Lyngbyaureidamides A and B, two anabaenopeptins from the cultured freshwater cyanobacterium Lyngbya sp. (SAG 36.91)

Article abstract of DOI:10.1016/j.phytochem.2011.09.017

Two anabaenopeptin-type peptides, lyngbyaureidamides A and B, together with two previously reported peptides lyngbyazothrins C and D, were isolated from the cultured freshwater cyanobacterium Lyngbya sp. (SAG 36.91). Their structures were determined by spectroscopic and chemical methods. Lyngbyazothrins C and D were also able to inhibit the 20S proteasome with IC₅₀ values of 7.1 μM and 19.2 μM, respectively, while lyngbyaureidamides A and B were not active at 50 μM.

Full text of DOI:10.1016/j.phytochem.2011.09.017

Phytochemistry 74 (2012) 173–177
Contents lists available at SciVerse ScienceDirect
Phytochemistry
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Lyngbyaureidamides A and B, two anabaenopeptins from the cultured
freshwater cyanobacterium Lyngbya sp. (SAG 36.91)
⇑
Jiachen Zi, Daniel D. Lantvit, Steven M. Swanson, Jimmy Orjala
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St., (M/C 781), Chicago, IL 60612, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 March 2011
Received in revised form 28 July 2011
Available online 5 December 2011
Two anabaenopeptin-type peptides, lyngbyaureidamides A and B, together with two previously reported
peptides lyngbyazothrins C and D, were isolated from the cultured freshwater cyanobacterium Lyngbya
sp. (SAG 36.91). Their structures were determined by spectroscopic and chemical methods. Lyngbyazoth-
rins C and D were also able to inhibit the 20S proteasome with IC50 values of 7.1
tively, while lyngbyaureidamides A and B were not active at 50 M.
Ó 2011 Elsevier Ltd. All rights reserved.
lM and 19.2 lM, respec-
l
Keywords:
Cyanobacterium
Lyngbya sp. (SAG 36.91)
Peptide
20S Proteasome
1. Introduction
The activity was recovered in fraction 3, eluting with 40% isopropa-
nol in water and further purified by HPLC, which led to the isola-
tion of four peptides: lyngbyaureidamides A (1) and B (2) and
the previously reported peptides lyngbyazothrins C (3) and D (4)
(Fig. 1).
Lyngbyaureidamide A (1) was obtained as a yellowish gum. The
molecular formula was determined as C46H61N7O9 by HRMS (m/z
878.4426 [M+Na]+). The proton spectrum of 1 contained signals
for six amide protons (dH 8.92, 8.75, 7.13, 7.03, 6.61 and 5.93), a
Cyanobacteria are known to be a rich source of secondary
metabolites with diverse chemical structures and biological activ-
ities (Wagoner et al., 2007; Tan, 2007; Müeller et al., 2006; Luesch
et al., 2001; Singh et al., 2005; Jaiswal et al., 2008). Many of these
metabolites have been found to inhibit various proteases and we
have initiated a screening program to discover cyanobacterial
inhibitors of the 20S proteasome, the catalytic core of the major
proteolytic system in eukaryotic cells and an established antican-
cer target. One of the active extracts was Lyngbya sp. (SAG
36.91), which showed inhibitory activity against the 20S protea-
N-methyl group (dH 2.64) and six
a-protons (dH 4.95, 4.78, 4.16,
3.95, 3.87 and 3.77). The latter set correlated with six carbon
resonances (dC 54.9, 49.0, 53.4, 55.2, 57.3 and 56.1) in the HSQC
spectrum. Detailed 2D NMR spectroscopic analysis (Table 1 and
Fig. 2) indicated the presence of six amino acid residues, three of
which were the common amino acids Lys, Ile and Phe. The struc-
tures of three remaining non-standard amino acid residues were
determined by analysis of COSY, HSQC and HMBC data. The struc-
ture of N-MeAla was determined by the HMBC correlation from the
N-methyl singlet to the N-MeAla C-1 (dC 170.6), in combination
some at a concentration of 50 lg/mL. Bioassay-guided fraction-
ation led to the isolation of four cyclic peptides, including two
new anabaenopeptin-type peptides lyngbyaureidamides A (1)
and B (2) and the previously reported peptides lyngbyazothrins C
(3) and D (4). Lyngbyazothrins C (3) and D (4) were originally
reported as an inseparable mixture with antibacterial activity from
this strain (Zainuddin et al., 2009). Lyngbyazothrins C (3) and D (4)
were recently reported individually as portoamides A and B (Leão
et al., 2010).
with a COSY correlation between N-MeAla H-
b (dH 1.25). The presence of a homophenylalanine (Hph) residue
was deduced by the HMBC correlation from Hph H2- (dH 2.39)
to C-20/C-60 (dC 128.6) connecting the two partial structures estab-
lished by COSY correlations (Hph NH to H2- and a mono substi-
tuted aromatic ring C-10–C-60). Similarly, the presence of
homotyrosine (Hty) residue was deduced by the HMBC correla-
tions from Hty H2-
(dH 2.48 and 2.67) to C-20/C-60 (dC 129.5),
partial structure to the para-substituted
a and N-MeAla H3-
c
c
2. Results and discussion
a
Lyngbya sp. (SAG 36.91) was mass cultured in Z medium (Mian
et al., 2003). The organic extract was active in our 20S proteasome
assay and was fractionated using a diaion HP20SS resin column.
c
connecting Hty NH to H2-
c
aromatic ring (Hty C-10–C60).
The structure of 1 was established by interpretation of the
HMBC spectrum (Table 1 and Fig. 2). The correlations from the
⇑
Corresponding author. Tel.: +1 312 996 5583; fax: +1 312 996 7107.
E-mail address: orjala@uic.edu (J. Orjala).
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.09.017

174
J. Zi et al. / Phytochemistry 74 (2012) 173–177
OH
R
HO
-Hty2
O
O
N
L
O
OH
L
-Ile1
-Phe6
L
-N-MeAla3
D
OH OH
NH
NH
O
HN
N
OH HN
HN
N
O
O
O
HN
O
O
O
O
O
O
O
O
OH
O
HN
O
N
H
N
H
HN
N
NH2
R
HN
NH OH
D
-Lys5
-Hph4/ -Phe4
L
O
L
O
R
HN
O
O
O
1: benzyl
2: phenyl
N
N
H
R
3: OMe
4: H
Fig. 1. Structure of lyngbyaureidamide A (1) and B (2), lyngbyazothrins C (3) and D (4).
NH of Hph to C-1 of N-MeAla (dC 170.6), from HNMe- of N-MeAla to
C-1 of Hty (dC 171.8), from the NH of Hty to C-1 of Ile (dC 173.2),
from the NH of Ile to C-1 of Lys (dC 172.9) and from e-NH of Lys
have been obtained from several cyanobacterial species, including
Anabaena (Harada et al., 1995; Grach-Pogrebinsky and Carmeli,
2008), Aphanizomenon (Murakami et al., 2000), Lyngbya (Matthew
et al., 2008), Microcystis (Williams et al., 1996; Beresovsky et al.,
2006; Gesner-Apter and Carmeli, 2009; Zafrir-Ilan and Carmeli,
2010), Nodularia (Fujii et al., 1997), Oscillatoria (Sano and Kaya,
1995; Shin et al., 1997; Itou et al., 1999), Planktothrix (Sano et al.,
2001; Okumura et al., 2009), Schizothrix (Reshef and Carmeli,
2002), and Tychonema (Müeller et al., 2006), as well as from
sponges (Kobayashi et al., 1991a,b; Schmidt et al., 1997; Uemoto
et al., 1998; Robinson et al., 2007; Plaza et al., 2010). The exocylic
amino acid for all anabaenopeptin–type peptides reported to date
has L configuration and lyngbyaureidamide A (1) and B (2) are the
first examples of a D-configuration amino acid residue at this
position (D-Phe).
to C-1 of Hph (dC 171.5), indicated that these five residues formed
a 19-membered peptide ring, characteristic of the anabaenopeptin-
type peptides (Welker and von Dohren, 2006). Finally, analysis of
the correlations from H-
a of Lys and H-a
of Phe to the same sp2
carbon at dC 157.1 suggested that the remaining Phe residue was
attached to the 19-membered ring through an ureido group. This
ureido group also accounted for the remaining carbonyl moiety im-
plied by the molecular formula. The absolute configurations of
amino acid residues of 1 were determined by Marfey’s method
(Marfey, 1984). The result of the HPLC analysis showed that N-
MeAla, Hty, Ile and Hph all were of the L configuration, while Lys
and Phe both were D.
Lyngbyaureidamide B (2) was obtained as a yellow gum. The
HRMS quasimolecular peak at m/z 864.4255 [M+Na]+ suggested a
molecular formula of C45H59N7O9. The 1D and 2D NMR spectro-
scopic data of 2 were very similar to those of 1. The only difference
was the loss of the Hph moiety and the appearance of an additional
Phe moiety in 2. The placement of the second Phe moiety was
determined by analysis of the ROESY spectrum of 2. The correla-
All isolates were evaluated for their 20S proteasome inhibitory
activity (chymotrypsin-like activity). Lyngbyazothrins C (3) and D
(4) were found to be active with IC50 values of 7.1
19.2 M, respectively, while lyngbyaureidamide A (1) and B (2)
were found to be inactive at 50 M. None of the four compounds
showed cytotoxic activities against the human colon cancer cell
line designated HT-29 at 50 M. Lyngbyazothrins C (3) and D (4)
lM and
l
l
l
tions between NH of Phe4 (dH 8.75) and H-
4.78) and
-NH of Lys5 (dH 7.16) indicated that Hph had been re-
placed by Phe. This was confirmed by the HMBC correlations from
the NH of Phe4 to C-1 of N-MeAla3 (dC 170.6), and from -NH of Lys5
a
of N-MeAla3 (dH
were originally obtained as an inseparable mixture from this
cyanobacterial strain and shown to inhibit the growth of Esche-
richia coli (Zainuddin et al., 2009). The same two structures were
recently also reported as pure compounds, named portoamides A
and B, obtained from an Oscillatoria sp. (Leão et al., 2010). The
portoamides were found to act synergistically to inhibit the growth
of the green microalga Chlorella vulgaris. We here were also able to
separate lyngbyazothrins C (3) and D (4), the two major compo-
nents of Lyngbya sp. (SAG 36.91) by HPLC. Comparison of the
spectroscopic data confirmed that lyngbyazothrins C and D were
identical to portoamides A and B, respectively.
e
e
to C-1 of Phe4 (dC 171.5). The amino acid residues of 2 were defined
as L-Ile, L-Hty, L-N-MeAla, L-Phe, D-Phe and D-Lys by Marfey’s
method), indicating the replacement of L-Hph4 by L-Phe4. This
assignment was further supported by comparison of the NMR
spectroscopic data of lyngbyaureidamide B with those published
for other anabaenopeptins containing a L-Hty-L-N-MeAla-L-Phe-
D-Lys sequence as part of their 19-membered peptide ring
(Williams et al., 1996; Zafrir-Ilan and Carmeli, 2010). No significant
deviation (<0.8 ppm) was found for these 13C chemical shifts. In
addition, the NMR chemical shifts for Phe6 of lyngbyaureidamide
B closely matched those of Phe6 of 1. Together, this indicated L-
Phe to be in position 4.
3. Conclusions
Bioassay-guided investigation (proteasome inhibition) of the
cyanobacterium Lyngbya sp. (SAG 36.91) led to the isolation of four
peptides. The previously reported lyngbyazothrins C (3) and D (4)
accounted for the 20S proteasome inhibitory activity. In addition,
two new anabaenopeptin-type peptides, lyngbyaureidamide A (1)
and B (2) were obtained.
Anabaenopeptins, a group of cyclic hexapeptides, are character-
ized by a 19-membered peptide ring that is formed by cyclization
between the C-terminal amino acid and the
idue. The -amine of the lysine is further linked through an ureido
group to a side-chain amino acid. Anabaenopeptin-type peptides
e-amine of a lysine res-
a

J. Zi et al. / Phytochemistry 74 (2012) 173–177
175
Table 1
NMR spectroscopic data (d) of compounds 1 and 2 in DMSO-d6.
Unit
C/H No.
1
2
a
b
c
b
dC
dH
COSY
HMBC
C-1Lys
dC
dH
COSY
HMBC
C-1Lys
Ile1
NH
C-1
7.03
H
7.14
H
a
a
173.2
57.3
35.9
25.4
173.0
57.1
36.1
25.3
C
Cb
C
3.87
1.54
1.45
1.04
0.89
0.73
8.92
NH, Hb
3.97
1.77
1.56
1.17
1.01
0.82
8.94
NH, Hb
a
H , H , H0
C-1Ile
C-1Ile
H , H , H0
C-1Ile
C-1Ile
a
c
c
a
c
c
Hb, Hd
Hb, Hd
Hb
Hb, Hd
Hb, Hd
Hb
c
C0
Cd
NH
C-1
C
15.5
10.8
15.4
10.7
c
H
H
H
H
c
c
Hty2
a
a
171.8
49.0
33.8
171.4
49.2
33.7
4.78
1.93
1.80
2.67
2.48
NH, Hb
4.71
1.87
1.72
2.64
2.43
NH, Hb
a
Cb
H , H
H , H
a
C-1Hty
H , H
H , H
a
C-1Hty
a
c
c
a
c
c
C
30.9
Hb
Hb
C-10, 20, 60
31.0
Hb
Hb
C-10, 20, 60
c
C-10
131.2
129.5
115.5
156.0
131.7
129.5
115.6
156.0
C-20,60
C-30,50
C-40
7.04
6.69
H-30,50
H-20,60
7.01
6.67
H-30,50
H-20,60
40-OH
N-Me
C-1
9.26
2.64
9.26
1.77
N-MeAla3
28.8
170.6
54.9
C-1NMe–Ala, C , C-1Hty
27.4
170.6
54.7
C-1NMe–Ala, C , C-1Hty
a
a
C
Cb
NH
C-1
C
4.95
1.25
8.75
Hb
4.78
1.06
8.75
Hb
a
14.5
H
H
C-1NMe–Ala
C-1NMe–Ala
14.3
H
a
H
a
C-1NMe–Ala
C-1NMe–Ala
a
a
Hph4/Phe4
171.5
53.4
34.5
171.5
55.4
37.9
4.16
2.08
1.90
2.39
NH, Hb
H , H
4.35
3.29
2.78
NH, Hb
a
Cb
C-1Hph
H
a
C-1Phe4 and C-10, 20, 60
a
c
c
H , H
a
H
a
C
C-10
C-20,60
C-30,50
C-40
32.3
141.9
128.6
128.5
126.2
Hb
C-10, 20, 60
c
139.1
129.3
128.7
126.4
7.07
7.23
7.15
6.61
7.05
7.20
7.15
6.62
Lys5
NH
H
H
a
a
C-1
172.9
55.2
32.7
20.7
172.9
55.4
32.7
20.8
C
3.95
1.54
1.26
1.09
1.45
1.38
3.50
2.79
7.13
5.93
NH, Hb
CUreido
C-1Lys
3.96
1.59
1.29
1.13
1.48
1.43
3.56
2.81
7.16
5.96
NH, Hb
CUreido
C-1Lys
a
Cb
H , H
a
H , H
a
c
c
C
Hb
Hb
c
Cd
28.5
38.7
H
28.5
38.8
H
e
e
C
e
e
-NH, Hd
-NH, Hd
e
e
-NH, Hd
-NH, Hd
e
e-NH
H
C-1Hph
H
H
C-1Phe4
e
e
Phe6
NH
C-1
H
a
a
173.0
56.1
38.2
173.3
55.4
38.2
C
3.77
3.00
2.94
NH, Hb
CUreido
C-1Phe, and C-10, 20, 60
3.96
3.01
2.96
NH, Hb
CUreido
C-1Phe6 and C-10, 20, 60
a
Cb
H
H
a
a
H
H
a
a
C-10
140.1
130.1
127.6
125.6
157.1
139.8
130.0
127.9
126.0
157.3
C-20,60
C-30,50
C-40
7.06
7.12
7.07
7.11
7.19
7.14
Ureido
CUreido
a
b
c
Chemical shifts were determined by HSQC and HMBC experiments recorded at 600 MHz.
Recorded at 600 MHz.
DEPTQ experiment recorded at 226 MHz.
tra were obtained on a Shimadzu IT-TOF spectrometer. HPLC sep-
arations were performed on an instrument consisting of a Waters
600 controller, a Waters 600 pump, and a Waters 2487 dual wave-
length absorbance detector, with an Alltima (250 ꢀ 10 mm i.d.)
4. Experimental
4.1. General experimental procedures
preparative column packed with C18 (5
lm) or a Varian (250 ꢀ
The optical rotations were determined on a Perkin–Elmer 241
polarimeter. UV spectra were obtained on a Varian Cary 50 Bio
spectrophotometer. IR spectra were obtained on a Jasco FTIR-410
Fourier transform infrared spectrometer. 1H and 13C NMR spectra
were obtained on a Bruker Avance DRX600 MHz NMR spectrome-
ter with a 5 mm CPTXI Z-gradient probe and a Bruker Avance
II900 MHz NMR spectrometer with a 5 mm ATM CPTCI Z-gradient
probe, referenced to the corresponding solvent peaks. HRMS spec-
10 mm i.d.) semipreparative column packed with C8 (5 lm).
4.2. Biological material
Lyngbya sp. (SAG 36.91) from Culture Collection of Algae, Göt-
tingen, Germany) was grown in 21 L of aerated inorganic Z media
(Mian et al., 2003). Cultures were illuminated with fluorescent

176
J. Zi et al. / Phytochemistry 74 (2012) 173–177
4.3.4. Lyngbyazothrin D (4)
Yellow gum; [
ꢁ17.2 (c 0.1 MeOH); IR (neat) mmax 3307,
2971, 1664, 1605, 1470, 1207, 1128 cmꢁ1
a
]
D
;
1H and 13C NMR data
as described (Zainuddin et al., 2009; Leão et al., 2010). HRMS m/z
1524.7673 [M+Na]+ (calcd for C45H59N7O9Na, 1524.7602).
4.4. Racemization of D-Homotyrosine
LD-Hty was obtained from D-Hty via an acetylation–deacetyla-
tion process (Sealock, 1946). D-homotyrosine hydrobromide
(100 mg, 0.00036 mol; Chem-Impex International Inc., Wood Dale,
USA) and NaOH (46.6 mg, 0,00115 mol) were dissolved in H2O
(1.12 mL), and Ac2O (600 lL, Sigma–Aldrich Inc., Louis, USA) was
added in ten portions at 3 min intervals with continuous stirring.
The reaction mixture was stirred at 65 °C for 6 h, and then evapo-
rated in vacuo to give a thick syrup (132 mg), which was extracted
by acetone and concentrated in vacuo to afford crude diacetylated
LD-homotyrosine (99 mg) as yellow oil, confirmed by IT–TOF MS:
HRMS m/z 302.1004 [M+Na]+ (calcd for C14H17NO5Na, 302.1004).
The crude diacetylated LD-homotyrosine was dissolved in 2 N
Fig. 2. 1H–1H COSY and key HMBC correlations of 1.
lamps at 1.93 klx with an 18/6 h light/dark cycle. The temperature
of the culture room was maintained at 22 °C. After 7 weeks, the
biomass of cyanobacteria was harvested by centrifugation and
freeze-dried (yield 11.24 g).
NaOH (400 lL), and stirred at room temperature for 30 min. The
resulting solution was neutralized to pH 7 by 6 N HCl and evapo-
rated in vacuo to give a thick syrup which was extracted by acetone
and concentrated in vacuo to afford crude N-acetylated LD-
homotyrosine (78 mg) as yellow oil, confirmed by IT–TOF MS:
HRMS m/z 260.0898 [M+Na]+ (calcd for C12H15NO4Na, 260.0899).
The residue was hydrolyzed with 6 N HCl (2 mL) in a sealed thick
glass tube at 102 °C for 3.5 h. The solvent was removed under
reduced pressure to give crude LD-homotyrosine (70.1 mg) as a
4.3. Extraction and isolation
Freeze-dried Lyngbya biomass (11.24 g) was extracted, each for
two hrs with CH2Cl2/MeOH (200 mL ꢀ 5, 1:1 v/v) at room temper-
ature to yield a crude extract (984.5 mg). A portion of the crude
extract (849.8 mg) was fractionated using a Diaion HP-20 column
(10 g, 130 ꢀ 25 mm) with increasing amounts of i-PrOH:H2O
(0:100; 20:80; 40:60; 70:30; 80:20; 90:10; 100:0, fraction size
100 mL) to generate eight sub-fractions. Fraction 3 (eluted with
i-PrOH 40:60, v/v) was the most active fraction and showed
red solid, [
a
]
D
ꢂ0, confirmed by IT–TOF MS. LD-Homotyrosine:
HRMS m/z 196.0968 [M+H]+ (calcd for C10H14NO3, 196.0895).
4.5. Acid hydrolysis of 1 and 2, and Marfey’s analyses of the
hydrolyzates
78.4% inhibition against 20S proteasome at 50
(103.5 mg) was subjected to reversed-phase HPLC (Alltima C18
solvent gradient of MeOH:H2O
(35:65 ? 90:10) over 50 min to afford two subfractions (3a at
32–33 min, and 3b at 35–36 min). Subfraction 3a (8.9 mg) was fur-
lg/mL. This fraction
,
In separate reactions, compounds 1 (0.2 mg) or 2 (0.2 mg) were
dissolved in 6 N HCl (0.4 ml), and heated at 110 °C in a sealed vial
for 13 h. The cooled reaction mixture was evaporated to dryness
under reduced pressure. The amino acid mixture was resuspended
5
l
m, 250 ꢀ 10 mm) with
a
ther separated by reversed-phase HPLC (Varian C8,
5 lm,
in H2O (40
alanine amide (FDAA) in acetone (100
(100 L) was added to each reaction vessel. The reaction mixture
was stirred at 40 °C for 2 h and the resulting solution was neutral-
ized using 2 M HCl. The FDAA-amino acid derivatives, from
hydrolyzates, were compared with similarly derivatized standard
amino acids by HPLC analysis at 340 nm. The column (Alltima
l
L). A solution of 1% (1-fluoro-2,4-dinitrophenyl)-5-
L-
250 ꢀ 10 mm), using MeOH–H2O (55:45) as mobile phase, to yield
1 (1.6 mg, eluting after 112 min) and 2 (1.1 mg eluting after
110 min). Subfraction 3b (67.3 mg) was subjected to reversed-
l
L) and 1 M NaHCO3
l
phase HPLC (Varian C8, 5
H2O (65:35), to give 3 (20.5 mg, eluting after 183 min) and 4
l
m, 250 ꢀ 10 mm), eluted by MeOH–
(10.3 mg, eluting after 188 min).
RP-C8, 5
l
m, 250 ꢀ 4.6 mm) was eluted with a linear gradient of
4.3.1. Lyngbyaureidamide A (1)
(A) CH3CN containing 0.65% AcOH and (B) H2O containing 0.65%
AcOH from 20% to 42% (A) over 70 min followed by another gradi-
ent elution from 42% to 45% (A) over 20 min at flow rate: 0.5 mL/
min. The standards gave the following retention times in min:
48.2 for FDAA; 67.5 for L-Ile, 76.4 for D-Ile; 66.8 for L-allo-Ile,
75.9 for D-allo-Ile; 69.5 for L-Phe, 76.1 for D-Phe; 71.1 for L-Lys,
73.4 for D-Lys; 77.8 for L-Hph, 85.8 for D-Hph; 58.0 for L-Hty,
63.2 for D-Hty; 42.1 for L-N-MeAla, 47.2 for D-N-MeAla.
Yellowish gum; [
a
]
D ꢁ27.3 (c 0.09 MeOH); UV (MeOH) kmax (log
e
) 210 (4.21) nm; IR (neat) mmax 3295, 3250, 2932, 2863, 1643,
1548, 1503, 1452, 1250, 1007 cmꢁ1; For 1H and 13C NMR spectro-
scopic data, see Table 1; HRMS m/z 878.4426 [M+Na]+ (calcd for
C46H61N7O9Na, 878.4428).
4.3.2. Lyngbyaureidamide B (2)
Yellow gum; [
a
]
D ꢁ41.1 (c 0.06 MeOH); UV (MeOH) kmax (log
e)
210 (4.27) nm; IR (neat) mmax 3286, 2928, 1643, 1548, 1524, 1504,
1250, 1024 cmꢁ1; For 1H and 13C NMR spectroscopic data, see Table
1; HRMS m/z 864.4255 [M+Na]+ (calcd for C45H59N7O9Na,
864.4272).
4.6. 20S Proteasome assay
The proteasome assay was performed according to the protocol
provided with the BIOMOL 20S Proteasome Assay Kit for Drug
Discovery (BIOMOL International LP, Plymouth Meeting, PA, USA
Catalog Number AK740-0001). The protocol was modified such that
the 10 min incubation period was performed at 37 °C. Enzyme was
acquired from BostonBiochem (20S proteasome, human, Boston-
Biochem, USA) and substrate from BIOMOL (Suc-LLVY-AMC). This
substrate is specific for the chymotrypsin-like activity of the 20S
4.3.3. Lyngbyazothrin C (3)
Yellow gum; [
a
]
D
ꢁ14.6 (c 0.1 MeOH); IR (neat) mmax 3321,
2957, 1652, 1601, 1506, 1250, 1214 cmꢁ1
;
1H and 13C NMR data
as described (Zainuddin et al., 2009; Leão et al., 2010); HRMS m/z
1554.7771 [M+Na]+ (calcd for C74H109N13O22Na, 1554.7708).

J. Zi et al. / Phytochemistry 74 (2012) 173–177
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ios Pro microplate reader or a Hewlett Packard model AF10000 fluo-
rimeter with an excitation wavelength of 360 nm and an emission
wavelength of 460 nm. The positive control was bortezomib (IC50
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Acknowledgments
This research was supported by the National Cancer Institute
Grant P01CA125066. We thank Dr. A. Krunic for performing the
DEPTQ experiment.
Okumura, H.S., Philmus, B., Portmann, C., Hemscheidt, T.K., 2009. Homotyrosine-
containing cyanopeptolins 880 and 960 and anabaenopeptins 908 and 915 from
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anabaenopeptin-type peptides from the Palau sponge Theonella swinhoei. J. Nat.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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R.W.M., Crews, P., 2007. Probing the bioactive constituents from chemotypes of
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