Mortiamides A-D, Cyclic Heptapeptides from a Novel Mortierella sp. Obtained from Frobisher Bay

Article abstract of DOI:10.1021/acs.jnatprod.7b00383

Four new cyclic heptapeptides, mortiamides A-D (1-4), were obtained from a novel Mortierella sp. isolate obtained from marine sediment collected from the intertidal zone of Frobisher Bay, Nunavut, Canada. The structures of the compounds were elucidated by NMR spectroscopy and tandem mass spectrometry. The absolute configurations of the amino acids were determined using Marfey's method. Localization of l and d amino acids within each compound was ascertained by retention time comparison of the partial hydrosylate products of each compound to synthesized dipeptide standards using LC-HRMS. Compounds 1-4 did not exhibit any significant antimicrobial or cytotoxic activity.

Full text of DOI:10.1021/acs.jnatprod.7b00383

Article
pubs.acs.org/jnp
Mortiamides A−D, Cyclic Heptapeptides from a Novel Mortierella sp.
Obtained from Frobisher Bay
Alyssa L. Grunwald,† Fabrice Berrue,‡ Andrew W. Robertson,‡ David P. Overy,‡,§
and Russell G. Kerr*,†,‡
§
†Department of Biomedical Science and Department of Pathology and Microbiology, Atlantic Veterinary College, University of
Prince Edward Island, Charlottetown, PEI C1A 4P3, Canada
‡Department of Chemistry, University of Prince Edward Island, Charlottetown, PEI C1A 4P3, Canada
S
* Supporting Information
ABSTRACT: Four new cyclic heptapeptides, mortiamides A−D
(1−4), were obtained from a novel Mortierella sp. isolate obtained
from marine sediment collected from the intertidal zone of
Frobisher Bay, Nunavut, Canada. The structures of the compounds
were elucidated by NMR spectroscopy and tandem mass
spectrometry. The absolute configurations of the amino acids
were determined using Marfey’s method. Localization of L and D
amino acids within each compound was ascertained by retention
time comparison of the partial hydrosylate products of each
compound to synthesized dipeptide standards using LC-HRMS.
Compounds 1−4 did not exhibit any significant antimicrobial or
cytotoxic activity.
ungi are an excellent source of novel and bioactive natural
products, but limited investigations of arctic fungi have
distinct from all known taxa of the genus Mortierella (using
barcoding gene sequences derived from rDNA). Chemical
investigation of this isolate led to the isolation of four new
cyclic heptapeptides, mortiamides A−D (1−4), containing an
unusually high proportion of D amino acids. LC-HRMS was
used to identify partial hydrolysis products and aid in the
localization of these L and D amino acids within each
compound. The isolation, structure elucidation, and biological
activity testing of these compounds are reported within.
F
been undertaken. Arctic fungi are diverse and abundant1 and
represent a vast untapped resource for natural product
discovery. Unique stresses associated with arctic life including
low temperature, high ultraviolet radiation, and low nutrient
availability have led to unique adaptations within these fungi2
and may lead to the potential for the discovery of new natural
products.
Canada’s arctic has been greatly underexplored in terms of
fungal and associated natural product diversity. Frobisher Bay
located within Canada’s arctic is a 230 km inlet of the Labrador
Sea located on the southeastern corner of Baffin Island,
Nunavut. This inlet experiences some of the highest diurnal
tidal variations in the world (7−11 m) and represents an
extreme environment for microorganisms. Previous work
conducted in our lab from this region has resulted in the
isolation of two new natural products, iqalisetins A and B, from
a Tolypocladium species.3 As part of ongoing bioprospecting
efforts within Frobisher Bay, a new member within the genus
Mortierella was isolated from sediment samples from the
intertidal zone. The genus Mortierella falls within the order
Mortierellales, one of the largest orders within the phylum
Mucoromycota, and is commonly isolated from cold environ-
ments.4,5 Most notably, members within this genus are known
for the production of polyunsaturated fatty acids6,7 and for the
ability to biotransform various organic compounds.8 Very few
natural products have been reported from this genus.9−12
The new Mortierella isolate presented an anamorphic state
consistent with the genus and was found to be phylogenetically
RESULTS AND DISCUSSION
■
The fungal isolate Mortierella sp. RKAG 110 was obtained from
sediment collected from Frobisher Bay, Nunavut, Canada.
BlastN search results of several rDNA genes (ITS and nLSU)
placed this isolate within the genus Mortierella and suggested
that this isolate was unique to the genus as the closest match of
the ITS rDNA nucleotide sequence to an accessioned strain
was with that of Mortierella antarctica (NR_111580.1) with
96% sequence similarity. Production of a white rosette-like
phenotype and microscopic observation of spherical sporangia
in agar culture confirmed the isolate RKAG 110 to be a
member of the genus Mortierella. As the organism originated
from a rarely studied geographic location and was taxonomi-
cally novel, further chemical investigations were carried out.
The 1 L solid agar fermentation of isolate RKAG 110 was
extracted with EtOAc, dried, and partitioned between 80%
Received: May 2, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00383
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hydrolysis was undertaken to generate dipeptide fragments
which were derivatized with N-(5-fluoro-2,4-dinitrophenyl-5)-L-
alaninamide (FDAA) and analyzed by LC-HRMS.14 The
retention time of the partial hydrosylate product (FDAA-Phe-
Phe) was compared to synthesized dipeptide standards that
were derivitized in the same manner (FDAA-L-Phe-D-Phe and
FDAA-D-Phe-L-Phe) and revealed the retention time of the
partial hydrosylate fragment was consistent with the retention
time of the synthesized FDAA-D-Phe-L-Phe standard (Figure
2). Therefore, the final structure of 1 was assigned as [cyclo-(D-
Phe-L-Phe-D-Val-D-Val-D-Val-L-Leu-D-Val)].
Mortiamide B (2) (Table 1) was obtained as a white powder,
and HRESIMS supported a molecular formula of C47H63N7O7
requiring 20 degrees of unsaturation. TOCSY experiments
1
revealed the presence of seven H spin systems corresponding
to three Phe and four Val residues (Table 1). HMBC
correlations from Phe1-NH (δH 7.81) to Val4-CO (δC
170.7) and Val4-NH (δH 7.56) to Phe3-CO (δC 170.5)
established the three amino acid sequence Phe3-Val4-Phe1.
ROESY correlations from Phe3-NH (δH 7.70) to Val3-Hβ (δH
2.06), Val3-NH (δH 8.00) to Val2-Hβ (δH 2.29), Val2-NH (δH
7.78) to Val1-Hα (δH 4.07), Val1-NH (δH 8.24) to Phe2-Hα (δH
4.49), and Phe2-NH (δH 8.19) to Phe1-Hα (δH 4.61)
established the final sequence as cyclo-Phe1-Phe2-Val1-Val2-
Val3-Phe3-Val4 (Figure 1). Peptide 2 differed from 1 by the
replacement of the Leu residue with a Phe residue. Marfey’s
analysis13 of the total hydrosylate of 2 revealed the presence of
D-Val, L-Phe, and D-Phe in a 2:1 ratio, respectively. After partial
hydrolysis and derivatization, the retention time of the FDAA-
Phe-Phe dipeptide (tR 48.55 min) was consistent with the
retention time of the synthesized FDAA-D-Phe-L-Phe dipeptide
(tR 48.40 min) (Supporting Information, Figure S12). The
remaining Phe residue was assigned as L, and the final structure
of 2 was assigned as [cyclo-(D-Phe-L-Phe-D-Val-D-Val-D-Val-L-
Phe-D-Val)].
CH3CN and hexane. The CH3CN-soluble portion was
subjected to flash chromatography and reversed-phase HPLC,
yielding four new cyclic heptapeptides, mortiamide A (1) (4.1
mg), mortiamide B (2) (1.5 mg), mortiamide C (3) (3.2 mg),
and mortiamide D (4) (2.4 mg).
Mortiamide A (1) (Table 1) was obtained as a white powder,
and HRESIMS supported a molecular formula of C44H65N7O7,
requiring 16 degrees of unsaturation. The peptidic nature of the
compound was determined by the presence of seven amide
1
protons within the H NMR spectrum, while the 13C NMR
Mortiamide C (3) (Table 2) was obtained as a white powder,
and HRESIMS supported a molecular formula of C41H67N7O7
requiring 12 degrees of unsaturation. TOCSY experiments
spectrum revealed the presence of seven amide carbonyls
between δC 170.5 and 173.3 ppm and seven α-amino acid
carbon resonances between δC 53.4 and 60.3 (Table 1).
Additionally TOCSY experiments revealed the presence of
1
revealed the presence of seven H spin systems corresponding
1
to one Phe, one Ala, one Val, and four Ile residues (Table 2).
HMBC correlations from Ala-NH (δH 7.54) to Ile4-CO (δC
170.8) and Ile4-NH (δH 7.80) to Phe-CO (δC 170.6)
established the three amino acid sequence Phe-Ile4-Ala. ROESY
correlations from Phe-NH (δH 7.61) to Ile3-Hα (δH 4.00), Ile3-
NH (δH 7.89) to Val-Hα (δH 4.03), Val-NH (δH 7.67) to Ile2-
Hα (δH 4.31), Ile2-NH (δH 8.44) to Ile1-Hα (δH 4.27), and Ile1-
NH (δH 7.93) to Ala-Hα (δH 4.42) established the final
sequence as [cyclo-(Ala-Ile1-Ile2-Val-Ile3-Phe-Ile4)] (Figure 1),
which was confirmed by MS/MS experiments (Supporting
Information, Figure S7). The absolute configurations of the
amino acid residues were determined to be D-Ala, D-Val, L-Phe,
L-Ile, and D-Ile, whereby D-Ile and L-Ile were in a 3:1 ratio. Due
to the coelution of Ile and allo-Ile by RP-HPLC, we were
unable to definitively assign the absolute configuration of these
residues. By comparison of the FDAA-Ile-Ala (tR 37.41 min)
and FDAA-Ile-Ile (tR 42.56 min) partial hydrosylate products of
3 to synthesized dipeptide standards it was determined that the
retention times by LC-HRMS corresponded to FDAA-D-Ile-D-
Ala (tR 37.03 min) and FDAA-L-Ile-D-Ile (tR 41.85)
(Supporting Information, Figure S13). Because L-Ile had been
localized, the remaining Ile was assigned as D, resulting in the
final structure being assigned as [cyclo-(D-Ala-L-Ile-D-Ile-D-Val-
D-Ile-L-Phe-D-Ile)].
seven H spin systems corresponding to seven amino acid side
chains consisting of one leucine, two phenylalanine, and four
valine residues. Amino acid side chains were assigned using
COSY, HMBC, and TOCSY NMR spectra whereby con-
nectivity was determined using HMBC, ROESY, and ESI-MSn
analysis. HMBC correlations from Val1-NH (δH 8.20) to Phe2-
CO (δC 171.4) and Val2-NH (δH 7.86) to Val1-CO (δC
172.4) established the three amino acid sequence Phe2-Val1-
Val2. HMBC correlations between Val4-NH (δH 7.64) and Leu-
CO (δC 171.6) established the two amino acid sequence
Leu-Val4. ROESY correlations from Val2-Hα (δH 4.01) to Val3-
NH (δH 8.29) and Val3-Hα (δH 4.03) to Leu-NH (δH 7.38)
connected both sequences via a Val residue, resulting in the six
amino acid sequence Phe2-Val1-Val2-Val3-Leu-Val4. ROESY
correlations from Val4-Hα (δH 4.12) to Phe1-NH (δH 7.72)
and Phe1-Hα (δH 4.63) to Phe2-NH (δH 7.64) resulted in the
final sequence of the heptapeptide being assigned as [cyclo-
(Phe1-Phe2-Val1-Val2-Val3-Leu-Val4)] (Figure 1), which was
subsequently confirmed using MS/MS experiments (Support-
ing Information, Figure S5).
The absolute configuration of 1 was determined using
Marfey’s method.13 The peptide was found to contain L-Leu, D-
Val, L-Phe, and D-Phe (Supporting Information, Figure S11). In
order to localize L-Phe and D-Phe within the structure, a partial
B
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1
Table 1. H (600 MHz) and 13C (150 MHz) NMR Data of Mortiamides A (1) and B (2) in DMSO-d6
1
2
position
D-Phe1
δC, type
δH (J, Hz)
171.1, C
position
D-Phe1
δC, type
171.1, C
δH (J, Hz)
CO
NH
α
CO
NH
α
7.72, d (8.1)
7.81, d (7.8)
53.4, CH
36.8, CH2
4.63, m
53.3, CH
36.8, CH2
4.61, m
βA
βB
1′
2.98, dd (13.7, 7.3)
2.76, dd (13.7, 7.5)
βA
βB
1′
2.97, dd (6.2, 13.5)
2.77, dd (8.1, 13.5)
137.5, C
137.7, C
2′
3′
4′
CO
NH
α
129.1, CH
128.2, CH
126.4, CH
171.4, C
7.10, m
7.21, m
7.18, m
2′
3′
4′
CO
NH
α
128.9, CH
129.0, CH
126.2, CH
171.4, C
7.09, m
7.21, m
7.17, m
L-Phe2
L-Phe2
a
8.29
8.19, d (5.7)
4.49, m
55.2, CH
36.3, CH2
4.47, m
55.2, CH
36.3, CH2
a
βA
βB
1′
2.88, dd (13.6, 7.6)
2.81, dd (13.6, 8.0)
βA
βB
1′
2.91
2.83, dd (13.6, 8.2)
137.2, C
137.4, C
2′
3′
4′
CO
NH
α
129.1, CH
128.2, CH
126.5, CH
172.4, C
7.19, m
7.23, m
7.23, m
2′
3′
4′
CO
NH
α
129.0, CH
128.0, CH
126.2, CH
171.8, C
7.21, m
7.24, m
7.17, m
D-Val1
D-Val2
D-Val3
L-Leu
D-Val1
D-Val2
D-Val3
L-Phe3
b
8.20, d (7.0)
4.06, m
8.24
a
58.4, CH
29.3, CH
17.0, CH3
19.1, CH3
173.3, C
58.5, CH
29.3, CH
17.1, CH3
18.8, CH3
171.4, C
4.07
β
2.10, m
β
2.10, m
γ1
γ2
CO
NH
α
0.73, d (6.9)
0.62, d (6.9)
γ1
γ2
CO
NH
α
0.75, d (6.9)
0.65, d (6.9)
7.86, d (5.3)
4.01, m
7.78, d (7.8)
a
60.3, CH
28.3, CH
19.4, CH3
18.9, CH3
170.5, C
59.7, CH
28.3, CH
19.0, CH3
18.8, CH3
172.4, C
4.03
β
2.34, m
β
2.29, m
γ1
γ2
CO
NH
α
1.03, d (6.6)
γ1
γ2
CO
NH
α
0.99, d (6.7)
0.89, d (6.7)
a
0.91
a
8.29
8.00, d (5.2)
a
59.0, CH
29.1, CH
18.9, CH3
16.8, CH3
171.6, C
4.03, m
2.22, m
58.5, CH
29.3, CH
18.7, CH3
16.5, CH3
170.5, C
4.03
β
β
2.06, m
a
γ1
γ2
CO
NH
α
0.91
γ1
γ2
CO
NH
α
0.76, d (6.9)
0.63, d (6.9)
0.84, d (6.9)
b
7.38, d (4.4)
4.09, m
7.70
58.8, CH
38.8, CH2
24.0, CH3
22.7, CH3
21.1, CH3
55.4, CH
35.8, CH2
4.39, m
β
1.55, m
βA
βB
1′
2′
3′
3.08, dd (13.8, 6.2)
2.90, m
γ
1.65, m
a
δ1
δ2
0.90
137.9, C
0.82, d (6.6)
128.9, CH
128.0, CH
126.2, CH
170.7, C
7.29, m
7.24, m
7.17, m
4′
D-Val
CO
NH
α
170.5, C
D-Val
CO
NH
α
7.64, d (9.3)
4.12, m
7.56, d (7.8)
a
58.8, CH
30.7, CH
17.7, CH3
19.0, CH3
58.5, CH
29.7, CH
17.2, CH3
18.7, CH3
4.06
β
1.91, m
β
1.88, m
a
γ1
0.69, d (6.8)
0.64, d (6.8)
γ1
γ2
0.51
a
γ2
0.50
a
b
Overlapping signals. Broad signals.
Mortiamide D (4) (Table 2) was obtained as a white
8.32) to Val-CO (δC 171.3) and Val-NH (δH 7.35) to Phe2-
CO (δC 170.9) established the three amino acid sequence
Phe2-Val-Leu. HMBC correlations between Ile1-NH (δH 7.44)
and Ala-CO (δC 171.9) established a two amino acid
sequence of Ala-Ile1. Both peptide segments were connected via
an Ile residue supported by ROESY correlations from Phe2-NH
powder, and HRESIMS supported a molecular formula of
C44H65N7O7 requiring 16 degrees of unsaturation. TOCSY
1
experiments revealed the presence of seven H spin systems
corresponding to one Ala, one Leu, one Val, two Phe, and two
Ile residues (Table 2). HMBC correlations from Leu-NH (δH
C
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Compounds 1−4 were assayed for biological activity against
methicillin-resistant Staphyloccocus aureus (ATCC 33591),
vancomycin-resistant Enterococcus faecium (EF379), Staph-
ylococcus warneri (ATCC 17917), Pseudomonas aeruginosa
(ATCC 14210), and Candida albicans (ATCC 14035). No
significant activity was observed for any compound tested at a
concentration of 128 μg/mL. Compounds 1−4 were also tested
for cytotoxicity against keratinocyte, fibroblast, HTB-26, and
MCF-7 breast cancer cell lines. None of the tested compounds
exhibited activity against these cell lines at 128 μg/mL.
Cyclic heptapeptides have previously been reported from
many microorganisms including fungi and bacteria15−17 as well
as macroorganisms including tunicates, sponges, and
plants18−20 and exhibit a wide range of activities.16,17,21,22
Mortiamides A−D are new members within the cyclic
heptapeptide family. They are characterized by the presence
of seven hydrophobic amino acids, whereby five of these
residues are in the D-configuration. The asymmetry of these
molecules is conserved across each of the peptides, whereby site
2 and 6 always contain amino acids in the L-configuration and
sites 1, 3, 4, 5, and 7 contain amino acids in the D-configuration.
The occurrence of a large number of D amino acids is relatively
rare for cyclic peptides and makes the mortiamides intriguing
members within the cyclic peptide family. Overall, this report
adds to a growing body of evidence that Canada’s arctic is a
resource for the discovery of new taxa of microbes that have the
ability to produce structurally novel natural products.
Figure 1. Key COSY, HMBC, and ROESY correlations of 1−4.
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, on a Thermo Nicolet 6700 FT-IR spectrometer.
NMR spectra were obtained on a 600 MHz Bruker Avance III NMR
spectrometer, where chemical shifts (δ) are reported in ppm and
referenced to the DMSO-d6 residual solvent signal (δH 2.50 and δC
39.51) or CDCl3 residual solvent signal (δH 7.26 and δC 77.16). LC-
HRMS using a Thermo Gold UPLC column were recorded on Accela
Thermo equipment with hyphenated MS-ELSD-UV detection
(Thermo LTQ Exactive fitted with an ESI source, PDA, and LT-
ELSD Sedex 80). Tandem mass spectra were measured on a Thermo
Velos Orbitrap mass spectrometer using a collision-induced
dissociation energy of 35 eV at a rate of 2 μL/min. Automated flash
chromatography was performed on a Teledyne Combiflash Rf200
using C18 RediSep columns or prepacked RediSep Rf Gold normal-
phase silica flash columns (24 g). HPLC purifications were carried out
on a Waters autopurification system coupled with an evaporative light-
scattering detector and UV detector. Silica gel on TLC Al foil plates
(Sigma-Aldrich) was used to monitor dipeptide synthesis reaction
progress and assess purity of compounds. All reported Rf values were
determined using the same plates. All protected dipeptides were
visualized under 254 nm UV light or using potassium permanganate
dip (1.5 g KMnO4, 10 g K2CO3, 125 mg NaOH, 200 mL H2O)
followed by heating. All amino acid standards were purchased from
Sigma-Aldrich (St. Louis, MO, USA). All reagents were purchased
from commercial sources and used without further purification unless
otherwise stated. ACS grade benzene and anhydrous ether were used
for benzylation reactions and purifications. All solvents used for
coupling reactions and chromatographic methods were HPLC grade.
Isolation and Cultivation of Mortierella sp. RKAG 110. A
marine sediment core was collected using a sterile sediment sampler
(LaMotte) at low tide at a depth of 30 cm in Frobisher Bay, Nunavut,
Canada (63.72804° N 68.41989° W) in August 2011. The core was
transferred to a sterile 50 mL conical tube, brought back to the lab on
ice, and stored at −80 °C until processing. The sediment was passed
through a series of sieves (104 and 51 μM) and separated based on
Figure 2. Comparison of the FDAA-derivatized partial hydrosylate of
1 compared to FDAA-derivatized D-Phe-L-Phe and L-Phe-D-Phe
standards by LC-MS.
(δH 8.13) to Ile2-Hα (δH 3.98) and Ile2-NH (δH 8.38) to Ile1-
Hα (δH 4.28), resulting in the six amino acid sequence Ala-Ile1-
1
Ile2-Phe2-Val-Leu. Due to overlapping H signals from Ala-NH
(δH 8.22) and Phe1-NH (δH 8.22), placement of Phe1 could not
be definitively assigned using ROESY or HMBC correlations.
In order to support the molecular formula and requirement for
unsaturation, the remaining phenylalanine residue was used to
cyclize the peptide sequence by peptide bonds between the Ala
and Leu residues, resulting in the final sequence as [cyclo-(Leu-
Phe1-Ala-Ile1-Ile2-Phe1-Val-)] (Figure 1). The final sequence of
the peptide was confirmed by MS/MS experiments (Support-
ing Information, Figure S8). The absolute configurations of the
amino acids within the peptide were definitively assigned using
the advanced Marfey’s method and consisted of D-Ala, D-Val, L-
Phe, D-Leu, and D-Ile, allowing the final structure to be assigned
as [cyclo-(D-Leu-L-Phe D-Ala-D-Ile-D-Ile-L-Phe-D-Val)] (Sup-
porting Information, Figure S14).
D
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1
Table 2. H (600 MHz) and 13C (150 MHz) NMR Data of Mortiamides C (3) and D (4) in DMSO-d6
3
4
position
δC, type
δH (J, Hz)
position
δC, type
δH (J, Hz)
D-Ala
CO
NH
α
172.9, C
D-Leu
CO
NH
α
171.8, C
b
7.54, d (7.4)
4.42, m
8.32
47.9, CH
17.3, CH3
51.9, CH
39.1, CH2
23.9, CH
22.6, CH3
22.1, CH3
170.5, C
4.01, m
β
1.18, d (6.8)
β
1.42, m
γ
1.22, m
δ1
δ2
CO
NH
α
0.78, d (6.6)
a
0.72
L-Ile1
CO
NH
α
172.2, C
L-Phe1
7.93, d (7.6)
4.27, m
8.22, d (7.0)
4.26, m
57.4, CH
35.1, CH
24.3, CH2
55.4, CH
36.4, CH2
β
1.71, m
βA
βB
1′
2′
3′
3.16, m
γ1A
γ1B
γ2
1.49, m
2.78, dd (13.7, 10.9)
1.11, m
138.2, C
a
14.7, CH3
10.6, CH3
0.81
129.0, CH
128.1, CH
126.2, CH
171.9, C
7.24, m
7.23, m
7.19, m
a
δ
0.83
4′
D-Ile2
CO
NH
α
172.2, C
D-Ala
D-Ile1
D-Ile2
L-Phe2
CO
NH
α
b
8.44
8.22, d (7.1)
4.31, m
56.0, CH
35.5, C
4.31, m
48.5, CH
17.5, CH3
β
2.00, m
β
1.20, d (7.2)
γ1A
γ1B
γ2
25.6, CH2
1.28, m
1.22, m
14.3, CH3
11.2, CH3
0.87, d (7.0)
a
δ
0.81
CO
NH
α
172.4, C
D-Val
CO
NH
α
173.5, C
7.44, d (6.9)
4.28, m
b
7.67
57.3, CH
36.1, CH
25.4, CH2
59.5, CH
28.2, CH2
18.7
4.03, m
β
1.92, m
β
2.25, m
γ1A
γ1B
γ2
δ
1.34, m
γ1
γ2
0.94, d (6.7)
0.85, d (7.1)
1.09, m
a
18.8, C
15.0, CH3
11.4, CH3
171.5, C
0.85
a
0.86
D-Ile3
CO
NH
α
170.7, C
CO
NH
α
b
7.89, d (8.8)
4.00, m
8.38
57.8, CH
35.9, C
58.6, CH
35.8, C
3.98, m
β
1.78, m
β
1.72, m
γ1A
γ1B
γ2
δ
23.5, CH2
1.10, m
γ1
γ2
δ
23.6, CH2
15.3, CH3
11.2, CH3
1.02, m
1.02, m
0.63, d (6.7)
a
15.1, CH3
11.1, CH3
170.6, C
0.72, m
0.66
0.68, t (7.4)
L-Phe
CO
NH
α
CO
NH
α
170.9, C
b
b
7.61
8.13
55.4
4.40, m
55.2, CH
36.5, CH2
4.44, m
βA
βB
1′
2′
3′
35.9, CH2
3.03, dd (13.8, 6.2)
2.85, dd (13.8, 9.2)
βA
βB
1′
2′
3′
3.13, m
2.94, dd (10.1, 13.7)
137.5, C
129.0, CH
128.1, CH
126.3, CH
170.8
138.0, C
7.30, m
7.25, m
7.19, m
129.0, CH
128.1, CH
126.2, CH
171.3, C
7.25, m
7.27, m
7.19, m
4′
4′
D-Ile4
CO
NH
α
D-Val
CO
NH
α
7.80, d(8.5)
4.30, m
7.35, d (6.7)
4.00, m
56.0, CH
36.6, CH
25.1, CH2
58.4, CH
29.5, CH
18.2, CH3
18.8, CH3
β
1.81, m
β
2.02, m
a
γ1A
γ1B
γ2
δ
0.99, m
γ1
γ2
0.73
a
0.86, m
0.68
14.0, CH3
11.3, CH3
0.64, d (6.9)
a
0.74
a
b
overlapping signals. Broad signals.
E
DOI: 10.1021/acs.jnatprod.7b00383
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
particle size in order to increase the rate of isolation of fungi
originating from vegetative propagules embedded within substrate
particles as opposed to dormant spores. The separated particles were
resuspended in sterile H2O containing 0.2 g/L chloramphenicol and
18 g/L Instant Ocean and were serially diluted (100- and 1000-fold
dilutions). A 10 μL aliquot from each dilution was pipetted into each
well of a 48-well plate containing YM and cottonseed oil agar and
incubated at 4 or 22 °C for three months. Emerging fungal colonies
were purified to obtain axenic cultures of isolate (RKAG 110).
Identification of the fungus was performed by observation of the
culture phenotype (both macro- and micromorphology) and sequence
homology of the rDNA genes, including the ITS1-5.8S-ITS2 region,
and the nLSU as described previously22 (sequence data are deposited
in GenBank with the accession numbers KY886144 and KY886145).
Extraction and Purification. Isolate RKAG 110 was inoculated
into 15 mL of YM liquid seed medium at 22 °C and agitated at 200
rpm for 5 days. The seed culture (200 μL) was used to inoculate the
isolate onto 150, 100 × 15 mm Petri plates containing 20 mL of solid
PDA agar and grown for 21 days at 22 °C. The solid agar cultures were
roughly cut up, pooled, and extracted with EtOAc. The extract was
evaporated to dryness and partitioned between 80% CH3CN and
100% hexane. The CH3CN layer was collected and evaporated to
dryness in vacuo to give a CH3CN extract (820 mg).
dinitrophenyl-5)-L-alaninamide (0.4 mg in 380 μL of acetone) was
added to the reaction mixture and stirred at 37 °C for 2 h. The
reaction was quenched with 1 N aqueous HCl (80 μL).13 MeOH was
added, and the sample was analyzed by LC-HRMS using a Hypersil
Gold 100 Å column (Thermo, 1.9 μm C18 50 mm × 2.1 mm) and a
flow rate of 400 μL/min. The following method was used: 0−55 min
95% H2O/0.1% formic acid (solvent A) and 5% CH3CN/0.1% formic
acid (solvent B) to 60% solvent A and 40% solvent B, 55−57 min 60%
solvent A:40% solvent B to 100% solvent B, 57−60 min 100% solvent
B. Retention times were compared to derivatized amino acid standards
to determine the amino acid configurations. Partial hydrolysis of the
mortiamides occurred with 6 N HCl at 80 °C for 2 h. The partial
hydrolysis of the compound was monitored by LC-MS for the mass of
the required hydrosylate products. Retention times for the derivatized
amino acid standards are as follows L-Ala 23.07 min, D-Ala 27.71 min,
L-Val 32.24 min, D-Val 38.33 min, L-Phe 39.09 min, D-Phe 43.87 min,
L-Leu 38.64 min, D-Leu 44.17 min, L-Ile 37.42 min, D-Ile 43.49 min, L-
allo-Ile 37.58 min, D-allo-Ile 43.52 min, L-Ile-D-Ala 31.20 min, D-Ile-D-
Ala 37.03 min, L-Phe-D-Phe 43.71 min, D-Phe-L-Phe 48.40 min, L-Ile-D-
Ile 41.85 min, D-Ile-L-Ile 47.69 min, and D-Ile-D-Ile 46.76 min.
Antimicrobial Assays. Compounds 1−4 were tested for
antimicrobial activity according to Clinical Laboratory Standards
Institute testing standards in a 96-well plate microbroth dilution assay
in triplicate against the human microbial pathogens methicillin-
resistant Staphyloccocus aureus ATCC 33591 (MRSA), vancomycin-
resistant Enterococcus faecium EF379 (VRE), Staphylococcus warneri
ATCC 17917, Pseudomonas aeruginosa ATCC 14210, and Candida
albicans ATCC 14035 as previously described.26 Optical density was
recorded at Tzero and Tfinal using a Thermo Scientific Varioskan Flash
plate reader to determine growth inhibition after incubation for 22 h.
Cytotoxicity Assays. Compounds 1−4 were tested for cytotox-
icity using human foreskin BJ fibroblast cells (ATCC CRL-2522),
adult human epidermal keratinocytes (HEKa), Vero kidney cells
(ATCC CL-81), human breast adenocarcinoma cells (ER−) (ATCC
HTB-26), and human breast adenocarcinoma cells (ER+) (ATCC
MCF-7) in triplicate in a 96-well cell culture plate as described
previously.26 Cell viability was determined 24 h after treatment using
the redox dye Alamar Blue to extrapolate cell viability. Fluorescence
was monitored using a Thermo Scientific Varioskan Flash plate reader
at 560/12 excitation, 590 nm emission at both time zero and 4 h after
Alamar Blue addition.
The extract was fractionated using automated reversed-phase flash
chromatography with a linear gradient from 20% aqueous MeOH to
100% MeOH over 15 min on a 15.5 g C18 column (High Performance
GOLD RediSep Rf) with a flow rate of 30 mL/min. The semipure
fraction eluting at 9.5 min was subjected to reversed-phase HPLC
using a Gemini 110A C18 column (5 μm 250 × 10 mm, Phenomenex)
and 20 min isocratic elution with 65% aqueous CH3CN (0.1% formic
acid), resulting in the purification of 1 (4.1 mg), 2 (3.2 mg), 3 (2.4
mg), and 4 (2 mg).
Mortiamide A (1): white powder; [α]25 +26 (c 0.1, MeOH); IR
D
(film) νmax 3309, 2962, 2934, 2875, 1652, 1533, 1456, 1386, 1027
1
cm−1; H and 13C NMR Table 1; (+) HRESIMS m/z 804.5024 [M +
H]+ (calcd for C44H65N7O7, 804.5019).
Mortiamide B (2): white powder; [α]25 +11 (c 0.1, MeOH); IR
D
(film) νmax 3329, 2963, 1651, 1592, 1531, 1456, 1385, 1352, 1026
1
cm−1; H and 13C NMR Table 1; (+) HRESIMS m/z 838.4851 [M +
H]+ (calcd for C47H63N7O7, 838.4862).
Mortiamide C (3): white powder; [α]25 +11 (c 0.1, MeOH); IR
D
(film) νmax 3330, 2963, 2936, 2875, 1646, 1531, 1468, 1392, 1029
1
cm−1; H and 13C NMR Table 2; (+) HRESIMS m/z 770.5189 [M +
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00383.
■
H]+ (calcd for C41H67N7O7, 770.5175).
S
Mortiamide D (4): white powder; [α]25 +20 (c 0.1, MeOH); IR
D
(film) νmax 3329, 2962, 2931, 1652, 1603, 1531, 1465, 1392, 1027
1
cm−1; H and 13C NMR Table 2; (+) HRESIMS m/z 804.5021 [M +
H]+ (calcd for C44H65N7O7, 804.5019).
Dipeptide synthesis methodology, 1D and 2D NMR
spectra, HRMS and MS/MS data for mortiamides A−D
(PDF)
Synthesis of Dipeptide Standards. Benzylation of amino acids
was accomplished according to literature precedent.23,24 All dipeptide
coupling reactions were carried out using benzyl ester protected amino
acids and purchased N-Boc-protected amino acids according to
modified literature precedent.25
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 902 566 0565. Fax: +1 902 566 7445. E-mail: rkerr@
upei.ca.
■
Deprotection of all dipeptides was accomplished first by hydro-
genolysis with ∼20% w/w of 10% Pd/C in CH2Cl2 under a H2
atmosphere for 24−48 h at room temperature, to produce the
corresponding free carboxylic acid. Reactions were filtered through a
Celite plug, dried in vacuo, and used without further purification. Boc
removal was accomplished by treatment with a 5 mL mixture of 1:1
CH2Cl2/trifluoroacetic acid (TFA) for 1 h at room temperature with
stirring. Residual CH2Cl2 and TFA were removed in vacuo. To ensure
TFA removal, samples were dried, reconstituted in CH2Cl2, and dried
again. This process was repeated several times (4×) to ensure
complete TFA removal, yielding the fully deprotected trifluoroacetic
acid salt of the corresponding dipeptide. Reaction progress was
monitored by TLC analysis. Deprotected dipeptides were used
without further purification or extensive characterization.
ORCID
Alyssa L. Grunwald: 0000-0001-9688-0003
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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 Canada Inc., Innovation
Absolute Configuration Assignments. Mortiamides A−D (0.25
mg each) were hydrolyzed with stirring in 6 N HCl (60 μL) at 80 °C
for 6 h and neutralized with 1 M NaHCO3 solution. N-(5-Fluoro-2,4-
F
DOI: 10.1021/acs.jnatprod.7b00383
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
PEI, and the Jeanne and Jean-Louis Lev
also acknowledge Nunavut Tunngavik Inc., for permission to
collect sediment samples and J. Shirley (Nunavut Research
Institute) for assistance with field work. Lastly, we acknowledge
NMR services provided by Dr. C. Kirby and M. Fischer
(AAFC).
Article
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Carr, G. S.; Vansteeland, M.; Kerr, R. G. Mycology 2014, 5, 130−144.
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DOI: 10.1021/acs.jnatprod.7b00383
J. Nat. Prod. XXXX, XXX, XXX−XXX