In situ aldehyde generation for aldol addition reactions catalyzed by d-fructose-6-phosphate aldolase

Article abstract of DOI:10.1016/j.molcatb.2012.02.001

In situ coupling of aldehyde generation, by a mild alcohol oxidation, with an enzymatic aldol addition reaction, mediated by d-fructose-6-phosphate aldolase (FSA) has been investigated as an approach to improve the performance of the process. Four sustainable oxidation methods compatible with the activity and stability of the enzymatic aldol addition have been assayed. Among them, the laccase/O₂/2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and alcohol oxidase gave the best results for the N-Cbz-aminoethanol to N-Cbz-glycinal (53%) and furfuryl alcohol to furfural (89%), respectively, followed by the aldol addition with hydroxyacetone catalyzed by FSA A129S mutant.

Full text of DOI:10.1016/j.molcatb.2012.02.001

Journal of Molecular Catalysis B: Enzymatic 84 (2012) 102–107
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
In situ aldehyde generation for aldol addition reactions catalyzed by
d-fructose-6-phosphate aldolase
Maria Mifsud∗, Anna Szekrényi, Jesús Joglar, Pere Clapés
Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Catalun˜a, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), Jordi Girona 18-26, 08034
Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 17 February 2012
In situ coupling of aldehyde generation, by a mild alcohol oxidation, with an enzymatic aldol addi-
tion reaction, mediated by d-fructose-6-phosphate aldolase (FSA) has been investigated as an approach
to improve the performance of the process. Four sustainable oxidation methods compatible with
the activity and stability of the enzymatic aldol addition have been assayed. Among them, the
laccase/O2/2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and alcohol oxidase gave the best results for
the N-Cbz-aminoethanol to N-Cbz-glycinal (53%) and furfuryl alcohol to furfural (89%), respectively,
followed by the aldol addition with hydroxyacetone catalyzed by FSA A129S mutant.
Keywords:
One-pot two-step oxidation/aldol reaction
Biocatalytic oxidation
Laccase/O2/TEMPO
Alcohol oxidase
d-Fructose-6-phosphate aldolase
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
(bio)/catalytic methods [5] employing oxygen or hydrogen perox-
ide as highly attractive oxidants in terms of minimum energy use
The aldol addition reaction is one of the most powerful meth-
ods of forming carbon–carbon bonds [1]. Biocatalysis by means of
aldolases offers a unique stereoselective and green tool to perform
this transformation [2,3].
and waste generation. In this sense, tremendous advances in tran-
sition metal catalysis [6–9], and organocatalysis [10,11] applied to
oxidation reactions have been achieved.
Moreover, biocatalysis is emerging as an additional pillar for
environmentally benign oxidation catalysis in water [12,13]. Thus,
biocatalytic approaches such as laccase/O2/mediator system (LMS),
alcohol oxidase (AO) and chloroperoxidase (CPO) are a priori good
candidates.
Examples for the oxidase/aldolase coupling reactions are
described in the literature [14–16]. In 1994 Fessner et al. devel-
oped an approach for the preparation of complex carbohydrates
by enzymatic aldolization in which both the aldol donor and
the acceptor components were generated in situ by air oxidation
using microbial oxidases [14]. More recently, Siebum et al. briefly
discussed the oxidation of 4-pentenol by alcohol oxidase (AO)
followed by the condensation reaction catalyzed by 2-deoxyribose-
5-phosphate aldolase (DERA) [16]. Similarly Wong and co-workers
integrated the galactose oxidase (GO) catalyzed oxidation of glyc-
erol to l-glyceraldehyde coupled with an aldolase reaction to
produce l-fructose [15]. Their main drawback is that the oxidases
usually lack of a broad substrate selectivity, therefore limiting their
synthetic applications. Moreover, in some instances the need of
metal cofactors used by the oxidases and class II aldolases such
as l-rhamnulose-1-phosphate (RhuA), may be incompatible. Thus,
alternatives must be provided to make the oxidation and aldol addi-
tion compatible to in situ transformations.
The aldol addition reaction involves an acceptor aldehyde that
must be stable throughout the reaction as well as during its prepa-
ration, purification and manipulation conditions. Aldehydes are
strong electrophiles that can undergo a number of side reactions
with other nucleophiles present in the medium, including self-
polymerization reactions and oxidation to acids. Moreover, they
can deactivate or inhibit the aldolase either by non-selective bind-
ing or by irreversible Schiff base formation with the amino group
of the catalytic lysine residue in Class I aldolases. To overcome
this problem an in situ aldehyde generation by the corresponding
alcohol oxidation through a sequential reaction under conditions
compatible with the enzymatic aldol addition would be ideal
(Fig. 1). The combination of both concepts, biocatalysis and multi-
step sequential, will have an economic and environmental benefit
in the chemical production.
Oxidation is a central reaction in organic chemistry [4]. Sev-
eral oxidizing reagents including permanganate and dichromate
have been traditionally employed to accomplish this transfor-
mation. However, many methodologies use reaction conditions
that are incompatible with the enzymatic activity and stability.
Recently, much attention has been focused on the development of
In this work, we endeavored to study the coupling of an in situ
aldehyde generation by a green oxidation methodology with an
enzymatic aldol addition reaction. We proposed to investigate
∗
Corresponding author. Tel.: +34 93 4006112; fax: +34 93 2045904.
E-mail address: mmgqbm@cid.csic.es (M. Mifsud).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.02.001

M. Mifsud et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 102–107
103
2.2. HPLC analyses
HPLC analyses were performed with a XBridgeTM C18, 5 mm,
4.6 mm × 250 mm column from Waters (Massachusetts, USA). The
solvent system used was solvent A (H2O in the presence of 0.1%
(v/v) trifluoroacetic acid (TFA)) and solvent B (MeCN/H2O 4:1 in the
presence of 0.095% (v/v) TFA), gradient elution from 10 to 100% B in
30 min, flow rate 1 mL/min, detection 215 nm. The amount of prod-
uct was quantified from the peak areas using an external standard
methodology.
Fig. 1. Alcohol oxidation/aldol addition reaction cascade strategy.
2.3. Absorption of laccase onto Celite®
Enzyme (50 mg, 650 U) was dissolved in acetate buffer (1 mL,
50 mM, pH 4.5). To this solution Celite® (1 g) was added, and after
mixing thoroughly the solid was dried under vacuum overnight.
The lyophilized powder was directly use for the oxidation reaction.
four suitable catalysts for the alcohol oxidation, that were ini-
tially assumed compatible with the activity and stability of the
enzymatic aldol addition step and they would only require the
modification of the pH. Moreover, we avoided to use oxidore-
ductases using nicotinamide cofactors. Instead, our choices were
systems that utilize oxygen or simple hydrogen peroxide, which
makes them attractive and competitive for preparative organic
chemistry.
2.4. Preparation of Pd/Au-TiO2
Pd/Au-TiO2 was prepared following the procedure described
in the literature [9]. An aqueous solution of HAuCl4·3H2O (5 mL,
100 mg, 0.25 mmol, 0.05 M) and an aqueous solution of PdCl2 (1 mL,
90 mg, 0.5 mmol, 0.5 M) were simultaneously added to TiO2 (1.9 g).
The paste formed was grown and dried at 80 ◦C for 16 h and calcined
in static air at 400 ◦C. A characterization has been done, including
TEM, elemental analysis, and BET surface area. The metal nanopar-
ticles (2.5 nm average) were identified by transmission electron
microscopy (TEM) technique. The final metal content was deter-
mined by inductive coupled plasma (ICP) and the amount of Pd
and Au was according with the predicted. The Pd/Au-TiO2 catalyst
has a BET area of 54.4 m2/g, which is in agreement with the area
reported by Hutchings et al. [9].
First, the system laccase/O2/2,2,6,6-tetramethylpiperidine-N-
oxyl (TEMPO) as mediator was selected. This has the advantage
that TEMPO, which is regenerated by the laccase, is the oxidant
agent and therefore the methodology is not restricted by the sub-
strate selectivity of the enzyme [17–19]. TEMPO is already known
to efficiently catalyze the oxidation of alcohols to carbonyl prod-
ucts [20,21]. Second, the alcohol oxidase (AO), a flavin-dependent
alcohol oxidizing enzyme in the presence of O2 will be tested as
well [22]. AO oxidizes a range of (aliphatic) primary alcohols to
the corresponding aldehyde with the concomitant production of
H2O2 [23]. Under these conditions, catalase must be added to elim-
inate the hydrogen peroxide formed during the reaction, which is
known to deactivate the enzymes. Third, chloroperoxidase (CPO)
from Caldaromyces fumago can catalyze the oxidation of primary
alcohols to aldehydes by the use of H2O2 as oxidant [24–26].
Peroxidases are generally restricted to oxidation of phenols to
quinones via free radicals intermediates, but CPO is the exception
to the general rule [25,27]. Fourth, a solvent free oxidation cata-
lyst based on Au/Pd-TiO2 [9] in the presence of O2 or H2O2 was
also tested. In terms of green chemistry, heterogeneous catalysis
can enormously contribute to the oxidation reaction performance
in a sustainable way. Solvent free oxidation reaction with hetero-
geneous catalyst could be also a good strategy for the multi-step
reaction.
2.5. One-pot two-step furfuryl alcohol oxidation by
laccase/O2/TEMPO and aldol addition catalyzed by FSA
2.5.1. Oxidation reaction
Reactions were conducted in 15 mL glass pressure vials. Fur-
furyl alcohol (69 ␮L, 0.8 mmol, 160 mM) and TEMPO (11.7 mg,
0.075 mmol, 15 mM) were dissolved in acetate buffer (5 mL, 50 mM,
pH 4.5). This solution was aerated during 5 min. Then, laccase (4 mg,
50 U) or (24 mg, 325 U) depending on the experiment was added.
The reaction was stirred in the presence of O2 (2 atm) at 25 ◦C. Sam-
ples (50 ␮L) were withdrawn at 1, 3, 5, 7, and 24 h, diluted with
MeOH (950 ␮L) and subsequently analyzed by HPLC.
We propose class I d-fructose-6-phosphate aldolase (FSA) from
Escherichia coli as catalyst for the aldol addition step. FSA has
the advantage of accepting unphosphorylated didhydroxyacetone
(DHA) analogues as the donor simplifying the process since there
is no need to handle the phosphoester moiety [28,29].
2.5.2. Aldol addition reaction
To the reaction mixture resulting from the oxidation reaction
(367 ␮L, 0.05 mmol, 100 mM final aldehyde concentration) was
adjusted until pH 7 with Na2CO3. To this solution, HA (133 ␮L of
563 mM solution, 0.075 mmol; final HA concentration 150 mM) and
FSA A129S (2.4 mg lyophilized powder, 1.9 mg protein, 18 U) were
added. Reactions (500 ␮L total volume) were performed using 1 mL
eppendorf tubes stirred with a vortex mixer (800 rpm) at 25 ◦C.
Samples (50 ␮L) were withdrawn at 1, 3, 5 and 7 h, diluted with
MeOH (950 ␮L) and subsequently analyzed by HPLC. The aldol addi-
tion product was identified by HPLC comparing with an authentic
sample characterized in a scale up reaction.
2. Experimental
2.1. Materials
Furfuryl alcohol, N-Cbz-aminoethanol, hydroxyacetone, hydro-
gen peroxide (30%), palladium(II) choride, gold(III) chloride
trihydrate, laccase from Trametes versicolor EC (1.10.3.2), alco-
hol oxidase from Pichia pastoris EC (1.1.3.13), galactose oxidase
from Dactylium dendroides EC (1.1.3.9), and catalase from bovine
liver were purchased from Sigma–Aldrich, CPO from Saccharomices
fumago was purchased from Codexis, TEMPO was purchased from
Evonik. Pd/Au-TiO2 [9], FSA A129S and FSA A129S/A165G were
obtained according to the literature [30].
2.5.2.1. Scale up of the aldol addition of HA to furfural by FSA A129S.
General procedure: Reactions were conducted in 250 mL erlenmeyer
flasks with 20 mL total reaction volume using the following reac-
tant and enzyme concentration: Furfural (164 ␮L, 2 mmol, 100 mM
final concentration), HA (204 ␮L, 3 mmol, 150 mM final concentra-
tion) and FSA A129S (72 mg lyophilized powder, 57.6 mg protein,

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M. Mifsud et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 102–107
541 U) in triethanolamine (TEA) 500 mM pH 7.4 buffer. After 3 h
reaction 50 ␮L sample was diluted with MeOH (950 ␮L) and the
reaction was stopped by adding 20 mL of MeOH. Then, the MeOH
was evaporated under vacuum and the precipitated enzyme fil-
tered off. The filtrate was purified by column chromatography on
AmberliteTM XADTM 16 (Rohm and has) was packed into a glass
column (45 cm × 1.5 cm). The AmberliteTM XADTM 16 was equili-
brated initially with H2O and then the crude material (i.e. filtrate)
was dissolved in water (50–70 mL) and loaded onto the column.
Impurities were washed away with H2O (2–3 column volumes). The
fractions were analyzed by HPLC. Pure fractions were pooled and
lyophilized obtaining the corresponding aldol adduct (4) (192 mg,
1.1 mmol, 58% isolated yield). The product was analized by NMR
and compared with the one reported in the literature [31]. 1H NMR
(400 MHz, D2O) ı = 7.54 (d, J = 0.8, Hz, 1H), 6.48 (dd, J = 4, 10 Hz, 1H),
6.46 (d, 7 Hz, 1H), 5.21 (d, J = 3.7 Hz, 1H), 4.66 (d, J = 3.7 Hz, 1H),
3.96 (m, 1H), 3.46 (m, 1H), 2.30 (s, 3H). 13C NMR (100 MHz, D2O)
ı = 212.3, 153.8, 142.9, 110.5, 107.9, 78.7, 67.8 and 26.1
Fig. 2. Cascade reaction furfuryl alcohol to furfuraldehyde and subsequent aldol
addition reaction with hydroxyacetone.
50 mM, pH 4.5)/AcOEt (1 mL). This solution was aerated during
5 min. Then, laccase (4 mg, 50 U) was added. The reaction was
stirred in the presence of O2 (2 atm) at 25 ◦C. Samples (50 ␮L) were
withdrawn at 1, 3, 5, 7, and 24 h, diluted with MeOH (950 ␮L) and
subsequently analyzed by HPLC.
2.10. One-pot two-step N-Cbz-aminoethanol oxidation by
laccase/O2/TEMPO and aldol addition catalyzed by FSA
2.10.1. Oxidation reaction
2.6. One-pot two-step furfuryl alcohol oxidation by alcohol
oxidase and aldol addition catalyzed by FSA
Reactions were conducted in 50 mL open vials. N-Cbz-
aminoethanol (234 mg, 1.2 mmol, 80 mM), TEMPO (70 mg,
0.45 mmol, 30 mM) or (140 mg, 0.9 mmol, 60 mM) depending
on the experiment and hexadecyltrimethylammonium bromide
(40 mg, 0.11 mmol, 7.3 mM) were dissolved in acetate buffer
(10 mL, 50 mM, pH 4.5) and toluene (5 mL). Then, laccase (12 mg,
150 U) or (75 mg, 975 U) was added. The reaction was continu-
ously aerated by a steel filter stone system and stirred at 25 ◦C.
Toluene (1 mL) was added to the reaction every 30 min to replace
the amount evaporated by the aereation. Samples (50 ␮L) were
withdrawn at 1, 3, 5, 7, and 24 h, diluted with MeOH (950 ␮L) and
subsequently analyzed by HPLC.
2.6.1. Oxidation reaction
Reactions were conducted in 15 mL glass pressure vials. Fur-
furyl alcohol (86 ␮L, 200 mM) was dissolved in acetate buffer (5 mL,
50 mM, pH 7.5). This solution was aerated during 5 min. Then, cata-
lase (2 mg, 7618 U) and alcohol oxidase (21 ␮L, 30 U) were added.
The reaction was stirred in the presence of O2 (2 atm) at 25 ◦C. Sam-
ples (50 ␮L) were withdrawn at 1, 3, 5, 7, and 24 h, diluted with
MeOH (950 ␮L) and subsequently analyzed by HPLC.
2.6.2. Aldol addition reaction
To the reaction mixture resulting from the oxidation reaction
(278 ␮L, 0.05 mmol, 100 mM final aldehyde concentration) was
added HA (150 ␮L of a 500 mM solution), acetate buffer (72 ␮L,
50 mM, pH 7.5) and FSA A129S (2.4 mg lyophilized powder, 1.9 mg
protein, 18 U). Reactions 500 ␮L (total volume) were performed
using 1 mL eppendorf tubes stirred with a vortex mixer at 25 ◦C
Samples (50 ␮L) were withdrawn at 1, 3, 5, and 7 h, diluted with
MeOH (950 ␮L) and subsequently analyzed by HPLC.
2.10.2. Aldol addition reaction
The previous reaction mixture containing the aldehyde (430 ␮L,
0.022 mmol, 45 mM final aldehyde concentration) hydroxyacetone
(HA) (70 ␮L of 500 mM solution, 0.035 mmol; 70 mM final HA con-
centration) and FSA (2.4 mg lyophilized powder, 0.96 mg protein,
2.4 U) were added. Reactions 500 ␮L (total volume) were performed
using 1 mL eppendorf tubes stirred with a vortex mixer at 25 ◦C
Samples (50 ␮L) were withdrawn at 1, 3, 5, and 7 h, diluted with
MeOH (950 ␮L) and subsequently analyzed by HPLC.
2.7. Furfuryl alcohol oxidation reaction by CPO
The aldol adduct was identified by HPLC comparing with an
authentic sample characterized in a previous work [29].
Furfuryl alcohol (2.15 ␮L, 0.025 mmol, 25 mM) and H2O2 (2.8 ␮L,
0.025 mmol, 25 mM) were dissolved in acetate buffer (1 mL, 50 mM,
pH 4.5). To this solution CPO (10 ␮L, 187 U) was added. The reaction
was stirred in the presence of O2 (2 atm) at 25 ◦C. Samples (50 ␮L)
were withdrawn at 1, 3, 5, 7 and 24 h, diluted with MeOH (950 ␮L)
and subsequently analyzed by HPLC.
3. Results and discussion
3.1. Selection of the oxidation methodology
Preparation of furan derivatives by carbon chain extension
through aldol addition is of great interest for the transformation of
biomass-derived molecules into added value compounds [31–33].
The consecutive oxidation/aldol addition reaction of furfuryl alco-
hol to furfuraldehyde and subsequent aldol addition reaction with
hydroxyacetone catalyzed by FSA was first tested as a model pro-
cess (Fig. 2). HA as well as the furfuryl alcohol contain primary
alcohol functions prone to be oxidized, consequently a catalytic oxi-
dation competition between both compounds can be anticipated.
Thus, a one-pot cascade reaction method where all the reagents
are added at the starting point could not be operated. Instead of
that, the method of choice was a one-pot multi-step consecutive
reaction in which the reagents are added at various points.
2.8. Furfuryl alcohol oxidation reaction by Au/Pd-TiO2
Reactions were conducted in 15 mL glass pressure vials. Furfuryl
alcohol (1.1 g, 11.5 mmol) was aerated during 5 min. Then Au/Pd-
TiO2 (20 mg) was added. The reaction was stirred in the presence
of O2 (2 atm) at 25 ◦C. Samples (50 ␮L) were withdrawn at 1, 3, 5, 7
and 24 h, diluted with MeOH (950 ␮L), and subsequently analyzed
by HPLC.
2.9. N-Cbz-aminoethanol oxidation by laccase/O2/TEMPO system
Reactions were conducted in 15 mL glass pressure vials. N-
Cbz-aminoethanol (10 mg, 0.05 mmol, 25 mM) and TEMPO (2.5 mg,
0.016 mmol, 8 mM) were dissolved in acetate buffer (2 mL, 50 mM,
pH 4.5), acetate buffer (1 mL, 50 mM, pH 4.5)/CH2Cl2 (1 mL), acetate
buffer (1 mL, 50 mM, pH 4.5)/hexane (1 mL) or acetate buffer (1 mL,
First, the oxidation step using the four selected methods was
separately evaluated. The oxidation with laccase/O2/TEMPO sys-
tem reached high yields (Table 1, entries 1 and 2). The reactions
were performed at room temperature, buffered at pH 4.5 under O2

M. Mifsud et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 102–107
105
Table 1
oxidation, the aldol addition was started just adding HA and the
FSA A129S. After 6 h of reaction 80% of conversion to aldol adduct
was achieved.
Furfuryl alcohol oxidation with different systems.
Entry
Oxidation system (reaction time, h)
Units
Yield [%]
1
2
3
4
5
Laccase/TEMPO (7)a
Laccase/TEMPO (24)a
AOb
325
50
30
187
–
89
83
89
47
0
3.3. Synthesis of 1,4,5-trideoxy-1,4-imino-d-arabinitol (5-DDAB)
by a chemo-enzymatic one-pot two-step consecutive reactions
CPOc
d
Au/Pd-TiO2
Our ongoing project on the chemo-enzymatic synthesis of
iminocyclitols has led us to apply the concept of oxidation/aldol
addition to their synthesis. The key step in the strategy developed
was the aldol addition reaction of dihydroxyacetone or analogues to
N-carbobenzyloxy-aminoaldehydes (N-Cbz-aminoaldehydes) cat-
alyzed by FSA [29]. Therefore, we first studied the oxidation of
N-Cbz-2-aminoethanol itself being the precursor to the N-Cbz-
glycinal (6). Aldol addition of hydroxyacetone to 6 leads to the aldol
adduct (7), precursor of 1,4,5-trideoxy-1,4-imino-d-arabinitol (5-
DDAB) (8) (Fig. 3) [36,37].
Considering the results obtained thus far for the furfuryl alco-
hol oxidation we selected the most successful methodologies to
prepare the N-Cbz-glycinal (6): laccase/O2/TEMPO and alcohol oxi-
dase approaches. In a preliminary screening of both strategies,
we found that laccase/O2/TEMPO system gave some aldehyde
formation whereas alcohol oxidase was not able to accept the N-
Cbz-2-aminoethanol as substrate.
As starting point for the oxidation of N-Cbz-2-aminoethanol by
laccase/O2/TEMPO the reaction was performed in acetate buffer at
pH 4.5. Under these conditions (Table 2, entry 1) a large amount of
undesired carboxylic acid derivate was obtained which arises from
the over-oxidation of the aldehyde formed during the reaction and
its amount rises on increasing the reaction time and the conversion
of the alcohol.
The oxidation of alcohols to aldehydes catalyzed by
TEMPO/bleach-systems using bromide as cocatalyst, proceeds
commonly in dichloromethaneas organic media [38]. Since the
oxidation agent was in both cases the TEMPO, we hypothesize that
the organic media would prevent the over-oxidation and therefore
an organic-buffer two phase system was envisaged. With this
strategy it was intended to remove the aldehyde from the water
phase preventing the acid formation. Dichloromethane, hexane
and ethyl acetate were the solvents of choice, and the reactions
were performed under the same conditions as in the aqueous
phase (Table 2, entries 2–4). As expected, the two phase systems
improved the aldehyde formation but still the over-oxidation
increased with the conversion. Ethyl acetate gave the best results
in terms of aldehyde produced (42%).
a
Reaction condition: 160 mM furfuryl alcohol, 15 mM TEMPO, 5 mL acetate buffer
50 mM, pH 4.5, 25 ◦C.
b
Reaction condition: 200 mM furfuryl alcohol, 2 mg catalase, 5 mL acetate buffer
50 mM, pH 7.5, 25 ◦C, 24 h.
c
Reaction condition: 25 mM furfuryl alcohol, 25 mM H2O2, 1 mL acetate buffer
50 mM, pH 4.5, 25 ◦C, 5 h.
d
Reaction condition: 1 mL furfuryl alcohol, 20 mg Au/Pd-TiO2, 25 ◦C, 24 h.
(2 atm). As summarized in Table 1, similar results can be obtained
with approximately three-fold less amount of laccase, but with
longer reaction times. Since the aldol additions are carried out at
pH 7, a modification of the reaction pH was necessary for the aldol
addition step when using this oxidation system.
Alcohol oxidase rendered also good conversions to furfuralde-
hyde. The oxidation was carried out in a slightly basic aqueous
solution under oxygen atmosphere and catalase was added to
decompose the H2O2 formed during the reaction avoiding deacti-
vation of the enzymes. One of the main advantages of this oxidation
system is that the pH of the oxidation step is the same as that of
the aldol addition, simplifying the procedure.
Furfuryl alcohol oxidation reaction was also performed with
chloroperoxidase (CPO) from C. fumago [26]. The reaction was car-
ried out under mild conditions with H2O2 as oxidizing agent and
aqueous buffer as solvent. Unfortunately, less than 50% conver-
sion to aldehyde was achieved after 7 h. This result may be due
to the enzyme’s deactivation by the H2O2. CPO instability towards
H2O2 and poor operational stability has already been reported in
the literature [34].
The solvent free oxidation reaction catalyzed by Au/Pd-TiO2 was
not effective [9] in the oxidation of furfuryl alcohol.
3.2. Oxidation/condensation one-pot two-step consecutive
reactions
The results obtained thus far revealed that laccase/O2/TEMPO
system and alcohol oxidase could be good candidates for the
cascade aldehyde generation/aldol addition reaction. Taken into
account our previous experience on FSA we choose the mutant
A129S [35] to carry out the aldol addition reaction of hydroxyace-
tone to the in situ generated furfuraldehyde.
The aldol reaction was first coupled with the oxidation per-
formed by laccase/O2/TEMPO system. Both enzymes have different
optimum operational pH, for that reason the pH was readjusted
from 4.5 until 7 with sodium carbonate just before the enzymatic
aldol addition. The reaction was performed at room temperature,
and under these conditions, 50% conversion to the corresponding
aldol adduct was reached after 5 h.
It is known that enzyme immobilization can overcome in some
instances the deactivation caused by the organic solvent. We envis-
aged the immobilization of laccase by a straightforward absorption
onto Celite, as reported previously for proteases, lipases and alcohol
dehydrogenase [39,40]. A reaction with immobilized laccase was
performed under the same reaction conditions as those for using
the enzyme in solution (Table 2, entry 5). With Celite-laccase prepa-
ration no acid formation was observed, but after 24 h of reaction
only 10% conversion to aldehyde was achieved, which not improved
substantially after 48 h (14%). It is likely that most of the laccase
activity was lost during the adsorption process or that the water
A more straightforward process was obtained when the oxida-
tion was catalyzed by the alcohol oxidase. In this case, after the
Fig. 3. 5-DDAB synthesis.

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M. Mifsud et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 102–107
Table 2
N-Cbz-2-aminoethanol oxidation by the laccase/TEMPO system.
Entry
Reaction media
Conversion 5 h [%]
21
Aldehyde/acid 5 h [%]
Aldehyde 5 h [%]
12
4
26
9
Conversion 24 h [%]
Aldehyde/acid 24 h [%]
Aldehyde 24 h [%]
1
2
3
4
5
Buffer
55/45
100/0
58/42
100/0
–
76
12
66
53
10
19/81
95/5
54/46
80/20
100/0
14
11
36
42
10
CH2Cl2:buffera (1:1)
4
Hexane:buffera (1:1) 45
AcOEt:buffera (1:1)
AcOEt:buffera (1:1)b
9
–
Reaction conditions: 25 mM N-Cbz-aminoethanol, 8 mM TEMPO, 2 atm O2, 2 mL acetate buffer 50 mM, pH 4.5.
a
1 mL acetate buffer 50 mM, pH 4.5: 1 mL solvent, 25 U laccase/mL.
Laccase immobilized.
b
Table 3
N-Cbz-aminoethanol oxidation by continuous aeration and monophase system.
Entry
Oxidation conditions
Conversion 5 h [%]
Acid 5 h [%]
Conversion 24 h [%]
Acid 24 h [%]
1
2
3
30 mM TEMPO 10 U enzyme/mL
30 mM TEMPO 65 U enzyme/mL
60 mM TEMPO 65 U enzyme/mL
21
37
80
10
10
33
50
91
–
25
38
–
Reaction conditions: 80 mM N-Cbz-aminoethanol, O2 bubbling, 10 mL toluene: 5 mL acetate buffer 50 mM, pH 4.5, 40 mg hexadecyltrimethylamonium bromide.
content (50%, v/v) was not optimal for the activity [39]. Therefore,
the oxidation reaction was performed in ethyl acetate at aw = 0.753
[41,42]. However, under these conditions no oxidation activity was
observed. Other support materials such as polyamide (Accurel PA6
powder), which gave excellent activities with proteases were also
tested for the laccase absorption but with similar results to those
obtained with Celite [40].
It is reported in the literature [43] that when a two phase sys-
tem was employed in the laccase/O2/TEMPO system, the partition
of the various components between the different phases may play
a key role in the oxidation process. TEMPO, the alcohol and the
aldehyde were mainly partitioned in the organic phase while lac-
case and the oxoammonium (i.e. oxidized TEMPO) are in the water
phase. Laccase needs to react with TEMPO in order to produce
the oxoammonium oxidant and it was assumed that this takes
place at the water phase or at the organic-aqueous interphase.
The oxidation of the alcohol by the oxoammonium cation takes
place at the organic phase or at the organic-aqueous interphase
too. Hence, it was hypothesized that the use of a system with large
interfacial areas would probably benefit the laccase/O2/TEMPO sys-
tem. For that reason, we tested a ternary system, which consisted
of toluene/buffer/hexadecyltrimethylamonium bromide (CTAB)
(1:2:0.008). CTAB is a cationic surfactant widely used in micromi-
cellar systems with application in enzyme catalyzed reactions [44].
enzymatic aldol addition reaction mediated by FSA was assayed.
Mild reaction conditions were employed making the system attrac-
tive from the environmental point of view. The concept was
demonstrated by the oxidation of furfuryl alcohol to furfuralde-
hyde followed by the aldol addition of hydroxyacetone. This was
further extended to the synthesis of a alpha-glucosidase inhibitor,
(5-DDAB), by modifying the reaction conditions of the oxida-
tion step. In aqueous media, treatment of N-Cbz-2-aminethanol
with laccase/O2/TEMPO system, lead to N-Cbz-glycine as major
product. Organic-water two phase systems partially avoid the over-
oxidation, and in ethyl acetate-buffer two phase system the yield
of aldehyde reached 42%. Further improvements were achieved by
using an organic two-phase system in the presence of a surfac-
tant. Thus in toluene/buffer/hexadecyltrimethylamonium bromide
(CTAB) (1:2:0.008) the aldehyde formation was improved up to
53%.
Acknowledgements
This work was supported by the Spanish MINECO project (CTQ
2009-07359) (PIM2010EEI-00607), Generalitat de Catalunya (2009
SGR 00281) and COST (CM0701). A. Szekrényi acknowledges the
CSIC for a JAE Predoctoral scholarship. M. Mifsud thanks the CSIC
for a JAE Doc postdoctoral contract.
Under these conditions,
a significant improvement was
achieved (Table 3). After 5 h reaction with 30 mM TEMPO and 65
units of laccase a 37% of conversion was reached with only 10%
of acid (Table 3, entry 1). Leading the reaction run for 24 h, 91% of
conversion with 62% of selectivity towards aldehyde formation was
achieved (Table 3, entry 2). In this case, aeration was sufficient for
the reaction to proceed and, since it caused solvent evaporation,
1 mL of toluene was added every 30 min.
On increasing the amount of TEMPO up to 60 mM, the conver-
sion raised to 80% with 54% of aldehyde in 5 h (Table 3, entry 3),
leading us to couple this reaction with the aldol addition reac-
tion. The aldol addition of HA to N-Cbz-glycinal formed in situ was
performed. First the pH of the reaction media was adjusted to 7
with sodium carbonate using FSA A129S/A165G as the optimum
biocatalyst for this reaction [30]. The aldehyde was quantitatively
converted to the adduct 7 after 3 h.
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