Systematic methodology for the development of biocatalytic hydrogen-borrowing cascades: Application to the synthesis of chiral α-substituted carboxylic acids from α-substituted α,β-unsaturated aldehydes

Article abstract of DOI:10.1039/c4ob02282c

Ene-reductases (ERs) are flavin dependent enzymes that catalyze the asymmetric reduction of activated carbon-carbon double bonds. In particular, α,β-unsaturated carbonyl compounds (e.g. enals and enones) as well as nitroalkenes are rapidly reduced. Conversely, α,β-unsaturated esters are poorly accepted substrates whereas free carboxylic acids are not converted at all. The only exceptions are α,β-unsaturated diacids, diesters as well as esters bearing an electron-withdrawing group in α- or β-position. Here, we present an alternative approach that has a general applicability for directly obtaining diverse chiral α-substituted carboxylic acids. This approach combines two enzyme classes, namely ERs and aldehyde dehydrogenases (Ald-DHs), in a concurrent reductive-oxidative biocatalytic cascade. This strategy has several advantages as the starting material is an α-substituted α,β-unsaturated aldehyde, a class of compounds extremely reactive for the reduction of the alkene moiety. Furthermore no external hydride source from a sacrificial substrate (e.g. glucose, formate) is required since the hydride for the first reductive step is liberated in the second oxidative step. Such a process is defined as a hydrogen-borrowing cascade. This methodology has wide applicability as it was successfully applied to the synthesis of chiral substituted hydrocinnamic acids, aliphatic acids, heterocycles and even acetylated amino acids with elevated yield, chemo- and stereo-selectivity. A systematic methodology for optimizing the hydrogen-borrowing two-enzyme synthesis of α-chiral substituted carboxylic acids was developed. This systematic methodology has general applicability for the development of diverse hydrogen-borrowing processes that possess the highest atom efficiency and the lowest environmental impact. This journal is

Full text of DOI:10.1039/c4ob02282c

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Systematic methodology for the development
of biocatalytic hydrogen-borrowing cascades:
application to the synthesis of chiral α-substituted
carboxylic acids from α-substituted
Cite this: Org. Biomol. Chem., 2015,
13, 223
α,β-unsaturated aldehydes†
Tanja Knaus,‡a Francesco G. Mutti,‡b Luke D. Humphreys,c Nicholas J. Turnerb and
Nigel S. Scrutton*a
Ene-reductases (ERs) are flavin dependent enzymes that catalyze the asymmetric reduction of activated
carbon–carbon double bonds. In particular, α,β-unsaturated carbonyl compounds (e.g. enals and enones)
as well as nitroalkenes are rapidly reduced. Conversely, α,β-unsaturated esters are poorly accepted sub-
strates whereas free carboxylic acids are not converted at all. The only exceptions are α,β-unsaturated
diacids, diesters as well as esters bearing an electron-withdrawing group in α- or β-position. Here, we
present an alternative approach that has a general applicability for directly obtaining diverse chiral α-sub-
stituted carboxylic acids. This approach combines two enzyme classes, namely ERs and aldehyde de-
hydrogenases (Ald-DHs), in a concurrent reductive-oxidative biocatalytic cascade. This strategy has several
advantages as the starting material is an α-substituted α,β-unsaturated aldehyde, a class of compounds
extremely reactive for the reduction of the alkene moiety. Furthermore no external hydride source from a
sacrificial substrate (e.g. glucose, formate) is required since the hydride for the first reductive step is liber-
ated in the second oxidative step. Such a process is defined as a hydrogen-borrowing cascade. This
methodology has wide applicability as it was successfully applied to the synthesis of chiral substituted
hydrocinnamic acids, aliphatic acids, heterocycles and even acetylated amino acids with elevated yield,
chemo- and stereo-selectivity. A systematic methodology for optimizing the hydrogen-borrowing two-
enzyme synthesis of α-chiral substituted carboxylic acids was developed. This systematic methodology
has general applicability for the development of diverse hydrogen-borrowing processes that possess the
highest atom efficiency and the lowest environmental impact.
Received 11th August 2014,
Accepted 29th October 2014
DOI: 10.1039/c4ob02282c
www.rsc.org/obc
reactions using enzymes one pot allow this goal to be achieved
as intermediate isolation and purification steps are
Introduction
Nowadays, there is an urgent demand for new chemical reac- avoided and energy consumption is minimized.4,5 The major
tions and processes that possess an elevated atom efficiency as challenge is to perform cascade reactions wherein an oxidative
well as a low environmental impact.1–3 Multi-step chemical and a reductive step are running simultaneously without any
compartmentalization.6 One of the early examples of two-step
concurrent oxidative-reductive enzymatic cascades was the de-
aManchester Institute of Biotechnology, Faculty of Life Sciences, University of
racemization and the stereoinversion of secondary alcohols
using stereocomplementary alcohol dehydrogenases (ADHs).7
Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
E-mail: nigel.scrutton@manchester.ac.uk
However, this cascade was operated by four enzymes constitut-
ing two redox independent steps (i.e. non-interconnected); as a
consequence, redox equivalents were supplied at the expense
of formate and molecular oxygen as sacrificial co-substrates,
hence generating hydrogen peroxide and carbon dioxide as
waste. A similar concept was applied to the two-step oxidative-
reductive combination of an enzyme with an artificial metal-
enzyme or a metal catalyst.8,9 In contrast, a two-step redox
bManchester Institute of Biotechnology, School of Chemistry, University of
Manchester, 131 Princess Street, Manchester, M1 7DN, UK
cGSK Medicines Research Centre, Gunnel’s Wood Road, Stevenage, Herts,
SG1 2NY, UK
†Electronic supplementary information (ESI) available: Synthesis of substrates
and reference compounds, sources and cloning/expression/purification con-
ditions for ERs and Ald-DHs used in this study, analytical methods as well as
chiral GC and HPLC chromatograms. See DOI: 10.1039/c4ob02282c
‡These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 223–233 | 223

View Article Online
Paper
Organic & Biomolecular Chemistry
self-sufficient biocatalytic network has been recently presented
for the amination of primary alcohols.10 In this case, the redox
equivalents liberated in the first oxidative step were consumed
in the second reductive step in the form of NAD(P)H. As the
cofactor acts as a shuttle of hydride within the catalytic cycle,
such a process is also defined as a hydrogen-borrowing
cascade. Another example is biocatalytic redox isomerisation
of allylic alcohols.11 Recently, a hydrogen-borrowing biocata-
lytic synthesis of α-substituted carboxylic acids from α-substi-
tuted α,β-unsaturated aldehydes was presented.12 However, the
two-step cascade was run using crude preparations of the
enzymes (cell extracts) and thereby efficient internal recycling
of the nicotinamide cofactor was not demonstrated as other
enzymes may contribute to the overall process. Furthermore, a
general strategy for setting up a hydrogen-borrowing biocata-
lytic cascade has not been presented until to date. Thus, in
this work, we decided to study a systematic approach for
Scheme 1 Concurrent redox self-sufficient two enzyme cascade reac-
tion. The redox equivalents required in the first reductive step are pro-
vided by the second oxidative step in form of NAD(P)H.
α,β-unsaturated esters requires an external source of hydride
that is provided by the NAD(P)H cofactor. Most of the recent
publications report the reduction of activated α,β-unsaturated
esters using suprastoichiometric NAD(P)H or catalytic NAD(P)H
in presence of glucose as the sacrificial substrate for cofactor
regeneration. Nevertheless, the use of catalytic amounts of
cofactor with GDH/glucose, somehow, worsened conversion
and stereoselectivity. Additionally, the asymmetric carbon–
carbon double bond reduction of some classes of α,β-unsatu-
rated esters is not applicable. For instance, α-substituted
cinnamic acid methyl esters bearing an additional α-cyano
moiety decomposed or polymerized under the reaction con-
ditions for the enzymatic reduction.33 In this work, we con-
ceived an alternative approach that has general applicability to
directly access optically active α-substituted carboxylic acids
(Scheme 1). The approach combines two enzyme classes,
namely ERs and aldehyde dehydrogenases (Ald-DHs), in a con-
current reductive-oxidative biocatalytic cascade.6
developing
a
successful biocatalytic hydrogen-borrowing
cascade. The asymmetric hydrogen-borrowing biocatalytic
synthesis of α-substituted carboxylic acids from α-substituted
α,β-unsaturated aldehydes was chosen as the case study. The
development of such a cascade is not trivial since the enzymes
involved in the reductive and in the oxidative steps have to
operate concomitantly with high activity and stability, and
under the same reaction conditions (T, pH, type of cofactor,
type of buffer, cosolvent, etc.). Moreover, enzyme concen-
trations and kinetics have to be thoroughly studied in order to
maximize chemo- and stereoselectivity.
Ene-reductases (ERs) from the “Old Yellow Enzyme Family”
(OYEs) are flavin dependent enzymes that, in the last decade,
have been applied extensively in biocatalysis.13–16 These
enzymes catalyze the asymmetric reduction of carbon–carbon
double bonds that are activated by conjugation with an elec-
tron-withdrawing substituent. In particular, α,β-unsaturated
carbonylic compounds (e.g. enals and enones) as well as
nitroalkenes are rapidly reduced by the ERs, affording quanti-
tative yields in most cases.17,18,19–22 In contrast, alkenes
bearing a single conjugated ester moiety or a free carboxylic
group are poorly reduced or not converted at all.23–25
Reduction of acid derivatives is restricted to only a few families
such as α,β-unsaturated, 1–2 substituted diesters or diacids
(e.g. fumarate, maleate, citraconate, mesaconate) or β-cyano-,
β-alkoxy- and β-aryloxy-α,β-unsaturated esters.26–30 More
recently, it has been shown that an electron-withdrawing
halogen group in the α-position can significantly increase the
reactivity of α,β-unsaturated esters towards bio-reduction.31–33
The requirement of an additional activating group, signifi-
cantly narrows the substrate scope for the biocatalytic asym-
metric reduction of α,β-unsaturated esters, whereas the same
reaction on α,β-unsaturated acids is completely inapplicable.
This is a potential limitation given that most developed syn-
thetic routes to pharmaceuticals and agrochemicals require
optically active α-substituted carboxylic acids as intermediates.
Therefore, synthetic routes involving an ER would require a
further hydrolytic step to obtain the carboxylic moiety from
This strategy has several advantages: (I) the starting
material is an α-substituted α,β-unsaturated aldehyde, a class
of compounds extremely reactive for the carbon–carbon
double bond reduction; (II) no external source of hydride from
Chart 1 Systematic steps for designing a successful hydrogen-borrow-
the related ester. Crucially, the direct reduction of activated ing cascade reaction.
224 | Org. Biomol. Chem., 2015, 13, 223–233
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
a sacrificial substrate (e.g. glucose, formate) is required; (III)
the cascade reaction has the highest atom efficiency as the
only additional reagent is water and no waste is produced; (IV)
the process takes advantage of the intrinsic stereoselectivity of
the ER to enable transfer of the hydride predominantly onto
one of the prochiral faces of the alkene moiety.
This challenging two enzyme hydrogen-borrowing cascade
was developed following the systematic strategy as depicted in
Chart 1. Finally, the potential utility of this cascade sequence
was demonstrated in the production of diverse important
intermediates for the chemical industry such as chiral substi-
tuted hydrocinnamic acids, aliphatic acids, heterocycles and
even acetylated amino acids.
Table 1 Asymmetric bioreduction of 1a using OYEs
Entry
ER
Conv.a [%]
1b [%]
1c [%]
ee 1bb [%]
1
2
3
4
5
6
7
8
PETNR
TOYE
OYE2
OYE3
XenA
XenB
LeOPR1
NerA
>99c
>99c
>99c
>99c
66c
78
87
75
71
56
82
85
79
71
89
93
22
13
25
29
10
18
15
21
29
11
7
rac
rac
28 (S)
28 (S)
18 (R)
rac
rac
rac
18 (S)
rac
>99c
>99c
>99c
>99c
>99d
>99d
9
10
11
GluOx
YqjM
MR
rac
a Achiral GC (DB-Wax). b Chiral HPLC (Chiralsil OJ-H). c Reaction time
24 h. d Reaction time 6 h. Experimental conditions: reaction volume =
1 mL, 50 mM KPi pH 7.0, 30 °C, [ER] = 2 µM, [1a] = 5 mM, [NADPH] =
10 µM, [GDH] = 10 U, [glucose] = 300 mM; extraction with MTBE
(2 × 500 µL).
Results and discussion
Since the stereogenic centre is introduced into the final
product in the first reductive step of the cascade (Scheme 2),
we initially tested a wide panel of eleven different ERs that are
originated from various sources such as bacteria, yeasts and
plants.34–42 The parameter for this initial screening of the ERs
was the stereoselectivity for the reduction of the carbon–
carbon double bond of the target substrate α-methyl-trans-
cinnamaldehyde (1a) (Chart 1). The starting reaction conditions
were taken from a survey of the recent literature.21,35,43,44
Asymmetric reduction is commonly conducted using
NADPH as cofactor that is recycled by glucose dehydrogenase
(GDH) at the expense of glucose as the sacrificial co-substrate.
Surprisingly, although it is known that α-chiral aldehydes are
prone to racemization in aqueous solution,45 the majority of
the procedures reported in the literature for this biocatalytic
reaction envisage 24 h reaction time. Therefore these reaction
conditions and this reaction time were employed in the initial
experiment. Quantitative conversion was achieved with the ERs
tested, with XenA being the only exception (Table 1). However,
the reduced aldehyde (1b) was obtained either in a racemic
form or with poor enantiomeric excess (up to 28% (S)). More-
over, a relevant amount of 1b (from 7% to 29%) was further
reduced to the corresponding alcohol (1c).
Fig. 1 Progress curve for the biotransformation of 1a by (A) YqjM and
■
In order to ascertain the origin of the poor enantiomeric (B) OYE2. Concentration of α-methyl-trans-cinnamaldehyde 1a
(
),
●
α-methylhydro-cinnamaldehyde 1b ( ), α-methylhydrocinnamic alcohol
1c ( ) and (R)-α-methyl-hydrocinnamaldehyde ( ) and (S)-α-methyl-
hydro-cinnamaldehyde ( ). Experimental conditions: reaction volume =
1 mL, 50 mM KPi pH 7.0, 30 °C, [ER] = 2 µM, [1a] = 5 mM, [NADPH] =
excess, biocatalytic reduction was studied in more detail using
two selected ERs. OYE2 was chosen since it showed the
highest ee (28% (S)) and better chemoselectivity compared to
OYE3. YqjM was also selected because it shows high chemo-
□
◆
○
11 mM; extraction with MTBE (2 × 500 µL). Conversion measured by
selectivity (89%), although the product was obtained in achiral GC (DB-Wax); ee measured by chiral HPLC (Chiralsil OJ-H).
racemic form. In this experiment, the impact of the reaction
time was evaluated on the conversion and ee. Additionally,
NADPH was used in stoichiometric amount (i.e. without a re-
cycling system) to avoid any possible cross-activity from GDH.
Fig. 1 shows that the reduction of the alkene moiety is com-
plete after two hours using YqjM (Fig. 1A) and in less than
30 min using OYE2 (Fig. 1B). OYE2 performs the reduction of
the carbon–carbon double bond with perfect stereoselectivity
as witnessed by the ee of >99% after the first few minutes of
the reaction. Hence, the origin of the poor enantioselectivity
for the reduction with OYE2 stems from the spontaneous
chemical racemization of 1b in aqueous buffer. Furthermore,
Scheme 2 Hydrogen borrowing cascade reaction.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 223–233 | 225

View Article Online
Paper
Organic & Biomolecular Chemistry
as the reaction is essentially complete after 15 min, prolonging
the reaction time is not advantageous as this depletes the ee of
1b. In contrast, the optical purity of 1b was found to be extre-
mely poor at early time points when the reduction was carried
out with YqjM.
Table 3 1 h bioreduction of 1a using NADH as cofactor
pH 7
pH 8
Entry
pH 9
Conv.a
[%]
Conv.a
[%]
eeb
[%]
Conv.a
[%]
eeb
[%]
ER
In another experiment, the same reaction under the same
reaction conditions was repeated using catalytic NADPH and
GDH/glucose for cofactor regeneration (Fig. S3 ESI†). In con-
trast to the experiment using OYE2 with stoichiometric
NADPH, the use of catalytic NADPH in presence of GDH led to
the formation of the alcohol 1c as side-product in significant
amounts (22% after 11 h). Therefore ERs must be in general
chemoselective enzymes for the reduction of 1a; further
reduction to 1c is attributed to a promiscuous activity of the
GDH (a few purified GDHs were tested). The reduction with
stoichiometric NADPH and YqjM also led to the formation of a
minor quantity of alcohol 1c (8%) after 19 h. However, this is
attributed to the presence of some impurities (e.g. alcohol
dehydrogenases) in the YqjM protein solution (Fig. S1 ESI†).
Therefore, it is important to avoid the use of GDH and employ
highly purified ERs to carry out biocatalytic reduction of sub-
strate 1a. Even taking this into account one step reduction
does not lead to high conversion and ee, because of spon-
taneous chemical racemization. The hydrogen-borrowing
approach described here also has the advantage of solving this
problem, since the aldehyde 1b is promptly oxidized to the
corresponding non-enolisable acid 1d.
1
2
3
4
5
6
7
8
PETNR
TOYE
OYE2
OYE3
XenA
XenB
2
50
99
15
6
n.d.
16 (S) 32
90 (S) 48
97 (S)
n.d.
n.d.
19 (R) 12
7 (R) 47
50 (S) 12
16 (R) 32
2
n.d.
rac
83 (S) n.m.
95 (S) n.m.
n.d.
n.d.
38 (R) n.m.
14 (R) n.m.
39 (S) n.m.
38 (R) n.m.
n.m.
n.m.
9
5
4
n.m.
n.m.
7
LeOPR1 19
NerA
GluOx
YqjM
MR
46
19
29
78
9
10
11
5 (R)
71
8 (R)
n.m.
a Achiral GC (DB-Wax). b Chiral HPLC (Chiralsil OJ-H); n.d. not
determined; n.m. not measurable. Experimental conditions: reaction
volume = 1 mL, 50 mM KPi (pH 7.0 and 8.0) and 50 mM Tris/HCl pH
9.0, 30 °C, [ER] = 2 µM, [1a] = 5 mM, [NADH] = 10 µM, [GDH] = 10 U,
[glucose] = 300 mM; extraction with MTBE (2 × 500 µL).
Table 2 clearly shows that the reaction rate for Ald-DH-BOV as
well as Ald-DH-HL gradually increases passing from pH 6, 7,
8 and reaching the maximum at pH 9. Moreover, Ald-DH-EC is
active at a broader pH range. Also at pH 7 – which is the pre-
ferred pH value for ERs – an acceptable reaction rate could be
observed.
In the next step (Chart 1) the activity of the ERs was reana-
lyzed at different pH values and using NADH as cofactor, as
this is the preferred cofactor for the Ald-DHs. This step is vital
for the set-up of a successful hydrogen-borrowing cascade, as
it aims at identifying suitable conditions for the combination
of both enzyme classes. The reaction time was set to 1 h to
minimize chemical racemization (Table 3). In all the cases at
pH 9 no activity for the ERs could be measured. Only at pH 7,
using OYE2 was possible to achieve quantitative conversion
and a good enantiomeric excess (90% (S); Table 3, entry 3). All
the other ERs showed imperfect ee and/or low conversion
under these reaction conditions. Unfortunately, none of the
ERs tested could provide the (R) enantiomer in high enantio-
enriched form. As a consequence OYE2 was selected as the
best ER and the Ald-DH from E. coli as the best dehydrogenase
for performing the cascade reaction.
Moving along Chart 1, the further steps are aimed at opti-
mizing the cascade to maximize conversion, chemo- and
stereoselectivity and reducing the reaction time. The para-
meters investigated are the concentrations of the enzymes and
of the cofactor. The first combination of both enzymes – ER
and Ald-DH – was performed with the optimized conditions
identified in the single experiments ([OYE2] = [Ald-DH-EC] =
2 µM, [NADH] = 10 µM). Unfortunately, after 6 h reaction time,
the conversion was only 45% (Fig. 2 and Table S3 ESI†).
Nevertheless we noted that the Ald-DH preferred to oxidize
the saturated aldehyde 1b, rather than the unsaturated starting
material 1a, as the main product was α-methyl-hydro cinnamic
acid (1d). Based on these results, the object parameters
The second branch of Chart 1 relates to the oxidative step
catalyzed by an Ald-DH. The oxidation of 1b to the corres-
ponding carboxylic acid (1d) was performed using three
different aldehyde dehydrogenases, namely the Ald-DHs from
bovine lens (Ald-DH-BOV),46,47 horse liver (Ald-DH-HL)48,49
and E. coli (Ald-DH-EC).50 The enzymes were tested for the
conversion of 1b. The parameters explored experimentally
(Chart 1) were reaction pH values (step 1) and the requirement
for
a
cofactor NADP+/NAD+ (step 2). Ald-DH-BOV and
Ald-DH-HL were shown to be strictly selective for NAD+; no
conversion was observed with NADP+ as cofactor (Table 2,
entries 2 and 4; Table S2 ESI† for detailed time study).
Conversely, Ald-DH-EC is able to accept both cofactors but the
reaction proceeded faster with NAD+ (Table 2, entries 5 and 6).
Table 2 Conversion of 1b to 1d after 23 h reaction timea
Entry
Ald-DH
Cofactor
pH 6
pH 7
pH 8
pH 9
1
2
3
4
5
6
BOV
BOV
HL
HL
EC
NAD+
20%
n.m.
7%
n.m.
7%
52%
n.m.
32%
n.m.
>99%
44%
63%
n.m.
32%
n.m.
>99%
66%
74%
n.m.
51%
n.m.
>99%
70%
NADP+
NAD+
NADP+
NAD+
EC
NADP+
3%
a n.m. not measurable. Experimental conditions: reaction volume =
1 mL, 50 mM KPi (pH 6.0, 7.0, 8.0) and 50 mM Tris/HCl pH 9.0, 30 °C,
reaction time = 23 h, [Ald-DH] = 2 µM, [1b] = 5 mM, [NAD+] = 7 mM;
extraction with MTBE (2 × 400 µL), derivatization with (trimethylsilyl)
diazomethane to methylester. Conversion measured by achiral GC
(DB-Wax).
226 | Org. Biomol. Chem., 2015, 13, 223–233
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
saturated aldehyde intermediate 1b, rather than the unsatu-
rated starting material 1a.
In almost all the cases quantitative conversion was achieved
within 6 h reaction time. The only exceptions were when the
concentration of both enzymes was reduced to 2 µM, using
10 µM or 100 µM NADH (Table 4, entries 9 and 16). The
increased enzyme concentrations (up to 10 µM for the ER and
5 µM for the Ald-DH, respectively) afforded quantitative con-
version, albeit only 10 µM of NADH were employed (Table 4,
entry 15). The ees were excellent in all the cases, ranging from
97% to 99%. Therefore, the saturated aldehyde intermediate
(1b) is quickly oxidized by the Ald-DH. It is also worth noting
that very low quantities of the unsaturated carboxylic acid 1e
were formed (from 2% to 8%).
At this stage of the work we did not have any information
regarding the kinetics of the process. Hence the progress of
the reaction was monitored as a function of time. Specifically,
the concentrations of the starting material (1a), of the inter-
mediate (1b), of the final product (1d) and of the side product
(1e) as well as the ees, were determined as a function of the
time (Fig. 3). At this step of our work flow (Chart 1), the
chemo- and, in particular, the stereoselectivity are maximized.
In fact, the cascade reaction must run the minimum time
required to achieve full conversion. Longer reaction times lead
to lower ees, due to spontaneous racemization, and dimin-
ished productivity. For this experiment, the best conditions
from the previous step were taken (Table 4, entry 14).
Fig. 2 First experiment one-pot two enzyme cascade reaction for the
conversion of 1a to 1d. Concentration of α-methylcinnamaldehyde 1a
■
●
(
), α-methylhydrocinnamic acid 1d ( ), α-methylhydrocinnamaldehyde
◆
□
1b ( ) and α-methylcinnamic acid 1e ( ). Experimental conditions: reac-
tion volume = 1 mL, 50 mM KPi pH 7.0, 30 °C, [OYE2] = 2 µM, [Ald-
DH-EC] = 2 µM, [1a] = 5 mM, [NADH] = 10 µM; two-step selective
extraction with MTBE: (I) under basic conditions (aldehydes) and
(II) acidic conditions and derivatization with (trimethylsilyl)diazomethane
(acids (ester)); IS = 2-phenylethanol. Conversion was measured by
achiral GC (DB-Wax).
(ER, Ald-DH, NAD+ concentration, time) were varied for
improving the outcome of the cascade (Table 4). The reaction
was stopped after 6 hours. The substrate was kept at 5 mM
concentration whereas: (I) the amount of the cofactor was
increased and (II) the concentration of the two enzymes was
varied to investigate the influence of the ratio [ER]/[Ald-DH].
The latter is a crucial point because the concentrations of the
two enzymes have to be carefully balanced. In fact, the Ald-DH
must quickly oxidize the saturated aldehyde to avoid racemiza-
tion of the aldehyde product; also the Ald-DH must oxidize the
The reaction was extremely efficient since it was complete
after 90 min despite the low concentrations of the enzymes
used (Fig. 3, Table S4 ESI;† [OYE2] = 10 µM, [Ald-DH-EC] =
5 µM). The concentration of the intermediate 1b remained
constant with time (ca. 3%). The cinnamic acid by-product 1e
was mainly produced at the beginning of the reaction. This is
not surprising as the cinnamic aldehyde 1a is present at
Table 4 Optimization of the one-pot two enzyme cascade reaction for
the conversion of 1a
ee (S)-
NADH
Entry [µM]
OYE2
[µM]
Ald-DH Conv.a
EC [µM] [%]
1e
[%]
1d
[%]
1db
[%]
1
2
3
4
5
6
7
8
500
500
500
500
250
250
250
250
100
100
100
100
50
2
2
10
10
2
2
10
2
10
2
10
2
10
2
10
2
10
5
5
5
>99
>99
>99
>99
>99
>99
>99
>99
90
>99
>99
>99
>99
>99
>99
45
2
6
2
4
5
7
4
6
5
6
5
6
5
5
6
8
98
94
98
96
95
93
96
94
82
94
95
94
95
95
94
36
98
98
99
99
98
98
99
99
97
98
99
99
99
99
99
n.d.
2
10
10
2
9
10
11
12
13
14
15
16
2
10
10
10
10
10
2
Fig. 3 Time study optimized one-pot two enzyme cascade reaction for
the conversion of 1a to 1d. Concentration of α-methylcinnamaldehyde
■
●
□
25
10
10
1a ( ), α-methylhydrocinnamic acid 1d ( ), α-methylcinnamic acid 1e ( ),
◆
α-methylhydro-cinnamaldehyde 1b
(
)
and enantiomeric excess of
2
○
α-methylhydrocinnamic acid (S)-1d ( ). Experimental conditions: reac-
tion volume = 1 mL, 50 mM KPi pH 7.0, 30 °C, [OYE2] = 10 µM,
[Ald-DH-EC] = 5 µM, [NADH] = 25 µM, [1a] = 5 mM; two-step selective
extraction with MTBE: (I) under basic conditions (aldehydes) and
(II) acidic conditions and derivatization with (trimethylsilyl)-diazomethane
(acids (ester)); IS = 2-phenylethanol. Conversion was measured by
achiral GC (DB-Wax) and ee by chiral HPLC (Chiralsil OJ-3).
a Achiral GC (DB-Wax). b Chiral HPLC (Chiralsil OJ-3); n.d. = not
determined. Experimental conditions: reaction volume = 1 mL, 50 mM
KPi pH 7.0, 30 °C, [1a] = 5 mM, reaction time = 6 h; two-step selective
extraction with MTBE: (I) under basic conditions (aldehydes) and (II)
acidic
conditions
and
derivatization
with
(trimethylsilyl)-
diazomethane (acids (ester)); IS = 2-phenylethanol.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 223–233 | 227

View Article Online
Paper
Organic & Biomolecular Chemistry
almost 5 mM concentration, whereas the concentration of the
For example, enantiopure 1d is a precursor for the pro-
intermediate 1b is almost negligible during the first few duction of biological active compounds that possess muscle
minutes of the reaction. However, after having identified the relaxant activity.54,55 2d constitutes the main core of drugs pos-
suitable reaction conditions, the concentration of cinnamic sessing antagonist activity for the acetylcholine M1 receptor of
acid 1e remained constant during the reaction time and always human.56 3d is commonly employed for the production of
below 5%. This is an indication that the Ald-DH-EC is signifi- herbicidals,57 antibacterial58 as well as anticancer compounds
cantly more active towards the saturated aldehyde 1b rather (e.g. leukemia, lung and prostate carcinoma).59–61 Optically
than the unsaturated one 1a, which is the crucial point for active compound 5d and derivatives thereof are precursors
enabling this hydrogen-borrowing biocatalytic cascade reac- of enantiopure aminochromane derivatives; these can be
tion. Moreover, the cascade showed a perfect stereoselectivity obtained through the conversion of the carboxylic moiety into
(>99% ee).
the isocyanate intermediate, followed by Curtius rearrange-
Productivity is an important factor for a successful process ment. The optically active aminochromanes are key intermedi-
and this objective is considered in the last step of the proposed ates of many antidepressant drugs such as robalzotan,62
Chart 1. The studied parameter was the substrate concen- ebalzotan63 as well as DBH inhibitors such as chromanyl-imid-
tration. Applying the optimized reaction conditions from the azolethiones.64 Finally, our strategy can give access to
previous round and increasing the substrate concentration to enantioenriched unnatural amino acids. For instance N-acetyl-
10 mM, 97% conversion was still obtained after 6 h reaction ated (S)-para-chloro phenylalanine 4d was obtained. Through
time with high chemoselectivity (93%) and excellent stereo- the carbon carbon coupling using the chloro group on the aro-
selectivity (99%; Table S5 ESI†). Increasing the substrate con- matic ring, more complex structures possessing various bio-
centration up to 25 mM required the prolongation of the logical activities can be achieved.65–68 (S)-para-chloro
reaction time up to 24 h and doubling of the amount of phenylalanine derivatives also possess antimycobacterial (e.g.
enzyme in order to achieve >99% conversion (chemoselectivity Mycobacterium tuberculosis),69 antifungal70 as well as anti-
= 96%, ee = 96%). However, bio-catalytic reductions with ERs cancer activity.71 Additionally, N-derivatized (S)-para-chloro
are commonly performed at 5 mM substrate concentration phenylalanine is also used for the production of herbicides.72
since these enzymes are plagued by substrate and/or product
For each substrate, the steps described in Chart 1 were
inhibition.51–53 The proposed two-step biocatalytic cascade we applied as described for 1a. For instance, in the case of
have developed here also allows alleviation of this problem α-phenylcinnamaldehyde (2a), two ERs, namely PETNR and
because the product of the bioreduction (1b) is immediately XenB, showed the highest activity/stereoselectivity for the
removed by the Ald-DH in the second step.
reduction of the carbon–carbon double bond of the unsaturated
The hydrogen-borrowing biocatalytic cascade was therefore aldehyde; the Ald-DH from bovine lens was found to be the
successfully developed for the conversion of the test substrate most efficient enzyme for the oxidation of the aldehyde inter-
1a into 1d with quantitative conversion, improved productivity mediate (2b) to the corresponding acid (2d) (Table S6 and S7
and excellent chemo- and stereoselectivity. Finally, we wanted ESI†). Fig. 5 depicts the final time studies, after the overall pro-
to investigate if this strategy for developing a hydrogen-borrow- cedure described in Chart 1, for the conversion of 2a into 2d
ing cascade has a more general validity and it is therefore using PETNR and XenB in combination with the aldehyde dehy-
applicable on a broad range of substrates. Thus, a panel of drogenase from bovine lens (Fig. 5A and B, and Table S8 and
structurally diverse α,β-unsaturated aldehydes, valuable syn- S9 ESI† for detailed information). Hence the substrate bearing
thons for the synthesis of chiral active pharmaceutical ingredi- the bulky phenyl-group in the alpha position was also accepted
ents, were selected (Fig. 4).
by PETNR and XenB with high stereoselectivity. In the hydro-
gen-borrowing cascade reaction using PETNR/Ald-DH-BOV, the
conversion reached 92% after 4 h, with 91% chemo-selectivity
and a perfect ee (97%). The combination of XenB/Ald-DH-BOV
resulted in 91% conversion after 5 h (95% chemoselectivity)
and excellent ee of 99%. Further changes on the reaction con-
ditions (e.g. concentration of NADH and AldDH) did not
improve the chemoselectivity but had a negative impact on the
stereoselectivity (Table S10 ESI†).
Using trans-2-methyl-2-pentenal (3a) as substrate, OYE2 and
Ald-DH-EC were selected as the best performing ER and
Ald-DH, respectively (Table S11 and S12 ESI†). Fig. 6 (Table S13
ESI†) displays the time study for the one-pot concurrent
cascade reaction. Full conversion was obtained after 60 min,
showing an ee of 98%. Using the Ald-DH from E. coli, 13% of
the unsaturated acid side product 3e were formed. Again, any
change in the reaction condition resulted in the same chemo-
selectivity but lower ee (96%). However, the chemoselectivity
Fig. 4 Schematic view of the broad substrate scope applicability of the
hydrogen-borrowing cascade combining ERs and Ald-DHs.
228 | Org. Biomol. Chem., 2015, 13, 223–233
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 7 Progress curves for the one-pot two enzyme cascade reaction
of 4a by OYE2 and Ald-DH-BOV. Concentration of (Z)-N-(1-(4-chloro-
■
(
phenyl)-3-oxoprop-1-en-2-yl)acetamide 4a
), 2-acetyl-4-chloro-
●
DL-phenylalanine 4d ( ), (Z)-2-acetamido-3-(4-chlorophenyl)acrylic acid
□
◆
4e ( ), N-(1-(4-chlorophenyl)-3-oxopropan-2-yl)acetamide 4b ( ) and
○
enantiomeric excess of 2-acetyl-4-chloro-L-phenyl-alanine (S)-4d ( ).
Experimental conditions: reaction volume = 1 mL, 50 mM KPi pH 7.0,
30 °C, [OYE2] = 10 µM, [Ald-DH-BOV] = 10 µM, [NADH] = 50 µM, [4a] =
5 mM; extraction under acidic conditions with EtOAc (2 × 400 µL) and
derivatization with (trimethylsilyl)diazomethane to methylester. Con-
version measured by achiral GC (HP5) and ee value by chiral GC (DEX-CB).
All three Ald-DHs have been tested for the oxidation of (Z)-
N-(1-(4-chlorophenyl)-3-oxoprop-1-en-2-yl)acetamide (4a) as
well as the related saturated aldehyde N-(1-(4-chlorophenyl)-3-
oxopropan-2-yl)acetamide (4b) to the corresponding carboxylic
acids. Two independent experiments showed that the Ald-DHs
clearly preferred to oxidize the saturated substrate (4b); after
5 h reaction time as the conversion for 4b was much higher
than for 4a (Table S16 and S17 ESI†).
OYE2 and Ald-DH-BOV were combined for performing the
cascade reaction starting from 4a, yielding 99% conversion,
95% ee and 81% chemoselectivity after 3 h (Fig. 7, Table S20
ESI†). Increasing the concentration of the Ald-DH from 10 µM
to 50 µM slightly increased the chemoselectivity (83%) and the
ee value (96%). In general, the ee was never higher than 96%
since the few initial minutes of the reaction. Thus, for 4a, the
ee is limited by the intrinsic stereoselectivity of the ER
(Table S21 ESI†).
Fig. 5 Progress curves for the one-pot two enzyme cascade reaction
of 2a by (A) PETNR and Ald-DH-BOV and (B) XenB and Ald-DH-BOV.
■
Concentration of α-phenylcinnamaldehyde 2a
(
), α-phenylhydro-
●
□
cinnamic acid 2d ( ), α-phenyl-cinnamic acid 2e ( ), α-phenylhydro-
◆
cinnamaldehyde 2b
(
)
and enantiomeric excess of α-phenyl-
○
hydrocinnamic acid (R)-2d ( ). Experimental conditions: reaction volume
= 1 mL, 50 mM KPi pH 7.0, 30 °C, [PETNR] & [XenB] = 25 µM,
[Ald-DH-BOV] = 4 µM, [NADH] = 50 µM, [2a] = 5 mM; two-step selective
extraction with MTBE: (I) under basic conditions (aldehydes) and (II) acidic
conditions and derivatization with (trimethylsilyl)diazomethane (acids
(ester)); IS = 2-phenylethanol. Conversion was measured by achiral GC
(DB-Wax) and ee by chiral HPLC (Chiralsil OJ-3).
The last substrate investigated was 2H-chromene-3-carb-
aldehyde (5a). After 2 h reaction time, quantitative conversion
was obtained for the one-pot two enzymes cascade reaction
using OYE2 or GluOx in combination with Ald-DH-EC result-
ing in 81% chemoselectivity and 95% ee (S-enantiomer, Fig. 8,
Table S25 and S26 ESI†).
Fig. 6 Progress curves for the one-pot two enzyme cascade reaction
of 3a by OYE2 and Ald-DH-EC. Concentration of trans-2-methyl-2-pen-
The best results for the one-pot two-enzyme cascade reac-
tion for substrates 1a–5a are summarized in Table 5. Although
the substrates investigated are structurally diverse, a suitable
adjustment of the reaction conditions allowed for obtaining
quantitative conversion in four cases out of five. Chemo-
selectivity varied from good to excellent. Moreover, the chemo-
selectivity might be improved if other Ald-DHs will be
discovered or engineered to accept only the saturated aldehyde
intermediate. The stereoselectivity was also in general elevated,
although there is an urgent demand for stereocomplementary
ERs that would lead to the opposite enantiomer with equally
high ees.
■
●
tenal 3a ( ), 2-methylpentanoic acid 3d ( ), trans-2-methyl-2-pentenoic
□
◆
acid 3e ( ), 2-methyl-pentanal 3b ( ) and enantiomeric excess of
○
2-methylpentanoic acid (S)-3d ( ). Experimental conditions: reaction
volume = 1 mL, 50 mM KPi pH 7.0, 30 °C, [OYE2] = 10 µM, [Ald-DH-EC]
= 3 µM, [NADH] = 25 µM, [3a] = 5 mM; extraction with MTBE (2 × 400
µL) under acidic conditions and derivatization with (trimethylsilyl)-diazo-
methane to methylester. Conversion was measured by achiral GC
(DB1701) and ee by chiral GC (Restek Rt-ßDEXsm).
was improved (only 8–9% of 3c were formed) when the Ald-DH
from bovine lens (Ald-DH-BOV) was combined with OYE2
remaining the ee still perfect (>98%, Table S14 ESI†).
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 223–233 | 229

View Article Online
Paper
Organic & Biomolecular Chemistry
As previously mentioned, substrates 1a and 3a have been
tested in a concomitant work by another group.12 It is impor-
tant to point out that the overall strategy depicted in Chart 1
was not developed and the enzymes were simply mixed together
without any additional accurate study. As a consequence, in
this study, substrate 1a and 3a were indeed quantitatively con-
verted but at the expense of the ees that were only 64% and
78%, respectively. In contrast, our study demonstrates that it is
possible to reach up to 99% stereoselectivity after selecting the
best suitable ER and systematically tuning the reaction para-
meters (Table 5, entry 1 and 3). Additionally, our procedure gen-
erally allowed for reduction of the reaction time from 24 h to
few hours. The substrate concentration was increased as well.
Both factors lead to a dramatic increase of the productivity.
Substrate 5a was instead the object of a two-step cascade com-
bining ERs with alcohol dehydrogenases to give enantio-
enriched β-substituted alcohols.45 The alcohol was recovered
with 91% ee, whereas in our study the related carboxylic acid
was obtained with 95% ee (Table 5, entry 5). Therefore, our pro-
posed methodology allows one to obtain the highest chemo-
selectivity and ee for a given hydrogen-borrowing cascade.
Finally, 1d was produced on a preparative scale (100 mg of
starting material 1a) resulting in quantitative conversion after
2 h reaction time with 96% chemoselectivity and >98% ee (S).
After work-up, the isolated yield was 88%.
Fig. 8 Progress curves for the one-pot two enzyme cascade reaction
of 5a by (A) OYE2 and Ald-DH-EC and (B) GluOx and Ald-DH-EC. Con-
■
centration of 2H-chromene-3-carbaldehyde 5a
(
), 3,4-dihydro 2H
●
benzopyran-3-carboxylic acid 5d ( ), 2H-chromene-3-carboxylic acid
□
◆
5e ( ), chromane-3-carbaldehyde 5b ( ) and enantiomeric excess of
○
3,4-Dihydro 2H-benzopyran-3-carboxylic acid (S)-5d ( ). Experimental
conditions: reaction volume = 1 mL, 50 mM KPi pH 7.0, 30 °C, [OYE2] &
[GluOx] = 10 µM, [Ald-DH-EC] = 3 µM, [NADH] = 100 µM, [5a] = 5 mM;
extraction under acidic conditions with EtOAc (2 × 400 µL) and derivati-
zation with (trimethylsilyl)diazomethane to methylester. Conversion
measured by achiral GC (DB-Wax) and ee value by chiral GC (Restek
Rt-ßDEXsm).
Materials and methods
Synthesis of substrates and reference compounds, sources
and cloning/expression/purification conditions for ERs and
Ald-DHs used in this study, analytical methods as well as
chiral GC and HPLC chromatograms can be found in the ESI.†
Table 5 Summary results for the optimized one-pot two enzyme cascade reaction for the conversion of 5 mM substrate
Fig. Substrate
1
ER
Ald-DH
NADH [µM] Conv. [%] Reac. time [h] Chemoselectivity [%] ee [%] Config.
OYE2 10 µM
EC 5 µM
25
>99
1.33
95
99
(S)
2
XenB 25 µM
BOV 4 µM
50
91
5
95
>98
(R)
3
4
OYE2 10 µM
OYE2 10 µM
BOV 3 µM
25
50
>99
99
12
92
81
>98
95
(S)
(S)
BOV 10 µM
6
5
OYE2 10 µM
GluOx 10 µM EC 3 µM
EC 3 µM
100
100
>99
>99
2.5
2.5
81
82
95
95
(S)
(S)
230 | Org. Biomol. Chem., 2015, 13, 223–233
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Materials
second step the carboxylic acids under acidic conditions (2 ×
400 µL MTBE or EtOAc). The acids were derivatized using the
protocol described below.
α-Methyl-trans-cinnamaldehyde (1a), α-methylhydrocinnamic
acid (1d), α-methylcinnamic acid (1e), α-phenylhydrocinnamic
acid (2d), α-phenylcinnamic acid (2e), trans-2-methyl-2-pente-
nal (3a), 2-methylpentanal (3b), 2-methylpentanoic acid (3d),
Derivatization of carboxylic acids to methylester
trans-2-methyl-2-pentenoic
acid
(3e),
2-acetyl-4-chloro-
To the extracts (800 µL) MeOH (200 µL) and (trimethylsilyl)dia-
zomethane (10 µL) were added and the reaction was shaken
at 30 °C, 160 rpm for 60 min. The excess of the derivatiza-
tion reagent was destroyed by the addition of acetic acid
(2 µL) and the reaction was again shaken at 30 °C for
another 25 min prior to analysis of the compounds by GC or
HPLC.
DL-phenylalanine (4d), 3,4-dihydro 2H-benzopyran-3-carboxylic
acid (5d) and (trimethylsilyl)diazomethane (2 M in hexanes)
were purchased from Sigma Aldrich (Gillingham, Dorset, UK).
2H-Chromene-3-carbaldehyde (5a) was purchased from
Thermo Fisher Scientific (Loughborough, UK). Glucose de-
hydrogenase (GDH, CDX-901) was bought from Codexis
(Redwood City, CA, USA). Synthetic genes for the Ald-DHs were
bought from GenScript (Piscataway, NJ, USA).
α-methylhydrocinnamaldehyde
cinnamic alcohol (1c), α-phenylcinnamaldehyde (2a), α-phenyl-
hydrocinnamaldehyde (2b), (Z)-N-(1-(4-chloro-phenyl)-3-
oxoprop-1-en-2-yl)acetamide (4a) and chromane-3-carb-
aldehyde (5b) were chemically synthesized as described in the
ESI.†
N-(1-(4-chlorophenyl)-3-oxopropan-2-yl)acetamide (4b) was
obtained through a biotransformation of the related unsatu-
rated substrate (4a) using ER and glucose as the final reducing
reagent. (Z)-2-acetamido-3-(4-chloro-phenyl)-acrylic acid (4e)
and 2H-chromene-3-carboxylic acid (5e) were obtained through
a biotransformation of the related substrates (4a and 5a,
respectively) using Ald-DH and stoichiometric amounts of
NAD+ (see ESI†).
(1b),
α-methylhydro-
Conclusions
In summary, we have developed a systematic strategy to set up
an efficient two-step hydrogen-borrowing cascade in terms of
conversion, chemoselectivity, stereoselectivity, reaction time
and substrate concentration. The biocatalytic cascade pos-
sesses the highest atom efficiency since the hydride consumed
in the first reductive step is produced in the following oxidative
step. The only additional reagent is a water molecule and no
waste (e.g. gluconolactone, carbon dioxide, etc.) is produced.
This approach was applied to the conversion of α-substituted
α,β-unsaturated aldehydes into the related optically active
saturated carboxylic acids. Our methodology proved to be suc-
cessful towards a panel of diverse substrates and can be
applied for the production of chiral substituted cinnamic
acids, aliphatic acids, heterocycles and even acetylated amino
acids. Future work should aim at further improving the
chemoselectivity by selecting or engineering other aldehyde
dehydrogenases or reversing the stereoselective outcome
of the reaction by engineering stereocomplementary ene-
reductases.
General procedure for biotransformations
The substrates were dissolved as stock solutions in ethanol or
DMSO, and then diluted to 5 mM in the reaction (2% final co-
solvent concentration). Standard reactions (1.0 mL) were per-
formed in phosphate buffer (50 mM KH2PO4/K2HPO4,
pH 7.0) and reactions were shaken at 30 °C at 160 rpm in an
orbital shaker. The reaction was terminated by the extraction
with EtOAc or MTBE (2 × 500 µL) under acidic conditions, the
extracts were dried using anhydrous MgSO4 and analyzed by
GC or HPLC as described in the ESI† to determine the conver-
sion and enantiomeric excess. (I) Asymmetric bioreduction using ER
Abbreviations
Ene-reductase
ERs: 1 mL reaction volume consisted of aldehydes 1a to 5a Ald-DH Aldehyde dehydrogenase
(5 mM), ER (2 to 4 μM), NAD(P)H (11 mM). If the GDH re-
cycling system was applied, the reaction mixture consisted of
NAD(P)H (10 µM), glucose (15 mM), and GDH (10 U). (II)
Acknowledgements
Oxidation of aldehydes using Ald-DHs: The reaction mixture con- The work was funded by the UK Biotechnology and Biological
sisted of substrate 1b to 5b (5 mM), Ald-DH (2 to 5 μM) and Sciences Research Council (BBSRC; BB/K0017802/1) and Glaxo-
NAD(P)+ (6 to 7 mM). After extraction with MTBE or EtOAc (2 × SmithKline. N.S.S. received funding as an Engineering and
400 µL) under acidic conditions and drying with anhydrous Physical Sciences Research Council (EPSRC) Established
MgSO4, the carboxylic acids were derivatized to the corres- Career Fellow (EP/J020192/1) and a Royal Society Wolfson
ponding methylester and analyzed by GC or HPLC. (III) One- Merit Award. F.G.M. has received funding from the European
pot two enzyme cascade reaction: 1 mL reaction mixture con- Union’s Seventh Framework Programme FP7/2007–2013 under
sisted of substrate (1a to 5a, 5 mM), ER (2–25 µM), Ald-DH grant agreement no. 266025 (BIONEXGEN) and no. 115360
(2–10 µM) and cofactor NADH (10–500 µM). A two-step extrac- (CHEM21). Prof. Peter Macheroux (University of Technology,
tion protocol was performed, extracting the aldehydes under Graz) is gratefully acknowledged for providing the plasmid
basic conditions with MTBE or EtOAc (2 × 500 µL) and in a DNA for XenA, XenB and NerA, LeOPR1 and YqjM.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 223–233 | 231

View Article Online
Paper
Organic & Biomolecular Chemistry
23 P. Ferraboschi, P. Grisenti, R. Casati, A. Fiecchi and
E. Santaniello, J. Chem. Soc., Perkin Trans. 1, 1987, 1743–
1748.
Notes and references
1 R. A. Sheldon, I. W. C. E. Arends and U. Hanefeld, in Green
Chemistry and Catalysis, Wiley-VCH Verlag GmbH & Co. 24 S. Koul, D. H. G. Crout, W. Errington and J. Tax, J. Chem.
KGaA, 2007, pp. 1–47.
Soc., Perkin Trans. 1, 1995, 2969–2988.
2 P. J. Dunn, Chem. Soc. Rev., 2012, 41, 1452–1461.
3 R. A. Sheldon, Chem. Soc. Rev., 2012, 41, 1437–1451.
4 E. Ricca, B. Brucher and J. H. Schrittwieser, Adv. Synth.
Catal., 2011, 353, 2239–2262.
5 P. A. Santacoloma, G. Sin, K. V. Gernaey and J. M. Woodley,
Org. Process Res. Dev., 2010, 15, 203–212.
6 J. H. Schrittwieser, J. Sattler, V. Resch, F. G. Mutti and
W. Kroutil, Curr. Opin. Chem. Biol., 2011, 15, 249–256.
7 C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux
and W. Kroutil, J. Am. Chem. Soc., 2008, 130, 13969–13972.
8 V. Köhler, Y. M. Wilson, M. Dürrenberger, D. Ghislieri,
E. Churakova, T. Quinto, L. Knörr, D. Häussinger,
F. Hollmann, N. J. Turner and T. R. Ward, Nat. Chem.,
2013, 5, 93–99.
25 M. Utaka, S. Konishi, A. Mizuoka, T. Ohkubo, T. Sakai,
S. Tsuboi and A. Takeda, J. Org. Chem., 1989, 54, 4989–
4992.
26 C. Stueckler, M. Hall, H. Ehammer, E. Pointner, W. Kroutil,
P. Macheroux and K. Faber, Org. Lett., 2007, 9, 5409–5411.
27 E. Brenna, F. G. Gatti, A. Manfredi, D. Monti and
F. Parmeggiani, Catal. Sci. Technol., 2013, 3, 1136–1146.
28 C. Stueckler, C. K. Winkler, M. Bonnekessel and K. Faber,
Adv. Synth. Catal., 2010, 352, 2663–2666.
29 C. K. Winkler, D. Clay, N. G. Turrini, H. Lechner,
W. Kroutil, S. Davies, S. Debarge, P. O’Neill, J. Steflik,
M. Karmilowicz, J. W. Wong and K. Faber, Adv. Synth.
Catal., 2014, 356, 1878–1882.
30 C. Stueckler, C. K. Winkler, M. Hall, B. Hauer,
M. Bonnekessel, K. Zangger and K. Faber, Adv. Synth.
Catal., 2011, 353, 1169–1173.
9 F. G. Mutti, A. Orthaber, J. H. Schrittwieser, J. G. de Vries,
R. Pietschnig and W. Kroutil, Chem. Commun., 2010, 46,
8046–8048.
31 E. Brenna, G. Fronza, C. Fuganti, D. Monti and
F. Parmeggiani, J. Mol. Catal. B: Enzym., 2011, 73, 17–21.
10 J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber,
J. Pfeffer, T. Haas and W. Kroutil, Angew. Chem., Int. Ed., 32 E. Brenna, F. G. Gatti, A. Manfredi, D. Monti and
2012, 51, 9156–9159. F. Parmeggiani, Org. Process Res. Dev., 2012, 16, 262–268.
11 S. Gargiulo, D. J. Opperman, U. Hanefeld, I. W. C. 33 G. Tasnadi, C. K. Winkler, D. Clay, N. Sultana,
E. Arends and F. Hollmann, Chem. Commun., 2012, 48,
6630–6632.
W. M. F. Fabian, M. Hall, K. Ditrich and K. Faber, Chem. –
Eur. J., 2012, 18, 10362–10367.
12 T. Winkler, H. Gröger and W. Hummel, ChemCatChem, 34 C. E. French and N. C. Bruce, Biochem. J., 1994, 301, 97–
2014, 6, 961–964. 103.
13 H. S. Toogood and N. S. Scrutton, Curr. Opin. Chem. Biol., 35 B. V. Adalbjornsson, H. S. Toogood, A. Fryszkowska,
2014, 19, 107–115.
14 C. K. Winkler, G. Tasnadi, D. Clay, M. Hall and K. Faber,
J. Biotechnol., 2012, 162, 381–389.
C. R. Pudney, T. A. Jowitt, D. Leys and N. S. Scrutton, Chem-
BioChem, 2010, 11, 197–207.
36 P. A. Karplus, K. M. Fox and V. Massey, FASEB J., 1995, 9,
1518–1526.
15 H. Toogood, D. Mansell, J. M. Gardiner and N. S. Scrutton,
in Comprehensive Chirality, ed. N. J. Turner, 2012, vol. 1, 37 D. S. Blehert, B. G. Fox and G. H. Chambliss, J. Bacteriol.,
pp. 216–255. 1999, 181, 6254–6263.
16 H. S. Toogood, J. M. Gardiner and N. S. Scrutton, Chem- 38 J. Straßner, A. Fürholz, P. Macheroux, N. Amrhein and
CatChem, 2010, 2, 892–914. A. Schaller, J. Biol. Chem., 1999, 274, 35067–35073.
17 Y. Fu, K. Castiglione and D. Weuster-Botz, Biotechnol. 39 J. R. Snape, N. A. Walkley, A. P. Morby, S. Nicklin and
Bioeng., 2013, 110, 1293–1301. G. F. White, J. Bacteriol., 1997, 179, 7796–7802.
18 D. J. Mansell, H. S. Toogood, J. Waller, J. M. X. Hughes, 40 N. Richter, H. Groger and W. Hummel, Appl. Microbiol. Bio-
C. W. Levy, J. M. Gardiner and N. S. Scrutton, Adv. Synth.
Catal., 2013, 3, 370–379.
19 E. Burda, T. Ress, T. Winkler, C. Giese, X. Kostrov,
technol., 2011, 89, 79–89.
41 T. B. Fitzpatrick, N. Amrhein and P. Macheroux, J. Biol.
Chem., 2003, 278, 19891–19897.
T. Huber, W. Hummel and H. Groger, Angew. Chem., Int. 42 C. E. French, S. Nicklin and N. C. Bruce, J. Bacteriol., 1996,
Ed., 2013, 52, 9323–9326. 178, 6623–6627.
20 H. S. Toogood, A. Fryszkowska, V. Hare, K. Fisher, 43 A. Fryszkowska, H. Toogood, M. Sakuma, J. M. Gardiner,
A. Roujeinikova, D. Leys, J. M. Gardiner, G. M. Stephens
and N. S. Scrutton, Adv. Synth. Catal., 2008, 350,
2789–2803.
G. M. Stephens and N. S. Scrutton, Adv. Synth. Catal., 2009,
351, 2976–2990.
44 M. Hall, C. Stueckler, H. Ehammer, E. Pointner,
G. Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W. Kroutil,
P. Macheroux and K. Faber, Adv. Synth. Catal., 2008, 350,
411–418.
21 K. Durchschein, S. Wallner, P. Macheroux, W. Schwab,
T. Winkler, W. Kreis and K. Faber, Eur. J. Org. Chem., 2012,
4963–4968.
22 Y. Yanto, H. H. Yu, M. Hall and A. S. Bommarius, Chem. 45 E. Brenna, F. G. Gatti, L. Malpezzi, D. Monti, F. Parmeggiani
Commun., 2010, 46, 8809–8811.
and A. Sacchetti, J. Org. Chem., 2013, 78, 4811–4822.
232 | Org. Biomol. Chem., 2015, 13, 223–233
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
46 H. H. Ting and M. J. Crabbe, Biochem. J., 1983, 215, 351–359. 61 M. Reis, R. J. Ferreira, M. M. M. Santos, D. J. V. A. dos
47 H. H. Ting and M. J. Crabbe, Biochem. J., 1983, 215, 361–
Santos, J. Molnár and M.-J. U. Ferreira, J. Med. Chem., 2013,
56, 748–760.
368.
48 J. Eckfeldt, L. Mope, K. Takio and T. Yonetani, J. Biol. 62 J. L. Evenden, E. M. Hammarberg, H. S. Hansson,
Chem., 1976, 251, 236–240.
49 J. H. Eckfeldt and T. Yonetani, Arch. Biochem. Biophys.,
1976, 173, 273–281.
S. E. Hellberg, L. G. Johansson, J. R. M. Lundkvist,
S. B. Ross, D. D. Sohn and S. O. Thorberg, U.S. Patent
5616610, 1997.
50 J. E. Jo, S. Mohan Raj, C. Rathnasingh, E. Selvakumar, 63 H.-J. Federsel, Org. Process Res. Dev., 2000, 4, 362–369.
W. C. Jung and S. Park, Appl. Microbiol. Biotechnol., 2008, 64 A. Beliaev, D. A. Learmonth and P. Soares-da-Silva, J. Med.
81, 51–60.
51 E. Brenna, F. G. Gatti, D. Monti, F. Parmeggiani and 65 S. Kousteni, The Trustees of Columbia University in the
A. Sacchetti, Chem. Commun., 2012, 48, 79–81. city of New York, WO2013/74889, 2013.
52 D. J. Bougioukou, A. Z. Walton and J. D. Stewart, Chem. 66 M. D. Gershon and K. Margolis, Lexicon Pharmaceuticals,
Commun., 2010, 46, 8558–8560. Inc., WO2013/148978, 2013.
53 E. Brenna, F. G. Gatti, D. Monti, F. Parmeggiani and 67 G. Karsenty, P. F. Duciy, V. K. Yadav, D. Landry and
Chem., 2006, 49, 1191–1197.
A. Sacchetti, ChemCatChem, 2012, 4, 653–659.
54 G. Maksay, E. Palosi, Z. Tegyey and L. Otvos, J. Med. Chem.,
1981, 24, 499–502.
55 G. Maksay, Z. Tegyey, V. Kemeny, I. Lukovits, L. Otvos and
E. Palosi, J. Med. Chem., 1979, 22, 1436–1443.
56 M. S. Poslusney, C. Sevel, T. J. Utley, T. M. Bridges,
R. D. Morrison, N. R. Kett, D. J. Sheffler, C. M. Niswender,
J. S. Daniels, P. J. Conn, C. W. Lindsley and M. R. Wood,
Bioorg. Med. Chem. Lett., 2012, 22, 6923–6928.
57 N. B. Carter, L. M. D. Turnbull, P. J. Crowley and
M. R. Cordingley, Syngenta Limited, WO2008/9908, 2008.
58 Z. Feng, J. H. Kim and S. F. Brady, J. Am. Chem. Soc., 2010,
132, 11902–11903.
S. X. Deng, The Trustees of Columbia University in the city of
New York, WO2010/56992, 2010.
68 Q. Liu, Q. Yang, W. Sun, P. Vogel, W. Heydorn, X.-Q. Yu,
Z. Hu, W. Yu, B. Jonas, R. Pineda, V. Calderon-Gay,
M. Germann, E. O’Neill, R. Brommage, E. Cullinan,
K. Platt, A. Wilson, D. Powell, A. Sands, B. Zambrowicz
and Z.-c. Shi, J. Pharmacol. Exp. Ther., 2008, 325,
47–55.
69 E. C. Dykhuizen, J. F. May, A. Tongpenyai and L. L. Kiessling,
J. Am. Chem. Soc., 2008, 130, 6706–6707.
70 K. Takiguchi, K. Yamada, M. Suzuki, K.-i. Nunami,
K. Hayashi and K. Matsumoto, Agric. Biol. Chem., 1989, 53,
77–82.
59 B. Kongkathip, S. Akkarasamiyo, K. Hasitapan, P. Sittikul, 71 T. Murata, M. Umeda, S. Yoshikawa, K. Urbahns, J. Gupta
N. Boonyalai and N. Kongkathip, Eur. J. Med. Chem., 2013,
60, 271–284.
and O. Sakurai, Bayer Healthcare AG, WO2004/43926,
2004.
60 H.-Y. Hung, E. Ohkoshi, M. Goto, K. F. Bastow, 72 M. B. Aspinall, W. R. Mound, J. S. Wailes,
K. Nakagawa-Goto and K.-H. Lee, J. Med. Chem., 2012, 55,
5413–5424.
W. G. Whittingham, J. Williams, C. L. Winn and
P. A. Worthington, Syngenta Limited, WO2009/81112, 2009.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 223–233 | 233