3-Hydroxy-4-methyldecanoic Acid-Containing Cyclotetradepsipeptides from an Endolichenic Beauveriasp.

Article abstract of DOI:10.1021/acs.jnatprod.0c01305

An investigation of an endolichenicBeauveriasp. led to the discovery of seven new cyclotetradepsipeptides, beauveamides A-G (2-8), along with the known beauverolide Ka (1). All incorporate a 3-hydroxy-4-methyldecanoic acid (HMDA) moiety in their structures. Their configuration was determined through Marfey’s,J-based configuration analysis, and NMR computational methods, representing the first time that the stereostructures of HMDA-moiety-containing cyclotetradepsipeptides have been established. Compounds1and2exhibited protecting effects on HEI-OC1 cells at 10 μM, while14, and5could stimulate glucose uptake in cultured rat L6 myoblasts at 50 μM. Compound1showed dose-dependent activity in both L6 myoblasts and myotubes.

Full text of DOI:10.1021/acs.jnatprod.0c01305

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Article
3
‑Hydroxy-4-methyldecanoic Acid-Containing
Cyclotetradepsipeptides from an Endolichenic Beauveria sp.
§
§
Yuan-Fei Zhou, Kun Hu, Fang Wang, Jian-Wei Tang, Liang Zhang, Han-Dong Sun, Xiang-Hai Cai,
and Pema-Tenzin Puno*
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sı Supporting Information
ABSTRACT: An investigation of an endolichenic Beauveria sp.
led to the discovery of seven new cyclotetradepsipeptides,
beauveamides A−G (2−8), along with the known beauverolide
Ka (1). All incorporate a 3-hydroxy-4-methyldecanoic acid
(
HMDA) moiety in their structures. Their configuration was
determined through Marfey’s, J-based configuration analysis, and
NMR computational methods, representing the first time that the
stereostructures of HMDA-moiety-containing cyclotetradepsipep-
tides have been established. Compounds 1 and 2 exhibited
protecting effects on HEI-OC1 cells at 10 μM, while 1, 4, and 5
could stimulate glucose uptake in cultured rat L6 myoblasts at 50
μM. Compound 1 showed dose-dependent activity in both L6
myoblasts and myotubes.
yclic depsipeptides are cyclopeptides containing a
myoblasts and myotubes. Herein, the isolation, structural
characterization, and biological evaluation of compounds 1−8
are presented.
C
hydroxylated carboxylic acid moiety, forming at least
1
one lactone bond in the backbone. They have various
1a
biological activities, and some exhibit significant value as
2
3
pharmaceuticals (e.g., cyclosporin A and surotomycin ) and
RESULTS AND DISCUSSION
Compound 1 was isolated as a white powder. The molecular formula
■
4
agrochemicals (e.g., iturin A ). As a subclass of cyclic
depsipeptides, cyclotetradepsipeptides usually consist of three
amino acid residues and a fatty acid moiety. Previous reports
have demonstrated the genus Beauveria to be the main source
C H N O was deduced from the HRESIMS data, implying 15
3
7
50
4
5
1
13
degrees of unsaturation. Analysis of the H NMR and C NMR data
revealed the presence of 50 hydrogens and 37 carbon resonances
including four amide carbonyls, six sp3 methines, nine sp3
methylenes, four methyls, four exchangeable protons, and 14 aromatic
5
of cyclotetradepsipeptides. To date, six cyclotetradepsipep-
tides containing a 3-hydroxy-4-methyldecanoic acid (HMDA)
motif have been reported (Figure S1a), but with no
1
1
carbons. An isoleucine unit was established by H− H COSY
correlations of H-2 (δ 4.97)/H-3 (δ 1.84), H -5 (δ 0.94)/H-3,
and H -6 (δ 0.84)/H -4 (δ 1.19 and 1.54), together with the
5
c,6
H
H
3
H
stereochemistry.
Endolichenic fungi are the source of
7
3
H
2
H
novel bioactive natural products. In the present research, an
endolichenic Beauveria sp. was isolated from Gypsoplaca
macrophylla (Zahlbr.) Timdal, a lichen reported as the source
HMBC correlations from H -6 to C-3 (δ 37.9), from H -5 to C-2
3
C
3
(
δC 59.8), and from H-3 to C-1 (δC 169.7). The existence of a
phenylalanine-derived unit can be deduced from key HMBC
correlations from H -9 (δ 3.63 and 3.90) to C-8 (δ 57.8), as well
as to carbons (δ 140.9, C-10; δ 130.0, C-11/C-15) in a
8
of a rare pentacyclic sesterterpenoid, gypmacrophin A. An
2
H
C
investigation on the cultures of this fungus led to the discovery
of seven new cyclotetradepsipeptides, beauveamides A−G (2−
), along with the known beauverolide Ka (1), all containing
C
C
monosubstituted benzene. The presence of a tryptophan-derived
unit can be inferred from key HMBC correlations from H -18 (δ
2
H
8
3
.40 and 3.67) to C-16 (δ 172.4), C-17 (δ 55.6), C-19 (δ 110.8),
C C C
an HMDA moiety. Their configurational determination was
completed through the combination of Marfey’s, J-based
configuration analysis, and NMR computation methods,
representing the first time the configurations of HMDA
moiety-containing cyclotetradepsipeptides were established.
Bioactivity evaluation indicated that 1 and 2 exhibited
protective effects on HEI-OC1 cells at a concentration of 10
μM. Furthermore, 1, 4, and 5 could stimulate glucose uptake in
cultured rat L6 myoblasts at a concentration of 50 μM. Of
note, compound 1 showed dose-dependent activity in both L6
from H -18 to carbons (δ 110.8, C-19), and C-26 (δ 124.5) in a
2
C
C
monosubstituted indole, as well as from H-24 (δ 7.58) to C-20 (δC
H
Received: December 4, 2020
Published: March 23, 2021
©
2021 American Chemical Society and
American Society of Pharmacognosy
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Article
1
28.1) and C-22 (δ 119.1). HMDA motif can be detected through
configuration, an NOE correlation between 2-NH and H -4 is
expected to be observed (Figure 2). According to the two-
C
2
HMBC correlations from H -37 (δ 0.82) to C-35 (δ 31.9) and C-
3
H
C
3
6 (δ 22.8), from H -31 (δ 0.69) to C-29 (δ 76.2), C-30 (δ 36.3)
C 3 H C C
and C-32 (δ 30.8), and from H-28a (δ 2.71) to C-27 (δ 171.5), C-
C
H
C
2
9 and C-30. The sequence of amino acids and the HMDA moiety
was established by key HMBC correlations from H-17 (δ 5.20) to C-
H
2
7 (δ 171.5), from H-8 (δ 4.19) to C-16 (δ 172.4), from H-2 to
C H C
C-7 (δ 171.0).
C
Compound 1 was found to share the same planar structure with
beauverolide Ka, which was determined using mass spectroscopy in
the original report (Figure 1); no NMR or crystallographic data had
Figure 2. Part of the 1D NOE spectrum of 1 (DMSO-d ) obtained
6
through selective irradiation upon 2-NH. Modeling of the two
possible dimethine systems (both in “anti” arrangement) in the Ile
fragment of 1.
Figure 1. (Left) Key HMBC (red arrows) and 1H− H COSY
correlations (blue bold lines) of BVKA (1). (Right) Unresolved chiral
centers (highlighted by gray circles) of BVKA (1).
1
dimensional NOESY experiment of 1 (Figure S17), a correlation
could be observed between 2-NH and H -5 but not between 2-NH
3
and H -4. Moreover, an additional one-dimensional NOE NMR
2
6
c
been collected to support its configuration. Thus, beauverolide Ka
1) was used as a model compound to study the stereochemistry of
this series of cyclotetradepsipeptides.
experiment imposing selective irradiation on 2-NH further
strengthened the above observation (Figure 2) and supported a 3S
configuration; i.e., the isoleucine motif should be assigned as D-allo-
Ile.
(
The beauverolide Ka (1) was subjected to acid hydrolysis and
subsequent derivatization with Marfey’s reagent [Nα-(2,4-dinitro-5-
fluorophenyl)-L-alaninamide (FDAA)]. The retention time and m/z
values of the derivatized amino acid residues of 1 were compared with
those of authentic amino acids, which illustrated the presence of an L-
Phe residue, an L-Trp residue, and an isoleucine residue being either
D-Ile or D-allo-Ile (Figure S10). The configuration of the isoleucine
motif was not fully resolved, as distinguishing the authentic D-Ile or D-
allo-Ile by their retention times on HPLC was not possible, despite
the various chromatographic conditions that were tested (Figure S9).
As isoleucine contains the typical dimethine system, it was expected
that the J-based configuration analysis method may be useful for its
To resolve the stereochemistry of the HMDA fragment, 4 possible
candidates of 1 (1a−1d) generated by truncating C-34−C-37 and
altering the chirality of C-29 and C-30 (Figure S99) were subjected to
quantum chemical calculations of NMR chemical shifts and spin−spin
coupling constants (SSCC). As a result, the calculated NMR chemical
shifts of all isomers were quite consistent with their experimental
counterpart, but they were not distinguishable (Tables S7 and S8). To
obtain more accurate experimental SSCCs, H-28b was selectively
decoupled to turn the doublet of triplets of H-29 into a doublet of
doublets, and H-28a (doublet of doublets) turned into a doublet peak
3
3
3
(Figure S21). Thus, the J
, J
, and J
were
H‑29/H‑28a
H‑29/H‑30
H‑29/H‑28b
9
configuration determination. According to the J-based configuration
clearly measured as 10.0, 4.9, and 4.9 Hz, respectively. Fortunately,
the calculated SSCCs of 1a with 29S, 30S configurations were found
to match their experimental counterparts very well (Table 1), while
those of other diastereomers deviated from the experimental values.
Taken together, beauverolide Ka (1) was finally identified as cyclo-(D-
allo-Ile−L-Phe−L-Trp−29S, 30S-HMDA).
10
analysis protocol, the large coupling constant (9.6 Hz) observed
between H-2 and H-3 indicated that the two hydrogens tended to
adopt an “anti” arrangement. The two possible stereoisomers, 3S-1
and 3R-1, cannot be distinguished through further use of JC/H
values; however, relevant NOE correlation can be useful: If C-3
possesses an S configuration, an NOE correlation between 2-NH and
2
,3
The methods for configurational determination include the
following aspects: (1) The amino acid composition can be
H -5 is expected to be observed, whereas if C-3 possesses an R
3
1
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in their respective NMR spectra (Figures S53, S74, and S95). Thus,
Table 1. Experimental SSCCs of 1−8 and Calculated SSCCs
of 1a−1d (Unit = Hz)
the Ile fragments in 4, 6, and 8 were all assigned as D-allo-Ile. The
3
3
HMDA fragments in 2−8 gave similar SSCCs ( J
, JH‑29/H‑30,
H‑29/H‑28a
number
3JH‑29/H‑30
3JH‑29/H‑28a
3JH‑29/H‑28b
and J
3
) to those in 1 (Table 1) in their respective H NMR
1
H‑29/H‑28b
a
spectra. Thus, the HMDA fragments in 2−8 can be determined to
have the same configurations with 1; i.e., both C-29 and C-30 were
assigned as S. Thus far, the complete structures of 1−8 were all
determined and depicted as shown.
1
4.9
4.8
10.2
10.1
8.2
4.1
4.1
4.1
4.1
4.2
4.1
4.3
4.1
10.0
9.5
5.2
4.6
4.4
12.4
11.1
12.4
12.4
11.0
12.4
11.0
4.9
4.8
7.7
4.6
9.3
4.1
4.1
4.1
4.1
4.2
4.1
4.3
4.1
a
1
1
1
1
1
2
3
4
5
6
7
8
a
a
b
a
c
Beauveamide A (2) was obtained as a white amorphous powder.
The molecular formula C H N O was determined from the
a
d
b
3
7
50
4
5
1
HRESIMS data, indicating 15 degrees of unsaturation. The H and
C NMR spectra of 2 are similar to those of 1, except that two methyl
b
b
b
b
b
b
b
13
groups (δ 14.9, δ 0.94 and δ 11.2, δ 0.84) in 1 were replaced by
C
H
C
H
two methyl doublets (δC 22.3, δH 0.88 and δC 22.4, δH 0.98) in 2.
Detailed analysis of the 1D and 2D NMR data of 2 revealed that the
Ile residue in 1 was replaced by a Leu residue in 2, which was
supported by HMBC correlations from H -3 (δ 1.20) to C-5 and C-
2
H
6, and from H-2 (δH 5.30) to C-4 (δ 41.9). Moreover, the HMBC
C
12.4
correlations from H-17 (δ 5.20) to C-27 (δ 171.4), from H-8 (δ
a
b
H
C
H
Measured or calculated in DMSO-d . Measured in pyridine-d .
6
5
4.19) to C-16 (δ 172.5), and from H-2 to C-7 (δ 171.8) supported
C
C
the planar structure of 2 as cyclo-(Leu−Phe−Trp−HMDA) (Figure
3). Subsequently, the absolute configurations of the amino acid
residues of 1 were defined by the advanced Marfey’s method, in the
same manner as described for 1. HPLC-MS analysis of the resulting
hydrolysates revealed the presence of D-Leu, L-Phe, and L-Trp (Figure
S26). Hence, 2 was assigned as cyclo-(D-Leu−L-Phe−L-Trp−29S,
30S-HMDA).
determined by the Marfey’s method; (2) the configuration of the Ile
fragment and the HMDA chain can be determined mainly through
analysis of coupling constants, based on the J-based configuration
analysis method and quantum chemical calculation. These methods
were then applied to the stereochemical study of beauveamides A−G
(
2−8). The planar structures, as well as the amino acid residues of 2−
, were characterized through careful analyses of their NMR spectra
and the application of Marfey’s method. For compounds 4, 6, and 8
8
Beauveamide B (3) showed a sodium adduct ion at m/z 639.3530
in the HRESIMS analysis, corresponding to the molecular formula
3
1
13
containing Ile fragments, large J
SSCCs (9.9, 9.9, and 9.9 Hz,
C H N O and 15 degrees of unsaturation. The H and C NMR
H‑2/H‑3
36 48 4 5
respectively) as well as 2-NH/H -5 NOE correlations were observed
spectra of 3 closely resembled those of 1 and 2, revealing their
3
1
1
Figure 3. Key HMBC (red arrows) and H− H COSY correlations (blue bold lines) of beauveamides A−G (2−8).
1
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Figure 4. (a) Protecting effects of 1−8 on HEI-OC1 cells at 10 μM. (b) Effects of 1−8 on glucose uptake in cultured L6 myoblasts at 50 μM after a
2
4 h treatment. (c) Dose−effect relationship of 1 in cultured L6 myoblasts after a 24 h treatment. (d) Dose-dependent manner of 1 in cultured L6
myotubes after a 24 and a 48 h treatment. Note: ** p < 0.01 vs blank control (Con), * p < 0.05 vs blank control; insulin (Ins, 0.2 μM) or
metformin (Met, 500 μM) were used as positive controls in glucose uptake assay.
structural similarities. The obvious difference was that the C-4
formulated as cyclo-(D-allo-Ile−L-Lyr−L-Trp−29S, 30S-HMDA) and
cyclo-(D-Leu−L-Lyr−L-Trp−29S, 30S-HMDA), respectively.
Beauveamide E (6) was isolated as a colorless powder. It had the
methylene in 2 disappeared in 3. The correlations from H-3 (δ 2.05)
H
to C-1 (δ 169.6), from H -4 (δ 0.97) to C-2 (δ 61.0), and from
C
3
H
C
H -5 (δ 0.97) to C-2 in the HMBC spectrum of 3 suggested that the
molecular formula C35H N O as deduced by (+)-HRESIMS,
49 3 6
3
H
1
13
indicating 13 degrees of unsaturation. H and C NMR spectra
disclosed 49 hydrogens and 35 carbon resonances, including four
amide carbonyls, six sp3 methines, nine sp3 methylenes, four methyls,
five exchangeable protons, and 12 aromatic carbons including two
benzenes, indicating the existence of phenylalanine-derived and
tyrosine-derived units. The monosubstituted indole was further
identified by HMBC correlations from H -9 (δ 3.67 and δH 3.93)
Leu residue in 2 was replaced by a Val residue in 3 (Figure 3).
Detailed comparison of the 1D and 2D NMR spectroscopic data of 2
and 3 assigned the planar structure of 3 as cyclo-(Val−Phe−Trp−
HMDA). HPLC-MS analysis of the resulting hydrolysates of 3
revealed the presence of D-Val, L-Phe, and L-Trp (Figure S36). Based
on the methods used for 1, the configuration C-29 and C-30 were
both assigned as S. Hence, 3 was defined as cyclo-(D-Val−L-Phe−L-
Trp−29S, 30S-HMDA).
2
H
to C-11/15 (δ 130.1), from H-11/15 (δ 7.06) to C-13 (δ 126.2),
C
H
C
and from H-12/14 (δ 7.35) to C-13. The monosubstituted benzene
H
Beauveamides C (4) and D (5) were both obtained as white
amorphous powders, and they were found to have the same molecular
formula C H N O , according to their HRESIMS data. In
was deduced from H -18 (δ 3.09 and δH 3.31) to C-20/24 (δC
2
H
130.9) and from H-20/24 (δH 7.11) to C-19 (δC 127.9). Careful
3
7
50
4
6
interpretations of the HMBC correlations from H -6 (δ 0.84) to C-3
3
H
comparison with 1 and 2, compounds 4 and 5 both had a 16 Da
increase in molecular weight, while possessing the same degree of
unsaturation. Comparison made between the NMR data of 1 and 4
revealed that they were structurally similar, except that an olefinic
(
δ 37.9), from H -5 (δ 0.94) to C-2 (δ 59.8), and from H-3 (δ
C 3 H C H
1
.84) to C-1 (δ 169.7) enabled the formation of an isoleucine unit
C
(
Figure 3). The amino acid sequence and HMDA unit of 6 were
established as cyclo-(Ile−Phe−Tyr−HMDA) by the HMBC
methine carbon (δ 126.1) in 1 was substituted by a hydroxy group
C
correlations from H-17 (δ 5.00) to C-25 (δ 171.4), from H-8
H
C
and turned into a nonprotonated carbon in 4 (δC 157.8, C-13), as
(δH 4.23) to C-16 (δ 171.9), and from H-2 (δ 4.96) to C-7 (δC
C
H
supported by the HMBC correlations fromH-11/15 (δ 6.97) to C-
H
170.9). According to methods found in 1, the amino acids should be
D-allo-Ile, L-Phe, and L-Lyr (Figure S67), and the C-27 and C-28
should be determined as S. Therefore, 6 was established as cyclo-(D-
allo-Ile−L-Phe−L-Tyr−27S, 28S-HMDA).
1
3 and from H-12/14 (δH 7.07) to C-13 (Figure 3). HPLC-MS
analysis of the hydrolysates in combination with JBCA analysis
revealed the presence of D-allo-Ile, L-Lyr, and L-Trp in 4 (Figure S46).
Analysis of the NMR data of 5 suggested that 2 and 5 shared the same
relationship as that between 1 and 4; that is, the place of the L-Phe
residue in 2 was taken by L-Lyr in 5. Therefore, 4 and 5 were
Beauveamide F (7) was deduced to have the molecular formula of
C H N O according to HRESIMS data, indicative of 13 degrees of
3
5
49
3
6
unsaturation. Comparison of 1D and 2D NMR data of 6 and 7
1
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revealed their structural similarities. The main differences were that
those signals ascribed to the methyl group of Ile (δ 14.9, δ 0.94 and
(0.4% glucose, 1% malt extract, and 0.4% yeast extract). The
inoculated medium was incubated on a rotary shaker at 28 °C and
170 rpm for 5 days to prepare a seed culture. Fermentation was
carried out on solid rice medium in 50 Fernbach flasks (500 mL, 90
mL of distilled water was added to 80 g of rice and kept overnight
before autoclaving). Each flask was inoculated with 5.0 mL of spore
inoculum and incubated in a static incubator at 28 °C for 40 days.
Extraction and Purification. The medium was overlaid and
extracted with EtOAc, and the solvent was evaporated in vacuo to
afford a crude extract (167 g). The extraction was subjected to
column chromatography on silica gel with a CHCl −Me CO gradient
C
H
δ 11.2, δ 0.84) in 6 were replaced by two doublet methyls (δ 22.2,
C
H
C
δH 0.85 and δC 22.4, δH 0.92), revealing that the place of the Ile
residue in 6 was taken by a Leu residue in 7 (Figure 3). HPLC-MS
analysis of the hydrolysates of 7 revealed the presence of D-Leu, L-Phe,
and L-Tyr (Figure S78). Therefore, 7 was identified as cyclo-(D-Leu−
L-Phe− L-Tyr−27S, 28S-HMDA).
Beauveamide G (8) was obtained as a white powder, and its
HRESIMS spectrum revealed the molecular formula C H N O and
3
7
49
3
7
1
13
1
3 degrees of unsaturation. The H and C NMR spectra of 8 closely
3
2
resembled those of 6, revealing their structural similarities, the
system (1:0, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1) to yield seven fractions, A−G.
obvious difference being that a new C-4 methylene at δ 157.9 was
Fraction B (CHCl −Me CO 9:1, 60 g) was chromatographed on RP-
C
3
2
present in 8. The HMBC correlations from H-11/H-15 (δ 7.08) to
18 eluted with a MeOH−H O gradient (30:70−100:0) to yield A−D
H
2
C-13 (δ 157.9) and from H-12/H-14 (δH 7.10) to C-13 suggested
fractions to afford fractions B1−B6. Fraction B5 was purified by
C
that the Phe residue in 6 was replaced by a Tyr residue in 8 (Figure
preparative HPLC (15 mL/min, detector UV λ = 202 nm, MeCN−
max
3). Detailed comparison of the 1D and 2D NMR spectroscopic data
H O 80:20) to yield subfractions B5-1−8. Subfraction B5-3 was
2
of 6 and 8 assigned the planar structure of 8 as cyclo-(Ile−Tyr−Tyr−
HMDA). The D-allo-Ile, L-Tyr, L-Tyr (Figure S88), and 27S, 28S-
HMDA were decided according to Marfey’s method, JBCA analysis,
and coupling constants. Hence, 8 was decided as cyclo-(D-allo-Ile−L-
Tyr−L-Tyr−27S, 28S-HMDA).
purified by semipreparative HPLC (3 mL/min, detector UV λ
=
max
202 nm, MeOH−H O 85:15) to yield 1 (10.8 mg, retention time =
2
8.5 min), 2 (7.5 mg, retention time = 8.9 min), and 3 (3.6 mg,
retention time = 8.0 min). Subfraction B5-2 was purified by
semipreparative HPLC (3 mL/min, detector UV λmax = 202 nm,
Hearing loss can significantly impact a person’s quality of life. It is
estimated that about 466 million people suffer from hearing loss
worldwide today, and the number is expected to exceed 900 million
MeOH−H O 80:20) to yield 8 (2.8 mg, retention time = 7.2 min), 4
2
(3.8 mg, retention time = 7.8 min), and 5 (2.2 mg, retention time =
8.3 min). Subfraction B5-4 was purified by semipreparative HPLC (3
11
by 2050. Furthermore, diabetes is a metabolic disease that causes
high blood sugar, which in turn causes many serious body problems,
including cardiovascular disease, kidney disease, and the above-
mL/min, detector UV λmax = 202 nm, MeOH−H O 85:15) to yield 6
2
(4.0 mg, retention time = 10.9 min) and 7 (2.5 mg, retention time =
11.5 min).
12
mentioned hearing loss, etc. Stimulating the absorption of glucose
into surrounding tissues is an important mechanism for eliminating
Preparation and Analysis of Marfey’s Derivatives. Com-
pounds 1−8 (0.5 mg each) were hydrolyzed in HCl (6 N, 600 μL) at
110 °C for 24 h. After cooling, the solutions were evaporated to
13
glucose, which is a way to treat diabetes. Hence, to discover natural
compounds bearing hearing protective and glucose uptake stimulating
activities is very meaningful.
dryness and redissolved in H O (50 μL). To each peptide acid
2
hydrolysate solution (or to 50 μL of a 50 mM solution of the
Thus, all above compounds were evaluated for their protective
effects on an auditory hybridoma cell line, the HEI-OC1 cells, at a
concentration of 10 μM; 1, 2, and 6 were found to exhibit activity
respective amino acid standard), NaHCO (1 M; 20 μL) was added;
3
then, a 1% (w/v) solution (100 μL) of FDAA in acetone was also
added. The mixture was incubated for 1 h at 40 °C. The reaction was
stopped by addition of HCL (1 N, 20 μL). The derivatized peptide
hydrolysate solutions were diluted by addition of 300 μL of MeCN,
and the derivatized amino acid standards solutions were diluted by
addition of 500 μL of MeCN. An aliquot of these solutions (10 μL for
1−8 and 2 μL for the standards) was analyzed by HPLC-UV-MS
(Figure 4a, Table S1). Compounds 1, 4, and 5 could stimulate
glucose uptake in cultured rat L6 myoblasts at a concentration of 50
μM, and 8 showed moderate activity (Figure 4b, Table S2). Of note,
compound 1 showed dose-dependent activity in both L6 myotubes
and L6 myogenic cells (Figures 4c,d, Tables S3−S5).
(
Zorbax SB-C column, 2.1 mm × 30 mm, 5 μm, 30 °C, 1 mL/min;
8
elution system: MeCN/0.1% aqueous formic acid, 0−20 min 40%
MeCN, 20−25 min 100% MeCN). Retention times (min) of the
FDAA amino acid derivatives used as standards were as follows: L-Val
(7.7 min, m/z = 370), D-Val (11.5 min, m/z = 370), L-Leu (12.0 min,
m/z = 384), D-Leu (18.9 min, m/z = 384), L-Ile (11.1 min, m/z =
384), D-Ile (17.9 min, m/z = 384), L-allo-Ile (11.0 min, m/z = 384), D-
allo-Ile (17.9 min, m/z = 384), L-Phe (11.8 min, m/z = 418), D-Phe
(16.9 min, m/z = 418), L-Tyr (3.3 min, m/z = 434), D-Tyr (6.2 min,
m/z = 434), L-Trp (10.5 min, m/z = 457), D-Trp (13.1 min, m/z =
457). Due to the close retention times observed for D-Ile and D-allo-
Ile, JBCA was used in above study. Retention times of the observed
peaks in the HPLC trace of the FDAA-derivatized hydrolysis products
of 1 were as follows: D-allo-Ile (17.9 min), L-Phe (11.7 min), L-Trp
(10.5 min). For 2, they were as follows: D-Leu (18.9 min), L-Phe
(11.7 min), L-Trp (10.5 min). For 3, they were as follows: D-Val (11.5
min), L-Phe (11.7 min), L-Trp (10.5 min). For 4, they were as follows:
D-allo-Ile (17.9 min), L-Tyr (3.3 min), L-Trp (10.5 min). For 5, they
were as follows: D-Leu (17.9 min), L-Tyr (3.3 min), L-Trp (10.5 min).
For 6, they were as follows: D-allo-Ile (17.9 min), L-Phe (11.7 min), L-
Tyr (3.3 min). For 7, they were as follows: D-Leu (17.9 min), L-Phe
(11.7 min), L-Tyr (3.3 min). Lastly, for 8, they were as follows: D-allo-
Ile (17.9 min), L-Tyr (3.3 min), L-Tyr (3.3 min) (Figures S2−S9).
Computational Method. Conformational searching of 1a−1d
were undertaken with the CREST code (version 2.8) using the default
EXPERIMENTAL SECTION
■
General Experimental Procedure. Optical rotations were
measured with a JASCO P-1020 polarimeter. UV spectra were
obtained using a Shimadzu UV-2401 PC spectrophotometer. A
Tensor 27 spectrophotometer was used for scanning IR spectroscopy
with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker
DRX-600 spectrometers with TMS as internal standard. Chemical
shifts (δ) are expressed in parts per million (ppm) with reference to
the solvent signals. LC-MS and HRESIMS were performed on an API
QSTAR spectrometer. Preparative HPLC was performed on an
Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (21.2 mm ×
50 mm) column. Semipreparative HPLC was performed on an
Agilent 1200 liquid chromatograph with a Zorbax SB-C18 (9.4 mm ×
50 mm) column. Column chromatography was performed with silica
gel (100−200 mesh, Qingdao Marine Chemical, Inc., Qingdao,
People’s Republic of China). Fractions were monitored by TLC, and
compounds were visualized by heating silica gel plates sprayed with
2
2
1
0% H SO in EtOH.
2 4
Fungal Material, Identification, and Fermentation. The
fungal strain of Beauveria sp. was isolated from fresh stems of
Gypsoplaca macrophylla (Zahlbr.) Timdal collected from Baima
Mountain, Yunnan Province, People’s Republic of China, in
September 2016. Fungal identification was based on sequence
analysis (GenBank Accession No. MG345074.1) of internal tran-
scribed spacer (ITS) regions of the rDNA. After being kept on PDA
medium at 28 °C for 7 days, Beauveria sp. was inoculated into three
14
iMTD-GC procedure. The flexibility of the compound led to
relatively large conformer ensembles; thus, in order to balance the
number and diversity of conformers subjected to subsequent process,
1
5
2
50 mL Erlenmeyer flasks, each flask containing 100 mL of medium
conformers were clustered using the molclus program: When the
1
248
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J. Nat. Prod. 2021, 84, 1244−1253

Journal of Natural Products
pubs.acs.org/jnp
Article
Table 2. H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 1 and 2 (δ in ppm, J in Hz)
1
a
b
a
1
1
2
no.
δC type
δH
δC type
δH
δC type
δH
1
2
3
4
169.7, C
59.8, CH
37.9, CH
26.2, CH2
169.5, C
59.0, CH
37.1, CH
25.8, CH2
170.0, C
53.1, CH
25.1, CH2
41.9, CH
4.97, overlapped
1.84, dtt (9.6 6.6 3.2)
1.19, m
4.14, t (9.6)
1.59, m
1.07, m
5.30, m
1.20, m
1.69, m
1
.54, m
1.28, m
5
6
2
7
8
9
14.9, CH3
11.2, CH3
0.94, d (6.6)
0.84, t (7.3)
7.34, d (9.6)
15.2, CH3
11.4, CH3
0.83, d (6.8)
0.84, overlapped
6.93, d (9.4)
22.3, CH3
22.4, CH3
0.88, d (5.8)
0.98, d (5.9)
7.56, overlapped
-NH
171.0, C
57.8, CH
35.0, CH2
170.6, C
55.7, CH
34.7, CH2
171.8, C
57.8, CH
35.2, CH2
4.19, ddd (10.5 7.0 3.2)
3.63, m
3.88, m
2.87, dd (13.8 9.6)
3.16, dd (13.8 5.0)
4.19, ddd (10.6 7.0 3.3)
3.62, m
3.86, dd (13.7 3.3)
3
.90, dd (13.7 3.2)
1
1
1
1
8
1
1
1
0
140.9, C
139.6, C
140.7, C
1/15
2/14
3
-NH
6
130.0, CH
128.6, CH
126.1, CH
6.97, m
7.16, m
7.17, m
10.45, d (7.0)
129.6, CH
128.4, CH
126.3, CH
6.87, m
7.11, m
7.10, m
8.50, d (7.4)
130.0, CH
128.6, CH
126.2, CH
6.99, m
7.16, m
7.17, m
10.33, d (7.0)
172.4, C
55.6, CH
26.8, CH2
171.8, C
55.8, CH
26.1, CH2
172.5, C
55.8, CH
26.8, CH2
7
8
5.20, q (7.6)
3.40, dd (14.2 7.2)
4.20, q (7.6)
2.98, dd (14.5 8.0)
3.08, dd (14.5 7.5)
5.20, q (7.6)
3.44, dd (14.2 7.2)
3.67, m
3
.67, m
1
2
2
2
2
2
2
2
2
1
2
2
9
0
1
2
3
4
5
110.8, C
128.1, C
110.2, C
127.5, C
110.8, C
128.1, C
118.9, CH
119.1, CH
121.7, CH
111.9, CH
137.5, C
7.54, m
6.99, m
7.23, m
7.58, d (8.1)
118.6, CH
118.8, CH
121.4, CH
111.9, CH
136.6, C
7.46, d (7.9)
6.97, m
7.06, t (7.5)
7.35, d (8.1)
118.9, CH
119.1, CH
121.7, CH
111.9, CH
137.5, C
7.55, m
6.99, m
7.23, m
7.59, d (8.1)
5-NH
6
7-NH
7
8
11.92, s
7.27, d (2.3)
10.10, d (7.0)
10.85, s
7.09, m
8.31, d (7.4)
11.93, s
7.27, d (2.3)
10.05, d (7.2)
124.5, CH
124.2, CH
124.4, CH
171.5, C
36.9, CH2
170.5, C
36.0, CH2
171.4, C
37.1, CH2
2.71, dd (12.8 12.4)
2.25, dd (13.6 10.0)
2.45, dd (13.6 4.9)
4.88, dt (10.0 4.9)
1.98, m
0.79, d (6.6)
1.00, m
2.73, dd (13.2 11.1)
2.86, dd (13.2 4.1)
5.30, dt (11.1 4.1)
2.08, m
0.71, d (6.9)
0.98, m
2
.84, dd (12.8 4.1)
29
30
31
32
76.2, CH
36.3, CH
15.3, CH3
30.8, CH2
5.28, dt (12.4 4.1)
2.00, m
0.69, d (6.9)
1.01, m
76.4, CH
35.5, CH
15.8, CH3
31.0, CH2
76.2, CH
36.2, CH
15.4, CH3
31.1, CH2
1
.47, m
1.33, m
1.46, m
33
34
35
36
37
27.6, CH2
29.7, CH2
31.9, CH2
22.8, CH2
14.1, CH3
1.15, m
1.18, m
1.14, m
1.20, m
27.2, CH2
29.5, CH2
31.7, CH2
22.5, CH2
14.4, CH3
1.21, m
1.22, m
1.00, m
1.32, m
27.4, CH2
29.7, CH2
31.8, CH2
22.8, CH2
14.1, CH3
1.15, m
1.18, m
1.14, m
1.20, m
0.82, t (7.3)
0.84, overlapped
0.82, t (7.2)
a
b
Measured in pyridine-d . Measured in DMSO-d .
5
6
−
ΔG /RT
i
difference between the distance matrices of two conformers is within
.2, they are put in the same cluster. Those conformers within an
ni
∑j nj
e
pi =
=
−ΔG /RT
j
0
∑j e
energy window of 2 kcal/mol were subjected to DFT geometry
optimization at the M06-2X/def2-SVP level of theory with Grimme’s
DFT-D3 dispersion correction. Frequency analysis of all optimized
conformations was undertaken at the same level of theory to ensure
they were true local minima on the potential energy surface. Then,
energies of all optimized conformations were evaluated at the M06-
th
where P is the population of the i conformer, n is the number of
i
i
th
molecules in i conformer, ΔG is the relative Gibbs free energy (kcal/
mol), T is the room temperature (298.15 K) here, and R is the ideal
gas constant (0.0019858995). Those conformers with a population of
over 2% were subjected to subsequent NMR calculations.
NMR shielding constants were calculated with the GIAO method
at the mPW1PW91-SCRF/6-31+G(d,p) level with IEFPCM solvent
model in DMSO solvent. The shielding constants obtained were
converted into chemical shifts by referencing to TMS at 0 ppm (δcal
= σTMS − σcal), where the σTMS was the shielding constant of TMS
calculated at the same level. For each possible candidate, the
2
X-D3/def2-TZVP level of theory. Gibbs free energies of each
conformers were calculated by adding “Thermal correction to Gibbs
Free Energy” obtained by frequency analysis to electronic energies
obtained at the M06-2X-D3/def2-TZVP level of theory. Room-
temperature (298.15 K) equilibrium populations were calculated
according to Boltzmann distribution law
1
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J. Nat. Prod. 2021, 84, 1244−1253

Journal of Natural Products
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Article
1
13
Table 3. H (600 MHz) and C (150 MHz) NMR Data of Compounds 3−5 (δ in ppm, J in Hz)
a
a
a
3
4
5
no.
δC type
δH
δC type
δH
δC type
δH
1
2
3
4
169.6, C
61.0, CH
31.4, CH
18.9, CH3
170.4, C
60.4, CH
38.5, CH
26.8, CH2
170.2, C
53.1, CH
25.1, CH2
41.9, CH
4.89, m
2.05, m
5.01, t (9.9)
1.87, dtt (9.9 6.7 3.3)
1.20, m
5.30, t (9.6)
1.69, m
1.73, m
0.97, overlapped
0.97, overlapped
7.41, d (9.8)
1
.55, m
5
6
2
7
8
9
19.1, CH3
15.5 CH3
11.8, CH3
0.98, d (6.7)
0.88, t (7.3)
7.40, d (9.9)
22.2, CH3
22.4, CH3
0.86, d (6.0)
0.93, d (6.2)
7.66, d (9.6)
-NH
171.1, C
57.8, CH
35.0, CH2
171.8, C
58.8, CH
34.8, CH2
172.5, C
58.1, CH
34.4, CH2
4.19, m
3.64, m
4.24, ddd (10.5 7.0 3.2)
3.69, m
4.24, ddd (10.8 7.1 4.0)
3.61, m
3
.88, dd (13.7 3.3)
3.92, dd (13.7 3.2)
3.84, dd (13.7 3.2)
1
1
1
1
8
1
1
1
0
140.8, C
131.6, C
131.8, C
1/15
2/14
3
-NH
6
129.9, CH
128.6, CH
126.1, CH
6.97, m
7.16, m
7.17, m
10.42, d (7.0)
131.6, CH
116.7, CH
157.8, C
6.97, m
7.07, m
131.0, CH
116.1, CH
157.2, C
6.98, m
7.04, m
10.50, d (7.0)
10.29, d (7.1)
172.4, C
55.6, CH
26.8, CH2
173.0, C
56.2, CH
27.4, CH2
171.5, C
56.0, CH
26.8, CH2
7
8
5.20, q (7.6)
3.40, dd (14.2 7.2)
5.28, q (7.6)
3.47, dd (14.2 7.2)
3.72, m
5.22, q (7.7)
3.48, dd (14.3 7.5)
3.71, dd (14.3 8.2)
3
.67, m
1
2
2
2
2
2
2
2
2
1
2
2
9
0
1
2
3
4
5
110.8, C
128.6, C
111.4, C
128.8, C
110.8, C
128.1, C
118.9, CH
119.1, CH
121.6, CH
111.9, CH
137.5, C
7.55, m
6.99, m
7.23, m
7.59, d (8.1)
119.5, CH
119.7, CH
122.2, CH
112.5, CH
138.1, C
7.60, m
7.00, m
7.21, m
7.52, d (8.1)
118.9, CH
119.1, CH
121.6, CH
111.8, CH
137.5, C
7.58, m
6.99, m
7.16, m
7.51, d (8.1)
5-NH
6
7-NH
7
8
11.93, s
11.98, s
7.17, m
10.10, d (7.2)
11.94, s
7.32, d (2.3)
10.03, d (7.2)
124.5, CH
125.1, CH
124.4, CH
10.08, d (7.2)
171.1, C
37.1, CH2
172.1, C
37.5, CH2
171.0, C
37.0, CH2
2.71, dd (12.8 12.4)
2.75, dd (12.8 12.4)
2.88, dd (12.8 4.1)
5.33, dt (12.4 4.1)
2.06, m
0.73, d (6.9)
1.01, m
2.74, dd (13.1 11.0)
2.87, dd (13.1 4.2)
5.32, dt (11.0 4.2)
2.11, m
0.73, d (6.9)
1.00, m
2
.84, dd (12.8 4.1)
29
30
31
32
76.2, CH
36.4, CH
15.3, CH3
30.9, CH2
5.28, dt (12.4 4.1)
2.02, m
0.71, d (6.9)
0.98, m
76.8, CH
36.9, CH
15.9, CH3
31.4, CH2
76.2, CH
36.2, CH
15.4, CH3
31.1, CH2
1
.48, m
1.53, m
1.49, m
33
34
35
36
37
27.4, CH2
29.6, CH2
31.8, CH2
22.7, CH2
14.1, CH3
1.15, m
1.18, m
1.14, m
1.20, m
28.2, CH2
30.3, CH2
32.5, CH2
23.4, CH2
14.7, CH3
1.22, m
1.27, m
1.18, m
1.25, m
27.4, CH2
29.6, CH2
31.8, CH2
22.7, CH2
14.1, CH3
1.20, m
1.15, m
1.14, m
1.21, m
0.82, t (7.2)
0.82, t (7.3)
0.83, t (7.3)
a
Measured in pyridine-d5.
parameters a and b of the linear regression δcal = aδexp + b; the
correlation coefficient, R ; the mean absolute error (MAE) defined as
compounds. At the same time, the suspended cells were seeded at an
initial density of 1 × 10 cells/mL just before the addition of the
2
5
Σn |δcal − δexp|/n; and the corrected mean absolute error, CMAE,
compounds (1−8). Each cell line was exposed to a test compound at
concentration 10 μM in DMSO in triplicate for 48 h. After 48 h of
treatment, the culture solution was removed; fresh culture solution
was added, followed by addition of reagent CCK8. Then, cell viability
defined as Σn |δcorr − δexp|/n, where δcorr = (δcal − b)/a, were
16
calculated. The theoretical calculation of coupling constants was
performed at the B972/pcJ-1 level with IEFPCM solvent model in
17
19
DMSO solvent. The geometry optimization, single-point energy
calculation, NMR shielding constant calculation, and SSCC
was assessed by the OD value. The mean OD value of three wells in
the indicated groups was used to calculate cell viability as follows: cell
viability = (Atreatment − Ablank)/(Acontrol− Ablank) (where A is the
absorbance).
18
calculation were all completed in Gaussian 09 program.
Evaluation of the Protective Effects on HEI-OC1 Cells. The
human HEI-OC1 cell lines were grown in DMEM media (HyClone,
USA) supplemented with 10% fetal bovine serum (HyClone, USA) at
Glucose Uptake Assays. The glucose uptake effect of test
compounds was evaluated on L6 myoblasts and L6 myotubes as
20
3
7 °C in 5% CO . In short, 100 μL of adherent cells was seeded into
described in a previous publication. L6 myoblasts were cultured in
2
each well and incubated for 12 h before the addition of the test
10% FBS DMEM under 5% CO environment at 37 °C. After the
2
1
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J. Nat. Prod. 2021, 84, 1244−1253

Journal of Natural Products
pubs.acs.org/jnp
Article
1
13
Table 4. H (600 MHz) and C (150 MHz) NMR Data of Compounds 6−8 (δ in ppm, J in Hz)
a
a
a
6
7
8
no.
δC type
δH
δC type
δH
δC type
δH
1
2
3
4
169.7, C
59.8, CH
37.9, CH
26.2, CH2
170.1, C
53.1, CH
25.1, CH2
41.9, CH
170.4, C
60.4, CH
38.4, CH
26.8, CH2
4.96, t (9.9)
1.84, dtt (9.9 6.6 3.2)
1.14, m
5.29, m
1.67, m
1.70, m
4.99, t (9.9)
1.87, dtt (9.9 6.6 3.2)
1.19, m
1
.49, m
1.53, m
5
6
2
7
8
9
14.9, CH3
11.1, CH3
0.94, d (6.6)
0.84, m
7.31, d (9.9)
22.2, CH3
22.4, CH3
0.85, d (5.8)
0.92, d (5.8)
7.56, overlapped
15.5, CH3
11.8, CH3
0.97, d (6.6)
0.87, m
7.38, d (9.9)
-NH
170.9, C
57.9, CH
35.0, CH2
170.7, C
57.8, CH
35.2, CH2
171.7, C
58.8, CH
34.8, CH2
4.23, ddd (10.6 7.0 3.2)
3.67, m
4.24, td (7.1 3.2)
3.63, dd (13.7 10.5)
3.88, dd (13.7 3.2)
4.28, ddd (10.5 7.0 3.2)
3.68, m
3.94, dd (13.7 3.2)
3
.93, dd (13.7 3.2)
1
1
1
1
8
1
1
1
0
140.8, C
140.7, C
131.6, C
1/15
2/14
3
-NH
6
130.1, CH
128.8, CH
126.2, CH
7.06, m
7.35, m
7.22, m
10.30, d (7.0)
130.0, CH
128.0, CH
126.3, CH
7.10, m
7.33, m
7.21, m
10.17, d (7.1)
131.8, CH
116.9, CH
157.9, C
7.08, m
7.10, m
10.32, d (7.0)
171.9, C
56.4, CH
35.8, CH2
172.0, C
56.7, CH
35.8, CH2
172.5, C
57.2, CH
36.4, CH2
7
8
5.00, m
3.09, dd (13.6 7.0)
4.98, m
3.13, dd (13.6 7.2)
3.33, dd (13.6 8.8)
5.06, m
3.15, dd (13.6 7.3)
3.36, dd (13.6 8.7)
3
.31, dd (13.6 8.9)
1
2
2
2
1
2
2
9
127.9, C
128.0, C
128.6, C
0/24
1/23
2
7-NH
5
130.9, CH
116.3, CH
157.7, C
7.11, m
7.07, m
130.8, CH
116.3, CH
157.7, C
7.12, m
7.08, m
131.5, CH
116.8, CH
158.3, C
7.15, m
7.25, m
10.08, d (7.2)
10.01, d (7.3)
10.08, d (7.2)
171.4, C
37.0, CH2
170.7, C
37.1, CH2
172.0, C
37.5, CH2
6
2.74, t (12.8 12.4)
2.76, dd (13.3 11.0)
2.87, dd (13.3 4.3)
5.28, dt (11.0 4.3)
2.09, m
0.72, d (6.9)
1.00, m
2.78, dd (12.8 12.4)
2.88, t (12.8 4.1)
5.33, dt (12.4 4.1)
2.06, m
0.73, d (6.9)
1.04, m
2
.85, dd (12.8 4.1)
27
28
29
30
76.1, CH
36.3, CH
15.3, CH3
30.9, CH2
5.28, dt (12.4 4.1)
2.02, m
0.70, d (6.9)
1.01, m
76.2, CH
36.2, CH
15.4, CH3
31.1, CH2
76.8, CH
36.9, CH
15.9, CH3
31.5, CH2
1
.47, m
1.46, m
1.53, m
31
32
33
34
35
27.6, CH2
29.7, CH2
31.9, CH2
22.8, CH2
14.1, CH3
1.15, m
1.18, m
1.14, m
1.20, m
27.4, CH2
29.6, CH2
31.8, CH2
22.8, CH2
14.1, CH3
1.14, m
1.18, m
1.14, m
1.20, m
28.2, CH2
30.3, CH2
32.5, CH2
23.4, CH2
14.7, CH3
1.18, m
1.19, m
1.18, m
1.24, m
0.84, t (7.3)
0.82, t (7.2)
0.85, t (7.3)
a
Measured in pyridine-d5.
cells grew to 60% density, they were digested with trypsin and planted
in 96-well plates. For differentiation, L6 myoblasts were transferred to
Dulbecco’s modified Eagle’s medium with 2% FBS in tissue culture
plates for 5−6 days. Compounds (1−8) and different concentrations
of 1 were added in plates, incubated for 24 or 48 h, and reacted
accurately at 37 °C for 15 min, and the absorbance was read at 492
nm; the glucose value of each well was calculated. Insulin (0.2 μM)
and metformin (500 μM) were used as positive control.
210 (−0.14), 223 (0.68), 235 (−0.08) nm; IR (ν ): 3318, 2926,
−1 +
max
1725, 1655, and 1639 cm . HRESIMS at m/z 639.3530 ([M + Na] ,
1
13
calcd for 639.3517). H and C NMR data, see Table 3.
2
2
Beauveamide C (4). [α] : −44.5 (MeOH, c 0.06); UV (MeOH)
D
λmax (log ε): 220 (3.41), 280 (2.68) nm; ECD (MeOH) λmax (Δε):
210 (−0.29), 223 (0.29), 235 (−0.26) nm; IR (ν ): 3315, 2960,
max
−
1
2919, 2928, 1724, 1673, and 1640 cm . HRESIMS at m/z 647.3818
+
1
13
([M + H] , calcd for 647.3803). H and C NMR data, see Table 3.
2
2
22
Beauverolide Ka (1). [α] : −1.0 (MeOH, c 0.10); UV (MeOH)
Beauveamide D (5). [α] : −14.4 (MeOH, c 0.11); UV (MeOH)
D
D
λmax (log ε): 219 (3.49), 278 (2.91) nm; ECD (MeOH) λmax (Δε):
λmax (log ε): 222 (3.66), 280 (2.81) nm; ECD (MeOH) λmax (Δε):
2
2
08 (−0.39), 223 (0.62), 235 (−0.06) nm; IR (ν ): 3419, 2959,
200 (1.34), 238 (0.24) nm; IR (ν ): 3424, 2960, 2927, 1663, 1630,
max
max
−
1
−1
+
919, 2851, 1725, 1261, and 1030 cm . HRESIMS at m/z 653.3673
and 1543 cm . HRESIMS at m/z 669.3644 ([M + Na] , calcd for
+
1
13
1
13
(
[M + Na] , calcd for 653.3670). H and C NMR data, see Table 2.
669.3623). H and C NMR data, see Table 3.
2
2
D
22
Beauveamide A (2). [α] : −15.8 (MeOH, c 0.04); UV (MeOH)
Beauveamide E (6). [α] D: +2.6 (MeOH, c 0.15); UV (MeOH)
λmax (log ε): 220 (3.06), 282 (2.38) nm; ECD (MeOH) λmax (Δε):
λmax (log ε): 225 (2.19), 280 (1.63) nm; ECD (MeOH) λmax (Δε):
2
2
10 (−0.13), 223 (0.15), 235 (−0.11) nm; IR (ν ): 3318, 2960,
200 (−0.17), 218 (−0.12) nm; IR (ν ): 3399, 3317, 2958, 2918,
max
max
−
1
−1
923, 2853, 1724, 1673, and 1261 cm . HRESIMS at m/z 653.3515
2850, 1735, 1439, and 1041 cm . HRESIMS at m/z 630.3515 ([M +
Na] , calcd for 630.3514). H and C NMR data, see Table 4.
Beauveamide F (7). [α] D: +1.6 (MeOH, c 0.12); UV (MeOH)
+
1
13
+
1
13
(
[M + Na] , calcd for 653.3514). H and C NMR data, see Table 2.
2
2
D
22
Beauveamide B (3). [α] : −14.2 (MeOH, c 0.09); UV (MeOH)
λmax (log ε): 220 (3.43), 280 (2.82) nm; ECD (MeOH) λmax (Δε):
λmax (log ε): 225 (1.96), 280 (1.39) nm; ECD (MeOH) λmax (Δε):
1
251
https://doi.org/10.1021/acs.jnatprod.0c01305
J. Nat. Prod. 2021, 84, 1244−1253

Journal of Natural Products
pubs.acs.org/jnp
Article
2
1
00 (−0.12), 218 (−0.09) nm; IR (ν ): 3428, 2958, 2919, 2851,
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, People’s Republic of China; orcid.org/0000-
max
−
1
+
728, 1668, and 1260 cm . HRESIMS at m/z 630.3531 ([M+ Na] ,
1
13
calcd for 630.3514). H and C NMR data, see Table 4.
2
2
D
Beauveamide G (8). [α] : −9.0 (MeOH, c 0.10); UV (MeOH)
0002-6723-6988
λmax (log ε): 225 (2.96), 280 (2.47) nm; ECD (MeOH) λmax (Δε):
2
1
03 (−0.81), 208 (−0.52) nm; IR (ν ): 3410, 3304, 2924, 1644,
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01305
max
−
1
+
530, 1517, and 1260 cm . HRESIMS at m/z 624.3656 ([M + H] ,
1
13
calcd for 624.3643). H and C NMR data, see Table 4.
Author Contributions
Y.-F.Z. and K.H. contributed equally to this work.
§
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01305.
Notes
The authors declare no competing financial interest.
1
13
1
1
ACKNOWLEDGMENTS
LC-MS, H and C NMR, HSQC, HMBC, H− H
COSY, ROESY, HRESIMS, IR, ORD, UV, and ECD
spectra of compounds 1−8 and computational data of
■
We thank Li-Song Wang from the Key Laboratory for Plant
Biodiversity and Biogeography of East Asia, Kunming Institute
of Botany, for supplying lichen materials. This project was
supported financially by the National Natural Science
Foundation of China (Nos. 81874298, 81673329, and
1
a−1d (PDF)
AUTHOR INFORMATION
Corresponding Author
Pema-Tenzin Puno − State Key Laboratory of Phytochemistry
and Plant Resources in West China, and Yunnan Key
Laboratory of Natural Medicinal Chemistry, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming
■
8
1903520), the CAS “Light of West China” Program and
CAS Interdisciplinary Innovation Team (Pema-Tenzin Puno),
and the Yunnan Science Fund for Distinguished Young
Scholars (2019FJ002).
REFERENCES
■
6
0
50201, Yunnan, People’s Republic of China; orcid.org/
000-0001-5212-3000; Email: punopematenzin@
mail.kib.ac.cn
(
1) (a) Wang, X.; Gong, X.; Li, P.; Lai, D.; Zhou, L. Molecules 2018,
3, 169. (b) Feng, L.; Wang, J.; Liu, S.; Zhang, X. J.; Bi, Q. R.; Hu, Y.
Y.; Wang, Z.; Tan, N. H. J. Nat. Prod. 2019, 82, 1434−1441.
c) Koecher, S.; Resch, S.; Kessenbrock, T.; Schrapp, L.; Ehrmann,
M.; Kaiser, M. Nat. Prod. Rep. 2020, 37, 163−174.
2) (a) Emmel, E. A.; Verweij, C. L.; Durand, D. B.; Higgins, K. M.;
2
(
Authors
Yuan-Fei Zhou − State Key Laboratory of Phytochemistry and
Plant Resources in West China, and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, People’s Republic of China; University of Chinese
Academy of Sciences, Beijing 100049, People’s Republic of
China
Kun Hu − State Key Laboratory of Phytochemistry and Plant
Resources in West China, and Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, Yunnan,
People’s Republic of China
(
Lacy, E.; Crabtree, G. R. Science 1989, 246, 1617−1620. (b) Pirsch, J.
D.; Miller, J.; Deierhoi, M. H.; Vincenti, F.; Filo, R. S. Transplantation
1997, 63, 977−983.
(3) (a) Lee, C. H.; Patino, H.; Stevens, C.; Rege, S.; Chesnel, L.;
Louie, T.; Mullane, K. M. J. Antimicrob. Chemother. 2016, 71, 2964−
2
971. (b) Daley, P.; Louie, T.; Lutz, J. E.; Khanna, S.; Stoutenburgh,
U.; Jin, M.; Adedoyin, A.; Chesnel, L.; Guris, D.; Larson, K. B.;
Murata, Y. J. Antimicrob. Chemother. 2017, 72, 3462−3470.
4) (a) Yu, G. Y.; Sinclair, J. B.; Hartman, G. L.; Bertagnolli, B. L.
(
Soil Biol. Biochem. 2002, 34, 955−963. (b) Arrebola, E.; Jacobs, R.;
Korsten, L. J. Appl. Microbiol. 2010, 108, 386−395.
(
5) (a) Mochizuki, K.; Ohmori, K.; Tamura, H.; Shizuri, Y.;
Fang Wang − State Key Laboratory of Phytochemistry and
Plant Resources in West China, and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, People’s Republic of China
Jian-Wei Tang − State Key Laboratory of Phytochemistry and
Plant Resources in West China, and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, People’s Republic of China
Liang Zhang − State Key Laboratory of Phytochemistry and
Plant Resources in West China, and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, People’s Republic of China
Han-Dong Sun − State Key Laboratory of Phytochemistry and
Plant Resources in West China, and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, People’s Republic of China
Nishiyama, S.; Miyoshi, E.; Yamamura, S. Bull. Chem. Soc. Jpn. 1993,
66, 3041−3046. (b) Namatame, I.; Tomoda, H.; Tabata, N.; Si, S. Y.;
Omura, S. J. Antibiot. 1999, 52, 7−12. (c) Matsuda, D.; Namatame, I.;
Tomoda, H.; Kobayashi, S.; Zocher, R.; Kleinkauf, H.; Omura, S. J.
Antibiot. 2004, 57, 1−9.
(6) (a) Briggs, L. H.; Fergus, B. J.; Shannon, J. S. Tetrahedron 1966,
22, 269−278. (b) Isogai, A.; Kanaoka, M.; Matsuda, H.; Hori, Y.;
Suzuki, A. Agric. Biol. Chem. 1978, 42, 1797−1798. (c) Grove, J. F. J.
Chem. Soc., Perkin Trans. 1 1980, 2878−2880. (d) Jegorov, A.;
Sedmera, P.; Matha, V.; Simek, P.; Zhradnickova, H.; Landa, Z.; Eyal,
J. Phytochemistry 1994, 37, 1301−1303. (e) Omura, S.; Tomoda, H.;
Sunazuka, T.; Nagai, K.; Takahashi, T.; Doi, T. WO 2005047311A1,
2005.
(7) (a) Paranagama, P. A.; Wijeratne, E. M. K.; Burns, A. M.;
Marron, M. T.; Gunatilaka, M. K.; Arnold, A. E.; Gunatilaka, A. A. L.
J. Nat. Prod. 2007, 70, 1700−1705. (b) Gao, H.; Zou, J.; Li, J.; Zhao,
H. In Studies in Natural Products Chemistry; Atta ur, R., Ed.; Vol. 48;
Elsevier, 2016; pp 347−397. (c) Xie, F.; Luan, X. Y.; Gao, Y.; Xu, K.;
Lou, H. X. J. Nat. Prod. 2020, 83, 1623−1633. (d) Yuan, W. H.; Teng,
M. T.; Yun, Y. F.; Jiang, N.; Ma, L.; Sun, S.-S.; Yuan, B.; Tang, J.; Wu,
Q. Y.; Li, Q.; Zhang, P.; Morris-Natschke, S. L.; Lee, K. H. J. Nat.
Prod. 2020, 83, 1716−1720.
Xiang-Hai Cai − State Key Laboratory of Phytochemistry and
Plant Resources in West China, and Yunnan Key Laboratory
(8) Zhou, Y. F.; Shi, H. X.; Hu, K.; Tang, J. W.; Li, X. R.; Du, X.;
Sun, H. D.; Wang, L. S.; Pu, J. X. Molecules 2017, 22, 1675−1683.
1
252
https://doi.org/10.1021/acs.jnatprod.0c01305
J. Nat. Prod. 2021, 84, 1244−1253

Journal of Natural Products
pubs.acs.org/jnp
Article
(
9) (a) Randazzo, A.; Bifulco, G.; Giannini, C.; Bucci, M.; Debitus,
C.; Cirino, G.; Gomez-Paloma, L. J. Am. Chem. Soc. 2001, 123,
1
0870−1087. (b) Rodríguez, J.; Jiménez, C.; Blanco, M.; Tarazona,
G.; Fernández, R.; Cuevas, C. Org. Lett. 2016, 18, 5832−5835. (c) Li,
W.-S.; Yan, R. J.; Yu, Y.; Shi, Z.; Mándi, A.; Shen, L.; Kurtán, T.; Wu,
J. Angew. Chem., Int. Ed. 2020, 59, 13028−13036.
(
10) (a) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (b) Bifulco, G.;
Dambruoso, P.; Gomez-Paloma, L.; Riccio, R. Chem. Rev. 2007, 107,
3
(
744−3779.
11) Organization WH: Deafness and Hearing Loss Geneva; World
Health Organization: Switzerland, 2018; p 2001.
12) (a) Hong, B. N.; Kang, T. H. Neurosci. Lett. 2014, 566, 309−
14. (b) Huo, X.; Gao, L.; Guo, L.; Xu, W.; Wang, W.; Zhi, X.; Li, L.;
(
3
Ren, Y.; Qi, X.; Sun, Z.; Li, W.; Ji, Q.; Ran, X.; Su, B.; Hao, C.; Lu, J.;
Guo, X.; Zhuo, H.; Zhang, D.; Pan, C.; Weng, J.; Hu, D.; Yang, X.; Ji,
L. Lancet Diabetes Endocrinol. 2016, 4, 115−124. (c) Afkarian, M.;
Zelnick, L. R.; Hall, Y. N.; Heagerty, P. J.; Tuttle, K.; Weiss, N. S.; de
Boer, I. H. JAMA-J. Am. Med. Assoc. 2016, 316, 602−610.
(
13) Krook, A.; Wallberg-Henriksson, H.; Zierath, J. R. Med. Sci.
Sports Exercise 2004, 36, 1212−1217.
14) Pracht, P.; Bohle, F.; Grimme, S. Phys. Chem. Chem. Phys. 2020,
2, 7169−7192.
15) Tian, L. molclus program, Version 1.8.8, http://www.keinsci.
com/research/molclus.html (accessed 2019-08-15).
16) (a) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J. Chem. Rev.
012, 112, 1839−1862. (b) Willoughby, P. H.; Jansma, M. J.; Hoye,
(
2
(
(
2
T. R. Nat. Protoc. 2014, 9, 643−660.
(
(
17) Jensen, F. Theor. Chem. Acc. 2010, 126, 371−382.
18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.;
Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,
R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar,
S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox,
J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.
D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.
Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2010.
(
19) Kalinec, G. M.; Webster, P.; Lim, D. J.; Kalinec, F. Audiol.
Neuro-Otol. 2003, 8, 177−189.
20) Zhang, W.; Hong, D.; Zhou, Y.; Zhang, Y.; Shen, Q.; Li, J. Y.;
Hu, L. H.; Li, J. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 1505−
512.
(
1
1
253
https://doi.org/10.1021/acs.jnatprod.0c01305
J. Nat. Prod. 2021, 84, 1244−1253