Pompanopeptins A and B, new cyclic peptides from the marine cyanobacterium Lyngbya confervoides

Article abstract of DOI:10.1016/j.tet.2008.02.035

A new 3-amino-6-hydroxy-2-piperidone (Ahp) containing peptolide, pompanopeptin A (1), and a novel N-methyl-2-amino-6-(4′-hydroxyphenyl)hexanoic acid (N-Me-Ahpha) containing cyclic pentapeptide connected with a sixth amino acid residue via a rare ureido linkage, pompanopeptin B (2), were isolated from the marine cyanobacterium Lyngbya confervoides collected from the southeastern coast of Florida. Their planar structures were determined by a combination of NMR spectroscopic analysis and mass spectrometry. The absolute configurations were established using advanced Marfey's method and chiral HPLC analysis of the chemical degradation products. Compound 1 selectively inhibited trypsin over elastase and chymotrypsin, with an IC₅₀ value of 2.4 μM; selectivity is conferred by an arginine residue in the cyclic core.

Full text of DOI:10.1016/j.tet.2008.02.035

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 4081e4089
www.elsevier.com/locate/tet
Pompanopeptins A and B, new cyclic peptides from the marine
cyanobacterium Lyngbya confervoides
Susan Matthew a, Cliff Ross b,y, Valerie J. Paul b, Hendrik Luesch a,
*
a Department of Medicinal Chemistry, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, United States
b Smithsonian Marine Station, 701 Seaway Drive, Fort Pierce, FL 34949, United States
Received 3 January 2008; received in revised form 10 February 2008; accepted 11 February 2008
Available online 15 February 2008
Abstract
A new 3-amino-6-hydroxy-2-piperidone (Ahp) containing peptolide, pompanopeptin A (1), and a novel N-methyl-2-amino-6-(40-hydroxy-
phenyl)hexanoic acid (N-Me-Ahpha) containing cyclic pentapeptide connected with a sixth amino acid residue via a rare ureido linkage, pom-
panopeptin B (2), were isolated from the marine cyanobacterium Lyngbya confervoides collected from the southeastern coast of Florida. Their
planar structures were determined by a combination of NMR spectroscopic analysis and mass spectrometry. The absolute configurations were
established using advanced Marfey’s method and chiral HPLC analysis of the chemical degradation products. Compound 1 selectively inhibited
trypsin over elastase and chymotrypsin, with an IC50 value of 2.4 mM; selectivity is conferred by an arginine residue in the cyclic core.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Natural product; Cyanobacteria; Lyngbya confervoides; Trypsin inhibitor
1. Introduction
(Ahp) moiety. The same extract yielded another new cyclic
peptide (2) with a rare ureido unit, which has structural resem-
blance to several marine derived compounds, mozamide,6
brunsvicamides,7 and metabolites from freshwater cyano-
bacteria, anabaenopeptins.8 Compound 2 is structurally dis-
tinct from other natural products due to the occurrence of a
novel N-methyl-2-amino-6-(40-hydroxyphenyl)hexanoic acid
(N-Me-Ahpha) unit. Here we report the isolation and structure
elucidation of 1 and 2, which we named as pompanopeptins
A (1) and B (2) due to the proximity of the collection sites
to Pompano Beach, FL. Compound 1 was evaluated for its
biological activity against several serine endopeptidases and
demonstrated selective in vitro trypsin inhibition when com-
pared to elastase and chymotrypsin inhibitory activities. Tryp-
sin is a proteolytic enzyme that catalyzes the cleavage of
peptide bonds on the carboxyl side of either arginine or lysine.
The imbalance of trypsin activation within the pancreatic
acinar cells presumably leads to the development of acute
pancreatitis.9 Additionally, an increase in trypsin activity has
been associated with conditions like inflammation and
angiogenesis.10
Marine cyanobacteria have emerged as an exceptionally
rich source of structurally distinct and biologically active me-
tabolites, which include cyclic peptides and depsipeptides.1
The present study is part of our exploration to discover novel
and biologically active metabolites from marine cyanobacteria
from coastal Florida. Recent investigation of the marine cya-
nobacterium Lyngbya confervoides collected in Florida led
to the isolation of new analogues of dolastatin 13 with elastase
and chymotrypsin inhibitory activities, and lyngbyastatins
4e6.2,3 Re-investigation of a large collection of this cyanobac-
terium has now resulted in the isolation of a new trypsin inhib-
itor (1), which has structural resemblance to freshwater
cyanobacterial isolates, micropeptins from Microcystis sp.,4,5
all of which contain a 3-amino-6-hydroxy-2-piperidone
* Corresponding author. Tel.: þ1 352 273 7738; fax: þ1 352 273 7741.
E-mail address: luesch@cop.ufl.edu (H. Luesch).
y
Present address: Department of Biology, University of North Florida, 1
UNF Drive, Jacksonville, FL 32224, United States.
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.02.035

4082
S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
N,O-diMe-Br-Tyr
Ba
Thr Met(O)
O
Val
concentrated in vacuo, defatted with hexanes and partitioned
between n-BuOH and H2O. Fractionation of the n-BuOH sol-
uble material using HP-20 resin followed by several purifica-
tion steps by using reversed-phase HPLC resulted in the
isolation of pompanopeptins A (1) and B (2).
Br
O
O
H
O
O
N
N
N
H
H
O
O
N
N
O
HN
O
H
N
Pompanopeptin A (1) was isolated as a colorless, amor-
phous solid with a molecular formula of C46H73BrN10O12S
deduced on the basis of HRAPCI/ESIMS [m/z (MþH)þ
1069.4400; calcd for C46H7479BrN10O12S, 1069.4392]. The
isotopic distribution of [MþH]þ ions in the ratio of 1:1 at
m/z 1069 and 1071 revealed the presence of one bromine
atom in the molecule. The structure of 1 was deduced through
1H and 13C NMR in combination with HSQC, HMBC, COSY,
TOCSY, and ROESY experiments. NMR analysis in DMSO-d6
established the presence of valine (Val), N,N-disubstituted-
isoleucine (Ile), threonine (Thr), arginine (Arg), butanoic
acid (Ba), and three modified amino acids (Table 1). In agree-
ment with literature values, one unusual spin system was
attributed to an Ahp unit.2,3 The second modified unit
consisted of a methine (dH 4.57) and two methylenes (dH
2.03/1.92 and dH 2.79/2.69) based on COSY and TOCSY anal-
ysis, and the corresponding methylene carbons both showed
HMBC correlations to a methyl singlet (Table 1). This data
is in agreement with a methionine sulfoxide [Met(O)] residue
as in somamide A11 and symplostatin 2.12 The doubling of sig-
nals in the ratio of 1:1 for the Met(O) unit in 1 suggested the
occurrence of a mixture of R and S sulfoxide diastereomers as
reported earlier11,12 (Table 1). The sulfoxide is presumably an
artifact formed by oxidation of the methionine-containing nat-
ural product during isolation.12 The last unit was an aromatic
S
H
O
Ile
O
HO
HN
NH2
NH
Ahp
Arg
1
OH
Htyr-1
N
N-Me-Ahpha
HO
Val
NH
O
O
NH
O
HN
O
O
O
O
HN
Htyr-2
HO
N
H
N
H
OH
Ile
Lys
2
2. Results and discussion
1
amino acid; the H NMR spectrum (Table 1) showed signals
L. confervoides was collected during the summer of 2005
near Pompano Beach and Ft. Lauderdale, Florida. The
freeze-dried sample was lyophilized and extracted with
EtOAceMeOH (1:1). The combined fractions were
characteristic of a 1,3,4-trisubstituted aryl ring system [dH
7.40, 1H, d (1.5); dH 7.01, 1H, d (8.5); dH 7.18, 1H, dd (8.5,
1.5)]. 13C NMR and HMBC data revealed that a Br substituent
Table 1
NMR spectral data for pompanopeptin A (1) in DMSO-d6 (500 MHz)
Unit
C/H
no.
dH
dC, multa
COSY
HMBCb
ROESYc
H-3, NH
(J in Hz)
Val
1
2
172.3, qC
56.1, CH
4.70, dd (9.0, 5.5)
H-3, NH
1, 4, 5, 1
(N,O-diMe-Br-Tyr)
4, 5
3
2.05, m
30.7, CH2
19.3, CH3
17.5, CH3
H-2, H3-4, H3-5
H-2, H3-4, H3-5
H-3, H3-5
4
0.86, d (7.0)
0.74, d (7.0)
7.63, d (9.0)
H-3
H-3
H-2
2, 3, 5
2, 3, 4
5
H-3, H3-4, H-3 (Thr)
H-2, H-2 (N,O-diMe-Br-Tyr)
NH
1
N,O-diMe-Br-Tyr
169.3, qC
60.4, CH
2
5.05, dd (11.5, 2.5)
H-3a, H-3b
H-3a, H-3b, N-Me, NH
(Val), H-2 (Ile)
3a
3b
3.21, dd (ꢀ13.5, 11.5)
2.78, dd (ꢀ13.5, 2.5)
32.9, CH2
H-2, H-3b
H-2, H-3a
H-2, H-3b, H-5, H-9
H-2, H-3a, H-5, H-9
4
131.2, qC
133.5, CH
111.1, qC
154.5, qC
112.9, CH
130.1, CH
30.2, CH3
56.3, CH3
169.6, qC
5
7.40, d (1.5)
4, 6, 7, 9
H-3a, H-3b, H-9
6
7
8
7.01, d (8.5)
7.18, dd (8.5, 1.5)
2.73, s
H-9
H-8
4, 6, 7, 9
5, 8
7-OMe
9
H-3a, H-3b, H-5
H-2, 6-OH (Ahp)
H-8
N-Me
7-OMe
1
1, 2, 1 (Ile)
7
3.76, s
Ile
(continued on next page)

S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
4083
Table 1 (continued)
Unit
C/H
no.
dH
dC, multa
COSY
H-3
HMBCb
ROESYc
(J in Hz)
2
4.37, d (10.5)
54.1, CH
1, 3, 6, 6 (Ahp)
H-3, H-2 (N,O-diMe-Br-Tyr),
H-6 (Ahp)
3
1.79, m
1.09, m
32.9, CH
23.7, CH2
H-2, H-4a, H-4b, H3-6
H-3, H-4b/H3-5
H-3, H-4a, H3-5
H-4a, H-4b
H-2, H-6
H-6
4a
4b
5
0.63, m
0.61, t (6.6)
ꢀ0.16, d (6.0)
3, 4
3, 4
H-6
H-6
10.2, CH3
13.6, CH3
169.1, qC
48.9, CH
21.7, CH2
6
H-3
2, 3, 4
H-3, H-4a, H-4b, H3-5
Ahp
2
3
4.44, m
2.57, m
1.74, m
1.76, m
1.73, m
4.92, br s
H-4a, H-4b, NH
H-4b, H-5b, NH
H-4b, 6-OH, NH
H-3, H-4a
4a
4b
5a
5b
6
H-3, H-4b, H-5a/5b
H-3, H-4a, H-5a/5b
H-4a, H-4b, H-6, 6-OH
H-4a, H-4b, H-6, 6-OH
H-5a/5b, 6-OH
29.6, CH2
73.9, CH
H-5b, H-6, 6-OH
H-3, H-5a, H-6, 6-OH
H-4a, H-5a/H-5b, 6-OH,
H-2 (Ile)
6-OH
6.13, br s
H-5a/5b, H-6
H-3
H-5a/H-5b, H-6, N-Me
(N,O-diMe-Br-Tyr)
H-3, H-4a, 2-NH (Arg)
NH
1
7.35, d (8.5)
2, 1 (Arg)
Arg
170.0, qC
51.7, CH
26.3, CH2
2
3a
4.29, br
2.01, m
1.42, m
H-3a, H-3b, 2-NH
H-2, H-3b, H2-4
H-2, H-3a, H2-4
H-3a, H-3b, H2-5
H2-4, 5-NH
H-2
H-3b/H2-4, 2-NH
H2-4
H-2, H2-4
3b
4
1.44, m (2H)
3.07, m (2H)
8.50, d (9.5)
7.49, t (5.5)
7.66, brd
e
25.0, CH2
40.1, CH2
H-2, H-3a/3b, H2-5
H2-4, 5-NH
5
2-NH
H-2, NH (Ahp), H-2 (Thr)
H2-5, 6-NH2
5-NH
5-NH
6-NH2
H2-5
6-NH
C]N
1
156.6, qC
169.2, qC
55.2, CH
71.8, CH
17.7, CH3
Thr
2
4.59, br
NH
1, 3, 1 (Met (O))
4, 1 (Val)
2, 3
H-3, NH, 2-NH (Arg)
H-2, H3-4, H3-5 (Val)
3
5.52, br q (6.0)
1.22, d (6.0)
8.06, d (9.0)
H3-4
H-3
H-2
4
H-3
H-2
NH
1
1 (Met (O))
Met(O)
171.8, qC
51.2, CH
25.0, CH2
2
4.57, br
2.03, m
H-3a, H-3b, NH
H-3a/3b, NH
H-2, H-4a/4b
H-2, H-4a/4b
H-3a/3b, S-Me
H-3a/3b, S-Me
H-4a/4b
3a
3b
4a
4b
S-Me
NH
1
H-2, H-3b, H-4a, H-4b
H-2, H-3a, H-4a, H-4b
H-3a, H-3b, H-4b
H-3a, H-3b, H-4a
1.92, m
2.79, m
f
48.9, 48.8, CH2
2.69, m
2.55, 2.54,f s
8.09, d (7.5)
37.9, 38.0,f CH3
3, 4
1 (Ba)
H-2
H-2, H2-2 (Ba)
Ba
172.3. qC
37.0, CH2
18.6, CH2
13.7, CH3
2
2.13, t (7.0) (2H)
1.52, hex (7.0) (2H)
0.86, t (7.0)
H2-3
H2-2, H3-4
H2-3
1, 3, 4
1, 2, 4
1, 2, 3
H2-3, H3-4, NH (Met (O))
3
4
a
Deduced from 13C NMR (125 MHz), HSQC, and HMBC.
Protons showing long-range correlation with indicated carbon.
Refers to protons within the same unit unless indicated otherwise.
Tentative assignment. No correlation observed in 2D NMR spectra.
Not observed.
b
c
d
e
f
Indicates diastereomers at chiral S*.
was located in the meta position (dC-6 111.1) and that the para
carbon was oxygenated (dC-7 154.5). O-Methylation of the
aromatic ring accounted for the low-field chemical shift of
one methyl singlet (dH 3.76) and the observed HMBC to the
carbon resonating at 154.5. Further COSYand HMBC analysis
verified that the moiety is tyrosine-derived and that the nitro-
gen was also methylated (Table 1). These chemical shifts were
consistent with a N,O-diMe-30-Br-Tyr unit data reported for
several largamides having a similar moiety.13
A strong ROESY cross-peak was observed between
a-protons of N,O-diMe-Br-tyrosine and isoleucine, suggesting
a cis-amide bond. The 1H NMR spectrum of 1 also displayed a
significantly shielded methyl doublet (H3-6, Ile, dH ꢀ0.16)
thereby revealing the spatial proximity of Ile to the shielding re-
gion of the aromatic portion of the N,O-diMe-Br-Tyr unit. The
HMBC analysis supported by ROESY correlations enabled us
to sequence two partial units as Val-(N,O-diMe-Br-Tyr)-Ile-
Ahp-Arg and Thr-Met(O)-Ba. The connectivity between Thr

4084
S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
and Arg was ascertained based on ROESY cross-peaks between
the a-proton of Thr and the NH of Arg (dH 4.59, Thr H-2 and dH
8.50, Arg NH), characteristic for a trans-amide linkage. The
ester linkage between Thr and Val was inferred by the HMBC
correlation from the deshielded b-proton of Thr (dH 5.52) to
the quaternary carbonyl (dC 172.3) of Val, thus concluding
that 1 is a cyclic depsipeptide. The proposed depsipeptide struc-
ture shown for 1 accounts for the molecular formula and is also
consistent with the IR spectrum, exhibiting bands at 1736 and
1656 cmꢀ1 rationalized by carbonyl stretch vibrations of ester
and amide functionalities, respectively. The IR spectrum also
supports the presence of a methionine sulfoxide moiety by dis-
playing bands at 1056 and 1022 cmꢀ1, a region characteristic
for sulfoxide stretch vibrations.
The absolute configuration of C-3 (Ahp) in 1 was deduced as
S by chiral HPLC analysis, after detecting a peak for the liber-
ated L-glutamic acid (Glu) upon oxidation with CrO3 oxida-
tion2,3 and subsequent acid hydrolysis. Further analysis of the
ROESY correlations and 1He1H coupling constant data (Table
1) and comparison with related Ahp-containing metabolites
suggested that the relative configuration of the two chiral centers
is the same and thus the absolute configuration of the Ahp unit is
3S,6R.2,3 Absolute configurations of all other amino acid units in
1 except N,O-diMe-Br-Tyr were established as L by chiral HPLC
analysis of the acid hydrolyzate. To deduce the configuration of
the N,O-diMe-Br-Tyr residue, an authentic standard of the L-iso-
mer was synthesized from Boc-OMe-L-Tyr followed by a series
of reaction steps involving N-methylation,14 N-deprotection,
and bromination (Scheme 1).15 The amino acid standard and
the hydrolyzate of 1 were subsequently derivatized with
L-FDLA and DL-FDLA for advanced Marfey’s analysis with
the aid of LCeMS, revealing L-configuration of this residue as
well.16,17
(Lys), two homotyrosine (Htyr) units, and a novel N-Me-2-
amino-6-(40-hydroxyphenyl)hexanoic acid (N-Me-Ahpha).
The occurrence of this unusual amino acid N-Me-Ahpha was
hitherto unknown, whereas its demethylated form has been
encountered once before, in largamide C.13 HMBC analysis
established the linear sequence of five amino acids as
(N-Me-Ahpha)-Htyr2-Lys-Val-Htyr1, all of which were linked
by trans-amide bonds based on ROESY correlations between
the signals for a-protons and NH protons in adjacent residues.
ROESY cross-peaks between the signals for H-2 of N-Me-
Ahpha and H-2 of Htyr-1 established the cis-amide linkage
between these two residues and consequently the cyclic
pentapeptide core in 2 as cyclo[Val-Htyr1-(N-Me-Ahpha)-
Htyr2-Lys]. The remaining NMR data were in accordance
with the literature reports for previously known compounds
having similar cyclic cores connected to the side chain through
a rare ureido carbonyl linkage.6e8 In compound 2 an HMBC
correlation observed from both the protons at dH 4.18 (Lys
H-2) and dH 4.28 (Ile H-2) to an unassigned sp2 carbon signal
(dC 158.6) suggested that NH (Ile) was joined to the 2-NH
(Lys) through an ureido moiety. The connection between Lys
and Ile was further ascertained by ROESY correlations be-
tween 2-NH (Lys, dH 6.79) and H-2 (Ile, dH 4.28), H-2 (Lys,
dH 4.18) and NH (Ile, dH 6.30), and 2-NH (Lys, dH 6.79)
and NH (Ile, dH 6.30). To account for the molecular formula
1
requirements and the broad singlet in the H NMR spectrum
of 2 at dH 12.8, the side chain terminated with an Ile carbox-
ylic acid functionality. The NMR assignments reported for the
related anabaenopeptins8 are consistent with the NMR assign-
ments given in Table 2 for 2.
Chiral HPLC analysis of the acid hydrolyzate of 2 revealed
that both Htyr units and Val have L configurations. Addition-
ally, according to chiral HPLC analysis, ozonolysis of 2 with
oxidative workup followed by acid hydrolysis generated
N-Me-(S)-2-aminopimelic acid and L-Glu in the hydrolyzate,
confirming the presence of N-Me-L-Ahpha and L-Htyr, re-
spectively, in 2. N-Me-2-aminopimelic acid standards for the
analysis were synthesized by selective N-methylation14 as dis-
cussed in Section 3.5.1 using Boc-(ꢁ)-2-aminopimelic acid as
starting material (Scheme 2). The configuration of Lys re-
mained uncertain due to its early elution profile during chiral
HPLC; hence the hydrolyzate was further subjected to
Marfey’s analysis, thereby confirming the presence of a D-isomer.
However, as reported for related compounds,6,7,18 the terminal
amino acid (Ile) could not be detected under normal acid
hydrolysis conditions. Hydrazinolysis19 of 2, for the character-
ization of carboxyl-terminal amino acids, liberated solely Ile
residue, which was identified as L-Ile by chiral HPLC analysis.
The cyclic core of pompanopeptin A (1) bears similarities
to micropeptins 478-A and B4 where Ile was replaced by
Val and N-Me-30-Cl-Tyr by N,O-diMe-30-Br-Tyr. Other closely
related cyclodepsipeptides, such as micropeptins 90,20
SD999,5 and E1992,21 containing an Arg unit in the cyclic
core have been reported to inhibit trypsin with IC50 values
of 2.0e4.2 mg/mL. Based on the co-crystal structure of
A90720A22 with trypsin, the Arg residue confers selectivity
for trypsin over other proteases. Expectedly, pompanopeptin
OMe
OMe
OMe
Br
(i) CH3I/NaH/THF, 24 h
(ii) TFA/CH2Cl2, 3 h
(iii) Br2
Boc
N
MeHN
MeHN
R
COOH
3a
COOH
COOH
3d
3c
3a R = H
3b R = Me
(i)
Scheme 1. Synthesis of standard N,O-diMe-30-Br-L-Tyr (3d).
Pompanopeptin B (2) was isolated as a colorless, amor-
phous solid with a molecular formula of C51H71N7O11 de-
duced on the basis of HRAPCI/ESIMS [m/z (MþH)þ
958.5319; calcd for C51H72N7O11, 958.5290]. 1H NMR analy-
sis of 2 in DMF-d7 suggested the presence of three
para-substituted phenolic residues, which together with six
deshielded NH protons and one N-Me singlet were indicative
of a peptide framework with three aromatic amino acid units.
A detailed analysis of the structure deduced through 1D (1H,
13C NMR, and APT) and 2D (HMQC, HMBC, COSY,
TOCSY, and ROESY) experiments revealed the presence of
six amino acid residues: isoleucine (Ile), valine (Val), lysine

S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
4085
Table 2
NMR spectral data for pompanopeptin B (2) in DMF-d7 (500 MHz)
Unit
Val
C/H no.
dH (J in Hz)
dC, multa
COSY
HMBCb
1
174.2, qC
59.8, CH
30.9, CH
2
4.03, dd (8.0, 6.5)
1.94, m
H-3, NH
H-2, H3-4, H3-5
1, 3, 4, 5
1, 2, 4, 5
2, 3, 5
3
4
1.02, d (6.8)
0.94, d (6.8)
7.22, d (6.5)
19.82, CH3
19.79, CH3
H-3
H-3
H-2
5
2, 3, 4
2, 3, 1 (Lys)
NH
1
Htyr-1
173.3, qC
50.0, CH
34.6, CH2
2
4.90, ddd (10, 5.1, 3.2)
2.05, m
1.83, m
H-3a, H-3b, NH
3
4
3a
3b
4a
4b
5
H-2, H-3b, H-4a, H-4b
H-2, H-3a, H-4a, H-4b
H-3a, H-3b, H-4b
H-3a, H-3b, H-4a
2.82, m
2.59, m
32.0, CH2
132.3, qC
130.2, CH
116.1, CH
156.9, qC
6/10
7/9
8
7.05, d (8.5)
6.79, d (8.5)
H-7/9
H-6/10
4
OH
NH
1
9.37, s
9.03, d (5.1)
7/9
2, 3, 1 (Val)
H-2
N-Me-Ahpha
170.9, qC
60.9, CH
28.7, CH2
2
4.78, dd (8.5, 5.5)
1.91, m
1.73, m
H-3a, H-3b
1, 3, 4, N-Me, 1 (Htyr-1)
2, 4
3a
3b
4a
4b
5
H-2, H-3b, H-4a, H-4b
H-2, H-3a, H-4a, H-4b
H-3a, H-3b, H-4b
H-3a, H-3b, H-4a
H-4b, H2-6
2, 4
2, 3, 5, 6
1.62, m
1.26, m
26.8, CH2
1.61, m (2H)
2.50, m (2H)
32.6, CH2
35.3, CH2
133.4, qC
130.0, CH
115.9, CH
156.8, qC
4
4
6
H2-5
7
8/12
9/11
10
OH
N-Me
1
7.01, d (8.5)
6.755, d (8.5)
H-9/11
H-8/12
9/11
9.33, s
2.72, s
29.0, CH3
172.5, qC
54.1, CH
35.4, CH2
2, 1 (Htyr-1)
Htyr-2
2
4.39, ddd (11.5, 8.9, 3.0)
2.18, m
1.91, m
H-3a, H-3b, NH
1
2, 3
3a
3b
4a
4b
5
H-2, H-3b, H-4a, H-4b
H-2, H-3a, H-4a, H-4b
H-3a, H-3b, H-4b
H-3a, H-3b, H-4a
2, 3
2, 3, 5, 6/9
2.45, m
2.35, m
32.5, CH2
132.9, qC
130.2, CH
115.9, CH
156.8, qC
6/10
7/9
8
6.97, d (8.5)
6.751, d (8.5)
H-7/9
H-6/10
4
OH
NH
1
9.31, s
8.94, d (8.9)
7/9
2, 3, 1 (N-Me-Ahpha)
H-2
Lys
173.9, qC
55.9, CH
32.9, CH2
2
4.18, ddd (8.3, 6.0, 4.5)
1.76, m
1.68, m
H-3a, H-3b, 2-NH
H-2, H-3b, H-4a
H-2, H-3a, H-4a
H-3a, H-3b, H-4b/H2-5
H-3a, H-3b, H-4a
H-4a, H-4b/5b, H-6a, H-6b
H-4a, H-4b, H-6a, H-6b
H2-5, H-6b, 6-NH
H2-5, H-6a, 6-NH
H-2
1, 3, 4, 1 (ureido)
3a
3b
4a
4b
5a
5b
6a
6b
2-NH
6-NH
C]O
1
2
6
1.23, m
1.39, m
21.5, CH2
29.5, CH2
39.1, CH2
1.41, m
1.36, m
3.66, m
2.72, m
6.79, d (8.3)
7.46, dd (8.2, 2.1)
1, 2, 1 (ureido)
6, 1 (Htyr-2)
H-6a, H-6b
Ureido
Ile
158.6, qC
174.7, qC
58.1, CH
38.5, CH
25.8, CH2
2
4.28, dd (9.0, 5.0)
1.83, m
1.46, m
H-3, NH
1, 3, 4, 6, 1 (ureido)
1, 2, 5, 6
2, 3, 5, 6
3
H-2, H-4a, H-4b, H3-6
H-3, H-4b, H3-5
H-3, H-4a, H3-5
H-4a, H-4b
4a
4b
5
1.18, m
0.88, t (7.4)
2, 3, 5, 6
3, 4, 6
12.1, CH3
(continued on next page)

4086
S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
Table 2 (continued)
Unit
C/H no.
dH (J in Hz)
dC, multa
COSY
HMBCb
6
NH
0.91, d (6.8)
6.30, d (9.0)
12.8, br s
16.3, CH3
H-3
H-2
2
2, 1 (ureido)
COOH
a
Deduced from 13C NMR (150 MHz), HMQC, and HMBC.
Protons showing long-range correlation with indicated carbon.
b
Boc
R2
O
N
NHMe
OH
R1O
OR1
HO
O
(i) 1 N MeOH/HCl, rt, 24 h
(ii) CH3I/NaH//THF, 24 h
(iii) 6 N HCl, 110 °C, 36 h
O
O
4a
4d
4a R1 = H, R2 = H
4b R1 = Me, R2 = H
4c R1 = Me, R2 = Me
(i)
(ii)
Scheme 2. Synthesis of N-Me-(ꢁ)-2-aminopimelic acid (4d).
A (1) also exhibited a comparable trypsin inhibitory activity
with an IC50 value of 2.4ꢁ0.4 mg/mL. Pompanopeptin B (2)
is structurally related to carboxypeptidase-A inhibitors ana-
baenopeptins I and J isolated from the cyanobacterium Apha-
nizomenon flos-aquae8 in which L-Leu/L-Phe and N-Me-L-Ala
residues were replaced by L-Htyr and N-Me-L-Ahpha, respec-
tively. Further biological evaluation will be carried out in due
course.
3.2. Biological material
Samples of L. confervoides were recollected during a persis-
tent cyanobacterial bloom, which occurred near Fort Lauder-
dale, Florida, USA (26ꢂ01.14140N, 80ꢂ05.99730W;
26ꢂ15.1340N, 80ꢂ03.9080W)23 at a depth of 15 m in August
2005. A voucher specimen is retained at the Smithsonian
Marine Station.
3.3. Extraction and isolation
3. Experimental
The freeze-dried organism was extracted with EtOAce
MeOH (1:1) to afford a crude extract, which was then defatted
with hexanes and partitioned between n-BuOH and H2O. The
combined n-BuOH extract (6.3 g) was applied on a diaion
HP-20 resin and subsequently fractionated with water and in-
creasing concentrations of MeOH, and then with MeCN. The
fraction eluting with 75% aqueous MeOH to 100% MeOH
(864 mg) was subjected to reversed-phase preparative HPLC
(Phenomenex LUNA-C18 10 m, 100ꢃ21.20 mm, 10.0 mL/
min; UV detection at 220 and 240 nm) using a MeOHeH2O
linear gradient (30e100% over 40 min and then 100%
MeOH for 10 min). A fraction eluting between tR 30 and
32 min was collected and subjected to repeated semiprepara-
tive reversed-phase HPLC (YMC-Pack ODS-AQ, 250ꢃ
10 mm, 2.0 mL/min; UV detection at 220 and 240 nm) using
MeOH in 0.05% aqueous TFA (60e90% in 25 min, 90e
100% in 10 min), to give 1 (tR 32.0 min, 5.3 mg). A fraction
that eluted at tR 16.0e23.0 min from the preparative HPLC
was separated by repeated semipreparative HPLC using the
same gradient as described above to yield 2 (tR 21.0 min,
1.8 mg).
3.1. General
Optical rotations were measured on a PerkineElmer 341
polarimeter. UV spectra were recorded on a SpectraMax M5
(Molecular Devices). IR spectra were obtained on a Bruker
Vector 22 instrument. H, 13C, and 2D NMR spectra for 1
were recorded in DMSO-d6 (dH 2.49, dC 39.5 for residual sol-
vent signals) on a Varian Unity Inova 500 spectrometer, oper-
ating at 500 MHz (1H) or 125 MHz (13C). H NMR and 2D
1
1
NMR data for 2 were acquired on a Bruker Avance 500 spec-
trometer and 13C NMR and APT spectra acquired on a Bruker
Avance 600 MHz spectrometer operating at 150 MHz, using
DMF-d7 as solvent (dH 8.02, dC 162.9 for residual solvent sig-
nals). 1H and 13C NMR spectra for synthetic compounds were
recorded on a Varian Mercury 400 MHz spectrometer, operat-
ing at 400 and 100 MHz, respectively, using D2O (dH 4.80 for
residual solvent signal, dC 67.0 for spiked in 1,4-dioxane) or
CDCl3 (dH 7.26, dC 77.0 for residual solvent signals).
HMQC/HSQC experiments were optimized for 1JCH¼145 Hz,
n
and HMBC experiments were optimized for JCH¼7 Hz.
HRMS data were obtained using an Agilent LCeTOF mass
spectrometer equipped with an APCI/ESI multimode ion
source detector. HPLCeMS analysis was performed using
Agilent 1100 HPLC system equipped with Thermo Finnigan
LCQ Advantage ion trap mass spectrometer via an ESI inter-
face. Boc-(ꢁ)-2-aminopimelic acid standard was procured
from AnaSpec Inc. (San Jose, CA).
3.3.1. Pompanopeptin A (1)
Colorless, amorphous solid; [a]2D0 ꢀ42.5 (c 0.20, MeOH);
UV (MeOH) lmax (log 3) 210 (4.06), 280 (sh) (3.11) nm;
IR (film) nmax 3357, 2966, 1736, 1656, 1535, 1203, 1136,
1
1056, 1022 cmꢀ1; for H NMR, 13C NMR, COSY, HMBC,

S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
4087
and ROESY data, see Table 1; HRAPCI/ESIMS m/z
[MþH]þ 1069.4400, 1071.4400, ion cluster (1:1) (calcd
for C46H7479BrN10O12S, 1069.4392; C46H7481BrN10O12S,
1071.4371).
2.70 (s, 3H); 13C NMR (100 MHz, D2O/pH¼2) d 171.4,
155.3, 134.2, 130.4, 128.0, 113.5, 111.4, 62.9, 56.7, 34.2,
32.3; APCI/ESIMS m/z 288/290 (1:1, [MþH]þ ion cluster),
HRAPCI/ESIMS m/z [MþH]þ 288.0232 (calcd for
C11H1579BrNO3, 288.0235).
3.3.2. Pompanopeptin B (2)
Colorless, amorphous solid; [a]2D0 ꢀ24.0 (c 0.10, MeOH);
UV (MeOH) lmax (log 3) 220 (4.07), 280 (sh) (3.37) nm; IR
(film) nmax 3331, 2935, 1720e1630, 1549, 1515, 1451,
1239 cmꢀ1; for 1H NMR, 13C NMR, COSY, HMBC, and RO-
ESY data, see Table 2; HRAPCI/ESIMS m/z [MþH]þ
958.5319 (calcd for C51H72N7O11, 958.5290).
3.4.2. Acid hydrolysis and chiral HPLC analysis of 1
A sample of compound 1 (0.2 mg) was dissolved in 6 N
HCl (0.6 mL) and heated at 110 ꢂC for 24 h. The hydrolyzate
was concentrated to dryness, re-suspended in H2O (100 mL),
filtered, and subjected to chiral HPLC analysis (Phenomenex
Chirex 3126 N,S-dioctyl-(D)-penicillamine, 250ꢃ4.60 mm,
5 mm; solvent, 2 mM CuSO4 or 2 mM CuSO4eMeCN
(95:5); flow rate, 1.0 mL/min; UV detection 254 nm). The ab-
solute configurations of the amino acid units in 1 (tR, min)
were established as L-Arg (7.5), L-Met(O) (13.5, 14.5) (solvent
2 mM CuSO4); L-Thr (7.8), L-Val (19.2), L-Ile (43.5) (solvent
95:5) by comparison of the retention times tR (min) with those
of standard amino acids. The retention times tR (min) for the
other standard amino acid isomers were 11.4 (D-Arg), 16.5,
18.5 (D-Met(O)) (solvent 2 mM CuSO4); 8.4 (D-Thr), 9.8
(L-allo-Thr), 10.2 (D-allo-Thr), 25.8 (D-Val), 37.5 (L-allo-Ile),
47.0 (D-allo-Ile), 57.5 (D-Ile) (solvent 95:5).
3.4. Determination of absolute configuration of amino
acids in 1
3.4.1. Synthesis of standard N,O-diMe-30-Br-L-Tyr (3d)
Boc-OMe-L-Tyr (3a) (300 mg, 1.02 mmol, 1 equiv) was
dissolved in THF (3.06 mL) and the mixture was cooled to
0 ꢂC. To this solution were added CH3I (8.13 mmol, 8.0 equiv)
and dry NaH (73.1 mg, 3.05 mmol, 3 equiv). The solution was
stirred at room temperature under N2 for 24 h. The reaction
was quenched by addition of H2O, acidified, and partitioned
with EtOAc and H2O. The organic layer was neutralized
with saturated aqueous NaHCO3 and sequentially washed with
H2O, 5% Na2S2O3, and H2O, and then dried, filtered, and
concentrated to afford Boc-N,O-diMe-L-Tyr (3b)14 as a pale
yellow oil (280 mg, 88% yield) {HRAPCI/ESIMS m/z
[MþNa]þ 332.1470 (calcd for C16H23NO5Na, 332.1474)}.
Boc deprotection was achieved by treating 3b with TFA in
CH2Cl2 and stirring for 3 h at room temperature. The reaction
product 3c was then applied on a Dowex 50WX4-50 ion ex-
change resin and eluted with 4 N NH4OH and dried. Compound
3c was obtained as a colorless, amorphous solid (190 mg, 100%
yield); [a]2D0 þ7.5 (c 0.30, H2O); IR (film) nmax 2961, 2873,
2360, 2341, 1735, 1671, 1613, 1514, 1466, 1442, 1250, 1181,
3.4.3. Oxidation and chiral HPLC analysis of 1
Compound 1 (0.5 mg) was dissolved in glacial acetic acid
(0.5 mL), followed by the addition of CrO3 (2 mg) and stirred
at room temperature for 2 h. The resulting product was applied
onto a C18 SPE cartridge eluting with H2O, dried, and sub-
jected to acid hydrolysis and chiral HPLC analysis using
2 mM CuSO4eMeCN (95:5). A peak detected in the hydroly-
zate at tR 65.0 min corresponded to the generated L-Glu,
whereas standard D-Glu eluted at tR 70.2 min under identical
condition.
3.4.4. Determination of absolute configuration of N,O-
diMe-30-Br-Tyr in 1 by advanced Marfey’s analysis
1143, 1031, 835 cmꢀ1
;
1H NMR (400 MHz, D2O/pH¼2)
d 7.17 (d, 2H, J¼8.8 Hz), 6.92 (d, 2H, J¼8.8 Hz), 4.17 (t,
1H, J¼6.2 Hz), 3.75 (s, 3H), 3.24 (dd, 1H, J¼14.9, 6.2 Hz),
3.17 (dd, 1H, J¼14.9, 6.2 Hz), 2.68 (s, 3H); 13C NMR
(100 MHz, D2O/pH¼2) d 171.1, 159.0, 131.1, 126.2, 115.0,
62.6, 55.8, 34.4, 32.3; HRAPCI/ESIMS m/z [MþH]þ
210.1128 (calcd for C11H16NO3, 210.1130).
Aliquots of hydrolyzate of 1 and N,O-diMe-30-Br-L-Tyr
standard (3d) were each treated with 1 M NaHCO3 (10 mL)
and a 1% solution of either L-FDLA or DL-FDLA (1-fluoro-
2,4-dinitrophenyl-5-leucinamide) in acetone and heated at
80 ꢂC for 3 min. The solutions were cooled, neutralized with
2 N HCl (5 mL), dried and dissolved in H2OeMeCN (1:1),
and analyzed by LCeMS (Phenomenex Synergi 4u Hydro-
RP 80A, 2ꢃ150 mm, 4 mm; flow rate, 0.15 mL/min; UV
detection 340 nm) using a step gradient of aqueous MeCN
containing 0.1% HCOOH and increasing MeCN content
(10e50% for 15 min, 50e70% for 55 min, then 70e95% for
60 min). LCeMS analysis of hydrolyzate of 1 of L-FDLA
derivatives detected the mono adduct of N,O-diMe-Br-L-Tyr
(tR 28.5 min, [MþH]þ m/z 568/570) and also the di-L-FDLA
derivative (tR 53.0 min, [MþH]þ m/z 862/864) arising because
of partial O-demethylation under acid hydrolysis conditions.
In contrast, mono and/or di-FDLA derivatives of the corre-
sponding D-isomer were not detected, which would have
been eluted at tR 29.4 and 55.1 min, respectively. These reten-
tion times were inferred from derivatization of acid-treated
Compound 3c was then subjected to bromination.15 Briefly,
Br2 (6.5 mL) was added dropwise to an ice-cooled solution of
3c (25 mg, 0.12 mmol) in 98% formic acid (150 mL) with
vigorous stirring. After stirring for 6 h, the colorless paste
was dissolved in 3 N HCl (150 mL), boiled for 1 h, and con-
centrated to dryness in vacuo. The residue was dissolved in
boiling water, filtered, and neutralized (pH¼7) with 6 N
NH4OH to obtain 3d as a colorless amorphous powder
(27 mg, 75% yield). [a]2D0 ꢀ10.4 (c 0.08, H2O); IR (film)
nmax 3133, 3038, 1736, 1604, 1499, 1403, 1282, 1258, 1150,
1
1054, 1018 cmꢀ1; H NMR (400 MHz, D2O/pH¼2) d 7.47
(d, 1H, J¼2.0 Hz), 7.22 (dd, 1H, J¼8.4, 2.0 Hz), 7.03 (d,
1H, J¼8.4 Hz), 4.11 (t, 1H, J¼6.0 Hz), 3.85 (s, 3H), 3.23
(dd, 1H, J¼14.6, 6.0 Hz), 3.16 (dd, 1H, J¼14.6, 6.0 Hz),

4088
S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
(6 N HCl, 110 ꢂC, 24 h) N,O-diMe-Br-L-Tyr with D-FDLA,
yielding a chromatographic standard for the L-FDLA-derivat-
ized D-amino acid on a nonchiral column because of the enan-
tiomeric relationship.
After evaporation of the solvent, the ozonolysis product was
subjected to acid hydrolysis as described above. The resultant
mixture was dissolved in H2O and analyzed by chiral HPLC as
described in Section 3.4.2. Since L-Glu (tR 65 min) and
N-Me-(S)-2-aminopimelic acid (tR 82 min) were detected in
the hydrolyzate, they must have been derived from the parent
amino acids L-Htyr and N-Me-L-Ahpha, respectively. The cor-
responding enantiomers were not present in the hydrolyzate
since no peak matched the retention times of standards (sol-
vent 95:5): D-Glu (tR 70.2 min) and N-Me-(R)-2-aminopimelic
acid (tR 106 min).24 Additionally, L-Val (19.2) was detected
but not D-Val (25.8).
3.5. Determination of absolute configuration of amino
acids in 2
3.5.1. Synthesis of N-Me-(ꢁ)-2-aminopimelic acid (4d)
Boc-(ꢁ)-2-aminopimelic acid (4a) (140 mg, 0.51 mmol)
was dissolved in 1 N methanolic HCl (1.25 mL) and stirred
for 24 h at room temperature. The resulting mixture after evap-
oration was basified (pH¼8) with a saturated aqueous solution
of NaHCO3, extracted with EtOAc, filtered, and dried to give
crude Boc-(ꢁ)-2-aminopimelic acid dimethylester (4b)
(105 mg), a portion of which (55 mg) was methylated using
CH3I and NaH as described above for N,O-diMe-Br-Tyr to
yield Boc-N-Me-(ꢁ)-2-aminopimelic acid dimethylester (4c)
(26 mg, 42% overall yield). IR (film) nmax 2953, 1741, 1696,
3.5.4. Hydrazinolysis of pompanopeptin B (2)
A solution of 2 (0.1 mg) in anhydrous hydrazine (0.5 mL)
was heated at 125 ꢂC for 18 h. After cooling, the excess hydr-
azine was evaporated in vacuo. Analysis of the hydrazinolysis
product of 2 by chiral HPLC as above (solvent mixture 95:5)
detected only L-Ile (43.5). The retention times for the other Ile
isomers were mentioned earlier under Section 3.4.2.
1437, 1392, 1367, 1328, 1157, 1014, 870, 774 cmꢀ1 1H
;
NMR (400 MHz, CDCl3, 1:1 conformers) d 4.72 (dd, 0.5H,
J¼10.8, 4.8 Hz), 4.36 (dd, 0.5H, J¼10.6, 4.8 Hz), 3.68 (s,
3H), 3.64 (s, 3H), 2.80 (s, 1.5H), 2.74 (s, 1.5H), 2.30 (t, 2H,
J¼6.8 Hz), 1.96e1.86 (m, 1H), 1.80e1.58 (m, 3H), 1.44 (s,
4.5H), 1.41 (s, 4.5H), 1.33e1.26 (m, 2H); 13C NMR
(100 MHz, CDCl3) d 173.9, 172.4 (172.2*), 156.3 (155.5*),
80.3 (80.0*), 59.0 (57.4*), 52.01 (51.98*), 51.5, 33.8, 31.1
(30.4*), 28.8 (28.5*), 28.3 (ꢃ3), 25.5, 24.4 (* indicates dou-
bling due to conformers); HRAPCI/ESIMS m/z [MþNa]þ
340.1742 (calcd for C15H27NO6Na, 340.1736).
3.5.5. Absolute configuration of Lys in 2 by Marfey’s
analysis
A portion of the hydrolyzed ozonolysis product from 2 was
derivatized with L-FDLA as described earlier. The reaction
mixture was dissolved in H2OeMeCN (1:1) and subjected to
reversed-phase HPLC (Alltech Altima HP C18 HL 54,
250ꢃ4.6 mm; flow rate, 1 mL/min; PDA detection from
200e500 nm) using a linear gradient of MeCN in 0.1% aque-
ous TFA (30e70% MeCN over 50 min). The retention times
(tR, min) of the derivatized amino acids in the hydrolyzate
of 2 matched with those of D-Lys (42.8), L-Val (23.7), and
L-Glu (16.2) and not with that of L-Lys (40.7), D-Val (32.6),
and D-Glu (17.6).
The product 4c (10 mg) was subjected to acid hydrolysis
(1 mL, 6 N HCl, 110 ꢂC, 36 h) to yield N-Me-(ꢁ)-2-aminopi-
melic acid (4d) as a colorless amorphous solid after concentra-
tion to dryness (5.5 mg, 92% yield). IR (film) nmax 3385, 2957,
1722, 1631, 1464, 1410, 1231, 1024, 983 cmꢀ1 1H NMR
;
(400 MHz, D2O) d 3.81 (dd, 1H, J¼8.0, 6.0 Hz), 2.71 (s,
3H), 2.39 (t, 2H, J¼7.4 Hz), 2.00e1.88 (m, 2H), 1.68e1.58
(m, 2H), 1.48e1.30 (m, 2H); 13C NMR (100 MHz, D2O)
d 182.6, 179.0, 62.3, 33.7, 32.0, 28.9, 24.2, 23.8; HRAPCI/
ESIMS m/z [MþH]þ 190.1078 (calcd for C8H16NO4,
190.1079).
3.6. Protease inhibition assays
Compound 1 was tested for serine protease-inhibitory activ-
ity. Specifically, a dilution series of 1 was incubated with tryp-
sin (porcine pancreas-Sigma T0303), a-chymotrypsin (bovine
pancreas-Sigma C4129), or porcine pancreatic elastase (Elas-
tase-high purity; EPC, EC134) in the presence of chromogenic
substrates (N-a-benzoyl-DL-arginine 4-nitroanilide hydrochlo-
ride for trypsin, N-succinyl-Gly-Gly-Phe-p-nitroanilide for
chymotrypsin, N-succinyl-Ala-Ala-Ala-p-nitroanilide for elas-
tase). Activity was determined as described previously.3
3.5.2. Acid hydrolysis and chiral HPLC analysis of 2
A sample of compound 2 (0.1 mg) was dissolved in 6 N
HCl (0.6 mL) and heated at 110 ꢂC for 24 h. The hydrolyzate
was treated in a similar manner and analyzed by chiral HPLC
as described in Section 3.4.2. The retention time (tR, min) of
the amino acids detected in the hydrolyzate of 2 matched
with those of L-Val (11.5) and L-Htyr (43.5), but not with those
of D-Val (13.8) and D-Htyr (63.5) (solvent 90:10).
Acknowledgements
This article was developed under the auspices of the Florida
Sea Grant College Program with support from the National Oce-
anic and Atmospheric Administration, Office of Sea Grant, U.S.
Department of Commerce, Grant No. NA06OAR4170014. The
authors gratefully acknowledge NSF for funding through the
External User Program of the National High Magnetic Field
Laboratory (NHMFL), which supported our NMR studies for
2 at the Advanced Magnetic Resonance Imaging and
3.5.3. Ozonolysis of pompanopeptin B (2)
Compound 2 (0.2 mg) was dissolved in 3 mL CH2Cl2e
MeOH (1:1) and subjected to ozonolysis by bubbling ozone
through the solution for 30 min at room temperature. The sol-
vent was evaporated, and the product was suspended in
H2O2eHCOOH (1:1), stirred, and heated for 1 h at 80 ꢂC.

S. Matthew et al. / Tetrahedron 64 (2008) 4081e4089
4089
8. Murakami, M.; Suzuki, S.; Itou, Y.; Kodani, S.; Ishida, K. J. Nat. Prod.
2000, 63, 1280e1282.
Spectroscopy (AMRIS) facility in the McKnight Brain Institute
of the University of Florida (UF). We thank L. Fisher and K.
Banks (Broward County Environmental Protection Depart-
ment), and A. Capper and R. Ritson-Williams (Smithsonian Ma-
rine Station) for assistance with collections, S. Sivendran (UF)
for assistance in synthesis, J. Johnson (Department of Chemis-
try, UF) for LCeMS analysis, W. Yoshida (University of
Hawaii) for providing NMR data for 1, and J. Rocca (AMRIS)
for assistance in acquiring NMR data for 2. HRMS analyses
were performed at the UCR Mass Spectrometry Facility, De-
partment of Chemistry, University of California at Riverside.
This is contribution #727 from the Smithsonian Marine Station
at Fort Pierce.
9. Hirota, M.; Ohmuraya, M.; Baba, H. J. Gastroenterol. 2006, 41,
832e836.
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