Enantioselective synthesis of the unnatural enantiomers of the fungal sesquiterpenoids acorenone and trichoacorenol

Article abstract of DOI:10.1002/ejoc.201100688

The volatiles released by several strains of Trichoderma fungi were collected using a closed-loop stripping apparatus and analysed by GC-MS. Most of the investigated strains released the two structurally related spirocyclic sesquiterpenoids trichoacorenol, previously identified in Trichoderma koningii (Huang et al., 1995), and acorenone. A new enantioselective synthesis of the enantiomer of the natural spirocyclic sesquiterpene acorenone and the first enantioselective synthesis of the related compound ent-trichoacorenol have been completed by a chiral-pool approach starting from (+)-(R)-pulegone using ring-closing metathesis as the key step. The absolute configuration of natural trichoacorenol from Trichoderma harzianum sp. 714 has been assigned by chiral GC-MS analysis and is the same as that in T. koningii. A new enantioselective synthesis of the unnatural enantiomers of the two spirocyclic sesquiterpenes acorenone and trichoacorenol has been accomplished from (+)-(R)-pulegone using ring-closing metathesis in a key step. The synthetic material has been used to determine the absolute configuration of trichoacorenol, the principal volatile component in headspace extracts of Trichoderma harzianum. Copyright

Full text of DOI:10.1002/ejoc.201100688

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DOI: 10.1002/ejoc.201100688
Enantioselective Synthesis of the Unnatural Enantiomers of the Fungal
Sesquiterpenoids Acorenone and Trichoacorenol
Nelson L. Brock[a] and Jeroen S. Dickschat*[a]
Keywords: Terpenoids / Total synthesis / Methathesis / Natural products / Volatiles
The volatiles released by several strains of Trichoderma fungi
clic sesquiterpene acorenone and the first enantioselective
were collected using a closed-loop stripping apparatus and
synthesis of the related compound ent-trichoacorenol have
analysed by GC–MS. Most of the investigated strains re- been completed by a chiral-pool approach starting from (+)-
leased the two structurally related spirocyclic sesquiterpen-
oids trichoacorenol, previously identified in Trichoderma
koningii (Huang et al., 1995), and acorenone. A new enantio-
selective synthesis of the enantiomer of the natural spirocy-
(R)-pulegone using ring-closing metathesis as the key step.
The absolute configuration of natural trichoacorenol from
Trichoderma harzianum sp. 714 has been assigned by chiral
GC–MS analysis and is the same as that in T. koningii.
zymes involved such as pectinases.[5] Besides these direct ef-
fects, Trichoderma is able to induce localized or systemic
resistance to fungal pathogens in plants.[6]
To date, little information has accumulated regarding the
molecular elicitors that participate in the interaction be-
tween the plants and their microbial symbionts. Some de-
tails are known for the symbiotic system between plants
and plant growth-promoting rhizobacteria that also have
the potential to induce systemic resistance. Herein, plant-
growth promotion is mediated by the volatiles acetoin (1)
and 2,3-butanediol (2, Figure 1).[7]
Introduction
Trichoderma spp. are asexually reproducing fungi com-
mon in soil, root, and foliar ecosystems. In addition, marine
representatives have frequently been isolated especially from
sediments or sponges.[1] In soil, Trichoderma belong to the
most frequently occurring fungi that are present in nearly
all temperate and tropical regions. The sexual teleomorph
is classified as the genus Hypocrea, but many strains do not
have a known sexual stage. The individuals of the asexual
form can exhibit an exceptionally high level of genetic di-
versity resulting in the production of a wide range of small-
molecule secondary metabolites with antibiotic activities
and extracellular enzymes such as chitinases.[2]
These metabolites and exoenzymes provide the molecular
basis for interactions between the plants and Trichoderma
as opportunistic, avirulent plant symbionts. The symbiotic
system is characterized by enhanced plant root growth and
thereby crop productivity as a result of an interaction of
the fungi with the plant’s nutrient-uptake system.[3]
Trichoderma can solubilize nutrients otherwise unavailable
to plants such as metal oxides and calcium phosphate by
reduction or release of siderophores as iron chelators.[4]
Further, the symbiont is beneficial to the plants by a myco-
parasitic effect towards a range of plant-pathogenic fungi,
as the extracellular chitinases released by Trichoderma are
toxic to the pathogen.[2] In addition, Trichoderma prevents
the penetration of leaf surfaces by plant-pathogenic fungi
such as Botrytis cinerea by inhibiting or degrading the en-
Figure 1. Plant-growth-promoting volatiles from rhizobacteria (1
and 2) and structures of trichoacorenol (3a) and acorenone (4).
This example demonstrates that volatiles can play impor-
tant roles with respect to interspecies communication. How-
ever, most research on secondary metabolites includes the
preparation of culture extracts from laboratory liquid cul-
tures followed by concentration steps during which volatile
metabolites are easily lost, and as a consequence volatiles
are frequently overlooked. The analysis of volatile metabo-
lites is a promising approach to identify new bioactive com-
pounds even from strains that are otherwise well studied in
terms of their secondary metabolism. These considerations
provided the motivation to analyse the volatiles released by
several strains of Trichoderma. Most of the strains investi-
gated produced two structurally related spirocyclic sesqui-
terpenes, trichoacorenol (3a) and acorenone (4).
[a] Institut für Organische Chemie, Technische Universität
Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
Fax: +49-531-3915272
E-mail: j.dickschat@tu-bs.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100688.
Eur. J. Org. Chem. 2011, 5167–5175
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N. L. Brock, J. S. Dickschat
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Compound (–)-3a has been previously isolated from
For the unambiguous identification and complete struc-
Trichoderma koningii, and its relative configuration has tural elucidation with respect to the relative and absolute
been determined by NOE experiments[8] and correlation of configurations of the tentatively identified compounds, an
its oxidation product acorenone with synthetic refer- enantioselective total synthesis was carried out. Scheme 1
ences.[9,10] The absolute configuration of (–)-3a has been shows the retrosynthetic analysis for 3a and 4. The ketone
correlated from its oxidation product (–)-4 to natural (–)- 4 can be obtained by the oxidation of 3a or its epimer 3b
acorenone from Acorus calamus.[11] Several synthetic ap- that are both available from ring-closing metathesis of the
proaches to prepare racemic 4[12] and also a few enantiose- diastereomeric dienes A. A mixture of these diastereomeric
lective routes[13] have been published. In addition, the race- alcohols can arise from the aldehyde B through a Grignard
mic alcohol rac-3a has been obtained during a formal syn- reaction. Compound B is a hydrogenated and twofold one-
thesis of rac-4 from its epimer acorenone B.[14] However, carbon-elongated homologue of the methyl ester C, which
an enantioselective synthesis of 3a has not been performed. can be constructed in analogy to literature methods starting
Furthermore, the structurally related compound α-acoradi- from (–)-(S)-pulegone.[12,20]
ene, the major aggregation pheromone of Gnatocerus
cornutus, has been synthesized, and its structure was revised
after total synthesis.[15–17] This clearly demonstrates the
need for total syntheses of natural products to fully and
unambiguously determine their structures. Here we report
a new enantioselective synthesis of the optical antipodes of
natural trichoacorenol and acorenone from (R)-pulegone.
The synthetic material was used to assign the relative and
absolute configurations of natural trichoacorenol from T.
harzianum, which is in agreement with the published struc-
ture of (–)-3a from T. koningii.
Results and Discussion
The volatiles emitted from agar plate cultures of different
Trichoderma strains were collected using a closed-loop
stripping apparatus (Figure S1, Supporting Information)
and analysed by GC–MS. These analyses resulted in the
identification of two sesquiterpenoids produced in large
Scheme 1. Retrosynthetic analysis of 3a and 4.
amounts by several Trichoderma strains, which were iden-
tified by comparison of their mass spectra to spectral librar-
ies as 3a and 4. Especially large amounts of these com-
As (+)-(R)-pulegone (ca. 3 j per g) is much cheaper than
(–)-(S)-pulegone (ca. 200 j per g), the synthesis of the
pounds were emitted by Trichoderma harzianum 714 (Fig-
enantiomers ent-3a and ent-4 was approached. The syn-
ure 2). The full results of the analyses and investigations on
thetic route to these sesquiterpenes was carried out as
the biosynthesis of 3a by feeding experiments with deuter-
shown in Scheme 2. The known pulegenic acid [6, dia-
ated mevalonolactone isotopomers have been published re-
stereomeric ratio (1R,2R)/(1S,2R) = 60:40] derived from
(+)-(R)-pulegone (5) was converted into puleganolide [7,
cently.[18,19]
diastereomeric ratio (3aS,6aR)/(3aR,6aS) = 60:40] under
acidic conditions.[20] Alkylation of the lactone 7 with lith-
ium diisopropylamide (LDA) and commercially available 2-
(bromomethyl)-1,3-dioxolane gave 8 in a diastereomeric ra-
tio of (3aS,6aR)/(3aR,6aS) = 80:20. The diastereoisomers
were separated by column chromatography, and the re-
quired diastereoisomer (3aS,6aR)-8 was converted into the
acid 9 by ring opening of the lactone moiety by treatment
with potassium tert-butoxide (KOtBu) in boiling N,N-di-
methylformamide (DMF). The acid 9 was esterified with
diazomethane to afford the corresponding methyl ester 10,
which was hydrogenated with a platinum catalyst on char-
coal to give 11. Deprotection under acidic conditions re-
leased the aldehyde 12, which was converted to 13 by a Wit-
tig methylenation. The alcohol 14 was afforded by re-
duction with LiAlH4 and transfomed into the mesylate 15.
Figure 2. Total ion chromatogram of a headspace extract from
Trichoderma harzianum 714.
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Enantioselective Synthesis of Acorenone and Trichoacorenol
The nucleophilic substitution with sodium cyanide in hexa- 3b, which were both oxidized with Dess–Martin
methylphosphoric triamide at 110 °C yielded 16, which was periodinane (DMP) to ent-4. This route included 14 steps
reduced to the aldehyde 17 with diisobutylaluminium hy- for the synthesis of ent-4 with an overall yield of 5% based
dride (DiBAL-H). The alcohols 18a and 18b (dia- on 6.
stereomeric ratio 60:40) were obtained by a CeCl3-mediated
The relative configurations of the hydroxy groups of ent-
Grignard reaction with isopropenylmagnesium bromide 3a and ent-3b were determined by 2D NMR spectroscopy.
and were separated by column chromatography.[21] Subse- Figure 3 shows the key NOESY correlations for both syn-
quent ring-closing metathesis of both diastereomeric thetic diastereomers (for NOESY spectra and carbon num-
alcohols was performed with a Grubbs–Hoveyda II catalyst bering of ent-3a and ent-3b see Supporting Information).
and resulted in the formation of ent-3a and its epimer ent- The configuration at C-3 of compound (3S)-ent-3a was de-
duced from the NOESY couplings of 3-H with the iso-
propyl group (12-H, 13-H). Additional cross peaks are vis-
ible with the OH proton, the HR proton at C-2, and the
methyl group bonded to C-4 (11-H). In contrast, the 3-H
proton of (3R)-ent-3b shows a strong NOESY coupling
with the methyl group bonded to C-10 (15-H), and ad-
ditionally to the HS proton at C-2, the methyl protons 11-
H, and OH.
Figure 3. Key NOESY correlations of ent-3a and ent-3b.
To determine the absolute configuration of natural 3a,
rac-3a was synthesized using the same approach as for ent-
3a starting from rac-citronellal that was converted into rac-
pulegone by a literature procedure.[22] GC–MS analyses of
the natural and the synthetic compounds on a chiral sta-
tionary phase are presented in Figure 4. The enantiomers
of rac-3a were separated on a chiral Hydrodex-6-TBDMS
fused-silica capillary column (Figure 4A). Synthetic ent-3a
proved to be the second enantiomer eluted (Figure 4B),
whereas 3a from T. harzianum eluted with the same reten-
tion time as the first enantiomer (Figure 4C). This was con-
firmed by analysis of a mixture of the racemic and the natu-
ral compound (Figure 4D), and a mixture of enantiomer-
ically pure ent-3a and the natural product (Figure 4E). In
conclusion, the synthetic material is the opposite enantio-
mer to natural 3a, clearly establishing the absolute configu-
ration of trichoacorenol from T. harzianum as
(1S,3R,7S,10S)-3a by correlation of ent-3a to (R)-pulegone.
To confirm that 3a from T. harzianum has the same abso-
lute configuration as trichoacorenol from T. koningii, the
Scheme 2. Synthesis of ent-3a and ent-4.
Eur. J. Org. Chem. 2011, 5167–5175
specific rotation of ent-3a was measured. The reported op-
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Experimental Section
General Synthetic Methods: Chemicals were purchased from Acros
Organics (Geel, Belgium) or Sigma–Aldrich Chemie GmbH (Ste-
inheim, Germany) and used without further purification. Solvents
were purified by distillation and dried according to standard meth-
ods. Oxygen- and/or moisture-sensitive reactions were carried out
under N2 in vacuum-heated flasks with dry solvents. TLC was per-
formed on 0.20 mm Macherey–Nagel silica gel plates (Polygram
SIL G/UV254). Column chromatography was performed with
Merck silica gel 60 (0.040–0.063 mm) using standard flash-chroma-
tographic methods. NMR spectra were recorded with Bruker
DRX-400 (400 MHz), AV III-400 (400 MHz), and AV II-600
(600 MHz) spectrometers and were referenced against TMS (δ =
0.00 ppm) for 1H NMR and CHCl3 (δ = 77.16 ppm) for 13C NMR
spectroscopy. Chemical shifts are reported in parts per million
(ppm), multiplicities are abbreviated as follows: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br. = broad, and
coupling constants J are given in Hertz (Hz). IR spectra were re-
corded with a Bruker Tensor 27 ATR spectrometer. UV spectra
were recorded with a Varian Cary 100 Bio spectrometer. Specific
rotations were measured using a Dr. Kernchen Propol Digital
Automatic Polarimeter. GC–MS analyses for the synthetic com-
pounds were carried out with an HP6890 GC system connected to
an HP5973 Mass Selective Detector fitted with a BPX-5 fused-
silica capillary column (25 mϫ0.22 mm i.d., 0.25 μm film, SGE
Inc., Melbourne, Australia) under the following conditions: inlet
pressure: 77.1 kPa, He 23.3 mLmin–1; injection volume: 1 μL; in-
jector: 250 °C; transfer line: 300 °C; electron energy: 70 eV. The GC
was programmed as follows: 50 °C (5 min isothermic), increasing
at 10 °Cmin–1 to 320 °C, and operated in split mode; carrier gas
(He): 1.0 mLmin–1. Retention indices I were determined from a
homologous series of n-alkanes (C8 – C32). Polar compounds with
adverse chromatographical behaviour were transformed into their
trimethylsilyl derivatives by treatment with N-methyl-N-(trimethyl-
Figure 4. Determination of the absolute configuration of natural
3a from T. harzianum by chiral GC. The chromatograms show
analyses of (A) synthetic (rac)-3a, (B) synthetic ent-3a, (C) a head-
space extract from T. harzianum, (D) a mixture of synthetic (rac)-
3a and a headspace extract from T. harzianum, and (E) synthetic
ent-3a and a headspace extract from T. harzianum.
tical rotation for natural 3a from T. koningii is [α]2D6 = –5.2
(c = 0.12, CHCl3),[8] and the specific rotation of the syn-
thetic material ent-3a is [α]1D7 = +5.5 (c = 0.12, CHCl3), con- silyl)trifluoroacetamide (MSTFA)[23] prior to GC analysis. Chiral
GC analyses were performed using a Hydrodex-6-TBDMS fused-
silica capillary column (30 mϫ0.25 mm i.d., 0.25 μm film, Mach-
erey–Nagel). The GC was programmed as follows: 10 min at 50 °C
increasing with 2 °Cmin–1 to 160 °C and then with 10 °Cmin–1 to
220 °C.
firming that both species produce the same enantiomer of
3a. Furthermore, the specific rotation of synthetic ent-4,
[α]1D7 = +44.0 (c = 0.02, CHCl3), exhibited the opposite sign
as reported for the oxidation product 4 obtained from natu-
ral 3a from T. koningii, [α]2D6 = –22.0 (c = 0.047, CHCl3),
and for natural 4 isolated from Acorus calamus, [α]2D5 = –28
(CCl4).[8,13c]
(2R)-2-Methyl-5-(propan-2-ylidene)cyclopentanecarboxylic
Acid
(6):[20] To
a
suspension of (+)-(R)-pulegone [(R)-5] (12.2 g,
80.1 mmol, 1.0 equiv.) and NaHCO3 (2.04 g) in dry Et2O (200 mL)
was added Br2 (12.7 g, 79.6 mmol, 1.0 equiv.) at 0 °C. After stirring
at 0 °C for 30 min, the mixture was concentrated in vacuo. An
aqueous KOH solution (0.8 m, 350 mL) was added, and the mixture
was stirred under reflux for 3 h. The reaction mixture was washed
with ethyl acetate, acidified with an aqueous H2SO4 solution (2 m),
extracted with diethyl ether, dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
Conclusions
The enantioselective synthesis of the unnatural enantio-
mers of the two main compounds in the headspace extract
of Trichoderma harzianum and other fungal species from
this genus, (–)-acorenone and (–)-trichoacorenol, has been silica gel (hexane/ethyl acetate
=
5:1) to give the acid
6
{diastereomeric mixture [(1R,2R)/(1S,2R) 60:40], 4.79 g,
=
accomplished starting from (+)-(R)-pulegone. As the key
step in the formation of the spirocyclic carbon backbone
an olefin metathesis was used. The same synthesis was per-
formed to prepare the racemic compound. The enantiomers
of the racemate of trichoacorenol were separated on a chiral
GC phase, and by comparison of the natural compound
from T. harzianum with the synthetic material the absolute
configuration of the natural product was unambiguously es-
tablished as (+)-trichoacorenol, which is the same as that
from T. koningii.[8]
28.5 mmol, 36%} as a colorless oil. Rf = 0.41; I = 1348 (major),
1
1326 (minor). H NMR (400 MHz, CDCl3): δ = 11.43 (br. s, 2 H,
3
3
2ϫ COOH), 3.40 (d, JH,H = 8.0 Hz, 1 H, CH), 2.97 (d, JH,H
=
5.1 Hz, 1 H, CH), 2.48 – 2.18 (m, 5 H, 2ϫ CH, 2ϫ CH2), 2.05 –
1.97 (m, 1 H, CH2), 1.85 – 1.70 (m, 2 H, CH2), 1.68 (s, 3 H, CH3),
1.67 (s, 3 H, CH3), 1.647 (s, 3 H, CH3), 1.645 (s, 3 H, CH3), 1.33 –
3
1.15 (m, 2 H, CH2), 1.092 (d, JH,H = 6.9 Hz, 3 H, CH3), 1.086 (d,
3JH,H = 6.8 Hz, 3 H, CH3) ppm. 13C NMR (100 MHz, CDCl3): δ
= 182.1 (Cq), 180.5 (Cq), 134.5 (Cq), 133.8 (Cq), 126.7 (Cq), 126.5
(Cq), 55.4 (CH), 52.7 (CH), 40.8 (CH), 38.9 (CH), 33.7 (CH2), 32.6
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