Preparation of halohydrin β-blocker precursors using yeast-catalysed reduction

Article abstract of DOI:10.1016/S0957-4166(00)00425-0

The preparation of halohydrin β-blocker precursors using yeast-catalysed reduction of α-haloketones was performed. The influence in the yield and e.e. of several process variables was analysed. The (S)-enantioselectivity observed with Saccharomyces cerevisiae can be changed to (R)-enantioselectivity using methyl vinyl ketone as selective inhibitor (25 mM). Using resting fresh cells better yields and e.e.s are observed than using growing cells. Yarrowia lipolytica 1240 resting cells gave 87% yield of (S)-1-chloro-3(1-naphthyloxy)propan-2-ol (99% e.e.). Pichia mexicana 11105 resting cells gave 85% yield of (R)-1-chloro-3(1-naphthyloxy)propan-2-ol (precursor of propranolol) (95% e.e). The reduction process is applied to other α-haloketones, a lower e.e. being obtained the closer the size of the ketone substituents.

Full text of DOI:10.1016/S0957-4166(00)00425-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 4651–4660
Preparation of halohydrin b-blocker precursors using
yeast-catalysed reduction
Fernando Mart´ınez, Carmen Del Campo, J. V. Sinisterra* and Emilio F. Llama
Departamento de Q. Orga´nica y Q. Farmace´utica, F. de Farmacia, Universidad Complutense, 28040 Madrid, Spain
Received 8 September 2000; accepted 23 October 2000
Abstract
The preparation of halohydrin b-blocker precursors using yeast-catalysed reduction of a-haloketones
was performed. The influence in the yield and e.e. of several process variables was analysed. The
(S)-enantioselectivity observed with Saccharomyces cerevisiae can be changed to (R)-enantioselectivity
using methyl vinyl ketone as selective inhibitor (25 mM). Using resting fresh cells better yields and e.e.s
are observed than using growing cells. Yarrowia lipolytica 1240 resting cells gave 87% yield of (S)-1-
chloro-3(1-naphthyloxy)propan-2-ol (99% e.e.). Pichia mexicana 11105 resting cells gave 85% yield of
(R)-1-chloro-3(1-naphthyloxy)propan-2-ol (precursor of propranolol) (95% e.e). The reduction process is
applied to other a-haloketones, a lower e.e. being obtained the closer the size of the ketone substituents.
© 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
The halohydrin 1 is the key intermediate in the preparation of homochiral b-blockers with the
aryloxy-2-propanolamine structure. The resolution of (RS)-1 using lipases, both by esterification
or by deacylation of acylated ( )-1 has been used as a standard alternative to prepare pure
homochiral adrenergic b-blockers.1 However, even the highly enantioselective lipase-catalysed
process can only provide 50% of the starting racemate in optically active form. Alternatively, an
enantiotope selective method, such as reduction of a prochiral ketone 2, might quantitatively
yield an optically active product with yields greater than 50%. The stereoselective reduction of
prochiral ketones 2 has been carried out by using chiral complexes of Rh(I).2 Knowing this
process is expensive and very difficult to scale up, a cheap alternative is the use of redox enzymes
from whole cells. In this way stereoselective reduction of ketones has been carried out with
oxidoreductases from Bacillus sp. or Staphylococcus sp.,3 or with alcohol dehydrogenases from
Alcaligenes eutrophus,4 Zygosaccharomyces rouxii, Saccharomyces cerevisiae (baker’s yeast),5 etc.
* Corresponding author.
0957-4166/00/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00425-0

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F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
In the in vitro enzymatic reductions of prochiral ketones to chiral alcohols, the recycling of
redox cofactors is still difficult to achieve in a low cost and effective way. The use of whole cells
offers the advantage that the cofactor is recycled in vivo and avoids the additional costs of the
enzyme purification. So this biotechnological process has the potential to be scaled up.
Some studies about the variables that control the biotransformation of prochiral ketones have
been reported5 due to the presence of several enzymes in the whole cell. In the case of the yeast
S. cerevisiae four of these enzymes have been identified and purified.6 These enzymes have
opposite enantioselectivities6 and can be inhibited by different substances, showing different
activity–concentration of substrate profile.7 These different behaviours are explained because
they are enzymes involved in different metabolic pathways.8
In the present paper we have performed the stereoselective reduction of 2 to the halohydrin
1 with good yield and e.e. using different microorganism strains and experimental conditions.
2. Results and discussion
The 1-aryloxy-3-halopropan-2-ones 2a–d were easily prepared by o-alkylation of the corre-
sponding phenols with racemic epihalohydrin, followed by oxidation of 1a–d (Table 1).
Table 1
Preparation of 1-aryloxy-3-halopropan-2-onesa
Ar
X
(RS)-1a–d yield (%)c
2a–d yield (%)c
A
B
C
D
1-Naphthyl
1-Naphthyl
Phenyl
Chloro
Bromob
Chloro
Chloro
80
82
85
90
92
70
78
95
p-Acetamidophenyl
a Reagents: (i) (a) pyridine, (b) XH, CHCl3; (ii) oxalyl chloride, DMSO, Et3N.
b Pyridinium fluorochromate (PFC).
c Yields referred to isolated pure products.
The reduction of 2 using two chiral commercial chemical reductants such as (−)-diisopinocam-
phenyl borane chloride or (R)-alpine-borane gave moderate yields of 1, such as 18 or 36% with
very low e.e. 16 or 8% (S>R).
The effect of the reaction conditions on enantiotope selectivity of the baker’s yeast (S.
cerevisiae, type II, Sigma) reduction of 1-chloro-3-(1-naphthyloxy)propan-2-one 2a was investi-
gated (Table 2).
We can observe that an increase in the amount of baker’s yeast used increases the yield but
the e.e. value remains nearly constant (entries 5, 7 and 9). This result indicates that we are in the
lineal zone ‘amount of biocatalyst versus yield’, useful for catalytic studies. Small variations in
e.e. and yield are observed using phosphate buffers with different pH (entries 9, 10 and 11). This
behaviour is different from the one observed in the reduction of b-keto esters described by Hunt
et al.5a using Z. rouxii or of ketones using alcohol dehydrogenase from Thermoanaerobium

F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
4653
Table 2
Effect of the reaction conditions on the selectivity of reduction of the 1-chloro-3-(1-naphthyloxy)propan-2-one
2aa
Entry
Temperature (°C)
S. cere6isiae (g)
Time (h)
pH
Yieldb (%)
e.e. (%)c S\R
1
2
3
4
5
6
7
8
9
18
25
25
25
30
30
30
30
30
30
30
6.5
1
7
24
1
7
7
7
7
7
7
7
7
7
6
8
6
14
25
9
18
29
31
44
45
56
52
60
51
49
68
40
39
44
42
45
45
55
1.68
1.68
6.5
1.68
1.68
3.36
3.36
6.5
6
24
24
48
24
24
24
10
11
6.5
6.5
a Reaction conditions: 2a, 100 mg; media (0.1 M sodium phosphate buffer), 25 ml; saccharose 4 g.
b Yields referred to isolated pure products.
c Absolute configurations and e.e. determined by chiral phase HPLC. The absolute configuration was assigned on
the basis of the specific rotation.1b,c
Brokii,9 where a clear variation was observed. This behaviour may be explained because S.
cerevisiae has four enzymes with different pH profile and different thermal stability.6
An increase in the reaction time produces an increase in the yield (entries 2 and 3) but a very
small diminution in the e.e. value.
The stereoselectivity of the process increases with the pH value of the phosphate buffer
(entries 10 and 11) (Table 2). The ability to switch the stereoselectivity by changing pH has been
observed in the oxidation of 1,2-cyclohexanediol by Gluconobacter oxidants, resulting from
catalysis by two enzymes of opposite enantioselectivity with different optimum pH.10 Hunt et
al.5a described the same phenomenon with the yeast Pichia capsulata in the reduction of
4-halo-2-ketoesters but with smaller variation than those described by Adlercreutz.10 The
increase in the enantioselectivity with pH has been analysed by De Souza,11 who indicates that
the production of NAD(P)H increases with the pH because at pH<7 the baker’s yeast cells
experience oxidative stress.
To explore the scope of the stereoselective reduction, several haloketones 2a–d were used
under the conditions which gave the best selectivity with 2a (Table 3). In all cases, the e.e. was
not very high, especially with 2c and 2d, the substrates with the smallest substituent, because the
greater the difference in the size of substituents of carbonyl group the greater the stereoselectiv-
ity of the process.5c,8
In all cases, an (S)-stereopreference was observed. This disagrees with Prelog’s rule of
stereocontrol, generally described for the Baker’s yeast reductions8–10 with addition of hydride
by the Re-face.

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F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
Table 3
Saccharomyces cere6isiae type II mediated biotransformation of 1-aryloxy-3-halopropan-2-ones 2a–da
Compound
Yield (%)b
Conf. of 1a–d
e.e. (%)c
2a
2b
2c
2d
52
41
54
58
S\R
S\R
S\R
S\R
55
50
18
22
a Reaction conditions: 2a–d, 100 mg; S. cere6isiae type II 6.5 g; media (0.1 M sodium phosphate buffer, pH 8) at
30°C, 25 ml; saccharose 4 g.
b Yields referred to isolated pure products.
c Absolute configurations and e.e. determined by chiral phase HPLC. The absolute configuration of 1b was assigned
on the basis of its conversion to propanolol using isopropylamine,1b 1c and 1d on the basis of their specific
rotations.1d
Nevertheless, this statement is not exclusive because the stereocontrol of the process depends
on the nature, the size and the electronic characteristics of the substituents of CꢀO bond, as
described by several workers5c,12,13 and to the presence of several isoenzymes with different
stereoselectivity, pH and temperature profiles.6 The polar characteristic of chlorine may be the
origin of this enantioselectivity, as described by Waagen et al. for 1-hydroxy-2-propanones
(giving R>S stereoselectivity as in our case) and for 1-aryloxy-2-propanones (giving S>R, the
opposite stereoselectivity) or by Nakamura13 (methyl ketones gave the Prelog (S)-alcohol and
trifluormethyl ketones gave the anti-Prelog (R)-alcohol).
2.1. Selective poisoning of the reductases
Because (S)-1a will give the (R)-adrenergic-b-blocker, propranolol, we tried to change the
enantioselectivity of the reduction process. In the literature the selective poisoning of Baker’s
yeast isoenzymes has been described using ethyl chloroacetate 3 or methyl vinyl ketone 4. The
results obtained by using 25 mM of inhibitor and 16.8 mM of substrate are shown in Table 4.
The experiments were performed in optimum experimental conditions according to Table 2.

F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
4655
Table 4
Alteration of the enantioselectivity in the reduction of 2a or c by the presence of some dehydrogenase
inhibitorsa
Compound
Inhibitor
1 Yieldb (%)
1 (R) (%)
1 (S) (%)
1 (e.e. (%))
1 Conf.c
2a
–
3
4
–
3
4
52
28
35
54
37
41
22
47
75
41
48
79
78
53
25
59
52
21
56
6
50
18
4
S\R
S\R
R\S
S\R
S\R
R\S
2c
58
a Reaction conditions: 2a and c 100 mg; S. cere6isiae type II 6.5 g; media (0.1 M sodium phosphate buffer, pH 8)
at 30°C, 25 ml; saccharose (4 g); inhibitor 3 or 4, 25 mM.
b Yields referred to isolated pure products.
c Absolute configurations and e.e. determined by chiral phase HPLC.
Other experiments, with higher inhibitor concentrations than 25 mM were performed, but a
strong reduction in the yield was observed. No reduction of 2 was observed at inhibitor
concentration ]50 mM.
We observed that the reduction of 2a or c in the presence of ethyl chloroacetate 3 produces
a reduction in the yield and an increase in the percentage of (R)-1 and a moderated diminution
in the amount of (S)-1. The same behaviour has been described by Egri et al.5c in the reduction
of 1-acetoxy-3-phenoxypropan-2-one. When 4 is used the yield decreases, but the enantioprefer-
ence changes due to the dramatic reduction of the percentages of (S)-1. Taking into account the
selective poisoning work carried out by Nakamura et al.6 with pure isolated oxidoreductase
enzymes from Baker’s yeast, we can say that our main alcohol ((S)-isomer)) is produced by a
D
-isoenzyme (probably
D
-enzyme-29) by means of an anti-Prelog pathway; and the (R)-isomer
is produced by
(Scheme 1).
L
-enzymes that should be less active than
D
-enzymes in our commercial strain
Scheme 1.
These
D
-enzymes that give (S)-1 are in our case very sensitive to the poisoning by 3 and
especially for 7. The poisoning produces a reduction in the yield according to Nakamura et al.6
and a strong diminution in the percentage of (S)-stereomer. Therefore the (R)-counterpart
becomes the main alcohol changing the apparent stereoselectivity.

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F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
2.2. Screening with other yeasts
As an alternative we performed the reduction using other microorganisms. The reductions
were directly performed in the growth cell culture medium or using fresh resting cells (Table 5).
Fresh resting cells gave better yields and e.e. values than growing cells because of secondary
metabolic reactions of growing cells that transform the substrate or the product.14
Table 5
Microbial reduction of 2a to alcohol 1a in water
Entry
Microorganism
Culturea
Conf.d
Fresh cellsb
Conf.d
Yieldc (%)
e.e.d
Yield (%)
e.e.d
1
2
3
4
5
Saccharomyces cere6isiae type II
Saccharomyces cere6isiae 1317
Saccharomyces bayanus 1969
Yarrowia lipolytica 1240
15
17
18
25
23
S
80
74
54
99
10
30
27
40
87
85
S
83
75
88
99
95
R
R
S
R
R
S
Pichia mexicana 11015
R
R
a Reductions carried out directly in the growth medium (48 h entry 1, 2, 4, 5 and 72 h entry 3). Reaction conditions:
2a, 40 mg; 120 h.
b Reductions carried out with fresh cells suspended in a 0.1 M phosphate buffer (pH 7.0) at 28°C, in an aqueous
reactor with a final volume of 20 ml; 2a, 7.5 mg; substrate dissolved in benzene, saccharose 600 mg.
c Molar conversions determined after 48 h by HPLC on the crude mixture.
d Absolute configurations and e.e. determined by chiral phase HPLC and specific rotation. (S)-(+)-1a (99%)
[h]2D5=+9.3 (c=1.9, EtOH); (R)-(−)-1a (95%) [h]2D5=−8.8 (c=1.9, EtOH).
The stereochemistry is different in each strain but it is not altered by the physical situation of
the biocatalyst—growing cells or resting cells. Resting cells of Yarrowia lipolytica 1240 yielded
the corresponding (S)-halohydrin in 99% e.e, whereas Pichia mexicana 11015 gave the (R)-halo-
hydrin in 95% e.e. These strains are the most interesting biocatalysts because the (R)-b-blocker
(Y. lipolytica 1240) or the (S)-b-blocker (P. mexicana) can easily be obtained by substitution of
Cl by NH-iPr. This methodology overcomes the shortfalls in terms of the yield and/or the
enantioselectivity obtained using lipase.1b,c Besides these strains improve on the results obtained
with S. cerevisiae (Table 2).
Presently, a more detailed study is in progress in order to immobilise the resting cells to enable
an industrial catalytic process to be scaled up.
3. Experimental
3.1. Chemicals
1-Naphthol, epichlorohydrin, epibromohydrin, phenol, 4-acetamidophenol, pyridinium
fluorochromate (PFC) were purchased from Aldrich Chemical Company. S. cerevisiae (Baker’s
yeast) type II was purchased from Sigma. S. cerevisiae 1317, S. cerevisiae 1969, Y. lipolytica 1240
and P. mexicana 11015 were kindly supplied by Spanish Type Culture Collection (CECT;
Valencia Spain).

F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
4657
3.2. Analytical methods
The enantiomeric excess (e.e.) and the absolute configuration of 1 were determined by HPLC
using a ConstaMetric 4100 system equipped with a chiral column (Chiracel OD), UV–vis detector
and a Knauer chiral detector. Mobile phases: 1a, c, and d, hexane–isopropanol–diethylamine=
70:30:0.1 (v/v/v), 0.5 ml min−1; 1b, hexane–isopropanol–diethylamine=60:40:0.1 (v/v/v), 0.4 ml
1
min−1. H and 13C NMR spectra were determined on a Bruker AC-250 MHz spectrometer; all
spectra were taken in CDCl3 solution and chemical shift values are expressed in ppm values from
TMS as internal standard. Optical rotations were measured on a Perkin–Elmer 241 polarimeter.
Optical density was measured on a Cecil 1021 spectrophotometer at 600 nm.
3.3. Preparation of 1-aryloxy-3-halopropan-2-ols (rac)-1a–d
A mixture of phenol (0.02 mol), epichlorohydrin (0.1 mol) and pyridine (0.002 mol) were stirred
at room temperature for 24 h. Then, the unreacted epichlorohydrin and pyridine were removed
in vacuo. The mixture was cooled and HCCl3 (10 ml) and 5 ml of HCl 35% were added, and the
mixture stirred for 1 h at room temperature. Afterwards, 10 ml of water were added and the organic
phase was removed and washed again with 10 ml of H2O. The organic phase was dried over
anhydrous CaCl2 and concentrated in vacuo.
3.3.1. (RS)-1-Chloro-3-(1-naphthyloxy)-2-propanol 1a
1H NMR (CDCl3, 250 MHz) l: 2.86 (d, 1H, OH), 3.85 (m, 2H, CH2ꢁCl), 4.23 (m, 2H, CH2ꢁO),
4.35 (m, 1H, CH), 6.82 (d, 1H, ArꢁH), 7.36 (t, 1H, ArꢁH), 7.49 (m, 3H, ArꢁH), 7.80 (m, 1H,
ArꢁH), 8.18 (m, 1H, ArꢁH) ppm; 13C NMR (CDCl3) l: 46.5 (CH2ꢁCl), 68.8 (CHꢁOH), 70.1
(CH2ꢁO), 105.2 (C2, Ar), 121.2 (C4, Ar), 121.7 (C8, Ar), 125.5 (C9, Ar), 125.6 (C7, Ar), 125.9 (C3,
Ar), 126.7 (C6, Ar), 127.7 (C5, Ar), 134.6 (C10, Ar), 153.9 (C1, Ar) ppm. Elemental anal. calcd
for C13H13O2Cl: C, 67.97; H, 5.54. Found: C, 67.72; H, 5.55.
3.3.2. (RS)-1-Bromo-3-(1-naphthyloxy)-2-propanol 1b
1H NMR (CDCl3, 250 MHz) l: 2.6 (d, 1H, OH), 3.75 (m, 2H, CH2ꢁBr), 4.23 (m, 2H, CH2ꢁO),
4.34 (m, 1H, CH), 6.81 (d, 1H, ArꢁH), 7.36 (t, 1H, ArꢁH), 7.49 (m, 3H, ArꢁH), 7.80 (m, 1H,
ArꢁH), 8.18 (m, 1H, ArꢁH) ppm; 13C NMR (CDCl3) l: 35.6 (CH2ꢁBr), 69.4 (CHꢁOH), 69.7
(CH2ꢁO), 105.1 (C2, Ar), 121.2 (C4, Ar), 121.7 (C8, Ar), 125.5 (C9, Ar), 125.6 (C7, Ar), 125.9 (C3,
Ar), 126.7 (C6, Ar), 127.7 (C5, Ar), 134.6 (C10, Ar), 153.8 (C1, Ar) ppm. Elemental anal. calcd
for C13H13O2Br: C, 55.55; H, 4.68. Found: C, 55.58; H, 4.75.
3.3.3. (RS)-1-Chloro-3-phenyloxy-2-propanol 1c
1H NMR (CDCl3, 250 MHz) l: 2.65 (d, 1H, OH), 3.74 (m, 2H, CH2ꢁCl), 4.06 (m, 2H, CH2ꢁO),
4.19 (m, 1H, CH), 6.90 (d, 2H, Ar-H), 6.97 (t, 1H, ArꢁH), 7.29 (m, 2H, ArꢁH) ppm; 13C NMR
(CDCl3) l: 46.0 (CH2ꢁCl), 68.4 (CHꢁOH), 69.9 (CH2ꢁO), 114.6 (C2, C6, Ar), 121.5 (C4, Ar), 129.7
(C3, C5, Ar), 158.2 (C1, Ar) ppm. Elemental anal. calcd for C9H11O2Cl: C, 57.92; H, 5.94. Found:
C, 57.82; H, 5.95.
3.3.4. (RS)-1-Chloro-3-(p-acetamidophenyloxy)-2-propanol 1d
1H NMR (CDCl3, 250 MHz) l: 2.14 (s, 3H, CH3), 2.51 (d, 1H, OH), 3.73 (m, 2H, CH2ꢁCl),
4.03 (m, 2H, CH2ꢁO), 4.19 (m, 1H, CH), 6.85 (d, 2H, ArꢁH), 7.05 (s, 1H, NH), 7.37 (d, 2H,

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F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
ArꢁH) ppm; 13C NMR (CDCl3) l: 24.9 (CH3), 46.0 (CH2ꢁCl), 68.8 (CHꢁOH), 69.9 (CH2ꢁO),
115.0 (C2, C6, Ar), 129.7 (C3, C5, Ar), 132.7 (C4, Ar), 154.0 (C1, Ar), 167.6 (CONH) ppm.
Elemental anal. calcd for C11H14NO3Cl: C, 54.22; H, 5.87; N, 5.79. Found: C, 57.82; H, 5.95; N,
5.77.
3.4. Preparation of 1-aryloxy-3-halopropan-2-ones 2a–d
The alcohols 1a, c and d was oxidised as follows: A solution of freshly distilled oxalyl chloride
(4.6 mmol) in CH2Cl2 (10 ml) was cooled to −70°C and DMSO (9.2 mmol) in CH2Cl2 (3 ml) was
added over 5 min. The reaction mixture was stirred for a further 10 min at the same temperature
and the alcohol (4.2 mmol) in CH2Cl2 (5 ml) was added over 5 min. After additional stirring for
15 min, Et3N (0.3 ml) was added and the cooling bath was removed. At room temperature H2O
(10 ml) was added and the organic phase was separated from the water phase which was
extracted with CH2Cl2 (3×25 ml). The organic phase was dried over anhydrous CaCl2,
concentrated under vacuum and chromatographed on silica gel.
3.4.1. 1-Chloro-3-(1-naphthyloxy)-2-propanone 2a
1H NMR (CDCl3, 250 MHz) l: 4.53 (s, 2H, CH2ꢁCl), 4.92 (s, 2H, CH2ꢁO), 6.68 (d, 1H,
ArꢁH), 7.35 (t, 1H, ArꢁH), 7.51 (m, 3H, ArꢁH), 7.81 (m, 1H, ArꢁH), 8.25 (m, 1H, ArꢁH) ppm;
13C NMR (CDCl3) l: 47.0 (CH2ꢁCl), 71.9 (CH2ꢁO), 104.9 (C2, Ar), 121.6 (C4, Ar), 122.0 (C8,
Ar), 125.2 (C9, Ar), 125.6 (C7, Ar), 125.9 (C3, Ar), 126.9 (C6, Ar), 127.8 (C5, Ar), 134.7 (C10, Ar),
153.1 (C1, Ar), 199 (CꢀO) ppm. Elemental anal. calcd for C13H11O2Cl: C, 66.53; H, 4.72. Found:
C, 66.47; H, 5.75.
3.4.2. 1-Chloro-3-phenyloxy-2-propanone 2c
1H NMR (CDCl3, 250 MHz) l: 4.41 (s, 2H, CH2ꢁCl), 4.75 (s, 2H, CH2ꢁO), 6.89 (d, 2H,
ArꢁH), 7.0 (t, 1H, ArꢁH), 7.31 (m, 2H, ArꢁH) ppm; 13C NMR (CDCl3) l: 46.0 (CH2ꢁCl), 72.5
(CH2ꢁO), 114.3 (C2, C6, Ar), 121.5 (C4, Ar), 129.6 (C3, C5, Ar), 157.2 (C1, Ar), 198.4 (CꢀO) ppm.
Elemental anal. calcd for C9H9O2Cl: C, 58.63; H, 4.94. Found: C, 58.65; H, 4.95.
3.4.3. 1-Chloro-3-(p-acetamidophenyloxy)-2-propanol 2d
1H NMR (CDCl3, 250 MHz) l: 2.15 (s, 3H, CH3), 4.39 (m, 2H, CH2ꢁCl), 4.73 (m, 2H,
CH2ꢁO), 6.84 (d, 2H, ArꢁH), 7.10 (s, 1H, NH), 7.41 (d, 2H, ArꢁH) ppm; 13C NMR (CDCl3) l:
25.1 (CH3), 48.0 (CH2ꢁCl), 72.8 (CH2ꢁO), 115.0 (C2, C6, Ar), 129.9 (C3, C5, Ar), 133.7 (C4, Ar),
154.2 (C1, Ar), 167.9 (CONH), 197.5 (CꢀO) ppm. Elemental anal. calcd for C11H12NO3Cl: C,
54.72; H, 5.07; N, 5.84. Found: C, 54.75; H, 5.05; N, 5.74.
3.5. Preparation of 1-bromo-3-(1-naphthyloxy)-2-propanone 2b
The alcohol 1b was oxidised as follows: A mixture of pyridinium fluorochromate (4.83 mmol)
and CH2Cl2 (20 ml) was stirred for 10 min at room temperature. Alcohol 1b (3.22 mmol) in
CH2Cl2 (10 ml) was then added. The solution was stirred for 6 h. The solution was decanted and
washed with three portions of ether. The combined organic solution was passed through Celite
and the solvent was removed and chromatographed on silica gel.

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4659
3.5.1. 1-Bromo-3-(1-naphthyloxy)-2-propanone 2b
1H NMR (CDCl3, 250 MHz) l: 4.26 (d, 2H, CH2ꢁBr), 4.94 (d, 2H, CH2ꢁO), 6.71 (d, 1H,
ArꢁH), 7.36 (t, 1H, ArꢁH), 7.52 (m, 3H, ArꢁH), 7.82 (m, 1H, ArꢁH), 8.28 (m, 1H, ArꢁH) ppm;
13C NMR (CDCl3) l: 31.5 (CH2ꢁBr), 71.0 (CH2ꢁO), 104.7 (C2, Ar), 121.5 (C4, Ar), 121.7 (C8,
Ar), 125.3 (C9, Ar), 125.4 (C7, Ar), 125.7 (C3, Ar), 126.7 (C6, Ar), 127.5 (C5, Ar), 134.5 (C10, Ar),
153.6 (C1, Ar), 198.8 (CꢀO) ppm. Elemental anal. calcd for C13H11O2Br: C, 55.91; H, 4.03.
Found: C, 55.88; H, 4.08.
3.6. Reduction of ketones with S. cerevisiae type II (Sigma). General procedure
A suspension of 2 (0.5 mmol), baker’s yeast type II (6.5 g) and saccharose (4 g) in water/0.1
M phosphate buffer was stirred at 30°C for 24 h. After extraction with ether, the extract was
washed with water, dried (MgSO4) and evaporated to dryness. Chromatography of the residue
1
on silica gel with dichloromethane as eluent yielded the alcohol 1. H and 13C NMR spectra
were identical with racemic samples.
3.7. Reduction with S. cerevisiae type II in the presence of inhibitors
A suspension of compound 2 (0.5 mmol), baker’s yeast type II (6.5 g), ethyl chloroacetate, 3
or methyl vinyl ketone, 4 (16.8 mM) and saccharose (4 g) in water/0.1 M phosphate buffer was
stirred at 30°C for 24 h. After extraction with ether, the extract was washed with water, dried
(MgSO4) and evaporated to dryness. Chromatography of the residue on silica gel with
1
dichloromethane as eluent yielded the alcohol 1. H and 13C NMR spectra were identical with
racemic samples.
3.8. Microorganisms, media and culture conditions
All the yeasts were grown at 28°C on media with glucose (10 g), peptone (3 g), yeast extract
(3 g) and 0.1 M phosphate buffer (pH 7.0) for 48–72 h. All the cultures were grown in 250 ml
flasks containing 50 ml of medium. Microbial reductions were directly performed on the media
of culture or with centrifuged and washed cells (see Table 5).
3.9. Biotransformation conditions
3.9.1. Reduction using the whole cells in the culture medium
The reduction of 2a Y. lipolytica 1240 is reported as an example: A solution of ketone (40 mg)
in benzene (200 ml) was added to a 250 ml flask containing 50 ml of culture of Y. lipolytica 1240
(48 h) (OD=2.14, no of cells=72.08×106 ml−1). After 120 h ethyl acetate (5 ml) was added. The
mixture was centrifuged and decanted to give pure (R)-(−)-1-chloro-3-(1-naphthyloxy)-2-propra-
nol 1a in 25% yield and 99% e.e.
3.9.2. Reduction using fresh resting cells
The reduction of 2a using Y. lipolytica 1240 is reported as an example: A solution of ketone
(7.5 mg) in benzene (200 ml) was added to a stirred suspension (20 ml) of fresh cells of Y.
lipolytica 1240 resuspended in 0.1 M phosphate buffer (pH 7.0) at 28°C and saccharose (600
mg). After 48 h ethyl acetate (5 ml) was added. The mixture was centrifuged and decanted to
give pure (S)-(+)-1-chloro-3-(1-naphthyloxy)-2-propanol 1a in 87% yield and 99% e.e.

4660
F. Mart´ınez et al. / Tetrahedron: Asymmetry 11 (2000) 4651–4660
Acknowledgements
This paper has been supported by a grant from the Ministerio de Educacio´n y Ciencia de
Espan˜a BIO97-0514.
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