Ene reductase-catalysed synthesis of (R)-profen derivatives

Article abstract of DOI:10.1002/adsc.201100743

Enantiomerically pure (R)-profen derivatives and intermediates are synthesised utilising the enzyme YqjM, an ene reductase from Bacillus subtilis. After optimisation of the reaction conditions, the chemoenzymatic approach was applied for the first time in the synthesis of (R)-flurbiprofen methyl ester. Copyright

Full text of DOI:10.1002/adsc.201100743

UPDATES
DOI: 10.1002/adsc.201100743
Ene Reductase-Catalysed Synthesis of (R)-Profen Derivatives
Jçrg Pietruszkaa,* and Melanie Schçlzela
a
Institut fꢀr Bioorganische Chemie der Heinrich-Heine-Universitꢁt Dꢀsseldorf im Forschungszentrum Jꢀlich, Stetternicher
Forst, Geb. 15.8, 52426 Jꢀlich, Germany
Fax : (+49)-2461-61-6196; phone: (+49)-2461-61-4158; e-mail: j.pietruszka@fz-juelich.de
Received: September 25, 2011; Revised: November 16, 2011; Published online: February 23, 2012
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201100743.
Abstract: Enantiomerically pure (R)-profen deriva-
tives and intermediates are synthesised utilising the
enzyme YqjM, an ene reductase from Bacillus sub-
tilis. After optimisation of the reaction conditions,
the chemoenzymatic approach was applied for the
first time in the synthesis of (R)-flurbiprofen
methyl ester.
enzymatic routes,[4] and in particular lipase-catalysed
(dynamic) kinetic resolutions.[5] While other classes of
enzymes have been utilised, applications of ene reduc-
tases are still elusive. Representatives of the enzyme
class are, for example, the well investigated old
yellow enzyme (OYE)[6] and its homologue YqjM.[7]
Due to their ability to reduce C=C double bonds that
are activated by electron-withdrawing groups, ene re-
ductases allow for the creation of up to two new ste-
reogenic centres by a formal trans-addition of hydro-
gen. Ene reductases have already been used for the
biotransformation of a wide range of different sub-
strates and are an emerging useful tool for the synthe-
sis of chiral building blocks.[8]
Keywords: asymmetric catalysis; asymmetric syn-
thesis; ene reductase; enzymes; profens; reduction
2-Arylpropionic acids are an important class of non-
In this context and in view of our ongoing interest
steroidal anti-inflammatory drugs (NSAIDs). Several in chemoenzymatic approaches towards key building
representatives, e.g., ibuprofen (1), naproxen (2) and blocks for natural product syntheses,[9] we herein
ketoprofen (3), are applied as pharmaceuticals with report a biocatalytic approach towards 2-arylpropion-
the pharmacological active enantiomer being (S)-con- ic acid esters (Scheme 1), applied in the synthesis of
figured (Figure 1). As an exception (R)-flurbiprofen enantiomerically pure (R)-flurbiprofen methyl ester.
(4) was considered and discussed as potential reducer
of levels of b-amyloids, the causal agents for plaque the substrates for the biotransformation: starting from
formation in Alzheimerꢂs disease.[1,2]
the commercially available substituted phenylacetic
The first step in our route was the preparation of
In order to obtain enantiomerically pure 2-arylpro- acids (8), the esters (9) were obtained by methylation
pionic acids considerable interest was directed to ste- in excellent yields (96–99%). By means of a Knoeve-
reoselective chemical methods[3] as well as to chemo-
Scheme 1. Alternative retrosynthesis of profens.
Scheme 2. Synthesis of substrates. Reagents and conditions:
a) MeOH, cat. H2SO4, b) paraformaldehyde, K2CO3, DMF,
Figure 1. Examples from the profen family.
708C (yields: see Table 1).
Adv. Synth. Catal. 2012, 354, 751 – 756
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
751

Jçrg Pietruszka and Melanie Schçlzel
UPDATES
Table 1. Substrate syntheses.
Table 2. Optimisation of the enzymatic reduction of enoate
7a. Constant reaction parameters: Substrate 7a (5 mM, from
500 mM stock solution in solvent), KPi buffer (1 mL),
NADP (100 mM), glucose (20 mM), GDH, YqjM, 258C,
24 h, 300 rpm.
Entry
Ar
Yield of 9 [%]
Yield of 7 [%]
a
b
c
d
e
f
Ph
p-tolyl
99
99
–
96
96
98
97
99
66
60
75
57
30
35
31
42
p-isobutyl-C6H4
o-Br-C6H4
m-Br-C6H4
p-Br-C6H4
m-F-C6H4
3-F-4-OH-C6H3
Entry
Conditions
Conversion
ee (6a)
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
KPi 20 mM, DMF[a]
KPi 50 mM, DMF[a]
KPi 100 mM, DMF[a]
KPi 20 mM, DMSO[a]
KPi 50 mM, DMSO[a]
KPi 100 mM, DMSO[a]
KPi 20 mM, THF
47%
55%
47%
73%
79%
85%
92%
89%
90%
95%
93%
81%
97%
87%
74%
13%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
g
h
KPi 50 mM, THF
KPi 100 mM, THF
KPi 20mM, 2-Me-THF
KPi 50 mM, 2-Me-THF
KPi 100 mM, 2-Me-THF
1 vol% 2-Me-THF
2 vol% 2-Me-THF
5 vol% 2-Me-THF
10 vol% 2-Me-THF
Scheme 3. Enzymatic reduction of profen derivative 6a on
an analytical scale (results: see Table 2).
nagel condensation with p-formaldehyde the sub-
strates for enzymatic reduction (7) could be synthes-
ised (Scheme 2, Table 1);[10] the reaction conditions
were not optimised.
[a]
Formation of side product.
The second step was the investigation of the enzy- carboxylic acid and the racemic reference compound
matic reduction by the known ene reductase YqjM was prepared by Pd-catalysed hydrogenation.
from Bacillus subtilis, a homologue of the old yellow
First, different reaction parameters were evaluated
enzyme (EC 1.6.99.1).[7] The unsubstituted methyl 2- on an analytical scale. Buffer concentration was
phenylacrylate was used as a model compound for an varied (20, 50, and 100mM) and different co-solvents
optimisation approach of the enzymatic reduction (dimethylformamide, dimethyl sulfoxide, tetrahydro-
(Scheme 3).
furan and 2-methyltetrahydrofuran) were tested
For the assignment of product 6, suitable ee analyt- (Table 2). The best results were obtained when using
ics were established by means of gas chromatographic a 20mM KPi buffer and 2-methyltetrahydrofuran as
separation using Hydrodex b-3P as stationary phase. co-solvent, whereby a higher concentration leads to
The absolute configurations were confirmed by com- a decrease in conversion. The product (R)-6a was ob-
parison of the retention times (Figure 2). The (S)-con- tained with 97% conversion and excellent enantiose-
figured 2-arylpropionic acid ester 6a was readily ob- lectivity (ee >99%).
tained after methylation of the commercially available
In a second step after successful optimisation, the
enzymatic reduction was performed for the different
substrates on a semi-preparative scale. For this pur-
pose a suitable cofactor regenerating system was re-
quired. In full agreement to previous investigations,
the incorporation of a glucose dehydrogenase (oxidis-
ing glucose) revealed an effective system
(Scheme 4).[9a,11]
As determined by gas chromatographic separation
of starting material and product, for products 6a–h
good to excellent conversions were reached as well as
excellent enantioselectivity for all cases; with excep-
tion of compound 7d which was not accepted as sub-
strates (Table 3). While the phenyl substrate 7a is
readily converted, the reactions with the p-tolyl sub-
strate 7b and with p-isobutyl substituent 7c proceeded
much slower due to the increased steric bulk of the
aryl groups. Up-scaling was also possible: Methyl 2-
phenylacrylate (7a) was completely converted within
Figure 2. GLC trace for the enzymatic reduction of enoate
7a. Reaction conditions: Hydrodex-b-3P, H2 (0.6 bar), 1108C
(5ꢂ iso), 28CminÀ1 to 1358C, (0ꢂ iso), 108CminÀ1 to 1508C
(3ꢂ iso), tR [(S)-6a]=9.88 min, tR [(R)-6a]=10.17 min.
752
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 751 – 756

Ene Reductase-Catalysed Synthesis of (R)-Profen Derivatives
Scheme 4. Semi-preparative scale. Reagents and conditions:
substrate 5 mM (500 mL stock solution in 2-Me-THF),
50 mL KPi buffer (20 mM, pH 7), NADP (100 mM), glucose
(20 mM), GDH (~5 U), YqjM (5 U), 258C, 24 h (results:
see Table 3).
Scheme 5. Synthetic route towards (R)-flurbiprofen methyl
ester. Reagents and conditions: a) 0.25 mmol substrate 7h
(500 mM solution in 2-Me-THF), 100 mM NADP, 20 mM
glucose, 50 mL KPi buffer (20 mM, pH 7), YqjM 5 U, GDH
~5 U, 68%; b) DIPA, (CF3SO2)2O, CH2Cl2, À158C to room
Table 3. Results of the biotransformation in semi-prepara-
tive scale.
temperature, 64%; c) Ph-B(OH)2, K3PO4, PdACTHNUGTRNEUNG(PPh3)4 (6
Entry
Ar
Conversion [%]
ee [%]
mol%), 1,4-dioxane, 1008C, 92%.
a
b
c
d
e
f
Ph
p-tolyl
100
73
40
n.c.
56
71
>99 (R)
>99
> 99 (R)
–
>99
>99
p-isobutyl-C6H4
o-Br-C6H4
m-Br-C6H4
p-Br-C6H4
m-F-C6H4
3-F-4-OH-C6H3
g
h
82
100
>99
>99
24 h and a 77% yield was obtained after work-up and
purification (see Supporting Information for details).
Next, we applied the approach in the chemoenzy-
matic synthesis of flurbiprofen methyl ester 11
(Scheme 5). The first step was the enzymatic reduc-
tion of methyl 2-(3-fluoro-4-hydroxyphenyl)acrylate
(7h) providing product 6h in 68% yield and an enan-
tiomeric excess >99% as determined by GLC
(Figure 3). After the biocatalytic step, a triflate was
introduced leading to substrate 10, ready for the
Suzuki–Miyaura coupling producing to the biarylic
system of flurbiprofen (4).[12] Both steps gave good to
very good yields (64% and 92%).
Figure 3. GLC trace for the enzymatic reduction of enoate
7h to propionate 6h. Reaction conditions: Hydrodex-b-
TBDAc, H2 (0.6 bar), 1408C (0.5ꢂ iso), 158CminÀ1 to 1738C
(10ꢂ iso), tR [(S)-6h]=5.19 min, tR [(R)-6h]=5.25 min.
Ensuring that no racemisation occurred during
steps b and c (Scheme 5), the products were again an-
alysed. The racemic reference compounds were ob-
tained by performing the reaction steps with racemic
starting material rac-6h which was produced by Pd-
catalysed hydrogenation of enoate 7h. The absolute
configurations were confirmed by comparison of the
retention times (Figure 3 and Figure 4) as well as the
optical rotations with literature data.[13]
In conclusion, we have described the application of
an ene reductase (YqjM) for the reduction of novel
substrates ultimately yielding (R)-profen derivatives.
The selectivity was excellent in all cases (>99%), but
Figure 4. GLC trace of esters rac-11 and (R)-11 (Scheme 5).
Reaction conditions: Hydrodex-b-TBDAc, H2 (0.6 bar),
608C (5ꢂ iso), 58CminÀ1 to 1508C (5ꢂ iso), tR [(S)-11]=
the reactivity decreased with increasing bulk of the ar- 5.94 min, tR [(R)-11]=9.09 min.
Adv. Synth. Catal. 2012, 354, 751 – 756
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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753

Jçrg Pietruszka and Melanie Schçlzel
UPDATES
3’), 174.3 (C-1); MS (EI, 70 eV): m/z=198 (25) [M+], 139
(100) [MÀC2H3O2 ], 109 (9) [MÀC4H7O2 ].
omatic moiety. As a first example, an alternative che-
moenzymatic synthesis of (R)-flurbiprofen methyl
ester 11 was successfully performed.
+
+
Synthetic Route towards Flurbiprofen Methyl Ester
(11)
Experimental Section
Synthesis of compound 10: To a solution of compound 6h
(33.0 mg, 0.17 mmol, 1 equiv.) and 30.0 mL (0.20 mmol,
1.2 equiv.) N,N-diisopropylamine in anhydrous dichlorome-
thane (500 mL) was added 34.0 mL trifluoromethanesulfonic
anhydride at À158C. The reaction mixture was stirred for
10 min at À158C and was then warmed to room tempera-
ture. The reaction progress was monitored by GC/MS. After
complete conversion was detected, the solution was washed
with water, dried over MgSO4 and the solvent was removed
under reduced pressure. The crude product was purified by
flash column chromatography on silica gel (eluting with
EtOAc/petroleum ether 10:90) to afford product 10 as a col-
ourless oil; yield: 35.9 mg (0.12 mmol, 64%). 1H NMR
Representative Procedure for the Syntheses of
substrates 7
Synthesis of enoate 7h: Under an atmosphere of dry nitro-
gen paraformaldehyde (4.08 mmol, 1.5 equiv.) was added to
a solution of methyl 2-(3-fluoro-4-hydroxyphenyl)acetate
(2.72 mmol, 1 equiv.) and potassium carbonate (2.72 mmol,
1 equiv.) in anhydrous dimethylformamide (15 mL). The re-
action mixture was heated to 708C and the reaction progress
was monitored by GC/MS. After the complete conversion
was detected, water was added and the reaction mixture was
extracted with EtOAc. The combined organic layer was
washed with water and brine, dried over MgSO4, and the
solvent was removed under reduced pressure. The crude
product was purified by flash column chromatography on
silica gel (eluting with EtOAc/petroleum ether 1:9) to afford
product 7h as a light yellow solid; yield: 226 mg (1.15 mmol,
3
(600 MHz, CDCl3): d=1.51 (d, 3H, J3,2 =7.2 Hz, 3-H), 3.69
3
(s, 3H, Me), 3.74 (q, 1H, J2,3 =7.2 Hz, 2-H), 7.15 (d, 1H,
4
3J6’,5’ =8.2 Hz, 6’-H), 7.24 (dd, 1H, 3J2’,F =10.6 Hz, J2’,6’
=
3
4
2.0 Hz, 2’-H), 7.28 (t, 1H, J5’,6’ =8.2 Hz, J5’,F =8.2 Hz, 5’-H);
13C NMR (151 MHz, CDCl3): d=18.5 (C-3, Me), 44.9 (C-2),
1
52.4 (Me), 116.8 (d, 2J2’,F =18.5 Hz, C-2’), 118.6 (d, JC,F
=
1
42%). H NMR (600 MHz, DMSO-d6): d=3.76 (s, 3H, Me),
320.7 Hz, CF3), 123.5 (C-6’), 124.2 (d, 3J5’,F =3.7 Hz, C-5’),
3
5.98 (s, 1H, 3-Ha), 6.14 (s, 1H, 3-Hb), 6.94 (t, 1H, J5’,6’
=
2
3
135.7 (d, J4’,F =13.7 Hz, C-4’), 142.8 (d, J1’,F =6.05 Hz, C-1’),
8.5 Hz, 4J5’,F =8.5 Hz, 5’-H), 7.09 (ddd, 1H, 3J6’,5’ =8.5 Hz,
1
153.5 (d, J3’,F =253.9 Hz, C-3’), 173.7 (C-1); MS (EI, 70 eV):
4J6’,2’ =2.2 Hz, 5J6’,F =1.1 Hz, 6’-H), 7.27 (dd, 1H, J2’,F
=
3
m/z=330 (48) [M+], 271 (100) [MÀC2H3O2 ], 197 (10)
+
4
12.7 Hz, J2’,6’ =2.2 Hz, 2’-H); 13C NMR (151 MHz, DMSO-
+
+
[MÀCF3SO2 ], 153 (34) [MÀC2H3F3SO2 ], 138 (26)
2
d6): d=52.3 (Me), 115.7 (d, J2’,F =19.6 Hz, C-2’), 116.9 (d,
+
+
[MÀC3H3F3SO4 ], 109 (31) [MÀC5H6F3SO4 ].< br/>
Synthesis of compound 11: Under an atmosphere of dry ni-
trogen a solution of triflate 10 (30 mg, 0.09 mmol), phenyl-
boronic acid (26.8 mg, 0.22 mmol), K3PO4 (57.3 mg,
3J5’,F =2.3 Hz, C-5’), 124.8 (d, J6’,F =3.3 Hz, C-6’), 126.5 (C-
4
3
2
3), 129.5 (d, J1’,F =6.7 Hz, C-1’), 139.7 (C-2), 143.7 (d, J4’,F
=
1
14.3 Hz, C-4’), 150.5 (d, J3’,F =236.9 Hz, C-3’), 167.2 (C-1);
MS (EI, 70 eV): m/z=196 (80) [M+], 166 (5) [MÀOCH3 ],
+
0.27 mmol) and PdACHTNUGTRNEG(UN PPh3)4 (6 mol%) in 1,4-dioxane (500 mL)
+
+
137 (100) [MÀC2H3O2 ], 109 (17) [MÀC4H7O2 ].
was heated at 1008C for 3 h. After cooling to room tempera-
ture water was added and the reaction mixture was extract-
ed with EtOAc. The combined organic phase was dried over
MgSO4 and the solvents were removed under reduced pres-
sure. The crude product was purified by flash column chro-
matography on silica gel (eluting with EtOAc/petroleum
ether 10:90) to afford product 11 as a colourless oil; yield:
Representative Procedure for the Enzymatic
Reduction
Synthesis of compound 6h: A solution of enoate 7h (49 mg,
0.25 mmol) in 500 mL 2-methyltetrahydrofuran was mixed
with 50 mL 20mM KPi buffer (pH 7) and NADP (3.94 mg,
0.005 mmol), 1 mmol glucose, 1 mg GDH[11] (lyophilised
powder, ~5 U) and 2 mg YqjM[7] (lyophilised powder, ~5 U)
were added. The reaction was run for 24 h at 258C and com-
plete conversion was detected by GLC and verified by
NMR of the crude product after work-up. The reaction mix-
ture was thoroughly extracted with MTBE, the organic
phase dried over MgSO4, filtered and the solvents were re-
moved under reduced pressure. The crude product was puri-
fied by flash column chromatography on silica gel (eluting
with EtOAc/petroleum ether 20:80) to afford product 6h as
1
21.4 mg (0.08 mmol, 92%). H NMR (600 MHz, CDCl3): d=
3
1.54 (d, 3H, J3,2 =7.1 Hz, 3-H), 3.70 (s, 3H, Me), 3.76 (q,
3
3
1H, J2,3 =7.1 Hz, 2-H), 7.12 (br d, 1H, J2’,F =11.6 Hz, 2’-H),
3
3
7.15 ( br d, 1H, J6’,5’ =8.0 Hz, 6’-H), 7.36 (br t, 1H, J4’’,3’’
8.0 Hz, 4’’-H), 7.39 (br dd, 1H, J5’,6’ =8.0 Hz, J5’,F =8.0 Hz,
=
3
4
3
3
5’-H), 7.43 (br dd, 2H, J3’’,2’’ =7.2 Hz, J3’’,4’’ =8.0 Hz, 3’’-H),
7.53 (br d, 2H, J2’’,3’’ =7.2 Hz, 2’’-H); 13C NMR (151 MHz,
3
CDCl3): d=18.4 (C-3), 44.9 (C-2), 52.2 (Me), 115.2 (d,
4
3J2’,F =23.7 Hz, C-2’’), 123.5 (d, J6’,F =3.3 Hz, C-6’), 127.7 (C-
4’’), 127.9 (d, 2J4’,F =13.5 Hz, C-4’), 128.4 (C-3’’), 128.9 (d,
3
4J2’’,F =2.9 Hz, C-2’’), 130.8 (d, J5’,F =3.9 Hz, C-5’), 135.5 (C-
3
1
1’’), 141.8 (d, J1’,F =7.7 Hz, C-1’), 159.7 (d, J3’,F =248.4 Hz,
C-3’), 174.4 (C-1); MS (EI, 70 eV): m/z=258 (52) [M+], 199
a
colourless solid; yield: 33.0 mg (0.17 mmol, 68%).
1H NMR (600 MHz, DMSO-d6): d=1.35 (d, 3H, J3,2
=
3
(100) [MÀC2H3O2 ], 183 (16) [MÀPh+], 170 (8)
+
3
+
7.2 Hz, 3-H), 3.59 (s, 3H, Me), 3.71 (q, 1H, J2,3 =7.2 Hz, 2-
H), 6.87–6.92 (m, 2H, 5’-H, 6’-H), 7.05 (dd, 1H, J=1.3 Hz,
3J2’,F =12.2 Hz, 2’-H), 9.78 (s, 1H, O-H); 13C NMR
(151 MHz, DMSO-d6): d=18.4 (C-3), 43.3 (C-2), 51.7 (Me),
[MÀC4H7O2 ].
2
3
115.0 (d, J2’,F =18.7 Hz, C-2’), 117.6 (d, J5’,F =3.2 Hz, C-5’),
4
3
123.4 (d, J6’,F =3.1 Hz, C-6’), 131.8 (d, J1’,F =5.9 Hz, C-1’),
2
1
143.7 (d, J4’,F =12.03 Hz, C-4’), 150.7 (d, J3’,F =240.7 Hz, C-
754
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 751 – 756

Ene Reductase-Catalysed Synthesis of (R)-Profen Derivatives
2001, 12, 1431–1434; e) M. T. Reetz, S. Prasad, J. D.
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Acknowledgements
We gratefully acknowledge the Ministry of Innovation, Sci-
ence and Research of the German Federal State of North
Rhine-Westphalia (NRW) and the Heinrich Heine University
Dꢀsseldorf (HHU; scholarship within the CLIB-Graduate
Cluster Industrial Biotechnology for M.S. and technology
platform ꢁExpressOꢂ), the Deutsche Forschungsgemeinschaft,
and the Forschungszentrum Jꢀlich GmbH for the support of
our projects. Generous gifts of enzymes from evocatal
GmbH, Prof. Dr. W. Hummel (IMET, HHU), Prof. Dr. Mar-
tina Pohl and Dr. Daniel Minçr (IBG 1, FZJ) as well as MSc
Thomas Classen and Saskia Schuback (IBOC, HHU) were
greatly appreciated. We are indebted to Stefanie Fischer for
preliminary experiments.
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
755

Jçrg Pietruszka and Melanie Schçlzel
UPDATES
[11] The GDH was kindly provided by evocatal GmbH.
YqjM was overexpressed in E. coli and purified as de-
scribed previously (see ref.[7]). The protein concentra-
tion was determined from a protein solution by spec-
trometric measurement of the absorption at 280 nm
with the Nanodrop 2000c (Thermo Scientificꢆ), assum-
ing that 1 absorption unit equals to 1 mg/mL. Activity
of YqjM was determined by a photometrical assay fol-
lowing the consumption of NADPH at 340 nm at 308C
for 1 min in a Shimadzu UV-spectrometer (UV-1800)
with a tempered cell positioner (CPS-240 A). Assay
conditions: 20 mM KPi buffer (790 mL), pH 6.5, 1 mM
0.15 mM NADPH (100 mL of a 1.5 mM solution) and
a defined amount of protein (via Nanodrop measure-
ment) in solution (10 mL). One unit is defined as the
amount of enzyme that converts 1 mmol substrate per
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2-cyclohexen-1-one (100 mL of
a 10 mM solution),
756
asc.wiley-vch.de
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 751 – 756