Natural Hydroxamate-Containing Siderophore Acremonpeptides A-D and an Aluminum Complex of Acremonpeptide D from the Marine-Derived Acremonium persicinum SCSIO 115

Article abstract of DOI:10.1021/acs.jnatprod.9b00545

Four new hydroxamate-containing natural product cyclopeptides designated acremonpeptides A-D (1-4), together with Al(III)-acremonpeptide D (5) were obtained from the marine fungus Acremonium persicinum SCSIO 115. The planar structures of 1-5 were established on the basis of HRMS as well as 1D and 2D NMR data sets. Moreover, the amino acid absolute configurations were determined using Marfey's method. Compounds 1-5 all feature three 2-amino-5-(N-hydroxyacetamido)pentanoic acid (N⁵-hydroxy-N⁵-acetyl-l-ornithine) metal ion chelating moieties. Beyond their discovery and structure elucidation, in vitro bioassays revealed acremonpeptides A (1), B (2), and Al(III)-acremonpeptide D (5) as moderate antiviral agents for herpes simplex virus 1 with EC₅₀ values of 16, 8.7, and 14 μM, respectively.

Full text of DOI:10.1021/acs.jnatprod.9b00545

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Natural Hydroxamate-Containing Siderophore Acremonpeptides A−
D and an Aluminum Complex of Acremonpeptide D from the
Marine-Derived Acremonium persicinum SCSIO 115
Minghe Luo,† Ruochen Zang,‡ Xin Wang,‡ Ziming Chen,† Xiaoxian Song,§ Jianhua Ju,*,†,⊥
and Hongbo Huang*,†,⊥
†CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica,
RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 Xingang
Road West, Guangzhou 510301, China
‡Innovative Marine Drug Screening and Evaluation Center, Qingdao National Laboratory for Marine Science and Technology, 23
Xianggang Road East, Qingdao 266100, China
§Chongqing Center For Drug Safety Evaluation, Chongqing Academy of Chinese Materia Medica, 34 Nanshan Road, Chongqing
400065, China
⊥University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
S
* Supporting Information
ABSTRACT: Four new hydroxamate-containing natural
product cyclopeptides designated acremonpeptides A−D
(1−4), together with Al(III)−acremonpeptide D (5) were
obtained from the marine fungus Acremonium persicinum
SCSIO 115. The planar structures of 1−5 were established on
the basis of HRMS as well as 1D and 2D NMR data sets.
Moreover, the amino acid absolute configurations were
determined using Marfey’s method. Compounds 1−5 all
feature three 2-amino-5-(N-hydroxyacetamido)pentanoic acid
(N5-hydroxy-N5-acetyl-L-ornithine) metal ion chelating moi-
eties. Beyond their discovery and structure elucidation, in vitro bioassays revealed acremonpeptides A (1), B (2), and Al(III)−
acremonpeptide D (5) as moderate antiviral agents for herpes simplex virus 1 with EC50 values of 16, 8.7, and 14 μM,
respectively.
espite its very low abundance in earth’s aerobic
typical sideromycins isolated from Streptomyces, were originally
identified on the basis of their potent abilities to target both
Gram-positive and Gram-negative bacteria.7,8 These findings
have inspired significant drug delivery efforts to exploit
siderophoric uptake systems in “Trojan horse” approaches
wherein cell permeability limits of specific agents can be
surpassed. Simply put, a target cell’s need to import iron (and
things associated with it) can be used to override permeability
factors that might otherwise restrict a drug’s cellular entry;
drugs can be smuggled into a target cell by virtue of their
association with cellularly valuable iron.9 This logic has
inspired dramatically increased reports of the discovery,
synthesis, and utilization of siderophores over the last 10−20
years.2
D
environments, iron (generally Fe2+ or Fe3+) is an
important nutritional element for almost all organisms on
the planet. To facilitate iron acquisition and bioavailability,
microorganisms have evolved elegant siderophore-based
systems wherein organic agents enhance the availability and
handling of iron in living systems.1,2 Natural product
siderophores are generally low-molecular-mass (500−1500
Da) iron chelators that enable the uptake of soluble iron(III)
from both terrestrial and aquatic environments.3 Structurally
speaking, these agents are typically biosynthesized by non-
ribosomal peptide synthetases (NRPSs) and are functionalized
with metal-chelating groups such as phenols, catechols,
hydroxamates, carboxylates, and mixed-type (heterocyclic)
metal binders such as oxazolines and thiazolines.2,4,5 The
sideromycins are excellent examples of these NRPS-derived
compounds.6 Produced by Actinomycetes and displaying
antibiotic activities, the sideromycins are readily taken up by
cells via the exploitation of specific siderophore/Fe uptake
systems.6 As a result, the sideromycins are characterized by
very low minimal inhibitory concentrations (MIC) values in
antimicrobial assays. For instance, ferrimycins and albomycins,
Although seawater is abundant in some metal ions such as
sodium and potassium, iron levels in seawater are extremely
low. Indeed, there are numerous examples of marine
microorganisms that biosynthesize and use siderophores just
Received: June 14, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00545
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
as their terrestrial counterparts do in order to acquire iron from
their habitat.10 In the course of our continuing investigations
into bioactive secondary metabolites from marine-derived
microorganisms, we have reported the cytotoxic heptapeptide
cordyheptapeptides C−E from the marine fungus Acremonium
persicinum SCSIO 115.11 Recently, this strain was cultured on
solid rice medium and HPLC-DAD analysis of the culture
extracts revealed an array of compounds showing similar UV
absorptions. Subsequent chemical investigations led to the
purification of new cyclohexapeptide siderophores herein
named acremonpeptides A−D (1−4) and Al(III)−acre-
monpeptide D (5). We report here the isolation, structure
elucidation, and antiviral activities of these marine fungal
metabolites.
127.9, 129.7 (2 × C), and 130.4 (2 × C) revealed the presence
of a phenyl group in 1. COSY and HSQC data sets revealed
1H−1H spin systems consistent with one −CH−CH2−CH−
(CH3)2, two −CH−CH2−, and three −CH−(CH2)2−CH2−
moieties. HMBC correlation analyses confirmed the presence
of one phenylalanine (Phe), one leucine (Leu), one serine
(Ser), and three ornithine (Orn) residues within the structure
of 1 (Figure 1). Further HMBC correlations from the protons
at the terminal methylenes in the Orn units to the acetyl
carbonyls (δH 3.57−3.75/δC 173.8 and δH 3.57−3.75/δC
173.7) confirmed the connectivity of the three acetyl groups
(previously established) to the 5-N atom of each Orn via
amide bonds. Furthermore, there were three hydroxy groups in
1 as dictated in large part by the molecular formula. The three
hydroxy groups were linked to each Orn 5-N atom to form
three hydroxylamine groups, which was determined by
comparison of the 13C NMR chemical shift value for C-5 of
the Orn unit in 1 (δC‑5 48.5) with those of the N5-hydroxy-Orn
unit (δC‑5 ∼ 46) and unsubstituted Orn unit (δC‑5 38.6) in
known compounds.5,12−14 The elucidated benzene ring and
nine carbonyl moieties accounted for 13 degrees of
unsaturation. The remaining unsaturation and absence of
other functionalities that might account for it suggested that 1
might be a cyclic scaffold, specifically a cyclohexapeptide. The
HMBC spectrum gave corrections of δH 4.53/δC 175.0, δH
4.07/δC 172.1, δH 4.43/δC 173.4, δH 4.44/δC 173.3, δH 4.13/δC
174.7, and δH 4.03/δC 172.8, establishing the sequence of
amino acid residues as cyclo-(Phe-Leu-Ser-AcN(OH)Orn1-
AcN(OH)Orn2-AcN(OH)Orn3) for 1. Rigorous review of the
literature revealed reports of an identical planar structure in a
Japanese patent, but the amino acid configurations had not
been disclosed.15 Accordingly, we determined the amino acid
absolute configurations for 1 using Marfey’s method. HPLC
analyses of the DAA derivatives of the HCl and HI
hydrolysates of 1 confirmed that 1 is composed of L-Phe, L-
Leu, L-Ser, and L-Orn (Figures S31 and S36). Consequently,
cyclohexapeptide 1 was elucidated as shown and named
acremonpeptide A.
RESULTS AND DISCUSSION
■
The fungus A. persicinum SCSIO 115 was cultured on
autoclaved rice medium for 30 days. The mycelium and
medium were extracted with MeOH, and the combined
extracts were subsequently subjected to a combination of silica
gel column chromatography (CC), Sephadex LH-20 CC, and
semipreparative HPLC to yield compounds 1−5. The
structures of 1−5 were identified on the basis of HRESIMS
in addition to 1D and 2D NMR data analyses, as well as
Marfey’s method for determinations of the absolute config-
urations.
The molecular formula C39H61N9O12 for acremonpeptide B
(2) was determined on the basis of HRESIMS. Signals at m/z
848.4519 ([M + H]+) and m/z 870.4331 ([M + Na]+),
indicated a 16 mass unit deficiency relative to 1 suggesting one
O atom difference between structures 1 and 2. Comparisons of
1
the H and 13C NMR spectroscopic data of 2 with those of 1
(Tables 1 and 2) revealed that the hydroxymethyl signals at δH
3.76, 3.99, and δC 63.0 in 1 were replaced by methyl signals at
δH 1.37 and δC 18.4 in 2 suggesting that 2 harbors an Ala
residue within the shared cyclohexapeptide scaffold where
structure 1 houses a Ser residue. Detailed analyses of HMBC
correlations of 2 confirmed the presence of the Ala residue and
the sequence of all amino acid residues (Figure 1). Application
of Marfey’s method revealed that 2 is composed of L-Phe, L-
Leu, L-Ala, and L-Orn (Figures S32 and S36). On the basis of
these data, compound 2 was established to be cyclo-(-L-Phe-L-
Leu-L-Ala-L-AcN(OH)Orn1-L-AcN(OH)Orn2-L- AcN(OH)-
Orn3-) and named acremonpeptide B.
Acremonpeptide A (1) was obtained as a white solid with a
molecular formula of C39H61N9O13 as established by the
HRESIMS signal at m/z 886.4288 ([M + Na]+), indicating 14
1
degrees of unsaturation. The H and 13C NMR signals for 1
(Tables 1 and 2) unveiled the presence of three acetyl groups
(δH 2.12, δC 20.3, δC 173.8; δH 2.09, δC 20.4, δC 173.7; δH 2.11,
δC 20.3, and δC 173.8) with the aid of the HMBC correlations
of δH 2.12/δC 173.8, δH 2.09/δC 173.7, and δH 2.11/δC 173.8.
The remaining six carbonyl resonances at δC 172.1, 172.8,
173.3, 173.4, 174.7, and 175.0, together with six α-methine
carbon resonances at δC 54.0, 55.5, 55.9, 56.2, 57.1, and 57.4
revealed 1 to be a hexapeptide. The 1H NMR resonances at δH
7.24 (2H, d, J = 7.5 Hz), 7.30 (2H, t, J = 7.5 Hz), and 7.22
(1H, d, J = 7.5 Hz), as well as the 13C NMR signals at δC 139.1,
The HRESIMS spectrum for acremonpeptide C (3) showed
a protonated molecule peak at m/z 924.4831 ([M + H]+) and
a sodium adduct ion peak at m/z 946.4647 ([M + Na]+),
1
indicating a molecular formula of C45H65N9O12. The H and
13C NMR data of 3 were similar to those of 1, except that
signals attributed to the Ser residue in 1 were absent and
appeared to be replaced with a set of signals consistent with a
B
DOI: 10.1021/acs.jnatprod.9b00545
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1
Table 1. H NMR (600 MHz) Spectroscopic Data for Compounds 1−5 in CD3OD
δH, multi. (J in Hz)
position
Phe
acremonpeptide A (1)
acremonpeptide B (2)
acremonpeptide C (3)
acremonpeptide D (4)
Al(III)−acremonpeptide D (5)
2
3
4.53, dd (11.1, 4.3)
3.37, dd (13.8, 4.3);
3.05, dd (13.8, 11.1)
4.48, dd (9.0, 4.2)
3.36, dd (13.5, 4.2);
3.15, dd (13.5, 9.0)
4.49, t (8.2)
3.17, overlapped;
3.28, dd (13.5, 8.2)
4.45, dd (8.3, 5.5)
3.30, dd (12.5, 5.5);
3.17, dd (12.5, 8.3)
4.06, dd (11.4, 4.2)
3.57, d (13.8); 3.53, overlapped
5, 9
6, 8
7
7.24, d (7.5)
7.30, t (7.5)
7.22, t (7.5)
7.23, d (7.2)
7.27, t (7.2)
7.19, t (7.2)
7.28−7.30, overlapped
7.28−7.30, overlapped
7.22, t (6.6)
7.26, overlapped
7.28, overlapped
7.20, t (6.6)
7.18, d (7.2)
7.26, t (7.2)
7.20, overlapped
Leu
2
3
4.07, dd (8.5, 6.3)
1.27, m; 1.40, m
1.34, m
4.01, t (7.2)
1.52, m; 1.37, m
1.31, m
3.90, dd (7.0, 5.7)
1.59, m; 1.43, m
1.20, m
3.86, m
1.44, m; 1.30, m
0.96, br s
3.67, dd (8.4, 6.0)
0.99, m; 1.05, m
0.64, m
4
5
6
0.78, d (6.0)
0.83, d (6.0)
Ser
0.77,d (6.0)
0.81, d (6.0)
Ala
0.75, d (6.4)
0.80, d (6.4)
Phe
0.63, d (6.0)
0.67, d (6.0)
Trp
0.53, d (6.0)
0.56, d (6.0)
Trp
2
3
4.43, dd (9.3, 4.7)
3.99, dd (11.4, 4.7);
3.76, dd (11.4, 9.3)
4.27, q (7.2)
1.37, d (7.2)
4.43, t (7.2)
3.17, overlapped;
3.10, dd (13.3, 7.2)
4.55, t (6.8)
3.28, d (6.8)
4.57, m
2.87, br d (13.8);
3.37, dd (13.8, 4.0)
4
7.19, s
7.43, s
5
6
7
7.28−7.30, overlapped
7.28−7.30, overlapped
7.22, t (6.6)
7.65, d (7.8)
7.04, t (7.8)
7.11, t (7.8)
7.35, d (7.8)
7.96, d (7.8)
7.05, t (7.8)
7.14, t (7.8)
7.37, d (7.8)
8
9
10
7.28−7.30, overlapped
7.28−7.30, overlapped
AcN(OH)
Orn1
2
3
4.44, dd (7.6, 3.8)
4.36, br s
4.21, t (5.0)
4.60, br s
4.55, m
1.90−2.10, m; 1.64−1.85, m
1.90−2.09, m;
1.95−2.08, m;
1.85−1.95, m;
1.85−1.95, m;
1.67−1.88, m
1.66−1.90, m
1.76−1.62, m
1.76−1.62, m
4
1.70−1.85, m;
1.56−1.68, m
1.70−1.90, m;
1.32−1.60, m
1.63−1.85, m;
1.40−1.55, m
1.50−1.73, m;
1.30−1.50, m
1.80−1.95, m; 1.60−1.75, m
5
3.53−3.75, m
3.57−3.66, m
3.56−3.66, m
3.40−3.60, m
3.50−3.80, m
Me
2.12, s
2.12, s
2.11, s
2.10, s
2.15, s
AcN(OH)
Orn2
2
3
4.13, dd (10.4, 3.8)
4.08, t (6.6)
4.09, t (6.0)
4.15, m
4.39, dd (13.0, 2.4)
1.90−2.10, m; 1.64−1.85, m
1.90−2.09, m;
1.95−2.08, m;
1.85−1.95, m;
1.75−1.90, m;
1.67−1.88, m
1.66−1.90, m
1.76−1.62, m
1.50−1.66, m
4
1.70−1.85, m;
1.56−1.68, m
1.70−1.90, m;
1.32−1.60, m
1.63−1.85, m;
1.40−1.55, m
1.50−1.73, m;
1.30−1.50, m
1.80−1.95, m; 1.60−1.75, m
5
3.53−3.75, m
3.57−3.66, m
3.56−3.66, m
3.40−3.60, m
3.50−3.80, m
Me
2.09, s
2.09, s
2.08, s
2.09, s
2.17,s
AcN(OH)
Orn3
2
3
4.03, t (7.0)
4.18,dd (8.9, 3.2)
4.15, t (6.0)
4.23, m
4.34, dd (11.0, 9.4)
1.90−2.10, m; 1.64−1.85, m
1.90−2.09, m;
1.95−2.08, m;
1.85−1.95, m;
1.75−1.90, m;
1.67−1.88, m
1.66−1.90, m
1.76−1.62, m
1.50−1.66, m
4
1.70−1.85, m;
1.56−1.68, m
1.70−1.90, m;
1.32−1.60, m
1.63−1.85, m;
1.40−1.55, m
1.50−1.73, m;
1.30−1.50, m
1.80−1.95, m; 1.60−1.75, m
5
3.53−3.75, m
3.57−3.66, m
3.56−3.66, m
3.40−3.60, m
3.50−3.80, m
Me
2.11, s
2.10, s
2.09, s
2.10, s
2.16, s
second Phe residue (Tables 1 and 2). All indications supported
a Phe for Ser replacement in 3 relative to structure 1. Indeed,
HMBC corrections of δH 4.49/δC 173.6, 173.1, δH 3.90/δC
173.0, 173.6, δH 4.43/δC 173.8, 173.0, δH 4.21/δC 174.1, 173.8,
δH 4.09/δC 174.1, and δH 4.15/δC 173.1, 174.1 confirmed the
amino acid sequence of 3 to be cyclo-(-Phe1-Leu-Phe2-
AcN(OH)Orn1-AcN(OH)Orn2-AcN(OH)Orn3-). As with 1
and 2, application of Marfey’s method revealed 3 to be
composed of L-Phe, L-Leu, and L-Orn (Figures S33 and S36).
Acremonpeptide D (4) was isolated as a white solid. The
molecular formula of 4 was determined to be C47H66N10O12
based on the HRESIMS signals at m/z 963.4932 ([M + H]+)
1
and m/z 985.4735 ([M + Na]+). Analyses of the H and 13C
NMR signals of 4 made clear the presence of Phe, Leu, and
three AcN(OH)Orn residues (Tables 1 and 2). Further
comparisons of the 1D NMR data with those of 1, revealed the
absence of Ser in 4, as had been noted for compounds 2 and 3.
Additionally, five aromatic methine carbon signals at δC 112.6,
119.6, 120.1, 122.7, and 124.9, three nonprotonated sp2
C
DOI: 10.1021/acs.jnatprod.9b00545
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Table 2. 13C NMR (150 MHz) Spectroscopic Data for Compounds 1−5 in CD3OD
position
1
2
3
4
5
Phe
CO
2
3
4
172.8, C
172.8, C
173.1, C
173.1, C
171.8, C
57.1, CH
38.2, CH2
139.1, C
57.0, CH
37.6, CH2
139.1, C
57.4, CH
37.9, CH2
138.9, C
57.3, CH
37.8, CH2
138.9, C
58.8, CH
35.4, CH2
140.3, C
5, 9
6, 8
7
130.4, CH
129.7, CH
127.9, CH
130.4, CH
129.6, CH
127.8, CH
130.6, CH
129.7, CH
128.0, CH
130.5, CH
129.6, CH
127.7, CH
130.3, CH
129.6, CH
127.7, CH
Leu
CO
2
3
4
175.0, C
55.5, CH
41.2, CH2
25.7, CH
22.5, CH3
23.0, CH3
Ser
174.9, C
55.3, CH
40.8, CH2
25.7, CH
22.5, CH3
23.1, CH3
Ala
173.6, C
55.2, CH
40.8, CH2
25.6, CH
22.1, CH3
23.5, CH3
Phe
173.9, C
55.1, CH
40.7, CH2
25.4, CH
22.0, CH3
23.3, CH3
Trp
174.9, C
55.5, CH
41.0, CH2
25.3, CH
22.4, CH3
22.9, CH3
Trp
5
6
CO
2
3
4
5
6
7
8
172.1, C
56.2, CH
63.0, CH2
174.5, C
50.9, CH
18.4, CH3
173.0, C
57.4, CH
38.3, CH2
138.6, C
130.5, CH
129.7, CH
127.9, CH
129.7, CH
130.5, CH
173.6, C
56.5, CH
28.4, CH2
124.9, CH
110.9, C
128.8, C
119.6, CH
120.1, CH
122.7, CH
112.6, CH
138.1, C
172.0, C
53.6, CH
30.5, CH2
125.9, CH
111.5, C
128.4, C
120.5, CH
120.6, CH
123.2, CH
112.7, CH
138.4, C
9
10
11
AcN(OH)Orn1
CO
173.4, C
173.2, C
173.8, C
174.3, C
171.9, C
2
3
4
5
54.0, CH
30.8, CH2
23.9, CH2
48.5, CH2
173.8, C
54.7, CH
30.3, CH2
24.7, CH2
48.5, CH2
173.8, C
55.7, CH
29.2, CH2
24.5, CH2
48.6, CH2
173.7, C
56.5, CH
29.2, CH2
24.4, CH2
48.6, CH2
173.8, C
54.5, CH
26.6, CH2
23.2, CH2
50.3, CH2
163.7, C
MeCO
MeCO
20.3, CH3
20.6, CH3
20.5, CH3
20.5, CH3
15.8, CH3
AcN(OH)Orn2
CO
173.3, C
174.6, C
174.1, C
173.5, C
171.9, C
2
3
4
5
55.9, CH
28.1, CH2
25.0, CH2
48.5, CH2
173.7, C
57.0, CH
28.7, CH2
24.1, CH2
48.6, CH2
173.9, C
55.9, CH
29.1, CH2
24.7, CH2
48.5, CH2
173.7, C
56.4, CH
29.2, CH2
24.7, CH2
48.5, CH2
173.9, C
60.3, CH
26.3, CH2
27.7, CH2
50.2, CH2
163.9, C
MeCO
MeCO
20.4, CH3
20.5, CH3
20.5, CH3
20.5, CH3
16.7, CH3
AcN(OH)Orn3
CO
174.7, C
173.2, C
174.1, C
173.7, C
172.9, C
2
3
4
5
57.4, CH
28.9, CH2
24.7, CH2
48.5, CH2
173.8, C
55.4, CH
29.1, CH2
24.7, CH2
48.5, CH2
173.7, C
56.3, CH
28.9, CH2
24.6, CH2
48.6, CH2
173.7, C
55.5, CH
29.5, CH2
24.5, CH2
48.5, CH2
173.8, C
55.1, CH
28.7, CH2
23.0, CH2
49.2, CH2
163.9, C
MeCO
MeCO
20.3, CH3
20.5, CH3
20.5, CH3
20.5, CH3
16.3, CH3
hybridized carbon signals at δC 110.9, 128.8, and 138.1, one
methylene signal at δC 28.4 and one α-methine signal at δC
56.5 were observed. Four coupled aromatic 1H NMR signals at
δH 7.04 (t, J = 7.8 Hz), 7.11 (t, J = 7.8 Hz), 7.35 (d, J = 7.8
Hz), and 7.65 (d, J = 7.8 Hz) indicated the presence of a 1,2-
disubstituted benzene, which was supported by the COSY data
(Figure 1). Also, a singlet signal at δH 7.19 defined the
presence of an isolated aromatic proton. The HMBC
correlations from the four coupled protons (δH 7.04, 7.11,
7.35, and 7.65) and the isolated aromatic proton at δH 7.19, as
well as from the α-proton at δH 4.55 suggested the presence of
a tryptophan (Trp) residue (Figure 1). The amino acid
sequence of 4 was assigned as cyclo-(-Phe-Leu-Trp-AcN-
(OH)Orn1-AcN(OH)Orn2-AcN(OH)Orn3-) based on
HMBC corrections of δH 4.45/δC 173.1 and 173.9, δH 3.86/
δC 173.9 and 173.6, δH 4.55/δC 173.6 and 174.3, δH 4.60/δC
D
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closely related to the known agent AS2488059, which
originates from the same species as the titled fungus,14 and
readily complexes with Al(III) (ASP2397), Fe(III)
(AS2488053), and Ga(III) (AS2529132) ions. Notably,
AS2488059 and its derivatives display in vitro antifungal
activities against the clinical isolate Aspergillus fumigatus
FP1305. The most potent antibiotic in the hydroxamate
siderophore family is albomycin. Albomycin was initially
isolated from Streptomyces griseus in 1947,16 completely
elucidated in 1982,17 and generated by total synthesis by He
and co-workers in 2018.18 Importantly, albomycin is more
potent than penicillin G and vancomycin against clinical
isolates of Streptococcus pneumoniae and Staphylococcus aureus.
Inspired by these realizations and shared structural properties,
we assayed acremonpeptides A−D (1−4) and Al(III)−
acremonpeptide D (5) for antimicrobial activities against a
panel of bacteria and fungi. Somewhat surprisingly, 1−5
proved inactive in these assays at concentrations up to 100 μg/
mL. We then evaluated 1−5 for antiviral activities against
herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus
(VSV) by measuring the inhibition of the green fluorescent
protein (GFP) expression in HSV-1 and VSV infected Vero
cells (Figure 2A). We found that acremonpeptides A (1), B
(2), and Al(III)−acremonpeptide D (5) showed antiviral
activities against HSV-1 with EC50 values of 16, 8.7, and 14
μM, respectively (Table 3). Further mechanism of action
Figure 1. Key COSY and HMBC correlations of acremonpeptides A−
D (1−4).
174.3 and 173.5, δH 4.15/δC 173.5 and 173.7, and δH 4.23/δC
173.7 and 173.1. The absolute configurations of the amino acid
residues in 4 were determined to be L-Phe, L-Leu, L-Trp, and L-
Orn on the basis of Marfey’s method analyses (Figures S34 and
S36).
Table 3. Antiviral Activities of Compounds 1−5 against
Herpes Simplex Virus 1 (EC50, μM)
Compound 5 displayed a molecular formula of
C47H63AlN10O12 based on the HRESIMS peak at m/z
compound
EC50 value
16
8.7 1.2
1
2
3
4
5
6
1
987.4514 ([M + H]+). The H and 13C NMR spectroscopic
data of 5 were very similar to those obtained for 4 (Tables 1
and 2). However, the 13C NMR chemical shifts of the three
acetyl group methyls were noted to shift from δC 20.5 in 4 to
δC 15.8, 16.3, and 16.7 in 5, respectively (Table 2), consistent
with aluminum ion chelation by 4 to generate 5; a similar
phenomenon has been demonstrated with conversion of
known compound AS2488059 into ASP2397.14 As anticipated,
the application of Marfey’s method confirmed the presence of
L-Phe, L-Leu, L-Trp, and L-Orn residues in 5 (Figures S35 and
S36). Compound 5 was thus identified as shown and named
Al(III)−acremonpeptide D, the aluminum complex of 4.
Compounds 1−5 are natural hydroxamate-containing side-
rophores containing N5-hydroxy-N5-acetyl-L-ornithine units
derived from L-ornithine. Thus far, all siderophores originating
from fungi are hydroxamate-based; the only exception known
is the polycarboxylate rhizoferrin.2 The structures of 1−5 are
27
24
14
2
3
2
Ganciclovir (GCV)
0.025
(MOA) studies revealed that 1, 2, and 5 inhibit the
transcription of the UL42 gene in HSV; UL42 codes for a
polypeptide essential to viral DNA replication (Figure 2B).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
obtained with an MCP 500 polarimeter (Anton Paar). UV spectra
were recorded with a U-2600 spectrometer (Shimadzu). NMR spectra
were acquired with a DD2 spectrometer (Agilent) at 600 MHz for 1H
nuclei and 150 MHz for 13C nuclei. Chemical shifts (δ) are given in
ppm with reference to TMS. HRESIMS spectra were obtained using a
Figure 2. (A) Observed GFP expression in HSV-1 infected Vero cells incubated with compounds 1−5; (B) compounds 1−5 inhibited the
transcription of UL42 gene in HSV. Ganciclovir (GCV) was used as a positive control and NTC as a negative control.
E
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Maxis quadrupole-time-of-flight mass spectrometer (Bruker). Column
chromatography (CC) was performed with silica gel (200−300 mesh,
Yantai Jiangyou Silica Gel Development Co., Ltd.). Medium-pressure
liquid chromatography (MPLC) was performed with a CHEETAH
100 (Bonna-Agela) flash chromatography system. Preparative HPLC
was carried out using a QuikSep-50 system (H&E Co., Ltd.) with an
Innoval C18 column (250 × 21.2 mm, 5 μm, Bonna-Agela). Natural
sea salt is a commercial product from Guangdong Province Salt
Industry Group Co., Ltd., China.
CD3OD) data, Tables 1 and 2; HRESIMS m/z 987.4514 [M + H]+
(calcd for C47H64AlN10O12, 987.4515).
Marfey’s Analysis. Compounds 1 (0.87 mg), 2 (0.61 mg), 3
(1.20 mg), 4 (0.58 mg), and 5 (0.60 mg) were separately dissolved
into 1 mL of HCl (6 N) and heated at 110 °C for 18 h. The
hydrolysates were dried and resuspended in 100 μL of H2O. Then,
100 μL of 1% (w/v) 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide
(FDAA) in acetone and 50 μL of NaHCO3 (1 N) were added. The
mixtures were heated at 60 °C for 1 h, and then neutralized with 50
μL of HCl (1 N). MeOH was added to the quenched reaction to
afford a total volume of 500 μL. Each sample of ∼ 0.5 mg of D- and L-
amino acid standards were dissolved in 1 mL of NaHCO3 (1 N); and
50 μL of amino acid solution was treated with FDAA in the same
fashion. Derivatization products (20 μL) were then analyzed by
HPLC with a Phenomenex column (Prodigy ODS (2), 150 × 4.6
mm, 5 μm) using an elution system consisting of solvent A (0.1%
TFA in H2O) and solvent B (0.1% TFA in CH3CN). Samples were
eluted with a linear gradient from 10% to 90% solvent B over the
course of 30 min at a flow rate of 1 mL/min and UV detection of 340
nm. The retention times for DAA derivatives of the D-Phe, L-Phe, D-
Leu, L-Leu, D-Ser, L-Ser, D-Ala, L-Ala D-Tyr, and L-Tyr were 23.30,
19.75, 24.25, 20.35, 7.82, 6.85, 13.45, 10.45, 21.85, and 19.15 min,
respectively. The Phe and Leu residues in 1−5 were all assigned as L-
Phe and L-Leu, respectively, the Ser residue in 1 was assigned as L-Ser,
the Ala residue in 2 was assigned as L-Ala, and the Trp residue in 4
and 5 was assigned as L-Trp (Figures S31−S35).
For the determination of Orn residue, hydrolyses of compounds 1
(0.58 mg), 2 (0.61 mg), 3 (0.60 mg), 4 (0.59 mg), and 5 (0.60 mg)
were conducted using 45% HI in place of HCl (6 N) to reduce N5-
acetyl-N5-hydroxyornithine to ornithine for FDAA derivatization.5
Similarly, D- and L-ornithine standards were treated with FDAA
solution according to the aforementioned method. All the FDAA
derivatization products were analyzed using the above-mentioned
HPLC system with a linear gradient elution from 10% to 80% solvent
B over the course of 40 min. The retention times for DAA derivatives
(derivatization at the α-amine, the δ-amine, and both amines) of the
D-Orn and L-Orn were 10.12, 12.20, and 21.13 min and 11.92, 12.23,
and 22.32 min, respectively (Figure S36). The Orn residue present in
1−5 was determined to be L-Orn.
Antiviral Assays. Briefly, African green monkey kidney Vero cells
were infected with recombinant HSV-1 in which a reporter gene green
fluorecence protein (GFP) was inserted, at multiple of infection
(MOI) of 1.0. Cells were treated with compounds at a final
concentration of 10 μM as indicated in the figure or with equal
amounts of DMSO, respectively. Ganciclovir (GCV), a clinically used
anti-HSV medicine, was used as a positive control. Infected cells were
monitored by a fluorescence microscope. Viral loads in infected cells
were measured by TCID50 assay, and viral gene transcriptions were
measured by qRT-PCR assays. To estimate EC50 for each compound,
infected cells were treated with compounds at different concentrations
in triplicate and viral loads were monitored at 72 h postinfection.
Antimicrobial Assays. Compounds 1−5 were tested for their
antimicrobial activities by the method previously reported.19,20 Nine
bacteria including Bacillus thuringiensis BT01, B. thuringiensis W102, S.
aureus ATCC 29213, methicillin-resistant S. aureus shhs-A1 (a clinical
isolate), Enterococcus faecalis ATCC 29212, Acinetobacter baumannii
ATCC 19606, Klebsiella pneumoniae ATCC 13883, Escherichia coli
ATCC 25922, and drug resistant E. coli E11 (a clinical isolate), as well
as two fungi, Candida albicans ATCC 96901 and C. albicans CMCC
(F) 98001, were used.
Fungal Material, Fermentation, and Isolation. The fungal
strain A. persicinum SCSIO 115 was isolated from a marine sediment
sample collected in the South China Sea.11 SCSIO 115 was cultured
in potato dextrose broth medium supplemented with 3% sea salt at 28
°C on a rotary shaker at 200 rpm for 2 days as seed cultures, and then
each 50 mL seed culture was transferred into a 2000 mL Erlenmeyer
flask containing rice solid medium (300 g of rice, 0.2% yeast extract,
0.2% bacterial peptone, and 3% sea salt). The flasks were cultured
statically at room temperature for 30 days. A total of 4.5 kg harvested
medium and mycelium were extracted with 5 L MeOH (3 times) to
afford a residue following solvent evaporation. The residue was
divided into nine fractions (Fr.A1-Fr.A9) by silica gel CC using a
gradient elution of petroleum ether/EtOAc/MeOH (10:0:0, 8:2:0,
6:4:0, 4:6:0, 2:8:0, 0:10:0, 0:9:1, 0:8:2, 0:7:3, v/v). Fr.A9 was purified
by MPLC with an ODS column eluting with MeOH/H2O (0−20
min, 10:90−50:50; 20−45 min, 50:50−100:0; 45−75 min, 100:0; v/
v) at a flow rate of 15 mL min−1 to get Fr.B1−B11. Fr.B(4−6) were
combined and subjected to MPLC using an ODS column eluting with
MeOH/H2O (0−30 min, 10:90−50:50; 30−70 min, 50:50−90:10;
70−100 min, 100:0; v/v) at a flow rate of 15 mL min−1 to obtain
Fr.C1−C11. Fr.C (7−8) were combined and purified with preparative
HPLC equipment with an Innoval C18 column (250 × 21.2 mm, 5
μm), eluting with a mixture of MeOH/H2O (0−20 min, 40:60−
60:40) at a flow rate of 9 mL min−1 to yield compounds 1 (20.3 mg)
and 2 (18.5 mg) at tR 8.3 min and tR 8.6 min, respectively. Fr.C10 was
prepared by preparative HPLC using an elution system of CH3CN/
H2O (0−20 min, 45:55−85:15) at a flow rate of 9 mL min−1 to yield
4 (42.5 mg) and 3 (9.6 mg) at tR 11.8 and 12.3 min, respectively.
Fr.B(9−11) were combined and subjected to silica gel CC eluting
with mixtures of EtOAc/MeOH (100:0, 98:2, 96:4, 94:6, 92:8, 90:10,
95:15, 80:20, v/v) to get Fr.D1-Fr.D10. Fr.D9 was further purified by
preparative HPLC eluting with CH3CN/H2O (0−20 min, 50:50−
80:20) at a flow rate of 9 mL min−1 to yield 5 (8.5 mg) at tR 13.9 min.
Acremonpeptide A (1). White powder; [α]25D −27 (0.33, MeOH);
UV (MeOH) λmax (log ε) 205 (4.60) nm; 1H NMR (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data, Tables 1 and 2;
HRESIMS m/z 864.4459 [M + H]+ (calcd for C39H62N9O13,
864.4462), m/z 886.4288 [M + Na]+ (calcd for C39H61N9NaO13,
886.4281).
Acremonpeptide B (2). White powder; [α]25D −24 (0.62, MeOH);
UV (MeOH) λmax (log ε) 203 (4.50) nm; 1H NMR (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data, Tables 1 and 2;
HRESIMS m/z 848.4519 [M + H]+ (calcd for C39H62N9O12,
848.4512), m/z 870.4331 [M + Na]+ (calcd for C39H61N9NaO12,
870.4332).
Acremonpeptide C (3). White powder; [α]25D −48 (0.86, MeOH);
UV (MeOH) λmax (log ε) 203 (4.72) nm; 1H NMR (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data, Tables 1 and 2;
HRESIMS m/z 924.4831 [M + H]+ (calcd for C45H66N9O12,
924.4825), m/z 946.4647 [M + Na]+ (calcd for C45H65N9NaO12,
946.4645).
Acremonpeptide D (4). White powder; [α]25 −53 (0.33,
D
ASSOCIATED CONTENT
■
MeOH); UV (MeOH) λmax (log ε) 206 (4.75), 216 sh (4.67), 281
(3.85) nm; 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz,
CD3OD) data, Tables 1 and 2; HRESIMS m/z 963.4932 [M + H]+,
(calcd for C47H67N10O12, 963.4934), m/z 985.4735 [M + Na]+,
(calcd for C47H66N10NaO12, 985.4754).
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00545.
Al(III)−Acremonpeptide D (5). White powder; [α]20 +59 (0.58,
D
1D and 2D NMR spectra, HRESIMS spectra of
compounds 1−5, and HPLC chromatograms associated
MeOH); UV (MeOH) λmax (log ε) 203 (4.64), 218 (4.56), 289
(3.74) nm; 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz,
F
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(19) Gui, C.; Zhang, S.; Zhu, X.; Ding, W.; Huang, H.; Gu, Y.; Duan,
Y.; Ju, J. J. Nat. Prod. 2017, 80, 1594−1603.
(20) Zhang, S.; Gui, C.; Shao, M.; Kumar, P. S.; Huang, H.; Ju, J.
Nat. Prod. Res. 2019, 1.
with Marfey’s reaction analyses for absolute config-
uration determinations (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*Telephone/Fax: +86-20-89023028. E-mail: jju@scsio.ac.cn
(J.J.).
*Telephone/Fax: +86-20-34066449. E-mail: huanghb@scsio.
ac.cn (H.H.).
ORCID
Hongbo Huang: 0000-0002-5235-739X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported in part by the National Natural
Science Foundation of China (U1501223, U1706206,
81425022), Natural Science Foundation of Guangdong
Province (2016A030312014), the Chinese Academy of
Sciences (XDA13020302), Institution of South China Sea
Ecology and Environmental Engineering, Chinese Academy of
Sciences (ISEE2018PY04), and Chongqing Municipal Bureau
of Health Science and Technology of Traditional Chinese
Medicine Project (ZY20132062). We gratefully acknowledge
Ms. A. Sun and Ms. Y. Zhang in the analytical facility center of
the SCSIO for recording MS data.
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