Evaluation of a new protocol for enzymatic dynamic kinetic resolution of 3-hydroxy-3-(aryl)propanoic acids

Article abstract of DOI:10.1039/c5ob01380a

The application of tandem metal-enzyme dynamic kinetic resolution (DKR) is a powerful tool for the manufacture of high-value chemical commodities. A new protocol of kinetic resolution based on irreversible enzymatic esterification of carboxylic acids with orthoesters was introduced to obtain optically active β-hydroxy esters. This procedure was combined with metal catalyzed racemization of the target substrate providing both (R) and (S) enantiomers of ethyl 3-hydroxy-3-(4-nitrophenyl)propanoate with a high yield of 89% at 40°C. A substantial influence of the enzyme type, organic co-solvent, and metal catalyst on the conversion and enantioselectivity of the enzymatic dynamic kinetic resolution was noted.

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Evaluation of the new protocol to enzymatic dynamic kinetic
resolution of 3-hydroxy-3-(aryl)propanoic acids
Received 00th January 20xx,
Accepted 00th January 20xx
Dominik Koszelewski, Małgorzata Zysk, Anna Brodzka, Anna Żądło, Daniel Paprocki and Ryszard
Ostaszewski*
DOI: 10.1039/x0xx00000x
The application of tandem metal-enzyme dynamic kinetic resolution (DKR) is a powerful tool for the manufacture of high-
value chemical commodities. The new protocol of kinetic resolution based on irreversible enzymatic esterification of
carboxylic acids with orthoesters was introduced to obtain optically active β-hydroxy ester. This procedure was combined
with metal catalyzed racemization of the target substrate providing both (R) and (S) enantiomers of ethyl 3-hydroxy-3-(4-
nitrophenyl)propanoate with high yield 89% at 40 °C. A substantial influence of enzyme type, organic co-solvent, and
metal catalyst on conversion and enantioselectivity of the enzymatic dynamic kinetic resolution was noted.
www.rsc.org/
alcohols the racemization predominantly occurs through
hydrogen transfer process.11 Several problems must be
addressed, e.g. interference of transition metals with enzyme
Introduction
The synthesis of enantiomerically pure chemical commodities
is of great importance for both academia and pharmaceutical
industry.1 Large amount of natural products are chiral, non-
racemic compounds which biological activity is highly
dependent on their absolute configuration of stereogenic
centers. Nowadays, the most convenient way to obtain
enantiomerically pure compounds is still based on kinetic
resolution (KR). The major disadvantage of these methods is
the great loss of yield due to the nature of the kinetic
resolution process, as no more than 50% yield could be
theoretically achieved while maintaining high enantiomeric
purity. If the racemization of the slowly reacting enantiomer
can occur concurrently with the kinetic resolution, then
theoretically 100% of racemic mixture can be converted into
one enantiomer, what is known as dynamic kinetic resolution
(DKR).2 Over the past decade, the procedures employing
enzymes have been intensively explored for the efficient DKR.3
The racemization of a substrate can occur through various
pathways, such as: thermal racemization,4 base-catalyzed
racemization,5 acid-catalyzed racemization via Schiff bases,6
enzyme-catalyzed racemization,7 racemization via redox and
radical reactions.8 The classical and commonly applied tandem
metal-enzyme DKR is based on sec-alcohol racemization and
enzymatic acylation of preferred enantiomer.9 It is well known,
that the several complexes of rhodium, iridium and ruthenium,
among others, are capable to catalyze rapid racemization of
various alcohol substrates, but only few have proved
structure, selection of an appropriate solvent and temperature
to achieve efficient enzymatic DKR.12
The DKR procedure can be used also for functionalized
alcohols, which are versatile building blocks in organic
synthesis.13 Enantiomerically enriched active β-hydroxy esters
and the derivatives thereafter are synthetically important
chiral synthons, of which the chiral β-hydroxy-β-
arylpropionates are precursors of Fluoxetine,14 β-lactam
antibiotics,15 Tuckolidean HMG CoA reductase-inhibitor.16
Chiral β-hydroxy esters are also used as a starting materials in
the preparation of enantiomerically pure β-blockers.17 In view
of these medicinal importance, synthetic studies of these
compounds have attracted considerable interest. Several
chemical procedures have been explored to provide the routes
to them. Two common approaches include the aldol
reactions18 as well as catalytic asymmetric hydrogenation.19
Enzyme
Acyl donor
O
OH
O
OAc
R1O
R
R1O
R
Fast
Ru(II)-catalyzed
racemization
Enzyme
Acyl donor
O
OH
R
O
OAc
R1O
R1O
R
Slow
R1= Me, Et
compatible with an enzymatic reaction.10 In the case of Figure 1 DKR of racemic β-hydroxy esters.
The enzymatic methods are available as well. They comprise
the asymmetric reduction of ketones20 and the lipase-
catalyzed kinetic resolution of racemic β-hydroxy esters.21 All
of these chemical and enzymatic methods have several
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disadvantages. In particular, the enzymatic kinetic resolution In the reactions catalyzed by Amano AK polarity of the solvent
has a serious limitation that the theoretical maximum yield is did not influence the yield of the reaction, but strongly
50% for the wanted enantiomer. New methods providing affected enantioselectivity E (Table 1, entries 1-4) leading in
enantiomerically pure compounds based on tandem metal- tert-butyl methyl ether (TBME) to the optically pure product
enzyme dynamic kinetic resolution are still of high interest due (S)-2a with high enantioselectivity >200 (Table 1, entry 4).
to the concepts of asymmetric synthesis. Bäckvall and co- Additionally, different ethoxy group donors were tested;
workers have extensively studied DKR of α- and β-hydroxy (diethoxymethyl)benzene, 1-(triethoxymethoxy)ethane and
esters (Figure 1). Authors applied transesterification in 1,1,1-triethoxyethane using TBME as a solvent (Table 1, entries
cyclohexane using immobilized PS-C as the biocatalyst and p- 5-7). In all cases the ester (S)-2a was obtained substantially
chlorophenyl acetate as the acyl donor.22 Under these lower enantioselectivity, exceptionally reaction with
conditions appropriate products were obtained in moderate to (diethoxymethyl)benzene lead to product (S)-2a with slightly
good yields and enantioselectivity. Nevertheless, this DKR higher isolated yield 41% (Table 1, entry 5).
reaction requires high temperature, long time (up to seven
days) and anhydrous conditions. There still remains some Table
1 Kinetic resolution of racemic 3-hydroxy-3-(4-
limitations in DKR of β-hydroxy esters: firstly, the Shvo’s nitrophenyl)propanoic acid (1a) with Amano AK in various
diruthenium complexes23 are not easily available because of organic solvents, the partition coefficient (LogP) and dielectric
the expense; secondly, the racemic temperature is mostly constant (εr)a
higher than the optimum reaction temperature for enzyme,
which may decrease the enzyme activity last but not least, the
boiling point of the acyl donor p-chlorophenyl acetate is high
what hinders product purification. The former method relies
on the enzymatic acylation of β-hydroxy moiety, an obvious
drawback of it is necessity to perform an additional not trivial
step to recover hydroxy group towards further
functionalization. Thus, it seems significant to find a method to
overcome these limitations.
LogP
(εr)
yielde
(%)
eef
(%)
Eg
Entry Solvent
2.30
(2.38)
2.70
(2.20)
4.25
(2.80)
1
2
3
toluene
29
31
37
51 (S)
5
cyclohexane
isopentylether
82 (S) 14
96 (S) 87
Results and Discussion
Herein, we report our results on the novel approach to the
synthesis of optically active β-hydroxy esters applying
corresponding β-hydroxy acids which are readily available
from corresponding aldehydes in straightforward synthesis.18
We first investigated the enzymatic kinetic resolution of
selected racemic β-hydroxy acids. Recently we have shown
that orthoesters can be successfully used for enzymatic
esterification of carboxylic acids.24 It is well known that
orthoesters trap the water through hydrolysis and therefore
prevent the reverse reaction, and at the same time provide the
nucleophile for the esterification.25 To the best of our
knowledge presented approach has been never used for
kinetic resolution of racemic β-hydroxy acids
37
41
15
17
99 (S)
38 (S)
90 (S)
78 (S)
>200
4
5b
6c
7d
1.40
(2.60)
3
TBME
22
9
aReaction conditions: 0.1mmolof 1a, ethoxy group donor 2
equiv., solvent (2.0 mL), Amano AK (5 mg).
b(diethoxymethyl)benzene.
c1-(triethoxymethoxy)ethane.
d1,1,1-triethoxyethane. eIsolated yield. fDetermined by the
chiral HPLC (Chiralpak IA). gEnantioselectivity, E=ln[1-
c(1+eep)]/ln[1-c(1-eep)]29
Separation of two enantiomers from racemic mixture is of
great importance, particularly in the pharmaceutical industry,
because of the different pharmacological activities and
pharmacokinetic characteristics of each enantiomer.30
Addressing this problem we were interested in finding enzyme
which can provide opposite (R)-enantiomer of the studied
ester 2a. Screening of biocatalysts was performed using over
20 commercially available enzymes. Only four out of these
tested catalysts were active in kinetic resolution of β-hydroxy
acid 1a; immobilized lipase from Candida antarctica (Novozym
435), immobilized lipase from Pseudomonas cepacia (Amano
PS-C I), lipase from wheat germ and domestic made goose liver
acetone powder (GLAP) (Table 2).
For the initial studies under EKR lipase from Pseudomonas
fluorescence (Amano AK), triethyl orthobenzoate and toluene
as a solvent were employed. All reactions were conducted at
40 °C for 48 hours in 0.1 mmol scale and obtained esters
were isolated before subjected to further HPLC analysis (Table
1). Racemic 3-hydroxy-3-(4-nitrophenyl)propanoic acid (1a
2
)
which ester 2a is a progenitor for the anti-malarial drugs26 and
hypoxia-selective cytotoxic agent (HSAs)27 has been used as a
model substrate for kinetic resolution.
As shown in Table 1, Amano AK enzyme seems to have a
potential for the kinetic resolution of racemic β-hydroxy acid
1a leading to enantiomerically enriched ester (S)-2a with 51%
ee and 29% isolated yield (Table 1, entry 1). The choice of the
proper organic solvent can be critical, because selectivity and
activity of used enzyme may change significantly.28
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In all cases the strong influence of solvent lipophilicity (LogP) reaction with acid 1a was filtered off and applied again (five
as well as polarity (εr) on the reaction course has been times) without loss of activity. Lipase from Pseudomonas
observed.
cepacia (Amano PS-C I) was more effective in polar solvents
leading to ester (S)-2a with low enantioselectivity (Table 2,
Table
2
Kinetic resolution of racemic 3-hydroxy-3-(4- entries 5 and 6). In the reactions catalyzed by lipase from
nitrophenyl)propanoic acid (1a) in different organic solvents wheat germ the yield increases with the polarity of the solvent
with various enzymesa
and was the highest in solvent with lowest partition
coefficient. On the other hand enantioselectivity of the
reaction catalyzed by this enzyme in different solvents
remained very low (Table 2, entries 9-12). GLAP were most
effective in nonpolar solvents considering enantioselectivity,
the yield of the reaction remained similar in almost all cases
leading to ester 2a with 94% enantiomeric excess what
corresponds to enantioselectivity value 56 (Table 2, entries 17-
20). Novozym 435, lipase from wheat germ and GLAP provided
product 2a with (R)-configuration. Next, the series of
yieldc eed
Entry
Enzyme
Solvent
TBME
Ee
(%)
(%)
42
19 (R)
-
1
experiments with different β-hydroxy acids 1b-i were
isopentylether 43
51 (R)
50 (R)
4
4
2
performed (Scheme 1). Applying previously used enzymes in
TMBE and toluene, it turned out that only Novozyme 435 in
toluene at 40 °C provides corresponding enantiomerically
Novozym 435
cyclohexane
toluene
39
41
31
3
enriched esters 2b
the groups attached to the phenyl ring. In case of three other
enzymes products 2b has not been observed on TLC after 72
-i with low enantioselectivities regardless of
95 (R) 78
76 (S) 10
4
TBME
5
-i
hours. Obtained results are shown in Table 3.
isopentylether 32
31 (S)
rac
2
-
6
Amano PS-C I
Table 3 Kinetic resolution of rac-3-hydroxy-3-arylpropanoic
acids1b-
ia
cyclohexane
toluene
28
29
45
7
rac
-
8
TBME
rac
-
9
Wheat germ
lipase
isopentylether 31
9 (R)
21 (R)
rac
1
2
-
10
11
12
13
14
15
16
cyclohexane
toluene
19
22
39
yieldb
(%)
eec
(%)
Entry
1
R
Product
Ed
4-CNC6H4
2b
2c
2d
2e
2f
29
18
42
31
39
<1
42
45
58 (R)
83
5
13
3
7
6
-
TBME
rac
-
2
3
4
5
6
7
8
2,4-(NO2)2C6H3
4-F-2-NO2C6H3
4-CF3-2-NO2C6H3
4-MeOC6H4
2-BrC6H4
isopentylether 47
rac
-
GLAPb
52
cyclohexane
35
33 (R)
2
62
toluene
37
94 (R) 56
60 (R)
-
aReaction conditions: 0.1mmolof 1a, triethyl orthobenzoate 2
equiv., solvent(2.0 mL), enzyme (native 5 mg or immobilized
10 mg). bDomestic made goose liver acetone powder. cIsolated
yield.d Determined by the chiral HPLC (Chiralpak IA).e
Enantioselectivity, E=ln[1-c(1+eep)]/ln[1-c(1-eep)].29
2g
2h
2i
2-FC6H4
68 (R)
63 (R)
4
4
Ph
aReaction conditions: 0.1mmolof
1, triethyl orthobenzoate 2
As shown in Table 2, Novozym 435 catalyzed esterification of
equiv., toluene (2.0 mL), Novozym 435 (10 mg). bIsolated yield.
cDetermined by the chiral HPLC (CHIRALPAK IA, OD-H, OB).
dEnantioselectivity, E=ln[1-c(1+eep)]/ln[1-c(1-eep)].29
acid 1a
, in contrast to Amano AK in TBME solvent
corresponding ester 2a was obtained as a racemate (Table 2,
entry 1). Also reactions performed in isopentyl ether or
cyclohexane showed very low enantioselectivity below 5
(Table 2, entries 2 and 3). The highest enantioselectivity 78 has
been observed applying toluene as a solvent what lead to
desired product 2a with 41% yield and high 95% enantiomeric
excess (Table 2, entry 4). Finally, the catalyst used in the
Obtained results applying Novozym 435 as biocatalyst indicate
that there are specific substrate requirements which cannot be
easily determined. The results indicated that the most suitable
substrates for the enzymatic reactions are these with strong
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electron withdrawing group, which offer the best compromise racemize in the presence of Amano AK lipase (Table 4, entry 1).
between conversion and selectivity (Table 3, entry 7). In case Under conditions proposed by Williams et al.12a significant
of derivative 1c with two strong electron withdrawing nitro racemization of (S)-2aproceeded(Table 4, entry 2).Also, in the
groups corresponding ester 2c was isolated with substantially presence of triethyl orthobenzoate and ruthenium catalyst
lower yield and 83% enantiomeric excess (Table 1, entry 7). exclusively, slight decrease in enantiomeric excess was
Moreover, the size of substituent attached to the ortho observed (Table 4, entries3 and 6).
position of the phenyl ring cannot exceed the size of the active
pocket in the enzyme (Table 1, entries 3 and 4).
Table
has nitrophenyl)propanoate ((S)-2a
2 is
4
Racemization of ethyl (S)-3-hydroxy-3-(4-
a
To the best of our knowledge, DKR of β-hydroxy acids
1
)
not been reported but corresponding β-hydroxy esters
well recognized and is based on enzymatic acylation of
hydroxyl group.22
In the next step we investigated the racemization of (S)-3-
hydroxy-3-(4-nitrophenyl)propanoic acid ((S)-1a). Optically
active acid (S)-1a (
ꢀαꢁꢃꢂꢄ= −30.2) obtained via hydrolysis of
Entry
Conditions
Amano AK
yieldb (%)
eec (%)
ester (S)-2a has been placed in TMBE at 40 °C together with
rhodium(II) acetate (Rh2(OOCCH3)4). This catalysts has been
used with success by Williams et al. in enzymatic DKR of 1-
phenylethanol.12a Applying rhodium(II) acetate together with
KOH, o-phenanthroline and acetophenone caused partial
1
2
3
4
5
6
7
>99
97
99 (S)
63(S)
94 (S)
99 (S)
99 (S)
96 (S)
99 (S)
PhCOMe, o-phenanthroline
Rh(II)
>99
>99
>99
>99
>99
racemization of (S)-1a(
ꢀαꢁꢃꢂꢄ= −12.7) within 24 hours in TMBE
Amano AK, Rh(II)
at 40 °C. Detailed studies shown that full racemization of (S)-1a
within 4 hours in TMBE at 40 °C can be achieved by applying
nothing but rhodium(II) acetate. Since the earliest
preparations of Rh2(O2CR)4L2 complexes, it was recognized that
rhodium(II) acetate readily exchange the bridging acetate
groups in the presence of other carboxylic acids what may
explain high efficiency of Rh(II) catalyst in DKR of β-hydroxy
acid.33 The exchanges of the bridging acetate groups with acid
1a can be followed by Uv-Vis spectroscopy. Hypsochromic shift
together with decreasing in infelicity can be observed in case
of formed rhodium complex with acid 1a (Fig. 1). Presented
absorption spectra were recorded after 2 hours of agitation at
ambient temperature in CDCl3 using the same concentration of
PhC(OEt)3, Amano AK
PhC(OEt)3, Rh(II)
PhC(OEt)3, Amano AK, Rh(II)
aReaction conditions: 0.1mmolof (S)-2a, ethoxy group donor 2
equiv., TMBE (2.0 mL), Amano AK (5 mg), metal catalyst (10
mol%). bIsolated yield. cDetermined by the chiral HPLC
(Chiralpak IA).
Surprisingly, application of lipase from Amano AK suppresses
the racemization of product (S)-2a (Table 4, entries 4, 5 and 7).
Presence of triethyl orthobenzoate does not promote the
racemization course of ester (S)-2a which after all was isolated
with any loss in enantiomer excess (Table 5, entry 4 and 5). In
all cases substrate 2a was recovered quantitatively.
acid 1a
.
Figure 1 Absorption spectra of free and rhodium complex with
acid 1a recorded in CDCl3.
The next step our studies was to combine the racemization
and the enzymatic transformation and run the reaction in one
pot. Further experiments were assigned with Amano AK lipase
and rhodium(II) acetate in four different organic solvents at 40
°C for 72 hours (Table 4).In all cases product (S)-2a was
obtained with higher yield (more than 67%) comparing to EKR.
The type of the solvent strongly influences the yield and
enantiomeric excess (Table 5, entries 1-3). Enantiomerically
pure ester (S)-2a with 89% isolated yield was provided from
the reaction performed in TBME which was also the best
solvent for kinetic resolution applying lipase from Amano AK
(Table 5, entry 4). Lipase Amano AK was easily recovered by
filtration and rhodium catalyst by recrystallization from
methanol34 with 90% yield and reused up to 3 times, without
loss of activity. In the course of the studied reaction acylation
of the free hydroxyl group of ester 2a by a second hydroxy
ester molecule did not take place what was verified by mass
spectroscopy.
Some more aspects of racemization process has been studied
extensively. In order to determine the influence of rhodium
catalyst, enzyme and triethyl orthobenzoate on the
racemization of enantiomerically pure ester (S)-2a additional
experiments were performed (Table 4). Ester (S)-2a does not
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Table 5 Dynamic kinetic resolution of racemic3-hydroxy-3-(4- Table
6
Racemization of ethyl (S)-3-hydroxy-3-(4-
nitrophenyl)propanoic acid (1a) catalyzed by Amano AK and nitrophenyl)propanoate ((S)-2a) with various metal catalystsa
Novozym 435a
yieldb(%)
78
eec (%)
48 (S)
84 (S)
56 (S)
99 (S)
97 (R)
48 (R)
50 (R)
9 (R)
Entry
Metal catalyst
yieldb (%)
91
eec (%)
Entry
1
Enzyme
Solvent
35 (S)
1
2
3
4
[Pd(PPh3)4]/dppf
PdCl2(dppf)
toluene
88
69
82
41 (S)
55 (S)
2
3
4
5
6
7
8
cyclohexane
73
Amano AK
Ru(p-cymene)Cl2]2
Shvo’s catalyst
isopentylether 67
49 (S)
TBME
89
78
87
aReaction conditions: 0.1 mmol of (S)-2a
,
triethyl
orthobenzoate 2 equiv., TMBE (2.0 mL), Amano AK (5 mg),
metal catalyst (10 mol%), t-BuOK (10 mol%). bIsolated yield.
cDetermined by the chiral HPLC (Chiralpak IA).
toluene
cyclohexane
Novozyme
435
isopentylether 91
TBME 69
To show the applicability of this concept, other β-acids 1c and
1i were subjected to DKR with Novozyme 435 leading to
corresponding esters (R)-2c and (R)-2i with 98% yield and
enantiomeric excess values identical with those obtained via
enzymatic kinetic resolution (Table 3, entries 2 and 8). Ethyl
(R)-3-hydroxy-3-phenylpropanoate (2i) is a precursor of the
antidepressant Fluoxetine also known by the trade names
Prozac.35
Reaction conditions: 0.1 mmol of 1a, triethyl orthobenzoate 2
equiv., solvent (2.0 mL), enzyme (Amano AK 5 mg or Novozym
435 10 mg), metal catalyst (10 mol%). bIsolated yield.
cDetermined by the chiral HPLC (Chiralpak IA).
Further experiments were assigned with immobilized lipase
from Candida antarctica (Novozyme 435).
As can be notice, appropriate combination of enzyme, metal
and organic solvent is required for successful enzymatic DKR
procedure. Further studies under optimization and
amplification of herein proposed protocol are still underway.
In spite of described limitations proposed approach diversifies
already knew methods and may lead to optically pure β-
hydroxy ester with very high yields.
Considering strong influence of organic solvent on the reaction
course observed in EKR the next set of experiments were
performed in different organic solvents with Novozym 435. In
the case of reactions with rhodium catalyst the most effective
were cyclohexane and isopentyl ether leading to optically
enriched (48% and 50% ee) ester (R)-2a with 87% and 91%
yield, respectively (Table 5, entries 6 and 7). Only in case of
using TBME the yield and enantiomeric excess of the reaction
were lower and dropped to 69% and 9%, respectively (Table 6,
entry 8). Nevertheless, ester (R)-2a was obtained with isolated
yield 78% and very high enantiomeric excess 97%(Table 6,
entry 1).
Conclusions
In summary, we have established the new protocol to
enantiomerically enriched β-hydroxy esters based on
enzymatic kinetic (EKR).Afterwards this protocol has been
extended to tandem metal-enzyme dynamic kinetic resolution
(DKR) of β-hydroxy acids providing optically pure product. The
whole process does not require anhydrous conditions,
moreover dehydration of the product is not observed. The role
of organic solvent and biocatalyst is crucial. Change of
biocatalyst and polarity of the solvent makes possible to
control (in respect to enantioselectivity) EKR and DKR. Under
optimal conditions both enantiomers of ester 2a were
obtained with very high isolated yield. It was demonstrated
that the high enantioselectivity of the enzyme is compatible
with the rhodium catalyzed racemization of β-hydroxy acids.
The protocol reported here should be a useful complement to
known methods. Moreover, the easy recovery of both catalyst
makes this process suitable for up scaling.
Also other commonly used in DKR metal catalysts(e.g. Shvo’s
Catalyst,31[Pd(PPh3)4]/dppf,
cymene)Cl2]2/dppb)32 were tested. In all cases, reaction
performing under DKR conditions leads to enhancement in the
reaction yield (Table 6).
PdCl2(dppf),
and
[Ru(p-
At the same time the enantiomeric excess of the ester 2b was
much lower than in EKR procedure what was anticipated due
to literature reports about DKR of the β-hydroxy esters.12,22
Finally, we applied this transformation concept on
a
preparative scale. Thus, 212 mg (1 mmol) of racemic 3-
hydroxy-3-(4-nitrophenyl)propanoic acid (1a) was transformed
into optically pure ester (S)-2a within 72 h, and with 94% yield
of isolated product. Scaling up to 10 mmol of the substrate 1a
is possible and also very effective.
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Experimental
Notes and references
‡We would like to dedicate this paper to professor Kurt Faber on
occasion of his 62th birthday.
Materials and general methods
All the chemicals were obtained from commercial sources and
the solvents were of analytical grade. Amano lipase PS-C I from
Pseudomonas cepacia (immobilized on ceramic) and Amano
lipase AK from Pseudomonas fluorescens were purchased from
Sigma-Aldrich. Novozym 435 was purchased from Novo
Nordisk. Goose liver was converted to the acetone powder
(GLAP) by the method of Connors et al.36 Column
chromatography was performed on Merck silica gel 60/230-
400 mesh. The enantioselectivity parameter E was calculated
using equation: E=ln[1-c(1+eep)]/ln[1-c(1-eep)]. E calculations
were performed for isolated yields of corresponding products
1
a) M.C. Nunez, M.E. Garcia-Rubino, A. Conejo-Garcia, O.
Cruz-Lopez, M. Kimatrai, M. A. Gallo, A. Espinosa, J.M.
Campos, Curr. Med. Chem. 2009, 16, 2064-2074; b) R.N.
Patel, Coord. Chem. Rev. 2008, 252
Murakami, Top. Curr. Chem. 2007, 269, 273-299; d) R.N.
Patel, Curr. Opin. DrugDisc. Dev. 2006, , 741-764; e) R.N.
, 659-701; c) H.
9
Patel, Curr. Org. Chem. 2006, 10, 1289-1321.
a) R. Noyori, M. Tokunaga, M. Kitamura, Bull. Chem. Soc. Jpn.
1995, 68, 36-56; b) R.S. Ward, Tetrahedron: Asymmetry1995,
2
6
, 475-1490.
3
4
5
a) O. Pamies, J. E. Bäckvall, Trends Biotechnol. 2004, 22, 130-
135; b) H. Pellissier, Tetrahedron2011, 67, 3769-3802.
K.R. Wilson, R.E. Pincock, J. Am. Chem. Soc. 1975, 97, 1474;
b) L.G. Hutchins, R.E. Pincock, J. Org. Chem. 1982, 47, 607.
S.K. Hsü, C.K. Ingold, C.L. Wilson, J. Chem. Soc. 1938, 78-81;
b) M. Aurally, G. Lamaty, Bull. Soc. Chim. Fr. 1980, 389; c) S.
Borg, G. Estenne-Bouhtov, K. Luthman, J. Csoregh, W.
Hesselink, U. Hacksell, J. Org. Chem. 1995, 60, 3112.
a) J.C. Clark, G.H. Phillipps, M.R. Steer, J. Chem. Soc. Perkin
Trans. 11976, 475.
2a-i. Enzymatic reactions were performed in a vortex
(Heidolph Promax 1020) equipped with incubator (Heidolph
Inkubator 1000). To prove the ability of the established
protocol each reaction was repeated at least three times.
6
7
General procedure for enzymatic kinetic resolution of racemic 3-
hydroxy-3-(aryl)propanoic acids
a) D. Koszelewski, B. Grischek, S.M. Glueck, W. Kroutil, K.
Faber, Chem. Eur. J. 2011, 17, 378-383; b) K. Soda, T. Osumi,
MethodsEnzymol. 17B1971, 629; c) D. Roise, K. Soda, T. Yagi,
C.T. Walsh, Biochemistry1984, 23, 5195.
To the solution of acid 1a-i (0.1 mmol) in organic solvent (2
mL), triethyl orthobenzoate (2 equiv.) and enzyme (native 5
mg or immobilized 10 mg) were added in 5 mL screwed vial.
The reaction mixture was stirred for 48 hours at 40 °C. After
cooling, crude product was purified by column
chromatography (ethyl acetate/hexanes). The 1H NMR data
were in accordance with those recorded for racemates; (S)-(-)-
8
9
a) P.M. Dinh, J.A. Howarth, A.R. Hudnott, J.M.J. Williams, W.
Harris, TetrahedronLett. 1996, 37, 7623; b) J.V. Allen, J.M.J.
Williams, TetrahedronLetters1996, 37, 1859; c) E.J. Ebbers,
G.J.A. Ariaans, J.P.M. Houbiers, A. Bruggink, B. Zwanenburg,
Tetrahedron1997, 53, 9417-9476.
a) S. Akai, K. Tanimoto, Y. Kanao, M.Egi, T. Yamamoto, Y.
Kita, Angew. Chem. Int. Ed. 2006, 118, 2654-2657; b) S. Akai,
K. Tanimoto, Y. Kanao, M. Egi, T. Yamamoto, Y. Kita, Angew.
Chem. Int. Ed. 2006, 45, 2592-2595; c) S. Akai, R. Hanada,
N.Fujiwara, Y. Kita, M. Egi, Org. Lett. 2010, 12, 4900-4903; d)
M. Egi, K. Sugiyama, M. Saneto, R. Hanada, K. Kato, S. Akai,
Angew. Chem. Int. Ed. 2013, 52, 3654-3658.
2a
(c 1.0, CHCl3, 95% ee); Lit (R)-enantiomer: ꢀαꢁꢃꢂꢄ= +23.1 (c 1.0,
CHCl3);37(+)-2b ꢀαꢁꢂꢃꢄ= +23.0 (c 1.00 CHCl3, 58% ee); (+)-2c
ꢀαꢁꢃꢂꢄ= +25.2 (c 1.00 CHCl3, 83% ee); (+)-2d ꢀαꢁꢃꢂꢄ= +16.3 (c
1.00 CHCl3, 52% ee); (+)-2e
ꢀαꢁꢃꢂꢄ= +12.8 (c 1.00 CHCl3, 62%
ee); (R)-(+)-2f
ꢀαꢁꢃꢂꢄ= +21.5 (c 1.00 CHCl3, 60% ee), Lit (R)-
enantiomer: ꢀαꢁꢂꢃꢄ = +28.8 (c 1.0, CHCl3);38(R)-(+)-2h ꢀαꢁꢂꢃꢄ
+34.6 (c 1.00 CHCl3, 68% ee), Lit (S)-enantiomer: ꢀαꢁꢃꢂꢄ= -49.7 (c
: :
ꢀαꢁꢃꢂꢄ= −59.5 (c 1.5, CHCl3, 99% ee), (R)-(+)-2a ꢀαꢁꢃꢂꢄ= +57.5
:
:
:
:
:
10 a) O. Verho, J.E. Bäckvall, J. Am. Chem. Soc. 2015, 137, 3996-
4009; b) V. Gotor, I. Alfonso, E. Garcia-Urdiales, Asymmetric
Organic Synthesis with Enzymes, Wiley-VCH, Weinheim, 2008
pp. 92-94; c) J. H. Lee, K. Han, M. -J. Kim, J. Park, Eur. J. Org.
Chem. 2010, 999-1015.
:
=
2.71, CHCl3);39(R)-(+)-2i ꢀαꢁꢂꢃꢄ= +40.2 (c 1.00 CHCl3, 63% ee), Lit
:
(R)-enantiomer: ꢀαꢁꢃꢂꢄ= +48.9 (c 0.89, CHCl3).40
11 a) M.C. Warner, J.E. Bӓckvall, Acc. Chem. Res. 2013, 46, 2545-
2555; b) B. Martín-Matute, M. Edin, K. Bogár, F. B. Kaynak,
J.E. Bäckvall, J. Am. Chem. Soc. 2005, 127, 8817-25; c) B.
Martín-Matute, J.B. Aberg, M. Edin, J.E. Bäckvall,
Chemistry2007, 13, 6063-6072.
General procedure for enzymatic dynamic kinetic resolution of
racemic 3-hydroxy-3-(aryl)propanoic acids
To the solution of acid
1 (0.1 mmol) in organic solvent (2 mL),
12 a) P.M. Dinh, J.A. Howarth, A.R. Hudnott, J.M.J. Williams, W.
Harris, Tetrahedron Lett. 1996, 42, 7623-7626; b) B.A.
triethyl orthobenzoate (2 equiv.), enzyme (native 5 mg or
immobilized 10 mg) and metal catalyst (10 mol%) were added
in 5 mL vial. The reaction mixture was stirring for 72 hours at
40 °C. After cooling, crude product was purified by column
chromatography (ethyl acetate/hexanes).
Persson, A.L.E. Larsson, M. Le Ray, J.E. Bӓckvall, J. Am. Chem.
Soc. 1999, 121, 1645-1650; c) L. Borén, B. Martín-Matute, Y.
Xu, A. Córdova, J.E. Bäckvall, Chemistry2005, 12, 225-232; d)
M.J. Kim, Y.I. Chung, Y.K. Choi, H.K. Lee, D. Kim, J. Park, J. Am.
Chem. Soc. 2003, 125, 11494-11495; e) J.V. Allen, J.M.J.
Williams, Tetrahedron Lett. 1996, 37, 1859-1862; f) A.L.E.
Larsson, B.A. Persson, J.E. Bӓckvall, Angew. Chem. Int. Ed.
1997, 36, 1211-1212.
Acknowledgements
13 O. Pamies, J.E. Bӓckvall, Chem. Rev. 2003, 103, 3247-3261.
14 a) R. Chenevert, G. Fortier, Chem. Lett. 1991, 1603-1606; b)
E.J. Corey, G.A. Reichard, Tetrahedron Lett. 1989, 30, 5207-
5210; c) A. Kumar, D. H. Ner, S.Y. Dike, Tetrahedron Lett.
1991, 32, 1901-1904.
15 L. Banfi, G. Cascio, C. Ghiron, G. Guanti, E. Manghisi, E.
Narisano, R. Riva, Tetrahedron1994, 50, 11983-11994; b) P.
Swaren, I. Massova, J.R. Bellettine, A. Bulychev, L.
This
work was
partially
supported
by
project
”Biotransformations for pharmaceutical and cosmetics
industry” No. POIG.01.03.01-00-158/09-01 also partially
supported by Polish National Science Center project No.
2013/11/B/ST5/02199.
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JournalName
ARTICLE
Maveyraud, L.P. Kotra, M.J. Miller, S. Mobashery, J.P. 36 W. M. Connors, A. Pihl, A. L. Dounce, E. I. Stotz, Biol.
Samama, J. Am. Chem. Soc. 1999, 121, 5353-5359. Chem.1950, 184, 29.
16 S. Colle, C. Taillefumier, Y. Chapleur, R. Liebl, A. Schmidt, 37 C. Xu, C. Yuan, Tetrahedron2005, 61, 2169-2186.
Bioorg. Med. Chem. 1999,
17 K. Wunsche, U. Schwaneberg, U.T. Bornscheuer, H.H. Meyer,
Tetrahedron: Asymmetry1996, , 2017-2022.
18 a) K. Wadhwa, J. G. Verkade, J. Org. Chem. 2009, 74, 4368-
7
, 1049-1057.
38 J. Brem, M. Naghi, M. -I. Tosa, Z. Boros, L. Poppe, F. -D.
Irimie, C.Paizs, Tetrahedron: Asymmetry2011, 22, 1672-1679.
7
39 N. A. Salvi, S. Chattopadhyay, Bioorg. Med. Chem. 2006, 14
4918-4922.
,
4371; b) C.W. Downey, M.W. Johnson, D.H. Lawrence, A.S. 40 T. Kitanosono, P. Xu, S. Kobayashi, Chem. Asian J. 2014,
9
,
Fleisher, K.J. Tracy, J. Org. Chem. 2010, 75, 5351-5354; c) P.V.
Ramachandran, P.B. Chanda, Org. Lett. 2012, 14, 4346-4349;
d) R.O. Duthaler, P. Herold, W. Lottenbach, K. Oertle, M.
Riediker, Angew. Chem. Int. Ed. 1989, 28, 495-497; e) C.
Mioskowski, G. Solladie, Tetrahedron1979, 36, 227-236; f)
A.I. Meyers, G. Knaus, Tetrahedron Lett. 1974, 15, 1333-
1336; g) J.Baudoux, P. Lefebvre, R.Legay, M.-
C.Lasne,J.Rouden, Green Chem. 2010, 12, 252-259.
179-188.
19 a) R. Noyori, Asymmetric Catalysis in Organic Chemistry,
Wiley& Sons, New York, 1994, pp. 62; b) T. Ireland, G.
Grossheimann, J. C. Wieser, P. Knochel, Angew. Chem. Int.
Ed. 1999, 38, 3212-3214; c) V.V. Ratovelomanana, J.P. Genet,
J. Organomet. Chem. 1998, 567, 163-171; d) K. Everaere, J.F.
Carpentier, A. Mortreux, M. Bulliard, Tetrahedron:
Asymmetry1999, 10, 4663-4666; e) Y. Sun, X. Wan, M.Guo,
D. Wang, X. Dong, Y. Plan, Z. Zhang, Tetrahedron:
Asymmetry2004, 15, 2185-2188; f) J.H. Xie, X.Y. Liu, X.H.
Yang, J.B. Xie, L.X. Wang, X.L. Zhou, Angew. Chem. Int. Ed.
2012, 51, 201-203.
20 S.K. Padhi, D. Titu, N.G. Pandian, A. Chadha,
Tetrahedron2006, 62, 5133-5140.
21 a) N.W. Boaz, J. Org. Chem. 1992, 57, 4289-4292; b) P.
Borowiecki, M. Bretner, Tetrahedron: Asymmetry2013, 24
925-936.
22 a) F.F. Huerta, Y.R.S. Laxmi, J.E. Bӓckvall, Org. Lett. 2000,
,
2,
1037-1040; b) F.F. Huerta, Y.R.S. Laxmi, J.E. Bӓckvall, Org.
Lett. 2001, , 1209-1212.
23 Y. Shvo, D. Czarkie, Y. Rahamim, J. Am. Chem. Soc. 1986, 108
3
,
7400-7402.
24 a) A. Brodzka, D. Koszelewski, R. Ostaszewski, J. Mol. Catal.
B: Enzymatic2012, 82, 96-101; b) A. Brodzka, D.Koszelewski,
M. Cwiklak, R. Ostaszewski, Tetrahedron: Asymmetry2013,
24, 427-433.
25 R. Morrone, M. Piattelli, G. Nicolosi, Eur. J. Org. Chem. 2001,
1441-1443.
26 A. J. Lin, R. E. Miller, J. Med. Chem. 1995, 38, 764-770.
27 R. T. Mulcahy, J. J. Gipp, J. P. Schmidt, C. Joswig, R. F. Borc, J.
Med. Chem. 1994, 37, 1610-1615.
28 G. Gavvea, S. Riva, Angew. Chem. Int. Ed. 2000, 39, 2226-
2254.
29 C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem.
Soc. 1982, 104, 7294-7299.
30 B. Testa, W. F. Trager, Chirality1990, 2, 129-133.
31 B. L. Conley, M. K. Pennington-Boggio, E.Boz, T. J. Williams,
Chem. Rev. 2010, 110, 2294-2312.
32 a) Y. Ahn, S. -B. Ko, M. -J. Kim, J. Park, Coordin. Chem. Rev.
2008, 252, 647-658; b) S. Agrawal, E. Martínez-Castro, R.
Marcos, B. Martín-Matute, Org. Lett. 2014, 16, 2256−2259
33 a) F. A. Cotton, R. A. Walton, Multiple Bonds Between Metal
Atoms, 2nd ed.; Oxford Univ. Press: New York, 1993; b) S.
Garcia-Granda, P. Lahuerta, J. Latorre, M. Martinez, E.Peris,
M. Sanau, M. A. Ubeda, J. Chem. Soc., Dalton Trans. 1994,
539-544; c) P. Lahuerta, E. Peris, Inorg. Chem. 1992, 31
4547; d) J. Kitchens, L. Bear, J. Inorg. Nucl. Chem. 1969, 31
2415; e) T. R. Jackand J. Powell, Can. J. Chem. 1975, 53, 2558;
,
,
f) A. Dobson, S. D. Robinson, Platinum Metal Rev. 1976, 20
56-63.
34 S. Wolfe, S. Ro, Z. Shi, Can. J. Chem. 2001, 79, 1259-1271.
,
35 R. Chenevert, G. Fortier, R. B. Rhlid Tetrahedron 1992, 48,
6769-6776.
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