Asymmetric epoxidation of chalcone catalyzed by reusable poly-l-leucine immobilized on hydrotalcite

Article abstract of DOI:10.1016/j.jcat.2011.05.026

Nanohybrid materials based on polyamino acids immobilized onto inorganic materials are of interest for their potential applications in protein engineering, biomedicine and catalysis. We developed an efficient and eco-friendly new protocol for the immobilization of synthesized poly-l-leucine (PLL) onto rehydrated hydrotalcite (HT_r). To do this, we synthesized different PLLs containing both C-terminal and N-terminal groups and compared them with a commercial PLL_c. These synthetic polypeptides were immobilized onto HT_r in water as the liquid medium with less than 30 min of ultrasound treatment. The obtained PLLs/HT_r synzyme showed excellent activity and enantioselectivity when used as a catalyst in the asymmetric Juli-Colonna epoxidation reaction of chalcone. Moreover, these nanohybrid materials based on PLL_S did not require any pre-activation time, which were easily separated from the reaction media and, unlike the commercial PLLc-supported catalyst, were reusable, exhibiting high stability after five consecutive runs without any apparent deactivation.

Full text of DOI:10.1016/j.jcat.2011.05.026

Journal of Catalysis 282 (2011) 65–73
Contents lists available at ScienceDirect
Journal of Catalysis
journal homepage: www.elsevier.com/locate/jcat
Asymmetric epoxidation of chalcone catalyzed by reusable poly-L-leucine
immobilized on hydrotalcite
Ronald-Alexander Miranda a,b, Jordi Llorca c, Francisco Medina a,b, Jesús E. Sueiras a,b, Anna M. Segarra a,b,
⇑
a Departament d’Enginyeria Química, Universitat Rovira i Virgili, Av. Països Catalans, 26, Campus Sescelades, Tarragona 43007, Spain
b EMaS-Centro de Investigación en Ingeniería de Materiales y Micro/nanoSistemas, Campus Sescelades, Tarragona 4300, Spain
c Institut de Tècniques Energètiques and Center for Research in NanoEngineering, Universitat Politècnica de Catalunya (CRnE-UPC), Diagonal 647, ed. ETSEIB, Barcelona 08028, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Nanohybrid materials based on polyamino acids immobilized onto inorganic materials are of interest for
their potential applications in protein engineering, biomedicine and catalysis. We developed an efficient
and eco-friendly new protocol for the immobilization of synthesized poly-L-leucine (PLL) onto rehydrated
Received 15 April 2011
Revised 26 May 2011
Accepted 27 May 2011
Available online 2 July 2011
hydrotalcite (HTr). To do this, we synthesized different PLLs containing both C-terminal and N-terminal
groups and compared them with a commercial PLLc. These synthetic polypeptides were immobilized onto
HTr in water as the liquid medium with less than 30 min of ultrasound treatment. The obtained PLLs/HTr
synzyme showed excellent activity and enantioselectivity when used as a catalyst in the asymmetric
Julià–Colonna epoxidation reaction of chalcone. Moreover, these nanohybrid materials based on PLLS
did not require any pre-activation time, which were easily separated from the reaction media and, unlike
the commercial PLLc-supported catalyst, were reusable, exhibiting high stability after five consecutive
runs without any apparent deactivation.
Keywords:
Synzyme
Nanohybrid
Julià–Colonna epoxidation
Asymmetric epoxidation
Poly-L-leucine
Hydrotalcite-like materials
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
ous biphasic system using organic bases and anhydrous urea-
hydrogen peroxide [12] and a PAA on silica (PaaSiCat) as catalyst
Polyamino acids (PAA) are a kind of natural polymer with
important applications in biotechnology [1], bio-nanomaterials
[2] and catalysis [3]. These biomolecules are usually synthesized
[13]. Under PaaSiCat conditions, the reaction was reduced to 4 h
and yielded optically active epoxides with high enantioselectivity
(up to 97% ee). In spite of these improvements, the potential indus-
trial application of these protocols still faces some problems re-
lated to the time-consuming catalyst pre-activation, which has to
be carried out separately and causes difficulties in the workup pro-
cess. Another important enhancement was done by Geller et al.
[14], who presented a modified triphasic protocol whereby the
use of a cocatalyst such as tetrabutylammonium bromide (TBAB)
accelerates the hydrogen peroxide anion phase transfer through
by ring-opening polymerization of a-amino acid and N-carboxyan-
hydrides (NCA) initialized by nucleophilic species or bases [4],
(Scheme 1). Tertiary amines are able to activate NCA and prepare
PAA with high molecular weight without incorporating the initia-
tor in the polymer structure [5,6].
Synthetic PAA such as poly-L-leucine (PLL) are known as excel-
lent catalysts for the asymmetric Julià–Colonna epoxidation reac-
tion of acyclic (E)-enones (e.g. chalcone) [3,7] (Scheme 2). The
the tri-phase interface. As a result, by using poly-L-leucine-1,3-
obtained enantiomerically enriched
tile intermediates for the synthesis of several important chiral
drugs such as taxol (cancer chemotherapy) [8], (+)-clausenamide
a
,b-epoxy ketones are versa-
diaminopropane as catalyst, reaction times were improved by up
to 30 min without any separate pre-activation being required.
Although the Julià–Colonna epoxidation mechanism is not at all
(antiamnaesic agent) [9], statine (cholesteroL-lowering drug) [10]
clear, several studies have suggested that the five terminal
L-leu-
and (+)-fenoprofen (rheumatoid arthritis drug) [11].
cine residues of -helical PLL play an important role in its catalytic
a
The original triphasic protocol [3] (PAA/aqueous phase/organic
phase) had two important problems: the lengthy reaction time
and the difficulty of recovering the gel- or paste-like catalyst. To
overcome these limitations, Roberts et al. developed a non-aque-
activity and enantioselectivity [15]. These results also suggest that
the C-terminal group does not participate in the catalytic process
[7,16].
Different methodologies for immobilizing AAs and oligoamino
2þ
acids onto hydrotalcite-like compounds (HTs),
M
M3xþðOHÞ2
1ꢀx
nꢀ
ðA
Þ
ꢁ yH2O, have been developed [17]. However, to the best of
x=n
⇑
Corresponding author at: Departament d’Enginyeria Química, Universitat
Rovira i Virgili, Av. Països Catalans, 26, Campus Sescelades, Tarragona 43007,
Spain. Fax: +34 977 559621.
our knowledge, there are no examples in the literature of PAA
being immobilized onto a HT. The positive charge of HTs layers is
the result of the brucite-like layers [Mg(OH)2] with edge-sharing
E-mail address: anamaria.segarra@urv.cat (A.M. Segarra).
0021-9517/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcat.2011.05.026

66
R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
The precursor salts, Mg(NO3)2ꢁ6H2O (purity 99%) and Al
(NO3)3ꢁ9H2O (purity 98%), were purchased from Sigma–Aldrich.
2.2. Analysis and characterization
FTIR spectra were recorded on a Nicolet Nexus Fourier trans-
form instrument equipped with a DTGS KBr detector. For each
spectrum, 100 scans in the 4000–400 cmꢀ1 range were recorded
with a resolution of 4 cmꢀ1, using the standard KBr disk (15 mg
of sample in 100 mg of KBr). High-resolution transmission electron
microscopy (HRTEM) was performed with a JEOL 2010F instrument
equipped with a field emission source and working at an accelera-
tion voltage of 200 kV. The point-to-point resolution of the micro-
scope was 0.19 nm, and the resolution between lines was 0.14 nm.
1H NMR spectra were recorded on a Varian 400 spectrometer. 13C
and 27Al-MAS-NMR spectra were recorded at room temperature
on a Varian Mercury spectrometer equipped with a 7 mm BB-CP
MAS probe at a working frequency of 100.6 and 104.21 MHz,
respectively. The spectra were recorded using the cross-polariza-
tion pulse sequence at room temperature under magic angle spin-
ning at a spinning rate of 5.5 kHz. A total of 4000 scans were
collected with a sweep width of 100 MHz and a pulse width of
0.2 s. Powder X-ray diffraction (XRD) patterns of the samples were
obtained using a Siemens D5000 diffractometer with nickel-fil-
Scheme 1. Synthesis of PAA by ring-opening polymerization of NCA.
octahedral hydroxyl occupied by bivalent (Mg2+) but also trivalent
(Al3+) cations and must be balanced by anions located in the inter-
layer. These can be exchanged by other organic or inorganic anions
[18]. Hydrotalcite-like materials can reconstruct the original lay-
ered structure after being calcined by rehydration in a CO2-free
atmosphere. Rehydrated hydrotalcite, also called meixnerite, con-
tains interlayer OHꢀ anions, which provide significant Brønsted ba-
sic properties [19,20].
In this context, and with a view to industrial application, we
have synthesized different PLL with a C-terminal group (PLLs). After
immobilizing those onto meixnerite, we obtained more stable PLL-
supported chiral synzymes. The nanohybrid catalysts were tested
in the Julià–Colonna epoxidation of chalcone under phase-transfer
catalyst/triphasic conditions. In addition, they were recovered by
simple filtration and reused in several consecutive runs without
any pre-activation or deactivation.
tered CuKa radiation. XRD analyses of all the samples were per-
formed in thin films. The patterns were recorded for 2h angles
between 3° and 70°. X-ray diffraction was used to determine the
basal spacing of the free supports and the supports with the ad-
sorbed complexes. The basal spacing of each sample was calculated
from the (0 0 1) reflection in its X-ray pattern. This basal spacing
was associated with the distance between (0 0 1) layers (d001
)
and was located at angles (2h) between 5° and 7°. The thermo-
gravimetric analysis (TGA) was performed using a Perkin Elmer
TGA7 thermobalance.
MALDI/MS measurements were performed on a Voyager-DE
STR, from Applied Biosystems, operating in reflectron positive ion
mode. The instrumental conditions were as follows: accelerating
voltage 20 kV; reflectron potential 66%; and delay time 185 ns.
Spectra were typically the sum of 100 laser shots. Dithranol was
used as matrix (saturated solution in THF). After 2 mg of copolymer
2. Experimental
2.1. General
was dissolved in 1 mL of trifluoroacetic acid:chloroform (1:1), 5
of this solution was added to the same volume of the matrix solu-
tion and 2.5 l of a doping agent (1 mg of potassium trifluoroace-
tate in 1 ml of THF). About 1 l of the resulting solution was
ll
All reactions and manipulations were conducted under an
atmosphere of dry argon. The L-leucine-NCA was prepared as
l
l
previously reported [21]. This compound was characterized by
1H NMR (400 MHz, CDCl3): d = 0.94–1.05 (m, 6H), 1.66–1.78 (m,
1H), 1.80–1.86 (m, 2H), 4.33–4.38 (m, 1H), 7.05 (s, 1H) ppm, where
chemical shifts are reported relative to tetramethylsilane.
Anhydrous 1,4-dioxane and triethylamine from Aldrich were
used as precursors for synthesizing the PLL.
Chalcone, tetrabutylammonium bromide (TBAB), hydrogen
peroxide, toluene, tetrahydrofurane (THF), NaOH and MgSO4 from
Sigma–Aldrich were used as reactants and solvents in the catalytic
test.
deposited on a stainless steel sample holder and allowed to dry be-
fore being introduced into the mass spectrometer. Three indepen-
dent measurements were carried out for each sample. External
mass calibration was done using Calmix 1 and Calmix 2 from a
Sequazyme Peptide Mass Standards Kit (Applied Biosystems). The
acquisition mass range was 200–50,000 Da.
2.3. Synthesis of PLL
The commercially available PLL (PLLc) was poly-
diaminopropane supplied by Across Organics (product No.
327582500) [15].
L
-leucine-1,3-
The different varieties of as-synthesized poly-
were synthesized by means of the polycondensation method, with
triethylamine used as initiator [22]. The -leucine-NCA (0.4 g) was
L-leucine (PLLS)
L
Scheme 2. Asymmetric Julià–Colonna epoxidation of chalcone.

R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
67
dissolved in dry 1,4-dioxane (7 mL) under Ar atmosphere and stir-
red at the indicated temperature (r.t or 60 °C). After 15 min, the
corresponding amount of triethylamine (monomer/initiator ratio
(M/I) = 10, 5 or 2.5) was added. The reactor was closed with a
freshly prepared calcium chloride drying tube. The reaction was
stopped after 4 days. Milli-Q water (14 mL) was added as a workup
solvent, and the mixture was stirred for 2 more hours. Finally, the
obtained solid was filtered and dried at 60 °C under vacuum. The
samples were labeled PLLXY (where X is the M/I ratio, X = 1 for
M/I = 10, X = 2 for M/I = 5 and X = 3 for M/I = 2.5; and Y is the tem-
perature of synthesis, nothing for room temperature and Y = 60 for
60 °C). All obtained polymers were characterized by 13C-MAS-NMR
(400 MHz) (d = 20.09, 35.24, 51.64, 71.540, 111.819, 131.91,
151.52, 171.80, 191.50, 211.11, 230.91 ppm) as well as by
MALDI-TOF and ESI-TOF mass spectroscopy.
1.3 mg) and NaOH 2 M (0.18 mL, 10 equiv.) in toluene (0.5 mL).
The reaction mixture was stirred at room temperature for the indi-
cated time. The reaction mixture was quenched with ethyl acetate
(1 mL). The liquid phase was separated by centrifugation, and the
catalyst (solid phase) was washed several times with toluene. The
liquid phase was finally extracted into ethyl acetate. The organic
fraction was dried over MgSO4. The solvent was then removed by
evaporation under reduced pressure. To study the reusability of
the catalyst, the catalytic solid was kept in the tube and the reagents
were then added for another run. The product was identified by
1H NMR (400 MHz, CDCl3): d = 4.01 (s, 1H), 4.23 (s, 1H), 7.32–7.45
(m, 7H), 7.54 (d, 1H), 7.94 (d, 2H) ppm. The ee of trans-(2R,3S)-
epoxy-1,3-diphenyl-propan-1-one formed by
L-leu/HT catalyst
was determined by chiral HPLC using a ChiralPak IA column. The
mobile phase was 25% hexane in ethanol, at a flow rate of 1 mL/
min. The wavelength reading was 254 nm. The retention times were
tmajor = 7.6 min and tminor = 10.6 min.
2.4. Synthesis of HTs
Mg-Al HT (molar ratio 2:1) was prepared by the standard
co-precipitation method at room temperature as follows. The
appropriate amounts of Mg(NO3)2ꢁ6H2O and Al(NO3)3ꢁ9H2O were
dissolved in 150 cmꢀ3 of distilled water and added dropwise into
a glass vessel which initially contained 200 cmꢀ3 of deionized
water. The pH was kept at 10 by adding a 2 M NaOH solution. Both
solutions were mixed by means of vigorous stirring. The suspen-
sion was stirred overnight at room temperature. The precipitated
solid was filtered and washed several times with water and dried
at 110 °C to yield the as-synthesized hydrotalcite (HT-as). The solid
was calcined in air by heating at 10 °C/min up to 450 °C over 6 h to
obtain the corresponding mixed oxides and then reconstructed in
water (1 mL/mg HT) decarbonated by sonication for 30 min,
stirred for 30 min and sonicated again for 30 min under inert
atmosphere [23,24]. After drying at 40 °C, the sample was labeled
HTr. Total rehydration of the meixnerite was confirmed by
27Al-MAS-NMR and DRX.
3. Results and discussion
3.1. Catalyst preparation and characterization
Nanohybrid materials based on PLL immobilized onto rehy-
drated hydrotalcite (HTr) were synthesized as potential catalysts
in an asymmetric Julià–Colonna epoxidation reaction. The immobi-
lization of polyamino acids onto cationic-charged layered materi-
als such as hydrotalcite is favoured by the presence of
carboxylate group in the bioguest. The N-terminal group is also
a
important in the catalytic process. The commercially available
poly-L-leucine (PLLC) shows two N-terminal groups. For this reason,
various poly-
L
-leucines (PLLS) with both N-terminal and C-terminal
groups were synthesized. Fig. 1 shows the molecular structures of
PLLC and PLLS.
PLLS catalysts were synthesized by means of the polycondensa-
tion method using triethylamine as initiator. The influence of the
temperature and monomer/initiator molar ratio (M/I) was investi-
gated, and all results were compared with PLLC. Table 1 summa-
rizes the results.
It was demonstrated that under the studied conditions, differ-
ent synthesis temperatures and M/I molar ratio variations do not
affect the molecular weight (Mw) of PLLS, which in all cases was
close to that of PLLC. PLLS synthesized at room temperature had
the distribution of degree of polymerization (DP) shown in Table
1. The dimmers, trimmers and tetramers presence in the synthe-
sized polymers (Table 1, entries 2–4) were removed by washing
the PLL with chloroform after synthesis (Table 1, entry 5). Similar
chemical shifts and vibration frequencies were observed for PLLC
2.5. Preparation of the chiral synzymes PLL/HTr
The PLL260 (poly-
60 °C) and the commercial poly-
L
-leucine synthesized using an M/I ratio of 5 at
-leucine (PLLC) were immobilized
L
as follows. Water or THF solutions (5 mL) of each PLL (100 mg)
were prepared and added to a suspension of the solid HTr
(300 mg) in water or THF, respectively. The immobilization process
was then performed using three different protocols. In the first
protocol (called method 1), the mixture was stirred for 2 days at
a temperature of 80 °C (water) or 60 °C (THF); in the second proto-
col (called method 2), the mixture was stirred at room temperature
for 1 h; and in the third protocol (called method 3), the PLL was
immobilized under ultrasound treatment for 30 min. After the
immobilization process, the obtained PLL/HTr nanohybrid materi-
als were separated by filtration, washed several times with THF
(the indicated material having been washed with chloroform)
and dried under vacuum. The samples obtained were called
IPLL260-XY or PLLC-XY, where X is the method used (A, B or C)
and Y is the solvent employed (W for water or T for THF). All nano-
hybrid materials were characterized by XRD, 13C-MAS-NMR and
MALDI-TOF MS. Anchored PLL amounts were calculated by TG
analysis.
(a)
(b)
2.6. Standard conditions for the catalytic asymmetric epoxidation of
chalcone
The asymmetric epoxidation reaction was performed in a tube of
10 mL. Chalcone (0.036 mmol, 7.7 mg), and H2O2 (30 wt.%, 40.6 lL,
11.9 equiv.) was added to a suspension of PLLC, PLLS or the immobi-
lized polymers (200 wt.% of PLLX, 100 mg) with TBAB (0.004 mmol,
Fig. 1. Molecular structures of (a) PLLC and (b) PLLS.

68
R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
Table 1
(Fig. 2). The two types of hydrotalcite structure showed different
Poly-L-leucine synthesis conditions and specifications.
interlayer spaces. While the interlayer space remained unchanged
with respect to the meixnerite in one structure (7.7 Å), the space
increased in size in the other one (11.2 Å). This indicates that only
a part of the PLL is located between the layers of the HTr. The PXRD
patterns also confirmed that a lower degree of immobilization of
PLLS is obtained with THF used as medium (Fig. 2d, f and h). More-
over, the material crystallinity of the sonicated samples decreased
during the immobilization process as the amount of PLL immobi-
lized onto the HTr increased (Fig. 2b–d).
Entrya
Material
T (°C)
M/I ratio
DPbc
Mwc
PDc
1
2
3
4
5
6
7
PLLC
PLL1
PLL2
–
–
10
5
2.5
10
10
5
5–39
2–39
2–39
2–37
5–42
5–42
5–38
2343.4
2212.4
2405.5
2249.1
2274.7
2455.8
2303.4
1.26
1.57
1.51
1.46
1.47
1.43
1.49
r.t.
r.t.
r.t.
r.t.
60
60
PLL3
PLL1d
PLL160
PLL260
a
b
c
See details of the synthesis conditions in Section 2.
Determined by ESI-TOF MS analyses.
Determined by MALDI-TOF analysis with DHB matrix.
Fig. 3 shows the HRTEM images of the nanohybrids IPL260-1T
,
IPL260-1W, IPL260-3W and IPLC-3W. Nanohybrid materials synthesized
by method 1 have indistinguishable morphology (Fig. 3a and b).
Nevertheless, the incorporation of the PLL into the hydrotalcite
structure was confirmed by increments in the basal spacing by
up to 11.1 Å.
d
PLL was washed several times with chloroform after synthesis.
Nanohybrids synthesized by method 3 showed similar behavior
(Fig. 3c and d). Lattice fringes at 7.5 and 3.7 Å were more intense
and corresponded to the (0 0 3) and (0 0 6) crystallographic planes
of the hydrotalcite structure, whereas the spacing at 11.1 Å indi-
cates the presence of intercalated polymer.
and PLLS. These results are in agreement with what was expected.
The MALDI-TOF MS spectra of PLLS showed two sets of peaks that
correspond to the Na+ and K+ cationized nꢂ
L-Leu + H/OH species.
This indicates the presence of a C-terminal group in the PLLs in-
stead of the two N-terminal groups in the PLLC.
The largest amount of immobilized PLL was obtained using
water as medium. This can be explained by the fact that hydrotal-
cite-like compounds are hydrophilic materials. This property per-
mits the interaction of the water molecules with both surface
and interlayer space of the hydrotalcite and facilitates anionic
interchange between the carboxylate end group of PLL and the
hydroxide groups of HTr. When THF was used as immobilization
medium, similar behavior was observed, although the interaction
with the interlayer space of HTr was found to be more difficult.
Moreover, the absence of C-terminal groups in the PLLC decreases
the prevalence of the immobilization process that occurs by inter-
action between carbonyl groups of the amino acid residues with
the OH- groups on the surface and in the edges of the HTr layers
and which deforms the initial hydrotalcite structure.
As Table 1 shows, the PLLC and the synthesized polyamino acid
PLL260 had similar Mw and distribution of DP. For this reason,
PLL260 was chosen as a model in the present study.
We studied the immobilization process of the commercial and
synthesized PLL onto rehydrated hydrotalcite solids with
a
2:1 Mg/Al molar ratio (HTr). The HTr was synthesized by the calci-
nation–rehydration method under ultrasounds. Three different
protocols were developed to immobilize the PLL onto HTr (see pro-
tocols detailed in Section 2). The effect of water and THF as immo-
bilization media was also studied (Table 2).
Method 1, which involves exfoliation of the starting HTr using
water as medium, showed the highest amount of immobilized
PLL (Table 2, entry 5), which indicates that nanohybrids based on
PLLC exhibit a low degree of immobilization (Table 2, entry 9).
These results demonstrate the important role played by the C-
terminal group in the immobilization process of PLL onto HTr. It
was also found that the use of ultrasonic treatment increased the
amount of immobilized PLL. These results are in agreement with
Medina et al., who showed that sonication maximizes accessibility
to the OH- groups of HTs [24].
3.2. Catalytic activity and selectivity
The commercial (PLLC), synthesized (PLLS) and immobilized PLL
were used as catalysts in the asymmetric epoxidation reaction of
chalcone under the standard conditions reported by Geller et al.
with TBAB as a phase transfer cocatalyst (PTC) [14]. Chalcone
was chosen as a test compound substrate in order to draw compar-
PXRD patterns of all of the synthesized nanohybrids showed
peaks corresponding to (0 0 3) and (0 0 6) reflection planes of mei-
xnerite phase (JCPDS: 22-700) obtained after rehydration of mag-
nesium and aluminum mixed oxides. The same samples, except
IPL260-2T (Fig. 2f), had new diffraction peaks corresponding to
(0 0 3) reflection planes of hydrotalcite with immobilized PLL
h
Table 2
Nanohybrid materials based on PLLs synthesis conditions.
g
f
Entry
Material
Methoda
PLL
Immobilization
medium
Immobilization
ratiob
e
d
1
2
3
4
5
6
7
8
9
IPL260-1W
IPL260-1T
IPL260-2W
IPL260-2T
IPL260-3W
IPL260-3T
IPLC-1W
1
1
2
2
3
3
1
2
3
PLL260
PLL260
PLL260
PLL260
PLL260
PLL260
PLLC
Water
THF
Water
THF
Water
THF
Water
Water
Water
0.196
0.082
0.130
0.085
0.225
0.180
0.071
0.037
0.076
c
b
a
IPLC-2W
IPLC-3W
PLLC
PLLC
4
10
20
30
40
50
60
70
2-θ/degrees
a
Method 1: Magnetic stirring for 2 days with indicated temperature. Method 2:
Magnetic stirring for 1 h at room temperature. Method 3: Ultrasound treatment for
30 min.
Fig. 2. XRD patterns of nanohybrid PLL/HTr materials: (a) HTr, (b) IPLC-3W, (c)
IPL260-3W, (d) IPL260-3T, (e) IPL260-2W, (f) IPL260-2T, (g) IPL260-1W and (h) IPL260-1T
Basal peaks of (0 0 3) plane with immobilized PLL. d Basal peaks of (0 0 3) and
.
b
Immobilization ratio = mg PLL/mg HTr. Calculated by thermogravimetric (TG)
analysis.
(0 0 6) planes of the starting HTr material.

R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
69
Fig. 3. HRTEM images of (a) IPL260-1T, (b) IPL260-1W, (c) IPL260-3W and (d) IPLC-3W
.
isons with the results published in the literature for the asymmet-
ric epoxidation process.
93%, respectively (Table 3, entry 8). This is because the oligomers
with fewer than five monomers do not have catalytic properties,
as proposed by Berkessel et al. [15a]. In order to optimize the reac-
tion conditions, further epoxidation reactions were carried out to
maximize the activity of all PLLS. Table 4 shows the results.
Interestingly, for all PLLS, the optimized methodology reduced
the reaction time to 30 min. In addition, the synthesized catalysts
were highly active yet did not require previous chloroform wash-
ing. These interesting results can be explained according to the
mechanistic studies of Colonna et al. [26] (Scheme 4).
3.2.1. Catalytic test of PLLS and PLLC
The manner in which PLL is prepared turned out to be of cru-
cial importance for all of the developed Julià–Colonna protocols
[25]. The PLL synthesized by different protocols were therefore
tested using the standard test reaction (Scheme 3). The results
were compared with those of PLL under the same conditions
(Table 3).
Colonna et al. found that both hydrogen peroxide and substrate
concentration exhibit inhibitory behavior due to the competition
of different pathways. Nevertheless, under our conditions, kinetic
saturation could be overcome by favouring the pathway that in-
volves the formation of the PLL:HOO- complex. This rationalization
might explain why PLL synthesized at room temperature had very
low conversion rates under the Geller conditions while the conver-
sion rate was up to 94% under optimized conditions with 94%
enantiomeric excess.
In agreement with the Geller studies, the polymers synthesized
under thermal treatment (PLL160 and PLL260) were considerably
more active in asymmetric epoxidation than the ones synthesized
at room temperature and slightly more active than the PLLC. The
PLLS synthesized at room temperature showed lower activity due
to the presence of oligomers with a smaller sized chain (<5 mono-
mers). Nevertheless, after the dimmers, trimmers and tetramers
were eliminated by means of chloroform washing, the conversion
rate and enantioselectivity increased significantly, up to 99% and
Scheme 3.

70
R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
Table 3
the epoxidation of phenyl trans-styryl sulfone (Entry 3 and 4, Table
5) and phenyl trans-4-phenyl-3-buten-2-one (Entry 5 and 6, Table
5) are two and four time lower, respectively, than in the Geller
experiment. In addition, the NaOH/TBAB ratio is different. Taken
together these facts could explain the poorer activity observed
with the PLL260 and IPL260-3W catalysts.
Asymmetric epoxidation reaction synthesized by poly-L-leucine.
Entrya
Catalyst
–
Conv. (%)b
e.e. (%)c
1
2
<2
<2
–
–
L-leu
3
4
5
6
NCA
PLLC
<2
85
27
45
37
99
91
90
–
95
87
85
91
93
94
92
PLL1
PLL2
PLL3
PLL1
PLL160
PLL260
3.2.3. Catalytic test and reusability of nanohybrid materials
We studied the potential of IPL260-3W and IPLC-3W nanohybrid
materials in an epoxidation reaction of chalcone in which both
materials were synthesized following the same immobilization
procedure. We evaluated the new synzymes under optimized con-
ditions at 1 h of reaction (Fig. 4). The activity of the basic catalyst
Mg/Al 2:1 dehydrated under ultrasounds (HTr) was also tested in
the epoxidation reaction of chalcone in a previous work [30] pre-
senting a very poor activity (6%).
7
8d
9
10
a
Standard conditions: Chalcone 7.7 mg (0.036 mmol), catalyst 100 mg (200 wt.%
of PLLX), H2O2 97.2 L (30 wt.%, 28.5 equiv.), NaOH 2 M 75.6 L (4.2 equiv.), TBAB
l
l
1.3 mg (0.004 mmol) in toluene (0.5 mL); 1.5 h reaction time, room temperature.
Total diastereoselectivity towards trans-1,2-epoxy-1,3-diphenyl-propane-1-ona.
b
Determined by 1H NMR.
Enantiomeric excess towards the trans-(2R, 3S)-epoxychalcone determined by
c
Fig. 4 shows that the activity and selectivity of the IPLC-3W and
IPL260-3W nanohybrid materials decreases slightly in the first run as
chiral HPLC.
d
Using PLL previously washed with chloroform.
compared to the corresponding non-immobilized PLLC and PLL260
.
In the case of the nanohybrid based on synthesized PLL, compara-
ble values for activity and stereoselectivity were obtained from the
second run. These remained constant after the organic phase was
removed by simple centrifugation, and epoxidation recycling was
repeated for at least five consecutive runs. A remarkable difference
in reusability was observed when the nanohybrid material was
prepared from PLLC. The conversion rate and enantiomeric excess
decreased significantly upon reuse. This was due to leaching of
the PLLC because no basal peaks of the (0 0 3) and (0 0 6) planes
of the PLL intercalated into the hydrotalcite-like compound were
observed in the XRD pattern of the IPLC-3W after five consecutive
runs (Fig. 5). The decrease in activity using PLLC-3W as catalyst con-
firmed the poor stabilization of PLLC onto HTr, probably due to the
absence of C-terminal groups in the PLLC.
After the final run, used catalysts were characterized by HRTEM.
These results show differences between the fresh and reused
IPL260-3W and IPLC-3W catalysts (Figs. 3 and 6). The interlayer space
of the HTr in the IPL260-3W is better preserved after five consecutive
runs (Fig. 6a). Nevertheless, the HRTEM image of reused IPLC-3W
catalyst also reveals that the lattice fringe at 11.1 Å observed in
the fresh catalyst disappeared and, interestingly, the (0 0 3) crys-
tallographic plane of the HT structure was centered at 7.6 Å
(Fig. 6b). These results are in agreement with the DRX analysis of
the reused IPL260-3W and IPLC-3W catalysts (Fig. 5), which indicates
that the polymer without any C-terminal groups is no longer inter-
calated between the HT layers.
Table 4
Optimization of asymmetric epoxidation reaction conditions catalyzed by poly-
L-leucine.
Entrya
Catalyst
–
Conv. (%)
ee (%)
1
2
<4
<4
–
–
L
-leu
3
4
5
6
7
8
9
NCA
PLLC
<4
97
98
98
98
94
95
–
92
90
92
93
94
93
PLL1
PLL2
PLL3
PLL160
PLL260
a
Standard conditions: Chalcone 7.7 mg (0.036 mmol), catalyst 100 mg (200 wt.%
of PLLX), H2O2 40.6 L (30 wt.%, 11.9 equiv.), NaOH 2 M 0.18 mL (10 equiv.), TBAB
l
1.3 mg (0.004 mmol) in toluene (0.5 mL); 0.5 h reaction time, r.t.
3.2.2. Variation of the starting enone
To widen the scope of the new catalysts (PLL260 and IPL260-3W),
some further epoxidation reactions were carried out to broaden
the substrate range using the same conditions as chalcone (Table
5). As test compounds substrates were chosen that exhibited low
reactivity under standard triphasic conditions [27–29] and much
faster conversions under new triphasic/PTC conditions [14].
The new catalysts in the triphasic/PTC conditions show slightly
higher activity when the reaction was performed using the non-
immobilized catalyst. And, they show similar enantioselectivities
in either PLL260 or IPL260-3W catalysts.
In order to understand the lower activity during the first run
with the IPL260-3W catalyst, TGA-DTA analysis of the pure HTr, fresh
IPL260-3W, IPL260-3W after first run and IPL260-3W after second con-
secutive run has been performed. The pure hydrotalcite shows
weight loss between 100 and 200 °C to the loss of interlayer water
and at 395 °C, which corresponds to the dehydroxylation of the
brucite-like layers. The pure synthesized poly-aminoacid discom-
poses between 330 and 400 °C. For the fresh IPL260-3W compounds,
On the other hand, the trans-1,4-diphenyl-2-butene-1,4-dione
shows activities comparable with the Geller and coworker results,
but the enantioselectivities are significantly lowers (Entry 1 and 2,
Table 5). It is important take into account that the reaction times in
Scheme 4. Mechanism of PLL-catalyzed asymmetric epoxidation reaction.

R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
71
Table 5
Epoxidation of different enones.
Entrya
1b
Product
Conv. (%)d
>99 (>99)
ee (%)e
62 (92)
Time referent experiment (min)
8
2c
3b
99
39 (65)
61
62 (64)
60
4c
5b
32
30 (82)
64
19 (68)
120
6c
22
13
a
Standard conditions: 0.036 mmol substrate, 200 wt.% of catalyst, H2O2 40.6 lL (30 wt.%, 11.9 equiv.), NaOH 2 M 0.18 mL (10 equiv.), TBAB 1.3 mg (0.004 mmol) in toluene
(0.5 mL); 0.5 h reaction time, r.t.
b
PLL260
IPL60-3W
.
c
.
d Determined by 1H NMR.
e Enantiomeric excess towards the trans-(2R,3S)-epoxychalcone determined by chiral HPLC and 1H NMR; results of Ref. [14] experiments are given in parenthesis.
Fig. 4. Reusability of IPL260-3W and IPLC-3W. Reaction conditions: 10 equiv. NaOH 2M, 11.9 equiv. H2O2 (30 wt.%), 1 h reaction time. Total diastereoselectivity towards
trans-1,2-epoxy-1,3-diphenyl-propane-1-ona.
and 185 °C. The second step involves a gradual weight loss proba-
bly due to the polycondensation [31] of the polymer immobilized
and dehydroxylation of the brucite-like layers (185–225 °C). And
the thirds step shows a weight loss in TGA (225–650 °C) with an
endothermic maximum in DTA at 407 °C corresponding to the
dehydroxylation of the host layers as well as decomposition of
poly-aminoacid located in the interlayers. Weight losses due to re-
agents or by-products remained in the solid phase was not ob-
served by TGA-DTA analysis.
a
b
4
10
20
30
40
50
60
70
2-θ
The Table 6 shows the total weight loss from the TGA analysis
for all the compounds. The initial content of PLL in the fresh
IPL260-3W corresponds to 9.25% w/w. After the first run and sev-
eral washes with diethyl acetate and toluene, the content of PLL
decreases 1.57%, while after the second consecutive run, there
are no significant leaching losses of the PLL. This fact could ex-
plain the different activity observed during the first run of the
Fig. 5. PDRX patterns of (a) IPLC-3w and (b) IPL260-3w after five consecutive runs.
Basal peaks of (0 0 3) plane of hydrotalcite with immobilized PLL.
the thermal effect is characterized by a three-step decomposition
of poly-aminoacid. In TGA of fresh IPL260-3W, the first weight loss
corresponds to the interlayer water and takes place between 100

72
R.-A. Miranda et al. / Journal of Catalysis 282 (2011) 65–73
Fig. 6. HRTEM images of (a) IPL260-3W and (b) IPLC-3W after five consecutive runs.
Acknowledgments
Table 6
Total weight loss by TGA.
The authors would like to thank the Spanish Ministry of Science
Compound
Total weight loss (%w/w)
and Innovation (CTQ2009-12520-C03-02; NYAM) and Rovira i Vir-
gili University (2009-AIRE10) for financial support. R.-A. Miranda
expresses his thanks to Rovira i Virgili University for his predoc-
toral fellowship. F. Medina and J. Llorca thank the Catalan Govern-
ment for the ICREA ACADEMIA award.
HTr
57.09
47.84
49.41
50.38
Fresh IPL260-3W
1st run IPL260-3W
2nd run IPL260-3W
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