Optimisation, scope and advantages of the synthesis of chiral phenylethanols using whole seeds of Bauhinia variegata L. (Fabaceae) as a new and stereoselective bio-reducer of carbonyl compounds

Biocatalysis and Biotransformation

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20

Optimisation, scope and advantages of the

synthesis of chiral phenylethanols using whole

seeds of Bauhinia variegata L. (Fabaceae) as a

new and stereoselective bio-reducer of carbonyl

compounds

Gabriela I. Demmel , Daniela L. Bordón , Ana M. Vázquez , María F. Decarlini ,

Gustavo M. Ruiz , Juan J. Cantero , Laura I. Rossi & Mario L. Aimar

To cite this article: Gabriela I. Demmel , Daniela L. Bordón , Ana M. Vázquez , María F. Decarlini ,

Gustavo M. Ruiz , Juan J. Cantero , Laura I. Rossi & Mario L. Aimar (2020): Optimisation, scope

and advantages of the synthesis of chiral phenylethanols using whole seeds of Bauhiniaꢀvariegata

L. (Fabaceae) as a new and stereoselective bio-reducer of carbonyl compounds, Biocatalysis and

Biotransformation, DOI: 10.1080/10242422.2020.1789115

To link to this article: https://doi.org/10.1080/10242422.2020.1789115

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Published online: 07 Jul 2020.

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BIOCATALYSIS AND BIOTRANSFORMATION

https://doi.org/10.1080/10242422.2020.1789115

RESEARCH ARTICLE

Optimisation, scope and advantages of the synthesis of chiral

phenylethanols using whole seeds of Bauhinia variegata L. (Fabaceae) as a

new and stereoselective bio-reducer of carbonyl compounds

a

b

a

c

ꢀ

Gabriela I. Demmel , Daniela L. Bordon , Ana M. Vazquez , Marıa F. Decarlini , Gustavo M. Ruiz ,

Juan J. Cantero^d, Laura I. Rossi^e,fand Mario L. Aimar^b

a

b

ꢀ

Catedra de Tecnologıa Quımica, Facultad de Ciencias Quımicas, Universidad Catolica de Cordoba, Cordoba, Argentina; Catedra de

c

ꢀ

Quımica Aplicada, Facultad de Ciencias Exactas, Fısicas y Naturales, Universidad Nacional de Cordoba, Cordoba, Argentina; Herbario

Marcelino Sayago, Facultad de Ciencias Agropecuarias, Universidad Catolica de Cordoba, Cordoba, Argentina; Facultad de

d

ꢀ

Agronomıa y Veterinaria, Departamento de Biologıa Agrıcola, Universidad Nacional de Rıo Cuarto, Rıo Cuarto, Argentina;

e

ꢀ

Departamento de Quımica Organica, Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba, Cordoba, Argentina.;

f

ꢀ

Instituto de Investigaciones en Fısico-Quımica de Cordoba, (INFIQC-CONICET), Cordoba, Argentina

ABSTRACT

ARTICLE HISTORY

Received 3 January 2020

Revised 27 May 2020

Accepted 22 June 2020

With the aim of finding new methods for environmentally friendly synthesis of chiral phenyle-

thanols, a screening was carried out to identify seeds that could be used as a biocatalyst cap-

able of reducing stereoselectively prochiral ketones. As a result, seeds of Bauhinia variegata L.

(Fabaceae) were identified as being an efficient and stereoselective biological reducer of aceto-

phenone to produce (S)-1-phenylethanol (conversion of 98% and 99 e.e.%). Then, to optimise

the reductive process, the effects of some variables such as temperature, load of substrate, pH,

co-solvent, and reuse and storability of the seeds as a function of time were established.

Utilising the optimal reaction conditions, nineteen substituted acetophenones were reduced to

their corresponding chiral alcohols with a conversion ranging from 30% to 98% and enantio-

meric excess of between 65% and >99%, and in addition, useful key intermediates were also

obtained by the synthesis of drugs. The scope and advantages of this new biocatalytic synthetic

method are also discussed.

KEYWORDS

Bioreduction; seeds;

Bauhinia variegata;

phenylethanols; key

intermediates

RESEARCH HIGHLIGHTS

ꢀ A screening was carried out to identify seeds that could be used as a biocatalyst

ꢀ Seeds of Bauhinia variegata have been identified as an efficient biocatalyst to reduce car-

bonyl compounds.

ꢀ Acetophenone and substituted acetophenones were reduced with high stereoselectivity.

ꢀ Some key intermediates were synthetised using this methodology.

ꢀ Seeds can be stored for twenty-four months without loss of activity.

Introduction

implies that the synthetic procedures used need to be

highly stereoselective in order to obtain a single

enantiomer in its pure form, which consequently

requires a great effort to be applied to the difficult

task of identifying stereoselective synthetic methodol-

ogies that allow chiral key intermediaries to be

obtained for the production of bioactive molecules.

In recent years, the utilisation of environmentally

friendly methodologies for producing the chemical

products used in the pharmaceutical industries has

gained increasing attention due to the increasingly

stringent environmental regulations (Roschangar et al.

The recognition of chiral structures is perhaps one of

the most amazing and important characteristics of the

metabolism of living creatures, and as a consequence,

often only one stereoisomer can exhibit biological

activity in therapeutic processes, while the other

remains inactive or even produces adverse effects (Wu

and Xiao 2007; Mansfield et al. 2004). For this reason,

the biological activity of compounds used as pharma-

ceuticals, agrochemicals or flavouring substances gen-

erally depends on the configuration of the chiral

centre (Nguyen et al. 2006). This special characteristic

ꢀ

ꢀ ꢀ

Catedra de Quımica Aplicada, Facultad de Ciencias Exactas, Fısicas y Naturales, Universidad

CONTACT Mario L. Aimar

mlaimar@unc.edu.ar

ꢀ

Nacional de Cordoba, Velez Sarsfield 1611, Cordoba, X5016GCA, Argentina

Supplemental data for this article can be accessed here.

ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2

G. I. DEMMEL ET AL.

2015). As a result, stereoselective synthetic processes

that can be performed in an environmentally friendly

way are becoming ever more important. In this sense,

biocatalytic systems have several singular characteris-

tics that meet many of the requirements imposed by

green chemistry (Patel 2018). In addition, they provide

more environmentally benign processes due to water

being used as the solvent, and involve energy-efficient

operations as a result of the fact that the reactions

can be performed at ambient temperature (Tao and

Xu 2009). It should also be noted that the crucial

advantage in utilising bioreagents for organic transfor-

mations is because they can be used on a sustainable

basis, rather than causing a depletion of resources

(Cordell et al. 2007). However, due to prolonged reac-

tion times and to their relatively low productivity com-

pared to classical chemical methodologies, their

widespread application is still limited (Hoyos et al.

2017). Thus, increasing attention is now being paid to

developing robust biocatalysts that can carry out proc-

esses at a high substrate loading in large-scale bio-

transformations in a short period of time in order to

achieve economic feasibility and greater competitive-

ness (Ni and Xu 2012).

Chiral 1-phenylethanols (1-PE) are very useful build-

ing blocks for the synthesis of complex and biologic-

ally active molecules, and are mostly produced by the

stereoselective reduction of asymmetric ketones

(Chanysheva et al. 2019). In recent years, several vege-

tables have been used as sources of enzymes capable

of carrying out the reduction of prochiral ketones in a

highly efficient manner and with excellent stereo-

selectivity (Patil 2015). Using this methodology, several

species of plants have been used as stereoselective

biological reducers to obtain chiral 1-PEs (Villa et al.

capable of reducing prochiral ketones stereoselec-

tively. This enabled us to identify the seeds of

Bauhinia variegata (L.) (Fabaceae) as being efficient

and stereoselective biological reducers of acetophe-

none (AP) to produce chiral (S)-1-PE. Then, the princi-

pal variables of the reductive process were optimised,

and by utilising these optimal reaction conditions use-

ful intermediates were obtained. The scope and

advantages of the method presented here are dis-

cussed below.

Material and methods

General

Acetophenone and substituted acetophenones were

purchased from Sigma-Aldrich S.A. (Argentina). NADPH

tetrasodium salt was purchased from Santa Cruz

Biotechnology, Inc. (R)-1-phenylethanol, (S)-1-phenyle-

thanol (1), (S)-2-chloro-1-phenylethanol (2), (S)-1-(2⁰-

chlorophenyl)ethanol (3), (S)-1-(3⁰-Chlorophenyl) etha-

nol (4), (S)-1-(4⁰-chlorophenyl)ethanol (5), (S)-1-(2⁰,4-

ꢀdichlorophenyl)ethanol (6), (S)-1-(2⁰-nitrophenyl)etha-

nol (7), (S)-1-(3⁰-nitrophenyl)ethanol (8), (S)-1-(4⁰-nitro-

phenyl)ethanol (9), (S)-1-(4⁰-fluorophenyl)ethanol (10),

(S)-1-(4⁰-trifluoromethylphenyl)ethanol (11), (S)-1-(4⁰-

bromophenyl)ethanol (12), (S)-1-(4⁰-cyanophenyl)etha-

nol (13), (S)-1-(4⁰-methylphenyl)ethanol (14), (S)-1-(3⁰-

hydroxyphenyl)ethanol (16), (S)-1-(thiophen-2-yl)etha-

nol (20) and (S)-1-(pyridin-2-yl)ethanol (21) were

synthesised using methodologies previously reported

ꢀ

in the literature (Aimar et al. 2014, Bordon et al. 2015,

Decarlini et al. 2017), and were used as a standard

reference in the chiral GC analysis.

GC-FID and GC-MS analyses

~

1998; Yadav et al. 2002; Cordell et al. 2007; Assunc¸ao

et al. 2008; Bennamane et al. 2018), but only a few

reports have described the use of seeds as biocatalysts

(Xie et al. 2009; Bizerra et al. 2010; Alves Ferreira et al.

2012; Bertini et al. 2012; Carvalho da Silva et al. 2018;

The chromatographic conditions of all substrates and

products used have been previously established and

reported by our research team (Aimar et al. 2014;

ꢀ

Bordon et al. 2015; Decarlini et al 2017). For more

ꢀ

Almeida de Sousa et al. 2019; Atak et al. 2019; Solıs

details, see Supplementary material.

et al. 2019). Moreover, it should be mentioned that we

have only found one published report employing

whole seeds (Kumaraswamy and Ramesh 2003). With

respect to this, it is important to mention that whole

seeds have the advantage of being easily stored and

preserved, which could be a valuable feature as the

biggest drawback related to the use of plants as bio-

catalysts is their seasonal availability.

Biocatalysts

For this study, seeds of Phaseolus vulgaris, Lupinus

albus, Sinapis alba and Lens culinaris were purchased

from a local supermarket. Other seeds used in this

study were collected during the period August –

December at different locations in the province of

ꢀ

In the present work, a screening using thirty seven

seeds was carried out with the aim of identifying

those that could be used as a source of enzymes

Cordoba, Argentina. All seed samples were identified

by a botanical expert. Before use, the seeds were

washed with tap water to remove traces of dirt, after

BIOCATALYSIS AND BIOTRANSFORMATION

3

Table 1. Seeds as biocatalyst in the stereoselective reduction of AP.^a

OH

O

whole seeds

water, 25°C, 100 rpm

CH₃

*

Entry

Family

Scientific name

Conversion %^b,c

e.e. %^b,c

1

2

3

4

5

6

7

8

Anacardiaceae

Apiaceae

Schinus areira L.

Coriandrum sativum L.

Araujia brachystephana (Griseb.) Fontella & Goyder

Mandevilla pentlandiana (A. DC.) Woodson

Helianthus annuus L.

Handroanthus impetiginosus (Mart. ex DC.) Mattos

Tecoma stans (L.) Juss. ex Kunth var. stans

Buglossoides arvensis (L.) I.M. Johnst.

Descurainia erodiifolia (Phil.) Prantl ex Reiche

Sinapis alba L.

Jodina rhombifolia (Hook. & Arn.) Reissek

Cucumis melo L.

Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir.

Bauhinia forficata Link subsp. pruinosa (Vogel) Fortunato & Wunderlin

Bauhinia variegata L.

Erythrostemon gilliesii (Wall. ex Hook.) Klotzsch var. gilliesii

Cicer arietinum L.

Lens culinaris Medik.

Leucaena leucocephala (Lam.) de Wit

Lupinus albus L.

Phaseolus vulgaris L.(red dark bean)

Phaseolus vulgaris L. (red light bean)

Phaseolus vulgaris L.(White bean)

Phaseolus vulgaris L.(red bean)

Senna subulata (Griseb.) H.S. Irwin & Barneby

Vachellia aroma (Gillies ex Hook. & Arn.) Seigler & Ebinger

Vachellia astringens (Gillies ex Hook. & Arn.) Speg.

Racosperma melanoxylon (R. Br.) Pedley

Linum narbonense L.

0

28

34

60

1

–

42 (S)

54 (S)

72 (S)

64 (S)

–

Apocynaceae

Asteraceae

Bignoniaceae

Boraginaceae

Brassicaceae

Cervantesiaceae

Cucurbitaceae

Fabaceae

Linaceae

Meliaceae

Oleaceae

Poaceae

Rosaceae

Sapotaceae

Solanaceae

0

–

9

24

0

48

24

0

98

0

1.5

64

0

12

8

0

48

0

40

2

8

1

0

13

11 (S)

–

92 (S)

91 (S)

–

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

–

99 (S)

–

99 (S)

96 (S)

–

63 (S)

54 (S)

–

32 (S)

–

Melia azedarach L

Fraxinus excelsior L

Menodora integrifolia (Cham. & Schltdl.) Steud.

Bromus catharticus Vahl var. catharticus

Eriobotrya japonica (Thumb.) Lindl.

Mespilus germanica L.

–

84 (S)

45 (S)

33 (R)

–

Manilkara zapota (L.) P.Royen

Solanum argentinum Bitter & Lillo

–

71 (S)

^a100 lL of AP in 80 mL of sterile water, 10 g of whole seeds of B. variegata, with shaking at 100 rpm and ambient temperature. ^bConversion (%) and

enantiomeric excess (e.e.%) were determined by chiral GC-FID. ^cAbsolute configurations were assigned by comparing the retention times of chiral GC

data with reports in the literature and through the use of pure (S)-1-PE and (R)-1-PE as a reference (Aimar et al. 2014, Decarlini et al. 2017).

which, they were disinfected by immersion in a 0.5%

sodium hypochlorite solution for 5 min. The seeds

were then washed again, but this time with deionised

sterile water.

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. The

products were purified by column chromatography, as

ꢀ

previously described (Aimar et al. 2014; Bordon et al.

2015; Decarlini et al 2017).

Bioreductions in optimal conditions

A seed sample (10 g) was placed in a disinfected

Erlenmeyer flask (250 mL) with sterile deionised water

(80 mL), and then ketone (300 mg) was dissolved in

8 mL of dimethyl sulfoxide (DMSO), which was added

to the suspension. The reaction was carried out by

stirring on an orbital shaker (100 rpm) at 40 ^ꢁC, with

the flask being closed and protected from direct light.

Finally, the crude reaction was filtered through cotton

Preparative-scale reactions

For the preparative-scale reaction, 2.4 g of AP, 80 g of

seeds of Bauhinia variegata, 64 mL of DMSO and

640 mL of sterile distilled water were placed in a disin-

fected 2 L conical flask (final concentration of the sub-

strate: 28.4 mM). Then, this flask was put on an orbital

shaker at a shaking speed of 100 rpm at 40 ^ꢁC for 48 h.

4

G. I. DEMMEL ET AL.

The progress of the reaction was determined by chiral

GC-FID, at the end of which, the crude reaction was

extracted with ethyl acetate (5 ꢂ 100 mL) and the

organic solution was dried using anhydrous magne-

sium sulphate. Then, the filtrate organic solution was

concentrated to dryness in a rotary evaporator, and

the extracted crude reaction products were filtered on

a short column with silica gel, as described in previous

1PE. Related to this, it is important to mention that

Villa et al. (1998), on conducting studies with cell cul-

tures of H. annus and C. melo, did not observe any

reduction of AP.

A poor performance was observed in the cases of

A. brahystephana, M. pentlandiana, H. impetiginosus, D.

erodiifolia, V. aroma, V. astringens, L. narbonense, M.

integrifolia, B. catharticus and S argentinum (Table 1,

entries 3–4, 6, 9, 29, 32–33 and 37 respectively) under

our reaction conditions, but with a slight preference

for the formation of (S)-1-PE.

ꢀ

reports (Aimar et al. 2014; Bordon et al. 2015; Decarlini

et al 2017).

According to data presented in Table 1 (entry 15),

Bauhinia variegata L. was the biocatalyst with the best

characteristics as a bio-reducer, obtaining for (S)-1-PE

a conversion of 98% and 99 e.e.%, and this represents

the first report of the seeds of this plant being effect-

ively used for enantioselective bioreduction of AP. In

contrast, it is interesting to note that B. forficata (Table

1, entry 14) was unable to produce any reduction of

AP under our reaction conditions.

In spite of some reports in the literature noting a bio-

reductive behaviour in the same species of bean, includ-

ing Phaseolus vulgaris (Kumaraswamy and Ramesh

2003; Xie et al. 2009; Atak et al. 2019), S. alba (Almeida

de Sousa et al. 2019) and the seeds of L. culinaris (Alves

Ferreira et al. 2012), in our reaction system the seeds of

these species were totally inert (Table 1, entries 10, 18,

21–24). Similarly, for our reaction conditions, the seeds

of C. sativum were totally inactive (Table 1, entry 2).

However, Andrade et al. (2006) carried out studies with

the roots of this plant as the biocatalyst, and although

the reaction did not have a good stereoselectivity, this

plant was able to reduce AP.

It is important to mention that in our investigation

seventeen out of eighteen of the tested seeds pro-

duced (S)-1-PE and observed Prelog’s rule, with E.

japonica (Table 1, entry 34) being the only exception,

which revealed a slight preference for the formation

of the R enantiomer (33 e.e.%). In this sense, it should

be mentioned that Orden et al. (2009) reported the

formation of the R enantiomer using hairy root cul-

tures of Raphanus sativus. However, it should be

pointed out that obtaining the anti-Prelog product is

surprisingly rare when using plants as biocatalysts.

Finally, considering the results achieved through

the screening using different seeds, it was decided to

continue our studies using the seeds of B. variegata.

Reduction of substituted acetophenones with

seeds of Bauhinia variegata

The same optimal proportions used in the scaling

down of acetophenone were used to reduce substi-

tuted acetophenones. The final product was processed

in the same way as described in previous studies.

After purification, all products were identified by com-

paring their GC retention times and MS, ¹H and ¹³C

NMR spectra with data from the literature (Mathre

et al. 1993; Nakamura and Matsuda 1998; Nakamura

et al. 1999; Ohkuma et al. 2000; Yadav et al. 2002;

Comasseto et al. 2006; Orden et al. 2009; Cazetta

2014; Aguirre-Pranzoni et al. 2015).

Results and discussion

Screening of seeds

As a part of the evaluation process to find environmen-

tally friendly reaction systems using vegetables as the

biological reducers, a total of 37 whole seeds were

assayed in order to determine their potential capabil-

ities to transform stereoselectivity acetophenone (AP)

to chiral 1-PE. The selected seeds and the results

obtained in this screening are shown below in Table 1.

As can be seen in Table 1 (entries 3–6, 9, 11–12, 15,

19–20, 26–27, 29, 32–34 and 37), only eighteen out of

a total of thirty seven seeds tested showed a reduc-

tion of AP to (S)-1-PE, but at different rates of conver-

sion and stereoselectivity, and in addition, only one

revealed a slight preference for the formation of (R)-1-

PE (entry 34; E. japonica).

A very good stereoselectivity (ꢃ90%) and good

conversion in the reduction of AP were obtained with

the seeds of J. rhombifolia (Table 1, entry 11) and L.

albus (Table 1, entry 20), while C. melo (Table 1, entry

12) and L. leucocephala (Table 1, entry 19) revealed a

very good stereoselectivity, but with a low conversion

efficiency. Moreover, H. annuus showed a good per-

centage of conversion and an appreciable stereo-

selectivity (Table 1, entry 5) in the formation of (S)-

Optimisation of the bioreduction process

Bauhinia variegata L. (Fabaceae) is a flowering plant

that is native to Southeast Asia and grows from India

BIOCATALYSIS AND BIOTRANSFORMATION

5

to China, with its common names including mountain

ebony and orchid tree. This tree can grow up to

10–15 metres tall and loses its leaves in the dry sea-

son (Sinou et al. 2009; Wang et al 2014). These leaves

are 10–20 centimetres in size, and are bilobed at the

base and apex. The flowers are white, white and pink

or bright pink, and are 8–12 centimetres in diameter

with five petals and an orchid-like appearance. The

fruit is a seedpod of 15–30 centimetres in length and

contains several seeds. The seedpod dries completely

on the tree, and when mature begins to release the

seeds. Almost all parts are used in traditional medicine

for the treatment of various ailments, such as asthma,

ulcers, leprosy, piles, snake bites and liver complaints

(Modh et al. 2011), and its extracts have been found

to have antibacterial and antifungal activity (Rajkapoor

et al. 2003).

Figure 1. Effect of AP concentration for 48 h of reaction.

In Argentina, it is also known as “cow hoof” due to

the characteristic shape of its bilobed leaves being

similar to a cow’s footprint, but it is cultivated in this

country only for ornamental use.

of 15.5 mM. However, higher concentrations of AP pro-

duced a more pronounced reduction in the percent-

age of conversion (25.9 mM; conversion of 43%). It is

interesting to note that for all the concentrations of

AP tested, the enantiomeric excess remained approxi-

mately constant (98%). Considering the conversion

rates, the best AP concentration for bio-reduction was

observed at 10.3 mM with 48 h of reaction at ambient

temperature.

Despite the conversion and enantioselectivity of

asymmetric synthesis of alcohols using biocatalytic

methodologies being significantly influenced by sev-

eral critical parameters, the optimisation of the reac-

tion conditions of the reduction process of carbonyl

compounds using a vegetable as the biocatalyst has

been very infrequently studied (Kazici et al. 2016;

Almeida de Sousa et al. 2019; Atak et al. 2019). Thus,

in the present study, in order to optimise the reduc-

tion of AP to (S)-1-PE promoted by seeds of Bauhinia

variegata, the effects of some parameters were

studied, including temperature, pH, and those of the

co-solvent and its concentration and load of AP on

the reaction system.

Effect of temperature

Temperature is a critical factor in biocatalytic proc-

esses because the enzymes are strongly influenced by

this parameter (Nakamura et al. 2003). Nevertheless,

we have only found two reports in the literature that

have described the effect of temperature on a biocata-

lytic process promoted by seeds (Almeida de Sousa

et al. 2019; Kazici et al. 2016). Thus, to determine the

effect of this parameter on the production of (S)-1-PE,

assays were performed at 20, 25, 30, 35, 40 and 45 ^ꢁC

with 100 lL of AP (concentration in the reaction

media: 10.3 mM) dissolved in 0.5 mL of DMSO (co-solv-

ent) and 80 mL of sterile water, with 10 g of whole

seeds of B. variegata, and shaken at 100 rpm. In order

to obtain the kinetic profiles at different temperatures,

samples were taken at regular intervals of time and

analysed by chiral GC-FID.

Effect of AP concentration

To determine the effect of the substrate concentration

on the formation of (S)-1-PE, assays were carried out

with 100, 120, 150, 170, 200, 220 and 250 lL AP dis-

solved in 0.5 mL of DMSO (co-solvent) and 80 mL of

sterile water (AP concentrations from 10.3 to 25.9 mM),

with 10 g of whole seeds of B. variegata, shaken at

100 rpm at ambient temperature. This reaction was

monitored for 48 h, and analysed by chiral GC-FID and

GC-MS.

As shown in Figure 1, an increase in the concentra-

tion of AP of between 10.3 and 15.5 mM in the reac-

tion medium had only a slight effect on the reductive

process, with the observed conversion being 98% at

10.3 mM, and decreasing to 91% for a concentration

It can be observed in the results shown in Figure

2(A) that when the temperature was increased from

20^ꢁto 40 ^ꢁC an increase in the velocity of the reaction

occurred, with the reaction time decreasing from 48 h

(25 ^ꢁC) to 20 h (40 ^ꢁC). To visualise better the effect of

temperature on transformation speed, the kinetic

6

G. I. DEMMEL ET AL.

Figure 2. (A) Conversion as a function of temperature. (B) Velocity constant obtained by adjust of kinetic date to first order equa-

tion as a function of temperature.

profiles were adjusted to a first order equation using

the Origin Pro software and the velocity constants

were calculated.

Effect of co-solvents

In the present study, we carried out an evaluation

using different organic co-solvents, with assays being

conducted testing eleven co-solvents at a final con-

centration of 1.25% v/v (1 mL) and a final volume of

reaction of 80 mL of sterile water. As a control, a reac-

tion without co-solvent was made. All these assays

were performed at 40 ^ꢁC using 100 lL of AP, with 10 g

of whole seeds of B. variegata, at 100 rpm for 48 h

(concentration of the substrate 10.3 mM with 125 g/L

of seeds). Samples were taken at the end of the reac-

tion and analysed by chiral GC-FID, with Figure 3 dis-

playing the co-solvent effect on the performance of

the reaction.

As can be seen in Figure 3, when the reaction was

carried out in the absence of a co-solvent the percent-

age conversion was 92% with an enantiomeric excess

of 98%, while when DMSO was used as the co-solvent,

a slight positive effect on the conversion (98%) was

observed with 99 e.e.%. However, the other ten co-sol-

vents tested in our experiments had an inhibitory

effect on the conversion percentage and were detri-

mental to the formation of (S)-1-PE to varying degrees,

compared with the reaction without a co-solvent.

While propylene glycol and glycerol had a low inhibi-

tory effect on the conversion, the stereoselectivity of

the reaction was the same as that of the reaction

without co-solvent. In contrast, a pronounced inhibi-

tory effect on the conversion was observed with 1-

butanol, cyclohexanol and benzyl alcohol.

It can be seen in Figure 2(B) that there was a posi-

tive linear relationship between the calculated velocity

constant and the reaction temperature up to 40 ^ꢁC,

but when the temperature reached 45 ^ꢁC a slight

negative effect on the speed of the reaction was

observed. In addition, it is noteworthy that the e.e. %

remained constant (98%) at all temperatures, and that

the reaction was stereoselective for the formation of

(S)-1-PE.

It is interesting to note that in the studies reported

by Kazici et al. (2016) using a freeze-dried carrot as

the biocatalyst, the optimum working temperature

was found to be 33 ^ꢁC with a reaction time of 48 h,

while Almeida de Sousa et al. (2019) reported 30 ^ꢁC as

the optimal temperature with a reaction time of 72 h.

As B variegata seeds can be used at a higher working

temperature (40 ^ꢁC), this translates into a shorter reac-

tion time (20 h), which clearly represents a compara-

tive advantage. In fact, 40 ^ꢁC turned out to be the

most suitable temperature for carrying out the reduc-

tive process, which was within the stability zone for

seeds of B. variegata. Moreover, it is interesting to

observe that B. variegata grows in warm climates, and

ꢀ

in Argentina (province of Cordoba) begins to fructify

in early summer. Then, with the progress of the warm

season, the seeds fall from the plant at a time of year

when the environmental temperatures are between

30–40 ^ꢁC. Due to this fact, it is perhaps possible to

understand the capacity of the seeds to resist high

temperatures during the procedures carried out for

their use as biocatalysts. However, to date, we have

no evidence to justify this hypothesis.

When 1-butanol was used as the co-solvent, a very

high stereoselectivity (>99 e.e.%) to the formation of

(S)-1-PE was revealed, but with a very low conversion

(5%), while cyclohexanol showed both a marked loss

of stereoselectivity (75 e.e.%) and conversion (4%). It is

BIOCATALYSIS AND BIOTRANSFORMATION

7

Figure 3. Effect of different co-solvents on the conversion and

enantiomeric excess.

Figure 4. Effect of different concentrations of DMSO on the

conversion and enantiomeric excess.

also noteworthy that benzyl alcohol produced a fully

inhibitory effect on the reaction at the concentration

used. The other co-solvents tested showed practically

the same stereoselectivities as the reaction without

co-solvent. In agreement, it should be noted that

Decarlini et al. (2017) reported similar beneficial results

when DMSO was used as a co-solvent, in a reaction

system that used a strain of G. candidus as a biocata-

lyst in AP stereoselective reduction. Thus, DMSO was

selected as being the best co-solvent.

percentage of conversion, with this decreasing until a

minimum occurred when 5 ml of DMSO were added

to the reaction media. However, after this point, when

the quantity of DMSO was increased (ꢃ6 mL), the con-

version rose until reaching a transformation percent-

age of 98% with 8 mL of DMSO. Moreover, when

either 8, 9 or 10 ml of DMSO were used as the co-solv-

ent, the reaction was always practically quantitative

with an excellent stereoselectivity. These results are

very encouraging because some substrates with a very

low solubility in water require appreciable quantities

of co-solvents to obtain a high solubilisation in the

reaction media. Thus, 8 mL of DMSO was selected as

the best quantity of co-solvent. Finally, it is important

to note that we have not found any reports in the lit-

erature which have determined the effects of the con-

centration of a co-solvent on the performance of a

reaction using seeds as the biocatalyst.

Effect of DMSO concentration

DMSO proved to be beneficial in the conversion of AP

to (S)-1-PE. However, it is also important to determine

the optimal amount of co-solvent to be used, as it is

known that increasing the amount of a co-solvent can

increase the risk of a toxic effect. For this reason, we

carried out a study using different concentrations of

DMSO in the reaction media, and tested eleven differ-

ent concentrations of DMSO (0.5; 1–10 mL) in 80 mL of

sterile water. In addition, a reaction without co-solvent

was used as a control. All these assays were per-

formed at 40 ^ꢁC using 100 lL of AP with 10 g of whole

seeds of B. variegata, at 100 rpm for 48 h. Samples

were taken at the end of the reaction and analysed by

chiral GC-FID, and Figure 4 shows the effect of the

concentration of DMSO on the performance of

the reaction.

Effect of AP concentration

Due to the fact that our initial trials with different con-

centrations of AP showed that only a relatively small

amount was tolerated by the seeds of B. variegata, a

new set of experiments was performed using a higher

concentration of substrate. To carry this out, AP con-

centrations ranging from 18.9 to 37.9 mM were used

with 10 g of whole seeds, 8 mL of DMSO, 80 mL of

sterile water, and 100 rpm of shaking for 48 h at 40 ^ꢁC.

The resulting samples were analysed by quiral GC, and

the results are displayed in Figure 5, which show the

As can be seen in Figure 4, the different concentra-

tions of DMSO used in our trials did not modify the

stereoselectivity of the reaction, but an unusual

behaviour was observed with respect to the

8

G. I. DEMMEL ET AL.

Figure 6. Effect of added of buffer and pH.

Figure 5. Effect of AP concentration.

possibly different effects. On the one hand, a change

of the ionic force produced a strong reduction in the

conversion. This can be observed on comparing the

results obtained using sterile water (conversion of 98%

and 98 e.e. %) with those using a solution of 100 mM

NaCl (conversion of 52% and 97 e.e.%) and with a pH

5.0 phosphate buffer solution (conversion of 52% and

97 e.e.%). On the other hand, the conversion declined

markedly when the pH of the solution changed from

5.0 to 8.0, thereby revealing an effect due to the con-

centration of hydrogen ions in the reaction media (at

pH ¼ 5.0, a conversion of 52% and 97 e.e.% were

observed, while at pH ¼ 8.0, there was a conversion

of 23% and 95 e.e.%). Additionally, it is worth men-

tioning that the pH measurements taken during the

assays using water as a solvent showed pH values of

between 6.0 ꢄ 6.4 in the reaction media.

variations in the conversion and e.e. % for different AP

concentrations.

It can be observed that when the AP concentration

increased from 18.9 to 28.4 mM the conversion

decreased slightly, while for higher concentrations of

AP (>28.4 mM), the conversion decayed more quickly.

However, there was no significant variation in the e.e.

% of (S)-1-PE resulting from a change in the AP con-

centration, regardless of the amount of substrate

used. Thus, as these experiments showed that under

these conditions it was possible to increase the AP

concentration from 10.3 mM to 28.4 mM in the reac-

tion media without a marked loss in the efficiency of

the stereoselective reduction of AP, subsequent tests

were made using an AP concentration of 28.4 mM.

Effect of pH

The enantiomeric excess remained approximately

constant for all experiments, demonstrating that

changes in the concentration of the hydrogen ions

had no strong effects on the stereoselectivity of the

reaction. In another study, Kazici et al. (2016) reported

that 7.0 was the optimal working pH for reductions

carried out using freeze-dried carrot as the biocatalyst.

Moreover, in agreement with our findings, in their

report no variations in enantiomeric excess were

observed when the experiments were performed at

different pH values.

Finally, taking into account these results and con-

sidering the strong effect observed on the percentage

conversion, the optimal reaction media for the synthe-

sis of (S)-1-PE was obtained by utilising sterile water

as the solvent.

Here, in order to determine the pH effect, assays were

carried out at pH 5.0, 6.0, 6.5, 7.0, 7.5 and 8.0 using

80 mL of sterile 100 mM phosphate buffer, 300 lL of

AP, 10 g of whole seeds of B. variegata and 8 mL of

DMSO at 40 ^ꢁC, with shaking at 100 rpm for 48 h (the

concentration of the substrate used was 28.4 mM with

125 g/L of whole seeds). Then, samples were taken

and analysed by chiral GC-FID. In addition, a reaction

using a solution of 100 mM of NaCl was carried out,

and the results were compared to the reaction using

sterile water.

Figure 6 shows the pH effect on the reaction using

a phosphate buffer solution. Here, it can be observed

that the conversion was significantly affected by

changes in pH, which could have occurred by two

BIOCATALYSIS AND BIOTRANSFORMATION

9

Figure 7. Recyclability of the seeds of B. variegata. (A) Without extensive prewashing before reuse. (B) With extensive prewashing

with water before reuse.

(28 mM) was added and a new set of four reactions

was carried out with and without the addition of

NAPH. These results are shown in Table 2.

Recyclability of the seeds

The recycling performance of the seeds of B. variegata

was investigated by using the biocatalytic reduction of

AP under the optimum conditions described above as

the model reaction. Between each cycle of the reac-

tion, the seeds were recovered by filtration and

washed with sterile water, before being reused in the

next run. After 48 h of reaction, chiral GC-FID was

used to analyse the results, which are displayed in

Figure 7.

In Figure 7(A), it can be observed that the reuse of

the seeds of B. variegata had a strong negative impact

on its biocatalytic capacity. Immediately after the first

cycle, the conversion began to decrease and fell to a

conversion of just 6% (last cycle). In parallel, the

enantiomeric excess also began to decrease, but in a

less marked way (from 98 e.e. % in the first use to 93

e.e.% in the last reuse). In addition, another recycling

study was carried out, but with a more thorough

washing of the seeds with water before reuse. These

results are displayed in Figure 7(B), where it can be

observed that an extensive prewash of seeds before

reuse led to a greater reduction in the conversion per-

centage, for example, from 80% to 66% after the

second cycle.

As can be seen in Table 2 (entries 1 and 2),

when the reactions were carried out using super-

natant liquid either with or without the addition of

NADPH the formation of (1) was practically not

observed. In addition, when resuspended seeds were

used with or without added NADPH (Table 2, entry

3 and 4), only the reaction with the addition of this

cofactor (entry 4) recovered the performance

observed previously.

It is important to note that the results show that

the enzymes responsible for the transformation are

dependent of NADPH, and moreover, that this cofactor

is lost by successive washes during the recycling pro-

cess, thereby decreasing the reaction efficiency.

Furthermore, the results also demonstrate that the

reaction occurs within the seed and not within the

solution (Table 2, entries 3 and 4 vs. 1 and 2), due to

the fact that no solubilisation in the reaction media of

the enzymes responsible for this transformation

was observed.

Finally, it is noteworthy that although several stud-

ies of reuse using microorganisms as biocatalysts

have been published (Ni and Xu 2012), few reports

have described the reuse of plants as biocatalysts

(Alves Ferreira et al. 2012; Takemoto and

Tanaka 2001).

In view of these results, a new set of experiments

was performed using seeds soaked in the reaction

medium (48 h at 100 rpm and 40 ^ꢁC with 9% v/v of

DMSO) but without the addition of the substrate. The

supernatant liquid was separated by filtration and the

seeds were recovered. Then, this supernatant liquid

was divided into two parts, and the two portions of

the seed were washed with sterile distilled water and

resuspended in fresh reaction media. In all cases, AP

Storability of the seeds

The seasonal availability of vegetables is a distinct dis-

advantage when they are used as biocatalysts, since

10

G. I. DEMMEL ET AL.

Table 2. Recycling reactions^awith or without addition

of NADPH.

Entry

Reaction media

Supernatant liquid Without NADPH traces

With NADPH^c

traces

Resuspended seeds Without NADPH 66

With NADPH^c

99

Conversion %^be.e.%^b

1

2

3

4

nd^d

nd

96 (S)

98 (S)

^aReaction conditions: AP ¼ 28.4 nM, DMSO ¼ 9% v/v, water, 40 ^ꢁC,

100 rpm, 48 hs. ^bPercentage of Conversion and enantiomeric excess were

c

determined by chiral GC analysis. Added NADPH in a final concentration

of 0.24 mM. ^dNot determined.

their use is limited to the productive period. B. varie-

gata fructifies once a year, with the appearance of the

fruits being observed in the months of November-

December. The seedpod then matures gradually and

remains on the plant until falling at the end of

February. For this reason, in order to try to eliminate

this dependence on the seasonal production of the

seeds, we investigated ways to conserve them. To

carry this out, once collected the seeds were stored

dry inside a dark plastic container with a lid at room

temperature, with the results being displayed below in

Figure 8.

It can be observed in Figure 8 that the conversion

percentage (94–98%), and the enantiomeric excess

remained approximately constant (96–98%) over the

first 24 months (the end of which represents the cur-

rent time as this is an ongoing study). Thus, the

results reported here show that it is possible to store

the seeds of B. variegata at room temperature over a

prolonged period, during which time they can be

used as a common chemical reagent. Related to this,

we have not found any other stability studies in the

literature which were carried out on vegetables to

determine how long they can be stored without losing

their biocatalytic capacity.

Figure 8. Stability studies of the capability to reduce AP of

the seeds of B. variegata as a function of storage time.

Advantages and scope of the seeds of Bauhinia

variegata as a stereoselective bioreducer

Having optimised the reaction conditions for obtaining

(S)-1-PE, we carried out a biocatalytic reduction of sub-

stituted APs. With this objective in mind, several AP

derivatives with different substituents (fluorine, tri-

fluoromethyl, chlorine, bromine, nitro, cyano, hydroxy,

methyl, and methoxy) on the benzene ring were

selected to establish the advantages, limitations and

scope of using the bioreductive process promoted by

the seeds of B. variegata. Additionally, 2-chloroaceto-

phenone, 2-acetylthiophene and 2-acetylpyridine were

also assayed, with the results obtained displayed

below in Table 3.

It can be seen in Table 3 that two effects were

observed, depending on the position and nature of

the substituent. On the one hand, there appeared to

be a relationship between the reactivity and the elec-

tronic nature of the substituent groups. In fact, while

the seeds of B. variegata reduced both substituted APs

with donor or attractor electron groups, the substrates

with electron attractor groups were more reactive

than those having donating groups (as can be seen in

Table 3, on comparing entry 1 with 5, 10, 13–14, 16

and 19), with this difference being observed in the

conversion percentages and the reaction times.

Moreover, similar to that observed by Alves Ferreira

et al. (2012), under our reaction conditions the elec-

tron attractor groups appeared to have a positive

effect on the reductive process (Table 3, entries 3–13

vs. 14, 16 and 19). Consequently, the percentage of

Preparative scale bioreduction of AP

To try to demonstrate the viability of this biocatalytic

system, a large scale reaction was performed. For this

purpose, 2.4 g of AP (28.4 mM) dissolved in 64 mL of

DMSO and 80 g mass of seeds of Bauhinia variegata

were placed in a 2 L disinfected conical flask contain-

ing 640 mL of sterile water. The reaction was shaken

at 100 rpm for 48 h at 40 ^ꢁC, and 1.07 g of (S)-1-PE

were obtained after purification (44% yield, 98 e.e.%).

It should be noted that these extraction and purifica-

tion procedures were not optimised.

BIOCATALYSIS AND BIOTRANSFORMATION

11

Table 3. Ability of seeds of B. variegata to reduce different substituted prochiral ketones.

OH

O

whole seeds of B. variegata

R₁

28.4 mM of substrate, 9 % v/v DMSO,

R₂

water, 40°C, 100 rpm

*

Time

(days)

Entry

1^d

2

3

4

5

6

7

8

R₁

H-

R₂

Conversion %^a

e.e.%^a,b

Yield %^c

phenyl-

2

8

2

5

2

95

98 (S)

65 (R)

95 (S)

>99 (S)

67 (S)

78 (S)

91 (S)

>99 (S)

96 (S)

94 (S)

97 (S)

–

99 (S)

–

95 (S)

99 (S)

93 (S)

96 (S)

44

63

74

44

73

7

22

15

31

46

50

17

64

30

–

33

–

57

12

51

21

Cl-

H-

92

99

93

75

29

90

93

95

78

92

96

86

nr^e

49

Nr

98

30

88

98

2⁰-chlorophenyl-

3⁰-chlorophenyl-

4⁰-chlorophenyl-

2⁰,4⁰-dichlorophenyl-

2⁰-nitrophenyl-

3⁰-nitrophenyl-

4⁰-nitrophenyl-

4⁰-fluorophenyl-

4⁰-trifluorophenyl-

4⁰-bromophenyl-

4⁰-cyanophenyl-

4⁰-methylphenyl-

2⁰-hydroxyphenyl-

3⁰-hydroxyphenyl-

4⁰-hydroxyphenyl-

3⁰-methoxyphenyl-

4⁰-methoxyphenyl-

2-thyenyl-

9

10

11

12

13

14

15

16

17

18

19

20

21

2-pyridyl-

^aPercentage of Conversion and enantiomeric excess were determined by chiral GC analysis. ^bAbsolute configurations are shown between parentheses and

were assigned by comparing the retention time of chiral GC data with the reports in the literature and by comparison of the sign of optical rotations

c

e

with the literature values. Isolated yield after purification by chromatographic column. ^dScale reaction. No reaction.

conversion decreased markedly when 3⁰-hydroxy- and

4⁰-methoxyacetophenone were used as the substrates,

compared to the reaction performed with AP (Table 3,

entry 1 vs. 16 and 19). However, it is worth mention-

ing that a very good conversion percentage for the

formation of (14) was obtained when 4⁰-methylaceto-

phenone was used, but with longer reaction times

(Table 3, entry 1 vs. 14). In addition, 3⁰-methoxyaceto-

phenone (Table 3, entry 18) produced an excellent

conversion, and with only a very slight decrease in

stereoselectivity for the formation of the correspond-

ing chiral alcohol, compared to the reaction with AP.

In line with the observations described above,

Chanysheva et al (2019) recently demonstrated, using

Daucus carota and Petroselimum crispum, that the

nature of the substituent has a profound effect on the

relative reactivity of substituted acetophenones and

the enantioselectivity of the biocatalytic reduction.

Electron-withdrawing substituents enhance the initial

rate of reaction and product yields, while electron-

donating substituents decrease these. In this study,

correlations were established to characterise the

nature between the initial reaction rate and the sub-

stituent constant (rþ) in the aromatic ring. Moreover,

comparison of the q constants of bioreduction cata-

lysed by D. carota and P. crispum revealed that the

sensitivity of the reduction to the nature of the sub-

stituents was more significant in the case of D. carota.

It is also worth emphasising the results obtained

when hydroxyacetophenones were subjected to

reduction with the seeds of B. variegata, with the reac-

tion failing completely when 2⁰- and 4⁰-hydroxyaceto-

phenone were used under our reaction conditions

(Table 3, entries 15 and 17 respectively). In contrast,

3⁰-hydroxy-, 3⁰-methoxy and 4⁰-methoxyacetophenone

were transformed into corresponding chiral alcohols

with good conversions and excellent enantioselectiv-

ities (Table 3, entries 16, 18–19). In agreement, similar

results were reported by Decarlini et al. (2017), and

this marked difference in the behaviour of the hydrox-

yacetophenones might be explained by the formation

of a quinone intermediate in the reaction media by

deprotonation of the phenolic hydroxyl. Moreover, the

fact that 3⁰-methoxyacetophenone showed a greater

facility to produce the corresponding chiral alcohol,

while 4⁰-methoxyacetophenone was also reduced but

to a lesser extent, reinforces the hypothesis of the for-

mation a quinone intermediate under our reac-

tion conditions.

The importance of the results obtained by the

stereoselective reduction of 3⁰-hydroxy- and 3⁰-

methoxyacetophenone should be highlighted because

12

G. I. DEMMEL ET AL.

both (S)-1-(3⁰-methoxyphenyl)ethanol (18) and (16)

are key intermediates in the synthesis of rivastigmine

conversion in our reaction conditions (Table 3, entry

2). However, the percentage of enantiomeric excess in

the formation of (2) was lower than in the reaction

with AP, showing that the introduction of a chlorine

atom on the methyl group could have a strong

steric effect.

ꢀ

(anti-Alzheimer drug) (Mangas-Sanchez et al. 2009;

Yan et al. 2013; Sethi et al 2013a, 2013b). Moreover,

the chiral alcohol (18) can be used as a key intermedi-

ate in the synthesis of the experimental calcimimetic

agent NPS-R-568, a potent candidate for the treatment

of primary and secondary hyperparathyroidism (Sahin

2017). Enantiopure (18) is also a key chiral synthon for

the preparation of cycloalkyl[b]indoles, which can be

used for the treatment of a general allergic response

(Hillier et al. 2004, 2005).

An effect exerted by the relative position of the

substituents with respect to the carbonyl group can

be observed in Table 3 for the reduction of nitroaceto-

phenone derivatives (Table 3, entries 7–9). The nitro

substituent in the 2⁰-position had a marked negative

effect on the percentage reduction, compared to AP,

when the seeds of B. variegata were used as the bio-

catalyst. This much lower formation of (7) may have

been due to a steric effect resulting from the proxim-

ity of the nitro substituent to the carbonyl group

(Table 3 entry 1 vs. 7). It is also important to mention

that a similar effect for 2⁰-nitroacetophenone has been

previously reported (Aimar et al. 2014) when fruits of

glossy privet were used as the biocatalyst to produce

the corresponding (S)-alcohol.

As a counterpart, conversions to the corresponding

(S)-nitrophenylethanols were comparable to the AP

reduction when the nitro group was further from the

carbonyl group (Table 3, entry 1 vs. 8 and 9).

Moreover, this hypothesis is reinforced by the vari-

ation in the values of enantiomeric excess, which

increased when the nitro group is positioned further

away from the carbonyl group in the nitroacetophe-

nones series.

It should also be pointed out that in investigations

carried out by Zhu et al. (2005), it was established that

electronic properties, steric factors, and the ability to

form a hydrogen bond to the substituent at the ortho-

position, play a significant role in determining both

the activity and enantioselectivity of ketoreductase-

catalysed reductions.

Very good conversions (92–99%) and excellent

enantioselectivities (>99–95%) were obtained in the

formation of the corresponding (S)-alcohols when 2⁰-,

3⁰- and 4⁰-chloro, 4⁰-fluor, 4⁰-bromo, 4⁰-cyanoacetophe-

none, and 2-acetylpyridine (Table 3, entries 3–5, 10,

12–13 and 20) were used as the substrates. Moreover,

although 4⁰-trifluoromethylacetophenone produced a

slightly lower conversion than previous substrates

(78%; Table 3, entry 11), the reaction was completely

stereoselective (>99 e.e.%) for the formation of (11).

It is important to emphasise that, in contrast with

that observed with the fruits of L. Lucidum (Aimar

et al. 2014), the seeds of B. variegata showed a very

good efficiency and stereoselectivity for reducing 2-

acetylpyridine to (19) (Table 3, entry 19). Similarly,

when 2-acetylthiophene was used as the substrate

(Table 3, entry 20), the seeds of B. variegata were

more efficient, but with a somewhat lower stereo-

selectivity in the formation of (20) than the roots of E.

ꢀ

horridum (Bordon et al. 2015). Furthermore, the times

in which the reaction occurred in both cases were

shorter than those reported in the above-mentioned

investigation. As observed by other authors using veg-

etables as biocatalysts (Yadav et al. 2002; Xie et al.

2009; Alves Ferreira et al. 2012), the products we

obtained complied with Prelog’s rule.

It is necessary to point out that although we have

not yet conducted studies to try to establish the

group of enzymes responsible for enabling this trans-

formation, it is likely that the alcohol dehydrogenase-

ketoreductase enzyme, which is dependent on nico-

tinamide cofactors as redox equivalents (Blanchard

and van de Weghe, 2006; Xie et al. 2009), is involved

in the realisation of this transformation. However, fur-

ther investigation is needed to test this hypothesis.

Finally, it needs to be clarified that the procedure

carried out for the isolation of the products was not

optimised, which resulted in the observed differences

between the percentages of conversion and the iso-

lated yields. The low yields obtained during purifica-

tion of the synthesised compounds were a result of it

being difficult to break the emulsions formed during

the extraction procedures, which significantly compli-

cated the extraction procedure and induced a notable

decline in the performance of the system. In this

sense, efforts have been made to solve this problem

No steric effects were observed when chloroaceto-

phenone derivatives were used as substrates. While 2⁰-

chloroacetophenone produced a practically quantita-

tive conversion with 95 e.e.% of (3) (Table 3, entry 3),

3⁰- and 4⁰-chloroacetophenone (Table 3, entries 4 and

5) gave excellent but slightly lower conversions and

with the same percentage of enantiomeric excess. In

addition, in line with other studies (Decarlini et al

2017), 2-chloroacetophenone produced an excellent

BIOCATALYSIS AND BIOTRANSFORMATION

13

by adding salt or heating the crude reaction to try to

break the emulsions, but it has not yet been possible

to improve the extraction process of the final product.

Since the appearance of other products promoted

by secondary reactions was not observed during our

studies, it is probable that the differences found

between the conversion percentages and the yields of

the isolated products may have been due to the phe-

nomenon of absorption on the biomass. Currently, our

research team is carrying out studies aimed at elimi-

nating the inert substances contained in the seeds,

which may have been hindering the purification of

the synthesised products, as well as attempting to iso-

late, purify and characterise the enzyme responsible

for the biocatalytic process.

Disclosure statement

No potential conflict of interest was reported by the authors.

The authors alone are responsible for the content and writ-

ing of the paper.

Funding

ꢀ

This work was financially supported by Secretarıa de Ciencia

ꢀ

y Tecnologıa de la Universidad Nacional de Cordoba, Grant

number 33620180100619CB01.

ORCID

Mario L. Aimar

http://orcid.org/0000-0001-8547-7198

References

Aguirre-Pranzoni C, Bisogno F, Orden AA, Kurina-Sanz M.

2015. Lyophilized Rhodotorula yeast as all-in-one redox

biocatalyst: access to enantiopure building blocks by sim-

ple chemoenzymatic one-pot procedures. J Mol Catal B

Enzym. 114:19–24.

Conclusions

Herein, we have presented an optimised methodology

for the production of chiral alcohols using whole

seeds of Bauhinia variegata as the biocatalyst, which

were very efficient in reducing ketones to their corre-

sponding chiral alcohols, some of which are important

key intermediates in the synthesis of commercial

drugs. Thus, the results presented here represent a

new and potential non-traditional use for the seeds of

this plant. Moreover, and as demonstrated by our

results, using whole seeds as the biocatalyst has the

advantage that they can be stored as a “stable bag of

enzymes”, thereby making this process independent

of seasonal availability.

In addition, the appreciable bioreduction potential

of the seeds of B. variegata, this makes them candi-

dates for major studies in order to extend their use as

an environmentally friendly tool for the synthesis of

other chiral key intermediates. Further investigations

are currently ongoing in order to try to expand the

applications of this novel, inexpensive and environ-

mentally friendly biocatalytic agent.

ꢀ

Aimar ML, Bordon DL, Formica SM, Cantero JJ, Vazquez AM,

Velasco MI, Rossi LI. 2014. Fruits of the glossy privet

(Ligustrum lucidum – Oleaceae) as biocatalysts for produc-

ing chiral aromatic alcohols. Biocatal Biotransform.

32(5–6):348–357.

Almeida de Sousa EY, Maia da Silva FF, Oliveira de Souza JM,

Alves Ferreira D, Gomes de Lemos TL, Queiroz Monte FJ.

2019. Preparation of chiral alcohols by enantioselective

reduction of prochiral ketones with Sinapis alba seeds as

biocatalyst. Ind Crops Prod. 141:111729.

~

Alves Ferreira D, Costa da Silva R, da Costa Assunc¸ao JC, de

Mattos MC, Gomes de Lemos TL, Queiroz Monte FJ. 2012.

Lens culinaris: a new biocatalyst for reducing carbonyl and

nitro groups. Biotechnol Bioproc E. 17(2):407–412.

Andrade LH, Utsunomiya RS, Omori AT, Porto ALM,

Comasseto JV. 2006. Edible catalysts for clean chemical

reactions: bioreduction of aromatic ketones and biooxida-

tion of secondary alcohols using plants. J Mol Catal B

Enzym. 38(2):84–90.

~

Assunc¸ao JCC, Machado LL, Lemos TLG, Cordell GA, Monte

FJQ. 2008. Sugar cane juice for the bioreduction of car-

bonyl compounds. J Mol Catal B Enzym. 52–53:194–198.

€

Atak GB, Bayraktar E, Mehmetoglu U. 2019. Optimization of

the asymmetric synthesis of (S)-1-phenylethanol using

Ispir bean as whole-cell biocatalyst. Green Process Synth.

8(1):525–532.

Acknowledgments

Bennamane M, Razi S, Zeror S, Aribi-Zouioueche L. 2018.

Preparation of chiral phenylethanols using various vegeta-

bles grown in Algeria. Biocatal Agric Biotechnol. 14:52–56.

Bertini LM, Gomes de Lemos TL, Alves LA, Queiroz Monte FJ,

de Mattos MC, de Oliveira M. 2012. Soybean (Glycine max)

as a versatile biocatalyst for organic synthesis. Afr J

Biotechnol. 11(30):7766–7770.

Mario L. Aimar gratefully acknowledges the financial support

ꢀ

from Secretarıa de Ciencia y Tecnologıa de la Universidad

ꢀ

Nacional de Cordoba. Gabriela I. Demmel thankfully

ꢀ

acknowledges financial support from Universidad Catolica de

ꢀ

Cordoba. Laura I. Rossi is a researcher of CONICET. We also

thank Dr. Paul Hobson, native speaker, for the revision of

this manuscript. This work is dedicated to the teaching car-

ꢀ

Bizerra AMC, de Gonzalo G, Lavandera I, Gotor-Fernandez V,

ꢀ

eer Dr. Jose Luis Cabrera.

de Mattos MC, de Oliveira MCF, Gomes de Lemos TL,

14

G. I. DEMMEL ET AL.

Gotor V. 2010. Reduction processes biocatalyzed by Vigna

unguiculata. Tetrahedron Asymmetry. 21(5):566–570.

rivastigmine based on lipase-catalyzed processes. J Org

Chem. 74(15):5304–5310.

Blanchard N, van de Weghe P. 2006. Daucus carota L. medi-

ated bioreduction of prochiral ketones. Org Biomol Chem.

4(12):2348–2353.

Mansfield P, Henry D, Tonkin A. 2004. Single-enantiomer

drugs: elegant science, disappointing effects. Clin

Pharmacokinet. 43(5):287–290.

ꢀ

Bordon DL, Villalba LD, Aimar ML, Cantero JJ, Vazquez AM,

Formica SM, Krapacher CR, Rossi LI. 2015. Weeds as bioca-

talysts in the stereoselective synthesis of chiral phenyle-

thanols used as key intermediates for pharmaceuticals.

Biocatal Agric Biotechnol. 4(4):493–499.

Mathre DJ, Thompson AS, Douglas AW, Hoogsteen K, Carroll

JD, Corley EG, Grabowski E. 1993. A practical process for

the preparation of Tetrahydro-l-methyl-3,3-diphenyll H, 3H

-pyrrolo1[,2-c]-[1,3,2]oxazaborole-Borane. A highly enantio-

selective stoichiometric and catalytic reducing agent. J

Org Chem. 58(10):2880–2888.

Modh KM, Parmar PT, Panigrahi B, Anand IS, Patel CN. 2011.

Pharmacognostical and phytochemical evaluation of

leaves of Bauhinia variegata Linn. Pharmacognosy J. 3(24):

45–49.

Carvalho da Silva RA, Marques de Mesquita B, Frota de

Farias I, Garcia do Nascimento PG, Gomes de Lemos TL,

Queiroz Monte FJ. 2018. Enzymatic chemical transforma-

tions of aldehydes, ketones, esters and alcohols using

plant fragments as the only biocatalyst: Ximenia ameri-

cana grains. Mol Catal. 445:187–194.

Nakamura K, Inoue Y, Matsuda T, Misawa I. 1999.

Stereoselective oxidation and reduction by immobilized

Geotrichum candidum in an organic solvent. J Chem Soc

Perkin Trans 1(16):2397–2402.

~

ꢀ

Cazetta T. 2014. Desracemizac¸ao de alcoois secundarios por

~

estereoinversao e reduc¸ao de metilenocetonas por micro-

organismos. Tese de Doutorado apresentada ao Instituto

ꢀ

~

ꢀ

de Quımica da UniCamp para obtenc¸ao do tıtulo de

Nakamura K, Matsuda T. 1998. Asymmetric reduction of

ketones by the acetone powder of Geotrichum candidum.

J Org Chem. 63(24):8957–8964.

Nakamura K, Yamanaka R, Matsuda T, Harada T. 2003.

Recent developments in asymmetric reduction of ketones

with biocatalysts. Tetrahedron Asymmetry. 14(18):

2659–2681.

^

Doutora em Ciencias, pages 28, 58 and 92. Universidad

Estadual de Campinas. [Accessed 2019 Aug 20]. http://

repositorio.unicamp.br/jspui/handle/REPOSIP/250620.

Chanysheva AR, Vorobyova TE, Zorin VV. 2019. Relative

reactivity of substituted acetophenones in enantioselec-

tive biocatalytic reduction catalyzed by plant cells of

Daucus carota and Petroselinum crispum. Tetrahedron.

75(36):130494.

Nguyen LA, He H, Pham-Huy C. 2006. Chiral Drugs: An

Overview. Int J Biomed Sc. 2(2):85–100.

Ni Y, Xu JH. 2012. Biocatalytic ketone reduction: a green and

efficient access to enantiopure alcohols. Biotechnol Adv.

30(6):1279–1288.

Ohkuma T, Koizumi M, Yoshida M, Noyori R. 2000. General

asymmetric hydrogenation of hetero-aromatic ketones.

Org Lett. 2(12):1749–1751.

Comasseto JV, Assis LF, Andrade LH, Schoenlein-Crusius IH,

Porto A. 2006. Biotransformations of ortho-, meta- and

para-aromatic nitrocompounds by strains of Aspergillus

terreus: Reduction of ketones and deracemization of alco-

hols. J Mol Cat B Enzym. 39(1–4):24–30.

Cordell GA, Lemos TLG, Monte FJQ, de Mattos MC. 2007.

Orden AA, Magallanes-Noguera C, Agostini E, Kurina-Sanz M.

2009. Anti-prelog reduction of ketones by hairy root cul-

tures. J Mol Catal B Enzym. 61(3–4):216–220.

Patel RN. 2018. Biocatalysis for synthesis of pharmaceuticals.

Bioorg Med Chem. 26(7):1252–1274.

Patil D. 2015. Biocatalysis using plant material: a green

access to asymmetric reduction. Int J ChemTech Res. 8(8):

318–324.

Rajkapoor B, Jayakar B, Murugesh N. 2003. Antitumor activity

of Bauhinia variegata on dalton’s ascetic lymphoma. J

Ethnopharmacol. 89(1):107–109.

Roschangar F, Sheldon RA, Senanayake CH. 2015.

Overcoming barriers to green chemistry in the pharma-

ceutical industry – the Green Aspiration Level^TMconcept.

Green Chem. 17(2):752–768.

Sahin E. 2017. Debaryomyces hansenii as a new biocatalyst in

the asymmetric reduction of substituted acetophenones.

Biocatal Biotransform. 35(5):363–371.

Sethi MK, Bhandya SR, Maddur N, Shukla R, Kumar A,

Jayalakshmi Mittapalli V. 2013a. Asymmetric synthesis of

an enantiomerically pure rivastigmine intermediate using

ketoreductase. Tetrahedron Asymmetry. 24(7):374–379.

Sethi MK, Bhandya SR, Maddur N, Shukla R, Kumar A,

Jayalakshmi Mittapalli V. 2013b. Chemo-enzymatic synthe-

sis of optically pure rivastigmine intermediate using alco-

hol dehydrogenase from baker’s yeast. J Mol Catal B

Enzym. 91:87–92.

Vegetables as chemical reagents.

478–492.

J Nat Prod. 70(3):

ꢀ

Decarlini MF, Aimar ML, Vazquez AM, Vero S, Rossi LI, Yang

P. 2017. Fungi isolated from food samples for an efficient

stereoselective production of phenylethanols. Biocatal

Agric Biotechnol. 12:275–285.

Hillier MC, Desrosiers JN, Marcoux JF, Grabowski E. 2004.

Stereoselective carbon-carbon bond formation via the

Mitsunobu displacement of chiral secondary benzylic alco-

hols. Org Lett. 6(4):573–576.

Hillier MC, Marcoux JF, Zhao DL, Grabowski EJJ, Mckeown

AE, Tillyer RD. 2005. Stereoselective formation of carbon-

ꢄ carbon bonds via S_N2-displacement: synthesis of substi-

tuted cycloalkyl[b]indoles. J Org Chem. 70(21):8385–8394.

ꢀ

Hoyos P, Hernaiz MJ, Alcantara AR. 2017. Biocatalyzed pro-

duction of fine chemicals. In Reference Module in Life

Sciences. Moo-Yung, M., Ed.; Pergamon: Oxford, UK; pp.

1–38.

€

Kazici HC, Bayraktar E, Mehmetoglu U. 2016. Optimization of

the asymmetric synthesis of chiral aromatic alcohol using

freeze-dried carrots as whole-cell biocatalysts. Green

Process Synth. 5:131–137.

Kumaraswamy G, Ramesh S. 2003. Soaked Phaseolus aureus

L: an efficient biocatalyst for asymmetric reduction of pro-

chiral aromatic ketones. Green Chem. 5(3):306–308.

ꢀ

Mangas-Sanchez J, Rodrıguez-Mata M, Busto E, Gotor-

ꢀ

Fernandez V, Gotor V. 2009. Chemoenzymatic synthesis of

BIOCATALYSIS AND BIOTRANSFORMATION

15

Sinou C, Forest F, Lewis GP, Bruneau A. 2009. The genus

Bauhinia s. l. (Leguminosae): a phylogeny based on the

plastid trnL-trnF region. Botany. 87(10):947–960.

Guangxi, South China and their biogeographic implica-

tions. BMC Evol Biol. 14(:88 116.

Wu XF, Xiao JL. 2007. Aqueous-phase asymmetric transfer

hydrogenation of ketones - a greener approach to chiral

alcohols. Chem Commun. 24(24):2449–2466.

Xie Y, Xu JH, Lu WY, Lin GQ. 2009. Adzuki bean: A new

resource of biocatalyst for asymmetric reduction of aro-

matic ketones with high stereoselectivity and substrate

tolerance. Bioresour Technol. 100(9):2463–2468.

Yadav JS, Nanda S, Thirupathi Reddy P, Bhaskar Rao A. 2002.

Efficient enantioselective reduction of ketones with

Daucus carota root. J Org Chem. 67(11):3900–3903.

Yan PCh, Zhu GL, Xie JH, Zhang XD, Zhou QL, Li YQ, Shen

WH, Che DQ. 2013. Industrial scale-up of enantioselective

hydrogenation for the asymmetric synthesis of rivastig-

mine. Org Process Res Dev. 17(2):307–312.

ꢀ

Solıs A, Martınez RS, Cervantes F, Perez HI, Manjarrez N, Solıs

M. 2019. Reduction of substituted benzaldehydes, aceto-

phenone and 2-acetylpyridine using bean seeds as crude

reductase enzymes. Biocatal Biotransform. 37(2):152–157.

Takemoto M, Tanaka K. 2001. Synthesis of optically active-

phenylpyridylmethanols by Camellia sinensis cell culture. J

Mol Catal B Enz. 15(4–6):173–176.

Tao J, Xu JH. 2009. Biocatalysis in development of green

pharmaceutical processes. Curr Opp Chem Biol. 13(1):

43–50.

Villa R, Molinari F, Levati M, Aragozzini F. 1998.

Stereoselective reduction of ketones by plant cell cultures.

Biotechnol Letters. 20(12):1105–1108.

Wang Q, Song Z, Chen Y, Shen S, Li Z. 2014. Leaves and

fruits of Bauhinia (Leguminosae, Caesalpinioideae,

Cercideae) from the Oligocene Ningming Formation of

Zhu D, Rios BE, Rozzell JD, Hua L. 2005. Evaluation of sub-

stituent effects on activity and enantioselectivity in the

enzymatic reduction of aryl ketones. Tetrahedron

Asymmetry. 16(8):1541–1546.

Article Doi

Optimisation, scope and advantages of the synthesis of chiral phenylethanols using whole seeds of Bauhinia variegata L. (Fabaceae) as a new and stereoselective bio-reducer of carbonyl compounds

DOI: 10.1080/10242422.2020.1789115

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