Efficient asymmetric synthesis of 1-cyano-tetrahydroisoquinolines from lipase dual activity and opposite enantioselectivities in α-aminonitrile resolution

Article abstract of DOI:10.1002/chem.201402615

Dual promiscuous racemization/amidation activities of lipases leading to efficient dynamic kinetic resolution protocols of racemic α-aminonitrile compounds are described. α-Amidonitrile products of high enantiomeric purity could be formed in high yields. Several lipases from different sources were shown to exhibit the dual catalytic activities, where opposite enantioselectivities could be recorded for certain substrates. Dynamic chemistry: Dual promiscuous racemization/amidation activities of lipases leading to efficient dynamic kinetic resolution protocols of racemic α-aminonitrile compounds are described. α-Amidonitrile products of high enantiomeric purity could be formed in high yields. Several lipases from different sources were shown to exhibit the dual catalytic activities, in which opposite enantioselectivities could be recorded for certain substrates (see scheme).

Full text of DOI:10.1002/chem.201402615

DOI: 10.1002/chem.201402615
Communication
&
Dynamic Chemistry
Efficient Asymmetric Synthesis of 1-Cyano-
tetrahydroisoquinolines from Lipase Dual Activity and Opposite
Enantioselectivities in a-Aminonitrile Resolution
Morakot Sakulsombat,[a] Pornrapee Vongvilai,[a, b] and Olof Ramstrçm*[a]
organic synthesis. These enzymes are commercially available
Abstract: Dual promiscuous racemization/amidation activ-
ities of lipases leading to efficient dynamic kinetic resolu-
tion protocols of racemic a-aminonitrile compounds are
described. a-Amidonitrile products of high enantiomeric
purity could be formed in high yields. Several lipases from
different sources were shown to exhibit the dual catalytic
activities, where opposite enantioselectivities could be re-
corded for certain substrates.
from many sources, show high tolerance to many reaction con-
ditions, and display broad substrate specificities.[13,14] Lipases
are also known to be promiscuous enzymes, and have been
used in a variety of transformations,[15–28]
A particularly useful methodology to enantiopure com-
pounds relies on dynamic kinetic resolution (DKR), a process
based on the combination of in situ racemization and kinetic
resolution.[29–33] The main challenge in the accomplishment of
efficient DKR processes is finding racemization and kinetic-res-
olution processes that are mutually compatible under suitable
reaction conditions.
Enzyme-catalyzed chemical reactions have witnessed concur-
rent growth on both the laboratory and industrial scales in
recent years, generally due to high reaction efficiencies and
low negative environmental impact.[1–6] Enzymes are recog-
nized as efficient catalysts for synthetic transformations, typi-
cally associated with high chemo-, regio-, and enantioselectivi-
ties, and have been used in preparations of key intermediates
of high enantiomeric purities in, for example, the synthesis of
pharmaceutically active species.[4,7] Although enzymes are
highly specific to their catalytic transformations and substrate
acceptances, it is highly challenging for enzymologists and or-
ganic chemists to extend the enzyme catalytic scope in organ-
ic synthesis. Owing to directed evolution methodologies, en-
zymes can, for example, often be modified to better suit cer-
tain transformations, reaction conditions, or classes of com-
pounds.[8] Furthermore, discovery of new activities for a specific
enzyme, known as enzyme catalytic promiscuity, has become
increasingly important, generally based on detailed under-
standing of the catalytic mechanisms of the enzymes.[3,6,9–13]
Lipases, belonging to the hydrolase class of enzymes, pos-
sess several advantageous features and are widely applied in
Recently, we have reported a promiscuous dual activity of
the lipase from Burkholderia cepacia, where the enzyme dis-
played both amidation and racemization activities towards N-
methyl a-aminonitriles.[24] This resulted in the dynamic kinetic
resolution of the corresponding N-methyl a-acetamidonitriles
of high enantiomeric purities in high yields, where both steps
were catalyzed by the same enzyme. Asymmetric synthesis of
a-aminonitriles and their derivatives is of high interest in or-
ganic synthesis, because these compounds can be transformed
into optically active a-amino acids and pharmaceutically inter-
esting compounds.[34,35]
Herein, the scope of the promiscuous dual function of lipas-
es in dynamic kinetic asymmetric resolution protocols is pre-
sented. Unexpectedly, it could be shown that lipases from dif-
ferent sources exhibited significantly different activities toward
a-aminonitriles, providing the desired amide products with op-
posite configurations (Scheme 1). In addition, cyclic substrates
based on the 1,2,3,4-tetrahydroisoquinoline motif proved to be
[a] Dr. M. Sakulsombat, Dr. P. Vongvilai, Prof. Dr. O. Ramstrçm
KTH Royal Institute of Technology Department of Chemistry
Teknikringen 30, 10044 Stockholm (Sweden)
E-mail: ramstrom@kth.se
[b] Dr. P. Vongvilai
Present address: BioNet-Asia Co., Ltd. 19 Soi Udomsuk 37
Sukhumvit 103 road, Bangkok 10260 (Thailand)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402615.
ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of Creative Commons Attri-
bution NonCommercial-NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
Scheme 1. Promiscuous dual activity of lipases resulting in dynamic kinetic
asymmetric resolution of a-aminonitriles in a one-pot process, in which ste-
reospecific amidation operates in sequel to racemization.
Chem. Eur. J. 2014, 20, 11322 – 11325
11322 ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
especially applicable to the concept, resulting in products of
high enantiopurity in high yields.
1H NMR spectroscopy and chiral HPLC. Unexpectedly, amide
products 2a–c formed under the same reaction conditions
using the two different lipase preparations, Novozyme 435 and
lipase PS-C I, provided the opposite absolute configuration, re-
spectively, in all cases (entries 1–6, Table 1). For the enzyme-
catalyzed reactions using Novozyme 435, amide products (À)-
2a, and (À)-2c were produced in very good to excellent yields
(89–94%) and very good ee values (83–89%). Amide product
(À)-2b was formed at lower yield (30%) under these condi-
tions, but with an excellent enantiopurity (97% ee). Nitrile 3
was, in this case, formed as a by-product, resulting from the re-
action between aminonitrile 1b and (4-methoxyphenyl)metha-
nimine, in turn formed from compound 1b during the enzy-
matic reaction.[37] For the somewhat larger amide product (À)-
2d, the contrary effect was instead recorded, quantitatively
formed from rac-1d but with lower resulting enantiomeric
excess (37%). This is likely due to an impaired racemization
step in this case due to the larger substrate structure.
The lipase-catalyzed racemization and asymmetric amidation
was first applied to the transformation of a-aminonitriles 1a–
d. 1-Amino-2-(4-fluorophenyl) acetonitrile 1c was selected as
a candidate for the initial lipase screening (entries 5–8, Table 1).
Four different lipase preparations were evaluated: Novo-
zyme 435 (immobilized lipase CAL-B from Candida antarctica)
lipase PS from Burkholderia cepacia, lipase PFL from Pseudomo-
nas fluorescens, and immobilized PS-C I from Burkholderia cepa-
cia. Of these, only reactions catalyzed by Novozyme 435 and
lipase PS-C I provided good conversions (95%) to amide prod-
ucts 2c at room temperature (entries 5–6), whereas no prod-
ucts were formed by using the PS and PFL preparations. Rais-
ing the temperature to 408C resulted in only marginally im-
proved conversions in the latter cases (11 and 4%).
Different acyl donors: phenyl acetate, 2,2,2-trifluoroethyl
acetate (TFEA),[36] and ethyl acetate, were also applied to the
enzyme-catalyzed reactions. The enantiomeric excess (ee) of
amide product 2c from the reactions using ethyl acetate was
slightly higher than using the other acyl donors. Moreover,
100 mg of lipase PS-C I and 50 mg of Novozyme 435 were suf-
ficient to catalyze the transformation of aminonitriles 1a–d to
the corresponding amide products 2a–d in similar reaction
times.
Using lipase preparation PS-C I, very similar yields/conver-
sions as for Novozyme 435 were obtained, and amide products
(+)-2a, (+)-2c, and (+)-2d were produced in 89–92% yield,
whereas product (+)-2b was formed at a lower rate (35%
yield). In the latter reaction from compound rac-1b, by-prod-
uct 3 was again formed. However, the enantiomeric excesses
of the products, of opposite configuration compared to the
products formed using Novozyme 435, were lower
After initial optimization of the dual lipase-catalyzed racemi-
zation and asymmetric amidation, the enzymatic reactions of
a-aminonitriles 1a–d were performed in tert-butyl methyl
ether (TBME) at room temperature by using ethyl acetate as
acyl donor. The results of the reactions were followed by
than for the CAL-B-catalyzed reactions (0–37% ee).
This effect is likely due to lower rates of the racemiza-
Table 1. Catalytic activities and stereoselectivities of lipase-catalyzed racemization and
asymmetric amidation of compounds 1a–d.[a]
tion step using PS-C I, compared with higher rates of
the asymmetric amidation step. Attempts to improve
the results by using silica gel as additive in the enzy-
matic reaction using lipase preparation PS-C I did not
result in any enhancement in these cases.[38]
To further evaluate the lipase catalytic activities in
racemization and asymmetric amidation, cyclic ami-
nonitrile structures were subsequently probed. Thus,
1-cyano-1,2,3,4-tetrahydroisoquinolines 4a and b,
representing important intermediates for isoquinoline
alkaloid syntheses,[39] were applied as aminonitrile
substrate candidates in the DKR process. Stereoselec-
tive acylation of this class of compounds, for exam-
ple, accomplished by using metal-based catalysts,[40]
organocatalysts,[41,42] and chiral auxiliaries,[43] results in
optically active isoquinoline Reissert-type products. In
the present case, Novozyme 435, PS-C I, PS, and PFL
were evaluated as racemizing and resolving agents,
and the enzymatic reactions were performed in TBME
at 408C by using phenyl acetate as acyl donor. The
Entry Product Lipase
Loading [mg] Time [days] Conversion [%][b] ee [%][c]
1
2
3[d]
4[e]
5
2a
2b
2c
Novozyme 435 50
PS-C I 100
Novozyme 435 50
PS-C I 100
Novozyme 435 50
9
10
12
12
10
10
4
98 (94)
97 (92)
33 (30)
38 (35)
95 (89)
95 (90)
11
(À) 83
(+) 15
(À) 97
(+) 52
(À) 89
(+) 37
(+) 56
(+) 62
(À) 37
0
6
PS-C I
PS
PFL
Novozyme 435 50
PS-C I 100
100
100
100
7[f]
8[f]
9
4
4
2d
10
10
quant. (93)
95 (89)
10
[a] Reactions carried out with compound 1 (0.05 mmol), ethyl acetate (3 equiv),
TMSCN (0.01 equiv), and lipase in TBME at RT. [b] Followed by chiral HPLC analysis
and 1H NMR spectroscopy. [c] Determined by chiral HPLC analysis on an OJ column;
see the Supporting information. [d] 63% a-Benylideneamino-a-phenylacetonitrile was
formed. [e] 59% a-Benylideneamino-a-phenylacetonitrile was formed. [f] Reactions
performed at 408C.
1
reactions were followed by H NMR spectroscopy and
chiral HPLC (Table 2).
To compare the catalytic activities, the different
enzyme preparations were first applied in equal
Chem. Eur. J. 2014, 20, 11322 – 11325
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11323 ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
These results are indicative of higher racemization rates with
higher enzyme loading, thereby improving the product enan-
tiopurity.
Table 2. Catalytic activities and stereoselectivities of lipase-catalyzed rac-
emization and asymmetric amidation of cyclic compounds 4a and b by
using different lipase preparations.[a]
Lipase-catalyzed racemization and asymmetric amidation of
6,7-dimethoxy-1-cyano-1,2,3,4-tetrahydroisoquinoline 4b using
different enzyme preparations displayed the same trend as the
results for compound 4a (entries 10–16, Table 2). Novo-
zyme 435 and lipase PS-C I (100 mg) provided complete trans-
formations to compound 5b within three days in 97 and 54%
ee, respectively, with the same product configuration (en-
tries 10 and 13). Similarly, lipase-preparation PFL exhibited
fairly good asymmetric transformation of compound 4b with
65% yield (60% ee) in three days. The result from lipase PS, on
the other hand, showed poor activities with respect to both
transformation and stereoselectivity, providing only 30% con-
version without any enantiomeric preference of product 5b
(entry 15, Table 2). The amount of enzyme loading for Novo-
zyme 435 was also varied, and the results indicate that 100 mg
of Novozyme 435 was optimal for the asymmetric transforma-
tion of compound 4b to 5b. When increasing the amount of
lipase preparation PS-C I to 200 mg, amide product 5b was
produced at quantitative conversion with 86% ee.
Entry Product Lipase
Loading
[mg]
Time
[days]
Conversion ee
[%][b]
[%][c]
1
5a
Novozyme 435 100
3
3
3
2
2
2
2
3
3
2
3
3
2
2
3
3
quant. (95)
quant.
quant.
95
99
quant.
97
95
95
87
39
47
60
75[d]
7
2
3
50
25
4
PS-C I
100
200
400
100
100
100
5
6
7
8
PS
26
9
PFL
68
48
97
73
64
54
86
0
10
11
12
13
14
15
16
5b
Novozyme 435 100
quant. (92)
quant.
quant.
97
quant.
30
50
25
100
200
100
100
PS-C I
In conclusion, it has been demonstrated that combined,
dual-function, lipase-catalyzed racemization and asymmetric
amidation can be efficiently used to produce different a-ami-
donitrile products in good yield and enantiopurity. The dual
function appears to be a general feature for several lipases,
where preparation using lipases from Candida, Burkholderia,
and Pseudomonas sp. all resulted in product formation. The
lipase preparations Novozyme 435, and PS-C I were most effi-
cient in the present cases. In addition, it could be shown that
the enantiopreferences of the enzyme preparations varied, and
noncyclic a-amidonitrile products of opposite configuration
were formed by using Novozyme 435 and PSC I, respectively.
Cyclic substrates were also evaluated, and 1-cyano-1,2,3,4-tet-
rahydroisoquinolines 4a–b were efficiently transformed by
using different lipase preparations. Novozyme 435 gave the
best results with excellent yield and enantiopurity. The product
configurations did not vary between the enzymes in this case.
These results showed that dual-function lipase promiscuity re-
sulted in simultaneous racemization and asymmetric amidation
of a-aminonitriles, thus providing a useful synthetic method to
optically active a-aminonitrile amide derivatives.
PS
PFL
65
60
[a] Reactions carried out with compound 1 (0.05 mmol), phenyl acetate
(3 equiv, 0.15 mmol), TMSCN (0.01 equiv), and lipase in TBME at 408C
[b] Followed by chiral HPLC analysis and 1H NMR spectroscopy; isolated
yields in parentheses [c] Determined by chiral HPLC analysis on an OD-H
column; see the Supporting information [d] SiO2 (10 mg) was added.
amounts (100 mg) under the same reaction conditions. In gen-
eral, the enzyme-catalyzed reaction rates from compounds 4a
and b were considerably higher than those for compounds
1a–d. Among the lipase preparations, Novozyme 435 and PS-
C I proved again to be better than either PS or PFL, in which
the reactions from compound 4a reached completion within
three days, providing Reissert-type product 5a in 99 and 39%
ee, respectively. In contrast to the results obtained for the non-
cyclic a-aminonitriles, the same absolute configuration were
obtained with both enzyme preparations (entries 1 and 4,
Table 2). Lipase preparations PFL and PS also exhibited dual
catalytic activities in the resolution of compound 4a, but their
activities were less efficient. Attempts to improve the results
by using silica gel were again performed for the enzymatic re-
action using lipase preparation PS-C I.[38] In this case, this re- Acknowledgements
sulted in higher enantiomeric excess of amide product 5a,
leading to 97% conversion and 75% ee (entry 7, Table 2). The
amount of enzyme loading was also screened in the enzymatic
reactions using Novozyme 435 and PS-C I. With decreasing
amounts of Novozyme 435, the catalytic rate remained un-
changed, but the enantiomeric purities decreased. For lipase
preparation PS-C I, the catalytic rate and the stereoselectivities
decreased dramatically upon lower enzyme loading. However,
the opposite effect was recorded with increased enzyme load-
ing, from 100 to 400 mg, when the enantiomeric excess of
product 5a increased from 39 to 60% (entries 4–6, Table 2).
This work was supported in part by the Swedish Research
Council and the Royal Institute of Technology.
Keywords: amidation
· asymmetric synthesis · dynamic
chemistry · enzymes · lipases · racemization
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Received: March 14, 2014
Published online on July 23, 2014
Chem. Eur. J. 2014, 20, 11322 – 11325
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11325 ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim