Tiglicamides A-C, cyclodepsipeptides from the marine cyanobacterium Lyngbya confervoides

Article abstract of DOI:10.1016/j.phytochem.2009.09.010

The Floridian marine cyanobacterium Lyngbya confervoides afforded cyclodepsipeptides, termed tiglicamides A-C (1-3), along with their previously reported analogues largamides A-C (4-6), all of which possess an unusual tiglic acid moiety. Their structures

Full text of DOI:10.1016/j.phytochem.2009.09.010

Phytochemistry 70 (2009) 2058–2063
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Phytochemistry
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Tiglicamides A–C, cyclodepsipeptides from the marine cyanobacterium
Lyngbya confervoides
Susan Matthew a, Valerie J. Paul b, Hendrik Luesch a,
*
a Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610, USA
b Smithsonian Marine Station, 701 Seaway Drive, Fort Pierce, Florida 34949, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2009
Received in revised form 13 August 2009
Available online 6 October 2009
The Floridian marine cyanobacterium Lyngbya confervoides afforded cyclodepsipeptides, termed tiglica-
mides A–C (1–3), along with their previously reported analogues largamides A–C (4–6), all of which pos-
sess an unusual tiglic acid moiety. Their structures were deduced by one- and two-dimensional NMR
combined with mass spectrometry and the absolute configurations established by chiral HPLC and Mar-
fey’s analysis of the degradation products. Compounds 1–3 moderately inhibited porcine pancreatic elas-
Keywords:
tase in vitro with IC50 values from 2.14 to 7.28 lM. Compounds 1–6 differ from each other by one amino
Marine cyanobacteria
Lyngbya confervoides
Cyclodepsipeptides
Nonribosomal peptide synthesis
Elastase inhibitors
acid residue within the cyclic core structure, suggesting an unusually relaxed substrate specificity of the
nonribosomal peptide synthetase that is the putative biosynthetic enzyme responsible for the corre-
sponding amino acid incorporation.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
astatins 4–6 (Matthew et al., 2007; Taori et al., 2007), pompano-
peptin A (Matthew et al., 2008), along with largamides D–H
Cyanobacteria are a group of microorganisms with the distinc-
tion of being the most ancient known organisms on Earth (Schopf,
1996). The secondary metabolites produced by cyanobacteria are
extremely diverse in their structural motifs, with modified poly-
peptides and peptide–polyketide hybrids being the most commonly
encountered. Most of these peptides are thought to be biosynthe-
sized by either nonribosomal polypeptide synthetases (NRPS) or
mixed polyketide synthase–NRPS pathways (Tan, 2007); however,
several peptidic natural products were recently shown to be made
ribosomally (McIntosh et al., 2009). The secondary metabolites pro-
duced by cyanobacteria exhibit a broad spectrum of biological activ-
ities affecting a variety of bacterial, viral, fungal and mammalian
targets. Among marine cyanobacteria, the genus Lyngbya is consid-
ered to be the most prolific producer of natural products with over
200 compounds reported (Blunt and Munro, 2008).
Here we describe the isolation, structure elucidation and bio-
logical evaluation of three new analogues of largamides A–C (4–
6) (Plaza and Bewley, 2006), which we named tiglicamides A–C
(1–3), from a recollection of the Floridian marine cyanobacterium
Lyngbya confervoides that also afforded compounds 4–6 (Matthew
et al., 2009). Our previous chemical investigations of the same spe-
cies already yielded several structurally unrelated secondary
metabolites, including serine protease inhibitors, namely lyngby-
(Plaza and Bewley, 2006). Due to the structural homology to larga-
mides A–C (4–6), which are moderate inhibitors of porcine pancre-
atic elastase (Matthew et al., 2009), we tested tiglicamides A–C (1–
3) for activity against this enzyme.
Among the five main classes of proteolytic enzymes (aspartic,
serine, cysteine, metallo- and threonine), the serine proteases con-
stitute the most extensively studied enzyme family. Serine prote-
ases are known to regulate important biological processes, which
makes them attractive therapeutic targets (Ilies et al., 2002). Elas-
tase is a serine protease implicated in adult respiratory distress
syndrome (ARDS), rheumatoid arthritis, pulmonary emphysema,
cystic fibrosis and chronic bronchitis. Despite extensive research
efforts, there are relatively few elastase inhibitors in advanced
stages of development; however, one of them, sivelestat (ONO-
5046), has already been launched in Japan for the treatment of
acute lung injury associated with systemic inflammatory response
syndrome (SIRS) (Abbenante and Fairlie, 2005). The investigation
of natural products from marine cyanobacteria as a source of novel
serine protease inhibitors may eventually aid the development of
more promising therapeutic leads.
2. Results and discussion
The marine cyanobacterium L. confervoides collected near Ft.
Lauderdale (Florida, USA) was extracted with organic solvents
and the organic extract subjected to HP-20 chromatographic
* Corresponding author. Tel.: +1 (352) 273 7738; fax: +1 (352) 273 7741.
E-mail address: luesch@cop.ufl.edu (H. Luesch).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.09.010

S. Matthew et al. / Phytochemistry 70 (2009) 2058–2063
2059
Table 1
fractionation, and several HPLC purifications to yield compounds
1–3 as colorless, amorphous solids. The structures of 1–3 (Fig. 1)
were determined by a combination of NMR (1H, COSY, TOCSY, ROESY,
HSQC and HMBC) spectroscopic analyses and mass spectrometry.
Compound 1 was isolated as a colorless amorphous solid. A
pseudomolecular [M+Na]+ ion peak at m/z 928.4032 in the HR-
ESI/APCI-MS suggested a molecular formula of C45H59N7O13, which
was in agreement with the putative molecular composition based
on NMR spectroscopic data. A detailed 2D NMR analysis in DMF-d7
(Table 1) enabled identification of the five common amino acids
alanine (Ala), valine (Val), glutamic acid (Glu), tyrosine (Tyr), thre-
onine (Thr), the two modified amino acid residues homotyrosine
(Htyr) and 2-amino-2-butenoic acid (Abu) and a tiglic acid (Tig)
unit in 1, thus indicating a striking resemblance to largamides
AꢀC (4–6) (Fig 1). The presence of the tiglic acid unit is corrobo-
rated by the appearance of two deshielded methyl groups (dH
1.81 and 1.73) and one olefinic methine proton (dH-3 6.44, br q)
in the 1H NMR spectrum; the latter further showed correlations
to a quaternary carbon at d 132.4 (C-2, Tig) and a carbonyl carbon
at d 169.1 (C-1,Tig) in the HMBC spectrum. In addition, the olefinic
proton d 6.44 (H-3) showed a ROESY cross peak to a methyl reso-
nating at d 1.73 (H3-3) and lacked a correlation to the methyl at
d 1.81 (H3-2), indicating E geometry of the double bond and con-
firming a tigloyl group in 1 as in 4–6. The geometry of the Abu unit
1H and 13C NMR spectroscopic assignments for tiglicamide A (1) (600 MHz, DMF-d7).
Unit
Htyr
C/H No.
dH (J in Hz)
dC, mult.
HMBCa
1
2
171.4, qC
50.6, CH
4.61, br m
1, 3
3
4
5
6/10
7/9
8
2.12, m; 1.82, m
2.47, m (2H)
33.9, CH2
30.8, CH2
132.2, qC
129.8, CH
115.1, CH
156.3, qC
2, 3
2, 3, 5, 6/10
7.04, d (8.0)
6.71, d (8.0)
4
4, 5
OH
NH
1
2
3
9.31, s
7.72, d (9.4)
7/9
1 (Glu)
Glu
171.2, qC
52.9, CH
26.7, CH2
30.7, CH2
174.9, qC
4.55, br m
2.50, m; 2.19, m
2.56, m; 2.47, m
2, 4, 5
2, 3, 5
4
5
OH
NH
1
2
3
4
NH
1
2
3
NH
1
2
3
4
NH
1
2
Not observed
7.55, d (8.6)
1 (Abu)
Abu
163.8, qC
129.5, qC
128.7, CH
12.4, CH3
6.57, br q (6.8)
1.78, d (7.0)
10.21, s
1, 4
1, 2, 3
1 (Ala)
Ala
Thr
175.5, qC
50.3, CH
16.4, CH3
4.36, br q (6.7)
1.40, d (6.7)
8.86, br s
3
1, 2
1 (Thr)
170.2, qC
55.5, CH
73.1, CH
15.9, CH3
4.78, br m
5.39, br q
1.19, d (5.9)
7.89, d (8.2)
1
1
2, 3
1 (Tyr)
Tyr
172.4, qC
55.4, CH
37.8, CH2
4.77, br m
1, 1 (Val)
2, 4, 5/9
3
3.08, dd (ꢀ13.2, 3.9)
2.84, dd (ꢀ13.2, 9.6)
4
128.2, qC
130.5, CH
115.1, CH
156.7, qC
5/9
6/8
7
7.12, d (7.8)
6.75, d (7.8)
3, 6/8, 7
5/9, 7
OH
NH
1
2
3
4
5
NH
1
2
9.35, s
8.08, br d (7.5)
6/8, 7
Val
Tig
171.8, qC
58.8, CH
31.3, CH
19.3, CH3
17.9, CH3
4.30, br dd
2.04, m
0.76, d (6.3)
0.73, d (6.3)
7.29, d (8.8)
1, 3, 4, 5
1, 2, 4, 5
2, 3, 5
2, 3, 4
1 (Tig)
169.1, qC
132.4, qC
130.1, CH
13.4, CH3
12.2, CH3
3
4
5
6.44, br q (6.3)
1.73, br d (6.3)
1.81, br s
1, 4
2, 3
1, 2, 3
a
Protons showing long-range correlation with indicated carbon.
was deduced as Z based on a ROESY cross peak between the Abu
NH (dH 10.21) and Abu methyl group (dH 1.78). HMBC analysis sup-
ported by ROESY correlations unambiguously established the lin-
ear sequence of the amino acid units and tiglic acid moiety
(Table 1). The deshielded proton signal at dH 5.39 (Thr) was indic-
ative of a lactone functionality which arises from ester linkage of 1
from the carbonyl of Htyr and the hydroxyl group of Thr. The IR
spectrum of 1, displaying absorptions at 1722 and 1652 cmꢀ1 char-
acteristic of amide and ester functionalities, respectively, sup-
ported the proposed depsipeptide structure.
A [M+Na]+ peak at m/z 898.3935 in the HR-ESI/APCI-MS of 2 in
conjunction with 2D NMR data suggested a molecular formula of
C44H57N7O12 for compound 2. The 1H NMR spectrum indicated that
compound 2 is closely related to 1. 2D NMR analysis (1H, COSY,
HSQC, HMBC, ROESY) provided further evidence for the presence
of Ala, Val, Thr, Glu, and two aromatic amino acid residues, only
Fig. 1. Structures of tiglicamides A–C (1–3), largamides A–C (4–6) and their
corresponding methyl esters 4a–6a.

2060
S. Matthew et al. / Phytochemistry 70 (2009) 2058–2063
one of which was para-substituted (Tyr) in 2. The Htyr unit in 1
was replaced by a Phe residue in 2 (Table 2), which accounted
for the 30 mass unit difference (CH2O) compared with 1. The pres-
ence of the Phe residue in the cyclic core was confirmed based on
an HMBC correlation of the Phe NH proton to C-1 of the Glu residue
(Table 2).
two additional methyl singlets at dH 2.55/2.52 (1:1), which inte-
grated together for one methyl group (S–Me). A detailed 2D NMR
analysis (COSY, TOCSY, HSQC, HMBC, ROESY) indicated that com-
pound 3 contained only one aromatic amino acid residue and dif-
fers from 1 or 2 only by the presence of a methionine sulfoxide
(Met(O)) unit instead of Htyr or Phe, respectively (Table 2). The
Met(O) residue was verified by 2D NMR spectroscopic analysis,
where the two methyl singlets at dH 2.55/2.52 showed HSQC corre-
lations to the doubled signals at dC 38.7/37.6 and HMBC correla-
tions to dC 50.56/50.60. This doubling of signals in the ratio of
HR-ESI/APCI-MS analysis for compound 3 provided an [M+Na]+
peak at m/z 898.3591, suggesting
a molecular formula of
C40H57N7O13S. The 1H NMR of 3 displayed signals characteristic
for 1 and 2 with the exception of the fewer aromatic signals and
Table 2
NMR spectroscopic data for tiglicamides B (2) and C (3) in DMF-d7 (600 MHz).
Unit
C/H No.
Tiglicamide B (2)
dH (J in Hz)
Tiglicamide C (3)
dC, mult.
HMBCa
dH (J in Hz)
dC, mult.
HMBCa
Pheb/Met(O)c
1
2
3
170.9, qC
52.9, CH
37.6, CH2
170.7, qC
4.77, br m
1, 3
2, 4, 5/8
4.80, br m
2.31/2.26d, m
1.96/1.86d, m
2.81/2.75d, m
2.71/2.63d, m
–
50.0, CH
1
2, 4
3.29, dd (ꢀ13.6, 4.9)
24.85, 24.90d, CH2
2.76, dd (ꢀ13.6, 10.3)
4
138.5, qC
50.56, 50.60d, CH2
3
5/9
6/8
7
S-Me
NH
1
2
3
4
5
7.29, m
7.27, m
7.19, m
–
129.7, CH
128.3, CH
126.4, CH
–
3, 6/9, 7
3, 5/8, 7
5/8, 6/9
–
–
–
–
–
–
2.55, 2.52d, s
7.71, d (9.4)
38.7, 37.6d, CH3
4
7.84, d (9.4)
2, 1 (Glu)
1(Glu)
Glu
171.1, qC
52.4, CH
27.0, CH2
30.6, CH2
174.9, qC
171.7, qC
53.6, CH
26.9, CH2
30.9, CH2
175.0, qC
4.42, br m
2.44, m; 2.10, m
2.50, m; 2.41, m
3, 4
2, 4, 5
2, 3, 5
4.48, br m
2.47, m; 2.18, m
2.60, m; 2.48, m
3, 4
1, 2, 4, 5
2, 3, 5
OH
NH
1
2
3
Not observed
7.60, d (9.0)
Not observed
2, 1 (Abu)
1, 4
7.49, d (8.6), 7.48, d (8.6)e
1 (Abu)
1, 4
Abu
163.9, qC
130.9, qC
129.0, CH
164.9, qC
130.6, qC
129.3, CH
6.65, q (7.0)
6.54, qd (7.0, 1.3)/6.53
qd (7.0, 1.3)e
4
NH
1
2
3
NH
1
2
3
4
1.80, d (7.0)
10.23, s
12.6, CH3
1, 2, 3
1, 1 (Ala)
1.77, dq (7.1, 1.3)
10.25, s
12.6, CH3
1, 2, 3
1, 1 (Ala)
Ala
Thr
175.7, qC
50.2, CH
16.4, CH3
176.1, qC
50.6, CH
16.7, CH3
4.39, br m
1.42, d (6.8)
8.89, br s
1, 3
1, 2
1, 2, 3
4.34, br m
1, 3
1, 2
1, 3
1.40, d (7.0)
8.90, d (3.0)/8.89, d (3.2)e
170.4, qC
55.2, CH
72.9, CH
15.7, CH3
170.3, qC
55.5, CH
73.9, CH
16.1, CH3
4.77, br m
5.45, br q
1.18, d (5.0)
7.92, d (8.2)
1
4.80, br m
1
5.429, qd (6.4, 2.9)/5.427, qd (6.4, 2.9)e
1.20, d (6.4)
1, 1 (Met(O))
2, 3
2, 1 (Tyr)
2, 3
2, 1 (Tyr)
NH
1
2
7.95, d (8.1)
Tyr
172.7, qC
55.3, CH
37.7, CH2
172.8, qC
55.8, CH
38.1, CH2
4.77, br m
3
4.79, br m
1, 3, 1 (Val)
1, 2, 4, 5/9
3
3.08, dd (ꢀ13.7, 4.5)
2, 4, 5/9
3.06, dd (ꢀ14.0, 4.8)
2.83, dd (ꢀ13.7, 10.1)
2.83, dd (ꢀ14.0, 10.0)
4
128.5, qC
130.5, CH
115.2, CH
157.0, qC
128.4, qC
131.1, CH
115.6, CH
157.3, qC
5/9
6/8
7
7.13, d (7.8)
6.73, d (7.8)
3, 6/8, 7
5/9, 7
7.12, d (8.5)
6.71, d (8.5)
3, 6/8, 7
5/9, 7
OH
NH
1
2
3
4
5
NH
1
2
9.37, br s
8.09, d (8.1)
6/8, 7
1 (Val)
9.35, s
8.12, d (8.5)
6/8, 7
2, 1 (Val)
Val
Tig
171.8, qC
58.8, CH
31.4, CH
19.4, CH3
18.0, CH3
172.0, qC
59.2, CH
31.5, CH
19.7, CH3
18.3, CH3
4.28, br t (7.3)
2.02, m
0.74, d (6.8)
0.72, d (6.8)
7.26, d (7.3)
1, 3, 4, 5, 1 (Tig)
1, 2, 4, 5
2, 3, 5
2, 3, 4
2, 1 (Tig)
4.29, dd (6.8, 2.7)/4.28, dd (6.8, 2.7)e
2.02, m
0.75, d (6.8)
0.72, d (6.8)
7.27, d (8.6)
1, 3, 4, 5, 1 (Tig)
1, 2, 4, 5
2, 3, 5
2, 3, 4
1, 2, 1 (Tig)
169.2, qC
132.5, qC
130.3, CH
13.5, CH3
12.3, CH3
169.3, qC
132.7, qC
130.6, CH
13.7, CH3
12.4, CH3
3
4
5
6.43, br q (6.8)
1.70, br d (6.8)
1.79, br s
1, 4, 5
2, 3
1, 2, 3
6.43, qq (7.0, 1.5)
1.72, dq (7.0, 1.5)
1.80, br s
1, 4, 5
2, 3
1, 2, 3
a
b
c
Protons showing long-range correlation with indicated carbon.
Refers to compound 2.
Refers to compound 3.
Signal doubling because of diastereomers at chiral S*.
Signal doubling observed.
d
e

S. Matthew et al. / Phytochemistry 70 (2009) 2058–2063
2061
Table 3
4. Experimental
Inhibition of porcine pancreatic elastase.
IC50 in
l
Ma
IC50 in
l
Mb
IC50 in
l
Mc
4.1. General experimental procedures
1
2
3
2.14 0.19
6.99 0.74
7.28 0.95
4
5
6
1.41 0.28
0.53 0.19
1.15 0.46
4a
5a
6a
2.94 0.19
2.23 0.08
2.16 0.13
Optical rotation was measured on a Perkin–Elmer 341 polarim-
eter. UV spectra were recorded using a SpectraMax M5 (Molecular
Devices). 1H and 2D NMR spectra for 1 and 2 were recorded in
DMF-d7 on a Bruker 600 MHz spectrometer equipped with a 1-
mm high-temperature superconducting cryogenic probe and 3
was recorded in 5 mm cryogenic probe operating at 600 MHz and
150 MHz using residual solvent signals as the internal standard
(dH 8.02, dC 162.7). HSQC experiments were optimized for
1JCH = 145 Hz, and HMBC experiments were optimized for
nJC,H = 7 Hz for 1 and 2 and 10 Hz for 3. HRMS data were obtained
using an Agilent LC-TOF mass spectrometer equipped with an
APCI/ESI multimode ion source detector (UCR Mass Spectrometry
Facility, University of California at Riverside), and low resolution
mass spectra were obtained on a A3200 Q TRAP LC/MS/MS (hybrid
triple quadrupole linear ion trap mass spectrometer, Applied Bio-
systems, USA) with an electrospray ionization (ESI) interface oper-
ated in positive mode. HPLC-based compound isolation was
performed on a Shimadzu LC-20AT prominence LC with peak
detection by a Shimadzu SPD-M20A prominence diode array
detector.
a
b
c
n = 4.
n = 4 (taken from Matthew et al., 2009).
n = 3.
1:1 suggested the occurrence of epimeric R and S sulfoxides. The IR
absorption at 1037 cmꢀ1 also supported the sulfoxide assignment.
The sulfoxide is most likely an isolation artifact formed by oxida-
tion of the methionine-containing natural product (Matthew
et al., 2008; Gunasekera et al., 2008; Harrigan et al., 1999).
The absolute configuration of all amino acid units in compounds
1–3 was deduced by chiral HPLC of the acid hydrolysis products,
which indicated D configuration for glutamic acid and tyrosine
and L configuration for all other amino acids. However, none of
the chiral HPLC conditions employed were successful in resolving
the D/L-isomeric peaks for Phe. Hence the acid hydrolyzate of 2
was subjected to Marfey’s analysis (Fujii et al., 1997), establishing
L configuration for Phe in 2.
Compounds 1–3 were tested for serine protease-inhibitory
activity. They showed moderate activity against porcine pancreatic
elastase in vitro with IC50 values ranging from 2.14 to 7.28 lM
4.2. Marine cyanobacterial samples
(Table 3). While the described compounds are two to three orders
of magnitudes less potent against the same enzyme or other mam-
malian elastases than lyngbyastatins 4–7 (Matthew et al., 2007;
Taori et al., 2007) or ONO-5046 (Kawabata et al., 1991), prelimin-
ary data suggested some selectivity towards elastase. The activities
of two other serine proteases tested (chymotrypsin, trypsin) were
not compromised by compounds 1–3 at concentrations up to
Samples of L. confervoides were collected at approximately 15 m
depth from reefs near the Port Everglades Inlet, Fort Lauderdale,
Florida, USA (26°05.99020N, 80°05.01840W) in August 2004 and
May and August 2005. S. Golubic identified the cyanobacterium
(Paul et al., 2005) and its 16S rDNA gene sequence has been re-
ported (Paul et al., 2005; Sharp et al., 2009).
50 lM. These results are consistent with those previously reported
for their analogues (4–6) (Matthew et al., 2009). Since we also iso-
lated the corresponding largamide methyl esters 4a–6a (presum-
ably isolation artifacts), we were able to probe the effect of
methylation at that position. Compounds 4a–6a retained
low-micromolar inhibitory activity (Table 3), indicating that the
carboxylic acid residue is not a requisite element for elastase-
inhibitory activity (Matthew et al., 2009).
4.3. Extraction and isolation
The freeze-dried organisms collected through 2004–2005
(ꢁ2700 g dry weight) were extracted with EtOAc–MeOH (1:1) to
afford a crude extract (ꢁ400 g) which was suspended in H2O
(500 mL) then defatted with hexanes (500 mL ꢂ 3; ꢁ2 g). The con-
centrated aqueous layer enriched with salt was further partitioned
between n-BuOH (250 mL ꢂ 3) and H2O. The combined n-BuOH ex-
tract (12 g) was applied on a Diaion HP-20 (Supelco) resin (120 g),
and subsequently fractionated with H2O and increasing concentra-
tions of MeOH, and then with MeCN and finally with CH2Cl2 to
yield 8 fractions [Fr. 1: H2O (100%, 2 L, ꢁ6.8 g); Fr. 2: H2O:MeOH
(80:20, 1 L, 854 mg); Fr. 3: H2O:MeOH (50:50, 1 L, 272 mg); Fr. 4:
H2O:MeOH (50:50–25:75, 1 L, 400 mg); Fr. 5: H2O:MeOH (25:75–
0:100, 1 L, 430 mg); Fr. 6: MeOH (100%, 1 L, 950 mg); Fr. 7: MeCN
(100%, 1 L, 490 mg), Fr. 8: CH2Cl2 (100%, 1 L, 457 mg)]. Fractions 5
and 6 were subjected to reversed-phase preparative HPLC (Phe-
3. Conclusion
The L. confervoides that yielded tiglicamides A–C (1–3) is a par-
ticularly prolific source of secondary metabolites (Matthew et al.,
2007, 2008, 2009; Taori et al., 2007), with already 13 previously re-
ported structures belonging to six different structural families
(Sharp et al., 2009). The similarity to the largamides A–C (4–6)
and, specifically, the variability of the last amino acid position
(N ? C) suggests relaxed substrate specificity of the corresponding
putative biosynthetic NRPS enzyme, allowing the incorporation of
at least six different amino acids, viz. Htyr (1), Phe (2), Met (3), Leu
(4), 2-amino-5-(40-hydroxy-phenyl)pentanoic acid (Ahppa) (5),
and 2-amino-5-(40-hydroxy-phenyl)hexanoic acid (Ahpha) (6) at
that position. Largamides A–C (4–6) were major metabolites in this
cyanobacterium, while tiglicamides A–C (1–3) were only minor
metabolites. It is unclear if the differing yields are reflective of
the relative efficiencies of substrate activation or due to genetic
heterogeneity of the cyanobacterial samples and consequently bio-
synthetic enzymes, although the 16S rDNA sequence was identical
for at least three distinct collections at different times (Sharp et al.,
2009). Detailed genetic studies of this intriguing cyanobacterium
will provide novel insights into the biosynthesis of compounds
1–6 and other co-produced L. confervoides metabolites.
nomenex Luna-C18 10
l, 100 ꢂ 21.20 mm, 5.0 mL/min; PDA detec-
tion at 200–400 nm) using a MeOH–0.05% aqueous TFA linear
gradient (40–100% over 30 min and then MeOH for 15 min). The
largamide- and tiglicamide-rich fractions eluting between tR 15–
25 min were collected and subjected to repeated semipreparative
reversed-phase HPLC (Phenomenex Synergi 4u Hydro-RP,
250 ꢂ 10 mm, 2.0 mL/min; PDA detection at 200–400 nm) using
two sequential linear gradients of MeOH in 0.05% aqueous TFA
(60–90% over 25 min, 90–100% over 10 min) to give semi-pure
compounds 1–6 eluting between tR 16–21 min. The final purifica-
tion of the compounds was achieved by means of a Phenomenex
Luna Phenyl-hexyl column 10 ꢂ 250 mm, using the same HPLC
conditions as described above to afford tiglicamide A (1), tR
16.0 min (1.2 mg), tiglicamide B (2), tR 21.8 min (0.8 mg), and

2062
S. Matthew et al. / Phytochemistry 70 (2009) 2058–2063
tiglicamide C (3), tR 13.1 min (0.4 mg) together with known com-
pounds largamide A (4), tR 20.3 min (8.0 mg), largamide B (5), tR
18.2 min (8.0 mg), largamide C (6), tR 20.0 min (12.0 mg) and
methyl esters of largamides AꢀC (4aꢀ6a) [4a, tR 23.3 min
(2.5 mg); 5a, tR 21.1 min (6.6 mg); 6a, tR 22.8 min (3.7 mg)]. These
methyl esters were presumably isolation artifacts generated from
4–6, respectively.
APCI-MS m/z [M+Na]+ 898.3935 (calcd for C44H57N7O12Na,
898.3963).
4.8. Tiglicamide C (3)
Colorless, amorphous solid; ½a D20
ꢀ56 (c 0.04, MeOH); UV
ꢃ
(MeOH) kmax (log
e) 220 (4.27), 280 (3.18) nm; IR (film) mmax
2945 br, 1730, 1670, 1559, 1458, 1055, 1037 cm–1; for 1H NMR,
13C NMR, and HMBC spectroscopic data, see Table 2; HR-ESI/
APCI-MS m/z [M+Na]+ 898.3591 (calcd for C40H57N7O13SNa,
898.3633).
4.4. Absolute configuration of amino acid units in compounds 1–3 by
chiral HPLC analysis
Compounds 1, 2, and 3 (0.1 mg each) were treated with 6 N HCl
(0.3 mL) and heated at 116 °C for 24 h. A portion of each hydroly-
zate was evaporated to dryness and the residues were resuspended
4.9. Largamide A methyl ester (4a)
in H2O (100
nomenex, Chirex 3126 N,S-dioctyl-(D)-penicillamine, 250 mm ꢂ
4.60 mm, m; solvent, 2 mM CuSO4–MeCN; UV detection
lL) and then subjected to chiral HPLC analysis (Phe-
Colorless, amorphous solid; ½a D20
ꢀ53 (c 0.03, MeOH); UV
ꢃ
(MeOH) kmax (log e
) 220 (4.30), 280 (3.24) nm; for 1H NMR spectro-
5
l
scopic data, see Table S1; HR-ESI/APCI-MS m/z [M+Na]+ 878.4277
254 nm; flow rate 1.0 mL/min). The absolute configuration of the
common amino acid units in 1–3 (tR, min) were established as L-
Ala (10.4), L-Thr (11.2) (solvent 2 mM CuSO4), L-Val (17.9), D-Glu
(58.0), and D-Tyr (61.0) (solvent 2 mM CuSO4–MeCN, 95:5) by
comparison with those of authentic standards. The retention times
for the standards were as follows (tR, min) D-Ala (14.6), L-allo-Thr
(15.5), D-Thr (13.5), D-allo-Thr (17.4) (solvent 2 mM CuSO4), D-Val
(22.8), L-Glu (53.5), and L-Tyr (54.0) (solvent 2 mM CuSO4–MeCN,
95:5). Acid hydrolyzates of 1–3 also yielded peaks for L-Htyr
(39.5 min; solvent 2 mM CuSO4–MeCN, 90:10), L/D-Phe (49.5/
49.5 min; solvent 2 mM CuSO4–MeCN, 85:15), and L-Met(O)
(13.6, 14.7; solvent 2 mM CuSO4), indicating the presence of L-
Htyr, L/D-Phe, and L-Met(O) units in 1, 2, and 3, respectively. The
standard for D-Htyr eluted at tR 58.5 min (solvent 2 mM CuSO4–
MeCN, 90:10) and for D-Met(O) at tR 16.5, 18.5 min (solvent
2 mM CuSO4). However the peaks for L/D-isomers of Phe did not
resolve under the above mentioned chiral HPLC conditions; hence
the hydrolyzate of 3 was further subjected to Marfey’s analysis.
(calcd for C42H61N7NaO12, 878.4276).
4.10. Largamide B methyl ester (5a)
Colorless, amorphous solid; ½a D20
ꢀ68 (c 0.17, MeOH); UV
ꢃ
(MeOH) kmax (log e
) 220 (4.29), 280 (3.49) nm; for 1H NMR spectro-
scopic data, see Table S1; HR-ESI/APCI-MS m/z [M+Na]+ 956.4353
(calcd for C47H63N7NaO13, 956.4382).
4.11. Largamide C methyl ester (6a)
Colorless, amorphous solid; ½a D20
ꢀ60 (c 0.10, MeOH); UV
ꢃ
(MeOH) kmax (loge
) 220 (4.43), 280 (3.45) nm; for 1H NMR spectro-
scopic data, see Table S1; HR-ESI/APCI-MS m/z [M+Na]+ 970.4533
(calcd for C48H65N7NaO13, 970.4538).
4.12. Serine protease inhibition assays
4.5. Absolute configuration of Phe in 2 by Marfey’s analysis
Compounds 1–3 were tested for serine protease-inhibitory
activity against porcine pancreatic elastase,
a-chymotrypsin from
A portion of the acid hydrolyzate of compound 2 was treated
with 1 M NaHCO3 (5 lL) and 1% v/v solution of 1-fluoro-2,4-dini-
trophenyl-5-L-leucinamide (L-FDLA) in acetone and heated at
40 °C for 60 min. The reaction mixture was then cooled, acidified
bovine pancreas, or trypsin from porcine pancreas, in the presence
of chromogenic substrates (N-succinyl-Ala-Ala-Ala-p-nitroanilide
for elastase, N-succinyl-Gly-Gly-Phe-p-nitroanilide for chymotryp-
sin, N-a-benzoyl-DL-arginine 4-nitroanilide hydrochloride for
with 2 N HCl (10
250 L). Aliquots were subjected to reversed-phase HPLC (Alltech
Alltima HP C18 HL 5
lL), dried and dissolved in H2O–MeCN (1:1,
trypsin). Activity was determined by measuring the increase in
absorbance at 405 nm over 30 min in a microplate reader using
phenylmethylsulfonyl fluoride (PMSF) and lyngbyastatin 4 as posi-
tive controls [Matthew et al., 2007]. All assays were performed in
quadruplicate (1–3) or triplicate (4a–6a) and IC50 values were ex-
pressed as mean S.D.
l
l
, 250 ꢂ 4.6 mm, PDA detection at 200–
500 nm; flow rate 0.8 mL/min) using a linear gradient of MeOH
in 0.1% (v/v) aqueous TFA (50–100% MeOH over 40 min). The
retention times (tR, min) of the derivatized amino acids in the
hydrolyzate of compound
2 matched with those of L-Phe
(25.8 min) and not of D-Phe (33.0 min).
Acknowledgments
4.6. Tiglicamide A (1)
This work was funded by NIGMS grant P41GM086210. We
thank L. Fisher and K. Banks (Broward County Environmental
Protection Department) and C. Ross, A. Capper, S. Reed and
R. Ritson-Williams (Smithsonian Marine Station) for assistance
with collections and C. Ross for assistance with extractions of the
cyanobacterium. The 600 MHz 1-mm triple-resonance HTS
cryogenic probe was developed through collaboration between
the University of Florida, NHMFL, and Bruker Biospin. This is con-
tribution #794 from the Smithsonian Marine Station at Fort Pierce.
Colorless, amorphous solid; ½a D20
ꢀ50 (c 0.07, MeOH); UV
ꢃ
(MeOH) kmax (log
e) 220 (4.28), 280 (3.34) nm; IR (film) mmax
3330 br, 2926, 1722, 1666, 1615 br, 1430, 1124 cm-1; for 1H
NMR, 13C NMR, and HMBC spectroscopic data, see Table 1; HR-
ESI/APCI-MS m/z [M+Na]+ 928.4032 (calcd for C45H59N7O13Na,
928.4069).
4.7. Tiglicamide B (2)
Colorless, amorphous solid; ½a D20
ꢃ
ꢀ45 (c 0.04, MeOH); UV
Appendix A. Supplementary data
(MeOH) kmax (log
e) 220 (4.26), 280 (3.15) nm; IR (film) mmax
3330 br, 2930, 1722, 1652, 1610, 1514, 1444 cm–1; for 1H NMR,
13C NMR, and HMBC spectroscopic data, see Table 2; HR-ESI/
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2009.09.010.

S. Matthew et al. / Phytochemistry 70 (2009) 2058–2063
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