Indolic metabolites from the new marine bacterium Roseivirga echinicomitans KMM 6058T

Article abstract of DOI:10.1007/s10600-006-0260-x

N-Acetyl-(1) and N,N-diacetyl-(2) tryptamines were isolated from the butanol extract of culture medium of the new marine bacterium Roseivirga echinicomitans KMM 6058^T. The structures of the compounds were proved using mass spectrometry, UV, PMR, and ¹³C NMR spectroscopy and by comparing these data with mass and NMR spectra of synthetic samples of 1 and 2. Compound 2 was isolated from a natural source and synthesized for the first time. The cytotoxic activity of the compounds was studied using Erlich carcinoma tumor cells, murine erythrocytes, and sperm and egg cells of the sea urchin Strongylocentrotus intermedius.

Full text of DOI:10.1007/s10600-006-0260-x

Chemistry of Natural Compounds, Vol. 42, No. 6, 2006
INDOLIC METABOLITES FROM THE NEW MARINE BACTERIUM
T
KMM 6058
Roseivirga echinicomitans
G. K. Oleinikova,1* O. I. Ivchuk,2 V. A. Denisenko,1 E. L. Chaikina,1
N. I. Menzorova,1 O. I. Nedashkovskaya,1 and T. A. Kuznetsova1
UDC 547.752:577.19:
615.331
N-Acetyl- (1) and N,N-diacetyl- (2) tryptamines were isolated from the butanol extract of culture medium of
T
Roseivirga echinicomitans
the new marine bacterium
KMM 6058 . The structures of the compounds were
13
proved using mass spectrometry, UV, PMR, and C NMR spectroscopy and by comparing these data with
mass and NMR spectra of synthetic samples of 1 and 2. Compound 2 was isolated from a natural source and
synthesized for the first time. The cytotoxic activity of the compounds was studied using Erlich carcinoma
Strongylocentrotus intermedius.
tumor cells, murine erythrocytes, and sperm and egg cells of the sea urchin
Key words: acetyltryptamine, NMR spectroscopy, marine bacterium
biological activity.
, indolic metabolites,
Roseivirga echinicomitans
Marine bacteria are good sources for producing physiologically active compounds [1, 2]. During a search of such
T
compounds, we investigated the new marine bacterium
KMM 6058 , which is associated with the
Roseivirga echinicomitans
sea urchin
. The bacterium cultivated in liquid medium produced compounds that exhibited
Strongylocentrotus intermedius
cytotoxic activity toward murine erythrocytes.
Fractional extraction followed by column chromatography over silica gel isolated from bacterium culture (2 L) two
compounds (0.2 mg each), 1 with 0.2 and 2 with 0.8 using hexane:ethylacetate (1:1). The UV spectra of the compounds
R
R
f
f
had three absorption maxima at 274.4, 281.4, and 290.2 nm, which are characteristic of indole [3]. The PMR spectrum of 1
contained two broad singlets for amide protons at δ 8.05 and 5.5 ppm that were assigned to protons on nitrogens of the indole
H
ring and a side chain, respectively[4]. Furthermore, signals of four protons appeared at 7-7.65 ppm as two doublets with SSCC
7.8 and 9.0 Hz and two multiplets. The position of the signals at weak field and their multiplicities were consistent with a
disubstituted benzene ring in 1. A signal at weak field (7.05 ppm, doublet, J = 2.0) was assigned to the C-2 proton in the indole
ring [4]. The spectrum also contained two signals for two protons at 3.0 ppm (triplet) and two protons at 3.6 ppm (multiplet),
which is characteristic of an R C–CH –CH –NH– system [4]. A 3H singlet at 1.92 ppm was assigned to an acetyl methyl.
3
2
2
130
143
116
R
R
13, 14
15, 16
1
4
N
3
9
10
12
5
2
H
8 N
1H
1, 2
1: R1 = H, R2 = COCH3
2: R1 = R2 = COCH3
1)PacificInstituteofBioorganicChemistry, Far-Eastern Division, Russian AcademyofSciences, 690022, Vladivostok,
pr. 100-Letiya Vladivostoka, 159, fax (4232) 31 40 50, e-mail: oleingk@mail.ru; 2) Far-Eastern State University, 690000,
Vladivostok, ul. Sukhanova, 8, Russian Federation. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 581-584,
November-December, 2006. Original article submitted October 5, 2006.
0009-3130/06/4206-0713 ©2006 Springer Science+Business Media, Inc.
713

TABLE 1. PMR and 13C NMR Spectra of 1 and 2 (δ, ppm, J/Hz, CDCl3)
1H
13C
Atom
1H-1H-COSY
HMBC
3
1
2
1
2
NH
2
3
4
5
6
7
8
9
8.05 br.s
7.05 (d, 2.0)
8.05 br.s
7.01 (d, 2.8)
-
-
129.9
113.1
118.7
119.6
122.6
111.2
136.4
127.4
25.3
39.8
-
122.3
112.4
118.7
119.7
122.3
111.3
136.2
127.2
24.8
46.0
-
173.4
26.4
173.4
26.4
-
-
7.61 (d, 7.8)
7.67 (d, 7.4)
5
4, 6
5, 7
7.14 m
7.22 m
7.39 (d, 9.0)
7.15 m
7.22 m
7.38 (d, 7.9)
-
-
-
-
10
11
3.0 (t, 6.6)
3.8 m
5.5 br. s
3.05 (t, 7.0)
3.95 (t, 8.3)
3, 11
10, 13
11
12-NH
13-CO
14-CH3
15-CO
16-CH3
-
-
11a
-
170.0
23.3
-
1.92 s
-
-
2.35 s
-
2.35 s
13
15
-
The PMR spectrum of 2 was practically identical to that of 1. However, it contained a signal for an amide proton at
5.5 ppm. Signals for four protons at 3.05 and 3.95 ppm appeared as triplets, which is characteristic of an acyclic
R C–CH –CH –NR system [4]. A strong singlet corresponding to six protons of two acetyl methyls was shifted to weak field
3
2
2
2
(2.35 ppm compared to 1.92 for 1). PMR spectra of the indole part of the compounds agreed completely with spectra of indolic
metabolites isolated from the bacterium Xenorhabdus bovienii A2 [5] and the marine cyanobacterium Lyngbya majuscula [6].
Electron-impact mass spectra (EIMS) of 1 and 2 gave molecular weights of 202 and 244 Da, respectively, which differ
by 42 mass units (COCH group). Both spectra contained the same set of fragment ions with m/z 116, 130, and 143 (Fig. 1).
3
The PMR and mass spectra suggested that 1 and 2 were mono- and diacetyltryptamine, respectively.
In order to confirm the proposed structures, 1 and 2 were synthesized by acetylation of tryptamine with acetic
13
anhydride. TLC, UV, mass, and PMR spectra confirmed that the natural and synthetic compounds were identical. C NMR
spectra were obtained and HMBC, HSQC, COSY, and DEPT experiments(Table1) wereperformed for thesynthetic compounds
and enabled signals for C and H atoms in 1 and 2 to be completely assigned.
13
The C NMR spectrum of 1 confirmed the presence of a carbamide (δ 170 ppm). Eight signals in the range 110-
C
140 ppm, three of which were quaternaryand five of which were tertiary, were characteristic of indole. The spectrum contained
two signals for CH of a side chain at 39.8 and 25.3 ppm and a signal for an acetyl methyl at 23.4 ppm. The C atoms and H
2
atoms corresponding to them were unambiguouslyassigned using HSQC, COSY, and HMBC correlation spectra. The bonding
sequence of C atoms in 1 was also determined (Table 1).
13
The C NMR spectrum of2 was almost identical to that of 1. The signals for the indole C atoms coincided completely.
However, the signal for the CH bonded to N was shifted to weak field at 46.2 ppm and the singlet for the methyl was twice as
2
strong and also shifted to weak field at 26.4 ppm. The spectrum contained a signal for carbamide C atoms at weak field at
173.6 ppm that was twice as strong, confirming the presence of two equivalent acetyls on the N in the side chain. The C atoms
and H atoms corresponding to them were completely assigned using HSQC, COSY, and HMBC correlation spectra. The
bonding sequence of the C atoms in 2 was also confirmed (Table 1).
Thus, it was established that the isolated natural compounds 1 and 2 were mono- (1) and diacetyltryptamine (2).
The ability of bacteria to acetylate tryptamine to the diacetyl derivative was observed for the first time. The bacterium
T
R. echinicomitans KMM 6058 is the onlynatural source of diacetyltryptamine. Tryptamine itself can be generated in bacteria
from endogeneous tryptophan [7]. Monoacetylated tryptamine was isolated previously from the actinobacterium Streptomyces
staurospores AM-2282 [8] and the marine bacterium Cytophaga sp. AM 13.1 [9].
The alcohol extract of sea urchin does not contain similar compounds, which confirms that the bacterium produced
1 and 2.
714

TABLE 2. Biological Activity of 1 and 2
1*
Compound
2*
3*
4*
sperm
egg cells
1
2
>50
7.5
>50
15
0
100
>50
>6.25<12.5
>100
100
______
1*, concentration (µg/mL) producing 50% destruction of membrane integrity of sperm and egg cells of sea urchin; 2*, percent
inhibition ofdivision offertilized egg cells ofsea urchin at a compound concentration of50 µg/mL; 3*, compound concentration
(µg/mL) causing 100% hemolysis of murine erythrocytes; 4*, compound concentration (µg/mL) causing 50% death of Erlich
carcinoma tumor cells.
*
Table 2 shows that 1 and 2 are weakly cytotoxic toward Erlich carcinoma tumor cells (4 ). The hemolytic activities
*
of 1 and 2 (3 ) showed that 2 is more highly cytotoxic than 1. Both compounds were checked for the ability to destroy
membranes of sperm and egg cells of S. intermedius. The percent destruction of membranes was determined from the change
of esterase activityusing diacetylfluorescein (DAF) as substrate [10]. Compound 2 caused 50% membrane destruction ofsperm
and egg cells at concentrations of 7.5 and 15 µg/mL, respectively. Compound 1 at 50 µg/mL had no effect on the membrane
integrity of these cells. The difference in the cytotoxic activities of these compounds was also noted using fertilized sea-urchin
*
egg cells as the biological model (2 ). At 50 µg/mL, 1 did not inhibit development of embryos whereas 2 caused 100%
inhibition of division. Both compounds showed no antimicrobial activitytoward gram-positive and gram-negative bacteria and
yeast-like fungi.
EXPERIMENTAL
T
A culture of R. echinicomitans KMM 6058 was isolated from gonads ofS. intermedius [11], specimens of which were
obtained at the Marine Experimental Station of PIBOC FED RAS in Troits Bay (October 2005).
3
Centrifugation was performed on a MLW K-23 apparatus (Germany) (V = 100 mL) at v = 4.5×10 rpm for 30 min with
cooling; HPLC, on a Beckman—Altex (USA) chromatograph, 4.6 mm × 25 cm column, Silosorb600, 12 µm; TLC, on silica-gel
plates (CTX-1A, 5-17 µm, 100×50) using hexane:ethylacetate (1:1) with development by anisaldehyde [12] and H SO .
2
4
UV spectra in methanol were obtained on a Specord M 40 UV—Vis spectrophotometer (Germany).
13
PMR and C NMR spectra in CDCl were recorded on Bruker DRX-500 (500 and 125 MHz) and Bruker DPX-300
3
(300 and 75 MHz) instruments with TMS internal standard. Structures of the studied compounds were established and signals
13
in PMR and C NMR spectra were assigned based on DEPT, COSY, HSQC, and HMBC experiments.
Electron-impact mass spectra (EIMS) were obtained in an LKB-9000S instrument (Sweden) using direct sample
introduction and ionizing potentials 15 and 70 eV.
The cell model for determining membrane integrity were sperm and egg cells of the sea urchin S. intermedius. The
percent destruction of membranes was determined using esterase activity and diacetylfluorescein (DAF) as substrate [10].
Fluorescence was estimated on a Microplate Fluorescence Reader FL 800 (Finland) using E /E 425/528 nm.
X
x
m
Analysis of Alcohol Extract of S. intermedius. Sea urchins (20 specimens) were extracted by ethanol (1 L) at room
temperature for 30 d. The resulting extract was concentrated at reduced pressure and analyzed byTLC on silica-gel plates with
development by anisaldehyde.
Bacterium Cultivation. Bacterium culture was fermented on rockers in 250-mL Ehrlenmeyer flasks for 7 d at 22°C
in standard medium containing peptone (5 g), yeast extract (2.5 g), glucose (1 g), K HPO (0.2 g), MgSO (0.05 g), tapwater
2
4
4
(500 mL), and seawater (500 mL).
Preparation of Total Extracts. Culture (2 L) was centrifuged. The microbial mass was separated. Culture liquid
(CL) was successively extracted with hexane, ethylacetate, and butanol. The extracts were concentrated at reduced pressure.
The initial extracts were dissolved in a minimum amount of water and reextracted with hexane, ethylacetate, and butanol to
produce total hexane, ethylacetate, and butanol extracts of CL.
715

Isolation of Indolic Compounds from Total Second Butanol CL Extract. The butanol extract was separated by
repeated column chromatography over silica gel using hexane:ethylacetate (100:0—0:100) and HPLC using a normal column
and hexane:ethylacetate (1:1) to isolate 1 and 2 (0.2 mg each).
Synthesis of Acyltryptamines. Tryptamine (40 mg) was boiled in acetic anhydride (1.5 mL) for 1.5 h at 140°C. The
excess of acetic anhydride was distilled in vacuo. The resulting solid was chromatographed over silica gel using
hexane:ethylacetate (100:0—0:100) followed by HPLC over a normal column using hexane:ethylacetate (1:1) to isolate two
compounds that were identical chromatographically to natural 1 (20 mg) and 2 (10 mg).
N-Acyltryptamine(1), C H N O, colorlesscompound. UVspectrum (MeOH, λ , nm): 274.4, 281.4, 290.2. Mass
12 14
2
max
+
+
+
spectrum (EI, m/z): 202 [M] , 144 [M - NHCOCH ] , 130 [M - CH NHCOCH ] , 116 [C H N]. Table 1 gives the PMR and
3
2
3
8 6
13
C NMR spectra.
N,N-Diacyltryptamine (2), C H N O , colorless compound. UV spectrum (MeOH, λ , nm): 274.4, 281.4, 290.2.
14 16
2
2
max
+
+
+
Mass spectrum (EI, m/z): 244 [M] , 144 [M - N(COCH ) ] , 130 [M - CH N(COCH ) ] , 116 [C H N]. Table 1 gives the PMR
3 2
2
3 2
8 6
13
and C NMR spectra.
Determination of Hemolytic Activity. Hemolytic activity was determined using a suspension of blood erythrocytes
from white mongrel mice of optical density 1.0 at 700 nm in 66 mM phosphate buffer at pH 7.4 containing NaCl (120 mM) and
KCl (4 mM). The erythrocyte suspension (200 µL) was treated with the studied compound at concentrations of 50, 25, 12.5,
and 6.25 µg/mL and incubated at 37°C for 60-180 min. Lysis (100%) was observed visually [13].
Determination of Cytotoxic Activity. Cytotoxic activity was determined using an alcohol solution (0.001 mL) of the
studied compound at various concentrations to treat a suspension (0.1 mL) of Erlich carcinoma tumor cells in medium
6
199 (1×10 cells/mL). The vitality of the tumor cells was determined after incubation at 37°C for 2 h using coloration by an
isotonic solution of trypan blue (0.17%) and a microscope. The concentration that inhibited tumor-cell vitality by 50% (IC )
50
was determined [14].
Determination of Membrane Destruction of S. intermedius Sperm Cells. A suspension (300 µL) of sperm
7
3
7
(2.5×10 cells/mL) or egg (3.5×10 cells/mL) cells in seawater (2.5×10 cells/mL) was treated with the tested compounds at
various concentrations. Microplates were held at 22 C for 30 min, treated with DAF (3 µL, 1 mg/mL acetone), and held again
for 30 min. The fluorescence was estimated. The control was seawater for which the background fluorescence was determined
before adding DAF.
Activities of compounds toward fertilized sea-urchin egg cells were determined using the literature method [15].
Antimicrobial activity was determined by a diffusion method in agar in Petri dishes. The test cultures were the bacteria
Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and the yeast-like fungus Candida
albicans from the American Type Culture Collection (ATCC) and the Collection of Marine Microorganisms (KMM) of the
PIBOC FED RAS.
ACKNOWLEDGMENT
We thank O. S. Radchenko of the PIBOC FED RAS for help with synthesizing the acetyltryptamines. The work was
supported by grants of the RFBR No. 06-04-48578 and 05-04-48211; state contracts No. 02.445.11.7263, 02.452.11.7041, and
02.445.11.7280 of the Federal Agency for Science and Innovation of the RF Ministry of Education and Science; and a grant
of the FED RAS Presidium No. 06-III-A-05-121.
REFERENCES
1.
2.
3.
4.
5.
6.
J. W. Blunt, B. R. Copp, M. G. Munro, P. T. Northcote, and M. R. Prinsep, Nat. Prod. Rep., 22, 15 (2005).
W. Gul and M. T. Hamann, Life Sci., 422 (2005).
J. A. Joule, G. Smith, and K. Mills, Heterocyclic Chemistry, 3rd Ed., Chapman and Hall, New York (1994).
B. Riemer, O. Hofer, and H. Greger, Phytochemistry, 45, 337 (1997).
J. Li, G. Chen, and J. M. Webster, J. Nat. Prod., 58, 1081 (1995).
L. T. Tan, T. Okino, and W. H. Gerwick, J. Nat. Prod., 67, 952 (2000).
716

7.
T. A. Dvornikova, Microbiological Synthesis and Transformation of Indolic Compounds [in Russian], Shtiintsa,
Kishinev (1980).
8.
9.
10.
11.
Y. Shu-Wei and G. A. Cordell, J. Nat. Prod., 60, 44 (1997).
M. Shaaban, R. P. Maskey, I. Wagner-Dobler, and H. Laatsch, J. Nat. Prod., 65, 1660 (2002).
B. Rotman and B. W. Papermaster, Proc. Nat. Acad. Sci. USA, 55, 134 (1966).
O. I. Nedashkovskaya, S. Bum Kim, A. M. Lysenko, M. S. Park, V. V. Mikhailov, K. S. Bac, and H. Y. Park, Int. J.
Syst. Evol. Microbiol., 55, 1797 (2005).
12.
13.
14.
A. A. Lur′e, Chromatographic Materials (Handbook) [in Russian], Khimiya, Moscow (1978).
N. G. Prokof′eva, E. B. Shentsova, S. I. Stekhova, et al., Izv. Akad. Nauk, Ser. Biol., 493 (1999).
N. G. Prokof′eva, N. K. Utkina, E. L. Chaikina, and A. E. Makarchenko, Comp. Biochem. Physiol. B: Biochem, Mol.
Biol., 139, 169 (2004).
15.
N. G. Prokof′eva, M. M. Anisimov, N. M. Rebachuk, M. M. Kisileva, and N. D. Pokhilo, Izv. Akad. Nauk, Ser. Biol.,
645 (2002).
717