Protease inhibitors from a water bloom of the cyanobacterium Microcystis aeruginosa

Article abstract of DOI:10.1021/np900340t

Bioassay-guided fractionation of the polar extract of a Microcystis aeruginosa water bloom biomass yielded 10 micropeptins and one anabaenopeptin. Eight of the micropeptins, micropeptins HU1069 (1), HU989 (2), HU1021 (3), HU1041 (4), HU975 (5), HU895A (6)

Full text of DOI:10.1021/np900340t

J. Nat. Prod. 2009, 72, 1429–1436
1429
Protease Inhibitors from a Water Bloom of the Cyanobacterium Microcystis aeruginosa
Shiri Gesner-Apter 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 June 4, 2009
Bioassay-guided fractionation of the polar extract of a Microcystis aeruginosa water bloom biomass yielded 10
micropeptins and one anabaenopeptin. Eight of the micropeptins, micropeptins HU1069 (1), HU989 (2), HU1021 (3),
HU1041 (4), HU975 (5), HU895A (6), HU909 (7), and HU895B (8), are new, while two, micropeptins 478-A (10) and
478-B (11), were previously isolated from a bloom of M. aeruginosa from Japan. The new anabaenopeptin HU892 (9)
belongs to the relatively rare subgroup, presenting an aliphatic amino acid at the carboxylic end of the peptide and
N-methylhomoaromatic amino acid at the second position. The structures of the compounds were determined by 1D
and 2D NMR techniques and mass spectrometric data. The isolated micropeptins inhibited trypsin with IC50’s that
varied between 0.7 and 5.2 µM and unexpectedly inhibited chymotrypsin with IC50’s that varied between 2.8 and 72.0
µM. The SAR of these micropeptins is discussed.
The micropeptins are a group of cyclic depsipeptides that are
produced by fresh water and marine cyanobacteria.1 They usually
accompany, together with the other four groups of protease
inhibitors, the hepatotoxic microcystins.2 The micropeptins compose
the most diverse group of the five known groups of protease
inhibitors that are produced by water bloom forming cyanobacteria.
Over eighty different micropeptin variants are known from Ana-
baena, Planktothrix (Oscillatoria), Nostoc, Microcystis, Symploca,
and Lyngbya spp. of cyanobacteria.3 The micropeptins may be
divided into three subgroups: (i) those that inhibit trypsin-type serine
proteases (i.e., trypsin, plasmin, thrombine); (ii) those that inhibit
chymotrypsin-type serine proteases (i.e., chymotrypsin, elastase);
and (iii) those that exhibit cytotoxic activity along with inhibition
of chymotrypsin-type serine proteases. This selectivity is gained
by the nature of the amino acid that occupies the fifth position from
the carboxylic end of the peptide chain. Basic amino acids (i.e.,
arginine and lysine) at this position select for inhibition of trypsin-
type serine protease, while aliphatic, aromatic, and other neutral
amino acids select for inhibition of chymotrypsin-type serine
proteases.4 Dehydroaminobutyric acid-containing micropeptins
exhibit cytotoxic activity and inhibit chymotrypsin-type serine
proteases.5 As part of our continuing interest in the chemical
ecology of cyanobacterial water blooms and the search for novel
drugs for human diseases, we examined the extracts of a Microcystis
aeruginosa bloom collected in October 2004 from Hulda Reservoir,
Israel. The extract of this bloom (IL-342) afforded 11 protease
inhibitors belonging to two groups: 10 micropeptins of which eight
are new natural products and two are known from a bloom of
Microcystis aeruginosa from Japan,6 together with a novel ana-
baenopeptin. The structure elucidation and biological activity of
the new compounds are presented below.
Results and Discussion
The 10 micropeptins isolated in this research work vary in one
d6, revealed four doublet NH signals and one triplet proton signal
between δH 7.0 and 8.5 ppm, pointing to six amino acid residues
(taking into account the NMe aromatic amino acid and the Ahp
residue that accounts for two amino acids). Analysis of the 1D (1H,
13C, and DEPT) (see Tables 1 and 2) and 2D (COSY, TOCSY,
ROESY, HMQC, and HMBC) NMR data (see Table 5, in the
Supporting Information) revealed the six amino acid units and a
hydroxy acid unit, namely, isoleucineI, N,O-diMe-o-chlorotyrosine,
N,N-disubstituted isoleucineII, amino-hydroxy-piperidone (Ahp),
arginine, threonine, and glyceric acid, that build micropeptin
HU1069 (1). The structure of the Ahp is suggested on the basis of
the COSY and HMQC spectra, the HMBC correlation between Ahp
amino acid, valine versus isoleucine, in the carboxylic end of the
peptide, and in the substitution of three other acid units: NMe-
tyrosine versus NMe-o-chlorotyrosine or N,O-diMe-o-chloroty-
rosine, amino hydroxy piperidone versus amino methoxy piperi-
done, and glyceric acid versus its mono- and disulfated derivatives.
Micropeptin HU1069 (1) was isolated as a glassy material with
a molecular formula of C41H64ClN9O18S2 based on the HR MALDI
TOF quasi-molecular ion at m/z 910.4486/912.4485 (3:1) corre-
sponding to [M + H - 2SO3]+. The 1H NMR spectrum, in DMSO-
* To whom correspondence should be addressed. Tel: ++972-3-6408550.
Fax: ++972-3-6409293. E-mail: carmeli@post.tau.ac.il.
10.1021/np900340t CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/03/2009

1430 Journal of Natural Products, 2009, Vol. 72, No. 8
Table 1. 1H NMR Data of Compounds 1-8 in DMSO-d6
Gesner-Apter and Carmeli
a
compound position
1b
2c
3c
4b
5b
6b
7c
8c
Ile/Val 2
3
4
4.87, dd
1.83, m
1.05, m
1.26, m
0.90, t
4.78, dd
1.81, m
1.11, m
1.24, m
0.87, t
4.86, dd
1.84, m
1.08, m
1.25, m
0.89, t
4.70, m
2.05, m
0.73, d
4.58, dd
2.02, m
0.75, d
4.51, dd
1.99, m
0.76, d
4.49, dd
1.95, m
0.84, d
4.65, brd
1.79, m
1.09, m
1.20, m
0.87, t
5
0.85, d
0.85, d
0.85, d
0.88, d
6
NH
Tyr der. 2
3
0.69, d
7.78, d
5.05, brd
2.76, dd
3.24, m
7.24, s
0.70, d
7.56, d
5.06, dd
2.78, dd
3.23, dd
7.24, brs
0.69, d
7.43, d
5.05, dd
2.68, dd
3.25, dd
6.99, d
6.62, d
6.62, d
6.99, d
2.70, s
0.71, d
7.60, d
5.04, dd
2.65, dd
3.19, dd
7.13, s
7.54, d
5.02, dd
2.75, m
3.20, m
7.12, d
7.64, d
5.05, dd
2.78, dd
3.25, dd
7.24, s
7.75, d
5.03, dd
2.78, dd
3.21, dd
7.24, s
6.99, d
5.07, dd
2.79, dd
3.25, dd
7.25, s
5
6
8
9
7.04, d
7.15, d
2.72, s
7.04, d
7.14, d
2.71, s
6.83, d
6.96, d
2.72, s
7.05, d
7.14, d
2.73, s
7.05, d
7.14, d
2.72, s
7.05, d
7.14, d
2.73, s
6.84, d
6.96, d
2.70, s
NMe
OMe/OH
Ile 2
3
4
3.78, s
3.77, s
4.37, d
9.14, s
9.94, s
3.77, s
3.76, s
4.35, d
3.77, s
9.94, brs
4.40, d
4.38, d
1.79, m
0.63, m
1.09, m
0.63, brd
-0.16, d
4.43, q
1.73, m
2.55, brq
1.73, m
4.45, m
1.74, m
0.63, m
1.05, m
0.63, brd
-0.12, d
4.43, m
1.73, m
2.60, brq
1.73, m
4.39, d
1.77, m
0.64, m
1.10, m
0.64, brd
-0.10, d
4.45, m
1.74, m
2.60, brq
1.73, m
1.77, m
4.92, brs
7.27, d
6.06, d
4.30, m
1.44, m
2.05, m
1.44, m
3.02, m
7.49, brt
8.53, d
4.63, dd
5.45, dq
1.21, d
7.74, d
4.72, t
4.37, d
1.80, m
0.61, m
1.10, m
0.61, brd
-0.15, d
4.44, q
1.73, m
2.61, brq
1.75, m
1.79, m
4.92, brs
7.28, d
6.16, d
4.31, m
1.43, m
2.03, m
1.43, m
3.07, m
7.51, brt
8.63, d
4.66, d
5.50, q
1.20, d
7.64, d
4.25, ddd
3.82, dd
3.97, dd
6.09, d
4.41, m
1.86, m
0.61, m
1.10, m
0.61, brd
-0.16, d
4.45, m
1.73, m
2.43, brq
1.70, m
2.10, m
4.43, d
1.81, m
0.62, m
1.12, m
0.62, brd
-0.15, d
4.45, ddd
1.73, m
2.58, brq
1.76, m
1.83, m
4.91, brs
7.35, d
1.78, m
0.64, m
1.10, m
0.62, brd
-0.15, d
4.43, m
1.75, m
2.60, brq
1.20, m
1.78, m
4.91, brs
7.33, d
6.23, brs
4.28, m
1.48, m
2.00, m
1.46, m
3.07, m
7.61, brt
8.68, d
1.80, m
0.64, m
1.10, m
0.64, brd
-0.08, d
4.45, m
1.75, m
2.55, m
1.77, m
5
6
Ahp/Amp 3
4
5
6
NH
OH/OMe
Arg 2
3
4.92, brs
7.37, d
6.04, d
4.32, m
1.44, m
2.04, m
1.44, m
3.04, m
7.46, brt
8.55, d
4.63, d
5.43, q
1.22, d
7.78, d
4.74, dd
3.91, dd
3.98, dd
4.92, brs
7.35, d
5.96, d
4.31, m
1.44, m
2.05, m
1.44, m
3.04, m
7.43, brt
8.53, d
4.63, d
5.42, q
1.21, d
7.75, d
4.73, t
4.92, brs
7.39, d
6.11, d
4.30, m
1.48, m
2.02, m
1.46, m
3.08, m
7.60, brt
8.62, d
4.65, d
4.59, q
1.20, d
7.46, d
4.07, ddd
3.49, ddd
3.60, ddd
5.74, d
7.21, d
3.03, s
6.09, d
4.35, m
1.42, m
2.01, m
1.42, m
3.06, m
7.46, brt
8.59, d
4.64, d
5.46, q
1.20, d
7.46, d
4.28, m
1.47, m
2.00, m
1.50, m
3.08, m
7.63, brt
8.69, d
4.70, d
5.55, brq
1.20, d
4
5
6-NH
NH
Thr 2
3
4.68, d
5.53, brq
1.21, d
4
NH
GA 2
3
7.62, d
7.63, d
4.25, ddd
3.85, dd
3.97, dd
6.07, d
4.06, ddd
3.46, ddd
3.58, ddd
5.81, d
4.08, ddd
3.49, ddd
3.60, ddd
5.79, d
3.91, dd
3.98, dd
3.92, dd
3.98, dd
2-OH
3-OH
4.78, t
4.75, t
4.77, t
a Complete NMR data are available in the Supporting Information (coupling constant, HMBC and ROESY correlations). b 500 MHz for 1H. c 400
1
MHz for H.
H-6 and C-2, and comparison of the chemical shifts of the carbon
and proton signals with similar moieties in the known micropeptin
478-B (11).6 The glyceric acid residue was established as 2,3-
disulfated glyceric acid on the basis of it proton and carbon chemical
shifts, which are almost identical with that of micropeptin 478-B
(11).6 All proton and carbon signals of the latter residues were fully
assigned by the COSY, TOCSY, HMQC, and HMBC data (see
Table 5, in the Supporting Information). The amino acid sequence
of 1 was determined from HMBC correlations of the NH proton
of an amino acid with the carbonyl of an adjacent amino acid (Ile-
N,O-diMe-o-chloroTyr and Ahp-Arg), the NMe of N,O-diMe-o-
chloroTyr with IleII-carbonyl, and H-2 of IleII with C-6 of the Ahp
residue and through NOE correlations between R-NH of Arg and
H-2 and H-3 of Thr and Thr H-2 and H-2 of GA. The ester bond
was assigned by a NOE correlation between H3-4 of threonine and
H-2 of isoleucine. Acid hydrolysis of 1 and derivatization with
Marfey’s reagent,7 followed by HPLC analysis, demonstrated the
L-configuration of the isoleucine residues, N,O-diMe-o-chloroty-
rosine (by comparison with synthetic material), arginine, and
threonine residues. Jones oxidation8 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 J-values
of H-6, <1 Hz, which points to an equatorial orientation of this
proton, the chemical shift of the pseudoaxial H-4, δH 2.54 brq,
which is downfield shifted by the axial hydroxy group, and NOE
correlation between H-4pax and the 6-OH. The configuration of
the glyceric acid was determined to be D (R), by comparison of the
retention time of the glyceric acid from the hydrolysate with the
retention time of authentic samples of D- and L-glyceric acid on a
chiral HPLC column.
Micropeptin HU989 (2) was isolated as a glassy material. It
1
presented H and 13C NMR data similar to those of micropeptin
HU1069 (1). The only notable difference was located in the glyceric
acid residue; H-2 in 2 is upfield shifted by 0.5 ppm relative to that
of 1 and coupled with an acidic proton resonating at 6.07 ppm,
while C-2 in 2 is upfield shifted by 3.8 ppm relative to that of 1.
These differences are in accordance with a 3-sulfated glyceric acid.
The molecular formula of 2, C41H64ClN9O15S, was deduced from
the high-resolution MALDI TOF quasi-molecular ion, [M + Na]+,
at m/z 1012.3863/1014.3863 (3:1). As for 1, analysis of the 1D
(1H, 13C, and DEPT) (see Tables 1 and 2) and 2D (COSY, TOCSY,
ROESY, HMQC, and HMBC) NMR data of 2 (see Table 6, in the
Supporting Information) allowed the full assignment of the fol-
lowing acid residues: isoleucineI, N,O-diMe-o-chlorotyrosine, N,N-

Protease Inhibitors from Microcystis aeruginosa
Journal of Natural Products, 2009, Vol. 72, No. 8 1431
Table 2. 13C NMR Data of Compounds 1-8, 10, and 11 in DMSO-d6
compound position
mult.c
1a
2b
3b
4a
5a
6a
7b
8b
10b
11b
Ile/Val 1
2
3
4
5
6
qC
CH
CH
CH2/CH3
CH3
CH3
qC
172.9
54.3
37.4
26.2
11.8
14.8
169.5
60.7
173.0
54.5
37.4
26.4
11.8
15.0
169.5
60.9
172.9
54.3
37.4
26.2
11.7
14.8
169.4
60.9
172.6
56.2
31.1
17.7
19.4
173.0
54.5
37.4
26.4
11.8
15.0
169.5
60.9
172.6
56.5
30.7
18.0
19.3
172.4
56.5
31.1
18.1
19.1
172.8
54.4
37.1
26.1
11.5
14.9
169.2
60.7
172.5
55.3
37.7
24.5
11.1
16.0
168.9
60.4
172.5
55.2
37.7
24.5
11.1
16.0
168.9
60.4
Tyr-der. 1
2
169.6
60.6
169.2
60.5
169.0
60.6
CH
3
4
5
6
7
8
9
CH2
qC
CH
qC/CH
qC
CH
33.2
33.2
33.6
33.2
33.2
32.9
32.8
33.2
34.8
34.8
131.5
130.9
121.5
153.8
113.3
129.7
29.9
56.4
169.9
54.3
33.1
23.9
10.4
13.9
169.1
48.9
22.0
30.5
74.2
131.2
131.0
121.7
154.0
113.5
129.8
30.7
56.6
170.1
54.5
33.3
24.0
10.6
14.1
169.6
49.3
22.0
30.1
74.4
127.6
130.6
115.5
156.4
115.5
130.6
30.4
129.3
130.8
120.0
152.2
116.8
129.4
30.4
131.2
131.0
121.7
154.0
113.5
129.8
30.7
56.6
170.1
54.5
33.3
24.0
10.6
14.1
169.6
49.3
22.0
30.1
74.4
130.9
130.6
121.4
153.7
113.2
129.4
30.4
56.2
169.7
54.2
32.9
23.7
10.2
13.9
169.4
49.1
21.6
29.8
74.1
130.7
130.5
121.4
153.6
113.2
129.4
30.3
56.2
169.9
53.9
32.5
23.6
10.1
13.5
169.0
49.2
21.5
23.6
83.1
131.8
130.6
120.0
152.3
116.8
129.2
30.4
129.1
130.6
119.8
152.0
116.7
129.0
30.1
129.1
130.6
119.8
152.0
116.7
129.0
30.1
CH
NMe
OMe
Ile 1
2
3
4
5
6
Ahp/Amp2
3
4
5
CH3
CH3
qC
CH
CH
CH3
CH2
CH3
qC
170.0
54.3
33.9
23.9
10.5
14.0
169.5
48.9
21.9
29.8
74.1
169.9
54.4
33.2
24.0
10.5
13.9
169.2
48.9
21.8
29.9
74.2
169.9
54.4
33.3
23.9
10.4
13.9
169.5
49.2
21.8
29.9
74.2
169.7
54.2
32.9
23.7
10.2
13.8
169.2
48.8
21.8
29.7
73.9
169.7
54.2
32.9
23.7
10.2
13.8
169.2
48.8
21.8
29.7
73.9
CH
CH2
CH2
CH
6
OMe
Arg 1
2
3
4
5
7
Thr 1
2
3
4
GA 1
2
3
CH3
qC
CH
CH2
CH2
CH2
qC
qC
CH
CH
CH3
qC
55.3
170.2
52.2
27.6
25.1
170.2
51.3
24.8
24.8
43.0
156.7
169.7
55.1
72.1
17.5
169.6
74.6
66.5
170.3
52.0
26.4
25.4
41.0
157.0
169.4
54.6
72.7
18.1
172.2
70.8
68.6
169.6
51.2
24.8
24.8
45.9
156.7
169.7
55.0
72.1
17.4
172.9
74.6
66.5
170.3
51.8
31.0
24.8
48.8
156.7
169.4
55.1
71.9
17.7
169.8
74.7
66.5
170.3
52.0
26.4
25.4
41.0
157.0
169.4
54.6
72.2
18.1
172.2
70.8
68.6
170.1
52.3
27.5
25.2
45.6
156.8
169.2
54.2
72.3
18.1
172.7
72.7
63.8
170.1
52.1
26.5
25.3
40.5
156.9
169.4
54.4
72.6
18.0
169.2
72.7
63.9
170.3
51.2
26.7
24.6
40.3
156.5
168.8
54.4
72.1
17.6
171.7
70.4
68.2
170.3
51.2
26.7
24.7
40.3
156.5
168.8
54.3
71.7
17.7
171.1
74.5
66.3
45.5
156.7
169.0
54.2
72.3
17.8
172.6
72.6
63.7
CH
CH2
a 125 MHz for 13C. b 100 MHz for 13C. c Multiplicity and assignment from a HSQC experiment.
disubstituted isoleucineII, Ahp, arginine, threonine, and 3-sulfoglyc-
eric acid. The amino acid sequence of 2 was determined from
HMBC correlations of the NH proton of an amino acid with the
carbonyl of an adjacent amino acid, IleI- N,O-diMe-o-chloroTyr,
Ahp-Arg, and Thr-GA and of the NMe of N,O-diMe-o-chloroTyr
with IleII-carbonyl and by NOE correlations between 6-OH of Ahp
and H-2 of isoleucineII, R-NH of Arg and H-2 of Thr, and Thr H-2
and H-2 of GA. The ester linkage between the carbonyl of
isoleucineI and the oxygen of threonine was assigned by an HMBC
correlation between H-3 of threonine and the carbonyl of isoleucineI.
The configurations of the stereogenic centers of 2 were assigned
as L-isoleucine residues, L-N,O-diMe-o-chlorotyrosine, L-arginine,
L-threonine, 3S,6R-Ahp, and D-glyceric acid by the same procedure
described for 1.
Micropeptin HU1021 (3) was isolated as an amorphous, white
solid. HR MALDI-TOF MS measurements for the compound (m/z
964.4083 [M + Na - SO3]+), coupled with the NMR data,
furnished a molecular formula of C40H63N9O18S2 for 3. The 1H and
13C NMR data of 3 differed from that of 1 only in the signals of
the aromatic residue. These data suggested that 3 contains a
p-substituted phenol moiety instead of the o-chloro-p-methoxy
phenyl moiety in 1. Analysis of the 1D (1H, 13C, and DEPT) (see
Tables 1 and 2) and 2D (COSY, TOCSY, ROESY, HMQC, and
HMBC) NMR data of 3 (see Table 7, in the Supporting Information)
allowed the full assignment of the following acid residues:
isoleucineI, NMe-tyrosine, N,N-disubstituted isoleucineII, Ahp,
arginine, threonine, and disulfated glyceric acid. HMBC correlations
of the NH proton of an amino acid with the carbonyl of an adjacent
amino acid, IleI-NMe-Tyr with Thr-GA, the NMe of NMe-Tyr with
IleII-carbonyl, C-6 of Ahp with H-2 of isoleucineII, and the carbonyl
of isoleucineI with H-3 of threonine, as well as NOE correlations
between R-NH of Ahp and H-2 of Arg and between R-NH of Arg
and H-2 of Thr, established the connectivity between all of the
acid residues that build 3. The nature of all of the chiral centers of
3 was established as described above for 1, namely, two L-isoleucine
residues, L-NMe-tyrosine, L-arginine, L-threonine, 3S,6R-Ahp, and
D-glyceric acid.
Micropeptin HU1041 (4) presented 1H NMR data similar to those
of micropeptin 478-B (11).6 The only notable difference was present
in the aliphatic region, where the triplet methyl resonating at δH
0.89 ppm and the methylene resonating at δH 1.01 and 1.22 ppm,
in 11, are substituted with a doublet methyl at δH 0.84 ppm, in 4.
The molecular formula of 4, C39H60ClN9O18S2, was determined from
its high-resolution MALDI TOF quasi-molecular ion, [M + Na -
SO3]+, at m/z 984.3421/986.3420 (3:1). Thorough analysis of the
1D (1H, 13C, and DEPT) (see Tables 1 and 2) and 2D (COSY,
TOCSY, ROESY, HMQC, and HMBC) NMR data of 4 (see Table
8, in the Supporting Information) allowed the full assignment of
the following acid residues: valine, NMe-o-chlorotyrosine, N,N-
disubstituted isoleucine, Ahp, arginine, threonine, and disulfated
glyceric acid. The sequence and the chirality of the stereocenters
of the cyclic depsipeptide were established in a similar way to that
described above, for 1-3, to establish structure 4 for micropeptin
HU1041.

1432 Journal of Natural Products, 2009, Vol. 72, No. 8
Gesner-Apter and Carmeli
a
Table 3. NMR Data of Anabaenopeptin HU892 (9) in DMSO-d6
position
δC, mult.b
δH, mult., J (Hz)
LR C-H correlationsc
ROESY correlationsd
Ile 1
170.7, qC
58.1, CH
36.0, CH
24.2, CH2
Ile-2, Lys-ε-NH
Ile-3,4a,4b,5,6,NH
Ile-2,4a,5,6
2
3
4
4.18, dd (9.7, 6.0)
1.99, m
0.95, m
Lys-ε-NH,Ile-3,4a,5,6,NH
Ile-2,4a,4b,5,6,NH
Ile-2,3,4b,5,6,NH
Ile-3,4a,5,6
Ile-2,5,6
1.30, m
5
6
11.5, CH3
16.1, CH3
0.76, t (7.8)
0.77, d (7.1)
8.18, d (9.7)
Ile-3,4a,4b
Ile-2,3,4a,4b
Ile-2,3,4a,4b,NH
Ile-2,3,4a,4b,NH
Lys-NH-ε,Ile-2,3,4a,5,6
NH
NMeHty 1
2
3
169.4, qC
59.5, CH
30.8, CH2
Ile-2NH NMeHty-2
NMeHty-NMe,3a,3b, 4a,4b
NMeHty-2,4a,4b
4.60, dd (6.2, 8.3)
1.70, m
1.99, m
2.19, dt (4.9, 12.4)
2.30, dt (5.1, 12.4)
Ile-NH, NMeHty-3a,3b,4b
NMeHty-2,3b,4a,4b,NMe
NMeHty-2,3a,4a,4b,NMe
NMeHty-3a,3b,4b
4
31.4, CH2
NMeHty-3a,3b,6,6′
NMeHty-3a,3b,4a
5
131.6, qC
129.0, CH
115.4, CH
155.7, qC
28.7, CH3
NMeHty-4a,4b,7,7′
NMeHty-4a,4b,6,6′
NMeHty-6,6′,OH
NMeHty-6,6′,7,7′,OH
NMeHty-2,4a
6,6′
7,7′
8
NMe
OH
Hph 1
2
6.95, d (8.3)
6.66, d (8.3)
NMeHty-7,7′
NMeHty-6,6′
2.57, s
9.16, s
NMeHty-3a,3b
172.3, qC
48.2, CH
33.2, CH2
NMeTyr-NMe,Hph-2,3b,NH
Hph-4a,4b,3b,NH
Hph-2,4a,4b
4.70, m
1.75, m
2.03, m
2.70, m
2.81, m
Hph-3a,3b,4a,6,6′,NH
Hph-2,3b,4b
Hph-2,3a,4b
Hph-4b
3
4
31.6, CH2
Hph-3a,3b,4a
5
141.3, qC
128.6, CH
128.5, CH
126.3, CH
Hph-3b,4a,4b,6,6′,7,7′,8
Hph-4a,4b,7,7′
Hph-6,6′,8
6,6′
7,7′
8
NH
Val 1
2
3
4
5
7.25, m
7.23, m
7.19, m
8.98, d (4.3)
Hph-8
Hph-6,6′,7,7′
Val-2,Hph-2,3a,3b,4a,4b
Hph-7,7′
172.9, qC
58.0, CH
30.1, CH
19.2, CH3
18.7, CH3
Hph-NH,Val-2,3
Val-3,4,5,NH
Val-2,4,5,NH
Val-2,3,5
3.97, t (6.8)
1.90, m
0.88, d (6.9)
0.87, d (6.9)
6.81, d (6.1)
Val-3,4,5,NH,Hph-NH
Val-2,4,5,NH
Val-2,3,5,NH
Val-2,3,4,NH
Val-2,3,4,5,Lys-2
Val-2,3,4
NH
Lys 1
2
Hph-NH, Val-2,3
Val-NH,Lys-2
Lys-3a,3b,NH
Lys-2,NH
170.7, qC
58.1, CH
36.0, CH2
3.91, dt (10.7, 6.4)
1.61, m
Lys-3a,3b,4a,4b,R-NH, Val-NH
Lys-2,4a,4b,R-NH
3
1.66, m
1.30, m
1.44, m
1.40, m
1.43, m
2.81, m
3.44, m
Lys-2,NH
Lys-2,6b
Lys-2,4a,4b,R-NH
4
5
6
24.2, CH2
28.3, CH2
38.3, CH2
Lys-2,3a,3b,5a,5b,6b,R-NH,ꢀ-NH
Lys-2,3a,3b,5a,5b,6b,R-NH,ꢀ-NH
Lys-4a,4b,6a,6b,R-NH,ꢀ-NH
Lys-4a,4b,6a,6b,R-NH,ꢀ-NH
Lys-5a,5b,6a,R-NH,ꢀ-NH
Lys-4a,4b
Lys-5a,5b
R-NH
ꢀ-NH
CO
Arg 1
2
6.53, d (6.4
7.09, dd (6.4, 4.1)
Lys-5a,5b,4a,4b,6b,R-NH
Lys-2,3a,3b,4a,4b,5a,5b,6b
157.5, qC
174.3, qC
52.1, CH
29.5, CH2
Arg-2,R-NH,Lys-2,R-NH
Arg-2,3a,3b,R-NH
Arg-3a,3b,4,R-NH
Arg-2,4,5,R-NH
4.09, dt (12.6, 7.7)
1.53, m
1.72, m
1.46, m
3.11, t (6.0)
Arg-3a,3b,4,R-NH
Arg-2,3b,4,R-NH
Arg-2,3a,4,R-NH
Arg-2,3a,3b,5
Arg-4,δ-NH
-
3
4
5
6
25.6, CH2
40.5, CH2
156.9, qC
Arg-2,3a,3b,5
Arg-3a,3b,4,δ-NH
Arg-5
R-NH
δ-NH
NH, NH2
6.42, d (7.7)
7.57, t (5.3)
7.30, brm
Arg-2,3a,3b
Arg-5
a 500 MHz for 1H, 125 MHz for 13C. b Multiplicity and assignment from a HSQC experiment. c Determined from HMBC experiment, JCH ) 8 Hz,
n
recycle time 1 s, presented as a correlation of a proton with carbon in the row. d Selected NOEs from a ROESY experiment.
Micropeptin HU975 (5) was isolated as a glassy material with a
molecular formula of C40H62ClN9O15S based on the HR MALDI
TOF quasi-molecular ion at m/z 998.3615/1000.3615 (3:1) corre-
sponding to [M + Na]+. These HRMS data suggested an additional
methyl unit and one less sulfate unit when 5 is compared with 4.
Indeed, when the NMR data of 5 and 4 were compared, (i) a
difference was noticed in the glyceric acid residue; H-2 in 5 is
upfield shifted by 0.47 ppm relative to that of 4 and coupled with
an acidic proton resonating at 6.09 ppm, while C-2 in 5 is upfield
shifted by 4.1 ppm relative to that of 4, in accordance with a
3-sulfated glyceric acid (in 5), and (ii) in the aromatic residue an
additional aromatic methoxy group (δH 3.77 s and δC 56.4 q) is
found in the spectra of 5. The analysis of the 1D (1H, 13C, and
DEPT) (see Tables 1 and 2) and 2D (COSY, TOCSY, ROESY,
HMQC, and HMBC) NMR data of 5 (see Table 9, in the Supporting
Information) allowed the full assignment of the following acid
residues: valine, N,O-diMe-o-chlorotyrosine, N,N-disubstituted iso-
leucine, Ahp, arginine, threonine, and 3-sulfoglyceric acid. The
amino acid sequence of 5 was determined from HMBC correlations
of the NH proton of an amino acid with the carbonyl of an adjacent

Protease Inhibitors from Microcystis aeruginosa
Journal of Natural Products, 2009, Vol. 72, No. 8 1433
Table 4. Protease Inhibition Data
compound
key structure components
trypsin IC50 µM
chymotrypsin IC50 µM
2
4
7
micropeptin HU1069 (1)
micropeptin HU989 (2)
micropeptin HU1021 (3)
micropeptin HU1041 (4)
micropeptin HU975 (5)
micropeptin HU895A (6)
micropeptin HU909 (7)
micropeptin HU895B (8)
micropeptin 478-A (10)
micropeptin 478-B (11)
1Ile, NMe, OMe-o-Cl-Tyr, Ahp, disulfo-GA
1.7
0.7
2.2
1.2
5.2
3.5
1.1
0.9
0.7
2.4
>100
18.2
>100
>100
24.0
19.6
2.8
5.4
5.2
72.0
2
4
7
1Ile, NMe, OMe-o-Cl-Tyr, Ahp, 3-sulfo-GA
1Ile, 2NMe-Tyr, 4Ahp, 7disulfo-GA
1Val, 2NMe-o-Cl-Tyr, 4Ahp, 7disulfo-GA
1Val, 2NMe, OMe-o-Cl-Tyr, 4Ahp, 73-sulfo-GA
1Val, NMe, OMe-o-Cl-Tyr, Ahp, GA
2
4
7
1Val, NMe, OMe-o-Cl-Tyr, Amp, GA
2
4
7
2
4
7
1Ile, NMe-o-Cl-Tyr, Ahp, GA
1Ile, 2NMe-o-Cl-Tyr, 4Ahp, 73-sulfo-GA
2
4
7
1Ile, NMe-o-Cl-Tyr, Ahp, disulfo-GA
amino acid, Val-N,O-diMe-o-chloroTyr, Arg-Thr, and Thr-GA, the
NMe of N,O-diMe-o-chloroTyr with Ile-carbonyl, and H-2 of Ile
with C-6 of Ahp and by NOE correlation between R-NH of Ahp
and R-NH of Arg. The ester linkage between the carbonyl of valine
and the oxygen of threonine was assigned by an HMBC correlation
between H-3 of threonine and the carbonyl of valine. The
configurations of the stereogenic centers of 5 were assigned as
L-valine, L-isoleucine, L-N,O-diMe-o-chlorotyrosine, L-arginine,
L-threonine, 3S,6R-Ahp, and D-glyceric acid by the same procedure
described for 1-4. On the basis of the above given arguments,
structure 5 was assigned to micropeptin HU975.
the Amp residue seemed to be similar to that of the Ahp residue,
in the rest of the micropeptins, since 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 the substitution of a hydroxy
group with a methoxy group. C-5 is upfield shifted by 6 ppm
and C-6 is downfield shifted by 9 ppm in 7 relative to 6. The
pseudoeqatorial H-5 in 7 is downfield shifted by ca. 0.4 ppm,
relative to 6, while H-6 is upfield shifted by ca. 0.5 ppm. An
attempt to determine the relative configuration of the Amp from
J values and NOE correlations from the NMR data of 7 in
DMSO-d6 failed due to overlapping of H-3peq and H-4pax and
the absence of NOE correlation between H-3pax and 6-OMe.
This obstacle was overcome by dissolving 7 in pyridine-d5 and
running 1H, COSY, and ROESY experiments for it, which
established the J values and through-space conectivities in the
Amp residue. The protons of this spin system were assigned as
follows: H-3pax (4.94 ddd, 13.6,8.8,4.0), 3-NH (7.80 d 8.8),
H-4pax (2.70 brq 13.6), H-4peq (1.86 m), H-5peq (1.84 m),
H-5pax (1.49 brt 13.6), H-6peq (4.45 brs), and 6-OMe (3.01 s).
The trans diaxial relationships, between H-3pax, H-4pax, and
H-5pax, were established by the COSY correlations and J values
(13.6 Hz). H-5pax presented NOEs with H-3pax, H-5peq, and
H-6peq, while H-4pax presented NOEs with 3-NH, H-4peq, and
H-5peq, thus establishing the relative configuration of the Amp,
which is similar to that of the Ahp, 3S*,6R*. The amino acid
sequence of 7 was determined from HMBC correlations of the
NH proton of an amino acid with the carbonyl of an adjacent
amino acid, Val-N,O-diMe-o-chloroTyr, Arg-Thr, and Thr-GA,
the NMe of N,O-diMe-o-chloroTyr with Ile-carbonyl, and H-2
of Ile with C-6 of Amp and by a NOE correlation between R-
NH of Amp and H-2 of Arg. The ester linkage between the
carbonyl of valine and the oxygen of threonine was assigned by
an HMBC correlation between H-3 of threonine and the carbonyl
of valine. The configurations of the chiral centers of 7 were
assigned as L-valine, L-isoleucine, L-N,O-diMe-o-chlorotyrosine,
L-arginine, L-threonine, 3S,6R-Amp, and D-glyceric acid by the
same procedure described for 1-6. On the basis of the above
given arguments, structure 7 was assigned to micropeptin
HU909.
Micropeptin HU895A (6), a glassy transparent material,
exhibited a HR MALDI TOF quasi-molecular ion at m/z
934.3868/936.3869 (3:1) corresponding to [M + K]+, for which
the molecular formula C40H62ClKN9O12 was calculated with 14
degrees of unsaturation. It presented 1H and 13C NMR data
similar to those of micropeptin HU975 (5). The only notable
differences were located in the glyceric acid residue; H-2 in 6
is upfield shifted by 0.2 ppm relative to that of 5, while H-3
and H-3′ in 6 are upfield shifted by ca. 0.4 ppm, relative to that
of 5, and coupled with an acidic proton resonating at 4.78 ppm.
C-3 in 6 is upfield shifted by 4.6 ppm relative to that of 5, and
C-2 in 6 is downfield shifted by 2.1 ppm relative to that of 5.
These differences are in accordance with a nonsubstituted
glyceric acid in 6. Analysis of the 1D (1H, 13C, and DEPT) (see
Tables 1 and 2) and 2D (COSY, TOCSY, ROESY, HMQC, and
HMBC) NMR data of 6 (see Table 10, in the Supporting
Information) allowed the full assignment of the following acid
residues: valine, N,O-diMe-o-chlorotyrosine, N,N-disubstituted
isoleucine, Ahp, arginine, threonine, and glyceric acid. The
amino acid sequence of 6 was determined from HMBC correla-
tions of the NH proton of an amino acid with the carbonyl of
an adjacent amino acid, Val-N,O-diMe-o-chloroTyr, Arg-Thr,
and Thr-GA, the NMe of N,O-diMe-o-chloroTyr with Ile-
carbonyl, and H-2 of Ile with C-6 of Ahp and by NOE correlation
between R-NH of Ahp and R-NH of Arg. The ester linkage
between the carbonyl of valine and the oxygen of threonine was
assigned by an HMBC correlation between H-3 of threonine and
the carbonyl of valine. The configurations of the stereogenic
centers of 6 were assigned as L-valine, L-isoleucine, L-N,O-diMe-
o-chlorotyrosine, L-arginine, L-threonine, 3S,6R-Ahp, and D-
glyceric acid by the same procedure described for 1-5, to
establish structure 6 for micropeptin HU895A.
Micropeptin HU895B (8) was isolated as a glassy material
with a molecular formula of C40H63ClN9O12 based on the HR
MALDI TOF quasi-molecular ion at m/z 896.5133/898.5133 (3:
1) corresponding to [M + H]+. Its molecular formula was found
to be identical with that of micropeptin 895A (6) and similar to
its NMR spectra. A singlet methoxy signal in the proton NMR
spectrum of 6, δH 3.76 ppm, was missing from the spectrum of
8, and instead a phenolic singlet proton appeared at δH 9.94
ppm. In the aliphatic region of the proton NMR spectrum the
doublet methyl signal of 6, δH 0.85 ppm, is substituted by a
triplet methyl signal, δH 0.88 ppm, in the spectrum of 8. These
differences are in accordance with a structure of 8 that is similar
to that of 6, where 8 is a des-O-methyl analogue, with isoleucine
replacing valine. Analysis of the 1D (1H, 13C, and DEPT) (see
Tables 1 and 2) and 2D (COSY, TOCSY, ROESY, HMQC, and
1
Micropeptin HU909 (7) presented H NMR data similar to
those of micropeptin HU895A (6). The only notable difference
was found around 3 ppm, where a singlet methyl resonating at
δH 3.03 ppm appeared in the spectrum of 7. This additional
methyl group was also reflected in the molecular formula of 7,
C41H64ClN9NaO12, which was determined from its high-resolution
MALDI TOF quasi-molecular ion, [M + Na]+, at m/z 932.4220/
934.4220 (3:1). The NMR data of 7 (see Table 11, in the
Supporting Information) allowed the full assignment of the
following acid residues: valine, N,O-diMe-o-chlorotyrosine, N,N-
disubstituted isoleucine, amino-methoxy-piperidone (Amp), argi-
nine, threonine, and glyceric acid. The relative configuration of

1434 Journal of Natural Products, 2009, Vol. 72, No. 8
Gesner-Apter and Carmeli
HMBC) NMR data of 8 (see Table 12, in the Supporting
Information) allowed the full assignment of the following acid
residues: isoleucineI, N-Me-o-chlorotyrosine, N,N-disubstituted
isoleucineII, Ahp, arginine, threonine, and glyceric acid. The
amino acid sequence of 8 was determined from HMBC correla-
tions of the NH proton of an amino acid with the carbonyl of
an adjacent amino acid, IleI-N-Me-o-chloroTyr, Ahp-Arg, Arg-
Thr, and Thr-GA, the NMe of N-Me-o-chloroTyr with IleII-
carbonyl, and H-2 of IleII with C-6 of Ahp. The ester linkage
between the carbonyl of isoleucine and the oxygen of threonine
was assigned by an HMBC correlation between H-3 of threonine
and the carbonyl of isoleucine. The configurations of the
stereogenic centers of 8 were assigned as L-isoleucine (×2), L-N-
Me-o-chlorotyrosine, L-arginine, L-threonine, 3S,6R-Ahp, and
D-glyceric acid by the same procedure described for 1-7. On
the basis of the above given arguments, structure 8 was assigned
to micropeptin HU895B.
compounds 1, 3, and 11, in Table 4, respectively). The lowest
inhibition concentrations of chymotrypsin were measured for
glyceric acid at position 7, while 3-sulfoglyceric acid derivatives
were less potent and disulfoglyceric acid derivatives exerted much
higher IC50’s (see compounds 2, 5-8, 10, and 11, in Table 4,
respectively). It is important to mention that a related group of
compounds containing a NMe-phenylalanyl moiety at the second
position do not inhibit chymotrypsin at similar concentrations, thus
suggesting that the o-chlorophenol moiety is responsible for this
unusual behavior.14 The reason for the biosynthesis of such an array
of compounds by toxic bloom forming cyanobacteria remains as
yet unsolved, although some evidence points toward protection of
the microcystins from proteolytic enzymes.15
Experimental Section
Instrumentation. Optical rotation values were obtained on a Jasco
P-1010 polarimeter at the sodium D line (589 nm). UV spectra were
obtained on a Varian Cary 5000 UV-vis-NIR spectrophotometer.
NMR spectra were recorded on a Bruker ARX-500 spectrometer at
The new anabaenopeptin HU892 (9), isolated from this M.
aeruginosa bloom material, belongs to a relatively rare subgroup
of the anabaenopeptins, presenting an aliphatic amino acid at
the carboxylic end of the peptide and an N-methyl-homoaromatic
amino acid at the second position. In a recent article, we
described three new anabaenopeptins and summarized the
different variation in the structure of the anabaenopeptins.9 Since
then, two additional, closely related (to 9) anabaenopeptins,
anabaenopeptins 908 and 915, were described in the literature,10
and the configuration of the lysine residue of the braunsvicam-
ides11 was established by total synthesis to be D, in accordance
with all of the other cyanobacterial-derived anabaenopeptins.12
Anabaenopeptin HU892 presented a proton NMR spectrum
characteristic of the anabaenopeptins where the two amide
protons of the urea bridge resonate around δH 6.5 ppm. It
presented a HR MALDI TOF quasi-molecular ion at m/z
893.5284, [M + H]+, in accordance with a molecular formula
of C45H68N10O9. Analysis of the 1D (1H, 13C, and DEPT) and
2D (COSY, TOCSY, ROESY, HMQC, and HMBC) NMR data
of 9 (see Table 3) revealed the six amino acid units, namely,
isoleucine, N-Me-homotyrosine (N-Me-Hty), homophenylalanine
(Hph), valine, and lysine, of anabaenopeptin HU892 (9). The
amino acid sequence of 9 was determined from HMBC correla-
tions of the NH proton of an amino acid with the carbonyl of
an adjacent amino acid, Ile-N-Me-Hty, Hph-Val, and Val-Lys
and the NMe of N-Me-Hty with Hph-carbonyl. The closure of
the peptide ring was established through an HMBC correlation
between Lys-ꢀ-NH and the carbonyl of Ile, while the occurrence
of the urea bridge is suggested on the basis of the HMBC
correlation between the urea carbonyl (δC 157.5 ppm) and the
H-2 and R-NH of the lysine and arginine residues.13 Acid
hydrolysis of 9 and derivatization with Marfey’s reagent,7
followed by HPLC analysis, demonstrated the L-configuration
of the isoleucine, valine, homophenylalanine, and arginine
residues and D-configuration for the lysine residue. The config-
uration of the N-Me-homotyrosine was not determined. On the
basis of this data, structure 9 was established for anabaenopeptin
HU892.
1
500.13 MHz for H and 125.76 MHz for 13C and a Bruker Avance
1
400 spectrometer at 400.13 MHz for H and 100.62 MHz for 13C.
DEPT, COSY-45, gTOCSY, gROESY, gHSQC, gHMQC, gHMBC,
gNHHSQC, and gNHHMBC spectra were recorded using standard
Bruker pulse sequences. High-resolution MS were recorded on an
Applied Biosystems Voyager System 4312 instrument. HPLC
separations were performed on an ISCO HPLC system (model 2350
pump and model 2360 gradient programmer) equipped with an
Applied Biosystem diode-array detector and Merck-Hitachi HPLC
system (model L-4200 UV-vis detector and model L-6200A
Intelligent pump).
Biological Material. Microcystis sp., TAU strain IL-342, was
collected on October 25, 2004, from a water reservoir next to Kibbutz
Hulda, Israel. A sample of the cyanobacterium is deposited at the culture
collection of Tel Aviv University.
Isolation Procedure. The freeze-dried cells (135 g) were extracted
with 7:3 MeOH/H2O. The crude extract (18.0 g) was evaporated to
dryness and separated on an ODS (YMC-GEL, 120A, 4.4 × 6.4
cm) flash column with increasing amounts of MeOH in water.
Fraction 2 (1:9 MeOH/H2O, 1.24 g) was subjected to a Sephadex
LH-20 column in 1:1 MeOH/H2O to obtain 12 fractions. The
combined fractions 9-11 (380.3 mg) were separated again on a
Sephadex LH-20 column in 1:1 CHCl3/MeOH to give 12 fractions.
The combined fractions 3-6 were subjected to a reversed-phase
HPLC (YMC-Pack C-8 250 mm × 20.0 mm, DAD at 210 nm, flow
rate 5.0 mL/min) in 7:3 0.1% TFA in H2O/CH3CN to obtain five
fractions. Fraction 5 (9.6 mg) was subjected to a reversed-phase
HPLC (YMC-Pack C-8, 250 mm × 20.0 mm, DAD at 210 nm, flow
rate 5.0 mL/min) in 65:35 0.1% TFA in H2O/CH3CN to obtain six
fractions. Micropeptin HU1069 (1) (1.8 mg, 0.0013% yield based
on the dry weight of the cyanobacteria) was eluted from the column
with a retention time of 21.3 min, and micropeptin HU989 (2) (1.0
mg, 0.0007% yield based on the dry weight of the cyanobacteria)
was eluted from the column with a retention time of 39.3 min.
Fraction 5 (2:3 MeOH/H2O, 314.4 mg) from the initial flash column
was subjected to a Sephadex LH-20 column in 1:1 MeOH/H2O to
obtain 14 fractions. The combined fractions 5-8 (142.8 mg) were
subjected to a reversed-phase HPLC (YMC-Pack C-8, 250 mm ×
20.0 mm, DAD at 210 nm, flow rate 5.0 mL/min) in 8:2 0.1% TFA
in H2O/CH3CN to obtain five fractions. Fraction 3 from this HPLC
separation (4.9 mg) was separated on a C-18 reversed-phase HPLC
column (YMC-Pack C-18, 250 mm × 20.0 mm, DAD at 210 nm,
flow rate 5.0 mL/min) with 8:2 0.1% TFA in H2O/CH3CN to obtain
pure micropeptin HU1021 (3) (1.3 mg, 0.001% yield) with a
retention time of 24.8 min. Fraction 4 (6.0 mg) was purified on the
same column with the same conditions to afford pure micropeptin
HU1041 (4) (1.5 mg, 0.0011% yield) with a retention time of 28.6
min. Fraction 5 (10.4 mg) was separated on the C-8 HPLC column
with the same conditions to afford micropeptin 478-A (10) (1.0 mg,
0.0007% yield, tR 28.9 min) and micropeptin 478-B (11) (1.0 mg,
0.0007% yield, tR 52.8 min). Fraction 6 (1:1 MeOH/H2O, 293.1 mg)
from the initial flash column was subjected to a C-18 reversed-
phase HPLC column (YMC-Pack C-18, 250 mm × 20.0 mm, DAD
at 210 nm, flow rate 5.0 mL/min) in 3:1 0.1% TFA in H2O/CH3CN
Compounds 1-8, 10, and 11 exhibit potent inhibitory activity
against trypsin (IC50 0.7-5.2 µM), as expected for micropeptins
that contain arginine at position 5. Nevertheless, some of the
compounds (2, 5-8, 10, 11) also exhibit moderate (to potent)
inhibition of chymotrypsin. Comparison of the potency of trypsin
inhibition by the various compounds reveals that isoleucine at
position 1 correlates to lower IC50’s than valine (see compounds 2
and 10 versus 5 and 4, in Table 4, respectively); 3-sulfo-GA at
position 7 is more potent than GA, which in turn is more potent
than disulfo-GA (see compounds 10, 8, and 11, in Table 4,
respectively), and N,O-diMe-o-chlorotyrosine is more potent than
NMe-tyrosine, which is more potent than NMe-o-chlorotyrosine (see

Protease Inhibitors from Microcystis aeruginosa
Journal of Natural Products, 2009, Vol. 72, No. 8 1435
to obtain 10 fractions. Fraction 4 contained micropeptin 478-B (11)
(4.0 mg, 0.003% yield, tR 34.9 min). Fraction 8 from this column
(19.5 mg) was separated on a reversed-phase HPLC (YMC-Pack
C-8, 250 mm × 20.0 mm, DAD at 210 nm, flow rate 5.0 mL/min)
with 55:45 0.1% TFA in H2O/CH3CN to obtain pure micropeptin
HU975 (5) (2.0 mg, 0.0015% yield, tR 34.9 min), micropeptin
HU895A (6) (2.5 mg, 0.0019% yield, tR 41.4 min), and micro-
peptin HU909 (7) (4.9 mg, 0.0036% yield, tR 54.5 min). Fraction 7
(3:2 MeOH/H2O, 372.0 mg) from the initial flash column was
subjected to a Sephadex LH-20 column in 1:1 MeOH/H2O to obtain
10 fractions. The combined fractions 5 and 6 (117.7 mg) were
subjected to reversed-phase HPLC (YMC-Pack C-8, 250 mm × 20.0
mm, DAD at 210 nm, flow rate 5.0 mL/min) in 8:2 0.1% TFA in
H2O/CH3CN to yield seven fractions. Fraction 7 (18.6 mg) from
this separation was purified on the same column with 7:3 0.1% TFA
in H2O/CH3CN to obtain micropeptin HU895B (8) (1.8 mg, 0.0013%
yield, tR 34.8 min). Fraction 8 (7:3 MeOH/H2O, 354.2 mg) from
the initial chromatography was subjected to a Sephadex LH-20
column in 1:1 CHCl3/MeOH to obtain 10 fractions. The combined
fractions 3-5 (154.4 mg) were separated again on a reversed-phase
HPLC column (YMC-Pack C-8, 250 mm × 20.0 mm, DAD at 210
nm, flow rate 5.0 mL/min) in 28:72 0.1% TFA in H2O/CH3CN to
yield nine fractions. Anabaenopeptin HU892 (9) (6.6 mg, 0.0045%
yield) was obtained from this separation with a retention time of
56.2 min.
L-isoleucine 50.0 min, and L-N,O-diMe-tyrosine 54.1 min. Retention
time of D-glyceric acid on a chiral column: 3.5 min.
Micropeptin HU909 (7): transparent oil; [R]25D -53 (c 2.3, MeOH);
UV (MeOH) λmax (log ꢀ) 226 (4.15), 280 (3.46) nm; 1H and 13C NMR
(see Tables 1 and 2); HR MALDI TOF MS m/z 932.4220/934.4220
(3:1) [M + Na]+ (calcd for C41H6435ClN9NaO12, 932.4255). Retention
times of AA Marfey derivatives: L-arginine 23.1 min, L-glutamic acid
26.4 min, L-threonine 27.6 min, L-valine 40.6 min, L-isoleucine 47.2
min, and L-N,O-diMe-o-chlorotyrosine 51.9 min. Retention time of
D-glyceric acid on a chiral column: 4.9 min.
Micropeptin HU895B (8): transparent oil; [R]25 -108 (c 0.5,
D
1
MeOH); UV (MeOH) λmax (log ꢀ) 220 (4.21), 280 (3.35) nm; H and
13C NMR (see Tables 1 and 2); HR MALDI TOF MS m/z 896.4235/
898.4234 (3:1) [M + H]+ (calcd for C40H6335ClN9O12, 896.4279).
Retention times of AA Marfey derivatives: L-arginine 22.6 min,
L-glutamic acid 25.8 min, L-threonine 26.9 min, L-isoleucine 45.9 min,
and L-NMe-o-chlorotyrosine 59.8 min. Retention time of D-glyceric
acid on a chiral column: 4.0 min.
Anabaenopeptin HU892 (9): transparent oil; [R]25 -36 (c 4.2,
D
1
MeOH); UV (MeOH) λmax (log ꢀ) 225 (4.28), 281 (3.24) nm; H and
13C NMR (see Table 3); HR MALDI TOF MS m/z 893.5284 [M +
H]+ (calcd for C45H68N10O9, 893.5243). Retention times of AA Marfey
derivatives: L-arginine 26.2 min, D-lysine 42.8 min, L-valine min,
L-isoleucine 51.5 min, and L-homophenyalanine 56.8 min.
Determination of the Absolute Configuration of the Amino Acids.
Portions of compounds (0.5 mg) 1-9 were dissolved in 6 N HCl (1
mL). The reaction mixture was then placed in a sealed glass bomb
at 110 °C for 20 h. After removal of HCl, by repeated evaporation
in vacuo, the hydrolysate was resuspended in water (40 mL). A
solution of (1-fluoro-2,4-dinitrophenyl)-5-L-alanine amide (FDAA)
(4.2 mmol) in acetone (150 mL) and 1 N NaHCO3 (20 mL) were
added to each reaction vessel, and the reaction mixture was stirred
at 40 °C for 2 h. A 2 N HCl solution (10 mL) was added to each
reaction vessel, and the solution was evaporated in vacuo. The
N-[(2,4-dinitrophenyl)-5-L-alanine amide]-amino acid derivatives,
from hydrolysates, were compared with similary derivatized standard
amino acids by HPLC analysis: Knauer GmbH Eurospher 100 C18,
10 m, 4.6 × 300 mm, flow rate 1 mL/min, UV detection at 340 nm,
linear gradient elution from 9:1 50 mM triethylammonium phosphate
(TEAP) buffer (pH 3)/CH3CN to 1:1 TEAP/CH3CN within 60 min.
The determination of the absolute configuration of each amino acid
was confirmed by spiking the derivatized hydrolysates with the
derivatized authentic amino acids.
Determination of the Absolute Configuration of Glyceric Acid.
The acid hydrolysates (1 mL) of compounds 1-8 were extracted
with ethyl ether (1 mL × 3) to separate the glyceric acid from the
amino acids mixture. The ether was removed and the residue
dissolved in MeOH (1 mL). The MeOH solution was analyzed on
an Astec Chirobiotic HPLC column, 250 × 4.6 mm flow rate 1 mL/
min, UV detection at 210 nm, linear elution with 1:49 1%
triethylammonium acetate (TEAA) buffer (pH 4)/MeOH. The
authentic samples were spiked with a standard mixture of L- and
D-glyceric acid.
Protease Inhibition Assay. Trypsin and chymotrypsin were
purchased from Sigma Chemical Co. Trypsin was dissolved in 50
mM Tris-HCl/100 mM NaCl/1 mM CaCl2 to prepare a 1 mg/mL
solution. Chymotrypsin was dissolved in 50 mM Tris-HCl/100 mM
NaCl/1 mM CaCl2/1 mM HCl to prepare a 1 mg/mL solution. A 2
mM solution of N-benzoyl-D,L-arginine-p-nitroanilide (for trypsin)
and Suc-Gly-Gly-p-nitroanilide (for chymotrypsin) in the appropriate
buffer solution was used as a substrate solution. The test sample
was dissolved in ethanol and diluted with the same buffer solution
that was used for the enzyme and substrate. A 100 µL buffer
solution, 10 µL of enzyme solution, and 10 µL of the test solution
were added to each microtiter plate well and preincubated at 37 °C
for 5 min. Then, 100 µL of substrate solution was added to begin
the reaction. The absorbance of the well was immediately measured
at 405 nm. The developed color was measured after incubation at
37 °C for 30 min.
Micropeptin HU1069 (1): transparent oil; [R]25 -43.1 (c 1.7,
D
1
MeOH); UV (MeOH) λmax (log ꢀ) 226 (4.38), 280 (3.36) nm; H and
13C NMR (see Tables 1 and 2); MALDI TOF MS m/z 1012.29/1014.29
(3:1) [MNa - SO3]+, HR MALDI TOF MS m/z 910.4486/912.4485
(3:1) [MH - 2SO3]+ (calcd for C41H6535ClN9O12, 910.4436). Retention
times of AA Marfey derivatives: L-arginine 24.5 min, L-glutamic acid
29.7 min, L-threonine 28.9 min, L-isoleucine 48.6 min, and L-N,O-diMe-
o-chlorotyrosine 53.2 min. Retention time of D-glyceric acid on a chiral
column: 4.8 min.
Micropeptin HU989 (2): transparent oil; [R]25 -77 (c 0.9,
D
MeOH); UV (MeOH) λmax (log ꢀ) 226 (4.17), 280 (3.30) nm; 1H
and 13C NMR (see Tables 1 and 2); HR MALDI TOF MS m/z
1012.3863/1014.3863 (3:1) [M
+
Na]+ (calcd for C41H64-
35ClN9NaO15S, 1012.3823). Retention times of AA Marfey deriva-
tives: L-arginine 24.7 min, L-glutamic acid 27.6 min, L-threonine
29.4 min, L-isoleucine 48.4 min, and L-N,O-diMe-o-chlorotyrosine
54.8 min. Retention time of D-glyceric acid on a chiral column: 4.0
min.
Micropeptin HU1021 (3): transparent oil; [R]25 -150 (c 0.4,
D
1
MeOH); UV (MeOH) λmax (log ꢀ) 229 (4.23), 277 (3.63) nm; H and
13C NMR (see Tables 1 and 2); MALDI TOF MS m/z 1012.29/1014.29
(3:1) [M + Na - SO3]+; HR MALDI TOF MS m/z 964.4083 [M +
Na - SO3]+ (calcd for C40H63N9NaO15S, 964.4057). Retention times
of AA Marfey derivatives: L-arginine 17.9 min, L-glutamic acid 19.5
min, L-threonine 21.4 min, L-isoleucine 42.3 min, and L-NMe-tyrosine
48.4 min. Retention time of D-glyceric acid on a chiral column: 4.1
min.
Micropeptin HU1041 (4): transparent oil; [R]25 -57 (c 0.5,
D
1
MeOH); UV (MeOH) λmax (log ꢀ) 230 (4.07), 282 (3.53) nm; H and
13C NMR (see Tables 1 and 2); HR MALDI TOF MS m/z 984.3421/
986.3420 (3:1) [M + Na - SO3]+ (calcd for C39H6035ClN9NaO15S,
984.3510). Retention times of AA Marfey derivatives: L-arginine 20.4
min, L-glutamic acid 24.8 min, L-threonine 25.9 min, L-valine 39.9 min,
L-isoleucine 44.5 min, and L-NMe-o-chlorotyrosine 59.9 min. Retention
time of D-glyceric acid on a chiral column: 4.0 min.
Micropeptin HU975 (5): transparent oil; [R]25D -26 (c 2.2, MeOH);
UV (MeOH) λmax (log ꢀ) 226 (4.09), 280 (3.31) nm; 1H and 13C NMR
(see Tables 1 and 2); HR MALDI TOF MS m/z 998.3615/1000.3615
(3:1) [M + Na]+ (calcd for C40H6235ClN9NaO15S, 998.3667). Retention
times of AA Marfey derivatives: L-arginine 20.5 min, L-glutamic acid
25.4 min, L-threonine 25.3 min, L-valine 39.4 min, L-isoleucine 45.4
min, and L-N,O-diMe-o-chlorotyrosine 50.3 min. Retention time of
D-glyceric acid on a chiral column: 4.2 min.
Micropeptin HU895A (6): transparent oil; [R]25 -65 (c 1.7,
D
1
Acknowledgment. A. Sacher, The Mass Spectrometry Laboratory
of The Maiman Institute for Proteome Research of Tel Aviv University,
is acknowledged for obtaining the MALDI TOF mass spectra. This
research was supported by The Israel Science Foundation grant 037/
02.
MeOH); UV (MeOH) λmax (log ꢀ) 226 (4.11), 280 (3.21) nm; H and
13C NMR (see Tables 1 and 2); HR MALDI TOF MS m/z 934.3868/
936.3869 (3:1) [M + K]+ (calcd for C40H6235ClKN9O12, 934.3838).
Retention times of AA Marfey derivatives: L-arginine 25.7 min,
L-glutamic acid 29.8 min, L-threonine 30.6 min, L-valine 44.4 min,

1436 Journal of Natural Products, 2009, Vol. 72, No. 8
Gesner-Apter and Carmeli
1
Supporting Information Available: H and 13C NMR spectra of
(7) Marfey’s reagent: 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. Marfey,
P. Carlsberg Res. Commun. 1984, 49, 591–596.
(8) Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon, B. C. L.
J. Chem. Soc. 1946, 39–45.
all new compounds as well as full NMR data. This material is available
free of charge via the Internet at http://pubs.acs.org.
(9) Grach-Pogrebinsky, O.; Carmeli, S. Tetrahedron 2008, 64, 10233–
10238.
(10) Okumura, H. S.; Philmus, B.; Portmann, C.; Hemscheidt, T. K. J.
Nat. Prod. 2009, 72, 172–176.
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F. C.; Saxena, K.; Prinz, H.; Schwalbe, H.; Janning, P.; Waldmann,
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NP900340T