Ochraceopetalin, a mixed-biogenetic salt of polyketide and amino acid origins from a marine-derived aspergillus ochraceopetaliformis fungus

Article abstract of DOI:10.3390/md19080413

Ochraceopetalin (1), a mixed-biogenetic salt compound and its component 2 were isolated from the culture broths of a marine-derived fungus, Aspergillus ochraceopetaliformis. Based on combined spectroscopic and chemical analyses, the structure of 1 was determined to be a sulfonated diphenylether-aminol-amino acid ester guanidinium salt of an unprecedented structural class, while 2 was determined to be the corresponding sulfonated diphenylether. Ochraceopetaguanidine (3), the other guanidine-bearing aminol amino acid ester component, was also prepared and structurally elucidated. Compound 1 exhibited significant cytotoxicity against K562 and A549 cells.

Full text of DOI:10.3390/md19080413

marine drugs
Article
Ochraceopetalin, a Mixed-Biogenetic Salt of Polyketide and
Amino Acid Origins from a Marine-Derived Aspergillus
ochraceopetaliformis Fungus
Sung Chul Park 1,†, Jung-Ho Lee 1,†, Ji-Yeon Hwang , Oh-Seok Kwon , Lijuan Liao 1,2, Dong-Chan Oh 1,
Ki-Bong Oh * and Jongheon Shin *
1
1
3
,
1,
1
Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim,
Gwanak, Seoul 151-742, Korea; sungchulpark@snu.ac.kr (S.C.P.); anilove@snu.ac.kr (J.-H.L.);
yahyah7@snu.ac.kr (J.-Y.H.); ideally225@snu.ac.kr (O.-S.K.); lxycxf@snu.ac.kr (L.L.);
dongchanoh@snu.ac.kr (D.-C.O.)
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University,
San 56-1, Sillim, Gwanak, Seoul 151-921, Korea
2
3
*
Correspondence: ohkibong@snu.ac.kr (K.-B.O.); shinj@snu.ac.kr (J.S.); Tel.: +82-2-880-4646 (K.-B.O.);
+82-2-880-2484 (J.S.)
†
Equal contribution.
Abstract: Ochraceopetalin (
from the culture broths of a marine-derived fungus, Aspergillus ochraceopetaliformis. Based on com-
bined spectroscopic and chemical analyses, the structure of was determined to be a sulfonated
diphenylether-aminol-amino acid ester guanidinium salt of an unprecedented structural class, while
was determined to be the corresponding sulfonated diphenylether. Ochraceopetaguanidine ( ), the
1), a mixed-biogenetic salt compound and its component 2 were isolated
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
1
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Park, S.C.; Lee, J.-H.;
Hwang, J.-Y.; Kwon, O.-S.; Liao, L.;
Oh, D.-C.; Oh, K.-B.; Shin, J.
2
3
other guanidine-bearing aminol amino acid ester component, was also prepared and structurally
elucidated. Compound 1 exhibited significant cytotoxicity against K562 and A549 cells.
Ochraceopetalin, a Mixed-Biogenetic
Salt of Polyketide and Amino Acid
Origins from a Marine-Derived
Aspergillus ochraceopetaliformis Fungus.
Mar. Drugs 2021, 19, 413. https://
doi.org/10.3390/md19080413
Keywords: organic salt; diphenylether; Aspergillus ochraceopetaliformis; marine-derived fungi
1
. Introduction
Metabolites of polyketide pathways are widely recognized as an important class of
fungal natural products due to their significant structural and functional diversities, as well
as their wide taxonomical distribution [ ]. Attributed from the potent bioactivity, several
Academic Editor: Hee Jae Shin
Received: 5 July 2021
Accepted: 24 July 2021
Published: 26 July 2021
1,2
fungal polyketides have been used as lead compounds in the pharmaceutical industry,
which culminates to occupy 20% of the major commercial drugs by these compounds, such
as lovastatin and griseofulvin [
distinctive from other subclasses of fungal polyketides in terms of their formation by the
condensation of two polyketide derivatives [ ]. Amidst their wide natural distribution,
diphenyl ethers are frequently obtained from marine microorganisms, especially from
filamentous fungi of the genera Aspergillus and Penicillium [ ]. These compounds possess
diverse bioactivities, including antibacterial, cytotoxic, enzyme inhibitory and antiviral
activities [ ]. Accordingly, these compounds are increasingly applied in cosmetics, pharma-
ceutical intermediates and chemical products, which in turn have led to extensive studies
on their biosynthesis [ ]. Recently, a detailed biosynthetic pathway with the gene cluster
and key enzyme system was unveiled for diorcinol, which is a representative of fungal
diphenyl ethers, in a marine-derived A. nidulans strain [6,9].
In the process of searching for bioactive compounds from marine-derived fungi,
we recently reported the structures of two new sortase A-inhibitory depsihexapeptides
from an Aspergillus ochraceopetaliformis fungus, collected from underwater sediment off
the coast of Jeju-do, Korea [10]. However, LC–UV and LC–ESI–MS analyses, aided by an
3–5]. Tetraketide-derived diphenyl ethers are structurally
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.0/).
Mar. Drugs 2021, 19, 413. https://doi.org/10.3390/md19080413
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Mar. Drugs 2021, 19, 413
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in-house spectral library of fungal extracts, revealed the presence of a natural product
with distinct structural motifs in a liquid culture broth of this strain. More interestingly,
the same analyses of this strain from a rice-based semi-solid culture broth revealed the
presence of a structurally related but fragmented compound, prompting an extensive
chemical investigation.
Herein, we report the structures of ochraceopetalin (
aminol-amino acid guanidinium salt and its aromatic component (
process of isolation and structure determination, ochraceopetaguanidine (
bearing aminol amino acid, was structurally elucidated as the other component of
1
), a novel sulfonated diphenylether-
) (Figure 1). In the
), a guanidine-
2
3
1.
Organic salts coupled with organic counterions are occasionally found in marine organisms:
psammaplins, bromotyrosines and dihydroxystyrenes, from an association of Poecillastra
sp. and Jaspis sp. sponges [11
sesterterpene sulfates, from a Ircinia sp. sponge [15]; iodotyramine derivative, from the
Didemnum rubem ascidian [16 17]. To the best of our knowledge, would be the first of this
–13]; suvanines, from a Coscinoderma mathewsi sponge [14];
,
1
kind to be obtained from marine-derived fungi. Furthermore, its mixed-biogenetic salt
formation, that is derived from polyketide and amino acid pathways, would be remarkably
unprecedented. Compound
1 exhibited noticeably stronger cytotoxicity toward the human
cancer cell lines K562 and A549 than the other compounds.
Figure 1. The structures of 1–4.
2. Results and Discussion
The molecular formula of compound 1 obtained from the YMM liquid culture broth
was deduced to be C H N O S, with 11 degrees of unsaturation, by positive HR–ESI–MS
27
40
4
8
+
analysis ([M + H] m/z 581.2628, calcd 581.2640). This positive mode MS analysis also
gave a highly conspicuous cluster of C H N O (m/z 271.2123, calcd 271.2129), while a
13
27
4
2
similar analysis in negative mode gave a conspicuous cluster of C H O S (m/z 309.0433,
14
13
6
calcd 309.0427), whose sum corresponded well with the molecular formula of
phenomenon of significant discrepancy attributed from the opposite MS ionization modes
strongly suggested the presence of ionized partial structures, thus the salt nature of . In
1. This
1
−
1
addition, a strong absorption band was observed in the IR spectrum at 1456 cm , which
corresponds with a sulfate group, as suggested by the MS-indicated sulfur atom.
The 13C NMR data of compound
1
showed signals of a carbonyl carbon (
δ
C
170.5)
2
1
and thirteen sp carbons (
δ
160.2–102.7) (Table 1). Based on the combined H NMR and
C
HSQC data, the chemical shifts and splitting patterns ( H
protons were indicative of aromatic moieties. In addition, the odd numbers of sp carbons
suggested the presence of an electron-deficient functionality, such as an imine or guanidine.
δ
6.74–6.16, all s) of corresponding
2

Mar. Drugs 2021, 19, 413
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3
The NMR data of
1
also contained three sp methines (
δC/δ
73.3/2.74, 53.0/3.63 and
H
26.8/1.94), of which the former two carbons would bear nitrogen, according to their carbon
and proton chemical shifts. Among the remaining four aliphatic methylene and six methyl
signals in the NMR data, the carbon and proton chemical shifts indicated the presence of
an oxymethylene (δC/δ 64.4/4.19 and 4.12), a nitrogenous methylene (δC/δ 42.7/3.30
H
H
and 3.05) and two nitrogenous methyls (δ /δH 41.0/2.21 × 2).
C
Table 1. The 13C and H NMR data of compounds 1–3
1
a,b
(δH and δC in ppm).
No.
1
2
3
δC, Type
154.5, C
108.1, CH
156.6, C
113.9, CH
139.3, C
115.8, CH
157.6, C
102.7, CH
158.4, C
109.8, CH
140.1, C
111.2, CH
21.0, CH3
21.1, CH3
δ
(J in Hz)
δC, Type
δ
(J in Hz)
δC, Type
δ (J in Hz)
H
H
H
1
2
3
4
5
6
7
8
9
1
1
1
1
1
9
1
2
3
4
5
154.5, C
108.1, CH
156.7, C
113.9, CH
139.3, C
116.0, CH
157.6, C
102.8, CH
158.6, C
109.9, CH
140.1, C
111.2, CH
21.1, CH3
21.1, CH3
6.61, s
6.49, s
6.74, s
6.16, s
6.25, s
6.61, s
6.49, s
6.74, s
6.16, s
6.25, s
0
1
2
3
4
6.34, s
2.23, s
2.18, s
9.47, s
3.30, m, 3.05, m
1.78, m, 1.57, m
1.78, m, 1.57, m
3.63, d (7.0)
4.19, dd (11.5, 5.0)
6.34, s
2.23, s
2.18, s
9.49, br s
-OH
0
42.7, CH2
24.8, CH2
29.0, CH2
53.0, CH
64.4, CH2
42.7, CH2
24.8, CH2
29.0, CH2
52.9, CH
64.6, CH2
3.30, m, 3.05, m
1.78, m, 1.57, m
1.78, m, 1.57, m
3.63, m
4.19, dd (11.5, 4.5)
4.12, dd (11.5, 6.5)
0
0
0
0
4
.12, dd (11.5, 6.5)
0
0
0
0
6
7
8
9
1
1
1
1
170.5, C
170.4, C
73.3, CH
26.8, CH
19.0, CH3
19.5, CH3
41.0, CH3
41.0, CH3
160.2, C
2.74, d (10.5)
1.94, dhep (10.5, 6.5)
0.82, d (6.5)
0.92, d (6.5)
2.21, s
73.2, CH
26.8, CH
19.2, CH3
19.2, CH3
41.0, CH3
41.0, CH3
160.4, C
2.74, d (10.5)
1.94, dhep (10.5, 6.5)
0.82, d (6.5)
0.92, d (6.5)
2.21, s
0
0
0
1
2
3
0
0
2.21, s
2.21, s
a
Data were obtained in DMSO-d6. b Data were measured at 125/500 (1) and 150/600 (2 and 3) MHz for δC/δH, respectively.
1
1
The gross structure of
iments. First, all of the aromatic protons ( H
couplings, placing them at meta- or para-positions to each other. The HMBC correla-
tions of these and two benzylic methyl protons ( H 2.23, H -13 and 2.18, H -14) with
neighboring carbons readily constructed two 1,3-dioxygenated-5-methyl-benzene moieties
1
was determined by combined H- H COSY and HMBC exper-
δ
6.74–6.16, all s) lacked direct proton–proton
δ
δ
H
3
3
(
(
C-1–C-6 and C-13, C-7–C-12 and C-14), accounting for eight degrees of unsaturation
Figure 2). Despite the identical proton–carbon correlations, the noticeable differences in
1
13
both the H and C chemical shifts revealed different oxygenated functionalities between
aromatic rings. Aided by the negative mode ESI–MS cluster of C H O S and the IR
14
13
6
−
1
absorption band of the sulfate group at 1456 cm , the partial structure was interpreted as
-hydroxy-1-(sulfooxy)-3,7-diphenylether, which is discussed later.
9

Mar. Drugs 2021, 19, 413
4 of 11
Figure 2. Key correlations of COSY (bold) and HMBC (arrows) experiments for 1.
Meanwhile, the COSY data showed the proton spin couplings of an isobutyl group
0 0
(δC/δ 73.3/2.74, 26.8/1.94, 19.0/0.82 and 19.5/0.92; C-7 –C-10 ) which was confirmed
H
by the HMBC correlations among these. Then, additional proton–carbon correlations of
0
0
0
δ 170.5, C-6 ) and two
C
the C-7 methine and C-8 methine groups placed a carbonyl (
0 0
methyl groups (
δC/
δ
41.0/2.21
×
2, C-11 and C-12 ) at the adjacent positions. Because
H
the latter methyl groups were determined to be N-CH by their chemical shifts, nitrogen
must be connected to the methyl groups and C-7 . Thus, an N,N-dimethyl-valine residue
3
0
was identified. The COSY data also showed a linear array of four methylenes and a
methine (
δC/δ 42.7/3.30 and 3.05, 24.8/1.78 and 1.57, 29.0/1.78 and 1.57, 53.0/3.63 and
H
0
0
1
13
6
4.4/4.19 and 4.12; C-1 –C-5 ). Aided by their H and C NMR chemical shifts and the
0
0
0
crucial HMBC correlation at H -1 /C-4 , a pyrrolidine moiety was identified for C-1 –C-
2
0
0 0
. Similarly, the C-5 methylene was connected to the C-6 carbonyl by an ester linkage,
4
0
0
according to the HMBC correlation at H -5 /C-6 . Thus, a prolinol moiety was defined for
2
0
0
the C-1 –C-5 portion.
The 13C NMR data of
1
had a remaining nonprotonated carbon (
δ
160.2), which was
C
0
0
placed at the prolinol nitrogen (C-13 ) by the crucial HMBC correlation with H -1 . The
2
significant deshielding of this carbon must be attributed to the remaining two nitrogen
atoms from the MS data. Thus, in conjunction with the C H N O cluster observed in
1
3
27
4
2
0
the positive ESI–MS analysis, the functionality of C-13 was assigned to the guanidinium
group. Thus, the C-1 –C-13 portion was defined as 1-(aminoiminomethyl)-prolinol, N,N-
0
0
dimethylvaline ester and the overall structure of
1, designated as ochraceopetalin, was
determined to be a mixed-biogenetic salt.
The proposed structure of
1, including the salt nature, was confirmed by a series of
pH-dependent degradations (Figure 3). When
1
was placed in a mildly acidic solution, a
compound separated into an organic layer. A combination of HR–ESI–MS and 1D- and 2D-
0
NMR analyses readily identified this compound as diorcinol (
5-methylphenol] of diverse fungal origins [
aqueous solution to basic conditions brought another compound (3) into an organic layer.
The molecular formula of this compound was deduced to be C H N O by HR–FAB–MS
4
), a well-known 3,3 -oxybis
[
4,6,8]. Changing the pH of the remaining
13
26
4
2
+
analysis ([M + H] m/z 271.2135, calcd 271.2129) and the positive mode MS analysis of
identical to that of . Furthermore, the combined 1D- and 2D-NMR data of were identical
to those of the cation portion of , which was designated as ochraceopetaguanidine and
identified as 1-(aminoiminomethyl)-prolinol, N,N-dimethylvaline ester. Thus, the structure
of was unambiguously defined as a mixed-biogenetic salt derived from polyketide and
amino acid pathways. The sulfate [18 19] and guanidinium salts of was also supported
by the comparison of 13C NMR data between the simple salts bearing these functionalities
and corresponding neutral groups (Figure S27). Compound as a single salt compound
3 was
1
3
1
1
,
1
1

Mar. Drugs 2021, 19, 413
5 of 11
was also supported by extensive HPLC analyses, in which
1
was always eluted as single
peak under diverse chromatographic conditions (Figure S28).
Figure 3. Sequential degradation and configurational assignments of
1
. Reagents and conditions: (
) PGME, PyBOP, HOBT, N-methylmorpholine, rt, 3 h; (e) NaHCO , L-FDAA,
3
a) H O, TFA, rt, 3 h;
2
◦
(b
) NH OH, rt, 10 min; (
c) NaOH, 100 C, 3 h; (
d
4
◦
acetone, 80 C, 15 min; ∆δ values (∆δ = δ − δ ) obtained for (S)- and (R)-PGME amide derivatives 3aS and 3aR.
S
R
The production of
1 was further investigated under different culture conditions.
After culturing the same strain in a static semi-solid YMM-rice medium, chromatographic
separation of the culture broth yielded compound
2
−
. The molecular formula of
H] m/z 309.0433, calcd 309.0427),
2
was
−
defined to be C H O S by HR–ESI–MS analysis ([M
14
14
6
which was identical to the data of
D- and 2D-NMR data of were identical to the negative ion portion of
addition, was slowly converted to diorcinol ( ) during prolonged storage, emphasizing
their structural relationship. Thus, the structure of was determined to be 1-(sulfooxy)-
diorcinol, a sulfate-bearing diphenyl ether of polyketide origin.
Ochraceopetalin ( ) and ochraceopetaguanidine ( ) commonly bear two stereogenic
centers in the amino acid portion. For the configurational assignments of these, firstly, a
1
in the negative mode MS analysis. Furthermore, the
1
2
1
(Table 1). In
2
4
2
1
3
basic hydrolysis of
3 gave N,N-dimethylvaline and prolinol. The absolute configuration at
0
C-7 of the valine residue was assigned using the PGME method [20]. Treatments with (S)-
and (R)-PGME produced the corresponding PGME amides, respectively. The ∆δH values
between these unambiguously assigned a D-configuration (Figure 3). For the C-4 center
0
of prolinol, the absolute configuration was assigned by using the Marfey reaction [21].
After the L-FDAA-prolinol adduct was prepared by condensation with L-FDAA, its HPLC
retention time was compared with those of authentic L-FDAA-L-prolinol and L-FDAA-D-

Mar. Drugs 2021, 19, 413
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prolinol, which resulted in assigning the D-configuration. Thus, both amino acid-derived
units of
1 were found to possess D-configurations. In addition, the guanidinium salt
13
of
1
was supported by the C NMR data of 3c, the TFA salt of
3
, whose guanidinium
0
bearing C-13 showed very similar chemical shift with
respectively) (Figure S29). At the same time, the carbon chemical shifts of C-13 of
3
(
δ
C
161.47 and 160.39 for 3c and
3
,
0
1
and
3
also showed similarity with 0.15 ppm and 0.13 ppm differences in DMSO-d6 (
δ
160.24
C
and
1
δ
160.39 for
1
and
and 3, respectively), respectively (Figures S30–S31, Table S2).
In addition to the structure determination, the structure of
3
, respectively) and MeOH-d solvent (
δ
161.95 and δ 162.08 for
C
4
C
C
1
and 2 led to an interesting
argument. Diorcinol (4) is widely recognized as a natural product of diverse fungi, such
as Aspergillus versicolor GH-2 [22], A. tennesseensis [23] and Cordyceps sp. [24]. The detailed
biosynthesis via orsellinic acid, including the gene cluster and enzymatic reactions, was
recently determined for a marine-derived A. nidulans [
and consecutively (eight times of cultivation) raise the possibility that, in the case of this
strain (strain number FJ120) of A. ochraceopetaliformis, the natural product of the fungus
is not diorcinol ( ), but 1-(sulfooxy)-diorcinol ( ), or its salt ( ). Alternatively, it would
be possible that naturally produced was sulfonated to or by a biotransformation
during the cultivation. It would be also possible to transform by an abiotic process
6,9]. However, our results of finding
1
2
4
2
1
4
1
2
1
during the cultivation or isolation procedures. To clarify this, time-dependent cultivation
and ESI–MS analyses of the broths revealed two interesting phenomena. The first is that
1
and
2
were independently produced after 8 and 6 weeks of cultivation, respectively,
was undetected by LC–ESI–
under given culturing conditions, while the production of
4
MS analysis (Figures S24 and S25). The second interesting phenomenon is that distinct
products according to the culturing conditions could be attributed to the differences in pH
in the culture media,
derivatives would be fully answered only by the extensive biosynthetic study on this strain.
Compounds were tested using a number of bioassays. In cytotoxicity tests, all
four compounds were either significantly inhibitory ( , IC50 9.5 and 6.8 M), or less active
, IC50 11–25 M) against the human cancer cell lines K562 and A549 (Figure S26 and
Table S1). Although its cytotoxicity was not remarkable, was noticeably more potent
than its components . These microbial compounds failed to inhibit both Gram-positive
and Gram-negative bacteria and pathogenic fungi (MIC > 128 g/mL). They were also
1, pH 9.0, and 2, pH 5.0. This question of biosynthesis of diorcinol
1–4
1
µ
(
2–
4
µ
1
2–4
µ
inactive against the microbial enzymes sortase A (SrtA), a transpeptidase responsible for
the anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria and
isocitrate lyase (ICL), a key enzyme in the glyoxylate cycle.
In summary, ochraceopetalin (
were isolated from a marine-derived fungus Aspergillus ochraceopetaliformis. Based on the
results of combined spectroscopic and chemical analyses, the structure of was determined
1), a mixed-biogenetic salt compound and its component
2
1
to be a sulfonated diphenylether-aminol-amino acid guanidinium of an unprecedented
structural class. Compounds 2 and 3, obtained from the different culturing condition and
chromatographic processes, respectively, were defined to be the corresponding components
of
1, 1-(sulfooxy)-diorcinol and 1-(aminoiminomethyl)-prolinol, N,N-dimethylvaline ester,
respectively. Compound 1 exhibited significant cytotoxicity against K562 and A549 cells.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured on a JASCO P1020 polarimeter (Jasco, Tokyo, Japan)
using a 1 cm cell. UV spectra were acquired with a Hitachi U-3010 spectrophotometer
Hitachi High-Technologies, Tokyo, Japan). IR spectra were recorded on a JASCO 4200
(
1
13
FT-IR spectrometer (Jasco, Tokyo, Japan) using a ZnSe cell. H and C NMR spectra were
measured in DMSO-d , CDCl , or MeOH-d solutions on Bruker Avance -400, -500, -600,
6
3
4
or -800 instruments (Bruker, Billerica, MA, USA), with solvent peaks at
δ 2.50/δ 39.50,
H C
δ
H
7.26/δ 77.16 and δ 3.31/δ 49.00, respectively, as their internal standards. High-
C H C
resolution ESI mass spectrometric data were obtained at the National Instrumentation

Mar. Drugs 2021, 19, 413
7 of 11
Center for Environmental Management (Seoul, Korea) and were acquired using an AB
Sciex 5600 QTOF HR-MS instrument (Sciex, Washington, DC, USA). High-resolution FAB
mass spectrometric data were obtained at the Korea Basic Science Institute (Daegu, Korea)
and were acquired using a JEOL JMS 700 mass spectrometer (Jeol, Tokyo, Japan) with meta-
nitrobenzyl alcohol (NBA) as the matrix. Low-resolution ESI–MS data were recorded on an
Agilent Technologies 6130 quadrupole mass spectrometer with an Agilent Technologies
1200 series HPLC. Semi-preparative and analytical HPLC separations were performed on
a Spectrasystem p2000 equipped with a Spectrasystem RI-150 refractive index detector
and a UV-Vis-151 detector (Gilson, Middleton, WI, USA). All the solvents used were of
spectroscopic grade or distilled from glass prior to use.
3.2. Fungal Material
The fungal strain Aspergillus ochraceopetaliformis (strain number FJ120) was isolated
from marine sediments collected from Jeju-do, Korea, in July 2007 [10]. The isolate was
identified using standard molecular biology protocols by DNA amplification and sequenc-
ing of the ITS region. Genomic DNA extraction was performed using Intron’s i-genomic
BYF DNA Extraction Mini Kit, according to the manufacturer’s protocol. The nucleotide
sequence was deposited in the GenBank database under accession number KF384187. The
18S rDNA sequence of this strain exhibited 100% identity (588/588) with that of Aspergillus
ochraceopetaliformis strain RKI08-134 (GenBank accession number FJ797698).
3.3. Fermentation
The fungal strain was cultured on solid YPG media (5 g of yeast extract, 5 g of peptone,
1
0 g of glucose, 24.8 g of sea salt and 16 g of agar in 1 L of distilled water) for 7 days. An
agar plug (1 cm 1 cm) was inoculated in 250 mL flask containing 100 mL of YPG media.
×
After 7 days of growth, 10 mL of each culture was transferred to 2.8 L Fernbach flasks
containing YMM media (5 g of yeast extract, 5 g of malt extract, 10 g of mannitol and 24.8 g
of sea salt in 1000 mL of distilled water). In total, 30 L of YMM media was prepared and
◦
cultivated under static conditions for 8 weeks at 30 C.
The large-scale cultivation of YPG-based seed culture was also performed on semi-
solid YMM-rice media (1 g of yeast extract, 1 g of malt extract, 2 g of mannitol and 200 g of
◦
rice in 200 mL of artificial seawater) in 2.8 L Fernbach flasks at 30 C in the static condition
for 6 weeks.
3.4. Extraction and Isolation
The entire culture was filtered and extracted with EtOAc (20 L
evaporated in vacuo to afford a brown organic gum (4.3 g). The extract was separated by
C18 reversed-phase vacuum flash chromatography using sequential mixtures of H O and
×
3). The solvent was
2
MeOH (five fractions of H O-MeOH, gradient from 80:20 to 0:100), acetone and, finally,
2
1
EtOAc as the eluents. Based on the results of H NMR and LR–ESI–MS analyses, the fraction
eluted with H O-MeOH 40:60 (850 mg) was subjected to semi-preparative reversed-phase
2
HPLC (YMC-ODS-A column, 250
×
10 mm; H O-MeOH, 50:50, 1.7 mL/min), affording
2
compound (t = 29.1 min). Compound
1
1 was further purified by analytical HPLC (YMC-
R
ODS-A column, 250 × 4.6 mm; H O-MeCN, 75:25, 0.7 mL/min; t = 22.0 min; 11.1 mg).
2
R
The broth (4.2 kg) from semi-solid YMM-rice media was extracted with methanol
(
21 L × 3) and dichloromethane (21 L × 3). The combined extracts (111.1 g) were dried in
vacuo and partitioned between H O (84.6 g) and n-BuOH (26.4 g). The organic layer was
2
repartitioned between H O-MeOH (15:85, 14.2 g) and n-hexane (12.2 g). The H O-MeOH
2
2
layer was evaporated to obtain an organic extract (14.2 g). The extract was fractionated by
C18 reversed-phase vacuum flash chromatography using a sequential mixture of MeCN
and H2O as eluents (H O-MeCN, from 80:20 to 50:50), MeOH, acetone and, finally, EtOAc.
2
1
On the basis of the result of H NMR and LC-MS profile, the fraction (1240 mg) eluted
with H O-MeCN (70:30) was purified by semi-preparative reversed-phase HPLC (YMC-
2
ODS-A column, 250
×
10 mm; H O-MeOH, 55:45, 1.7 mL/min) to yield compound 2
2

Mar. Drugs 2021, 19, 413
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(tR = 34.5 min). Compound
2
was further purified by analytical HPLC (YMC-ODS-A col-
umn, 250 × 4.6 mm; H O-MeCN, 80:20, 0.7 mL/min; t = 15.6 min; 13.7 mg).
2
R
Ochraceopetalin (
1
): white amorphous solid; [
α
]
25D -5 (c 0.4, MeOH); UV (MeOH) λmax
−
1 1
13
(log
ε
) 209 (3.17), 275 (2.01) nm; IR (ZnSe) νmax 3347 (br), 2946, 1456, 1031 cm ; H and
C
+
NMR data, Table 1; HR–ESI–MS m/z 581.2628 [M + H] (calcd for C H N O S, 581.2640).
27
41
4
8
1
-(Sulfooxy)-diorcinol (2): yellow amorphous solid; UV (MeOH) λmax (log ε) 210 (3.16), 275
−
1
1
13
(2.19) nm; IR (ZnSe) νmax 3359 (br), 2833, 1455, 1033 cm ; H and C NMR data, Table 1;
−
HR–ESI–MS m/z 309.0437 [M − H] (calcd for C H O S, 309.0438).
14
13
6
3
.5. pH-Dependent Hydrolysis of Compound 1
Firstly, compound (9.7 mg) was dissolved in water (0.05% TFA, 5 mL). The solution
was stood at room temperature for 3 h. The aqueous solution was partitioned with n-BuOH
1
(
5 mL) through a separate funnel. Removal of n-BuOH in vacuo yielded pure compound
4
(
3.5 mg). To the aqueous fraction, 0.05% of NH OH (5 mL) was added. After removing
4
water, purification by analytical HPLC (YMC-ODS-A column, 250
×
4.6 mm; 0.7 mL/min;
H O-MeCN, 70:30) afforded compound 3 (t = 21.1 min; 4.2 mg).
2
R
Ochraceopetaguanidine (
3
): white amorphous solid; [α]25D -4 (c 0.4, MeOH); UV (MeOH)
−
1
1
13
λmax (log
ε
) 205 (2.39) nm; IR (ZnSe) νmax 3362 (br), 2360, 1636 cm ; H and C NMR
+
data, Table 1; HR-FAB-MS m/z 271.2135 [M + H] (calcd for C H N O , 271.2129).
13
27
4
2
): colorless gum; H and 13C NMR spectra, Figures S17 and S18; HR–ESI–MS
1
Diorcinol (
4
+
m/z 231.1012 [M + H] (calcd for C14H15O3, 231.1016).
3.6. Basic Hydrolysis of Compound 3
Compound
3
(3.5 mg) was dissolved in 1 N NaOH (1 mL) and the solution was stirred
◦
at 100 C for 3 h. The separation by analytical HPLC (YMC-ODS-A column, 250 × 4.6 mm;
0
.7 mL/min; H O-MeCN, 65:35) afforded compounds 3a (N,N-dimethylvaline, tR = 15.4 min;
2
2
.2 mg) and 3b (prolinol, t = 23.8 min; 2.8 mg) as pure compounds.
R
1
N,N-Dimethylvaline (3a): white amorphous solid; H NMR (DMSO-d , 400 MHz)
δ 3.16
H
6
0
0
0
0
(
1H, br s, 6 -OH), 2.66 (1H, d, J = 9.1 Hz, H-7 ), 2.29 (6H, s, H-11 , H-12 ), 1.90 (1H, dhep,
0
+
0
0
J = 9.1, 6.8 Hz, H-8 ), 0.92 (3H, d, J = 6.6 Hz, H-10 ), 0.83 (3H, d, J = 6.6 Hz, H-9 ); LR–ESI–MS
m/z 146.2 [M + H] .
1
Prolinol (3b): pale yellow oil; H NMR (CDCl , 400 MHz)
δ
3.81 (2H, OH, NH), 3.37 (1H,
3
H
0
0
0
dd, J = 11.0, 4.0 Hz, H-5 ), 3.20 (1H, dd, J = 11.0, 7.0 Hz, H-5 ), 3.02 (1H, m, H-4 ), 2.71 (1H,
m, H-1 ), 2.68 (1H, m, H-1 ), 1.63–1.52 (3H, m, H-2 ,3 ), 1.22 (1H, m, H-2 ); LR–ESI–MS m/z
0
0
0
0
0
+
1
02.1 [M + H] .
3
.7. Preparation of (S)- and (R)-PGME Amides of 3a
To a dry DMF solution (500 L) of compound 3a (0.5 mg, 6.9 mM) and (S)-PGME
1.5 mg, 33 mM), PyBOP (8.5 mg, 33 mM), HOBT (2.2 mg, 33 mM) and N-methylmorpholine
100 L) were added. After stirring the mixture for 3 h at room temp, a 5% HCl solution
µ
(
(
µ
and EtOAc were added to the reaction mixture. The EtOAc layer was subsequently
washed with saturated NaHCO solution and brine. The organic layer was dried over
3
anhydrous Na SO . After removing the solvent under vacuum, the residue was purified
2
4
by reversed-phase HPLC (YMC-ODS column, 250
S)-PGME amide 3aS (0.2 mg). Compound 3aR (0.2 mg), the (R)-PGME amide of 3a, was
prepared from (R)-PGME in a similar fashion. The molecular formulae of 3aS and 3aR
were confirmed as C H N O based on LR–ESI–MS data.
×
4.6 mm; H O-MeCN, 25:75) to give
2
(
16
24
2
3
1
(S)-PGME amide of 3a
(3aS): white amorphous solid; H NMR (CD OD, 800 MHz) δ
3 H
7
(
.393–7.338 (5H, m, PGME-Ar), 5.495 (1H, s, PGME-H-1), 3.697 (3H, s, PGME-OMe), 2.716
0 0 0 0
1H, d, J = 9.0 Hz, H-7 ), 2.240 (6H, s, H-11 , H-12 ), 2.050 (1H, dhep, J = 9.3, 6.7 Hz, H-8 ),
0
0
+
0.975 (3H, d, J = 6.7 Hz, H-10 ), 0.931 (3H, d, J = 6.7 Hz, H-9 ); LR–ESI–MS m/z 293.4 [M + H] .

Mar. Drugs 2021, 19, 413
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1
(R)-PGME amide of 3a
(3aR): white amorphous solid; H NMR (CD OD, 800 MHz) δ
3 H
7
.373–7.335 (5H, m, PGME-Ar), 5.481 (1H, s, PGME-H-1), 3.704 (3H, s, PGME-OMe), 2.739
0
0
0
0
(1H, d, J = 9.2 Hz, H-7 ), 2.345 (6H, s, H-11 , H-12 ), 2.037 (1H, dhep, J = 9.4, 6.7 Hz, H-8 ),
0
0
+
0.945 (3H, d, J = 6.7 Hz, H-10 ), 0.810 (3H, d, J = 6.7 Hz, H-9 ); LR–ESI–MS m/z 293.4 [M + H] .
3
.8. Marfey’s Analysis of 3b
◦
Compound 3b (0.5 mg) was dissolved in 12 N HCl (0.5 mL) and heated at 110 C for
1
6 h. The solution and traces of HCl were removed by repeated drying under vacuum with
distilled water. To the hydrolysate, 1 N NaHCO (100 L) and 1% L- or D-FDAA (50 L)
in acetone were added. The mixture was stirred at 80 C for 15 min. After quenching the
reaction by the addition of 2 N HCl (50 L), the residue was analyzed using HPLC with
an analytical column (YMC-ODS column, 250 4.6 mm; H O-MeCN, 60:40). As standard
µ
µ
3
◦
µ
×
2
compounds, L- and D-prolinol were also prepared using this method. The retention times
of the L-FDAA-derivatized L-prolinol was 19.686 min and that of L-FDAA-derivatized D-
prolinol was 20.160 min. The retention time of the L-FDAA-derivatized 3b was 20.222 min,
leading to assignment of the D-configuration.
3.9. Antibacterial, Antifungal, Enzyme-Inhibitory and Cytotoxic Assays
3.9.1. Antibacterial Activity Assay
To investigate the antibacterial activity of isolated compounds, a series of minimal
inhibitory concentration (MIC) tests was performed according to the Clinical and Lab-
oratory Standards Institute (CLSI) guide methods [25]. Three species of Gram-positive
bacteria (S. aureus strain Newman, E. faecalis ATCC19433 and E. faecium ATCC19434) and
three species of Gram-negative bacteria (K. pneumoniae ATCC10031, S. enterica ATCC14028
and E. coli ATCC25922) were selected as test strains. The MIC of each test compound
against six bacterial strains was determined in liquid culture using Mueller–Hinton broth
4
(
0
inoculum concentration, 5
.06–128
reference compounds.
×
10 cfu/mL) with the compound (concentration range of
◦
µ
g/mL) after 24 h incubation at 37 C. Ampicillin and tetracycline were used as
3.9.2. Antifungal Activity Assay
The antifungal activity of isolated compounds was estimated against C. albicans
ATCC10231, A. fumigatus HIC6094, T. rubrum NBRC9185 and T. mentagrophytes IFM40996
according to the guidelines in CLSI document M38 [26]. The growth of test fungi was
4
monitored in RPMI 1640 broth (inoculum concentration: 10 cells/mL) with the compound
concentration range of 0.06–128 µg/mL. Amphotericin B was used as a positive control.
3.9.3. ICL Inhibition Assay
The purification of recombinant ICL from the genomic DNA of C. albicans ATCC10231
and the evaluation of the effect of isolated compounds on ICL were carried out by the
method described previously [27]. The formation of glyoxylate phenylhydrazone from
isocitrate and phenylhydrazine substrates was monitored spectrophotometrically at 324 nm.
3-Nitropropionic acid was used as a positive control.
3.9.4. SrtA Inhibition Assay
The preparation of recombinant SrtA from S. aureus ATCC6538p and the evaluation
of the effect of isolated compounds on SrtA were performed according to a previously
◦
described procedure [27]. The enzyme reaction was carried out at 37 C for 1 h with
3
00
µ
L of buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl , pH 7.5), 55
µg of purified
2
SrtA, 0.75
µg of synthetic peptide dabcyl-QALPETGEE-edans and test samples at various
concentrations. The increase in the fluorescence intensity was recorded by a fluorescence
spectrophotometer (excitation, 350 nm; emission, 495 nm). Curcumin and berberine
chloride were used as positive controls.

Mar. Drugs 2021, 19, 413
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3.9.5. Cytotoxic Assay
The effects of compounds
1–
4
on cell viability were analyzed by MTT assay [27].
A549 (lung cancer) and K562 (leukemia) cells were purchased from the Korean Cell Line
Bank (KCLB), Seoul, Korea. Both cell lines were cultured in RPMI-1640 medium with
L-glutamine, 10% fetal bovine serum and 1% penicillin/streptomycin. All the cells were
◦
cultured at 37 C in a humidified atmosphere with 5% CO . For the assay, each cell
2
4
(
5 × 10 cells/mL) was seeded in 96-well plates (100
µ
L/well). After 24 h, they were
. After 24 h of compound treatment,
g/mL) was added to each well and incubated for 4 h. The formazan product was
dissolved with 250 L of DMSO. Absorbances of each well were detected at the wavelength
treated with various concentrations of compounds
MTT (250
1–4
µ
µ
of 595 nm using an ELISA microplate reader (BioTek, Winooski, VT, USA) to determine the
cell viability by quantifying the production of formazan. The IC50 values were calculated
using a nonlinear regression analysis (percent survival versus concentration). Doxorubicin
was used as a positive control.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
1
0.3390/md19080413/s1, Figures S1–S5, S7–S18, S30–S31: annotated 1D NMR and selected 2D NMR
1
spectra of
1
–4
, Figure S6: HR–ESI–MS of
1
, Figures S19–S22: H NMR spectra of 3a
,
3b
,
3aS and 3aR
,
Figure S23: The LC/MS chromatogram data of 3b-L-FDAA adduct, Figures S24–S25: The time-scale
LC–MS analysis of YMM liquid media extracts and YMM-rice-semi-solid media extracts, Figure S26:
MTT assay against K562 and A549 cancer cells, Table S1: Results of cytotoxicity tests, Figure S27:
The 13C NMR (25 MHz, D O) spectra of guanidine and guanidine hydrochloride from Scifinder,
2
1
3
13
Figure S28: The HPLC analysis of 1, Figure S29: C NMR spectrum of 3c, Table S2: The C NMR
data of 1–3 and 3c.
Author Contributions: S.C.P., J.-H.L. and L.L. carried out the cultivation, isolation and structural
elucidation; K.-B.O. performed the antimicrobial and enzymatical bioassays and fungal identification;
J.-Y.H. and O.-S.K. performed the cytotoxicity tests; J.S. and D.-C.O. reviewed and evaluated all the
data; J.S. and K.-B.O. supervised the research work and prepared the paper. All authors have read
and agreed to the published version of the manuscript.
Funding: This study was supported by the BK21 Plus program and the National Research Foun-
dation (NRF, grant No. 2021R1A4A2001251) funded by the Ministry of Science, ICT and Future
Planning, Korea.
Conflicts of Interest: The authors declare no conflict of interest.
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