Leveraging Peptaibol Biosynthetic Promiscuity for Next-Generation Antiplasmodial Therapeutics

Article abstract of DOI:10.1021/acs.jnatprod.0c01370

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC50 < 0.30 μg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 μg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1-14 and 28-43), along with 22 known metabolites (15-27 and 44-52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC50 = 0.10 μM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC50 > 25 μM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.

Full text of DOI:10.1021/acs.jnatprod.0c01370

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Article
Leveraging Peptaibol Biosynthetic Promiscuity for Next-Generation
Antiplasmodial Therapeutics
§
§
Jin Woo Lee, Jennifer E. Collins, Karen L. Wendt, Debopam Chakrabarti,* and Robert H. Cichewicz*
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ABSTRACT: Malaria remains a worldwide threat, afflicting over 200
million people each year. The emergence of drug resistance against
existing therapeutics threatens to destabilize global efforts aimed at
controlling Plasmodium spp. parasites, which is expected to leave vast
portions of humanity unprotected against the disease. To address this
need, systematic testing of a fungal natural product extract library
assembled through the University of Oklahoma Citizen Science Soil
Collection Program has generated an initial set of bioactive extracts
that exhibit potent antiplasmodial activity (EC50 < 0.30 μg/mL) and
low levels of toxicity against human cells (less than 50% reduction in
HepG2 growth at 25 μg/mL). Analysis of the two top-performing
extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both
contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial
actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1−14 and 28−43), along
with 22 known metabolites (15−27 and 44−52). While several compounds displayed promising activity profiles, one of the new
metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC = 0.10 μM against multi-drug-
5
0
resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC50
5 μM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for
translating peptaibols into a bioactive scaffold worthy of further development.
>
2
alaria is a devastating infectious disease caused by
Plasmodium spp. parasites, transmitted through the bite
artemisinin resistance among Plasmodium parasites. Consider-
ing the healthcare burden imposed by malaria, emerging
threats presented by the parasite, and the yet unanswered
demand for safer and more effective therapeutics, there is a
critical need to identify new antiplasmodial scaffolds for lead
development.
M
of female Anopheles spp. mosquitoes. This disease is prevalent
throughout tropical and sub-Saharan regions, with pregnant
women and children facing the highest risk of mortality.
Despite recent improvements afforded by artemisinin combi-
nation therapy (ACT), malaria remains a major health and
economic burden to developing countries, with 229 million
The historic success achieved from applying natural
products to the field of malaria treatment provides hope for
future discovery efforts based on natural scaffolds. To this end,
peptides are one class of natural products that offer promising
indications for focused development. To date, several types of
naturally occurring peptides have been tested for their
antiplasmodial properties. For example, insect-derived peptides
have been shown to possess inhibitory activities against the
1
cases reported in 2019 alone. Approved treatments for malaria
consist of several chemical scaffolds including (i) sesquiterpene
peroxides (e.g., artemisinin derivatives and analogues), (ii)
quinoline ring derivates (e.g., quinine, quinidine, primaquine,
mefloquine, atovaquone, chloroquine), (iii) antifolates (e.g.,
pyrimethamine, sulfadoxine), and (iv) repurposed antibiotics
(
e.g., clindamycin, doxycycline, tetracycline) (Figure 1).
6
Plasmodium parasite, including cecropins from giant silk
Notably, this group of therapeutics is dominated by natural
products, which have been a mainstay of antimalarial agents for
many decades. Unfortunately, growing levels of clinical
resistance have been observed for all the compounds listed,
and their administration is often limited due to an assortment
7,8
moths that inhibit the growth of P. falciparum. Other
examples include scorpine and meucine-25, obtained from
Received: December 21, 2020
Published: February 10, 2021
2−4
of dose-limiting side effects.
Of particular concern is the
spread of artemisinin-resistant parasites throughout large
portions of the Greater Mekong Subregion in Southeast
5
Asia. This has led to a precarious healthcare situation, as
monitoring efforts have revealed increasing levels of
©
2021 American Chemical Society and
American Society of Pharmacognosy
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03
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Figure 1. Structural classification of antimalarial drugs.
scorpion venom, which exhibit antimalarial effects against both
efrapeptins, and zervamicins, which inhibit P. falciparum with
EC50 values ranging from 0.45 to 6.16 μM.
9
,10
16
P. falciparum and P. berghei.
An additional intriguing
example is gambicin, which is isolated from Anopheles spp., and
it exhibits inhibitory effects against P. berghei ookinetes.
Recognizing the value offered by fungi and their natural
products, we initiated a program to identify chemical matter
that could serve as new scaffolds for antiplasmodial
pharmacophore development. These efforts focused on
utilizing a portion of the >66 000 isolates in the University
of Oklahoma Natural Products Discovery Group screening
library, which is composed primarily of fungi obtained through
the Citizen Science Soil Collection Program. This report
focuses on our team’s collaborative efforts identifying,
purifying, characterizing, and testing a diverse selection of
bioactive peptaibols and lipopeptaibols. These compounds
provide valuable insights for the development of fungal natural
products as antiplasmodial agents.
11
Microorganisms represent another prospective source for the
discovery of peptidic natural products with antiplasmodial
activities. Gallinamide A from a marine cyanobacteria and
rhabdopeptide/xenortide-like peptides from the bacterium
Xenorhabdus innexi exhibit potent antiprotozoal activities
against P. falciparum, with EC values of 8.4 and 0.09−3.2
17
50
1
2,13
μM, respectively.
Fungi are a widely recognized source of novel peptidic
molecules, but these compounds have been given scant
attention as a resource for the creation of antiplasmodial
leads. Among the handful of interesting antiplasmodial natural
products reported from fungi are cyclic tetrapeptides such as
apicidin A, which inhibits class I histone deacetylase enzymes
and is lethal to P. falciparum with a minimum inhibitory
RESULTS AND DISCUSSION
■
3
An in vitro biological screening system was used to test over
000 fungus-derived natural product samples from the
1
4
concentration (MIC) of 0.19 μM. Additionally, the
kozupeptins obtained from Paracamarosporium sp. were
shown to possess potent antiplasmodial activities against
both chloroquine-sensitive and chloroquine-resistant P.
falciparum strains, with EC values in the range of 0.15−
University of Oklahoma Citizen Science Soil Collection for
inhibition of the asexual stage of the intraerythrocytic life cycle
of P. falciparum. This process included a counter screen against
the HepG2 human cell line, to identify substances that afford
selective toxicity against P. falciparum. Based on those tests, a
subset of 38 extracts that exhibited potent (EC50 < 0.30 μg/
mL) and selective (>50% growth in HepG2 at 25 μg/mL)
5
0
1
5
1
.46 μM. Further examples of inspiring fungus-derived
antiplasmodial peptidic natural products are found scattered
among several groups of compounds including antiamoebin,
5
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Table 1. Inhibitory Effects of 18-AA Peptaibols (1−5 and 15−19)
a
a
compound
MW
R1
R2
R3
R4
R5
P. falciparum EC50 (μM)
HepG2 EC50 (μM)
selectivity index (SI)
trichorzin NPDG A (1)
trichorzin NPDG B (2)
trichorzin NPDG C (3)
trichorzin NPDG D (4)
trichorzin NPDG E (5)
trichorzin HA I (15)
trichorzin HA II (16)
trichorzin HA V (17)
trichorzin HA VI (18)
trichorzin HA VII (19)
1703
1704
1731
1745
1759
1703
1717
1731
1745
1745
L-Ala
Aib
Aib
Aib
L-Val
L-Val
L-Val
L-Ile
D-Iva
Aib
L-Gln
L-Glu
L-Gln
L-Gln
L-Gln
L-Gln
L-Gln
L-Gln
L-Gln
L-Gln
1.14 ± 0.09
1.66 ± 0.09
0.89 ± 0.10
0.61 ± 0.11
0.87 ± 0.04
1.18 ± 0.05
0.45 ± 0.06
0.75 ± 0.09
0.92 ± 0.10
0.59 ± 0.06
17.42 ± 1.70
>25
15
>15
14
16
7
18
28
20
20
19
Aib
D-Iva
Aib
D-Iva
D-Iva
D-Iva
Aib
12.30 ± 1.48
9.61 ± 0.63
6.23 ± 0.19
21.40 ± 0.29
12.80 ± 1.57
14.67 ± 0.25
17.99 ± 0.74
11.10 ± 0.92
D-Iva
D-Iva
Aib
D-Iva
Aib
L-Ile
L-Val
L-Val
L-Val
L-Val
L-Val
Aib
Aib
Aib
D-Iva
D-Iva
D-Iva
D-Iva
D-Iva
D-Iva
D-Iva
D-Iva
L-Val
a
Results are expressed as means from triplicate experiments.
Figure 2. ECD spectra of peptaibols and lipopeptaibols: (A) peptaibols composed of 18 amino acid residues (1−5); (B) peptaibols composed of
4 amino acid residues (6−13); (C) peptaibols composed of 11 amino acid residues (14); (D) lipopeptaibols composed of 7 amino acid residues
28−30); (E) lipopeptaibols composed of 11 amino acid residues (31−36); and (F) lipopeptaibols composed of 15 amino acid residues (37−43).
1
(
activity against P. falciparum were identified. The top-
performing extracts were examined by LC-MS to gain an
understanding of each sample’s natural product profiles. Two
of the samples provided evidence that peptidic natural
products were the dominant types of metabolites in the
extracts. The gene sequence data for the ribosomal internal
transcribed spacer (ITS) regions of the fungi were analyzed by
BLAST comparisons to sequences contained in GenBank with
one fungal isolate identified as a probable Trichoderma sp.
determined that the bioactive metabolites from the isolates
should be examined in tandem since their respective LC-MS
data indicated both fungi were prodigious producers of
nonoverlapping sets of natural products. Doing so afforded
the unique opportunity to comparatively test dozens of
compounds in parallel. Thus, we proceeded to generate a
mini-library of peptides from the two fungal isolates using a
chemistry- and bioassay-guided purification processes. Those
efforts afforded a set of 30 peptaibols (1−14 and 28−43) that
could not be matched to previously described natural products,
along with 22 known compounds (15−27 and 44−52) for
testing against P. falciparum.
(
100% match to Trichoderma harzianum), and the other isolate
was found to be a probable Hypocrea sp. (99.6% match to
Hypocrea pachybasioides). Exploratory bioassay-guided fractio-
nation confirmed that both extracts contained combinations of
known and presumptively new peptaibols and lipopeptaibols
that were responsible for the observed bioactivity. We
Structure Characterization of Peptaibols from T.
harzianum. Compound 1 was obtained as a colorless solid,
and its molecular formula (C H N O ) was ascertained
7
9
138 20 21
5
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from the HRESIMS data. The 1H NMR spectrum of 1
data for 3 provided evidence that this peptaibol contained 18
contained signals representing both amide protons (δ 7 to 10
amino acid residues and was structurally similar to the reported
H
2
4,25
ppm) and α-protons derived from amino acid residues (δ 4 to
metabolite trichorzin HA II (16).
The key difference
H
13
7
5
ppm), while the C, HSQC, and HMBC NMR spectra of 1
exhibited 79 carbon signals including 20 amide carbonyls
Figure S1, Supporting Information). The HSQC, HMBC, and
between the metabolites was the Aib residue in 16 had
changed to an Iva residue in 3. This was supported by the C
1
3
(
NMR data, which contained two signals at δ 7.44 and 7.40
C
COSY spectrum of 1 led to the tentative identification of
several structural features consistent with other Trichoderma-
characteristic for the Iva γ-methyl groups. Subsequent analysis
of the HMBC and ROESY, as well as MS/MS data, aided in
confirming the identities and placement of each amino acid
residue including both Iva residues (i.e., Iva and Iva ). To
confirm the absolute configurations of the chiral amino acids,
Marfey’s analysis was employed, thus affirming that the
1
8−23
derived peptaibol natural products.
This included
7
16
evidence for 18 amino acid residues consisting of a
combination of 10 proteogenic amino acids [two alanines
(
(
Ala), two glycines (Gly), two leucines (Leu), two glutamines
Gln), valine (Val), and proline (Pro)] and eight non-
1
2
3
4
structure of the metabolite was Ac-Aib -Gly -L-Ala -Aib -
5
6
7
8
9
10
11
12
13
proteogenic amino acids that are frequently associated with
fungal NRPS chemistry [six α-aminoisobutyric acids (Aib),
isovaline (Iva), and leucinol (Leuol)]. Analysis of the NMR
data revealed additional features of prototypical peptaibol
chemistry, including C-terminal (reduction of Leu to a Leuol
residue) and N-terminal (acetylated Aib residue) modifica-
Aib -L-Gln -D-Iva -L-Val -Aib -Gly -L-Leu -Aib -L-Pro -L-
1
4
15
16
17
18
Leu -Aib -D-Iva -L-Gln -L-Leuol . This compound was
assigned the trivial name trichorzin NPDG C (3). The ECD
spectrum of 3 revealed negative Cotton effects at 208 and 225
nm, which supported its right-handed helical conformation
(Figure 2A).
1
2
3
tions. The linear sequence of peptaibol 1 (Ac-Aib -Gly -Ala -
Aib -Ala -Gln -Aib -Val -Aib -Gly -Leu -Aib -Pro -Leu -
Upon purification, compound 4 was observed to be a
colorless solid that was assigned the molecular formula
C H N O based on interpretation of its HRESIMS data.
4
5
6
7
8
9
10
11
12
13
14
1
5
16
17
18
Aib -Iva -Gln -Leuol ) was determined by HMBC correla-
tions between amide carbons and amide protons, as well as
ROESY correlations between α-protons. The sequence of 1
was further confirmed by interpretation of ESIMS/MS
fragmentation data, which revealed the metabolite was
structurally similar to trichorzin HA I (15) from T.
8
2
144 20 21
Analysis of the 1D and 2D NMR data for 4 revealed that its
amino acid sequence was similar to that of trichorzin HA V
24,25
13
(17).
One important difference noted in the C NMR
spectrum of 4 was that it contained two signals characteristic
for the methyl groups of an isoleucine (δ 16.1 and 10.8) rather
C
2
4,25
harzianum.
1
A comparison of the structures of metabolites
than the signals attributed to a valine in 17. Subsequent
5
16
and 15 revealed that 1 contained both Ala and Iva
probing of the metabolite by MS/MS provided evidence
1
2
3
4
5
residues, whereas those positions were occupied by Aib
supporting the sequence of 4 as Ac-Aib -Gly -Ala -Aib -Iva -
5
6
7
8
9
10
11
12
13
14
15
16
residues in 15. The inclusion of the Ala residue in 1 is an
Gln -Aib -Ile -Aib -Gly -Leu -Aib -Pro -Leu -Aib -Iva -
1
7
18
unusual structural feature compared to other trichorzin
peptaibols (Table 1). The absolute configurations of the
amino acids bearing stereogenic centers were analyzed using
Gln -Leuol . The absolute configurations of the amino acid
residues were investigated using Marfey’s method, which
succeed in resolving most of the amino acid configurations
except for the Ile residue. After several failed attempts to
chromatographically resolve the products of DL-Ile and DL-allo-
Ile by Marfey’s method, a GITC (2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl isothiocyanate) derivatization method was
used, leading to the conclusion that the metabolite contained
2
6,27
Marfey’s method,
which revealed that amino acid residues
3
, 5, 6, 8, 11, 13, 14, 17, and 18 were L-configured, whereas
1
6
Iva was D-configured. The electronic circular dichroism
ECD) spectrum of 1 exhibited negative Cotton effects at 208
and 225 nm, which supported a right-handed helical
(
2
8−30
31
1
conformation (Figure 2A).
peptaibol 1 was established to be Ac-Aib -Gly -L-Ala -Aib -L-
Ala -L-Gln -Aib -L-Val -Aib -Gly -L-Leu -Aib -L-Pro -L-
Leu -Aib -D-Iva -L-Gln -L-Leuol , and it was given the
trivial name trichorzin NPDG A (1).
Thus, the structure of
L-Ile. Thus, the structure of 4 was determined to be Ac-Aib -
1
2
3
4
2
3
4
5
6
7
8
9
10
11
Gly -L-Ala -Aib -D-Iva -L-Gln -Aib -L-Ile -Aib -Gly -L-Leu -
5
6
7
8
9
10
11
12
13
12
13
14
15
16
17
18
Aib -L-Pro -L-Leu -Aib -D-Iva -L-Gln -L-Leuol , and it
was assigned the trivial name trichorzin NPDG D (4). A
right-handed helical conformation was deduced for 4 based on
the negative Cotton effects at 208 and 225 nm observed in its
ECD spectrum (Figure 2A).
1
4
15
16
17
18
Compound 2 was obtained as a colorless solid, and its
HRESIMS data were consistent with the molecular formula
C H N O . Based on analysis of its 1D and 2D NMR, the
Compound 5 was purified as a colorless solid, and it was
assigned the molecular formula C H N O based on
7
9
137 19 22
structure of 2 was determined to be similar to that of trichorzin
8
3
146 20 21
2
4,25
HA I (15)
with one key difference: a glutamic acid (Glu)
analysis of the HRESIMS data. The 1D and 2D NMR data for
5 indicated that the compound was a peptaibol consisting of 18
17
residue replaced Gln . This conjecture was supported by
analysis of the mass fragmentation data for 2, confirming the
amino acid residues similar to those reported in trichorzin HA
17
24,25
presence and placement of Glu . The absolute configurations
of the amino acids bearing stereogenic centers were confirmed
by Marfey’s method. Accordingly, the structure of peptaibol 2
VI (18).
Results from the MS/MS fragmentation analysis,
1
3
as well as the detection of methyl signals in the C NMR
spectrum characteristic for Ile residues (δ 15.9 and 10.9), led
C
1
2
3
4
5
6
was determined to be Ac-Aib -Gly -L-Ala -Aib -Aib -L-Gln -
to the conclusion that the Val residue in 18 was switched to an
Ile residue in 5. To determine the absolute configurations of
amino acid residues in 5, both Marfey’s and GITC methods
were employed. Based on the results of those analyses, the
7
8
9
10
11
12
13
14
15
Aib -L-Val -Aib -Gly -L-Leu -Aib -L-Pro -L-Leu -Aib -
1
6
17
18
Aib -L-Glu -L-Leuol , and it was assigned the trivial name
trichorzin NPDG B (2). The ECD spectrum of 2 revealed
negative Cotton effects at 208 and 225 nm, which indicated its
right-handed helical conformation (Figure 2A).
1
absolute configuration of 5 was determined to be Ac-Aib -
2
3
4
5
6
7
8
9
10
11
Gly -L-Ala -Aib -D-Iva -L-Gln -D-Iva -L-Ile -Aib -Gly -L-Leu -
1
2
13
14
15
16
17
18
Compound 3 was obtained as a colorless solid, and its
HRESIMS data were used to assign this metabolite the
Aib -L-Pro -L-Leu -Aib -D-Iva -L-Gln -L-Leuol , and the
compound was given the trivial name trichorzin NPDG E (5).
A right-handed helical conformation was assigned to 5 based
13
molecular formula C H N O . The C NMR and HMBC
81
142 20 21
5
06
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1
2
on the appearance of negative Cotton effects at 208 and 225
nm observed in its ECD spectrum (Figure 2A).
established its absolute configuration as Ac-D-Iva -L-Asn -L-
3
4
5
6
7
8
9
10
11
12
Leu -Aib -L-Pro -L-Ser -L-Ile -Aib -L-Pro -Aib -L-Leu -Aib -
1
3
14
Compound 6 was obtained as a colorless solid. The
HRESIMS data were consistent with the molecular formula
L-Pro -L-Leuol , and it was assigned the trivial name harzianin
NPDG C. The ECD data for 8 were examined (Figure 2B),
and the results were consistent with a right-handed helical
conformation.
Compound 9 was obtained as a colorless solid, and its
HRESIMS data were consistent with the molecular formula
C H N O . Based on the substantial similarities among the
MS, H NMR, HSQC, and HMBC data for 9 and co-occurring
metabolites 8 and harzianin HC XII (23), it was concluded
that one of the Aib residues had been replaced by an Iva in 9
i.e., δ 8.44, 8.28, and 8.19 for NH of Aib residues and δH
.80 and 7.89 attributed to NH of Iva residues). Examination
of the MS/MS data identified a fragment ion at m/z 1032 (Ac-
Iva ), securing the position of the new Iva residue. HMBC
and ROESY data, along with Marfey’s and GITC analyses
Figure S9), were used to resolve the structure of 9 as Ac-D-
Iva -L-Asn -L-Leu -Aib -L-Pro -L-Ser -L-Ile -Aib -L-Pro -D-Iva -
L-Leu -Aib -L-Pro -L-Leuol , and the compound was given
the trivial name harzianin NPDG D. Evidence concerning the
solution conformation of 9 were obtained through the analysis
of ECD data and were found to be consistent with expectations
for a right-handed helix (Figure 2B).
Compound 10 was obtained as a colorless solid, and its
HRESIMS data were used to assign it the molecular formula
C H N O . Based on the observation that compound 10
contained one less oxygen atom than co-occurring metabolite
, the HSQC data for both compounds were comparatively
probed, revealing that a signal attributable to a hydroxymethyl
group in 7 (δ 62.1) had been replaced by a new methyl group
δC 16.8) in 10. Considering how this change would manifest
1
13
C H N O . The H and C NMR data for 6 supported
6
8
117 15 17
the results of the mass spectrometry experiment indicating that
the compound was a peptaibol; however, it was smaller than
13
metabolites 1−5. The C NMR of 6 revealed 15 carbonyl
resonances (δ 171−178 ppm), while the HSQC, HMBC, and
C
70 121 15 17
1
COSY spectra suggested the compound likely contained 14
amino acid residues including Asn, Ser, Aib, Pro, Val, Iva, Leu,
and Leuol (Figure S6). The sequence of amino acid residues in
32
6
was established using HMBC and ROESY correlation data
(
H
1
2
3
4
5
6
7
and confirmed to be Ac-Iva -Asn -Leu -Aib -Pro -Ser -Val -
9
8
9
10
11
12
13
14
Aib -Pro -Aib -Leu -Aib -Pro -Leuol . The tentative struc-
ture of 6 was further supported by MS/MS data, which
contained several intense fragment ion peaks at m/z 454 (Ac-
10
4
5
14
8
Aib ) and 963 (Pro -Leuol ), m/z 822 (Ac-Aib ) and 595
(
9
14
12
13
(
Pro -Leuol ), and m/z 1202 (Ac-Aib ) and 215 (Pro -
1
2
3
4
5
6
7
8
9
10
1
4
Leuol ). We conjectured that these abundant ions stemmed
from the cleavage of one or both MS-labile bonds associated
with the Aib and Pro residues. Notably, the same MS data
pattern was observed for compound 20, which had been
obtained from the same fungal isolate and dereplicated as the
metabolite harzianin HC I. Comparison of the MS and NMR
data for the two compounds indicated that the Aib residue in
11
12
13
14
32
2
2
20 had been changed to Iva in 6. Marfey’s method was used
6
9
119 15 16
to secure the absolute configurations of the amino acids
bearing stereogenic centers in 6, which led to the conclusion
7
1
2
that the structure of the metabolite was Ac-D-Iva -L-Asn -L-
3
4
5
6
7
8
9
10
11
12
Leu -Aib -L-Pro -L-Ser -L-Val -Aib -L-Pro -Aib -L-Leu -Aib -
1
3
14
C
L-Pro -L-Leuol . Metabolites 6 was given the trivial name
harzianin NPDG A. An examination of the ECD data for 6
supported its conformation as a right-handed helix based on
the appearance of negative Cotton effects at 205 and 230 nm
Figure 2B).
Compound 7 was isolated as a colorless solid, and its
molecular formula was determined to be C H N O based
(
itself in the structure of 10, it was determined that the most
probable solution involved substituting the Ser residue in 7
with an Ala residue in metabolite 10. Subsequent analysis of
data from HMBC, ROESY, and MS/MS experiments
confirmed this change, as well as helped to establish the
amino acid sequence of 10. Marfey’s and GITC methods were
used to establish the absolute configurations of its amino acid
residues (Figure S10). Accordingly, the structure of peptaibol
2
8−30
(
69
119 15 17
on interpretation of its HRESIMS data. Analysis of the MS/
MS and 1D and 2D NMR for 7 led to the realization that this
32
peptaibol was structurally similar to harzianin HC XI (22).
A
1
2
3
4
5
1
0 was determined to be Ac-Aib -L-Gln -L-Leu -Aib -L-Pro -L-
key structural difference between the two compounds was
supported by the presence of a fragment ion at m/z 256 (Ac-
6
7
8
9
10
11
12
13
14
Ala -L-Ile -Aib -L-Pro -Aib -L-Leu -Aib -L-Pro -L-Leuol ,
and it was given the trivial name harzianin NPDG E.
Compound 10 was determined to have a right-handed helical
conformation as determined via analysis of its ECD data
(Figure 2B).
1
2
13
Aib -Gln ) in 7 and C NMR chemical shift data that led to
the conclusion that the Asn residue in 22 was replaced by a
Gln. Using Marfey’s and GITC methods, the absolute
1
2
configuration of 7 was determined to be Ac-Aib -L-Gln -L-
3
4
5
6
7
8
9
10
11
12
Compound 11 was purified as a colorless solid, and it was
assigned the molecular formula C H N O based on
Leu -Aib -L-Pro -L-Ser -L-Ile -Aib -L-Pro -Aib -L-Leu -Aib -
1
3
14
L-Pro -L-Leuol , and it was given the name harzianin NPDG
B. The ECD data for 7 supported its conformation as a right-
handed helix (Figure 2B).
70 121 15 16
interpretation of its HRESIMS data. An examination of the 1D
and 2D NMR of 11 revealed that it was structurally similar to
compound 10; however, one of the Aib residues had been
replaced by an Iva residue. Further scrutiny of the HMBC data
indicated that the new Iva residue was positioned at the
acetylated N-terminus of 11. A combination of ROESY and
MS/MS data was used to secure the sequence of 11, while
Marfey’s and GITC methods were used to establish its
Compound 8 was obtained as a colorless solid, and its
molecular formula (C H N O ) was established from
69
119 15 17
13
HRESIMS data. Examination of the C NMR and HSQC data
for 8 revealed the metabolite shared many structural features
with 6; however, key differences were observed including the
7
resonances attributable to Val in 6 were replaced in 8 by peaks
1
2
3
4
5
corresponding to an Ile residue. Further investigation of the
HMBC and ROESY correlation data for compound 8, as well
as results from its MS/MS fragmentation pattern, supported
absolute configuration as Ac-D-Iva -L-Gln -L-Leu -Aib -L-Pro -
6
7
8
9
10
11
12
13
14
L-Ala -L-Ile -Aib -L-Pro -Aib -L-Leu -Aib -L-Pro -L-Leuol .
Compound 11 was assigned the trivial name harzianin NPDG
F. Metabolite 11 was proposed to possess a right-handed
helical conformation based on data acquired from an ECD
experiment (Figure 2B).
1
2
3
the proposed amino acid residue sequence Ac-Iva -Asn -Leu -
4
5
6
7
8
9
10
11
12
13
14
Aib -Pro -Ser -Ile -Aib -Pro -Aib -Leu -Aib -Pro -Leuol .
Further investigation of 8 using Marfey’s and GITC methods
5
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32
32
32
Compound 12 was purified as a colorless solid. HRESIMS
analysis revealed a doubly charged quasimolecular ion that was
consistent with the molecular formula C H N O . Analysis
of 1D and 2D NMR spectroscopic data for 12 revealed its high
degree of similarity to 9 with the exception that the Ser
(21), harzianin HC XI (22), harzianin HC XII (23),
32 32
harzianin HC XIV (24),3 harzianin HC X (25), and
2
harzianin HC XV (26) ], and one harzianin-HB-type
7
0
121 15 16
33
peptaibol [harzianin HB I (27) ]. This terminology is
intended to help avoid further confusion associated with the
circuitous naming history of these structurally diverse
metabolites. The co-production of so many different peptaibol
types from a single fungus is not entirely unusual; however, the
combination of peptaibol diversity and abundance observed
from this Trichoderma isolate offers a striking example of
nature’s capacity for biosynthetic promiscuity and structural
experimentation.
Structure Elucidation of Peptaibols and Lipopeptai-
bols Isolated from H. pachybasioides. Compound 28 was
obtained as a colorless solid, and its molecular formula was
determined to be C H N O based on interpretation of its
6
residue had been replaced by an Ala residue. Marfey’s and
GITC methods were used to establish the identities and
absolute configurations of the amino acid residues in peptaibol
1
2
3
4
5
6
7
8
1
2 as Ac-D-Iva -L-Asn -L-Leu -Aib -L-Pro -L-Ala -L-Ile -Aib -L-
9
10
11
12
13
14
Pro -D-Iva -L-Leu -Aib -L-Pro -L-Leuol , and it was given
the trivial name harzianin NPDG G. Analysis of the ECD
spectrum of 12 revealed Cotton effects (Figure 2B) that were
consistent with the metabolite possessing a right-handed
helical conformation.
Compound 13 was obtained as a colorless solid, and its
HRESIMS data were consistent with the molecular formula
C H N O . Based on the analysis of its 1D and 2D NMR
3
7
69
7
8
1
HRESIMS data. An examination of the H NMR data yielded
7
1
123 15 16
spectroscopy data, the structure of 13 was proposed to be
similar to 12. The major difference was that the Asn residue in
evidence supporting the occurrence of several putative NH (δH
2
8.0−10.0 ppm) and α-protons (δ 3.7−5.0 ppm) that were
H
12 was replaced by a Gln residue in 13. Further investigation
indicative of seven amino acids in 28 (Figure S15). Analysis of
1
3
of the MS/MS, HMBC, and ROESY data helped secure the
amino acid sequence of 13, while the absolute configurations
of its chiral amino acid residues were established using
Marfey’s and GITC methods. Thus, the structure of 13 was
the C, HMBC, and COSY NMR data supported the presence
of two Aib, two Gly, one Val, one Ile, and one Leuol residue,
which left eight carbon atoms unaccounted for in 28.
Examination of the COSY and HMBC correlation data
attributable to the remaining carbon atoms led to the
conclusion that they formed an n-octanoyl moiety. A
combination of HMBC and ROESY correlation data along
with MS/MS fragmentation results were employed to
1
2
3
4
5
6
determined to be Ac-D-Iva -L-Gln -L-Leu -Aib -L-Pro -L-Ala -L-
7
8
9
10
11
12
13
14
Ile -Aib -L-Pro -D-Iva -L-Leu -Aib -L-Pro -L-Leuol , and it
was given the trivial name harzianin NPDG H (13). The ECD
spectrum of 13 exhibited Cotton effects similar to the other co-
occurring metabolites (Figure 2B), which were consistent with
a peptaibol bearing a right-handed helical conformation.
Compound 14 was obtained as a colorless solid, and it was
assigned the molecular formula C H N O based on
1
2
3
4
construct the sequence of 28: n-Oct-Aib -Gly -Val -Aib -
5
6
7
Gly -Ile -Leuol . The absolute configurations of the chiral
amino acid residue were determined by Marfey’s and GITC
methods, which established the structure of metabolite 28 as n-
Oct-Aib -Gly -L-Val -Aib -Gly -L-Ile -L-Leuol . Notably metab-
olite 28, which was given the trivial name hypocrin NPDG A,
is a notable addition to an uncommon subfamily of n-Oct-
peptaibols (lipopeptaibols); it contains a smaller number of
amino acid residues (seven amino acids) as compared to the
more commonly encountered members of the group, which
59
104 12 13
1
3
1
2
3
4
5
6
7
interpretation of its HRESIMS data. The C NMR and
HMBC data for 14 confirmed the presence of 59 unique
carbon atoms including 11 amide carbonyl resonances (δC
∼
170.0−180.0). Those data indicated that metabolite 14
contained fewer amino acid residues compared to peptaibols
−13. Further analysis of the 1D and 2D NMR data set
revealed that 14 was structurally similar to harzianin HB I
1
34−37
contain 11 amino acids.
Interpretation of the ECD data
3
3
1
2
(27), but the Aib -Asn residues in 27 had been replaced by
for 28 indicated that this compound did not exhibit a right-
handed helical conformation (Figure 2D) like the other
metabolites from the Trichoderma isolate. We speculated that
the reduced length of the amino acid residue chain was too
short for this compound to adopt distinctive secondary
1
2
Iva -Gln in 14. The HMBC and ROESY correlation data were
further probed, thus establishing the sequence of 14, while
Marfey’s and GITC methods were used to confirm the
absolute configurations of its chiral amino acids resulting in the
1
2
3
4
5
6
38−40
proposed structure Ac-D-Iva -L-Gln -L-Leu -L-Ile -Aib -L-Pro -
structural features, and instead, it remained a random coil.
7
8
9
10
11
D-Iva -L-Leu -Aib -L-Pro -L-Leuol . Metabolite 14 was given
the trivial name harzianin NPDG I, and it was determined to
possess a right-handed helical conformation based on
Compound 29 was isolated as a colorless solid, and its
molecular formula was established as C H N O based on
3
7
69
7
8
interpretation of its HRESIMS data. The molecular formula of
29 was identical to that for metabolite 28, which provided an
initial indication that these compounds might be structurally
related. An inspection of the 1D and 2D NMR data revealed
28−30
interpretation of the results of ECD data (Figure 2C).
Based on our investigation, natural products 1−14 had not
been previously reported; however, these metabolites
possessed structural features enabling their assignments to
previously described families/subfamilies of peptaibols. For the
sake of simplicity and clarity, we have used the predominant
familial name of each peptaibol and added the cognominal
term “NPDG” (Natural Products Discovery Group) to retain
information pertaining to the structural relationship of each
compound to the known co-occurring metabolites reported
from this fungal genus, which included five trichorzin-HA-type
3
6
3
that the Val and Ile residues in 28 had been replaced by Leu
6
and Val residues in 29. Using Marfey’s method, the absolute
configurations of the chiral amino acids were established,
resulting in the structure elucidated as n-Oct-Aib -Gly -L-Leu -
Aib -Gly -L-Val -L-Leuol . Lipopeptaibol 29 was given the
trivial name hypocrin NPDG B. Analysis of the ECD data
indicated that compound 29 did not possess a defined
secondary structure and was instead a random coil (Figure
2D).
1
2
3
4
5
6
7
2
4,25
peptaibols [trichorzin HA I (15),
trichorzin HA II
trichorzin HA VI
24,25
2
4,25
24,25
(
(
16),
18),
trichorzin HA V (17),
and trichorzin HA VII (19),
Compound 30 was obtained as a colorless solid, and its
HRESIMS data were consistent with the molecular formula
C H N O . In comparison to metabolite 29, compound 30
2
4,25
], seven harzianin-
2
3
HC-type peptaibols [harzianin HC I (20), harzianin HC III
3
8
71
7
8
5
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contained the equivalent of one additional −CH − unit. Upon
36 appeared to contain no Val or Ile residues at positions 3, 7,
or 10. Instead, these amino acid residues had been replaced by
Leu residues. Upon determining the sequence of 36, Marfey’s
method was used to determine the absolute configurations of
the chiral amino acids. Based on interpretation of data from
those experiments, the structure of 36 was established as n-
2
examining the HMBC and ROESY data, it was determined that
these atoms were incorporated into what had formerly been
the Val residue in compound 29, resulting in a new Ile residue
in 30. Further examination of the structure of the molecule
using 1D and 2D NMR experiments, MS/MS fragmentation
data, and Marfey’s and GITC methods yielded evidence
1
2
3
4
5
6
7
8
9
10
Oct-Aib -Gly -L-Leu -Aib -Gly -Gly -L-Leu -Aib -Gly -L-Leu -
1
2
3
4
11
supporting the structure of 30 as n-Oct-Aib -Gly -L-Leu -Aib -
L-Leuol , and it was given the trivial name hypocrin NPDG I.
5
6
7
Gly -L-Ile -L-Leuol . Lipopeptaibol 30 was given the trivial
name hypocrin NPDG C. The ECD spectrum of 30 offered no
evidence supporting the presence of a defined secondary
structure for this metabolite (Figure 2D).
Analysis of the ECD spectrum for 36 revealed negative Cotton
effects at 206 and 223 nm, indicative of a right-handed helical
conformation (Figure 2E).
Compound 37 appeared as a colorless solid, and its
molecular formula (C H N O ) was assigned based on
Compound 31 was purified as a colorless solid, and it was
assigned the molecular formula C H N O based on
6
3
113 15 16
analysis of its HRESIMS data. Compared to metabolites 30−
49
89 11 12
1
3
interpretation of its HRESIMS data. Examination of the 1D
and 2D NMR data for 31 confirmed the presence of four
additional amino acid residues in this metabolite compared to
co-occurring metabolites 28−30. A combination of HMBC
and ROESY correlation data, MS/MS fragmentation analysis,
and Marfey’s method was used to establish the structure of 31
36, compound 37 was larger, with the C NMR and HMBC
data supporting the presence of 15 putative amide carbonyls
(δ ∼170−180 ppm). Using 1D and 2D NMR data, as well as
C
MS/MS fragmentation data, the sequence of 37 was
established, while Marfey’s and GITC methods were used to
verify the absolute configurations of the chiral amino acid
residues. Based on interpretation of the data from these
experiments, the structure of lipopeptaibol 37 was determined
1
2
3
4
5
6
7
8
9
as n-Oct-Aib -Gly -L-Val -Aib -Gly -Gly -L-Val -Aib -Gly -L-
1
0
11
Val -L-Leuol , and the metabolite was given the trivial
name hypocrin NPDG D. Compound 31 was subjected to
ECD spectroscopy, which revealed negative Cotton effects at
1
2
3
4
5
6
7
8
9
to be n-Oct-Aib -Gly -L-Val -Aib -Gly -Gly -L-Val -Aib -Gly -
1
0
11
12
13
14
15
Gly -L-Val -Aib -Gly -L-Ile -L-Leuol , and it was given the
trivial name hypocrin NPDG J. The ECD data for 37 were
consistent with those expected for a peptide possessing a right-
handed helical conformation based on the appearance of
negative Cotton effects at 206 and 222 nm (Figure 2F).
Compounds 38, 39, and 40 were purified as colorless solids
that shared the same molecular formula (C H N O )
2
06 and 223 nm that were indicative of a right-handed helical
conformation (Figure 2E).
Compound 32 was obtained as a colorless solid, and
interpretation of its HRESIMS data supported the molecular
formula C H N O . Inspection of the 1D NMR data for 32
5
0
91 11 12
revealed many of the chemical shifts in this compound were
64
115 15 16
similar to those found in lipopeptaibol 31. An investigation of
based on interpretation of their HRESIMS data. Analysis of the
1D and 2D NMR data indicated that compounds 38, 39, and
40 possessed similar amino acid patterns consisting of n-Oct-
3
2 using HMBC and ROESY correlation data, MS/MS
fragmentation analysis, and Marfey’s method led to its
assignment as n-Oct-Aib -Gly -L-Leu -Aib -Gly -Gly -L-Val -
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
Aib -Gly -(Val or Leu) -Aib -Gly -Gly -(Val or Leu) -Aib -
8
9
10
11
9
10
11
12
13
14
15
Aib -Gly -L-Val -L-Leuol , and it was given the trivial name
hypocrin NPDG E. The ECD spectrum of 32 was dominated
by two negative Cotton effects characteristic for compounds
that adopt right-handed helical conformations (Figure 2E).
Compounds 33−35 were obtained as colorless solids, and all
three compounds were determined to share the molecular
formula C H N O based on interpretation of their
Gly -Gly -(Val or Leu) -Aib -Gly -Ile -L-Leuol . Focusing
on 2D NMR correlation data for the variable portions of each
compound (amino acid residue positions 3, 7, and 11), it was
3
7
determined that lipopeptaibol 38 was composed of Leu , Val ,
and Val , lipopeptaibol 39 comprised Val , Leu , and Val ,
and lipopeptaibol 40 comprised Val , Val , and Leu . With the
sequences of 38−40 established, Marfey’s and GITC methods
were used to secure the absolute configurations of the chiral
amino acid, resulting in the following structure assignments: n-
1
1
3
7
11
3
7
11
5
1
93 11 12
HRESIMS data. A preliminary examination of the 1D and
D data for each of the three compounds revealed that they
2
1
2
3
4
5
6
7
8
9
10
were structurally similar to one another and were analogues of
co-occurring lipopeptaibols 31 and 32. Using this information
to guide the structure determination process, the structures of
Oct-Aib -Gly -L-Leu -Aib -Gly -Gly -L-Val -Aib -Gly -Gly -L-
1
1
12
13
14
15
1
2
Val -Aib -Gly -L-Ile -L-Leuol (38), n-Oct-Aib -Gly -L-
3
4
5
6
7
8
9
10
11
12
Val -Aib -Gly -Gly -L-Leu -Aib -Gly -Gly -L-Val -Aib -
1
3
14
15
1
2
3
4
3
3−35 were solved in parallel using a combination of 1D and
Gly -L-Ile -L-Leuol (39), and n-Oct-Aib -Gly -L-Val -Aib -
5
6
7
8
9
10
11
12
13
14
2
D NMR data, MS/MS fragmentation analysis, and Marfey’s
Gly -Gly -L-Val -Aib -Gly -Gly -L-Leu -Aib -Gly -L-Ile -L-
1
5
and GITC methods. The resulting structures of the
Leuol (40). These compounds were assigned the trivial
names hypocrins NPDG K (38), L (39), and M (40). The
ECD spectra for peptaibols 38−40 exhibited negative Cotton
effects at 206 and 222 nm (Figure 2F), indicative of right-
handed helical conformations. Additionally, colorless block-
shaped crystals of compound 40 were obtained and subjected
to single-crystal X-ray diffraction analysis, which served to
independently confirm the structure and conformation of this
lipopeptaibol (Figure 3).
1
2
3
compounds were determined to be n-Oct-Aib -Gly -L-Leu -
4
5
6
7
8
9
10
11
Aib -Gly -Gly -L-Val -Aib -Gly -L-Leu -L-Leuol (33), n-Oct-
1
2
3
4
5
6
7
8
9
10
Aib -Gly -L-Leu -Aib -Gly -Gly -L-Val -Aib -Gly -L-Ile -L-
1
1
1
2
3
4
5
6
Leuol (34), and n-Oct-Aib -Gly -L-Val -Aib -Gly -Gly -L-
7
8
9
10
11
Leu -Aib -Gly -L-Ile -L-Leuol (35), which were assigned
the trivial names hypocrins NPDG F, G, and H, respectively.
Compounds 33−35 were probed using ECD spectroscopy,
which revealed Cotton effect patterns similar to lipopeptaibols
3
1 and 32. Accordingly, a right-handed helical conformation
Compounds 41, 42, and 43 were obtained as colorless
solids, which shared the molecular formula C H N O
117 15 16
based on analyses of their HRESIMS data. Comparisons of the
1D and 2D NMR data and MS/MS fragmentation results for
41−43 versus 38−40 led to the determination that the
structures of these metabolites were similar; however, each
was assigned to 33−35 (Figure 2E).
6
5
Compound 36 was obtained as a colorless solid. The
HRESIMS data for 36 were interpreted to support the
molecular formula C H N O . Examination of the 1D and
52
95 11 12
2
D NMR revealed that unlike compounds 31−35, metabolite
5
09
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conformations based on the negative Cotton effects at 206 and
2
22 nm (Figure 2F).
To the best of our knowledge, natural products 28−43 had
not been previously reported, but these lipopeptaibols are
structurally similar to other well-established families and
subfamilies of peptidic fungal natural products. As previously
described (vide supra), we attempted to match the new
metabolites described in this report with existing compound
names, while avoiding the perpetuation of awkward alphanu-
meric codes, by adding the term “NPDG” to each new name.
In addition to metabolites 28−43, several additional peptaibols
and lipopeptaibols were purified from the H. pachybasioides
extract and dereplicated by comparisons to reported
spectroscopic data. The compounds dereplicated in this
41
manner included trikoningin KB I (44), trichogin A IV
4
2
43
44
45
(
45), trichogin GB IX (46), trichosporin B IIId (47),
44
trichosporin B IIIa (48), trichosporin B IVc (49),
trichosporin B VIb (50), trichosporin B VIa (51), and
trichosporin B VIIa (52).
46
46
Figure 3. Helical structure of 40 as determined by single-crystal X-ray
diffraction analysis.
46
Testing the Antiplasmodial Activities of Fungal-
Derived Peptidic Natural Products. The 52 peptaibols
and lipopetaibols purified from T. harzianum and H.
pachybasioides, along with two additional fungal peptides
from the University of Oklahoma Natural Products Discovery
Group pure compound library [efrapeptins F (53) and G (54)
member of the new compound set (41−43) possessed new
Leu residues in place of the Val residues in 38−40. The
structures of the lipopeptaibols were established based on
interpretation of 1D and 2D NMR data in combination with
results from Marfey’s and GITC experiments to give n-Oct-
47
were previously procured from a Tolypocladium sp. isolate ],
were tested for their antiplasmodial activities. Tests were
conducted using the multi-drug-resistant P. falciparum line
Dd2, while a cytotoxicity counter screen was performed using
human (HepG2) hepatocytes. All of the compounds exhibited
varying degrees of inhibition of P. falciparum, with several
compounds (10, 11, 13, 14, 48, 51, and 53) demonstrating
potent, submicromolar activities (Tables 1−8).
1
2
3
4
5
6
7
8
9
10
Aib -Gly -L-Val -Aib -Gly -Gly -L-Leu -Aib -Gly -Gly -L-
1
1
12
13
14
15
1
2
Leu -Aib -Gly -L-Ile -L-Leuol (41), n-Oct-Aib -Gly -L-
3
4
5
6
7
8
9
10
11
12
Leu -Aib -Gly -Gly -L-Val -Aib -Gly -Gly -L-Leu -Aib -
1
3
14
15
1
2
3
4
Gly -L-Ile -L-Leuol (42), and n-Oct-Aib -Gly -L-Leu -Aib -
5
6
7
8
9
10
11
12
13
14
Gly -Gly -L-Leu -Aib -Gly -Gly -L-Val -Aib -Gly -L-Ile -L-
1
5
Leuol (43). The compounds were given the trivial names
hypocrins NPDG N (41), O (42), and P (43). Lipopeptaibols
Further exploration of the data to determine how particular
molecular features of the individual metabolites (e.g., presence
or absence of specific amino acid residues and the order in
4
1−43 were subjected to ECD experiments, and the
compounds were determined to possess right-handed helical
Table 2. Inhibitory Effects of 14-AA Peptaibols (6−13 and 20−26)
a
a
compound
MW
R1
R2
R3
R4
R5
P. falciparum EC50 (μM)
HepG2 EC50 (μM)
SI
harzianin NPDG A (6)
harzianin NPDG B (7)
harzianin NPDG C (8)
harzianin NPDG D (9)
harzianin NPDG E (10)
harzianin NPDG F (11)
harzianin NPDG G (12)
harzianin NPDG H (13)
harzianin HC I (20)
harzianin HC III (21)
harzianin HC XI (22)
harzianin HC XII (23)
harzianin HC XIV (24)
harzianin HC X (25)
harzianin HC XV (26)
1415
1429
1429
1443
1413
1427
1427
1441
1401
1415
1415
1429
1399
1413
1427
D-Iva
Aib
L-Asn
L-Gln
L-Asn
L-Asn
L-Gln
L-Gln
L-Asn
L-Gln
L-Asn
L-Asn
L-Asn
L-Asn
L-Asn
L-Gln
L-Gln
L-Ser
L-Ser
L-Ser
L-Ser
L-Ala
L-Ala
L-Ala
L-Ala
L-Ser
L-Ser
L-Ser
L-Ser
L-Ala
L-Ala
L-Ala
L-Val
L-Ile
L-Ile
L-Ile
L-Ile
L-Ile
L-Ile
L-Ile
L-Val
L-Val
L-Ile
L-Ile
L-Ile
L-Val
L-Ile
Aib
Aib
Aib
0.62 ± 0.06
1.75 ± 0.12
0.58 ± 0.06
0.52 ± 0.04
0.29 ± 0.02
0.32 ± 0.01
0.62 ± 0.01
0.33 ± 0.09
0.71 ± 0.08
0.56 ± 0.07
0.63 ± 0.08
0.52 ± 0.05
0.42 ± 0.03
1.32 ± 0.08
0.63 ± 0.08
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>40
>14
>43
>48
>86
>78
>40
>76
>35
>45
>40
>48
>60
>19
>40
D-Iva
D-Iva
Aib
D-Iva
Aib
D-Iva
D-Iva
D-Iva
Aib
Aib
D-Iva
D-Iva
Aib
Aib
Aib
Aib
Aib
Aib
Aib
D-Iva
Aib
D-Iva
Aib
D-Iva
D-Iva
a
Results are expressed as means from triplicate experiments.
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Table 3. Inhibitory Effects of 11-AA Peptaibols (14 and 27)
a
a
compound
MW
R1
R2
P. falciparum EC50 (μM)
HepG2 EC50 (μM)
SI
harzianin NPDG I (14)
harzianin HB I (27)
1188
1160
D-Iva
Aib
L-Gln
L-Asn
0.10 ± 0.01
0.72 ± 0.13
>25
>25
>250
>35
a
Results are expressed as means from triplicate experiments.
Table 4. Inhibitory Effects of 7-AA Lipopeptaibols (28−30)
general, the lipopeptaibols (groups E, F, and G) were less
potent against P. falciparum compared to the other groups of
metabolites. In contrast, the efrapeptins (group H) showed
tremendous potency and selectivity; however, with only two
representatives available from this group, it is not known if our
results are generalizable to all efrapeptins or might be outliers.
Taken together, the peptaibols (groups A, B, C, and D)
exhibited consistent inhibitory activity against P. falciparum.
However, a more notable property of many peptaibols,
especially groups A and B, was the selectivity exhibited toward
the parasite versus human cells. Several of these metabolites
HepG2
P. falciparum
EC
50
a
a
compound
MW
R1
R2
EC (μM)
(μM)
SI
50
hypocrin
NPDG A
739 L-Val L-Ile
739 L-Leu L-Val
753 L-Leu L-Ile
1.12 ± 0.05
3.00 ± 0.15
2.90 ± 0.22
>25
>22
(28)
(
e.g., compounds 10, 11, 13, 14, and 24) exhibited selectivity
hypocrin
NPDG B
>25
>25
>8
>9
index values (SI = EC50 for HepG2 cells/EC50 for P.
falciparum) that were > 60. The only compound from another
group to achieve such a remarkable threshold was 53 from
group H (SI > 64). These results indicate that peptaibols and
lipopeptaibols are capable of eliciting divergent toxicity profiles
across dissimilar organisms such as humans (Chordata) and
Apicomplexa. These results provide a reasonable basis for
optimism that further investigation of peptailbol and lip-
opeptaibol chemistry may offer additional potent inhibitors of
Plasmodium spp. that exhibit little or no toxicity toward
humans.
(29)
hypocrin
NPDG C
(30)
a
Results are expressed as means from triplicate experiments.
which the amino acids appeared) impacted biological activity
proved unfruitful at detecting decisive structure−activity
trends. However, some notable patterns emerged when the
data were considered in aggregate based on their classification
into eight groups: peptaibols composed of 11 amino acid
residues (group A), peptaibols composed of 14 amino acid
residues (group B), peptaibols composed of 18 amino acid
residues (group C), peptaibols composed of 20 amino acid
residues (group D), lipopeptaibols composed of seven amino
acid residues (group E), lipopeptaibols composed of 11 amino
acid residues (group F), lipopeptaibols composed of 15 amino
acid residues (group G), and efrapeptins (group H). Data
visualization using a scatter plot (Figure 4) revealed that, in
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation measure-
ments were made using a Rudolph Research Autopol III automatic
polarimeter. NMR spectra were obtained on Varian NMR
■
1
spectrometers (400 and 500 MHz for H and 100 and 125 MHz
13
for C). HRESIMS data were obtained on an Agilent 6538 high-
mass-resolution QTOF mass spectrometer. ECD spectra were
obtained on a JASCO J-715 circular dichroism spectrometer. Vacuum
Table 5. Inhibitory Effects of 11-AA Lipopeptaibols (31−36, 44, and 45)
a
a
compound
MW
R1
R2
R3
P. falciparum EC50 (μM)
HepG2 EC50 (μM)
SI
hypocrin NPDG D (31)
hypocrin NPDG E (32)
hypocrin NPDG F (33)
hypocrin NPDG G (34)
hypocrin NPDG H (35)
hypocrin NPDG I (36)
trikoningin KB I (44)
trichogin A IV (45)
1023
1037
1051
1051
1051
1065
1037
1065
L-Val
L-Leu
L-Leu
L-Leu
L-Val
L-Leu
L-Val
L-Leu
L-Val
L-Val
L-Val
L-Val
L-Leu
L-Leu
L-Val
L-Leu
L-Val
L-Val
L-Leu
L-Ile
2.68 ± 0.10
2.53 ± 0.31
0.89 ± 0.13
3.14 ± 0.13
1.93 ± 0.12
2.38 ± 0.06
0.91 ± 0.05
0.93 ± 0.06
>25
>25
>25
>25
>25
>25
10.73 ± 1.47
8.09 ± 1.07
>9
>10
>28
>8
>13
>11
12
L-Ile
L-Leu
L-Ile
L-Ile
9
a
Results are expressed as means from triplicate experiments.
5
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Table 6. Inhibitory Effects of 15-AA Lipopeptaibols (37−43 and 46)
a
a
compound
MW
R1
R2
R3
P. falciparum EC50 (μM)
HepG2 EC50 (μM)
SI
hypocrin NPDG J (37)
hypocrin NPDG K (38)
hypocrin NPDG L (39)
hypocrin NPDG M (40)
hypocrin NPDG N (41)
hypocrin NPDG O (42)
hypocrin NPDG P (43)
trichogin A IV (46)
1335
1349
1349
1349
1363
1363
1363
1377
L-Val
L-Leu
L-Val
L-Val
L-Val
L-Leu
L-Leu
L-Leu
L-Val
L-Val
L-Leu
L-Val
L-Leu
L-Val
L-Leu
L-Leu
L-Val
L-Val
L-Val
L-Leu
L-Leu
L-Leu
L-Val
L-Leu
0.58 ± 0.06
2.27 ± 0.20
1.55 ± 0.22
2.53 ± 0.10
2.10 ± 0.14
0.79 ± 0.09
0.83 ± 0.13
0.58 ± 0.05
>25
>25
>25
>25
>25
23.55 ± 0.63
>25
>43
>11
>16
>10
>12
30
>30
27
15.88 ± 0.64
a
Results are expressed as means from triplicate experiments.
Table 7. Inhibitory Effects of 20-AA Peptaibols (47−52)
a
a
compound
MW
R1
R2
R3
P. falciparum EC50 (μM)
HepG2 EC50 (μM)
SI
trichosporin B IIId (47)
trichosporin B IIIa (48)
trichosporin B IVc (49)
trichosporin B VIb (50)
trichosporin B VIa (51)
trichosporin B VIIa (52)
1936
1950
1950
1964
1964
1978
L-Ala
L-Ala
Aib
L-Val
L-Leu
L-Val
L-Ile
Aib
Aib
Aib
0.56 ± 0.06
0.32 ± 0.02
0.58 ± 0.03
0.52 ± 0.06
0.37 ± 0.02
0.72 ± 0.02
9.28 ± 0.84
5.77 ± 0.53
6.53 ± 0.74
3.35 ± 0.25
2.93 ± 0.21
3.78 ± 0.24
17
18
11
6
8
5
L-Ala
Aib
D-Iva
Aib
L-Ile
Aib
L-Ile
D-Iva
a
Results are expressed as means from triplicate experiments.
Table 8. Inhibitory Effects of Peptides in the Pure Compound Library (53 and 54)
compound
M.W.
R
P. falciparum EC50 (μM)a
HepG2 EC50 (μM)a
SI
Source
efrapeptin F (53)
efrapeptin G (54)
1635
1649
H
CH3
0.39 ± 0.06
0.46 ± 0.05
>25
>25
>64
>54
fungus: Tolypocladium sp.
fungus: Tolypocladium sp.
column chromatography was performed over silica gel (VWR, 40−60
μm, 6 Å) and HP20ss gel (Sorbtech). The preparative HPLC system
was equipped with Shimadzu SCL-10A VP pumps and a system
controller using a Gemini 5 μm C18 column (210 Å, 250 × 21.2 mm)
with a flow rate of 10 mL/min. The semipreparative HPLC were
conducted on a Waters system (1525 binary pumps and Waters 2998
photodiode array detectors) using a Gemini 5 μm C18 (110 Å, 250 ×
Galveston, Texas, USA, while the Hypocrea sp. isolate (PA4898 RBM-
5) was obtained from a soil sample collected in the vicinity of
Gilbertsville, Pennsylvania, USA. The fungi were identified by
collecting mycelium and subjecting the samples to homogenization
in TE buffer (10 mM Tris HCl, 0.1 mM EDTA, pH 8.0) with
zirconium oxide beads in a Bullet blender (MidSci #BBY24M). The
DNA was collected, and the ITS region (i.e., ITS1, 5.8S, and ITS2
regions) amplified by PCR for sequencing. The resulting sequence
data were compared to fungal sequences contained in GenBank,
which led to 100% identity matches to isolates described as
Trichoderma harzianum (isolate from Galveston, Texas) and Hypocrea
pachybasioides (isolate from Gilbertsville, Pennsylvania). Sequence
10 mm), F5 (110 Å, 250 × 10 mm), or biphenyl column (110 Å, 250
×
10 mm) with a flow rate of 4 mL/min. All solvents used were of
ACS grade or better.
Fungal Isolates and Fermentation. The Trichoderma sp. isolate
(TX3005 RBM-20) was obtained from a soil sample collected near
5
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[
F5, isocratic MeCN−H O (42.5:57.5), flow rate: 4 mL/min] to give
2
compounds 21 (2.5 mg, t = 11 min) and 6 (2.3 mg, t = 12 min).
R
R
Compound 22 (5 mg, t = 12 min) was purified from subfraction H1-
R
4
(20 mg) by semipreparative HPLC [F5, isocratic MeCN−H O
2
(42.5:57.5), flow rate: 4 mL/min]. Subfraction H1-5 (35 mg) was
subjected to semipreparative HPLC [F5, isocratic MeCN−H O
2
(
42.5:57.5), flow rate: 4 mL/min], yielding compounds 7 (3 mg, t =
R
14 min), 23 (6 mg, tR = 15 min), and 8 (6 mg, tR = 16 min).
Compound 9 (10 mg, t = 7 min) was purified from subfraction H1-6
R
(
(
35 mg) by semipreparative HPLC [C , isocratic MeCN−H O
1
8
2
42.5:57.5), flow rate: 4 mL/min]. Subfraction H1-8 (30 mg) was
further subjected to semipreparative HPLC [F5, isocratic MeCN−
H O (45:55), flow rate: 4 mL/min] to yield compounds 24 (2 mg, t
2
R
=
8 min), 25 (2 mg, t = 10 min), and 27 (3 mg, t = 12 min).
R
R
Subfraction H1-9 (30 mg) was purified by semipreparative HPLC
F5, isocratic MeCN−H O (47.5:52.5), flow rate: 4 mL/min] to give
[
2
compounds 10 (2 mg, t = 8 min) and 11 (2 mg, t = 11 min).
Subfraction H1-10 (20 mg) was further purified by semipreparative
R
R
Figure 4. Scatter plot displaying antiplasmodial activity and selectivity
afforded by 54 peptidic fungal natural products including peptaibols
composed of 11 amino acid residues (14 and 27; group A), peptaibols
composed of 14 amino acid residues (6−13 and 20−26; group B),
peptaibols composed of 18 amino acid residues (1−5 and 15−
HPLC [F5, isocratic MeCN−H O (47.5:52.5), flow rate: 4 mL/min]
2
to yield compounds 26 (5 mg, t = 10 min), 12 (5 mg, t = 11 min),
R
R
and 14 (1.5 mg, t = 13 min). Compound 13 (3 mg, t = 12 min) was
R
R
purified from subfraction H1-11 (25 mg) by semipreparative HPLC
1
5
9;group C), peptaibols composed of 20 amino acid residues (47−
[
F5, isocratic MeCN−H O (47.5:52.5), flow rate: 4 mL/min].
2
2; group D), lipopeptaibols composed of seven amino acid residues
Subfraction H2 (100 mg) was subjected to semipreparative HPLC
F5, isocratic MeCN−H O (45:55), flow rate: 4 mL/min], yielding
(
28−30; group E), lipopeptaibols composed of 11 amino acid
residues (31−36 and 44−45; group F), lipopeptaibols composed of
5 amino acid residues (37−43 and 46; group G), and efrapeptins
53 and 54; group H).
[
2
compounds 1 (2 mg, t = 8 min), 15 (25 mg, t = 9 min), and 2 (5
R
R
1
(
mg, tR = 10 min). Subfraction H3 (450 mg) was subjected to
semipreparative HPLC [F5, isocratic MeCN−H O (45:55), flow rate:
2
4
mL/min] to give compounds 16 (25 mg, t = 11 min) and 3 (5 mg,
R
data were deposited in GenBank (T. harzianum: GenBank accession
no. MK558706, and H. pachybasioides: GenBank accession no.
MK883713). Based on our standard natural product dereplication
procedures, extracts prepared from the two fungal isolates provided
evidence [i.e., λmax = 190−210, m/z values in the range of 700−2000,
and t = 9−12 min (LC-MS method: C , gradient system from 10%
tR = 12 min). Subfraction H5 (170 mg) was further purified by
semipreparative HPLC [F5, isocratic MeCN−H O (47.5:52.5), flow
2
rate: 4 mL/min] to give compounds 17 (2 mg, t = 9 min), 19 (3 mg,
R
t = 10 min), 18 (8 mg, t = 11 min), and 4 (3 mg, t = 12 min).
R
R
R
Compound 5 (5 mg, t = 11 min) was obtained from subfraction H6
R
R
18
by semipreparative HPLC [F5, isocratic MeCN−H O (1:1), flow
2
to 100% MeCN in H O with 0.1% formic acid over 13 min, flow rate:
2
rate: 4 mL/min].
0
.4 mL/min)] that peptidic natural products were the dominant type
Trichorzin NPDG A (1): colorless solid; [α] 2D 5 +14 (c 0.1, MeOH);
of metabolite in the samples under investigation.
1
ECD (MeOH, c 0.1) λmax (Δε) 208 (−157), 225 (−94.1); H NMR
To prepare the isolates for chemical studies, fungi were recovered
from cryogenic storage (stored in a vial at −80 °C as mycelium with
13
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
2+
Table S3; HRESIMS m/z 852.5261 [M + 2H] (calcd for
C H N O , 852.5245; mass error −1.9 ppm).
2
0% aqueous glycerol). Following recovery on Czapek agar plates (30
7
9
140 20 21
g sucrose, 2 g NaNO , 1 g K HPO , 0.5 g MgSO ·7H O, 0.5 g KCl,
0
3
2
4
4
2
25
D
Trichorzin NPDG B (2): colorless solid; [α] −6 (c 0.1, MeOH);
.01 g FeSO ·7H O, 0.05 g chloramphenicol, 1 L DI H O), lawns of
4 2 2
2
1
ECD (MeOH, c 0.1) λmax (Δε) 208 (−301), 225 (−175); H NMR
fungal mycelium were aseptically cut into small pieces (∼1 cm ) for
use as the scale-up culture inoculum. Scale-up cultures were carried
out by charging mycobags (Unicorn Bags, Plano, TX, USA) with
monolayers of Cheerios breakfast cereal supplemented with a 0.3%
sucrose solution and 0.005% chloramphenicol. The pieces of
mycelium were aseptically added to three mycobags per fungal
isolate, and the cultures were grown at room temperature for 4 weeks.
Extraction and Isolation of T. harzianum. Fungal biomass was
extracted with 2 L of EtOAc (×3) at room temperature, the organic
solvent layers were recovered, and the solvent was removed under
vacuum. The EtOAc-soluble material was combined for further
processing (34 g, fraction A). Fraction A was subjected to silica gel
vacuum column chromatography with elution performed using
dichloromethane (fraction B), dichloromethane−MeOH (10:1)
13
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
2
+
Table S4; HRESIMS m/z 853.0184 [M + 2H] (calcd for
C H N O , 853.0166; mass error −2.1 ppm).
7
9
139 19 22
2
5
Trichorzin NPDG C (3): colorless solid; [α] +10 (c 0.1, MeOH);
D
1
ECD (MeOH, c 0.1) λmax (Δε) 208 (−326), 225 (−202); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
Table S5; HRESIMS m/z 866.5419 [M + 2H] (calcd for
1
3
(
5
5
2
+
C H N O , 866.5402; mass error −2.0 ppm).
81 144 20 21
2
5
Trichorzin NPDG D (4): colorless solid; [α] +12 (c 0.1, MeOH);
ECD (MeOH, c 0.1) λmax (Δε) 208 (−332), 225 (−200); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
D
1
1
3
(
5
5
2
+
Table S6; HRESIMS m/z 873.5493 [M + 2H] (calcd for
C H N O , 873.5480; mass error −1.5 ppm).
82 146 20 21
2
5
Trichorzin NPDG E (5): colorless solid; [α] +4 (c 0.1, MeOH);
ECD (MeOH, c 0.1) λmax (Δε) 208 (−249), 225 (−147); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
(fraction C), and MeOH (fraction D). Fraction D (10 g) was also
D
1
further fractionated by HP20ss gel vacuum column chromatography
into five samples: fractions E (30% MeOH), F (50% MeOH), G (70%
MeOH), H (90% MeOH), and I (100% MeOH). Fraction H (5 g)
was further subjected to preparative HPLC (C , gradient elution with
1
3
(
5
5
2
+
Table S7; HRESIMS m/z 880.5585 [M + 2H] (calcd for
21, 880.5558; mass error −3.1 ppm).
C H N O
83 148 20
1
8
2
5
8
5−100% MeOH in H O over 15 min using a 10 mL/min flow rate)
Harzianin NPDG A (6): colorless solid; [α]
+12 (c 0.1, MeOH);
D
2
1
to afford seven subfractions (H1−H7). Subfraction H1 (300 mg) was
ECD (MeOH, c 0.1) λmax (Δε) 205 (−163), 230 (−17.5); H NMR
1
3
subjected to preparative HPLC (C , gradient elution with 85−100%
(500 MHz, pyridine-d
Table S8; HRESIMS m/z 1438.8705 [M + Na] (calcd for
15NaO17, 1438.8644; mass error −4.2 ppm).
5
) and C NMR (100 MHz, pyridine-d ), see
5
1
8
+
MeCN−H O over 15 min using a 10 mL/min flow rate) to afford 12
2
subfractions (H1-1−H1-12). Compound 20 (2 mg, t = 10 min) was
C H N
68 117
R
2
5
purified from subfraction H1-2 (10 mg) by semipreparative HPLC
Harzianin NPDG B (7): colorless solid; [α]
D
−4 (c 0.1, MeOH);
1
[
F5, isocratic MeCN−H O (42.5:57.5), flow rate: 4 mL/min].
ECD (MeOH, c 0.1) λmax (Δε) 205 (−211), 230 (−19.9); H NMR
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
2
1
3
Subfraction H1-3 (20 mg) was obtained by semipreparative HPLC
5
5
5
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Article
Table S9; HRESIMS m/z 715.9541 [M + 2H]2+ (calcd for
C H N O , 715.9527; mass error −2.0 ppm).
mg, t = 12 min), and 36 (3 mg, t = 13 min). Fraction J (90 mg) was
R R
further purified by semipreparative HPLC [biphenyl, isocratic
6
9
121 15 17
2
5
Harzianin NPDG C (8): colorless solid; [α] +30 (c 0.1, MeOH);
MeCN−H O (45:55), flow rate: 4 mL/min] to give compounds 50
D
2
1
ECD (MeOH, c 0.1) λmax (Δε) 205 (−184), 230 (−21.2); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
Table S10; HRESIMS m/z 715.9541 [M + 2H] (calcd for
(25 mg, t = 9 min) and 51 (2 mg, t = 10 min). Compound 52 (35
R
R
1
3
(
mg, tR = 10 min) was purified from fraction L (60 mg) by
5
5
2
+
semipreparative HPLC [F5, isocratic MeCN−H O (47.5:52.5), flow
2
C H N O , 715.9527; mass error −2.0 ppm).
rate: 4 mL/min]. Fraction M (500 mg) was fractionated into five
subfractions using HP20ss gel vacuum column chromatography:
subfractions M1 (30% MeOH), M2 (50% MeOH), M3 (70%
MeOH), M4 (90% MeOH), and M5 (100% MeOH). Fraction M4
(180 mg) was separated into five subfractions (M4-1−M4-5) by
6
9
121 15 17
2
5
Harzianin NPDG D (9): colorless solid; [α] +36 (c 0.1, MeOH);
D
1
ECD (MeOH, c 0.1) λmax (Δε) 205 (−175), 230 (−19.5); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
Table S11; HRESIMS m/z 722.9621 [M + 2H] (calcd for
1
3
(
5
5
2
+
C H N O , 722.9605; mass error −2.2 ppm).
preparative HPLC (C , gradient elution with 85−100% MeOH in
7
0
123 15 17
18
2
5
Harzianin NPDG E (10): colorless solid; [α] −6 (c 0.1, MeOH);
H O over 15 min, flow rate: 10 mL/min). Subfraction M4-3 (20 mg)
D
2
1
ECD (MeOH, c 0.1) λmax (Δε) 205 (−196), 230 (−13.4); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
was further subjected to semipreparative HPLC [F5, isocratic
1
3
(
MeCN−H O (1:1), flow rate: 4 mL/min] to yield compounds 38
5
5
2
2
+
Table S12; HRESIMS m/z 707.9569 [M + 2H] (calcd for
C H N O , 707.9552; mass error −2.4 ppm).
(1.5 mg, t = 11 min), 39 (3 mg, t = 12 min), and 43 (2 mg, t = 13
R
R
R
min). Subfraction M4-4 (30 mg) was subjected to semipreparative
69
121 15 16
2
5
Harzianin NPDG F (11): colorless solid; [α] +40 (c 0.1, MeOH);
HPLC [F5, isocratic MeCN−H O (52.5:47.5), flow rate: 4 mL/min]
D
2
1
ECD (MeOH, c 0.1) λmax (Δε) 205 (−119), 230 (−8.5); H NMR
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
Table S13; HRESIMS m/z 714.9647 [M + 2H] (calcd for
to give compounds 37 (3 mg, t = 7 min), 42 (4 mg, t = 9 min), and
R
R
1
3
(
46 (6 mg, t = 11 min). Compounds 40 (6 mg, t = 8 min) and 41 (7
5
5
R
R
2
+
mg, tR = 9 min) were obtained from subfraction M4-5 by
C H N O , 714.9631; mass error −2.2 ppm).
semipreparative HPLC [biphenyl, isocratic MeCN−H O (1:1), flow
7
0
123 15 16
2
2
5
Harzianin NPDG G (12): colorless solid; [α] +38 (c 0.1, MeOH);
rate: 4 mL/min).
D
1
Hypocrin NPDG A (28): colorless solid; [α] 2D 5 −14 (c 0.1, MeOH);
ECD (MeOH, c 0.1) λmax (Δε) 205 (−149), 230 (−14.2); H NMR
1
3
1
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
ECD (MeOH, c 0.1) λ (Δε) 215 (+21.5); H NMR (500 MHz,
5
5
max
2
+
13
Table S14; HRESIMS m/z 714.9644 [M + 2H] (calcd for
pyridine-d ) and C NMR (100 MHz, pyridine-d ), see Table S17;
HRESIMS m/z 740.5278 [M + H] (calcd for C H N O ,
5
5
+
C H N O , 714.9631; mass error −1.8 ppm).
7
0
123 15 16
37 70
7
8
2
5
Harzianin NPDG H (13): colorless solid; [α] +6 (c 0.1, MeOH);
740.5280; mass error 0.3 ppm).
Hypocrin NPDG B (29): colorless solid; [α] +6 (c 0.1, MeOH);
D
1
25
ECD (MeOH, c 0.1) λmax (Δε) 205 (−147), 225 (−9.4); H NMR
D
1
3
1
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
ECD (MeOH, c 0.1) λ (Δε) 215 (+5.6); H NMR (500 MHz,
5
5
max
2
+
13
Table S15; HRESIMS m/z 721.9717 [M + 2H] (calcd for
C H N O , 721.9709; mass error −1.1 ppm).
pyridine-d ) and C NMR (100 MHz, pyridine-d ), see Table S18;
5
5
+
HRESIMS m/z 740.5292 [M + H] (calcd for C H N O ,
7
1
125 15 16
37 70
7
8
2
5
Harzianin NPDG I (14): colorless solid; [α] +12 (c 0.1, MeOH);
740.5280; mass error −1.6 ppm).
D
1
25
ECD (MeOH, c 0.1) λmax (Δε) 207 (−36.8), 230 (−4.6); H NMR
Hypocrin NPDG C (30): colorless solid; [α] −4 (c 0.1, MeOH);
D
1
3
1
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
ECD (MeOH, c 0.1) λ (Δε) 215 (+8.0); H NMR (500 MHz,
5
5
max
+
13
Table S16; HRESIMS m/z 1211.7720 [M + Na] (calcd for
pyridine-d ) and C NMR (100 MHz, pyridine-d ), see Table S19;
5
5
+
C H N NaO , 1211.7738; mass error 1.5 ppm).
HRESIMS m/z 754.5453 [M + H] (calcd for C H N O ,
59
104 12
13
38 72 7 8
Extraction and Isolation of H. pachybasioides. Fungal biomass
754.5437; mass error −2.1 ppm).
2
5
was extracted with 2 L of EtOAc (×3) at room temperature, the
organic solvent layers were recovered, and the solvent was removed
under vacuum. The EtOAc-soluble material was combined for further
processing (21 g; fraction A). Fraction A was subjected to silica gel
vacuum column chromatography with elution performed using
dichloromethane (fraction B), dichloromethane−MeOH (10:1)
Hypocrin NPDG D (31): colorless solid; [α] −8 (c 0.1, MeOH);
D
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−44.6), 223 (−24.7); H NMR
1
3
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
+
Table S20; HRESIMS m/z 1024.6771 [M + H] (calcd for
C H N O , 1024.6765; mass error −0.6 ppm).
4
9
90 11 12
2
5
Hypocrin NPDG E (32): colorless solid; [α] −8 (c 0.1, MeOH);
D
1
(
fraction C), and MeOH (fraction D). Fraction D (5 g) was
ECD (MeOH, c 0.1) λmax (Δε) 205 (−60.4), 222 (−26.5); H NMR
13
subjected to preparative HPLC (C , isocratic 85% MeOH−H O,
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5 5
1
8
2
+
flow rate: 10 mL/min) to afford nine fractions (fractions E−M).
Table S21; HRESIMS m/z 1038.6919 [M + H] (calcd for
Fraction E (25 mg) was subjected to semipreparative HPLC
C H N O , 1038.6921; mass error 0.2 ppm).
5
0
92 11 12
2
5
D
[
biphenyl, isocratic MeCN−H O (1:1), flow rate: 4 mL/min] to
Hypocrin NPDG F (33): colorless solid; [α] −22 (c 0.1, MeOH);
2
1
give compounds 29 (2 mg, t = 9 min) and 28 (6 mg, t = 10 min).
ECD (MeOH, c 0.1) λmax (Δε) 205 (−76.3), 223 (−28.4); H NMR
R
R
13
Fraction F (20 mg) was subjected to semipreparative HPLC
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5 5
+
[
biphenyl, isocratic MeCN−H O (1:1), flow rate: 4 mL/min],
Table S22; HRESIMS m/z 1052.7061 [M + H] (calcd for
2
yielding compounds 31 (2 mg, t = 8 min), 32 (5 mg, t = 9 min),
C H N O , 1052.7078; mass error 1.6 ppm).
R
R
51 94 11 12
2
5
and 30 (25 mg, t = 11 min). Fraction H (300 mg) was separated into
Hypocrin NPDG G (34): colorless solid; [α] −24 (c 0.1, MeOH);
R
D
1
four subfractions (H1−4) by preparative HPLC (C , gradient elution
ECD (MeOH, c 0.1) λmax (Δε) 205 (−89.6), 222 (−36.0); H NMR
1
8
1
3
with 80−100% MeOH in H O over 20 min, flow rate: 10 mL/min).
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
2
5
5
+
Subfraction H1 (130 mg) was subjected to semipreparative HPLC
Table S23; HRESIMS m/z 1052.7062 [M + H] (calcd for
[
biphenyl, isocratic MeCN−H O (1:1), flow rate: 4 mL/min] to give
C H N O , 1052.7078; mass error 1.5 ppm).
2
51 94 11 12
2
5
compounds 44 (15 mg, t = 9 min) and 34 (37 mg, t = 10 min).
Hypocrin NPDG H (35): colorless solid; [α] −10 (c 0.1, MeOH);
R
R
D
1
Subfraction H2 (50 mg) was subjected to semipreparative HPLC
biphenyl, isocratic MeCN−H O (47.5:52.5), flow rate: 4 mL/min]
ECD (MeOH, c 0.1) λmax (Δε) 206 (−103), 223 (−48.8); H NMR
1
3
[
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
2
5
5
+
to obtain compounds 48 (6 mg, t = 10 min), 47 (6 mg, t = 11 min),
Table S24; HRESIMS m/z 1052.7076 [M + H] (calcd for
R
R
and 33 (3 mg, t = 16 min). Fraction I was fractionated into four
C H N O , 1052.7078; mass error 0.2 ppm).
R
51 94 11 12
2
5
subfractions (I1−4) by preparative HPLC (C , gradient elution with
Hypocrin NPDG I (36): colorless solid; [α] +8 (c 0.1, MeOH);
1
8
D
1
MeOH in H O over 15 min, flow rate: 10 mL/min). Compound 49
ECD (MeOH, c 0.1) λmax (Δε) 206 (−42.3), 223 (−65.7); H NMR
2
1
3
(
4 mg, t = 11 min) was purified from subfraction I1 (15 mg) by
(500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
R
5
5
+
semipreparative HPLC [F5, MeCN−H O (45:55), flow rate: 4 mL/
min]. Subfraction I3 (65 mg) was further subjected to semi-
Table S25; HRESIMS m/z 1088.7073 [M + Na] (calcd for
C H N NaO , 1088.7054; mass error −1.7 ppm).
2
5
2
95 11
12
2
5
preparative HPLC [biphenyl, isocratic MeCN−H O (1:1), flow
Hypocrin NPDG J (37): colorless solid; [α] +6 (c 0.1, MeOH);
D
1
2
rate: 4 mL/min], yielding compounds 35 (2 mg, t = 11 min), 45 (2
ECD (MeOH, c 0.1) λmax (Δε) 206 (−94.6), 222 (−65.7); H NMR
R
5
14
https://dx.doi.org/10.1021/acs.jnatprod.0c01370
J. Nat. Prod. 2021, 84, 503−517

Journal of Natural Products
pubs.acs.org/jnp
Article
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
X-ray Crystallographic Analysis of Compound 40. Crystals of
compound 40 were obtained from MeOH−water (5:1). A colorless,
block-shaped crystal of dimensions 0.060 × 0.104 × 0.186 mm was
selected for structural analysis. Intensity data were collected using a
D8 Quest κ-geometry diffractometer with a Bruker Photon II cmos
area detector and an Incoatec Iμs microfocus Mo Kα source (λ =
0.710 73 Å). The sample was cooled to 100(2) K. Cell parameters
were determined from a least-squares fit of 9954 peaks in the range
2.19° < θ < 23.11°. A total of 50 593 data points were measured in the
range 2.090° < θ < 19.027° using φ and ω oscillation frames. The data
were corrected for absorption by the empirical method, giving
minimum and maximum transmission factors of 0.5817 and 0.7453.
The data were merged to form a set of 23 614 independent data with
R(int) = 0.0917 and a coverage of 99.4%. Crystallographic data for 40
have been deposited at the Cambridge Crystallographic Data Centre
(deposition number: CCDC 1967991). Copies of these data can be
obtained free of charge from the CCDC via www.ccdc.cam.ac.uk.
Parasite Culture Conditions. The P. falciparum culture was
5
5
+
Table S26; HRESIMS m/z 1358.8427 [M + Na] (calcd for
C H N NaO , 1358.8382; mass error −3.3 ppm).
Hypocrin NPDG K (38): colorless solid; [α] +2 (c 0.1, MeOH);
63
113 15
16
2
5
D
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−71.9), 222 (−49.7); H NMR
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
+
Table S27; HRESIMS m/z 1372.8540 [M + Na] (calcd for
C H N NaO , 1372.8538; mass error −0.1 ppm).
64
115 15
16
2
5
Hypocrin NPDG L (39): colorless solid; [α] −12 (c 0.1, MeOH);
D
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−99.9), 222 (−64.8); H NMR
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
+
Table S28; HRESIMS m/z 1350.8716 [M + H] (calcd for
C H N O , 1350.8719; mass error 0.2 ppm).
Hypocrin NPDG M (40): colorless solid; [α]D +6 (c 0.1, MeOH);
64
116 15 16
2
5
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−188), 222 (−116); H NMR
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
+
Table S29; HRESIMS m/z 1372.8555 [M + Na] (calcd for
C H N NaO , 1372.8538; mass error −1.2 ppm).
64
115 15
16
2
5
48
Hypocrin NPDG N (41): colorless solid; [α] −4 (c 0.1, MeOH);
maintained following a modified Trager and Jensen protocol. The
multi-drug-resistant P. falciparum line Dd2 was grown in RPMI 1640
D
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−152), 222 (−89.7); H NMR
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
supplemented with 25 mM HEPES pH 7.4, 26 mM NaHCO , 2%
5
5
3
+
Table S30; HRESIMS m/z 1386.8720 [M + Na] (calcd for
dextrose, 15 mg/L hypoxanthine, 25 mg/L gentamicin, and 0.5%
Albumax II in human A+ erythrocytes. Cultures were maintained at
4% hematocrit at 37 °C with 5% CO2.
C H N NaO , 1386.8695; mass error −1.8 ppm).
65
117 15
16
2
5
Hypocrin NPDG O (42): colorless solid; [α] −12 (c 0.1, MeOH);
D
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−93.9), 222 (−53.4); H NMR
Phenotypic Screen for Antiplasmodial Activity. Antiplasmo-
dial EC50 results were determined using a fluorescence-based SYBR
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
+
49
Table S31; HRESIMS m/z 1386.8722 [M + Na] (calcd for
C H N NaO , 1386.8695; mass error −1.9 ppm).
Green I assay performed using asynchronous cultures. For
screening, parasites were diluted to 1% parasitemia and 2%
hematocrit, then incubated with serial dilutions of compounds in
microtiter plates for 72 h at standard growth conditions. Plates were
subsequently frozen at −80 °C to facilitate lysis. After thawing, plates
were incubated with 1× SYBR Green I in lysis buffer (20 mM Tris-
HCl, 0.08% saponin, 5 mM EDTA, 0.8% Triton X-100) for 45 min at
room temperature. Fluorescence was measured at an excitation
wavelength of 485 nm and emission wavelength of 530 nm in a
Synergy Neo2 multimode reader (BioTek, Winsooki, VT, USA).
Relative fluorescence units (RFUs) were normalized based on
chloroquine-treated and no treatment controls. Serial dilutions of
compounds were prepared in RPMI with final assay conditions of
≤0.2% DMSO.
65
117 15
16
2
5
Hypocrin NPDG P (43): colorless solid; [α] −10 (c 0.1, MeOH);
D
1
ECD (MeOH, c 0.1) λmax (Δε) 206 (−46.0), 222 (−28.3); H NMR
1
3
(
500 MHz, pyridine-d ) and C NMR (100 MHz, pyridine-d ), see
5
5
+
Table S32; HRESIMS m/z 1386.8685 [M + Na] (calcd for
C H N NaO , 1386.8695; mass error 0.7 ppm).
Preparation of Amino Acid Standards and Absolute
Configuration Analysis of Amino Acid Residues. Peptaibols
0.3 mg) were hydrolyzed in 300 μL of 6 N HCl at 110 °C overnight.
After cooling to room temperature, the hydrolysate neutralized by 2 N
NaOH was evaporated to dryness and the remaining residue dissolved
in 100 μL of water and 1 M NaHCO (30 μL). A solution of N-α-
2,4-dinitro-5-fluorophenyl)-L-alaninamide (L-FDAA, Marfey’s re-
agent, Sigma, 100 μL, 1% in acetone) was added to each reaction
vial. The reaction mixture was heated to 45 °C for 1 h, quenched by
adding 1 N HCl (30 μL), and mixed with CH CN (1 mL). Samples
65
117 15
16
(
3
(
Human Cell Cytotoxicity Assay. Mammalian cell cytotoxicity
was assessed in HepG2 human hepatocytes using an MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophen-
yl)-2H-tetrazolium)-based cytotoxicity assay. For cytotoxicity testing,
∼2250 cells were seeded into each well of a 384-well microtiter plate,
and the cells were incubated for 24 h. Serial dilutions of the
compounds were added in triplicate starting at 25 μM (final
concentration). This maximum concentration was chosen to maintain
a consistently high concentration without sacrificing compound
solubility, while also keeping DMSO concentration below toxic levels.
Cells were further incubated for an additional 48 h at 37 °C in an
3
consisting of 5 μL of the FDAA derivatives were taken for LC/MS
analysis (Kinetex C , 2.6 μm, 100 Å, 75 × 3.0 mm, flow rate: 0.4 mL/
min), which was performed at room temperature. Aqueous CH CN
18
3
containing 0.1% formic acid was used as the mobile phase in gradient
mode (10−50% CH CN in H O over 30 min). The D- and L-amino
3
2
acid authentic standards were prepared similarly. The following
retention times (min) were observed for the L-FDAA derivatives of
the standards: 11.1 (L-Ser), 11.7 (D-Ser), 12.1 (L-Asp), 13.2 (D-Asp),
1
1
2
3.5 (L-Glu), 14.6 (D-Glu), 14.6 (L-Ala), 16.7 (D-Ala), 15.2 (L-Pro),
6.1 (D-Pro), 19.0 (L-Val), 21.8 (D-Val), 19.2 (L-Iva), 20.3 (D-Iva),
1.0 (L-Leuol), 24.2 (D-Leuol), 21.6 (L-Ile), 24.2 (D-Ile), 21.6 (L-allo-
Ile), 24.2 (D-allo-Ile), 22.0 (L-Leu), and 24.5 (D-Leu).
Absolute Configuration Analysis of Isoleucine Residues.
Hydrolysates of the metabolites were prepared as described for the
Marfey’s analysis (vide supra), and the residue was dissolved in H O
100 μL). Aliquots consisting of 200 μL of 5% trimethylamine in
acetone and 200 μL of 1% GITC solution in acetone were added to
atmosphere containing 5% CO . Zero percent growth control wells
2
were incubated with 5% Triton X-100 for 5 min prior to MTS
addition. Following the addition of MTS to all wells, the plates were
incubated for an additional 4 h under the same environmental
conditions before taking absorbance measurements. Absorbance
values were recorded at 490 nm using a Synergy Neo2 multimode
reader (BioTek), and values were normalized based on Triton X-100-
lysed and no treatment controls.
2
(
the hydrolysate. Reaction vials were maintained at room temperature
ASSOCIATED CONTENT
(
∼25 °C) for 15 min before being quenched by adding 200 μL of 5%
■
acetic acid. For LC/MS analyses, 5 μL aliquots of the GITC
derivatives were prepared and tested at room temperature (Kinetex
C , 2.6 μm, 100 Å, 75 × 3.0 mm, flow rate: 0.4 mL/min, 10−30%
sı
*
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01370.
18
CH CN−H O with 0.1% formic acid for 0−50 min). Standards
3
2
consisting of authentic L-isoleucine and L-allo-isoleucine were
1
D NMR, 2D NMR, HRESIMS, MS/MS fragment
prepared in the same fashion. The following retention times were
analysis, and ECD spectrum for new peptaibols 1−14
and 28−43 (PDF)
observed for the GITC derivatives of the standards: t (min) 48.5 (L-
R
allo-Ile) and 49.0 (L-Ile).
5
15
https://dx.doi.org/10.1021/acs.jnatprod.0c01370
J. Nat. Prod. 2021, 84, 503−517

Journal of Natural Products
pubs.acs.org/jnp
Article
Crystallographic information file for compound 40
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Vical, F.; Fairhurst, R. M.; Menard, D.; Fidock, D. A. Science 2015,
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(
47, 428−31.
5) Imwong, M.; Dhorda, M.; Tun, K. M.; Thu, A. M.; Phyo, A. P.;
AUTHOR INFORMATION
Corresponding Authors
■
Proux, S.; Suwannasin, K.; Kunasol, C.; Srisutham, S.; Duanguppama,
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Robert H. Cichewicz − Natural Products Discovery Group,
Institute for Natural Products Applications and Research
Technologies, Department of Chemistry and Biochemistry,
Stephenson Life Sciences Research Center, University of
Oklahoma, Norman, Oklahoma 73019, United States;
orcid.org/0000-0003-0744-4117; Email: rhcichewicz@
ou.edu
Debopam Chakrabarti − Division of Molecular Microbiology,
Burnett School of Biomedical Sciences, University of Central
Florida, Orlando, Florida 32826, United States;
Email: Debopam.Chakrabarti@ucf.edu
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Hernan
(
́
̈
Authors
Jin Woo Lee − Natural Products Discovery Group, Institute
for Natural Products Applications and Research
Technologies, Department of Chemistry and Biochemistry,
Stephenson Life Sciences Research Center, University of
Oklahoma, Norman, Oklahoma 73019, United States
Jennifer E. Collins − Division of Molecular Microbiology,
Burnett School of Biomedical Sciences, University of Central
Florida, Orlando, Florida 32826, United States;
orcid.org/0000-0002-1195-4630
Karen L. Wendt − Natural Products Discovery Group,
Institute for Natural Products Applications and Research
Technologies, Department of Chemistry and Biochemistry,
Stephenson Life Sciences Research Center, University of
Oklahoma, Norman, Oklahoma 73019, United States
(
7
(
(
.
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(18) Jaworski, A.; Kirschbaum, J.; Bru
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01370
̈
ckner, H. J. Pept. Sci. 1999, 5,
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Spectrom. 1984, 11, 569−82.
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19) Przybylski, M.; Dietrich, I.; Manz, I.; Bruckner, H. Biomed. Mass
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Author Contributions
J. W. Lee and J. E. Collins contributed equally to this work.
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Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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ckner, H. J. Chromatogr. A 1999, 862, 179−89.
Support for this project was provided by NIH grants
R21AI143052 and R01AI154777 to D.C. and R.H.C. An
LC-MS instrument used for this project was provided in part
by a Challenge Grant from the Office of the Vice President for
Research, University of Oklahoma, Norman Campus, and an
award through the Shimadzu Equipment Grant Program
(23) Jiao, W.-H.; Khalil, Z.; Dewapriya, P.; Salim, A. A.; Lin, H.-W.;
Capon, R. J. J. Nat. Prod. 2018, 81, 976−84.
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R.H.C.). A grant from the NSF (CHE-0130835) was used
for the purchase of the X-ray diffractometer. We thank Dr. D.
R. Powell for his technical assistance with the X-ray diffraction
analysis. We are grateful for the gift of soil samples provided by
citizen scientists J. Smith and T. Elliot that were used to obtain
fungi described in this report.
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