Article pubs.acs.org/Langmuir
Supramolecular Interaction-Assisted Fluorescence and Tunable Stimuli-Responsiveness of L‑Phenylalanine-Based Polymers Mridula Nandi,†,‡ Binoy Maiti,†,‡ Kambalapalli Srikanth,‡ and Priyadarsi De*,†,‡,§ †
Polymer Research Centre, ‡Department of Chemical Sciences, and §Centre for Advanced Functional Materials, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246 Nadia, West Bengal, India
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S Supporting Information *
ABSTRACT: Supramolecular host−guest interactions between randomly methylated β-cyclodextrin (RM β-CD) and side-chain phenylalanine (Phe) and Phe−Phe dipeptide-based homopolymers have been employed for the amplification of fluorescence emission of otherwise weakly fluorescent amino acid Phe. The host−guest complex has been characterized by 1 H and 13C NMR spectroscopy, two-dimensional rotatingframe overhauser spectroscopy, Fourier-transform infrared spectroscopy, UV−visible spectroscopy, and fluorescence spectroscopy. To gain insights into the origin of fluorescence in homopolymers, density functional theory calculations were performed where phenyl moieties inside the less polar core of β-CD were observed to form a π−π coupled complex resulting in an enhanced emission. Furthermore, the complex-forming ability of Phe, the guest molecule, has been employed in tuning the cloud point temperature (TCP) of statistical copolymers derived from side-chain Phe/Phe−Phe-based methacrylate monomers and N-isopropylacrylamide. By varying the co-monomer feed ratios in the statistical copolymer and hence the concentration of RM β-CD throughout the polymer chain, host−guest interaction-assisted broad tunability in TCP of the supramolecular polymeric complex has been achieved.
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INTRODUCTION The cooperative association between supramolecular chemistry and polymers have led to the fabrication of smart materials with well-defined suprastructures and properties capable of mimicking biological systems.1,2 On account of associated reversibility, the polymer conformations, properties, and functions can be adjusted via host−guest chemistry.3 Among several supramolecular hosts available, cyclodextrins (CDs) are the most intriguing molecules because of their good water solubility and commercial availability, compatibility with a wide range of hydrophobic guests, and ability to recognize selectively different guests, such as aromatic, aliphatic, and chiral molecules.4,5 CDmodified polymers, via host−guest interactions, have been used to develop molecular systems with specific functions in fields spanning water management,6 sensing,7 as well as biomedicine.8 Formation of an inclusion complex between host and guest moieties significantly alters the solubility9,10 and spectral properties11,12 of the hydrophobic guest molecules. Considerable attention has been paid on the use of CDs as a means of improving aqueous solubility and spectrofluorometric methods for the identification of various analytes.13 The altered microenvironment around a sequestrated hydrophobic guest inside the CD cavity prevents any nonradiative or fluorescence quenching process, thereby aiding in enhancement of fluorescence emission.14,15 For instance, complexation behavior of several dyes such as acridine red, fluorescein, crystal violet, and so forth with CDs and calix[n]arenesulfonates as molecular receptors revealed an increase in the fluorescence intensity © 2017 American Chemical Society
upon complexation with CDs while a decrease in the fluorescence intensity with the latter.16 A study by Al-Hassan et al.17,18 established that the orientation of the probe inside the CD and the cavity size direct the intensity, emission wavelength, and nature of the spectra of the fluorescent probes. Recently, Li et al. reported a fluorescent supramolecular hyperbranched polymer based on host−guest interactions. Even though devoid of any conventional chromophore, the diethylenetriamine-based hyperbranched polymer could emit wide-band fluorescence on account of inclusion complexation.19 Additionally, “stimuli”-responsive polymeric materials are extensively investigated with the interest to fabricate systems capable of monitoring temperature and pH, owing to their importance in several fields of science and engineering.20,21 Among various thermoresponsive polymers, the most explored is poly(N-isopropylacrylamide) (PNIPAM) with a lower critical solution temperature (LCST) of 32 °C, that is, on increase in temperature, it undergoes transformation from soluble to insoluble state.22,23 Till date for tuning thermoresponsiveness, polymers have been modified into various statistical copolymers,24 block copolymers,25 hyperbranched polymers,26 and graft copolymers. 27 Hereof, supramolecular interactions between the host and the guest provide a facile alternative Received: July 14, 2017 Revised: September 15, 2017 Published: September 18, 2017 10588
DOI: 10.1021/acs.langmuir.7b02431 Langmuir 2017, 33, 10588−10597
Article
Langmuir Scheme 1. Synthesis of Amino Acid/Dipeptide-Based Random Copolymers by RAFT Polymerization, Followed by Deprotection of the Boc Group
polymer chain.42 By varying the co-monomer feed ratios in the statistical copolymer and hence the concentration of RM β-CD throughout the polymer chain, host−guest interaction-assisted broad tunability in TCP of the dual thermo- and pH-responsive polymer has been achieved.
for tuning the phase transition temperature (also known as cloud point temperature (TCP)) of thermoresponsive polymers,28 eliminating otherwise tedious and extensive ways of copolymer preparation. Cavitands with hydrophilic exterior and hydrophobic cavity, such as CDs, encapsulate hydrophobic guests in thermoresponsive polymer chains and increase hydrophilicity and hence phase transition temperature. Consequently, several cavitands such as cucurbit[n]urils,29 pillar[n]arenes,30 calixarenes,31 cyclobis(paraquat-p-phenylene),32 crown ethers,33 as well as CDs have been investigated for supramolecular control over thermoresponsiveness. Covalent attachments of β-CD to the polymer main chain,34 as end groups,35 or through noncovalent36 attachment to the polymer chain have been studied for precise tuning of TCP. Although the above-mentioned systems were successful in altering LCST, most of them actually resulted in decrease in TCP of the thermoresponsive polymer. Moreover, there are very few systems with broad tunability of TCP reported in the literature that can function as sensors.37,38 Even though host−guest interaction between the phenylalanine (Phe)-based polymer and CDs has been reported,39,40 alteration in its spectral properties on complexation or its ability to tune the LCST of the thermoresponsive polymer on complexation has not been given much attention because hydrophobicity associated with the phenyl ring leads to decreased solubility in aqueous solution or reduction in TCP. Among the intrinsic fluorophores in proteins, aromatic Phe is weakly fluorescent with a very low molar extinction coefficient and quantum yield.41 Herein, we present a strategy for enhancing the fluorescence of the side-chain Phe-based homopolymer via the host−guest interaction with randomly methylated β-CD (RM β-CD). Further increased solubility of Phe/RM β-CD or dipeptide (Phe−Phe)/RM β-CD complex has been employed to tune the TCP of the thermoresponsive supramolecular polymeric complex from statistical copolymers derived from side-chain Phe/Phe−Phe-based methacrylate monomers and NIPAM. The side-chain amino acid/dipeptide-containing polymers are particularly advantageous because their synthesis involves a relatively straightforward method and free amine functionalities in the side chains undergo reversible protonation/deprotonation incurring pH responsivity to the
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EXPERIMENTAL SECTION
Materials. Trifluoroacetic acid (TFA, 99.5%) and tert-butyloxycarbonyl (Boc)-L-phenylalanine (Boc-F-OH, 99%) were obtained from Sisco Research Laboratories Pvt. Ltd., India. RM β-CD (≥97%) was bought from Tokyo Chemical Industries Co. Ltd., Japan. 4Dimethylaminopyridine (99%), anhydrous N,N-dimethylformamide (DMF, 99.9%), dicyclohexylcarbodiimide (99%), and 2-hydroxyethyl methacrylate (97%) were purchased from Sigma and used without any further purification. NIPAM (Sigma-Aldrich, 97%) was recrystallized twice from a mixture of toluene and hexanes prior to polymerization. 2,2′-Azobisisobutyronitrile (AIBN) (Sigma, 98%) was recrystallized twice from methanol. CDCl3 (99.8% D) and methanol-d4 (CD3OD, 99.8% D) from Cambridge Isotope Laboratories, Inc., USA were used for NMR study. The solvents such as hexanes (mixture of isomers), methanol (MeOH), acetone, ethyl acetate, tetrahydrofuran (THF), and dichloromethane (DCM) were purified by standard distillation procedures. The reversible addition fragmentation (RAFT) chain transfer agent (CTA) 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (CDP) was synthesized following a previously reported procedure.43 Boc-phenylalanine methacryloyloxyethyl ester (Boc-F-EMA)44 and Boc-Phe-Phe-oxyethyl methacrylate (Boc-FFEMA)45 were prepared by previously established synthetic procedures from our group. Characterization Techniques. Two-dimensional (2D) rotatingframe overhauser spectroscopy (ROESY) and 1H and 13C NMR spectra were obtained either on a Bruker AVANCE III 500 MHz spectrometer or on a JEOL 400 MHz spectrometer at 25 °C. The number-average molecular weight (Mn) and molecular weight distributions (dispersity (D̵ )) of homopolymers were determined in THF by a Waters gel permeation chromatography (GPC) instrument consisting of a model 515 high-performance liquid chromatography pump, a model 2414 refractive index (RI) detector, and two columns (PolarGel-M guard column (50 × 7.5 mm) and PolarGel-M analytical columns (300 × 7.5 mm)) at 30 °C. The THF mobile phase contained 0.05 w/v % butylhydroxy toluene, and the flow rate was fixed at 1.0 mL/min. A series of 10 near monodisperse poly(methyl methacrylate) (PMMA) standards (peak average molecular weight (Mp) values ranging from 1280 to 199 000 g/mol) were used to calibrate the instrument. For copolymers, Mn and D̵ values were determined in the 10589
DOI: 10.1021/acs.langmuir.7b02431 Langmuir 2017, 33, 10588−10597
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Langmuir
Table 1. Results from the Synthesis of Homopolymers (PF and PFF) and Various Copolymers from the RAFT Polymerization of Boc-F-EMA/Boc-FF-EMA with NIPAM in DMF at 70 °C polymer
[Boc-F/FF-EMA]/[NIPAM]
time (min)
conv.c (%)
% of Boc-F/FF-EMA in copolymerd
Mn,NMRd (g/mol)
Mn,GPCe (g/mol)
D̵ e
Mn,theof (g/mol)
PFa PFN5b PFN10b PFN15b PFFa PFFN5b PFFN10b PFFN15b
100:0 5:95 10:90 15:85 100:0 5:95 10:90 15:85
210 210 210 210 300 300 300 300
60 96 92 95 53 95 88 89
1.00 0.03 0.08 0.14 1.00 N.D 0.09 N.D
7000 11 600 12 000 15 400 8000 N.D 13 500 N.D
5800 15 000 16 700 17 500 5500 14 500 18 200 19 000
1.12 1.2 1.17 1.15 1.15 1.3 1.15 1.15
6100 12 500 13 000 14 900 7300 13 000 15 000 16 000
a
[Monomer]/[CDP]/[AIBN] = 25/1/0.1. b[Monomer]/[CDP]/[AIBN] = 100/1/0.1. cDetermined gravimetrically. dDetermined from the 1H NMR study. eMeasured by GPC using PMMA standards. fTheoretical number-average molecular weights; Mn,theo = (([monomer]/[CDP] × average molecular weight (MW) of the monomer × conv.) + (MW of CDP)). N.D: not determined. DMF solvent at 40 °C. Fourier-transform infrared (FT-IR) spectra were recorded on a PerkinElmer spectrum 100 FT-IR spectrometer on KBr pellets. UV−visible spectroscopic measurements were carried out on a PerkinElmer LAMBDA 35 spectrophotometer. For cloud point determination, a UV−vis spectrophotometer was coupled to a Peltier Temperature Programmer for temperature control. Fluorescence measurements were carried out using a FluoroMax-3 spectrofluorometer from HORIBA Jobin Yvon. Theoretical calculations were performed using the Gaussian 09 program.46 The structure and orientation of the polymer−β-CD complex were optimized using the B97D functional47 and 6-31G(d)48,49 basis set of density functional theory (DFT) methods. Homopolymer Synthesis. A typical RAFT polymerization was carried out as follows: Boc-F-EMA (400 mg, 1.61 mmol), CDP (17.1 mg, 0.04 mmol), AIBN (0.7 mg, 4.24 μmol; 44.9 mg stock solution of 5.6 mg AIBN in 503.2 mg DMF), and DMF (1.6 g) were taken in a 20 mL septa sealed vial equipped with a small magnetic stir bar, purged with dry nitrogen for 20 min and placed in a preheated reaction block at 70 °C. The polymerization was stopped by cooling the vial in an icewater bath and exposing the reaction mixture to air. After quenching, the reaction mixture was diluted with acetone and precipitated from hexanes. The corresponding homopolymer poly(Boc-F-EMA) (PF) was further reprecipitated around five times from acetone/hexanes and dried under high vacuum for 8 h at 35 °C to afford faint yellowish polymers. Monomer conversion was determined gravimetrically from the initial weight of the monomer taken and the weight of the purified polymer. A similar procedure was followed for the synthesis of the side-chain dipeptide-based polymer, poly(Boc-FF-EMA) (PFF) (Scheme S1). Copolymer Synthesis. A typical RAFT copolymerization process was as follows: Boc-F-EMA (104.5 mg, 0.28 mmol), NIPAM (595.5 mg, 5.26 mmol), CDP (22.4 mg, 0.06 mmol), AIBN (1.0 mg, 5.5 μmol, from stock solution), and DMF (2.8 g) were taken in a 20 mL septa sealed glass vial equipped with a small magnetic stir bar, purged with dry nitrogen for 20 min and placed in a preheated reaction block at 70 °C. After predetermined time, the reaction was stopped, copolymers were purified, and monomer conversions were determined following a procedure similar to those of homopolymers. A similar procedure was followed for the synthesis of copolymers of NIPAM and Boc-FF-EMA (Scheme 1). Deprotection of Boc-Protected Polymers. Typically, to a homogeneous solution of PF (0.16 g) in 1.0 mL of DCM, 0.5 mL of TFA was added dropwise under ice-water bath conditions. The solution was kept stirring for 2 h at room temperature. The Bocdeprotected polymer (DPF) was precipitated in diethyl ether and dried under vacuum for 8 h at 35 °C. Similarly, PFF was used to prepare the corresponding Boc-deprotected copolymer, DPFF. Also, various copolymers were deprotected under acidic conditions at room temperature. Preparation of the Supramolecular Inclusion Complex. For inclusion complex formation, to a homogeneous solution of DPF (50.0 mg, 0.13 mmol) in 1.0 mL of acetone taken in a 20 mL vial, a solution
of RM β-CD (149.0 mg, 0.13 mmol) in 2.0 mL of acetone was added. Following continuous stirring for 7 days at room temperature, the solution was dialyzed against MeOH using a Spectra/Por dialysis membrane (2 kDa molecular weight cutoff) to remove excess RM βCD. During dialysis, MeOH was changed at least 5−7 times in 2−8 h interval. A yellowish solid complex was obtained after rotary evaporation and drying under vacuum for 8 h at 35 °C. Similarly, complexation of RM β-CD with DPFF and the copolymers was carried out following the above-mentioned procedure. Determination of Phase Transition Temperature. For the measurement of TCP, the % transmittance (% T) of aqueous solution of the copolymer (2.0 mg/mL) was recorded at wavelength (λ) = 500 nm within the temperature range of 20−75 °C. The temperature was gradually increased at the heating rate of 2−5 °C/min, and prior to each measurement, the sample was equilibrated for 5 min. The TCP is defined as the temperature where 50% reduction in % T of the polymer solution was observed.
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RESULTS AND DISCUSSION Synthesis of Homo- and Copolymers. Previously, we have demonstrated the effectiveness of CDP for controlled RAFT polymerization of Boc-F-EMA,44 Boc-FF-EMA,45 and NIPAM.50 Thus, in this study, we have used CDP to prepare homo- and copolymers in the presence of AIBN in DMF at 70 °C. Two series of copolymers with different compositions were prepared by varying the co-monomer feed ratios, that is, [NIPAM]/[Boc-F/FF-EMA]. However, [CDP]/[AIBN] and [monomer]/[CDP] ratios were kept constant at 1/0.1 and 100/1, respectively. Homopolymers were coded as PF, PFF, and PNIPAM, which were prepared from Boc-F-EMA, Boc-FFEMA, and NIPAM, respectively. Copolymers were named as PFN or PFFN, where F, FF, and N signify Boc-F-EMA, BocFF-EMA, and NIPAM, respectively (Table 1). The numbers 5, 10, and 15 after PFN or PFFN in Table 1 represent the F or FF monomer feed ratios. For example, a copolymer of Boc-FFEMA and NIPAM with [Boc-FF-EMA]/[NIPAM] = 5/95 feed ratio is designated as PFFN5. To represent polymers after Boc deprotection, the letter “D” was introduced before PFFN5 and written as DPFFN5. The homo- and copolymers were characterized by 1H NMR study. In the 1H NMR spectrum of PF, PFF, and their copolymers (Figures S1 and S2), characteristic resonance signals for the different types of protons in the repeating unit of the polymer are assigned. For the PF and PFF, the degree of polymerization (DPn) was determined by comparing the integration areas from the terminal −CH2−CH2− protons (from the HOOC−CH2−CH2−C(CN)(CH3)− chain end) at 2.4−2.6 ppm and the repeating unit protons at 4.1−4.5 ppm 10590
DOI: 10.1021/acs.langmuir.7b02431 Langmuir 2017, 33, 10588−10597
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Langmuir
Figure 1. 1H NMR spectra of (A) PF in CDCl3, DPF in CD3OD, and the complex of DPF with RM β-CD in CD3OD and (B) PFN10 in CDCl3 and its Boc-deprotected form DPFN10 in CD3OD.
(for O−CH2−CH2−O− and the chiral proton). Then, the number-average molecular weight (Mn,NMR) values of homopolymers were calculated using respective DPn and molecular weight of the monomers. Compositions of copolymers were calculated from their 1H NMR spectra by comparing the peak intensity of methylene proton (−CH2−Ph) at 3.2−3.0 ppm to the total peak area at 4.44−3.89 ppm for the −O−CH2−CH2− O− (4H), the chiral proton of the Boc-F-EMA segment, and the −CH(CH3)2 proton of the NIPAM unit. The −CH2− CH2− (4H) protons of HOOC−CH2−CH2−C(CN)(CH3)− chain ends were clearly observed at 2.42−2.54 ppm in the case of PFNx (x = 5, 10, and 15) copolymers and can be used to determine Mn,NMR by the chain-end analysis method. First, the average number of Boc-F-EMA units (DPn,F) in the copolymer was calculated from the comparison of peak areas at 3.2−3.0 and 2.42−2.54 ppm. Similarly, comparison of the integration areas from the terminal group (4H) at 2.42−2.54 ppm and peak areas at 4.44−3.89 ppm (after subtracting the peak area contributed by the PF units in this region) gives the average number of NIPAM units (DPn,N) in the copolymer. Thus, Mn,NMR values of copolymers were determined using the following equation: Mn,NMR = [(DPn,F × MF) + (DPn,N × MN) + molecular weight of CDP], where DPn,F/N and MF/N are the average number of the particular monomer unit in the copolymer and the molecular weight of the monomer, respectively. GPC RI traces (Figure S3) of the homopolymers and copolymers are symmetric and unimodal with narrow D̵ values (
