Stereospecific synthesis of aszonalenins by using two recombinant prenyltransferases

Article abstract of DOI:10.1039/b902413a

In previous studies, two prenyltransferases were overproduced and characterised biochemically. AnaPT from Neosartorya fischeri is involved in the biosynthesis of acetylaszonalenin and was shown to catalyse the C3-prenylation of (R)-benzodiazepinedione (6)

Full text of DOI:10.1039/b902413a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Stereospecific synthesis of aszonalenins by using two recombinant
prenyltransferases
Wen-Bing Yin, Jun Cheng and Shu-Ming Li*
Received 4th February 2009, Accepted 6th March 2009
First published as an Advance Article on the web 3rd April 2009
DOI: 10.1039/b902413a
In previous studies, two prenyltransferases were overproduced and characterised biochemically. AnaPT
from Neosartorya fischeri is involved in the biosynthesis of acetylaszonalenin and was shown to catalyse
the C3-prenylation of (R)-benzodiazepinedione (6). CdpNPT from Aspergillus fumigatus catalysed the
N1-prenylation of different tryptophan-containing cyclic dipeptides. In this report, CdpNPT was found
to catalyse the C3-prenylation of 6 and its (S)-isomer (7). Interestingly, AnaPT and CdpNPT
introduced prenyl moieties from opposite sides of the indoline ring system. This feature was successfully
used for the chemoenzymatic synthesis of four aszonalenin stereoisomers by using 6 and 7 as substrates
and AnaPT and CdpNPT as catalysts. The stereoselectivity of the one-step reactions was about 100%
and the conversion rates reached 85–100%.
synthesis of aszonalenins is available in the literature. Here, we
Introduction
describe a novel strategy for stereospecific synthesis of all four
aszonalenin stereoisomers with a cis-configuration at positions C2
and C3 of the indoline ring by one-step reactions using purified
enzymes.
Aszonalenin (1) and its diastereomer epi-aszonalenin C (2) (Fig. 1),
as well as their N1-acetylated forms, have been identified in
different Aspergillus and Neosartorya strains.1–5 These compounds
belong to indole derivatives consisting of the amino acids trypto-
phan and anthranilic acid and carry a reverse prenyl moiety, i.e.
3¢-(3¢,3¢-dimethylallyl) (= 3¢-DMA), at position C3 of the indoline
ring. A five-membered ring is inserted between the indoline and
the seven-membered ring system. The absolute configuration of 1
and 2 was confirmed by chemical synthesis of a dihydro derivative
and by X-ray analysis of the acetylated form, respectively.6,7
Several natural products with similar structures are also found
in different fungi,5 e.g. roquefortine C in Penicillium roqueforti8
(3), amauromine in Amauroascus sp9 (4) and 5-N-acetylardeemin
(5) in Neosartorya fischeri var. brasiliensis10 (Fig. 1) as well as
epiamauromine in Aspergillus ochraceus9 and fructigenine A and
B in Penicillium fructigenum.11 All of these substances have a cis-
configuration between the two five-membered rings.
Results and discussion
In a previous study,5 we identified a biosynthetic gene cluster for
acetylaszonalenin from the fungus Neosartorya fischeri NRRL181
by genomic mining. A putative prenyltransferase gene from
this cluster, anaPT, was cloned and overexpressed in E. coli.
The overproduced recombinant protein AnaPT was purified to
homogeneity and characterised biochemically. AnaPT was found
to catalyse the prenylation of (R)-benzodiazepinedione (6) in the
presence of dimethylallyl diphosphate (DMAPP), resulting in the
formation of 1 with an absolute configuration of (2R,3S,11R)
(Scheme 1). As shown in Fig. 2A, the conversion of 6 to 1 was
nearly quantitative and only one enzymatic product was detected.
To get isomers of 1, we then synthesized the (S)-isomer 7 by
using L- instead of D-tryptophan (Experimental section) and
incubated it with AnaPT and DMAPP. As shown in Fig. 2B,
7 was also well accepted by AnaPT, with a conversion rate of
95.8%. Three enzymatic products with a ratio of 1.5:13.5:1 could
be detected at 8.5, 12.1, and 15.3 min, respectively. After isolation,
Due to their important biological activities,12,13 different strate-
gies were developed for the synthesis of roquefortine C,14 isoro-
quefortine C,15,16 amauromine17–19 and 5-N-acetylardeemin.18,19 In
these reports, many steps are essential for the desired compounds.
With the exception of (-)-dihydroaszonalenin,6 no report on the
Philipps-Universita¨t Marburg, Institut fu¨r Pharmazeutische Biologie,
Deutschhausstrasse 17A, D-35037 Marburg, Germany. E-mail: shuming.li@
Staff.uni-Marburg.de; Fax: +49-6421-2825365; Tel: +49-6421-2822461
1
the structures of the enzymatic products were elucidated by H
and 13C NMR (Tables 1 and 2) as well as by mass spectrometry.
Fig. 1 Examples of C3-prenylated indole derivatives from fungi.
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Scheme 1 Stereospecific chemoenzymatic synthesis of aszonalenins from benzodiazepinediones.
aszonalenin in this paper (Scheme 1). The two minor products
were identified as C2–3¢-DMA-(S)-benzodiazepinedione (8) and
N1–1¢-DMA-(S)-benzodiazepinedione (9), respectively (Fig. 3).
The presence of 8 and 9 could indicate that rearrangements could
be implicated in the formation of 2. It can be speculated that the
prenylation was taking place firstly at the N1-position of the indole
ring resulting in the formation of 9. 8 and 2 are rearrangement
products of 9, as proposed for 3-alkyl-1-allylindoles.20,21 In the
case of the natural substrate of AnaPT 6, the rearrangement steps
would be much more rapid than the prenylation, so only 1 was
detected in the incubation mixture. Incubation of 8 or 9 with
AnaPT in the presence or absence of DMAPP did not result in the
formation of any enzymatic products (data not shown), indicating
the independent formation of these compounds. However, the
possibility cannot be excluded that the conversion of 9 to 8 and
then to 2 takes place only as enzyme-bound intermediates. In
summary, the two known aszonalenin diastereomers 1 and 2 could
be successfully prepared from 6 and 7 by using AnaPT, respectively.
Theoretically, there are two further diastereomers with a cis-
configuration at positions C2 and C3, i.e. (2S,3R,11R)- (10)
and (2S,3R,11S)-aszonalenin (12) (Scheme 1). To obtain these
compounds, we carried out incubations with 6 and 7 in the
presence of CdpNPT and DMAPP. CdpNPT from Aspergillus
fumigatus was found to catalyse the reverse prenylation at position
N1 of the tryptophan-containing cyclic dipeptides.22 Only the
reversely N1-prenylated derivatives could be detected in the
reaction mixtures of cyclo-L-Trp-L-Trp, cyclo-L-Trp-L-Tyr, cyclo-
D-Trp-L-Tyr, cyclo-L-Trp-L-Phe, cyclo-L-Trp-L-Leu and cyclo-
L-Trp-Gly.22 In the reaction mixtures of cyclo-L-Trp-L-Pro and
cyclo-D-Trp-L-Pro, however, two additional products could be
detected in each (data not shown).22 One of them was identified
as the regularly prenylated derivative which was formed by
rearrangement of the reversely N1-prenylated derivative.22 The
structures of the second ones could not be determined in this
study. However, based on the information obtained from NMR
data (data not shown), we proposed that these compounds carry
probably a reverse prenyl moiety at position C3 and two fused
five-membered rings, similar to compounds shown in Fig. 1.
Considering the structural difference between cyclic dipeptides
containing proline and those containing other amino acids such
Fig. 2 HPLC analysis of enzymatic reaction mixtures of 6 with AnaPT
(A), 7 with AnaPT (B), 6 with CdpNPT (C), and 7 with CdpNPT (D).
The NMR spectra of the major product at 12.1 min corresponded
very well to those of epi-aszonalenin C (2),7 with a configuration
of (2R,3S,11S).
Because a retention of the configuration at position C11 was
expected during the prenyl transfer reaction, this compound
was identified as epi-aszonalenin C, termed as (2R,3S,11S)-
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Table 2 1H and 13C NMR data of the side products (in CDCl3) during the production of aszonalenins
Compound
Position
8
9
11
dH, multi., J in Hz
dC
dH, multi., J in Hz
dC
dH, multi., J in Hz
N-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
8.01, s
–
–
7.38, d, 7.9
7.05, t, 7.4
7.15, t, 7.6
7.32, d, 8.2
–
–
–
–
–
134.2
104.8
117.7
120.7
121.9
110.8
141.1
129.3
23.5
7.09, s
–
7.49, d, 7.9
7.08, t, 7.4
7.20, t, 7.8
7.30, d, 8.2
–
127.1
107.6
118.3
119.3
121.8
109.9
136.4
127.9
24.2
7.09, s
–
7.49, d, 8.0
7.08, m
7.21, t, 6.9
7.29, m
–
–
–
–
3.48, dd, 15.6, 6.8
3.44, dd, 15.6, 4.6
4.21, dt, 9.8, 5.6
6.10, d, 4.1
–
3.50, dd, 15.2, 5.7
3.22, dd, 15.2, 8.2
4.13, dt, 13.4, 5.5
6.08, d, 4.8
–
3.49, dd, 15.2, 6.4
3.21, dd, 15.1, 7.3
4.12, dt, 13.4, 6.3
6.02, d, 4.8
–
C-11
C-13
C-14
C-15
N-16
C-17
C-18
C-19
C-20
C-21
C-1¢
52.3
168.2
125.3
135.3
–
171.8
119.9
133.0
125.3
131.6
112.9
52.0
168.3
123.3
135.3
–
171.5
120.8
133.1
125.3
131.6
44.2
–
–
–
7.56, s
–
7.75, s
–
6.96, d, 7.9
7.50, td, 7.6, 1.3
7.26, t, 7.3
7.91, dd, 7.9, 1.3
4.65, d, 6.9
7.75, s
–
6.95, d, 8.0
7.50, td, 7.6, 1.3
7.26, m
7.91, d, 7.3
4.66, d, 6.7
6.96, d, 8.2
7.50, td, 7.9, 1.3
7.23, t, 7.6
7.85, dd, 7.9, 1.3
5.22, d, 18.0
5.19, d, 10.4
6.18, dd, 17.5, 10.4
–
C-2¢
C-3¢
C-4¢
C-5¢
146.1
38.9
27.7
27.7
5.36, t, 6.9
–
1.76, s
1.82, s
119.8
124.8
25.6
5.35, t, 6.0
–
1.76, s
1.82, s
1.54, s
1.55, s
18.1
For numbering please see Fig. 3.
Fig. 3 Structures of side products formed during the production of aszonalenins.
as tyrosine, phenylalanine, glycine or leucine, it could be speculated
that the fused ring system in the structures of cyclo-L-Trp-L-Pro
and cyclo-D-Trp-L-Pro could be responsible for the formation of
the C3-prenylated derivatives, which were absent in the incubation
mixtures of the other tryptophan-containing cyclic dipeptides.
Therefore, we expected that prenylation of 6 and 7 by CdpNPT
would produce 1 and 2 as in the case of AnaPT or their isomers
10 and 12, respectively. As shown in Fig. 2C and D, both 6 and
7 were accepted well by CdpNPT. In the case of 6, a conversion
rate of 84.8% could be reached and two products with a ratio of
10:1 were detected at 12.1 and 15.3 min, respectively (Fig. 2C).
The minor product of 6 was identified as N1–1¢-DMA-(R)-
benzodiazepinedione (11) (Table 2, Fig. 3). The 1H and 13C NMR
data of the predominant product 10 differed clearly from those
of 1. With an exception for the signal of NH-16, the spectra of
10 were almost identical to those of 2 (Table 1). In the case of 7,
NMR spectra of the enzymatic product 12 were almost identical
to those of 1 (Table 1).This indicates that the two products 10
and 12 could be enantiomers of 2 and 1, respectively, if the
stereochemistry at position C11 remained unchanged during the
prenylation. To confirm the stereochemistry of 10 and 12, CD
spectra of the four enzymatic products as well as those of the
two substrates were taken in ethanol and compared to each other
(Fig. 4). Fig. 4 shows clearly that the substance pairs 6 and 7, 1 and
12 as well as 2 and 10 have opposite Cotton effects. This proved
unequivocally that they are enantiomers of each other. 10 and 12
have not been isolated from nature yet. The results provided in
this study also proved that both AnaPT and CdpNPT catalyse the
formation of indoline derivatives carrying fused five-membered
rings with a cis-configuration. However, they introduce the ring
system from opposite sides. They are therefore complementary to
each other with regard to prenylation, which expands their usage
in chemoenzymatic synthesis.
1
the conversion rate was estimated to be 99.4%. The H and 13C
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Bacterial strains, plasmids and cultural conditions
Plasmids pWY225 and pHL525 were used for overproduction of
AnaPT and CdpNPT, respectively.
Escherichia coli XL1 Blue MRF¢ (Stratagene, Heidelberg,
Germany) was used for overexpression experiments and grown
in liquid or on solid Luria-Bertani medium with 1.5% (w/v) agar
◦
at 37 C. Carbenicillin (50 mg mL-1) was used for selection of
recombinant E. coli strains.
Preparation of AnaPT and CdpNPT
For preparation of AnaPT, E. coli XL1 Blue MRF¢ cells har-
bouring pWY22 were induced by 0.5 mM of IPTG at 37 ◦C.
His6-AnaPT was purified with Ni-NTA agarose to homogeneity
as judged by SDS-PAGE and a protein yield of 25 mg of purified
His6-tagged AnaPT per litre of culture was obtained.5
For preparation of CdpNPT, E. coli XL1 Blue MRF¢ cells
harbouring pHL5 were induced by 1 mM of IPTG at 37 ◦C. His6-
CdpNPT was purified with Ni-NTA agarose to homogeneity as
judged by SDS-PAGE and a protein yield of 5 mg of purified
His6-tagged CdpNPT per litre of culture was obtained.25
HPLC conditions for analysis and isolation of synthetic products
Fig. 4 CD spectra of reported compounds.
The enzymatic products of the incubation mixtures of AnaPT and
CdpNPT were analysed by HPLC on an Agilent series 1200 by
using a LiChrospher RP 18–5 column (125 ¥ 4 mm, 5 mm, Agilent)
at a flow rate of 1 mL min-1. 50% methanol in water (solvent A)
and methanol (solvent B) were used as solvents. For analysis of
enzymatic products, a linear gradient of 10–50% (v/v) solvent B
in solvent A in 15 min was used. The column was then washed
with 100% solvent B for 5 min and equilibrated with 10% (v/v)
solvent B for 5 min. Detection was carried out by a Photo Diode
Array detector and illustrated at 254 nm.
For isolation, the same HPLC equipment with a Multospher
120 RP-18 column (250 ¥ 10 mm, 5 mm, C + S Chromatographie
Service, Langenfeld, Germany) was used. A linear gradient of 30–
70% (v/v) solvent B in A in 12 min at a flow rate of 2.5 mL min-1
was used. The column was then washed with 100% solvent B for
8 min and equilibrated with 30% (v/v) solvent B for 5 min.
Conclusions
In conclusion, we have presented a new strategy for the synthesis
of four aszonalenin stereoisomers in one-step reactions by using
two soluble prenyltransferases, which could be easily produced
in E. coli and purified to homogeneity in high yields. The
stereoselectivity of the reactions was approximately 100% and the
reaction yields were found to be between 85% to 100%. More
importantly, AnaPT and CdpNPT accepted the same substrates,
but introduced the five-membered ring from different sides. As
a result, two compounds with different stereochemistry could
be obtained from one substrate by using different enzymes.
The results reported in this study provide an additional exam-
ple of chemoenzymatic synthesis or enzyme-catalysed chemical
transformations, which are now widely recognised as practical
alternatives to traditional organic synthesis and as convenient
solutions to certain intractable synthetic problems.23 The approach
described in this study could also find use in the synthesis of
C3-prenylated natural products, e.g. roquefortine D, amauromine,
fructigenines A and B by using AnaPT and the respective cyclic
dipeptides.
Circular dichroism spectra
The samples were dissolved in ethanol. CD spectra were recorded
on a JASCO J-810 CD spectropolarimeter by using Spectra
Manager Software (JASCO Inc). Spectral data were collected from
400 to 200 nm with a data pitch of 0.1 nm. A band width of 1 nm
was used with a detector response time of 0.5 sec. and scanning
speed of 200 nm/min. Each spectrum or data point was acquired
5 times, and mean values were used.
Experimental section
Chemicals
Synthesis of benzodiazepinediones
Dimethylallyl diphosphate was prepared according to the method
described for geranyl diphosphate by Woodside.24 D- and L-
tryptophan were purchased from Roth (Karlsruhe, Germany).
Isatoic anhydride was purchased from Fluka (Steinheim, Ger-
many).
(R)- (6) and (S)-benzodiazepinedione (7) were synthesized by
using D- or L-tryptophan, triethylamine and isatoic anhydride
according to the method described by Barrow and Sun,13 respec-
tively.
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Enzymatic synthesis of aszonalenins
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The reaction mixtures containing 6 or 7 (1 mM), DMAPP
(1 mM), CaCl2 (10 mM), Tris-HCl (50 mM, pH 7.5), glycerol
1.5% (v/v), AnaPT (0.25 mM) or CdpNPT (0.38 mM) were
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NMR spectroscopy, H-H-COSY, H-C-COSY as well as positive
and negative electrospray ionization (ESI) mass spectrometry
with a ThermoFinnigan TSQ Quantum. The mass spectrometer
was coupled with an Agilent HPLC series 1100 equipped with
a RP18-column (5 mm, 2 ¥ 250 mm, 5 mm). For separation,
the column was run with 10% (v/v) solvent B (CH3OH) in
solvent A (H2O) (each containing 0.1% (v/v) HCOOH) for
5 min, followed by a gradient from 10% (v/v) to 100% B over
30 min. After washing with 100% B for 10 min, the column was
equilibrated with 10% (v/v) B for 10 min. The flow rate was at
0.2 mL min-1.
From the incubation mixture of AnaPT with 6.1 mg of 6, 5.5 mg
of 1 was isolated. From the incubation mixture of AnaPT with
61 mg of 7, 40 mg of 2, 5 mg of 8 and 3 mg of 9 were isolated. 8 mg
of 10 and 0.8 mg of 11 were obtained from the incubation mixture
of CdpNPT with 12.2 mg of 6. 10 mg of 12 was obtained from the
incubation mixture of CdpNPT with 12.2 mg of 7. Positive and
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1: m/z: 374.3 ([M + 1]+), 372.5 ([M - 1]-); 2: m/z: 746.9 ([2M + 1]+),
374.2 ([M + 1]+), 744.8 ([2M - 1]-), 372.2 ([M - 1]-); 7: m/z: 611.1
([2M + 1]+), 306.3 ([M + 1]+); 8: m/z: [M + 1]+, 374.2, [M - 1]-,
372.1; 9: m/z: 374.3 ([M + 1]+), 372.2 ([M - 1]-); 11: m/z: 374.1
([M + 1]+), 396.1 ([M + Na]+);.12: m/z: 374.2 ([M + 1]+), 372.7
([M - 1]-).
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This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (SPP 1152: Evolution of metabolic di-
versity, to S.-M. Li). We thank Mark Schlegel for his help to take
the CD spectra.
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