Deracemization of diastereomerically pure syn- and anti-α-substituted β-hydroxyesters by Novozyme 435 lipase and determination of their absolute configuration by NMR spectroscopy

Article abstract of DOI:10.1016/j.mcat.2021.111578

Enantiomerically pure α-substituted β-hydroxyesters are important chiral building blocks for ligands, auxiliaries and β-lactam antibiotics. A two-step chemo-enzymatic procedure using lipase as biocatalyst is an efficient way to synthesize such products. To date, the methods described are limited to molecules that do not contain a chiral center adjacent to the racemic carbinol, and furthermore, they are limited to acylation. Here, we investigated the deracemization of diastereomerically pure syn- and anti-α-substituted β-hydroxyesters containing two stereo centers, using experimental methods under neat conditions and classical molecule dynamics (MD) simulation. A screening of free and immobilized commercial lipases identified immobilized lipase B from Candida antarctica (Novozyme 435) as the most appropriate biocatalyst for sterically demanding α-substituted β-hydroxyesters. Using Novozyme 435, reaction conditions were optimized and hydroxyesters (3S) or (3R) were achieved with enantiomeric excesses up to ≥99% ee and maximum overall yields of 80%. The absolute configuration of the enantiomers was eventually determined by ¹H-NMR spectroscopy after derivatization with MOSHER'S reagent (α-methoxy-α-trifluoromethylphenylacetic acid = MTPA).

Full text of DOI:10.1016/j.mcat.2021.111578

Molecular Catalysis 509 (2021) 111578
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.journals.elsevier.com/molecular-catalysis
Deracemization of diastereomerically pure syn- and anti- -substituted
α
β-hydroxyesters by Novozyme 435 lipase and determination of their
absolute configuration by NMR spectroscopy
a
,
b
,
*
a
a
a
Christian Trapp
, Kate ˇr ina Barkov a´ , Marek Jan Pecyna , Corinna Herrmann ,
a
a
b
Annett Fuchs , Dieter Greif , Martin Hofrichter
a
University of Applied Science Zittau/Goerlitz, Theodor-K o¨ rner-Allee 16, Zittau 02763, Germany
b
TU Dresden, International Institute Zittau, Markt 23, Zittau 02763, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Enantiomerically pure
α-substituted β-hydroxyesters are important chiral building blocks for ligands, auxiliaries
Candida antarctica Lipase B
Novozyme 435
and β-lactam antibiotics. A two-step chemo-enzymatic procedure using lipase as biocatalyst is an efficient way to
synthesize such products. To date, the methods described are limited to molecules that do not contain a chiral
center adjacent to the racemic carbinol, and furthermore, they are limited to acylation. Here, we investigated the
β-Hydroxyesters
Enzymatic kinetic resolution
NMR Spectroscopy
Catalysis
deracemization of diastereomerically pure syn- and anti-
α-substituted β-hydroxyesters containing two stereo
centers, using experimental methods under neat conditions and classical molecule dynamics (MD) simulation. A
screening of free and immobilized commercial lipases identified immobilized lipase B from Candida antarctica
Molecular docking
MOSHER’S reagent
(Novozyme 435) as the most appropriate biocatalyst for sterically demanding
α-substituted β-hydroxyesters.
Using Novozyme 435, reaction conditions were optimized and hydroxyesters (3S) or (3R) were achieved with
enantiomeric excesses up to ≥99% ee and maximum overall yields of 80%. The absolute configuration of the
enantiomers was eventually determined by 1H-NMR spectroscopy after derivatization with MOSHER’S reagent
(
α
-methoxy-α-trifluoromethylphenylacetic acid = MTPA).
1
. Introduction
achieved with lipases as enantioselective biocatalysts [12]. Notably,
such combined approaches are working without platinum metals (e.g.
ruthenium or iridium) unlike asymmetric hydrogenations [13].
Chiral β-hydroxyesters and α-substituted β-hydroxyesters represent
versatile building blocks, which can be used for the synthesis of auxil-
iaries [1] (e.g. 1,2- or 1,3-amino alcohols [4]), β-lactam antibiotics [2]
and pheromones [3]. They can be prepared biochemically or chemically.
Biochemical routes proceed via reduction with isolated enzymes [5] or
biotransformation with whole cells [6]. Main drawbacks of these ap-
proaches are insufficient solubility of nonpolar substrates in aqueous
solvents and poor stabilities of most enzymes in pure organic solvents
Meanwhile the use of lipases as catalysts in organic synthesis has led
to reliable synthetic routes that yield environmentally friendly products
with high stereo- and regioselectivities [14]. One of the most important
commercial lipases in organic synthesis is lipase B of the yeast Candida
antarctica (CALB). It is typically used in immobilized form on macro-
porous poly(methyl methacrylate) resin and commercially available
under the trade name Novozyme 435 (N435). The application of N435
has increased over the last two decades and includes reactions such as
transesterification, MICHAEL addition and epoxidation under mild and at
the same time selective conditions [15]. One example is the N435
catalyzed resolution of racemic proxyphylline, a cardiac stimulant,
vasodilator and bronchodilator, with a yield of 43% and ee up to >99%
(E = 90) at a 5-g scale [29]. A second example is the resolution of
racemic 1,2-propanediol using a packed-bed reactor filled with N435 to
obtain (R)-propanediol with an E-value of >200 [38]. The usage of N435
(
neat conditions) [7]. Chemical methods to obtain these chiral hydrox-
yesters use aldol-type reactions [8] or asymmetric hydrogenation [9].
Furthermore, suitable combinations of chemical and enzymatic steps
(
chemoenzymatic approaches) turned out to be appropriate reaction
setups. The latter start with LEWIS acid-mediated diastereoselective
reduction of analogous β-ketoesters using complexing and
non-complexing metal salts like zinc [10], titanium [11] or cerium [11].
Afterwards, chiral resolution of these pure diastereomers can be
*
Corresponding author.
E-mail address: christian.trapp@hszg.de (C. Trapp).
https://doi.org/10.1016/j.mcat.2021.111578
Received 13 December 2020; Received in revised form 10 April 2021; Accepted 13 April 2021
Available online 23 May 2021
2
468-8231/© 2021 Elsevier B.V. All rights reserved.

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
Fig. 1. Lipase-catalyzed kinetic resolution of
α
-substituted β-hydroxyesters in two steps. The formula scheme shows the resolution of syn-isomers as an example.
Table 1
Diastereoselective reduction of
α-substituted β-ketoesters into the corresponding syn- or anti-β-hydroxyesters.
1
2
1
2
syn-Hydroxyester
R
R
anti-Hydroxyester
R
R
1
3
5
Et
Et
Et
Ph
Bn
iPr
2
4
6
Et
Ph
Bn
iPr
tBu
tBu
in organic solvents is particularly relevant when reactants are poorly
soluble in water or when reversal of the hydrolysis reaction is desired.
However, the use of N435 is not limited to organic solvents; other re-
action media such as ionic liquids [16], supercritical fluids [17] or deep
eutectic solvents can be applied as well [18].
Starting material of the aforementioned lipase reactions was syn-
thesized by diastereoselective reduction of the corresponding
β-ketoesters.
syn-Selective reduction followed CRAM ´s chelate model, therefore
coordinating LEWIS acids, i.e. zinc and titanium salts, were used. The anti-
selective reduction, however, followed the model according to FELKIN
and ANH. In this case, a sterically hindered reducing agent (lithium
The resolution of racemic ethyl 3-hydroxybutanoate was already
described for the preparation of enantiomerically pure (3R)- and (3S)-
ethyl-3-hydroxybutanoate by Fishman et al.; they used vinyl acetate as
acylating agent and lipase B from Candida antarctica in a solvent-free
system giving 96% ee and 73% total yield [19]. Here, we were aiming
at extending this elegant kinetic resolution on bulky 3-hydroxybuta-
triethyl-borohydride, LiEt
3
BH) and a non-complexing LEWIS acid (cerium
chloride, CeCl ) were chosen as reactants. The pure diastereomers ob-
3
tained were used for screening four lipases (free or immobilized en-
zymes), whereat we had a closer look onto the influence of (i) the type of
noates with sterically hindered
α
-substituents (i.e. phenyl-, isopropyl-
α
-substituent, (ii) the absolute configuration in -position and (iii) the
α
or benzyl-groups as ethyl or tert-butyl esters). For this approach, lipases
were used as regio- and enantioselective biocatalysts to obtain the (3S)-
and (3R)-hydroxyesters shown in Fig. 1. Afterwards, each pure enan-
tiomer was used for the synthesis of chiral 4,5-disubstitued
oxazolidin-2-ones.
ester group (ethyl or tert-butyl ester) with regard to reaction rate and
enantioselectivity. Eventually, we identified a suitable acyl donor for
esterification and an adequate nucleophile for alcoholysis of the latter in
different organic solvents.
Table 2
1
Acylation of syn- and anti-β-hydroxyesters into the corresponding acyloxyesters.
1
2
1
2
3
Hydroxyester
R
R
Acyloxyester
R
R
R
Yield
syn-1
anti-2
syn-3
anti-4
syn-5
anti-6
Et
Ph
Ph
Bn
Bn
iPr
iPr
syn-7
Et
Ph
Ph
Bn
Bn
iPr
iPr
Pr
Pr
Pr
Pr
Me
Pr
95%
95%
88%
95%
88%
77%
Et
anti-8
syn-9
Et
Et
Et
tBu
Et
anti-10
syn-11
anti-12
tBu
Et
tBu
tBu
1
Only those acyloxyesters later used under optimized conditions are listed in Table 2.
2

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
2
. Results and discussion
acetate in n-hexane was used as irreversible acyl donor. The reaction
◦
was performed in an overhead shaker at 40 C. Conversion was deter-
Diastereomerically pure syn- or anti-
α
-substituted β-hydroxyesters 1 -
mined using GC-FID and enantioselectivity of the acetylation reaction
was analyzed by HPLC on chiral columns. Figs. 2 and 3 summarize the
results of the screening.
6
were obtained by chemical reduction with LEWIS acids in combination
-substituted
with complex borohydrides from the corresponding
α
β-ketoesters as previously reported [20] (Table 1). To obtain racemic
acyloxyester rac-7 - 12, required for preparative enzymatic acylation
and alcoholysis attempts as well as for comprehensive HPLC studies on
their separation, the afore-prepared hydroxyesters 1 - 6 were treated
with the appropriate acyl chloride in dry dichloromethane in the pres-
ence of pyridine. Under these esterification conditions, acyloxyesters
rac-7 - 12 could be synthesized in high yields (77-95%).
N435 and PSL B accomplished good performance for compounds rac-
1 - 5 over 72 h. In some cases, conversion reached more than 50%
indicating unselective acylation over time; noteworthy, 72% of com-
pound rac-3 was converted by PSL B. For hydroxyester rac-6, moderate
conversion was only observed with N435 (max. 29%); the other lipases
failed. The performance of CRL and iPCL was generally insufficient.
Fig. 3 shows the enantiomeric access (ee values) in relation to the con-
versions of Fig. 1. For all compounds, the highest ee values were ob-
tained with N435, followed by PLS B. In the case of hydroxyester rac-1,
an excellent ee of ≥99% was achieved with N435 at 59% conversion.
Owing to these results, we decided to use N435 in all further experi-
ments. This enzyme preparation has also the advantage of being
recoverable due to lipase immobilization on an acrylic resin and it is
stable over several reaction cycles [21].
2
.1. Lipase catalyzed acylation of
α-substituted β-hydroxyesters
In a recent lipase screening using compound 1 as substrate (data not
shown), we have identified four potent lipases for kinetic resolution.
These are Novozyme 435 (N435, immobilized lipase from Candida
antarctica B = CAL B), immobilized lipase from Pseudomonas cepacia
All tested lipases showed preference for the (3R)-enantiomer (as we
will discuss below). To increase reaction rate and enantioselectivity, we
decided to optimize the reaction conditions, particularly for compounds
rac-5 and 6. At first, we studied the influence of the solvent system. In
(
iPCL), lipase from Pseudomonas sp. Typ B (PSL B) and lipase from
Candida rugosa (CRL). Thus, we decided to study these lipases more in
detail (particularly with respect to overall conversion and enantiose-
lectivity) using our compounds rac-1 - 6 as target substrates. First, vinyl
Fig. 2. Conversion of
α-substituted β-hydroxyesters by different lipases after 72 h. Reactions were performed in duplicate.
Fig. 3. Enantiomeric excess (ee) of
α-substituted β-hydroxyesters obtained with four lipases after 72 h. Reactions were performed in duplicate.
3

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
Table 3
Table 5
Influence of the solvent on acylation of rac-1 with vinyl butanoate and Novo-
Influence of the acyl donor concentration on the conversion of rac-4. Reactions
zyme 435.
were performed in duplicate.
a
a
b
Solvent
log P
22]
Conversion
after
Conversion
after
ee-Value after 24
Acyl Donor
Conversion after 6 h
ee value of (2S,3S)-4
[
h [%]
vinyl acetate
28%
47%
45%
42%
98%
90%
2
h [%]
24 h [%]
vinyl butanoate
vinyl hexanoate
1
.4-
dioxane
-1.10
<1
<1
n/a
acetone
THF
-0.23
0.49
2.00
2.50
3.50
5.10
<1
<1
n/a
n/a
n/a
91
<1
2
chloroform
toluene
n-hexane
n-nonane
<1
<1
Table 6
10.4
32.9
32.1
44.0
47.7
48.5
Results of kinetic resolution of racemic
α
-substituted β-hydroxyesters by N435
99
(CAL B). Reactions were performed in duplicate.
98
a
b
c
Hydroxyester
Reaction
Time [h]
Conversion
[%]
ee-Value
Yield
[%]
E
a) measured by gas chromatography.
[%]
b) of hydroxyester (2R,3S)-1, measured by chiral HPLC.
syn-isomers
(2R,3S)-
hydroxyester
rac-1
rac-3
24
9
54
50
≥ 99
44
41
40
83
Table 4
99
97
≥100
57
Influence of the acyl donor concentration on the conversion of rac-1.
rac-5
96
53
anti-isomers
(2S,3S)-
hydroxyester
Cosubstrate Concentration
mM]
Conversion after 1 h
[%]
Decrease of
conversion
[
rac-2
11
51
≥ 99
44
≥100
6
3 mM VA (1 eq.)
VA as solvent
3 mM VB (1 eq.)
VB as solvent
3 mM VH (1 eq.)
VH as solvent
5.5
92%
92%
84%
rac-4
rac-6
24
55
45
> 99
45
36
67
0.4
d
466
83
n/a
6
34.5
2.7
a) measured by GC-FID.
b) hydroxyesters measured on HPLC with chiral columns.
6
24.5
3.8
c) after column chromatography (theoretical maximum of 50%).
d) not chemo selective.
general, a log P value >2 is recommended for enzymatic reactions, since
The effect was particularly pronounced when small acid moieties with
low log P values were present (log P: VA = 0.5, VB = 1.6, VH = 2.5).
Decrease of conversion was observed at concentrations above 250 mM
of the vinyl donor. At lower concentrations, vinyl donors just acted as
reactants without affecting the solution matrix.
the enzyme conformation strongly depends on the reaction medium
[
22]. According to literature, the turnover of lipase CAL B tends to in-
crease with decreasing polarity [23]. Seven nonpolar and polar aprotic
solvents were tested using the hydroxyester rac-1 as model substrate
(
Table 3).
Table 3 summarizes the solvent effects on enzymatic conversion and
ee. The higher the log P, the higher was the reaction rate. Conversions in
Further effects of the carboxylic acid chain length were studied with
regard to the enantioselectivity of rac-4 (Table 5). Vinyl butanoate gave
the best results with respect to conversion and enantioselectivity after 6
h reaction time. For the remaining hydroxyester, (2S,3S)-4, an ee value
of 98% was ascertained; the ee increased to ≥99 % when the time was
extended. The same tendency was observed for the hydroxyesters rac-1 -
1
,4-dioxane, acetone, THF and chloroform failed (≤ 2%). In addition to
unfavorable log P, swelling of the PMMA resin matrix of N435 may have
affected the enzyme performance in polar solvents by blocking the
matrix pores, which eventually stopped lipase reaction. Another
disturbance may come from viscosity of the solvent, which causes an
opposing effect to polarity and hence could have affected conversion
3
and 6 (results not shown). The only exception was rac-5 that gave the
best enantioselectivity with vinyl acetate. Nevertheless, the reaction rate
with vinyl butanoate was the highest for all compounds.
[
24].
After 2 h, conversion turned out to be highest in alkane solvents (n-
Based on these results, we studied kinetic resolution of syn- and anti-
α
-substituted β-hydroxyesters, using the hitherto optimized conditions
hexane, n-nonane); obviously, a high log P value had a positive effect on
the enzymatic reaction. This finding and the fact that n-hexane is the
solvent of choice was confirmed by good to very good ee values for the
remaining alcohol (2R,3S)-1 formed in n-hexane (99% ee) within 24 h
◦
(
40 C, n-hexane, N435, 0.15 mg/mg hydroxyester, and VB as an acyl
donor). Samples were taken from the reaction mixtures after defined
times, diluted with n-hexane and analyzed by GC-FID and HPLC (on an
appropriate chiral column). The latter was only used for the remaining
hydroxyesters. However, HPLC analysis of the corresponding enantio-
meric acyloxyesters from lipase-catalyzed kinetic resolution was not
successful. Results of the up-scaled kinetic resolution are summarized in
Table 6. Excellent results were obtained for five out of six hydroxyesters
(
for comparison, 98% ee in n-nonane and 91% ee in toluene). Moreover,
n-hexane has the advantage over longer-chain alkanes of being easier to
remove from the reaction mixture by evaporation. On the other hand,
reactants with low boiling point, such as ethyl-3-hydroxy-2-
isopropylbutanoate (5), cannot be removed under vacuum. Not least,
n-hexane was the main component of the HPLC eluent, which facilitated
the measurement of reaction controls because matrix effects could be
excluded.
(
rac-1 - 5), demonstrating the catalytic suitability and broad substrate
specificity of immobilized CAL B (N435).
For compound rac-6, we observed the formation of an interesting
side product. After some time (>100 h), acetaldehyde appeared in the
reaction solution either as the result of acylation or as hydrolysis product
of residual water associated with the enzyme. Acetaldehyde in turn
reacted with rac-6 to form a hemiacetal that contained a new secondary
alcohol moiety (Fig. 4). The latter again favored acylation of the
hemiacetal of rac-6 over that of rac-6 itself. Therefore, the yield was only
Since it was reported that the acyl donor influences the performance
of the reaction catalyzed by CAL B as well [25], the acylation of rac-1
was performed under already established conditions (i.e. in n-hexane at
◦
4
0 C, concentration of the racemic substrate 63 µM) with three vinyl
esters (vinyl acetate, VA; vinyl butanoate, VB and vinyl hexanoate, VH)
as donors. Results (Table 4) revealed a significant difference in reactivity
depending on the carboxylic acid chain length. The best results were
obtained with vinyl butanoate as acylation agent. Then, we tried to use
the acyl donor as solvent instead of n-hexane. This, however, led to a
dramatic decrease in reactivity (up to 92%) for all hydroxyesters 1 - 6.
3
6% instead of the expected 50%.
Chiral secondary alcohols with stereocenters directly linked to the
hydroxyl functionality were enantioselectively resolved by CAL B with
the highest efficiency. The two substituents of such a secondary alcohol
4

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
Fig. 4. Proposed side reaction during the kinetic resolution of rac-6.
twice the reaction time than its diastereomer rac-2 did to achieve a
similar result. This could be explained by better fitting of hydroxyester
(
2R,3R)-2 inside the active center so that the positioning of the substrate
is optimal for the acylation to occur. In docking studies, more productive
binding poses and shorter distances between substrate methyl group and
tryptophane 104 (which lines the stereospecific pocket) were found for
hydroxyester (2R,3R)-2 (Fig. 5). The influence of the ester (ethyl or tert-
butyl) as part of the large substituent was marginal, since these groups
protrude from the active site of the enzyme (Fig. 6).
2
.2. Lipase catalyzed alcoholysis of α-substituted β-acyloxyesters
Fig. 5. Docking poses of (2S,3R)-1 (gold) and (2R,3R)-2 (magenta) in the active
site of N435. The hydroxyester (2R,3R)-2 is fitted more easily so that the
positioning of the substrate is optimal for the acylation to occur.
Eventually, we obtained substantial amounts of enantiomerically
pure (2R,3S)-1, 3, 5 and (2S,3S)-2, 4, 6
-substituted β-hydroxyesters.
α
The corresponding acyloxyesters enriched during kinetic resolution,
(2S,3R)-7, 9, 11 and (2R,3R)-8, 10, 12, were cleaved by alcoholysis.
Since alcohols are not involved in the formation of the intermediate
acyl-lipase complex (comprising acylated serine), the deacylation rate is
determined by the diffusion of alcohol molecules into the active site of
the enzyme [27]. Thus, small aliphatic alcohols diffuse faster into the
active site than larger/bulky ones, i.e. the reactivity of the system may
decrease with increasing chain length of the alcohol. In addition,
nucleophilicity of alcohols decreases with increasing chain length,
which can further contribute to decreased reactivity [28]. Accordingly,
short-chain alcohols such as ethanol or methanol should ensure highest
conversion rates with acylated serine in the active site of lipase [21a].
are referred to as the medium and large sized substituents. In preferred
substrates, the medium sized substituent is placed in a small cavity near
the catalytic serine (the stereospecificity pocket), while the large sub-
stituent will be directed towards the entrance of the enzyme active site.
This stereospecificity pocket can only accommodate an ethyl group (or
smaller substituent), which explains the low reactivity towards non-
preferred configurations with a large substituent longer than the ethyl
chain [26].
Conversion of the hydroxyesters rac-1 and rac-2 indicated that the
stereocenter in
α
-position effected the reaction rate of the secondary
alcohol moiety. Under the same reaction conditions, rac-1 required
Fig. 6. Docking poses of (2R,3R)-4 in the active site of N435 depicted as sticks (A) and space-filling model (B) model. The ester moieties of tert-butylester (2R,3R)-4 is
protruding from the active site so that the influence of the reaction rate of ethyl or tert-butylester was marginal.
Table 7
◦
Alcoholysis of rac-10 catalyzed by N435 (CAL B) in n-hexane at 40 C. Reactions were performed in duplicate.
Time [h]
Acyloxyester 10 [%]
Methanol
Ethanol
1-Butanol
1-Hexanol
1-Octanol
1-Decanol
0
100
64
100
70
ß
84
100
67
100
62
100
62
4
6
9
8
8
1
63
71
77
59
57
56
63
69
69
55
53
53
1
64
64
71
53
51
51
51
ee [%] (2R,3R)-4
≥ 99
≥ 99
≥ 99
≥ 99
≥ 99
≥ 99
after 164 h
5

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
Fig. 7. Alcoholysis of rac-10 to obtain enantiomerically pure (2R,3R)-4.
the acylation reaction (3 mg/ 0.1 mmol substrate). Results of respective
alcoholyses are given in Table 8.
Table 8
Results of N435-catalyzed alcoholysis of enantiomerically enriched
They indicate high conversion along with excellent ee values be-
tween 97 and ≥99%. However, substantial shortcomings of these
enzymatic deacylations are the long reaction time ranging from several
days to weeks as well as the demand for large amounts of immobilized
CAL B (N435). This gave rise to unwanted side reactions such as trans-
esterification of ethyl esters.
α
-substituted β-acyloxyesters.
a
b
c
Acyloxyester
Reaction time
d]
Conversion
[%]
ee-Value
Yield
[%]
[
[%]
syn-isomers
(2S,3R)-hydroxyester
(
(
2S,3R)-7
2S,3R)-9
35
33
77
85
≥ 99
65
56
97
(
2R,3R)-10 showed the highest alcoholysis rate (79% conversion
anti-isomers
(2R,3R)-hydroxyester
(
(
2R,3R)-8
23
13
82
89
99
48
70
within 6 days), despite of its sterically demanding tert-butyl group in the
acyloxyester molecule. Interestingly, transesterification was not
observed in this case, which may be due to steric hindrance of the nu-
2R,3R)-10
≥ 99
a) measured by GC-FID.
cleophiles. Furthermore, we observed that the configuration in -posi-
α
b) measured hydroxyester on HPLC with chiral columns.
c) after column chromatography (max. 50%).
tion, besides that of carbinol, affected the reactivity as well. Thus,
enriched (2R,3R)-10 showed higher reactivity compared to the diaste-
reomer (2S,3R)-7. This tendency was already ascertained in the acyla-
tion of the pair rac-1 and rac-2 (Table 6).
However, deacylation is more difficult to accomplish than acylation as
alcohols can inhibit CAL B (N435). Thus, small alcohols – as polar sol-
vents – tend to detach the essential layer of water molecules (des-
olvation), which covers and protects the protein. In consequence, lipase
conformation is going to change resulting in diminished catalytic ac-
tivity [29]. Moreover, alcohols can act as unspecific inhibitors of CAL B
and may even form dead-end complexes with the lipase protein [30].
N435 catalyzed hydrolysis of acyloxyesters was studied using the
The isopropyl substituted enantiomers (2S,3R)-10 and (2R,3R)-11
enriched by kinetic resolution did not show any conversion under the
reaction conditions described above. Probably, the substrate was steri-
cally too demanding and did hardly diffuse into the active side of N435
(
CAL B). The reaction times of preceding acylation of 5 and 6 support
this assumption. Hence, acyloxyesters (2S,3R)-10 and (2R,3R)-11 were
◦
chemically saponified with aqueous potassium hydroxide.
alcohols (1 eq.) listed in Table 7 as reactants at 40 C in n-hexane; rac-10
was used as model substrate (Fig. 7). Samples were taken from the re-
action mixtures over time and analyzed by GC-FID. After 164 h, re-
actions were stopped by separating the immobilized lipase via filtration.
Afterwards, the ratio of enantiomeric hydroxyesters was analyzed by
chiral HPLC.
2
.3. Determination of the absolute configuration by NMR-spectroscopy
Finally, the absolute configurations of separated enantiomer pairs of
each
31]. To this end, (R)-MOSHER´S acid chloride [(R)-MTPA-Cl] was used to
derivatize the enantiomerically pure hydroxyesters (2R,3S)-1, 3, 5 and
2S,3S)-2, 4, 6. As reference, racemic hydroxyesters 1 - 6 were also
α-substituted β-hydroxyester were determined by MOSHER ´S method
Results of alcoholysis tests are summarized in Table 7. With ethanol
and methanol, a rapid incipient reaction was observed, which slowed
down to constant product levels of 64% and 70%, respectively, after 48
h. In the case of longer-chain alcohols (1-hexanol, 1-octanol, 1-decanol),
the reaction equilibrium was more on the side of products with turn-
overs around 50% after 164 h. 1-Butanol turned out to be the worst
nucleophile for N435-catalyzed alcoholysis. Notably, all obtained
hydroxyesters of type (2R,3R)-4 were enantiomerically pure (ee ≥ 99%).
The procedure was successfully transferred to acyloxyesters rac-7
and 9, for which similar reaction rates were observed. In consequence,
we decided to use 1-decanol as nucleophilic alcohol for deacylation of
the enantiomerically enriched acyloxyesters (2S,3R)-7, 9 and (2R,3R)-8,
[
(
esterified with (R)-MTPA-Cl. After derivatization and purification by
liquid column chromatography, all (S)-MTPA-esters were analyzed by
1
H-NMR spectroscopy to determine the characteristic chemical shift of
each enantiomer. The derivatizing reactions were performed in dry
dichloromethane supplemented with (R)-MTPA-Cl, the respective
hydroxyester and pyridine to obtain the corresponding MTPA-esters
1
3-24 in quantitative yields (Fig. 8).
The phenyl-ring is known to impose an anisotropic, magnetic
shielding effect on protons residing above (or below) the plane of the
aryl ring. This shielding results in a more up-field chemical shift for the
affected protons in the 1H-NMR spectrum. Fig. 9 shows the mostly
1
0. Maximum reaction rate was determined at an enzyme load of 500
mg N435/ 0.1 mmol substrate, which is two orders higher than that for
Fig. 8. Derivatization of
α-substituted β-hydroxyesters with (R)-MTPA-Cl.
6

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
Fig. 9. Most populated conformer of the (R)-MTPA ester of rac-1. Arrows in respective formula representations (red moieties) mark shielding effects of the
phenyl group.
populated rotamer where the methyl group of (2R,3S)-1 is syn-peri-
planar to the phenyl ring of (R)-MPTA, resulting in a high-field shift.
Thus, the methyl group of (2S,3R)-1 was found at lower field, while the
rest of the molecule shifted to higher field. These shielding effects
decreased with increasing distance to the aryl ring of MTPA.
equipped with a VF-5ms column (30 m × 0.25 mm × 0.25 µm, Agilent)
◦
and GC parameters were chosen as follows: inlet - 250 C, flow - 1.2 mL/
◦
◦
◦
min (N
2
), oven -70 C to 270 C (10 C/min), detector temperature -
◦
300 C. Enantiomeric excesses (% ee) of the hydroxyesters were deter-
mined by HPLC on a Thermo Fisher Scientific Ultimate 3000 system,
equipped with a UV detector and chiral Phenomenex columns (Lux 5 µm
i-Amylose-1, LC Column 250 mm x 4.6 mm; Lux 5 µm Cellulose-3, LC
Column 250 mm x 4.6 mm) eluted with mixtures of n-hexane/ iso-
propanol as the mobile phase in the appropriate ratios given in the
supporting information. MM2 force field was calculated with the Soft-
ware “Chem3D” (version 17.0).
To quantify the difference in chemical shift, following equations
were used for syn-hydroxyesters and anti-isomers, respectively:
Δδ(2S,3R)/(2R,3S) = δ(2S,3R) ꢀ δ(2R,3S) and Δδ(2R,3R)/(2S,3S) = δ(2R,3R)
ꢀ
δ(2S,3S). The methyl group in γ-position was used for calculation, since it
is not superimposed with other signals and just appears as a duplet. All
examined MTPA-esters from hydroxyesters 1 - 7 showed a positive dif-
ference in chemical shift in the range from 0.05 to 0.10 ppm of the
γ-methyl group for enantiomers in (3S)-configuration. These findings
strongly indicate that N435 (CAL B) exhibits (3R)-stereo-preference to-
4
.2. Chemical material
wards racemic
α-substituted β-hydroxyesters, which is in accordance
Commercial grade reagents and solvents were used without further
with the steric binding model of KAZLAUSKAS et al.[32]
purification. Solvents were dried and purified as recommended [33].
Lipases including immobilized CAL B (Novozymes 435 = N435) were
purchased from Sigma Aldrich. Specific features of Novozymes lipases
are reported in the brochure “Immobilized lipases for biocatalysis”
3
. Conclusions
We describe a reliable lipase-catalyzed approach for the preparation
[
21a]. Enzymatic reactions were discontinuously performed in an
of enantiomerically pure
α-substituted β-hydroxyesters to be used as
ꢀ 1
overhead shaker (15 min ).
potential chiral building blocks. Four free or immobilized lipases were
tested for enantioselective acylation with respect to conversion and
enantioselectivity. Our method includes a two-step kinetic resolution
using immobilized lipase B from Candida antarctica (CAL B, Novozyme
4.3. Computational
4
35 = N435) as most appropriate biocatalyst that could be recovered
To analyze favorable ligand binding geometries for the substrates
AutoDock Vina 1.1.2 were carried out [34]. Ligand molecules were
prepared with ChemDraw (version 17.0 and saved in PDB format. Gas-
teiger partial charges were calculated with AutoDockTools 1.5.6rc3
[35], and the final ligand files were prepared in PDBQT format.
after reaction (even if the re-use of the enzyme was not quantitatively
investigated). The influence of enzymatic reaction parameters, such as
solvent type, acyl donor and its concentration as well as alcohol chain
length, on the stereochemical performance of kinetic resolution was
studied and optimized. Under the latter conditions, we obtained high
The crystallographic structure of lipase from Candida antarctica B
(CAL B, PDB code 5A71) was downloaded from RCSB Protein Data Bank
(PDB, http://www.rcsb.org/pdb/). Chain A of the crystallographic
structure was chosen (open conformation of CAL B [36] and rotated to
adjust fitting in searching grid box of AutoDockTools using PyMOL [37]
The pdb file was prepared by AutoDockTools, all nonstandard
(nonprotein) molecules including the two sugar units (NAG) and crystal
waters were removed, the polar hydrogen atoms were added, and Gas-
teiger charges were calculated. With use of AutoDockTools, a searching
grid box was set in the appropriate size before docking. The box center
was set at catalytic Ser105 of CAL B. Docking was performed in a 40 ×
yields of both enantiomers of all
99%). Beyond that, we demonstrated the limits of lipase-catalyzed
resolution for demanding substrates such as the
-isopropyl β-hydrox-
α-substituted β-hydroxyesters (up to
≥
α
yesters; in this case, the reaction time of acylation was unsatisfactory
and alcoholysis did not occur. We determined the absolute configuration
of all enantiomerically pure β-hydroxyesters according to the method of
MOSHER, which proved the (3R)-stereo-preference of Novozyme 435
(
N435) towards racemic
α-substituted β-hydroxyesters.
4
. Experimental
5
0 × 40 unit grid box centered on the enzyme active site (center_x =
-39.285, center_y = 29.55, center_z = -23.294) with a grid spacing of
.325 Å. Docking was performed with an exhaustiveness level of 100.
4
.1. Analytical and general procedures
0
1H-NMR spectra were recorded at 400 MHz and for 13C at 101 MHz
The docking results of each ligand were ranked based on free binding
energy (kcal/mol). The best twenty poses (modes) within an energy
range of 3 kcal/mol were selected according to AutoDock Vina scoring
functions mainly based on binding energies and show mutual affinity
(kcal/mol). Each selected binding mode was manually inspected in
order to select only productive conformations where the substrate as-
sumes a Near Attack Conformation (NAC) compatible with the attack of
the catalytic serine (Ser105) to the carbon atom of the acyl group of the
ligand. Each docking was repeated ten times for each substrate. The
analyses of interactions of docked ligand conformations in the CAL B
active site were performed and visualized with PyMOL.
with a Bruker Ascend NMR-spectrometer. As reference peak, TMS or the
deuterated solvent was used. All reactions were performed in oven-dried
glassware under a positive pressure of nitrogen and stirring with a
magnet. Liquid column chromatography was carried out on silica gel
(
40-63 µm mesh) using a solvent mixture of ethyl acetate and n-hexane
as eluent. Thin layer chromatography (TLC) was performed using 0.25-
mm E. Merck precoated silica gel plates (60F-254). Diastereomeric pu-
rity was determined by gas chromatography (GC) on an Agilent 7890A
system with flame ionization detector (FID). The GC device was
7

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
3
3
4
6
.4. Diastereoselective syntheses of
α
-substituted β-hydroxyesters rac-1 -
(sext, J = 7.4 Hz, 2H, CH
2
-CH
2
-CH
3
), 1.31 (d, J = 6.4 Hz, 3H, HOCH-
3
CH
3
), 1.29 (s, 9H, C(CH
3
)
3
), 0.93 (t, J = 7.4 Hz, 3H, CH
2 2 3
-CH -CH )
1
3
C-NMR (101 MHz, CDCl
138.71 (C-1 aromat.), 129.01 (C-3 aromat.), 128.30 (C-2 aromat.),
126.36 (C-4 aromat.), 80.68 (C(CH -C), 36.37
), 70.85 (β-C), 53.50 (
CH -CH -CH ), 34.34 (Ph-CH ), 27.87 (C(CH ), 18.42 (CH -CH
CH ), 17.80 (HOCH-CH ), 13.69 (CH -CH -CH
3
) δ in ppm: 172.68 (C=O), 171.37 (C=O),
Diastereoselective reduction starting from the corresponding
α
-substituted β-ketoesters was reported recently [20].
3
)
3
α
(
2
2
3
2
3
)
3
2
2
-
4
.5. General procedure for chemical acylation of -substituted
α
3
3
2
2
3
)
syn-Ethyl-3-acetoxy-2-isopropylbutanoate (11)
β-hydroxyesters rac-1 - 6
Yield: 474 mg (88%)
1
3
3
H-NMR (400 MHz, CDCl
3
) δ in ppm: 5.18 (dq, J = 7.6 Hz, J = 6.4
In a dry 50-mL reaction vessel, the respective hydroxyester (1 eq.)
was dissolved in absolute DCM (2.5 mL/mmol hydroxyester) and mixed
3
Hz 1H, β-CH), 4.22 - 4.11 (m, 2H, O-CH
-CH), 2.03 (s, 1H, CO-CH ), 2.03 - 1.94 (m, 1H, CH(CH ) ), 1.27 (t, J
2
-CH
3
), 2.49 (d, J = 7.8 Hz, 1H,
3
◦
α
with pyridine (3 eq.). Acid chloride (2 eq.) was added at 0 C. The
3
3 2
3
=
7.1 Hz, 3H, O-CH
2
-CH
3
), 1.27 (d, 3H, d, J = 6.2 Hz, 3H, HOCH-CH
3
),
mixture was stirred overnight at room temperature and then acidified
with HCl 1N to pH 3. Afterwards, the same amount of water was added.
The phases were separated and the aqueous layer was extracted two
3
3
0
.98 (d, J = 6.8 Hz, 3H, CH(CH
3
)
2
), 0.91 (d, J = 6.7 Hz, 3H, CH(CH
) δ in ppm: 172.43 (C=O), 170.23 (C=O),
-CH ), 56.28 ( -C), 21.24 (CO-CH ), 21.24
), 16.64 (HOCH-CH ), 14.39 (O-CH -CH
3 2
) )
1
3
C-NMR (101 MHz, CDCl
9.50 (β-C), 60.15 (O-CH
), 20.51 (CH(CH
3
6
2
3
α
3
2 4
more times with DCM. The organic phases were dried over Na SO ,
(
CH(CH
3
)
2
3
)
2
3
2
3
)
filtered and concentrated under vacuum. The crude product was purified
anti-tert-Butyl-3-(butyryloxy)-2-isopropylbutanoate (12)
by column chromatography (n-hexane: ethyl acetate = 95:5) using silica
Yield: 205 mg (77 %)
gel to obtain the
α-substituted β-hydroxyesters rac-7 - 12.
1
3
3
H-NMR (400 MHz, CDCl
3
) δ in ppm: 5.18 (dq, J = 7.9 Hz, J = 6.3
syn-Ethyl-3-(butyryloxy)-2-phenylbutanoate (7)
3
Hz 1H, β-CH), 2.29 – 2.19 (m, 3H,
α
-CH, CH
2
-CH
2
-CH
3
), 1.95 (oct, J =
Yield: 129 mg (95%)
3
1
6.8 Hz, 1H, CH(CH ) ), 1.64 (sext, J = 7.4 Hz, 2H, CH -CH -CH ), 1.45
H-NMR (400 MHz, CDCl
3
) δ in ppm: 7.36 - 7.24 (m, 5H, aromat.),
3 2
2
2
3
3
3
3
3
(s, 9H, C(CH ) ), 1.24 (d, J = 6.3 Hz, 3H, HOCH-CH ), 0.99 (d, J = 6.9
5
.56 (dq, J = 9.0 Hz, J = 6.2 Hz 1H, β-CH), 4.21 - 4.07 (m, 2H, O-CH
2
-
3 3
3
3
3
3
Hz, 3H, CH(CH ) ), 0.94 (d, J = 7.2 Hz, 3H, CH(CH ) ), 0.9 (t, J = 7.2
CH
3
), 3.71 (d, J = 9.0 Hz, 1H,
α
-CH), 2.11 - 1.97 (m, 2H, CH
2
-CH
2
-CH
3
),
3 2
3 2
1
.43 - 1.30 (m, 2H, CH
2
-CH
2
-CH
3
), 1.31 (d, 3J = 6.2 Hz, 3H, HOCH-
Hz, 3H, CH -CH -CH )
2
2
3
1
3
), 1.22 (t, 3J = 7.1 Hz, 3H, O-CH
), 0.72 (t, 3J = 7.4 Hz, 3H,
C-NMR (101 MHz, CDCl ) δ in ppm: 172.80 (C=O), 171.44 (C=O),
CH
CH
3
2
-CH
3
3
8
0.45 (C(CH
3
)
3
), 69.53 (β-C), 57.98 (
α
2 2 3
-C), 36.46 (CH -CH -CH ), 28.14
2
2 3
-CH -CH )
1
3
3 3 3 2 3 2 3 2
(C(CH ) ), 27.07 (CH(CH ) ), 21.21 (CH(CH ) ), 18.59 (CH(CH ) ),
C-NMR (101 MHz, CDCl
35.51 (C-1 aromat.), 128.78 (C-3 aromat.), 128.39 (C-2 aromat.),
27.61 (C-4 aromat.), 70.49 (β-C), 61.03 (O-CH -CH ), 57.37 ( -C),
6.25 (CH -CH -CH ), 18.79 (CH -CH -CH ), 18.26 (HOCH-CH ), 14.06
-CH ), 13.39 (CH -CH -CH
3
) δ in ppm: 172.57 (C=O), 171.28 (C=O),
1
8.45 (CH
2
-CH
2
-CH
3
), 18.11 (HOCH-CH
3 2 2 3
), 13.68 (CH -CH -CH )
1
1
3
2
3
α
4.6. General procedure for lipase screening of rac-1 - 6
2
2
3
2
2
3
3
(
O-CH
2
3
2
2
3
)
anti-Ethyl-3-(butyryloxy)-2-phenylbutanoate (8)
The respective lipase (6 mg) was weighed into a 1.5-mL vial and
suspended in n-hexane (1.2 mL). The hydroxyester (0.6 µmol) and vinyl
acetate (0.54 mmol, 50 µL) were added and allowed to react for 72 h in
Yield: 129 mg (95%)
1
H-NMR (400 MHz, CDCl
3
) δ in ppm: 7.37 - 7.27 (m, 5H, aromat.),
3
3
◦
5
.50 (dq, J = 10.5 Hz, J = 6.2 Hz 1H, β-CH), 4.19 - 4.04 (m, 2H, O-CH
2
-
-
an overhead shaker at 40 C. After 72 h, reaction was stopped by filtering
CH
CH
3
2
), 3.68 (d, 3J = 10.5 Hz, 1H,
α
-CH), 2.26 (t, 3J = 7.4 Hz, 2H, CH
2
the solution (45-µm pore size) to remove the enzyme. Enzymatic con-
version was followed by gas chromatography and the enantiomeric ra-
tios were determined by HPLC on chiral columns.
3
3
-CH
3
), 1.65 (sext, J = 7.3 Hz, 2H, CH
2
-CH
2
-CH
3
), 1.21 (t, J = 7.1
3
3
Hz, 3H, O-CH
2
-CH
.4 Hz, 3H, CH -CH
C-NMR (101 MHz, CDCl
35.00 (C-1 aromat.), 128.85 (C-3 aromat.), 128.63 (C-2 aromat.),
28.02 (C-4 aromat.), 71.72 (β-C), 60.91 (O-CH -CH ), 57.84 ( -C),
6.45 (CH -CH -CH ), 18.45 (CH -CH -CH ), 17.79 (HOCH-CH ), 14.11
-CH ), 13.63 (CH -CH -CH
3
), 1.04 (d, J = 6.2 Hz, 3H, HOCH-CH
3
), 0.95 (t, J =
7
2
2
-CH
3
)
1
3
3
) δ in ppm: 172.61 (C=O), 171.51 (C=O),
4.7. General procedure for enantioselective acylation of rac-1 - 6 with
Novozyme 435
1
1
3
2
3
α
2
2
3
2
2
3
3
Hydroxyesters 1 - 6 (1 eq.) were weighed into a sealed reaction
vessel, the vinyl donor (1.3 eq.) was added and the solution diluted with
n-hexane (1 mL/ 33 mg hydroxyester). Immobilized CAL B (0.15 mg/mg
hydroxyester) was supplemented and the suspension allowed reacting
(
O-CH
2
3
2
2
3
)
syn-Ethyl-2-benzyl-3-(butyryloxy)butanoate (9)
Yield: 514 mg (88%)
1
◦
H-NMR (400 MHz, CDCl
3
) δ in ppm: 7.28 - 7.26 (m, 2H, aromat.),
discontinuously in an overhead shaker at 40 C. Reaction control was
7
2
.21 - 7.14 (m, 3H, aromat.), 5.19 - 5.13 (m, 1H, β-CH), 4.08 - 3.97 (m,
performed by gas chromatography. Enantioselectivity was proven by
chiral HPLC. After completing the reaction, the immobilized enzyme
was separated by filtration and washed several times with n-hexane.
Afterwards, the reaction solution was concentrated under vacuum and
the acylated product purified by column chromatography (n-hexane:
ethyl acetate = 9:1 to 8:2) on silica gel.
3
H, O-CH
2
-CH
3
), 2.96 - 2.84 (m, 3H,
α
-CH, Ph-CH
2
), 2.28 (t, J = 7.4 Hz,
-CH -CH ), 1.29 (d,
), 1.10 (t, J = 7.1 Hz, 2H, O-CH -CH ), 0.96
-CH -CH
C-NMR (101 MHz, CDCl
) δ in ppm: 172.87 (C=O), 172.35 (C=O),
38.66 (C-1 aromat.), 128.77 (C-3 aromat.), 128.40 (C-2 aromat.),
26.43 (C-4 aromat.), 70.51 (β-C), 60.49 (O-CH -CH ), 57.37 ( -C),
6.41 (CH -CH -CH ), 34.71 (Ph-CH ), 18.45 (CH -CH -CH ), 17.72
), 14.07 (O-CH -CH ), 13.67 (CH -CH -CH
3
2
3
H, CH -CH
2
2
-CH
3
), 1.66 (sext, J = 7.3 Hz, 2H, CH
2
2
3
3
J = 6.4 Hz, 3H, HOCH-CH
3
2
3
3
(
t, J = 7.4 Hz, 2H, CH
2
2
3
)
1
3
3
1
1
3
(2R,3S)-Ethyl-3-hydroxy-2-phenylbutanoate ((2R,3S)-1)
Vinyl butanoate was used as vinyl donor. After 24 h of reaction,
(2R,3S)-1 was obtained as colorless oil.
2
3
α
2
2
3
2
2
2
3
(
HOCH-CH
3
2
3
2
2
3
)
Yield: 222 mg (44%); ee ≥ 99%
anti-tert-Butyl-2-benzyl-3-(butyryloxy)butanoate (10)
(2S,3S)-Ethyl-3-hydroxy-2-phenylbutanoate ((2S,3S)-2)
Vinyl butanoate was used as vinyl donor. After 10 h of reaction,
(2S,3S)-2 was obtained as colorless oil.
Yield: 304 mg (95%)
1
H-NMR (400 MHz, CDCl
3
) δ in ppm: 7.28 - 7.24 (m, 2H, aromat.),
3
7
.21 - 7.15 (m, 3H, aromat.), 5.13 (p, 1H, J = 6.4 Hz, 2H, β-CH), 2.99 -
.77 (m, 3H, -CH, Ph-CH ), 2.31 - 2.19 (m, 2H, CH -CH -CH ), 1.65
Yield: 87 mg (44%); ee ≥ 99%
2
α
2
2
2
3
(2R,3S)-Ethyl-2-benzyl-3-hydroxybutanoate ((2R,3S)-3)
8

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
Vinyl butanoate was used as vinyl donor. After 9 h of reaction,
2R,3S)-3 was obtained as colorless oil.
Yield: 207 mg (41%); ee = 99%
2S,3S)-tert-Butyl-2-benzyl-3-hydroxybutanoate ((2S,3S)-4)
× 20 mL) and the collected organic phases were washed with brine. The
(
2 4
etheric phase was dried over Na SO , filtered and concentrated under
vacuum. The crude product was purified by column chromatography (n-
hexane: ethyl acetate = 8:2) on silica gel to obtain (2R,3R)-6 as colorless
oil.
(
Vinyl butanoate was used as vinyl donor. After 24 h of reaction,
2S,3S)-4 was obtained as colorless solid.
(
Yield: 155 mg (72%); ee = 98%
Yield: 227 mg (45%); ee ≥ 99%
(
2R,3S)-Ethyl-3-hydroxy-2-isopropylbutanoate ((2R,3S)-5)
4
.9. General procedure for acylation reactions with (R)-MTPA-Cl
Vinyl acetate was used as vinyl donor. After 96 h of reaction, (2R,3S)-
was obtained as colorless oil.
5
In a 1.5-mL vial, the respective hydroxyester (66 µmol, 1 eq.) was
Yield: 201 mg (40%); ee = 97%
dissolved in absolute DCM (1 mL) and mixed with dry pyridine (22 µL,
(
2S,3S)-tert-Butyl-3-hydroxy-2-isopropylbutanoate ((2S,3S)-6)
2
70 µmol, 3 eq.) under nitrogen. (R)-MTPA-Cl (32 µL, 171 µmol, 2 eq.)
Vinyl acetate was used as vinyl donor. After 466 h of reaction,
was added under stirring at room temperature. The obtained mixture
was further stirred for two h (ethyl ester) or overnight (tert-butylester).
After the reaction had been completed, the mixture was partitioned
between diethyl ether (3 mL) and water (1 mL). The two phases were
separated in a separatory funnel. The aqueous phase was extracted with
diethyl ether (3 mL) and the combined organic phases were washed
again with a small amount of water. The organic phase was dried over
(
2S,3S)-6 was obtained as colorless oil.
Yield: 179 mg (36%); ee = 83%
4
.8. General procedure for enantioselective alcoholysis of 7 - 10 with
Novozyme 435 (N435)
Acyloxyester 7 - 10 (1 eq.) was weighed into a sealed reaction vessel
and 1-decanol (1.1 eq.) was added. The solution was diluted with n-
hexane (15 mL/ mmol acyloxyester). Immobilized CAL B (N435, 5 g/
mmol acyloxyester) was added to the mixture and the suspension was
2 4
Na SO , filtered and the solvent removed under vacuum. The crude (S)-
MTPA ester was further purified by column chromatography (n-hexane:
ethyl acetate = 9:1) on silica gel. The MTPA esters were obtained as
colorless oils in quantitative yields.
◦
allowed to react in an overhead shaker at 40 C discontinuously. Reac-
(2R,3S)-1-(S)-MTPA (13):
1
tion progress was controlled by gas chromatography. After stagnation of
the conversion, additional 1-decanol (0.2 eq.) was added. When no more
conversion was observed, the mixture was filtered and the immobilized
enzyme washed several times with n-hexane (for re-use). The filtrate was
concentrated under vacuum and the hydroxyester purified by column
chromatography (n-hexane: ethyl acetate = 9:1 to 8:2) using silica gel.
H-NMR (400 MHz, DMSO-d
6
) δ in ppm: 7.46 – 7.39 (m, 1H), 7.39 –
3 3
3
7.27 (m, 7H), 7.14 (d, J = 7.6 Hz, 2H), 5.73 (dq, J = 8.4 Hz, J = 6.1
3
3
Hz, 1H), 4.16 – 4.04 (m, 2H), 4.02 (d, J = 8.4 Hz, 1H), 3.12 (d, J = 1.2
3
3
Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H)
1
3
C-NMR (101 MHz, DMSO-d
6
) δ in ppm: 170.67, 165.03, 134.95,
1
131.59, 129.74, 128.90, 128.62, 128.50, 127.91, 126.76, 123.41 (q, JC-
2
(
2S,3R)-Ethyl-3-hydroxy-2-phenylbutanoate ((2S,3R)-1)
F
= 290 Hz), 84.13 (q, JC-F = 24 Hz), 73.48, 60.96, 55.37, 54.81, 17.90,
13.85
(2S,3R)-1-(S)-MTPA (14):
After 35 days of reaction (2S,3R)-1 was obtained as colorless oil.
Yield: 156 mg (65%); ee ≥ 99%
1
(
2R,3R)-Ethyl-3-hydroxy-2-phenylbutanoate ((2R,3R)-2)
H-NMR (400 MHz, DMSO-d
6
) δ 7.47 – 7.33 (m, 4H), 7.30 – 7.14 (m,
3
After 23 days of reaction (2R,3R)-2 was obtained as colorless oil.
Yield: 18 mg (48%); ee = 99%
7H), 5.81 – 5.73 (m, 1H), 4.08 – 4.00 (m, 3H), 3.24 (s, 3H), 1.30 (d, J =
3
6.3 Hz, 3H), 1.10 (t, J = 7.1 Hz, 3H)
1
3
(
2S,3R)-Ethyl-2-benzyl-3-hydroxybutanoate ((2S,3R)-3)
C-NMR (101 MHz, DMSO-d
6
) δ in ppm: 170.53, 164.84, 134.48,
1
After 33 days of reaction (2S,3R)-3 was obtained as colorless oil.
Yield: 117 mg (56%); ee = 97%
131.27, 129.62, 129.04, 128.44, 128.27, 127.66, 126.87, 123.51 (q, JC-
2
F
= 290 Hz), 84.06 (q, JC-F = 24 Hz), 73.07, 60.80, 55.14, 54.97, 18.03,
13.81
(2S,3S)-2-(S)-MTPA (15):
(
2R,3R)-tert-Butyl-2-benzyl-3-hydroxybutanoate ((2R,3R)-4)
After 13 days of reaction (2R,3R)-4 was obtained as colorless solid.
Yield: 143 mg (70%); ee ≥ 99%
1
H-NMR (400 MHz, DMSO-d
6
) δ in ppm: 7.53 – 7.43 (m, 5H), 7.42 –
3
(
2S,3R)-Ethyl-3-hydroxy-2-isopropylbutanoate ((2S,3R)-5)
7.28 (m, 5H), 5.63 (dq, J = 10.6 Hz, 6.2 Hz, 1H), 4.14 – 3.95 (m, 3H),
3
3
In a round bottom flask, acyloxyester (2S,3R)-11 (268 mg; 1.24
mmol) was dissolved in ethanol (10 mL) and aqueous KOH (1 N; 4.96
mmol; 4.96 mL; 4 eq.) was added at room temperature. The mixture was
stirred until no more starting material was observed (reaction control by
GC). After completion of the reaction (after about 3 h), ethanol was
removed under vacuum. The residue was extracted with diethyl ether (3
3.47 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H), 1.00 (d, J = 6.3 Hz, 3H)
13
C-NMR (101 MHz, DMSO-d
6
) δ in ppm: 171.26, 165.03, 134.37,
1
131.58, 129.95, 129.06, 128.59, 128.35, 128.25, 127.01, 123.05 (q, JC-
2
F
= 290 Hz), 84.13 (q, JC-F = 27 Hz) 74.41, 60.87, 55.35, 55.18, 16.59,
13.77
(2R,3R)-2-(S)-MTPA (16):
1
×
20 mL) and the collected organic phases were washed with brine. The
H-NMR (400 MHz, DMSO-d
6
) δ in ppm: 7.52 – 7.42 (m, 5H), 7.41 –
3
3
3
etheric phase was dried over Na SO , filtered and concentrated under
2
4
7.31 (m, 5H), 5.63 (dq, J = 10.7 Hz, J = 6.3 Hz, 1H), 3.95 (d, J = 10.5
3
3
vacuum. The crude product was purified by column chromatography (n-
hexane: ethyl acetate = 8:2) on silica gel to obtain (2S,3R)-5 as colorless
oil.
Hz, 1H), 3.92 – 3.79 (m, 2H), 3.49 (d, J = 1.1 Hz, 3H), 1.10 (d, J = 6.3
3
Hz, 3H), 0.98 (t, J = 7.1 Hz, 3H)
1
3
C-NMR (101 MHz, DMSO-d
6
) δ in ppm: 171.27, 165.03, 134.38,
1
Yield: 155 mg (72%); ee = 70%
131.58, 129.95, 129.06, 128.60, 128.35, 128.25, 127.01, 123.10 (q, JC-
2
(
2R,3R)-tert-butyl-3-hydroxy-2-isopropylbutanoate ((2R,3R)-6)
F
= 290 Hz), 84.14 (q, JC-F = 27 Hz), 74.41, 60.88, 55.35, 55.19, 16.59,
13.78
(2S,3R)-3-(S)-MTPA(17):
In a round bottom flask, acyloxyester (2S,3S)-12 (284 mg; 1.04
mmol) was dissolved in ethanol (15 mL) and aqueous KOH (1 N; 5.21
mmol; 5.21 mL; 5 eq.) was added at room temperature. The mixture was
1
H-NMR (400 MHz, DMSO-d
6
) δ in ppm: 7.49 (s, 5H), 7.31 – 7.24 (m,
◦
3 3
stirred at 50 C until no more starting material was observed (reaction
2H), 7.23 – 7.15 (m, 3H), 5.37 (qd, J = 6.3 Hz, J = 5.2 Hz, 1H), 3.94 (q,
3
3
3
control by GC). After completion of the reaction (after 3 h), ethanol was
removed under vacuum. The residue was extracted with diethyl ether (3
J = 7.1 Hz, 2H), 3.51 (s, 1H), 3.08 (dt, J = 9.4 Hz, J = 5.4 Hz, 1H),
3
3
2.92 – 2.81 (m, 2H), 1.30 (d, J = 6.4 Hz, 3H), 0.98 (t, J = 7.1 Hz, 3H)
9

C. Trapp et al.
Molecular Catalysis 509 (2021) 111578
13C-NMR (101 MHz, DMSO-d
31.41, 129.93, 128.71, 128.62, 128.33, 127.16, 126.41, 123.16 (q, JC-
) δ in ppm: 171.50, 165.06, 138.40,
6
Table 9
1
1
Chemical shifts of γ-CH
3
groups of MTPA-Esters 13-24.
2
= 289 Hz), 84.17 (q, JC-F = 27 Hz), 73.54, 60.26, 55.30, 50.81, 33.05,
1
2
F
MTPA
Ester
R
R
δ
Δδ
1
6.40, 13.72
(γ-CH
3
)
(γ-CH
3
)
[
ppm]
[ppm]
(
2R,3S)-3-(S)-MTPA (18):
1
13
Et
Ph
Ph
Bn
Bn
iPr
iPr
1.25
1.30
1.00
1.10
1.30
1.39
1.34
1.42
1.27
1.35
1.25
1.33
0.05
H-NMR (400 MHz, DMSO-d
6
) δ in ppm: 7.52 – 7.44 (m, 5H), 7.29 –
1
1
4
5
7
2
2
.15 (m, 3H), 7.12 – 7.04 (m, 2H), 5.37 – 5.27 (m, 1H), 3.91 – 3.84 (m,
Et
0.10
0.09
0.08
0.08
0.08
3
3
H), 3.50 (s, 3H), 2.98 (dt, J = 8.4 Hz, J = 6.3 Hz, 1H), 2.76 – 2.67 (m,
16
17
18
19
3
3
H), 1.39 (d, J = 6.4 Hz, 3H), 0.96 (t, J = 7.1 Hz, 3H)
Et
1
3
C-NMR (101 MHz, DMSO-d
6
) δ in ppm: 171.36, 165.05, 138.33,
1
tBu
Et
1
31.46, 129.94, 128.71, 128.63, 128.33, 127.08, 126.40, 123.16 (q, JC-
2
0
2
F
= 289 Hz), 84.17 (q, JC-F = 27 Hz), 73.54, 60.22, 55.30, 51.11, 33.05,
21
22
1
6.95, 13.72
2
3
tBu
(
1
2S,3S)-4-(S)-MTPA(19):
24
H-NMR (400 MHz, DMSO-d
6
) δ 7.54 – 7.43 (m, 5H), 7.33 – 7.24 (m,
3
2
2
H), 7.24 – 7.18 (m, 3H), 5.28 (p, J = 6.5 Hz, 1H), 3.49 (s, 2H), 2.98 –
2
3
3
.84 (m, 2H), 2.73 (dd, J = 13.4 Hz, J = 10.7 Hz, 1H), 1.34 (d, J = 6.3
Hz), 80.27, 72.59, 56.26, 55.08, 27.59, 26.37, 20.75, 18.39, 17.25
Table 9
Hz, 3H), 1.12 (s, 9H)
13C-NMR (101 MHz, DMSO-d
6
) δ in ppm: 170.66, 165.18, 137.99,
1
2
1
31.36, 129.93, 129.07, 128.60, 128.15, 127.20, 126.40, 123.16 (q, J =
Supplementary material
87.7 Hz), 84.15 (q, J = 27.0 Hz), 74.21, 55.30, 52.01, 34.32, 27.31,
7.15
Supplementary data to this article are available.
(
1
2R,3R)-4-(S)-MTPA (20):
H-NMR (400 MHz, DMSO-d
6
) δ 7.48 (s, 5H), 7.30 – 7.08 (m, 5H),
3
2 3
Declaration of Competing Interest
5
.26 (p, J = 6.4 Hz, 1H), 3.33 (s, 3H), 2.91 (ddd, J = 10.8 Hz, J = 6.9
3 2 3
Hz, J = 5.0 Hz, 1H), 2.77 (dd, J = 13.9 Hz, J = 4.8 Hz, 1H), 2.64 (dd,
2
3
3
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
J = 13.6 Hz, J = 10.8 Hz, 1H), 1.42 (d, J = 6.4 Hz, 3H), 1.07 (s, 9H)
1
3
C-NMR (101 MHz, DMSO-d
31.32, 129.91, 128.98, 128.63, 128.14, 127.13, 126.34, 123.16 (q, JC-F
287.7 Hz), 84.15 (q, JC-F = 27.0 Hz), 80.25, 74.04, 55.15, 51.87,
3.94, 27.26, 17.22
2R,3S)-5-(S)-MTPA (21):
6
) δ in ppm: 170.35, 165.09, 138.08,
1
=
Acknowledgments
3
(
1
This research has been funded by the European Social Fund (ESF,
project no. 100310513) and the Free State of Saxony (grant 100316181
for Christian Trapp). In addition, we would like to thank the members of
the project group BioPlatZ HSZG.
H-NMR (400 MHz, DMSO-d
6
) δ in ppm: 7.59 – 7.34 (m, 5H), 5.36 (p,
3
3
3
3
J = 6.5 Hz, 1H), 4.09 (dq, J = 10.8 Hz, J = 7.1 Hz, 1H), 3.99 (dq, J =
3
3
3
1
0.9 Hz, J = 7.1 Hz, 1H), 2.59 (dd, J = 8.1 Hz, J = 6.8 Hz, 1H), 2.00 –
3
3
1
3
.83 (m, 1H), 1.27 (d, J = 6.4 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H), 0.96 (d,
3
J = 6.8 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H)
1
3
C-NMR (101 MHz, DMSO-d
29.91, 128.59, 127.15, 123.14 (q, JC-F = 288 Hz), 84.15 (q, JC-F = 27
Hz), 72.46, 60.04, 55.18, 54.99, 27.11, 20.17, 19.51, 15.60, 13.92
2S,3R)-5-(S)-MTPA (22):
6
) δ in ppm: 171.42, 165.07, 131.36,
Supplementary materials
1
2
1
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.mcat.2021.111578.
(
1
H-NMR (400 MHz, DMSO-d
6
) δ 7.53 – 7.44 (m, 5H), 5.37 – 5.27 (m,
3
3
3 3
1
H), 4.06 (dq, J = 10.8 Hz, J = 7.1 Hz, 1H), 3.94 (dq, J = 10.8 Hz, J
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