Enzymatic resolution of methyl (1RS)-N-tBoc-6-hydroxy-3,4-dihydro-1H-isoquinoline-1-carboxylate by Seaprose S

Article abstract of DOI:10.1016/j.tetasy.2007.09.011

An efficient biocatalytic process has been developed for the resolution of methyl (1RS)-N-tBoc-6-hydroxy-3,4-dihydro-1H-isoquinoline-1-carboxylate rac-1 by means of Seaprose S-mediated enantioselective hydrolysis to afford (1S)-2 and (1R)-1 in 87% and 93% isolated yield, 101% and 96% potency, and ee >99.8% and >99.5%, respectively.

Full text of DOI:10.1016/j.tetasy.2007.09.011

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 18 (2007) 2147–2154
Enzymatic resolution of methyl (1RS)-N-tBoc-6-hydroxy-3,4-dihydro-
1
H-isoquinoline-1-carboxylate by Seaprose S
a,ꢀ
b
a
b
b
Iqbal S. Gill, Ellen Kick, Kate Richlin-Zack, Wu Yang, Yufeng Wang
a,
*
and Ramesh N. Patel
a
Process Research and Development Department, Bristol-Myers Squibb Pharmaceutical Research Institute,
New Brunswick, NJ, United States
Discovery Chemistry Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Hopewell, NJ, United States
b
Received 28 June 2007; accepted 6 September 2007
Abstract—An efficient biocatalytic process has been developed for the resolution of methyl (1RS)-N-tBoc-6-hydroxy-3,4-dihydro-1H-iso-
quinoline-1-carboxylate rac-1 by means of Seaprose S-mediated enantioselective hydrolysis to afford (1S)-2 and (1R)-1 in 87% and 93%
isolated yield, 101% and 96% potency, and ee >99.8% and >99.5%, respectively.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
acylation of alcohols and amines, are among the most uti-
lized biocatalytic methods for the preparation of enantio-
merically pure alcohols, amines, amides, acids, and
Biocatalysis has become a widely used tool in the pharma-
ceutical industry for the synthesis of chiral intermediates
via asymmetric syntheses and kinetic resolutions, mediated
most commonly by hydrolase, lyase, oxidoreductase, and
9
–17
esters.
This derives from the early recognition of lipases
as highly active and robust enzymes with broad substrate
specificities, unique stereo-, chemo- and regio-selectivities,
and the ability to perform a range of synthetically useful
reactions under a variety of conditions, together with their
decades-long industrial application in the pharmaceutical,
1
–8
transaminase enzymes.
Lipase-catalyzed transforma-
tions of carboxylic acids, alcohols, and amines, especially
the enantioselective hydrolysis of esters and amides, and
the stereoselective esterification of carboxylic acids and
1
8–20
fine-chemicals, and food sectors.
O
O
O
N
O
N
O
O
N
Seaprose S
O
HO
O
+
O
O
O
OH
OH
OH
(
RS)-1
(S)-2
(R)-1
Figure 1. Biocatalytic hydrolytic resolution of methyl (1RS)-N-tBoc-6-hydroxy-3,4-dihydro-1H-isoquinoline-1-carboxylate rac-1.
*
Corresponding author. Fax: +1 732 227 3994; e-mail: ramesh.patel@bms.com
Present address: Codexis, Inc., Redwood City, CA, United States.
ꢀ
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.09.011

2148
I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
Herein, we report the screening of enzymes for the enantio-
selective hydrolysis of racemic methyl N-tBoc-6-hydroxy-
(S)-selective hydrolysis with no detectable cleavage of the
undesired (1R)-ester. Interestingly, the large majority of
the lipase, protease, peptidase, and acylase biocatalysts ap-
peared to be (S)-selective, while the esterase enzymes
showed either no significant enantioselectivity or only
slight (R)-preference. Biocatalyst screening for hydrolysis
in monophasic aqueous–organic media (comprising 20%,
40%, 60% or 80% v/v of ethanol, propan-2-ol, butan-1-
ol, acetonitrile, dimethoxymethane or THF in 50 mM
phosphate buffer, pH 8.0), or transesterification in organic
media (ethanol, propan-1-ol or butan-1-ol containing 0–
10% v/v buffer, or acetonitrile, THF, methyl-THF, diethyl
ether, MTBE, dichloromethane or toluene containing
0.1 M propan-1-ol and 0–10% v/v of buffer) were unsuc-
cessful, with no significant reaction (<2%) being detected
with any enzyme (native or immobilized) under any of
the conditions tried. Similarly, hydrolysis in biphasic
aqueous–organic media (buffer with an equal volume of
t-butanol, t-amyl alcohol, methyl-tetrahydrofuran, 1,2-
dimethoxyethane, diethyl ether, diisopropyl ether, methyl
3
,4-dihydro-1H-isoquinoline-1-carboxylate 1. The (R)-iso-
mer is a key intermediate for the synthesis of potent ago-
2
1,22
nists for a Liver X Receptor (LXR) discovery program.
We also report the screening of hydrolase biocatalysts for
the resolution of (RS)-1, and the development of a highly
enantioselective hydrolytic resolution process for the prep-
aration of (1S)-2 and (1R)-1 (Fig. 1).
2
. Results and discussion
About 140 native and immobilized hydrolase enzymes were
screened for the enantioselective hydrolysis of (RS)-1 at a
ꢀ1
substrate concentration of 1 g L in aqueous buffer con-
taining 1% v/v of DMSO (Tables 1–3), 303 K, 40 h. Only
a few enzymes showed significant activity toward the sub-
strate, most notably Protease P and Seaprose S (a more
concentrated form of Protease P), which mediated highly
Table 1. Lipase-mediated enantioselective hydrolysis of (RS)-1
ꢀ1
Biocatalyst
[Biocatalyst] (g L
)
Conversion (%)
Selectivity
ee
P
(%)
Amano Lipase-AK
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
40
20
41
4
9
—
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
—
(S)
(S)
(S)
—
—
—
—
—
—
(S)
—
0
38
3
9
5
9
6
5
5
Amano Lipase-AP12
Amano Lipase-AY30
Amanop Lipase-D
Amano Lipase-F
Amano Lipase-FAP15
Amano Lipase-G
Amano Lipase-GC20
Amano Lipase-M
Amano Lipase-MAP10
Amano Lipase-N
Amano Lipase-PS
Amano Lipase-PS30
Amano Lipase-R
Biocatalysts Lipase-ANL
Biocatalysts Lipase-CCL
Biocatalysts Lipase-RJL
Boehringer Chirazyme-L3
Enzymatix Lipase-B1
Enzymatix Lipase-F5
Europa Lipase-4
Europa Lipase-13
Europa Lipase-14
Europa Lipase-21
Julich Lipase-RN
10
16
6
12
14
15
17
9
6
5
8
1
4
10
17
9
4
53
0
3
3
14
0
0
0
0
0
5
5
0
0
0
0
4
4
0
0
0
27
1
30
31
17
2
50
32
35
28
45
13
20
19
18
39
47
19
19
27
35
30
7
Julich Lipase-RO
Meito Sangyo Lipase-AL
Meito Sangyo Lipase-MY
Meito Sangyo Lipase-OF
Meito Sangyo Lipase-PL
Meito Sangyo Lipase-QLM
Meito Sangyo Lipase-SL
Meito Sangyo Lipase-TL
Meito Sangyo Lipase-UL
Sigma Lipase-CRL
—
—
—
(S)
(S)
—
—
—
Sigma Lipase-PPL
Sepracor Lipase-OF
(S)
—
53
ꢀ1
ꢀ1
Conditions: substrate [10 lL, 100 g L of (RS)-1 in DMSO] was added to a vigorously stirred solution/suspension of biocatalyst (1 mL, 20 or 40 g L of
biocatalyst in 0.1 M sodium phosphate, pH 8), and the mixture stirred at 400 rpm, 303 K, 40 h. The percentage conversion refers to the overall extent of
hydrolysis. The selectivity refers to the configuration of the major hydrolysis product. ee values were determined by chiral HPLC.

I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
Table 2. Esterase-mediated enantioselective hydrolysis of (RS)-1
2149
ꢀ1
Biocatalyst
[Biocatalyst] (g L
)
Conversion (%)
Selectivity
ee
P
(%)
NA
Biocatalysts Lipomod-200
Boehringer Chirazyme-L2-c2
Boehringer Chirazyme-L5
Amano Lipase PS30/Accurel
Novo Lipolase-30T
100
100
100
100
100
100
100
20
20
20
20
20
20
20
20
20
20
20
20
20
27
51
43
38
80
83
58
48
45
47
45
36
71
64
68
73
59
61
58
68
14
(S)
(R)
(R)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Novo Lipozym-IM60
Novo Novozym-435
Fluka B. thermoglucisdasus Esterase
Fluka B. stearothermophilus Esterase
Fluka C. lipolytica Esterase
Fluka M. miehei Esterase
Fluka R. oryzae Esterase
Fluka S. cerevisiae Esterase
Fluka S. diastatochromogenes Esterase
Fluka T. lanuginosus Esterase
Julich Esterase-BS1
Julich Esterase-BS2
Julich Esterase-BS3
Julich Esterase-SD
Julich Esterase-PF
Immobilized Captopril Esterase
100
ꢀ1
ꢀ1
Conditions: substrate [10 lL, 100 g L of (RS)-1 in DMSO] was added to a vigorously stirred solution/suspension of biocatalyst (1 mL, 20 or 100 g L of
biocatalyst in 0.1 M sodium phosphate, pH 8), and the mixture stirred at 400 rpm, 303 K, 40 h. The percentage conversion refers to the overall extent of
hydrolysis. The selectivity refers to the configuration of the major hydrolysis product. ee values were determined by chiral HPLC.
t-butyl ether, dichloromethane, hexane or heptane) gave
very poor conversions (<5%) presumably due to unfavor-
able substrate partitioning and/or poor enzyme stability.
enantioselectivity (Table 6). Similarly, the optimal DMSO
concentration was found to be 1–2% v/v, with higher levels
leading to reductions in rate, conversion, and enantioselec-
tivity. In an effort to circumvent the limitation of substrate
load and improve its availability and reduce oiling-out, the
hydrolysis of (1RS)-1 was examined in the presence of Cel-
ite 561 and R633 filter-aids, microcrystalline and fibrous
cellulose, and Amberlite XAD-4, 7, 8 and 16, and Diaion
HP20, HP2MG resins, and using up to 10% v/v of DMSO
as a cosolvent. However, there was no significant visible
improvement of substrate dispersion in any case, with sub-
strate crusting apparent after just one day, and the inclu-
sion of adsorbent resulted in significantly decreased
conversions (results not shown). With regards to reaction
temperature, it was found that the results were optimal at
about 293–303 K, with reaction rates dropping signifi-
cantly below 303 K and resulting in incomplete conver-
sions, while oiling-out of the substrate and rapid
inactivation of the biocatalyst compromised selectivities
and yields above 313 K. It should be noted that the corre-
sponding ethyl, prop-1-yl and 2-hydroxyethyl esters (pre-
pared in quantitative yields by stirring 0.2 M of (1RS)-1,
in the corresponding alcohol with 5 mol % of ytterbium
or scandium triflate as catalyst, 50 ꢁC, 3 d) gave rather
poor results, with severe oiling-out and low conversions
[42–76% hydrolysis of (1S)-1] being observed.
Further experiments were carried out to optimize Protease
P and Seaprose S-mediated hydrolysis in aqueous milieu,
with the focus on buffer type and concentration, substrate
load, enzyme-to-substrate ratio, type and concentration of
the cosolvent, and reaction temperature. Buffer screening
over pH 5–9 showed that 0.1 M sodium phosphate, pH
7
.5–8.0, gave the best results in terms of both conversion
and enantioselectivity (Table 4). Thus, low rates and
incomplete reactions were observed in the absence of buffer
and when buffering with 50 mM phosphate and at pH val-
ues below 7.5, while high background (non-selective)
hydrolysis and low ee values were incurred at pH values
ꢀ
1
above 8.0. The effect of substrate loading over 1–5 g L
,
with biocatalyst-to-substrate ratios of 5–20 for Protease P
and 2.5–7.5 for Seaprose S was then examined (Table 5).
ꢀ
1
It was found that a substrate input of 1 g L with a Pro-
ꢀ1
tease P load of 15–20 g L
or a Seaprose S load of
consistently provided near-quantitative and
ꢀ
1
7
.5 g L
highly enantiospecific hydrolysis of (1S)-1, with no detect-
able cleavage of (1R)-1. Although some experiments con-
ꢀ1
ducted with 2 and 5 g L
of substrate provided good
conversions and enantioselectivities, reproducibility was
lacking due to oiling-out and crusting of the substrate/
product compromising conversions and selectivity, despite
increasing the buffer strength to 0.2 M and/or the DMSO
concentration to 15% v/v. The inclusion of cosolvents (eth-
anol, propan-2-ol, acetonitrile, acetone, tetrahydrofuran,
and dimethoxymethane) at 5% or 10% v/v (in addition to
ꢀ1
Having optimized the reaction conditions (1 g L of sub-
strate, Seaprose S as catalyst, 2% v/v DMSO, 100 mM
sodium phosphate, pH 8.0, rt), the work-up of the reac-
tion mixture was then examined. Initial efforts at process-
ing the reaction by way of adjusting the pH to 9–9.5 and
extracting (1R)-1 with MTBE, DCM, ethyl acetate or hex-
ane, followed by acidification of the aqueous phase to pH
1
% v/v of DMSO) led to drastic reductions in hydrolysis
rates and conversions, together with significant drops in

2150
I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
Table 3. Protease, peptidase and acylase-mediated enantioselective hydrolysis of (RS)-1
ꢀ1
Biocatalyst
[Biocatalyst] (g L
)
Conversion (%)
Selectivity
P
ee (%)
Amano Acylase
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
40
40
14
3
0
1
11
6
47
50
2
0
1
16
0
3
0
1
1
5
1
3
15
7
6
4
3
2
1
2
3
1
0
1
1
(S)
(S)
—
15
2
0
0
17
8
>99.8
>99.8
3
0
0
20
0
4
0
0
0
3
0
4
22
9
8
5
3
0
0
0
3
0
0
0
0
Amano D-Aminoacylase
Amino Acid Protease-A
Amano Acid Protease-II
Amano Protease-A
Amano Protease-M
Amano Protease-P (6K)
Amano Seaprose S
Amano Protease-S
Amano Newlase-F
Amano Peptidase-R
Amano Umamizyme
Julich Esterase-RO
Novo Flavorzyme-M6
Novo Neutrase-1
Novo Protames
Novo Semiacylase
Sigma PLE
Sigma Pig Acylase
Sigma a-Chymotrypsin
Sigma Subtilisin
Sigma Pronase-E
Sigma Proteinase-K
Sigma Trypsin
—
(S)
(S)
(S)
(S)
(S)
—
—
(S)
—
(S)
—
—
—
(R)
—
(S)
(S)
(S)
(S)
(S)
(S)
—
—
—
(S)
—
—
Sigma B. polymyxa Protease
Sigma Serratiopeptidase
Sigma A. saitoi Peptidase
Sigma Rhizopus Protease
Sigma S. caespitosus Protease
Sigma Bacillus Protease-N
Sigma Bacillus Proteinase
Fluka Ficin
—
—
Fluka Papain
ꢀ1
ꢀ1
Conditions: substrate [10 lL, 100 g L of (RS)-1 in DMSO] was added to a vigorously stirred solution/suspension of biocatalyst (1 mL, 20 or 40 g L of
biocatalyst in 0.1 M sodium phosphate, pH 8), and the mixture stirred at 400 rpm, 303 K, 40 h. The percentage conversion refers to the overall extent of
hydrolysis. The selectivity refers to the configuration of the major hydrolysis product. ee values were determined by chiral HPLC.
2
–2.5 and solvent extraction of (1S)-2 were hindered by
of alternative polystyrene/polyacrylate resins or ion
exchangers, such as Amberlites XAD4, XAD7 and
XAD8, Lewatits 1064 and 1163, Mitsubishi HP2MG
and Ambersorb-359, although enabling the selective
adsorption of (1R)-1 at pH 9–9.5 in several cases, was
precluded by difficulties encountered in recovering the ad-
sorbed (S)-acid.
extensive emulsification during the extraction of the (S)-
acid. This phenomenon was traced to the presence of
the biocatalyst, and the use of temperature cycling, addi-
tion of salts and the application of different solvents (bu-
tan-1-ol, pentan-1-ol, methyltetrahydrofuran and pentan-
3
-one) failed to overcome emulsification. However, it
was found that the simple addition of Celite filter aid dur-
ing extraction of (1S)-2 was sufficient to enable efficient
recovery of the desired acid without significant emulsion
issues. It should be mentioned that extraction with resins
was examined as a more facile and cost-efficient alterna-
tive to solvent extraction—thus, Amberlite XAD16 was
found to adsorb >98% of acid and ester from the reaction
mixture acidified to pH 2–2.5, and both (1R)-1 and (1S)-2
were recovered in >95% yield upon back-extraction with
MTBE. However, all attempts to separate the (S)-acid
from the (R)-ester by extraction with base or pad-filtra-
tion through silica or alumina failed. High pH extractions
were confounded by poor recoveries and the presence of
polar impurities derived from the biocatalyst, resulting
in severe contamination of the acid. On the other hand,
very poor separations and/or recoveries of the acid, and
less so the ester, were incurred with pad-filtration or di-
rect elution of the Amberlite adsorbate. The application
To assess the practicality of the developed resolution and
work-up processes, the Seaprose S-mediated resolution
was performed on a 2 g scale and the reaction mixture pro-
cessed. Complete hydrolysis of (1S)-1 was obtained after
3 d of reaction with a biocatalyst-to-substrate ratio of 8,
with no detectable cleavage of the (1R)-1, and sequential
extractions with MTBE provided crude (1S)-2 with 94%
recovery, 68% potency and ee >99.8%, and crude (1R)-1
with 96% recovery, 91% potency and ee >99.5%. Polishing
by way of trituration with aqueous acetic acid enabled a
facile and effective purification of the acid, furnishing enan-
tiopure (1S)-2 in high potency (100–102%) and moderate
recovery (87% mass recovery). Aqueous trituration with
bicarbonate and acetic acid provided a significant, but
not complete purification of (1R)-1, delivering enantiopure
ester of moderate potency (95–96% potency) and good
recovery (93% mass recovery).

I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
2151
Table 4. Effect of buffer type and pH on the Protease P-mediated (S)-enantioselective hydrolysis of (RS)-1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
[
Protease P] (g L
)
[Substrate] (g L
)
Buffer
[Buffer] (mM) pH Initial rate (mmol h kg )
P
Conversion (%) ee (%)
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
None
—
50
—
2.9
6.6
6.4
8.1
8.9
6.9
7.6
7.8
7.9
8.8
3.8
4.2
4.7
4.8
7.2
7.4
8.0
7.6
8.1
8.7
28
44
47
50
50
41
49
52
55
63
38
44
47
52
48
51
54
44
46
52
98.6
99.2
99.7
>99.8
>99.8
>99.8
>99.8
99.1
88.6
79.3
>99.8
99.1
98.0
92.7
99.3
94.7
90.9
98.8
98.2
94.1
Sodium phosphate
Sodium phosphate
Sodium phosphate
Sodium phosphate
Sodium metaphosphate
Sodium metaphosphate
Sodium metaphosphate
Sodium metaphosphate
Sodium metaphosphate
Sodium bicarbonate
Sodium bicarbonate
Sodium bicarbonate
Sodium bicarbonate
Tris
7.5
7.0
7.5
8.0
7.0
7.5
8.0
8.5
9.0
7.0
7.5
8.0
8.5
7.5
8.0
8.5
7.0
7.5
8.0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Tris
Tris
Triethanolamine
Triethanolamine
Triethanolamine
ꢀ
1
ꢀ1
Conditions: substrate [10 lL, 100 g L of (RS)-1 in DMSO] was added to a vigorously stirred suspension of Protease P (1 mL, 20 g L of biocatalyst in
water, or 0.05 or 0.1 M buffer), and the mixture stirred at 400 rpm, 303 K, 40 h. Initial rates were measured at 1 h and are quoted as mmol of product
formed per h per kg-enzyme. The percentage conversion refers to the overall extent of hydrolysis. ee values were determined by chiral HPLC.
Table 5. Effects of substrate, biocatalyst and DMSO loads on the Protease P and Seaprose S-mediated (S)-selective hydrolysis of (RS)-1
Biocatalyst
[Biocatalyst]
[Substrate]
[DMSO]
(% v/v)
[Buffer] (mM)
Initial rate
(mmol h kg
Conversion (%)
P
ee (%)
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
(g L
)
(g L
)
)
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Protease P
Seaprose S
Seaprose S
Seaprose S
Seaprose S
Seaprose S
Seaprose S
Seaprose S
Seaprose S
Seaprose S
5
10
1.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
5.0
10.0
2.0
2.0
2.0
2.0
10.0
15.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
5.0
10.0
100
100
100
100
100
200
100
100
100
100
100
200
100
100
100
100
100
100
100
100
100
100
100
100
7.4–8.0
8.2–8.7
8.0–9.2
7.8–8.4
4.1–6.3
3.8–4.2
4.8–8.0
4.5–7.3
3.8–8.2
3.0–6.5
5.9–7.8
3.5–7.3
8.1
37–39
42–45
49–50
49–50
38–44
27–32
35–39
31–33
27–36
18–27
22–25
17–22
19
99.1–99.5
99.6–>99.8
99.8–>99.8
1.0
1.0
1.0
2.0
2.0
2.0
2.0
2.0
5.0
5.0
5.0
5.0
5.0
5.0
1.0
1.0
1.0
2.0
2.0
2.0
2.0
2.0
2.0
15
20
40
40
60
40
40
25
50
50
100
100
100
5
7.5
10
5
7.5
10
15
15
15
>99.8
99.4–>99.8
99.1 >99.8
99.5–>99.8
99.0–99.3
99.2–99.7
98.2–99.3
99.4–99.8
99.7–>99.8
>99.8
>99.8
99.6
99.7–>99.8
>99.8
99.4–>99.8
99.1–99.5
99.2–>99.8
99.3–99.8
98.4
7.4
6.7
25
22
8.5–9.1
9.1–9.5
8.8–9.8
4.8–7.5
6.1–7.7
5.5–8.9
5.7
40–42
49–50
50–51
28–34
35–38
31–40
36
6.1
5.3
46
31
>99.8
>99.8
ꢀ
1
Conditions: substrate [20–150 lL, 50–250 g L of (RS)-1 in DMSO] was added to a vigorously stirred suspension of Protease P or Seaprose S (1 mL, 5–
ꢀ1
1
00 g L of biocatalyst in 0.1 or 0.2 M sodium phosphate, pH 8.0), and the mixture stirred at 400–600 rpm, 303 K, 40 h. Initial rates were measured at 1 h
and are quoted as mmol of product formed per h per kg-enzyme. The percentage conversion refers to the overall extent of hydrolysis. ee values were
determined by chiral HPLC.
3
. Conclusion
mercial biocatalyst, the presence of surface-active protein
contaminants therein, and the low solubilities of the sub-
strate and product. These lead to the requirement for a
high biocatalyst-to-substrate ratio (8:1 w/w), a low sub-
The developed resolution process exhibits significant scale-
up issues deriving from the low activity of the crude com-

2152
I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
Table 6. Effects of cosolvents on the Seaprose S-mediated (S)-selective hydrolysis of (RS)-1
ꢀ1
[
Biocat] (g L
)
[Substrate] [DMSO] (% v/v) Cosolvent (% v/v)
[Buffer] (mM) Initial rate
P
Conversion (%) ee (%)
ꢀ1
ꢀ1
ꢀ1
(g L
)
(mmol h kg
)
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
3.0
4.0
5.0
6.0
8.0
10.0
—
100
100
100
100
100
100
100
100
200
100
100
100
100
100
100
100
100
100
100
100
9.6
2.9
3.5
3.7
2.8
50
22
28
31
16
12
18
<2
3
8
<2
7
5
>99.8
>99.8
>99.8
99.3
97.4
99.2
98.6
—
—
97.7
—
97.3
96.9
>99.8
>99.8
99.2–99.5
98.8–99.3
98.2–99.4
99.2–>99.8
98.7–99.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5% ethanol
5% propan-2-ol
5% acetonitrile
5% acetone
5% tetrahydrofuran
5% dimethoxymethane
10% ethanol
10% propan-2-ol
10% acetonitrile
10% acetone
10% tetrahydrofuran
10% dimethoxymethane
—
—
—
—
—
—
—
2.4
2.1
<0.2
<0.2
0.3
<0.2
0.5
0.3
8.4–8.8
49–50
44–47
39–41
34–42
30–38
29–34
23–41
7.3–8.2
6.2–6.7
5.3–6.0
4.2–5.5
4.0–5.1
3.8–4.4
ꢀ1
ꢀ1
Conditions: substrate [10 lL, 100 g L of (RS)-1 in DMSO] was added to a vigorously stirred suspension of Seaprose S (1 mL, 8 g L of biocatalyst in
.1 or 0.2 M sodium phosphate, pH 8.0, containing 0–9% v/v of DMSO and 0–10% v/v of cosolvent), and the mixture stirred at 400–600 rpm, 303 K, 40 h.
0
Initial rates were measured at 1 h and are quoted as mmol of product formed per h per kg-enzyme. The percentage conversion refers to the overall extent
of hydrolysis. ee values were determined by chiral HPLC.
ꢀ
1
strate load (1 g L ), a long reaction time (3 d), and the
need for high-volume solvent extraction procedures for
the recovery of (1S)-2 and (1R)-1, and make the process
unsuitable for industrial implementation. However, it is
envisaged that these issues can be overcome with the use
of an immobilized, high-activity cloned biocatalyst, which
could enable the resolution of high concentrations
ment of commercially available N-tBoc-6-hydroxy-3,4-
dihydro-1H-isoquinoline-1-carboxylic acid with 1 equiv of
LiOHÆH O in THF for 40 min followed by the addition
2
of 1 equiv of Me SO . The reaction mixture was heated
2
4
at reflux for 3 h. After cooling the reaction vessel to room
temperature, the THF was removed and the residue was di-
luted with CH Cl . The organic layer was washed with
2
2
ꢀ
1
(
>10 g L ) of substrate in monophasic or biphasic aque-
H O and brine, then dried over MgSO , filtered and con-
2 4
ous–organic media, and thus enable sufficiently high pro-
ductivities and recovery efficiencies for economic scale-up.
Efforts are currently underway to isolate and clone the bio-
catalyst and develop such a process. In addition, the
deployment of in situ racemization of (1S)-2 is also being
investigated in order to enable conversions beyond 50%,
although preliminary data indicate that thermal and acid/
base-mediated approaches result in substantial degradation
centrated under reduced pressure. The residue was purified
by column chromatography with a gradient of 0–50% ethyl
acetate in hexanes to yield (RS)-1 as a pale, yellowish foam.
Enantiopure methyl N-tBoc-6-hydroxy-3,4-dihydro-1H-
isoquinoline-1-carboxylate isomers (R)-1 and (S)-1 were
isolated by chiral preparative HPLC using chiralpak OD
20l column (5 · 50 cm, eluting with 5% (EtOH/MeOH
(50:50))/heptane, 50 mL/min flow rate). The retention
times were 12.79 min for (S)-1 and the 14.43 min for (R)-
(
hydrolysis, decarboxylation, and ring cleavage).
1. Characterization data was consistent with the data
described below (Section 4.5). The stereochemistry assign-
ment was made based on a crystal structure with LXRb
and a tetrahydroisoquinoline analog prepared from (R)-1
4
. Experimental
4
.1. General
(
21).
.3. Analytical methods
Reactions were followed by chiral RP-HPLC on a Shima-
Enzymes were purchased from Novozymes, Amano En-
zyme Company, Biocatalysts, Boehringer, Julich, Meito
Sangyo and Sigma–Aldrich. All other chemicals and sol-
vents were purchased from Aldrich, Fluka and Sigma
and were of AR quality or higher.
4
ꢀ
1
dzu LC-10 system. Samples were diluted to 0.1–0.2 g L
with methanol containing 0.5% v/v of acetic acid and fil-
tered (0.2 lm PTFE), prior to HPLC analysis. Samples
were analyzed as follows: Chiralpak AS-RH (5 lm,
4
6
.2. Preparation of racemic and enantiopure methyl N-tBoc-
2
1
-hydroxy-3,4-dihydro-1H-isoquinoline-1-carboxylate
4
.6 · 150 mm) column; elution with 0–20% v/v B over
Racemic methyl N-tBoc-6-hydroxy-3,4-dihydro-1H-iso-
quinoline-1-carboxylate (RS)-1 was prepared by the treat-
15 min, then 20–25% v/v B over 15–45 min; solvent A
was 8:2 v/v water–methanol containing 0.05% v/v TFA

I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
2153
and solvent B was 8:2 v/v acetonitrile–methanol containing
.05% v/v TFA; 0.7 mL/min flow rate; ambient column
temperature; 50 lL injection; 220 nm and 254 nm detec-
tion. The retention times were 9.5 min for the (S)-acid,
dium hydroxide, the mixture extracted with MTBE
(2 · 1 L), and the organic extract evaporated at 25 ꢁC to
furnish the crude (R)-ester as a pale-brown viscous oil
(1.06 g). This was triturated with aqueous sodium bicar-
bonate (100 mL, 25 mM) to give an off-white semi-solid,
which was recovered and stirred with aqueous acetic acid
(100 mL, 0.5% v/v) at rt, 1 h, then washed with water
(3 · 10 mL), and dried under vacuum over Drierite, potas-
sium hydroxide, and charcoal, at room temperature for
0
2
8.4 min for the (S)-ester, and 29.5 min for the (R)-ester.
NMR spectra were recorded on a Bruker-300 or Jeol-400
spectrometers using deutero-chloroform as solvent. Optical
rotations were recorded on a Perkin–Elmer 241 digital
polarimeter at 20 ꢁC using the sodium D line.
20 h to yield methyl (1R)-N-tBoc-6-hydroxy-3,4-dihydro-
4
(
.4. Screening of biocatalysts for the resolution of methyl
1RS)-N-tBoc-6-hydroxy-3,4-dihydro-1H-isoquinoline-1-
carboxylate (1RS)-1
1H-isoquinoline-1-carboxylate as an off-white semi-solid:
0.87 g, 96% potency (HPLC), 93% yield, ee >99.5%;
2
D
0
+
½aꢁ ¼ þ18:2 (c 0.2, methanol); LC–MS (ESI ) m/z
1
(
%) = 308 ([M+H], 100); H NMR (CDCl ) d 1.39 (s,
3
4
.4.1. Hydrolysis of (1RS)-1 in aqueous media. Substrate
9H, tBoc), 2.90–2.97 (m, 2H, 4-H), 3.28–3.34 (m, 2H, 3-
ꢀ
1
(
10 lL, 100 g L of rac-1 in DMSO) was added to vigor-
H), 3.72 (s, 3H, OCH ), 5.74 (s, 1H, 1-H), 6.47 (d, 1H,
3
ously stirred biocatalyst solution/suspension (1 mL, 20–
J = 6.4 Hz, 7-H), 6.88–7.04 (m, 2H, 5-H + 8-H), 9.35 (br
ꢀ
1
13
100 g L of biocatalyst in 0.1 M sodium phosphate, pH
6, 7, 8 or 9), and the suspension stirred at 400 rpm,
303 K, 40 h.
s, 1H, OH) ppm; C NMR (CDCl ) d 28.4, 28.6, 37.4,
3
52.6, 62.4, 79.8, 113.9, 115.7, 126.8, 127.0, 135.6, 154.9,
155.1, 171.0 ppm. The pH of the aqueous phase from the
MTBE extraction was adjusted to 2.2–2.3 with aqueous
sulfuric acid, and Celite (300 g) followed by MTBE (2 L)
added, and the mixture filtered. The filter cake was washed
with MTBE (2 · 1 L), the organic phase separated from the
combined filtrates, the aqueous phase extracted with
MTBE (2 · 1 L), and the combined organic phases evapo-
rated at 25 ꢁC, then dried under vacuum at 25 ꢁC to furnish
the crude (S)-acid as a pale-yellow solid (0.98 g). This was
triturated with aqueous acetic acid (100 mL, 0.5% v/v), the
suspension filtered, the filter cake washed with water
(4 · 4 mL), then dried under vacuum over Drierite, potas-
sium hydroxide and charcoal, at rt, 20 h to furnish (1S)-
N-tBoc-6-hydroxy-3,4-dihydro-1H-isoquinoline-1-carbox-
ylic acid as a soft white solid: 0.62 g, 101% potency
(HPLC), 87% yield, ee >99.8%; mp 123–127 ꢁC (decomp.);
4
.4.2. Hydrolysis of (1RS)-1 in monophasic aqueous–organic
ꢀ
1
media. Substrate (10 lL, 100 g L of rac-1 in DMSO),
was added to vigorously stirred biocatalyst solution/sus-
ꢀ1
pension (1 mL, 20–100 g L
of biocatalyst in 0.1 M
sodium phosphate, pH 8, containing 20–95% v/v of
ethanol, propan-1-ol, propan-2-ol, butan-1-ol, acetonitrile,
dimethoxymethane or THF), and the mixture stirred at
4
00 rpm, 303 K, 40 h.
4
.4.3. Hydrolysis of (1RS)-1 in biphasic aqueous–organic
ꢀ
1
media. Biocatalyst (0.5 mL, 10–50 g L of biocatalyst in
.1 M sodium phosphate, pH 8) was added to the substrate
0
ꢀ
1
solution (0.5 mL, 2, 5 or 10 g L of rac-1 in t-butanol,
t-amyl alcohol, methyl-THF, THP, 1,2-dimethoxyethane,
DEE, DIPE, MTBE, DCM, hexane or heptane), and the
mixture stirred at 500–600 rpm, rt, 40 h.
2
D
0
+
½aꢁ ¼ ꢀ19:2 (c 0.2, methanol); LC–MS (ESI ) m/z
1
(%) = 294 ([M+H], 100), 316 ([M+Na], 68); H NMR
(
CDCl ) d 1.40 (s, 9H, tBoc), 2.92–2.96 (m, 2H, 4-H),
3
4
.4.4. Transesterification of (1RS)-1 in organic media. Bio-
3.22–3.30 (m, 2H, 3-H), 5.78 (s, 1H, 1-H), 6.49 (d, 1H,
catalyst (10 or 50 mg) was added to substrate solution
J = 6.2 Hz, 7-H), 6.88–7.06 (m, 2H, 5-H + 8-H), 9.31 (br
ꢀ
1
13
(
0.5 mL, 5 or 10 g L of rac-1 in ethanol, propan-1-ol or
s, 1H, OH) ppm; C NMR (CDCl ) d 28.7, 28.9, 37.6,
3
butan-1-ol containing 0%, 2%, 5% or 10% v/v buffer, or
in acetonitrile, THF, methyl-THF, DEE, MTBE, DCM
or toluene containing 0.1 M propan-1-ol and 0%, 1%,
65.0, 80.3, 114.2, 116.1, 127.0, 127.5, 136.0, 155.2, 155.8,
171.3 ppm.
2
%, 5% or 10% v/v of buffer), and the suspension stirred
at 400 rpm, rt, 40 h.
References
4
.5. Seaprose S-mediated enantioselective hydrolysis of
1
. Pesti, J. A.; DiCosimo, R. Curr. Opin. Drug Discovery Dev.
003, 6, 884–901.
methyl (1RS)-N-tBoc-6-hydroxy-3,4-dihydro-1H-isoquino-
line-1-carboxylate (1RS)-1
2
2
3
. Patel, R. N. Curr. Org. Chem. 2006, 10, 1289–1321.
. Faber, K.; Kroutil, W. Curr. Opin. Chem. Biol. 2005, 9, 181–
187.
Seaprose S (16 g) was mixed with sodium phosphate buffer
(
20 mL, 0.1 M, pH 8.0) to form a paste, this was diluted
4. Sheldon, R. A.; Lau, R. M.; Sorgedrager, M. J.; van
Rantwijk, F.; Seddon, K. R. Green Chem. 2002, 4, 147–151.
with a buffer (1.92 L) and the resulting solution transferred
to a 2 L glass bottle. The solution was stirred at 250 rpm
with an overhead PTFE half-moon paddle stirrer, and
DMSO (30 mL), followed by rac-1 (2 g of ester dissolved
in 30 mL of DMSO) added over 2 min, and stirring contin-
ued at room temperature for 3 days. Chiral HPLC analysis
indicated complete hydrolysis of the (S)-ester without any
detectable cleavage of the (R)-enantiomer. The pH of the
reaction mixture was adjusted to 9.2–9.5 using 0.5 M so-
5
6
. Turner, N. J. Curr. Opin. Chem. Biol. 2004, 8, 114–119.
. Alphand, V.; Carrea, G.; Wohlgemuth, R.; Furstoss, R.;
Woodley, J. M. Trends Biotechnol. 2003, 21, 318–323.
. Robertson, D. E.; Bornscheuer, U. T. Curr. Opin. Chem. Biol.
7
8
2
005, 9, 164–165.
. Ishige, T.; Honda, K.; Shimizu, S. Curr. Opin. Chem. Biol.
005, 9, 174–180.
2
9. Homann, M. J., Morgan, W. B., Zaks, A. 2002, 7pp. US
6410306 B1 20020625 Patent Application: US 2000-512247

2
154
I. S. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 2147–2154
17. Hidalgo, A.; Bornscheuer, U. T. In Biocatalysis in Pharma-
2
0000224. Priority: US 99-121749 19990226. CAN 137:46194
AN 2002:483022.
0. Svendsen, A. V., Jesper; B. J., De Maria, L PCT Int.
Appl. 2006, 17pp. WO 2006084470 A2 20060817 AN 2006:
18102.
1. Bae, H.-A.; Lee, K.-W.; Lee, Y.-H. J. Mol. Catal. B: Enzym.
006, 40, 24–29.
2. Gogoi, S.; Argade, N. P. Tetrahedron: Asymmetry 2006, 17,
27–932.
3. Miyazawa, T.; Minowa, H.; Yamada, T. Biotechnol. Lett.
006, 28, 295–299.
ceutical and Biotechnology Industries; Patel, R., Ed.; CRC
Press: FL, 2006; pp 159–179.
18. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic
Synthesis: Regio- and Stereoselective Biotransformations;
Wiley-VCH: Weinheim, 1999.
19. Miyazawa, T.; Imagawa, K.; Minowa, H.; Miyamoto, T.;
Yamada, T. Tetrahedron 2005, 61, 10254–10261.
20. Breitgoff, D., Essert, T., Laumen, K., Schneider, M. P. In
F.E.C.S. 3rd Int. Conf., Chem. Biotechnol. Biol. Act. Nat.
Prod. [Proc.], 1985; 1987; Vol. 2, pp 127–147.
1
8
1
1
1
1
2
9
2
4. Schulze, B.; De Vroom, E. In Enzyme Catalysis in Organic
Synthesis, 2nd ed; Drauz, K., Waldmann, H., Eds.; Wiley-
VCH Verlag GmbH: Weinheim, Germany, 2002; Vol. 2, pp
21. Yang, W., Wang, Y., Kick, E.K. PCT Int. Appl. 2007, 111pp.
WO 2007047991 A1 20070426 AN 2007:458738.
22. For recent LXR reviews see: (a) Bradley, M. N.; Tontonoz, P.
Drug Discovery Today: Therap. Strat. 2005, 2, 97–103; (b)
Bennett, D. J.; Cooke, A. J.; Edwards, A. S. Recent Patents
Cardiovascular Drug Discovery 2006, 1, 21–46; (c) Bruemmer,
D.; Law, R. E. Curr. Drug Targets: Cardiovascular Haematol.
Disorders 2005, 5, 533–540; (d) Jaye, M. Curr. Opin. Invest.
Drugs (Thomson Current Drugs) 2003, 4, 1053–1058.
7
16–740.
1
1
5. Berglung, P.; Hult, K. In Stereoselective Biocatalysis; Patel,
R., Ed.; Marcel and Dekker: NY, 2000; pp 633–658.
6. Deussen, H-J.; Zundel, M.; Valdois, M.; Lehmann, S. J.;
Weil, V.; Hjort, C. M.; Oestergaard, P. R.; Marcussen, E.;
Ebdrup, S. Org. Process Res. Dev. 2003, 7, 82–88.