Keronopsamides, a new class of pigments from marine ciliates

Article abstract of DOI:10.1002/ejoc.200900905

New pigments with an unprecedented skeleton, named keronopsamides A-C, were isolated from, the marine ciliate Pseudokeronopsis riccii. The structure of the most abundant: secondary metabolite, keronopsamide A, was established through extended Nuclear Magnetic Resonance (NMR) analysis and mass spectrometric (MS) data obtained by using electrospray (ESI) and. matrix assisted laser desorption (MALDI) ionizations. Structures of the minor analogues (keronopsamide B and C) were inferred from 1H NMR, chemical correlation, and LC-MS measurements. The analysis of NOE dipolar couplings and quantum chemical calculations, carried out by density functional theory (DFT) on the pre-ferred, rotamers of keronopsamide A, suggested a fully planar structure with, amide bond, in anti stereochemistry capable to break down the extended conjugation of the whole π electronic system. Although the presence of a 2-substituted 3,4dibromo-pyrrole moiety is reminiscent of previously isolated metabolites isolated from, a cell extract of Pseudakeronopsis rubra (keronopsins), keronopsamides show a new molecular skeleton formally derived from a peptide-like condensation of a 3-pyrrolepropenoic acid with an unsaturated bromotyramine.

Full text of DOI:10.1002/ejoc.200900905

FULL PAPER
DOI: 10.1002/ejoc.200900905
Keronopsamides, a New Class of Pigments from Marine Ciliates
Graziano Guella,*[a,b] Rita Frassanito[a] Ines Mancini[a] Tommaso Sandron,[a]
Letizia Modeo,[c] Franco Verni,[c] Fernando Dini,[c] and Giulio Petroni[c]
Keywords: Natural products / Marine ciliates / Pseudokeronopsis riccii / Cortical granules / Keronopsamides / Dyes/pig-
ments / Structure elucidation
New pigments with an unprecedented skeleton, named
keronopsamides A–C, were isolated from the marine ciliate
Pseudokeronopsis riccii. The structure of the most abundant
secondary metabolite, keronopsamide A, was established
through extended Nuclear Magnetic Resonance (NMR)
analysis and mass spectrometric (MS) data obtained by using
electrospray (ESI) and matrix assisted laser desorption
(MALDI) ionizations. Structures of the minor analogues
carried out by density functional theory (DFT) on the pre-
ferred rotamers of keronopsamide A, suggested a fully planar
structure with amide bond in anti stereochemistry capable to
break down the extended conjugation of the whole π elec-
tronic system. Although the presence of a 2-substituted 3,4-
dibromo-pyrrole moiety is reminiscent of previously isolated
metabolites isolated from a cell extract of Pseudokeronopsis
rubra (keronopsins), keronopsamides show a new molecular
(keronopsamide B and C) were inferred from 1H NMR, chem- skeleton formally derived from a peptide-like condensation
ical correlation, and LC-MS measurements. The analysis of
NOE dipolar couplings and quantum chemical calculations,
of a 3-pyrrolepropenoic acid with an unsaturated bromotyr-
amine.
stances, which has been inferred from their toxic effect on
certain protists.[8] Toxicity of blepharismin and stentorin in
the light seems to be mainly related to their capabilities to
generate singlet molecular oxygen (1O2) both in situ and in
the medium. More recently, a novel pigment structurally
related to stentorin, called maristentorin, has been isolated
from the ciliate Maristentor dinoferus,[9] a benthic marine
species of coral reefs harbouring zooxanthellae.[10] Stentor
amethystinus is another ciliate which produces secondary
metabolites: six different mycosporine like amino acids
(MAAs)[11] have been found in a single strain of this mor-
phospecies collected from an Argentinian lake. MAAs have
also been found in M. dinoferus[12] and in several Chlorella-
bearing ciliates.[13]
In addition to stentorins and blepharismins, charac-
terized by the hypericin-like skeleton, ciliate taxa compris-
ing the cosmopolitan and ubiquitous genus Pseudokeron-
opsis (Spirotrichea, Stichotrichia)[14] produce a second class
of pigments,[15] called keronopsins 3–6 (Scheme 1), wherein
a β-bromine-substituted pyrrole is linked to a sulfate pyrone
through an extended conjugated chain. In particular, keron-
opsins have been isolated from cell cultures of the marine
morphospecies Pseudokeronopsis rubra.[15]
Introduction
Among Earth’s biota, secondary metabolites are largely
produced as expression of evolutionary adaptive purposes,
thus characterizing often entire taxa to such a degree to
represent reliable chemotaxonomic tools.[1] Our investi-
gations of various taxa comprising the marine ciliate Eu-
plotes, marked this protist as a fruitful source of new terpe-
noids,[2] among which euplotin C deserves particular men-
tion being involved in several interesting biological activi-
ties.[3] Even freshwater ciliate taxa produce secondary me-
tabolite: raw extracts of Tetrahymena pyriformis turned out
to contain interesting polycyclic triterpenes,[4] whereas
bioactive alkyl-resorcinols, referred to as “climacostol
family”, have been isolated from Climacostomum virens.[5]
In this context, the photoactive phenanthroperylene-
quinones stentorin (1) and blepharismin C (2) (Scheme 1)
isolated from the ciliates Stentor coeruleus[6] and Blepharisma
japonicum[7] are also noteworthy.
These pigments have been demonstrated to act as both
photoreceptors for abrupt light-avoidance responses (al-
though the signaling cascades leading to ciliary stroke re-
versal still remain an intriguing issue) and defense sub-
In this paper we report on the occurrence of pigments
belonging to a third unrecorded class, produced by the
newly described marine ciliate morphospecies, Pseudokeron-
opsis riccii (strain Oxsard2) collected from the Tyrrhenian
Sea. The structural elucidation of these new pigments,
named keronopsamides, are described and a possible bioge-
netic route for the biosynthesis of the most abundant sec-
ondary metabolite produced by P. riccii is given.
[a] Dipartimento di Fisica, Università di Trento,
via Sommarive 14, 38100 Povo (TN), Italy
Fax: +39-0461-882009
E-mail: graziano.guella@.unitn.it
[b] Istituto di Biofisica, CNR,
via alla Cascata 56/C, 38100 Povo (TN), Italy
[c] Dipartimento di Biologia, Università di Pisa,
via A. Volta 4/6, 56126 Pisa, Italy
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Figure 1. In vivo photomicrograph of Pseudokeronopsis riccii.
[keronopsamide A (7)], and two minor ones corresponding
to more polar metabolites absorbing at 379 nm [keronops-
amides B (8) and C (9)].
The most abundant metabolite keronopsamide A (7)
(Scheme 2) was obtained in pure form by following the
standard purification procedures we recently reported for
the isolation of ciliate metabolites.[2,3]
Scheme 1. Structures of pigments isolated from freshwater and ma-
rine ciliates.
Results and Discussion
The marine ciliate Pseudokeronopsis riccii shows an ellip-
tical body shape (ca. 225ϫ50 µm in vivo), with rounded
front and rear edges, the latter slightly narrower. The cell
body is very flexible and manifests a colored cortical sur-
face owing to the presence of embedded reddish circular
cortical granules ca. 1.0 µm in diameter, arranged in ro-
settes, small bunches, and rows docking the somatic and
oral ciliature. Cytoplasm includes few scattered colored
granules whose concentration does not enhance in the rear
body edge, as it occurs in other species of the genus. Scat-
Scheme 2. Structures of the new alkaloids keronopsamides A (7),
B(8), and C(9) isolated from Pseudokeronopsis riccii. Arbitrary
numbering on structure is followed in NMR assignments (see
Table 1 and Exp. Sect.)
Its molecular formula was established by MALDI-TOF
tered throughout the cytoplasm other colorless, circular, and ESI-MS measurements, the latter carried out through
cortical granules occur, ca. 1.5 µm in diameter, previously a coupled HPLC-MS system. In the negative ion-mode
called “blood cell-shaped granules” (Figure 1).
MALDI spectrum of 7 a clear 1:3:3:1 quartet at the nom-
We feel confident to exclude any symbiotic relationship inal masses of 487:489:491:493 was present, hence suggest-
of P. riccii, since the extensive transmission electron micro- ing in the putative parent ion [M – H]– the presence of 3
scope analysis of specimens comprising the experimental Bromine atoms. The same 1:3:3:1 intensity pattern, al-
strain Oxsard2 produced negative results. A detailed de- though shifted of two Da (489:491:493:495), was observed
scription of Pseudokeronopsis riccii will be available in a in the corresponding positive ion-mode ESI spectrum in
forthcoming paper (by Modeo et al.).
agreement with the expected parent ion as [M + H]+. More-
When the ethanol raw extract of the cells of Pseudokeron- over, the presence in the same mass spectrum of similar
opsis riccii (strain Oxsard2) was subjected to liquid clusters attributable to electrostatic sodium ion adducts ([M
chromatography coupled to both Diode Array and ESI-MS + Na]+ at m/z 511:513:515:517) and potassium ion adducts
detectors (HPLC-PDA-ESI/MS), the chromatogram ([M + K]+ at m/z 527:529:531:533), gives further support
showed one major peak strongly absorbing at 357 nm to the parent ion attribution. Finally, high resolution mass
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Keronopsamides, a New Class of Pigments from Marine Ciliates
measurements carried out by TOF analyzers (reflector to the stabilized acylium ion at the monoisotopic mass
mode, internal calibration) on the monoisotopic peak of the 276 Da via collision induced dissociation of the protonated
[M – H]– parent ion at m/z 487 provided the molecular com- amide bond (Scheme 3); as expected, this fragment ion
position of 7 as C15H11Br3N2O2 which implies ten degrees showed the characteristic isotopic cluster as a 1:2:1 triplet
of unsaturation. On the one hand, the analysis of 1D and due to the presence of two bromine atoms.
1
2D H NMR spectra revealed the presence of i) one trisub-
stituted 1,2,4 benzene ring conjugated to one disubstituted
carbon–carbon double bond, ii) one isolated disubstituted
carbon–carbon double bond, and iii) one isolated (amide or
ester) carbonyl group; on the other, 13C NMR and HMBC
measurements, revealed the presence of a trisubstituted pyr-
role ring (δH 7.20 J coupled with N-H exchangeable proton
at δH = 8.73), thus fulfilling the overall number of unsatura-
tions. The 2-bromo-3-hydroxy phenyl moiety (arbitrary
numbering as defined in Scheme 2) was established by the
δH of aromatic protons, by their characteristic J coupling
pattern (see Table 1), and by their correlation with the cor-
responding δC values from HSQC experiment. The HMBC
spectrum not only confirmed this partial structure, but also
allowed us to set up the substituent at position 6 as a disub-
stituted C(7)=C(8) double bond with E stereochemistry
(J7,8 = 14.7 Hz). Actually, C(7) shows 3J hetero-correlations
Scheme 3. The collisional activated dissociation (MS/MS) of the
protonated molecular ion (ESI, positive ion mode) of keronops-
amide A leading to the fragment ion at m/z 276.
both with H–C(1) and H–C(5) while C(6) resulted hetero-
correlated with H–C(8). However, the remaining
C(5Ј)=C(6Ј) E double bond (J5Ј,6’ = 15.8 Hz) must be linked
to the α position [C(4Ј)] of the pyrrole ring since H–C(6Ј)
shows 3J hetero-correlation with C(4Ј), whereas H–C(5Ј) re-
The structure of the minor keronopsamide B (8) was es-
sulted coupled to C(3Ј). Thus, the two moieties defined for tablished by MALDI-TOF high resolution mass spectro-
1
keronopsamide A must be joined together as reported in metric measurements and by the analysis of its H NMR
3
structure 7 (Scheme 2) on the basis of the strong J hetero- spectra. In particular, the molecular formula of keronops-
correlations of C(7Ј) with H-C(5Ј) on one side and with H- amide B (8), C15H10Br3N2O5S, was inferred by MALDI-
C(8) on the other. Taking into account the proton coupling TOF negative ion mass measurements, hence indicating the
pattern of H-C(8), the molecular formula, and the value of presence of a sulfate ester (80 Da mass shift). Remarkably,
the 13C resonance for C(7Ј), it was unambiguously estab- in both MALDI and ESI spectra of 8 we observed relevant
lished that the bridge between these two partial structures losses of sulfur trioxide neutral molecules leading to the
must be represented by an unsaturated amide bond. As ad- same mass spectrum previously described for 7. The batho-
ditional support, the ESI(+) MS/MS fragmentation of the chromic effect on the Visible band (shifted from 358 nm in
protonated molecular ion at the nominal mass 489 Da led 7 to 379 nm in 8) and NMR analysis suggest that the phen-
Table 1. NMR spectroscopic data of keronopsamide A (7) in CD3COCD3 at 298 K (1H at 400 MHz, 13C at 100 MHz).
C-Atom
δH (ppm), J [Hz]
δC (ppm) (HSQC)
Long-range H/C correlations
(HMBC)
Relevant proton dipolar
couplings (NOESY)
1
2
3
4
5
6
7
8
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
7Ј
HO-
7.48 d (2.1)
–
–
6.95 d (8.6)
7.26 dd (2.1, 8.6)
–
6.16 d (14.7)
7.53 dd (10.5, 14.7)
7.20 d (3.0)
–
–
–
130.9 d
111.0 s
153.8 s
117.8 d
126.5 d
131.8 s
111.7 d
124.0 d
122.9 d
101.5 s
104.4 s
128.2 s
129.5 d
118.4 d
163.9 s
–
C-3, C-5 and C-7
C-2, C-3 and C-6
C-1, C-3 and C-7
H-7
C-1, C-5 and C-8
C-6 and C-7Ј
C-2Ј, C-3Ј and C-4Ј
H-1, H-5 and –NHCO
–
7.56 d (15.4)
6.67 d (15.4)
C-3Ј, C-6Ј and C-7Ј
C-4Ј and C-7Ј
–NHCO
8.04 br. s
HNCO-
HN-pyrrole
9.55 d (10.5)
8.73 brd (3.0)
–
–
H-7, H-6Ј and H-5Ј
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olic OH group of 7 is esterified by sulfuric acid in structure bution in this compound, ab initio quantum chemical calcu-
8 as indicated by the strong downfield effect (about lation on both the tautomers was carried out by using
0.7 ppm) on H–C(4). The final proof was obtained by Hartree–Fock level of theory followed by Density Func-
NMR observation that sulfate ester 8 afforded, in quantita- tional (DFT) minimizations. In agreement with our experi-
tive yields and fast reaction times, pure 7 when treating its mental evidences, these calculations pointed out that 7 must
deuterated methanol solution with a small amount of tri- exist in only one tautomeric form (amidic form). Actually,
chloroacetic acid; this result is in agreement with the ex- by using the 6-311G basis set, DFT calculations suggested
pected hydrolytic instability of oxygen-arylsulfates in acidic this form at room temperature is about 18 kcal/mol more
media. Stereoisomeric structure 9 for keronopsamide C was stable than the “imidic” form, thus ruling out any contri-
1
ascertained mainly by its H NMR spectrum which was al- bution of the latter.
most superimposable to that of 8 with the exception for δH
Moreover, geometry minimization pointed out that
3
and J values of H–C(5Ј) and H–C(6Ј). These protons were keronopsamide A has a perfectly planar shape in all the
found as doublets (J5Ј–6Ј = 12.1 Hz, Z stereochemistry) at stable conformers adopted by 7 following rotation of 180°
δH = 6.76 and 5.75 respectively, in sharp contrast to the around every single bond of the chain. Dipolar couplings
corresponding downfield doublets (J5Ј-6Ј = 15.6 Hz, E as detected by NOESY experiments (Table 1) indicated that
stereochemistry) at δH = 7.52 and 6.50 for 8.
the amide-proton must be spatially close to H–C(7) and H–
C(6Ј); it follows that the anti conformation of the amide
bond must be much more populated than the syn confor-
mation since the expected NOE effect between NH and H–
Tautomers and Conformational Equlibria
Due to the stabilization of the whole electronic structure C(6Ј) has not been found in the NOESY spectra of 7. The
of keronopsamide A through the extended π electrons con- observed NOE effects ruled out also the contributions of
jugation, the molecule would be somehow expected to the conformer obtained by 180° rotation around the C8–N
adopt an “imidic acid” tautomeric form in place of the clas- single bond (dihedral angle C7–C8–N–C7Ј near zero) be-
sical “amide form” as above described for 7. Nevertheless, cause therein a strong NOE of NH with H-8 would have
structure 7 exists only in the “amide form” as firmly estab- been expected. On the other hand, a small contribution
lished by several experimental evidences. First of all, in per- must be represented by conformers obtained through 180°
deuterated dry acetone the tautomeric proton appears as a rotation around the C6Ј–C7Ј single bond (7c and 7d in
broad doublet (JNH-H8 = 10.3 Hz) due to the coupling of Scheme 4) because the NH amidic proton shows a small
the nitrogen-linked proton with the “vicinal” H–C(8), an but detectable dipolar coupling with H–C(5Ј).
outcome which cannot be explained for the “imidic oxygen-
Concerning the existence of conformational isomers for
linked proton” in the alternative tautomer. Furthermore, rotation around the C6–C7 bond, the strong and almost
the presence in the infrared spectrum of a strong absorption equally intense NOE effects of H-7 with H-1 and H-5 speak
at 1640 cm–1 is attributable to classical amide II band. Fi- for almost populated conformers 7a and 7b. Finally, the
nally, the relatively low wavelength value (350 nm) of the absence of any detectable dipolar coupling of the pyrrole
electronic absorption is not in agreement with an extended NH with both H–C(5Ј) and H–C(6Ј) is a strong evidence
conjugation of the whole 16π electron system expected for that this proton undergoes a fast chemical exchange which
the imide form. In order to understand the electronic distri- hinders the occurrence of any dipolar nuclear contact.
Scheme 4. Optimized geometry and conformational equilibrium of the most stable rotamers, 7a–7d, of keronopsamide A.
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Keronopsamides, a New Class of Pigments from Marine Ciliates
Scheme 5. Proposed biogenetic Scheme for the biosynthesis of keronopsamide A isolated from marine ciliates of the genus Pseudokeron-
opsis.
Thus, from our experimental evidences, only four con- amide A, which actually adsorbs at wavelengths (maximum
formers for keronopsamide A are present in solution at λ = 350 nm), lower than expected for a chromophore
(Scheme 4): the two major conformers 7a and 7b due to consisting of eight conjugated double bonds.
rotation around the C(6)–C(7) bond, and the two minor
conformers 7c and 7d due to rotation around the C(6Ј)–
C(6Ј) bond. The relative weight of the four rotamers was
Biogenetic Considerations
estimated by integration of the relevant NOESY maps lead-
The biosynthesis of keronopsamide A may be supposed
(Scheme 5) to occur from condensation of the amine group
of 3-bromotyrosine 11 (in turn derived from the electro-
philic bromination of tyrosine 10) with the β,βЈ-dibromi-
nated pyrrolepropenoic acid 13 (in turn obtained by bromi-
nation of 12), leading to the intermediate 14. Oxidative de-
carboxylation of the intermediate 14 eventually provided
keronopsamide A (7).
ing to the following molar ratio 7a/7b:(7c+7d)
=
0.54:0.38:(0.08).
In order to obtain a theoretical evaluation of these con-
formational equilibria, a full conformational search was
carried out at first with the molecular mechanics GMMX
program[16] allowing free rotation around the C1–C7, C8–
N, N–C7Ј, C4Ј–C5Ј, and C6Ј–C7Ј bonds. The minimized
rotamers were used as initial input structures; geometry was
thereafter refined first at HF/321G, and later at DFT/
B3LYP 6-31G level of theory.[17] Quantum-chemical calcu-
lations were in fair agreement with experiment results sug-
gesting not only that conformers with amide bond in syn
Conclusions
A new alkaloid pigment, keronopsamide A (7), and two
conformation were at least 5 kcal/mol higher in total energy stereoisomeric sulfate esters, keronopsamide B (8) and
than anti conformers but also that the contribution of rota- keronopsamide C (9), were isolated from a massive cell cul-
mers around C8–N was negligible being at least 12 kcal/mol ture of the marine ciliate Pseudokeronopsis riccii. Their
higher in energy than the corresponding 180° rotamers 7a- structures were elucidated combining the usual experimen-
d. Calculations supported the experimental evidences as tal approach relying on NMR spectroscopic data with
well, pointing out a similar weight for rotamers 7a and 7b; molecular mechanics and quantum chemical calculations.
in fact, two almost equally populated rotamers were evalu- Keronopsamides are the first example of secondary metab-
ated by our DFT calculations (difference of total energy olites so far isolated from marine and freshwater ciliates
between 7a and 7b: about 0.2 kcal/mol). Finally, for the belonging to a new, third class of pigments. These newly
analysis of rotamers around C4Ј–C5Ј, where the experiment discovered secondary metabolites join the first class pig-
could not indicate any preference, calculations suggested a ments based on the phenanthroperylenequinone skeleton
large prevalence (about 95%) of the conformer defined by (i.e. stentorins and blepharismins) and the few representa-
the torsional angle NH–C4Ј–C5Ј–C6Ј close to 180°.
tives of the second class pigments based on the keronopsin
In conclusion, NMR measurements and QM calcula- skeleton (i.e. keronopsins) already known.
tions suggest that keronopsamide A has an entirely planar
The new pigments herein described are very likely local-
structure and mainly exists in solution as a fast equilibrat- ized in the cell colored cortical granules, which is in line
ing 3:2 mixture of two C6–C7 rotamers 7a and 7b, as de- with the localization of the other cited pigments. Moreover,
picted in Scheme 4. According to DFT/B3LYP/6- any possibility of symbiont involvement in keronopsamide
311G+(2d,p) level of theory for 7a, the 16 π electrons are production could be excluded because extended TEM ob-
not allowed to delocalize along the whole molecule. Even if servation on cells did not provide any evidence of symbiotic
the amide bond has a significant double bond character, association.
the molecule appears to contain two isolated π electronic
Finally, another rather interesting result of this work,
clouds, with the amide bond acting as a boundary for the which deserves consideration by both ciliatologists and nat-
extended conjugation of the π electronic system. This is ural product chemists concerning perspectives of future
somehow reflected in the Visible spectrum of keronops- studies, is definitely the “secondary metabolite diversity”
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Molecular mechanics calculations were carried out by the com-
puter program GMMX as implemented in PCMODEL 7.0.[16] All
the minimized structures falling in a strain-energy window of
3.0 kcal/mol were saved and finally minimized with both MMX
and MM3 force fields keeping only those falling in a 2.0 kcal/mol.
discovered within the genus Pseudokeronopsis: actually, the
two solely up to now physiologically investigated species,
P. rubra and P. riccii, produce different pigments along dif-
ferent biogenetic routes. Studies are in progress to investi-
gate biological activities as well as the physiological/ecologi-
cal role of keronopsamides.
Ab initio calculations were performed with the Gaussian 03 pro-
gram suite[17] utilizing unrestricted hybrid density functional/mo-
lecular orbital theory with a Lee–Yang–Parr correlation func-
tional[18] a Becke 3-parameter exchange functional (for example,
B3LYP)[19] and basis sets of 6-311+G(d,p).[20]
Experimental Section
General Methods: NMR spectra (1H NMR, 13C NMR, DQCOSY,
NOESY, HSQC, and HMBC) for 7 were recorded on a Bruker–
Avance 400 MHz NMR spectrometer by using a 5 mm BBI probe
with 90° proton pulse length of 8 µs at a transmission power of
0 db. HPLC/DAD measurements were performed using a high-
pressure liquid chromatograph (Hewlett–Packard model HP1100
series, Agilent Technologies Sales & Services GmbH & Co. KG,
76337 Waldbronn, Germany). A Rheodyne 7725 (Rohnert Park,
CA, USA) injection valve equipped with a 20 µL internal loop for
the injections was used. The analyses were performed at room tem-
perature on a Agilent ZORBAX Eclipse XDB-C8 150ϫ4.6 mm,
3.5 µm; eluent: pump A CH3OH/NH4C2H3O2 28 m 70:30, pump
B CH3OH, gradient: 35–100% of B in 40 min, 0.8 mL min. The
photodiode array (PDA) detector (Agilent 1100 series) was set at
the wavelengths of 215, 254, and 450 nm. All solvents used were of
analytical grade (Merck, Darmstadt, Germany) or HPLC grade
(Riedel-de Haën–Sigma Chemical Co, St. Louis, MO, USA). LC/
ESI-MS analysis was made on reverse phase column (Agilent Zor-
bax eclipse XDB-C18 4.6ϫ150mm ϫ3.5 µm) with CH3CN/H2O
(Riedel-de Haën) 7:3, 0.9 mL/min, split UV/MS 7/3, λ 250 nm,
5 µL injected, mounted on a Hewlett–Packard HP1100 HPLC-UV
Diode Array system mated with an Esquire LC Bruker–Daltonics
ion trap mass spectrometer. Mass spectra were obtained with an
ESI source in negative-ion mode. MS conditions: source temp.
300 °C, nebulizing gas N2, 4 L/min, positive ion mode, ISV 4 kV,
OV 38.3 V, scan range 100–1000 m/z. To analyze the mass and UV
the following software has been used: DataAnalysis (Version 3.0,
Bruker Daltonik GmbH) and LC/MSD ChemStation (Agilent
Technologies), respectively.
Ciliate Strain Collection, Culturing, and Isolation of the Metabo-
lites: Pseudokeronopsis riccii (Spirotrichea, Stichotrichia) is a new
species whose formal description using a multidisciplinary ap-
proach (morphological-ultrastructural description plus 18S rRNA
gene sequencing) will be elsewhere reported (Modeo et al., in prep-
aration). A monoaxenic culture of the clonal strain Oxsard2 was
established under laboratory conditions since spring 1999. A single
specimen was isolated separately from a naturally occurring P. riccii
population, sampling marine tidal pools (1–2 m deep) along the
Tyrrhenian coast of Sardinia, near “Torre di Piscinni” (Cagliari,
Italy, N 38° 54Ј 15.57”, E 8° 46Ј 40.67ЈЈ). The analytical, clonal,
massive cultures were grown by feeding them twice a week on a
clonal line of the diatom Pheodactylym tricornutum (culture me-
dium, 33 ‰ salinity) and maintained in a laboratory incubator at
19Ϯ1 °C in a 12 h light/dark regimen (200 µmol photons m–2 s–1).
The massive cultures (ca. 2ϫ106 cells per liter) were frequently
cleaned by means of filtering (diameter of the gauze pores: 100 µm)
and bimonthly processed for secondary metabolite extraction as
hereby described. After a 5–7 d starving period, cells of 5–7 liter
massive cultures were harvested through the following two step pel-
letting procedure at room temperature: 1. some preliminary centri-
fugations at 200 g to lower the massive cultures to ca. 200 mL with-
out endangering the cells; 2. a single last centrifugation at 920 g to
reach a pellet varying from 0.3 to 0.6 mL. Then, cells were sus-
pended in absolute ethanol and stored at –20 °C. Through the
massive culturing up to 2.9 mL of closely packed, ethanol preserved
ciliates (ca. 5ϫ107 cells) were obtained for the secondary metabo-
lite analysis. Cells concentrated by filtration were extracted by etha-
nol at room temperature. The extraction was repeated until the
glass filter appeared colorless, then the extracts were combined,
reduced to dryness by rotary evaporation and then partitioned be-
tween ethyl acetate and water. Organic extract was initially purified
by flash-chromatography [RP-18 LiChroCART250-4 (5 µm
Merck), acetonitrile/water gradient elution]. Metabolites of inter-
est, contained in fractions 4–6 and 10–14 were further purified by
HPLC (Si-60 Merck LiChrosphere CN column, n-hexane/iPrOH,
gradient elution), giving pure 7 (tR = 7.6 min, 1.8 mg). Keronops-
amide B (8) and keronopsamide C (9) were obtained in almost pure
form by using RP-HPLC (Merck Lichrosphere RP18), gradient
MALDI-TOF measurements were performed on Bruker Daltonics
Ultraflex MALDI-TOF-TOF mass spectrometer equipped with a
reflector unit. The acceleration voltage was set at 20 kV. For de-
sorption of the components, a nitrogen laser beam (λ = 337 nm)
was focused on the template. The laser power level was adjusted to
obtain high signal-to-noise ratios, while ensuring minimal fragmen-
tation of the parent ions. All measurements were carried out in the
delayed extraction mode, allowing the determination of monoiso-
topic mass values (m/z; mass-to-charge ratio). After crystallization
at ambient conditions, both positive and negative ion spectra were
acquired in the reflectron mode, giving mainly singly protonated
molecular ions ([M + H]+) or sodiated adducts ([M + Na]+) and
deprotonated molecular ions ([M – H]–), respectively. Samples were
directly applied onto the stainless-steel spectrometer plate as 1 µL
droplets, followed by the addition of 1 µL of DHB-matrix solution
(0.5  of 2,5-DHB in methanol containing 0.1% TFA). Every mass
spectrum represents the average of about 100 single laser shoots.
elution with acetonitrile/ water from 3:7 to 7:3 in 30 min (tR
=
5.1 min, 0.1 mg for 8, and tR = 11.8 min, 0.2 mg for 9). In the same
chromatographic conditions the retention time of keronopsamide
A (7) is 18.1 min.
Keronopsamide A (7): Yellow amorphous solid. NMR spectroscopic
data (see Table 1, solvent: [D6]acetone). 1H NMR (CD3OD): δ =
3
Infrared spectra were recorded by using a FT-IR Equinox 55
Bruker spectrometer (ATR configuration) at 1 cm–1 resolution in
7.90 (s, 1 H, NHCO), 7.50 (d, J5Ј,6Ј = 15.8 Hz, 1 H, 5Ј-H), 7.44
3
(d, J1,5 = 2.1 Hz, 1 H, 1-H), 7.43 (d, J7,8 = 14.7 Hz, 1 H, 8-H),
the absorption region ∆ν 4000–1000 cm–1. A thin solid layer is ob-
7.19 (dd, J1,5 = 2.1, J4,5 = 8.5. Hz 1 H, 5-H), 7.04 (s, 1Ј-H), 6.83
˜
3
tained by evaporation of a methanol solution of keronopsamide A.
The instrument was purged with a constant dry air flux and clean
ATR crystal as background was used. Spectra processing was made
using Opus software package.
(d, J4,5 = 8.5 Hz, 1 H, 4-H), 6.49 (d, J5Ј,6Ј = 15.8 Hz, 1 H, 6Ј-H),
3
6.18 (d, J7,8 = 14.7 Hz, 1 H, 7-H) ppm. 13C NMR (CD3OD): δ =
165.8 (s, NHCO), 154.2 (s, C-3), 131.8 (s, C-6), 131.2 (d, C-1), 131.1
(d, C-5Ј), 127.7 (s, C-4Ј), 126.5 (d, C-5), 123.1 (d, C-8), 122.8 (d,
432
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Eur. J. Org. Chem. 2010, 427–434

Keronopsamides, a New Class of Pigments from Marine Ciliates
D. Cervia, D. Martini, M. Garcia-Gil, G. Di Giuseppe, G.
Guella, F. Dini, P. Bagnoli, Apoptosis 2006, 11, 829–843; c) D.
Cervia, M. Garcia-Gil, E. Simonetti, G. Di Giuseppe, G.
Guella, P. Bagnoli, F. Dini, Apoptosis 2007, 12, 1349–1363; d)
P. Ramoino, C. Usai, S. Maccione, F. Beltrame, A. Diaspro,
M. Fato, G. Guella, F. Dini, Aquat. Toxicol. 2007, 85, 67–75.
I. Abe, M. Rohmer, J. Chem. Soc., Chem. Commun. 1991, 902–
903.
M. E. Masaki, T. Harumoto, M. N. Terazima, A. Miyake, Y.
Usuki, H. Iio, Tetrahedron Lett. 1999, 40, 8227–8229.
N. Tao, M. Orlando, J. Hyon, M. Gross, P. Song, J. Am. Chem.
Soc. 1993, 115, 2526–2528.
a) G. Checcucci, R. S. Shoemaker, E. Bini, R. Cerny, N. Tao,
J. Hyon, D. Gioffre, F. Ghetti, F. Lenci, P. Song, J. Am. Chem.
Soc. 1997, 119, 5762–5763; b) M. Maeda, H. Naoki, T. Mats-
uoka, Y. Kato, H. Kotsuki, K. Utsumi, T. Tanaka, Tetrahedron
Lett. 1997, 38, 7411–7414; c) D. Spitzner, G. Höfle, I. Klein, S.
Pohlan, D. Ammermann, L. Jaenicke, Tetrahedron Lett. 1998,
39, 4003–4006.
a) M. E. Masaki, S. Hiro, Y. Usuki, T. Harumoto, M. N. Teraz-
ima, F. Buonanno, A. Miyake, H. Iio, Tetrahedron 2004, 60,
7041–7048; b) Y. Muto, T. Matsuoka, A. Kida, Y. Okano, Y.
Kirino, FEBS Lett. 2001, 508, 423–426.
P. Mukherjee, D. B. Fulton, M. Halder, X. Han, D. W. Arm-
strong, J. W. Petrich, C. S. Lobban, J. Phys. Chem. B 2006, 110,
6359–6364.
a) C. S. Lobban, M. Schefter, A. G. B. Simpson, X. Pochon, J.
Pawlowski, W. Foissner, Mar. Biol. 2002, 140, 411–423; b) C. S.
Lobban, M. Schefter, A. G. B. Simpson, X. Pochon, J. Pawlow-
ski, W. Foissner, Mar. Biol. 2002, 141, 207–208; c) W. Miao,
A. G. B. Simpson, C. Fu, C. S. Lobban, J. Eukaryot. Microbiol.
2005, 54, 11–15.
B. Tartarotti, G. Baffico, P. Temporetti, H. E. Zagarese, J.
Plankton Res. 2004, 26, 753–762.
R. Sommaruga, K. Whitehead, J. M. Shick, C. S. Lobban, Pro-
tist 2006, 157, 185–191.
B. Sonntag, M. Summerer, R. Sommaruga, Freshwater Biol.
C-1Ј), 117.8 (d, C-4), 116.6 (d, C-6Ј), 113.6 (d, C-7), 111.1 (s, C-2),
105.1 (s, C-3Ј), 101.5 (s, C2Ј) ppm. FT-IR (neat): ν = 3420 (strong,
˜
NH and OH stretching), 1683 (strong, C=O stretching) cm–1. UV
(MeOH): λmax (ε) = 357 nm (29100 mol–1 dm3 cm–1) MALDI-TOF
(negative ion mode) HR-MS: m/z 486.8220Ϯ0.010 (calcd. for
C15H11Br3N2O2: 486.8298). ESI-MS (positive ion mode detection)
m/z: [M + H]+ [M + Na]+ and [M + K]+ as 1:3:3:1 quartets at
[4]
[5]
[6]
[7]
nominal
masses
489:491:493:495,
511:513:515:517
and
527:529:531:533, respectively. ESI-MS (negative ion mode detec-
tion) m/z: [M – H]– as 1:3:3:1 quartet at nominal masses
487:489:491:493.
Keronopsamide B (8): 1H NMR (CD3OD): δ = 7.57 (d, J1,5
=
2.1 Hz, 1 H, 1-H), 7.56 (d, 3J7,8 = 14.7 Hz, 1 H, 8-H), 7.52 (d, 3J5Ј,6Ј
= 15.8 Hz, 1 H, 5Ј-H), 7.51 (d, J4,5 = 8.5 Hz, 1 H, 4-H), 7.30 (dd,
3
J1,5 = 2.1, J4,5 = 8.5. Hz 1 H, 5-H), 7.04 (s, 1Ј-H), 6.50 (d, J5Ј,6Ј
=
3
15.8 Hz, 1 H, 6Ј-H), 6.18 (d, J7,8 = 14.7 Hz, 1 H, 7-H) ppm. UV
(MeOH): λmax (ε) = 379 nm (31000 mol–1 dm3 cm–1). ESI-MS
(negative ion mode detection) m/z: [M – H]– and [M – SO3 – H]–
as 1:3:3:1 quartets at nominal masses 567:569:571:573 and
487:489:491:493, respectively. Compound 8 afforded pure 7 when
treated in NMR tube (CD3OD) with a catalytic amount of tri-
chloroacetic acid.
[8]
[9]
3
Keronopsamide C (9): 1H NMR (CD3OD): δ = 7.56 (d, J7,8
=
[10]
14.7 Hz, 1 H, 8-H), 7.54 (d, J1,5 = 2.1 Hz, 1 H, 1-H), 7.50 (d, J4,5
= 8.5 Hz, 1 H, 4-H), 7.30 (dd, J1,5 = 2.1, J4,5 = 8.5. Hz 1 H, 5-H),
3
3
7.04 (s, 1Ј-H), 6.76 (d, J5Ј,6Ј = 12.1 Hz, 1 H, 5Ј-H), 5.75 (d, J5Ј,6Ј
3
= 12.1 Hz, 1 H, 6Ј-H), 6.18 (d, J7,8 = 14.7 Hz, 1 H, 7-H) ppm.
ESI-MS (negative ion mode detection) m/z: [M – H]– and [M –
SO3 – H]– as 1:3:3:1 quartets at nominal masses 567:569:571:573
and 487:489:491:493, respectively.
[11]
[12]
[13]
[14]
Supporting Information (see also the footnote on the first page of
this article): 400 MHz NMR (CD3COCD3) spectra of keronops-
amide A 7. 1H NMR (Figure S1), COSY full spectrum (Figure S2),
HSQC (Figure S3), HMBC (Figure S4), and NOESY (Figure S5).
2007, 52, 1476–1485.
a) H. Berger, Monograph of the Urostyloidea (Ciliophora, Hy-
potricha). Monographiae Biologicae, H. J. Dumont (Eds.),
Springer, 2006, Vol. 85; b) X. Hu, W. Song, Acta Protozool.
2001, 40, 107–115; c) X. Hu, A. Warren, T. Suzuki, Acta Proto-
zool. 2004, 43, 351–368; d) W. Lueken, G. Uhlig, T. Kruppel,
J. Protozool. 1989, 36, 350–353; e) X. Shi, X. Hu, A. Warren,
G. Liu, Acta Protozool. 2007, 46, 41–54; f) W. Song, N. Wilbert,
A. Warren, Acta Protozool. 2002, 41, 145–162; g) W. Song, A.
Warren, D. Roberts, N. Wilbert, L. Li, S. Ping, X. Hu, H. Ma,
Acta Protozool. 2002, 45, 271–287; h) R. C. Wanick, I. D. Silva-
Neto, Zootaxa 2004, 587, 1–11; i) E. Wirnsberger, H. F. Larsen,
G. Uhlig, Eur. J. Protistol. 1987, 23, 76–88; j) Z. Yi, Z. Chen,
A. Warren, D. Roberts, K. A. S. Al-Rasheid, M. Miao, S. Gao,
C. Shao, W. Song, J. Zool. 2008, 275, 268–275.
Acknowledgments
This research was supported by the Italian Department of Environ-
ment (Ministero dell’Ambiente e della Tutela del Territorio e del
Mare), projects DPN-2008-005185 and 27/02/08-21/12/07. Ad-
ditional support was provided by funds of University of Trento,
Department of Physics and University of Pisa, Department of Bio-
logy.
[1] J. C. Frisvad, B. Andersen, U. Thrane, Mycol. Res. 2008, 112,
231–240.
[15]
[16]
[17]
G. Höfle, S. Pohlan, G. Uhlig, K. Kabbe, D. Schumacher, An-
gew. Chem. Int. Ed. Engl. 1994, 33, 1495–1497.
PCMOD 7.0/GMMX version 1.5, Serena Software, P. O. Box
3076, Bloomington.
[2] a) F. Dini, G. Guella, P. Giubbilini, I. Mancini, F. Pietra, Nat-
urwissenschaften 1993, 80, 84–86; b) G. Guella, F. Dini, A. To-
mei, F. Pietra, J. Chem. Soc. Perkin Trans. 1 1994, 2, 161–166;
c) G. Guella, F. Dini, F. Erra, F. Pietra, J. Chem. Soc., Chem.
Commun. 1994, 22, 2585–2586; d) G. Guella, F. Dini, F. Pietra,
Helv. Chim. Acta 1995, 78, 1747–1754; e) G. Guella, F. Dini,
F. Pietra, Helv. Chim. Acta 1996, 79, 710–717; f) G. Guella, F.
Dini, F. Pietra, Helv. Chim. Acta 1996, 79, 2180–2189; g) G.
Guella, F. Dini, F. Pietra, Helv. Chim. Acta 1996, 79, 439–448;
h) G. Guella, F. Dini, F. Pietra, Angew. Chem. 1999, 111, 1217–
1220; Angew. Chem. Int. Ed. 1999, 38, 1134–1136; i) G. Guella,
E. Callone, I. Mancini, G. Uccello-Barretta, F. Balzano, F.
Dini, Eur. J. Org. Chem. 2004, 6, 1308–1317; j) G. Guella, E.
Callone, G. Di Giuseppe, R. Frassanito, F. Frontini, I. Man-
cini, F. Dini, Eur. J. Org. Chem. 2007, 31, 5226–5234.
M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, J. A. Montgomery, T. Vreven,
K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tom-
asi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega,
G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratch-
ian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E.
Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli,
J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Sal-
vador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D.
Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck,
K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Ba-
boul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Lia-
[3] a) D. Savoia, C. Avanzini, T. Allice, E. Callone, G. Guella, F.
Dini, Antimicrob. Agents Chemother. 2004, 48, 3828–3833; b)
Eur. J. Org. Chem. 2010, 427–434
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
433

G. Guella et al.
FULL PAPER
shenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T.
Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M.
Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W.
Wong, C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.02,
Gaussian, Inc., Wallingford CT, 2004.
[20]
a) R. Krishnan, J. S. Binkley, R. Seeger, J. A. Pople, J. Chem.
Phys. 1980, 72, 650–654; b) T. Clark, J. Chandrasekhar, G. W.
Spitznagel, P. v. R. Schleyer, J. Comput. Chem. 1983, 4, 294–
301; c) A. D. McLean, G. S. Chandler, J. Chem. Phys. 1980, 72,
5639–5648.
[18] C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 1988, 37, 785–789.
[19] a) A. D. Becke, J. Chem. Phys. 1993, 98, 5648–5652; b) B. Miel-
ich, A. Savin, H. Stoll, H. Preuss, Chem. Phys. Lett. 1989, 157,
200–206.
Received: August 10, 2009
Published Online: December 3, 2009
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