Biocatalytic Organic Synthesis of Optically Pure (S)-Scoulerine and Berbine and Benzylisoquinoline Alkaloids

Abstract

A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C–C bond formation catalyzed by berberine bridge enzyme (BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4–8 linear steps using either a Bischler–Napieralski cyclization or a C1–Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5–9 linear steps.

Introduction

Benzylisoquinolines and berbines(1) are two closely related classes of alkaloids encompassing more than 100 known structures. Both alkaloid families are associated with a broad range of biological activities: Many 1-benzyl-1,2,3,4-tetrahydroisoquinolines act as antispasmodic or hypotensive and some, such as norcoclaurine, coclaurine, and N-methylcoclaurine, possess anti-HIV activity in vitro.(2) Berbines show diverse biological activities such as analgesic, sedative, hypnotic, or anti-inflammatory effects,(3) and the non-natural derivative l-chloroscoulerine is currently investigated as a novel treatment of schizophrenia.(4) In addition, tetrahydroisoquinolines have recently been employed as chiral ligands for metal-catalyzed transfer-hydrogenation.(5)

Because of their biological significance, benzylisoquinolines and berbines have been targets for organic synthesis for a long time, and their asymmetric synthesis has been achieved by many different strategies.^6,7 However, a large number of steps and harsh reaction conditions are often required, resulting in limited overall yields and ee values. Furthermore, among the published procedures only few catalytic processes are found, with metal-catalyzed asymmetric hydrogenation,(8) intramolecular allylic amination or amidation,(9) and various metal- or organocatalyzed asymmetric alkylation reactions(10) representing the most notable exceptions. Despite the impressive progress in these areas, enantiomerically pure (ee > 99%) substances are rarely obtained. On the other hand, optically pure benzylisoquinoline(11) and berbine alkaloids are produced by a number of plants belonging mainly to the Berberidaceae and Papaveraceae families. However, isolation of the natural products is cumbersome, and biotransformations using plant cell cultures(12) afford minute amounts only. The production of benzylisoquinolines and related alkaloids from the morphine and sanguinarine pathways using recombinant enzymes in Escherichia coli and Saccharomyces cerevisiae has recently been reported,(13) but conversions were rather low (<15%) in these fermentative processes and product isolation was not reported. In addition, this approach is limited to a small number of target molecules and is therefore not as flexible as chemical and biocatalytic synthetic methods.

Biocatalytic steps in organic synthesis have already proven to be an efficient, highly stereoselective and flexible option in the preparation of many target compounds.(14) Of special interest are C–C bond-forming enzymes to set up the carbon framework of the organic molecules.(15) Berberine bridge enzyme (BBE) represents an outstanding biocatalyst enabling an aerobic oxidative C–C bond formation transforming benzylisoquinolines to berbines. BBE catalyzes the first committed step in the benzophenanthridine, protoberberine, and protopine biosynthesis pathways(16) in plants as it converts (S)-reticuline to (S)-scoulerine by intramolecular C–C coupling, forming the so-called “berberine bridge” (Scheme 1).

Scheme 1. C–C Bond Formation Leading to (S)-Scoulerine Catalyzed by Berberine Bridge Enzyme (BBE).

This transformation takes place via oxidative C–H activation of the substrate’s N-methyl group at the expense of molecular oxygen, a reaction unparalleled in organic synthesis.(17) BBE from Eschscholzia californica (California poppy) has been heterologously expressed in Pichia pastoris, and its X-ray crystal structure and molecular mechanism have been solved.^17,18

Recently, it has been shown that BBE accepts also non-natural substrates, whereby it transforms exclusively the (S)-enantiomer of racemic benzylisoquinolines to optically pure (S)-berbines. Since this reaction represents a highly enantioselective kinetic resolution, it provides access to the remaining optically pure (R)-substrates as well.(19)

In the present paper, we demonstrate the broad applicability of the enzyme to establish a novel synthetic route to optically pure (S)-berbines and (R)-benzylisoquinolines, including the first asymmetric total synthesis of naturally occurring (S)-scoulerine.

Results and Discussion

The synthesis of racemic 1-substituted tetrahydroisoquinolines 1 usually relies on one out of three different strategies: (i) formation of the C1–C8a bond of the isoquinoline core employing either the Pictet–Spengler(20) or the Bischler–Napieralski(21) cyclization, (ii) alkylation at position C1 of the isoquinoline via nucleophilic or electrophilic activation,(22) and (iii) formation of the C4–C4a bond by a Pomeranz–Fritsch reaction (Scheme 2).(23) The first two approaches are particularly appealing since the target molecule is disconnected at central bonds leading to simple starting materials. We focused first on the Bischler–Napieralski cyclization of amides 3a–g, since it offers a broad scope and mild reaction conditions. Therefore, the N-methylphenethylamines 4a–g and phenylacetic acid derivatives 5a and 5f were needed for synthesis of the amides 3a–g used as educts in the cyclization reaction.

Scheme 2. Strategies for the Construction of 1-Substituted 1,2,3,4-Tetrahydroisoquinolines.

Although N-methyl-(3,4-dimethoxyphenyl)ethylamine (N-methylhomoveratrylamine) 4a as well as N-methylphenethylamine 4g were commercially available, all other phenethylamines had to be synthesized. Compounds 4b–d were prepared from the corresponding phenylacetic acid derivatives 6b–d via conversion into the N-methylamides 7b–d followed by reduction (Scheme 3). The latter transformation was first attempted employing LiAlH₄ as reducing agent; however, only incomplete conversion was achieved even with a 3-fold excess of LiAlH₄ and prolonged reaction time under reflux heating (48 h). Fortunately, borane proved to be more efficient: the reduction of 7b with BH₃·THF led to full conversion as judged by TLC and GC–MS, giving 4b in 72% isolated yield.

Scheme 3. Synthesis of Phenethylamine Derivatives 4b–e.

Reagents and conditions: (a) (COCl)₂ (1.2 equiv), DMF cat., toluene, room temperature, 2 h, quant. (b) MeNH₂, aq NaOH, CH₂Cl₂, 0 °C to room temperature, 16 h, 78–95%. (c) BH₃·THF (5 equiv), THF, reflux, 16 h, 58–78%. (d) BnBr (1.0 equiv), K₂CO₃, argon, room temperature, 20 h, 89%. (e) MeNO₂ (3.2 equiv), NH₄OAc, HOAc, reflux, 5 h, 68%. (f) LiAlH₄ (5 equiv), THF, reflux, 20 h, 77%. (g) ClCO₂Et (1.2 equiv), Et₃N, CH₂Cl₂, 0 °C to room temperature, 3 h, 99%. (h) LiAlH₄ (5 equiv), THF, 0 °C to reflux, 4 h, 67%.

Compound 4e was obtained starting from cheap and readily available vanillin 8.(24) Benzylation followed by Henry-reaction with nitromethane and LiAlH₄-reduction afforded the primary amine derivative 9 in 47% overall yield (Scheme 3). In a first trial, the amine 9 was reacted with acid chloride 5a to give the corresponding amide. N-Methylation of this compound was attempted following a published procedure,(25) but unfortunately alkylation occurred not only at the nitrogen but also on the α-carbon of the amide, giving an undesired dimethylated product in 72% yield. In a second trial, cyclization of the secondary amide formed from 9 and 5a led to the desired tetrahydroisoquinoline; however, N-methylation employing methyl iodide in the presence of sodium hydride and triethylamine(26) did not lead to any conversion. Finally, the third attempt was successful: the monomethylation of 9 was performed prior to amide formation via conversion into a carbamate and LiAlH₄ reduction, giving the desired N-methylphenethylamine derivative 4e in 66% yield.

For the preparation of the phenylacetic acid building blocks two different approaches were investigated. Compound 5a was obtained from 3-hydroxyphenylacetic acid by selective monobenzylation of the dianion.(27) 3-Benzyloxy-4-methoxyphenylacetic acid 5f was also obtained by this method, requiring the commercially accessible acid 12 as starting material. Alternatively, 5f was synthesized from isovanillin 10 in a three-step sequence involving one-carbon homologation via the α-trichloromethylcarbinol 11 (Scheme 4).(28)

Scheme 4. Synthesis of Phenylacetic Acid Derivative 5f.

Reagents and conditions: (a) BnBr (1.0 equiv), K₂CO₃ (1.1 equiv), EtOH, argon, room temperature, 20 h, 91%. (b) CHCl₃ (3.6 equiv), KOH (1.3 equiv), DMF, argon, −10 °C, 2.5 h, 97%. (c) (PhSe)₂ (1.05 equiv), NaBH₄ (2.1 equiv), NaOH (6.0 equiv), ethanol, room temperature, 30 min, 40 °C, 18 h, 31%. (d) BnBr (1.1 equiv), KOH, NaI cat., EtOH, 100 °C, 16 h, 67%.

With the required building blocks (4 and 5) in hand, amide coupling was performed. The carboxylic acids 5 were converted into the corresponding acyl chlorides using oxalyl dichloride in toluene and connected with the amines under Schotten–Baumann conditions. Amides 3a–g were obtained in yields ranging from 63% to 97%. The best results were generally obtained when the acyl chloride was applied in slight excess.

Next, the Bischler–Napieralski cyclization of these amides was investigated to obtain the corresponding racemic tetrahydroisoquinolines 1. A broad range of reagents, employed in a wide variety of solvents, has been reported to effect this transformation.(21a) Cyclization of 3a employing PCl₅ in chloroform at room temperature followed by NaBH₄-reduction afforded the desired tetrahydroisoquinoline, albeit only in 13% yield. The best results were obtained using phosphorus oxychloride in refluxing acetonitrile, followed by NaBH₄-reduction in methanol. This sequence gave the tetrahydroisoquinolines in yields of 85–97%. Only the cyclization of 3g failed under these conditions, most likely owing to the lack of electron-donating substituents on the aromatic ring of the original amine building block. Although the Bischler–Napieralski reaction of nonactivated arenes is described in literature,^21a,29 no conversion was achieved in our case even under the most forcing reaction conditions employed (P₂O₅ in tetralin at 206 °C). Consequently, we had to change our strategy for the synthesis of 1g. Alkylation of a C1-lithiated tetrahydroisoquinoline derivative appeared promising and lithiation of tetrahydroisoquinoline carbamates employing t-BuLi has previously been described.(22a) Carbamate 14 and 3-benzyloxybenzyl bromide 16 were prepared and reacted following the published procedure to give the desired C–C coupling product in 29% yield (Scheme 5). By slightly changing the reaction conditions, i.e., higher temperature during the alkylation stage (see Experimental Section), this value could be improved to 51%, which approaches the reported yields obtained with less hindered nucleophiles.(22a) The alkylated carbamate was converted into the N-methyltetrahydroisoquinoline by LiAlH₄ reduction. This represents an improvement on the original report, where this transformation was achieved in a two-step deprotection/reductive amination sequence. In our case the carbamate moiety serves a triple purpose: it protects the nitrogen atom, directs the lithiation, and serves as precursor of the N-methyl group.

Scheme 5. Synthesis of 1-(3-Benzyloxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline 18.

Reagents and conditions: (a) Boc₂O (1.02 equiv), CH₂Cl₂, room temperature, 2 h, quant. (b) CBr₄ (1.05 equiv), PPh₃ (1.04 equiv), CH₂Cl₂, 0 °C to room temperature, 3 h, 94%. (c) t-BuLi (1.05 equiv), TMEDA (1.05 equiv), THF, −78 to −50 °C, 4 h, 51%. (d) LiAlH₄ (5 equiv), THF, 0 °C to reflux, 16 h, 74%.

The synthesis of racemic tetrahydroisoquinolines 1a–g (summarized in Table 1) was completed by hydrogenolytic cleavage of the benzyl ether protective groups, which proceeded quantitatively and generally gave the target compounds without the need for chromatographic purification. For instance, racemic reticuline rac-1f, as the most complex structure, was obtained with 16% overall yield, while the most efficient synthesis in terms of yield was achieved for rac-1d and rac-1b with 43% and 42% isolated overall yield, respectively.

Table 1. Overall Yields of Chemical Route to Racemic Tetrahydroisoquinolines rac-1a–g.

Finally, the racemic tetrahydroisoquinolines rac-1a–g were subjected to enantioselective oxidative ring closure catalyzed by BBE, leading to the untouched optically pure (R)-substrates and the optically pure (S)-berbine products 2a–g via kinetic resolution (Table 2). The reaction was performed employing 1 g/L BBE, 5 g/L catalase, and 20 g/L substrate in a toluene/buffer (70:30) biphasic mixture.(19) Under these conditions, substrate solubility is not an issue. Maximum conversion (50%) was achieved within 24 h in all cases, and the enantiomerically pure products (ee > 97%, HPLC) were obtained in good to excellent yields (Table 2). For instance, the kinetic resolution of racemic reticuline rac-1f yielded optically pure (R)-reticuline (R)-1f and optically pure (S)-scoulerine (S)-2f in 37% and 47% isolated yield.

Table 2. Yields of BBE-Catalyzed Oxidative Kinetic Resolution via C–C Bond Formation.

entry	substrate	c [%]a	yield (S)-2 [%]b	ee (S)-2 [%]c	yield (R)-1 [%]b	ee (R)-1 [%]c	Ed
1	rac-1ae	50	42	>97	50	>97	>200
2	rac-1be	50	36	>97	36	>97	>200
3	rac-1ce	50	39	>97	47	>97	>200
4	rac-1de	50	31	>97	46	>97	>200
5	rac-1e	50	22	>97	49	>97	>200
6	rac-1f	50	47	>97	37	>97	>200
7	rac-1g	50	46	>97	49	>97	>200

^aDetermined by HPLC on an achiral stationary phase.

^bIsolated yield (maximum theoretical yield = 50%).

^cDetermined by HPLC on a chiral stationary phase.

^dDetermined from the ee of substrate and product.

^eKinetic resolution from ref (19).

Conclusion

The combination of chemical synthesis of racemic 1-benzyl-1,2,3,4-tetrahydroisoquinolines with biocatalytic enantioselective intramolecular oxidative C–C coupling by BBE provided a new and efficient synthetic route to enantiomerically pure benzylisoquinoline and berbine alkaloids. The racemic substrates for BBE were prepared by two different pathways: either via Bischler–Napieralski cyclization or by alkylation of Boc-protected tetrahydroisoquinoline. BBE-catalyzed kinetic resolution proceeded with excellent enantioselectivity (E > 200), affording optically pure products in all cases. The overall chemoenzymatic synthesis resulted in yields of up to 20% for the benzylisoquinolines and 17% for the berbines, which represents a competitive alternative to the conventional asymmetric syntheses of these compounds.^{21d,21e,24,30} In particular, this novel synthetic route enabled the first asymmetric total synthesis of naturally occurring (S)-scoulerine, a sedative and muscle-relaxing agent,^3b,31 yielding 230 mg (7.4%) of the enantiomerically pure alkaloid over 9 linear steps.

Experimental Section

Synthesis of Amides 7b–d

A literature procedure(32) was adapted for our purpose: A solution of phenylacetic acid derivative 6b–d (20.0 mmol), oxalyl chloride (2.89 g, 22.8 mmol) and one drop of DMF in dry toluene (50 mL) was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure to give the acyl chloride (quant), which was used without further purification. A solution of the crude acyl chloride (20.0 mmol) in CH₂Cl₂ (40 mL) was cooled to 0 °C on an ice bath. A solution of amino methane (40% in H₂O; 4.11 g, 52.9 mmol) in 2 M aqueous NaOH (20 mL) was added dropwise over 1 h. The ice bath was removed and stirring was continued overnight. The phases were separated and the aqueous phase was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic phases were washed with 2 N HCl solution (100 mL), saturated NaHCO₃ solution (100 mL), and water (100 mL) and dried over Na₂SO₄. Evaporation of the solvent under reduced pressure yielded the amides 7b–d, which were used in the following transformation without further purification.

3-Methoxyphenyl-N-methylacetamide (7b)

Yield: 3.40 g (95%) as a pale yellowish solid. Mp: 41–44 °C. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.22. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.^19,33

N-Methyl-3,4,5-trimethoxyphenylacetamide (7c)

Yield: 3.74 g (78%) as a pale yellowish solid. Mp: 87–89 °C (lit.(34) 90.5–91.5 °C). TLC (petroleum ether/EtOAc = 1/1): R_f = 0.12. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19)

(3,4-Methylenedioxy)phenyl-N-methylacetamide (7d)

Yield: 3.68 g (95%) as a pale yellowish solid. Mp: 100–101 °C (lit.(35) 99–101 °C). TLC (petroleum ether/EtOAc = 1/1): R_f = 0.20. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.^19,32

Reduction of Amides 7b–d Giving Amines 4b–d

A literature procedure(32) was adapted for our purpose: BH₃·THF (1.0 M in THF; 100 mL, 100 mmol) was added to a solution of amide 7b–d (17.4–20.0 mmol) in anhydrous THF (100 mL) and the mixture was gently refluxed for 18 h under an argon atmosphere. The solution was allowed to cool to room temperature, and 6 N HCl solution (20 mL) was cautiously added. After stirring for 30 min, the resulting solution was concentrated under reduced pressure, basified by addition of 2 M NaOH solution (100 mL), and saturated with NaCl. The product was extracted into EtOAc (3 × 30 mL), and the combined organic phases were washed with brine, dried over Na₂SO₄, and evaporated under reduced pressure to give the crude product as a yellowish liquid. Flash chromatography (silica; CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1) afforded the pure amine 4b–d.

N-Methyl-3-methoxyphenethylamine (4b)

Yield: 2.23 g (72%) as a pale yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.21. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.^19,33

N-Methyl-3,4,5-trimethoxyphenethylamine (4c)

Yield: 3.54 g (78%) as a pale yellowish liquid, which crystallized upon standing to a pale yellowish solid. Mp: 175–177 °C (lit.(34) 178 °C). TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.37. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19)

N-Methyl-(3,4-methylenedioxy)phenethylamine (4d)

Yield: 1.81 g (58%) as a pale yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.20. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.^19,35

4-Benzyloxy-3-methoxybenzaldehyde(36)

K₂CO₃ (20.1 g, 0.146 mol) and benzyl bromide (22.5 g, 0.132 mol) were added to a solution of vanillin 8 (20.0 g, 0.131 mol) in ethanol (120 mL). The mixture was stirred for 20 h at room temperature under argon atmosphere. The solution was filtered through Celite and washed with CH₂Cl₂ (3 × 100 mL), and the solvent was evaporated under reduced pressure. The residue was taken up in CH₂Cl₂ (200 mL), washed with 5% NaOH solution (100 mL) and dried over K₂CO₃. Evaporation of the solvent under reduced pressure yielded 30.5 g of a yellow solid. Recrystallization from ethanol gave 4-benzyloxy-3-methoxybenzaldehyde (28.1 g, 89%) as a white solid. Mp: 61–63 °C (lit.(36) 61–62 °C). TLC (petroleum ether/EtOAc = 3/1): R_f = 0.62. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(36)

4-Benzyloxy-3-methoxy-β-nitrostyrene(37)

A solution of 4-benzyloxy-3-methoxy-benzaldehyde (22.7 g, 0.094 mol), nitromethane (18.4 g, 0.301 mol), and NH₄OAc (18.4 g, 0.239 mol) in AcOH (220 mL) was refluxed for 5 h. The mixture was poured into ice–water (300 mL), followed by addition of CH₂Cl₂ (150 mL) to dissolve the formed precipitate. The phases were separated, and the aqueous phase was extracted with CH₂Cl₂ (3 × 50 mL). The combined organic phases were washed with water (100 mL), half-saturated Na₂CO₃ solution (50 mL), and brine (50 mL), dried over Na₂SO₄, and evaporated under reduced pressure to give 21.4 g of a brown solid. Recrystallization from ethanol gave 4-benzyloxy-3-methoxy-β-nitrostyrene (18.1 g, 68%) as a yellow solid. Mp: 119–121 °C (lit.(36) 124–125 °C). TLC (petroleum ether/EtOAc = 3/1): R_f = 0.47. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(36)

4-Benzyloxy-3-methoxyphenethylamine (9)(36)

To a suspension of LiAlH₄ (8.05 g, 212 mmol) in dry THF (120 mL) under argon was added dropwise a solution of 4-benzyloxy-3-methoxy-β-nitrostyrene (12.0 g, 42.0 mmol) in dry THF (80 mL) over 1 h. The reaction mixture was refluxed for 16 h, then diluted with THF (100 mL), and cooled to 0 °C on an ice bath. To the vigorously stirred mixture were added water (8 mL), 15% NaOH solution (8 mL), and water (24 mL), the ice bath was removed, and stirring was continued for 1 h at room temperature. The resulting suspension was filtered through Celite, washed with THF, and evaporated under reduced pressure. The residue was dissolved in 10% HCl solution (20 mL) and washed with ether; afterward the aqueous layer was made basic and extracted with ether (3 × 50 mL). The combined organic phases were washed with water (20 mL) and brine (20 mL), dried over K₂CO₃, and evaporated under reduced pressure to give 8.28 g (77%) of 4-benzyloxy-3-methoxyphenethylamine as a yellowish liquid that crystallized upon standing to a yellowish solid. Mp: 63–65 °C (lit.(38) 59–61 °C). TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.27. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(36)

Ethyl 4-Benzyloxy-3-methoxyphenethylcarbamate

A literature procedure(39) was adapted for our purpose: To a solution of 4-benzyloxy-3-methoxyphenethylamine 9 (4.00 g, 15.5 mmol) in dichloromethane (120 mL) were added triethylamine (1.75 g, 17.3 mmol) and ethyl chloroformate (2.01 g, 18.4 mmol), and the mixture was stirred for 3 h at room temperature. Water (100 mL) was added, the phases were separated, and the aqueous phase was extracted with CH₂Cl₂ (2 × 30 mL). The combined organic phases were dried over Na₂SO₄ and evaporated under reduced pressure to give 5.06 g (99%) of ethyl 4-benzyloxy-3-methoxyphenethylcarbamate as a yellow liquid that crystallized upon standing to a yellowish solid. Mp: 80–81 °C. TLC (petroleum ether/EtOAc = 3/1): R_f = 0.23. ¹H NMR (CDCl₃, 300 MHz): δ 1.24 (3H, t, J = 7.1 Hz, OCH₂CH₃), 2.75 (2H, t, J = 7.1 Hz, Ar-CH₂CH₂-N), 3.41 (2H, dt, J₁ = 6.6 Hz, J₂ = 6.5 Hz, Ar-CH₂CH₂-N), 3.89 (3H, s, OCH₃), 4.12 (2H, q, J = 7.2 Hz, OCH₂CH₃), 4.71 (1H, br s, NH), 5.15 (2H, s, PhCH₂O), 6.66–6.86 (3H, m, Ar), 7.31–7.47 (5H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 14.7, 30.3, 35.8, 42.2, 56.0, 60.7, 71.1, 112.5, 114.3, 120.7, 127.3, 127.8, 128.5, 132.0, 137.3, 146.8, 149.7, 156.6. MS (EI, 70 eV): m/z = 329 (M⁺, 13), 240 (27), 137 (59), 91 (100).

4-Benzyloxy-3-methoxy-N-methylphenethylamine (4e)

A literature procedure(39) was adapted for our purpose: A solution of 4-benzyloxy-3-methoxyphenethylcarbamate (7.43 g, 22.6 mmol) in anhydrous THF (160 mL) under argon atmosphere was cooled to 0 °C on an ice bath. LiAlH₄ (4.33 g, 114 mmol) was added in portions to the stirred solution; afterward the ice bath was removed and the mixture was refluxed for 4 h. The suspension was diluted with THF (50 mL) and cooled to 0 °C on an ice bath. To the vigorously stirred mixture were added water (4.3 mL), 15% NaOH solution (4.3 mL) and water (12.9 mL), the ice bath was removed, and stirring was continued for 1 h at room temperature. The resulting suspension was filtered through Celite, washed with THF, dried over Na₂SO₄, and evaporated under reduced pressure to give 6.44 g of a brownish liquid. Flash chromatography (silica; CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1) afforded 4-benzyloxy-3-methoxy-N-methylphenethylamine (4.16 g, 67%) as an orange liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.22. ¹H NMR (CDCl₃, 300 MHz): δ 1.73 (1H, br s, NH), 2.44 (3H, s, NCH₃), 2.73–2.86 (4H, m, CH₂-CH₂-N), 3.90 (3H, s, OCH₃), 5.14 (2H, s, PhCH₂O), 6.68–6.84 (3H, m, Ar), 7.28–7.46 (5H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 35.8, 36.4, 53.3, 56.0, 71.2, 112.6, 114.3, 120.6, 127.3, 127.8, 128.5, 133.2, 137.4, 146.6, 149.6. MS (EI, 70 eV): m/z = 271 (M⁺, <1), 228 (46), 137 (18), 137 (18) 91 (58), 44 (100).

3-Benzyloxyphenylacetic Acid (5a)

A literature procedure(40) was adapted for our purpose: A solution of 3-hydroxyphenylacetic acid (6.02 g, 39.6 mmol), KOH (6.0 g, 107 mmol), and NaI (0.2 g, 1.4 mmol) in ethanol (200 mL) was heated to 90 °C. Benzyl bromide (8.01 g, 46.8 mmol) was added, whereupon the mixture was refluxed at 100 °C for 16 h. The resulting suspension was concentrated to 70 mL and poured into water (200 mL) to give a slightly brownish solution from which the product was precipitated by addition of conc hydrochloric acid. The precipitate was filtered and recrystallized from H₂O/AcOH (1/1, 70 mL) to yield 6.61 g (69%) of 3-benzyloxyphenylacetic acid as a white solid. Mp =124–125 °C (lit.(41) 119 °C). TLC (EtOAc): R_f = 0.60. The ¹H NMR data are in accordance with literature.(41)¹³C NMR (CDCl₃, 75 MHz): δ 41.1, 70.0, 113.7, 116.0, 122.0, 127.6, 128.0, 128.6, 129.7, 134.7, 136.9, 159.0, 177.6. MS (EI, 70 eV): m/z = 242 (M⁺, 9), 91 (100), 65 (10).

3-Benzyloxy-4-methoxyphenylacetic Acid (5f)

Method A

A literature procedure(40) was adapted for our purpose: A solution of 3-hydroxy-4-methoxyphenylacetic acid (2.00 g, 11.0 mmol), KOH (1.73 g, 30 mmol), and NaI (0.06 g, 0.4 mmol) in ethanol (60 mL) was heated to 90 °C. Benzyl bromide (2.59 g, 16.5 mmol) was added, whereupon the mixture was refluxed at 100 °C for 16 h. The resulting suspension was poured into water (110 mL) to give a brownish solution from which the product was precipitated by addition of conc hydrochloric acid. The precipitate was filtered and recrystallized from H₂O/AcOH (1/1, 35 mL) to yield 1.99 g (67%) of 3-benzyloxy-4-methoxyphenylacetic acid as an off-white solid. Mp =117–118 °C (lit.(42) 124–125 °C). TLC (petroleum ether/EtOAc = 3/1 + 1 drop of AcOH): R_f = 0.63. The ¹H NMR data are in accordance with literature.(41)¹³C NMR (CDCl₃, 75 MHz): δ 40.5, 56.1, 71.1, 111.9, 115.3, 122.2, 125.6, 127.5, 127.9, 128.5, 137.0, 148.2, 149.1, 177.7.

Method B

A literature procedure^28,43 was adapted for our purpose: To a stirred solution of isovanillin 10 (20.0 g, 0.131 mol) in ethanol (120 mL) were added K₂CO₃ (20.1 g, 0.145 mol) and benzyl bromide (22.5 g, 0.131 mol). The mixture was stirred for 20 h at room temperature under argon atmosphere. The solution was filtered through Celite and washed with CH₂Cl₂ (3 × 100 mL), and the solvent was evaporated under reduced pressure. The residue was taken up in CH₂Cl₂ (200 mL), washed with 5% NaOH solution (100 mL), and dried over K₂CO₃. Evaporation of the solvent under reduced pressure yielded 31.3 g of a yellow solid. Recrystallization from ethanol gave 3-benzyloxy-4-methoxybenzaldehyde (29.2 g, 91%) as a white solid. Mp: 62–63 °C (lit.(24) 61–62 °C). TLC (petroleum ether/EtOAc = 3/1): R_f = 0.29. The ¹H and ¹³C NMR data are in accordance with literature.(44) MS (EI, 70 eV): m/z = 242 (M⁺, 13), 91 (100), 65 (9).

A solution of 3-benzyloxy-4-methoxybenzaldehyde (29.0 g, 0.120 mol) and chloroform (35 mL) in DMF (120 mL) under argon atmosphere was cooled to −10 °C on an ice/NaCl bath. A solution of KOH (8.88 g, 0.158 mol) in methanol (30 mL) was added dropwise over 30 min and the resulting mixture was stirred for 2 h at −10 °C. The reaction was quenched with 1 N hydrochloric acid (270 mL) and stirred for an additional 30 min at −10 °C. Afterward, the mixture was allowed to warm to room temperature, toluene (100 mL) was added, and the phases were separated. The aqueous phase was extracted with toluene (2 × 100 mL), and the combined organic phases were washed with water (30 mL) and brine (30 mL) and dried over Na₂SO₄. Evaporation of the solvent under reduced pressure gave 42.9 g (97%) of 1-(3-benzyloxy-4-methoxyphenyl)-2,2,2-trichloroethanol 11 as a yellowish solid, which was used in the next step without further purification. TLC (petroleum ether/EtOAc = 3/1): R_f = 0.53. ¹H NMR (CDCl₃, 300 MHz): δ 3.71 (1H, br s, OH), 3.92 (3H, s, OCH₃), 5.09 (1H, s, CH–OH), 5.21 (2H, s, PhCH₂O), 6.89 (1H, d, J = 8.7 Hz, Ar), 7.14–7.21 (2H, m, Ar), 7.26–7.46 (5H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 55.9, 71.0, 84.1, 103.4, 110.6, 115.1, 122.6, 127.3, 127.4, 127.9, 128.6, 137.0, 147.1, 150.5. MS (EI, 70 eV): m/z = 360 (M⁺, 1), 243 (36), 91 (100).

Diphenyl diselenide (36.9 g, 0.118 mol) was dissolved in deoxygenated ethanol (300 mL; purged with argon for 1 h). NaBH₄ (9.0 g, 0.238 mol) was added in portions over 30 min, upon which the previously orange solution turned colorless. The resulting mixture was stirred for 30 min at room temperature before addition of 11 (40.8 g, 0.113 mol) followed by NaOH (27.1 g, 0.678 mol). The reaction was then stirred for 18 h at 40 °C. The solvent was evaporated under reduced pressure, and the solid residue was dissolved in water (200 mL). The pH of the solution was adjusted to 1.0 by addition of conc hydrochloric acid, and the product was extracted into EtOAc (5 × 100 mL). The combined organic phases were dried over Na₂SO₄, and the solvent was evaporated under reduced pressure to give an orange solid, which was recrystallized from petroleum ether/acetone to afford 5f (11.0 g, 31%) as an off-white solid. The spectroscopic and chromatographic data are identical to those of 5f obtained by method A.

Synthesis of Amides 3a–g

A literature procedure(45) was adapted for our purpose: A solution of phenylacetic acid derivative 5a or 5f (10.5–12.0 mmol), oxalyl chloride (1.95 g, 15.4 mmol), and one drop of DMF in dry toluene (40 mL) was stirred at room temperature under argon for 1 h. The solvent was evaporated under reduced pressure to give the acyl chloride (quant), which was used without further purification.

The amine 4a–g (10.0–13.5 mmol) was dissolved in CHCl₃ (30 mL). A 3% NaOH solution (150 mL) was added, and the mixture was cooled to 0 °C on an ice bath. A solution of the crude phenylacetyl chloride derivative (10.6–12.8 mmol) in chloroform (20 mL) was added dropwise over 1 h to the vigorously stirred mixture. The ice bath was removed, and stirring was continued for 16 h at room temperature. The phases were separated, and the aqueous phase was extracted with CHCl₃ (50 mL). The combined organic phases were washed with dilute HCl solution (100 mL) and then water (100 mL) and dried over Na₂SO₄. Evaporation of the solvent under reduced pressure yielded the crude amide 3a–g, which was purified by flash chromatography (silica; petroleum ether/EtOAc = 1/1). The product is obtained as a mixture of rotamers, to which NMR signals are assigned based on the peak intensities as well as the DEPT, COSY, and HSQC spectra.

2-(3-Benzyloxyphenyl)-N-(3,4-dimethoxyphenethyl)-N-methylacetamide (3a)

Yield: 2.97 g (64%) as an off-white solid. Ratio trans/cis = 1.15/1. Mp: 97–98 °C. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.55. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₆H₂₉NO₄ 419.2097; found 419.2099.

2-(3-Benzyloxyphenyl)-N-(3-methoxyphenethyl)-N-methylacetamide (3b)

Yield: 2.79 g (68%) as a pale yellowish liquid. Ratio trans/cis = 1.05/1. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.37. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₅H₂₇NO₃ 389.1991; found 389.1990.

2-(3-Benzyloxyphenyl)-N-methyl-N-(3,4,5-trimethoxyphenethyl)acetamide (3c)

Yield: 3.63 g (63%) as a pale yellowish liquid. Ratio trans/cis = 1.05/1. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.18. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₇H₃₁NO₅ 449.2202; found 449.2224.

2-(3-Benzyloxyphenyl)-N-(3,4-methylenedioxy)phenethyl-N-methylacetamide (3d)

Yield: 3.88 g (97%) as a pale yellowish liquid. Ratio trans/cis = 1.07/1. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.37. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₅H₂₅NO₄ 403.1783; found 403.1796.

2-(3-Benzyloxyphenyl)-N-(4-benzyloxy-3-methoxyphenethyl)-N-methylacetamide (3e)

Yield: 5.19 g (78%) as a pale yellowish liquid. Ratio trans/cis = 1.11/1. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.26. MS (EI, 70 eV): m/z = 495 (M⁺, 5), 240 (48), 197 (5), 149 (12), 91 (100). HRMS: calcd for C₃₂H₃₃NO₄ 495.2410; found 495.2440. trans-3e: ¹H NMR (CDCl₃, 300 MHz): δ 2.76 (2H, t, J = 7.5 Hz, CH₂-CH₂-N), 2.82 (3H, s, N-CH₃), 3.56 (2H, t, J = 7.5 Hz, CH₂-CH₂-N), 3.65 (2H, s, CH₂-CO), 3.81 (3H, s, OCH₃), 5.04 (2H, s, Ph-C H₂-O), 5.10 (2H, s, Ph-C H₂-O), 6.61–6.64 (1H, m, Ar), 6.74–6.90 (5H, m, Ar), 7.18–7.40 (11H, m, Ar). ¹³ C NMR (CDCl ₃, 75 MHz): δ 33.3 (CH₂), 36.6 (CH₃), 41.4 (CH₂), 50.3 (CH₂), 56.0 (CH₃), 69.9 (CH₂), 71.1 (CH₂), 112.6 (CH), 113.2 (CH), 114.3 (CH), 115.3 (CH), 120.7 (CH), 121.4 (CH), 127.3 (CH), 127.5 (CH), 127.8 (CH), 128.0 (CH), 128.5 (CH), 128.6 (CH), 129.7 (CH), 132.4 (C), 136.5 (C), 137.0 (C), 137.2 (C), 146.7 (C), 149.7 (C), 159.1 (C), 170.6 (C). cis-3e: ¹ H NMR (CDCl ₃, 300 MHz): δ 2.60 (2H, t, J = 7.2 Hz, CH₂-CH₂-N), 2.95 (3H, s, N-CH₃), 3.41 (2H, s, CH₂-CO), 3.42 (2H, t, J = 7.1 Hz, CH₂-C H₂-N), 3.84 (3H, s, OCH₃), 5.02 (2H, s, Ph-C H₂-O), 5.10 (2H, s, Ph-C H₂-O), 6.53–6.90 (2H, m, Ar), 6.74–6.90 (4H, m, Ar), 7.18–7.40 (11H, m, Ar). ¹³ C NMR (CDCl ₃, 75 MHz): δ 33.6 (CH₃), 34.3 (CH₂), 40.8 (CH₂), 52.2 (CH₂), 56.1 (CH₃), 69.9 (CH₂), 71.1 (CH₂), 112.5 (CH), 113.2 (CH), 114.5 (CH), 115.2 (CH), 120.7 (CH), 121.3 (CH), 127.3 (CH), 127.5 (CH), 127.9 (CH), 128.0 (CH), 128.5 (CH), 128.6 (CH), 129.7 (CH), 131.4 (C), 136.9 (C), 137.0 (C), 137.1 (C), 147.0 (C), 149.9 (C), 159.1 (C), 170.8 (C).

2-(3-Benzyloxy-4-methoxyphenyl)-N-(4-benzyloxy-3-methoxyphenethyl)-N-methylacetamide (3f)

Yield: 2.33 g (76%) as an off-white solid. Ratio trans/cis = 1.14/1. Mp: 126–127 °C. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.64. MS (EI, 70 eV): m/z = 525 (M⁺, 3), 240 (22), 149 (11), 105 (14), 91 (100). HRMS: calcd for C₃₃H₃₅NO₅: 525.2515; found 525.2523. trans-3f¹H NMR (CDCl₃, 300 MHz): δ 2.74 (2H, t, J = 7.5 Hz, CH₂-CH₂-N), 2.79 (3H, s, N-CH₃), 3.54 (2H, t, J = 7.7 Hz, CH₂-CH₂-N), 3.59 (2H, s, CH₂-CO), 3.84 (3H, m, OCH₃), 3.88 (3H, s, OCH₃), 5.13 (2H, s, Ph-C H₂-O), 5.16 (2H, s, Ph-C H₂-O), 6.53–6.86 (6H, m, Ar), 7.28–7.47 (10H, m, Ar). ¹³ C NMR (CDCl ₃, 75 MHz): δ 33.3 (CH₂), 36.5 (CH₃), 40.8 (CH₂), 50.2 (CH₂), 56.0 (CH₃), 70.9 (CH₂), 71.1 (CH₂), 112.0 (CH), 112.6 (CH), 114.2 (CH), 114.7 (CH), 120.7 (CH), 121.5 (CH), 127.3 (CH), 127.4 (CH), 127.8 (CH), 127.9 (CH), 128.5 (CH), 132.4 (C), 137.1 (C), 146.6 (C), 148.2 (C), 149.6 (C), 170.9 (C). cis-3f: ¹H NMR (CDCl₃, 300 MHz): δ 2.58 (2H, t, J = 7.2 Hz, CH₂-CH₂-N), 2.94 (3H, s, N-CH₃), 3.35 (2H, s, CH₂-CO), 3.39 (2H, t, J = 6.9 Hz, CH₂-CH₂-N), 3.87 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 5.13 (2H, s, Ph-CH₂-O × 2), 6.53–6.90 (2H, m, Ar), 6.74–6.90 (4H, m, Ar), 7.18–7.40 (11H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 33.6 (CH₃), 34.2 (CH₂), 40.8 (CH₂), 52.0 (CH₂), 56.0 (CH₃), 70.8 (CH₂), 71.1 (CH₂), 111.9 (CH), 112.6 (CH), 114.5 (CH), 114.5 (CH), 120.7 (CH), 121.3 (CH), 127.3 (CH), 127.4 (CH), 127.7 (CH), 127.8 (CH), 128.5 (CH), 131.4 (CH), 137.1 (CH), 137.3 (CH), 147.0 (CH), 148.6 (CH), 149.8 (CH), 171.2 (CH).

2-(3-Benzyloxyphenyl)-N-phenethyl-N-methylacetamide (3g)

Yield: 2.30 g (63%) as a pale yellowish liquid. Ratio trans/cis = 1.05/1. TLC (petroleum ether/EtOAc = 1/1): R_f = 0.51. trans-3g: ¹H NMR (CDCl₃, 300 MHz): δ 2.79–2.84 (5H, s + t overlap, N-CH₃ + CH₂-CH₂-N), 3.57 (2H, t, J = 7.5 Hz, CH₂-CH₂-N), 3.63 (2H, s, CH₂-CO), 5.03 (2H, s, Ph-C H₂-O), 6.73–6.88 (3H, m, Ar), 7.05–7.07 (1H, m, Ar), 7.14–7.42 (10H, m, Ar). ¹³ C NMR (CDCl ₃, 75 MHz): δ 33.7 (CH₂), 36.6 (CH₃), 41.4 (CH₂), 50.2 (CH₂), 69.9 (CH₂), 113.2 (CH), 115.3 (CH), 121.5 (CH), 126.3 (CH), 127.5 (CH), 128.0 (CH), 128.6 (CH), 128.8 (CH), 128.9 (CH), 129.7 (CH), 136.6 (C), 137.0 (C), 139.1 (C), 159.1 (C), 170.8 (C). cis-3g: ¹H NMR (CDCl₃, 300 MHz): δ 2.66 (2H, t, J = 7.3 Hz, CH₂-CH₂-N), 2.95 (3H, s, N-CH₃), 3.40 (2H, s, CH₂-CO), 3.44 (1H, t, J = 7.3 Hz, CH₂-CH₂-N), 5.01 (2H, s, Ph-CH₂-O), 6.73–6.88 (3H, m, Ar), 7.05–7.07 (1H, m, Ar), 7.14–7.42 (10H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 33.6 (CH₃), 34.7 (CH₂), 40.8 (CH₂), 52.0 (CH₂), 69.9 (CH₂), 113.3 (CH), 115.2 (CH), 121.4 (CH), 126.8 (CH), 127.5 (CH), 128.0 (CH), 128.5 (CH), 128.6 (CH), 128.9 (CH), 129.7 (CH), 136.9 (C), 137.0 (C), 138.3 (C), 159.1 (C), 170.7 (C).

Bischler–Napieralski Cyclization of Amides 3a–f

A literature procedure(46) was adapted for our purpose: A solution of amide 3a–f (7.0 mmol) and POCl₃ (21.0 mmol) in dry acetonitrile (60 mL) was refluxed for 3 h under argon atmosphere. The solvent and excess POCl₃ were evaporated under reduced pressure, and the residue was dissolved in dry methanol (50 mL), flushed with argon, and cooled to −5 °C on an ice/NaCl bath. NaBH₄ (50.0 mmol) was added in portions to the stirred mixture. The ice bath was then removed, and stirring was continued for 16 h at room temperature. The solvent was evaporated, and the residue was treated with half-saturated Na₂CO₃ solution (60 mL). The product was extracted with CH₂Cl₂ (3 × 30 mL) and the combined organic phases were dried over Na₂SO₄ and evaporated under reduced pressure to give the crude tetrahydroisoquinoline, which was purified by flash chromatography (silica; CH₂Cl₂/MeOH/NH₃(aq) = 96/3/1).

1-(3-Benzyloxybenzyl)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline

Yield: 2.78 g (94%) as a yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.75. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₆H₂₈NO₃ [(M – H)⁺] 402.2069; found 402.2071.

1-(3-Benzyloxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline

Yield: 2.43 g (94%) as a pale yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.61. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₅H₂₆NO₂ [(M – H)⁺] 372.1964; found 372.1974.

1-(3-Benzyloxybenzyl)-2-methyl-6,7,8-trimethoxy-1,2,3,4-tetrahydroisoquinoline

Yield: 2.89 g (85%) as a pale yellowish viscous liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.75. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₇H₃₀NO₄ [(M – H)⁺] 432.2175; found 432.2194.

1-(3-Benzyloxybenzyl)-6,7-methylenedioxy-2-methyl-1,2,3,4-tetrahydroisoquinoline

Yield: 3.59 g (97%) as a pale yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.76. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₅H₂₄NO₃ [(M – H)⁺] 386.1756; found 386.1740.

1-(3-Benzyloxybenzyl)-7-benzyloxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline

Yield: 4.36 g (88%) as a pale yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.28. ¹H NMR (CDCl₃, 300 MHz): δ 2.56 (3H, s, NCH₃), 2.58–2.90 (4H, m, CH₂), 3.10–3.20 (2H, m, CH₂), 3.68 (1H, dd, J₁ = 6.9 H, J₂ = 5.7 Hz, CH), 3.86 (3H, s, OCH₃), 4.76 (1H, d, J = 12.3 Hz, PhCH₂O), 4.86 (1H, d, J = 12.0 Hz, PhCH₂O), 5.01 (2H, s, Ph-CH₂O), 6.13 (1H, s, Ar), 6.60 (1H, s, Ar), 6.69–6.88 (3H, m, Ar), 7.20 (1H, t, J = 8.0 Hz, Ar), 7.28–7.45 (10H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 25.7, 41.1, 42.7, 47.0, 55.9, 64.6, 69.9, 70.8, 111.7, 112.3, 113.7, 116.4, 122.6, 126.5, 127.3, 127.5, 127.7, 127.9, 128.4, 128.6, 129.1, 129.4, 137.1, 137.1, 141.9, 145.6, 147.9, 158.7. MS (EI, 70 eV): m/z = 478 [(M – H)⁺, <1], 282 (100), 191 (30), 162 (18), 91 (37). HRMS: calcd for C₃₂H₃₂NO₃ [(M – H)⁺] 478.2382; found 478.2391.

1-(3-Benzyloxy-4-methoxybenzyl)-7-benzyloxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline

Yield: 1.98 g (97%) as a yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.56. ¹H NMR (CDCl₃, 300 MHz): δ 2.47 (3H, s, NCH₃), 2.50–2.58 (1H, m, CH₂), 2.66–2.83 (3H, m, CH₂), 2.97–3.12 (2H, m, CH₂), 3.55 (1H, dd, J₁ = 6.9 H, J₂ = 5.2 Hz, CH), 3.85 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 4.80 (1H, d, J = 12.3 Hz, PhCH₂O), 4.87 (1H, d, J = 12.3 Hz, PhCH₂O), 5.07 (2H, s, Ph-CH₂O), 6.10 (1H, s, Ar), 6.55–6.57 (2H, m, Ar), 6.64 (1H, d, J = 1.9 Hz, Ar), 6.77 (1H, d, J = 8.2 Hz, Ar), 7.26–7.38 (8H, m, Ar), 7.42–7.45 (2H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 25.8, 40.5, 42.7, 47.2, 55.9, 56.0, 64.6, 70.8, 70.9, 111.4, 111.6, 113.7, 115.7, 122.5, 126.8, 127.2, 127.3, 127.7, 127.7, 128.4, 128.5, 129.3, 132.4, 137.3, 145.6, 147.7, 147.8, 148.0. MS (EI, 70 eV): m/z = 507 [(M – 2H)⁺, <1], 282 (100), 191 (25), 162 (13), 91 (21). HRMS: calcd for C₃₃H₃₃NO₄ [(M–2H)⁺] 507.2410; found 507.2435.

tert-Butyl 3,4-Dihydro-2(1H)-isoquinolinecarboxylate (14)(22a)

A solution of di-tert-butyl dicarbonate (11.11 g, 50.9 mmol) in CH₂Cl₂ (20 mL) was added dropwise to a solution of 1,2,3,4-tetrahydroisoquinoline 13 (6.66 g, 50.0 mmol) in CH₂Cl₂ (30 mL). After stirring at room temperature for 2 h, the solvent was evaporated under reduced pressure to give 11.74 g (100%) of 14 as an orange liquid. TLC (petroleum ether/EtOAc = 3/1): R_f = 0.62. The ¹H NMR data are in accordance with literature.(22a)¹³C NMR (CDCl₃, 75 MHz): δ 28.5, 29.0, 40.7, 45.9, 85.2, 126.2, 126.3, 128.7, 134.8, 154.9. MS (EI, 70 eV): m/z = 218 [(M – CH₃)⁺, <1), 176 (100), 160 (24), 142 (9), 132 (70), 117 (13), 104 (52), 77 (13), 57 (78), 41 (22).

3-Benzyloxybenzyl bromide (16)

A literature procedure(47) was adapted for our purpose: A solution of 3-benzyloxybenzyl alcohol 15 (8.01 g, 37.4 mmol) and tetrabromomethane (13.1 g, 39.4 mmol) in CH₂Cl₂ (60 mL) was cooled to 0 °C on an ice/NaCl bath. Triphenylphosphine (10.22 g, 39.0 mmol) was added in portions to the stirred mixture, the cooling bath was removed, and the solution was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the liquid residue was poured into well-stirred petroleum ether (100 mL), resulting in the formation of a white precipitate. The solid was removed by filtration and washed with petroleum ether (3 × 50 mL), and the filtrate was evaporated under reduced pressure to give 18.4 g of an orange liquid. Flash chromatography (silica; petroleum ether → petroleum ether/EtOAc = 9/1) afforded 16 (9.75 g, 94%) as a white crystalline solid. Mp: 54–55 °C (lit.(48) 37–39 °C). TLC (petroleum ether/EtOAc = 3/1): R_f = 0.76. The ¹H and ¹³C NMR data are in accordance with literature.(48) MS (EI, 70 eV): m/z = 276 (M⁺, 8), 197 (15), 91 (100).

tert-Butyl 1-(3-Benzyloxybenzyl)-3,4-dihydro-2(1H)-isoquinolinecarboxylate (17)

A literature procedure(22a) was adapted for our purpose: A solution of tert-butyl 3,4-dihydro-2(1H)-carboxylate 14 (2.33 g, 10.0 mmol) and tetramethylethylene-diamine (1.22 g, 10.5 mmol) in anhydrous THF under argon atmosphere was cooled to −78 °C. tert-Butyl lithium solution (1.7 M in pentane; 6.2 mL, 10.5 mmol) was added dropwise over 30 min, resulting in a deep red solution, which was stirred at −78 °C for 30 min. A solution of 3-benzyloxybenzyl bromide (5; 2.77 g, 10.0 mmol) in anhydrous THF (10 mL) was added dropwise over 30 min. The mixture was then stirred for 3 h, during which time the temperature was allowed to rise to −50 °C. The resulting yellow suspension was quenched with saturated NH₄Cl solution (10 mL). Water (30 mL) was added, the phases were separated, and the aqueous phase was extracted with tert-butyl methyl ether (2 × 20 mL). The combined organic phases were dried over Na₂SO₄, and the solvent was evaporated under reduced pressure to give 5.52 g of an orange liquid. Flash chromatography (silica; petroleum ether → petroleum ether/EtOAc = 95/5) afforded 17 (2.21 g, 51%) as a colorless liquid. TLC (petroleum ether/EtOAc = 3/1): R_f = 0.59. MS (EI, 70 eV): m/z = 429 (M⁺, <1), 232 (16), 176 (57), 132 (100), 91 (38). HRMS: calcd for C₂₈H₃₁NO₃ (M⁺) 429.2304; found 429.2333. The product is obtained as a mixture of rotamers (ratio cis/trans = 2/1), to which NMR signals are assigned based on the peak intensities as well as the DEPT, COSY, and HSQC spectra. cis-17: ¹H NMR (CDCl₃, 300 MHz): δ 1.25 (9H, s, CH₃), 2.62–3.07 (4H, m, CH₂), 3.22–3.31 (1H, m, CH₂CH₂N), 4.19 (1H, ddd, J₁ = 13.1 Hz, J₂ = 5.6 Hz, J₃ = 3.5 Hz, CH₂CH₂N), 5.01 (2H, s, PhCH₂O), 5.22 (1H, dd, J₁ = 8.2 Hz, J₂ = 5.6 Hz, CH), 6.68–6.92 (3H, m, Ar), 7.03–7.19 (5H, m, Ar), 7.32–7.49 (5H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 28.1 (CH₃), 28.5 (CH₂), 37.0 (CH₂), 43.0 (CH₂), 56.7 (CH), 69.9 (CH₂), 79.6 (C), 112.7 (CH), 116.3 (CH), 122.4 (CH), 125.9 (CH), 126.7 (CH), 127.3 (CH), 127.5 (CH), 127.9 (CH), 128.6 (CH), 129.1 (CH), 129.3 (CH), 134.8 (C), 137.0 (C), 137.1 (C), 140.2 (C), 154.4 (C), 158.8 (C). trans-17: ¹H NMR (CDCl₃, 300 MHz): δ 1.42 (9H, s, CH₃), 2.62–3.07 (4H, m, CH₂), 3.22–3.31 (1H, m, CH₂CH₂N), 3.78 (1H, dt, J₁ = 11.3 Hz, J₂ = 5.2 Hz, CH₂CH₂N), 4.96 (2H, s, PhCH₂O), 5.38 (1H, t, J = 6.7 Hz, CH), 6.68–6.92 (3H, m, Ar), 7.03–7.19 (5H, m, Ar), 7.32–7.49 (5H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 28.4 (CH₂), 28.6 (CH₃), 39.4 (CH₂), 42.7 (CH₂), 55.5 (CH), 69.8 (CH₂), 79.5 (C), 113.0 (CH), 116.0 (CH), 122.5 (CH), 125.9 (CH), 126.6 (CH), 127.3 (CH), 127.5 (CH), 127.9 (CH), 128.4 (CH), 129.0 (CH), 129.3 (CH), 134.6 (C), 137.0 (C), 137.2 (C), 139.8 (C), 154.7 (C), 158.6 (C).

1-(3-Benzyloxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (18)

A solution of tert-butyl 1-(3-benzyloxybenzyl)-3,4-dihydro-2(1H)-isoquinolinecarboxylate 17 (3.55 g, 8.26 mmol) in anhydrous THF (160 mL) under argon atmosphere was cooled to 0 °C on an ice bath. LiAlH₄ (1.60 g, 42.2 mmol) was added in portions to the stirred solution; afterward the ice bath was removed and the mixture was refluxed for 16 h. The suspension was diluted with THF (50 mL) and cooled to 0 °C on an ice bath. Water (1.6 mL), 15% NaOH solution (1.6 mL), and again water (4.8 mL) were added to the vigorously stirred mixture, the ice bath was removed, and stirring was continued for 1 h at room temperature. The resulting suspension was filtered through Celite, washed with THF, dried over Na₂SO₄, and evaporated under reduced pressure to give 2.89 g of a yellow liquid. Flash chromatography (silica; CH₂Cl₂/MeOH/NH₃(aq) = 98/1/1) afforded 18 (2.09 g, 74%) as a yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.56. ¹H NMR (CDCl₃, 300 MHz): δ 2.54 (3H, s, NCH₃), 2.67–2.83 (2H, m, CH₂), 2.88–2.98 (2H, m, CH₂), 3.14–3.27 (2H, m, CH₂), 3.87 (1H, t, J = 6.2 Hz, CH), 5.04 (2H, s, PhCH₂O), 6.78–6.89 (4H, m, Ar), 7.06–7.24 (4H, m, Ar), 7.36–7.46 (5H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 26.1, 41.5, 42.9, 47.1, 65.0, 69.9, 112.4, 116.2, 122.4, 125.4, 126.0, 127.6, 127.9, 128.0, 128.6, 128.8, 129.0, 134.4, 137.3, 137.9, 141.7, 158.6. MS (EI, 70 eV): m/z = 342 [(M – H)⁺, <1], 146 (100), 131 (6), 91 (10). HRMS: calcd for C₂₄H₂₄NO [(M – H)⁺] 342.1858; found 342.1851.

Hydrogenolytic Deprotection Affording Tetrahydroisoquinolines 1a–g

A literature procedure(24) was adapted for our purpose: A mixture of benzyl-protected tetrahydroisoquinoline (5.75–9.16 mmol), Pd 10% on activated charcoal (0.20–0.30 g), acetic acid (12.5–20.0 mmol), and dry methanol (50 mL) was stirred under H₂ atmosphere (balloon) for 16 h. The mixture was filtered through Celite, washed with methanol (100 mL), and evaporated under reduced pressure. The residue was dissolved in CH₂Cl₂ (30 mL) and washed with half-saturated NaHCO₃ solution (40 mL). The organic phase was dried over Na₂SO₄ and evaporated under reduced pressure to afford pure 1a–g.

6,7-Dimethoxy-1-(3-hydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (1a)

Yield: 2.07 g (98%) as an off-white solid foam. Mp: 127–128 °C (lit.(49) 135 °C). TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.53. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.^19,50 HRMS: calcd for C₁₉H₂₂NO₃ [(M – H)⁺] 312.1600; found 312.1589.

1-(3-Hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (1b)

Yield: 1.53 g (94%) as an off-white solid foam. Mp =113–116 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.51. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₁₈H₂₀NO₂ [(M – H)⁺] 282.1494; found 282.1499.

1-(3-Hydroxybenzyl)-2-methyl-6,7,8-trimethoxy-1,2,3,4-tetrahydroisoquinoline (1c)

Yield: 1.87 g (84%) as a highly viscous yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.51. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₀H₂₄NO₄ [(M – H)⁺]: 342.1705; found 342.1727.

1-(3-Hydroxybenzyl)-6,7-methylenedioxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (1d)

Yield: 2.19 g (81%) as a white solid foam. Mp: 143–145 °C (lit.(49) 145 °C). TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.45. The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₁₈H₁₈NO₃ [(M – H)⁺] 296.1287; found 296.1297.

1-(3-Hydroxybenzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (1e)

Yield: 2.11 g (98%) as an off-white solid foam. Mp: 103–106 °C (lit.(49) 111–113 °C). TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.25. The NMR data are in accordance with literature.(50) MS (EI, 70 eV): m/z = 298 [(M – H)⁺, <1), 192 (100), 177 (19), 148 (5). MS (EI, 70 eV): m/z = 298 [(M – H)⁺, <1), 192 (100), 177 (19), 148 (5). HRMS: calcd for C₁₈H₂₀NO₃ [(M – H)⁺] 298.1443; found 298.1450.

Reticuline (1f)

Yield: 0.90 g (70%) as an off-white solid foam. Mp: 83–84 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.29. The NMR data are in accordance with literature.(24) MS (EI, 70 eV): m/z = 328 [(M – H)⁺, <1], 192 (100), 177 (21). HRMS: calcd for C₁₉H₂₂NO₄ [(M – H)⁺] 328.1549; found 328.1571.

1-(3-Hydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (1g)

Yield: 1.25 g (94%) as an off-white solid foam. Mp: 129–130 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.47. ¹H NMR (CDCl₃, 300 MHz): δ 2.50 (3H, s, NCH₃), 2.73–3.05 (4H, m, CH₂), 3.17 (1H, dd, J₁ = 13.8 Hz, J₂ = 6.0 Hz, CH₂), 3.28–3.37 (1H, m, CH₂), 3.92 (1H, t, J = 6.5 Hz, CH), 6.60–6.63 (3H, m, Ar), 6.76 (1H, d, J = 7.8 Hz, Ar), 7.02–7.15 (4H, m, Ar). ¹³C NMR (CDCl₃, 75 MHz): δ 24.5 (CH₂), 41.7 (CH₂), 41.9 (CH₃), 46.0 (CH₂), 64.9 (CH), 113.9 (CH), 116.6 (CH), 121.1 (CH), 125.6 (CH), 126.4 (CH), 128.1 (CH), 128.9 (CH), 129.4 (CH), 133.2 (C), 136.8 (C), 140.9 (C), 156.7 (C). MS (EI, 70 eV): m/z = 252 [(M – H)⁺, <1], 146 (100), 131 (7). HRMS: calcd for C₁₇H₁₈NO [(M – H)⁺] 252.1388; found 252.1403.

BBE-Catalyzed Kinetic Resolution of 1a–g(19)

Substrate 1a–g (500 mg, 1.5–2.0 mmol) was dissolved in toluene (17.5 mL) and buffer (7.5 mL, 10 mM Tris-HCl, pH 9.0, 10 mM MgCl₂) containing BBE (1.5 mL enzyme solution, final concentration = 1 g/L = 0.017 mM) and crude catalase (125 mg, final concentration 5 g/L). The mixture was shaken in a light-shielded round-bottom flask (50 mL) at 200 rpm and 40 °C for 24 h. The reaction was stopped by phase separation, followed by extraction of the aqueous phase with ethyl acetate (3 × 10 mL). The combined organic phases were dried over Na₂SO₄ and evaporated under reduced pressure to give the crude product. Flash chromatography (silica; a–f, CH₂Cl₂/MeOH/NH₃(aq) = 96/3/1; g, CH₂Cl₂/MeOH/NH₃(aq) = 98/1/1) afforded pure (S)-2a–g and (R)-1a–g.

(S)-2,3-Dimethoxy-9-hydroxyberbine (S)-2a

Yield: 207 mg (42%) as an off-white solid foam. Mp: 90–95 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.78. [α]²⁰_D = −273.4 (CHCl₃, c = 1.0); lit.(50) (R) +176 (MeOH, c = 0.34). The ¹H and ¹³C NMR as well as MS data are in accordance with literature.^19,50 HRMS: calcd for C₁₉H₂₁NO₃ 311.1521; found 311.1519.

(R)-6,7-Dimethoxy-1-(3-hydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (R)-1a

Yield: 249 mg (50%) as an off-white solid foam. Mp: 151–153 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.53. [α]²⁰_D = −109.4 (CHCl₃, c = 1.0). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₁₉H₂₂NO₃ [(M – H)⁺] 312.1600; found 312.1591. The NMR data are in accordance with literature.(50)

(S)-9-Hydroxy-3-methoxyberbine (S)-2b

Yield: 177 mg (36%) as an off-white solid foam. Mp: 192–195 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.56. [α]²⁰_D = −280.6 (CHCl₃, c = 0.5). The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₁₈H₁₉NO₂ 281.1416; found 281.1415.

(R)-1-(3-Hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (R)-1b

Yield: 181 mg (36%) as a highly viscous yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.47. [α]²⁰_D = −76.3 (CHCl₃, c = 0.63). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₁₈H₂₀NO₂ [(M – H)⁺] 282.1494; found 282.1504.

(S)-9-Hydroxy-1,2,3-trimethoxyberbine (S)-2c

Yield: 194 mg (39%) as an off-white solid foam. Mp: 85–89 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.60. [α]²⁰_D = −226.5 (CHCl₃, c = 0.57). The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₂₀H₂₃NO₄ 341.1627; found 341.1623.

(R)-1-(3-Hydroxybenzyl)-2-methyl-6,7,8-trimethoxy-1,2,3,4-tetrahydroisoquinoline (R)-1c

Yield: 237 mg (47%) as highly viscous yellowish liquid. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.33. [α]²⁰_D = −75.4 (CHCl₃, c = 0.75). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₂₀H₂₄NO₄ [(M – H)⁺] 342.1705; found 342.1703.

(S)-9-Hydroxy-2,3-methylenedioxyberbine (S)-2d

Yield: 155 mg (31%) as an off-white solid foam. Mp: 177–180 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.50. [α]²⁰_D = −342.5 (CHCl₃, c = 0.63). The ¹H and ¹³C NMR as well as MS data are in accordance with literature.(19) HRMS: calcd for C₁₈H₁₇NO₃ 295.1208; found 295.1209.

(R)-1-(3-Hydroxybenzyl)-6,7-methylenedioxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (R)-1d

Yield: 231 mg (46%) as an off-white solid foam. Mp: 165–167 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.38. [α]²⁰_D = −83.2 (CHCl₃, c = 0.31). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₁₈H₁₈NO₃ [(M – H)⁺] 296.1287; found 296.1303.

(S)-2,9-Dihydroxy-3-methoxyberbine (S)-2e

Yield: 129 mg (22%) as an off-white solid foam. Mp: 135 °C (decomp.). TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.63. [α]²⁰_D = −281.5 (CHCl₃, c = 0.28); lit.(50) −129° (CHCl₃, c = 0.3). The ¹H and ¹³C NMR data are in accordance with literature.(50) MS (EI, 70 eV): m/z = 297 (M⁺, 100), 296 (92), 282 (15), 178 (60), 176 (82), 163 (16), 149 (19), 120 (24), 86 (52). HRMS: calcd for C₁₈H₁₉NO₃ 297.1365; found 297.1373.

(R)-1-(3-Hydroxybenzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (R)-1e

Yield: 247 mg (49%) as an off-white solid foam. Mp: 89–90 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.39. [α]²⁰_D = −29.1 (CHCl₃, c = 0.36); lit.(50) (S): +43 (MeOH, c = 0.5). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₁₈H₂₀NO₃ [(M – H)⁺] 298.1443; found 298.1453. The NMR data are in accordance with literature.(50)

(S)-Scoulerine (S)-2f

Yield: 232 mg (47%) as an off-white solid foam. Mp: 194–195 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.49. [α]²⁰_D = −248.3 (CHCl₃, c = 0.27); lit.(51) −315 (MeOH, c = 0.11). ¹H NMR (CDCl₃, 300 MHz): δ 2.63–2.70 (2H, m, CH₂), 2.83 (1H, dd, J₁ = 15.9 Hz, J₂ = 11.4 Hz, CH₂), 3.11–3.28 (3H, m, CH₂), 3.49–3.57 (2H, m, N-CH₂-Ar + CH), 3.87 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 4.25 (1H, d, J = 15.6 Hz, N-CH₂-Ar), 6.61 (1H, s, Ar), 6.68 (1H, d, J = 8.3 Hz, Ar), 6.75 (1H, d, J = 8.3 Hz, Ar), 6.84 (1H, s, Ar). ¹³C NMR (CHCl₃, 75 MHz): δ 29.2 (CH₂), 36.3 (CH₂), 51.6 (CH₂), 53.5 (CH₂), 55.9 (CH₂), 56.2 (CH₃), 59.2 (CH), 109.0 (CH), 110.6 (CH), 111.4 (CH), 119.3 (CH), 121.2 (C), 126.1 (C), 128.2 (C), 130.6 (C), 141.5 (C), 143.9 (C), 144.0 (C), 145.1 (C). MS (EI, 70 eV): m/z = 327 (M⁺, 55), 310 (8), 178 (100), 176 (32), 163 (13), 150 (48), 135 (27), 107 (16). HRMS: calcd for C₁₉H₂₁NO₄ 327.1471; found 327.1490.

(R)-Reticuline (R)-1f

Yield: 182 mg (37%) as an off-white solid foam. Mp: 74–75 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.29. [α]²⁰_D = −64.6 (CHCl₃, c = 0.26); lit.(52) −55 (EtOH, c = 0.44). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₁₉H₂₂NO₄ [(M – H)⁺] 328.1549; found 328.1600. The NMR data are in accordance with literature.(24)

(S)-9-Hydroxyberbine (S)-2g

Yield: 230 mg (46%) as an off-white solid foam. Mp: 103–104 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.58. [α]²⁰_D = −328.8 (CHCl₃, c = 1.0). ¹H NMR (CDCl₃, 300 MHz): δ 2.55–2.71 (2H, m, CH₂), 2.86 (1H, dd, J₁ = 16.3 Hz, J₂ = 11.5 Hz, CH₂), 3.10–3.20 (2H, m, CH₂), 3.26 (1H, dd, J₁ = 16.5 Hz, J₂ = 3.7 Hz, CH₂), 3.36 (1H, d, J = 15.6 Hz, N-CH₂-Ar), 3.62 (1H, dd, J₁ = 11.2 Hz, J₂ = 3.3 Hz, CH), 4.09 (1H, d, J = 15.6 Hz, N-CH₂-Ar), 6.21 (1H, d, J = 7.9 Hz, Ar), 6.58 (1H, d, J = 7.6 Hz, Ar), 6.78 (1H, t, J = 7.8 Hz, Ar), 7.04–7.21 (3H, m, Ar). ¹³C NMR (DMSO-d₆, 75 MHz): δ 29.1 (CH₂), 36.2 (CH₂), 51.3 (CH₂), 53.6 (CH₂), 59.4 (CH), 112.5 (CH), 120.5 (CH), 121.8 (C), 125.5 (CH), 126.1 (CH), 126.3 (CH), 126.8 (CH), 128.9 (CH), 134.4 (C), 135.9 (C), 137.5 (C), 152.4 (C). MS (EI, 70 eV): m/z = 251 (M⁺, 70), 132 (100), 130 (50), 130 (32), 91 (27). HRMS: calcd for C₁₇H₁₇NO 251.1310; found 251.1308.

(R)-1-(3-Hydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (R)-1g

Yield: 247 mg (49%) as an off-white solid foam. Mp: 110–111 °C. TLC (CH₂Cl₂/MeOH/NH₃(aq) = 90/9/1): R_f = 0.45. [α]²⁰_D = −58.4 (CHCl₃, c = 1.0). The ¹H and ¹³C NMR as well as MS data are in agreement with those obtained for the racemic compound. HRMS: calcd for C₁₇H₁₇NO [(M – 2H)⁺]: 251.1310; found 251.1338.

Determination of Absolute Configuration

Absolute configurations of benzylisoquinolines 1a–g and berbines 2a–g were assigned based on optical rotation, circular dichroism, and HPLC elution order analogies as previously described.(19)

Preparation of Racemic Reference Samples for Chiral HPLC Analysis

Racemic samples of berbines 2a–g for use as HPLC reference were prepared as previously described.(19)

Enzyme Expression and Purification

BBE expression was carried out in a 7 L glass fermenter according to the following protocol:

Preparation of Inoculum

Overnight cultures (ONCs) of Pichia pastoris colonies containing the BBE expression plasmid [pPICZα-BBE-ER] were grown in 50 mL of YPD medium containing 100 μg/mL zeocin (in 300 mL Erlenmeyer flasks) at 30 °C and 150 rpm for 20 h. The ONC was used to inoculate 300 mL of YPD medium (in 2 L baffled Erlenmeyer flasks) to an initial OD₆₀₀ of 1.0. The cultures were grown to an OD₆₀₀ of 10–15 at 30 °C and 150 rpm.

Preparation of Fermenter

Feeding flasks and tubing for base, antifoam, glycerol, and methanol addition as well as inoculum flasks were autoclaved. The fermenter was equipped with a calibrated pH electrode, a pO₂ electrode, and a sampling nozzle, filled with fermentation basal salts medium (3.5 L, see below), sterilized, and cooled to 30 °C. Trace salts solution (15 mL, for composition see below) and antifoam (Struktol J650, 1:10 dilution; 100 mL) were added, and the pH was adjusted to 5.0 by addition of 25% aqueous ammonia. The pO₂ electrode was calibrated using N₂ and air saturation for adjusting the 0% and 100% values, respectively.

Inoculation and Glycerol Batch Phase

The fermenter was inoculated with the shaking flask culture (300 mL, the initial OD₆₀₀ in the fermenter should be 1.0), and the batch was stirred overnight with automatic control of pO₂ (≥30%), pH (pH 5.0) and temperature (30 °C). The next morning, the culture had consumed all glycerol present in the medium (as indicated by a sharp rise in the pO₂ value).

Glycerol Fed-Batch Phase

A glycerol feed (50% w/v; containing 12 mL/L of trace salts solution) was started with an initial feed rate of 15 g/h, causing the pO₂ to drop. After about 5 min, when the pO₂ had reached again a value above 30%, the feed rate was raised continuously to 30 g/h over 30 min. Three hours later, the feed rate was raised continuously to 45 g/h over 30 min. This feed rate was maintained overnight. The next morning, 1000 g of 50% glycerol had been added in total. A sample was taken and analyzed for wet cell weight (WCW). A WCW of 280 g/L was reached at the end of the glycerol batch-phase.

Methanol Fed-Batch Phase (Induction Phase)

After approximately 1000 g of glycerol had been added, methanol adaptation was started by pumping 5 g of methanol feed (HPLC grade, methanol containing 12 mL/L of trace salts solution) into the fermenter. Subsequently, the glycerol feed was continuously reduced to 15 g/h over 1 h 59 min and finally reduced to zero within 1 min. During these 2 h of decrease of glycerol feed, the pH value was raised to 6.0 by addition of base. This pH adjustment is crucial for protein expression. As soon as the glycerol feed had been stopped, the methanol feed was started with an initial feed rate of 3 g/h. During the next 12 h, the feed rate was slowly raised to 9 g/h in several steps. This rate was maintained until the end of fermentation. Samples were taken every 24 h and analyzed for WCW and BBE activity. The activity rose over time, while the WCW stayed constant. After addition of 750 g of methanol in total (96 h of induction), pH and pO₂ control were disabled, and (NH₄)₂SO₄ was added to the batch to a final concentration of 1 M. The culture was aliquoted into centrifuge beakers (1 L), and the cells were pelleted by centrifugation (4000 rpm, 30 min, 4 °C). The supernatant was subjected to protein purification (see below).

Protein Purification

Hydrophobic Interaction Chromatography (HIC)

The fermentation supernatant was loaded onto a Phenyl Sepharose column (XK50/20, Phenyl Sepharose 6 High Sub) equilibrated with HIC start buffer (50 mM K₂HPO₄·3H₂O, 1 M (NH₄)₂SO₄, pH 7.5; filtered and degassed) with maximum flow rate and the column was washed with HIC start buffer until absorption (280, 375, 450 nm) and conductivity readings were constant. BBE was eluted using a gradient of HIC start buffer against 20% aqueous ethanol (100% start buffer to 50% in 25 min; 50% to 20% in 60 min; 20% to 0% start buffer in 60 min) and a flow rate of 6 mL/min. Fractions of 12 mL were collected, activity assays were performed and the active fractions were pooled and concentrated using the Centriprep centrifugal filtration system.

Gel Filtration (GF)

The pooled and concentrated fractions from HIC were loaded onto a Superdex 200 column (XK16/100, Superdex 200, prep grade) equilibrated with GF buffer (100 mM Tris-HCl, 150 mM NaCl, pH 8.0; filtered and degassed) using a 3 mL sample loop. BBE was eluted with GF buffer at a flow rate of 1 mL/min. Fractions of 3 mL were collected, activity assays were performed and the active fractions were pooled and concentrated as above. The protein solution was flash frozen by dripping into liquid nitrogen.

Activity Assay

A mixture of rac-reticuline solution (4 μL, 10 mM in reaction buffer/DMSO = 9/1), 4 μL BBE solution (protein purification fraction, fermentation supernatant, etc.), and BBE assay buffer (17 μL, 100 mM Tris-HCl, pH 9.0) were incubated at 37 °C for 10 min. The reaction was analyzed by TLC (silica; CH₂Cl₂/MeOH/NH₄OH = 90/9/1; visualization by UV irradiation).

Media and Feed Solutions

YPD Medium (for 1 L)

Bacto yeast extract (10 g) and Bacto peptone (20 g) were dissolved and autoclaved in 900 mL of H₂O; glucose (20 g) was dissolved and autoclaved in 100 mL of H₂O. The two solutions were mixed after autoclaving.

Basal Salts Medium (according to Hartner & Winkler; 3.5 L)

A total of 0.6 g CaSO₄·2H₂O, 8.1 g MgSO₄·7H₂O, 10 g K₂SO₄, 7 g KOH, 0.77 g NaCl, 112.8 g glycerol, 44.6 mL phosphoric acid, and water (bidest.) ad 3500 mL.

Trace Salts Solution (200 mL)

Dissolve 40 mg biotin, 16 mg NaI, 40 mg Na₂MoO₄·2H₂O, 4 mg H₃BO₃, and 146 mg CoCl₂·6H₂O in 100 mL H₂O (bidest). Additionally, 1.2 g CuSO₄·5H₂O, 590 mg MnCl₂·4H₂O, 4 g ZnCl₂, 13 g FeSO₄·7H₂O and 1 mL H₂SO₄ (conc) are dissolved in 100 mL H₂O (bidest.). The two solutions were mixed, filter-sterilized, and stored at 4 °C.

Glycerol Feed (1.5 L)

Mix 750 g glycerol, water (bidest.) ad 1500 mL; autoclave, add 18 mL trace salts solution.

Methanol Feed (1.5 L)

Mix 1.5 L HPLC grade MeOH and 18 mL trace salts solution.

Base (400 mL)

Ammonium hydroxide 25% solution.

Fermenter Settings

Temperature

Setpoint: 30 °C, Mode: auto

Stirrer

Mode: casc, Min: 25% (= 500 rpm), Max: 75% (= 1500 rpm), Ramp: 20%/sec

pH

Setpoint: 5.00, Mode: Auto, Pump: ---/base

pO₂

Setpoint: 30%, Mode: auto, Casc: stirr airfl, Parameter: HTime: 1 min, Dead: 0.5%, Stirr Min: 25%, Stirr Max: 75%, Airfl Min: 25%, Airfl Max: 100%

Foam

Mode: auto, Pump: afoam, Cycle: 0:10 m:s

Airflow

Mode: casc, Min: 25% (= 2.5 L/min), Max: 100% (= 10 L/min)

Supporting Information Available

General experimental information; analytical methods; ¹H and ¹³C NMR spectra, MS spectra, and HRMS results of new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.