Taxol Pathway 10-O-Acetyltransferase
A R T I C L E S
To 7,13-bis(triethylsilyl)baccatin III (287 mg, 0.35 mmol)
dissolved in dry N,N-dimethylformamide (2 mL) at 0 °C was added
imidazole (72 mg, 1.1 mmol), and the solution was stirred for 2
min. Chlorodimethylsilane (0.11 mL, 1.1 mmol) was added
dropwise; the reaction was stirred at 0 °C for 45 min, diluted with
EtOAc (30 mL), and washed with water (4 × 20 mL). The organic
phase was dried (Na2SO4) and concentrated under vacuum. The
product was purified from the crude mixture by PTLC (15:85 (v/
v) EtOAc:hexanes) to give 1-dimethylsilyl-7,13-bis(triethylsilyl)
baccatin III product (95% yield, 99% purity by 1H NMR analysis).
7 Hz, 7R), 5.57 (d, J ) 5 Hz, 2ꢀ), 6.22 (s, 10R), 7.46 (m), 7.57
(m), 8.06 (m) [m-H, p-H, o-H of OBz, respectively].
Synthesis of 13-Acetyl-4-DAB. To a solution of 4-DAB (20
mg, 0.04 mmol) in THF (2 mL) were added dimethylaminopyridine
(23 mg, 0.18 mmol), triethylamine (5 µL, 0.04 mmol), and acetic
anhydride (4 µL, 0.04 mmol). The solution was stirred for 15 min
at 0 °C, the reaction was concentrated in vacuo, and the 13-acetyl-
4-DAB was purified by PTLC (80:20 (v/v) EtOAc/hexanes, Rf )
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0.41) (65% yield, at >98% purity by H NMR analysis). ESI-MS
(positive ion mode), m/z: 587 (MH+), 604 (M + NH4+), 609 (M +
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Na+). H NMR (300 MHz, CDCl3) δ: 1.15 (s, CH3-16), 1.18 (s,
Sodium bis(2-methoxyethoxy)aluminum hydride (110 µL) was
added dropwise to a solution of 1-dimethylsilyl-7,13-bis(triethyl-
silyl)baccatin III (120 mg, 0.14 mmol) in dry tetrahydrofuran (THF)
(3 mL) at 0 °C. The reaction was stirred for 40 min and then
quenched with 1 mL of saturated sodium tartarate solution for 10
min. The solution containing crude product was diluted with EtOAc
(40 mL), washed with an equal amount of water, and dried
(Na2SO4). The organic layer was removed under vacuum, and the
crude product was purified by PTLC (20:80 (v/v) ethyl acetate:
hexane) to give 1-dimethylsilyl-4-deacetyl-7,13-bis(triethylsilyl)
CH3-17), 1.38 (s, CH3-19), 1.94 (s, CH3-18), 2.19 (s, OC(O)CH3
at 10ꢀ), 2.23 (s, OC(O)CH3 at 13R), 2.39 (m, 6R), 2.49 (m, 14R,
ꢀ), 2.59 (m, 6ꢀ), 2.69 (bs, 4R-OH), 3.28 (d, J ) 6 Hz, 3R), 3.98
(dd, J ) 6 Hz, J ) 6 Hz, 7R), 4.08 (d, J ) 9 Hz, 20ꢀ), 4.32 (d, J
) 9 Hz, 20R), 4.81 (dd, J ) 4 Hz, J ) 4 Hz, 5R), 5.62 (d, J ) 6
Hz, 2ꢀ), 5.94 (m, 13ꢀ), 6.29 (s, 10R), 7.46 (t, J ) 6 Hz, J ) 6
Hz), 7.57 (m), 7.80 (m) [m-H, p-H, o-H of OBz, respectively].
Synthesis of 7,13-Diacetyl-4-DAB. Analogous to the procedures
described above for the synthesis of 13-acetyl-4-DAB, to a solution
of 4-DAB (18 mg, 0.033 mmol) dissolved in THF (3 mL) were
added dimethylaminopyridine (19 mg, 0.16 mmol), triethylamine
(8.8 µL, 0.06 mmol), and acetic anhydride (9 µL, 0.09 mmol). The
reaction was stirred at 23 °C for 20 min, diluted with 10 mL of
EtOAc, and washed with water (pH 1.0). The organic layer was
concentrated, and the 7,13-diacetyl-4-DAB was purified by PTLC
(80:20 (v/v) ethyl acetate:hexanes, Rf ) 0.74), isolated in 75% yield,
and judged to be >99% pure by NMR. ESI-MS (positive ion mode),
m/z: 630 (MH+), 647 (M + NH4+), 652 (M + Na+). 1H NMR (300
MHz, CDCl3) δ: 1.12 (s, CH3-16), 1.15 (s, CH3-17), 1.7 (s, CH3-
19), 2.00 (s, CH3-18), 2.04 (s, OC(O)CH3 at 10ꢀ), 2.17 (s,
OC(O)CH3 at 7ꢀ), 2.21 (s, OC(O)CH3 at 13R), 2.45 (m, 6R, ꢀ),
2.48 (m, 14R, ꢀ), 2.68 (bs, 4R-OH), 3.42 (d, J ) 7 Hz, 3R), 4.14
(d, J ) 8 Hz, 20ꢀ), 4.29 (d, J ) 8 Hz, 20R), 4.82 (dd, J ) 3 Hz,
J ) 3 Hz, 5R), 5.19 (dd, J ) 7 Hz, J ) 7 Hz, 7R), 5.62 (d, J ) 6
Hz, 2ꢀ), 5.92 (m, 13ꢀ), 6.20 (s, 10R), 7.46 (t, J ) 9 Hz), 7.59 (m),
δ 8.00 (m) [m-H, p-H, o-H of OBz, respectively].
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baccatin III (70% yield, 99% purity by H NMR).
To a solution of 1-dimethylsilyl-4-deacetyl-7,13-bis(triethylsi-
lyl)baccatin III (56.6 mg, 0.068 mmol) in THF (2 mL) was added
pyridine (400 µL). The solution was stirred and cooled to 0 °C,
and 60% HF/pyridine solution (400 µL) was added dropwise over
20 min to remove the silyl protecting groups. The reaction was
warmed to room temperature and, after 6 h, diluted with EtOAc
(20 mL). The aqueous phase was removed, and the remaining
organic fraction was washed with sodium bicarbonate (0.4 M),
water, and brine (2 × 20 mL each), dried over sodium sulfate,
filtered, and finally concentrated under vacuum. The product was
purified by PTLC (80:20 (v/v) EtOAc/hexanes, Rf ) 0.24) to give
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the final product, 4-DAB (60% yield, 99% purity by H NMR
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analysis). H NMR (300 MHz, CDCl3) δ: 1.10 (s, CH3-16), 1.15
(s, CH3-17), 1.61 (s, CH3-19), 2.14 (s, CH3-18), 2.29 (s, OC(O)CH3
at 10ꢀ), 2.50 (m, 6R, ꢀ), 2.58 (m, 14R, ꢀ), 3.50 (bs, 4R-OH), 3.63
(d, J ) 6 Hz, 3R), 4.09 (dd, J ) 6 Hz, J ) 6 Hz, 7R), 4.18 (d, J
) 8 Hz, 20R), 4.43 (d, J ) 8 Hz, 20ꢀ), 4.67 (bt, 13ꢀ), 4.85 (dd, J
) 4 Hz, J ) 4 Hz, 5R), 5.64 (d, J ) 6 Hz, 2ꢀ), 6.34 (s, 10R), 7.50
(m), 7.64 (m), 8.09 (m) [m-H, p-H, o-H of OBz, respectively].
Synthesis of 7-Acetyl-4-DAB. To a solution of 4-DAB (20 mg,
0.037 mmol) in N,N-dimethylformamide (2 mL) were added
imidazole (25 mg, 0.37 mmol) and triethylsilyl chloride (12.4 µL,
0.074 mmol), and the reaction was stirred at 45 °C for 3 h under
nitrogen to obtain a 1:1 mixture of starting material and 13-
triethylsilyl-4-DAB, which was purified by PTLC (80:20 (v/v)
EtOAc/hexanes). To a solution of this silylated product (6 mg, 0.01
mmol) in tetrahydrofuran (2 mL) was added dimethylaminopyridine
(5.6 mg, 0.05 mmol), triethylamine (1.3 µL, 0.01 mmol), and acetic
anhydride (172 µL, 0.2 mmol). The solution was stirred for 3 h at
23 °C and concentrated in vacuo; the sample was then loaded onto
a PTLC plate and eluted with 80:20 (v/v) EtOAc:hexanes to give
7-acetyl-13-triethylsilyl-4-DAB (99% yield). To the latter product
(7 mg, 0.01 mmol) in THF (2 mL) and pyridine (60 µL) at 0 °C
under nitrogen was added (dropwise) a 60% solution of HF in
pyridine (60 µL) over 5 min, and the reaction was warmed to room
temperature. After 2 h, the solution was diluted with EtOAc (10
mL), quenched with water (5 mL), and extracted with EtOAc (2 ×
5 mL). The organic extracts were dried (Na2SO4) and evaporated
in vacuo. The 7-acetyl-4-DAB was purified by PTLC (80:20 (v/v)
EtOAc/hexanes, Rf ) 0.59) and isolated in 95% yield, 94% purity
by 1H NMR analysis. ESI-MS (positive ion mode), m/z: 587 (MH+),
604 (M + NH4+), 609 (M+Na+). 1H NMR (300 MHz, CDCl3) δ:
1.02 (s, CH3-16), 1.13 (s, CH3-17), 1.66 (s, CH3-19), 2.04 (s,
OC(O)CH3 at 7ꢀ), 2.12 (s, CH3-18), 2.18 (s, OC(O)CH3 at 10ꢀ),
2.44 (m, 6R), 2.49 (m, 14R, ꢀ), 2.64 (m, 6ꢀ), 3.68 (d, J ) 6 Hz,
3R), 4.2 (d, J ) 8 Hz, 20ꢀ), 4.30 (d, J ) 8 Hz, 20R), 4.62 (bd,
13ꢀ), 4.84 (dd, J ) 3 Hz, J ) 4 Hz, 5R), 5.21 (dd, J ) 7 Hz, J )
Synthesis of 13-Acetylbaccatin III. 13-Acetylbaccatin III was
synthesized similarly to a procedure described for the synthesis of
13-butyrylbaccatin III,11 except acetic anhydride was used in place
of butyryl chloride as the acyl donor. ESI-MS (positive ion mode),
m/z: 629 (MH+), 646 (M + NH4+), 651 (M + Na+). 1H NMR (300
MHz, CDCl3) δ: 1.13 (s, CH3-16), 1.21 (s, CH3-17), 1.65 (s, CH3-
19), 1.89 (d, J ) 2 Hz, CH3-18), 2.19 (s, OC(O)CH3 at 10ꢀ), 2.22
(s, OC(O)CH3 at 4R), 2.23 (m, 6R, ꢀ), 2.31 (s, OC(O)CH3 at 13R),
2.54 (m, 14R, ꢀ), 3.81 (d, J ) 7 Hz, 3R), 4.14 (d, J ) 9 Hz, 20R),
4.29(d, J ) 9 Hz, 20ꢀ), 4.42 (dd, J ) 7 Hz, J ) 7 Hz, 7R), 4.95
(dd, J ) 2 Hz, J ) 2 Hz, 7R), 5.64 (d, J ) 7 Hz, 2ꢀ), 6.16 (m,
13ꢀ), 6.28 (s, 10R), 7.46 (m), 7.59 (m), 8.05 (m) [o-H, p-H, m-H
of OBz, respectively].
Expression of Acyltransferases and Screening Enzyme
Activity with 4-DAB Compounds. The Taxus cuspidata cDNA
clones of the five acyltransferases on the paclitaxel pathway were
used in their original recombinant expression system as described
in previous studies12 or subcloned into a new expression vector
and transferred to a suitable bacterial host. For directional ligation
of a full-length acyltransferase into a new expression vector cut
with restriction enzymes PCR primers were used to install
appropriate 5′- and 3′-overhangs into the DNA amplicon by a
cohesive-end ligation method described previously.13 The 2R-O-
benzoyltransferase was expressed from pCWori+ in E. coli
JM109;14 the 5R-O-acetyltransferase8 was expressed from pSBET
(11) Loncaric, C.; Ward, A. F.; Walker, K. D. Biotechnol. J. 2007, 2, 266–
274.
(12) Jennewein, S.; Wildung, M. R.; Chau, M.; Walker, K.; Croteau, R.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9149–9154.
(13) Zeng, G. BioTechniques 1998, 25, 788.
(14) Walker, K.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13591–
13596.
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J. AM. CHEM. SOC. VOL. 130, NO. 50, 2008 17189