A three-enzyme system involving an ene-reductase for generating valuable chiral building blocks

Article abstract of DOI:10.1002/adsc.201101006

The use of ene-reductase (ERED) enzymes for the asymmetric reduction of olefins offers a green, renewable alternative to metal-catalysed asymmetric reduction. We report herein the first example of an ERED-catalysed enantiospecific reduction carried out at large scale using a carbonyl reductase (CRED) enzyme in the cofactor recycle. This reaction has been paired with a hydrolase-mediated regioselective ester hydrolysis to generate a valuable chiral building block using a straightforward one-pot process. Copyright

Full text of DOI:10.1002/adsc.201101006

FULL PAPERS
DOI: 10.1002/adsc.201101006
A Three-Enzyme System Involving an Ene-Reductase for
Generating Valuable Chiral Building Blocks
David Mangan,a,* Iain Miskelly,a and Thomas S. Moodya
a
Almac, Biocatalysis Group, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, U.K.
Fax : (+44)-28-9022-5544; phone: (+44)-28-3836-5912; e-mail: david.mangan@almacgroup.com or
daveymangan@gmail.com
Received: December 23, 2011; Revised: April 23, 2012; Published online: July 29, 2012
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201101006.
Abstract: The use of ene-reductase (ERED) en- gioselective ester hydrolysis to generate a valuable
zymes for the asymmetric reduction of olefins offers chiral building block using a straightforward one-pot
a green, renewable alternative to metal-catalysed process.
asymmetric reduction. We report herein the first ex-
ample of an ERED-catalysed enantiospecific reduc-
tion carried out at large scale using a carbonyl reduc- Keywords: asymmetric synthesis; biocatalysis; chiral-
tase (CRED) enzyme in the cofactor recycle. This re- ity; enzyme catalysis; hydrogenation; hydrolases; re-
action has been paired with a hydrolase-mediated re- duction
Introduction
Almac recently had the requirement to synthesise
monoacid (R)-1 shown in Figure 1 as an intermediate
The use of biocatalysis in the field of organic synthe- in a complex convergent synthesis. Retrosynthetic
sis continues to grow year on year for a number of analysis of (R)-1 revealed an opportunity for the use
reasons.[1] The high chemoselectivity, regioselectivity of ERED and hydrolase enzymes. This particular re-
and enantioselectivity exhibited by biocatalysts trans- ductive transformation has been shown to be possible
late into hard cost savings for the fine chemical and using enzymes from the Old Yellow family.[8a,b,e,9] This
pharmaceutical industry by increasing the net yields suggested that the target compound could be fur-
of enantiopure products.[2] The incredible fluctuation nished using an ERED enzyme to form the saturated
in the price of rare metals over the past decade, par- diester (R)-3 from the olefin 2, followed by a regiose-
ticularly palladium, platinum and rhodium is quite lective hydrolase-mediated ester hydrolysis as shown
rightly a cause for concern for the pharmaceutical in- in Scheme 1. To minimise operation and manufactur-
dustry.[3] The possibility of completely removing this ing costs, the process was designed to be a three-
uncertainty from future syntheses of chiral products is enzyme, one-pot process.
an attractive one.
The use of hydrolase enzymes for the resolution of
racemic mixtures and to carry out regiospecific hy- Results and Discussion
drolysis or esterification in complex molecules has
been well documented.[4] The synthesis of chiral sec- The required starting material 2 was formed from the
ondary alcohols is now a straightforward prospect commercially available diacid in 53% yield using es-
using CRED enzymes[5] while the synthesis of chiral
amines using transaminase[6] and, more recently,
monoamine oxidase[7] enzymes is increasingly achiev-
able.
ERED enzymes, on the other hand, must be con-
sidered in their infancy in terms of synthetic applica-
tions. Although a number of examples have appeared
in the literature these are generally carried out at
screening scale.[8]
Figure 1. Chiral acid target molecule 1.
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David Mangan et al.
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pH control (Scheme 2) – an excellent benefit when
carrying out large numbers of optimisation reactions
in parallel during process development. This also re-
moves the necessity for adapting large-scale chemical
reactors to include the circulation loops that are re-
quired for pH control. In addition, the irreversible
nature of the olefin reduction ensures that the equi-
librium issues associated with the use of CRED en-
zymes for cofactor recycle in biocatalytic ketone re-
ductions cannot play a role.
Scheme 1. Envisaged enzymatic route to monoacid 1.
Three selectAZyme CRED enzymes along with an
alcohol dehydrogenase enzyme from Sigma Aldrich
were trialed for this purpose with varying IPA load-
ings. The results are outlined in Table 1 and plotted
for clarity in Figure 2.
A131 was chosen for the role of the recycle enzyme
as its activity was far superior tothat of the highly pu-
rified alcohol dehydrogenase from bakerꢁs yeast.
The parameters that have been found to exert the
greatest influence on enzymatic activity are tempera-
ture, pH and organic co-solvent. The optimisation
study is complicated somewhat by the fact that each
of these parameters obviously need to be suited to
both enzymes.
The thermostabilities of ER-104 and A-131 were
assessed by carrying out standard experiments at
258C, 308C, 40 8C and 508C. The results are displayed
in Figure 3.
Scheme 2. Recycle systems for NAD(P)H in ERED synthet-
ic applications.
tablished chemistry.[10] This reaction was not opti-
It was evident that maximum activity was achieved
mised further. With 2 in hand, our attention turned to at 358C with a sharp drop in activity observed beyond
the identification of an active and selective enzyme this point signifying denaturation of one or both pro-
for the desired bioreduction.
teins. Subsequent optimisation reactions were carried
An ERED enzyme screen using the SelectAZyme
EESK-1300 kit was initiated. The panel was screened
against diester 2 under ideal screening conditions.
Two enzymes were found to afford the desired enan-
tiomer in good yield. It was decided to attempt to op-
timise the reaction with ER-104 as this enzyme pos-
sessed the higher activity at screening scale. Most ex-
amples of ERED reactions in the literature use the
glucose-glucose dehydrogenase (GDH) system to re-
cycle the NAD(P)H required for the reduction of the
alkene moiety.[8] It was our intention to attempt to re-
place this cofactor recycle system with a CRED
enzyme capable of converting IPA to acetone, an ap-
proach recently demonstrated by Glueck and co-
workers.[11] This has the advantage of not requiring
Figure 2. Assessing the suitability of a CRED/IPA recycle
system.
Table 1. Assessing the suitability of a CRED/IPA recycle system.
Entry
Enzyme
Reaction conversion after 20 h
2 equiv. IPA
4 equiv. IPA
10 equiv. IPA
20 equiv. IPA
200 equiv. IPA
1
2
3
4
A-201
A-161
A-132
0
29
28
100
100
39
70
100
100
23
99
100
100
2
28
8
13
68
86
ADH from Bakerꢁs yeast
2
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A Three-Enzyme System Involving an Ene-Reductase for Generating Valuable Chiral Building Blocks
addition of a suitable organic co-solvent. It has been
shown that this can minimise the inhibitory effect of
the substrate and/or product on a given enzyme. Al-
ternatively, the use of a water miscible co-solvent can
increase the availability of the substrate to the
enzyme resulting in an increased reaction rate. A
broad co-solvent screen for the desired biotransfor-
mation was therefore conducted using four water-mis-
cible solvents; DMF, DMSO, THF, acetonitrile and
six water-immiscible solvents; toluene, hexane, ethyl
acetate, 2-methyl-THF, diisopropyl ether and DCM at
a single solvent:aqueous buffer ratio (10% v/v). Tolu-
ene was found to be the most suitable co-solvent, in-
creasing the observed conversion after 20 h by
Figure 3. Thermostability assessment of ER-104 and A131.
out at 328C in an effort to maximise reaction rate a factor of 2.5 when compared to the standard reac-
without comprising enzyme stability. tion conditions without any co-solvent present. By
The optimum pH for the given biotransformation further optimising the ratio of toluene to aqueous
was assessed by carrying out standard experiments at buffer to 40% v/v, the observed conversion after 20 h
pH 6, 6.5, 7, 7.5, 8, and 8.5. The results are displayed was increased by a factor of 4 when compared to the
in Figure 4.
standard reaction conditions without any co-solvent
A clear trend emerged revealing that even mildly present. These modifications to the reaction condi-
acidic conditions hindered the desired reaction. Sub- tions ensured that acceptable conversion (53% after
sequent reactions were carried out at pH 8 in order to 20 h, 73% after 44 h) could be achieved at the syn-
ensure that the maximum reaction rate could be thetically viable volume efficiency of 0.2M (~30 vol-
maintained without approaching the alkalinity re- umes) with favourable enzyme and cofactor loadings
quired to hydrolyse the methyl ester moieties in 2 or (100% w/w ER-104 cell pellet, 12.5% w/w A131
3. Until this point, optimisation reactions were carried crude cell-free extract and 5% NAD cofactor). Ef-
out under highly favourable screening conditions in- forts to improve conversion beyond this level by fur-
volving dilute systems (0.062M, ~100 volumes with ther optimisation of reaction components met with
respect to substrate), however, it became pertinent to failure.
move towards synthetically useful concentrations.
It finally emerged that the acetone produced by the
Upon attempting a design of experiment (DOE) A131/IPA cofactor recycle system was inhibitory to
approach to assess the relative importance of the re- ER-104. Attempting to carry out the desired biore-
maining experimental parameters, namely, ERED, duction under standard conditions with one equiva-
CRED, NAD and IPA loading with respect to sub- lent of acetone present from the outset resulted in
strate along with substrate concentration, extremely a retarded reaction rate and a final conversion of only
poor product titres were obtained. This was attributed 25% after 24 h. It has been demonstrated that remov-
to the fact that these reactions were carried out at al of acetone from the equilibrium can drive the de-
0.2M–0.6M (10 to 30 volumes with respect to sub- sired reaction to completion.[12] This is a form of
strate) and that at this concentration, substrate/prod- ISPR, a concept first pioneered in the area of biotech-
uct inhibition of the enzyme was a crucial factor. This nology in the 1960s. The system shown in Figure 5
issue can, in some cases, be dealt with by conducting was employed in an attempt to remove the acetone
the desired reaction in a biphasic medium through the formed throughout the course of the reaction.
By air sparging through a flask containing a 3% v/v
solution of IPA in H2O, the IPA level in the reaction
flask can be maintained while the acetone is driven
off. Using this method, a conversion of 95% in 40 h
was achieved at the 5-g scale.
Following this achievement, a hydrolase screening
programme was initiated with the aim of identifying
a hydrolase enzyme that would selectively hydrolyse
the desired ester moiety in diester (R)-3, thereby fa-
cilitating a straightforward base-mediated separation
of the required final product, (R)-1, from a mixture of
2 and (R)-3 as shown in Scheme 3. It should be noted
that a number of groups has already had some success
with their investigations into the selective hydrolysis
Figure 4. Optimum pH assessment for biotransformation of
2 to (R)-3.
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David Mangan et al.
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Figure 6. Reaction profile for the stepwise conversion of di-
AHCTUNGERTGeNNUN ster 2 to monoester (R)-1.
Figure 5. IPA/H2O saturated air sparge removes acetone
formed during the course of the reaction.
A trial was immediately conducted to investigate
the possibility of the simultaneous addition of ER-
104, A131 and AH-33 at the beginning of the reaction
in an effort to remove the desired product as it was
formed. Unfortunately, no conversion was observed
in this case. A stepwise, one-pot process was carried
out at the 700-mg scale involving the addition of AH-
33 along with pH stat control to the reaction medium
after 45 h [75% conversion of 2 to (R)-3] which al-
lowed the facile isolation of (R)-1 in 71% yield after
a further 20 h reaction time. It should be noted that
the ISPR technique was not employed here as it was
found to be unsuitable to reactions performed at this
scale.
A 70-g scale reaction was subsequently carried out
combining the high yielding olefin reduction with
ISPR and the selective hydrolase-mediated product
isolation. The reaction profile for the two-step, one-
pot biotransformation is shown in Figure 6.
Scheme 3. Product isolation/purification using hydrolase
technology.
of diesters closely related to compounds 2 and (R)-
3.[13]
A conversion of 98% was obtained after 44 h and
following addition of AH-33 and concomitant pH
48 SelectAZyme hydrolase enzymes (HESK 4800) control through the automated addition of NaOH for
were screened for this purpose. a further 22 h, quantitative conversion of 3 to (R)-
There were clearly six possible products (shown in 1 was observed. The desired monoacid (R)-1 was sub-
Scheme 3) that could be formed from the mixture of sequently isolated in 89% yield. Although there was
compounds 2 and (R)-3. Following overnight screen- no negative impact on reaction rate observed when
ing reactions, an organic extraction from the aqueous scaling it to 70 g and it is envisaged that no further
reaction medium at pH 7.5 served to remove the un- optimisation would be required to achieve kg scale
reacted diesters. Acidification to pH 2 followed by ex- synthesis, it is worth noting that a thorough cost-bene-
traction directly into CDCl3 and subsequent analysis fit analysis would have to be carried out to ascertain
1
by H NMR allowed the product distribution to be as- the economic viability of the process from a manufac-
sessed qualitatively. In order to ascertain whether hy- turing standpoint.
drolysis to diacids 6 or (R)-8 had been effected, the
aqueous layer was lyophilised and the residue ob-
tained dissolved in D2O and subsequently analysed by Conclusions
1H NMR. Of 48 enzymes screened, 18 exhibited con-
version. Of these successful hits, a single hydrolase This study has demonstrated that ERED enzymes are
enzyme, AH-33 (Amano Proleather FG), was identi- applicable to preparative reactions at the 70-g scale.
fied for the completely regioselective hydrolysis to This should help to encourage the synthetic organic
the desired monoacid, (R)-1.
chemistry community to consider the use of these en-
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Adv. Synth. Catal. 2012, 354, 2185 – 2190

A Three-Enzyme System Involving an Ene-Reductase for Generating Valuable Chiral Building Blocks
zymes for the asymmetric reduction of olefins. In ad-
dition, the use of a CRED/IPA cofactor recycle
system in place of the traditional GDH/glucose
system has greatly simplified the optimisation of the
desired reaction, obviating the need for pH control
while an ISPR technique has been used effectively to
circumnavigate the inhibitory effect of acetone that
was encountered with ERED-04. Finally, a hydrolase-
mediated regioselective ester hydrolysis has provided
the required functional handle for further manipula-
tion of the chiral building block generated while also
providing a method for the facile separation of prod-
uct from unreacted starting material.
ACHTUNGTRENN(UNG R/S)-Dimethyl 2-Methylsuccinate (3)
Olefin 2 (30 mg, 0.189 mmol) was dissolved in ethanol
(2 mL). The reaction mixture was hydrogenated at atmos-
pheric pressure using 10% Pd/C (5 mg) as catalyst. After
14 h, the mixture was filtered through a celite pad and con-
centrated under vacuum to furnish the title compound as
1
a clear oil; yield: 22 mg (71%). H NMR 400 MHz (CDCl3):
d=1.22–1.24 (d, 3H, J=7.2 Hz), 2.39–2.45 (dd, 1H, J=
6.06 Hz, J=16.5 Hz), 2.72–2.79 (dd, 1H, J=8.15 Hz, J=
16.5 Hz), 2.91–2.94 (m, 1H), 3.69 (s, 3H), 3.71 (s, 3H);
13C NMR 100 MHz (CDCl3): d=17.0, 35.7, 37.4, 51.7, 51.9,
172.3, 175.7.
Screening Conditions for the Enzymatic Reduction of
Olefin 2
Experimental Section
A solution of olefin 2 (5 mg) in DMSO (50 mL) was added
to a mixture of 0.1M KH2PO4 buffer at pH 7 (1.5 mL) and
NAD+ (2 mg). Lyophilised ERED biocatalyst (10 mg), glu-
cose (50 mg) and GDH (3 mg) were added; the vial sealed
and shaken overnight at 308C. Methyl tert-butyl ether
(1.5 mL) was added to the vial and the organic layer was
separated, filtered through a cotton wool plug containing
anhydrous MgSO4 and analysed by 1H NMR to ascertain
conversion. Enantioselectivity was assessed by chiral HPLC
as discussed in analytical methods.
Chemicals and Enzymes
Chemicals were purchased from Alfa Aesar. All enzymes
were obtained from Almac. The term ꢂvolumesꢁ is used
throughout this paper to describe the quantity of solvent
used in a given experiment with respect to mass input of
substrate. For example, 100 g of substrate in 1 L equates to
10 volumes.
Analytical Methods
Screening Conditions for the Enzymatic Hydrolysis of
1H NMR spectra were recorded at 400 MHz on a Bruker
AV-400 spectrometer; shifts are relative to internal TMS. A
chiral HPLC method was developed to analyse the enantio-
selectivity observed in the enzymatic bioreduction of 2 to 3:
Chiralpak OD-H column (250ꢃ4.6 mm), isocratic mobile
phase 7/3 hexane/2-propanol with 0.1% TFA, flow rate
1 mLminÀ1 at 258C, detection at 210 nm, retention times
(S)-3, tR =3.3 min; (R)-3, tR =3.7 min; 2, tR =4.5 min.
Diester (R)-3
A 1:1 mixture of 2 and (R)-3 (5 mg) was added to a suspen-
sion of hydrolase (10 mg) in 0.1M KH2PO4 buffer at pH 7
(1.5 mL). The reaction was shaken for 16 h at 308C. The re-
action mixture was extracted with MtBE (2ꢃ1 mL) to
remove unreacted diester (R)-3. 2M HCl (60 mL) and
CDCl3 (1 mL) were added to the reaction mixture and the
organic layer was separated, filtered through a cotton wool
plug containing anhydrous Na2SO4 and analysed directly by
1H NMR. In order to ascertain whether hydrolysis to diacids
7 or 8 had occurred, the aqueous phase was lyophilised and
the resulting residue redissolved in D2O and analysed by
1H NMR.
Citraconic Acid Dimethyl Ester (2)
A suspension of K2CO3 (335 g, 2.43 mol) in DMF (1.5 L)
was slurried for 1 hour. To this, a solution of citraconic acid
(150 g, 1.15 mol) in DMF (150 mL) was added. The resul-
tant mixture was stirred at 258C for 30 min. The appearance
of the suspension changed from a chalk white to pale
yellow. At this point, iodomethane (288 mL, 656 g, 4.62 mol)
was added over 1 hour. The reaction vessel was sealed and
stirring continued at 258C for 60 h. H2O (7.5 L) was added
and the product was extracted with MtBE (1ꢃ2.4 L, 2ꢃ
1.2 L). The combined organic extracts were washed with
H2O (3ꢃ1.2 L), saturated NaHCO3 (1 L) and brine
(500 mL), dried over Na2SO4 and concentrated under
vacuum to afford citraconic acid dimethyl ester (2) as
a clear oil; yield: 96 g (53%). Purity was determined to be
98.4% by 1H NMR w/w analysis against an internal stan-
Process Development Reaction Conditions for the
Optimisation of the Conversion of 2 to 3
Process development experiments were carried out using
a Radleyꢁs Carousel parallel reactor at 400 rpm. All reac-
tions were scaled up/down as required to run at a standar-
dised 20 mL total reaction volume in order to eliminate ex-
traneous environmental factors. Biphasic samples (1 mL)
were withdrawn from the reaction media while stirring was
maintained. MtBE (2 mL) was added, and the sample was
shaken vigorously. The organic layer was separated, dried
over anhydrous Na2SO4, and filtered through cotton wool.
The samples were analysed directly by GC as described in
Analytical Methods.
1
dard. H NMR 400 MHz (CDCl3): d=2.06–2.07 (d, 3H, J=
1.6 Hz), 3.73 (s, 3H), 3.83 (s, 3H), 5.86–5.87 (d, 1H, J=
1.6 Hz); 13C NMR 100 MHz (CDCl3): d=20.5, 51.8, 52.4,
120.6, 145.7, 165.4, 169.4.
(R)-Dimethyl 2-Methylsuccinate [(R)-3]
A solution of olefin 2 (5.00 g, 31.65 mmol) in toluene
(40 mL) was added to a suspension of ERED-04 cell paste
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David Mangan et al.
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(5 g) in pH 8 Tris HCl buffer (100 mL, 0.1M) containing
NAD+ (250 mg, 0.38 mmol) while stirring at 308C. Isopropyl
alcohol (2.60 mL, 2.03 g, 33.78 mmol) was added. In addi-
tion, polypropylene glycol (1 mL) was added to prevent ex-
cessive foaming. The reaction mixture was stirred mechani-
cally using an overhead stirrer at 308C for 59 h while sparg-
ing at 2 LminÀ1 to remove acetone formed. The depletion of
2-propanol was counterbalanced by passing the pressurised
airflow through a solution of 2-propanol in water (3% v/v)
before passing through the reaction mixture. The reaction
tiveness Program for Northern Ireland. The authors would
like to thank Dr. Alan Thompson for his expert assistance
with mass spectral analysis.
1
was monitored by in situ H NMR sampling. Complete con-
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afford the title compound as an oil with an enantiomeric
excess of 99.8% as determined by HPLC analysis; yield:
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A solution of olefin 2 (70 g, 0.443 moles) in toluene
(560 mL) was added to a suspension of ERED-04 cell paste
(70 g) in pH 8 Tris buffer (1.4 L, 0.1M) containing NAD+
(3.5 g, 5.28 mmol) while stirring at 308C. Isopropyl alcohol
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8.1 Hz, J=16.8 Hz), 2.91–3.03 (m, 1H), 3.70 (s, 3H);
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Acknowledgements
This study was part financed by the European Regional De-
velopment Fund under the European Sustainable Competi-
2190
asc.wiley-vch.de
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 2185 – 2190