Exploring mutasynthesis to increase structural diversity in the synthesis of highly oxygenated polyketide lactones

Article abstract of DOI:10.1039/c4ob00717d

The enantioselective synthesis of (2R,3R,4E,8E)-3-hydroxy-2,4,8- trimethyldeca-4,8-dienolide (5) by ring-closing metathesis is described. This compound is an analogue of 3,4-dihydroxy-2,4,6,8-tetramethyldec-8-enolide (4) which is a rare 11-membered lactone produced by the fungus, Botrytis cinerea. Mutasynthetic studies with compound 5 using two mutants of B. cinerea led to the isolation of four new highly oxygenated 11-membered lactones (11-14) in which compound 5 has been stereoselectively epoxidized and hydroxylated at sites that were not easily accessible by classical synthetic chemistry.

Full text of DOI:10.1039/c4ob00717d

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Exploring mutasynthesis to increase structural
diversity in the synthesis of highly oxygenated
polyketide lactones†
Cite this: Org. Biomol. Chem., 2014,
12, 5304
J. M. Botubol-Ares,a M. J. Durán-Peña,a A. J. Macías-Sánchez,a J. R. Hanson,b
I. G. Colladoa and R. Hernández-Galán*a
The enantioselective synthesis of (2R,3R,4E,8E)-3-hydroxy-2,4,8-trimethyldeca-4,8-dienolide (5) by
ring-closing metathesis is described. This compound is an analogue of 3,4-dihydroxy-2,4,6,8-tetra-
methyldec-8-enolide (4) which is a rare 11-membered lactone produced by the fungus, Botrytis cinerea.
Mutasynthetic studies with compound 5 using two mutants of B. cinerea led to the isolation of four new
highly oxygenated 11-membered lactones (11–14) in which compound 5 has been stereoselectively
epoxidized and hydroxylated at sites that were not easily accessible by classical synthetic chemistry.
Received 4th April 2014,
Accepted 23rd May 2014
DOI: 10.1039/c4ob00717d
www.rsc.org/obc
Introduction
Botrytis cinerea is a grey powdery phytopathogenic mould that
affects a large number of commercial crops.1 Its major phyto-
toxic metabolites are a family of sesquiterpenes with the
botryane carbon skeleton2 and two groups of polyketides
exemplified, on the one hand, by the botcinic and botcineric
acids (1) and their cyclic derivatives, the botcinins (2),3 and on
the other hand, by botrylactone (3).4 A related lactone,
3,4-dihydroxy-2,4,6,8-tetramethyldec-8-enolide (4), has been
isolated5 from a mutant strain of B. cinerea. This lactone
possesses the same carbon chain as botrylactone with identical
functional groups and stereochemistry in the C1–C4 fragments
as both the botcinins and botrylactone. This structural hom-
ology and the presence of compound 4 in cultures of the
strains of B. cinerea which also produce large amounts of the
botcinins suggest that they have a common biosynthetic
origin. Compound 4 may be a ‘shunt’ metabolite in this
pathway (Fig. 1).
Fig. 1 Some polyketides produced by B. cinerea.
polyketides.7 The genes responsible for the formation of the
per-methylated tetraketide core of both botcinins (2) and botry-
lactones (3) are all included in the same cluster comprising
genes BcBOA1 to BcBOA6. The latter encodes the polyketide
synthase (PKS).6
Naturally-occurring medium-sized lactones (8 to 11-mem-
bered lactones) are a relatively small number of metabolites
which have nevertheless attracted considerable attention
because of the range of biological activities which they
possess.8 In particular there are only a few examples of
11-membered lactones that are found in nature.9 Apart from
compound 4 and some complex pyrrolizidine and daphniphyl-
lum alkaloids, there are only reports of two insect phero-
mones, ferrulactone I10 and suspensolide,11 and four fungal
metabolites including the aspercyclides A–C.12 The latter have
anti-inflammatory activity and have potential to be used in the
treatment of allergic disorders such as asthma.
The sequencing of the B. cinerea genome led to the develop-
ment of genetically modified strains lacking the genes which
code for the enzymes that are involved in the biosynthesis of
the secondary metabolites produced by this fungus.6 Studies
of the metabolites produced by these mutants permitted the
identification of the genes involved in the production of the
aDepartamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz,
11510 Puerto Real, Cádiz, Spain. E-mail: rosario.hernandez@uca.es
bDepartment of Chemistry, University of Sussex, Brighton, Sussex BN1 9QJ, UK.
E-mail: j.r.hanson@sussex.ac.uk
†Electronic supplementary information (ESI) available: Copies of the 1H NMR
and 13C NMR spectra for all key intermediates and final products, 2D NMR of
11–14 as well as nOe spectra of (E)-5 and 11–14. See DOI: 10.1039/c4ob00717d
5304 | Org. Biomol. Chem., 2014, 12, 5304–5310
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
The relative thermodynamic instability of 11-membered lac-
tones has made their synthesis and that of their derivatives
difficult even using Mitsunobu lactonization13 or an intramole-
cular Reformatsky reaction.14 Recently, ring-closing metathesis
(RCM) has been developed to provide an efficient method for
the preparation of some 11-membered lactones from acyclic
precursors.15 However RCM reactions in the macrocyclic series
tend to give mixtures of the (E) and (Z) isomers of cyclic
olefins. A reliable general method for controlling the geometry
of the new double bond is yet to be found.16 Consequently
there are few examples of synthetic analogues whose biological
activity has been thoroughly evaluated. Although botrylactone
was described by Welmar et al.17 as a powerful antibiotic
whilst the botcinins have been reported18 to show antifungal
activity against Magnaporthe grisea, the causal agent of rice
blast, nothing is known of the biological activity of compound
4 or its close relatives because of the small amount of material
that has been isolated.
Mutasynthesis is a very interesting strategy that combines
chemical synthesis with biosynthetically-patterned biotrans-
formations using genetically engineered microorganisms.19
The availability of mutants of B. cinerea blocked in the early
stages of the pathway but retaining some of the later stages
leading to these 11-membered lactones has allowed us to
explore the use of mutasynthesis to generate modified
11-membered lactones that are analogues of compound 4 in
order to evaluate their biological activity.
Scheme 2 Stereoselective synthesis of 5.
Oxidation of 8 with TEMPO-BAIB (2,2,6,6-tetramethyl piperidi-
nyloxy-bis(acetoxy)iodobenzene)21 afforded the aldehyde 7.
This was then treated with the boron Z-enolate of the N-propio-
nyl oxazolidinone (−)-(R)-922 to give the syn-aldol product 10 in
70% yield and 88% de.‡ Exocyclic cleavage of the oxazolidi-
none 10 with allyl alcohol and 4 eq. of allylmagnesium
bromide at −20 °C generated the ester 6 with a yield of 67%.
Finally an RCM reaction of 6 under high dilution conditions
catalysed by the second-generation ruthenium complex A23
in dry, degassed, refluxing dichloromethane produced the
11-membered lactone 5 with a good regioselectivity and yield
(9 : 1 E/Z ratio, 68% yield).§‡ This is in agreement with mole-
cular mechanics calculations (MM2) which predict that the
(E)-alkene is the most stable regioisomer.24
The lactone 5 was then incubated with two mutant strains
of B. cinerea, bcbot2Δ and bcboa6Δbcbot2Δ (bcΔΔd1). The first
mutant, which was obtained by deactivating the botryane bio-
synthesis gene BcBOT2 that encodes a sesquiterpene cyclase,25
does not produce the botryanes but does produce a significant
amount of botcinic acid (1a) and its relatives. The second
mutant, bcΔΔd1,6 is a double mutant which was obtained by
deactivating the genes BcBOT2 and BcBOA6 and is conse-
quently unable to produce the three characteristic families of
metabolites: the botryanes, the botcinins and the botry-
lactone.6 Incubation of compound 5 with both mutant strains
using liquid surface-culture conditions gave four new 11-mem-
bered lactones 11–14 (Fig. 2). Their distribution and % yields
are shown in Table 1. The strain bcbot2Δ gave a higher conver-
sion of compound 5. The metabolites 11, 12 and 13 were
Results and discussion
We chose compound 5 as a simplified analogue of compound
4 although the stereochemistry of the alcohol at C-3 is
different. However it could be easily synthesized in sufficient
quantity for the mutasynthesis experiments. The retrosynthetic
analysis of the lactone is shown in Scheme 1. An RCM cycliza-
tion of the ester 6 and a syn aldol reaction of the aldehyde 7
play key roles in the stereoselective construction of the
11-membered lactone ring.
The synthetic sequence leading to the lactone 5 is shown in
Scheme 2. 2-Methyl-2-vinyloxirane was treated with commer-
cially available 2-methylallyl magnesium chloride in the pres-
ence of CuI to produce the allylic alcohol 8 in a yield of 95%.20
‡Ratio valuated using 1H-NMR.
§It is noteworthy that with the first-generation ruthenium catalyst
PhCHvRuCl2(PCy3)2, a linear dimer was obtained after 16 h at reflux in CH2Cl2.
Scheme 1 Retrosynthetic analysis of lactone 5.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 5304–5310 | 5305

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 2 Metabolites isolated in mutasynthesis.
Table 1 Distribution and yields of isolated metabolites
Strains
Metabolites (% yields)
bcbot2Δ
bcΔΔd1
5 (3), 11 (5), 12 (4), 13 (19), 14 (1)
5 (23), 11 (15), 12 (7), 13 (7)
Fig. 3 MM2 minimized models with decisive nOe correlations for com-
pounds 11–14.
produced in both cases but there was no apparent incorpor-
ation of compound 5 into the botcinin/botrylactone pathway.
The structures of the metabolites were established from their The 13C NMR spectrum contained 13 signals. A comparison
1H and 13C NMR spectra using a combination of 1D and 2D with compound 5 showed that the resonance for C-7 [δC 38.2 (t)]
NMR experiments (see ESI†).
had changed to δC 77.2 (d) in compound 12. There were COSY
Compound 11 was obtained as a colourless oil whose correlations between δH 4.18 (H-7) and the signals at δH 2.46
HRMS possessed a molecular ion at m/z 240.1353 corres- and 2.30 which were assigned to H-6α and H-6β respectively.
ponding to the molecular formula C13H20O4. The 13C NMR There were nOe effects which were observed (Fig. 3)
spectrum contained resonances at δC 81.6, 62.9, 59.8 and between the signals at δH 4.18 (H-7), 5.62 (H-9), 5.02 (H-5) and
58.6 ppm (CH, CH2, C, and CH carbons respectively) and at H-6β, together with δH 4.70 (H-10β) and H-9. These were con-
δC 174.4 ppm (C carbon) consistent with the presence of four sistent with the R configuration at C-7 and hence compound
C–O and one lactone CvO carbon atoms in the molecule. The 12 was (2R,3R,4E,7R,8E)-3,7-dihydroxy-2,4,8-trimethyldeca-4,8-
1
main difference between the H NMR spectra of compounds 5 dienolide.
and 11 was the absence of the olefinic proton H-9 and the
The HRMS data for compounds 13 and 14 showed that they
appearance of a double-doublet (J 10.0 and 4.2 Hz) at both had a molecular formula, C13H20O5. Their 13C NMR
δH 3.01 ppm in compound 11. The presence of an epoxide at spectra contained five C–O signals at δC 81.4 (CH, C-3), 76.9
this position was confirmed by an HSQC correlation of H-9 (CH, C-7), 62.6 (C, C-8), 62.2 (CH2, C-10) and 56.6 (CH, C-9)
with the signal at δC 58.6 ppm (C-9) and HMBC correlations ppm for compound 13 and δC 80.9 (CH, C-3), 65.9 (CH, C-6),
between H-9 and signals at δC 59.8 (C-8) and δC 62.9 ppm 62.8 (CH2, C-10), 58.6 (CH, C-9) and 58.0 (C, C-8) ppm for 14.
(C-10). The stereochemistry of the oxirane ring was established These compounds possessed similar 1H NMR spectra in which
by nOe experiments which were rationalized on the basis of the olefinic C–H of compound 5 had been replaced by epoxy
the lowest energy optimized conformer derived from MM2 cal- C–H signals at δH 3.09 (dd, J 10.0, 4.0 Hz) and δH 3.05 (dd, J
culations (Fig. 3).24 In particular there were interactions 10.2, 4.2 Hz) respectively. However compound 13 possessed a
between H-9 and δH 5.17 (H-5), δH 2.16–2.08 (H-6β), and new CH(OH) signal at δH 3.22 (dd, J 11.3, 5.2 Hz) (H-7) which
δH 1.08 (H-7β) and between the signals at δH 1.75 (4-Me) and showed COSY correlations with signals at δH 2.50 (H-6α) and
δH 1.31 (8-Me). There were also nOe effects involving the signals δH 2.32 (H-6β). Compound 14 possessed a new CH(OH) signal
at δH 3.99 (H-3β) and δH 1.28 (2-Meβ). These were consistent at δH 4.64 (triplet J 11.2 of doublets J 4.8 Hz) (H-6) which
with the epoxidation of compound 5 on the β-face of the showed COSY correlations with signals at δH, 2.48 (H-7β),
ring.26 Based on the known absolute configuration of com- δH 1.12 (H-7α) and 5.20 (H-5). The nOe effects, shown in Fig. 3,
pound 5 and the MM2 calculations, compound 11 has the were in accord with the optimized structures obtained by MM2
absolute configuration (2R,3R,4E,8S,9S).
calculations24 and, bearing in mind the origin of the metab-
The HRMS (m/z 239.1282, M − H+) of compound 12 olites, led to the absolute stereochemistry for compounds 13
was consistent with the molecular formula
C
13H20O4. and 14 as 2R,3R,4E,7R,8S,9S and 2R,3R,4E,6R,8S,9S respectively.
5306 | Org. Biomol. Chem., 2014, 12, 5304–5310
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
These mutasynthetic transformations have produced deriva- with SiMe4 as the internal reference at room temperature.
tives that were hydroxylated at C-6 and C-7 and regioselectively J values are given in Hz. Chemical shifts were referenced to
epoxidized at C-8/C9. However there was no hydration of epoxi- CDCl3 (δH 7.25, δC 77.0). NMR assignments were made using a
dation of the C4/C5 double bond. Consequently additional combination of 1D and 2D techniques. Multiplicities are
synthetic work is necessary in order to develop an efficient described using the following abbreviations: s = singlet, d =
route for the synthesis of closer analogues of compound 4.
doublet, t = triplet, q = quarter; quint = quintuplet; sext =
Compounds 11 and 13 did not show any significant sextuplet; m = multiplet, br = broad. High-Resolution Mass
anti-bacterial activity at a concentration of 200 ppm against Spectroscopy (HRMS) was recorded with a double-focusing
Escherichia coli, Bacillus subtilis or Streptococcus faecalis.27 magnetic sector mass spectrometer in positive ion mode or
Compounds 11 and 13 did not show any significant phytotoxic with a QTOF mass spectrometer in positive ion electrospray
activity when tested in vivo on sterilized leaves of Phaseolus mode at 20 V cone voltage. HRESIMS/MS experiments were
vulgaris at a concentration of 500 ppm.28
performed with a QTOF mass spectrometer at 20 V cone
voltage and 10 eV collision energy.
Microorganisms
Conclusions
B. cinerea mutants, bcbot2Δ and bcΔΔd1, were previously
The regioselective ring-closing metathesis of the triene 6 has obtained by deactivating the genes encoding the sesquiterpene
provided an efficient method for the asymmetric synthesis of synthase BcBOT2,25 the synthase BcBOT2 and the polyketide
(2R,3R,4E,8E)-3-hydroxy-2,4,8-trimethyldeca-4,8-dienolide (E)-5 synthase BcBOA6,6 respectively, and are maintained in the
in 5 steps with 26% overall yield from simple starting BIOGER strain collection INRA (Grignon, France). Conidial
materials. This compound is an advanced analogue of 3,4- stock suspensions of these strains were maintained in glycerol
dihydroxy-2,4,6,8-tetramethyldec-8-enolide (4) which is
a
(80%) at −40 °C.
scarce metabolite of B. cinerea. Compound 5 was biotrans-
formed by two mutant strains of B. cinerea, bcbot2Δ and
bcΔΔd1, into four new highly oxygenated 11-membered lac-
Synthesis of the substrates
(E)-2,6-Dimethylhepta-2,6-dien-1-ol (8). 2-Methylallylmagne-
tones. These mutasynthetic steps involved regioselective epoxi- sium chloride (58.4 mL of
a 0.5 M solution in THF,
dation of the C8/C9 double bond and hydroxylation at either 29.2 mmol) at −30 °C was added to a stirred solution of
the C-6 (minor) or C-7 (major) positions. This has proved to be 2-methyl-2-vinyloxirane (2 mL, 20.9 mmol) and CuI (199 mg,
a valuable and versatile approach to increasing the structural 1.04 mmol) in dry THF (22 mL) at −30 °C. The mixture was
diversity of 11-membered lactones. Further experiments are in stirred at −30 °C and, after 3 h, was treated with a saturated
progress to develop a synthesis method of compound 4.
ammonium chloride solution (60 mL) and then allowed to
warm to room temperature. The aqueous layer was extracted
three times with diethyl ether (90 mL). Combined extracts were
washed with 1 N HCl (200 mL), saturated sodium bicarbonate
(200 mL), water (200 mL), and brine (200 mL) and then dried
over anhydrous sodium sulphate. Finally, the solvent was con-
Experimental
General procedures
Unless otherwise noted, materials and reagents were obtained centrated under reduced pressure and the crude was purified
from commercial suppliers and were used without further by silica gel column chromatography eluted with pentane–
purification. Dichloromethane was freshly distilled from CaH2 Et2O (80 : 20) to yield compound 8 (2756 mg, 94%) as a colour-
and tetrahydrofuran was dried over sodium and benzophe- less oil. Spectroscopic data of compound 8 were identical to
none and freshly distilled before use. Air- and moisture-sensi- those described in the literature.20
tive reactions were performed under an argon atmosphere.
(E)-2,6-Dimethylhepta-2,6-dienal (7). TEMPO (890 mg,
Purification by semipreparative and analytical HPLC was per- 5.6 mmol) and (diacetoxyiodo)benzene (BAIB, 18.3 g,
formed with a Hitachi/Merck L-6270 apparatus equipped with 83.7 mmol) were added at 0 °C to a solution of (E)-2,6-
a differential refractometer detector (RI-7490). A LiChrospher® dimethylhepta-2,6-dien-1-ol (8) (3.9 g, 27.9 mmol) in dry
Si 60 (5 µm) LiChroCart® (250 mm × 4 mm) column and a CH2Cl2 (62 mL). The mixture was stirred for 6 h and the
LiChrospher® Si 60 (10 µm) LiChroCart® (250 mm × 10 mm) solvent was concentrated under reduced pressure. The crude
column were used in isolation experiments. Silica gel (Merck) was purified by silica gel column chromatography eluted with
was used for column chromatography. TLC was performed pentane–Et2O (96 : 4) to yield the aldehyde 7 (3339 mg, 87%)
on a Merck Kiesegel 60 F254, 0.25 mm thick. Melting points as a yellow oil. IR (film) νmax/cm−1 2925, 2852, 2769 (CHO),
were measured with a Reichert-Jung Kofler block and are 1702 (CO), 1420, 1102, 1090; 1H NMR (400 MHz, CDCl3)
uncorrected. Optical rotations were determined with a digital δH 1.71 (6H, s, 2-Me, 6-Me), 2.20 (2H, t, J 7.2, 5-H), 2.49 (2H, dq,
polarimeter. Infrared spectra were recorded on a FT-IR spectro- J 7.2, 0.6, 4-H), 4.70 (1H, br s, 7a-H), 4.77 (1H, br s, 7b-H), 6.47
photometer and reported as wave number (cm−1). 1H and (1H, tq, J 7.2, 1.0, 3-H), 9.38 (1H, s, 1-H); 13C NMR (100 MHz,
13C NMR measurements were recorded on Varian Unity 400 MHz, CDCl3) δC 9.1 (q, 2-Me), 22.2 (q, 6-Me), 26.8 (t, C4), 36.0 (t, C5),
Agilent 500 MHz and Varian Inova 600 MHz spectrometers 110.8 (t, C7), 139.3 (s, C2), 143.9 (s, C6), 153.9 (d, C3), 195.0
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 5304–5310 | 5307

View Article Online
Paper
Organic & Biomolecular Chemistry
(s, C1); HRMS (CI+): calcd for C9H14O [M]+ 138.1044, found and the solvent was concentrated under reduced pressure. The
138.1028. crude product was purified by silica gel column chromato-
(4R,2″R,3″R,4″E)-4-Benzyl-3-[3-hydroxy-2,4,8-trimethylnona- graphy eluted with petroleum ether–Et2O (90 : 10) to yield the
4,8-dienoyl]-oxazolidin-2-one (10). n-Dibutylboron triflate ester 6 (405 mg, 67%) as a yellow oil. [α]2D0 +3.7° (c 0.42 in
(5.2 mL of a 1.0 M solution in CH2Cl2, 5.2 mmol) was added CHCl3); IR (film) νmax/cm−1 3448 (OH), 2924, 1736 (CO), 1458,
1
dropwise at 0 °C to a stirred solution of (−)-(4R)-4-benzyl-3-pro- 1376, 1170, 1032, 888; H NMR (400 MHz, CDCl3) δH 1.15 (3H,
pionyloxazolidin-2-one ((−)-(R)-9) (1020 m, 4.36 mmol) in dry d, J 7.1, 2-Me), 1.60 (3H, s, 4-Me), 1.71 (3H, s, 8-Me), 2.04 (2H,
CH2Cl2 (6.3 mL) under argon atmosphere conditions. The t, J 7.6, 7-H), 2.13–2.20 (2H, m, 6-H), 2.25 (d, J 4.4, 3-OH), 2.70
mixture was stirred for 5 min and then N,N′-diisopropylethyla- (1H, dq, J 7.1, 4.4, 2-H), 4.27 (1H, t, J 4.4, 3-H), 4.57 (2H, dt,
mine was added dropwise (0.96 mL, 5.7 mmol). After complete J 5.6, 1.4, 1′-H), 4.66 (1H, br s, 9b-H), 4.70 (1H, br s, 9a-H), 5.23
addition, the mixture was stirred at 0 °C for 15 min. The yellow (1H, ddd, J 10.4, 2.8, 1.4, 3′b-H), 5.31 (1H, ddd, J 17.4, 2.8, 1.4,
solution was re-cooled to −78 °C and a solution of (E)-2,6- 3′a-H), 5.47 (1H, t, J 7.0, 5-H), 5.90 (1H, ddt, J 17.4, 10.4, 5.6,
dimethylhepta-2,6-dienal (7) (1220 mg, 8.71 mmol) was added 2′-H); 13C NMR (100 MHz, CDCl3) δC 11.3 (q, 2-Me), 12.7 (q,
dropwise in dry CH2Cl2 (2.0 mL). The mixture was stirred at 4-Me), 22.5 (q, 8-Me), 25.8 (t, C6), 37.4 (t, C7), 43.0 (d, C2), 65.2
−78 °C and was then allowed to warm to 0 °C and stirred for (t, C1′), 76.8 (d, C3), 110.0 (t, C9), 118.3 (t, C3′), 126.6 (d, C5),
an additional hour. The reaction was quenched with a mixture 132.0 (d, C2′), 134.0 (s, C4), 145.4 (s, C8), 175.1 (s, C1); HRMS
of phosphate buffer (5 mL of a 1.0 M solution at pH 7) and (CI+): calcd for C15H24O3 [M]+ 252.1725, found 252.1718.
MeOH (15 mL) at 0 °C and the mixture was stirred for 5 min.
RCM of 6. (1,3-Bis(2,4,6-trimethylphenyl)-2-imidazolidinyli-
Finally, a solution of 2.4 : 1 MeOH–H2O2 (30%, 15 mL) was dene)dichloro(phenylmethylene)(tricyclohexyl-phosphine)
added slowly at 0 °C and stirred for an additional hour. The ruthenium (Grubbs ruthenium catalyst A, 121.3 mg,
solvent was concentrated under reduced pressure and the 0.14 mmol) was added to a refluxed and stirred solution of
residue was re-dissolved in diethyl ether. The aqueous layer ester 6 (180.0 mg, 0.71 mmol) in degassed and dry CH2Cl2
was extracted three times with diethyl ether (35 mL). Com- (420 mL) under argon conditions. The reaction mixture was
bined extracts were washed with brine (200 mL), dried over stirred until consumption of the starting material (20 h). The
anhydrous sodium sulphate, filtered, the solvent evaporated crude was filtered over a pad of silica gel and washed with
and the crude was purified by silica gel column chromato- ethyl acetate (400 mL). The solvent was removed under
graphy eluted with petroleum ether–ethyl acetate (75 : 25) to reduced pressure to give a crude that was purified by silica gel
yield compound 10 (1126 mg, 70%) as a white solid. m. column chromatography eluted with ether petroleum–ethyl
p. 37 °C (from CH2Cl2); [α]D20 −41.3° (c 0.65 in CHCl3); IR (film) acetate (90 : 10). Final purification was carried out by semi-pre-
νmax/cm−1 3524 (OH), 3176, 2935, 1781 (CO), 1698 (CO), 1456, parative HPLC (ether petroleum–ethyl acetate 85 : 15; flow =
1
1385, 1208, 1107, 886, 747; H NMR (400 MHz, CDCl3) δH 1.16 3.0 mL min−1) to yield a mixture 9 : 1 of (E)-5 (108.6 mg, 68%)
(3H, d, J 6.8, 2′-Me), 1.60 (3H, s, 4′-Me), 1.70 (3H, s, 8′-Me), and (Z)-5.¶
2.05 (2H, t, J 7.4, 7′-H), 2.18 (2H, q, J 7.4, 6′-H), 2.77 (1H, dd,
(2R,3R,4E,8E)-3-Hydroxy-2,4,8-trimethyldeca-4,8-dienolide
J 13.4, 9.2, CHHPh), 2.88 (d, J 2.8, 3′-OH), 3.24 (1H, dd, J 13.4, ((E)-5). Colourless oil; tR = 28 min; [α]2D0 +116° (c 0.42 in
3.2, CHHPh), 3.95 (1H, dq, J 6.8, 3.6, 2′-H), 4.18 (2H, m, 5a-H, CHCl3); IR (film) νmax/cm−1 3442 (OH), 2918, 2850, 1731 (CO),
5b-H), 4.33 (1H, br s, 3′-H), 4.64–4.66 (2H, m, 4-H, 9′a-H), 4.69 1455, 1374, 1259, 1164, 1024, 904, 794; 1H NMR (500 MHz,
(1H, br s, 9′b-H), 5.52 (1H, t, J 7.4, 5′-H), 7.17–7.33 (5H, m, CDCl3) δH 1.25 (3H, d, J 6.5, 2-Me), 1.61 (3H, s, 4-Me), 1.71 (3H,
Harom); 13C NMR (100 MHz, CDCl3) δC 10.4 (q, 2′-Me), 13.3 (q, s, 8-Me), 1.94–2.02 (1H, m, 7b-H), 2.02–2.11 (1H, m, 6b-H),
4′-Me), 22.3 (q, 8′-Me), 25.7 (t, C6′), 37.4 (t, C7′), 37.6 (t, 2.13–2.19 (1H, dt, J 11.6, 4.0, 7a-H), 2.41 (1H, dq, J 12.2, 4.0,
CH2Ph), 40.4 (d, C2′), 55.2 (d, C4), 66.0 (t, C5), 75.1 (d, C3′), 6a-H), 2.64 (1H, dq, J 10.0, 6.5, 2-H), 3.93 (1H, d, J 10.0, 3-H),
109.9 (t, C9′), 125.7 (d, C5′), 127.3 (Carom), 128.8 (2C, Carom), 4.33 (1H, t, J 10.6, 10b-H), 4.68 (1H, dd, J 10.6, 6.2, 10a-H), 5.02
129.3 (2C, Carom), 133.6 (s, C4′), 135.0 (s, Carom), 145.3 (s, (1H, dd, J 11.7, 4.0, 5-H), 5.50 (1H, m, 9-H); 13C NMR
C8′), 152.9 (s, C2), 176.9 (s, C1′); HRMS (CI+): calcd for (125 MHz, CDCl3) δC 10.2 (q, 4-Me), 13.8 (q, 2-Me), 15.6 (q,
C22H29NO4 [M]+ 371.2097, found 371.2088.
8-Me), 25.7 (t, C6), 38.2 (t, C7), 44.8 (d, C2), 60.9 (t, C10), 82.2
(2R,3R,4E)-Allyl 3-hydroxy-2,4,8-trimethylnona-4,8-dienoate (d, C3), 122.2 (d, C9), 127.9 (d, C5), 136.4 (s, C4), 143.6 (s, C8),
(6). Allylmagnesium bromide (4.9 mL of a 1.0 M solution in 175.0 (s, C1); HRMS (CI+): calcd for C13H20O3 [M]+ 224.1412,
Et2O, 4.9 mmol) was added at 0 °C to alcohol (15 mL) under found 224.1397.
argon conditions in a Schlenk flask. The allylic mixture was
stirred for 10 min and re-cooled at −20 °C. Then, a solution of
10 (886 mg, 2.40 mmol) in allylic alcohol (3 mL) was slowly
added. When TLC monitoring indicated the completion of the
reaction (3 h), a saturated ammonium chloride solution
comprising (per L of distilled water) glucose (50.0 g), yeast
(20 mL) was added and then allowed to warm to room temp-
erature. The aqueous layer was extracted three times with
Mutasynthesis experiments
General methods. B. cinerea was grown on a surface culture
in Roux bottles on a Czapek-Dox medium (150 mL per flask)
extract (1.0 g), KH2PO4 (5.0 g), NaNO3 (2.0 g), MgSO4·7H2O
diethyl ether (50 mL). Combined extracts were washed with
brine (100 mL), dried over anhydrous sodium sulphate, filtered ¶(Z)-5 could not be purified.
5308 | Org. Biomol. Chem., 2014, 12, 5304–5310
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
(0.5 g), and FeSO4·7H2O. The pH of the medium was adjusted (c 0.47 in CHCl3); IR (film) νmax/cm−1 3431 (OH), 2922, 2855
to 7.0 with aqueous NaOH (4 M). Each Roux bottle was inocu- (CH), 1716 (CO), 1448, 1373, 1254, 1166, 1019, 848, 741;
lated with 2 × 106 fresh conidia or six uniform discs of 0.9 cm 1H NMR (400 MHz, CDCl3) δH 1.28 (3H, d, J 6.8, 2-Me), 1.34
diameter mycelia of four-day old culture on malta agar. A (3H, s, 8-Me), 1.77 (3H, d, J 1.6, 4-Me), 2.28–2.35 (1H, m, 6b-H),
filter-sterilised aqueous solution of the labeled precursor or a 2.50 (1H, dt, J 13.6, 11.8, 6a-H), 2.69 (1H, dq, J 9.8, 6.8, 2-H), 3.09
solution of (E)-5 in ethanol was fed at a carefully determined (1H, dd, J 10.0, 4.0, 9-H), 3.22 (1H, dd, J 11.8, 5.2, 7-H), 3.56
optimum time. Roux bottles were incubated at 25 2 °C in (1H, dd, J 10.8, 10.0, 10b-H), 3.97 (1H, d, J 9.8, 3-H), 4.89 (1H,
daylight under static conditions for the optimum period of dd, J 10.8, 4.0, 10a-H), 5.12 (1H, dd, J 11.8, 3.2, 5-H); 13C NMR
time. The culture medium and mycelia were then separated by (100 MHz, CDCl3) δC 10.2 (q, 8-Me), 10.5 (q, 4-Me), 13.6 (q,
filtration. The broth was separated with NaCl and extracted 2-Me), 32.5 (t, C6), 43.4 (d, C2), 56.6 (d, C9), 62.2 (t, C10), 62.6
with ethyl acetate (3×) and dried over anhydrous Na2SO4. The (s, C8), 76.9 (d, C7), 81.4 (d, C3), 122.5 (d, C5), 137.6 (s, C4), 174.4
organic extract obtained was evaporated under reduced (s, C1); HRMS (CI+): calcd for C13H20O5 [M]+ 256.1311, found
pressure to dryness.
256.1310.
Feeding of (2R,3R,4E,8E)-3-hydroxy-2,4,8-trimethyldeca-4,8-
(2R,3R,4E,6R,8S,9S)-8,9-Epoxy-3,6-dihydroxy-2,4,8-trimethyl-
dienolide ((E)-5) to B. cinerea bcbot2Δ and bcΔΔd1. Com- dec-4-enolide (14). Colourless oil; tR = 38 min, petroleum
pound (E)-5 (120 mg), dissolved in EtOH (960 µL), was distribu- ether–ethyl acetate (30 : 70), flow = 0.8 mL min−1; [α]D20 +46.3°
ted among 6 Roux bottles containing a 4-day old culture of (c 0.11 in CHCl3); IR (film) νmax/cm−1 3422 (OH), 2921, 2856
B. cinerea bcbot2Δ or bcΔΔd1 and grown for a further 6 days. (CH), 1719 (CO), 1458, 1387, 1259, 1172, 1028, 932, 824; 1H
Filtration, ethyl acetate extraction and column chromatography, NMR (600 MHz, CDCl3) δH 1.12 (1H, t, J 11.2, 7b-H), 1.30 (3H,
followed by analytical HPLC purification, gave 11, 12, 13 and d, J 6.6, 2-Me), 1.32 (3H, s, 8-Me), 1.83 (3H, d, J 1.2, 4-Me), 2.48
14 in the yields shown in Table 1.
(1H, dd, J 11.2, 4.8, 7a-H), 2.72 (1H, dq, J 10.2, 6.6, 2-H), 3.05
(2R,3R,4E,8S,9S)-8,9-Epoxy-3-hydroxy-2,4,8-trimethyldec-4- (1H, dd, J 10.2, 4.2, 9-H), 3.49 (1H, dd, J 10.8, 10.2, 10b-H),
enolide (11). Colourless oil; tR = 27 min, petroleum ether– 4.00 (1H, dd, J 10.2, 1.2, 3-H), 4.64 (1H, dt, J 11.2, 4.8, 6-H),
ethyl acetate (77 : 23), flow = 0.8 mL min−1; [α]D20 +184° (c 0.34 4.90 (1H, dd, J 10.8, 4.2, 10a-H), 5.20 (1H, d, J 11.2, 5-H); 13C
in CHCl3); IR (film) νmax/cm−1 3448 (OH), 1734 (CO), 1458, NMR (150 MHz, CDCl3) δC 10.7 (q, 4-Me), 13.5 (q, 2-Me), 17.1
1
1165, 1022, 887, 764; H NMR (400 MHz, CDCl3) δH 1.08 (3H, (q, 8-Me), 43.7 (t, C7), 45.6 (d, C2), 58.0 (s, C8), 58.6 (d, C9),
dt, J 13.4, 4.8, 7b-H), 1.28 (3H, d, J 6.6, 2-Me), 1.31 (3H, s, 8- 62.8 (t, C10), 65.9 (d, C6), 80.9 (d, C3), 129.4 (d, C5), 139.0 (s,
Me), 1.75 (3H, t, J 1.6, 4-Me), 2.08–2.16 (2H, m, 6b-H, 7a-H), C4), 174.1 (s, C1); HRMS (CI+): calcd for C13H21O5 [M + H]+
2.37–2.48 (1H, m, 6a-H), 2.72 (1H, dq, J 10.0, 6.6, 2-H), 3.01 257.1389, found 257.1387.
(1H, dd, J 10.0, 4.2, 9-H), 3.53 (1H, dd, J 10.8, 10.0, 10b-H),
3.99 (1H, dd, J 10.0, 1.8, 3-H), 4.86 (1H, dd, J 10.8, 4.2, 10a-H),
5.17 (1H, ddd, J 12.0, 3.2, 1.6, 5-H); 13C NMR (100 MHz,
CDCl3) δC 10.3 (q, 4-Me), 13.6 (q, 2-Me), 15.9 (q, 8-Me), 24.3 (t,
Acknowledgements
C6), 37.0 (t, C7), 43.6 (d, C2), 58.6 (d, C9), 59.8 (s, C8), 62.9 (t,
This research was supported by grants from MICINN
C10), 81.6 (d, C3), 126.9 (d, C5), 136.2 (s, C4), 174.4 (s, C1);
(AGL2012-39798-C02-01) and from the Junta de Andalucía
HRMS (CI+): calcd for C13H20O4 [M]+ 240.1362, found
(P07-FQM-02689). José Manuel Botubol is grateful to the Junta
240.1353.
de Andalucia for his research fellowship. We gratefully
(2R,3R,4E,7R,8E)-3,7-Dihydroxy-2,4,8-trimethyldeca-4,8-die-
acknowledge Dr Muriel Viaud from the UMR BIOGER, INRA
nolide (12). Colourless oil; tR = 28 min, petroleum ether–ethyl
(Versailles, France) and P. Tudzynski from Munster University
acetate (53 : 47), flow = 0.8 mL min−1; [α]D20 +219° (c 0.31 in
(Germany) for the supply of B. cinerea mutant strains. The use
CHCl3); IR (film) νmax/cm−1 3395 (OH), 2923, 1707 (CO), 1458,
of NMR and mass spectrometry (QTOF) facilities at the Servicio
1354, 1258, 1168, 1021, 935, 851, 750; 1H NMR (400 MHz,
Centralizado de Ciencia y Tecnología (SCCYT) of the University
CDCl3) δH 1.24 (3H, d, J 6.6, 2-Me), 1.64 (3H, t, J 1.2, 4-Me),
of Cádiz is acknowledged.
1.75 (3H, d, J 1.2, 8-Me), 2.25–2.32 (1H, m, 6b-H), 2.46 (1H,
ddd, J 13.6, 12.0, 11.2, 6a-H), 2.61 (1H, dq, J 10.2, 6.6, 2-H),
3.90 (1H, d, J 10.2, 3-H), 4.18 (1H, dd, J 11.2, 5.6, 7-H), 4.37
(1H, t, J 10.2, 10b-H), 4.70 (1H, dd, J 10.2, 6.0, 10a-H), 5.02
(1H, dd, J 12.0, 2.4, 5-H), 5.62 (1H, dd, J 10.2, 6.0, 9-H);
Notes and references
13C NMR (100 MHz, CDCl3) δC 10.1 (q, C8), 10.2 (q, C4), 13.8
(q, C2), 34.5 (t, C6), 44.0 (d, C2), 60.1 (t, C10), 77.2 (d, C7), 81.9
(d, C3), 121.2 (d, C9), 124.7 (d, C5), 137.3 (s, C4), 145.2 (s, C8),
174.8 (s, C1); HRMS (CI+): calcd for C13H19O4 [M − H]+
239.1283, found 239.1283.
(2R,3R,4E,7R,8S,9S)-8,9-Epoxy-3,7-dihydroxy-2,4,8-trimethyl-
dec-4-enolide (13). Colourless oil; tR = 31 min, petroleum
ether–ethyl acetate (53 : 47), flow = 0.8 mL min−1; [α]D20 +94.1°
1 Y. Elad, B. Williamson, P. Tudzynski and N. Delen, in Botrytis:
Biology, pathology and control, Springer, 2007.
2 I. G. Collado, A. J. Macías-Sánchez and J. R. Hanson, Nat.
Prod. Rep., 2007, 24, 674–686.
3 H. Tani, H. Koshino, E. Sakuno, H. G. Cutler and
H. Nakajima, J. Nat. Prod., 2006, 69, 722–725.
4 J. Moraga, C. Pinedo, R. Durán-Patrón, I. G. Collado and
R. Hernández-Galán, Tetrahedron, 2011, 67, 417–420.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 5304–5310 | 5309

View Article Online
Paper
Organic & Biomolecular Chemistry
5 J. L. Reino, R. Durán-Patrón, M. Daoubi, I. G. Collado and
R. Hernández-Galán, J. Org. Chem., 2006, 71, 562–565.
6 B. Dalmais, J. Schumacher, J. Moraga, P. Le Pecheur,
B. Tudzynski, I. G. Collado and M. Viaud, Mol. Plant
Pathol., 2011, 12, 564–579.
J. Org. Chem., 2003, 68, 1878–1885; (e) J. Pospisil,
C. Mueller and A. Fuerstner, Chem. – Eur. J., 2009, 15,
5956–5968; (f) M. Lejkowski, P. Banerjee, S. Schueller,
A. Muench, J. Runsink, C. Vermeeren and H.-J. Gais, Chem.
– Eur. J., 2012, 18, 3529–3548.
7 M. Massaroli, J. Moraga, K. Bastos Borges, J. Ramírez- 16 A. Fürstner, K. Radkowski, C. Wirtz, R. Goddard,
Fernández, M. Viaud, I. G. Collado, R. Durán-Patrón and
R. Hernández-Galán, ChemBioChem, 2013, 14, 132–136.
8 G. Rousseau, Tetrahedron, 1995, 51, 2777–2849.
9 H. M. C. Ferraz, F. I. Bombonato, M. K. Sano and
L. S. Longo Jr., Quim. Nova, 2008, 31, 885–900.
C. W. Lehmann and R. Mynott, J. Am. Chem. Soc., 2002,
124, 7061–7069.
17 K. Welmar, R. Tschesche and E. Breitmaier, Chem. Ber.,
1979, 112, 3598–3602.
18 H. Tani, H. Koshino, E. Sakuno and H. Nakajima, J. Nat.
Prod., 2005, 68, 1768–1772.
10 J. W. Wong, V. Verigin, A. C. Oehlschlager, J. H. Borden,
H. D. Pierce Jr., A. M. Pierce and L. Chong, J. Chem. Ecol., 19 J. Kennedy, Nat. Prod. Rep., 2008, 25, 25–34.
1983, 9, 451–474. 20 D. Yang and M. Xu, Org. Lett., 2001, 3, 1785–1788.
11 (a) T. Chuman, J. Sivinski, R. R. Heath, C. O. Calkins, 21 A. De Mico, R. Margarita, L. Parlanti, A. Vescovi and
J. H. Tumlinson, M. A. Battiste, R. L. Wydra, L. Strekowski G. Piancatelli, J. Org. Chem., 1997, 62, 6974–6977.
and J. L. Nation, Tetrahedron Lett., 1988, 29, 6561–6563; 22 D. A. Evans, J. Bartroli and T. L. Shih, J. Am. Chem. Soc.,
(b) W. P. Ponce, J. L. Nation, T. C. Emmel, B. J. Smittle and 1981, 103, 2127–2129.
P. E. A. Teal, J. Chem. Ecol., 1993, 19, 3045–3056; 23 (a) P. Schwab, M. B. France, J. W. Ziller and R. H. Grubbs,
(c) J. R. Rocca, J. L. Nation, L. Strekowski and M. A. Battiste,
J. Chem. Ecol., 1992, 18, 223–244; (d) I. S. Lima, P. E. House
and R. R. Nascimento, J. Braz. Chem. Soc., 2001, 12,
196–201.
Angew. Chem., Int. Ed. Engl., 1995, 34, 2039–2041;
(b) P. Schwab, R. H. Grubbs and J. W. Ziller, J. Am. Chem.
Soc., 1996, 118, 100–110.
24 Energy minimization of compounds (Z)-5, (E)-5 and 11–14
were carried out on the basis of MM2 force field from CS
Chem 3D Ultra 12.0. For an overview regarding the use of
MM2 for minimizations, see: (a) D. A. Evans, C. E. Masse
and J. Wu, Org. Lett., 2002, 4, 3375–3378; (b) Z.-J. Zhan,
S.-P. Yang and J.-M. Yue, J. Org. Chem., 2004, 69, 1726–
1729; (c) A. Sharma, A. Mishra, R. P. Prajapat, S. Jain and
A. Bhandari, J. Chem. Pharm. Res., 2010, 2, 682–689;
(d) K. Simon, J. Wefer, E. Schöttner and T. Lindel, Angew.
Chem., Int. Ed., 2012, 51, 10889–10892.
12 S. B. Singh, H. Jayasuriya, D. L. Zink, J. D. Polishook,
A. W. Dombrowski and H. Zweerink, Tetrahedron Lett.,
2004, 45, 7605–7608.
13 (a) M. A. Battiste, J. R. Rocca, R. L. Wydra, J. H. Tumlinson
III and T. Chuman, Tetrahedron Lett., 1988, 29, 6565–6567;
(b) K. Mori and Y. Nakazono, Liebigs Ann. Chem.,
1988, 167–174; (c) B. A. Cheskis, N. A. Shpiro and
A. M. Moiseenkov, Russ. Chem. Bull., 1989, 38, 2389–2393;
(d) G. H. Del Vecchio and A. C. Oehlschlager, J. Org. Chem.,
1994, 59, 4853–4857.
25 C. Pinedo, C.-M. Wang, J. M. Pradier, B. Dalmais,
M. Choque, P. Le Pecheur, G. Morgant, I. G. Collado,
D. Cane and M. Viaud, ACS Chem. Biol., 2008, 3, 791–801.
14 T. Moriya, Y. Handa, J. Inanaga and M. Yamaguchi, Tetra-
hedron Lett., 1988, 29, 6947–6948.
15 For the synthesis of 11-membered lactones via RCM, see: 26 A. Rose, K. R. Hanson, K. D. Wilkinson and M. J. Wimmer,
(a) H. El Sukkari, J.-P. Gesson and B. Renoux, Tetrahedron Proc. Natl. Acad. Sci. U. S. A., 1980, 77, 2439–2441.
Lett., 1998, 39, 4043–4046; (b) S. C. Cho, P. H. Dussault, 27 R. Durán-Patrón, R. Hernández-Galán, L. G. Rebordinos,
A. D. Lisec, E. C. Jensen and K. W. Nickerson, J. Chem.
Soc., Perkin Trans. 1, 1999, 193–196; (c) J. D. Winkler,
J. M. Cantoral and I. G. Collado, Tetrahedron, 1999, 55,
2389–2400.
J. M. Holland, J. Kasparec and P. H. Axelsen, Tetrahedron, 28 L. Rebordinos, J. M. Cantoral, M. V. Prieto, J. R. Hanson
1999, 55, 8199–8214; (d) M. D. Ronsheim and C. K. Zercher,
and I. G. Collado, Phytochemistry, 1996, 42, 383–387.
5310 | Org. Biomol. Chem., 2014, 12, 5304–5310
This journal is © The Royal Society of Chemistry 2014