Fusaristatin C, a Cyclic Lipodepsipeptide from Pithomyces sp. RKDO 1698

Article abstract of DOI:10.1021/acs.jnatprod.8b00787

A new cyclic lipodepsipeptide, fusaristatin C (1), was obtained from the fungus Pithomyces sp. RKDO 1698, which was isolated from the Caribbean octocoral Eunicea fusca. The 2D structure of fusaristatin C was elucidated using NMR spectroscopy and mass spectrometry, while the absolute configuration of the sole chiral amino acid residue (l-serine) was determined using Marfey's method. 3-Hydroxy-2,11-dimethyltetradecanoic acid (HDMT) was cleaved from 1, and the absolute configuration at the C-3 position was determined using Mosher's ester analysis. Subsequent J-based configuration analysis of 1 allowed for assignment of the C-2 configuration. Fusaristatin C exhibited no antimicrobial activity or cytotoxicity.

Full text of DOI:10.1021/acs.jnatprod.8b00787

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Fusaristatin C, a Cyclic Lipodepsipeptide from Pithomyces sp. RKDO
1698
Logan W. MacIntyre,† Douglas H. Marchbank,‡,§ Hebelin Correa,§ and Russell G. Kerr*,†,‡,§
‡
†Department of Biomedical Sciences and Department of Chemistry, University of Prince Edward Island, 550 University Avenue,
Charlottetown, PE, Canada, C1A 4P3
§Nautilus Biosciences Croda, Regis and Joan Duffy Research Centre, 550 University Avenue, Charlottetown, PE, Canada, C1A 4P3
S
* Supporting Information
ABSTRACT: A new cyclic lipodepsipeptide, fusaristatin C
(1), was obtained from the fungus Pithomyces sp. RKDO
1698, which was isolated from the Caribbean octocoral
Eunicea fusca. The 2D structure of fusaristatin C was
elucidated using NMR spectroscopy and mass spectrometry,
while the absolute configuration of the sole chiral amino acid
residue (L-serine) was determined using Marfey’s method. 3-
Hydroxy-2,11-dimethyltetradecanoic acid (HDMT) was
cleaved from 1, and the absolute configuration at the C-3 position was determined using Mosher’s ester analysis. Subsequent
J-based configuration analysis of 1 allowed for assignment of the C-2 configuration. Fusaristatin C exhibited no antimicrobial
activity or cytotoxicity.
espite a long-standing recognition that marine environ-
from E. fusca. It was observed that when fermented in yeast
Dments harbor unique fungal taxa, organisms from these extract sucrose (YES) broth, the EtOAc extract of RKDO 1698
contained predominantly compound 1 in very high abundance.
Using UHPLC-HRMS, we observed a protonated molecule
(m/z 482.3219 [M + H]+) of 1 that after database queries
(AntiBase 2014) yielded no matches of fungal origin within a
10 ppm mass accuracy window.8 This strongly suggested that 1
was an unreported natural product; moreover, its abundance
and chromatographic resolution by UHPLC rendered it a good
candidate for expedient purification and structure elucidation.
Pithomyces sp. RKDO 1698 was fermented at a larger scale in
glass culture tubes (total 1.56 L) to obtain higher quantities of
1, and the mycelia and cell-free broth (CFB) were extracted
separately with EtOAc. Interestingly, the mycelia extract was
highly enriched in 1, whereas this metabolite was barely
detected in the CFB extract (Figure S2), suggesting that
perhaps RKDO 1698 sequesters 1 in nature. Compound 1 was
purified from the mycelia extract using automated reversed-
phase flash chromatography, and 240 mg of 1 was recovered.
The chemical structure of 1 was elucidated using a
combination of NMR spectroscopy, mass spectrometry, and
chemical derivatization.
sources have only relatively recently gained widespread
attention as sources of natural products with new chemical
scaffolds. For example, 1000 new natural products have been
isolated from marine-derived fungi during the period spanning
from 1970 to 2010.1 In a previous study by our research group
investigating the microbial diversity of the octocoral Eunicea
fusca, an understudied microbial habitat, we isolated and
identified the fungal strain Pithomyces sp. RKDO 1698.2
Reports of secondary metabolites from the genus Pithomyces
date as early as 1960 when P. chartarum collected from
pastures in New Zealand was shown to produce sporidesmin, a
hepatotoxic metabolite that causes facial eczema in grazing
sheep.3 This discovery stimulated additional prospecting
efforts that afforded numerous depsipeptide metabolites
including the sporidesmolides, pithomycolide, pimaydolide,
and angolide (Figure S1).4−7 Herein we report the purification
and chemical structure elucidation of a new cyclic lip-
odepsipeptide from RKDO 1698, fusaristatin C (1), which
we categorized as a new member of the fusaristatin family of
natural products (Chart 1). It is accompanied by fusaristatin A
(2; Fusarium sp. YG-45), fusaristatin B (3; Fusarium sp. YG-
45), and congeners topostatin (4; Thermomonospora alba) and
YM-17032 (5; unidentified fungus).
Purified 1 was obtained as an amorphous, white solid, and
ESI+ HRMS analysis supported a molecular formula of
C25H43N3O6, which requires six degrees of unsaturation
(Figure S3). The 2D structure of 1 (Figure 1) was elucidated
Pithomyces sp. RKDO 1698 was isolated from the tissue of E.
fusca, which was collected on a reef in Crab Cove, south
Florida, USA, in June 2009. Nucleotide BLAST search results
of rDNA genes (ITS and nLSU) placed this isolate within the
genus Pithomyces.2 RKDO 1698 was fermented under various
media conditions as part of a broader unpublished study
examining the secondary metabolic potential of fungi isolated
1
using H, 13C, COSY, TOCSY, HSQC, HMBC, and ROESY
NMR spectra (Figures S4−S10). The peptidic nature of 1 was
apparent from three amide proton signals (ranging from 7.06
Received: September 14, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00787
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Journal of Natural Products
Note
Chart 1
correlations involving only the protons indicated in Figure 1
were discernible due to overlapping signals. HDMT is
connected to (Dha-β-Ala-Ser) through amide and ester
linkages at the N-terminus and C-terminus, respectively;
therefore, 1 may be classified as a cyclic lipodepsipeptide.
The position of the C-11 methyl branch within HDMT was
3
assigned on the basis of J HMBC correlations between both
CH3-11/C-14 and C-12. Four degrees of unsaturation were
accounted for within (Dha-β-Ala-Ser), one by the HDMT
amide and another by cyclization of (Dha-β-Ala-Ser) with
HDMT. In addition to the discussed NMR evidence, MS/MS
fragmentation (Figure S14) supports the proposed 2D planar
structure of 1.
It was expected that with the exception of C-11 the
configurations of all stereogenic carbons within 1 could be
readily determined. The Ser residue was first addressed using
Marfey’s method.9 The acid hydrolysate of 1 in addition to L-
Ser and DL-Ser was derivatized with 1-fluoro-2,4-dinitrophenyl-
5-L-alanine amide (L-FDAA) and then analyzed by UHPLC-
HRMS. By comparing the retention time of Ser-L-FDAA
derived from 1 to that of the derivatized amino acid standards,
1 was found to contain L-Ser (Figure S15). Insoluble material
generated during the hydrolysis of 1 was extracted from the
acidified hydrolysate with hexanes and found to contain
HDMT (6) by UHPLC-HRMS (m/z 271.2274 [M − H]−),
which we suspected would be suitable for configuration
analysis of C-3 (Figure S16). Given the potential for
epimerization of 6 at C-2, the acid hydrolysis was repeated
on a larger scale using DCl in D2O/CD3OD to introduce a
deuterium label into any epimerized product. Fortunately, a
deuterium-labeled 6 (m/z 272.2341 [M − H]−) was not
observed by UHPLC-HRMS, indicating that epimerization did
not occur and that cleaved 6 retained the same C-2
configuration as 1 (Figure 2). Compound 6 was purified
using automated normal-phase flash chromatography, and its
structure was elucidated using 1D and 2D NMR spectroscopy
(Figures S17−S21).
Figure 1. Key COSY, HMBC, and ROESY NMR correlations for
fusaristatin C (1).
to 8.91 ppm) in the 1H NMR spectrum and their
corresponding carbonyl signals in the 13C spectrum (ranging
from 163.6 to 170.6 ppm; Table 1). It was revealed in the
COSY spectrum that only two of these protons belong to
1
distinct H−1H spin systems, which suggested the presence of
one α-unsaturated amino acid residue within a tripeptide
moiety. Amino acid side chains were assigned using COSY,
TOCSY, HSQC, and HMBC spectra, and the tripeptide was
found to contain a dehydroalanine (Dha), β-alanine (β-Ala),
and serine residue. Dha, an uncommon and nonproteinogenic
amino acid, was identified based on unusually high 1H and 13
C
resonances at the α-position (137.0 ppm) and β-position
(5.40/5.61 and 111.7 ppm), which are characteristic of an
exocyclic methylene group. The order of amino acid residues
was assigned using HMBC and ROESY correlations involving
amide protons; it was determined to be (Dha-β-Ala-Ser). The
nonpeptidic portion of 1 exhibited a molecular formula of
C16H30O2 and was identified as a 3-hydroxy-2,11-dimethyl-
tetradecanoic acid (HDMT). Although this group is expected
to contain one continuous 1H−1H spin system, COSY
Mosher’s ester analysis was carried out on a sub-milligram
quantity of 6 in order to determine the C-3 configuration of 1.
B
DOI: 10.1021/acs.jnatprod.8b00787
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Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data (1H 600 MHz, 13C 151
MHz, DMSO-d6) for Fusaristatin C (1)
position
CO
δC, type
169.9,
δH, (J in Hz)
Ser
C
NH
α
8.25, d (8.7)
4.48, m
3.51, m
53.8,
60.9,
CH
CH2
β
3.58, m
β-OH
CO
NH
α
4.96, t (5.5)
a
β-Ala
170.6,
36.0,
C
7.06, dd (8.0, 4.8)
2.12, dt (13.7, 3.5)
2.30, m
3.19, m
3.62, m
CH2
CH2
C
β
35.4,
b
Dha
CO
NH
α
163.6,
8.91, s
137.0,
111.7,
C
CH2
β
5.40, s
5.61, s
c
HDMT
1
2
172.2,
43.4,
14.6,
75.5,
28.8,
C
Figure 2. Determination of C-2 and C-3 configurations in fusaristatin
C (1). (A) Mosher’s ester analysis of 3-hydroxy-2,11-dimethylte-
tradecanoic acid (HDMT; 6) cleaved from 1 with ΔδSR displayed in
ppm. Configuration analysis (J-based) of eight possible gauche
rotamers of (B) 2S,3S-1 and (C) 2R,3S-1. The magnitudes of relevant
CH
CH3
CH
CH2
2.68, m (7.1)
1.07, d (7.1)
4.91, td (7.5, 4.4)
1.49, m
2-CH3
3
4
3
1.62, m
1.09, m
1.27, m
1.22, m
1.24, m
1.24, m
1.22, m
2J and J values (S = small and L = large) were predicted for each
1
rotamer and compared to experimental values obtained using H and
5
31.0,
CH2
HETLOC NMR [3JH2H3 = 4.4 Hz (S), 2JH2C3 = −4.0 Hz (L), 3JH3CH3
= 1.8 Hz (S)]. Rotamer iv agrees best with experimental coupling
constants and led to the assignment of 2R,3S-1. Predicted NOEs
denoted with black arrows (for iv only) agree with correlations
observed in a 1D selective-gradient ROESY spectrum of 1.
d
6
29.4,
29.0,
28.7,
26.3,
36.3,
CH2
CH2
CH2
CH2
CH2
d
7
d
8
d
9
10
1.06, m
1.22, m
1.34, m
0.81, d (6.7)
1.23, m
extracted from the HETLOC spectrum on account of
insufficient signal-to-noise, and thus our relative configuration
assignment may be subject to interpretation.
11
11-CH3
12
13
14
31.9,
19.3,
31.3,
22.1,
14.0,
CH
CH3
CH2
CH2
Compound 1 bears strong structural resemblance to the
fusaristatins, topostatin, and YM-170320. The name fusar-
istatin C was chosen in an effort to provide consistent naming
for 1−5.13−17 Compounds 1−5 are comprised of a tripeptide
core containing an N-terminal Dha residue that is cyclized via
an α-methyl-β-hydroxy acyl moiety, but each contains a
different C-terminal amino acid. Fusaristatin C is structurally
unique in that it lacks the characteristic arachidic acid skeleton
and 3-aminoisobutyric acid residue that 2−5 possess.
Compound 1 instead contains an HDMT moiety and β-Ala
residue, respectively. It is worth noting that although YM-
170320 was not reported as a cyclic structure, we speculate
that perhaps it exists as such in its natural state and that it was
methanolyzed during purification because analogous degrada-
tion was observed by UHPLC-HRMS during the purification
of 1 with acidic mobile phases.
1.25, m
0.85, t (7.1)
CH3
a
b
c
β-Ala: β-alanine. Dha: dehydroalanine. HDMT: 3-hydroxy-2,11-
dimethyltetradecanoyl group. Overlapping signals may be inter-
changed.
d
Values for ΔδSR are displayed in Figure 2A and are consistent
with the S configuration at C-3 (Figure S22). The relative
configuration of C-2 and C-3 was deduced using J-based
configuration analysis (Figure 2B and C).10−12 The magni-
3
2
3
tudes of JHH in addition to various JCH and JCH values were
predicted for rotamers of 2R,3S-1/2S,3S-1 and then compared
1
to experimentally determined values for 1 obtained using H
and HETLOC NMR experiments (Figure S13). Since JH2H3
3
was measured as 4.4 Hz, rotamers i, iii, iv, and v represent
plausible configurations of 1. Rotamer iv is most consistent
with the measured values of 2JH2C3 (−4.0 Hz) and 3JH3CH3 (1.8
Hz), leading to the assignment of 2R,3S-1. The 2R,3S
configuration is also supported by comparison of predicted
NOEs to data obtained using a 1D selective-gradient ROESY
experiment. As predicted for rotamer iv, we observed a ROESY
correlation between H-3/CH3-2 and H-3/H-2, whereas strong
correlations between H-4a/H-2 and H-4b/H-2 were not
observed (Figures S11 and S12). It should be noted that
additional JCH values pertinent to this analysis could not be
Compound 1 was tested for antimicrobial activity in
microbroth dilution assays against methicillin-resistant Staph-
ylococcus aureus (MRSA), S. warneri, vancomycin-resistant
Enterococcus faecium (VRE), Pseudomonas aeruginosa, Proteus
vulgaris, Mycobacterium tuberculosis, and Candida albicans. It
was also tested for cytotoxicity against three healthy cell lines
(Vero, HEKa, and BJ) and three cancer cell lines (HTB-22,
HTB-26, and HCT-116). Compound 1 exhibited no
antimicrobial activity at the highest concentration tested
(128 μg/mL or 265 μM) and was not cytotoxic toward any
of the six cell lines tested. Dose−response curves and IC50
C
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Journal of Natural Products
Note
streptomycin sulfate, and 100 μg/mL chloramphenicol in filtered
seawater), a white filamentous fungal strain (RKDO 1698) was
isolated. The fungus was taxonomically identified using sequence
homology of rDNA genes, including the ITS1-5.8S-ITS2 region and
the nLSU region. A seed culture of Pithomyces sp. RKDO 1698 was
prepared by inoculating 13 mL of SMY broth (10 g/L peptone, 40 g/
L maltose, 10 g/L yeast extract, and 18 g/L Instant Ocean in
deionized H2O) contained in a 60 mL glass culture tube with
cryopreserved mycelia. The seed culture was fermented for 7 days on
a rotary shaker (200 rpm at 22.0 °C), after which it was homogenized
with glass beads using a vortex mixer. The homogenized seed culture
was used to inoculate 120 glass culture tubes containing 13 mL of
YES broth (20 g/L yeast extract,150 g/L sucrose, and 0.5 g/L
MgSO4·7H2O in deionized H2O) each with approximately 50 μL.
The culture tubes were distributed between two racks that were
adjusted such that the tubes lay at 45° with respect to the horizontal
and were fermented without agitation at 22.0 °C for 14 days.
Extraction and Purification. After 14 days of fermentation, all
culture tubes contained a mat of mycelia on the broth surface. The
mats were removed from their respective tubes, pooled, and then
rinsed with deionized H2O. The remaining broth from each tube was
also pooled. Both the mycelia and broth were extracted separately
with 1.5 L of EtOAc in 3 L Fernbach flasks. The extracts were dried in
vacuo to yield 2.91 g (mycelia) and 0.60 g (broth) of dried material,
which was analyzed by UHPLC-HRMS. As 1 was contained almost
entirely in the mycelia extract, this extract was chosen for further
purification. The mycelia extract was fractionated by automated
reversed-phase flash chromatography (Teledyne ISCO CombiFlash
Rf) using a 120 g C18 column (Redisep Rf High Performance GOLD)
and eluted using the following elution method: 95% H2O (solvent
A)/5% CH3OH (solvent B) from 0 to 5 min, linear gradient from 5%
solvent B to 100% B from 5 to 40 min and 100% solvent B from 40 to
50 min (flow rate = 60 mL/min). The eluent was detected by UV
(227 nm), and 1 eluted between 38.9 and 40.7 min. All fractions
within this window were dried in vacuo and analyzed by UHPLC-
HRMS and those that were deemed pure were pooled to afford 240
mg of 1.
values are provided in Figure S23. Screening protocols are also
provided in the Supporting Information.
In conclusion, we report a new Pithomyces cyclic lip-
odepsipeptide, fusaristatin C, which shares structural features
with several natural products of both fungal and bacterial
origin. With the exception of the C-11 position, we assigned
the absolute configuration using chemical derivatization and J-
based configuration analysis. Fusaristatin C did not exhibit
biological activity against the microbial pathogens or cell lines
tested. This is the first report of a natural product from a coral-
derived Pithomyces sp.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation was
measured on a Rudolph Autopol III polarimeter using a 50 mm
microcell (1.2 mL). Infrared (IR) spectra were recorded using
attenuated total reflectance on a Thermo Nicolet 6700 FT-IR
spectrometer. All NMR spectra (unless specified) were acquired on a
Bruker Avance III NMR spectrometer (1H: 600 MHz, 13C: 151 MHz)
equipped with a 5 mm cryoprobe and located at Agriculture and Agri-
Food Canada (AAFC) in Charlottetown, PE, Canada. All chemical
shifts are reported in ppm and referenced to residual solvent signals
[1H (DMSO-d6): 2.50 ppm, 13C (DMSO-d6): 39.51 ppm, 1H
(CD3OD): 3.31 ppm, and 13C (CD3OD): 49.00 ppm]. The
HETLOC and 1D-ROESY NMR experiments were acquired on a
400 MHz NMR with the dipsi2etgpjcsix1 pulse program. Default
acquisition parameters were used except for the following
modifications: td (f2) = 6144; td (f1) = 512; ns = 128; sw (f2 and
f1) = 4.80 ppm; o1p (f2 and f1) = 2.9 ppm; cnst2 = 145.0; cnst16 =
5.0; gpz1 = 13.0%; gpz2 = 19.0%; gpz3 = 30.0%. The HETLOC
spectrum was zero-filled to 8192 and 2048 in the f2 and f1
dimensions, respectively. Tandem mass spectra were acquired by
direct infusion on a Thermo Velos Orbitrap mass spectrometer using
a collision-induced dissociation energy of 30 eV at a rate of 2 μL/min.
UHPLC-HRMS analyses (unless specified) were carried out using a
Thermo Accela chromatograph equipped with HRMS-ELSD-UV
detection: a Thermo LTQ Exactive fitted with an ESI source, Sedex
80 LT-ELSD, and Thermo PDA, respectively. The following
chromatographic conditions were used: Kinetex core−shell 100 Å
UHPLC column (2.1 × 50 mm, 1.7 μm, Phenomenex), mobile-phase
flow rate of 0.5 mL/min, injection volume of 10 μL (all samples were
prepared in CH3OH), and a linear gradient from H2O/CH3CN (95:5,
0.1% formic acid) at 0.2 min to 100% CH3CN (0.1% formic acid) at
4.8 min, which was held until 8.0 min before returning to H2O/
CH3CN (95:5, 0.1% formic acid) for a 1.5 min equilibration. The
following HRMS parameters were used: positive ionization mode,
mass resolution of 30 000, mass range of m/z 190 to 2000, spray
voltage of 2.0 kV, capillary temperature of 300 °C, S-lens RF voltage
of 60.0%, maximum injection time of 10 ms, and 1 microscan. The
system was controlled by Thermo Xcalibur software modules. All
reagents were purchased from commercial sources and used without
further purification. All solvents used for purification were of HPLC
grade or higher.
Fusaristatin C (1): amorphous, white solid; [α]24.4D = +1.4 (c 0.28,
CH3OH); UV (CH3CN/H2O) λmax 230 nm; IR νmax = 1623, 1658,
1730, 2850, 2920, 2954, 3278, 3322, 3346, and 3457 cm−1; see Table
1
1 for H and 13C NMR data; ESI+ HRMS m/z 482.3219 [M + H]+
+
(calcd for C25H44N3O6 , 482.3225).
Amino Acid Configuration Using Marfey’s Method. To a vial
containing dried 1 (5 mg, 0.01 mmol) was added 6 M HCl (1 mL),
and then the mixture heated to 80 °C for 1 h with stirring. The
reaction mixture was dried in vacuo, and a portion of the resulting
hydrolysate (1 mg) was transferred to a separate vial, to which 150 μL
of deionized H2O, 300 μL of L-FDAA (10 mg/mL in acetone), and 70
μL of aqueous NaHCO3 (1 M) were added. The reaction mixture was
heated to 37.0 °C for 2 h, quenched with 70 μL of HCl (1 M), and
then dried in vacuo. The same process was carried out with L-serine (1
mg, 0.01 mmol) and DL-serine (1 mg, 0.01 mmol) to afford L-FDAA-
derivatized serine standards. L-FDAA-derivatized hydrolysate and
standards were suspended in CH3OH (500 μg/mL) for UHPLC-
HRMS analysis. The following chromatographic conditions were
used: Thermo Hypersil Gold 100 Å UHPLC column (1.9 μm C18, 50
mm × 2.1 mm), 95% H2O with 0.1% (v/v) formic acid (solvent A):
5% CH3CN with 0.1% (v/v) formic acid (solvent B) from 0 to 55 min
followed by a linear gradient from 5% B to 40% B from 55 to 57 min
and then 40% solvent B from 57 to 60 min, flow rate of 400 μL min−1
and injection volume of 10 μL.
Configuration Analysis of 3-Hydroxy-2,11-dimethyltetrade-
canoic Acid. Compound 1 (50 mg, 0.1 mmol) was dissolved in 5 mL
of DCl (3 M in a 3:1 mixture of D2O/CD3OD) and stirred at 80.0 °C
for 1 h. The reaction mixture was extracted three times with 5 mL of
hexanes, and the pooled extracts were dried in vacuo to afford 39 mg
of material. The entire extract was fractionated by automated flash
chromatography using a 12 g silica column (InnoFlash, Canadian Life
Sciences) and eluted with 100% EtOAc for 10 min (flow rate = 30
mL/min). All fractions were dried in vacuo, and those containing 6
Isolation and Fermentation of Pithomyces sp. RKDO 1698.
E. fusca samples were collected by hand using scuba at a depth of 12.0
m from a reef in Crab Cove, south Florida, USA (26°18.736′ N,
80°03.583′ W) on June 2, 2009. Healthy branches were aseptically
removed from a coral colony, brought to the ocean surface, and stored
at 18−22 °C (ocean temperature). Approximately 4 h postcollection,
the branches were aseptically cut into 0.5−1.0 cm sections, transferred
to separate sterile 50 mL conical tubes, and then washed three times
with filter-sterilized seawater (0.22 μm cellulose acetate filters from
Corning) to remove loosely associated surface bacteria. Branch
samples were homogenized in filter-sterilized seawater and then
separated according to particle size (≥500, ≥213, ≥104, ≥51, and
<51 μm) using an adapted particle-filtration apparatus.18 From a
particle filtrate plated onto YM media (10 g/L agar, 5 g/L malt
extract, 1 g/L yeast extract, 50 μg/mL tetracycline, 50 μg/mL
D
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Journal of Natural Products
Note
were identified by UHPLC-HRMS. It was determined that 6 elutes
between 1 and 2 min, and 4 mg of pure material was collected. NMR
data for 6 are provided in Table S1.
(10) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
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The configuration at C-3 of 6 was determined using Mosher’s ester
analysis. To a stirred solution of HDMT (0.3 mg, 0.001 mmol) and
anhydrous pyridine (0.005 mL, 0.06 mmol, 60 equiv) in 300 μL of
anhydrous CDCl3 was added S-(+)-MTPA-Cl (3S, 0.03 mL, 0.16
mmol, 160 equiv). The reaction mixture was stirred for 1 h at room
temperature and then dried in vacuo, after which it was suspended in
200 μL of CDCl3 for NMR analysis. In an entirely analogous fashion,
the S-MTPA ester of HDMT was prepared from R-(+)-MTPA-Cl.
(12) Williamson, R. T.; Boulanger, A.; Vulpanovici, A.; Roberts, M.
A.; Gerwick, W. H. J. Org. Chem. 2002, 67 (23), 7927−7936.
(13) Shiono, Y.; Tsuchinari, M.; Shimanuki, K.; Miyajima, T.;
Murayama, T.; Koseki, T.; Laatsch, H.; Funakoshi, T.; Takanami, K.;
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(14) Keitarou, S.; Nagao, K.; Monnai, Y.; Yagi, A.; Uyeda, M. J.
Antibiot. 1998, 51 (11), 991−998.
(15) Keitarou, S.; Yahara, S.; Kido, Y.; Nagao, K.; Hatano, Y.; Uyeda,
M. J. Antibiot. 1998, 51 (11), 999−1003.
ASSOCIATED CONTENT
* Supporting Information
■
(16) Keitarou, S.; Yamaizumi, M.; Tateishi, S.; Monnai, Y.; Uyeda,
M. J. Antibiot. 1999, 52 (5), 460−465.
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00787.
(17) Sugawara, T.; Tanaka, A.; Tanaka, K.; Nagai, K.; Suzuki, K.;
Suzuki, T. J. Antibiot. 1998, 51 (4), 435−438.
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UHPLC-HRMS and MS/MS data, NMR spectra,
bioactivity data, and additional figures (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-902-566-0565. Fax: 1-902-566-7445. E-mail: rkerr@
upei.ca.
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ORCID
Logan W. MacIntyre: 0000-0002-8680-5912
Russell G. Kerr: 0000-0002-2557-4321
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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The authors gratefully acknowledge financial support from the
Natural Sciences and Engineering Council of Canada
(NSERC), the Canada Research Chair Program, Atlantic
Canada Opportunities Agency, Canada Foundation for
Innovation, Nautilus Biosciences Croda, Innovation PEI, and
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the Jeanne and Jean-Louis Levesque Foundation. We also
acknowledge Dr. R. Pike (UPEI) for collection of coral
samples and microbial isolation, M. Lantegine (Nautilus), K.
McQuillan (Nautilus), and T. Clark (UNB) for performing
bioassays, and Dr. C. Kirby (AAFC) and M. Fischer (AAFC)
for the NMR services they provided.
REFERENCES
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DOI: 10.1021/acs.jnatprod.8b00787
J. Nat. Prod. XXXX, XXX, XXX−XXX