Biocatalytic oxidative C-C bond formation catalysed by the berberine bridge enzyme: Optimal reaction conditions

Article abstract of DOI:10.1002/adsc.201100233

Berberine bridge enzyme (BBE) catalyses the oxidative formation of an intramolecular C-C bond using (S)-reticuline as the natural substrate to form (S)-scoulerine as the product. To allow application of the enzyme on a preparative scale for the synthesis

Full text of DOI:10.1002/adsc.201100233

FULL PAPERS
DOI: 10.1002/adsc.201100233
À
Biocatalytic Oxidative C C Bond Formation Catalysed by the
Berberine Bridge Enzyme: Optimal Reaction Conditions
Verena Resch,a Joerg H. Schrittwieser,a Silvia Wallner,b Peter Macheroux,b
and Wolfgang Kroutila,*
a
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
Fax : (+43)-316-380-9840; e-mail: wolfgang.kroutil@uni-graz.at
Institute of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria
b
Received: May 30, 2011; Published online: August 26, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100233.
Abstract: Berberine bridge enzyme (BBE) catalyses substrate concentration of at least 20 gLÀ1. In addi-
À
the oxidative formation of an intramolecular C C tion, the enzyme works in a broad operational
bond using (S)-reticuline as the natural substrate to window concerning pH and temperature. High con-
form (S)-scoulerine as the product. To allow applica- versions can be reached between pH 8 and 11 and
tion of the enzyme on a preparative scale for the from 30 to 508C, respectively. The enantioselective
À
synthesis of novel optically pure berbine and isoqui- oxidative C C bond formation was demonstrated on
noline derivatives, an organic solvent is required to a preparative scale (500 mg) in a kinetic resolution
solubilise the barely soluble substrates. It was shown leading to optically pure products (>97% ee).
that BBE tolerates a broad variety of organic co-sol-
vents. Ideally the enzymatic enantioselective oxida-
À
À
tive C C bond formation can be performed in 70% Keywords: alkaloids; biotransformations; C C bond
vvÀ1 toluene concentration, which allowed a soluble formation; enzyme catalysis; oxidation
Introduction
bridging a phenol moiety to an N-methyl group of
(S)-reticuline to yield (S)-scoulerine at the expense of
Employing biocatalysts for synthetic organic chemis- molecular oxygen (Scheme 1). The enzyme contains a
try gains constantly increasing significance.[1–4] Focus- bicovalently bonded flavin responsible for the reac-
[5]
ing on C C bond formation, aldolases and transke- tion.[21]
À
tolases,[6–9] hydroxynitrile lyases,[5,10–12] ThDP-depen-
Recently, it could be shown that recombinant BBE
dent enzymes[5–15] as well as laccases and peroxidas- from Eschscholzia californica (California poppy)[22,23]
es[16–18] belong to the most commonly employed bio- can be employed efficiently for the preparation of
catalysts. Berberine bridge enzyme (BBE), first novel optically pure isoquinoline and berbine alka-
identified in 1963,[19,20] catalyses in nature an outstand- loids via enantioselective oxidative C C bond forma-
À
ing oxidative intramolecular C C bond formation by tion.[24] The enzyme displayed perfect enantioselectivi-
À
Scheme 1. Natural reaction catalysed by the berberine bridge enzyme (BBE).
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ty, thus only a single substrate enantiomer of a race-
As a first approach to evaluate the tolerance of the
mate was converted. Unfortunately, potential sub- enzyme towards organic solvents various water misci-
strates of BBE, isoquinolines, are only barely soluble ble, water immiscible solvents and ionic liquids were
in buffer at
a
useful preparative concentration tested at a concentration of 10% vvÀ1 at 6.5 mM sub-
(>50 mM); consequently an organic solvent is re- strate concentration (Figure 1). In most tested water
quired to solubilise the substrate. Mainly lipases and miscible and immiscible organic solvents high conver-
proteases have been described to show a significant sion was achieved. Low conversions were obtained in
tolerance toward organic solvents beside a few other the presence of selected water miscible organic sol-
enzymes like alcohol dehydrogenases.[25] Nevertheless, vents (THF, pyridine), some immiscible solvents
the solvent tolerance can be improved, for instance, (CH2Cl2, EtOAc, CHCl3) and one tested water immis-
by protein engineering[26–31] or immobilisation.[32–36]
cible ionic liquid [trioctylmethylammonium bis(tri-
In this study we report in detail on the influence of fluoromethylsulfonyl)imide=TOMAbisTMSI]. Mod-
the organic solvent, pH, temperature, and buffer on erate to high conversion was achieved employing
À
the conversion of the oxidative C C bond formation water miscible ionic liquids (Ammoeng 102,
catalysed by BBE from Eschscholzia californica.
T-2HMAMS, BMimAc, EMimAc).
Due to the promising tolerance of the enzyme to-
wards organic solvents, the organic solvent concentra-
tion was increased to 20% v vÀ1 (Figure 2). Since a
number of common organic solvents were superior
Results and Discussion
For studying the influence of organic solvents and compared to ionic liquids, the ionic liquids were not
other relevant parameters a racemic non-natural tested further.
model substrate 1 (Scheme 2) was chosen.[22] The
At 20% vvÀ1 of co-solvent all water miscible co-sol-
model substrate was designed considering the natural vents seemed to affect the enzyme, except for metha-
substrate reticuline as a template, but omitting the nol and DMSO, whereby DMSO was the best. All
methoxy group of reticuline in the 4’-position and in- water immiscible solvents tested led to perfect conver-
troducing a methoxy instead of the hydroxy group in sion for an enantioselective kinetic resolution (50%).
position 7. Before starting with the organic solvents
Consequently DMSO and toluene were chosen for
some basic pre-experiments were performed: hydro- further studies to reach even higher co-solvent con-
gen peroxide is formed as a side product, which might centrations. Toluene was chosen since it is less toxic
harm the enzyme. To test this, experiments were per- than benzene and more commonly employed than di-
formed with and without catalase. In presence of cata- phenyl ether. On increasing the concentration of co-
lase a 4-fold higher conversion (37 vs. 9.5%) was ach- solvent up to 40% v vÀ1, DMSO caused a significantly
ieved than without catalase. Additional studies reduced conversion at 30% vvÀ1 and complete loss of
showed that MgCl2 has a stabilising effect on the activity at 40% vvÀ1 (Figure 3). In contrast, toluene
enzyme. For this reason catalase and MgCl2 (10 mM) still led to perfect conversion at 40% vvÀ1.
were added to the reaction mixtures.
À
Scheme 2. Enantioselective oxidative C C bond formation of the non-natural racemic substrate 1 catalysed by berberine
bridge enzyme (BBE).
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Adv. Synth. Catal. 2011, 353, 2377 – 2383

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Biocatalytic Oxidative C C Bond Formation Catalysed by the Berberine Bridge Enzyme
Figure 1. Conversion in the presence of various solvents 10% (vvÀ1). Reaction conditions: 2 gLÀ1 substrate 1 (6.5 mM), BBE
(0.0017 mM), Tris-HCl 50 mM+MgCl2 10 mM, pH 9, 10% vvÀ1 organic solvent, 5 gLÀ1 crude catalase, 4 h, 408C; TOMA-
bisTMSI: trioctylmethylammonium bis(trifluoromethylsulfonyl)imide, T-2-HMAMS: tris-(2-hydroxyethyl)-methylammonium
methyl sulfate, BMimAc: 1-butyl-3-methylimidazolium acetate, EMimAc: 1-ethyl-3-methylimidazolium acetate.
Figure 2. Conversion in the presence of 20% vvÀ1 organic solvents. Reaction conditions: 2 gLÀ1 substrate 1 (6.5 mM),
0.1 gLÀ1 BBE, Tris-HCl 50 mM+MgCl2 10 mM, pH 9, 20% vvÀ1 organic solvent, 5 gLÀ1 crude catalase, 4 h, 408C.
Encouraged by these results, the concentration of
Product was detected at solvent concentrations up
toluene was further increased up to 100% (Figure 4). to 99% vvÀ1. Just in toluene alone no conversion was
For comparison the related experiments were per- detected. This experiment was performed with freeze-
formed in the presence of benzene. These experi- dried enzyme; additional tests ensured that the
ments were already performed at a substrate concen- freeze-dried enzyme is active.
tration of 13 mM, thus the concentration was doubled
compared to previous studies.
In the case of benzene full conversion (50%) was
achieved at a solvent concentration of 40 to 60%
vvÀ1, while in the case of toluene also at 80% vvÀ1
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Figure 5. Conversion at varied pH values ranging from 6 to
13. Reaction conditions: 2 gLÀ1 substrate 1 (6.5 mM), BBE
(0.0017 mM), Tris-HCl 50 mM+MgCl2 10 mM, pH 6–13, co-
solvent: DMSO 10% vvÀ1, toluene 70% vvÀ1, 5 gLÀ1 crude
catalase, 4 h, 408C.
Figure 3. Comparison of DMSO and toluene at solvent con-
centrations from 10 to 40% vvÀ1. Reaction conditions:
2 gLÀ1 substrate 1 (6.5 mM), BBE (0.0017 mM), Tris-HCl
50 mM+MgCl2 10 mM, pH 9, co-solvent, 5 gLÀ1 crude cata-
lase, 4 h, 408C.
Figure 4. Conversion in the presence of toluene and benzene
reaching from 30% to 100% (vvÀ1). Reaction conditions:
4 gLÀ1 substrate 1 (13 mM), BBE (0.0017 mM), Tris-HCl
50 mM+MgCl2 10 mM, pH 9, co-solvent, 5 gLÀ1 crude cata-
lase, 4 h, 408C.
Figure 6. Conversion at varied temperature. Reaction condi-
tions: 2 gLÀ1 substrate 1 (6.5 mM), BBE (0.0017 mM), Tris-
HCl 50 mM+MgCl2 10 mM, pH 9, co-solvent: DMSO 10%
vvÀ1, toluene 70% vvÀ1, 5 gLÀ1 crude catalase, 4 h.
perfect conversion was found. A toluene concentra- sults. Thus, neither the buffer salt nor the salt concen-
tion of 70% vvÀ1 was considered as ideal for an opti- tration had any significant effect on the conversion
mal performance.
(data not shown).
In addition to the type of co-solvent also the opti-
The published assays for the BBE were performed
mal pH of the reaction medium had to be elucidated. at 378C[37,38] and 458C.[39] This was also the reason
In the literature the pH optimum for the aqueous why all studies described here until now were per-
buffer is reported to be pH 9.[20] Testing toluene in a formed at 408C as a compromise. To get a clear pic-
bi-phasic system and DMSO for comparison in a ture for the temperature optimum, the conversion
mono-phasic system revealed that the pH optimum was determined at temperatures between 108C and
was actually rather broad ranging from pH 8 to pH 11 708C employing toluene as co-solvent but also
(Figure 5). At pH 12 only in the case of DMSO (10% DMSO for comparison.
vvÀ1) was a significant conversion detected, while in
the case of toluene almost no conversion was ob- ature range between 30–508C (Figure 6). Neverthe-
served. less, highest conversion was achieved at 408C in the
Four different buffer salts (Tris-HCl, phosphate cases of DMSO and toluene, respectively.
High conversion was reached over a broad temper-
buffer, Ches-HCl and triethylamine-HCl) were evalu-
Having identified optimised reaction conditions, a
tated at varied buffer concentration (10–50 mM, time study was performed at 20 gLÀ1 (65 mM) sub-
2 gLÀ1 substrate); all buffer systems led to similar re- strate concentration to clarify when the reaction
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Adv. Synth. Catal. 2011, 353, 2377 – 2383

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Biocatalytic Oxidative C C Bond Formation Catalysed by the Berberine Bridge Enzyme
Experimental Section
General Remarks
Substrate 1 was synthesised as previously described.[22] For
the expression of BBE from Eschscholzia californica in
Pichia pastoris and its purification see Supporting Informa-
tion.
Analytics
Determination of conversion: Conversions were measured
by HPLC: Shimadzu, Communication Bus Module CBM-
20A, column oven CTO-20 AC, degasser DGU-20 A5,
liquid chromatograph LC-20 AD, auto sampler SIL-20 AC,
diode array detector SPD-M20, C18 column (Phenomenex,
LUNA C18, 0.46 cmꢁ25 cm, 5 mm). Eluent: acetonitrile/
methanol/ammonium formate (30 mM, pH 2.8) 15:18:67
(isocratic), flow rate: 0.5 mLminÀ1, detection wavelength:
280 nm. Retention times: 3: 10.0 min, 1: 13.2 min, 2:
16.6 min.
Determination of optical purity: The HPLC system de-
scribed above was equipped with a chiral column (Chiralcel
OJ from Daicel Chemical Industries, 0.46 cmꢁ25 cm, Lot.
No.: OJ00CE-NK006). Eluent: n-heptane (0.1% formic
acid)/2-propanol (0.1% formic acid) 70:30, flow rate:
0.5 mLminÀ1 (isocratic), detection wavelength: 280 nm. Re-
tention times substrate 1: (S): 14.2 min, (R):18.8 min
À
Figure 7. Time course of enantioselective oxidative C C
bond formation of substrate 1 catalysed by BBE. Reaction
conditions: 20 gLÀ1 substrate 1 (65 mM), BBE (0.017 mM),
Tris-HCl 50 mM+MgCl2 10 mM, pH 9, 70% vvÀ1 toluene,
5 gLÀ1 crude catalase, 4 h.
reaches full conversion (50%) (Figure 7). For this
study the enzyme concentration was increased to
0.017 mM to reach completion within a reasonable
time.
Using the optimised conditions the reaction
reached 49% conversion within 12 h. The space time
yield is therefore 10 gLÀ1 dÀ1. For the turnover
number a value of 1849 was calculated.
Performing a preparative transformation, 500 mg of
substrate 1 (65 mM) were shaken for 24 h under opti-
mised conditions leading to 50% conversion. Work-up
of the reaction yielded 249 mg (50%) of optically
pure (R)-1 (ee>97%) and 207 mg (42%) of optically
pure (S)-2 (ee>97%) besides 8% of regioisomer 3.
For chiral analysis of product 2 the same system as men-
tioned above was used but employing as chiral phase Chiral-
pak AD from Daicel Chemical Industries (0.46 cmꢁ25 cm,
Lot. No.: AD00CE-AB081). Eluent: n-heptane/2-propanol
70:30, flow rate: 0.5 mLminÀ1, detection wavelength:
280 nm. Retention times: 18.4 min (S), 11.0 min (R).
Representative Procedure for Solvent Study
A stock solution of substrate rac-1 in dichloromethane
(2 mgmLÀ1) was prepared and aliquots containing 1 mg of
substrate were transferred into glass vials (4 mL). Dichloro-
methane was removed under constant air flow. The sub-
strate was redissolved in the appropriate organic solvent
(50 mL). For the addition of the enzyme a stock solution of
BBE (0.1 mgmLÀ1) and catalase (0.05 mgmLÀ1) was pre-
pared in Tris-HCl buffer (50 mM, pH 9, 10 mM MgCl2).
After adding the enzyme stock solution (450 mL) to the sub-
strate in organic solvent (final concentration: substrate
2 gLÀ1, BBE 0.1 gLÀ1, catalase 5 gLÀ1), the glass vials were
closed with screw caps containing sealing gaskets and sam-
ples were shaken in an Incubator Mini Shaker (VWR,
rotary, orbit 3 mm) with 200 rpm at 408C for four hours.
The reaction was extracted with ethyl acetate (3ꢁ300 mL)
and the combined organic layers were dried (Na2SO4). The
organic solvents were removed under constant air flow and
the residue was redissolved in methanol (1 mL, Roth, Roti-
solv HPLC grade, Lot. P717.1) for HPLC analysis. This pro-
cedure was repeated with 20% vvÀ1 (100 mL) for selected
co-solvents.
Conclusions
Berberine bridge enzyme (BBE) was shown to toler-
ate organic solvents, especially toluene, DMSO, ben-
zene and diphenyl ether. Ideally, the enzymatic enan-
À
tioselective oxidative C C bond formation was per-
formed at 70% vvÀ1 toluene concentration, which al-
lowed a substrate concentration of 20 gLÀ1. Its excel-
lent stability in the presence of organic solvents
renders the enzyme suitable for application in organic
synthesis to prepare non-natural optically pure alka-
loids. Additionally, the enzyme works in a broad op-
erational window concerning pH and temperature.
High conversions were obtained between pH 8 and 11
and from 30 to 508C, respectively, with still good ac-
tivity remaining at 608C.
The reaction was demonstrated successfully on a
preparative scale (500 mg) leading to optically pure
products (ee>99%).
Influence of Catalase
The experiments were performed similar as described
above. Substrate concentration: 8 gLÀ1, BBE amount:
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0.1 gLÀ1, catalase amount: 5 gLÀ1, reaction time: 24 h, co-
solvent: methanol 10% vvÀ1, buffer: Tris-HCl 50 mM+
MgCl2 10 mM, pH of the buffer: 9, reaction temperature:
408C. The work-up was performed as described above.
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pH Study
The experiments were performed similar as described
above. A Tris-HCl buffer (50 mM+MgCl2 10 mM) with pH
values from 6 to 13 was used (the pH was measured before
addition of solvent). As co-solvent 10% vvÀ1 of DMSO and
toluene, respectively, was employed. Conditions: 2 gLÀ1 of
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Time Study
The experiments were performed using 20 gLÀ1 of substrate
in 70% vvÀ1 toluene and 30% vvÀ1 Tris-HCl buffer, 1 gLÀ1
of BBE and 5 gLÀ1 catalase in a total volume of 0.5 mL.
Samples were taken at 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h.
Work-up and analysis were performed as described above.
Preparative Transformation
Substrate
1
(500 mg, 1.6 mmol, final concentration:
20 gLÀ1 =65 mM) was shaken in a mixture of toluene 70
vvÀ1 (17.5 mL) and buffer (7.5 mL, Tris-HCl, 50 mM, pH 9,
10 mM MgCl2) with BBE (1.5 mL enzyme solution, final
concentration: 1 gLÀ1 =0.017 mM) and catalase (125 mg
crude preparation). The mixture was shaken in a light-
shielded round bottom flask (50 mL) in an Incubator Mini
Shaker (VWR, rotary, orbit 3 mm) at 200 rpm and 408C for
24 h. Reaction work-up: the phases were separated separa-
tion followed by extraction of the aqueous phase with ethyl
acetate (3ꢁ10 mL). The combined organic phases were
dried (Na2SO4) and the organic solvents were removed
under reduced pressure. The crude product was purified by
silica gel chromatography (silica gel 60, 0.040–0.063 mm,
Merck, Lot.: 1.09385.9025; eluent: CH2Cl2/MeOH/NH4OH
97:2:1)
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For analytical details, preparation of enzyme and product
characterization see Supporting Information.
Acknowledgements
[25] For a review see: N. Doukyua, H. Ogino, Biochem.
This study was financed by the Austrian Science Fund (FWF
Project P20903-N17 and P22115-N17). Financial support by
NAWI Graz is acknowledged.
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