Biocatalytic organic synthesis of optically pure (S)-scoulerine and berbine and benzylisoquinoline alkaloids

Article abstract of DOI:10.1021/jo201056f

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.

Full text of DOI:10.1021/jo201056f

ARTICLE
pubs.acs.org/joc
Biocatalytic Organic Synthesis of Optically Pure (S)-Scoulerine and
Berbine and Benzylisoquinoline Alkaloids
Joerg H. Schrittwieser,† Verena Resch,† Silvia Wallner,‡ Wolf-Dieter Lienhart,† Johann H. Sattler,†
Jasmin Resch,† Peter Macheroux,‡ and Wolfgang Kroutil*,†
†Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
‡Institute of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria
S Supporting Information
b
ABSTRACT: A chemoenzymatic approach for the asymmetric
total synthesis of the title compounds is described that employs
an enantioselective oxidative CÀC bond formation catalyzed by
berberine bridge enzyme (BBE) in the asymmetric key step.
This unique reaction yielded enantiomerically pure (R)-benzyl-
isoquinoline 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 4À8 linear steps using either a BischlerÀ
Napieralski cyclization or a C1ÀCR 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 5À9 linear steps.
’ INTRODUCTION
plant cell cultures12 afford minute amounts only. The produc-
tion of benzylisoquinolines and related alkaloids from the mor-
phine 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
CÀC bond-forming enzymes to set up the carbon framework of
the organic molecules.15 Berberine bridge enzyme (BBE) repre-
sents an outstanding biocatalyst enabling an aerobic oxidative
CÀC bond formation transforming benzylisoquinolines to ber-
bines. BBE catalyzes the first committed step in the benzophenan-
thridine, protoberberine, and protopine biosynthesis pathways16 in
plants as it converts (S)-reticuline to (S)-scoulerine by intramolecular
CÀC coupling, forming the so-called “berberine bridge” (Scheme 1).
This transformation takes place via oxidative CÀH 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
Benzylisoquinolines and berbines1 are two closely related
classes of alkaloids encompassing more than 100 known struc-
tures. Both alkaloid families are associated with a broad range of
biological activities: Many 1-benzyl-1,2,3,4-tetrahydroisoquino-
lines 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 proce-
dures 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 reactions10 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 benzylisoquinoline11 and berbine alkaloids are
produced by a number of plants belonging mainly to the Berber-
idaceae and Papaveraceae families. However, isolation of the
natural products is cumbersome, and biotransformations using
Received: May 24, 2011
Published: July 08, 2011
r
2011 American Chemical Society
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Scheme 1. CÀC Bond Formation Leading to (S)-Scoulerine
5f were needed for synthesis of the amides 3aÀg used as educts
in the cyclization reaction.
Catalyzed by Berberine Bridge Enzyme (BBE)
Although N-methyl-(3,4-dimethoxyphenyl)ethylamine
(N-methylhomoveratrylamine) 4a as well as N-methylphenethy-
lamine 4g were commercially available, all other phenethyla-
mines had to be synthesized. Compounds 4bÀd were prepared
from the corresponding phenylacetic acid derivatives 6bÀd via
conversion into the N-methylamides 7bÀd followed by reduc-
tion (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
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.
BH3 THF led to full conversion as judged by TLC and GCÀMS,
3
giving 4b in 72% isolated yield.
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 R-
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 triethylamine26 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 reduc-
tion, giving the desired N-methylphenethylamine derivative
4e in 66% yield.
’ RESULTS AND DISCUSSION
The synthesis of racemic 1-substituted tetrahydroisoquino-
lines 1 usually relies on one out of three different strategies: (i)
formation of the C1ÀC8a bond of the isoquinoline core employ-
ing either the PictetÀSpengler20 or the BischlerÀNapieralski21
cyclization, (ii) alkylation at position C1 of the isoquinoline via
nucleophilic or electrophilic activation,22 and (iii) formation of
the C4ÀC4a bond by a PomeranzÀFritsch 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 BischlerÀ
Napieralski cyclization of amides 3aÀg, since it offers a broad
scope and mild reaction conditions. Therefore, the N-methyl-
phenethylamines 4aÀg and phenylacetic acid derivatives 5a and
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
Scheme 2. Strategies for the Construction of 1-Substituted 1,2,3,4-Tetrahydroisoquinolines
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Scheme 3. Synthesis of Phenethylamine Derivatives 4bÀea
a 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, 78À95%. (c) BH3 THF (5 equiv), THF, reflux, 16 h, 58À78%. (d) BnBr (1.0 equiv), K2CO3, argon, room temperature, 20 h, 89%.
3
(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%.
Scheme 4. Synthesis of Phenylacetic Acid Derivative 5fa
a 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, À10 °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%.
Scheme 5. Synthesis of 1-(3-Benzyloxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline 18a
a 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, À78 to À50 °C, 4 h, 51%. (d) LiAlH4 (5 equiv), THF, 0 °C to reflux,
16 h, 74%.
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 R-trichloro-
methylcarbinol 11 (Scheme 4).28
With the required building blocks (4 and 5) in hand, amide
coupling was performed. The carboxylic acids 5 were converted
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into the corresponding acyl chlorides using oxalyl dichloride in
toluene and connected with the amines under SchottenÀ
Baumann conditions. Amides 3aÀg 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 BischlerÀNapieralski cyclization of these amides
was investigated to obtain the corresponding racemic tetrahy-
droisoquinolines 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 acet-
onitrile, followed by NaBH4-reduction in methanol. This se-
quence gave the tetrahydroisoquinolines in yields of 85À97%.
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
BischlerÀNapieralski 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 tetrahydro-
isoquinoline derivative appeared promising and lithiation of
tetrahydroisoquinoline carbamates employing t-BuLi has pre-
viously been described.22a Carbamate 14 and 3-benzyloxybenzyl
bromide 16 were prepared and reacted following the published
procedure to give the desired CÀC coupling product in 29%
yield (Scheme 5). By slightly changing the reaction conditions,
i.e., higher temperature during the alkylation stage (see Experi-
mental 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/reduc-
tive 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.
Table 1. Overall Yields of Chemical Route to Racemic
Tetrahydroisoquinolines rac-1aÀg
The synthesis of racemic tetrahydroisoquinolines 1aÀg
(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
Table 2. Yields of BBE-Catalyzed Oxidative Kinetic Resolution via CÀC Bond Formation
entry
substrate
c [%]a
yield (S)-2 [%]b
ee (S)-2 [%]c
yield (R)-1 [%]b
ee (R)-1 [%]c
Ed
1
2
3
4
5
6
7
rac-1ae
rac-1be
rac-1ce
rac-1de
rac-1e
rac-1f
50
50
50
50
50
50
50
42
36
39
31
22
47
46
>97
>97
>97
>97
>97
>97
>97
50
36
47
46
49
37
49
>97
>97
>97
>97
>97
>97
>97
>200
>200
>200
>200
>200
>200
>200
rac-1g
a Determined by HPLC on an achiral stationary phase. b Isolated yield (maximum theoretical yield = 50%). c Determined by HPLC on a chiral
stationary phase. d Determined from the ee of substrate and product. e Kinetic resolution from ref 19.
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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.
TLC (petroleumether/EtOAc = 1/1):Rf = 0.12. The 1H and13C NMRas
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: 100À101 °C (lit.35 99À
101 °C). TLC (petroleum ether/EtOAc = 1/1): Rf = 0.20. The 1H and 13
C
Finally, the racemic tetrahydroisoquinolines rac-1aÀg 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 2aÀg via kineticresolution
(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)-scou-
lerine (S)-2f in 37% and 47% isolated yield.
NMR as well as MS data are in accordance with literature.19,32
Reduction of Amides 7bÀd Giving Amines 4bÀd. A litera-
ture procedure32 was adapted for our purpose: BH3 THF (1.0 M in
3
THF; 100 mL, 100 mmol) was added to a solution of amide 7bÀd
(17.4À20.0 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 Â 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
4bÀd.
’ CONCLUSION
N-Methyl-3-methoxyphenethylamine (4b). Yield: 2.23 g
(72%) as a pale yellowish liquid. TLC (CH2Cl2/MeOH/NH3(aq) =
The combination of chemical synthesis of racemic 1-benzyl-
1,2,3,4-tetrahydroisoquinolines with biocatalytic enantioselective
intramolecular oxidative CÀC coupling by BBE provided a new
and efficient synthetic route to enantiomerically pure benzylisoqui-
noline and berbine alkaloids. The racemic substrates for BBE were
prepared by two different pathways: either via BischlerÀNapieralski
cyclization or by alkylation of Boc-protected tetrahydroisoquino-
line. 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,e,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 enantio-
merically pure alkaloid over 9 linear steps.
1
90/9/1): Rf = 0.21. The H 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: 175À177 °C (lit.34 178 °C). TLC (CH2Cl2/MeOH/
NH3(aq) = 90/9/1): Rf = 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) =
1
90/9/1): Rf = 0.20. The H and 13C NMR as well as MS data are in
accordance with literature.19,35
4-Benzyloxy-3-methoxybenzaldehyde36. 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 atmo-
sphere. The solution was filtered through Celite and washed with
CH2Cl2 (3 Â 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-methoxybenzalde-
hyde (28.1 g, 89%) as a white solid. Mp: 61À63 °C (lit.36 61À62 °C).
TLC (petroleum ether/EtOAc = 3/1): Rf = 0.62. The 1H and 13C NMR
as well as MS data are in accordance with literature.36
’ EXPERIMENTAL SECTION
Synthesis of Amides 7bÀd. A literature procedure32 was adapted
for our purpose: A solution of phenylacetic acid derivative 6bÀd
(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 Â 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 7bÀd, which were used in
the following transformation without further purification.
4-Benzyloxy-3-methoxy-β-nitrostyrene37. 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 iceÀwater
(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 Â 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: 119À121 °C (lit.36 124À125 °C). TLC (petroleum
ether/EtOAc = 3/1): Rf = 0.47. The 1H and 13C NMR as well as MS data
are in accordance with literature.36
3-Methoxyphenyl-N-methylacetamide (7b). Yield: 3.40 g
(95%) as a pale yellowish solid. Mp: 41À44 °C. TLC (petroleum
ether/EtOAc = 1/1): Rf = 0.22. The 1H and 13C NMR as well as MS data
are in accordance with literature.19,33
4-Benzyloxy-3-methoxyphenethylamine (9)36. To a suspen-
sion 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-nitrostyr-
ene (12.0 g, 42.0 mmol) in dry THF (80 mL) over 1 h. The reaction
N-Methyl-3,4,5-trimethoxyphenylacetamide (7c). Yield: 3.74
g (78%) as a pale yellowish solid. Mp: 87À89 °C (lit.34 90.5À91.5 °C).
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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-methoxyphe-
nethylamine as a yellowish liquid that crystallized upon standing to a
yellowish solid. Mp: 63À65 °C (lit.38 59À61 °C). TLC (CH2Cl2/
MeOH/NH3(aq) = 90/9/1): Rf = 0.27. The 1H and 13C NMR as well as
MS data are in accordance with literature.36
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 =124À125 °C (lit.41 119 °C). TLC (EtOAc): Rf = 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 procedure40 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 hydro-
chloric acid. The precipitate was filtered and recrystallized from H2O/
AcOH (1/1, 35 mL) to yield 1.99 g (67%) of 3-benzyloxy-4-methox-
yphenylacetic acid as an off-white solid. Mp =117À118 °C (lit.42
124À125 °C). TLC (petroleum ether/EtOAc = 3/1 + 1 drop of
AcOH): Rf = 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.
Ethyl 4-Benzyloxy-3-methoxyphenethylcarbamate. A lit-
erature procedure39 was adapted for our purpose: To a solution of
4-benzyloxy-3-methoxyphenethylamine 9 (4.00 g, 15.5 mmol) in di-
chloromethane (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 Â
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 crys-
tallized upon standing to a yellowish solid. Mp: 80À81 °C. TLC
Method B. A literature procedure28,43 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 Â 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: 62À63 °C
(lit.24 61À62 °C). TLC (petroleum ether/EtOAc = 3/1): Rf = 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).
1
(petroleum ether/EtOAc = 3/1): Rf = 0.23. H 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), 6.66À6.86 (3H, m, Ar), 7.31À7.47 (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 procedure39 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 chromatog-
raphy (silica; CH2Cl2/MeOH/NH3(aq) = 90/9/1) afforded 4-benzyl-
oxy-3-methoxy-N-methylphenethylamine (4.16 g, 67%) as an orange
liquid. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.22. 1H NMR
(CDCl3, 300 MHz): δ 1.73 (1H, br s, NH), 2.44 (3H, s, NCH3),
2.73À2.86 (4H, m, CH2-CH2-N), 3.90 (3H, s, OCH3), 5.14 (2H, s,
PhCH2O), 6.68À6.84 (3H, m, Ar), 7.28À7.46 (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).
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 À10 °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 À10 °C. The reaction was
quenched with 1 N hydrochloric acid (270 mL) and stirred for an
additional 30 min at À10 °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 Â 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
1
(petroleum ether/EtOAc = 3/1): Rf = 0.53. H NMR (CDCl3, 300
MHz): δ 3.71 (1H, br s, OH), 3.92 (3H, s, OCH3), 5.09 (1H, s,
CHÀOH), 5.21 (2H, s, PhCH2O), 6.89 (1H, d, J = 8.7 Hz, Ar),
7.14À7.21 (2H, m, Ar), 7.26À7.46 (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 deoxyge-
nated 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
3-Benzyloxyphenylacetic Acid (5a). A literature procedure40
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
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The Journal of Organic Chemistry
ARTICLE
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 Â 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.
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), 6.53À6.90 (2H, m, Ar), 6.74À6.90 (4H, m, Ar),
13
7.18À7.40 (11H, m, Ar).
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).
Synthesis of Amides 3aÀg. A literature procedure45 was adapted
for our purpose: A solution of phenylacetic acid derivative 5a or 5f
(10.5À12.0 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 4aÀg (10.0À13.5 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 (10.6À12.8 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 3aÀg, 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: 97À98 °C. TLC (petroleum ether/EtOAc =
1/1): Rf = 0.55. The 1H and 13C NMR aswellasMS data are inaccordance
withliterature.19 HRMS:calcdfor C26H29NO4 419.2097; found419.2099.
2-(3-Benzyloxyphenyl)-N-(3-methoxyphenethyl)-N-methy-
lacetamide (3b). Yield: 2.79 g (68%) as a pale yellowish liquid. Ratio
trans/cis = 1.05/1. TLC (petroleum ether/EtOAc = 1/1): Rf = 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-Benzyloxy-4-methoxyphenyl)-N-(4-benzyloxy-3-meth-
oxyphenethyl)-N-methylacetamide (3f). Yield: 2.33 g (76%) as
an off-white solid. Ratio trans/cis = 1.14/1. Mp: 126À127 °C. TLC
(petroleum ether/EtOAc = 1/1): Rf = 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-3f 1H 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,
13
Ph-C H2-O), 6.53À6.86 (6H, m, Ar), 7.28À7.47 (10H, m, Ar).
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 Â 2),
6.53À6.90 (2H, m, Ar), 6.74À6.90 (4H, m, Ar), 7.18À7.40 (11H, m, Ar).
13CNMR (CDCl3, 75 MHz): δ33.6 (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): Rf = 0.51. trans-3g: 1H NMR (CDCl3,
300 MHz): δ2.79À2.84 (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),
6.73À6.88 (3H, m, Ar), 7.05À7.07 (1H, m, Ar), 7.14À7.42 (10H, m, Ar). 13 C
2-(3-Benzyloxyphenyl)-N-methyl-N-(3,4,5-trimethoxy-
phenethyl)acetamide (3c). Yield: 3.63 g (63%) as a pale yellowish
liquid. Ratio trans/cis = 1.05/1. TLC (petroleum ether/EtOAc = 1/1):
Rf = 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):
Rf = 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):
Rf = 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
NMR (CDCl , 75 MHz): δ 33.7 (CH2), 36.6 (CH3), 41.4 (CH2), 50.2
3
(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), 6.73À6.88 (3H, m, Ar), 7.05À7.07 (1H, m, Ar), 7.14À
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).
BischlerÀNapieralski Cyclization of Amides 3aÀf. A litera-
ture procedure46 was adapted for our purpose: A solution of amide 3aÀf
(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 À5 °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 Â 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
495.2440. trans-3e: H 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), 6.61À6.64 (1H, m, Ar), 6.74À6.90
(5H, m, Ar), 7.18À7.40 (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),
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The Journal of Organic Chemistry
ARTICLE
1-(3-Benzyloxybenzyl)-6,7-dimethoxy-2-methyl-1,2,3,4-
tetrahydroisoquinoline. Yield: 2.78 g (94%) as a yellowish liquid.
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 Â 50 mL), and the filtrate was evaporated under
reduced pressure to give 18.4 g of an orange liquid. Flash chromatog-
raphy (silica; petroleum ether f petroleum ether/EtOAc = 9/1)
afforded 16 (9.75 g, 94%) as a white crystalline solid. Mp: 54À55 °C
(lit.48 37À39 °C). TLC (petroleum ether/EtOAc = 3/1): Rf = 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).
TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.75. The 1H and 13
C
NMR as well as MS data are in accordance with literature.19 HRMS:
calcd for C26H28NO3 [(M À H)+] 402.2069; found 402.2071.
1-(3-Benzyloxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tet-
rahydroisoquinoline. Yield: 2.43 g (94%) as a pale yellowish liquid.
TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.61. The 1H and 13
C
NMR as well as MS data are in accordance with literature.19 HRMS:
calcd for C25H26NO2 [(M À 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): Rf = 0.75.
1
The H and 13C NMR as well as MS data are in accordance with
tert-Butyl 1-(3-Benzyloxybenzyl)-3,4-dihydro-2(1H)-iso-
quinolinecarboxylate (17). A literature procedure22a 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 À78 °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 À78 °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 À50 °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 Â 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 f petroleum ether/EtOAc =
95/5) afforded 17 (2.21 g, 51%) as a colorless liquid. TLC (petroleum
ether/EtOAc = 3/1): Rf = 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
literature.19 HRMS: calcd for C27H30NO4 [(M À 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): Rf = 0.76. The 1H
and 13C NMR as well as MS data are in accordance with literature.19
HRMS: calcd for C25H24NO3 [(M À 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): Rf =
0.28. 1H NMR (CDCl3, 300 MHz): δ 2.56 (3H, s, NCH3), 2.58À2.90
(4H, m, CH2), 3.10À3.20 (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), 6.69À6.88 (3H, m, Ar), 7.20 (1H, t, J = 8.0 Hz, Ar),
7.28À7.45 (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 À H)+, <1], 282
(100), 191 (30), 162 (18), 91 (37). HRMS: calcd for C32H32NO3 [(M
À 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): Rf =
0.56. 1H NMR (CDCl3, 300 MHz): δ 2.47 (3H, s, NCH3), 2.50À2.58
(1H, m, CH2), 2.66À2.83 (3H, m, CH2), 2.97À3.12 (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), 6.55À6.57 (2H, m,
Ar), 6.64 (1H, d, J = 1.9 Hz, Ar), 6.77 (1H, d, J = 8.2 Hz, Ar), 7.26À7.38
(8H, m, Ar), 7.42À7.45 (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 À
2H)+, <1], 282 (100), 191 (25), 162 (13), 91 (21). HRMS: calcd for
C33H33NO4 [(MÀ2H)+] 507.2410; found 507.2435.
1
spectra. cis-17: H NMR (CDCl3, 300 MHz): δ 1.25 (9H, s, CH3),
2.62À3.07 (4H, m, CH2), 3.22À3.31 (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), 6.68À6.92 (3H,
m, Ar), 7.03À7.19 (5H, m, Ar), 7.32À7.49 (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),
1
140.2 (C), 154.4 (C), 158.8 (C). trans-17: H NMR (CDCl3, 300
MHz): δ 1.42 (9H, s, CH3), 2.62À3.07 (4H, m, CH2), 3.22À3.31 (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), 6.68À6.92 (3H, m, Ar),
7.03À7.19 (5H, m, Ar), 7.32À7.49 (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-tetrahydroisoqui-
noline (18). A solution of tert-butyl 1-(3-benzyloxybenzyl)-3,4-dihy-
dro-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
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): Rf = 0.62. The 1H NMR data are in accordance with literature.22a 13
C
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 [(MÀ 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 procedure47 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.
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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): Rf = 0.56.
1H NMR (CDCl3, 300 MHz): δ 2.54 (3H, s, NCH3), 2.67À2.83 (2H,
m, CH2), 2.88À2.98 (2H, m, CH2), 3.14À3.27 (2H, m, CH2), 3.87 (1H,
t, J = 6.2 Hz, CH), 5.04 (2H, s, PhCH2O), 6.78À6.89 (4H, m, Ar),
7.06À7.24 (4H, m, Ar), 7.36À7.46 (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 À H)+, <1], 146 (100), 131 (6),
91 (10). HRMS: calcd for C24H24NO [(M À H)+] 342.1858; found
342.1851.
Hydrogenolytic Deprotection Affording Tetrahydroiso-
quinolines 1aÀg. A literature procedure24 was adapted for our
purpose: A mixture of benzyl-protected tetrahydroisoquinoline (5.75À
9.16 mmol), Pd 10% on activated charcoal (0.20À0.30 g), acetic acid
(12.5À20.0 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 1aÀg.
1-(3-Hydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoqui-
noline (1g). Yield: 1.25 g (94%) as an off-white solid foam. Mp:
129À130 °C. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.47.
1H NMR (CDCl3, 300 MHz): δ 2.50 (3H, s, NCH3), 2.73À3.05 (4H,
m, CH2), 3.17 (1H, dd, J1 = 13.8 Hz, J2 = 6.0 Hz, CH2), 3.28À3.37 (1H,
m, CH2), 3.92 (1H, t, J = 6.5 Hz, CH), 6.60À6.63 (3H, m, Ar), 6.76 (1H,
d, J = 7.8 Hz, Ar), 7.02À7.15 (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 À H)+, <1], 146 (100), 131 (7). HRMS:
calcd for C17H18NO [(M À H)+] 252.1388; found 252.1403.
BBE-Catalyzed Kinetic Resolution of 1aÀg19. Substrate
1aÀg (500 mg, 1.5À2.0 mmol) was dissolved in toluene (17.5 mL)
and buffer (7.5 mL, 10 mM Tris-HCl, pH 9.0, 10 mM MgCl2) contain-
ing 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 Â 10 mL). The combined organic phases were dried over
Na2SO4 and evaporated under reduced pressure to give the crude
product. Flash chromatography (silica; aÀf, CH2Cl2/MeOH/NH3(aq)
= 96/3/1; g, CH2Cl2/MeOH/NH3(aq) = 98/1/1) afforded pure (S)-
2aÀg and (R)-1aÀg.
(S)-2,3-Dimethoxy-9-hydroxyberbine (S)-2a. Yield: 207 mg
(42%) as an off-white solid foam. Mp: 90À95 °C. TLC (CH2Cl2/
MeOH/NH3(aq) = 90/9/1): Rf = 0.78. [R]20D = À273.4 (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.
6,7-Dimethoxy-1-(3-hydroxybenzyl)-2-methyl-1,2,3,4-tetra-
hydroisoquinoline (1a). Yield: 2.07 g (98%) as an off-white solid
foam. Mp: 127À128 °C (lit.49 135 °C). TLC (CH2Cl2/MeOH/NH3-
(aq) = 90/9/1): Rf = 0.53. The 1H and 13C NMR as well as MS data are
in accordance with literature.19,50 HRMS: calcd for C19H22NO3 [(M À
H)+] 312.1600; found 312.1589.
1-(3-Hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetra-
hydroisoquinoline (1b). Yield: 1.53 g (94%) as an off-white solid
foam. Mp =113À116 °C. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1):
Rf = 0.51. The 1H and 13C NMR as well as MS data are in accordance
with literature.19 HRMS: calcd for C18H20NO2 [(M À H)+] 282.1494;
found 282.1499.
(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: 151À153 °C. TLC (CH2Cl2/MeOH/NH3(aq) =
90/9/1): Rf = 0.53. [R]20D = À109.4 (CHCl3, c = 1.0). The 1H and 13
C
NMR as well as MS data are in agreement with those obtained for the
racemic compound. HRMS: calcd for C19H22NO3 [(M À H)+]
312.1600; found 312.1591. The NMR data are in accordance with
literature.50
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): Rf = 0.51.
(S)-9-Hydroxy-3-methoxyberbine (S)-2b. Yield: 177 mg
(36%) as an off-white solid foam. Mp: 192À195 °C. TLC (CH2Cl2/
MeOH/NH3(aq) = 90/9/1): Rf = 0.56. [R]20D = À280.6 (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-tet-
rahydroisoquinoline (R)-1b. Yield: 181 mg (36%) as a highly
viscous yellowish liquid. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1):
Rf = 0.47. [R]20D = À76.3 (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 À 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: 85À89 °C. TLC (CH2Cl2/
MeOH/NH3(aq) = 90/9/1): Rf = 0.60. [R]20D = À226.5 (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):
Rf = 0.33. [R]20D = À75.4 (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 À H)+] 342.1705; found
342.1703.
1
The H and 13C NMR as well as MS data are in accordance with
literature.19 HRMS: calcd for C20H24NO4 [(M À 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: 143À145 °C (lit.49 145 °C). TLC (CH2Cl2/MeOH/NH3-
(aq) = 90/9/1): Rf = 0.45. The 1H and 13C NMR as well as MS data are
in accordance with literature.19 HRMS: calcd for C18H18NO3 [(M À
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: 103À106 °C (lit.49 111À113 °C). TLC (CH2Cl2/
MeOH/NH3(aq) = 90/9/1): Rf = 0.25. The NMR data are in
accordance with literature.50 MS (EI, 70 eV): m/z = 298 [(M À H)+,
<1), 192 (100), 177 (19), 148 (5). MS (EI, 70 eV): m/z = 298 [(M À
H)+, <1), 192 (100), 177 (19), 148 (5). HRMS: calcd for C18H20NO3
[(M À H)+] 298.1443; found 298.1450.
Reticuline (1f). Yield: 0.90 g (70%) as an off-white solid foam. Mp:
83À84 °C. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.29. The
NMR data are in accordance with literature.24 MS (EI, 70 eV): m/z =
328 [(M À H)+, <1], 192 (100), 177 (21). HRMS: calcd for
C19H22NO4 [(M À H)+] 328.1549; found 328.1571.
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(S)-9-Hydroxy-2,3-methylenedioxyberbine (S)-2d. Yield:
155 mg (31%) as an off-white solid foam. Mp: 177À180 °C. TLC
(R)-1-(3-Hydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroiso-
quinoline (R)-1g. Yield: 247 mg (49%) as an off-white solid foam.
Mp: 110À111 °C. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf =
0.45. [R]20D = À58.4 (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 À 2H)+]: 251.1310; found 251.1338.
Determination of Absolute Configuration. Absolute config-
urations of benzylisoquinolines 1aÀg and berbines 2aÀg were assigned
based on optical rotation, circular dichroism, and HPLC elution order
analogies as previously described.19
(CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.50. [R]20 = À342.5
D
1
(CHCl3, c = 0.63). The H 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: 165À167 °C. TLC (CH2Cl2/MeOH/
NH3(aq) = 90/9/1): Rf = 0.38. [R]20D = À83.2 (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 À H)+] 296.1287; found 296.1303.
Preparation of Racemic Reference Samples for Chiral
HPLC Analysis. Racemic samples of berbines 2aÀg for use as HPLC
reference were prepared as previously described.19
(S)-2,9-Dihydroxy-3-methoxyberbine (S)-2e. Yield: 129 mg
(22%) as an off-white solid foam. Mp: 135 °C (decomp.). TLC
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 [pPICZR-
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 10À15 at 30 °C and 150 rpm.
(CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.63. [R]20 = À281.5
D
(CHCl3, c = 0.28); lit.50 À129° (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: 89À90 °C. TLC (CH2Cl2/MeOH/NH3-
(aq) = 90/9/1): Rf = 0.39. [R]20D = À29.1 (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 À H)+] 298.1443; found 298.1453. The NMR data
are in accordance with literature.50
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 fermenta-
tion 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.
(S)-Scoulerine (S)-2f. Yield: 232 mg (47%) as an off-white solid
foam. Mp: 194À195 °C. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1):
Rf = 0.49. [R]20D = À248.3 (CHCl3, c = 0.27); lit.51 À315 (MeOH, c =
0.11). 1H NMR (CDCl3, 300 MHz): δ 2.63À2.70 (2H, m, CH2), 2.83
(1H, dd, J1 = 15.9 Hz, J2 = 11.4 Hz, CH2), 3.11À3.28 (3H, m, CH2),
3.49À3.57 (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: 74À75 °C. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf
= 0.29. [R]20D = À64.6 (CHCl3, c = 0.26); lit.52 À55 (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
À H)+] 328.1549; found 328.1600. The NMR data are in accordance
with literature.24
Inoculation and Glycerol Batch Phase. The fermenter was inocu-
lated 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 (g30%), 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,
(S)-9-Hydroxyberbine (S)-2g. Yield: 230 mg (46%) as an off-
white solid foam. Mp: 103À104 °C. TLC (CH2Cl2/MeOH/NH3(aq) =
1
90/9/1): Rf = 0.58. [R]20 = À328.8 (CHCl3, c = 1.0). H NMR
D
(CDCl3, 300 MHz): δ 2.55À2.71 (2H, m, CH2), 2.86 (1H, dd, J1 = 16.3
Hz, J2 = 11.5 Hz, CH2), 3.10À3.20 (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), 7.04À7.21 (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.
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ARTICLE
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 Sephar-
ose column (XK50/20, Phenyl Sepharose 6 High Sub) equilibrated with
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: wolfgang.kroutil@uni-graz.at.
HIC start buffer (50 mM K2HPO4 3H2O, 1 M (NH4)2SO4, pH 7.5;
’ ACKNOWLEDGMENT
3
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.
This study was financed by the Austrian Science Fund (FWF
Project P20903-N17 and P22115-N17). The authors would like
to thank Bernd Werner for acquiring the NMR spectra. Financial
support by NAWI Graz is acknowledged.
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3
3
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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)
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