Micropeptins from an Israeli fishpond water bloom of the cyanobacterium microcystis sp

Article abstract of DOI:10.1021/np900546u

Seven new natural products, micropeptin MZ845 (1), micropeptin MZ859 (2), micropeptin MZ939A (3), micropeptin MZ925 (4), micropeptin MZ939B (5), micropeptin MZ1019 (6), and micropeptin MZ771 (7), as well as two known micropeptins, cyanopeptolin S (8) and cyanopeptolin SS (9), were isolated from the hydrophilic extract of the cyanobacterium Microcystis sp. that was collected from a fishpond in Kibbutz Ma'ayan Tzvi, Israel, in July 2006. The structures of the pure natural products were elucidated using spectroscopic methods, including UV, 1D and 2D NMR, and MS techniques. The absolute configuration of the chiral centers of the compounds was determined using Marfey's method for HPLC. The inhibitory activity of the compounds was determined for the serine proteases: trypsin, chymotrypsin, thrombin, and elastase. These micropeptins inhibited trypsin with IC₅₀'s that varied between 0.6 and 24.2 μM. The SAR of these micropeptins is discussed.

Full text of DOI:10.1021/np900546u

352
J. Nat. Prod. 2010, 73, 352–358
Micropeptins from an Israeli Fishpond Water Bloom of the Cyanobacterium Microcystis sp.†
Ella Zafrir and Shmuel Carmeli*
Raymond and BeVerly Sackler School of Chemistry and Faculty of Exact Sciences, Tel-AViV UniVersity, Ramat AViV, Tel-AViV 69978, Israel
ReceiVed September 6, 2009
Seven new natural products, micropeptin MZ845 (1), micropeptin MZ859 (2), micropeptin MZ939A (3), micropeptin
MZ925 (4), micropeptin MZ939B (5), micropeptin MZ1019 (6), and micropeptin MZ771 (7), as well as two known
micropeptins, cyanopeptolin S (8) and cyanopeptolin SS (9), were isolated from the hydrophilic extract of the
cyanobacterium Microcystis sp. that was collected from a fishpond in Kibbutz Ma’ayan Tzvi, Israel, in July 2006. The
structures of the pure natural products were elucidated using spectroscopic methods, including UV, 1D and 2D NMR,
and MS techniques. The absolute configuration of the chiral centers of the compounds was determined using Marfey’s
method for HPLC. The inhibitory activity of the compounds was determined for the serine proteases: trypsin, chymotrypsin,
thrombin, and elastase. These micropeptins inhibited trypsin with IC50’s that varied between 0.6 and 24.2 µM. The
SAR of these micropeptins is discussed.
Cyanobacteria produce numerous natural products that are not
used for primary metabolism, and their role in the producing
organism is usually unknown. Microcystis spp. are responsible for
water blooms that frequently produce potent hepatotoxins, and thus
their environmental, toxicological, biological, and chemical proper-
ties have been extensively studied.1 Extracts of toxic strains of
Microcystis have been shown to be a rich source of unique modified
peptides, such as microginins,2 micropeptins,3 aeruginosins,4 ana-
baenopeptins,5 and microviridines,6 all of which are protease
inhibitors. The micropeptins are the most abundant group of serine
protease inhibitors from cyanobacteria, composed of more than 100
different members.7 The selectivity of the micropeptins for the
inhibition of different serine proteases is mainly influenced by
the nature of the amino acid occupying the fifth position from the
carboxy terminus. Basic amino acids (i.e., arginine and lysine) at
this position select for inhibition of trypsin-like serine proteases,
while aliphatic, aromatic, and other neutral amino acids select for
inhibition of chymotrypsin-like serine proteases.8 As part of our
ongoing research on the chemistry and chemical ecology of
cyanobacterial blooms in water bodies, a biomass (TAU strain IL-
361) of the cyanobacterium Microcystis sp. was collected, in the
summer of 2006, from a fishpond in Kibbutz Ma’ayan-Tzvi, Israel.
The isolation and structure elucidation of micropeptins isolated from
this cyanobacterial bloom biomass, as well as their biological
activity and some SAR, is discussed below.
Inspection of the NMR spectroscopic data of 1-7 reveals signals
1
indicative of the micropeptin family. In the H NMR spectrum a
methyl singlet at ca. 2.70 ppm of an NMe moiety, a hydroxy of
the amino hydroxy piperidone (Ahp) moiety at 6.10 ppm, a quartet
signal at 5.40 ppm, and a doublet methyl at 1.21 ppm of the
1
O-substituted Thr were all visible. Comparison of the H NMR
spectra of all of the nine members of this micropeptin family
revealed their similarity: a doublet methyl (IIIle-6) at ca. -0.25 ppm,
a triplet amide proton signal at ca. 7.50 ppm (Arg-δ-NH), a very
broad signal of the guanidine moiety of Arg around 6.50-7.00 ppm,
and overlapping signals of a monosubstituted phenyl moiety in the
aromatic region (7.15-7.30 ppm) of the spectrum. The 13C NMR
spectrum revealed some other characteristic signals including a
guanidine carbon at 156 ppm of the Arg moiety and three (two in
the case of 7) methine carbons adjacent to oxygen at around 73
ppm, in accordance with Ahp, Thr, and glyceric acid. The NMR
data suggested a structure composed of the same seven acid units
(except for 7, which contains six acid units). The differences
between the nine micropeptins were located at the amino hydroxy
piperidone moiety and in the substitutions of the glyceric acid
moiety. Five of the new micropeptins had an amino methoxy
piperidone (Amp) moiety instead of an Ahp moiety, which was
characterized by the appearance of a singlet methoxy group at 3.00
ppm, instead of the hydroxy group at 6.10 ppm. One of the
micropeptins (7) was truncated and did not contain the glyceric
acid moiety, while the remaining eight micropeptins differed in
the substitution pattern of the glyceric acid (GA) by sulfate groups;
two of the compounds possessed a sulfate substituent on position
2 of the glyceric acid. Such derivatives have not yet been described
in the literature.
Results and Discussion
The cyanobacterium biomass was freeze-dried and extracted with
70% MeOH in H2O. The extract that inhibited trypsin was flash-
chromatographed on an ODS column. Two fractions eluted from
the column with 40% and 50% MeOH in H2O exhibited protease
inhibitory activity and were further separated on a reversed-phase
HPLC column. Seven new protease inhibitors, micropeptin MZ845
(1) (first identified by Martin Welker at al.9 using an HPLC-MS
method), micropeptin MZ859 (2), micropeptin MZ939A (3),
micropeptin MZ925 (4), micropeptin MZ939B (5), micropeptin
MZ1019 (6), and micropeptin MZ771 (7), along with two previously
described natural products, cyanopeptolin S10 (8) and cyanopeptolin
SS11 (9), were isolated from the cyanobacterial extract, their
structures were elucidated, and their biological activities were
studied.
Micropeptin MZ845 (1) was isolated as an amorphous, white
solid. The molecular formula of 1, C40H63N9O11, was deduced from
the high-resolution MALDI-TOF-MS measurements of its proto-
nated molecular cluster ion at m/z 846.4641. Examination of the
NMR spectra of 1 in DMSO-d6 revealed its peptide nature, i.e.,
seven carboxylic carbons in the 13C NMR spectrum (see Table
1
2) and four amide doublet protons in the H NMR spectrum (see
Table 1). Taking into account the NMe-aromatic amino acid and
the N,N-disubstituted-amino acid of the micropeptins, this mi-
cropeptin was composed of six amino acids. The Ahp and glyceric
acid structure was suggested on the basis of the COSY and HSQC
spectra, which showed a singlet of a hydroxy group at 6.20 ppm
of the Ahp moiety and two hydroxy signals at 4.76 ppm (t) and
5.80 ppm (brd) for GA. Analysis of the COSY, TOCSY, and HSQC
2D NMR spectra allowed the assignment of the side chains of two
isoleucine units, a threonine, an arginine, a glyceric acid, and two
† Dedicated to the late Dr. Richard E. Moore of the University of Hawaii
at Manoa for his pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: ++972-3-6408550.
Fax: ++972-3-6409293. E-mail: carmeli@post.tau.ac.il.
10.1021/np900546u  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/22/2009

Micropeptins from the Cyanobacterium Microcystis sp.
Journal of Natural Products, 2010, Vol. 73, No. 3 353
Chart 1
a
1
Table 1. Comparison of the H NMR Data of Compounds 1-7 in DMSO-d6
1c
2b
3c
4b
5c
6c
7c
position
δH, mult.
δH, mult.
δH, mult.
δH, mult.
δH, mult.
δH, mult.
δH, mult.
IIle
2
3
4a
4b
5
6
NH
2
3a
4.57, dd
1.74, m
1.27, m
1.06, m
0.80, t
0.83, d
7.77, d
5.12, dd
3.29, m
2.79, dd
7.26, d
7.22, d
7.19, m
2.73, s
4.37, d
1.74, m
1.07, m
0.60, m
0.60, brd
-0.25, d
4.44, m
2.61, m
1.75, m
1.75, m
1.75, m
4.91, brs
7.32, d
6.20, brd
4.29, m
2.02, m
1.47, m
1.47, m
3.08, brq
7.53, t
8.60, d
4.66, d
5.52, q
1.21, d
7.64, d
4.07, q
3.60, m
3.49, m
5.80, brd
4.76, t
4.58, dd
1.72, m
1.34, m
1.13, m
0.84, t
0.87, d
7.00, d
5.16, dd
3.30, m
2.80, dd
7.26, m
7.22, m
7.19, m
2.73, s
4.42, d
1.80, m
1.05, m
0.59, m
0.59, brd
-0.28, d
4.47, m
2.43, dd
1.74, m
2.10, m
1.67, m
4.44, m
7.19, m
3.03, s
4.29, m
2.00, m
1.45, m
1.45, m
3.08, m
7.45, t
4.60, dd
1.73, m
1.33, m
1.11, m
0.84, t
0.86, d
6.95, d
5.15, dd
3.32, m
2.79, dd
7.24, m
7.21, m
7.17, m
2.73, s
4.42, d
1.76, m
1.05, m
0.58, m
0.58, brd
-0.29, d
4.46, m
2.42, dd
1.74, m
2.06, m
1.66, m
4.43, m
7.15, d
3.02, s
4.31, m
2.05, m
1.42, m
1.41, m
3.07, m
7.44, t
4.72, dd
1.76, m
1.26, m
1.00, m
0.81, t
0.84, d
7.61, d
5.13, dd
3.29, m
2.81, dd
7.25, m
7.23, m
7.20, m
2.73, s
4.38, d
1.74, m
1.05, m
0.60, m
0.60, brd
-0.26, d
4.44, m
2.59, m
1.73, m
1.73, m
1.73, m
4.91, brs
7.30, d
4.70, dd
1.78, m
1.34, m
1.11, m
0.85, t
0.86, d
6.89, d
5.17, dd
3.30, m
2.81, dd
7.25, m
7.22, m
7.19, m
2.74, s
4.44, m
1.79, m
1.07, m
0.60, m
0.60, brd
-0.27, d
4.44, m
2.40, dd
1.74, m
2.04, m
1.67, m
4.44, m
7.17, d
3.02, s
4.26, m
2.00, m
1.47, m
1.44, m
3.06, m
7.47, t
4.70, m
1.78, m
1.33, m
1.06, m
0.84, m
0.86, d
6.88, d
5.16, d
3.30, m
2.81, dd
7.25, m
7.23, m
7.20, m
2.74, s
4.44, m
1.80, m
1.06, m
0.60, m
0.59, brd
-0.29, d
4.47, m
2.40, m
1.74, m
2.07, m
1.66, m
4.43, m
7.17, d
3.02, s
4.30, m
2.02, m
1.43, m
1.43, m
3.07, m
7.44, t
8.53, d
4.63, d
5.46, q
1.21, d
7.75, d
4.72, m
3.99, m
3.91, dd
4.85, dd
1.77, m
1.30, m
1.10, m
0.84, t
0.87, d
6.89, d
NMePhe
5.16, dd
3.29, m
2.82, dd
7.26, m
7.21, m
7.20, m
2.74, s
4.44, m
1.81, m
1.05, m
0.59, brd
0.59, brd
-0.27, d
4.45, m
2.33, m
1.77, m
2.10, m
1.65, m
4.45, m
7.24, m
3.01, s
4.36, m
2.06, m
1.45, m
1.46, m
3.10, m
7.49, m
8.93, d
4.14, brs
5.60, q
1.40, d
3b
5,5′
6,6′
7
NMe
2
3
4a
4b
5
IIIle
6
3
Ahp/Amp
4pax
4peq
5peq
5pax
6
NH
OH/OMe
2
3a
3b
4
5
5-NH
NH
2
3
4
NH
2
3a
3b
2-OH
3-OH
6.06, d
Arg
4.28, m
2.02, m
1.46, m
1.46, m
3.06, m
7.46, t
8.53, brs
4.63, m
5.46, q
1.22, d
7.83, d
4.65, m
3.63, dd
3.63, dd
8.63, d
4.68, d
5.54, q
1.21, d
7.66, d
4.07, m
3.60, m
3.48, m
5.80, d
4.76, t
8.58, d
4.65, d
5.50, q
1.20, d
7.64, d
4.24, dd
3.97, dd
3.84, dd
6.09, brs
8.56, d
4.63, d
5.48, q
1.22, d
7.83, d
4.65, d
3.63, m
3.63, m
Thr
GA
8.40, brs
4.77, t
4.73, dd
a Complete NMR data are available in the Supporting Information (coupling constants, HMBC and ROESY correlations). b 500 MHz. c 400 MHz.
II
short fragments in accordance with a phenylalanine moiety and an
amino hydroxy piperidone (Ahp). The structure of the side chains
of the latter two amino acids and the assignment of the carboxya-
mide carbons to the side chains were achieved by analysis of the
results of a 1H-13C HMBC experiment (see Table 4 in the
Supporting Information). The sequence of the amino acids of the
peptide, Ile, NMePhe, Ile, Ahp, Arg, Thr, and GA was assigned
on the basis of HMBC correlations between the carboxyl of
NMePhe and IIle NH, the carboxyl of IIIle and the NMe of NMePhe,
the C-6 methine of Ahp and H-2 of the Ile, the carboxyl of Arg
and NH of Ahp, the carboxyl of Thr and NH of Arg, the carboxyl
I
of Ile and H-3 of Thr (the lactone linkage), and the carboxyl of
GA and the NH of Thr. The amino acid sequence could also be
assembled from the ROESY data (see Table 4 in the Supporting
Information). The relative configuration of Ahp-C-6 (R*) is based
on the J-values of H-6 (<1 Hz), which point to an equatorial
orientation of this proton, the chemical shift of the pseudoaxial H-4
(δH 2.61), which is downfield shifted by the axial hydroxy group,
I
II

354 Journal of Natural Products, 2010, Vol. 73, No. 3
Zafrir and Carmeli
Table 2. Comparison of the 13C NMR Data of Compounds 1-7 in DMSO-d6
1b
2b
3b
4c
5b
6b
7b
position
mult.a
δC
δC
δC
δC
δC
δC
δC
d
IIle
1
2
3
4
5
6
1
2
qC
173.0
55.5
37.3
24.8
11.1
15.9
169.3
60.6
34.3
137.8
128.7
129.7
126.8
30.3
169.8
54.3
33.1
23.8
10.4
14.0
169.8
49.2
21.6
29.9
74.2
172.7
55.4
37.7
24.9
10.8
15.9
169.2
60.8
34.2
137.7
128.8
129.7
126.9
30.3
169.7
54.1
32.8
23.8
10.3
13.7
169.2
49.4
21.6
23.8
83.3
55.4
170.4
52.2
27.7
25.3
40.5
156.8
169.1
54.4
72.4
18.0
172.8
72.7
63.7
172.7
55.4
37.9
25.0
11.0
16.0
169.1
60.8
34.2
137.8
128.8
129.7
126.8
30.3
169.8
54.0
32.8
23.8
10.4
13.7
169.2
49.3
21.6
23.8
83.2
55.4
170.4
51.9
27.3
25.2
40.2
156.7
169.2
54.5
72.7
17.9
171.9
70.6
68.4
-
172.4
55.4
38.1
25.0
11.3
16.0
169.0
60.8
34.4
137.8
128.8
129.7
126.8
30.2
169.7
54.0
32.8
23.8
10.3
13.7
169.3
49.3
21.7
23.8
83.1
55.4
170.4
51.6
27.1
25.0
40.5
156.7
169.4
55.0
71.9
17.5
170.4
77.0
62.3
172.5
55.5
38.2
24.9
11.4
16.0
169.2
60.8
34.3
137.9
128.8
129.8
126.9
30.3
169.8
54.0
32.8
23.8
10.4
13.7
169.0
49.2
21.8
23.7
83.1
55.5
170.4
51.4
26.9
25.0
40.2
156.7
169.3
55.0
71.8
17.3
169.7
74.7
66.5
172.2
55.1
37.7
24.8
11.3
16.1
169.2
60.9
34.3
137.6
128.9
129.7
126.9
30.2
169.6
53.7
32.9
23.7
10.2
13.6
169.3
49.1
21.9
23.7
83.2
55.5
169.7
52.5
27.8
25.2
40.5
156.9
167.1
55.7
69.7
17.8
CH
CH
CH2
CH3
CH3
qC
CH
CH2
qC
CH
CH
CH
CH3
qC
CH
CH2
CH2
CH3
CH3
qC
CH
CH2
CH2
CH
CH3
qC
CH
CH2
CH2
CH2
qC
55.7
37.8
24.7
11.5
16.1
169.0
60.7
34.4
137.9
128.7
129.8
126.8
30.3
NMePhe
3
4
5,5′
6,6′
7
NMe
1
2
3
4
5
6
2
3
4
5
6
OMe
1
2
3
4
5
6
1
2
3
4
1
2
IIIle
170.0
54.0
33.2
23.8
10.5
14.0
d
Ahp/Amp
-
49.1
21.8
29.8
73.9
Arg
170.2
52.3
27.7
25.3
40.3
156.8
169.2
54.4
72.3
18.1
172.8
72.7
63.9
170.3
51.6
25.7
25.0
39.9
d
-
Thr
GA
qC
169.4
55.6
71.9
17.7
172.0
77.0
62.4
CH
CH
CH3
qC
CH
CH2
3
a Multiplicity and assignment from an HSQC experiment. b 100 MHz. c 125 MHz. d Absent from spectrum due to limited amount of material that was
isolated.
correlations established the connectivity of H-3 through H-6, and
carbons 3 through 6 were assigned by correlations in the HSQC
experiment. The amide carbonyl resonating at 169.2 ppm was
assigned as C-2 of the Amp by its HMBC correlation with H-3,
and the cyclic structure of Amp is suggested on the basis of the
HMBC correlation of H-6 with the latter carbonyl. The HMBC
correlation of the methoxy at δH 3.03 (s) with C-6, δC 83.3 ppm,
established its attachment to C-6. The relative configuration of the
Amp residue was the same as that of the Ahp residue, because
the proton and carbon chemical shifts of positions 2, 3, and 4 of
the Ahp and Amp were almost identical. The chemical shifts of
carbons 5 and 6 were changed, as expected, from a substitution of
a hydroxy group with a methoxy group. C-5 was upfield shifted
by 6 ppm and C-6 was downfield shifted by 9 ppm in 2 relative to
1. The pseudoequatorial H-5 in 2 was downfield shifted by ca. 0.4
ppm relative to 1, while H-6 was upfield shifted by ca. 0.5 ppm.
H-4pax presented NOEs with H-5peq and 3-NH and H-5peq presented
NOEs with H-4pax and OMe, while H-3pax presented NOEs with
H-5pax and H-4peq and H-6peq presented NOEs with H-5peq (Figure
1). This established the relative configuration of the Amp to be the
same as to that of Ahp, (3R*,6R*). The sequence of the amino
acids of the peptide, IIle-NMePhe-IIIle-Amp-Arg-Thr-GA, was
assigned on the basis of HMBC correlations between the carboxy
of an amino acids and the amide proton of the adjacent acid in the
pairs NMePhe-IIle, Arg-Amp, Thr-Arg, and GA-Thr; between the
carboxy of IIIle and the NMe of NMePhe; between C-6 of Amp
Figure 1. Relative configuration and NOEs of the Ahp and Amp
moieties.
and the NOE correlation observed between H-4pax and 6-OH (see
Figure 1). On the basis of the results discussed above, the planar
structure of micropeptin MZ845 was assigned as 1.
Micropeptin MZ859 (2) was isolated as an amorphous, white
solid. The molecular formula of 2, C41H65N9O11, was deduced from
the high-resolution MALDI-TOF-MS measurements of its proto-
nated molecular ion cluster at m/z 860.4893. Comparison of the
NMR data (see Tables 1 and 2) of micropeptin MZ859 (2) and
micropeptin MZ845 (1) revealed that the difference between them
was located in the piperidone moiety, where a methyl ether
substituent in 2 replaced the hydroxy in 1. Analysis of the COSY,
TOCSY, HSQC, and HMBC 2D NMR data allowed the assignment
of the side chains of the micropeptin as two isoleucines, a threonine,
an arginine, a glyceric acid, and an NMe-phenylalanine (see Table
5 in the Supporting Information). The structure of the amino
methoxy piperidone (Amp) was deduced as follows: COSY
II
I
and H-2 of Ile; and between the carboxy of Ile and H-3 of Thr

Micropeptins from the Cyanobacterium Microcystis sp.
Journal of Natural Products, 2010, Vol. 73, No. 3 355
(the lactone linkage). The amino acid sequence could also be
assembled from the ROESY data (see Table 5 in the Supporting
Information). The planar structure 2 was thus assigned to micropep-
tin MZ859.
in the piperidone moiety, where an Amp moiety appears in 5,
instead of an Ahp moiety in 4 (see Tables 1 and 2). Analysis of
the COSY, TOCSY, HSQC, and HMBC 2D NMR spectra allowed
the assignment of the side chains of the amino acids of this
micropeptin, as two isoleucines, a threonine, an arginine, a
2-sulfated glyceric acid, an Amp, and an NMe-phenylalanine (see
Table 8 in the Supporting Information). The sequence of the amino
acids was determined by HMBC and ROESY 2D NMR experi-
ments. The HMBC correlations were observed between the carboxy
of an amino acid and the amide proton of the adjacent amino acid
for NMePhe-IIle, Arg-Amp, Thr-Arg, and GA-Thr; between the
carboxy of IIIle and the NMe of NMePhe; between C-6 of Amp
Micropeptin MZ939A (3) was isolated as an amorphous, white
solid. The molecular formula of 3, C41H65N9O14S, was deduced from
the high-resolution MALDI-TOF-MS measurements of its sodiated
molecular cluster ion at m/z 962.4260. The difference in the 1D
NMR spectrum between micropeptin MZ939A (3) and micropeptin
MZ859 (2) was located in the glyceric acid residue (see Tables 1
and 2). In 3, H-3a and H-3b were downfield shifted by ca. 0.36
ppm, relative to those of 2, and were not coupled to an alcoholic
proton. In addition, C-3 in 3 was downfield shifted by 4.7 ppm
relative to that of 2. These differences were in accordance with a
3-sulfated glyceric acid on the basis of the comparison with the
NMR chemical shifts of the 3-sulfated glyceric acid residue in
cyanopeptolin S.10 Analysis of the COSY, TOCSY, HSQC, and
HMBC 2D NMR spectra allowed the assignment of all the side
chains that assemble this micropeptin as two isoleucine units, a
threonine, an arginine, a 3-sulfated glyceric acid, an NMe-
phenylalanine, and an amino methoxy piperidone (Amp) as
described for 2 (see Table 6 in the Supporting Information). The
sequence of the amino acids was determined by HMBC and ROESY
2D NMR experiments. HMBC correlations were observed between
the carboxy of each amino acid and the amide proton of the adjacent
amino acid in the pairs NMePhe-IIle, Arg-Amp, and Thr-Arg;
between the carboxy of IIIle and the NMe of NMePhe; between
C-6 of Amp and H-2 of IIIle; between the carboxy of GA and NH
II
and H-2 of Ile; and between the carboxy of Ile and Thr H-3 (the
lactone linkage). The relative configuration of C-6 of Amp was
established in the same way as for 2. On the basis of the results
discussed here, the planar structure of micropeptin MZ939B was
assigned as 5.
Micropeptin MZ1019 (6) was isolated as an amorphous, white
solid. High-resolution ESIMS measurements in the positive mode
gave a sodiated cluster ion [M + Na - SO3]+ at m/z 962.4274,
derived from a natural loss of sulfur trioxide, due to a low stability
of the protonated sulfate moiety.12 The molecular formula of 6,
C41H65N9O17S2, was deduced from the negative mode HR-ESIMS
measurment, which presented a molecular ion [M - H]- at m/z
1018.3912. Micropeptin MZ1019 (6) differs from micropeptin
MZ939B (5) in the glyceric acid residue (see Tables 1 and 2). In
6, H-3a and 3b were downfield shifted by 0.30-0.36 ppm relative
to that of 5 and not coupled to an alcoholic proton. In addition,
C-3 in 6 was downfield shifted by 4.2 ppm relative to that of 5.
These differences are in accordance with 2,3-disulfated glyceric
acid, on the basis of a comparison with the NMR chemical shifts
of the 2,3-disulfated glyceric acid residue in cyanopeptolin SS (9).11
Analysis of the COSY, TOCSY, HSQC, and HMBC 2D NMR
spectra allowed the assignment of the side chains of the amino acids
that are contained in 6: two isoleucines, a threonine, an arginine, a
disulfated glyceric acid, an Amp, and an NMe-phenylalanine (see
Table 9 in the Supporting Information). The sequence of the amino
acids was determined by HMBC and ROESY 2D NMR experi-
ments. The HMBC correlations were observed between the carboxy
of the amino acid and an amide proton of the adjacent amino acid
in Arg-Ahp and GA-Thr; between C-6 of Amp and H-2 of IIIle;
and between the carboxy of IIIle and the NMe of NMePhe. The
ROESY correlations were observed between H-2 of NMePhe and
IIle-NH; between Thr-2 and Arg-NH; and between Thr-NH and
IIle-2 (the lactone linkage). The relative configuration of C-6 of
Amp was established in the same way as for 2. On the basis of the
results discussed, the planar structure of micropeptin MZ1019 was
assigned as 6.
I
of Thr; and between the carboxy of Ile and Thr H-3 (the lactone
linkage). The structure and relative configuration of the Amp moiety
was established as for 2. On the basis of the results discussed, the
planar structure of micropeptin MZ939A was assigned as 3.
Micropeptin MZ925 (4) was isolated as an amorphous, white
solid. The molecular formula of 4, C40H63N9O14S, was deduced from
the high-resolution MALDI-TOF-MS measurements of its proto-
nated fragment ion [M + H - SO3]+ at m/z 846.4741, which derives
from a natural loss of sulfur trioxide, due to the low stability of
the protonated sulfate moiety.12 The difference between micropeptin
MZ925 (4) and micropeptin MZ845 (1) was located in the glyceric
acid residue (see Tables 1 and 2). In 4, H-2 was downfield shifted
by 0.58 ppm relative to that of 1 and not coupled to an alcoholic
proton. In addition C-2 in 4 was downfield shifted by 4.3 ppm
relative to that of 1. Analysis of the COSY, TOCSY, HSQC, and
HMBC 2D NMR data allowed the assignment of all the side chains
that assemble this micropeptin as two isoleucine units, a threonine,
an arginine, a 2-sulfated glyceric acid, an NMe-phenylalanine, and
an amino hydroxy piperidone (Ahp) (see Table 7 in the Supporting
Information). Due to the poor 13C and HMBC NMR data, as a
consequence of the limited amount of material that was isolated,
the sequence of the amino acids was determined mainly on the
basis of a ROESY experiment and the comparison of relevant
chemical shifts with that of the related micropeptins 1 and 5. HMBC
correlations were observed between the carboxy of an amino acid
and the amide proton of the adjacent amino acid of NMePhe-IIle
and Arg-Ahp and between the carboxy of IIIle and the NMe of
NMePhe. ROESY correlations were observed between H-2 of an
amino acid and the NH of the adjacent amino acid in Thr-Arg and
GA-Thr, between H-2 of IIle and NH of Thr (the lactone linkage),
and between H-2 of IIIle and H-6 of Ahp. The relative configuration
of C-6 of Ahp was established in the same way as for 1. On the
basis of the results discussed above, the planar structure of
micropeptin MZ925 was assigned as 4.
Micropeptin MZ771 (7) was isolated as an amorphous, white
solid. The molecular formula of 6, C38H61N9O8, was deduced from
the high-resolution MALDI-TOF-MS measurements of its proto-
nated molecular cluster ion at m/z 772.4764. Examination of the
NMR spectrum of 7 in DMSO-d6 revealed that the Thr moiety in
7 was different from all of the known micropeptins. In the 1H NMR
spectrum, H-2 was upfield shifted by 0.5 ppm, H-3 and H3-4 were
downfield shifted by 0.14 and 0.19 ppm, respectively, and the amide
proton was substituted by a two-proton signal at 8.40 ppm. In the
13C NMR spectrum C-2 was downfield shifted by 0.7 ppm, while
C-1, -3, and -4 were upfield shifted by 2.2, 2.1, and 0.5 ppm,
respectively. The signals for a glyceric acid were missing from both
1
of the H and 13C NMR spectra of 7 (see Tables 1 and 2). These
findings were in accordance with a nonsubstituted threonine moiety.
Analysis of the COSY, TOCSY, HSQC, and HMBC 2D NMR data
allowed the assignment of the side chains of the amino acids in
this micropeptin: two isoleucines, a threonine, an arginine, an Amp,
and an NMe-phenylalanine (see Table 10 in the Supporting
Information). The sequence of the amino acids was determined by
Micropeptin MZ939B (5) was isolated as an amorphous, white
solid. The molecular formula of 5, C41H65N9O14S, was deduced from
the high-resolution ESI-MS measurements of its protonated frag-
ment ion [M + H]+ at m/z 940.4472. The only difference between
micropeptin MZ939B (5) and micropeptin MZ925 (4) was located

356 Journal of Natural Products, 2010, Vol. 73, No. 3
Zafrir and Carmeli
Table 3. Comparison of the Relative Trypsin Inhibition Potency
of 1-9 as a Function of the Glyceric Acid and Piperidone
Substitution
thrombin. Comparison of the potency of trypsin inhibition by 1-9
(see Table 3) reveals that the Amp derivatives are more potent than
the Ahp derivatives, and GA at position 7 is the most potent of the
GA derivatives. Recently we described a related family of mi-
cropeptins, which differed from 1-9 in the aromatic amino acid.
It presents a substituted NMeTyr moiety instead of NMePhe in 1-9.
This related family of compounds exhibit both trypsin and chy-
motrypsin inhibition, most probably due to the chlorine substitution
on the tyrosine aromatic ring.18
Ahp
Amp
GA-substitution
diol
compound (IC50 µM)
1 (2.6)
compound (IC50 µM)
2 (0.6)
3-sulfate
2-sulfate
disulfate
8 (4.1)
4 (24.2)
9 (2.9)
3 (1.5)
5 (7.4)
6 (1.6)
Experimental Section
analysis of the HMBC and ROESY 2D NMR experiments. HMBC
correlations were observed between the carboxy of an amino acid
and an amide proton of the adjacent amino acid of NMePhe-IIle,
Arg-Amp, and Thr-Arg; between the carboxy of IIIle and the NMe
of NMePhe, between C-6 of Amp and H-2 of IIIle, and between
the carboxy of IIle and Thr H-3 (the lactone linkage). The relative
configuration of C-6 of Amp was established in the same way as
for 2. On the basis of the results discussed, the planar structure of
micropeptin MZ771 was assigned as 7.
General Experimental Procedures. Optical rotations were deter-
mined on a JASCO P-1010 polarimeter. UV spectra were recorded on
an Agilent 8453 spectrophotometer. NMR spectra were recorded on a
Bruker ARX-500 spectrometer at 500.13 MHz for 1H and 125.76 MHz
for 13C and a Bruker Avance 400 spectrometer at 400.13 MHz for H
1
and 100.62 MHz for 13C. DEPT, COSY-45, gTOCSY, gROESY,
gHSQC, gHMBC, and gHMBC spectra were recorded using standard
Bruker pulse sequences. High-resolution MS were recorded on an
Applied Biosystems Voyager System 4312 instrument and Waters
MALDI Synapt System. 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 multiwavelength
detector) and on a Merck HPLC system (model L-6200A pump and
model L-4200 UV-vis detector). ELISA was used for the protease
inhibition assay, ELx808, BIO-TEK Instruments, Inc.
To determine the absolute configuration of the chiral centers of
micropeptins 1-7, Marfey’s method for HPLC was used. This
involved acid hydrolysis to liberate the free amino acids, followed
by derivatization with Marfey’s reagent13 and HPLC analysis. In
order to liberate glutamic acid from the Ahp/Amp residues, a small
amount of 1-7 was oxidized with Jones’ reagent,14 following the
same procedure. These two procedures demonstrated the L-
configuration of all amino acids. The observed J-value (0-1 Hz)
between H-2 and H-3 of the substituted threonine in 1-7 suggested
that, as in the case of all known micropeptins, it should be threonine
and not allo-threonine.15 The possibility that D- or L-allo-Ile is
present in these cyclic depsipeptides was ruled out by the
comparison of the 13C chemical shifts of C-5 and C-6 of the
isoleucine residues in known micropeptins. For the Ile at the carboxy
terminus of the peptide the measured chemical shifts in L-allo-Ile
(established for oscillamide J by chiral-GC-MS16) are 11.4 (C-5)
and 14.3 (C-6) ppm, while for L-Ile (established for nostopeptins
A and B by chiral-GC-MS17) they are 11.3 (C-5) and 16.1 (C-6)
ppm, in accordance with the values measured for 1-7. In the case
of the N,N-disubstituted-Ile, only values for L-Ile are available, 10.3
(C-5) and 13.9 (C-6) ppm (established for nostopeptins A and B
by chiral-GC-MS17), which are similar to those measured for
compounds 1-7. Ether extracts of the acid hydrolysates followed
by chiral HPLC analysis demonstrated the D-configuration for the
glyceric acid moieties of micropeptins 1-6. This established the
absolute stereostructures, 1-7, for the new micropeptins.
To rule out the possibility that compounds 1-7 are isolation
artifacts of cyanopeptolins SS (9), either derived from methoxylation
of the Ahp moiety or hydrolysis of the sulfate esters, we dissolved
9 in a 1:1 methanol/0.1% aqueous TFA solution and allowed it to
stand at RT for a week. Comparison of the proton NMR and
negative ESIMS spectra of the sample, before and after the treatment
with the aqueous acidic methanol solution, verified our previous
findings that the micropeptins are stable under these conditions;
namely, the sulfate esters do not hydrolyze and the Ahp does not
convert to the Amp residue.
Micropeptins 1-9 were isolated through serine proteases (trypsin
and chymotrypsin) inhibition assay-guided separation. The inhibi-
tory activities of pure 1-9 were determined for the serine proteases
trypsin (see Table 3), thrombin, chymotrypsin, and elastase.
Thrombin was mildly inhibited by micropeptin MZ859 (2), with
an IC50 of 52.9 µM, and cynopeptolin SS (9) with an IC50 of 45.2
µM, but not by the rest of the compounds. Micropeptin MZ771
(7) was inactive in these inhibition assays. Micropeptins 1-9 were
inactive in the chymotrypsin and elastase inhibition assays.
Compounds 1-9 exhibit potent to moderate inhibitory activity
against trypsin (IC50 0.6-24.2 µM), as expected for micropeptins
that contain arginine at position 5, but almost no activity against
Biological Material. Microcystis sp., TAU strain IL-361, was
collected in July 2006, from a fishpond at Kibbutz Ma’ayan-Tzvi, Israel.
The cell mass was frozen after it was collected and lyophilized. A
sample of the cyanobacterium is deposited at the culture collection of
Tel-Aviv University.
Isolation Procedure. Lyophilization of the first batch of IL-361 cells
produced 0.54 kg of dry cells. Extraction with 7:3 MeOH/H2O (3 × 2
L) and evaporation yielded 20 g of extract. The extract was chromato-
graphed, in 5 g portions, on a reversed-phase (ODS) flash column
(YMC-GEL, 120A, 4.4 × 6.4 cm) eluted with increasing percentage
of MeOH in H2O. Fraction 5 (2:3 MeOH/H2O; 190 mg) was further
separated on a Sephadex LH-20 column with 7:3 MeOH/H2O to obtain
10 fractions (fractions 5a-j). Fraction 6 from the initial RP column
(1:1 MeOH/H2O; 220 mg) was separated on a Sephadex LH-20 column
eluted with 7:3 MeOH/H2O to obtain 11 fractions (fractions 6a-k).
Fractions 5c-g and 6d-f were combined (a, 188 mg) and separated
again on a Sephadex LH-20 column eluted with 1:1 MeOH/H2O to
obtain 10 fractions (a1-a10). Fractions a5-a8 were combined, and
the combined fraction (b, 106 mg) was separated on a reversed-phase
HPLC column (YMC-Pack C-8 250 mm × 20.0 mm, DAD at
maximum absorbance, flow rate 5.0 mL/min) eluted with 4:1 0.1% TFA
in H2O/acetonitrile (ACN) to obtain four fractions (b1-b4). Fraction
b3 was further separated on a RP HPLC column eluted with 2:3 ACN/
0.1% TFA in H2O to obtain 10 fractions (b3a-j), seven of which were
found to be pure compounds. Compound 9 (b3c, 1.9 mg) was eluted
from the column with a retention time of 23.7 min (it was isolated
twice more from other fractions, adding to a total weight of 6.4 mg,
0.0015% yield based on the dry weight of the cyanobacterium).
Compound 4 (b3d, 1.4 mg) was eluted from the column with a retention
time of 30.2 min (it was reisolated from another fraction, adding to a
total weight of 1.1 mg, 0.0005% yield). Compound 8 (b3e, 3.4 mg)
was eluted from the column with a retention time of 32.6 min (it was
reisolated five more times from other fractions, adding to a total weight
of 8.9 mg, 0.002% yield). Compound 6 (b3f, 1.4 mg, 0.0003% yield)
was eluted from the column with a retention time of 35.6 min.
Compound 1 (b3g, 1.7 mg) was eluted from the column with a retention
time of 37.5 min (it was reisolated four more times from other fractions,
adding to a total weight of 7.7 mg, 0.0017% yield). Compound 5 (b3h,
0.9 mg) was eluted from the column with a retention time of 49.3 min
(it was reisolated one more time, adding to a total of 2.1 mg, 0.0006%
yield). Compound 2 (b3j, 2.6 mg) was eluted from the column with a
retention time of 64.4 min (it was reisolated three more times from
other fractions, adding to a total weight of 24.1 mg; the total yield is
0.0045%). Fraction b3j was further separated on the same RP HPLC
column with 4.7:5.3 ACN/0.1% TFA in H2O to obtain six fractions
(b3j1-6). Compound 3 (b3j-5, 1.2 mg, 0.0002% yield) was eluted
from the column with a retention time of 19.6 min. A second batch of
lyophilized cells (1.1 kg) was extracted with 7:3 MeOH/H2O (3 × 1.5

Micropeptins from the Cyanobacterium Microcystis sp.
Journal of Natural Products, 2010, Vol. 73, No. 3 357
L) and then evaporated in Vacuo to produce 42 g of extract. The extract
was separated on an ODS Flash column with an increasing amount of
MeOH in H2O. Fraction 5′ (2:3 MeOH/H2O; 540 mg) was further
separated on a Sephadex LH-20 column with 7:3 MeOH/H2O to obtain
11 fractions (5′k-u). Fraction 6 from the RP column (1:1 MeOH/H2O;
720 mg) was separated on a Sephadex LH-20 column with 7:3 MeOH/
H2O to obtain 11 fractions (6′l-w). Fractions 5′k-n and 6′l-o were
combined (c, 300 mg) and separated again on a Sephadex LH-20
column eluted with 1:1 MeOH/H2O to obtain 10 fractions (c1-c10).
Fractions c5-c10 were combined with fractions 5′m and 6′p. The
combined fraction (d, 503 mg) was separated on a Sephadex LH-20
column eluted with 1:1 MeOH/H2O to obtain 11 fractions (d1-d11).
Fractions d5-d7 were combined (e, 188 mg) and separated on a RP
HPLC column YMC-Pack C-8 eluted with 5:15 ACN/0.1% TFA in
H2O to obtain six fractions (e1-e6), fraction e4 was found to be a
pure compound, and compound 7 (2.6 mg, 0.0006% yield) was eluted
from the column with a retention time of 43.4 min.
min (D-Thr 27.1 min), L-Glu 27.9 min (D-Glu 29.5 min), L-Ile 40.0
min (D-Ile 44.3 min), and L-NMe-Phe 42.0 min.
Cyanopeptolin S (8): amorphous, white solid; HR MALDI-TOF-
MS m/z 948.4117 [M + Na]+ (calcd for C40H63N9NaO14S, 948.4107).
Identical in all respects with the published data.10
Cyanopeptolin SS (9): amorphous, white solid; HR-ESIMS m/z
1004.3729 [M - H]- (calcd for C40H62N9O17S2, 1004.3705). Identical
in all respects with the published data.11
Determination of the Absolute Configuration of the Amino
Acids. Compounds 1-7 (0.3 mg each) were hydrolyzed in 6 M 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, 1 M) 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 ACN 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 50 mM triethylammonium phosphate (TEAP) buffer, pH 3/ACN,
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 Ahp and Amp
Derivatives. Compounds 1-7 (0.1 mg each) were oxidized with Jones’
reagent (1 drop from a solution of 1.34 g of K2CrO7, 1 mL of H2SO4
in 8 mL of H2O) in 0.5 mL of acetone at 0 °C for 10 min. The mixture
was allowed to warm to room temperature, and a few drops of MeOH
were added. The bluish residue that developed was filtered, and the
solvent was evaporated in Vacuo. The resultant products were treated
as described above.
Micropeptin MZ845 (1): amorphous, white solid; [R]23 -160 (c
D
0.05, MeOH); UV (MeOH) λmax (log ε) 203 (4.20), 250 (2.88) nm; 1H
and 13C NMR (see Tables 1 and 2); HR MALDI-TOF-MS m/z 846.4641
[M + H]+ (calcd for C40H63N9O11, 846.4720). Retention time of amino
acids (AA) Marfey’s derivatives: L-Arg 18.2 min (D-Arg 18.6 min),
L-Thr 23.3 min (D-Thr 26.9 min), L-Glu 25.4 min (D-Glu 27.0 min),
L-Ile 39.8 min (D-Ile 44.1 min), and L-NMe-Phe 41.8 min. Retention
time of D-glyceric acid on the chiral column 3.8 min (L-glyceric acid
3.4 min).
Micropeptin MZ859 (2): amorphous, white solid; [R]22 -38 (c
D
0.53, MeOH); UV (MeOH) λmax (log ε) 203 (5.04), 254 (2.71) nm; 1H
and 13C NMR (see Tables 1 and 2); HR MALDI-TOF-MS m/z 860.4893
[M + H]+ (calcd for C41H66N9O11, 860.4876). Retention time of AA
Marfey’s derivatives: L-Arg 18.3 min (D-Arg 18.4 min), L-Thr 21.1
min (D-Thr 24.6 min), L-Glu 23.1 min (D-Glu 24.9 min), L-Ile 40.5
min (D-Ile 44.3 min), and L-NMe-Phe 42.3 min. Retention time of
D-glyceric acid on the chiral column 3.7 min (L-glyceric acid 3.4 min).
Determination of the Absolute Configuration of Glyceric Acid.
Extraction of the acid hydrolysates of compounds 1-6 with ethyl ether
separated the glyceric acid 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
210 nm, linear elution 1:49 1% triethylamine, 1% acetic acid (TEAA)
buffer, pH 4/MeOH. The glyceric acid from the micropeptins was
compared with standard D,L-glyceric acids.
Micropeptin MZ939A (3): amorphous, white solid; [R]23 -119
D
(c 0.23, MeOH); UV (MeOH) λmax (log ε) 203 (4.22), 250 (2.93) nm;
1H and 13C NMR (see Tables 1 and 2); HR MALDI-TOF-MS m/z
962.4260 [M + Na]+ (calcd for C41H65N9NaO14S, 962.4260). Retention
time of AA Marfey’s derivatives: L-Arg 18.3 min (D-Arg 18.5 min),
L-Thr 25.1 min (D-Thr 28.5 min), L-Glu 25.9 min (D-Glu 27.4 min),
L-Ile 41.2 min (D-Ile 44.9 min), and L-NMe-Phe 42.9 min. Retention
time of D-glyceric acid on the chiral column 3.7 min (L-glyceric acid
3.3 min).
Protease Inhibition Assays. Trypsin, thrombin, chymotrypsin, and
elastase were purchased from Sigma Chemical Co. Trypsin (1 mg/
mL) and chymotrypsin (10 mg/mL) were dissolved in 0.05 M Tris-
HCl/100 mM NaCl/1 mM CaCl2, pH 7.5 buffer solution. Benzoyl-L-
arginine-p-nitroanilide hydrochloride (BAPNA), the trypsin substrate,
was dissolved in a solution of 1:9 DMSO/Tris-buffer (0.85 g/mL). Suc-
Gly-Gly-p-nitroanilide (SGGPNA), the substrate for chymotrypsin, was
dissolved in Tris buffer (1 mg/mL). Test samples were dissolved in
DMSO (1 mg/mL). A 100 µL buffer solution, 10 µL of enzyme solution,
and 10 µL of sample solution were added to each microtiter plate well
and preincubated at 37 °C for 10 min. Substrate solution (10 µL) was
added, and the kinetics of the reaction were measured at 405 nm, 37
°C, for 30 min. Elastase (75 mg/mL) and thrombin (0.5 g/mL) were
dissolved in 0.2 M Tris-HCl, pH 8 buffer solution. Z-Gly-Pro-Arg-
4MꢀNA-acetate salt, the thrombin substrate, was dissolved in Tris buffer
(0.5 mg/mL). N-Suc-Ala-Ala-Ala-p-nitroanilide, the elastase substrate,
was dissolved in Tris buffer (1 mg/mL). The test samples were dissolved
in DMSO (1 mg/mL). For elastase, 150 µL of buffer solution, 10 µL
of enzyme solution, and 10 µL of sample solution were added to each
microtiter plate well and preincubated at 30 °C for 20 min. Substrate
solution (30 µL) was added, and the kinetics of the reaction were
measured at 405 nm, 37 °C, for 20 min. For thrombin, 170 µL of buffer
solution, 10 µL of enzyme solution, and 10 µL of sample solution were
added to each microtiter plate well and preincubated at 25 °C for 5
min. Substrate solution (10 µL) was added, and the kinetics of the
reaction were measured at 405 nm, 37 °C, for 20 min.
Micropeptin MZ925 (4): amorphous, white solid; [R]22 -89 (c
D
0.13, MeOH); UV (MeOH) λmax (log ε) 203 (5.04), 254 (3.00) nm; 1H
and 13C NMR (see Tables 1 and 2); HR MALDI-TOF-MS m/z 846.4741
[M + H - SO3]+ (calcd for C40H64N9O11, 846.4720). Retention time
of AA Marfey’s derivatives: L-Arg 17.2 min (D-Arg 17.6 min), L-Thr
22.9 min (D-Thr 26.4 min), L-Glu 24.7 min (D-Glu 26.5 min), L-Ile
39.4 min (D-Ile 43.5 min), and L-NMe-Phe 41.3 min. Retention time
of D-glyceric acid on the chiral column 3.8 min (L-glyceric acid 3.4
min).
Micropeptin MZ939B (5): amorphous, white solid; [R]23D -80 (c
1
0.2, MeOH); UV (MeOH) λmax (log ε) 203 (4.07), 252 (2.83) nm; H
and 13C NMR (see Tables 1 and 2); HR-ESIMS m/z 940.4472 [M +
H]+ (calcd for C41H66N9O14S, 940.4450). Retention time of AA
Marfey’s derivatives: L-Arg 18.5 min (D-Arg 18.6 min), L-Thr 23.4
min (D-Thr 26.8 min), L-Glu 26.2 min (D-Glu 27.7 min), L-Ile 40.6
min (D-Ile 46.0 min), and L-NMe-Phe 41.8 min. Retention time of
D-glyceric acid on the chiral column 3.7 min (L-glyceric acid 3.3 min).
Micropeptin MZ1019 (6): amorphous, white solid; [R]23 -44 (c
D
0.18, MeOH); UV (MeOH) λmax (log ε) 202 (4.13), 227 (3.66), 277
1
(3.03) nm; H and 13C NMR (see Tables 1 and 2); HR-ESIMS m/z
962.4274 [M + Na - SO3]+ (calcd for C41H65N9NaO14S, 962.4269),
m/z 1018.3912 [M - H]- (calcd for C41H64N9O17S2, 1018.3862).
Retention time of AA Marfey’s derivatives: L-Arg 18.0 min (D-Arg
18.2 min), L-Thr 22.8 min (D-Thr 26.2 min), L-Glu 24.7 min (D-Glu
26.5 min), L-Ile 39.4 min (D-Ile 43.3 min), and L-NMe-Phe 41.4 min.
Retention time of D-glyceric acid on the chiral column 3.7 min (L-
glyceric acid 3.4 min).
Micropeptin MZ771 (7): amorphous, white solid; [R]23 -176 (c
Acknowledgment. We thank A. Sacher and K. Shereshevsky, The
Mass Spectrometry Laboratory of The Maiman Institute for Proteome
Research of Tel-Aviv University, for the MALDI mass spectra
measurements and N. Tal, the Mass Spectrometry Facility of the School
of Chemistry, Tel-Aviv University, for the measurements of the HR
D
0.05, MeOH); UV (MeOH) λmax (log ε) 202 (3.58), 248 (2.14) nm; 1H
and 13C NMR (see Tables 1 and 2); HR MALDI-TOF-MS m/z 772.4764
[M + H]+ (calcd for C38H62N9O8, 772.4716). Retention time of AA
Marfey’s derivatives: L-Arg 18.5 min (D-Arg 18.7 min), L-Thr 23.4

358 Journal of Natural Products, 2010, Vol. 73, No. 3
Zafrir and Carmeli
(7) Martin, C.; Oberber, L.; Ino, T.; Konig, W. A.; Busch, M.; Weckesser,
J. J. Antibiot. 1993, 46, 1550–1556.
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ESI mass spectra. This research was supported by the Israel Science
Foundation grant 037/02.
Supporting Information Available: Tables 4-10, summarizing the
1H, 13C, HMQC or HSQC, HMBC, and ROESY data of 1-7, as well
as the 1H and 13C NMR spectra of compounds 1-9, are available free
of charge via the Internet at http://pubs.acs.org.
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