Using pure halohydrin dehalogenase (HheC), competing activities observed in
whole-cell preparations were eliminated and halohydrins could be resolved via
enantioselective ring-closure with excellent selectivities yielding (R)-epoxides
and nonreacted (S)-halohydrins (Scheme 2.235) [2051, 2052]. A stereocomplementary enzyme (HheA) showing opposite stereoselectivity could be
identified [2053].
Subsequent studies revealed that the natural nucleophile halide (Cl, Br, I) could
be replaced by nonnatural analogs, such as azide [2054], nitrite [2055], cyanide
[2056], (thio)cyanate and formate by maintining the exquisite regioselectivity of
nucleophilic attack at the less hindered oxirane carbon atom. Whereas the reaction
rates observed with cyanide, (thio)cyanate, and formate were comparable to those
using halide, azide and nitrite proved to be much better nucleophiles
[2057]. Nonlinear and nonanionic nucleophiles, such as H 2 S, acetate, PO 4
3À
, CO 3
2À ,
BO 3
3À , and F
À were unreactive. The use of N-nucleophiles opened the way to
prepare 1,2- and 1,3-aminoalcohols using azide or cyanide via the corresponding
1-azido-2-ols and 1-cyano-2-ols, respectively (Scheme 2.236).
Arg149
O
R
HO Ser132
HN
H 2 N
NH 2
Tyr145
O
H
H
O Tyr145
NH 2
H 2 N
HN
Ser132
HO
Arg149
R
Hal
O
Halide
binding
site
Hal
Halide
binding
site
Hal = Cl, Br, I
Scheme 2.234 Catalytic mechanism of halohydrin dehalogenase from Agrobacterium
radiobacter
O
R
OH
Cl
R
OH
Cl
R
rac
+
Halohydrin
dehalogenase HheC
(S)
(R)
- HCl
R
Enantioselectivity (E)
(E)-Me-CH=CH>200
(E)-Et-CH=CH177
(E)-n-C 4 H 9 -CH=CH>200
(E)-Ph-CH=CH>200
2-Furyl
>200
2-Thiophenyl
65
Scheme 2.235 Kinetic resolution of halohydrins using halohydrin dehalogenase
260
2 Biocatalytic Applications
whole-cell preparations were eliminated and halohydrins could be resolved via
enantioselective ring-closure with excellent selectivities yielding (R)-epoxides
and nonreacted (S)-halohydrins (Scheme 2.235) [2051, 2052]. A stereocomplementary enzyme (HheA) showing opposite stereoselectivity could be
identified [2053].
Subsequent studies revealed that the natural nucleophile halide (Cl, Br, I) could
be replaced by nonnatural analogs, such as azide [2054], nitrite [2055], cyanide
[2056], (thio)cyanate and formate by maintining the exquisite regioselectivity of
nucleophilic attack at the less hindered oxirane carbon atom. Whereas the reaction
rates observed with cyanide, (thio)cyanate, and formate were comparable to those
using halide, azide and nitrite proved to be much better nucleophiles
[2057]. Nonlinear and nonanionic nucleophiles, such as H 2 S, acetate, PO 4
3À
, CO 3
2À ,
BO 3
3À , and F
À were unreactive. The use of N-nucleophiles opened the way to
prepare 1,2- and 1,3-aminoalcohols using azide or cyanide via the corresponding
1-azido-2-ols and 1-cyano-2-ols, respectively (Scheme 2.236).
Arg149
O
R
HO Ser132
HN
H 2 N
NH 2
Tyr145
O
H
H
O Tyr145
NH 2
H 2 N
HN
Ser132
HO
Arg149
R
Hal
O
Halide
binding
site
Hal
Halide
binding
site
Hal = Cl, Br, I
Scheme 2.234 Catalytic mechanism of halohydrin dehalogenase from Agrobacterium
radiobacter
O
R
OH
Cl
R
OH
Cl
R
rac
+
Halohydrin
dehalogenase HheC
(S)
(R)
- HCl
R
Enantioselectivity (E)
(E)-Me-CH=CH>200
(E)-Et-CH=CH177
(E)-n-C 4 H 9 -CH=CH>200
(E)-Ph-CH=CH>200
2-Furyl
>200
2-Thiophenyl
65
Scheme 2.235 Kinetic resolution of halohydrins using halohydrin dehalogenase
260
2 Biocatalytic Applications