Tailored sol-gel immobilized lipase preparates for the enzymatic kinetic resolution of heteroaromatic alcohols in batch and continuous flow systems

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Tailored sol–gel immobilized lipase preparates for

the enzymatic kinetic resolution of heteroaromatic

alcohols in batch and continuous ﬂow systems†

Cite this: RSC Adv., 2017, 7, 52977

a

b

Madalina Elena Moisa,

˘ ˘

Cristina Georgiana Spelmezan,

Cristina Paul,

˘

a

Judith Hajnal Bartha-Vari,

Laszlo Csaba Bencze,

Florin Dan Irimie,

´

a

b

a

*

Csaba Paizs,

Francisc Peter

and Monica Ioana Tosa

¸

´

Tailored immobilized lipases from Candida antarctica B and Pseudomonas ﬂuorescens, with improved

thermal and operational stability, were prepared through ﬁne tuning of the structure of the sol–gel

matrix, using various binary or ternary precursor mixtures for the EKR of various chiral heteroaromatic

secondary alcohols with benzofuran, benzo[b]thiophen, phenothiazine and 2-phenylthiazol moieties.

The operational stability in batch process was studied for ﬁve selected systems by performing reuse

experiments, using the conversion, enantiomeric excesses and enantiomeric ratio as parameters,

demonstrating the dependence of the sol–gel lipase preparate performance on the structure of both

biocatalyst and substrate. The resolution of the benzofuranic substrates with the best performing

biocatalysts was studied in continuous-ﬂow mode, using the productivity as a criterion. The speciﬁc

reaction rates under continuous-ﬂow operation (r_ﬂow) were higher than those obtained in batch mode

(r_batch) in both cases, sustaining its usefulness for further process development.

Received 12th September 2017

Accepted 9th November 2017

DOI: 10.1039/c7ra10157k

rsc.li/rsc-advances

immobilization, the protein is multipoint attached to a carrier

by covalent bonds, directly or through a spacer. One of the

Introduction

benets of this method is that enzyme leaching is not possible,

but the activity could be signicantly modied, decreasing in

most of the cases.

With the aims of stabilizing enzymes and improving enzymatic

activity and selectivity, the development of eﬀective immobili-

zation methods has become crucial for the synthetic applica-

tions of lipases. Moreover, the processes performed with

immobilized catalysts oﬀer as advantages continuous produc-

tion, the absence of the biocatalyst in the product stream and

the possibility to reuse the biocatalyst. In this way, the synthetic

and production processes could be more selective and are for

certain more procient economically and environmentally.

Accordingly, immobilization methods such as binding to

a carrier, cross-linking, and encapsulation in an organic or

inorganic polymeric matrix have been intensively studied.^1–8

While the adsorption on solid supports (such as Celite, a dia-

tomaceous earth) is a simple, inexpensive and easy to perform

method, suitable for applications in organic solvents, the

enzyme leaching from the support is generally the most

important disadvantage of this procedure.⁹In the covalent

For enzymes which are easily deactivated (for instance by

covalent immobilization), encapsulation could be the most

useful method. In the case of sol–gel encapsulation inert glasses

with improved catalytic properties (porosity, thermal and

mechanical stability, and tunable hydrophobicity) are prepared.

The synthesis can be performed by acid- or base-catalyzed

hydrolysis of the organic silane precursors, followed by

condensation of the resulted intermediates in the presence of

an enzyme.^10–12When some protective additives (crown ether,

isopropyl alcohol, etc) were used in the sol–gel immobilization

process, for certain substrates a supplementary increase of the

lipase activity in esterication and enzymatic kinetic resolution

(EKR) processes was already reported.^13–15

Lipases are most frequently used for the synthesis of chiral

intermediates by kinetic resolution in an immobilized form.^16,17

Using continuous-ow reactors, the performances of

enzyme-catalyzed reactions are generally signicantly

improved, including the reduction of reaction time, as

compared to the classical batch method. The packed-bed

reactor (a column containing the immobilized biocatalyst

which is ushed with the reaction mixture using a pump,

allowing ow and residence time control) is usually the rst and

simplest choice.^18–21

^aBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and

´

Chemical Engineering, Babe¸s-Bolyai University of Cluj-Napoca, Arany Janos 11,

RO-400028, Cluj-Napoca, Romania. E-mail: mtosa@chem.ubbcluj.ro

^bBiocatalysis Group, Faculty of Industrial Chemistry and Environmental Engineering,

University Politehnica Timi¸soara, C. Telbisz 6, RO-300001, Timi¸soara, Romania

† Electronic supplementary information (ESI) available: Protocols for lipases

immobilization, chemical synthesis of racemic compounds, chromatographic

chiral separation conditions, lipase and solvents screening results, recyclability

studies results. See DOI: 10.1039/c7ra10157k

This journal is © The Royal Society of Chemistry 2017

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In the present research, several immobilized lipase pre-

The silane precursors, methyl- (MeTMOS), vinyl- (VTMOS),

parates (lipases from Candida antarctica B and from Pseudo- octyl- (OcTMOS), and phenyl-trimethoxysilane (PhTMOS) were

monas uorescens immobilized by sol–gel entrapment, purchased from Merck (Germany), excepting tetra-methoxysilane

adsorption on Celite or their combination) were tested in (TMOS), a product of Fluka. The other materials and additives

transesterication reactions of various heteroaromatic used for immobilization, tris-(hydroxymethyl)-aminoethane, iso-

secondary alcohols, useful chiral building blocks for the phar- propyl alcohol, Celite 545, acetone and hydrochloric acid were

maceutical industry.^22–25Previous studies^15,26–28already stated products of Merck, while sodium uoride and the ionic liquid

the versatility of the sol–gel immobilization method and the 1-octyl-3-methyl-imidazolium tetrauoroborate [Omim]BF₄were

possibility to ne tune the composition of the binary or ternary purchased from Fluka.

precursors mixture to achieve high activity and selectivity, but

Vinyl acetate and the solvents used for enzymatic reactions

these studies were carried out on model secondary alcohols were of the highest grade from Sigma-Aldrich or Merck and

˚

(1-phenylethanol or 2-alkanols). The present study demon- stored over molecular sieves (4 A). Solvents employed in

strates for the rst time the eﬃciency of the tailored binary and chemical synthesis were used as purchased (methanol > 99.8%)

ternary sol–gel immobilized lipase preparates for the EKR of or dried over molecular sieve (dichloromethane) or sodium

various heteroaromatic alcohols. The recycling capacity of the (diethyl ether). Other reagents were purchased from Sigma-

best performing biocatalysts was also investigated and two Aldrich or Merck.

continuous-ow procedures were developed by testing the

A Heidolph Vibramax 110 shaker equipped with incubator

most important reaction parameters: temperature, substrate module (Heidolph, Germany) was used for all batch experiments.

concentration, and ow rate.

Continuous-ow experiments were performed in a stainless

steel column (50 ꢂ 2.1 mm) using the thermostat and the pump

modules of an Agilent 1200 Series HPLC instrument.

Experimental

Materials and methods

Chemical synthesis

The synthesis of the investigated racemic substrates and prod-

ucts was rst performed using known methods^29–33from the

corresponding prochiral heteroaryl-methyl-ketones (1a–c,f) by

chemical reduction with sodium borohydride or from the car-

baldehydes (1d,e,g–i) by Grignard reaction. Racemic ethanols

rac-2a–i were next transformed with acetyl chloride (1.2 equiv.)

and a catalytic amount of 1% DMAP/pyridine in dry CH₂Cl₂into

the corresponding racemic acetates rac-3a–i (Scheme 1a). The

obtained NMR data for rac-2,3a–i are in agreement with those

already reported.^29–33

Thin layer chromatography (TLC) was performed on Merck

Kieselgel 60F254 sheets. Spots were visualized by treatment

with 5% ethanolic phosphomolybdic acid solution followed by

heating. Preparative chromatographic separations were per-

formed using column chromatography on Merck Kieselgel 60

(63–200 mm).

¹H-NMR and ¹³C-NMR were recorded in CDCl₃at 25 ^ꢀC using

a Bruker spectrometer operating at 300 or 600 MHz and 75 or

151 MHz, respectively.

Chiral HPLC (Agilent 1200 Series instrument) and GC (Agi-

lent 77890A instrument) were employed for the quantitative

analysis of the enzymatic kinetic resolutions, by periodically

taking samples (50 mL) and analyzing them by chiral chroma-

tography: GC (compounds rac-2,3a,b) or HPLC (compounds rac-

2,3c–i). The type of chiral columns used for the enantiomeric

separations, separation conditions and retention times for each

individual enantiomer are presented in Table 1S.† The absolute

conguration of products was assigned according to previously

reported data, based on the retention time of a reference

enantiomerically enriched compound.^29–33

The enantiomeric excesses of the substrate ee_Sand product

ee_Pwere determined from peak areas of GC or HPLC chro-

matograms. The enantiomeric ratio (E) was calculated with the

known equation E ¼ ln[(1 ꢁ c)(1 ꢁ ee_S)]/ln[(1 ꢁ c) (1 + ee_S)],

using the calculated conversion c ¼ ee_S/(ee_S+ ee_P).³⁴

Lipase B from Candida antarctica was purchased as solid

lyophilized powder from C-Lecta (Leipzig, Germany, product

code 20606-1). Lipase AK (beige-brown powder, from Pseudo-

monas uorescens) and lipase A from Candida antarctica were

bought from Sigma-Aldrich (Germany, product code 534730

and 65986 respectively). Novozyme 435 (lipase from Candida

antarctica B, immobilized on acrylic resin) was also purchased

from Sigma Aldrich.

Immobilization of lipases

The immobilization protocols for the sol–gel entrapment of

lipases B from Candida antarctica and Pseudomonas uorescens

(Amano-AK) were accomplished as previously described

(Table 1), with NaF as catalyst for simultaneous hydrolysis and

polycondensation (Method 1)²⁷or using HCl for the formation

of a prepolymer (Method 2).³⁵The combined method of sol–gel

entrapment and adsorption was also carried out as described

elsewhere.^26–28Method 3 was simple adsorption on Celite 545

(see the ESI† for detailed description of all immobilization

Scheme 1 (a) Chemical synthesis of racemic substrates; (b) EKR

mediated by lipases.

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Table 1 Immobilized lipases description

Preparate code^a

Immobilization method

Silane precursors^b(molar ratio)

CaL-B 1 BS

CaL-B 1 BSC

CaL-B 1 TS

CaL-B 1 TSC

CaL-B 1 TS*

CaL-B 2 TS

CaL-B 2 TSC

CaL-B Celite

AK 1 BS

Method 1

Method 1 + adsorption on Celite 545

Method 1

Method 1 + adsorption on Celite 545

Method 1

Method 2

Method 2 + adsorption on Celite 545

Method 3 (adsorption on Celite 545)

Method 1

VTMOS : TMOS (4 : 1)

PhTMOS : VTMOS : TMOS (1.6 : 0.4 : 1)

PhTMOS : MeTMOS : TMOS (1.6 : 0.4 : 1)

—

OcTMOS : TMOS (1 : 1)

AK 1 BSC

AK 1 TS

AK 1 TSC

AK 2 TSC

Method 1 + adsorption on Celite 545

Method 1

Method 1 + adsorption on Celite 545

Method 2 + adsorption on Celite 545

OcTMOS : TMOS (1 : 1)

PhTMOS : VTMOS : TMOS (1.6 : 0.4 : 1)

PhTMOS : MeTMOS : TMOS (1.6 : 0.4 : 1)

a

b

Lipases source: CaL-B-lipase B from Candida antarctica, AK-lipase from Pseudomonas uorescens. VTMOS (vinyltrimethoxysilane), PhTMOS

(phenyltrimethoxysilane), MeTMOS (methyltrimethoxy-silane), OcTMOS (octyltrimethoxysilane) and TMOS (tetramethoxysilane).

methods). The protein loading values, calculated for 1 g bio- temperatures (25–65 ^ꢀC) at ow rates of 0.1–0.5 mL min^ꢁ1. The

catalyst, were 17.4 mg for sol–gel entrapment and adsorption on residence time (RT) was calculated using the volume of n-

Celite and 10.7 mg for the entrapment combined with adsorp- hexane in the lipase-lled reactor (measuring the weights of the

tion, respectively. They were determined as described previ- dry and wet lipase-lled column) and the ow rate. Samples (50

ously.²⁷The immobilization method and the silane precursors mL) were taken periodically, diluted with n-hexane (500 mL) and

used for every preparate selected for the present study are analyzed by HPLC. The steady-state condition was reached in

described in Table 1.

30–90 min, depending on the used ow. Between experiments,

the lipase-lled column was washed with n-hexane at 0.5

mL min^ꢁ1for 20 min and stored overnight at 4 ^ꢀC.

Enzymatic O-transesterications

Lipase-catalyzed O-acylation of rac-2c,d in batch mode. To

a solution of rac-2c,d (10 mmol) in n-hexane (1 mL), vinyl acetate

(0.75 equiv. and 3 equiv., respectively) and the immobilized

lipase (5 mg) were added and shaken at 700 rpm at 65 ^ꢀC until

the maximum conversion was reached (48% aer 14 h for rac-2c

and 50% aer 2.5 h for rac-2d). Samples were collected as

previously mentioned and subjected to HPLC analysis.

All experiments were performed in triplicate and the average

values were further used.

Analytical-scale lipase-mediated O-acylation of racemic

hetero-aryl-ethanols rac-2a–i. The immobilized lipase (5 mg for

the 17.4 mg protein per g biocatalyst preparates and 8 mg for

the 10.7 mg protein per g biocatalyst preparates), the tested

substrate (10 mmol), solvent (1 mL) and vinyl acetate (30 mmol,

2.77 mL) were combined in a reaction vial and shaken at 700 rpm

at 25 ^ꢀC until z50% conversion was reached. For the reactions

performed in absence of solvent, vinyl acetate was added in

excess (250 mL). Samples (50 mL) were taken periodically from

the reaction vials and diluted with 950 mL n-hexane/2-propanol

for HPLC analysis or 950 mL CH₂Cl₂for GC analysis and then

ltered before analysis.

Reuse experiments. The rst cycle of enzymatic trans-

esterication was performed as described above. Aer each cycle

the enzyme was washed three times with 1 mL n-hexane, and

immediately reused in the next cycle. Samples for GC or HPLC

analysis were collected aer each cycle as previously mentioned.

Lipase-catalyzed O-acylation of rac-2c,d in continuous-ow

packed-bed microreactors. Continuous-ow experiments were

performed in a thermostated stainless steel column (50 ꢂ

2.1 mm) lled with immobilized lipase (107 mg of AK 1 BS and

51 mg of CaL-B 1 TS*) attached to an Agilent 1200 Series HPLC

pump. Before performing the reactions, the microreactor was

washed with n-hexane at 0.5 mL min^ꢁ1for 20 min. The solution

Results and discussion

Chemical synthesis of racemic heteroaryl-ethanols rac-2a–i

and their corresponding acetates rac-3a–i

Racemic heteroaryl-ethanols rac-2a–c,f were obtained starting

from the corresponding prochiral heteroaryl-methyl-ketones

(1a–c,f) by chemical reduction with sodium borohydride,

while in the case of rac-2d,e,g–i the Grignard reaction of car-

baldehydes 1d,e,g–i as starting materials was used (Scheme 1a).

Racemic ethanols rac-2a–i were used next as substrates in

lipase-catalyzed transesterication reactions (Scheme 1b) and

also for the synthesis of the corresponding racemic acetates

rac-3a–i (see ESI,† Section 2), used for establishing the chro-

matographic separation methods (see ESI,† Section 3).

Enzymatic kinetic resolution of racemic ethanols rac-2a–i

through O-acylation with vinyl acetate and lipase preparates

in organic solvents

of the substrate rac-2c or rac-2d (10–100 mM) and vinyl acetate Eﬃcient enzymatic procedures for the synthesis of enantio-

(0.75 equiv. or 3 equiv.) in n-hexane was pumped through the merically enriched (R)- and (S)-ethanols through enzymatic

lipase-lled

microreactor

thermostated

at

diﬀerent kinetic resolution of the racemic ethanols or their esters

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(generally acetates) with commercially available lipases have investigated in several solvents with diﬀerent polarities, such as

been already described.^29–33

n-hexane, toluene, diisopropyl ether, methyl-tert-butyl ether,

An optimal immobilization method can improve the bio- dichloromethane and acetonitrile (see ESI, Tables 10S–18S†).

catalyst eﬃciency and the operational stability, allowing the The reactions were evaluated using the conversion c, enantio-

process operation in continuous ow reactors, which can meric excesses (of the untransformed alcohol ee_Sand of the

provide increased productivity. Lipase encapsulation in sol–gel formed ester ee_P), and enantiomeric ratio E as parameters.

matrices leads to enzyme preparates with high mechanical and

As a preliminary general conclusion of the screening, all

thermal stability and also high porosity. Moreover the surface lipase preparates demonstrated high enantiomer selectivity

properties of the sol–gel matrix can be tailored for an optimal towards the racemic substrates in neat vinyl acetate, except

interaction of enzymes with particular substrates by choosing some L-AK preparates (AK 1 TSC and AK 2 TSC in the acylation

the appropriate silane precursors and synthesis method.

reaction of rac-2b, E ¼ 25–58 see Table 3S,† entries 14 and 15 in

The immobilizations were accomplished by adsorption on ESI†), while the conversion was inuenced by the structure of

Celite 545, sol–gel entrapment in mesoporous silica materials substrate, as well as by the immobilization method. A more

obtained from binary (BS) or ternary silane (TS) precursor detailed study of the inuence of diﬀerent immobilization

mixtures, and sol–gel entrapment combined with adsorption on conditions was not the subject of this work.

Celite 545, as described in the experimental part (Table 1). Two

The optimal lipase preparates are summatively presented in

sol–gel entrapment methods were employed, one that uses NaF Table 2. Some particular information obtained for each

as catalyst for simultaneous hydrolysis and polycondensation substrate is discussed.

(Method 1) and another one that uses a sol prepolymer obtained

with HCl (Method 2).

The rst tested substrate, rac-1-(benzo[b]thiophen-2-yl)

ethan-1-ol rac-2a, was quantitatively resolved (c y 50%) with

The items used in the present work were selected from all tested CaL-B preparates and almost all L-AK preparates in

numerous preparates, according to their activities on model neat vinyl acetate (Table 2S in ESI†). In the given reaction

substrates (rac-2-hexanol and rac-1-phenylethanol, data not conditions, this substrate was easily acylated regardless to the

shown), with the aim to explore several immobilization tech- immobilization method, but it is noteworthy that the catalytic

niques based on entrapment and adsorption, as well as support eﬃciency of the immobilized preparates was higher (c ¼ 45–

matrices with diﬀerent composition and morphology. In order 50%, E [ 200) compared to the reaction catalyzed by native

to be able to compare the catalytic activities of the tested lipase CaL-B, with only 16% conversion aer the same reaction time

preparates, the same enzyme content was used in all experi- (9.5 hours, as presented in Table 2S, entry 1 in ESI†). Since

ments. Vinyl acetate was employed as solvent and as irreversible almost all immobilized preparates studied in the acylation

acyl donor as the liberated vinyl alcohol undergoes spontaneous reaction of rac-2a gave very good results, for the solvent

tautomerization to acetaldehyde, shiing the reaction equilib- screening one candidate from each of the two forms of lipases

rium towards product formation.³⁶

(CaL-B and AK) was selected (CaL-B 1 TSC and AK 1 TSC). The

In order to nd the possible applications of the tested lipase best results were obtained with CaL-B 1 TSC in n-hexane (c y

preparates in the chemoenzymatic synthesis of heteroaromatic 50% aer 1 h, see Table 10S, entry 1 in ESI†), therefore we tested

compounds, rst the selected substrates rac-2a–i (40 mM) were the other CaL-B preparates in the optimal solvent, n-hexane as

acylated in neat vinyl acetate in the presence of immobilized well. The results (Table 17S in ESI†) indicated that almost all

biocatalysts (see ESI, Tables 2S–9S†). Furthermore since the biocatalysts aﬀorded maximum conversion of the substrate in

nature of the solvent could signicantly inuence the selectivity 1 h, except for CaL-B 1 BS (c ¼ 42%, Table 17S, entry 1 in ESI†)

and activity of the immobilized enzyme, the enzymatic acylation and CaL-B 2 TSC (c ¼ 24%, Table 17S, entry 6 in ESI†).

of racemic ethanols (10 mM) with vinyl acetate (3 equiv.) in

The most eﬃcient biocatalysts (see Table 2) for the acetyla-

presence of the previously selected optimal biocatalysts was tion of the racemic alcohols rac-2b,d,i in neat vinyl acetate

Table 2 Lipase mediated O-acylation of rac-2a–i (40 mM) in neat vinyl acetate at 25 ^ꢀC^a

c^b,d(%)

b,c

c

Entry

Substrate rac-2a–i

Optimal biocatalyst

Reaction time (h)

ee_S(%)

ee_P(%)

1

2

rac-2a

rac-2b

All CaL-B preparates

CaL-B 1 TS*

CaL-B Celite

AK 1 BS

CaL-B 1 TS*

CaL-B Celite

All preparates

AK 1 BS

9.5

132

>99

<1

>99

50

82 (ꢃ1.7)

83 (ꢃ2.1)

94 (ꢃ1.)

91 (ꢃ0.9)

96 (ꢃ2.8)

<1

89 (ꢃ1.4)

52 (ꢃ1.2)

75 (ꢃ0.5)

45 (ꢃ0.5)

46 (ꢃ0.5)

49 (ꢃ0.5)

48 (ꢃ0)

49 (ꢃ0.9)

<1

47 (ꢃ0.5)

34 (ꢃ0.5)

43 (ꢃ0)

3

4

rac-2c

rac-2d

136

24

144

11

5

6

7

8

rac-2e^e

rac-2g

rac-2h

rac-2i

CaL-B Celite

CaL-B 2 TSC

178

40

a

E [ 200. ^bExperiments were performed in triplicate and standard deviations from average values are given in brackets. ^cDetermined from peak

areas of GC or HPLC chromatograms. Calculated using the formula c ¼ [ee_S/(ee_S+ ee_P)]. ^eNo product was detected even aer 6 days.

d

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based on reaction conversion, proved to be the CaL-B preparates

In the case of rac-1-(10-ethyl-10H-phenothiazin-1-yl)ethan-1-

obtained by sol–gel entrapment in ternary silane precursor ol, rac-2e, a sterically hindered secondary alcohol, even sol–gel

systems with or without deposition on Celite (CaL-B 1 TS* and preparates containing lipase A from C. antarctica, previously

CaL-B 2 TSC, with 43–48% conversion), depending on the reported as an eﬃcient biocatalyst³²were inactive (the acetate

substrate structure, but the adsorbed CaL-B lipase was similarly was not detected even aer 6 days, see Table 6S in ESI†).

good with 46–49% conversion. Although the entrapped pre-

The resolution of rac-1-(10-ethyl-10H-phenothiazin-4-yl)

parates have the overall advantage of less enzyme leaking, in ethan-1-ol, rac-2h with CaL-B immobilized preparates in neat

almost anhydrous organic solvents the adsorbed enzyme could vinyl acetate occurred very slowly, but in a selective manner

also exhibit high stability, as it was recently demonstrated for (Table 8S in ESI†). The best results were obtained with CaL-B on

Burkholderia cepacia lipase.³⁷

Celite 545 (c ¼ 34% aer 178 h, E [ 200, entry 7, Table 2).

Lipase from Pseudomonas uorescens (Amano AK), already Based on the solvent screening, performed in the same manner,

reported as eﬃcient,²⁹demonstrated excellent selectivity for the n-hexane was also selected as optimal solvent since the reaction

EKR of rac-2c in neat vinyl acetate (entry 3 in Table 2); the rate signicantly increased and the maximum 50% conversion

optimal biocatalyst was obtained by sol–gel immobilization in was obtained aer only 27 h (entry 7 in Table 15S of ESI†). As

a binary silane precursor system (AK 1 BS).

already reported,^31,32during the enzymatic reactions and

The most appropriate solvent for the transesterication particularly during the work-up of the reaction mixture, the

reactions of rac-2a–d,i proved to be n-hexane in all cases (E [ instability of the phenothiazinic compounds (ethanol and

200 at c ¼ 46–50% aer 1–28 hours, depending on the substrate, acetate) was observed, mainly for rac-2h.

see Table 3).

It is noticeable that the best performing biocatalysts have

In an eﬀort to nd a general enzymatic route for the shown comparable biocatalytic power with the extensively used

synthesis of enantiomerically enriched phenothiazinic- Novozyme 435 in the transesterication reactions of rac-

ethanols, an enzyme and solvent screening was next per- 2a,b,d,g,i in neat vinyl acetate (Tables 2S, 3S, 5S, 7S and 9S†).

formed on the model substrate rac-2g and the best reaction Even though the acylation reaction of rac-2d in neat vinyl

conditions (lipase preparate and solvent) were applied to the acetate occurred faster with Novozyme 435 (c ¼ 50% aer 16 h,

other phenothiazinic substrates. Accordingly, the enzymatic entry 9, Table 5S†) than with any of the sol–gel CaL-B preparates

kinetic resolution of rac-2g with CaL-B and L-AK immobilized (c ¼ 48% aer 136 h with CaL-B 1 TS*, entry 10, Table 5S†), the

preparates in neat vinyl acetate was rstly investigated. AK 1 enantiomeric excess of the product was smaller (ee_P¼ 97%

BS was the only appropriate enzyme preparate in neat vinyl versus ee_P> 99%). In terms of conversion Novozyme 435 proved

acetate (47% conversion aer 11 h, entry 14 in Table 7S in to be more eﬃcient in the acylation reaction of rac-2h in neat

ESI†). When performing the resolution of rac-2g with this vinyl acetate (c ¼ 47% aer 42 h, entry 6, Table 8S†), in

biocatalyst and vinyl acetate (3 equiv.) in the selected solvents, comparison to all the tested sol–gel preparates (c ¼ 14% aer

the maximum conversion was obtained in n-hexane (34% 42 h with CaL-B 1 TS*, entry 3, Table 8S†). It must be taken into

aer 2 h and 50% aer 9 h, entries 1 and 4 in Table 14S consideration that Novozyme 435 is fragile under stirring in

of ESI†). These reaction conditions were next applied to the batch mode, oen resulting in the breakage of the particles and

other phenothiazinic substrates rac-2e,f,h, leading to very loss of enzyme activity. Consequently, this biocatalyst might not

good results in case of rac-2f (50% c and E [ 200 aer be a suitable candidate for repeated use. Biocatalysts obtained

8 h, entry 2 in Table 18S of ESI†), while for rac-2e and rac- through sol–gel entrapment, on the other hand, do not have

2h further investigations were performed (Tables 6S–8S in this disadvantage and have proved in our previous work^27,35,38

ESI†).

and in other studies^11,15,39remarkable recycling stability.

Table 3 Lipase mediated acylation of rac-2a–i (10 mM) with vinyl acetate (3 equiv.) in n-hexane at 25 ^ꢀC^a

b,c

c

Entry

Substrate rac-2a–i

rac-2a

Biocatalyst

Reaction time (h)

ee_S(%)

ee_P(%)

c^b,d(%)

1

2

CaL-B 1 BSC

CaL-B Celite

CaL-B 1 TS*

CaL-B Celite

AK 1 BS

CaL-B 1 TS*

CaL-B Celite

AK 1 BS

1

5

>99

50

rac-2b

92 (ꢃ1.6)

48 (ꢃ0.5)

>99

50

3

4

rac-2c

rac-2d

21

19

2

8

9

86 (ꢃ1.2)

94 (ꢃ2.1)

92 (ꢃ2.5)

>99

46 (ꢃ0.5)

49 (ꢃ0.5)

48 (ꢃ0.8)

50

5

6

7

8

rac-2f

rac-2g

rac-2h

rac-2i

AK 1 BS

CaL-B Celite

CaL-B 2 TSC

>99

50

27

28

99 (ꢃ0.5)

50 (ꢃ0)

95 (ꢃ2.2)

49 (ꢃ0.8)

E [ 200. ^bExperiments were performed in triplicate and standard deviations from average values are given in brackets. ^cDetermined from peak

a

d

areas of GC or HPLC chromatograms. Calculated using the formula c ¼ [ee_S/(ee_S+ ee_P)].

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cycle. Between cycles the catalyst was just washed with anhy-

drous n-hexane three times and thereaer immediately reused.

As presented in Fig. 1A and B, the immobilized lipases were

eﬃcient biocatalysts for the EKR of the benzothiophenic

derivative rac-2a in ten consecutive reaction cycles (Tables 19S

and 20S†). The adsorbed CaL-B was even more eﬀective than the

binary sol–gel preparate containing vinyl pending groups, but

by extending the reaction time to more than one hour the

entrapped biocatalyst was also able to give almost complete

conversion of the substrate with high enantiomer purity.

For the EKR of rac-2c the most active immobilized form of AK

was obtained by immobilization of this lipase in a binary sol–gel

material as well, but containing octyl groups instead of vinyl, to

supply the organic functionality. The recycling results (Fig. 2A)

show a signicant decrease of the conversion in only three

reaction cycles (from 42 to 23% c, Table 21S in the ESI†), while

the enantiopurity of the product remained high. The same

biocatalyst was also subjected to recyclability studies in the

acylation reaction of rac-2g. In this case the conversion started

to drop only aer 5 acylation cycles (Fig. 2B, Table 22S in ESI†).

A rigorous comparative evaluation of this enzyme preparate

Operational stability of the immobilized lipase preparates at

repeated use in batch processes

Long-term catalytic eﬃciency is a key requirement for any

industrial biocatalyst and immobilization should have positive

impact on the operational stability of the enzyme. The sol–gel

entrapped lipases demonstrated excellent reuse eﬃciency in the

acylation reaction of 2-octanol, showing the signicant poten-

tial of this technique to provide highly eﬃcient solid-phase

biocatalysts for prolonged exploitation.²⁷

However, the reuse capability of the biocatalyst must be

tested for every individual application, since it depends on the

structure of the substrate, the composition, and characteristics

of the reaction system, as well as on the operational parameters.

For this reason, the reusability of the optimal immobilized

lipase preparates was studied in the enantioselective acylations

of rac-2a,c,d,g,i with vinyl acetate, in n-hexane (Fig. 1–4, Tables

19S–24S in ESI†).

Each reaction was repeated with the same enzyme preparate

up to 10 times and has been allowed to proceed to ꢄ50%

conversion, or as long as the preparate remained active and

enantioselective, before the catalyst was subjected to the next

Fig. 1 Multiple use eﬃciency of the selected biocatalysts for the EKR of rac-1-(benzo[b]thiophen-2-yl)ethan-1-ol rac-2a (10 mM) with vinyl

acetate (3 equiv.) in n-hexane at 25 ^ꢀC with: (A) CaL-B 1 BSC and (B) Cal-B on Celite 545 (after 40 min reaction time). Error bars represent

standard deviations from average.

Fig. 2 Multiple use eﬃciency of AK 1 BS as selected biocatalyst for the EKR of: (A) rac-1-(benzofuran-2-yl)ethan-1-ol rac-2c (10 mM) with vinyl

acetate (3 equiv.) in n-hexane at 25 ^ꢀC (after 20 hours reaction time) and (B) rac-1-(10-ethyl-10H-phenothiazin-3-yl)ethan-1-ol rac-2g (10 mM)

with vinyl acetate (3 equiv.) in n-hexane at 25 ^ꢀC (after 8 hours reaction time). Error bars represent standard deviations from average.

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sol–gel matrix and the catalytic eﬃciency, the presence of bulky

and mid-polar phenyl groups could have a specic inuence on

the structural organization and hydrophobicity of the formed

silica network, resulting in improved performance, as it was

also observed in our previous studies.^15,40

The operational stability of a biocatalyst obtained by the

immobilization method involving the formation of a sol pre-

polymer in acid catalysis (Method 2) was investigated for the

EKR of the phenyl-thiazolyl derivative rac-2i.

As results from Fig. 4, this immobilized CaL-B lipase was not

suitable for repeated use, although in the screening study

demonstrated comparable catalytic eﬃciency and enantiose-

lectivity with the best performing preparates obtained with the

method using NaF as catalyst (Method 1). The most important

drawback was the quick decline of the catalytic performance, as

shown by the low conversion values (falling from 49 to 17% c in

the second cycle, see Table 24S in ESI†). The stability of this type

of sol–gel matrix is probably not appropriate for the employed

reaction conditions.

Fig. 3 Multiple use eﬃciency of the selected biocatalyst CaL-B 1 TS*

for the EKR of rac-1-(benzofuran-3-yl)ethan-1-ol rac-2d (10 mM) with

vinyl acetate (3 equiv.) in n-hexane at 25 ^ꢀC (after 20 hours reaction

time). Error bars represent standard deviations from average.

Lipase-mediated acylation of racemic heteroaromatic

ethanols in continuous-ow packed-bed microreactors

The best performing immobilized lipase preparates were also

tested in continuous-ow experiments using packed-bed micro-

reactors. From all CaL-B preparates, CaL-B 1 TS* was selected and

from AK preparates, lipase AK 1 BS was selected. The two

benzofuranyl-ethanols rac-2c,d were chosen as substrates (Fig. 5).

According to preliminary results of experiments performed

in continuous-ow system, under identical reaction conditions,

an increase in the amount of vinyl acetate for the acylation

ꢀ

reaction of rac-2c (catalysed by AK 1 BS, in n-hexane, at 65 C

and 0.5 mL min^ꢁ1, substrate concentration 10 mM) did not

have a major impact on the conversion (20% c with 0.75 equiv.

of vinyl acetate and 22% c with 3 equiv. of vinyl acetate), while

for the acylation reaction of rac-2d (catalysed by CaL-B 1 TS*, in

Fig. 4 Multiple use eﬃciency of the selected biocatalyst CaL-B 2 TSC

for the EKR of rac-1-(2-phenyl-thiazol-4-yl)-ethanol rac-2i (10 mM)

with vinyl acetate (3 equiv.) in n-hexane at 25 ^ꢀC (after 28 hours

reaction time). Error bars represent standard deviations from average.

n-hexane, at 35 C and 0.5 mL min^ꢁ1, substrate concentration

ꢀ

10 mM) rising the amount of acyl donor led to a signicant

increase in conversion (8% c with 0.75 equiv. of vinyl acetate

and 23% c with 3 equiv. of vinyl acetate). For this reason, in all

further experiments involving rac-2c 0.75 equiv. of vinyl acetate

were used and for rac-2d, 3 equiv. of the same acylating agent

were employed.

performance is not possible, since the time needed to reach the

maximum conversion strongly depends on the substrate struc-

ture (20 h for rac-2c and 8 h for rac-2g). These results conrm

that the recycling capacity of a biocatalyst depends on the

structure of the substrate.

Although rac-2d is a positional isomer of rac-2c, it was

demonstrated to be a substrate with diﬀerent behaviour in the

EKR process. In this case, CaL-B lipase was the most eﬃcient

biocatalyst (not Amano AK, as for rac-2c) and the best immo-

bilized enzyme showed high eﬃciency throughout multiple use

(Fig. 3, Table 23S in ESI†).

Firstly, the continuous-ow acylation reaction was investi-

gated at diﬀerent temperatures (25–65 ^ꢀC) using a 10 mM

The ternary sol–gel matrix was obtained from tetramethoxy-

silane as building block of the inorganic structure, with phenyl-

trimethoxysilane and methyl-trimethoxysilane (at 4 : 1 relative

molar ratio), supplying the pending organic functional groups

of the hybrid material. The presence of relatively high phenyl

group concentration was benecial for the long-term catalytic

eﬃciency of this biocatalyst, which did not show notable

decline of conversion and enantioselectivity throughout 10

Fig. 5 Continuous-ﬂow lipase mediated kinetic resolution of racemic

recycles. Although it is diﬃcult to correlate the structure of the benzofuranyl-ethanols rac-2c,d.

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substrate solution at 0.5 mL min^ꢁ1ow rate (Fig. 6A). For both the ow rate was gradually decreased from 0.5 to 0.1 mL min^ꢁ1

,

lipase preparates, an increase in conversion was observed by maintaining the temperature at 65 ^ꢀC, which led to almost

increasing the temperature, the maximum value being reached complete conversion of the substrates (48% and 50%, Fig. 6B).

at 65 ^ꢀC. Further increase of the reaction temperature was However it must be taken into consideration that even though

limited by the boiling point of the employed solvent. In the case the conversion increases, the productivity of the continuous-

of CaL-B 1 TS*-mediated acylation of rac-2d, almost a linear ow system can decrease at lower ow rates. This however

increase in conversion with respect to temperature was depends on the magnitude of conversion increase and ow rate

observed, while for AK 1 BS-catalyzed acylation almost a 10% decrease, since the rate of product formation is not a linear

ꢀ

increase in conversion was seen from 45 to 50 C.

function on conversion.

This experiment revealed that the immobilized lipase B from

Furthermore, in an eﬀort to enhance the productivity of the

C. antarctica is more eﬃcient towards rac-2d than is the continuous-ow packed-bed microreactor, diﬀerent substrate

immobilized lipase from Pseudomonas uorescens towards rac- concentrations were investigated at 65 ^ꢀC at 0.1 (Fig. 6C) and 0.5

2c, as a two times higher conversion was reached at 65 ^ꢀC (41% mL min^ꢁ1ow rate (Tables 4 and 5).

vs. 20%). The continuous-ow reactions proceeded with excel-

It was noted that for CaL-B 1 TS*-mediated trans-

lent enantioselectivities (E [ 200) throughout the investigated esterication of rac-2d, there was no signicant decline in

ꢀ

temperature domain (25–65 C).

conversion in the range of 10–50 mM substrate concentration at

With the aim to improve the conversion of the enantiose- 0.1 mL min^ꢁ1

.

lective acylation of the investigated racemic ethanols rac-2c,d,

Even when a 100 mM substrate concentration was employed,

the conversion was still high (44%), proving the eﬃciency of

this biocatalyst. In the case of the immobilized preparate AK 1

BS, the conversion of the acylation reaction of rac-2c decreased

as the substrate concentration increased and at 100 mM the

conversion was half of the initial value. At higher substrate

concentrations (10–100 mM) the enantiomeric excesses of the

products were maximum (>99%), aﬀording excellent enantio-

selectivities of the continuous-ow acylation reactions.

The maximum productivity achieved by lipase AK 1 BS was

37.4 mmol of product min^ꢁ1g^ꢁ1at 65 ^ꢀC, 0.5 mL min^ꢁ1ow rate

and 100 mM substrate concentration (Table 4, entry 20), while

the maximum productivity of CaL-B 1 TS* was 157.5 mmol of

product min^ꢁ1g^ꢁ1at 65 ^ꢀC, 0.5 mL min^ꢁ1ow rate and 50 mM

substrate concentration (Table 5, entry 18).

The long term operational stability of the two selected bio-

catalysts was also studied using a 10 mM substrate concentra-

tion and 0.1 mL min^ꢁ1ow rate. The obtained results showed

that the immobilized preparate AK 1 BS maintained 81% of its

initial activity at 65 ^ꢀC aer 44 h of continuous operation (data

not shown).

At 55 ^ꢀC the enzyme exhibited remarkable stability as almost

95% of the initial activity was kept aer 100 h. The other bio-

catalyst, CaL-B 1 TS* proved to be even more stable, the results

showing practically no loss of activity aer 100 h at 65 ^ꢀC

(Fig. 7). The enantioselectivities of both lipase-mediated

continuous-ow reactions were not aﬀected during long term

operation of the biocatalysts.

Another goal was to compare the productivities of the

continuous-ow systems with those obtained in batch mode by

calculating the specic reaction rates (eqn (1) and (2)):

ꢀ

ꢁ

n_P

mmol

r_batch

¼

(1)

t ꢂ m_emin ꢂ g

ꢀ

½Pꢅ ꢂ f mmol

r_flow

(2)

m_e

min ꢂ g

Fig. 6 Eﬀect of temperature (A), ﬂow rate (B) and substrate concen-

tration (C) on the continuous-ﬂow enzymatic acylation of rac-2c with

AK 1 BS ( ) and rac-2d with CaL-B 1 TS*( ) with vinyl acetate (0.75

and 3 equiv., respectively) in n-hexane.

The specic reaction rate of a batch reaction (r_batch) can be

calculated from the amount of product formed (n_p, mmol), the

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Table 4 Inﬂuence of temperature, ﬂow rate and substrate concentration on the continuous-ﬂow kinetic resolution of rac-2c with vinyl acetate

(0.75 equiv.) in n-hexane mediated by AK 1 BS

Flow rate

Residence

Substrate

concentration [mM]

Temperature

[^ꢀC]

Productivity^d

[mL min^ꢁ1

]

time^a[min]

ee_S(%)

[mmol min^ꢁ1g^ꢁ1

]

b,c

Entry

1

2

3

4

5

6

7

8

0.5

0.4

0.3

0.2

0.1

0.5

0.1

0.5

0.1

0.5

0.1

0.5

0.1

0.5

0.1

0.17

0.21

0.28

0.42

0.85

0.17

0.85

0.17

0.85

0.17

0.85

0.17

0.85

0.17

0.85

10

20

30

40

50

100

25

35

45

50

55

60

65

2 (ꢃ0.5)

0.9

2.3

7.0

8.4

9.3

8.2

9.0

6.5

4.5

15.9

8.0

22.4

12.3

26.2

16.4

32.7

17.3

37.4

22.4

5 (ꢃ1.2)

5 (ꢃ1.4)

17 (ꢃ0.5)

17 (ꢃ1.7)

22 (ꢃ2.4)

25 (ꢃ0.5)

27 (ꢃ1.2)

47 (ꢃ1.6)

54 (ꢃ2.4)

91 (ꢃ3.1)

21 (ꢃ1.7)

76 (ꢃ1.2)

19 (ꢃ2.1)

78 (ꢃ1.7)

15 (ꢃ1.7)

78 (ꢃ2.1)

16 (ꢃ0.5)

57 (ꢃ2.5)

9 (ꢃ1.2)

9

10

11

12

13

14

15

16

17

18

19

20

21

31 (ꢃ0.5)

volume of liquid inside the packed bed microreactor

a

b

Calculated with the formula RT ¼

½minꢅ. Experiments were performed in triplicate and

flow rate

c

d

standard deviations from average values are given in brackets. Determined from peak areas of HPLC chromatograms. Calculated using the

ꢀ

ꢁ

½Pꢅ ꢂ f mmol

average values of the conversions with the formula r_flow

¼

.

m_e

min ꢂ g

Table 5 Inﬂuence of temperature, ﬂow rate and substrate concentration on the continuous-ﬂow kinetic resolution of rac-2d with vinyl acetate

(3 equiv.) in n-hexane mediated by CaL-B 1 TS*

Flow rate

Residence

Substrate

concentration [mM]

Temperature

[^ꢀC]

Productivity^d

[mL min^ꢁ1

]

time^a[min]

ee_S(%)

[mmol min^ꢁ1g^ꢁ1

]

b,c

Entry

1

2

3

4

5

6

7

8

0.5

0.4

0.3

0.2

0.1

0.5

0.1

0.5

0.1

0.5

0.1

0.5

0.1

0.5

0.1

0.25

0.31

0.42

0.62

1.25

0.25

1.25

0.25

1.25

0.25

1.25

0.25

1.25

0.25

1.25

10

20

30

40

50

100

25

35

45

50

55

60

65

22 (ꢃ2.1)

30 (ꢃ0.5)

42 (ꢃ2.1)

49 (ꢃ2.4)

58 (ꢃ2.1)

63 (ꢃ0.5)

69 (ꢃ1.7)

72 (ꢃ0.8)

78 (ꢃ2.4)

91 (ꢃ0.9)

>99

72 (ꢃ1.2)

95 (ꢃ2.5)

49 (ꢃ2.2)

>99

30 (ꢃ0.8)

91 (ꢃ0.5)

47 (ꢃ2.5)

95 (ꢃ2.4)

14 (ꢃ1.7)

78 (ꢃ1.7)

17.7

22.6

29.5

32.5

36.4

38.4

40.4

33.1

26.0

18.9

9.8

82.7

19.3

97.4

29.5

90.6

37.8

157.5

48.2

118.1

86.6

9

10

11

12

13

14

15

16

17

18

19

20

21

volume of liquid inside the packed-bed microreactor

a

b

Calculated with the formula RT ¼

½minꢅ. Experiments were performed in triplicate and

flow rate

c

d

standard deviations from average values are given in brackets. Determined from peak areas of HPLC chromatograms. Calculated using the

ꢀ

ꢁ

½Pꢅ ꢂ f mmol

average values of the conversions with the formula r_flow

¼

.

m_e

min ꢂ g

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rac-2a,d), average (5 cycles for rac-2g) or modest (approx. 55%

enzymatic activity recovered aer three cycles for rac-2c, 35%

enzymatic activity recovered aer two cycles for rac-2i).

Furthermore for the resolution of the racemic benzofuranyl-

ethanols (rac-2c,d) the best performing biocatalysts were

studied in continuous-ow mode at diﬀerent temperatures, ow

rates and substrate concentrations, using the productivity as

criterion. The calculated specic reaction rates under

continuous-ow operation (r_ow) were in both cases higher than

those obtained in batch mode (r_batch), 4.5 and 1.1 mmol min^ꢁ1g^ꢁ1

for AK 1 BS preparate and 9.8 and 6.7 mmol min^ꢁ1g^ꢁ1for CaL-B 1

TS* preparate, respectively, demonstrating the advantages of this

operating system. Moreover, these biocatalysts showed very good

stability under long term continuous operation at high temper-

Fig. 7 Long term operational stability of immobilized preparates AK 1

BS and CaL-B 1 TS* in the continuous-ﬂow acylation reactions of rac-

2c,d (10 mM) with vinyl acetate (0.75 and 3 equiv., respectively) in n-

hexane at 55 ^ꢀC and 65 ^ꢀC, respectively and 0.1 mL min^ꢁ1ﬂow rate.

Error bars represent standard deviations from average.

ꢀ

atures (65 C). In conclusion the use of tailored sol–gel immo-

bilized biocatalysts in packed-bed reactors under continuous

ow regime represents a good strategy to perform lipase cata-

lyzed kinetic resolutions of heteroaromatic ethanols and oﬀers

a good background for process development.

reaction time (t, min), and the mass of the applied enzyme

(m_e, g). Similarly the specic reaction rate of a continuous-ow

reaction (r_ow) can be determined from the concentration of the

product ([P], mmol mL^ꢁ1), the ow rate (f, mL min^ꢁ1), and the

mass of the applied enzyme (m_e, g).

Conﬂicts of interest

There are no conicts to declare.

A rigorous comparison between these two can be made at the

same degree of conversions, using identical experimental

conditions (substrate and acyl donor concentrations, solvent,

and temperature).⁴¹

In the case of AK 1 BS-catalyzed batch and continuous-ow

reactions a signicant diﬀerence in the specic reaction rates

values was observed, this biocatalyst aﬀording a 4 times higher

Acknowledgements

This work was supported by a grant of the Romanian National

Authority for Scientic Research, CNDI–UEFISCDI, project

number PN-II-PT-PCCA-2013-4-0734.

productivity under continuous-ow operation (r_batch

¼

Notes and references

1.1 mmol min^ꢁ_ꢀ¹g^ꢁ1, r_ow¼ 4.5 mmol min^ꢁ1g^ꢁ1, at 48%

conversion, 65 C, 10 mM substrate concentration, 0.75 equiv.

of vinyl acetate and 0.1 mL min^ꢁ1ow rate). On the other hand,

CaL-B 1 TS* proved to be eﬃcient in both systems, nevertheless,

the specic reaction rate obtained in continuous-ow mode was

almost 1.5 times higher than the one obtained in batch mode

(r_batch¼ 6.7 mmol min^ꢁ1g^ꢁ1, r_ow¼ 9.8 mmol min^ꢁ1g^ꢁ1, at 50%

´

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in biotechnology, Humana Press, New York, 2006, vol. 22.

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The possibility to obtain tailored immobilized biocatalysts for

the EKR of some heteroaromatic secondary alcohols through

entrapement in various sol–gel matrix, starting from binary or

ternary precursor mixtures, was demonstrated. Using the

conversion c, enantiomeric excesses ee_Pand ee_Sand enantio-

meric ratio E in order to characterize the enzymatic process

eﬃciency, the optimal conditions (enzyme preparates and

solvent) were found for each tested substrate.

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126.

´

Moreover the operational stability in batch process was

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9 V. V. Pace, J. V. Sinisterra and A. R. Alcantara, Curr. Org.

Chem., 2010, 14, 2384.

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1996, 49, 527.

52986 | RSC Adv., 2017, 7, 52977–52987

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13 M. T. Reetz, P. Tielmann, W. Wiesenhofer, W. Konen and

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´

F. Peter, Process Biochem., 2011, 46, 52.

´

29 C. Paizs, M. I. To¸sa, V. Bodai, G. Szakacs, I. Kmecz,

´

14 P. Tielmann, H. Kierkels, A. Zonta, A. Ilie and M. T. Reetz,

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RSC Adv., 2017, 7, 52977–52987 | 52987

Article Doi

Tailored sol-gel immobilized lipase preparates for the enzymatic kinetic resolution of heteroaromatic alcohols in batch and continuous flow systems

DOI: 10.1039/c7ra10157k

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Article abstract of DOI:10.1039/c7ra10157k

Full text of DOI:10.1039/c7ra10157k

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