5. The methylation step was initiated upon addition of 10% v/v of EcMAT lysate, 2% v/v of EcMTAN lysate, and 10% v/v of MxSafC lysate in a total volume of 10 mL containing 20 mM MgCl2 and 200 mM KCl. The reaction was incubated overnight at 37 °C and 180 rpm. After this time an aliquot was taken to calculate the yield by HPLC against the product standard (see calibration curve below). The reaction was centrifuged, the supernatant was basified with NaHCO3 and extracted with EtOAc (3 x 10 mL). The organic fractions were collected, evaporated under vacuum and the residue purified using preparative HPLC (method B). Fractions containing the desired product were freeze-dried to give 6-OMe-10 as white powder (TFA salt, 4.9 mg, 30% yield, 89% yield by HPLC). An impurity (10%) was also identified by NMR corresponding to the regioisomeric product methylated at O-7. Only signals for the major isomer are shown. 1

6-OMe-11
(k) (l) Figure S5. Representative HPLC chromatograms from the single step methylation reaction (preliminary screening) using SAM. For each methyltrasferase: yellow dots correspond to starting material, full dots correspond to 6-OMe product, light dots correspond to 7-OMe products, purple dots correspond respectively to the dimethoxy products.