Chiral recognition of macromolecules with cyclodextrins: pH- and thermosensitive copolymers from N-isopropylacrylamide and N-acryloyl-D/L- phenylalanine and their inclusion complexes with cyclodextrins

Article abstract of DOI:10.3762/bjoc.7.27

In the present work we report the enantioselective recognition of water soluble stimuli-responsive polymers bearing phenylalanine moieties via host-guest interaction with β-cyclodextrin and randomly-methylated-β- cyclodextrin (RAMEB-CD). We synthesised N-

Full text of DOI:10.3762/bjoc.7.27

Chiral recognition of macromolecules with
cyclodextrins: pH- and thermosensitive copolymers
from N-isopropylacrylamide and
N-acryloyl-D/L-phenylalanine and their
inclusion complexes with cyclodextrins
Sabrina Gingter, Ella Bezdushna and Helmut Ritter*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2011, 7, 204–209.
Institute of Organic Chemistry and Macromolecular Chemistry,
Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, D-40225
Düsseldorf, Germany
doi:10.3762/bjoc.7.27
Received: 11 November 2010
Accepted: 09 January 2011
Published: 14 February 2011
Email:
Helmut Ritter* - h.ritter@uni-duesseldorf.de
Editor-in-Chief: J. Clayden
*
Corresponding author
©
2011 Gingter et al; licensee Beilstein-Institut.
Keywords:
License and terms: see end of document.
amino acid; chiral recognition; cyclodextrins; LCST; stimuli-responsive
polymers
Abstract
In the present work we report the enantioselective recognition of water soluble stimuli-responsive polymers bearing phenylalanine
moieties via host-guest interaction with β-cyclodextrin and randomly-methylated-β-cyclodextrin (RAMEB-CD). We synthesised
N-acryloyl-D/L-phenylalanine monomers (2D, 2L) which were then copolymerised under free radical conditions with N-isopropyl-
acrylamide (NIPAAm). The resulting copolymers 3D and 3L exhibit a lower critical solution temperature (LCST) of 25 °C. As a
further benefit, the presence of a free carboxylic group in the copolymer system gives a high sensitivity to the pH value in respect to
the LCST value. The enantioselective recognition of the side groups of copolymers 3D and 3L and their solubility behaviour were
investigated by dynamic light scattering and 2D NMR spectroscopy, respectively.
Introduction
Chiral recognition has attracted much interest not only as a sep- chiral polymeric systems by use of cyclodextrins. One of the
aration technique but also in the pharmaceutical industry where first published methods was the enantioselective inclusion of
the supply of pure enantiomers for drug purposes is of great isotactic polylactides with cyclodextrin (CD) rings [2].
importance [1]. Thus, it is inherently important to find reliable
recognition systems for chirality in various kinds of molecules. Cyclodextrins (CDs) are cyclic oligosaccharides comprising six,
To date only a few papers exist dealing with recognition of seven or eight α-1,4 linked chiral glucopyranose units. They
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Beilstein J. Org. Chem. 2011, 7, 204–209.
easily include suitable molecules with hydrophobic segments
3-6]. Due to their asymmetric centres, CDs have the ability to
[
discriminate between suitable enantiomers. They form dia-
stereomeric complexes with slightly different stabilities [5-10].
Nevertheless, these small energy differences are usually suffi-
cient to achieve an adequate chromatographic resolution [11].
These small energy differences can even be detected by thermal
titration or spectroscopic methods, which have been achieved,
e.g., for natural amino acids or their N-protected derivatives
[
12-14]. In this connection we have recently shown that in
CD-complexed racemic mixture of N-methylacrylamidophenyl-
alanine, only the D-enantiomer is preferably polymerised,
whereas the L-enantiomer accumulates in the solution [15].
We also showed recently a chiral recognition of polymer at-
tached tryptophan with CD [16]. In this paper we now wish to
report the chiral recognition of polymeric attached
phenylalanine (Phe) with randomly-methylated-β-CD
(
RAMEB-CD) and native β-CD.
Scheme 1: Synthesis of copolymers 3D, 3L and CD-complexes 4D, 4L.
Results and Discussion
Copolymers 3D and 3L comprising N-isopropylacrylamide example the case of 2D where interactions of protons from the
(
NIPAAm), N-acryloyl-D-phenylalanine (2D) and N-acryloyl-L- phenyl group with protons of the CD cavity are shown, while no
phenylalanine (2L) were copolymerised under free radical interactions with protons of the outer rim are apparent. Further-
conditions with NIPAAm in a molar ratio of 1:20 and 1:10. more, there is a second interaction of the protons of the double
Compounds 2D or 2L were obtained under Schotten–Baumann bond of 2D with the CD cavity. Therefore, it can be concluded,
conditions using acryloyl chloride. Both copolymers were that the complex composition with 2D or 2L has a stoi-
prepared under similar conditions (Scheme 1). The molar chiometry of 1:2.
weight distribution of polymers 3D and 3L were determined by
gel permeation chromatography (GPC) after silylation of the To assure for further investigations that the complexation also
free carboxylic groups with trimethylchlorosilane.
takes place in the copolymer, additional ROESY experiments
were carried out (Figure 2).
The resulting copolymer 3D, 3L (1:20) is soluble in water below
the critical solution temperature (LCST). However, the pres- The NMR experiments clearly show that the complexation takes
ence of D- or L-Phe significantly affects the LCST value of place in monomer and polymer solutions, for both enantiomers
pure poly(NIPAAm) (32 °C), since the hydrophilic/hydro- (also see Supporting Information File 1).
phobic balance of the polymer is changed [17,18]. As noted
above, incorporation of the hydrophobic phenylalanine moieties Furthermore, both a shift of proton signals and the duplication
into the polymer reduces the LCST to 25 °C for the 3D, 3L of proton signals can be detected when the spectra of the com-
(
1:20) copolymer, whereas the 3D, 3L (1:10) copolymer is rela- plex is compared to the pure compounds 2D and 2L (Figure 3).
tively hydrophobic and not water-soluble. Surprisingly, the
polymers 3D, 3L (1:10) become water-soluble due to host–guest Figure 3 shows the specific shifts of signals, which occur on
interaction with RAMEB-CD yielding 4D and 4L.
complexation. CDs being chiral molecules themselves effect a
downfield shift of 0.1 ppm of the aromatic proton signal since
To confirm the formation of the proposed inclusion complexes the protons are magnetically shielded by the CD cavity. Addi-
between the copolymers and CD, which was added in excess, tionally, each of the aromatic and double bond proton signals,
the corresponding monomers 2D and 2L were used as models to respectively, change from singlets to doublets on complexation.
prove complexation. Thus, 2D ROESY NMR spectroscopy was Obviously, the supramolecular structure changes and former
performed to show the correlation between the protons of the magnetically identical protons become non-equivalent protons
β-CD and the protons of the guest 2D. Figure 1 indicates as an and appear as doublets. This can easily be observed in case of
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Beilstein J. Org. Chem. 2011, 7, 204–209.
Figure 1: 2D NMR ROESY spectrum of monomer 2D with RAMEB-CD.
Figure 2: 2D NMR ROESY experiment showing the correlation between protons of the phenyl moiety of 4D with the β-CD protons.
aromatic protons at 7.31–7.14 ppm and the proton signal of the unffected. The described shift of proton signals can be detected
double bond at 5.6 ppm and 6.0 ppm, respectively. The signal for both enantiomers, but with different intensities as can be
of the proton next to the double bond (6.3 ppm) remains observed in Figure 4.
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Beilstein J. Org. Chem. 2011, 7, 204–209.
of colour. Taking this into account, the addition of a compet-
itive guest molecule should displace the included phen-
olphthalein and turn the colour of the solution from colourless
to pink. Accordingly, the addition of 2D followed by stirring for
one hour caused the solution to regain its characteristic pink
colour as Figure 5 shows.
Figure 3: NMR shifts of the complexed monomer 2D.
Figure 5: Complex formation with phenolphthalein and phenylalanine
as competitor.
To prove further the influence of complexation of chiral poly-
mers 3D and 3L with chiral RAMEB-CD, we employed
dynamic light scattering to measure particle size and hydro-
dynamic diameters of the supramolecular structures (Figure 6).
Evidently, the 3D and 3L copolymers possess nearly identical
mean coil sizes as expected from the fact that the synthesis of
both polymers was carried out under nearly identical conditions.
However, the chiral RAMEB-CD causes hydrodynamic
Figure 4: Comparison of 1H NMR spectra of 2D and 2L complexed
with β-CD.
As expected, we found that 1H NMR spectra of complexed diameters which are affected in dramatically different ways for
compounds 2D and 2L with β-CD show differences in the split- the polymer attached enantiomers.
ting pattern of the proton signals (Figure 4). Therefore we
conclude that the complex stability is different for 2D and 2L Conclusion
and 4D and 4L, respectively.
In this work, we have succeeded in showing enantioselective
recognition of copolymers containing D- or L-phenylalanine
To further prove the complexation process of β-CD as host and moieties by use of DLS and NMR spectroscopy. The results
the Phe derivatives 2D and 2L as guests, we visualized the com- clearly indicate that enantioselective recognition of the poly-
plexation utilising phenolphthalein [19], i.e., the complexation meric attached chiral amino acid takes place due to host–guest
and decomplexation of a dye with β-CD in basic medium can be interaction with CD. The D-enatiomer of polymeric attached
employed as a method to prove these processes [20]. In basic Phe shows a larger increase in hydrodynamic diameter than the
media, phenolphthalein exhibits its characteristic pink colour, L-enantiomer on CD-complexation. In addition, differences in
caused by its planar configuration and electron delocalisation. the splitting pattern of 1H NMR spectra of CD-complexed
On adding cyclodextrin to the solution, phenolphthalein monomers were detected. The complexed 2D exhibits a
becomes colourless in the complexed state. Hydrogen bond for- distinctive splitting pattern compared to 2L. We also demon-
mation is accompanied with a conformational change as the strated that this complexation of phenylalanine containing
planar state of phenolphthalein is reversed which leads to loss polymer can be visualised by the use of phenolphthalein.
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Beilstein J. Org. Chem. 2011, 7, 204–209.
scale relative to tetramethylsilane was calibrated to the solvent
value δ = 4.79 ppm for D2O and 2.51 ppm for DMSO-d6.
Copolymer compositions were determined by 1H NMR.
Infrared (IR) spectra were recorded on a Nicolet 6700 FT-IR
spectrometer equipped with a diamond single bounce ATR
accessory at room temperature. Turbidity measurements were
determined using a TP1 turbidity photometer over a tempera-
ture range of 15 to 70 °C. During continuous stirring, the trans-
parency of the sample was determined by a voltage controlled
semiconductor laser and a silicium-photodiode at a wavelength
of 500 nm and a heating or cooling rate of 1 °C·min−1. All crit-
ical temperatures were detected by determination of the
temperature where the transparency of the solution was 50% of
the initial value. The hydrodynamic diameters of the copoly-
mers were determined by dynamic light scattering (DLS) in
backscattering mode on a Malvern Zetasizer Nano ZS ZEN
3
600 with a laser wavelength of 633 nm and a detection angle
of 173°. Measured solutions contained 1 mg/mL substance in
water and were performed in a polystyrene cuvette with a layer
thickness of 1 cm. Measurement results are calculated by the
non-negative least-squares (NNLS) algorithm. Depending on
measurement, number- or volume-averaged diameters are used
for characterization. Each experiment was performed at least
five times. Molecular weights and molecular weight distribu-
tions were measured by size exclusion chromatography (SEC)
with a Viscotek GPCmax VE2001 system that contained a
column set with one Viscotek TSK guard column HHR-H
Figure 6: Hydrodynamic diameters of the copolymers and their corres-
ponding complexes with RAMEB-CD a) L-phenylalanine containing
copolymer b) D-phenylalanine containing copolymer (1 mg/mL, pH 4).
6
7
.0 mm (ID) × 4 cm (L) and two Viscotek TSK GMHHR-M
.8 mm (ID) × 30 cm (L) columns at 60 °C. N,N-Dimethylform-
Experimental
Materials
amide (DMF, 0.1 M LiCl) was used as eluent at a flow rate of
1 mL·min−1. A Viscotek VE 3500 RI detector and a Viscotek
All reagents used were commercially available (Aldrich Co, Viscometer model 250 were used. The system was calibrated
Acros Organics) and were used without further purification. with polystyrene standards with a molecular range from 580 D
Randomly methylated β-cyclodextrin (RAMEB-CD) and to 1,186 kD.
β-cyclodextrin were obtained from Wacker Chemie GmbH,
Burghausen, Germany, and were used after drying overnight Polarimetry measurements were performed at T = 20 °C in
under vacuum (oil pump) in the presence of P4O10. D- and 0.1 N sodium hydroxide solution (λ = 590 nm).
L-phenylalanine (98.5%) were purchased from Alfa Aesar
GmbH & CoKG, Germany. Acryloyl chloride (97%) and N-iso- Synthesis of D/L-acryloylphenylalanine 2D, 2L
propylacrylamide (NIPAAm, 97%) were obtained from Aldrich, 3 g (18 mmol) D/L-phenylalanine were dissolved in 30 mL of
Germany, and used as received. Azobisisobutyronitrile (AIBN) sodium hydroxide solution 1.44 g (3.6 mmol) and 1.8 mL
(
96%) and N,N-dimethylformamide (DMF) were purchased (18 mmol) of acryloyl chloride were added dropwise with stir-
from Fluka, Germany. Dimethylsulfoxide-d6 (99.9 atom % D) ring at 0 °C. The mixture was stirred for 3 h at 0 °C and then at
and deuterium oxide D2O were obtained from Deutero GmbH, 25 °C overnight. The product was then precipitated. To
Germany.
complete the precipitation, the solution was acidified with
00 mL of ice cold 1 N HCl, the solid separated by filtration,
washed with water and lyophilised.
1
Measurements
1
H NMR was performed using a Bruker Avance DRX 500
spectrometer operating at 200.13 MHz or 500 MHz for protons Yield: 50%, FT-IR (diamond cm−1): ν = 3341(NH amide), 1709
in DMSO-d6 or deuterium oxide (99.9%) as solvents. The δ (C=O), 1648 (C=O amide I), 1594 (Ar), 1532 (NH amide II),
208

Beilstein J. Org. Chem. 2011, 7, 204–209.
6
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495, 1455, 1437, 1251, 1222, 1112, 1065, 990, 914, 1H NMR
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.30 (dd, J = 10.0, 17.1 Hz, 1H), 6.06 (dd, J = 2.4 Hz, 17.1 Hz,
H), 5.60 (dd, J = 2.4 Hz, 9.9 Hz, 1H), 4.53 (J = 4.9 Hz, 9.5 Hz,
H), 3.12 (dd, J = 4.9 Hz, 13.8 Hz, 1H), 2.91 (dd, J = 9.6 Hz,
3.8 Hz, 1H), Polarimetry: λ = 590 nm, T = 25 °C, 2D [α]D20 =
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2
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.2 g (54 mmol) of N-isopropylacrylamide (NIPAAm) were
1
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3
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doi:10.1002/(SICI)1522-2683(19990901)20:13<2614::AID-ELPS2614>
3
.0.CO;2-Q
(
AIBN) was dissolved in 2 mL of DMF and the two solutions of
1
5.Schwarz-Barac, S.; Ritter, H.; Schollmeyer, D.
NIPAAm and AIBN were divided equally and added to the two
flasks containing the monomer solution. The flasks were then
flushed with nitrogen for at least 15 min. Both flasks were
heated in the same oil bath for 23 h at a constant temperature of
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0 °C, the reactions stopped and the products obtained by addi-
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products were purified by dialysis in a micropore 1000. Yield:
3
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0%, 1H NMR (500 MHz, DMSO-d6) δ (ppm) 8.06–6.49 (m,
H), 3.84 (s, 10H), 2.14 (dd, J = 365.4 Hz, 367.1 Hz, 9H)
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doi:10.1021/ma100812q
1
1
713 (C=O), 1641 (NH amide II), 1538 (Ar), 1457, 1386, 1256,
172, 1095, SEC (DMF): Mn (3L) 16000 g/mol; PDI (3L) 3.6,
Mn (3D) 7000 g/mol, PDI (3D) 2.
License and Terms
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Creative Commons Attribution License
Supporting Information
Supporting Information File 1
(http://creativecommons.org/licenses/by/2.0), which
Additional 2D NMR ROESY and 1H NMR data.
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-7-27-S1.pdf]
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
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