The Butenolide Signaling Molecules SRB1 and SRB2 Induce Lankacidin and Lankamycin Production in Streptomyces rochei

Article abstract of DOI:10.1002/cbic.201200149

New signaling molecules that induce lankacidin and lankamycin production in Streptomyces rochei were extracted from the culture filtrate and purified by Sephadex LH20 and silica gel chromatography with the help of bioassay. Chiral HPLC and ESI-MS analyses indicated the presence of two active components-SRB1 and SRB2-and their molecular formulas were established to be C₁₅H₂₄O₅ and C₁₆H₂₆O₅, respectively. By extensive NMR analysis, SRB1 and SRB2 were determined to be 2-(1′-hydroxy-6′-oxo-8′-methylnonyl)-3-methyl-4-hydroxybut-2-en-1,4-olide and 2-(1′-hydroxy-6′-oxo-8′-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4-olide, respectively. These structures were finally confirmed by chemical synthesis and the absolute configuration at C-1′ was determined to be R in each case. The synthetic 1′R isomers induced production of lankacidin and lankamycin at around 40 nM concentrations. SRB1 and SRB2 are therefore distinct from the well-known 2,3-disubstituted γ-butyrolactone molecules such as A-factor, virginia butanolide, and SCB1 and and belong, like avenolide, recently isolated from Streptomyces avermitilis, to the γ-butenolide family.

Full text of DOI:10.1002/cbic.201200149

DOI: 10.1002/cbic.201200149
The Butenolide Signaling Molecules SRB1 and SRB2 Induce
Lankacidin and Lankamycin Production in Streptomyces
rochei
Kenji Arakawa,* Naoto Tsuda, Akihiro Taniguchi, and Haruyasu Kinashi[a]
New signaling molecules that induce lankacidin and lankamy-
cin production in Streptomyces rochei were extracted from the
culture filtrate and purified by Sephadex LH20 and silica gel
chromatography with the help of bioassay. Chiral HPLC and
ESI-MS analyses indicated the presence of two active compo-
nents—SRB1 and SRB2—and their molecular formulas were
established to be C15H24O5 and C16H26O5, respectively. By exten-
sive NMR analysis, SRB1 and SRB2 were determined to be 2-(1’-
hydroxy-6’-oxo-8’-methylnonyl)-3-methyl-4-hydroxybut-2-en-
1,4-olide and 2-(1’-hydroxy-6’-oxo-8’-methyldecyl)-3-methyl-4-
hydroxybut-2-en-1,4-olide, respectively. These structures were
finally confirmed by chemical synthesis and the absolute con-
figuration at C-1’ was determined to be R in each case. The
synthetic 1’R isomers induced production of lankacidin and
lankamycin at around 40 nm concentrations. SRB1 and SRB2
are therefore distinct from the well-known 2,3-disubstituted g-
butyrolactone molecules such as A-factor, virginia butanolide,
and SCB1 and and belong, like avenolide, recently isolated
from Streptomyces avermitilis, to the g-butenolide family.
Introduction
The filamentous soil bacteria
Streptomyces are characterized
by the ability to produce a wide
variety of secondary metabolites,
including antibiotics. In many
Streptomyces species, antibiotic
production and morphological
differentiation are controlled by
small diffusible signaling mole-
cules.[1,2] The well-studied family
of signaling molecules identified
up to date have 2,3-disubstitut-
ed g-butyrolactone skeletons
and are active at nanomolar con-
centrations.[3] The most exten-
sively characterized molecule is
Scheme 1. Structures of Streptomyces signaling molecules. A) Known signaling molecules: the 2,3-disubstituted g-
butyrolactone type (A-factor, SCB1, SCB3, virginia butanolide A), the furan type (methylenomycin furan), and the
4-monosubstituted butenolide type (avenolide). B) The new 2,3-disubstituted butenolides SRB1 (1) and SRB2 (2)
isolated from S. rochei 7434AN4.
A-factor
(2-isocapryloyl-3R-hy-
droxymethyl-g-butyrolactone;
Scheme 1A), which is responsi-
ble for streptomycin production
and morphological differentia-
tion in Streptomyces griseus.[4] When A-factor produced in a
growth-dependent manner reaches a critical concentration, it
binds to a specific receptor protein (ArpA), and the A-factor/
ArpA complex then dissociates from the promoter region of
adpA, a global transcriptional activator gene. The de-repressed
gene product AdpA binds to its target genes to activate strep-
tomycin production and morphological differentiation.[5] At the
top of this regulatory cascade, the afsA gene encodes a key
enzyme in A-factor biosynthesis.[6,7]
myces species, including Streptomyces coelicolor A3(2),[8] Strep-
tomyces virginiae,[9] and Streptomyces lavendulae FRI-5.[10] The g-
butyrolactone-type signaling molecules have been classified
[a] Prof. Dr. K. Arakawa, N. Tsuda, A. Taniguchi, Prof. Dr. H. Kinashi
Department of Molecular Biotechnology
Graduate School of Advanced Sciences of Matter
Hiroshima University
1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530 (Japan)
E-mail: karakawa@hiroshima-u.ac.jp
Additional g-butyrolactone-type signaling molecules and
their specific receptors have been identified in several Strepto-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200149.
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K. Arakawa et al.
into three major groups based on the reduction state and the
stereochemistry at C-1’ (Scheme 1A): 1) the 1’-keto type (e.g.,
A-factor in S. griseus), 2) the (1’R)-hydroxy type [SCB1 in S. coeli-
color A3(2)], and 3) the (1’S)-hydroxy type (virginia butanolides
in S. virginiae).
antibiotic-inducing activity. The active fractions were combined
and purified on Sephadex LH20 with methanol as eluent. The
active fractions were then collected and further purified by
series of silica gel chromatographic separations with the fol-
lowing three solvent systems to give a single spot on TLC (Rf =
0.20 in hexane/EtOAc 1:1 v/v): CHCl3/methanol (50:1–30:1 v/v),
toluene/EtOAc (2:1–1:1 v/v), and hexane/EtOAc (2:1–1:1 v/v).
No other fractions showed any inducing activity, indicating
that the purified component is an inducing factor of lankacidin
and lankamycin in S. rochei.
New signaling molecules have recently been identified in
S. coelicolor and Streptomyces avermitilis. A furan-type signaling
molecule— methylenomycin furan (Scheme 1A)—was shown
to induce methylenomycin production in S. coelicolor.[11] A 4-
monosubstituted butenolide named avenolide (Scheme 1A) in-
duces avermectin production in S. avermitilis with a minimum
inducing concentration of 4 nm.[12] Signaling molecules of vari-
ous types are thus involved in controlling antibiotic biosynthe-
sis in Streptomyces species.
The active component was further analyzed by chiral HPLC
(TCI Chiral MB-S column; macroporous silica gel coated with
optically active N-substituted polymaleimides) with aqueous
acetonitrile (10%) as mobile phase (flow rate 1.0 mLminÀ1
)
Streptomyces rochei strain 7434AN4 contains three linear
plasmids (pSLA2-L, -M, and -S) and produces two structurally
unrelated polyketide antibiotics: lankacidin and lankamycin
(Figure S1 in the Supporting Information).[13] Lankacidin and
lankamycin inhibit peptide synthesis synergistically by target-
ing neighboring sites in the ribosome.[14] The biosynthetic
gene clusters of these antibiotics are located on the giant
linear plasmid pSLA2-L.[15–17] In addition, many regulatory
genes have been identified on pSLA2-L, including a synthesis
gene of the signaling molecule (srrX), six tetR-type repressor
genes (srrA-F), and three SARP (Streptomyces antibiotic regula-
tory protein) genes (srrY, srrZ, and srrW). We revealed that srrX
and srrA constitute the signaling molecule/receptor system in
S. rochei.[18] Extensive mutational and transcriptional analyses
revealed that the SARP gene srrY is a target of SrrA and that
the signaling pathway goes from srrX through srrA to srrY, lead-
ing to lankacidin and lankamycin production.[19] Furthermore, it
was shown that SrrY directly activates transcription of the
second SARP gene srrZ for LM production.[20] In spite of these
accumulated data on the regulatory cascade of antibiotic pro-
duction in S. rochei 7434AN4, the signaling molecules them-
selves had not been isolated. Here we report the isolation,
structural elucidation, and biological activities of the signaling
molecules termed “SRBs” (Streptomyces rochei butenolides),
which induce lankacidin and lankamycin production at nano-
molar concentrations.
with detection at 210 nm. In contrast with the results obtained
with HPLC and TLC, two peaks were detected: at 9.1 min
(SRB1) and 17.3 min (SRB2; Figure 1A). Because of the limited
amounts of the two compounds available (total ꢀ250 mg; esti-
mated later by UV absorbance), however, structural analysis
was carried out as a mixture. ESI-MS analysis also indicated the
presence of SRB1 and SRB2 in 1:1 ratio (Figure 1B). Their mo-
lecular formulas were established to be C15H24O5 for SRB1 and
C16H26O5 for SRB2, suggesting that SRB1 and SRB2 are homo-
logues with a difference consisting of one methylene unit.
With regard to the HPLC chromatogram of the SRBs (Fig-
ure 1A), it is noteworthy that their retention times differed by
8.2 min. A similar relationship was observed in the S. coelicolor
signaling molecules SCB1 and SCB3 (Scheme 1A): SCB1 (iso-
type C8 side chain) eluted at 22–23 min, whereas SCB3 (antei-
so-type C9 side chain) eluted at 40–48 min.[21]
Structural elucidation of SRBs
The 13C NMR and HMBC spectra of the mixture of SRB1 and
SRB2 (Figure 1D) revealed two carbonyl groups (C-1 and C-6’;
dC =170.7 and 213.1 ppm) and one C=C double bond (C-2 and
C-3; dC =129.4 and 157.1 ppm) for each compound. No olefinic
protons were observed in the 1H NMR spectrum (Figure 1C),
suggesting that the C=C double bond was fully substituted.
The existence of cyclic skeletons in the SRBs was therefore in-
ferred from the degree of unsaturation. A strong absorbance
at 1750 cmÀ1 in the IR spectrum indicated the presence of an
a,b-unsaturated g-lactone ring. The singlet H-5 protons at dH =
2.08 ppm were assigned to a methyl group at C-3 (dC =157.1)
by HMBC of the H-5 protons to C-3 (Figure 1E-i). The H-5 pro-
tons showed long-range HMBC correlations with the C-4 me-
thine carbon (dC =98.7 ppm) and the C-2 quaternary carbon
(dC =129.4 ppm). The C-4 carbon is bound to a highly de-
shielded broad-singlet proton (dH =5.88 ppm), indicating that
C-4 is hydroxylated. Furthermore, the broad-singlet H-4 proton
showed a HMBC correlation with the C-1 carbonyl carbon (dC =
170.7 ppm). The H-5 protons showed moderate NOEs with the
deshielded H-1’ proton (dH =4.50 ppm) of the alkyl side chain
as well as with H-4. From these data, a 2,3-disubstituted g-hy-
droxybutenolide ring, in which C-3 was substituted with
a methyl group and C-2 with an alkyl side chain, was deduced
as a partial structure (Figure 1E-i). The broad-singlet signal of
Results
Isolation of SRBs
S. rochei KA61, a disruptant of the SARP-family activator gene
srrY, was used to isolate the SRB molecules. This strain does
not produce lankacidin or lankamycin, due to a shutdown of
the regulatory cascade downstream of srrY, which improves
the isolation efficiency of the SRB molecules without the dis-
turbance of antibiotics. A 160 L culture of strain KA61 was
grown in a 200 L jar fermenter at 288C for 43 h. The culture fil-
trate was passed through an Amberlite XAD16 column to
absorb hydrophobic metabolites, including SRBs. The column
was extracted with EtOAc, and the resulting oil (18 g) was puri-
fied by silica gel chromatography with hexane/EtOAc (2:1 to
1:2 v/v). Each fraction was subjected to bioassay to check its
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Butenolide Signaling Molecules
Figure 1. Spectral analysis of natural SRBs. A) Chiral HPLC analysis. Elution profiles were monitored by UV absorbance at 210 nm. B) ESI-MS spectrum.
C) 1H NMR spectrum. D) HMBC spectrum and the key cross-peaks. E) 1H,1H COSY and HMBC correlations: i) in the butenolide moieties in both SRBs, ii) in
the C10 alkyl chain moiety of SRB1, and iii) in the C11 alkyl chain moiety of SRB2. The dH and dC values are shown underlined and in italics, respectively.
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K. Arakawa et al.
H-4 implied that the two compounds exist as mixtures of inter-
converting epimers at C-4. These assignments showed a good
agreement with those of known 2,3-disubstituted g-hydroxy-
butenolides (R=isopropyl or sec-butyl; Figure 1E-i) previously
isolated from Streptomyces antibioticus Tꢁ99.[22]
Grignard reagent isobutylmagnesium bromide to give the C10
unit 5 in 81% yield, and the newly generated hydroxy group
was then oxidized with PCC to afford ketone 6 in 82% yield.
Protection of the ketone group in 6 with ethylenedioxybis(tri-
methylsilane), removal of the benzyl group, and subsequent
oxidation with PCC afforded aldehyde 7 in 64% yield (three
steps).
The common partial structure of the two SRBs indicated that
the alkyl side chains branched at C-2 have the formulas
C10H19O2 for SRB1 and C11H21O2 for SRB2. The C-6’ carbonyl
carbon at dC =213.1 ppm was assigned to a ketone and
showed HMBC correlations with proton signals at dH =2.26 (d),
2.18 (dd), and 2.37–2.40 ppm (m) (Figure 1D). These signals
correspond to the methylene protons: dH =2.26 ppm at dC =
52.0 ppm (C-7’ of SRB1; Figure 1E-ii), dH =2.18 and 2.37 ppm at
dC =50.1 ppm (C-7’ of SRB2; Figure 1E-iii), and dH =2.40 ppm
at dC =42.6 ppm (C-5’ of SRB1 and SRB2; Figure 1E-ii and -iii).
These spectral data indicate that the methylene groups (C-5’
and C-7’) on the two sides of C-6’ have different chemical envi-
ronments.
Enantiomerically pure 3-methyl-4-(l-menthyloxy)but-2-en-
1,4-olide (8) was prepared by condensation of propanal with
glyoxylic acid and l-menthol, followed by two-stage recrystalli-
zation from petroleum ether.[24,25] The key coupling reaction
between the anion derived from 8 [with lithium diisopropyla-
mide (LDA)] and the aldehyde 7 provided a diastereomeric
mixture of 9a and 9b in 2:1 ratio and in 31% yield. The large
protecting group (l-menthyl) at the C-4 hydroxy function of 8
prevented removal of the more acidic H-5 proton,[26] leading to
the production of the desired 2,3-disubstituted g-(l-menthylox-
y)butenolide. The diastereomeric mixture of 9a and 9b was
separated by repeated flash chromatography runs with
hexane/EtOAc. Each of the purified compounds had 94% dia-
stereomeric purity as judged from the two well-separated
hemiacetal H-4 proton signals in the 1H NMR spectra (dH =
5.69 ppm for 9a and dH =5.71 ppm for 9b). The C-1’ configura-
tions of these compounds were determined from the Dd
(dSÀdR) values of their (S)- and (R)-a-methoxy-a-(trifluorometh-
yl)-a-phenylacetate (MTPA) ester derivatives.[27] Characteristic
H-4 and H-5 proton signals in the g-butenolide moiety of 9a
showed distinct negative Dd values (À25 and À98 Hz, respec-
tively), whereas the H-2’ proton showed a slightly positive
value (+5 Hz). The C’-1 configuration of 9a was thus deter-
mined to be R, whereas that of 9b is S. Finally, removal of the
l-menthyl and 1,3-dioxolane groups in 9a and 9b was per-
formed simultaneously with boron tribromide to afford SRB1a
(1a) and SRB1b (1b) in 52 and 63% yields, respectively.
SRB2a (2a) and SRB2b (2b, Scheme 2B) were synthesized in
a similar way as for 1a and 1b but a different Grignard
reagent, (S)-(2-methylbutyl)magnesium chloride, was used for
construction of the C11 unit 10.
With regard to SRB1, the doublet H-7’ proton (dH =
1
2.26 ppm) showed a H,1H COSY correlation with the proton at
dH =2.10 ppm, which in turn connected to the two methyl
groups H-9’ and H-9’’ at dH =0.89–0.90 ppm. A partial structure
of the alkyl chain of SRB1 was thus elucidated as shown in Fig-
ure 1E-ii.
With regard to SRB2, the double–doublet H-7’a proton (dH =
1
2.18 ppm) showed H,1H COSY correlations with signals at dH =
2.37 ppm and dH =1.90 ppm, the former being assigned to
a geminal H-7’b proton by HMQC experiment. The latter
proton (dH =1.90 ppm) connected to C-8’ showed a further
1H,1H COSY correlation with the H-9’ methyl protons (dH =
0.86 ppm; Figure 1E-iii). In addition, the C-8’ carbon (dC =
30.7 ppm) showed HMBC correlations with the double-doublet
H-7’a proton and the H-10’ methyl protons (dH =0.86 ppm),
1
the latter of which showed a H,1H COSY correlation with the
H-9’’ methylene protons (dH =1.2–1.3 ppm). These assignments
led to a partial structure of the alkyl chain of SRB2 as illustrat-
ed in Figure 1E-iii. Although further assignments of the three
additional methylene groups corresponding to C2’–C4’ of SRB1
and SRB2 were difficult due to overlapping signals, SRB1 and
SRB2 were assigned as 2-(1’-hydroxyl-6’-oxo-8’-methylnonyl)-3-
methyl-4-hydroxybut-2-en-1,4-olide (1) and 2-(1’-hydroxyl-6’-
oxo-8’-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4-olide (2).
Each compound contains a 2,3-disubstituted g-hydroxybuteno-
lide skeleton, which is distinct from those of the known g-bu-
tyrolactone molecules such as A-factor, virginia butanolide,
and SCB1 (Scheme 1).
The retention times of SRB1 and SRB2 (9.1 and 17.3 min) on
a chiral HPLC column (mobile phase; 10% aqueous acetonitrile
containing 0.1% trifluoroacetic acid) at
a flow rate of
1.0 mLminÀ1 were identical to those of the synthetic 1’R iso-
mers 1a and 2a, whereas the synthetic 1’S isomers 1b and 2b
eluted slightly earlier, at 8.7 and 16.6 min (Figure 2A). Further-
more, the natural SRB mixture showed a positive optical rota-
tion value ([a]2D2 =+17.5), which was in good agreement with
those of the synthetic 1’R isomers (+18.4 for 1a, +22.4 for
2a). The synthetic 1’S isomers exhibited negative values of op-
tical rotation (À9.32 for 1b, À5.71 for 2b). Consequently, the
absolute C-1’ configurations in SRB1 and SRB2 were both de-
termined to be R (Scheme 1B). As shown in Figure 2B, spectra
of the natural SRBs showed good agreement with those of the
Synthesis of SRB molecules
To confirm the proposed structures of 1 and 2 and to deter-
mine their C-1’ configurations, the 1’R isomers (1a and 2a)
and the 1’S isomers (1b and 2b) were synthesized as shown in
Scheme 2.
1
synthetic compounds 1a and 2a. In the H and 13C NMR spec-
For the synthesis of the alkyl chains of 1a and 1b
(Scheme 2A), the readily available 1-(benzyloxy)hexan-6-ol
(3)[23] was oxidized with pyridinium chlorochromate (PCC) to
give aldehyde 4 in 81% yield, 4 was then coupled with the
tra of the synthetic SRBs (1a, 1b, 2a, 2b), small separate reso-
nances (Dd<0.03 ppm in 1H NMR and Dd<0.3 ppm in
13C NMR) due to the presence of interconverting epimers at C-
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Butenolide Signaling Molecules
Scheme 2. Synthesis of S. rochei butenolides: A) SRB1, and B) SRB2. a) Pyridinium chlorochromate, CH2Cl2; b) isobutylmagnesium bromide, THF; c) TMSO-
(CH2)2OTMS, TMSOTf, CH2Cl2; d) H2, Pd-C (10%), EtOAc; e) LDA, THF/HMPA, and then 7; f) BBr3, CH2Cl2; g) (S)-(2-methylbutyl)magnesium chloride, THF; h) LDA,
THF/HMPA, and then 12.
4 are observed (Table 1). These hemiacetalic epimers intercon-
vert rapidly[28] and were inseparable even by chiral HPLC.
cannot exclude the possibility that the low inducing activities
of 1b and 2b might be due to contamination by 1a and 2a.
The average yield of SRBs in strain KA61 was around 6 mg per
liter of the culture. The metabolic profiles of strain KA20,
a srrX–srrB double mutant, separately supplemented with SRB1
and SRB2 were comparable (Figure 3), suggesting that SRB1
and SRB2 play equal roles in inducing lankacidin and lankamy-
cin production in S. rochei.
Biological activities of SRB molecules
The minimum concentrations of the synthetic SRBs necessary
for antibiotic production were determined by bioassay. The 1’R
isomers 1a and 2a showed inducing activities at 42 and
40 nm, whereas the 1’S isomers 1b and 2b were active at 660
and 630 nm, respectively. The values for 1a and 2a corre-
sponded to that for the natural SRBs (ca. 50 nm). The 1’S iso-
mers (1b and 2b) showed much lower activities (around 6%)
than the 1’R isomers, suggesting that the 1’R configuration is
important for antibiotic-inducing activity. In this respect, we
Discussion
Two signaling molecules, SRB1 and SRB2, which induce pro-
duction of lankacidin and lankamycin in S. rochei 7434AN4,
were isolated and their structures were determined. The SRB
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K. Arakawa et al.
Figure 2. Determination of the C-1’ configurations in natural SRBs. A) Chiral HPLC analysis of: i) natural SRBs (1 and 2), ii) co-injection of natural SRBs and syn-
thetic 1a, iii) co-injection of natural SRBs and synthetic 2a, iv) synthetic 1a, v) 1b, vi) 2a, and vii) 2b. Elution profiles were monitored by UV absorbance at
210 nm. B) 1H NMR spectra of: i) natural SRBs, ii) synthetic 1a, and iii) 2a (dH =0.75–2.50 ppm).
molecules each contain a 2,3-disubstituted g-hydroxybuteno-
Table 1. NMR data for 1a and 2a in CDCl3.[a]
lide skeleton. Structurally related butenolides have been isolat-
ed from S. antibioticus Tꢁ99 as antibiotics, which exhibited
Pos.
SRB1a (1a)
SRB2a (2a)
dC (mult.)[b]
dH (mult.,
J [Hz])[b]
dC (mult.)[b]
dH (mult.,
J [Hz])[b]
moderate antimicrobial activity against Pseudomonas aerugino-
sa.[22] About 60% of Streptomyces species use g-butyrolactone-
type signaling molecules to induce antibiotic production,[29]
and fourteen molecules of this type have previously been iden-
tified.[3,21] New types of signaling molecules have recently been
isolated from several Streptomyces species. A furan-type signal-
ing molecule, methylenomycin furan, was identified as an
autoregulator for methylenomycin production in S. coelicolor
A3(2).[11] In addition, avenolide, a new 4-monosubstituted g-bu-
tenolide-type molecule, was identified as an inducing factor
for avermectin production in S. avermitilis.[12] It is noteworthy
that the avaR gene (sav2269) in S. avermitilis, which encodes
a homologue of the AfsA-family proteins, is not involved in
avenolide production. We have not determined the stereo-
chemistry at C-8’ in SRB2. Nevertheless the S configuration of
C-8’ in 2 seems more plausible in view of the following evi-
dence. 8-Methyldecanoyl-ACP, a possible biosynthetic inter-
mediate of 2, is synthesized by condensation of four malonyl-
CoA units with a starter (S)-2-methylbutyryl-CoA, derived from
l-isoleucine, in primary fatty acid biosynthesis. Hafner et al.
1
2
3
4
171.4/171.5 (s)
129.9/130.0 (s)
157.6/157.6 (d)
98.7/98.9 (d)
11.5/11.6 (d)
66.4/66.5 (d)
35.6/35.7 (t)
24.7/24.8 (t)
23.0/23.1 (t)
42.7/42.9 (t)
211.6/212.5 (s)
51.9/52.0 (t)
–
–
–
171.4/171.5 (s)
129.9/130.0 (s)
157.6/157.7 (d)
98.7/98.9 (d)
11.5/11.6 (q)
66.4/66.5 (d)
35.5/35.7 (t)
24.7/24.8 (t)
23.0/23.2 (t)
–
–
–
5.85 (brs)
2.07/2.09 (s)
4.49 (m)
1.70–1.85 (m)
1.25, 1.36 (m)
1.53 (m)
2.40 (dt, 3.4, 7.1) 42.7/42.9 (t)
–
2.26 (d, 7.1)
5.85 (brs)
2.08/2.09 (s)
4.48 (m)
1.70–1.85 (m)
1.22, 1.35 (m)
1.53 (m)
2.40 (m)
–
2.18 (dd, 7.9, 8.3),
2.37 (m)
1.90 (m)
1.18, 1.28 (m)
0.87 (d, 6.7)
0.86 (d, 7.0)
5
1’
2’
3’
4’
5’
6’
7’
211.8/212.8 (s)
50.0/50.1 (t)
8’
9’
10’
9’’
24.7 (d)
2.09 (m)
0.89 (d, 6.8)[c]
–
0.90 (d, 6.7)[c]
30.9 (d)
29.5 (t)
11.3 (q)
22.5/22.6 (d)
–
22.5/22.6 (d)
19.3/19.4 (q)
[a] Spectra were recorded at 500 MHz for 1H and 125 MHz for 13C. [b] The
residential solvent signal (dC =77 ppm) and internal standard tetramethyl-
silane (dH =0 ppm) were used as references. [c] Assignments are ex-
changeable.
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Butenolide Signaling Molecules
C2=C3 double bond for activity. The function(s) of the C-4
hemiacetal hydroxy and the C-6’ ketone groups together with
the chain length should also be studied in due course. It was
reported that higher concentrations of SCB1 (>1000 nm) inhib-
ited antibiotic production in S. coelicolor, but did not affect
growth and morphological development.[31] A similar effect on
antibiotic production was also observed for virginiae butano-
lides in S. virginiae.[33] In contrast, no inhibitory effect on
antibiotic production and morphological differentiation was
observed for SRBs in S. rochei even at a concentration of
6700 nm. We have previously observed a negative effect of
srrX on spore formation on the basis of gene inactivation ex-
periments.[18] However, the present work clearly indicates that
SRBs themselves have no inhibitory effect on spore formation.
It might therefore be possible that the SrrX protein has a regu-
latory function in morphological differentiation.
With regard to the biosynthesis of SRB1 and SRB2, we
speculated that srrX encodes a key enzyme like afsA in S. gri-
seus, based on the following evidence. 1) SRB1 and SRB2 re-
semble possible intermediates of the g-butyrolactone signaling
molecules. 2) The culture extract of the srrX mutant has no an-
tibiotic-production-inducing activity in S. rochei. 3) No other
positive signals were detected in S. rochei DNA when srrX was
used as a probe for hybridization. Consistently with these spec-
ulation, several possible genes for SRB biosynthesis were
found around srrX (orf85) on pSLA2-L: an NAD-dependent de-
hydrogenase gene srrG (orf81), a phosphatase gene srrP
(orf83), a P450 hydroxylase gene srrO (orf84), and a thioesterase
gene srrH (orf86). Analogously to the biosyntheses of A-factor[7]
and virginia butanolides,[34] SrrX could be responsible for cou-
pling between a C3 unit and a C12 or C13 b-keto acid derived
from fatty acid biosynthesis, followed by spontaneous intramo-
lecular aldol condensation to form a butenolide skeleton. The
butenolide intermediate might then be further modified by
biosynthetic enzymes including SrrG to synthesize SRBs. The
SrrG protein is homologous to BarS1, which is involved in the
reduction of the C-1’ ketone group to produce virginia butano-
lides in S. virginiae.[35] Our preliminary data showed that a muta-
tion of srrG abolished lankacidin and lankamycin production in
S. rochei (unpublished result). Comprehensive analyses of the
SRB biosynthetic pathway by gene inactivation and feeding
experiments are in progress in our laboratory.
Figure 3. Effects of SRB1 and SRB2 on lankacidin and lankamycin production.
A) TLC analysis of metabolites. i) Parent 51252; ii) strain KA20; iii) strain KA20
supplemented with 1a (840 nm); iv) strain KA20 supplemented with 2a
(840 nm). Lane LC is a standard sample of lankacidin C. B) HPLC analysis of
metabolites. Elution profiles were monitored by UV absorbance at 230 nm.
Closed diamonds and circle indicate the peaks of lankacidin C and lankacidi-
nol A, respectively.
reported that the branched-chain 2-oxo acid decarboxylase is
specific to the S isomer of 3-methyl-2-oxopentanoic acid for
the synthesis of anteiso-type fatty acids and avermectins in
S. avermitilis.[30]
The minimum concentrations of SRB1 (1=1a) and SRB2
(2=2a) for induction of antibiotic production in S. rochei were
42 nm and 40 nm, respectively. These values were lower than
those of SCB1 (128–256 nm) for actinorhodin and undecylpro-
digiosin production in S. coelicolor[31] and higher than those of
A-factor (around 1 nm) for streptomycin in S. griseus[6] and of
virginia butanolides (around 3 nm) for virginiamycin in S. virgin-
iae.[32] The synthetic 1’S isomers 1b and 2b exhibited signifi-
cantly weaker activities than the 1’R isomers 1a and 2a, indi-
cating that the stereochemistry at C-1’ is crucial for induction
of activity. Our preliminary experiments demonstrated that 2,3-
dihydro-SRB1, prepared from 1a by catalytic hydrogenation,
showed no ability to induce antibiotic production in S. rochei
at a concentration of 33.5 mm, indicating the importance of the
Experimental Section
Strains and culture conditions: S. rochei wild-type strain 7434AN4
and strain 51252, which carries only pSLA2-L, were described previ-
ously.[13,15] Strain KA61, a disruptant of the SARP-family transcrip-
tional activator gene srrY, was used as an SRB producer.[20] Strain
KA20, a double mutant of srrX and the transcriptional repressor
gene srrB, was used as an SRB indicator strain.[18] YM medium
[yeast extract (0.4%), malt extract (1.0%), d-glucose (0.4%), pH 7.3]
was used for SRB synthesis and bioassay.
Spectroscopic instruments: NMR spectra were recorded with
a JEOL LA-500 spectrometer fitted with a field gradient accessory.
CDCl3 (99.8 atom% enriched, Acros) was used as a solvent. Chemi-
cal shifts were recorded as d values based on a resident solvent
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K. Arakawa et al.
signal (dC =77.0 ppm) or on the internal standard signal of tetra-
methylsilane (dH =0 ppm). ESI and atmospheric pressure chemical
ionization (APCI) mass spectra were measured with a LTQ Orbi-
trap XL mass spectrometer (Thermo Fisher Scientific, USA). Optical
rotations were measured with a JASCO DIP-370 polarimeter. IR
spectra were recorded with a JASCO FT/IR-5300 spectrometer. UV
spectra were obtained with an Ultrospec 3300pro UV/visible spec-
trophotometer (Amersham Biosciences).
1.45 (m, 2H; H-3), 1.61–1.68 (m, 4H; H-2 and H-4), 2.35 (t, J=
7.4 Hz, 1H; H-5a), 2.43 (dt, J=1.8, 7.4 Hz, 1H; H-5b), 3.47 (t, J=
6.4 Hz, 2H; H-1), 4.50 (s, 2H; PhCH2), 7.25–7.37 (m, 5H; PhCH2),
9.75 ppm (t, J=1.8 Hz, 1H; H-6); 13C NMR (CDCl3): d=21.9 (C-3),
24.5 (C-4), 29.4 (C-2), 43.7 (C-5), 70.0 (C-1), 202.7 ppm (C-6);
13C NMR (CDCl3, benzyl resonances): d=72.9 (PhCH2), 127.5, 127.6,
128.4, 138.6 ppm; IR (neat): n˜ =2938, 2861, 1725, 1454, 1364, 1101,
739, 698 cmÀ1; HRMS (ESI): m/z calcd for C13H18O2Na: 229.1199
[M+Na]+; found: 229.1199.
Isolation of SRB molecules: An inoculum of strain KA61 was
grown by use of a two-stage seed culture system. The first-stage
seed culture (50 mL) was used as an inoculum for 4 L medium and
was grown at 288C and 90 rpm for 28 h. The resulting second-
stage seed culture was inoculated into YM medium (160 L) and cul-
tivated at 288C and 110 rpm with an airflow rate of 0.5 vvÀ1 minÀ1
(air volume/liquid volume per minute) for 43 h. The culture super-
natant was passed through a column of Amberlite XAD16 resin
(5 kg, Sigma) at a flow rate of 10 LhÀ1 to absorb hydrophobic com-
pounds, including SRBs. The resin was extracted three times with
equal volumes of EtOAc. The combined organic phase was dried
(Na2SO4), filtered, and concentrated to dryness. The resulting resi-
due (18 g) was purified by silica gel chromatography with hexane/
EtOAc (1:1 to 1:2, v/v). Each fraction was subjected to bioassay
with use of strain KA20 as a test organism (see below). Active frac-
tions were collected and purified on Sephadex LH-20 (GE Health-
care) with methanol and further purified by a series of silica gel
column chromatographic separations with three different solvent
systems [CHCl3/MeOH (50:1–30:1 v/v), toluene/EtOAc (2:1–1:1 v/v),
and hexane/EtOAc (2:1–1:1 v/v)]. The purified sample was analyzed
by ESI-MS and NMR (Figure 1). The 1H and 13C NMR assignments
were supported by 1H,1H COSY, HMQC, and HMBC experiments
(see the Supporting Information).
1-(Benzyloxy)-8-methylnonan-6-ol (5): 1-Bromo-2-methylpropane
(9.00 mL, 83.4 mmol) was added at 08C to a suspension of magne-
sium (2.00 g, 82.3 mmol) and iodine (160 mg) in THF (40 mL), and
the mixture was stirred at 08C for 2 h. A solution of aldehyde 4
(2.82 g, 13.7 mmol) in THF (10 mL) was added dropwise to the mix-
ture at 08C, and the mixture was stirred at room temperature for
2.5 h. Saturated aqueous NH4Cl (50 mL) was added, and the mix-
ture was extracted twice with EtOAc. The combined organic
phases were washed with brine, dried (Na2SO4), filtered, and con-
centrated to dryness. The residue was purified over silica gel with
hexane/EtOAc (7:1–5:1 v/v) to give 5 (2.93 g, 81%) as a colorless
oil.
1
Diastereomer mixture: H NMR (CDCl3): d=0.91 (t, J=7.0 Hz, 6H; H-
9 and H-9’), 1.22 (m, 1H; H-7a), 1.32–1.46 (m, 7H; H-3,-4,-5,-7b),
1.63 (t, J=7.0 Hz, 2H; H-2), 1.76 (m, 1H; H-8), 3.47 (t, J=6.7 Hz,
2H; H-1), 3.66 (brs, 1H; H-6), 4.50 (s, 2H; PhCH2), 7.26–7.34 ppm
(m, 5H; PhCH2); 13C NMR (CDCl3): d=22.0 (C-9), 23.5 (C-9’), 24.6 (C-
8), 25.4 (C-4), 26.3 (C-3), 29.7 (C-2), 38.0 (C-5), 46.8 (C-7), 69.9 (C-6),
70.3 ppm (C-1); 13C NMR (CDCl3, benzyl resonances): d=72.8
(PhCH2), 127.5, 127.6, 128.3, 138.6 ppm; IR (neat): n˜ =3397, 2932,
2863, 1454, 1366, 1101, 735, 698 cmÀ1; HRMS (ESI): m/z calcd for
C17H28O2Na: 287.1982 [M+Na]+; found: 287.1981.
Compound 1 (SRB1): HRMS (ESI): m/z calcd for C16H26O5Na: 321.1673
[M+Na]+; found: 321.1671.
1-(Benzyloxy)-8-methylnonan-6-one (6): A mixture of alcohol 5
(2.90 g, 11.0 mmol), PCC (3.75 g, 17.4 mmol), and sodium acetate
(290 mg, 3.54 mmol) in CH2Cl2 (60 mL) was stirred at room temper-
ature for 2 h. The mixture was diluted with ether (100 mL), and fil-
tered through a pad of Celite. The filtrate and washings were con-
centrated in vacuo. The residue was purified over silica gel with
hexane/EtOAc (10:1 v/v) to give 6 (2.37 g, 82%) as a colorless oil.
1H NMR (CDCl3): d=0.90 (d, J=6.4 Hz, 6H; H-9 and H-9’), 1.34–1.40
(m, 2H; H-3), 1.55–1.66 (m, 4H; H-2 and H-4), 2.13 (m, 1H; H-8),
2.26 (d, J=7.0 Hz, 2H; H-7), 2.37 (t, J=7.4 Hz, 2H; H-5), 3.46 (t, J=
6.4 Hz, 2H; H-1), 4.49 (s, 2H; PhCH2), 7.25–7.35 ppm (m, 5H;
PhCH2); 13C NMR (CDCl3): d=22.6 (C-9 and C-9’), 23.5 (C-4), 24.6 (C-
8), 25.8 (C-3), 29.5 (C-2), 43.2 (C-5), 51.8 (C-7), 70.1 (C-1), 211.0 ppm
(C-6); 13C NMR (CDCl3,benzyl resonances): d=72.9 (PhCH2), 127.5,
127.6, 128.3, 138.6 ppm; IR (neat): n˜ =2936, 2866, 1711, 1454, 1366,
Compound 2 (SRB2): HRMS (ESI): m/z calcd for C15H24O5Na: 307.1516
[M+Na]+; found: 307.1513.
Optical rotation of natural SRBs (mixture): [a]2D2 =+17.5 (c=0.012,
CHCl3).
SRB assay: The srrX–srrB double mutant KA20 was used as a highly
sensitive SRB indicator, because this strain, like the srrB mutant,
produces two antibiotics when SRBs are added. A seed culture of
strain KA20 (100 mL) supplemented with an aliquot of chromato-
graphic fraction (10 mL) was grown in YM liquid medium (5 mL) at
288C for 24 h. Antibiotic production was analyzed by HPLC and
TLC as follows. The crude extract was placed on a COSMOSIL 5C18-
MS-II column (4.6ꢂ250 mm, Nakarai Tesque, Japan) and eluted
with acetonitrile/sodium phosphate (10 mm) buffer (3:7 v/v, pH 8.2)
at a flow rate of 1.0 mLminÀ1. The eluate was monitored at 230 nm
with a JASCO MD-2010 multi-wavelength photodiode array detec-
tor. Lankacidin C and lankacidinol A were detected at 8.5 and
16.9 min, respectively. TLC was developed with a mixture of CHCl3/
methanol (15:1 v/v) and baked after spraying with anisaldehyde/
H2SO4.
1103, 737, 698 cmÀ1
; HRMS (ESI): m/z calcd for C17H26O2Na,
285.1825 [M+Na]+; found: 285.1822.
5-(2’-Isobutyl-1’,3’-dioxolan-2’-yl)pentanal (7): Trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf, 300 mL, 1.66 mmol) was added at
À788C to a solution of ketone 6 (2.30 g, 8.77 mmol) and ethylene-
dioxybis(trimethylsilane) (4.20 mL, 17.1 mmol) in CH2Cl2 (80 mL)
and the mixture was stirred at À788C for 1 h and at room temper-
ature for 7 h. Pyridine (1 mL) and water (10 mL) were added at
08C, and the mixture was extracted twice with CH2Cl2. The com-
bined organic phases were washed with brine, dried (Na2SO4), fil-
tered, and concentrated to dryness. The residue was purified over
silica gel with hexane/EtOAc (20:1 v/v) to give the corresponding
dioxolane derivative (2.43 g, 90%) as a colorless oil. A mixture of
the dioxolane derivative (2.40 g, 7.83 mmol) and Pd-C (10%,
750 mg) in EtOAc (60 mL) was stirred under hydrogen at room
1-(Benzyloxy)hexan-6-al (4): A mixture of 1-(benzyloxy)hexan-6-ol
(3,[23] 8.00 g, 38.4 mmol), pyridinium chlorochromate (PCC, 12.8 g,
59.5 mmol), and sodium acetate (1.40 g, 17.1 mmol) in CH2Cl2
(200 mL) was stirred at room temperature for 1.5 h. The mixture
was diluted with ether (300 mL) and filtered through a pad of
Celite. The filtrate and washings were concentrated in vacuo. The
residue was purified over silica gel with hexane/EtOAc (7:1 v/v) to
give 4 (6.45 g, 81%) as a colorless oil. 1H NMR (CDCl3): d=1.40–
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Butenolide Signaling Molecules
temperature for 24 h. The mixture was passed through a pad of
Celite. The filtrate and washings were concentrated in vacuo. The
residue was purified over silica gel with hexane/EtOAc (4:1–2:1 v/v)
to give the deprotected alcohol (1.61 g, 95%) as a colorless oil.
Compound 9b: [a]2D8 =À99.5 (c=0.760, CHCl3); 1H NMR (CDCl3):
d=0.91 (d, J=6.4 Hz, 3H; Me2CH), 0.93 (d, J=6.8 Hz, 3H; Me2CH),
1.28–1.42 (m, 2H; C-3’), 1.50 (d, J=6.1 Hz, 2H; iPrCH2), 1.59–1.72
(m, 5H; H-2’a,4’,5’), 1.75 (m, 1H; Me2CH), 1.83 (m, 1H; H-2’b), 1.99
(s, 3H; H-5), 3.91 [m, 4H; (CH2OÀ)2], 4.48 (t, J=7.3 Hz, 1H; H-1’),
5.71 ppm (s, 1H; H-4); 1H NMR (CDCl3, menthyl resonances): d=
0.80 (d, J=7.0 Hz, 3H), 0.86 (m, 1H), 0.87 (d, J=7.1 Hz, 3H), 0.96
(d, J=6.7 Hz, 3H), 1.05 (m, 2H), 1.22–1.27 (m, 1H), 1.28–1.42 (m,
1H), 1.59–1.72 (m, 2H), 2.10–2.15 (m, 2H), 3.62 ppm (dt, J=4.3,
11 Hz, 1H); 13C NMR (CDCl3): d=11.5 (C-5), 23.5 (C-4’), 24.0 (Me2CH),
24.1 (Me2CH), 25.7 (C-3’), 36.4 (C-2’), 37.2 (C-5’), 45.1 (iPr-CH2), 64.5
((CH2O-)2), 66.7 (C-1’), 100.7 (C-4), 111.8 (C-2’’), 130.7 (C-2), 155.4 (C-
3), 171.4 ppm (C-1); 13C NMR (CDCl3, menthyl resonances): d=15.7,
20.8, 22.2, 23.1, 25.1, 31.4, 34.2, 40.4, 47.7, 79.5 ppm; IR (neat): n˜ =
2953, 2870, 1759, 1456, 1370, 1333, 1096, 947 cmÀ1; HRMS (ESI):
m/z calcd for C27H46O6Na: 489.3187 [M+Na]+; found: 489.3184.
A
mixture of this alcohol (1.61 g, 7.44 mmol), PCC (3.40 g,
15.8 mmol), and sodium acetate (280 mg, 3.41 mmol) in CH2Cl2
(60 mL) was stirred at room temperature for 4 h. The mixture was
diluted with ether (50 mL) and filtered through a pad of Celite. The
filtrate and washings were concentrated in vacuo. The residue was
purified over silica gel with hexane/EtOAc (5:1 v/v) to give 7
(1.20 g, 75%) as a colorless oil. Overall yield 64% (three steps).
1H NMR (CDCl3): d=0.94 (d, J=6.7 Hz, 6H; 2ꢂCH3), 1.37–1.44 (m,
2H; H-4), 1.51 (m, 2H; iPrCH2), 1.60–1.67 (m, 4H; H-3 and H-5), 1.75
(m, 1H, Me2CH), 2.43 (m, 2H; H-2), 3.91 [m, 4H; (CH2OÀ)2],
9.76 ppm (t, J=1.9 Hz, 1H; H-1); 13C NMR (CDCl3): d=22.3 (C-3),
23.4 (C-4), 24.0 (Me2CH), 24.1 (Me2CH), 37.1 (C-5), 43.9 (C-2), 45.2
(iPrCH2), 64.6 [(CH2OÀ)2], 111.7 (C-2’), 202.6 ppm (C-1); IR (neat): n˜ =
2953, 2872, 1711, 1466, 1366, 1090, 949 cmÀ1; HRMS (APCI): m/z
calcd for C12H23O3: 215.1642 [M+H]+; found: 215.1642.
SRB1a (1a): BBr3 solution in CH2Cl2 (10%, 400 mL, 420 mmol) was
added at À788C to a solution of 9a (82 mg, 0.18 mmol) in CH2Cl2
(3.0 mL), and the mixture was stirred at À788C for 1.5 h. Saturated
aqueous NaHCO3 (2 mL) was carefully added, and the mixture was
extracted twice with EtOAc. The combined organic phases were
washed with brine, dried (Na2SO4), filtered, and concentrated to
dryness. The residue was purified by silica gel chromatography
with hexane/EtOAc (1:1 v/v) to give SRB1a (1a, 26 mg, 52%) as
a colorless oil.
(1’R)-2-(1’-hydroxyl-5’-(2’’-isobutyl-1’’,3’’-dioxolan-2’’-yl)pentyl)-3-
methyl-4-(l-menthyloxy)but-2-en-1,4-olide (9a) and (1’S)-2-(1’-
hydroxyl-5’-(2’’-isobutyl-1’’,3’’-dioxolan-2’’-yl)pentyl)-3-methyl-4-
(l-menthyloxy)but-2-en-1,4-olide (9b): A solution of n-butyllithi-
um (1.65m in hexane, 2.80 mL, 4.62 mmol) was added dropwise at
08C to a solution of diisopropylamine (640 mL, 4.55 mmol) in THF
(10 mL). After the system had been stirred for 30 min, hexamethyl-
phosphoric triamide (HMPA, 4 mL) was added dropwise at 08C. A
solution of readily available 3-methyl-4-(l-menthyloxy)but-2-en-1,4-
olide (8, 1.15 g, 4.55 mmol)[24,25] in THF (12 mL) was added drop-
wise at À788C, and the mixture was further stirred at the same
1
Mixture of C-4 epimers: [a]D24 =+18.4 (c=0.860, CHCl3); H NMR and
13C NMR assignments are listed in Table 1; IR (neat): n˜ =3389, 2957,
2872, 1752, 1705, 1464, 1370, 1335, 1088, 955, 758 cmÀ1; UV/Vis
(MeOH): lmax (loge)=210 nm (3.98 molÀ1 dm3 cmÀ1); HRMS (ESI):
m/z calcd for C15H24O5Na: 307.1516 [M+Na]+; found: 307.1519.
temperature for 30 min.
A solution of aldehyde 7 (1.05 g,
4.90 mmol) in THF (10 mL) was then added dropwise at À788C
over 10 min, and the mixture was further stirred at the same tem-
perature for 1.5 h. Saturated aqueous NH4Cl (10 mL) was added,
and the mixture was extracted twice with CH2Cl2. The combined
organic phases were washed with brine, dried (Na2SO4), filtered,
and concentrated to dryness. The residue was purified by silica gel
chromatography with hexane/EtOAc (4:1 v/v) to give a mixture of
9a and 9b (2:1, 660 mg, 31%) as a colorless oil; these were further
separated by repeated flash chromatography runs. The absolute
configurations at C-1’ were established by the modified Mosher
method.[27] The differences in chemical shifts (Dd) were obtained
by subtracting the d values for the (R)-MTPA ester from their coun-
terparts for the (S)-MTPA ester (dSÀdR).
SRB1b (1b): Compound 9b (23 mg, 50 mmol) was treated in the
same manner as described for the preparation of 1a to give SRB1b
(1b, 8.9 mg, 63%) as a colorless oil.
Mixture of C-4 epimers: [a]2D5 =À9.32 (c=0.400, CHCl3); 1H NMR
(CDCl3): d=0.89 (d, J=6.4 Hz, 3H; H-9’ or H-9’’), 0.90 (d, J=6.4 Hz,
3H; H-9’’ or H-9’), 1.25 (m, 1H; H-3’a), 1.36 (m, 1H; H-3’b), 1.53 (m,
2H; H-4’), 1.69–1.81 (m, 2H; H-2’), 2.08/2.09 (s, 3H; H-5), 2.09 (m,
1H; H-8’), 2.26 (d, J=7.1 Hz, 2H; H-7’), 2.40 (dt, J=2.2, 7.1 Hz, 2H;
H-5’), 4.46 (m, 1H; H-1’), 5.85 ppm (brs, 1H; H-4); 13C NMR (CDCl3):
d=11.5/11.6 (C-5), 22.5/22.6 (C-9’ and C-9’’), 22.9/23.2 (C-4’), 24.5/
24.7 (C-8’), 24.7/24.8 (C-3’), 35.6/35.7 (C-2’), 42.6/42.8 (C-5’), 51.9/
52.0 (C-7’), 66.4/66.6 (C-1’), 98.5/98.8 (C-4), 129.9/130.3 (C-2), 157.1/
157.2 (C-3), 171.1/171.2 (C-1), 211.4/212.6 ppm (C-6’); IR (neat): n˜ =
3389, 2957, 2872, 1752, 1705, 1464, 1370, 1335, 1088, 955,
1
Compound 9a: [a]2D7 =À58.8 (c=1.10, CHCl3); H NMR (CDCl3): d=
0.91 (d, J=6.7 Hz, 3H; Me2CH), 0.93 (d, J=6.8 Hz, 3H; Me2CH),
1.28–1.42 (m, 2H; C-3’), 1.50 (d, J=6.1 Hz, 2H; iPrCH2), 1.59–1.63
(m, 5H; H-2’a,-4’,-5’), 1.75 (m, 1H, Me2CH), 1.83 (m, 1H; H-2’b), 1.97
(s, 3H; H-5), 2.86 (brs, 1H; C1’ÀOH), 3.91 [m, 4H; (CH2OÀ)2], 4.46 (t,
758 cmÀ1
;
UV/Vis
(MeOH):
lmax
(loge)=210 nm
(4.02 molÀ1 dm3 cmÀ1); HRMS (ESI): m/z calcd for C15H24O5Na:
307.1516 [M+Na]+; found: 307.1515.
1
J=6.7 Hz, 1H; H-1’), 5.69 ppm (s, 1H; H-4); H NMR (CDCl3, menthyl
(8S)-1-(Benzyloxy)-8-methyldecan-6-ol (10): (S)-(+)-1-Chloro-2-
methylbutane (9.00 mL, 75.1 mmol) was added at room tempera-
ture to a suspension of magnesium (2.05 g, 84.3 mmol) and iodine
(135 mg) in THF (35 mL), and the mixture was stirred at 708C for
2 h. A solution of aldehyde 4 (2.78 g, 13.5 mmol) in THF (10 mL)
was added dropwise at 08C to the mixture, which was stirred at
room temperature for 1 h. Saturated aqueous NH4Cl (30 mL) was
added, and the mixture was extracted twice with EtOAc. The com-
bined organic phases were washed with brine, dried (Na2SO4), fil-
tered, and concentrated to dryness. The residue was purified over
silica gel with hexane/EtOAc (7:1–5:1 v/v) to give 10 (2.55 g, 68%)
as a colorless oil.
resonances): d=0.81 (d, J=6.8 Hz, 3H), 0.86 (m, 1H), 0.88 (d, J=
7.1 Hz, 3H), 0.96 (d, J=6.4 Hz, 3H), 1.02 (m, 2H), 1.22–1.27 (m, 1H),
1.28–1.42 (m, 1H), 1.64–1.70 (m, 2H), 2.08–2.14 (m, 2H), 3.62 ppm
(dt, J=4.3, 11 Hz, 1H); 13C NMR (CDCl3): d=11.5 (C-5), 23.5 (C-4’),
24.0 (Me2CH), 24.1 (Me2CH), 25.7 (C-3’), 36.6 (C-2’), 37.2 (C-5’), 45.2
(iPr-CH2), 64.6 ((CH2O-)2), 66.9 (C-1’), 100.9 (C-4), 111.9 (C-2’’), 130.6
(C-2), 155.4 (C-3), 171.4 ppm (C-1); 13C NMR (CDCl3, menthyl reso-
nances): d=15.9, 20.8, 22.2, 23.2, 25.3, 31.4, 34.2, 40.5, 47.7,
79.6 ppm; IR (neat): n˜ =2953, 2870, 1759, 1458, 1370, 1331, 1094,
947, 756 cmÀ1; HRMS (ESI): m/z calcd for C27H46O6Na: 489.3187
[M+Na]+; found: 489.3182.
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K. Arakawa et al.
Diastereomer mixture: 1H NMR (CDCl3): d=0.85–0.92 (m, 6H; H-9’
and H-10), 1.11 (m, 1H; H-9a), 1.25–1.50 (m, 10H; H-3,-4,-5,-7,-8,-
9b), 1.63 (t, J=6.7 Hz, 2H; H-2), 3.47 (t, J=6.7 Hz, 2H; H-1), 3.68
(m, 1H; H-6), 4.50 (s, 2H; PhCH2), 7.25–7.34 ppm (m, 5H; PhCH2);
13C NMR (CDCl3): d=11.1/11.3 (C-10), 18.8/19.8 (C-9’), 25.3/25.5 (C-
4), 26.3 (C-3), 29.0/30.4 (C-9), 29.7 (C-2), 30.8/31.1 (C-8), 37.7/38.3
(C-5), 44.6/44.8 (C-7), 69.6/69.9 (C-6), 70.3 ppm (C-1); 13C NMR
(CDCl3, benzyl resonances): d=72.8 (PhCH2), 127.5, 127.6, 128.3,
138.7 ppm; IR (neat): n˜ =3397, 2930, 2857, 1454, 1101, 735,
698 cmÀ1; HRMS (ESI): m/z calcd for C18H30O2Na: 301.2138 [M+Na]+;
found: 301.2142.
(C-2’’), 130.6 (C-2), 155.4 (C-3), 171.4 ppm (C-1); 13C NMR (CDCl3,
menthyl resonances): d=15.9, 20.8, 22.2, 23.2, 25.4, 31.5, 34.2, 40.5,
47.7, 79.6 ppm; IR (neat): n˜ =2955, 2926, 1755, 1456, 1372, 1331,
; HRMS (ESI): m/z calcd for C28H48O6Na:
503.3343 [M+Na]+; found: 503.3339.
1096, 949, 758 cmÀ1
Compound 13b: [a]2D2 =À74.4 (c=0.860, CHCl3). 1H NMR (CDCl3):
d=0.86 (t, J=7.3 Hz, 3H; MeCH2), 0.93 (d, J=6.4 Hz, 3H; MeCH),
1.14–1.20 (m, 1H; MeCH2a), 1.31–1.42 (m, 6H; H-3’, -4’, MeCH2b,
and MeCHCH2a), 1.50 (m, 1H; MeCH), 1.59–1.71 (m, 4H; H-2’a, -5’,
and MeCHCH2b), 1.82 (m, 1H; H-2’b), 1.98 (s, 3H; H-5), 2.39 (m, 1H;
C1’OH), 3.91 [m, 4H; (CH2OÀ)2], 4.47 (t, J=7.3 Hz, 1H; H-1’),
5.71 ppm (s, 1H; H-4); 1H NMR (CDCl3, menthyl resonances): d=
0.80 (d, J=6.7 Hz, 3H), 0.86 (m, 1H), 0.87 (d, J=7.1 Hz, 3H), 0.96
(d, J=6.7 Hz, 3H), 1.10 (m, 2H), 1.26 (m, 1H), 1.31–1.42 (m, 1H),
1.59–1.71 (m, 2H), 2.12 (m, 2H), 3.62 ppm (dt, J=4.3, 11 Hz, 1H);
13C NMR (CDCl3): d=11.3 (MeCH2), 11.5 (C-5), 20.4 (MeCH), 23.5 (C-
4’), 25.7 (C-3’), 30.3 (MeCH), 30.6 (MeCH2), 36.4 (C-2’), 37.2 (C-5’),
43.1 (MeCHCH2), 64.5/64.6 [(CH2OÀ)2], 66.8 (C-1’), 100.7 (C-4), 112.0
(C-2’’), 130.7 (C-2), 155.4 (C-3), 171.4 ppm (C-1); 13C NMR (CDCl3,
menthyl resonances): d=15.7, 20.8, 22.2, 23.1, 25.2, 31.4, 34.2, 40.4,
47.7, 79.5 ppm; IR (neat): n˜ =2955, 2872, 1761, 1456, 1372, 1331,
1096, 949 cmÀ1; HRMS (ESI): m/z calcd for C28H48O6Na: 503.3343
[M+Na]+; found: 503.3341.
(8S)-1-(Benzyloxy)-8-methyldecan-6-one (11): The alcohol 10
(2.50 g, 8.98 mmol) was treated in the same manner as described
for the preparation of 6 to give 11 (1.53 g, 62%) as a colorless oil.
[a]2D6 =+2.40 (c=0.500, CHCl3); 1H NMR (CDCl3): d=0.86 (d, J=
6.7 Hz, 3H; H-9’), 0.87 (t, J=7.4 Hz, 3H; H-10), 1.18 (m, 1H; H-9a),
1.30 (m, 1H; H-9b), 1.37 (m, 2H; H-3), 1.55–1.65 (m, 4H; H-2 and H-
4), 1.91 (m, 1H; H-8), 2.18 (dd, J=8.0, 16 Hz, 1H; H-7a), 2.35–2.42
(m, 3H; H-5 and H-7b), 3.46 (t, J=6.7 Hz, 2H; H-1), 4.49 (s, 2H;
PhCH2), 7.25–7.35 ppm (m, 5H; PhCH2); 13C NMR (CDCl3): d=11.3
(C-10), 19.4 (C-9’), 23.5 (C-4), 25.8 (C-3), 29.5 (C-9), 29.6 (C-2), 30.8
(C-8), 43.2 (C-5), 49.9 (C-7), 70.2 (C-1), 211.2 ppm (C-6); 13C NMR
(CDCl3, benzyl resonances): d=72.9 (PhCH2), 127.5, 127.6, 128.3,
138.6 ppm; IR (neat): n˜ =2936, 2861, 1713, 1456, 1366, 1103, 737,
698 cmÀ1; HRMS (ESI): m/z calcd for C18H28O2Na: 299.1982 [M+Na]+;
found: 299.1983.
SRB2a (2a): Compound 13a (20 mg, 42 mmol) was treated in the
same manner as described for the preparation of 1a to give SRB2a
(2a, 8.4 mg, 70%) as a colorless oil.
5-(2’-((2“S)-2”-methylbutyl)-1’,3’-dioxolan-2’-yl)pentanal
(12):
1
Compound 11 (1.50 g, 5.43 mmol) was treated in the same manner
as described for the preparation of 7 to give 12 (720 mg, 58% in
three steps) as a colorless oil. [a]2D7 =+5.92 (c=1.09, CHCl3);
1H NMR (CDCl3): d=0.86 (t, J=7.4 Hz, 3H; MeCH2), 0.93 (d, J=
6.4 Hz, 3H; MeCH), 1.17 (m, 1H; MeCH2a), 1.36–1.44 (m, 4H; H-4
and MeCHCH2), 1.51 (m, 1H; MeCH), 1.60–1.67 (m, 5H; H-3, -5, and
MeCH2b), 2.44 (dt, J=1.9, 7.4 Hz, 2H; H-2), 3.91 [m, 4H; (CH2OÀ)2],
9.76 ppm (t, J=1.9 Hz, 1H; H-1); 13C NMR (CDCl3): d=11.3 (MeCH2),
20.5 (MeCH), 22.3 (C-3), 23.4 (C-4), 30.4 (MeCH), 30.6 (MeCH2), 37.1
(C-5), 43.1 (MeCHCH2), 43.9 (C-2), 64.6/64.7 [(CH2OÀ)2], 111.9 (C-2’),
202.6 ppm (C-1); IR (neat): n˜ =2957, 2876, 1725, 1462, 1377, 1138,
1082, 949 cmÀ1; HRMS (APCI): m/z calcd for C13H25O3: 229.1798
[M+H]+; found: 229.1796.
Mixture of C-4 epimers: [a]D22 =+22.4 (c=0.800, CHCl3); H NMR and
13C NMR assignments are listed in Table 1; IR (neat): n˜ =3397, 2959,
2932, 2876, 1750, 1705, 1460, 1381, 1337, 1090, 957, 756 cmÀ1; UV/
Vis (MeOH): lmax (loge)=210 nm (4.06 molÀ1 dm3 cmÀ1); HRMS (ESI):
m/z calcd for C16H26O5Na: 321.1673 [M+Na]+; found: 321.1676.
SRB2b (2b): Compound 13b (20 mg, 42 mmol) was treated in the
same manner as described for the preparation of 1a to give SRB2b
(2b, 6.8 mg, 55%) as a colorless oil.
Mixture of C-4 epimers: [a]2D4 =À5.71 (c=0.700, CHCl3); 1H NMR
(CDCl3): d=0.86 (d, J=7.0 Hz, 3H; H-9’’), 0.87 (t, J=6.7 Hz, 3H; H-
10’), 1.15–1.20 (m, 1H; H-9’a), 1.21–1.32 (m, 2H; H-3’), 1.30–1.40
(m, 1H; H-9’b), 1.51–1.59 (m, 2H; H-4’), 1.65–1.80 (m, 1H; H-2’a),
1.81–1.93 (m, 2H; H-2’b and H-8’), 2.08/2.09 (s, 3H; H-5), 2.17 (d,
J=7.9, 8.0 Hz, 1H; H-7’a), 2.35–2.42 (m, 3H; H-5’ and H-7’b), 3.03
(brs, 1H; C1’OH), 4.49 (m, 1H; H-1’), 5.85 ppm (brs, 1H; H-4);
13C NMR (CDCl3): d=11.3/11.3 (C-10’), 11.5/11.6 (C-5), 19.3/19.4 (C-
9’’), 23.0/23.2 (C-4’), 24.6/24.8 (C-3’), 29.5/29.5 (C-9’), 30.9 (C-8’),
35.6 (C-2’), 42.7/42.9 (C-5’), 50.0/50.1 (C-7’), 66.4/66.5 (C-1’), 98.7/
98.9 (C-4), 129.9/130.1 (C-2), 157.4/157.5 (C-3), 171.3/171.4 (C-1),
211.7/212.8 ppm (C-6’); IR (neat): n˜ =3397, 2959, 2932, 2876, 1750,
1705, 1460, 1381, 1337, 1090, 957, 756 cmÀ1; UV/Vis (MeOH): lmax
(loge)=210 nm (4.03 molÀ1 dm3 cmÀ1); HRMS (ESI): m/z calcd for
C16H26O5Na: 321.1673 [M+Na]+; found: 321.1676.
(1’R)-2-(1’-hydroxyl-5’-(2“-((2”’S)-2’-methylbutyl-1“,3”-dioxolan-
2“-yl)pentyl)-3-methyl-4-(l-menthyloxy)but-2-en-1,4-olide (13a)
and (1’S)-2-(1’-hydroxyl-5’-(2”-((2“’S)-2’-methylbutyl-1”,3“-dioxo-
lan-2”-yl)pentyl)-3-methyl-4-(l-menthyloxy)but-2-en-1,4-olide
(13b): Compound 12 (670 mg, 2.93 mmol) was treated in the same
manner as described for the preparation of 9 to give a mixture
(2:1) of 13a and 13b (656 mg, 54%) as a colorless oil, which was
also further separated by flash chromatography.
Compound 13a: [a]2D3 =À72.4 (c=1.27, CHCl3); 1H NMR (CDCl3):
d=0.86 (t, J=7.3 Hz, 3H; MeCH2), 0.93 (d, J=6.8 Hz, 3H; MeCH),
1.14–1.20 (m, 1H; MeCH2a), 1.31–1.42 (m, 6H; H-3’, -4’, MeCH2b,
and MeCHCH2a), 1.50 (m, 1H; MeCH), 1.59–1.63 (m, 4H; H-2’a, -5’,
and MeCHCH2b), 1.84 (m, 1H; H-2’b), 1.97 (s, 3H; H-5), 2.85 (d, J=
9.2 Hz, 1H; C1’OH), 3.91 [m, 4H; (CH2OÀ)2], 4.46 (q, J=8.2 Hz, 1H;
Chiral HPLC analysis: Natural SRB1 and SRB2 (1 and 2) and syn-
thetic 1a, 1b, 2a, and 2b were dissolved in acetonitrile
(30 mgmLÀ1) and an aliquot of each sample (10 mL) was analyzed
by HPLC with a Chiral MB-S column (macroporous silica gel coated
with optically active N-substituted polymaleimides, 4.6ꢂ250 mm,
Tokyo Chemical Industry, Co., Ltd., Japan) with aqueous acetonitrile
(10%) containing trifluoroacetic acid (0.1%) as solvent at a flow
rate of 1.0 mLminÀ1, and with detection at 210 nm. Natural SRB1
(1) and SRB2 (2) were eluted at 9.1 and 17.3 min. Synthetic 1a, 1b,
2a, and 2b were eluted at 9.1, 8.7, 17.3, and 16.6 min, respectively.
1
H-1’), 5.69 ppm (s, 1H; H-4); H NMR (CDCl3, menthyl resonances):
d=0.81 (d, J=7.1 Hz, 3H), 0.86 (m, 1H), 0.87 (d, J=7.3 Hz, 3H),
0.96 (d, J=6.7 Hz, 3H), 1.10 (m, 2H), 1.25 (m, 1H), 1.31–1.42 (m,
1H), 1.64–1.71 (m, 2H), 2.10 (m, 2H), 3.62 ppm (dt, J=4.3, 11 Hz,
1H); 13C NMR (CDCl3): d=11.3 (MeCH2), 11.5 (C-5), 20.4 (MeCH), 23.5
(C-4’), 25.7 (C-3’), 30.3 (MeCH), 30.6 (MeCH2), 36.6 (C-2’), 37.2 (C-5’),
43.1 (MeCHCH2), 64.5/64.7 [(CH2OÀ)2], 66.9 (C-1’), 100.9 (C-4), 112.0
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Butenolide Signaling Molecules
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shi, Mol. Microbiol. 2003, 48, 1501–1510.
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We thank Daisuke Kajiya and Tomoko Amimoto (Natural Science
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grateful to Dr. Keigo Omura (Ikeda Tohka Industries Co., Ltd.) for
large-scale fermentation. This work was supported by a Grant-in-
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Received: March 1, 2012
Published online on && &&, 0000
ChemBioChem 0000, 00, 1 – 12
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FULL PAPERS
K. Arakawa,* N. Tsuda, A. Taniguchi,
H. Kinashi
Control of antibiotic production: Two
signaling molecules—SRB1 and SRB2—
that induce production of lankacidin
and lankamycin in Streptomyces rochei
7434AN4 were isolated and their struc-
tures were determined. Each contains
a 2,3-disubstituted g-hydroxybutenolide
skeleton, and the stereochemistry at C-
1’ is crucial for inducing activity.
&& – &&
The Butenolide Signaling Molecules
SRB1 and SRB2 Induce Lankacidin and
Lankamycin Production in
Streptomyces rochei
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ChemBioChem 0000, 00, 1 – 12
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These are not the final page numbers!