218
G. A. B. Vieira et al. / Tetrahedron: Asymmetry 20 (2009) 214–219
EtOAc (3 ꢁ 20 mL). The organic phases were combined, dried over
Na2SO4, and the solvent was evaporated under reduced pressure.
The reaction crude was finally purified by flash chromatography
(5% EtOAc/hexane) to yield 446.1 mg of optically active 1 as a col-
0.1 M buffer of pH 7.0. Then, 0.74 mg of NAD+ was added as cofac-
tor and 2 L (1U) of glucose dehydrogenase for cofactor regenera-
tion. The reaction mixture was shaken at 140 rpm and 30 °C, and
l
after 24 h the reaction mixture was extracted with EtOAc
orless oil (88%). Rf (20% EtOAc/hexane): 0.36; IR (NaCl):
m
3360,
(2 ꢁ 500
lL) and dried over Na2SO4.
2930, 2865, 1453, 1383, 1070, 932 cmꢀ1
;
1H NMR (CDCl3,
0
00
300.13 MHz): d S-cis 3.72 (m, 1H, H1 ), 1.59–1.49 (m, 1H, H1 ),
4.6.7. ADH A
3
1.47–1.41 (m, 8H, 2H2 + 2H3 + 2H5 + 2H6), 1.15 (d, JHH = 6.3 Hz,
3H, H2 ), 1.18–1.13 (td, JHH = 7.0, 1.1 Hz, 2H, H1, and H4), 0.83 (d,
JHH = 6.6 Hz, 6H, H2 , and H3 ); S-trans d 3.51 (m, 1H, H1 ), 1.59–
1.49 (m, 1H, H1 ), 1.47–1.41 (m, 8H, 2H2 + 2H3 + 2H5, and 2H6),
In an eppendorf tube were added 1.5 mg (9 U) of enzyme,
3
0
30.0
l
L of 2, and 800
l
L of Tris–HCl 0.1 M buffer of pH 7.0. Then,
3
0.74 mg of NADP+ was added as cofactor and 200
lL of 2-propanol
00
00
0
00
for cofactor regeneration. The reaction mixture was shaken at
140 rpm and 30 °C, and after 24 h the reaction mixture was ex-
3
3
0
1.15 (d, JHH = 5.9 Hz, 3H, H2 ), 1.18–1.13 (td, JHH = 7.0, 1.1 Hz,
2H, H1, and H4), 0.84 (d, JHH = 6.8 Hz, 6H, H2 , and H3 ); 13C NMR
tracted with EtOAc (2 ꢁ 500
4.7. Catalytic hydrogenation of ketone 2
suspension of (1.0 g, 6,1 mmol) over [1,5-HDRhCl]2
lL) and dried over Na2SO4.
3
00
00
0
(CDCl3, 75.5 MHz): d S-cis 72.2 (C1 ), 43.3 (C1), 42.0 (C4), 32.8
(C1 ), 26.3 (C3), 26.1 (C5), 25.6 (C2), 24.6 (C6), 21.1 (C2 ), 20.4 (C2
and C3 ); S-trans d 69.4 (C1 ), 45.2 (C1), 44.0 (C4), 29.4 (C3), 29.3
00
0
00
00
0
00
00
00
0
(C5), 29.0 (C1 ), 28.7 (C2), 28.4 (C6), 20.4 (C2 and C3 ), 19.5 (C2 );
A
2
MS (ESI+, m/z): 152 [(MꢀH2O), 19%], 45 [(Mꢀ PrCyhex)+, 100%].
(26,5 mg, 0.061 mmol) and TBAHS (849 mg, 2.5 mmol) in a mixture
of hexane (20 mL), buffer KPi pH 7.4 (5 mL), and THF (3 mL) was
charged with H2 (80 atm) and the mixture was stirred at room
temperature in an autoclave. After 12 h, the reaction mixture was
filtered in Celite and the filtrate was then extracted with EtOAc
(3 ꢁ 20 mL). Organic phases were combined, dried over Na2SO4,
and the solvent was evaporated under reduced pressure. The reac-
tion crude was finally purified by flash chromatography (5% EtOAc/
hexane) yielding a mixture of compounds: 331.8 mg of 1, 220.1 mg
of 3, and 164.0 mg of 6. Compound 6 was isolated as a colorless oil.
i
4.6. Procedure for the bioreduction of ketone 2 depending on
the ADH used in the enzymatic process
4.6.1. ADH T
In an eppendorf tube were added 132
12.0 L of 2, and 800 L of Tris–HCl 0.1 M buffer of pH 7.0. Then,
0.17 mg of NADP+ was added as cofactor and 200
L of 2-propanol
lL (50 U) of enzyme,
l
l
l
for cofactor regeneration. The reaction mixture was shaken at
140 rpm and 30 °C, and after 24 h the reaction mixture was ex-
Rf (20% EtOAc/hexane): 0.59; IR (NaCl): m 2930, 2861, 2361, 1710,
tracted with EtOAc (2 ꢁ 500
lL) and dried over Na2SO4.
1598, 1451, 1366, 1243, 1177 cmꢀ1
;
1H NMR (CDCl3,
3
0
300.13 MHz): d cis 2.48 (q, JHH = 5.0 Hz, 1H, H1), 2.13 (s, 3H, H2 ),
0
4.6.2. ADH LB
2.04–1.91 (m, 3H, H1 + H2, and H6), 1.56–1.41 (m, 4H,
In an eppendorf tube were added 38.5
12.0 L of 2, and 800 L of Tris–HCl 0.1 M buffer of pH 7.0. Then,
0.17 mg of NADP+ was added as cofactor and 200
L of 2-propanol
l
L (50 U) of enzyme,
H2 + H3 + H5, and H6), 1.32–1.21 (m, 3H, H3 + H4, and H5), 0.83 (d,
3
00
00
l
l
JHH = 6.8 Hz, 6H, H2 , and H3 ); trans d 2.29–2.20 (m, 1H, H1),
0
0
l
2.21 (s, 3H, H2 ), 2.04–1.91 (m, 3H, H1 + H2, and H6), 1.56–1.41
for cofactor regeneration. Reaction mixture was shaken at 140 rpm
and 30 °C, and after 24 h the reaction mixture was extracted with
(m, 4H, H2 + H3 + H5, and H4), 1.32–1.21 (m, 3H, H3 + H4, and H5),
3
0.85 (d, JHH = 7.4 Hz, 6H, H2
,
and H3 ); 13C NMR (CDCl3,
00
00
0
00
EtOAc (2 ꢁ 500
lL) and dried over Na2SO4.
75.5 MHz): cis d 211.2 (C1 ), 51.7 (C1), 42.8 (C4), 30.7 (C1 ), 27.9
0
00
00
(C2 ), 26.5 (C3 and C5), 25.9 (C2 and C6), 20.0 (C2 and C3 ); trans d
0
00
4.6.3. ADH CP
212.4 (C1 ), 51.7 (C1), 43.3 (C4), 32.7 (C1 ), 28.9 (C3 and C5), 28.6
+
+
0
00
00
In an eppendorf tube were added 3
of 2, and 800 L of Tris–HCl 0.1 M buffer of pH 7.0. Then, 0.74 mg of
NAD+ was added as cofactor and 200
L of 2-propanol for cofactor
l
L (1 U) of enzyme, 30.0
lL
(C2 and C6), 27.9 (C2 ), 19.7 (C2 and C3 ); MS (ESI , m/z): 168 (M ,
i
l
6%), 43 [(Mꢀ PrCyhex)+, 100%].
l
regeneration. The reaction mixture was shaken at 140 rpm and
4.8. 1-(4-Isopropylcyclohexyl)ethyl acetate ( )-7
30 °C, and after 24 h the reaction mixture was extracted with
EtOAc (2 ꢁ 500
lL) and dried over Na2SO4.
Over a solution under a nitrogen atmosphere of alcohol ( )-1
(20.5 mg, 0.12 mmol) in dry CH2Cl2 (1.2 mL), Et3 N (32.2
0.24 mmol), DMAP (1.5 mg, 0.012 mmol), and Ac2O (22.5
l
l
L,
L,
4.6.4. ADH PR2
In an eppendorf tube were added 7.7
30.0 L of 2, and 800 L of Tris–HCl 0.1 M buffer of pH 7.0. Then,
0.74 mg of NADP+ was added as cofactor and 200
L of 2-propanol
for cofactor regeneration. The reaction mixture was shaken at
140 rpm and 30 °C, and after 24 h the reaction mixture was ex-
l
L (1 U) of enzyme,
0.24 mmol) were successively added. The reaction mixture was
stirred at room temperature for 2 h, until complete consumption
of the starting material was observed by TLC analysis (20% EtOAc/
hexane), then the solvent was evaporated under reduced pressure.
The reaction crude was finally purified by flash chromatography
(5% EtOAc/hexane) yielding 18 mg of ( )-7 as a colorless oil (71%).
l
l
l
tracted with EtOAc (2 ꢁ 500
lL) and dried over Na2SO4.
Rf (20% EtOAc/hexane): 0.67; IR (NaCl):
m 2933, 2864, 2361, 1736,
4.6.5. ADH RS1
1454, 1371, 1246, 1116, 1042, 950 cmꢀ1
;
1H NMR (CDCl3,
0
0
In an eppendorf tube were added 3.6
30.0 L of 2, 54 mg of glucose, and 1 mL of Tris–HCl 0.1 M buffer
of pH 7.0. Then, 0.74 mg of NAD+ was added as cofactor and 2
(1U) of glucose dehydrogenase for cofactor regeneration. The reac-
tion mixture was shaken at 140 rpm and 30 °C, and after 24 h the
l
L (1 U) of enzyme,
300.13 MHz): d R-cis 4.93 (m, 1H, H1 ), 2.03 (s, 3H, H4 ), 1.81–1.37
00
l
(m, 11H, H1 + 2H2 + 2H3 + H4 + 2H5 + 2H6, and H1 ), 1.18 (d,
JHH = 6.5 Hz, 3H, H2 ), 0.85 (d, JHH = 6.6 Hz, 6H, H2 , and H3 ); R-
trans d 4.71 (m, 1H, H1 ), 2.02 (s, 3H, H4 ), 1.81–1.37 (m, 11H,
H1 + 2H2 + 2H3 + H4 + 2H5 + 2H6, and H1 ), 1.15 (d, JHH = 6.3 Hz,
3H, H2 ), 0.84 (d, JHH = 6.8 Hz, 6H, H2 , and H3
75.5 MHz): d R-cis 170.8 (C3 ), 72.3 (C1 ), 42.1 (C4), 40.4 (C1), 32.7
3
3
0
00
00
lL
0
0
3
00
3
13C NMR (CDCl3,
0
00
00
reaction mixture was extracted with EtOAc (2 ꢁ 500
lL) and dried
)
0
0
over Na2SO4.
00
00
00
(C1 ), 29.2 (C2), 28.5 (C6), 26.1 (C3), 25.0 (C5), 20.4 (C2 and C3 ),
0
0
0
4.6.6. ADH BS2
17.9 (C2 ); R-trans d 170.8 (C3 ), 74.6 (C1 ), 43.9 (C4), 42.6 (C1), 29.2
00
00
00
In an eppendorf tube were added 1.0
L of 2, 54 mg of glucose, 35 mg of CaCO3, and 1 mL of Tris–HCl
l
L (1 U) of enzyme, 30.0
(C1 ), 29.2 (C2), 28.5 (C6), 26.1 (C3), 25.4 (C5), 19.7 (C2 and C3 ),
+
+
i
+
0
l
17.1 (C2 ); MS (ESI , m/z): 152 [(MꢀAcOH) , 19%], 43 [( Pr) , 100%].