Polyphosphazenes as tunable and recyclable supports to immobilize alcohol dehydrogenases and lipases: Synthesis, catalytic activity, and recycling efficiency

Article abstract of DOI:10.1021/bm100091a

The polyphosphazene {NP[O₂C₁₂H_7.5 (NH ₂)_0.5]}_n, prepared by reacting {NP[O ₂C₁₂H_7.5(NO₂)_0.5]} with the Lalancette's reagent, was used for attaching enzymes such as alcohol dehydrogenase (ADH-A) and lipase (CAL-B). The resulting new biocatalysts exhibited great potential as tunable supports for enzymatic reactions in both aqueous and organic media. The material with immobilized ADH-A was as efficient as the commercial enzyme to perform stereoselective bioreductions of ketones in aqueous solutions and could be used for the reduction of various aliphatic and aromatic ketones up to 60 °C and recycled several times without significant loss of activity even after three months of storage. The biocatalyst obtained with CAL-B was more efficient than the free enzyme for kinetic resolutions in organic solvents and exhibited a moderately good capability of reutilization.

Full text of DOI:10.1021/bm100091a

Biomacromolecules 2010, 11, 1291–1297
1291
Polyphosphazenes as Tunable and Recyclable Supports To
Immobilize Alcohol Dehydrogenases and Lipases: Synthesis,
Catalytic Activity, and Recycling Efficiency
An´ıbal Cuetos,† Mar´ıa L. Valenzuela,‡ Iva´n Lavandera,† Vicente Gotor,*,† and
Gabino A. Carriedo*,§
Departamento de Qu´ımica Orga´nica e Inorga´nica, Instituto Universitario de Biotecnolog´ıa de Asturias,
University of Oviedo, 33006 Oviedo, Spain, Departamento de Ciencias Qu´ımica, Facultad de Ecolog´ıa y
Recursos Naturales, Universidad Andre´s Bello, Av. Republica 275, Santiago, Chile, and Departamento de
Qu´ımica Orga´nica e Inorga´nica, University of Oviedo, 33006 Oviedo, Spain
Received January 26, 2010; Revised Manuscript Received February 26, 2010
The polyphosphazene {NP[O2C12H7.5(NH2)0.5]}n, prepared by reacting {NP[O2C12H7.5(NO2)0.5]} with the Lalancette’s
reagent, was used for attaching enzymes such as alcohol dehydrogenase (ADH-A) and lipase (CAL-B). The
resulting new biocatalysts exhibited great potential as tunable supports for enzymatic reactions in both aqueous
and organic media. The material with immobilized ADH-A was as efficient as the commercial enzyme to perform
stereoselective bioreductions of ketones in aqueous solutions and could be used for the reduction of various
aliphatic and aromatic ketones up to 60 °C and recycled several times without significant loss of activity even
after three months of storage. The biocatalyst obtained with CAL-B was more efficient than the free enzyme for
kinetic resolutions in organic solvents and exhibited a moderately good capability of reutilization.
phazene derivative that was subsequently activated with glut-
araldehyde and then reacted with the enzyme solution. Later,
an invertase was supported on spherical particles of
[NP(OCH2CF3)2]n, first displacing the trifluoroethoxy moieties
with NaOCH2CH2NH2 and then anchoring the enzyme, obtain-
ing good activities for this preparation.9 Finally, an urease was
encapsulated on a hydrogel derived from poly[bis(methoxy-
ethoxyethoxy)phosphazene] through irradiation with γ-rays to
cross the polymer and trap the biocatalyst.10
Biocatalytic reactions have recently gained more relevance
due to the mild conditions employed and the highly chemo-,
regio-, and stereoselective transformations obtained to synthesize
natural products and drugs.11 Among all available enzymes,
probably hydrolases and oxidoreductases are the most explored
catalysts.12 Thus, lipases are a class of hydrolases that have
widely been used to achieve kinetic resolutions through hy-
drolysis, esterification, and aminolysis reactions, among others.13
Candida antarctica lipase B (CAL-B) has been used in organic
solvents to perform the kinetic resolution of alcohols and
especially amines.14 In the case of oxidoreductases, alcohol
dehydrogenases (ADHs, also called carbonyl reductases) have
been employed to catalyze the stereoselective reduction of
prochiral ketones with remarkable selectivities.15 For instance,
the nicotinamide adenine dinucleotide (NADH)-dependent
alcohol dehydrogenase ADH-A from Rhodococcus ruber DSM
44541 has been isolated and overexpressed in Escherichia coli
and can be applied to the asymmetric reduction of alkyl- and
aryl-substituted ketones or diketones.16
Introduction
Polyphosphazenes are ideal compounds to tune their physical
and chemical properties by selecting the appropriate function-
alities linked to the phosphorus atom.1 Because of their highly
valuable characteristics, mainly their chemical and temperature
resistance, these derivatives are potentially useful for biotech-
nological applications and, in fact, novel materials derived from
this type of polymer have been recently designed and applied
to important fields such as biomedicine.2 The synthesis of
functionalized polyphosphazenes by secondary reactions on
pendant side groups is a fruitful alternative to the classical
macromolecular substitution with nucleophiles carrying the
desired functional groups.1,3 The latter is a more advantageous
approach in cases like the sulfonation of aryloxyphosphazenes4
or the introduction of pendant PPh2 ligands.5
In the past few years, an increasing number of catalytic
processes for industry have been described. The new require-
ments that society demands concerning greener and safer
methodologies, make the immobilization of (bio)catalysts a very
important issue aiming to better recycling efficiency and more
economical and environmental friendly processes.6 Thus, the
chemical or physical fixation of enzymes on supporting carriers
can be achieved by different methods.7 In this context, poly-
phosphazenes have scarcely been used as support of biomac-
romolecules, and in the particular case of biocatalysts, only a
few examples can be mentioned.2b In a first article, Allcock
and Kwon covalently immobilized tripsine and glucose-6-
phosphate dehydrogenase on a support of modified [NP(OPh)2]n
on an alumina carrier.8 The process consisted of a nitration-
reduction protocol to obtain the corresponding polyaminophos-
To improve the stability of lipases in organic solvents, they
have been adsorbed/immobilized in many supports. In case of
immobilization of ADHs, fewer reports have been shown and
most of these processes were based on adsorption techniques.
Only very few examples using covalent binding are described.17
There is only one previous report where ADH-A has been
covalently anchored to amino-functionalized materials, including
porous glass beads, magnetic particles, and nanodiamonds.18
* To whom correspondence should be addressed. E-mail: vgs@fq.uniovi.es
(V.G.); gac@fq.uniovi.es (G.A.C.).
† Instituto Universitario de Biotecnolog´ıa de Asturias, University of
Oviedo.
‡ Universidad Andre´s Bello.
§ Departamento de Qu´ımica Orga´nica e Inorga´nica, University of Oviedo.
10.1021/bm100091a
 2010 American Chemical Society
Published on Web 04/01/2010

1292 Biomacromolecules, Vol. 11, No. 5, 2010
Scheme 1. Synthesis of Polyphosphazene 3
Cuetos et al.
Synthesis of {NP[O2C12H7.5(NH2)0.5]}n (3). First, the Lalancette’s
reagent22 was prepared (in a well ventilated hood). Air and humidity
must be strictly avoided. A three-necked 250 mL flask, filled with a
dried N2 atmosphere and equipped with a condenser and a bubbler with
paraffin oil, was cooled to 0 °C (ice bath). Then the following were
added in the order NaBH4 (0.63 g, 16.7 mmol), sulfur (1.78 g, 55
mmol), and recently well-dried and distilled (twice) THF (40 mL;
CARE, exothermic reaction with evolution of H2S). The mixture was
stirred for 1 h to give a transparent yellow solution. The volatiles were
evaporated in a vacuum, and the residue was washed with diethyl ether
(2 × 50 mL) and dried overnight in a vacuum.
Glutaraldehyde was employed as linker and, although very good
immobilization yields were achieved, the activities were lower.
Until three biocatalytic cycles could be performed with a
minimum loss of enzymatic activity.
In earlier reports, we described the synthesis of phosphazene
polymers with phosphorus-dioxy-biphenyl moieties (or the chiral
binaphthyl analogues) in the repeating units, and a number of
copolymers with monodentated aryloxydes carrying different
functional groups.19 Recently, the regioselective and controlled
direct nitration of the homopolymer [NP(O2C12H8)]n (1), which
is readily available,20 was achieved by taking advantage of the
unusual high solubility and stability of this precursor in
concentrated sulfuric acid, to obtain in one pot a simple
procedure for the derivatives {NP[O2C12H8-x(NO2)x]}n (2), with
x ranging from 0.2 to 2 in excellent yields (Scheme 1).21 As
polymers 2, with Mw of the order of 105, are well characterized
To the prepared Lalancette’s reagent, THF (50 mL) and a solution
of {NP[O2C12H7.5(NO2)0.5]}n (2, x ) 0.5; 2 g, 9.96 mmol) in THF (50
mL) were added and the final volume was increased by adding 100
mL of THF. The mixture was then refluxed with stirring for 24 h (the
formation of a solid in the walls of the flask was observed). After
cooling to room temperature, 10% v v-1 aqueous HCl (5 mL) was added
and stirring was continued for 7 h. The resulting mixture was filtered
and the solid was stirred with 10% aqueous NaOH (100 mL) for 24 h.
The solid was separated by filtration, washed with plenty of water until
neutral pH, and dried at 40 °C under vacuum for 3 days. Yield: 1.51 g
(80%). IR (KBr) cm-1: 3435, 3368 m.br (ν NH), 3065 w (ν CH arom.),
1624 br.m, 1502 m, 1478 m (ν CC arom., δ NH), 1384 sh.m (typical
of biphenoxyphosphazenes, not assigned), 1347 m, 1266 s, 1247 s, 1191
vs (ν NP), 1096 s (ν P-OC), 1041 w, 1013 w (not assigned), 941-922
s.br (δ POC), 870 w, 819 v.w (not assigned), 785 s, 751 s (δ CH arom.),
607 m, 536 m.br (other); 1H NMR (ppm, DMSO-d6): 6-8 v.br,
(aromatic protons), 4.9 v.br (NH2); 13C NMR (ppm, DMSO-d6, all peaks
are broad): 151 (C2), 149 (C5-NH2), 132, 131, 128, 125 (C1, C3, C4,
and C6); 31P NMR (ppm, DMSO-d6): -6.3 br; Anal. (Calcd.): C 57.0
(60.9), 3.40 (3.59), 8.50 (8.87). Sulfur retained 1%; TGA (from ambient
to 900 °C): Continuous loss from 300-800 °C. Final residue 44%
1
materials (by H, 13C, 31P, and IR spectra) with well-defined
chemical composition, and with limited but sufficient solubility
in THF,21 they were selected as very advantageous candidates
to be converted into the NH2 derivatives {NP[O2C12H7.5-
(NH2)0.5]}n (3), useful to support enzymes. Herein we wish to
report the use of 3 to immobilize an alcohol dehydrogenase
(ADH-A) for bioreductions on aqueous solutions and a lipase
(CAL-B) to achieve kinetic resolutions on organic solvents.
Experimental Section
General. Ketones, racemic alcohols, racemic acetate 17, alcohol
dehydrogenase from Rhodococcus ruber ADH-A and Candida antarc-
tica lipase B were purchased from commercial sources. One unit (U)
of ADH-A reduces 1.0 µM of acetophenone to 1-phenylethanol per
minute at pH 7.5 and 30 °C in the presence of NADH. Polyphosphazene
1 was synthesized as previously described.20 Polyphosphazene 2 was
synthesized as previously described.21 Tetrahydrofuran (THF) was
treated with KOH and distilled twice from Na in the presence of
benzophenone.
(under N2), 7% (under air); DSC: Tg ) 129 °C, ∆Cp ) 0.11 J g-1 K-1
.
Synthesis of 5 and 6. Polyphosphazene 3 (60 mg) was added to a
saturated solution of (NH4)2SO4 (9 mL) and a solution of glutaraldehyde
(1 mL, 2.5% v v-1) in phosphate buffer (50 mM, pH 7), affording a
suspension. Then it was mixed under magnetic stirring for 2 h at 50
°C. The solid obtained was filtered off, washed with phosphate buffer
50 mM pH 7 (3 × 1 mL), and dried under vacuum, affording 4 (61
mg, 87%).
The infrared (IR) spectra were recorded with a Perkin-Elmer FT
Paragon 1000 spectrometer. NMR spectra were recorded on Bruker
1
NAV-400, DPX-300, AV-400 and AV-600 instruments. The H and
13C{1H} NMR spectra in deuterated dimethylsulfoxide (DMSO-d6) are
given in δ relative to trimethylsilane (TMS; DMSO at 2.51 and 40.2
ppm, respectively). 31P{1H} NMR are given in δ relative to external
85% aqueous H3PO4. The C, H, N, analyses were performed with an
Elemental Vario Macro. Tg was measured with a Mettler DSC Toledo
822 differential scanning calorimeter equipped with a TA 1100
computer. Thermal gravimetric analyses (TGA) were performed on a
Mettler Toledo TG 50 TA 4000 instrument. The polymer samples were
heated at a rate of 10 °C min-1 from ambient temperature to 900 °C
under constant flow of nitrogen or under air. Gas chromatography (GC)
analyses were performed on a Hewlett-Packard 6890 Series II chro-
matograph. The degree of enzymatic conversion corresponded to the
measured area of the product in the GC chromatogram among the sum
of GC areas of the substrate and product. The enantiomeric excesses
were calculated dividing the subtraction of the GC area for the major
enantiomer minus the GC area for the minor enantiomer among the
sum of the GC areas for both enantiomers.
Subsequently, 4 (20 mg) was added to a 1 mL of tris(hydroxym-
ethyl)aminomethane (Tris ·HCl) buffer 50 mM pH 7.5 with ADH-A
(1 mg) or CAL-B (10 µL), affording a heterogeneous mixture that was
magnetically stirred at 5 °C for 15 h. Afterward, the polymer was
filtered, washed with Tris ·HCl buffer 50 mM pH 7.5 (4 × 0.5 mL),
and dried under vacuum, affording 5 or 6 (20 mg).
These formulations were obtained as fine powders, and no control
of the particle size (micrometers) could be achieved, so the effects of
this parameter in the catalytic activity could not be evaluated.
Enzymatic Reduction of Ketones Employing 5 and 2-Propanol. A
total of 20 mg of 5 were placed in an eppendorf tube and then 600 µL
of Tris ·HCl buffer 50 mM pH 7.5, 1 mM NADH, the corresponding
ketone (35 mM), and 2-propanol (32 µL, 5% v v-1) were added. The
heterogeneous mixture was shaken under orbital shaking at 30 °C and
250 rpm for 24 h. Afterward, this mixture was extracted with ethyl
acetate (2 × 0.6 mL), and the organic phase was dried over Na2SO4.

Polyphosphazenes as Tunable and Recyclable Supports
Biomacromolecules, Vol. 11, No. 5, 2010 1293
Scheme 2. General Strategy To Covalently Link a Biocatalyst to 3
Finally, conversion and enantiomeric excess was measured by achiral
and chiral GC, respectively.
300-800 °C with a final residue of 44% (under N2) or 7%
(under air). The glass transition was not very clearly observed
in the differential scanning calorimetry (DSC) trace (second
Study of the Stability of 5 over a 3 Month Period. A total of 20
mg of 5 were placed in an eppendorf tube and then 600 µL of Tris ·HCl
buffer 50 mM pH 7.5, 1 mM NADH, acetophenone (2.5 µL, 35 mM),
and 2-propanol (32 µL, 5% v v-1) were added. The heterogeneous
mixture was shaken under orbital shaking at 30 °C and 250 rpm for
24 h. Afterward, this mixture was extracted with ethyl acetate (2 ×
0.6 mL), and the organic phase was dried over Na2SO4. Finally,
conversion was measured by achiral GC. To avoid material loss, the
second cycle was performed in the same eppendorf tube, adding to the
polymer the rest of reagents, as mentioned before. After 3 consecutive
cycles, dry polyphosphazene was stored for 30 days at -20 °C to
achieve the following catalytic reactions. To reuse it, the support was
simply allowed to warm up to room temperature.
Kinetic Resolution of 1-Phenylethanol Employing 6 and Vinyl
Acetate. Compound 6 (20 mg) was introduced in an erlenmeyer flask
with dry tert-butyl methyl ether (TBME; 0.82 mL). Afterward,
1-phenylethanol (10 mg, 0.08 mmol) and vinyl acetate (22 µL, 0.24
mmol) were added. The heterogeneous mixture was shaken under orbital
shaking and kept under nitrogen for 24 h at 30 °C and 250 rpm. Then,
the catalyst was filtered and washed with CH2Cl2 (2 × 1 mL). Finally,
conversion and enantiomeric excesses were measured by achiral and
chiral GC, respectively.
heating run), but a value Tg ) 129 °C (∆Cp ) 0.11 J g-1 K-1
)
could be estimated.
Immobilization of ADH-A on Polyphosphazene 3. To study
the use of polymer 3 as support of biocatalysts, the reactivity
of the amino groups with the appropriate linkers applied to the
subsequent covalent immobilization of a biomacromolecule was
considered. Glutaraldehyde, with two reactive aldehyde moieties
is one of the connectors more frequently used to achieve
covalent unions6 and, therefore, was first considered as linker
between the phosphazene chain and the enzyme (Scheme 2).
Because of the low solubility of 3 in most of the solvents tested
(H2O, THF, CH2Cl2, and CHCl3) and no swelling was observed
in any case, it was also envisaged that the resulting materials
could be very appropriate to perform heterogeneous enzymatic
transformations in both aqueous and organic media.
In a first approach, we decided to anchor the alcohol
dehydrogenase from Rhodococcus ruber DSM 44541 ADH-A,
known for its high stability and versatility to perform biore-
ductions on carbonyl groups in aqueous media.16 Due to the
simplicity of reaction conditions, for the first step, we reproduced
the methodology described by Liese and co-workers.18
Thus, 3 was treated with the linker glutaraldehyde (2 equiv)
at room temperature for 2 h. To remove any excess connector,
the polymer was washed with buffer. The H NMR spectrum
Results and Discussion
1
Synthesis of {NP[O2C12H7.5(NH2)0.5]}n (3) by Reduction
of 2 (x ) 0.5). The synthesis of polymer 3 (Scheme 1) was
achieved in 80% yield starting from the readily available
polyphosphazene 1,20 by selective nitration with a sulfonitric
mixture at room temperature during 1.5 h,21 followed by
reduction with Lalancette’s reagent22 under reflux of tetrahy-
drofuran (see Experimental Section). The composition and
structure of the product was confirmed by the analytical and
spectroscopic data. The IR showed the expected bands at 3435
and 3368 cm-1 (ν NH) and the broadness of the band at 1624
cm-1 due to the overlapping with the NH-bending absorption.
of the product in DMSO-d6, where it was partially soluble,
showed the absence of glutaraldehyde. Only when the reaction
was performed at 50 °C for 2 h, a singlet at 9.63 ppm was
observed showing the formation of 4 (see Supporting Informa-
tion). The particles of 4 were then reacted with the enzyme
solution under magnetic stirring at 4 °C during 30 min, and
then the remaining nonreacting aldehyde residues were treated
with glycine and the final product was washed with buffer to
afford the enzyme attached 5. The activity of the immobilized
biocatalyst in 5 was tested using as a model reaction the
stereoselective reduction of acetophenone 7 into 1-(S)-phenyl-
ethanol 8 in buffer, using 2-propanol to recycle the nicotinamide
cofactor in a “substrate-coupled” approach (Scheme 3).23 The
observed enzymatic conversion (13%) was much lower than
the obtained with the commercial ADH-A (around 85%),24 but
1
The presence of the NH2 group was also observed in the H
NMR (DMSO-d6) as a very broad signal centered at 4.9 ppm
and also in a weak signal at 149 ppm in the 13C NMR. The low
solubility in THF prevented the measurement of the Mw. The
TGA (from rt to 900 °C), showed a continuous weight loss from

1294 Biomacromolecules, Vol. 11, No. 5, 2010
Cuetos et al.
Scheme 3. ADH-A-Catalyzed Reduction of Acetophenone in a
“Substrate-Coupled” Approach
Table 2. Optimization of the Immobilization of ADH-A on
Polyphosphazene 4a
enzymatic
conv.b (%)
entry
reaction conditions
1
2
3
4
5
initial conditions
43
48
85
44
67
using satd (NH4)2SO4
reaction time 12 h at 0 °C
reaction at rt
orbital shaking at 20 °C
a For reaction conditions, see Supporting Information. b Measured by
GC. In all cases, enantiopure (S)-8 was obtained.
ammonium sulfate (entry 5) afforded a better anchoring of
glutaraldehyde as shown by the higher enzymatic activity of
the catalyst. These observations can be explained considering
that the ammonium salt helps to extend the phosphazene chains
facilitating the accessibility of the functionalized sites to the
incoming reagents. It is known that reproducible size exclusion
chromatograms (SEC) for polyphosphazenes are obtained in the
presence of ammonium salts27 that favor extended conformations.
Table 1. Optimization of the Anchoring of Glutaraldehyde to
Polyphosphazene 3a
enzymatic
,
entry
reaction conditions
initial conditions
excess glutaraldehyde
reaction in DMSO
reaction with Et3N
reaction with satd (NH4)2SO4
use of 1,4-diaminobutane as spacer arm
conv.b c (%)
1
2
3
4
5
6
13
12
12
17
43
46
The use of 1,4-diaminobutane as a spacer arm to increase
the distance of the reacting groups to the phosphazene main
chain, lowering the steric hindrance and minimizing undesirable
interactions between the other functional groups on the carrier
surface, was also investigated. It has recently been found that
immobilized Candida rugosa lipase on silica presented a
stability improvement using similar linkers.28 Thus, starting from
4, a diamine was inserted as shown in Scheme 4. Due to the
higher nucleophility of the aliphatic amines, the coupling
reactions were done at room temperature. The new biocatalytic
preparation showed a good activity (entry 6), but the enhance-
ment was similar to the one achieved with the saturated solution
of (NH4)2SO4. Therefore, the simpler conditions of entry 5 were
chosen as the best ones to perform the first step to the
preparation of the supported biocatalyst (Scheme 2). The
percentage of glutaraldehyde introduced in the polymer, as
a For reaction conditions, see Supporting Information. b Measured by
GC. In all cases, enantiopure (S)-8 was obtained. c In all cases, buffer
used to wash 5 showed remaining enzymatic activity, demonstrating that
ADH-A was employed in excess when was immobilized on polyphos-
phazene 4.
the result clearly demonstrated the anchoring of the enzyme to
the polyphosphazene.25 Interestingly, enantiopure (S)-8 was
obtained as in the case of commercial ADH-A, showing that
selectivity was not affected by the immobilization.
Following these results, the immobilization protocol was
optimized. First, the anchoring of glutaraldehyde to 3 was
performed by varying several reaction parameters (Table 1). The
products of each experiment were equally treated with the
enzyme and the glycine solution, and the supported catalysts
were tested with the same model reaction (Table 1).
No improvement was observed using an excess of glutaral-
dehyde (10 equiv) at 50 °C or in the presence of DMSO to
make the reaction homogeneous, and only a very slight increase
in conversion was noted employing Et3N as catalyst for the
coupling reaction (entries 2-4). However, as in other recent
examples,26 the employment of a saturated aqueous solution of
1
estimated by H NMR spectroscopy was around 8% (Scheme
2, approximately x ) 0.04).
To optimize the enzyme immobilization protocol (second step,
Scheme 2), mixtures of polyphosphazene 4 and ADH-A were
stirred by varying the reaction conditions, like reaction times,
temperature, and type of shaking. The effects on the final activity
of the preparations are shown in Table 2.
Scheme 4. Use of 1,4-Diaminobutane as Spacer Arm To Immobilize ADH-A on the Polyphosphazene

Polyphosphazenes as Tunable and Recyclable Supports
Biomacromolecules, Vol. 11, No. 5, 2010 1295
Scheme 5. Use of NaBH4 To Reduce the Imine Bonds in Polyphosphazene 5
Table 3. Bioreduction of Several Ketones Employing Immobilized
ADH-Aa
Due to the good results obtained for the first step with
(NH4)2SO4, we stirred a mixture of 4 and ADH-A in a saturated
solution of (NH4)2SO4 during 30 min at 0 °C, but only a slight
improvement was observed (entry 2). However, a great activity
enhancement was achieved when ADH-A was mixed in buffer
at 0 °C with magnetic stirring during 12 h (entry 3). The activity
(85% conv.) was comparable to the one obtained with the
commercial ADH-A.29 The lower conversion observed at room
temperature during 12 h with magnetic stirring (entry 4) proved
that the immobilization was better performed at low temperature.
Because it is known that magnetic stirring can be harmful to
enzymes, the orbital shaking at 20 °C was essayed (entry 5).
The activity was better in comparison with the anchoring at
the same temperature with magnetic stirring, but lower than that
obtained at 0 °C. This can be attributed to the fact that when
incubating the enzyme directly with the unmodified polyphos-
phazene 3 using orbital shaking, this led to a preparation with
catalytic activity (around 15% conv.), evidencing unspecific
adsorptions of the biocatalyst to the polymer.
enzymatic
conv.b (%)
e.e.c d
,
entry
ketone
product
(config.)
1
2
3
4
9
11
13
15
10
12
14
16
78
85
83
99
98% (S)
99% (S)
99% (R)e
99% (R)e
a For reaction conditions, see Supporting Information. b Measured by
GC. c Measured by chiral GC. d e.e. ) enantiomeric excess. e Change in
Cahn-Ingold-Prelog (CIP) priority.
A final step using a glycine solution to block the nonreacting
aldehydes was employed in all cases to obtain the supported
catalyst. In an attempt to simplify the protocol, we examined
the effects of this step on the enzymatic activity. The ADH-A
immobilized on the polyphosphazene without the treatment with
glycine exhibited a similar conversion (82%), demonstrating that
this step was not necessary.
Finally, considering the potential hydrolytic instability of the
imine bond present in the catalyst 5 that could diminish its
activity within time, a mild reduction agent such as sodium
borohydride (NaBH4) was employed (Scheme 5). The material
obtained by treating 5 with a solution of NaBH4 in water during
30 min at 0 °C showed a lower enzymatic conversion (62%),
suggesting the partial inactivation of the biocatalyst during the
reduction. In another experiment, after incubation of 5 in buffer
at 30 °C during 24 h, filtration and wash, the buffer used to
wash the polymer displayed no enzymatic activity, showing that
imine bonds in 5 were stable during its use as catalyst. Therefore,
the reduction protocol was not further considered.
As a consequence, the anchoring of ADH-A to polyphos-
phazene 3 (Scheme 2) was done using the conditions shown in
Tables 1 (entry 5) and 2 (entry 3), and no glycine and reduction
steps were necessary. Considering that the reaction of the
enzyme with the aldehyde is almost quantitative,18 the final
enzymatic preparation possess approximately y ) 0 and z )
0.04 (Scheme 2).
Several ketones were selected to investigate the effects of
the immobilization of ADH-A on the catalytic selectivity (Table
3). We observed that aliphatic (9 and 11) and aromatic (13 and
15) ketones were selectively reduced to the corresponding
enantiopure Prelog alcohols with similar conversions as the ones
obtained with the commercial ADH-A.
higher temperatures,30 and more importantly, the possibility of
recycling exists, therefore, minimizing the cost of the processes.
We observed that the enzymatic activity of both commercial
ADH-A (already described as a very thermostable enzyme31)
and 5 remained unchanged after heating in buffer for 72 h at
temperatures between 20 and 60 °C (see Supporting Informa-
tion). Only at 80 °C for 1 h, a loss of 80-90% of the activity
was observed in both cases. Therefore, the immobilization of
the alcohol dehydrogenase to the polyphosphazene had no
positive or negative effect on the thermal stability. Recycling
is a key advantage of supported biocatalysts. In the case of
ADHs, this is especially beneficial because this enzyme works
in aqueous media, where proteins are highly soluble. Therefore,
we studied the recycling capacity of 5 applied to the bioreduction
of acetophenone. We observed that the immobilized enzyme
could be used after six catalytic cycles without appreciable loss
of activity (in all cases between 95-100% of remaining
activity). We also found that storing the catalyst for 30 days at
-20 °C after using it in three consecutive catalytic cycles of
reduction of ketone 7, it could be reutilized to perform three
other bioreductions (Figure 1). As can be noted, the immobilized
ADH-A was used after 3 months of storage without loss of
enzymatic activity, showing the high stability of this preparation
and its great potential as biocatalyst for stereoselective biore-
ductions in aqueous media.
Immobilization of CAL-B on Polyphosphazene 3. The use
of polyphosphazene 3 as an adequate precursor to anchor
biocatalysts was extended to other enzymes that work in organic
media. Thus, Candida antarctica lipase B was selected as a
stable enzyme since it has been shown as a very stable catalyst
for many selective transformations over secondary alcohols or
amines.14 First, we tried to covalently link CAL-B employing
the optimized conditions previously described for ADH-A. This
product 6 (Scheme 2) was tested in the acylation reaction of
The enzyme immobilization presents several advantages: the
protein may become thermally stabilized, allowing its use at

1296 Biomacromolecules, Vol. 11, No. 5, 2010
Cuetos et al.
highly efficient for the stereoselective bioreductions of several
aliphatic and aromatic ketones in aqueous media showing a good
thermal stability and an excellent recycling capacity, being
equally active after three months of storage. For the immobilized
CAL-B, this material showed a higher activity than the pure
lipase to achieve kinetic resolutions on organic solvents and a
moderately good capability of reutilization.
Acknowledgment. A.C. thanks Universidad de Oviedo for
a predoctoral fellowship. I.L. thanks Principado de Asturias for
personal funding (Clar´ın Program). Financial support from the
Spanish Ministerio de Ciencia e Innovacio´n (MICINN, Projects
CTQ2007-61126andCTQ2007-61188)isgratefullyacknowledged.
Figure 1. Stability study of 5 vs time in the bioreduction of acetophe-
none. Three consecutive catalytic cycles were performed and then 5
was kept for 30 days at -20 °C until the next three catalytic cycles
were done.
Supporting Information Available. Experimental proce-
dures, analytics, and H NMR and TGA copies are provided.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
Scheme 6. Enzymatic Acylation of 1-Phenylethanol Using
Immobilized CAL-B (6) and Vinyl Acetate
References and Notes
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