Two new threonine-containing metabolites from fungus Curvularia inaequalis strain HS-FG-257

Article abstract of DOI:10.1080/14786419.2020.1784168

Two new threonine-containing metabolites, N-[4-hydroxy-3-prenyl-benzoyl]-L-threonine (1) and N-[2,2-dimethyl-2H-chromene-6-carbonyl]-L-threonine (2), were isolated from the fermentation broth of the soil fungus Curvularia inaequalis strain HS-FG-257. Their structures were elucidated through the interpretation of HR-ESIMS and extensive NMR spectroscopic data. Both compounds exhibited no cytotoxic activity against the test cell lines A549 and HCT-116.

Full text of DOI:10.1080/14786419.2020.1784168

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two new threonine-containing metabolites from
fungus Curvularia inaequalis strain HS-FG-257
Shao-Yong Zhang, Jian-Song Li, Hui Zhang, Huan Qi, Rui-Jun Wang, Chou-Fei
Wu, Li-Qin Zhang, An-Liang Chen, Ji-Dong Wang & Zhi-Kui Hao
To cite this article: Shao-Yong Zhang, Jian-Song Li, Hui Zhang, Huan Qi, Rui-Jun Wang, Chou-
Fei Wu, Li-Qin Zhang, An-Liang Chen, Ji-Dong Wang & Zhi-Kui Hao (2020): Two new threonine-
containing metabolites from fungus Curvulariaꢀinaequalis strain HS-FG-257, Natural Product
Research, DOI: 10.1080/14786419.2020.1784168
To link to this article: https://doi.org/10.1080/14786419.2020.1784168
View supplementary material
Published online: 25 Jun 2020.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1784168
SHORT COMMUNICATION
Two new threonine-containing metabolites from fungus
Curvularia inaequalis strain HS-FG-257
a
b
c
a
a
Shao-Yong Zhang , Jian-Song Li , Hui Zhang , Huan Qi , Rui-Jun Wang , Chou-
a
a
d
a
b
Fei Wu , Li-Qin Zhang , An-Liang Chen , Ji-Dong Wang and Zhi-Kui Hao
a
Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life
b
Science, Huzhou University, Huzhou, China; Institute of Applied Biotechnology, School of Medicine
and Pharmaceutical Engineering, Taizhou Vocational and Technical College, Taizhou, China;
c
Zhejiang Key Laboratory of Antifungal Drugs, Zhejiang Hisun Pharmaceutical Co., Ltd, Taizhou,
d
China; Provincial Joint Engineering Laboratory of Biopesticide Preparation, School of Forestry &
Biotechnology, Zhejiang Agricultural and Forestry University, China
ABSTRACT
ARTICLE HISTORY
Two new threonine-containing metabolites, N-[4-hydroxy-3-
prenyl-benzoyl]-L-threonine (1) and N-[2,2-dimethyl-2H-chromene-
Received 12 January 2020
Accepted 7 June 2020
6
-carbonyl]-L-threonine (2), were isolated from the fermentation
KEYWORDS
broth of the soil fungus Curvularia inaequalis strain HS-FG-257.
Their structures were elucidated through the interpretation of HR-
ESIMS and extensive NMR spectroscopic data. Both compounds
exhibited no cytotoxic activity against the test cell lines A549 and
HCT-116.
Curvularia inaequalis;
threonine-containing
metabolites; structure
elucidation
1. Introduction
Fungi are a rich source of secondary metabolites and some of these secondary metab-
olites are important pharmaceuticals, such as penicillin, cyclosporin and statins (Keller
et al. 2005). Although many fungi-derived secondary metabolites have been described,
the sequencing of fungal genomes has discovered that the potential of fungi for the
production of bioactive molecules is still largely untapped (Greco et al. 2019). During
the course of the screening program for new fungal metabolites, three new
CONTACT J.-D. Wang
02752@zjhu.edu.cn; Z.-K. Hao
haozhikui123@126.com
Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2020.1784168.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
S.-Y. ZHANG ET AL.
Figure 1. The structures of compounds 1 and 2.
compounds, curvularone A, 4-hydroxyradianthin, and 4-hydroxy-3-(3-methyoxy-3-meth-
ylbutyl)-benzoic acid, have been obtained from fermentation broth of the soil fungus
Curvularia inaequalis strain HS-FG-257 (Pang et al. 2013; Pang et al. 2012). In further
studies concerning the chemical diversity of the constituents of Curvularia inaequalis
strain HS-FG-257, two new threonine-containing metabolites, N-[4-hydroxy-3-prenyl-
benzoyl]-L-threonine (1) and N-[2,2-dimethyl-2H-chromene-6-carbonyl]-L-threonine (2),
were isolated from the n-BuOH soluble part of its fermentation broth. Here, we
describe the isolation and chemical characterization of the two new compounds.
2. Results and discussion
2.1. Structure determination
25
Compound 1 was obtained as colorless oil with a specific rotation of [a] þ25 (c 0.08,
D
EtOH) and UV (EtOH) kmax (log e): 257 (3.72) nm. Its HR-ESIMS showed a molecular ion
þ
peak at m/z 308.1518 [M þ H] (calcd for C H NO , 308.1492), indicating a molecular
16
22
5
ꢀ
1
formula of C H NO . IR absorption bands at 3360, 1728 and 1637 cm suggested
the presence of hydroxyl, carbonyl and olefinic functionalities. The H NMR data of 1
Table S1, Supplementary material) displayed signals for the protons of a 1,2,4-trisub-
1
6
21
5
1
(
stituted benzene ring [d 7.74 (1H, d, J ¼ 1.6 Hz, H-2), 7.66 (1H, dd, J ¼ 8.2, 1.6 Hz, H-6)
H
and 6.91 (1H, d, J ¼ 8.2 Hz, H-5)], an olefinic methine proton [d 5.34 (1H, t, J ¼ 7.2 Hz,
H
H-9)], two oxygen or nitrogen-bearing methine protons [d 4.66 (1H, dd, J ¼ 8.3,
H
0
0
2
.8 Hz, H-2 ) and 4.44 (1H, m, H-3 )], a methylene [d 3.36 (2H, d, J ¼ 7.2 Hz, H -8)], two
H
2
methyls connected with double bond [d 1.72 (6H, s, H -11, H -12)] and a doublet for
H
3
3
0
13
aliphatic methyl [d 1.25 (3H, d, J ¼ 6.3 Hz, H -4 )]. The C NMR spectrum (Table S1,
H
3
Supplementary material), complemented by DEPT and HSQC experiments, showed 16
2
signals including two carbonyl carbons at d 172.5, 167.6, four sp methines at d
30.0, 127.1, 123.2, 115.2, four sp quaternary carbons at d 158.7, 132.7, 128.7, 126.5,
C
two sp methines (one of which contained oxygen) at d 68.0, 58.6, one sp methylene
C
C
2
1
3
3
C
at d 28.9, and three methyls at d 25.7, 20.6, 17.7. The observed cross peak of H -8
C
C
2
1
1
and H-9 in the H- H COSY spectrum (Figure S1, Supplementary material) together
with the HMBC correlations (Figure S1, Supplementary material) from H -11, H -12 to
3
3
d 123.2 (C-9) and d 132.7 (C-10) revealed the presence of an isoprenyl group. The
C
C
HMBC correlations from H -8 to d 128.7 (C-3), 158.7 (C-4) and from H-2 to d 28.9 (C-
2
C
C
8) indicated the isoprenyl group was substituted at C-3. An ester or amide carbonyl
was situated at C-1, deduced from the long-range couplings from H-2 and H-6 to dC

NATURAL PRODUCT RESEARCH
3
0 0 0
67.6 (C-7) in the HMBC spectrum. The correlated signals of H-2 /H-3 /H -4 in the
3
1
1
1
0
0
H- H COSY spectrum and the HMBC correlation between H-2 and d 172.5 (C-1 ) in
C
1
13
0
0
combination with the H and C NMR data of C-1 -C-4 suggested the presence of a
threonine residue (Qi et al. 2019). The C NMR chemical shift at d 58.6 (C-2 ) and the
H- H COSY correlation of H-2 with the active hydrogen (d 7.21) indicated the active
13
0
C
1
1
0
H
0
hydrogen was a NH group. The long-range coupling from H-2 to C-7 established the
0
13
linkage of C-2 and C-7 via the NH group. Considered the downfield C NMR data of
C-4 (d 158.7) and the molecular formula C H NO of 1, a phenolic hydroxyl was situ-
C
16 21
5
ated at C-4. The presence of a phenolic hydroxyl was supported by the broad singlet
1
at d 8.83 (1H, br s) in the H NMR spectrum. Accordingly, the planar structure of 1
H
was elucidated as shown in Figure 1.
The absolute configuration of the threonine residue in 1 was defined by the
advanced Marfey’s method applied to the acidic hydrolysate of 1 (Peter 1984; Soumini
et al. 2016) and the configuration of the threonine moiety was determined as L.
25
Compound 2 was isolated as colorless oil with a positive rotation value ([a] þ47,
D
EtOH) and UV (EtOH) k
(log e): 237 (3.95) nm. Its molecular formula was established
max
þ
as C H NO by HR-ESIMS at m/z 306.1325 [M þ H] . The IR spectrum revealed car-
1
6
19
5
ꢀ
1
ꢀ1
bonyl absorption at 1747 cm and hydroxyl group absorption at 3179 cm . Analysis
1
1
1
of H NMR spectrum (Table S1, Supplementary material) and the H- H COSY correla-
tions (Figure S1, Supplementary material) of 2 indicated a 1,2,4-trisubstituted benzene
ring [d 7.74 (1H, d, J ¼ 8.3 Hz, H-6), 7.67 (1H, s, H-2), 6.80 (1H, d, J ¼ 8.3 Hz, H-5)], one
H
cis-double bond [d 6.48 (1H, d, J ¼ 9.8 Hz, H-8) and 5.80 (1H, d, J ¼ 9.8 Hz, H-9)], two
H
0
oxygen or nitrogen-bearing methine protons [d 4.68 (1H, m, H-2 ) and 4.45 (1H, m, H-
H
0
3
)], two aliphatic methyl singlets [d 1.43 (6H, s, H -11, H -12)] and one doublet for
H 3 3
0
13
aliphatic methyl [d 1.25 (3H, d, J ¼ 6.3 Hz, H -4 )]. The C and DEPT NMR data (Table
H
3
2
S1, Supplementary material) exhibited two carbonyl carbons, three sp quaternary car-
2
3
bons, five sp methines, one oxygen-bearing sp quaternary carbon, one oxygen-bear-
ing sp methine, one nitrogen-bearing sp methine and three methyls. The cross peaks
of H-2 /H-3 /H-4 in the H- H COSY spectrum and the HMBC correlation (Figure S1,
Supplementary material) from H-2 to d 172.4 (C-1 ) suggested the presence of a
3
3
0
0
0
1
1
0
0
C
threonine residue in 2. HMBC correlations of H -11/d 28.2 (C-12) and H -12/d 28.2
3
C
3
C
(C-11) showed the presence of gem-dimethyls. The gem-dimethyls and the cis-double
bond were connected through an oxygenated aliphatic quaternary carbon (d 77.1)
C
deduced from the long-range couplings from H -11 and H -12 to d 132.1 (C-9) and
3
3
C
1
13
d 77.6 (C-10). Except for the threonine moiety, the remaining H and C NMR data
C
showed close similarities to those of 2,2-dimethyl-2H-chromene-6-carboxylic acid (Ayer
and Trifonov 1994; Baldoqui et al. 1999), indicated the presence of the 2,2-dimethyl-
2
H-chromene-6-carbonyl substructure. A NH group was deduced from the chemical
0
1
1
0
shift at d 58.6 (C-2 ) and the H- H COSY cross signal between H-2 and the active
C
hydrogen (dH 7.29). The threonine residue and the 2,2-dimethyl-2H-chromene-6-car-
bonyl moiety were connected via the NH group evidenced by the HMBC correlation of
0
H-2 /d 167.1 (C-7).
C
The configuration of the threonine moiety of 2 was determined as L using the
advanced Marfey’s method after acidic hydrolysis.

4
S.-Y. ZHANG ET AL.
25
N-[4-hydroxy-3-prenyl-benzoyl]-L-threonine (1). Colorless oil; [a] þ25 (c 0.08, EtOH);
D
UV (EtOH) k
nm (log e): 257 (3.72) nm; IR (KBr) v : 3360, 2977, 1728, 1637, 1538,
max
max
ꢀ
1
1
13
1490, 1278, 1153, 1116 cm
;
H and
C NMR spectral data see Table S1,
þ
Supplementary material; HR-ESIMS: m/z 308.1518 [M þ H]
(calcd for
C H NO , 308.1492).
1
6
22
5
25
N-[2,2-dimethyl-2H-chromene-6-carbonyl]-L-threonine (2). Colorless oil; [a] þ47 (c
D
0
1
.17, EtOH); UV (EtOH) k
nm (log e): 237 (3.95) nm; IR (KBr) v : 3179, 2976, 1747,
max max
ꢀ
1
1
13
630, 1533, 1485, 1363, 1276, 1199, 1124 cm ; H and C NMR spectral data see
þ
Table S1, Supplementary material; HR-ESIMS: m/z 306.1325 [M þ H] (calcd for
C H NO , 306.1336).
1
6
20
5
2.2. Cytotoxic activities
The cytotoxic activities of compounds 1 and 2 against human lung adenocarcinoma
cell line A549 and human colon cancer cell line HCT-116 were evaluated. But, none of
them exhibited cytotoxic activity against the test cell lines.
3
. Experimental
.1. Fermentation,extraction and isolation
The fermentation of the soil fungus Curvularia inaequalis (Dematiaceae) strain HS-FG-
57 was the same as those described previously (Pang et al. 2013) and 30 L fermenta-
3
2
tion broth was obtained.
The fermentation broth (30 L) was filtered to obtain the mycelial cake and the
supernate. The mycelial cake was extracted with MeOH (3 L) and the supernate was
subjected to a Diaion HP-20 resin column eluting with 95% EtOH (5 L). The MeOH
extract and the 95% EtOH eluates were combined and concentrated under reduced
ꢁ
pressure to about 1 L at 50 C. The residual aqueous extract was successively extracted
with EtOAc followed by n-BuOH extraction. The n-BuOH phase was concentrated
under reduced pressure to yield a mixture (18 g). The mixture was applied on a silica
gel (Qingdao Haiyang Chemical Group, Qingdao, Shandong, China; 100–200 mesh) col-
umn and successively eluted with a stepwise gradient of CHCl /MeOH (49:1–1:1, v/v)
3
to offer three fractions (Fr.1 to Fr.3) based on the TLC profiles. The Fr.2 (1.3 g) was sep-
arated by preparative HPLC (Shimadzu LC-8A, Shimadzu-C18, 5 lm, 250 ꢂ 20 mm inner
ꢀ
1
diameter; 20 mL min ; 220/254 nm; Shimadzu, Kyoto, Japan) eluting with MeOH/H O
2
(
3:7, v/v) to give Fr.2-1 (t 16.9 min, 105 mg). Fr.2-1 was further purified by semi-pre-
R
parative HPLC (Agilent 1100, Zorbax SB-C18, 5 lm, 250 ꢂ 9.4 mm inner diameter;
ꢀ
1
1
3
2
.5 mL min ; 220 nm; Agilent, Palo Alto, CA, USA) eluting with a solvent system of
5% aqueous CH CN containing 0.1% acetic acid to yield compounds 1 (t 14.2 min,
3 mg) and 2 (t 17.0 min, 14 mg).
3
R
R
3
.2. Acid hydrolysis and marfey’s method
Compound 1 (1 mg) in 2 M HCl (7.4%, 1 mL) was heated at 100 C in a sealed vial for
ꢃ 3 h, after which the hydrolysate was concentrated to dryness under a stream of
ꢁ
2

NATURAL PRODUCT RESEARCH
5
dry N . The hydrolysate was then treated with 1 M NaHCO (1 mL) and -FDAA (10%
2
3
D
ꢁ
solution in acetone, 2 mL) at 40 C for 1 h, after which the reaction was neutralized
with 1 M HCl (3.7%), filtered (0.45 lm PTFE) prior to HPLC analysis. Likewise, the deriva-
tives of compound 2 (1 mg) and the standard L-threonine (5 mg) were prepared
according to themethod described above. An aliquot (5 lL) of the analytes were
ꢁ
injected into an Agilent Eclipse XDB-C18 column (5 lm, 250 ꢂ 9.4 mm, 30 C) with a
1
mL/min, 30 min linear gradient elution from 30% to 70% CH CN/H O with 0.1% tri-
3 2
fluoroacetic acid in H O. The threonine content of analytes was assessed by UV
2
(338 nm). Under these conditions, the derivative of standard L-threonine gave a peak
at t (min) ¼ 14.47(Figure S20, Supplementary material), while the derivatives of the
R
liberated threonine residues of 1 and 2 showed peaks at 14.46 and 14.37 min (Figure
S20, Supplementary material), respectively. The threonine moieties of the two new
compounds 1 and 2 were identified as L-threonine.
3.3. Cytotoxic bioassay
The cytotoxicity of compounds 1 and 2 against the human tumor cell lines A549, and
HCT-116 were assayed using the CCK-8 colorimetric method as described in our previ-
ous paper (Wang et al. 2009).
4. Conclusion
Two new threonine-containing metabolites, N-[4-hydroxy-3-prenyl-benzoyl]-L-threo-
nine(1) and N-[2,2-dimethyl-2H-chromene-6-carbonyl]-L-threonine (2), were obtained
from the n-BuOH soluble fraction of the fermentation broth of the soil fungus
Curvularia inaequalis strain HS-FG-257. It revealed the structural diversity of the sec-
ondary metabolites derived from this fungus. Furthermore, the structures of 1 and 2
also suggested that the fungus Curvularia inaequalis strain HS-FG-257 has the potential
to produce the hybrids of threonine and aromatic carboxylic acid compounds, which
is worth further study.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Key Research and Development Programs of Zhejiang Province
(Grant No:2020C02028), (Grant No:2019C02030), (Grant No:2019C02024) and the Zhejiang
Provincial Natural Science Foundation of China (HX2019069, HX2019078).
References
Ayer WA, Trifonov LS. 1994. Aromatic compounds from liquid cultures of Lactarius deliciosus. J
Nat Prod. 57(6):839–841.

6
S.-Y. ZHANG ET AL.
Baldoqui DC, Kato MJ, Cavalheiro AJ, Bolzani VS, Young MC, Furlan M. 1999. A chromene and
prenylated benzoic acid from Piper aduncum. Phytochemistry. 51(7):899–902.
Greco C, Keller NP, Rokas A. 2019. Unearthing fungal chemodiversity and prospects for drug dis-
covery. Curr Opin Microbiol. 51:22–29.
Keller NP, Turner G, Bennett JW. 2005. Fungal secondary metabolism - from biochemistry to
genomics . Nat Rev Microbiol. 3(12):937–947.
Pang YW, Wang JD, Xiang WS, Liu QF, Wang XJ. 2012. A new benzoic acid derivative from a soil
fungus Curvularia inaequalis strain HS-FG-257. Nat Prod Res Dev. 24:1331–1334.
Pang YW, Zhang LJ, Fang JS, Liu QF, Zhang H, Xiang WS, Wang JD, Wang XJ. 2013. Two new
antitumor constituents from a soil fungus Curvularia inaequalis (strain HS-FG-257). J Antibiot.
66(5):287–289.
Peter M. 1984. Determination of D-amino acids.II.use of a bifunctional reagent, 1,5-difluoro-2,4-
dinitrobenzene. Carlsberg Res Commun. 49:591–596.
Qi H, Ma Z, Xue ZL, Yu Z, Xu QY, Zhang H, Yu XP, Wang JD. 2019. A new O-cinnamoyl threonine
derivative from gene adpA overexpression strain Streptomyces sp. HS-NF-1222A. Nat Prod Res.
DOI:10.1080/14786419.2019.1573234.
Soumini V, Pritesh P, Leith JF, Ranjala R, Angela AS, Zeinab K, Robert JC. 2016. C3 and 2D C3
marfey’s methods for amino acid analysis in natural products. J Nat Prod. 79:421–427.
Wang JD, Zhang H, Ying LP, Wang CX, Jiang N, Zhou Y, Wang HB, Bai H. 2009. Five new epothi-
lone metabolites from Sorangium cellulosum strain So0157-2. J Antibiot. 62(9):483–487.