Substrate Specificity of a Taxane 2R-O-Benzoyltransferase
Journal of Natural Products, 2010, Vol. 73, No. 2 157
5.22 (dd, J ) 3.6, 8.7 Hz, H-7), 6.12 (t, J ) 8.8 Hz, H-13), 6.16 (s,
H-10); LC-ESIMS (positive ion mode) m/z 589.20 [M + Na]+.
Synthesis of 7-O-Acetylbaccatin III. To a stirred solution of
baccatin III (100 mg, 170 µmol) in dry THF (3 mL) at 23 °C under N2
were added Ac2O (5 equiv), 4-DMAP (5 equiv), and Et3N (24 µL, 172
µmol). The reaction was monitored by TLC, and upon completion, the
reaction was diluted with EtOAc (50 mL) and quenched with H2O (10
mL). The mixture was stirred for 15 min, and the aqueous fraction
was separated and extracted with EtOAc (2 × 25 mL). The combined
organic fractions were washed with saturated CuSO4, brine, 0.1 M HCl,
and H2O and dried over anhydrous Na2SO4. The organic solvent was
evaporated, and the crude product was purified by silica gel flash column
chromatography (40:60 to 60:40 (v/v), linear gradient of EtOAc in
hexanes) to yield pure 7-O-acetylbaccatin III (104 mg, 97% yield): 1H
NMR (500 MHz, CDCl3) δ 1.06 (s, H-16), 1.11 (s, H-17), 1.77 (s,
H-19), 1.80 (m, H-6ꢀ), 2.01 (s, C(O)CH3 at C-7), 2.08 (s, H-18), 2.15
(s, C(O)CH3 at C-10), 2.26 (s, C(O)CH3 at C-4), 2.59 (ddd, J ) 2.5,
8.5, 14.2 Hz, H-6R), 3.98 (d, J ) 6.5 Hz, H-3), 4.13 (d, J ) 8.5 Hz,
H-20R), 4.29 (d, J ) 8.5 Hz, H-20ꢀ), 4.83 (br t, H-13), 4.95 (d, J )
8.5 Hz, H-5), 5.58 (dd, J ) 3.5, 7.0 Hz, H-7), 5.60 (d, J ) 3.5 Hz,
H-2), 6.25 (s, H-10), 7.46-8.08 (aromatic protons) (cf. Scheme 1 for
position of proton and the number of protons at a particular position);
LC-ESIMS (positive ion mode), m/z 651.20 [M + Na]+.
Synthesis of 7-O-Acetyl-13-oxobaccatin III. To a stirred solution
of 7-O-acetylbaccatin III (80 mg, 127 µmol) in dry CH2Cl2 (10 mL) at
23 °C under N2 was added activated MnO2 powder (1 g). The reaction
was monitored by TLC, and upon completion of the reaction, the
mixture was filtered to remove excess MnO2. The filtrate was diluted
with EtOAc (15 mL) and quenched with H2O (10 mL). The aqueous
fraction was separated and extracted with EtOAc (2 × 25 mL). The
combined organic fractions were washed with brine and H2O and dried
over anhydrous Na2SO4. The organic solvent was evaporated, and the
crude product was purified by silica gel flash column chromatography
(30:70 to 60:40 (v/v), linear gradient of EtOAc in hexanes) to yield
pure 7-O-acetyl-13-oxobaccatin III (79 mg, 100% yield): 1H NMR (500
MHz, CDCl3) δ 1.18 (s, H-16), 1.19 (s, H-17), 1.74 (s, H-19), 1.78
(ddd, J ) 1.5, 3.0, 12.5 Hz, H-6ꢀ), 2.01 (s, H-18), 2.09 (s, C(O)CH3
at C-7), 2.17 (s, C(O)CH3 at C-10), 2.19 (s, C(O)CH3 at C-4), 2.58
(ddd, J ) 7.5, 9.5, 14.5 Hz, H-6R), 2.64 (d, J ) 19.5 Hz, H-14ꢀ), 2.92
(d, J ) 19.5 Hz, H-14R), 4.00 (d, J ) 6.5 Hz, H-3), 4.08 (d, J ) 7.0
Hz, H-20R), 4.30 (d, J ) 8.5 Hz, H-20ꢀ), 4.90 (d, J ) 8.5 Hz, H-5),
5.56 (dd, J ) 7.5, 10.5 Hz, H-7), 5.63 (d, J ) 7.0 Hz, H-2), 6.33 (s,
H-10), 7.46-8.03 (aromatic protons) (cf. Scheme 1 for position of
proton and the number of protons at a particular position); LC-ESIMS
(positive ion mode), m/z 644.06 [M + NH4]+.
Synthesis of [13-3H]-7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III.
To a stirred solution of [13-3H]-7,13-O,O-diacetylbaccatin III (21 mg,
0.59 mCi, 30 Ci/mol) in dry THF (5 mL) at 0 °C under N2 was added
(dropwise) bis(2-methoxyethoxy)aluminum hydride (Red-Al) (>65 wt
% in toluene, 3 equiv). After stirring for 2.5 h, the reaction was
quenched by dropwise addition of saturated NH4Cl at 0 °C, and the
mixture was stirred for 10 min, warmed to room temperature, and
diluted with EtOAc (5 mL) and H2O (5 mL). The aqueous phase was
separated and extracted again with EtOAc (2 × 5 mL). The combined
organic fractions were washed with brine and H2O and then dried over
anhydrous Na2SO4. The solvent was evaporated, and the crude product
was purified by silica gel PTLC (60:40 (v/v), EtOAc/hexanes) to yield
pure [13-3H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (6.6 mg, 0.35
mCi, 99% radiochemical purity by radio-HPLC, 30 Ci/mol specific
activity). The LC-ESIMS (positive ion mode) fragmentation profile and
1
the H NMR spectrum of authentic unlabeled 7,13-O,O-diacetyl-2-O-
debenzoylbaccatin III, obtained as described,6 were identical to those
obtained for the [3H]-labeled material.
Synthesis of Aroyl/alkyl CoA Thioesters. Several aroyl CoA donors
(heteroaroyls and variously substituted benzoyls) were synthesized via
a previously described method that proceeds through a mixed ethyl
carbonate anhydride.5 Briefly, Et3N (3.0 µL, 30 µmol) was added to a
solution of the a carboxylic acid (27 µmol) in 5:2 CH2Cl2/THF (v/v,
1.4 mL) under N2 gas. The mixture was stirred for 10 min at 23 °C,
ethyl chloroformate (2.9 µL, 30 µmol) was added in one portion, and
the reaction was stirred for 1 h at 23 °C. The solvents were evaporated
under reduced pressure, and the residue was dissolved in 0.5 mL of
t-BuOH. Coenzyme A as the sodium salt (23 mg, 30 µmol dissolved
in 0.5 mL of 0.4 M NaHCO3) was added to the solution, and the mixture
was stirred for 0.5 h at 23 °C and then quenched with dropwise addition
of 1 M HCl, to adjust the pH to 3-5. The solvents were evaporated
under vacuum, and the residue was dissolved in H2O (5 mL, pH 5)
and loaded onto a C18 silica gel column (10% capped with TMS) that
was washed with 100% MeOH (50 mL) and pre-equilibrated with
distilled H2O (100 mL, pH 5). The sample was washed with H2O (100
mL, pH 5) and then eluted with 15% MeOH in H2O (50 mL, pH 5).
The fractions containing aroyl CoA, as determined by TLC, were
combined, and the solvent was evaporated to yield pure product
(95-99% yield; data reported elsewhere22). The syntheses of cyclo-
hexanoyl CoA and phenylacetyl CoA have not been previously reported,
1
and their H NMR data are included in the Supporting Information.
TBT Activity Assay and Protein Purification. An assessment of
the relative quantity of solubly expressed wtTBT and mTBT in E. coli
(described above) revealed that the recombinant mTBT enzyme
partitioned into the soluble enzyme fraction approximately 5-fold better
than the wild-type form. Therefore, an aliquot (0.9 mL) of the soluble
mTBT preparation as a crude mixture, described in the previous section,
was separately incubated at 31 °C with [13-3H]-7,13-O,O-diacetyl-2-
O-debenzoylbaccatin III (33 µM, 1 µCi) and 1 mM benzoyl CoA, and
the total volume was adjusted to 1 mL with assay buffer. After 4 h,
the reaction was quenched and extracted with EtOAc (4 × 2 mL), the
respective organic fractions were combined, the solvent was evaporated,
and the residue was dissolved in 50 µL of MeCN. A 25 µL aliquot
was loaded onto a reversed-phase column (Econosphere C18, 5 µm,
250 × 4.6 mm, Alltech, Mentor, OH) and eluted at 1 mL/min with a
linear gradient of solvent A:solvent B [solvent A: 97.99% H2O with
2% MeCN and 0.01% H3PO4 (v/v); solvent B: 99.99% MeCN with
0.01% H3PO4 (v/v)] from 70:30 (v/v) to 40:60 (v/v) over 31 min, then
to 0:100 (v/v) over another 2 min, and returning to the initial conditions
over 7 min with a 5 min hold on an Agilent 1100 HPLC system (Agilent
Technologies, Wilmington, DE). The HPLC was connected in series
with a UV detector and a Packard Radiomatic Flow-One Beta 150TR
radioactivity detector (Perkin-Elmer, Shelton, CT), which mixed the
effluent with 3a70B Complete Counting Cocktail (Research Products
International, Mount Prospect, IL). The UV absorbance and radioactivity
profiles of the biosynthetic product isolated from the assays containing
crude enzyme extracts of cells expressing tbt mutant were compared
to those of the control assays containing extracts of cells transformed
with vector without an insert.
Synthesis of [13-3H]-7-O-Acetylbaccatin III. To a stirred solution
of 7-O-acetyl-13-oxobaccatin III (7 mg, 11 µmol) in dry THF (5 mL)
at 0 °C under N2 were added sodium [3H]borohydride (333 µmol,
specific activity of 150 Ci/mol) dissolved in a minimum amount of
0.01 M NaOH. The reaction mixture was warmed to room temperature,
and the progress of the reaction was monitored by TLC. After 3 h,
more 7-O-acetyl-13-oxobaccatin III (14.0 mg, 22 µmol) was added to
the reaction, which was stirred for 3 h, diluted with EtOAc (5 mL),
and quenched with H2O (5 mL). The mixture was stirred for 15 min,
and the aqueous fraction was separated and extracted with EtOAc (2
× 5 mL). The combined organic fractions were dried over anhydrous
Na2SO4, the solution was filtered, the filtrate was concentrated in vacuo,
and the crude product was purified on silica gel PTLC (60:40 (v/v),
EtOAc/hexanes) to yield pure [13-3H]-7-O-acetylbaccatin III (20 mg,
0.61 mCi, 95% radiochemical purity by radio-HPLC).
Synthesis of [13-3H]-7,13-O,O-Diacetylbaccatin III. To a stirred
solution of [13-3H]-7-O-acetylbaccatin III (20 mg, 0.61 mCi) in dry
THF (5 mL) at 23 °C under N2 were added Ac2O (100 equiv), 4-DMAP
(40 equiv), and Et3N (24 µL, 172 µmol), and the starting material was
depleted after 28 h, as determined by TLC monitoring of the reaction
progress. An aliquot of unlabeled 7-O-acetylbaccatin III (12 mg, 19
µmol) was added to the reaction and stirred in the solution for 3 h.
The mixture was then diluted with EtOAc (5 mL), quenched with H2O
(5 mL), and stirred for 15 min; the aqueous fraction was separated and
extracted with EtOAc (2 × 5 mL). The combined organic fractions
were washed with saturated CuSO4, brine, 0.1 M HCl, and H2O and
dried over anhydrous Na2SO4. The organic solvent was evaporated,
and the crude product was purified on silica gel PTLC (60:40 (v/v),
EtOAc/hexanes) to yield pure [13-3H]-7,13-O,O-diacetylbaccatin III (21
mg, 0.59 mCi, 97% radiochemical purity by radio-HPLC).
Once active mTBT was verified by radiochemically guided assays,
plasmid p28PK-TBT encoding the mutant tbt was expressed in large-
scale (4 L), transformed E. coli cell cultures. After 16 h, the cells were
harvested and processed as before except that, instead of 3-(N-
morpholino)-2-hydroxypropanesulfonic acid buffer, the cell pellet was
resuspended in 50 mM Na3PO4 buffer (pH 8.0), which was compatible