Enzyme-catalyzed C - C bond formation using 2-methyltetrahydrofuran (2-MTHF) as (co)solvent: Efficient and bio-based alternative to DMSO and MTBE

Article abstract of DOI:10.1039/c0gc00590h

The enzymatic carboligation of aldehydes (C-C bond formation) catalyzed by benzaldehyde lyase (BAL) affords chiral α-hydroxy-ketones under mild reaction conditions in aqueous media. To enhance substrate and product availability under aqueous conditions, processes are often set-up using either DMSO as co-solvent, or MTBE as second organic phase. Although efficient, DMSO leads to difficulties in separation during downstream processing, with wastewater formation. MTBE provides a cleaner and straightforward work-up, but its petrochemical origin, together with its poor degradability, gives rise to environmental concerns. Herein it is reported that 2-methyltetrahydrofuran (2-MTHF) is a promising candidate to substitute DMSO or MTBE in lyase-catalyzed reactions. 2-MTHF can be derived from bio-based resources (e.g. levulinic acid), and it is abiotically degraded by air. When BAL is added to buffer-2-MTHF (5% v/v) mixtures, enzyme remains stable with a half-life of 178 ± 8 h, with productivities (benzoin synthesis) of 10 g benzoin L^-1h^-1. Several BAL-catalyzed aldehyde carboligations were assessed under those conditions, leading in all cases to high isolated yield (quantitative in majority), and to high enantioselectivity (up to >99%). Furthermore, preliminary results obtained with two phase systems in the BAL-catalyzed benzoin synthesis afforded 60 g benzoin L^-1 in 24 h (ee > 99%). Therefore, 2-MTHF may be a valuable (co)solvent, not only to tackle environmental concerns, but also in terms of practical, efficient biocatalysis.

Full text of DOI:10.1039/c0gc00590h

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Enzyme-catalyzed C–C bond formation using 2-methyltetrahydrofuran
(2-MTHF) as (co)solvent: efficient and bio-based alternative to DMSO and
MTBE
Saravanakumar Shanmuganathan,a Dessy Natalia,a Anne van den Wittenboer,a Christina Kohlmann,a
Lasse Greiner*a,b and Pablo Dom´ınguez de Mar´ıa*a
Received 21st September 2010, Accepted 21st October 2010
DOI: 10.1039/c0gc00590h
The enzymatic carboligation of aldehydes (C–C bond formation) catalyzed by benzaldehyde lyase
(BAL) affords chiral a-hydroxy-ketones under mild reaction conditions in aqueous media. To
enhance substrate and product availability under aqueous conditions, processes are often set-up
using either DMSO as co-solvent, or MTBE as second organic phase. Although efficient, DMSO
leads to difficulties in separation during downstream processing, with wastewater formation.
MTBE provides a cleaner and straightforward work-up, but its petrochemical origin, together
with its poor degradability, gives rise to environmental concerns. Herein it is reported that
2-methyltetrahydrofuran (2-MTHF) is a promising candidate to substitute DMSO or MTBE in
lyase-catalyzed reactions. 2-MTHF can be derived from bio-based resources (e.g. levulinic acid),
and it is abiotically degraded by air. When BAL is added to buffer-2-MTHF (5% v/v) mixtures,
enzyme remains stable with a half-life of 178 8 h, with productivities (benzoin synthesis) of 10 g
benzoin L-1h-1. Several BAL-catalyzed aldehyde carboligations were assessed under those
conditions, leading in all cases to high isolated yield (quantitative in majority), and to high
enantioselectivity (up to >99%). Furthermore, preliminary results obtained with two phase
systems in the BAL-catalyzed benzoin synthesis afforded 60 g benzoin L-1 in 24 h (ee > 99%).
Therefore, 2-MTHF may be a valuable (co)solvent, not only to tackle environmental concerns, but
also in terms of practical, efficient biocatalysis.
1. Introduction
Biocatalysis is being increasingly accepted as an alternative
for the preparation of chiral building blocks. Key-factors are
the high regio- and enantioselectivities often reported, together
with the applied mild reaction conditions. Furthermore, once
enzyme genes are cloned and overexpressed, biocatalysts can be
produced “on demand” via cost-effective fermentative routes.1
Thiamine-diphosphate dependent lyases (ThDP-Lyases) are
a versatile group of enzymes that enantioselectively catalyze
the carboligation of aldehydes (C–C bond formation) to afford
chiral a-hydroxy-ketones (Scheme 1).2 These compounds are
useful building blocks for pharmaceutical and fine chemical
applications.2a
An outstanding example of ThDP-lyases is benzaldehyde
lyase from Pseudomonas fluorescens (BAL, EC.4.1.2.38). BAL
catalyzes the enantioselective carboligation of both aromatic
and aliphatic aldehydes.2,3 Several process-development stud-
ies concerning BAL-catalyzed synthetic systems (e.g. biphasic
Scheme
1
Enantioselective C–C bond formation catalyzed by
thiamine-diphosphate dependent enzymes.2–4
media, whole-cell approach, etc.) have demonstrated the po-
tential of this enzyme at an industrial scale as well.2,4 Apart
from BAL, recently other lyase-based reactions were reported,
e.g. enantioselective Stetter-type 1,4-additions,5 or aldehyde-
ketone and ketone-ketone carboligations to afford enatiop-
ure/enantiomerically enriched tertiary alcohols.6 Likewise, the
cofactor of these enzymes, thiamine diphosphate, has also
been the model for the development of many organocatalytic
umpolung carboligations.7
Biocatalytic reactions are often conducted in aqueous media,
as the more compatible milieu for enzymes (natural catalysts).
Yet, that approach normally decreases synthetic productivities,
since organic compounds are often poorly soluble in aqueous
conditions. To overcome this, the use of either a co-solvent
(e.g. DMSO, 2-propanol, tert-butanol, etc.), or a second organic
phase (toluene, MTBE, etc.) is widely employed in biocatalysis.1
For ThDP-lyases, the use of DMSO as co-solvent and outstand-
aInstitute of Technical and Macromolecular Chemistry (ITMC), RWTH
Aachen University, Worringerweg 1, 52074, Aachen, Germany.
E-mail: dominguez@itmc.rwth-aachen.de; Fax: +49 241 8022177;
Tel: +49 241 8020468
bDECHEMA e.V. Karl-Winnacker-Institut, Theodor-Heuss-Allee 25,
60486, Frankfurt am Main, Germany. E-mail: greiner@dechema.de;
Fax: +49 697564388; Tel: +49 697564337
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ing stabilizer of those enzymes is well-known.8 In addition,
MTBE has been reported as a proper organic solvent to set-
up biphasic reactions involving ThDP-lyases, leading to high
productivities (ca. 80–100 g L-1) in short reaction times (up to
24 h).4
Productivities are certainly a crucial parameter when novel
biocatalytic approaches are assessed. However, environmental
issues must also be taken into account. In this respect, the use
of DMSO leads to a considerable wastewater formation during
work-up. On the other hand, the use of MTBE as a second phase
provides a more straightforward extractive work-up, and may
positively influence equilibrium positions.9 However, MTBE
has a petrochemical origin, with a poor biodegradability in
the environment, leading to its accumulation and subsequent
pollution of water reservoirs.10
Therefore, there is the need for novel biocatalytic strategies
that can provide environmentally-friendly processes, while at
the same time being economically sound for industrial needs.
In this respect 2-methyltetrahydrofuran (2-MTHF) may be
an alternative as (co)solvent. Albeit a profound toxicological
assessment of 2-MTHF is pending – and thus 2-MTHF should
not (yet) be regarded as a “green solvent” – it can be derived
from biomass (Scheme 2),11 provides a straightforward work-up
(b.p. ca. 80 ◦C), and it is abiotically degraded in air.12
the set-up of a biphasic system. As first goal the activity of BAL
in 2-MTHF was assessed. Traditional activity assay methods
for BAL reported in the open literature (e.g. spectrophotometry,
HPLC, or enzyme-coupled approaches),2,8 though more or less
reliable, are often time-consuming and not straightforward.
Therefore, firstly a more convenient spectrophotometric pro-
tocol for BAL assays – based on the furoin formation BAL-
catalyzed carboligation of 2 furfurals – was set-up. Furoin
absorbance was measured at 320 nm where substrate absorbance
(furfural) is negligible and product formation can be monitored
directly. In this new protocol, one unit of activity was defined
as the amount of BAL that catalyzes the formation of 1 mmol
furoin per minute under standard conditions (30 ◦C, pH 8) (see
experimental for details). By means of this method, a kinetic
characterization of BAL was performed under three reaction
conditions: pure buffer, buffer with 5% v/v of DMSO, and buffer
with 5% v/v of 2-MTHF (Fig. 1).
Scheme 2 Concept for the bio-based production of 2-MTHF.11
Fig. 1 BAL activity as a function of 2-furaldehyde concentration. (᭜)
Buffer; (ꢀ) Buffer/DMSO; (ꢀ) Buffer/2-MTHF.
2-MTHF has found applications in the preparation of
Grignard reagents, cross-coupling reactions,12 in enantiose-
lective 1,4-additions,13 in classic organometallic chemistry,14
organocatalysis,15,16 as well as in other synthetic procedures.17,18
In biocatalysis there is only one example dealing with lipase-
catalyzed acylations in 2-MTHF as solvent,19 with lipases as
exemplary robust enzymes.20 In general, the quest of green
solvents is presently an important area of research.21
Herein, we report for the first time the use of 2-MTHF
as (co)solvent in ThDP-lyase-catalyzed reactions. The aim of
this work is to show that the combination of 2-MTHF with
lyases may provide a promising scenario where enzyme stability,
activity, and reaction productivity can be achieved with the use
of bio-based, easily-degradable solvents.
BAL showed enzymatic activities in all buffer/co-solvent
systems studied. Interestingly, the enzymatic performance in
buffer/2-MTHF (5% v/v) displayed a slightly higher vmax than
these observed for pure buffer or buffer/DMSO (5% v/v).
Affinities of BAL (KM) were however slightly lower than values
in pure buffer. Overall, it was clear that BAL was active in
the novel reaction media buffer/2-MTHF (5% v/v). Since
kinetically BAL provided a promising framework (Fig. 1), later
on the stability of BAL in the presence of 2-MTHF was assessed.
BAL was dissolved in phosphate buffer. Stabilities were assessed
in pure buffer, and using either 2-MTHF (5% v/v) or DMSO (5%
v/v) as cosolvents. Samples were stored and tested for residual
activity. Deactivation kinetics are depicted in Fig. 2.
In agreement with literature, BAL stability is greatly enhanced
when DMSO is used as co-solvent, and thus only 10% loss of
activity was observed within 250 h of incubation (Fig. 2).8 Yet,
as previously stated, using DMSO as co-solvent in enzymatic
reactions is not the preferred option for practical biocatalysis,
due to problems in the downstream processing. Remarkably,
BAL stability in the presence of 2-MTHF is slightly better than
data observed for pure buffer systems. Thus, for buffer/2-MTHF
(5% v/v) system, a half-life of 178 8 h was estimated, assuming
first order deactivation kinetics. Parameter estimation for a
2. Results and discussion
2.1. BAL characterization and stability in 2-MTHF
Several phosphate buffer solutions containing different amounts
of 2-MTHF were prepared. In accordance with literature12 2-
MTHF was completely miscible with the buffer in mixtures
containing up to 5% v/v of 2-MTHF. At higher proportions
a second phase was formed. Therefore, 2-MTHF may easily be
used both as co-solvent (up to 5% v/v), as well as solvent for
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enantioselectivities in both cases. Likewise, further experiments
with the buffer/2-MTHF (5% v/v) medium showed that quan-
titative benzoin yields could even be obtained after 1 h reaction,
with a productivity of 0.05 mol L-1 benzoin with 200 U BAL
(Fig. 3). This gives a space-time-yield of 10 g benzoin L-1 h-1.
Fig. 2 Deactivation kinetics of BAL in different conditions: (᭹) Pure
buffer (᭺: disregarded for estimation of half life, see text); (᭜) Buffer/2-
MTHF (5% v/v); (ꢀ) Buffer/DMSO (5% v/v).
second order exponential decay, as discussed in the literature,22
did not lead to meaningful parameter values. Interestingly,
the mechanism of deactivation changes with the addition of
2-MTHF from apparently zero order in buffer. Clearly, the
interaction of BAL with 2-MTHF may be subjected to further
investigation. From a practical perspective 2-MTHF provides
a promising framework of BAL stability from which synthetic
applications could be envisaged, provided that industrial batch
biocatalytic processes typically run in less than 24 h reaction
time.
Fig. 3 Isolated yield (benzoin formation) after 1 h reaction time, as a
function of the amount of enzyme employed (95 mmol L-1 benzaldehyde,
buffer/2-MTHF 5% v/v).
Therefore, taking those productivities together with the
stability of BAL in 2-MTHF, it becomes clear that 2-MTHF is
not only a bio-based alternative for organic solvents, but also an
efficient reaction medium for conducting biocatalysis with high
productivities. Encouraged by the promising results, the system
buffer/2-MTHF (5% v/v) was extended to other aldehydes as
substrates in BAL-catalyzed reactions (Table 1).
2.2. BAL-catalyzed carboligations using 2-MTHF as
(co)solvent
As previously stated, BAL shows a broad substrate spectrum, be-
ing able to enantioselectively carboligate aromatic and aliphatic
aldehydes, as well as in a cross-condensation fashion (e.g.
aromatic and aliphatic aldehydes).2–4,8 Therefore, to fully assess
the possibilities of 2-MTHF as a (co)solvent, examples of this
type of reaction were checked. First of all, benzoin formation
(condensation of two benzaldehyde molecules) was studied.
Again, three systems were compared: pure buffer and additions
of either DMSO (5% v/v) or 2-MTHF (5% v/v) as co-solvent
(Scheme 3).
Both aliphatic and aromatic aldehydes exhibit high conver-
sion in BAL-catalyzed reactions, when 2-MTHF (5% v/v) is
used as co-solvent. Gratifyingly, cross-condensation reactions
(carboligation of two different aldehydes) already showed close
to quantitative conversions within 1 h as well. In agreement
with literature, to obtain a pure crossed-product, a surplus of the
acceptor aldehyde was added, to shift the equilibrium created by
the benzoin.2–4,8 Concerning enantioselectivities of the afforded
a-hydroxy-ketones, values remained the same as those reported
with DMSO or MTBE.2–4,8 Therefore, the use of 2-MTHF did
not affect the high enantioselectivity usually displayed by BAL.
Furthermore, 2-MTHF was also assessed as an extractive
solvent during the work-up procedure. Taking again the benzoin
synthesis as the model reaction, the work-up procedure was con-
ducted with three different organic solvents: dichloromethane,
ethyl acetate, and 2-MTHF. In all cases quantitative yields
of benzoin (99%) were isolated. This may provide additional
environmental benefits, since more toxic organic solvents (e.g.
dichloromethane) can be easily replaced by other more benign
bio-based derivatives (e.g. ethyl acetate or 2-MTHF). Likewise,
fewer emulsion problems and phase separation are expected with
2-MTHF.17
Scheme 3 BAL-catalyzed benzoin condensations in different reaction
media: Pure phosphate buffer (pH 8), and buffer with either DMSO or 2-
MTHF as co-solvent (5% v/v). Conditions: benzaldehyde 95 mmol L-1,
200 U BAL, room temperature.
As observed, the addition of a co-solvent to the aqueous buffer
is crucial for achieving a proper performance in BAL-catalyzed
reactions, in agreement with literature.2–4,8 Importantly, both
DMSO and 2-MTHF were equally useful for the enzymatic
performance, leading to quantitative isolated yields and high
Finally, the substrate loading was increased, taking again the
benzoin synthesis as the model reaction. The aim was to evaluate
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Table 1 Summary of results obtained in BAL-catalyzed carboligation of aldehydes using 2-MTHF (5% (v/v) as co-solvent in potassium phosphate
buffer (50 mmol L-1, pH 7.0, MgSO4 (2.5 mmol L-1), and ThDP (0.15 mmol L-1). 16 h reaction time
Substrate(s)
Product
Isolated yield
ee (R)
>99%
>99%
92%
95%
99%
93%
52%
>99%
95%
98%
whether practical biocatalysis could be set-up with 2-MTHF as
second phase. To this end, an initial solution of 95 mmol L-1
benzaldeyde in buffer/2-MTHF (5% v/v) was prepared. Later
on a mixture of benzaldehyde/2-MTHF was carefully dosed by
using a micro syringe pump, reaching a final 2-MTHF volume
of 10 mL and 1 M benzaldehyde. 250 U of BAL were added (in
two portions, 100 U at the beginning and 150 U at 6 h reaction
time). After 24 h work-up was performed, 60 g benzoin L-1
was isolated (60% yield) with high enantiomeric excess (>99%).
Although reaction conditions are non-optimized (in terms of
volumes, amount of substrate, dosing rate, etc.) achieved yields
were already comparable to the outcomes of biphasic systems
reported in the literature, in which yields of 80–100 g L-1 were
provided.4 Therefore, 2-MTHF can be successfully used for
BAL-catalyzed reactions, not only as co-solvent (5% v/v), but
also as a second organic phase in biphasic set-ups, if higher
yields or different reactions conditions are desired.
conditions production of 60 g benzoin L-1 in 24 h was achieved.
Furthermore, 2-MTHF can also be used as an extractive agent
during work-up, with similar performances as ethyl acetate or
dichloromethane. Overall, results show that 2-MTHF may be
not only a promising alternative in terms of environmental
concerns – being a bio-based, abiotically-degradable derivative
– but also that 2-MTHF may be an important option to set-up
practical biocatalytic concepts. Research dealing with 2-MTHF
in other enzymatic reactions involving organic (co)solvents may
be a promising research line for the future as well.
4. Experimental
4.1. Chemicals and enzyme production
All compounds were purchased from Sigma-Aldrich, and were
used directly. Benzaldehyde lyase from Pseudomonas fluorescens
was cloned in E. coli, overexpressed, and produced by fer-
mentation as described elsewhere.2–4,8 After fermentation and
purification, BAL was lyophilized and stored at -20 ◦C until
use.
3. Conclusions
The potential of 2-MTHF as (co)solvent in BAL-catalyzed
enzymatic reactions has been assessed. Enzymatic asymmetric
syntheses of a-hydroxy-ketones can be efficiently performed in
buffer systems with the addition of 5% v/v of 2-MTHF (as
co-solvent), leading to quantitative yields, and (in many cases)
high enantioselectivity. Likewise, when 2-MTHF is applied in
excess to form a second phase (as solvent), under non-optimized
4.2. BAL characterization
Furoin absorbance was measured at 320 nm. At that range, 2-
furaldehyde absorbance is negligible and therefore the analytic
tool was reliable. In this new protocol, one unit of activity was
defined as the amount of BAL that catalyzes the formation of
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1 mmol furoin per minute under standard conditions (30 ◦C,
pH 8). 2-Furaldehyde 5 mmol L-1 in phosphate buffer (pH 8)
which contained 2.5 mmol L-1 MgSO4 and 0.25 mmol L-1
ThDP were used as a substrate. The formation of 2,2-furoin
over time was followed with spectrophotometry at 320 nm using
a multiplate reader (PowerWave, BioTek Instruments) with a
quartz 96 well plate (Helima).
7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): 202.3, 133.9, 133.3,
128.8, 128.7, 69.3, 22.3.
4.6. Cross condensation benzaldehyde and
2,2-dimethoxyacetaldehyde
Benzaldehyde (105 mg, 1.0 mmol) was dissolved in a mixture
of 5% v/v 2-methyltetrahydrofuran and potassium phosphate
buffer (35 mL, 50 mmol L-1, pH 7.0, containing MgSO4
(2.5 mmol L-1) and ThDP (0.15 mmol L-1). After addition of
BAL (20 mg) the reaction mixture was gently stirred for 1 h. To
this solution, dimethoxyglyoxal was added in a three different
intervals (1 h) with concentrations not more than 95 mmol L-1
(947 mg, 6 mmol) and stirred for 16 h. The reaction mixture
was extracted with ethyl acetate (3 ¥ 10 mL) and the organic
layers washed with water (3 ¥ 10 mL) and brine (3 ¥ 10 mL),
and were dried over Na2SO4. Evaporation of the solvent gave
yellow oil. The yield was calculated from NMR; yield: 149 mg
(95%, (R)-enantiomer 99% ee). HPLC: (Chiralpak IA) Rt (R) =
2.1 min; Rt (S) = 34.5 min. NMR data consistent with literature.4
1H-NMR (400 MHz, CDCl3): 3.32 (s, 3H), 3.35 (s, 3H), 4.40 (d,
J = 4, 1H), 5.06 (br. t, 1H), 7.38–7.42 (m, 2H), 7.51–7.56 (m,
1H), 7.89–7.91 (m, 2H); 13C-NMR (100 MHz, CDCl3): 199.8,
134.9, 133.8, 129.1, 128.4, 76.7, 73.7, 56.5.
4.3. Analytics
NMR spectra were recorded on a Bruker DPX 400, or on
a Bruker AMX 300. Chemical shifts d are reported in ppm
relative to CHCl3 (1H: d = 7.27) and CDCl3 (13C: d = 77.0) as
internal standard. Enantiomeric excesses were determined by
chiral phase SFC analysis (Chiralcel IA column, UV detection
at 254 nm). Benzoin: eluent: CO2/2-propanol = 90 : 10, flow
◦
4.0 mL min-1, 40 C. (R)-2-Hydroxy-phenylpropan-1-one: elu-
ent: CO2/2-propanol = 90 : 10, flow 4.0 mL min-1, 40 ◦C. (R)-2-
Hydroxy-3,3-dimethoxy-1-phenylpropan-1-one: eluent: CO2/2-
propanol = 90 : 10, flow 4.0 mL min-1, 40 ◦C. Furoin: eluent:
CO2/2-propanol = 90 : 10, flow 3.0 mL min-1, 40 ◦C.
4.4. Benzoin
Benzaldehyde (199 mg, 1.8 mmol) was dissolved in a mixture
of 5% v/v 2-methyltetrahydrofuran and potassium phosphate
buffer (20 mL, 50 mmol L-1, pH 7.0, containing MgSO4
(2.5 mmol L-1) and ThDP (0.15 mmol L-1). After addition
of BAL (20 mg) the reaction mixture was gently stirred for
16 h. The reaction mixture was extracted with ethyl acetate
or 2-methyltetrahydrofuran (3 ¥ 10 mL), and the organic layer
washed with water (3 times 10 mL) and brine (3 times 10 mL),
and were dried over Na2SO4. Evaporation of the solvent and
purification of the crude product by crystallization afforded (R)-
2-hydroxy-1,2-diphenylethan-1-one as a colorless solid; yield:
198 mg (99%, (R)-enantiomer 99% ee); HPLC: (Chiralpak IA)
4.7. Furoin
Furaldehyde (96 mg, 1.0 mmol) was dissolved in a mixture of 5%
v/v 2-methyltetrahydrofuran and potassium phosphate buffer
(20 mL, 50 mmol L-1, pH 7.0, containing MgSO4 (2.5 mmol L-1)
and ThDP (0.15 mmol L-1). After addition of BAL (20 mg)
the reaction mixture was allowed to stir for 16 h. The reaction
mixture was extracted with ethyl acetate (3 ¥ 10 mL) and the
organic layers washed with water (3 ¥ 10 mL) and brine (3
¥ 10 mL), and were dried over Na2SO4. Evaporation of the
solvent and purification of the crude product by crystallization
afforded (R)-2-hydroxy-1,2-diphenylethan-1-one as a colorless
solid; yield: 88 mg (92%, (R)-enantiomer 99% ee). HPLC
1
Rt (R) = 5.0 min; H NMR (400 MHz, CDCl3): 7.83 (d, J =
7.0 Hz, 2H), 7.33–7.19 (m, 8H), 5.88 (d, J = 6.0 Hz, 1H), 4.58
(d, J = 7.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): 198.9, 139.0,
133.9, 133.5, 129.2, 129.4, 129.1, 128.7, 128.6, 76.2.
1
(Chiralpak IA): Rt (S) = 3.9 min; Rt (R) = 4.7 min. H NMR
(400 MHz, CDCl3): 7.54 (m, 1H), 7.30 (m, 1H), 7.17 (d, J =
4.0 Hz, 1H), 6.46 (dd, J = 2.0, 4.0 Hz, 1H), 6.33–6.27 (m, 2H),
5.72 (d, J = 7.0 Hz, 1H), 4.12 (d, J = 7.0 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6): 184.2, 151.2, 149.6, 147.7, 143.1, 120.2,
112.6, 110.8, 109.1, 69.3.
4.5. Cross condensation benzaldehyde and acetaldehyde
Benzaldehyde (105 mg, 1.0 mmol) was dissolved in a mixture
of 5% v/v 2-methyltetrahydrofuran and potassium phosphate
buffer (20 mL, 50 mmol L-1, pH 7.0, containing MgSO4
(2.5 mmol L-1) and ThDP (0.15 mmol L-1). After addition of
BAL (20 mg) the reaction mixture was gently stirred for 1 h. To
this solution, acetaldehyde was added in three different intervals
(1 h) with concentration of 97 mmol L-1 (261 mg, 6 mmol) it
was allowed to stir for 16 h. The reaction mixture was extracted
with ethyl acetate (3 ¥ 10 mL) and the organic layers washed
with water (3 ¥ 10 mL) and brine (3 ¥ 10 mL), and were dried
over Na2SO4. Evaporation of the solvent and purification of
the crude product by crystallization afforded (R)-2-hydroxy-1-
phenylpropan-1-one as a colorless solid; yield: 149 mg (99%, (R)-
enantiomer 99% ee). HPLC: (Chiralpak IA) Rt (R) = 2.61 min;
1H NMR (400 MHz, CDCl3): 7.85 (d, J = 8.0 Hz, 2H), 7.56–7.40
(m, 3H), 5.13 (q, J = 7.0 Hz, 1H), 3.75 (br. s, 1H), 1.37 (d, J =
4.8. BAL-carboligation of butyraldehyde
Butyraldehyde (128 mg, 1.7 mmol) was dissolved in a mixture
of 5% v/v 2-methyltetrahydrofuran and potassium phosphate
buffer (20 mL, 50 mmol L-1, pH 7.0, containing MgSO4
(2.5 mmol L-1) and ThDP (0.15 mmol L-1). After addition
of BAL (20 mg) the reaction mixture was allowed to stir for
16 h. The reaction mixture was extracted with ethyl acetate
(3 ¥ 10 mL) and the organic layers washed with water (3 ¥
10 mL) and brine (3 ¥ 10 mL), and were dried over Na2SO4.
Evaporation of the solvent gave the crude product as yellow oil.
(R)-5-Hydroxyoctan-4-one; yield: 121 mg (95%, (R)-enantiomer
52% ee). 1H NMR (400 MHz, DMSO-d6): 5.11 (d, J = 5.6 Hz,
1H), 3.78 (m, 1H), 2.39 (m, 2H) 1.51–1.15 (m, 6H), 0.78 (t,
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J = 7.5 Hz, 3H), 0.74 (t, J = 7.5 Hz, 3); 13C-NMR (100 MHz,
CDCl3): 13.71, 13.84, 17.07, 18.54, 35.77, 39.69, 76.23, 212.22.
7 Recent reviews on umpolung organocatalytic biomimetic C–C bond
formation: (a) P. Dom´ınguez de Mar´ıa and S. Shanmuganathan,
Curr. Org. Chem., 2010. Accepted for publication; (b) D. Enders and
A. A. Narine, J. Org. Chem., 2008, 73, 7857–7870; (c) D. Enders,
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Acknowledgements
Financial support from DFG training group 1166 “BioNoCo”
(“Biocatalysis in Non-conventional Media”) is gratefully ac-
knowledged. Furthermore, this work was performed as part of
the Cluster of Excellence “Tailor-Made Fuels from Biomass”,
which is funded by the Excellence Initiative of the German
Research Foundation to promote science and research at
German universities.
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