Identification of Cyclic Depsipeptides and Their Dedicated Synthetase from Hapsidospora irregularis

Article abstract of DOI:10.1021/acs.jnatprod.6b00808

Seven cyclic depsipeptides were isolated from Hapsidospora irregularis and structurally characterized as the calcium channel blocker leualacin and six new analogues based on the NMR and HRESIMS data. These new compounds were named leualacins B-G. The absolute configurations of the amino acids and 2-hydroxyisocaproic acids were determined by recording the optical rotation values. Biological studies showed that calcium influx elicited by leualacin F in primary human lobar bronchial epithelial cells involves the TRPA1 channel. Through genome sequencing and targeted gene disruption, a noniterative nonribosomal peptide synthetase was found to be involved in the biosynthesis of leualacin. A comparison of the structures of leualacin and its analogues indicated that the A₂ and A₄ domains of the leualacin synthetase are substrate specific, while A₁, A₃, and A₅ can accept alternative precursors to yield new molecules.

Full text of DOI:10.1021/acs.jnatprod.6b00808

Article
pubs.acs.org/jnp
Identification of Cyclic Depsipeptides and Their Dedicated
Synthetase from Hapsidospora irregularis
Shuwei Zhang,† Yixing Qiu,†,‡ Thomas B. Kakule,§ Zhenyu Lu,⊥ Fuchao Xu,† John G. Lamb,⊥
Christopher A. Reilly,⊥ Yong Zheng,∥ Shing Wo Simon Sham,# Wei Wang,*,‡ Lijiang Xuan,∥
Eric W. Schmidt,§ and Jixun Zhan*,†,‡
†Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, Utah 84322, United States
‡TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine,
Changsha, Hunan 410208, People’s Republic of China
§Department of Medicinal Chemistry, L. S. Skaggs Pharmacy Institute, and ⊥Department of Pharmacology and Toxicology, University
of Utah, Salt Lake City, Utah 84112, United States
∥State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road,
Shanghai 202203, People’s Republic of China
#Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322, United States
S
* Supporting Information
ABSTRACT: Seven cyclic depsipeptides were isolated from Hapsidospora irregularis and structurally characterized as the calcium
channel blocker leualacin and six new analogues based on the NMR and HRESIMS data. These new compounds were named
leualacins B−G. The absolute configurations of the amino acids and 2-hydroxyisocaproic acids were determined by recording the
optical rotation values. Biological studies showed that calcium influx elicited by leualacin F in primary human lobar bronchial
epithelial cells involves the TRPA1 channel. Through genome sequencing and targeted gene disruption, a noniterative
nonribosomal peptide synthetase was found to be involved in the biosynthesis of leualacin. A comparison of the structures of
leualacin and its analogues indicated that the A2 and A4 domains of the leualacin synthetase are substrate specific, while A1, A3,
and A5 can accept alternative precursors to yield new molecules.
Current clinic calcium channel blockers are mainly divided
yclic depsipeptides are a group of natural products that
Chave shown a wide range of biological activities and show into three groups: phenylalkylamines (e.g., verapamil),
immunosuppressant,1,2 antibiotic,3,4 anti-inflammatory,5,6 and
benzothiazepines (e.g., diltiazem), and dihydropyridines (e.g.,
cilnidipine). Compound 1 represents a new type of calcium
channel blocker. It is of significant interest to find more
analogues of 1 for bioactivity screening. To this end, we
reinvestigated the chemical constituents of H. irregularis. Herein
we report the isolation and structure elucidation of six new
depsipeptides from this fungus. The absolute configurations of
amino and α-hydroxy acids were determined by hydrolysis of
antitumor7−9 properties. They are assembled from proteino-
genic/nonproteinogenic amino acids and hydroxy acids
through amide and ester linkages. Fungi are an important
source of biologically active molecules. Leualacin (1) was
previously isolated from the fungus Hapsidospora irregularis
FERM BP-2511 as a new cyclic depsipeptide and potent
calcium channel blocker, which inhibits the binding of H-
the parent depsipeptides and measurement of the optical
nitrendipine to porcine heart membranes in vitro and lowers the
blood pressure of spontaneous hypertensive rats.10,11 Calcium
channel blockers block the entry of calcium into cells. These
agents are used to treat hypertension, angina pectoris,
myocardial infarction, and abnormal heart rhythms.12,13
rotation values of the isolated moieties. The leualacin
synthetase gene was discovered by genome sequencing. Its
Received: September 4, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00808
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
2) with those previously reported.11 Compound 2 was obtained
as an off-white powder. The molecular formula was elucidated
1
as C32H49N3O7 based on HRESIMS analysis as well as its H
and 13C NMR data (Figures S4 and S5; Tables 1 and 2), which
indicated that 2 has an additional CH2 group compared to 1. By
1
comparing the H NMR data of 2 with those of 1, one CH at
δH 2.64−2.69 (1H, m) in 2 replaced the CH2 at 2.54−2.61
(2H, m) in 1. Compound 2 has an additional methyl group.
1
Analysis of its HSQC, HMBC, and H−1H COSY spectra
(Figure 1 and Figures S6−S8) revealed that 2 contains one unit
of N-methyl-L-phenylalanine (N-Me-L-Phe), two units of 2-
hydroxyisocaproic acid (2-HICA or leucic acid), and one unit
of leucine (Leu) that are also present in 1. However, the β-
alanine (β-Ala) moiety in 1 was replaced with 3-amino-
isobutyric acid (3-AIB) based on the specific COSY signals of
the spin system of NH−CH2−CH−CH3 and the HMBC
correlations of CH3 at δH 1.21 (3H, d, 7.1) and CH at δH 2.64−
2.69 (1H, m) to the carbonyl group at δC 174.9. The
connection order of the five moieties was determined by
HMBC correlations of α-CH of the N-Me-L-Phe moiety at δH
4.63 (1H, dd, 2.5, 10.5), β-CH2 at δH 3.60−3.71 (2H, m), and
NH at δH 7.45 (1H, dd, 5.0, 5.5) of the 3-AIB unit to the
carbonyl carbon of the N-Me-L-Phe moiety at δC 170.0, α-CH
of the 3-AIB moiety at δH 2.64−2.69 (1H, m) and α-CH of the
R-HICA moiety at δH 5.12 (1H, dd, 5.0, 7.6) to the carbonyl
group of the 3-AIB moiety at δC 174.9, α-CH of R-HICA, α-
CH at δH 4.50 (1H, ddd, 3.0, 9.7, 10.8) and NH at δH 6.32 (1H,
d, 9.4) of the Leu moiety to the carbonyl group of the R-HICA
unit at δC 171.0, α-CH of the Leu moiety and α-CH of the S-
HICA moiety at δH 4.73 (1H, dd, 2.1, 10.5) to the carbonyl
group of the Leu moiety at δC 174.1, and α-CH of the S-HICA
moiety, α-CH of the N-Me-L-Phe moiety, and NCH3 of the N-
Me-L-Phe moiety at δH 2.87 (3H, s) to the carbonyl group of
the S-HICA moiety at δC 170.1. Thus, 2 was identified as a new
compound that is identical to 1 except for a 3-AIB moiety that
replaces β-Ala in 1. It was named leualacin B.
Compounds 3 and 4 were obtained as an off-white powder.
HRESIMS analysis revealed that 3 and 4 have the same
molecular formula of C30H45N3O7. Thus, they have one CH2
less than 1. A comparison of the NMR data of 3 (Figures S9
and S10) and 1 and analysis of the 2D NMR spectra (Figures
S11−S13) revealed that 3 does not contain a methyl group at
the amino group of the L-Phe moiety. Instead, an NH signal
was found at δH 6.54 (1H, d, 8.6), which was confirmed by its
1H−1H COSY correlation to the α-CH proton of the L-Phe
moiety at δH 4.69−4.77 (1H, m) as well as its HMBC
correlation to the carbonyl group of the S-HICA moiety at δC
involvement in leualacin biosynthesis was confirmed by double
crossover gene disruption through Agribacterium-mediated
transformation. Production of the six leualacin analogues
revealed that this giant fungal noniterative peptide synthetase
has relaxed substrate specificity, which presents great potential
for precursor-directed biosynthesis of new cyclic depsipeptides.
1
170.1. Further analysis of the HSQC, HMBC, and H−1H
COSY spectra (Figure 1) indicated that 3 is also a cyclic
depsipeptide composed of five units, including β-Ala, R-HICA,
L-Leu, and S-HICA and L-Phe. Unlike 3, compound 4 was
found to have the methyl group at the amino group of the L-
Phe moiety (Figures S14−S18). Except for the leucine unit, the
1H and 13C NMR data of the other four units in compound 4
were nearly identical to those of compound 1. Furthermore, the
1H−1H COSY spectrum revealed a spin system of NH−CH−
CH−(CH3)2. The HMBC correlations of the CH at δH 4.43
(1H, d, 4.1) and CH at δH 2.35 (1H, qqd, 4.1, 6.5, 6.9) of this
spin system to the carbonyl group at δC 174.1 showed that the
L-Leu moiety in 1 was replaced with valine (Val) in this
compound. The connection of the five units was confirmed by
RESULTS AND DISCUSSION
■
H. irregularis FERM BP-2511 was grown in K-2 broth at 30 °C
for 8 days, and HPLC analysis revealed the production of 1−7,
in addition to the previously characterized cephalosporin P1,
isocephalosporin P1, Sch210971, and Sch210972 (Figure
S1).14,15 The strain was cultured in K2 broth with and without
0.5% tyrosine (3 L each), from which 1 and six new cyclic
depsipeptides were isolated (2−7).
The molecular formula of 1 was determined to be
C31H47N3O7 based on HRESIMS analysis, which is the same
as that of leualacin. It was further confirmed to be leualacin by a
comparison of its NMR data (Figures S2 and S3; Tables 1 and
B
DOI: 10.1021/acs.jnatprod.6b00808
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H NMR Data of Compounds 1−7 (1−6 Recorded in CDCl3, 7 Recorded in CD3OD, δH in ppm, J in Hz)
position
1
2
3
4
5
6
7
L-Phenylalanine
L-Tyrosine
α
4.63−4.67, m
4.63, dd (2.5, 10.5)
4.69−4.77, m
4.65, dd (3.4, 11.4)
4.61, dd (3.0, 11.0)
4.61−4.67, m
4.52, dd (6.2,
8.3)
α-NH
6.54, d (8.6)
6.8, d (8.3)
N/A
N-CH3 2.87, s
2.87, s
2.87, s
2.87, s
β
2.94 dd (10.5,
14.4)
2.95, dd (10.9, 12.4)
3.12−3.23, m
2.94, dd (11.7, 14.4)
2.88, dd (11.5, 14.5)
3.05−3.10, m
2.99 dd (8.6,
14.1)
3.38, dd (2.7,
14.3)
3.30, dd (2.52, 12.2)
3.41, dd (3.4, 14.1)
3.31, dd (3.0, 14.5)
3.12, dd (6.2,
14.1)
o
7.10, d (7.5)
7.26−7.31, m
7.23−7.25, m
7.07, d (6.9)
7.23−7.29, m
7.23, t (6.9)
7.17, d (7.1)
7.23−7.32, m
7.23, t (6.9)
β-Alanine
7.09, d (6.9)
7.22−7.31, m
7.29, t (7.6)
6.91, d (8.5)
6.75, d (8.5)
6.96, d (7.9)
6.74, d (7.9)
6.99, d (8.6)
6.99, d (8.6)
m
p
β-Alanine
R-3-Aminoisobutyric
acid (R-3-AIB)
α
2.54−2.61, m
2.64−2.69, m
1.21, d (7.1)
3.60−3.71, m
2.53−2.67, m
2.57, dd (3.8, 8.3)
2.54−2.57, m
2.49−2.52, m
2.44−2.59, m
α-CH3
β
3.31−3.39, m
3.98−4.09, m
7.45−7.48, m
3.82−3.94, m
3.25−3.34, m
7.10−7.15, m
4.04, dt (3.8, 13.7)
3.33−3.42, m
7.44−7.49, m
3.97−4.10, m
3.34−3.42, m
7.49, d (6.8)
3.80−3.84, m
3.21−3.25, m
7.55−7.57, m
3.73−3.80, m
3.22−3.30, m
N/A
β-NH
7.45, dd (5.0, 5.5)
(R)-2-hydroxyisocaproic acid (R-HICA)
α
5.13−5.18, m
5.12, dd (5.0, 7.6)
5.19, dd (3.8,
9.9)
5.21, dd (6.2, 7.9)
5.17, dd (6.4, 7.6)
5.17−5.21, m
5.17−5.24, m
β
1.68−1.85, m
1.59−1.71, m
1.68−1.88, m
1.58−1.73, m
1.66−1.90, m
1.60−1.77, m
1.72−1.87, m
1.59−1.70, m
L-Valine
1.71−1.85, m
1.60−1.69, m
L-Leucine
1.62−1.83, m
1.54−1.68, m
1.61−1.78, m
1.64−1.74, m
L-Valine
γ
L-Leucine
α
4.48−4.56, m
4.50, ddd (3.0, 9.7,
10.8)
4.08−4.18, m
4.43, d (4.1)
4.48−4.55, m
4.13−4.17, m
3.90, d (8.9)
α-NH
6.16, d (9.8)
6.32, d (9.4)
6.89, d (6.6)
6.41, d (9.6)
6.32, d (9.6)
7.41, d (6.6)
N/A
β
1.57−1.72, m
1.48−1.73, m
1.54−1.73, m
2.35, qqd (4.1, 6.5,
6.9)
1.50−1.81, m
1.50−1.70, m
2.08−2.16, m
γ
1.65−1.86, m
1.61−1.76, m
1.60−1.77, m
1.64−1.82, m
1.54−1.65, m
(S)-2-Hydroxyisocaproic acid (S-HICA)
α
4.65−4.70, m
4.73, dd (2.1, 10.5)
5.07, dd (3.6,
6.1)
4.66, dd (2.3, 11.3)
4.72, dd (1.8, 11.0)
5.15−5.17, m
1.45−1.57, m
5.17−5.24, m
1.29−1.54, m
β
1.35−1.59, m
1.36−1.48, m
1.35−1.54, m
1.37−1.52, m
1.41−1.50, m
−0.38 - −0.47, m −0.32 - −0.40, m
1.35−1.59, m 1.36−1.48, m
−0.38 to −0.47, m
1.23−1.40, m
−0.21 to −0.27, m
1.43−1.60, m
γ
1.33−1.52, m
1.30−1.43, m
1.43−1.49, m
R/S-2-Hydroxyisocaproic acid (R/S-HICA) and δ-H of L-leucine (L-Leu)/γ-H of L-valine (L-Val)
R-HICA
R-HICA/L-Leu
0.92, d (5.5)
0.87, d (5.9)
S-HICA
S-HICA
R-HICA
R-HICA/L-Leu
S-HICA
R-HICA
0.56, d (6.5)
0.67, d (6.5)
S-HICA/
0.84, d (5.9)
0.86, d (6.8)
R-HICA/
0.91, d (6.5)
0.95, d (6.1)
L-Val
0.91, d (5.9)
0.92, d (6.2)
L-Leu
0.95, d (6.5)
0.99, d (6.2)
L-Val
0.90, d (6.2)
0.9, d (6.4)
S-HICA
L-Leu
0.66, d (6.2)
0.56, d (6.4)
L-Leu
0.91, d (6.5)
1.01, d (6.9)
S-HICA
0.91, d (5.9)
0.97, d (5.5)
R-HICA
0.97, d (6.5)
1.00, d (6.5)
S-HICA
0.89, d (6.5)
0.93, d (6.5)
0.92, d (6.2)
0.93, d (6.2)
0.95, d (6.5)
1.00, d (5.9)
0.62, d (6.4)
0.73, d (6.2)
0.56, d (6.5)
0.67, d (6.5)
0.83, d (5.8)
0.87, d (5.2)
0.86, d (5.8)
0.89, d (5.8)
its HMBC spectrum (Figure 1). Both 3 and 4 are new
compounds and were named leualacins C and D, respectively.
The molecular formula of 5 was determined to be
C31H47N3O8 according to the HRESIMS data, suggesting that
it has one more oxygen atom than 1. The NMR spectra of 5
(Figures S19−S23) were similar to those of 1, except the
difference in the N-Me-L-Phe moiety. The aromatic proton
2D NMR spectra, 5 was characterized as a new leualacin
analogue that has the same structure as 1 except that the N-Me-
L-Phe was replaced with N-methyl-L-tyrosine (N-Me-L-Tyr).
This compound was named leualacin E. Compound 6 also has
eight oxygen atoms in its molecular formula of C30H45N3O8
based on the HRESIMS, but it has one less CH2 than 5. The
NMR spectra (Figures S24−S28) also showed that it has a Tyr
unit, and the detailed 2D NMR signals confirmed it contains
the same units as those in 5 with the same connection order,
except that there is no methyl group at the amino group of the
L-Tyr moiety. This was confirmed by the 1H−1H COSY
correlation (Figure 1) between NH at δH 6.83 (1H, d, 8.3) and
the α-CH of L-Tyr at δH 4.61−4.67 (1H, m). 6 was named
leualacin F.
1
signals at δH 6.91 (2H, d, 8.5) and 6.75 (2H, d, 8.5) in the H
NMR spectrum and carbon signals at δC 127.6 (C), 130.4 (2C,
CH), 115.9 (2C, CH), and 155.7 (C) in the 13C NMR
spectrum (Tables 1 and 2) indicated that there is a 1,4-
disubstituted phenyl ring, with a hydroxy as one of the
substituents. The HMBC correlations (Figure 1) revealed the
presence of an N-Me-L-Tyr moiety. According to the 1D and
C
DOI: 10.1021/acs.jnatprod.6b00808
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Data of Compounds 1−7 (1−6 Recorded in CDCl3, 7 Recorded in CD3OD, δC in ppm)
position
1
2
3
4
5
6
7
L-Phenylalanine
L-Tyrosine
168.8 (s)
64.0 (d)
CO
173.5 (s)
63.9 (d)
170.0 (s)
63.3 (d)
171.4 (s)
54.4 (d)
168.5 (s)
63.8 (d)
172.0 (s)
54.8 (d)
173.6 (s)
α
56.5 (d)
N-CH3
30.3 (q)
30.2 (q)
30.2 (q)
30.3 (q)
β
34.4 (t)
34.4 (t)
37.8 (t)
34.3 (t)
33.3 (t)
37.2 (t)
37.6 (t)
γ
138.1 (s)
129.8 (2C, d)
129.5 (2C, d)
127.4 (d)
137.5 (s)
129.7 (2C, d)
129.4 (2C, d)
127.3 (d)
136.9 (s)
138.0 (s)
129.7 (2C, d)
129.4 (2C, d)
127.3 (d)
128.7 (s)
130.7 (2C, d)
116.4 (2C, d)
155.9 (s)
127.6 (s)
128.8 (s)
o
129.5 (2C, d)
128.9 (2C, d)
127.1 (d)
β-Alanine
171.8 (s)
130.4 (2C, d)
115.9 (2C, d)
155.7 (s)
131.5 (2C, d)
116.4 (2C, d)
157.6 (s)
m
p
β-Alanine
CO
α
R-3-Aminoisobutyric acid (R-3-AIB)
174.8 (s)
34.7 (t)
174.9 (s)
40.4 (d)
13.6 (q)
41.4 (t)
173.5 (s)
34.8 (t)
173.2 (s)
34.5 (t)
171.8 (s)
34.8 (t)
172.2 (s)
35.6 (t)
34.8 (t)
α-CH3
β
34.9 (t)
36.1 (t)
34.3 (t)
34.7 (t)
36.1 (t)
37.1 (t)
(R)-2-Hydroxyisocaproic acid (R-HICA)
CO
170.6 (s)
70.9 (d)
37.9 (t)
24.4 (d)
171.0 (s)
72.8 (d)
37.8 (t)
24.4 (d)
171.0 (s)
71.7 (d)
39.0 (t)
24.6 (d)
170.7 (s)
72.9 (d)
37.4 (t)
24.3 (d)
L-Valine
174.1 (s)
57.8 (d)
29.0 (d)
171.0 (s)
72.9 (d)
37.5 (t)
24.3 (d)
L-Leucine
174.6 (s)
51.6 (d)
38.6 (t)
25.2 (d)
171.2 (s)
71.9 (d)
39.5 (t)
24.6 (d)
173.0 (s)
73.9 (d)
41.3 (t)
25.9 (d)
L-Valine
171.0 (s)
61.9 (d)
30.5 (d)
α
β
γ
L-Leucine
CO
171.6 (s)
51.5 (d)
38.9 (t)
25.4 (d)
174.1 (s)
51.6 (d)
38.5 (t)
25.2 (d)
171.2 (s)
53.2 (d)
39.2 (t)
25.2 (d)
170.6 (s)
53.2 (d)
39.1 (t)
25.1 (d)
α
β
γ
(S)-2-Hydroxyisocaproic acid (S-HICA)
CO
168.6 (s)
72.9 (d)
37.6 (t)
24.2 (d)
171.7 (s)
70.6 (d)
37.9 (t)
24.2 (d)
170.1 (s)
74.5 (d)
40.4 (t)
24.7 (d)
171.2 (s)
70.8 (d)
37.9 (t)
24.4 (d)
172.1 (s)
71.0 (d)
37.7 (t)
24.4 (d)
170.8 (s)
74.1 (d)
40.6 (t)
24.6 (d)
173.3 (s)
74.3 (d)
42.4 (t)
25.6 (d)
α
β
γ
δ-C of L-Leucine and R/S-HICA, γ-C of L-valine
R-HICA
20.9 (q)
23.1(q)
S-HICA/L-
Leu
R-HICA
S-HICA
21.7 (q)
23.1 (q)
R-HICA/L-
Leu
R-HICA
R-HICA
R-HICA
R-HICA
22.2/22.9 (q)
L-Leu
22.4/23.0 (q)
L-Val
22.3/23.0 (q)
L-Leu
21.7 (q)
22.7/23.5 (q)
L-Val
22.9 (q)
20.8/22.9 (q)
S-HICA
17.2/19.6 (q)
S-HICA
20.8/22.8 (q)
S-HICA
S-HICA/L-Leu
23.1 (2C, q)
21.8 (q)
20.2/19.6 (q)
S-HICA
23.2 (q)
23.1 (q)
22.5 (q)
21.3 (q)
21.2/22.9 (q)
23.3 (q)
23.2 (q)
21.8 (2C, q)
20.8/23.0 (q)
21.1/22.9 (q)
22.2/23.3 (q)
21.7 (q)
The molecular formula of 7 was determined to be
C29H43N3O8 by HRESIMS analysis, which is one CH2 unit
less than 6. Analysis of the 2D NMR spectra of 7 (Figure 1 and
Figures S29−S33; Tables 1 and 2) revealed that it contains the
N-Me-L-Tyr, β-Ala, R-HICA, and S-HICA moieties that are also
present in 6. However, 7 has a Val moiety in the same position
as the L-Leu moiety in 6. It was named leualacin G.
The absolute configuration of the major product 1 has been
previously determined11 to consist of β-Ala, R-HICA, L-Leu, S-
HICA, and N-Me-L-Phe. 2−7 are new compounds that have
one or two new moieties that substitute the β-Ala, L-Leu, or N-
Me-L-Phe moiety in 1. In order to determine the absolute
configurations of these new moieties, we hydrolyzed the
representative compounds 2, 4, and 6 at the ester bonds using 1
N NaOH (Supporting Information) to get two fragments and
analyzed the products by LC-MS. The two fragments were
separated by HPLC and then treated with 6 N HCl to
hydrolyze the amide bonds, from which individual units of
these compounds were obtained (Figure S34). The optical
rotations of these units were recorded (Table S1). Their
absolute configurations were determined by comparing the
recorded optical rotations with those of standards or reported.
The results showed the moieties in 2−7 are L-Phe11 or L-Tyr,16
β-Ala or R-3-AIB,17 R-HICA, S-HICA,11,18 and L-Leu19 or L-
Val.20
Compound 1 blocks H-nitrendipine binding to porcine heart
membranes in vitro and can lower the blood pressure of
spontaneous hypertensive rats.5 Calcium is a versatile and
critical second messenger in all cells. Stimulation and inhibition
of changes in intracellular calcium content caused by 1−7 were
assessed in lobar bronchial epithelial cells. TRP ion channels in
lung epithelial cells are involved in innate immunity and various
other processes. Calcium influx was elicited by 3 and 6 at 100
μM, which was correlated with activation of the human TRPA1
and to a lesser extent M8 channels, but not TRPV1, -V3, or -V4
(Table S2). Activation of TRPA1 is associated with pulmonary
irritation, cough, and edema. Elsewhere, TRPA1 signals pain
and is associated with allergic sensitization including asthma
and dermatitis.21−23
D
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1
Figure 1. Selected H−1H COSY and HMBC correlations for 2−7.
Figure 2. Proposed biosynthesis of 1 by a noniterative NRPS.
Compounds 1−7 belong to a large group of structurally
diverse natural products, nonribosomal peptides (NRPs). They
are synthesized by nonribosomal peptide synthetases (NRPSs).
These synthetases are modular enzymes that consist of different
catalytic domains, such as condensation (C), adenylation (A),
thiolation (T), methylation (MT), epimerization (E), and
oxidation (Ox). To understand how 1−7 are synthesized, the
genome of H. irregularis was sequenced. Because the structure
of 1 contains different amino acid/hydroxy acid units, it is
proposed that this molecule is assembled by a noniterative
NRPS that is often present in bacteria. Annotation of the
sequenced genome revealed the presence of a large noniterative
NRPS (Figure 2, GenBank accession number KU994894) that
consists of five A domains, five T domains, and five C domains.
Additionally, there is an E domain sitting between the T2 and
C3 domains. An N-MT domain is embedded in the A5 domain.
The domain organization of this 6521-aa enzyme (717 kDa,
named leualacin synthetase, or LACS) is colinear with the
structure of 1, and thus it represents a possible NRPS involved
in leualacin biosynthesis. Although the signature sequences of
the five A domains (Table S3) could be extracted from the
amino acid sequence of LACS using NRPPSpredictor224 based
on the reported bacterial NRPSs, it is hard to conclusively
predict the substrate of each A domain due to the lack of
information about genuine signature sequences in fungal
NRPSs. In addition, there are several novel precursors used
in the biosynthetic process of 1−7, such as HICA, which has
not been reported as a direct precursor for NRP biosynthesis.
Although cryptophycins also have an HICA unit, it was
reported that the A4 domain of the dedicated PKS/NRPS
selects α-ketoisocaproic acid, which is reduced by a
ketoreductase domain in the same module to yield the HICA
unit.25 We predicted the substrate of each A domain based on
the domain organization of LACS and the structure of 1. A1−A5
were proposed to select and activate β-Ala, S-HICA, L-Leu, S-
HICA, and L-Phe. The E domain in the module converts S-
HICA to R-HICA, while the MT domain in module 5
methylates the NH2 group of the L-Phe moiety. The C-terminal
E
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Figure 3. Disruption of lacs in H. irregularis. (A) Strategy for targeted disruption of lacs. (B) Verification of the correct mutant by PCR. (C) HPLC
analysis of the products of the wild type and mutant on the oatmeal agar plates at 210 nm. (i) Wild type; (ii) mutant; (iii) standard of 1; (iv)
standard of 2.
HRESIMS were measured on an Agilent G6224A TOF mass
spectrometer.
condensation-like (CT) domain catalyzes the cyclization and
concomitant release of the final products.
Strains, Media, and Culture Conditions. Hapsidospora irregu-
laris FERM BP-2511 was purchased from the International Patent
Organism Depositary, Japan. It was routinely grown in K2 agar
medium consisting of potato dextrose agar (3.9%, w/v), sucrose (2%,
w/v), casamino acid (1%, w/v), KH2PO4 (0.5%, w/v), and MgSO4
(0.125%, w/v) at 30 °C. E. coli XL-1 Blue (Stratagene) was used for
routine cloning and plasmid propagation. It was routinely grown at 37
°C on LB agar plates or in liquid LB medium supplemented with
appropriate antibiotics (ampicillin, 50 μg/mL; kanamycin, 50 μg/mL).
Agrobacterium tumefaciens LBA4404 was used for the introduction of
the disruption plasmid into H. irregularis FERM BP-2511.
Production and Purification of Cyclic Depsipeptides. Single
colonies were obtained by streaking the fungus on K2 agar medium at
30 °C for 8 days. A single colony was picked and precultured in 4 mL
of K2 broth, then cultured in 50 mL of K2 broth consisting of potato
dextrose broth (2.4%, w/v), sucrose (2%, w/v), casamino acid (1%, w/
v), KH2PO4 (0.5%, w/v), and MgSO4 (0.125%, w/v) at 250 rpm and
30 °C for 7 days. The culture was centrifuged at 3500 rpm for 5 min.
The supernatant was extracted with 50 mL of ethyl acetate three times,
and the cells were extracted three times with 50 mL of methanol. The
ethyl acetate and methanol extracts were combined and dried under
reduced pressure. The residue was redissolved in 1 mL of methanol for
analysis on an Agilent 1200 HPLC (HPLC conditions are shown in
Figure S1).
The strain was also grown in 3 L of K2 broth and 3 L of K2 plus
0.5% L-tyrosine (to enhance the production of 5−7) for the isolation
of 1−7. After 8 days, the cultures were combined and centrifuged at
3500 rpm for 5 min. The supernatant was combined and loaded to an
HP-20 column (10 cm × 50 cm), and the column was eluted with 4 L
of water. The cells were extracted with 500 mL of methanol three
times with sonication for 40 min. After centrifugation at 3500 rpm for
5 min, the methanol extract was diluted with 3 volumes of water and
the solution was loaded to the HP-20 column as the 25% eluant. The
column was then successively eluted with 4 L of 50% aqueous
methanol, 75% aqueous methanol, and 100% methanol. These
fractions were analyzed by HPLC, and the target compounds were
found in the 75% and 100% methanol fractions. These two fractions
were separated on the Agilent 1220 HPLC with an Agilent ZORBAX
SB-C18 column (5 μm, 21.2 mm × 150 mm), eluted with a gradient of
methanol−water (0−15 min, 70%; 15−18 min, 70−80%; 18−25 min,
80%; 25−27 min, 80−90%; 27−35 min, 90%, v/v) with 0.1% formic
acid at a flow rate of 3 mL min−1 to obtain six fractions, A−F. LC-MS
analysis indicated that fraction A contained 7, fraction B contained 5
and 6, fraction D contained 3 and 4, fraction E contained 1, and
fraction F contained 1 and 2. Then fractions A−F were repurified by
HPLC with an Agilent ZORBAX SB-C18 column (5 μm, 4.6 mm ×
250 mm) at 1 mL min−1 and detected at 210 nm. Fraction A was
eluted with 40% acetonitrile−water to get pure 7 (18 mg). Fraction B
was eluted with 55% acetonitrile−water to get pure 5 (33 mg) and 6
(59 mg). Fraction D was eluted with 60% acetonitrile−water to get
pure 3 (35 mg) and 4 (35 mg). Fraction E was eluted with 70%
To find out whether this NRPS is involved in the
biosynthesis of 1, we conducted a gene disruption experiment.
Two fragments (3.5 and 4.0 kb) of lacs were amplified from the
genome of H. irregularis and ligated into the binary donor
vector pAG1-H3 as the left and right arms, respectively. The
resulting plasmid pAG1-H3-KO (Figure 3A) was introduced
into H. irregularis through Agrobacterium tumefaciens-mediated
transformation. Correct transformants were then subjected to
PCR screening using the genome- and vector-specific primers
A−D (Figure 3A). As shown in Figure 3B, a 4534-bp fragment
was amplified from the correct double crossover mutant with
primers A and B and a 5026-bp fragment with primers C and D.
Because primers B and C were specifically from the hygromycin
resistance gene (HygR) in the vector, no PCR products were
obtained from the wild type. This correct mutant was then
grown on the oatmeal agar modified from WSH agar5 for
product analysis. As shown in Figure 3C, when grown in the
oatmeal medium, the wild type produces 1 and 2 as the major
metabolites (traces i−iii). In contrast, the mutant failed to
produce 1 or any analogues (trace (iv), indicating the essential
role of LACS in leualacin biosynthesis.
H. irregularis is a fungus of the family Pseudeurotiaceae.26 In
this work, six new cyclic depsipeptides were characterized,
which significantly expanded the spectrum of natural products
from this fungus. These structures consist of two HICA units
with different configurations and three amino acid moieties.
The main difference among the seven cyclic depsipeptides lies
in the amino acid units, while the two HICA units remain the
same. This indicated that A2 and A4 of LACS, which activate S-
HICA, are more substrate specific. In contrast, A1, A3, and A5
are more flexible. The structures of 5−7 showed that A5 can use
L-Tyr instead of L-Phe in 1. Similarly, A1 was found to take 3-
AIB to yield 2, while the discovery of 4 and 7 indicated that A3
activates both L-Val and L-Leu. Thus, LACS is a highly flexible
NRPS that synthesizes a variety of leualacin analogues in the
presence of different precursors.
EXPERIMENTAL SECTION
■
General Experimental Procedures. 1D NMR spectra were
recorded in CDCl3 or CD3OD at 25 °C on a JEOL ECX-300
1
instrument (300 MHz for H NMR and 75 MHz for 13C NMR). 2D
NMR spectra were collected on a JEOL ECX-300 or a Bruker
AvanceIII HD Ascend-500 (HMBC spectrum of compound 7)
instrument. The chemical shift (δ) values are given in parts per
million (ppm). The coupling constants (J values) are reported in hertz
(Hz). LRESIMS were acquired on an Agilent 6130 LC-MS, and
F
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Article
methanol−water to get pure 1 (450 mg). Fraction F was eluted with
85% methanol−water to get pure 2 (39 mg). The purified compounds
were subjected to spectral analyses for structure determination (Tables
1 and 2).
Treatment solutions were prepared in LHC9 (TRPV1, M8, and V4)
or calcium assay buffer (TRPA1, V3, LOBAR) at 3× concentration,
and 25 μL was added to 50 μL of media on the cells in 96-well plates.
Changes in cellular fluorescence were monitored for 1 min at 37 °C.
Data were quantified as the maximum rate of change in fluorescence
intensity (max ΔF/s) vs media-only treatment (negative control) and,
in HEK-293 or normal BEAS-2B cells, were represented as the
percentage of response relative to the specific TRP agonist control
(TRPA1-2,4-di-tert-butylphenol at 250 μM, M8-icilin at 25 μM, V1-
capsaicin at 10 μM, V3-carvacrol at 300 μM, and V4-GSK1016790A at
30 nM). A minimum of three replicates were used for all treatments.
Knockout of lacs in H. irregularis FERM BP-2511 through
Agrobacterium-Mediated Transformation. H. irregularis FERM
BP-2511 was cultured in 50 mL of K2 broth at 30 °C and 250 rpm for
6 days. The mycelia were collected by filtration and stored at −80 °C
for 2 h. Then 200 mg of the mycelia was ground in liquid nitrogen. A 3
mL amount of the lysis buffer consisting of 0.5 M NaCl, 10 mM Tris-
HCl (pH = 7.5), 10 mM EDTA, and 1% SDS was added into the
mycelia at 65 °C. The extraction of genomic DNA was done with
cetyltrimethylammonium bromide extraction buffer28 followed by
purification through phenol/chloroform extraction and precipitation
with 2-propanol.29 RNA was removed by adding RNase.
Two DNA fragments were amplified by PCR from the lacs gene
using the genomic DNA of H. irregularis FERM BP-2511 as the
template. A 3600-bp fragment upstream of the disruption region of the
lacs gene was amplified using the primers BP2511-128-NRPS-L-AvrII-
3 (5′-aaCCTAGGacagcctgggtttcgagaac-3′, AvrII site in bold) and
BP2511-128-NRPS-L-KpnI-5 (5′-aaGGTACCagagcctgtgacttccagac-3′,
KpnI site in bold) and cloned into pJET1.2 for sequencing. After
sequence validation, this left-arm gene fragment was excised with AvrII
and KpnI and inserted into pAG1-H3 between the same sites. A 3976-
bp fragment downstream of the disruption region of the lacs gene was
amplified using the primers BP2511-128-NRPS-R-SbfI-3 (5′-
aaCCTGCAGGgtgtcgctctttcggtcgtg-3′, SbfI site in bold) and
BP2511-128-NRPS-R-SpeI-5 (5′-aaACTAGTcgagcccgacgttggctgca-3′,
SpeI site in bold) and cloned into pJET1.2 for sequencing. After
sequence validation, the right-arm gene fragment was excised with SbfI
and SpeI and inserted into pAG1-H3-right arm between the same
sites. This disruption plasmid was designed to replace a portion of the
lacs gene with the hygromycin phosphotransferase gene of pAg1-H3
upon double homologous recombination with the H. irregularis
genome.
Compound 1: off-white powder; [α]1D9 −97.8 (c 1.0, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 574.3493 [M
+ H]+ (calcd for C31H48N3O7, 574.3487).
Compound 2: off-white powder; [α]1D9 −99.0 (c 0.3, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 588.3648 [M
+ H]+ (calcd for C32H50N3O7, 588.3643).
Compound 3: off-white powder; [α]1D9 −48.2 (c 1.17, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 560.3332 [M
+ H]+ (calcd for C30H46N3O7, 560.3330).
Compound 4: off-white powder; [α]1D9 −68.0 (c 0.25, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 560.3333 [M
+ H]+ (calcd for C30H46N3O7, 560.3330).
Compound 5: off-white powder; [α]1D9 −96.1 (c 0.62, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 590.3440 [M
+ H]+ (calcd for C31H48N3O8, 590.3436).
Compound 6: off-white powder; [α]1D9 −46.0 (c 0.50, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 576.3284 [M
+ H]+ (calcd for C30H46N3O8, 576.3279).
Compound 7: off-white powder; [α]1D9 −52.9 (c 0.34, MeOH); H
1
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 562.3119 [M
+ H]+ (calcd for C29H44N3O8, 562.3123).
Determination of Absolute Configurations of the Five Units
in 1−7 (Table S1 and Figure S34). A 25 mg amount of 6 was
dissolved in 600 μL of 30% aqueous acetonitrile, and 100 μL of 1 N
NaOH in water was added under stirring. The compound was
hydrolyzed at 40 °C for 1 h. The reaction mixture was neutralized with
1 N HCl. The hydrolysis was confirmed by LC-MS, and two fragments
(with a MW of 366 and 245, respectively) were purified on an Agilent
1220 HPLC with an Agilent ZORBAX SB-C18 (5 μm, 4.6 mm × 250
mm) at 1 mL min−1, eluted with 35% acetonitrile−water and
monitored at 210 nm. The two fragments were further hydrolyzed
with 6 N HCl at 115 °C for 12 h. After neutralizing with NaOH, the
products were analyzed by LC-MS and compared with the standards.
Then the target products were purified on the same HPLC eluted with
a gradient of acetonitrile−water from 0% to 100% in 30 min. The
optical rotations of the purified amino acids and hydroxy acids were
recorded on a Rudolph Autopol IV polarimeter with a 10 cm cell at 19
°C. Compounds 2 and 4 were hydrolyzed and purified according to
the same procedure for 6. The absolute configurations were
determined by comparing the optical data with those reported in
the literature. The optical rotations of compounds 1−7 were also
recorded on the same equipment (Table S1).
Transient Receptor Potential Channel Assay. Fluorometric
cell-based Ca2+ flux assays were performed using a BMG Labtech
NOVOStar fluorescence plate reader equipped with a plate-to-plate
reagent delivery system. Human embryonic kidney-293 (HEK-293)
cells that stably overexpress human TRPA1, M8, TRPV3, or V4 have
been previously described27 and were grown to confluence in 2% (w/
v) gelatin-coated 96-well plates in DMEM [F12 media supplemented
with 5% fetal bovine serum, 1× penicillin/streptomycin (Invitrogen),
and geneticin (300 μg/mL)]. Assays using TRPV1 overexpressing and
normal BEAS-2B cells have also been described.1 Lobar bronchial
epithelial cells (Lifeline Cell Technology, donor lot #1344) were
grown to 80−90% confluence in BronchiaLife B/T medium (Lifeline
Cell Technology).
Agrobacterium-mediated transformation was performed according to
the previously reported procedure.30 We used the modified WSH agar
(1.0% crushed oatmeal, 0.1% KH2PO4, 0.1% NaNO3, 0.1% MgSO4,
0.5% glycerol, 0.1% MES, 2.0% agar) plates supplemented with 200
μg/mL hygromycin and 500 μg/mL kanamycin (final concentrations)
to select the correct transformants at 30 °C. The growing fungal
spores were transferred onto new oatmeal plates with 500 μg/mL
carbenicillin and 200 μg/mL hygromycin for the second-round
selection.
The spores of the selected mutant were cultured in YM broth (yeast
extract, 4 g/L; malt extract, 10 g/L; glucose, 4 g/L) for 7 days to
extract DNA according to the extract protocol for the wild type. Four
genome- and vector-specific primers (A−D) were designed to confirm
the correct mutant. A 4534-bp fragment was amplified from the
correct double crossover mutant with primers
A (5′-
ATGTATGACAATGGCCATCATAC-3′) and B (5′-CGGAGACGC-
TGTCGAACTTT-3′), and a 5,026-bp fragment was amplified with
primers C (5′-AGCTTGACTATGAAAATTCCGTCAC-3′) and D
(5′-CACCAGTCATGTAGACCCTTCC-3′). The genomic DNA of
wild-type H. irregularis FERM BP-2511 was used as the template to
amplify the same fragments by the same primers, but no target bands
were obtained.
Cells were prepared for assay by replacing the growth media with a
1:1 solution of LHC-9 and Fluo 4-Direct (Invitrogen) reagent
containing Fluo 4-AM, pluronic F-127, probenecid, and a proprietary
quencher dye. Cells were then incubated at 37 °C for 1 h in a cell
culture incubator or at room temperature (BEAS-2B cells). Cells were
subsequently washed by replacing the loading solution with LHC-9
containing 1 mM water-soluble probenecid (Invitrogen) and 750 μM
Trypan Red (ATT Bioquest). All cell types were incubated for an
additional 30 min at 37 °C to allow for Fluo 4-AM cleavage and
activation as well as equilibration to both the test compounds and Fluo
4.
The mutant and wild-type strains were cultured on the same
oatmeal plate without any antibiotics at 30 °C for 7 days. The cultures
were harvested and extracted three times with methanol. After drying,
the extracts were redissolved in 1 mL of methanol for analysis on an
Agilent 1200 HPLC with an Agilent XDB-C18 column (5 μm, 4.6 mm
× 250 mm), eluted with a gradient of acetonitrile−water (0−10 min,
G
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00808.
■
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S
Optical rotation values of the parent depsipeptides and
hydrolyzed units (Table S1), Ca2+ flux data for 1−7
(Table S2), extracted signature sequences of the A
domains of LACS (Table S3), HPLC analysis of the
metabolites of Hapsidospora irregularis FERM BP-2511
(Figure S1), NMR spectra of compounds 1−7 (Figures
S2−S33), and the hydrolysis of compounds 2, 4, and 6
(Figure S34) (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel: +1 435 797 8774. Fax: +1 435 797 2769. E-mail:
wangwei402@hotmail.com.
■
*E-mail: jixun.zhan@usu.edu.
ORCID
Wei Wang: 0000-0003-0876-2205
Lijiang Xuan: 0000-0002-0593-3921
Jixun Zhan: 0000-0003-0200-9183
(26) Malloch, D.; Cain, R. F. Can. J. Bot. 1970, 48, 1815−1825.
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Light, A. R.; Yost, G. S.; Veranth, J. M.; Reilly, C. A. Chem. Res. Toxicol.
2011, 24, 950−959.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a Utah State University SPARC
grant, a National Science Foundation Award CHE-1429195,
and NIH grants ES017431 and GM107557. The authors thank
Dr. S. T. Lee at the USDA Poisonous Plant Research Lab for
helping measure the optical rotation data, and Dr. R. Shin at the
University of Alabama at Birmingham for collecting the HMBC
spectrum of 2.
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DOI: 10.1021/acs.jnatprod.6b00808
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