Journal of Natural Products
Note
which lies in the presumption that amino acids are liberated
from the peptide with retention of configuration, precluding one
to consider the possibility of the presence of its enantiomer
The 40% MeCN fraction was purified by RP-HPLC on a COSMOSIL
C -MS-II column with gradient elution from 15% MeCN to 100%
5
18
MeCN in the presence of 0.5% AcOH to afford 1 (4.5 mg).
1
6
15
−53 (c 0.14,
D
buried under an impurity peak.
1
13
62.39397 [M + Na] (calcd for C H N O Na, 662.39656).
Because the exocyclic amino acid residue attached through
the ureido bond was basic, homophymamide A was expected to
inhibit carboxypeptidase B, which cleaves the C-terminal residue
by recognizing a basic side chain; the enzyme recognizes the
+
6
29 53 9 7
Marfey’s Analysis of Ala, N-Me-Leu, Lys, and Arg. Compound
(50 μg) was hydrolyzed in 6 N HCl (500 μL) at 110 °C for 16 h. The
1
solution was dried under reduced pressure and dissolved in H O (100
2
1
peptide but cannot cleave the ureido bond. As expected,
μL), to a 50 μL portion, to which 1% L-FDAA in acetone (100 μL) and
homophymamide A inhibited carboxypeptidase B with an IC
value 0.59 μM.
1 M NaHCO
0 °C for 1 h. After neutralization with 2 N HCl (20 μL) and drying
under reduced pressure, the reaction mixture was analyzed by LCMS:
in H O (40 μL) were added. The solution was heated to
3 2
5
0
1
6
4
Homophymamide A (1) is a heterodetic cyclic peptide, a one-
residue-lower homologue of the anabaenopeptin class. Two-
residue-lower homologues, namalides, have also been reported
−1
flow rate of 0.5 mL min on a COSMOSIL MS-II column (2.6 × 150
mm) at 40 °C. Gradient elution was performed using solvent A (0.5%
acetic acid in H O) and solvent B (0.5% acetic acid in MeCN) with the
3
,17
2
from a marine sponge and cyanobacteria.
Because the
following linear gradient combination: 10% B for 5 min then 10% B to
biosynthetic gene cluster for konbamide, an anabaenopeptin
class peptide from the marine sponge Theonella swinhoei, is
encoded in the symbiotic bacteria ‘Candidatus Entotheonella
1
00% B for 25 min. The D-DAA derivatives were prepared in the same
manner.
Retention times for standard amino acids (min): L-DAA-L-Arg (7.6),
18
factor’, we suspect the bacterial origin of homophymamide A.
D-DAA-L-Arg (8.3), L-DAA-L-Lys (14.5), D-DAA-L-Lys (15.2), L-DAA-
L-Ala (10.7), D-DAA-L-Ala (11.9), L-DAA-N-Me-L-Leu (14.7), D-DAA-
N-Me-L-Leu (15.2).
EXPERIMENTAL SECTION
■
C3 Marfey’s Analysis of Ile. For the differentiation of four isomers
General Experimental Procedures. Optical rotations were
measured on a Jasco P-2100 polarimeter. NMR spectra were measured
on a JEOL alpha 600 NMR spectrometer and referenced to the solvent
of Ile, we employed the C Marfey’s method. Derivatization of amino
3
acids is conducted as mentioned above. The reaction mixture was
−1
analyzed by LCMS at a flow rate of 1.0 mL min on a Zorbax
peak: δH 2.49 and δC 39.5 for DMSO-d . ESI mass spectra were
StableBond C column at 50 °C. Gradient elution was performed using
6
3
recorded on either a JEOL JMS-T100LC mass spectrometer or a Sciex
TripleTOF 5600 mass spectrometer. LCMS experiments were
performed on a Shimadzu LC-20AD solvent delivery system interfaced
to a Bruker amaZon SL mass spectrometer. HPLC experiments were
performed on a Shimadzu HPLC system equipped with an LC-20AT
solvent delivery system. The results of the enzyme inhibitory assay were
recorded with a Molecular Devices SPECTRA max M2.
solvent A (H O with 5% MeCN that includes 1% formic acid) and
2
solvent B (MeOH with 5% MeCN that includes 1% formic acid) with
the following linear gradient program: 45% B to 60% B for 35 min.
Retention times for standard amino acids (min): L-DAA-L-Ile (10.1),
D-DAA-L-Ile (17.6), L-DAA-L-allo-Ile (9.6), D-DAA-L-allo-Ile (16.6)
Boc-D-Lys(Fmoc)-OH Allyl Ester. Boc-D-Lys(Fmoc)-OH (1 g) in
DMF (10 mL) was stirred with cesium carbonate (0.7 g) for 1 h. To the
solution was added allyl bromide (0.56 mL) in DMF (3 mL) dropwise
followed by stirring for 1 h. After confirmation of the completion of the
Sponge Material. The sponge specimen was collected by dredging
at a depth of ca. 200 m at the seamount “Sango-Sone” (30°07′08″ N,
129°42′03″ E), southern Japan, in June 2017. Sponge description:
reaction by TLC, the solution was diluted by H O (250 mL) and
2
external morphology roughly turbinate with thick body wall; oscules
dispersed on upper part of the sponge; surface smooth and
subectosomal water canals visible; color pale brown in life, cream in
ethanol; skeleton composed of ectosomal pseudodiscotriaene,
choanosomal monocrepid desma, oxea, and one type of amphiaster;
pseudodiscotriaene varying in shape from circular to circular with
irregular margins; axial canal of pseudodiscotriaene only present in
rhabd; monocrepid desma having complex branching shape; oxea thin
and slender, slightly curved at midpoint, tapering to sharp tips;
amphiaster, almost a straight shaft with several short bud-like rays
around both extremities.
acidified by addition of 2 M KHSO (50 mL). The resulting solution
4
was extracted with CH Cl (3 × 30 mL), and the organic layer was
2
2
washed with H O and brine, dried with MgSO , and filtered. The
2
4
resulting solution was dried under reduced pressure and lyophilized to
remove remaining DMF to yield the allyl ester (904 mg, 83.3%).
4-Nitrophenyloxycarbonyl-D-Lys(Fmoc)-OH Allyl Ester (2).
Boc-D-Lys(Fmoc)-OH allyl ester (904 mg) was dissolved in CH Cl /
2
2
EtOH (1:1, 20 mL) in the presence of p-toluenesulfonic acid (1 equiv).
After stirring for 3 h at 40 °C, the mixture was evaporated to dryness.
This procedure was repeated five times to completely deprotect the Boc
group, which was confirmed by TLC. The resulting white solid was
dissolved in CH Cl and added to a suspension of 4-nitrophenyl-
Morphological characters of the specimen analyzed in this study
2
2
19
match the diagnosis of Homophymia Vacelet & Vasseur, 1971, except
for the difference of ectosomal spicules: pseudophyllotriaene in
Homophymia. And here we tentatively assigned our specimen to the
genus Homophymia because there is no other sponge genus similar to
the present specimen in the family Neopeltidae that is diagnosed by
possession of monaxial ectosomal megasclere and monocrepid
chloroformate (1.3 equiv) and pyridine (8 equiv) in CH Cl and stirred
2 2
in an ice salt bath. The temperature was allowed to increase to 4 °C, and
the mixture continued stirring for 3 h. When completion of the reaction
was confirmed by TLC, the resulting mixture was acidified with 1 M
KHSO . The layers were separated, the aqueous layer was further
4
extracted with CH Cl (3 × 30 mL), and the combined organic layers
2
2
19
desma. Up to now, three species are known from the genus
were dried under reduced pressure. The resulting yellow oil was
subjected to silica open column chromatography and eluted with 2:1
hexane/EtOAc to remove 4-nitrophenylchloroformate and 2:3 hexane/
EtOAc to obtain the target compound 2 (927 mg, 91%).
20
Homophymia, but none of them are similar to our specimen especially
for the shape of ectosomal megascleres and amphiasters. The specimen
used for identification (NSMT-Po-250) is deposited at National
Museum of Nature and Science, Tokyo. The sponge (130 g) was kept
frozen until extraction.
Solid Phase Peptide Synthesis of Homophymamide A. Fmoc-
Arg(Pbf)-Alko resin was purchased from Watanabe Chemical
Industries. We conducted synthesis with six columns each in 0.03
mmol scale. The experiments for one column are described below.
General Procedure for Deprotection and Coupling of
Protected Amino Acids. Fmoc was cleaved by treatment with 20%
piperidine in DMF (1.5 mL) for 1 h. After deprotection, the resin was
washed with DMF (5 × 1.5 mL), CH Cl (5 × 1.5 mL), and DMF (5 ×
Extraction and Isolation. The sponge was homogenized in EtOH
and filtered, and the solution was dried under reduced pressure. The
extract was partitioned between CHCl and H O, and the aqueous layer
3
2
was further partitioned between n-BuOH and H O. The n-BuOH layer
2
was dried under reduced pressure and subjected to ODS flash column
chromatography with stepwise elution of 20% MeCN (AcOH 0.5%),
2
2
4
0% MeCN (AcOH 0.5%), 60% MeCN (AcOH 0.5%), 80% MeCN
1.5 mL). Coupling reagent (Fmoc-protected amino acid 4 equiv,
HBTU 3 equiv, DIPEA 8 equiv in DMF 3 mL) was added to the washed
(AcOH 0.5%), 100% MeCN (AcOH 0.5%), and CHCl /MeOH (1:1).
3
1
851
J. Nat. Prod. 2021, 84, 1848−1853