Eight micropeptins from a Microcystis spp. bloom collected from a fishpond near Kibbutz Lehavot HaBashan, Israel

Article abstract of DOI:10.1016/j.tet.2013.09.054

Eight new micropeptin-type cyclic depsipeptides, micropeptins LH920, LH1021, LH1048, LH1062, LH911A, LH911B, LH911C, and LH925, along with five known micropeptins, three known anabaenopeptins and two known microcystins (LR and RR), were isolated from a wa

Full text of DOI:10.1016/j.tet.2013.09.054

Tetrahedron 69 (2013) 10108e10115
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Eight micropeptins from a Microcystis spp. bloom collected from
a fishpond near Kibbutz Lehavot HaBashan, Israel
*
Margarita Vegman, 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 23 June 2013
Received in revised form 1 September 2013
Accepted 16 September 2013
Available online 25 September 2013
Eight new micropeptin-type cyclic depsipeptides, micropeptins LH920, LH1021, LH1048, LH1062,
LH911A, LH911B, LH911C, and LH925, along with five known micropeptins, three known anabaeno-
peptins and two known microcystins (LR and RR), were isolated from a water bloom biomass of Mi-
crocystis spp. assembly that was collected from a fishpond near Kibbutz Lehavot HaBashan, Israel. The
structures of the pure compounds were elucidated using 1D and 2D NMR techniques, as well as high-
resolution mass spectrometry. The stereochemistry of the new natural products was determined using
Marfey’s method for amino acids. The inhibitory activity of the compounds was determined for the
serine proteases, trypsin, and chymotrypsin.
Keywords:
Micropeptins
Natural products
Protease inhibitors
Cyanobacteria
Microcystis
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Evidence accumulated over the past 30 years suggest that these
groups of protease inhibitors affect the ability of these cyanobac-
The micropeptins are cyclic depsipeptides characterized by the
unique 3-amino-6-hydroxy-piperidone (Ahp) moiety and a lactone
ring. The Ahp moiety is formed by cyclization of glutamyl-g-alde-
teria to survive in their ecological niche by preventing the de-
toxification of microcystins,6 thus having a negative impact on
population growth and on survival of zooplankton species.7 The
involvement of the micropeptins and anabaenopeptins in the lysis
of cultured cyanobacteria cell lines was demonstrated and may
imply their role in strains dynamic in water blooms.8 As part of our
ongoing research on the chemistry and chemical ecology of cya-
nobacteria blooms in water bodies,9 a biomass of yellow-brown
bloom material composed of an assembly of several Microcystis
spp. (TAU IL-376) was collected in November 2007 from a fishpond
near Kibbutz Lehavot HaBashan, Israel. The extract of this bloom
material afforded eight new micropeptins; micropeptins LH920 (1),
LH1021 (2), LH1048 (3), LH1062 (4), LH911A (5), LH911B (6), LH911C
(7), and LH925 (8) along with the known, micropeptins MZ939A10
and SF909,11 cyanopeptolins S,12 SS,13 and 1020,14 anabaenopeptins,
A,15 B,15 and F,16 and microcystins LR17 and RR.18 The isolation and
structure elucidation of the new secondary metabolites and their
biological activity are discussed below.
hyde to the amide nitrogen of the neighboring amino acid. The
lactone ring contains six amino-acid residues and derives from
a cyclization of the C-terminal carboxyl usually to the side-chain
hydroxyl of threonine1 or, in a single case, to the hydroxyl of 3-
hydroxy-4-methylproline (Hmp).2 A side-chain of one to four
amino-, hydroxyl- or fatty acids is attached to the amide nitrogen of
the lactone forming amino acid. So far, 131 micropeptins have been
isolated3 from fresh and brackish water toxic-bloom forming gen-
era of cyanobacteria4 and from filamentous bloom-forming marine
cyanobacteria.5 Most of the micropeptin-type metabolites display
inhibitory activity against serine proteases. The selectivity of the
inhibitory activity to the trypsin clade or chymotrypsin clade of
enzymes is governed by the nature of the amino acid at the fifth
position from the C-terminus of the peptide, where basic amino
acids select for the trypsin clade and lipophilic or aromatic amino
acids select for the chymotrypsin clade. The micropeptins usually
appear, in blooms and cultures, as a mixture of several isoforms
with comparable protease inhibition potency, which does not ex-
plain their co-existence in the same bloom or their ecological role.
2. Results and discussion
The lyophilized Microcystis biomass was extracted and sepa-
rated in two batches. The 7:3 MeOH/H2O extract from each batch
was separated on a reversed phase (ODS) open column followed by
separation on Sephadex LH-20 and repeated preparative reversed-
phase HPLC to afford 1e8 and the 10 known metabolites mentioned
* 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 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.09.054

M. Vegman, S. Carmeli / Tetrahedron 69 (2013) 10108e10115
10109
above. Micropeptin LH920 (1) was isolated as a transparent glassy
material exhibiting an HRESIMS sodiated quasi-molecular ion at m/
z 943.4576, which was consistent with a molecular formula of
a micropeptin-type compound, i.e., a broad singlet hydroxyl signal
at dH 5.99, a downfield shifted quartet ester-oxymethine at dH 5.37,
a singlet NMe group at dH 2.75, and a doublet methyl signal at dH
1.16.19 Taking into account the NMe-aromatic amino acid and the
N,N-disubstituted-amino acid of the micropeptins, the five amide
doublet protons revealed that this micropeptin was composed of
seven amino acids and a fatty acid. Analysis of the COSY, TOCSY, and
HSQC 2D NMR experiments (Table S1 in Supplementary data)
allowed the assignment of the side chains of a valine, a glutamine,
C
46H64N8O12 and 19 degrees of unsaturation. Examination of the
NMR spectra of 1 in DMSO-d6 (Tables 1 and 2 and Table S1 in
Supplementary data) revealed its peptide nature, i.e., nine car-
boxylic carbons in the 13C NMR spectrum and four doublet and one
triplet amide protons in the 1H NMR spectrum. Some characteristic
signals in the 1H NMR spectrum of 1 suggested that it was
Table 1
1H NMR data of compounds 1e8 in DMSO-d6
Moiety
1Val/Ile
Position
1a
2b
3a
4a
5b
6a
7b
8a
2
3
4
4.70, dd
2.04, m
0.72, d
4.62, dd
2.04, m
0.72, d
4.69, dd
2.05, m
0.71, d
4.67, dd
2.04, m
0.70, d
4.58, dd
2.01, m
0.75, d
4.60, dd
1.74, m
1.05, m
1.25, m
0.80, t
0.83, d
7.72, d
5.06, dd
2.79, dd
3.29, m
7.23, d
7.26, t
4.60, m
1.74, m
1.03, m
1.25, m
0.80, t
0.83, d
7.71, d
5.12, dd
2.79, br t
3.29, m
7.22, d
7.25, t
4.62, m
1.75, m
1.04, m
1.25, m
0.80, t
0.83, d
7.69, d
5.12, dd
2.80, dd
3.29, m
7.22, d
7.25, t
5
6
NH
2
3
0.86, d
d
0.84, d
d
0.85, d
d
0.84, d
d
0.85, d
d
7.41, d
4.91, br d
2.70, br t
3.09, br d
7.00, d
6.77, d
9.36, s
2.75, s
4.77, d
1.80, dd
2.86, dd
d
7.48, d
4.87, m
2.69, br t
3.11, br d
6.99, d
6.76, d
9.32, s
2.74, s
4.74, dd
1.79, dd
2.85, br t
d
7.39, d
4.88, br d
2.70, br t
3.10, br d
6.98, d
6.76, d
9.32, s
2.74, s
4.74, d
1.79, dd
2.85, br t
d
7.44, d
4.86, br d
2.68, br t
3.08, br d
6.98, d
6.76, d
9.34, br s
2.74, s
4.72, dd
1.78, m
2.84, br t
d
7.66, d
5.12, dd
2.79, br t
3.30, m
7.22, d
7.25, t
7.18, t
2.74, s
4.37, d
1.74, m
2NMeTyr/Phe
5,50
6,60
7-OH/7
NMe
2
7.18, t
2.72, s
4.31, m
1.92, m
7.19, t
2.72, s
4.37, d
1.75, m
7.18, t
2.72, s
4.36, d
1.75, m
3Phe/Ile/Val
3
4
0.60, m
1.06, m
0.59, br d
ꢀ0.26, d
d
ꢀ0.19, d
0.60, m
1.04, m
0.59, br d
ꢀ0.26, d
d
0.60, m
1.06, m
0.59, br d
ꢀ0.26, d
d
5,50/5
6,60/6
7
3
4
6.84, d
7.19, t
7.13, t
6.82, d
7.17, t
7.13, t
6.82, d
7.16, t
7.13, t
6.82, d
7.16, t
7.12, t
0.46, d
d
d
4Ahp
3.60, m
1.58, m
2.39, br q
1.53, m
1.65, m
5.05, br s
5.99, br s
7.05, d
4.15, ddd
1.57, m
2.05, m
1.98, m
d
3.60, m
1.60, m
2.38, br q
1.54, m
1.68, m
5.04, br s
6.05, br d
7.06, d
4.13, ddd
1.58, m
2.05, m
1.94, m
d
3.60, m
1.57, m
2.39, br q
1.53, m
1.68, m
5.04, br s
6.00, d
7.07, d
4.21, m
1.36, m
1.88, m
1.37, m
3.02, m
3.59, m
1.57, m
2.36, m
1.54, m
1.65, m
5.04, br s
6.06, d
7.09, d
4.19, m
1.35, m
1.86, m
1.36, m
3.02, m
4.43, m
1.72, m
2.60, br q
1.73, m
4.45, m
1.73, m
2.61, br q
1.72, m
1.77, m
4.91, br s
6.11, br s
7.26, d
4.30, m
1.45, m
2.03, m
1.44, m
3.06, m
4.43, m
1.72, m
2.61, br q
1.73, m
4.44, m
1.73, m
2.61, br q
1.74, m
5
6
4.90, br s
6.12, d
7.27, d
4.31, m
1.42, m
2.04, m
1.41, m
3.06, m
4.90, br s
6.14, br s
7.26, d
4.28, m
1.46, m
2.02, m
1.27, m
1.46, m
1.53, m
2.82, m
8.53, br d
8.20, br s
2.53, br t
4.62, m
5.49, br q
1.21, d
7.67, d
4.26, m
3.82, dd
3.98, dd
6.09, d
d
4.90, br s
6.11, d
7.28, d
4.31, m
1.48, m
2.05, m
1.25, m
1.49, m
1.59, m
2.95, m
8.54, d
d
6-OH
NH
2
5Gln/Arg/Lys
3
4
5
6
NH
NH2/NH/
NMe
2
3
4
NH
2
3
d
d
d
d
d
d
8.43, d
6.63, br s
7.16, br s
4.61, d
5.37, br q
1.16, d
7.90, d
d
8.43, d
8.47, d
7.77, br t
8.46, d
7.76, br t
8.57, d
7.46, br t
8.59, d
7.63, br s
6.66, br s
7.12, br s
4.60, d
5.38, br q
1.17, d
7.89, d
4.41, dd
3.99, ddq
2.71, s
4.61, m
5.49, br q
1.21, d
7.67, d
4.26, m
3.82, dd
3.97, dd
6.07, d
d
6Thr
4.56, d
5.38, q
1.14, d
7.83, d
4.38, dt
1.73, m
1.88, m
2.18, m
d
4.53, d
5.39, q
1.15, d
7.87, d
4.39, dt
1.76, m
1.89, m
2.32, m
d
4.64, d
5.49, q
1.20, d
7.64, d
4.25, m
3.82, dd
3.97, dd
6.09, d
d
4.65, d
5.49, br q
1.20, d
7.62, d
4.25, m
3.82, dd
3.98, dd
6.06, d
d
7Thr/Glu/Ga
d
4/3-OH
5
NH
d
1.03, d
4.88, d
7.65, d
d
d
d
7.99, d
d
8.00, d
3.55, s
d
d
d
d
d
OMe
d
d
d
d
d
7/8Gly
2
3.78, dd
3.85, dd
8.06, t
2.11, t
1.51, m
1.25, m
1.26, m
0.85, t
d
3.72, dd
3.78, dd
8.06, t
2.10, t
1.48, tt
1.22, m
1.24, m
0.83, t
d
d
d
d
d
d
NH
2
3
4
5
6
7
8
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
8/9Hex/Oct
2.11, m
1.48, m
1.23, m
1.23, m
1.22, m
1.24, m
0.83, t
2.11, m
1.47, m
1.22, m
1.22, m
1.21, m
1.23, m
0.83, t
d
d
a
400 MHz.
500 MHz.
b

10110
M. Vegman, S. Carmeli / Tetrahedron 69 (2013) 10108e10115
Table 2
13C NMR data of compounds 1e8 in DMSO-d6
Moiety
1Val/Ile
Position
1a
2b
3a
4a
5b
6a
7b
8a
1
2
3
4
5
6
1
2
172.0, s
56.0, d
30.9, d
17.4, q
19.4, q
d
172.2, s
56.2, d
30.8, d
17.5, q
19.4, q
d
172.1, s
55.9, d
30.5, d
17.3, q
19.4, q
d
172.1, s
56.0, d
30.8, d
17.4, q
19.4, q
d
172.7, s
56.4, d
31.0, d
17.9, q
19.4, q
d
172.8, s
55.5, d
37.4, d
24.8, t
11.1, q
16.0, q
169.1, s
60.6, d
34.3, t
137.9, s
129.7, dꢁ2
128.6, dꢁ2
126.7, d
30.2, q
169.8, s
55.9, d
27.4, d
18.1, q
18.1, q
d
172.9, s
55.5, d
37.5, d
24.6, t
11.2, q
16.0, q
169.2, s
60.6, d
34.3, t
137.9, s
129.7, dꢁ2
128.7, dꢁ2
126.8, d
30.3, q
169.8, s
54.2, d
33.1, d
23.6, t
10.4, q
14.0, q
d
172.8, s
55.5, d
37.5, d
24.7, t
11.2, q
16.0, q
169.2, s
60.6, d
34.3, t
137.8, s
129.7, dꢁ2
128.7, dꢁ2
126.8, d
30.3, q
169.8, s
54.3, d
33.1, d
23.8, t
10.4, q
14.0, q
d
2NMeTyr/Phe
169.3, s
61.0, d
33.0, t
127.6, s
130.5, dꢁ2
115.5, dꢁ2
156.4, s
30.4, q
170.5, s
50.4, d
35.5, t
136.9, s
129.6, dꢁ2
127.9, dꢁ2
126.3, d
169.1, s
48.9, d
21.7, t
29.4, t
73.9, d
169.8, s
51.9, d
26.5, t
31.6, t
173.8, s
d
169.5, s
61.0, d
32.9, t
127.7, s
130.6, dꢁ2
115.5, dꢁ2
156.4, s
30.5, q
170.5, s
50.4, d
35.4, t
136.9, s
129.6, dꢁ2
127.9, dꢁ2
126.4, d
168.9, s
48.9, d
21.7, t
29.4, t
73.9, d
169.8, s
52.0, d
26.5, t
31.6, t
173.8, s
d
169.3, s
61.1, d
33.0, t
127.6, s
130.5, dꢁ2
115.5, dꢁ2
156.4, s
30.5, q
170.5, s
50.4, d
35.4, t
136.9, s
129.5, dꢁ2
127.8, dꢁ2
126.3, d
169.0, s
48.8, d
21.7, t
29.4, t
73.9, d
169.8, s
51.5, d
27.5, t
25.0, t
40.3, t
d
169.4, s
61.1, d
33.0, t
127.6, s
130.5, dꢁ2
115.5, dꢁ2
156.4, s
30.5, q
170.5, s
50.5, d
35.5, t
136.9, s
129.6, dꢁ2
127.9, dꢁ2
126.3, d
168.2, s
48.9, d
21.7, t
29.4, t
73.9, d
169.7, s
51.5, d
27.4, t
25.1, t
40.3, t
d
169.3, s
60.7, d
34.4, t
137.9, s
129.8, dꢁ2
128.7, dꢁ2
126.8, d
30.5, q
169.9, s
54.3, d
33.1, d
23.8, t
10.5, q
14.0, q
d
3
4
5,50
6,60
7
NMe
1
2
3Phe/Ile/Val
3
4
5,50/5
6,60/6
7
2
3
4
5
6
1
2
3
4
5
6
d
4Ahp
169.5, s
49.1, d
21.7, t
29.9, t
74.2, d
170.3, s
51.9, d
27.3, t
25.2, t
40.5, t
d
169.4, s
49.0, d
21.6, t
29.8, t
74.1, d
170.2, s
52.0, d
27.3, t
25.1, t
40.3, t
d
169.5, s
49.1, d
21.7, t
29.9, t
74.1, d
170.4, s
51.8, d
29.4, t
22.4, t
24.7, t
48.3, t
32.6, q
169.1, s
54.6, d
72.1, d
18.0, q
172.0, s
70.7, d
68.3, t
d
169.5, s
49.1, d
21.7, t
29.9, t
74.1, d
170.4, s
51.6, d
29.4, t
22.3, t
23.2, t
56.8, t
42.4, qꢁ2
169.2, s
54.7, d
72.1, d
18.0, q
172.0, s
70.7, d
68.3, t
d
5Gln/Arg/Lys
7/NMe
d
d
157.0, s
169.2, s
54.7, d
72.1, d
17.8, q
172.5, s
52.0, d
27.3, t
d
156.8, s
169.2, s
54.9, d
72.0, d
18.0, q
172.3, s
51.8, d
27.1, t
d
156.7, s
169.2, s
54.5, d
72.3, d
17.9, q
171.9, s
70.6, d
68.4, t
d
156.9, s
169.1, s
54.5, d
72.2, d
17.9, q
171.9, s
70.7, d
68.4, t
d
6Thr
1
2
3
4
1
2
3
4
5
169.3, s
54.8, d
72.1, d
18.0, q
d
169.3, s
55.0, d
72.0, d
18.1, q
170.9, s
57.7, d
67.0, d
19.7, q
d
7Thr/Glu/Ga
d
d
d
d
174.5, s
d
173.1 s
51.5, q
d
d
d
d
d
OMe
d
d
d
d
d
d
7/8Gly
1
2
1
2
3
4
5
6
7
8
170.0, s
42.1, t
172.9, s
35.3, t
25.0, t
31.0, t
22.0, t
14.0, q
d
169.3, s
42.1, t
172.7, s
35.3, t
25.1, t
31.0, t
22.1, t
14.0, q
d
d
d
d
d
d
d
d
d
d
d
d
8/9Hex/Oct
172.6, s
35.2, t
25.4, t
28.6, t
28.7, t
31.3, t
22.2, t
14.1, q
172.7, s
35.2, t
25.4, t
28.6, t
28.7, t
31.4, t
22.2, t
14.1, q
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
a
100 MHz.
125 MHz.
b
a threonine, a glycine, a hexanoic acid, two short fragments in
agreement with N,N-disubstituted aromatic amino acids, a 1,4-
disubstituted phenol ring, a monosubstituted phenyl ring, an
amino hydroxy piperidone (Ahp) moiety, and n-pentyl.
carboxamide of Gly and the NH of Thr and the carboxamide of HA
and NH of Gly, and the NOE correlations between H-2,3a,3b of N,N-
disubstituted-Phe and Ahp H-6. The ester linkage between Thr and
Val was established through the HMBC correlation of Thr H-3 and
the carboxyl of Val. Acid hydrolysis of 1 and derivatization with
The latter fragments were extended to the complete structures,
and the carboxamide carbons were assigned to the side chains, by
analysis of the correlation map of an 1He13C HMBC experiment
(Table S1 in Supplementary data). This procedure established the
structures of Val, NMeTyr, N,N-disubstituted Phe, Ahp, Gln, 3-O-
substituted-Thr, Gly, and hexanoic acid (HA) residues. The sequence
of the acid residues of the peptide: Val, NMeTyr, N,N-disubstituted-
Phe, Ahp, Gln, Thr, Gly, and HA was assigned on the basis of HMBC
and ROESY correlations as follows: HMBC correlations between the
carboxamide of NMeTyr and Val-NH, the carboxamide of N,N-di-
substituted-Phe and the NMe of NMeTyr, the carboxamide of Gln
and NH of Ahp, the carboxamide of Thr and NH of Gln, the
Marfey’s reagent,20 followed by HPLC analysis, demonstrated the
L-
configuration of valine, NMe-tyrosine (by comparison with syn-
thetic standard), phenylalanine, threonine, and glutamic acid resi-
dues. Jones oxidation21 of 1, followed by a similar hydrolysis,
derivatization and HPLC analysis, determined the 3S-configuration
for the Ahp residue (oxidation and subsequent hydrolysis liberated
L-glutamic acid from Ahp). The configuration of C-6 of the Ahp was
determined as R on the basis of the coupling constants of H-6
(<1 Hz), which points to an equatorial orientation of this proton,
the chemical shift of the pseudoaxial H-4 (dH 2.39 br q J¼12.8 Hz),
which is shifted downfield by the axial 6-hydroxy-group, the trans

M. Vegman, S. Carmeli / Tetrahedron 69 (2013) 10108e10115
10111
diaxial relationship with H-3 and the NOE correlation between H-
Ahp, the carboxamide of 3-O-substituted-Thr and NH of Gln, the
carboxamide of Thr and H-2 and NH of 3-O-substituted-Thr, the
carboxamide of Gly and H-2 and NH of Thr, and the carboxamide of
HA and NH of Gly, and correlation of 3-O-substituted-Thr H-3 with
the carboxyl of Val established the sequence and point of cycliza-
tion of 2. A similar procedure to that applied for 1 assigned the
absolute configuration of all of the amino-acid residues, in micro-
peptin LH1021, to be L. Based on the results discussed above the
structure of micropeptin LH1021 was established as 2.
4pax and the 6-OH.11 Marfey’s analysis using 1-fluoro-2,4-
dinitrophenyl-5-
threonine from
L L-
-alanine amide (FDAA)20 fails to distinguish
L-allo-threonine and L-isoleucine from L-allo-iso-
leucine and thus additional evidence was needed to support the
assignment of the absolute configuration of C-3 of threonine. The
observed J-value (0e1 Hz) between H-2 and H-3 of the N,O-di-
substituted threonine in 1, suggested that, as in the case of all
known micropeptins, it should be L-threonine and not L-allo-thre-
onine.22 On the basis of the arguments described above the struc-
Micropeptins LH1048 (3) and LH1062 (4) were isolated as glassy
solids that presented high-resolution ESIMS protonated quasi-
ture of micropeptin LH920 was established as 1.
Micropeptin LH1021 (2) was isolated as a glassy solid presenting
molecular ions, m/z 1049.5670 and 1063.5840, which corre-
a
high-resolution ESIMS sodiated quasi-molecular ion, m/z
sponded to the molecular formulas,
C52H76N10O13 and
1044.5032, which corresponds to the molecular formula,
C50H71N9O14 and 20 degrees of unsaturation. The 1H NMR spectrum
of 2 resembled that of 1 except for the presence of seven extra
protons; a doublet amide proton (dH 7.65); an exchangeable doublet
proton (dH 4.88); two protons next to electronegative atoms (dH
4.41, dd and 3.99, ddq) and a doublet methyl (dH 1.03). Correlations
of the later protons in the COSY spectrum revealed an additional
threonine moiety relative to 1. The chemical shifts of the 1H (Table
1) and 13C (Table 2) NMR signals of the remaining amino acids and
the fatty acid that compose 2, were almost identical to those of 1.
Analysis of the COSY, TOCSY, HSQC, and HMBC 2D NMR
experiments (Table S2 in Supplementary data) established the
structures of Val, NMeTyr, N,N-disubstituted-Phe, Ahp, Gln, 3-O-
substituted-Thr, Thr, Gly, and HA residues. HMBC correlations be-
tween the carboxamide of NMeTyr and Val-NH, the carboxamide of
N,N-disubstituted-Phe and the NMe of NMeTyr, the carboxamide
and C-6 of Ahp and Phe-H-2, the carboxamide of Gln and NH of
C
53H78N10O13, respectively, and 20 degrees of unsaturation, each.
Their 1D NMR spectra (Tables 1 and 2) were almost identical and
indicated an extra methoxy-group in 4, in accordance with the
mass spectral data. When the 1H NMR spectra (Table 1) of 3 and 4
were compared with that of 1, the absence of the primary amide
protons of Gln from the spectra of 3 and 4 was noticed and instead
a broad signal integrating for two protons and a very broad ex-
changeable signal appeared at 3.02 ppm and around 7.00 ppm,
respectively, in the spectra of 3 and 4. Analysis of the COSY, TOCSY,
HSQC, and HMBC 2D NMR experiments of 3 and 4 (Tables S3 and S4
in Supplementary data) established the structures of Val, NMeTyr,
N,N-disubstituted-Phe, Ahp, Arg, 3-O-substituted-Thr, Glu (Glu-
OMe, in 4), and octanoic acid (OA) for both compounds. For 3,
HMBC correlations between the carboxamide of NMeTyr and Val-
NH, the carboxamide of N,N-disubstituted-Phe and H-2 and NMe
of NMeTyr, the carboxamide of Arg and NH of Ahp, the carboxamide
of 3-O-substitued-Thr and NH of Arg, the carboxamide of Glu and

10112
M. Vegman, S. Carmeli / Tetrahedron 69 (2013) 10108e10115
H-2 and NH of 3-O-substituted-Thr, and the carboxamide of OA and
NH of Glu and NOE correlations (from ROESY experiment) between
Ahp H-6 and Phe H-3 and H-30 established the sequence of the
peptide. Correlation of 3-O-substituted-Thr H-3 with the carboxyl
of Val established the point of cyclization of 3. For 4, a similar
pattern of correlations was observed except for the HMBC corre-
lation of the carboxamide of 3-O-substituted-Thr and the NH of Arg.
The connectivity between these two was established by the ob-
servation of NOE correlation of 3-O-substituted-Thr H-2 with the
amide proton of Arg, in the ROESY map. The absolute configuration
of all of the amino acids that compose micropeptins LH1048 and
LH1062 were determined as L, by the same procedure applied for 1.
Based on these arguments, the structures of micropeptins LH1048
and LH1062 were established as 3 and 4, respectively.
N,N-disubstituted-Val and the NMe of NMePhe, C-6 of Ahp and N,N-
disubstituted-Val H-2, the carboxamide of Arg and NH of Ahp, the
carboxamide of 3-O-substituted-Thr and NH of Arg, the carbox-
amide of 3-sulfo-GA and H-2 and NH of 3-O-substituted-Thr, and
correlation of 3-O-substituted-Thr H-3 with the carboxyl of Ile
established the sequence and point of cyclization of 6. This analysis
defined that the Val and Ile switched positions in 5 and 6. The
absolute configuration of the chiral centers of the amino acids of 6
was established by application of Marfey’s procedure,20 as de-
scribed for 5, leading to the assignment of structure 6 to micro-
peptin LH911B.
Micropeptin LH911C (7), shared the same nominal mass with 5
and 6, but its HRESIMS exhibited a sodiated quasi-molecular ion at
m/z 934.4233, consistent with a molecular formula of C41H65N7O14
S
Micropeptin LH911A (5) was isolated as a transparent glassy
solid, which presented a high-resolution ESIMS sodiated molecular
cluster ion, m/z 934.3964, corresponding to the molecular formula,
and 13 degrees of unsaturation, thus differing from 5 and 6. Cor-
respondingly, 7 displayed 1D NMR spectra (Tables 1 and 2) signif-
icantly different from that of 5 and 6, indicating the replacement of
C
39H61N9O14S and 14 degrees of unsaturation. Examination of the
the Val unit by additional Ile (in 7) and the Arg by u-NMeLys. In-
NMR spectra of 5 in DMSO-d6 (Tables 1 and 2 and Table S5 in
Supplementary data) revealed that this micropeptin markedly
differed in the composition of its acid residues from those of 1e4.
Analysis of the COSY, TOCSY, HSQC, and HMBC 2D NMR experi-
ments of 5 (Table S5 in Supplementary data) established the
structure of Val, NMePhe, N,N-disubstituted-Ile, Ahp, Arg, 3-O-
substituted-Thr, and 3-sulfo-glyceric acid (3-sulfo-GA).12 The car-
boxamide of the arginine residue was assigned based on an NOE
correlation between the Ahp amide proton and Arg H-2, and an
HMBC correlation of the Ahp amide proton with the carboxamide
that resonated at 170.3 ppm. HMBC correlations between the car-
boxamide of NMePhe and Val-NH, the carboxamide of N,N-di-
substituted-Ile and the NMe of NMePhe, C-6 of Ahp, and N,N-
disubstituted-Ile H-2, the carboxamide of Arg and NH of Ahp, the
carboxamide of 3-O-substituted-Thr and NH of Arg, the carbox-
amide of 3-sulfo-GA and H-2 and NH of 3-O-substituted-Thr, and
correlation of 3-O-substituted-Thr H-3 with the carboxyl of Val
established the sequence and point of cyclization of 5. The absolute
configuration of the chiral centers of the amino acids of 5 was
established by application of Marfey’s procedure,20 as described for
terpretation of the COSY, TOCSY, HSQC, and HMBC 2D NMR ex-
periments of 7 (Table S7 in Supplementary data) revealed the
presence of Ile, NMePhe, N,N-disubstituted-Ile, Ahp,
u-NMeLys, 3-
O-substituted-Thr, and 3-sulfo-GA. The carboxamide of the lysine
residue was assigned based on an NOE correlation between the Ahp
amide proton and
u-NMeLys H-2, and an HMBC correlation of the
Ahp amide proton with the carboxamide that resonated at
170.4 ppm. HMBC correlations between the carboxamide of NMe-
Phe and Ile-NH, the carboxamide of N,N-disubstituted-Ile and the
NMe of NMePhe, C-6 of Ahp and N,N-disubstituted-Ile H-2, the
carboxamide of
u-NMeLys and NH of Ahp, the carboxamide of 3-
sulfo-GA and H-2 and NH of 3-O-substituted-Thr, and correlation
of 3-O-substituted-Thr H-3 with the carboxyl of Ile, and NOE cor-
relation of
u-NMeLys with the a-amide proton and H-2 of 3-O-
substituted-Thr, established the sequence and point of cyclization
of 7. Application of Marfey’s procedure,20 as described for 5,
assigned structure 7 to micropeptin LH911C.
Micropeptin LH925 (8) was isolated as a transparent glassy
solid, which presented a high-resolution ESIMS sodiated quasi-
molecular ion, m/z 948.4383, corresponding to the molecular for-
mula, C42H67N7O14S and 13 degrees of unsaturation. Micropeptin
LH925 presented 1D NMR spectra almost identical with those of 7.
The only differences observed in the 1H NMR spectra were the
downfield shift of the lysine H2-6 and NMe by 0.13 and 0.18 ppm, in
1. This procedure established the
L configuration of all of the a-
carbons of the amino acids in 5 and allowed determining the 6R
configuration of the aminal carbon of Ahp, as described for 1. As
mentioned above, Marfey’s analysis fails to distinguish
L-threonine
from -allo-threonine and -isoleucine from -allo-isoleucine. The
L
L
L
8, the absence of the u-NH in 8, and the doubled integration of the
observed J-value (0e1 Hz) between H-2 and H-3 of the N,O-di-
substituted threonine in 5, suggested that, as in the case of all
NMe signal in 8. In the 13C NMR spectra, 8 differed from 7 in the
upfield shift of the lysine C-5 signal (by 1.5 ppm) and the downfield
shift of C-6 and NMe signals (by 8.5 and 9.6 ppm, respectively).
These differences were in accordance with the presence of a N,N-
known micropeptins, it should be L
-threonine.22 In the case of the
N,N-disubstituted-Ile, the 13C NMR chemical shifts measured for
compound 5, 10.5 (C-5) and 14.0 (C-6) ppm, were found to be
dimethyl group at the
u-amine of Lys in 8. Interpretation of the
similar to those reported for
L
-Ile (10.3 and 13.9 ppm, C-5 and C-6,
COSY, TOCSY, HSQC, and HMBC 2D NMR experiments of 8 (Table S8
in Supplementary data) revealed the presence and the sequence of
respectively),23 while different from those reported for allo-Ile (12.2
and 14.1 ppm, C-5 and C-6, respectively).24 On the basis of the ar-
guments described above the structure of micropeptin LH911A was
established as 5.
Ile, NMePhe, N,N-disubstituted-Ile, Ahp,
u,u-NMe2Lys, 3-O-
substituted-Thr, and 3-sulfo-GA. Application of Marfey’s pro-
cedure,20 as described for 5, assigned structure 8 to micropeptin
LH925.
Micropeptin LH911B (6) presented 1D NMR spectra (Tables 1
and 2) almost identical with those of 5. It displayed a quasi-
molecular ion at m/z 910.3989 [MꢀH]ꢀ in the HRESIMS, which
correspond to the molecular formula, C39H61N9O14S, and 14 degrees
of unsaturation, identical to that of 5. Analysis of the COSY, TOCSY,
The crude cyanobacteria extract exhibited significant inhibition
of the serine proteases trypsin and chymotrypsin at a concentration
of 1 mg/mL. The activity-guided purification of the proteases
inhibiting components of the extract revealed that micropeptins
1e4 were responsible for the inhibition of chymotrypsin, while
micropeptins 3e8 were responsible for the inhibition of trypsin
(see Table 3). The selectivity and potency of 1 and 2 against chy-
motrypsin is comparable with that of micropeptin 10325 (IC50
HSQC, and HMBC 2D NMR experiments of
6 (Table S6 in
Supplementary data) established the structures of Ile, NMePhe,
N,N-disubstituted-Val, Ahp, Arg, 3-O-substituted-Thr, and 3-sulfo-
GA, as in 5. The carboxamide of the arginine residue was assigned
based on an NOE correlation between the Ahp amide proton and
Arg H-2, and an HMBC correlation of the Ahp amide proton with the
carboxamide that resonated at 170.2 ppm. HMBC correlations be-
tween the carboxamide of NMePhe and Ile NH, the carboxamide of
1.1 m mM). Micropeptin 103
M) and micropeptin MM97826 (IC50 4.6
contains the same sequence as 1 except of NMeTrp instead of
NMeTyr, in 1 and micropeptin MM978 contains the same sequence
as 1 except of Ile instead of Val and Thr instead of Gly. Micropeptins

M. Vegman, S. Carmeli / Tetrahedron 69 (2013) 10108e10115
10113
Table 3
inhibited trypsin and chymotrypsin in at 0.45 mg/mL were each
further separated on a Sephadex LH-20 column eluted with a 1:1
chloroform/methanol or 2:1:1 petroleum ether/chloroform/meth-
anol solutions to obtain fractions that contain different mixture of
the micropeptins and accompanying compounds. These fractions
were separated repeatedly on different preparative HPLC columns
Protease inhibition properties of 1e8a
Compound
Trypsin
Chymotrypsin
Micropeptin LH920 (1)
Micropeptin LH1021 (2)
Micropeptin LH1048 (3)
Micropeptin LH1062 (4)
Micropeptin LH911A (5)
Micropeptin LH911B (6)
Micropeptin LH911C (7)
Micropeptin LH925 (8)
d
4.7
1.1
5.3
3.0
d
d
2.0
3.0
1.9
3.1
11.7
>45.5
[YMC ODS-A, 10
250 mmꢁ20.0 mm; Phenomenex, Cosmosil C-8,
m
m, 250 mmꢁ20.0 mm; YMC C-8, 10
m
m
m,
m,
5
d
250 mmꢁ25.0 mm; DAD at maximum absorbance or 210 nm, flow
rate 5.0 mL/min] in a variety of MeOH/water or acetonitrile/water
containing 0.1% trifluoroacetic acid mixtures to the pure com-
pounds. The pure compounds isolated (weight and yield, % of crude
extract weight) were: micropeptin LH920 (1, 9.9 mg, 0.00059%);
micropeptin LH1021 (2, 10.8 mg, 0.00064%); micropeptin LH1048
(3, 16.0 mg, 0.00095%); micropeptin LH1062 (4, 2.0 mg, 0.00012%);
micropeptin LH911A (5, 3.0 mg, 0.00018%); micropeptin LH911B (6,
6.0 mg, 0.00036%); micropeptin LH911C (7, 8.7 mg, 0.00052%);
micropeptin LH925 (8, 4.9 mg, 0.00029%); micropeptin SF909
(1.2 mg, 0.00007%); micropeptin MZ939A (2.0 mg, 0.00012%);
cyanopeptolin S (98.6 mg, 0.00587%); cyanopeptolin SS (6.5 mg,
0.00037%); cyanopeptolin 1020 (8.6 mg, 0.00051%); microcystin LR
(2.8 mg, 0.00017%); microcystin RR (8.3 mg, 0.00049%); anabae-
d
d
a
IC50, mM.
LH1048 (3) and LH1062 (4) inhibited both trypsin and chymo-
trypsin in a similar potency. They share this unique property with
micropeptin HU90927 (IC50 chymotrypsin 2.8
mM, trypsin 1.1 mM)
and related micropeptins from the same bloom, all of which display
remarkably different amino acid sequences. Interestingly, cyano-
peptolin 1020 that was isolated along with 3 and 4 from the same
extract was reported to potently inhibit trypsin (IC50 0.67 nM), but
not chymotrypsin.14 Micropeptins LH911A (5) and B (6) were found
to be somewhat less potent against trypsin than the known cya-
nopeptolin S12 (IC50 0.2
mM) that was also isolated in the current
nopeptin
A (2.3 mg, 0.00013%), anabaenopeptin B (4.6 mg,
0.00027%); and anabaenopeptin F (6.7 mg, 0.00040%).
study. Cyanopeptolin S contains two Ile residues, instead of Val and
Ile in 5 and 6, and identical with them in the rest of the sequences.
The N-methylated lysine containing, 7 and 8, displayed lower po-
tency to trypsin than 5 and 6 suggesting that the methylation
resulted in higher steric hindrance and thus lowering their binding
affinity to the active pocket of the enzyme.
3.4. Micropeptin LH920 (1)
29
Transparent glassy material; [
a]
ꢀ26 (c 0.41, MeOH); UV
D
(MeOH) lmax (log ) 202 (4.38), 227 (3.80), 279 (3.07) nm; For NMR
3
data see Tables 1 and 2 and S1; HRESIMS m/z 943.4576 (MNaþ,
calcd for C46H64N8NaO12 m/z 943.4541). Retention time of AA
3. Experimental section
3.1. Instrumentation
Marfey derivatives:
31.9 min), -glutamic acid 31.7 min (
valine 39.5 min ( -Val 39.5, -Val 42.7 min),
44.5 min ( -Phe 44.5, -Phe 46.6 min), -NMe-tyrosine 48.7 min (
NMeTyr 48.7 min).
L
-threonine 29.6 min
(
D
L
-Thr 29.6,
-Glu 32.5 min),
-phenylalanine
D-Thr
L
L-Glu 31.7,
L-
L
D
L
L
D
L
L-
Mass spectra were recorded on a Waters MALDI Synapt in-
strument. UV spectra were recorded on an Agilent 8453 spectro-
photometer. 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, 100.62 MHz for 13C. DEPT, COSY-45, gTOCSY,
gROESY, gHSQC, gHMQC, gHMBC, spectra were recorded using
standard Bruker pulse sequences. HPLC separations were per-
formed on a JASCO HPLC system (model PU-2080-plus pump,
model LG-2080-04 Quaternary gradient unit and model MD-2010-
plus Multiwavelength detector) and on a Merck Hitachi HPLC sys-
tem (model L-7100 pump and model L-7450A Diode Array de-
tector). Protease inhibition assays were measured on an ELx808,
BIO-TEK Instrument ELISA Reader.
3.5. Micropeptin LH1021 (2)
29
Transparent glassy material; [
a]
ꢀ34 (c 0.35, MeOH); UV
D
(MeOH) lmax (log ) 202 (4.51), 226 (3.89), 279 (3.11) nm; For NMR
3
data see Tables 1 and 2 and S2; HRESIMS m/z 1044.5032 (MNaþ,
calcd for C50H71N9NaO14 m/z 1044.5018). Retention time of AA
Marfey derivatives:
31.9 min), -glutamic acid 31.4 min (
valine 39.6 min ( -Val 39.6, -Val 42.7 min),
44.0 min ( -Phe 44.0, -Phe 46.2 min), -NMe-tyrosine 48.5 min (
NMeTyr 48.5 min).
L
-threonine 29.4 min
(
D
L
-Thr 29.4,
-Glu 32.2 min),
-phenylalanine
D-Thr
L
L-Glu 31.4,
L-
L
D
L
L
D
L
L-
3.6. Micropeptin LH1048 (3)
30
3.2. Biological material
Glassy white solid; [
lmax (log
Tables 1 and 2 and S3; HRESIMS m/z 1049.5670 (MHþ, calcd for
52H77N10O13 m/z 1049.5672). Retention time of AA Marfey de-
rivatives: -threonine 29.6 min ( -Thr 29.6, -Thr 32.1 min), -argi-
nine 29.9 min ( -Arg 29.9, -Arg 29.1 min), -glutamic acid 31.7 min
-Glu 31.7, -Glu 32.4 min), -valine 39.8 min ( -Val 39.8, -Val
42.9 min), -phenylalanine 44.3 min ( -Phe 44.3, -Phe 46.4 min),
NMe-tyrosine 48.7 min ( -NMeTyr 48.7 min).
a
]
ꢀ28 (c 0.34, MeOH); UV (MeOH)
D
3
) 202 (4.40), 227 (3.80), 279 (3.04) nm; For NMR data see
Microcystis sp., TAU strain IL-376, was collected from a fishpond
near Kibbutz Lehavot HaBashan, Israel on November 4, 2007.
Samples of the cyanobacteria are deposited at the culture collection
of Tel Aviv University.
C
L
L
D
L
L
D
L
(L
D
L
L
D
3.3. Isolation procedure
L
L
D
L-
L
The freeze-dried cells (IL-376, 1680 g) were extracted with 7:3
MeOH/H2O (3ꢁ3 L). The active crude extract (92.0 g, full inhibition
of trypsin and chymotrypsin at 1 mg/mL) was evaporated to dry-
ness and separated, in 10 portions, on an ODS (YMC-GEL, 120A,
4.4ꢁ6.4 cm) flash column with increasing amounts of MeOH in
water. Six of the fractions (3:7 MeOH/H2O to 9:1 MeOH/H2O) that
3.7. Micropeptin LH1062 (4)
30
Glassy white solid; [
lmax (log
Tables 1 and 2 and S4; HRESIMS m/z 1063.5840 (MHþ, calcd for
a
]
ꢀ36 (c 0.19, MeOH); UV (MeOH)
D
3 ) 202 (4.36), 227 (3.80), 279 (3.20) nm; For NMR data see

10114
M. Vegman, S. Carmeli / Tetrahedron 69 (2013) 10108e10115
C53H79N10O13 m/z 1063.5828). Retention time of AA Marfey de-
rivatives: -threonine 29.4 min ( -Thr 29.4, -Thr 32.0 min), -ar-
ginine 29.9 min ( -Arg 29.9, -Arg 29.1 min), -glutamic acid
31.6 min ( -Glu 31.6, -Glu 32.4 min), -valine 39.9 min ( -Val 39.9,
-Val 42.9 min), -phenylalanine 44.5 min ( -Phe 44.5, -Phe
46.5 min), -NMe-tyrosine 48.6 min ( -NMeTyr 48.6 min).
resultant product was dissolved in 6 N aqueous HCl (1 mL) and
placed in a sealed glass bomb at 110 ꢂC for 16 h. After the removal of
the hydrochloric acid, by repeated evaporation in vacuo, the hy-
L
L
D
L
L
D
L
L
D
L
L
drolyzate was resuspended in water (250
(1-fluoro-2,4-dinitrophenyl)-5- -alanine amide (FDAA) in acetone
(1.2 mol per 1 mol AA) and 1 N aqueous NaHCO3 (20 L per AA)
were added to each reaction vessel and the reaction mixtures were
stirred at 70 ꢂC for 2 h. A 2 N HCl solution (10
L per AA) was added
to each reaction vessel and the solutions were evaporated in vacuo.
The N-[(-dinitrophenyl)-5- -alanine amide]-amino acid derivatives,
mL). A 0.03 M solution of
D
L
L
D
L
L
L
m
3.8. Micropeptin LH911A (5)
m
29
Glassy white solid; [
lmax (log
HRESIMS m/z 934.3964 (MNaþ, calcd for C39H61N9NaO14S m/z
934.3956). Retention time of AA Marfey derivatives: -threonine
29.6 min ( -Thr 29.6, -Thr 32.3 min), -arginine 30.1 min (
30.1, -Arg 29.3 min), -glutamic acid 32.0 min ( -Glu 32.0,
32.8 min), -valine 39.9 min ( -Val 39.9, -Val 42.9 min),
leucine 43.2 min ( -Ile 43.2, -Ile 46.2 min), -NMe-phenylalanine
43.9 min ( -NMePhe 43.9 min).
a]
ꢀ63 (c 0.20, MeOH); UV (MeOH)
L
D
3
) 202 (4.46) nm; For NMR data see Tables 1 and 2 and S5;
from hydrolyzates, were compared with similarly derivatized
standard amino-acids by HPLC analysis: LiChrospher 60 RP-select B,
L
5
m
m, 4.0ꢁ250 mm, flow rate: 1 mL/min, UV detection at 340 nm,
L
D
L
L-Arg
linear gradient elution from 100%, 0.1% TFA in H2O to 1:1, 0.1% TFA
in H2O/ACN, within 60 min. The determination of the absolute
configuration of each amino acid was confirmed by spiking the
derivatized hydrolyzates with the derivatized authentic amino
acids.
D
L
L
D
-Glu
L
L
D
L-iso-
L
D
L
L
3.9. Micropeptin LH911B (6)
3.13. Protease inhibition assays
29
Glassy white solid; [
a
]
ꢀ61 (c 0.21, MeOH); UV (MeOH)
D
The procedures used to determine the inhibitory activity of the
new compounds against trypsin and chymotrypsin were described
in a previous paper.26
lmax (log ) 202 (4.22) nm; For NMR data see Tables 1 and 2 and S6;
3
HRESIMS m/z 910.3989 (MꢀHꢀ, calcd for C39H60N9O14
S m/z
910.3980). Retention time of AA Marfey derivatives: L-threonine
29.7 min (
30.1, -Arg 29.3 min),
32.7 min), -valine 39.8 min (
leucine 43.1 min ( -Ile 43.1, -Ile 46.3 min),
43.9 min ( -NMePhe 43.9 min).
L
-Thr 29.7,
D
-Thr 32.3 min),
-glutamic acid 31.8 min (
-Val 39.8, -Val 42.9 min),
-NMe-phenylalanine
L
-arginine 30.1 min (
L
-Arg
-Glu
Acknowledgements
D
L
L-Glu 31.8,
D
L
L
D
L-iso-
N. Tal, the Mass Spectrometry Facility of the School of Chemistry,
Tel Aviv University, for the measurements of the HRESI mass
spectra. This research was supported by the Israel Science Foun-
dation grant 776/06.
L
D
L
L
3.10. Micropeptin LH911C (7)
29
Supplementary data
Glassy white solid; [
lmax (log
and
and S7; HRESIMS m/z 934.4233 (MNaþ, calcd for
41H65N7NaO14S m/z 934.4208). Retention time of AA Marfey de-
rivatives: -threonine 29.5 min ( -Thr 29.5, -Thr 32.1 min), -glu-
tamic acid 31.7 min ( -Glu 31.7, -Glu 32.5 min), -valine 39.9 min (
-Val 42.9 min), -isoleucine 43.1 min ( -Ile 43.1, -Ile
-NMe-phenylalanine 43.8 min ( -NMePhe 43.8 min),
-NMeLys, 44.8 min).
a
]
D
ꢀ79 (c 0.47, MeOH); UV (MeOH)
3
) 203 (4.33), 258 (2.36) nm; For NMR data see Tables 1
The 1H, 13C, HMQC or HSQC, HMBC COSY, and ROESY NMR
spectra, table of full NMR data, and HRESIMS data of compounds
1e8. Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.tet.2013.09.054. These data include MOL
files and InChIKeys of the most important compounds described in
this article.
2
C
L
L
D
L
L
D
L
L-
Val 39.9,
46.2 min),
D
L
L
D
L
L
u-
NMe-lysine 44.8 min (
L
References and notes
3.11. Micropeptin LH925 (8)
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Glassy white solid; [
lmax (log
HRESIMS m/z 949.4421 (MNaþ, calcd for C42H68N7NaO14S m/z
949.4443). Retention time of AA Marfey derivatives: -threonine
30.1 min ( -Thr 30.1, -Thr 32.7 min), -glutamic acid 31.8 min ( -Glu
31.8, -Glu 32.7 min), -isoleucine 43.9 min ( -Ile 43.9, -Ile
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a]
ꢀ78 (c 0.28, MeOH); UV (MeOH)
D
3 ) 203 (4.38) nm; For NMR data see Tables 1 and 2 and S7;
L
L
D
L
L
D
L
L
D
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8 mL of H2O) in 1 mL of acetone at 0 ꢂC for 10 min. The mixture was
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