One-pot chemoenzymatic synthesis of trolline and tetrahydroisoquinoline analogues

A highly efficient one-pot asymmetric route to tetrahydroisoquinoline alkaloids including the natural product trolline is described.

Analytical HPLC method Protein Purity Test Protein sample was analysed by SDS-12% polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-Protein Precast Gels (Bio-Rad company). Briefly, the protein composition was prepared by mixing 10 µL loading buffer, 10 µL DTT and 20 µL testing sample. The prepared mixture as well as a Ladder protein marker (10-25 kDa, New England Biolabs) were boiled at 95 o C for 15 min and spun at 13000 rpm for 10 min. The supernatants were loaded to the gels and run (200 V for 40 min). The gel was then washed with distilled water and treated with instant blue overnight. A ProteinSimple camera and AlphaImager MiNi software were used to take a picture of the gel.
Chemical Reagent Flash silica chromatography was carried out using Geduran ® Si 60 (40-63 µm). Thin layer chromatography (TLC) used aluminium backed silica gel plates from Merck Keiselgel and were visualised using ultra-violet light (254 nm). The solvents and chemicals were purchased from Sigma Aldrich, Alfa Aesar, Santa Cruz Biotechnology and were used as supplied unless indicated otherwise.
Chemical Characterization Melting points were tested using Stuart (SMP11) and DigiMelt MPA161 melting point machines. The value of [α] ! was recorded using PerkinElmer Model 343 Polarimeter machine in the solvent indicated. 1 H and 13 C NMR spectra were recorded on Bruker Avance III 600, Bruker Avance III 400, Bruker Avance 300 spectrometer instruments at the field indicated. Chemical shifts (in ppm) were determined relative to tetramethylsilane (TMS) and referenced to residual protonated solvent. Coupling constants (J) were measured in Hertz (Hz) Table 1 in the main text).
For enzyme reactions, HEPES buffer (100 mM HEPES, pH 7.5) was added with a final concentration of DMSO (1% v/v), dopamine 1 (10 mM), methyl 4-oxobutanoate 5 (15 mM), sodium ascorbate (10 mM) and △29TfNCS (wild type, 0.1 mg/mL) in a 1.5 mL capped microcentrifuge tube. The reactions were flushed with argon, sealed and shaken for 3 h at 37 o C, 500 rpm. Reactions were monitored by taking 100 µL sample diluting with water/HCl (10 µL HCl + 440 µL water) and analysed by HPLC following the analytical HPLC method. Then 800 µL of the reaction mixtures were treated with 80 µL HCl (1 M) and 120 µL sodium carbonate solution (1 M) and shaken for 2 h at 60 o C, 500 rpm. Similar procedures were used with methyl 5-oxopentanoate 7 and methyl 6-oxohexanoate 10. For amines 13, 14 and 15 reacted with 5, the first reactions (step a, 0.5 mg/mL WT-△29TfNCS) were 6 h (13 and 15) or 18 h (14) and the second reactions (cyclisations) were for 2 h (13 and 15) and 4 h (14). HPLC yields were calculated based on standard curves as above (see Table 1 in the main text).  Figure  3).  Figure 2. Note that dopamine depletion was used to monitor the reactions to reduce complexity of the assay due to linear and cyclic products being formed.

Screening of reaction conditions for second-step (cyclization
Computational docking Computational docking calculations were conducted using AutoDock Vina 4 , implemented through UCSF Chimera and the Opal web service. Ligand structures were prepared and energy minimised on the molview.org server prior to docking. PDB structure 5NON was prepared by converting selenomethionines to methionines, removing ligands and adding hydrogens as previously. 5 The docking prameters were: center_x = -16.99; center_y = -5.81; center_z = 15.42; size_x = 16.56; size_y = 14.22; size_z = 22.15; energy_range = 3; exhaustiveness = 8; num_modes = 10. Ten binding modes were predicted for each ligand, and only top ranked modes which showed interactions in line with the dopamine-first mechanism 2,5 were analysed. To a solution of δ-valerolactone (10.0 g, 99.9 mmol) in methanol (30 mL), concentrated sulfuric acid (0.4 mL) was added and the mixture was stirred for 2 h at 65 o C. It was then cooled to 0 o C and H 2 O (30 mL) was added. The solution was adjusted to pH 8.0 by adding aq. Na 2 CO 3 (1 M). The mixture was extracted with ether (60 mL, then 2 × 30 mL), washed with brine (2 × 30 mL), dried (Na 2 SO 4 ) and concentrated under vacuum to give the alcohol as a colourless oil (5.14 g, 39%), which was used directly in the next step. 1  To a solution of ε-caprolactone (10.0 g, 87.6 mmol) and methanol (30 mL), concentrated sulfuric acid (0.4 mL) was added, and the mixture was heated at reflux for 2 h. It was then cooled to room temperature, and water (30 mL) was added. The solution was adjusted to pH 8.0 by adding 1 M aq. S15 Na 2 CO 3 . Then the mixture was extracted with ether (3 × 30 mL), washed with brine (2 × 20 mL), dried (Na 2 SO 4 ) and concentrated under vacuum to give the alcohol as a colourless oil (10.0 g, 78%), which was used directly in the next step. 1
(i) Small-scale (1 mL) KPi reaction. The procedure described on pS11 was used and reaction yields determined by HPLC. Trolline 2 was formed in 97% yield in a para:ortho isomeric ratio of 18:1 (see Supplementary Figure 8B).

(ii) Preparative scale reaction for product isolation and characterisation.
To a solution of dopamine hydrochloride (76 mg, 0.40 mmol), ascorbic acid (70 mg, 0.40 mmol), KPi buffer (20 mL, pH 6, 0.3 M) and CH 3 CN (20 mL) was added methyl 4-oxobutanoate 5 (64 µL, 0.60 mmol). The mixture was heated at 60 o C and stirred for 18 h under argon. The pH of the solution was then adjusted to 7.5 by adding aq. Na 2 CO 3 (1 M) and stirred for another 4 h at 60 o C under argon. The product was extracted with ethyl acetate (3 × 30 mL), the organic layer dried (Na 2 SO 4 ) and evaporated to give a yellow solid. The residue was re-suspended in aq. HCl (10 mL, 1 M) and dimethyl carbonate (DMC) (10 mL), and the aqueous layer was washed with DMC (3 × 5 mL). The DMC fractions were combined and washed with aq. HCl (3 × 5 mL, 1 M). The aqueous phase was co-evaporated with methanol at 55 o C to obtain a white solid, (±)-trolline 2 (71 mg, 81%) in a para:ortho isomeric ratio of 34:1. The characterization data is given below for the (S)-isomer.

Synthesis of (-)-trolline (S)-2. (i) Small-scale (1 mL) NCS reaction.
The procedure described on pS11 was used and reaction yield determined by HPLC. Trolline (S)-2 was was formed in 75% yield (see Supplementary Figure 8D) and no minor isomeric product was observed. The ee was 95% by chiral HPLC (of intermediate (S)-6: see Supplementary Figure 6A and Table S2).

Synthesis of 9.
(i) Small-scale (1 mL) KPi reaction. The procedure described on pS11 was used and reaction yield determined by HPLC. Compound 9 was formed in 89% yield in a para:ortho isomeric ratio of 9:1 (see Supplementary Figure 9B).

(ii) Reaction for product isolation and characterisation.
To a solution of dopamine hydrochloride (19 mg, 0.10 mmol), ascorbic acid (18 mg, 0.10 mmol), KPi buffer (5 mL, pH 6, 0.3 M) and CH 3 CN (5 mL) was added methyl 5-oxopantanoate 7 (22 µL, 0.15 mmol; 93% purity). The mixture was stirred at 60 o C for 18 h under argon. The pH of the solution was then adjusted to 7.5 by adding aq. Na 2 CO 3 (1 M) and stirred for another 4 h at 60 o C under argon. The reaction mixture was purified directly by preparative HPLC following semi-preparative HPLC method C to obtain a white solid 9 (16 mg, 69%) in a para:ortho isomeric ratio of 50:1 by NMR. The characterization data is given below for the (S)-isomer.

S18
The procedure described on pS11 was used and reaction yield determined by HPLC. Compound (S)-9 was was formed in 96% yield (see Supplementary Figure 9D) and no minor isomeric product was observed. The ee was determined to be 96% by chiral HPLC (of intermediate (S)-8:see Supplementary Figure 6B and Table S2). (ii) Preparative scale reaction for product isolation and characterisation. Dopamine hydrochloride (76 mg, 0.40 mmol) and sodium ascorbate (80 mg, 0.40 mmol) were added to HEPES buffer (38.9 mL, pH 7.5, 100 mM). Methyl 5-oxopantanoate 5 (80 µL, 0.60 mmol) in DMSO (0.4 mL) was then added to the solution, followed by WT-△29TfNCS (final concentration 0.1 mg/mL). The mixture was stirred at 37 o C under argon. After 6 h, the reaction was quenched with aq. HCl (4 mL, 1 M) and adjusted to pH 7.5 by adding aq. Na 2 CO 3 (1 M). The mixture was then stirred for another 4 h at 60 o C under argon and extracted with ethyl acetate (4 × 20 mL). The organic layer was washed with brine (3 × 40 mL), dried (Na 2 SO 4 ) and evaporated to give the crude product. The residue was resuspended in aq. HCl (10 mL, 1 M) and DMC (10 mL), and the aqueous layer was washed with DMC

Synthesis of 11. (i) Small-scale (1 mL) KPi reaction.
The procedure described on pS11 was used and reaction yield determined by HPLC. Compound 11 was formed in 72% yield in a para:ortho isomeric ratio of 7:1 (see Supplementary Figure 10A).

Synthesis of (S)-11. (i) Small-scale (1 mL) NCS reaction.
The procedure described on pS11 was used and reaction yield determined by HPLC. Compound (S)-11 was was formed in 92% yield (see Supplementary Figure 10B) and no minor isomeric product was observed. The ee was determined to be >99.5% by chiral HPLC (see Supplementary Figure 6C and Table S2).

(ii) Reaction for product isolation and characterisation.
Dopamine hydrochloride (10.0 mg, 0.053 mmol) and sodium ascorbate (11.0 mg, 0.056 mmol) were added to HEPES buffer (4.86 mL, pH 7.5, 100 mM). Methyl 6-oxopantanoate 7 (11 µL, 0.075 mmol) in DMSO (50 µL) was added to the solution, followed by WT-△29TfNCS (final concentration 0.1 mg/mL) to make the total volume 5 mL. The mixture was stirred at 37 o C under argon. The reaction was quenched after 3 h by adding HCl (0.5 mL, 1 M) and centrifuged for 10 min at 4000 rpm to remove the protein. The supernatant was purified by preparative HPLC following semi-preparative HPLC method A. The purified product was further washed with diethyl ether (3 × 2 mL) and dried under high vacuum to obtain a white solid (S)-11 as the TFA salt (13 mg, 63%). No minor isomeric product was observed. The characterisation data was identical to that above for the racemate and

S21
vacuum. The residue was dissolved in methanol (20 mL) and evaporated to obtain the crude product, which was purified by SiO 2 column chromatography (CH 2 Cl 2 and MeOH (with 5% Et 3 N), 25:1 to 5:1), giving 3-methoxyphenethylamine as a pale yellow oil (440 mg, 68%). 1  3-Methoxyphenethylamine (350 mg, 2.31 mmol) was added to anhydrous dichloromethane (10 mL) and the reaction was stirred at -78 o C. Then boron tribromide (5.1 mL, 5.1 mmol; 1 M) was added dropwise. The mixture was warmed to room temperature and stirred for 12 h. Methanol (10 mL) was added dropwise and the reaction was stirred for another 3 h, then concentrated under vacuum. The residue was dissolved in methanol (10 mL) and evaporated under vacuum, which was repeated several times. The product 13 was obtained as the HBr salt (502 mg, 99%). 1

8-
(i) Small-scale (1 mL) KPi reaction. The procedure described on pS11 was used and reaction yield determined by HPLC. Compound 16 was formed in 97% yield in a para:ortho isomeric ratio of 6:1 (see Supplementary Figure 11A).

Synthesis of (S)-16. (i) Small-scale (1 mL) NCS reaction.
The procedure described on pS11 was used and reaction yield determined by HPLC. Compound (S)-16 was was formed in 51% yield (see Supplementary Figure 11C) and no minor isomeric product was observed. The ee was determined to be 93% by chiral HPLC (see Supplementary Figure 6D and Table  S2). △29TfNCS (final concentration 0.5 mg/mL) to a total volume of 5 mL. The mixture was stirred at 37 o C under argon. After 18 h, the reaction was quenched with aq. HCl (0.5 mL, 1 M) and adjusted to pH 7.5 by adding aq. Na 2 CO 3 (0.75 mL, 1 M). The mixture was then stirred for another 4 h at 50 o C under argon. The reaction mixture was then centrifuged to remove proteins, and purified by preparative HPLC following semi-preparative HPLC method B to obtain (S)-16 (1.9 mg, 19%) as a yellow solid. The characterization data were consistent with that described above and [α] ! !" -20 (c 0.2, MeOH), but no minor regioisomer was present (see spectra).
In addition, experiments were performed to investigate the possible racemisation of the products such as (S)-16. When compound (S)-16 was stirred at 60 o C for 4 h stirred at 60 o C for 4 h in HEPES buffer (and 1% DMSO) under Ar with sodium ascorbate, Na 2 CO 3 (1 M) and at pH 7-8, the ee afterwards was the same within experimental error to that before being subjected to these reaction conditions (0 to 1% ee difference). Figure 11. Chromatogram of KPi and NCS mediated synthesis (1 mL scale) of 8hydroxy-1,5,6,10b-tetrahydropyrrolo [2,1-a] isoquinolin-3(2H)-one 16.

Synthesis of 18.
(i) Small-scale (1 mL) KPi reaction. The procedure described on pS11 was used and reaction yield determined by HPLC. Compound 18 was formed in 100% yield in a para:ortho isomeric ratio of 13:1 (see Supplementary Figure 12A).

S24
To a solution of 2-(4-fluoro-3-hydroxyphenyl)ethylamine (HBr salt) 15 (47 mg, 0.20 mmol), ascorbic acid (36 mg, 0.20 mmol), KPi buffer (10 mL, pH 6, 0.3 M) and CH 3 CN (10 mL) was added methyl 4oxobutanoate (32 µL, 0.31 mmol). The mixture was stirred at 60 for 18 h under argon. Then 1 M Na 2 CO 3 was added to adjust the pH to 7.5 and the reaction was stirred for another 3 h at 60 o C. The mixture was extracted with ethyl acetate (3 × 10 mL), the organic layer was dried (Na 2 SO 4 ) and evaporated. The residue was re-suspended in aq. HCl (10 mL, 1 M) and DMC (10 mL), and the aqueous layer was washed with DMC (2 × 5 mL). The DMC fractions were combined and washed with aq. HCl (2 × 5 mL, 1 M). The aqueous phase was evaporated to give 18 (25 mg, 57%), in a para:ortho ratio of 10:1, as a yellow solid. The spectroscopic characterization data was consistent with that given below for the NCS generated compound.
(i) Small-scale (1 mL) NCS reaction. The procedure described on pS11 was used and reaction yield determined by HPLC. Compound (S)-18 was was formed in 57% yield (see Supplementary Figure 12C) and no minor isomeric product was observed. The ee was determined to be 87% by chiral HPLC (see Supplementary Figure 6E and Table  S2).

(ii) Preparative scale reaction for product isolation and characterisation.
To a solution of Metaraminol (+)-bitartrate salt 14 (64 mg, 0.20 mmol), KPi buffer (18 mL, pH 7, 1 M) and CH 3 CN (2 mL) was added methyl 4-oxobutanoate (32 µL, 0.31 mmol). The mixture was stirred at 60 o C for 18 h under argon. Then 1 M Na 2 CO 3 was added to adjust the pH to 7.5 and the reaction was stirred for another 4 h at 50 o C. The reaction mixture was then centrifuged, and the supernatant was purified directly by C 18 preparative HPLC following the semi-preparative HPLC method A, giving the product as a mixture of (S)-17, (R)-17 and an ortho-product (Ratio, 100:76:47, Supplementary Figure  14A) (5.0 mg, 11% combined yield).