Journal of Natural Products
Article
(12.2), Hty (11.0), Arg (3.9), N-MeLys (7.8). The retention times
(min) of the derivatives prepared from the hydrolysate of spumigin J
(1) were as follows: L-FDLA-Hpla (13.0), Hty (14.4), N-MeLys (7.2).
The retention times (min) of the derivatives from the hydrolysate of
spumigin A (2) were noted as L-FDLA-Hpla (12.8), Hty (14.3), Arg
(3.9).
compound, and docking experiments were performed using the
previously defined hydrogen-bonding constraint with postdocking
minimization of the resulting poses. Up to three poses per compound
state were retained for rescoring using MM-GBSA. Default options
were used elsewhere.
Analysis of the 4-MePro configuration was performed under similar
conditions using 25−65% MeCN with 0.1% HCOOH over 40 min,
YMC Hydrosphere C18, 2.0 × 150 mm. The synthesized
diastereoisomers of 4-MePro were used as standards for the analysis.
The retention times (tR, min) of the Marfey’s derivatives of the four
synthesized diastereoisomers of 4-methylproline were as follows: 2S,4S
(11.75−11.76), 2S,4R (11.86−11.88), 2R,4S (12.84), 2R,4R (13.00−
13.02). The retention times (min) of the derivatives from the
hydrolysate of spumigin J (1) were as follows: 2S,4S (11.74), 2S,4R
(11.86), 2R,4S (12.85), 2R,4R (13.02). The retention times (min) of
spumigin A (2) hydrolysate derivatives were as follows: 2S,4S (11.72),
2S,4R (11.87), 2R,4S (12.92), and 2R,4R (13.02).
Cysteine and Serine Protease Inhibitory Activity Assays.
Cathepsin B was purchased from Sigma Chemical Co., and the Z-Phe-
Arg-MCA substrate from Peptide Institute, Inc. The cathepsin B
inhibitory activity was determined using the modified method of
Greenspan et al.20 The cathepsin B enzyme was dissolved in 0.1 M
phosphate buffer with 1.33 mM EDTA·2Na, 2.7 mM DTT, and 0.03%
Brij-35, adjusted to pH 8.2 for the preparation of a 10 U/mL enzyme
solution. A 200 μg/mL solution of Z-Phe-Arg-MCA in the buffer was
used as the substrate. The buffer (80 μL), enzyme (50 μL), and test
solution (20 μL) were added to each 96-plate well and preincubated at
37 °C for 10 min, and substrate (50 μL) was added to start the
reaction. The fluorescence was measured directly at 390 nm as
excitation wavelength and 460 nm as the emission wavelength at an
incubation temperature of 37 °C before and after 30 min.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional information concerning 1H, 13C, and 2D-NMR
1
spectra of spumigins J (1) and A (2); H NMR spectra of 4-
methylproline stereoisomers. This material is available free of
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81-11-706-4519. Fax: +81-11-706-4867. E-mail: okino@
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is a result of the financial support from the Hokkaido
University Clark Foundation (540-1) and Global COE
Research Grant for Young Scientists (B-0904-52). A.R.J.A.
would like to thank the Ministry of Education, Culture, Sports,
Science, and Technology Japan (MEXT) for the fellowship.
NMR runs were done by Dr. Y. Kumaki of Graduate School of
Science, Hokkaido University.
The thrombin enzyme, from bovine plasma, was purchased from
Sigma-Aldrich, while the Bz-Phe-Val-Arg·pNA HCl from Bachem was
considered as a substrate in the thrombin inhibitory activity assay. The
thrombin inhibitory activity assay was determined by the modified
method of Laszlo21 and Yamaguchi22 et al. Two stock solutions were
prepared before the assay experiment. The 0.15 M, pH 8.2 Tris-
imidazole buffer was prepared as follows: (I) 1.211 g of Tris and 0.681
g of imidazole in 200 mL of 0.1 M HCl; (II) 1.211 g of Tris, 0.681 g of
imidazole, and 0.585 g of NaCl in 300 mL of distilled water. Solutions
I and II were mixed proportionally to make a final pH of 8.2. The
enzyme previously dissolved in Tris-imidazole buffer was used to make
up 5.32 U/mL thrombin solutions. Substrate (2.0 mg) was diluted
with 200 μL of DMSO and a 20-fold buffer. The enzyme (90 μL) and
sample (20 μL) solutions were added to each well of a 96-well plate
and preincubated for 5 min at 37 °C. A substrate solution (90 μL) was
added to start the reaction, and the absorbance was read at 405 nm
before and after 30 min with incubation at 37 °C.
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dx.doi.org/10.1021/np300282a | J. Nat. Prod. 2012, 75, 1546−1552