Article
pubs.acs.org/jnp
MDN-0104, an Antiplasmodial Betaine Lipid from Heterospora
chenopodii
Jesús Martín,† Gloria Crespo,† Víctor González-Menéndez,† Guiomar Pérez-Moreno,‡
Paula Sánchez-Carrasco,‡ Ignacio Pérez-Victoria,*,† Luis M. Ruiz-Pérez,‡ Dolores González-Pacanowska,‡
Francisca Vicente,† Olga Genilloud,† Gerald F. Bills,†,§ and Fernando Reyes*,†
†
Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Avenida del
Conocimiento 34, Parque Tecnológico de Ciencias de la Salud, E-18016, Granada, Spain
‡
Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Avenida del Conocimiento s/n, Parque Tecnológico de Ciencias de la
Salud, E-18100 Armilla, Granada, Spain
§
Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science
Centre at Houston, Houston, Texas 77054, United States
S Supporting Information
*
ABSTRACT: Bioassay-guided fractionation of the crude
fermentation extract of Heterospora chenopodii led to the
isolation of a novel monoacylglyceryltrimethylhomoserine (1).
The structure of this new betaine lipid was elucidated by
detailed spectroscopic analysis using one- and two-dimensional
NMR experiments and high-resolution mass spectrometry.
Compound 1 displayed moderate in vitro antimalarial activity
against Plasmodium falciparum, with an IC50 value of 7 μM.
This betaine lipid is the first monoacylglyceryltrimethylhomoserine ever reported in the Fungi, and its acyl moiety also represents
a novel natural 3-keto fatty acid. The new compound was isolated during a drug discovery program aimed at the identification of
new antimalarial leads from a natural product library of microbial extracts. Interestingly, the related fungus Heterospora
dimorphospora was also found to produce compound 1, suggesting that species of this genus may be a promising source of
monoacylglyceryltrimethylhomoserines.
E
Indian coastline. Betaine lipid 3 was the major component of
the mixture in which the related DGTS 4 was also present.8
ther-linked glycerolipids containing a betaine moiety occur
naturally in algae, bryophytes, fungi, and some primitive
protozoa and photosynthetic bacteria.1−7 They are not found in
flowering plants, but have been detected in some sporeproducing plants.3 These complex lipids contain a polar group
linked by an ether bond at the sn-3 position of the glycerol
moiety, with fatty acids esterifying the sn-1 and sn-2 positions.
Three related classes of these lipids differing in the
permethylated hydroxyamino acids linked to diacylglycerols
through an ether bond have been described. They all possess a
positively charged trimethylammonium group and a negatively
charged carboxyl group and therefore are zwitterionic at neutral
pH. The three types of betaine lipids include 1,2-diacylglyceryl3-O-4′-(N,N,N-trimethyl)homoserine (DGTS), 1,2-diacylglyceryl-3-O-2′-(hydroxymethyl)(N,N,N-trimethyl)-β-alanine
(DGTA), and 1,2-diacylglyceryl-3-O-carboxy-(hydroxymethyl)choline (DGCC).7 Of these, the diacylglyceryltrimethylhomoserines are by far the most common in nature, and
taxonomic studies suggest that they may have been the
evolutionary progenitor of betaine lipids.3 On the other hand,
only two monoacylglyceryltrimethylhomoserines have been
reported to date (2, 3) and were isolated as an inseparable
mixture from the green alga Ulva fasciata collected from the
© XXXX American Chemical Society and
American Society of Pharmacognosy
Malaria is an infectious disease caused by parasites belonging
to the genus Plasmodium. Two billion people live in areas at risk
Received: July 21, 2014
A
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from the disease, and annually up to one million people die
from malaria infection.9,10 The discovery and development of
new drugs for the treatment of malaria is thus of major
importance. Historically, medicinal plants have played a very
important role in combating this disease. The malaria-active
metabolites quinine and artemisinin represent the best-known
examples of natural products that have led to the development
of numerous antimalarial drugs.11 However, the prevalence of
resistance of the malaria parasite to known drugs and the lack
of efficacy of some of these have highlighted the urgent need
for the discovery and development of new antimalarials
developed from unique chemical structures based on different
modes of action.12,13 Microbial secondary metabolites cover a
broad chemical space14,15 and offer an excellent opportunity for
finding possible antiplasmodium leads.16 Fundación MEDINA
has initiated a drug discovery program aimed at the
identification of new antimalarial leads from its proprietary
library of microbial (fungi, actinomycetes, and other bacteria)
natural product extracts. In this article, we report the isolation
and structure elucidation of MDN-0104, a novel monoacylglyceryltrimethylhomoserine (1) containing as the acyl moiety a
new unusual 3-keto fatty acid. Its in vitro antimalarial activity
against Plasmodium falciparum is also reported herein. This new
betaine lipid has been isolated from a fermentation broth of the
fungus Heterospora chenopodii (Ascomycota, Pleosporales,
Leptosphaeriaceae).
Table 1. NMR Spectroscopic Data (500 MHz, CD3OD, at 24
°C) for MDN-0104 (1)
position
1
67.2, CH2
a 4.14, dd
(11.1, 5.5)
b 4.10, dd
(11.1, 4.9)
3.89, quintet
(5.3)
a 3.46, dd
(10.0, 4.5)
b 3.41, dd
(10.0, 5.9)
69.3, CH
3
73.4, CH2
176.6,
54.0,
200.7,
135.7,
144.2,
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
17′
18′
19′
1″
RESULTS AND DISCUSSION
In one of our screening campaigns, in vitro antiplasmodial
activity against P. falciparum Pf 3D7 was detected in the acetone
extract corresponding to the microfermentation of the fungus
H. chenopodii in 1 mL of STP medium, one of eight media
included in our nutritional array microfermentations.17−21 The
strain employed (CBS 109836) was originally isolated from
Atriplex prostrata collected at Wissenkerke, Zeedijk Oosterschelde, near Westnol (Netherlands) and deposited at the
Fungal Biodiversity Centre by G. Verkley. Interestingly, H.
chenopodii was first described as Phyllosticta chenopodii22 and
recently reclassified in the genus Heterospora after molecular
and phylogenetic studies, which led to the conclusion that
Phoma should be restricted to Didymellaceae.23 This revision
also led to the redisposition of another Phoma-like anamorph
associated with plants of the Chenopodiaceae, P. heteromorphospora, a sister species of H. chenopodii, which was transferred
to the genus Heterospora as H. heteromorphospora.23 LC-UV-MS
analysis of the corresponding extract did not identify any
known bioactive microbial metabolite included in our in-house
library.21,24−26 A bioassay-guided chromatographic fractionation strategy was then pursued to isolate the bioactive
compound. The culture was scaled up to 600 mL in a static
fermentation of the same medium. Fractionation of the acetone
extract of the broth on SP-207ss resin followed by semipreparative HPLC led to the isolation of a new monoacylglyceryltrimethylhomoserine, MDN-0104 (1), as the
compound responsible for the biological activity.
Compound 1 was assigned a molecular formula of
C29H53NO7 using HRESIMS and MS/MS. No hit was retrieved
when searching this formula in the Dictionary of Natural
Products database,27 suggesting the novelty of the compound.
Analysis of the 1H NMR and 13C NMR spectra (Table 1)
alongside the HSQC spectrum allowed the identification of
signals corresponding to two olefinic carbons (one quaternary
plus a methine), two aliphatic methines, 14 methylene groups
δH (J in Hz)
2
1′
2′
3′
4′
5′
■
δC, type
2″
C
C
C
C
CH
29.8, CH2
29.6, CH2
30.4, CH2
30.5,a CH2
30.7,a CH2
30.7,a CH2
30.7,a CH2
30.5,a CH2
33.0, CH2
23.7, CH2
14.4, CH3
25.1, CH3
25.1, CH3
12.6, CH3
68.6, CH2
29.0, CH2
6.43, td (7.2,
0.9)
2.24, m
1.45, t (6.8)
1.33, m
1.41, m
1.41, m
1.41, m
1.41, m
1.41, m
1.29, m
1.32, m
0.90, t (6.9)
1.41, s
1.41, s
1.76, br s
a 3.64, dt
(9.5, 4.7)
b 3.55, td
(9.5, 4.3)
a 2.23, m
b 2.08, m
3″
4″
N-CH3
a13
77.7, CH
171.8, C
52.4, CH3
3.75, dd
(11.3)
COSY
HMBC (1H to 13C)
1b, 2
1′, 2, 3
1a, 2
1′, 2, 3
1a, 1b, 3a,
3b
3b, 2
1, 3
1, 2, 1″
3a, 2
1, 2, 1″
6′, 19′
3′, 6′, 7′, 19′
5′, 7′, 19′
6′, 8′
7′, 9′
8′, 10′
9′, 11′
10′, 12′
11′, 13′
12′, 14′
13′, 15′
14′, 16′
15′
4′, 5′, 7′, 8′
5′, 6′, 8′, 9′
6′, 7′, 9′, 10′
7′, 8′, 10′, 11′
8′, 9′, 11′, 12′
9′, 10′, 12′, 13′
10′, 11′, 13′, 14′
11′, 12′, 14′, 15′
12′, 13′, 15′, 16′
13′, 14′, 16′
14′, 15′
1′, 2′, 3′, 18′
1′, 2′, 3′, 17′
3′, 4′, 5′, 6′
3, 2″, 3″
5′, 6′
1a″, 2a″,
2b″
1b″, 2a″,
2b″
2b″, 1a″,
1b″, 3″
2a″, 1a″,
1b″, 3″
2a″, 2b″
3.22, s
3, 2″, 3″
1″, 3″, 4″
1″, 3″, 4″
1″, 2″, 4″, N-CH3
3″
C assignments may be interchanged.
including three oxygenated methylenes, seven methyls, and
three carbonyl signals (δC 200.7, 176.6, and 171.8 ppm). A
strong singlet signal corresponding to three equivalent methyl
groups at δH 3.22 ppm in 1H NMR spectrum, with correlation
in HSQC at δC 52.4 ppm, suggested the presence of a
trimethylammonium group. In-depth analysis of the 2D NMR
data including additional COSY and HMBC spectra led to the
identification of a trimethylhomoserine unit with 13C NMR
signals at δC 68.6 (C-1″), 29.0 (C-2″), 77.7 (C-3″), 171.8 (C4″), and 52.4 (N-CH3) ppm. Similarly, a glycerol moiety was
also revealed with 13C NMR signals at δC 67.2 (C-1), 69.3 (C2), and 73.4 (C-3) ppm. The HMBC correlation (Figure 1)
between the methylene protons of the homoserine moiety at
position C-1″ with the methylene carbon of glycerol at δC 73.4
ppm (C-3) and vice versa suggested that the homoserine unit
was attached to the glycerol moiety at C-3 via an ether bond.
The carbonyl carbon at δC 176.6 ppm (C-1′) displayed an
HMBC correlation with the glycerol methylene at position C-1,
B
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Figure 1. Key HMBC (solid arrows) and NOESY (dashed arrow) correlations observed for 1.
two chiral centers of 1 based on biosynthetic arguments. Our
hypothesis assumes that the new monoacylglyceryltrimethylhomoserine and the common diacylglyceryltrimethylhomoserines share the same biosynthetic pathway. The
coexistence of the monoacylglyceryltrimethylhomoserines 2
and 3 with the related DGTS 4 in the green alga Ulva fasciata8
supports such a hypothesis. The biosynthesis of DGTS
(Scheme 1) has been characterized in phosphate-starved cells
suggesting the acylation of this methylene, which was also
confirmed by the low-field chemical shift of both H-1
diastereotopic protons (δH 4.14 and 4.10 ppm). On the other
hand the glyceryl methine proton (H-2) only had HMBC
correlations with the other two glyceryl methylenes (C-1 and
C-3). Thus, compound 1 was identified as a
monoacylglyceryltrimethylhomoserine. The connectivity of
the fatty acid moiety was established starting with the key
HMBC correlations (Figure 1) observed for the carbonyl
carbon of the ester group (δC 176.6 ppm). Apart from the
already mentioned correlation with the corresponding glyceryl
methylene, this carbonyl displayed an additional correlation
with the singlet signal corresponding to two isochronous
methyl groups at δH 1.41 ppm. The HMBC correlation
observed for these methyl groups at their own frequency (δC
25.1 ppm) confirmed their geminal location at position α with
respect to the ester carbonyl. Those methyl groups also gave
HMBC correlations with the quaternary carbon they are
directly bonded to (δC 54.0 ppm) and with a ketone carbonyl at
δC 200.7 ppm, indicating the 3-oxo (i.e., β-keto) nature of the
fatty acid. The ketone carbonyl also showed HMBC
correlations with a broad methyl singlet at δH 1.76 ppm and
with the only olefinic proton found at δH 6.43 ppm (triple
doublet). The methyl singlet displayed additional HMBC
correlations with two olefinic carbons (the methine at δC 144.2
ppm and the quaternary carbon at δC 135.7 ppm) but no
correlation with any aliphatic methylene, confirming its location
at position α with respect to the keto functionality and
indicating that this carbonyl was α,β-unsaturated. The COSY
spectrum clearly displayed two long-range correlations for this
methyl group, one due to allylic coupling with the olefinic
proton and the second due to homoallylic coupling with the
methylene vicinal to the unsaturation. Analysis of the 2D NMR
spectra revealed that the methylene directly bonded to the
unsaturation (C-6′) was linked to a nonbranched aliphatic
chain of nine methylenes and a terminal methyl group (C-7′ to
C-16′). The analysis of the NOESY spectrum allowed an E
configuration to be established for the double bond based on
the observation of a correlation between H-6′ and H3-19′
(Figure 1). The structure of the acyl moiety therefore
corresponds to (E)-2,2,4-trimethyl-3-oxohexadec-4-enoic acid,
which represents a new 3-keto fatty acid. Thus, the planar
structure of 1 was established as 1-O-[(E)-2′,2′,4′-trimethyl-3′oxohexadec-4′-enoyl]glycero-3-O-4″-(N,N,N-trimethyl)homoserine and given the name MDN-0104 (according to our
proprietary compound database). ESIMS/MS experiments in
positive ionization mode further confirmed the planar structure
of this new monoacylglyceryltrimethylhomoserine (see Supporting Information). Likewise, the maximum absorbance
observed at 239 nm in the UV (DAD) spectrum is in
agreement with the expected value for the corresponding π →
π* transition of the α,β-unsaturated enone present in the
structure.28
MDN-0104 (1) was isolated as an optically active material.
We have assigned a tentative S absolute configuration to the
Scheme 1. Biosynthetic Hypothesis for MDN-0104 (1)
of the purple bacterium Rhodobacter sphaeroides.29 Heterologous expression experiments have identified the two enzyme
systems, BtaA and BtaB, essential to the biosynthesis.30 A
parallel system, the BTA1 gene, has also been characterized in
the eukaryotic alga Chlamydomonas reinhardtii and consists of
two domains, corresponding to the bacterial BtaA and BtaB
proteins.31 Furthermore, many fungal genomes encode an
enzymatic system homogolous to BTA1 for DGTS biosynthesis, and fungi synthesize DGTS during phosphorus (P)
limitation possibly as a common strategy among many
C
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eukaryotes for conserving P under P-limiting conditions.32
However, the medium in our fermentation system supplied an
excess of P for fungal growth, suggesting that such enzymes
might be under a different regulatory control. On the other
hand, the eukaryotic alga C. reinhardtii synthesizes DGTS to the
exclusion of phosphatidylcholine regardless of P availability.33
Due to the unusual nature of the fatty acid moiety in MDN0104 (1), it seems reasonable that the parent DGTS from
which 1 derives is not a mere structural substitute for
phosphatidylcholine in the fungal membranes. Rather, betaine
lipid 1 must play some ecological role34 in H. chenopodii, which
would explain its production in P-replete media. In any case,
the parent DGTS is likely synthesized via an enzymatic system
homologous to BTA1 (regardless of its regulatory control). In
such a system, the first enzymatic domain (BtaA-like) transfers
the 3-amino-3-carboxypropyl group of S-adenosyl-L-methionine
to the 3-hydroxyl of a 1,2-diacyl-sn-glycerol to form the
intermediate diacylglycerylhomoserine. The second enzyme
domain (BtaB-like) transfers methyl groups from S-adenosyl-Lmethionine in three successive steps to form the final product
diacylglyceryl-N,N,N-trimethylhomoserine. The starting 1,2-sndiacylglycerol in such a route has an S configuration in the
glyceryl methine. On the other hand, the homoserine unit is
transferred from S-adenosyl-L-methionine.30 This renders an
absolute S configuration for both chiral centers of the parent
DGTS. Monoacylglyceryltrimethylhomoserines may derive
from DGTS by regioselective enzymatic hydrolysis of the
fatty acid at position sn-2 (Scheme 1). Such hydrolysis would
be analogous to the selective hydrolysis of phosphatidylcholine
catalyzed by phospholipase A2 to render lysophosphatidylcholine.35 It cannot be ruled out that a similar specialized lipase
must be involved in the regioselective hydrolysis of DGTS. This
would lead to the following lipid name for MDN-0104: 1-O[(E)-2′,2′,4′-trimethyl-3′-oxohexadec-4′-enoyl]-sn-glycero-3-O4″-(N,N,N-trimethyl)-L-homoserine.
MDN-0104 (1) exhibited activity against P. falciparum
Pf 3D7 using the previously described LDH growth inhibition
in vitro assay36 with an IC50 of 7.00 ± 0.25 μM. Chloroquine
gave an IC50 of 5.5 nM when tested under the same conditions.
The structural resemblance of MDN-0104 (1) to lysophosphatidylcholine may explain the observed bioactivity. It is
possible that 1 induces a membrane disruption due to its
analogous zwitterionic detergent structure.37,38 Some biological
effects of lysophosphatidylcholine may simply be due to its
ability to diffuse readily into membranes, altering their
curvature and indirectly affecting the properties of membrane
proteins.37 In fact, lysophosphatidylcholine has inspired the
design of synthetic alkyl-lysophospholipids, such as miltefosine,
which have recently found use as antiprotozoal drugs.39 Even
more interesting, hexadecyltrimethylammonium bromide, a
detergent compound structurally similar to miltefosine, was
recently found to exhibit potent in vitro activity against P.
falciparum via an antimalarial mechanism based on the
inhibition of the parasite choline kinase.40 Thus, it cannot be
ruled out that MDN-0104 displays antiplasmodial activity due
to inhibition of the same enzyme.
Finally, we investigated whether other fungal species
described in the genus Heterospora were able to produce
MDN-0104 (1). To this end, a strain of the sister species
Heterospora dimorphospora was chosen.23 The epi-type strain
employed (CBS 345.78) from Chenopodium quinoa was grown
under the same conditions. LC-UV-MS analysis of its
corresponding acetone extract revealed that this strain also
produced compound 1, although much less than H. chenopodii.
Additionally, we found that the new betaine lipid was also
produced by another strain of H. chenopodii (CBS 115.96)
(data not shown). This result suggested that the species of
Heterospora lineage could be a promising source of
monoacylglyceryltrimethylhomoserines.
In conclusion, a new betaine lipid, MDN-0104 (1), that
displays moderate antiplasmodial activity has been isolated
from fermentation broths of H. chenopodii. It has also been
found that H. dimorphospora produces this secondary
metabolite, suggesting the potential of Heterospora species as
a source of this type of betaine lipids. The novel compound 1 is
the first monoacylglyceryltrimethylhomoserine ever reported in
the Fungi, and its acyl moiety also represents a novel natural 3keto fatty acid. Its discovery during a research program aimed at
the identification of new antimalarial leads from MEDINA’s
natural product library of microbial extracts again confirms the
value of microbial secondary metabolites as a potential source
of novel chemical entities that could be further developed into
new antimalarial drugs. The production of 1 in one among
eight growth conditions is yet another example of the value of
microfermentation arrays as a strategy for identifying nutritional
conditions to produce novel compounds.20
■
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was
determined with a JASCO P-2000 polarimeter. IR spectrum was
measured with a JASCO FT/IR-4100 spectrometer equipped with a
PIKE MIRacle single reflection ATR accessory. NMR spectra were
recorded on a Bruker Avance III spectrometer (500 and 125 MHz for
1
H and 13C NMR, respectively) equipped with a 1.7 mm TCI
MicroCryoProbe, using the signal of the residual solvent as internal
reference (δH 3.31 and δC 49.0 ppm for CD3OD). LC-UV-MS analysis
was performed on an Agilent 1100 single quadrupole LC-MS system,
using a Zorbax SB-C8 column (2.1 × 30 mm, 5 μm), maintained at 40
°C and with a flow rate of 300 μL min−1. Solvent A consisted of 10%
acetronitrile and 90% water with 1.3 mM trifluoroacetic acid and
ammonium formate, and solvent B was 90% acetronitrile and 10%
water with 1.3 mM trifluoroacetic acid and ammonium formate. The
gradient started at 10% B and went to 100% B in 6 min, was kept at
100% B for 2 min, and returned to 10% B for 2 min to initialize the
system. Full diode array UV scans from 100 to 900 nm were collected
in 4 nm steps at 0.25 s/scan. The eluting solvent was ionized using the
standard Agilent 1100 electrospray ionization source adjusted to a
drying gas flow of 11 L min−1 at 325 °C and a nebulizer pressure of 40
psig. The capillary voltage was set to 3500 V. Mass spectra were
collected as full scans from 150 m/z to 1500 m/z, with one scan every
0.77 s, in both positive and negative modes. Database searching was
performed using an in-house-developed application where the DAD
(UV−vis) spectra, retention time, and positive and negative mass
spectra of the samples are compared to the corresponding UV-LC-MS
data of known microbial metabolites stored in the proprietary database
(Fundación MEDINA reference library containing annotated metabolite data obtained under identical conditions to those for the samples
under analysis; the library includes 380 fungal metabolites and 450
metabolites from bacteria and actinomycetes).21,24−26 HRESIMS and
MS/MS spectra were acquired using a Bruker maXis QTOF mass
spectrometer coupled to the same HPLC system as described above.
The mass spectrometer was operated in positive ESI mode. The
instrumental parameters were 4 kV capillary voltage, drying gas flow of
11 L min−1 at 200 °C, and nebulizer pressure of 2.8 bar. TFA-Na
cluster ions were used for mass calibration of the instrument prior to
sample injection. Prerun calibration was by infusion with the same
TFA-Na calibrant. Acetone used for extraction was of analytical grade.
Solvents employed for isolation were of HPLC grade.
Strain and Fermentation. Heterospora chenopodii (CBS 109836)
was purchased from the Centraalbureau voor Schimmelcultures (www.
D
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cbs.knaw.nl). The strategy and protocols for microfermentation of
fungi on nutritional arrays have been described previously.19,20,41 To
scale up the microfermentation to 600 mL, 10 mycelial discs were used
to inoculate 50 mL of SMYA (Difco neopeptone 10 g, maltose 40 g,
Difco yeast extract 10 g, agar 4 g, distilled H2O 1 L). After 7 days’
incubation at 22 °C and 220 rpm, 0.3 mL aliquots of this culture were
used to inoculate STP medium (sucrose 75 g, tomato paste 10 g, Difco
malt extract 10 g, soy flour 1 g, (NH4)2SO4 1 g, KH2PO4 9 g, distilled
H2O 1 L) distributed among 60 × 10 mL in 40 mL flat-sided tissue
culture tubes (TPP Techno Plastic Products AG) with 10 cm2 growth
area. The inclined TPP tubes were incubated statically at 22 °C and
70% relative humidity for 21 days. Fungal growth was removed from
tubes and pooled before extraction.
Extraction and Isolation. The initial 1 mL microfermentations
were extracted with an equal volume of acetone, and the acetone was
removed by vacuum evaporation as described previously.19,41 The
scaled up fermentation broth (600 mL) was extracted with acetone
(600 mL) under continuous shaking at 220 rpm for 1 h. The mycelium
was then separated by centrifugation, and the supernatant (ca. 1.2 L)
was concentrated to ca. 600 mL under a stream of nitrogen. This
solution was loaded (with continuous 1:1 water dilution, discarding
the flow-through) on a column packed with SP-207ss reversed-phase
resin (brominated styrenic polymer, 65 g) previously equilibrated with
water. The loaded column was further washed with water (ca. 1 L) and
afterward eluted at 8 mL min−1 on an automatic flash-chromatography
system (CombiFlash Rf, Teledyne Isco) using a linear gradient from
10% to 100% acetone in water (in 12.5 min) with a final 100% acetone
step (for 15 min) collecting 11 fractions of 20 mL. Fractions were
concentrated to dryness on a centrifugal evaporator, and fraction 6,
containing the active compound, was further purified by reversedphase semipreparative HPLC (Agilent Zorbax SB-C8, 9.4 × 250 mm, 7
μm; 3.6 mL min−1, UV detection at 210 nm) with a linear gradient of
water−CH3CN from 5% to 100% CH3CN over 37 min to yield
compound 1 (7.7 mg) eluting at 31 min.
Compound 1 (1-O-[(E)-2′,2′,4′-trimethyl-3′-oxohexadec-4′enoyl]-sn-glycero-3-O-4″-(N,N,N-trimethyl)-L-homoserine): white,
amorphous solid; [α]20D +9.4 (c 0.23, MeOH); IR (ATR) νmax
3358, 2955, 2923, 2854, 1735, 1666, 1627, 1465, 1388, 1361, 1269,
1123, 1042 cm−1; for 1H and 13C NMR data see Table 1; HRESIMS
m/z 528.3907 [M + H]+ (calcd for C29H54NO7, 528.3895); MS/MS
(see Supporting Information).
Biological Activity. Parasites of the P. falciparum strain 3D7 were
grown in fresh group 0 positive human erythrocytes, obtained from the
Centro Regional de Transfusión Sanguı ́nea-SAS (Granada, Spain), and
suspended at 5% hematocrit in RPMI 1640 containing 2% human
serum, 0.2% NaHCO3, 0.5% Albumax II, 150 μM hypoxanthine, and
12.5 μg/mL gentamicin. Flasks were incubated at 37 °C, under a 5%
CO2 and 95% air mixture. Stock cultures were synchronized with 5%
sorbitol, and 96 h later parasites were mostly late ring stages and early
trophozoites. The stock culture was then diluted with complete
medium and nonparasitized erythrocytes to yield a hematocrit of 2%
and a parasitemia of 0.25%. The extracts, fractions, and pure
compounds were evaluated in 384-well plates. Each plate also included
negative (no additions) and positive controls with 100 nM
chloroquine. Parasite growth inhibition assays and 50% inhibitory
concentration (IC50) determinations were measured using the LDH
assay as previously described.36
■
*(F. Reyes) Tel: +34 958993965, ext 7006. Fax: +34
958846710. E-mail: fernando.reyes@medinaandalucia.es.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research has been supported by grants from the Junta de
Andalucı ́a (BIO-199, BIO-023, BIO-024, BIO-025, P09-CVI5367), the VI PN de I+D+I 2008-2011, ISCIII-Subdirección
General de Redes y Centros de Investigación Cooperativa
(RICET FIS Network: RD12/0018/0017 and RD12/0018/
0005), and a Marie Curie Career Integration Grant (I.P.-V.)
[PCIG-GA-2011-293762]. We thank C. Moreno, F. Muñoz, L.
Rodrı ́guez, and J. Cantizani for technical assistance and
suggestions. The polarimeter, HPLC, IR, and NMR equipment,
and plate reader used in this work were purchased via grants for
scientific and technological infrastructures from the Ministerio
de Ciencia e Innovación [Grants No. PCT-010000-2010-4
(NMR), INP-2011-0016-PCT-010000-ACT6 (polarimeter,
HPLC, and IR), and PCT-01000-ACT7, 2011-13 (plate
reader)].
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ASSOCIATED CONTENT
S Supporting Information
*
NMR, HR-MS/MS, and DAD (UV−vis) spectra for compound
1 and photographs of the microorganism in culture are available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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