Biosynthesis of phoslactomycins: Cyclohexanecarboxylic acid as the starter unit

Article abstract of DOI:10.1016/S0040-4020(03)01205-5

Phoslactomycins (PLMs) A-F, produced by actinomycetes are polyketide-type antibiotics derived from a hydroxycyclohexanecarboxylic acid or a cyclohexanecarboxylic acid starter unit. Feeding experiments with [2- ¹³C]shikimic acid indicated that the C-18 carbon of PLMs comes from C-5 of shikimate. Further feeding studies of cis and trans-3-hydroxy[7- ¹³C]cyclohexanecarboxylic acid, [7-¹³C]- and [ ²H₁₁]cyclohexanecarboxylic acid have suggested that the starter unit in the PLM biosynthesis is not cis-3-hydroxycyclohexanecarboxylate but cyclohexanecarboxylate and that PLM-B is produced initially, and subsequently converted to other analogs by hydroxylation and acylation.

Full text of DOI:10.1016/S0040-4020(03)01205-5

Tetrahedron 59 (2003) 7465–7471
Biosynthesis of phoslactomycins: cyclohexanecarboxylic
acid as the starter unit
Yasuyo Sekiyama,a Nadaraj Palaniappan,b Kevin A. Reynoldsb and Hiroyuki Osadaa
,
*
aAntibiotics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
bDepartment of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University,
800 E. Leigh St. Suite 212, Richmond, VA 23219, USA
Received 15 May 2003; revised 30 July 2003; accepted 31 July 2003
Abstract—Phoslactomycins (PLMs) A–F, produced by actinomycetes are polyketide-type antibiotics derived from a hydroxycyclohex-
anecarboxylic acid or a cyclohexanecarboxylic acid starter unit. Feeding experiments with [2-13C]shikimic acid indicated that the C-18
carbon of PLMs comes from C-5 of shikimate. Further feeding studies of cis and trans-3-hydroxy[7-13C]cyclohexanecarboxylic acid,
[7-13C]- and [2H11]cyclohexanecarboxylic acid have suggested that the starter unit in the PLM biosynthesis is not cis-3-
hydroxycyclohexanecarboxylate but cyclohexanecarboxylate and that PLM-B is produced initially, and subsequently converted to other
analogs by hydroxylation and acylation.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
characterized as possessing an a,b-unsaturated d-lactone,
amino group, phosphate ester, cyclohexane ring and
absolute configuration 4S,5S,8R,9R,11R,16R,18S (deter-
mined by Shibata et al.13).
Polyketides are a large and structurally diverse group of
natural products, which are constructed via successive
condensation of simple extender units such as malonyl CoA
with various starter units such as activated short-chain fatty
acids, alicyclic acids, aromatic acids, glycerol metabolites,
and amino acids.1 Natural products biosynthesized using a
cyclohexanecarboxylic acid (CHC) derivative as the starter
unit are found in microbial metabolites and exhibit
important biological activities. For example, manumycin
group antibiotics (e.g. askamycin),2 and the immunosup-
pressive macrolides (e.g. rapamycin,3 FK5064) were all
isolated from cultures of actinomycetes. v-Cyclohexyl fatty
acids are found in thermoacidophilic bacterial membranes.5
Phoslactomycins (PLMs) A–F, isolated from several
Streptomyces strains including S. sp. HK-8036 – 8 as
antifungal antibiotics, belong to this group of compounds.
Similar to the structurally-related compound, fostriesin,9
PLMs exhibit type 2A serine/threonine protein phosphatase
(PP2A) specific inhibitory activity.10 Leustroducsins, which
differ from PLMs only at the acyl moiety bound to the
cyclohexane ring, and PLM-F were reported to be inducers
of a colony stimulating factor in bone marrow stromal
cells.11 Most recently, the PP2A inhibition activity of PLMs
has been shown to inhibit tumor metastasis through
augmentation of natural killer cells.12 Their structures are
Biosynthetically, PLMs can be considered as polyketides
elongated from an unusual starter unit, hydroxycyclohex-
anecarboxylic acid or CHC (1). The biosynthetic studies of
ansatrienin A,14,15 v-cyclohexyl fatty acids,16 FK-52017,18
and rapamycin19,20 have revealed that these cyclohexane
moiety was biosynthesized from shikimic acid (2) via a
series of dehydrations and double bond reductions
(Scheme 1). The CHC pathway and (1R,3R,4R)-3,4-
dihydroxycyclohexanecarboxylic acid (DHCHC, 3) path-
way diverge at the first step, suggesting that these two
related pathways evolved independently. Since PLM
producers make two types of PLMs i.e. PLMs with a
hydroxyl substituent at C-18, which is cis to C-16 alkyl
substituent (PLM-A and PLMs C–F), and PLM-B, which
has no hydroxyl substituent on the cyclohexane ring,
Keywords: phoslactomycin; phosphatase inhibitor; cyclohexanecarboxylic
acid; biosynthesis.
*
Corresponding authors. Tel.: þ81-48-467-9542; fax: þ81-48-462-4669;
e-mail: hisyo@postman.riken.go.jp
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)01205-5

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Y. Sekiyama et al. / Tetrahedron 59 (2003) 7465–7471
Scheme 1. Proposed conversion of shikimic acid (2) to cyclohexanecarboxylic acid derivatives and possible biosynthetic pathways of PLMs.
cis-3-hydroxycyclohexanecarboxylic acid (4) and/or 1
could be envisioned as starter units. Several possible
biosynthetic routes can be considered in the biosynthesis
of PLMs (Scheme 1). In this paper, we report the results of
labeling studies with Streptomyces sp. HK-803, which led to
the conclusion that 1 served as the starter unit in all cases,
and that the resulting PLM-B is likely converted by
hydroxylation and acylation to the other PLM analogs.
comparison, the corresponding trans isomer 5. The (^)-
[7-13C]4 (containing 5% of trans isomer (^)-[7-13C]5,
estimated by integrating the methine H NMR signals at
1
2. Results
2.1. Feeding experiment with [2-13C]shikimic acid
We initiated feeding studies of shikimic acid (2) in order to
both confirm its intermediacy and establish a biosynthetic
correlation of shikimate carbon and cyclohexane carbon of
PLMs. [2-13C]2 was synthesized according to the procedure
described by Moore et al.14 and fed to Streptomyces sp. HK-
803. LC–MS analysis of the resulting PLM mixture showed
that (MHþ1)þ peaks of PLM-A to F and (MHþ2)þpeak of
PLM-E were enhanced (Table 1), indicating that 2 was
efficiently incorporated into PLMs. The major components,
PLM-E and F were purified by HPLC and analyzed by 13C
NMR. 13C NMR spectrum of PLM-E showed signals at d
30.2 (C-30 or 70) and d 33.1 (C-21), while that of PLM-F
showed a signal at d 33.1 (C-21) (.10 fold enrichment,
consistent with the LC–MS analysis, Fig. 1(c) and (f)). In
addition to demonstrating the shikimic acid origin of the
cyclohexane moiety of PLMs, these results confirmed that
C-21 of PLMs was originated from C-2 of 2, thereby
indicating that C-18 of PLMs was derived from C-5 of 2
(Scheme 2).
2.2. Feeding experiments with potential starter units
Since the majority of the PLMs produced in a fermentation
have a hydroxyl substituent at C-18, which is cis to C-16
alkyl substituent, we next carried out an incorporation study
with cis-3-hydroxycyclohexane carboxylic acid (4), and for
Figure 1. 13C NMR spectra (125 MHz). (a) PLM-B derived from [7-13C]1;
(b) non-labeled PLM-B; (c) PLM-E derived from [2-13C]2; (d) PLM-E
derived from [7-13C]1; (e) non-labeled PLM-E; (f) PLM-F derived from
[2-13C]2; (g) PLM-F derived from [7-13C]1; (h) non-labeled PLM-F.

Y. Sekiyama et al. / Tetrahedron 59 (2003) 7465–7471
7467
Table 1. Mass spectral analysis of PLMs obtained from a feeding experiment with [2-13C]221
Molecular related ions
(m/z)
Unlabeled PLMs
Relative intensity obtained
from [7-13C]2
PLM-A
600
601
602
100
29
10
100
48 (16%)
16
PLM-B
PLM-C
PLM-D
PLM-E
514
515
516
100
31
7
100
47 (14%)
10
614
615
616
100
35
9
100
61 (21%)
18
628
629
630
100
38
10
100
52 (12%)
14
641
642
643
644
100
35
11
0
100
87 (43%)
35
9
PLM-F
642
643
644
100
38
10
100
66 (22%)
17
C-3) was prepared by reduction of methyl 3-oxo[7-13C]cy-
clohexanecarboxylate (7), which was obtained by cyanation
of 2-cyclohexenone22 with K13CN, with NaBH4.23 The (^)-
[7-13C]5 (containing 10% of cis isomer (^)-[7-13C]4,
were incorporated into the cyclohexane moiety of PLMs
(Scheme 3).
Finally, [2H11]1, (showing a deuterium distribution by MS
of 10% D9, 28% D10, 62% D11 species) was synthesized
from sodium benzoate by reduction with deuterated Raney
nickel,26 and fed to the PLM producer. Analysis of the
resulting PLM mixture by LC–MS revealed the occurrence
of the undecadeuterio species (MHþ11)þ of PLM-B, and
decadeuterio species (MHþ10)þ of PLMs A, C–F,
providing additional evidence that 1 was incorporated as
such, and used as the starter unit (Table 3).
1
estimated by integrating the methine H NMR signals at
C-3) was synthesized by reduction of 7 with K-selectride.24
LC–MS analysis of the PLM mixture obtained from feeding
of (^)-[7-13C]4 showed that 4 was poorly converted into
PLMs. On the other hand, (MHþ1)þ peaks of PLMs A–F
and (MHþ2)þpeak of PLM-E obtained from (^)-[7-13C]5
were significantly enhanced (Table 2). The specific uptake
of 5 indicated that the starter unit of PLMs is not 4, but 1
biosynthesized from 5. Support for this hypothesis was
obtained by an incorporation study of 1. The requisite
substrate, [7-13C]1 was synthesized from cyclohexanone
and K13CN via cyclohexanecarbonitrile.25 LC–MS analysis
of the resulting PLM mixture showed remarkable incorpor-
ation of 1 into PLMs (Table 2). The 13C NMR spectra of the
purified PLM-B, E and F obtained from feeding [7-13C]1
showed enhanced signals at d 139.9 (C-15), d 138.2 (C-15)
and 177.3 (C-10), and d 138.2 (C-15), respectively, (.20
fold enrichment, consistent with the LC–MS analysis,
Fig. 1(a), (d), and (g)). The 13C NMR spectra of PLM-B, E
and F obtained from feeding [7-13C]5 were essentially
identical to those from 1. These results indicated that 1 and 5
3. Discussion
PLM producers make two types of PLMs, i.e. PLMs with an
acyloxy side chain at C-18, which is cis to C-16 alkyl
substituent (PLM-A and PLMs C-F), and PLM-B, which has
Scheme 3. Incorporation of [7-13C]4, [7-13C]5 and [7-13C]1 into PLMs B, E
and F.
Scheme 2. Incorporation of [2-13C]2 into PLMs E and F.

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Y. Sekiyama et al. / Tetrahedron 59 (2003) 7465–7471
Table 2. Mass spectral analysis of PLMs obtained from feeding experiments of [7-13C]4, [7-13C]5 and [7-13C]121
Molecular related ions
(m/z)
Unlabeled PLMs
Relative intensity obtained from
(^)-[7-13C]4
(^)-[7-13C]5
[7-13C]1
100
68 (28%)
19
PLM-A
PLM-B
PLM-C
PLM-D
PLM-E
600
601
602
100
29
10
100
38 (8%)
8
73
100 (52%)
28
514
515
516
100
31
7
100
40 (8%)
10
93
100 (43%)
33
80
100 (48%)
27
614
615
616
100
35
9
100
43 (7%)
10
74
100 (50%)
29
100
63 (22%)
20
628
629
630
100
38
10
100
42 (4%)
10
72
100 (50%)
30
100
65 (21%)
19
641
642
643
644
100
35
11
0
100
46 (13%)
15
0
45
100 (78%)
76
21
74
100 (68%)
91
29
PLM-F
642
643
644
100
38
10
100
42 (4%)
9
71
100 (51%)
31
100
63 (20%)
18
Table 3. Mass spectral analysis of PLMs obtained from a feeding experiment with [2H11]1
Molecular related ions
(m/z)
Unlabeled PLMs
Relative intensity obtained
from [2H11]1
PLM-A
600
601
602
609
610
611
100
29
10
100
38
9
7
16
5
PLM-B
PLM-C
PLM-D
PLM-E
PLM-F
514
515
516
524
525
526
100
31
7
100
31
7
13
23
7
614
615
616
623
624
625
100
35
9
100
34
8
6
12
4
628
629
630
637
638
639
100
38
10
100
37
9
10
21
6
641
642
643
649
650
651
100
35
11
100
37
9
7
12
7
642
643
644
651
652
653
100
38
10
100
37
9
7
15
5

Y. Sekiyama et al. / Tetrahedron 59 (2003) 7465–7471
7469
no hydroxyl substituent on cyclohexane ring. cis-3-
Hydroxycyclohexanecarboxylic acid (4) and CHC (1) can
thus be considered as potential starter units. If 4 is utilized
for PLM biosynthesis, PLM-B could be produced by
dehydration and reduction of 18-hydroxylated precursor
before acylation, or biosynthesized from 1 formed by
dehydration and reduction of 4 (Scheme 1). In this case, 4
can be biosynthesized via reduction of 1,2-unsaturated
intermediate (6) from 1-Re face, as in the biosynthesis of the
DHCHC moiety of FK-52017,18 and rapamysin,19,20 or
hydroxylation of 1 (Scheme 1). Alternatively, 1 could be
used to initiate biosynthesis of all PLMs (Scheme 1). The
product of the polyketide synthase might then be PLM-B,
which could be converted to other analogs by hydroxylation
and acylation (Scheme 1). The specific uptake of 1 and 5
indicated that the starter unit of PLMs is not 4, but 1
biosynthesized from 5. The moderate enrichment of PLMs
observed in the feeding experiment of 4 can be attributed to
contamination of 4 with 5% of 5 and/or inefficient
dehydration of 4 by actynomycetes to produce 1 as shown
in biosynthesis of v-cyclohexyl fatty acids in Alicycloba-
cillus acidocaldarius.16 The fact that all deuterium atoms of
[2H11]1 were incorporated into PLMs has confirmed that 1
was incorporated in an intact form and used as the starter
unit directly. A pathway from shikimic acid (2) to the
coenzyme A activated CHC has been delineated in the
ansatrienin producer, S. collinus.14–16 This pathway has
been shown to have 5 as an intermediate in this process
(Scheme 1). The labeling of PLMs by 1, 4 and 5
demonstrated in this work is consistent with the same
pathway in Streptomyces sp. HK-803. A homologous set of
genes to those, which encode a CHC–CoA biosynthetic
pathway in S. collinus might also be predicted.
chromatography. Flash column chromatography was per-
formed on Merck silica gel (60, 230–400 mesh). All
reactions were performed under argon atmosphere.
Reagents and solvents were purchased at highest commer-
cial quality and used without further purification. K13CN
(99% 13C) was purchased from Sigma-Aldrich Co. D2O
(.99.8% D) was purchased from Merck Co.
4.1.1. [2-13C]Shikimic acid ([2-13C]2). This compound was
prepared according to the procedure described by Moore
et al.14 1H NMR (500 MHz, CD3OD) d 2.18 (1H, dm,
1
1JH–H¼18.4 Hz), 2.69 (1H, dm, JH–H¼18.3 Hz), 3.63–
3.71 (1H, m), 3.98 (1H, dd, J¼12.4, 5.5 Hz), 4.32–4.40
1
(1H, m), 6.79 (1H, dm, JC–H¼161.3 Hz, H-2); 13C NMR
(125 MHz, CD3OD) d 31.6, 67.3 (d, 1JC–C¼43.9 Hz, C-3),
68.4 (d, 3JC–C¼1.9 Hz, C-5), 72.7, 130.7 (d,
1JC–C¼68.7 Hz, C-1), 138.8 (enhanced signal, C-2), 170.0.
4.1.2. (6)cis-3-Hydroxy[7-13C]cyclohexanecarboxylic
acid ([7-13C]4). The mixture of 2-cyclohexenone (97.0%,
500 mg, 5.04 mmol), triethylamine hydrochloride (1.04 g,
7.55 mmol), K13CN (412 mg, 6.23 mmol), MeOH
(2.00 mL) and water (1.00 mL) was stirred under reflux at
708C for 9 h. The reaction mixture was diluted with
saturated aqueous NaHCO3 and CHCl3. The separated
aqueous layer was repeatedly extracted with CHCl3. The
combined organic layer was dried over Na2SO4, and
concentrated under reduced pressure. Column chromatog-
raphy of the resulting crude product with hexane–ether
(2:1) gave 3-oxo[7-13C]cyclohexanecarbonitrile (8)
(430 mg, 69%) as a colorless oil: 1H NMR (270 MHz,
CDCl3) d 1.70–2.23 (4H, m), 2.30–2.48 (2H, m), 2.50–
2.73 (2H, m), 2.93–3.12 (1H, m); 13C NMR (67.5 MHz,
3
CDCl3) d 23.8 (d, JC–C¼4.46 Hz, C-5), 28.1 (d,
1
In conclusion, we have demonstrated that CHC (1) is the
starter unit in the biosynthesis of all PLMs. The resulting
product from the polyketide synthase would then be
hydroxylated, before or after conversion to PLM-B, to
provide the other PLM analogs. Additional support for this
hypothesis has recently come through the cloning and
sequencing of the PLM biosynthetic gene cluster. Disrup-
tion of a cytochrome P450 monooxygenase gene within this
cluster results in a strain, which generates only PLM-B.27
2JC–C¼2.23 Hz, C-6), 28.6 (d, JC–C¼56.6 Hz, C-1),
2
40.7, 43.2 (d, JC–C¼2.23 Hz, C-2), 120.16 (enhanced
3
signal, C-7), 205.18 (d, JC–C¼3.92 Hz, C-3).
The mixture of 8 (430 mg, 3.46 mmol), MeOH (16.0 mL) and
H2SO4 (95.0%, 1.95 mL, 34.7 mmol) was stirred under reflux
for 15 h. The reaction mixture was diluted with saturated
aqueous NaHCO3 and CHCl3. The separated aqueous layer
was repeatedly extracted with CHCl3. The combined organic
layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. Column chromatog-
raphy of the resulting crude product with hexane–EtOAc
(10:1) gave methyl 3-oxo[7-13C]cyclohexanecarboxylate (7)
(301 mg, 55%) as a colorless oil: 1H NMR (270 MHz, CDCl3)
d 1.65–1.94 (2H, m), 1.99–2.18 (2H, m), 2.25–2.44 (2H, m),
4. Experimental
4.1. General procedures and materials
3
2.55 (2H, dd, JH–H¼7.97 Hz, JC–H¼3.11 Hz), 2.71–2.89
NMR spectra were recorded on JEOL ECP-500 or EX-270
spectrometers and chemical shifts (d) were referenced to the
residual undeuterated solvents as internal standards. LC–
MS spectra [positive turbo-ion spray ionization mode;
HPLC: Hewlett–Packard Series 1100; column:
150£2.1 mm RP-18 3.5 mm column from Waters; mobile
phase: 40:60 CH3CN–water (containing 0.05% HCO2H) at
a flow rate 0.2 mL/min] were taken on a Perkin–Elmer
SCIEX API 2000 pneumatically assisted electrospray
triplequadrapole mass spectrometer. Melting points were
measured by a Yanagimoto micro melting point apparatus,
and were uncorrected. Analytical TLC was carried out on
Merck silica gel 60F-254 plates (0.25 mm precoated).
Merck silica gel (60, 70–230 mesh) was used for column
(1H, m), 3.70 (3H, d, 3JC–H¼3.78 Hz); 13C NMR (67.5 MHz,
3
CDCl3) d 24.3 (d, JC – C¼5.00 Hz, C-5), 27.6 (d,
2JC–C¼1.62 Hz, C-6), 40.7, 42.88 (d, 1JC–C¼58.3 Hz, C-1),
2
2
42.93 (d, JC–C¼1.69 Hz, C-2), 51.9 (d, JC–C¼2.77 Hz,
OMe), 174.1 (enhanced signal, C-7), 208.6 (d,
3JC–C¼4.46 Hz, C-3).
NaBH4 (90.0%, 32 mg, 0.76 mmol) was added to a solution
of 7 (301 mg, 1.92 mmol) in MeOH (7.68 mL) at 08C. The
reaction mixture was stirred at room temperature for 15 min
and diluted with 2N HCl and ether. The separated aqueous
layer was repeatedly extracted with ether. The combined
organic layer was washed with saturated aqueous NaHCO3

7470
Y. Sekiyama et al. / Tetrahedron 59 (2003) 7465–7471
and brine, dried over Na2SO4, and concentrated under
reduced pressure. Flash column chromatography of the
resulting crude product with hexane–ether (4:1) gave
methyl (^)-cis-3-hydroxy[7-13C]cyclohexanecarboxylate
4.1.4. [7-13C]Cyclohexanecarboxylic acid ([7-13C]1).
Cyclohexanone (200 mg, 2.04 mmol) and 2,4,6-triisopro-
pylbenzenesulfonyl hydrazide (761 mg, 2.55 mmol) were
stirred together in MeOH (3.00 mL) solution at room
temperature for 1.5 h. K13CN (405 mg, 6.12 mmol) was
then added and the reaction mixture was stirred under reflux
for 4 h. The reaction mixture was diluted with saturated
aqueous NaHCO3 and CH2Cl2. The separated aqueous layer
was repeatedly extracted with CH2Cl2. The combined
organic layer was dried over Na2SO4, and concentrated
under reduced pressure to give [7-13C]cyclohexanecarboni-
trile (11) (178 mg, 79%) as a pale yellow oil which was used
for the next step without further purification: 1H NMR
(300 MHz, CDCl3) d 1.15–1.96 (10H, m), 2.56–2.71 (1H,
m); 13C NMR (75 MHz, CDCl3) d 24.0 (d, 3JC–C¼3.08 Hz,
1
(9) (181 mg, 59%). 9: colorless oil; H NMR (300 MHz,
CDCl3) d 1.15–1.49 (4H, m), 1.70–2.01 (4H, m), 2.13–
2.24 (1H, m), 2.29–2.45 (1H, m), 3.62 (1H, tt, J¼9.90,
3
4.20 Hz), 3.67 (3H, d, JC – H¼3.90 Hz); 13C NMR
3
(75 MHz, CDCl3) d 23.1 (d, JC–C¼5.55 Hz, C-5), 28.0
2
2
(d, JC–C¼1.88 Hz, C-6), 34.9, 37.6 (d, JC–C¼1.88 Hz,
1
C-2), 41.7 (d, JC – C¼58.1 Hz, C-1), 51.7, (d,
2JC–C¼2.48 Hz, OMe), 69.7 (d, JC–C¼4.95 Hz, C-3),
3
175.5 (enhanced signal, C-7).
Sodium hydroxide (97.0%, 175 mg, 4.24 mmol) was added
to the solution of 9 (335 mg, 2.10 mmol) in H2O (22.3 mL).
The mixture was stirred at room temperature for 12 h and
then continuously extracted with ether. The separated
aqueous layer was acidified with 6N HCl and continuously
extracted with ether. The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure
to afford [7-13C]4 (214 mg, 70%) as a white solid; mp 129–
1
C-3), 25.2, 27.9 (d, JC–C¼55.0 Hz, C-1), 29.4 (d,
2JC–C¼2.48 Hz, C-2), 122.6 (enhanced signal, C-7).
The mixture of 11 (325 mg, 2.95 mmol), EtOH (10.8 mL),
KOH (85%, 1.95 g, 29.5 mmol) was stirred under reflux for
6 h. The mixture was diluted with ether and H2O. The
separated organic layer was repeatedly extracted with 15%
aqueous NaOH. The combined aqueous layer was acidified
with 6N HCl and extracted with ether. The organic layer
was washed with brine, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Column chromatog-
raphy of the resulting crude product with hexane–ether
(5:1) afforded [7-13C]1 (204 mg, 54%) as a colorless oil: 1H
NMR (270 MHz, CDCl3) d 1.24–2.00(10H, m), 2.33(1H, tdt,
JH–H¼10.9 Hz, 2JC–H¼7.1 Hz, JH–H¼3.6 Hz), 11.4(1H, bs);
1
1308C; H NMR (500 MHz, acetone-d6) d 1.09–1.37 (4H,
m), 1.42–1.60 (0.2H, m, for 5), 1.73–1.93 (3H, m), 2.16
(1H, dm, J¼12.4 Hz), 2.27–2.37 (1H, m), 2.70–2.78
(0.05H, m, H-1 of 5), 3.54 (1H, tt, J¼10.8, 4.13 Hz),
3.96–4.01 (0.05H, m, H-3 of 3); 13C NMR (125 MHz,
acetone-d6) d 23.4 (d, 3JC–C¼4.76 Hz, C-5), 28.3, 35.2, 38.3,
41.5 (d, 1JC–C¼56.3 Hz, C-1), 69.0 (d, 3JC–C¼5.73 Hz, C-3),
175.6 (enhanced signal, C-7). Anal. calcd for C163CH12O3:
13
13
3
Cþ C, 57.92; H, 8.33. Found: Cþ C, 57.76; H, 8.32.
13C NMR (67.5 MHz, CDCl3) d 25.4, (d, JC–C¼3.85 Hz,
2
C-3), 25.8, 28.8 (d, JC–C¼1.08 Hz, C-2), 43.0
4.1.3. (6)trans-3-Hydroxy[7-13C]cyclohexanecarboxylic
acid ([7-13C]5). K-selectride (7.64 mL, 1.0 M solution in
THF, 7.64 mmol) was added dropwise to a solution of 7
(600 mg, 3.82 mmol) in THF (39.0 mL) at 2208C and the
mixture was stirred at the same temperature for 30 min. The
reaction mixture was diluted with saturated aqueous NH4Cl
and ether. The separated aqueous layer was repeatedly
extracted with ether. The combined organic layer was dried
over Na2SO4, and concentrated under reduced pressure. Flash
column chromatography of the resulting crude product with
hexane–ether (2:1) gavemethyl(^)-trans-3-hydroxy[7-13C]-
cyclohexanecarboxylate(10)(682 mg, 56%)asa colorless oil:
1H NMR (270 MHz, CDCl3) d 1.35–1.94 (8H, m), 2.70–2.83
(1H, m), 4.02–4.13 (1H, m); 13C NMR (67.5 MHz, CDCl3) d
(1JC–C¼55.0 Hz, C-1), 182.6 (enhanced signal, C-7).
4.1.5. [2H11]Cyclohexanecarboxylic acid ([2H11]1). This
compound was prepared according to the procedure
described by Pojer.26 1H NMR (500 MHz, CDCl3) d
1.14–1.31 (0.3H, m), 1.41 (0.3H, bs), 1.70 (0.2H, bs),
1.88 (0.2H, bs), 2.30 (0.2H, bs), 11.52 (1H, bs); 13C NMR
(125 MHz, CDCl3), d 23.6–25.4 (m), 27.2–28.6 (m), 41.8–
2
42.8 (m), 182.4; H NMR (77 MHz, CHCl3) d 1.08–1.31
(3D), 1.39 (2D), 1.69 (2D), 1.87 (2D), 2.27 (1D); ESI MS
(direct infusion, negative turbo-ion spray ionization mode,
Perkin–Elmer SCIEX API 2000 pneumatically assisted
electrospray triplequadrapole mass spectrometer) m/z 136
(16.7), 137 (45.1), 138 (100), 139 (8.1).
2
20.4 (d, 3JC–C¼3.38 Hz, C-5), 28.6 (d, JC–C¼1.69 Hz,
2
C-6), 33.4, 36.1 (d, JC–C¼1.69 Hz, C-2), 38.3 (d,
4.2. Fermentation
2
1JC–C¼57.2 Hz, C-1), 52.1, (d, JC–C¼2.77 Hz, OMe),
66.6 (d, 3JC–C¼3.31 Hz, C-3), 176.6 (enhanced signal, C-7).
Streptomyces sp. HK-803 was maintained at 158C on SY
agar slants (0.1% yeast extract, 1.0% soluble starch, 0.1%
N–Z amine type A, 1.5% agar, pH 7.0). A loopful of spore
was inoculated into a 500 mL cylindrical flask containing
70 mL of the production medium (2.0% glucose, 0.1% beef
extract, 1.0% soybean flour, 0.2% NaCl, 0.005% K2HPO4,
0.2% phenylalanine, pH 7.0). The inoculated cultures were
incubated at 288C in the dark for 96 h on a rotary shaker at
170 rpm.
The ester 10 (682 mg, 4.28 mmol) was hydrolyzed to
[7-13C]5 (461 mg, 74%) in the same manner as described for
1
[7-13C]4. [7-13C]5: white solids; mp 101–1028C; H NMR
(500 MHz, acetone-d6) d 1.14–1.42 (0.4H, m, for 4), 1.47–
1.64 (4H, m), 1.71–1.90 (4H, m), 2.18–2.24 (0.1H, H-2
of 4), 2.32–2.42 (0.1H, m, H-1 of 4), 2.75–2.84 (1H, m),
3.58 (0.1H, tt, J¼11.0, 4.13 Hz, H-3 of 4), 3.99–4.06
(1H, m); 13C NMR (125 MHz, acetone-d6) d 19.8 (d,
3JC – C¼3.81 Hz, C-5), 28.4, 32.9, 35.9, 37.4 (d,
4.3. Feeding experiments with labeled compounds
3
1JC–C¼56.3 Hz, C-1), 65.0 (d, JC–C¼2.86 Hz, C-3),
176.3 (enhanced signal, C-7); Anal. calcd for C163CH12O3:
Labeled compounds were dissolved in EtOH and added
directly to each flask 30 h after inoculation in the amount
13
13
Cþ C, 57.92; H, 8.33. Found: Cþ C, 57.65; H, 8.36.

Y. Sekiyama et al. / Tetrahedron 59 (2003) 7465–7471
7471
indicated in parenthesis: [7-13C]1 (10 mg), [7-13C]2
(30 mg), [7-13C]4 (20 mg), [7-13C]5 (20 mg), [2H11]1
(10 mg).
1983, 36, 1595–1600. (b) Stampwala, S. S.; Bunge, R. H.;
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4.4. Isolation of PLMs
The cultures were worked up, as previously described.7
Preparative HPLC was performed on a system equipped
with a Waterse 600 pump and a Waters 996 photodiode
array detector using a following conditions: column, Senshu
Pak PEGASIL ODS (20£250 mm); mobile phase: 40:60
CH3CN–water (containing 0.05% HCO2H) at a flow rate
9.0 mL/min; detection at UV 235 nm.
10. Usui, T.; Marriott, G.; Inagaki, M.; Swarup, G.; Osada, H.
J. Biochem. 1999, 125, 960–965.
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Acknowledgements
13. Shibata, T.; Kurihara, S.; Yoda, K.; Haruyama, H. Tetrahe-
dron 1995, 51, 11999–12012.
This work was supported by a Special Postdoctoral
Researchers Program (Y. S.) and Special Project Funding
for Basic Science (Chemical Biology Research from
RIKEN) and a grant from the NIH (AI51629-01) (K. A. R.).
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