Cystargolides, 20S proteasome inhibitors isolated from Kitasatospora cystarginea

Article abstract of DOI:10.1021/np501060k

Two novel β-lactone-containing natural products, cystargolides A (1) and B (2), were isolated from the actinomycete Kitasatospora cystarginea. The production of these two natural products was highlighted using a methodology associating liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis and the statistical analysis tool principal component analysis (PCA). Their structures were elucidated by interpretation of NMR experiments and tandem mass spectrometry. The absolute configurations of the amino acid residues were determined using Marfey's method, and the relative configurations of the β-lactone substituents were determined on the basis of the vicinal ³J_HH coupling value. Due to the presence of the β-lactone, 1 and 2 were evaluated for their ability to inhibit the human 20S proteasome. 1 and 2 both inhibited the 20S proteasome in vitro with IC₅₀ values of 0.35 and 0.93 μM, respectively.

Full text of DOI:10.1021/np501060k

Article
pubs.acs.org/jnp
Cystargolides, 20S Proteasome Inhibitors Isolated from Kitasatospora
cystarginea
Krista A. Gill,† Fabrice Berrue,†,‡ Jennifer C. Arens,§ Gavin Carr,‡ and Russell G. Kerr*,†,‡,§
́
†Department of Chemistry, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3
‡Nautilus Biosciences Canada Inc., DRC 550 University Avenue, Charlottetown, PE, Canada C1A 4P3
§Department of Biomedical Sciences, Atlantic Veterinary College, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3
S
* Supporting Information
ABSTRACT: Two novel β-lactone-containing natural products, cystargolides A (1) and B (2), were isolated from the
actinomycete Kitasatospora cystarginea. The production of these two natural products was highlighted using a methodology
associating liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis and the statistical analysis tool
principal component analysis (PCA). Their structures were elucidated by interpretation of NMR experiments and tandem mass
spectrometry. The absolute configurations of the amino acid residues were determined using Marfey’s method, and the relative
configurations of the β-lactone substituents were determined on the basis of the vicinal 3JHH coupling value. Due to the presence
of the β-lactone, 1 and 2 were evaluated for their ability to inhibit the human 20S proteasome. 1 and 2 both inhibited the 20S
proteasome in vitro with IC50 values of 0.35 and 0.93 μM, respectively.
ctinomycetes have been described as the most prolific
source of bioactive microbial natural products, making
Japan, and was reported to produce the antifungal peptide
cystargin.10,11 It has also recently been reported to produce the
cyclic lipopeptide natural product cystargamide.12 The
cystargolides belong to the β-lactone-containing class of natural
products, which are a broad class that have a variety of
biological activities.13 Tetrahydroxylipstatin (THL, Orlistat) is a
derivative of the natural product lipstatin and is a β-lactone-
containing compound that is approved by the FDA as a
pancreatic lipase inhibitor used to treat obesity.14a,b Other
members of this class include belactosin A, salinosporamide A,
and omuralide, which are all reported to have protease
inhibitory activity.15−18 The ubiquitin−proteasome pathway
plays a major role in eukaryotic cellular protein degradation.19
This system adjusts the level of proteins involved in regulating
cellular processes such as signal transduction, immune
responses, and cell cycle progression.20−22 Inhibitors of the
proteasome have gained attention for their ability to block cell
cycle progression and cause apoptosis, which makes them
useful antiproliferative agents.23 Bortezomib and carfilzomib are
A
them a valuable resource for the discovery of new secondary
metabolites.1−3 Members of the genus Kitasatospora, which are
classified as rare Actinobacteria, have been shown to produce a
wide variety of natural products including bafilomycins,
kitasetaline, and more recently satosporins.4−6 Members of
this rare and underexplored genus have the potential to
produce multiple natural products, which was revealed by
genome sequencing of Kitasatospora setae, showing the
presence of 24 putative secondary metabolite gene clusters.7
This makes members of this genus ideal microorganisms to
investigate for their ability to produce structurally unique and
bioactive secondary metabolites. Consequently, 12 strains of
the genus Kitasatospora were fermented, and the chemical
composition of each extract was assessed using an LC-HRMS-
based metabolomics approach, in which principal component
analysis (PCA) allowed us to rapidly identify the presence of
putatively novel chemical entities.8,9 Using this approach,
cystargolides A and B were discovered from Kitasatospora
cystarginea.
K. cystarginea NRRL-B16505 is a soil-dwelling bacterium that
was originally isolated in 1988 from Yamaguchi Prefecture,
Received: December 29, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Prioritization of K. cystarginea B16505 based on LC-HRMS/PCA dereplication. PCA scores plot (PC-1 vs PC-2) showed replicates of K.
cystarginea (16505) as outliers from the other 11 strains. PCA loadings plot (PC-1 vs PC-2) showed that the presence of bucket values m/z
=357.2021, tR = 2.8 and m/z = 371.2177, tR = 3.0 are contributing to the observed variation between the chemical profiles.
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for 1
proteasome inhibitors that have been approved by the FDA for
treatment of multiple myeloma, and several other proteasome
inhibitors are currently in clinical trials.24,25 Herein is reported
the isolation and structure elucidation of two novel β-lactone-
containing natural products, cystargolides A (1) and B (2), that
exhibit promising inhibitory activity in a 20S proteasome assay.
To the best of our knowledge, the cystargolides are the first
examples of β-lactone-containing metabolites with a 3-
isopropyl-4-oxo oxetate-2-carboxyl moiety.
Recorded in DMSO-d6
δH (J in
1H−1H
HMBC
position
δC, type
Hz)
COSY
(H→C)
NOESY
1
2
172.8, C
56.4, CH
4.16, dd
(7.1,
7.1)
3, NHa
1, 3, 4, 5, 1′ 3, 4, NHa
3
4
5
36.0, CH
1.78, m
2, 4, 5
1, 2, 4, 5, 6
3, 5
2
15.3, CH3 0.86, m
24.5, CH2 1.43, m
1.19, m
3
3, 6
3, 6
5
2, 3, 4, 6
2, 3, 4, 6
3, 5
RESULTS AND DISCUSSION
■
Metabolomic Screening and Purification. Twelve
strains of Kitasatospora spp. were cultured in triplicate for 72
h in a nutrient lean medium.26 Fermentation broths were
extracted with ethyl acetate, and the resulting organic extracts
were analyzed by LC-HRMS. The data processing prior to
statistical analysis was performed according to the previously
described procedure.8 Principal component analysis was used to
discriminate the secondary metabolomes of each bacterial strain
and highlighted K. cystarginea NRRL-B16505 as a producer of
putatively novel natural products.12 The visualization of the
score plot PC-1 vs PC-2 rapidly indicated that the three
replicate extracts were ungrouping from the rest of the samples,
while the corresponding loadings plot revealed the variables
(retention time−m/z) responsible for the observed discrim-
ination in Figure 1.
6
10.9, CH3 0.82, m
NHa
8.12, d
(8.0)
2
1, 2, 3, 1′
2, 5, 2′, 3′
1′
2′
170.5, C
57.2, CH
4.37, dd
(8.0,
8.0)
3′, NHb
1′, 3′, 4′,
5′, 1″
3′, NHa,
NHb, 4′
3′
30.7, CH
2.01, m
2′, 4′, 5′
1′, 2′, 4′
2′, 3′, 5′
2′, 3′, 4′
2′
4′
5′
18.8, CH3 0.87, m
17.4, CH3 0.81, m
3′
3′
2′
NHb
8.53, d
(9.0)
2′, 3′, 1″,
2′, 3′, 2″
2″
1″
2″
167.3, C
70.0, CH
5.01, d
(4.0)
3″
1″, 3″, 4″,
5″
NHb, 3″,
5″, 6″
3″
62.6, CH
3.50, dd
(4.1,
8.1)
2″, 5″
1″, 2″, 4″,
5′, 2″, 5″,
5″, 6″, 7″
7″
Two metabolites defined by the buckets (3.0 min, m/z
371.2177 [M + H]+) and (2.8 min, m/z 357.2021 [M + H]+)
were therefore identified as compounds solely produced by the
strain K. cystarginea (16505). Database searches (Anti-
Base2012)27 for the exact masses of these protonated adducts
returned no matches, which led to a further chemical
investigation of K. cystarginea metabolomes. A 3 L scale-up
fermentation was undertaken, culture broths were extracted
with ethyl acetate, and the resulting organic extract was
fractionated by C18 liquid chromatography and further purified
using RP-HPLC with a phenyl hexyl stationary phase to yield
2.5 and 3.1 mg of cystargolides A (1) and B (2), respectively.
Structure Elucidation. Cystargolide A (1) was isolated as a
pale yellow powder, and HRESIMS supported a molecular
formula of C18H30N2O6 (m/z 371.2177 [M + H]+, Δ = 0.1
ppm), indicating five degrees of unsaturation. The NMR data
(Table 1) revealed the presence of four carbonyls accounting
for four of the five degrees of unsaturation in addition to two
amide proton signals, which suggested that compound 1 was a
peptide. Two amino acids were recognized as isoleucine and
4″
5″
169.8, C
26.4, CH
2.11, m
3″, 6″, 7″ 2″, 3″, 4″,
6″, 7″
5″
2″, 3″
6″
7″
19.0, CH3 1.00, d
(6.7)
3″, 5″, 7″
19.2, CH3 0.97, d
(6.7)
5″
3″, 5″, 6″
valine by interpretation of the 1H−1H COSY correlations
identifying the two peptidic spin systems NHa (δH 8.12) to H3-
6 (δH 0.82) and NHb (δH 8.53) to H3-5′ (δH 0.81).
In a similar manner, COSY correlations assigned the third
spin system H-2″ (δH 5.01) to H3-7″ (δH 0.97). Key HMBC
correlations between H-2″/C-1″ (δC 167.3), C-4″ (δC 169.8);
H-3″ (δH 3.50)/C-1″, C-4″; and H-5″ (δH 2.11)/C-4″
unambiguously located the two carbonyl functional groups at
positions C-1″ and C-4″, respectively. The deshielded
resonances at δH 5.01 (H-2″) and δC 69.9 (C-2″) suggested
that C-2″ was attached to an oxygen atom. The connectivity
B
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revealed a trans-substituted β-lactone (see the Supporting
Information).
20S Proteasome Inhibition. The presence of a β-lactone
ring in the structures of 1 and 2 suggested that these natural
products may be proteasome inhibitors, and so they were
evaluated for their ability to inhibit the human 20S proteasome
in an enzyme assay. Varying concentrations of 1 and 2 were
incubated with purified human 20S proteasome. To determine
the chymotrypsin-like activity, the fluorogenic substrate Suc-
LLVY-AMC was added, and fluorescence was recorded to
measure the rate of enzyme activity. A dose−response curve
was used to calculate IC50 values (Figure 2), which were
determined to be 0.36 0.017 and 0.93 0.032 μM for 1 and
2, respectively.
between the isoleucine, valine, and the third spin system was
determined based on the key HMBC correlations H-2 (δH
4.16), NHa/C-1 (δC 172.8); H-2, NHa, H-2′ (δH 4.37)/C-1′
(δC 170.5); H-2′, NHb, H-2″, H-3″/C-1″; and NHb/C-2″ (δC
70.0). NOESY data supported the amino acid sequence
assignment and showed strong correlations between NHa (δH
8.12)/H-2′ (δH 4.37) and NHb (δH 8.53)/H-2″ (δH 5.01),
which was also supported by tandem mass spectrometry and
the specific fragmentation pattern described in the Supporting
Information.
The fifth degree of unsaturation was attributed to cyclization.
Two options were therefore available: a macrolactone
cyclization between C-1 and the C-2″ hydroxy or a β-lactone
between C-4″ and the C-2″ hydroxy. The presence of an IR
absorption at 1835 cm−1 was characteristic of a β-lactone
carbonyl stretching band and therefore strongly supports
cyclization between C-4″ and the hydroxy at C-2″.28 The
lack of HMBC correlation between H-2″ and C-1 also
supported a β-lactone cyclization. The presence of the β-
lactone was further confirmed by the instability of 1 in mild
acidic conditions, resulting in polymerization through con-
densation between two or three monomers.29 To determine the
relative configuration at the C-2″ and C-3″ positions on the β-
Figure 2. Dose−response curve showing the effects on the rate of Suc-
LLVY-AMC cleavage by human 20S proteasome (AFU/s Ex 360 nm,
Em 460 nm) in the presence of increasing concentrations of
cystargolides A and B and 0.5 μM epoxomicin as a positive control.
No inhibitor and blank values show maximal and minimal rates of
substrate cleavage.
Cystargolide A (1) was observed to be a more potent
inhibitor than 2, suggesting that the additional methylene
results in a small change to the bulkiness of the peptide side
chain that increases the interaction with the 20S proteasome.
The ability of 1 and 2 to inhibit chymotrypsin-like activity of
the proteasome is not surprising given their similarity to the
natural products belactosins A and C. The β-lactone-containing
belactosins, A and C, inhibit chymotrypsin-like activity of the
rabbit 20S proteasome both with an IC50 value of 0.21 μM.16
This is similar to the value observed for 1 and slightly more
potent than 2. Several medicinal chemistry and structure
activity relationship studies of the belactosins have resulted in
synthetic analogues and hybrids with increased potency and cell
permeability, leading to improved compounds for the develop-
ment of antiproliferative agents.33−35 The clinically used agents
carfilzomib and bortezomib inhibit the proteasome in vitro with
an IC50 value of 5 nM and a Ki value of 0.6 nM,
respectively.24,25 The great potency of these compounds leads
to their development for clinical use, which suggests that the
cystargolides may benefit from further structure optimization to
increase their potency. The cystargolides represent excellent
leads for continued development as inhibitors of the ubiquitin−
proteasome pathway and cell proliferation.
3
3
lactone moiety, the JHH was used. A small JH2″‑H3″ of 3.8 Hz
revealed that H-2″ and H-3″ have a trans configuration; thus
the relative configuration of the ring was determined to be
(2R*, 3S*).30,31
The absolute configurations of the amino acids in
cystargolide A were determined using Marfey’s analysis.32
The peptides were hydrolyzed in HCl, derivatized using L-
FDAA, and compared to authentic derivatized amino acid
standards using LC-HRMS. The derivatized hydrolysate of 1
gave peaks at 28.8 and 34.0 min (see Supporting Information),
which corresponded to L-Ile (34.0 min) and L-Val (28.7 min).
Mosher’s method was attempted to determine the absolute
configuration of the β-lactone moiety after hydrolysis of
cystargolides with 1 N NaOH. Unfortunately, neither R nor S
Mosher’s derivatives were detected by LC-HRMS analysis,
which is most likely due to the instability of the β-lactone
moiety, leading to polycondensation of 1 and 2.
Cystargolide B (2) was isolated as a pale yellow powder, and
HRESIMS supported a molecular formula of C17H28N2O6 (m/z
357.2021 [M + H]+, Δ = 0.2 ppm), which suggested the
absence of one methylene group. The NMR spectra of 2 closely
resembled those of 1, while HSQC correlations revealed the
absence of two signals, δH 1.43 (H2-5a) and 1.19 (H2-5b),
which are in agreement with valine replacing isoleucine. The
interpretation of COSY, HMBC, and tandem mass spectrom-
etry data further confirmed these observations and indicates the
replacement of the isoleucine with a second valine residue.
Marfey’s analysis indicated that both valines possess an L-
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Rudolph Autopol III polarimeter using a 50 mm
microcell (1 mL). Infrared spectra were recorded using attenuated
total reflectance, with samples deposited as a thin film on a Bruker
Alpha FT-IR spectrometer. NMR spectra were obtained on a 600
MHz Bruker Avance III spectrometer equipped with a 1.7 mm inverse
3
configuration, and the JHH coupling constants similarly
C
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probe. Chemical shifts (δ) are reported in ppm and were referenced to
the DMSO-d6 residual peaks at δH 2.50 ppm and δC 39.51 ppm, and
coupling constants (J) are reported in Hz with the abbreviations (s)
singlet, (d) doublet, (t) triplet, (q) quartet, and (m) multiplet. LC-
HRMS data were recorded on Accela Thermo equipment with
hyphenated MS-ELSD-UV detection: Thermo LTQ Exactive fitted
with ESI source, PDA, and LT-ELSD Sedex 80. High-resolution mass
spectra were measured on a Thermo Orbitrap Velos mass
spectrometer. MS/MS analysis was conducted in negative mode by
direct infusion of cystargolides A and B at a rate of 2 μL/min using an
ESI source and a collision-induced dissociation energy of 35 eV.
HPLC purifications were carried out on a Thermo Surveyor coupled
with a Sedex 55 evaporative light-scattering detector. L-Valine, L/D-
valine, L-isoleucine, L/D isoleucine, L-allo-isoleucine, and D-allo-
isoleucine were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Small-Scale Fermentations and Statistic Analysis. K.
cystarginea was obtained from the agriculture research service culture
collection (NRRL B-16505) along with 11 other organisms classified
as Kitasatospora spp. in the collection. Two seed cultures of each
organism were fermented in a seed medium containing 10 g of glucose
and 10 g of yeast extract per liter for 48 h, each at 200 rpm and 30 °C.
A 10 mL portion of a lean production medium containing 1.6 g/L
dextrin, 0.8 g/L galactose, 0.8 g/L maltose, 0.8 g/L bacto-soytone, 0.4
g/L glucose, and 0.3 g/L ammonium sulfate was then inoculated in
triplicate with 750 μL of seed culture and fermented for 72 h under the
same conditions.26 Medium blanks were also included as negative
controls. The cultures were then extracted twice with 10 mL of ethyl
acetate (EtOAc), and the organic fractions were combined and
evaporated to dryness. A 10 μL (0.5 mg/mL in MeOH) amount of the
fermentation extracts was analyzed by LC-HRMS using a Thermo
LTQ Exactive HPLC system with a Core Shell 100 Å C18 column
(Kinetex, 1.7 μm 50 × 2.1 mm). A linear solvent gradient from 95%
diH2O 0.1% formic acid (solvent A) and 5% acetonitrile 0.1% formic
acid (solvent B) to 100% solvent B over 4.8 min followed by an
isocratic elution at 100% solvent B for 3.2 min with a flow rate of 500
μL/min was used. Eluent was detected by ESIMS monitoring at m/z
190−2000 in positive mode, ELSD, and 200−600 nm UV. LC-HRMS
profiles were analyzed using principal component analysis and cluster
analysis as previously described with the omission of intensity
standardization.8 In brief, mzMine 2 was used for peak picking of
LC-HRMS profiles, set with an intensity threshold of 1E4, followed by
deisotoping, bucketing alignment, artifact suppression, and statistical
analysis using The Unscrambler (Camo Software).
1H and 13C NMR see Table 1; (+) HRESIMS m/z 371.2177 [M +
H]+ (calcd for C18H31N2O6, 371.21766).
Cystargolide B (2): pale yellow powder; [α]25 −28 (c 0.12,
D
MeOH); IR νmax 3299, 2964, 1838, 1723, 1648, 1529, 1467, 1157,
1
1024 cm−1; H NMR (600 MHz, DMSO-d6) δH 8.53 (NHb, d, 9.0),
8.11 (NHa, d, 8.1), 5.01 (H-2″, d, 3.8), 4.38 (H-2′, dd 7.7, 7.7), 4.12
(H-2, dd, 6.6, 6.6), 3.51 (H-3″, dd, 8.1, 3.9), 2.12 (H-5″, m), 2.06 (H-
3, m), 2.01 (H-3′, m), 1.00 (H3-6″, d, 6.7), 0.97 (H3-7″, d, 6.5), 0.89
(H3-4, m), 0.88 (H3-5, m), 0.86 (H3-4′, m), 0.82 (H3-5′, m); 13C
NMR (150 MHz, DMSO-d6) δC 172.5 (C, C-1), 170.5 (C, C-1′),
169.7 (C, C-4″), 167.0 (C, C-1″), 69.9 (CH,C-2″), 62.5 (CH, C-3″),
57.2 (CH, C-2′), 57.2 (CH, C-2), 30.7 (CH, C-3′), 29.4 (CH, C-3),
26.4 (CH, C-5″), 19.3 (CH3, C-7″), 19.2 (CH3, C-6″), 18.9 (CH3, C-
4′), 17.6 (CH3, C-5′), 18.8 (CH3, C-4), 17.7 (CH3, C-5); (+)
HRESIMS m/z 357.2021 [M + H]+ (calcd for C17H29N2O6,
357.20201).
Stereochemical Analysis by Marfey’s Method. Amino acid
configurations were determined by Marfey’s analysis of hydrolyzed
cystargolides A (1) and B (2).30 To separate microconical vials, 20 μL
of 1 and 2 (10 mg/mL in MeOH) were added then dried. HCl (250
μL of 6M) was added to each vial along with a stir bar and heated to
70−80 °C for 75 min. Once the reaction mixtures had cooled, 1 mL of
1 N NaHCO3 followed by 20 μL of 1-fluoro-2,4-dinitrophenyl-L-
alanine (L-FDAA) (10 mg/mL in acetone) were added to each
reaction vial. The reactions were heated at 30−40 °C for 1 h before
quenching with 100 μL of 6 M HCl. The reaction mixture was reduced
in volume under air, then diluted to 1 mL with 50:50 MeOH/H2O for
LCMS analysis. Derivatized amino acids (10 μL) were analyzed by LC-
HRMS with a Hypersil Gold 100 Å column (Thermo, 1.9 μm C18 50
× 2.1 mm). Compounds were eluted using a linear gradient from 95%
diH2O 0.1% formic acid (solvent A) and 5% acetonitrile 0.1% formic
acid (solvent B) to 60% solvent A and 40% solvent B over 55 min
followed by a rapid increase to 100% solvent B over 2 min, then a hold
for 3 min. A flow rate of 400 μL/min was used. Eluent was detected by
ESIMS monitoring m/z 120−800 in positive mode and UV (200−600
nm). Retention times were compared to those of authentic derivatized
standards to determine the amino acid configurations. Retention times
of derivatized standards were as follows: L-Val 28.70 min, D-Val 35.03
min, L-Ile 34.02 min, D-Ile 40.44 min, L-allo-Ile 34.27 min, and D-allo-
Ile 40.52 min.
20S Proteasome Inhibition Assay. 1 and 2 were tested for
proteasome inhibition using purified human erythrocyte 20S
proteasome (Enzo Life Sciences: BML-PW8720-0020). The 20S
proteasome was diluted to a final concentration of 3 μg/mL in assay
buffer (50 mM Tris/HCl, pH 7.5, 25 mM KCl, 10 mM NaCl, 1 mM
MgCl2, and 0.03% SDS) and incubated with inhibitors at varying
concentrations at 30 °C for 10 min. To determine the chymotrypsin-
like activity, the reaction was initiated by the addition of the
fluorogenic substrate Suc-LLVY-AMC (Enzo Life Sciences, BML-
9802-9090) at a final concentration of 75 μM. The rate of cleavage of
the substrate was determined by measuring the fluorescence using a
Spectra Max M2 (Molecular Devices) plate reader at an excitation
wavelength of 360 nm and emission of 460 nm. The fluorescence was
recorded every 15 s for 30 min, and the linear regression between 15
and 30 min was used to calculate the rate of substrate cleavage (AFU/
s). Control wells were included that contained no inhibitor to show
the maximum substrate cleavage rate, 0.5 μM epoxomicin (IC50 = 4
nM)36 as a positive control, and no enzyme (blank) to show the
minimum response. The IC50 values, the concentration required to
reduce the enzyme response by 50%, were calculated by Prism 6.0
(GraphPad software) using a nonlinear regression dose−response,
Extraction and Isolation. Two seed cultures of K. cystarginea
NRRL-B16505 were fermented as previously described for 48 h each,
at 200 rpm and 30 °C. A 3 L amount of the lean production medium
(4 × 750 mL) was then fermented with a 6.6% inoculum from seed
cultures, for 72 h under the same conditions.26 Each fermentation was
extracted with 500 mL and then 300 mL of EtOAc, which were then
partitioned twice with equal volume of diH2O. After evaporation, the
combined extract was dissolved in 10 mL of 80% aqueous acetonitrile
and partitioned with an equal volume of hexanes. The acetonitrile
fraction was separated by reversed-phase flash chromatography using
CombiFlash Rf (Teledyne ISCO) with a 15.5 g C18 column (High
Performance GOLD, RediSep Rf) and eluted with a linear gradient
from 50:50 diH2O/MeOH to 100% MeOH over 15 min followed by
100% MeOH for 5 min. A flow rate of 30 mL/min was used, and
eluent was detected by UV (214 nm). A mixture of 1 and 2 eluted at
6.0−8.5 min.
Cystargolides were purified using a Luna 110 Å phenyl hexyl
column (5 μm, 250 × 10.00 mm, Phenomenex). diH2O 0.1% formic
acid (solvent A) and MeOH 0.1% formic acid (solvent B) were used
with a flow rate of 3 mL/min. The mixture was separated using a linear
gradient increasing from 50% solvent B to 80% solvent B over 17 min,
followed by a linear increase to 100% solvent B over 2 min and 100%
solvent B for 12 min. 1 and 2 eluted at 10.2 and 12.1 min, respectively,
which were detected by ELSD and UV (220 and 254 nm).
variable slope model based on triplicate measurements
deviation.
standard
ASSOCIATED CONTENT
■
S
* Supporting Information
Cystargolide A (1): pale yellow power; [α]25D −18 (c 0.29, MeOH);
IR νmax 3294, 2962, 1835, 1719, 1646, 1534, 1465, 1201, 1025 cm−1;
HRMS, NMR, IR spectra and Marfey’s analysis chromato-
graphs are included in the Supporting Information. This
D
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(22) King, R. W.; Deshaies, R. J.; Peters, J. M.; Kirschner, M. W.
Science 1996, 274, 1652−1659.
material is available free of charge via the Internet at http://
pubs.acs.org.
(23) Adams, J. Nat. Rev. Cancer 2004, 4, 349−360.
(24) Paramore, A.; Frantz, S. Nat. Rev. Drug Discovery 2003, 2, 611−
612.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 902 566 0565. Fax: +1 902 566 7445. E-mail: rkerr@
upei.ca.
■
(25) Kisselev, A. F.; van der Linden, W. A.; Overkleeft, H. S. Chem.
Biol. 2012, 19, 99−115.
(26) Tamamura, T.; Sawa, T.; Isshiki, K.; Masuda, T.; Homma, Y.;
Iinuma, H.; Naganawa, H.; Takeuchi, T.; Umezawa, H. J. Antibiot.
1985, 38, 1664−1669.
Notes
The authors declare no competing financial interest.
(27) Laatsch, H. AntiBase Version 4.0-The Natural Compound
Identifier; Wiley-VCH Verlag GmbH and Co. KGaA, 2012.
(28) Pommier, A.; Pons, J. M. Synthesis 1992, 5, 441−459.
(29) Hall, H. K. J. Macromolecules 1969, 2, 488−498.
(30) Riccio, R.; Bifulco, G.; Cimino, P.; Bassarello, C.; Gomez-
Paloma, L. Pure Appl. Chem. 2003, 75, 295−308.
(31) Hill, E. A.; Roberts, J. D. J. Am. Chem. Soc. 1967, 89, 2047−
2049.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge financial support from
Natural Sciences and Engineering Council of Canada
(NSERC), the Canada Research Chair Program, the University
of Prince Edward Island, the Atlantic Innovation Fund,
Nautilus Biosciences Canada Inc., and the Jeanne and Jean-
(32) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
(33) Kawamura, S.; Unno, Y.; List, A.; Mizuno, A.; Tanaka, M.;
Sasaki, T.; Arisawa, M.; Asai, A.; Groll, M.; Shuto, S. J. Med. Chem.
2013, 56, 3689−3700.
́
Louis Levesque Foundation. The authors also acknowledge
NMR services provided by Dr. C. Kirby and M. Fischer
(AAFC).
(34) Korotkov, V. S.; Ludwig, A.; Larionov, O. V.; Lygin, A. V.; Groll,
M.; de Meijere, A. Org. Biomol. Chem. 2011, 9, 7791−7798.
(35) Nakamura, H.; Watanabe, M.; Ban, H. S.; Nabeyama, W.; Asai,
A. Bioorg. Med. Chem. Lett. 2009, 19, 3220−3224.
(36) Kim, K. B.; Myung, J.; Sin, N.; Crews, C. M. Bioorg. Med. Chem.
Lett. 1999, 9, 3335−3340.
REFERENCES
(1) Berdy, J. J. Antibiot. 2005, 58, 1−26.
(2) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335.
(3) Demain, A. L.; Sanchez, S. J. Antibiot. 2009, 62, 5−16.
(4) Werner, G.; Hagenmaier, H.; Drautz, H.; Baumgartner, A.;
■
Zahner, H. J. Antibiot. (Tokyo) 1984, 37, 110−117.
̈
(5) Aroonsri, A.; Kitani, S.; Ikeda, H.; Nihira, T. J. Biosci. Bioeng.
2012, 114, 56−58.
(6) Arens, J. C.; Berrue,
15, 3864−3867.
́
F.; Pearson, J. K.; Kerr, R. G. Org. Lett. 2013,
(7) Ichikawa, N.; Oguchi, A.; Ikeda, H.; Ishikawa, J.; Kitani, S.;
Watanabe, Y.; Nakamura, S.; Katano, Y.; Kishi, E.; Sasagawa, M.;
Ankai, A.; Fukui, S.; Hashimoto, Y.; Kamata, S.; Otoguro, M.;
Tanikawa, S.; Nihira, T.; Horinouchi, S.; Ohnishi, Y.; Hayakawa, M.;
Kuzuyama, T.; Arisawa, A.; Nomoto, F.; Miura, H.; Takahashi, Y.;
Fujita, N. DNA Res. 2010, 17, 393−406.
́
(8) Forner, D.; Berrue, F.; Correa, H.; Duncan, K.; Kerr, R. G. Anal.
Chim. Acta 2013, 805, 70−9.
(9) Hou, Y.; Braun, D. R.; Michel, C. R.; Klassen, J. L.; Adnani, N.;
Wyche, T. P.; Bugni, T. S. Anal. Chem. 2012, 84, 4277−4283.
(10) Kusakabe, H.; Isono, K. J. Antibiot. (Tokyo) 1988, 41, 1758−
1762.
(11) Uramoto, M.; Itoh, Y.; Sekiguchi, R.; Shin-ya, K.; Kusakabe, H.;
Isono, K. J. Antibiot. (Tokyo) 1988, 41, 1763−1768.
(12) Gill, K. A.; Berrue, F.; Arens, J. C.; Kerr, R. G. J. Nat. Prod. 2014,
77, 1372−1376.
(13) Bottcher, R.; Sieber, S. A. Med. Chem. Commun. 2012, 3, 408−
417.
(14) (a) Weibel, E. K.; Hadvary, P.; Hochuli, E.; Kupfer, E.;
Lengsfeld, H. J. Antibiot. 1987, 40, 1081−1085. (b) Guerciolini, R. Int.
J. Obes. Relat. Metab. Disord. 1997, 21 (Suppl. 3), S12−S23.
(15) Asai, A.; Hasegawa, A.; Ochiai, K.; Yamashita, Y.; Mizukami, T.
J. Antibiot. (Tokyo) 2000, 53, 81−83.
(16) Asai, A.; Tsujita, T.; Sharma, S. V.; Yamashita, Y.; Akinaga, S.;
Funakoshi, M.; Kobayashi, H.; Mizukami, T. Biochem. Pharmacol.
2004, 67, 227−234.
(17) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Angew. Chem. 2003, 42, 355−357.
(18) Umezawa, H.; Aoyagi, T.; Uotani, K.; Hamada, M.; Takeuchi,
T.; Takahashi, S. J. Antibiot. 1980, 33, 1594−1596.
(19) Orlowski, M. Biochemistry 1990, 29, 10289−10297.
(20) Fuchs, S. Y. Cancer Biol. Ther. 2002, 1, 337−341.
(21) Muchamuel, T.; Baslet, M.; Aujay, M. A.; Suzuki, E.; Kalim, K.
W.; Lauer, C.; Sylvain, C.; Ring, E. R.; Shields, J.; Jiang, J.; Shwonek,
P.; Parlati, F.; Demo, S. D.; Bennett, M. K.; Kirk, C. J.; Groettrup, M.
Nat. Med. 2009, 15, 781−787.
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DOI: 10.1021/np501060k
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