Covalently Immobilized Lipases are Efficient Stereoselective Catalysts for the Kinetic Resolution of rac-(5-Phenylfuran-2-yl)-β-alanine Ethyl Ester Hydrochlorides

Article abstract of DOI:10.1002/ejoc.201700174

Lipase-catalyzed enzymatic resolution of several new, exotic and variously substituted rac-(5-phenylfuran-2-yl)-β-alanine ethyl esters was investigated. Given the structural instability of unsubstituted rac-(5-phenylfuran-2-yl)-β-alanine ethyl ester, we u

Full text of DOI:10.1002/ejoc.201700174

DOI: 10.1002/ejoc.201700174
Full Paper
Enzymatic Hydrolysis
Covalently Immobilized Lipases are Efficient Stereoselective
Catalysts for the Kinetic Resolution of rac-(5-Phenylfuran-2-yl)-
ꢀ-alanine Ethyl Ester Hydrochlorides
Botond Nagy,[a,b] Zsolt Galla,[a] László Csaba Bencze,[b] Monica Ioana Toșa,[b] Csaba Paizs,[b]
Enikő Forró,[a] and Ferenc Fülöp*[a]
Abstract: Lipase-catalyzed enzymatic resolution of several preparative-scale (S)-selective hydrolysis of several racemic ꢀ-
new, exotic and variously substituted rac-(5-phenylfuran-2-yl)- amino ester hydrochlorides in NH4OAc buffer (20 mM, pH 5.8)
ꢀ-alanine ethyl esters was investigated. Given the structural in- at 30 °C. Enzymatic resolutions were performed with covalently
stability of unsubstituted rac-(5-phenylfuran-2-yl)-ꢀ-alanine bound lipase AK from Pseudomonas fluorescens and lipase PS
ethyl ester, we used the stable hydrochloride salt of this rac- from Burkholderia cepacia on Immobead T2-150 as catalyst.
heteroaryl-3-aminopropanoic acid ethyl ester as potential sub- Seven out of eight new resolution products were successfully
strate to increase the scope of the reaction. Optimization exper- isolated and appropriately characterized.
iments revealed an efficient procedure for both analytical- and
Introduction
protected- and non-protected ꢀ-amino esters,[16] N-acylation,[17]
and transesterification reactions.[18]
Natural and unnatural ꢀ-amino acids are valuable and versatile
building blocks of pharmaceutically important compounds and
biologically active natural products,[1] making them especially
valuable for drug research and development.[2] Chiral ꢀ-amino
acids are present in the structure of anticancer agents taxol[3]
and bleomycin,[4] dolastatins and many others.[5] Furthermore,
they are key structural elements of ꢀ-peptides and peptidomi-
metics[6] because peptides containing ꢀ-amino acids show re-
markable stability against protease-type hydrolases.[7]
A large number of chemical and biochemical methods have
been developed for the asymmetric synthesis of ꢀ-amino ac-
ids.[8] Ammonia lyases and aminomutases,[9] pyrimidine-catabo-
lism enzymes,[10] hydantoinases,[11] ꢀ-transaminases,[12] and ω-
transaminases[13] have been reported to be efficient biocata-
lysts for the preparation of enantiomerically pure ꢀ-amino acids.
Lipases, however, have gained greater popularity and become
the most widely used group of biocatalysts because of their
ability to accept a wide range of substrates and their stability
in organic solvents.[14] Indeed, they catalyze several asymmetric
transformations of non-natural amino acid derivatives including
enantioselective ring cleavage of ꢀ-lactams,[15] hydrolysis of N-
Phenylfuran-2-yl moieties are involved as building blocks in
the structure of mPGES-1 inhibitors based on dihydropyrimidin-
2(1H)-one,[19] CXCR2/CXCR1 receptor antagonists,[20] and an-
thrax lethal factor inhibitors.[21] They also exhibit radical-scav-
enging activity and cytoprotective effects against oxygen-
ases.[22] These heteroaromatic structures are not unknown to
our research group. We have already described highly stereo-
selective, lipase- or baker's yeast-mediated procedures for the
kinetic resolution of racemic 1-(5-phenylfuran-2-yl)ethanols,
ethane-1,2-diols, and ethanones.[23] Recently,
L-(5-phenylfuran-
2-yl)-α-alanines were also synthesized by a sequential multi-
enzyme process.[24]
In this work, we disclose our results on the lipase-catalyzed
resolution of several new, exotically substituted phenylfuran-
based ꢀ-amino esters (rac-3a–d), by applying enantioselective
hydrolysis. The real challenge to overcome has been the low
solubility of substrates in organic solvents.
Results and Discussion
Synthesis of 3-Amino-3-(5-phenylfuran-2-yl)propionic Acid
Ethyl Esters rac-3a–d
[a] Institute of Pharmaceutical Chemistry, University of Szeged
6701 Szeged, Eötvös u. 6, Hungary
Racemic 3-amino-3-(5-phenylfuran-2-yl)propionic acid ethyl
ester hydrochlorides (rac-3a–d·HCl) were prepared according to
Scheme 1. 5-Phenylfuran-2-carbaldehyde 1a was obtained by
Suzuki–Miyaura coupling reaction between phenylboronic acid
and 5-bromofuran-2-carbaldehyde in the presence of a catalytic
amount of tetrakis(triphenylphosphine)palladium(0). Substi-
tuted phenylfuran derivatives 1b–d, in turn, were synthesized
E-mail: fulop@pharm.u-szeged.hu
http://www2.pharm.u-szeged.hu/gyki/index.php/en/
[b] Faculty of Chemistry and Chemical Engineering,
Biocatalysis and Biotransformation Research Centre
400028 Cluj-Napoca, Arany János str. 11, Romania
https://www.researchgate.net/profile/Csaba_Paizs
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.201700174.
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Scheme 1. Chemical synthesis of racemic substrate molecules. Reagents and conditions: (i) Phenylboronic acid, Pd(PPh3)4, Na2CO3, toluene/EtOH; (ii) NaNO2,
HCl, 0–5 °C; (iii) Furan-2-carbaldehyde, CuCl2, room temp., overnight; (iv) Ammonium acetate, CH2(COOH)2, AcOH, reflux; (v) SOCl2, EtOH, –10 °C.
by Meerwein arylation, coupling furan-2-carbaldehyde with the hydrophobic acrylic resin (Novozyme 435), lipase PS from Burk-
corresponding aryl-diazonium halides in the presence of CuCl2. holderia cepacia immobilized on diatomaceous earth (LPS-Diat),
The slightly modified Rodionov-type transformation of alde-
hydes into ꢀ-amino acids and a final esterification step of the
latter compounds with ethanol induced by thionyl chloride af-
forded the desired rac-3a–d·HCl.
lipase from Mucor miehei, pancreatic porcine lipase PPL, lipase
AK from Pseudomonas fluorescens immobilized on Celite (LAK-
Cel), and lipase from Candida rugosa (CRL) were tested at 45 °C
for the kinetic resolution of the in situ released rac-3a. In 19-
It is important to note that the liberation of rac-3a–d from hour reactions, lipase A from Candida antarctica and lipase from
the synthesized rac-3a–d·HCl was detected. However, the sub-
sequent isolation and purification procedures resulted in the
complete degradation of rac-3a–d, impairing their further
chemical transformation or their use as substrate in enzymatic
kinetic resolutions.
Mucor miehei proved to be inactive. PPL, Novozyme 435 and
CRL, in turn, showed some activities (conversions between 35–
40 %) but very low enantioselectivities (enantiomeric excesses
below 70 % in all cases). Whereas LAK-Cel (4.8 % w/w protein
content) displayed satisfactory activity and moderate enantio-
selectivity, to our delight, however, excellent enantioselectivities
(E >> 200) were observed for LPS-Diat (6.7 % w/w protein con-
tent), as shown in Table 1, entries 1 and 6. Therefore, LPS-Diat
was selected for further studies.
Enzymatic Kinetic Resolution through Enantioselective
Hydrolysis
Distilled water and several organic solvents, such as methyl
tert-butyl ether (MTBE), diisopropyl ether (DIIPE), 1,4-dioxane
and 2-methyltetrahydrofuran were investigated as potential re-
action media. Whereas excellent reaction rates, but very low
enantioselectivities were observed in acyclic ethers (Table 1, en-
tries 3 and 4), no catalytic activities were found when cyclic
ethers were used. Although the biocatalytically formed (S)-2a
precipitated as (S)-2a·HCl, the reproducibility of the small-scale
enzymatic reactions was unsatisfactory because of the multiple
heterogeneity of the reaction mixture.
Because of the structural instability of the substrates, we investi-
gated the lipase-mediated biotransformation of the more stable
racemic phenylfuran-2-yl-ꢀ-alanine hydrochlorides rac-3a–
d·HCl. This decision was based on our very recent results on
enzyme-catalyzed dynamic kinetic resolution of racemic hydro-
chloric salts of 1,2,3,4-tetrahydroisoquinoline-1-carboxylates.[25]
Specifically, our attention focused on the development of an
efficient enzyme-assisted enantioselective ester hydrolysis of
rac-3a–d·HCl (Scheme 2).
First, an enzyme screening was conducted in toluene con-
taining 0.5 equiv. water with rac-3a·HCl as a model compound.
NEt3 (2–10 equiv.) was also added into the reaction mixture for
the in situ deprotonation of rac-3a·HCl. Various enzymes,
namely lipase A from Candida antarctica immobilized on Celite,
lipase B from Candida antarctica immobilized by adsorption on
Given that all ꢀ-amino acid hydrochlorides rac-2a–d·HCl and
ꢀ-amino ester hydrochlorides rac-3a–d·HCl are water-soluble
compounds, we turned our attention to the development of a
reliable enzymatic kinetic resolution of rac-3a–d·HCl in aqueous
media. Promising results were obtained for the unsubstituted
Scheme 2. Lipase-catalyzed enantioselective hydrolysis of ( )-3a–d.
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Table 1. Preliminary results for the enzymatic hydrolysis of ꢀ-amino esters ( )-3a–d.[a]
Entry
Substrate[a]
Enzyme[a]
Solvent[a]
Temp.
[°C]
Time
[h]
ees
eep
Conv.
[%]
E
Specific enzyme activity
μmolsubstrate/(mgprotein×min)
[%][c]
[%][c]
1
2
3
4
5
6
7
8
9
rac-3a
rac-3a
rac-3a
rac-3a
rac-3a
rac-3a
rac-3b
rac-3c
rac-3d
LPS-Diat
LAK-Cel
LPS-Diat
LPS-Diat
LPS-Diat
LPS-Diat
LAK-Cel
LAK-Cel
LAK-Cel
toluene[b]
toluene[b]
MTBE[b]
DIIPE[b]
H2O
H2O
H2O
H2O
H2O
45
45
45
45
45
30
30
30
30
7
90
25
99
99
95
96
83
40
25
99
99
27
45
95
99
96
78
85
48
20
78
69
50
49
47
34
22
>>200
254
7
8.6 × 10–3
3.2 × 10–3
65 × 10–3
57.5 × 10–3
62.5 × 10–3
30.6 × 10–3
20.4 × 10–3
7.4 × 10–3
4.8 × 10–3
11
1.5
1.5
1
2
4
12
146
>>200
102
12
8
8
16
[a] Reaction conditions: substrate (0.015 mmol/mL), enzyme preparation (30 mg/mL), solvent (1 mL). [b] 5 equiv. NEt3, 0.5 equiv. H2O as reactant. [c] Based
on HPLC analysis (see the Supporting Information).
ester ( )-3a·HCl with LPS-Diat in water at 45 °C (Table 1, en- face provides high stability for the enzyme preparation. The
tries 5 and 6). The poor enantiomeric excess of product (S)- obtained LPS-IM and LAK-IM enzyme preparations, both with
2a·HCl at 45 °C can be attributed to the chemical hydrolysis of 9.4 % protein content, were found to be both highly stereo-
the amino ester, which can occur at high reaction temperature. selective and stable biocatalysts. Small-scale reusability tests for
Indeed, aqueous solutions of ( )-3a·HCl incubated at 4, 15, 20, LPS-IM and LAK-IM revealed that after 12 reaction cycles, both
25, 30, and 45 °C without enzyme revealed that approximately biocatalysts retained more than 90 % of their initial activity.
6 % chemical hydrolysis product was generated above 30 °C
after 1 h incubation time. For this reason, 30 °C was selected as
the standard reaction temperature for further studies.
It is well known that enzyme stability, activity, and selectivity
are strongly influenced by the ionic strength and pH of the
solution.[26] Therefore, several buffers in various concentrations
were tested as reaction medium instead of distilled water to
improve the enzymatic kinetic resolution of rac-3a–d·HCl. Phos-
phate-citrate buffers and acetate at different concentration (20–
LPS-Diat displayed low and continuously decreasing activi-
ties towards phenyl-substituted derivatives ( )-3b–d·HCl (con-
version values were below 20 % in all cases after 24 h reaction
time; data not shown). Therefore, a second enzyme-screening
test was performed in water, and immobilized lipase AK on Cel-
ite (LAK-Cel) was found to be the most promising biocatalyst
(Table 1, entries 7–9), although the stability of the enzyme
preparation still diminished in time.
500 mM) in the pH range of 4–6 were tested to enhance the
operational parameters of LPS-IM and LAK-IM. It is important to
note that in solutions with pH >6, deprotonation of rac-3a–
d·HCl and subsequent decomposition of the released rac-3a–d
was observed. Optimal biotransformation of rac-3a–d·HCl oc-
curred in 20 m
pH 5.8, as shown in Table 2.
M ammonium acetate/acetic acid buffer at
The significant decrease in the enzyme activity noted above
can be ascribed to the structural instability of the enzyme prep-
arations. LPS and LAK immobilized on the surface of Diatomite
or Celite by adsorption are susceptible to desorption in aque-
ous media; indeed, water-soluble proteins were spectrophoto-
metrically detected in the reaction mixtures by using the Brad-
ford method. Moreover, free LAK and LPS tested in small-scale
experiments proved to be completely inactive.
Enzymatic reaction velocities calculated as μmolsubstrate
/
(mgprotein×min)[27] generally revealed a slightly increased enzy-
matic activity for covalently immobilized enzyme preparations
(LAK-IM and LPS-IM) compared with physically adsorbed biocat-
alysts (LAK-Cel and LPS-Diat) as shown in Table 1 and Table 2.
Nevertheless, the low structural stability of LAK-Cel and LPS-
To enhance the stability of LPS and LAK, both enzymes were Diat in water impaired their efficacy for preparative-scale bio-
covalently bonded on Immobead T2-150 (see the Experimental transformation of ( )-3a–d. Except for ortho-nitro derivative ( )-
Section), which is a hydrophobic support of methacrylate co- 3c, covalently immobilized lipase AK from Pseudomonas fluores-
polymers with epoxy groups having an average particle size cens (LAK-IM) provides high selectivities, conversions and
of 0.15–0.30 mm. Multipoint covalent attachment between the enantiomer excesses for all tested compounds (Table 2, entries
oxirane groups and the free amino groups on the enzyme sur- 2, 4 and 6 vs. Table 1, entries 2, 7, 8 and 9). Lipase LPS, bonded
Table 2. LPS- and AK-catalyzed hydrolysis of substituted 5-phenylfuran-ꢀ-amino esters ( )-3a–d.[a]
Entry
Substrate
Enzyme[b]
Time
[h]
ees
eep
Conv.
[%]
E
Specific enzyme activity
μmolsubstrate/(mgprotein×min)
[%][b]
[%][b]
1
2
3
4
5
6
7
rac-3a
rac-3b
rac-3b
rac-3c
rac-3c
rac-3d
rac-3d
LPS-IM
LAK-IM
LPS-IM
LAK-IM
LPS-IM
LAK-IM
LPS-IM
6
6
2
6
6
9
6
99
99
99
65
72
99
99
95
93
94
93
88
95
95
51
51
51
41
45
51
51
206
145
170
54
34
206
206
75.4 × 10–3
75.4 × 10–3
226.1 × 10–3
60.6 × 10–3
66.5 × 10–3
50.2 × 10–3
75.4 × 10–3
[a] Reaction conditions: substrate (0.015 mmol/mL), enzyme preparation (3 mg/mL), 20 m
M
NH4OAc, pH 5.8, 30 °C. [b] Based on HPLC analysis (see the
Supporting Information).
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Table 3. Preparative-scale transformations.
Entry
Substrate
Time
[h]
Conv.
[%]
(R)-2·HCl
Yield
(S)-2·HCl
Yield
ee
ee [%][b]
[α]2D2
[α]2D2
[%][a]
[%][b]
[%][a]
1
2
3
4
( )-3a
( )-3b
( )-3c
( )-3d
6
6
6
9
50
50
48
50
–
–
–
57
93
36
72
95
94
86
95
–15.5 (c 6; 5 % HCl[c])
–3.5 (c 2; 5 % HCl[c])
–77 (c 2; CH3COOH)
–6.1 (c 1.6; CH3COOH)
67
30
52
93
80
94
+2.7 (c 3; 5 % HCl[c])
+60 (c 1.5; CH3COOH)
+9.7 (c 2; CH3COOH)
[a] Isolated yield calculated as percentage of the 50 % theoretical yield of the kinetic resolution. [b] Based on HPLC analysis (see the Supporting Information).
[c] 5 % aqueous HCl solution.
on Immobead T2-150 (LPS-IM), also afforded significantly in- IM and/or LAK-IM enzyme preparations proved to be excellent,
creased activity toward substituted 5-phenylfuran-2-yl ꢀ-amino (S)-selective biocatalysts for the asymmetric hydrolysis of ( )-
esters ( )-3b–d·HCl in comparison with the LPS-Diat.
3a–d·HCl in NH4OAc buffer (20 mM, pH 5.8) at 30 °C with high
enantioselectivities (E > 145), leading to enantiomers (R)-2b–d
(ee > 99 %) and (S)-2a–d (ee = 93 %). The stable hydrochloride
salts of ( )-3a–d were reliable substrates for their stereoselect-
ive biocatalytic hydrolysis in water, offering new possibilities for
exploring enzymatic kinetic resolution of other unstable race-
mates.
Preparative-Scale Biotransformations of Compounds
( )-3a–d·HCl
Preparative-scale hydrolysis of ( )-3a–d·HCl were carried out
under the optimal conditions developed for the small-scale bio-
transformation (0.015 mg/mL substrate, 3 mg/mL enzyme,
20 m
M NH4OAc pH 5.8, 30 °C). The reactions were stopped at
Experimental Section
the time indicated in Table 3, by removing the enzyme through
filtration. The unreacted (R)-3a–d·HCl and the formed (S)-2a–
d·HCl were separated by using preparative C18 HPLC column
(see the Experimental Section) and characterized as their hydro-
chloride salt. As an exception, purification of compound (R)-2a
failed. Isolated yields, conversions, enantiomeric excesses and
specific rotation values for the enantiomers are given in Table 3.
For stability reasons, (R)-3a–d·HCl esters were hydrolyzed by
Materials and Methods: All starting materials and reagents were
purchased from Sigma–Aldrich or Alfa-Aesar and used as received.
Lipases from Pseudomonas fluorescens (LAK), from Burkholderia
cepacia (LPS), from Candida rugosa (CRL), from Mucor miehei, from
porcine pancreas (PPL) and lipase A from Candida antarctica were
obtained from Sigma–Aldrich. Lipases from Pseudomonas fluores-
cens immobilized on Celite (LAK-Cel) was prepared by us.[28] Lipase
from Burkholderia cepacia immobilized on diatomaceous earth (LPS-
heating in aqueous hydrochloric acid solution and character- Diat) was a gift from Amano Enzyme Europe Ltd. Lipase B from
Candida antarctica (Novozyme 435) was the product of Novozymes,
Denmark. Immobead T2-150 was from ChiralVision, The Nether-
lands.
ized as amino acid hydrochloride salts.
The Absolute Configuration of the Resolution Products
Covalent Immobilization of Lipase PS from Burkholderia cepacia
and Lipase AK from Pseudomonas fluorescens on Immobead T2-
150: Into a solution of Amano lipase from Burkholderia cepacia (LPS,
40 mg, 56 μg enzyme in 1 mg of liophylized preparation) or Amano
lipase from Pseudomonas fluorescens (LAK, 40 mg, 53 μg enzyme in
1 mg of liophylized preparation) and Tween-80 (6.5 μL) in PBS buffer
The absolute configuration of the novel enantiopure ꢀ-amino
ester (+)-3d was determined by a detailed 1H NMR study of
both diastereomers formed with (R)- and (S)-Mosher acids. Thus,
the unreacted ethyl 3-amino-3-[5-(4-bromophenyl)furan-2-yl]-
propanoate (+)-3d was N-acylated with (R)- and (S)-MTPA-Cl and
the resulting diastereomers were differentiated by their 1H NMR
spectra (see the Supporting Information). Hereby, the (R)-(+)-3d
absolute configuration was assigned for the unreacted enantio-
mer in the enzyme-catalyzed hydrolysis of rac-3d.
Based on the same sense of their specific rotation with (R)-
(+)-3d and (S)-(–)-2d, the absolute configurations for the other
enzymatic resolution products were assigned as (R)-(+)-3a–c
and (S)-(–)-2a–c.
(20 m
M Na2HPO4, 150 mM NaCl, pH 7, 6 mL) Immobead T2-150
(20 mg) was added and the mixture was shaken overnight at room
temperature at 1350 rpm. The mixture was filtered and the biocata-
lyst was washed with water (3 × 10 mL) until no protein trace was
detected in the filtrate. Finally, the immobilized enzymes were
freeze-dried. The amount of immobilized enzyme on the support
was determined as the difference between the total mass of the
pure enzyme in the solution before immobilization (2.12 mg of LAK
in 40 mg of liophylized powder and 2.24 mg of LPS in 40 mg of
liophylized powder) and in the unified filtrates after immobilization
(0.043 mg of LAK and, 0.16 mg of LPS respectively) determined
spectrophotometrically by using the Bradford method. Immobiliza-
tion yield: 92.9 % for LAK and 97.9 % for LPS, enzyme loading: ca.
94 μg protein/mg immobilized preparation for both, LPS-IM and
Conclusions
A synthetic method for the preparation of four novel, very LAK-IM.
exotic phenylfuran-2-yl-ꢀ-alanine esters was developed. The ob-
tained stereoisomers were successfully resolved in their hydro-
chloride salt form through lipase-catalyzed enantioselective
Analytical-Scale Enzymatic Hydrolysis: A mixture of racemic sub-
strate (0.015 mol) and lipase (3 mg of enzyme preparation/mL) was
shaken in 20 m NH4OAc pH 5.8 buffer solution (1 mL) at 30 °C.
M
hydrolysis in aqueous media. The covalently immobilized LPS- Samples (6 μL) taken periodically were diluted with the mobile
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phase (30 μL), filtered and injected on chiral CROWNPAK CR (+)
HPLC column by using a mixture of HClO4 aqueous solution (pH 2.3)
and ACN 70:30 (v/v) as mobile phase (except when indicated other-
wise; see the Supporting Information).
Preparative-Scale Enzymatic Hydrolysis: Racemic compounds
3a–d (0.6 mmol) were dissolved in 20 mM NH4OAc pH 5.8 buffer
solution (40 mL). LPS-IM for ( )-3a (120 mg) and LAK-IM for ( )-3b–
d (120 mg), were added and the mixture was shaken at 30 °C.
After completion of the reaction (times indicated in Table 3), the
immobilized enzyme was filtered out and the solvent was removed
by freeze drying. The unreacted ꢀ-amino ester enantiomers (R)-3a–
d and the obtained ꢀ-amino acid enantiomers (S)-2a–d were
separated on achiral ZORBAX SB-C18 preparative column
(21.2 × 250 mm) by using a mixture of aqueous HCl solution
(100 m
Information).
M
) and MeOH as eluent (see Table 3 and the Supporting
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Acknowledgments
B. N. acknowledges financial support from the Székely Forerun-
ner Fellowship Award and from the Sectorial Operational Pro-
gram for Human Resources Development 2007-2013, cofi-
nanced by the European Social Fund (project POSDRU/159/1.5/
S/132400). L. C. B. thanks the Romanian National Authority for
Scientific Research and Innovation, CNCS-UEFISCDI, project
number PN-II-RU-TE-2014-4-1668 for financial support. The au-
thors also acknowledge the receipt of OTKA Grants K-108943
and K-115731 for financial support.
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Received: February 6, 2017
Eur. J. Org. Chem. 2017, 2878–2882
www.eurjoc.org
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