162 J . Org. Chem., Vol. 65, No. 1, 2000
Matsuda et al.
g), trichloroacetyl chloride (19 mmol, 3.4 g), and benzene (32
mL) in 96% yield: 1H NMR (CDCl3) δ 7.46-7.54 (m, 2H, Ph),
7.60-7.68 (m, 1H, Ph), 8.23-8.28 (m, 2H, Ph); IR (neat) 1713
7.58 (m, 2H, Ph); 19F NMR (CDCl3-CFCl3) δ -79 (d, J ) 6.2
Hz); IR (Kbr) 1127, 1181, 3366 cm-1. Anal. Calcd for C8H6-
OBrF3: C, 37.68; H, 2.37. Found: C, 37.68; H, 2.32.
cm-1
.
(S)-1,1,1-Tr iflu or o-3-p h en yl-2-p r op a n ol ((S)-4F b). Com-
pound 4F a (412 mg, 2.19 mmol) was converted to (S)-4F b (307
mg) by APG4. GC conditions: CPCD 140 °C. R: 6.5 min, S:
7.0 min. [R]25D -45.5 (c 0.95, CHCl3) (lit.13 [R]23D -28.2 (c 0.88,
En zym e P r ep a r a tion s a n d Sep a r a tion of th e En zym es
by An ion -Exch a n ge Colu m n Ch r om a togr a p h y. APG4
(acetone powder of Geotrichum candidum IFO 4597, a micro-
bial dried-cell preparation dehydrated using acetone) prepared
as described previously,2a was used for the APG4 reductions.
For the separation of the enzymes, APG4 (5.0 g) was homog-
enized with HEPES buffer (4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid-NaOH, pH 7.2, 0.1 M, 50 mL), ME
(2-mercaptoethanol, 0.04%), DTT (dithiothreitol, 1 mM), and
PMSF (R-toluenesulfonyl fluoride, 1 mM) and sonically disin-
tegrated. The cell-free extract (40 mL) obtained by centrifuga-
tion (30 000 × g, 2 °C, 30 min) was added to PMSF (1 mM)
and DFP (diisopropyl fluorophosphate 1 mM) and dialyzed
against HEPES (pH 7.2, 10 mM, 750 mL × 2) containing ME
(0.04%) and DTT (1 mM). The supernatant (53 mL) obtained
by centrifugation (30 000 × g, 2 °C, 30 min) and filtration
through Sterivex-GV (0.22 µm, Millipore) was applied to an
anion-exchange column (FPLC system, Pharmacia MonoQ-HR
10/10 column) and eluted with the above buffer and a linear
KCl gradient of 0 M for 15 min, 0.0075 M/min for 40 min, and
0.07 M/min for 10 min. The injection volume was 5 mL, the
flow rate was 2 mL/min, and the fraction size was 2 mL. The
enzyme activity was assayed at 30 °C by measuring the change
in absorbance at 340 nm due to the oxidation of NADPH at a
substrate concentration of 2.5 mM in Tris-HCl buffer (pH 7.0,
0.1 M). One unit of enzyme oxidizes 1 µmol NADPH to NADP+/
min. The B enzyme eluted around fraction number 36 was
collected and used for the preparative-scale synthesis.
Effect of Su bstitu en ts a t th e R-P osition of Acetop h e-
n on e on th e En a n tioselectivity of th e AP G4 Red u ction .
A ketone (0.08 mmol), NADP+ (0.007 mmol), and cyclopentanol
(0.65 mmol) were added to a suspension of APG4 (20 mg) in
MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 7.0, 0.1
M, 3 mL). The mixture was shaken at 130 rpm and 30 °C for
20 h, and the resulting mixture was put on Extrelut and eluted
with ether. The chemical yield and ee of the product were
determined by GC, HPLC, or NMR analysis of the ether
extract.
1
CHCl3) 98% ee (S)); H NMR (CDCl3) δ 2.15 (d, 1H, OH, J )
5.4 Hz), 2.84 (q, 1H, CH2, J ) 10.0, 14.2 Hz), 3.06 (q, 1H, CH2,
J ) 3.0, 14.2 Hz), 4.05-4.24 (m, 1H, CH), 7.22-7.40 (m, 5H,
Ph); 19F NMR (CDCl3-CFCl3) δ -80 (d, J ) 6.4 Hz); IR (neat)
1130, 1169, 3432 cm-1. Anal. Calcd for C9H9OF3: C, 56.48; H,
4.77. Found: C, 56.55; H, 4.80.
(+)-2,2,2-Tr iflu or o-1-(3-th ien yl)eth a n ol ((+)-6F b). Com-
pound 6F a (984 mg, 5.46 mmol) was converted to (+)-6F b (862
mg) by APG4. GC conditions: DEX 140 °C. (+): 6.4 min, (-):
7.0 min. [R]26 +31.6 (c 0.46, MeOH); 1H NMR (CDCl3) δ 2.60
D
(d, 1H, OH, J ) 4.8 Hz), 5.07-5.19 (m, 1H, CH), 7.17-7.19
(m, 1H), 7.35-7.39 (m, 1H), 7.45-7.47 (m, 1H) cm-1 13C NMR
;
(CDCl3) δ 69.4 (q, CH, J ) 33.1 Hz), 124.1 (q, CF3, J ) 281
Hz), 124.8, 126.1, 126.6, 134.9; 19F NMR (CDCl3-CFCl3) δ -79
(d, J ) 6.2 Hz); IR (neat) 1127, 1167, 3395 cm-1; HRMS for
(C6H5OF3S)+ calcd 182.0013, found 182.0023.
(R)-2-F lu or o-1-p h en yleth a n ol ((R)-11b). Compound 11a
(200 mg, 1.45 mmol) was converted to (R)-11b (189 mg) by
APG4. GC conditions: DEX 130 °C. S: 8.8 min, R: 9.6 min.
[R]24 -52.3 (c 1.08, MeOH) (lit.5b [R]23 -76.2 (c 3, MeOH)
D
D
95.4% ee (R)); 1H NMR (CDCl3) δ 2.65 (d, 1H, OH, J ) 2.2
Hz), 4.42 (octet, 1H, CH2, J ) 8.1, 9.5, 49 Hz), 4.50 (octet, 1H,
CH2, J ) 3.4, 9.5, 47 Hz), 5.00 (m, 1H, CH), 7.37 (m, 5H, Ph);
13C NMR (CDCl3) δ 73.0 (d, CH, J ) 19.8 Hz), 87.2 (d, CH2F,
J ) 174 Hz), 126.3 (Ph), 128.4 (Ph), 128.6 (Ph), 138.1 (d, Ph,
J ) 8.3 Hz); 19F NMR (CDCl3-CFCl3) δ -221 (dt, J ) 14.1,
48 Hz); IR (neat) 1011, 3397 cm-1. Anal. Calcd for C8H9OF:
C, 68.56; H, 6.47. Found: C, 68.56; H, 6.64.
(R)-2,2-Diflu or o-1-p h en yleth a n ol ((R)-12b). Compound
12a (240 mg, 1.54 mmol) was converted to (R)-12b (240 mg)
by APG4. GC conditions: DEX 140 °C. S: 6.7 min, R: 7.2 min.
[R]24 -11.5 (c 1.00, CH2Cl2) (lit.5b [R]23 -14.27 (c 3, CH2Cl2)
D
D
1
84.66% ee (R)); H NMR (CDCl3) δ 2.62 (d, 1H, OH, J ) 3.2
Hz), 4.81 (m, 1H, CH), 5.76 (dt, 1H, CHF2, J (d) ) 4.8 Hz, J (t)
) 56 Hz), 7.41 (m, 5H, Ph);13C NMR (CDCl3) δ 73.6 (t, CHOH,
J ) 24.5 Hz), 115.8 (t, CHF2, J ) 245 Hz), 127.1 (Ph), 128.7
(Ph), 129.0 (Ph), 135.8 (t, Ph, J ) 3.4 Hz); 19F NMR (CDCl3-
CFCl3) δ -128.1 (dd, J ) 4.2, 56 Hz), -128.0 (dd, J ) 3.7, 56
Hz); IR (neat) 1071, 3410 cm-1. Anal. Calcd for C8H8OF2: C,
60.76; H, 5.10. Found: C, 60.67; H, 5.27.
Gen er a l P r oced u r e for th e Red u ction of Keton es by
AP G4 on a P r ep a r a tive Sca le. Fluorinated and/or chlori-
nated ketones were reduced using NAD+ or NADP+ and
2-propanol or cyclopentanol for 20 h as described previously.2a
(S)-2,2,2-Tr iflu or o-1-p h en ylet h a n ol ((S)-1F b ). Com-
pound 1F a (205 mg, 1.18 mmol) was converted to (S)-1F b (175
mg) by APG4. GC conditions: DEX 130 °C. S: 9.6 min, R: 10.2
(R)-2,2-Dich lor o-1-p h en yleth a n ol ((R)-17b). Compound
17a (461 mg, 2.44 mmol) was converted to (R)-17b (63.1 mg)
by APG4. GC conditions: CPCD 150 °C. S: 19.8 min, R: 20.9
min. [R]21 -11.8 (c 1.26, CH2Cl2) (lit.14 [R]22 -15.0 (c 3,
min. [R]24 +30.4 (c 1.56, CHCl3); +25.1 (c 0.81, CCl4) (lit.11
D
[R]20 -25.1(c 3, CCl4) >99% ee (R)); 1H NMR (CDC l3) δ 2.63
D
D
D
1
(d, 1H, OH, J ) 4.6 Hz), 5.02 (dq, 1H, CH, J (d) ) 4.5 Hz, J (q)
) 6.8 Hz) and 7.37-7.51 (m, 5H, Ph); 19F NMR (CDCl3-CFCl3)
δ -79 (d, J ) 6.6 Hz); IR (neat) 1128, 1173, 3407 cm-1. Anal.
Calcd for C8H7OF3: C, 54.55; H, 4.01. Found: C, 54.24; H, 4.09.
(S)-2,2,2-Tr iflu or o-1-(p-ch lor oph en yl)eth an ol ((S)-2Fb).
Compound 2F a (429 mg, 2.06 mmol) was converted to (S)-2F b
(350 mg) by APG4. GC conditions: CPCD 140 °C. S: 13.7 min,
R: 14.5 min. [R]24 +33.1 (c 0.914, EtOH) (lit.12 [R]20 -19.0
CH2Cl2) 45.7% ee (R)); H NMR (CDCl3) δ 2.87 (d, 1H, OH, J
) 4.0 Hz), 4.98 (dd, 1H, CH, J ) 3.8, 5.4 Hz), 5.82 (d, 1H,
CHCl2, J ) 5.4 Hz), 7.36-7.44 (m, 5H, Ph); IR (KBr) 3362
cm-1. Anal. Calcd for C8H8OCl2: C, 50.29; H, 4.22. Found: C,
50.26; H, 4.23.
(S)-2-Ch lor o-2,2-d iflu or o-1-p h en ylet h a n ol ((S)-19b ).
Compound 19a (390 mg, 2.05 mmol) was converted to (S)-19b
(322 mg) by APG4. GC conditions: G-TA 125 °C. R: 11.4 min,
S: 11.7 min; [R]25 +22.2 (c 1.90, CHCl3) (lit.15 [R]21 -13.82
D
D
1
(c 1.05, EtOH) 82.5% ee (R)); H NMR (CDCl3) δ 2.88 (s, 1H,
D
D
OH), 5.00 (q, 1H, CH, J ) 6.5 Hz), 7.35-7.45 (m, 4H, Ph); 19
F
1
(c 1.01, CHCl3) 73% ee (R)); H NMR (CDCl3) δ 2.91 (d, 1Η,
ÃΗ, J ) 4.6 Hz), 5.06 (dq, 1H, CH, J ) 4.4, 7.5 Hz), 7.39-
7.50 (m, 5H, Ph); 19F NMR (CDCl3-CFCl3) δ -65 (dd, J ) 8.8,
165 Hz), -63 (dd, J ) 7.2, 165 Hz); IR (neat) 3420 cm-1. Anal.
Calcd for C8H7OClF2: C, 49.90; H, 3.66. Found: C, 49.60; H,
3.68.
NMR (CDCl3-CFCl3) δ -79 (d, J ) 6.4 Hz); IR (KBr) 1128,
1179, 3382 cm-1. Anal. Calcd for C8H6OClF3: C, 45.63; H, 2.87.
Found: C, 45.81; H, 2.99.
(S)-2,2,2-Tr iflu or o-1-(p-br om oph en yl)eth an ol ((S)-3Fb).
Compound 3F a (504 mg, 1.99 mmol) was converted to (S)-3F b
(406 mg) by APG4. GC conditions: CPCD 150 °C. S: 13.9 min,
R: 14.8 min. [R]24 +30.25 (c 0.862, EtOH) (lit.12 [R]20 -21.5
Gen er a l P r oced u r e for th e Red u ction of Keton es by
th e B En zym e in a P r ep a r a tive Sca le. A ketone (0.80
D
D
1
(c 1.04, EtOH) 82.2% ee (R)); H NMR (CDCl3) δ 2.73 (s, 1H,
OH), 4.99 (q, 1H, CH, J ) 6.5), 7.33-7.37 (m, 2H, Ph), 7.51-
(13) Shimizu, M.; Sugiyama, K.; Fujisawa, T. Bull. Chem. Soc. J pn.
1996, 69, 2655.
(11) Holzwarth, G.; Hsu, E. C.; Mosher, H. S.; Faulkner, T. R.;
Moscowitz, A. J . Am. Chem. Soc. 1974, 96, 251.
(12) Ohno, A.; Nakai, J .; Nakamura, K.; Goto, T.; Oka, S. Bull.
Chem. Soc. J pn. 1981, 54, 3486.
(14) Weidmann, R.; Schoofs, A. R.; Horeau. A. C. R. Acad. Sci. Ser.
II 1982, 294, 319.
(15) Kitazume, T.; Asai, M.; Tsukamoto, T.; Yamazaki, T. J . Fluorine
Chem. 1992, 56, 271.