Isolation, structure elucidation, and biological activity of altersolanol p using staphylococcus aureus fitness test based genome-wide screening

Article abstract of DOI:10.1021/np400759f

Bacteria continue to evade existing antibiotics by acquiring resistance by various mechanisms, leading to loss of antibiotic effectiveness. To avoid an epidemic from infections of incurable drug-resistant bacteria, new antibiotics with new modes of action are desperately needed. Using a genome-wide mechanism of action-guided whole cell screening approach based on antisense Staphylococcus aureus fitness test technology, we report herein the discovery of altersolanol P (1), a new tetrahydroanthraquinone from an unknown fungus from the Hypocreales isolated from forest litter collected in Puerto Rico. The structure was elucidated by high-resolution mass spectrometry and 2D NMR spectroscopy. Relative stereochemistry was established by NOESY correlations, and absolute configuration was deduced by the application of MPA ester-based methodology. Observed ¹H and ¹³C NMR shifts were well aligned with the corresponding chemical shifts predicted by DFT calculations. Altersolanol P exhibited Gram-positive antibacterial activity (MIC range 1-8 μg/mL) and inhibited the growth of Gram-negative Haemophilus influenzae (MIC 2 μg/mL). The isolation, structure elucidation, and antibacterial activity of altersolanol P are described.

Full text of DOI:10.1021/np400759f

Article
pubs.acs.org/jnp
Isolation, Structure Elucidation, and Biological Activity of
Altersolanol P Using Staphylococcus aureus Fitness Test Based
Genome-Wide Screening
John Ondeyka,†,§ Alexei V. Buevich,† R. Thomas Williamson,† Deborah L. Zink,† Jon D. Polishook,†
James Occi,† Francisca Vicente,‡,⊥ Angela Basilio,‡,⊥ Gerald F. Bills,‡,∥ Robert G. K. Donald,†,▽
John W. Phillips,†,○ Michael A. Goetz,†,§ and Sheo B. Singh*,†
†Merck Research Laboratories, Rahway, New Jersey 07065 and Kenilworth, New Jersey 07033, United States
‡CIBE, Merck Sharp & Dohme de Espana, S. A. Josefa Valcar
́
cel, Madrid, Spain
S
* Supporting Information
ABSTRACT: Bacteria continue to evade existing antibiotics by
acquiring resistance by various mechanisms, leading to loss of
antibiotic effectiveness. To avoid an epidemic from infections of
incurable drug-resistant bacteria, new antibiotics with new modes
of action are desperately needed. Using a genome-wide
mechanism of action-guided whole cell screening approach
based on antisense Staphylococcus aureus fitness test technology,
we report herein the discovery of altersolanol P (1), a new tetrahydroanthraquinone from an unknown fungus from the
Hypocreales isolated from forest litter collected in Puerto Rico. The structure was elucidated by high-resolution mass
spectrometry and 2D NMR spectroscopy. Relative stereochemistry was established by NOESY correlations, and absolute
1
configuration was deduced by the application of MPA ester-based methodology. Observed H and 13C NMR shifts were well
aligned with the corresponding chemical shifts predicted by DFT calculations. Altersolanol P exhibited Gram-positive
antibacterial activity (MIC range 1−8 μg/mL) and inhibited the growth of Gram-negative Haemophilus influenzae (MIC 2 μg/
mL). The isolation, structure elucidation, and antibacterial activity of altersolanol P are described.
from different geographical regions and habitat (including
marine microbes) in combination with improved high-
throughput fermentation methods.3 These approaches led to
the discovery of a series of natural products4−6 including
platensimycin7,8 and platencin9,10 using an fabH/fabF-sensi-
tized strain and a number of other new natural products using
an rpsD-sensitized antisense strain.11−15
In the past few years, we have extended our antisense-based
screening approach to include a Staphylococcus aureus fitness
test (SaFT), in which 245 antisense-containing S. aureus strains
are used simultaneously to elucidate the mechanism of action of
antibacterial compounds.16 As described earlier, this approach
allows the grouping of compounds and crude natural product
extracts based on SaFT profile comparison in the SaFT profile
database,16,19 which when combined with high resolving power
accurate mass measurement Fourier transform ion cyclotron
resonance mass spectrometry (FTICR MS) provides a robust
dereplication tool,17,18 as illustrated by the discovery of
kibdelomycin and kibdelomycin A.19,20
ntibiotic-resistant bacteria are ever more common and
Apose serious threats. Infections caused by methicillin-
resistant Staphylococcus aureus (MRSA) alone are responsible
for about 18 000 deaths per year in the United States.1 While
the MRSA report has drawn significant attention, infections by
a number of drug-resistant Gram-negative bacteria such as
Pseudomonas aeruginosa and Acinetobacter baumannii are
perhaps even more alarming. Incremental improvements in
the lead classes of antibiotics discovered during the Golden Age
of antibiotic discovery provide most of the currently used
antibiotics. Further progress to improve existing antibiotics is
proving to be challenging, and returns on investment in
research have significantly diminished. The best way to alleviate
this problem is through the discovery of new lead classes of
antibacterial agents. Natural products are prolific sources of
antibacterial leads. One of the biggest challenges in natural
products discovery is how to avoid rediscovery of known
compounds and therefore improve the odds for success. In
order to address this challenge, new screening technologies and
new sources of natural products are needed. We have
attempted to address both of these questions through two
approaches. First, we introduced an S. aureus target-based
antisense whole-cell screening approach that utilizes strains
hypersensitive to certain target-based inhibitors and allows for
differentiation of antibacterial agents at the crude extract stage.2
Second, we expanded our efforts to isolate microorganisms
Acetone extracts of fermentations of the fungal strain (F-
166,863) screened in the SaFT assay produced a distinct SaFT
Special Issue: Special Issue in Honor of Otto Sticher
Received: September 16, 2013
Published: January 15, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Article
profile that did not match other compounds in the profile
database. Bioassay-guided fractionation using a simple S. aureus
growth inhibition assay led to the isolation of altersolanol P (1),
a new member of the altersolanol family of compounds. The
isolation, structure elucidation by 2D NMR, mass spectral
analysis, DFT calculations, absolute configuration, and
antibacterial activity of altersolanol P are described.
eight carbons were assigned as quaternary (Table 1). Of these,
two were indicative of quinone-type carbonyls, one an
1
Table 1. H (600 MHz) and 13C (150 MHz) NMR
Assignment of Altersolanol P (1) in CDCl3
δC,
no.
type
CH2
ppm
δH in ppm, mult (J in Hz)
HMBC (H→C)
1
21.0 H1, 2.82, dddd (20.3, 8.2, 5.8, 2, 3, 9, 1a, 4a
2.3)
H1′, 2.62, dddd (20.3, 5.6,
2, 3, 9, 1a, 4a
5.5, 1.5)
2
CH2
30.9 H2, 2.06, dt (13.8, 5.6)
1, 3, 4, 11, 1a
H2′, 1.66, ddd (13.5, 8.2, 5.9) 1, 3, 4, 11, 1a
3
4
C
69.0
CH
69.6 4.26, s
1a, 4a, 2, 3, 10,
11
5
6
7
8
9
CH
CH
CH
C
119.2 7.62, m
136.4 7.61, m
124.6 7.26, m
161.5
7, 10, 9a
8, 10a
RESULTS AND DISCUSSIONS
■
The producing strain (F-166,863, Figure S1, Supporting
Information) was isolated from forest leaf litter collected at
Bosque de Tabanuco, Caribbean National Forest, in Puerto
Rico using a particle filtration method.21 In culture, the fungus
produced simple to sparsely branched phialidic conidiogenous
cells on the aerial hyphae, which in turn produced elliptical,
single-celled hyaline conidia, and therefore would fit the
definition of the form genus Acremonium. However, a revision
of generic concepts among fungi classified as Acremonium has
confirmed the long-recognized phylogenetic heterogeneity of
the genus and has designated A. alternatum as the type species
for the genus.22 Sequence homology searches in public
databases (www.fungalbarcoding.org, NCBI) indicated that
the fungus was unrelated to A. alternatum but instead exhibited
affinities to various genera in the Cordycipataceae, Clavicipi-
taceae, Ophiocordycipitaceae, Nectriaceae, and other families of
the Hypocreales. However, overall sequence homologies to
authentically named taxa were low, and any clear association
with a monophyletic lineage within the Hypocreales was weak.
The most similar D1−D2 regions of the 28S rDNA sequences
were those of Metacordyceps chlamydosporia (95% similarity).
For the intertranscribed spacer rDNA sequences, the most
similar matches were several Cosmospora species (88%
similarity). Such low similarity scores and its ambiguous
association with any known family within the relatively well-
studied Hypocreales indicate that this fungus may belong to a
yet uncharacterized phylogenetic lineage (see Figure S2,
Supporting Information).
The submerged production stage fermentation broth of this
fungal species was extracted with an equal volume of acetone
and was partitioned with EtOAc. Chromatographic separation
of the EtOAc extract by gel permeation on Sephadex LH20
followed by reverse-phase HPLC led to the isolation of
homogeneous altersolanol P (18 mg, 18 mg/L) as a gum.
Electrospray ionization (ESI) mass spectral analysis of 1
yielded m/z 297 [M + Na]+ and m/z 273 [M − H]+. ESI
FTICR MS analysis of 1 yielded a fragment ion at m/z
257.0807 due to loss of a molecule of water, [M + H]+ − H2O
with an accurate mass measurement providing the formula
C15H13O4. The UV spectrum of 1 showed absorption maxima
at 210 and 270 nm, and the IR spectrum showed absorption
bands for hydroxy groups (3400 cm−1) and a conjugated
ketone (1660 cm−1). The 13C NMR spectrum of 1 in CDCl3
showed 15 distinct resonances. Analysis of the DEPT spectrum
suggested the presence of a methyl, two methylene, and an
oxymethine group and three olefinic methines. The remaining
5, 8, 9a, 10a
C
189.8
10
C
185.9
1a
C
146.2
4a
C
142.4
9a
C
114.8
10a
11
C
132.0
CH3
25.4 1.34, s
2.89, brs
1, 2, 3, 4
3-OH
4-OH
8-OH
4.08, d (2.8)
11.98, s
3, 4
6, 7, 8, 9, 9a
oxygenated downfield-shifted olefinic carbon, and another an
oxygen-bearing aliphatic carbon. COSY correlations suggested
three contiguous aromatic methines. They also indicated that
both methylene moieties were coupled only to each other. The
oxygenated methine appeared as a singlet (δH 4.26, δC 69.6).
The methyl singlet (δH 1.34) showed HMBC correlations to C-
2 (δC 30.9), C-3 (δC 69.0), and C-4 (δC 69.6), thus establishing
the connectivity of the upfield C1−C4 carbons. H-4 exhibited
HMBC correlations to C-1a (δC 146.2), C-4a (δC 142.4), C-3,
and C-10 (δC 185.9), establishing connectivity to the central
ring. The HMBC correlations of H-5 to C-10; H-6 to C-8 (δC
161.5); H-7 to C-9a (δC 114.8); and H-1 to C-9 established the
tetrahydroanthraquinone structure 1 for altersolanol P.
To confirm resonance assignments of 1, proton and carbon
chemical shifts were analyzed by the density functional theory
(DFT) method with the MPW1PW91 functional and 6-
31G(d,p) basis set. Prior to chemical shift calculations, the
molecular geometry of 1 was optimized by the DFT
calculations at the B3LYP/6-31G(d) level of theory. All
quantum mechanical calculations in the present study were
carried out with the Gaussian 09 software package.23 Excellent
agreement between experimental and calculated chemical shifts
was confirmed by nearly ideal linear regressions described by
the 1.065 slope, 0.07 ppm intercept coefficient, 0.9970
1
correlation coefficient, and 0.27 ppm standard error for H
chemical shifts (Figure 1a) and by the 0.970 slope, 3.28 ppm
intercept coefficient, 0.9994 correlation coefficient, and 1.89
ppm standard error for 13C chemical shifts (Figure 1b).
Interestingly, the intramolecular hydrogen bond between the
phenolic OH at C-8 and the oxygen of the quinone carbonyl at
C-9 was well reproduced by the DFT calculations. Thus, the
predicted 13.02 ppm shift for the δH OH-8 proton and 187.5
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1
Figure 1. Comparison of the DFT-calculated (MPW1PW91/6-31G(d,p)) vs measured H (a) and 13C (b) chemical shifts for altersolanol P (1).
Figure 2. Conformational equilibrium of altersolanol P. Red arrows show NOEs used for stereochemical analysis. Analysis of trans-3JHH couplings
between the H-1 and H-2 protons (Table 2) predicted the 65:35 ratio of A to B.
ppm for the δC C-9 carbonyl carbon were in good agreement
with experimental values of 11.97 and 189.8 ppm, respectively.
In the case of no intramolecular hydrogen bond, the DFT-
calculated prediction for the C-8 hydroxyl δH is 4.8 ppm and
that for the C-9 carbonyl δC is 180.3 ppm. The only outlier in
the correlation plot of proton chemical shifts is the C-4
hydroxyl (δH 4.08 experimental vs δH 5.15 calculated). This
discrepancy can be explained on the basis of conformational
analysis, which is discussed below.
The relative stereochemistry between C-3 and C-4 was
established by 2D NOESY data. The cis-orientation of the H-4
and CH3-11 was confirmed by a strong NOE between the H-4
and H-2′ and between CH3-11 and H-2′ protons (Figure 2).
Simultaneous observation of long-range NOEs between the
CH3-11 and H-1′ and between the H-4 and H-2′ also suggested
that 1 undergoes fast conformational exchange between
conformations A and B with pseudoaxial conformations of H-
4 and CH3-11, respectively (Figure 2).
method was used at the B3LYP/6-311+G(d,p) level of theory.
Specifically, the unconstructed basis set with additionally
tightened s and d functions for calculating Fermi contact
components was used as previously described.24 Prior to J-
coupling calculations, the conformation geometries were
optimized by DFT at the B3LYP/6-31G(d) level of theory.
Then we applied least-squares minimization to find proba-
bilities pA and pB, which produced the best fit of both trans-3J-
couplings (Jcalc) to their corresponding experimental values
(Jexp). Relatively small, 0.17 Hz root-mean-square deviation of
that analysis ensured high accuracy of estimated pA and pB
values, whose 65:35 ratio is well consistent with the observed
NOEs. Results of a conformational analysis of 1 are
summarized in Table 2.
The discrepancy between experimental and calculated proton
chemical shifts of the C-4 hydroxyl is now explained by the
conformational equilibrium of 1. The correlation plots in Figure
1 were based on chemical shifts of the dominant conformation
A. Conformational equilibrium had little effect on chemical
shifts except for the C-4 hydroxyl. Thus, taking into
consideration the 65:35 ratio of conformations A and B and
the DFT-predicted chemical shift for OH-4 in A and B, 5.15
and 2.97 ppm, respectively, the average value of δH OH-4 is
estimated to be 4.39 ppm, which correlated much better with
the experimental observed value of 4.08 ppm (see complete
table of chemical shifts for conformations A and B (Supporting
Information)).
The ratio of conformations A and B in 1 was estimated from
the analysis of experimental trans-3JH1′,H2 and trans-3JH1,H2′
couplings. Each of these couplings is an average value that
can be described by eq 1:
Jexp = p JA + p JB
(1)
A
B
where Jexp is the experimentally observed J-coupling, JA and JB
are corresponding J-couplings in conformations A and B, and
pA and pB are probabilities of conformations A and B. Since
probabilities are normalized (pA + pB = 1), only one of them
need be determined. To calculate JA and JB couplings, the DFT
The absolute configuration of altersolanol P (1) was
determined thorough derivatization with (R)-(−)-methoxyphe-
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Table 2. Conformational Analysis of Altersolanol P Based on
Experimental and DFT-Calculated (B3LYP/6-311+G(d,p))
trans-3JHH Couplings
Table 3. In Vitro Antibacterial Activity and Spectrum of
a
Altersolanol P (MIC, μg/mL)
b
strain
strain no.
1
penicillin G
S. aureus Smith strain
MB2865
MB2865
4 (2)
>64
<0.06
0.12
S. aureus Smith strain 50% human
serum
c
S. pneumoniae
CL2883
CL2883
CL8516
MB964
8
4
<0.015
<0.015
4
d
S. pneumoniae
E. faecalis VSE
B. subtilis
1
a
2
<0.06
32
JA
JB
exptl
calcd
E. coli (wt)
MB 2884
MB 5746
MB 4572
>64
32
2
J
J
H1,H2′, Hz
H1′,H2, Hz
12.1
1.6
1.9
8.2
8.37
5.77
E. coli (envA/tolC)
H. influenzae
C. albicans
a
8
12.8
5.6
0.06
>64
b
RMSD, Hz
0.17
MY1055
8
pA:pB
65:35
b
MIC (minimum inhibitory concentration). All strains were tested in
cation-adjusted Mueller Hinton broth (CAMHB) medium, unless
mentioned otherwise, under Clinical Laboratory Standard Institute
a
b
Jcalc were calculated as pAJA + pBJB. Root-mean-square deviation
(RMSD) was calculated as (∑(Jcalc − Jexp)2/2)1/2
.
c
(CLSI) guidelines. CAMHB + 2.5% lysed horse blood medium.
d
nylacetic acid (MPA) using PS-carbodiimide, a solid-phase
resin-supported carbodiimide reagent (Biotage Inc. Charlotte,
NC, USA).25−28 The MPA derivative was chosen due to the
larger anisotropic chemical shift changes observed in secondary
alcohols relative to the more traditional MTPA derivative.29 In
addition, it has been demonstrated that a variable-temperature
NMR analysis protocol obviates the need of forming both
diastereomeric derivatives.30 Variable-temperature NMR anal-
ysis of derivative 2 and comparison with Riguera’s sp vs ap
conformational model29 revealed that the stereochemistry at C-
3 is R (Figure 3).
Isosensitet medium.
antibacterial activity against S. aureus (MIC 12.5−25 μg/mL)
and Pseudomonas aeruginosa (MIC 12.5−100 μg/mL).31,32
Biological Activity. Altersolanol P (2) showed inhibition of
growth of S. aureus with MIC values of 2−4 μg/mL (Table 3).
It inhibited the growth of Streptococcus pneumoniae with an
MIC value of of 4−8 μg/mL regardless of the medium used. It
showed strong activity against Bacillus subtilis (MIC 2 μg/mL)
and Enterococcus faecalis (MIC 1 μg/mL). Altersolanol P was
highly effective against Haemophilus influenzae (MIC 2 μg/mL)
but was inactive vs the other Gram-negative bacteria, e.g.,
Escherichia coli (MIC >64 μg/mL). This is due to either lack of
cell penetration or drug efflux as evidenced by the enhanced
susceptibility (MIC 30 μg/mL) against envA/tolC E. coli strain.
This strain of E. coli has been rendered more permeable and
reduced efflux by mutations both in the cell membrane and in
the efflux pump. The antibacterial activity of altersolanol K was
negatively affected by human serum. This compound also
inhibited the growth of Candida albicans (MIC 8 μg/mL),
indicating lack of selectivity for prokaryotic cells. Altersolanols
A−K with additional substitutions at C-1, C-2, C-4, and C-6
have been reported from various species of Alternaria with
In summary, we have described the discovery of a new
member of the altersolanol family of tetrahydroanthraquinones
by means of new assay technology. Altersolanol P is perhaps
one of the simpler members of the family and showed broad-
spectrum Gram-positive activity and activity against H.
influenzae, one of the Gram-negative bacteria.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation was
recorded with a Perkin-Elmer 241 polarimeter. UV spectra were
recorded on a Perkin-Elmer Lambda 35 spectrometer. IR spectra was
recorded with a Perkin-Elmer Spectrum One FT-IR spectropho-
tometer. All NMR spectra were recorded with a Varian Unity 500 (1H,
500 MHz; 13C, 125 MHz) spectrometer in CDCl3 using a 3 mm NMR
probe. Chemical shifts are reported in δ (ppm) using residual solvent
1
signals (δH 7.26 and δC 77.5 ppm). H, 13C, COSY, DEPT, HMQC,
and HMBC spectra were measured using standard Varian pulse
Figure 3. Variable-temperature 1H NMR data in CDCl3 for the (R)-MPA ester of altersolanol P acquired at (a) 300 K, (b) 282.5 K, (c) 273.8 K, and
(d) 260 K. The Δδ300K/260K values were −7.7, −22.5, and −22.6 ppb for H-2, H-2′, and CH3-11, respectively.
500
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sequences. Low resolving power nominal mass measurement spectral
data were recorded on an Agilent 1100 MSD with electrospray
ionization, and high resolving power accurate mass measurement
EIMS was obtained on a Thermo Finnigan LTQ-FTMS instrument.
An Agilent HP 1100 instrument was used for analytical HPLC.
Producing Organism. The producing strain (F-166,863) was
isolated from forest leaf litter collected at Bosque de Tabanuco,
Caribbean National Forest, in Puerto Rico using a particle filtration
method. Frozen stock cultures of F-166,863 in 10% glycerol (−80 °C)
broth dilution method as previously described.34 The cultures were
incubated at 37 °C for 20 h before activity was determined. MIC is
defined as the lowest concentration of antibiotic that inhibited visible
growth.
ASSOCIATED CONTENT
■
S
* Supporting Information
Photographs of the fungus, phylogenetic tree, H, 13C, COSY,
1
1
are maintained in the collection of Fundacion MEDINA. Total
́
NOESY, HSQC, and HMBC NMR spectra of 1, and H and
genomic DNA was extracted from mycelia grown on YM agar. The
28S rDNA fragments, containing D1−D2 and ITS regions, were PCR
amplified, and sequence alignments and phylogenetic analysis was
performed as previously described.18,33
HSQC NMR spectra of MPA ester 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Fermentation. The strain was fermented by inoculating several
agar plugs with mycelia into a seed flask (50 mL of medium in a 250
mL unbaffled flask). The formulation for the seed medium was as
follows (g/L, unless specified): corn steep liquor, 5.0; tomato paste,
40.0; oat flour, 10.0; glucose, 10.0; trace element mix, 10.0 mL per
liter; agar, 4.0; with pH adjusted to 6.8 with NaOH prior to
sterilization. The formulation for the trace element mix consisted of
(g/L) FeSO4·7H2O, 1.0; MnSO4·H2O, 1.0; CuCl2·2H2O, 0.025;
CaCl2, 0.1; H3BO3, 0.056; (NH4)6Mo6O24·4H2O, 0.019; ZnSO4·
7H2O, 0.2, prepared in 0.6 N HCl. The flasks were incubated at 22 °C
at 80% relative humidity and shaken on a rotary shaker at 220 rpm.
When the inoculum flasks had grown for 4 days, a 1 mL aliquot was
used to inoculate each flask of production medium (50 mL of medium
in a 250 Erlenmeyer flask). The production media formulation
consisted of (g/L) mannitol, 75.0; oat flour, 15.0; yeast extract, 5.0; L-
glutamic acid, 4.0; MES, 16.2. The pH was adjusted to 6.0 with NaOH
prior to sterilization. The flasks were incubated at 22 °C at 80%
relative humidity on a rotary shaker at 220 rpm for 14 days.
Corresponding Author
*E-mail: sheo.singh.215@gmail.com.
Present Addresses
§Natural Products Discovery Institute, 3805 Old Easton Road,
Doylestown, Pennsylvania 18902, USA.
⊥Fundacion
́ ́
MEDINA, Centro de Excelencia en Investigacion
́
de Medicamentos Innovadores en Andalucia, Avenida Con-
ocimiento 3, Parque Tecnologico Ciencias de la Salud, 18100
Armilla, Granada, Spain.
́
∥The Brown Foundation of Molecular Medicine, University of
Texas Health Science Center, Houston, Texas 77030, USA.
▽Pfizer Vaccine Research, 401 N. Middletown Road, Pearl
River, New York 10965, USA.
○Allen Institute for Brain Science, Seattle, Washington 98103,
USA.
Isolation of Altersolanol P (1). One liter of fermentation broth was
extracted with 1 L of acetone by shaking on a reciprocating shaker for
1 h and subsequently filtering through a Celite plug. The acetone was
removed under reduced pressure, and the remaining aqueous residue
was extracted with 220 mL of ethyl acetate. The organic extract was
concentrated under reduced pressure to give 100 mg of gum, which
was dissolved in 3 mL of MeOH and charged to an 80 cm3 Sephadex
LH20 column in MeOH. The active fractions were pooled and
concentrated to afford 62.5 mg of gum. This material was dissolved in
400 μL of a 1:1 MeOH/DMSO mixture and chromatographed on a
Zorbax RX C8 column (21.2 × 250 mm) in two equal portions eluting
with a 35 min gradient of 10−80% aqueous CH3CN + 0.1% TFA at a
flow rate of 12 mL/min. Fractions eluting at 15−16 min from each run
were pooled and lyophilized to yield 18 mg (18 mg/L) of altersolanol
P (1) as an orange gum.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge generous help from Tom J. Novak
for obtaining accurate mass data on the MPA-ester, Gary E.
Martin for proofreading the manuscript, and Jesus Martin for
initial dereplication.
Altersolanol P: [α]23 +10.0 (c 0.68, MeOH); UV (MeOH) λmax
D
DEDICATION
212 (log ε 4.30), 250 (4.06), 256 (4.06), 271 (4.11), 418 (3.72) nm;
IR (ZnSe) νmax 3400, 2971, 2934, 1660, 1638, 1615, 1576, 1456, 1418,
1364, 1331, 1284, 1242, 1177, 1058, 1031, 1007, 976, 931, 914 cm−1;
1H and 13C NMR data, see Table 1; ESIMS m/z 297 [M + Na]+ and
m/z 273 [M − H]+; HRESIFTMS m/z 257.0808 ([M + H]+ − H2O,
calcd for C15H12O4 + H, 275.0816).
■
Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich,
Switzerland, for his pioneering work in pharmacognosy and
phytochemistry.
REFERENCES
■
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501
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