Biotransformation of a biosynthetic intermediate mimic of nonactin by Streptomyces griseus

Article abstract of DOI:10.1515/HC.2011.021

A mimic of a plausible biosynthetic intermediate of bishomononactic acid, one of the monomers of macrotetrolide antibiotics (polynactin), was synthesized as a thiol ester. FAB-MS analysis showed that fermentation of polynactin producing Streptomyces griseus with the compound, afforded the unnatural polynactins containing bishomononactic acid(s). Copyright by Walter de Gruyter.

Full text of DOI:10.1515/HC.2011.021

Heterocycl. Commun., Vol. 17(3-4), pp. 93–98, 2011 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/HC.2011.021
Biotransformation of a biosynthetic intermediate mimic of
nonactin by Streptomyces griseus
Tadaatsu Hanadatea, Kentaro Takaia, Naoki Abe,
mimic of B or B’ could also be incorporated into the enzyme
system of this pathway, to produce new tetramer analogs
effectively. Thus, a racemic analog with a larger isopropyl
substituent 3, which would lead to macrotetrolides B-G and α
or new analogs, was designed for the biotransformation. This
paper describes the synthesis and biotransformation of 3.
Teiko Yamada, Shigefumi Kuwahara and Hiromasa
Kiyota*
Graduate School of Agricultural Science, Tohoku
University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai
981-8555, Japan
*Corresponding author
e-mail: kiyota@biochem.tohoku.ac.jp
Results and discussion
Scheme 2 shows the synthetic route towards 3. Regioselective
deprotonation of methyl isopropyl ketone, followed by the
addition of commercially available aldehyde 4, afforded aldol
product 5. Stereoselective reduction of the keto group in 5 was
accomplished using Me4NHB(OAc)3 (Evans et al., 1986) to
give 6 (anti/syn=95:5) in 86% yield after separation of the ste-
reoisomers. The anti-relationship of the dihydroxy functional-
ity was determined by 13C NMR analysis of the corresponding
acetonide (16, vide infra). Protection of the two hydroxy groups
by TBS groups 7, deprotection of the Bn group (8), followed
by Swern oxidation, afforded aldehyde 9, which was subjected
to the modified Horner-Wadsworth-Emmons reaction (Rathke
and Nowak, 1985) to give 10 (E/Z=9:1, separated by SiO2 chro-
matography). The geometry of the isomers was determined by
observation of a strong NOE correlation between 3-H and 2-Me
for (Z)-10. Hydrolysis of the ethoxycarbonyl group furnished
carboxylic acid 11. On the other hand, synthesis of the thiol frag-
ment suffered from oxidative dimerization of products, such as
the formation of 13 from 12. Thus, thiol 14 was prepared in situ
by the reduction of 13 and was subjected to condensation with
11 to afford the thiol ester 15 in 73% yield. Finally, removal of
the two TBS groups gave the desired substrate 3.
In order to determine the stereochemistry of the anti-diol
moiety, 6 was converted to an acetonide 16 (Scheme 3). Then,
the C2-symmetrical nature of the dioxane ring of 16 was elu-
cidated by 13C NMR chemical shift of the geminal methyl
groups (24.3 and 24.6 ppm), and that of the quaternary car-
bon (100.2 ppm) which is typical for this kind of structure
(Kocienski, 2005). In a similar manner as described for the
TBS derivatives, 16 was converted to 3.
Priestley has reported the synthesis of the inhibitor 18 of
polynactin biosynthesis (Earle and Priestley, 1997). To stop
the polyketide biosynthesis upstream toward A, B and B’, we
also prepared the analog 20 in a different manner (Scheme 4).
Aldol reaction of acetone with 4 afforded 21 (Nogawa et al.,
2006), which was converted to aldehyde 22 with anti-oxygen
functions. Treatment of 22 with Ohira reagent (Ohira, 1989)
gave the intermediate product 23, the chain elongation of
which gave carboxylic acid 24. Deprotection of the thioester
derivative 25 furnished Priestley’s diol 20.
Abstract
A mimic of a plausible biosynthetic intermediate of bisho-
mononactic acid, one of the monomers of macrotetrolide
antibiotics (polynactin), was synthesized as a thiol ester.
FAB-MS analysis showed that fermentation of polynactin pro-
ducing Streptomyces griseus with the compound, afforded the
unnatural polynactins containing bishomononactic acid(s).
Keywords: antibiotics; bishomononactic acid; biotransfor-
mation; polynactin; Streptomyces griseus; synthesis.
Introduction
The macrotetrolide ionophore antibiotic ‘polynactin’ family
1, which has been isolated from various Streptomyces species
(Keller-Schierlein and Gerlach, 1967; Ando et al., 1971), is
composed of both enantiomers of nonactic acid 2a, homonon-
actic acid 2b or bishomononactic acid 2c arranged in an alter-
nating order (Figure 1).
The macrolides and monomers show a wide variety of bio-
logical activities (Zizka, 1998) such as antimicrobial, fungi-
cidal, acaricidal, immunosuppressive etc. During the course of
the synthetic studies of new tetramer analogs, we synthesized
macrotetrolides α 1j (Hanadate et al., 2001; Takai et al., 2011)
and β 1k (Takai et al., 2006), however, the synthesis needed
over 30 chemical steps. On the other hand, a mixture of the
parts 1a-1e is used as an acaricide by effective fermentative
production. Recently, their unique biosynthetic pathway was
revealed (Rong et al., 2010) (Scheme 1). Assembly of the
various acyl CoA fragments led to diketone A. Successive
anti-reduction affords the enantiomeric pair diols B and B’,
which are cyclized to give nonactic acid thiol esters C and C’,
respectively. These monomers are alternatively assembled to
form nonactin 1a. Very recently, fermentative production of
polynactin congeners by addition of various acyl CoA deriva-
tives was reported (Rezanka et al., 2010). We assumed that a
a Co-first author.
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94 T. Hanadate et al.
R2
O
O
OH
5
a)
OBn
O
b)
e)
OBn
OBn
O
O
O
O
O
R3
O
(+)
(–)
4
O
O
O
R1
TBSO
OTBS
*
OH OH
(–)
O
(+)
O
c)
OR
*
*
*
O
R4
6
7 R=Bn
8 R=H
OTBS
d)
(anti/syn : 95:5)
Polynactin (1)
TBSO
TBSO
*
OTBS
R1
R2
R3
R4
f)
OR
(1a)
(1b)
(1c)
(1d)
(1e)
(1f)
Nonactin
Monactin
Dinactin
Me
Me
Me
Me
Et
Me
Me
Me
Me
Et
Me
Et
Et
Et
Et
*
*
*
Me
Et
O
10 R=Et (E/Z=9:1)
11 R=H
9
g)
Trinactin
Tetranactin
Et
Et
Et
Macrotetrolide B
Et
i-Pr
Me
Me
Me
Et
i-Pr
i-Pr
i-Pr
Me
(1g)
(1h)
(1i)
Et
i-Pr
Et
C
D
G
HS
NH2
h)
Et
Et
i-Pr
12
O
(1j)
(1k)
Macrotetrolide α
Macrotetrolide β
i-Pr i-Pr i-Pr
CF3 CF3 CF3 CF3
i-Pr
O
i)
C7H15
N
H
13
C7H15
N
O
OH
H
S
SH
14
2
HO
R
O
TBSO
*
OTBS
*
O
(+)-Nonactic acid
[(+)-2a] R = Me
[(+)-2b] R = Et
[(+)-2c] R = i-Pr
S
N
(+)-Homononactic acid
C7H15
(+)-Bishomononactic acid
H
O
15
j)
Figure 1 Polynactin antibiotics.
OH OH
O
S
N
Biotransformation was performed using Streptomyces gri-
seus ETH 7796. The pre-incubated strain (Bacto-tryptone,
yeast extract, and maltose media, 30°C, 2 days) was incubated
with and without the compounds (A: control, B: 3, C: 3+20,
*
*
C7H15
H
O
3
Scheme 2 Synthesis of the substrate for biotransformation-1.
a) LDA, MeC(=O)i-Pr (88%). b) Me4NHB(OAc)3, AcOH, MeCN
(86%). c) TBSCl, imidazole, DMF (88%). d) H2, Pd/C, MeOH
(89%). e) Swern oxi. f) (EtO)2P(=O)CHMeCO2Et, LiBr, Et3N, THF
(78%, 2 steps). g) 2 m aqueous KOH. h) i. 15% aqueous NaOH, Et2O,
10°C. ii. C7H15COCl (74%). i) Bu3P, N2, DMF, H2O then 11, EDCI,
DMAP (73% from 10). j) BF3·Et2O, CH2Cl2, 0°C to room tempera-
ture (90%).
O
O
–O2C
SCoA CoAS
c
a
d
–O2C
e
CoAS
–
b
CO2
O
CoAS
O
Condensation
O
O
e
CoAS
and D: 20, each 17 mm) for 6 days by a rotary shaker, and each
culture broth was filtrated through a Celite pad. The pad was
washed with acetone and the washings were combined with
the filtrate. Each combined organic layer was concentrated in
vacuo and silica gel chromatography gave the crude polynac-
tin mixture (Table 1) which was analyzed by FAB-MS (Figure
2). Addition of the inhibitor 20 (entries 3 and 4) significantly
diminished the yield of polynactin, indicating that 20 actually
inhibits the biosynthesis. Unexpectedly, addition of the mimic
3 also decreased the yield (entry 2) as compared with con-
trol (entry 1). The mimic 3, with a bulky isopropyl substitu-
ent, would partly act as an inhibitor as well as the substrate.
Figure 2 (A) indicates that the control fraction contained an
almost equal amount of nonactin (1a, m/z 759) and monactin
(1b, m/z 773). The signal (m/z 787) is for dinactin 1c. On the
contrary, the signals corresponding to 1a and 1b decreased
in Figure 2 (B), and the larger peaks (m/z 787, 801, and 815)
were predominant. These peaks could be assigned to unnatu-
ral polynactin with R1–4=Me3 and i-Pr (m/z 787 [M+Na]+),
R1–4=Me2, Et and i-Pr (m/z 801 [M+Na]+), and R1–4=Me2 and
c
a
d
b
O
A
Reduction
OH OH
OH OH
CoAS
CoAS
CoAS
O
O
B
B'
O
oxy-Michael
addition
O
HO
OH
+
CoAS
O
C
O
C'
Condensation
Nonactin (1a)
O
OH OH
S
N
*
*
H
O
Substrate for biotransformation (3)
Scheme 1 Biosynthetic pathway of polynactin antibiotics.
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Biotransformation of a biosynthetic intermediate mimic of nonactin 95
24.6
24.3 ppm
Table 1 Biotransformation of the synthetic compounds by S.
griseus ETH 7796.
100.2
O
O
O
*
O
*
a
b
6
*
*
Entry
Substrates
Yield (mg/100 ml broth)
OBn
OH
16
17
1
2
3
4
None
3
3 + 20
20
67
5
<1
2
O
*
O
c
e
3
OR
*
O
18 R=Et
19 R=SCH2CH2NHCOC7H15
Gemini 2000 (300 MHz for 1H and 75 MHz for 13C) spectrometer in
CDCl3 with tetramethylsilane (δH 0 ppm) and CHCl3 (δC 77.00 ppm)
as internal standards. Mass spectra were recorded with a Jeol JMS–
700 spectrometer. Merck silica gel 60 (70–230 mesh) was used for
column chromatography. Merck silica gel 60 F254 (0.50 mm thick-
ness) was used for preparative TLC. Streptomyces griseus ETH 7796
was purchased from Deutsche Sasmmlung von Mikroorganismen
und Zellkulturen GmbH.
d
Scheme 3 Synthesis of the substrate for biotransformation-2.
a) 2,2-dimethoxypropane, PPTS (quant.). b) H2, Pd/C, NaHCO3,
EtOH (54%). c) i. Swern oxi. ii. Ph3P=CMeCO2Et, toluene, reflux
(98%). d) i. 1 m aqueous KOH-MeOH-THF. ii. 14, EDCI, DMAP,
CH2Cl2 (61%). e) PPTS, MeOH (quant.).
8-Benzyloxy-5-hydroxy-2-methyloctan-3-one (5)
i-Pr2 or Me, Et and i-Pr (m/z 815 [M+Na]+) considering the
amount of other natural polynactin congeners. A weak signal
872 [M+Na]+ corresponding to macrotetrolide α 1j was also
detected. Although a total amount of polynactin production
decreased, the designed mimic 3 would be incorporated by
the enzyme system.
In conclusion, diol 3, a mimic of biosynthetic intermediate
of macrotetrolide antibiotics, was synthesized as a racemate
for biotransformation. Although partly acting as a biosyn-
thetic inhibitor, 3 could be incorporated into the enzyme sys-
tem to produce polynactin congeners.
To a solution of LDA [lithium diisopropylamide (ca. 88 mmol, pre-
pared from diisopropylamine (12 ml, 88 mmol) and BuLi (1.6 m in
hexane, 55 ml, 88 mmol)] in dry THF (tetrahydrofuran, 100 ml) was
added dropwise 3-methylbutan-2-one (6.7 g, 78 mmol) in dry THF
(20 ml) at -78°C over 1 h, and the mixture was stirred for 1.5 h.
Then, to this was added dropwise a solution of 4 (14 g, 78 mmol) in
dry THF (30 ml) over 30 min, and the mixture was stirred for 1 h.
The reaction mixture was poured into a saturated aqueous NH4Cl
solution and extracted with ether. The combined extract was washed
with water and brine, dried with MgSO4 and concentrated in vacuo.
The residue was chromatographed on silica gel. Elution with hexane/
EtOAc (3:1) gave 5 (18 g, 69 mmol, 88%) as a colorless oil. FT-IR:
νmax 3439 (s, OH), 1704 (s, C=O), 1095 (s), 736 (s), 697 cm-1 (s);
NMR: δH 1.07 (3H, d, J=6.7 Hz, i-Pr), 1.08 (3H, d, J=7.2 Hz, i-Pr),
1.50–1.85 (4H, m), 2.56–2.64, 3.35 (1H, d, J=3.3 Hz, OH), 3.51 (2H,
t, J=6.0 Hz, H-8), 4.00–4.10 (1H, m, 5-H), 4.51 (2H, s, CH2Ph),
7.32–7.36 (5H, m, Ph). NMR: δC 138.4, 128.3, 127.6, 127.5, 72.7,
70.1, 67.3, 46.5, 41.2, 33.3, 25.6, 17.7. HR-FAB MS: m/z calcd. for
C16H25O3 [M+H]+ 265.1804; found 265.1811.
Experimental
General
FT-IR spectra were recorded for films on a Jasco 4100 spectrometer
(ATR, Zn-Se). 1H and 13C NMR spectra were recorded with a Varian
O
O
OH
A
B
R=Me3, i-Pr
a
b
OBn
OBn
Monactin
(1b)
773
787
4
21
R=Me2, i-Pr2
(Me, Et, i-Pr)
759
TBSO
*
OTBS
*
TBSO
*
OTBS
*
Nonactin
(1a)
c
d
O
801
22
23
R=Me2, i-Pr2
(Me, Et, i-Pr)
OH OH
TBSO
*
OTBS
*
f
Dinactin
(1c)
787
*
*
O
OR
O
815
S
O
24 R=H
25 R=SCH2CH2NHCOC7H15
e
C7H15
N
20
773
759
Scheme 4 Synthesis of the biosynthesis inhibitor.
750 760 770 780 790 800 810 820
760 770 780 790 800 810 820 830
m/z
m/z
a) i. LDA, acetone, THF, -78°C (74%). ii. b) i. Me4NBH(OAc)3,
AcOH, MeCN (86%, anti/syn=95:5). ii. TBSCl, imidazole, DMF
(80%). iii. H2, Pd/C, MeOH, iv. Swern oxi. c) Ohira reagent, K2CO3,
MeOH (72%, 3 steps). d) BuLi, CO2(s), THF. e) 13, Bu3P, DMF, H2O
then EDCI, DMAP (54%, 2 steps). f) AcOH-THF-H2O (85%).
Figure 2 Parts of FAB-MS charts of the products of microbial
transformation.
(A) Control; (B) +3.
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96 T. Hanadate et al.
(3SR,5RS)-8-Benzyloxy-2-methyloctane-3,5-
diol (6)
yellow oil. FT-IR: νmax 1713 (s, C=O), 1652 (w, C=C), 1471 (m),
1367 (m), 1254 (s), 1053 (s), 834 (s), 772 cm-1 (s); NMR: δH 0.04
(3H, s, SiMe), 0.05 (3H, s, SiMe), 0.06 (3H, s, SiMe), 0.07 (3H, s,
SiMe), 0.83 (3H, d, J=6.9 Hz, i-Pr), 0.85–0.88 (3H, m, i-Pr), 0.880
(9H, s, t-Bu), 0.889 (9H, s, t-Bu), 1.29 (3H, t, J=7.1 Hz, CH2CH3),
1.45 (1H, ddd, J=14.7, 7.4, 5.2 Hz), 1.52–1.61 (3H, m), 1.70 (1H,
dquint, J=3.0, 6.9 Hz), 1.84 (3H, d, J=1.1 Hz, 2-Me), 2.16–2.28 (2H,
m, 4-H), 3.62 (1H, ddd, J=7.4, 4.7, 3.1 Hz), 3.76 (1H, dt, J=5.5,
6.6 Hz), 4.19 (2H, q, J=7.1 Hz, CH2CH3), 6.76 (1H, dt, J=7.4,
1.4 Hz, 3-H); NMR: δC 168.4, 142.2, 127.9, 74.2, 70.0, 60.3, 40.7,
36.6, 33.1, 25.8, 25.8, 24.2, 18.0, 17.9, 17.4, 16.9, 14.2, 12.2, -4.1,
-4.3, -4.4. HR-FAB MS: m/z calculated for C26H55O4Si2 [M+H]+
487.3639; found 487.3645.
To a solution of Me4NBH(OAc)3 (10 g, 38 mmol) in dry CH3CN
(11 ml) was added anhydrous AcOH (11 ml) and the mixture was
stirred at 20°C for 30 min. After the mixture was cooled to -40°C, a
solution of 5 (2.0 g, 7.6 mmol) in dry CH3CN (4 ml) was added. After
being stirred at -40°C for 18 h, the mixture was treated with 0.5 m
aqueous solution of NaOH and allowed to warm to 20°C with stir-
ring. The mixture was extracted with EtOAc and the combined ex-
tract was washed with a saturated aqueous NH4Cl solution and brine,
dried with MgSO4 and concentrated in vacuo. The residue was chro-
matographed on silica gel. Elution with hexane/EtOAc (3:1) gave 6
(1.7 g, 6.5 mmol, 86%) as a colorless oil. FT-IR: νmax 3399 (s, OH),
1096 (m), 735 (m), 687 cm-1 (m); NMR: δH 0.87 (3H, d, J=6.9 Hz,
i-Pr), 0.92 (3H, d, J=6.9 Hz, i-Pr), 1.46–1.66 (4H, m), 1.71 (1H,
sep, J=6.6 Hz, 2-H), 3.18 (1H, br, OH), 3.50 (2H, t, J=6.0 Hz, H-8),
3.61 (1H, ddd, J=8.8, 5.8, 3.0 Hz, 5-H), 3.85–3.94 (1H, m, 3-H),
4.50 (2H, s, CH2Ph), 7.24–7.37 (5H, m, Ph); NMR: δC 138.1, 128.4,
127.7, 127.7, 73.5, 72.9, 70.4, 69.0, 39.4, 34.47, 33.6, 26.3, 18.4,
17.9. HR-FAB MS: m/z calculated for C16H27O3 [M+H]+ 267.1960;
found 267.1967.
Bis(2-octanamidoethyl) disulfide (13)
A mixture of 12 (2.5 g, 32 mmol), 15% aqueous NaOH (75 ml) and
ether (25 ml) was stirred at 10°C for 1 h, then treated with octanoyl
chloride (6.8 ml, 40 mmol) and the mixture was stirred at 10°C for
2 h. The mixture was diluted with 1 m aqueous HCl and extracted
with ether. The extract was washed with brine, dried with MgSO4 and
concentrated in vacuo. The residue was crystallized from hexanes
to give 13 (4.8 g, 12 mmol, 74%) as a white powder. FT-IR: νmax
3299 (s, NH), 3059 (w), 1635 (s, C=O), 1542 (s), 1469 (m), 1421
(m), 1204 (m), 1041 (w), 682 cm-1 (w); NMR: δH 0.86–0.90 (6H,
m, 8-H), 1.2–1.4 (16H, m, 4,5,6,7-H), 1.6–1.7 (4H, m, 3-H), 2.14
(4H, t, J=7.7 Hz, 2-H), 2.83 (4H, t, J=6.3 Hz, SCH2), 3.58 (4H, q,
J=6.6 Hz, NHCH2), 6.25 (2H, br, NH); NMR: δC 169.7, 38.7, 37.6,
36.2, 31.6, 29.1, 28.9, 25.7, 22.5, 13.9. HR-FAB MS: m/z calculated
for C20H41O2N2S2 [M+H]+ 405.2609; found 405.2607.
(4RS,6SR)-1-Benzyloxy-4,6-bis(t-
butyldimethylsilyloxy)-7-methyloctane (7)
A solution of 6 (2.0 g, 7.6 mmol), imidazole (2.4 g, 30 mmol) and
TBSCl (3.0 g, 20 mmol) in DMF (50 ml) was stirred at 20°C for
16 h. The mixture was diluted with ether, washed with water, dried
with MgSO4 and concentrated in vacuo. The residue was chromato-
graphed on silica gel. Elution with hexane/EtOAc (15:1) gave 7
(3.3 g, 6.7 mmol, 88%) as a colorless oil. FT-IR: νmax 1471 (w), 1361
(w), 1253 (m), 1057 (s), 833 (s), 771 cm-1 (s); NMR: δH 0.02 (3H,
s, SiMe), 0.03 (6H, s, SiMe), 0.04 (3H, s, SiMe), 0.79–0.85 (6H, m,
i-Pr), 0.86 (18H, s, t-Bu), 1.37–1.56 (4H, m), 1.60–1.73 (3H, m),
3.44 (2H, t, J=6.3 Hz, 1-H), 3.61 (1H, quint, J=3.6 Hz), 3.73 (1H,
quint, J=5.7 Hz), 4.48 (2H, s, CH2Ph), 7.22–7.28 (1H, m, Ph), 7.31
(2H, s, Ph), 7.32 (2H, s, Ph); NMR: δC 138.8, 128.4, 127.7, 127.5,
74.2, 72.8, 70.5, 70.2, 40.8, 34.4, 33.2, 25.8, 25.6, 25.2, 18.0, 18.0,
17.4, 16.9, -4.0, -4.3. HR-FAB MS: m/z calculated for C28H55O3Si2
[M+H]+ 495.3690; found 495.3692.
S-2-Octanamidoethyl (2E,6RS,8SR)-6,8-bis
(t-butyldimethylsilyloxy)-2,9-dimethyldec-2-enethioate
(15)
In the same manner as described for 18, ester 10 (19.5 mg,
0.0400 mmol) was converted to 11. NMR: δH 0.04 (3H, s, SiMe),
0.05 (3H, s, SiMe), 0.06 (3H, s, SiMe), 0.07 (3H, s, SiMe), 0.83 (3H,
d, J=6.9 Hz, i-Pr), 0.85–0.88 (3H, m, i-Pr), 0.879 (9H, s, t-Bu), 0.889
(9H, s, t-Bu), 1.40–1.78 (5H, m), 1.84 (3H, s, 2-Me), 2.17–2.32 (2H,
m, 4-H), 3.58–3.65 (1H, m), 3.72–3.81 (1H, m), 6.91 (1H, t, J=7.4 Hz,
3-H). The carboxylic acid 11 was condensed with thiol 14 [FT-IR:
ν
max 3302 (s, NH), 3059 (w), 1638 (s, C=O), 1550 cm-1 (s); NMR: δH
(4RS,6SR)-4,6-Bis(t-butyldimethylsilyloxy)-7-
methyloctan-1-ol (8)
0.88 (3H, t, J=5.8 Hz, 8-H), 1.2–1.4 (8H, m, 4,5,6,7-H), 1.6–1.7 (2H,
m, 3-H), 2.20 (2H, t, J=8.2 Hz, 2-H), 2.68 (2H, t, J=7.1 Hz, SCH2),
3.45 (2H, q, J=6.1 Hz, NHCH2), 5.84 (1H, br, NH); prepared from
the sulfide 13] to give 15 (19 mg, 0.029 mmol, 73% from 10) as a
pale yellow oil. FT-IR: νmax 1658 (m, C=O), 1471 (w), 1254 (m),
1068 (m), 837 (m), 774 cm-1 (m); NMR: δH 0.05 (6H, br s, SiMe),
0.07 (3H, s, SiMe), 0.08 (3H, s, SiMe), 0.82–0.88 (6H, m, i-Pr), 0.88
(9H, s, t-Bu), 0.89–0.90 (9H, m, t-Bu), 1.24–1.64 (15H, m), 1.89
(3H, s, 2-Me), 2.15 (2H, t, J=7.7 Hz), 2.20–2.31 (2H, m), 3.08 (1H, t,
J=6.0 Hz, SCH2), 3.46 (2H, q, J=6.0 Hz, NCH2), 3.58–3.68 (1H, m),
3.72–3.80 (1H, m), 5.84 (1H, br, NH), 6.77 (1H, dt, J=6.0, 2.4 Hz,
3-H). HR-FAB MS: m/z calculated for C34H70O4NSSi2 [M+H]+
644.4564; found 644.4557.
In a similar manner as described for 17, compound 7 (3.0 g,
6.1 mmol) was converted to 8 (2.2 g, 5.4 mmol, 89%) as a colorless
oil. FT-IR: νmax 3389 (m, OH), 1471 (w), 1387 (m), 1254 (m), 1053
(s), 907 (s), 833 (s), 772 (s), 733 cm-1 (s); NMR: δH 0.04 (3H, s,
SiMe), 0.05 (3H, s, SiMe), 0.07 (3H, s, SiMe), 0.08 (3H, s, SiMe),
0.83 (3H, d, J=7.1 Hz, i-Pr), 0.86 (3H, d, J=7.1 Hz, i-Pr), 0.884 (6H,
s, t-Bu), 0.889 (6H, s, t-Bu), 0.891 (6H, s, t-Bu), 1.42–1.76 (7H, m),
2.07 (1H, br, OH), 3.56–3.66 (4H, m), 3.79 (1H, quint, J=5.4 Hz);
NMR: δC 74.3, 70.2, 63.1, 40.4, 34.3, 33.0, 28.0, 25.8, 25.8, 18.0,
17.9, 17.4, 16.8, -4.1, -4.3, -4.3, -4.4. HR-FAB MS: m/z calculated
for C21H49O3Si2 [M+H]+ 405.3220; found 405.3213.
S-2-Octanamidoethyl (2E,6RS,8SR)-6,8-hydroxy-2,9-
dimethyldec-2-enethioate (3)
Ethyl (2E,6RS,8SR)-6,8-bis(t-butyldimethylsilyloxy)-
2,9-dimethyldec-2-enoate (10)
From 15: a solution of 15 (10 mg, 0.016 mmol) and BF3·OEt2 (2
drops) in CHCl3 (2 ml) was stirred at 0°C for 1 h. The mixture was
diluted with a saturated aqueous NaHCO3 solution and extracted
In a similar manner as described for 18, compound 8 (4.4 g,
11 mmol) was converted to 10 (4.1 g, 8.6 mmol, 78%) as a pale
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Biotransformation of a biosynthetic intermediate mimic of nonactin 97
with ether. The extract was washed with brine, dried with MgSO4
mixture was added 17 (966 mg, 4.67 mmol) in dry THF (25 ml) and
the resulting mixture was stirred for 30 min. Then Et3N (3.30 ml,
23.40 mmol) was added and the mixture was allowed to warm to
20°C. After 30 min, the mixture was diluted with water and extract-
ed with EtOAc. The combined extract was washed with water and
brine, dried with MgSO4 and concentrated in vacuo to give aldehyde
(990 mg, ca. 4.63 mmol) as a pale yellow oil. The aldehyde was used
in the next step without further purification.
A solution of the aldehyde (990 mg, ca. 4.63 mmol) and
Ph3P=CMeCO2Et (2.52 g, 6.95 mmol) in dry toluene (90 ml) was
stirred at reflux for 20 h. The mixture was concentrated in vacuo and
the residue was chromatographed on silica gel. Elution with hexanes/
EtOAc (7:1) gave 18 (1.31 g, 4.39 mmol, 98%) as a pale yellow oil.
Rf=0.66 (SiO2, hexane/EtOAc=3:1); FT-IR: νmax 2984 (s), 2936 (s),
1711 (s, C=O), 1651 (w), 1464 (w), 1378 (m), 1263 (m), 1225 (s),
1171 (m), 1135 (m), 1105 (m), 1020 (m), 907 (w), 744 cm-1 (w);
NMR: δH 0.85 (3H, d, J=6.6 Hz, i-Pr), 0.91 (3H, d, J=6.6 Hz, i-Pr),
1.29 (3H, t, J=7.1 Hz, CH2CH3), 1.33 (3H, s, acetonide), 1.34 (3H,
s, acetonide), 1.47–1.69 (5H, m), 1.84 (3H, s, 2-Me), 2.22–2.31 (2H,
q, J=7.6 Hz, 4-H), 3.36–3.46 (1H, m), 3.66–3.78 (1H, m), 4.17 (2H,
q, J=7.1 Hz, CH2CH3), 6.75 (1H, dt, J=7.4, 1.4 Hz, 3-H); NMR: δC
141.8, 100.3, 71.7, 66.1, 60.4, 36.5, 34.6, 32.8, 24.7, 24.5, 24.2, 18.7,
17.5, 14.2, 12.3. HR-FAB MS: m/z calculated for C17H31O4 [M+H]+
347.1470; found 299.2225.
and concentrated in vacuo. The residue was chromatographed on
preparative TLC. Development with hexanes/EtOAc (4:1) gave 3
(5.8 mg, 0.014 mmol, 90%) as a pale yellow oil.
From 19: a solution of 19 (1.34 g, 2.93 mmol) and PPTS (300 mg)
in MeOH (50 ml) was stirred at 20°C for 4.5 h, and the reaction
mixture was concentrated in vacuo. The residue was diluted with
EtOAc, washed with saturated aqueous NaHCO3 solution and brine,
dried with MgSO4 and concentrated in vacuo. The residue was chro-
matographed on silica gel. Elution with hexanes/EtOAc (1:2) gave
3 (1.22 g, 2.93 mmol, quantitative). NMR: δH 0.80–1.00 (9H, m),
1.20–1.40 (8H, m), 1.52–1.80 (9H, m), 1.89 (3H, s, 2-Me), 2.15
(2H, t, J=7.8 Hz, CH2C=O), 2.24–2.50 (2H, m, 4-H), 3.07 (2H, t,
J=6.0 Hz, SCH2), 3.42–3.50 (3H, m, NCH2, OH), 3.66–3.74 (1H,
m), 3.90–4.00 (1H, m), 5.84 (1H, br, NH), 6.77 (1H, dt, J=6.0,
2.4 Hz, 3-H). HR-FABMS m/z: calculated for C22H42O4NS [M+H]+
416.2835; found 416.2838.
(4RS,6SR)-1-Benzyloxy-4,6-isopropylidenedioxy-7-
methyloctane (16)
A solution of 6 (30 mg, 0.11 mmol) and PPTS (pyridinium p-tolue-
nesulfonate, 10 mg) in 2,2-dimethoxypropane (3 ml) was stirred for
2 h at 0°C. The reaction mixture was diluted with a saturated aqueous
NaHCO3 solution and concentrated in vacuo. The residue was diluted
with ether, washed with water and brine, dried with MgSO4 and con-
centrated in vacuo. The residue was chromatographed on silica gel.
Elution with hexanes/EtOAc (3:1) gave 16 (33 mg, 0.11 mmol, quan-
titative) as a pale yellow oil. Rf=0.70 (SiO2, hexane/EtOAc=3:1);
FT-IR: νmax 3031 (w), 2984 (s), 2937 (s), 2871 (s), 1586 (w), 1496
(w), 1454 (m), 1377 (s), 1224 (s), 1171 (m), 1100 (s), 1071 (m), 998
(m), 931 (w), 908 (w), 735 (m), 697 cm-1 (m); NMR δH 0.84 (3H, d,
J=6.6 Hz, i-Pr), 0.91 (3H, d, J=6.6 Hz, i-Pr), 1.31 (6H, s, acetonide),
1.46–1.81 (7H, m), 3.35–3.54 (3H, m), 3.67–3.78 (1H, m), 4.50 (2H,
s, Bn), 7.26–7.34 (5H, m, Ph); NMR: δC 138.7, 128.4, 127.7, 127.6,
100.2 (acetonide), 72.9, 71.7, 70.2, 66.6, 36.5, 32.9, 32.4, 25.8, 24.5
(acetonide), 24.3 (acetonide), 18.7, 17.5. HR-FAB MS: m/z calcu-
lated for C19H31O3 [M+H]+ 307.2273; found 307.2270.
S-2-Octanamidoethyl (2E,6RS,8SR)-6,8-
isopropylidenedioxy-2,9-dimethyldec-2-enethioate
(19)
A mixture of 18 (1.42 g, 4.76 mmol), 1 m aqueous KOH (20 ml),
THF (15 ml) and MeOH (10 ml) was stirred for 20 h. The mixture
was concentrated and the residue was neutralized with 1 m aqueous
HCl, and extracted with EtOAc. The combined extract was washed
with brine, dried with MgSO4 and concentrated in vacuo to give car-
boxylic acid (1.17 g, ca. 4.33 mmol). The carboxylic acid was used
in the next step without further purification.
A mixture of the carboxylic acid (1.17 g, ca. 4.33 mmol), thiol
14 (1.76 g, 8.65 mmol), EDCI·HCl [1-(3-dimethylaminopropyl)-3
-ethylcarbodiimide hydrochloride, 1.66 g, 8.66 mmol] and DMAP
(4-dimethylaminopyridine, 60.0 mg, 0.50 mmol) in dry CH2Cl2
(10 ml) was stirred for 18 h at room temperature. The reaction mix-
ture was diluted with a saturated aqueous NH4Cl solution and ex-
tracted with EtOAc. The combined extract was washed with brine,
dried with MgSO4 and concentrated in vacuo. The residue was chro-
matographed on silica gel. Elution with hexanes/EtOAc (3:1) gave
19 (1.33 g, 2.92 mmol, 61%) as a pale yellow oil. Rf=0.26 (SiO2,
hexane/EtOAc=3:1); FT-IR: νmax 3289 (w, NH), 2985 (m), 2955
(s), 2929 (s), 2871 (m), 1654 (s, C=O), 1544 (m), 1462 (w), 1378
(m), 1224 (s), 1111 (w), 1066 (w), 1020 (w), 909 (w), 659 cm-1 (w);
NMR: δH 0.83–0.92 (24H, m), 1.24–1.35 (15H m), 1.48–1.71 (5H,
m), 1.88 (3H, s, 2-Me), 2.15 (2H, t, J=Hz), 2.31 (2H, q, J=Hz), 3.09
(2H, t, J=6.3 Hz), 3.36–3.50 (3H, m), 3.68–3.78 (1H, m), 5.88 (1H,
br, NH), 6.75–6.82 (1H, t, 2-H, J=Hz, H-3).
(4RS,6SR)-4,6-isopropylidenedioxy-7-methyloctan-
1-ol (17)
A suspension of 16 (2.58 g, 8.42 mmol), NaHCO3 (0.70 g) and 10%
Pd/C (0.70 g) in EtOH (80 ml) was stirred for 3 h under hydrogen at-
mosphere (1 atm). The mixture was filtrated and the filtrate was con-
centrated in vacuo. The residue was chromatographed on silica gel.
Elution with hexanes/EtOAc (3:1) gave 17 (0.98 g, 4.53 mmol, 54%)
as a pale yellow oil. Rf=0.27 (SiO2, hexane/EtOAc=1:1); FT-IR: νmax
3419 (m, OH), 2985 (s), 2938 (s), 2874 (s), 1470 (w), 1378 (m), 1224
(s), 1169 (m), 1114 (w), 1038 (m), 996 (m), 929 (w), 907 (w), 854
(w), 669 cm-1 (w); NMR: δH 0.85 (3H, d, J=6.9 Hz, i-Pr), 0.93 (3H,
d, J=6.6 Hz, i-Pr), 1.34 (3H, s, acetonide), 1.36 (3H, s, acetonide),
1.50–1.72 (7H, m), 2.44 (1H, br, OH), 3.37–3.48 (1H, m), 3.57–3.70
(2H, m), 3.72–3.82 (1H, m); NMR: δC 100.4, 71.8, 67.1, 62.7, 36.7,
32.6, 29.1, 24.5, 24.2, 18.6, 17.4. HR-FAB MS: m/z calculated for
C12H25O3 [M+H]+ 217.1804; found 217.1806.
Pre-incubation of Streptomyces griseus ETH 7796
Cultures of S. griseus ETH 7796 were maintained on Emerson agar
at 4°C. Mycelium scraped from an agar slant was inoculated in a
solution of glucose (0.02 g), yeast extract (0.02 g), malt extract
(0.05 g) and CaCO3 (0.01 g) mixed in distilled water (5 ml) in a
10 ml test tube, and incubated at 30°C for 48 h on an orbital shaker
at 125 rpm (medium A). The medium A and a solution of maltose
(3 g) in distilled water (10 ml) were sterilized and transferred into
Ethyl (2E,6RS,8SR)-6,8-isopropylidenedioxy-2,9-
dimethyldec-2-enoate (18)
To a solution of (COCl)2 (0.520 ml, 6.07 mmol) in dry THF (50 ml)
was added a solution of DMSO (0.862 ml, 12.1 mmol) in dry THF
(15 ml) at -78°C, and the mixture was stirred for 15 min. To this
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98 T. Hanadate et al.
a 500 ml Sakaguchi flask containing a sterilized solution of Bacto-
tryptone (0.8 g), yeast extract (0.4 g), NaNO3 (0.3 g), and CaCO3
(0.2 g) in distilled water (90 ml). This mixture was incubated at 30°C
for 48 h at 125 rpm (medium B). The production medium comprised
of Bact-tryptone (0.8 g), yeast extract (0.4 g), NaNO3 (0.3 g), CaCO3
(0.2 g), MnSO4 (0.04 g), ZnSO4 (0.005 g) and distilled water (90 ml)
was sterilized at 120°C for 20 min in a 500 ml Sakaguchi flask, and
to this mixture was added a sterilized solution of maltose (3 g) in
distilled water (10 ml), and medium B (5% v/v). The incubation was
performed at 30°C for 96 h at 125 rpm (medium C).
Evans, D. A.; Chapman K. T.; Carreira, E. M. Directed reduc-
tion of β-hydroxy ketones employing tetramethylammonium
triacetoxyborohydride. J. Am. Chem. Soc. 1988, 110, 3560–
3578.
Hanadate, T.; Kiyota, H.; Oritani, T. Synthesis of macrotetrolide
α, a designed polynactin analog composed of (+)- and (-)-
bishomononactic acid. Biosci. Biotechnol. Biochem. 2001, 65,
2118–2120.
Keller-Schierlein, W.; Gerlach, H. Makrotetrokide. Fortschr. Chem.
Org. Naturst. 1967, 26, 161–189 and references cited therin.
Kocienski, P. J. Diol protecting groups. In Protecting Groups, 3rd
Edition. Stuttgart: Georg Thieme Verlag, 2005; pp. 120–185.
Nogawa, M.; Sugawara, S.; Iizuka, R.; Shimojo, M.; Ohta, H.;
Hatanaka, M., Matsumoto, K. Enantioselective microbial hydro-
lysis of dissymmetrical cyclic carbonates with disubstitution.
Tetrahedron 2006, 62, 12071–12083.
Ohira, S. Methanolysis of dimethyl (1-diazo-2-oxopropyl) phospho-
nate. Generation of (diazomethyl) phosphonate and reaction with
carbonyl compounds. Synth. Commun. 1989, 19, 561–564.
Rathke, M. W.; Nowak, M. The Horner-Wadsworth-Emmons modi-
fication of the Wittig reaction using triethylamine and lithium or
magnesium salts. J. Org. Chem. 1985, 50, 2624–2626.
Rezanka, T.; Prell, A.; Spizek, J.; Sigler, K. Pilot-plant cultivation
of Streptomyces griseus producing homologues of nonactin by
precursor-directed biosynthesis and their identification by LC/
MS-ESI, J. Antibiot. 2010, 63, 524–529.
Feeding experiments
Compounds 3, 20, 3+20, and none were dissolved in distilled water-
EtOH (5:1) and the solution was administrated portionwise into me-
dium C over 3 days, to a final concentration in the broth of 17 mm.
Then each flask was incubated at 30°C for 6 days at 125 rpm. Each
medium was filtered through a Celite pad and the pad was washed
with water. The washings were extracted with acetone for 16 h, and
the extract was concentrated in vacuo. The residue was extracted with
EtOAc and the combined organic layers were dried with MgSO4,
and concentrated in vacuo. The residue was chromatographed on
preparative TLC. Development with hexanes/EtOAc (2:1) furnished
polynactin congeners.
Rong, J.; Nelson, M. E.; Kusche, B.; Priestley, N. D. Nonactin
biosynthesis: unexpected patterns of label incorporation from
4,6-dioxoheptanoate show evidence of a degradation pathway
for levulinate through propionate in Streptomyces griseus. J. Nat.
Prod. 2010, 73, 2009–2012.
Takai, K.; Hanadate, T.; Abe, M.; Ono, Y.; Yamada, T.; Kuwahara, S.;
Kiyota, H. Synthesis of macrotetrolide α, a designed polynactin
analog composed of bishomononactic acids. Tetrahedron 2011,
67, 7066–7072.
Acknowledgements
Financialsupportbygrant-in-aidfromJapanSocietyforthePromotion
of Science (No. 17580092 and 19580120), TheAgricultural Chemical
Research Foundation, Intelligent Cosmos Foundation and The Naito
Foundation is gratefully acknowledged.
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Received May 30, 2011; accepted June 23, 2011; previously
published online August 25, 2011
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