Journal of Natural Products
Article
methyl-5-decanol (5) (a combined weight of 1.5 g). A portion of this
mixture (30 mg) was converted to the mixture of (S)-(−)-MTPA ester
(6a and 7a) with (R)-(−)-MTPACl (50 μL) in pyridine−CH2Cl2
(1:1, 500 μL). After the addition of H2O, the mixture was extracted
with CHCl3, and the organic layer was purified by RP-HPLC. The 2D
NMR data showed that the product was a 2:1 mixture of 6a and 7a.
The corresponding (R)-(+)-MTPA esters (6b and 7b) were prepared
in the same manner.
HDAC Assay. Measurements of HDAC activities of human sirtuins
including SIRT1, -2, and -3 were carried out using fluorogenic peptide
substrates. Recombinant sirtuin proteins were incubated with a
fluorescent peptide (100 μM Ac-RHKK(Ac)-MCA for SIRT1 and
SIRT2; 100 μM Ac-VSTPVK(Ac)-MCA for SIRT3) and NAD (final
concentration 1 mM) in 20 μL of assay buffer (50 mM Tris-HCl [pH
9.0], 4 mM MgCl2, 0.2 mM DTT). After 60 min at 37 °C, trypsin
(final concentration 20 mg/mL) was added and samples were
incubated for an additional 15 min at 37 °C. The fluorescence of
released amino methyl coumarin (λex: 370 nm and λem: 460 nm) was
measured using a fluorescence plate reader (Molecular Devices).
Enzymatic activity assays for HDAC1, -4, and -6 were performed as
described previously.34
(S)-MTPA ester of 6a: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz)
δH 7.71, 7.40, 5.31 (1H, m, H-5), 1.67 (2H, m, H-6), 1.53 (1H, m, H-
4), 1.48 (1H, m, H-4), 1.42 (1H, m, H-2), 1.38 (2H, m, H-7), 1.33
(1H, m, H-3), 1.33 (2H, m, H-8), 1.09 (1H, m, H-2), 0.93 (3H, t, H-
10), 0.87 (3H, t, H-1), 0.85 (3H, d, 3-Me); 13C NMR (CDCl3−
C5D5N) δC 76.0 (CH, C-5), 40.7 (CH2, C-4), 34.3 (CH2, C-6), 31.7
(CH2, C-9), 30.8 (CH, C-3), 28.7 (CH2, C-2), 25.0 (CH2, C-7), 22.6
(CH2, C-8), 19.0 (CH3, 3-Me), 14.1 (CH3, C-10), 11.2 (CH3, C-1);
ESIMS m/z 411 [M + Na]+.
ASSOCIATED CONTENT
■
S
* Supporting Information
(S)-MTPA ester of 7a: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz)
δH 7.71, 7.40, 5.31 (1H, m, H-5), 1.78 (1H, m, H-4), 1.60 (2H, m, H-
6), 1.46 (1H, m, H-3), 1.32 (1H, m, H-2), 1.31 (1H, m, H-4), 1.26
(2H, m, H-8), 1.23 (2H, m, H-7), 1.22 (1H, m, H-2), 0.95 (3H, d, 3-
Me), 0.91 (3H, t, H-1), 0.90 (3H, t, H-10); 13C NMR (CDCl3−
C5D5N) δC 76.0 (CH, C-5), 41.0 (CH2, C-4), 34.5 (CH2, C-6), 32.1
(CH2, C-9), 31.0 (CH, C-3), 30.0 (CH2, C-2), 24.6 (CH2, C-7), 22.6
(CH2, C-8), 19.3 (CH3, 3-Me), 14.1 (CH3, C-10), 11.2 (CH3, C-1) ;
ESIMS m/z 411 [M + Na]+.
The Supporting Information is available free of charge on the
NMR data for 1, 2, 3, 8a, 8b, 9, 10, 11, 12a, 12b, 13a,
13b, a mixture of 6a and 6b, and a mixture of 7a and 7b,
and tandem FABMS data for 3 and 9 (PDF)
(R)-MTPA ester of 6b: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz)
δH7.71, 7.40, 5.31 (1H, m, H-5), 1.59 (2H, m, H-6), 1.58 (2H, m, H-
4), 1.46 (1H, m, H-2), 1.46 (1H, m, H-3), 1.25 (2H, m, H-8), 1.21
(2H, m, H-7), 1.16 (1H, m, H-2), 0.93 (3H, d, 3-Me), 0.89 (3H, t, H-
1) 0.87 (3H, t, H-10); 13C NMR (CDCl3−C5D5N) δC 76.0 (CH, C-
5), 41.0 (CH2, C-4), 33.9 (CH2, C-6), 31.7 (CH2, C-9), 31.1 (CH, C-
3), 29.2 (CH2, C-2), 24.6 (CH2, C-7), 22.7 (CH2, C-8), 19.5 (CH3, 3-
Me), 14.1 (CH3, C-10), 11.2 (CH3, C-1) ; ESIMS m/z 411[M + Na]+.
(R)-MTPA ester of 7b: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz)
δH 7.71, 7.40, 5.31 (1H, m, H-5), 1.69 (1H, m, H-4), 1.71 (1H, m, H-
6), 1.60 (1H, m, H-6), 1.37 (2H, m, H-7), 1.32 (2H, m, H-8), 1.24
(1H, m, H-4), 1.23 (1H, m, H-3), 1.21 (1H, m, H-2), 1.12 (1H, m, H-
2), 0.91 (3H, t, H-10), 0.88 (3H, d, 3-Me), 0.79 (3H, t, H-1); 13C
NMR (CDCl3−C5D5N) δC 76.0 (CH, C-5), 40.9 (CH2, C-4), 34.9
(CH2, C-6), 31.7 (CH2, C-9), 31.1 (CH, C-3), 30.1 (CH2, C-2), 25.1
(CH2, C-7), 22.7 (CH2, C-8), 18.9 (CH3, 3-Me), 14.1 (CH3, C-10),
11.4 (CH3, C-1); ESIMS m/z 411 [M + Na]+.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166. E-
Author Contributions
‡K. Takada and Y. Imae contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural
Products” (23102007) and JSPS KAKENHI Grant Numbers
25252037, 25712024, 25660163, 15K14800, and 15K14799
from The Ministry of Education, Culture, Sports, Science and
Technology, Japan. We thank Professor T. Kusumi, Professor
Emeritus, Tokushima University, for valuable discussion and
Ms. A. Nakata, RIKEN, for conducting the enzyme inhibitory
assay.
Preparation of 13a and 13b. Compound 12a was dissolved in 10
M NaOH−MeOH (1:9, 500 μL) and left at rt for 1 h to afford the
20,28-bis-MTPA ester as detected by LCMS. After neutralization with
1 N HCl the solution was extracted with CHCl3. The organic layer was
dried and the residue was dissolved in MeOH. To this solution was
added NaIO4, and the solution stirred at rt for 2 h. The resulting
aldehyde as detected by LCMS was dissolved in 90% HCOOH−35%
H2O2 (2:1, 500 μL) and left at rt. The product was dried and
redissolved in MeOH. To this solution was added TMS diazomethane
in n-hexane, and the reaction mixture was dried and purified by RP-
HPLC (80−100% MeCN) to afford the bis-MTPA ester 13a. Bis-
MTPA ester 13b was prepared in the same manner.
REFERENCES
■
(1) Witt, O.; Deubzer, H. E.; Milde, T.; Oehme, I. Cancer Lett. 2009,
277, 8−21.
1
13a: H and 13C NMR, Table S4.
(2) Dutnall, R.; Pillus, L. Cell 2001, 105, 161−164.
(3) Taunton, J.; Hassig, C.; Schreiber, S. Science 1996, 272, 408−411.
(4) Weichert, W.; Roeske, A.; Gekeler, V.; Beckers, T.; Ebert, M. P.
A.; Pross, M.; Dietel, M.; Denkert, C.; Roecken, C. Lancet Oncol. 2008,
9, 139−148.
1
13b: H and 13C NMR, Table S4.
Preparation of Recombinant Human HDAC Proteins.
Purification of recombinant GST-fused SIRT2 catalytic domain was
performed as described previously.32 Purification of the recombinant
His-tagged SIRT3 protein was performed as described in the
literature.33 For preparation of the SIRT1 protein, pCold TF DNA-
SIRT1 was introduced into Escherichia coli BL21 (DE3). The
expression of recombinant proteins was induced with 0.3 mM
isopropyl-β-D-galactopyranoside (IPTG) at 16 °C for 24 h.
Purification of the (His)6-fused proteins was carried out using the
nickel affinity (GE Healthcare) interaction, followed by anion-
exchange chromatography (GE Healthcare). Purifications of the
zinc-dependent HDACs including HDAC1, -4, and -6 were performed
as described previously.34
(5) Weichert, W.; Roeske, A.; Gekeler, V.; Beckers, T.; Stephan, C.;
Jung, K.; Fritzsche, F. R.; Niesporek, S.; Denkert, C.; Dietel, M.;
Kristiansen, G. Br. J. Cancer 2008, 98, 604−610.
(6) Weichert, W.; Roeske, A.; Niesporek, S.; Noske, A.; Buckendahl,
A.; Dietel, M.; Gekeler, V.; Boehm, M.; Beckers, T.; Denkert, C. Clin.
Cancer Res. 2008, 14, 1669−1677.
(7) Inoue, S.; Mai, A.; Dyer, M. J. S.; Cohen, G. M. Cancer Res. 2006,
66, 6785−6792.
(8) Keshelava, N.; Davicioni, E.; Wan, Z.; Ji, L.; Sposto, R.; Triche, T.
J.; Reynolds, C. P. J. Natl. Cancer Inst. 2007, 99, 1107−1119.
F
J. Nat. Prod. XXXX, XXX, XXX−XXX