Covalent Immobilization of Pseudomonas stutzeri Lipase on a Porous Polymer: An Efficient Biocatalyst for a Scalable Production of Enantiopure Benzoin Esters under Sustainable Conditions

Article abstract of DOI:10.1021/op500326k

The immobilization of lipase from Pseudomonas stutzeri (lipase TL) by covalent bonding to a porous polymer is described for the first time. The immobilized enzyme was characterized in terms of optimal pH and thermal stability, and its catalytic efficiency was tested in the kinetic resolution (KR) of symmetrical and unsymmetrical benzoins (1,2-diaryl-2-hydroxyethanone structures). Reactions were performed in the green solvent 2-MeTHF, reaching maximum conversion and enantiomeric excess, with a significant increase of productivity due to the possibility of reuse of the catalyst. Moreover, the immobilization allowed the development of an adequate scaling up of this KR process permitting a further rise in the catalytic efficiency. Finally, the dynamic kinetic resolution of benzoin (DKR) was carried out by the combination of the immobilized lipase and a ruthenium catalyst (Shvo's catalyst) in 2-MeTHF, reaching conversions up to 90%, maintaining its excellent enantioselectivity during six catalytic cycles.

Full text of DOI:10.1021/op500326k

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Full Paper
Covalent Immobilization of Pseudomonas stutzeri lipase on a
porous polymer: an efficient biocatalyst for a scalable production
of enantiopure benzoin esters under sustainable conditions
María J. Hernáiz, Antonio Aires-Trapote, Pilar Hoyos, Andrés
Alcántara, Aitana Tamayo, Juan Rubio, and Angel Rumbero
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op500326k • Publication Date (Web): 06 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Covalent Immobilization of Pseudomonas stutzeri
lipase on a porous polymer: an efficient biocatalyst
for a scalable production of enantiopure benzoin
esters under sustainable conditions.
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Antonio Aires-Trapote,†,‡ Pilar Hoyos,† Andrés R. Alcántara,† Aitana Tamayo,§ Juan Rubio,§
Angel Rumbero*,‡ and María J. Hernáiz.*†
†Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, Complutense
University of Madrid, Campus de Moncloa, 28040 Madrid, Spain. Fax: (+34) 9139 41822; Tel:
(+34) 91139 41821
‡Department of Organic Chemistry, Faculty of Science, Autonoma University of Madrid,
Campus de Cantoblanco.28049ꢀMadrid. Spain.
§Department of Chemistry Physics of Surfaces and Processes, Instituto de Cerámica y Vidrio
(CSIC), Kelsen, nº 5, 28049ꢀMadrid. Spain.
KEYWORDS: Covalent immobilization, Lipase, Pseudomonas stutzeri, Benzoins, Dynamic
Kinetic resolution, 2ꢀMeTHF
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ABSTRACT:
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The immobilization of lipase from Pseudomonas stutzeri (Lipase TL®) by covalent bonding to a
porous polymer is described for the first time. The immobilized enzyme was characterized in
terms of optimal pH and thermal stability, and its catalytic efficiency was tested in the kinetic
resolution (KR) of symmetrical and unsymmetrical benzoins (1,2ꢀdiarylꢀ2ꢀhydroxyethanone
structures). Reactions were performed in a green solvent 2ꢀMeTHF, reaching maximum
conversions and enantiomeric excesses, with a significant increase of productivity due to the
possibility of reuse of the catalyst. Moreover, the immobilization allowed the development of an
adequate scaling up of this KR process permitting a further rise in the catalytic efficiency.
Finally, the dynamic kinetic resolution of benzoin (DKR) was carried out by the combination of
the immobilized lipase and a ruthenium catalyst (Shvo´s catalyst) in 2ꢀMeTHF, reaching
conversions up to 90%, maintaining its excellent enantioselectivity during 6 catalytic cycles.
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INTRODUCTION
Optically pure benzoins (1,2ꢀdiarylꢀ2ꢀhydroxyethanone structures) are very useful building
blocks in the synthesis of different drugs as antidepressant, astringent, antiꢀinflammatory,
carminative, diuretic, expectorant, sedatives and antibacterial.1, 2Benzoins and its derivatives are
used as intermediates for the synthesis of organic compounds and as a catalyst in photo
polymerization, which are used as anticratering in powder coating. It has been reported that the
benzoin can be used in skin disorders as an antibacterial and antifungal agent.3 Benzoin has also
been used as photo initiators in polymeric reaction. Benzoin also used as a starting material for
preparing complexes like Schiff base compound. Schiff base complexes of transition metals are
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of greater importance in medicine, biochemistry and industries among others.4 Due to the
increasing interest towards these molecules, different strategies have been developed for
obtaining benzoin derivatives in an enantioselective manner. In this context, although several
attempts have been carried out by using asymmetric synthesis,5 biocatalysis is a real alternative,
because more sustainable processes can be implemented by using this approach6 In this sense,
enzymatic kinetic resolution (KR) of racemic mixtures has proven to be a useful method to
obtain these chiral benzoins.7 Additionally, the coupling of enzymes and metal catalysts in a
dynamic kinetic resolution (DKR) process has overcome the intrinsic limitation of the maximum
theoretical yield of 50% obtained in a KR process, making possible the complete transformation
of racemic substrates to optically pure products.8
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As it is well known, the application of lipases in the resolution of racemic mixtures is widely
spread, due to their regioꢀ, stereoꢀ and chemoselectivity and their stability in organic solvents.9
Recently we have reported the DKR of benzoins, a special type of bulky secondary alcohols, by
using a commercial Lipase TL® (from Pseudomonas stutzeri) coupled to an in situ racemization
of the remnant substrate through a ruthenium catalyzed redox process (Shvo’s catalyst),
obtaining high conversions and excellent enantiomeric excess.7,10
On the other hand, it is widely accepted that enzyme immobilization onto solid materials offers a
great number of advantages over native enzymes such as the enhanced stability, simplicity in
separation, and the capability in recovery and reuse,11 thus making these immobilized
biocatalysts very useful specially for industrial applications.12 There are many examples on
lipase immobilization in literature, being covalent bonding to a solid support one of the preferred
methodologies;12a nevertheless, for lipase TL, only physical adsorption on hydrophobic support13
and entrapment in silicon spheres14 had been described so far.
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Thus, in this work we described the first example of a covalent immobilization of lipase TL from
Pseudomonas stutzeri onto a new porous polymer activated with epoxy groups, showing how the
best immobilized derivative can be usefully applied as a recoverable and reusable catalyst in the
green bioꢀsolvent 2ꢀMeTHF for KR and DKR of benzoins with high yield and stereoselectivity.
The excellent activities displayed by the immobilized biocatalyst allowed an initial scalingꢀup
the process, testing two types of bioreactors (batch and column), and opening the way for further
implementation of the methodology.
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RESULTS AND DISCUSSION
Thus, we have carried out the first covalent immobilization of commercial lipase from
Pseudomonas stutzeri (Lipase TL®). In these study two different supports were tested,
commercial Eupergit C, used for comparative purposes, and poly(GMAꢀcoꢀHDDMA). Eupergit
C is a spherical acrylic polymer (surface area: 4.5 m2/g; pore size: 10 nm; pore volume: 0.06
mL/g), made by copolymerization of N,N’ꢀmethylenꢀbis(methacrylamide), glycidyl
methacrylate, allyl glycidyl ether, and methacrylamide. This epoxideꢀcontaining copolymer (0.6
mmol epoxy groups per g dry weight15), is particularly suitable for covalent immobilization of
enzymes for industrial applications because of its reactorꢀcompatibility.16 The methacrylic
copolymer described in this paper, Poly(GMAꢀcoꢀHDDMA, surface area: 27 m2/g; pore size: 26
nm; pore volume: 0.15 mL/g), also contains epoxide groups as the reactive components, although
displaying a higher density of reactive epoxy groups (4.3 mmol epoxy groups per g dry weight)
compared to Eupergit C.
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Synthesis and characterization of the porous poly(GMA-co-HDDMA).
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Porous poly(GMAꢀcoꢀHDDMA) copolymer was synthesized by solution polymerization as
previously described17, and characterized using different techniques:
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i) Porosity Volume Distribution Analysis (PVD). Nitrogen adsorption provides a good estimation
of the Specific Surface Area (SSA), Pore Volume (PV) and Median Pore Size (MPS) of any
support, however it is also possible to determine its pore volume distribution (PVD) for a given
pore size (radius) range. The porosity analysis (see Table S1 in supporting information) showed
that the presence of macro pores contributes significantly to the volume of the support porosity.
ii) FT-IR spectrum. The infrared spectrum of the synthesized copolymer was used to verify the
presence of epoxy groups (Fig. S1a in supporing information). From the FTꢀIR spectra, epoxy
groups are clearly present in the copolymer as indicated by the occurrence of typical stretching
vibrations of epoxy groups at 1258, 908, and 844 cm−1. On the other hand, those stretching
vibrations of C=O and C–O at 1720 and 1144 cm−1, respectively, confirms the presence of ester
groups in the structure of the copolymer coming from GMA monomer.
iii) Thermal characterization. Thermo gravimetric analysis (TGA) shows that this copolymer has
excellent thermal stability up to 190 ºC, while for higher temperature the material begins to
decompose and weight loss becomes evident (see Fig. S1b in supporting information).
iv) Scanning electronic microscopy (SEM). From the SEM photographs (Fig. 1) the internal
structure presents numerous interconnected cavities (pores) of different sizes that are aggregated
in larger clusters.
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Figure 1. SEM photographs showing the internal morphology of porous poly(GMAꢀcoꢀ
HDDMA).
Enzyme Immobilization
Optimization of enzyme loading
The first variable studied which could affect the immobilization efficiency was the relative
amounts of polymer and enzyme used in the coupling process. Thus, different enzymatic
amounts were used, keeping a fixed quantity of supporting copolymer (Table S2 in supporting
information). As can be seen, using a 1:100 ratio of protein to polymer, 100% binding of protein
to poly(GMAꢀcoꢀHDDMA) copolymer was observed. Nevertheless, when the amount of enzyme
added to the immobilization mixture was increased, up to 40% of the protein added did not get
linked to the support. On the other hand, for Eupergit C at the optimum molar ratio of enzyme to
polymer (1:100), around 30% less protein was bound compared to poly(GMAꢀcoꢀHDDMA)
copolymer. This finding could be explained taking into account the lower density of reactive
groups for Eupergit C already mentioned, so that a larger quantity of polymer should be used to
achieve the same enzymatic loading.
Increasing the initial amount of enzyme from 1 to 2 mg (entry 2) led to a derivative (TLꢀPGcHꢀ
2) with higher enzymatic loading but displaying approximately the same stationary effectiveness
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factor. Nevertheless, this value decreased when the amount of enzyme was increased up to 3 mg
(entry 3). This fact is suggesting the presence of diffusional problems for the substrate upon
reaching the enzyme molecules on the derivative having a higher enzymatic loading, maybe as
consequence of enzyme clustering. Anyhow, all the derivatives on poly(GMAꢀcoꢀHDDMA)
were more active than that one obtained with Eupergit C (TLꢀEuC), which only showed 45% of
catalytic efficiency compared to the native enzyme, and for which diffusional restrictions have
been frequently described.15,18 Based upon results. all the following experiments were conducted
with the best immobilized derivative, TLꢀPGcHꢀ2 (entry 2).
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Effect of pH on enzyme activity
The study of pH effect on the activity of free lipase from Pseudomonas stutzeri and TLꢀPGcHꢀ2
was performed in the pH range from 6 to 10 (Fig.S2 in supporting information). TLꢀPGcHꢀ2
exhibited a shift in the optimal pH of about 0.5 units toward acidic pH values, indicative of the
activated matrix behaving as a polycation. Similar results have been reported for different
enzymes immobilized on Eupergit C, such as pectinlyase,19 galactosidases18 or lipases.20
Thermal stability of free and immobilized enzyme
The immobilized enzyme preparations can be stored at 4°C for at least one month without
appreciable deactivation (less than 5%). Thermal stability experiments were performed as
described in the experimental section, and Figure 2 shows the thermal stability behavior of the
commercial and immobilized enzymes.
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a)
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b)
Figure 2 Thermal stability of free and immobilized lipase TL from Pseudomonas stutzeri. a)
37°C b) 50 °C.
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As can be seen, native enzyme shows a clear deactivation with halfꢀlife times of 14 and 9 h at 37
and 50 ºC, respectively. This loss of activity upon heating at 50 ºC had been previously described
by Hoyos et al.20a Equally, those derivatives on Poly(GMAꢀcoꢀHDDMA) show a higher
thermostability compared to those obtained with Eupergit C. It has been described that enzymes
are immobilized on Eupergit C® through their different reactive groups (amino, sulfhydryl,
hydroxyl, phenolic), and this fact can block the substrate accessibility to the active site, or may
establish multipointꢀbinding or can even lead to enzyme denaturation20a but this deleterious
effect is not observed in our immobilized derivatives.
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Kinetic Resolution and Dynamic Kinetic Resolution of benzoins.
Substrates employed in the enzymatic resolution (Table 1) were purchased from Sigma Aldrich
or synthesized previously by our research group.20
Kinetic Resolution of benzoins
Lipase TLꢀcatalyzed benzoin kinetic resolution (KR) was selected as test reaction to measure the
activity of both immobilized and free enzyme (Scheme 1).
Substrates (1a-i) were chosen because of the different size of the substituent at both sides of the
acyloin core, in order to study the ability of the immobilized enzyme to distinguish between
them. The enzymatic kinetic resolution of compounds 1a-i was carried out in 2 MeꢀTHF at room
temperature, employing vinyl butyrate as acyl donor and crude or immobilized lipase. The
reaction progress was monitored by HPLC in order to calculate the initial rate of the enzymatic
process, as reported in the experimental section.
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Table 1. Kinetic resolutions of different benzoinsa.
b
reaction
Conv.b
(%)
50
eep
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R1
R2
biocatalyst
Ec
time (h)
(%)
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
95
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free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
free enzyme
TLꢀPGcHꢀ2
6
6
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
113
1a
1b
1c
1d
1e
1f
Phenyl
Phenyl
50
50
4ꢀMethoxyphenyl
4ꢀIsopropylphenyl
Phenyl
4ꢀMethoxyphenyl
4ꢀIsopropylphenyl
4ꢀEthoxyphenyl
Phenyl
50
49
48
50
49
49
4ꢀEthoxyphenyl
Phenyl
48
50
3,4ꢀdichlorophenyl
Phenyl
49
49
1g
1h
1i
3,4ꢀdichlorophenyl
2ꢀFuryl
49
48
2ꢀFuryl
47
95
125
48
>99
>99
>200
>200
3ꢀThienyl
3ꢀThienyl
48
aReaction conditions: 0.094 mmol of substrate were solved in 1.5 mL of 2ꢀMeTHF and vinyl
butyrate (0.3 mmol) and Lipase TL (20 mg of free enzyme (0.336 U) or 200 mg of immobilized
enzyme (0.269 U)) were added. The mixture was stirred at room temperature under inert
atmosphere. Enzyme units were calculated through hydrolysis of pꢀnitrophenylꢀpalmitate as
described in the experimental section. bDetermined by HPLC analysis using Chiralcell OD®
column. cEnantiomeric ratio, ratio of specific constants (apparent secondꢀorder rate constant
kcat/Km) for both enantiomers.7,10
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Scheme 1. Kinetic resolutions of different benzoins catalyzed by lipase from Pseudomonas
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stutzeri.
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As shown in Table 1, in most cases maximum conversions (around 50%) were reached after 8 h;
in all cases, excellent enantiomeric excess values were obtained both with the crude enzyme and
TLꢀPGcHꢀ2, clearly indicating that the immobilization procedure had not altered the exquisite
enantioselectivity of this lipase in the stereorecognition of benzoins. Thus, enantiomerically pure
butyrates (S)-2a-i were easily isolated and purified by silica column chromatography, and
absolute configurations were assigned to be S according to a correlation of the positive optical
rotations values of these compounds with data from literature.21
Re-use of the immobilized lipase TL in the kinetic resolution of benzoin.
The potential for the reꢀuse of the supported enzyme in the KR of benzoin was investigated.
After the first assay, TLꢀPGcHꢀ2 was recovered, washed and reꢀassayed with fresh substrate
mixture under the same experimental conditions, and this procedure was repeated 15 times,
always using the green solvent 2ꢀMeTHF, in which we have already reported the highest yields
for KR of benzoins.7,10
The experimental results shown in Figure 3 illustrate the excellent reusability in 2ꢀMeTHF for
the immobilized enzyme, which retained 74% of its initial activity after the 15th catalytic cycle
(Figure 3).
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Figure 3. Operational stability of TLꢀPGcHꢀ2.
Scale up of the kinetic resolution of benzoin
Based on the excellent results obtained with the immobilized lipase, KR of benzoin was scaledꢀ
up 5 times (from 20 to 100 mg) up to 10 ml volume, testing two types of bioreactors (batch and
column). For both experiments, 3 g of TLꢀPGcHꢀ2 were used. In the case of the batch reactor, a
final conversion of 49% after 2 h was observed, with an enantiomeric excess of the product over
99%, resulting in a productivity of 2.46·10ꢀ3 mmol product·hꢀ1 (mg enzyme)ꢀ1. Similar results
were obtained in the same process on a smaller scale (2.5·10ꢀ3 mmol product·hꢀ1 (mg enzyme)ꢀ1),
therefore confirming the absence of any diffusional limitation for the immobilized derivative.
For the KR protocol in a column reactor (see experimental section), the retention time observed
for the reaction mixture in the column was 60 minutes, yielding a 50% conversion with an
enantiomeric excess above 99%. The optimal reaction time was 90 minutes (after that time,
product (S)-2a and remnant (R)-1a were not detected in the reaction eluent). In this kinetic
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resolution process the productivity obtained was 3.3·10ꢀ3 mmol product·hꢀ1 (mg enzyme)ꢀ1,
slightly higher than that one obtained employing in a batch reactor. Very recently, benzoin DKR
in batch and continuous mode has been reported with Accurelꢀadsorbed lipase TL22 not only in
2ꢀMeTHF, but also in other green biosolvents such as 1,2ꢀdioxolane and remarkably cyclopentyl
methyl ether (CPME, a very attractive new alternative for replacing traditional organic solvents),
also with excellent results22.
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Dynamic Kinetic Resolution of benzoins
Once the optimal conditions were selected for the kinetic resolution, and it was proven that TLꢀ
PGcHꢀ2 derivative showed a good thermostability at 50 ºC (Figure 2b), the dynamic kinetic
resolution (DKR) was the next logical step to confirm the real capability of the immobilized
enzyme. Nevertheless, in order to avoid the described interference of acetaldehyde produced
from vinyl esters during KR with the hydrogen transfer catalyst,23 trifluoroethyl butyrate was
used as acyl donor, as depicted in Scheme 2. The Shvo’s catalyst, (1ꢀ
hydroxytetraphenylcyclopentadienyl (tetraphenylꢀ2,4ꢀcyclopentadienꢀ1ꢀone)ꢀαꢀ
hydrotetracarbonyldiruthenium (II)), was chosen as the catalyst of the in-situ racemization via a
reversible oxidation of the remnant benzoin (R)-1a into bencil 3 and its ulterior reduction to
regenerate (rac)-1a.
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Scheme 2. Dynamic kinetic resolution process.
As previously commented, we had formerly studied the combination of the Shvo´s catalyst and
native lipase from Ps. stutzeri in the resolution of symmetrical24 and asymmetrical benzoins,7
developing a threeꢀstep DKR in order to minimize lipase deactivation at 50 ºC. This temperature,
relatively high for a biocatalytic process mediated by a mesophylic lipase, is needed to promote
the required splitting of the Shvo’s catalyst into the two monomers required for the in situ
racemization of the remnant substrate.10a Therefore, immobilization was the rational procedure
for increasing thermostability of the lipase: in that sense we initially described how the
entrapment of Lipase TL in silicon elastomer (LipTLꢀSS derivative) allowed the development of
a oneꢀpot DKR at 60 ºC in THF leading to 87% yield an 99% eep in the first catalytic cycle and a
fourꢀtimes recycling of the catalyst.24b
In order to improve these results, we have now developed a one pot process (Scheme 2) to afford
a rapid DKR of (rac)-1a in 20 h at a lower temperature (50 ºC), finding an almost quantitative
conversion (95%) of product with an enantiomeric excess higher than 99% (although some traces
of the correspondent dicarbonyl intermediated 3 were detected by HPLC analysis). For reusing
TLꢀPGcHꢀ2 derivative in consecutive DKR reactions, the immobilized enzyme matrices were
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washed with 2ꢀMeTHF until no remaining substrate or product was detectable in the solvent and
another DKR cycle was started. The results obtained in the reꢀuse of immobilized enzyme in
benzoin DKR process are shown in the Figure 4.
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Figure 4. Repetitive DKR of benzoin with trifluoroethyl butyrate catalyzed by TLꢀPGcHꢀ2.
As can be seen, the new immobilized enzyme was recycled five times (6 catalytic cycles), so this
mean that the same amount of TLꢀPGcHꢀ2 was working 114 h (24 h, initial cycle, plus (5x18h),
5 reuses) at 50ºC, still retaining a 78% of residual activity. To calculate the operational
productivity of TLꢀPGcHꢀ2, in Table 2 we show the results of the catalytic performance of TLꢀ
PGcHꢀ2 compared to both native and silicon elastomerꢀtrapped lipase TL (LipTLꢀSS).
As can be seen, although the overall productivity obtained with covalent derivative TLꢀPGcHꢀ2
is slightly lower than that obtained with LipTLꢀSS, in general terms TLꢀPGcHꢀ2 results a better
catalyst considering the lower operational temperature required (50 vs 60º C) and the improved
mechanical properties of the covalent immobilized derivative, making it suitable for both batch
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and column engineering designs. AnsorgeꢀSchumacher and coworkers14 also reported a good
stability of their immobilized derivative in the same repetitive reaction using 2ꢀMeTHF as
solvent, better than using toluene, although only three reaction cycles were carried out.
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Table 2. Repetitive DKR of benzoin with trifluoroethyl butyrate
Reaction parameters
Cycle
Catalyst
Overall
productivityb
2
Conversion (%)
eep
99
99
Productivitya
1
commercial LipTL, THFc
commercial LipTL, 2ꢀ
MeTHFd
92
85
2
4
4
LipTLꢀSS, THFe
87
95
81
99
99
99
15
8
15
8
TLꢀPGcHꢀ2, 2ꢀMeTHF
LipTLꢀSS, THFe
2
14
29
TLꢀPGcHꢀ2, 2ꢀMeTHF
90
99
7.5
15.5
3
4
5
LipTLꢀSS, THFe
TLꢀPGcHꢀ2, 2ꢀMeTHF
LipTLꢀSS, THFe
80
87
78
82
80
74
99
99
99
99
99
99
14
7.3
13
43
22.8
56
TLꢀPGcHꢀ2, 2ꢀMeTHF
TLꢀPGcHꢀ2, 2ꢀMeTHF
TLꢀPGcHꢀ2, 2ꢀMeTHF
6.9
6.7
6.5
29.7
36.4
42.9
6
a
mg of product/mg protein of the individual catalytic cycle. For immobilized derivatives, the amount of protein is
calculated from the enzymatic loading
b mg of product/mg protein considering also the previous cycles
c T = 50ºC (data taken from literature22
d T = 50ºC (data taken from literature 7
e T = 60ºC, THF as solvent (data taken from literature 10b
)
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Chemometric Parameters
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To calculate the usual chemometric parameters to quantify the “greenness” of the reactions,
equations defined in literature were used25 using always racemic benzoin 1a as standard
substrate. These parameters are EꢀFactor (E)26 Eꢀfactor Based Molecular (EMW)27, Atom
Economy (AE)28, Mass Intensity (MI)29, Material Recovery Parameter (MRP)27, Stoichiometric
Factor (SF)27, and, finally, Reaction Mass Efficiency (RME)27, which can be used as a threshold
metric for gauging the true “greenness” of reactions, considering the “golden ratio”, a RME
value ≥0.61830.
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Benign index (BI) has been recently described to also assess the overall “greenness” of chemical
reactions and synthesis plans25b, and takes into account the following potentials for
environmental harm: acidification−basification (ABP), ozone depletion (ODP), global warming
(GWP), smog formation (SFP), inhalation toxicity (INHTP), ingestion toxicity (INGTP),
inhalation carcinogenicity (INHCP), ingestion carcinogenicity (INGCP), bioconcentration
(BCP), abiotic resource depletion (ARDP), cancer potency (CPP), persistence (PER), and
endocrine disruption (EDP). Although the software automatically calculates BI with data
implemented, not many of the parameters included in it can be calculated for the (D)KR of
benzoins, so that we have only considered those parameters directly derived by implementing
data obtained from logP and H (Henry’s law constant, HLC), as reported from ChemDraw
(Table S3 and Figures S3 and S4 in supporting information).
As can be seen, RME value is smaller than 0.61830, the golden value for real sustainability.
Also, MI parameter for KR increased from 2.8 up to 91.3 (Eꢀfactor from 1. 8 to 90.3, 32,6ꢀfold
rise) upon committing all reaction solvents, catalysts, and byproducts, while for DKR MI
increased from 2.8 to a very similar value of 87.9 (31.4 rise). This means that moving from KR
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to DKR does not denote a significant growth of production of waste material, while the
productivity enhance is really noteworthy. Furthermore, we should also take into account that,
because of the good reusability of TLꢀPGcꢀ2H both in KRs and DKRs, really the immobilized
enzyme cannot be considered as “normal waste”, because it can be employed in several
successive reaction runs.
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Scale up of the dynamic kinetic resolution of benzoin
The DKR of benzoin was scaled up from 40 mg to 400 mg in order to study the applicability of
the lipaseꢀcopolymer particles in a batch reactor. As it has been previously described, increasing
the reaction volume allows the decrease of the ruthenium catalyst loading, as the presence of
traces of molecular oxygen, which causes its deactivation, is reduced at larger scales. Thus,
catalyst loading was decreased from 10 % mol to 2.5 % mol. After 24 h, 92 % yield was
achieved, maintaining an excellent 99 % enantiomeic excess.
CONCLUSION
All results presented in this work shows how lipase from Pseudomonas stutzeri (Lipase TL®)
immobilized on poly(GMAꢀcoꢀHDDA) polymer could be usefully applied as a recoverable and
reusable biocatalyst for the KR and DKR of benzoins in 2ꢀMeTHF with high yield and
enantioselectivity in all cases. The first scaling up presented here is a proofꢀofꢀconcept of the
applicability of this immobilized derivative, which can be easily operated both in batch and
column design.
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EXPERIMENTAL SECTION
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General
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Lipase TL® (from Pseudomonas stutzeri) was purchased from Meito & Sangyo Co., Ltd. Shvo’s
catalyst (1ꢀhydroxytetraphenylcyclopentadienyl(tetraphenylꢀ2,4ꢀcyclopentadienꢀ1ꢀone)ꢀαꢀ
hydrotetracarbonyldiruthenium (II), 98%) was obtained from Strem Chemicals Inc.
(Newburyport, MA, USA). ). Glycidyl methacrylate (GMA), 1,6ꢀhexanediol dimetacrylate
(HDDMA), cyclohexanol and 2,2´ꢀazobis(isobutyronitrile) (AIBN) were purchased from Sigmaꢀ
Aldrich. All other chemicals were from analytical grade. UV–visible spectra were recorded on a
UVꢀ2401 PC Shimadzu. HPLC analyses were performed with a chiral column Chiralcel® ODꢀH
(UV detector) at room temperature; mobile phase nꢀhexane/2ꢀpropanol 90/10, at a flow rate of 1
mL/min. NMR spectra were performed on a Bruker ACꢀ300 MHz: chemical shifts (δ) are
reported in parts per million (ppm) relative to CHCl3 (1H: δ 7.27 ppm) and CDCl3 (13C: δ 77.0
ppm). Polymerization procedure. Porous poly(GMAꢀcoꢀHDDMA) copolymer was synthesized
by solution polymerization as previously described17 in a cylindrical reactor (15x160 mm) at 70
rpm under argon overlay at 50ºC for 16 h. Cyclohexanol was employed as porogenic diluent
(60% respect the reaction mixture). The GMA/HDDMA ratio employed was 60/40 and the freeꢀ
radical initiator percentage (in the reaction mixture) was 5%. After the reaction was completed,
the polymeric product was filtered under vacuum and washed with acetone, water and methanol
several times to remove lowꢀmolecularꢀweight products and then dried in at 70 ºC in vacuum till
constant weight.
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Characterization of epoxy-functionalized polymers
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i) Surface Area and Pore Size Analysis. Specific Surface Area (SSA), Pore Volume (PV) and
Median Pore Size (MPS) were determined by nitrogen gas adsorptionꢀdesorption a 77 K by using
a TriꢀStar 3000 instrument (Micromeritics, USA). Samples were degassed at 120 ºC for 18 h.
SSA values were calculated using the BET equation14 in the nitrogen partial pressure range of
0.05ꢀ0.35. PV and MPS were obtained by using the adsorption branch of the nitrogen isotherms
according to the BJH method in the nitrogen partial pressure range of 0.35ꢀ0.99.31
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(ii) FT-IR spectroscopy. A PerkinꢀElmer 681ꢀFourier transform infrared spectrophotometer with
a resolution of 1 cmꢀ1 in the transmission mode was used to study the infrared absorption. The
synthesized polymers (2.0 mg) were milled with potassium bromide (100 mg) and pressed into a
solid disk of 1.2 cm diameter prior to the infrared measurement.
(iii) Scanning electron microscopy (SEM). Surface morphology of copolymers particles was
observed by using FieldꢀEmission Scanning Electron Microscopy (FEꢀSEM). Specimen
preparation was done as follows: dried copolymers particles were mounted on stubs and sputterꢀ
coated with gold. Micrographs were taken on a HitachiꢀS4700 FEꢀSEM instrument.
(iv) Thermal Characterization. Thermo Gravimetric Analysis (TGA) was performed using an
AQꢀ500 TA Instruments equipment. For each essay 4ꢀ5 mg of polymer were used. The heating
rate was set at 5 ºC/min and all the experiments were carried out under a constant nitrogen flow
of 20 ml/min.
(v) Polymers epoxidation degree. Quantitative determination of epoxide groups in the polymers
was carried out by chemical titration.32 The polymer (2.0 g) was reꢀsuspended in CH2Cl2 (30
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mL) and treated with a 20 wt % solution of tetraethylammonium bromide in glacial acetic acid,
prepared previously. After addition of 6ꢀ8 drops of crystal violet indicator solution in acetic acid,
the mixture was titrated with 0.1 N perchloric acid solution in acetic acid. Hydrobromic acid
formed in situ by the exchange reaction between perchloric acid and tetraethylammonium
bromide reacted instantaneously with the epoxide group, leading to bromohydrin formation. The
end point of the titration was determined by the change of the color of the solution from a sharp
blue to green.
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Enzyme immobilization. Poly(GMAꢀcoꢀHDDMA) (100 mg) was added to the lipase solution
(different concentrations were tested; 1, 2 and 3 mg) in 1 mL of 0.05 M phosphate buffer, pH 7.
The reaction was carried out for 24 h at 20 ºC with gentle shaking (200 rpm). After 24 h of
incubation, polymer particles were collected on a sinteredꢀglass filter and the solution was
removed by suction under vacuum. The copolymer particles were washed thoroughly on the
same filter with 10 ml of 0.05 M phosphate buffer, pH 7 (the volume was divided into five
aliquots and one aliquot was used for each washing step). The excess of epoxy groups on the
matrix were blocked by incubation with 3 M glycine solution for 16 h at 25 °C. The immobilized
lipaseꢀcopolymer samples were stored at 4 ºC until use.
Determination of the amount of immobilized lipase. Protein concentration was determined using
Bradford’s method33 following the manufacturer’s protocol (BioꢀRad) and bovine serum albumin
as standard. The coupling yield (%) of the lipase was determined by the difference between the
initial lipase amount present in the lipase coupling solution and the final lipase amount present in
the remaining coupling solution.
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Enzyme assays. Hydrolytic activity of free and immobilized enzyme were analyzed
spectrophotometrically measuring the increment in the absorption at 410 nm promoted by the
hydrolysis of pꢀnitrophenol palmitate (pNPP). The reaction mixture consisted of 0.1 mL of
diluted enzyme sample (or 10 mg immobilized copolymer particles), 2.0 mL of 0.05 M sodium
phosphate buffer (pH 7.0) and 0.25 mL 10 mM pNPP in 2ꢀpropanol, incubated at 25ºC for 1 min.
The absorbance was measured at 410 nm. A lipase unit was defined as the amount of enzyme
necessary to hydrolyze 1 µmol of pNPP per minute under the described conditions.
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pH and thermal stability of free and immobilized enzyme. The pH stability of free and
immobilized lipase was studied by incubating the enzyme at 25 ºC in buffers of varying pH in
the range of 6ꢀ10 for 20 min and then determining the catalytic activity. Residual activities were
calculated as the ratio of the activity of enzyme after incubation to the activity at the optimum
reaction pH.
Thermal stability experiments were performed with free and immobilized enzymes which were
incubated in the absence of substrate at 37 °C and 50 °C. The immobilized enzymes were placed
in 100 mM sodium phosphate buffer (pH 6.0) and the specific enzymatic activities were
measured at different storage times.
General procedure for benzoin kinetic resolution (KR). Substrate (20 mg, 0.094 mmol) was
dissolved in 1.5 mL of dry 2ꢀMeTHF. Lipase TL (20 mg of the commercial preparation or 200
mg of lipase–copolymer particles) and vinyl butyrate (38 µl, 0.3 mmol) were added to the
solution and the mixture was stirred at room temperature for 10 h. At fixed reaction times,
samples (20 µL) were taken from the reaction medium, reꢀdissolved in 1 mL of nꢀhexane, and
filtered through Millex®ꢀGV (PVDF, 0.22 µm pore size) to stop the enzymatic reaction. As 2ꢀ
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MeTHF (polar solvent) may damage the chiral column (Chiralcel ODꢀH®), each sample was
evaporated under vacuum and the solid collected was reꢀdissolved in 1 mL of nꢀhexane/2ꢀ
propanol (50:50, v/v) before analyzing by HPLC to measure conversion and enantiomeric excess
values. After 10 h the reaction mixture was filtered, and 2ꢀMeTHF was evaporated before
products were purified by column chromatography (SiO2, nꢀhexane/ ethyl acetate, 5:1). The
following compounds were isolated and characterized.
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For evaluating potential biocatalyst reuses for KR, 400 mg of lipaseꢀcopolymer particles were
used, choosing 3 hours as final time. Lipaseꢀcopolymer particles were washed with 2ꢀMeTHF
until no remaining product was detected by HPLC analysis, and these recycled lipaseꢀcopolymer
particles were used in a new KR process.
Analysis of reaction products (S)ꢀ2ꢀoxoꢀ1,2ꢀdiphenylethyl butyrate (2a), (S)ꢀ1,2ꢀbis(4ꢀ
methoxyphenyl)ꢀ2ꢀoxoethyl butyrate (2b), (S)ꢀ1,2ꢀbis(4ꢀisopropylphenyl)ꢀ2ꢀoxoethyl butyrate
(2c), (S)ꢀ1ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀoxoꢀ2ꢀphenylethyl butyrate (2d), (S)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀ
oxoꢀ1ꢀphenylethyl butyrate (2e), (S)ꢀ1ꢀ(3,4ꢀdichlorophenyl)ꢀ2ꢀoxoꢀ2ꢀphenylethyl butyrate (2f),
(S)ꢀ2ꢀ(3,4ꢀdichlorophenyl)ꢀ2ꢀoxoꢀ1ꢀphenylethyl butyrate (2g), (S)ꢀ1,2ꢀdiꢀ2ꢀfurylꢀ2ꢀoxoethyl
butyrate (2h) and (S)ꢀ2ꢀoxoꢀ1,2ꢀdiꢀ3ꢀthienylethyl butyrate (2i), was carried out by NMR, and all
data were in accordance with those ones previously published (NMR data in Supporting
information).34
Scale-up of the kinetic resolution process of the benzoin. The process of scaleꢀup was carried
out in two different types of reactors, batch reactor and column reactor, using an initial substrate
concentration 5 times higher than that one used for analytical scale. Therefore, also biocatalyst
amount was increased from 200 mg up to 3 g of lipaseꢀcopolymer catalyst.
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Scale-up in batch reactor. Racemic benzoin (100 mg, 0.47 mmol) was dissolved in 10 mL of dry
2ꢀMeTHF. Lipase–copolymer particles (3 g) and vinyl butyrate (190 µl, 1.5 mmol) were added
to the solution and the mixture was stirred at room temperature for 10 h. At fixed reaction times,
20 µL aliquots of supernatant were taken from the reaction medium, redissolved in 1 mL of nꢀ
hexane, and filtered through Millex®ꢀGV (PVDF, 0.22 µm pore size) to stop the enzymatic
reaction an analyzed as described above.
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Scale-up in column reactor. Lipaseꢀcopolymer particles (3 g) were packed with 2ꢀMeTHF in a
chromatography column (40 cm x 1 cm), giving rise to a filling length of 21 cm. Then, this
column reactor was loaded with racemic benzoin (100 mg, 0.47 mmol) dissolved in 10 mL of dry
2ꢀMeTHF. The column reactor was set up with a peristaltic pump to run the reaction and the
eluent passed through the column where a fraction collector at the end of the column collected
the eluted samples. 20 µL of each sample were evaporated under vacuum and the solid collected
was reꢀdissolved in 1 mL of nꢀhexane/2ꢀpropanol (50:50, v/v) before analyzing by HPLC to
measure conversion and enantiomeric excess values.
Dynamic Kinetic Resolution of benzoin. Racemic benzoin (40 mg, 0.19 mmol), lipaseꢀ
copolymer particles (400 mg) and Shvo´s catalyst (20 mg, 0.02 mmol) were added to a 25 mL
flask. Anhydrous 2ꢀMeTHF (1,5 ml) was incorporated and the reaction was started by the
addition of trifluoroethyl butyrate (92 µl, 0.6 mmol). The mixture was stirred at 50 ºC under
argon atmosphere for 24 h. Conversion and enantiomeric excess were determined by HPLC
analysis, following a similar protocol to that described in the previous section. After 24 h the
reaction was finished and the mixture was filtered. In the case of the reuses, the final time was 18
hours and then the reaction mixture was filtered and the lipaseꢀcopolymer particles were washed
with 2ꢀMeTHF until no remaining product was detected by HPLC analysis, and those recycled
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lipaseꢀcopolymer particles were used in a new benzoin DKR process. 2ꢀMeTHF was evaporated
and the products were purified by column chromatography (SiO2, nꢀhexane/ ethyl acetate, 5:1)
and characterized.
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Scale-up in batch reactor. Racemic benzoin (400 mg, 1.88 mmol), lipase copolymers particles (6
g) and Shvo´s catalyst (51 mg, 0.047 mmol) were added to a 50 mL flask. Anhydrous 2ꢀMeTHF
(15 mL) and trifluoroethyl butyrate (851 µL, 5.64 mmol) were added, and the reaction was
stirred at 50 ºC under argon atmosphere. After 24 h the reaction mixture was filtered, conversion
and enantiomeric excess were determined by HPLC analyses and the product (S)-2a (488 mg,
1.72 mmol) was purified as described above.
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*María J. Hernáiz: mjhernai@ucm.es (Tel: +34 913941821)
Angel Rumbero: angel.rumbero@uam.es
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. All authors contributed equally.
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The authors declare no competing finalcial interest.
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ACKNOWLEDGMENT
This work was supported by a research project of the Spanish MINECO (Ministerio de Ciencia e
Innovación de España) CTQ2012ꢀ32042.
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Table of Contents Graphic
O
R2
R1
OH
rac
O
O
CF3
Biosolvent
One-pot process
High yield and enantioselectivity
Reusable biocatalyst
O
R2
O
Scaling up the process
in batch or column design.
R1
R2
R1
OH
OH
O
O
O
CF3
O
CF3
TL-PGcH-2
O
R2
R1
O
S
O
O
R2
R1
OH
R
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