Polyketides and nitrogenous metabolites from the endophytic fungus Phomopsis sp. D15a2a

Article abstract of DOI:10.1016/j.tetlet.2019.151325

Three new polyketides, phomopones A?C (1–3), one new cyclic tetrapeptide, 18-hydroxydihydrotentoxin (4), and a new amide, 6-hydroxyenamidin (5) together with a known derivative, enamindin (6) were obtained from the endophytic fungus Phomopsis sp. D15a2a i

Full text of DOI:10.1016/j.tetlet.2019.151325

Journal Pre-proofs
Polyketides and Nitrogenous Metabolites from the Endophytic Fungus Pho-
mopsis sp. D15a2a
Haiqian Yu, Simon-Patrick Höfert, Mariam Moussa, Christoph Janiak, Werner
E.G. Müller, Blessing O. Umeokoli, Haofu Dai, Zhen Liu, Peter Proksch
PII:
S0040-4039(19)31112-8
DOI:
https://doi.org/10.1016/j.tetlet.2019.151325
TETL 151325
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 August 2019
22 October 2019
24 October 2019
Please cite this article as: Yu, H., Höfert, S-P., Moussa, M., Janiak, C., Müller, W.E.G., Umeokoli, B.O., Dai, H.,
Liu, Z., Proksch, P., Polyketides and Nitrogenous Metabolites from the Endophytic Fungus Phomopsis sp. D15a2a,
Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.151325
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Polyketides and Nitrogenous Metabolites from the Endophytic
Fungus Phomopsis sp. D15a2a
Haiqian Yu a, Simon-Patrick Höfert b, Mariam Moussa a, Christoph Janiak b, Werner E.
G. Mꢀller c, Blessing O. Umeokoli a d, Haofu Dai e, Zhen Liu a,*, Peter Proksch a,*
a Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-Universität Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
b Institute of Inorganic and Structural Chemistry, Heinrich-Heine-Universität Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
c
Institute of Physiological Chemistry, Universitätsmedizin der Johannes-Gutenberg-Universität
Mainz, 55128 Mainz, Germany
d
Department of Pharmaceutical and Medicinal Chemistry, Nnamdi Azikiwe University, Awka,
Nigeria
e
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture,
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural
Sciences, Haikou 571101, China
*Corresponding authors.
Tel.: +49 211 81 14163; fax: +49 211 81 11923;
e-mail: zhenfeizi0@sina.com (Z. Liu),
proksch@uni-duesseldorf.de (P. Proksch)
1

ABSTRACT
Three new polyketides, phomopones A−C (1−3), one new cyclic tetrapeptide,
18-hydroxydihydrotentoxin (4), and a new amide, 6-hydroxyenamidin (5) together
with a known derivative, enamindin (6) were obtained from the endophytic fungus
Phomopsis sp. D15a2a isolated from the plant Alternanthera bettzickiana. The
structures of the new compounds were elucidated by 1D, 2D NMR and HRMS data.
The absolute configurations of the isolated metabolites were determined either by
X-ray crystallography, Marfey’s method or by converting the compounds to Mosher
esters.
Keywords: Phomopsis sp.; endophytic fungus; polyketide; peptide.
2

Introduction
Fungi of genus Phomopsis have been reported as endophytes, saprobes, plant
pathogens, animal pathogens and are even known to infect humans.1 They have been
rather well investigated due to their production of structurally diverse metabolites.
Phomopsis. sp. TJ507A, an endophyte obtained from the medicinal plant Phyllanthus
glaucus (Phyllanthaceae), yielded a series of protoilludane, illudalane and botryane
sesquiterpenoids, of which some showed β-site amyloid precursor protein cleaving
enzyme 1 (BACE1) inhibitory activities.2 From the endophytic fungus Phomopsis sp.
YE3250, seven polyoxygenated cyclohexenoids, phomopoxides A−G, were isolated
with promising α-glycosidase inhibitory activity.3 Four furanones, phomopsolidones
A−D, were isolated from Phomopsis sp. DC275 and two of them exhibited
antibacterial activity against Bacillus subtilis with MIC values of 0.1 ng.4 In this study,
the endophytic fungus Phomopsis sp. D15a2a, that was isolated from leaves of
Alternanthera bettzickiana (Amaranthaceae) collected in Anambra state of Nigeria,
was investigated with regard to its secondary metabolites. The EtOAc extract of
Phomopsis sp. following fermentation on solid rice medium yielded three new
polyketides,
phomopones
A−C
(1−3),
a
new
cyclic
tetrapeptide,
18-hydroxydihydrotentoxin (4), and a new amide, 6-hydroxyenamidin (5) in addition
to a known derivative, enamindin (6) (Figure 1).5
3

Figure 1. Structures of isolated compounds from Phomopsis sp.
Results and Discussion
Compound 1 had the molecular formula of C10H14O3 with four degrees of
unsaturation as established from the HRESIMS data. The 13C NMR data of 1 (Table 1)
showed one carbonyl at δC 199.9 (C-1), two olefinic carbons at δC 173.6 (C-5) and
109.6 (C-6), two oxygenated methines at δC 75.5 (C-9) and 72.4 (C-2), a methyl at δC
20.5 (C-10) and four aliphatic methylenes, accounting for two degrees of unsaturation.
The remaining two degrees of unsaturation suggested a bicyclic structure for
compound 1. Two spin systems from C-2 to C-4 and from C-7 to C-10 were
established from the COSY correlations between H-2/ H-3ab/H-4ab and between
H-7ab/H-8ab/H-9/Me-10 (Figure 2). The HMBC correlations from H-2 and H-3ab to
C-1, from H-3ab to C-5, from H-4ab to C-5 and C-6, from H-7ab to C-1, C-5 and C-6,
and from H-8ab to C-6 indicated the presence of an α,β-unsaturated ketone and
linkages between C-1/C-2, C-4/C-5, and C-6/C-7. The location of a hydroxy group at
4

C-2 and an ether linkage between C-5 and C-9 were deduced from the molecular
formula of 1, the chemical shifts of C-2, C-5 and C-9 in addition to the weak HMBC
correlation from Me-10 to C-5. Thus, the planar structure of 1 was elucidated as
shown and the trivial name phomopone A was given to 1. The relative and absolute
configuration of 1 was determined by X-ray diffraction analysis (Figure 3).
Figure 2. COSY and key HMBC correlations of compound 1.
Figure 3. Molecular structure of compound 1 from X-ray diffraction analysis.
The molecular formula of compound 2 was determined as C11H16O4 by the
HRESIMS data. The NMR data of 2 (Table 1) were similar to those of 1 except for
the replacement of an aliphatic methylene by an oxygenated methine (δC 67.6 and δH
4.14) and an additional methoxy group (δC 56.6 and δH 3.37) in 2. The COSY
correlations between the protons of this additional methine and H-8ab, H-8ab/H-9,
and between H-9/Me-10, together with the HMBC correlations from the protons of
this additional methine to C-1 (δC 199.1), C-5 (δC 176.2) and C-6 (δC 110.9) indicated
5

the location of this additional oxygenated methine group at C-7. Furthermore, the
attachment of the additional methoxy group at C-7 was confirmed by the HMBC
correlations from the protons of the methoxy group to C-7 and in turn from H-7 to the
carbon of the methoxy group. The remaining structure of 2 was elucidated to be
identical to that of 1 after detailed analysis of the 2D NMR spectra of 2. Compound 2
is suggested to share the same absolute configurations at C-2 and C-9 as 1 in
consideration of the close biogenetic relationship between both compounds. The large
3
3
3
value of JH-9/H-8b (12.6 Hz) and the small values of JH-9/H-8a (2.4 Hz), JH-7/H-8a (2.4
3
Hz) and JH-7/H-8b (3.0 Hz) indicated diaxial orientation of H-9 and H-8b and
equatorial orientation of H-8a and H-7, implying that H-7 and H-9 were on different
sides of the ring. Thus, the configuration of 2 was tentatively assigned as
2S*,7R*,9R*.
Compound 3 shared the same molecular formula as 2 as evident from the
HRESIMS data. Comparison of the H and 13C NMR data of 2 and 3 (Table 1)
1
revealed that both compounds exhibit almost identical signals in the cyclic ether ring
whereas the chemical shifts of the oxygenated methine in the cyclohexenone ring in 3
were obviously shifted (δC 67.2 and δH 4.38 in 3 while δC 72.5 and δH 4.01 in 2). In
addition, the protons of this oxygenated methine showed HMBC correlations to C-5
and C-6 in 3 rather than to C-1 in 2, confirming the attachment of a hydroxy group at
C-4 in 3 rather than at C-2 in 2. Based on the similar chemical shifts and coupling
constants, 3 was suggested to share the same (7R*,9R*) configuration as 2. The
6

relatively small coupling constants between H-4 and H-3ab (7.2 and 4.4 Hz) in 3 were
more comparable to those of H-4b (5.0 and 3.3 Hz) rather than to those of H-4a (12.0
and 5.0 Hz) in 2, suggesting equatorial orientation of H-4 in 3 and hence (4S*)
configuration for 3.
7

Table 1. 1H and 13C NMR Data of Compounds 1−3.
1 a
2 b
3 a
position
δC, type
δH, (J in Hz)
δC, type
199.1, C
72.5, CH
δH, (J in Hz)
δC, type
199.3, C
34.4, C
δH, (J in Hz)
1
2
199.9, C
72.4, CH
4.03, dd (12.0, 5.2)
4.01, dd (12.0, 5.0)
2.54, ddd (16.9, 7.9, 4.7)
2.38, ddd (16.9, 8.8, 4.9)
3
4
30.8, CH2 2.23, dddd (12.0, 5.2, 5.2, 3.2)
1.84, dddd (12.0, 12.0, 12.0, 5.0)
28.3, CH2 2.59, br ddd (17.5, 12.0, 5.2)
2.42, br ddd (17.5, 5.0, 3.2)
173.6, C
30.1, CH2 2.23, dddd (12.0, 5.0, 5.0, 3.3)
1.86, dddd (12.0, 12.0, 12.0, 5.0)
28.4, CH2 2.64, ddd (17.7, 12.0, 5.0)
2.48, ddd (17.7, 5.0, 3.3)
176.2, C
30.6, CH2 2.20, dddd (13.3, 7.9, 4.9, 4.4)
1.92, dddd (13.3, 8.8, 7.2, 4.7)
67.2, CH
4.38, dd (7.2, 4.4)
5
6
7
175.4, C
112.7, C
67.4, CH
109.6, C
110.9, C
18.2, CH2 2.25, m
67.6, CH
4.14, dd (3.0, 2.4)
4.22, dd (3.1, 2.3)
2.22, m
8
28.6, CH2 1.95, dddd (13.8, 5.8, 4.6, 2.8)
1.53, dddd (13.8, 9.3, 9.3, 5.6)
34.0, CH2 2.12, ddd (14.5, 2.4, 2.4)
1.39, ddd (14.5, 12.6, 3.0)
33.9, CH2 2.13, ddd (14.5, 2.3, 2.3)
1.40, ddd (14.5, 12.6, 3.1)
9
75.5, CH
4.23, dqd (9.3, 6.4, 2.8)
71.8, CH
4.34, dqd (12.6, 6.3, 2.4)
71.8, CH
4.30, dqd (12.6, 6.3, 2.3)
10
20.5, CH3 1.33, d (6.4)
20.8, CH3 1.39, d (6.3)
56.6, CH3 3.37, s
20.7, CH3 1.45, d (6.3)
56.8, CH3 3.36, s,
7-OMe
a Measured in CD3OD at 600 (1H) and 150 MHz (13C); b Measured in CD3OD at 300 (1H) and 75 MHz (13C).
8

Compound 4 was isolated as a white powder. Its molecular formula of
C22H32N4O5 with 9 degrees of unsaturation was deduced from the HRESIMS data.
1
The H NMR data of 4 displayed signals of five methyls, three methylenes, three
aliphatic methines, and five aromatic methines. The presence of carbonyls at δC 169
−172 together with α-protons/carbons at δH 4.78 (H-5a), 4.49 (H-16), 4.44 (H-7), 4.03
(H-2), 3.37 (H-5b), and δC 61.8 (C-7), 55.9 (C-2), 46.8 (C-16), 44.2 (C-5) suggested
1
13
compound 4 to be a peptide. The H and C NMR data of 4 (Table 2) resembled
those of dihydrotentoxin6, 7 except for the replacement of an aliphatic methine of the
latter by an oxygenated carbon of the former at δC 68.1 (C-18) and an additional
hydroxy group at δH 4.14 (18-OH). The HMBC correlations from Me-19 (δH 0.92)
and Me-20 (δH 0.90) to C-18 and C-17 (δC 44.9), from 18-OH to C-17, C-18, C-19 (δC
29.5) and C-20 (δC 28.9), and from H-17ab (δH 2.11 and 1.35) to C-15 (δC 171.6),
along with the COSY correlations between H-17ab/H-16/16-NH, indicated the
replacement of leucine as in dihydrotentoxin by a γ-OH-Leu residue in 4. Detailed
analysis of 2D NMR data of 4 confirmed that the remaining structure of 4 was
identical to that of dihydrotentoxin. Thus, compound 4 was identified as
18-hydroxydihydrotentoxin, representing a new cyclotetrapeptide. The absolute
configuration of 4 was determined by Marfey’s reaction employing the rule that
D-FDAA-D-amino acid or L-FDAA-L-amino acid elute earlier during HPLC analysis
compared to L-FDAA-D-amino acid or D-FDAA-L-amino acid.8,9 Comparison of the
retention time of the resulting L-FDAA- and D-FDAA-amino acid derivatives
[L-FDAA-N-Methyl-Ala (35.66 min), D-FDAA-N-Methyl-Ala (38.86 min);
9

L-FDAA-18-hydroxy-Leu (47.64 min), D-FDAA-18-hydroxy-Leu (56.15 min);
L-FDAA-N-Methyl-Phe (85.17 min), D-FDAA-N-Methyl-Phe (85.80 min)] revealed
that all amino acid residues in 4 were of the L-form.
Table 2. 1H and 13C NMR Data of Compound 4.
amino acid
position
δC, type
170.4, C
55.9, C
δH, (J in Hz)
N-Methyl-Ala
1
2
4.03, q (7.1）
1.34, d (7.1）
3
15.4, CH3
2-NCH3
29.5, CH3 2.65, s
170.4, C
Gly
4
5
44.2, CH2 4.78, br d (15.4)
3.37, d (15.4)
5-NH
8.04, br s
N-Methyl-Phe
6
7
8
169.2, C
61.8, CH
33.7, CH2 3.41, d (14.6)
2.91, dd (14.6, 11.0)
4.44, d (11.0)
9
137.9, C
10, 14
11, 13
12
128.1, CH 7.20, d (7.6)
128.6, CH 7.28, t (7.6)
126.5, CH 7.19, d (7.6)
30.6, CH3 2.69, s
171.6, C
7-NCH3
15
γ-OH-Leu
16
46.8, CH
4.49, m
17
44.9, CH2 2.11, dd (14.3, 4.9)
1.35, m
18
68.1, C
19
29.5, CH3 0.92, s
28.9, CH3 0.90, s
8.14, br s
20
16-NH
18-OH
4.14, s
Measured in DMSO-d6 at 600 (1H) and 150 MHz (13C).
10

The molecular formula of compound 5 was determined to be C8H13NO5
according to the HRESIMS data, indicating the presence of an additional oxygen
1
atom when compared to the co-isolated known compound, enamindin (6).5 The H
13
and C NMR data of 5 (Table 3) were similar to those of enamindin (6). However,
the presence of an oxygenated carbon at δC 73.3 (C-6) and the observation that the
methyl groups (Me-7 and Me-8) resonated as a singlet in 5 rather than as a doublet in
6 indicated the presence of an additional hydroxy group at C-6. This was further
confirmed by the HMBC correlations from Me-7 and Me-8 (both at δH 1.23) to C-6
and C-5 (δC 78.9), and from H-5 (δH 3.92) to C-7 (δC 25.9), C-8 (δC 25.5), and C-4 (δC
173.9). The remaining structure of 5 including the configuration of the double bond
was elucidated to be identical to enamindin (6) after detailed analysis of the 2D NMR
spectra. Thus, compound 5 was determined to be 6-hydroxyenamidin. Due to the
limited amount of 5, Mosher’s reaction was carried out for 6, whose absolute
configuration had not been reported in the previous publication.5 Calculation of
differences of chemical shifts between the resulting methylated (R)- and (S)-MPA
esters of 6 led to the assignment of the absolute configuration of C-5 as R (Figure 4).
Considering the close biogenetic relationship and similarity of optical rotation
between 5 and 6, compound 5 was suggested to have the same R configuration at C-5.
Figure 4. ΔδRS (δR – δS) values of methylated (R)- and (S)-MPA esters of 6.
11

Table 3. 1H and 13C NMR Data of Compound 5.
δC, type
δH, (J in Hz)
1
2
3
4
5
6
7
8
171.1, C
99.0, CH
5.18, d (9.0)
137.3, CH 7.44, d (9.0)
173.9, C
78.9, CH
73.3, C
3.92, s
25.9, CH3 1.23, s
25.5, CH3 1.23, s
Measured in CD3OD at 300 (1H) and 75 MHz (13C).
All isolated compounds were tested for their cytotoxicity against the L5178Y
mouse lymphoma cell line but proved to be inactive when tested at a concentration of
10 μM.
In conclusion, three new polyketides, phomopones A−C (1−3), a new cyclic
tetrapeptide, 18-hydroxydihydrotentoxin (4), and a new amide, 6-hydroxyenamidin (5)
together with a known derivative, enamindin (6) were isolated from the rice medium
fermentation of Phomopsis sp. The absolute configurations of those compounds were
determined by X-ray crystallography, Marfey’s method or Mosher’s reaction.
Acknowledgements
This work is funded by the Deutsche Forschungsgemeinschaft (DFG, German
Research Foundation) – 270650915/GRK 2158 and the Manchot Foundation.
Supplementary Data
Supplementary data (UV, MS and NMR spectra of 1−5 as well as X-ray data of
1) associated with this article can be found in the online version.
12

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 The endophytic fungus Phomopsis sp. yielded five new compounds.
 X-ray diffraction analysis was performed for the polyketide phomopone A.
 Marfey’s method was applied for 4, a new cyclic tetrapeptide.
 The absolute configurations of amides were determined by Mosher’s reaction.
14

15

Declaration of interests
☒ The authors declare that they have no known competing financial
interestsorpersonalrelationshipsthatcouldhaveappearedtoinfluence
the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
16