Deracemisation of benzylisoquinoline alkaloids employing monoamine oxidase variants

Article abstract of DOI:10.1039/c4cy00642a

Chemo-enzymatic deracemisation was applied to obtain the (S)-enantiomer of 1-benzylisoquinolines from the racemate in high isolated yield (up to 85%) and excellent optical purity (ee > 97%). The one-pot deracemisation protocol encompassed enantioselective oxidation by a monoamine oxidase (MAO-N) and concomitant reduction of the resulting iminium species by ammonia-borane. The challenge was the oxidation at the sterically demanding chiral centre. Recently developed variants of MAO-N, featuring an enlarged active-site pocket, turned out to be suitable biocatalysts for these substrates. In contrast to previous MAO-N variants, which preferentially converted the (S)-enantiomer, the MAO-N variant D11 used in the present study was found to oxidise all tested benzylisoquinoline substrates with (R)-enantiopreference. The structural determinants of enantioselectivity were investigated by means of protein-ligand docking simulations. The applicability of the deracemisation system was demonstrated on preparative scale (150 mg) for three benzylisoquinoline alkaloids (natural as well as non-natural), including the hypotensive and antispasmodic agent (S)-reticuline.

Full text of DOI:10.1039/c4cy00642a

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Deracemisation of benzylisoquinoline alkaloids
employing monoamine oxidase variants†
Cite this: Catal. Sci. Technol., 2014,
4, 3657
Joerg H. Schrittwieser,a Bas Groenendaal,b Simon C. Willies,b Diego Ghislieri,b
Ian Rowles,b Verena Resch,a Johann H. Sattler,a Eva-Maria Fischereder,a
a
a
b
*
Barbara Grischek, Wolf-Dieter Lienhart, Nicholas J. Turner
a
*
and Wolfgang Kroutil
Chemo-enzymatic deracemisation was applied to obtain the (S)-enantiomer of 1-benzylisoquinolines
from the racemate in high isolated yield (up to 85%) and excellent optical purity (ee > 97%). The one-pot
deracemisation protocol encompassed enantioselective oxidation by a monoamine oxidase (MAO-N)
and concomitant reduction of the resulting iminium species by ammonia-borane. The challenge was the
oxidation at the sterically demanding chiral centre. Recently developed variants of MAO-N, featuring an
enlarged active-site pocket, turned out to be suitable biocatalysts for these substrates. In contrast to previous
MAO-N variants, which preferentially converted the (S)-enantiomer, the MAO-N variant D11 used in the
present study was found to oxidise all tested benzylisoquinoline substrates with (R)-enantiopreference.
The structural determinants of enantioselectivity were investigated by means of protein–ligand docking
simulations. The applicability of the deracemisation system was demonstrated on preparative scale
(150 mg) for three benzylisoquinoline alkaloids (natural as well as non-natural), including the hypotensive
and antispasmodic agent (S)-reticuline.
Received 19th May 2014,
Accepted 1st July 2014
DOI: 10.1039/c4cy00642a
www.rsc.org/catalysis
developed.4 The majority of the established syntheses rely on
chiral auxiliaries in the asymmetric key step,5 and out of
Introduction
1-Benzylisoquinoline alkaloids are produced by various higher
plant species, for instance members of the Papaveraceae and
Berberidaceae families.1 Simple 1-benzyl-1,2,3,4-tetrahydro-
isoquinolines constitute intermediates in the biosynthesis
of – among others – the benzophenanthridine, protopine, and
morphinan alkaloids, and as such they usually do not accu-
mulate to substantial amounts in the plant tissue.2 As a con-
sequence, the availability of benzylisoquinolines from natural
sources is limited, yet pharmacological studies have found
diverse and potent biological effects for these substances,
ranging from sedative, antispasmodic and hypotensive
properties to anti-cancer and anti-HIV activity.3 Prompted by
these interesting features, many different strategies for the
asymmetric synthesis of these natural products have been
the few catalytic procedures that have been reported, only
asymmetric hydrogenation6 and transfer hydrogenation7 have
proven to be of general use.8
In Nature, benzylisoquinolines are produced in optically
pure form by highly stereoselective enzymes.2 Using these
biocatalysts in synthetic organic chemistry can open up novel
routes to enantiomerically pure alkaloids, often with favourable
yields and enantioselectivities.9 For instance, the broad appli-
cability of norcoclaurine synthase (NCS) for the preparation
of (S)-benzylisoquinolines by stereoselective Pictet–Spengler
cyclisation has recently been demonstrated,10 and berberine
bridge enzyme has been used to access (R)-benzylisoquinolines
via oxidative kinetic resolution of the racemate.11 Herein, we
describe the deracemisation of 1-benzylisoquinoline deriva-
tives using engineered variants of monoamine oxidase from
Aspergillus niger as a novel biocatalytic entry to benzylisoquin-
olines that offers high yields and excellent enantioselectivity.
The simultaneous one-pot combination of enantioselective
oxidases with chemical reducing agents has been shown to allow
the deracemisation of amino acids, keto acids, alcohols, and
amines via a “cyclic” oxidation/reduction cascade (Scheme 1).12
Monoamine oxidase from Aspergillus niger (MAO-N) has
been engineered for the deracemisation of selected amines,
and variants optimised for the conversion of α-chiral
primary,13 secondary,14 and tertiary15 amines have been
a Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz,
Heinrichstraße 28/II, 8010 Graz, Austria. E-mail: wolfgang.kroutil@uni-graz.at
b School of Chemistry, University of Manchester, Manchester Institute of
Biotechnology, 131 Princess Street, Manchester, M1 7DN, UK.
E-mail: nicholas.turner@manchester.ac.uk
† Electronic supplementary information (ESI) available: Results of MAO-N
activity screenings for variants D5 and D9, detailed results and procedures for
protein expression and purification, experimental procedures for substrate
synthesis including full characterisation data for all new compounds, analytical
methods, copies of NMR and MS spectra (and, where applicable HPLC traces of
chiral separations) for new chemically synthesised compounds and products of
biotransformations. See DOI: 10.1039/c4cy00642a
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Results and discussion
Activity and enantioselectivity screenings
In a recent study aimed at producing berbine alkaloids via a
cascade deracemisation system, we have identified four
benzylisoquinolines that are accepted as substrates by
MAO-N D11.21 To gain more insight into the scope of this trans-
formation, we screened MAO-N variants D5, D9 and D11 against
a panel of 15 racemic 1-benzyl-1,2,3,4-tetrahydroisoquinolines
using a previously reported13b colorimetric assay on hydrogen
peroxide formation. The substrates (Fig. 2) can be classified
into three groups according to their substitution pattern:
compounds 1a and 1b lack any substituents on the iso-
quinoline ring, and are thus the sterically least demanding
substrates. Compounds 2a–f share 6,7-dimethoxy substitution
on the isoquinoline ring as the common structural feature,
but carry different functionalities on the benzyl moiety.
Compounds 3a–g vary in the substitution on both the iso-
quinoline and the benzyl ring system, although they all fea-
ture a 3′-hydroxyl group.
Scheme 1 Deracemisation of amines via a “cyclic” oxidation/reduction
cascade.
reported. The fifth-generation variant MAO-N D5 has proven
suitable for preparation of the naturally occurring tetrahydro-
isoquinoline alkaloid (R)-crispine A (Fig. 1).16
Efforts to extend the scope of MAO-N to accommodate
more bulky substrates have recently led to the development
of variants (named D9 and D11) with a more spacious active-
site pocket.17 These biocatalysts show increased activity on
crispine A,18 and have also been used in the chemo-enzymatic
asymmetric synthesis of indole alkaloids (e.g., leptaflorine
and harmicine) and the acetylcholine receptor antagonist
solifenacin (Fig. 1).19 Notably, the deracemisation of 1-phenyl-
1,2,3,4-tetrahydroisoquinoline employing MAO-N D11 (carried
out en route to solifenacin) afforded the (S)-enantiomer in
98% ee, which represented the first example of (R)-enantio-
preference in amine oxidations by MAO-N.20 This stereo-
chemical switch prompted us to investigate the activity and
enantioselectivity of MAO-N variants for benzylisoquinolines,
as a structurally related class of alkaloids. Herein, we show
that MAO-N D11 generally oxidises benzylisoquinolines with
preference for the (R)-enantiomer, and that the substitution
pattern on the isoquinoline ring determines the extent of
enantioselectivity. Furthermore, we report on the optimisa-
tion of biocatalyst preparation, which was required for
achieving satisfactory activity, and on the application of
MAO-N variant D11 to the preparative-scale deracemisation
of three benzylisoquinoline alkaloids.
In the colorimetric assay,13b MAO-N D5 proved inactive on
all substrates except for the positive controls rac-crispine A
and rac-1-phenylethylamine (Fig. S1, ESI†), while variant D9
converted five of the tested compounds, although at very
limited rates (Fig. S2, ESI†).22 Variant D11 showed fair
activity (1–14 U mg−1 at 1 mM substrate concentration) on
eight benzylisoquinolines, and the rate of oxidation deter-
mined for the smallest substrate, 1a, even exceeded that for
crispine A (Fig. 3).
Encouraged by these results, we proceeded to investigate
the enantioselectivity of MAO-N D11 in the oxidation of those
benzylisoquinolines that showed good activity. To this end,
bacterial cells expressing the enzyme were incubated with the
substrates for 24 h in the absence of a reducing agent, and
the conversion as well as the ee of the remaining substrate
were determined by GC and/or HPLC analysis. As shown in
Table 1, the (S)-enantiomer was in excess in all samples,
Fig. 1 Examples of alkaloids that have been obtained in optically pure form
using MAO-catalysed deracemisation. The 1-phenyltetrahydroisoquinoline
moiety of solifenacin is highlighted in blue.
Fig. 2 Substrates used in the activity screening of MAO-N variants.
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Fig. 4A). (S)-1a adopts a binding mode in which the benzyl
substituent points into the active site, while in the docked
conformation of the (R)-enantiomer the benzyl ring points
towards amino acids Thr246 and Tyr365, located near the
active-site entrance channel. These results are in good agree-
ment with recently reported docking studies of various
C1-substituted tetrahydro-β-carbolines to MAO-N D11.19b
In contrast, only the (R)-enantiomer of 3a was successfully
docked into the enzyme structure, with the substituents on
the isoquinoline ring tightly accommodated in a large pocket
created by the Trp430Gly mutation (Fig. 4B, blue structure).
When docking of (S)-3a is forced in a position analogous to
(S)-1a, the 6-methoxy and 7-hydroxy substituents produce
steric clashes with the side chains of Thr246 and Phe382,
respectively, which seems to prevent (S)-3a from binding in a
productive conformation. Consequently, the isoquinoline
substitution pattern governs the enantioselectivity in MAO-N
catalysed benzylisoquinoline oxidation.
Fig. 3 Results of substrate screening with MAO-N variant D11. Activity
(criterion: mean > 5 standard deviations) was found for substrates
1a, 1b, 2a–c, 3a, 3c, and 3f (blue) as well as the positive controls
rac-1-phenylethylamine (4) and rac-crispine A (5, both green). Error
ranges represent standard deviations of triplicate experiments.
Table 1 Results of oxidation reactions employing E. coli cells expressing
MAO-N variant D11
Improvement of biocatalyst preparation
After investigating the substrate scope and enantioselectivity
of the MAO-N-catalysed oxidation of benzylisoquinolines, we
sought to optimise the preparative aspect of this transforma-
tion. Although six of the eight substrates were oxidised with
high enantioselectivity, the conversions after 24 h of reaction
time (≤29%, Table 1) suggested that higher biocatalyst load-
ings would be required. The low expression level of MAO-N
(below 0.5 mg of protein per g of wet cell weight) was identi-
fied as the main source of the limited catalytic activity, and
hence we decided to test alternative expression hosts and cul-
tivation conditions in order to optimise protein production
and thus the specific activity of the cells. The key to success
in this regard was the use of E. coli C43 (DE3) as expression
host24 in combination with cultivation under auto-inducing
conditions (for details on the optimisation study, see ESI†).25
These modifications resulted in a more than 3-fold improve-
ment in the specific activity of the whole cell biocatalyst com-
pared to the previously reported expression protocol, along
with a ~5-fold increase in cell yield (Tables S4 and S5, ESI†).
Substrate
Conversion [%]
eea [%]
Eb
1a
1b
2a
2b
2c
3a
3c
3f
39c
26d
9d
36 (S)
24 (S)
10 (S)
2 (S)
5
7
>200
n.d.e
n.d.e
>200
>200
>200
2d
6d
7 (S)
29d
15d
26d
40 (S)
18 (S)
35 (S)
a
Enantiomeric excess of residual substrate determined by HPLC
analysis on a chiral stationary phase. b Determined from conversion (c)
and substrate ee (eeS) according to the equation: E = ln[(1 − c)·(1 − eeS)]/
ln[(1 − c)·(1 + eeS)].23 c Determined by GC analysis. Determined by
d
HPLC analysis using an internal standard. e Not determined due to low
conversion.
indicating that the MAO-N variant preferentially oxidised the
(R)-enantiomer of the substrates. Interestingly, this enantio-
preference was excellent for six out of the eight compounds
tested (E > 200), while the other two – 1a and 1b – were
converted with strikingly low selectivity (E = 4.9 and 6.5,
respectively).
Protein–ligand docking simulations
To elucidate the dramatic differences in enantiopreference of
MAO-N towards the investigated benzylisoquinolines, pro-
tein–ligand docking simulations were carried out using the
X-ray crystal structure of MAO-N D11 (PDB: 3ZDN)19a as
receptor molecule and substrates 1a and 3a as ligands (for
details, see Experimental section). Consistent with the experi-
mental results, both enantiomers of 1a were docked in con-
formations that allow productive interaction with the FAD
prosthetic group (i.e., the lone pair on N2 of the isoquinoline
ring pointing towards C4a of the flavin cofactor, and the
hydrogen atom at C1 positioned close to N5 of FAD; see
Fig. 4 Docked structures of (A) (R)-1a (blue) and (S)-1a (green), and
(B) (R)-3a (blue) and (S)-3a (green, forced docking) in the active site
of MAO-N D11. Active-site residues are shown in grey and the FAD
cofactor is shown in orange. The steric clash between the methoxy
group of (S)-3a and the side chain of Thr246 is illustrated using space-
filling models.
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Furthermore, it was found that lyophilised cells could be
used instead of wet cells with no loss of specific activity
(corrected for the weight loss caused by lyophilisation), and
hence we chose to use lyophilised cells prepared by the auto-
induction protocol for all further experiments.
deracemisation was only achieved for two substrates (3a and 3f).
The study was therefore repeated employing 100 mg mL−1 of
lyophilised cells. Under these conditions, borane was quickly
depleted in the samples where oxidation took place with low
enantioselectivity, and had to be supplemented as the reac-
tion proceeded. Using the higher amount of MAO-N express-
ing cells, four substrates (2a, 3a, 3c, 3f) could be deracemised
to enantiomeric purity (ee > 97% by HPLC) within 2–9 days.
In two cases (1a, 1b) the ee levelled out at a lower value (55%
and 68%, respectively), and two substances (2b, 2c) were still
turned over too slowly to reach a plateau within 9 days of
reaction time (Fig. 5). Addition of fresh biocatalyst after 7 days
(where applicable) assured that the enantiomeric excess
did not level out because of enzyme inactivation. Enantio-
selectivity values E of 3 and 5 were determined from the final
ee reached in the deracemisation reactions of substrates 1a
and 1b, respectively.26 These values are comparable with
those calculated from the substrate ee and conversion
observed in the oxidation reactions (Table 1).
Time-course experiments
Time-course studies of deracemisation reactions were carried
out for all substrates to evaluate the stability of the biocata-
lyst under the process conditions (37 °C, 10% v/v DMSO,
40 mM NH3·BH3; for details see Experimental section) and
also to confirm the enantioselectivity values determined in
the oxidation experiments. An initial set of experiments using
30 mg mL−1 cell mass revealed a good long-term performance
of the system (as indicated by the linear increase in substrate
ee over a period of 90 h; Fig. 5, green squares), but complete
Preparative-scale deracemisation reactions
Finally, we scaled up three deracemisation reactions to a pre-
parative batch size, converting 0.5 mmol (142–165 mg) of the
racemic benzylisoquinoline substrates. As expected based on
the time-study results, the deracemisation was completed
within 48 h for substrates 3a and 3f, while 3c required 72 h
for the reaction to reach optical purity. The deracemised
alkaloids were recovered in high yields and enantiomerically
pure form after column chromatography (Table 2), which
demonstrates the synthetic potential of the investigated trans-
formation. In particular, the deracemisation of reticuline 3f,
in combination with the previously reported preparation of
the racemate,11d establishes an asymmetric total synthesis of
this sedative,3h hypotensive,3g and antispasmodic3j natural
product in 9 linear steps and with an overall yield of 13%.
Table 2 Results of preparative-scale deracemisation reactions employing
ammonia-borane and lyophilised E. coli cells containing overexpressed
MAO-N variant D11
Substrate
Reaction time [h]
Yielda [%]
eeb
Fig. 5 Time studies of deracemisation reactions employing 30 mg mL−1
(green squares) or 100 mg mL−1 (blue circles) of lyophilised E. coli cells
containing overexpressed MAO-N variant D11. Mind the different time
scales for 1a, 3a, 3c and 3f vs. 1b and 2a–c. Arrows indicate time points
where fresh biocatalyst (20 mg mL−1) was added. The intermediate
iminium species was not detected in any experiment.
3a
3c
3f
48
72
48
77
85
80
>97 (S)
>97 (S)
>97 (S)
a
b
Isolated yield after column chromatography. Enantiomeric excess
determined by HPLC analysis.
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Substrates 1b, 2b, and 3a–f were synthesised as previously
described.11a,c,d Laudanosoline 3g is commercially available.
Synthetic procedures and full characterisation data for all
other substrates are provided in the ESI.†
Catalase (Lot 81H7146, from bovine liver), lysozyme from
chicken egg white (cat. L6876, min. 40 000 U mg−1 protein),
and horseradish peroxidase (cat. P6782, type VI-A, 250–330 U
mg−1 solid) were purchased from Sigma-Aldrich. Pyrogallol
red (cat. 207540050) was obtained from Acros. The MAO-N
variants D5, D9, and D11 were heterologously expressed in
E. coli as described in the ESI.†
Conclusions
In summary, the scope of chemo-enzymatic deracemisation by
combining monoamine oxidase (MAO-N) from Aspergillus niger
and ammonia-borane has been extended to (S)-benzylisoquin-
olines, thereby establishing a novel biocatalytic synthetic route
to these alkaloids. Previous studies had found that MAO-N
variant D11 can oxidise amines with (S)- or (R)-enantio-
selectivity, depending on the substrate structure. In our pres-
ent report, benzylisoquinoline alkaloids are identified as an
entire class of substrates that are oxidised by MAO-N D11
with (R)-enantiopreference, although not all investigated
compounds were converted with high selectivity. Insights
into the structural determinants of enantioselectivity were
gained by relating the results of extensive substrate screening
to in silico docking simulations.
The analytical methods used for determination of conver-
sion and enantiomeric excess are described in the ESI.†
Activity screening of MAO-N variants
Stock solutions of the test substrates (20 mM; final concen-
tration in the reaction mixture 1 mM) were prepared in
DMSO. For each colorimetric reaction, the substrate stock
solution (5 μL) was mixed with potassium phosphate buffer
(20 μL; 100 mM, pH 7.7), pyrogallol red solution (50 μL; 0.3
mM in buffer), horseradish peroxidase solution (5 μL; 1 mg
mL−1 in buffer) and purified MAO-N solution (20 μL; 0.4–1.1
mg mL−1) in a 96-well microtitre plate. In addition, reactions
with rac-1-phenylethylamine (1 mM) and rac-crispine A
(1 mM) as well as negative controls (lacking substrate and MAO,
respectively) were set up. All reactions, including the blanks,
were performed in triplicate. Reactions were followed by mea-
suring the absorbance at 550 nm every minute for a period of
4 h using a Molecular Devices SpectraMax M2 plate reader.
For the faster transformations (full conversion in <30 min),
reactions with diluted enzyme solution were also performed,
so as to obtain at least 20 data points in the linear range of
absorbance decrease. Slopes were determined by applying a
linear fit to the linear range of the absorbance curve using
the built-in function of the plate reader's Molecular Devices
Softmax Pro v5.0 software. Slopes were corrected for sponta-
neous decolourisation of pyrogallol red (rate obtained from
the blank samples) and the specific MAO activity was calcu-
lated using the formula given below.
Improvement of the MAO-N expression level proved
important for achieving a satisfactory specific activity of the
whole-cell biocatalyst, which enabled reactions on a prepara-
tive scale. Three benzylisoquinolines were deracemised to
optical purity (>97% by HPLC) and were recovered from pre-
parative biotransformations in good yields.
Overall, our study once more demonstrates the versatility
of monoamine oxidase from Aspergillus niger as a biocatalyst
and highlights its potential for the asymmetric synthesis of
alkaloids.
Experimental section
General methods and materials
1H- and 13C-NMR spectra were recorded using a 300 MHz
instrument. Chemical shifts are given in parts per million
(ppm) relative to TMS (δ = 0 ppm) and coupling constants (J)
are reported in Hertz (Hz). Melting points were determined
in open capillary tubes and are uncorrected. Thin layer chro-
matography was carried out on silica gel 60 F254 plates and
compounds were visualised either by spraying with Mo
reagent [100 g L−1 (NH4)6Mo7O24·4H2O, 4 g L−1 Ce(SO4)2·4H2O,
in 10% H2SO4] or by UV. Unit resolution GC–MS analyses were
performed using electron impact (EI) ionisation at 70 eV and
quadrupole mass selection. High resolution MS analyses were
performed using electron impact (EI) ionisation at 70 eV
and TOF mass selection. Optical rotation values [α]2D0 were
measured at 589 nm (Na line) using a cuvette of 1 dm path
length.
Unless otherwise noted, reagents and organic solvents
were obtained from commercial suppliers in reagent grade
quality and used without further purification. Toluene,
methanol and acetonitrile used for anhydrous reactions were
dried over appropriate molecular sieves (4 Å for toluene, 3 Å
for MeOH and MeCN) for at least 48 hours. THF used for
anhydrous reactions was distilled from potassium/benzophenone
directly before use. For anhydrous reactions, flasks were
oven-dried and flushed with dry argon just before use. Stan-
dard syringe techniques were applied to transfer dry solvents
and reagents in an inert atmosphere of dry argon.
ODY
A 
LcP
A [U mg−1] MAO activity; ΔOD [min−1] slope of absorbance
decrease; Y dilution factor of MAO solution (5 in this case);
ε [L mmol−1 cm−1] extinction coefficient of pyrogallol red
(30.9 at 550 nm); L [cm] path length of sample (0.31 in this case);
cP [mg mL−1] protein concentration of MAO stock solution.
Biotransformations
Oxidation of benzylisoquinolines catalysed by MAO-N D11.
Frozen cells of E. coli BL21 (DE3) [pET16b MAO-N (D11)]
(50 mg) were thawed and resuspended in potassium phos-
phate buffer (450 μL; 100 mM, pH 7.7) in Eppendorf vials
(2 mL). Substrate (5 μmol, final concentration 10 mM) was
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dissolved in DMSO (50 μL; containing 4 mg mL−1 2,3-dimethoxy-
9-hydroxyberbine as internal standard) and added to the cell
suspension. The reaction mixture was shaken at 37 °C and
150 rpm for 24 h. Samples using 1a, 2a and 2c as substrate
were basified by addition of 2 M aq. NaOH solution (100 μL).
Afterwards, all samples were extracted with EtOAc (2 × 500 μL)
and dried over Na2SO4. The solvent was evaporated under a
stream of air, the sample was re-dissolved in HPLC-grade
methanol (700 μL) and conversion as well as substrate ee were
determined by GC-FID and/or HPLC analysis.
EtOAc and analysed for substrate ee. HPLC analysis indicated
complete deracemisation, and the reaction mixture was
aliquoted into Falcon tubes (50 mL) and centrifuged (1800× g,
45 min, rt) to remove the cell mass. The supernatant was
extracted with EtOAc (3 × 20 mL) and the cell pellets were
suspended in EtOAc, centrifuged again (1800× g, 10 min, rt)
and the EtOAc phase was combined with the extracts. The
combined organic phases were dried over Na2SO4 and the
solvent was evaporated under reduced pressure to give the
crude benzylisoquinoline as a yellowish liquid. Column chro-
matography (silica; CH2Cl2/MeOH/NH3(aq) = 96/3/1) afforded
(S)-3a, (S)-3c, and (S)-3f, respectively.
Deracemisation of benzylisoquinolines by MAO-N D11 and
NH3·BH3 (time-course experiments)
Using 30 mg mL−1 lyophilised cells. In Falcon tubes (15 mL),
lyophilised cells of E. coli C43 (DE3) [pET16b MAO-N (D11)]
from auto-induction cultures (75 mg) were resuspended in
potassium phosphate buffer (2.25 mL; 100 mM, pH 7.7).
Borane-ammonia complex (3.1 mg, final concentration 40 mM)
and a solution of substrate (25 μmol, final concentration
10 mM) in DMSO (250 μL) were added to the cell suspension.
The reaction mixture was shaken at 37 °C and 150 rpm for
90 h. Samples (500 μL) were taken after 18, 26 and 90 h and
samples with 1a, 2a and 2c as substrate were basified by
addition of 2 M aq. NaOH solution (50 μL). Afterwards, all
samples were extracted with EtOAc (2 × 500 μL) and dried
over Na2SO4. The solvent was evaporated under a stream of
air, the sample was re-dissolved in HPLC-grade methanol
(700 μL) and the substrate ee was determined by HPLC analysis.
Using 100 mg mL−1 lyophilised cells. In Falcon tubes
(15 mL), lyophilised cells of E. coli C43(DE3) [pET16b MAO-N
(D11)] from auto-induction cultures (250 mg) were resuspended
in potassium phosphate buffer (2.25 mL; 100 mM, pH 7.7).
Borane-ammonia complex (3.1 mg, final concentration 40 mM)
and a solution of substrate (25 μmol, final concentration 10 mM)
in DMSO (250 μL) were added to the cell suspension. The
reaction mixture was shaken at 37 °C and 150 rpm for 216 h.
Additional borane-ammonia complex (1.5 mg) was added
after 24, 48, 96, and 168 h. Fresh cell lyophilisate (50 mg)
was added after 200 h. Samples (500 μL) were taken after 24,
48, 72, 96, 168 and 216 h and samples with 1a, 2a and 2c as
substrate were basified by addition of 2 M aq. NaOH solution
(50 μL). Afterwards, all samples were extracted with EtOAc
(2 × 500 μL) and dried over Na2SO4. The solvent was evapo-
rated under a stream of air, the sample was re-dissolved in
HPLC-grade methanol (700 μL) and the substrate ee was
determined by HPLC analysis.
(S)-1-(3-Hydroxybenzyl)-7-hydroxy-6-methoxy-2-methyl-
1,2,3,4-tetrahydroisoquinoline (S)-3a. 115 mg (77%) as a white
solid foam. mp: 88–90 °C. TLC (CH2Cl2/MeOH/NH3(aq) =
90/9/1): Rf = 0.33. [α]2D5 = +37.8 (CHCl3, c = 0.28); lit. (ref. 27)
1
+43 (MeOH, c = 0.5). H-NMR (DMSO-d6, 300 MHz): δ = 2.30
(3H, s, NCH3), 2.40–2.66 (3H, m, CH2), 2.73 (1H, dd, J1 = 14.4 Hz,
J2 = 5.1 Hz, CH2), 2.87 (1H, dd, J1 = 14.3 Hz, J2 = 7.0 Hz, CH2),
3.02–3.10 (1H, m, CH2), 3.56 (1H, t, J = 6.0 Hz, CH), 3.71 (3H, s,
OCH3), 6.43 (1H, s, Ar), 6.52–6.60 (4H, m, Ar), 6.99 (1H, t, J = 7.9
Hz, Ar), 8.54 (1H, s, OH), 9.11 (1H, s, OH). 13C-NMR (DMSO-d6,
75 MHz): δ = 24.9, 41.3, 42.8, 47.1, 55.9, 64.2, 112.3, 113.0, 114.8,
116.8, 120.5, 124.9, 129.0, 130.4, 142.1, 144.7, 146.3, 157.3.
MS (EI, 70 eV): m/z = 298 (M+–H, <1), 192 (100), 177 (21).
(S)-1-(3-Hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-
tetrahydroisoquinoline (S)-3c. 121 mg (85%) as a yellowish
oil. TLC (CH2Cl2/MeOH/NH3(aq) = 90/9/1): Rf = 0.40. [α]D25
=
+61.9 (CHCl3, c = 1.0) lit. (ref. 11d) (R): −76.3 (CHCl3, c =
0.63). 1H-NMR (CDCl3, 300 MHz): δ = 2.40 (3H, s, NCH3),
2.61–2.93 (4H, m, CH2), 3.06 (1H, dd, J1 = 13.8 Hz, J2 = 5.7
Hz, CH2), 3.15–3.23 (1H, m, CH2), 3.68 (3H, s, OCH3), 3.79
(1H, t, J = 6.5 Hz, CH), 6.46–6.54 (6H, m, Ar), 6.99 (1H, dd,
J1 = 8.7 Hz, J2 = 7.8 Hz, Ar). 13C-NMR (CDCl3, 75 MHz): δ =
24.8, 41.7, 45.8, 55.2, 64.5, 111.9, 113.3, 113.8, 116.4, 121.2,
128.9, 129.1, 129.3, 129.4, 134.4, 140.9, 156.7, 157.9. MS (EI,
70 eV): m/z = 282 (M+–H, <1), 176 (100), 132 (11).
(S)-1-(3-Hydroxy-4-methoxybenzyl)-7-hydroxy-6-methoxy-2-
methyl-1,2,3,4-tetrahydro-isoquinoline (S)-3f. 132 mg (80%)
as a white solid foam. mp: 75–76 °C. TLC (CH2Cl2/MeOH/
NH3(aq) = 90/9/1): Rf = 0.31. [α]2D5 = +58.2 (CHCl3, c = 1.0) lit.
(ref. 28) +57 (CHCl3, c = 1.2). 1H-NMR (CDCl3, 300 MHz):
δ = 2.38 (3H, s, NCH3), 2.48–2.55 (1H, m, CH2), 2.64–2.74
(3H, m, CH2), 2.95 (1H, dd, J1 = 14.1 Hz, J2 = 6.1 Hz, CH2),
3.06–3.13 (1H, m, CH2), 3.60 (1H, t, J = 6.2 Hz, CH), 3.77 (6H,
2 × s overlap, 2 × OCH3), 6.30 (1H, s, Ar), 6.46 (1H, s, Ar),
6.51 (1H, dd, J1 = 8.2 Hz, J2 = 2.1 Hz, Ar), 6.64–6.68 (2H, d + t
overlap, Ar). 13C-NMR (CDCl3, 75 MHz): δ = 25.0, 40.9, 42.4,
46.7, 55.8, 55.9, 64.5, 110.4, 110.6, 113.7, 115.6, 120.9, 125.2,
130.2, 133.2, 143.3, 145.0, 145.2, 145.3. MS (EI, 70 eV): m/z =
328 (M+–H, <1), 192 (100), 177 (21).
Deracemisation of benzylisoquinolines by MAO-N D11 and
NH3·BH3 (preparative scale). In an Erlenmeyer flask (250 mL),
lyophilised cells of E. coli C43 (DE3) [pET16b–MAO-N(D11)]
from auto-induction cultures (5.0 g) were resuspended in
phosphate buffer (45 mL; 100 mM K–Pi, pH 7.7). NH3·BH3
(62 mg, 2 mmol) and a solution of substrate (142–165 mg,
0.5 mmol) in DMSO (5 mL) were added and the mixture was
shaken at 37 °C and 150 rpm. After 24 h (and in case of 3c
after 48 h), further NH3·BH3 (31 mg, 1 mmol) was added and
shaking was continued. After the appropriate time (3a, 3f: 48
h; 3c: 72 h), a sample (250 μL) was taken, extracted with
Protein–ligand docking simulations
Docking simulations were performed using the Discovery
Studio 3.1 software from Accelrys with the MAO-N D11 X-ray
3662 | Catal. Sci. Technol., 2014, 4, 3657–3664
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crystal structure (PDB: 3ZDN) as template.19a Substrates were
docked into the active site using the protocol Ligand Fit with
default settings and the CHARMm force field. “Forced docking”
of (S)-3a was performed by positioning the substrate into the
active site, followed by energy minimisation.
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Acknowledgements
This study was financed by the Austrian Science Fund (FWF
project P20903-N17 and P22115-N17). The authors would like
to thank Prof. Bauke Dijkstra and Dr. Niels van Oosterwijk
(University of Groningen) for providing E. coli C43 (DE3) and
Bernd Werner (University of Graz) for acquiring NMR spectra.
Financial support by the European Commission (Marie Curie
Networks for Initial Training fellowship, project “BIOTRAINS”
FP7-PEOPLE-ITN-2008-238531; project: KyroBio, grant agree-
ment no. 289646), by the COST Action CM1303 “SysBioCat”,
by NAWI Graz and by OeFG is gratefully acknowledged.
J. H. Sattler has been supported by the Austrian BMWFJ,
BMVIT, SFG, Standortagentur Tirol and ZIT through the
Austrian FFG-COMET-Funding Program. NJT is grateful for
receipt of a Royal Society Wolfson Research Merit Award.
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3664 | Catal. Sci. Technol., 2014, 4, 3657–3664
This journal is © The Royal Society of Chemistry 2014