continued for 90 min. The mixture was centrifuged, the pellet
was dissolved in Tris (50 cm3), and the resulting solution was
dialyzed against 50 mM Tris, pH 8.0, (3 × 4 dm3). The solution
was then heated at 70 ЊC for 1 h, centrifuged, and lyophilized
yielding 2.12 g of SADH as a tan powder with the specific
activity of 27 U mgϪ1 solid.
(R)-Hept-1-yn-3-ol (8h). Yield 30.3 mg (27%), 42% ee, Rf 0.19
(10% ether–pet. ether), [α]D20 ϩ3.8 (c = 5.8, CHCl3) (lit.16 [α]D20
1
Ϫ5.5 (c = 0.9, CHCl3), 70% ee for (S)-enantiomer), H NMR
(250 MHz, CDCl3) δ 4.37 (dt, J = 7.3 Hz, 2.1 Hz, 1H), 2.47 (d,
J = 2.1 Hz, 1H), 1.87 (br s, 1H), 1.72 (m, 2H), 1.41 (m, 4H), 0.92
(t, J = 7.0 Hz, 3H).
Determination of minimum enzyme concentration required for
complete conversion
(R)-6-Methylhept-1-yn-3-ol (8k). Yield 61.8 mg (49%), 80%
ee, Rf 0.21 (10% ether–pet. ether), [α]D20 ϩ9.2 (c = 1.2, CHCl3),
ϩ13.8 (c = 2.0, dioxane), H NMR (300 MHz, CDCl3) δ 4.35
(dt, J = 6.6 Hz, 1.9 Hz, 1H), 2.47 (d, J = 1.9 Hz, 1H), 2.03 (br s,
1H), 1.71 (m, 2H), 1.57 (m, 1H), 1.33 (m, 2H), 0.89 (d, J = 6.5
Hz, 6H).
1
SADH (2.0, 13.5, 27, 54, 81, 108 U) and NADP (0.1 mg, 0.12
µmol) were dissolved in 50 mM Tris, pH 8.0, (0.85 cm3). After
pre-incubating the solution for 10 min at 50 ЊC, a solution of 5c
(16.8 mg, 0.10 mmol) or 7c (9.6 mg, 0.10 mmol) in PriOH (0.15
cm3) was added in one portion. The reactions were monitored
by extracting an aliquot (40 mm3) with CH2Cl2 (40 mm3)
followed by GC analysis (40–125 ЊC, 2 ЊC minϪ1 for 7c, 40–
175 ЊC, 2 ЊC minϪ1 for 5c). The presence or absence of enzyme
activity was assessed by incubating an aliquot (0.2 cm3) with
pentan-2-one (1.7 mg, 0.02 mmol) for 10 min and checking for
the presence of pentan-2-ol by GC.
(R)-Methyl 4-hydroxyhex-5-ynoate (6a). Yield 49.8 mg (35%),
82% ee, Rf 0.20 (20% ethyl acetate–hexanes), [α]D20 ϩ9.9 (c = 1.8,
CHCl3), 1H NMR (250 MHz, CDCl3) δ 4.49 (dt, J = 6.0 Hz, 2.0
Hz, 1H), 3.69 (s, 3H), 2.55 (m, 2H), 2.49 (d, J = 2.1 Hz, 1H),
2.21–1.98 (m, 3H).
(R)-Ethyl 4-hydroxyhex-5-ynoate (6b). Yield 79.6 mg (51%),
90% ee, Rf 0.18 (15% ethyl acetate–hexanes), [α]D20 ϩ17.9 (c = 2.0,
Enzymatic reduction of ethynyl ketones and ethynylketoesters
with SADH
1
CHCl3), IR (neat) νmax 3443, 3291, 2112, 1732 cmϪ1; H NMR
(300 MHz, CDCl3) δ 4.49 (dt, J = 6.1 Hz, 2.1 Hz, 1H), 4.14 (q,
J = 7.1 Hz, 2H), 2.52 (m, 2H), 2.48 (d, J = 1.7 Hz, 1H), 2.21 (br
s, 1H), 2.04 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 173.9, 97.6, 84.0, 73.4, 61.3, 60.7, 32.1, 29.8,
14.1.
SADH (1000 U) and NADP (1 mg, 1.2 µmol) were dissolved in
50 mM Tris, pH 8.0, (8.5 cm3). After pre-incubation for 10 min
at 50 ЊC, the appropriate ketone (5 or 7) (1.0 mmol) was added
in one portion and the mixture was kept at 50 ЊC. When com-
plete conversion was obtained (GC), the reaction mixture was
saturated with NaCl and extracted with Et2O (3 × 4 cm3). Due
to their tendency to form emulsions, the extractions had to be
centrifuged (10 min at 4000 rpm) prior to separation. The com-
bined extracts were dried with Na2SO4, the solvent was removed
in vacuo, and the residue chromatographed.
(R)-Isopropyl 4-hydroxyhex-5-ynoate (6c). Yield 150 mg
(88%), >98% ee, Rf 0.21 (15% ethyl acetate–hexanes), [α]D20 ϩ13.1
(c = 2.0, CHCl3), IR (neat) νmax 3442, 3293, 2114, 1731 cmϪ1; 1H
NMR (250 MHz, CDCl3) δ 5.02 (m, 1H), 4.49 (dt, J = 6.1 Hz,
2.0 Hz, 1H), 2.51 (m, 3H), 2.03 (m, 2H), 1.24 (d, J = 6.4 Hz,
6H); 13C NMR (75 MHz, CDCl3) δ 173.2, 84.0, 73.3, 68.1, 61.2,
32.2, 30.2, 21.7.
Determination of optical purity of the alcohols
The alcohol (0.02 mmol) was dissolved in CDCl3 (0.3 cm3) in an
NMR tube and a 50 mM solution of Eu(hfc)3 in CDCl3 (0.5
(R)-Ethyl 5-hydroxyhept-6-ynoate (6d). Yield 129 mg (76%),
97% ee, Rf 0.22 (20% ethyl acetate–hexanes), [α]D20 ϩ16.9
(c = 1.66, CCl4), ϩ7.8 (c = 3.8, CHCl3) (lit.17 [α]D20 Ϫ16.4
1
cm3) was added in portions of 0.1 cm3. A H NMR spectrum
was taken after each addition, and the two signals correspond-
ing to the diastereotopic methanol protons were integrated. If
only one signal was visible, the enantiomeric excess was
assumed to be greater than 98%.
1
(c = 3.30, CCl4), 96% ee for (S)-enantiomer); H NMR (300
MHz, CDCl3) δ 4.38 (dt, J = 6.2 Hz, 1.8 Hz, 1H), 4.12
(q, J = 7.2 Hz, 2H), 2.46 (d, J = 2.2 Hz, 1H), 2.35 (t, J = 7.0
Hz, 2H), 2.24 (br s, 1H), 1.78 (m, 4H), 1.24 (t, J = 7.1 Hz,
3H).
Spectral data of isolated products
(S)-Pent-1-yn-3-ol (8b). Yield 30.3 mg (36%), 80% ee, Rf 0.20
(10% ether–pet. ether), [α]D20 Ϫ19.2 (c = 12, dioxane) (lit.15 [α]D25
ϩ23.15 (c = 2, dioxane), 86% ee for (R)-enantiomer), 1H NMR
(300 MHz, CDCl3) δ 4.32 (dt, J = 6.5 Hz, 2.0 Hz, 1H), 2.46 (d,
J = 1.8 Hz, 1H), 1.74 (m, 3H), 1.02 (t, J = 7.4 Hz, 3H).
(R)-Isopropyl 5-hydroxyhept-6-ynoate (6e). Yield 139 mg
(76%), >98% ee, Rf 0.24 (20% ethyl acetate–hexanes), [α]D20 ϩ10.3
(c = 2.6, CHCl3), IR (neat) νmax 3433, 3291, 2112, 1727 cmϪ1; 1H
NMR (300 MHz, CDCl3) δ 5.01 (m, 1H), 4.40 (dt, J = 6.0 Hz,
2.6 Hz, 1H), 2.47 (d, J = 2.6 Hz, 1H), 2.33 (t, J = 6.9 Hz, 2H),
2.03 (br s, 1H), 1.78 (m, 4H), 1.23 (d, J = 6.2 Hz, 6H); 13C NMR
(75 MHz, CDCl3) δ 173.0, 84.5, 73.0, 67.7, 61.7, 36.7, 34.0, 21.8,
20.4.
(S)-4-Methylpent-1-yn-3-ol (8c). Yield 49.1 mg (50%), >98%
ee, Rf 0.22 (10% ether–pet. ether), [α]D20 Ϫ15.7 (c = 13, dioxane)
(lit.15 [α]D20 ϩ13.8 (c = 2, dioxane), 86% ee for (R)-enantiomer),
1H NMR (300 MHz, CDCl3) δ 4.17 (dd, J = 5.7 Hz, 2.0 Hz,
1H), 2.45 (d, J = 2.3 Hz, 1H), 1.89 (m, 2H), 1.01 (m, 6H).
(R)-Methyl 6-hydroxyoct-7-ynoate (6f). Yield 115 mg (68%),
>98% ee, Rf 0.22 (20% ethyl acetate–hexanes), [α]D20 ϩ8.8 (c = 2.2,
(S)-Hex-1-yn-3-ol (8e). Yield 27.5 mg (28%), 51% ee, Rf 0.22
(10% ether–pet. ether), [α]D20 Ϫ4.5 (c = 1.5, CHCl3) (lit.15 [α]D20
CHCl3), IR (neat) νmax 3434, 3289, 2111, 1734 cmϪ1 1H
;
NMR (300 MHz, CDCl3) δ 4.36 (dt, J = 6.5 Hz, 2.1 Hz, 1H),
3.65 (s, 3H), 2.45 (d, J = 2.5 Hz, 1H), 2.32 (t, J = 7.4 Hz, 2H),
2.22 (br s, 1H), 1.79–1.60 (m, 4H), 1.50 (m, 2H); 13C NMR
(75 MHz, CDCl3) δ 174.1, 84.8, 73.0, 62.0, 51.6, 37.1, 33.9,
24.5 (2×).
1
ϩ9.0 (c = 1.0, CHCl3), 75% ee for (R)-enantiomer), H NMR
(250 MHz, CDCl3) δ 4.39 (dt, J = 6.5 Hz, 2.2 Hz, 1H), 2.47 (d,
J = 2.1 Hz, 1H), 1.71 (m, 3H), 1.50 (m, 2H), 0.97 (t, J = 7.0 Hz,
3H).
(R)-5-Methylhex-1-yn-3-ol (8f). Yield 22.4 mg (20%), 50% ee,
Rf 0.20 (10% ether–pet. ether), [α]D20 ϩ18.3 (c = 1.2, dioxane)
(lit.15 [α]D25 ϩ28.8 (c = 3, dioxane), 88% ee for (R)-enantiomer),
1H NMR (300 MHz, CDCl3) δ 4.42 (dt, J = 7.4 Hz, 2.3 Hz, 1H),
2.46 (d, J = 2.1 Hz, 1H), 1.87 (m, 1H), 1.60 (m, 3H), 0.95 (d,
J = 6.7 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3 H).
Acknowledgements
We thank Professor J. Gregory Zeikus and Dr Douglas S.
Burdette for generously providing the microorganisms express-
ing SADH.
2824
J. Chem. Soc., Perkin Trans. 1, 2000, 2821–2825