A chemoenzymatic scalable route to optically active (R)-1-(pyridin-3-yl)-2- aminoethanol, valuable moiety of β3-adrenergic receptor agonists

Article abstract of DOI:10.1016/j.bmc.2005.09.046

Enantiomerically pure (R)-2-chloro-1-(pyridin-3-yl)ethanol has been prepared by kinetic resolution performed in the presence of Candida antarctica SP435-L lipase immobilized on a macroporous polyacrylate resin (Novozym 435). It was converted into (R)-1-(pyridin-3-yl)-2-aminoethanol, left-hand side of β₃-adrenergic receptor ligands.

Full text of DOI:10.1016/j.bmc.2005.09.046

Bioorganic & Medicinal Chemistry 14 (2006) 1207–1214
A chemoenzymatic scalable route to optically active
(R)-1-(pyridin-3-yl)-2-aminoethanol, valuable moiety
of b -adrenergic receptor agonists
3
Maria Grazia Perrone, Ernesto Santandrea, Erika Giorgio, Laura Bleve,
*
Antonio Scilimati and Paolo Tortorella
Dipartimento Farmaco-Chimico, Universit a` degli Studi di Bari, Via E.Orabona 4, 70125 Bari, Italy
Received 26 July 2005; revised 13 September 2005; accepted 20 September 2005
Available online 10 October 2005
Abstract—Enantiomerically pure (R)-2-chloro-1-(pyridin-3-yl)ethanol has been prepared by kinetic resolution performed in the
presence of Candida antarctica SP435-L lipase immobilized on a macroporous polyacrylate resin (Novozym 435 ). It was converted
ꢀ
into (R)-1-(pyridin-3-yl)-2-aminoethanol, left-hand side of b
2005 Elsevier Ltd. All rights reserved.
3
-adrenergic receptor ligands.
ꢁ
1
. Introduction
the numerous reaction types (hydrolysis, esterification,
transesterification, amidation, etc.) mediated by these
enzymes, along with their high stability in aqueous solu-
tions and in organic solvents, has made hydrolases
important catalysts for the preparation of enantiomeri-
cally pure building blocks, as valuable precursors of fine
b -Adrenergic receptor agonists are potential drugs for
the treatment of obesity. These agents activating such
specific receptors, located on the surface of adipocytes,
stimulate lipolysis and increase in metabolic rate. A
number of different structural classes of b -adrenoceptor
agonists have been developed. Among these, pyridyl-
ethanolamines (A, Fig. 1) were found to be potent and
3
1
2
2
8–35
chemicals and pharmaceuticals.
3
1
Enantioselectivity of hydrolase-induced reactions, so far
reported, for a direct preparation of optically active
b-aminoalcohols depends dramatically on the nature
of the substrate, and the enantiomeric excess of the
reaction products is often not very high. An exception
3
–5
selective human b -adrenergic receptor agonists.
3
For instance, L-770,644 was selected for pre-clinical
6
evaluation due to its favourable oral bioavailability.
The activity of such compounds is strictly dependent on
,8
is the kinetic resolution of propranolol, a b -block-
er, for which suitable reaction conditions (hydrolysis
in phosphate buffer/DMF/Tween 80 medium in the pres-
1
7
36,37
the absolute configuration at C which must be (R).
1
Pure enantiomers of b-adrenenoceptor ligands have
9
–17
been prepared by ÔchemicalÕ asymmetric synthesis,
ence of porcine pancreas esterase) and computer-aided
38,39
1
8–20
crystallization of diastereoisomeric derivatives,
pre-
and enzymatic pro-
cedures that in the last 20 years have become more and
substrate design (B, Fig. 2), not widely usable,
identified [in the series of aryloxy propanolamines the
were
2
1–25
parative chiral chromatography
higher activity resides in the (S)-enantiomer].
2
6–28
more attractive.
In this field, hydrolases are widely
used as chiral catalysts for asymmetric acyl transfer
reactions between a number of acyl-acceptors and
Hence, enzyme-catalysed kinetic resolution towards the
preparation of b-aminoalcohols is usually performed on
their precursors or derivatives, such as b-chlorohyd-
2
9
-
donors. The broad range of accepted substrates,
4
0–44
43–45
46
rins,
b-bromohydrins,
b-azidohydrins, oxaz-
48–54
4
7
olidines and oxazolidinones.
Keywords: Pyridylethanolamines; Kinetic resolution; Candida antarc-
tica SP435-L lipase; b -Adrenergic receptor agonist.
Corresponding author. Tel.: +39 080 544 2762; fax: +39 080 544
231; e-mail: ascilimati@farmchim.uniba.it
Herein, we report lipase-mediated kinetic resolution of
rac-2-chloro-1-(pyridin-3-yl)ethanol [rac-3], to afford
(R)-3, precursor of (R)-1-(pyridin-3-yl)-2-aminoethanol
3
*
2
0
968-0896/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.046

1208
M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
OH
OH
H
N
NHR
O
O
S
N
N
N
H
O
N
N N
N
A
L-770,644
Figure 1.
strategy to obtain the highly enantiomerically enriched
R)-3 was chosen.
O
(
O
NH
O
N
O
OH
O
Reduction of 2 in the presence of yeasts with reductive
properties such as BakerÕs yeast and Kluyveromyces
marxianus CBS 6556 was performed as a first attempt
to prepare (R)-3. As expected, BakerÕs yeast induced
O
propranolol
B
6
6,67
Figure 2.
the formation of (S)-3 (44% yield and ee = 59%).
It is known that K. marxianus KCTC 7155 mediated
the conversion of prostereogenic ketones into the corre-
(
nists.
5), valuable moiety of b -adrenergic receptor ago-
68–70
3
sponding (R)-alcohols.
the reducing capabilities of K. marxianus CBS
Since we have investigated
6
,55–59
6
4,65
6
556,
we used this yeast in the bioreduction of 2.
Unfortunately, this reaction afforded again (S)-3 (11%
2
. Results and discussion
yield and ee = 44%) (Scheme 1).
3-Chloroacetylpyridine hydrochloride (2), prepared in
good yield (75%) by reacting commercially available 3-
acetylpyridine (1) with N-chlorosuccinimide in acetic
(
9
S)-3 was previously obtained in quantitative yield and
9.8% ee by using enzymes from other yeasts or recom-
70,71
binant Escherichia coli HB101 (pNTSSIG).
6
0
acid saturated with gaseous hydrochloric acid, was
quantitatively reduced to the corresponding rac-2-
chloro-1-(pyridin-3-yl)ethanol [rac-3] with NaBH4
As a further attempt to prepare (R)-2-chloro-1-(pyridin-
3-yl)ethanol [(R)-3] in higher yield and with a methodol-
ogy suitable for a scale-up, rac-3 was incubated in
hexane, in the presence of different lipases and vinyl
acetate as acyl donor (Scheme 2). Lipases isolated from
the following sources were screened: Penicillium cyclopi-
um (G), Rhizopus delemar (Rd), Humicola sp. (CE-10),
6
1
(
Scheme 1).
(
R)-3 was prepared, as a reference sample for HPLC
analysis and absolute configuration assignment, in
3% enantiomeric excess and 4% yield in 4 days of
reaction time, by treating
with (ꢀ)-B-chloro-
diisopinocampheylborane [(ꢀ)-DIP-Cl] (Scheme 1),
widely used reagent in organic synthesis for asymmetric
9
2
5
5
a
OH
OH
OAc
6
2
reduction of prostereogenic ketones.
Cl
lipase
Cl
Cl
+
vinyl acetate,
hexane
Since our research group is interested in the develop-
ment of biocatalytic procedures and biotransformations
to prepare enantiomerically pure active ingredients
N
N
N
rac - 3
(R) - 3
(S) - 4
3
1,63–65
for pharmaceuticals,
a chemoenzymatic synthetic
Scheme 2. Lipase-mediated resolution of rac-3.
OH
Cl
a
b
c
NaBH
4
room temp.
N
rac - 3
yield= 99%
O
O
OH
Cl
Cl
NCS/HCl
AcOH
(-)-DIP-Cl
-25˚C
.
HCl
N
N
N
yield= 4%
ee = 93%
1
2
(R) - 3
OH
Cl
yeast
30-37˚C
yield= 11-44%
ee = 44-59%
N
S) - 3
(
Scheme 1. Preparation of 3-chloroacetylpyridine hydrochloride (2) and its reduction by NaBH
4
, (ꢀ)-DIP-Cl or yeast.

M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
1209
Candida cylindracea (Type VII CCL), Candida rugosa
CRL), Mucor javanicus (MAP), Pseudomonas fluores-
cens (PF), Aspergillus niger (AP), Pseudomonas sp. (K-
0), Pseudomonas sp. (K-10 immobilized on XAD-8),
Candida antarctica (SP435-L), Humicola lanuginosa
R10-Amano), Pseudomonas sp. (AK, immobilized on
XAD-8), Candida cylindracea (AY-Amano 30), Mucor
meihei (M-Amano), Candida cylindracea (OF-360) and
Rhizopus niveus (N conc).
Lipase from Candida antarctica is well recognized for
carrying out highly enantioselective acylations of race-
(
7
3–79
mic secondary alcohols.
Recovery of enzymes from
1
reaction solutions and often separation of the enzymes
from substrates and products are generally difficult.
These problems can be successfully tackled by immobi-
lization of the enzyme or by performing the reaction in
organic solvents in which the enzyme results not soluble.
For these reasons, further investigation of the reaction
conditions was carried out for lipase SP435-L from
Candida antarctica (Table 2), a lipase immobilized on
(
Lipases from Aspergillus niger (AP), Penicillium cyclopi-
um (G), Pseudomonas fluorescens (PF), and Candida ant-
arctica (SP435-L) were chosen for the detailed
investigation, which led to the results reported in
Table 1.
ꢀ
a macroporous polyacrylate resin (Novozym 435 ).
An immobilized enzyme is definitely more suitable for
an automated multi-gram scale reaction.
Performing the kinetic resolution using the reaction con-
ditions A and B (Table 2), in which lipase/substrate ratio
and acyl-donor/substrate ratio are, respectively, 1:10
and 8.5:1, rac-3 concentration was determinant for
enantioselectivity: by doubling substrate concentration,
the enantioselectivity factor E goes from 62 (A) to
>100 (B). Notwithstanding the high enantioselectivity
factor value observed, in both conditions the unreacted
recovered substrate showed only a modest enantiomeric
enrichment (ee = 24% and 16%, respectively), of course
due to the low extent of conversion (C up to 20%).
Reaction times did not exceed 2 days for all the selected
lipases, except for that from Aspergillus niger (4 days)
for which a very low enantioselectivity (E = 4), the low-
est enantiomeric excesses for both unreacted substrate
(
8%) and product (30%), and a 21% extent of conversion
were observed. C = 22% was obtained in the presence of
lipase from Penicillium cyclopium which mediated the
transesterification reaction with a similar enantioselec-
tivity factor value (E = 5), and 17% ee for the unreacted
substrate and 60% ee for the ester 4. Enantioselectivity
was greatly improved when lipase from Pseudomonas
fluorescens (E = 56) and lipase from Candida antarctica
SP435-L (E = 47) were employed: enantiomeric excess
ranged between 64% and 70% for (R)-3 and it was
Since the enzyme reaction with acetaldehyde formed in
the transesterification reaction could be responsible for
8
0
activity and enantioselectivity lowering, we reduced
the vinyl acetate/substrate ratio from 8.5:1 to 5:1
(C, Table 2). Enantiomeric excess of the unreacted
93% for (S)-4, while conversion was about twofold
higher.
a
Table 1. Summary of the results of the lipase-mediated kinetic resolution of rac-3
b
Ee-3 (%)
c
Ee-4 (%)
d
C (%)
e
E
Lipase source
Reaction time (days)
(
Absolute configuration)
Aspergillus niger (AP)
4
2
2
2
8 (R)
17 (R)
70 (R)
64 (R)
30 (S)
60 (S)
93 (S)
93 (S)
21
22
43
41
4
5
Penicillum cyclopium (G)
Pseudomonas fluorescens (PF)
Candida antarctica (SP435-L)
56
47
a
b
c
All the reactions were carried out as described in Section 4: screening conditions.
Enantiomeric excesses of the remaining unreacted substrate 3 were determined by HPLC.
Enantiomeric excesses of the product 4 were determined by HPLC.
d
Conversion was determined according to the following equation: C = ee
s
/(ee
s
+ ee
p
), where ee
s
is the enantiomeric excess of remaining unreacted
72
p
alcohol 3 and ee is the enantiomeric excess of the produced ester 4.
e
72
E, enantioselectivity factor value calculated according to the following equation: E = ln[(1 ꢀ C)(1 ꢀ ee
s
)]/ln[(1 ꢀ C)(1 + ee
s
)].
a
Table 2. Scale-up of kinetic resolution of rac-3 mediated by lipase from Candida antarctica (SP435-L)
b
c
d
Reaction conditions Lipase/rac-3 (w/w) Vinyl acetate/rac-3 (mol ratio) rac-3 Concn. (g/100 mL) Ee-3 (%) Ee-4 (%) C (%)
e
E
A
B
C
D
E
F
1:10
1:10
1:10
1:1
8.5:1
8.5:1
5:1
1
2
2
2
2
2
24
16
96
95
95
97
97
87
20
14
12
37
49
53
62
>100
48
13
57
8.5:1
8.5:1
8.5:1
>100
>100
>100
1.5:1
1.5:1
93
>98
a
b
c
All the reactions here described were incubated at 37 ꢂC for 6 days, with the exception of reaction F that took 7 days.
Enantiomeric excesses of the unreacted substrate 3 were determined by HPLC.
Enantiomeric excesses of the product 4 were determined by HPLC.
d
Conversion determined according to the following equation: C = ee
s
/(ee
s
+ ee ), where ee
p
s
is the enantiomeric excess of remaining unreacted alcohol
72
3
p
and ee is the enantiomeric excess of the produced ester 4.
e
72
E, enantioselectivity factor calculated according to the following equation: E = ln[(1 ꢀ C)(1 ꢀ ee
s
)]/ln [(1 ꢀ C)(1 + ee
s
)].

1210
M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
OH
OH
din-3-yl)ethanol [(R)-5]. The lipase from Candida
antarctica (SP435-L) is one of the most often used
Cl
NH2
1
) 30% NH
4
OH / MeOH, reflux
8
4
.2HCl
enzymes in preparative organic chemistry since it rep-
resents a valid alternative to the use of Ôclassical chemi-
calsÕ to promote enantioselective reactions. The use of
immobilized enzymes has also some advantages because
they can be recovered by simply filtering the reaction
medium and reused several times. Further studies are
currently in progress to optimize the reaction condi-
tions, mainly reaction time, enzyme–substrate ratio
and temperature.
N
2) 37% HCl / EtOH
N
(
R)-3
(R)-5
Scheme 3. Preparation of (R)-1-(pyridin-3-yl)-2-aminoethanol [(R)-5].
substrate was still low (13%, C = 12%) and the reaction
had the lowest enantioselectivity factor value (E = 48).
To increase the extent of conversion, a higher enzyme/
substrate ratio was used (D, Table 2) and ee = 57% for
the alcohol after 6 reaction days (E > 100). This result
prompted us to still increase the amount of the enzyme
The obtained product (R)-5 constitutes the left-hand
side of the class of pyridylethanolamines endowed with
b3-adrenoceptor agonist activity. Such compounds are
potential thermogenetic agents that might be used for
the treatment of obesity, which still represents an open
field of pharmaceutical research.
(
enzyme/substrate = 1.5:1, E, Table 2). In these condi-
tions, after the same reaction time, an almost enantio-
pure alcohol was obtained with the desired absolute
configuration (ee = 93%). The enantioselectivity factor
was again very high (E > 100). Hence, this procedure al-
lowed the preparation of (R)-2-chloro-1-(pyridin-3-
yl)ethanol [(R)-3] with the same enantiomeric purity
4. Experimental
4.1. General methods
(
(
ee = 93%) as in the reduction of 2 with (ꢀ)-DIP-Cl
Scheme 1), but with a yield 10-fold higher (40% yield),
even by costless and environmentally friendly
procedure.
Melting points were taken on an electrothermal appara-
tus and are uncorrected. H NMR spectra were recorded
1
in CDCl on a VARIAN Mercury 300 MHz or BRU-
3
Enantiomerically pure (R)-3 (ee > 98%) was obtained
just by increasing to seven days the reaction time (row
F in Table 2).
KER AM 500 MHz spectrometer and chemical shifts
are reported in parts per million (d). Absolute values
of the coupling constant (J) are reported. IR spectra
were recorded on a Perkin-Elmer 681 spectrometer.
Reaction progress was monitored by TLC and GC anal-
ysis. Thin-layer chromatography (TLC) was performed
on silica gel sheets with a fluorescent indicator (Stato-
crom SIF, 60 F254 MERK). GC analyses were per-
formed by using a HP-5MS column (5% phenyl
methyl siloxane; 30 m · 0.321 mm · 0.25 lm) on an
Agilent 6850 SERIES GC SYSTEM. GC–MS analyses
were performed on a HEWLETT PACKARD 6890–
5793MSD, and microanalyses on a Elemental Analyzer
1106-Carlo Erba instrument. ESI-MS analyses were per-
formed on an Agilent 1100 LC/MSD trap system VL.
Optical rotations were measured on a Perkin-Elmer
model 341 polarimeter. The enantiomeric excesses and
absolute configurations of the reaction products were
determined by the HPLC analysis performed on a Per-
kin-Elmer 200 series with a UV–vis detector 785A on
a commercially available Chiralcel OD (Daicel) in
isocratic conditions employing n-hexane/2-propa-
nol = 95:5, flow rate 1 mL/min, k = 254 nm. The extent
of conversion was determined according to the following
equation: C = ee /(ee + ee ), where ee is the enantio-
It has been recently reported that polymeric matrix can
8
1
affect the enantioselectivity of the reaction. Its swelling
properties might, in fact, influence substrate and prod-
uct diffusion ability. For this reason, enantiomeric
excess of compounds 3 and 4 obtained from the suspen-
sion and those obtained by washing with methanol the
immobilized lipase recovered by filtration were deter-
mined. Ee of 3 and 4 recovered from both the polymeric
s
matrix and the reaction medium were, respectively, iden-
tical. Thus, it seems that in our conditions the presence
of the polymer in the reaction suspension has no impact,
at least apparently, on the enantioselectivity showed by
Candida antarctica lipase in catalyzing the kinetic reso-
lution of racemic 2-chloro-1-(pyridin-3-yl)ethanol (3),
probably due to a rapid dynamic equilibrium that oc-
curs between the polymeric matrix and the reaction
8
2
medium.
(R)-2-Chloro-1-(pyridin-3-yl)ethanol [(R)-3] was, then,
converted into the desired (R)-1-(pyridin-3-yl)-2-amino-
8
3
ethanol [(R)-5], a useful intermediate for the prepara-
tion of b -adrenergic receptor agonist (Scheme 3).
s
s
p
s
3
meric excess of the remaining unreacted alcohol 3 and
ee is the enantiomeric excess of the produced ester 4
p
determined by HPLC. The ee and the extent of conver-
s
3. Conclusions
sion (C) were used to calculate the enantioselectivity
factor values (E).
Kinetic resolution of rac-2-chloro-1-(pyridin-3-yl)etha-
nol [rac-3] was achieved by a transesterification reaction
performed in the presence of a lipase from Candida ant-
arctica (SP435-L) immobilized on a macroporous poly-
acrylate resin (Novozym 435 ). (R)-3, obtained with
ee > 98%, was then converted into (R)-2-amino-1-(pyri-
4.2. Materials
Lipases used were supplied from either Amano Enzyme
Co. Meito Sangyo, or Sigma Chemical Co. In particular,
lipase from Candida antarctica (SP435-L) was a gift of
ꢀ

M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
1211
Novozymes (Denmark). The bakerÕs yeast used to re-
duce 2 was supplied by Lievitalia. Kluyveromyces marxi-
anus CBS 6556 was cultivated under aerobic conditions
in a medium containing 0.3% yeast extract, 0.3% malt
extract, 0.5% peptone and 1% glucose. Agar-agar (2%)
was added to the same medium for cell preservation
on agar slants.
NMR (75 MHz, CD OD, d): 148.04, 147.24, 137.98,
3
135.07, 123.71, 71.04, 48.80. GC–MS (70 eV) (m/z) (rel
3
7
+
35
+
int.) 159 [M( Cl) , 2], 157 [M( Cl) , 5], 108 (100), 80
(28), 78 (10), 53 (11), 51 (9).
a
Chromatographic data:
0
Compound
tR
k
a
0
(
(
ꢀ)-(R)-3
+)-(S)-3
31.72
35.41
9.47 ðk Þ
1.13
1
All other chemicals and solvents were of the highest
quality grade commercially available and purchased
from Aldrich Chemical Co. or Sigma Chemical Co.
0
10.69 ðk Þ
Stationary phase: Chiralcel OD (Daicel). Mobile phase: n-hexane/iso-
2
a
R
propanol = 95:5; flow rate, 1 mL/min; UV-k, 254 nm; t , retention
0
0
time (min); k and k , capacity factors of (ꢀ)-(R)-3 and (+)-(S)-3,
4
.3. Synthesis of 3-chloroacetylpyridine hydrochloride
6
1
2
0
0
respectively; a, separation factor. Capacity factors (k ) were calculated
by using as tR the retention time of the mobile phase prepared
(
2)
ꢁ
8
replacing i-PrOH with i-PrOH-d revealed by a refractive index
Gaseous HCl (28 g, 0.77 mol) was slowly introduced
into glacial acetic acid (51 mL) kept at 0 ꢂC. The temper-
ature was raised to 20 ꢂC and 3-acetylpyridine (4.5 mL,
detector.
4
.5. (R)-3 prepared with (ꢀ)-B-chloro-
0
.04 mol) was added dropwise. N-chlorosuccinimide
55
diisopinocampheylborane [(ꢀ)-DIP-Cl]
20
(
6.85 g, 0.05 mol) was added to the solution. The mix-
ture was stirred overnight at room temperature, then
cooled to 15 ꢂC and filtered. The solid was washed with
acetic acid and with ethyl acetate, and dried in high vac-
uum affording a white powder in 76% yield. Mp 200–
Oil (4% yield). ½aꢂ = ꢀ34.47 (c 0.55, CH OH).
D
3
Ee = 93%.
4.6. Bioreduction procedures of 3-chloroacetylpyridine
hydrochloride (2)
2
2
1
1
6
1
01 ꢂC (dec). FT-IR (KBr): 3116, 3084, 3055, 3017,
975, 2930, 2869, 2860-2200, 2109, 1988, 1873, 1717,
628, 1607, 1548, 1465, 1377, 1345, 1258, 1234, 1194,
176, 1107, 1065, 1011, 938, 902, 807, 680, 658,
4.6.1. BakerÕs yeast-mediated reduction of 2. To bakerÕs
ꢀ
1
1
yeast (250 g) dispersed in tap water (180 mL) 3-chloro-
acetylpyridine hydrochloride (2, 200 mg) in absolute
ethanol (5 mL) was added. The suspension was stirred
at 250 rpm and kept at 37 ꢂC. Reaction progress was
monitored by the GC analysis: 0.5 mL samples were
extracted by adding ethyl acetate (1 mL) and then cen-
trifuged at 10,000g for 5 min; the supernatant (organic
phase) was separated, dried over anhyd Na SO and
24 cm . H NMR (300 MHz, DMSO-d , d): 14.50–
6
+
3.20 (br s, 1H, NH : exchanges with D O); 9.33–9.32
2
(
d, J = 1.9 Hz, 1H, aromatic proton); 9.02–9.00 (dd,
J = 5.4 and 1.5 Hz, 1H, aromatic proton); 8.77–8.73
m, 1H, aromatic proton); 8.02–7.98 (dd, 1H, J = 8.1
and 5.4 Hz, aromatic proton); 5.32 (s, 2H, CH Cl).
(
2
1
3
C NMR (75 MHz, DMSO-d , d): 190.26, 148.88,
6
2
4
1
45.32, 142.15, 132.49, 127.00, 48.74. Anal. Calcd for
C H Cl NO: C, 43.78; H, 3.67; N, 7.29. Found: C,
then analysed by GC and/or TLC. The reaction was
stopped after 3 h. NaOH (1 N) was added to the mixture
to alkaline pH. The mixture so obtained was extracted
several times with EtOAc. The extracts were combined
and dried over anhyd Na SO . A yellow oil (44% yield,
7
7
2
4
3.75; H, 3.70; N, 7.29.
4
[
.4. Synthesis of rac-2-chloro-1-(pyridin-3-yl)ethanol
6
rac-3]
1
2
4
ee = 59%) was obtained after removal of the solvent
under reduced pressure.
To an ice-bath cooled solution of 3-chloroacetylpyridine
hydrochloride (2) (3 g, 15.7 mmol) in water (10 mL) and
methanol (20 mL) was slowly added NaBH (775 mg,
4
4.6.2. Kluyveromyces marxianus CBS 6556-mediated
reduction of 2. Cells preserved on agar slants at 4 ꢂC
were used to inoculate 250-mL flasks containing the cul-
tivation medium (100 mL). The flasks were incubated
aerobically at 30 ꢂC on an orbital shaker and stirred at
250 rpm. 250-mL Flasks containing the cultivation
medium (100 mL) were then inoculated with 5 mL of
the 24 h-old suspension and incubated under the same
conditions for 24 h. 1 L-Flask containing the cultivation
medium (400 mL) was then inoculated with 5 mL of the
latter suspension and incubated for 24 h, and 2 (152 mg)
dissolved in ethanol (1 mL) was added when the culture
had the optical density of 7.2. Reaction progress was
monitored by the GC analysis: 0.5 mL samples were
extracted by adding ethyl acetate (1 mL) and then cen-
trifuged at 10,000g for 5 min. The supernatant (organic
phase) was separated, dried over anhyd Na SO and
2
0
0 mmol). The resulting reaction mixture was stirred at
ꢂC for 30 min, warmed at room temperature and stir-
red for further 2 h. Solvent was removed under reduced
pressure. The residue was dissolved in Na CO and
2
3
extracted with ethyl acetate. The organic layer was dried
over anhyd Na SO and the solvent was evaporated un-
2
4
der reduced pressure to afford a viscous yellow oil in
quantitative yield. FT-IR (neat): 3600–3000, 2956,
2
1
7
8
1
924, 2853, 1596, 1581, 1479, 1426, 1341, 1319, 1257,
217, 1192, 1168, 1081, 1046, 1029, 975, 956, 874, 811,
ꢀ
1
1
45, 710, 644 cm . H NMR (500 MHz, CDCl , d):
3
.56 (s, 1H, aromatic proton); 8.51–8.50 (d, J = 4.7 Hz,
H, aromatic proton); 7.78–7.77 (d, J = 7.9 Hz, 1H, aro-
matic proton); 7.33–7.30 (dd, J = 4.7 and 7.9 Hz, 1H,
aromatic proton); 4.96–4.94 (dd, J = 8.1 and 4.2 Hz,
1
D O); 3.76–3.72 (dd, J = 4.2 and 11.2 Hz, 1H, CH Cl);
H, CHOH); 4.00–3.50 (s, 1H, OH: exchanges with
2
4
then analysed by GC and TLC. The reaction was
stopped after 113 h (ꢃ5 days). The content of the flask
2
2
1
3
3
.69–3.65 (dd, J = 8.1 and 11.2 Hz, 1H, CH Cl).
2
C

1212
M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
3
(70 eV) m/z (rel. int.) 201 [M( Cl) , 0.2], 199
[M( Cl) , 0.4], 163 (21), 150 (6), 140 (6), 121 (73), 108
(100), 104 (30), 78 (16), 51 (15), 43 (36).
7
+
was centrifuged. 1 N NaOH was added to the mixture to
alkaline pH. The reaction mixture was extracted several
times with EtOAc. The extracts were combined and
dried over anhyd Na SO . A yellow oil (11% yield,
3
5
+
2
4
ee = 44%) was obtained after removal of the solvent un-
der reduced pressure.
4.7.1.2. (R)-2-Chloro-1-(pyridin-3-yl)ethanol [(R)-3].
20
Oil (40% yield, ee > 98%). ½aꢂ ꢀ40.4 (c 0.90, CH OH).
D
3
4
dihydrochloride [(R)-5]
.8. Preparation of (R)-1-(pyridin-3-yl)-2-aminoethanol
6
4.7. Lipase-catalyzed acylation of rac-2-chloro-1-(pyri-
din-3-yl)ethanol 3: screening conditions
0
NH OH (9.5 mL) was added to a solution of (R)-3
4
Crude lipase (20 mg) and vinyl acetate (0.1 mL) were
added to a suspension of racemic alcohol (20 mg) in hex-
ane (1 mL). The heterogeneous mixture was incubated
at 37 ꢂC and stirred at 250 rpm. The reaction progress
was monitored by TLC. The enantiomeric excesses of
the product formation 4 and remaining unreacted
substrate 3 were determined analysing directly the
suspension by chiral chromatography.
(426 mg, 2.71 mmol) in methanol (12 mL) kept at
room temperature. The reaction mixture was stirred
for 3 h under reflux and then concentrated under re-
duced pressure. n-Butanol was added to the residue
and removed as azeotropic mixture (n-butanol,
remaining water and excess ammonia) by distillation
under reduced pressure. 37% HCl (0.5 mL) was added
to the solution and it was concentrated under reduced
pressure. The product was recrystallized from absolute
ethanol obtaining white crystals20 in 47% yield.
a
Chromatographic data:
0
Compound
tR
k
a
Mp = 198.3–199.2 ꢂC (ethanol). ½aꢂ ꢀ45.31 (c 0.6,
D
H O). IR (KBr): 3600–3300, 3209, 3158, 3119, 3098,
3044, 3017, 2960, 2932, 2882, 1633, 1609, 1547,
0
2
(
(
(
(
ꢀ)-(R)-4
+)-(S)-4
ꢀ)-(R)-3
+)-(S)-3
25.39
27.39
35.34
40.69
7.46 ðk Þ
1.09
1
0
8.13 ðk Þ
2
1
6
490, 1470, 1440, 1259, 1135, 1044, 997, 808,
0
10.78 ðk Þ
1.16
ꢀ1
1
1
85 cm . H NMR (300 MHz, DMSO-d , d): 14.35–
0
6
12.53 ðk Þ
+
2
13.20 (br s, 1H, NH : exchanges with D O); 8.89–
2
a
Stationary phase: Chiralcel OD (Daicel). Mobile phase: n-hexane/iso-
8.88 (d, J = 1.6 Hz, 1H, aromatic proton); 8.86–8.84
(d, J = 5.6 Hz, 1H, aromatic proton); 8.58–8.56 (d,
J = 8.0 Hz, 1H, aromatic proton); 8.45–8.10 (br s, 3H,
R
propanol = 95:5; flow rate, 1 mL/min; UV-k, 254 nm; t , retention
0
0
time (min); k and k , capacity factors of (ꢀ)-(R)-4, (+)-(S)-4, (ꢀ)-(R)-
1
2
0
3
, (+)-(S)-3, respectively; a, separation factor. Capacity factors (k )
were calculated by using as t the retention time of the mobile phase
þ
CH
and 5.6 Hz, 1H, aromatic proton); 7.20–5.80 (br s, 1H,
2
NH : exchange with D O); 8.06–8.01 (dd, J = 8.0
2
ꢁ
3
R
8
prepared replacing i-PrOH with i-PrOH-d revealed by refractive
OH: exchanges with D O); 5.18–5.14 (dd, J = 7.7 and
2
index detector.
þ
3
3
.8 Hz, 1H, CHOH); 3.23–3.16 (m, 1H, CH
2
NH );
3
þ
3
13
.08–2.99 (m, 1H, CH NH ). C NMR (75 MHz,
2
ꢀ
4
.7.1. Gram-scale Novozym 435 -catalyzed acylation of
rac-2-chloro-1-(pyridin-3-yl)ethanol (3). Lipase from
DMSO-d , d): 143.93, 141.88, 141.81, 140.88, 127.49,
6
+
66.86, 45.27. MS-ESI m/z (%): 139 [M+H] (100%).
ꢀ
Candida antarctica (SP435-L, Novozym 435 ) (2.4 g)
and vinyl acetate (8 mL) were added to a suspension
of racemic alcohol (1.6 g) in hexane (100 mL). The het-
erogeneous mixture was incubated at 37 ꢂC and stirred
at 250 rpm. The reaction was monitored by TLC and
stopped at the time indicated in the Table 1. The mixture
was filtered through a sintered glass funnel to recover
the lipase. The hexane was removed under reduced pres-
sure. Product (S)-4 and remaining unreacted substrate
Acknowledgments
Work carried out under the framework of the National
Project ÔProgettazione, Sintesi e Valutazione Biologica
di Nuovi Farmaci CardiovascolariÕ supported by the
Ministero dellÕIstruzione, dellÕUniversit a` e della Ricerca
(MIUR, Rome). Thanks are also due to the University
of Bari, CNR-Istituto di Chimica dei Composti Organo-
Metallici-ICCOM/Sezione di Bari (Italy) and to Ms. E.
Perrone who performed the preliminary experiments.
(R)-3 were separated by chromatography (silica gel,
eluent: petroleum ether/ethyl acetate = 8:2).
4
.7.1.1. (S)-3-[(1-Acetoxy-2-chloro)ethyl]pyridine [(S)-
]. Oil (41% yield, ee = 97%). [a]D +63.28 (c 1, CHCl3).
IR (neat): 3037, 2961, 1747, 1594, 1579, 1481, 1429,
4
References and notes
1
7
373, 1325, 1231, 1045, 1024, 986, 941, 812, 748,
ꢀ
1
1
12 cm . H NMR (300 MHz, CDCl , d): 8.60–8.59
3
1. Strosberg, A. D. The b -Adrenoreceptor; Taylor & Francis:
3
New York, 2000.
. Weber, A. E.; Mathvink, R. J.; Perkins, L.; Hutchins, J.
(
d, J = 2.2 Hz, 1H, aromatic proton); 8.57–8.55 (dd,
J = 4.9 and 1.8 Hz, 1H, aromatic proton); 7.69–7.65
dt, J = 8.0 and 1.8 Hz, 1H, aromatic proton); 7.31–
2
E.; Candelore, M. R.; Tota, L.; Strader, C. D.; Wyvratt,
M. J.; Fisher, M. H. Bioorg. Med. Chem. Lett. 1998, 8,
(
7
5
.26 (dd, J = 8.0 and 4.9 Hz, 1H, aromatic proton);
.97–5.92 (dd, J = 7.0 and 5.2 Hz, 1H, CH); 3.82–3.750
1
101.
3
. Choi, J. G.; Chun, H. G.; Ha, J. D.; Han, S. S.; Kang, S.
G.; Kim, S. S.; Yang, S. D.; Yum, E. G. Patent
KR2002020288, 2002; CAN 142:113899.
(dd, J = 11.6 and 7.0 Hz, 1H, CH ); 3.74 –3.69 (dd,
2 7
J = 11.6 and 5.2 Hz, 1H, CH ); 2.11 (s, 3H, CH ). C
1
3
2
3
NMR (75 MHz, CDCl , d): 169.94, 150.32, 148.54,
4. Hattori, K.; Tomishima, Y.; Nakajima, Y.; Imanishi, M.
Patent WO2003076397, 2003; CAN 139:261169.
3
1
34.78, 133.06, 123.74, 73.06, 46.18, 21.10. GC–MS

M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
1213
5
. Taniguchi, K.; Kayakiri, H.; Fujii, N.; Hamashima, H.;
Sakurai, M.; Washizuka, K.; Tomishima, Y.; Hamada,
K.; Yamamoto, N.; Ishikawa, H.; Tanimura, N. Patent
WO2001036375, 2001; CAN 135:5445.
. Shih, T. L.; Candelore, M. R.; Cascieri, M. A.; Chiu, S. L.;
Colwell, L. F.; Deng, L.; Feeney, W. P.; Forrest, M. J.;
Hom, G. J.; MacIntyre, D. E.; Miller, R. R.; Stearns, A.;
Strader, C. D.; Tota, L.; Wyvratt, M. J.; Fisher, M. H.;
Weber, A. E. Bioorg. Med. Chem. Lett. 1999, 9, 1251.
. Oriowo, M. A.; Chapman, H.; Kirkham, D. M.; Sennitt,
M. V.; Ruffolo, R. R.; Cawthorne, M. A. J. Pharmacol.
Exp. Ther. 1996, 277, 22.
34. Virsu, P.; Liljeblad, A.; Kanerva, A.; Kanerva, L. T.
Tetrahedron: Asymmetry 2001, 12, 2447.
35. Xu, D.; Li, Z.; Ma, S. Tetrahedron Lett. 2003, 44, 6343.
36. Chen, C. S.; Gou, D.-M.; Shieh, W.-R.; Liu, Y.-C.
Tetrahedron 1993, 49, 3281.
37. Ball, J. B.; Nero, T. L.; Iacovidis, D.; Tung, L.; Jackaman,
G.; Louis, W. J. J. Med. Chem. 1992, 35, 4676.
38. Di Bono, G.; Scilimati, A. Synthesis 1995, 6, 699.
39. Kamal, A.; Rao, M. V. Tetrahedron: Asymmetry 1991, 2,
751.
40. Bevinakatti, H. S.; Banerji, A. A. J. Org. Chem. 1991, 56,
5372.
41. Kita, Y.; Takebe, Y.; Murata, K.; Naka, T.; Akai, S.
J. Org. Chem. 2000, 65, 83.
42. Kim, K.-W.; Song, B.; Choi, M.-Y.; Kim, M. J. Org. Lett.
2001, 3, 1507.
6
7
8
9
. Tanaka, N.; Tamai, T.; Makaiyama, H.; Hirabayashi, A.;
Muranaka, H.; Hishikawa, T.; Kobayashi, Y.; Akahane,
S.; Akahane, M. J. Med. Chem. 2003, 46, 105.
. Rozzell, J. D., Jr. Patent US6207862, 2001; CAN
1
34:252087.
0. Rozzell, J. D., Jr. Patent US5834261, 1998; CAN
30:3114.
1. Yanagi, T.; Kikuchi, T. Patent JP2004018377, 2004; CAN
40:111272.
43. P a` mis, O.; B a¨ ckvall, J.-E. J. Org. Chem. 2002, 67, 9006.
44. Mart `ı n-Matute, B.; Edin, M.; Bog a` r, K.; Kaynak, F. B.;
B a¨ ckvall, J.-E. J. Am. Chem. Soc. 2005, 127, 8817.
45. Ader, U.; Schneider, M. P. Tetrahedron: Asymmetry 1992,
3, 521.
1
1
1
1
1
2. Raghavan, S.; Rajender, A.; Joseph, S. C.; Rasheed, M.
A.; Ravi Kumar, K. Tetrahedron: Asymmetry 2004, 15,
46. Ader, U.; Schneider, M. P. Tetrahedron: Asymmetry 1992,
3, 205.
3
65.
47. Binet, J.; Manoury, P. Synthelabo Patent DE 3,341,983,
1984; Baldwin, J. J.; Claremon, D. A. Merck and Co. Inc.
(USA) Patent GB 2,209,749, 1989.
1
3. Mizusuna, A.; Yoshihara, H.; Miyata, R. Patent
JP2003327564, 2003. CAN 139:381243.
1
4. Bartoli, G.; Cupone, G.; Dalpozzo, R.; De Nino, A.;
Maiuolo, L.; Procopio, A.; Tagarelli, A. Tetrahedron Lett.
48. Hamaguchi, S.; Hasegawa, J.; Kawaharada, H.; Watan-
abe, K. Agric. Biol. Chem. 1984, 48, 2055.
2
002, 43, 7441.
49. Hamaguchi, S.; Asada, M.; Hasegawa, J.; Watanabe, K.
Agric. Biol. Chem. 1984, 48, 2331.
50. Kan, K.; Miyama, A.; Hamaguchi, S.; Ohashi, T.;
Watanabe, K. Agric. Biol. Chem. 1985, 49, 207.
51. Hamaguchi, S.; Yamamura, H.; Hasegawa, J.; Watanabe,
K. Agric. Biol. Chem. 1984, 48, 1509.
1
5. Hitchcock, S. R.; Squire, M. D.; Ferrence, G. M.; Burwell,
A. S. Abstract of Papers, 224th ACS National Meeting,
Boston, MA, USA, August 18–22, 2002.
6. Schilling, G. GIT Labor-Fachzeitschrift 2002, 46, 905.
7. Hamada, T.; Torii, T.; Izawa, K.; Ikariya, T. Tetrahedron
1
1
2004, 60, 7422.
8. Zhang, G.; Liao, Y.; Wang, Z.; Nohaira, H.; Hirose, T.
Tetrahedron: Asymmetry 2003, 14, 3297.
9. Ozaki, E.; Endou, T.; Yamaguchi, Y.; Hamanaka, M.
Patent WO2001017944, 2001; CAN 134:193127.
0. Desantis, N.; Fedeli, F. Patent WO9824755, 1998; CAN
52. Hamaguchi, S.; Asada, M.; Hasegawa, J.; Watanabe, K.
Agric. Biol. Chem. 1985, 49, 1661.
53. Hamaguchi, S.; Ohashi, T.; Watanabe, K. Agric. Biol.
Chem. 1986, 50, 375.
1
1
2
2
2
2
2
2
2
54. Kan, K.; Miyama, A.; Hamaguchi, S.; Ohashi, T.;
Watanabe, K. Agric. Biol. Chem. 1985, 49, 1669.
55. Naylor, M. E.; Colandrea, V. J.; Candelore, M. R.;
Cascieri, M. A.; Colwell, L. F., Jr.; Deng, L.; Feeney, W.
P.; Forrest, M. J.; Hom, G. J.; MacIntyre, D. E.; Strader,
C. D.; Tota, L.; Wang, P.-R.; Wyvratt, M. J.; Fisher, M.
H.; Weber, A. E. Bioorg. Med. Chem. Lett. 1998, 8, 3087,
and references cited therein.
56. Naylor, E. M.; Parmee, E. R.; Colandrea, V. J.; Perkins,
L.; Brockunier, L.; Candelore, M. R.; Cascieri, M. A.;
Colwell, L. F., Jr.; Deng, L.; Feeney, W. P.; Forrest, M. J.;
Hom, G. J.; MacIntyre, D. E.; Strader, C. D.; Tota, L.;
Wang, P.-R.; Wyvratt, M. J.; Fisher, M. H.; Weber, A. E.
Bioorg. Med. Chem. Lett. 1999, 9, 755.
1
29:54111.
1. Yamazaki, S.; Tanimura, T.; Uchikura, K. Chromatogra-
phy 1998, 19, 201.
2. Tivert, A. M.; Backman, A. J. Planar Chromatogr.-
Modern TLC 1989, 2, 472.
3. Delee, E.; Le Garrec, L.; Jullien, L.; Beranger, S.; Pascal,
J. C.; Pinhass, H. Chromatographia 1987, 24, 357.
4. Connell, R. D.; Lease, T. G.; Baryza, J. Patent
US6469031, 2002; CAN 137:310812.
5. Connell, R. D.; Lease, T. G.; Baryza, J. Patent
WO9932476, 1999. CAN 131:58750.
6. Clariana, J.; Garcia-Granda, S.; Gotor, V.; Gutierrez-
Fernandez, A.; Luna, A.; Moreno-Manas, M.; Vallribera,
A. Tetrahedron: Asymmetry 2000, 11, 4549.
7. Patel, R. N. Stereoselective Biocatalysis; Dekker: New
York, 2000.
57. Mathvink, R. J.; Barritta, A. M.; Candelore, M. R.;
Cascieri, M. A.; Deng, L.; Tota, L.; Strader, C. D.;
Wyvratt, M. J.; Fisher, M. H.; Weber, A. E. Bioorg. Med.
Chem. Lett. 1999, 9, 1869.
2
2
2
8. Gotor, V. Bioorg. Med. Chem. 1999, 7, 2189.
9. Asano, Y.; Kaga, H. J. Mol. Cat. B: Enzymatic, 2004, 30,
58. Feng, D. D.; Biftu, T.; Candelore, M. R.; Cascieri M. A.;
Colwell, L. F., Jr.; Deng, L.; Feeney, W. P.; Forrest, M.
J.; Hom, G. J.; MacIntyre, D. E.; Miller, R. R.; Stearns,
R. A.; Strader, C. D.; Tota, L.; Wyvratt, M. J.; Fisher,
M. H.; Weber, A. E. Bioorg. Med. Chem. Lett. 2000, 10,
1427.
59. Mathvink, R. J.; Tolman, J. S.; Chitty, D.; Celore, M. R.;
Cascieri, M. A.; Colwell, L. F.; Deng, L.; Feeney, W. P.;
Forrest, M. J.; Hom, G. J.; MacIntyre, D. E.; Miller, R.
R.; Stearns, R. A.; Tota, L.; Wyvratt, M. J.; Fisher, M. H.;
Weber, A. E. J. Med. Chem. 2000, 43, 3832.
241: Selected Abstracts from the 7th Japanese Symposium
on Chemistry of Biocatalysis.
3
3
0. Roberts, S. M. Biocatalysis for Fine Chemicals Synthesis;
Wiley: New York, 1999.
1. Ferrorelli, S.; Franchini, C.; Loiodice, F.; Perrone, M. G.;
Scilimati, A.; Sinicropi, M. S.; Tortorella, P. Tetrahedron:
Asymmetry 2001, 12, 853.
3
2. Paizs, C.; Tosa, M.; Majdik, C.; T a¨ htinen, P.; Dan Irime,
F.; Kanerva, L. T. Tetrahedron: Asymmetry 2003, 14, 619.
3. Gedey, S.; Liljeblad, A.; L a´ z a´ r, L.; F u¨ l o¨ p, F.; Kanerva, L.
T. Tetrahedron: Asymmetry 2001, 12, 105.
3
60. Duquette, J.; Zhang, M.; Zhu, L.; Reeves, R. S. Org.
Process Res. Dev. 2003, 7, 285.

1214
M. G. Perrone et al. / Bioorg. Med. Chem. 14 (2006) 1207–1214
6
1. Perrone, M. G.; Santandrea, E.; DellÕUomo, N.; Gian- 73. Jaeger, K.-E.; Reetz, M. T. Trends Biotechnol. 1998, 16,
nessi, F.; Milazzo, F. M.; Sciarroni, A. F.; Scilimati, A.;
Tortorella, V. Eur. J. Med. Chem. 2005, 40, 143.
2. Procter, G. Sintesi Asimmetrica, EdiSES 2000, Napoli,
396.
74. Orrenius, C.; Haeffner, F.; Rotticci, D.; Ohrner, N.;
Norin, T.; Hult, K. Biocatal. Biotrans. 1998, 16, 1.
75. Frykman, H.; Ohrner, N.; Norin, T.; Hult, K. Tetrahedron
Lett. 1993, 34, 1367.
76. Orrenius, C.; Norin, T.; Hult, K.; Carrea, G. Tetrahedron:
Asymmetry 1995, 6, 3023.
6
6
162–163.
3. Perrone, M. G.; Santandrea, E.; Scilimati, A.; Tortorella,
V.; Capitelli, F.; Bertolasi, V. Tetrahedron: Asymmetry
2004, 15, 3501.
6
4. Perrone, M. G.; Santandrea, E.; Scilimati, A.; Syldatk, C.;
Tortorella, V.; Capitelli, F.; Bertolasi, V. Tetrahedron:
Asymmetry 2004, 15, 3511.
77. Rotticci, D.; Orrenius, C.; Hult, K.; Norin, T. Tetrahe-
dron: Asymmetry 1997, 8, 359.
78. Overmeyer, A.; Schrader-Lippelt, S.; Kasche, V.; Brunner,
G. Biotechnol. Lett. 1999, 21, 65.
79. Rotticci, D.; Haeffner, F.; Orrenius, C.; Norin, T.; Hult,
K. J. Mol. Catal. B: Enzym. 1998, 5, 267.
80. Weber, H. K.; Stecher, H.; Faber, K. Biotechnol. Lett.
1995, 17, 803.
6
5. Perrone, M. G.; Santandrea, E.; Scilimati, A.; Syldatk, C.;
Tortorella, V. Tetrahedron: Asymmetry 2005, 16, 1473.
6. Servi, S. Synthesis 1990, 1.
6
6
7. Faber, K. Biotransformations in Organic Chemistry;
Springer-Verlag: Berlin, 2000.
6
8. Koh, H. Y.; Han, Y.-S.; Cha, J.-H.; Kim, W.-H.; Lee,
J.-S.; Yun, H.-C. Patent US 170844 2003.
81. Heinsman, N. W. J. T.; Schro e¨ n, C. G. P. H.; van der
Padt, A.; Franssen, M. C. R.; Boom, R. M.; vanÕt Riet, K.
Tetrahedron: Asymmetry 2003, 14, 2699.
82. Jacobsen, E. E.; Andresen, L. S.; Anthonsen, T. Tetrahe-
dron: Asymmetry 2005, 16, 847.
6
9. Cha, J.-H.; Pae, A. N.; Choi, K. I.; Cho, Y. S.; Kim,
W.-H.; Han, Y.-S.; Yun, H. C.; Lee, J.; Koh, H. Y.;
Lee, E. Biotechnol. Lett. 2002, 24, 1695.
7
7
0. Rozzell, J. D. ACS Symposium Series 2001, 776, 191.
1. Taoka, N.; Kizaki, N.; Kawano, S.; Horikawa, M.;
Yasohara, Y. Patent WO2003033468 2003; CAN
83. Hu, B.; Ellingboe, J.; Gunawan, I.; Han, S.; Largis, E.;
Li, Z.; Malamas, M.; Mullvey, R.; Oliphant, A.; Sum,
F.-W.; Tillett, J.; Wong, V. Bioorg. Med. Chem. Lett.
2001, 11, 757.
138:337992.
7
2. Chen, C. S.; Wu, S. H.; Girdaukas, G.; Sih, C. J. J. Am.
Chem. Soc. 1987, 109, 2812.
84. Anderson, E. M.; Larsson, K. M.; Kirk, O. Biocatal.
Biotrans. 1998, 16, 181.