New reagent for oxidative phenol coupling. The transformation of the monocyclic spermine base (S)-dihydroxyverbacine to the bicyclic alkaloid (S,S,S)-aphelandrine by cell free extract of barley seedlings

Article abstract of DOI:10.1016/S0040-4039(01)00670-0

The soluble protein fraction of barley seedlings (Hordeum vulgare, Gramineae) in the presence of O₂ stereoselectively catalyzes the intramolecular phenol coupling of the monocyclic spermine base (S)-dihydroxyverbacine to the bicyclic aphelandri

Full text of DOI:10.1016/S0040-4039(01)00670-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4139–4141
New reagent for oxidative phenol coupling. The transformation
of the monocyclic spermine base (S)-dihydroxyverbacine
to the bicyclic alkaloid (S,S,S)-aphelandrine by cell free
extract of barley seedlings
Lenka Nezbedov a´ , Manfred Hesse, Konstantin Drandarov and Christa Werner*
Organisch-chemisches Institut der Universit a¨ t Z u¨ rich, Winterthurerstrasse 190, 8057 Z u¨ rich, Switzerland
Received 16 March 2001; accepted 20 April 2001
Abstract—The soluble protein fraction of barley seedlings (Hordeum vulgare, Gramineae) in the presence of O stereoselectively
2
catalyzes the intramolecular phenol coupling of the monocyclic spermine base (S)-dihydroxyverbacine to the bicyclic aphelandrine
in preparative yield. The expected microsomal-bond cytochrome P-450 enzyme system does not contribute to this reaction. The
nature of the involved biocatalyst still remains uncertain. The catalytic potential of the cell free extract of barley seedlings suggests
its possible use as an efficient tool for large scale stereoselective chemoenzymatic synthesis of aphelandrine type alkaloids. © 2001
Elsevier Science Ltd. All rights reserved.
The oxidative phenol coupling contributes to the bio-
genesis of a number of natural compounds namely
alkaloids (benzylisoquinolines, Amarylidaceae alka-
loids, colchicine etc.), flavonoids, lignins, antibiotics
The macrocyclic spermine alkaloid aphelandrine (1) is
10–12
present in Aphelandra plants (Acanthaceae).
Recent
studies confirmed that the last step in the aphelandrine
(1) biosynthesis is the regioselective intramolecular phe-
nol oxidative coupling of its terminal precursor (S)-
(
vancomycine) etc. This reaction involves oxidative gen-
1
3
eration of phenolate radicals and their further intra- or
dihydroxyverbacine (2) (Scheme 1).
It was
intermolecular recombination to form new CꢀO and/or
demonstrated that in Aphelandra plants this reaction is
1
CꢀC bonds. Cytochrome P-450 enzymes often partici-
catalyzed by a membrane-bound cytochrome P-450
14
pate in these coupling reactions as highly stereo- and
enzyme, requiring NADPH and O2.
2
–4
regioselective biocatalysts but other oxidases, peroxi-
5–7
dases or laccases are also catalysts in certain cases.
The similarities in the benzofuran moieties in both
+)-hordatine A (4) and aphelandrine (1) motivated us
(
The polyamine alkaloid (+)-hordatine A (4), isolated
to introduce, instead of coumaroylagmatine (3), (S)-
dihydroxyverbacine (2) as a substrate for phenol cou-
pling in the presence of the cell free extract of barley
seedlings.
8
from barley (Hordeum vulgare L.), is a product of the
phenol oxidation reaction. Obviously, (+)-hordatine A
(
4) is formed by intermolecular coupling of two cou-
maroylagmatine (3) molecules (Scheme 1). Until now,
the enzyme involved in this reaction has not been well
established. Hordatine A (4) has been enzymatically
prepared from coumaroylagmatine (3) using cell free
15
(
−)-(S)-Dihydroxyverbacine (2), synthesized recently,
was used as a substrate for the enzymatic assays and
three different enzyme fractions from barley seedlings
were tested as biocatalysts. The enzyme fractions were
prepared as follows: 8-day-old seedlings were washed
9
extracts from the shoots of barley seedlings. The same
reaction catalyzed by a horseradish peroxidase leads to
8
the racemate. This is indirect evidence that the specific
(
222 g fr. wt.) and frozen for 1 h at −20°C. Roots were
peroxidases might be involved in this phenol
taken away and the upper parts were cut into small
pieces and homogenized in 300 ml of 0.1 M K/Na
phosphate buffer (pH 7.4), containing 0.6 M mannitol,
8
,9
coupling.
1
0 mM mercaptoethanol, 5 mM EDTA, 1 mM PMSF
*
0
Corresponding author. Fax: +1-635-68-12; e-mail: cwerner@
oci.unizh.ch
and 4 g Amberlite XAD-4. The homogenate was
filtrated through four layers of cheesecloth, and cen-
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00670-0

4
140
L. Nezbedo6 a´ et al. / Tetrahedron Letters 42 (2001) 4139–4141
Scheme 1.
trifuged for 20 min at 20 000g. The pellet was separated
from the supernatant, washed several times with buffer
Based on the results obtained with the enzymes from
14
Aphelandra squarrosa Nees roots, the formation of
aphelandrine (1) was expected in the microsomes (frac-
tion 2). The analysis of the enzymatic assay with the
microsomal fraction from the barley seedlings contain-
ing cytochrome P-450 did not show any formation of
aphelandrine (1). However, in the HPLC-ESI-MS of
the assay containing the soluble protein fraction 3 a
and used immediately or shock frozen in liquid N and
2
stored at −80°C until use (fraction 1). The supernatant
was centrifuged for 90 min at 200 000g to obtain the
microsomal pellet (fraction 2). After removing the
microsomes, the supernatant was concentrated by
ultrafiltration using 10K ultrafiltration membranes (sol-
uble protein fraction 3).
+
[M+H] quasi molecular peak at m/z 493 was detected
showing the same retention time as the natural aphe-
landrine (1) (m/z 493). Additionally, the MS/MS frag-
mentation pattern was identical with that of the natural
aphelandrine (1), used as a standard. This reaction was
strictly dependent on the presence of the protein. Heat
denatured enzyme did not yield any product formation.
A barley variety, devoid of endogenous hordatine A (4)
were also unable to produce (11S,17S,18S)-aphe-
landrine (1).
All three fractions were used for enzyme assays. The
enzyme assays for attempted phenol coupling reaction
contained 0.99 mM 2, 0.1 M K/Na phosphate buffer
(
pH 7.4) and 400 ml protein suspension in a total
volume of 500 ml. In the case of microsomes (fraction 2)
mM NADPH was added into the incubation mixture.
5
The mixtures were incubated for 1 h at 25°C with
gentle shaking in the presence of air. The reactions were
stopped by adding 100 ml AcOH in 20 ml MeOH and
centrifuged for 10 min at 3000g. The supernatants were
evaporated to dryness (30°C), the residues dissolved in
Thus, it was shown that, although being an unnatural
substrate, (S)-dihydroxyverbacine (2) was transformed
stereospecifically by the soluble protein fraction 3 into
(11S,17S,18S)-aphelandrine (1) in a surprisingly high
yield of 25% (determined by HPLC using aphelandrine
(1) as external standard). No other products where
detected in the enzymatic assay. The unchanged start-
ing (S)-dihydroxyverbacine is recoverable from the
reaction mixture.
0
.1 N aq. HCl and extracted ×5 with EtOAc. The water
layers were adjusted to pH 9 (K CO ) and extracted ×5
with CHCl . The combined organic extracts were dried
2
3
3
(
Na SO ), evaporated and analyzed by on-line coupled
2 4
HPLC–UV(DAD)–ESI-MS and by HPLC–UV(DAD)–
®
ESI-MS/MS (Waters Symmetry C column 150×2 mm
8
−
1
at 20°C; flow rate 0.2 ml min ; DAD detector setting
at 280 and 309 nm; mobile phase, A: 1% AcOH in H O,
2
solvent B: 1% AcOH in CH CN; linear gradient 97:3
Presumably both (S)-dihydroxyverbacine (2) and cou-
maroylagmatine (3) are substrates of one and the same
enzyme, present in the soluble fraction of the barley
homogenate. Thus, the transformation of (S)-dihydroxy-
verbacine (2) to (11S,17S,18S)-aphelandrine (1) is the
3
(
(
A:B) to 0:100 within 70 min; R (17S)-aphelandrine
t
1)=11.3 min; R (17R)-orantine=2.3 min). The ESI-
t
MS detector (ion trap instrument) was interfaced
directly to the output of the UV detector.

L. Nezbedo6 a´ et al. / Tetrahedron Letters 42 (2001) 4139–4141
4141
indirect evidence for the (S,S)-configuration of hor-
datine A (4). Unfortunately the absolute configuration
of hordatine A (4) is so far unknown.
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1
The present reaction opens the possibility for the
chemoenzymatic preparation of aphelandrine (1)-type
alkaloids. Barley seeds offer big advantages compared
with Aphelandra plants as they are easily available, the
material can be used 8 days after planting and the
reaction does not require co-factors as NADPH. The
whole cell-free extract from barley can be used for this
transformation without preliminary separation. Further
investigations in this direction are in progress.
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We thank the members of our mass department for
measuring the spectra, especially Dr. L. Bigler. This
work was supported by the Swiss National Science
Foundation, which is gratefully acknowledged.
.