Aqueous micro and nanoreactors based on alternating copolymers of phenylmaleimide and vinylpyrrolidone bearing pendant l-proline stabilized with PEG grafted chains

Article abstract of DOI:10.1002/pola.28487

Proline may work efficiently in water as catalyst of aldol reactions if it is hydrophobically activated. In this work, we have maximized this hydrophobic activation by the preparation of linear alternating copolymers of hydrophobic phenylmaleimide and a v

Full text of DOI:10.1002/pola.28487

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Aqueous Micro and Nanoreactors Based on Alternating Copolymers of
Phenylmaleimide and Vinylpyrrolidone Bearing Pendant L-Proline
Stabilized with PEG Grafted Chains
Anselmo del Prado,1 Mercedes Pintado-Sierra,2 Marta Juan-y-Seva,1 Rodrigo Navarro,2
1
1
1
ꢀ
ꢀ
Helmut Reinecke, Juan Rodrıguez-Hernandez, Carlos Elvira,
2
1
ꢀ
Alfonso Fernandez-Mayoralas, Alberto Gallardo
1
ꢀ
ꢀ
Instituto de Ciencia y Tecnologıa de Polımeros, CSIC, Juan de la Cierva 3, Madrid 28006, Spain
2
ꢀ
ꢀ
Instituto de Quımica Organica General, CSIC, Juan de la Cierva 3, Madrid 28006, Spain
Correspondence to: A. Gallardo (E-mail: gallardo@ictp.csic.es)
Received 24 October 2016; accepted 2 December 2016; published online 00 Month 2017
DOI: 10.1002/pola.28487
ABSTRACT: Proline may work efficiently in water as catalyst of
aldol reactions if it is hydrophobically activated. In this work,
we have maximized this hydrophobic activation by the prepa-
ration of linear alternating copolymers of hydrophobic phenyl-
maleimide and a vinylpyrrolidone derivative bearing proline.
These copolymers were water soluble above pH 5.0 and, unlike
the free proline, exhibited efficient catalysis at pH 7.0. More-
over, they catalyzed and presented enantioselectivity in an
aggregated form at pH 4.0 (close to the isoelectric point, IEP,
of the polymer). This enantioselectivity has been related to the
exclusion of water at this IEP. To control the size and stabilize
the aggregates, PEG grafted copolymers were prepared by the
incorporation of a PEG-macromer (2–10 mol%), which rendered
stable nano-aggregates in water at the IEP. At this pH they cat-
alyzed the aldol reaction in a higher rate than the non-grafted
C
V
polymer, but the enantioselectivity was decreased.
2017
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2017, 00, 000–000
KEYWORDS: catalysis; copolymerization; functionalization of
polymers; radical polymerization; stimuli-sensitive polymers
enzymes generally creates a hydrophobic “pocket” at the active
centre. This interaction has been achieved by anchoring L-pro-
line to hydrophobic porous solid resins,7,8 by copolymerizing
L-proline based monomers with hydrophobic units such as sty-
INTRODUCTION Polymer chemistry has different tools that
make possible tailoring properties and structure of polymeric
materials. Thus, for instance, when two complementary moie-
ties -for example catalyst and co-catalyst- must participate
simultaneously in a process, they can be easily combined in a
common macromolecular backbone just by copolymerizing
two monomeric precursors containing those moieties. Even
more, polymer chemistry offers the possibility of obtaining
alternating copolymers, which may maximize the interaction
between the two required moieties. In this work, this strategy
has been combined with the use of polyethylenglycol (PEG)-
macromers to obtain aqueous nanoreactors containing
L-proline for asymmetric organocatalysis.
rene,9 or designing more complex macromolecular structures
10–12
like the described by O’Reilly and coworkers
and coworkers13,14
and Meijer
In this work, we propose to optimize the hydrophobic nature
of the proline moiety surroundings by preparing alternating
copolymers of units containing respectively L-proline and
aromatic groups. Specifically, we have synthesized a vinylpyr-
rolidone derivative bearing protected L-proline, which has
been copolymerized with an aromatic maleimide, a 1,2-
disubstituted alkene, by radical polymerization. Vinylpyrroli-
done (VP) and 1,2-disubstituted vinylics, such as maleic
derivatives, have been polymerized in the past to obtain
alternating copolymers.15,16 This alternation is due both to
steric and electronic activation reasons: on the one hand, VP
belongs to the group of low activated monomers,17 which
Although L-proline is considered as an enzyme mimic of
aldolase in polar media,1–3 its catalytic activity in water is
very low.4,5 Good efficiencies in water may indeed be
achieved through “hydrophobic activation,”6 linking L-proline
derivatives to hydrophobic moieties, which actually raises
mimicking to a higher level since 3D structure of natural
Additional Supporting Information may be found in the online version of this article.
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means that a growing macroradical ended in VP is much
more reactive toward a maleic derivative; on the other hand,
these 1,2-disubstituted monomers have strong difficulties to
homopolymerize because of steric hindrance, which means
that a growing macroradical ended in maleic derivative can
only propagate by reacting with VP.
and tetramethylsilane (TMS) as internal standard. Chemical shift
values (d) are reported in parts per million (ppm) relative to
TMS. Coupling constants (J values) are reported in Hertz (Hz),
and spin multiplicities are indicated by the following symbols: s
(singlet), d (doublet), t (triplet), q (quartet), dd (doublet of dou-
blets), dt (doublet of triplets), ddd (doublet of doublet of dou-
blets), td (triplet of doublets), and m (multiplet or unresolved).
The 1H NMR spectra of the polymers were measured on an Inova
300 spectrometer (300 MHz) using CDCl3 or D2O as solvents at
room temperature and TMS as internal standard. 1H NMR spectra
of the in situ polymerizations were recorded on a VARIAN
NMR system (400 MHz) using DMF-d7 as solvent at 60 8C and
hexamethyldisiloxane (HMSO) as reference.
This hydrophobic activation may turn the L-proline catalyst
efficient in water but, however, may not be enough to
achieve enantioselectivity. To do so, it has been reported that
water must be excluded from the active centre,9,13 because
water alters the highly organized transition states that are
thought to be responsible for the stereoselectivity.18–20
A
strategy described previously in our group to exclude water
is to work at the isoelectric point (IEP), where the charge
neutralization makes the macromolecules to precipitate and
accordingly to exclude water.21 One disadvantage of this
strategy may be the formation of large aggregates at the IEP,
which may lead to slow catalyst because of the limited diffu-
sion of reactants. In this work, we have used another tool of
polymer chemistry to address this issue by including in the
copolymerization PEG macromers, which will lead to graft
copolymers with PEG branches able to stabilize nanometer
size aggregates in water and thus providing the possibility of
reducing diffusion problems.
High resolution mass spectra (HRMS) were recorded in an Agi-
lent 1200 Series LC system (equipped with a binary pump, an
autosampler, and
a column oven) coupled to a 6520
quadrupole-time of flight (QTOF) mass spectrometer. Acetoni-
trile/water (75:25, v:v) was used as mobile phase at 0.2 mL
min21. The ionization source was an ESI interface working in
the positive-ion mode. Fourier Transform Infrared (FTIR)
spectra were recorded on a Perkin Elmer RX-1 instrument
with a Universal Attenuated Total Reflectance (ATR) device
using diamond/ZnSe as internal reflection elements.
Diastereomeric and enantiomeric excess were calculated by
HPLC Dionex P680 with DAD detector (lecture at 254 nm),
using a chiral chromatographic column Daicel Chiralpak AD-H.
In summary, we propose the preparation of alternating
copolymers of a VP unit bearing pendant L-proline (VPcprol)
with phenylmaleimide (PMI) and the grafting to those
backbones of PEG chains by including a small amount of
PEG-methacrylate (PEGMA) in the reaction. The hypotheses
are that the alternation will maximize the hydrophobic
interaction making the catalysts highly efficient in water, and
that the PEG chains will offer colloidal stabilization at the
IEP affording the enantioselective reaction to occur on small
entities and therefore allowing a fast reaction.
Gel permeation chromatography (GPC) analyses were carried
out using a Perkin Elmer chromatographic system equipped
with a Waters model 2414 refractive index detector, using
Styragel (300 3 7.8 mm, 5 lm nominal particle size) HR3 and
HR5 water columns. DMF with 1 wt% LiBr was used as eluent.
Measurements were performed at 70 8C at a flow rate of
0.7 mL/min using a polymer concentration of 4 mg/mL. The
calibration was performed with monodispersed polystyrene
standards in the range of 2.0 and 9000.0 kDa.
EXPERIMENTAL
DLS experiments were carried out using a Malvern Zetasizer
(Zetasizer NS Malvern Instruments, Malvern, UK), working at a
scattering angle of 1738 relative to the source. This apparatus is
equipped with a 4 mW He/Ne laser emitting at 633 nm, a mea-
surement cell, an auto-correlator and a photomultiplier. The
measurements were carried out in the fully automatic mode.
Intensity autocorrelation functions were analyzed by a General
Purpose Algorithm (integrated in the Malvern Zetasizer soft-
ware) to determine values of zeta potential (in mV) and zeta
average diameter (in nm). The measurements were carried out
using aqueous solutions of the polymers of 0.5 mg/mL. The
influence of the pH on the size was evidenced measuring the
polymer samples in pH 7.0 and pH 4.0.
Materials
Vinylpyrrolidone (Sigma-Aldrich) was distilled under reduced
pressure before use. 2,20-Azobis(isobutyronitrile) (AIBN) 98%
(Sigma-Aldrich) was recrystallized in ethanol. Polyethylengly-
col methyl ether methacrylate average Mn 1.100 (Sigma-
Aldrich) was used as received, as well as all other chemicals,
which were purchased from commercial suppliers.
Methods
Thin layer chromatography (TLC) was performed on alumi-
num sheets 60 F254 Merck silica gel and compounds were
visualized by irradiation with UV light and/or by treatment
with a solution of Ninhydrin in n-BuOH/EtOH or H2SO4 (5%)
in EtOH followed by heating. Flash chromatography was per-
formed using thick walled columns, using silica gel (Merck 60)
or deactivated aluminum oxide, Brockmann II (Aldrich).
The turbidity change of the aqueous solutions of the polymers
(1 mg/mL) as a function of pH was monitored measuring the
absorbance at 600 nm in a UV-vis Lambda 35 spectrophotom-
eter (Perkin Elmer Instruments). The initial polymer solution
was freshly prepared in an aqueous solution of 0.15 M of
NaCl, increasing the pH up to 9.0 with an aqueous solution of
Monomers 1H and 13C NMR spectra were recorded on a VARIAN
NMR system (400 and 500 MHz for 1H, and 100 and 125 MHz for
13C, respectively) using CDCl3 as solvent at room temperature
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SCHEME 1 Synthesis of VPcprol-Boc2 via copper(I)-catalyzed azide alkyne cycloaddition (CuAAC).
NaOH 1M to ensure the total deprotonation of the L-proline
moieties. A standard aqueous solution 0.1 M of HCl was
delivered stepwise. The pH changes were monitored with a
SCHOTT Instruments hadylab pH-Meter.
(2S,4R)-Di-tert-butyl 4-p-toluenesulfonylpyrrolidine-1,2-dicarboxylate
(TsO-prol-Boc2). p-Toluenesulfonyl chloride (3.25 g, 16.70 mmol)
was added over a solution of HO-pz-Boc2 (4.0 g, 13.92 mmol) in
dry pyridine (17 mL) at 0 8C in an ice bath. The mixture was stirred
at 0 8C during 1 h, and then it allowed to warm up to room temper-
ature, keeping the stirring for further 24 h. Afterwards, solvent was
removed at low pressure and the residue was purified by silica gel
flash chromatography using hexane/ethyl acetate (5:1) as eluent,
yielding a yellowish oil. Yield 75%.
Synthesis of Monomers
The synthetic approach to prepare the protected L-proline
vinylpyrrolidone monomer is depicted in Scheme 1. The
compounds 3-propargyl-N-vinylpyrrolidone (VPgyl) and phe-
nylmaleimide (PMI) were prepared according to previously
reported procedures.22,23
1H NMR (CDCl3, 400 MHz): d 5 7.79–7.77 (m, 2H, Ar-H),
737–7.34 (m, 2H, Ar-H), 5.04–4.98 (m, 1H, NCHCOO),
4.26–4.22 (m, 1H, TsOCH), 3.61–3.51 (m, 2H, TsOCHCH2N),
2.57–2.36 (m, 4H, Ar-CH3 and TsOCHCHHCH), 2.17–2.02
(m, 1H, TsOCHCHHCH), 1.44–1.41 (m, 18H, COOtBu 32)
ppm.
(2S,4R)-Di-tert-butyl
4-hydroxypyrrolidine-1,2-dicarboxylate
(HO-prol-Boc2). (2S,4R) N-(tert-butoxycarbonyl)-4-hydroxy-L-
proline (5.0 g, 21.60 mmol), tetrabutylammonium bromide
(3.5 g, 11.00 mmol), K2CO3 (59.76 g, 432.40 mmol), and
tert-butyl bromide (49.55 mL, 432.4 mmol) were dissolved
in N,N-dimethylacetamide (90 mL). The mixture was stirred
at 55 8C during 24 h. Then, distilled water was added to the
mixture until a clear solution was obtained. The mixture was
extracted with diethylether (3 3 100 mL). The organic
phases were collected and dried over anhydrous Na2SO4.
The solid was removed by simple filtration and the solvent
was evaporated under reduced pressure, yielding an oil that
was purified silica gel flash chromatography using a mixture
of hexane/ethyl acetate (1:1) as eluent. Yield 70%.
13C NMR (CDCl3, 100 MHz): d 5 171.42 (CHCOOtBu), 153.86
and 153.55 (NCOOtBu), 145.39 (CSO2), 133.68 and 133.44
(CH3C), 130.21 (CHAC(CH3)ACH), 127.90 (CHAC(SO2)ACH),
81.76 (CHCOOC(CH3)3), 80.70 (NCOOC(CH3)3), 79.26 and
78.46 (CHCOO), 58.09 (TsOCH), 52.26 and 51.86
(TsOCHCH2N), 37.42 and 36.10 (TsOCHCH2CH), 28.39 and
28.10 (C(CH3)3 32), 21.84 (CH3) ppm.
FTIR (cm21): 2979, 2934, 2878, 1740, 1702, 1598, 1479,
1457, 1397, 1366, 1257, 1221, 1175, 1151, 1129, 1098, 1052,
1019, 993, 961, 900, 841, 816, 771.
1H NMR (CDCl3, 400 MHz): d 5 4.44 (s, 1H, NCHCOO), 4.30–4.23
(m, 1H, HOCH), 3.60–3.39 (m, 2H, HOCHCH2N), 2.30–2.20 (m, 1H,
HOCHCHHCH), 2.06–1.97 (m, 1H, HOCHCHHCH), 1.54–1.31 (m,
18H, COOtBu 32) ppm.
HRMS (ESI1): calc. m/z: 464.1717 (M 1 Na)1, found: 464.1725.
(2S,4S)-Di-tert-butyl 4-azidopyrrolidine-1,2-dicarboxylate (N3-
prol-Boc2). Sodium azide (0.88 g, 13.58 mmol) was added to a
solution of TsO-prol-Boc2 (3 g, 6.79 mmol) in DMF/H2O
(20:14 mL), and left stirring at 60 8C during 48 h. Then, the
reaction was quenched adding CHCl3 (20 mL) and saturated
NaCl (20 mL). Organic phase was separated, and the aqueous
phase was extracted with CHCl3 (2 3 10 mL). The organic
phases were collected and dried over anhydrous Na2SO4. The
solid was removed by simple filtration and the solvent was
removed at low pressure and the residue was purified by
flash chromatography in silica gel, using hexane/ethyl acetate
(7:2) as eluent, yielding an oil. Yield 82%.
13C NMR (CDCl3, 100 MHz): d 5 172.27 (CHCOOtBu), 154.34
(NCOOtBu), 81.26 (CHCOOC(CH3)3), 80.31 (NCOOC(CH3)3), 70.26
and 69.37 (CHCOO), 58.63 (HOCH), 54.72 (HOCHCH2N), 39.27
and 38.53 (HOCHCH2CH), 28.45 and 28.11 (C(CH3)3 32) ppm.
FTIR (cm21): 3432, 2978, 2935, 2878, 1740, 1702, 1675, 1479,
1457, 1394, 1366, 1256, 1220, 1148, 1128, 1086, 1053, 992,
973, 938, 914, 854, 841, 771.
HRMS (ESI1): calc. m/z: 310.1625 (M 1 Na)1, found m/z:
310.1633.
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1H NMR (CDCl3, 400 MHz): d 5 4.32–4.16 (m, 2H, NCHCOO
and N3CH), 3.73–3.63 (m, 1H, N3CHCHHN), 3.50–3.40 (m, 1H,
N3CHCHHN), 2.47–2.38 (m, 1H, N3CHCHHCH), 2.16–2.14 (m,
1H, N3CHCHHCH), 1.48–1.43 (m, 18H, COOtBu 32) ppm.
HRMS (ESI1): calc. m/z: 462.2711 (M 1 H)1, found: 462.2699.
Synthesis of Polymers
Alternating Copolymer
Protected copolymer poly(VPcprol-Boc2-alt-PMI) 50:50 was
prepared by free radical polymerization in 1,4-dioxane at 60 8C
for 24 h using AIBN as initiator. The total concentration of
monomer and AIBN was 1 and 1.5 3 1022, respectively. Reac-
tions were carried out in the absence of oxygen by gently bub-
bling nitrogen for 20–30 min before sealing the system. After
24 h, the reaction mixture was poured into diethylether, and
the resulting precipitate was dried under vacuum overnight.
The polymer was obtained as a white solid. To determine the
monomer composition, the relative areas of the corresponding
monomeric units were used applying the following equations:
13C RMN (CDCl3, 100 MHz): d 5 170.85 and 170.54 (CHCOOtBu),
153.91 and 153.74 (NCOOtBu), 81.70 (CHCOOC(CH3)3), 80.41
(NCOOC(CH3)3), 59.34 and 58.40 (CHCOO), 58.24 (N3CH), 51.30
and 50.97 (N3CHCH2N), 36.24 and 35.20 (N3CHCH2CH), 28.43
and 28.08 (C(CH3)3 32) ppm.
FTIR (cm21):2979, 2934, 2889, 2105, 1746, 1702, 1479,
1457, 1479, 1457, 1395, 1367, 1258, 1220, 1152, 1117,
1055, 1002, 907, 844, 770.
HRMS (ESI1): calc. m/z: 335.1717 (M 1 Na)1, found: 335.1725.
A
7:826:851HVPcprol2Boc215HPMI
5:223:853HVPcprol2Boc2
(1)
(2)
(2S,4S)-Di-tert-butyl-4-(4-((2-oxo-1-vinylpyrrolidin-3-yl)methyl)-
1H-1,2,3-triazol-1-yl)pyrrolidin-1,2-dicarboxylate (VPcprol-Boc2).
On the one hand, VPgyl (2.00 g, 13.4 mmol) and sodium
ascorbate (0.53 g, 2.68 mmol) were dissolved in THF
(10 mL). On the other hand, CuSO4ꢀ5H2O (1.02 g, 4.02 mmol)
were dissolved in 6 mL of distilled water. Both solutions
were added at the same time to a third solution of N3-prol-Boc2
(4.18 g, 13.4 mmol) in 20 mL of THF. The mixture was stirred
at 30 8C during 24 h. Then, the reaction was quenched with
5 mL of MeOH. 50 mL of a solution of Na2CO3 0.1 M and 50 mL
de CHCl3 were added to the mixture. The organic phase was
separated, and the aqueous phase was extracted with CHCl3 (2
3 30 mL). All the organic phases were combined and dried
over anhydrous Na2SO4. The solid residue was removed by sim-
ple filtration and the solvent removed at low pressure. The
yielding residue was purified by flash chromatography with sil-
ica gel, using a gradient solution of hexane/ethyl acetate from
1:1 to 0:1 as eluent. Yield 75%.
A
To confirm the tendency to alternation of these monomers, the
copolymerization was carried out in situ by dynamic H NMR
1
ARRAY in DMF-d7 using VPcprol-Boc2/PMI 4:3 as monomer
feed ratio. The experiment was carried out using a Varian 400
NMR equipment. To perform quantitative experiments, the fol-
lowing conditions were used: a pulse sequence of 7 ms equiva-
lent to a 90 tip angle and a 120 s delay time were applied to
allow for the total relaxation of the protons and to process the
individual data. The spinning rate of the samples was 7 Hz.
The sample temperature was maintained at 60 8C using the
heat controller of the NMR equipment. To determine the mono-
mer consumption the relative areas of the corresponding
monomers were used applying the following equations:
H
VPcprol2Boc25A7:2527:10
(3)
1H NMR (CDCl3, 500 MHz): d 5 7.50 (s, 1H, C-CH-N), 7.07
(dd, 1H, N-CH@ CH2, J 5 16.0 and 9.0 Hz), 5.10–5.02 (m, 1H,
N@ NACHACH2AN), 4.46–4.10 (m, 4H, NACH@ CH2,
NACHACOOtBu and N@ NACHACHHAN), 3.85–3.75 (m, 1H,
N@ NACHACHHAN), 3.41–3.17 (m, 3H, COANACH2 and
COACHACHH), 2.98–2.86 (m, 3H, COACHACHH, COACH and
CHHACHACOOtBu), 2.59–2.26 (m, 2H, CHHACHACOOtBu
and COANACH2ACHH), 3.04–2.89 (m, 1H, COANACH2ACHH),
1.45–1.41 (m, 18H, COOtBu 32) ppm.
A7:3027:25
H
PMI5
(4)
2
Hmonomer
À
Á
monomer ð%Þ5
(5)
H
PMI1HVPcprol2Boc2 t50
where A is the area of the corresponding peak or group of
peaks, and monomer is VPcprol-Boc2 or PMI.
PEG Grafted Terpolymers
Protected PEG grafted terpolymers poly(VPcprol-Boc2-co-
PMI-co-PEGMA)-X% (where X is the mol% of PEGMA macro-
monomer in the feed ratio) were prepared using the same
conditions as the alternating copolymer explained above. In
this case, X% was 2%, 5%, and 10%. Poly(VPcpro-Boc2-co-
PMI-co-PEGMA) polymers were obtained as a white solid. To
determine the monomer composition, the following equation
was used in addition to eqs 1 and 2:
13C NMR (CDCl3, 125 MHz): d 5 173.95 (NCO), 170.54
(CHCOOtBu), 153.66 (NCOOtBu), 145.16 (CACHAN), 129.28
(NACH@CH2), 120.76 (CACHAN), 94.71 (NACH@ CH2), 81.87
(NCOOC(CH3), 80.77 (CHCOOC(CH3), 58.12 (CHCOOC(CH3),
57.76 and 57.08 (N@ NACHACH2AN), 51.36 and 51.09 (N@
NACHACH2AN), 42.77 (COANACH2), 42.43 (COACH), 36.29
and 35.30 (CH2ACHACOOtBu), 28.24 (NCOOC(CH3)3), 27.89
(CHCOOC(CH3)3), 26.55(COACHACH2), 23.35 (NACH2ACH2)
ppm.
A
3:823:35 91HPEGMA 1 2HVPcprol2Boc2
(6)
FTIR (cm21): 3138, 2978, 2933, 2888, 1742, 1698, 1633,
1551, 1479, 1456, 1393, 1327, 1270, 1222, 1153, 1117, 1042,
981, 956, 913, 844, 771.
General Deprotection Procedure
The synthesized polymers were dissolved in a mixture of
dichloromethane/trifluoroacetic acid (1:1) (2 mL per 100 mg
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SCHEME 2 Preparation of the alternating copolymer.
of polymer) and the mixture was then stirred for 24 h. After-
wards, the solvent was removed at low pressure, and the
resulting polymers were purified by dialysis in distilled
water using membranes of cut-off 1000 Da, followed by
freeze drying. All the polymers were obtained as white solids
in yields above 95%.
To confirm the alternating structure of the copolymers, a
copolymerization reaction with a slight excess of the VP
derivative was followed using dynamic 1H NMR ARRAY and
multiple spectra were collected during the reaction, follow-
ing procedures previously reported.24,25 The slight excess of
the L-proline derivative was used for the clarity of the graph-
ical presentation, and also to analyze qualitatively the differ-
ent polymerization rates before and after the consumption of
PMI. The consumption of both monomers, VPcprol-Boc2 and
PMI, as a function of their conversion are depicted in Figure
2 using the eqs 3, 4, and 5. An alternating incorporation
might occur if both monomers were equally consumed
throughout the reaction, regardless of the initial feed molar
ratio. For example, a growing chain ending in VPcprol-Boc2
will preferentially react with activated PMI, providing a new
growing radical ended in PMI. This would then only react
with VPcprol-Boc2 because of steric reasons, until one of the
monomers was completely consumed. As is shown in Figure
2, the slopes for consumption of both monomers are very
similar, indicating that the two monomers are consumed at
almost the same rate. The slight deviation between both
slopes indicates that the alternation is high but not complete.
After the consumption of PMI monomer, the polymerization
rate decrees drastically, obtaining only a 5% VPcprol-Boc2
conversion in a few hours.
Asymmetric Aldol Reaction
To a solution of polymer (30 mol%, that is amount of L-pro-
line on the polymer relative to p-nitrobenzaldehyde reagent)
in phosphate buffer (pH 7.0 or 4.0) (it could require sonica-
tion for complete solubilization), p-nitrobenzaldehyde (x, 1
eq.) and cyclohexanone (y, 5 eq.) were added. The mixture
was stirred at room temperature for 24 h. After the specified
time elapsed, 0.2 M NaOH (1 mL) was added to the mixture,
and was extracted with CH2Cl2 (3 3 1 mL). Then, organic
layers were combined, dried over anhydrous Na2SO4 and sol-
id was removed by simple filtration. The solvent was
removed at low pressure, obtaining a yellowish residue that
was redissolved in 1 mL of CH2Cl2. From this solution, an
aliquot of 100 lL was taken, the solvent was evaporated
under reduced pressure and the residue dissolved in a mix-
ture of hexane/isopropanol (4:1) for HPLC analysis.
RESULTS AND DISCUSSION
Synthesis and Catalytic Evaluation of the
Alternating Copolymer
VPcprol-Boc2 was copolymerized with PMI, at an equimolar
ratio, by conventional radical reaction (see Scheme 2).
The L-proline derivative was copolymerized in the protected
form because of two reasons: (1) free amines may act as
transfer agents in the radical process, and (2) the protected
monomer exhibited an improved solubility in organic sol-
vents allowing us to find a common solvent for the polymeri-
zation reaction. Both units copolymerized properly, being the
final copolymer composition 0.50 – calculated from the 1H
NMR spectra as indicated in the Experimental section. After
deprotection with TFA the copolymer is soluble in water at
pH 7.0. Figure 1 shows the spectra of the protected and the
free form of the copolymer in CDCl3 and D2O, respectively.
Figure 1 shows the disappearance of the Boc groups at 1.6
ppm. Molecular weights of the copolymers were character-
ized by GPC in the protected form, using polystyrene stand-
ards as reference. Number average molecular weights of
polymers were 81 kDa. The dispersity index was 1.7.
FIGURE 1 1H NMR spectra of: above, the protected alternating
poly(VPcprol-Boc2-alt-PMI) copolymer and below, the deprotected
alternating copolymer, poly(VPcprol-alt-PMI).
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TABLE 1 Aldol Reaction Between x and y Using Copolymer
Poly(VPcprol-alt-PMI) as Catalyst (30 mol%) at pH 7.0 and 4.0,
and Characteristics of the Entities in Solution (Size and f
Potential)
Poly(VPcprol-
alt-PMI)
L-Proline
pH 7
pH 4
pH 7
pH 4
Conversion (%)a,b
Anti/synb
4 h
n.d.
99
24
91
5.5
4
n.d.
9
n.d.
0
FIGURE 2 Quantity of VPcprol-Boc2 and PMI in the polymeriza-
tion reaction versus conversion monitored by dynamic 1H NMR
ARRAY.
24 h
4 h
n.d.
1.5
n.d.
2
n.d.
2.2
n.d.
14
–
n.d.
-
24 h
4 h
ee (%)b
83
81
n.d.
n.d.
–
The solubility of the alternating copolymer was investigated
both by Dynamic Light Scattering (DLS) and turbidimetry
measurements. According to these results, the copolymer
24 h
Size (nm)c
f potential (mV)c
5.2
238
microns
21
–
–
a
Determined by 1H NMR.
Determined by HPLC.
b
c
Features of polymer entities.
n.d., No determined.
was soluble in aqueous solutions at pH values above 5.0.
Below this pH, as evidenced in the turbidimetry curve
depicted in Figure 5, the copolymer becomes insoluble
because coulombic interactions between opposite charges
are maximal at this interval, that is, the range around the
isoelectric point (IEP), whose value is in agreement with the
previously reported for linear polymers containing pendant
L-proline (IEP 5 3.8).9,26 It has to be noted that IEP of free L-
proline is 6.3. This remarkable shift of the IEP to lower val-
ues in the polymeric form may be related to the differences
in accessibility for ionisable groups, the weak amino base
and the weak carboxylic acid. Above IEP the net charge is
negative since the stoichiometry is lost allowing linear mac-
romolecular chains to expand, solvate and eventually dis-
solve. In agreement with the turbidimetry measurements,
DLS also confirmed the aggregation and precipitation at pH
values near to the IEP (see Figure 3 and Table 1). On the
one hand, the relaxation time significantly decreased by
increasing the pH indicating the formation of smaller entities
that upon CONTIN analysis evidenced the formation of
smaller objects with sizes in the range of 8–30 nm.
The copolymer was tested as a catalyst in the aldol reaction
between p-nitrobenzaldehyde (x, 1 eq.) and cyclohexanone
(y, 5 eq.) in phosphate buffer pH 7.0 and 4.0. as a model
reaction (scheme of Table 1). Table 1 shows the results
obtained at these pHs.
FIGURE 3 Correlation curves (a) and size distribution curves
(b) for the poly(VPcprol-alt-PMI) copolymer solutions at two
different pH values, that is 4.0 and 7.0. Approaching the iso-
electric point (IEP) the solubility of the copolymer decreased
while remains soluble at neutral to basic pH values.
These two pHs were chosen to test the catalyst in the solu-
ble and in the non-soluble forms, namely, at pH 7.0 and at
6
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FIGURE 5 Absorbance at 600 nm versus the pH for the
copolymer.
At pH 4.0, in despite of the large size of the aggregates, the
reaction is quite efficient, exhibiting after 4 and 24 h a 24 and
91% of conversion, respectively. Equally, the reaction rate
decreases compared with pH 7.0 due both to the limited diffu-
sion of the reactants and the accessibility to the catalytic
FIGURE 4 Conversion versus time of the aldol reaction of p-
nitrobenzaldehyde with cyclohexanone at pH 4.0 catalyzed by
poly(VPcprol-alt-PMI) (above), and the evolution of the anti/syn
ratio (᭿) and ee (᭹) with conversion in the same reaction
(below).
the pH close to the IEP, respectively. As it has been described
before, at pH 7.0 the L-proline moieties are negatively charged
and the macromolecules are extended and soluble, being the
active catalytic centers solvated by water molecules. At this
pH the entities have average sizes of 5.2 nm, which is compat-
ible with unimolecular negatively charged species. At the IEP,
on the contrary, the charge cancellation makes the polymer to
precipitate and water is expected to be excluded from the
aggregates in view of the hydrophobic nature of PMI. Large
and non-charged aggregates are found in solution.
Table 1 shows that the copolymer is active at pH 7.0, unlike free
L-proline. This result confirms the initial hypothesis related to
the hydrophobic interaction of PMI in an alternate sequence dis-
tribution. Reactions are non stereoselective, which is in agree-
ment with previous result and it is related to the charge
solvation that cause water to be located in the active center,
probably influencing the transition states and preventing
stereoselectivity.9 The chains may be seen as “hydrophobic”
poly-anions with columbic repulsions between carboxylate
units and solvation of the ions with water molecules.
FIGURE 6 Correlation curves (a) and size distribution curves
(b) for the copolymer solutions with variable amount of
PEGMA. The measurements were carried out at pH 4.0 in a
50mM PBS solution.
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TABLE 2 Characteristics of the PEG Grafted Copolymers
Molecular
Weight
Composition
(pH 4.0)
Mn (kDa)b
ꢁ
Size (nm)
f Potential (mV)
a
a
b
-
D
Polymer
F/f
F/f
(VPcprol-Boc2)
(PEGMA)
Poly(VPcprol-co-PMI-co-PEGMA)22%
Poly(VPcprol-co-PMI-co-PEGMA)25%
Poly(VPcprol-co-PMI-co-PEGMA)210%
0.49/0.47
0.47/0.47
0.45/0.46
0.02/0.02
0.05/0.06
0.10/0.09
181.8
170.3
205.0
1.6
1.7
1.8
15.8
13.5
21.2
26
216
217
a
b
Determined by 1H NMR using eqs 1, 2, and 6.
Determined by GPC.
centers. It is remarkable that at this pH, where the catalytic
polymer is not soluble, the reaction is stereoselective: anti/syn
diastereoselectivities were 5.5 and 4.0, with enantiomeric
excesses of 83% and 81% after 4 and 24 h, respectively. This is
in agreement with the proposed exclusion of water from the
aggregates and with previous results of literature on linear poly-
mers,9 as well as on solid supports.6–8 Figure 4 shows a more
detailed study of this reaction at pH 4.0. The reproducibility of
the enantioselectivity has been studied carrying out four repli-
cates at 24 h (see data in Table S1, Supporting Information). The
results were anti/syn of 4.3 6 0.4 and ee of 74 6 6.
the protected form, using polystyrene standards as reference.
Number average molecular weights (between 170 and 205
kDa) of polymers and dispersity indexes (1.6–1.8) can be
found in Table 2 as well.
The resulting copolymers incorporate grafted PEG chains
that may be able to stabilize smaller aggregates at pH 4.0.
DLS and turbidimetry analysis shown in Figures 5, 6, and
Table 2 confirmed this hypothesis. Turbidimetry curves indi-
cated that the incorporation of an increasing amount of
PEGMA within the polymer structure resulted in a lower
absorbance of the solution at pH 4.0. As a result, the copoly-
mer with 10 mol% of PEGMA is transparent independently
of the solution pH. Similarly, DLS shows that the relaxation
times decreased by incorporation of PEGMA.
Synthesis and Catalytic Evaluation of
the Grafted Copolymers
As shown in Table 1 and Figure 3, the size of the aggregates of
the alternating copolymer poly(VPcprol-alt-PMI) at pH 4.0 are
larger than 1 micron. As it was mentioned in the Introduction,
this rather large size may hinder the diffusion of reactants. To
obtain smaller aggregates the macromer PEGMA was included
in the reaction at molar percentages of 2%, 5%, and 10%.
Spectra of the protected and deprotected copolymers can be
found at Supporting Information (Fig. S1). The copolymer com-
positions were calculated from the 1H NMR spectra as indicat-
ed in the Experimental section, and are quoted in Table 2. The
molar fractions were found to be very close to the feed mono-
mer compositions, which indicate a proper incorporation of
the three monomers to the macromolecular chains. Molecular
weights of the copolymers were also characterized by GPC in
As a result, the size of the objects formed in solution at pH
4.0 is below than 21 nm incorporating only 2% of PEGMA. It
is interesting to note, that the average diameters values
observed comprised between 13 and 21 nm are in the range
of micelle-type entities.
Thus, most probably the long PEG chains of the PEGMA form
the shell of micelles in which proline and VP form the core
of the aggregates.
The catalytic studies with these grafted copolymers at pH 4.0
showed a clear higher reaction rate and a decrease on stereo-
selectivity, when compared with the use of Poly(VPcprol-
alt-PMI) (see Table 3). Both effects are related to the smaller
size of the aggregates that, on one side increases the reaction
rate but on the other permit the presence of water molecules
near the catalytic center.
TABLE 3 Aldol Reaction Between x and y Using Copolymers
Poly(VPcprol-co-PMI-co-PEGMA)-X% as Catalyst (30 mol%) at
pH 4.0 and 4 h of Reaction
Conv.
(%)a,b
Anti/
synb
ee (%)b
CONCLUSIONS
Catalyst
These studies have confirmed that the catalytic action of sup-
ported proline is efficient in water when there is “hydrophobic
activation” through the formation of linear and water soluble
(above pH 5.0) alternating copolymers of hydrophobic and
proline-containing units, in agreement with previous studies
in literature. As the alternation optimizes the hydrophobic
nature of the proline surroundings, it is proposed that the
“hydrophobic activation” is maximized. At pH 4.0, very close to
the IEP and to a scenario of charge cancellation, the copolymer
precipitates forming micrometric aggregates and it is able to
Poly-(VPcprol-alt-PMI)
24
93
5.5
1.8
83
5
Poly-(VPcprol-co-PMI-
co-PEGMA)22%
Poly-(VPcprol-co-PMI-
co-PEGMA)25%
96
97
2
1
3
Poly-(VPcprol-co-PMI-
co-PEGMA)210%
1.4
a
Determined by 1H NMR.
Determined by HPLC.
b
8
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9 E. G. Doyagu€ez, G. Corrales, L. Garrido, J. Rodriꢀguez-
Hernaꢀndez, A. Gallardo, A. Fernaꢀndez-Mayoralas, Macromole-
cules. 2011, 44, 6268–6276.
form enantioselective products, which has been related to the
exclusion of water as it has also been indicated previously in
literature. The grafting of PEG chains to the linear copolymers
has reduced the size of the aggregates to 15–22 nm, which are
compatible with micelle-type entities. Grafting increased the
reaction rate, which must be related to the reduction of the
aggregate size and the easier reactants diffusion. However,
enantioselectivity was lost, in agreement with the presence of
water molecules in the active center.
10 A. Lu, P. Cotanda, J. P. Patterson, D. A. Longbottom, R. K.
O’Reilly, Chem. Commun. 2012, 48, 9699–9701.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge support from the grants
ref. MAT-2010-20001, MAT2015-65184-C2-2-R and MAT2013-
42957-R (Ministry of Economy and Competitiveness-MINECO).
A. del Prado acknowledges the Ministry of Economy and Com-
petitiveness, Spain, for the FPI grant. R. Navarro gratefully
acknowledges funding from the Spanish Research Council (CSIC)
and European Social Fund (ESF) through the JAE-doc program.
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