New apocarotenoids and β-carotene cleavage in Blakeslea trispora

Article abstract of DOI:10.1039/c1ob05712j

Mixed cultures of strains of opposite sex ("mated" cultures) of Blakeslea trispora contain trisporic acids and other apocarotenoids, some of which mediate the sexual responses of this fungus and other Mucorales. In mated cultures of the wild-type strains

Full text of DOI:10.1039/c1ob05712j

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PAPER
New apocarotenoids and b-carotene cleavage in Blakeslea trispora†
Alejandro F. Barrero,*a M. Mar Herrador,a Pilar Arteaga,a Jesu´s Gil,a Jose-Antonio Gonza´lez,a
Eugenio Alcaldeb and Enrique Cerda´-Olmedo*b
Received 5th May 2011, Accepted 17th June 2011
DOI: 10.1039/c1ob05712j
Mixed cultures of strains of opposite sex (“mated” cultures) of Blakeslea trispora contain trisporic acids
and other apocarotenoids, some of which mediate the sexual responses of this fungus and other
Mucorales. In mated cultures of the wild-type strains F986 and F921 we identified eleven
apocarotenoids: two C18 trisporoids, three C15 compounds with a monocyclofarnesane skeleton, a C13
compound, and five C7 compounds with a 2-methylhexane skeleton. Six of them are new natural
products and two others are new for Blakeslea. Their structures were established by NMR and mass
spectra and those of the C7 and C13 compounds were confirmed by chemical synthesis. The finding of
these compounds and the presence of approximately equimolecular amounts of the C18, C15, and C7
families led to the conclusion that b-carotene is initially split in three fragments by cleavage of its 13,14
and 11¢,12¢ double bonds.
Introduction
Results and discussion
b-Carotene (1, Fig. 1) is a natural pigment, antioxidant, and pro-
vitamin A with many applications in the alimentary, pharmaceuti-
cal, and cosmetic industries.1 It is obtained commercially by either
chemical synthesis or biotechnology, particularly from the fungus
Blakeslea trispora (syn. Choanephora trispora, Mucoromycotina,
Mucorales, Choanephoraceae). The wild-type strains of this
fungus belong to either the (+) or the (-) sex, and many pairs
of strains of opposite sex, cultured together (“mated” cultures”),
increase their b-carotene content and start the morphological
program of the sexual cycle. These physiological effects were
attributed to apocarotenoids such as trisporic acid C (2) and
similar compounds present in mated cultures of Blakeslea.2
The culture media of Blakeslea contain apocarotenoids with 18
carbons (2–14),2a,3 called trisporoids, or with 15 carbons (15–17),4
often called apotrisporoids because they were presumed to derive
from the former.5 On the other hand two apocarotenoids with 7
carbons (23, 24) have been found recently in cultures from another
Mucoral, Phycomyces blakesleeanus.6a
Results
We analyzed the agar medium where the strains F986, sexually
(+), and F921, sexually (-) of B. trispora had been cultured
together for three days. A clean medium, obtained by freezing
and squeezing the agar and centrifuging the resulting liquid, was
brought to pH 8 with NaOH and extracted with ethyl acetate. This
“neutral extract” was fractionated by semi-preparative normal-
phase HPLC. The remaining water solution was brought to
pH 2 with HCl and extracted with ethyl acetate. This “acid
extract” was fractionated in the same way after methylation with
(trimethylsilyl)diazomethane (TMSCHN2).
We isolated eleven apocarotenoids: three C15 (18 and 19, as
methyl esters, and 17), two C18 (the 9E and 9Z isomers of 2, both
as methyl esters), five C7 (20–24), and one C13 apocarotenoid (25,
as methyl ester) (Fig. 2). The structures of five of them (17, 23, 24,
and the 9E and 9Z isomers of 2) were determined by comparing
their spectroscopic data with those reported in the literature.4c,6
Apocarotenoid 25 is a new natural product, but its methyl ester
(25a) has been described as an intermediate in the synthesis of
trisporic acid B.7 The five remaining apocarotenoids (18–22) are
new to science.
Apocarotenoid 22 is the most abundant C7 apocarotenoid in
our cultures. Its molecular formula C7H12O2 was deduced from
HRFABMS. Its IR spectrum showed the absorption bands of a
hydroxyl group at 3447 cm-1 and of a conjugated diene at 1600
and 1650 cm-1. The 13C NMR spectrum showed seven signals: two
primary alcohol signals at d 63.6 and 68.3, a methyl signal at d
14.1, and four signals for disubstituted and trisubstituted double
bonds at d 137.9 (C), 131.8 (CH), 127.1 (CH) and 123.8 (CH).
The 2-methyl-2,4-hexadiene skeleton was established by the direct
We have studied the apocarotenoids in cultures of our standard
wild types of B. trispora. The structure of the identified products
and their relative amounts indicate that the apocarotenoids of
B. trispora derive from an asymmetrical double cleavage of b-
carotene.
aDepartment of Organic Chemistry, Institute of Biotechnology, University
of Granada, Avda. Fuente Nueva, s/n, 18071, Granada, Spain. E-mail:
afbarre@ugr.es; Fax: +34 958243318; Tel: +34 958243318
bDepartment of Genetic, Faculty of Biology, University of Sevilla, Reina Mer-
cedes, s/n, 41071, Sevilla, Spain. E-mail: eco@us.es; Tel: +34 954624107
† Electronic supplementary information (ESI) available: NMR spectra of
all compounds. See DOI: 10.1039/c1ob05712j
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Fig. 1 Apocarotenoids isolated from cultures of different strains of Blakeslea trispora.
Table 1 1H and 13C NMR data of 20–22a
20
21
22
C dH
dC
dH
dH
dC
1 9.62 d (8.0)
2 6.09 dd (8.0; 15.1) 121.7 —
3 7.65 dd (11.6; 15.1) 148.7 7.03 d (11.2)
194.1 9.48 s
4.02 s
—
6.02 d (11.0)
68.3
137.9
123.8
4 6.48 d (11.6)
5 —
6 4.18 br s
7 1.92 s
131.7 6.85 dd (11.2, 15.1) 6.43 dd (11.0; 15.1) 131.8
152.6 6.44 dt (4.6; 15.1) 5.78 dt (5.5; 15.1) 127.1
67.0 4.31 d (4.6)
14.6 1.82
4.15 d (5.5)
1.73
63.6
14.1
a
d in ppm. J in Hz in parentheses.
NMR spectrum of compound 21 lacked the other CH2OH signal
(d 4.02, s) of compound 22, but showed a CHO signal (d 9.48, s).
The proposed structures for 20–22 were confirmed by chemical
synthesis (Scheme 1).
An efficient synthesis of acetoxyester 27 from the commercial
prenol 26 (3-methylbut-2-en-1-ol) has been reported.6a Reduction
of 27 with excess diisobutylaluminium hydride (DIBAL) produced
22 with a yield of 86%, whereas the chemoselective saponification
of the acetoxyl group with K2CO3 produced the hydroxyester
28 with a yield of 96%. Protection of 28 with triisopropylsilyl
chloride (TIPSCl) and reduction with DIBAL produced to 29 with
a yield of 95%. Oxidation of 29 with the Dess–Martin reagent
and removal of the silyl protection with tetrabutylammonium
fluoride (TBAF) produced 20 with a yield of 66%. Acetylation
of 29 with Ac2O/pyridine and removal of the silyl protection
with TBAF produced 30 with a yield of 78%. Oxidation of
30 with the Dess–Martin reagent and saponification with 1 M
NaOH produced 21 with a yield of 60%. The spectroscopic data
of the synthetic products coincided with those of the natural
products.
Fig. 2 Apocarotenoids isolated from mated cultures of the B. trispora
wild-type strains F986 and F921.
1
coupling of the three olefinic protons in the H NMR spectrum
at d 6.43 (dd, J1 = 11.0 Hz, J2 = 15.1 Hz), 6.02 (d, J = 11.0 Hz),
and 5.78 (dt, J1 = 5.5 Hz, J2 = 15.1 Hz). The E stereochemistry
of the disubstituted double bond was deduced from its coupling
constant (15.1 Hz) and that of the trisubstituted one, from the
chemical shifts at C-1 (d 68.3) and C-7 (d 14.1) in the 13C NMR
spectrum.8 We conclude that compound 22 is (2E,4E)-2-methyl-
2,4-hexadiene-1,6-diol.
Compound 20 and 21 shared most of their 1H NMR signals with
compound 22 (Table 1). The 1H NMR spectrum of compound 20
lacked one of the CH2OH signals (d 4.15, d, J = 5.5 Hz) of com-
pound 22, but showed a CHO signal (d 9.62, d, J = 8.0 Hz). The 1H
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Scheme 2 Synthesis of apocarotenoid 25a.
The reaction of the methyl ester of the trisporic acid C
(2a) with m-chloroperoxybenzoic acid (MCPBA) produced the
regioselective epoxidation of the 9,10 double bond. Treatment of
the crude product with periodic acid produced 25a with an overall
yield of 90%. Its spectroscopic data coincided with those of the
methyl ester of the natural product.
A semiquantitative analysis based on the 1H NMR signals from
spectra of the “neutral extract” and “acid extract” indicated that
the three groups of apocarotenoids (trisporoids with 18 carbons,
cyclofarnesoids with 15 carbons, and methylhexadienes with 7
carbons) were found in approximately equimolar amounts. For
this summation the small amount of the only C13 apocarotenoid
can be disregarded.
Small amounts of compounds 23 and 24 and of three other
compounds were detected by their UV absorption in the chro-
matograms of the wild type F921, but not in those of strain SB64,
a mutant derived from it and completely devoid of b-carotene. This
confirms that 23 and 24 are apocarotenoids, as shown already for
Phycomyces.6a
Scheme 1 Synthesis of the apocarotenoids 20–22.
Table 2 1H and 13C NMR data of 17, 18a, 19aa
17
18a
19a
C
dH
dC
dH
dC
dH
dC
47.0
34.5
—
33.6
—
197.8
133.0
152.2
1
—
41.2 —
46.9 —
34.5 1.91–1.96 m
2.36–2.41 m
33.5 2.45–2.53 m
— 2.45–2.53 m
197.4 —
132.5 —
152.3 —
2a
2b
3a
3b
4
5
6
1.69–1.73 m 33.9 1.92–1.97 m
2.23–2.29 m 2.37–2.43 m
2.53–2.60 m 31.8 2.50–2.54 m
—
—
2.53–2.60 m
—
2.50–2.54 m
—
—
—
199.0 —
135.5 —
157.4 —
7
8
6.22 d (16.3) 124.4 6.34 s
6.26 d (16.3) 140.3 6.34 s
124.6 6.30 d (16.5) 125.2
139.8 6.35 d (16.5) 139.1
9
—
135.5 —
135.9 —
137.9
10
11
12
5.76 t (6.8) 132.8 5.77 dd (1.1; 6.6) 134.1 5.67 br t (6.9) 128.7
Discussion
4.34 d (6.8)
1.86
59.4 4.33 d (6.6)
13.8 1.83 s
59.6 4.72 d (6.9)
12.5 1.85 s
176.3 —
61.2
12.6
176.3
—
There are four reasons to think that the compounds described
here derive from b-carotene: the structural similarity between
them and three segments of the b-carotene molecule (Fig. 3); the
approximate equimolar amounts of trisporoids, cyclofarnesoids,
and methylhexadienes; the early label experiments by Austin
et al.,2b and the comparisons6a of wild-type strains with mutants
devoid of b-carotene.
These arguments indicate that the three families of apoc-
arotenoids of Blakeslea result from the double oxidative cleavage
of b-carotene at its 13,14 and 11¢,12¢ double bonds. This mech-
anism was already shown for Phycomyces6a and is likely to be
common to all Mucorales.
Our results with Blakeslea and the previous ones with
Phycomyces argue strongly against the hypothesis2b that trisporic
acids derive from b-carotene via retinal. This hypothesis was
already suspicious because of the failure of the efforts to
identify retinal in Phycomyces cultures. Our results also reject the
alternative hypothesis that b-carotene is cut twice at its 13,14 and
13¢,14¢ double bonds to produce the first trisporoid and that C15
apocarotenoids result from the secondary loss of three carbon
atoms of the side chain.
13a 3.44 d (11.0) 69.5 —
13b 3.73 d (11.0)
14
15
OH 1.53 br s
OMe —
Ac
Ac
—
—
—
—
1.14
1.87
21.7 1.54 s
12.5 1.94 s
23.0 1.49 s
12.3 1.92 s
23.1
12.5
—
52.6
170.9
21.0
—
—
—
—
—
3.69 s
—
—
—
52.5 3.67 s
—
—
—
—
—
2.06 s
—
a
d in ppm. J in Hz in parentheses.
The molecular formula of methyl ester of apocarotenoid 18
(18a) was C16H22O4 according to its HRFABMS. Its structure
was established from its NMR data (Table 2), which indicated a
COOMe group attached to C1, instead of the CH2OH group of
the well known apotrisporol (17).
The spectroscopic data of methyl ester of 19 (19a) led us to the
conclusion that 19a was the acetyl derivative of 18a (Table 2).
Compound 25 is the first C13 apocarotenoid isolated in the
Mucorales. It is found in very small amounts in the cultures
and could have been formed from a C15 or C18 apocarotenoid by
spontaneous oxidative breakage or a retroaldol reaction. It was
identified by comparison of spectral data of its methyl ester 25a
with those published for a synthetic compound.7 The structure
was confirmed by semisynthesis (Scheme 2).
The two C7 compounds with an aldehyde group and a hydroxy
group 20 and 21 were the least abundant and probably represent
metabolic intermediates. The likely biosynthetic pathway would
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Fig. 3 The three families of apocarotenoids in Blakeslea trispora. According to our results, b-carotene, 1, must be split at the points indicated by the
wavy lines to produce compounds with 18 carbons (left), 7 carbons (center), and 15 carbons (right). The example given for each group is one of the
compounds reported here.
start with the reduction of one end of the initial dialdehyde, not
detected. Phycomyces oxidizes the other end to a carboxylic group,
while Blakeslea has the additional capability to form a diol.
The metabolism of the apocarotenoids is not well fixed or
conserved in the Mucorales, not even in strains assigned to the
same species. Thus, we have not found fourteen apocarotenoids
previously identified in other strains of B. trispora, but have added
eight new ones to the list.
Extraction and fractionation of apocarotenoids
For the extraction and fractionation of apocarotenoids see Polaino
et al.6a
An initial extract of 500 mL (from 1 L of medium of mated
cultures F921 ¥ F986) yielded neutral and acid dry extracts of 114
and 246 mg, respectively.
The neutral extract (70 mg) was fractionated by semi-
preparative HPLC. The fraction (10.2 < RT < 15.4 min, 5 mg)
contained 20. The fraction (15.4 < RT < 17.8 min, 20 mg)
contained a 2 : 1 : 12 mixture of 20:21:22. The fraction (17.8 <
RT < 21.5 min, 5.2 mg) contained a mixture 3 : 1 mixture of 22:17.
The fraction (21.5 < RT < 25.4 min, 11 mg) contained 17.
The acid fraction was methylated with TMSCHN2 and then it
was also fractionated by semi-preparative HPLC.
Conclusions
The Mucorales produce a large diversity of apocarotenoids,
depending not only on the taxonomic species, but on the particular
strains. Of the eleven apocarotenoids found in mated cultures of B.
trispora strains F986 and F921, six are new natural products and
two are new for Blakeslea, while fourteen other apocarotenoids
reported from other strains of Blakeslea have not been found
now. The eleven apocarotenoids include two C18 compounds,
three C15 compounds, five C7 compounds, and a small amount
of one C13 compound. The initial step in the biosynthesis of the
apocarotenoids is a double cleavage of b-carotene at its 13,14 and
11¢,12¢ double bonds, which gives rise to the heads of the C18, C15,
and C7 families of apocarotenoids. This mechanism, already seen
in Phycomyces, is likely to be general to the Mucorales.
(2E,4E)-6-Hydroxy-5-methyl-2,4-hexadienal
(20). Yellow
1
syrup. H NMR (500 MHz, CO(CD3)2): see Table 1; 13C NMR
(125 MHz, CO(CD3)2): see Table 1; m/z (HRMS(FAB)) 149.0580
(M + Na. C7H10O2Na requires 149.0579).
(2E,4E)-6-Hydroxy-2-methyl-2,4-hexadienal
(21). Yellow
syrup. 1H NMR (500 MHz, CO(CD3)2): see Table 1; m/z
(HRMS(FAB)) 149.0580 (M
149.0579).
+ Na. C7H10O2Na requires
(2E,4E)-2-Methyl-2,4-hexadiene-1,6-diol
(22). Colourless
syrup. 1H NMR (500 MHz, CDCl3): see Table 1; 13C NMR (125
MHz, CDCl3): see Table 1; m/z (HRMS(FAB)) 151.0734 (M +
Na. C7H12O2Na requires 151.0735).
Experimental
General details
For general details see Polaino et al.6a
Apotrisporol (17). Colourless syrup. It has been identified by
spectroscopic data (see Table 2). These data were consistent with
those previously reported.4c
Strains and culture conditions
Strains F986 and F921 are wild-type (+) and (-) strains of
Blakeslea (Choanephora) trispora, respectively, and were obtained
from VKM (All-Russian Collection of Microorganisms, Moscow,
Russia). Plates containing 25 ml minimal agar medium9 were
inoculated with 5 ¥ 103 spores of each sex and incubated in the
dark at 30 ◦C for three days.
Methylation of the acid fraction
TMSCHN2 2 M in Et2O (232 mL) was added under stirring to a
solution of the acid fraction (70 mg) in C6H6:MeOH (4 : 1 v/v) (2.2
mL) at 0 ◦C. The solution was left for 5 min at room temperature
and the solvent was evaporated at low pressure to obtain a methyl
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ester mixture (80 mg) that was fractionated by semi-preparative
HPLC. The fraction (10.5 < RT < 13.1 min, 3.4 mg) contained
19a. The fraction (13.4 < RT < 15.9 min, 9 mg) contained a 2 : 1
mixture of 23a and 24a. The fraction (16.1 < RT < 16.5 min, 9.5
mg) contained 2a(9E). The fraction (16.5 < RT < 17.0 min, 3.9
mg) contained a 2 : 2 : 1 mixture of 2a(9E), 2a(9Z) and 25a. The
fraction (17.0 < RT < 18.4 min, 11.9 mg) contained a 5 : 1 mixture
of 2a(9Z) and 25a. The fraction (18.4 < RT < 20.7 min, 10 mg)
contained 2a(9Z). The fraction (20.7 < RT < 23.1 min, 4 mg)
contained a 2 : 1 mixture of 2a(9Z) and 18a. The fraction (23.1 <
RT < 25.6 min, 6 mg) contained 18a.
(43 mL) were added to a solution of 27 (1.4 g, 6.65 mmol) in
THF (84 mL) and MeOH (25 mL). The mixture was stirred for
40 min at room temperature. Then the solvent was evaporated
under low pressure to obtain a residue that was extracted
with Et2O. The organic phase was washed with brine, dried
over anhydrous Na2SO4 and filtered. The residue obtained after
removing the solvent under low pressure was chromatographed in
a silica gel column (petroleum ether (bp 30–40 ◦C)/Et2O, 50/50,
v/v) to obtain 28 (1.08 g, 96%).
Ethyl (2E,4E)-6-hydroxy-5-methyl-2,4-hexadienoate (28). Yel-
low oil. IR nmax(film)/cm-1 3435, 1641, 1308, 1276, 1160, 1035 and
983; dH(300 MHz; CDCl3; Me4Si) 7.62 (1H, dd, J 11.7 and 15.2,
H-3), 6.29 (1H, d, J 11.7, H-4), 5.91 (1H, d, J 15.2, H-2), 4.24 (2H,
q, J 7.1, OCH2CH3), 4.19 (2H, s, H-6), 1.92 (3H, s, H-7), 1.72 (1H,
br s, OH) and 1.33 (3H, t, J 7.1, OCH2CH3); dC(75 MHz; CDCl3;
Me4Si) 167.5 (C, C-1), 147.2 (C, C-5), 140.0 (CH, C-3), 121.8 (CH,
C-4), 121.0 (CH, C-2), 67.6 (CH2, C-6), 60.4 (CH2, OCH2CH3),
14.7 (CH3, OCH2CH3)a and 14.4 (CH3, C-7)a (aSignals with the
same letter are exchangeable); m/z (HRMS(FAB)) 193.0844 (M +
Na. C9H14O3Na requires 193.0841).
(2E,4E)-6-hydroxy-5-methylhexa-2,4-dienoic acid (23, as methyl
ester) and (2E,4E)-6-hydroxy-2-methylhexa-2,4-dienoic acid (24,
as methyl ester). Their spectroscopic data coincided with those
previously reported.6a
1
Apotrisporic acid (18, as methyl ester). Colourless syrup. H
NMR (500 MHz, CDCl3): see Table 2; 13C NMR (125 MHz,
CDCl3): see Table 2; m/z (HRMS(FAB)) 301.1414 (M + Na.
C16H22O4Na requires 301.1416).
Apotrisporic acid 11-acetate (19, as methyl ester). Colourless
syrup. 1H NMR (500 MHz, CDCl3): see Table 2; 13C NMR (125
MHz, CDCl3): see Table 2; m/z (HRMS(FAB)) 343.1518 (M +
Na. C18H24O5Na requires 343.1521).
Preparation of hydroxy-ether 29. Imidazole (1.07 g,
15.73 mmol) and TIPSCl (1.45 g, 7.42 mmol) were added to a
solution of 28 (1.07 g, 6.29 mmol) in dry DMF (1.1 mL) under
argon and stirring at room temperature. After 2 h, the mixture
was fractionated in t-BuOMe : H2O (2 : 1). The organic phase
was washed successively with HCl 2 N and brine and dried over
anhydrous Na2SO4. Once the solvent was evaporated under low
pressure we obtained a crude product (2.14 g), which was◦dissolved
in dry DCM (121 mL) under argon and chilled at -20 C. After
adding DIBAL (1 M in hexane, 16 mL) the solution was stirred for
20 min and then, H2O (49 mL) was added. The mixture was stirred
at room temperature for 20 min and filtered through a layer of silica
gel/anhydrous Na2SO4 (2/1, w/w). The layer was washed with
Et2O and the organic phase was evaporated under low pressure
to afford a crude product, which was chromatographed over silica
gel column (petroleum ether (bp 30–40 ◦C)/Et2O, 85/15, v/v) to
obtain 29 (1.7 g, 95%).
(7E, 9E)-Trisporic acid C (2(9E)), as methyl ester). Colourless
syrup. Its spectroscopic data coincided with those previously
reported.6c
(7E,9Z)-Trisporic acid C (2(9Z)), as methyl ester). Colourless
syrup. Its spectroscopic data coincided with those previously
reported.6b,c
Methyl (E)-1,3-dimethyl-4-oxo-2-(3-oxo-1-butenyl)-2-cyclohe-
xene-1-carboxylate (25a). Colourless syrup. Its spectroscopic
data coincided with those previously reported.7
Preparation of 25a
m-CPBA (64 mg, 2.27 mmol) in DCM (0.8 mL) was added
dropwise to a mixture of 7 (50 mg, 0.16 mmol) in dichloromethane
(DCM) (2.6 mL) and 0.5 M NaHCO3 (0.5 mL) under stirring
and argon at 0 ◦C. After 2.5 h, the mixture was diluted with t-
butyl methyl ether and washed with 0.5 M NaHCO3, a saturated
solution of sodium thiosulfate and brine, successively. The organic
phase was dried over anhydrous Na2SO4 and filtered. The residue
obtained (50 mg) after removing the solvent under low pressure
was dissolved in THF (2.6 mL) and H5IO6 (35.7 mg, 0.16 mmol)
was added at room temperature. After 10 min, water (10 mL) was
added to the mixture and tetrahydrofuran (THF) was removed
under low pressure. The aqueous phase was extracted with t-butyl
methyl ether and the organic phase was dried over anhydrous
Na2SO4 and filtered. The residue obtained after removing the
solvent under low pressure was chromatographed in a silica gel
column (hexane : t-butyl methyl ether, 1/1, v/v) to obtain 25 (36
mg, 90%).
(2E,4E)-6-Triisopropylsilyloxy-5-methyl-2,4-hexadien-1-ol (29).
Colourless syrup. IR nmax(film)/cm-1 3327, 2942, 2865, 1641, 1462,
1385, 1113, 1085, 1065, 995 and 882; dH(500 MHz; CDCl3; Me4Si)
6.50 (1H, dd, J 11.2 and 15.1, H-3), 6.15 (1H, d, J 11.2, H-4),
5.80 (1H, dt, J 6.0 and 15.1, H-2), 4.20 (2H, d, J 6.0, H-1), 4.14
(2H, s, H-6), 1.72 (3H, s, H-7) and 1.04–1.18 (21H, m, TIPS);
dC(125 MHz; CDCl3; Me4Si) 138.3 (C, C-5), 130.8 (CH, C-3),
127.7 (CH, C-2), 122.2 (CH, C-4), 67.9 (CH2, C-6), 63.8 (CH2, C-
1), 18.1 (6CH3, TIPS), 13.9 (CH3, C-7) and 12.1 (3CH, TIPS);
m/z (HRMS(FAB)) 307.2071 (M + Na. C16H32O2Na requires
307.2070).
Preparation of hydroxy-aldehyde 20. The Dess–Martin reagent
(1.01 g) was added to a solution of 29 (800 mg, 2.82 mmol)
in dry DCM (31 mL) at room temperature under argon. The
mixture was left for 50 min under stirring. A saturated solution of
Na2S2O3 and NaHCO3 was then added dropwise to the mixture
and extracted with Et2O. The organic phase was washed with
brine, dried over anhydrous Na2SO4, and filtered. The residue
obtained after evaporating the solvent under low pressure was
dissolved in petroleum ether (bp 30–40 ◦C)/Et2O (3/1, v/v) and
Preparation of (2E,4E)-6-hydroxy-5-methyl-2,4-hexadienal (20)
Saponification of 27. Preparation of ethyl (2E,4E)-6-hydroxy-
5-methyl-2,4-hexadienoate (28). K2CO3 (20 g) and K2CO3 2 M
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filtered through silica gel. The residue (713 mg) obtained after
evaporating the solvent under low pressure was dissolved in dry
THF (37 mL) and mixed with TBAF (1 M in THF, 16 mL), stirred
at room temperature for 65 min, diluted with Et2O (15 mL), and
washed with brine. The organic phase was dried over anhydrous
Na2SO4 and filtered. The residue obtained after removing the
solvent under low pressure was chromatographed in a silica gel
column (petroleum ether (bp 30–40 ◦C)/Et2O, 35/65, v/v) to
obtain 20 (234 mg, 66%).
Preparation of (2E,4E)-2-methyl-2,4-hexadiene-1,6-diol (22)
DIBAL (1 M in hexane, 2 mL) was added to a cold (-20 ◦C)
solution of 30 (100 mg, 0.78 mmol) in dry CH2Cl2 (9 mL) under
argon with stirring. After 20 min, water (3 mL) was added to
the mixture, and it was stirred at room temperature for 20 min.
The mixture was filtered through a layer of silica gel/anhydrous
Na2SO4 (2/1, w/w). The layer was washed with Et2O and the
organic phase was evaporated under low pressure to afford a
crude product, which was chromatographed over silica gel column
◦
(petroleum ether (bp 30–40 C)/Et2O, 35/65, v/v) to obtain 22
Preparation of (2E,4E)-6-hydroxy-2-methyl-2,4-hexadienal (21)
(65 mg, 86%).
Preparation of hydroxy-acetate 30. A mixture of 29 (730 mg,
2.57 mmol), dry pyridine (11 mL) and acetic anhydride (0.5 mL)
was left at room temperature for 2 h and then was worked up as
usual to give a crude product (835 mg), which was dissolved in dry
THF (33 mL), mixed with TBAF (1 M in THF, 2.2 mL), stirred
at room temperature under argon for 15 min, diluted with Et2O
(15 mL), and washed with brine. The organic phase was dried
over anhydrous Na2SO4 and filtered. The residue obtained after
removing the solvent under low pressure was chromatographed
in a silica gel column (petroleum ether (bp 30–40 ◦C)/Et2O, 1/1,
v/v) to obtain 30 (340 mg, 78%).
Acknowledgements
This research was financed by Junta de Andaluc
´ıa (Grants FQM
340, CVI 910, and P08-CVI-03901), and the Spanish Government
(Grants BIO2009-12486 and BIO2009-11131), with support from
the European Union Feder program.
Notes and references
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(2E,4E)-6-Hydroxy-5-methyl-2,4-hexadienyl
acetate
(30).
Colourless syrup. IR nmax(film)/cm-1 3420, 2917, 2861, 1737,
1662, 1445, 1380, 1365, 1235, 1113, 1071, 1023 and 971; dH(400
MHz; CDCl3; Me4Si) 6.51 (1H, dd, J 11.0 and 15.2, H-3), 6.05
(1H, d, J 11.0, H-4), 5.71 (1H, dt, J 6.3, 15.2, H-2), 4.58 (2H, d,
J 6.3, H-1), 4.03 (2H, s, H-6), 2.06 (1H, br s, OH), 2.03 (3H, s,
COCH3) and 1.75 (3H, s, H-7); dC(100 MHz; CDCl3; Me4Si) 171.9
(C, COCH3), 140.0 (C, C-5), 131.0 (CH, C-3), 126.8 (CH, C-2),
123.9 (CH, C-4), 68.3 (CH2, C-6), 65.4 (CH2, C-1), 21.1 (CH3,
COCH3) and 14.2 (CH3, C-7); m/z (HRMS(FAB)) 193.0838 (M
+ Na. C9H14O3Na requires 193.0841).
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Preparation of hydroxy-aldehyde 21. The Dess–Martin reagent
(1.05 g) was added to a solution of 30 (340 mg, 2.00 mmol)
in dry DCM (11 mL) at room temperature under argon. The
mixture was left for 15 min under stirring. A saturated solution of
Na2S2O3 and NaHCO3 was then added dropwise to the mixture
and extracted with Et2O. The organic phase was washed with brine,
dried over anhydrous Na2SO4, and filtered. The residue obtained
after evaporating the solvent under low pressure was dissolved
in petroleum ether (bp 30–40 ◦C)/Et2O (3/1, v/v) and filtered
through silica gel. The residue (245 mg) obtained after evaporating
the solvent under low pressure was dissolved in EtOH (7.60 mL)
and NaOH 1 M (3.80 mL) was added dropwise at 0 ◦C. The
mixture was then left at room temperature for 3 h. The solution
was neutralized with HCl 1 N (3.80 mL) and the solvent was
evaporated under low pressure. The residue was extracted with
EtOAc and the solvent was evaporated under low pressure to
obtain 21 (151 mg, 60%).
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The Royal Society of Chemistry 2011
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