Multistep oxidase-lyase reactions: Synthesis of optically active 2-hydroxyketones by using biobased aliphatic alcohols

Article abstract of DOI:10.1002/cctc.201300093

Enzymatic multistep reactions are presently an important research field, from which integrated and efficient synthetic protocols can be created, accompanied by a diminished waste formation (avoiding downstream units operations). This article explores the benzaldehyde lyase (BAL) catalyzed crossed carboligation of benzaldehyde with different aliphatic aldehydes to afford optically active α-hydroxyketones. To this end, different biobased aliphatic alcohols were insitu oxidized to aldehydes by oxidase from Hansenula sp. and subsequently carboligated with benzaldehyde by BAL in the same reactor system. For short nonbranched aliphatic alcohols, moderate to high conversions in carboligations (15-99%) with excellent enantioselectivities (98-99%, R), were achieved. Both enzymes also exhibited activities at high concentrations of benzaldehyde (up to 200mM) and with butanol as cosolvent, albeit at the cost of lower conversions, presumably owing to kinetic reasons. After needed optimization of the biocatalyst (e.g., through genetic evolution, whole-cell setup) and the process setup (e.g., stepwise addition of substrates, reaction time), the herein reported concept might provide promising entries in the field of asymmetric synthesis, delivering useful building blocks starting from biobased materials, and in an integrative manner.

Full text of DOI:10.1002/cctc.201300093

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DOI: 10.1002/cctc.201300093
Multistep Oxidase–Lyase Reactions: Synthesis of Optically
Active 2-Hydroxyketones by Using Biobased Aliphatic
Alcohols
Marꢀa Pꢁrez-Sꢂnchez, Christoph R. Mꢃller, and Pablo Domꢀnguez de Marꢀa*[a]
Enzymatic multistep reactions are presently an important re-
search field, from which integrated and efficient synthetic pro-
tocols can be created, accompanied by a diminished waste for-
mation (avoiding downstream units operations). This article ex-
plores the benzaldehyde lyase (BAL) catalyzed crossed carboli-
gation of benzaldehyde with different aliphatic aldehydes to
afford optically active a-hydroxyketones. To this end, different
biobased aliphatic alcohols were in situ oxidized to aldehydes
by oxidase from Hansenula sp. and subsequently carboligated
with benzaldehyde by BAL in the same reactor system. For
short nonbranched aliphatic alcohols, moderate to high con-
versions in carboligations (15–99%) with excellent enantiose-
lectivities (98–99%, R), were achieved. Both enzymes also ex-
hibited activities at high concentrations of benzaldehyde (up
to 200 mm) and with butanol as cosolvent, albeit at the cost of
lower conversions, presumably owing to kinetic reasons. After
needed optimization of the biocatalyst (e.g., through genetic
evolution, whole-cell setup) and the process setup (e.g., step-
wise addition of substrates, reaction time), the herein reported
concept might provide promising entries in the field of asym-
metric synthesis, delivering useful building blocks starting from
biobased materials, and in an integrative manner.
Introduction
Biocatalysis has been established in the last decades as a pow-
erful tool for the synthesis of a broad array of optically active
chemicals. The main reasons for such interest are the exquisite
regio-, enantio-, and chemoselectivity that enzymes often dis-
play for many substrates. Aligned to this, advances in molecu-
lar biology have enabled the cost-effective provision of large-
scale, reproducible, and genetically improved biocatalysts.[1]
Adding to that selectivity, enzymatic processes have been typi-
cally addressed as “green” processes, owing to the mild (aque-
ous) reaction conditions in which those reactions are conduct-
ed. In recent years, however, considering the wastewater pro-
duction that biocatalytic process actually create, investigations
have also focused on the use of biobased cosolvents (e.g.,
2-methyltetrahydrofuran, 2-MeTHF),[2] ionic liquids, and deep-
eutectic solvents,[3] as well as solvent-free and nonaqueous
processes.[4] The combination of the high efficiency and selec-
tivity of enzymes with a more environmentally friendly process
setup may certainly boost even more the interest and the
scope of biocatalysis on an industrial level.
cusing on lipases, oxidoreductases, transaminases, and lyases.[6]
Among these examples, the combination of oxidoreductases[7]
and lyases[8] led to promising entries for the synthesis of highly
valuable optically active alcohols with diminished waste forma-
tion, starting from accessible substrates (e.g., aldehydes, ke-
tones). The enantioselective reduction of ketones is an industri-
ally proven useful method to afford chiral alcohols under ex-
tremely mild reaction conditions.[1b,4c,7] Likewise, the enzymatic
enantioselective CÀC bond formation represents an outstand-
ing alternative to create novel optically active compounds
starting from aldehydes as inexpensive substrates.[8] On this
basis, several examples on combinations of enzymatic oxida-
tions and CÀC bond formation have been reported, either in
two-pot or in one-pot setups.[6d,9]
Following these considerations, aiming at combining sus-
tainable chemistry with biocatalysis, the present paper ex-
plores the use of (biobased) aliphatic alcohols as substrates for
the production of optically active a-hydroxyketones. To this
end, the oxidase-catalyzed oxidation of aliphatic alcohols—to
form aldehydes—would be in situ coupled with a lyase-cata-
lyzed enantioselective CÀC bond formation, to yield the de-
sired chiral a-hydroxyketones in a one-pot, two-steps process.
Besides the introduction of biobased alcohols,[10] the envisaged
approach would avoid the use of highly volatile and hazardous
aliphatic aldehydes, as they would be only formed in situ in
millimolar amounts. To validate the concept, benzaldehyde
In this respect, another emerging approximation to provide
“greener” biocatalysis is the setup of multistep catalytic pro-
cesses, involving the performance of several enzymes in the
same reactor, and, therefore, diminishing the waste formation
upon the reduction of downstream processing units.[5] Several
outstanding examples have been reported in recent years, fo-
[a] Dr. M. Pꢀrez-Sꢁnchez, C. R. Mꢂller, Dr. P. Domꢃnguez de Marꢃa
Institut fꢂr Technische und Makromolekulare Chemie (ITMC)
RWTH Aachen University
lyase
(BAL)
was
used
for
the
enantioselective
CÀC bond formation, the core step of the process. BAL is a thia-
mine diphosphate dependent enzyme (ThDP lyase) with
a broad substrate acceptance, that is, it accepts aromatic and
aliphatic aldehydes, to afford a wide range of optically active
Worringerweg 1, 52074 Aachen (Germany)
Fax: (+49)241-8022177
E-mail: dominguez@itmc.rwth-aachen.de
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BAL in the presence of such al-
cohols. Thus, aiming at extend-
ing the reaction scope, galactose
oxidase from Dactylium den-
droides was assessed, as this
enzyme has shown activity for
the oxidation of diols.[15b,16] Yet,
no conversions could be ob-
served for any of the branched
or allylic alcohols, and only
quantitative amounts of benzoin
Scheme 1. Conceptual approach of an oxidase–lyase system to afford optically active a-hydroxyketones in a multi-
step process starting from benzaldehyde[10] and from biobased aliphatic alcohols. Catalase is added to eliminate
the formed hydrogen peroxide.
were
achieved.
Likewise,
a
2,2,6,6-tetramethylpyrimidine
a-hydroxyketones.[8,11] Very recently, diastereoselective reac-
Table 1. One-pot
multistep
Product
process
to
afford
optically
active
tions catalyzed by BAL were reported as well.[12] The use of
BAL in multistep reactions has been so far scarcely ad-
dressed, with preliminary examples on lipase–BAL and ox-
idase–BAL combinations.[6c,d] The overall reaction process is
depicted in Scheme 1.
a-hydroxyketones.[a]
Aliphatic alcohol
Conversion[b] ee[c] Benzoin
[%]
[%] conversion[b] [%]
CH3OH
>99[d]
–
–
–
Results and Discussion
Following our previous work,[6d] in which oxidases were able
to oxidize methanol to produce, in situ, formaldehyde for
the further BAL-catalyzed hydroxymethylation, in the first
set of experiments different aliphatic alcohols were tested
as substrates for the oxidase from Hansenula sp. In the
same pot, benzaldehyde was added for the subsequent
BAL-catalyzed carboligation to afford the crossed chiral
a-hydroxyketones. The one-pot multistep reactions pro-
ceeded in aqueous solution, and as cosolvent the biobased
2-MeTHF was used,[2b] as it has proven to be a useful cosol-
vent for BAL[13] and for oxidases.[6d] The results are depicted
in Table 1.
>99
15[e]
91[e]
99
98
98
81
–
–
–
–
–
–
–
99
99
–
–
–
–
–
–
99
99
As observed, oxidase from Hansenula sp. was successfully
applied for the oxidation of short-chain aliphatic alcohols
(from methanol to butanol) to afford aldehydes in catalytic
amounts, which were then in situ carboligated by BAL to
afford the optically active a-hydroxyketones, in excellent
conversions for methanol, ethanol, and butanol. Further-
more, BAL displayed outstanding enantioselectivities in all
cases (98–99%, see Experimental Section for details), in
agreement with previous literature, in which BAL-catalyzed
carboligations with benzaldehyde as the donor always lead
to highly enantioselective processes.[8,11–14] In the case of
propanol, a lower conversion was observed, with benzoin
formed by BAL as reversible product (Scheme 1), which
rather indicates a lower oxidase-catalyzed rate for this sub-
strate than deactivation of the enzymes in the combined reac-
tor system. The same assumption may be made for hexanol, al-
lylic alcohol, and the branched substrates (Table 1), though ac-
tivity of oxidase from Hansenula sp. for a broad number of
short-chain alcohols has been reported.[15] Herein, however,
albeit no crossed carboligation could be observed, the forma-
tion of benzoin in quantitative amounts assured the activity of
[a] Reaction conditions: benzaldehyde (6 mm), aliphatic alcohol (53.5 mm),
BAL (1 mgmLÀ1, 20 U), ThDP (0.15 mm), alcohol oxidase from Hansenula sp.
(0.25 mgmLÀ1, 8 U), catalase from bovine liver (12 U, 0.35 mgmLÀ1), FAD
(0.1 mm), phosphate buffer pH 8.0 (50 mm), 2-MeTHF (5vol.%), MgSO4
(2.5 mm). Absolute configuration assigned to (R) for BAL-catalyzed carboliga-
tions.[8,11,12] [b] Determined by 1H NMR. [c] Determined by chiral HPLC (see Ex-
perimental Section). [d] In agreement with previous literature.[6d] [e] Traces (5–
10%) of the other crossed product (benzaldehyde as acceptor and the
formed aliphatic aldehyde as donor) were observed by 1H NMR (see also
below, Figure 1).
N-oxide (TEMPO) catalyzed organocatalytic alcohol oxidation
was attempted,[17] aiming at combining, in one pot, the orga-
nocatalytic oxidation of aliphatic alcohols with subsequent
BAL-catalyzed carboligations. Disappointingly, attempts to
obtain compatible reaction conditions for both catalysts
(TEMPO–BAL) were unsuccessful. In any case, given the diversi-
ty of oxidases in nature,[7] as well as the possibilities that mo-
lecular biology techniques offer nowadays for the genetic
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design of improved enzymes,[1] it may be expected that novel
biocatalysts can be found for the oxidation of more challeng-
ing aliphatic alcohols. In addition, apart from oxidases, other
oxidative enzymes (e.g., oxidoreductases) could be envisaged
for the analogous processes of alcohol-to-aldehydes as well.[7]
In a second set of experiments, the reaction conditions for
the one-pot multistep strategy were further studied. To this
end, the BAL-catalyzed carboligation of benzaldehyde with
butyraldehyde, using butanol as the substrate, was chosen as
a model reaction. The results are depicted in Figure 1.
all reactions. Likewise, apart from using 2-MeTHF as a cosolvent
for the reaction (to fully dissolve the substrates in the aqueous
media), in another set of experiments, butanol was directly
used as both cosolvent and substrate for the reaction. Other
aliphatic alcohols (e.g., isopropanol) have also been used as
cosolvents for lyases[11d] and for other biocatalytic reactions in
general.[18] The results of our experimental setup are depicted
in Figure 2.
Figure 2. Influence of the benzaldehyde loading in the conceptual one-pot
oxidase–lyase setup. Reaction conditions: 1:5 ratio benzaldehyde/butanol in
the set of 2-MeTHF as cosolvent; BAL (20 U, 1 mgmLÀ1), ThDP (0.15 mm), al-
cohol oxidase from Hansenula sp. (8 U, 0.25 mgmLÀ1), catalase from bovine
liver (12 U, 0.358 mgmLÀ1), FAD (0.1 mm), phosphate buffer (50 mm, pH 8.0),
cosolvent (5 vol.%, 2-MeTHF or butanol,), MgSO4 (2.5 mm).
Figure 1. Kinetic profile obtained for the oxidase–lyase one-pot reaction
using benzaldehyde and butanol as substrates. Reactions conditions: benzal-
dehyde (6 mm), butanol (53.5 mm), BAL (20 U, 1 mgmLÀ1), ThDP (0.15 mm),
alcohol oxidase from Hansenula sp. (8 U, 0.25 mgmLÀ1), catalase from
bovine liver (12 U, 0.358 mgmLÀ1), FAD (0.1 mm), phosphate buffer (50 mm,
pH 8.0), 2-MeTHF (5 vol.%), MgSO4 (2.5 mm). Product profile determined by
1H NMR.
Remarkably, the oxidase–lyase system remained active at
higher benzaldehyde loadings (even up to 200 mm benzalde-
hyde in the case of butanol as the cosolvent), yet at the cost
of significantly decreasing the conversion in the crossed aro-
matic–aliphatic product. The lower conversion may be partly
explained by the influence of the butanol concentration. Com-
pared to the use of cosolvent 2-MeTHF with a lower excess of
aliphatic alcohol, if butanol was used as the cosolvent and
was, thus, present in a higher excess, higher conversions to
the crossed a-hydroxyketone were observed at a benzaldehyde
concentration of 100 mm. Although the combined one-pot
two-step approach was assumed to be feasible and promising
at first sight, the observed results clearly pointed out the need
for further optimization both at the enzyme level, as well as
from the process development point of view (e.g., optimal
loading of enzymes, stepwise addition of different substrates).
Once these further improvements are installed, it may be ex-
pected that higher conversions of a valuable building block
can be achieved.
As observed (Figure 1), the production of benzoin proceed-
ed extremely fast and selectively in the first minutes of the re-
action, consistent with previous literature reporting BAL-cata-
lyzed benzoin condensations.[8,11–14] Subsequently, upon enzy-
matic oxidation of butanol to form butyraldehyde, the crossed
carboligation product was formed, reaching approximately
90% conversion in 5–6 h. Interestingly, the other crossed car-
boligation product—with benzaldehyde as the acceptor and
butyraldehyde as the donor—was observed in low concentra-
tions as well. To improve the biocatalytic selectivity over the
desired cross-condensation product, the setup of directed evo-
lution rounds[1a] might lead to improved variants of lyases, dis-
playing higher bias for using benzaldehyde as donor substrate.
As another option, reaction kinetics could be controlled. For in-
stance, by stopping the reaction at short reaction times
(ꢀ2 h), a mixture of the desired crossed product with benzoin
was obtained. As both compounds could be easily separat-
ed—benzoin precipitates as a solid in aqueous solutions—and
Conclusions
Herein, we explored the possibility of using (biobased) aliphat-
ic alcohols for the in situ enzymatic production of aldehydes
and the subsequent lyase-catalyzed carboligation to afford val-
uable a-hydroxyketones typically with high conversions and
always with excellent enantioselectivity. The system allows the
use of either biobased cosolvents such as 2-MeTHF or directly
alcohols (e.g., butanol) as both substrate and cosolvent for the
(R)-benzoin was actually
a substrate for BAL as well
(Scheme 1), after separation of the crossed aromatic–aliphatic
compounds, the remnant benzoin could be recycled again in
the reaction to afford more of the desired product.
Finally, the influence of the benzaldehyde loadings in the re-
action was assessed by using an excess of aliphatic alcohol in
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production of the crossed a-hydroxyketones. Moreover, the
possibilities of affording important optically active building
blocks from biorenewable resources represent an important
option for future synthetic processes, in which the replace-
ment of petroleum-based resources will be more important.
For industrial application, however, extensive process optimiza-
tion is needed to enable higher productivities and substrate
loadings. A possibility for such further improvements would be
the genetic design of oxidase and lyase variants that could cat-
alyze the oxidation–carboligation steps in a faster and proper
way, or the construction of a designer bug (whole cell) overex-
pressing all the enzymes for a simplified and even further inte-
grated process. Apart from these genetic considerations, other
kinetic aspects related to the reaction setup might also be
modified, such as optimal enzyme loadings, step-wise addition
of substrates, operation at low reaction times and in repetitive
batches. We hope that this work will trigger other research
groups to undertake more studies on the setup of enzymatic
processes with a focus on biobased substrates.
Scheme 2. Synthesis of racemates 3.
dehyde (30.0 mmol, 1.05 equiv.) was added slowly within 30 min
into the flask. After 1.5 h the reaction was allowed to reach RT.
After stirring for another hour the reaction was quenched by
adding 60 mL saturated NH4Cl solution. After neutralization of the
reaction mixture with 10% aqueous HCl solution, the aqueous
phase was extracted with EtOAc (3ꢅ60 mL) and subsequently
washed with water (1ꢅ60 mL) and brine (1ꢅ60 mL). The combined
organic layers were dried over Na2SO4. After the removal of the
drying agent and solvent, the raw product was purified by flash
chromatography (ethyl acetate/petroleum ether 1:20).
Synthesis of 3:[19] 2 (7.85 mmol) in dry diethylether (10 mL) was
charged to
a flask under an argon atmosphere. 3HF·NEt3
(17.28 mmol, 2.2 equiv.) was added and the reaction mixture was
stirred for 2 h at RT. Then, saturated NaHCO3 solution was added
until neutralization. The aqueous phase was extracted with diethyl-
ether (3ꢅ20 mL) and washed with water (2ꢅ20 mL) and brine
(1ꢅ20 mL). The organic layer was dried over MgSO4. After the re-
moval of drying agent and solvent, the raw product was purified
by flash chromatography (ethyl acetate/petroleum ether 1:20).
Experimental Section
Chemicals
All compounds were purchased from Sigma–Aldrich and were
used directly. Benzaldehyde lyase form Pseudomonas fluorescens
was cloned and overexpressed in Escherichia coli cells, and pro-
duced by fermentation.[13] After fermentation, BAL was lyophilized
and stored at À208C until use. BAL characterization was performed
by using benzaldehyde as the substrate and benzoin formation
was monitored as a control reaction, as reported elsewhere.[13] Al-
cohol oxidase from Hansenula sp. and catalase from bovine liver
were purchased from Sigma–Aldrich.
Determination of enantiomeric excess
Enantiomeric excesses were determined by HPLC, using a Chiralcel
OD-H column (n-heptane/isopropanol 99:1, l=250 nm), flow
0.5 mLminÀ1. Major enantiomer tR =31.4 min, minor enantiomer
tS =22.6 min (Figure 3).
Standard oxidase–lyase protocol
Benzaldehyde (6.36 mg, 0.6 mmol) was dissolved in a 10 mL mix-
ture of 5 vol.% 2-MeTHF, phosphate buffer (50 mm, pH 8.0) con-
taining MgSO4 (2.5 mm), FAD (0.1 mm), ThDP (0.15 mm), and ali-
phatic alcohol (53.5 mm). After addition of BAL (10 mg, 20 U), alco-
hol oxidase from Hansenula sp. (3.5 mg, 8 U) and catalase from
bovine liver (3.5 mg, 12 U), the reaction system was covered with
an air-filled balloon and the mixture was gently stirred for 16 h.
The reaction mixture was extracted with ethyl acetate (3ꢅ20 mL),
and the organic layer washed with water (3ꢅ20 mL) and brine
(1ꢅ20 mL) and dried over Na2SO4. The solvent was evaporated in
NMR data of products
NMR spectra were recorded on a Bruker DPX400. Chemical shifts d
are reported in ppm relative to CHCl3 (1H: d=7.27) and CDCl3 (13C:
d=77.0) as an internal standard.
2-hydroxy-1-phenylpropan-1-one: 1H NMR (300 MHz, CDCl3): d=
7.86 (d, J=7.6 Hz, 2H), 7.55 (t, J=7.4 Hz, 1H), 7.43 (t, J=7.6 Hz,
2H), 5.10 (q, J=7.0 Hz, 1H), 1.38 ppm (d, J=7.0 Hz, 3H);
1
1
2-hydroxy-1-phenylbutan-1-one: H NMR (300 MHz, CDCl3): d=7.90
vacuum. Reactions were followed by H NMR and the enantiomeric
(d, J=7.9 Hz, 2H), 7.60 (d, J=7.5 Hz, 1H), 7.49 (d, J=7.5 Hz, 2H),
5.05 (q, J=3.86 Hz, 1H), 1.99–1.91 (m, 1H), 1.65–1.56 (m, 1H),
0.93 ppm (t, J=7.4 Hz, 3H); 13C NMR(300 MHz, CDCl3): d=202.1,
133.9, 128.8, 128.5, 174.0, 28.78, 8.9 ppm.
excesses were determined by chiral-phase HPLC (Chiralpak IA
column, UV detection at 210 nm).
Synthesis of racemates
2-hydroxy-1-phenylpentan-1-one: 1H NMR (300 MHz, CDCl3): d=
7.91 (d, J=7.2 Hz, 2H), 7.61 (d, J=7.3 Hz, 1H), 7.50 (d, J=7.4 Hz,
2H), 5.09 (m, 1H), 1.85–1.39 (m, 4H), 0.91 ppm (t, J=7.2 Hz, 3H);
13C NMR(300 MHz, CDCl3): d=202.2, 130.2, 128.8, 128.5, 72.9, 37.9,
18.3, 13.8 ppm.
The reaction scheme is shown in Scheme 2.
Synthesis of 2: Isopropylamine (3.1 g, 31.4 mmol) and dry THF
(60 mL) were added into a flask under argon atmosphere at
À788C. Butyl lithium (32.8 mmol, 1.15 equiv.) was added dropwise
and the reaction mixture was allowed to reach RT within 15 min.
The flask was cooled down again to À788C and a-
(trimethylsilyloxy)phenylacetonitrile 1 (5.8 g, 28.6 mmol) was added
dropwise. The reaction mixture was stirred for 15 min until the al-
1
Benzoin: H NMR (300 MHz, CDCl3): d=7.85–7.19 (m, 10H), 5.88 (d,
J=6.1 Hz, 1H), 4.48 ppm (d, J=6.1 Hz, 1H); 13C NMR(300 MHz,
CDCl3): d=198.9, 139.0, 133.9, 129.2, 129.1, 187.7, 128.6,
127.7 ppm.
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Figure 3. HPLC chromatogram of a) (R,S)-2-hydroxy-1-phenylpentan-1-one;
b) (R)-2-hydroxy-1-phenylpentan-1-one obtained by oxidase–lyase carboliga-
tion.
Keywords: alcohols · aldehydes · asymmetric synthesis ·
enzyme catalysis · green chemistry
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Received: February 1, 2013
Published online on May 24, 2013
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