Journal of Natural Products
Article
(
IR) spectra were recorded on a Thermo Scientific Nicolet iS5 FT-IR
The residue was purified by flash column chromatography eluting
with EtOAc/hexanes (1:4) to afford 4.51 g (64%) of 4 as a white
foam.
1
13
spectrometer. H and C NMR spectra were run on a Bruker AV-III
00, Bruker AVIIIHD 500, or Bruker Avance 400 spectrometer in
5
CDCl at 298 K. High-resolution mass spectra were obtained on a
16-Allyloxytabersonine (27). To a solution of 4 (100 mg, 0.280
mmol) in DMF (2 mL) were added K CO (157 mg, 1.14 mmol) and
allyl bromide (0.074 mL, 0.85 mmol). The reaction mixture was
3
Waters LCT Premier XE LC/MS system. Single-crystal X-ray
diffraction was performed using a Bruker KAPPA APEX II DUO
diffractometer with Mo Kα radiation obtained from a sealed
molybdenum tube with a TRIUMPH monochromator. The sample
was mounted on a MiTeGen loop with Paratone N oil and cooled to
2
3
heated to 60 °C and stirred for 1 h. The reaction mixture was cooled
to rt, and aqueous NaHCO (5 mL) was added. The reaction mixture
3
was extracted with EtOAc (3 × 5 mL), washed with brine (3 × 5 mL),
173 K using an Oxford Cryostream low-temperature device. The
dried (Na SO ), and concentrated under reduced pressure. The
2
4
structure was solved using direct methods and refined using full-
matrix least-squares (SHELXTL). Additional experimental and
sample details are given in the crystallographic tables (see Supporting
Information). Chemical shifts are indicated in ppm relative to
tetramethylsilane (TMS, δ = 0.00) and referenced to residual solvent
residue was purified by flash column chromatography eluting with
EtOAc/hexanes (1:20) to afford 76 mg (68%) of 27 as a white foam.
N-Troc-Tabersonine (28). To a solution of tabersonine (6) (1.98
g, 5.90 mmol) and DMAP (1.44 g, 11.8 mmol) in THF (30 mL) and
DMF (10 mL) was added NaH (1.17 g, 29.5 mmol). The reaction
mixture was stirred for 1 h at 0 °C. TrocCl (2.43 mL, 17.7 mmol) was
added dropwise, and the reaction mixture was stirred an additional 3 h
1
13
signals ( H, 7.26 ppm, C centerline of CDCl triplet, 77.0 ppm).
3
Flash column chromatography was performed according to the
procedure of Still using ICN Silitech 32−63 D 60 Å silica gel with the
at rt. The reaction mixture was poured into aqueous NaHCO (24
3
5
9
indicated solvents. Thin layer chromatography was performed on
Merck 60 F254 silica gel plates. Detection was performed using UV
light, KMnO4 stain, and an iodine chamber, or PMA stain and
subsequent heating.
mL), extracted with CHCl (3 × 24 mL), washed with brine (3 × 24
3
mL), dried over Na SO , and concentrated under reduced pressure.
2
4
The residue was purified by flash column chromatography eluting
with EtOAc/hexanes (1:6) to afford 2.80 g of 28 (92%) as a white
foam.
Ester 20. To a solution of 19 (1.10 g, 2.56 mmol) in THF (10
mL) was added LHMDS (1.0 M in THF, 3.8, 3.8 mmol) at −78 °C,
and the mixture stirred for 1 h. A solution of ethyl methacrolein (9)
N-Troc-Pachysiphine (29). To a solution of N-Troc-tabersonine
(28) (2.00 g, 3.91 mmol) in CH Cl (14 mL) was added TFA (2.30
2
2
(
0.430 g, 3.81 mmol) in THF (2 mL) was added at −78 °C and
g, 19.5 mmol) at −10 °C. The reaction mixture was stirred for 15 min.
stirred for 2 h. The dry ice cooling bath was replaced with an ice−
water cooling bath, and a solution of allyl bromide (1.56 g, 12.7
mmol) in DMF (60 mL) was added. The ice bath was removed, and
the reaction was stirred for 14 h. The reaction was quenched with
saturated aqueous NH Cl and diluted with H O (10 mL), followed by
m-CPBA (2.0 g, 13.3 mmol) dissolved in CH Cl (10 mL) was added
2
2
dropwise at −10 °C. The reaction mixture was gradually warmed to rt
and stirred overnight. The reaction was quenched with aqueous
Na SO (24 mL) and aqueous NaHCO (24 mL), extracted with
2
3
3
CH Cl (3 × 24 mL), dried over Na SO , and concentrated under
4
2
2
2
2
4
extraction with EtOAc (3 × 20 mL). The combined organic layers
reduced pressure. The residue was purified by flash column
chromatography eluting with EtOAc/hexanes (1:3) to afford 800
mg of N-Troc-pachysiphine (29) (38%) as a white foam.
were washed with brine (5 × 20 mL), dried over Na SO , filtered, and
2
4
concentrated under reduced pressure. The resulting residue was
purified by flash column chromatography eluting with EtOAc/hexane
N -Troc-Pachysiphine N -Oxide (30). To a solution of N-Troc-
a
b
(
0:100 → 40:60) to afford 1.43 g of 20 as a white foam (96%, dr =
0:1).
pachysiphine (29) (655 mg, 1.24 mmol) in CH Cl (2 mL) at 0 °C
2 2
1
was added m-CPBA (643 mg, 3.73 mmol) dissolved in CH Cl (2
2 2
Extraction and isolation of (−)-Tabersonine (6). To a ground
powder of Voacanga africana seeds (100 g) was added 1% aqueous
mL) dropwise. The reaction mixture was stirred for 30 min. The
reaction was quenched with aqueous NaHCO (5 mL), extracted with
3
H SO (1 L), and the mixture was stirred for 24 h at rt. The acidic
CH Cl (3 × 5 mL), washed with brine (3 × 5 mL), dried over
2
4
2
2
solution was filtered over a pad of Celite. To this solution was added
sodium chloride (100 g), and the mixture was aged for 4 h. The
solution was filtered over another pad of Celite, extracted with CHCl3
Na SO , and concentrated under reduced pressure. The residue was
2 4
purified by column chromatography in 5% MeOH/CHCl to afford
3
240 mg (36%) of 30 as a white foam.
(
2 × 500 mL), dried over Na SO , and concentrated under reduced
Dimer 32. To a solution of N -Troc-pachysiphine N -oxide (30)
2
4
a
b
pressure. The residue was recrystallized from acetone at 0 °C,
redissolved in chloroform, washed with 10% aqueous NaOH (40
mL), H O (20 mL), and brine (20 mL), extracted with CHCl , dried
(85.0 mg, 0.156 mmol) in CH Cl (2 mL) at 0 °C was added TFAA
2 2
(0.22 mL, 1.6 mmol). The reaction mixture was stirred for 15 min,
upon which a solution of 16-allyloxytabersonine (27) (22 mg, 0.055
mmol) in CH Cl (2 mL) was added dropwise. The reaction mixture
2
3
over Na SO , and concentrated under reduced pressure. The crude
2
4
2
2
residue was purified by column chromatography eluting with EtOAc/
hexanes (1:6) to afford 6 (1.30 g, 1%) as a white foam.
was stirred at 0 °C for 1 h, then warmed to rt and stirred an additional
16 h. Aqueous NaHCO (5 mL) was added, and the reaction mixture
3
16-Hydroxytabersonine (4). A Petri plate with CSM-Ura-Ade
was extracted with CH Cl (3 × 5 mL), washed with brine (3 × 5
2
2
medium was streaked with yeast (T16H in pYedp60) and incubated
mL), dried over Na SO , and concentrated under reduced pressure.
2 4
at 30 °C for 48 h. YPG medium (500 mL) containing yeast extract
The residue was purified by flash column chromatography eluting
with EtOAc/hexanes (1:1) to afford 40 mg (78%) of 32 as a white
foam.
(
1%, 5.0 g), peptone (2%, 10.0 g), and glucose (2%, 10.0 g) in H O
2
was autoclaved. The media was inoculated with yeast, covered with
cheesecloth, and incubated at 30 °C at 250 rpm for 36 h. The media
was then poured into 16 Falcon tubes (30 mL each) and centrifuged
at 10 °C at 3000 rpm for 10 min. The yeast pellets were then washed
N-Troc-Melodinine K (33). Argon was bubbled through a
solution of 32 (50 mg, 0.054 mmol) in CH Cl (2 mL) at rt for 20
2
2
min. Pd(PPh ) (62 mg, 0.054 mmol) was added, and the reaction
3
4
with H O (20 mL each) and centrifuged again at 10 °C at 3000 rpm
mixture was stirred at rt for 1 h. Pyrrolidine (0.02 mL, 0.27 mmol)
was then added, and the reaction was stirred at rt for 1 h. The
resulting mixture was passed through a short plug of silica in 100%
EtOAc. Aqueous NaHCO3 (5 mL) was added, and the reaction
mixture was extracted with CH Cl (3 × 5 mL), washed with brine (3
2
for 10 min. Five 2 L round-bottom flasks each containing YPGal
medium (1 L) containing yeast extract (1%, 10.0 g), peptone (2%,
2
0.0 g), and galactose (2%, 20.0 g) in H O were autoclaved. The yeast
2
pellets were inoculated into each flask evenly, covered with
cheesecloth, and incubated at 30 °C at 250 rpm for 16 h. Then, to
each flask was added tabersonine hydrochloride salt (1.50 g, 4.03
mmol) in DMSO (10 mL), followed by the addition of galactose (2%,
2
2
× 5 mL), dried over Na SO , and concentrated under reduced
2
4
pressure. The crude residue 33 was isolated as a brown solid and used
directly in the next step.
2
0.0 g), covered with cheesecloth, and incubated at 30 °C at 250 rpm
(−)-Melodinine K (1). To a solution of crude N-Troc-melodinine
K (33) from the previous step dissolved in THF (2 mL) were added
activated zinc dust (105 mg, 1.61 mmol) and aqueous KH PO
for 96 h. The media was then stirred with EtOAc (1 L) for 1 h,
extracted with EtOAc (2 × 1 L), filtered, washed with brine (1 × 100
mL), dried over Na SO , and concentrated under reduced pressure.
2
4
solution (1.0 M, 0.09 mL). The reaction mixture was heated to 60 °C
2
4
G
J. Nat. Prod. XXXX, XXX, XXX−XXX