Eight novel serine proteases inhibitors from a water bloom of the cyanobacterium Microcystis sp.

Article abstract of DOI:10.1016/j.tet.2010.09.067

Eight new secondary metabolites, micropeptin MM836 (1), micropeptin MM850 (2), micropeptin MM916 (3), micropeptin MM932 (4), micropeptin MM978 (5), anabaenopeptin MM823 (6), anabaenopeptin MM850 (7), and anabaenopeptin MM913 (8), as well as the known anabaenopeptin B (9) were isolated from the hydrophilic extract of the cyanobacterium Microcystis sp. that was collected from a fishpond in Kibbutz Ma'agan Michael, Israel, in September 2006. The structure of the pure natural products was established by spectroscopic methods including 1D and 2D NMR, UV, and MS techniques. The absolute configuration of the chiral centers of the compounds was determined using Marfey's method. The inhibitory activity of the compounds was determined against the serine proteases, trypsin, chymotrypsin, thrombin and elastase.

Full text of DOI:10.1016/j.tet.2010.09.067

Tetrahedron 66 (2010) 9194e9202
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Eight novel serine proteases inhibitors from a water bloom of the cyanobacterium
Microcystis sp.
*
Ella Zafrir-Ilan, Shmuel Carmeli
Raymond and Beverly Sackler School of Chemistry and Faculty of Exact Sciences, Tel Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 March 2010
Received in revised form 6 September 2010
Accepted 20 September 2010
Available online 24 September 2010
Eight new secondary metabolites, micropeptin MM836 (1), micropeptin MM850 (2), micropeptin
MM916 (3), micropeptin MM932 (4), micropeptin MM978 (5), anabaenopeptin MM823 (6), anabaeno-
peptin MM850 (7), and anabaenopeptin MM913 (8), as well as the known anabaenopeptin B (9) were
isolated from the hydrophilic extract of the cyanobacterium Microcystis sp. that was collected from
a fishpond in Kibbutz Ma’agan Michael, Israel, in September 2006. The structure of the pure natural
products was established by spectroscopic methods including 1D and 2D NMR, UV, and MS techniques.
The absolute configuration of the chiral centers of the compounds was determined using Marfey’s
method. The inhibitory activity of the compounds was determined against the serine proteases, trypsin,
chymotrypsin, thrombin and elastase.
Keywords:
Cyanobacteria
Microcystis
Micropeptins
Anabaenopeptins
Natural products
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
for the other amino acids and exhibit mainly carboxypeptidase
inhibition.8
Freshwater cyanobacteria produce a diverse mixture of toxic
and non-toxic metabolites. In most cases, the non-toxic secondary
metabolites are cyclic and non-cyclic modified peptides, usually
members of five distinct families: micropeptins,1 anabaeno-
peptins,2 aeruginosins,3 microginins,4 and microviridins,5 which
inhibit proteolytic enzymes. Proteases are involved in a variety of
biological processes and are implicated in the pathogenesis of many
human diseases, including cancer and Alzheimer’s disease. Thus,
there are many proteases that are validated drug targets and the
discovery of new protease modulators is important for the de-
velopment of pharmacological tools, as well as potential thera-
peutics.6 The micropeptins are the most diverse group of cyclic
depsipeptides in cyanobacteria and are composed of more then 115
members7 that inhibit various serine proteases. They are charac-
terized by a lactone ring consisting of six amino acid residues, of
which one is 3-amino-6-hydroxy-2-piperidone (Ahp). Anabaeno-
peptins are cyclic hexapeptides, which are characterized by the
2. Results and discussion
As part of our continuing interest in the chemical ecology of
cyanobacterial water blooms and the search for novel drugs for
human diseases, we investigated the extracts of a Microcystis
sp. bloom material, collected from a fishpond in Kibbutz Ma’agan
Michael, Israel, in September 2006. The crude extract was found
to inhibit the serine protease, chymotrypsin. The active extract
was flash-chromatographed on an ODS column to afford three
fractions, which exhibited chymotrypsin inhibitory activity and
were thus further purified on a reversed phase HPLC column.
Eight new protease inhibitors, micropeptin MM836 (1), micro-
peptin MM850 (2), micropeptin MM916 (3), micropeptin MM932
(4), micropeptin MM978 (5), anabaenopeptin MM823 (6), ana-
baenopeptin MM850 (7), and anabaenopeptin MM913 (8) along
with the known anabaenopeptin B (9)2,9 were isolated and
characterized from the extract of this cyanobacterium. The
structure elucidation and biological activity of these metabolites
are discussed below (Scheme 1).
cyclization of the C-terminal amino acid carboxyl to the
e-amine
residue of the N-terminal- -lysine, while the -amine of this lysine
D
a
moiety is linked through an ureido-bridge to the side chain of an-
other amino acid.8 All anabaenopeptins described to date from
Micropeptin MM836 (1) was isolated as a glassy material. The
molecular formula of 1, C43H60N6O11, was deduced from a high-
resolution ESI mass measurement of its sodiated molecular ion
cluster at m/z 859.4249. Examination of the 1D NMR spectra in
DMSO-d6 (see Tables 1 and 2) revealed its peptide nature and
similarity to the micropeptin family of cyanobacteria natural
cyanobacteria, contain D-lysine, have an L-absolute configuration
* Corresponding author. Tel.: þ972 3 6408550; fax: þ972 3 6409293; e-mail
address: carmeli@post.tau.ac.il (S. Carmeli).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.067

E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
9195
NH2
O
HN
OR1
HN
NH O O
OH
N
N
O
O
NH O O
O
O
O
O
H
N
O O
NH
N
O O
NH
N
N
H
N
H
OH
R2O
O
O
O
R3
OH
OH
Micropeptin MM978 (5)
Micropeptin MM836 (1) R1=R2=R3=H
Micropeptin MM850 (2) R1=CH3, R2=R3=H
Micropeptin MM916 (3) R1=R3=H, R2=SO3
-
-
Micropeptin MM932 (4) R1=H, R2=SO3 , R3=OH
OH
OH
O
HO
X=
OCH3
O
O
H
N
H
N
H
N
H
N
H
N
H
N
R2
N
H
N
H
O
O
O
O
OR1
N
O
O
O
N
O
O
O
O
O
NH
HN
NH
HN
NH
Y=
N
NH2
HO
H
Anabaenopeptin MM913 (8)
Anabaenopeptin MM823 (6) R1=H, R2=X
Anabaenopeptin MM850 (7) R1=CH3, R2=Y
Anabaenopeptin B (9) R1=H, R2=Y
Scheme 1.
products. In particular, seven carbonyl carbons at dC 168e173, four
amide protons dH 7.0e8.5, and seven -protons of amino acids at dH
established based on correlations observed between the oxy-
methine (H-6) of Ahp and H-2 of N,N-disubstituted Phe, and be-
tween the oxymethine (H-3) of Thr and H-2 of Ile in the ROESY
spectrum. The amino acid sequence could also be fully assembled
from the ROESY data (see Fig. 1 and Table 5 in the Supplementary
data).
a
3.6e5.0 were observed, in addition to a broad singlet attributed to
the hydroxyl function of 3-amino-6-hydroxy-2-piperidinone (Ahp,
dH 6.04), an amide NMe (dH 2.76; dC 29.9), and an oxymethine
quartet due to a threonine ester (dH 5.39). Analysis of the COSY,
TOCSY, and HSQC 2D NMR experiments (see Table 5 in the
Supplementary data) allowed the assignment of the side chains
of an isoleucine, a leucine, a O-acylated threonine, a glyceric acid,
two aliphatic spin systems of aromatic amino acids, and the spin
system of an Ahp moiety. Further analysis of the 2D HMBC spec-
trum allowed the full assignment of the N,N-disubstituted phe-
nylalanine, the N-Me-phenylalanine and the Ahp moiety as well as
the ascription of the carbonyl signals to the individual amino acids.
The sequence of the amino acids in 1 was deduced from the HMBC
and ROESY correlations. Key HMBC correlations (see Fig. 1), ob-
served from the carbonyl of one amino acid to the NH or NMe of
the neighboring amino acid residues included NMePhe and Ile, N,N-
disubstituted Phe and NMePhe, Leu and Ahp, Thr and Leu, and
glyceric acid and Thr. The remaining connectivities were
4Ahp
HN
OH
5Leu
O
N
3N,N-disubst.Phe
NH O O
O
O
7GA
O O
NH
N
N
H
HO
O
6Thr
OH
2NMePhe
1Ile
NOE correlation
HMBC correlation
Micropeptin MM836 (1)
Fig. 1. Key HMBC and ROESY correlations in 1.

9196
E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
Table 1
Comparison of 1H NMR data of micropeptins 1e5 in DMSO-d6
a
Compound
Position
1Ile
1c
2b
3c
4b
5b
dH, mult., J(Hz)
dH, mult., J(Hz)
dH, mult., J(Hz)
dH, mult., J(Hz)
Position
1Ile
dH, mult., J(Hz)
2
3
4.60 dd 9.7,5.3
1.73 m
4.61 m
1.73 m
4.62 dd 9.6, 5.6
1.76 m
4.62 dd 9.5, 5.1
1.74 m
2
3
4.62 dd 9.5, 5.2
1.77 m
4a
4b
5
6
NH
2
3a
3b
5,50
6,60
7
NMe
OH
2
3a
3b
5,50
6,60
7
1.23 m
1.03 m
1.28 m
1.08 m
1.23 m
1.03 m
1.23 m
1.03 m
4a
4b
5
6
NH
2
3a
3b
5,50
6,60
7
1.22 m
1.02 m
0.78 t 7.0
0.82 d 7.5
0.81 t 7.4
0.82 d 6.7
7.54 d 9.7
5.00 br d 12.5
3.24 br d 14.5
2.84 dd 14.5, 12.5
7.23 d 7.5
7.40 t 7.5
7.31 t 7.5
2.76 s
0.83 t 6.6
0.85 d 7.2
6.81 d 9.6
5.03 dd 11.5, 2.5
3.23 dd 14.1, 2.5
2.89 dd 14.1, 11.5
7.24 d 7.8
7.40 t 7.8
7.30 t 7.8
2.77 s
0.81 t 7.5
0.83 d 7.5
7.50 d 9.6
5.02 dd 12.3, 2.2
3.24 m
0.80 t 7.7
0.82 d 6.6
7.49 d 9.5
4.89 dd 12.1, 2.2
3.10 dd 13.9, 2.2
2.70 dd 13.9, 12.1
7.00 d 8.1
6.76 d 8.1
d
7.50 d 9.5
2NMePhe/
2NMeTyr
2NMeTyr
4.87 br d 11.5
3.10 br d 13.5
2.67 dd 13.5, 11.5
6.99 d 7.7
2.83 m
7.24 d 7.3
7.38 t 7.3
7.29 t 7.3
2.76 s
6.76 d 7.7
2.73 s
9.32 s
NMe
OH
2.72 s
9.31 br s
d
d
d
3N,N-
disubst.
Phe
4.71 m
4.75 dd 11.1, 4.2
2.86 dd 13.9, 11.1
1.59 dd 13.9, 4.2
6.78 d 7.0
7.15 t 7.0
7.12 t 7.0
3.56 m
4.72 dd 10.8, 3.1
2.81 m
1.67 m
6.76 d 7.2
7.16 t 7.2
7.12 t 7.2
3.60 m
4.73 dd 11.7, 4.4
2.85 dd 15.4, 11.7
1.79 dd 15.4, 4.4
6.83 d 7.3
7.16 t 7.3
7.12 m
3N,N-2
disubst.
Phe
4.74 dd 12.0, 3.0
2.84 dd 14.5, 12.0
1.79 dd 14.5, 3.3
6.82 d 7.5
7.16 dd 7.5, 7.0
7.12 t 7.0
2.85 dd 13.7, 11.1
1.67 dd 14.5, 2.6
6.76 d 7.4
7.17 t 7.4
7.12 t 7.4
3.59 m
3a
3b
5,50
6,60
7
4Ahp/Amp
3
3.60 m
4Ahp
3
3.59 m
4pax
4peq
5pax
5peq
6
NH
OH/OMe
2
3a
3b
4
5
6
NH
2
3
4
NH
2
3a
3b
2-OH
3-OH
2.41 br q 14.3
1.57 m
1.50 m
2.21 br q 12.2
1.57 m
1.33 m
2.00 ddt 14.3, 2.5, 3.0
4.59 br s
6.96 d 8.9
3.04 s
4.19 ddd 12.0, 8.8, 3.7
1.70 m
1.29 m
2.40 br q 12.8
1.58 m
1.48 m
2.42 br q 14.2
1.57 m
1.54 m
1.68 m
5.04 br s
4pax
4peq
2.37 br q 13.4
1.60 m
1.53 m
1.67 m
5.03 br s
7.04 d 8.7
6.04 br s
4.12 ddd 12.2, 8.6, 4.1
2.00 m
1.60 m
5pax
1.67 m
1.66 m
5peq
5.02 br s
7.03 d 9.0
6.09 br s
4.19 m
1.71 m
1.30 m
5.02 br s
7.02 d 8.8
6.04 d 3.1
4.20 ddd 10.5, 9.1, 2.0
1.70 m
6
7.03 d
NH
OH
2
3a
3b
4a
4b
NH2
NH
2
3
4
NH
2
3
6.04 br d 2.9
4.19 ddd 12.6, 8.8, 3.6
1.69 m
1.29 m
1.45 m
0.83 d 5.9
0.73 d 6.2
8.41 d 8.8
4.59 br d 9.5
5.38 br q 6.6
1.16 d 6.6
7.65 d 9.5
4.26 ddd 8.1, 5.1, 2.6
3.99 dd 11.7, 2.6
3.65 dd 11.7, 8.1
5.96 d 5.1
d
5Leu
5Gln
1.32 m
1.48 m
1.43 m
1.43 m
1.98 m
1.96 m
0.84 d 6.6
0.73 d 6.4
8.41 d 8.7
4.59 br d 10.0
5.39 br q 6.7
1.17 d 6.7
7.58 d 10.0
4.04 dt 4.0, 5.4
3.56 m
0.84 d 6.5
0.72 d 6.6
8.41 d 8.8
4.61 br d 9.9
5.39 br q 6.7
1.16 d 6.7
7.58 d 9.9
4.03 ddd 6.0, 5.5, 4.3
3.57 m
3.43 dt 11.1, 5.8
5.69 d 5.5
4.67 t 5.8
d
0.83 d 6.5
0.74 d 6.4
8.39 d 9.1
4.59 br d 9.8
5.39 br q 6.6
1.17 d 6.6
7.65 d 9.1
4.26 ddd 8.2, 5.1, 2.6
3.99 dd 10.8, 2.6
3.66 dd 10.8, 8.2
5.98 d 5.1
d
7.12 br s, 6.64 br s
8.43 d 8.6
4.59 br d 9.0
5.37 br q 6.6
1.17 d 6.6
6Thr
7GA
6Thr
7Thr
7.73 d 9.0
4.38 dd 8.9, 4.2
3.99 ddq 4.2, 4.6, 6.2
1.04 d 6.2
7.73 d 8.9
4.83 br d 4.6
2.18 t 7.2
3.44 m
4
5.74 d 5.4
4.70 t 6.0
d
NH
3-OH
2
d
d
8Hex
d
d
d
d
3
4
1.49 tt 7.2 7.2
1.22 m
d
d
d
d
d
d
d
d
5
1.25 m
d
d
d
d
6
0.84 t 7.3
a
Complete NMR data is available in the Supplementary data.
500 MHz.
400 MHz.
b
c
The relative configuration of Ahp as 3S
*
,6R
*
was established by
instead of hydroxyl (dH 6.04, 1H, br s), in 1. Analysis of the 2D NMR
experiments (see Table 6 in the Supplementary data) allowed the
assignment of the full planar structure of 2. The proton and carbon
chemical shifts of positions 2, 3, and 4 of the Ahp of 1 and Amp of 2
were almost identical (see Tables 1 and 2) while those of carbons 5
and 6 were considerably different. C-5 was shifted upfield by 6 ppm
and C-6 was shifted downfield by 9 ppm, in 2 relative to 1. H-5peq
(pseudoeqatorial) was shifted downfield by 0.33 ppm while H-5pax
(pseudoaxial) and H-6 were upfield shifted by 0.17 and 0.44 ppm,
respectively, in 2 relative to 1. In the ROESY spectrum, H-4pax dis-
comparison of its 1H and 13C NMR data with those of micropeptin
MZ8457 and the arguments described below for compound 3. The
signals observed for H2-5 of the Ahp moiety in 1 (as well as in 3 and
4) appeared at a higher field (1.67 ppm and 1.50 ppm) than in most
of the known micropeptins,1 most probably due to the weak an-
isotropic affect of the neighboring Phe aromatic ring. Based on the
arguments presented above planar structure, 1, was assigned to
micropeptin MM836.
Micropeptin MM850 (2) was isolated as a glassy colorless ma-
terial. The molecular formula of 2, C44H62N6O11, was deduced from
a high-resolution MALDI-TOF-MS measurement of its sodiated
molecular ion cluster at m/z 873.4405. Comparison of the 1H NMR
data (see Table 1) of 2 and 1 revealed that they differ in the sub-
stitution of C-6 of the Ahp moiety; a methoxyl (dH 3.03, 3H, s) in 2
played NOE’s with H-5peq and 3-NH, 6-OMe correlated with H-5peq
,
while H-5pax showed NOEs with H-3pax and H-4peq (see Fig. 2). This
established the relative configuration of the Amp to be identical to
that of the Ahp moiety in 1, 3S
*
,6R . The possibility that 2 is an
*
isolation artifact was excluded by us in the past, for cyanopeptolin

E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
9197
Table 2
Comparison of the 13C NMR data of micropeptins 1e5 in DMSO-d6
a
Position
1Ile
1c
2b
3c
4b
Position
1Ile
5b
1
2
3
4
5
6
1
2
3
4
5,50
6,60
7
NMe
1
2
3
4
5,50
6,60
7
2
3
4
5
6
OMe
1
2
3
4
5
6
1
172.4 s
54.2 d
37.4 d
24.5 t
11.3 q
16.0 q
168.7 s
60.7 d
33.5 t
137.6 s
129.7 d
128.6 d
126.4 d
29.9 q
170.3 s
50.3 d
35.3 t
136.2 s
129.5 d
127.9 d
126.0 d
168.0 s
48.5 d
21.5 t
29.2 t
73.8 d
d
172.1 s
55.2 d
38.0 d
24.8 t
11.2 q
16.0 q
168.8 s
60.8 d
33.8 t
137.9 s
129.6 d
128.8 d
126.9 d
30.4 q
170.3 s
50.0 d
34.7 t
136.5 s
129.4 d
127.9 d
126.4 d
168.7 s
51.3 d
21.5 t
23.3 t
82.8 d
55.4 q
170.4 s
50.5 d
39.2 t
24.3 d
23.3 q
21.1 q
169.0 s
54.1 d
72.3 d
17.7 q
172.7 s
72.8 d
64.0 t
d
172.3 s
55.3 d
37.7 d
24.3 t
11.3 q
16.0 q
168.9 s
60.7 d
33.8 t
138.0 s
129.6 d
128.7 d
126.7 d
30.4 q
170.4 s
50.2 d
35.3 t
136.7 s
129.4 d
127.9 d
126.3 d
169.0 s
48.8 d
21.5 t
29.4 t
73.9 d
d
172.1 s
55.2 d
37.7 d
24.6 t
11.4 q
16.1 q
169.0 s
61.0 d
33.0 t
127.9 s
130.5 d
115.4 d
156.3 s
30.4 q
170.5 s
49.7 d
35.4 t
136.8 s
129.5 d
127.7 d
126.3 d
169.0 s
51.3 d
21.2 t
29.4 t
73.9 d
d
1
2
3
4
5
6
1
2
3
4
5,50
6,60
7
NMe
1
2
3
4
5,50
6,60
7
2
3
4
5
172.3 s
55.5 d
37.5 d
24.6 t
11.4 q
16.1 q
169.3 s
61.0 d
32.9 t
127.7 s
130.6 d
115.5 d
156.4 s
30.4 q
170.6 s
50.5 d
35.4 t
136.9 s
129.6 d
127.9 d
126.4 d
168.9 s
48.9 d
21.7 t
2NMePhe/
2NMeTyr
2NMeTyr
3N,N-disubst.-Phe
3N,N-disubst.-Phe
4Ahp/Amp
4Ahp
29.5 t
73.9 d
d
6
5Leu
169.9 s
50.3 d
38.9 t
24.3 d
23.4 q
21.1 q
169.0 s
54.0 d
71.9 d
18.0 q
172.4 s
72.8 d
64.0 t
d
170.3 s
50.5 d
39.0 t
24.6 d
23.3 q
21.2 q
169.1 s
54.2 d
72.2 d
17.9 q
171.1 s
70.8 d
68.5 t
d
170.5 s
50.6 d
39.0 t
24.2 d
23.3 q
21.2 q
169.1 s
54.2 d
72.2 d
17.9 q
171.7 s
70.8 d
68.5 t
d
5Gln
1
2
3
4
5
6
1
2
3
4
1
2
3
4
1
2
3
4
5
6
169.8 s
52.0 d
26.5 t
31.7 t
173.8 s
d
6Thr
7GA
6Thr
7Thr
8Hex
169.4 s
54.9 d
72.1 d
18.0 q
171.2 s
58.0 d
66.9 d
19.8 q
172.8 s
35.3 t
2
3
4
1
2
3
4
d
d
d
d
d
d
d
d
d
d
d
d
31.0 t
25.2 t
22.1 t
14.1 q
d
d
d
d
d
d
d
d
d
d
d
d
a
Complete NMR data is available in the Supplementary data.
125 MHz.
100 MHz.
b
c
pax
Me
pax
SS, by verifying its stability in conditions similar to those used in
the isolation procedure.7 Based on these arguments planar struc-
ture, 2, was assigned to micropeptin MM850.
H
O
6
peq
H
H
N
4
peq
H
3
peq
H
N
H
O
Micropeptin MM916 (3) was isolated as a glassy colorless ma-
terial. Its molecular formula, C43H60N6O14S, was determined by
high-resolution ESIMS (m/z 939.3826, [MþNa]þ). Comparison of
the 1H and 13C NMR data (see Tables 1 and 2) of 3 and 1 revealed
that the difference between them was located in the GA moiety.
In 3, C-1 and C-2 were shifted downfield by 1.3 and 2.0 ppm,
respectively, while C-3 was shifted upfield by 4.5 ppm, relative to 1,
indicating the presence of a 3-sulfo-glyceric acid moiety in 3, as in
the case of micropeptin MZ845.7 Analysis of COSY, TOCSY, HSQC,
and HMBC 2D NMR experiments (see Table 7 in the Supplementary
data) allowed the assignment of all signals to the individual amino-
and hydroxy-acid residues in 3. The sequence of the amino acids
was deduced from HMBC and ROESY correlations in a similar
manner as described above for 1, with the exception that the ester
linkage between Thr and Ile was evident from the HMBC correlation
of the oxymethine H-3 of Thr to the carbonyl of Ile. The relative
pax
H
pax
Amp
H
pax
pax
H
O
6
peq
H
4
peq
H
peq
H
H
O
N
N
3
H
pax
H
pax
Ahp
pax - pseudo axial, peq - pseudo equatorial
Fig. 2. NOE correlations of Ahp moiety in 3 and Amp moiety in 2.

9198
E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
Table 3
Comparison of 1H and 13C NMR data of anabaenopeptins 6e8 in DMSO-d6
a
Compound
Position
1Phe
6c
7b
8b
dC, mult.
dH, mult. J (Hz)
dC, mult.
dH, mult. J (Hz)
Position
1Ile
dC, mult.
dH, mult. J (Hz)
1
2
171.0 s
55.1 d
37.7 t
d
d
171.1 s
55.1 d
37.7 t
d
d
1
2
3
4a
4b
5
6
NH
1
170.7 s
58.1 d
36.1 d
24.3 t
4.37 ddd 12.7, 8.3, 3.2
3.32 m
2.78 m
d
4.38 ddd 12.3, 8.9, 2.8
3.31 m
2.77 m
d
4.17 dd 9.5, 7.0
2.01 m
1.30 m
3a
3b
4
5,50
6,60
7
NH
1
2
138.4 s
129.0 d
128.4 d
126.2 d
d
138.5 s
129.1 d
128.5 d
126.3 d
d
0.96 m
7.05 d 7.2
7.18 t 7.2
7.14 t 7.2
8.68 d 8.3
d
7.06 d 7.4
7.19 t 7.4
7.15 m
8.67 d 8.9
d
11.5 q
16.1 q
0.74 t 7.4
0.76 d 6.4
8.10 d 9.5
2NMeHt
169.5 s
59.5 d
30.9 t
2NMeAla
170.0 s
54.4 d
14.0 q
27.3 q
171.1 s
48.9 d
33.4 t
d
170.0 s
54.5 d
14.0 q
27.2 q
171.0 s
48.9 d
33.4 t
d
2
4.66 dd 7.7, 5.9
2.04 m
1.79 m
2.31 dd 13.1, 4.5
2.24 dd 13.1, 4.2
4.79 q 6.8
1.06 d 6.8
1.76 s
4.79 q 6.6
1.06 d 6.6
1.77 s
3a
3b
4a
4b
5
6,60
7,70
8(OH)
NMe
1
3
NMe
1
28.8 t
3Hty
d
d
2
4.72 dt 8.0, 4.8
1.86 m
1.71 m
2.62 ddd 13.9, 9.9, 4.0
2.42 ddd 13.9, 10.8, 6.6
d
4.73 dt 4.2, 6.0
1.86 m
1.70 m
2.61 ddd 13.4, 12.2, 2.5
2.42 ddd 13.4, 11.5, 6.1
d
131.6 s
129.1 d
115.4 d
155.7 s
28.2 q
172.4 s
48.5 d
33.1 d
3a
3b
4a
4b
5
6,60
7,70
8(OH)
NH
1
2
3
4
5
NH
1
2
6.96 d 6.6
6.66 d 6.6
9.18 s
30.7 t
d
30.7 t
d
2.57 s
131.2 s
129.2 d
115.3 d
155.7 s
d
131.2 s
129.2 d
115.3 d
155.7 s
d
3Hph
7.00 d 8.3
6.66 d 8.3
9.17 s
8.91 d 4.8
d
7.00 d 8.3
6.66 d 8.3
9.18 s
8.92 d 4.2
d
2
4.71 m
2.00 m
1.78 m
2.81 m
2.66 m
3a
3b
4a
4b
5
6,60
7,70
8
31.7 t
4Val
172.8 s
58.1 d
30.2 d
19.4 q
19.1 q
d
172.8 s
58.1 d
30.2 d
19.1 q
19.2 q
d
3.89 dd 8.8, 7.8
1.96 m
0.93 d 6.8
1.03 d 6.8
7.01 d 7.8
d
3.92 m
1.99 m
1.03 d 7.2
0.92 d 6.6
7.00 m
141.3 s
130.0 d
128.6 d
126.3 d
7.08 d 7.8
7.23 t 7.8
7.18 t 7.8
8.95 d 4.6
NH
1
2
5Lys
172.3 s
54.9 d
31.9 t
20.5 t
d
172.2 s
55.0 d
31.9 t
20.5 t
d
d
4Ile
173.1 s
55.9 d
36.4 d
25.7 t
3.92 dt 5.2, 7.2
1.61 m
1.33 m
1.16 m
1.44 m
3.57 m
2.79 m
7.13 m
6.42 d 7.2
d
3.91 m
1.62 m
1.28 m
1.17 m
1.43 m
3.58 m
2.79 m
4.20 dd 5.0, 5.9
1.78 m
1.34 m
3
3
4a
4b
5
6a
6b
4a
4b
5
6
NH
1
2
3
4
5
1.12 m
28.3 t
38.4 t
d
28.3 t
38.4 t
d
11.8 q
14.6 q
0.84 t 7.5
0.79 d 7.0
6.72 d 5.9
e
-NH
d
d
7.18 t 7.4
6.46 d 7.2
d
5Lys
172.4 s
55.0 d
30.9 t
20.6 t
29.3 t
38.2 t
NH
CO
d
d
3.85 dt 5.2, 6.1
1.62 m
1.22 m
1.22 m
3.47 m
Urea
157.3 s
174.0 s
51.9 d
27.2 t
d
157.3 s
173.5 s
52.3 d
29.0 t
d
6OMeGlu/1
6OMeArg/2
d
d
4.07 dt 4.1, 8.0
1.96 m
1.77 m
2.32 m
d
4.15 dt 5.4, 8.2
1.66 m
1.53 m
1.47 m
3.08 m
d
3a
3b
4
5
6
OMe
NH
5-NH
6
e-NH
7.15 dd 8.1, 4.8
6.56 d 6.1
29.9 t
172.8 s
d
25.2 t
40.4 t
156.8 s
52.0 q
d
NH
CO
1
2
3a
Urea
6Tyr
157.3 s
173.8 s
54.2 d
37.1 t
d
51.5 q
d
3.57 s
6.38 d 8.0
d
3.58 s
6.55 d 8.1
7.54 t 5.5
4.26 dt 4.8, 7.6
2.86 dd 13.7, 4.8
2.75 dd 13.7, 7.3
d
d
3b
4
127.5 s
130.3 d
115.2 d
156.2 s
5,50
6,60
7 (OH)
NH
6.94 d 6.2
6.65 d 6.2
9.23 s
6.22 d 7.9
a
Complete NMR data is available in the Supplementary data (coupling constant, HMBC, and ROESY correlations).
500 MHz for 1H and 125 MHz for 13C.
b
c
400 MHz for 1H and 100 MHz for 13C.
configuration of the chiral centers of the Ahp moiety, 3S
*
,6R
*
,
in 3 was substituted by a tyrosine in 4, as evident from the two
doublet protons at 6.76 (2H) and 7.00 (2H) ppm, a phenolic OH at
9.32 ppm, two aromatic methine carbons at 115.4 ppm, and
a phenolic quaternary carbon at 156.3 ppm. Correlations from the
HMBC spectrum (see Table 8 in the Supplementary data) allowed
the full assignment of the phenol ring and its attachment, through
the quaternary carbon resonating at 127.9 ppm, to a methylene
was based on the NOE correlations (see Fig. 2 and Table 7 in
the Supplementary data) between H-4pax and 6-OH, H-4pax and
3-NH, and between H-3pax and H-5pax, which are characteristic to
this moiety.7 On the basis of these arguments, planar structure 3
was thus assigned to micropeptin MM916.
Micropeptin MM932 (4) was isolated as a glassy colorless ma-
terial. Its molecular formula was determined as C43H60N6O15S by
high-resolution negative ESI mass analysis of its molecular ion at
m/z 931.3785 [MꢀH]ꢀ. Comparison of the 1H and 13C NMR data
(see Tables 1 and 2) of 4 and 3 revealed that one phenylalanine unit
(dC 33.0, dH 3.10 and 2.70) and a methine (dC 61.0, dH 4.89). The
correlation of the methine with the N-methyl (dH 2.73) determined
the structure of this unit as NMe-tyrosine. This established that the
NMe-phenylalanine in 3 was replaced by an NMe-tyrosine in 4.

E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
HO
9199
Analysis of COSY, TOCSY, HSQC, and HMBC 2D NMR experiments
4Val
(see Table 8 in the Supplementary data) allowed the assignment of
all signals, the sequence and relative stereochemistry of the chiral
centers, in a similar fashion as described for 3. Consequently, the
planar structure, 4, was assigned to micropeptin MM932.
O
O
H
N
H
N
H
N
3Hty
N
OH
H
O
O
O
N
O
O
5Lys
Micropeptin MM978 (5) was isolated as a glassy colorless ma-
terial exhibiting a molecular formula of C49H70N8O13 as determined
by high-resolution ESI mass analysis of its sodiated molecular ion
cluster at m/z 1001.4977. Comparison of the 1D NMR spectra of 4
and 5 suggested that both share the same 1Ile-2NMeTyr-N,N-dis-
ubst.3Phe-4Ahp fragment but differ in the rest of the acid sequence.
Analysis of the COSY, TOCSY, HSQC, and HMBC 2D NMR experi-
ments (see Table 9 in the Supplementary data) allowed the as-
signment of amino hydroxy piperidone (Ahp), glutamine,
hexanoate, isoleucine, N,N-disubstituted-phenylalanine, NMe-ty-
rosine, O-acetylated threonine, and threonine residues. The se-
quence of the amino acids was deduced from HMBC correlations
observed between the carbonyl of one amino acid to the NH or NMe
of the neighboring amino acid residue. This established the con-
nectivity between NMePhe and Ile, N,N-disubstituted Phe and
NMePhe, Gln and Ahp, 1Thr and Gln, 2Thr and 1Thr, and hexanoic
acid and 2Thr. The carbonyl of Ahp exhibited correlation with H-2 of
N,N-disubstituted-Phe, and the lactone linkage was deduced from
the correlation of Ile carboxyl and H-3 of 1Thr. The amino acid se-
quence could also be exclusively assembled from the ROESY data
(see Table 9 in the Supplementary data). The relative configuration
2NMeAla
OCH3
6OMeGlu
O
NH
HN
1Phe
NOE correlation
HMBC correlation
Anabaenopeptin MM823 (6)
Fig. 3. Key HMBC and NOE correlations in 6.
the cyclic portion of 7 was identical with that of 6, and that 7 is the
methyl ester of anabaenopeptin B (9), which was also isolated from
the extract of this bloom material. Thus, the planar structure, 7, was
assigned to anabaenopeptin MM850.
Anabaenopeptin MM913 (8) was isolated as a glassy material
with a molecular formula of C49H67N7O10 deduced from HR ESI
mass measurement of its molecular ion ([MþH]þ) at m/z
914.5031. Comparison of its 1D NMR spectra in DMSO-d6 to those
of compounds 6 and 7 (see Table 3) revealed that 8 belonged to
the anabaenopeptins since it contained the urea bridge but its
characteristic NMe group (dH 2.57) resonate downfield of the
expected chemical shift (dH w1.77). Analysis of the 1D and 2D
NMR experiments (see Tables 3 and 12 in the Supplementary
data) allowed the full assignment of five amino acid residues,
two isoleucines, an NMe-homotyrosine, a lysine, a tyrosine as
well as the side chain of homophenylalanine. The remaining
carbonyl still unaccounted for was assigned as C-1 of the latter
amino acid. The amino acid sequence of 8 was deduced from the
analysis of the HMBC and ROESY 2D NMR experiments. HMBC
correlations observed between the carbonyl of an amino acid and
the amide proton or NMe of the adjacent amino acid, for the
following pairs: NMeHty and 1Ile, Hph and NMeHty and, 4Ile and
Hph. In addition ROESY correlations were observed between the
of Ahp C-3 and C-6, was assigned as 3S
*
,6R , as described for
*
compound 3. The planar structure, 5, was assigned to micropeptin
MM978.
Anabaenopeptin MM823 (6) was isolated as a glassy colorless
material. Its molecular formula, C41H57N7O11, was deduced by the
analysis of high-resolution ESI mass measurements of its sodiated
molecular ion at m/z 846.4056. The 1D NMR spectra of 6 in
DMSO-d6 (see Table 3) revealed the peptide nature of this com-
pound; i.e., carboxamide carbons dC 170e174, in the 13C NMR
spectrum, amide protons dH 6.30e9.00 and protons
a to carbonyl
in amino acids dH 3.90e4.70. The alliance of 6 with the anabae-
nopeptins was evident from its NMR data, i.e., the chemical shifts
of the NMe at dH 1.76 (s), dC 27.3 (q) and of the urea bridge,
a carbon at dC 157.3 and amide protons at dH 6.38 and 6.42.
Analysis of the COSY, TOCSY, HSQC, and HMBC 2D NMR experi-
ments (see Table 10 in the Supplementary data) allowed the as-
signment of valine, NMeAla, lysine, phenylalanine, homo-tyrosine,
a
-protons of NMeHty and Hph and the amide proton of 4Ile and
the -proton of Lys. The ring closure was based on the NOEs
between -NH of Lys and the amide and
-protons of 1Ile. The
urea bridge was assigned through HMBC correlation of the
proton of Lys with the urea carbonyl (dC 157.3) and NOE corre-
lations of Lys- -NH with Tyr amide and protons. Based on these
a
and
d
-OMe-glutamic acid. The sequence of the amino acids in 6
e
a
was established through HMBC correlations (see Fig. 3) of the
carbonyl of an amino acid with the amide proton or NMe of the
adjacent amino acid, for the following pairs: Lys-Val, Val-Hty, Hty-
NMeAla, and NMeAla-Phe. The correlation between the carbonyl
a-
a
a
arguments the planar structure, 8, was assigned to anabaeno-
peptin MM913.
of Phe and
e
-NH of Lys established the closure of the peptide cycle,
-amide
while correlations of the urea carbonyl (dC 157.3) and the
a
The absolute configuration of the chiral centers of compounds
1e8 was determined using Marfey’s method. This involved
acid hydrolysis that liberated the amino acids, followed by
derivatization with Marfey’s reagent10 and HPLC analysis. A small
amount of material of micropeptins 1e5 was initially oxidized
with Jones’ reagent,11 in order to liberate a glutamic acid from the
Ahp/Amp residues, followed by Marfey’s procedure. The described
protons of Lys and OMeGlu established the urea bridge. The amino
acid sequence could also be assembled from the NOE data (see
Fig. 3 and Table 10 in the Supplementary data). Based on this
evidence the planar structure, 6, was assigned to anabaenopeptin
MM823.
Anabaenopeptin MM850 (7) was isolated as a glassy material
exhibiting a molecular formula of C42H62N10O9 that was de-
termined from analysis of its high-resolution ESIMS protonated
molecular ion at m/z 851.4794. The 1D NMR spectra of 7 in DMSO-
d6 were similar to that of 6, suggesting its association with the
anabaenopeptins. Comparison of the NMR data (see Table 3) of
anabaenopeptin MM850 (7) and anabaenopeptin MM823 (6)
revealed that the difference between them consisted in the sub-
stitution of the urea bridge with an OMeArg in 7, instead of the
OMeGlu in 6. Analysis of the one and two-dimensional NMR
experiments (see Table 11 in the Supplementary data) revealed that
procedures established the
D-absolute configuration of Lys in the
anabaenopeptins 6e8 and
L-stereochemistry of all the remaining
amino acids in compounds 1e8. The Ile units in 1e5 were estab-
lished by their carbon chemical shifts to be Ile (contrary to alloIle).7
Diethyl-ether extraction of the acid hydrolyzate of micropeptins
1e4, followed by chiral HPLC analysis, demonstrated a D-absolute
configuration for the glyceric acid moieties. This established the full
absolute configuration for compounds 1e8.
The inhibitory activity of 1e9 that was determined against
the serine proteases chymotrypsin, elastase, trypsin, and thrombin

9200
E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
is summarized in Table 4. Comparison of the IC50’s of the micro-
peptins revealed that the affinity of the micropeptins to the cata-
lytic binding pocket of chymotrypsin decreases when the
hydroxy group at position 3 of 7GA is substituted with a sulfate
group, and when the aromatic ring, of residue-2, is substituted by
a para-hydroxy group. The opposite is observed for the affinity to
the catalytic binding pocket of elastase, which increased when
residue-2 is a tyrosine instead of a phenylalanine. The inhibition of
serine proteases by the anabaenopeptins is much milder then those
of the micropeptins. Comparison of the inhibitory activity of the
anabaenopeptins revealed that an OMeArg in the ureido-bridge of 7
decreased the affinity to the catalytic binding pocket of chymo-
trypsin (when compared to OMeGlu in 6 and Arg in 9) and in-
creased the affinity to the catalytic binding pocket of elastase
(compared to OMeGlu in 6).
pump and model MD-2010-plus diode array detector), a Merck
Hitachi (model L-6200 intelligent pump, model L-4200 UVevis)
and an Agilent 1100 series HPLC system. The proteases inhibition
assays were preformed on an ELx808, BIO-TEK Instruments Eliza
reader.
4.2. Biological material
Microcystis sp., TAU strain IL-362, was collected in September
2006, from a fishpond at Kibbutz Ma’agan Michael, Israel. The cell
mass was frozen immediately after collection and lyophilized. A
sample of the cyanobacterium is deposited at the culture collection
of Tel Aviv University.
4.3. Isolation procedure
Lyophilization of the first batch of IL-362 cells produced 1 Kg of
dry cells. Extraction with 7:3 MeOH/H2O (4ꢁ2L) and evaporation
yielded 41 g of crude extract. The crude extract was chromato-
graphed in 5 g portions, on a reversed phase (ODS) flash column
(YMC-GEL, 120A, 4.4ꢁ6.4 cm) eluted with increasing percentage of
MeOH in water. Fraction 7 (3:2 MeOH/H2O) 484 mg, was further
separated on a Sephadex LH-20 column with 1:1 MeOH/CHCl3 to
obtain twelve fractions (fractions 7ael). Fractions 7eeh were
combined (a, 169 mg) and separated on a reversed-phase HPLC
column (YMC-Pack C-8, 250 mmꢁ20.0 mm, DAD at maximum ab-
sorbance, flow rate 5.0 mL/min) eluted with 1:1 H2O/acetonitrile
(MeCN) to obtain seven fractions (a1ea7). Fraction a7, pure com-
pound 1 (4.0 mg, 0.0004% yield based on the dry weight of the
bacterium, retention time 32.5 min). Fraction a1 was further
separated on the same RP HPLC column with 3:7 MeCN/0.1% TFA
in H2O to obtain four fractions (a1aed). Fraction a1d, pure com-
pound 7 (2.4 mg, 0.0002% yield, retention time 56.2 min). Fraction 8
from the initial RP separation (7:3 MeOH/H2O) 480 mg, was further
separated on a Sephadex LH-20 column with 1:1 MeOH/CHCl3 to
obtain eleven fractions (fractions 8aek). Fractions 8dei were
combined (b, 97 mg) and separated on a reversed-phase HPLC
column (YMC-Pack C-8, 250 mmꢁ20.0 mm, DAD at maximum ab-
sorbance, flow rate 5.0 mL/min) with 3:2 H2O/MeCN to obtain
seven fractions (b1eb7). Fraction b3, pure compound 5 (8.8 mg,
retention time 17.2 min, it was isolated once more from another
fraction, 3.8 mg, total yield 0.0012%). Fraction b1 and b5 were
combined (c, 71 mg) and further separated on the same RP HPLC
column with 7:13 ACN/0.1% TFA in H2O to obtain eleven fractions
(c1ec11), two of which were found to be pure compounds. Com-
pound 9 (c4, 3.3 mg, 0.0003% yield, retention time 15.6 min).
Compound 4 (c6, 3.4 mg, 0.0003% yield, retention time of 17.6 min).
Fraction c10 (7.8 mg) was further separated on the same RP HPLC
column, with 1:1 MeCN/0.1% TFA in H2O to obtain five fractions
(c10aee). Fraction c10d, pure compound 2 (4.0 mg, retention time
19.6 min, it was isolated once more from another fraction, 1.0 mg,
total yield 0.0005%). A second batch of lyophilized cells (0.5 Kg) was
extracted with 7:3 MeOH/H2O (3ꢁ1.5 L) and then evaporated in
vacuo to produce 42 g of crude extract. It was separated on an ODS
Flash column with an increasing amount of MeOH in water. Fraction
6 from the RP column (1:1 MeOH/H2O) 491 mg was separated on
a Sephadex LH-20 column with 1:1 MeOH/H2O to obtain ten frac-
tions (6aej). Fractions 6cee were combined (d, 129 mg) and sepa-
rated on an RP HPLC column YMC-Pack C-8, with 7:13 MeCN/0.1%
TFA in H2O to obtain 16 fractions (d1e16), one of which was found
to be a pure compound. Compound 6 (d9, 3.7 mg, 0.0007% yield,
retention time 29.9 min). Fraction 7 from the RP column (1:1
MeOH/H2O) 256 mg, was separated on a Sephadex LH-20 column
with 1:1 MeOH:H2O to obtain fourteen fractions (7m-z). Fractions
7r-t were combined (e, 63 mg) and separated on an RP HPLC column
YMC-Pack C-8 with 23:27 MeCN: 0.1% TFA in H2O to obtain twelve
Table 4
Summary of the IC50’s of compounds 1e9 against serine proteasesa
Compound
Chymo-trypsin
Elastase
Trypsin
Thrombin
1
2
3
4
5
6
7
8
9
1.4
1.7
3.0
5.4
4.6
16.0
45.0
d
45.5
d
d
d
d
d
d
d
d
4.4
19.1
50.0
14.3
d
d
d
d
52.9
d
d
45.0
d
d
d
d
d
d
d
a
In mM.
3. Conclusions
The study describes the isolation of five new micropeptins
together with three new and one known anabaenopeptins from
the water bloom material of the cyanobacterium Microcystis sp.
This research expandes the library of the micropeptins and ana-
baenopeptins, and illuminates the variety of the metabolites
produced by these fresh water cyanobacteria. In particular, the
protease inhibition assays showed that several of the micropeptins
possess significant biological activity and selectivity toward dif-
ferent serine proteases. Anabaenopeptin MM913 (8), belongs to
a relatively rare subgroup of the anabaenopeptins, presenting an
N-methyl-homoaromatic amino acid at the second position. There
are nine more members of this subgroup of anabaenopeptins:
nodulapeptins A and B,12 anabaenopetins G, H,13 and T,14 oscil-
lamide C,15 anabaenopeptins 908 and 915,16 and anabaenopeptin
HU892.17
4. Experimental section
4.1. General experimental procedures
Low and high-resolution MALDI mass spectra were recorded on
an Applied Biosystems Voyager System 4312 instrument and low-
and high-resolution ESI mass spectra were recorded on a Waters
Synapt instrument. UV spectra were recorded on an Agilent 8453
spectrophotometer. Optical rotation values were obtained on
a Jasco P-1010 polarimeter at the sodium D line (589 nm). NMR
spectra were recorded on a Bruker DRX-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 1H, and 100.62 MHz for 13C. COSY-
45, gTOCSY, gROESY, gHSQC, gHMQC, and gHMBC spectra were
recorded using standard Bruker pulse sequences. HPLC separations
were preformed on a Jasco HPLC system (model PU-2080-plus

E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
9201
fractions (e1e12) two of which were pure compounds. Compound 3
(e3, 3.8 mg, 0.0008% yield, retention time 24.4 min) and compound
8 (e12, 3.1 mg, 0.0006% yield, retention time 37.3 min).
40.2 min (
L
-Ile 40.2,
D
-Ile 44.2 min),
L
-phenylalanine 39.9 (
L
-Phe
39.9, -Phe 42.8) min and
D
L-NMe-tyrosine 46.4 min.
4.3.6. Anabaenopeptin MM823 (6). Glassy white material; [
(c 0.2, MeOH); UV (MeOH) lmax (log
a]
24 þ5
D
4.3.1. Micropeptin MM836 (1). Glassy material; [
a
]
23 þ120 (c 0.05,
e
) 205 (4.41), 228 (4.41), 248
D
MeOH); UV (MeOH) lmax (log
e) 203 (4.12), 230 (3.24), 248 (2.55),
(3.50), 280 (3.41) nm; IR (KBr): 3200, 2935, 2875, 1735, 1654, 1543,
280 (1.29) nm; IR (KBr): 3450, 3021, 2950, 2933, 2875, 1735, 1632,
1546, 1384, 1200, 1036 cmꢀ1; 1H and 13C NMR (see Tables 1 and 2);
HR ESIMS m/z 859.4249 [MþNa]þ, (calcd for C43H60N6NaO11,
1458, 1384, 1259, 1106, 1035 cmꢀ1 1H and 13C NMR (see Table 4);
;
HR ESIMS m/z 846.4056 [MþNa]þ, (calcd for C41H57N7O11,
846.4014). Retention time of AA Marfey derivatives: L-glutamic acid
859.4218). Retention time of AA Marfey derivatives:
L
-threonine
-glutamic acid 32.7 min
-isoleucine 44.4 min ( -Ile 44.4, -Ile
-leucine 45.1 min ( -Leu 45.1, -Leu 47.9 min), -NMe-
phenylalanine 45.1 min, and -phenylalanine 45.5 min ( -Phe 45.5,
-Phe 47.6). Retention time of -glyceric acid on the chiral column
-glyceric acid 3.92 -glyceric acid 3.95 min).
32.7 min (
valine 41.0 min (
41.2 min, -phenylalanine 45.8 min (
and -lysine 46.6 min ( -Lys 46.2, -Lys 46.6 min).
L
-Glu 32.7,
D
-Glu 33.3 min),
-Val 44.1 min),
-Phe 45.8,
L-NMe-alanine 30.9 min,
L-
30.5 min (
-Glu 32.7,
47.5 min),
L
-Tre 30.5,
-Glu 33.5 min),
D
-Thr 33.0 min),
L
L-Val 41.0,
D
L-homo-tyrosine
(L
D
L
L
D
L
L
D
-Phe 48.0 min),
L
L
D
L
D
L
D
L
L
25
D
D
4.3.7. Anabaenopeptin MM850 (7). Glassy material; [
a
]
ꢀ54
D
was 3.95 min (
L
D
(c 0.17, MeOH); UV (MeOH) lmax (log e) 204 (4.29), 228 (3.82), 248
(3.22) nm; IR (KBr): 3255, 2927, 2862, 1658, 1546, 1204, 1136 cmꢀ1
;
23
4.3.2. Micropeptin MM850 (2). Glassy white material; [
(c 0.29, MeOH); UV (MeOH) lmax (log ) 204 (4.21), 248 (2.88) nm;
IR (KBr): 3420, 3029, 2960, 2934, 2874, 1735, 1656, 1527, 1441, 1201,
a
]
þ10
1H and 13C NMR (see Table 4); HR ESIMS m/z 851.4794 [MþH]þ,
D
e
(calcd for C42H63N10O9, 851.4779). Retention time of AA Marfey de-
rivatives:
alanine 30.9 min,
homo-tyrosine 41.0 min,
Phe 47.6 min), and -lysine 46.3 min (
L
-arginine 29.7 min (
-valine 41.1 min (
-phenylalanine 45.5 min (
L-Arg 29.7,
D
-Arg 30.7 min),
-Val 44.2 min),
-Phe 45.5,
L-NMe-
1054 cmꢀ1
;
1H and 13C NMR (see Tables 1 and 2); HR MALDI-TOF-
L
L
-Val 41.1,
D
L-
MS m/z 873.4405 [MþNa]þ, (calcd for C44H62N6NaO11, 873.4369).
L
L
D
-
Retention time of AA Marfey derivatives:
L-threonine 22.9 min
D
L
-Lys 45.9,
D-Lys 46.3 min).
(
D
L
-Thr 22.9,
-Glu 26.2 min),
-phenylalanine 40.0 min (
39.9 min ( -Leu 39.9, -leucine 43.9 min), and
47.3 min. Retention time of -glyceric acid on the chiral column was
3.97 min ( -glyceric acid 3.95, -glyceric acid 3.97 min).
D
-Thr 26.2 min),
-isoleucine 39.3 min (
-Phe 40.0,
L
-glutamic acid 24.5 min (
-Ile 39.3, -Ile 40.0 min),
-Phe 43.1 min), -leucine
-NMe-phenylalanine
L-Glu 24.5,
25
L
L
D
4.3.8. Anabaenopeptin MM913 (8). Glassy material;
(c 0.25, MeOH); UV (MeOH) lmax (log
(3.83), 280 (3.73) nm; IR (KBr): 3350, 2975, 2939, 1655, 1544, 1474,
[
a
]
ꢀ18
D
L
L
D
L
e) 206 (4.57), 228 (4.23), 248
L
D
L
D
1297, 1129, 1035 cmꢀ1 1H and 13C NMR (see Table 5); Positive HR
;
L
D
ESIMS m/z 914.5031 [MþH]þ, (calcd for C49H68N7O10, 914.5028).
Retention time of AA Marfey derivatives: L-NMe-homo-tyrosine
23
4.3.3. Micropeptin MM916 (3). Glassy white material; [
(c 0.29, MeOH); UV (MeOH) lmax (log ) 205 (4.35), 230 (3.72), 248
(3.36), 280 (3.16) nm; IR (KBr): 3420, 3360, 2961, 2933, 2874, 1735,
1670, 1533, 1451, 1384, 1204, 1138, 1007 cmꢀ1; 1H and 13C NMR (see
Tables 1 and 2); HR ESIMS m/z 939.3826 [MþNa]þ, (calcd for
C43H60N6NaO14S, 939.3786). Retention time of AA Marfey de-
a]
þ14
41.0 min (
44.5 min ( -Ile 44.5,
-Lys 46.3 min), -homo-phenylalanine 48.3 min (
-Hph 50.9 min), and -tyrosine 51.1 min ( -Tyr 51.1, -Tyr 52.8 min).
L-NMe-Hty 41.0,
D
-NMe-Hty 44.6 min),
-lysine 46.3 min (
-Hph 48.3,
L
-isoleucine
D
e
L
D
-Ile 47.6 min),
D
L
-Lys 45.8,
D
D
L
L
L
L
D
4.3.9. Anabaenopeptin B (9). Glassy white solid; Negative HR
ESIMS m/z 835.4508 [MꢀH]ꢀ, (calcd for C41H59N10O9, 835.4466);
Identical in all respects with the published data.2,9
rivatives:
tamic acid 32.6 min (
44.5 min ( -Ile 44.5, -Ile 47.6 min),
-Leu 48.1 min), -NMe-phenylalanine 45.3 min, and
nine 45.7 min ( -Phe 45.7, -Phe 47.7). Retention time of
acid on the chiral column was 3.92 min (
-glyceric acid 3.92 min).
L
-threonine 30.8 min (
L
-Thr 30.8,
-Glu 33.4 min),
-leucine 45.3 min (
-phenylala-
-glyceric
D
-Thr 33.2 min),
-isoleucine
-Leu 45.3,
L-glu-
L-Glu 32.6,
D
L
L
D
L
L
D
L
L
4.4. Determination of the absolute configuration of the
L
D
D
amino acids
L-glyceric acid 3.90,
D
Compounds 1e9 (0.3 mg each) were hydrolyzed in 6 M, HCl
(1 mL). The reaction mixture was maintained in a sealed glass vial at
110 ꢂC for 16 h. Theacidwasremoved invacuoand the residuewasre-
24
4.3.4. Micropeptin MM932 (4). Glassy white material; [
(c 0.19, MeOH); UV (MeOH) lmax (log
(3.14), 280 (3.07) nm; IR (KBr): 3400, 2960, 2927, 2874, 1735, 1656,
a]
þ26
D
e
) 203 (4.19), 228 (3.68), 248
suspended in 250
trophenyl)-5- -alanine amide] in acetone (115
NaHCO3 (1M,120 L)wereaddedtoeachreactionvessel.Thereaction
mixture was stirred at 40 ꢂC for 2 h. Then HCl (2 M, 60
L) was added
m
L of H2O. FDAA solution [(1-fluoro-2,4-dini-
L
m
L, 0.03 M) and
1544, 1442, 1384, 1204, 1139, 1010 cmꢀ1
;
1H and 13C NMR (see
m
Tables 1 and 2); Negative HR ESIMS m/z 931.3785 [MꢀH]ꢀ, (calcd
m
for C43H59N6O15S, 931.3759). Retention time of AA Marfey de-
to each reaction vessel and the solutionwas evaporated in vacuo. The
FDAA-amino acids derivatives from hydrolyzate were dissolved in
1 mL ACN and compared with standard FDAA-amino acids by HPLC
rivatives:
tamic acid 24.6 min (
39.5 min ( -Ile 39.5, -Ile 43.7 min),
-Leu 47.6 min), -phenylalanine 40.4 min (
-NMe-tyrosine 47.2 min. Retention time of
L
-threonine 22.8 min (
L
-Thr 22.8,
-Glu 26.3 min),
-leucine 44.2 min (
-Phe 40.4, -Phe 43.4),
-glyceric acid on
D
-Thr 26.3 min),
-isoleucine
-Leu 44.2,
L-glu-
L-Glu 24.6,
D
L
L
D
L
L
analysis: LiChrospherÒ 60, RP-select B (5
mm), flow rate 1 mL/min, UV
D
L
L
D
detection at 340 nm. For compounds 2, 4, and 5, a linear gradient
elution from 9:150 mM triethylammonium phosphate (TEAP) buffer,
pH 3:ACN, within 60 min, was used. For compounds 1, 3, 6, 7, and 8,
a linear gradientelution from 100%, 0.1% TFA in H2O to 1:1, 0.1% TFA in
H2O/ACN, within 60 min, was used. The absolute configuration of
each amino acid was determined by spiking the derivatized hydro-
lyzates with the standard FDAA-amino acids.
and
L
D
the chiral column 3.92 min (
3.92 min).
L-glyceric acid 3.90,
D-glyceric acid
24
4.3.5. Micropeptin MM978 (5). Glassy white material; [
(c 0.12, MeOH); UV (MeOH) lmax (log
(3.43), 280 (3.30) nm; IR (KBr): 3420, 3658, 2970, 2935, 2877, 1735,
a
]
D
þ57
e
) 203 (4.36), 228 (3.87), 248
1656, 1537, 1468, 1384, 1277, 1079, 1036 cmꢀ1
;
1H and 13C NMR
4.5. Determination of the absolute configuration of Ahp and
(see Table 3); HR ESIMS m/z 1001.4977 [MþNa]þ, (calcd for
Amp derivatives
C49H70N8NaO13, 1001.4960). Retention time of AA Marfey de-
rivatives:
L-threonine 24.4 min (
L-Thr 24.4,
D
-Thr 26.3 min),
L-glu-
Compounds 1e5 (0.1 mg each) were oxidized with Jones’ re-
tamic acid 26.5 min (L-Glu 26.5,
D-Glu 27.8 min),
L-isoleucine
agent (1 drop from solution of 1.34 g K2CrO7, 1 mL H2SO4 in 8 mL

9202
E. Zafrir-Ilan, S. Carmeli / Tetrahedron 66 (2010) 9194e9202
H2O) in 0.5 mL acetone at 0 ꢂC 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.
substrate solution was added and the kinetic of the reaction was
measured at 405 nm, 37 ꢂC for 20 min.
Acknowledgements
We thank Noam Tal, The Mass Spectrometry Laboratory of The
School of Chemistry, Tel Aviv University, for the ESI mass spectra
measurements, and Karin Shereshevsky, The Mass Spectrometry
Laboratory of The Maiman Institute for Proteome Research of Tel
Aviv University, for the MALDI mass spectra measurements.
This research was supported by the Israel Science Foundation grant
776/06.
4.6. Determination of the absolute configuration of glyceric
acid
Extraction of the acid hydrolyzates of compounds 1e4 with
diethyl-ether separated the glyceric acid from the amino acid
salts. The ether was removed in vacuo and the residue was dis-
solved in MeOH (1 mL). The MeOH solution was analyzed on an
Astec, ChirobioticÔ, LC Stationary Phases, 250ꢁ4.6 mm flow rate
1 mL/min, UV detection at 210 nm, linear elution 1:49 1% trie-
thylamine, 1% acetic acid (TEAA) aq buffer, pH 4: MeOH. The
absolute configuration of the glyceric acid from the micropeptins
Supplementary data
Tables 5e13, summarizing the 1H and 13C NMR, HMQC or HSQC,
HMBC, and ROESY data of 1e9, as well as, the 1H and 13C NMR
spectra of compounds 1e9, are available. Supplementary data as-
sociated with this article can be found in the online version, at
doi:10.1016/j.tet.2010.09.067. These data include MOL files and
InChIKeys of the most important compounds described in this
article.
was determined by spiking with a mixture of standard
acids.
L,D-glyceric
4.7. Proteases 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
References and notes
CaCl2, pH 7.5 buffer solution. Benzoyl-L-arginine-p-nitroanilide
1. Okino, T.; Murakami, M.; Haraguchi, R.; Munekata, J.; Matsuda, H.; Yamaguchi, K.
Tetrahedron Lett. 1993, 34, 8131e8134.
2. Harada, K.;Fujii, K.;Shimada, T.;Suzuki, M.;Sano, H.; Adachi, K.;Carmichael, W. W.
Tetrahedron Lett. 1995, 36, 1511e1514.
3. Ersmark, K.; Del Valle, J. R.; Hanessian, S. Angew. Chem., Int. Ed. 2008, 47,
1202e1223.
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 dis-
solved in Tris buffer (1 mg/mL). Test samples were dissolved in
DMSO (1 mg/mL). A 100
and 10 L sample solution were added to each micro titer plate well
and pre-incubated at 37 ꢂC for 10 min. Then, 10
L of substrate
solution was added and the kinetic of the reaction was measured at
405 nm, 37 ꢂC for 30 min. Elastase (75 mg/mL) and thrombin
(0.5 g/mL) were dissolved in 0.2 M TriseHCl, pH 8 buffer solution.
m
L buffer solution, 10
m
L enzyme solution
4. Ishida, K.; Kato, T.; Murakami, M.; Watanabe, M.; Watanabe, M. F. Tetrahedron
2000, 56, 8643e8656.
m
5. Reshef, V.; Carmeli, S. Tetrahedron 2006, 62, 7361e7369.
6. Southan, C. Drug Discovery Today 2001, 6, 681e688.
7. Zafrir, E.; Carmeli, S. J. Nat. Prod. 2010, 73, 352e358.
8. Grach-Pogrebinsky, O.; Carmeli, S. Tetrahedron 2008, 64, 10233e10238.
9. Murakami, M.; Shin, H. J.; Matsuda, H.; Ishida, K.; Yamaguchi, K. Phytochemistry
1997, 44, 449e452.
10. Marfey’s reagent: 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide Marfey, P.
Carlsberg Res. Commun. 1984, 49, 591e596.
11. Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon, B. C. L. J. Chem. Soc. 1946,
39e45.
m
Z-Gly-Pro-Arg-4MbNA-acetate salt, the thrombin substrate, was
dissolved in Tris buffer (0.5 mg/mL). N-Suc-Ala-Ala-Ala-p-nitro-
anilide, the elastase substrate, was dissolved in Tris buffer
(1 mg/mL). The test samples were dissolved in DMSO (1 mg/mL).
12. Fujii, K.; Sivonen, K.; Adachi, K.; Noguchi, K.; Sano, H.; Hirayama, K.; Suzuki, M.;
Harada, K. Tetrahedron Lett. 1997, 38, 5525e5528.
13. Itou, Y.; Suzuki, S.; Ishida, K.; Murakami, M. Bioorg. Med. Chem. Lett. 1999, 9,
1243e1246.
14. Kodani, S.; Suzuki, S.; Ishida, K.; Murakami, M. FEMS Microbiol. Lett. 1999, 178,
343e348.
15. Sano, T.; Usui, T.; Ueda, K.; Osada, H.; Kaya, K. J. Nat. Prod. 2001, 64, 1052e1055.
16. Okumura, H. S.; Philmus, B.; Portmann, C.; Hemscheidt, T. K. J. Nat. Prod. 2009,
72, 172e176.
For elastase, 150
sample solution were added to each micro titer plate well and pre-
incubated at 30 ꢂC for 20 min. Then, 30
L of substrate solution was
added and the kinetic of the reaction was measured at 405 nm,
37 ꢂC for 20 min. For thrombin, 170
L buffer solution,10 L enzyme
solution and 10 L sample solution were added to each micro titer
plate well and pre-incubated at 25 ꢂC for 5 min. Then, 10
L of
mL buffer solution,10 mL enzyme solution and 10 mL
m
m
m
m
m
17. Gesner-Apter, S.; Carmeli, S. J. Nat. Prod. 2009, 72, 1429e1436.