Chymotrypsin-catalyzed peptide synthesis in deep eutectic solvents

Article abstract of DOI:10.1002/ejoc.201300448

Deep eutectic solvents (DESs) are formed by mixing quaternary ammonium salts (e.g., choline chloride) and hydrogen-bond donors (e.g., glycerol or urea), which leads to biodegradable and readily available ionic solvents at room temperature. Analogous to other ionic liquids, DESs represent a promising reaction media if hydrophobic and hydrophilic substrates need to be combined. This paper assesses DESs as reaction media for chymotrypsin-catalyzed peptide synthesis. After careful determination of the reaction conditions (e.g., water content, enzyme loading), α-chymotrypsin displayed high activity for peptide synthesis in choline chloride/glycerol mixtures to afford productivities of ca. 20 g L^-1 h^-1 and with complete selectivity for the peptide, which is in contrast to the detrimental hydrolysis pathway observed in aqueous media. The nonimmobilized suspended enzyme could be reused several times by simple filtration with excellent to moderate activities. Overall, the results reported suggest that choline chloride based DESs may become promising neoteric solvents for peptide synthesis through biocatalysis. Copyright

Full text of DOI:10.1002/ejoc.201300448

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201300448
Chymotrypsin-Catalyzed Peptide Synthesis in Deep Eutectic Solvents
Zaira Maugeri,[a] Walter Leitner,[a] and Pablo Domínguez de María*[a]
Keywords: Biocatalysis / Enzymes / Peptides / Sustainable chemistry / Ionic liquids
Deep eutectic solvents (DESs) are formed by mixing quater-
nary ammonium salts (e.g., choline chloride) and hydrogen-
bond donors (e.g., glycerol or urea), which leads to biode-
gradable and readily available ionic solvents at room tem-
perature. Analogous to other ionic liquids, DESs represent
a promising reaction media if hydrophobic and hydrophilic
substrates need to be combined. This paper assesses DESs
as reaction media for chymotrypsin-catalyzed peptide syn-
thesis. After careful determination of the reaction conditions
(e.g., water content, enzyme loading), α-chymotrypsin dis-
played high activity for peptide synthesis in choline chloride/
glycerol mixtures to afford productivities of ca. 20 gL–1 h–1
and with complete selectivity for the peptide, which is in con-
trast to the detrimental hydrolysis pathway observed in aque-
ous media. The nonimmobilized suspended enzyme could be
reused several times by simple filtration with excellent to
moderate activities. Overall, the results reported suggest that
choline chloride based DESs may become promising neoteric
solvents for peptide synthesis through biocatalysis.
Besides their potential environmental impact, the cost as-
sociated with the synthesis and use of ILs has been typically
Introduction
Over the last decades, enzyme catalysis has been estab- a hurdle for their further practical use. In this respect, aim-
lished as a broadly applicable and useful technology for or- ing to keep the versatility of the properties that ILs display,
ganic synthesis, in particular for regio-, enantio-, and while at the same time providing more environmentally
chemoselective processes. Together with advances in molec- friendly and cost-effective options, deep eutectic solvents
ular biology – for genetic design and further accessibility of (DESs) have emerged as a novel generation of ionic solvents
biocatalysts on a large scale – an important reason for such with promising applications.[6–9] DESs are formed by mix-
increasing utility is the fact that many enzymes can operate ing quaternary ammonium salts (e.g., choline chloride as
under non-aqueous conditions, the so-called nonconven- the most commonly used) with compounds capable of
tional media, which opens the possibility of combining high forming hydrogen bonds with the first component (e.g.,
substrate loadings and biocatalysis with a clear focus on amines, amides, alcohols, or carboxylic acids, the so-called
industrial synthesis.[1] After the seminal work developed by HBD: hydrogen-bond donor). After mixing and upon gen-
Klibanov, mostly with hydrolases in organic media,[2] in re- tle stirring and warming (up to 100 °C), the HBD disrupts
cent years other enzymes such as oxidoreductases and the crystalline structure of the quaternary ammonium salt,
transaminases have been reported to display excellent ac- which decreases the freezing point (Tf) of the mixture and
tivities and selectivities in non-aqueous media, including generates (in some cases) liquids at room temperature. The
solvent-free systems or the use of different organic sol- classic example is the combination of choline chloride (m.p.
vents.[3] Moreover, a broad number of ionic liquids (ILs) 302 °C) with urea (m.p. 133 °C) in a 1:2 molar ratio to cre-
have also been successfully tested as useful (co)solvents for ate a room-temperature liquid DES (Tf = 12 °C).[6–9] The
enzyme-catalyzed reactions. The possibility of dissolving synthesis of DESs is often straightforward, does not require
challenging materials or substrates with different polarities further purification steps, and no byproducts are formed.
has emerged as an added value of these neoteric solvents.[4] As starting materials, biodegradable, readily available, and
Yet, recently the environmental impact of commonly used inexpensive compounds are often used, and thus, this route
ILs has been put under discussion.[5]
has a promising environmental prognosis.[9] Considering
these aspects, research into the applications of DESs in dif-
ferent chemical segments has flourished over the last dec-
ade.[6–9] With regard to applications in biocatalysis, several
enzyme-catalyzed processes with the use of DESs as sol-
vents have been reported, and they typically focus on lipase-
catalyzed (trans)esterifications and aminolysis reactions.[10]
Actually, urea is a well-known denaturing agent for pro-
teins, but lipases remain largely stable and active in choline
chloride/urea (ChCl:Ur) DESs.[10]
[a] Institut für Technische und Makromolekulare Chemie (ITMC),
RWTH Aachen University
Worringerweg 1, 52074 Aachen, Germany
Fax: +49-241-8022177
E-mail: dominguez@itmc.rwth-aachen.de
Homepage: www.tc.rwth-aachen.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300448
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Z. Maugeri, W. Leitner, P. Domínguez de María
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These potential advantages of DESs – biodegradability,
ease of preparation, availability – together with their capa-
bility of dissolving a broad range of complex natural prod-
ucts and molecules[8] might make them appealing neoteric
solvents for industrial reactions. An example of this might
be represented by peptide synthesis, as peptides and oligo-
peptides are added-value products with a broad range of
market-oriented applications.[11] In this respect, one of the
challenges for peptide synthesis is the reaction medium, as
hydrophobic and hydrophilic substrates typically need to be
combined at the same time. Therein, the use of aqueous
solutions is in many cases not recommended, as water often
shifts the equilibrium towards hydrolysis rather than to
peptide synthesis. For such purposes, non-aqueous ILs have
been used as solvents for the production of peptides with
the use of either chemo- or biocatalysts[12] such as prote-
ases.[13] Moreover, another approach involving the use of
proteases for peptide synthesis comprises the formation of
eutectic mixtures of substrates (e.g., two amino acids or
peptides) with the aid of adjuvants (e.g., small amounts of
ethanol or water), which leads to “solvent-free” mixtures
with high substrate loadings[14] and to “solid-to-solid” reac-
tions.[15] In the field of choline chloride based DESs, how-
ever, proteases have been scarcely explored.[16] Addressing
the potential advantages of DESs for biocatalytic industrial
processes, in general, and the added value that they may
bring to the arena of peptide synthesis, in particular, in this
paper the use of proteases for peptide synthesis in choline
chloride based DESs is explored.
Figure 1. DESs employed in this study (see the Supporting Infor-
mation for details).
To assess DESs in protease-catalyzed processes, α-chym-
otrypsin was used as the biocatalyst. As a prototypical reac-
tion, the synthesis of the protected N-Ac-Phe-Gly-NH2
peptide (APG, 3) in different DESs was used, starting from
N-acetylphenylalanine ethyl ester (APEE, 1) and glycin-
amide hydrochloride (GH, 2) as substrates (Scheme 1). The
reason for this election was the preponderance of APEE (1)
to undergo dissolution in hydrophobic solvents, whereas
GH (2) tends to be more soluble in hydrophilic solvents.[17]
The resulting dipeptide, APG (3), has also found some
therapeutic uses.[17]
Results and Discussion
The formation of the desired APG (3) dipeptide may be
Different choline chloride based DESs were prepared by hampered by detrimental hydrolysis to yield AP (4), espe-
gently mixing their components under mild conditions (up cially if significant amounts of water are pres-
to 100 °C). As HBDs, several biomass-derived compounds ent.[15a,15c,15f,17] However, some molecules of water are cru-
were used, namely, glycerol, urea, xylitol, and isosorbide cial for the activity of enzymes in nonconventional me-
(Figure 1).
dia.[2,3] Thus, the reaction conditions must chosen carefully
Scheme 1. Chymotripsin-catalyzed model reaction used in this paper.
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Enzyme-Catalyzed Peptide Synthesis in Deep Eutectic Solvents
to minimize the hydrolytic side reaction, while at the same
Once these bench reaction conditions were fixed
time enhancing the biocatalytic production of 3 and thus (ChCl:Gly, 10% water, v/v), the enzyme loading was sub-
taking full benefit of the neoteric nonconventional media sequently assessed to obtain insight into the reaction rate
(e.g., ease of dissolution of substrates with different polarit- of the synthetic process, and especially to gain more infor-
ies). Addressing these aspects, in a first set of experiments mation on the relationship between synthesis and hydroly-
the influence of water in the reaction media was assessed. sis, and to determine the best reaction conditions for fur-
As a prototypical DES, choline chloride/glycerol (ChCl:Gly, ther optimizations of the reaction. The results for two dif-
1:2 molar ratio) was used.[7c] The results are depicted in ferent enzyme loadings (20 and 40 mgmL–1) are depicted in
Figure 2.
Figure 3.
Figure 2. Effect of water (synthesis vs. hydrolysis) in α-chymotryp-
sin-catalyzed peptide synthesis by using N-acetylphenylalanine
ethyl ester (APEE, 1) and glycinamide hydrochloride (GH, 2) as
substrates. Reaction conditions: APEE (20 gL–1), GH (10 gL–1), α-
chymotrypsin (4 mgmL–1) ChCl:Gly DES [0–50% water (v/v)],
room temperature, 24 h. Conversions determined by 1H NMR
spectroscopy.
As can be observed (Figure 2), lower amounts of water
(up to 5% v/v) led to virtually no protease activity in
ChCl:Gly media, which suggests denaturation of the en-
zyme under those extremely dry conditions. Remarkably,
the addition of 10% water (v/v) enabled high enzymatic ac-
tivity, and peptide 3 was obtained in 90% yield. Excellent
results were also observed if up to 25% water (v/v) was
added. In this range (10–25% v/v), the hydrolytic reaction
was completely suppressed. At higher amounts of water
(50% v/v), hydrolysis was significantly observed. Obviously,
for practical considerations in peptide synthesis, a broad
range of water contents (10–25% v/v) should be available
to allow both excellent enzymatic activity and outstanding
selectivity for the synthetic reaction. Looking beyond and
envisaging this DES-based strategy as a useful concept for
other more difficult-to-dissolve substrates (e.g., oligopept-
ides), 10% water (v/v) was taken in further experiments. It
may be expected that a broad range of substrates may be
dissolved in such DES/water mixtures (90–10% v/v),
whereas at the same time excellent enzymatic operational
conditions (stability and activity) may be achieved. Once
Figure 3. α-Chymotrypsin-catalyzed peptide synthesis by using N-
acetylphenylalanine ethyl ester (APEE, 1) and glycinamide hydro-
chloride (GH, 2) as substrates. Reaction conditions: APEE
(20 gL–1), GH (10 gL–1), ChCl:Gly DES [10% water (v/v)], room
temperature with α-chymotrypsin at a loading of 20 mgmL–1 (top)
and 40 mgmL–1 (bottom) Conversions determined by 1H NMR
spectroscopy.
The synthetic reaction (peptide-bond formation) pro-
the reaction was finished, the downstream processing for ceeded fast with virtually full conversion and outstanding
the DES was conducted upon the addition of water (to selectivities (for synthesis) at short reaction times (4 h),
break the DES) and extraction with ethyl acetate.[18] Gentle whereby no hydrolytic reactions were observed. As ex-
warming of the DES/water mixture to remove the water led pected, significant amounts of hydrolysis product 4 were
to the recovery of the DES, as reported previously.[7a,7b] observed in both cases at longer reaction times (24 h), as
Likewise, considering further practical uses of the DES, well as at 8 h if higher enzyme loadings were used. There-
preliminary assessments in the literature suggest a promis- fore, considering the importance of fast reactions with high
ing diminishing corrosiveness of choline chloride based selectivities, for practical purposes the assessed reaction
DESs with the use of non-acidic HBDs (e.g., polyols).[19]
may be performed in short reaction times (4 h) to afford
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Z. Maugeri, W. Leitner, P. Domínguez de María
SHORT COMMUNICATION
complete selectivity for desired peptide 3. Under these con-
trolled conditions [water amount (10% v/v) and enzyme
loading (40 mgmL–1)], full conversion with excellent selec-
tivity for APG (3) were achieved.
In the next set of experiments, the substrate loading was
assessed. As a starting point, for the above-described reac-
tion conditions, a substrate loadings of APEE (1) of ca.
20 gL–1 was applied, which led to full conversion in 4 h.
Envisaging industrial conditions for peptide synthesis and
taking advantage of the capability of DESs to dissolve these
challenging substrates, the loading of APEE (1) was signifi-
cantly increased up to 170 gL–1. The results are depicted in
Figure 4.
Figure 5. Enzyme recycling. Reaction conditions: APEE (20 gL–1),
GH (10 gL–1), α-chymotrypsin (20 mgmL–1), ChCl:Gly DES [10%
water (v/v)], room temperature, 2 h. Conversions determined by
1H NMR spectroscopy.
cles, clear deactivation of the biocatalyst was observed. Tak-
ing into account that in this case the enzyme was not immo-
bilized, it can be expected that a more robust immobilized
system may lead to more stable catalysts for the reaction
proposed. In any case, the free enzyme already shows prom-
ising stability in DESs.
Finally, different choline chloride based DESs were as-
sessed for the protease-catalyzed reaction. The rationale be-
hind these studies was the assumption that proteasesϪfor
industrial purposesϪmay be used for a broad range of di-
verse substrates, including (protected) amino acids, oligo-
peptides, and so on. Some of these substrates might dissolve
well in some DESs, whereas other substrates might perform
better in different DESs (or in mixtures of them). Thus, the
preparation of several DESs with positive enzymatic activi-
ties might be an asset. Herein, four choline chloride based
DESs (Figure 1) with glycerol (ChCl:Gly, 1:2), urea
(ChCl:Ur, 1:2), isosorbide (ChCl:Is, 1:2), and xylitol
(ChCl:Xyl, 1:1) as HBDs were assessed. The results are de-
picted in Figure 6.
Figure 4. Peptide-catalyzed reactions at higher substrate loadings.
Reaction conditions: APEE (20–170 gL–1), GH (10–81 gL–1), α-
chymotrypsin (40 mgmL–1), ChCl:Gly DES [10% water (v/v)],
room temperature, 4 h. Conversions determined by 1H NMR spec-
troscopy.
Gratifyingly, the enzymatic process in the DES was re-
vealed to be quite robust, and thus, substrate loadings in
the range of 80 gL–1 maintained full conversion and perfect
synthetic selectivities under the (still) unoptimized condi-
tions. At higher substrate loadings, lower conversions were
gradually observed (at 4 h reaction time). By considering
these data together with the observations reported in Fig-
ure 3, it may be envisaged that a reaction time between 4
and 8 h should still allow room to enhance peptide pro-
duction without compromising the selectivity through the
expected concomitant hydrolysis route. In any case, the re-
sults clearly support the idea that DESs may be useful reac-
tion media for proteases under industrially oriented condi-
tions (high substrate loadings and conversions, short reac-
tion times, etc.), and thus, productivities of ca. 20 gL–1 h–1
with excellent selectivities are observed.
The reactions were performed by using free, nonimmobi-
lized α-chymotrypsin that remained suspended as a powder
in the DES media. With the goal of assessing whether reuse
of the enzyme was feasible, the suspended powder was reco-
vered upon filtration, washed with 2-propanol, and directly
used in the next catalytic cycle (see the Exp. Section for
details). The results are depicted in Figure 5.
As observed, the nonimmobilized suspended enzyme
could be directly used over 3–4 cycles while maintaining
excellent to moderate activities in the model reaction
studied, with complete selectivity for the synthesis of the
Figure 6. α-Chymotrypsin-catalyzed peptide synthesis by using N-
acetylphenylalanine ethyl ester (APEE, 1) and glycinamide hydro-
chloride (GH, 2) in four different DESs. Reaction conditions:
APEE (20 gL–1), GH (10 gL–1), α-chymotrypsin (20 mgmL–1),
DES [10% water (v/v)], room temperature, 4 h. Conversions deter-
1
peptide (vs. hydrolysis) in all cases. After those reaction cy- mined by H NMR spectroscopy.
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Enzyme-Catalyzed Peptide Synthesis in Deep Eutectic Solvents
[5]
[6]
M. Deetlefs, K. R. Seddon, Green Chem. 2010, 12, 17–30.
a) D. Carriazo, M. C. Serrano, M. C. Gutiérrez, M. L. Ferrer,
F. del Monte, Chem. Soc. Rev. 2012, 41, 4996–5014; b) Q.
Zhang, K. De Oliveria Vigier, S. Royer, F. Jerome, Chem. Soc.
Rev. 2012, 41, 7108–7146; c) C. Ruß, B. König, Green Chem.
2012, 14, 2969–2982.
a) Z. Maugeri, W. Leitner, P. Domínguez de María, Tetrahe-
dron Lett. 2012, 53, 6968–6971; b) Z. Maugeri, P. Domíng-
uez de María, RSC Adv. 2012, 2, 421–425; c) A. P. Abott, R. C.
Harris, K. S. Ryder, C. D’Agostino, L. F. Gladden, M. D.
Mantle, Green Chem. 2011, 13, 82–90; d) M. Avalos, R. Babi-
ano, P. Cintas, J. L. Jimenez, J. C. Palacios, Angew. Chem. 2006,
118, 4008–4012; Angew. Chem. Int. Ed. 2006, 45, 3904–3908;
e) A. P. Abott, D. Boothby, G. Capper, D. L. Davies, R. K.
Rasheed, J. Am. Chem. Soc. 2004, 126, 9142–9147; f) A. P.
Abott, G. Capper, D. L. Davies, R. K. Rasheed, V. Tambyra-
jah, Chem. Commun. 2003, 70–71; g) A. P. Abott, G. Capper,
D. L. Davies, H. L. Munro, R. K. Rasheed, V. Tambyrajah,
Chem. Commun. 2001, 2010–2011.
a) M. Francisco, A. van den Bruinhorst, M. C. Kroon, Green
Chem. 2012, 14, 2153–2157; b) J. van Spronsen, G. J. Witkamp,
F. Hollmann, Y. H. Choi, R. Verpoorte, WO2011155829, 2011;
c) Y. H. Choi, J. van Spronsen, Y. Dai, M. Verberne, F.
Hollmann, I. W. C. E. Arends, G. J. Witkamp, R. Verpoorte,
Plant Physiol. 2011, 156, 1701–1705.
M. Hayyan, M. A. Hashim, A. Hayvan, M. A. Al-Saadi, I. M.
AlNashef, M. E. S. MIrghani, O. K. Saheed, Chemosphere
2013, 90, 2193–2195.
As can be observed, different DESs led to positive results
in the protease-catalyzed synthesis. When glycerol or iso-
sorbide were used as the HBD, virtually full conversion was
reached, with complete selectivity for synthetic product 3.
Moreover, despite the denaturing effect of urea, ChCl:Ur
provided high conversions as well. Interestingly, the use of
a DES composed of choline chloride and xylitol provided
much lower conversion to the ester, together with signifi-
cant formation of the hydrolysis product.
[7]
Conclusions
In summary, chymotrypsin has been successfully assessed
in choline chloride based DESs for the synthesis of pept-
ides. DESs are biodegradable and can be straightforwardly
produced; at the same time, they enable the dissolution of
amino acids and peptides, which can open interesting op-
tions for industrial processes. Under the reported condi-
tions in a prototypical reaction, productivities of ca.
20 gL–1 h–1 were reached with completely controlled and di-
minished hydrolysis. Furthermore, the suspended nonim-
mobilized free enzyme could be reused over several cycles
before deactivation. The determination of proper enzyme
immobilization strategies might improve the recycling sig-
nificantly. The results demonstrate the successful applica-
tion of choline chloride based DESs to enzymatic processes
of significant industrial importance, which further substan-
tiates the synthetic and practical potential of these reaction
media for practical applications under the sustainable
chemistry principles.
[8]
[9]
[10]
a) E. Durand, J. Lecomte, B. Baréa, G. Piombo, E. Dubreucq,
P. Villeneuve, Process Biochem. 2012, 47, 2081–2089; b) H.
Zhao, G. A. Baker, S. Holmes, Org. Biomol. Chem. 2011, 9,
1908–1916; c) J. T. Gorke, F. Srienc, R. J. Kazlauskas, Chem.
Commun. 2008, 1235–1237.
[11]
a) T. Nuijens, A. H. M. Schepers, C. Cusan, J. A. W. Kruitjtzer,
D. T. S. Rijkers, R. M. J. Liskamp, P. J. L. M. Quaedflieg, Adv.
Synth. Catal. 2013, 355, 287–293; b) R. J. A. C. de Beer, T. Nui-
jens, L. Wiermans, P. J. L. M. Quaedflieg, F. P. J. T. Rutjes, Org.
Biomol. Chem. 2012, 10, 6767–6775; c) T. Nuijens, C. Cusan,
T. J. G. M. van Dooren, H. M. Moody, R. Merkx, J. A. W.
Kruijtzer, D. T. S. Rijkers, R. M. J. Liskamp, P. J. L. M. Quaed-
flieg, Adv. Synth. Catal. 2010, 352, 2399–2404; d) T. Nuijens,
C. Cusan, J. A. W. Kruijtzer, D. T. S. Rijkers, R. M. J. Liskamp,
P. J. L. M. Quaedflieg, J. Org. Chem. 2009, 74, 5145–5150; e)
T. Nuijens, C. Cusan, A. C. H. M. Schepers, J. A. W. Kruijtzer,
D. T. S. Rijkers, R. M. J. Liskamp, P. J. L. M. Quaedflieg, J.
Mol. Catal. B 2011, 71, 79–84.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures and copies of the NMR spectra of
the solvents (DESs), reagents, and products
Acknowledgments
Financial support from the Deutsche Forschungsgemeinschaft
(DFG) training group 1166 “BioNoCo” (Biocatalysis in Non-con-
ventional Media) is gratefully acknowledged.
[12]
[13]
A. A. Tietze, P. Heimer, A. Stark, D. Imhof, Molecules 2012,
17, 4158–4185.
a) P. Attri, P. Venkatesu, A. Kumar, Phys. Chem. Chem. Phys.
2011, 13, 2788–2796; b) G. W. Xing, F. Y. Li, C. Ming, L. Ran,
Tetrahedron Lett. 2007, 48, 4271–4274; c) J. A. Laszlo, D. L.
Compton, Biotechnol. Bioeng. 2001, 75, 181–186; d) M. Erbeld-
inger, A. J. Mesiano, A. J. Russell, Biotechnol. Prog. 2000, 16,
1129–1131.
a) I. Gill, R. Valivety, Org. Process Res. Dev. 2002, 6, 684–691;
b) X. Jorba, I. Gill, E. N. Vulfson, J. Agric. Food Chem. 1995,
43, 2536–2541; c) R. López-Fandino, I. Gill, E. N. Vulfson,
Biotechnol. Bioeng. 1994, 43, 1016–1023; d) R. López-Fandino,
I. Gill, E. N. Vulfson, Biotechnol. Bioeng. 1994, 43, 1024–1030;
e) I. Gill, E. N. Vulfson, Trends Biotechnol. 1994, 12, 118–122;
f) I. Gill, E. N. Vulfson, J. Am. Chem. Soc. 1993, 115, 3348–
3349.
a) M. Erbeldinger, P. J. Halling, X. Ni, AIChE J. 2001, 47, 500–
508; b) M. Erbeldinger, X. Ni, P. J. Halling, Biotechnol. Bioeng.
2001, 72, 69–76; c) M. Erbeldinger, U. Eichhorn, P. Kuhl, P. J.
Halling, Meth. Biotechnol. 2001, 15, 471–477; d) R. V. Ulijn,
M. Erbeldinger, P. J. Halling, Biotechnol. Bioeng. 2000, 69, 663–
638; e) M. Erbeldinger, X. Ni, P. J. Halling, Biotechnol. Bioeng.
1999, 63, 316–321; f) P. Björup, P. Adlercreutz, P. Clapes, Bio-
catal. Biotransform. 1999, 17, 319–345; g) M. Erbeldinger, X.
[1] a) U. T. Bornscheuer, G. Huisman, R. J. Kazlauskas, S. Lutz,
J. Moore, K. Robins, Nature 2012, 485, 185–194; b) K. H.
Drauz, H. Gröger, O. May, Enzyme Catalysis in Organic Syn-
thesis Wiley-VCH, Weinheim, Germany, 2012.
[14]
[15]
[2] A. M. Klibanov, Nature 2001, 409, 241–246.
[3] a) F. G. Mutti, W. Kroutil, Adv. Synth. Catal. 2012, 354, 3409–
3413; b) M. D. Truppo, H. Strotman, G. Hughes, ChemCat-
Chem 2012, 4, 1071–1074; c) A. Jakoblinnert, R. Mladenov, A.
Paul, F. Sibilla, U. Schwaneberg, M. B. Ansorge-Schumacher,
P. Domínguez de María, Chem. Commun. 2011, 47, 12230–
12232.
[4] a) P. Domínguez de María (Ed.), Ionic Liquids in Biotransfor-
mations and Organocatalysis: Solvents and Beyond, Wiley, Ho-
boken, New Jersey, 2012; b) P. Domínguez de María, Z. Maug-
eri, Curr. Opin. Chem. Biol. 2011, 15, 220–225; c) P. Lozano,
Green Chem. 2010, 12, 555–569; d) P. Domínguez de María,
Angew. Chem. 2008, 120, 7066–7075; Angew. Chem. Int. Ed.
2008, 47, 6960–6968; e) F. van Rantwijk, R. A. Sheldon, Chem.
Rev. 2007, 107, 2757–2785.
Eur. J. Org. Chem. 2013, 4223–4228
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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Ni, P. J. Halling, Biotechnol. Bioeng. 1998, 59, 68–72; h) M.
Erbeldinger, X. Ni, P. J. Halling, Enz. Microb. Technol. 1998,
23, 141–148; i) P. Björup, J. L. Torres, P. Adlercreutz, P. Clapes,
Bioorg. Med. Chem. 1998, 6, 891–901.
[18] Under unoptimized conditions, water (2 mL) was added to the
reaction media, and the mixture was extracted with ethyl acet-
ate, evaporated under vacuum, and studied by H NMR spec-
troscopy.
[16] H. Zhao, G. A. Baker, S. Holmes, J. Mol. Catal. B 2011, 72, [19] A. P. Abbott, G. Capper, K. J. McKenzie, A. Glidle, K. S. Ry-
1
163–167.
der, Phys. Chem. Chem. Phys. 2006, 8, 4214–4221.
Received: March 26, 2013
[17] H. Y. Ju, J. R. Too, C. Chang, C. J. Shieh, J. Agric. Food Chem.
2009, 57, 403–408.
Published Online: June 4, 2013
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