Direct biosynthetic cyclization of a distorted paracyclophane highlighted by double isotopic labelling of l-tyrosine

Article abstract of DOI:10.1039/c5ob00114e

The biosynthesis of pyrrocidines, fungal PK-NRP compounds featuring a strained [9]paracyclophane, was investigated in Acremonium zeae. We used a synthetic l-tyrosine probe, labelled with oxygen 18 as a reporter of phenol reactivity and carbon 13 as a tracer of incorporation of this exogenous precursor. The (¹⁸O)phenolic oxygen was incorporated, suggesting that phenol behaves as a nucleophile during the formation of the bent aryl ether.

Full text of DOI:10.1039/c5ob00114e

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Direct biosynthetic cyclization of a distorted
paracyclophane highlighted by double isotopic
Cite this: DOI: 10.1039/x0xx00000x
labelling of
L
-tyrosine†
a
a
b
a
Alexandre Ear, Séverine Amand, Florent Blanchard, Alain Blond, Lionel
a
a
a
Received 00th January 2012,
Accepted 00th January 2012
Dubost, Didier Buisson* and Bastien Nay*
DOI: 10.1039/x0xx00000x
The biosynthesis of pyrrocidines, fungal PK-NRP compounds featured by a strained
9]paracyclophane, was investigated in Acremonium zeae. We used a synthetic -tyrosine
probe, labelled with oxygen 18 as a reporter of phenol reactivity and carbon 13 as a tracer of
incorporation of this exogenous precursor. The ( O)phenolic oxygen was incorporated,
suggesting that the phenol behaves as a nucleophile during the formation of the bent aryl ether.
[
L
www.rsc.org/
1
8
Introduction
clues to the mixed polyketide–non ribosomal peptide nature of
4
(Scheme 1), originated from nine acetate units (AcCoA), five
The biosynthetic macrocyclization of bent [
n
]paracyclophanes methyls from
S
-adenosylmethionine (SAM) and one -tyrosine
L
6
is a challenging problem in biosynthetic studies as bending (Tyr). However the complexity of the mechanisms leading to
results in highly energetic benzene rings. A reasonable the full carboheterocyclic core of these compounds still
hypothesis suggests the transient loss of aromaticity during the questions us about the cyclization of the distorted
cyclization, followed by rearomatization and distortion [9]paracyclophane from a linear
triggered by tautomery or dehydration. This mechanism has constructed by
PKS-NRPS enzymatic complex.
been suggested for the biosynthesis of haouamine whose bent particular, the role of the phenolic oxygen in the closure of the
7]paracyclophane would be formed after oxidative phenol strained paracyclophane was unknown.
N-acyl tyrosine precursor
7,8
a
In
[
1
coupling. We report the first evidence that the phenolic oxygen
of tyrosine is directly involved in the formation of the bent
Results and Discussion
2
-4
[
9]paracyclophane of pyrrocidines 1-3 (Scheme 1), possibly
through enzymatic templation and without breaking the Biosynthetic mechanism hypotheses
aromatic character of the phenol ring.
With pyrrocidine A (
for the cyclization of linear precursor
1
), two mechanisms can be hypothesized
, ultimately leading to
5
benzene ring distortion in the [9]paracyclophane (Scheme 2),
either head-to-tail through remote activation at C-18, route (a),
or tail-to-head through oxidative activation of the double bond
at C-6–C-7, route (b), both leading to the electrophilic
9
cyclization of ring C. In route (a), a nucleophilic quench of the
allylic cation
the paracyclophane, while intramolecular Diels-Alder (IMDA)
reaction and C-H oxidation at C-2’ in would complete the
biosynthesis of . In route (b), oxidation of the phenol ring
would result in an electrophilic quinone methide ( ) whose
6 by the phenol would proceed with formation of
7
1
8
attack by the hydroxyl at C-6 would allow bypassing the
drawbacks of benzene distortion through a transient tetrahedral
Scheme 1 Structures of pyrrocidines A-C (
blocks of GKK1032A2 ( , in box).
1-3) and the biosynthetic building
ketal carbon in
9
. In this case, the aromaticity of the
3' hydride shift
4
paracyclophane would be recovered by the 2'
ꢀ
before final hydration at C-2'. It is worth noting that these
routes may also involve radical species. Obviously, the
An early biosynthetic study was performed on the parent
5
GKK1032A2 compound
4 isolated from a Penicillium, using
6
extensive isotopic labelling. With this work, Oikawa gave
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DOI: 10.1039/C5OB00114E
Journal Name
phenolic oxygen is conserved in route (a) but not in route (b)
where it is lost at the rearomatization step.
18OH
18OMe
B(OH)
2
blue light
[Ru(bpy) Cl ] H O
3
2
2
MeI, KOH
18O
2
, iPr NEt, DMF
2
DMSO
93%
H
H
9% (86% 18O
10 CHO
8
11 CHO
12 CHO
2'
enrichment)
NH
1
1'
O
18OMe
18OMe
H
BocHN 13COOH
4
3
2
O
(
b) [O]
(a)
[O]
6
5
13
15
, MeOH
quant.
(ee>99%)
O
1
8
(b) Tail-to-head
(a) Head-to-tail
Ac O, AcONa
then MeOH
8% b.r.s.m.
H
2
2
H
13CO
Me
13CO
NHAc
5
2
2
Me
4
3'
H
H
2'
2'
H
BI
14 NHAc
16
AH
NH
NH
O
HO
18OH
3
O
3
O
1) HBr (5N), NaI, reflux
2) HCl (6N), reflux
HO
6
5
5
O
6
O
TfO
P
Rh
1
3
16
13
16
quant.
P
1
8
18
1
3
CO H
2
(
ee>99%)
15
8
6
H
NH
3
Cl 17
1
2
) macrocyclization
) IMDA reaction
[O]
Scheme 3 Enantioselective synthesis of isotopically labelled L-tyrosine (17).
H
3'
3'
O
H
[H
2
O]
2'
H
AH
2'
O
NH
O
NH
O
6
O
6
Culture conditions for the optimal production of pyrrocidines
and crystallographic evidence of aromatic distortion
2
'-H-shift
[O] at 2'
H
H
3
H
C
H
5
1
5
O
O
then H
addition
2
O
B
A
The plant associated fungus Acremonium zeae NRRL 13540
H
9
H
7
3
(
syn. A. strictum
medium containing glucose and ground corn leaves. The three
pyrrocidines A-C ( ) were identified in the fungal extracts by
LC-HRMS and LC-HRMS/MS experiments. Supplementing
the medium with pulsed additions of unlabelled -tyrosine (3
additions at 48h, 72h and 96h before stopping the culture at
68h) greatly enhanced their production by ca. 10-fold and
allowed isolation of . The separation of and was not
initially possible in our chromatographic conditions. However,
) was grown in a plant-mimicking liquid
Scheme 2 Two hypothetic scenarios for the electrophilic cyclization leading to
pyrrocidine A ( ): (a) head-to-tail and (b) tail-to-head, following oxidative
cyclization of linear precursor into or , respectively.
1
1-3
5
6
8
3
a
L
To distinguish between these two routes, biosynthetic
experiments in the presence of labelled -tyrosine were
envisaged. Our strategy relied on a double labelling to test, in a
one-shot experiment, the possibility of these hypotheses.
Oxygen 18 was used to report on the phenol reactivity while
carbon 13 allowed tracing at the same time the incorporation of
-tyrosine in pyrrocidines, especially in the
eventuality O would be lost. Since such doubly labelled
tyrosine (17) was not commercially available, an isotope-
incorporating enantioselective total synthesis was undertaken.
L
1
3
1
2
1
3
1
was spontaneously converted into
for separation of from the epoxide
analysis of could be performed after crystallization from
3
upon storage, allowing
2
3. X-ray crystallographic
doubly labelled
L
1
8
3
L
-
1
4
CH CN (Figure 1), showing two molecules per asymmetric
3
unit, each exhibiting a [9]paracyclophane and displaying
different bending angles ꢀθ of 12.26° and 12.64° (ꢀθ
θ1).
= θ2 –
Synthesis of doubly labelled L-tyrosine
A key photoredox oxidation of commercially available 4-
formylphenylboronic acid 10 was performed in the presence of
[
4
Ru(bpy) Cl ]•6H O under blue led visible light irradiation at
3 2 2
1
8
10
20 nm (Scheme 3), using 97.1% enriched O (1 atm). The
2
1
8
resulting (4- O)-4-hydroxybenzaldehyde 11 was obtained with
1
8
8
6%
methylated into (4- O)-4-anisaldehyde 12 in 83% yield over
two steps. Knoevenagel reaction between 12 and -Boc-(1-
C)glycine 13 in the presence of Ac O and NaOAc, followed
O-enrichment due to slight air-dilution and was
1
8
N
Figure 1 X-ray crystallographic drawing of 3 (only one among the two molecules
per asymmetric unit is shown), showing the bent [9]paracyclophane.
1
3
2
by quenching with methanol, directly gave the
N-acetyl-p-
1
8
13
11
(
O)methoxy-(1- C)dehydrophenylalanine methyl ester 14
The enantioselective hydrogenation of 14 was undertaken
quantitatively in the presence of [( )-Et-
Duphos]Rh(cod)(OTf) 15 yielding -acetyl- -( O)methoxy-
-phenylalanine methyl ester 16 with >99%
enantiomeric excess. At last, deprotection in acidic media
provided doubly labelled -tyrosine hydrochloride 17 in
quantitative yield as a sole enantiomer (ee>99%), with 86%
.
In vivo isotopic labelling of pyrrocidines
The reporting amino acid 17 was added to a spore-seeded
culture broth of A. zeae, using the pulsed protocol described
before. After one week of culture, the mycelial ethyl acetate
extract was analyzed by UHPLC-MS under atmospheric
S,S
1
2
18
,
N
p
1
3
(
1- C)-L
pressure chemical ionization (APCI). In preliminary
a
L
1
8
experiment, it was observed that exogenous L-tyrosine (i.e.
labelled) was predominantly used in this biosynthesis. This
O
1
3
and 99% C enrichments.
2
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ART14ICLE
DOI: 10.1039/C5OB001 E
result was duplicated and showed incorporation of the labelled Possible biosynthetic mechanisms of the head-to-tail route
phenolic oxygen in the natural products, with m/z incremented
Even though paracyclophane cyclization could be easier on a
flexible intermediate like 18 which is the product of the PKS-
NRPS complex, it is most probable that the γ-lactam 19 is the
effective substrate for the cyclization (Scheme 5). Indeed it
by 3 amu for
1 and 2 at 491 and 493, respectively (pyrrocidine
C was not observed), corresponding to the protonated adducts
+
[
M+H] (Figure 2).
would be formed by a specialized enzyme, as hypothesized for
1
6
cytochalasins, catalyzing the Knoevenagel reaction of 18. In
cytochalasins, this enzyme also catalyzes an IMDA reaction
which is not possible in 19 due to the different topology and
reactivity of olefins. Instead, an oxidative polycyclization is
likely to occur after oxidation by a cytochrome P450 or a non-
heme iron(II) oxygenase. An electrophilic process involving a
cation like 21a (4,5-saturated) or 21b (4,5-unsaturated) could
lead to the paracyclophane 22a or 22b, respectively. The
cationic intermediates 21 could arise either from the terminal
oxidation of 19 at C-18 into the allylic alcohol 20 (Scheme 5,
pathway a), or from epoxidation of internal olefin C-6=C-7 into
2
3
and electrophilic cyclization into 24, then OH elimination
(
Scheme 5, pathway b). At last, a radical mechanism cannot be
excluded through phenolic radical 25a or 25b. The mechanism
involving radical species would involve H-abstraction at the C-
1
8 allylic position of the linear intermediate 25 (Scheme 5,
Figure 2 UHPLC-MS(APCI) of the labelling experiment performed in A. zeae,
1
8
13
17
showing incorporation of O and C into pyrrocidines A and B. Left: total ion pathway c).
chromatogram (TIC); Right: full MS spectra. (a) Control experiment; (b) Labelling
experiment showing production of both unlabelled and labelled pyrrocidines.
1
8
13
This ( O, C)-labelling pattern (Figure 2b) strongly supports
the hypothesis of a phenolic attack at C-6 on the acyl chain, as
suggested by the head-to-tail route (a) in Scheme 2 and thus the
direct formation of the paracyclophane, without the loss of
HN
1
4
6
18
3
2
H
5
7
O
O
18a/b
enzymatic Knoevenagel reaction
O
1
8
[O]
(
b)
(a) [O]
aromaticity for the phenol, resulting in the conservation of
O
HN
1
4
6
18
H
(
Scheme 4). However, a deeper analysis of the isotope
3
2
5
7
O
distribution of products showed irregular relative intensities of
peaks at m/z 488 (100) and 489 (relatively 62 observed for 34
expected for the isotope pattern of unlabelled natural
pyrrocidine A). While it is highly improbable that both
mechanisms co-occur during this biosynthesis, this increased
peak at m/z 489 may sign the loss of oxygen 18. In fact, this
O
19a/b
(
c)
(
a) allylic oxidation at C-18 (b) 6,7-epoxidation
(c) hydrogen
abstraction
at C-18
AH
O
H
6
7
H
O
18
NH
NH
H
O
4
23a/b
BI
4
O
O
1
8
HA
situation was logically explained by the 86% O enrichment of
6
6
7
O
O
cyclization
O
5
5
1
6
18
our doubly labelled tyrosine, containing 14% of the
O
H
H
AH
O
isotopomer, leading to higher peak intensity at m/z 489 for the
20a/b
ation
25a/b
O
Fe-O
1
3
labelled ( C)pyrrocidine A. Indeed simulation with an isotope
1
5
H
24a/b
distribution calculator (mMass), using the isotope enrichment
of precursor 17 and considering that is produced at a 71%
level of (as seen in Figure 2a), allowed reproducing and thus
demonstrating the origin of relative intensities of peaks at 488
100) and 489 (relatively 66 as simulated, Figure S3, Table S1).
NH
O
1
2
H O
2
O
[O]
H
(
2
1a/b
NH
O
5
H
cyclization
18OH
O
OH
NH
4
O
13C
C
A. zeae
18O
O
H
H
Me
H
H
O
22a (4,5-saturated)
22b (4,5-unsaturated)
IMDA
13CO
H
Me
Me
(18O,13C)-pyrrocidines
[O]
2
H
pyrrocidine A (1)
H
NH
3
Cl
Scheme 5 Hypothetical biosynthetic pathways involving a head-to-tail cyclization
compounds : 4,5-saturated; compounds : 4,5-unsaturated).
Me
1
7
(
a
b
Scheme 4 In vivo incorporation of labelled
L
-tyrosine 17 into pyrrocidines.
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Owing to the disfavouring
π
-bond polarization of the dienoyl
1
2
M. Matveenko, G. Liang, E. M. W. Lauterwasser, E. Zubia and D.
Trauner, J. Am. Chem. Soc., 2012, 134, 9291.
moiety in 20b and 25b, it is questionable if C-6 is electrophilic
toward the phenol, especially in pathways a and c. It suggests
that the bond C-4–C-5 in 20a and 25a could be saturated up to
the macrocyclization step. In such case, once the
(a) H. He, H. Y. Yang, R. Bigelis, E. H. Solum, M. Greenstein and G.
Carter, Tetrahedron Lett., 2002, 43, 1633; (b) R. Bigelis, H. He, H.
Y. Yang, L.-P. Chang and M. Greenstein, J. Ind. Microbiol.
Biotechnol., 2006, 33, 815; (c) H. He, H. Yang and R. Bigelis, Patent
US7129266B2 (2006).
paracyclophane has been formed, an additional
β-oxidation of
2
2a into the -enoyl derivative 22b would release a strained
E
and very reactive bridgehead dienophile (anti-Bredt olefin)
prompt to click with the diene in the IMDA reaction.
3
(a) D. T. Wicklow, S. Roth, S. T. Deyrup and J. B. Gloer, Mycol.
Res., 2005, 109, 610; (b) D. T. Wicklow, S. M. Poling and R. C.
Summerbell, Can. J. Plant Pathol., 2008, 30, 425; (c) D. T. Wicklow
and S. M. Poling, Phytopathology, 2009, 99, 109.
Conclusions
4
5
Y. Shiono, K. Shimanuki, F. Hiramatsu, T. Koseki, M. Tetsuya, N.
Fujisawa and K. Kimura, Bioorg. Med. Chem. Lett., 2008, 18, 6050.
K. Fumito, H. Atsuhiro, A. Katsuhiko, O. Tatsuhiro and H.
Mitsunobu, Patent JP 2001247574 (2001).
We demonstrate for the first time that
a
distorted
paracyclophane can directly be formed during a biosynthetic
1
8
process, especially through the key O-labelling of the phenol
oxygen which was conserved during the experiment. Such
6
7
H. Oikawa, J. Org. Chem., 2003, 68, 3552.
1
3
tyrosine reporter, also provided with a C tracer in case of
oxygen loss, may be useful to study the biosynthesis of other
(a) C. Hertweck, Angew. Chem. Int. Ed., 2009, 48, 4688; (b) D.
Boettger and C. Hertweck, ChemBioChem, 2013, 14, 28.
X.-W. Li, A. Ear and B. Nay, Nat. Prod. Rep., 2013, 30, 765.
1
8
19
mixed polyketides like macrocidins or cyclopeptides like
8
9
2
0
21
pandamine or mauritine A. Pyrrocidines and structurally
related compounds have been described in several fungal
As for the first cyclization of ring C in
7 and 8, both mechanisms
have been supported by synthetic works on the decahydrofluorene
core: (a) Head-to-tail, route (a) through a terminal allylic alcohol at
C-18: X.-W. Li, A. Ear, L. Roger, N. Riache, A. Deville and B. Nay,
Chem. Eur. J., 2013, 19, 16389; (b) Tail-to-head, route (b) through an
internal epoxide at C-6–C-7: K. C. Nicolaou, D. Sarlah, T. R. Wu and
W. Zhan, Angew. Chem. Int. Ed., 2009, 48, 6870.
8
genus. These biologically active compounds form a large
family of secondary metabolites, sharing common features with
cytochalasans (PKS-NRPS nature, polycyclic skeleton, linear
biosynthetic precursors) and thus deserving extensive studies to
understand their role and origin in nature. Our biosynthetic
study shows again how Nature is capable of exploiting the
molecular properties of reactive intermediates, sometimes up to
an extreme degree of efficiency as observed with the formation
of this distorted paracyclophane, to build an armada of
biologically active compounds.
1
1
1
1
0 Y.-Q. Zou, J.-R. Chen, X.-P. Liu, L.-Q. Lu, R. L. Davis, K. A.
Jørgensen and W.-J. Xiao, Angew. Chem. Int. Ed., 2012, 51, 784.
1 M. Takeda, J. Jee, A. M. Ono, T. Terauchi and M. Kainosho, J. Am.
Chem. Soc., 2009, 131, 18556.
2 M. J. Burk, J. E. Feaster, W. A. Nugent and R. L. Harlow, J. Am.
Chem. Soc., 1993, 115, 10125.
Acknowledgements
3 Spontaneous formation of
3
was observed from a mixture of
1 and 2,
We thank the Agricultural Research Service (NRRL collection,
USA), for providing us with Acremonium zeae NRRL 13540.
We thank Dr. Virginie Ratovelomanana-Vidal and Pierre-
Georges Echeverria from ENSCP (France) for hosting and
technical assistance in the asymmetric hydrogenation of 13, and
Ms. Assia Hessani from University Paris Descartes for HRMS.
This work was supported by the ANR (grant number ANR-12-
BS07-0028-01, SYNBIORG) and CNRS (interdisciplinary call
Physic-Chemistry-Biology 2011). NMR was funded by Région
Ile-de-France, MNHN and CNRS. We thank the French
Ministry of Research and the University Pierre et Marie Curie
Paris 6 (ED406) for granting AE with a PhD fellowship.
with concomitant disappearance of
1
, when stored in the freezer for
several months. This conversion had previously been observed by
Wicklow and co. (ref. 10c). Compound3 may arise from oxidation of
1
involving ground-state triplet oxygen, through a reactive biradical
intermediate leading to peroxide II. Such a mechanism was
suggested by Bartlett and Banavali for the oxygenation of strained
I
olefins: P. D. Bartlett and R. Banavali, J. Org. Chem., 1991, 56
,
6
043.
1
4 Crystallographic data for compound
3
were deposited with the
Cambridge Crystallographic Data Centre as number CCDC 1031563.
Drawings in Figure 1 were generated using the Mercury software.
5 The mMass calculator is available at: http://www.mmass.org/
Notes and references
Muséum National d’Histoire Naturelle and Centre National de la
a
1
1
1
Recherche Scientifique (joint unit UMR 7245 CNRS-MNHN), 57 rue
Cuvier (CP 54), 75005 Paris, France.
(downloaded on 16/01/2015).
6 R. Fujii, A. Minami, K. Gomi and H. Oikawa, Tetrahedron Lett.
2013, 54, 2999.
,
Corresponding authors: buisson@mnhn.fr, bnay@mnhn.fr
b
Institut de Chimie des Substances Naturelles (ICSN, CNRS). 1, Avenue
7 Such oxidations are common in the secondary metabolism, especially
with P450 enzymes which even can catalyze several successive
oxidations on the same substrate: L. M. Podust and D. H. Sherman,
Nat. Prod. Rep., 2012, 29, 1251.
de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
†
Electronic Supplementary Information (ESI) available: Experimental
1
13
procedures, characterization data, and H and C NMR spectra of
products. See DOI: 10.1039/c000000x/
4
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1
1
2
2
8 P. R. Graupner, A. Carr, E. Clancy, J. Gilbert, K. L. Bailey, J.-A.
Derby and B. C. Gerwick, J. Nat. Prod., 2003, 66, 1558.
9 D. C. Gournelis, G. G. Laskaris and R. Verpoorte, Nat. Prod. Rep.
,
1
997,14, 75.
0 M. Pais, F. X. Jarreau, X. Lusinchi and R. Goutarel, Ann. Chem.
Paris), 1966, 14, 83.
1 R. Tschesche, H. Welhelm and H.-W. Fehlhaber, Tetrahedron Lett.
972, 13, 2609.
(
,
1
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Graphical and short textual abstracts for
Direct biosynthetic cyclization of a distorted paracyclophane highlighted by double
isotopic labelling of L-tyrosine
Alexandre Ear, Séverine Amand, Florent Blanchard, Alain Blond, Lionel Dubost, Didier
Buisson and Bastien Nay
The biosynthesis of pyrrocidines was investigated using a double labelling of L-tyrosine
1
8
obtained by enantioselective total synthesis. It was found that the phenolic ( O)oxygen is
conserved all over the cyclization process, suggesting that the bent paracyclophane core is
directly installed during the biosynthesis through aryl ether bond formation.