Journal of Natural Products
Note
(4.6) 278 (4.2); 1H and 13C NMR [DMSO-d6−H2O (4:1)], Table 1
and Table S1; HRESIMS m/z 1725.5701 (calcd for
C74H9579BrN16O26Na, 1725.5684, Δ +1.7 mmu) .
for 4 h. The absorbance at 450 nm was measured with a microplate
reader. Three replicates were examined to determine the IC50 value
(Figure S28). A parallel analysis of adriamycin using MTT gave an
IC50 value of 2 μM. The purity of 1 used for the cytotoxicity test was
Marfey’s Analysis of Theonellamide I (1). Compound 1 (100
μg) was dissolved in 6 N HCl (200 μL) and heated at 110 °C for 3 h.
The mixture of theonellamides A, D, and E11 was hydrolyzed in the
same manner. The solvent was evaporated with a stream of N2 and
redissolved in 0.6 M NaHCO3 (100 μL). To the solution was added
3% FDNP-L-Val in EtOH (80 μL), and the mixture was kept at 55 °C
for 1 h.13 After neutralization with 3 N HCl (20 μL), the reaction
mixture was analyzed by LC-MS on a COSMOCORE 2.6PBr column
with gradient elution from MeCN−H2O (1:9) to MeCN−H2O (7:3)
containing 0.5% acetic acid for 32 min. Standard amino acids were
derivatized with either FDNP-L-Val or FDNP-D-Val and analyzed by
LC-MS. Standard amino acids of L-BrPhe and (2S,3R)-β-OHAsp were
obtained from the hydrolysate of the mixture of theonellamides A, D,
and E.11 Retention times of the amino acids and LC-MS charts are
Determination of the Absolute Configuration of the
Histidinoalanine Residue. The mixture of theonellamide A, D,
and E (500 μg) was dissolved in 6 N HCl (250 μL) and subjected to
acid hydrolysis (110 °C, 16 h). A half-portion of the hydrolysate was
derivatized with FDNP-L-Val and analyzed by LC-MS. In addition to
L-histidino-D-alanine, L-histidino-L-alanine was detected.11 The ratio
of the LD-isomer and LL-isomer was 3:1. The rest of the hydrolysate
was derivatized with FDNP-D-Val to prepare HPLC equivalents of D-
histidino-L-alanine and D-histidino-D-alanine.11 Retention times and
1
ca. 90%, as judged from the H NMR spectrum.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
Spectroscopic data (1H, COSY, HSQC, TOCSY,
HMBC, and NOESY) and the results of the Marfey’s
analysis of theonellamide I (1) (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
ORCID
Present Address
§School of Marine Bioscience, Kitasato University, Sagamihara,
Kanagawa 252-0373, Japan.
Determination of the Absolute Configuration of 3-Amino-
4-hydroxy-6-methyl-8-phenyl-5,7-octadienoic Acid Residue.
To a solution of 1 (100 μg) in MeCN−H2O (1:1, 1 mL) was added
10% Pd/C (2 mg), and the mixture was stirred under an atmosphere
of H2 for 18 h. The mixture was filtered through Celite, and the
solvent was removed in vacuo to afford a diastereomeric mixture of
tetrahydrotheonellamide I.18,19 The product was subjected to acid
hydrolysis, derivatization with FDNP-L-Val, and LC-MS analysis as
described above. The standard of a diastereomeric mixture of 3-
amino-6-methyl-8-phenyloctanoic acid was obtained from the mixture
of theonellamides A, D, and E and derivatized with either FDNP-D-
Val or FDNP-L-Val and then analyzed by LC-MS in the same manner.
Retention times and the LC-MS chart are shown in Table S5 and
Analysis of the Absolute Configuration of the Arabinose
Residue in Theonellamide I (1). Theonellamide I (1, 60 μg) was
dissolved in 6 N HCl (200 μL) and heated at 110 °C for 3 h. The
solvent was evaporated with a stream of N2, and the residue was
dissolved in 10% HCl in MeOH (200 μL). The mixture was heated at
100 °C for 1 h. After evaporation of the solvent, to the product was
added a solution of L-cysteine methyl ester hydrochloride in pyridine
(2 mg/mL; 100 μL) and the solution was heated at 60 °C for 1 h.
Then, a 5 μL portion of phenylisothiocyanate was added, and the
solution was heated for 1 h at 60 °C. The solvent was evaporated, the
product was dissolved in MeOH (100 μL), and the product was
analyzed by LC-MS on a COSMOSIL 2.5C18-MS-II column with
gradient elution from MeCN−H2O (1:9) to MeCN−H2O (3:17)
containing 0.5% acetic acid for 60 min. L- and D-Arabinose were
treated in the same manner. Retention times and the LC-MS chart are
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by JSPS KAKENHI Grant
Numbers 25252037, 16H04980, 17J09477, and 17H06403
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan. We thank Professor J. Piel, ETA Zurich, for
valuable discussion and R. Suo for experimental assistance.
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D
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