Water-soluble pendant copolymers bearing proline and permethylated β-cyclodextrin: PH-dependent catalytic nanoreactors

Article abstract of DOI:10.1021/ma301615a

To achieve efficient proline-based catalysis in water, proline has been supported in the past to porous and hydrophobic solid resins leading to heterogeneous systems. These solid resins provide a hydrophobic environment to the active centers, mimicking what happens in natural enzymes. However, a more realistic mimetic approach would be to carry out the aldol reaction in a homogeneous way, maintaining the hydrophobic environment, using for example properly designed noncross-linked polymer carriers. In this work, we report the synthesis and aqueous catalytic evaluation of a linear copolymer bearing both pendant proline and permethylated β-cyclodextrin (β-CD) groups. It was designed on the basis that the presence of the hydrophobic cavity of the β-CD could bring aromatic substrates into close proximity to the surrounding catalytic proline residues through host-guest interactions. The compound is water-soluble and catalyzes aldol reactions in this medium without the need for any extra organic solvent. We employed a model reaction between cylohexanone and p-nitrobenzaldehyde, and we observed a decrease of the reaction rate when a competing aromatic compound, known to form a strong inclusion complex with β-CD, was added. The copolymeric catalyst showed a pH-dependent behavior. At pH 7, the copolymer is found in solution as extended single chains with negative charge, catalyzing the reaction in a fast and nonstereoselective mode. At the isoelectric point (pH 3.8) where the positive and negative charges of the zwitterionic proline are canceled by forming charge complexes, the copolymer forms multichain hydrophobic nanoaggregates most probably stabilized by the permethylated β-CD. Although the reaction inside these nanoreactors is slower, it exhibits high stereoselectivity. It is proposed that the observed stereoselectivity is caused by the exclusion of water from the core of these homogeneous entities.

Full text of DOI:10.1021/ma301615a

Article
pubs.acs.org/Macromolecules
Water-Soluble Pendant Copolymers Bearing Proline and
Permethylated β‑Cyclodextrin: pH-Dependent Catalytic
Nanoreactors
Elisa G. Doyaguez,† Juan Rodríguez-Hernandez,‡ Guillermo Corrales,† Alfonso Fernandez-Mayoralas,†,*
́
́
̈
and Alberto Gallardo‡,*
†Instituto de Química Organ
́
ica General, CSIC, Juan de la Cierva 3, 28006, Madrid, Spain
‡Instituto de Ciencia y Tecnología de Polímeros, CSIC, Juan de la Cierva 3, 28006, Madrid, Spain
ABSTRACT: To achieve efficient proline-based catalysis in water,
proline has been supported in the past to porous and hydrophobic
solid resins leading to heterogeneous systems. These solid resins
provide a hydrophobic environment to the active centers,
mimicking what happens in natural enzymes. However, a more
realistic mimetic approach would be to carry out the aldol reaction
in a homogeneous way, maintaining the hydrophobic environment,
using for example properly designed noncross-linked polymer
carriers. In this work, we report the synthesis and aqueous catalytic
evaluation of a linear copolymer bearing both pendant proline and
permethylated β-cyclodextrin (β-CD) groups. It was designed on the basis that the presence of the hydrophobic cavity of the β-
CD could bring aromatic substrates into close proximity to the surrounding catalytic proline residues through host−guest
interactions. The compound is water-soluble and catalyzes aldol reactions in this medium without the need for any extra organic
solvent. We employed a model reaction between cylohexanone and p-nitrobenzaldehyde, and we observed a decrease of the
reaction rate when a competing aromatic compound, known to form a strong inclusion complex with β-CD, was added. The
copolymeric catalyst showed a pH-dependent behavior. At pH 7, the copolymer is found in solution as extended single chains
with negative charge, catalyzing the reaction in a fast and nonstereoselective mode. At the isoelectric point (pH 3.8) where the
positive and negative charges of the zwitterionic proline are canceled by forming charge complexes, the copolymer forms
multichain hydrophobic nanoaggregates most probably stabilized by the permethylated β-CD. Although the reaction inside these
“nanoreactors” is slower, it exhibits high stereoselectivity. It is proposed that the observed stereoselectivity is caused by the
exclusion of water from the core of these homogeneous entities.
perspective. Proline works properly in polar media but not in
water where it exhibits poor efficiency despite its good
solubility.9,10 Recent works have shown that good efficiencies
in water may be achieved through “hydrophobic activation”,
linking proline derivatives to hydrophobic moieties.11 Thus,
some of the hydrophobic porous solid resins exhibited excellent
catalytic properties in water and lead to aldol products in high
yields and stereoselectivity.12−14 These resins actually provide
hydrophobic environments next to the active proline mimicking
at least to some extent natural enzymes that generally make use
of a hydrophobic “pocket” at the active center.
This “hydrophobic activation” applies also to soluble
polymeric supports. Recently, Meijer et al. highlighted that in
order to create a versatile synthetic catalyst the polymer should
be compartmentalized to create a catalytic core.15 In line with
this study, we have reported recently that water-soluble linear
copolymers of hydroxyproline methacrylate and styrene (that
INTRODUCTION
■
It is well-known that proline catalyzes aldol reactions.1,2 Proline
derivatives such as hydroxyproline, bearing an extra functional
group for conjugation, have been used to support proline
mainly on polymeric entities.3,4 Some of the most representa-
tive polymeric supports are the solid resins. These catalysts are
basically porous beads of highly cross-linked polymers
(polystyrene-PS or acrylics),5 which obviously catalyze in a
heterogeneous mode. Noncross-linked polymer supports are
also feasible, such as the well-known linear polymer
polyethylenglycol (PEG), which is soluble in many different
media. The use of soluble polymeric carriers such as PEG
derivatives allows the reaction to be performed in homoge-
neous conditions.6 Besides, different strategies for separation
can be applied to these soluble supports profiting from their
macromolecular nature.7
The use of polymer supports (cross-linked or un-cross-
linked), not only facilitates the recovery and recycling of the
catalyst8 but it may also enlarge the application of proline-
catalyzed reactions from nonpolar solvents to water. Partic-
ularly relevant is the use of water from a green chemistry
Received: August 1, 2012
Revised: September 18, 2012
Published: September 25, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of the Monomers and Copolymer (Me* Stands for Permethylated)
have a tendency to form alternating sequences) catalyzes the
reaction in water with a high rate while the homopolymers
without styrene are inactive.16,17 The increased activity was
explained by hydrophobic interactions between the phenyl ring
of the styrene moiety and the substrates.16 A similar
hydrophobic substituent effect on proline catalysis in water,
supported by quantum mechanical calculations, has recently
been described by Schafmeistes et al.18 These copolymers
however did not exhibit stereoselectivity in pure homogeneous
mode (soluble individual chains), although aggregates obtained
by adding MgCl2 indeed worked stereoselectively, probably by
formation of hydrophobic regions where the reaction takes
place with the exclusion of water.16 This is in agreement with
the role of water since water alters the highly organized
transition states that are thought to be responsible for the
stereoselectivity.19−21 Unlike the coupling to preformed PS-
resins or PEG conjugates, the above-mentioned water-soluble
copolymers of hydroxyproline methacrylate and styrene were
prepared using a “bottom-up” methodology, that is, acrylic
proline-monomers were prepared in a first step and then
copolymerized. The first examples of the preparation of
supported prolines using this ‘bottom-up’ approach were
described recently by us17 and by Hansen et al.22 This
approach gives high flexibility to the synthesis since the
properties of the chains may be easily modulated just by
choosing the right comonomers in the right ratio. In this work,
we have used this flexibility to design a new family of water-
soluble polymer catalysts. Our previous studies indicated that
the presence of hydrophobic pockets is needed for the reaction
to proceed. Thus, in this work we have designed a new styrenic
monomer bearing permethylated β-cyclodextrin (β-CD) to be
copolymerized with a hydroxyproline methacrylate which has
been previously reported.17 It is well-known that cyclodextrins
have the ability to form inclusion complexes with organic
compounds in water, due to the hydrophobic character of their
internal cavity.23,24 There are a few examples describing
organocatalysts that combine the catalytic properties of proline
with the capabilities of cyclodextrin to form inclusion
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complexes.25−31 The permethylated structure of the CD,
instead of the nonpermethylated, has been chosen to tip the
amphiphilic balance in favor of the hydrophobicity. In this
work, the bottom-up synthesis and catalytic evaluation in water
of linear copolymers incorporating a 20 mol % of cyclodextrin,
are described.
spectrophotometer (Perkin-Elmer Instruments). The initial
polymer solution was freshly prepared in an aqueous solution
of 0.15 M of NaCl and 0.1 M of NaOH. A standard aqueous
solution 1 M of HCl was delivered stepwise. pH was monitored
with a Beckman 40 pH-Meter (Beckman Instruments,
Fullerton, CA).
Synthesis. The synthesis of the protected hydroxyproline
methacrylate 4 and the permethylated 6I-azido-6I-deoxi-β-
cyclodextrin (1)used as precursor of the styrenic compound
3are described elsewhere.16,28 The structure of the
monomers can be found in Scheme 1.
EXPERIMENTAL SECTION
■
General Remarks. 2,2′-Azobis(isobutyronitrile) (AIBN,
Merck) was recrystallized twice from ethanol. Other chemicals
purchased from commercial suppliers were of analytical purity
or purified by standard techniques. Thin-layer chromatography
(TLC) was performed on aluminum sheets 60 F254 Merck silica
gel and compounds were visualized by irradiation with UV light
and/or by treatment with a solution of Ce2MoO4 in water, a
solution of ninhydrin in n-BuOH/EtOH or H2SO4 (5%) in
EtOH followed by heating. Flash chromatography was
performed using thick walled columns, employing silica gel
(Merck 60: 0.040−0.063 nm). NMR (1H, 13C NMR) spectra
were recorded on a 300 MHz (Inova 300 or Bruker 300) and
400 MHz (Inova 400 or Mercury 400) spectrometers, using
CDCl3 or D2O as solvents at room temperature. Chemical shift
values are reported in parts per million (δ) relative to
Propargyl 4-Vinylbenzyl Ether (2). To a solution of
propargyl alcohol (224 mg, 4 mmol) in THF (5 mL), sodium
hydride (150 mg, 6.25 mmol) was added, and the mixture was
stirred for 15 min in an ice bath. After this time, reaction was let
to reach room temperature and 4-vinylbenzyl chloride (610 mg,
4 mmol) and tetrabutylammonium bromide (750 mg, 2.36
mmol) were added, and the reaction was stirred at room
temperature for 5 h. Then, methanol (0.5 mL) was added to
eliminate the excess of sodium hydride, and after, dichloro-
methane (40 mL). The mixture was washed with water (3 × 15
mL), and the organic phase was dried (Na2SO4) and
concentrated. The residue was purified by column chromatog-
raphy (hexane:EtOAc, 6:1) to give 2 (420 mg, 59%) as a
colorless oil.
1
tetramethylsilane (TMS) in H and CDCl3 (δ =77.0) in 13C
NMR. Coupling constants (J values) are reported in hertz
(Hz), and spin multiplicities are indicated by the following
symbol: s (singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). Diastereomeric and enantiomeric excess were
calculated by NMR and HPLC Dionex P680 with DAD
detector (lecture at 254 nm). Mass spectra were recorded on a
HP series 1100 MSD spectrometer or in an Agilent 6250
Accurate Mass Q-TOF spectrometer.
1H NMR (300 MHz, CDCl3, 298 K): δ 7.5−7.2 (m, 4H,
CH−Ar), 6.63 (dd, 1H, J = 10.9 Hz, 17.8 Hz, CHCH2), 5.78
(d, 1H, J = 15.7 Hz, CHCH2), 5.30 (d, 1H, J = 15.7 Hz,
CHCH2), 4.65 (s, 2H, ArCH2O), 4.20 (s, 2H, ArCH2OCH2),
2.51 (s, 1H, CCH). 13C NMR (75 MHz, CD3OD): δ 137.5
(C−Ar), 137.1 (C−Ar), 136.7 (CHCH2), 128.6 (CH−Ar),
126.5 (CH−Ar), 114.2 (CHCH2), 79.8 (CCH), 74.9
(CCH), 71.5 (ArCH2O), 57.2 (ArCH2OCH2). HRMS (ESI)
m/z (%): calcd for: m/z C12H12ONa [M + Na]+, 195.0786;
found, 195.0781. Anal. Calcd (%) for C12H12O: C, 83.69; H,
7.02. Found: C, 83.22; H, 6.73.
Monomer MeβCDSty (Compound 3). To a solution of
permethylated 6I-azido-6I-deoxi-β-cyclodextrin (1.970 mg,
0.673 mmol) in DMF:H2O (1:1, 42 mL) were added propargyl
4-vinylbenzyl ether (2.191 mg, 1.108 mmol), CuSO4·5H2O
(185 mg), and sodium L-ascorbate (156 mg, 0.873 mmol)
successively. The mixture was stirred at 80 °C for 35 min in a
microwave. After this time, the mixture was diluted with water,
and it was extracted with CH2Cl2 (4 × 10 mL). The organic
layer was dried (Na2SO4) and solvent was evaporated under
reduced pressure. The residue was purified by column
chromatography (EtOAc:MeOH, 20:1) to give 3 (0.87 g,
80%) as a white foam.
Gel permeation chromatography (GPC) analyses were
carried out using a Perkin-Elmer apparatus with an isocratic
pump serial 200 connected to a differential refractometric
detector (serial 200a). Two Resipore columns (Varian) were
conditioned at 70 °C and used to elute the samples (1 mg/mL
́
concentration) at 1 mL/min. HPLC-Grade N,N-dimethyl
formamide (DMF) supplemented with 0.1% v/v LiBr was
used as eluent. Calibration of SEC was carried out with
monodisperse standard polystyrene samples in the range of 2.9
× 103 to 480 × 103 obtained from Polymer Laboratories.
DLS experiments were carried out using a Malvern Zetasizer
(Zetasizer NS Malvern Instruments, Malvern, UK), working at
a scattering angle of 173° relative to the source. This apparatus
is equipped with a 4 mW He/Ne laser emitting at 633 nm, a
measurement cell, an autocorrelator and a photomultiplier. The
measurements were carried out in the fully automatic mode.
Intensity auto correlation functions were analyzed by a General
Purpose Algorithm (integrated in the Malvern Zetasizer
software) in order to determine values of ζ potential (in mV)
and ζ average diameter (in nm). The measurements were
carried out using a 50 mM PBS solution. To study the influence
of the polymer concentration on the aggregation we employed
three different concentrations 25 mM (5.5 mg/mL), 100 mM
(19.6 mg/mL), and 330 mM (73.3 mg/mL). The influence of
the pH on the size was evidenced measuring the polymer
samples in pH 7 and pH 3.8. Moreover, the isoelectric point
was determined by ζ potential measurements varying the
solution pH between 2.4 and 11.
1H NMR (400 MHz, CDCl3, 298 K): δ 7.63 (s, 1H, NCH
C), 7.35 (d, 2H, J = 8.19 Hz, CH−Ar), 7.27 (d, 2H, J = 8.19
Hz, CH−Ar), 6.67 (dd, 1H, J = 10.9 Hz, 17.6 Hz, CHCH2),
5.71 (dd, 1H, J = 0.98 Hz, 17.6 Hz, CHCH2 trans), 5.26 (d,
1H, J = 3.51 Hz, CH−anom), 5.22 (dd, 1H, J = 0.78 Hz, 10.9
Hz, CHCH2 cis), 5.15−5.05 (m, 6H, CH−anom), 4.63 (s,
2H, ArCH2OCH2), 4.55 (s, 2H, ArCH2O), 3.9−3.7 (m, 10H,
CH and CH2 CD), 3.6−3.5 (m, 32H, CH and CH2 CD, OMe),
3.5−3.4 (m, 34H, CH and CH2 CD, OMe), 3.4−3.2 (m, 14H,
CH and CH2 CD, OMe), 3.2−3.1 (m, 12H, CH and CH2 CD).
13C NMR (100 MHz, CDCl3): δ 144.5 (C-triazol), 137.2 (C−
Ar), 137.1 (C−Ar), 136.3 (CHCH2), 128.0 (CH−Ar), 126.2
(CH−Ar), 124.8 (CH−triazol), 113.9 (CHCH2), 99.1−98.7
(CH−anom), 82.0−81.0 (CH CD), 80.2−77.2 (CH CD), 72.2
(ArCH2O), 71.3−70.2 (CH2−CD), 63.6 (CH2 triazol), 61.6−
The turbidity change of the aqueous solutions of the
polymers (2 mg/mL) as a function of pH was monitored
measuring the absorbance at 600 nm in a UV−vis Lambda 35
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61.2 (CH CD), 59.1−58.3 (CH CD). HRMS (ESI) m/z (%):
calcd for C74H121N3O35Na [M + Na]+, 1634.7678; found,
1634.7691.
Table 1. Aldol Reaction between 7 and 8 Using Copolymer
Poly(HPrMA-stat-MeβCDSty) 80:20 as Catalyst (30 mol %)
at pH 7.0
General Polymerization Procedure. Protected copoly-
mer poly(pHPrMA-stat-MeβCDSty) 80:20 (compound 5) was
prepared by free radical polymerization in N,N-dimethyl
formamide (DMF) at 60 °C for 24 h using AIBN as initiator.
The total concentration of comonomers was 0.5 mol/L and the
initiator concentration was 0.025 mol/L. Example of recipe:
50.4 mg, 62.3 mg and 1.3 mg of 3, 4, and AIBN, respectively,
were dissolved in 0.5 mL of DMF. Reactions were carried out
in the absence of oxygen by gently bubbling nitrogen for 20−30
min before sealing the system. After 24 h, the reaction mixture
was poured into water, and the resulting precipitate was dried
under vacuum overnight. Poly(pHPrMA4n-stat-MeβCDStyn)
was obtained as a white solid (82%).
a
a
b
entry aldehyde 7 (mmol/L) time (h) yield (%)
anti:syn
ee (%)
1
2
3
4
25
4.5
7
43
55
19
21
70
91
56
1.5:1
1.1:1
1.5:1
1.8:1
1.4:1
1.4:1
>20:1
2
2
c
25
4.5
7
4
8
100
330
4.5
7
6
1H NMR (300 MHz, CDCl3, 298 K): δ 7.7−7.6 (wide
singlet, n × 1H, triazol), 7.2−7.0 (wide singlet, Ar), 7.0−6.8
(wide singlet, Ar), 5.5−5.3 (m, 4n × 1H, H-4pro), 5.2−5.0 (m,
n × 7H, CH-anom), 4.7−4.4 (m), 4.2−4.1 (m) 4.0−2.8 (m, n
× 96H, 20 × OCH3, CH-2I−VII, CH-3I−VII, CH-4I−VII, CH2-
6I−VII (CD), CH2CHAr; 4n × 6H, OCH2CH2CH2CH2-
CH2CH2NH, CH2-5pro), 2.4−2.2 (m, 4n × 1H, CH2-3Apro),
2.2−2.0 (m, 4n × 1H, CH2-3Bpro), 2.0−0.5 (m, n × 2H,
CH2CHAr; 4n × 31H, CH2CMe, OCH2CH2CH2CH2CH2-
18
96
20
a
b
Determined by NMR and HPLC. Determined by HPLC. They are
c
referred to the major isomer. Same conditions as entry 1 except that
sodium 2-naphthalenesulfonate (2 mol equiv with respect to the
aldehyde) was added.
Table 2. Aldol Reaction between 7 (100 mM) and 8 (200
mM) Using Copolymer Poly(HPrMA-stat-MeβCDSty) 80:20
as Catalyst (30 mol %) in PBS (50 mM) at pHs 3.8 and 7.0
t
CH2NH, CH3 Bu, CH3 methacryl). GPC data: Mn = 33000 g/
mol. PI (polydispersity index) = 2.8.
a
a
b
entry
1
pH
7.0
time (h)
yield (%)
anti:syn
ee (%)
General Deprotection Procedure. Poly(pHPrMA-stat-
MeβCDSty) (compound 5) was dissolved in 1:2 dichloro-
methane/trifluoroacetic acid (2 mL per 100 mg of polymer)
and the mixture was stirred for 24 h. After this time, the
reaction mixture was concentrated and diethyl ether was added.
The precipitate was decanted, washed with diethyl ether (3×),
and dried under vacuum. The solution was dialyzed in distilled
water for 1 week. Polymer was recovered by freezing and
lyophilization. Poly(HPrMA4n-stat-MeβCDStyn) (compound
6) was obtained as a white solid (100%).
4.5
7
67
91
26
41
15
22
6
1.4:1
1.4:1
>20:1
5:1
6
18
2
3.8
7.0
3.8
24
48
4.5
7
>99
>99
18
c
3
1.9:1
1.3:1
>20:1
>20:1
14
c
4
24
48
>99
>99
9
a
b
Determined by NMR and HPLC. Determined by HPLC. They are
c
1H NMR (300 MHz, D2O, 298 K): δ 8.1−7.8 (wide singlet,
n × 1H, triazol) 7.4−6.9 (wide singlet, n × 4H, Ar), 5.2−5.0
(m, n × 7H, CH-anom), 4.2−3.9 (m), 3.8−2.7 (m), 2.5−2.2
(m, 4n × 1H, CH2-3Apro), 2.2−2.0 (m, 4n × 1H, CH2-3Bpro),
2.0−0.5 (m, n x 2H, CH2CHAr, 4n × 13H, CH2CMe,
OCH2CH2CH2CH2CH2CH2NH, CH3 methacryl).
referred to the major isomer. Reaction was carried out in the presence
of sodium 2-naphthalenesulfonate (2 mol equiv with respect to the
aldehyde).
(2S,1′R)-2-(Hydroxy(4-nitrophenyl)methyl)-
cyclohexan-1-one (9). 1H NMR (300 MHz, CDCl3, 298 K):
δ (major isomer) 8.19 (d, J = 8.7 Hz, 2H, ArH), 7.49 (d, J = 8.7
Hz, 2H, ArH), 4.88 (d, J = 8.4 Hz, 1H, CHOH), 4.02 (wide
singlet, 1H), 2.63−2.54 (m, 1H), 2.50−2.33 (m, 1H), 2.31−
2.21 (m, 1H), 2.14−2.06 (m, 1H), 1.83−1.79 (m, 1H), 1.73−
1.52 (m, 3H), 1.49−1.28 (m, 1H). All spectroscopic data were
in agreement with reported values.32,33 Retention time (HPLC,
Daicel Chiralpak AD-H, hexane/i-PrOH = 80:20, flow rate 0.5
mL/min, λ = 254 nm): tR = 23.79 (syn, minor), tR = 25.40 (syn,
major), tR = 27.30 (anti, minor), 34.60 (anti, major).
General Procedure for Asymmetric Aldol Reaction. To
a solution of polymer (30 mol %, i.e. amount of proline on the
polymer relative to p-nitrobenzaldehyde reactant) in phosphate
buffer (50 mM, pH 7 or 3.8) (it could require sonication for
complete solubilization), p-nitrobenzaldehyde (7) and cyclo-
hexanone (8, 2 equiv) were added. Depending on the desired
concentration, the quantities of the substrates were as follows:
(1) 0.01 mmol of aldehyde (25 mM) and 0.02 mmol of ketone
in 0.4 mL of buffer; (2) 0.01 mmol of aldehyde (100 mM) and
0.02 mmol of ketone in 0.1 mL of buffer; (3) 0.033 mmol of
aldehyde (330 mM) and 0.165 mmol of ketone in 0.1 mL of
buffer. The mixture was stirred at room temperature for the
time indicated in Tables 1 and 2. After the specified time
elapsed, water was added (1 mL) and the mixture was extracted
with dichloromethane (3 × 1 mL). The organic phase was
concentrated under reduced pressure. Conversions and stereo-
selectivities are summarized in Tables 1 and 2. In the
experiments carried out with an inhibitor, sodium 2-
naphthalenesulfonate (2 equiv., 0.02 mmol) was also added
(Table 2).
RESULTS AND DISCUSSION
■
The structure of the two monomers as well as a schematic
detail of a dyad along the statistical copolymer are shown in
Scheme 1. The synthesis of the styrenic monomer MeβCDSty
(3) was carried out by a copper catalyzed azide−alkyne
Huisgen's cyclization between the permethylated β-cyclodextrin
(Meβ-CD) functionalized with azide 1 and the alkyne 2,
obtained by etherification of propargyl alcohol with 4-
vinylbenzyl chloride.
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The synthesis of the protected hydroxyproline methacrylate
pHPrMA (4, Scheme 1), to be used as comonomer, has been
described previously.17 This methacrylate contains an aliphatic
and flexible spacer with six carbons to facilitate the interaction
of the catalytic proline residue with the CD-cavity. The
hydrophobic nature of the spacer together with the
permethylated structure of the CD (instead of the hydroxylated
CD) has been chosen to tip the amphiphilic balance in favor of
the hydrophobicity. It has to be also noted that the MeβCD is
linked to the macromolecular backbone through a spacer with
two aromatic rings. Due to the large size of the cyclodextrin
moiety, a monomer ratio HPrMA: MeβCDSty of 4:1 (molar)
was chosen for this study to balance the proline load and the
presence of the hydrophobic cavity. A facile standard radical
copolymerization of the two units (see Experimental Section)
results in the statistical copolymers indicated in the Scheme,
which exhibit a tendency to form alternating sequences. This
tendency guarantees that most of the CD cavities are
surrounded by proline methacrylates, which is the catalytic
residue, thus optimizing the possible synergistic effect of both
structures. According to our previous analysis, the population of
(MeβCDSty)−(MeβCDSty) dyads is negligible.16 1H NMR
spectra of the copolymer before and after deprotection are
shown in Figure 1.
for the determination of the molar fraction of the copolymer
with relatively high accuracy, according to:
A2 = 96HMeβCDSty + 6HpHPrMA
A3 = 2HMeβCDSty + 31HpHPrMA
([1])
Using this formula, a molar fraction of MeβCDSty, fMeβCDSty
,
of 0.21 has been obtained, which is close to the molar fraction
of the feed, FMeβCDSty = 0.20, indicating that both components
are incorporated into the copolymers.
The molecular weight of the copolymer was characterized by
GPC in the protected form, using polystyrene standards as a
reference. The number average molecular weight and
polydispersity index were found to be 33000 g/mol and 2.8,
which are expected values in conventional radical polymer-
izations.
The copolymer, which is soluble in water, was catalytically
tested in an aqueous buffer at pH 7, in the aldol reaction
between p-nitrobenzaldehyde 7 and cyclohexanone 8 (2 mol
equiv with respect to the aldehyde) as a model reaction
(scheme of Table 1). Table 1 shows the results obtained at this
pH using different concentrations of the aldehyde.
The copolymer is active and the rate of the aldol reaction is
reasonably good; in diluted conditions (25 mM of aldehyde,
entry 1) it reaches a yield of 55% after 7 h. At higher
concentrations of reactants and catalyst (100 mM of aldehyde,
entry 2) the reaction is obviously faster with 91% conversion
after 7 h. Both reactions are non stereoselective. Since the
homopolymer poly(HPrMA) did not exhibit any significant
activity,17 these results must be attributed to the MeβCDSty
and its binding capacity. A complementary experiment has
evidenced the role of the CD cavity. The addition to the media
of the aromatic sodium 2-naphthalenesulfonate (entry 2),
which is known to form an inclusion complex with β-CD,34,35
decreases the reaction rate. Therefore, the cavity of the β-CD
plays a role in the catalytic function of the polymer in water,
probably by binding the aromatic aldehyde substrate and
bringing it close to the catalytic proline unit.
It is remarkable that under conditions where the catalytic
polymer is not soluble (at 330 mM of aldehyde, entry 4) the
reaction is much slower but stereoselective. This result can be
rationalized as the reaction takes place in water-free hydro-
phobic domains of a thick paste; the exclusion of water in these
regions favors the stereoselectivity of the reaction as discussed
below. On the other hand, at 25 and 100 mM of aldehyde the
copolymer is soluble and water may influence the process. The
cartoon shown in Scheme 2 is proposed to explain the results of
the soluble forms at pH 7. The data previously discussed
indicates that the CD cavity has an active role, probably
forming inclusion complexes with the aromatic aldehyde.
According to our previous studies16,17 the polymer has a net
negative charge at this pH 7; therefore the anions along the
chains and their solvation may cause H2O to be located in the
active center, probably influencing the transition states and
preventing stereoselectivity.
Figure 1. 1H NMR spectrum of 5 (bottom) and 6 (up), protected and
unprotected copolymeric forms respectively, see Scheme 1. 1 =
anomeric protons of the CD (7H). 2 and 3 = groups of peaks used to
determine the molar fractions as described in the text. ∗ and # = other
peaks of the pHPrMA/HPrMA and MeβCDSty units respectively.
The copolymer behavior in solution has been studied by
dynamic light scattering (DLS) experiments. Figure 2 shows
the correlation curves obtained for 25 mM and 100 mM at pH
7, which indicates the presence of soluble chains of around 10
nm in diameter.
These entities are probably individual extended chains since,
as mentioned above, the copolymer at this pH 7 has a net
negative charge,17 and the chains may be seen as “hydrophobic”
Due to the presence of tert-butyl groups and permethylated
cyclodextrin, pHPrMA and MeβCDSty have a high amount of
protons in two differential regions of the spectrum: 4.0−2.8
ppm (integral 2 of the figure has 96 and 6 protons of the
MeβCDSty and pHPrMA units respectively) and 2.0−0.5 ppm
(integral 3 of the figure corresponding to 2 and 31 protons of
the MeβCDSty and pHPrMA units, respectively). This allows
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polyanions with columbic repulsions between carboxylate units
and solvation of the ions with water molecules. The correlation
curve obtained for the sample at 330 mM (not shown here) did
not show a single decay but rather a multimodal decay,
evidencing the presence of polydisperse and aggregated
polymer chains. In this case, the reaction proceeds in a
nonhomogeneous media, which precludes us from performing
appropriate DLS analysis. Under these conditions, hydrophobic
areas where water can be excluded could explain the data
observed in Table 1. It has to be mentioned that this
explanation is in agreement with other previous results16 and
with the performance of some solid resins.12−14 Equally, the
reaction rate decreases due both to the limited diffusion of the
reactants and the accessibility to the catalytic centers. Similar
observations were previously obtained in styrene-proline
copolymers in which the addition of magnesium salts provokes
the aggregation of the copolymer and enhances the
enantioselectivity.16
Scheme 2. Tentative Scheme Showing the Solvation of the
Carboxylate Unit at pH 7 as Well as the Active Role of the
Cyclodextrin Cavity
It has been previously reported that the homopolymer poly-
HPrMA is insoluble in aqueous media at the isoelectric point
(IEP),17 which is around 3.5. The proline units are zwitterions
in a certain pH range where both the anionic and cationic
charges coexist. The pH at which the two charges cancel each
other is the IEP. Above and below the IEP, the net charge is
negative or positive, respectively, since the stoichiometry is lost
allowing linear macromolecular chains to expand, solvate, and
eventually dissolve. Figure 3 (top) shows the results of a
turbidimetry study of the homopolymer. Poly-HPrMA is
soluble at pHs far enough from IEP (i.e., 7) and precipitates
in the pH region between 4.5 and 2 approximately. The
copolymer poly(HPrMA-stat-MeβCDSty) 80:20, however, is
soluble in aqueous solutions at all pHs (see turbidimetry studies
Figure 2. Correlation curves (up) and size distribution curves
(bottom) for the copolymer solutions. (a) Solid line: 5.5 mg/mL
copolymer (7.5 mM pendant proline) and (b) Dashed line: 22.2 mg/
mL of copolymer (30 mM pendant proline). The measurements were
carried out at pH 7 in a 50 mM PBS solution.
Figure 3. Absorbance at 600 nm vs the pH for the copolymer
□
poly(HPrMA-stat-MeβCDSty) 80:20 ( , bottom) and the homopol-
□
ymer poly(HPrMA) (( , top) at ionic strength = 0.15. The graph
○
includes the ζ potential (mV) values ( ) as a function of the pH.
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in Figure 3 (bottom)). In spite of this solubility, the ionization
of the proline groups (carboxylic and amine) as a function of
the pH seems to be quite similar to the behavior of these
groups in the homopolymer, as it is shown in Figure 3 using ζ
potential measurements. These measurements reveals a charge
cancellation, this is, an IEP, at pH = 3.8, very close to the IEP
observed for the homopolymer. Thus, it seems that the
permethylated β-cyclodextrin stabilizes the hydrophobic
interpolymeric stoichiometric complexes near IEP avoiding
precipitation. This result is in agreement with some recent
studies reported in the literature on the stabilization properties
of amphiphilic permethylated β-cyclodextrin functionalized
with hydrophobic moieties.36,37
Scheme 3. Schematic Cartoon Showing the Proposed
Entities at pH 7 and 3.8, Individual Extended Chains and
Multichain Nano-Aggregates, Respectively
It is worth recalling that in the case of the homopolymer, the
global hydrophobia causes the interpolymer complexes to
precipitate. The presence of permethylated cyclodextrin,
however, seems to stabilize these complexes, thus avoiding
precipitation. The DLS analysis of the copolymeric entities at
this IEP, as compared to the analysis at pH 7, can be found in
Figure 4.
formation of large aggregates. A similar stabilization role of the
permethylated cyclodextrin has been reported recently.36,37
The copolymer was evaluated at pH 3.8, and the obtained
results were compared to those obtained at pH 7. As depicted
in Table 2 their behavior is remarkably different. A
concentration of 100 mM of aldehyde was used in this study.
At pH 3.8 the reaction rate decreases while the
enantioselectivity is almost absolute (see entries 1 and 2 in
Table 2). To explain this result, an ‘exclusion of water’
mechanism is proposed. Considering that the nonionizable
structure of the copolymer backbone is globally hydrophobic,
the interpolymer complexation and the formation of larger
aggregates are compatible with an expulsion of water from the
vicinities of the inner active centers, allowing the reaction to
occur in a stereoselective way. The decrease of the reaction rate
is in agreement with a poorer accessibility to these active sites.
Moreover, the addition of sodium 2-naphthalenesulfonate
decreases the reaction rate at both pHs (entries 3 and 4 in
Table 2). This again supports the proposed active role of the
CD cavity. Interestingly, the enantioselectivity of the reaction is
not affected by the presence of the inhibitor.
In conclusion, these studies provide new insights on the role
of water and the effect of pH changes on the aldol reactions
using supported proline. It has been confirmed that the reaction
is efficient in water when there is “hydrophobic activation”, in
this study the hydrophobic activation is supplied by the
component bearing CD. Besides, new evidence shows that a
hydrophobic microenvironment around the active proline
moiety is beneficial for stereoselectivity. As proline has two
ionizable groups (carboxylic and amine), which are solvated in
the ionized form, the exclusion of water can be achieved only
by “blocking” that solvation, i.e., forming stoichiometric charge
complexes at the IEP. This is in agreement with the previous
studies on copolymers of hydroxyproline methacrylate and
styrene at pH 7 using Mg2+ salts that strongly interact with the
major carboxylate groups present at this pH.16 The aggregates
formed upon addition of Mg2+ excluded water and exhibited
enantioselectivity as well.
Figure 4. Correlation curves (up) and size distribution curves
(bottom) for copolymer solutions containing 30 mM of pendant
proline in the copolymer obtained at two different pH values: 3.8
(solid line) and 7 (dashed line).
This study has found that the permethylated β-CD plays a
key role in stabilizing the hydrophobic complexes formed at the
IEP, which ensures the catalysis process in a homogeneous
manner.
Whereas at pH 7 the copolymers exist as individual entities
with average diameters of 10−12 nm, at pH 3.8 (the IEP)
objects with sizes 1 order of magnitude bigger than at pH 7
(around 100 nm) were observed. The solution is homogeneous
(transparent) but DLS shows that there are no single chains
anymore but homogeneous nanoaggregates. A model to explain
this behavior has been proposed in Scheme 3. We suggest that
the formation of hydrophobic domains upon charge
cancellation at the IEP (as observed for the homopolymer)
and a stabilization effect of the amphiphilic CD prevents the
AUTHOR INFORMATION
■
Corresponding Author
*(A.F.-M.) Fax: (+34)-91-564-48-53. E-mail: mayoralas@iqog.
csic.es. (A.G.) Fax: (+34)-91-564-48-53. E-mail: gallardo@ictp.
csic.es.
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(31) Breslow, R.; Dong, S. D. Chem. Rev. 1998, 98, 1997−2011.
Author Contributions
(32) Font, D.; Sayalero, S.; Bastero, A.; Jimeno, C.; Pericas
̀
, M. A.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Org. Lett. 2008, 10, 337−340.
(33) Guillena, G.; Hita, M. C.; Naj
́
era, C.; Vioz
́
quez, S. F. J. Org.
Chem. 2008, 73, 5933−5943.
Notes
(34) Inoue, Y.; Hakushi, T.; Liu, Y.; Tong, L. H.; Shen, B. J.; Jin, D.-
S. J. Am. Chem. Soc. 1993, 115, 475−481.
The authors declare no competing financial interest.
(35) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875−1917.
(36) Gervaise, C.; Bonnet, V.; Wattraint, O.; Aubry, F.; Sarazin, C.;
ACKNOWLEDGMENTS
■
̀
Jaffres, P. A.; Djedaïni-Pilard, F. Biochimie 2012, 94, 66−74.
(37) Bauer, M.; Fajolles, C.; Charitat, T.; Wacklin, H.; Daillant, J. J.
Phys. Chem. B 2011, 115, 15263−15270.
This work was supported by the Ministry of Science and
Innovation (MAT2010-20001, MAT2010-17016 and
CTQ2010-15418), the Comunidad de Madrid (S2009/PPQ-
1752), and the CSIC (201080E001 fellowship to E.G.D.).
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