Journal of Natural Products
NOTE
stenothricin13 and imacidinic acid,14 which show antibacterial
activity. However, to the best of our knowledge, 1 is the first
example of the N-phenylacetylated pentapeptide involving the
cysteic acid and lactone structures. Contrary to the characteristic
structure of 1, it did not show cytotoxic or antimicrobial activity.
The unique structure of 1 may have been biosynthesized by a
novel nonribosomal peptide synthetase gene. Thus, studies on
the detailed biosynthesis of 1 are now underway.
’ EXPERIMENTAL SECTION
Figure 3. Fragmentation pattern of the hydrolysate (2) of 1 in the
positive ion mode of HRESIMS/MS.
General Experimental Procedures. Optical rotation was oper-
ated on a Horiba SEPA-300 polarimeter. HRESIMS data were recorded
on a Waters LCT-Premier XE mass spectrometer. HRESIMS/MS data
were recorded on a Waters SYNAPT G2. UV spectra were measured on
a Beckman Coulter DU730 UV/vis spectrophotometer. FT-IR spectra
were obtained using a Horiba FT-720 spectrophotometer. 13C
1
(150 MHz) and H (600 MHz) NMR spectra were recorded on a
Varian NMR System 600 NB CL. Samples were measured in DMSO-d6,
and the solvent peak was used for spectra calibration (δC 39.7, δH 2.49
ppm). Reverse-phase medium-pressure liquid chromatography
(MPLC) was performed on a Purif-Pack ODS 100 column (Shoko
Scientific, Yokohama, Japan). Analytical reversed-phase HPLC was
carried out using a CAPCELL PAK C18 MGII column (5.0 μm,
4.6 i.d. ꢁ 150 mm; Shiseido, Tokyo, Japan) with a Waters 2996
photodiode array detector and a Waters 3100 mass detector. Preparative
reversed-phase HPLC was carried out using a CAPCELL PAK C18
MGII column (5.0 μm, 20 i.d. ꢁ 150 mm; Shiseido) with a Waters 2996
photodiode array detector and a Waters 3100 mass detector. Reagents
and solvents were of the highest grade available.
Isolation of Streptomyces sp. RI051-SDHV6. Streptomyces sp.
RI051-SDHV6 was isolated from a soil sample collected in Rishiri Island,
Hokkaido Prefecture, Japan, using the sodium dodecyl sulfate-yeast
extract (SDS-YE) method.15 To identify the genus of the strain RI051-
SDHV6, we compared the 16S rRNA gene sequences of RI051-SDHV6
to those available in the DNA Data Bank of Japan using the basic local
alignment search tool (BLAST) search and identified it as the genus
Streptomyces.
Fermentation. The seed medium was composed of 1% starch, 1%
Polypepton, 1% molasses, 1% meat extract, and 1.75% Sealife (pH 7.2
before sterilization). The production medium consisted of 2% glycerol,
1% molasses, 0.5% casein, 0.1% Polypepton, and 0.4% CaCO3 (pH 7.2
before sterilization). Strain RI051-SDHV6 was cultivated in 50 mL test
tubes containing 15 mL of the seed medium. The test tubes were shaken
on a reciprocal shaker (355 rpm) at 27 ꢀC for 3 days. Aliquots (2.5 mL)
of the culture were transferred to 500 mL Erlenmeyer flasks containing
100 mL of the production medium and cultured on a rotary shaker
(180 rpm) at 27 ꢀC for 5 days.
Purification of 1. The fermentation broth (2 L) of RI051-SDHV6
was centrifuged, and the collected mycelial cake was extracted with
acetone (500 mL). The extract was concentrated in vacuo, and the
residual aqueous concentrate was first washed with EtOAc and extracted
with n-BuOH, successively. The n-BuOH layer was concentrated
in vacuo. The dried residue (770 mg) was subjected to reversed-phase
MPLC using a H2OꢀMeOH stepwise solvent system (0%, 20%, 40%,
and 60% MeOH). The 40% MeOH eluate (38 mg) was further purified
by preparative reversed-phase HPLC using a CAPCELL PAK C18 MGII
column developed with 50% MeOHꢀH2O containing 0.1% formic acid
(flow rate: 10 mL/min) monitored by LC-MS to yield 1 (25.3 mg, tR =
15.9 min).
Figure 4. Absolute configuration of C-20 in 1 revealed by the modified
Mosher’s method. The differences in chemical shift values were obtained
by subtracting the (R)-MTPA ester values from (S)-MTPA ester values
(δΔ = δ(S)-MTPA ꢀ δ(R)-MTPA).
determined to be an N-phenylacetylated linear pentapeptide
arranged in the order β-alanine, two threonines, cysteic acid,
and valine.
The absolute configuration of the two threonines, the cysteic
acid, and the valine residues in 1 was clarified by Marfey’s
method8 applied for the acid hydrolysate of 1 in comparison
with standard amino acids. Retention times of the standard
NR-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA) deriva-
tives were as follows: L-threonine, 6.1 min; L-allo-threonine, 7.7
min; D-allo-threonine, 8.7 min; D-threonine, 10.7 min; L-valine,
6.3 min; D-valine, 12.2 min. Retention times of the standard
NR-(5-fluoro-2,4-dinitrophenyl)-L-valinamide (FDVA) deriva-
tives were as follows: D-cysteic acid, 6.6 min; L-cysteic acid,
7.3 min. The chromatogram of the FDAA or FDVA derivatives of
the acid hydrolysate showed peaks corresponding to L-threonine
(6.3 min), D-threonine (10.8 min), L-valine (6.2 min), and
D-cysteic acid (6.6 min). Since both L-threonine (6.3 min) and
D-threonine (10.8 min) were observed in the hydrolysate, we
adopted the modified Mosher’s method.9
The proton chemical shift of the methyl proton H-21 in the
(R)-MTPA ester derivative of 1 appeared at a lower field than
that of the (S)-MTPA ester derivative (Figure 4). On the other
hand, the proton chemical shift of the R-methine proton H-5 and
the amide proton 5-NH in the (R)-MTPA ester derivative was
observed at a higher field than that of the (S)-MTPA ester
derivative. Thus, the absolute configuration at the C-20 position
was deduced to be S, indicating that this threonine moiety is
D-threonine. Conclusively, the absolute structure of 1 was
established, as shown in Figure 1.
In conclusion, we herein isolated a unique peptide designated
JBIR-96 from Streptomyces sp. RI051-SDHV6. It has been
reported that antibiotic YF 044P-D,10 antibiotic L 174580,11
and antibiotic YM 4769012 are isolated from Streptomyces sp. as
phenylacetyl N-terminal masked peptides. Antibiotic YF 044P-
D, antibiotic L 174580, and antibiotic YM 47690 show inhibition
of proteases, aspartyl protease, rennin, and cathepsin L, respec-
tively. In addition, the cysteic acid of 1 is identical to those of
Determination of Amino Acid Configurations. Compound 1
(1.0 mg) was hydrolyzed in 0.2 mL of 6 N HCl at 110 ꢀC for 14 h. After
the reaction mixture was concentrated in vacuo, the residue was added to
0.1 M NaHCO3 (200 μL) with 0.2 mg of FDAA or FDVA. The solution
1346
dx.doi.org/10.1021/np200054s |J. Nat. Prod. 2011, 74, 1344–1347