A new spirotryprostatin from the marine isolate of the fungus Aspergillus fumigatus

Article abstract of DOI:10.1007/s10600-012-0166-8

The new spirocyclic diketopiperazine alkaloid spirotryprostatin F was isolated from the marine isolate of the fungus Aspergillus fumigatus. The structure of the compound was established using on NMR spectroscopy, high-resolution mass spectrometry, and acid hydrolysis. Spirotryprostatin F in low and ultralow doses (10^-6-10^-17 M) exhibited stimulating action on the growth of sprout roots of soy [Glycine max (L.) Merr.], buckwheat (Fagopyrum esculentum Moench), and corn (Zea mays L.). The dose-effect curve had a bimodal character.

Full text of DOI:10.1007/s10600-012-0166-8

Chemistry of Natural Compounds, Vol. 48, No. 1, March, 2012 [Russian original No. 1, January-February, 2012]
A NEW SPIROTRYPROSTATIN FROM THE MARINE
ISOLATE OF THE FUNGUS Aspergillus fumigatus
1*
2
Sh. Sh. Afiyatullov, O. I. Zhuravleva,
UDC 581.481:577.115:528.281
1
1
E. L. Chaikina, and M. M. Anisimov
The new spirocyclic diketopiperazine alkaloid spirotryprostatin F was isolated from the marine isolate of
the fungus Aspergillus fumigatus. The structure of the compound was established using on NMR spectroscopy,
high-resolution mass spectrometry, and acid hydrolysis. Spirotryprostatin F in low and ultralow doses
–6
–17
(10 –10 M) exhibited stimulating action on the growth of sprout roots of soy [Glycine max (L.) Merr.],
buckwheat (Fagopyrum esculentum Moench), and corn (Zea mays L.). The dose-effect curve had a bimodal
character.
Keywords: spirotryprostatin, marine isolate of the fungus Aspergillus fumigatus, NMR spectroscopy, soy, buckwheat,
and corn sprout roots.
Recent studies have shown that marine isolates of fungi and micromycetes are promising sources of both new and
known biologically active compounds [1]. Marine and terrestrial ecoforms of the fungus Aspergillus fumigatus are capable of
producing compounds with an amazing variety of structures. Terpenoids, peptides, indole alkaloids, anthraquinones, and
other compounds have been identified in its various extracts. The metabolites of this fungus exhibit antibacterial, fungicidal,
insecticidal, cytotoxic, and several other types of activity [2]. In continuation of the search for producers of biologically active
compounds among marine isolates of microscopic fungi, we studied A. fumigatus isolated from soft coral Sinularia sp. Herein
data on the isolation and structural identification of the new diketopiperazine alkaloid spirotryprostatin F (1) are reported and
its phytoregulating activity at low and ultralow concentrations is studied.
The fungus was cultivated for 21 d in a specially modified rice medium [3]. The dry EtOAc extract of the culture was
separated successively on a column of silica gel and then by normal-phase HPLC to produce pure 1.
+
The ESI-MS of 1 contained a peak for the cationic molecule at m/z 450 [M + Na] . The molecular formula of 1 was
13
determined as C H N O based on ES-TOF high-resolution mass spectral data and was confirmed by C NMR spectral
22 25
3 6
analysis. The UV spectrum of 1 had maxima at ꢀ
220 (log 4.24) and 274 (log 3.44) nm that were characteristic for
max
absorption of a substituted benzene ring. The IR spectrum exhibited absorption bands at 3444 (NH), 1715 (ꢁ-lactam CO),
–1
1686, and 1636 cm (CO, amide I). The PMR spectrum of 1 contained resonances for a tri-substituted benzene ring
(ꢂ 6.97, d, J = 8.5 Hz, H-4; 6.56, dd, J = 2.3, 8.5 Hz, H-5; 6.45, d, J = 2.3 Hz, H-7); an olefinic proton (ꢂ 4.89, m); an N–H
proton (ꢂ 8.1, s), a methoxyl (ꢂ 3.79, s, OCH ), and two methyls (ꢂ 1.62, s, 3H-21 and 1.11, s, 3H-22) in addition to resonances
3
for several methine and methylene groups. DEPT and HSQC spectra of 1 confirmed the presence of two methyls (ꢂ 18.0 and
25.3) and a methoxyl (ꢂ 55.5, OCH ) and indicated the presence of three amide carbonyls (ꢂ 181.3, C-2; 169.0, C-11; 165.1,
3
2
2
3
C-17), an sp C atom bonded to O (ꢂ 160.7, C-6), four sp (ꢂ 97.4, C-7; 107.6, C-5; 121.6, C-19; 127.3, C-4), and three sp
(ꢂ 75.5, C-8; 60.7, C-12; 57.5, C-18) methines, several quaternary C atoms and three methylenes.
Resonances of H and C atoms in NMR spectra of 1 practically coincided with the corresponding resonances in
spectra of spirotryprostatins C-E, which were recently isolated by Chinese researchers [4], with the exception of the C-2 and
C-7a resonances. Figure 1 illustrates COSY-45 spectra and HMBC correlations, which established the complete primary
structure of 1. ROESY correlations between H-4 and H-8 and H-19 and between 9-OH and H-12 and H-18 showed that
the first three protons were located on one side of the molecule whereas 9-OH, H-12, and H-18 were situated in the opposite
direction.
1) Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, Russian Federation,
690022, Vladivostok, fax: (4232) 31 40 50, e-mail: afiyat@piboc.dvo.ru; 2) Far-East Federal University, Russian Federation,
690000, Vladivostok, Ul. Sukhanova, 8. Translated from Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2012,
pp. 90–92. Original article submitted June 18, 2011.
©
0009-3130/12/4801-0095 2012 Springer Science+Business Media, Inc.
95

TABLE 1. NMR Spectra of Spirotryprostatin F (1) (CDCl , ꢂ, ppm, J/Hz)
3
Atom
HMBC
NOESY
7
ꢂÑ (DEPT)
ꢂH
1 (NH)
8.10 s
3, 3a, 7a
2
3
4
5
6
7
8
9
181.3 (C)
61.2 (C)
127.3 (CH)
107.6 (CH)
160.7 (C)
97.4 (CH)
75.5 (CH)
87.1 (C)
6.97 (d, J = 8.4)
6.56 (dd, J = 2.3, 8.5)
3, 5, 6, 7, 7a, 18*
3, 3a, 6, 7, 7a*
8, 18*, 19, 21
4, 6-OCH3
6.45 (d, J = 2.3)
4.82 s
3*, 3a, 4, 5, 6, 7a
2, 3, 3a, 17, 18
1 (NH), 6-OCH3
4
11
12
13
169.0 (C)
60.7 (CH)
27.6 (CH2)
4.61 (dd, J = 7.3, 9.6)
11, 13, 14*
11, 12, 14, 15
11, 12, 14, 15
12*, 13, 15
12, 13, 15
12, 13, 14, 17
9-OH, 13ꢃ, 14ꢃ
ꢃ
: 2.38 m
ꢄ: 2.10 m
12
14
23.2 (CH2)
12
ꢃ
: 1.97 m
ꢄ: 2.05 m
3.59 m
15
17
45.1 (CH2)
165.1 (C)
18
19
20
57.5 (CH)
121.6 (CH)
139.1 (C)
2, 8, 9*, 19, 20, 21*
3*, 18*, 20, 21, 22
4*, 9-OH, 21, 22
4, 21, 22
4.92 m
4.89 m
21
22
6-OCH3
9-OH
3a
25.3 (CH3)
18.0 (CH3)
55.5 (CH3)
19, 20, 22
19, 20, 21
6
19
1.62 s
1.11 s
3.79 s
7.11 s
5, 7
8*, 12, 18
9, 17
117.8 (C)
142.2 (C)
7a
______
*Weak HMBC and NOESY correlations.
HO
O
OH
O
H
16
1
N
2
N
9
7a
5
14
7
3a
12
3
18
N
11
H
19
CH O
3
O
21
22
1
Fig. 1. Structure and key HMBC correlations of 1.
Acid hydrolysis of 1 gave L-proline, which was identified by Marfey’s method using L-FDAA derivatives [5]. Thus, the
absolute stereochemistry of the new metabolite from A. fumigatus was established as 3S,8S,9R,12S,18S (Fig. 1). It was called
spirotryprostatin F (1).
Phytoregulating activity was found for 1 at low and ultralow concentrations on the growth of sprout roots of soy
[Glycine max (L.) Merr.], buckwheat (Fagopyrum esculentum Moench), and corn (Zea mays L.) (Fig. 2). However, the
studied test cultures reacted differently to the action of the alkaloid. This compound showed the most pronounced stimulating
–16
–14
–12
–7
action on the growth of corn sprout roots at concentrations of 10 (109%), 10 (120%), 10 (115%), and 10 (116%) M;
–13
–10
–8
–6
–10
–8
–6
soy, 10 (117%), 10 (114%), 10 (115%), and 10 (115%) M; buckwheat, 10 (117%), 10 (111%), and 10 (111%) M.
Dead zones were typically observed for this alkaloid between the stimulating doses where the effect corresponded to the control.
The positive control was heteroauxin, which exhibits both stimulating and inhibiting activity on the growth of roots
of the test cultures.
Our results agreed somewhat with the literature. Thus, it was observed in one of the early studies that a preparation
–13
–7
exhibited the same activity at doses differing by six orders of magnitude (10 and 10 M) for the action of a hydroperoxide
herbicide on plant cell culture whereas the effect was missing at intermediate concentrations [6]. Analogous trends were noted
for the action of ultralow doses of triethanolamine derivatives [7] and diterpene glycosides [8] on the growth of root sprouts of
monocotyledonous and dicotyledonous plants.
96

b
a
1
120
110
100
90
120
110
100
90
3
3
2
1
2
0
-16 -14 -12 -10
-8
-6
0
-16 -14
-12 -10
-8
-6
log of molar concentration
log of molar concentration
Fig. 2. Effect of spirotryprostatin F (a) and heteroauxin (b) on growth of sprout roots of soy (1), buckwheat (2), and corn (3).
The study of the action of ultralow doses is promising because it can provide a basis for creating new methods of
applying biologically active compounds in plant cultivation.
EXPERIMENTAL
Optical rotation was measured on a Perkin–Elmer 343 polarimeter (Germany). UV spectra were recorded in MeOH
13
on a Shimadzu UV-1601 PC spectrophotometer (Japan). PMR and C NMR spectra were recorded in CDCl with Me Si
3
4
internal standard on a Bruker DRX-700 spectrometer (700 and 175.6 MHz, respectively). IR spectra were obtained in CCl on
4
a Bruker OPUS Vector-22 spectrophotometer. HMBC correlations were optimized for 8 Hz, NOESY (350 ms). ESI-TOF
mass spectra were taken on an Agilent 6510 LCQ-TOF spectrometer (USA). The potential in the capillary, nozzle, and
skimmer and the collision energy were 3500, 215, and 65 V, and 35 V, respectively. Column chromatography used silica gel L
(40/100 ꢅm, Chemapol, Czechoslovakia). HPLC was performed on an Agilent 1100 Series chromatograph (USA).
Cultivation of A. fumigatus (KMM 4631). The strain was isolated from soft coral Sinularia sp. (Kunashir Is., Kuril
Islands, 52 m depth). Fermentation was carried out at 25°C in 10 Ehrlenmeyer flasks (500 mL) containing rice (20 g), yeast
extract (20 mg), KH PO (10 mg), and natural seawater (40 mL).
2
4
Isolation of Spirotryprostatin F. Fungus mycelium together with medium was ground and extracted (ꢆ3) with
EtOAc. The extract was evaporated. The solid (1.2 g) was chromatographed over a column (7 ꢆ 13 cm) of silica gel with
elution successively by hexane and hexane:EtOAc systems (stepwise gradient, 5:1ꢇ1:4). The fraction (210 mg) eluted by the
1:4 system was separated by HPLC over a Hypersil Si column (25 cm ꢆ 4.6 mm, 5 ꢅm) using CHCl :i-PrOH:MeOH (100:0.5:1.0)
3
to afford pure spirotryprostatin F (28 mg).
20
Spirotryprostatin F. C H N O , pale-yellow powder, [ꢃ] –52° (c 0.1, CHCl ). UV spectrum (MeOH,
22 25
3
6
D
3
–1
ꢀ
, nm, log ꢈ): 220 (4.24), 274 (3.44), 295 (3.36). IR spectrum (CCl , ꢉ, cm ): 3444, 2927, 2855, 2360, 1747, 1715, 1686,
1458. ESI-HR-MS (m/z): 450.1657 [M + Na] ; calcd: 450.1636. Table 1 presents the NMR spectra.
max
4
+
Acid Hydrolysis of Spirotryprostatin F. Compound 1 (1.5 mg) was placed in a glass ampul and dissolved in HCl
solution (6 N, 0.8 mL). The ampul was evacuated, heated at 105°C for 24 h, and cooled. The reaction mixture was diluted
with doubly distilled H O and concentrated in vacuo. The absolute configuration of the amino acid was determined by
2
Marfey’s method [5].
Determination of Phytoregulating Activity. We studied sprout roots of soy (G. max), buckwheat (F. esculentum),
and corn (Z. mays). Seeds from the 2010 harvest were obtained from Primorskii Agricultural Research Institute, RAAS
(Ussuriisk, Russia). We used a sprouting scheme in rolls of filter paper. Dry seeds were spread on strips of filter paper
(12 ꢆ 42 cm) that were previously moistened with test solution, rolled, placed into beakers with a small amount of test solution
(100 mL), and left for 3 d in a thermostat at 26–27°C. After incubation, the length of the main root of the sprouts was
measured. The controls were sprouts of the same culture grown in distilled H O. The positive control was heteroauxin. Test
2
results were estimated as the arithmetic mean of three repeated tests (20 seeds in each) and were expressed in percent of the
controls (M se). Results were processed statistically using the ORIGIN 7.0 computer program. The significance of the
results between control and test samples was estimated using the Student t-criterion (p < 0.05).
97

ACKNOWLEDGMENT
The work was supported financially by the Russian Foundation for Basic Research (Project No. 09-04-00388),
the Presidium of the RAS (Molecular and Cellular Biology Program), and a grant of the RF President (Project NSh-3531.2010.4).
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