Salinipyrones and pacificanones, mixed-precursor polyketides from the marine actinomycete Salinispora pacifica

Article abstract of DOI:10.1021/np0705155

Chemical examination of a phylogenetically unique strain of the obligate marine actinomycete Salinispora pacifica led to the discovery of four new polyketides, salinipyrones A and B (1,2) and pacificanones A and B (3, 4). These compounds appear to be derived from a mixed-precursor polyketide biosynthesis involving acetate, propionate, and butyrate building blocks. Spectral analysis, employing NMR, IR, UV, and CD methods and chemical derivatization, was used to assign the structures and absolute configurations of these new metabolites. Salinipyrones A and B displayed exactly opposite CD spectra, indicating their pseudoenantiomeric relationship. This relationship was shown to be a consequence of the geometric isomerization of one double bond. The phenomenon of polyketide module skipping is proposed to explain the unusual biosynthesis of the salinipyrones and the pacificanones.

Full text of DOI:10.1021/np0705155

570
J. Nat. Prod. 2008, 71, 570–575
Salinipyrones and Pacificanones, Mixed-Precursor Polyketides from the Marine Actinomycete
Salinispora pacifica
Dong-Chan Oh, Erin A. Gontang, Christopher A. Kauffman, Paul R. Jensen, and William Fenical*
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, UniVersity of California, San Diego, La Jolla,
California 92093-0204
ReceiVed September 25, 2007
Chemical examination of a phylogenetically unique strain of the obligate marine actinomycete Salinispora pacifica led
to the discovery of four new polyketides, salinipyrones A and B (1, 2) and pacificanones A and B (3, 4). These compounds
appear to be derived from a mixed-precursor polyketide biosynthesis involving acetate, propionate, and butyrate building
blocks. Spectral analysis, employing NMR, IR, UV, and CD methods and chemical derivatization, was used to assign
the structures and absolute configurations of these new metabolites. Salinipyrones A and B displayed exactly opposite
CD spectra, indicating their pseudoenantiomeric relationship. This relationship was shown to be a consequence of the
geometric isomerization of one double bond. The phenomenon of polyketide module skipping is proposed to explain
the unusual biosynthesis of the salinipyrones and the pacificanones.
The existence of actinomycetes indigenous to the marine
environment has been debated,1 but recent research results have
shown that the ocean is indeed a rich habitat for these chemically
prolific microorganisms. Although their ecological roles in the
marine environment remain largely unknown, some marine acti-
nomycetes are adapted to live in seawater, some are found in
association with invertebrate hosts, while others have been recov-
ered from the deepest ocean trenches. As an example, a salt-
dependent pelagic marine actinomycete belonging to the family
Nocardioidaceae was recently identified in surface sea water.2
Marine invertebrates, especially sponges, are now well-known as
hosts for diverse bacterial assemblages and represent rich sources
for novel actinobacteria.3 From marine sediments, we recently
reported the first obligate marine actinomycete genus, the Salin-
ispora,4 which has proven to be a prolific source of secondary
metabolites.5 Our continuing research on Salinispora diversity,
based upon comprehensive comparisons of 16S rDNA sequence
data, has resulted in the cultivation of a third Salinispora species
for which the name Salinispora pacifica has been proposed.6 An
investigation of the secondary metabolites produced by S. pacifica
led to the isolation of the structurally novel metabolites cyanospo-
rasides A and B, which raised intriguing questions about the genetic
capacity of Salinispora strains to produce enediyne antibiotics.7
Further genetic analysis of other S. pacifica strains collected from
Palau8 revealed a new phylotype (strain CNS-237) that differed
from the cyanosporaside-producing strains at only three nucleotide
positions (16S rRNA gene).9 Chemical screening of this strain by
LC-MS analysis indicated that a secondary metabolite profile was
quite different from those of the other S. pacifica phylotypes.
Here we report the isolation and structural determination of four
secondary metabolites from S. pacifica, strain CNS-237, cultivated
in a seawater-based medium. In culture, this strain produced the
related polyketides salinipyrones A and B (1, 2) and pacificanones
A and B (3, 4).
sp3 carbon, and seven carbon signals in the aliphatic region. An
observed IR absorption at 1655 cm-1 demonstrated the presence
of an unsaturated ester functionality, and this was confirmed by
the presence of an ester carbonyl carbon at δC 167.5 in the 13C
NMR spectrum. Since four double bonds (eight olefinic carbons)
and one ester carbonyl accounted for five of the six double-bond
equivalents inherent in the molecular formula, salinipyrone A must
be a monocyclic compound.
1
The H NMR spectrum of salinipyrone A (1) displayed five
distinct methyl signals. Two were aliphatic methyl groups; one was
observed as a triplet [δH 0.95] and one as a doublet [δH 1.03]. Three
olefinic methyls were also observed at δH 2.04, 1.93, and 1.87.
Two mutually coupled E vinyl protons were observed at δH 7.09
(d, 15.5 Hz) and 6.37 (d, 15.5 Hz). Interpretation of gHMQC and
1H-1H gCOSY spectral data allowed the assignment of the side-
chain part of the molecule (C-9 to C-13) with C-17 methyl
substitution at C-10 and connectivity of C-6 and C-7. Analysis of
the gHMBC spectrum allowed the connection of C-7 and C-8 by
observation of correlations from H3-16 to C-7 and C-8 and from
H-7 to C-8 and C-9. The R-pyrone ring system was constructed on
the basis of HMBC correlations from the olefinic methyl groups.
The two-bond and three-bond heteronuclear couplings from H3-14
to C-1, C-2, and C-3 and from H3-15 to C-3, C-4, and C-5
established the ester linkage and closed the pyrone ring. Finally,
HMBC signals from H-6 to C-5 completed the planar structure of
1 by connecting the R-pyrone ring with the side chain.
The geometries of the conjugated double bonds in 1 were
determined by coupling constant and 1D NOE analyses (Figure
1a). The large coupling constant (15.5 Hz) between H-6 and H-7
allowed the C-6–C-7 E-geometry to be assigned. NOE correlations
between H-7 and H-9 and between H-6 and H3-16 established the
C-8–C-9 E-olefin geometry. The relationship of the pyrone ring to
the chain was determined by an observed NOE correlation between
H3-15 and H-6.
The relative configurations of the two consecutive stereogenic
centers (C-10 and C-11) were first proposed by comparison of the
13C NMR chemical shifts of 1 with published values obtained from
synthetic diastereomers10 sharing common partial structures from
C-8 to C-13 with C-17 substitution (Figure 2). In this analysis of
13C NMR chemical shifts, the shift of the C-17 methyl group is
the most significantly distinguishable, as it is reported to be
dependent on the relative configuration of the methyl-hydroxy-
substituted stereogenic centers.11 The 13C NMR chemical shifts of
1 suggested that the relative configurations were 10S* and 11R*,
Results and Discussion
Salinipyrone A (1) was obtained as a viscous oil that analyzed
for the molecular formula C17H24O4 by ESIHRMS (obsd [M + H]+
at m/z 293.1744, calcd [M + H]+ 293.1747). This molecular formula
was supported by 1H and 13C NMR data (Table 1). The 13C NMR
spectrum of 1 showed nine sp2 carbon resonances, one oxygenated
* To whom correspondence should be addressed. Tel: (858) 534-2133.
Fax: (858) 534-1318. E-mail: wfenical@ucsd.edu.
10.1021/np0705155 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/06/2008

Mixed-Precursor Polyketides from Salinispora pacifica
Journal of Natural Products, 2008, Vol. 71, No. 4 571
Table 1. NMR Data for Salinipyrone A (1) in CD3OD
a
b
C/H
δH
mult (J in Hz)
δC
#H
COSY
HMBC
NOE
1
2
3
4
5
6
7
8
167.5
100.0
167.7
110.0
154.3
115.0
140.8
134.9
142.1
40.0
C
C
C
C
C
6.37
7.09
d (15.5)
d (15.5)
CH
CH
C
CH
CH
CH
CH2
7
6
5, 7, 8
5, 6, 8, 9
15, 16
9
9
5.80
2.67
3.38
1.47
1.38
0.95
1.93
2.04
1.87
1.03
br. d (10.0)
10, 16
9, 11, 17
10, 12
11, 13
11, 13
12
7, 10, 16, 17
9, 17
16, 13
11
7, 10, 11, 12, 17
9, 11, 12, 13, 17
9, 10, 12, 13, 17
9, 10, 11, 13
9, 10, 11, 13
10, 11, 12
10
11
12a
12b
13
14
15
16
17
dqd (10.5, 7.0, 5.0)c
ddd (8.5, 5.0, 4.0)c
78.0
28.6
dqd (14.0, 7.5, 4.0)c
ddq (14.0, 8.5, 7.5)c
11
t (7.5)
s
s
d (1.0)
d (7.0)
10.8
9.1
9.6
12.7
17.5
CH3
CH3
CH3
CH3
CH3
11, 12
1, 2, 3
3, 4, 5
7, 8, 9
9, 10, 11
6
6
9
10
9, 10, 11
a 500 MHz. b 75 MHz. c Acquired by homodecoupling experiments.
1
Figure 3. H NMR ∆δS-R values in ppm for the S- and R-MTPA
esters of 1 (a) and 2 (b) in CD3OD.
comparisons, we undertook a comprehensive examination of the
1D NOE data for the side-chain protons in 1. Careful consideration
of the expected NOE correlations from every possible rotamer (see
the Supporting Information) also indicated that the relative con-
figurations of C-10 and C-11 were S* and R*, respectively.
The overall absolute configuration of 1 was determined by
application of the modified Mosher method.12 Acylation of 1 with
R-(-) and S-(+)-R-methoxy-R-(trifluoromethyl)phenyl acetyl chlo-
ride (MTPA-Cl) furnished bis-S- and R-MTPA esters (6a and 6b),
Figure 1. 1D NOE correlations used to assign the double-bond
geometries in 1 (a) and 2 (b).
1
respectively. Analysis of H NMR data for these MTPA esters
allowed the assignment of the ∆δS-R values, which were positive
for H2-12 and H3-13, while those of H-6, H-7, H-9, H-10, H3-16,
and H3-17 were negative. This is sufficiently consistent to establish
the absolute configuration of C-11 as R (Figure 3a). On the basis
of the relative configuration of C-10 and C-11, the absolute
configuration of C-10 was assigned as S.
Salinipyrone B (2) was isolated as a viscous oil, which analyzed
for the same molecular formula, C17H24O4, as 1 (six degrees of
unsaturation) by ESI high-resolution mass spectrometry (obsd [M
+ H]+ at m/z 293.1747, calcd [M + H]+ 293.1747) in combination
with interpretation of 1H and 13C NMR data (Table 2). The 1H NMR
coupling patterns observed for salinipyrone B were identical to those
of 1, but the chemical shifts of the protons were slightly different.
13C NMR and gHMQC spectra recorded for 2 also displayed a high
degree of similarity to those of 1 except for one allylic methyl signal
[δC 20.5], which was relatively deshielded. As in 1, the gross
structure of salinipyrone B was constructed by analysis of gCOSY,
Figure 2. Comparison of 13C NMR chemical shifts (in CDCl3) of
salinipyrone A (1) with two synthetic model compounds (a and b)
to establish the relative configurations at C-10 and C-11 in 1.
respectively, although the differences in chemical shifts were small.
To confirm the relative configuration indicated by chemical shift

572 Journal of Natural Products, 2008, Vol. 71, No. 4
Oh et al.
Table 2. NMR Data for Salinipyrone B (2) in CD3OD
a
b
C/H
δH
mult (J in Hz)
δC
#H
COSY
HMBC
NOE
1
2
3
4
5
6
7
8
167.7
100.2
168.5
110.7
154.0
117.5
132.4
133.0
139.6
38.9
C
C
C
C
C
6.47
7.50
d (15.5)
d (15.5)
CH
CH
C
CH
CH
CH
CH2
7
6
5, 7, 8
5, 6, 8, 9
15, 16
10, 17
9
5.63
2.90
3.38
1.46
1.36
0.94
1.94
2.05
1.94
1.05
d (10.0)
10
7, 10, 16, 17
9, 17
16, 13
11
10, 16, 17
10
11
12a
12b
13
14
15
16
17
dqd (10.0, 7.0, 4.5)c
9, 11, 17
10, 12
11, 13
11, 13
12
7, 9, 11, 12, 13, 17
10, 12, 13, 17
10, 11, 13
10, 11, 13
10, 11, 12
ddd (8.5, 4.5, 4.0)c
78.2
28.6
dqd (14.0, 7.5, 4.0)c
ddq (14.0, 8.5, 7.5)c
11
t (7.5)
s
s
s
10.9
9.1
9.6
20.5
18.2
CH3
CH3
CH3
CH3
CH3
11, 12
1, 2, 3
3, 4, 5
7, 8, 9
9, 10, 11
6
6, 9
9, 10, 11
d (7.0)
10
a 500 MHz. b 75 MHz. c Acquired by homodecoupling experiments.
gHMQC, and gHMBC data. The geometries of the double bonds
were determined by coupling constant and 1D NOE analysis (Figure
1b). The only difference between salinipyrone B (2) and 1 is the
geometry of the C-8-C-9 double bond. NOE correlations between
H-9 and H3-16 and between H-7 and H-10 as well as H3-17 clearly
established the C-8-C-9 Z-geometry. This geometry is consistent
with the relatively deshielded chemical shift of the olefinic methyl
(C-16 δC 20.5) due to less steric compression compared to that of
the C-16 methyl for the C-8-C-9 E-geometry in 1 (C-16 δC in 1:
12.7).
The relative configurations at C-10 and C-11 in 2 were proposed
as S* and R* (identical to the relative configurations in 1 based on
the high degree of similarity of the 1H-1H coupling constants and
NOE correlations around the C-10 and C-11 stereogenic centers to
those in 1). The absolute configuration of C-11 in 2 was determined
as R by application of the modified Mosher method, which involved
conversion to the corresponding S- and R-MTPA esters (7a and
7b) with R- and S-MTPA chloride, respectively. In these experi-
ments, the configuration at C-10 was assigned as S (Figure 3b).
Interestingly, the specific rotation values ([R]D) of 1 and 2 were
unequal and showed opposite signs (-87 and +146 for 1 and 2,
respectively). This led us to acquire CD spectra to examine their
chiroptical properties. Even though these two compounds possess
identical stereogenic centers and are not enantiomers, their CD
spectra displayed characteristics similar to those of enantiomeric
structures (Figure 4). This is a clear example of the effects of
double-bond geometry on the sign of the optical rotation and upon
circular dichroic behavior. Inversions of optical rotation and CD
spectra have been previously reported in polyene amides13 and
norsesquiterpenes,14 respectively.
Figure 4. CD spectra of salinipyrones A and B (1 and 2).
tively. Long-range heteronuclear couplings from H-1, H-5, H3-15,
and H3-18 to C-6 allowed the oxygen-bearing quaternary carbon
to be placed between C-1 and C-5. Further, HMBC correlations to
the ketone carbon established the 1,3,5,6-tetra-alkyl-substituted
cyclohexanone moiety. The linkage between the side chain and the
cyclohexanone ring was secured by HMBC correlations from H-7
to C-6 and C-1, which allowed the planar structure of 3 to be
assigned.
Pacificanone A (3) was obtained as a viscous oil, the molecular
formula of which was assigned as C20H34O3 by interpretation of
ESIHRMS (obsd [M + Na]+ ) m/z 345.2392, calcd [M + Na]+
345.2400) combined with 1H and 13C NMR features (Table 3). The
proton NMR spectrum of 3 displayed E-coupled olefinic protons
[δH 6.30 (d, 16.0 Hz); 5.67 (d, 16.0 Hz)], one olefinic proton [δH
5.47 (d, 10.0 Hz)], one oxygenated methine signal [δH 3.35 (ddd,
8.5, 5.0, 4.0 Hz)], and five methyl groups including an olefinic
methyl group at δH 1.78. The 13C NMR spectrum showed one
ketone [δC 218.0], four olefinic carbons from δC 131 to 136, two
oxygenated carbons [δC 81.0; 78.1], and 13 aliphatic signals.
Analysis of 2D gCOSY, gHMQC, and gHMBC NMR spectra
allowed all protons and carbons to be assigned. This analysis led
to the conclusion that the side chain from C-7 to C-14, including
the C-19 and C-20 methyl groups, was identical to the side chain
of salinipyrone A (1). COSY correlations established the connectiv-
ity of C-17-C-16-C-3-C-4-C-5-C-18 and C-1-C-15, respec-
The relative configuration of the 1,3,5,6-tetra-alkyl-substituted
cyclohexanone was established by 1D NOE experiments (Figure
5a). NOE enhancements of H-3 and H-5 were observed upon
irradiation of H3-15, indicating that these two protons are in axial
positions relative to the axial C-15 methyl group. As a result, the
C-18 methyl group and the ethyl group [C-16-C-17] were assigned
at equatorial positions. NOE enhancements upon irradiation of both
H-7 and H-8 to H-1, H3-15, H-5, and H3-18 established the
equatorial position of the chain. Overall, these data defined a
substituted cyclohexanone ring in a rigid chair conformation with
1S*, 3S*, 5R*, and 6R* relative configurations.
The assignment of 11S* and 12R* configurations in 3, which
are identical to the relative configurations of C-10 and C-11 in 1,
was based on the high degree of identity of carbon chemical shifts
and proton coupling constants with those of the identical side chain
in 1.

Mixed-Precursor Polyketides from Salinispora pacifica
Journal of Natural Products, 2008, Vol. 71, No. 4 573
Table 3. NMR Data for Pacificanone A (3) in CD3OD
a
b
C/H
δH
mult (J in Hz)
δC
#H
COSY
HMBC
2, 3, 6, 15
NOE
1
2
3
4R
4ꢀ
5
2.28
mc
m
59.2
218.0
47.5
CH
C
CH
CH2
15
7, 8, 15
2.52
1.87
1.48
2.26
4a, 4b, 16
3, 4b, 5
3, 4a, 5
2, 4, 16, 17
4R, 5, 15, 16a, 16b, 17
3, 5, 16a, 16b, 17, 18
16a, 16b, 17, 18
ddd (13.0, 6.5, 4.5)
37.0
2, 3, 5, 6, 16, 18
2, 3, 5, 6, 16, 18
4, 6, 18
md
mc
34.9
81.0
CH
C
4a, 4b, 18
3, 4R, 7, 8, 15, 18
6
7
8
9
5.67
6.30
d (16.0)
d (16.0)
131.8
135.2
134.3
136.0
39.4
CH
CH
C
CH
CH
CH2
CH2
8
7
1, 5, 6, 8, 9
1, 6, 7, 9, 10, 19
1, 5, 15, 18, 19
1, 5, 10, 15, 18
10
11
12
13a
13b
14
15
16a
16b
17
18
19
20
5.47
2.61
3.35
1.46
1.36
0.93
1.15
1.74
1.20
0.90
0.87
1.78
1.00
d (10.0)
11
8, 11, 12, 19, 20
9, 10, 11, 13, 20
10, 11, 13, 14, 20
11, 12, 14
11, 12, 14
12, 13
8, 11, 12, 20
10, 12, 13a, 13b, 14, 20
11, 13a, 13b, 14. 20
11, 12, 14
11, 12, 14
11, 12, 13a, 13b
1, 3, 5, 7, 8
3, 4R, 4ꢀ, 17
3, 4R, 4ꢀ, 17
3, 4R, 4ꢀ, 16a, 16b
4R, 4ꢀ, 7, 8
7, 11
10, 11, 12
dqd (10.5, 6.5, 5.0)e
10, 12, 20
11, 13a, 13b
12, 13b, 14
12, 13a, 14
13a, 13b
1
3, 16b, 17
3, 16a, 17
16a, 16b
5
ddd (8.5, 5.0, 4.0)e
78.1
28.5
md
m
t (7.5)
d (7.5)
m
10.9
15.4
23.0
CH3
CH3
CH2
1, 2, 6
2, 3, 4, 17
2, 3, 4, 17
2, 16
4, 5, 6
8, 9, 10
m
t (7.5)
d (6.5)
s
11.9
15.2
13.1
17.7
CH3
CH3
CH3
CH3
d (7.0)
11
10, 11, 12
a 500 MHz. b 75 MHz. c Overlapping signals. d Overlapping signals. e Acquired by homodecoupling experiments.
Cotton effect at 240 nm in the CD spectrum of 4, which is derived
from the E,E-diene and hydroxy group interaction,16 led us to
propose the same absolute configuration for pacificanone A (3) at
relevant centers, except for the C-1 position. The C-1 configuration
in 3 is assumed to be S since the relative configuration is opposite
of that in 4.
Salinipyrones A and B appear to be R-pyrone polyketides that
are derived from incorporation of acetate and propionate precursors.
Propionate polyketide R-pyrones are well-known from marine
invertebrates such as sacoglossans17 and pulmonates,18 and on
occasion from actinomycetes.19 The pacificanones bear a unique
1,3,5,6-tetra-alkyl-substituted cyclohexanone ring, which is a
structural feature not previously reported in natural products. The
biosynthetic origin of the salinipyrones and the pacificanones seems
most likely via a type I polyketide biosynthetic pathway. The carbon
backbone of the pacificanones differs from that of the salinipyrones
in that an additional ethylmalonyl-CoA building block appears to
have been incorporated. This skeletal variation can be explained
by evoking a PKS module skipping mechanism, an event reported
in the biosynthesis of methymycin and pikromycin by Streptomyces
Venezulae.20 However, the possibility that the salinipyrones and
the pacificanones are derived from two separate PKS I modules,
while unlikely, cannot be excluded without further experimental
information.
The biological activity of compounds 1-4 is currently being
examined in diverse bioassays. In initial screening, the salinipyrones
and the pacificanones displayed no significant activity in a cancer
cell cytotoxicity assay (HCT-116 human colon cancer), nor did these
compounds show appreciable antibiotic activities against methi-
cillin-resistant Staphylococcus aureus, vancomycin-resistant
Enterococcus faecium, and amphotericin-resistant Candida albicans.
In an isolated mouse splenocyte model of allergic inflammation,
salinipyrone A displayed moderate inhibition of interleukin-5
production by 50% at 10 µg/mL without measurable human cell
cytotoxicity (HCT-116).
Figure 5. Key NOE correlations that defined the relative configura-
tions for substituents on the chair cyclohexanone rings in 3 (a) and
4 (b).
Pacificanone B (4), also isolated as a viscous oil, was analyzed
for the molecular formula C20H34O3 by ESI high-resolution mass
spectrometry (obsd [M + Na]+ m/z ) 345.2392, calcd [M + Na]+
345.2400). The 1D and 2D NMR data (Table 4) for 4 were virtually
identical to those observed from 3, suggesting that this compound
is a stereoisomer of 3 with the same planar structure. Proton 1D
1
NOE experiments and H coupling constant analysis revealed an
equatorial C-15 methyl group rather than the axial C-15 methyl
found in 3 (Figure 5b). The large vicinal coupling constant (12.0
Hz) for H-5 showed it was in an axial position. NOE correlations
from H-5 to H-1 and H-3 established their axial relationships, thus
also indicating an equatorial position for C-15. The relatively upfield
carbon chemical shift for C-15 [δC 8.6] in 4 compared to C-15 [δC
15.4] in 3 also supported this assignment.
The absolute configuration of pacificanone B (4) was determined
by application of the modified Mosher method on the alcohol
obtained by reduction of the ketone functionality. Treatment of
pacificanone B with sodium borohydride in MeOH for 30 min at 0
°C generated the alcohol 5, which was purified and structurally
defined by NMR methods. The ring hydroxy group in 5 was found
to be in an axial position on the basis of interpretation of chemical
shift and coupling constant data from the H-5 carbinol proton [δH
3.71 (br s, the width of the peak ≈ 10 Hz)].15 Mosher acylation of
5 with R- and S-MTPA-Cl yielded the bis-S- and R-MTPA esters
The discovery of the salinipyrones and the pacificanones is the
result of a comprehensive phylogenetic analysis approach. Fine-
scale phylogenetic analyses of the 16S rDNA sequences of
Salinispora strains revealed subtle diversity within the S. pacifica
clade, which prompted detailed chemical analyses of strain CNS-
237. This diversity-based approach led to the discovery of the
salinipyrones with unusual chiroptical properties and the pacifi-
canones, which appear to constitute a new example of module
1
1
(8a and 8b). Analysis of H chemical shifts in the 1D H NMR
and gCOSY spectra of the esters allowed calculation of ∆δS-R
values and established the absolute configurations of C-2 as S and
C-12 as R (Figure 6). These assignments, coupled with the
established relative configurations, allowed 1R, 3S, 5R, 6R, and
11S absolute configurations to be assigned. The identical positive

574 Journal of Natural Products, 2008, Vol. 71, No. 4
Table 4. NMR Data for Pacificanone B (4) in CD3OD
Oh et al.
a
b
C/H
δH
mult (J in Hz)
q (6.5)
δC
#H
COSY
HMBC
2, 3, 6, 15
NOE
3, 5, 7, 8, 15
1
2
3
4R
4ꢀ
5
2.64
54.2
214.6
52.6
CH
C
CH
CH2
15
2.40
1.94
1.48
2.12
m
4a, 4b, 16
3, 4b, 5
3, 4a, 5
2, 4, 16, 17
1, 4R, 5, 16a, 16b, 17
3, 5, 16a, 16b, 17, 18
16a, 16b, 17, 18
ddd (13.0, 6.0, 3.5)
38.4
2, 3, 5, 6, 16, 18
2, 3, 5, 6, 16, 18
4, 6, 18
mc
dqd (12.0, 6.5, 3.5)d
41.6
83.3
CH
C
4a, 4b, 18
1, 3, 4R, 7, 8, 18
6
7
8
9
5.52
6.22
d (16.0)
d (16.0)
134.9
135.1
134.1
135.4
39.4
CH
CH
C
CH
CH
CH2
CH2
8
7
1, 5, 6, 8, 9
1, 6, 7, 9, 10, 19
1, 5, 15, 18, 19
1, 5, 10, 15, 18
10
11
12
13a
13b
14
15
16a
16b
17
18
19
20
5.45
2.61
3.35
1.46
1.36
0.93
0.89
1.74
1.20
0.90
0.87
1.78
1.01
d (10.0)
11
8, 11, 12, 19, 20
9, 10, 11, 13, 20
10, 11, 13, 14, 20
11, 12, 14
11, 12, 14
12, 13
8, 11, 12, 20
10, 12, 13a, 13b, 14, 20
11, 13a, 13b, 14. 20
11, 12, 14
11, 12, 14
11, 12, 13a, 13b
1, 3, 5, 7, 8
3, 4R, 4ꢀ, 17
3, 4R, 4ꢀ, 17
3, 4R, 4ꢀ, 16a, 16b
4R, 4ꢀ, 7, 8
7, 11
10, 11, 12
dqd (10.5, 6.5, 5.0)d
10, 12, 20
11, 13a, 13b
12, 13b, 14
12, 13a, 14
13a, 13b
1
3, 16b, 17
3, 16a, 17
16a, 16b
5
ddd (8.5, 5.0, 4.0)d
78.2
28.5
mc
m
t (7.5)
d (6.5)
m
10.9
8.6
23.2
CH3
CH3
CH2
1, 2, 6
2, 3, 4, 17
2, 3, 4, 17
2, 16
4, 5, 6
8, 9, 10
m
t (7.5)
d (6.5)
s
12.2
15.5
13.1
17.7
CH3
CH3
CH3
CH3
d (6.5)
11
10, 11, 12
a 500 MHz. b 75 MHz. c Overlapping signals. d Acquired by homodecoupling experiments.
Isolation of Salinipyrones A and B (1, 2) and Pacificanones A
and B (3, 4). The crude extract (1.9 g) was fractionated by C18 vacuum
column chromatography eluting with a step gradient from 10 to 100%
MeOH in H2O. The 60% MeOH/H2O fraction (219 mg) was subjected
to reversed-phase HPLC with 75% aqueous MeOH (Waters Prep LC
4000 system, Waters preparative column C18 25 mm × 200 mm, 10
mL/min, UV detection at 254 nm). Pacificanones A and B were isolated,
as pure metabolites, with retention times of 21.7 and 23 min.
Salinipyrones A and B were subsequently purified from a complex
fraction that eluted at 14.7 and 13.5 min by normal-phase HPLC
(Lichrospher semipreparative column Si gel, 10 mm × 250 mm, 2 mL/
min, refractive index detection) with a solvent composed of EtOAc/
isooctane/MeOH, 47.5:47.5:5.
Figure 6. 1H NMR ∆δS-R values in ppm for the bis-S- and R-MTPA
esters of alcohol 5 in CD3OD.
skipping in a PKS I biosynthetic pathway. Application of compre-
hensive 16S rDNA phylogenetic analysis appears to be an effective
method to define diverse actinomycete taxa from marine ecosys-
tems, thus enhancing the rate of discovery of novel secondary
metabolites.
Salinipyrone A (1): yellow oil; [R]D -87 (c 0.33, CH3OH); IR (neat)
ν
max 3397, 1672, 1545, 1222 cm-1; UV (CH3OH) λmax (log ꢀ) 255 (4.5),
348 (4.0) nm; CD (CH3OH) (∆ꢀ) 252 (+7.9), 350 (-2.4) nm; 1H NMR
(500 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD), see Table 1;
HRESITOFMS [M + H]+ m/z 293.1744 (C17H25O4, calcd [M + H]+
293.1747).
Experimental Section
Salinipyrone B (2): yellow oil; [R]D +146 (c 0.21, CH3OH); IR
(neat) νmax 3380, 1664, 1548, 1220 cm-1; UV (CH3OH) λmax (log ꢀ)
256 (4.5), 351 (3.9) nm; CD (CH3OH) (∆ꢀ) 253 (–7.6), 349 (+2.9)
General Experimental Procedures. The optical rotations were
measured using a Rudolph Research Autopol III polarimeter with a 10
cm cell. UV spectra were recorded in a Varian Cary UV–visible
spectrophotometer with a path length of 1 cm. CD spectra were collected
in an AVIV model 215 CD spectrometer with a 0.5 cm long cell. IR
spectra were acquired in a Nicolet Magna 550 FT-IR series II FT-IR
spectrometer. 1H, 13C, and 2D NMR data were obtained on Varian Inova
500 and 600 MHz spectrometers. High-resolution mass spectra (HRES-
ITOFMS) were recorded at The Scripps Research Institute, La Jolla,
CA. Low-resolution LC/MS data were acquired using a Hewlett-Packard
series 1100 LC/MS system with a reversed-phase C18 column (Phe-
nomenex Luna, 4.6 mm × 100 mm, 5 µm) at a flow rate of 0.7 mL/
min.
Collection and Phylogenetic Analysis of Strain CNS-237. The
marine actinomycete strain CNS-237 was isolated from a sediment
sample collected on the island of Palau in 2004. The strain appears to
represent a new Salinispora species (proposed as Salinispora pacifica)
based on 16S rDNA analysis and DNA-DNA hybridization studies.
The full 16S rDNA sequence data have been deposited in GenBank
under the acquisition number DQ3128246.
1
nm; H NMR (500 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD),
see Table 2; HRESITOFMS [M + H]+ m/z 293.1747 (C17H25O4, calcd
[M + H]+ 293.1747).
Pacificanone A (3): colorless oil; [R]D -10 (c 0.16, CH3OH); IR
(neat) νmax 3414, 2960, 1698, 1519 cm-1; UV (CH3CN) λmax (log ꢀ)
239 (4.3) nm; CD (CH3CN) (∆ꢀ) 240 (+4.5), 290 (–1.7) nm; 1H NMR
(500 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD), see Table 3;
HRESITOFMS [M + Na]+ m/z 345.2392 (C20H34O3Na, calcd [M +
Na]+ 345.2400).
Pacificanone B (4): colorless oil; [R]D -3 (c 0.20, CH3OH); IR
(neat) νmax 3439, 2960, 1706, 1519 cm-1; UV (CH3CN) λmax (log ꢀ)
237 (4.3) nm; CD (CH3CN) (∆ꢀ) 220 (+3.6), 240 (+3.2), 290 (–2.1)
1
nm; H NMR (500 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD),
see Table 4; HRESITOFMS [M + Na]+ m/z 345.2392 (C20H34O3Na,
calcd [M + Na]+ 345.2400).
Reduction of 4 to Yield Alcohol 5. Pacificanone B (4, 2 mg) was
dissolved in 1 mL of dry MeOH. Two milligrams of NaBH4 was added
to the solution, and the reaction mixture was stirred for 30 min. Ten
milliliters of DCM was added, and the mixture was partitioned with
2.5% aqueous NH4Cl solution. The DCM/MeOH portion was then
washed again with brine. The organic layer was dried with MgSO4,
and the major product, alcohol 5, was purified by reversed-phase HPLC
(Alltech semipreparative column C18, 10 mm × 250 mm, 2 mL/min,
refractive index detection) with the isocratic solvent mixture 70%
CH3CN in H2O. Alcohol 5 (2 mg) was eluted at 31 min. The molecular
Fermentation and Extraction. Salinispora strain CNS-237 was
cultured at 27 °C with shaking at 250 rpm in twelve 2.8 L Fernbach
flasks each containing 1 L of the medium A1BFe+C (10 g of starch,
4 g of yeast extract, 2 g of peptone, 1 g of CaCO3, 40 mg of
Fe2(SO4)3 ·4H2O, 100 mg of KBr, and 1 L of sea water). After 6 days,
the whole culture (12 L) was extracted twice with EtOAc, and the
resulting extract was concentrated by rotary evaporation.

Mixed-Precursor Polyketides from Salinispora pacifica
Journal of Natural Products, 2008, Vol. 71, No. 4 575
formula of the reduction product was confirmed as C20H36O3 by
ESIHRMS analysis (obsd [M + Na]+ m/z 347.2557, calc m/z 347.2562).
Alcohol 5: colorless oil; [R]D +35 (c 0.02, CH3OH); IR (neat) νmax
m), 5.26 (1H, d, J ) 9.5 Hz), 5.34 (1H, d, J ) 15.5 Hz), 5.47 (1H, br.
s), 6.18 (1H, d, J ) 15.5 Hz), 7.38-7.64 (10H, m).
Bis-R-MTPA ester of 5 (8b): 1H NMR (500 MHz, CD3OD) δ 0.70
(3H, d, J ) 7.0 Hz), 0.77 (3H, t, J ) 7.0 Hz), 0.80 (3H, t, J ) 7.0 Hz),
0.93 (3H, d, J ) 7.0 Hz), 0.97 (3H, d, J ) 6.5 Hz), 1.59 (3H, m), 1.60
(1H, m), 1.62 (2H, m), 1.73 (3H, s), 1.83 (1H, m), 2.17 (1H, m), 2.90
(1H, m), 3.54 (3H, s), 3.57 (3H, s), 5.00 (1H, m), 5.28 (1H, d, J ) 9.5
Hz), 5.32 (1H, d, J ) 15.5 Hz), 5.45 (1H, br s), 6.14 (1H, d, J ) 15.5
Hz), 7.27-7.66 (10H, m).
1
3430, 2960, 1517 cm-1; UV (CH3CN) λmax (log ꢀ) 237 (4.3) nm; H
NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD), see
Table S1 for the full assignment; HRESITOFMS [M + Na]+ m/z
1
347.2557 (C20H36O3Na, calcd [M + Na]+ 347.2562); H NMR (600
MHz, CD3OD) δ 0.82 (3H, d, J ) 6.5 Hz), 0.93 (3H, t, J ) 7.5 Hz),
0.94 (3H, t, J ) 7.5 Hz), 1.00 (3H, d, J ) 6.5 Hz), 1.01 (3H, d, J )
6.5 Hz), 1.31 (1H, m), 1.36 (1H, m), 1.37 (2H, m), 1.42 (1H, m), 1.45
(1H, m), 1.50 (1H, m), 1.55 (1H, m), 1.75 (3H, s), 2.61 (1H, m), 3.34
(1H, m), 3.71 (1H, br. s), 5.32 (1H, d, J ) 16.0 Hz), 5.40 (1H, d, J )
10.0 Hz), 6.19 (1H, d, J ) 16.0 Hz).
Acknowledgment. This research is a result of financial support from
the National Cancer Institute, under grant CA44848 (to W.F.). We thank
W. K. Strangman and L. Zeigler for assistance with the bioassays.
MTPA Esters of 1, 2, and 5 (6a/6b, 7a/7b, and 8a/8b). Duplicate
(1.2 mg, 4.1 µmol) samples of dry salinipyrone A (1) were prepared
for both R- and S-MTPA acylation reactions. In separate vials, the
samples were dissolved in 1.5 mL of dry pyridine, a catalytic amount
of dry DMAP was added, and after stirring for 30 min, 20 µL (107
µmol) of R-MTPA chloride and S-MTPA chloride were added. The
reactions were monitored by LC/MS, which showed the clean conver-
sion to the S- and R-bis-MTPA esters, respectively. The acylation
products were purified by reversed-phase HPLC (Alltech C18 semi-
preparative column, 10 mm × 250 mm, 2 mL/min, UV 254 nm
detection, 20%–100% H2O/MeOH gradient over 40 min, 100% CH3OH
isocratic after 40 min). The bis-S-MTPA ester (6a) and bis-R-MTPA
ester (6b) of 1 were eluted at 58 min. The molecular formulas for 6a
and 6b were confirmed as C37H38F6O8 by ESI-LC/MS analysis ([M +
H]+ m/z 725 and [M + Na]+ m/z 747). For salinipyrone B, the same
procedure above was carried out to obtain the corresponding S- and
R-MTPA esters (7a and 7b).
Supporting Information Available: 1H, 13C, and 2D NMR spectra
of 1–5, H NMR data for the MTPA esters 6a and 6b, LC/MS traces
of CNS-237, and additional discussion about the relative configurations
of 1. This material is available free of charge via the Internet at http://
pubs.acs.org.
1
References and Notes
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Bis-S-MTPA ester of 1 (6a): 1H NMR (500 MHz, CD3OD) δ 0.94
(3H, d, J ) 7.0 Hz), 0.95 (3H, t, J ) 7.5 Hz), 1.67 (1H, m), 1.72 (1H,
m), 1.79 (3H, d, J ) 1.0 Hz), 1.80 (3H, s), 1.82 (3H, s), 2.95 (1H, m),
3.53 (3H, s), 3.66 (3H, s), 5.01 (1H, m), 5.59 (1H, d, J ) 10.0 Hz),
6.28 (1H, d, J ) 15.5 Hz), 6.97 (1H, d, J ) 15.5 Hz), 7.34–7.41 (3H,
m), 7.50–7.54 (5H, m), 7.66–7.68 (2H, m).
Bis-R-MTPA ester of 1 (6b): 1H NMR (500 MHz, CD3OD) δ 0.83
(3H, t, J ) 7.5 Hz), 1.03 (3H, d, J ) 7.0 Hz), 1.60 (1H, m), 1.67 (1H,
m), 1.80 (3H, s), 1.84 (3H, s), 1.86 (3H, d, J ) 1.0 Hz), 3.00 (1H, m),
3.50 (3H, s), 3.66 (3H, s), 5.01 (1H, m), 5.69 (1H, d, J ) 10.0 Hz),
6.38 (1H, d, J ) 15.5 Hz), 7.08 (1H, d, J ) 15.5 Hz), 7.37–7.42 (3H,
m), 7.48–7.55 (5H, m), 7.65–7.70 (2H, m).
Bis-S-MTPA ester of 2 (7a): 1H NMR (500 MHz, CD3OD) δ 0.94
(3H, t, J ) 7.5 Hz), 0.94 (3H, d, J ) 7.0 Hz), 1.66 (1H, m), 1.77 (1H,
m), 1.79 (3H, s), 1.80 (3H, s), 1.82 (3H, d, J ) 1.0 Hz), 3.12 (1H, m),
3.53 (3H, s), 3.66 (3H, s), 5.01 (1H, m), 5.46 (1H, d, J ) 10.5 Hz),
6.35 (1H, d, J ) 15.5 Hz), 7.33–7.45 (3H, m), 7.43 (1H, m), 7.48–7.55
(5H, m), 7.64–7.71 (2H, m).
(9) Jensen, P. R.; Williams, P. G.; Oh, D.-C.; Zeigler, L.; Fenical, W.
Appl. EnViron. Microbiol. 2007, 73, 1146–1152.
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Bis-R-MTPA ester of 2 (7b): 1H NMR (500 MHz, CD3OD) δ 0.90
(3H, t, J ) 7.5 Hz), 1.05 (3H, d, J ) 7.0 Hz), 1.60 (1H, m), 1.69 (1H,
m), 1.81 (3H, s), 1.86 (3H, s), 1.89 (3H, d, J ) 1.0 Hz), 3.17 (1H, m),
3.48 (3H, s), 3.66 (3H, s), 5.03 (1H, m), 5.54 (1H, d, J ) 11.0 Hz),
6.46 (1H, d, J ) 16.0 Hz), 7.37–7.43 (3H, m), 7.47–7.56 (5H, m),
7.49 (1H, d, m), 7.65–7.71 (2H, m).
Duplicate (1 mg) aliquots of alcohol 5, from pacificanone B (4), were
treated with R- and S-MTPA chloride in the same manner as shown above.
Bis-S- and R-MTPA esters (8a and 8b) of 5 (0.3 and 0.2 mg, respectively)
were purified by reversed-phase HPLC (Alltech C18 semipreparative
column, 10 mm × 250 mm, 2 mL/min, UV 230 nm detection, 20%
aqueous CH3CN gradient from 0 to 10 min, 20–100% CH3CN from 10 to
50 min, 100% CH3CN isocratic after 50 min) at the retention times at
63.9 and 62.9 min, respectively. ESI-LC/MS analysis resolved the bis-
MTPA esters (C40H50F6O7) at [M + Na]+ m/z 779.
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Bis-S-MTPA ester of 5 (8a): 1H NMR (500 MHz, CD3OD) δ 0.79
(3H, t, J ) 6.5 Hz), 0.87 (3H, d, J ) 7.5 Hz), 0.88 (3H, d, J ) 7.5
Hz), 0.89 (3H, t, J ) 6.5 Hz), 0.90 (3H, d, J ) 6.5 Hz), 1.60 (1H, m),
1.61 (1H, m), 1.62 (2H, m), 1.64 (2H, m), 1.74 (3H, s), 1.85 (1H, m),
2.02 (1H, m), 2.89 (1H, m), 3.51 (3H, s), 3.57 (3H, s), 5.00 (1H,
NP0705155