Protease inhibitors from microcystis aeruginosa bloom material collected from the dalton reservoir, israel

Article abstract of DOI:10.1021/np4006844

Nine new metabolites, aeruginosins DA495A (1), DA511 (2), DA642A (3), DA642B (4), DA688 (5), DA722 (6), and DA495B (7), microguanidine DA368 (8), and anabaenopeptin DA850 (9), were isolated along with the known micropeptins MZ924, MZ939A, and MZ1019, cyanopeptolins S and SS, microcin SF608, and aeruginazoles DA1497, DA1304, and DA1274 from bloom material of the cyanobacterium Microcystis aeruginosa collected from the Dalton reservoir, Israel, in October 2007. Their structures were elucidated by a combination of various spectroscopic techniques, primarily NMR and MS, while the absolute configurations of the asymmetric centers were determined by Marfey's and chiral-phase HPLC methods. Two of the new aeruginosins, DA511 (1) and DA495A (2), contain a new Choi isomer, (2S,3aS,6S,7aS)-Choi. The structure elucidation and biological activities of the new metabolites are described.

Full text of DOI:10.1021/np4006844

Article
pubs.acs.org/jnp
Protease Inhibitors from Microcystis aeruginosa Bloom Material
Collected from the Dalton Reservoir, Israel
Simi Adiv and Shmuel Carmeli*
Raymond and Beverly Sackler School of Chemistry and Faculty of Exact Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978,
Israel
S
* Supporting Information
ABSTRACT: Nine new metabolites, aeruginosins DA495A (1), DA511 (2), DA642A (3),
DA642B (4), DA688 (5), DA722 (6), and DA495B (7), microguanidine DA368 (8), and
anabaenopeptin DA850 (9), were isolated along with the known micropeptins MZ924,
MZ939A, and MZ1019, cyanopeptolins S and SS, microcin SF608, and aeruginazoles
DA1497, DA1304, and DA1274 from bloom material of the cyanobacterium Microcystis
aeruginosa collected from the Dalton reservoir, Israel, in October 2007. Their structures
were elucidated by a combination of various spectroscopic techniques, primarily NMR and
MS, while the absolute configurations of the asymmetric centers were determined by
Marfey’s and chiral-phase HPLC methods. Two of the new aeruginosins, DA511 (1) and
DA495A (2), contain a new Choi isomer, (2S,3aS,6S,7aS)-Choi. The structure elucidation and biological activities of the new
metabolites are described.
uring the past three decades, marine, freshwater, and
terrestrial cyanobacteria have been documented as
type serine proteases (i.e., microcin SF608 inhibits trypsin with
an IC50 value of 0.5 μg/mL15). It has been shown that extracts
of Microcystis aeruginosa, and other toxic bloom-forming
cyanobacteria, are more toxic than the pure microcystins
themselves, and the enhanced toxicity is attributed to the
presence of the different groups of protease inhibitors (i.e.,
aeruginosins, micropeptins, anabaenopeptins, and microvir-
idins) in the extracts.16,17 In the past decade attempts have been
made to reveal the purpose(s) for the production of the
microcystins and the accompanying protease inhibitors in
cyanobacteria and to study the relationships between these
groups of compounds. Inhibition of trypsin-type proteases and
lowering the amounts of active glutathione S-transferase
(GST)18 in the water flea Daphnia magna, a cyanobacteria
grazer, may imply that the areuginosins are produced in order
to prevent the detoxification of the microcystins in the
environment.19 As part of our continuing interest in the
chemical ecology of cyanobacterial water blooms and the search
for novel drugs for human diseases, we examined the extracts of
M. aeruginosa bloom material collected in October 2007 from
the Dalton reservoir in northern Israel.20 The extract of this
bloom material afforded 18 natural products: eight aerugino-
sins, aeruginosins DA495A (1), DA511 (2), DA642A (3),
DA642B (4), DA688 (5), DA722 (6), and DA495B (7) and
the known microcin SF608,15 two of which, aeruginosins
DA495A (1) and DA511 (2), contain a new Choi isomer,
2S,3aS,6S,7aS-Choi, microguanidine DA368 (8), anabaenopep-
tin DA850 (9), aeruginazoles DA1497, DA1304, and
DA1274,21 and the known micropeptins MZ924, MZ939A,
and MZ101922 and cyanopeptolins S23 and SS.24 In this paper,
D
prolific producers of biologically active natural products.1−3
Freshwater bloom-forming genera of cyanobacteria have been
extensively studied, mainly due to their competence to
biosynthesize toxins, which poses a serious threat to the health
of humans and domesticated and wild animals.4 The micro-
cystins are the most abundant family of hepatotoxic metabolites
found in freshwater bloom-forming cyanobacteria.5 These
toxins are usually accompanied by other groups of metabolites,
mainly by the following five groups of protease inhibitors:
micropeptins (139 isolated variants),6 anabaenopeptins (38
isolated variants),7 aeruginosins (38 isolated variants),8 micro-
ginins (32 isolated variants),9 and microviridins (15 isolated
variants).10 The aeruginosins are a group of linear modified
peptides, which attracted the attention of synthetic chemists
due to their modified peptidic nature and their capacity to
inhibit important proteolytic processes.11 They are charac-
terized by the presence of the proline-mimicking 2-carboxy-6-
hydroxyoctahydroindole (Choi) derivative at the third position,
a hydroxyphenyl lactic acid (Hpla) derivative at the N-terminus
of the modified peptide, a variable amino acid at the second
position, and an arginine derivative (if present) at the fourth
position. Analyses of the aeruginosin gene-cluster sequences
revealed that these linear modified peptides are biosynthesized
in cyanobacteria from amino acid precursors by nonribosmal
peptide synthetase (NRPS)-type enzyme assembly.12 To date,
three natural Choi stereoisomers (out of 16 possible) have been
characterized in the literature. The most frequent Choi isomer,
2S,3aS,6R,7aS-Choi (L-Choi), is contained in 33 known
compounds (including those isolated in the present research),8
2S,3aR,6R,7aR-Choi (L-3a,7a-diepi-Choi) in one isolated
compound,13 and 2R,3aR,6R,7aR-Choi (D-3a,7a-diepi-Choi) in
four published compounds.14 The aeruginosins inhibit trypsin-
Received: August 21, 2013
Published: November 21, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Article
Chart 1
we report the isolation, structure elucidation, and biological
activities of the nine new metabolites isolated from this bloom
material.
mid-spectrum (5.5−3.8 ppm) two pairs of exchangeable
doublet signals and four pairs of methine protons next to
electronegative atoms were observed. The aliphatic region
presented many overlapping signals that were too complex to
be interpreted. The 13C NMR spectrum (Table 1) revealed a
similar doubling of signals corresponding to three pairs of
amide/ester carbonyls, a pair of phenol sp2 carbons, 13 signals
of aromatic sp2 carbons consistent with a pair of phenol and a
pair of phenyl moieties, five pairs of methines in the midrange
of the spectrum (73 to 50 ppm), and several additional methine
and methylene signals in the aliphatic region. Both rotamers
were fully characterized (see Table 1 for the NMR data of the
cis rotamer and Tables S1a and S1b in the Supporting
Information for the full NMR data of the cis rotamer and the
trans rotamer, respectively), but for clarity only the structure
elucidation of the major cis rotamer is discussed below. The
nature of the cis and trans rotamers of the amide bond between
Phe and Choi was distinguished through the NOEs of Phe-H-2
with Choi-H-2 in the cis rotamer and of Phe-H-2 with Choi-H-
7a in the trans rotamer.25 The assignment of the Phe and Hpla
moieties was readily achieved through the interpretation of the
data from the COSY, TOCSY, HSQC, and HMBC 2D NMR
experiments (Tables 1, S1a, and S1b). The structure elucidation
of the 2-carboxyamide-6-hydroxyoctahydroindole (Choi-
amide) moiety was more challenging due to the overlapping
signals in the aliphatic region. Analyses of the COSY and
TOCSY correlations assembled the sequence of H-2 through
RESULTS AND DISCUSSION
■
Aeruginosins DA495A (1) and DA511 (2) were eluted closely
one after the other from a reversed-phase HPLC column. They
were isolated as colorless oils with comparable NMR spectra
and molecular weights that differ by 16 mass units. Both
appeared as ca. 55:45 mixtures, respectively, of two rotamers
characteristic of the aeruginosins.25 Full assignment of the H
1
and 13C NMR data suggested that 1 contained a phenylalanine
moiety at the second position of its short peptide backbone,
while 2 contained a tyrosine moiety, and the rest of their
structures were identical.
Aeruginosin DA495A (1) exhibited a positive HRESIMS
molecular adduct ion at m/z 518.2266 ([M + Na]+), in
agreement with the molecular formula C27H33N3NaO6 and 13
degrees of unsaturation. The interpretation of the NMR data in
DMSO-d6 was rather complicated due to the high similarity of
the chemical shifts of the proton and carbon signals of the
1
above-mentioned rotamers. The aromatic region of the H
NMR spectrum of 1 (Table 1) presented pairs of phenol signals
and three amide protons (two pairs of primary amide singlet
signals and a pair of doublet secondary amides), overlapping
signals indicating the presence of a phenyl moiety, and doublet
signals of two pairs of para-substituted phenol moieties. In the
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Table 1. NMR Data of the Major cis Rotamer of Aeruginosins DA495A (1) and DA511 (2) in DMSO-d6
Article
a
b
aeruginosin DA495A (1)
aeruginosin DA511 (2)
c
c
position
Hpla 1
δC, mult.
δH, mult., J (Hz)
position
Hpla 1
δC, mult.
δH, mult., J (Hz)
173.4, C
173.2, C
72.4, CH
39.9, CH2
2
3
72.5, CH
39.9, CH2
3.85, ddd (8.8, 6.1, 3.8)
2.60, dd (14.0, 3.6)
2.29, dd (14.0, 8.5)
2
3
3.84, ddd (9.7, 6.0, 3.7)
2.61, m
2.28, dd (14.0, 9.0)
4
128.7, C
130.3, CH
115.0, d
4
128.7, C
130.2, CH
114.9, d
5,5′
6,6′
7
6.84, d (8.4)
6.59, d (8.4)
5,5′
6,6′
7
6.86, d (8.4)
6.61, m
155.8, C
155.8, C
2-OH
7-OH
Phe 1
2
5.29, d (6.3)
9.07, s
2-OH
7-OH
Tyr 1
2
5.29, d (6.0)
9.09, s
170.1, C
51.7, CH
36.7, CH2
170.1, C
51.5, CH
37.0, CH2
4.28, dt (2.5, 8.4)
2.88, m
4.19, m
2.79, m
2.63, m
3
3
2.77, m
4
138.3, C
4
128.7, C
5,5′
6,6′
7
129.6, CH
128.2, CH
126.4, CH
7.19, m
5,5′
6,6′
7
130.3, CH
114.9, CH
155.7, C
6.99, m
6.62, m
7.22, m
7.16, m
NH
7.71, d (8.3)
NH
7.64, d (8.0)
9.06, s
7-OH
Choi 1 amide
174.3, C
59.6, CH
33.3, CH2
Choi 1 amide
174.2, C
59.4, CH
33.2, CH2
2
3
4.78, d (9.0)
2.32, m
2
3
4.76, d (8.8)
2.35, m
1.77, dd (12.2, 5.7)
2.23, m
1.75, dd (11.6, 5.5)
2.25, m
3a
4
32.8, CH
22.8, CH2
3a
4
32.7, CH
22.7, CH2
1.62, m
1.58, m
1.52, m
1.62, m
5
29.9, CH2
1.60, m
5
29.8, CH2
1.56, m
1.21, m
1.20, m
6
7
67.1, CH
36.3, CH2
3.29, m
6
7
67.0, CH
35.9, CH2
3.28, m
2.34, m
2.37, m (eq)
0.83, q (11.8)
4.01, ddd (11.8, 6.7, 6.1)
4.51, d (4.1)
7.73, s
0.84, q (12.0)
4.04, ddd (12.0, 6.3, 6.3)
4.49, d (4.3)
7.73, s
7a
56.7, CH
7a
56.3, CH
6-OH
NH2(a)
6-OH
NH2(a)
NH2(b)
NH2(b)
7.32, s
7.31, s
a
b
c
1
1
500 MHz for H, 125 MHz for 13C. 400 MHz for H, 100 MHz for 13C. Multiplicities and assignments from HSQC experiment.
H-7a, while the correlations in the HSQC spectrum assigned
the carbons directly attached to these protons (Tables 1 and
S1a). H-2 was correlated to the adjacent protons resonating at
δH 2.35 (a 9.0 Hz coupling constant) and 1.77 (<1 Hz) (COSY
correlations) and presented NOEs (ROESY experiment) with
both of them, suggesting dihedral angles of ca. 15° between H-
2 and the cis proton resonating at δH 2.35 (H-3ax) and of ca. 90°
between H-2 and the trans proton resonating at δH 1.77 (H-
3eq). H-3a was coupled to H-3eq with a coupling constant of 5.7
Hz and presented an NOE with H-2, suggesting that H-3a was
cis oriented to H-2 in the pyrrolidine ring. H-3a exhibited an
NOE and a 6.3 Hz coupling constant with H-7a, confirming
their cis relationships. The cis relationships of H-2, H-3a, and
H-7a in the pyrrolidine ring were thus confirmed by the mutual
coupling constants of the latter and by NOEs.26 In the
cyclohexane ring, H-6 exhibited large coupling constants (12.0
Hz, based on the COSY correlations as H-6 co-resonated with
the H2O signal in DMSO-d6) with H-5ax and H-7ax, suggesting
that it occupied an axial position. In the trans rotamer, the NOE
between H-7a and Tyr-H-2 suggested an exo orientation of Tyr
and an envelope conformation of the pyrrolidine ring where the
nitrogen was out of the ring plane, thus explaining the
multiplicity of H-2 (doublet of 9.0 Hz). A comparison of the
NMR data of Choi in 1 with those of microcin SF608
(containing L-Choi)15 and aeruginosin KT608A (containing D-
3a,7a-diepi-Choi)14 revealed the similarity of the Choi moiety in
1 with those of the D-3a,7a-diepi-Choi moiety of aeruginosin
KT608A. This suggested that the relative configuration of the
Choi moiety of 1 was consistent with either 2S,3aS,6S,7aS-Choi
or 2R,3aR,6R,7aR-Choi (L-6-epi-Choi or D-3a,7a-diepi-Choi,
respectively). The sequence of the subunits that compose 1,
Hpla-Phe-Choi-amide, was based on the HMBC correlation of
Hpla-CO with Phe-NH, the NOE correlation of Phe-H-2 and
Choi-H-2, and the HMBC correlations of Choi-CO with the
primary amide protons. These arguments established the
relative configuration of the Choi moiety and the planar
structure of 1. The application of Marfey’s method27 (L-FDAA)
and chiral-phase HPLC to determine the absolute config-
urations of the amino acids and the hydroxy acid, respectively,
revealed the presence of D-Phe and D-Hpla moieties in 1. The
retention time of Choi-L-FDAA (35.1 min) from 1 was
identical with that of the synthetic L-6-epi-Choi-L-FDAA (35.1
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a
Table 2. NMR Data of the Major trans Rotamer of Aeruginosins DA642A (3) and DA642B (4) in DMSO-d6
aeruginosin DA642A (3)
δH, mult., J (Hz)
aeruginosin DA642B (4)
b
c
b
position
δC, mult.
HMBC correlations
δC, mult.
δH, mult., J (Hz)
Cl-Hpla 1
172.6, C
172.9, C
2
3
71.9, CH
39.0, CH2
3.94, ddd (8.2, 6.0, 3.4)
2.69, dd (14.1, 3.4)
2.45, dd (14.1, 8.2)
71.8, CH
39.1, CH2
3.95, ddd (8.4, 6.3, 3.6)
2.67, dd (13.9, 3.6)
2.41, dd (13.9, 8.4)
Cl-Hpla-4
Cl-Hpla-2,4
4
130.0, C
130.7, CH
119.1, C
151.5, C
116.3 CH
129.2 CH
130.2, C
130.6, CH
119.1, C
151.4, C
116.2 CH
129.0 CH
5
7.09, d (1.6)
Cl-Hpla-3,6,7,9
7.08, d (1.8)
6
7
8
6.81, d (7.9)
6.86, dd (7.9, 1.6)
5.54, d (6.0)
9.88, s
Cl-Hpla-4,6
Cl-Hpla-5,7
Cl-Hpla-1
6.78, d (8.4)
6.83, dd (8.8, 1.8)
5.32, d (6.3)
9.81, s
9
2-OH
7-OH
Phe 1
2
Cl-Hpla-6,7
169.6, C
50.6, CH
38.4, CH2
169.8, C
51.1, CH
38.4, CH2
4.69, ddd (9.9, 8.2, 5.3)
2.86, dd (14.5, 5.3)
2.78, m
Phe-1
4.66, ddd (8.8, 7.7, 4.1)
2.91, dd (14.0, 4.1)
2.79, dd (14.0, 8.8)
3
Phe-1,2,4
Phe-1,2,4
4
137.0, C
137.4, C
5,5′
6,6′
7
129.6, CH
128.2, CH
126.6, CH
7.12, m
Phe-3,7
129.5, CH
128.3, CH
126.6, CH
7.22, m
7.23, m
7.23, m
7.19, m
Phe-5
7.19, m
NH
Choi 1
2
7.58, d (8.2)
Cl-Hpla-1
7.79, d (7.7)
171.4, C
59.9, CH
30.5, CH2
171.4, C
59.9, CH
30.6, CH2
4.22, dd (9.2, 8.4)
1.95, ddd
Phe-1, Choi-1,3
Choi-2,
4.24, dd (9.4, 8.3)
1.95, m
3
(12.4, 8.4, 6.8)
1.83, dt (9.2, 12.4)
2.20, dtt (12.4, 6.8, 6.1)
2.01, m
1.83, m
3a
4
36.6, CH
19.2, CH2
36.6, CH
19.2, CH2
2.21, m
2.02, m
1.39, m
Choi-3a,5,6,7a
1.41, m
5
26.2, CH2
64.1, CH
34.3, CH2
54.7, CH
1.42, m (2H)
3.86, brs
26.1, CH2
64.1, CH
34.3, CH2
54.6, CH
1.36, m (2H)
3.87, brs
1.68, m (2H)
4.44, m
6
7
1.67, m (2H)
4.40, dt (9.0, 6.8)
4.54, d (2.5)
3.06, m
Choi-3a,5,7a
Choi-2,3,3a
Choi-5,6
7a
6-OH
4.51, d (2.3)
3.07, m
Agm 1
38.1, CH2
25.9, CH2
26.5, CH2
40.5, CH2
156.8, C
Choi-1,2
38.1, CH2
25.9, CH2
26.5, CH2
40.5, CH2
156.8, C
2
1.41, m
1.41, m
3
1.43, m
Agm-2
1.43, m
4
3.09, m
Agm-3,5
3.09, m
5
1-NH
7.84, t (5.7)
7.48, t (5.5)
Choi-1, Agm-1
7.80, m
4-NH
7.47, t (5.3)
a
b
c
1
400 MHz for H, 100 MHz for 13C. Multiplicities and assignments from HSQC experiment. HMBC correlations, optimized for 8 Hz, are from
the proton(s) stated to the indicated carbon.
min) and was different from the L-Choi-L-FDAA (36.6 min,
synthetic) and the D-3a,7a-diepi-Choi-L-FDAA (31.0 min,
obtained from hydrolysis of aeruginosin KT608B14) standards
used for comparison. This procedure established this moiety as
a new fourth natural Choi isomer, 2S,3aS,6S,7aS-Choi (L-6-epi-
Choi) and the structure of aeruginosin DA495A (1) as a D-
Hpla-D-Phe-L-6-epi-Choi-amide.
Aeruginosins DA642A (3) and DA642B (4) were eluted
from the reversed-phase HPLC column closely one after the
other, presenting similar NMR properties (Table 2 and Figures
S3a and S4a) and identical molecular weights. Their specific
rotations had the same sign but differed in magnitude, −63 for
3 and −36 for 4. The structures of 3 and 4 were closely related
to that of microcin SF608, which was isolated along with those
from the aqueous MeOH extract. They appeared as ca. 1:3
mixtures of two rotamers of the amide bond between Phe and
Choi (29:71 ratio in 3 and 27:73 ratio in 4).25 The full
1
Aeruginosin DA511 (2) presented comparable H and 13C
NMR data (Tables 1, S2a, and S2b) to those of 1 and exhibited
a positive HRESIMS molecular adduct ion at m/z 534.2212
([M + Na]+) consistent with the molecular formula
C27H33N3NaO7 and 13 degrees of unsaturation. Analyses of
the 2D NMR data and the absolute configuration of the
asymmetric centers, as discussed above for 1, established the
structure of 2 as D-Hpla-D-Tyr-L-6-epi-Choi-amide.
1
assignment of the H and 13C NMR data suggested that they
present opposite configurations of their Hpla moiety.14
Aeruginosin DA642A (3) exhibited a positive HRESIMS
quasi-molecular ion at m/z 643.3016/645.3014 (3:1, [M +
H]+), which was consistent with the molecular formula
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a
Table 3. NMR Data of the Major Rotamer of Aeruginosins DA688 (5), DA722 (6), and DA495B (7) in DMSO-d6
aeruginosin DA688 (5)
aeruginosin DA722 (6)
aeruginosin DA495B (7)
b
b
b
position
δC, mult.
δH, mult., J (Hz)
δC, mult.
δH, mult., J (Hz)
δC, mult.
δH, mult., J (Hz)
Cl-Hpla 1
172.5, C
71.6, CH
38.7, CH2
172.3, C
71.3, CH
38.6, CH2
172.5, C
2
3
4.08, m
4.14, m
71.8, CH
38.8, CH2
4.06, m
2.83, dd (13.9, 3.7)
2.67, dd (13.9, 7.4)
2.85, dd (13.7, 2.7)
2.69, dd (13.7, 7.0)
2.80, dd (13.9, 3.8)
2.67, dd (13.9, 6.6)
4
129.7, C
130.7, CH
118.9, C
151.4, C
116.1 CH
129.2 CH
130.9, C
129.8, CH
121.7, C
147.5, C
121.7, C
129.8, CH
129.8, C
130.9, CH
118.9, C
151.6, C
116.2 CH
129.4 CH
5
7.16, brd (1.4)
7.17, s
7.12, brs
6
7
8
6.84, d (8.0)
6.95, dd (8.0, 1.4)
5.76, d (5.2)
9.87, s
6.83, d (8.3)
6.93, brd (8.3)
5.86, d (5.5)
9.87, s
9
7.17, s
2-OH
7-OH
Leu 1
2
5.83, d (5.5)
9.83, s
170.0, C
48.5, CH
41.3, CH2
170.0, C
48.6, CH
41.7, CH2
169.6, C
47.9, CH
42.4, CH2
4.43, ddd (7.7, 7.2, 3.3)
1.36, m
4.44, ddd (9.5, 8.0, 2.0)
1.35, m
4.52, ddd (9.1, 8.7, 2.1)
1.30, m
3
1.24, m
1.22, m
1.20, m
4
24.0, CH
23.4, CH3
21.6, CH3
1.47, m
24.2, CH
23.6, CH3
21.7 CH3
1.43, m
23.8, CH
23.6, CH3
21.6, CH3
1.21, m
5
0.81, d (6.0)
0.85, d (5.6)
7.51, d (7.2)
0.79, d (5.5)
0.83, d (5.5)
7.51, d (8.0)
0.78, d (5.8)
0.85, d (5.8)
7.37, d (8.7)
6
NH
Choi 1 sulfate
171.5, C
59.9, CH
30.5, CH2
171.7, C
60.0, CH
30.6, CH2
173.5, C
59.7, CH
30.6, CH2
2
3
4.17, dd (9.0, 8.4)
2.02, m
4.16, t (9.1)
2.00, m
4.12, dd (9.3, 8.2)
1.97, m
1.80, m
1.77, m
1.79, m
3a
4
35.9, CH
19.4, CH2
2.29, m
36.1, CH
19.5, CH2
2.30, m
36.3, CH
19.2, CH2
2.23, m
1.95, m
1.97, m
2.02, m
1.43, m
1.42, m
1.39, m
5
23.4, CH2
1.84, m
23.6, CH2
1.82, m
26.1, CH2
1.40, m
1.31, m
1.32, m
6
7
70.7, CH
31.6, CH2
4.37, brs
70.9, CH
31.8, CH2
4.35, brs
64.0, CH
33.7, CH2
3.91, brs
2.32, m
2.26, m
1.96, m
1.61, brt (12.0)
3.98, ddd (11.1, 5.7, 5.4)
3.04, m
1.60, brt (12.0)
3.96, ddd (11.0, 6.1, 5.8)
3.03, m
1.65, brt (12.0)
4.00, ddd (11.2, 6.5, 5.9)
7a
54.1, CH
37.9, CH2
25.8, CH2
25.9, CH2
40.2, CH2
156.6, C
54.3, CH
38.0, CH2
26.0, CH2
26.4, CH2
40.5, CH2
156.8, C
54.1, CH
c
Agm 1
4.48, d (2.8)
d
2
1.40, m
1.40, m
7.23, s
d
3
1.43, m
1.41, m
6.83, s
4
3.09, m
3.08, m
5
1-NH
7.71, t (5.2)
7.71, brt (5.5)
4-NH
7.47, m
7.43, brt (5.5)
a
b
c
d
1
400 MHz for H, 100 MHz for 13C. Multiplicities and assignments from HSQC experiment. 6-OH. NH2.
C32H4535ClN6O6 and 14 degrees of unsaturation. The 1H NMR
spectrum (Table 2) of 3 in DMSO-d6 was closely related to the
spectrum of microcin SF608, which differed from 3 by 34 mass
confirmed this suggestion. HMBC correlations of C-4 with H-3
and H-3′, COSY correlations of H-2 with H-3, H-3′, and 2-OH,
and an HMBC correlation of 2-OH with the carbonyl carbon
resonating at δC 172.6 established this moiety as an o-Cl-Hpla.
The application of Marfey’s method27 (L-FDAA) and the chiral-
phase HPLC, as described above for 1, revealed the presence of
L-Choi, L-Phe, and L-o-Cl-Hpla moieties in 3. On the basis of
the evidence discussed above and the close similarity to
microcin SF608 the structure of aeruginosin DA642A (3) was
established as L-o-Cl-Hpla-L-Phe-L-Choi-agmatine.
Aeruginosin DA642B (4) exhibited analogous NMR data
(Tables 2, S4a, and S4b) and the same molecular formula when
compared with the data of 3. Analyses of the data from the 2D
NMR experiments revealed that 4 was assembled from the
same four planar subunits, o-Cl-Hpla, Phe, Choi, and agmatine,
in the same linear order as 3. Some minor differences were
observed in the chemical shifts of the Hpla-2-OH, Phe-C-2, and
1
units. The main differences between the H NMR spectra of
the two compounds were located in the aromatic region of the
spectra where the para-substituted phenol spin system in
microcin SF608 was substituted by a nonsymmetric 1,2,4-
trisubstituted phenyl spin system in 3. The two rotamers of 3
were fully characterized (see Table 2 for the NMR data of the
major trans rotamer and Tables S3a and S3b for the cis and
trans rotamers). The trisubstituted phenyl moiety was
established, based on the COSY correlations, as an o,p-
disubstituted phenol, while the HMBC correlations (Table 2)
suggested that an alkyl substituent was attached to the ring in
the para position and a chlorine was attached in the ortho
position. A comparison of the carbon chemical shifts of the
latter moiety with that of the o-Cl-Hpla in aeruginosin KY60828
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Journal of Natural Products
Article
Phe-NH signals (Table 2 and Figures S3b and S4b) when the
1H and 13C NMR data of 3 and 4 were compared. These results
were similar to those previously found for aeruginosins
KT608A and KT608B,14 suggesting that either Phe or Hpla
differed in their absolute configurations in 3 and 4. As discussed
above for 1, NMR analysis and applying Marfey’s method27 (L-
FDAA) and chiral-phase HPLC revealed that the L-Phe and L-
Choi were identical with those of 3, while the o-Cl-Hpla moiety
was of the D-series in 4. This procedure established the
structure of aeruginosin DA642B (4) as D-o-Cl-Hpla-L-Phe-L-
Choi-agmatine.
Aeruginosin DA495B (7) was isolated as a glassy material,
which presented a positive HRESIMS molecular adduct ion at
m/z 518.2031/520.2043 (3:1, [M + Na]+), corresponding to
the molecular formula C24H34ClN3NaO6 and nine degrees of
1
unsaturation. The H NMR spectrum (Table 3) in DMSO-d6
revealed that 7 existed as a ca. 1:3 mixture of the cis and trans
rotamers of the amide bond between the Choi moiety and the
adjacent leucine moiety. The 13C NMR spectrum (Table 3) of
7 was comparable to that of 5 except for the missing guanidine
carbon, the shift of a methine carbon from 70.7 (in 5) to 64.0
(in 7) ppm, which indicated a Choi-6-OH moiety, and the
abolition of four methylene carbons from the aliphatic region
(Figure S7b). The two rotamers were fully characterized (Table
3 for the NMR data of the major trans rotamer and Tables S7a
and S7b for the full data of both rotamers). The structure
elucidation of the major, trans, rotamer is discussed below.
Analyses of the data from the COSY, TOCSY, HSQC, and
HMBC 2D NMR experiments assigned the structures of o-Cl-
Hpla (similar to that of 5), Leu, and Choi-amide moieties
(Table S7a). The sequence of the subunits was determined as
o-Cl-Hpla-Leu-Choi-amide on the basis of NOE correlations of
o-Cl-Hpla-H-2 with Leu-NH, of Leu-H-2 with Choi-H-7a, and
of Choi-H-2 with the primary amide protons and the HMBC
correlation o-Cl-Hpla-CO with the Leu-NH. Applying Marfey’s
method27 (L-FDAA) and chiral-phase HPLC revealed the
presence of L-Choi, D-Leu, and D-o-Cl-Hpla moieties in 7. On
the basis of the above evidence the structure of aeruginosin
DA495B (7) was established as D-o-Cl-Hpla-D-Leu-L-Choi-
amide.
Microguanidine DA368 (8) was isolated as a transparent,
glassy material that exhibited an HRESIMS molecular cluster
ion at m/z 369.2860 ([M + H]+), which matched the molecular
formula C19H37N4O3 and four degrees of unsaturation. The 1H
NMR spectrum of 8 in DMSO-d6 (Table 4) was relatively
broad, displaying a broad signal of three exchangeable protons
(based on the HSQC spectrum) in the aromatic region, three
vinyl protons and one exchangeable proton around 5 ppm, six
protons adjacent to electronegative substituents [δH 3.96 brd
(2H), 3.85 brs, 3.64 brd, 3.42 and 3.02 m], a nine-proton
singlet (δH 3.10), and two broad singlet methyl groups (δH 1.71
and 1.65), reminiscent of the characteristic protons of
microguanidine AL772, which was isolated and published by
us several years ago.30 The 13C NMR spectrum (Table 4)
revealed, among other signals, a carbonyl, guanidinium carbon,
two carbons adjacent to electronegative substituents, two
methylenes next to nitrogen, and a trimethylammonium residue
(δC 50.9, CH3). An interpretation of the COSY, TOCSY,
HSQC, and HMBC data (Table 4) allowed for the full
assignment of the structure of the oxidized geranyl moiety and
of the Nα,Nα,Nα-trimethyl-Nε-substituted arginine, which
were assembled to the complete structure through HMBC
correlations of H-5 with C-12 and of H-5 and H2-12 with C-7
(the guanidine carbon). This established the planar structure of
amphiphilic microguanidine DA368 as 8. The absolute
configurations of C-2 and C-17 were not determined due to
the small amount of material that was isolated.
Aeruginosin DA688 (5) was obtained as a pale yellow, glassy
material, which presented a positive HRESIMS molecular
adduct ion at m/z 711.2540/713.2585 (3:1, [M + Na]+),
corresponding to the molecular formula C29H45ClN6NaO9S
and 12 degrees of unsaturation. The 1H NMR spectrum (Table
3) in DMSO-d6 suggested that 5 existed as a ca. 1:2 mixture of
the cis and trans rotamers of the amide bond between the Choi
moiety and the adjacent leucine moiety. The two rotamers were
fully characterized (Table 3 for the NMR data of the major
trans rotamer and Tables S5a and S5b for the full data of both
rotamers). The structure elucidation of the major trans rotamer
is discussed below. Analyses of the data from the COSY,
TOCSY, HSQC, and HMBC 2D NMR experiments assigned
the structures of the agmatine, Leu, and o-Cl-Hpla (similar to
those of 3 and 4) moieties (Table S5a). The double doublet
proton resonating at δH 4.17 and the broad singlet at δH 4.37
were characteristic of a Choi-6-sulfate moiety.29 COSY
correlations established all of the expected connectivities
between the protons of the Choi moiety, except those of H2-
4 and H-3a. A comparison of the chemical shifts and
multiplicities of the Choi moiety of 5 with those of aeruginosin
98-A29 confirmed the suggested Choi-6-sulfate structure. The
sequence of the subunits of 5 was determined as o-Cl-Hpla-
Leu-Choi-6-sulfate-agmatine on the basis of the HMBC
correlation of o-Cl-Hpla-CO with Leu-NH, the NOE
correlation of Leu-H-2 and Choi-H-7a, and the HMBC
correlation of Choi-CO with agmatine-1-NH. The application
of Marfey’s method27 (L-FDAA) and chiral-phase HPLC
revealed the presence of L-Choi, D-Leu, and D-o-Cl-Hpla
moieties in 5. On the basis of the evidence discussed above, the
structure of aeruginosin DA688 (5) was established as D-o-Cl-
Hpla-D-Leu-L-Choi-6-sulfate-agmatine.
Aeruginosin DA722 (6) eluted from the HPLC column right
after 5 as a pale yellow, glassy material. It presented a negative
HRESIMS molecular ion at m/z 721.2203/723.2231/725.2165
(9:6:1, [M − H]−), which was characteristic of two chlorine
atoms and corresponded to a molecular formula of
C29H43Cl2N6O9S. The 1H NMR spectrum (Table 3) in
DMSO-d6 suggested that 6 existed as a ca. 1:2 mixture of the
cis and trans rotamers and was similar to that of 5. The major
difference between the 1H NMR spectra of 5 and 6 was noticed
in the aromatic region, where the three pairs of protons of the
o-Cl-Hpla in 5 were substituted by a pair of two-proton singlet
signals in 6, suggesting that the phenolic ring is symmetrically
substituted by two chlorine atoms. The rest of the molecule was
similar to that of 5. The application of Marfey’s method27 (L-
FDAA) and chiral-phase HPLC revealed the presence of L-
Choi, D-Leu, and D-o,o-diCl-Hpla moieties in 6. On the basis of
the evidence discussed above, the structure of aeruginosin
DA722 (6) was established as D-o,o-di-Cl-Hpla-D-Leu-L-Choi-6-
sulfate-agmatine.
Anabaenopeptin DA850 (9) presented an HRESIMS
molecular cluster ion at m/z 851.4774 ([M + H]+) and
NMR spectra in DMSO-d6 similar to that of anabaenopeptin
F.31,32 A complete structure elucidation of 9, based on the
interpretation of the COSY, TOCSY, HSQC, and HMBC data
(Table S8), revealed that indeed its planar structure is identical
with that of anabaenopeptin F, cyclo-(Phe-NMeAla-Hty-Ile-Lys-
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Marfey’s method27 (L-FDAA) to the hydrolysate of 9
established the absolute configuration of the amino acids that
build 9 as L-Phe, L-NMeAla, L-Hty, L-Ile, D-Lys, and L-Arg,
confirming that the difference between the isoleucine moieties
in both compounds is in the β-position. On the basis of these
arguments the structure of anabaenopeptine DA850 (9) was
established as cyclo-(L-Phe-L-NMeAla-L-Hty-L-Ile-D-Lys-Nε)-
Nα-CO-L-Arg-CO2H.
Biological Activities. The extracts of strain IL-374
exhibited significant inhibition of the serine protease trypsin
at a concentration of 1 mg/mL. The activity-guided purification
of the protease-inhibiting components of the extract revealed
that the known micropeptins MZ924, MZ939A, MZ1019,22
cyanopeptolin S,23 and cyanopeptolin SS24 and the new
aeruginosins DA642A (3), DA642B (4), DA688 (5), and
DA722 (6) were responsible for the inhibition of trypsin.
The inhibitory activities of 1−9 were determined for the
serine proteases trypsin and thrombin. Aeruginosin DA642A
(3) inhibited trypsin with an IC50 of 30.8 μM and thrombin
with an IC50 of 15.5 μM. Aeruginosin DA642B (4) inhibited
trypsin with an IC50 of 19.0 μM, but not thrombin at a
concentration of 45.5 μM. Aeruginosins DA688 (5) and
DA722 (6) inhibited trypsin with IC50 values of 9.5 and 7.3
μM, respectively, but not thrombin at a concentration of 45.5
μM. Aeruginosins DA495A (1) and DA511 (2), micro-
guanidine DA368 (8), and anabaenopeptin DA850 (9) did
not inhibit trypsin and thrombin at a concentration of 45.5 μM.
Table 4. NMR Data of Microguanidine DA369 (8) in
DMSO-d6
a
δH, mult., J
(Hz)
HMBC
correlations
NOE
d
b
c
position
δC, mult.
correlations
1
2
168.6, C
76.3, CH
3.64, d
(11.2)
1,3,11,11′,11″
4,4′11,11′,11″
3
22.4, CH2 1.81, m
1.66, m
5
4
23.8, CH2 1.77, m
1.52, m
2,4′,11,11′,11″
2,4
4′
5
46.6, CH2 3.42, t
(10.6)
5′
5′
7
3.08, m
156.0, C
7
5,12
8,9
7.49, brs
12
11−11″
12
50.9, CH3 3.10, s
2,11′,11″
5,7,13,14
2,4
45.6, CH2 3.91, d
(5.3)
5′,8,9,13,15,20
13
119.2, CH
5.07, brt
(5.3)
12,15,20
12,20
14
15
140.9, C
27.8, CH2 2.06, t
(7.4)
13,14,16,17,20 12,16,16′,17
16
17
33.4, CH2 1.52, m
1.45, m
15,17,18
15,17
15
14,15,17,18
73.5, CH
3.85, brs
15,16,17-
OH,19,19′
18
19
148.5, C
110.1, CH
4.89, s
4.75, s
17,21
17,17-
CONCLUDING REMARKS
■
OH,19′,21
Aeruginosins 1 and 2 compose a new subgroup of aeruginosins
that contain a new stereoisomer of the Choi core. This isomer,
2S,3aS,6S,7aS-Choi (L-6-epi-Choi), adds to the three known
isomers (2S,3aS,6R,7aS)-Choi (L-Choi),8 (2S,3aR,6R,7aR)-
Choi (L-3a,7a-diepi-Choi),13 and 2R,3aR,6R,7aR-Choi (D-
3a,7a-diepiChoi).14 A proposed biogenesis (Scheme S1) is
based on the one suggested by Ishida et al. for L-Choi
biosynthesis in aeruginoside 126.12 The variety of enzymatic
processes demonstrates the versatility of the biosynthetic
machinery utilized by cyanobacteria for the synthesis of
secondary metabolites. This strategy allows cyanobacteria to
produce peptide-mimicking building blocks that interfere with
the degradation of these short peptides.
19′
17,21
17,17-OH,19,21
2
20
23.3, CH3 1.71, s
18.1, CH3 1.65, s
13,14,15
17,18,19
16,17
12,13
21
19,19′
17,19,19′
17-OH
4.98, brs
a
b
500 MHz for H, 125 MHz for 13C. Multiplicities and assignments
from HSQC experiment. HMBC correlations, optimized for 8 Hz, are
from the proton(s) stated to the indicated carbon. Selected NOEs
1
c
d
from ROESY experiment.
1
Nε)-Nα-CO-Arg-CO2H. The H and 13C NMR spectra of 9
and anabaenopeptin F could be superimposed on one another,
except for the signals of their Ile moieties (Tables 5 and S8).
Such chemical shift differences were noticed by us in the
micropeptins where Ile or allo-Ile are situated at the C-terminus
of the peptide33 or at the fourth position.14 In anabaenopeptin
F, the latter amino acid was determined as L-allo-Ile.32 Applying
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1010 polarimeter. UV spectra were recorded
on an Agilent 8453 spectrophotometer. IR spectra were recorded on a
Bruker Vector 22 spectrometer. NMR spectra were recorded on a
Table 5. Comparison of the NMR Data of the Ile Moieties of
a
1
Anabaenopeptins DA850 (9) and F in DMSO-d6
Bruker DMX-500 spectrometer at 500.13 MHz for H and 125.76
MHz for 13C and a Bruker Avance 400 spectrometer at 400.13 MHz
for 1H and 100.62 MHz for 13C, DEPT, COSY-45, gTOCSY,
gROESY, gHSQC, and gHMBC spectra were recorded using standard
Bruker pulse sequences. Mass spectra were recorded on a Waters
MaldiSynapt instrument. HPLC separations were performed on a
JASCO HPLC system (model PU-2080 Plus pump, model LG-2080-
04 Quaternary Gradient unit, and model PU-2010 Plus multi-
wavelength detector), on a Merck Hitachi HPLC system (model L-
6200A pump and model L-4200 UV−vis detector), and on a Merck
Hitachi HPLC system (L-7000A intelligent pump and model L-6200
UV−vis detector). An Elisa ELx808 reader (Bio-Tek Instruments, Inc.)
was used for protease inhibition assays.
anabaenopeptin DA850 (9)
anabaenopeptin F
b
b
position
δC, mult.
δH, mult., J (Hz)
δC, mult.
δH, mult., J (Hz)
Ile 1
2
172.6, C
56.3, CH
172.7, C
56.6, CH
4.08, dd (7.5,
7.0)
3.97, dd (8.5,
7.3)
3
4
36.3, CH
1.77, m
35.7, CH
1.78, m
25.0, CH2 1.16, m
1.61, m
24.7, CH2 1.14, m
5
11.5, CH3 0.89, t (7.5)
14.9, CH3 0.88, d (6.5)
6.91, d (7.0)
10.3, CH3 0.82, t (7.3)
14.9, CH3 0.88, d (6.4)
6.97, d (7.3)
6
NH
Biological Material. Microcystis aeruginosa, TAU strain IL-374, was
collected in October 2007, from the Dalton water reservoir, Gush
Halav, Israel. Samples of the cyanobacteria are deposited at the culture
collection of Tel Aviv University.
a
b
1
500 MHz for H, 125 MHz for 13C. Multiplicities and assignments
from HSQC experiment.
2313
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Journal of Natural Products
Article
Isolation Procedure. The freeze-dried cells (IL-374, 1600 g) were
extracted with 7:3 MeOH/H2O (3 × 3 L). The extract was evaporated
to dryness (98.5 g) and separated on an ODS (YMC-GEL, 120A, 4.4
× 6.4 cm, 10 portions of 10 g each) flash column, eluting with
increasing amounts of MeOH in H2O. Fraction 6 (1:1 MeOH/H2O,
2.645 g) was subjected to a Sephadex LH-20 column eluting with 7:3
MeOH/H2O to obtain 14 fractions. Fractions 5−7 from the Sephadex
LH-20 column were again subjected to a Sephadex LH-20 column
eluting with 1:1 MeOH/H2O to obtain 14 fractions. Fraction 7 from
the second Sephadex LH-20 column (30.5 mg) was separated on a
reversed-phase HPLC column (Hibar, 5 μm, 250 mm × 25.0 mm,
DAD at 238 nm, flow rate 5.0 mL/min) eluting with 77:23 0.1%
aqueous TFA/CH3CN to obtain pure compounds 5 (1.1 mg,
retention time 32.0 min, 0.0001% yield based on the dry weight of
the bacteria) and 6 (0.9 mg, retention time 40.8 min, 0.0001% yield).
Fraction 9 from the second Sephadex LH-20 column (28.4 mg) was
separated on a reversed-phase HPLC column (Hibar, 5 μm, 250 mm ×
25.0 mm, DAD at 238 nm, flow rate 5.0 mL/min) eluting with 72:28
0.1% aqueous TFA/CH3CN to obtain pure compounds 1 (1.7 mg,
retention time 20.8 min, 0.0001% yield) and 2 (2.0 mg, retention time
22.6 min, 0.0001% yield). Combined fractions 7 and 8 from the initial
reversed-phase column (6:4 and 7:3 MeOH/H2O, 2.929 g) was loaded
on a Sephadex LH-20 column eluting with 1:1 CHCl3/MeOH to
obtain 14 fractions. Combined fractions 3 and 4 from this Sephadex
LH-20 column were subjected to an additional Sephadex LH-20
column eluting with 7:3 MeOH/H2O to obtain 14 fractions. Fractions
8−11 from the second Sephadex LH-20 column (95.4 mg) were
separated on a reversed-phase HPLC column (YMC-Pack ODS-A, 5
μm, 250 mm × 20.0 mm, DAD at 238 nm, flow rate 5.0 mL/min) in
1:1 0.1% aqueous TFA/MeOH to obtain pure compound 3 (4.0 mg,
retention time 17.2 min, 0.0003% yield), compound 4 (3.0 mg,
retention time 14.9 min, 0.0002% yield), compound 7 (2.4 mg,
retention time 16.5 min, 0.0002% yield), and microcin SF608 (4.5 mg,
retention time 21.3 min, 0.0003% yield). Fractions 4−7 from the
second Sephadex LH-20 column (379 mg) were separated on a
reversed-phase HPLC column (YMC-Pack C-8, 5 μm, 250 mm × 20.0
mm, DAD at 238 nm, flow rate 5.0 mL/min) in 1:1 0.1% aqueous
TFA/MeOH to obtain pure anabaenopeptin DA850 (9) (2.1 mg,
retention time 23.9 min, 0.0001% yield). Fraction 9 from the initial
reversed-phase column (4:1 MeOH/H2O, 1.842 g) was subjected to a
Sephadex LH-20 column eluting with 1:1 CHCl3/MeOH to obtain 13
fractions. Fractions 4−8 from the Sephadex LH-20 column were
subjected to an additional Sephadex LH-20 column eluting with 7:3
MeOH/H2O to obtain 15 fractions. Fractions 3−5 from the second
Sephadex LH-20 column (184.8 mg) were separated on a reversed-
phase HPLC column (YMC-Pack ODS-A, 5 μm, 250 mm × 20.0 mm,
DAD at 238 nm, flow rate 5.0 mL/min) in 1:1 H2O/CH3CN to obtain
five fractions, some of which contain aeruginazoles DA1497, DA1304,
and DA1274.21 Fraction 2 from the reversed-phase HPLC was
separated again by a reversed-phase HPLC column (YMC-Pack C-8, 5
μm, 250 mm × 20.0 mm, DAD at 238 nm, flow rate 5.0 mL/min) in
55:45 0.1% aqueous TFA/MeOH to obtain pure compound 8 (2.6
mg, retention time 29.0 min, 0.0002% yield). Fraction 5 from the
initial reversed-phase column was subjected to a Sephadex LH-20
column eluting with 1:1 CHCl3/MeOH to obtain 10 fractions.
Fractions 4−7 from the second Sephadex LH-20 column (211.5 mg)
were separated on a reversed-phase HPLC column (YMC-Pack C-8, 5
μm, 250 mm × 20.0 mm, DAD at 238 nm, flow rate 5.0 mL/min) in
55:45 0.1% aqueous TFA/MeOH to obtain micropeptin MZ1019 (2.2
mg, retention time 20.7 min, 0.0001% yield), cyanopeptolin SS (1.4
mg, retention time 30.0 min, 0.000 06% yield), micropeptin MZ924
(0.7 mg, retention time 39.7 min, 0.000 03% yield), micropeptin
MZ939A (1.8 mg, retention time 42.1 min, 0.000 11% yield), and
cyanopeptolin S (1.3 mg, retention time 51.2 min, 0.000 05% yield).
3.95, D-Hpla 4.24 min). Retention times of AA Marfey’s derivatives: D-
phenylalanine 46.9 min (L-Phe 44.9, D-Phe 46.9 min), L-6-epi-Choi
34.9 min (L-Choi 36.5, L-6-epi-Choi 34.9 min, D-3a,7a-diepi-Choi 31.0
min).
Aeruginosin DA511 (2): colorless oil; [α]25 −134 (c 0.19,
D
MeOH); UV (MeOH) λmax (log ε) 207 (4.06), 225 (3.95), 277 (3.41)
nm; IR (KBr) νmax 3460, 1684, 1205, 1141 cm−1; NMR data, see
Tables 1, S2a, and S2b; HRESIMS m/z 534.2212 [M + Na]+ (calcd for
C27H33N3NaO7, 534.2216). Retention time of hydroxy acid on chiral-
phase column: D-Hpla 4.28 min (L-Hpla 3.99, D-Hpla 4.28 min).
Retention times of AA Marfey’s derivatives: D-tyrosine 52.1 min (L-Tyr
50.4, D-Tyr 52.1 min), L-6-epi-Choi 35.1 min (L-Choi 36.6, L-6-epi-
Choi 35.1 min, D-3a,7a-diepi-Choi 31.0 min).
Aeruginosin DA642A (3): colorless oil; [α]25 −63 (c 0.31,
D
MeOH); UV (MeOH) λmax (log ε) 203 (4.42), 229 (3.72), 281 (3.18)
nm; IR (KBr) νmax 3450, 2935, 1678, 1204, 1138 cm−1; NMR data, see
Tables 2, S3a, and S3b; HRESIMS m/z 643.3016/645.3014 (3:1), [M
+ H]+ (calcd for C32H4535ClN6O6, 643.3011). Retention time of
hydroxy acid on chiral-phase column: L-Cl-Hpla 3.93 min (L-Cl-Hpla
3.93, D-Cl-Hpla 4.29 min). Retention times of AA Marfey’s derivatives:
L-phenylalanine 44.6 min (L-Phe 44.6, D-Phe 46.5 min), L-Choi 36.5
min (L-Choi 36.5, L-6-epi-Choi 34.9 min).
Aeruginosin DA642B (4): colorless oil; [α]25 −36 (c 0.28,
D
MeOH); UV (MeOH) λmax (log ε) 202 (4.32), 228 (3.63), 281 (3.01)
nm; IR (KBr) νmax 3450, 2934, 1678, 1205, 1138 cm−1; NMR data, see
Tables 2, S4a, and S4b; HRESIMS m/z 643.3014/645.3006 (3:1), [M
+ H]+ (calcd for C32H4535ClN6O6, 643.3011). Retention time of
hydroxy acid on chiral-phase column: D-Cl-Hpla 4.27 min (L-Cl-Hpla
3.93, D-Cl-Hpla 4.27 min). Retention times of AA Marfey’s derivatives:
L-phenylalanine 44.6 min (L-Phe 44.6, D-Phe 46.5 min), L-Choi 36.5
min (L-Choi 36.5, L-6-epi-Choi 35.2 min).
Aeruginosin DA688 (5): pale yellow, glassy material; [α]25D −160
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.19), 229 (3.78), 279
(3.34) nm; IR (KBr) νmax 2924, 1682, 1205, 1140 cm−1; NMR data,
see Tables 3, S5a, and S5b; HRESIMS m/z 711.2540/713.2585 (3:1),
[M + Na]+ (calcd for C29H4535ClN6NaO9S, 711.2555). Retention time
of hydroxy acid on chiral-phase column: D-Cl-Hpla 4.28 min (L-Cl-
Hpla 3.81, D-Cl-Hpla 4.28 min). Retention times of AA Marfey’s
derivatives: D-leucine 47.1 min (L-Leu 44.4, D-Leu 47.1 min), L-Choi
36.6 min (L-Choi 36.6, L-6-epi-Choi 35.3 min).
Aeruginosin DA722 (6): pale yellow, glassy material; [α]25D −135
(c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (4.23), 226 (3.68),
285 (3.04) nm; IR (KBr) νmax 2928, 1666, 1205 cm−1; NMR data, see
Tables 3 and S6a; HRESIMS m/z 721.2203/723.2231/725.2165
(9:6:1), [M − H]− (calcd for C29H4335Cl2N6O9S, 721.2189).
Retention time of hydroxy acid on chiral-phase column: D-di-Cl-
Hpla 4.31 min (L-di-Cl-Hpla 3.81, D-di-Cl-Hpla 4.31 min). Retention
times of AA Marfey’s derivatives: D-leucine 46.6 min (L-Leu 43.9, D-
Leu 46.6 min), L-Choi 36.4 min (L-Choi 36.4, L-6-epi-Choi 35.0 min).
Aeruginosin DA495B (7): colorless, glassy material; [α]25D −44 (c
0.13, MeOH); UV (MeOH) λmax (log ε) 202 (3.88), 227 (3.20), 280
(2.69) nm; IR (KBr) νmax 1685, 1206, 1140 cm−1; NMR data, see
Tables 3, S7a, and S7b; HRESIMS m/z 518.2031/520.2043 (3:1), [M
+ Na]+ (calcd for C24H3435ClN3NaO6, 518.2034). Retention time of
hydroxy acid on chiral-phase column: D-Cl-Hpla 4.20 min (L-Cl-Hpla
3.88, D-Cl-Hpla 4.20 min). Retention times of AA Marfey’s derivatives:
D-leucine 47.1 min (L-Leu 44.4, D-Leu 47.1 min), L-Choi 36.8 min (L-
Choi 36.8, L-6-epi-Choi 35.6 min).
Microguanidine DA368 (8): transparent, glassy material; [α]25
D
−78 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 202 (3.81), 223
(3.74), 278 (3.05); IR (KBr) νmax 3445, 1647, 1206, 1139 cm−1; NMR
data, see Table S8; HRESIMS m/z 369.2860 ([M + H]+ (calcd for
C19H37N4O3 m/z 369.2871).
Anabaenopeptin DA850 (9): white, glassy material; [α]25D −145
(c 0.15, MeOH); UV (MeOH) λmax (log ε) 202 (4.21), 273 (2.63); IR
(KBr) νmax 3448, 1664, 1206, 1139 cm−1; NMR data, see Table S9;
HRESIMS m/z 851.4774 [M + H]+ (calcd for C42H63N10O9,
851.4779). Retention times of AA Marfey’s derivatives: L-Phe 45.4
min (L-Phe 45.4, D-Phe 57.5 min), L-NMe-alanine 35.5 min (L-NMeAla
35.5, D-NMeAla 36.2 min), L-homotyrosine 40.8 min, L-isoleucine 44.2
Aeruginosin DA495A (1): colorless oil; [α]25 −48 (c 0.15,
D
MeOH); UV (MeOH) λmax (log ε) 201 (4.14), 225 (3.69), 277 (2.94)
nm; IR (KBr) νmax 3450, 1680, 1206, 1138 cm−1; NMR data, see
Tables 1, S1a and S1b in Supporting Information; HRESIMS m/z
518.2266 [M + Na]+ (calcd for C27H33N3NaO6, 518.2267). Retention
time of hydroxy acid on chiral-phase column: D-Hpla 4.24 min (L-Hpla
2314
dx.doi.org/10.1021/np4006844 | J. Nat. Prod. 2013, 76, 2307−2315

Journal of Natural Products
Article
min (L-Ile 44.2, D-Ile 47.2 min), D-lysine 46.1 min (L-Lys 45.7, D-Lys
46.1 min), and D-arginine 30.8 min (L-Arg 29.7, D-Arg 30.8 min).
Determination of the Absolute Configurations of the Amino
Acids. Compounds 1−7 and 9 (0.3 mg portions) were dissolved in 6
N HCl (1 mL). The reaction mixture was then placed in a sealed glass
bomb at 110 °C for 20 h. After the removal of HCl, by repeated
evaporation in vacuo, the hydrolysate was resuspended in H2O (250
μL). A solution of (1-fluoro-2,4-dinitrophenyl)-5-L-alanine amide
(FDAA) in acetone (1.2 equiv) and 1 N NaHCO3 (120 μL) was
added to each reaction vessel, and the reaction mixture was stirred at
70 °C for 3 h. A 2 N HCl solution (60 μL) was added to each reaction
vessel, and the solution was evaporated in vacuo. The N-
[(dinitrophenyl)-5-L-alanine amide]-amino acid derivatives, from
hydrolysates, were compared with similarly derivatized standard
amino acids by HPLC analysis: Knauer GmbH Eurospher 100 C18,
10 μm, 4.6 × 300 mm, flow rate 1 mL/min, UV detection at 340 nm,
linear gradient elution from 100%, 0.1% TFA in H2O to 1:1, 0.1% TFA
in H2O/CH3CN, within 60 min. The absolute configuration of each
amino acid was confirmed by spiking the derivatized hydrolysates with
the derivatized authentic amino acids.
Determination of the Absolute Configurations of the
Hydroxy Acids. Compounds 1−7 (0.25 mg portions) were dissolved
in 6 N HCl (1 mL), and the reaction mixture was then placed in a
sealed glass bomb at 110 °C for 20 h. The ethereal extract of the acid
hydrolysate of 1−7 was removed in vacuo, and the residue was
dissolved in MeOH (1 mL). The MeOH solution was analyzed on an
Astec, Chirobiotic, LC stationary phases, 250 × 4.6 mm flow rate 1
mL/min, UV detection at 210 nm, linear elution 1:9 1% triethylamine,
1% acetic acid aqueous buffer, pH 4/MeOH. The Hpla residues from
the aeruginosins were compared with standard L,D-Hpla.
ACKNOWLEDGMENTS
■
The authors thank N. Tal, the Mass Spectrometry Facility of
the School of Chemistry, Tel Aviv University, for the
measurements of the HRESI mass spectra and J. Bonjoch
Faculty of Pharmacy, University of Barcelona, Spain, for the
kind provision of the synthetic Choi isomers. This research was
supported by the Israel Science Foundation grant 776/06.
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ASSOCIATED CONTENT
■
S
* Supporting Information
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Full tabulated NMR data, 1D and 2D NMR spectra, and
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material is available free of charge via the Internet at http://
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AUTHOR INFORMATION
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Corresponding Author
*Tel: ++972-3-6408550. Fax: ++972-3-6409293. E-mail:
carmeli@post.tau.ac.il.
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Notes
The authors declare no competing financial interest.
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