Enzymatic chemical transformations of aldehydes, ketones, esters and alcohols using plant fragments as the only biocatalyst: Ximenia americana grains

Article abstract of DOI:10.1016/j.mcat.2017.11.033

The present study demonstrated the ability of Ximenia american as a biocatalyst in reduction, hydrolysis and esterification reactions. The reduction reactions of aldehydes and ketones, ester hydrolysis and esterification of alcohols were carried out with interesting results. Reduction of ketones afforded yields of 6–60% with ee in the range of 35–>99% and that of aldehydes in yields of 51–99%. On the other hand, ester hydrolysis afforded yields of 58–98% with ee in the range 34–87%, while esterification of alcohols in 18–99% yields. Experimental conditions for all reactions have been defined using standard substrates as indicated in results and discussion. Some of the products are the potential building blocks for the synthesis of molecules which are of pharmaceutical and agrochemical importance.

Full text of DOI:10.1016/j.mcat.2017.11.033

Molecular Catalysis 445 (2018) 187–194
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Enzymatic chemical transformations of aldehydes, ketones, esters and
alcohols using plant fragments as the only biocatalyst: Ximenia
americana grains
Romézio Alves Carvalho da Silva∗, Bruna Marques de Mesquita, Iolanda Frota de Farias,
Patrícia Georgiana Garcia do Nascimento, Telma Leda Gomes de Lemos,
Francisco José Queiroz Monte
Programa de Pós-Graduac¸ ão em Química, Universidade Federal do Ceará, Centro de Ciências, CEP 60455-760, Fortaleza, Ceará, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study demonstrated the ability of Ximenia american as a biocatalyst in reduction, hydroly-
sis and esterification reactions. The reduction reactions of aldehydes and ketones, ester hydrolysis and
esterification of alcohols were carried out with interesting results. Reduction of ketones afforded yields
of 6–60% with ee in the range of 35–>99% and that of aldehydes in yields of 51–99%. On the other hand,
ester hydrolysis afforded yields of 58–98% with ee in the range 34–87%, while esterification of alcohols
in 18–99% yields. Experimental conditions for all reactions have been defined using standard substrates
as indicated in results and discussion. Some of the products are the potential building blocks for the
synthesis of molecules which are of pharmaceutical and agrochemical importance.
Received 11 September 2017
Received in revised form
14 November 2017
Accepted 24 November 2017
Keywords:
Bicatalytic transformation
Ximenia americana grains
Reduction
© 2017 Published by Elsevier B.V.
Hydrolysis
Esterification
1. Introduction
ment. Depending on the vegetable used, either enantiomer may
be made available in high yield enantiomeric excess (ee), which
This work will continue our investigations [1–7] using locally
available vegetables as an alternative for developing countries.
Great attention has been paid to syntheses of pure compounds or
chiral synthons and are increasing in demand for the development
of complex pharmaceutical drugs, agrochemicals, and cosmetic
products. The ability of natural sources to induce chirality has been
known for a long time. Consequently, due to the increasing need for
chiral drugs, the interest in using enzymes has steadily increased,
with the consequent development in the field of biocatalysis. In
recent years, the use of the intact plant, instead of plant cells cul-
tures or isolated enzymes have been studied as potential agents
for biotransformation reactions. The use of the intact plant system
instead of plant cells or isolated enzymes exhibits several consid-
erable advantages. For example, the need for expensive cofactors
is eliminated since the plant automatically provides this require-
could be critical from a drug development/bioactivity evaluation
perspective. In addition, the lower time factor, the lower cost, and
the ease preparation of the reactive system make the use of the
whole plant an attractive economic alternative. Furthermore, there
is evidence that such systems can be used repeatedly (4–6 times)
without substantial loss of activity, thus reducing costs even fur-
ther. Although the cofactor regeneration is a limiting factor a few
cases, in this study it was observed that with the direct use of X.
americana grains in the esterification reactions, the biocatalytic
activity was maintained five times (about 120 h) using the same
material (grains) in the five reaction cycles. Thus, there was only a
minimal decrease from the first (99.9%) to the fifth reaction (90.0%)
(Fig. 2). Using intact plant systems, much of the research has been
developed to conduct reduction, hydrolysis, and esterification reac-
tions. As examples, racemic 2-methylcyclhexanone was completely
reduced to a 1: 1 mixture of (1S,2R) and (1S,2S) with >99% ee and
racemic 2-hydroxycyclohehanone was a 35:65 mixture of (1S,2R)
and (1S,2S) (>95% ee), respectively, using carrot (Daucus carota) root
material [8]; enantioselectivity was observed in the reduction of
acetophenone and 3-metoxiacetophenone, where the respective
produced alcohols were formed as the enantiomer with “S” con-
figuration, with ee values of 95 and 97% for Manihot Esculenta and
∗
Corresponding author.
E-mail addresses: romezioac@gmail.com (R.A.C. da Silva),
brunamarques1403@gmail.com (B.M. de Mesquita), iolanda frota91@hotmail.com
(I.F. de Farias), georgina.quimica@gmail.com (P.G.G. do Nascimento),
tlemos@dqoi.ufc.br (T.L.G. de Lemos), fmonte@dqoi.ufc.br (F.J. Queiroz Monte).
http://dx.doi.org/10.1016/j.mcat.2017.11.033
2468-8231/© 2017 Published by Elsevier B.V.

188
R.A.C. da Silva et al. / Molecular Catalysis 445 (2018) 187–194
94% and 96% for Manihot. dulcis, respectively. Regioselectivity was
observed in the reduction reaction of the aldehydes cinnamic and
2-metilcinnamic, with bioconversion of 87 and 92%, respectively,
using the two Manihot species [9–12].
In the case of esters hydrolysis reactions, the apple roots
(Malus pupila) converted racemic 1-phenyetyl acetate to the (S)-
1-phenyletyl alcohol in 98% yield [13]; the Manihot species were
very effective in the hydrolysis of the aliphatic ethyl butyrate ester
producing butyric acid in 91.5% and 94% yield, but they proceeded in
low yields (35.5 and 35.3%) with the aromatic ethyl benzoate ester
[10]; the catalytic activity for hydrolyzing reactions using orange
peels of Citrus aurantium L. was recently reported [6].
detector equipped with a chiral column CP-Chirasil-Dex CB [␤-
cyclodextrin (25 m x 0.25 mm x 0.25 ␮m)], using He as the carrier
gas (1.2 mL/min) in split mode; the injector and detector tempera-
tures were 200 and 220 ◦C, respectively; column temperature was
programmed at 80 ◦C (7 min) to 120 ◦C (10 min) at 6 ◦C/min, then
10 ◦C/min to 200 ◦C (10 min). The 1H and 13C spectra were recorded
in Brucker DPX 300 instrument with TMS as internal standard and
CDCl3 as solvent. Chemical shifts are reported in ␦ and coupling
constants (J) in Hertz (Hz).
2.2. Biocatalyst
Already, in the case of esterification reactions, a study was
recently reported [7] involving the use of orange peels (Citrus
aurantium) in esterification reactions of alcohols. Thus, the alco-
hol ( )-1-phenylethan-1-ol was used to produce the corresponding
ester ( )-1-phenylethyl acetate. In hexane, the reaction took place
with good rates of both conversion (65.05%) and ee (80%) of the
ester. Also was studied the behavior of the reaction as a function of
temperature. The results showed that running the reaction above
25 ◦C resulted in higher yields. For example, using substrate ( )-
1-phenylethan-1-ol, the yield and ee increased from 73.3 to 88.8%
and 88 to 99%, respectively, when the reaction temperature was
raised from 38 to 55 ◦C. Regarding the observed increase in forma-
tion of the isomer (R)-as well as the increase in conversion from
73.3 to 88.8% with increasing temperature, it is noteworthy that, in
the resolution of racemates by enantioselective esterification, the
optimal conversion rate is 50% [14]. Thus, conversions above 50%
indicate, in this case, that part of the (R)-enantiomer has been con-
verted to ester; as other examples, immobilized Manihot esculenta
preparation was used as a novel biocatalyst in the enantioselec-
tive acetylation of alcohols [15,16], and the Origanum, Mentha and
Salvia species, in the enantioselective acetylation of monoterpenes
[17].
Fresh grains of X. Americana were collected in the rural area
of the State of Ceará, Brazil. A voucher specimen (Ximenia amer-
icana L.), number 040411, has been deposited in the Herbarium
Prisco Bezerra, Departamento de Biologia da Universidade Federal
do Ceará, Brazil.
2.3. General procedures for biotransformations
The grains were washed with water and then maintained in a 5%
sodium hypochlorite aqueous solution for 20 min. Then, they were
washed with distilled water, dried at room temperature (28 ◦C)
for 24 h, and peeled with a sterilized cutter. To increase the con-
tact of the substrate with the biocatalyst, the external layer was
removed and the rest was shredded in blender, washed with ace-
tone, and filtered to eliminate excess water. The material was left at
room temperature for about 2 h to remove residual solvent, thereby
yielding the biocatalyst used in all experiments.
2.3.1. Protein contents/hydrolytic enzymatic activity
The suspension of crushed grains (3.0 g) of X. Americana in
distilled water (50 mL) was stirred for 72 h at 175 rpm at room
temperature. After filtration, the aqueous solution was subjected
to methodology for the determination of the estimated total pro-
tein content (19.77%) according to the Kjeldahl method, modified
by Nogueira and Souza [18]. The hydrolytic enzymatic activ-
ity of grains was performed by hydrolysis of the acetate ester
of p-nitrophenol (5 mM) in acetonitrile/water (95:5). One gram
of the grains was added to 10 mL of substrate (p-nitrophenol
acetate) under stirring at 175 rpm for 2 h at room temperature.
The content of p-nitrophenol was determined by calibration curve
(y = 0,2859x + 0.0037, R2 = 0.9881) in the spectrophotometer at
410 nm. Considering a unit of enzyme activity (U) defined as the
amount of biocatalyst (g) required to release 1 mol of p-nitrophenol
per minute, the enzyme activity found in the grains was 9.38 U
(1 ␮mol/g) [19].
In order to avoid the time-consuming and care-intensive prepa-
rations of plant cell cultures, we have been investigating the
possibility to directly use of parts of plants as biocatalysts in reduc-
tion reactions of ketones and aldehydes, hydrolysis of esters and
esterification of alcohols. Fresh grains of Ximenia americana has
therefore been used as biocatalyst and acetophenone (1), benzalde-
hyde (6), methylbenzoate (14) and, benzyl alcohol (19) were chosen
as model compounds.
2. Experimental
2.1. General methods
The solvents and reagents used were of analytical grade pur-
chased from Merck and Synth and were used without further
purification. Chemical reactions were monitored by thin-layer
chromatography (TLC), performed using precoated plates (Alu-
minum foil, silica gel 60 F254 Merck, 2–25 ␮m) and visualized
with UV light (254 and 365 nm) and/or vanilin-HClO4 0,75 M-
EtOH as the detecting agent. Merck 60 silica gel was utilized
(70–230 mesh) was used for flash chromatography. GC analysis
were performed on a Shimadzu GC–MS QP2010SE Plus instrument
equipped with capillary Rtx® -5MS (95% dimethylpolysiloxane and
5% diphenyl) column (30 m × 0.25 mm × id 0.25 m) using He as
carrier gas (1.0 mL/min); the injector and detector temperatures
were 250 and 280 ◦C, respectively; column temperature was pro-
grammed at 10 ◦C/min from 40 ◦C to 180 ◦C, then at 20 ◦C/min to
280 ◦C. Mass spectra were recorded in the range of 47–600 Da
(70 eV) with the detector in scan mode. The enantiomeric excess
(ee) of 1a–5a, 7a, 8a, 17a, 18a and 22a, were determined by
GC analysis using a Shimadzu GC 2010 instrument with a FID
2.4. Biocatalysed transformations
In separate experiments, ketones 1–8 [50 mg: 0.416 mmol (1),
0333 mmol (2), 0.362 mmol (3), 0.324 mmol (4), 0.264 mmol (5),
0.510 mmol (6), 0.390 mmol (7) and 0.290 mmol (8)] and aldehydes
9–13 [50 mg: 0.470 mmol (9), 0.367 mmol (10), 0.367 mmol (11),
0,328 mmol (12) and 0.320 mmol((13)] were added to a stirred
suspension of freshly grains (5.0 g) in 50 mL of distilled water
[pH 5.0 for ketones and 7.0 for aldehydes (pH 7.0 maintained by
a buffer solution of NaH2PO4 and Na2HPO4)] and the reaction
mixtures were stirred in orbital shaker (175 rpm) at room temper-
ature (30 ◦C) for 72 h. The progress of the reactions was monitored
by thin-layer chromatography (TLC) analysis. The final suspen-
sions were the filtered off and the filtrates were extracted AcOEt
(3 × 30.0 mL). The organic phases (dried with anh. Na2SO4) were
then evaporated under reduced pressure and the residues chro-
matographed on silica gel column in order to obtain the alcohols
(R,S)-1a (28.0 mg, 0.229 mmol), (R,S)-2a (36.0 mg, 0.236 mmol),

R.A.C. da Silva et al. / Molecular Catalysis 445 (2018) 187–194
189
Table 1
The esterification reaction was carried out five times consecutively
using the same material (biocatalyst).
Reduction of aromatic (1-5) and aliphatic (6-8) ketones with Ximenia americana
grains.
entry
substract
yield (%)
ee
configuration
1
2
3
4
5
6
7
8
acetophenone
54
60
6
48
53
14
43
29
48
89
99
91
>99
–
S
S
R
S
S
–
R
S
2.7. Compounds identity
3-methoxyacetophenone
4-fluoroacetophenone
4-chloroacetophenone
2,4-dichloroacetophenone
cyclohexanone
1a
– 5a and 7a, 8a: GC-FID – CP-Chirasil-Dex CB (␤-
cyclodextrin), 80 ◦C (7 min) to 120 ◦C (10 min) at 6 ◦C/min, then
10 ◦C/min to 200 ◦C (10 min); 6a: GC–MS: capillary Rtx® -5MS (95%
dimethylpolysiloxane and 5% diphenyl); injector 250 ◦C and detec-
tor 280 ◦C; column temperature at 10 ◦C/min from 40 ◦C to 180 ◦C,
then at 20 ◦C/min to 280 ◦C.
7-octanone
undecanone
81
35
ee: enantiomeric excess.
0.05 g substrate and 5.0 g X. amercana grains in H2O (50 mL), pH 5.0, 175 rpm, 30 ◦C,
72 h.
(
)-1-Phenylethan-1-ol (1a) – tR (S) 15.7 min; tR (R) 15.5. MS:
m/z 122 (M+), 107 (M-15, 100%), 79 (M-43, 100%), 43. 1H NMR
(300 MHz, CDCl3): ␦H 1.44 (d, 6.0 Hz, 3H), 4.80 (q, 6.0 Hz, 1H), 7.28
(sl, 5H). 13C NMR (75 MHz, CDCl3): ␦C 25.1 (CH3), 70.0 (HC O),
125.4 (2HC), 127.2 (HC ), 128.3 (2HC), 146.0 (C).
(R,S)-3a (13.0 mg, 0.093 mmol), (R,S)-4a (29.0 mg, 0.185 mmol),
(R,S)-5a (32.0 mg, 0.167 mmol), 6a (12.0 mg, 0.120 mmol), (R,S)-
7a (27.0 mg, 0.207 mmol) and (R,S)-8a (18.0 mg, 0.104 mmol)
from the ketones, and the alcohols 9a (54.0 mg0.499 mmol), 10a
(40.0 mg, 0.289 mmol), 11a (48.0 mg, 0.347 mmol), 12a (28.0 mg,
0.181 mmol) and 13a (33.0 mg, 0.208 mmol) from the aldehy-
des. In separate experiments, esters 14-18 [50 mg: 0.367 mmol
(14), 0.333 mmol (15), 0.333 mmol (16), 0.252 mmol (17) and
0.304 mmol (18) were added to a stirred suspension of fresh grains
(2.0 g) in 50 mL of water (pH 5.0) and the reaction mixtures were
stirred in orbital shaker (175 rpm) at room temperature (30 ◦C) for
72 h. According to the previous procedure (ketones and aldehy-
des), hydrolysis of 14-18 gave 14a (42 mg, 0.344 mmol), 15a (41 mg,
0.335 mmol), 16a (42 mg, 0.388 mmol), 17a (22 mg, 0.140 mmol)
and 18a (27 mg, 0.221 mmol). In separate experiments, alcohols 19-
24 [50 mg (50 ␮L): 0.462 mmol (19), 0.499 mmol (20), 0.409 mmol
(21), 0.361 (22), 0.361 mmol (23), and 0.383 mmol (24) were added
to a stirred suspensions of fresh grains (2.0 g) in hexane (10 mL), and
after addition of vinyl acetate (100 ␮L) the reaction mixtures were
stirred in orbital shaker (225 rpm) at room temperature (30 ◦C) for
24 h.
(
)-3-Methoxy-1-phenylethan-1-ol (2a) – tR (S) 19.7; tR (R)
19.5. MS: m/z 152 (M+), 137 (M-15), 109 (M-43, 100%), 94, 77, 43. 1H
NMR (300 MHz, CDCl3): ␦H 1.49 (d, 6.0 Hz, 3H), 4.86 (q, 6.0 Hz, 1H),
6.80 (dd, 8.0 and 2.3 Hz, 2H), 6.95 (m, 2H), 7.25 (t, 8.1 Hz, 1H). 13
C
NMR (75 MHz, CDCl3): ␦C 25.3 (CH3), 55.4 (H3C O), 75.5 (HC O),
111.3 (HC), 113.1 (HC), 117.9 (HC), 129.7 (HC), 147.8 (C), 160.0
(O C).
(
)-4-Fluoro-1-phenylethan-1-ol (3a) – tR (S) 14.6; tR (R) 14.3.
MS: m/z 140 (M+), 125 (M-15, 100%), 97, 96, 77, 43. 1H NMR
(300 MHz, CDCl3): ␦H 1.49 (d, 6.4 Hz, 3H), 4.80 (q, 6.4 Hz, 1H), 6.99
(t, 8.7 Hz, 2H) and 7.30 (dd, 8.4 and 5.5 Hz, 2H). 13C NMR (75 MHz,
CDCl3): ␦C 25.3 (CH3), 69.7 (HC O), 114.4/115.1 (2HC), 127.1/127.2
(2HC), 141.6/141.7 (C) and 160.5/163.8 (C).
(
)-4-Cloro-1-phenylethan-1-ol (4a) – tR (S) 19.9; tR (R) 19.6.
MS: m/z 156 (M) and 158 (M + 2), 141/143 (M-15, 100%), 121 (M-Cl),
77 and 43. 1H NMR (300 MHz, CDCl3): ␦H 1.41 (d, 6.4 Hz, 3H), 4.79
(q, 6.4 Hz, 1H), 7.24 (d, 8.6 Hz, 2H) and 7.28 (d, 8.6 Hz, 2H). 13C NMR
(75 MHz, CDCl3): ␦C 25.3 (CH3), 69.6 (HC O), 126.9 (2HC), 128.6
(2HC), 133.0 (C) and 144.5 (C).
According to the previous procedure, esterification of 19-
24 gave 19a (62 mg, 0.413 mmol), 20a (61 mg, 0.429 mmol),
21a (63 mg, 383 mmol), 22a (60 mg, 0.309 mmol), 23a (13 mg,
0.067 mmol) and 24a (25 mg, 0.145 mmol). The conversions and the
enantiomeric excess were determined by GC/MS and GC/FID anal-
ysis, respectively, and both were equipped with chiral columns.
Results in Table 1 (reduction of ketones), 2 (reduction of aldehy-
des), 3 (hydrolysis of esters), and 4 (esterification of alcohols). All
experiments were performed in duplicate. Identity of compounds
was confirmed by an analysis of their respective 1H and 13C NMR
spectra and chiral GC–MS/GC-FID analysis.
(
)-2,4—di-Cloro-1-phenylethan-1-ol (5a) – tR (S) 22.9; tR (R)
21.9. MS: m/z 190 (M) and 192 (M + 2), 175/177 (M-15, 100%),
147/149, 111/113, and 43. 1H NMR (300 MHz, CDCl3): ␦H 1.40 (d,
6.4 Hz, 3H), 5.17 (q, 6.4 Hz, 1H), 7.22 (dd, 8.4 and 2.0 Hz, 1H), 7.29
(d, 2.0 Hz, 1H) and 7.47 (d, 8.4 Hz, 1H). 13C NMR (75 MHz, CDCl3): ␦C
23.7 (CH3), 66.6 (HC O), 127.6 (2HC), 129.1 (HC), 132.2 (C), 133.4
(C) and 142.0 (C).
Cyclohexanol (6a) – tR 3.8. MS: m/z 100 (M+), 82 (M-H2O), 57
(100%), 44 and 41. 1H NMR (300 MHz, CDCl3): ␦H 1.10–2.65 (ms,
10H) and 3.60 (m, 1H, CH O). 13C NMR (75 MHz, CDCl3): ␦C 24.3 (2
CH2), 25.6 (CH), 35.6 (2 CH2) and 70.5 (HC O).
(
)-2–Octanol (7a) – tR (S) 12.7 and tR (R) 11.9 (acetates).
MS: m/z 130 (not registered), 129 (M-1), 115 (M-15), 97 and 45
([H3CCH OH]+, 100%). 1H NMR (300 MHz, CDCl3): ␦H 0.87 (t, 6.7 Hz,
3H), 1.37 (m, 11H), 1.55 (m, 2H), 3.61 (t, 6.4 Hz, 1H, CH-O). 13C NMR
(75 MHz, CDCl3): ␦C 14.2 (CH3), 22.8 (CH3), 25.9-32.9 (5 CH2) and
63.1 (HC O).
2.5. Standard acetylation of alcohols 7a and 8a: 7a’ and 8a’
In separate experiments, the esters 7a’ and 8a’ were
obtained by acetylation of 7a (125 mg) and 8a (125 mg)
with Ac2O (2 mL)/pyridine (1 mL) and catalytic amounts of 4-
dimetthylaminopyridine in CH2Cl2 (2 mL). After agitation at room
temperature for 2 h, the reaction mixture was treated with 0.4 M
CuSO4 and subjected to extraction with CH2Cl2 to afford 7a’ (99%)
and 8a’ (99%).
(
)-2-Undecanol (8a) – tR (S) 12.7 and tR (R) 11.9 (acetates).
MS: m/z 172 (not registered), 171 (M-1), 125, 111 (m/z 125-14), 97
(m/z 111-14), 83 (m/z 97-14), 55 (m/z 83-14), 45 ([H3CCH OH]+,
100%). 1H NMR (300 MHz, CDCl3): ␦H 0.83 (t, 6.6 Hz, 3H), 1.11 (d,
6.2 Hz, 3H), 1.22 (sl, 14H), 1.35 (m, 2H), 3.71 (m, 1H, CH-O). 13C NMR
(75 MHz, CDCl3): ␦C 14.1 (CH3), 23.8–39.4 (8 CH2), 68.0 (HC-O).
2.6. Reuse capacity
9a – 13a: GC–MS – capillary Rtx® -5MS (95% dimethylpolysilox-
ane and 5% diphenyl); injector 250 ◦C and detector 280 ◦C; column
temperature at 10 ◦C/min from 40 ◦C to 180 ◦C, then at 20 ◦C/min to
280 ◦C.
The alcohol 19 (50 mg, 0.462 mmol) was added to a stirred sus-
pensions of fresh grains (2.0 g) in hexane (10 mL), and after addition
of vinyl acetate (100 ␮L), the reaction mixtures were stirred in
an orbital shaker (225 rpm) at room temperature (30 ◦C) for 24 h.
Benzilic alcohol (9a) – tR 7.38. MS: m/z 108 (M+), 107 (M-H), 91
(M-OH), 79 (100%), 77, 51. 1H NMR (300 MHz, CDCl3): ␦H 2.38 (sl,

190
R.A.C. da Silva et al. / Molecular Catalysis 445 (2018) 187–194
1H), 4.66 (s, 2H, H2C O), 7.32 (sl, 5H). 13C NMR (75 MHz, CDCl3):
␦
65.4 (H2C O), 127.1 (2HC ), 127.5 (HC), 128.7 (2HC),141.0 (C).
C
3-Methoxy-benzilic alcohol (10a) – tR 10.62. MS: m/z 138 (M+,
100%), 137 (M−H), 121 (M-OH), 109, 94, 77. 1H NMR (300 MHz,
CDCl3): ␦H 3.81 (s, 3H, H3C O), 4.65, (s, 2H, H2C O), 6.85 (ddl, 1H),
6.92 (sl, 1H), 6.94 (dl, 1H), 7.28 (tl, 1H). 13C NMR (75 MHz, CDCl3):
Fig. 1. Reduction of acetophenone (1) with Ximenia americana grains.
␦
C
55.4 (H3C-O), 65.4 (H2C O), 112.4 (HC), 113.5(HC), 119.4 (HC),
129.8 (HC), 142.7 (C), 160.0 (O C).
3-Methoxy-benzyl-acetate (20a) – tR 11.44. MS: m/z 180 (M+),
138 (M-H2C
(300 MHz, CDCl3): ␦H 2.24 (s, CH3), 3.87 (s, H3C O), 5.18 (s, H2C O),
3-Methoxy-benzoic acid (10b) – tR 11.67. MS: m/z 152 (M+),
135 (M-OH, 100%), 107 (m/z 135-CO), 92, 77.
C
O, 100%), 121 (M-CH3CO2), 109, 91, 77, 43. 1H NMR
4-Methoxy-benzilic alcohol (11a) – tR 10.62. MS: m/z 138 (M+,
100%), 137 (M-H), 121 (M-OH), 109, 94, 77. 1H NMR (300 MHz,
CDCl3): ␦H 3.81 (s, 3H, CH3O), 4.59 (s, 2H, H2C O), 6.88 (d,
6.82 (m, 3H), 7.40 (dl, 1H). 13C NMR (75 MHz, CDCl3): ␦C 21.2 (CH3),
55.6 (H3C-O), 66.5 (H2C-O), 114.5 (HC), 121.6 (HC), 123.6 (HC),
130.2 (HC), 137.9 (C), 160.3 (O C), 171.0 (C O).
9.0 Hz, 2H), 7.28 (d, 2H, 9.0 Hz). 13C NMR (75 MHz, CDCl3): ␦C 55.4
((H3C O), 65.1 (H2C O), 113.9 (2HC), 129.0 (2HC), 133.3 (C), 159.3
(O C).
4-Methoxy-benzyl-acetate (21a) – tR 11.54. MS: m/z 180 (M+),
138 (M- M-H2C
C H
O), 121 (M-CH3CO2, 100%), 109, 91, 77, 43. 1
4-Methoxy-benzoic acid (11b) – tR 11.75. MS: m/z 152 (M+),
135 (M OH, 100%), 135 (M OH, 100%), 121 (M CH3O), 107, 93,
77.
NMR (300 MHz, CDCl3): ␦H 2.11 (s, 3H; CH3), 3.87 (s, 3H; H3C O),
5.12 (s, 2H; H2C O), 6.99 (d, 8.0 Hz, 2H), 7.55 (d, 8.0 Hz, 2H). 13C
NMR (75 MHz, CDCl3): ␦C 21.2 (CH3), 55.5 ((H3C O), 65.1 ((H2C-
O), 114.1 (2HC = ), 129.6 (2HC = ), 130.8 (C = ), 159.3 (O-C = ), 171.8
(C O).
3-Methoxy-4-hydroxy-benzilic alcohol (12a) – tR 11.76. MS:
m/z 154 (M+, 100%), 153 (M−H), 137 (M-OH), 125, 93, 65. 1H
NMR (300 MHz, CDCl3): ␦H 3.90 (s, 3H, H3CO), 4.56 (s, 2H, H2C O),
6.83–6.90 (m, 3H). 13C NMR (75 MHz, CDCl3): ␦C 56.5 ((H3C-O), 65.4
(H2C-O), 112.3 (HC), 116.1 (HC), 121.2 (HC), 134.3 (C), 147.0 (O C),
149.0 (O C); 13C NMR DEPT 135: ␦C 112.3, 116.1, 121.2 (3 CH), 65.4
(CH2), 56.5 (CH3).
1-Phenyl-ethan-1-acetoxy (22a) – tR (S) 14.32; tR (R) 13.66. MS:
m/z 164 (M+), 122 (M-H2C
C O), 104 (M-H3CCO2H), 100%), 77. 43.
1H NMR (300 MHz, CDCl3): ␦H 1.53 (d, 6.0 Hz, 3H), 20.2 (s, 3H), 5.87
(q, 6.0 Hz, 1H; HC O), 7.30 (sl, 5H). 13C NMR (75 MHz, CDCl3): ␦C
21.9 (CH3), 22.3 (CH3), 72.5 (HC O), 126.2 (2HC), 127.9 (HC), 128.8
(2HC), 141.8 (C), 170.4 (C O).
1-hydroxy-2-methoxy-benzene (12b) – tR 8.40. MS: m/z 124
Cyclohexil-acetate (23a) – tR 7.42. MS: m/z 142 (M+, no reg-
istered), 82 (M-H3CCO2H), 67, 54, 43 (100%). 1H NMR (300 MHz,
CDCl3): ␦H 1.10–2.30 (ms, 10H), 2.08 (s, 3H), 3.59 (m, 1H; HC O).
13C NMR (75 MHz, CDCl3): ␦C 24.3 (CH3), 25.6 (CH2), 33.1 (2 CH2),
35.6 (2 CH2), 70.4 (HC O), 171.0 (C O).
(M+), 109 (M-15, 100%), 81 (m/z 109-CO).
1-naphthol (13a) – tR 12.49. MS: m/z 158 (M+), 129 (100%0. 1
H
NMR (300 MHz, CDCl3): ␦H 5.12 (s, 2H, H2C O), 7.54–7.72 (m, 4H),
7.91–8.00 (ddl, 2H), 8.10 (dl, 1H). 13C NMR (75 MHz, CDCl3): ␦C 63.7
(H2C O), 123.9–136.7 (ten signals of aromatic carbons); 13C NMR
DEPT 135: ␦C 125.0–136.7 (7 CH), 63.7 (CH2).
Octyl-acetate (24a) – tR 9.91. MS: m/z 172 (M+, no registered),
112 (M-H3CCO2H), 98, 84, 70, 56, 43 (100%), 42, 41. 1H NMR
(300 MHz, CDCl3): ␦H 0.87 (t, 3H), 1.15–1.60 (ms, 6CH2), 2.00 (s,
3H), 4.87 (m, 2H). 13C NMR (75 MHz, CDCl3): ␦C 14.1 (CH3), 21.4
(CH3), 20.1 (CH2), 22.7 (CH2), 25.5 (CH2), 29.4 (CH2), 31.9 (CH2),
39.5 (CH2).
Benzoic acid (14a) – tR 9.59. MS: m/z 122 (M+, 95%), 105 (M-OH,
100%), 77, 51. 1H NMR (300 MHz, CDCl3): ␦H 7.50 (t, 7.5 Hz, 2H), 7.64
(t, 7.4 Hz, 1H), 8.20 (d, 7.3 Hz, 2H), 11.93 (sl, 1H). 13C NMR (75 MHz,
CDCl3): ␦C 128.6 (2HC), 129.5 (C), 130.4 (2HC), 134.0 (HC), 172.8
(CO).
Benzoic acid (15a) – tR 9.65. MS: m/z 122 (M+, 100%), 105 (M-
OH, 100%), 77, 51. 1H NMR (300 MHz, CDCl3): ␦H 7.44 (t, 7.4 Hz,
2H), 7.55 (tl, 7.3 Hz, 1H), 8.10 (d, 7.3 Hz, 2H), 11.9 (sl, 1H). 13C NMR
(75 MHz, CDCl3): ␦C 128.4 (2HC), 129.7 (2HC), 130.7 (C), 132.9 (HC),
172.3 (CO).
3. Results and discussion
3.1. Reduction of ketones
Benzilic alcohol (16a) – tR 7.38. MS: m/z 108 (M+, 85%)), 107
(M−H), 91 (M-OH), 79 (M-HCO, 100%), 77, 51. 1H NMR (300 MHz,
CDCl3): ␦H 2,0 (s, 1H), 4.66 (s, 2H), 7.29 (s, 5H). 13C NMR (75 MHz,
CDCl3): ␦C 65.5 ((H2C O), 127.1 (2HC), 127.7 (HC), 128.6 (2HC),
141.0 (C).
Chiral alcohols are one of the most well-known synthons and
can be obtained from the corresponding prochiral ketones by asym-
metric reduction. Thus, initially, as part of an evaluation process to
find reductases in the grains of X. americana, we selected acetophe-
none (1) (Fig. 1) as a model substrate and a know methodology
[20,21]. We studied the behavior of the reaction regarding temper-
ature, pH, time and biocatalyst/substrate ratio concentrations. It
was observed that running the reaction in less than 72 h and above
30 ◦C resulted in a lower yield. Regarding the biocatalyst/substrate
mass ratio, a positive effect on the isolated yield of the reaction was
observed when the grains/acetophenone ratio increased from 40/1
to 60/1, achieving a yield of 54% in the ratio of 100/1. Thus, analyzes
of the different reaction parameters in acetophenone reduction led
to a greater conversion (54%) using 5.0 g of the grains and 0.05 g of
acetophenone in water (50 mL, pH 5.0), under agitation (175 rpm),
at 30 ◦C, for 72 h. This result prompted us to test the ability of grains
of X. Americana for the reduction various compounds containing the
keto functionality (Scheme 1).
(
)-4-Cloro-1-phenylethan-1-ol (17a) – tR (S) 19.9; tR (R) 19.6.
MS: m/z 156 (M) and 158 (M + 2), 141/143 (M-CH3), 121 (M-Cl),
113/115, 77, 43. 1H NMR (300 MHz, CDCl3): ␦H 1.41 (d, 6.5 Hz, 3H),
4.79 (q, 6.5 Hz, 1H; CH O), 7.23 (d, 8.6 Hz, 2H), 7.29 (d, 8.6 Hz, 2H).
13C NMR (75 MHz, CDCl3): ␦C 25.3 (CH3), 69.7 (CH O), 126.9 (2HC),
128.6 (2HC), 133.0(C), 144.4 (C).
1-phenylethan-1-ol (18a) – tR (S) 15.7; tR (R) 15.4. MS: m/z 122
(M+), 107 (M-CH3, 100%), 79, 77, 51, 43. 1H NMR (300 MHz, CDCl3):
␦
H
1.48 (d, 6.4 Hz, 3H), 4.82 (q, 6.4 Hz, 1H; CH-O), 7.30 (sl, 5H). 13
C
NMR (75 MHz, CDCl3): ␦C 25.0 (CH3), 69.9 (CH O), 125.4 (2HC = ),
127.1 (HC = ), 128.3 (2HC = ), 145.9 (C = ).
Benzyl-acetate (19a) – tR 9.44. MS: m/z 150 (M+), 108 (M-
H2C
(s, 3H), 5.12 (s, 2H; H2C O), 7.34 (s, 5H). 13C NMR (75 MHz, CDCl3):
20.9 (CH3), 66.3 (H2C O), 128.2 (2HC), 128.3 (HC), 128.6 (2HC),
C
O, 100%), 91, 79, 77, 43. 1H NMR (300 MHz, CDCl3): ␦H 2.10
As shown in Table 1, incubations of ketones 1-8 with fresh grains
in water yielded the corresponding alcohols in yields ranging from
moderate (29%) to good (60%), except for 3 (6%) and 6 (14%). Aro-
matic ketones 1-5, except 3, yielded the corresponding alcohols in
␦
C
136.0 (HC), 170.9 (C O).

R.A.C. da Silva et al. / Molecular Catalysis 445 (2018) 187–194
191
Scheme 1. Reduction of aromatic and aliphatic ketones with X. americana grains.
similar and higher yields than aliphatic ketones 6-8. Incidentally,
the alcohols were obtained with high to excellent optical purity
(89–99%), with the exception of 1 (48%) and 8 (35%). Comparing
the reactivity of the aromatic ketones, it is interesting to note that
in the case of compound 3 [with the fluorine halogen (a strong
electron-withdrawing substituent)], with the low conversion (6%),
an influence on the steric course of the reduction was observed,
with formation of the (R)-enantiomer, possessing excellent optical
purity (99%).
shown in Table 2, incubations of aldehydes 9-13 with fresh grains
in water gave the alcohols 9a to 13a yields ranging from moderate
(51–61%), to good (75%), to excellent (98- > 99%).
As expected, and reported in previous work, aldehydes were
more reactive than ketones and benzaldehyde 9 was completely
reduced to corresponding alcohol 9a (>99%). With respect to the
product 9a, the chemical yield of the alcohols 10a − 12a decreased
moderately in the case of 10, slightly in 11, and drastically in 12;
This result was discrepant with the reduction of p-
fluoroacetophenone (3) with Daucus carota root regarding
bioconversion (80%) and the alcohol (S)-enantiomer [(S)-3a, ee
90%] [22], in agreement with the Prelog model for bioreduction.
Finally, it should also be noted that (S)-1a alcohol was obtained
using X. americana (54% yield and 48% ee) (present study) and Lens
culinaris (17% yield and 75% ee) [4] while the use of S. officinarum led
to the formation of the alcohol (R)-1a (36.5% yield and 57% ee) with
opposite stereochemistry as predicted by Prelog’ rule [1]. Products
1a – 5a and 7a – 8a were identified as (S) or (R)-enantiomers
by comparison of retention times (tR) and peak areas of alcohol
(R,S)-1a – 5a and acetates (R,S)-7a’- 8a’ by CG/DIC injection. All
alcohols have been fully characterized by MS and NMR (1H and
13C) spectral data (2.5. Compounds Identity).
3.2. Reductions of aldehydes
Scheme 2. Reduction of aldehydes with X. americana grains.
Aldehydes, similar to ketones, are important and accessible
substrates capable of biocatalytic reduction for the production of
important chemicals such as pharmaceuticals, cosmetics, and agro-
chemicals. Considering the results obtained with the ketones and
also that, in general, the aldehydes are more reactive than ketones,
we investigated five aromatic aldehydes (9-13) (Scheme 2) as sub-
strates in order to obtain this bioreduction. Using benzaldehyde
(9) as the standard substrate, the conditions established for the
reduction were the same as those used for the ketones [5.0 g of the
grains and 0.05 g of benzaldehyde in water (50 mL) under agitation
(175 rpm) at 30 ◦C for 72 h], except that the reaction was carried
out in a aqueous buffer/NaH2PO4/Na2HPO4 solution at pH 7.0. As
Table 2
Reduction of aldehydes (9-13) with X. americana grains.
entry
substract
yield (%)
9
benzaldehyde
>99
75
98
51
61
10
11
12
13
3-methoxybenzaldehyde
4-methoxybenzaldehyde
3-methoxy-4-hydroxybenzaldehyde
1-naphthaldehyde
0.05 g substrate and 5.0 g X. amercana grains in H2O (50 mL), pH 7.0, 175 rpm, 30 ◦C,
72 h.

192
R.A.C. da Silva et al. / Molecular Catalysis 445 (2018) 187–194
Scheme 3. Hydrolysis of esters with X. americana grains.
the latter with the presence of two electron-donating substituents
on the aromatic ring. However, in addition to the alcohols 10a (75%
yield), 11a (98% yield), and 12a (51% yield), the products 10b (25%
yield), 11b (1% yield), and 12b (49% yield) were obtained.
(Scheme 3). Using methyl benzoate (14) as the standard substrate,
the conditions established for the hydrolysis were the same as those
used for ketones, with the exception of the amount of biocatalyst
[2.0 g of the grains and 50 mg of methyl benzoate in water (50 mL)
under agitation (175 rpm) at 30 ◦C for 72 h].
The compounds (14a – 18a) obtained for the five esters all
resulted from hydrolysis reactions rather than products from
reduction. The enzymes ester hydrolysis of X. Americana was quite
effective with the unsubstituted aromatic esters 14 and 15 yielding
benzoic acid in 98% yield from each compound. The benzyl acetate
16 was also hydrolyzed to the benzyl alcohol in 98% yield, although,
with the other aliphatic esters (17 and 18), it proceeded only in a
relatively good yield (58 and 65%, respectively) (Table 3). Alcohols
17a and 18a were identified as (S)-enantiomers by comparison of
retention times (TR) and peak area of (R,S)-17a and (R,S)-18a by
CG/DIC injection. All products have been fully characterized by MS
and NMR (1H and 13C) spectral data (2.5. Compounds Identity).
Oxidation products, with formation of carboxylic acids, have
already been observed in the reactions of cinnamic and ␣-methyl
cinnamic aldehydes with L.culinaris (3-phenylpropen-1-oic acid,
29% yield) ([4] and S. officinarum (␣-methyl-3-phenylpropen-
1-oic acid, 56% yield) [1], respectively. Even so, the sub-
strates benzaldehyde (9), m-methoxybenzaldehyde (10) and
p-methoxybenzaldehyde (11) were reduced by X. americana in
yields comparable to those obtained with L. culinaris. In the case of
12b, it is likely that the 3-methoxy-4-hydroxy benzoic acid initially
originated by the same path as 10b and 11b, was decarboxylated.
These yields were determined by GC–MS analysis. All alcohols have
been fully characterized by MS and NMR (1H and 13C) spectral data
(2.5. Compounds Identity).
3.4. Esterification of alcohols
3.3. Hydrolysis of esters
Considering the results obtained with the esters and in order
to broaden the scope of use of X. Americana as a biocatalyst, we
are interested, at this point, in expanding these studies for the
esterification reaction of alcohols. Esters are widespread in nature.
Many of the structurally simple esters are pleasant smelling liq-
uids that contribute to fragrances and flavours of plants and fruits,
and are widely used in the food and cosmetic industries. The ester-
ification of alcohols, as well as phenols and amines, is of interest,
A commonly required functional group transformation is the
hydrolysis of an ester for chiral acid and alcohol, for which bio-
catalytic strategies have been developed [3]. Esterase activities
have been studied using a number of plant species. When esters
are used, although the dominant reaction is hydrolysis [4,10,23],
several cases were observed of enzymatic hydrolysis/oxidation
effects. One example is the hydrolysis/oxidation of racemic 1-
phenylethyl acetate to the (S)-alcohol 1-phenylethanol by apple
(Malus sylvestris) pulp in 40% yield with 15% of the acetophenone
also produced Mironowicz et al., 1998. In addition, the enzyme
effects of hydrolysis, oxidation and reduction are observed for
racemic 1-phenylethyl acetate by carrot roots, celery (Apium grave-
olens var. rapaceum), and horseradish (Amroracia lapatifolia) such
that the initially formed (S)-alcohol is oxidized to the ketone, leav-
ing (R)-alcohol [13].
Table 3
Hydrolysis of esters (14-18) with X. americana grains.
entry
substract
yield (%)
ee
config.
14
15
16
17
18
methylbenzoate
ethylbenzoate
benzylacetate
4-chloro-1-phenylethylacetate
1-phenylethylacetate
98
98
98
58
65
–
–
–
87
34
–
–
–
S
Thus, following our study, the next step was to observe the effi-
ciency of the enzymatic system against other types of carbonyl
groups, particularly organic esters. The selected compounds were
methyl benzoate (14), ethyl benzoate (15), benzyl acetate (16), p-
cloro-1-phenylethyl acetate (17) and 1-phenylethyl acetate (18)
S
ee: enantiomeric excess; config.: configuration.
0.05 g substrate and 2.0 g X. amercana grains in H2O (50 mL), pH 5.0, 175 rpm, 30 ◦C,
72 h.

R.A.C. da Silva et al. / Molecular Catalysis 445 (2018) 187–194
193
because it renders these substrates more soluble in usual organic
solvents and protects the inherent functional groups. Lipases and
hydrolytic enzymes, used as catalysts in esterification, transester-
ification, and hydrolysis reactions, are among the most commonly
used in organic chemistry.
peak area of (R,S)-22a by CG/DIC injection. All products have been
fully characterized by MS and NMR (1H and 13C) spectral data (2.5.
Compounds Identity).
The equilibrium of the forward reaction (hydrolysis) or reverse
reaction (synthesis) can be controlled by the amount of water
present in the reaction mixture. Immobile enzyme systems of
Saccharum officinarum and of Manihot esculenta have been used
to acetylate alcohols in good (68%) to high (93%) yields [2] and
in the enantioselective acetylation of racemic alcohols in poor
(ee 9%) to excellent (ee 99%) enantiomeric excess [15], respec-
tively. In addition, the enzymatic enantioselective esterification of
(
)-alcohols with Citrus aurantium peels occurred with conver-
sions and enantiomeric excess in the ranges of 28.8–70.2% and
52–97%, respectively [7]. Initially, having the benzylic alcohol (19)
(Scheme 4) as the standard substrate, we studied the behavior of
the reaction of esterification as a function of the same parameters of
the hydrolysis reaction, but in the presence of different acetylating
reagents.
With n-butyl acetate, no acetylation occurred, and with acetic
anhydride, the maximum conversion was 80%. An excellent yield
(>99%) in the enzymatic acetylation was achieved by varying three
of the reaction parameters which were solvent, stirring speed and
time, all in the presence of vinyl acetate as an acetylating reagent.
Thus, acetylation reactions with other related substrates (20–24)
were carried out under the same conditions of esterification of 19
[2.0 g of the grains, 50 mg (0.460 mmol)] of benzylic alcohol and
vinyl acetate (100 ␮L) in hexane (10 mL) under agitation (225 rpm)
at 30 ◦C for 24 h]. In general, it was observed that lipases were quite
efficient in acetylation of the alcohols (19-21, R,S-22 > 99% yields)
of an aromatic nature, but not very efficient with respect to the
purely aliphatic alcohols (23 e 24, 19 and 40% yields, respectively)
(Table 4).
To prove the efficiency of this biocatalyst, we have also inves-
tigated the feasibility of reusing under the optimal reaction
conditions found for acetylation of 19. As shown in Fig. 2, the grains
of X. americana can be used as biocatalyst up to five times with an
efficiency equal to or greater than 90%. Ester 22a was identified as
(R)-enantiomer (25% ee) by comparison of retention time (tR) and
Scheme 4. Esterification of alcohols with X. americana grains.
Table 4
Esterification of alcohols (19-24) with X. americana grains.
entry
substract
yield (%)
ee
config.
19
20
21
22
23
24
benzilic alcohol
>99
>99
>99
>99
18
–
–
–
25
–
–
–
–
R
–
–
3-Methoxy-benzil alcohol
3-Methoxy-benzil alcohol
1-Phenylethyl alcohol
cyclohexanol
1-octanol
40
–
ee: enantiomeric excess; config.: configuration.
0.05 g substrate and 2.0 g X. amercana grains in hexane (10 mL), vinyl acetate
(100 ␮L), pH 5.0, 225 rpm, 30◦, 24 h.
Fig. 2. Reuse of the biocatalyst (X. americana grains) in the acetylation reaction.

194
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synthetic chemical reactions using intact plant materials, and also
allows the expansion of the role of natural product chemistry by
enriching the value of green chemistry in industrial production
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tion of the aldehydes to the respective alcohols, the yields in all
cases were good to excellent (51- > 99%). In the case of ketones
in five of the eight substrates, the yields were moderate to good
(43–60%), with e.e. values of 81- > 99%. In ester hydrolysis, there
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Nacional do Desenvolvimento Científico e Tecnológico (CNPq) and
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