Amethystin, the coloring principle of Stentor amethystinus

Article abstract of DOI:10.1021/np5001363

Among the ciliates, Stentor amethystinus stands out for its conspicuous red-violet color compared to its blue- and red-colored relatives Stentor coeruleus and Blepharisma japonicum. Rich blooms in German lakes allowed us to collect sufficient organisms to isolate the pigments and elucidate the structure of the main component amethystin (4) by spectroscopic methods as a carboxy derivative of blepharismin. Depending on conditions, the carboxy group appears as an orthoester or as a mixture of the orthoester and small amounts of a hydroxylactone. Derivatives of both isomeric forms were obtained by acetylation and methylation supporting the proposed structures. On reaction of amethystin with base in the presence of oxygen, oxyamethystin and, under vigorous conditions, p-hydroxybenzoic acid were formed. In addition to 4, two homologues, an isomer of amethystin, and stentorin F (1b) were identified in the primary extract. Further, a biosynthetic scheme is proposed linking stentorin, blepharismin, and amethystin type compounds to the hypothetical protostentorin as a common intermediate.

Full text of DOI:10.1021/np5001363

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Amethystin, the Coloring Principle of Stentor amethystinus
Gerhard Hofle,*,† Silke Reinecke,† Uwe Laude,‡ and Dietrich Spitzner§
̈
†Helmholtz Center for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany
‡Institut fur Gewasserokologie und Binnenfischerei, Muggelseedamm 310, 12587 Berlin, Germany
̈
̈
̈
̈
̈
§Institut fur Bioorganische Chemie, Universitat Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
̈
S
* Supporting Information
ABSTRACT: Among the ciliates, Stentor amethystinus stands out for its
conspicuous red-violet color compared to its blue- and red-colored relatives
Stentor coeruleus and Blepharisma japonicum. Rich blooms in German lakes
allowed us to collect sufficient organisms to isolate the pigments and
elucidate the structure of the main component amethystin (4) by
spectroscopic methods as a carboxy derivative of blepharismin. Depending
on conditions, the carboxy group appears as an orthoester or as a mixture of
the orthoester and small amounts of a hydroxylactone. Derivatives of both
isomeric forms were obtained by acetylation and methylation supporting the
proposed structures. On reaction of amethystin with base in the presence of
oxygen, oxyamethystin and, under vigorous conditions, p-hydroxybenzoic
acid were formed. In addition to 4, two homologues, an isomer of
amethystin, and stentorin F (1b) were identified in the primary extract.
Further, a biosynthetic scheme is proposed linking stentorin, blepharismin, and amethystin type compounds to the hypothetical
protostentorin as a common intermediate.
and its stentorin-like structure was elucidated.11 Both compounds
Stentor and Blepharisma are heterotrich ciliates (protozoa) prevalent
were found as a mixture of homologues with R1, R2 = Et, i-Pr,
in lakes, ponds, and marine environments. Some species are brightly
R3 = H, Me. Surprisingly, the deep-red-colored marine ciliate
colored by pinkish, red, blue, violet, or brown pigments located
Pseudokeronopsis rubra contains an entirely different merocyanin-
like chromophore with pyrrole and pyrone end groups.12 Yet
another type of yellow-brown pigment is present in Loxodes
species.13 Its chemical nature is not known.
predominantly in granules that are arranged in stripes just below the
cell surface,1,2 The compounds responsible are structurally related
to hypericin, the coloring principle of St. John’s Wort (Hypericum
perforatum), which, most remarkably, is also stored in “granules”
embedded in the leaves and petals of the plant,3,4 and the fringelites,
fossil pigments from a Jurassic sea lily.4,5
All of these pigments appear to have two functions.14 They
are apparently deterrent, paralyzing, or even toxic for predators
such as other protozoa when they are ingested or released from
the granules (extrusomes) by mechanical stress.15−18 Experi-
ments with the isolated compounds indicated antimicrobial
activity14,19 and inhibition of the development of sea urchin
eggs17c and of the inward calcium transport of the ciliate
Euplotes.20 Another function of the pigments is that of photo-
receptors mediating the orientation of the organisms in a light
gradient.14,21 Whereas generally negative phototaxis was observed,
in the presence of algae as symbionts, positive phototaxis is also
observed to allow photosynthesis. Investigation of the photosignal
transduction pathway revealed the presence of stentorin associated
with a 50 kDa protein22 and of blepharismin associated with a
200 kDa protein.23 However, no further protein data have been
published so far. Other work indicates that there is no specific
blepharismin-binding protein.24 Thus, the primary signal,
protons released from the excited chromophore (photoacidity),25
Thus, stentorin C (1a), the blue pigment isolated from
Stentor coeruleus, has been identified as the 3,3′-diisopropyl
analogue6 of fringelite D5 with the alkylation pattern proven by
total synthesis.7 Maristentorin from Stentor dinoferus shows yet
another, though questionable, alkylation pattern.8a,b In contrast,
the pinkish blepharismin (2) from Blepharisma japonicum
resembles protohypericin with the gap in the ring system
bridged by a p-hydroxybenzylidene group.9,10 From cultures
kept in daylight, or on subsequent irradiation of blepharismins
(2) in the presence of oxygen, oxyblepharismins (3) were isolated,2
Received: February 12, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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singlet oxygen,26 or hydroxy radicals27 would have to reach the
target molecule by diffusion.
could be eliminated by filtration through a nylon stocking.
Surprisingly, HPLC analysis of the extracts showed a very low
yield of amethystin per specimen (3.3 ng) compared with the
bloom of 1995, which gave an estimated >10-fold higher yield.
Nevertheless, microscopic inspection of the organisms showed
the typical arrangement of dark pigment granules.
The electronic spectrum of 4 is very similar to that of
blepharismin (2),9 with only a 7 nm bathochromic shift of the
three bands in the visible region. The molecular weight was
consistently determined by FABMS and HRESIMS to be
724 mu, and the elemental composition, C42H28O12. This is two
hydrogen atoms less and one carbon and one oxygen atom
more than blepharismin C (2, R1, R2 = i-Pr, R3, R4 = H);11 thus,
amethystin (4) is a close relative of blepharismin C. From the
proton NMR spectra (Table 1) 4 is symmetrical with the
Table 1. NMR Spectroscopic Data of Amethystin (4) in
DMSO-d6 (75 and 300 MHz)
a
position
δC, type
δH (J in Hz)
HMBC
b
1
122.1, C
2
161.3, C
108.5, C
165.4, C
3
4
In general, the ciliates represent only a small percentage of
the total zooplankton, and organisms such as Blepharisma or
Pseudokeronopsis have to be cultivated to generate biomass for
in-depth chemical analysis or biosynthetic investigations by
precursor feeding.29 Fortunately Stentor, which is basically a
sessile organism that occurs only sparsely in the open water,
sometimes forms massive blooms that turn the water black.
Such a “black-spot” bloom of Stentor amethystinus was reported
in Lake Garda, Italy, in the summer of 2004.31 Our work on S.
amethystinus described below was initiated during such a bloom
in Germany in 1995. At that time we became interested in S.
amethystinus because of its conspicuous red-violet color, which
is not observed in other ciliates. In fact, the visible absorption
spectrum of organic extracts excluded the presence of a stentorin-
like chromophore, but showed similarity to blepharismins (2).
4-OH
5
14.19 s
14.74 s
3, 4, 5
7, 8, 9
107.3, C
182.0, C
100.1, C
164.5, C
6
7
8
8-OH
9
117.2, C
171.4, C
10
10-OH
11
120.0, C
b
12
124.1, C
b
13
124.7, C
b
14
126.9, C
15
57.3, C
16
118.5, C
16-OH
17
4.10 br s
RESULTS AND DISCUSSION
■
136.9, C
Large-scale collection from blooms in ponds in northeast and
southwest Germany using a plankton net produced a biomass
consisting of <95% of S. amethystinus. On contact of the wet
cell mass with organic solvents such as acetone, the pigments
are instantaneously released; they can be completely extracted
by EtOAc. Re-extraction of the brown cell debris with various
solvents, including extraction after previous acid or base hydro-
lysis, did not produce any more coloring matter. The organic
extracts were preseparated by Sephadex LH-20 chromatog-
raphy or by precipitation from ether solutions with petroleum
ether, followed by silica gel chromatography. Final purification
was achieved by RP-18 chromatography to yield 755 mg of
amethystin (4) as a dark red, crystalline powder. Solutions in
MeOH/H2O were red, but changed to red-violet at pH 8.
Contrary to blepharismin,30 amethystin solutions in MeOH
were perfectly stable when exposed to sunlight. The
cytotoxicity of 4 against the mouse fibroblast cell line L-929
was very low, with an MIC of 37 μg/mL in the dark; however,
this value decreased to 4 μg/mL in the presence of light, due to
a photodynamic effect.31
c
18
127.8, CH
114.3, CH
155.9, C
6.11 s
20
20
c
19
6.11 s
20
20-OH
21
8.92 br s
2.22 s
8.6, CH3
16.4, CH2
13.3, CH3
2, 3, 4
22
2.76 q (7,0)
1.14 t (7,0)
8, 9, 10, 23
22, 9
23
a
HMBC correlations are from the proton(s) stated to the indicated
b
c
carbon. Tentative assignment. In pyridine-d5: 6.53 (d, J = 7.5 Hz),
6.84 (d, J = 7.5 Hz).
isopropyl groups replaced by two ethyl groups tentatively
located at C-9, C-9′ and two methyl groups at C-3, C-3′. The
four protons of a p-hydroxyphenyl group appear as a singlet at
6.11 ppm but are split into two doublets at 6.53 and 6.84 ppm
in pyridine-d5. Four hydroxy protons in peri positions are
observed at 14.19 and 14.74 ppm and a phenylhydroxy is
observed at 8.92 ppm, whereas hydroxy signals from the bay
region are not observed as described also for blepharismin
(2a).9 An additional broad hydroxy signal at 4.10 ppm was also
present. The assignment of the carbon NMR spectrum was
greatly facilitated by comparison with that from 13C-labeled
Later collections from a small water basin in Darmstadt
(Germany) in the summer and fall of 2013 gave only low yields
of S. amethystinus together with an excess of copepods, which
B
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Figure 1. Possible structures of amethystin (4).
Figure 2. Chemical derivatives of amethystin (4).
blepharismin (2a).29 A significant shift was observed for the
benzylic methine carbon from 32 ppm in blepharismin to
57.3 ppm in amethystin (4). The C-15 proton in blepharismin
is replaced in 4 by an extra quaternary carbon observed at
118.5 ppm. This together with the elemental composition leads
tentatively to the orthoester structure A for amethystin (Figure 1).
It is corroborated by an IR spectrum showing only quinone
carbonyl signals. Disturbingly, with several other samples of 4, a
carbonyl band of low and variable intensity was observed at
1798 cm−1, an absorbance later found in the lactone derivative
5b (see below). We believe that, depending on the conditions
of purification and crystallization, a variable small proportion of
the lactone isomer B is formed. Similar orthoesters are well
known from terpenoid natural products where hydroxy and
carboxy groups in favorable steric alignment are present, as is
the case with amethystin. A well-known example is tetrodotox-
in, which has also been observed as the orthoester or the
hydroxylactone.32 Another ambiguity arose with the elemental
analysis, which for C, H, and O agreed very well with the free
acid C but not with A and B. However, because the IR did not
show a carboxy group, the crystalline amethystin analyzed must
be a monohydrate of A.
that in the 1H NMR spectrum showed doubling of the alkyl and of
most of the acetyl signals. Due to the H NMR shift difference
1
between the two C-methyl groups of 0.1 ppm, the asymmetry
must be located in the 3,3′ position. This is in good agreement
with the lactone structure 5a, which is formally derived from the
amethystin isomer B. Methylation with diazomethane gave
trimethyl ether 5b and as side products an isomer 6a and
tetramethyl ether 6b. The trimethyl ether 5b was characterized in
detail by 1H, 13C, and HMBC NMR spectra (Table S1). As with
the octaacetate, most proton and carbon signals were doubled.
The lactone group was proven by an IR band at 1796 cm−1 and
the chemical shift of 174.2 ppm for C-16. The assignment of
carbon signals was greatly facilitated by comparison with the data
from 13C-labeled blepharismin21 and by the HMBC spectrum. The
four hydroxy groups in peri positions are expectedly hydrogen
bonded, giving four individual signals in the range 13 to 14 ppm,
which show far ranging correlations with all carbons of the ring
system with the exception of those in the center part, C-1, C-1′, C-
11 to C-14, and C-11′ to C-14′. A free hydroxy group at C-20 is
indicated by a hydroxy signal at 8.29 ppm and the chemical shift of
156.2 ppm for C-20. This was proven by degradation in melting
KOH to give p-hydroxybenzoic acid. Thus, the O-methylation
must have occurred in the bay positions at C-10, C-10′, and
C-2′, which is corroborated by their HMBC correlation with the
O-methyl protons. The isomeric trimethyl ether must have the
orthoester structure 6a, as it shows a single set of NMR signals and
no lactone carbonyl. The third methoxy group, according to its
chemical shift of 3.24 ppm, and that of C-20 (155.8 ppm), are at
C-16. Another byproduct of methylation is tetramethyl ether 6b.
Its structure is nearly identical to 6a with the difference that the
C-20 hydroxy is also methylated (3.68 ppm).
Three minor biosynthetic byproducts of amethystin (4) were
partially characterized by HPLC/HRMS and proton NMR
spectra. They are lower and higher homologues by CH2 with an
1
undefined substitution pattern, whereas an isomer in the H
NMR spectrum in addition to ethyl and methyl groups shows
signals for an isopropyl group and an aryl hydrogen and, thus,
has the same substitution pattern as blepharismin BL-4/D.9,11
Another regular polar component in primary extracts amounting
to 3−6% of amethystin was identified by HPLC/UV/HRESIMS
as stentorin F (1b). It has the same elemental composition as
stentorin C (1a) from S. coeruleus;7 however, it has the same
In order to obtain a salt of the hypothetical free acid 4C and
take NMR spectra, 4 was dissolved in aqueous 3 N sodium
hydroxide. Immediately a deep blue color developed; however,
no defined NMR signals could be observed, indicating that a
radical reaction was occurring. After 20 h the reaction mixture
was acidified and extracted with EtOAc. Two predominant
products, a blue-violet one (7) and a red one, were observed by
1
substitution pattern as amethystin (4) according to the H NMR
data.
After attempts to prepare crystals of sufficient quality for an
X-ray structure analysis failed, independent proof of structure 4
was sought by derivatization. Thus, acetylation gave an octaacetate
C
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Scheme 1. Proposed Biosynthesis of the Ciliate Pigments Stentorin, Blepharismin, and Amethystin via the Hypothetical
a
Intermediate Protostentorin
a
R1, R2 = Et, i-Pr; R3, R4 = H, Me.
HPLC/MS and separated by preparative HPLC. For 7
HRESIMS gave a molecular weight of 667.1624, indicating an
elemental composition of C42H28O10, which corresponds to loss
of C2O2 compared to amethystin (4). The proton NMR
spectrum shows asymmetry in the benzodianthrone system and
low field signals for one of the C methyl groups (2.39 ppm)
and for the C-18 protons of the hydroxyphenyl group. From
that, stentorin-related structure 7 is derived, in which one of the
bay hydroxy groups is replaced by the p-hydroxyphenyl moiety.
For the second compound HRESIMS showed a molecular
weight of 739.1473, corresponding to an elemental composition
of C42H28O13. This is one oxygen more than the educt, and
consequently the compound was named oxyamethystin. Due to
the lack of material, only proton NMR spectra could be measured,
showing four conspicuous broad signals in the range 5.60 to 8.00
for the p-hydroxyphenyl group. From that, oxyamethystin is not
structurally related to oxyblepharismin, which is characterized by a
well-resolved AA′BB′ system at 6.45 and 6.65. Differing from
oxyblepharismin, the hydroxyphenyl in oxyamethystin must be
locked in an asymmetric position above the naphthodianthrone
ring system exposed to strong shielding and deshielding effects. Its
structure may be related to that of amethystin octaacetate (5a) and
trimethyl ether (5b), which show similar, however less
pronounced, shielding effects for the p-hydroxyphenyl protons.
The biosynthesis of the Stentor and Blepharisma pigments
appears to be closely related to that of hypericin. Both are
polyketides, which by dimerization of intermediate anthrones
form symmetrical naphtho- and benzodianthrone systems.
However, on closer inspection, profound differences turn up.
Whereas emodin33 and emodinanthrone,34 the supposed bio-
synthetic intermediates to hypericin, dimerize selectively through
the more activated dihydroxyphenyl moieties to protohypericin,
such selectivity is not available for an analogous intermediate to
stentorin (1), blepharismin (2), and amethystin (4) type
compounds (Scheme 1). Both phenyl rings of a corresponding
13,13′-bisanthranol would be equally active in radical or oxidative
coupling, resulting in isomeric mixtures with respect to the position
of the ethyl or isopropyl groups. Such isomers have never been
observed with 1, 2, and 4. To achieve this strict selectivity in the
dimerization, the corresponding enzyme must differentiate between
ethyl/isopropyl and hydrogen in both building blocks to be
coupled. After the second ring closure through C-11, and optionally
methylation at C-3 and C-3′, the so far hypothetical protostentorins
would be formed as a central branching point for further
modifications. Whereas protostentorin undergoes another ring
closure to give the stentorins, it is condensed with p-hydro-
xyphenylglyoxylic acid (or a biochemical equivalent) to give the
amethystins in S. amethystinus. These, by decarboxylation in
B. japonicum, eventually lead to the blepharismins. Interestingly, by
HPLC/MS analysis of extracts from B. japonicum and
S. amethystinus only small amounts of the corresponding stentorins,
but no protostentorin, could be detected. No amethystin was
detected in B. japonicum, and no blepharismin in S. amethystinus.
In conclusion, we have achieved the isolation of amethystin
in a crystalline state, which is responsible for the red-violet
coloration of S. amethystinus. Its benzodianthrone substructure
was elucidated by spectroscopic measurements as a carboxy
derivative of blepharismin (2) from B. japonicum. According to
D
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in May 1996. The suspension of the collected organisms in pond water
was filtered through a bed of Celite (8 × 30 cm). The column was
washed with H2O (2 L) and eluted with acetone (3 L) to yield a dark
red aqueous acetone fraction, which was concentrated in vacuo at
30 °C. The dark-colored, oily residue was lyophilized. The residue was
triturated with diethyl ether, and the suspension formed was diluted
with petroleum ether, chilled, and filtered. This procedure was
repeated four times to give 5.0 g of crude material, which was further
purified and separated by HPLC. A 4.4 g portion of the crude material
was separated in five batches on Lichroprep Si100, 15−25 μm silica gel
(70 mL) with acetone as eluent. The dark red fraction was collected
to give 1.55 g of crude 4. Final purification was achieved by RP18
chromatography in two batches on HD-SIL 35−70 μm (column
83 × 9 cm, MeOH/10 mM NH4OAc buffer, 9:1) to give 740 mg of
amethystin (4).
Amethystin (4): dark red to black needles (EtOAc); slow de-
composition above 200 °C without melting; UV (MeOH) λmax 579
(4.87), 538 (4.73), 494 (4.70), 355 (sh) (4.80), 333 (4.98), 287
(5.07), 254 (5.22), 224 (5.31) nm; after addition of base: 580, 539,
491 427, 339, 287, 257, 225 nm; after addition of acid: 575, 539, 486,
403, 354, 330, 285, 252, 225 nm; IR (KBr) νmax 1798 (weak to very
weak), 1614, 1590, 1419 cm−1; 1H and 13C NMR see Table 1; ESIMS
(−) m/z 723; HRESIMS (+) m/z 725.1672 (calcd for C42H29O12,
725.1659); anal. 67.92; H 4.07; O 28.00%, calcd for C42H30O13, C
67.99; H 3.80; O 27.96%; TLC Rf = 0.48 (silica gel, EtOAc/MeOH/
NMR measurements amethystin (4) exists in solution
exclusively as an orthoester, whereas in the crystal and in the
solid state the isomeric hydroxylactone form may be present to
a moderate extent. To prove independently the orthoester and
hydroxylactone structures, O-acetyl and O-methyl derivatives
were prepared and characterized by spectroscopy. Reaction
with base in the presence of oxygen produced oxyamethystin
of unknown structure and a degradation product carrying a
p-hydroxyphenyl residue at C-2.
S. amethystinus collected from rich blooms was found to
contain high concentrations of amethystin (4), whereas sparingly
occurring organisms appear uncolored and contain more than
10-fold less pigment. From that, it could be speculated that the
very small amounts of pigments are localized in the cilia28 responsible
for photodetection, while larger amounts are concentrated in
the granula during a bloom for defense.
Beyond that, a consistent biosynthetic scheme is proposed
including the ciliate pigments, stentorin, blepharismin, and
amethystin. As central intermediate, protostentorin is postulated
in analogy with the biosynthesis of hypericin via protohypericin.
The formation of protostentorin from two heptaketides by
dimerization is a highly regioselective process, as only the
smaller substituent H is found in the 3,3′-positions. Thus, the
corresponding enzyme must provide an extended recognition
site for both anthrone units. Apart from the search for early bio-
synthetic intermediates, genetic analysis of the biosynthetic
machinery in the three species will give a clue to this mechanism
and, moreover, explain the further species-specific modifications.
H2O, 80:15:10), Rf = 0.33 (RP-18, MeOH/H2O, 8:2); HPLC tR
=
6.15 min (Nucleosil C18, 4 × 250 mm, MeOH/10 mM NH4OAc
buffer, 85:15, diode array detection).
(c) Stentor amethystinus was collected from a small pond at the
Darmstadt University campus (49°52′9.1″ N, 8°40′51.5″ E) in the
summer and fall of 2013 and separated from an excess of copepods by
filtration through a nylon stocking. For analytical purposes a
suspension of approximately 20 000 organisms was filtered through a
bed of Celite and extracted as described above. HPLC/HRESIMS
analysis (WatersXBridge C18, 3,5 μm, 2.1 × 100 mm column, gradient
MeOH/5 mM NH4OAc buffer, 1:1, to 100% MeOH/5 mM NH4OAc
buffer in 20 min at 0.6 mL/min) revealed in addition to amethystin
(66 μg, tR = 13.5 min, 100%) three minor components with the
chromophore of amethystin. They were identified as lower (tR = 11.1
min, 1%) and higher (tR = 14.9 min, 3%) homologues and as an
isomer of amethystin (tR = 12.4 min 5%). For ESIMS data see
Supporting Information. A semipure sample of the isomer from the
large-scale isolation showed the following: 1H NMR (CD3OD) δ 1.28
(t, J = 7.5 Hz, 3H), 1.50 (d, J = 7.5, 3H), 1.55 (d, J = 7.5, 3H), 2.50 (s,
3H), 2.95 (br q, 2H), 3.91 (m, 1H), 5.80 (br s, 1H), 5.97 (br s, 1H),
6.30 (br s, 1H), 6.46 (br s, 1H), 6.75 (s, 1H).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined with a Buchi 510 apparatus. UV spectra were recorded
̈
on a Shimadzu UV-2102 PC scanning spectrometer; IR spectra, with a
Nicolet 20DXB FT-IR spectrometer. NMR spectra were recorded on
a Bruker ARX 300 or ARX-400 spectrometer. EI and DCI mass
spectra (reactant gas ammonia) were obtained on a Finnigan MAT 95
spectrometer, with high-resolution data acquired using peak matching
(M/DM = 10 000). ESI mass spectra were obtained with a Q-Tof II
from Micromass. HPLC/ESI/APCIMS: PE Sciex Api-2000 LC/MS.
HRESIMS data were recorded on a Maxis spectrometer (Bruker
Daltonics). Analytical RP HPLC was carried out on an Agilent 1100 HPLC
system equipped with a UV diode-array detector. HPLC conditions:
Nucleodur C-18, 5 μm, 2 × 125 mm column (Macherey-Nagel), solvent A:
5% MeCN in 5 mM of NH4Ac buffer; solvent B: 95% MeCN, 5 mM of
NH4Ac buffer, pH 5.5; gradient 5:95 to 95:5 in 30 min. For analytical TLC
aluminum sheets coated with silica gel Si 60 F254 and for preparative TLC
precoated silica gel Si 60 F254 plates of 0.25 and 0.5 mm layer thickness
(Merck) were used.
A more polar compound (tR = 2.4 min 6%) by UV and molecular
mass was identified as stentorin F (1b). It was isolated from the prerun
of the above separation (b) by two consecutive RP HPLC runs, first
with MeOH/50 mmol NH4OAc buffer, 9:1, then MeCN/H2O, 96:4,
and 0.1% TFA to give 16 mg of stentorin F (1b): TLC Rf = 0.62
(EtOAc/MeOH/H2O, 80:15:10); UV (MeOH) λmax 602, 551. 463,
1
437 sh, 342, 329, 296, 242 sh, 225 nm; H NMR (DMSO-d6) δ 1.20
Isolation of Amethystin (4). (a) Stentor amethystinus and
concomitant plankton were collected from the lake “Große Fuchskule”
in Neubrandenburg, Germany (53°06′20.6″ N, 12°59′05.2″ E) in
August 1995. The organisms were flushed with water from the
plankton net (60 μm mesh size) and immediately extracted by the
addition of four volumes of acetone, 1.92 L in total. From the
absorption at 580 nm the presence of 10 mg of amethystin was
estimated. The extract was concentrated in vacuo to give a dark yellow
water phase and a brown precipitate, which were extracted with
EtOAc. The organic layer was dried with Na2SO4 and evaportated to
dryness to give 380 mg of extract. A second batch of organisms yielded
250 mg of extract. Separation of the combined extracts on Sephadex
LH-20 (column 4 × 87 cm, MeOH) gave a dark red fraction of crude
amethystin (31 mg). Final purification was achieved by preparative
HPLC (Nucleosil RP18, column 2 × 25 cm, MeOH/5 mM NH4Ac
buffer 8:2), yielding 15 mg of 4 as a dark red, microcrystalline powder.
(b) Stentor amethystinus was collected from a small pond near
Ostrach, Germany (47°55′14.6″ N, 9°23′56.0″ E) as described above
(t, J = 7.5, 23-H3), 2.37 (br s, 21-H3), 2.50 (q, J = 7.6 Hz, 22-H2),
14.50 (br s, 4-OH, 8-OH); ESIMS (−) m/z 591 [M − H]− (100);
HRESIMS (+) m/z 593.1438 [M + H]+ (calcd for C34H25O10,
593.1448).
Derivatization. Amethystin Octaacetate (5a). (a) Amethystin
(4 mg) was dissolved in acetic anhydride (0.5 mL) and 13 μL of
concentrated sulfuric acid and heated for 1 min to 90 °C. Some
sodium acetate was added, and the solution evaporated to dryness.
The residue was distributed between EtOAc and phosphate buffer
pH 7. From the organic phase 1.1 mg of 5a was isolated by preparative
TLC (CH2Cl2/acetone, 95:5). (b) Amethystin (40 mg) was acetylated
with acetic anhydride (1 mL) and pyridine (0.5 mL) at room
temperature (rt). The reaction product was evaporated in vacuo at rt,
and the residue was dissolved in 5 mL of toluene and evaporated again.
This procedure was repeated four times. Then the residue was
dissolved in 3 mL of toluene/CH2Cl2 (10:1) and filtered through a
pad of kieselguhr, and the filtrate was evaporated to dryness. The yield
E
dx.doi.org/10.1021/np5001363 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
of 40 mg of crude peracetate was separated by preparative TLC
5.58 (br s, 1H), 6.31 (br s, 1H), 7.12 (br s, 1H), 8.00 (br s, 1H);
HRESIMS (−) m/z 739.1473 [M − H]− (100) (calcd for C42H27O12,
739.1452).
(CH2Cl2/ MeOH, 95:5) to give 11 mg of 5a as a dark yellow solid:
1
TLC Rf = 0.44 (CH2Cl2/acetone, 95:5); H NMR (CDCl3) δ 1.12
(t, J = 7.5, 3H), 1.17 (t, J = 7.5, 3H), 2.00 (s, 3H), 2.10 (s, 3H), 2.22
(s, 3H), 2,33 (s, 3H), 2.41 (s, 3H), 2.42 (s, 3H), 2.44 (s, 6H), 2.50
(s, 3H), 2.52 (s, 3H), 3.55−3.58 (m, 4H), 6.11 (br d, 1H), 6.41 (br d,
1H), 6.57 (br d, 1H), 6.68 (br d, 1H); EIMS m/z 1020 [M − 40]+
(66), 978 (64), 936 (70), 894 (100), 852 (73), 810 (48), 768 (100);
ESIMS (+) m/z 1083 [M+ Na]+ (100).
Amethystin Tri- and Tetramethyl Ether (5b, 6a, 6b). A 50 mg
amount of amethystin was added to 3 mL of diazomethane in diethyl
ether. To solubilize the amethystin, some MeOH and EtOAc were
added. After 10 min the solvents were evaporated and the residue was
separated into two batches by HPLC (Nucleosil 100, 7 μm, column
2 × 25 cm, CH2Cl2/petroleum ether, 95:5, detection 254 nm). The
center fraction (46 mg) was separated again to give pure 5b (22 mg)
as a dark red solid. A side fraction gave 3.4 mg of isomeric trimethyl
ether 6a, and another side fraction by preparative TLC (diethyl ether/
petroleum ether, 1:1) gave tetramethyl ether 6b (∼1 mg) and a
pentamethyl derivative (∼1 mg) of unknown structure.
ASSOCIATED CONTENT
* Supporting Information
The UV/vis spectrum of amethystin (4), analytical HPLC/UV/
■
S
1
HRESIMS of extracts (a) and (b) of S. amethystinus, and H
and 13C NMR data and spectra of stentorin (1b), amethystin
(4), amethystin octaacetate (5a), and trimethyl ether (5b) are
presented. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Gerhard.Hoefle@helmholtz-hzi.de. Tel: +49(0)531-
611287.
■
Notes
5b: TLC Rf = 0.42 (CH2Cl2/MeOH, 98:2); UV (MeOH) λmax 565
(4.11), 531 (4.02), 486 (4.15), 416 (3.77), 367 (3.80), 282 (4.43), 246
(4.56) nm; IR (KBr) νmax 1796, 1630, 1585 cm−1; NMR spectroscopic
data see Table S2, Supporting Information; DCIMS (NH3) m/z 767
[M + H]+ (100), 444 (60), 150 (80); HREIMS m/z 766.2029 (calcd
for C45H34O12, 766.2050), 645.1788 (calcd for C38H29O10, 645.1761).
6a: TLC Rf = 0.60 (CH2Cl2/MeOH, 98:2); UV (CH3OH) λmax
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank M. Plewka (plingfactory) for providing the
microscopic picture of S. amethystinus, Prof. H. Iio (Osaka
City University) for a sample of synthetic stentorin C, and F.
Sasse (HZI) for toxicity testing. Spectroscopic measurements
and large-scale isolation by C. Kakoschke, A. Teichmann, and
H. Steinmetz (HZI) are gratefully acknowledged. We further
thank S. Pinnow (Neuglobsow), D. Kramer (Darmstadt), and I.
Klein (University of Hohenheim) for their help with the
collection of S. amethystinus.
1
567, 531, 480, 363, 316 sh, 280 sh, 249, 226 nm; H NMR (CDCl3/
DMSO-d6, 8:2) δ 0.93 (t, J = 7.5 Hz, 6H), 1.97 (s, 6H), 2.5−2.8 (m,
4H), 3.02 (s, 6H), 3.24 (s, 3H), 5.88 (s, 4H), 13.40 (s, 2H), 13.94
(s, 2H); 13C NMR (CDCl3/DMSO-d6, 8:2) δ 185.6, 164.3, 163.8,
161.4, 157.3, 155.8, 127.6, 125.8, 125.2, 124.7, 123.4, 123.0, 118.7,
117.4, 108.4, 107.7, 105.2, 60.2, 55.9, 52.1, 16.1, 12.8, 7.5; HRESIMS
m/z 767.2115 [M + H]+ (100) (calcd for C45H35O12, 767.2128),
1533.4177 [2M + H]+ (33) (calcd for C90H69O24, 1533.4179).
6b: TLC: Rf = 0.67 (diethyl ether/petroleum ether, 1:1); UV
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