Whole seeds of Bauhinia variegata L. (Fabaceae) as an efficient biocatalyst for benzyl alcohol preparations from benzaldehydes

Article abstract of DOI:10.1080/10242422.2021.1956910

Whole seeds of Bauhinia variegata L. (Fabaceae) were utilized as a biological reducer to transform benzaldehyde into benzyl alcohol. The effects of some variables such as temperature, the load of substrate and co-solvent, were established to optimize the reductive process. Utilizing the optimal reaction conditions, a laboratory-scale reaction (final concentration of the substrate: 21.2 mM) was performed to obtain benzyl alcohol (conversion: 95%; isolated yield: 49%; productivity: 1.11 g L^?1 or 0.046 g L^?1h^?1 of benzyl alcohol). In addition, using these optimal conditions, fourteen substituted benzaldehydes were reduced, with a conversion achieved to their corresponding benzyl alcohols ranging from 62% to >99% (isolated yields from 7% to 70%). Moreover, useful building blocks by the synthesis of the drugs and important commercial products were also obtained. The scope, limitations and advantages of this new biocatalytic synthetic method are also discussed.

Full text of DOI:10.1080/10242422.2021.1956910

Biocatalysis and Biotransformation
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Whole seeds of Bauhinia variegata L. (Fabaceae)
as an efficient biocatalyst for benzyl alcohol
preparations from benzaldehydes
Gabriel I. Demmel, Daniela L. Bordón, Ana M. Vázquez, María F. Decarlini,
Mariángeles Díaz Panero, Laura I. Rossi & Mario L. Aimar
To cite this article: Gabriel I. Demmel, Daniela L. Bordón, Ana M. Vázquez, María F. Decarlini,
Mariángeles Díaz Panero, Laura I. Rossi & Mario L. Aimar (2021): Whole seeds of Bauhinia
variegata L. (Fabaceae) as an efficient biocatalyst for benzyl alcohol preparations from
benzaldehydes, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2021.1956910
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2021.1956910
RESEARCH ARTICLE
Whole seeds of Bauhinia variegata L. (Fabaceae) as an efficient biocatalyst
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for benzyl alcohol preparations from benzaldehydes
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Gabriel I. Demmel , Daniela L. Bordon , Ana M. Vazquez , Marıa F. Decarlini , Mariangeles Dıaz Panero ,
Laura I. Rossic,d
and Mario L. Aimarb
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Facultad de Ciencias Quımicas, Catedra de Tecnologıa Quımica, Universidad Catolica de Cordoba, Cordoba, Argentina; Facultad de
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Ciencias Exactas, Fısicas y Naturales, Catedra de Quımica Aplicada, Universidad Nacional de Cordoba, Cordoba, Argentina; Facultad
de Ciencias Quımicas, Departamento de Quımica Organica, Universidad Nacional de Cordoba, Cordoba, Argentina; Instituto de
Investigaciones en Fısico-Quımica de Cordoba (INFIQC-CONICET), Cordoba, Argentina
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ABSTRACT
ARTICLE HISTORY
Received 13 April 2021
Revised 5 July 2021
Accepted 13 July 2021
Whole seeds of Bauhinia variegata L. (Fabaceae) were utilized as a biological reducer to trans-
form benzaldehyde into benzyl alcohol. The effects of some variables such as temperature, the
load of substrate and co-solvent, were established to optimize the reductive process. Utilizing
the optimal reaction conditions, a laboratory-scale reaction (final concentration of the substrate:
21.2 mM) was performed to obtain benzyl alcohol (conversion: 95%; isolated yield: 49%; product-
ivity: 1.11 g Lꢁ1 or 0.046 g Lꢁ1hꢁ1 of benzyl alcohol). In addition, using these optimal conditions,
fourteen substituted benzaldehydes were reduced, with a conversion achieved to their corre-
sponding benzyl alcohols ranging from 62% to >99% (isolated yields from 7% to 70%).
Moreover, useful building blocks by the synthesis of the drugs and important commercial prod-
ucts were also obtained. The scope, limitations and advantages of this new biocatalytic synthetic
method are also discussed.
KEYWORDS
Seeds; biocatalysis; Bauhinia
variegata L.; benzaldehydes;
benzyl alcohols
1. Introduction
(Kumaraswamy and Ramesh 2003). For these reasons,
and as a direct consequence of the philosophical con-
ception of Green Chemistry, the synthetic community
has begun to pay greater attention to the use of bio-
processes applied to the synthesis of chem-
ical compounds.
Nowadays, access to chemical processes that can be
carried out in a manner compatible with the environ-
ment is a necessity of the first order (Roschangar et al.
2015). For this reason, the production of chemical
compounds through the use of biological processes is
gaining more and more prominence, because these
methodologies can be used on a sustainable basis
rather than by depleting natural resources (Cordell et
al. 2007).
In this sense, the biocatalytic reduction of carbonyl
compounds utilized to synthesize alcohols has estab-
lished itself as a very important alternative to classical
chemical procedures, not only because the reaction
performance is very high, but also because the reac-
tion media is usually water, so these systems generally
operate at ambient temperature with the biocatalyst
being renewable raw material (Patel 2018). For these
characteristics, the waste products generated at the
end of the process are generally more compatible
with the environment than classical chemical reac-
tions, which clearly represents an advantage
The bioreduction processes of carbonyl compounds
to obtain alcohols have been catalyzed using different
biological reducers. However, it is important to note
that most investigations have focussed on the search
for and improvement of methods that can provide
chiral secondary alcohols obtained from the biocata-
lytic reduction of prochiral ketones. For this reason,
the bioreduction of carboxylic acids (Strohmeier et al.
2019; Tramontina et al. 2020) and aldehydes (Li et al.
2013; Torrelo et al. 2015) has been proportionally less
well studied, despite the importance of primary alco-
hols as intermediaries in the synthesis of numerous
chemical compounds with industrial applications
(Akhtar et al. 2013; Tramontina et al. 2020).
Furthermore, the studies to date which have reported
on aldehyde reduction have been oriented in two
ꢀ ꢀ
ꢀ
Facultad de Ciencias Exactas, Fısicas y Naturales, Catedra de Quımica Aplicada, Universidad
CONTACT Mario L. Aimar
mlaimar@unc.edu.ar
ꢀ
ꢀ
Nacional de Cordoba, Cordoba, Argentina
ꢀ
This work is dedicated to the memory and teaching career of Prof. Dr. Hector R. Juliani.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
G. I. DEMMEL ET AL.
main directions (Hollmann et al. 2021). On the one
hand, the bioreduction of aldehydes has been carried
out on racemic mixtures of a-substituted aldehydes,
which when bioreduced can provide primary alcohols
with a chiral centre adjacent to the carbon atom that
contains the hydroxyl group (Giacomini et al. 2007;
Friest et al. 2010; Galletti et al. 2010; Gooding et al.
2010; Kalaitzakis and Smonou 2010a, 2010b). On the
other hand, studies have also been carried out in
order to develop chemoselective reduction of alde-
hydes in the presence of ketones and other functional
Ligustrum lucidum) (Kumaraswamy and Ramesh 2003;
Bertini 2012; Alves-Ferreira, da Costa, et al. 2012;
Alves-Ferreira, Da Silva, et al. 2012; Carvalho da Silva
ꢀ
et al. 2018; Bordon et al. 2021), and as of yet, there is
only one report published that has described the pos-
sibility of reducing benzaldehydes to their correspond-
ing benzyl alcohols using whole seeds as the
ꢀ
biocatalyst (Bordon et al. 2021).
It is important to highlight that the use of seeds as
biocatalysts does not imply a restriction related to the
seasonal availability of seeds, since they can be stored
ꢀ
groups (Sello et al. 2006; Suarez-Franco et al. 2010;
ꢀ
until being used (Bordon et al. 2021; Demmel et al.
Salvano et al. 2011; Li et al. 2013).
2021). This situation clearly contributes to their utiliza-
tion as a “stable bag of enzymes” capable of carrying
out chemical transformations of different compounds
as if they were classic chemical catalysts. Moreover,
the direct utilization of whole seeds further simplifies
the work methodology.
Whole cells of microorganisms (bacteria (Vitale et
al. 2020), fungus (Decarlini et al. 2017) and algae
(Mouad et al. 2011)), isolated enzymes from various
ꢁ
ꢀ
resources (Petrovicova et al. 2021), and more recently
ꢀ
plants (Bennamane et al. 2014, 2015; Pavokovic et al.
2017; Atak et al. 2019) and their cell cultures (Akakabe
and Naoshima 1994; Baldassarre et al. 2000; Kazici et
al. 2016, 2017), are among the biocatalytic systems
that have been used to carry out the bioreduction of
carbonyl compounds. However, it is necessary to rec-
ognize that the utilization of vegetables for these
methods is a relatively new methodology (Cordell et
al. 2007; Patil 2015). This way of working has recently
been termed “botanochemistry” (Vandenberghe et al.
2013), and it offers numerous advantages in terms of
bioavailability, cost and time, since complex processes
of preparation, extraction, purification and multiplica-
tion of the biocatalyst are not necessary, which is
often the case when working with microorganisms or
cell cultures.
As in the case of classical biocatalytic systems, the
use of different parts of plants as biocatalysts has
been mainly utilized to biocatalyze the stereoselective
reduction of ketones (Cordell et al. 2007; Patil 2015).
However, as mentioned above, this methodology has
also been applied, but less frequently, to carry out the
synthesis of primary alcohols obtained from aldehydes
(Daucus corota, Conium maculatum, Manihot esculenta,
Recently, our team has reported a study on the
excellent capability of whole seeds of Bauhinia varie-
gata L. (Fabaceae) to carry out the stereoselective
reduction of acetophenones (Demmel et al. 2021) and
following on from this investigation, we describe here
the results obtained using whole seeds of “Orchid
tree” to reduce different aromatic aldehydes to the
corresponding benzyl alcohols. In this way, the field of
application of this efficient and new biocatalyst has
been expanded to be able to obtain important key
intermediates used in the production of drugs and
chemical compounds of commercial interest.
2. Material and methods
2.1. General
Benzaldehyde (BzHO) and substituted BzHOs were
purchased from Sigma-Aldrich S.A. (Argentina). The
corresponding benzyl alcohols were synthesized by
reduction with NaBH4 (Sigma-Aldrich) in medicinal-
grade ethanol and used as a standard reference in the
gas chromatography (GC) analysis. Sterile deionized
water was used as the reaction media, and ethyl acet-
ate and hexane were purified by a simple distillation
prior to use. GC analyses were performed using a
Buck Scientific Model 910/310 instrument with a flame
ionization detector (FID), and GC-mass spectrometry
(MS) analyses were carried out on a Hewlett Packard
HP 5890 Series II gas chromatograph equipped with a
ꢀ
Manihot dulcis, Cocos nucıfera, Zea maiz, Musa sapien-
tum) (Yadav et al. 2002; Machado et al. 2006 and
2008; Fonseca et al. 2009; Salvano et al. 2011; Misra et
ꢀ
al. 2012; Luna et al. 2014; Solıs et al. 2019).
The possibility of using seeds as biocatalysts, more-
over, has been very little explored in the synthesis of
primary alcohols. In this sense, as far as we have been
able to determine, very few reports have described
the utilization of seeds as a biological reducer for car-
rying out the bioreduction of aldehydes (Glicine max,
Phaseolus vulgaris, Ximenia Americana, Lens culinaris,
1
Mass Detector HP 5970. The H nuclear magnetic res-
onance (NMR) and 13C NMR spectra were recorded on
a Bruker Avance II 400 MHz.

BIOCATALYSIS AND BIOTRANSFORMATION
3
which were dried in an oven at 105 ꢂC to constant
weight (AOAC 1980).
2.2. GC-FID and GC-MS analyses
GC separation was performed on a Restek RTX-5 capil-
lary column (dimethyl Crosslinked diphenyl polysilox-
ane, 30 m, 0.25 mm, 0.25 lm film thickness). The GC
general conditions used for the split analysis were:
145 mL/min; injector, 250 ꢂC; detector FID, 250 ꢂC; car-
rier gas, N2; and head pressure, 14.5 psi. The conver-
sion percentages of the reactions were determined by
GC using normalized peak areas without a correction
factor, and the GC-MS (70 eV) analyses were carried
using a Hewlett Packard HP-5ms capillary column
(Crosslinked 5% PhMeSiloxane, 30 m, 0.32 mm, 0.25 lm
film thickness).
2.3.6. Ash content
Ash content was determined gravimetrically by calcin-
ation of samples of 4.73 g of crushed seeds of B. varie-
gata in a muffle at 500 ꢂC to constant weight
(AOAC 1980).
2.4. General procedure for the biocatalytic
reductions of BzHO
A whole seed sample of B. variegata (10 g) was placed
in a disinfected Erlenmeyer flask (250 mL) with sterile
deionized water (80 mL), and then aldehyde
(100–300 mg) was dissolved in an appropriate quantity
of dimethyl sulfoxide (DMSO), which was added to the
suspension. The reaction was carried out by stirring on
an orbital shaker (100 rpm) at 20–45 ꢂC, with the flask
being closed and protected from direct light. Finally,
the crude reaction was filtered through cotton in
order to remove the seeds, and the solution was
extracted with ethyl acetate, with very gentle stirring
performed to avoid the formation of an emulsion.
2.3. Biocatalyst
Seeds of B. variegata L. were collected during the
period September–December in the city of Cordoba
ꢀ
and identified by a botanic expert. Before use, the
seeds were washed with tap water to remove traces
of dirt before being disinfected by immersion in a
0.5% sodium hypochlorite solution for 15 min. The
seeds were then washed again, but this time with
deionized sterile water.
2.3.1. Protein content
2.5. Preparative-scale reactions
Samples of 0.6 g of dried and crushed B. variegata
seeds were used to determine the protein content
according to the Kjeldahl method (Carvalho da Silva et
al. 2018).
For the preparative-scale reaction, 1.8 g of BzHO, 80 g
of seeds of B. variegata, 64 mL of DMSO and 640 mL
of sterile distilled water were placed in a disinfected
2 L conical flask (final concentration of the substrate:
2.56 g/L or 14.3 mM). Then, this flask was put on an
orbital shaker at a shaking speed of 100 rpm at 40 ꢂC
for 24 h, after which, a sample was taken and analyzed
by GC-FID (95% conversion). The crude reaction sys-
tem was extracted for 24 h with ethyl acetate using a
liquid-liquid extractor, and the organic solution was
dried using anhydrous magnesium sulphate. Then, the
organic solution was concentrated to dryness in a
rotary evaporator, and the crude reaction products
were filtered on a short column with silica gel (70–230
mesh) using hexane–ethyl acetate mixtures in variable
proportions as the eluent. Finally, 0.78 g of pure (1)
was isolated (yield of the isolated product: 49%).
2.3.2. Carbohydrate content
Samples of 1.0 g of dried and crushed B. variegata
seeds were used to determine the carbohydrate con-
tent according to the Phenol/Sulfuric acid colorimetric
method (DuBois et al. 1956).
2.3.3. Lipid content
Samples of 1.7 g of dried and crushed B. variegata
seeds were used to determine the lipid content using
a Soxhlet extractor with ethyl ether as the solvent
(AOAC 1980).
2.3.4. Fibre content
The solid residue after lipid content determination
(Soxhlet extraction) was used to determine the fibre
content by acidic and basic hydrolysis using samples
of 1.4 g (AOAC 1980).
2.6. Reduction of substituted BzHOs with whole
seeds of B. variegata
The same optimal proportions used in the scaling
down of BzHO were used to reduce substituted
BzHOs. The final products were extracted from reac-
tion media with ethyl acetate using a liquid-liquid
2.3.5. Moisture content
Moisture content was determined gravimetrically using
samples of 4.73 g of crushed seeds of B. variegata

4
G. I. DEMMEL ET AL.
extractor. The organic layer was separated and dried
using anhydrous magnesium sulphate, after which, the
organic solution was evaporated and the extracted
crude reaction products were filtered on a short col-
umn with silica gel (70–230 mesh) using hexane–ethyl
acetate mixtures in variable proportions as the eluent.
¼ 2.96 (s, 1H, OH), 4.82 (s, 2H, CH2), 7.53–7.56 (d, 2H,
ArH), 8.18–8.21 (d, 2H, ArH). 13C NMR (100 MHz, CDCl3)
d (ppm) ¼ 63.78; 123.61; 126.95; 148.62; 207.58. EIMS
m/z (rel. int. %) ¼ 154 (Mþþ1, 3), 153 (Mþ, 10), 136
(Mþꢁ17, 5), 124 (2), 107 (Mþꢁ46, 47), 89 (30), 77
(100), 63 (20), 51 (50), 50 (30).
2-Chlorobenzyl alcohol (5): GC-FID analysis condi-
tions: T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC
(7 min). GC Rt: 2-chlorobenzaldehyde: 10.38 min, 2-
chlorobenzyl alcohol: 12.32 min. 1H NMR (400 M Hz,
CDCl3) d (ppm) ¼ 2.31 (s, 1H, OH); 4.75 (s, 2H; CH2);
7.21–7.29 (m, 2H, ArH); 7.33–7.35 (m, 1H, ArH);
7.45–7.47 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3) d
(ppm) ¼ 62.78; 127.02; 128.72; 128.81; 129.34; 132.71;
138.19. EIMS m/z (rel. int. %) ¼ 144 (Mþþ2, 12), 142
(Mþ, 45), 125 (Mþꢁ17, 10), 113 (12), 107 (Mþꢁ36, 70),
105 (12), 89 (10), 79 (87), 77 (100), 63 (10), 51 (30),
50 (18).
2.7. Chromatographic and spectroscopic data
After purification, all products were identified by com-
paring their retention times obtained by CG-FID ana-
lysis against the control samples, and by comparing
1
their spectra of GC-MS, H and 13C NMR with literature
~
data (Machado et al. 2006; Assunc¸ao et al. 2008; Misra
et al. 2012; Alves-Ferreira, da Costa, et al. 2012; Alves-
Ferreira, Da Silva, et al. 2012; Luna et al. 2014; Solıs et
al. 2019).
ꢀ
Benzyl alcohol (1): GC-FID analysis conditions: T1 ¼
80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼180 ꢂC (7 min). GC
Rt: benzaldehyde: 6.12 min, benzyl alcohol: 7.65 min.
1H NMR (400 M Hz, CDCl3) d (ppm) ¼ 2.52 (s, 1H, OH),
4.68 (s, 2H, CH2), 7.32–7.38 (m, 5H, ArH). 13C NMR
(100 MHz, CDCl3) d (ppm) ¼ 65.26; 127.00; 127.68;
128.57; 140.85. EIMS m/z (rel. int. %) ¼ 109 (Mþþ1, 8),
108 (Mþ, 98), 107 (Mþꢁ1, 68), 91 (18), 78 (100), 78
(11), 77 (45), 51 (19), 50 (10).
3-Chlorobenzyl alcohol (6): GC-FID analysis conditions:
T1 ¼ 80ꢂC (5 min), DT ¼ 20ꢂC/min, T2 ¼ 180 ꢂC (7 min).
GC Rt: 2-chlorobenzaldehyde: 10.72min, 3-chlorobenzyl
1
alcohol: 11.93 min. H NMR (400 M Hz, CDCl3) d (ppm)
¼ 1.26 (s, 1H, OH), 4.68 (s, 2H, CH2), 7.20–7.29 (m, 4H,
ArH). 13C NMR (100 MHz, CDCl3) d (ppm) ¼ 64.57;
124.84; 126.97; 127.72; 129.81; 134.48; 142.83. EIMS m/z
(rel. int. %) ¼ 144 (Mþþ2, 20), 142 (Mþ, 70), 125 (Mþ–
17, 10), 113 (20), 107 (Mþ– 36, 83), 105 (12), 89 (10), 79
(78), 77 (100), 63 (7), 51 (20), 50 (12).
4-chlorobenzyl alcohol (7): GC-FID analysis condi-
tions: T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC
(7 min). GC Rt: 4-chlorobenzaldehyde: 10.69 min, 4-
chlorobenzyl alcohol: 11.99 min. 1H NMR (400 M Hz,
CDCl3) d (ppm) ¼ 1.88 (s, 1H, OH), 4.67 (s, 2H, CH2),
6.76–7.40–7.50 (m, 4H, ArH). 13C NMR (100 MHz, CDCl3)
d (ppm) ¼ 64.57; 128.27; 128.69; 133.39; 139.27. EIMS
m/z (rel. int. %) ¼ 144 (Mþþ2), 142 (Mþ, 28), 125
(Mþꢁ17, 5), 113 (12), 107 (Mþ– 35, 55), 79 (87), 77
(100), 63 (10), 51 (30).
2,4-diChlorobenzyl alcohol (8): GC-FID analysis condi-
tions: T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC
(7 min). GC Rt: 2,4-dichlorobenzaldehyde: 10.05 min,
2,4-dichlorobenzyl alcohol: 11.51 min. 1H NMR (400 M
Hz, CDCl3) d (ppm) ¼ 4.76 (s, 2H, CH2), 7.28–7.52 (m,
3H, ArH). 13C NMR (100 MHz, CDCl3) d (ppm) ¼ 62.21;
127.18; 128.16; 129.48; 133.44; 133.89; 136.74. EIMS m/
z (rel. int. %) ¼ 178 (Mþþ2, 10), 176 (Mþ, 26), 159
(Mþꢁ17, 7), 141 (Mþꢁ35, 41), 113 (46),111 (43), 77
(100), 75 (35), 63 (10), 50 (23).
2-Nitrobenzyl alcohol (2): GC-FID analysis conditions:
T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC (7 min).
GC Rt: 2-nitrobenzaldehyde: 12.64 min, 2-nitrobenzyl
1
alcohol: 12.86 min. H NMR (400 M Hz, CDCl3) d (ppm)
¼ 2.92 (s, 1H, OH); 4.97 (s, 2H, CH2); 7.45–7.49 (t, 1H,
ArH); 7.67 (t, 1H, ArH); 7.74 (d, 1H, ArH); 8.08 (d, 1H,
ArH). 13C NMR (100 MHz, CDCl3) d (ppm) ¼ 62.45;
124.98; 128.46; 129.87; 134.12; 136.81; 147.62. EIMS m/
z (rel. int. %) ¼ 153 (Mþ, 1), 136 (Mþ-17, 2), 135 (10),
107 (Mþꢁ46, 25), 91 (30), 79 (58), 78 (22), 77 (100), 65
(13), 51 (25), 50 (13).
3-Nitrobenzyl alcohol (3): GC-FID analysis conditions:
T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC (7 min).
GC Rt: 3-nitrobenzaldehyde: 12.86 min, 3-nitrobenzyl
1
alcohol: 14.50 min. H NMR (400 M Hz, CDCl3) d (ppm)
¼ 1.56 (s, 1H, OH); 4.83 (s, 2H, CH2), 7.54 (t, 1H, ArH);
7.70 (d, 1H, ArH); 8.15 (d, 1H, ArH); 8.26 (s, 1H, ArH).
13C NMR (100 MHz, CDCl3) d (ppm) ¼ 64.02; 121.54;
122.62; 129.45; 130.89; 132.56; 142.86. EIMS m/z (rel.
int. %) ¼ 154 (Mþþ1, 3), 153 (Mþ, 30), 136 (Mþꢁ17,
33), 124 (5), 107 (Mþꢁ46, 33), 89 (60), 77 (100), 63
(12), 51 (30), 50 (15).
4-Nitrobenzyl alcohol (4): GC-FID analysis conditions:
T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC (7 min).
GC Rt: 4-nitrobenzaldehyde: 14.65 min, 4-nitrobenzyl
4-Flurobenzyl alcohol (9): GC-FID analysis conditions:
T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC (7 min).
GC Rt: 4-fluorobenzaldehyde: 7.96 min, 4-flurobenzyl
1
1
alcohol: 15.96 min. H NMR (400 M Hz, CDCl3) d (ppm)
alcohol: 9.64 min. H NMR (400 M Hz, CDCl3) d (ppm)

BIOCATALYSIS AND BIOTRANSFORMATION
5
Table 1. Proximate composition of B. variegata seeds.
¼ 2.50 (s, 1H, ArH); 4.66 (s, 2H, CH2); 7.02–7.34 (m, 4H,
ArH). 13C NMR (100 MHz, CDCl3) d (ppm) ¼ 64.65;
115.29; 115.50; 128.74; 128.72; 136.53; 161.12; 167.55.
EIMS m/z (rel. int. %) ¼ 126 (Mþ, 100), 125 (Mþꢁ1,
67), 109 (Mþꢁ17, 30), 105 (30), 92 (100), 90 (27), 83
(10), 77 (30), 75 (13), 51 (14), 50 (9).
Entry
Constituents
% p/pa
1
2
3
4
5
6
Proteins
Carbohydrates
Lipids
Fibres
Moisture
Ash
5.2
1
61.8 2.2
18.1 0.3
3.8 1.3
5.5 0.9
5.6 0.9
aAll determinations were made in duplicate.
3-Hydroxybenzyl alcohol (10): GC-FID analysis condi-
tions: T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC
(7 min). GC Rt: 3-hydroxybenzaldehyde: 11.82 min, 3-
GC Rt: cinnamaldehyde: 11.52 min; cinnamyl alcohol:
1
1
hydroxybenzyl alcohol: 13.03 min. H NMR (400 M Hz,
13.22 min. H NMR (400 M Hz, CDCl3) d (ppm) ¼ 2.1 (s,
CDCl3) d (ppm) ¼ 5.07 (s, 2H, CH2); 5.74 (s, 1H, OH);
6.79–6.85 (m, 2H, ArH); 6.90 (d, 1H, ArH); 7.20–7.26 (m,
1H, ArH). 13C NMR (100 MHz, CDCl3) d (ppm) ¼ 66.20;
115.09; 115.33; 120.35; 129.88; 137.53; 155.96. EIMS m/
z (rel. int. %) ¼ 125 (Mþþ1, 7), 124 (Mþ, 100), 123
(Mþꢁ1, 27), 107 (Mþꢁ17, 25), 106 (Mþꢁ18, 21), 105
(23), 95 (98), 79 (18), 78 (50), 77 (89), 65 (23), 51 (19),
50 (9).
1H, OH), 4.31–4.33 (c, 1H), 6.33–6.64 (m, 1H), 7.24–7.44
(m, 5H, ArH). 13C NMR (100 MHz, CDCl3) d (ppm) ¼
63.14; 126.47; 127.69; 128.58; 128.66; 131.22; 136.71.
EIMS m/z (rel. int. %) ¼ 134 (Mþ, 50), 133 (Mþꢁ1, 10),
115 (37), 105 (40), 92 (100), 91 (75), 78 (70), 77 (50), 63
(18), 55 (17), 51 (40).
3. Results and discussion
4-Hydroxybenzyl alcohol (11): GC-FID analysis condi-
tions: T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC
(7 min). GC Rt: 4-hydroxybenzaldehyde: 11.78 min, 4-
3.1. Composition of B. variegata seeds
In order to characterize the seeds of B. variegata, a
proximal analysis was carried out to establish the con-
tent of proteins, lipids, carbohydrates, fibres, moisture
and ash, with the results shown below in Table 1.
It can be seen in Table 1 (entry 1) that the protein
content was 5.2%, which is significantly lower than
that observed for some other seeds used as biocata-
lysts. In this sense, Ximenia americana grains, for
example, presented 19.8% protein content (Carvalho
da Silva et al. 2018), while Sinapis alba ( De Sousa et
al. 2019) revealed a protein content of approximately
34.6%. In contrast, Lens culinaris (Alves-Ferreira et al.
2012) showed a low protein content (1.07–1.33%),
which was significantly lower than that found in the
seeds of B. variegata. Similarly, a low protein content
(1.2%) was reported for Cocos nucifera (Fonseca et al.
2009). Because enzymes are made up of proteins,
these results indicate that enzymes present in seeds
may be responsible for the biocatalytic process
(Almeida de Souza et al. 2019). On the other hand, a
high content of carbohydrates was observed (61.8% –
Table 1, entry 2), while the value of lipids, fibres, mois-
ture and ash content (Table 1, entries 3–6) were in
agreement with other results reported for the seeds of
B. variegata (Sharma et al. 2020).
1
hydroxybenzyl alcohol: 11.97 min. H NMR (400 M Hz,
CDCl3) d (ppm) ¼ 2.09 (s, 2H, CH2); 5.05 (s, 1H, OH);
7.00 (d, 2H, ArH); 7.81 (d, 2H, ArH), 9.85 (s, 1H, OH).
13C NMR (100 MHz, CDCl3) d (ppm) ¼ 66.42; 115.49;
130.36; 132.66; 162.08. EIMS m/z (rel. int. %) ¼ 125
(Mþþ1, 6), 124 (Mþ, 88), 123 (Mþꢁ1, 47), 107 (Mþꢁ17,
50), 106 (Mþꢁ18, 40), 105 (17), 95 (100), 79 (16), 78
(72), 77 (91), 65 (23), 51 (35), 50 (20).
4-Methylbenzyl alcohol (12): GC-FID analysis condi-
tions: T1 ¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC
(7 min). GC Rt: 4-methylbenzaldehyde: 9.31 min, 4-
methylbenzyl alcohol: 11.00 min. 1H NMR (400 M Hz,
CDCl3) d (ppm) ¼ 2.04 (s, 1H, OH); 2.34 (s, 3H, CH3);
4.63 (s, 2H, CH2); 7.16 (d, 2H, ArH); 7.24 (d, 2H, ArH).
13C NMR (100 MHz, CDCl3) d (ppm) ¼ 21.14; 65.25;
127.13; 129.24; 137.39; 137.89. EIMS m/z (rel. int. %) ¼
123 (Mþþ1, 7), 122 (Mþ, 86), 121 (Mþ– 1, 20), 107
(Mþꢁ15, 100), 93 (48), 91 (52), 79 (60), 77 (52), 65 (18),
51 (14), 50 (7).
Vanillyl alcohol (13): GC-FID analysis conditions: T1
¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 200 ꢂC (10 min).
GC Rt: vanillin: 12.45 min; vanillyl alcohol: 13.07 min.
1H NMR (400 M Hz, CDCl3) d (ppm) ¼ 3.90 (s, 3H,
OCH3), 4.60 (s, 2H, –CH2–), 5.60 (s, 1H, OH), 6.82–6.92
(m, 3H, ArH), 7.26 (s, 1H, ArH). 13C NMR (100 MHz,
CDCl3) d (ppm) ¼ 55.93; 65.44; 109.99; 114.29; 120.25;
132.96; 145.33; 146.69. EIMS m/z (rel. int. %) ¼ 154
(Mþ, 72), 153 (Mþꢁ1, 7), 137 (32), 125 (39), 93(88), 77
(21), 65 (100), 55 (36).
3.2. Kinetic study
Considering the results previously reported with aceto-
phenones (Demmel et al. 2021), and in order to
broaden the scope of the biocatalytic process pro-
moted by whole seeds of B. variegata, the capacity of
Cinnamyl alcohol (14): GC-FID analysis conditions: T1
¼ 80 ꢂC (5 min), DT ¼ 20 ꢂC/min, T2 ¼ 180 ꢂC (7 min).

6
G. I. DEMMEL ET AL.
Figure 2. Effect of BzHO concentration on the formation of 1
promoted by whole seeds of B. variegata.
Figure 1. Kinetics of the reduction of BzHO to 1 promoted by
the whole seeds of B. variegata.
soaked seeds of Phaseolus vulgaris to carry out the
reduction of BzHO, and observed a 100% conversion
for the formation of (1) for all varieties utilized in 24 h
of reaction at 25 ꢂC. In addition, Carvalho da Silva et
al. (2018) carried out studies using shredded grains of
Ximenia americana, with (1) being obtained with a
conversion of >99% for their reaction conditions.
this new biocatalyst to carry out the reduction BzHO,
and that of 14 substituted benzaldehydes derivatives
to obtain their corresponding benzyl alcohols (1–15)
was investigated.
Initially, a kinetic study was performed in order to
determine the time in which the reduction of BzHO to
(1) promoted by whole seeds of B. variegata reached
the maximum percentage of conversion. Reactions
were carried out in duplicate using 100 mg of sub-
strate dissolved in 80 mL of sterile milliQ water inside
an Erlenmeyer (250 mL), which had been previously
disinfected, and with the addition of 10 g of disin-
fected whole seeds (room temperature and 100 rpm).
The reactions were monitored for 24 h, taking samples
at regular time intervals, and their evolutions were
established by GC-FID and by GC-MS analyses. The
results are shown below in Figure 1.
ꢀ
Finally, Bordon et al. (2021) described the performance
of whole seeds of Ligustrum lucidum to carry out the
transformation from BzHO to (1) and obtained 90% of
conversion in 72 h of reaction at 25 ꢂC. It is important
ꢀ
to note that the study reported by Bordon et al.
(2021) is the only report that has described the use of
whole seeds as a biological reducer being capable of
transforming BzHO into (1).
Due to the encouraging results obtained when
using whole seeds of B. variegata to reduce BzHO
(Figure 1), it was decided to perform studies to deter-
mine the effect of some different variables on the per-
formance of the reaction.
As can be seen in Figure 1, the process of bioreduc-
tion of BzHO was completed in approximately 13 h of
reaction in a highly efficient way (ꢃ100% of conver-
sion) for the formation of (1).
3.3. Effect of BzHO concentration
It is important to mention that despite the fact that
the capacity of several vegetables to carry out the
reduction of BzHO has been previously reported, only
five studies have described the utilization of seeds as
biocatalysts for carrying out this reductive process. In
this sense, Bertini (2012) reported a study using
ground seeds of Glycine max (soybean), with BzHO
being transformed to (1) with 100% of conversion in
72 h. Moreover, Alves-Ferreira, da Costa, et al. (2012)
and Alves-Ferreira, Da Silva, et al. (2012) reported the
capability of ground seeds of Lens culinaris to reduce
several carbonyl compounds, and among these, BzHO
was reduced with a bioconversion of ꢄ99% in 72 h of
To determine the effect of substrate loading on the
production of (1), tests were carried out using differ-
ent concentrations of BzHO in the reaction medium.
The quantities used were 100, 150, 200, 250, 300, 350
and 400 lL in 80 mL of milliQ water (BzHO concentra-
tions ranging from 1.25 to 5.6 g/L) with 10 g of the
whole B. variegata seeds, which were stirred at
100 rpm at room temperature. The evolution of the
reaction was followed for 24 h, taking samples at dif-
ferent times and analyzing these by GC-FID. The
results can be observed in Figure 2.
As shown in Figure 2, when the amount of BzHO in
the reaction medium was increased by 50% (from 1.25
to 1.88 g/L), a pronounced decrease in the percentage
ꢀ
reaction. On the other hand, Solıs et al. (2017)
described the use of five varieties of ground and

BIOCATALYSIS AND BIOTRANSFORMATION
7
of (1) formation was observed, with the conversion
decreasing from values >99% at a substrate concen-
tration of 1.25 g/L to <65% when the substrate con-
centration was 1.88 g/L. Furthermore, the decrease in
the conversion percentage was more significant when
the substrate concentration was higher (with a conver-
sion of just 6% at 5.0 g/L of BzHO), thus evidencing a
strong negative effect of the increase in substrate
loading on the performance of the process. Due to
the results achieved in these experiments, it was
decided to continue with the studies using a concen-
tration of BzHO of 1.25 g/L in the reaction medium.
Figure 3. Kinetics of the reduction of BzHO mediated by
whole seeds of B. variegata determined at different
temperatures.
3.4. Effect of temperature
In order to determine the effect of temperature on
the production of (1) promoted by B. variegata seeds,
tests were carried out on working temperatures of 25,
30, 35, 40 and 45 ꢂC. The experiments were performed
using 100 lL of the substrate (1.25 g/L) in 80 mL of
milliQ water, plus the addition of 10 g of whole seeds
and under constant orbital shaking at 100 rpm. Under
these working conditions, the kinetic profiles of each
reaction were obtained by taking samples at regular
intervals of time and analyzing these by GC-FID in
order to establish the percentage of conversion. All
reactions were performed in duplicate, and the kinetic
profiles obtained are shown in Figure 3.
As can be seen in Figure 3, on increasing the tem-
perature from 25 to 45 ꢂC, a corresponding increase in
the reaction rate was observed, and consequently, the
reaction time at which the maximum percentage of
conversion was reached was reduced, which
decreased from 20 h (at 25 ꢂC – lower curve) to 10 h
(40 ꢂC – upper curve).
To visualize better the effect of temperature on
reaction rate, the kinetic profiles were fitted to a first-
order equation using Origin Pro 8 software, and the
rate constants were calculated for each temperature.
The results can be seen below in Figure 4.
As can be seen in Figure 4, when the reaction tem-
perature was higher, the rate at which BzHO was
reduced to (1) increased. Furthermore, the increase in
reaction speed was mostly observed in the tempera-
ture range 30–40 ꢂC.
The experiments carried out at 40ꢂ and 45 ꢂC were
the ones that presented the highest reaction rates,
with the experiments carried out at temperatures of
40 ꢂC showing the maximum value. In addition, it is
interesting to observe that in contrast with the results
obtained for the optimization of the temperature in
the stereoselective reduction of acetophenone
Figure 4. Rate constant (k) vs. reaction temperature.
(Demmel et al. 2021), in the reduction of BzHO the
increase in the value of the calculated rate constants
did not reveal a linear behaviour for the range
between 25–40 ꢂC. It is important to mention that no
studies have yet been described for carrying out opti-
mization of the temperature using seeds as biological
reducers in BzHO reduction procedures. In this sense,
using Phaseolus vulgaris (bean) seeds as a biocatalyst,
the formation of (1) was reported for a reaction tem-
perature at 25 ꢂC with a total process time of 24 h
ꢀ
ꢀ
ꢀ
(Solıs et al. 2017, Solıs et al. Solıs et al. 2019).
Finally, since it is possible to use B. variegata seeds
at a higher working temperature (40 ꢂC) than in previ-
ous reports, it was possible to carry out the reduction
of BzHO in a shorter reaction time (ꢃ10 h), with this
characteristic clearly representing
a
comparative
advantage. In fact, 40 ꢂC was the most suitable tem-
perature for carrying out the BzHO bio-reduction pro-
cess, and for this reason, this temperature was
selected to continue with our studies.

8
G. I. DEMMEL ET AL.
by GC-FID to determine the percentage conversion.
These results are shown below in Figure 5.
As can be seen in Figure 5, the use of different
DMSO concentrations in the reaction medium did not
have a significant effect on BzHO formation, and in all
3.5. Effect of DMSO concentration
As mentioned above, biocatalytic systems are more
environmentally friendly because the reaction medium
is usually water. However, this situation can be a
drawback when using organic substrates that are
insoluble or poorly soluble in the reaction media.
Although this limitation can be solved, in part, by
using co-solvents that facilitate the solubilization of
the substrates, the effect of the cosolvents must be
established, with the aim of preventing negative
effects on the enzymatic activity, and consequently,
reducing the performance of the reaction. As a coun-
terpart, some cosolvents can sometimes have a benefi-
cial effect on the regeneration of cofactors (Du et
al. 2014).
As demonstrated in previous studies using the
whole seed of B. variegata (Demmel et al. 2021),
DMSO was the only cosolvent that did not interfere
with the enzymatic activity in the reductive transform-
ation of acetophenone. Furthermore, a small improve-
ment in the performance of the reaction was
observed when using DMSO as a cosolvent.
In order to determine the effect of the concentra-
tion of DMSO in the reaction medium, essays with dif-
ferent concentrations of DMSO (0.5–10 mL for each
80 mL of water) were carried out. Additionally, an
experiment was also made without the addition of a
cosolvent (control test). All the tests were carried out
at 40 ꢂC using 100 lL of BzHO with 10 g of whole
seeds of B. variegata, at 100 rpm for 24 h (substrate
concentration 1.25 g/L with 125 g/L of seeds). Samples
were taken at the end of each reaction and analyzed
the tests
a
percentage of conversion >98%
was obtained.
It is important to mention that although BzHO is
relatively soluble in water, the results achieved in our
experiments provide a solution for substrates that
have a very low or zero solubility in the reaction
media and require appreciable amounts of a cosolvent
to carry out their solubilization. Due to the results
obtained in these studies, the addition of DMSO at a
final concentration of 9% v/v was selected as being
the optimal amount of cosolvent to use in the essays
that are shown in the following sections.
3.6. Effect of BzHO concentration
As a consequence of the results obtained in the previ-
ous essays, a new substrate loading study was again
performed, but this time using the addition of DMSO
as a cosolvent in order to determine if the tolerance
of the whole seeds of B. variegata could be improved.
To carry out this study, concentrations ranging
between 1.25 g/L and 5.6 g/L of BzHO were used with
10 g of whole seeds, 80 mL of milliQ water, and the
addition of 8 mL of DMSO. The system was maintained
at 40 ꢂC with orbital shaking (100 rpm) for 24 h. At the
end of the reaction, samples were taken, which were
Figure 5. Effect of different amounts of DMSO on the reduction of BzHO promoted by whole seeds of B. variegate.

BIOCATALYSIS AND BIOTRANSFORMATION
9
analyzed by GC-FID to determine the conversion per-
centage. These results are shown below in Figure 6.
As can be seen in Figure 6, when the BzHO concen-
tration was increased from 1.25 to 2.5 g/L, the conver-
sion percentage only decreased slightly due to the
addition of DMSO at 9% v/v, while for higher concen-
trations (>2.5 g/L), the conversion decreased more
markedly. Thus, it is clear that concentrations higher
than 2.5 g/L of the substrate can cause an inhibitory
effect on the catalytic activity of the enzymatic system
in the seeds of B. variegata. For this reason, 2.5 g/L of
BzHO was selected as the optimal concentration as
the loading ratio of the substrate per 112 g/L of seeds.
Finally, it should be mentioned that essays to opti-
mize the pH of the reaction media were not carried
out, because our laboratory team (Demmel et al.
2021) had reported that the use of a buffer to regulate
the concentration of hydrogen ions was detrimental
to the performance of the reaction of reduction of
acetophenone, probably due to a negative effect on
the enzymatic system caused by the modification of
the ionic strength of the medium.
3.7. Bioreduction of BzHO to laboratory scale
In order to demonstrate the capacity of the developed
biocatalytic system, a reaction was carried out using
the optimized working conditions but on a larger
scale. To accomplish this, 1.6 g of BzHO, 80 g of seeds
of B. variegata, which had been previously washed
and disinfected, 64 mL of DMSO, and 640 mL of sterile
distilled water were placed in a disinfected 2 L conical
flask (final concentration of the substrate: 21.2 mM).
The reaction was carried out for 24 h, keeping the
reaction system at 40 ꢂC under constant orbital shak-
ing of 100 rpm, after which, a sample was taken and
analyzed by GC-FID (95% conversion). The reaction
crude was filtered and the water reaction media was
extracted for 24 h with ethyl acetate, using a liquid-
Figure 6. Effect of the concentration of BzHO in the reaction
media on the production of (1) promoted by the seeds of B.
variegata with the addition of DMSO at 9% v/v.
Table 2. Ability of whole seeds of B. variegata to reduce different substituted BzHOs.
Substrate/product
Time
Conversion
(%)a
Entry
R1
R2
R3
(days)
Yield (%)b
1c
2
3
4
5
6
7
8
9
H–
NO2–
H–
H–
Cl–
H–
H–
Cl–
H–
HO–
H–
H–
H–
H–
H–
H–
NO2–
H–
H–
Cl–
H–
H–
H–
H–
HO–
H–
H–
CH3O–
H–
H–
H–
NO2–
H–
1
1
1
1
2
2
2
2
2
2
3
1
2
4
3
95
90
98
94
>99
92
49
71
42
54
70
45
47
30
41
H–
Cl–
Cl–
F–
H–
H–
HO–
CH3–
HO–
91
97
>99
nrd
>99
94
>99
73
10
11
12
13
14
15
50
41
25
7
C6H5–CH ¼ CH–
62
61
aConversion (%) determined by GC-FID. bIsolated yield by chromatographic column. Scale bioreduction reaction of BzHO. dnr ¼ No reaction.
c

10
G. I. DEMMEL ET AL.
liquid extractor. The organic solution was evaporated
and the residue was purified by a chromatographic
column, with 0.78 g of pure (1) being isolated (yield:
49%). This correlates to the productivity of 1.11 g Lꢁ1
or a space–time yield of 0.046 g Lꢁ1hꢁ1. It is important
to note that both the extraction and purification proc-
esses were not optimized.
reducing agent, reported better reactivities for those
benzaldehydes that presented substitutions in the
para position.
When the seeds of B. variegata were used to reduce
benzaldehydes with nitro substituents (Table 2, entries
2–4), the transformation was chemoselective, in add-
ition to being efficient (conversions >90%), since no
parallel reduction of the nitro to amine groups was
observed. In line with our results and working with
different vegetables, similar findings were observed by
3.8. Advantages, limitations and scope of using
whole seeds of B. variegata as a biocatalyst in
the preparation of substituted benzylic alcohols
ꢀ
Suarez-Franco et al. (2010) for this type of substituted
BzHOs. However, although Leyva et al. (2012) also
observed chemoselectivity in the reduction of nitro-
benzaldehydes, the conversions to the corresponding
nitrobenzylic alcohols (3) and (4) were significantly
lower (60 and 65% conversions respectively) when
compared to the conversion percentages obtained
using whole seeds of B. variegata as the biocatalyst
(Table 2 entries 3–4; 98 and 94% respectively).
It is also important to mention that Andrade et al.
(2006) observed when using Manihot esculenta,
Colocasia esculenta, Allium schoenoprasum and
Arracacia xanthorrhiza as biocatalysts to carry out
stereoselective reductions of nitroacetophenones, that
the nitro groups were reduced to amine in addition to
the formation of the corresponding alcohol.
Furthermore, Colocasia esculenta produced a 100%
reduction of nitro groups after six days of reaction. In
contrast, Alves-Ferreira, Da Silva, et al. (2012) found
that the seeds of Lens culinaris were capable of reduc-
ing substituted nitrobenzenes to their corresponding
anilines, but with different degrees of efficiency.
After achieving the optimization of the main variables
of the bioreduction process of the BzHO to synthesize
the alcohol (1), a study of the biocatalytic reduction of
substituted BzHOs was carried out. With this objective
in mind, several substituted BzHOs derivatives with
different substituents (fluorine, chlorine, nitro, hydroxy,
methyl, and methoxy) on the benzene ring were
selected in order to establish the advantages, limita-
tions and scope of using the whole seeds of B. varie-
gata as a biological reducer. In addition, vanillin and
cinnamaldehyde were also used as substrates. These
results are shown below in Table 2.
As can be seen in Table 2 (entries 1–13), the whole
seeds of B. variegata very efficiently reduced substi-
tuted benzaldehydes to their corresponding alcohols,
presenting conversion percentages higher than 90%,
except for vanillin and cinnamaldehyde, whose corre-
sponding alcohols were obtained in moderate conver-
sions. The only exception, with a lack of reactivity,
was 2-hydroxybenzaldehyde, which could not be
reduced under our working conditions (Table 2, entry
10). Also in Table 2 (entry 1) are shown the results
obtained for a scale bioreduction of BzHO, included
as a reference. Nevertheless, it can be seen from the
results obtained that no significant differences in
reactivity were observed between substrates contain-
ing electron-withdrawing substituents and those with
electron-donating substituents (Table 2, entries 3, 5, 8
and 9 versus 11–13). As a counterpart, it is important
A special mention should be made of the results
obtained in the bioreduction of benzaldehydes con-
taining chlorine as substituents. In this sense, the con-
version percentages were excellent (91 to >99%)
(Table 2, entries 5–7). However, and unlike that
observed for the seeds of B. variegata, when Leyva et
al. (2012) investigated the same substrates working
with the extract of Aloe vera leaves, the percentages
ꢀ
were significantly lower (65%). Moreover, when Solıs
ꢀ
to mention that Solıs et al. (2019) carried out studies
et al. (2019) reported the same reduction reactions
using one of the varieties of Phaseolus vulgaris seeds,
they obtained a 70% conversion in the formation of
(5), 16% for (6), and 97% for (7) (Table 2 entries 5–7).
The results achieved by reducing chlorine substi-
tuted benzaldehydes should be highlighted as being
important since both (7) and (8) are key intermediaries
in the synthesis of the commercial antifungals econa-
zole and miconazole (Cuevas et al. 2004). Furthermore,
(8) is also used as an intermediate in the synthesis of
oxiconazole (Soltani et al. 2009).
using different varieties of Phaseolus vulgaris seeds,
and observed that both the reducing activity and the
degree of conversion to the corresponding alcohol of
the substituted BzHOs studied depended on the acti-
vating/deactivating effects, electrophilicity of the sub-
stituents and steric hindrance, as well as on their
position on the benzene ring with respect to the car-
bonyl group. Furthermore, similar results using Aloe
vera leaves were observed by Leyva et al. (2012),
while Bertini (2012), using Glicine max seeds as a

BIOCATALYSIS AND BIOTRANSFORMATION
11
In another study, an excellent result was reported
in the reduction of 4-flurobenzaldehyde (Table 2, entry
9) to obtain the fluorine derivative (9), which is
important as this product is one of the key raw mate-
rials in the synthesis of the recently patented anti-
product (80%), while guaiacol was formed as a sec-
ondary product (20%).
It should be highlighted that alcohol (14) is used in
the treatment of some neurodegenerative diseases
such as Parkinson’s, due to its pharmacokinetic charac-
teristics and its bioactivity (Hsu et al. 2008), in addition
to it having anticonvulsant and anti-inflammatory
effects (Hsieh et al. 2000). Furthermore, (14) is a
potential chemical platform for the production of
thermoset epoxy resins obtained from renewable raw
materials (Hernandez et al. 2016).
Although the conversion percentage obtained dur-
ing the synthesis of (15) was only moderate (Table 2
entry 15; 62%), the results achieved were higher than
those described by Machado et al. (2008) working
with Manihot species. When Luna et al. (2014) carried
out the reduction of cinnamaldehyde with Passiflora
edulis, 41% of (15) was obtained, while by using Zea
mays leaves as a biocatalyst a conversion of 50% was
recorded for the formation of the same alcohol. In
cancer
agent
monofluorobenzyl
ester
of
norcantharidin carboxylate (Zhao et al. 2018; Li et al.
2020). Similarly, the 3-hydroxy derivative (11) (Table 2,
entry 11; >99%) is one of the necessary intermediates
for the synthesis of insecticides such as permethrin
and phenothrin (Naumann 1990), and the p-hydroxy
derivative (12) (Table 2, entry 12; 94%) is a key inter-
mediate in the synthesis of the commercial drug biso-
prolol, which is
a
cardioselective b1-adrenergic
blocking agent used in treating high blood pressure
(Soloviev et al. 2001).
In particular, an excellent result was achieved when
4-methylbenzaldehyde (Table 2, entry 13) was reduced
under our reaction conditions (conversion percentage
>99% for 2 days of reaction). In addition, 4-methylbe-
zyl alcohol is used in perfumery (Villa et al. 2008).
Although there are some reports in the literature that
have described the reduction of this aldehyde for the
formation of (13), such efficiency was not achieved
(Leyva et al. 2012).
With regard to obtaining (14) (Table 2, entry 14), it
should be mentioned that the reaction was carried
out with a longer reaction time than that carried out
on BzHO (4 days vs. 1 day) to obtain the corresponding
alcohol (Table 2 entry 14 vs. 1). Furthermore, the con-
version percentage was also lower (73%). However,
the efficiency of this reaction was similar to the values
reported by Luna et al. (2014) when working with the
leaves of Zea mays (64%) and also to the value
reported by Carvalho da Silva et al. (2018) using
Ximenia americana grains (51%). On the other hand,
Leyva et al. (2012) did not observe any formation of
(14) when the extract of Aloe vera leaves was used as
a biocatalyst. However, when the biocatalyst was
Saccharum officinale juice, the conversion percentages
ꢀ
addition, it should be mentioned that when Bordon et
al. (2021) used whole seeds of Ligustrum lucidum, a
very good percentage of conversion was obtained
with reaction times similar to those observed with the
seeds of B. variegata (3 days). Furthermore, it is import-
ant to highlight that the reductive process carried out
on cinnamaldehyde using whole seeds of B. variegata
as a biological reducer was chemoselective (Table 2
entry 15), and therefore, the double bond remained
unaltered in the final product. Related to this,
~
Assunc¸ao et al. (2008), when carrying out the same
transformation but using the juice of Saccharum offin-
cinale, not only obtained the product of interest, but
also the reduction of unsaturation in addition to the
concomitant oxidation of the aldehyde group to car-
boxylic acid. Due to this fact, the end product turned
out to be a mixture of (15), 3-phenylpropyl alcohol,
and 3-phenyl propionic acid. The reduction of cinna-
maldehyde is important because (15) is used as a key
intermediate in the synthesis of fluoxetine, atomoxe-
tine, and nisoxetine (Gao and Sharpless 1988), in add-
ition to dapoxetine (Sasikumar and Nikalje 2012) and
reboxetine (Henegar and Cebula 2007), which are all
used as commercial antidepressants.
It is important to mention that, as far as we have
been able to determine, the isolation and characteriza-
tion, using a plant-catalyzed methodology, of the
group of enzymes responsible for the bioreduction
processes of carbonyl groups has not been described
in detail. However, it is probable that the enzyme
~
obtained were approximately 90% (Assunc¸ao et
al. 2008).
Salvano et al. (2011) carried out similar studies
aimed at reducing vanillin using roots of Conium mac-
ulatum. On this occasion, although (14) was formed, it
was as a secondary product (27%) since the reaction
mainly transformed the substrate into 2-methoxyphe-
nol (guaiacol ꢁ 73%), possibly by the decarboxylation
ꢀ
of vanillin. A similar situation was observed by Bordon
et al. (2021) using whole seeds of Ligustrum ludidum.
However, in this study, (14) was formed as a majority
alcohol dehydrogenase
which is dependent on the nicotinamide cofactors as
– ketoreductases (AD-KR),

12
G. I. DEMMEL ET AL.
redox equivalents ( Blanchard and Van De Weghe
2006; Xie et al. 2009) , is involved in the realization of
this transformation. In this sense, it is worth mention-
ing that our research group has recently reported evi-
dence (Demmel et al. 2021) that Bauhinia variegata
seeds use NADPH as a redox cofactor during the bio-
catalysis process, which is lost by washing when the
seeds are recycled for use in consecutive reactions.
Furthermore, Xie et al. (2009) examined the bioreduc-
tion of alkyl aryl ketones employing a pre-concen-
trated enzymatic system derived from Adzuki beans
and showed that glucose may be employed as an effi-
cient cosubstrate in the NADPH recycling process. In
light of both findings, it is possible that some of the
sugars that are part of the high carbohydrate content
(61.8% – Table 1, entry 2) found in B. variegata seeds
are involved in the NADPH recycling process, which is
employed by the enzyme system of these seeds as
the redox equivalent to carry out the transformations
detailed in our research. These observations support a
mechanistic hypothesis via AD-KR enzymes (Xie et al.
2009). Nevertheless, it is clear that further studies are
needed to establish the specific characteristics of the
enzyme system and, in line with this, studies are cur-
rently being carried out with the objective of isolating
and characterizing the enzymes present in B. varie-
gata seeds.
Finally, the yield of the products, obtained after the
corresponding purification, showed differences with
respect to the conversion percentage determined by
GC-FID. This situation may be attributed to difficulties
experienced during the product extraction process,
due to the formation of emulsions that notably com-
plicated the isolation procedure of the final product,
which may be explained by the relatively high lipid
content (18.1% – Table 1, entry 3) observed in the
determinations made during characterization of seeds,
and could be the reason why the formation of stable
emulsions was observed. Thus, it is necessary to
emphasize that the purification method used to
obtain the synthesized benzyl alcohols has not
been optimized.
nature of the substituents on the aromatic ring of the
benzaldehydes used as substrates and, in general, the
conversion percentages for the formation of the prod-
ucts were excellent in most cases. In addition, the
methodology employed allows DMSO to be used as a
co-solvent at an appreciable concentration in the reac-
tion medium, which is a definite advantage when
working with substrates of poor or no solubility
in water.
It is noteworthy that the enzymatic systems present
in the B. variegata seeds turned out to be chemoselec-
tive, which permitted the substituted benzaldehydes
to be obtained with some functional groups under-
going a biotransformation process, under similar reac-
tion conditions to those reported in the literature.
Currently, sustainability in chemical processes is
being increasingly looked upon as a necessity in order
to eliminate or minimize the impact on the environ-
ment of the productive procedures used. Thus, the
biocatalytic processes promoted by whole seeds of
vegetables could be considered a valuable tool that
easily permits, in a cheap and compatible way with
the environment, valuable products to be obtained
with different applications. In this sense, the results
described in this work, using the whole seeds of B.
variegata as a biological reducer, allow us to offer new
biocatalytic environmentally friendly strategies for the
production of benzyl alcohols, which are used as key
intermediates for obtaining important bioactive mole-
cules with industrial application. Moreover, the simpli-
city, efficiency, fairly wide scope, and the observed
chemoselectivity of this biocatalytic approach are its
most advantageous features.
Finally, we believe that it is necessary to develop
procedures that use renewable inputs, in order to
evolve from a linear economy, consuming large
amounts of non-renewable natural resources and con-
sequently degrading the environment, to a resource-
efficient circular economy that is waste-free by design.
In line with this concept, further investigations using
whole seeds of B. variegata are currently ongoing in
order not only to understand the mechanism by
which this type of transformation occurs and in this
way, try to improve the productivity of the method-
ology but also to try to expand the applications of
this novel, efficient, inexpensive, renewable and envir-
onmentally friendly biocatalytic agent.
4. Conclusions
Our studies were performed with the objective of
identifying efficient environmentally friendly synthesis
methods and established the ability of B. variegata
seeds to carry out the reduction of different benzalde-
hydes to their corresponding benzyl alcohols. In this
sense, it should be mentioned that the developed
methodology was shown to be little influenced by the
Acknowledgement
The authors thank Dr. Paul Hobson, a native speaker, for the
revision of this manuscript.

BIOCATALYSIS AND BIOTRANSFORMATION
13
clementine mandarin (Citrus reticulata) fruit grown in
annaba or by ruthenium catalysis for access to both enan-
tiomers. Chirality. 27(3):205–210.
Disclosure statement
No potential conflict of interest was reported by the
author(s). Laura I. Rossi is a researcher of CONICET.
Bertini LM. 2012. Soybean (Glycine max) as a versatile bioca-
talyst for organic synthesis. African J Biotechnol. 11(30):
7766–7770.
Blanchard N, Van De Weghe P. 2006. Daucus carota L. medi-
ated bioreduction of prochiral ketones. Org Biomol Chem.
4(12):2348–2353. doi:10.1039/b605233a
Funding
ꢀ
The present work was financially supported by Secretarıa de
ꢀ
ꢀ
Ciencia y Tecnologıa de la Universidad Nacional de Cordoba
ꢀ
ꢀ
Bordon DL, Vazquez AM, Decarlini MF, Demmel GI, Rossi LI,
Aimar ML. 2021. Optimisation of the bioreduction process
of carbonyl compounds promoted by seeds of glossy pri-
vet (Ligustrum lucidum - Oleaceae) and its application to
the synthesis of key intermediates. Biocat Biotrans. 39(1):
1–15.
ꢀ
(SeCyT-UNC) [33620180100619CB] and Universidad Catolica
ꢀ
de Cordoba.
ORCID
Carvalho da Silva RA, Marques de Mesquita B, Frota de
Farias I, Garcia do Nascimento PG, Lemos TLG, Queiroz
Monte F. 2018. Enzymatic chemical transformations of
aldehydes, ketones, esters and alcohols using plant frag-
ments as the only biocatalyst: Ximenia americana grains.
Mol Catal. 445:187–194.
Laura I. Rossi
Mario L. Aimar
http://orcid.org/0000-0003-1670-6529
http://orcid.org/0000-0001-8547-7198
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