Enantioselective enzymatic acylation of 1-(3′-bromophenyl)ethylamine

Article abstract of DOI:10.1016/j.tetasy.2007.05.039

An efficient biocatalytic process has been developed for the resolution of 1-(3′-bromophenyl)ethylamine (RS)-1 by way of enantioselective lipase-mediated (R)-selective acylation with ethyl 2-methoxyacetate to afford (S)-amine (S)-1 and (R)-2″-methoxyaceta

Full text of DOI:10.1016/j.tetasy.2007.05.039

Tetrahedron: Asymmetry 18 (2007) 1330–1337
0
Enantioselective enzymatic acylation of 1-(3 -bromophenyl)ethylamine
a,
b
a,
*
Iqbal I. Gill, Jagbandhu Das and Ramesh N. Patel
a
Process Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, NJ, USA
b
Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Lawrenceville, NJ, USA
Received 6 April 2007; accepted 15 May 2007
0
Abstract—An efficient biocatalytic process has been developed for the resolution of 1-(3 -bromophenyl)ethylamine (RS)-1 by way of
00
enantioselective lipase-mediated (R)-selective acylation with ethyl 2-methoxyacetate to afford (S)-amine (S)-1 and (R)-2 -methoxyacet-
amide ((R)-2) in 91–95% and 90–92% isolated yield, respectively, and both with >99% ee.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
numerous reports of the enantioselective lipase-mediated
acylation of amines appeared in the 1990s and the field
has since gained significant industrial academic and signif-
Enantiopure amines are widely used in the pharmaceutical,
agrochemical and fine chemical industries as synthetic
intermediates, chiral auxiliaries, catalysts and resolving
2
0–29
icance, as described in a number of recent reviews.
1
–8
agents.
Amongst the methodologies that have been
Herein we report the highly enantioselective CALB/Novo-
0
developed for the industrial production of enantiopure
amines, lipase-mediated enantioselective acylation has been
increasingly utilized, despite many other available method-
ologies, such as hydroamination, hydrosilylation, asymmet-
ric hydrogenation and diastereomeric salt crystallization,
transaminase-mediated reductive amination or amine
oxidase or deaminase-mediated resolution.9–12
zym 435- and amano lipase PS-catalyzed acylation of 1-(3 -
bromophenyl)ethylamine (RS)-1 with ethyl 2-methoxyace-
tate as the acyl donor, to furnish the (S)-amine (S)-1 and
00
(R)-2 -methoxyacetamide (R)-2 in high yield (90–96%)
and high enantiopurity (ee >99%). The enantiomers of 1
and 2 were required with enantiopurities >99.5% as inter-
mediates in the synthesis of IB kinase (IKK) modulators
3
0–39
and other pharmaceuticals.
Lipases have long been recognized as highly active and sta-
ble enzymes endowed with broad substrate ranges together
with unique stereo-, chemo- and regio-selectivities and the
robustness to operate efficiently in a wide variety of sol-
vent, pH and temperature conditions. These attributes
have made lipases outstanding catalysts for synthetic bio-
transformations that involve the carboxyl group, such as
2
. Results and discussion
Initially, a range of hydrolases were evaluated (Table 1) at
the room-temperature enantioselective N-acylation of
RS)-1 with vinyl acetate, isopropenyl acetate and ethyl ace-
tate as acyl donors and methyl t-butyl ether (MTBE) as sol-
vent, with the focus on obtaining highly enantioenriched (ee
(
(
thio)esterification, transesterification, perhydrolysis and
aminolysis, wherein the water is replaced by an alcohol,
thiol, hydroperoxide or amine.13–15 The principle of
lipase-catalyzed aminolysis of carboxylic esters was first
>
(
99%) amide in high yield (>45% of starting material)
Scheme 1). While vinyl acetate proved to be too reactive
1
6
described in 1984 by Inada et al. in a seminal paper that
contributed greatly by initiating the synthetic application
and resulted in high levels (12–34%) of non-selective non-
enzymatic acetylation as well as the formation of several
side products, isopropenyl acetate and ethyl acetate fur-
nished fairly clean enzymatic N-acylations without signifi-
cant background reactions. Although a number protease,
acylase, esterase and amidase biocatalysts did provide sig-
nificant (R)- or (S)-enantioselectivity, conversions and/or
selectivities were generally low, with overall conversions
1
7–19
of lipases in organic media.
Following this publication,
*
Corresponding author. Tel.: +1 732 227 6213; fax: +1 732 227 3994;
e-mail: ramesh.patel@bms.com
Present address: Codexis, Inc., Redwood City, CA, USA.
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.039

I. I. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 1330–1337
1331
Table 1. Hydrolase-mediated enantioselective N-acylation of (RS)-1 with isopropenyl acetate
Biocatalyst
[Biocatalyst] (mg/mL)
Conversion (%, HPLC)
Amide product
ee (amide) (%, HPLC)
Amano Acylase
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
25
25
4
12
1
1
6
5
3
2
2
2
2
4
1
1
1
4
10
2
(R)-2
(R)-2
(R)-2
(S)-2
(R)-2
(S)-2
(S)-2
(R)-2
(R)-2
(S)-2
(S)-2
(RS)-2
(RS)-2
(S)-2
(S)-2
(S)-2
(S)-2
(R)-2
(S)-2
(S)-2
(RS)-2
40
23
6
71
1
1
4
14
2
1
9
0
0
21
12
33
1
2
2
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
Subtilisin-Celite R633
Alcalase-Celite R633
2
6
0
7
0
˚
Reactions were conducted with 50 mM (RS)-1, 100 mM (2 mol equiv) isopropenyl acetate and 125 mg/mL 4 A molecular sieves in dry MTBE, using
2
0 mg/mL (free-form) or 25 mg/mL (immobilized) biocatalyst, with stirring at 400 rpm at 40 ꢁC for 20 h. Conversions and enantioselectivities were
determined by HPLC.
NHR
NH2
Lipase
NH2
+
Br
Br
Br
Acyl Donor
(S)-1
(R)-2a: R = OAc
R)-2b: R = COCH OMe
(RS)-1
(
2
Scheme 1.
remaining below 15%, even after 3 days of incubation at
0 ꢁC. In contrast, several lipases (Table 2) were found to
effect the enantioselective N-acylation with isopropenyl ace-
tate as the acyl donor, notably amano lipase-AK, Europa
Lipase-14, Meito Sangyo Lipase-TL, Boehringer Chira-
zyme-L2, Amano Lipase-PS30 on Accurel and Novozym-
formation with certain batches of isopropenyl acetate. In
view of the poor reproducibility of isopropenyl acetate
reactions, and the lower cost of ethyl acetate and its re-
duced propensity towards side reactions, resolution with
the latter was studied under different solvent conditions
with the aim of improving the ee of (R)-2a to over 99%
(Table 3). Although hexane and dichloromethane did pro-
vide (R)-2a with >99.8% ee, the conversions were very low
(<10%), and further modifications of the reaction condi-
tions (solvent, additives, biocatalyst load, acyl donor-to-
substrate ratio and reaction temperature) did not afford
the desired combination of high enantiopurity for (S)-1
and (R)-2a together with a high conversion. Thus, although
highly enantiopure (S)-1 could be obtained via Chirazyme-
L2 and Novozym-435-mediated enantioselective acylation
of 0.5 M (RS)-1 in neat ethyl acetate, these conditions
afforded conversions over 60% resulting in a substantial
loss of enantiopurity in (R)-2a as well as the formation of
hydrophobic by-products. On the other hand, (R)-2a with
ee above 99.8% was obtained by running reactions to low
conversions, for example, 700 g/L (3.5 M) substrate and
451 g/L (5.1 M, 46 mol % excess) ethyl acetate, with 70 g/
L Novozym-435, provided 18% of (R)-2a with 95% ee after
20 h, but at the expense of very low ee for (S)-1.
4
4
35, all of which showed (R)-selectivity, whilst Boehringer
Chirazyme-L5 was the only lipase that showed the opposite
S)-selectivity. The best results were obtained with Chira-
zyme-L2 and Novozym-435, which provided close to
00% conversion of (R)-1 with very high enantioselectivity
>99.8% ee). In the case of ethyl acetate, Europa Lipase-
4, Meito Sangyo Lipase-TL, Boehringer Chirazyme-L2-
(
1
(
1
c2, Amano Lipase PS30/Accurel, Amano Lipase PS30 in
sol–gel, Novo Novozym-435 and CALB on Celite provided
(
R)-selective N-acylation, albeit with lower selectivities (Ta-
ble 3). The best results were obtained with Chirazyme-L2,
Novozym-435 and CALB on Celite, which furnished (R)-
2
a with 95–97% ee at 51–54% conversion.
Despite the good initial results with isopropenyl acetate,
subsequent experiments with this acyl donor gave poor
conversions and enantioselectivities, due to low enzymatic
acylation activity and/or extensive (up to 60%) by-product

1332
I. I. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 1330–1337
Table 2. Lipase-mediated enantioselective N-acylation of (RS)-1 with isopropenyl acetate
Lipase biocatalyst
[Biocatalyst] (mg/mL)
Conversion (%, HPLC)
Amide product
ee (amide) (%, HPLC)
Amano Lipase-A
Amano Lipase-AK
Amano Lipase-AP12
Amano Lipase-AY30
Amano 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
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
100
100
100
100
100
100
100
0
40
0
0
1
0
0
0
0
1
1
1
5
9
0
0
1
0
0
14
2
30
16
50
33
0
0
9
1
10
13
0
21
50
2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(S)-2
(R)-2
(R)-2
(R)-2
(R)-2
0
92
0
0
>99.8
0
0
0
0
>99.8
>99.8
>99.8
>99.8
>99.8
0
0
>99.8
0
0
84
>99.8
90
92
92
91
0
0
84
>99.8
70
73
0
88
93
>99.8
>99.8
45
>99.8
>99.8
>99.8
ꢀ23
96
Europa Lipase-13
Europa Lipase-14
Europa Lipase-21
Julich Lipase-RN
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
1
3
2
2
49
11
41
7
6
50
Sigma Lipase-PPL
Sepracor Lipase-OF
Biocatalysts Lipomod-200
Boehringer Chirazyme-L2-c2
Boehringer Chirazyme-L5
Amano Lipase PS30 on Accurel
Novo Lipolase-30T
40
77
>99.8
Novo Lipozym-IM60
Novo Novozym-435
˚
Reactions were conducted with 50 mM (RS)-1, 100 mM (2 mol equiv) isopropenyl acetate and 125 mg/mL 4 A molecular sieves in dry MTBE, using
25 mg/mL (free-form) or 100 mg/mL (immobilized) biocatalyst, with stirring at 400 rpm at 40 ꢁC for 20 h. Conversions and enantioselectivities were
determined by HPLC.
In view of the suboptimal results obtained with vinyl, iso-
propenyl and ethyl acetates, a variety of other acyl donors
were screened for the enantioselective (N)-acylation using
Novozym 435 as catalyst (Table 4). Of the acyl donors
tested, diethyl oxalate and ethyl methoxyacetate were nota-
ble in furnishing highly enantiopure (>99% ee) (S)-1 and
with (RS)-1, and low enzyme activity, presumably due to
inactivation of the enzyme by methanol released during
the reaction. A screening of solvents for the N-acylation
of (RS)-1 with ethyl 2-methoxyacetate revealed MTBE as
the best solvent, which despite promoting extensive precip-
itation of the corresponding (R)-2-methoxyacetamide
product gave almost complete conversion of (R)-1 and high
enantiopurities (>99% ee) for both (S)-1 and (R)-2b at sub-
strate loads up to 251 g/L (Table 5). Diethyl ether and hex-
ane were also promising solvents, although furnishing
lower enantiopurities (93–99%) for the desired (S)-amine,
while also incurring conversions above 50%. Incomplete
reactions were observed with dichloromethane and acetoni-
trile as solvents.
(
R)-2 with 0.6 mol equiv of the acyl donor at close to com-
plete (50%) the conversion with very little background
reaction (<1%). Ethyl 2-methoxyacetate was selected over
the former acyl donor since the formation of mono- and
bis-oxamides complicated work-up and recovery proce-
dures. It should be noted that methyl 2-methoxyacetate
was also tested but found to be unsuitable as an acyl donor
due to significant (>5%) non-selective background reaction

I. I. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 1330–1337
Table 3. Lipase-mediated enantioselective N-acylation of (RS)-1 with ethyl acetate
1333
Biocatalyst
[Biocatalyst]
Reaction medium (v/v)
Conversion
(%, HPLC)
Amide product
ee (amide)
(%, HPLC)
(
mg/mL)
Europa Lipase-14
25
25
Neat EtOAc
Neat EtOAc
Neat EtOAc
Neat EtOAc
Neat EtOAc
Neat EtOAc
Neat EtOAc
30
38
50
20
17
51
53
28
36
53
25
26
52
54
16
18
52
12
3
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
66
59
76
50
5
75
70
92
90
87
93
50
88
85
98
95
97
98
51
97
95
>99.8
>99.8
>99.8
70
0
98
98
89
94
97
88
>99.8
95
Meito Sangyo Lipase-TL
Boehringer Chirazyme-L2-c2
Amano Lipase PS30/Accurel
Amano Lipase PS30 in Sol–gel
Novo Novozym-435
100
100
100
100
100
25
CALB on Celite
Europa Lipase-14
Neat EtOAc + 4AMS
Neat EtOAc + 4AMS
Neat EtOAc + 4AMS
Neat EtOAc + 4AMS
Neat EtOAc + 4AMS
Neat EtOAc + 4AMS
Neat EtOAc + 4AMS
1:9 EtOAc–MTBE
1:9 EtOAc–MTBE
1:9 EtOAc–MTBE
1:9 EtOAc–MTBE
1:9 EtOAc–MTBE
1:9 EtOAc–MTBE
1:9 EtOAc–MTBE
1:9 EtOAc–DCM
1:9 EtOAc–DCM
1:9 EtOAc–DCM
1:9 EtOAc–DCM
1:9 EtOAc–DCM
1:9 EtOAc–DCM
1:9 EtOAc–DCM
1:9 EtOAc–hexane
1:9 EtOAc–hexane
1:9 EtOAc–hexane
1:9 EtOAc–hexane
1:9 EtOAc–hexane
1:9 EtOAc–hexane
1:9 EtOAc–hexane
Meito Sangyo Lipase-TL
Boehringer Chirazyme-L2-c2
Amano Lipase PS30/Accurel
Amano Lipase PS30 in Sol–gel
Novo Novozym-435
25
100
100
100
100
100
25
CALB on Celite
Europa Lipase-14
Meito Sangyo Lipase-TL
Boehringer Chirazyme-L2-c2
Amano Lipase PS30/Accurel
Amano Lipase PS30 in Sol–gel
Novo Novozym-435
25
100
100
100
100
100
25
51
54
2
2
10
1
CALB on Celite
Europa Lipase-14
Meito Sangyo Lipase-TL
Boehringer Chirazyme-L2-c2
Amano Lipase PS30/Accurel
Amano Lipase PS30 in Sol–gel
Novo Novozym-435
25
100
100
100
100
100
25
0
18
18
10
5
27
9
6
24
27
CALB on Celite
Europa Lipase-14
Meito Sangyo Lipase-TL
Boehringer Chirazyme-L2-c2
Amano Lipase PS30/Accurel
Amano Lipase PS30 in Sol–gel
Novo Novozym-435
25
100
100
100
100
100
CALB on Celite
96
˚
Reactions were conducted with 50 mM (RS)-1, with or without 125 mg/mL 4 A molecular sieves in the appropriate solvent mixture, using 25 mg/mL (free-
form) or 100 mg/mL (immobilized) biocatalyst, with stirring at 400 rpm at 40 ꢁC for 20 h. Conversions and enantioselectivities were determined by HPLC.
Two preparative-scale resolutions of (RS)-1 were per-
formed at a 0.19 and 0.25 molar scale using Novozym
Table 4. Novozym 435-mediated enantioselective N-acylation of (RS)-1
435 as catalyst, ethyl 2-methoxyacetate as acyl donor and
Acyl donor
Conversion Amide
ee (amine)
ee (amide)
MTBE as solvent. Both reactions were run close to comple-
tion with >99% conversion of (R)-amine and, respectively,
provided 23.7 and 29.7 g (92% and 90% isolated yield) of
(R)-2b as a white crystalline solid with >99% potency and
>99.8% ee, and 17.6 and 22.1 g (95% and 91% isolated
yield) of (S)-1 as a light yellow/orange oil with 100% and
(%, HPLC) product (%, HPLC) (%, HPLC)
Propyl acetate
Prop-2-yl acetate
Butyl acetate
Triacetin
Propyl butyrate
Ethyl valerate
Ethyl (S)-lactate
Ethyl glycolate
Diethyl oxalate
Ethyl
18
22
16
40
13
24
39
41
48
13
(S)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(S)-2
7
13
5
60
0
14
51
34
94
ꢀ4
>99.8
>99.8
>99.8
93
95
51
100% potency and 99.8% and 99.2% ee.
81
Candida antarctica lipase B (CALB) was also immobilized
on surface-treated Celite R633 (as described in Section 3)
and the immobilized enzyme evaluated in the resolution
process as an alternative to Novozym 435. The resolution
of 2.5 mmol of racemic amine at ca. 140 g/L in MTBE with
0.6 mol equiv of ethyl 2-methoxyacetate and a 10% w/w
load (with respect to amine substrate) of CALB, provided
>99.8
>99.8
23
trichloroacetate
Ethyl levulinate
Ethyl
6
49
(S)-2
(R)-2
ꢀ7
>99.8
99.1
99.2
2-methoxyacetate
(
S)-1 and (R)-2b in 95% and 96% isolated yield, 100% and
Reactions were conducted with 250 mM (RS)-1 and 150 mM (0.6 mol
equiv) of acyl donor in dry MTBE, using 10 mg/mL Novozym 435, with
stirring at 400 rpm at 40 ꢁC for 20 h. Conversions and enantioselectivities
were determined by HPLC.
100% potency and 99.3% and >99.8% ee, respectively, with
an overall conversion of >49% after 2 d. Thus, the in-house
prepared immobilized catalyst appeared to be comparable

1334
I. I. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 1330–1337
Table 5. Novozym 435-mediated enantioselective N-acylation of (RS)-1
Solvent
[Substrate] (M)
[Acyl donor] (M)
Conversion (%, HPLC)
ee (S)-1 (%, HPLC)
ee (R)-2 (%, HPLC)
Methyl t-butyl ether
Methyl t-butyl ether
Diethyl ether
Diethyl ether
Dichloromethane
Dichloromethane
Hexane
0.63
1.26
0.63
1.26
0.63
1.26
0.63
1.26
0.70
0.38
0.77
0.38
0.77
0.38
0.77
0.38
0.77
0.44
59
56
62
63
33
43
47
56
44
99
99
96
99
36
54
65
93
69
>99.8
>99.8
>99.8
>99.8
>99.8
>99.8
>99.8
>99.8
>99.8
Hexane
Acetonitrile
Reactions were conducted in dry solvent using 0.6 mol equiv of ethyl 2-methoxyacetate, with 10% w/w of Novozym 435 with respect to amine substrate,
with stirring at 400 rpm at 40 ꢁC for 20 h. Conversions and enantioselectivities were determined by HPLC. Precipitation of (R)-2-methoxyamide was
observed in the case of MTBE, diethyl ether and hexane, while no precipitation was observed for dichloromethane or acetonitrile.
to or better than Novozym 435, and may provide a more
economic alternative to the commercial catalyst.
amide, 13.9 min for (R)-2-methoxyacetamide and
16.7 min for (S)-methoxyacetamide. For chiral amine anal-
ysis, samples were diluted to 10 mM with methanol, an ali-
quot (20 lL) treated with a mixture of water (40 lL),
sodium bicarbonate (8 lL, 1 M) and Marfey’s reagent
(40 lL, 2% w/w in 1:1 acetonitrile–methanol) at 300 rpm,
40 ꢁC, 1 h. The solution was quenched with hydrochloric
acid (88 lL, 1 M), diluted to 0.2–0.5 mM with 1:1 aceto-
nitrile–water (0.98 mL) and filtered (PTFE, 0.45 lm) prior
to HPLC analysis as follows: Phenomenex Synergi Max-
RPA (4 lm, 0.2 · 5 cm) column; elution with 10–100% B
over 0–8 min, then hold at 100% B over 8–10 min; solvent
A was 8:2 water–methanol containing 0.05% TFA and the
solvent B was 8:2 acetonitrile–methanol containing 0.05%
TFA; 0.6 mL/min flow rate; 25 ꢁC column temperature;
A small-scale resolution of (RS)-1 was also effected with
Amano Lipase PS30 immobilized on Accurel polypropyl-
ene as catalyst. The resolution of 2.5 mmol of racemic
amine at ca. 140 g/L in MTBE with 0.6 mol equiv of ethyl
2
-methoxyacetate and a 40% w/w load (with respect to
amine substrate) of lipase, provided (S)-1 and (R)-2b in
1% and 91% isolated yield, 100% and >99% potency
9
and 98.4% and >99.8% ee, respectively, with an overall
conversion of >49% after 2 d. Thus, Amano Lipase PS30-
Accurel appeared to be a good alternative to CALB/Nov-
ozym-435, although it is required at 40% w/w of substrate
load as opposed to 10% w/w for the latter biocatalysts.
20 lL injection; 340 and 350 nm detection; the retention
times were for 7.6 min for (R)-amine and 8.0 min for (S)-
amine. NMR spectra were recorded on a Bruker-300 or
Jeol-400 spectrometers using deuterochloroform as solvent.
Optical rotations were recorded on a Perkin–Elmer 241
digital polarimeter at 20 ꢁC using the sodium D line.
3
. Experimental
3
.1. General
Immobilized Candida antartica lipase B (Novozym 435)
was purchased from Novozymes, Davis, CA. Other lipases,
esterases and proteases (Tables 1–3) were purchased from
Amano Enzyme Company, Biocatalysts, Boehringer,
Julich, Meito Sangyo and Sigma–Aldrich. Racemic and
enantiopure amine 1 was provided by Discovery Chemis-
try, Bristol-Myers Squibb Pharmaceutical Research Insti-
tute, Lawrenceville. Ethyl 2-methoxyacetyate and all
other chemicals and solvents were purchased from
Sigma–Aldrich.
3.3. Screening of hydrolases for the enantioselective acyl-
ation of (RS)-1
3.3.1. Immobilization of subtilisin and alcalase. Subtilisin
solution (1 mL, 50 mg/mL in 0.1 M potassium phosphate,
pH 7.0, containing 0.1 M sodium sulfate) was mixed with
Celite R633 (1 g) and the damp powder dried in air at rt
for 24 h. The immobilizate was obtained as a free-flowing
powder (yield 1.08 g). For Alcalase, the enzyme solution
(
(
0.8 mL) was mixed with a potassium phosphate stock
0.1 mL, 1 M, pH 7.0) and sodium sulfate stock (0.1 mL,
3
.2. Analytical methods
1
M). The solution was then mixed with Celite R633 (1 g)
Reactions were followed by chiral RP-HPLC on Shimadzu
LC-10 systems: For amide analysis, samples were diluted to
and the damp powder dried in air at rt for 24 h. The immo-
bilizate was obtained as a free-flowing powder, yield 1.16 g.
0
.2–0.5 mM with methanol and filtered (0.2 lm PTFE),
prior to HPLC analysis as follows: Daicel Chiralpak AS-
RH (5 lm, 0.46 · 15 cm) column; elution with 30% v/v B
over 0–10 min, then 30–60% v/v B over 10–20 min; solvent
A was 8:2 water–methanol containing 0.05% TFA and sol-
vent B was 8:2 acetonitrile–methanol containing 0.05%
TFA; 0.7 mL/min flow rate; ambient column temperature;
3.3.2. Enantioselective acylation of (RS)-1. In a typical
reaction, a mixture of (RS)-1 (50–500 mM), acyl donor
(0.3–1.0 M or neat), biocatalyst (10, 25 or 100 mg/mL, or
indicated amount) with or without molecular sieves (100–
150 mg/mL) in a 2 mL glass vial was incubated at 40 ꢁC,
with stirring at approximately 400 rpm for the indicated
time period.
2
0 lL injection; 210 and 220 nm detection; retention times
were 9.6 min for (R)-acetamide, 11.2 min for (S)-acet-

I. I. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 1330–1337
1335
3.4. Immobilized amano lipase PS30-mediated resolution
of (RS)-1
3.5.2. CALB-Celite-mediated resolution of (RS)-1. CALB-
Celite (0.41 g, 10% w/w of substrate input) was added to a
mixture of (RS)-1 (0.50 g, 2.5 mmol), ethyl 2-methoxyace-
tate (0.18 g, 1.5 mmol, 0.6 mol equiv) and MTBE (3 mL)
in a 6 mL vial, after which the mixture stirred at rt for
2 days. The reaction mixture [showing some precipitation
of (R)-2b] was diluted with ethanol (ca. 2.0 mL) to dissolve
the 2-methoxyacetamide precipitate, filtered, and the bio-
catalyst washed with 2:3 ethanol–MTBE. The combined
organic phases were evaporated at 40 ꢁC to yield a light-
orange semi-solid, which was suspended in water (3 mL)
and titrated with aqueous sulfuric acid (1 M) to pH 2.5.
The resulting suspension was stirred at rt for 0.5 h, and
then filtered. The filter cake was suspended in water
(2 mL). The pH was adjusted to 2.5 with sulfuric acid
(0.1 M) and the mixture then stirred at 200 rpm at rt for
0.5 h, after which it was filtered. The cake was washed with
aqueous sulfuric acid (2 · 2 mL, 10 mM), then water
(3 · 2 mL), filter dried in air at rt for 1 h, then dried under
vacuum over Drierite at rt for 18 h, to give (R)-2b as an off-
white crystalline solid: 0.33 g, 101% potency (HPLC), 96%
Amano lipase PS-30 was immobilized on Accurel polypro-
3
9
pylene as described earlier. Amano PS 30-Accurel (0.2 g,
0% w/w of substrate input) was added to a mixture of
4
(
(
RS)-1 (0.5 g, 2.5 mmol) and ethyl 2-methoxyacetate
0.18 g, 1.5 mmol, 0.6 mol equiv) and MTBE (3 mL) in a
6
2
mL vial, and the mixture stirred at rt for 2 days. After
days, the reaction mixture [showing extensive precipita-
tion of (R)-2b] was diluted with methanol (ca. 1.5 mL) to
dissolve the amide precipitate, then filtered to remove the
biocatalyst and washed with 3:1 MTBE–methanol. The
organic phases were combined and rotary evaporated at
4
0 ꢁC to furnish a light-orange semi-solid. This was sus-
pended in water (3 mL) and titrated with aqueous sulfuric
acid (1 M) to pH 2.5. The suspension was stirred at rt for
0
.5 h, and then filtered. The cake was suspended in water
(
(
2 mL) and the pH adjusted to 2.5 with sulfuric acid
0.1 M). The suspension was filtered, after which the cake
was washed with aqueous sulfuric acid (2 · 2 mL,
2
0
1
0 mM), then water (3 · 2 mL), and the cake filter dried
yield, ee >99.8%; ½aꢁ ¼ þ18:5 (c 2.0, methanol); LC–MS
D
in air at rt for 1 h, then dried under vacuum over Drierite
and NMR data identical to those of compound isolated
in Section 3.6.1. The aqueous extracts were pooled and
extracted with MTBE (3 · 3 mL), the pH of the aqueous
phase adjusted to 9.75 with sodium hydroxide (1 M), and
the resulting emulsion extracted with MTBE (3 · 3 mL).
The MTBE extract was washed with brine (3 · 3 mL),
dried over anhydrous magnesium sulfate, filtered, then
evaporated at 50 ꢁC to furnish (S)-1 as a pale yellow oil:
0.24 g, 102% potency (HPLC), 95% yield, ee 99.3%;
at rt, 24 h, to give (R)-2b as an off-white crystalline solid:
0
.31 g, 99% potency (HPLC), 91% yield, ee >99.8%;
20
½
aꢁ ¼ þ20:2 (c 2.7, methanol); LC–MS and NMR data
D
are identical to those of compound isolated in Section
3
.6.1. The aqueous acidic extracts were pooled and ex-
tracted with MTBE (3 · 3 mL), the pH of the aqueous
phase adjusted with sodium hydroxide (1 M) to pH 9.75,
the resulting emulsion extracted with MTBE (3 · 3 mL),
the organic layer washed with brine (3 · 3 mL) then dried
over anhydrous magnesium sulfate. Filtration and evapo-
ration at 50 ꢁC provided (S)-1 as a pale yellow oil: 0.23 g,
2
0
½aꢁ ¼ ꢀ25:6 (c 2.1, methanol); LC–MS and NMR data
D
identical to those of compound isolated in Section 3.6.1.
1
00% potency (HPLC), 91% yield, ee 98.4%;
3.6. Preparative-scale Novozym 435-mediated resolution of
(RS)-1
2
0
½
aꢁ ¼ ꢀ23:9 (c 2.8, methanol); LC–MS and NMR data
D
identical to those of compound isolated in Section 3.6.1.
3.6.1. Reaction 1. (RS)-1 (38 g, 190 mmol), ethyl 2-meth-
oxyacetate (13.5 g, 114 mmol, 0.6 mol equiv, 20 mol %
excess) and MTBE (115 mL) were added in sequence to a
3
.5. Immobilized CALB-mediated resolution of (RS)-1
.5.1. Immobilization of CALB on Celite R633. 3-Glyci-
250 mL PTFE flask, followed by Novozym-435 (3.8 g,
3
10% w/w of substrate input), and the flask sealed and sha-
ken at 200 rpm, 35 ꢁC, 42 h. The reaction mixture [showing
extensive precipitation of (R)-2b] was diluted with metha-
nol (50 mL) to dissolve the precipitate formed, filtered
and the biocatalyst washed with 3:1 MTBE–methanol.
The combined organic phases were evaporated at 40 ꢁC
to give a sticky light-orange solid, which was suspended
in water (150 mL) and titrated with aqueous sulfuric acid
(2 M) to pH 2.5, and the resulting suspension filtered.
The filter cake was suspended in water (50 mL) and the
pH adjusted to 2.5 with sulfuric acid (2 M), after which
the suspension filtered. The filter cake was washed with
aqueous sulfuric acid (50 mL, 10 mM), then water
(3 · 50 mL) and the cake filter dried in air at rt for 1 h, then
dried under vacuum over Drierite at rt for 24 h, to give (R)-
2b as an off-white crystalline solid: 23.7 g, 99% potency
(HPLC), 92% yield, ee >99.8%; mp 131–135 ꢁC (decomp);
doxypropyltrimethoxysilane (0.69 g, 2.1 mmol) in THF
2 mL) was added to a mixture of poly(ethylene glycol)-
bl-poly(propylene glycol)-bl-poly(ethylene glycol) (1 g,
.53 mmol, MW = 1900), zirconium(IV) chloride (20 mg)
(
0
and THF (4 mL), and the solution stirred at 400 rpm at
rt for 15 min, after which the temperature raised to 70 ꢁC
over 1 h and stirring continued at reflux for 20 h. A portion
of the solution (5.0 mL, ca. 1.25 g of solids) was diluted
with ethanol (6 mL) and applied onto Celite R-633 (10 g,
powdered). The wet powder was dried in air at rt for 5 h,
then cured at 120 ꢁC for 20 h to give ca. 11.3 g of modified
Celite R633. A solution of CALB (0.6 g in 4 mL of ice-cold
0
.2 M sodium phosphate, pH 7.0, containing 10 mM each
of calcium and magnesium acetates) was coated onto the
modified Celite R633 (4 g), the wet powder was dried in
air at rt for 5 h, then under vacuum, over Drierite at rt
for 20 h. This yielded 4.90 g of immobilized CALB (ca.
2
D
0
+
½aꢁ ¼ þ21:5 (c 3.0, methanol); LC–MS (ESI ) m/z
1
1
2.2% w/w CALB loading) as a free-flowing off-white pow-
(%) = 273 ([M+H], 100), 295 ([M+Na], 27); H NMR
der. The specific activity of the immobilizate was 5.3 times
that of lyophilized CALB in the resolution of (RS)-1.
(CDCl ) d 1.53 (d, 3H, 2-H), 3.35 (s, 3H, CH ), 3.62 (s,
3
3
0
2H, CH O), 5.02 (q, 1H, 1-H), 7.29 (d, 1H, 6 -H), 7.33
2

1336
I. I. Gill et al. / Tetrahedron: Asymmetry 18 (2007) 1330–1337
0
0
0
(
(
dd, 1H, 5 -H), 7.45 (d, 1H, 4 -H), 7.51 (s, 1H, 2 -H), 8.33
Acknowledgement
1
3
br d, 1H, NH) ppm; C NMR (CDCl ) d 22.6, 51.0, 57.2,
3
7
3.4, 123.1, 126.8, 130.5, 130.9, 132.3, 147.2, 170.4 ppm.
The author would like to acknowledge Dr. Robert Walter-
mire for reviewing this manuscript and making valuable
suggestions.
The aqueous acidic extracts were pooled and extracted with
MTBE (3 · 50 mL). The pH of the aqueous phase was
adjusted with sodium hydroxide (5 M) to pH 9.75. The
resulting emulsion was extracted with MTBE (3 · 50 mL)
and the combined MTBE extracts washed with brine
(
2 · 25 mL) then dried over anhydrous magnesium sulfate.
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[
1
1
1
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1
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