Biocatalytic enantioselective oxidative C-C coupling by aerobic C-H activation

Article abstract of DOI:10.1002/anie.201006268

Bridging the gap: The berberine bridge enzyme (BBE) was employed for the first preparative oxidative biocatalytic C-C coupling that leads to a new intramolecular bond. This unique transformation requires O₂ as sole stoichiometric oxidant and gives access to novel optically pure (S)-berbine 2 and (R)-1-benzyl-1,2,3,4-tetrahydroisoquinoline 1 alkaloid derivatives by kinetic resolution.

Full text of DOI:10.1002/anie.201006268

Communications
DOI: 10.1002/anie.201006268
À
Biocatalytic C C Coupling
À
Biocatalytic Enantioselective Oxidative C C Coupling by Aerobic
C H Activation**
À
Joerg H. Schrittwieser, Verena Resch, Johann H. Sattler, Wolf-Dieter Lienhart,
Katharina Durchschein, Andreas Winkler, Karl Gruber, Peter Macheroux, and
Wolfgang Kroutil*
Reactions that form carbon–carbon bonds are the basis for
organic chemistry, setting up the carbon framework of organic
À
molecules. In particular, the formation of C C bonds by
[1]
À
organo- or metal-catalyzed activation of C H bonds has
recently obtained increased attention.[2] However, to the best
À
of our knowledge, no biocatalytic oxidative C C bond-
forming reaction has yet been exploited for synthetic
Scheme 1. Natural reaction of BBE. Formation of the “berberine
À
bridge” by oxidative C C coupling at the expense of molecular oxygen.
purposes. To date, biocatalysis only offers a limited number
[3]
À
of enzymes for synthetic C C bond formation, such as
aldolases,[4] transketolases[4d,5] and hydroxynitrile lyases,[3,6]
and thiamine diphosphate (ThDP)-dependent enzymes.[3,7]
the mechanism.[10,14] Thus, BBE has been thoroughly inves-
tigated regarding its biochemical properties and catalytic
mechanism, while its potential as a biocatalyst for preparative
transformation of non-natural substrates has not been
explored yet. A major concern referred to the question
whether non-natural substrates could be transformed at all,
since plant enzymes may have strict substrate specificity.
Furthermore, no information on the enantioselectivity of the
catalyst for non-natural substrates was available. The bio-
chemical investigations of BBE were all performed on a
microgram[10] scale and at low substrate concentration
(0.5 mm),[15] both of which have to be significantly increased
for preparative applications.
To gain sufficient quantities of a non-natural model
substrate that can easily be prepared, racemic tetrahydro-
isoquinoline rac-1b (Scheme 2), which lacks the methoxy
group of reticuline in the 4’-position and possesses a methoxy
instead of the hydroxy group in position 7 of the benzyliso-
quinoline backbone, was prepared in five steps with 40%
overall yield (see the Supporting Information). As a first test,
reductive rate measurements were performed, which clearly
indicated that the non-natural substrate rac-1b was accepted
À
Other enzymatic C C bond-forming reactions have just
started to be investigated.[8] All of these enzymes are either
lyases or transferases, but not redox enzymes.[9]
In contrast, the berberine bridge enzyme (BBE) [EC
1.21.3.3] is a redox enzyme that converts (S)-reticuline 1a as
the natural substrate with a 1-benzyl-1,2,3,4-tetrahydroiso-
quinoline backbone to (S)-scoulerine 2a, a berbine derivative.
À
The transformation occurs through an intramolecular C C
coupling by activation of the methyl group attached to the
tertiary nitrogen atom at the expense of molecular oxygen
(Scheme 1).[10] The enzyme is found in plants, mainly from the
poppy family, where it plays a central role in the biosynthesis
of benzophenantridine alkaloids.[11,12] Only recently, BBE
from Eschscholzia californica (California poppy) could be
expressed efficiently in Pichia pastoris[13] to give a sufficient
quantity of the enzyme for crystallization and investigation of
[*] J. H. Schrittwieser,[+] V. Resch,[+] J. H. Sattler, W.-D. Lienhart,
K. Durchschein, Prof. Dr. W. Kroutil
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
Dr. A. Winkler, Prof. Dr. P. Macheroux
Institute of Biochemistry, Graz University of Technology
Petersgasse 12, 8010 Graz (Austria)
Prof. Dr. K. Gruber
Institute of Molecular Biosciences, University of Graz
Humboldtstraße 50/III, 8010 Graz (Austria)
[+] These authors contributed equally to this work.
[**] This study was financed by the Austrian Science Fund (FWF Project
P20903-N17 and P22115-N17). We thank the European Commis-
sion (MC-ITN Biotrains, grant agreement no.: 238531) for financial
support. We thank Christoph Gꢀbl for measuring CD spectra and
Bernd Werner for NMR measurements.
À
Scheme 2. Biocatalytic enantioselective oxidative C C coupling of non-
natural substrates by BBE led to optically pure (R)-2b–e and (S)-2b–e
as the main products by kinetic resolution.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201006268.
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 1068 –1071

by purified BBE at a promising rate of kred = 2 sÀ1. A first
preparative transformation (8 mg) under nonoptimized con-
ditions allowed to identify the product of the transformation
as 2b by comparison of the MS spectrum with literature
values.[16]
Subsequently, substrate rac-1b was used for further
optimization studies. It quickly became clear that as soon as
a higher substrate concentration was used and higher
conversions were achieved, the hydrogen peroxide formed
as byproduct inhibited or degraded the enzyme; therefore it
was necessary to add catalase to disproportionate the formed
hydrogen peroxide to water and molecular oxygen.[17]
Since substrate rac-1b was barely soluble in buffer,
various water-miscible as well as water-immiscible organic
solvents were tested for substrate solubilization. A substrate
concentration of 2 gLÀ1 (7 mm) was chosen for the studies of
organic cosolvents as a first step to increase the substrate
concentration compared to the biochemical studies. Testing
the solvents at 10% v/v, BBE showed an unexpectedly high
tolerance toward a broad range of organic solvents (Figure 1).
Figure 2. Enzymatic transformation of substrate rac-1b in the presence
of increasing amounts of toluene (%v/v).
performed by suspending freeze-dried BBE in toluene; it was
ensured that the enzyme was not damaged by freeze-drying:
reactions with rehydrated enzyme showed full activity.
Experiments to chemically synthesize 2b as reference
material by Pictet–Spengler[18] reaction from N-demethylated
1b and formaldehyde gave a mixture of the racemic
regioisomers 2b/3b in a 40:60 ratio according to GC-MS
analysis. The products were isolated in low yields of 30% and
15%, respectively. Obviously, the enzymatic reaction is
unique not only concerning the catalyzed transformation,
but also with respect to regioselectivity. Nevertheless, the
regioisomer 3b was also found as a minor side-product of the
enzymatic transformation in the solvent study. The formation
of 3b varied depending on 1) the type of solvent used and
2) solvent concentration. For instance, product 2b and
regioisomer 3b were formed in a ratio of 96:4 when employ-
ing toluene (99.4% v/v), while when using acetonitrile (10%
v/v) a ratio of 1:1 was obtained.[17]
In addition to the identification of a suitable cosolvent,
other reaction parameters were optimized such as buffer salt
and buffer concentration, pH value, temperature, shaking
celerity, and the amount of catalase. The most suitable buffer
was found to be 10 mm tris(hydroxymethyl)aminomethane
hydrochloride (Tris-HCl) at pH 9 containing 10 mm MgCl2.
Reactions were ideally performed in the dark[19] by employing
catalase, purified BBE (1 gLÀ1), and a two-phase toluene/
buffer mixture 70:30 v/v for a substrate concentration of
20 gLÀ1. Employing these conditions, the transformation of
rac-1b stops at 50% conversion after 12 h as shown in a time
study. This corresponds to a space–time yield of 20 gLÀ1 dÀ1
and an apparent turnover number of 1850.
The conversions obtained for the transformation of the
racemic substrate rac-1b never exceeded 50%, which already
indicated that the enzyme might possess excellent enantiose-
lectivity. Indeed, the analysis of the reactions by HPLC on a
chiral phase showed that only one single enantiomer was
transformed while the other one remained untouched, con-
sequently leading to an optically pure product. Thus, BBE
catalyzed the kinetic resolution of rac-1b to give optically
pure products (S)-2b and (R)-1b both in > 97% ee as
determined by HPLC, which corresponds to an enantioselec-
tivity E > 200.
Figure 1. Conversion c of substrate rac-1b by BBE in the presence of
organic solvents (10% v/v) in buffer.
For instance, dimethyl sulfoxide (DMSO) led to almost 50%
conversion, which was the best value obtained for all water-
miscible organic solvents tested. Nevertheless, also dioxane,
formamide, methanol, ethanol, and even hexamethyl phos-
phoric triamide (HMPA) were accepted; methanol was
already used in previous biochemical studies to solubilize
the natural substrate (S)-reticuline. On the other hand,
tetrahydrofuran (THF) led to low conversion. Similar low
conversions were achieved by employing some water-immis-
cible solvents like dichloromethane or ethyl acetate. Best
results were obtained by employing toluene, benzene, or
diphenyl ether in a two-phase system. Toluene was chosen for
further studies because of its lower toxicity compared to
benzene and because of an easier work-up procedure
compared to the water-miscible organic solvents. Testing the
tolerance of BBE toward increasing concentrations of toluene
showed that employing 80% v/v of toluene still led to 50%
conversion within 24 h at 4 gLÀ1 substrate concentration and
0.1 gLÀ1 BBE (Figure 2). Even at 99% v/v toluene the
enzyme was still remarkably active; however, in dry toluene
no conversion could be detected. The latter experiment was
To test whether other racemic benzylisoquinoline deriv-
atives would be transformed as well, substrates 1c–e were
Angew. Chem. Int. Ed. 2011, 50, 1068 –1071
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www.angewandte.org 1069

Communications
synthesized and subjected to BBE-catalyzed ring closure.
In summary, the berberine bridge enzyme from California
poppy was employed for a highly enantioselective biocatalytic
Substrate rac-1c bears a methylene bridge between the two
oxygen atoms at the isoquinoline part, rac-1d possesses only
one methoxy and rac-1e three methoxy groups. All three
benzylisoquinolines turned out to be good substrates that
were enantioselectively transformed into the corresponding
À
oxidative C C coupling reaction to prepare optically pure
berbine derivatives as well as optically pure tetrahydro-
benzylisoquinolines. The described reaction was successfully
performed on a 500 mg scale only requiring molecular oxygen
as oxidant and mild reaction conditions, thus it represents a
À
berbine derivative by oxidative C C coupling. Most of the
obtained products have never been described before, neither
in optically pure nor racemic form. Compounds 1c and 2c
have been described in racemic form only, and product 2b has
previously been described only after isolation of 5 mg of this
step towards cleaner and more selective organic transforma-
[30]
À
tions that expand the scope of C C bond formation.
compound (common name: manibacanine) from the stem Experimental Section
bark of Anila canelilla.[16] Thus, the biocatalytic C C coupling
À
Representative preparative C C coupling: Substrate 1b (500 mg,
1.6 mmol, final concentration: 20 gLÀ1 = 65 mm) was dissolved in
toluene (17.5 mL) and buffer (7.5 mL, Tris-HCl, 10 mm, pH 9, 10 mm
MgCl2) containing BBE (1.5 mL enzyme solution, final concentra-
tion: 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. The reaction was stopped by phase
separation followed by extraction of the aqueous phase with ethyl
acetate (3 ꢀ 10 mL). The combined organic phases were dried
(Na2SO4) and the organic solvents were removed under reduced
pressure. The crude product was purified by silica gel chromatog-
raphy (silica gel 60, 0.040–0.063 mm, Merck, Lot.: 1.09385.9025;
eluent: CH2Cl2/MeOH/NH4OH 97:2:1) to give 207 mg of (S)-1b
(42% yield, > 97% ee) and 249 mg (S)-2b (49% yield, > 97% ee) (for
full characterization (NMR spectra, HPLC data, optical rotation,
HRMS, and CD spectra) see the Supporting Information).
À
using BBE allowed to access novel optically pure alkaloids.
To demonstrate the applicability of the enzyme on a
preparative scale, all four non-natural substrates (1b–e) were
transformed on a 500 mg scale. All substrates could be fully
resolved within 24 h and the products of the kinetic resolution
(R)-1b–e and (S)-2b–e could be isolated with good to
excellent yield and excellent optical purity (Table 1).
[a]
À
Table 1: Preparative oxidative C C coupling employing BBE.
Subst.
c[b]
(R)-1
ee (1)[c]
(S)-2
ee (2)[c]
E[d]
[%]
[mg(%)]
[%]
[mg(%)]
[%]
1b
1c
1d
1e
50
50
50
50
249 (50)
231 (46)
181 (36)
237 (47)
>97
>97
>97
>97
207 (42)
155 (31)
177 (36)
194 (39)
>97
>97
>97
>97
>200
>200
>200
>200
Received: October 6, 2010
Published online: January 18, 2011
[a] Reactions were performed in the dark in toluene/buffer 70:30, pH 9,
at a substrate concentration of 20 gLÀ1, 1 gLÀ1 BBE, 0.05 gLÀ1 catalase,
408C, 24 h. [b] Conversion was measured by HPLC on an achiral C18
phase. Depending on the substrate converted, 4–10% of the regioisomer
3b–e were formed: 1b: 8%, 1c: 7%, 1d: 4%, 1e: 10%. [c] Enantiomeric
excess was measured by HPLC on a chiral phase. [d] E value determined
from the ee of the substrate and product.[20]
À
Keywords: alkaloids · asymmetric synthesis · C C coupling ·
.
À
C H activation · enzyme catalysis
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