Stereoselective Enzymatic Synthesis of cis-Pellitorine
FULL PAPER
Varian ProStar 510 column oven (50 °C). Chromatographic separ-
ation was carried out on an OmniSpher C18 column (250 ϫ
0.9, J ϭ 0.9 Hz, 1 H, C(5)ϪH], 5.82 [d, J ϭ 14.9 Hz, 1 H, C(2)ϪH],
6.08 [ddtd, J ϭ 11.5, J ϭ 10.8, J ϭ 1.4, J ϭ 0.6 Hz, 1 H, C(4)ϪH],
1
3
4.6 mm, particle size 5 µm) with gradients with acetonitrile and
7.56 [ddd, J ϭ 14.9, J ϭ 11.5, J ϭ 1.0 Hz, 1 H, C(3)ϪH] ppm.
NMR (50 MHz, CDCl ): δ ϭ 14.02 [CH , C(10)], 20.15 [2 ϫ CH
C(4Ј) ϩ C(3Ј)], 22.52 [CH , C(9)], 28.15 [CH , C(6)], 28.63 [CH,
C(2Ј)], 29.14 [CH , C(7) or C(8)], 31.41 [CH , C(7) or C(8)], 46.96
[CH , C(1Ј)], 123.78 [CH, C(2)], 126.28 [CH, C(4)], 135.76 [CH,
C
D
2
O (contains 0.01% TFA v/v). Solvent suppression was employed
3
3
3
,
by executing WET pulse sequence before every acquisition.
For preparative gas chromatography, a Gerstel MCS-system
HP5980 Series gas chromatograph equipped with a KAS 3 injector
and a flame ionization detector (300 °C) was used. Preparative sep-
arations were performed on a wide bore DB-WAX fused silica col-
2
2
2
2
2
C(3)], 140.07 [CH, C(5)], 166.34 [C, C(1)] ppm, MS (GC/EI MS):
m/z ϭ 29 (9%), 41 (16%), 53 (12%), 57 (12%), 67 (18%), 69 (14%),
f
umn (30 m ϫ 0.53 mm i.d./ d ϭ 1.0 µm). The column temperature 81 (49%), 96 (33%), 110 (12%), 113 (17%), 138 (5%), 151 (100%,
ϩ
was programmed from 80 °C to 230 °C at 4 °C/min. Helium carrier
gas was used with a column flow of 5 mL/min.
base peak), 166 (7%), 180 (10%), 208 (10%), 223 (29, M ·), HRMS:
ϩ
M
calcd. for C14H25NO, 223.1936; found, 223.1938.
High resolution mass spectra were recorded using a Finnigan MAT
8
200 (Finnigan MAT, Bremen, Germany) by flash evaporation of
(
2E,4E)-N-Isobutyldeca-2,4-dienamide (1a): (2E,4Z)-N-Isobutyl-
samples after dissolving in methanol (resolution Ͼ 8000). GC/EI-
MS were recorded on Finnigan SSQ 7000 (Finnigan MAT Bremen)
at 70 eV ionization energy, 220 °C ion source temperature applying
DB1 fused silica column (30 m, 0.25 mm i.d., 0.25 µm film).
Kovacs Indices (retention index, RI) have been determined by GC
on DB1 (20 m, 0.18 mm i.d., 0.18 µm film) and the GC oven tem-
perature was programmed from 60 °C to 300 °C at 12 °C/min.
Hydrogen carrier gas was used with a constant flow of approxi-
mately 1 mL/min.
deca-2,4-dienamide (15 g) (1b) was treated by iodine (2.13 g) in
toluene (50 mL) at room temperature for 30 h. The solvents were
evaporated in vacuo, and the oily residue recrystallized from petro-
leum ether (b.p. 60Ϫ95 °C). Yield 3.5 g (23%. GC: RI 1889, 94.9%,
RI 1848 3.7%, RI 1787 0.1%). H NMR (400 MHz, CDCl
1
3
): δ ϭ
0
.89 [t, J ϭ 6.9 Hz, 3 H, C(10)ϪH], 0.93 [d, J ϭ 6.7 Hz, 6 H,
C(4Ј)ϪH ϩ C(3Ј)ϪH], 1.37Ϫ1.22 [m, 4 H, C(8)-H, C(9)ϪH], 1.42
m, J ϭ 7.1 Hz, 2 H, C(7)ϪH], 1.80 [m, J ϭ 6.7 Hz, 1 H, C(2Ј)ϪH],
[
2
6
1
.14 [dt, J ϭ 7.0, J ϭ 6.4 Hz, 2 H, C(6)ϪH], 3.17 [dd, J ϭ 6.9, J ϭ
.1 Hz, 2 H, C(1Ј)ϪH], 5.50 (bs, 1 H, NϪH), 5.75 [d, J ϭ 14.9 Hz,
H, C(2)ϪH], 6.07 [dt, J ϭ 15.1, J ϭ 6.4 Hz, 1 H, C(5)ϪH], 6.13
Mixture of Different Diastereoisomers of Ethyl 2,4-Decadienoate
(3): Commercial ethyl (2E,4Z)-deca-2,4-dienoate (3b, 200 g) was
[dd, J ϭ 15.1, J ϭ 9.6 Hz, 1 H, C(4)ϪH], 7.19 [m, J ϭ 14.9, J ϭ
isomerized in toluene (650 mL) by treatment with iodine (2.5 g).
The resulting mixture was stirred for 48 h at room temperature,
washed with saturated Na
and water (200 mL), dried over Na
was evaporated, and the residue fractionated in vacuo. A fraction
13
9
1
.7 Hz, 1 H, C(3)ϪH] ppm. C NMR (100 MHz, CDCl
4.02 [CH , C(10)], 20.13 [2 ϫ CH , C(4Ј) ϩ C(3Ј)], 22.48 [CH
, C(7)], 28.64 [CH, C(2Ј)], 31.38 [CH , C(8)], 32.93
, C(6)], 46.83 [CH , C(1Ј)], 121.74 [CH, C(2)], 128.21 [CH,
3
): δ ϭ
3
3
2
,
2
S
2
O
3
solution (two 200 mL portions)
SO , filtered, and the solvent
C(9)], 28.49 [CH
CH
2
2
2
4
[
2
2
C(4)], 141.32 [CH, C(3)], 143.23 [CH, C(5)], 166.41 [C, C(1)] ppm,
MS (GC/MS EI): m/z ϭ 29 (14%), 41 (26%), 53 (20%), 55 (18%),
5
composed of 2E,4Z isomer 3b (30%, RI 1443), 2E,4E isomer 3a
(47%, RI 1483) and 2Z,4E isomer 3c (18%, RI 1429) was used for
7 (15%), 66 (16%), 67 (23%), 69 (20%), 81 (67%), 96 (65%), 110
the following enzyme screening.
(
(
16%), 113 (19%), 151 (100%, base peak), 166 (8%), 180 (8%), 208
10%), 223 (34, M ·).
ϩ
Screening of Enzymes: The ethyl 2,4-decadienoate mixture (54 µL,
4
0
8.7 mg, 0.25 mmol) and isobutylamine (20 µL, 14.7 mg,
.20 mmol) were mixed in a polyethylene vessel with an appropriate
(
2Z,4E)-N-Isobutyldeca-2,4-dienamide (1c): An enriched fraction
of diastereomer 1c (50%, 25% 1a and minor components) was ob-
tained by preparative GC of a pellitorine crop and analyzed by
NMR spectroscopy and GC/MS. H NMR (400 MHz, CDCl
only peaks for the 2Z,4E isomer 1c, correlated by a TOCSY experi-
ment): δ ϭ 0.9 [m, 3 H, C(10)ϪH], 0.94 [d, J ϭ 6.7 Hz, 6 H,
C(4Ј)ϪH ϩ C(3Ј)ϪH], 1.44Ϫ1.20 [m, C(7)-H, C(8)ϪH, C(9)ϪH],
solvent (heptane, triethylamine, toluene, 1 mL). Sodium sulfate
10 mg) and the enzyme (10 mg) were added, and the mixtures
(
1
3
,
shaken at 50 °C for 24 h. The mixtures were centrifuged, the super-
natant decanted and analyzed using GC. For further evaluation,
the same reaction was performed in 5 mL solvent using the
amounts of substrates and enzyme described earlier.
1
.81 [m, J ϭ 6.8 Hz, C(2Ј)ϪH], 2.00Ϫ2.24 [m, C(6)ϪH], 3.14 [dd,
J ϭ 6.8, J ϭ 6.1 Hz, 2 H, C(1Ј)ϪH], 5.46 [m, J ϭ 11.3 Hz, 1 H,
C(2)ϪH], 5.52Ϫ5.40 [bs, 1 H, NϪH], 5.97 [m, J ϭ 15.3, J ϭ 7.0,
J ϭ 0.8 Hz, 1 H, C(5)ϪH], 6.38 [ddd, J ϭ 11.7, J ϭ 11.2, J ϭ
(
2
4
mg
2E,4Z)-N-Isobutyldeca-2,4-dienamide (1b): Ethyl (2E,4Z)-deca-
,4-dienoate (3b) (10 g, 50.9 mmol), Chirazyme -2 (c.f., C2, lyo.,
Ϫ1
.7 g, activity of carrier fixed type 200 u g in contrast to 120 u
0
1
.8 Hz, 1 H, C(3)ϪH], 7.45 [dddt, J ϭ 15.3, J ϭ 11.2, J ϭ 1.5, J ϭ
.0 Hz, 1 H, C(4)ϪH] ppm. MS (GC/MS EI): m/z ϭ 41 (7%), 67
Ϫ1
[15]
for the simple protein ), and isobutylamine (4 g,
5
4.7 mmol) were stirred at 55 °C for 4 days. The mixture was di-
(
(
7%), 81 (17%), 96 (23%), 152 (100%, base peak), 166 (5%), 180
5%), 194 (2%), 208 (2%), 223 (13%, M ·).
luted by diethyl ether (100 mL) and filtered; the filtrate was evapo-
rated to dryness in vacuo (yield of raw material 15.2 g). The prod-
uct was stirred in 10% KOH/methanol (1:1 w/w) at room tempera-
ture for 45 min and extracted with diethyl ether. The organic phase
was dried over Na
dryness in vacuo. The saponified raw material was chromato-
ϩ
N-Isobutyl-4-(isobutylamino)dec-3-enamide (4): Ethyl (2E,4Z)-deca-
2,4-dienoate (3b, 20 g, 102 mmol), isobutylamine (8 g, 110 mmol)
and Chirazyme -2 (c.f., C2, lyo., 4.7 g) were stirred at 55 °C for 7
2 4
SO , filtered, and the filtrate was evaporated to
graphed on silica gel 60 using hexane/ethyl acetate 10:1 (v/v) as days. The crude reaction mixture was diluted with diethyl ether
1
eluent. Yield 9.1 g (40.7 mmol, 80%, GC: RI 1844, 99.4%).
NMR (200 MHz, CDCl ): δ ϭ 0.88 [t, J ϭ 6.5 Hz, 3 H, C(10)ϪH], crude product (15.1 g) containing 3b (4%), 1b (71%), 1a (2.5%),
.93 [d, J ϭ 6.7 Hz, 6 H, C(3Ј)-H ϩ C(4Ј)ϪH], 1.50Ϫ1.22 [m, 6 H,
C(7)ϪH, C(8)-H, C(9)ϪH], 1.81 [m, J ϭ 6.7 Hz, 1 H, C(2Ј)ϪH],
H
(100 mL), filtered, and the filtrate evaporated to dryness to yield a
3
0
and 4 (7.4%). The side product 4 was analyzed by GC/MS (RI DB
1 2088) and HPLC/ NMR spectroscopy starting from the crude
2.22Ϫ2.36 [m, 2 H, C(6)ϪH], 3.18 [dd, J ϭ 6.8, J ϭ 6.1 Hz, 2 H, batch. LC separation was performed with a gradient with D
2
O
C(1Ј)ϪH], 5.50 (bs, 1 H, NϪH), 5.79 [dtdd, J ϭ 10.8, J ϭ 7.8, J ϭ (containing 0.01 TFA% v/v) and acetonitrile (95:5 Ϫ 30 min Ϫ 5:95
Eur. J. Org. Chem. 2004, 5135Ϫ5140
www.eurjoc.org
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5139