Optimization of 2-alkoxyacetates as acylating agent for enzymatic kinetic resolution of chiral amines

Article abstract of DOI:10.1016/j.tet.2018.05.032

In this study, the activity of acetic acid esters modified with electron withdrawing 2-alkoxy-groups was investigated as acylating agent in kinetic resolution (KR) of racemic amines. A homologous series of the isopropyl esters of four 2-alkoxyacetic acids (2-methoxy-, 2-ethoxy-, 2-propoxy- and 2-butoxyacetic acids) were prepared and investigated for enantiomer selective N-acylation, catalyzed by lipase B from Candida antarctica, under batch and continuous-flow conditions. In the first set of experiments, isopropyl 2-propoxyacetate showed the highest effectivity with all of the four racemic amines [(±)-1-phenylethylamine, (±)-4-phenylbutan-2-amine, (±)-heptan-2-amine and (±)-1-methoxypropane-2-amine] in the set enabling excellent conversions (≥46%) and enantiomeric excess values (ee ≥ 99%) with each amines in continuous-flow mode KRs under the optimized reaction conditions. In a second set of experiments, KRs of five additional amines – being substituted derivatives of (±)-1-phenylethylamine – further demonstrated the usefulness of isopropyl 2-propoxyacetate – being the best acylating agent in the first set of KRs – in KRs leading to (R)-N-propoxyacetamides with high ee values (≥99.8%).

Full text of DOI:10.1016/j.tet.2018.05.032

Accepted Manuscript
Optimization of 2-alkoxyacetates as acylating agent for enzymatic kinetic resolution of
chiral amines
Márk Oláh, Dániel Kovács, Gabriel Katona, Gábor Hornyánszky, László Poppe
PII:
S0040-4020(18)30551-9
10.1016/j.tet.2018.05.032
TET 29539
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 6 March 2018
Revised Date: 1 May 2018
Accepted Date: 11 May 2018
Please cite this article as: Oláh Má, Kovács Dá, Katona G, Hornyánszky Gá, Poppe Láó, Optimization
of 2-alkoxyacetates as acylating agent for enzymatic kinetic resolution of chiral amines, Tetrahedron
(2018), doi: 10.1016/j.tet.2018.05.032.
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ACCEPTED MANUSCRIPT
Graphical Abstrac
Optimization of 2-alkyloxyacetates as
acylating agent for enzymatic kinetic
resolution of chiral amines
Leave this area blank for abstract info.
Márk Oláha, Dániel Kovácsa, Gabriel Katonac, Gábor Hornyánszkya,b and László Poppea,b,c
aBudapest University of Technology and Economics, Budapest, Hungary; bSynBiocat Ltd, Budapest, Hungary;
cBabeș-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania

ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Optimization of 2-alkoxyacetates as acylating agent for enzymatic kinetic resolution
of chiral amines
Márk Oláha, Dániel Kovácsa, Gabriel Katonac, Gábor Hornyánszkya,b and László Poppea,b,c,
a Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, Budapest H-1111, Hungary
b SynBiocat Ltd., Szilasliget u. 3, Budapest H-1172, Hungary
c Biocatalysis and Biotransformation Research Centre, Babeș-Bolyai University of Cluj-Napoca, Arany János Str. 11, Cluj-Napoca RO-400028, Romania
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In this study, the activity of acetic acid esters modified with electron withdrawing 2-alkoxy-
groups was investigated as acylating agent in kinetic resolution (KR) of racemic amines. A
homologous series of the isopropyl esters of four 2-alkoxyacetic acids (2-methoxy-, 2-ethoxy-,
2-propoxy- and 2-butoxyacetic acids) were prepared and investigated for enantiomer selective
N-acylation catalyzed by lipase B from Candida antarctica under batch and continuous-flow
conditions. In the first set of experiments, isopropyl 2-propoxyacetate showed the highest
effectivity with all of the four racemic amines [(±)-1-phenylethylamine, (±)-4-phenylbutan-2-
amine, (±)-heptan-2-amine and (±)-1-methoxypropane-2-amine] in the set enabling excellent
conversions (≥46%) and enantiomeric excess values (ee ≥99%) with each amines in
continuous-flow mode KRs under the optimized reaction conditions. In a second set of
experiments, KRs of five additional amines – being substituted derivatives of (±)-1-
phenylethylamine – further demonstrated the usefulness of isopropyl 2-propoxyacetate – being
the best acylating agent in the first set of KRs – in KRs leading to (R)-N-propoxyacetamides
with high ee values (≥99.8%).
Available online
Keywords:
Kinetic resolution
Primary amine
Acyl donor
Alkoxyacetic acid esters
Continuous-flow biotransformation
2018 Elsevier Ltd. All rights reserved
.
———
Corresponding author. Tel.: +36-1-463-3299; fax: +36-1-463-3297; e-mail: poppe@mail.bme.hu

2
Tetrahedron
1. Introduction
ACCEPTED MANUSCRIPT
The production of chiral organic molecules1 in enantiopure
form is an important objective from physiological,2 economic,
and environmental3 aspects as well. Among the possible
technological solutions, kinetic resolution (KR)4,5 is a widely
used method which can afford almost enantiopure compounds
with a theoretical yield of 50% starting from racemates.6 To
distinguish between the two enantiomers of a racemate by this
method, use of an adequate chiral auxiliary namely a chiral
reagent,7 a chiral metal-ligand complex8 or an enzyme9 is
fundamental. Moreover, in many cases an effective KR could be
extended to a dynamic kinetic resolution (DKR) method by an in
situ combination with racemization of the residual enantiomer to
raise the theoretical yield up to 100%.10
To ensure the required enantiomer selectivity, enzymes as
biocatalysts offer the necessary chiral environment due to their
inherent chirality deriving from their building blocks, the chiral
amino acids.11 This initiated numerous studies in the past few
decades to apply many classes of enzymes (such as hydrolases,12
transferases,13 oxidoreductases14 or lyases15) to perform synthetic
organic reactions.16 Since cofactor-free enzymatic methods are
preferred for the large-scale production of chemicals, lipase-
catalyzed KRs gained popularity in the enantiomer separation of
alcohols, amines and their derivatives.17,18
Besides the
advantageous catalytic properties of lipases (substrate specificity,
activity, selectivity), their enhanced stability and easy recovery is
an indispensable issue in an effective and industrially applicable
synthetic process. Thus, different immobilization methods were
developed to keep these enzymes in active and stable form over
numerous recycling steps.19-21 After the proper technique is
chosen to immobilize an enzyme influencing activity of the
enzyme, there are further parameters which influence the
productivity of lipase-catalyzed KRs of amines, e.g. the type of
solvent or the water content of reaction components.22
Scheme 1. KR of racemic amines rac-1a-i using isopropyl esters of 2-
alkoxyacetic acids 2A-D as acylating agent catalyzed by CaLB-CV-T2-150
in batch mode
2. Results and Discussion
2.1. Synthesis of 2-alkyloxy acid isopropyl esters
First, the desired homologous series of alkoxyacetic acid
isopropyl esters 2A-D being potential acylating agents for kinetic
resolution (KR) of racemic amines were synthesized (Scheme 2).
The corresponding 2-alkyloxyacetic acids 5A,B were already
available, while 5C,D were prepared by reacting sodium
2-chloroacetate 4 with the corresponding alcohols (R-OH) in the
presence of sodium. Esterification of the acids 5A-D with
2-propanol using p-toluenesulfonic acid catalysis, followed by
vacuum distillation led to isopropyl esters 2A-D (Scheme 2).
Many investigations emphasized the key role of the acyl donor
in KR of amines. In the first DKR of (±)-1-phenylethylamine,
ethyl acetate was applied as acylating agent by Reetz and
Schimossek in 1996.23 Later, it was found that the modification
of acylating agent’s carboxylic acid moiety with electron
withdrawing groups could provide enhanced catalytic activity in
enzymatic KRs. It is important to note that esters of strong acids
(pKa<2.0) as acylating agents in KR could facilitate not just the
enzymatic but the chemical acylation of substrate during enzymic
resolution thereby lowering the enantiomeric purity of product
acetamide even under ambient conditions.24-27
In order to further increase the productivity of enzymatic KR
processes for amines by enzymatic acylation, we decided to
extend our previous study on the esters of 2-ethoxyacetic acid as
enhanced acylating agents for lipase-catalyzed KR of (±)-1-
phenylethylamine rac-1a.28 In this study enzymatic KRs of rac-
1a and eight other racemic amines [(±)-4-phenylbutan-2-amine
Scheme 2. Synthesis of 2-alkyloxyacetic acids 5A-D and their isopropyl
esters 2A-D (a obtained commercially, b synthesized as described earlier27)
rac-1b,
(±)-heptan-2-amine
rac-1c,
(±)-1-
methoxypropane-2-amine rac-1d, (±)-1-(4-nitrophenyl)ethan-1-
amine rac-1e, (±)-1-(4-chlorophenyl)ethan-1-amine rac-1f,
2.2. Comparison the activity of 2-alkoxyacetic acid esters 2A-D
in kinetic resolution of chiral amines 1a-d in batch mode
(±)-1-(4-bromophenyl)ethan-1-amine
phenylpropan-1-amine rac-1h,
rac-1g,
(±)-1-
(±)-1-(3,4-
dimethoxyphenyl)ethan-1-amine rac-1i] catalyzed by
a
The biocatalytic applicability of four 2-alkoxyacetic acid
esters 2A-D as acylating agents in the CaLB-CV-T2-150-
catalyzed KRs of the selected four racemic amines 1a-d was
studied in batch mode using shake flasks by comparison of the
achievable conversion and enantiomeric excess (ee) values of
formed (R)-2-alkoxyacetamides (ee(R)-3(a-d)(A-D)) (Scheme 1).
covalently immobilized lipase B from Candida antarctica
(CaLB-CV-T2-150) were performed using as acylating agents an
extended set of isopropyl alkoxyacetates 2A-D [isopropyl 2-
propoxy- (2C) and 2-butoxyacetate (2D), in addition to the
already known isopropyl 2-methoxy- (2A) and 2-ethoxyacetate
(2B)] under batch and continuous-flow conditions (Scheme 1).

ACCEPTED MANUSCRIPT
Figure 1. CaLB-CV-T2-150-catalyzed batch mode KR of rac-1a-d (0.778 M) with various acylating agents 2A-D (1.0 equiv., 0.778 M) using shake flasks
[Panel A: KR of rac-1a, Panel B: KR of rac-1b, Panel C: KR of rac-1c, Panel D: KR of rac-1d; Conditions – CaLB-CV-T2-150: 15.0 mg, dry toluene: 1.0 mL,
reaction temperature: 30 °C, shaking: 750 rpm, conversion and ee determined by GC after sampling directly from reaction mixture and treated by acetic
anhydride; Markers: conversions with 2A (ꢀ), 2B (■), 2C (●), 2D (▲), ee values for (R)-3dA (◊), (R)-3dB (□), (R)-3dC (○), (R)-3dD (∆)]
Analysis of the conversion values (Figure 1, sections A-D)
indicated similar tendency with all the four esters 2A-D for each
of the amines rac-1a-d. The 2-methoxyacetate 2A showed the
lowest activity while increased number of carbon atoms in the
alkoxy moiety of esters 2B-D led to enhanced acylating activity.
Among these four isopropyl esters, the one with 2-propoxy
moiety 2C proved to be optimal in terms of reaction rate with
amines rac-1a-d. Isopropyl 2-propoxyacetate 2C overcome the
performance of the already applied isopropyl 2-methoxyacetate
2A29 or isopropyl 2-ethoxyacetate 2B28 as acylating agent in
CaLB-catalyzed KRs of these amines. Even isopropyl 2-
buthoxyacetate 2D enabled higher efficiency in enzymatic N-
acylation of amines rac-1a-d than isopropyl 2-methoxyacetate
2A.
After the first series of experiments in shake flasks, the effect
of temperature on the activity and the selectivity of KRs of the
amines rac-1a-d was investigated in continuous-flow mode using
packed-bed columns filled with CaLB-CV-T2-150 as biocatalyst
and applying isopropyl 2-propoxyacetate 2C as the optimal
acylating agent in the shake flask KRs. Thus, a solution of
racemic amine (rac-1a-d) and ester (2C) in dry toluene was fed
to CaLB-CV-T2-150-filled column by a syringe pump at
different flow rates (50, 100, 200 µL min -1) and the column was
thermostated to 30, 40, 50 and 60 °C in an HPLC column
thermostat (Scheme 3).
Besides activity, enantiomer selectivity of the enzyme in KRs
is another important parameter to be compared with the different
acylating agents 2A-D. Thus, ee values of formed (R)-2-
alkoxyacetamides (ee(R)-3(a-d)(A-D)) were determined to characterize
the influence of acylating agents 2A-D on the enantiomer
selectivity. In case of the two aromatic ring-containing amines
rac-1a,b and the aliphatic heptan-2-amine rac-1c of comparable
size, the enantiomer selectivity was remarkable with ee values
over 99% even at the highest conversions (Figure 1, panels A-C).
In contrast, KR of 1-methoxypropan-2-amine rac-1d showed
slightly lower but acceptable enantiomer selectivity (ee >95%,
Figure 1, panel D), even if the conversion with isopropyl 2-
propoxyacetate 2C (being the most active acylating agent for rac-
1d) reached 49.6% (the theoretical limit in a fully selective KR is
50%).
Scheme 3. Kinetic resolution of racemic primary amines rac-1a-d using
isopropyl 2-propoxyacetate 2C as acylating agent catalyzed by CaLB-CV-
T2-150 in continuous-flow mode Kinetic resolution of amines rac-1a-
d in continuous-flow mode using isopropyl 2-propoxyacetate 2C
as acylating agent

4
Tetrahedron
Table 1. Optimized reaction conditions for continuous-flow mode KR of
Data for the continuous-flow mode KRs of amines rac-1a-d
ACCEPTED MANUSCRIPT
-1
racemic amines rac-1a-d with isopropyl 2-propxyacetate 2C as acylating
with ester 2C at 200 µL min flow rate are presented in Figure
2. In the whole temperature range, conversions of the
1-methoxypropan-2-amine rac-1d were the highest and at 60 °C,
the conversion from rac-1d with 2C (crac-1d= 47%) approached
the theoretical limit of a fully selective KR process (c= 50%).
agent
Subst. a T [°C]
ν
c
Y(R)-3(a-d)C ee(R)-3(a-d)C
E
[µL min-1] [%]
[%]
45
49
46
43
[%]
99.7
99.2
99.1
99.9
[-]
rac-1a
rac-1b
rac-1c
rac-1d
60
30
40
60
50
100
50
46
49
48
47
»200
»200
»200
»200
200
a Conditions: rac-1a-d 0.63 M, 2C 0.38 M (0.6 equiv.), column filled with
CaLB-CV-T2-150 (216 mg).
In Table 2, a thorough comparison is listed for various esters
used as acylating agents in amine KRs involving ethyl acetate
being the most often used acylating agent in such processes. It is
clearly apparent that presence of electron withdrawing 2-alkyloxy
groups in the acetates significantly enhance their acylating ability
in enzymatic KR for rac-1a,b (ca. 7-18 times) as compared to the
non-activated ethyl acetate. Use of isopropyl instead of ethyl
esters as acylating agent, led to the products (R)-3a,b with higher
ee values due to lowering the degree of non-selective chemical
acylation as undesired side-reaction.28
Figure 2. Continuous-flow mode KR of racemic amines rac-1a-d at different
temperatures using isopropyl 2-propoxyacetate 2C as acylating agent
[conditions: rac-1a-d 0.63 M, 2C 0.38 M (0.6 equiv.), flow rate 200 µL
min-1, column filling: CaLB-CV-T2-150 216 mg, markers: 1a (ꢀ), 1b (■), 1c
(▲), 1d (●)].
To broaden the substrate scope, KRs of further five racemic
amines [(±)-1-(4-nitrophenyl)ethan-1-amine rac-1e, (±)-1-(4-
chlorophenyl)ethan-1-amine rac-1f, (±)-1-(4-bromophenyl)ethan-
1-amine rac-1g, (±)-1-phenylpropan-1-amine rac-1h, (±)-1-(3,4-
dimethoxyphenyl)ethan-1-amine rac-1i] were investigated in
batch mode using isopropyl 2-propoxyacetate (2C) as acylating
agent (Scheme 1). The additional set of substrates were variously
substituted derivatives of (±)-1-phenylethylamine rac-1a. The
conversions from rac-1e-i and rac-1a with the best acylating
agent 2C reflecting to the relative activities are directly compared
in PanelA of Figure 3. The enantiomeric excess values of (R)-3e-
iC and (R)-3aC reflecting to the relative selectivities of the KRs
from the additional five amines rac-1e-i are shown in Pane B of
Figure 3. Only the p-chlorophenyl derivative rac-1f showed
higher activity than the rac-1a while the conversions of the four
other substrates (rac-1e,g-i) were lower than that of rac-1a. The
enantiomer selectivity of CaLB-CV-T2-150 for the derivatives
containing a halogen (chlorine or bromine) rac-1f,g was a bit
lower than that for rac-1a as indicated by the ee values of their
products (R)-3fC and (R)-3gC. After 8 h of reaction time, the
(R)-propoxyacetamides (R)-3e-iC were isolated and characterized
with high ee values (≥99.8%) (Table 3).
The KRs of amines rac-1b and rac-1c in continuous-flow
mode behaved quite similar, as indicated by the progress of their
temperature-conversion curves. This can be explained by
considering that rac-1b and rac-1c are flexible amines of
comparable size. In this series of experiments, KR of amine
rac-1a being a relatively rigid molecule with the direct binding of
the rigid aromatic ring to the center of asymmetry proved to be
the less active substrate for the covalently immobilized CaLB-
CV-T2-150 biocatalyst.
After finding that in the 30 – 60 °C temperature range the
highest conversions from amines rac-1a-d with acylating agent
2C could be achieved at 60 °C, the flow rate was fine-tuned in
the range of 50–200 µL min-1 to reach nearly the theoretical
conversions for all substrates (c ≥46%) with high enantiomeric
purities (ee(R)-3(a-d)C ≥99.1%) (Table 1).
Table 2. Comparison of various acylating agents in KRs from racemic amines rac-1a,b using CaLB-catalyzed reactions in batch mode (30 °C, shaken flask)18,28
Subst.
Acylating agenta
Concentration [M]
Time
[h]
Biocatalyst
c
ee(R)-3a,b
E
[%]
[%]
[-]
Acylating
Substrate
CaLB-type
Amount [mg]
agent (equiv.)
0.770 (2.0)
0.770 (2.0)
0.231 (0.6)
0.231 (0.6)
0.770 (2.0)
0.231 (0.6)
0.778 (1.0)
0.231 (0.6)
0.770 (2.0)
0.778 (1.0)
0.778 (1.0)
0.778 (1.0)
10.2d
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1ab
rac-1bc
rac-1bb
ethyl acetate
0.385
0.385
0.385
0.385
0.385
0.385
0.778
0.385
0.385
0.778
0.778
0.778
0.200
0.778
1
1
1
1
1
1
1
1
1
1
1
1
4
4
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
CV-T2-150
Novozym 435
CV-T2-150
50.0
50.0
50.0
50.0
50.0
50.0
15.0
50.0
50.0
15.0
15.0
15.0
300.0
15.0
2.0
4.1
99.6
99.8
99.7
99.8
99.0
>99.9
99.4
>99.9
>99.9
99.6
99.7
99.6
86
>200
»200
»200
»200
>200
»200
>200
»200
»200
»200
»200
»200
41
isopropyl acetate
isopropyl acetate
0.9
ethyl 2-methoxyacetate
6.5
ethyl 2-ethoxyacetate
14.4
7.6
2A
2A
17.5
16.0
36.1
26.1
29.2
21.9
51
2B
2B
2B
2C
2D
ethyl acetate
2C
0.778 (1.0)
46.8
99.9
»200
a 2A: isopropyl 2-methoxyacetate, 2B: isopropyl 2-ethoxyacetate, 2C: isopropyl 2-propoxyacetate, 2D: isopropyl 2-butoxyacetate, b Data obtained by Oláh et al.28
c Data obtained by González-Sabín et al.19 d Used as solvent (15 mL)

ACCEPTED MANUSCRIPT
Figure 3. CaLB-CV-T2-150-catalyzed KR of rac-1a,e-i (0.778 M) with isopropyl 2-propoxyacetate 2C (1.0 equiv., 0.778 M) in shaken flasks in batch mode
[Panel A: conversion values of KRs from rac-1a,e-i; Panel B: ee values of formed (R)-N-propoxyacetamides (R)-3aC and (R)-3(e-i)C (results for KR of rac-1a
were also shown in Figure 1, Panel A); conditions – CaLB-CV-T2-150: 15.0 mg, dry toluene: 1.0 mL, reaction temperature: 30 °C, shaking: 750 rpm, conversion
and ee were determined by GC after sampling directly from reaction mixture and derivatized with by acetic anhydride; markers: 1a (◊), 1e (□), 1f (ꢀ), 1g (▲),
1h (●) and 1i (■).]
Table 3. KR of racemic amines rac-1e-i with isopropyl 2-propoxyacetate 2C
(Karlsruhe, Germany), Merck (Darmstadt, Germany) and used as
received.
as acylating agent in batch modea
Subst. a
c
Y(R)-3(e-i)C
[%]
40
ee(R)-3(e-i)C
[%]
E
4.2. Methods
[%]
42.3
47.8
22.2
22.6
24.9
[-]
rac-1e
rac-1f
rac-1g
rac-1h
99.9
»200
»200
»200
»200
»200
TLC was carried out using Kieselgel 60 F254 (Merck) sheets.
Spots were visualized under UV light (Vilber Lourmat VL-6.LC,
254 nm) or after treatment with 5% ethanolic phosphomolybdic
acid solution and heating of the dried plates. The NMR spectra
were recorded in CDCl3 on a Bruker Avance 500 spectrometer
46
99.9
19
99.8
19
99.8
rac-1i
a
24
99.8
Conditions: rac-1e-i (0.778 M), 2C (0.778 M, 1.0 equiv.), CaLB-CV-T2
(15.0 mg), dry toluene (1.0 mL), reaction time: 8 h, reaction temperature:
30 °C, shaking: 750 rpm.
1
operating at 300 MHz or 500 MHz for H and 125 MHz for 13C,
and signals are given in ppm on the δ scale. Infrared spectra were
recorded on
wavenumbers of bands are listed in cm–1. Optical rotation was
measured on Perkin–Elmer 241 polarimeter at the -line of
a Bruker ALPHA FT-IR spectrometer and
3. Conclusions
D
Our study focused on the optimization of isopropyl
2-alkoxyacetates as acylating agent in enzymatic kinetic
resolution of chiral amines with the robust lipase B from Candida
antarctica. The study with a homologous series of isopropyl
2-alkoxyacetates 2A-D – ranging from 2-methoxy to 2-butoxy
derivatives – in CaLB-catalyzed KRs of racemic aromatic and
aliphatic primary amines in batch and continuous-flow modes
revealed isopropyl 2-propoxyacetate 2C as the most efficient
acylating agent providing higher activity than the previously
applied 2-methoxy- or 2-ethoxyacetic acid esters. The most
efficient ester 2C was applied and optimized as acylating agent in
KRs of four racemic amines rac-1a-d under continuous-flow
conditions leading to the enantioenriched forms of the four
valuable amines of industrial interest in high enantiomeric purity
(99.1% ≤ ee ≤ 99.9%) at nearly 50% conversions (46% ≤ c ≤
49%). The KRs of amines with 2C were extended to five further
racemic amines rac-1e-i in batch mode to yield enantioriched
(R)-2-propoxy-N-acetmides (R)-3e-iC with high ee (≥99.8%).
The results indicated the increased potential and applicability of
the most efficient isopropyl 2-propoxyacetate 2C in lipase-
catalyzed KRs.
sodium. The polarimeter was calibrated with measurements of
both enantiomers of menthol. Samples (20 µL) from kinetic
resolution reactions were diluted with ethanol (1000 µL) treated
with acetic anhydride (30 µL, at 60 °C at 750 rpm in a
thermostatted shaker, for derivatization of unreacted amines into
N-acetamides), dried over Na2SO4 and analyzed in parallel by
two GC equipment: an Agilent 5890 equipped with a Hydrodex
β-TBDAc column (Macherey-Nagel; 25 m×0.25 mm×0.25 µm,
film of heptakis-(2,3-di-O-acetyl-6-O-t-butyldimethylsilyl)-β-
cyclodextrin and an Agilent 4890 equipped with column
Hydrodex β-TBDM column (Macherey-Nagel; 25 m × 0.25 mm
×
0.25 µm, film of heptakis-(2,3-di-O-methyl-6-O-t-
butyldimethylsilyl)-β-cyclodextrin) [FID (250 °C), injector
(250 °C), H2 (12 psi, split ratio: 1:50].
Conversion (c) and enantiomeric excess (ee) were determined
by GC. Conversion was calculated using the equation
c= eeS×(eeS+eeP)-1 (where eeS is the ee of the substrate and eeP is
the ee of the product).30 The specific reaction rates in continuous-
flow systems (rflow
) were calculated using the equation
rflow = [P] × v/mB (where [P] [mol mL−1] is the molar
concentration of the product, v [mL min−1] is the flow rate and
mB [g] is the mass of the applied biocatalyst). The yields of the
isolated (R)-amides were related to the corresponding racemates
of substrates.31 Enantiomeric ratio (E) was calculated from c and
enantiomeric excess of the product (eeP) using the equation
E= ln[1−c(1+eeP)]/ln[1−c(1−eeP)].32 Due to sensitivity of E value
above 100 to small deviations of experimental errors, E values
calculated in the range of 100–200 were given as >100, those in
the range of 200–500 as >200 and above 500 as »200.
4. Experimental Section
4.1. Materials
CaLB-CV-T2-150 (lipase
B from Candida antarctica,
covalently attached to dry acrylic beads of 150-300 µm particle
size) was the product of ChiralVision BV (Leiden, The
Netherlands). All other reagents and solvents were purchased
from Sigma Aldrich (Saint Louis, MO, USA), Alfa Aesar Europe

6
Tetrahedron
4.3. Synthesis of 2-alkoxy acids 5A-D and their isopropyl esters
Into a screw cap reaction vial were added a mixture of dry
ACCEPTED MANUSCRIPT
2A-D
toluene (1.0 mL), immobilized CaLB enzyme (15.0 mg, CaLB-
CV-T2-150), the corresponding racemic amine rac-1a-d (0.778
mmol) and the corresponding isopropyl 2-alkoxyacetate 2A-D
(1.0 equiv., 0.778 mmol). The reaction mixture was shaken (750
rpm) at 30 °C and monitored by taking samples (20 µL) after
different reaction times (0.25, 0.5, 1, 2, 3, 4, 6, 8 h). After 8 h, the
reactions were worked up.
2-Methoxy- and 2-ethoxyacetic acids 5A,B and their isopropyl
esters 2A,B were synthesized as described earlier.27
Sodium (7.00 g, 304 mmol) was dissolved in the
corresponding dry alcohol (n-propanol or n-butanol, 300 mL) at
60 °C. To the forming solution of sodium alcoholate in the
alcohol were added triethylbenzylammonium chloride (0.50 g, 18
mmol) and sodium chloroacetate 4 and the resulted mixture was
refluxed for 12 h. After the alcohol was evaporated in vacuum,
the residue was dissolved in water (200 mL) and 5N HCl was
added (to pH= 2). The solution was extracted with ethyl acetate
(3×100 mL). The organic phases were unified, washed with brine
(50 mL) and dried over Na2SO4. After removal of the solvent by
vacuum rotary evaporation, the residue was purified with vacuum
distillation to yield 2-propoxyacetic 2C or 2-butoxyacetic acid
2D as colorless oil.
The raw reaction mixtures of rac-1a-c and rac-1e-i were
filtered on glass filter, evaporated in vacuum, picked up in
dichloromethane (20 mL) and extracted with HCl (2×10 mL,
5N). The aqueous phase was extracted with dichloromethane (10
mL) and the combined organic phases were dried on Na2SO4 and
after rotary evaporation the corresponding (R)-2-alkoxyacetamide
[(R)-3(a-c)(A-D), (R)-3(e-i)C] was obtained as light yellow
crystal or oil.
From the raw KR reaction mixtures of rac-1d the CaLB-CV-
T2-150 was filtered off on a glass filter, the volatiles were
evaporated in vacuum. The formed amide [(R)-3d(A-D)] was
afforded as light yellow oil after preparative TLC.
2-Propoxyacetic acid 5C (15.40 g, 130 mmol, 76%): Bp 103 °C
20
(9 torr); nD 1.4231; dH (300 MHz, CDCl3) 10.05 (1H, s,
COOH), 4.13 (2H, s, CH2COOH), 3.51 (2H, t, J=6.6 Hz, CH2O),
1.75–1.54 (2H, m, CH3CH2), 0.94 (3H, t, J=7.4 Hz, CH3); dC (75
MHz, CDCl3) 175.54, 73.67, 67.70, 22.66, 10.33; nmax (liquid
film): 2965, 2939, 2879, 1734, 1430, 1203, 1121, 962, 675 cm-1.
Elemental analysis for C5H10O3: required: C 50.84, H 8.53, found
C 50.73, H 8.56.
For (R)-2-methoxy-N-(1-phenylethyl)acetamide (R)-3aA and
(R)-2-ethoxy-N-(1-phenylethyl)acetamide (R)-3aB the physical
properties agreed with the published data.28
(R)-2-Propoxy-N-(1-phenylethyl)acetamide (R)-3aC (47 mg,
0.212 mmol, 27%): ee= 99.8%, [α]D20= +70.6 (c=10 mg mL-1,
CH2Cl2); dH (500 MHz, CDCl3) 7.41–7.25 (5H, m, Ar), 6.83 (1H,
s, NH), 5.21 (1H, p, J=7.0 Hz, NHCH), 3.96 (2H, q, J=15.2 Hz,
CH2CONH), 3.50 (2H, dt, J=12.9, 7.4 Hz, CH2Et), 1.71–1.59
(2H, m, J=14.0, 7.3 Hz, CH2Me), 1.55 (3H, d, J=6.8 Hz,
CHCH3), 0.95 (3H, t, J=7.4 Hz, CH2CH3). Elemental analysis for
C13H19NO2: required: C 70.56, H 8.65, N 6.33, found C 70.62, H
8.59, N 6.28.
2-Butoxyacetic acid 5D (14.27 g, 108 mmol, 63%): Bp 111 °C
(7 torr); nD20 1.4258; dH (500 MHz, CDCl3) 7.29 (1H, s, COOH),
4.13 (2H, s, CH2COOH), 3.59 (2H, t, J=6.2 Hz, CH2O), 1.68–
1.58 (2H, m, J1=14.0 J2=6.8 Hz, CH2Et), 1.47–1.36 (2H, m,
J1=14.4 J2=7.2 Hz, CH3CH2), 0.96 (3H, t, J=7.0 Hz, CH3); dC
(126 MHz, CDCl3) 174.41, 71.86, 67.73, 31.45, 19.09, 13.78;
nmax (liquid film): 2959, 2935, 2873, 1725, 1430, 1242, 1201,
1121, 933, 673 cm-1. Elemental analysis for C6H12O3: required: C
54.53, H 9.15, found C 54.61, H 9.08.
(R)-2-Butoxy-N-(1-phenylethyl)acetamide (R)-3aD (42 mg,
0.178 mmol, 23%): ee= 99.8%, [α]D20= +72.9 (c= 10 mg mL-1,
CH2Cl2); dH (500 MHz, CDCl3) 7.42–7.25 (5H, m, Ar), 6.82 (1H,
s, NH), 5.21 (1H, p, J= 6.9 Hz, NHCH), 3.96 (2H, q, J= 15.2 Hz,
CH2CONH), 3.52 (2H, t, J= 6.6 Hz, CH2Pr), 1.70–1.57 (2H, m,
CH2Et), 1.54 (3H, d, J= 6.8 Hz, CHCH3), 1.48–1.33 (2H, m, J1=
14.9 J2= 7.9 Hz, CH2CH3), 0.95 (3H, t, J= 7.1 Hz, CH2CH3).
Elemental analysis for C14H21NO2: required: C 71.46, H 9.00,
N 5.95, found C 71.55, H 8.97, N 5.89.
The corresponding 2-alkoxyacetic acid [2-propoxy- 5C (2.00
g, 16.9 mmol), 2-butoxyacetic acid 5D (2.24 g, 16.9 mmol)] and
p-toluenesulfonic acid (0.05 equiv., 0.155 g, 0.845 mmol) were
dissolved in 2-propanol (30 mL) and refluxed for 12 h. After
evaporation of 2-propanol from the reaction mixture, the desired
isopropyl ester (2C or 2D) was obtained by vacuum distillation.
Isopropyl 2-propoxyacetate 2C (1.73 g, 10.8 mmol, 64%): Bp
60 °C (8 torr); nD20 1.4068; dH (300 MHz, CDCl3) 5.09 (1H, hept,
J=6.2 Hz, MeCHMe), 4.03 (2H, s, CH2COO), 3.48 (2H, t, J=6.7
Hz, CH2Et), 1.73–1.56 (2H, m, CH2Me), 1.26 (6H, d, J=6.3 Hz,
MeCHMe), 0.94 (3H, t, J=7.4 Hz, CH2CH3); dC (75 MHz,
CDCl3) 170.38, 73.68, 68.69, 68.53, 22.97, 22.00, 10.61; nmax
(liquid film): 2979, 2938, 2878, 1749, 1730, 1466, 1375, 1277,
1205, 1127, 1104, 957, 931, 726, 584, 411 cm-1. Elemental
analysis for C8H16O3: required: C 59.98, H 10.07, found C 60.03,
H 9.98.
(R)-2-Methoxy-N-(4-phenylbutan-2-yl)acetamide
(R)-3bA
(69 mg, 0.312 mmol, 40%): ee= 97.5%, [α]D20= +27.3 (c= 10 mg
mL-1, CH2Cl2); dH (500 MHz, CDCl3) 7.26 (5H, dt, J1= 41.1, J2=
6.9 Hz, Ar), 6.37 (1H, d, J= 6.3 Hz, NH), 4.19–4.07 (1H, m,
NHCH), 3.90 (2H, s, CH2CONH), 3.42 (3H, s, OCH3), 2.71–2.63
(2H, m, ArCH2), 1.82 (2H, dd, J1= 15.8, J2= 6.9 Hz, Ar CH2CH2),
1.22 (3H, d, J= 6.6 Hz, CHCH3). Elemental analysis for
C13H19NO2: required: C 70.56, H 8.65, N 6.33, found C 70.61,
H 8.59, N 6.28.
(R)-2-Ethoxy-N-(4-phenylbutan-2-yl)acetamide
(R)-3bB
Isopropyl 2-butoxyacetate 2D (2.04 g, 11.7 mmol, 68%): Bp
76 °C (8 torr); nD20 1.4012; dH (300 MHz, CDCl3) 5.18–5.01 (1H,
m, MeCHMe), 4.02 (2H, s, CH2COO), 3.52 (2H, t, J=6.6 Hz,
CH2Pr), 1.67–1.53 (2H, m, CH2Et), 1.49–1.31 (2H, m, CH2Me),
1.26 (6H, d, J=6.3 Hz, MeCHMe), 0.92 (3H, t, J=7.3 Hz,
CH2CH3); dC (75 MHz, CDCl3) 170.23, 71.63, 68.54, 68.36,
31.64, 21.83, 19.20, 13.87; nmax (liquid film): 2960, 2935, 2874,
1750, 1730, 1467, 1375, 1278, 1204, 1139, 1104, 956, 932, 725,
584, 410 cm-1. Elemental analysis for C9H18O3: required: C
62.04, H 10.41, found C 61.97, H 10.52.
(72 mg, 0.306 mmol, 40%): ee= 96.4%, [α]D20= +18.2 (c= 10 mg
mL-1, CH2Cl2), dH (500 MHz, CDCl3) 7.36–7.13 (5H, m, Ar),
6.39 (1H, s, NH), 4.17–4.06 (1H, m, NHCH), 3.98–3.89 (2H, m,
CH2CONH), 3.58 (2H, q, J= 7.0 Hz, CH2CH3), 2.71–2.63 (2H,
m, ArCH2), 1.86–1.76 (2H, m, ArCH2CH2), 1.26 (3H, t, J= 7.0
Hz, CHCH3), 1.23 (3H, d, J= 6.6 Hz, CH2CH3). Elemental
analysis for C14H21NO2: required: C 71.46, H 9.00, N 5.95, found
C 71.40, H 9.01, N 5.96.
(R)-2-Propoxy-N-(4-phenylbutan-2-yl)acetamide
(R)-3bC
(107 mg, 0.429 mmol, 55%): ee= 92.2%, [α]D20= +13.2 (c= 10
mg mL-1, CH2Cl2), dH (500 MHz, CDCl3) 7.34–7.15 (5H, m, J1=
4.4. Kinetic resolution of racemic amines rac-1a-i in shake flask

for C7H15NO3: required: C 52.16, H 9.38, N 8.69, found C 52.35,
37.0, J2= 10.3, J3= 5.9 Hz, Ar), 6.47 (1H, d, AJ=C2C6.4EHPzT, NEHD), MANUSCRIPT
4.18–4.07 (1H, m, CHNH), 3.95 (2H, s, CH2CONH), 3.48 (2H, t,
J= 6.6 Hz, CH2Et); 2.70–2.63 (2H, m, ArCH2), 1.87–1.76 (2H,
m, ArCH2CH2), 1.72–1.60 (2H, m, CH2CH3), 1.23 (3H, d, J= 6.6
Hz, CHCH3), 0.98–0.93 (3H, m, CH2CH3). Elemental analysis
for C15H23NO2: required: C 72.25, H 9.30, N 5.62, found C 72.35,
H 9.31, N 5.61.
H 9.44, N 8.61.
(R)-2-Ethoxy-N-(1-methoxypropan-2-yl)acetamide (R)-3dB
(33 mg, 0.187 mmol 24%): Rf= 0.45 (EtOAc), ee= 98.3%,
[α]D20= +9.7 (c= 10 mg mL-1, CH2Cl2), dH (500, MHz CDCl3)
6.71 (1H, s, NH), 4,20 (1H, dt, J1= 11.5, J2= 6.3 Hz, NHCH),
3.92 (2H, s, CH2CONH), 3.57 (2H, q, J= 7.0 Hz, CH2CH3), 3.42–
3.35 (5H, m, CH3OCH2), 1.25 (3H, t, J= 7.0 Hz, CH2CH3), 1.21
(3H, d, J= 6.8 Hz, CHCH3). Elemental analysis for C8H17NO3:
required: C 54.84, H 9.78, N 7.99, found C 55.04, H 9.82, N
8.01.
(R)-2-Butoxy-N-(4-phenylbutan-2-yl)acetamide (R)-3bD (112
mg, 0.426 mmol, 61%): ee= 92.6%, [α]D20= +11.1 (c= 10 mg
mL-1, CH2Cl2), dH (500 MHz, CDCl3) 7.33–7.16 (5H, m, J1=
11.0, J2= 10.3, J3= 6.3 Hz, Ar), 6.40 (1H, d, J= 22.6 Hz, NH),
4.16–4.07 (1H, m, CHNH), 3.94 (2H, s, CH2CONH), 3.52 (2H, t,
J= 6.6 Hz, CH2Pr), 2.71–2.63 (2H, m, ArCH2), 1.86–1.77 (2H,
m, ArCH2CH2), 1.68–1.57 (2H, m, CH2Et), 1.48–1.36 (2H, m,
CH2Me), 1.23 (3H, d, J= 6.6 Hz, CHCH3), 0.97 (3H, t, J= 7.4 Hz,
CH2CH3). Elemental analysis for C16H25NO2: required: C 72.97,
H 9.57, N 5.32, found C 72.95, H 9.58, N 5.33.
(R)-2-Propoxy-N-(1-methoxypropan-2-yl)acetamide (R)-3dC
(19 mg, 0.101 mmol, 13%): Rf= 0.50 (EtOAc), ee= 97.2%,
[α]D20= +5.2 (c= 10 mg mL-1, CH2Cl2), dH (500 MHz, CDCl3)
6.74 (1H, s, NH), 4.25–4.18 (1H, m, J1= 8.8 Hz, J2= 6.7 Hz, J3=
4.2 Hz, J4= 2.3 Hz, NHCH), 3.93 (2H, s, CH2CONH), 3.47 (2H,
t, J= 6.6 Hz, CH2Et), 3.41–3.36 (5H, m, CH3OCH2), 1.69–1.61
(2H, m, CH2CH3), 1.22 (3H, d, J= 6.8 Hz, CHCH3), 0.97 (3H, t,
J= 7.4 Hz, CH2CH3). Elemental analysis for C9H19NO3: required:
C 57.12, H 10.12, N 7.40, found C 57.25, H 10.03, N 7.34.
(R)-2-Methoxy-N-(heptan-2-yl)acetamide (R)-3cA (58 mg,
0.311 mmol, 40%): ee= 97.9%, [α]D20= +0.03 (c= 10 mg mL-1,
CH2Cl2), [α]D20= +0.43 (c= 39 mg mL-1, CH2Cl2), dH (500 MHz,
CDCl3) 6.28 (1H, s, NH), 4.04–3.95 (1H, m, CHNH), 3.85 (2H,
s, CH2CONH), 3.40 (3H, s, OCH3), 1.48–1.37 (2H, m, CH2Bu),
1.26 (6H, t, J= 13.9 Hz, 3×CH2), 1.13 (3H, d, J= 6.5 Hz,
CHCH3), 0.86 (3H, t, J= 6. Hz, CH3CH2). Elemental analysis for
C10H21NO2: required: C 64.13, H 11.30, N 7.48, found C 64.20,
H 11.35, N 7.46.
(R)-2-Butoxy-N-(1-methoxypropan-2-yl)acetamide (R)-3dD
(40 mg, 0.195 mmol, 25%): Rf= 0.60 (EtOAc), ee= 95.4%,
[α]D20= +9.3 (c= 10 mg mL-1, CH2Cl2), dH (500 MHz, CDCl3)
6.72 (1h, s, NH), 4.26–4.16 (1H, m, CHNH), 3.92 (2H, s,
CH2CONH), 3.51 (2H, t, J= 6.5 Hz, CH2Pr), 3.38 (5H, d, J= 7.2
Hz, CH3OCH2), 1.64–1.56 (2H, m, CH2Et), 1.45–1.36 (2H, m,
CH2CH3), 1.22 (3H, d, J= 6.8 Hz, CHCH3), 0.95 (3H, t, J= 7.4
Hz, CH2CH3). Elemental analysis for C10H21NO3: required: C
59.09, H 10.41, N 6.89, found C 58.93, H 10.54, N 6.79.
(R)-2-Ethoxy-N-(heptan-2-yl)acetamide (R)-3cB (66 mg,
0.327 mmol, 42%): ee= 96.5%, [α]D20= -3.29 (c= 10 mg mL-1,
CH2Cl2), dH (500 MHz CDCl3) 6.33 (1H, s, NH), 4.04–3.94 (1H,
m, CHNH), 3.89 (2H, s, CH2CONH), 3.54 (2H, q, J= 7.0 Hz,
OCH2), 1.49–1.38 (2H, m, CH2Bu), 1.28 (6H, d, J= 6.4 Hz,
3×CH2), 1.22 (3H, t, J= 7.0 Hz, CH3CH2O), 1.13 (3H, d, J= 6.6
Hz, CHCH3), 0.86 (3H, t, J= 6.7 Hz, CH3CH2). Elemental
analysis for C11H23NO2: required: C 65.63, H 11.52, N 6.96,
found C 65.57, H 11.48, N 6.95.
(R)-2-Propoxy-N-(1-(4-nitrophenyl)ethyl)acetamide (R)-3eC
(83 mg, 0.101 mmol, 40%): Rf= 0.71 (5% MeOH/CH2Cl2),
ee= 99.9%, [α]D20= +50.8 (c= 10 mg mL-1, CH2Cl2), dH (500
MHz, CDCl3) 8.19–8.13 (2H, m, 2×CH), 7.46 (2H, d, J= 8.6 Hz,
2×CH), 6.87 (1H, d, J= 6.9 Hz, NH), 5.19 (1H, p, J= 7.2 Hz,
CHNH), 3.97–3.87 (2H, m, CH2CONH), 3.50–3.42 (2H, m,
OCH2Et), 1.68–1.56 (2H, m, CH3CH2), 1.52 (3H, d, J= 7.0 Hz,
CH3CH), 0.97–0.89 (3H, m, CH3CH2). Elemental analysis for
C13H18N2O4: required: C 58.63, H 6.81, N 10.52, found C 58.49,
H 6.75, N 10.63.
(R)-2-Propoxy-N-(heptan-2-yl)acetamide (R)-3cC (80 g, 0.373
mmol, 48%): ee= 94.2%, [α]D20= -3.02 (c= 10 mg mL-1, CH2Cl2),
dH (500 MHz, CDCl3) 6.34 (1H, d, J= 6.3 Hz, NH), 4.03–3.94
(1H, m, NHCH), 3.88 (2H, s, CH2CONH), 3.43 (2H, t, J= 6.6 Hz,
OCH2Et), 1.66–1.56 (2H, m, OCH2CH2), 1.40 (2H, dd, J1= 17.0
J2= 10.1 Hz, CHCH2), 1.26 (6H, t, J= 11.2 Hz, 3×CH2), 1.12 (3H,
d, J= 6.6 Hz, CHCH3), 0.91 (3H, t, J= 6.1 Hz, OCH2CH2CH3),
0.85 (3H, t, J= 6.5 Hz, CH3). Elemental analysis for C12H25NO2:
required: C 66.93, H 11.70, N 6.50, found C 66.89, H 11.64,
N 6.44.
(R)-2-Propoxy-N-(1-(4-chlorophenyl)ethyl)acetamide (R)-3fC
(92 mg, 0.109 mmol, 46%): Rf= 0.71 (5% MeOH/CH2Cl2),
ee= 99.9%, [α]D20= +56.5 (c= 10 mg mL-1, CH2Cl2), dH (500
MHz, CDCl3) 7.33–7.19 (4H, m, 4×CH), 6.77 (1H, d, J= 6.7 Hz,
NH), 5.12 (1H, p, J= 7.1 Hz, CHNH), 3.92 (2H, q, J= 15.3 Hz,
CH2CONH), 3.44 (2H, td, J1= 6.6 J2= 1.2 Hz, OCH2Et), 1.61
(2H, dt, J1= 14.1 J2= 7.1 Hz, CH3CH2), 1.48 (3H, d, J= 7.0 Hz,
CH3CH), 0.96–0.88 (3H, m, CH3CH2). Elemental analysis for
C13H18ClNO2: required: C 61.05, H 7.09, N 5.48, found C 60.91,
H 7.11, N 5.41.
(R)-2-Butoxy-N-(heptan-2-yl)acetamide (R)-3cD (95 mg,
0.412 mmol, 53%): ee= 98.1%, [α]D20= -3.76 (c= 10 mg mL-1,
CH2Cl2); dH (500 MHz, CDCl3) 6.36 (1H, d, J= 7.0 Hz, NH),
4.03–3.96 (1H, m, NHCH), 3.90 (2H, s, CH2CONH), 3.49 (2H, t,
J= 6.5 Hz, OCH2Pr), 1.65–1.54 (2H, m, OCH2CH2Et), 1.42 (2H,
dd, J1= 13.1 J2= 5.9 Hz, OCH2Ch2CH2Me), 1.38 (2H, dd, J1=
14.9 J2= 7.4 Hz, CH2CH), 1.33–1.23 (6H, m, 3×CH2), 1.14 (3H,
d, J= 6.6 Hz, CHCH3), 0.93 (3H, t, J= 7.4 Hz,
OCH2Ch2CH2CH3), 0.87 (3H, t, J= 6.6 Hz, CH3). Elemental
analysis for C13H27NO2: required: C 68.08, H 11.87, N 6.11,
found C 69.98, H 11.93, N 6.06.
(R)-2-Propoxy-N-(1-(4-bromophenyl)ethyl)acetamide (R)-3gC
(44 mg, 0.062 mmol, 19%): Rf= 0.74 (5% MeOH/CH2Cl2),
ee= 99.8%, [α]D20= +50.0 (c= 10 mg mL-1, CH2Cl2), dH (500
MHz, CDCl3) 7.45 (2H, d, J= 8.4 Hz, 2×CH), 7.18 (2H, d, J= 8.3
Hz, 2×CH), 6.78 (1H, s, NH), 5.16–5.06 (1H, m, CHNH), 3.93
(2H, q, J= 15.3 Hz, CH2CONH), 3.45 (2H, t, J= 6.2 Hz,
OCH2Et), 1.61 (2H, td, J1= 14.2 J2= 7.2 Hz, CH3CH2), 1.48 (3H,
d, J= 6.9 Hz, CH3CH), 0.92 (3H, t, J= 7.3 Hz, CH3CH2).
Elemental analysis for C13H18BrNO2: required: C 52.01, H 6.04,
N 4.67, found C 51.92, H 6.07, N 4.58.
(R)-2-Methoxy-N-(1-methoxypropan-2-yl)acetamide (R)-3dA
(48 mg, 0.298 mmol, 30%): Rf= 0.40 (EtOAc) ee= 99.7%,
[α]D20= +15.5 (c= 10 mg mL-1, CH2Cl2), dH (500 MHz, CDCl3)
6.65 (1H, s, NH), 4.30–4.12 (1H, m, NHCH), 3.88 (2H, s,
CH2CONH), 3.42 (3H, s, COCH2OCH3), 3.38 (5H, d, J= 4.5 Hz,
CH3OCH2), 1.21 (3H, d, J= 6,8 Hz, CHCH3). Elemental analysis
(R)-2-Propoxy-N-(1-phenylpropyl)acetamide (R)-3hC (34 mg,
0.053 mmol, 14%): Rf= 0.69 (5% MeOH/CH2Cl2), ee= 99.8%,
[α]D20= +53.4 (c= 10 mg mL-1, CH2Cl2), dH (500 MHz, CDCl3)

8
Tetrahedron
7.37–7.21 (m, Ar), 6.84 (1H, d, J= 7.4 Hz, NH), 4.92 (1H, dd,
filled column was washed with the toluene (200 µL min-1, 30
ACCEPTED MANUSCRIPT
J1= 15.9 J2= 7.4 Hz, CHNH), 3.93 (2H, dd, J1= 36.1 J2= 15.3 Hz,
CH2CONH), 3.45 (2H, qd, J1= 6.8 J2= 2.8 Hz, OCH2Et), 1.88–
1.80 (2H, m, CH3CH2CH), 1.68–1.57 (2H, m, CH3CH2CH2), 1.48
(3H, d, J= 7.0 Hz, CH3CH), 0.96–0.90 (3H, m, CH3CH2CH),
0.89 (3H, t, J= 7.4 Hz, CH3CH2CH2). Elemental analysis for
C14H21NO2: required: C 71.46, H 9.00, N 5.95, found C 71.39, H
9.06, N 5.89.
min) and stored in refrigerator (4 °C).
4.6. Continuous-flow kinetic resolution of racemic amines
rac-1a-d with ester 2C at different reaction temperatures
The solution of racemic amine (rac-1a-d, 0.65 mmol mL-1)
and isopropyl 2-propoxyacetate (2C, 0.6 equiv., 0.39 mmol mL-1)
in dry toluene was pumped through the CaLB-CV-T2-150-filled
column thermostated to different temperatures (30, 40, 50 and
60 °C) at different flow rates (50, 100 and 200 µL min-1).
Sampling of the reactions was performed as described in section
4.5. After the stationary operation has been established, 10 mL of
reaction mixture was collected and worked up according to the
description in Section 4.4 to obtain the corresponding amides
(R)-3(a-d)C:
(R)-2-Propoxy-N-(1-(3,4-dimethoxyphenyl)ethyl)acetamide
(R)-3iC (53 mg, 0.071 mmol, 24%): Rf= 0.63 (5%
MeOH/CH2Cl2), ee= 99.8%, [α]D20= +70.9 (c= 10 mg mL-1,
CH2Cl2), dH (500 MHz, CDCl3) 6.94–6.81 (3H, m, 3×CH), 6.76
(1H, d, J= 7.4 Hz, NH), 5.13 (1H, p, J= 7.0 Hz, CHNH), 3.91
(8H, dd, J1= 17.0 J2= 4.5 Hz, CH2CONH, 2×OCH3), 3.45 (2H, t,
J= 6.5 Hz, CH2Pr), 1.61 (2H, dq, J1= 14.2 J2= 7.1 Hz, CH3CH2),
1.51 (3H, d, J= 6.9 Hz, CH2Et), 1.45–1.36 (2H, m, CH2CH3),
1.22 (3H, d, J= 6.8 Hz, CHCH3), 0.92 (3H, t, J= 7.4 Hz,
CH2CH3). Elemental analysis for C15H23NO4: required: C 64.04,
H 8.24, N 4.98, found C 64.11, H 8.19, N 5.01.
(R)-2-Propoxy-N-(1-phenylethyl)acetamide (R)-3aC, 45%,
ee(R)-3aC= 99.7%.
(R)-2-Propoxy-N-(4-phenylbutan-2-yl)acetamide
49%, ee(R)-3bC= 99.2%.
(R)-3bC,
4.5. General methods for kinetic resolution of racemic amines
rac-1a-d with ester 2C in continuous-flow mode
(R)-2-Propoxy-N-(heptan-2-yl)acetamide
ee(R)-3cC= 99.1%.
(R)-3cC,
46%,
Packed-bed columns for continuous-flow biotransformation
were made by filling CaLB-CV-T2-150 into stainless steel
columns (PTFE layer inside; inner diameter: 4 mm; total length:
70 mm; packed length: 65 mm; inner volume: 0.816 mL)
according to the filling process of ThalesNano Inc. The columns
were settled by silver metal [Sterlitech Silver Membrane from
Sigma-Aldrich, Z623237, pore size 0.45 µm; pure metallic silver,
99.97% with no extractable or detectable contaminants] and
PTFE [Whatman® Sigma-Aldrich, WHA10411311, pore size
0.45 µm] filter membranes. The sealings were made of PTFE.
The reaction mixtures were pumped through the CaLB-filled
columns by syringe pumps (Chemyx) and heated by an HPLC
column thermostat.
(R)-2-Propoxy-N-(1-methoxypropan-2-yl)acetamide (R)-3dC,
43%, ee(R)-3dC= 99.9%.
Acknowledgements
OM thank Pro Progressio Foundation (Budapest Hungary) for
the financial support. Financial support for project NEMSyB, ID
P37_273, Cod MySMIS 103413 funded by the Romanian
Ministry for European Funds, through the National Authority for
Scientific Research and Innovation (ANCSI) and co-funded by
the European Regional Development Fund, Competitiveness
Operational Program 2014-2020 (POC), Priority axis 1, Action
1.1 is also gratefully acknowledged.
Before use, the CaLB-CV-T2-150-filled columns were
washed with toluene (200 µL min-1, 30 min). After setting a new
reaction parameter (temperature, substrate concentration, flow
rate), samples were analyzed by GC every 10 min up to 60 min
from the start of the actual experiment. After the stationary
operation has been established (40 min after the start of the
experiment), samples were collected (20 µL sample was diluted
with ethanol to 1 mL) and analyzed by TLC and GC. When a
series of experiments was finished, the actual CaLB-CV-T2-150-
Supplementary Material
Please see the Supplementary material to find details of the
preparation of the racemic 2-alkoxyacetamides for GC analysis,
GC methods, representative chromatograms and spectra (1H, 13C
NMR, FT-IR).
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