Bromine-and chlorine-containing aeruginosins from microcystis aeruginosa bloom material collected in Kibbutz Geva, Israel

Article abstract of DOI:10.1021/np3005612

Five new natural products, aeruginosins GE686 (1), GE766 (2), GE730 (3), GE810 (4), and GE642 (5), were isolated along with four known aeruginosins, 98C, 101, KY642, and DA688, from bloom material of the cyanobacterium Microcystis aeruginosa collected from a fish pond in Kibbutz Geva, Israel, in August 2007. Their structures were elucidated by a combination of various spectroscopic techniques, primarily NMR and MS, while the absolute configurations of the stereogenic centers were determined by Marfey's and chiral-phase HPLC methods. Two of the new aeruginosins, aeruginosins GE686 (1) and GE766 (2), contain the unprecedented d-m-Br-m′-Cl-p-hydroxyphenyllactic acid derivative. The structures and biological activities of the five new metabolites are described.

Full text of DOI:10.1021/np3005612

Article
pubs.acs.org/jnp
Bromine- and Chlorine-Containing Aeruginosins from Microcystis
aeruginosa Bloom Material Collected in Kibbutz Geva, Israel
Shira Elkobi-Peer, Raya Faigenbaum, 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: Five new natural products, aeruginosins GE686
(1), GE766 (2), GE730 (3), GE810 (4), and GE642 (5), were
isolated along with four known aeruginosins, 98C, 101, KY642,
and DA688, from bloom material of the cyanobacterium
Microcystis aeruginosa collected from a fish pond in Kibbutz
Geva, Israel, in August 2007. Their structures were elucidated
by a combination of various spectroscopic techniques, primarily
NMR and MS, while the absolute configurations of the
stereogenic centers were determined by Marfey’s and chiral-
phase HPLC methods. Two of the new aeruginosins,
aeruginosins GE686 (1) and GE766 (2), contain the
unprecedented D-m-Br-m′-Cl-p-hydroxyphenyllactic acid derivative. The structures and biological activities of the five new
metabolites are described.
he aeruginosins are a group of linear modified peptides
characterized by the presence of a hydroxyphenyllactic
elucidation, and biological activity of the five new aeruginosins
isolated from this bloom material.
T
acid (Hpla) derivative at the N-terminus of the modified
peptide, a variable lipophilic amino acid at the second position,
a 2-carboxy-6-hydroxyoctahydroindole (Choi) derivative at the
third position, and an arginine derivative (if any) at the fourth
position.1 They are produced mainly by aquatic bloom-forming
cyanobacteria and possess serine-protease inhibitory activity.2
To date, 27 variants of this group of protease inhibitors have
been isolated and characterized from cyanobacteria extracts.3 A
wealth of additional aeruginosins have been identified by
tandem mass spectrometry analyses of cyanobacteria bloom
materials.4 Three related metabolites, suomilide5 and banya-
sides A and B,6,7 were isolated from cyanobacteria bloom
materials, while five other related metabolites, dysinosins A−D
and chlorodysinosin A, were isolated from the marine sponge
Lamellodysidea chlorea.8 These linear modified peptides are
biosynthesized in cyanobacteria from amino acids or amino acid
precursors by a nonribosomal peptide synthetase (NRPS)-type
enzyme assembly, which allows the incorporation of different
precursors at the N-terminus and the second positions, but
allows only stereochemical and substituent changes at the third
and fourth positions.9 As part of our continuing interest in the
chemical ecology of cyanobacteria water blooms and the search
for novel drugs for human diseases, we examined the extracts of
a Microcystis aeruginosa bloom material collected in August
2007 from a fishpond in Kibbutz Geva, Israel. The 70%
aqueous MeOH extract of the lyophilized bloom material
yielded five new aeruginosins, aeruginosins GE686 (1), GE766
(2), GE730 (3), GE810 (4), and GE642 (5), along with four
known ones, aeruginosins 98C (6),10 101 (7),10 KY642 (8),11
and DA688.12 In this article we report the isolation, structure
The nine natural products were isolated from the 70%
aqueous MeOH extract through separation on a reversed-phase
open column, size exclusion chromatography on Sephadex LH-
20, and repeated chromatography on various reversed-phase
HPLC columns.
Aeruginosin GE686 (1) was isolated as a yellowish glassy
material, which exhibited an HRESIMS complex molecular ion
adduct at m/z 687.2285/689.2256/691.2254 (5:8:3 [M + H]+),
characteristic of a molecule that contains both bromine and
chlorine atoms and corresponds to the molecular formula
1
C29H44BrClN6O6. The H and 13C NMR spectra of 1 were
characteristic of the aeruginosins and displayed two signals for
each proton and carbon, as 1 appeared as a 13:1 mixture of the
trans and cis rotamers.3 The 1H NMR spectrum (Table 1) of 1
presented a broad signal of a phenol, three pairs of amide
protons (a pair of doublets and two pairs of broad triplets), two
pairs of singlet signals in the aromatic region, two pairs of
exchangeable doublet signals, four pairs of methine protons
next to the electronegative atoms in the 5.5−3.8 ppm region,
and two broad signals around 3.05 ppm corresponding to four
protons. The aliphatic region was too complicated to be
interpreted except for a pair of doublet methyl groups and a
pair of distorted broad doublet methyl groups. The 13C NMR
spectrum (Table 1) revealed three pairs of amide/ester
carbonyls, two pairs of sp2 carbons characteristic of phenol
and/or guanidine carbons, five signals of aromatic sp2 carbons,
Received: August 17, 2012
Published: November 15, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Chart 1
five pairs of methine carbons between 73 and 50 ppm, and
several additional methine, methylene, and methyl signals in the
aliphatic region. The structure elucidation is based on the data
of the major trans rotamer (Table 1) since the correlations of
the cis rotamer were weak and resulted in only partial
substructures. The signals of the aromatic residue in the
NMR spectra of 1 in DMSO-d6 presented two singlet protons
(δH 7.20 and 7.32 s) and six carbons (δC 111.7, 121.4, 131.6,
and 148.4 C's, 129.8 and 132.9 CH's), suggesting that the
tetrasubstituted aromatic ring was not symmetric. The chemical
shifts of the quaternary carbons suggested their substitution by
bromine (δC 111.7), chlorine (δC 121.4), carbon (δC 131.6),
and phenol (δC 148.4) groups (by comparison with the
chemical shift of mixed halo-substituted N-acetyl-tyrosine
derivatives).13 HMBC correlations (Table S1 in the Supporting
Information) established the aromatic ring as an ortho-bromo,
ortho′-chloro, para-substituted phenol. The aromatic protons
(δH 7.20 and 7.32 s) presented HMBC correlations with a
methylene carbon (δC 38.5). Through COSY correlations the
protons of this methylene (HSQC) were found to be part of an
ABMX spin system of H-3,3′,2 and 2-OH of the novel
disubstituted Hpla moiety, m-Br-m′-Cl-p-hydroxyphenyllactic
acid. The carbonyl of this moiety was established through
HMBC correlations of H-2,3,3′ with the carbonyl resonating at
δC 171.7. The second residue that was established included the
doublet methyl group (δH 0.57) and the distorted doublet
methyl (δH 0.85, brd) that were correlated in the HSQC
spectrum with two carbon signals (δC 13.8 and 12.0,
respectively). These NMR signals were established as part of
an isoleucine moiety. Methyl-5 of the Ile appeared as a
distorted doublet due to the resonance of one of the
methylene-4 protons at the same chemical shift (δH 0.85 m,
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1
Table 1. H and 13C NMR Data of Aeruginosin GE686 (1)
proton resonating at 4.12 ppm, identified as the α-proton of an
amino acid (δC 60.1 ppm). This methine proton showed COSY
connectivity with methylene protons resonating at 1.74 and
1.94 ppm (δC 30.8 ppm). The latter two protons could be
further correlated with a methine proton at 2.26 ppm (δC 36.2
ppm). This methine showed three additional COSY
correlations with a methine proton resonating at 4.05 ppm
(δC 54.0 ppm, characteristic of a methine adjacent to a nitrogen
atom) and protons of a methylene resonating at 1.41 and 2.02
ppm (δC 19.1 ppm). The protons at 1.41 and 2.02 ppm show
COSY and HMBC correlations to further methylene protons at
1.42 ppm (δC 26.0 ppm), which in turn showed a COSY
correlation to a methine proton at 3.90 ppm (δC 64.1 ppm),
assigned as a carbinol moiety, and reinforced by a COSY
correlation and an HMBC correlation with an acidic proton at
4.52 ppm, the OH proton. The hydroxymethine proton at 3.90
ppm was connected (through COSY correlation) to protons of
a methylene resonating at 1.72 and 1.92 ppm (δC 33.7 ppm),
which were, in turn, connected to the methine at 4.05 ppm.
These correlations closed a hexanol ring and identified the
amino acid as Choi. The chemical shifts of the protons and
carbons of the identified moiety were almost identical with
those of the L-Choi moiety of aeruginosin KY642, which was
isolated from the same bloom material and previously
characterized by us from another bloom material.11 The
relative configurations of the asymmetric carbons of the Choi
were deduced from the results of a ROESY experiment as
follows: Choi-2 (δH 4.12 ppm) possessed a NOE correlation
a
and Aeruginosin GE766 (2) in DMSO-d6
aeruginosin GE686 (1)
aeruginosin GE766 (2)
δH, mult., J
(Hz)
δH, mult., J
(Hz)
b
b
position
δC, mult.
δC, mult.
bromo, chloro, 171.7, C
Hpla 1
171.9, C
2
3
71.5, CH
38.5, CH2 2.86, dd
(14.0, 3.4)
4.14, m
71.3, CH
4.12, m
39.5, CH2 2.87, dd
(14.0, 3.4)
3′
2.76, dd
(14.0, 5.3)
2.71, dd
(14.0, 5.3)
4
131.6, C
129.8, CH
121.4, C
148.4, C
111.7, C
132.9, CH
131.2, C
130.2, CH
121.3, C
149.0, C
111.5, C
132.6, CH
5
7.20, s
7.22, s
6
7
8
9
7.32, s
7.34, s
2-OH
7-OH
Ile
6.04, d (5.3)
9.77, brs
5.95, d (5.3)
168.6, C
51.6, CH
168.6, C
52.5, CH
2
4.54, dd (8.3,
4.0)
4.47, dd (6.6,
3.3)
3
38.1, CH
1.48, m
37.6, CH
1.51, m
4
26.3, CH2 1.03, m
0.85, m
26.0, CH2 1.10, m
0.90, m
4′
5
12.0, CH3 0.85, brd
(4.0)
11.9, CH3 0.85, t (5.6)
6
13.8, CH3 0.57, d (6.6)
7.25, d (8.3)
13.7, CH3 0.63, d (6.4)
NH
7.35, d
(10.0)
Choi 1
171.6, C
171.5, C
2
60.1, CH
4.12, t (9.2)
59.9, CH
4.16, t (9.2)
3
30.8, CH2 1.94, m
1.74, m
30.6, CH2 2.02, m
1.74, m
3′
3a
4
36.2, CH
2.26, m
35.9, CH
2.28, m
19.1, CH2 2.02, m
1.41, m
19.3, CH2 1.98, m
1.42, m
4′
5
26.0, CH2 1.42, m
23.3, CH2 1.85, m
1.32, m
6
64.1, CH
3.90, brs
70.8, CH
4.35, brs
7
33.7, CH2 1.92, m
1.72, m
31.6, CH2 2.24, m
1.68, m
7′
7a
54.0, CH
4.05, dt
(10.8, 5.6)
54.0, CH
4.00, dt
(10.0, 4.8)
Figure 1. Relative configuration of L-Choi deduced from NOE
correlations.
6-OH
4.52, brd
(2.0)
with a proton at 1.94 ppm (Choi-3). Choi-2 and -3 presented
NOE correlations with Choi-3a, which in turn had an NOE
correlation with Choi-7a. This set of NOEs was possible if all of
these protons are pointing to the same face of the pyrrolidine
residue. Proton 7a showed additional NOE correlation to a
proton at 1.92 ppm (Choi-7), which in turn had a correlation
with the hydroxy proton at 4.52 ppm. These correlations,
together with the correlation of the hydroxy proton with the
axial Choi-4 (δH 2.02 ppm), suggested that the hydroxy was
axial in the cyclohexane ring. Choi-6 appeared as a broad singlet
at 3.90 ppm, in accordance with its equatorial orientation. On
the basis of these arguments the relative configuration of the
Choi residue was suggested to be 2S*,3aS*,6R*,7aS*.
Interpretation of the data from the COSY, TOCSY, HSQC,
and HMBC 2D NMR experiments enabled the assignment of
the agmatine moiety (Table S1 in the Supporting Information).
The four residues were connected through HMBC correlations
(Hpla-CO with Ile-2, and NH and Choi-CO with Agm-1-NH)
Agm 1
38.1, CH2 3.07, m
2.98, m
38.4, CH2 3.03, m
1′
2
26.2, CH2 1.41, m
26.4, CH2 1.41, m
40.6, CH2 3.08, m
25.8, CH2 1.42, m
26.3, CH2 1.44, m
40.3, CH2 3.08, m
3
4
1-NH
4-NH
7.92, t (5.4)
7.85, t (5.4)
7.55, t (5.0)
7.54, brt
(5.0)
5
156.9, C
156.7, C
5-NH,NH2
6.90, brm
6.95, brm
a
b
400.13 MHz for 1H, 100.62 MHz for 13C. Multiplicity and
assignment from HSQC experiment.
based on the HSQC correlations). Interpretation of the data
from the COSY, TOCSY, HSQC, and HMBC 2D NMR
experiments enabled the assignment of the Choi residue (Table
S1 in the Supporting Information). An amide carbon resonating
at 171.6 ppm showed an HMBC correlation with a methine
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1
and ROESY correlations (Ile-2 with Choi-7a) to establish the
planar structure of 1. Applying Marfey’s method14 (L-FDAA)
revealed the presence of L-Choi and D-Ile/alloIle moieties in 1.
Chiral-phase HPLC chromatography and comparison of the
Hpla derivative obtained from the hydrolysis of 1 with the
corresponding synthetic Hpla derivatives synthesized according
to the procedure developed for N-acetyl-tyrosine13 established
it as D-m-Br-m′-Cl-p-Hpla (9) in 1. Despite the report in the
literature,15 we could not separate D-Ile from D-alloIle,
regardless of what column or mobile phase mixture we used.
In the case of the micropeptins, our previous experience shows
that the proton and carbon chemical shifts of Ile are highly
sensitive to the conformation and configuration of its
asymmetric centers.16 The chemical shifts of the carbons of
the isoleucine residue in 1 were similar to those of the D-alloIle
in aeruginosin 98-C10 (its structure was established by X-ray
diffraction, and it was isolated in this study as well). These
arguments suggest that D-alloIle and not D-Ile is incorporated
into 1. On the basis of the evidence discussed above, the
structure of aeruginosin GE686 (1) was established as D-m-Br-
m′-Cl-p-Hpla-D-alloIle-L-Choi-agmatine.
matched the molecular formula C29H45Br2N6O6. The H and
13C NMR spectra of 3 (Table 2) were similar to those of 1 and
appeared as a ca. 12:1 mixture of the trans and cis rotamers
around the Ile-CO-Choi-N bond.3 The differences observed in
the NMR spectra of 3, when compared with those of 1, were
the appearance of only one singlet proton signal (δH 7.35 s,
2H) in the aromatic region of the 1H NMR spectrum and only
four carbon signals (δC 111.5, 132.9, and 149.2 C's, 133.5
CH's) in the aromatic region of the 13C NMR spectrum of 3.
This observation suggested that the Hpla moiety is symmetric
and substituted in both ortho positions by two bromine atoms.
The rest of the moieties that compose 3 were similar to that of
1. An analysis of the 2D NMR spectra of 3 (Table S3 in the
Supporting Information) established the structure of the m,m-
di-Br-p-Hpla (10), Ile, Choi, and agmatine moieties. Applying
the same methodology described above established the
structure of aeruginosin GE730 (3) as D-m,m-di-Br-p-Hpla-D-
alloIle-L-Choi-agmatine.
Aeruginosin GE810 (4) was isolated as an amorphous solid
presenting an HRESIMS complex molecular adduct ion at m/z
833.1169/835.1136/837.1133 (1:2:1, [M + Na]+), correspond-
1
ing to the molecular formula C29H45Br2N6NaO9S. Its H and
13C NMR spectra (Table 2) resembled those of 3, and as in the
case of 1 and 2, it seems that 4 is the Choi-6-sulfate derivative
of 3. An analysis of the COSY, HSQC, HMBC, and ROESY
spectra of 4 (Table S4 in the Supporting Information) verified
this suggestion, while Marfey’s analysis14 (L-FDAA) and chiral-
phase HPLC chromatography revealed the presence of L-Choi,
D-alloIle, and D-m,m-di-Br-p-Hpla (10) moieties in 4. On the
basis of these results the structure of aeruginosin GE810 (4)
was established as D-m,m-di-Br-Hpla-D-alloIle-L-Choi-6-sulfate-
agmatine.
Figure 2. Synthetic Hpla derivatives.
Aeruginosin GE642 (5) was isolated as a glassy material,
which exhibited an HRESIMS complex molecular ion adduct at
m/z 643.2772/645.2777/647.2780 with (9:6:1, [M + H]+),
which is characteristic of a molecule that contains two chlorine
atoms11 and corresponds to the molecular formula
Aeruginosin GE766 (2) was isolated as an amorphous, white
material that presented an HRESIMS complex molecular ion
adduct at m/z 789.1649/791.1637/793.1608 (5:8:3, [M +
Na]+ ) corresponding to the molecular formula
C29H44BrClN6NaO9S. The 80 mass unit differences between
2 and 1 could be explained by a substitution of a hydroxy group
in 1 with a sulfate group in 2. The 1H and 13C NMR spectra of
2 (Table 1) were almost identical with those of 1 and appeared
as a ca. 12:1 mixture of the trans and cis rotamers around the
Ile-CO-Choi-N bond.3 The major differences were the absence
of the Choi-6-OH proton, the downfield shift of Choi-H-6 to
δH 4.35 brs, and the downfield shift of Choi-C-6 to δC 70.8 in
the NMR spectra of 3. All of these differences were in
accordance with a substitution of Choi-6-OH with a sulfate
group similar to the Choi-6-sulfate of aeruginosin 98-C.11 The
interpretation of the COSY, HSQC, HMBC, and ROESY 2D
NMR spectra (Table S2 in the Supporting Information)
established the structure of m-Br-m′-Cl-p-Hpla, Ile, Choi-6-
sulfate, and agmatine. Applying Marfey’s method14 (L-FDAA)
and chiral-phase HPLC chromatography revealed the presence
of L-Choi, D-Ile/alloIle, and D-m-Br-m′-Cl-p-Hpla (9) moieties
in 2. The chemical shifts of the carbons of the isoleucine
residue were almost identical with those of the D-alloIle of
aeruginosin 98-C,11 suggesting that 2 contains D-alloIle. ROESY
and HMBC correlations, as described for 1, established the
structure of aeruginosin GE766 (2) as D-m-Br-m′-Cl-p-Hpla-D-
alloIle-L-Choi-6-sulfate-agmatine.
1
C29H44Cl2N6O6. The H and 13C NMR spectra of 5 were
characteristic of the aeruginosins and displayed a 5:1 mixture of
trans and cis rotamers.3 The 1H NMR spectrum (Table 2) of 5
presented three pairs of amide protons (a pair of doublets and
two pairs of broad triplets), a pair of singlet signals in the
aromatic region, two pairs of exchangeable doublet signals, four
pairs of methine protons next to electronegative atoms in the
5.5−3.8 ppm region, and two broad signals around 3.05 ppm
corresponding to four protons. The aliphatic region was too
complicated to be interpreted except for two pairs of doublet
methyl groups. The 13C NMR spectrum (Table 2) revealed
three pairs of amide/ester carbonyls, two pairs of sp2 carbons
characteristic of phenol and/or guanidine carbons, three signals
of aromatic sp2 carbons, five pairs of methine carbons between
73 and 50 ppm, and several additional methine, methylene, and
methyl signals in the aliphatic region. The structure elucidation
is based on the data of the major trans rotamer, as the
correlations of the cis rotamer were weak and resulted in only
partial substructures. The interpretation of the data from the
COSY, TOCSY, HSQC, and HMBC 2D NMR experiments
enabled the assignment of the Leu and agmatine moieties
(Table S5 in the Supporting Information). The structure of the
m,m-dichloro-Hpla was elucidated as follows: COSY correla-
tions established the connectivity of the ABMX spin system of
H-3,3′,2 and 2-OH. The two-proton singlet aromatic signal at
δH 7.11 (δC 129.8) suggested the existence of a symmetric
Aeruginosin GE730 (3), a yellowish, amorphous solid,
presented an HRESIMS complex molecular adduct ion at m/
z 731.1768/733.1755/735.1782 (1:2:1, [M + Na]+), which
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Table 2. H and 13C NMR Data of Aeruginosins GE730 (3), GE810 (4), and GE642 (5) in DMSO-d6
a
b
a
aeruginosin GE730 (3)
aeruginosin GE810 (4)
aeruginosin GE642 (5)
c
b
b
position
δC, mult.
δH, mult., J (Hz)
δC, mult.
δH, mult., J (Hz)
δC, mult.
δH, mult., J (Hz)
di-Br/Cl- Hpla 1
171.7, C
71.5, CH
38.4, CH2
171.9, C
71.3, CH
37.8, CH2
171.7, C
71.3, CH
38.6, CH2
2
4.14, m
4.13, m
2.86, m
2.70, m
4.10, m
3
2.86, dd (14.0, 3.6)
2.76, dd (14.0, 6.0)
2.83, dd (14.4, 4.0)
2.77, dd (14.4, 6.4)
3′
4
132.9, C
133.5, CH
111.5, C
149.2, C
132.3, C
133.2, CH
111.4, C
149.0, C
130.8, C
129.8, CH
121.7, C
147.5, C
5,5′
6,6′
7
7.35, s
7.36, s
7.11, s
2-OH
7-OH
Ile/Leu
2
6.03, d (6.0)
9.60, brs
5.92, d (5.0)
5.96, d (4.0)
9.70, brs
168.6, C
51.6, CH
38.1, CH
168.9, C
52.4, CH
37.5, CH
169.6, C
47.9, CH
42.4, CH2
4.54, m
1.48, m
4.45, dd (8.5, 4.0)
1.52, m
4.52, m
1.32, m
1.20, m
1.20, m
3
4
26.2, CH2
1.02, m
25.8, CH2
1.10, m
24.1, CH
4′
0.85, m
0.91, m
5
12.1, CH3
13.8, CH3
0.85, brd (4.0)
0.57, d (6.6)
7.25, d (9.2)
11.9, CH3
13.7, CH3
0.85, t (6.5)
0.63, d (6.5)
7.35, d (8.0)
23.5, CH3
21.5, CH3
0.76, d (6.5)
0.85, d (6.0)
7.35, d (8.4)
6
NH
Choi 1
171.6, C
60.1, CH
30.7, CH2
171.5, C
59.9, CH
30.6, CH2
171.5, C
60.1, CH
30.8, CH2
2
4.12, t (9.2)
1.94, m
4.16, dd (9.5, 8.5)
2.00, m
4.12, m
1.98, m
1.76, m
2.27, m
2.02, m
1.41, m
1.42, m
3
3′
3a
4
1.74, dt (16.8, 12.0)
2.26, m
1.76, dt (11.5, 10.5)
2.27, m
36.2, CH
19.1, CH2
35.8, CH
19.3, CH2
36.2, CH
19.1, CH2
2.02, m
1.98, m
4′
5
1.41, m
1.42, m
26.0, CH2
64.1, CH
33.7, CH2
1.42, m
23.3, CH2
70.7, CH
31.6, CH2
1.85, m
26.0, CH2
64.0, CH
33.7, CH2
1.32, m
6
3.90, brs
4.35, brs
3.92, brs
6-OH
4.52, brd (1.2)
1.92, m,
4.49, brs
7
2.18, m
1.98, m
7′
1.72, m
1.68, m
1.72, m
7a
54.0, CH
38.1, CH2
4.05, dt (10.8, 5.6)
3.07, m
53.9, CH
38.2, CH2
4.01, dt (10.5, 6.0)
3.08, m
54.0, CH
38.1, CH2
4.06, dt (11.6, 6.0)
3.03, m
Agm 1
1′
2.98, m
3.02, m
2
26.3, CH2
26.4, CH2
40.6, CH2
1.41, m
26.2, CH2
26.5, CH2
39.8, CH2
1.42, m
26.0, CH2
26.4, CH2
40.5, CH2
1.42, m
3
1.41, m
1.41, m
1.41, m
4
3.08, m
3.08, m
3.07, m
1-NH
4-NH
5
7.92, t (5.0)
7.51, brt (4.5)
7.82, brt (5.0)
7.56, brm
7.83, brt (5.2)
7.67, brt (4.9)
156.9, C
156.7, C
156.8, C
5-NH,NH2
6.95, brm
6.95, brm
6.95, brm
a
b
c
1
1
400.13 MHz for H, 100.62 MHz for 13C. 500.13 MHz for H, 125.76 MHz for 13C. Multiplicity and assignment from HSQC experiment.
tetrasubstituted aromatic ring. This was supported by the
appearance of only four pairs of aromatic carbons in the region
of 148 to 121 ppm. The aromatic proton presented HMBC
correlations with the carbon signals resonating at δC 147.5,
121.7, which were thus assigned as C-7 and C-6,6′, and with C-
3 (δC 38.6). On the basis of a comparison with the chemical
shifts of the same residue in aeruginosin KY64211 (that
contains Ile instead of Leu) the remaining aromatic carbon (δC
130.8) was assigned as C-4 of this residue, m,m-di-Cl-p-Hpla.
The structure of the Choi moiety was established mainly based
on the COSY correlations. The four characteristic protons of
this moiety, H-2 (δH 4.12 m), H-3a (δH 2.27, m), equatorial H-
6 (δH 3.92, brs), and H-7a (δH 4.06, m), could be sequentially
extended to the entire Choi spin system through the COSY
correlations. HSQC correlations assigned the carbons of the
Choi moiety, which were identical with the chemical shifts of
the protons and carbons of the same moiety in aeruginosin
KY642.11 The four subunits were assembled through ROESY
correlations of Hpla-H-2 and Leu-NH and of Leu-H-2 and
Choi-H-7a and HMBC correlation of Choi-CO and Agm-1-
NH. Applying Marfey’s method14 (L-FDAA) and chiral-phase
HPLC chromatography revealed the presence of L-Choi, D-Leu,
and D-m,m-di-Cl-p-Hpla (11) moieties in 5. On the basis of the
evidence discussed above the structure of aeruginosin GE642
(5) was established as D-m,m-di-Cl-p-Hpla-D-Leu-L-Choi-
agmatine.
BIOLOGICAL ACTIVITY
■
The extracts of strain IL-377 exhibited significant inhibition of
the serine protease trypsin at a concentration of 1 mg/mL. The
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Isolation Procedure. The freeze-dried cells (IL-377, 473 g) were
extracted with 7:3 MeOH/H2O (3 × 3L). The crude extract (74 g)
was evaporated to dryness and separated on an ODS (YMC-GEL,
120A, 4.4 × 6.4 cm) flash column with an increasing concentration of
MeOH in H2O. Fraction 6 (1:1 MeOH/H2O, 1.553 g) was subjected
to a Sephadex LH-20 column in 1:1 CHCl3/MeOH to obtain seven
fractions. Fractions 5−7 from the Sephadex LH-20 column (45 mg)
were separated on a reversed-phase HPLC (Cosmosil 5C-8 MS, 250
mm × 20.0 mm, DAD at 238 nm, flow rate 5.0 mL/min) in 78:22
0.1% aqueous TFA/CH3CN to obtain five pure compounds,
aeruginosin DA688 (5.8 mg, retention time 19.3 min, 0.0013% yield
based on the dry weight of the bacteria), aeruginosin 98C (5.1 mg,
retention time 21.3 min, 0.0011% yield based on the dry weight of the
bacteria), aeruginosin 101 (4.3 mg, retention time 25.1 min, 0.0009%
yield based on the dry weight of the bacteria), aeruginosin GE766 (2)
(4.6 mg, retention time 27.5 min, 0.0010% yield based on the dry
weight of the bacteria), and aeruginosin GE810 (4) (4.3 mg, retention
time 30.1 min, 0.0009% yield based on the dry weight of the bacteria).
Fractions 7 and 8 from the RP separation (6:4 and 7:3 MeOH/H2O,
1.43 g) were separated on a reversed-phase HPLC (Cosmosil 5C-8
MS, 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 aeruginosin GE642 (5)
(1.9 mg, retention time 18.7 min, 0.0004% yield based on the dry
weight of the bacteria), aeruginosin KY642 (2.2 mg, retention time
19.4 min, 0.0005% yield based on the dry weight of the bacteria),
aeruginosin GE686 (1) (3.6 mg, retention time 21.6 min, 0.0008%
yield based on the dry weight of the bacteria), and aeruginosin GE730
(3) (4.6 mg, retention time 27.5 min, 0.0010% yield based on the dry
weight of the bacteria).
activity-guided purification of the trypsin-inhibiting compo-
nents of the extract revealed that the known as well as the new
aeruginosins were responsible for the inhibition of trypsin. The
inhibitory activities of 1−5 were determined for the serine
proteases trypsin and thrombin (Table 3). These results
indicate that compounds containing 6-OH-Choi inhibit trypsin
and thrombin more potently than their 6-sulfated counterparts.
Table 3. Summary of the IC50's of Compounds 1−5 against
Trypsin and Thrombin
compound
trypsin
thrombin
aeruginosin GE686 (1)
aeruginosin GE766 (2)
aeruginosin GE730 (3)
aeruginosin GE810 (4)
aeruginosin GE642 (5)
3.2 μM
12.2 μM
2.3 μM
18.2 μM
8.5 μM
12.8 μM
>45.5 μM
12.9 μM
>45.5 μM
>45.5 μM
CONCLUDING REMARKS
■
Cyanobacteria present high biosynthetic versatility, which is
reflected in the synthesis of modified peptides such as the
aeruginosins. In the aeruginosins the biosynthetic versatility is
demonstrated in the ability to synthesize at least three different
isomers of the core modified amino acid Choi, (2S,3aS,6R,7aS)-
Choi (L-Choi),10 (2S,3aR,6R,7aR)-Choi (L-3a,7a-diepiChoi),16
and 2R,3aR,6R,7aR-Choi (D-3a,7a-diepiChoi),3 as well as the
ability to synthesize different derivatives of arginine at the C-
termius and to modify the configuration of C-2 and the
substituents of the aromatic ring of the Hpla residue in the N-
termius of these linear peptides.10 Alternation of the
configuration of Hpla-C-2 occurs even in isomers from the
same organisms (i.e., aeruginosins KT608A and B3), and mono-
and dihalogenation of the phenolic ring of Hpla with chlorine
and bromine increase the diversity of this group of linear
peptides.10 The isolation of aeruginosins GE686 (1) and
GE766 (2) emphasized again how versatile the biosynthetic
machinery of cyanobacteria is, allowing the substitution of the
same aromatic ring with chlorine and bromine. Such versatility
of biosynthetic enzymes is seen only for the selective
halogenases of red and brown algae, which produce
halogenated terpenes17 and polyketides,18 and rarely by bacteria
for the production of halogenated phenols such as 2,4-dibromo-
6-chlorophenol.19
Aeruginosin GE686 (1): [α]25 −23.8 (c 0.42, MeOH); UV
D
(MeOH) λmax (log ε) 205 (4.32), 248 (3.53), 291 (3.23), 308 (3.23)
nm; IR (KBr) 2932, 1675, 1203, 1138 cm−1; for NMR data see Tables
1 and S1 (in the Supporting Information); HRESIMS m/z 687.2285/
689.2256/691.2254 (5:8:3) [M + H]+ (calcd for C29H4581Br35ClN6O6,
689.2252). Retention times of AA Marfey’s derivatives: D-Br,Cl-Hpla
3.3 min (L-Br,Cl-Hpla 3.0, D-Br,Cl-Hpla 3.3 min), D-alloIle 47.4 min
(L-Ile 44.6, D-Ile/D-alloIle 47.4 min), L-Choi-6α-OH 38.8 min (L-Choi-
6α-OH 38.8, L-Choi-6β-OH 37.7 min).
Aeruginosin GE766 (2): [α]25 −8.8 (c 0.23, MeOH); UV
D
(MeOH) λmax (log ε) 204 (4.00), 247 (3.14), 290 (2.85), 310 (2.73)
nm; IR (KBr) 2927, 1677, 1384, 1206, 1138 cm−1; for NMR data see
Tables 1 and S2 (in the Supporting Information); HRESIMS m/z
789.1649/791.1637/793.1608 (5:8:3) [M + Na]+ (calcd for
C29H4481Br35ClN6NaO9S 791.1640). Retention times of AA Marfey’s
derivatives: D-Br,Cl-Hpla 3.4 min (L-Br,Cl-Hpla 3.0, D-Br,Cl-Hpla 3.4
min), D-alloIle 53.3 min (L-Ile 49.2, D-Ile/D-alloIle 53.3 min), L-Choi-
6α-OH 41.9 min (L-Choi-6α-OH 41.9, L-Choi-6β-OH 41.5 min).
Aeruginosin GE730 (3): [α]25 −39.1 (c 0.31, MeOH); UV
D
(MeOH) λmax (log ε) 207 (4.29), 247 (3.44), 292 (2.99), 310 (3.05)
nm; IR (KBr) 2932, 1675, 1204, 1138 cm−1; for NMR data see Tables
2 and S3 (in the Supporting Information); HRESIMS m/z 731.1768/
733.1755/735.1782 (1:2:1, [M + H]+, calcd for C29H4579Br81BrN6O6
733.1747). Retention times of AA Marfey’s derivatives: D-di-Br-Hpla
4.1 min (L-di-Br-Hpla 3.8, D-di-Br-Hpla 4.1 min), D-alloIle 45.9 min (L-
Ile 43.0, D-Ile/D-alloIle 45.9 min), L-Choi-6α-OH 36.2 min (L-Choi-
6α-OH 36.2, L-Choi-6β-OH 34.9 min).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation values were
obtained on a Jasco P-1010 polarimeter at the sodium D line (589
nm). UV spectra were recorded on an Agilent 8453 spectropho-
tometer. NMR spectra were recorded on a Bruker ARX-500
Aeruginosin GE810 (4): [α]25 −39.0 (c 2.7, MeOH); UV
1
spectrometer at 500.13 MHz for H and 125.76 MHz for 13C and a
D
1
(MeOH) λmax (log ε) 202 (3.95), 289 (2.64), 344 (1.08) nm; IR
(KBr) 2929, 1679, 1206, 1139 cm−1; for NMR data see Tables 2 and
S4 (in the Supporting Information); HRESIMS m/z 833.1169/
Bruker Avance 400 spectrometer at 400.13 MHz for H and 100.62
MHz for 13C. DEPT, COSY-45, gTOCSY, gROESY, gHSQC,
gHMQC, and gHMBC spectra were recorded using standard Bruker
pulse sequences. Mass spectra were recorded on a Waters MaldiSynapt
instrument. HPLC separations were performed on an ISCO HPLC
system (model 2350 pump and model 2360 gradient programmer)
equipped with an Applied Biosystem Inc. diode-array detector. An
Eliza ELx808 reader (BIO-TEK Instruments, Inc.) was used for
protease inhibition assays.
835.1136/837.1133 (1:2:1) [M
+
Na]+ (calcd for
C29H4479Br2N6NaO9S, 833.1134). Retention times of AA Marfey’s
derivatives: D-di-Br-Hpla 3.4 min (L-di-Br-Hpla 3.1, D-di-Br-Hpla 3.4
min), D-alloIle 51.3 min (L-Ile 47.5, D-Ile/D-alloIle 51.3 min), L-Choi-
6α-OH 40.3 min (L-Choi-6α-OH 40.3, L-Choi-6β-OH 38.7 min).
Aeruginosin GE642 (5): [α]25 −19.5 (c 0.11, MeOH); UV
D
(MeOH) λmax (log ε) 202 (3.75), 249 (2.50), 291 (2.95) nm; IR
(KBr) 1683, 1383, 1206 cm−1; for NMR data see Tables 2 and S5 (in
the Supporting Information); HRESIMS m/z 643.2772/645.2777/
647.2780 (9:6:1) [M + H]+ (calcd for C29H4535Cl2N6O6, 643.2778).
Biological Material. Microcystis aeruginosa, TAU strain IL-377, was
collected in August 2007 from a commercial fishpond in Kibbutz Geva,
Israel. Samples of the cyanobacteria are deposited at the culture
collection of Tel Aviv University.
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Retention times of AA Marfey’s derivatives: D-di-Cl-Hpla 3.29 min (L-
di-Cl-Hpla 2.99, D-di-Cl-Hpla 3.29 min), D-leucine 46.89 min (L-Leu
43.8, D-Leu 46.9 min), L-Choi-6α-OH 36.3 min (L-Choi-6α-OH 36.3,
L-Choi-6β-OH 35.0 min).
(1H, dd, J = 14.0, 4.0 Hz, H-3′), 7.18 (2H, s, H-5, H-9), 9.70 (1H, s, 7-
OH); 13C NMR (DMSO-d6, 125 MHz) δ175.3 (C, C-1), 71.1 (CH,
C-2), 38.9 (CH2, C-3), 132.0 (C, C-4), 129.8 (CH × 2, C-5,5′), 122.1
(C × 2, C-6,6′), 147.7 (C, C-7); HRESIMS m/z 248.9725, [M − H]−
(calcd for C9H735Cl2O4, 248.9721).
Preparation of m-Bromo-m′-chloro-D,L-p-hydroxyphenyllac-
tic Acid (9) (ref 13). To a stirring solution of hydroxyphenyllactic
acid (10 mg) in 2 mL of CH3CN was added 0.1 equiv of TsOH in
CH3CN (2 mL). After 5 min of stirring 1.1 equiv (8.1 mg) of N-
chlorosuccinimide in CH3CN (2 mL) was added in one portion. The
reaction was left to stir at room temperature for 6 h. Then 1.1 equiv
(10.8 mg) of N-bromosuccinimide in CH3CN (2 mL) was added in
one portion. The reaction was left to stir at room temperature for 18 h.
For the workup, the CH3CN solution was diluted with EtOAc (20
mL) and washed three times with a 5% aqueous solution of Na2S2O3
(25 mL), followed by three washes with water (25 mL) and then with
brine (25 mL). After evaporation of the solvents under vacuum, the
solid was subjected to a preparative reversed-phase HPLC column
(YMC-pack ODS-A, 5 μm, 250 × 20 mm, DAD at 238 nm, flow rate
5.0 mL/min) eluted with 1:1 0.1% aqueous TFA/MeOH to afford m-
bromo-m′-chloro-D,L-p-Hpla (9, 7.2 mg, 44% yield, retention time 21.7
min) and m,m-dichloro-D,L-p-Hpla (11, 2.9 mg, 21% yield, retention
time 23.5 min).
Determination of the Absolute Configuration of the Amino
Acids. Compounds 1−5 (0.3 mg each) were hydrolyzed in 6 N HCl
(1 mL). The reaction mixture was maintained in a sealed glass bomb at
110 °C for 16 h. The acid was removed in vacuo, and the residue was
resuspended in 250 μL of H2O. FDAA solution [(1-fluoro-2,4-
dinitrophenyl)-5-L-alanine amide] in acetone (115 μL, 0.03 M) and
NaHCO3 (120 μL, 1M) were added to each reaction vessel. The
reaction mixture was stirred at 40 °C for 2 h. Then HCl (2 M, 60 μL)
was added to each reaction vessel, and the solution was evaporated in
vacuo. The FDAA-amino acid derivatives from the hydrolysate were
dissolved in 1 mL of CH3CN and compared with standard FDAA-
amino acids by HPLC analysis: LiChrospher 60, RP-select B (5 μm),
flow rate 1 mL/min, UV detection at 340 nm, linear gradient elution
from 9:1 0.1% aqueous TFA buffer, pH 3: CH3CN to CH3CN, within
60 min. The absolute configuration of each amino acid was determined
by spiking the derivatized hydrolysates with a D,L-mixture of the
standard derivatized amino acids.
Determination of the Absolute Configuration of Hydroxy
Phenyl Lactic Acid Derivatives. Extraction of the acid hydrolysates
of compounds 1−5 with ethyl ether separated the Hpla derivatives
from the amino acid salts. The ether was removed in vacuo, and the
residue was dissolved in MeOH (1 mL). The MeOH solution was
analyzed on an Astec, Chirobiotic, LC stationary phase, 250 × 4.6 mm,
flow rate 1 mL/min, UV detection at 277 nm, linear elution with 1:19
1% aqueous triethylamnium acetate buffer, pH 4: MeOH. The Hpla
derivative from the aeruginosins was compared with corresponding
synthetic standard D,L-Hpla derivatives.
m-Bromo-m′-chloro-D,L-p-hydroxyphenyllactic acid (9): yel-
lowish solid; 1H NMR (DMSO-d6, 500 MHz) δ 4.08 (1H, dd, J = 4.0,
8.0 Hz, H-2), 5.58 (1H, brs, 2-OH), 2.68 (1H, dd, J = 14.0, 8.0 Hz, H-
3), 2.84 (1H, dd, J = 14.0, 4.0 Hz, H-3′), 7.33 (1H, d, J = 1.5 Hz, H-5),
7.22 (1H, d, J = 1.5 Hz, H-9), 9.75 (1H, s, 7-OH); 13C NMR (DMSO-
d6, 125 MHz) δ 175.3 (C, C-1), 71.0 (CH, C-2), 38.7 (CH2, C-3),
132.4 (C, C-4), 132.9 (CH, C-5), 112.0 (C, C-6), 148.6 (C, C-7),
121.8 (C, C-8), 130.5 (CH, C-9); HRESIMS m/z 292.9211, [M −
H]− (calcd for C9H779Br35ClO4, 292.9216).
Preparation of m,m-Dibromo-D,L-p-hydroxyphenyllactic
Acid (10) (ref 13). To a stirring solution of hydroxyphenyllactic
acid (10 mg) in 2 mL of CH3CN was added 2.0 equiv (19.6 mg) of N-
bromosuccinimide in CH3CN (2 mL) in one portion. The reaction
was left to stir at room temperature for 18 h. For the workup, the
CH3CN solution was diluted with EtOAc (20 mL) and washed three
times with a 5% aqueous solution of Na2S2O3 (25 mL), followed by
three washes with water (25 mL) and then with brine (25 mL). After
evaporation of the solvents under vacuum, the solid was subjected to a
preparative reversed-phase HPLC column (YMC-pack ODS-A, 5 μm,
250 × 20 mm, DAD at 238 nm, flow rate 5.0 mL/min) eluted with 1:1
0.1% aqueous TFA/MeOH to afford m-bromo-D,L-p-Hpla (1.5 mg,
13% yield, retention time 18.9 min) and m,m-dibromo-D,L-p-Hpla (10,
10.9 mg, 58% yield, retention time 25.5 min).
m,m-Dibromo-D,L-p-hydroxyphenyllactic acid (10): yellowish
solid; 1H NMR (DMSO-d6, 500 MHz) δ 4.08 (1H, dd, J = 4.0, 8.0 Hz,
H-2), 5.58 (1H, brs, 2-OH), 2.70 (1H, dd, J = 13.5, 8.0 Hz, H-3), 2.84
(1H, dd, J = 13.5, 4.0 Hz, H-3′), 7.36 (2H, s, H-5, H-9), 9.60 (1H, s, 7-
OH); 13C NMR (DMSO-d6, 125 MHz) δ 175.3 C, C-1), 71.2 (CH, C-
2), 38.5 (CH2, C-3), 132.7 (C, C-4), 133.5 (CH × 2, C-5,5′), 112.0 (C
× 2, C-6,6′), 149.4 (C, C-7); HRESIMS m/z 338.8696, [M − H]−
(calcd for C9H779Br80BrO4, 338.8691).
Preparation of m,m-Dichloro-D,L-p-hydroxyphenyllactic Acid
(11) (ref 13). To a stirring solution of hydroxyphenyllactic acid (10
mg) in 2 mL of CH3CN was added 2.2 equiv (16.1 mg) of N-
chlorosuccinimide in CH3CN (2 mL) in one portion. The reaction
was left to stir at room temperature for 18 h. For the workup, the
CH3CN solution was diluted with EtOAc (20 mL) and washed three
times with a 5% aqueous solution of Na2S2O3, (25 mL) followed by
three washes with water (25 mL) and then with brine (25 mL). After
evaporation of the solvents under vacuum, the solid was subjected to a
preparative reversed-phase HPLC column (YMC-pack ODS-A, 5 μm,
250 × 20 mm, DAD at 238 nm, flow rate 5.0 mL/min) eluted with 1:1
0.1% aqueous TFA/MeOH to afford m-chloro-D,L-p-Hpla (1.4 mg,
12% yield, retention time 13.8 min) and m,m-dichloro-D,L-p-Hpla (11,
5.5 mg, 41% yield, retention time 18.2 min).
Protease Inhibition Assays. The procedures used to determine
the inhibitory activity of the new compounds on trypsin and thrombin
were described in a previous paper.21
ASSOCIATED CONTENT
* Supporting Information
■
S
1D and 2D NMR spectra and HR MS data of compounds 1−5;
tables of full NMR data of 1−5. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: ++972-3-6408550. Fax: ++972-3-6409293. E-mail:
carmeli@post.tau.ac.il.
Notes
The authors declare no competing financial interest.
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. We thank 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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