Structure and absolute configuration of aspergilumamide A, a Novel lumazine peptide from the mangrove-derived fungus aspergillus sp.

Article abstract of DOI:10.1002/hlca.201400197

A novel lumazine peptide, aspergilumamide A (1), as well as a known analog penilumamide (2), were isolated from the mycelia of a marine-derived fungus Aspergillus sp. (33241), obtained from the mangrove Bruguiera sexangula var. rhynchopetala collected from the South China Sea. The structure of 1 was identified by comprehensive spectroscopic analysis, including 1D- and 2D-NMR, ESI-MS, and MS/MS experiments. The absolute configuration of 1 was determined by Marfey's method.

Full text of DOI:10.1002/hlca.201400197

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Helvetica Chimica Acta – Vol. 98 (2015)
Structure and Absolute Configuration of Aspergilumamide A, a Novel
Lumazine Peptide from the Mangrove-Derived Fungus Aspergillus sp.
by Cai-Juan Zhenga), Lu-Yong Wua), Xiao-Bao Lia), Xin-Ming Songa), Zhi-Gang Niua),
Xiao-Ping Songa), Guang-Ying Chen*a), and Chang-Yun Wang*b)
a) Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal
University, Haikou 571158, P. R. China
(phone: þ 86-898-65889422; fax: þ 86-898-65889422 ext. 571158; e-mail: chgying123@163.com)
b) Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy,
Ocean University of China, Qingdao 266003, P. R. China
(phone: þ 86-532-82031536; fax: þ 86-532-82031381 ext. 266003; e-mail: changyun@ouc.edu.cn)
A novel lumazine peptide, aspergilumamide A (1), as well as a known analog penilumamide (2),
were isolated from the mycelia of a marine-derived fungus Aspergillus sp. (33241), obtained from the
mangrove Bruguiera sexangula var. rhynchopetala collected from the South China Sea. The structure of 1
was identified by comprehensive spectroscopic analysis, including 1D- and 2D-NMR, ESI-MS, and MS/
MS experiments. The absolute configuration of 1 was determined by Marfeyꢀs method.
Introduction. – Endophytic fungi have proved to be productive sources of
structurally diverse and biologically active secondary metabolites [1]. Marine-derived
fungi in the genus Aspergillus are known to be major contributors to bioactive meta-
bolites, such as cytotoxic tetrapeptide asperterrestide A from A. terreus [2], antifouling
benzylazaphilones from Aspergillus sp. [3], antiviral butenolides from Aspergillus
terreus [4], and antibacterial cytochalasins from Aspergillus elegans [5]. In the course of
our investigation on new bioactive natural products from marine fungi isolated from
mangrove, the fungus Aspergillus sp. (33241), obtained from the mangrove Bruguiera
sexangula var. rhynchopetala, attracted our attention. The AcOEt extracts of the
mycelia of a Aspergillus sp. fungus exhibited strong antibacterial activity against a panel
of pathogenic bacteria. Chemical investigation of the bioactive extracts led to the
discovery of a noval lumazine (¼ pteridine-2,4(1H,3H)-dione) peptide aspergiluma-
mide A (1), together with the known analog penilumamide (2) [6] (Fig. 1). Aspergilu-
mamide A (1) is a peptide with an unusual starter unit, 1,3-dimethyllumazine-6-car-
boxylic acid, coupled to glutamine and anthranilic acid methyl ester. Only two reports
on the 1,3-dimethyllumazine-6-carboxamide moiety within natural products [6][7] are
available. The planar structure and absolute configuration of 1 was established by a
combination of spectroscopic methods and acid hydrolysis. Herein, we report the
isolation, structure elucidation, and biological activity of the isolated compounds.
Results and Discussion. – Aspergilumamide A (1) was obtained as white
amorphous powder. Its molecular formula, C22H23O7N7 (15 degrees of unsaturation),
was determined by HR-ESI-MS, combined with 1H- and 13C-NMR data (Table). In the
ꢁ 2015 Verlag Helvetica Chimica Acta AG, Zꢂrich

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Fig. 1. Structures of compounds 1 and 2 from Aspergillus sp.
1H-NMR spectrum (Table), the signals, together with the coupling constants, at d(H)
8.68 (dd, J ¼ 8.4, 1.2), 8.01 (dd, J ¼ 8.0, 1.2), 7.55 (ddd, J ¼ 8.4, 7.6, 1.2), and 7.11 (ddd,
J ¼ 8.0, 7.6, 1.2), indicated the presence of an ortho-disubstituted benzene moiety. Four
exchangeable H-atom signals, observed in the low-field at d(H) 11.54 (s), 8.94 (d, J ¼
8.2), 5.99 (s), and 5.68 (s), were assigned to NH. In the downfield, there was also an
olefinic H-atom signal at d(H) 9.42 (s). In the highfield region, signals of a CH group
(d(H) 4.86 – 4.91 (m)), three Me groups (d(H) 3.81 (s), 3.77 (s), and 3.56 (s)), two CH2
groups (d(H) 2.51 – 2.54 (m) and 2.34 – 2.36 (m), and d(H) 2.48 – 2.51 (m)) were also
observed. The 13C-NMR data of 1 exhibited signals of three Me and two CH2 groups,
one CH group, ten olefinic or aromatic C-atoms, and six either C¼O groups or other
related C-atoms at d(C) 174.3, 169.4, 167.7, 162.5, 159.2, and 150.3. Detailed analysis of
the 1H- and 13C-NMR data of 1 (Table), together with a literature search, revealed that
1 was closely related to the known lumazine peptide penilumamide (2) [6]. The
1
significant difference in the H-NMR spectra of 1 and 2 was the presence of two H-
atom signals for NH2 at d(H) 5.99 (s) and 5.68 (s) in 1 instead of a Me singlet at d(H)
2.57 (s) in 2. Furthermore, in the 13C-NMR spectrum, the appearance of a C¼O signal at
Table. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; CDCl3) of 1. d in ppm, J in Hz. Atom numbering
as indicated in Fig. 1.
Position d(H)
d(C)
Position d(H)
3’
d(C)
1
169.4 (C)
2
150.3 (C)
4’
2.34 – 2.36 (m), 2.51 – 2.54 (m) 28.1 (CH2)
3
4
4a
5
6
5’
6’
7’
2.48 – 2.51 (m)
32.0 (CH2)
174.3 (C)
159.2 (C)
125.2 (C)
5.99 (s), 5.68 (s)
11.54 (s)
1’’
2’’
3’’
4’’
5’’
6’’
7’’
8’’
9’’
140.6 (C)
147.9 (CH)
140.1 (C)
7
8
9.42 (s)
8.68 (dd, J ¼ 8.4, 1.2)
120.6 (CH)
134.6 (CH)
123.1 (CH)
130.9 (CH)
115.6 (C)
7.55 (ddd, J ¼ 8.4, 7.6, 1.2)
7.11 (ddd, J ¼ 8.0, 7.6, 1.2)
8.01 (dd, J ¼ 8.0, 1.2)
8a
9
10
11
1’
2’
149.5 (C)
29.8 (Me)
29.2 (Me)
162.5 (C)
3.77 (s)
3.56 (s)
167.7 (C)
52.3 (Me)
8.94 (d, J ¼ 8.2)
4.86 – 4.91 (m)
3.81 (s)
54.6 (CH)

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d(C) 174.3 in 1 instead of a Me signal at d(C) 37.9 in 2. These spectroscopic features,
combined with the HR-ESI-MS data, indicated two compounds with different amino
acid residues, glutamine in 1 and methionine sulfoxide in 2 (Fig. 1). The planar
1
structure of 1 was further confirmed by the H,1H-COSY and HMBC experiments
(Fig. 2). Additional evidence for the structure of 1 was provided by the ESI-MS and
MS/MS experiments, verifying the structure elements through interpretation of
fragmentation patterns (Fig. 3). The observed ion peaks and a proposed interpretation
were presented in Fig. 3.
Fig. 2. 1H,1H-COSY (—) and key HMB (H ! C) correlations of 1
Fig. 3. Proposed MS/MS fragmentation pattern of 1

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To determine the absolute configuration of the glutamine residue, 1 was hydrolyzed
with 6.0m HCl at 1058 for 12 h. The hydrolysates of 1, as well as the standard l-Glu and
d-Glu, were derivatized with Marfeyꢀs reagent [8], 1-fluoro-2,4-dinitrophenyl-5-l-
alanine amide (FDAA). Analysis of the FDAA derivatives of the hydrolysates from 1
by HPLC, compared with the FDAA derivatives from l-Glu and d-Glu, revealed the
presence of an l-Glu moiety in 1.
Aspergilumamide A (1) is a novel lumazine peptide that combines a lumazine
system with l-glutamine and anthranilic acid ester. Although lumazines are well-
known metabolites, this is the first example of the unique combination of a 1,3-
dimethyllumazine-6-carboxylic acid, a glutamine group, and an anthranilic acid methyl
ester moiety within a natural product. The proposed biosynthetic pathway for 1 started
with folic acid that is well-known to undergo microbial degradation [9] to lumazine-6-
carboxylic acid, and then with the stepwise addition of glutamine and finally anthranilic
acid.
Compounds 1 and 2 were evaluated for their antibacterial activities against six
terrestrial pathogenic bacteria, including Staphylococcus epidermidis, S. aureus,
Escherichia coli, Bacillus subtilis, B. cereus, and Micrococcus luteus, and two marine
pathogenic bacteria, Vibrio parahaemolyticus and Listonella anguillarum by the
microplate assay method [10]. Cytotoxicities against four human tumor cell lines (SPC-
A-1, BEL-7402, SGC-7901, and K562 were tested) by MTT (¼ 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide) method [11]. Both compounds showed no
potent antibacterial or cytoxic activities.
Conclusions. – In this study, a noval lumazine peptide aspergilumamide A (1) with
an unusual starter unit 1,3-dimethyl-lumazine-6-carboxylic acid, coupled to glutamine
and anthranilic acid methyl ester, and a known analog penilumamide (2) were isolated
from the mycelia of a mangrove-derived fungus Aspergillus sp. (33241). The structure
and the absolute configuration of 1 was determined by spectroscopic analysis and
Marfeyꢀs method.
This work was supported by the National Natural Science Foundation of China (Nos. 21462015 and
21162009), the International S&T cooperation Program of China (ISTCP) (2014DFA40850); the Open
Research Fund Program of Key Laboratory of Marine Drugs (Ocean University of China), Ministry of
Education (No. 2013062051), the Open Research Fund Program of Guangxi Key Laboratory of Marine
Biotechnology (GLMBT-201305), and Foundation for Young Teachers of Hainan Normal University
(Nos. QN 1433 and QN 1337).
Experimental Part
General. TLC: Precoated silica gel GF 254 plates (Yantai Zifu Chemical Group). Column
chromatography (CC): silica gel (SiO2, 200 – 300 mesh; Qingdao Marine Chemical Group), octadecyl-
silyl (ODS) gel (SiO2, 45 – 60 mm; Unicorn, Merck KGaA, DE-Darmstadt) and Sephadex LH-20
(Amersham Biosciences Inc., Piscataway, NJ, USA). Prep. HPLC: Agilent 1260 prep-HPLC system with
an Agilent C18 anal. HPLC column (4.6 ꢀ 250 mm, 5 mm) and semi-prep. column (9.4 ꢀ 250 mm, 7 mm).
Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: Beckman DU 640 spectrophoto-
meter; lmax (log e) in nm. IR Spectra: Nicolet Nexus 470 spectrophotometer; ˜n in cmꢁ1. NMR Spectra:
Bruker 400 MHz spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Q-TOF
Ultima Global GAA076 LC mass spectrometer; in m/z.

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Fungal Material. The fungal strain, Aspergillus sp. 33241, was isolated from the mangrove Bruguiera
sexangula var. rhynchopetala collected in the South China Sea in August, 2012. The strain was deposited
with the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of
Chemistry and Chemical Engineering, Hainan Normal University of P. R. China, Hainan, with the access
code 33241.
Identification of Fungus. This fungus was identified according to its morphological characteristics and
molecular-biological protocol. Its 577 base-pair ITS sequence had 99% sequence identity to that of
Aspergillus sp. F 62 (FJ755829). The sequence data have been submitted to GenBank, with an accession
No. KF 924560, and the fungal strain was identified as Aspergillus sp.
Extraction and Isolation. The fungus Aspergillus sp. (33241) was cultivated in 20 l potato glucose
liquid medium (15 g of glucose and 33 g of sea salt in 1 l of potato infusion, in 1-l Erlenmeyer flasks, each
containing 300 ml of culture broth) at r.t. without shaking for 4 weeks. The AcOEt extract of the mycelia
was purified repeatedly by CC (SiO2 and Sephadex LH-20K) and reversed-phase (RP) HPLC to obtain
aspergilumamide A (1) and the known analog penilumamide (2).The fungus cultures were filtered
through cheesecloth, and the mycelia was extracted with MeOH to obtain MeOH extracts, and then
MeOH extracts were extracted with AcOEt three times to obtain AcOEt extracts (20.5 g). The AcOEt
extracts were subjected to CC (SiO2; petroleum ether (PE)/AcOEt 100 :0 – 0 :100) to give six fractions,
Fr. 1 – Fr. 6. Fr. 4 was purified by CC (SiO2; PE/AcOEt 3 :1, then subjected to CC (Sephadex LH-20;
CHCl3/MeOH 1:1), and further purified by semi-prep. HPLC (62% MeOH/H2O) to afford compounds 1
(3.8 mg) and 2 (20.0 mg)
Aspergilumamide A (¼ Methyl 2-({N2-[(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydropteridin-6-yl)car-
bonyl]-l-glutaminyl}amino)benzoate; 1). White powder. [a]2D5 ¼ þ28 (c ¼ 0.18, MeOH). UV (MeOH):
222 (2.16), 249 (1.70), 318 (0.51), 334 (0.47). IR (KBr): 3436, 2924, 1647, 1246, 1089. 1H- and 13C-NMR:
Table. ESI-MS: 520.0 ([M þ Na]þ). HR-ESI-MS: 520.1554 ([M þ Na]þ, C22H23N7NaOþ7 ; calc. 520.1551).
Determination of Absolute Configuration of 1 by Marfeyꢀs Method. A soln. of 1 (1.5 mg) in 6m HCl
(1 ml) was heated to 1058 for 12 h. The soln. was then evaporated to dryness, and the residue was
redissolved in H2O (250 ml). A 50-ml portion of the hydrolysate was then placed in a 1-ml reaction vial and
treated with a 1% soln. of FDAA (200 ml) in acetone, followed by 1.0m NaHCO3 (40 ml). The mixture was
heated at 458 for 1 h, cooled to r.t., and then acidified with 2.0m HCl (20 ml). In a similar fashion, l-Glu
and d-Glu were derivatized with FDAA separately. The FDAA derivatives of the hydrolysates, and l-
Glu and d-Glu were subjected to HPLC analysis (Waters C18 column; 5 mm, 4.6 ꢀ 250 mm; 1.0 ml/min)
at 308, using the following gradient program: solvent A (H2O þ 0.1% TFA), solvent B (MeCN), linear
gradient: 20 – 60% aq. MeCN with 0.1% TFA (linear gradient for 60 min), 308; UV detection at 340 nm.
The retention times (tR) for the FDAA-l-Glu and FDAA-d-Glu are 15.37 and 16.74 min, resp.; tR of the
FDAA derivative of hydrolysates of 1 was 15.37 min.
Antibacterial Activity Assay. Antibacterial activity was evaluated by the conventional broth dilution
assay [10]. Six terrestrial pathogenic bacteria, including Staphylococcus epidermidis, S. aureus,
Escherichia coli, Bacillus subtilis, B. cereus, and Micrococcus luteus, and two marine pathogenic bacteria,
Vibrio parahaemolyticus and Listonella anguillarum, were used, and ciprofloxacin was used as a positive
control.
Cytotoxicity Assay. The cytotoxicity against human lung cancer (SPC-A-1), human liver cancer
(BEL-7402), human gastric cancer (SGC-7901), and leukemia (K562) cell lines were evaluated by using
MTT assay [11].
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Received June 11, 2014