A Mild Procedure for Enone Preparation Catalysed by Bovine Serum Albumin in a Green and Easily Available Medium

Article abstract of DOI:10.1007/s10562-018-2386-4

Abstract: A simple and mild procedure to obtain α,β-unsaturated ketones from acetone and a set of benzaldehydes is described. The approach applies bovine serum albumin (BSA) catalysis and water or ethanol, this mild reaction medium contrasting with the strong reaction conditions of the classic aldol condensation. Except for the assayed nitrobenzaldehydes, high enone yields (88–97%) were attained. In addition to its mildness, further advantages of this procedure are the use of a green catalyst exhibiting an efficient reuse and the use of eco-friendly and cheap solvents. In order to gain a deeper understanding of the involved catalytic mechanism, computational experiments on BSA structural analysis and molecular docking were carried out.

Full text of DOI:10.1007/s10562-018-2386-4

Catalysis Letters
https://doi.org/10.1007/s10562-018-2386-4
A Mild Procedure for Enone Preparation Catalysed by Bovine Serum
Albumin in a Green and Easily Available Medium
Sebastián M. Ardanaz1 · Ana J. Velez Rueda2 · Gustavo Parisi2 · Adolfo M. Iribarren1 · Luis E. Iglesias1
Received: 27 February 2018 / Accepted: 1 April 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A simple and mild procedure to obtain α,β-unsaturated ketones from acetone and a set of benzaldehydes is described. The
approach applies bovine serum albumin (BSA) catalysis and water or ethanol, this mild reaction medium contrasting with
the strong reaction conditions of the classic aldol condensation. Except for the assayed nitrobenzaldehydes, high enone
yields (88–97%) were attained. In addition to its mildness, further advantages of this procedure are the use of a green catalyst
exhibiting an efficient reuse and the use of eco-friendly and cheap solvents. In order to gain a deeper understanding of the
involved catalytic mechanism, computational experiments on BSA structural analysis and molecular docking were carried out.
Graphical Abstract
Keywords Bovine serum albumin · Catalytic promiscuity · Cross aldol condensation · Enones
1 Introduction
The cross aldol condensation, also known as
Claisen–Schmidt reaction, is a carbon–carbon bond-
Electronic supplementary material The online version of this
forming reaction conducting to α,β-unsaturated carbonyl
compounds (3a–f), which are formed by dehydration of
the intermediate aldol addition product 2a–f (Scheme 1).
Although it is a well-known transformation, it still receives
attention because of the synthetic versatility of the α,β-
unsaturated carbonyl moiety; for instance, aromatic enones
are precursors of diverse pharmacologically active prod-
ucts [1, 2]. However, conventional aldol condensation
is usually carried out in a strong and hot basic medium,
which affords subproducts such as bis α,β-unsaturated
carbonyl compounds and Michael addition aducts [3, 4].
To circumvent this problem, reported alternatives include
article (https://doi.org/10.1007/s10562-018-2386-4) contains
supplementary material, which is available to authorized users.
* Luis E. Iglesias
leiglesias@unq.edu.ar
1
Laboratory of Biocatalysis and Biotransformation,
Department of Science and Technology, National University
of Quilmes, Roque Sáenz Peña 352, 1876 Bernal,
Provincia De Buenos Aires, Argentina
2
Structural Bioinformatics Group, Department of Science
and Technology, National University of Quilmes, Roque
Sáenz Peña 352, 1876 Bernal, Provincia De Buenos Aires,
Argentina
Vol.:(0123456789)
1 3

S. M. Ardanaz et al.
Scheme 1 BSA-catalysed aldolic reaction of benzaldehydes 1a–f with acetone
microwave irradiation or metal ion catalysis, but many of
them involve hazardous or expensive reagents and solvents
[5].
substitued benzaldehydes, giving the corresponding α,β-
unsaturated carbonylic compounds in good and high yields
[19].
Biocatalytic procedures are currently integrated into
synthetic chemistry because they provide environmentally
friendly and sustainable transformations, carried out under
mild reaction conditions. Focusing on aldol reactions, aldo-
lases usually display high stereoselectivity but limited aldol
donor acceptance [6]. Catalytic promiscuity, the ability of
an enzyme to catalyse reactions mechanistically different
to those involved in vivo [7, 8], has expanded the synthetic
opportunities of biocatalysts. Several hydrolases, mainly
commercially available lipases and proteases, have exhib-
ited promiscuous activity and variable enantioselectivities in
the cross aldol addition [9–12]; recently, an enzyme isolated
from Daucus carota root has also been reported to catalyse
enantioselectively the cross aldol addition of acetone to a set
of benzaldehydes [13]. In relation to the cross aldol conden-
sation, the enzyme 4-oxalocrotonate tautomerase (4-OT) has
displayed promiscuous activity in the aldol addition of ben-
zaldehyde to acetaldehyde and its subsequent dehydration to
cinnamaldehyde; further engineering of 4-OT has conducted
to cinnamaldehyde in 50% yield [14]. Additionally, prepa-
ration of enones using a d-aminoacylase and imidazole as
co-catalyst has also been reported [15].
Taking into account the commented interest on mild,
green and inexpensive procedures to get α,β-unsaturated
carbonyl compounds and considering the above outlined
antecedents, we studied the BSA-catalysed cross aldol con-
densation of a set of benzaldehydes (1a–f, Scheme 1) with
acetone in non-expensive solvents and in this work we report
the obtained results.
2 Materials and Methods
2.1 General
Bovine serum albumin (BSA) was purchased from Sigma-
Aldrich (fraction V) and used straight from the bottle except
for control experiments (Table 1). All reagents and solvents
were of analytical grade and purchased from commercial
suppliers. Benzaldehyde was purified by filtration through
basic aluminium oxide, followed by distillation. Absolute
ethanol was employed and prior to use, acetone was dried
over potassium carbonate and distilled.
In addition to enzymes, non-enzymatic proteins such as
albumin are also able to promote organic reactions even
if they do not possess a true catalytic site. In particular,
commercially available bovine serum albumin (BSA) has
been applied to catalyse different reactions such as the
Morita–Baylis–Hillman reaction [16] and other carbon–car-
bon bond-forming reactions, ketone reduction, sulfide oxi-
dation and it has been defined as a promiscuous biocata-
lyst [17]. In regard to the aldol addition step in cross aldol
reactions, BSA exhibited aldolase activity with moderate
enantioselectivity in the addition of acetone to 6-methoxy-
2-naphtaldehyde in neutral aqueous solution; benzaldehyde
and other substitued benzaldehydes were less efficiently
recognised by the biocatalyst [18]. On the other hand, in
the ionic liquid 1-butyl-3-methyl imidazolium bromide,
BSA promoted the condensation of acetone with a set of
Table 1 Control experiments for BSA-catalysed benzalacetone (3a)
formation
Entry
Biocatalyst
Conversiona
1
2
3
–
BSA
0
54
1
BSA pretreated with urea at
100 °Cb
Reactions were carried out in acetone/water 1:1 at 30 °C and a
BSA/1a (mass ratio)=5.2 (See Sect. 2)
aDetermined by HPLC at 120 h
bPrior to the assay, BSA (100 mg) was stirred in 8 M aqueous urea
(2 ml) for 24 h at 100 °C, then evaporated under reduced pressure. To
remove urea, the resulting residue was washed with acetone/water 1:1
(3×5 ml) and dried
1 3

A Mild Procedure for Enone Preparation Catalysed by Bovine Serum Albumin in a Green and Easily…
Table 2 Cosolvent content effect in BSA-catalysed benzalacetone
Reactions were monitored by thin-layer chromatography
with Silicagel 60 F254 plates (Merck); plates were developed
using hexane/ethyl acetate 70:30 v/v and visualised with UV
light. Flash column chromatography was performed employ-
ing 0.040–0.063 mm Silicagel 60 (Merck).
(3a) formation
Entry
Acetone:cosolvent ratio
Conver-
siona (%)
1
2
3
4
5
6
7
8
Acetone
21
33
37
40
51
31
34
39
41
44
56
59
54
10
HPLC analyses were carried out using a C18 column
(length: 150 mm; internal diameter: 4.6 mm; particle size:
5 μm). Analysis of aliquots from biotransformations of
1a–c,e,f was done applying a 4 min methanol/water gradi-
ent from 60:40 v/v (2 min) to 75:25 v/v (1 min) at a flow rate
of 0.9 ml min−1 and detection at 274 nm. Retention times
were: 1a: 3.9 min; 3a: 5.6 min; 1b: 4.6 min; 3b: 6.3 min; 1c:
7.1 min; 3c: 9.1 min; 1e: 3.8 min; 2e: 2.9 min; 3e: 4.9 min;
1f: 3.6 min; 2f: 3.0 min; 3f: 5.1 min: Samples involving 1d
were analysed by a 3 min gradient of a mixture of the same
solvents from 60:40 v/v (2 min) to 70:30 v/v (1 min) at the
same flow rate and detection at 254 nm. Retention times
were: 1d: 2.9 min; 2d: 2.4 min; 3d: 3.7 min.
Acetone/ethanol 90:10
Acetone/ethanol 85:15
Acetone/ethanol 75:25
Acetone/ethanol 50:50
Acetone/ethanol 25: 75
Acetone/ethanol 15:85
Acetone/ethanol 5:95
Acetone/water 95:5
Acetone/water 90:10
Acetone/water 80:20
Acetone/water 75:25
Acetone/water 50:50
Acetone/water 25:75
9
10
11
12
13
14
Aldols 2d–f were analysed by GC using a chiral col-
umn CP-Chirasil-Dex (25 m × 0.32 mm × 0.25 µm). Oven
temperature was set at 100 °C (1 min), increased to 185 °C
(5 min) and maintained at the final temperature for 25 min.
1H-NMR and 13C-NMR spectra were recorded on a
Bruker Avance II spectrometer at 500 and 125 MHz, respec-
tively; chemical shifts (δ) are reported in ppm from TMS
and CDCl3 as solvent. The following abbreviations were
used to indicate the multiplicity: s, singlet; d, doublet; t,
triplet; m, multiplet; br, broad signal.
Reactions were carried out at 30 °C and at a (BSA/1a) mass
ratio=5.2. (See Sect. 2)
aDetermined by HPLC at 120 h
v/v. The mixtures were shaken at 45 °C and 200 rpm for
the below reported time and then, BSA was filtered off and
washed with acetone (5 ml). The filtrates were concentrated
to remove acetone under reduced pressure and extracted
with ethyl acetate (4×5 ml), the organic phases were dried
over sodium carbonate, filtered and the solvent eliminated
in vacuo. Silicagel flash column chromatography of the
resulting residue, using the below indicated elution solvents,
afforded products 2d–f and 3a–f.
2.2 BSA‑Catalyzed Aldol Condensation
of Compounds 1a–f
2.2.1 General Procedure for BSA‑Catalysed Aldol
Condensation in Analytical Scale
2.2.2.1 4‑(1‑Hydroxy‑3‑oxobutyl)benzonitrile
(2d) Reac-
tion time: 72 h; elution solvent hexane/ethyl acetate 80:20
v/v, yield: 54%; Rf = 0.39 (hexane/ethyl acetate 70:30). 1H
NMR: δ 7.63 (d, J=8.3 Hz, 2H, Ar–H), 7.48 (d, J=8.3 Hz,
2H, Ar–H), 5.21 (t, J=6.2 Hz, 1H, H1), 3.68 (br, 1H, OH),
2.84 (d, J=6.2 Hz, 2H, H2), 2.21 (s, 3H, CH3). 13C NMR: δ
208.50 (CO), 148.14, 132.35, 126.35 (Ar-C), 118.73 (CN),
111.29 (Ar–C), 69.05(C4), 51.56 (C3), 30.74 (CH3).
Experiments reported in Tables 1, 2, 3 and 4 involved a solu-
tion of the aldehyde (1a–f, 0.05 mmol; for instance, 5 μl
of 1a), the indicated solvent or mixture of solvents (1 ml)
and BSA at the indicated BSA/1a–f mass ratio (for instance,
54 mg for BSA/1a = 10.4). The resulting mixtures were
shaken at 200 rpm and at the indicated temperature; aliquots
from the biotransformations were withdrawn at different
times and after centrifugation, analysed by TLC and HPLC.
2.2.2.2 4‑Hydroxy‑4‑(4′‑nitrophenyl)‑2‑butanone(2e) Reac-
tion time: 48 h; elution solvent hexane:ethyl acetate 80:20
v/v, yield: 41%; Rf = 0.17 (hexane/ethyl acetate 70:30). 1H
NMR: δ 8.18 (d, J=8.7 Hz, 2H, Ar–H), 7.54 (d, J=8.7 Hz,
2H, Ar–H), 5.26 (t, J=6.3 Hz, 1H, H4), 3,63 (br, 1H, OH),
2.87 (d, J=6.1 Hz, 2H, H2), 2.22 (s, 3H, CH3). 13C NMR δ
208.53 (CO), 150.15, 147.27, 126.45, 123.74 (Ar–C), 68.90
(C4), 51.53 (C3), 30.72 (CH3).
2.2.2 BSA‑Catalysed Preparation of Products 2d–f and
3a–f
Following the above reported analytical procedure, BSA-
catalysed preparative biotransformations were conducted
by adding the biocatalyst (540 mg) to a solution of the
corresponding aldehyde 1a–f (0.5 mmol) in acetone/water
1:1 v/v (10 ml). To obtain product 2d from 1d, acetone/
ethanol 1:1 v/v was employed instead of acetone/water 1:1
2.2.2.3 4‑Hydroxy‑4‑(3′‑nitrophenyl)‑2‑butanone (2f) Reac-
tion time: 96 h; elution solvent hexane:ethyl acetate 80:20
1 3

S. M. Ardanaz et al.
Conversion (%)a
Table 3 Effect of temperature
and BSA mass on BSA-
catalysed benzalacetone (3a)
formation
Entry
Solvent
(BSA/1a)b
T (°C)
24 h
48 h
120 h
1
2
3
4
5
6
7
8
9
Acetone/ethanol 1:1
Acetone/ethanol 1:1
Acetone/water 1:1
Acetone/water 1:1
Acetone/water 1:1
Acetone/water 1:1
Acetone/water 1:1
Acetone/water 1:1
Acetone/water 1:1
5.2
5.2
5.2
5.2
10.4
10.4c
10.4d
10.4e
15.6
30
45
30
45
45
45
45
45
45
15
22
15
46
74
ndf
nd
nd
82
27
47
34
80
96
65
66
2
51
58
54
85
97
nd
nd
nd
98
97
aDetermined by HPLC
bBiocatalyst/substrate mass ratio
cBSA pretreated with urea at 30 °C (as described in Table 1)
dBSA pretreated with H2O at 100 °C
eBSA pretreated with urea at 100 °C
fnd not determined
Table 4 BSA-catalysed aldol
reaction of benzaldehydes 1a–f
with acetone
Entry
Substrate
T (°C)
t (h)
Conversion to
Conversion to
Enone
Cetol
enone 3a–f (%)a
ketol 2a–f (%)a
yield (%)b yield
(%)b
1
2
3
4
5
6
7
8
9
1a
1b
1c
45
45
45
45
60
45
45
60
45
45
48
120
72
72
96
96
48
72
168
96
96
96
88
33
63
97
24
32
35
37
–
–
–
66
37
–
47
67
53
61
93
87
65
25
53
84
18
24
26
34
–
–
–
54
25
–
41
58
45
44
1d
1d
1dc
1e
1e
1ec
1f
10
Reactions were carried out in acetone/water 1:1 at a (BSA /1a–f) mass ratio=10.4 (See Sect. 2)
aDetermined by HPLC
bAfter silicagel column chromatography
cIn acetone:ethanol 1:1
v/v, yield: 44%; Rf = 0.17 (hexane/ethyl acetate 70:30). 1H
NMR δ 8.24 (m, 1H, Ar–H), 8.13 (ddd, J=8.2 Hz, 2.3 Hz,
1.0 Hz, 1H, Ar-H), 7.72 (m, 1H, Ar–H), 7.53 (t, J=7.9 Hz,
1H, Ar–H), 5.27 (t, J=6.2 Hz, 1H, H4), 2.90 (d, J=6.2 Hz,
2H, H3), 2.24 (s, 3H, CH3). 13C NMR δ 208.63 (CO),
148.36, 144.96, 131.86, 129.51, 122.55, 120.72 (Ar–C),
68.77 (C4), 51.56 (C3), 30.72 (CH3).
200.22 (CO), 146.97 (C4), 135.88, 129.46, 129.02, 128.06
(Ar–C), 126.52 (C3), 26.78 (CH3).
2 . 2 . 2 . 5 (E)‑4‑(4′‑Methoxyphenyl)‑3‑buten‑2‑one
(3b) Reaction time: 120 h; elution solvent dichlorometh-
ane, yield: 87%; Rf = 0.50 (hexane/ethyl acetate 70:30).
1H NMR: δ 7.51 (m, 3H, 2 Ar-H, H4), 6.94 (d, J = 8.8 Hz,
2H, Ar–H), 6.63 (d, J = 16.2 Hz, 1H, H3), 3.87 (s, 3H,
OCH3), 2.38 (s, 3H, CH3). 13C NMR: δ 198.41 (CO),
161.61 (Ar–C), 143.25 (C4), 129.96, 127.06 (Ar–C),
125.03 (C3), 114.44 (Ar-C), 55.41 (OCH3), 27.41 (CH3).
2.2.2.4 (E)‑4‑Phenyl‑3‑buten‑2‑one (3a) Reaction time:
48 h; elution solvent hexane:ethyl acetate 97:3 v/v, yield:
1
93%; Rf = 0.57 (hexane/ethyl acetate 70:30). H NMR
δ 7.56 (m, 3H, 2 Ar-H, H4), 7.42 (m, 3H, Ar–H), 6.75
(d, J = 16.3 Hz, 1H, H3), 2.41 (s, 3H, CH3). 13C NMR δ
1 3

A Mild Procedure for Enone Preparation Catalysed by Bovine Serum Albumin in a Green and Easily…
2.2.2.6 (E)‑4‑(4′‑Chlorophenyl)‑3‑buten‑2‑one (3c) Reac-
tion time: 72 h; elution solvent hexane:ethyl acetate 95:5
v/v, yield: 65%; Rf = 0.57 (hexane/ethyl acetate 70:30). 1H
NMR: δ 7.47 (m, 3H, 2 Ar–H, H4), 7.37 (d, J=8.5 Hz, 2H,
filtrate analysed by HPLC. BSA was washed with acetone
(3 × 5 ml), dried and reused in subsequent reaction turns
using a similar protocol. In the second assay, BSA (540 mg)
was added to a solution of p-formylbenzonitrile (1d,
0.5 mmol) in acetone/ethanol 1:1 v/v (10 ml) and shaked at
45 °C and 200 rpm for 72 h. To recover BSA for reuse, the
same procedure above described was applied.
Ar–H), 6.69 (d, J=16.3 Hz, 1H, H3), 2.38 (s, 3H, CH3). 13
C
NMR: δ 198.06 (CO), 141.86 (C4), 136.43, 132.92, 129.39,
129.26 (Ar–C), 127.48 (C3), 27.69 (CH3).
2.2.2.7 4‑[(1E)‑3‑Oxo‑1‑buten‑1‑yl]benzonitrile (3d) Reac-
tion time: 72 h; elution solvent hexane:ethyl acetate 80:20
v/v, yield: 84%; Rf = 0.50 (hexane/ethyl acetate 70:30). 1H
NMR: δ 7.71 (d, J=8.5 Hz, 2H, Ar–H), 7.64 (d, J=8.2 Hz,
2H, Ar–H), 7.50 (d, J=16.3 Hz, 1H, H1), 6.80 (d, J=16.3,
1H, H2), 2.42 (s, 3H, CH3). 13C NMR: δ 197.62 (CO),
140.63, 138.82, 132.68, 129.79, 128.56 (Ar–C, C3, C4),
118.31 (CN), 113.54 (Ar–C), 27.98 (CH3).
2.5 Computational Experiments on BSA Structural
Analysis and Molecular Docking
The protein data bank (PDB) structure of bovine serum albu-
min (ID:40r0) complexed with naproxen was taken as refer-
ence for the bioinformatic study. The numbers of cavities,
as well as their properties, were estimated using Fpocket
[20]. The pKa values of the protein ionizable residues were
estimated using PROPKA [21] and linked to the cavity resi-
dues using our own scripts written in Python. The molecular
docking was performed at the assayed pH using Autodock
Vina software [22]. The conformations of the ligand–protein
complexes obtained with Autodock Vina software were then
used to estimate interatomic contacts using RING (Residue
Interaction Network Generator, [23]).
2.2.2.8 (E)‑4‑(4′‑Nitrophenyl)‑3‑buten‑2‑one (3e) Reaction
time: 72 h; elution solvent hexane:ethyl acetate 80:20 v/v,
yield: 18%; Rf = 0.50 (hexane/ethyl acetate 70:30). 1H NMR
δ 8.26 (d, J=8.8 Hz, 2H, Ar–H), 7.71 (d, J=8.6 HZ, 2H,
Ar–H), 7.55 (d, J=16.3 Hz, 1H, H4), 6.83 (d, J=16.3, 1H,
H3), 2.43 (s, 3H, CH3).13C NMR: δ 197.53 (CO), 148.58,
140.67, 140.06 (Ar–C, C4), 130.38 (C3), 128.81, 124.20
(Ar–C), 28.05 (CH3).
3 Results and Discussion
2.2.2.9 (E)‑4‑(3′‑Nitrophenyl)‑3‑buten‑2‑one
(3f) Reac-
tion time: 96 h; elution solvent hexane:ethyl acetate 80:20
v/v, yield: 34%; Rf = 0.42 (hexane/ethyl acetate 70:30). 1H
NMR δ 8.41 (t, J=1.9 Hz, 1H, Ar-H), 8.25 (ddd, J=8.2 Hz,
2.2 Hz, 1.0 Hz, 1H, Ar–H), 7.87 (d, J=7.7 Hz, 1H, Ar–H),
7.62 (t, J=8.0 Hz, 1H, Ar–H), 7.56 (d, J=16.3 Hz, 1H, H4),
6.85 (d, J=16.3 Hz, 1H, H3), 2.43 (s, 3H, CH3). 13C NMR δ
197.58 (CO), 148.72 (Ar–C), 140.18 (C4), 136.28, 133.77,
130.06 (Ar–C), 129.39 (C3), 124.69, 122.60 (Ar-C), 28.05
(CH3).
First studies were carried out using benzaldehyde (1a,
Scheme 1) as model substrate and involved a screening of
several experimental parameters. In order to assess results
properly, control experiments without BSA and with previ-
ously treated BSA were carried out (Table 1). In absence of
BSA (Entry 1) no benzalacetone formation was observed,
ruling out a non-catalysed aldol condensation; compared
with untreated BSA (Entry 2), BSA pretreated with urea
at 100 °C (Entry 3) rendered BSA inactive. These control
experiments confirm then that BSA promotes benzalacetone
formation and indicate that the native form of the protein
displays the highest catalytic activity.
2.3 BSA‑Catalyzed Dehydration of 2e
BSA (54 mg) was added to a solution of 4-hydroxy-4-(4′-
nitrophenyl)-2-butanone (2e, 0.05 mmol) in acetone/water
1:1 v/v (1 ml) and shaken at 45 °C and 200 rpm for 48 h.
Aliquots from the biotransformation were withdrawn at dif-
ferent times and after centrifugation, analysed by HPLC.
The solvent is a key parameter in a biotransformation and,
as above stated, a green and cheap reaction medium such
as ethanol or water (Table 2) is important when planning
a reaction; thus, the solvent effect on benzalacetone (3a)
production was first studied. It can be seen from Table 2
that ethanol improves the conversion to benzalacetone, the
highest value (51%) being attained at 50% ethanol (Entry
5). Results also show that addition of water to acetone also
increased appreciably enone conversion except for the high-
est tested water proportion (Entry 14). It is to point out that
all the assayed (co)solvent/acetone ratios involved high
molar acetone/benzaldehyde (A/B) ratios favoring the for-
mation of benzalacetone (A/B = 278 at 100% acetone and
A/B=69 at 75% (co)solvent). Moreover, although water is
2.4 Assays of BSA Reusability
To study the recyclability of BSA, experiments involving
two substrates were carried out. In the first assay, BSA
(540 mg) was added to a solution of benzaldehyde (1a,
0.5 mmol) in acetone/water 1:1 v/v (10 ml) and shaked
at 45 °C and 200 rpm for 48 h. The mixture was then fil-
tered off to recover the biocatalyst and an aliquot from the
1 3

S. M. Ardanaz et al.
Table 5 BSA-mediated
dehydration of ketol 2e
formed in the dehydration step yielding the enone, decrease
in conversion to benzalacetone (10%) at 75% water [water/
benzaldehyde (W/B) molar ratio = 850] should not to be
explained by equilibrium inhibition due to high water pro-
portion, since the highest conversions (54, 56 and 59%) were
also attained at high water proportions (20, 25 and 50%
water, W/B = 227–567). Furthermore, benzaldehyde was
soluble in all assayed water/acetone ratios. Consequently, the
effect of the water content on conversion to benzalacetone
should not be related to solubility or stoichiometric reasons
but to the impact of water on BSA catalytic activity.
The optimal water contents for maximum product con-
version from a given substrate and a given transformation
is usually very dependent on the solvent [10, 24]; this has
also been reported for biotransformations involving BSA.
In the Henry reaction, for instance, high and quantitative
conversions to the addition product were reached with
nitromethane-water mixtures containing 5–98% water, but
no conversion took place in anhydrous nitromethane [25].
In a BSA-catalysed tandem Michael addition and intramo-
lecular cyclization in ethanol [26], high product yields were
obtained only at 0, 10 and 20% water, while higher water
contents afforded lower yields. In contrast, in a related BSA-
catalysed one-pot cyclization, high product yield could only
be attained in ethanol/water 3:7, while no conversion was
observed in absolute ethanol [27].
Entry Biocatalyst Conver-
sion to 3e
(%)a
24 h 48 h
1
2
–
5
13
3
18
BSAb
aDetermined by HPLC
bReaction was carried out at
45 °C in acetone/water 1:1 and
at a (BSA/1a) mass ratio=10.4
(see Sect. 2)
Fig. 1 Reciclability of BSA in aldol condensation of 1a and 1d.
Reaction runs for 1a were carried out in acetone/water 1:1 at 45 °C
and at a BSA/1a mass ratio=10.4 for 48 h (see Sect. 2); for 1d, in
acetone/ethanol 1:1 at 45 °C and at a BSA/1d mass ratio=10.4 for
72 h. Conversion to enone was determined by HPLC
In our case, since similar conversions were reached at 20,
25 and 50% water (Table 2), the latter was chosen in order to
minimise acetone use (Entry 13), according to the trend in
chemo- and chemoenzymatic processes to replace organic
solvents by economical and environmentally friendly water
[28, 29]. Further experiments were then conducted at ace-
tone–water 1:1.
Scale-up of the biotransformations allowed isolation of
the products and their spectroscopic characterization (see
Sect. 2) and showed the applicability of this methodology
on a preparative scale.
Regarding the effect of temperature, (Table 3) its impact
on benzalacetone production was more marked in the reac-
tions carried out in acetone/water than in acetone/ethanol; in
the former medium, a conversion to benzalacetone over 80%
could be reached (Entry 4). The influence of BSA mass was
also tested: by a two- and threefold increase of the biocata-
lyst mass, almost quantitative conversions of benzaldehyde
to benzalacetone were obtained (Entries 5 and 6).
Additionally, in order to know if the dehydration step of
the aldol condensation is promoted by BSA, we decided to
do some experiments with the ketol 2e (Table 5). Although
BSA activity in some β-elimination reactions has been
reported [30, 31], to the best of our knowledge, no previous
work has reported experiments studying the role of BSA in
the dehydration of β-hydroxycarbonyl compounds. Results
in Table 5 suggest that 2e dehydration giving enone 3e is
promoted by BSA, ruling out a non-catalysed process. It can
also be mentioned that chiral gas chromatography analyses
of ketols 2d–f indicated that these compounds were obtained
as racemic mixtures.
Aiming at widening the substrate scope of the biotransfor-
mation, a set of substituted benzaldehydes (1b–f, Scheme 1,
Table 4) was tested under the most satisfactory experimental
conditions found during the previously discussed screen-
ing (1a, Entry 1). High conversions in the range 88–97%
were obtained for three of the assayed substrates (1a–c);
the replacement of acetone/water 1:1 by acetone/ethanol
1:1 as the reaction medium changed drastically the product
mixture profile of 1d and allowed high conversions of the
enone 3d. In contrast, nitrobenzaldehydes 1e and 1f afforded
mixtures of the corresponding intermediate ketol and the
enone (Entries 7–10).
Finally, the reciclability of the biocatalyst was also con-
sidered (Fig. 1), studying the reuse of BSA in the aldol con-
densation of benzaldehyde (1a) and p-cyanobenzaldehyde
(1d) (see Sect. 2). BSA showed good potential for reciclabil-
ity since in both reactions no significant decrease in conver-
sion to products was observed even after ten consecutive
reaction runs.
1 3

A Mild Procedure for Enone Preparation Catalysed by Bovine Serum Albumin in a Green and Easily…
Focusing now on the catalytic mechanism, the mode of
action of BSA is generally assigned to Lys-222, a residue
which has an abnormal low pKa and is buried in a pocket,
the hydrophobic binding site IIA [17, 32]. The aldol addi-
tion has been reported to be accelerated by BSA through an
enzyme-like mechanism and inhibited by warfarin, a ligand
of the site IIA; moreover, the catalytic activity is conserved
in a polypeptide derived from the site IIA sequence. These
results enforce the evidence that the observed catalysis is
not due to impurities in commercial BSA preparations [18].
In order to further study the structural characteristics that
would make possible the catalysis and to explore the struc-
tural environment of the binding site IIA, we performed
some computational experiments. For that purpose, an
open structure of bovine serum albumin (PDB ID:40r0) was
taken as reference and using the Fpocket software [33], we
explored the biochemical characteristics of albumin pockets.
We found a major cavity (having a volume of 2849.9 Å3)
showing also the highest polarity and containing an abnor-
mally acidic lysine residue (Lys 221 according to our PDB
numeration; pKa = 7.7) and an abnormally acid histidine
(His 241 according to our PDB numeration, pKa= 1.37).
To the best of our knowledge, no previous works reported
the existence of this histidine residue that could be involved
in the catalysis, since shifts in acid-base behavior have been
observed in residues contributing to the catalytic mecha-
nisms of several enzymes [34]. The mentioned major cav-
ity merges with a smaller one having a volume of 1127.1
Å3, showing the highest hydrophobicity score according to
Fpocket and connected to the protein surface through a large
tunnel, possibly allowing the transit of substrates between
the protein surface and the cavities. Taking into account pre-
vious work on BSA catalytic promiscuity, the results herein
reported in BSA-catalysed aldol condensation of the series
of benzaldehydes 1a–f and the BSA structural characteristics
above mentioned, we performed molecular docking explora-
tions focusing on these cavities. Assayed substrates showed
an average of 28 interatomic and Van der Waals contacts
with the cavities residues Arg 256, Arg 217, Lys 221, Glu
292 and Arg 199 (see Fig. 2 and Supplementary Material);
interestingly, only nitrobenzaldehydes 1e and 1f showed
absence of contacts with the cavities residues.
Fig. 2 Benzaldehyde (1a; shown in blue) bound to the major cavity
of BSA binding site IIA. Cavity residues including Arg 256, Arg 217,
Glu 292 and Arg 199 are shown in yellow; abnormally acidic Lys 221
(pKa=7.7) and abnormally acid His 241 (pKa=1.37), in red
and hot basic media), exhibiting potential for molecules car-
rying base labile substituents. This method avoids also the
use of hazardous or expensive reagents and solvents reported
in alternative strategies for the aldol condensation. The
potential benefits of an efficient biocatalyst reuse are also
remarkable.
Acknowledgements The authors acknowledge UNQ and ANPCyT,
Argentina (PICT 2013-0232, LEI) for financial support. GP, AMI
and LEI are members of the Scientific Research Career of CONICET
(Argentina); SMI is research fellow of CONICET and AJVL, research
fellow of ANPCyT.
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