Benzaldehyde lyase (BAL)-catalyzed enantioselective CC bond formation in deep-eutectic-solvents-buffer mixtures

Article abstract of DOI:10.1016/j.molcatb.2014.06.003

Deep-eutectic-solvents (DES) have emerged in the last decades as promising bio-based and biodegradable neoteric solvents for biocatalysis, with examples covering different enzymes (mostly hydrolases) and whole-cells (baker's yeast). This paper explores fo

Full text of DOI:10.1016/j.molcatb.2014.06.003

Accepted Manuscript
Title: Benzaldehyde Lyase (BAL)-catalyzed enantioselective
C-C bond formation in Deep-Eutectic-Solvents–Buffer
Mixtures
Author: Zaira Maugeri Pablo Dom ´ı nguez de Mar ´ı a
PII:
S1381-1177(14)00176-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.06.003
MOLCAB 2974
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
24-5-2014
6-6-2014
7-6-2014
´
´
Please cite this article as: Z. Maugeri, P. Dominguez de Maria, Benzaldehyde
Lyase (BAL)-catalyzed enantioselective C-C bond formation in Deep-Eutectic-
SolventsndashBuffer Mixtures, Journal of Molecular Catalysis B: Enzymatic (2014),
http://dx.doi.org/10.1016/j.molcatb.2014.06.003
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Benzaldehyde Lyase (BAL)-catalyzed enantioselective C-C bond formation in
Deep-Eutectic-Solvents–Buffer Mixtures
a
a,b
Zaira Maugeri , Pablo Domínguez de María *
a
:
Institut für Technische und Makromolekulare Chemie (ITMC),
RWTH Aachen University. Worringerweg 1. 52074 Aachen, Germany.
b
:
Present address: Sustainable Momentum, SL. Ap. Correos 3517. 35004, Las Palmas
de Gran Canaria; Canary Islands; Spain; www.sustainable-momentum.net
Corresponding Author:
Dr. Pablo Domínguez de María.
Tel.: +34 609 565237.
E-mail: dominguez@itmc.rwth-aachen.de;
dominguez@sustainable-momentum.net
1
Page 1 of 22

GRAPHICAL ABSTRACT.
O
O
O
Benzaldehyde Lyase
DES-Buffer
R1 Conversion up to 99 %
ee up to 99 % (R)
+
R1
R1
H
R1
H
OH
Deep-Eutectic-Solvents
O
HO
OH
H N
NH2
2
OH
N
+
OH
OH
Cl-
HO
OH
OH
OH
2
Page 2 of 22

HIGHLIGHTS
•
•
•
Lyases catalyze C-C bond forming reactions in deep-eutectic-solvents – buffer
mixtures.
Choline chloride-urea DES can be tolerated up to 70 % (v/v) with full
conversion.
DES may be tuned to enable novel opportunities for lyases in non-conventional
media.
3
Page 3 of 22

Summary.
Deep-eutectic-solvents (DES) have emerged in the last decades as promising
bio-based and biodegradable neoteric solvents for biocatalysis, with examples
covering different enzymes (mostly hydrolases) and whole-cells (baker´s yeast). This
paper explores for the first time the use of Benzaldehyde Lyase (BAL), a thiamine-
diphosphate dependent lyase (ThDP-Lyase) able to catalyze the carboligation of
aldehydes (C-C bond formation) in different DES-buffer mixtures. By using choline
chloride – glycerol DES, BAL remains fully active with excellent enantioselectivity at
60:40 DES-buffer (v/v), whereby a significant denaturation is observed at 70:30
mixtures. Remarkably, the use of choline chloride – urea DES as reaction media leads
to full conversions with BAL at such solvent – buffer proportions, suggesting that the
design of both the biocatalyst and the neoteric solvent may provide useful novel
reactive systems for biocatalysis in non-conventional media.
Key-words: Lyases; Deep-eutectic-solvents; Biocatalysis; non-conventional
media
4
Page 4 of 22

1
.- Introduction
Over the last decades enzyme catalysis has established as a powerful technology
in organic synthesis based on the excellent regio-, stereo-, and chemo-selectivities
obtained, typically under environmentally-friendly conditions [1]. Although natural
media for biocatalysis are aqueous solutions, many enzymes can operate under the so-
called non-conventional media, such as neat substrates, organic solvents, supercritical
fluids, gas phase or ionic liquids (ILs) [2]. The use of enzymes in these solvents opens
the possibility of combining hydrophobic substrates and products – often sparingly
soluble in water –, with a focus on practical applications. Specially, an increasing
interest has been observed recently for ILs, with a broad number of academic
applications disclosed [3]. ILs might dissolve challenging substrates or with different
polarities, while being enzyme-friendly with comparable or higher catalytic activities
than the observed in organic solvents and, in some cases, enhanced thermal and
operational stabilities. Yet, the typical high prices of ILs may represent a hurdle for
their broader implementation at practical scale. Moreover, concerns raised about the
toxicity and poor biodegradability of some of them may hamper even further such
interest [4]. Remarkably, over the last decade a new class of neoteric solvents, called
deep-eutectic-solvents (DES), has emerged. DES are essentially made by mixing a
quaternary ammonium salt (in some cases replaced with a quaternary phosphonium
salt) with hydrogen-bond-forming molecules. Choline chloride is the most employed
quaternary ammonium salt because of its low cost and biodegradability, whereas as a
hydrogen-bond-donors (HBD) different molecules such as alcohols, amides, amines,
carboxylic acids, etc., have been successfully used [5]. DES are often relatively cheap
(due to the inexpensive starting materials and ease of their preparation), and most of
5
Page 5 of 22

their components are biodegradable and non-toxic, as several recent papers have
shown [6]. These features, together with the possibility of having tailored solvents by
changing one or both components, make DES certainly appealing for a broad number
of applications. Many promising examples using DES in electrochemistry, material
and organic synthesis, as well as extracting agents and separation media have been
reported [7,8]. Several attempts were also done in biocatalysis where DES have been
used as (co)-solvents mainly for hydrolases (lipases, proteases and epoxide
hydrolases) catalyzed reactions [9]. Concerning DESs and microorganisms, the
stability of lyophilized E. coli was proven in DES [10], and recently the first example
of baker´s yeast whole-cell biotransformations in these media was published [11].
Furthermore, DES display excellent dissolving properties, enhancing the solubility of
complex functionalized molecules. Applications of DES in peptide synthesis have
successfully appeared, even using high substrate loadings [9g].
As a continuation of our research program on DES and biocatalysis in non-
conventional media [5e,7c,8a,b,9f,9g,11], the focus of this work was to explore for the
first time the use of thiamine-diphosphate dependent lyases (ThDP-lyases) in different
mixtures of DES-aqueous solutions. ThDP-lyases are a versatile group of enzymes
catalyzing asymmetric aldehyde carboligations to afford chiral α-hydroxy ketones,
relevant compounds used as building blocks for pharmaceutical (stimulants, anti-
depressants, fungicides, anti-cancer drugs) and fine chemical applications [12]. A
prominent example is benzaldehyde lyase (BAL) from Pseudomonas fluorescens, an
enzyme that carboligates both aromatic and aliphatic aldehydes with high-to-excellent
enantioselectivities and diastereoselectivities [12,13]. Several process-development
studies concerning BAL-catalyzed synthetic systems have demonstrated the potential
of this enzyme for industrially-sound reaction conditions.
6
Page 6 of 22

2.- Results & Discussion.
The introduction of DES as reaction media in lyase-catalyzed reactions might
lead to novel options for biocatalytic process development. To assess the new line,
different BAL-catalyzed C-C bond forming reactions were chosen. Given the broad
substrate acceptance of BAL [12e,13a,f], both aliphatic and aromatic aldehydes were
included in the study (Scheme 1).
[Scheme 1]
As prototypical DES, choline chloride was used as the quaternary ammonium
salt, whereas different HBDs such as glycerol, urea and xylitol were chosen as bio-
based complements. To form the DES, choline chloride was gently mixed together
with the correspondent HBD in the adequate proportion (Table 1), and at 80ºC for one
hour.
[Table 1]
For the lyase assessment in DES-aqueous media, BAL as free enzyme was used
in a lyophilized form (see experimental part for details). Since a certain amount of
water is necessary for the enzyme to keep its catalytically active conformation – as
also observed for other enzymes in DES media [9d,e,g] –, in a first set of experiments
the effect of water was investigated by using different DES-buffer mixtures as
7
Page 7 of 22

reaction media. Apart from the beneficial effect for the enzyme, some amounts of
water lead to much less viscous DES solutions. In this case, glycerol-based DES was
used as model DES. Results are depicted in Figure 1.
[Figure 1]
As it can be observed in Figure 1, BAL resulted to be highly sensitive to the
amount of DES added. Thus, at 10 vol.% water (in DES) its rapid denaturation
occurred, and no conversions in the carboligation were observed. Moderate
conversions for all substrates were obtained for DES containing 30 vol.% water.
Remarkably, virtually full conversions were reached with DES containing 40 vol.%
water, thus showing for the first time the activity of lyases in those new reaction
media. It must be mentioned that aromatic α-hydroxy ketones formed – benzoin and
furoin – were not soluble in none of those conditions. With regard to the
enantioselectivities, values resulted similar to those ones previously reported in
literature [13a,f], with the exception of furoin where a lower enantiomeric excess was
observed (Table 2). Presumably, some racemization might occur in the novel media
with this typically unstable product.
[Table 2]
Having observed that at 60:40 glycerol-based DES-buffer virtually full
conversions were achieved with BAL, further studies at 70:30 DES-buffer were
conducted. The motivation behind these studies was to probe that BAL could actually
perform C-C bond reactions in even more non-aqueous solutions than the mentioned
8
Page 8 of 22

60:40 one (Figure 1). Experiments aimed to verify whether BAL remained stable in
the DES in presence of 30% water amount, albeit at lower kinetic rates than for
mixtures with higher buffer amounts. In this case, test reactions were performed with
valeraldehyde as substrate at longer reaction times (over 72 hours) and the results are
depicted in Figure 2. As it can be observed, BAL did not reach full conversions not
even at longer reaction times, thus clearly demonstrating that under these conditions
(G-DES), BAL was rapidly deactivated by the non-conventional media. Therefore, it
can be concluded that for such glycerol-based DES, BAL requires more amounts of
water than other enzymes (e.g. hydrolases [9d,e,g,], or even whole-cells [11], where
catalytic activities are observed in more anhydrous media. Further studies (e.g.
addition of polymers as enzyme stabilizers), or genetic design of lyases might lead to
more robust biocatalysts for such reaction conditions.
[Figure 2]
Once observed that glycerol-based DES were not so compatible for BAL in
largely anhydrous solutions, other alternatives were searched. In this respect, one of
the advantages of neoteric solvents is their capacity of being finely tuned according to
the desired applications. Therefore, in the quest of more BAL-friendly neoteric
solvents for carboligation reactions, other DES such as ChCl-urea (U-DES) and ChCl-
xylitol (X-DES) were assessed using the same challenging reaction conditions
(30 vol.% water) (Figure 3). Quite remarkably, while X-DES led to similar results as
G-DES did – thus showing a rapid BAL deactivation –, leading to only ca. 80%
conversion, the use of U-DES led to full conversions under the same reaction
conditions, thus suggesting that the enzyme may actually be more stable in this
9
Page 9 of 22

solvent. Albeit U-DES can intuitively be an enzyme-unfriendly derivative – as urea is
a well-known denaturating agent for proteins –, the (unexpected) potential of U-DES
for different enzyme-catalyzed reactions has been reported by other groups as well
[9a-e,g]. More research is needed to understand the relationship between U-DES and
enzymes for biocatalytic reactions.
[Figure 3]
3
.- Conclusions.
This paper has successfully explored for the first time the use of ThDP-lyases to
perform enantioselective C-C bond carboligation reactions in different DES-buffer
mixtures. Different substrates were effectively carboligated by BAL in glycerol-based
DES containing 40 % buffer, with high stereoselectivities and conversions. With the
set-up of more anhydrous DES-aqueous media a rapid denaturation of the enzyme
was observed. However, by making use of the large tuneability of DES, the use of
urea-based containing DES enabled the biocatalytic carboligation even at 70:30 DES-
buffer media. Overall our results suggest that DES may become a potentially useful
non-conventional media for lyases. More research is needed to understand the actual
role of the different HBDs in the stability of the enzyme, what could provide insights
on how more stable and active lyase variants can be designed.
4- Experimental.
4
.1.- Materials. All chemicals were used without any further purification. Choline
chloride, glycerol, ethyl acetate, thiamine-diphosphate, magnesium sulfate,
1
0
Page 10 of 22

butyraldehyde, valeraldehyde, benzaldehyde, and furaldehyde were purchased from
Sigma; xylitol from Aldrich; urea from KMF Laborchemie Handels GmbH;
Benzaldehyde lyase from Pseudomonas fluorescens was cloned in E. coli,
overexpressed, and produced by fermentation. After fermentation and purification,
BAL was lyophilized and stored at -20°C until use.
4.2.- DES’s synthesis. In a 20 mL vial the quaternary ammonium salt choline chloride
and the hydrogen bond donor were mixed together in a ratio of 1:2 (mol:mol) for
glycerol and urea and 1:1 (mol:mol) for xylitol respectively and heated up at the
temperature of 80°C until a clear and homogenous liquid was formed (time within a
range of 15- 60 minutes).
4
.3.- Standard biocatalytic protocol in DES-phosphate buffer mixtures. The phosphate
buffer (pH 8) was prepared by dissolving 7.49 g of K HPO with 0.95 g of KH PO
and 300 mg of MgSO in 1 L water. The pH was adjusted to 8.00 with phosphoric
2
4
2
4
4
acid or 2.0 M potassium hydroxide solution. As example the reaction with 40 vol.%
phosphate buffer is considered. A solution of ThDP was prepared by dissolving 2 mg
of ThDP in 16 mL of phosphate buffer. 2 mg of BAL were dissolved in 400 μL of
phosphate buffer containing ThDP and then this mixture was added to 600 μL of DES
in a G8 vial. The aldehyde (4.5 μL of butyraldehyde, 5.3 μL of valeraldehyde, 5.1 μL
of benzaldehyde or 4.1 μL of 2-furaldehyde) was finally added to the solution and
stirred at room temperature at 300 rpm. After a precise interval of time the reaction
was stopped, 2 mL of water were added and then the mixture was extracted with ethyl
acetate (3 x 2 mL). The organic phases were collected and the ethyl acetate was
evaporated under reduced pressure.
1
4
.4.- Analytics. The product was then analyzed by H-NMR. The enantiomeric
excesses for the 5-hydroxy-4-octanone and 6-hydroxy-5-decanone were determined
1
1
Page 11 of 22

by a GC equipped with the chiral column CP Chirasil Dex (250 μm × 0.250 μm × 25
m) and a flame ionization detector. The following temperature program was used for
the analysis: 50°C (0 min) - 3°C/min - 160°C (10 min). The temperature of the
injector and split ratio were 250°C and 40:1 respectively. The carrier gas was
hydrogen and the flow rate was 2.0 mL/min. The enantiomeric excesses for the
benzoin and furoin were determined by HPLC equipped with a Chiralpak AD-H
column (n-heptane:2-propanol = 90:10, λ = 240 nm), 0.5 mL/min. The results are
reported in Table 31, 32 and 33.
Acknowledgements.
Financial support from DFG training group 1166 “BioNoCo” (“Biocatalysis in
Non-conventional Media”). Mrs. Hannelore Eschmann and Ms. Julia Wurlitzer are
acknowledged for analytical measurements.
5
.- References
[1] a) K. Faber, Biotransformations in Organic Chemistry, Springer Berlin
Heidelberg, 2011 d) U.T. Bornscheuer, G. Huisman, R.J. Kazlauskas, S. Lutz, J.
Moore, K. Robins, Nature 485 (2012) 185-194;
[2] a) A. S. Bommarius, B. R. Riebel, Protein Science Encyclopedia, Wiley-
VCH Verlag GmbH & Co, KGaA Weinheim, 2008, pp. 339–372; b) A. Jakoblinnert,
R. Mladenov, A. Paul, F. Sibilla, U. Schwaneberg, M.B. Ansorge-Schumacher, P.
Domínguez de María, Chem. Commun. 47 (2011) 12230-12232.
1
2
Page 12 of 22

[3] a) P. Domínguez de María, Ionic Liquids in biotransformations and
organocatalysis: Solvents and beyond, John Wiley & Sons, Hoboken, 2012; b) P.
Domínguez de María, Angew. Chem. Int. Ed. 47 (2008) 6960-6968;
[4] a) M. Deetlefs, K. R. Seddon, Green Chem. 12 (2010) 17–30; b) A. Romero,
A. Santos, J. Tojo, A. Rodríguez, J. Hazard. Mater. 151 (2008) 268–273; c) S.
Wellens, B. Thijs, K. Binnemans, Green Chem. 15 (2013) 3484-3485.
[5] a) A. P. Abbott, G. Capper, D. L. Davies, R. K. Rasheed, V. Tambyrajah,
Chem. Commun. (2003) 70–71; b) A. P. Abbott, D. Boothby, G. Capper, D. L.
Davies, R. K. Rasheed, J. Am. Chem. Soc. 126 (2004) 9142–9147; c) Y. Fukaya, Y.
Iizuka, K. Sekikawa, H. Ohno, Green Chem. 9 (2007) 1155–1157; e) Z. Maugeri, P.
Domínguez de María, RSC Adv. 2 (2012) 421-425.
[6] a) K. D. Weaver, H. J. Kim, J. Sun, D. R. MacFarlane, G. D. Elliott, Green
Chem. 12 (2010) 507–513;
[7] Reviews on DES: a) Q. Zhang, K. De Oliveira Vigier, S. Royer, F. Jérôme,
Chem. Soc. Rev. 41 (2012) 7108–7146; b) C. Ruß, B. König, Green Chem. 14 (2012)
2
969–2982; c) P. Domínguez de María, Z. Maugeri, Curr. Op. Chem. Biol. 15 (2011)
20-225.
2
[8] a) Z. Maugeri, W. Leitner, P. Domínguez de María, Tetrahedron Lett. 53
(2012) 6968–6971; b) M. Krystof, M. Pérez-Sánchez, P. Domínguez de María,
ChemSusChem (2013) 1–6; c) F. S. Oliveira, A. B. Pereiro, L. P. N. Rebelo, I. M.
Marrucho, Green Chem. 15 (2013) 1326–1330; d) K. Pang, Y. Hou, W. Wu, W. Guo,
W. Peng, K. N. Marsh, Green Chem. 14 (20129 2398–2401; e) A. P. Abbott, P. M.
Cullis, M. J. Gibson, R. C. Harris, E. Raven, Green Chem. 9 (2007) 868–872; f) M.
1
3
Page 13 of 22

Hayyan, F. S. Mjalli, M. A. Hashim, I. M. AlNashef, Fuel Process. Technol. 91
2010) 116–120; g) K. Shahbaz, F. S. Mjalli, M. A. Hashim, I. M. ALNashef, J. Appl.
Sci. 10 (2010) 3349–3354.
(
[9] a) J. T. Gorke, F. Srienc, R. J. Kazlauskas, Chem. Commun. (2008) 1235–
1237; b) D. Lindberg, M. de la Fuente Revenga, M. Widersten, J. Biotechnol. 147
(2010) 169–171; c) H. Zhao, G. A. Baker, S. Holmes, Org. Biomol. Chem. 9 (2011)
1
908–1916; d) H. Zhao, G. A. Baker, S. Holmes, J. Mol. Catal. B-Enzym. 72 (2011)
63–167; e) E. Durand, J. Lecomte, B. Baréa, G. Piombo, E. Dubreucq, P. Villeneuve,
1
Process Biochem. 47 (2012) 2081–2089; f) C. Lehmann, F. Sibilla, Z. Maugeri, W. R.
Streit, P. Domínguez de María, R. Martinez, U. Schwaneberg, Green Chem. 14 (2012)
2719-2726; g) Z. Maugeri, W. Leitner, P. Domínguez de María, Eur. J. Org. Chem.
(2013) 4223–4228.
[10] M.C. Gutiérrez, M.L. Ferrer, L. Yuste, F. Rojo, F. del Monte, Angew.
Chem. Int. Ed. 49 (2010) 2158-2162.
[11] Z. Maugeri, P. Domínguez de María, ChemCatChem. (2014) DOI:
1
3
0.1002/cctc.201400077.
[12] b) M. Pohl, G. A. Sprenger, M. Müller, Curr. Opin. Biotech. 15 (2004)
35–342; c) P. Hoyos, J.-V. Sinisterra, F. Molinari, A. R. Alcántara, P. Domínguez de
María, Accounts Chem. Res. 43 (2010) 288–99; d) M. Müller, D. Gocke, M. Pohl,
FEBS J. 276 (2009) 2894–2904; e) A. Cosp, C. Dresen, M. Pohl, L. Walter, C. Röhr,
M. Müller, Adv. Synth. Catal. 350 (2008) 759–771.
[13] a) P. Domínguez de María, M. Pohl, D. Gocke, H. Gröger, H. Trauthwein,
T. Stillger, L. Walter, M. Müller, Eur. J. Org. Chem. (2007) 2940–2944; b) C. R.
1
4
Page 14 of 22

Müller, M. Pérez-Sánchez, P. Domínguez de María, Org. Biomol. Chem. 11 (2013)
2
000–2004; c) M. Pérez-Sánchez, P. Domínguez de María, ChemCatChem 4 (2012)
17–619; f) S. Shanmuganathan, D. Natalia, A. van den Wittenboer, C. Kohlmann, L.
6
Greiner and P. Domínguez de María, Green Chem. 12 (2010) 2240–2245; (g) P.
Domínguez de María, T. Stillger, M. Pohl, M. Kiesel, A. Liese, H. Gröger and H.
Trauthwein, Adv. Synth. Catal. 350 (2008) 165–173; (i) P. Domínguez de María, T.
Stillger, M. Pohl, S. Wallert, K. H. Drauz, H. Gröger, H. Trauthwein and A. Liese, J.
Mol. Catal. B: Enzym., 38 (2006) 43–47.
1
5
Page 15 of 22

SCHEME AND FIGURE CAPTIONS
Scheme 1. Selected BAL-catalyzed enantioseletive C-C bond formations in DES-
aqueous mixtures.
Figure 1. BAL-catalyzed carboligation of butyraldehyde (BuA), valeraldehyde
(VaA), benzaldehyde (BeA) and 2-furaldehyde (FuA) in different G-DES-buffer
mixtures (10-100 vol.% phosphate buffer). Reaction conditions: aldehyde 50 mM,
-
1
-1
BAL 2 mg·mL , ThDP 0.05 mg·mL , phosphate buffer 50 mM pH 8 with
1
2
.5 mM MgSO
4
, RT, 24 hours. Conversions determined by H-NMR.
Figure 2. BAL-catalyzed carboligation of valeraldehyde in G-DES-phosphate buffer
-
1
mixture 70:30 vol./vol. Reaction conditions: aldehyde 50 mM, BAL 2 mg·mL ,
-
1
4
ThDP 0.05 mg·mL , phosphate buffer 50 mM pH 8 with 2.5 mM MgSO , RT.
1
Conversions determined by H-NMR.
Figure 3. BAL-catalyzed carboligation of valeraldehyde in DES-phosphate buffer
-
1
mixture 70:30 vol./vol. Reaction conditions: aldehyde 50 mM, BAL 2 mg·mL ,
-
1
4
ThDP 0.05 mg·mL , phosphate buffer 50 mM pH 8 with 2.5 mM MgSO , RT, 24 h.
1
Conversions determined by H-NMR.
1
6
Page 16 of 22

Scheme 1.
O
BAL - ThDP, Mg2+
DES/Buffer
2
O
OH
Butyraldehyde
(R)-5-Hydroxy-4-octanone
O
BAL - ThDP, Mg2+
DES/Buffer
2
2
O
OH
Valeraldehyde
(
R)-6-Hydroxy-5-decanone
O
O
BAL - ThDP, Mg2+
DES/Buffer
OH
Benzaldehyde
(R)-Benzoin
O
O
BAL - ThDP, Mg2+
DES/Buffer
O
O
O
2
OH
2
-Furaldehyde
(R)-Furoin
1
7
Page 17 of 22

Figure 1.
BuA
VaA
BeA
FuA
100
80
60
40
20
0
10
30
40
100
Water amount (vol.%)
1
8
Page 18 of 22

Figure 2.
100
8
0
0
0
0
0
6
4
2
24
48
72
Time (h)
1
9
Page 19 of 22

Figure 3.
100
8
0
0
0
0
0
6
4
2
G-DES
U-DES
X-DES
DES
2
0
Page 20 of 22

Table 1.
Quaternary ammonium salt Hydrogen bond donor ChCl:HBD
Name
(HBD)
molar ratio
N+
Cl-
OH
HO
HO
OH
1:2
G-DES
Choline chloride
Glycerol
O
N+
HO
Cl-
1:2
1:1
U-DES
X-DES
H
2
N
NH
2
Choline chloride
Urea
OH
N+
HO
OH
HO
Cl-
OH OH
Choline chloride
Xylitol
Table 1. Selected ChCl-based DESs used in the carboligation reactions
performed with BAL. Proportion of components was selected according to our
previous studies [5e].
2
1
Page 21 of 22

Table 2.
Substrate
Conversion (%)
ee %
52 (R)
27 (R)
ee* %
50 (R)
30 (R)
Butyraldehyde
Valeraldehyde
Benzaldehyde
96
98
95
75
> 99 (R) > 99 (R)
63 (R) 93 (R)
2
-Furaldehyde
Table 2. Observed conversions and enantiomeric excesses (ee) in the BAL-
catalyzed carboligations using G-DES-phosphate buffer mixture 60:40 vol./vol.
1
Conversions determined by H-NMR and ee by GC (see experimental part for
details). ee*: enantiomeric excesses reported in literature, where DMSO or 2-
methyltetrahydrofuran (2-MeTHF) were used as co-solvents [13a,f].
2
2
Page 22 of 22