Metabolism of deuterated isomeric 6,7-dihydroxydodecanoic acids in Saccharomyces cerevisiae - Diastereo- and enantioselective formation and characterization of 5-hydroxydecano-4-lactone (=4,5-dihydro-5-(1-hydroxyhexyl)furan-2(3H)-one) isomers

Article abstract of DOI:10.1002/hlca.200390189

The chemical synthesis of deuterated isomeric 6,7-dihydroxydodecanoic acid methyl esters 1 and the subsequent metabolism of esters 1 and the corresponding acids 1a in liquid cultures of the yeast Saccharomyces cerevisiae was investigated. Incubation experiments with (6R,7R)- or (6S,7S)-6,7-dihydroxy(6,7-²H₂)dodecanoic acid methyl ester ((6R,7R)- or (6S,7S)-(6,7-²H₂)-1, resp.) and (±)-threo- or (±)-erythro-6,7-dihydroxy(6,7-²H ₂)dodecanoic acid ((±)-threo- or (±)-erythro-(6,7- ²H₂)-1a, resp.) elucidated their metabolic pathway in yeast (Tables 1-3). The main products were isomeric ²H-labeled 5-hydroxydecano-4-lactones 2. The absolute configuration of the four isomeric lactones 2 was assigned by chemical synthesis via Sharpless asymmetric dihydroxylation and chiral gas chromatography (Lipodex E). The enantiomers of threo-2 were separated without derivatization on Lipodex E; in contrast, the enantiomers of erythro-2 could be separated only after transformation to their 5-O-(trifluoroacetyl) derivatives. Biotransformation of the methyl ester (6R,7R)-(6,7-²H₂)-1 led to (4R,5R)- and (4S,5R)-(2,5- ²H₂)-2 (ratio ca. 4:1; Table 2). Estimation of the label content and position of (4S,5R)-(2,5-²H₂)-2 showed 95% label at C(5), 68% label at C(2), and no ²H at C(4) (Table 2). Therefore, oxidation and subsequent reduction with inversion at C(4) of 4,5-dihydroxydecanoic acid and transfer of ²H from C(4) to C(2) is postulated. The 5-hydroxydecano-4-lactones 2 are of biochemical importance: during the fermentation of Streptomyces griseus, (4S,5R)-2, known as L-factor, occurs temporarily before the antibiotic production, and (-)-muricatacin (=(4R,5R)-5-hydroxy-heptadecano-4-lactone), a homologue of (4R,5R)-2, is an anticancer agent.

Full text of DOI:10.1002/hlca.200390189

Helvetica Chimica Acta Vol. 86 (2003)
2349
Metabolism of Deuterated Isomeric 6,7-Dihydroxydodecanoic Acids in
Saccharomyces cerevisiae Diastereo- and Enantioselective Formation and
Characterization of 5-Hydroxydecano-4-lactone (ˆ4,5-Dihydro-5-
(1-hydroxyhexyl)furan-2(3H)-one) Isomers
by Leif-A. Garbe* and Roland Tressl
Technische Universit‰t Berlin, Institut f¸r Biotechnologie, Chemisch-technische Analyse, Seestrasse 13,
D-13353 Berlin
(phone: ‡49-30-45080-231; fax: ‡49-30-314-27544; e-mail: Leif-A.Garbe@TU-Berlin.de)
The chemical synthesis of deuterated isomeric 6,7-dihydroxydodecanoic acid methyl esters 1 and the
subsequent metabolism of esters 1 and the corresponding acids 1a in liquid cultures of the yeast Saccharomyces
cerevisiae was investigated. Incubation experiments with (6R,7R)- or (6S,7S)-6,7-dihydroxy(6,7-2H2)dodecanoic
acid methyl ester ((6R,7R)- or (6S,7S)-(6,7-2H2)-1, resp.) and (Æ)-threo- or (Æ)-erythro-6,7-dihydroxy(6,7-
2H2)dodecanoic acid ((Æ)-threo- or (Æ)-erythro-(6,7-2H2)-1a, resp.) elucidated their metabolic pathway in yeast
(Tables 1 3). The main products were isomeric 2H-labeled 5-hydroxydecano-4-lactones 2. The absolute
configuration of the four isomeric lactones 2 was assigned by chemical synthesis via Sharpless asymmetric
¾
dihydroxylation and chiral gas chromatography (Lipodex E). The enantiomers of threo-2 were separated
¾
without derivatization on Lipodex E; in contrast, the enantiomers of erythro-2 could be separated only after
transformation to their 5-O-(trifluoroacetyl) derivatives. Biotransformation of the methyl ester (6R,7R)-(6,7-
2H2)-1 led to (4R,5R)- and (4S,5R)-(2,5-2H2)-2 (ratio ca. 4 :1; Table 2). Estimation of the label content and
position of (4S,5R)-(2,5-2H2)-2 showed 95% label at C(5), 68% label at C(2), and no 2H at C(4) (Table 2).
Therefore, oxidation and subsequent reduction with inversion at C(4) of 4,5-dihydroxydecanoic acid and
transfer of 2H from C(4) to C(2) is postulated. The 5-hydroxydecano-4-lactones 2 are of biochemical
importance: during the fermentation of Streptomyces griseus, (4S,5R)-2, known as L-factor, occurs temporarily
before the antibiotic production, and (À)-muricatacin (ˆ(4R,5R)-5-hydroxy-heptadecano-4-lactone),
a
homologue of (4R,5R)-2, is an anticancer agent.
Introduction. The b-oxidation of fatty acids in the yeast Saccharomyces cerevisiae
occurs inside the peroxisomes and is well-described [1][2]. In contrast, little is known
about the metabolism of oxygenated fatty acids in yeast. Common enzymes introducing
O-atoms into fatty acids are lipoxygenases, dioxygenases, and monooxygenases. In the
mammalian cell, the cytochrome-P450-dependent monooxygenases transforming (Z)-
unsaturated fatty acids into cis-epoxy acids are well-investigated, and further metabolic
products are dihydroxy-substituted fatty acids [3]. Enzymes that utilize hydroperoxide
as O-source are summarized in the terms −epoxygenases× or −peroxygenases× [4].
Epoxy fatty acids are chiral compounds. They can be found in mammalian and
microbial cells but also in many plants, where they are involved in defense mechanisms
[5]. Rice plants, e.g., utilize linoleic acid (ˆ(9Z,12Z)-octadeca-9,12-dienoic acid) to
produce (‡)-coronaric acid (ˆ(9R,10S,12Z)-9,10-epoxyoctadec-12-enoic acid) [6],
seeds from Euphorbia sp. contain optically active (‡)-vernolic acid ((9Z,12S,13R)-
12,13-epoxyoctadec-9-enoic acid) [7].

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Incubation experiments with enantiomerically pure (‡)-vernolic acid were
performed with Sporobolomyces odorus [8], and (4R,5R)-5-hydroxydecano-4-lactone
((4R,5R)-2) was formed as the final product with 93.8% ee. The reference enantiomers
of 2 were synthesized from amino acids and analyzed by achiral GC as derivatives
obtained by reaction with (1R)-1-phenylethyl isocyanate. In analogous experiments,
(Æ)-vernolic acid was converted to (4R,5R)-2 with 74% ee., and 6,7-dihydroxydode-
canoic acid (1a) was characterized as an intermediate [9]. The yeast Saccharomyces
cerevisiae metabolized (Æ)-vernolic acid to mixtures of threo- and erythro-2 (Scheme 1)
[10].
Scheme 1. Biotransformation of (Æ)-Vernolic Acid in Cultures of Saccharomyces cerevisiae. The isomeric 5-
hydroxydecano-4-lactones 2 were the main products in the culture broth.
The 5-hydroxydecano-4-lactones 2 are of biochemical importance. During the
fermentation of Streptomyces griseus, the enantiomer (4S,5R)-2 occurs temporarily
before the antibiotic production and is known as L-factor in the literature [11][12]. Due
to the biological activity of 2, many contributions have been published concerning the
chemical and chemoenzymatic synthesis of the four isomers of 2 [13 17]. Sharpless
asymmetric dihydroxylation (Sharpless AD) [18] has been utilized to synthesize (‡)-
epimuricatacin and (À)-muricatacin (muricatacin ˆ 5-hydroxyheptadecano-4-lactone)
[19][20].
Investigations of Blee and Schuber demonstrated the hydrolysis of (Æ)-coronaric
acid by purified soybean epoxide hydrolase to (9R,10R,12Z)-9,10-dihydroxyoctadec-
12-enoic acid [21]. Therefore, the configuration of epoxy fatty acids cannot be deduced

Helvetica Chimica Acta Vol. 86 (2003)
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by analysis of the corresponding dihydroxy fatty acids or further degradation products
(e.g., lactones). To get more insight into the biotransformation of 12,13-epoxyoleates to
5-hydroxydecano-4-lactones 2, deuterated 6,7-dihydroxydodecanoic acids 1a and their
methyl esters 1 were used as precursors, i.e., (Æ)-threo- and (Æ)-erythro-(6,7-2H2)-1a
and (6R,7R)- and (6S,7S)-(6,7-2H2)-1.
Results. 1. Synthesis of Isomeric Methyl 6,7-dihydroxy(6,7-2H2)dodecanoates 1,
Corresponding Acids 1a, and 5-Hydroxydecano-4-lactones (2). The enantiomeric
methyl esters (6R,7R)- and (6S,7S)-(6,7-2H2)-1 were synthesized by Sharpless AD [18]
of methyl (6E)-(6,7-2H2)dodec-6-enoate. The enantiomers (6R,7R)- and (6S,7S)-1
could not be separated by chiral GC or HPLC. The Sharpless AD of methyl (E)-
alkenoates of different C-chain lengths usually showed high stereoselectivity. Thus, AD
of methyl (4E)-dec-4-enoate and lactonization of methyl 4,5-dihydroxydecanoate
yielded (4R,5R)- and (4S,5S)-2 with >98% ee.; the hydroxy-4-lactone enantiomers
could be separated by chiral GC. −AD-Mix a× and −AD mix b× are reported to yield high
amounts of threo-dihydroxy fatty acids, e.g., methyl 9,10-dihydroxyoctadecanoate with
>95% ee. [22]. Therefore, we assume a high ee. also for the Sharpless AD of methyl
(6E)-(6,7-2H2)dodec-6-enoate yielding esters (6S,7S)- and (6R,7R)-(6,7-2H2)-1.
Racemic isomeric acids (Æ)-threo- and (Æ)-erythro-(6,7-2H2)-1a were synthesized
by standard dihydroxylation procedures of (6Z)-(6,7-2H2)dodec-6-enoic acid (threo: 1.
H2O2/HCOOH 2. NaOH; erythro: OsO4). Deuteration of the (6Z)- and (6E)-double
bond was >97% (1H-NMR).
Diastereoisomers of 2 were analyzed on achiral (DB-1, DB-Wax) and enantiomers
¾
of 2 on chiral (Lipodex E) GC phases. Enantiomers (4R,5R)- and (4S,5S)-2 were
synthesized by Sharpless AD [18] from methyl (4E)-dec-4-enoate. Acid-catalyzed
lactonization of methyl 4,5-dihydroxydecanoates gave 2 with high ee. (> 98%; chiral
GC). Dess-Martin oxidation of (4R,5R)-2 led to (4R)-5-oxodecano-4-lactone, and a
NaBH4 reduction yielded an analytic reference, i.e., (4R,5S)-2. An analogous experi-
ment with (4S,5S)-2 generated (4S,5R)-2. The diastereoisomers of 2 were separated on
DB-1 after formation of their 5-O-(trimethylsilyl)ethers in the order erythro-2
¾
derivative > threo-2 derivative. On chiral GC Lipodex E phases, the free isomers
of 2 were eluted in the order (4S,5S)-2 > (4R,5R)-2 > (Æ)-erythro-2 (no resolution of
the erythro-enantiomers), and their 5-O-(trifluoroacetyl) derivatives in the order
(4R,5S)-2 > (4S,5R)-2 > (Æ)-threo-2 (no resolution of threo-enantiomers) (see below,
Fig.4 ).
2. Incubation Experiments. The results of the incubations of racemic acid (Æ)-threo-
(6,7-2H2)-1a and enantiomeric esters (6R,7R)- and (6S,7S)-(6,7-2H2)-1 with S.cerevisiae
are summarized in Tables 1 3. During the course of the fermentation of the racemic
acids 1a and enantiomeric methyl esters 1, the substrates were metabolized to
deuterated hydroxy-oxo acids and 5-hydroxydecano-4-lactones as major products. One
cycle of b-oxidation of 1 or 1a would lead to 4,5-dihydroxydecanoic acids, which were
not detectable in the fermentation broth. b-Oxidation to 2,3-dihydroxyoctanoate was
not observed.
In incubation experiments of (Æ)-threo-(6,7-2H2)-1a, four isomeric 5-hydroxy-4-
lactones 2 were characterized as doubly labeled isotopomers with a yield of 3 4%
(Table 1). The ratio threo-2/erythro-2 was analyzed as 92 :8. Chiral analysis of the

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2
Table 1. Total Amounts [ppm], Relative Ratios, and Distribution of the H-Label of Isomeric-5-Hydroxy(2Hx)-
decano-4-lactones 2 after Incubation of 150 ppm of (Æ)-threo-6,7-Dihydroxy(6,7-2H2)dodecanoic Acid 1a in
Liquid Cultures of Saccharomyces cerevisiae. R1 ˆ Me(CH2)3.
Amount
Ratio
2H-Label at C(2)
2H-Label at C(4)
2H-Label at C(5)
2.85 ppm
72.9%
0%
95%
95%
0.76 ppm
19.4%
n.d.
95%
95%
0.02 ppm
0.5%
n.d.
n.d.
n.d.
0.28 ppm
7.2%
86%
0%
95%
products indicated (4R,5R)-2 as major metabolite (72.9%) besides 19.4% of (4S,5S)-2,
7.2% of (4S,5R)-2, and only 0.5% of (4R,5S)-2.
The main products formed from methyl ester (6R,7R)-(6,7-2H2)-1 were (4R,5R)-2
and (4S,5R)-2. They accumulated in a ratio of 78.5 :21.5 with a total yield of 44%
(Table 2). The separation of the isomers of 2 showed unequivocally the inversion of the
configuration at C(4) from (4R) to (4S).
This inversion might be explained by oxidation to a keto acid followed by a
reduction step. We identified isomeric a-ketols, i.e., methyl 6-hydroxy-7-oxododeca-
noate and methyl 7-hydroxy-6-oxododecanoate (see below, Fig.1 ), during the
metabolism of 1 and 1a by GC/MS as major products. They had a maximum in
concentration after 168 h, and their amount decreased until the end of fermentation.
But no reduction of the a-ketols into erythro-1 and no 2H-depletion at position C(6) or
C(7) of the substrate 1 or 1a was observed by GC/MS. The identification of a doubly
labeled (4S,5R)-2 is not in agreement with an oxidation/keto acid reduction step.
3. Analysis of Isotopomers. The 2H-content of the substrates 1 and 1a (methylated,
silylated) was analyzed during the incubation period by GC/EI-MS by means of the
2
characteristic fragments m/z 173/174 and 217/218 (see Fig.1 ) [23]. No significant H-
depletion was observed for (6,7-2H2)-1 and -1a. The label content of the side chain of 2
(position C(5)) was measured by analyzing m/z 173/174 (1H1/2H1) (a-cleavage)
comparable to silylated dihydroxy fatty acid esters. The 2H-content of the lactone ring
was quantified by m/z 158/159 (1H1/2H1). The Me3Si-group migration in 5-O-
(trimethylsilyl)derivatives to the lactone moiety is revealed by Fig.2 . The Me3Si-

Helvetica Chimica Acta Vol. 86 (2003)
2353
Table 2. Total Amounts [ppm], Relative Ratios, and Distribution of the 2H-Label of Isomeric-5-Hydroxy(2Hx)-
decano-4-lactones 2 after Incubation of 150 ppm of (6R,7R)-6,7-Dihydroxy(6,7-2H2)dodecanoic Acid Methyl
Ester 1 in Liquid Cultures of Saccharomyces cerevisiae. R1 ˆ Me(CH2)3.
Amount
Ratio
2H-Label at C(2)
2H-Label at C(4)
2H-Label at C(5)
40.6 ppm
78.5%
0%
96%
97%
0 ppm
0%
0 ppm
0%
11.1 ppm
21.5%
68%
0%
98%
group rearrangement from C(5) to the lactone ring under EI-MS conditions could be
established by several experiments. The nonderivatized hydroxy-lactones 2 showed a
H-rearrangement from OHÀC(5) to the g-lactone moiety (m/z 85 ‡ 1). This H-
transfer was secured by O2HÀC(5) shifts in 2H2O (data not shown). In contrast, 5-O-
(trifluoroacetyl) derivatives of 2 did not rearrange and showed a-cleavage, as
established by the results of Fig.3 . These EI-MS results were confirmed by measure-
ments with unlabeled references.
In the case of the incubation of ester (6R,7R)-(6,7-2H2)-1, we identified doubly
labeled (4R,5R)-2 and unexpectedly doubly labeled (4S,5R)-2. The inversion at the
chiral center C(4) by the oxidation/keto reduction pathways with intermediate a-ketols
2
(Fig.1 ) would be a suitable explanation. But, instead of total H-loss, we detected a
68% lactone-ring label and a 98%-labeled side chain (C(5)) for (4S,5R)-2 in this
experiment (see above, Table 2). The amounts of (4R,5R)-2 and (4S,5R)-2 increased
during fermentation in a distinct ratio of 4 :1 and showed concentration maxima at the
end of the fermentation. The initial 2H-contents of the side chain (C(5)) were retained
in both lactones. The lactone-ring label of (4S,5R)-2 was decreased from initially 86%
to 68%. In contrast to that, the ring label content of (4R,5R)-2 did not change and was
96%.
2H-Depletion at C(4) and C(3) is not favored but plausible at the acidic position
C(2). This 2H-exchange could be demonstrated in model experiments with (2,2-
2H2)decano-4-lactone. Therefore, we postulate a (2,5-2H2)-labeled (4S,5R)-2, which is
in agreement with the postulated metabolism (see below, Scheme 2).

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Helvetica Chimica Acta Vol. 86 (2003)
Fig. 1. Low-Resolution EI-MS of a) methyl ester (6R,7R)-(6,7-2H2)-1 (synthesized, Me3Si ether), b) methyl 6-
hydroxy-7-oxo(6-2H1)dodecanoate (postulated), and c) methyl 7-hydroxy-6-oxo(7-2H1)dodecanoate (postulat-
ed). Both a-ketols were isolated from culture broth of incubation experiment of 1 and 1a in S.cerevisiae .
TMS ˆ Me3Si.
Incubation experiments of methyl ester (6S,7S)-(6,7-2H2)-1 showed the degradation
into four hydroxy-4-lactones 2 with 6.8% yield (Table 3). (4S,5S)-(4,5-2H2)-2 was
characterized as the main product (92.6%). Inversion to erythro-2 comparable to that

Helvetica Chimica Acta Vol. 86 (2003)
2355
Fig. 2. Low-Resolution EI-MS of a) (4,5-2H2)-2 (Me3Si ether) and b) (2Hx)-2 (Me3Si ether) with significant
2
lactone-ring H-depletion, isolated from cultures of S. cerevisiae after incubation of methyl ester (6R,7R)-(6,7-
2H2)-1. TMS ˆ Me3Si.
described in the experiment with methyl ester (6R,7R)-(6,7-2H)-1 was not observed.
The minor amounts of (4R,5R)-2 and (4S,5R)-2 might be explained by a small amount
of (6R,7R)-(6,7-2H2)-1 present in the incubation substrate.
In incubation experiments of acid (Æ)-erythro-(6,7-2H2)-1a, four isomeric 5-
hydroxy-4-lactones 2 were characterized as doubly labeled isotopomers with a yield
of 3.1%. The ratio erythro-2/threo-2 was 95 :5. Chiral analysis of the products indicated
the presence of 65.3% of (4S,5R)-(4,5-2H2)-2, 29.2% of (4R,5S)-(4,5-2H2)-2, and minor
amounts of threo-2 (Table 4). In contrast to the case of the (Æ)-threo-1 incubations, the
(4S)-isomer was predominantly lactonized.
Discussion. The biotransformations of monohydroxy fatty acids [24][25] have
been well-investigated, whereas the degradation of dihydroxy fatty acids are mostly
unknown. The 6,7-dihydroxydodecanoic acids 1a, which are formed as intermediates
during the vernolic acid metabolism, are transformed by one cycle of b-oxidation into
4,5-dihydroxydecanoyl-CoA, which is immediately lactonized to 2. A further degra-
dation into 2,3-dihydroxyoctanoyl-CoA, hexanoyl-CoA, and 2-hydroxyacetyl-CoA has
not been observed [26]. In addition, the substrates 1 and 1a are oxidized to the

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Helvetica Chimica Acta Vol. 86 (2003)
Fig. 3. Low-Resolution EI-MS of a) (4,5-2H2)-2 isolated from cultures of S. cerevisiae after incubation of
methylester (6R,7R)-(6,7-2H2)-1, b) synthesized 2, and c) 5-O-(trifluoroacetyl) derivative of synthesized 2
corresponding a-ketols in oscillating concentrations which disappear at the end of the
fermentation. The (Æ)-threo- and (Æ)-erythro-6,7-dihydroxydodecanoic acids 1a were
efficiently metabolized by S.cerevisiae , and only 3 4% was converted to the
hydroxylactones 2. In corresponding incubations of the methyl esters 1 of the threo-
enantiomers, 10 40% of the substrates were transformed into hydroxylactones, and
high amounts of 1 were not metabolized. The racemic dihydroxy acid threo-1a formed
threo-2/erythro-2 in a ratio of 92 :8 with (4R,5R)-2 as the main product. The

Helvetica Chimica Acta Vol. 86 (2003)
2357
¾
Fig. 4. Chiral gas chromatographic resolution (Lipodex E) of isomeric 2 with and without trifluoroacetic acid
(TFA) derivatization
metabolism of the corresponding acid (Æ)-erythro-1a showed a comparable pattern,
with an erythro-2/threo-2 ratio of 95 :5 and (4S,5R)-2 as the major product.
Lactonization of threo-4,5-dihydroxy acids is preferred with (4R)-configuration,
whereas from erythro-4,5-dihydroxy acids, the (4S)-configured center was predom-
inantly esterified. The hydroxy-4-lactones and the substrates possessed comparable 2H-
contents.
The result of the (6R,7R)-(6,7-2H2)-1 metabolism was of particular interest because
the presence of 25% of (4S,5R)-(2,5-2H2)-2 was observed in the fermentation broth.
2
During fermentation, the H-content of the lactone ring of (4S,5R)-(2,5-2H2)-2 was
2
decreasing from 86% to 68%, whereas the H-content of (4R,5R)-(4,5-2H2)-2 did not
change. The enantiomer (4S,5R)-2 is the bioactive L-factor in Streptomyces griseus
fermentation. Therefore, we postulate a degradation pathway of methyl ester (6R,7R)-
2
(6,7-2H2)-1 according to Scheme 2, wherein the transfer of H is catalyzed by an enone
reductase. Isomeric a-ketols were the products with the highest concentration in all
experiments, and their amounts continuously oscillated during fermentation. But direct

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Table 3. Total Amounts [ppm], Relative Ratios, and Distribution of the 2H-Label of Isomeric-5-Hydroxy(2Hx)-
decano-4-lactones 2 after Incubation of 150 ppm of (6S,7S)-6,7-Dihydroxy(6,7-2H2)dodecanoic Acid Methyl
Ester 1 in Liquid Cultures of Saccharomyces cerevisiae. R1 ˆ Me(CH2)3.
Amount
Ratio
2H-Label at C(2)
2H-Label at C(4)
2H-Label at C(5)
0.3ppm
3.7%
0%
95%
97%
7.4 ppm
92.6%
n.d.
95%
97%
0.06 ppm
0.8%
n.d.
n.d.
n.d.
0.23ppm
2.9%
79%
0%
94%
Table 4. Total Amounts [ppm], Relative Ratios, and Distribution of the 2H-Label of Isomeric-5-Hydroxy(2Hx)-
decano-4-lactones 2 after Incubation of 150 ppm of (Æ)-erythro-6,7-Dihydroxy(6,7-2H2)dodecanoic Acid 1a in
Liquid Cultures of Saccharomyces cerevisiae. R1 ˆ Me(CH2)3.
Amount
Ratio
2H-Label at C(2)
2H-Label at C(4)
2H-Label at C(5)
0.13ppm
3.6%
75%
0%
92%
0.07 ppm
1.9%
n.d.
n.d.
n.d.
1.05 ppm
29.2%
0%
97%
96%
2.35 ppm
65.3%
0%
93%
96%

Helvetica Chimica Acta Vol. 86 (2003)
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Scheme 2. Postulated Metabolism of Methyl Ester (6R,7R)-(6,7-2H2)-1 in Liquid Cultures of Saccharomyces
cerevisiae. MFP ˆ Multifunctional protein.
reduction of a-ketols to 2H-depleted erythro-1 or threo-1 or -1a could not be detected.
Therefore, a BaeyerÀVilliger-type oxidation of the a-ketols might be involved in the
pathway.
Experimental Part
1. General. LC ˆ liquid chromatography. 1H- and 13C-NMR Spectra: Bruker AMX-500 spectrometer
(Karlsruhe, Germany); chemical shifts d in ppm rel. to SiMe4 (ˆ0 ppm) as external standard, J in Hz. GC/MS:

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Helvetica Chimica Acta Vol. 86 (2003)
fused silica DB-1 capillary column (poly(dimethylsiloxane)), 60 m  0.32 mm i.d., 0.25 mm film (J & W Scientific
¾
Folsome, CA), temp. program 4 min at 808, then 48/min to 2808, 100 kPa He (4.0); or Lipodex E (octakis(3-O-
butyryl-2,6-di-O-pentyl)-g-cyclodextrin, 50 m  0.25 i.d., 0.25 mm film (Macherey & Nagel, D¸ren, Germany),
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-ionization Varian Mat-8230 mass spectrometer, ionization
energy 70 eV; m/z (intensity in %).
2. (Æ)-threo- and (Æ)-erythro-6,7-Dihydroxy(6,7-2H2)dodecanoic Acids 1a and Methyl (6R,7R)- and
Methyl (6S,7S)-6,7-Dihydroxy(6,7-2H2)dodecanoates 1.
2.1. Dodec-6-ynenitrile. At 08 under N2, 2.5m BuLi in hexane (22 ml) was added dropwise to hept-1-yne
(4.4 g, 45 mmol) in abs. THF (40 ml) and stirred for 2 h. Then 1,4-dibromobutane (19.5 g, 90 mmol) in abs.
DMPU (ˆ 3,4,5,6-tetrahydro-1,3-dimethylpyrimidin-2(1H)-one; 100 ml) was rapidly added at 08. The mixture
was stirred at 208 for 24 h, refluxed for 2 h, cooled to 108, mixed with ice/H2O (60 ml), and extracted with
petroleum ether (3 Â 50 ml). The combined extracts were washed with brine (50 ml), dried (Na2SO4), and
‡
evaporated: crude 1-bromoundec-5-yne (14.0 g). GC/MS: 151 (3, [M À Br] ), 134 (2), 121 (2), 109 (25), 95 (10),
93 (15), 91 (15), 82 (10), 81 (60), 79 (30), 77 (25), 69 (15), 68 (15), 67 (90), 65 (15), 55 (45), 54 (55), 53 (35), 52
(20), 51 (15), 43(15), 42 (10), 41 (100), 40 (10), 39 (60).
At 808 under N2, the crude 1-bromoundec-5-yne was added dropwise to NaCN (8.2 g, 160 mmol) in abs.
DMSO (80 ml). The mixture was stirred at 808 for 2 h and at 208 for 24 h. After addition of dist. H2O (100 ml),
the mixture was extracted with Et2O (2 Â 50 ml), the combined Et2O phase washed with H2O (100 ml), 6m HCl
(30 ml), H2O (2 Â 50 ml), and brine (50 ml) and evaporated, and the crude product purified by LC silica gel
(50 g), petroleum ether/AcOEt 18 :2 (500 ml): 6.2 g (77% based on hept-1-yne) of dodec-6-ynenitrile. GC/MS:
‡
176 (3, [M À 1] ), 162 (5), 148 (10), 134 (60), 120 (30), 107 (25), 106 (25), 95 (25), 94 (15), 93 (35), 91 (20), 83
(5), 82 (10), 81 (50), 80 (25), 79 (44), 77 (30), 68 (10), 67 (75), 66 (10), 65 (21), 56 (10), 55 (35), 54 (30), 53
(30), 52 (25), 51 (24), 50 (10), 43 (15), 42 (17), 41 (100), 40 (20), 39 (60).
2.2. Dodec-6-ynoic Acid. At 208 under N2, Na2O2 (6.2 g, 80 mmol) was added to dodec-6-ynenitrile (6.2 g,
35 mmol) in abs. DMSO (40 ml). The mixture was stirred for 24 h at 208 and, after addition of H2O (80 ml), 2 h
at 808. The mixture was cooled to 208, acidified with 6m HCl and extracted with Et2O (2 Â 50 ml). The combined
Et2O extract was washed with brine (50 ml), dried (Na2SO4), and evaporated, and the crude acid purified by LC
(silica-gel (50 g), petroleum ether/AcOEt 18 :2 (100 ml), petroleum ether/AcOEt/AcOH 18 :2 :0.2 (200 ml)
and petroleum ether/AcOEt/AcOH 15 :5 :0.2 (400 ml)): 4.6 g (67%) of dodec-6-ynoic acid. GC/MS
‡
‡
(corresponding methyl ester): 210 (2, M ), 178 (5, [M À MeOH] ), 161 (2), 154 (25), 136 (15), 135 (8), 122
(15), 121 (17), 107 (15), 96 (8), 95 (30), 94 (55), 93 (40), 91 (30), 82 (18), 81 (56), 80 (100), 79 (90), 78 (20), 77
(30), 74 (25), 69 (8), 68 (15), 67 (64), 66 (15), 65 (22), 59 (39), 57 (12), 55 (48), 54 (28), 53 (26), 52 (24), 51
(20), 43 (30), 42 (18), 41 (88), 40 (15), 39 (45).
2.3. (6Z)-(6,7-2H2)Dodec-6-enoic Acid. At 08, 5% Pd/BaSO4 (60 mg) was stirred in MeOH (80 ml).
2
Quinoline (0.75 ml) was added, and the mixture was evacuated and aerated with H2 gas (Messer-Griesheim,
Germany, 2.7). Addition of dodec-6-ynoic acid (6.7 mmol) in MeOH (5 ml) started the consumption of the
theoretical volume of 150 ml of 2H2. The catalyst was filtered off and the filtrate diluted with Et2O (50 ml) and
H2O (50 ml) and acidified with 2m HCl. The Et2O phase was washed (brine), dried (Na2SO4), evaporated and
the crude acid purified by LC (silica gel (50 g), petroleum ether/AcOEt/AcOH 18 :2 :0.2 (400 ml)): 1.2 g (89%)
‡
of (6Z)-6,7-2H2)dodec-6-enoic acid. GC/MS (corresponding methyl ester): 214 (22, M ), 183(30), 182 (40), 165
(15), 153 (8), 141 (10), 140 (61), 139 (20), 138 (13), 126 (10), 125 (22), 124 (12), 111 (25), 110 (25), 99 (45), 98
(72), 97 (85), 96 (35), 88 (20), 87 (46), 86 (15), 85 (57), 84 (85), 83 (70), 82 (40), 81 (25), 75 (25), 74 (100), 71
(32), 70 (52), 69 (70), 68 (35), 59 (40), 57 (55), 56 (88), 55 (76), 54 (18), 44 (15), 43 (62), 40 (52), 41 (69), 40
(15), 39 (20).
2
2.4. (6E)-(6,7-2H2)Dodec-6-enoic Acid. At 08 under N2, LiAl2H4 (1.88 g, 44.6 mmol; >98% H atom) was
suspended in abs. diethylene glycol dimethyl ether (ˆdiglyme; 50 ml), and dodec-6-ynoic acid (1.45 g, 7.4 mmol)
in diglyme (5 ml) was added very slowly and stirred for 15 min. The mixture was heated to 1508 for a minimum
of 72 h, hydrolyzed at 108 with 2H2O (15 ml; >98% 2H atom; Campro Scientific, The Netherlands), heated to
608 for 30 min, acidified with 2m HCl, and extracted with petroleum ether (3Â). The combined org. phase was
washed with H2O (50 ml) and brine (50 ml), dried (Na2SO4), and evaporated, and the crude alcohol submitted
to LC (silica gel (50 g), petroleum ether/AcOEt 18 :2 (500 ml); 0.93g (67%) of (6E)-(6,7-2H2)dodec-6-en-1-ol.
At 208, pyridinium chlorochromate (2.1 g, 10 mmol) and anh. NaOAc (80 mg) were dissolved in CH2Cl2
(20 ml). Then (6E)-(6,7-2H2)dodec-6-en-1-ol (0.93g, 5 mmol) was added and stirred for 3h. After addition of
petroleum ether (20 ml), the mixture was filtered through a silica-gel (10 g) column, which was washed with
‡
CH2Cl2 (50 ml). Evaporation gave 0.83g (90%) of (6E)-(6,7-2H2)dodec-6-enal. GC/MS: 166 (15, [M À 18] ),

Helvetica Chimica Acta Vol. 86 (2003)
2361
140 (8), 123(10), 114 (8), 113(7), 112 (9), 111 (5), 110 (4), 109 (6), 99 (25), 98 (30), 97 (24), 96 (15), 95 (15), 85
(15), 84 (24), 83(40), 82 (25), 81 (25), 80 (20), 71 (25), 70 (48), 69 (42), 68 (26), 67 (20), 57 (55), 56 (85), 55
(70), 54 (15), 44 (20), 43 (78), 42 (75), 41 (100), 40 (27), 39 (35).
t
At 208, BuOH (8 ml) and 2-methylbut-2-ene (2 ml) were mixed with a freshly prepared soln. of NaClO2
(0.33 g, 3.7 mmol) and NaH2PO4 (0.38 g, 3.1 mmol) in H2O (4 ml). Crude (6E)-(6,7-2H2)dodec-6-enal (0.83g,
4.5 mmol) was added and stirred for 18 h. After evaporation, the residue was dissolved in H2O (20 ml), acidified
with 2m HCl (20 ml), and extracted with Et2O (3 Â 50 ml). The combined Et2O phases were washed (sat.
Na2S2O3 soln. (20 ml) and brine (50 ml)) and the crude acid was purified by LC (silica gel (50 g), petroleum
ether/AcOEt/AcOH 18 :2 :0.2 (400 ml)): 0.7 g (47% based on dodec-6-ynoic acid) of (6E)-(6,7-2H2)dodec-6-
enoic acid. 1H-NMR (corresponding methyl ester, CDCl3): 0.75 (t, J ˆ 7, Me); 1.1 1.3( m, 8 H, CH2); 1.49
(quint., J ˆ 7, CH2CH2COOMe); 1.85 (m, CH2CHˆCHCH2); 2.19 (t, J ˆ 7, CH2COOMe); 3.53 (s, COOMe);
0.07 H for signal of HÀC(6) and HÀC(7), i.e., labeling 97%. 13C-NMR (CDCl3): 13.97 (C(12)); 22.46 (C(11));
24.34; 28.96; 29.17; 31.32; 31.92; 32.30; 33.86; 51.33 (MeO); 128.99 (t, J ˆ 22, C(7)); 130.42 (t, J ˆ 22, C(6));
‡
174.14 (C(1)). GC/MS (corresponding methyl ester): 214 (11, M ), 182 (19), 165 (15), 164 (15), 153(10), 140
(35), 125 (25), 112 (20), 111 (20), 99 (30), 98 (40), 97 (60), 96 (28), 87 (35), 86 (15), 85 (40), 84 (55), 83 (50), 82
(30), 81 (27), 75 (25), 74 (100), 71 (30), 70 (40), 69 (55), 68 (29), 67 (18), 59 (60), 57 (55), 56 (80), 55 (75), 54
(25), 44 (15), 43 (80), 42 (77), 41 (95), 40 (30), 39 (35), 31 (15).
2.5. (Æ)-threo-Dihydroxy Acids threo-1a: General Procedure. At 08, H2O2 (10 ml; 30% in H2O), conc.
formic acid (50 ml), and (Z)-alkenoic acid (4 mmol) were stirred for 5 min, then for 1 h at 408 and overnight at
208. The mixture was acidified with 6m HCl and extracted with Et2O (3Â). The combined extract was washed
with brine (50 ml), dried (Na2SO4), and evaporated. The crude product was hydrolyzed with 1m NaOH (40 ml)
at 1008 for 1 h. Acidification of the mixture (6m HCl), addition of solid NaCl (5 g), and extraction with Et2O
(3Â), re-extraction of the combined Et2O phases with brine (50 ml), drying (Na2SO4), and evaporation
afforded the crude dihydroxy fatty acid, which was purified by LC (silica gel (50 g), petroleum ether/AcOEt
18 :2 (100 ml), petroleum ether/AcOEt/AcOH 18 :2 :0.2 (200 ml), and petroleum ether/AcOEt/AcOH
15 :5 :0.2 (400 ml)): 2.4 mmol (60%) of the threo-dihydroxy acid threo-1a, containing ca. 2% of the erythro-
diastereoisomer (by TLC and GC).
(Æ)-threo-6,7-Dihydroxy(6,7-2H2)dodecanoic Acid ((Æ)-threo-(6,7-2H2)-1a): 1H-NMR (CDCl3): 0.70 (t, J ˆ
7, Me ) ; 1.0 1.5 ( m, 14 H, CH2); 2.13( t, J ˆ 7, CH2COOH); 0.06 H for signal of HÀC(6) and HÀC(7), i.e.,
labeling 97%. 13C-NMR (CDCl3): 13.60 (C(12)); 22.28 (C(11)); 24.56; 25.38; 30.47; 31.02; 31.60; 32.80; 33.71;
73.59, 73.75, 73.89, 74.06 (m, C(6), C(7)); 176.44 (C(1)).
2.6. (Æ)-erythro-Dihydroxy Acid erythro-1a: General Procedure. (Z)-Alkenoic acid in tBuOH was oxidized
with stoichiometric amounts of OsO4. The mixture was worked up as described in Exper.25. . (Æ)-erythro-
dihydroxy acid erythro-1a containing ca. 4% (GC) of the threo-diastereoisomer.
1
(Æ)-erythro-6,7-Dihydroxy(6,7-2H2)-dodecanoic Acid: ((Æ)-erythro-(6,7-2H2)-1a): H-NMR (CDCl3): 0.70
(t, J ˆ 7, Me); 1.0 1.5 (m, 14 H, CH2); 2.13( t, J ˆ 7, CH2COOH); 0.06 H for signal of HÀC(6) and HÀC(7), i.e.,
labeling 97%. 13C-NMR (CDCl3): 13.58 (C(12)); 22.28 (C(11)); 24.56; 25.16; 25.35; 31.59; 32.50; 32.79; 33.72;
72.97, 73.25, 73.41, 73.61 (m, C(6), C(7)); 176.42 (C(1)).
2.7. Methyl (4E)-Dec-4-enoate was synthesized from commercially available (4E)-dec-4-enal by NaClO2
oxidation (see Exper.24. ) and MeOH/HCl methylation.
2.8. Asymmetric Dihydroxylation (AD): Formation of Dihydroxyalkanoic Acid Methyl Esters 1. AD was
performed according to the Sharpless AD method [18]. Methyl (E)-alkenoates yielded (S,S)-dihydroxy fatty
acid methyl esters with −AD-Mix a× and (R,R)-dihydroxy fatty acid methyl esters with −AD-Mix b×. In a typical
procedure, tBuOH (5 ml), H2O (5 ml), −AD-Mix a× or −AD Mix b× (1.4 g), and methansulfonamide
(MeSO2NH2; 95 mg, 1 mmol) were mixed at 208 and chilled to 08. Methyl (E)-alkenoate (1 mmol) was added
and stirred overnight at 08. For workup, Na2SO3 (1.5 g, 12 mmol) was added and the mixture warmed to 208 and
extracted with AcOEt (3Â). Washing with 2n NaOH (30 ml) and brine (50 ml)), drying (Na2SO4), and
evaporation afforded the crude product, which was purified by LC (silica gel (50 g), petroleum ether/AcOEt
18 :2 (200 ml), and petroleum ether/AcOEt 15 :5 (400 ml)): >0.9 mmol (> 90%) of methyl dihydroxyalka-
noates 1.
Methyl (6R,7R)-6,7-Dihydroxy(6,7-2H2)-dodecanoate ((6R,7R)-(6,7-2H2)-1) (from −AD-Mix b×): 1H-NMR
(CDCl3): 0.75 (t, J ˆ 7, Me); 1.1 1.6 (m, 14 H, CH2); 2.19 (t, J ˆ 7, CH2COOMe); 3.52 (s, COOMe); 0.05 H for
signal of HÀC(6) and HÀC(7), i.e., labeling 97%. 13C-NMR (CDCl3): 13.94 (C(12)); 22.52 (C(11)); 24.72;
25.08; 25.23; 31.78; 32.91; 33.28; 33.84; 51.46 (MeO); 73.29, 73.47, 73.63, 73.81, 73.95 (m, C(6), C(7)); 174.27
(C(1)).
The enantiomer (6S,7S)-(6,7-2H2)-1 showed the same NMR spectrum.

2362
Helvetica Chimica Acta Vol. 86 (2003)
3. 5-Hydroxydecano-4-lactones (ˆ4,5-Dihydro-5-(1-hydroxyhexyl)furan-2(3H)-ones) 2. (4R,5R)- and
(4S,5S)-2 were prepared from methyl (4E)-dec-4-enoate by Sharpless AD and lactonization of methyl 4,5-
dihydroxydecanoate in CH2Cl2/0.5m HCl 4 :1 overnight at 208. Enantiomers (4R,5R)- and (4S,5S)-2 were
oxidized at OHÀC(5) with −Dess-Martin periodinane× [27] to yield enantiomers (4R)- and (4S)-5-oxodecano-4-
lactone, resp., which were reduced with NaBH4 in Et2O to give (4R,5R)/(4R,5S)-2 and (4S,5S)/(4S,5R)-2, resp.
NMR-Signal assignments were performed by H,H-COSY and H,C-HETCOR.
(4R,5R)-2 (threo): 1H-NMR (CDCl3): 2.46, 2.55 (2m, CH2(2)); 2.07, 2.18 (2m, CH2(3)); 4.36 (ddd, J ˆ 7, 7, 5,
HÀC(4)); 3.51 (br. m, HÀC(5)); 1.46 (m, CH2(6)); 1.18 1.52 (m, CH2(7), CH2(8), CH2(9)); 0.82 (t, J ˆ 7,
Me(10)); 2.30 (br. d, J ˆ 7, OH ÀC(5)). 13C-NMR (CDCl3): 177.48 (C(1)); 28.62 (C(2)); 23.96 (C(3)); 82.98
(C(4)); 73.45 (C(5)); 32.82 (C(6)); 25.07 (C(7)); 31.58 (C(8)); 22.44 (C(9)); 13.92 (C(10)).
1
(4S,5S)-2 (threo): H- and 13C-NMR identical to those of the enantiomer.
(Æ)-erythro-2: 1H-NMR (CDCl3): 2.47, 2.54 (2m, CH2(2)); 2.08, 2.21 (2m, CH2(3)); 4.38 (ddd, J ˆ 7, 7, 4,
HÀC(4)); 3.86 (br. m, HÀC(5)); 1.35 (m, CH2(6)); 1.18 1.51 (m, CH2(7), CH2(8), CH2(9)); 0.83( t, J ˆ 7,
Me(10)); 2.95 (br. d, J ˆ 7, OH ÀC(5)). 13C-NMR (CDCl3): 177.97 (C(1)); 28.64 (C(2)); 20.90 (C(3)); 83.04
(C(4)); 71.14 (C(5)); 31.80 (C(6)); 25.03 (C(7)); 31.58 (C(8)); 22.40 (C(9)); 13.87 (C(10)).
4. Yeast Strain, Culture Conditions, Sampling, and Workup. Yeast (Saccharomyces cerevisiae IfG 06136) was
obtained from the culture collection of the −Institut f¸r G‰rungstechnologie×, Berlin. The organism was stored
(48) on worth agar-agar slants. For metabolic experiments, cells were cultivated in a medium (200 ml, pH 5.5)
consisting of glucose (6.0 g/l), MgSO4 (3.0 g/l), (NH4)2SO4 (2.5 g/l), KH2PO4, l-alanine (2.5 g/l), and yeast
extract (3.0 g/l) on a horizontal shaker at 178 and 100 rpm. The pre-culture (10 ml) was inoculated into 200 ml of
fresh culture medium, and 30 mg (150 ppm) of each substrate, (i.e., (6R,7R)-(6,7-2H2)-1, (6S,7S)-(6,7-2H2)-1,
(Æ)-threo-(6,7-2H2)-1a and (Æ)-erythro-(6,7-2H2)-1a, in 250 ml of EtOH were immediately administered to
different flasks. At certain times, i.e., after 24, 48, 72, 96, 120, 144, 168, 216, and 268 h, aliquots (10 ml) of the
culture broth were taken, internal standards (5 ppm of decan-1-ol, octano-5-lactone, and heptadecanoic acid)
were added, the mixture was extracted with Et2O (2 Â 20 ml), the extract dried (Na2SO4), methylated with
diazomethane, and converted to derivatives (silyl ether or trifluoroacetate) under standard conditions [28] if
necessary.
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Received November 28, 2002