Metabolism of deuterated erythro-dihydroxy fatty acids in Saccharomyces cerevisiae: Enantioselective formation and characterization of hydroxylactones

Article abstract of DOI:10.1002/hlca.200890129

Epoxides of fatty acids are hydrolyzed by epoxide hydrolases (EHs) into dihydroxy fatty acids which are of particular interest in the mammalian leukotriene pathway. In the present report, the analysis of the configuration of dihydroxy fatty acids via their respective hydroxylactones is described. In addition, the biotransformation of (±)-erythro-7,8- and -3,4-dihydroxy fatty acids in the yeast Saccharomyces cerevisiae was characterized by GC/EI-MS analysis. Biotransformation of chemically synthesized (±)-erythro-7,8- dihydroxy(7,8-²H₂)tetradecanoic acid ((±)-erythro- 1) in the yeast S. cerevisiae resulted in the formation of 5,6-dihydroxy(5,6- ²H₂)dodecanoic acid (6), which was lactonized into (5S,6R)-6-hydroxy(5,6-²H₂)dodecano-5-lactone ((5S,6R)-4) with 86% ee and into erythro-5-hydroxy(5,6-²H₂)dodecano-6- lactone (erythro-8). Additionally, the α-ketols 7-hydroxy-8-oxo(7- ²H₁)tetradecanoic acid (9a) and 8-hydroxy-7-oxo(8- ²H₁)tetradecanoic acid (9b) were detected as intermediates. Further metabolism of 6 led to 3,4-dihydroxy(3,4- ²H₂)decanoic acid (2) which was lactonized into 3-hydroxy(3,4-²H₂)decano-4-lactone (5) with (3R,4S)-5=88% ee. Chemical synthesis and incubation of (±)-erythro-3,4-dihydroxy(3,4- ²H₂)decanoic acid ((±)-erythro-2) in yeast led to (3S,4R)-5 with 10% ee. No decano-4-lactone was formed from the precursors 1 or 2 by yeast. The enantiomers (3S,4R)- and (3R,4S)-3,4-dihydroxy(3- ²H₁)nonanoic acid ((3S,4R)- and (3R,4S)-3) were chemically synthesized and comparably degraded by yeast without formation of nonano-4-lactone. The major products of the transformation of (3S,4R)- and (3R,4S)-3 were (3S,4R)- and (3R,4S)-3-hydroxy(3-²H ₁)nonano-4-lactones ((3S,4R)- and (3R,4S)-7), respectively. The enantiomers of the hydroxylactones 4, 5, and 7 were chemically synthesized and their GC-elution sequence on Lipodex E chiral phase was determined.

Full text of DOI:10.1002/hlca.200890129

Helvetica Chimica Acta – Vol. 91 (2008)
993
Metabolism of Deuterated erythro-Dihydroxy Fatty Acids in Saccharomyces
cerevisiae: Enantioselective Formation and Characterization of
Hydroxylactones
by Leif-A. Garbe*, Katja Morgenthal1), Katrin Kuscher2), and Roland Tressl
Technische Universität Berlin, Institut für Biotechnologie, Molekularanalytik, Seestrasse 13,
D-13353 Berlin
(phone: þ 49-30-45080-231; fax: þ 49-30-314-27544; e-mail: Leif-A.Garbe@TU-Berlin.de)
Epoxides of fatty acids are hydrolyzed by epoxide hydrolases (EHs) into dihydroxy fatty acids which
are of particular interest in the mammalian leukotriene pathway. In the present report, the analysis of the
configuration of dihydroxy fatty acids via their respective hydroxylactones is described. In addition, the
biotransformation of (Æ)-erythro-7,8- and -3,4-dihydroxy fatty acids in the yeast Saccharomyces cerevisiae
was characterized by GC/EI-MS analysis. Biotransformation of chemically synthesized (Æ)-erythro-7,8-
dihydroxy(7,8-2H2)tetradecanoic acid ((Æ)-erythro-1) in the yeast S. cerevisiae resulted in the formation
of 5,6-dihydroxy(5,6-2H2)dodecanoic acid (6), which was lactonized into (5S,6R)-6-hydroxy(5,6-2H2)do-
decano-5-lactone ((5S,6R)-4) with 86% ee and into erythro-5-hydroxy(5,6-2H2)dodecano-6-lactone
(erythro-8). Additionally, the a-ketols 7-hydroxy-8-oxo(7-2H1)tetradecanoic acid (9a) and 8-hydroxy-7-
oxo(8-2H1)tetradecanoic acid (9b) were detected as intermediates. Further metabolism of 6 led to 3,4-
dihydroxy(3,4-2H2)decanoic acid (2) which was lactonized into 3-hydroxy(3,4-2H2)decano-4-lactone (5)
with (3R,4S)-5 ¼ 88% ee. Chemical synthesis and incubation of (Æ)-erythro-3,4-dihydroxy(3,4-
2H2)decanoic acid ((Æ)-erythro-2) in yeast led to (3S,4R)-5 with 10% ee. No decano-4-lactone was
formed from the precursors 1 or 2 by yeast. The enantiomers (3S,4R)- and (3R,4S)-3,4-dihydroxy(3-
2H1)nonanoic acid ((3S,4R)- and (3R,4S)-3) were chemically synthesized and comparably degraded by
yeast without formation of nonano-4-lactone. The major products of the transformation of (3S,4R)- and
(3R,4S)-3 were (3S,4R)- and (3R,4S)-3-hydroxy(3-2H1)nonano-4-lactones ((3S,4R)- and (3R,4S)-7),
respectively. The enantiomers of the hydroxylactones 4, 5, and 7 were chemically synthesized and their
GC-elution sequence on Lipodexꢀ E chiral phase was determined.
Introduction. – Epoxides are ubiquitous in the environment, and epoxide hydro-
lases (EHs) are vital to many organisms through their roles in detoxification,
metabolism of epoxides, and processing of signaling molecules. It has been suggested
that inhibitors of soluble epoxide hydrolases (sEHs) have therapeutic efficacy in the
treatment and management of acute inflammatory diseases [1]. In mammals, sEH
metabolizes vasodilatory epoxyeicosatrienoic acids derived from arachidonic acid into
their dihydroxy derivatives. Therefore, sEH is hypothesized as a main effector of
angiotensin II-induced hypertension [2]. The transformation of oleic acid and linoleic
acid epoxides into fruit and dairy product flavors, especially chiral g-lactones (5-alkyl-
1
)
)
Current Address: Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14476 Golm.
Current Address: Istituto di Ricerca in Biomedicina, Institute for Research in Biomedicine,
CH-6500 Bellinzona.
2
ꢁ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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dihydro-2H-furanones) which also show bioactivity with pheromone character has
been documented in plants and microorganisms [3– 6].
Generally, the hydrolysis of epoxides proceeds under inversion of one epoxide C-
atom, whereby cis- or erythro-epoxides yield vicinal threo-diols and trans- or threo-
epoxides yield vicinal erythro-diols [7]. In contrast, a yeast EH has been characterized
converting leukotriene A4 ((5S,6S)-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid)
into (5S,6S)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, showing retention of
the initial epoxide configuration. In the literature, chiral gas chromatographic (GC)
separations of threo-hydroxylactones have been described [4][5]. Therefore, the
absolute configuration of threo-3,4-, 4,5- or 5,6-dihydroxy fatty acids can be analyzed by
Lipodexꢀ E gas chromatography of the respective 3-hydroxy-4-, 5-hydroxy-4-, or 6-
hydroxy-5-lactones. In addition, the hydroxylactones themselves are valuable since
they show biological activity e.g. as mosquito oviposition pheromone [8], as a sex
pheromone in the parasitic wasp Nasonia vitripennis [9], or may be used as chiral
intermediates in natural product synthesis.
To gain more insight into the metabolism of erythro-dihydroxy fatty acids and the
formation of erythro-hydroxylactones, (Æ)-erythro-7,8-dihydroxy(7,8-2H2)tetradecano-
ic acid ((Æ)-erythro-1), (Æ)-erythro-3,4-dihydroxy(3,4-2H2)decanoic acid ((Æ)-erythro-
2), (3S,4R)-3,4-dihydroxy(3-2H1)nonanoic acid ((3S,4R)-3)), and (3R,4S)-3,4-dihy-
droxy(3-2H1)nonanoic acid ((3R,4S)-3) were chemically synthesized, and their bio-
transformation by Saccharomyces cerevisiae was investigated.
Results. – 1. Chemical Synthesis: The (Æ)-erythro-7,8-dihydroxy(7,8-2H2)tetrade-
canoic acid ((Æ)-erythro-1) and (Æ)-erythro-3,4-dihydroxy(3,4-2H2)decanoic acid ((Æ)-
erythro-2) were synthesized via catalytic deuteration (Pd/BaSO4, 2H2) [6] of tetradec-7-
ynoic acid and dec-3-ynoic acid, respectively, followed by OsO4 dihydroxylation [10]
(Scheme 1,a).
(5S,6S)-6-Hydroxydodecano-5-lactone ((5S,6S)-4) was synthesized from methyl
(5E)-dodec-5-enoate by ꢂSharpless asymmetric dihydroxylationꢃ (AD-Mix a) [11] and
acid catalyzed lactonization according to [4]. Oxidation (Dess – Martin periodinane
[12]) of (5S,6S)-4 and reduction with NaBH4 yielded an analytical reference containing
(5S,6S)- and (5S,6R)-4 (Scheme 1,b). Analogously, synthesis of (5R,6R)-4 using AD-
Mix b and subsequent oxidation/reduction yielded a reference mixture of (5R,6R)- and
(5R,6S)-4.
In a similar procedure, the enantiomers (3R,4S)- and (3S,4R)-3-hydroxydecano-4-
lactone ((3R,4S)- and (3S,4R)-5) were synthesized from methyl (3E)-dec-3-enoate
(Scheme 1,c).
(3R,4S)- and (3S,4R)-3,4-dihydroxynonanoic acid ((3R,4S)- and (3S,4R)-3) were
synthesized by ꢂSharpless epoxydationꢃ [13] of (Æ)-oct-1-en-3-ol. Reaction with l-(þ)-
t
tartrate and BuOOH yields (2R,3S)-1,2-epoxyoctan-3-ol (¼(1S)-1-[(2R)-oxiran-2-
yl]hexan-1-ol), subsequent treatment with KCN [14] gave (3R,4S)-3,4-dihydroxyno-
nanenitrile which was subjected to basic saponification resulting in (3R,4S)-3
(Scheme 2). The enantiomer (3S,4R)-3 was synthesized accordingly with d-(ꢀ)-tartrate.
For the synthesis of (3R,4S)- and (3S,4R)-3,4-dihydroxy(3-2H1)nonanoic acid
2
((3R,4S)- and (3S,4R)-3), (Æ)-oct-1-in-3-ol was deuterated with Pd/BaSO4 and H2
resulting in (Æ)-(1,2-2H2)-oct-1-en-3-ol and further treated as described above.

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Scheme 1. Synthesis of a) Deuterated erythro-Dihydroxy Fatty Acids, b) 6-Hydroxydodecano-5-lactone
Enantiomers, and c) 3-Hydroxydecano-4-lactones

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Scheme 2. Chemical Synthesis of (3R,4S)-3 via ꢁSharpless Epoxydationꢂ
However, basic saponification of 3,4-dihydroxy(2,3-2H2)nonanenitrile caused a com-
plete depletion of the 2H label at C(2) resulting in the singly deuterated enantiomers of
2
(3- H1)-3. For the synthesis of (3R,4S)- and (3S,4R)-(1-13C)-3, the reaction with (2R,3S)-
or (2S,3R)-1,2-epoxyoctan-3-ol (Sharpless epoxidation) was carried out with K13CN
(99% 13C). Subsequent saponification gave the enantiomers of erythro-(1-13C)-3.
The 3,4- and 5,6-dihydroxy acids 3, 2, and 6 were lactonized in CH2Cl2/0.5m HCl
(4 :1) into 3-hydroxynonano-4-lactone (7, see Formula in Fig. 1), 3-hydroxydecano-4-
lactone (5), and 6-hydroxydodecano-5-lactone (4), respectively [15].
2. Analysis. 2.1. GC Separations. The erythro-enantiomers (5R,6S and 5S,6R)- of 6-
hydroxydodecano-5-lactone (4), 3R,4S and 3S,4R)- of 3-hydroxydecano-4-lactone (5)
and of 3-hydroxynonano-4-lactone (7), respectively, separated by gas chromatography
with a chiral phase (Lipodexꢀ E) and the elution sequence was determined (Table 1).
Enantiomers of 5-hydroxydodecano-6-lactones (8) were also separated on Lipodexꢀ E,
but the absolute configuration could not be assigned. On an achiral phase (poly-
methylsiloxane phase, DB-1), the diastereoisomers of 6-O-(trimethylsilyl)-4 and of 5-
O-(trimethylsilyl)-8, respectively, co-eluted.
Table 1. Gas Chromatographic Lipodexꢀ E Elution Order of Hydroxylactones. TFA: 6-O-trifluoroacetic
acid ester, w.d.: without derivatization.
Hydroxylactone
Elution sequence
Derivative
erythro-6-hydroxydodecano-5-lactone (4)
erythro-3-hydroxydecano-4-lactone (5)
erythro-3-hydroxynonano-4-lactone (7)
1. (5R,6S)-
1. (3S,4R)-
1. (3S,4R)-
2. (5S,6R)-
2. (3R,4S)-
2. (3R,4S)-
TFA
w.d.
w.d.
2.2. EI-MS of 4. The erythro-6-hydroxydodecano-5-lactones (4) showed an EI-MS
fragmentation pattern comparable to the previously characterized threo-6-hydroxydo-
decano-5-lactones (4) [4]. The 2H-content at C(6) (m/z 188/187) and of the lactone ring
C(5) (m/z 173/172) was analyzed by EI-MS of the C(6)-OH trimethylsilyl ethers of 4:
2
no H depletion could be observed during fermentation of [7,8-2H2]-1.
2.3. EI-MS of 7 and 5. The EI mass spectra of the 3-hydroxy-4-lactones unlabeled 7
and [1-13C]-7, unlabeled 5 and [3,4-2H2]-5, as well as of the 3-O-(trimethylsilyl) ethers of
[3-2H1]-7 and [1-13C]-7 are shown in Figs. 1 – 3. The base peak fragment of 7 with m/z 83

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997
and the abundant peak at m/z 101 (Fig. 1,a) is in accordance with a rearrangement of
the lactone ring under EI conditions, whereby the furan O-atom at the alkyl chain is
recorded as [Me(CH2)4CHO]þ (m/z 101) which eliminated H2O (neutral loss, m/z 83).
These fragment ions of 7 do not carry the isotope labels at 13C(1) or 2HꢀC(3), and the
label content only exhibits with the molecular ion signals m/z 173and 155 ( Fig. 1,b).
The rearrangement of 7 is confirmed by the EI fragmentation pattern of unlabelled and
(3,4-2H2)-5. The abundant fragment ions m/z 115 and 97 of unlabelled 5 (Fig. 2,a) and
the fragmentation pattern of (3,4-2H2)-5 with m/z 116 and 98 (Fig. 2,b) is in accord with
the ions [Me(CH2)5C(2)HO]þ and subsequent neutral loss of H2O. The EI mass spectra
of erythro-5 and threo-5 [4] are identical.
Fig. 1. EI-MS Spectra of a) unlabeled 3-hydroxynonano-4-lactone (7) and b) [1-13C]-7
The EI mass spectra of the 3-O-(trimethylsilyl) ethers of 3-hydroxy(3-2H1)nonano-
4-lactone ((3-2H1)-7) (Fig. 3,a) and 13C-labeled 7 (Fig. 3,b) exhibit major MS fragment
peaks at m/z 117/116 and 102/101 which agree with the increments
[ꢀC(2)HꢀC(3)(2)HOSiMe3]þ and [ꢀC(2)H2C(3)(2)HOSiMe2]þ. All 3-O-(trimethyl-
silyl) ethers of 3-hydroxy-4-lactones with different alkyl-chain length exhibit this EI-
MS fragmentation pattern. Another rearrangement of 3-O-(trimethylsilyl)-(3-2H1)-7

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Fig. 2. EI-MS Spectra of a) unlabeled 3-hydroxydecano-4-lactone (5) and b) [3,4-2H2]-5
.
leads to the fragment ion m/z 188. Here, the radical cation Mþ of 3-O-(trimethylsilyl)-
(3-2H1)-7 eliminates a Me radical from the TMS group, and keten (H2C¼C¼O) from
.
the CO group, resulting in the fragment ion m/z 188 (Mþ ꢀ 15 ꢀ 42) which is bearing
the O-atoms at C(3) and C(4). The signal m/z 173is in accord with the fragment ion
[Me(CH2)5CHOꢀSiMe3]þ (cf. Fig. 3,a). These fragmentations were elucidated also by
18O-labeled isotopomers of 5 (data not shown).
2.4. EI-MS of 8. As stated in Section 2.1, erythro- and threo-5-O-(trimethylsilyl) 5-
hydroxydodecano-6-lactones (8) co-eluted on DB1 (polymethylsiloxane) GC capillary
columns. In contrast to the diastereoisomers of 4, 5 and 7, the EI-MS fragment ion
intensities of the 5-O-(trimethylsilyl) ethers of erythro- and threo-8 were not identical
(data not shown). Unlabeled 5-O-(trimethylsilyl) threo-8 showed fragment ions with
m/z 130 and 201 [4] whereas 5-O-(trimethylsilyl) erythro-8 exhibited an EI-MS
spectrum similar to the EI-MS of 6-O-(trimethylsilyl)-4 with fragment signals at m/z
172 and 157.
3. Incubation Experiments. All substrates were administered to liquid cultures of the
yeast Saccharomyces cerevisiae as free acids. Aliquots were taken from the culture
broth at indicated times, worked up, derivatized, and analyzed by GC/EI-MS.

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Fig. 3. EI-MS Spectra of a) 3-O-(trimethylsilyl)-3-hydroxy(3-2H1)nonano-4-lactone and b) 3-O-(trime-
thylsilyl)-3-hydroxy(1-13C)nonano-4-lactone
3.1. Metabolism of 1. The results of the biotransformation of (Æ)-erythro-7,8-
dihydroxy(7,8-2H2)tetradecanoic acids (1) are summarized in Table 2 and illustrated in
Fig. 4. A possible reaction pathway is shown in Scheme 3. The substrate was nearly
consumed at the end of the fermentation time. During fermentation, two isomeric a-
ketols 7-hydroxy-8-oxo(7-2H1)tetradecanoic acid (9a) and 8-hydroxy-7-oxo(8-2H1)tet-
radecanoic acid (9b) (EI mass spectra are shown in Fig. 5) and hydroxylactones were
identified as major products. In contrast to the biotransformation of (Æ)-threo-7,8-
dihydroxy(7,8-2H2)tetradecanoic acids by S. cerevisiae [4], no decano-4-lactone could
be detected as metabolite. The concentration of the a-ketols 9a/b changed during
fermentation but neither conversion into threo-dihydroxy fatty acids nor depletion of
the 2H-content of 1 was observed. The configuration of 6-hydroxydodecano-5-lactones
(4) was assigned by GC on a chiral phase (Lipodexꢀ E) as the 6-acetoxy-4 derivative.
During fermentation, (5S,6R)-4 was determined with 86% ee. The erythro-5-
hydroxydodecano-6-lactones (8) separated on Lipodexꢀ E and exhibited a similar
enantiomeric distribution, but the absolute configuration of enantiomeric 8 could not

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Table 2. Biotransformation of 150 ppm (Æ)-erythro-7,8-Dihydroxy(7,8-2H2)tetradecanoic Acid (1).
Formation of a-ketols 9a/b and hydroxylactones 4, 8, and 5 during fermentation of S. cerevisiaea) .
Analyte (2H-labeled)
Time [h]
72
96
144
264
4.0
Substrate
(Æ)-erythro-1
80.6
42.1
43.2
Metabolites
Isomeric methyl C14-a-ketols (9a/b)
Methyl 5,6-dihydroxydodecanoate (6)
6-Hydroxydodecano-5-lactone (4)
5-Hydroxydodecano-6-lactone (8)
Methyl 3,4-dihydroxydecanoate (2)
3-Hydroxydecano-4-lactone (5)
Decano-4-lactone
28.5
11.8
26.2
23.3
0
0.2
6.4
15.6
19.1
0
35.9
10.1
30.3
21.7
0
0.1
6.3
19.4
8.0
2.2
11.4
0
1.4
0
0.4
0
5.6
0
Methyl 2-hydroxyoctanoate
0
0
0.2
1.2
a) Concentrations in ppm.
be determined. The configuration of erythro-3-hydroxydecano-4-lactones (5) was
assigned by reference enantiomers. The concentration of 5 increased until the end of
the fermentation and (3R,4S)-5 was formed with 88% ee (Table 3).
3.2. Metabolism of 2. Incubation of (Æ)-erythro-3,4-dihydroxy(3,4-2H2)decanoic
acid (2) yielded 3-hydroxydecano-4-lactone as major product with low optical purity
((3S,4R)-5 ¼ 10% ee). According to the metabolism of erythro-1, no decano-4-lactone
was formed from precursor erythro-2, and no transformation into threo-isomers was
observed (Table 4).
3.3. Metabolism of 3. Incubation of (3R,4S)- and (3S,4R)-3,4-dihydroxy(3-
2H1)nonanoic acids ((3R,4S)- and (3S,4R)-3), respectively, was performed to inves-
tigate differences in the metabolism of the dihydroxy fatty acid enantiomers. Both
enantiomers of 3 were comparably degraded by S. cerevisiae, and 3-hydroxy(3-
2H1)nonano-4-lactones (7) were formed as major products in similar concentrations
(Table 5). As observed before, no nonano-4-lactone was formed from the precursors
(3R,4S)- and (3S,4R)-7 by yeast, respectively.
Discussion. – Naturally occurring unsaturated fatty acids usually exhibit the C¼C
bond with cis-geometry. Oxidations, e.g. by epoxygenases [16] or chemically by
peracids, lead to erythro-epoxides. Generally, the hydrolysis of erythro-epoxides
proceeds under inversion of the configuration resulting in vicinal threo-diols which has
been studied, e.g., from soy bean epoxide hydrolases [17]. Therefore, (Æ)-erythro-9,10-
epoxy stearic acid [3][6] was transformed via threo-diols in fruits and Sporidiobolus
salmonicolor into aroma active enantiomers of dodecano-4-lactone. The biotransfor-
mation of (Æ)-threo-9,10-dihydroxyoctadecanoic acids also lead to dodecano-4-lacton
in S. cerevisiae, however, only (9S,10S)-9,10-dihydroxyoctadecanoic acid served as
(4R)-dodecano-4-lactone precursor whereas the (9R,10R)-enantiomer did not [4]. As
intermediates, threo-6-hydroxytetradecano-5-lactones and threo-5-hydroxy-tetradeca-
no-6-lactones were identified. These results indicate a more complex degradation

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Fig. 4. GC-Chromatogram (methylated, silylated, DB-1 stationary GC phase) of metabolites isolated
from the biotransformation of erythro-7,8-dihydroxytetradecanoic acid (1) (IS ¼ internal standard:
heptadecanoic acid). Traces of (Æ)-threo-3,4-dihydroxydecanoic acid ((Æ)-threo-2) and (Æ)-threo-3-
hydroxydecano-4-lactone ((Æ)-threo-5) were added to emphasize the difference to the erythro-
diastereoisomers.
pathway in S. cerevisiae within g-lactone synthesis. In addition, S. cerevisiae was shown
to hydrolyze the threo-epoxide leukotriene A4 ((5S,6S)-5,6-oxido-7,9-trans-11,14-cis-
eicosatetraenoic acid (LTA4)) into the threo-diol (5S,6S)-dihydroxy-7,9-trans-11,14-cis-
eicosatetraenoic acid, demonstrating retention of the configuration within epoxide
hydrolysis [18]. The degradation of erythro-6,7-dihydroxydodecanoic acids by S.
cerevisiae has been reported elucidating differences in their metabolism to threo-6,7-
dihydroxydodecanoic acids. (Æ)-erythro-6,7-dihydroxydodecanoic acid were trans-
formed into (4S,5R)- and (4R,5S)-5-hydroxydecano-4-lactone in a 69 :31 ratio, whereas
(Æ)-threo-6,7-dihydroxydodecanoic acid yielded (4R,5R)-, (4S,5S)-, and (4S,5R)-5-
hydroxydecano-4-lactone in a 73:19 :8 ratio. Biotransformation of (6 R,7R)-6,7-

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Scheme 3. Degradation of [7,8-2H2]-(Æ)-erythro-1 and Formation of Hydroxylactones 4, 8, and 5 by S.
cerevisiae
dihydroxydodecanoic acid gave (4R,5R)- and (4S,5R)-5-hydroxydecano-4-lactone in a
78 :22 ratio [5]. These results confirm a conversion of the threo-isomer into the erythro-
isomer and the predominant lactonization of the (4S,5R)-5-hydroxydecano-4-lactone.
Therefore, the degradation of (Æ)-erythro-7,8-dihydroxy(7,8-2H2)tetradecanoic acid
((Æ)-erythro-1) and (Æ)-erythro-3,4-dihydroxy(3,4-2H2)decanoic acid ((Æ)-erythro-2),
respectively, was investigated in yeast under biotransformation conditions. The main
metabolites were 3-hydroxydecano-4-lactone (5), 5-hydroxydodecano-6-lactone (8),
and 6-hydroxydodecano-5-lactone (4) as well as isomeric a-ketols. The hydoxylactone
structures were confirmed by chemical synthesis, their EI-MS fragmentations were
elucidated by means of isotopically labeling (2H and 13C), and their elution order by gas
chromatography with a chiral phase (Lipodexꢀ E) was proven by synthetic
enantiomers (except 5-hydroxydodecano-6-lactones (8)). These hydroxylactones can

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Fig. 5. Postulated EI-MS spectra of a-ketols a) 7-hydroxy-8-oxo(7-2H1)tetradecanoic acid (9a) and of b)
8-hydroxy-7-oxo(8-2H1)tetradecanoic acid (9b) (methylated, silylated) isolated from cultures of S.
cerevisiae after incubation of (Æ)-erythro-7,8-dihydroxy(7,8-2H2)tetradecanoic acid (1)
Table 3. Isotopomeric and Enantiomeric 6-Hydroxydodecano-5-lactone (4) and 3-Hydroxydecano-4-
lactone (5) Isolated from Liquid Cultures of S. cerevisiae after Incubation of 150 ppm (Æ)-erythro-7,8-
Dihydroxy(7,8-2H2)tetradecanoic Acid (1)
(5R,6S)-4 [%]
(5S,6R)-4 [%]
(3R,4S)-5 [%]
(3S,4R)-5 [%]
[5,6-2H2]-4
[3,4-2H2]-5
14
86
88
12
serve as chiral building blocks and exhibit bioactivity themselves. The erythro-6-
hydroxy-5-lactones are of particular interest, (5R,6S)-6-acetoxyhexadecan-5-olide is
the major component of the mosquito oviposition attractant pheromone [8], and its
chemical synthesis was performed utilizing the ꢂSharpless asymmetric epoxydationꢃ, as
described previously [19]. In addition, hydroxylactones with anti-tumor and anti-
malarial activities have been published [20]. The lactonization of erythro-3-hydroxy-
decano-4-lactone proceeded with favor to the (3S,4R)-enantiomer starting from

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Helvetica Chimica Acta – Vol. 91 (2008)
Table 4. Biotransformation of 150 ppm (Æ)-erythro-3,4-Dihydroxy(3,4-2H2)decanoic Acid (2). Forma-
tion of 3-hydroxydecano-4-lactone (5)a)b) during fermentation of S. cerevisiae.
Analyte (2H-labeled)
Time [h]
24
48
3.5
16.0
144
312
Substrate: (Æ)-erythro-2
8.7
23.9
2.7
5.4
8.6
13.2
Metabolite: 3-Hydroxydecano-4-lactone (5)
a) Concentrations in ppm. b) (3R,4S)-5 :(3S,4R)-5 ¼ 45 : 55.
Table 5. Biotransformation of 150 ppm (3R,4S)- and of 150 ppm (3S,4R)-3,4-Dihydroxy(3-2H1)nonano-
ic Acid (3). Formation of 3-hydroxynonano-4-lactone (7)a) during fermentation of S. cerevisiae after
144 h incubation time.
Analyte (2H-labeled)
(3R,4S)-[3-2H1]-3
(3S,4R)-[3-2H1]-3
Substrate (3R,4S)-3 or (3S,4R)-3
Metabolites
5.2
7.2
3-Hydroxynonano-4-lactone (7)
Nonano-4-lactone
16.8
0
26.9
0
a) Concentrations in ppm.
substrates 2 and 3. Using the ꢂFischer projectionꢃ, the configuration of (3S,4R)-5 and of
(4S,5R)-5-hydroxydecano-4-lactone at C(4) is d. S. cerevisiae might prefer lactonization
of erythro-diols with d-configuration at C(4). However, biotransformation of 1 ended
up with (3R,4S)-3-hydroxydecano-4-lactone (5) exhibiting l-configuration at C(4),
and therefore the inverse configuration was formed with 88% ee. Within this
biotransformation of 1, (5S,6R)-6-hydroxydodecano-5-lactone (4) was analyzed with
86% ee, thus eliminating higher concentrations of (5S,6R)-5,6-dihydroxydodecanoic
acid (6) from the metabolism via b-oxidation. The yeast b-oxidation exclusively occurs
inside the peroxisomes and the enzyme cascade degrades d-3-hydroxyacylCoA,
whereas l-3-hydroxyacylCoA has to be metabolized alternatively [21]. The d-
configuration at C(3) of (3S,4R)-3,4-dihydroxydecanoic acid (2) accords with the b-
oxidation stereoselectivity. Therefore, we assume the accumulation of (3R,4S)-3,4-
dihydroxydecanoic acid during transformation of 1 finally leading to (3R,4S)-3-
hydroxydecano-4-lactone (5). Since the configuration of the dihydroxy acids cannot be
determined by GC on chiral phases, their configuration can only be postulated by
analyzing the hydroxylactones. The stereoselective synthesis of chiral hydroxylactones
from (Æ)-erythro-dihydroxy fatty acids by S. cerevisiae is of preparative interest
because the precursors are easily available by catalytic OsO4 dihydroxylation of natural
cis-configured fatty acids.
Additionally to the characterization of hydroxylactones, a main focus was the
disclosure of the metabolic fate of erythro-dihydroxy fatty acids. During fermentation,
isomeric a-ketols were identified, but, no degradation products were characterized.
Generally, ketones can undergo a Baeyer – Villiger oxidation with peracids, and fatty
acid metabolism in yeasts proceeds inside the peroxisomes where H2O2 is present. The
genome of the yeast Saccharomyces cerevisiae was unsuccessfully screened for a DNA

Helvetica Chimica Acta – Vol. 91 (2008)
1005
sequence that is comparable to the bacterial Baeyer – Villiger monooxygenase
(BVMO) encoding gene. Therefore, a bacterial BVMO gene was overexpressed in S.
cerevisiae [22]. However, the yeast Sporobolomyces odorus endogenously metabolizes
the g- and d-lactone precursors 4- and 5-hydroxy decanoic acids via 4- and 5-
oxodecanoic acids, which are further metabolized by a Baeyer – Villiger type oxidation.
The Baeyer – Villiger esters have been characterized [23] and we assume a comparable
metabolic fate of the a-ketols 9a/b.
Experimental Part
1. General. LC: Liquid chromatography; GC/EI-MS: Gas chromatography/electron impact mass
spectrometry: fused silica DB-1 cap. column (poly(dimethylsiloxan)), 60 m ꢁ 0.32 mm i.d., 0.25 mm film
(J&W Scientific, Folsome, CA), temp. program 4 min at 808, then 48/min to 2808, 100 kPa He (4.0); or
Lipodexꢀ E (octakis-(3-O-butyryl-2,6-di-O-pentyl-)-g-cyclodextrin, 50 m ꢁ 0.25 i.d., 0.25 mm film (Ma-
cherey und Nagel, D-Düren)), temp. program 10 min at 708, then 48/min to 2208, 100 kPa He (4.0); Carlo
Erba-Fractovap 4160 coupled by a heated transfer line to a double focusing electron impact (EI)
1
ionization Varian-Mat 8230 mass spectrometer, ionization energy 70 eV; m/z (intensity in %). H- and
13C-NMR spectra: AMX-500 spectrometer (Bruker, D-Karlsruhe); chemical shifts d in ppm rel. to Me4Si
(0 ppm) as external standard, J in Hz, signal assignment by H,H-COSY and H,C-HETCOR.
2. Dihydroxy Fatty Acids. 2.1. (Æ)-erythro-7,8-Dihydroxy(7,8-2H2)tetradecanoic Acid ((Æ)-erythro-
1). As described for erythro-6,7-dihydroxy(6,7-2H2)dodecanoic acid in [5]. 1,5-Dibromopentane (46 g,
0.2 mol) was coupled with lithiated oct-1-yne, and the resulting 1-bromotridec-6-yne was substituted with
NaCN (19.6 g, 0.4 mol) to give tetradec-7-ynenitrile (9.2 g, 45 mmol) in a 45% overall yield.
Saponification of the nitrile with Na2O2 afforded tetradec-7-ynoic acid which was hydrogenated (Pd
2
5% on BaSO4) with H2 gas to give (7Z)-(7,8-2H2)tetradec-7-enoic acid. Dihydroxylation using OsO4
yielded (Æ)-erythro-7,8-dihydroxy(7,8-2H2)tetradecanoic acid ((Æ)-erythro-1, 5.5 g, 25 mmol, 55% yield
based on tetradec-7-ynenitrile). Mass spectra of the intermediates were identical to the data of threo-1 in
1
2
the literature [4]. H-NMR (CDCl3): 3.21 – 3.38 (m, HꢀC(7), HꢀC(8), 0.16 H, i.e. 92% H-labeling);
2.22 (t, J ¼ 7, CH2COOH); 1.05 – 1.51 (m, CH2, 18 H); 0.81 (t, J ¼ 7, Me). GC/MS: 405 (3, [M ꢀ Me]þ),
305 (30, [232 þ TMS]þ), 232 (100), 188 (90), 147 (20), 128 (20), 73 (80).
2.2. (Æ)-erythro-3,4-Dihydroxy(3,4-2H2)decanoic Acid ((Æ)-erythro-2). As described for (Æ)-threo-
3,4-dihydroxy(3,4-2H2)decanoic acids in [4]. In brief, but-3-yn-1-ol and dihydropyran were coupled to 2-
(but-3-ynyloxy)tetrahydropyran which was lithiated (BuLi) and coupled with 1-bromohexane in DMPU
to 2-(decynyloxy)tetrahydropyran. The THP ether was cleaved with TsOH in MeOH to dec-3-yn-1-ol.
Jones oxidation (CrO3/H2SO4) of dec-3-yn-1-ol to dec-3-ynoic acid and subsequent hydrogenation (Pd,
BaSO4) with H2 gas yielded (3Z)-(3,4-2H2)dec-3-enoic acid (1.21 g, 7 mmol, 15% based on 3-butyn-1-
2
ol). According to the synthesis of (Æ)-erythro-1, 1.21 g (3Z)-(3,4-2H2)dec-3-enoic was dihydroxylated
with stoichiometric amounts of OsO4 in tBuOOH. LC of the crude dihydroxy acid gave (Æ)-erythro-3,4-
dihydroxy(3,4-2H2)decanoic acid ((Æ)-erythro-6, 0.7 g 3.4 mmol, 49%). 1H-NMR (CDCl3): 3.29 – 3.38
(m, HꢀC(3), HꢀC(4), 0.10 H, i.e. 95% 2H-labeling); 2.43(br. s, CH2(2)); 1.15 – 1.60 (m, CH2(5) to
CH2(9)); 0.83( t, J ¼ 7, Me ) .
2.3. Asymmetric Dihydroxylation (AD). Formation of methyl threo-3,4-dihydroxydecanoates and
methyl threo-5,6-dihydroxydodecanoates. AD was performed by the ꢂSharpless ADꢃ method as described
essentially in [5]. Methyl (E)-alkanoates yielded (S,S)-dihydroxy fatty acid methyl esters with ꢂAD-Mix
aꢃ and vice versa.
Methyl (3E)-Dec-3-enoate. Under N2, malonic acid (propanedioic acid, 10.4 g, 0.1 mol) was
dissolved in dry DMSO (50 ml), and octanal (6.4 g, 50 mmol) was added. Subsequently, a freshly
prepared soln. of piperidine (85 mg, 1 mmol) and AcOH (60 mg, 1 mmol) in 0.5 ml dry DMSO was
added and stirred for 20 min at r.t. The mixture was heated to 858 for 4 h, cooled to r.t., poured on 100 ml
ice water and extracted three times with Et2O (each 50 ml). The org. phase was washed (H2O), dried
(Na2SO4), and evaporated. The crude (3E)-dec-3-enoic acid was subjected to methylation (HCl/MeOH)

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Helvetica Chimica Acta – Vol. 91 (2008)
and purification by LC (100 g SiO2, petroleum ether (PE)/ethyl acetate (AcOEt) 90 :10) yielded methyl
(3E)-dec-3-enoate (7.4 g 40 mmol, 80% based on octanal) similar to products reported in [4].
Methyl (5E)-dodec-5-enoate. The synthesis has been reported previously [4].
2.4. (3R,4S)- and (3S,4R)-3,4-Dihydroxy(1-13C)nonanoic acid ((3R,4S)-3 and (3S,4R)-3). 1,2-
Epoxyoctan-3-ol ( ¼ 1-(oxiran-2-yl)hexan-1-ol). At ꢀ 208, Ti(OiPr)4 (14.3g, 50 mmol), ( þ)-l- or (ꢀ)-d-
diethyl tartrate (12.4 g, 60 mmol) and oct-1-en-3-ol (6.3 g, 50 mmol) were stirred in 150 ml abs. CH2Cl2,
and abs. tert-butyl hydroperoxide (2.7 g, 30 mmol) was added. The homogenous soln. was stored at ꢀ 218
for 21 d. For work up, acetone (ꢀ218, 350 ml) and H2O (15 ml) were added and the mixture was stirred
until r.t. was reached. The mixture was centrifuged (5000 ꢁ g), the supernatant was chilled to ꢀ 208,
stirred, and a soln. of tartaric acid (10% in H2O, 125 ml) was added slowly and warmed up to r.t. during
90 min. The phases were separated and the org. phase was washed (H2O, 100 ml), dried (Na2SO4), and
evaporated. The resulting oil was dissolved in Et2O (375 ml), and at 08, 1m NaOH (150 ml) was added
and the mixture was stirred for 30 min. The phases were separated, and the Et2O phase was washed three
times with brine (each 50 ml), evaporated and purified by LC (50 g SiO2, 500 ml PE/AcOEt (18 :2)):
2.8 g (80%) of (1S)-1-[(2R)-oxiran-2-yl]hexan-1-ol (from (þ)-l-diethyl tartrate) and 2.7 g (78%) of
(1R)-1-[(2S)-oxiran-2-yl]hexan-1-ol (from d-(ꢀ)-diethyl tartrate) were isolated, resp., as well as the
.
remaining enantiomers (3R)- and (3S)-oct-1-en-3-ol (data not shown). GC/MS: 144 (1, Mþ ), 101 (15),
83 (80), 73 (35), 57 (30), 55 (100), 45 (30), 43 (40), 41 (45).
erythro-3,4-Dihydroxy(1-13C)nonanoic Acid: At 208, (1S)-1-[(2R)-oxiran-2-yl]hexan-1-ol or (1R)-1-
[(2S)-oxiran-2-yl]hexan-1-ol (500 mg, 7.5 mmol) were dissolved in 15 ml MeOH, K13CN (0.5 g, 7.5 mmol)
were added, and the mixture was stirred overnight. The solvent was evaporated, the residue dissolved in
H2O (10 ml) and acidified at 08 with 1m HCl. The residue was extracted twice with Et2O (each 50 ml),
dried (Na2SO4), and evaporated, whereby (2R,3S)- or (2S,3R)-3,4-dihydroxy(1-13C)nonanenitrile was
isolated: 3,4-dihydroxy-(1-13C)nonanoic acid nitrile (3-O-,4-O-bis-trimethylsilylether). GC/MS: 228 (5),
210 (15), 187 (5), 173(25), 144 (15), 143(18), 142 (10), 116 (100), 101 (40), 75 (70), 73(60), 55 (20), 45
(20), 43(20), 41 (20).
The crude nitrile was dissolved in H2O (5 ml) and NaOH (0.6 g, 15 mmol) and stirred for 24 h. The
mixture was heated to 808 and a moderate N2 stream was bubbled through the soln. until no NH3 was
detected. The mixture was acidified with 1m H3PO4 to pH 5, extracted three times with Et2O (each
30 ml), and purified by LC (50 g SiO2, 500 ml PE/AcOEt/AcOH (15 :5 :0.2)): 200 mg (30% based on the
epoxide) (3R,4S)-3,4-dihydroxy(1-13C)nonanoic acid ((3R,4S)-3 and 210 mg (32%) (3S,4R)-3,4-
dihydroxy(1-13C)nonanoic acid (3S,4R)-3).
(3R,4S)- and (3S,4R)-3,4-Dihydroxy(3-2H1)nonanoic Acid. The deuterated enantiomers of erythro-
3,4-dihydroxynonanoic acids were synthesized according to the [1-13C]-labeled compounds. (1,2-2H2)-oct-
1-en-3-ol was synthesized by hydrogenation (Pd (BaSO4), 2H2) of oct-1-yne-3-ol. (1,2-2H2)-oct-1-en-3-ol:
GC/MS: 112 (2, [M ꢀ H2Oþ), 101 (3), 99 (4), 83(7), 74 (15), 60 (20), 59 (100), 58 (25), 43(30), 41 (20).
Methyl (3R,4S)-3,4-Dihydroxy[3-2H1]nonanoate (bis(trimethylsilyl) ether). GC/MS: 332 (1, [M ꢀ
15]þ), 318 (3), 249 (25), 244 (5), 243(5), 173(75); 147 (20), 103(29), 73(100), 45 (10).
3. Hydroxylactones. 3,4- And 5,6-dihydroxy fatty acids or their corresponding methyl esters (1 –
100 mg) were lactonized in 0.5m HCl/CH2Cl2 overnight at 208. The phases were separated, the CH2Cl2
phase was neutralized (NaHCO3), dried (Na2SO4), and evaporated.
(3S,4R)- and (3R,4S)-3-Hydroxydecano-4-lactone ((3S,4R)- and (3R,4S)-5). The enantiomers
(3R,4R)- and (3S,4S)-5 were oxidized at OHꢀC(3) with ꢂDess – Martin periodinaneꢃ [12] to yield
enantiomers (4R)- and (4S)-3-oxodecano-4-lactone, resp., which were reduced with NaBH4 in Et2O to
give (3R,4R)/(3R,4S)-5 and (3S,4S)/(3S,4R)-5, resp. Accordingly, (5R,6R)- and (5S,6S)-6-hydroxydo-
decano-5-lactones (4) were oxidized into (5R)- and (5S)-6-oxododecano-5-lactones, resp., and NaBH4
reduced to (5R,6R)/(5R,6S)-4 and (5S,6S)/(5S,6R)-4, resp. The diastereomeric lactones could be
separated by LC (SiO2, PE/AcOEt 75 :25).
(Æ)-erythro-6-Hydroxydodecano-5-lactone (4). 1H-NMR (CDCl3): 4.18 – 4.24 (m, HꢀC(5)); 3.72 –
3.80 (m, HꢀC(6); 2.52 – 2.58 (m, HaꢀC(2)); 2.36 – 2.44 (m, HbꢀC(2)); 1.68 – 1.97 (m, CH2(3),
CH2(4)); 1.40 (q, J ¼ 7, CH2(7)); 1.18 – 1.32 (m, CH2, 8 H); 0.83( t, J ¼ 7, Me ) . 13C-NMR: 171.93
(C(1)); 83.51 (C(5)); 72.19 (C(6)); 31.64 (C(7)); 31.63; 29.70 (C(2)); 29.12; 25.80; 22.50; 21.04 (C(4));
18.22 (C(3)); 13.99 (C(12)).

Helvetica Chimica Acta – Vol. 91 (2008)
1007
(3S,4R)-3-Hydroxy(1-13C)nonano-4-lactone ((3S,4R)-7): 1H-NMR (CDCl3): 4.27 – 4.35 (m,
HꢀC(4)); 4.16 – 4.24 (m, HꢀC(3)); 2.42 – 2.49 (m, HaꢀC(2)); 2.71 – 2.83( m, HbꢀC(2)); 1.47 – 1.62
(m, CH2(5)); 1.30 – 1.46 (m, CH2(6)); 1.20 – 1.30 (m, CH2, 4 H); 0.89 (t, J ¼ 6.5, Me). 13C-NMR: 176.3
(C(1)); 88.40 (C(4)); 71.18 (C(3)); 37.43 (d, J ¼ 49 (C(2)); 32.78 (C(5)); 31.26; 24.75; 22.29 (C(6)); 13.82
(C(9)).
4. Yeast Strain, Culture Conditions, Sampling and Work Up. Yeast (Saccharomyces cerevisiae IfG
06136) was obtained from the culture collection of the ꢂInstitut für Gärungstechnologieꢃ, Berlin. The
organism was stored (48) on Wort agar-agar slants. For metabolic experiments cells were cultivated in a
medium (200 ml) consisting of 6.0 g/l glucose, 3.0 g/l MgSO4, 2.5 g/l (NH4)2SO4, 2.5 g/l KH2PO4, 2.5 g/l
l-alanine and 3.0 g/l yeast extract (pH 5.5) on a horizontal shaker at 178 and 100 rpm. The pre-culture
(10 ml) was inoculated into 200 ml fresh culture medium and 30 mg (Æ)-erythro-7,8-dihydroxy(7,8-
2H2)tetradecanoic acid ((Æ)-erythro-1), 30 mg (Æ)-erythro-3,4-dihydroxy(3,4-2H2)decanoic acid ((Æ)-
erythro-2), 30 mg (3R,4S)-3,4-dihydroxy(3-2H1)nonanoic acid ((3R,4S)-3), or 30 mg (3S,4R)-3,4-
dihydroxy(3-2H1)nonanoic acid ((3S,4R)-3), each in 250 ml EtOH were immediately administered to
different flasks. At certain times (24, 48, 72, 96, 120, 144, 168, 216, 268, 312 h), aliquots (10 ml) of the
culture broth were taken, internal standards (5 ppm of 1-decanol, d-octalactone and heptadecanoic acid)
were added, extracted with Et2O (2 ꢁ 20 ml), dried (Na2SO4), methylated with diazomethane and
converted into derivatives (silyl-ether or trifluoroacetyl-ester) under standard conditions [24] if
necessary.
REFERENCES
[1] K. R. Schmelzer, L. Kubala, J. W. Newman, I.-H. Kim, J. P. Eiserich, B. D. Hammock, Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 9772.
[2] O. Jung, R. P. Brandes, I.-H. Kim, F. Schweda, R. Schmidt, B. D. Hammock, R. Busse, I. Fleming,
Hypertension 2005, 45, 759.
[3] M. Schçttler, W. Bohland, Helv. Chim. Acta 1996, 79, 1488.
[4] L.-A. Garbe, R. Tressl, Helv. Chim. Acta 2004, 87, 180.
[5] L.-A. Garbe, R. Tressl, Helv. Chim. Acta 2003, 86, 2349.
[6] T. Haffner, R. Tressl, Lipids 1998, 33, 47.
[7] S. S. Gill, B. D. Hammock, Biochem. Biophys. Res. Commun. 1979, 89, 965.
[8] B. R. Laurence, J. A. Pickett, J. Chem. Soc., Chem. Commun. 1982, 59.
[9] J. Ruther, L. M. Stahl, S. Steiner, L. A. Garbe, T. Tolasch, J. Exp. Biol. 2007, 210, 2163.
[10] M. Hamberg, R. P. Herman, U. Jacobsson, Biochim. Biophys. Acta – Lipids and Lipid Metabolism
1986, 879, 410.
[11] R. A. Johnson, K. B. Sharpless, in ꢂCatalytic Asymmetric Synthesisꢃ, Ed. I. Oijama, Wiley VCH, New
York, 1993, p. 227.
[12] D. B. Dess, J. C. Martin, J. Org. Chem. 1983, 48, 4155.
[13] T. Katsuki, K. B. Sharpless, J. Am. Chem. Soc. 1980, 102, 5974.
[14] C. H. Behrens, S. Y. Ko, K. B. Sharpless, F. J. Walker, J. Org. Chem. 1985, 50, 5687.
[15] J. Turk, T. Stump, W. Conrad-Kessel, R. R. Seabold, B. A. Wolf, Methods Enzymol. 1990, 187, 175.
[16] R. Croteau, P. E. Kolattukudy, Arch. Biochem. Biophys. 1975, 170, 61.
´
[17] E. Blee, F. Schuber, Biochem. J. 1992, 282, 711.
[18] F. Kull, E. Ohlson, J. Z. Haeggstrçm, J. Biol. Chem. 1999, 274, 34683.
[19] K. Mori, T. Otsuka, Tetrahedron 1983, 39, 3267.
[20] E. A. Couladouros, A. P. Mihou, Tetrahedron Lett. 1999, 40, 4861.
[21] S. A. Filppula, R. T. Sormunen, A. Hartig, W.-H. Kunau, J. K. Hiltunen, J. Biol. Chem. 1995, 270, 27453.
[22] J. D. Stewart, K. W. Reed, M. M. Kayser, J. Chem. Soc., Perkin Trans. 1 1996, 755.
[23] L.-A. Garbe, R. Tressl, Chem. Biodivers. 2004, 1, 900.
[24] T. Haffner, R. Tressl, J. Agric. Food Chem. 1996, 44, 1218.
Received February 15, 2008