Psychrophilin E, a new cyclotripeptide, from co-fermentation of two marine alga-derived fungi of the genus Aspergillus

Article abstract of DOI:10.1080/14786419.2014.880911

Chemical investigation of the mycelial extract of a mixed culture of two marine alga-derived fungal strains of the genus Aspergillus has yielded one new cyclotripeptide, psychrophilin E (1), the recently reported oxepin-containing alkaloids, protuboxepin A (2) and oxepinamide E (3), together with three other polyketide derivatives (4-6). The chemical structure and relative and absolute configurations of psychrophilin E (1) were unambiguously established based on HRMS, 1D, 2D NMR and chiral-phase HPLC analysis of its hydrolysate. All the isolated compounds were assessed for their anti-proliferative activity against four different human cancer cell lines and some of them revealed selective activities. 2014

Full text of DOI:10.1080/14786419.2014.880911

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Psychrophilin E, a new cyclotripeptide,
from co-fermentation of two marine
alga-derived fungi of the genus
Aspergillus
ad
a
b
Sherif S. Ebada , Thomas Fischer , Alexandra Hamacher , Feng-
c
a
b
c
Yu Du , Yoen Ok Roth , Matthias U. Kassack , Bin-Gui Wang &
a
Eckhard H. Roth
a
BIO-MAR, Merowingerplatz 1a, D-40225 Düsseldorf, Germany
b
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-
Heine Universität, Universitätsstrasse 1, Geb. 26.23, D-40225
Düsseldorf, Germany
c
Key Laboratory of Experimental Marine Biology, Institute of
Oceanology, Chinese Academy of Sciences, Nahnhai Road 7,
Qingdao 266071, P.R. China
d
Department of Pharmacognosy and Phytochemistry, Faculty of
Pharmacy, Ain-Shams University, Organization of African Unity
Street 1, 11566 Cairo, Egypt
Published online: 31 Jan 2014.
To cite this article: Sherif S. Ebada, Thomas Fischer, Alexandra Hamacher, Feng-Yu Du, Yoen
Ok Roth, Matthias U. Kassack, Bin-Gui Wang & Eckhard H. Roth (2014) Psychrophilin E, a
new cyclotripeptide, from co-fermentation of two marine alga-derived fungi of the genus
Aspergillus, Natural Product Research: Formerly Natural Product Letters, 28:11, 776-781, DOI:
10.1080/14786419.2014.880911
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Natural Product Research, 2014
Vol. 28, No. 11, 776–781, http://dx.doi.org/10.1080/14786419.2014.880911
Psychrophilin E, a new cyclotripeptide, from co-fermentation
of two marine alga-derived fungi of the genus Aspergillus
ad
Sherif S. Ebada *, Thomas Fischer , Alexandra Hamacher , Feng-Yu Du , Yoen Ok Roth ,
a
b
c
a
b
Matthias U. Kassack , Bin-Gui Wang and Eckhard H. Roth
c
a
a b
BIO-MAR, Merowingerplatz 1a, D-40225 D u¨ sseldorf, Germany; Institut f u¨ r Pharmazeutische und
Medizinische Chemie, Heinrich-Heine Universit a¨ t, Universit a¨ tsstrasse 1, Geb. 26.23, D-40225 D u¨ sseldorf,
Germany; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of
c
d
Sciences, Nahnhai Road 7, Qingdao 266071, P.R. China; Department of Pharmacognosy and
Phytochemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 1,
1
1566 Cairo, Egypt
(
Received 16 July 2013; final version received 26 December 2013)
Chemical investigation of the mycelial extract of a mixed culture of two marine alga-
derived fungal strains of the genus Aspergillus has yielded one new cyclotripeptide,
psychrophilin E (1), the recently reported oxepin-containing alkaloids, protuboxepin
A (2) and oxepinamide E (3), together with three other polyketide derivatives (4–6).
The chemical structure and relative and absolute configurations of psychrophilin E (1)
were unambiguously established based on HRMS, 1D, 2D NMR and chiral-phase
HPLC analysis of its hydrolysate. All the isolated compounds were assessed for their
anti-proliferative activity against four different human cancer cell lines and some of
them revealed selective activities.
Keywords: Aspergillus; co-fermentation; marine-derived fungi; anti-proliferative
1
. Introduction
Marine-derived fungi continue to be a prolific source providing a plethora of hitherto
unprecedented chemical scaffolds and/or new metabolites with potent pharmacologically
significant activities (Rateb & Ebel 2011). This notion was proven by the increasing research
efforts directed towards marine-derived fungi during the last few decades, which yielded a
sixfold increase in the number of identified marine fungal metabolites in 2007 compared with
those reported from 1965 to 2005 (Imhoff et al. 2011).
Recently, co-culturing of two or more marine-derived fungal strains in order to imitate their
natural environment and/or to induce other biosynthetic interactions has gained a considerable
attention aiming at identifying novel chemical skeletons produced through different biosynthetic
pathways such as aspergicin, a potent anti-microbial alkaloid recently reported from a mixed
culture of two marine-derived mangrove epiphytic fungal strains of the genus Aspergillus (Zhu
et al. 2011).
Psychrophilins (Psp’s) are a class of cyclic nitrotripeptides comprising four metabolites that
have been reported from psychrotolerant fungal species of the genus Penicillium (Dalsgaard
et al. 2004a, 2004b; 2005). The core structural elements of these peptides are one anthranilic acid
(2-aminobenzoic acid) and one tryptophan residue together with variable amino acid residues
including L-proline (Psp A) (Dalsgaard et al. 2004a), L-valine (Psp B) (Dalsgaard et al. 2004b),
*
Corresponding author. Emails: sherif_elsayed@pharma.asu.edu.eg, selsayed@bio-mar.de
q 2014 Taylor & Francis

Natural Product Research
777
L-alanine (Psp C) (Dalsgaard et al. 2004b) and L-leucine (Psp D) (Dalsgaard et al. 2005). During
our ongoing search for bioactive secondary metabolites from marine-derived fungi through co-
cultures, we have studied an EtOAc extract of a mixed culture of two marine alga-derived fungal
strains belonging to the genus Aspergillus which were derived from the brown algae Sargassum
sp. collected off the North Sea shores in Germany. The two fungi were cultivated as a co-culture
under static conditions at 288C for four weeks till the dark brown pigment of conidia was noticed
and then EtOAc was added to the culture mixture to end the fermentation process and to extract
the whole biomass.
2
. Results and discussion
Chemical investigation of EtOAc extract yielded one new psychrophilin congener (Psp E, 1),
two recently reported oxepin-containing pyrimidine alkaloids, protuboxepin A (2) (Lee et al.
2
011) and oxepinamide E (3) (Lu et al. 2011), together with three mycotoxins, namely
sterigmatocystin (4) (Seto et al. 1974; Shao et al. 2007), 5-methoxysterigmatocystin (5) (Seto
et al. 1974; Shao et al. 2007; Cai et al. 2011) and aversin (6) (Shao et al. 2007).
2
D
5
Psychrophilin E (1), ½aꢀ þ 32.0 (c 0.2, CHCl ), IR (film): 3296, 2926, 2851, 1693, 1623,
3
2
603, 1524, 1453, 1363, 754 cm , UV (MeOH) l
1
1
white amorphous solid featuring in C NMR spectrum and DEPT experiment the existence of
¼ 222, 246, 306 nm, was isolated as a
max
1
3
2
25 carbon signals differentiated into nine quaternary carbons including four carbonyl sp
3
carbons at d 166.2, 167.4, 170.1 and 172.4, eleven tertiary carbons including two sp at d 51.0
C
C
and 59.8, four secondary carbons, and one primary methyl carbon (d 23.0). The molecular
C
formula of 1 was established to be C H N O on the basis of HR-ESI-MS revealing ion peak at
2
5 24 4 4
þ
m/z 445.1870 [M þ H] (calcd for C H N O 445.1876) implying the presence of 16 degrees
2
5 25 4 4
of unsaturation in the compound. In addition, UV spectrum of 1 in MeOH indicated the presence
of aromatic and indole moieties in the compound as revealed by psychrophilins A–D (Dalsgaard
et al. 2004a, 2004b; 2005). Based on the literature search, a match was found in psychrophilins, a
class of cyclic nitrotripeptides. Further investigation was conducted through IR spectrum,
2
1
showing an intense stretching vibration at 1693 cm indicating the presence of amide carbonyl
groups and missing those characteristic stretching vibrations of the nitro functionality at 1553
2
1
and 1360 cm as for psychrophilins A–D (Dalsgaard et al. 2004a, 2004b; 2005). These findings
suggested that 1 is a cyclic tripeptide resembling the psychrophilins except in the absence of the
nitro group (Figure 1).
Structural elucidation was continued through extensive 2D NMR spectral analyses including
1
H– H COSY and HSQC (see Supplementary materials, Figures S9 and S10) which confirmed
1
the existence of an ortho-substituted benzene and indole ring systems similar to those in
psychrophilins A–D representing an anthranilic acid (Abz) and tryptophan (Try) residues in
0
00
addition to two aliphatic spin systems of the types X–CH(X )–CH –CH –CH –X and
2
2
2
0 00
XNH–CH(X )–CH –X which were attributed to proline and tryptophan residues,
2
–
respectively. The arrangement of amino acid residues was confirmed through the HMBC
spectrum (see Supplementary materials, Figure S11) which revealed correlations between H-11
and C-12 (d 166.2), 18-NH to C-19 (d 167.4) and H-20 to C-1 (d 172.4) together with the
C
C
C
correlations of 2-NH, H-2 and a singlet methyl resonance at d 2.04 (s, 3H) to C-24 (d 170.1)
H
C
which established the amino acid sequence of psychrophilin E (1) as cyclo-(N-acetyltryptophan-
prolyl-O-aminobenzoyl).
The absolute configuration of 1 was determined through implementing chiral-phase gas
chromatographic (GC) analysis of methyl N-(trifluoroacetyl)proline derivative obtained from
hydrolysis, using methanolic HCl followed by acetylation using trifluoroacetic acid anhydride
(TFAA) compared with those of D- and L-proline standards (Stenerson & Lee 2011).
Comparison of GC retention times of the derivatised proline with D- and L-proline standards

7
78
S.S. Ebada et al.
O
HN 24
O
25
Pro
6
Try
O
N
O
N
O
22
1
3
N
NH
NH
20
11
N
N
O
O
1
9
N
O
9
OH
O
12
HN
O
17
1
4
3
2
Abz
1
O
H
R
H 4
O
O
1'
O
OH
H
5
O
O
O
O
O
H
8
1
OH
O
O
6
4
5
R= H
R= OCH3
Figure 1. Structures of compounds 1–6.
unambiguously proved proline to possess the L-configuration assigning S configuration to C-20.
The absolute configuration of C-2 cannot be directly established due to the destruction of
N-acetyltryptophan during acid hydrolysis.
However, the ROESY spectrum (see Supplementary materials, Figures S14 and S15) of
psychrophilin E (1) revealed key correlations which were used to determine the most likely
configuration. Using the 3D models of the minimal energy conformation of (2R, 20S) and (2S,
2
0S) psychrophilin E (1) together with the ROESY correlations (see Supplementary material,
Figure S15) between the a-proton in N-acetyltryptophan (H-2) and H-3b; H-2 and H-23a/b; and
the correlations between H-3b and H-6; H-3a and H-11, the absolute stereochemistry of C-2 was
assigned as R configuration and hence (2R, 20S) for psychrophilin E (1) unlike the absolute
configuration reported for psychrophilins A–D (Dalsgaard et al. 2004a, 2004b; 2005).
All the isolated compounds were assessed for their in vitro anti-proliferative activities using
a microplate (MTT) assay (M u¨ ller et al. 2004) against four different human tumour cell lines,
namely K562 (leukaemia), HCT116 (colon), A2780 (ovary) and A2780CisR (cisplatin-resistant
mutant). Among the tested compounds (Table 1), psychrophilin E (1), sterigmatocystin (4) and
5
-methoxysterigmatocystin (5) exhibited selective anti-proliferative activities in particular
towards HCT116 (colon) cell line with IC50 values of 28.5, 10.3 and 4.4 mM, respectively,
compared with cisplatin as a positive control (IC50 ¼ 33.4 mM). In addition, 5-methoxyster-
Table 1. IC50 values of selected compounds against four different human tumour cell lines.
IC50 (mM)
Compound
K562
HCT116
A2780
A2780CisR
Psychrophilin E (1)
Sterigmatocystin (4)
5-Methoxysterigmatocystin (5)
Cisplatin
67.8
57.0
13.4
7.8
28.5
10.3
4.4
27.3
30.6
51.0
0.8
49.4
95.5
.140
8.4
33.4

Natural Product Research
779
igmatocystin (5) showed significant anti-proliferative activity against K562 (leukaemia) cell line
with IC50 of 13.4 mM compared with cisplatin (IC50 ¼ 7.8 mM).
3
3
. Experimental
.1. General experimental procedures
w
Optical rotation was recorded using a Perkin-Elmer-341 MC polarimeter (Perkin-Elmer ,
Waltham, MA, USA) at 589 nm using a cylinder with 10 cm length at 208C. IR spectrum was
w
recorded using Perkin-Elmer Spectrum One IR-Spectrometer (Perkin-Elmer , Waltham, MA,
USA). 1D and 2D NMR spectra were measured using Bruker ARX 500 and AVANCE DRX 600
w
NMR spectrometers (Bruker , Billerica, MA, USA). MS (ESI) and HRMS (ESI) were determined
w
with a ThermoFinnigan LCQ DECA (Thermo , Waltham, MA, USA) and Micromass QTOF 2
w
Waters , Milford, MA, USA) mass spectrometers, respectively. Solvents were distilled prior to
(
use, and spectral grade solvents were used for spectroscopic measurements.
For HPLC analysis, a Dionex P580 HPLC system coupled to a photodiode array detector
UVD340U) was used. Routine detection was conducted at 225, 250, 275 and 350 nm. The
(
separation column (125 £ 4 mm ID) was pre-filled with C-18 Eurosphere, 5 mM (Knauer, Berlin,
Germany). Separation was performed implementing a linear gradient from 10% MeOH to 100%
MeOH over 40 min. For preparative HPLC separations, a separation column (250 £ 8 mm ID)
pre-filled with C-18 Eurosphere, flow rate 3.0 mL/min and UV detection at 240 and 350 nm; the
elution system comprised a linear gradient of CH OH and nanopure H O. TLC was performed
3
2
w
using Polygram , plastic sheets pre-coated with silica gel 60 F
w
Germany). Chiral GC analysis was carried out on Shimadzu GC-2010 Plus equipped with
capillary CP-chirasil Dex CB column using helium as carrier gas and an isothermal program at
(Macherey-Nagel, D u¨ ren,
54
2
1208C for 15 min. Minimal energy conformations of psychrophilin E (1) was calculated using
w
ChemDraw Ultra 12.0 based on 3D models of the two conformers (2R, 20S) and (2S, 20S).
3
.2. Isolation and cultivation of the microbial material
Two fungal strains of the genus Aspergillus (BM-05 and BM-05ML) were isolated from a brown
algal species belonging to the genus Sargassum collected off Helgoland, North Sea, Germany in
2001. The two fungi were cultivated together as a co-culture under static conditions at 288C for
four weeks in ten 1-L Erlenmeyer flasks containing the culture medium (500 mL/flask)
composed of peptone from soya (4 g/L), maize starch (10 g/L), MgSO (3.6 g/L), NaCl (20 g/L),
4
yeast extract (2 g/L) and CaCO (1.8 g/L) using demineralised water. The culture medium was
3
sterilised in 1- L Erlenmeyer flasks at 1218C for 20 min. Agar plugs from plated culture were
seeded into production medium (10 £ 500 mL). The fermentation time was reached when dark
brown pigment of conidia was noticed.
3
.3. Extraction and isolation
At the end of the incubation period, the mycelium and medium were extracted four times with
ethyl acetate. The obtained extract was concentrated under vacuum to yield residual gum in 6.5 g.
This residue was subjected to vacuum liquid chromatography on a silica gel column
implementing a step gradient of n-hexane–EtOAc and then CH Cl –CH OH yielding nine
2
2
3
fractions (V1–V9) each of 500 mL. These fractions were dried under reduced pressure and
examined using analytical HPLC system and TLC on pre-made silica gel plates using a CH Cl –
CH OH-based solvent system. Further purification of fractions V2 (240 mg), V3 (210 mg) and
2
2
3
V8 (160 mg) was obtained separately through column chromatography using Sephadex LH20 as
stationary phase and eluted with CH Cl :CH OH (2:8). Final purification steps were achieved
2
2
3

7
80
S.S. Ebada et al.
through preparative reversed-phase HPLC using a C18 column (MeOH–H O, linear gradient,
2
1
0% MeOH ! 100% MeOH over 40 min) to yield 1 (14.0 mg), 2 (3.0 mg), 3 (4.0 mg), 4
(
10.0 mg), 5 (2.0 mg) and 6 (1.0 mg). Chemical structures of the isolated known compounds were
characterised based on spectrometric and spectroscopic analyses, including mass spectrometry,
HRMS, 1D and 2D NMR spectroscopy, and by comparing their NMR spectroscopic data with
the literature values (Shao et al. 2007; Cai et al. 2011; Lee et al. 2011; Lu et al. 2011).
3
.3.1. Psychrophilin E (1)
2
D
5
White amorphous solid; ½aꢀ þ 32.0 (c 0.2, CHCl ). IR (film): 3296, 2926, 2851, 1693, 1623,
3
2
603, 1524, 1453, 1363, 754 cm , UV (MeOH) l
1
1
13
1
¼ 222, 246, 306 nm. H and C NMR
max
(
500 MHz, CDCl ): 1.23 (m, 1H, H-22b), 1.52 (m, 1H, H-22a), 1.69 (m, 1H, H-21b), 2.02 (m,
3
1
H, H-21a), 2.04 (s, 3H, H-25), 2.14 (m, 1H, H-23b), 2.89 (t, J ¼ 12.2 Hz, 1H, H-3a), 3.27 (dd,
J ¼ 16.7, 9.2 Hz, 1H, H-23a), 3.35 (dd, J ¼ 12.2, 5.4 Hz, 1H, H-3b), 4.55 (dd, J ¼ 8.3, 2.7 Hz,
H, H-20), 5.08 (ddd, J ¼ 13.1, 7.9, 5.5 Hz, 1H, H-2), 6.49 (brd, 1H, NH-Ac-Try), 6.86 (s, 1H,
1
H-11), 7.38 (t, J ¼ 7.3 Hz, 2H, H-7 and H-15), 7.43 (t, J ¼ 7.6 Hz, 1H, H-8), 7.57 (d, J ¼ 7.6 Hz,
1
1
2
1
H, H-17), 7.57 (t, J ¼ 7.3 Hz, 1H, H-16), 7.67 (d, J ¼ 7.6 Hz, 1H, H-6), 7.82 (d, J ¼ 7.6 Hz,
1
3
H, H-14), 8.50 (brs, 1H, NH-Abz), and 8.62 (d, J ¼ 8.1 Hz, 1H, H-9); C NMR: 23.0, 24.7,
6.3, 29.3, 47.7, 51.0, 59.8, 116.4, 117.2, 118.3, 123.7, 124.5, 125.6, 126.1, 126.3, 128.1, 129.6,
31.7, 132.5, 133.4, 135.5, 166.2, 167.4, 170.1, and 172.4; HRESIMS m/z 445.1870 [M þ H]
þ
(
calcd for C H N O 445.1876).
25 25 4 4
3
.4. Acid hydrolysis of psychrophilin E (1)
The compound (0.5 mg) was refluxed at 958C for 6 h with 6 N methanolic HCl. After cooling, the
sample was evaporated under reduced pressure to dryness. Then, the remaining residue was
dissolved in 1.0 mL of methylene chloride and 100 mL of TFAA and then it was refluxed at 608C
for 20 min. The sample was then cooled and the remaining liquid was evaporated at room
temperature. The residue obtained was dissolved in methylene chloride for chiral gas GC
analysis. The same procedure was conducted for an unequal known mixture of D- and L-proline
standards. The configuration of proline was determined by an isothermic GC analysis using CP-
chirasil Dex CB column at 1208C over 15 min. Retention times (in min) for the standards were
proline, R, 7.12 min, and S, 7.24 min. Analysis of the derivative gave a retention time of
7
.24 min, determining an S configuration for the proline residue.
3
.5. Cell proliferation assay
Anti-proliferative activity was tested in vitro against human chronic myelogenous leukaemia
K562), human colon carcinoma (HCT116), human ovarian cancer (A2780) and cisplatin-
(
resistant human ovarian cancer (A2780CisR) cell lines using a microplate-based 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and compared with that of
untreated controls as previously described (M u¨ ller et al. 2004). All experiments were carried out
in triplicate using cisplatin (CDDP) as positive control.
4
. Conclusions
Psychrophilins A–D were all reported from marine-derived psychrotolerant fungal species of
the genus Penicillium, cultivated in the dark at 20–258C, psychrophilin E (1) was obtained from
a mixed culture of two marine alga-derived fungal strains of the genus Aspergillus which were
cultivated at 288C. In addition, psychrophilin E (1) exhibited acetylation of a-amino group in
tryptophan residue into an N-acetyl moiety, which was oxidised in psychrophilins A–D into a

Natural Product Research
781
nitro group (Dalsgaard et al. 2004a, 2004b; 2005). Furthermore, LC–MS screening of the fungal
extract and purified fractions did not reveal the existence of other detectable psychrophilin
derivatives which implies that this co-culture may have played a role in modifying the
biosynthetic pathway to acetylation rather than oxidation of a-amino group in tryptophan
residue.
Interestingly, the additional methoxy group in 5 potentiated its activity towards HCT116 and
K562 cell lines (Table 1) as compared with sterigmatocystin (4) with IC50 values of 4.4 and
13.4 mM with 5 and 10.3 and 57.0 mM with 4, respectively. These results may strengthen the
significance of 5-methoxy group as a functional group important for anti-proliferative activity of
compound (5).
Supplementary material
Supplementary material relating to this article is available online alongside Table S1 and Figures
S1–S15.
Acknowledgements
We gratefully acknowledge the Ministry of Economics and Technology, Germany for funding our research
(
AiF-ZIM). We are also indebted to Prof. Dr J. Pietruszka and Dipl.-Ing. (FH) Rainer Goldbaum (Institute
for Bioorganic Chemistry, HHU D u¨ sseldorf) for their kind assistance on the analytical measurements. B.-G.
W. acknowledges the support of a project from NSFC (30910103914).
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