Diphenylpyrenylamine-functionalized polypeptides: secondary structures, aggregation-induced emission, and carbon nanotube dispersibility

In this study we prepared—through ring-opening polymerization of γ-benzyl-l-glutamate N-carboxyanhydride (BLG-NCA) initiated by N,N-di(4-aminophenyl)-1-aminopyrene (pyrene-DPA-2NH2)—poly(γ-benzyl-l-glutamate) (PBLG) polymers with various degrees of polymerization (DP), each featuring a di(4-aminophenyl)pyrenylamine (DPA) luminophore on the main backbone. The secondary structures of these pyrene-DPA-PBLG polypeptides were investigated using Fourier transform infrared spectroscopy and wide-angle X-ray diffraction, revealing that the polypeptides with DPs of less than 19 were mixtures of α-helical and β-sheet conformations, whereas the α-helical structures were preferred for longer chains. Interestingly, pyrene-DPA-2NH2 exhibited weak photoluminescence (PL), yet the emission of the pyrene-DPA-PBLG polypeptides was 16-fold stronger, suggesting that attaching PBLG chains to pyrene-DPA-2NH2 turned on a radiative pathway for the non-fluorescent molecule. Furthermore, pyrene-DPA-2NH2 exhibited aggregation-caused quenching; in contrast, after incorporation into the PBLG segments with rigid-rod conformations, the resulting pyrene-DPA-PBLG polypeptides displayed aggregation-induced emission. Transmission electron microscopy revealed that mixing these polypeptides with multiwalled carbon nanotubes (MWCNTs) in DMF led to the formation of extremely dispersible pyrene-DPA-PBLG/MWCNT composites. The fabrication of MWCNT composites with such biocompatible polymers should lead to bio-inspired carbon nanostructures with useful biomedical applications.


Introduction
Polypeptides are protein-like polymers composed of repeating a-amino acid residues connected through peptide bonds (-CONH-); they are readily degraded into the corresponding aamino acids in the human body. Polypeptides are attracting much attention for biomedical applications because of their excellent biodegradability and good biocompatibility in vivo; 1 for example, they may be used as gene vectors, 2 drug carriers, 3 and tissue engineering materials. 4 Notably, polypeptides can form stable hierarchically ordered structures, including rigidrod-like a-helical and b-sheet conformations, both in solution and in the solid state. 1,5 The a-helical and b-sheet conformations are stabilized primarily through intramolecular and intermolecular hydrogen bonds, respectively. 6 Among the polypeptides, poly(g-benzyl-L-glutamate) (PBLG) has been widely investigated as a synthetic polypeptide that degrades in vivo to L-glutamic acid, one of the essential amino acids for the human body. PBLG can be synthesized through ring-opening polymerization (ROP) of g-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) or multilateral peptide monomers. 5 Attractive properties can result when PBLG polypeptides are conjugated with other functional peptide monomers. 7 The continued design of new supramolecular polypeptides will inevitably lead to a diverse range of prospective applications.
Carbon nanotubes (CNTs) are unique one-dimensional (1D) structures that are composed essentially of sheets of carbon atoms that are arranged in hexagons and rolled into tubes. They can be categorized in two basic forms: single-wall carbon nanotubes (SWCNTs), which feature a single roll of hexagonal carbon atoms, and multiwall carbon nanotubes (MWCNTs), which are single tubes masked into wider tubes, which are also encased into other tubes. As a result of their p-electron systems, CNTs have unique mechanical, optoelectronic, and thermal properties. 8 Nanocomposites containing CNTs are receiving increasing attention for their potential applications in electronic devices, 9 high-performance composites, 10 sensors, 11 and biological materials. 12 Considerable efforts have been devoted to developing covalent and noncovalent techniques for controlling the aggregation of CNTs and improving their dispersion in various nanocomposites. 13 For example, CNTs covalently modied with polypeptides on their surfaces have been examined for their biological applications. 14 Yao et al. used the "gra-from" approach to synthesize polypeptide-modied MWCNTs (PBLG-MWCNT) through ROP of amino-functional MWCNTs with the BLG-NCA monomer. 15 In addition, the synthesis of polypeptide-modied SWCNTs (PBLG-SWCNT) has been performed through the addition of azido-terminated PBLGs to SWCNTs. 16 Nevertheless, such covalent modication can change the hybridization of the CNT's carbon atoms from sp 2 to sp 3 , possibly weakening the mechanical, electronic, or optical properties. 17 In contrast, noncovalent modication of CNTs involves the physical absorption of small surfactants or conjugated polymers to the CNT surface. This approach can enhance the dispersion of CNTs, while maintaining the mechanical and optoelectronic properties. The most common noncovalent modications involve mussel inspired surface and pyrene modications. In the former modication, MWCNTs with polydopamine (PDA) coating have been prepared via mussel inspired chemistry. The dopamine molecules are rstly self-polymerized into PDA under weakly alkaline aqueous conditions and then the formed PDA strongly adhered and coated on MWCNTs surface. Aer that the PDA coating can be further reacted with thiol, amino and acrylamide, acrylate and thiocarbonylthio-modied polymers through Michael addition reaction, 18 atom transfer radical polymerization (ATRP), 19 single-electron transfer living radical polymerization (SET-LRP), 20 and reversible addition-fragmentation chain transfer polymerization (RAFT) 21 to form surface-modied MWCNTs. However, this modication needs many steps, long reaction times. In the later modication, pyrene units are interacted strongly with the surface of CNTs through p-stacking, thereby producing homogenously dispersed pyrene/CNT composites. 22 This method has many further advantages include short reaction time, high yield, and operation simplicity. Highly dispersed polypeptide/CNT composites are of interest for their biophysical and biomedical applications.
Pyrene is a uorogenic unit displaying variable photophysical properties, making it useful as a uorophore for labeling in probes for nucleic acids 23 and metal ions. 24 The attractive properties of pyrene include its appearance of delayed uorescence, ready functionalization, high propensity for forming excimers, and distinct solvatochromic phenomena. In recent years, a great number of pyrene derivatives have been prepared, including oligothiophenes with pyrenyl side/end groups, 25 tetraphenylpyrene, 26 hexapyrenylbenzene, 27 ethynylene-conjugated pyrene, 28 and dipyrenebenzenes, 27 in addition to uorene-pyrene, carbazolepyrene, and uorene-carbazole-pyrene systems. 29,30 Furthermore, polypyrenes, 31 pyrene-starbursts, 32 and pyrene-dendrimers 33 have been investigated for organic electronic applications (e.g., organic light-emitting devices) that take advantage of their emissive properties. Moreover, alkyl pyrenes have been incorporated as photophysical probes into synthetic peptide skeletons; their photouorescence properties have been applied to investigate peptide conformations 34 and the tertiary and quaternary structures of peptides. 35 The structures and aggregation behavior of peptides containing pyrene units can be examined by monitoring the changes in their emission spectra. Although pyrene is a blue-emitting chromophore, it emits weakly when aggregated or at high concentrations, due to strong intermolecular p-stacking of these planar molecules; this phenomenon is known as aggregation-caused quenching (ACQ). 36 The aggregation of pyrene-containing peptides leads to changes in their emission spectra-namely, decreases in the molecular extinction coef-cients and red-shis of the absorption maxima. 37,38 Therefore, it would be interesting to discover new uorescent pyrene-containing peptides that emit blue light in the aggregation state, as well as in the solution, with high efficient quantum yield.
Triarylamines have been studied extensively for their high charge mobilities and excellent photonic and electronic properties, and have been applied as building blocks in the construction of light-emitters, hole-transporters, and photoconductors. 39 Among the developed triarylamines, triphenylamine (TPA) derivatives are uorescent in solution, but are less emissive when aggregated in the solid state; therefore, they are ACQ chromophores. 40,41 For example, tetraphenylbiphenyl-4,4 0 -diamine (TPA-dimer) exhibits a strong emission in tetrahydrofuran (THF), but the emission intensity is 5.5-fold lower in the solid state. Recently, we reported the incorporation of TPA into polytyrosine (PTyr) through the ROP of the NCA amino acid at room temperature. The emission intensity of TPA itself decreased upon increasing its concentration in THF-typical ACQ behavior. In contrast, the TPA emission transformed from ACQ to aggregation-induced emission (AIE) 42,43 aer the incorporation of TPA into the main chain of polytyrosine. 44 To the best of our knowledge, di(4-aminophenyl) pyrenylamine, which combines the structural features of pyrene and TPA, has never previously been incorporated into polypeptide main chains, despite the interesting properties of pyrene and TPA units. Herein, we report the use of N,N-di(4aminophenyl)-1-aminopyrene (pyrene-DPA-2NH 2 ) as an initiator for the synthesis of a series of pyrene-DPA-PBLG polypeptides formed through ROP of the BLG-NCA monomer (Scheme 1). We characterized the resulting polypeptides using Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass-analyzed laser desorption/ionization time-of-ight (MALDI-TOF) mass spectrometry. We studied of the secondary structures of these polypeptides using FTIR spectroscopy and wide-angle X-ray diffraction (WAXD). We also applied photoluminescence spectroscopy to investigate the uorescence properties of pyrene-DPA-2NH 2 and pyrene-DPA-PBLG. Moreover, we used transmission electron microscopy (TEM) and uorescence spectroscopy to investigate the interactions and dispersibility of our new polypeptides when complexed with multiwalled carbon nanotubes (MWCNTs).
In a 100 mL two-neck round-bottomed ask equipped with a stirring bar, a solution of 1-aminopyrene (1.5 g, 7.0 mmol) and K 2 CO 3 (3.8 g, 28 mmol) in DMSO (30 mL) was stirred under N 2 atmosphere for 10 min. 4-Fluoronitrobenzene (1.5 mL, 14 mmol) was added and then the mixture was heated at 140 C for 24 h under a N 2 atmosphere. The cooled mixture was poured into MeOH (200 mL) slowly; the precipitated product was ltered off and washed thoroughly with MeOH and hot water. The crude product was recrystallized (DMF/MeOH) to afford brown needles (  In a 100 mL two-neck round-bottomed ask equipped with a stirring bar, pyrene-DPA-2NO 2 (2.0 g, 4.4 mmol) and 10% Pd/C (0.10 g) were suspended in EtOH (60 mL) and THF (10 mL) under a N 2 atmosphere. The suspension was heated at 90 C for 15 min before hydrazine monohydrate (6.5 mL) was added slowly. The mixture was stirred at 90 C for 36 h and then it was ltered to remove the Pd/C. The ltrate was cooled, giving yellow crystals, which were ltered off and dried under vacuum at 70 C (1.35 g, 75%); mp: 227-229 C (DSC). FTIR (KBr, cm À1 ): 3206-3387 (N-H stretch). 1   Pyrene-DPA-PBLG BLG-NCA (1.0 g, 3.8 mmol) was weighed in a dry-box under N 2 , transferred to a three-neck round-bottom ask, and then dissolved in dry DMF (20 mL). The solution was stirred at 0 C for 15 min prior to the introduction of a solution of pyrene-DPA-2NH 2 (various ratios) in DMF (3 mL) using a N 2 -purged syringe. Aer stirring at 0 C for 72 h, the mixture was poured into diethyl ether (Et 2 O). The precipitate was puried three times through dissolution into MeOH and reprecipitation from Et 2 O, giving a pale-yellow powder that was dried under vacuum at 40 C overnight. Pyrene-DPA-PBLG(24): T g ¼ 24.2 C; FTIR (KBr, cm À1 ): 3304,3062,2959,1736,1653,1548,1449,1163,746,698,610

Dispersion of MWCNTs and pyrene-DPA-PBLG
MWCNTs were dispersed into DMF (5 mL) through sonication for 2 h. A solution of pyrene-DPA-PBLG in DMF (1 mL) was then added dropwise into the MWCNT dispersion. The mixture was sonicated for 2 h and then stirred at room temperature for 24 h. The mixture was centrifuged (5000 rpm, 60 min) and then the supernatant was subjected to ultraltration through PALL disc membrane lters (FP-450 PVDF lters) to give MWCNT/pyrene-DPA-PBLG composites. These composites were redispersed in various solvents through sonication for 2 min.
Characterization FTIR spectra were measured using a Bruker Tensor 27 FTIR spectrometer; samples were prepared using the KBr disk method; 64 scans were collected at room temperature at a spectral resolution of 4 cm À1 ; the sample lms were suitably thin to obey the Beer-Lambert law. 1 H and 13 C NMR spectra were recorded using an Agilent VMRS-600 NMR spectrometer at 600 and 150 MHz, respectively; CDCl 3 and DMSO-d 6 were used as solvents, and tetramethylsilane (TMS) as the external standard. The molecular weights of the synthesized polypeptides were obtained from MALDI-TOF mass spectra, recorded using a Bruker Daltonics Autoex III spectrometer (operating parameters: ion source 1, 19.06 kV; ion source 2, 16.61 kV; lens, 8.78 kV; reector 1, 21.08 kV; reector 2, 9.73 kV). Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were performed under a N 2 atmosphere using Q-20 and Q-50 thermogravimetric analyzers (TAs), respectively; for DSC, the samples were places in a sealed aluminum pan and heated from 40 to 200 C (heating rate: 10 C min À1 ); for TGA, the samples were heated from 30 to 800 C (heating rate: 20 C min À1 ). WAXD patterns were measured using the wiggler beam line BL17A1 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan; a triangular bent Si (111) single crystal was used to obtain a monochromatic beam having a wavelength (l) of 1.24Å; the samples were annealed at 180 C for 2 h, and then cooled to room temperature, prior to measurement. UV-Vis absorption spectra were recorded using an Ocean Optics DT 1000 CE 376 spectrophotometer. PL spectra were recorded using a Lab-Guide X350 uorescence spectrometer, with a 450 W Xe lamp as the continuous light source; a small quartz cell (dimensions: 0.2 Â 1.0 Â 4.5 cm 3 ) was used to adjust the solution sample. TEM images for the samples were recorded using a JEOL-2100 transmission electron microscope operated at an accelerating voltage of 200 kV.

Results and discussion
Synthesis of pyrene-DPA-2NH 2 Pyrene-DPA-2NH 2 , used as a monomer initiator for the ROP, was synthesized according to the synthetic strategy outlined in Scheme 1. First, the dinitro-compound (pyrene-DPA-2NO 2 ) was prepared through N,N-diarylation of 1-aminopyrene with 4-uoronitrobenzene in the presence of K 2 CO 3 at 140 C in DMSO. Reduction of pyrene-DPA-2NO 2 with hydrazine hydrate in the presence of a catalytic amount of 10% Pd/C in EtOH at 90 C provided the diamine-initiator compound pyrene-DPA-2NH 2 . FTIR and 1 H and 13 C NMR spectroscopic analyses conrmed the chemical structures of pyrene-DPA-2NO 2 and pyrene-DPA-2NH 2 . Fig. 1(A) and (B) present the FTIR spectra of pyrene-DPA-2NO 2 and pyrene-DPA-2NH 2 , respectively. The spectrum of pyrene-DPA-2NO 2 exhibits two sharp signals at 1302 and 1579 cm À1 for the NO 2 group, while the spectrum of pyrene-DPA-2NH 2 exhibits three sharp signals at 3206, 3302, and 3387 cm À1 for symmetric and asymmetric NH stretching. Fig. 2(A) and (B) present the 1 H NMR spectra of pyrene-DPA-2NO 2 and pyrene-DPA-2NH 2 , respectively. The spectrum of the dinitro compound features characteristic signals for nitro-substituted aromatic rings at 7.37 and 8.30 ppm, with signals for the pyrene protons in the range 7.94-8.46 ppm. The spectrum of the diamine featured a broad signal at 4.81 ppm, attributed to the NH 2 groups, in addition to signals for the aromatic protons. The 13 C NMR spectrum of pyrene-DPA-2NO 2 features [ Fig. 3(A)] a signal for the C-NO 2 unit at 142.35 ppm; for pyrene-DPA-2NH 2 [ Fig. 3(B)], the signal for the C-NH 2 unit appears at 144.25 ppm. These features are all consistent with the high-yield synthesis of the diamine-functionalized initiator pyrene-DPA-2NH 2 .

Synthesis of pyrene-DPA-PBLG
As illustrated in Scheme 1, pyrene-DPA-PBLG polypeptides, with two PBLG chains linked to a pyrene-DPA unit, were synthesized at room temperature through ROP of the BLG-NCA monomer initiated by the diamine pyrene-DPA-2NH 2 . The successful formation of these polypeptides was elucidated from their FTIR, 1 H and 13 C NMR, and MALDI-TOF mass spectra. The FTIR spectrum of BLG-NCA [ Fig. 1(C)] features two signals at 1787 and 1883 cm À1 , attributed to the two modes of anhydride C]O stretching, in addition to a signal for the ester at 1718 cm À1 . Aer polymerization of BLG-NCA [ Fig. 1(D) and S1 †], the two anhydride peaks disappeared and a new absorption appeared at 3299 cm À1 for NH stretching. In addition, absorption peaks emerged for the side chain ester, amide I, and amide II groups of pyrene-DPA-PBLG at 1728-1736, 1649-1653, and 1545-1548 cm À1 , respectively. Fig. 2(C) presents the 1 H NMR spectrum of BLG-NCA. The signals of the NH group and methylene protons (H d ) appeared at 6.78 and 5.13 ppm, respectively. The formation of pyrene-DPA-PBLG was conrmed from its 1 H NMR spectrum [ Fig. 2(D) and S2 †]: the aromatic protons (H a , H b ) of pyrene-DPA-2NH 2 (at 6.47 and 6.66 ppm) were shied downeld (to 6.56-6.61 and 7.56-7.75 ppm) aer formation of the PBLG chains. Moreover, signals for the pyrene moiety and methylene protons (H d ) were present in the spectrum of pyrene-DPA-PBLG. Fig. 3(C) displays the 13 C NMR spectrum of the BLG-NCA monomer; signals for the three C]O groups (C h , C i , C g ) and three CH 2 groups (C a , C b , C d ) appeared at 169.50, 172.56, 151.80, 26.90, 29.92, and 67.31 ppm, respectively. In addition, the signal of the amino acid a-carbon atoms (C c ) appeared at 56.90 ppm. Fig. 3(D) presents the 13 C NMR spectrum of pyrene-DPA-PBLG (6); the signals of the two anhydride C]O groups and the ester C]O group were absent, but two new signals appeared at 171.45 and 171.77 ppm for the ester C]O and amide carbon atoms. The signals at 65.22 and 55.69 ppm represent the methylene carbon atom (C d ) and the amino acid a-carbon atom (C c ) of the a-helical conformation, respectively.
We calculated the molecular weights of the polypeptides pyrene-DPA-PBLG from their 1 H NMR and MALDI-TOF mass spectra. Taking pyrene-DPA-PBLG(9) as an example, the integration ratio between the aromatic protons H f and the methylene protons H d provided a number-average molecular weight of 2370 g mol À1 (Table 1), in good accordance with the value (2367 g mol À1 ) calculated from the MALDI-TOF mass spectrum [ Fig. 4(C)]. As indicated in Fig. 4 and Table 1, the molecular weights of all the pyrene-DPA-PBLG polypeptides, determined from the 1 H NMR and MALDI-TOF mass spectra, correlated well. Moreover, the mass difference between pairs of adjacent peaks in all of the MALDI-TOF spectra of the synthesized polypeptides was m/z 219, consistent with a BLG repeating unit. Taken together, our results from FTIR, 1 H and 13 C NMR, and MALDI-TOF spectroscopy conrmed the successful preparation of the pyrene/TPA uorophores-containing pyrene-DPA-PBLG polypeptides; Table 1 summarizes the results.

Secondary structures of pyrene-DPA-PBLG polypeptides
Polypeptides are attractive materials because they can form various secondary structures. It has reported recently that the degree of polymerization strongly affects the secondary structure of a polypeptide. 47 To study this behavior more deeply, we prepared a series of pyrene-DPA-PBLG polypeptides having degrees of polymerization (DPs) of 6, 9, 19, and 24. We then used FTIR spectroscopy to obtain information about the secondary structure of each prepared polypeptide. As displayed in Fig. 5, we analyzed the FTIR spectra using the secondderivative technique, 48 which revealed that the a-helical secondary structure of a pyrene-DPA-PBLG polypeptide was characterized by an amide I band at 1651 cm À1 . For pyrene-DPA-PBLG polypeptides possessing a b-sheet secondary structure, the amide I band appeared at 1624 cm À1 , in addition to a band located at 1691 cm À1 representing the random coil structure. The free C]O groups in the side chains of pyrene-DPA-PBLG were represented by a signal at 1735 cm À1 . The quantities of the a-helical, b-sheet, and random coil structures in the pyrene-DPA-PBLG polypeptides were calculated using the deconvolution technique, with a series of Gaussian distributions tted to each amide I region; Table 2 summarizes the results.
Papadopoulos et al. reported that PBLG with a low DP (<18) was present as a mixture of a-helical and b-sheet structures, while the a-helical secondary structure was favored at a high DP (>18). 49 Similarly, we found that pyrene-DPA-PBLG polypeptides with DPs of 24 and 19 existed mainly as a-helical structures (100 and 96.6%, respectively). In contrast, both secondary structures were observed for pyrene-DPA-PBLG peptides having DPs of 9 and 6 ( Fig. 5, Table 2). Thus, we could dene the secondary structures of the polypeptides by controlling their DPspotentially useful for a wide range of specic applications.
We recorded WAXD patterns from the synthesized polypeptides to conrm their secondary structures. As illustrated in Fig. 6, the WAXD patterns of pyrene-DPA-PBLG(24) and pyrene-DPA-PBLG (19) revealed the presence of only a-helical structures, whereas the patterns of pyrene-DPA-PBLG(9) and pyrene-DPA-PBLG(6) revealed both secondary structures, in agreement with the FTIR spectroscopic data. The rst strong signal in the diffraction patterns of pyrene-DPA-PBLG(9) and pyrene-DPA-PBLG(6) appeared at a value of q of 4.5 nm À1 , attributable to the distance (d ¼ 1.38 nm) between the backbones in the antiparallel b-sheet structure. Another diffraction peak at a value of q of 16.8 nm À1 (d ¼ 0.37 nm) represented the intermolecular distance between neighboring polypeptide backbone chains in one lamella. In the WAXD patterns of Paper pyrene-DPA-PBLG(24) and pyrene-DPA-PBLG (19), the primary diffraction signal at 4.5 nm À1 , corresponding to the b-sheet secondary structure, had disappeared, with only a strong diffraction signal (q*) appearing at 5.37 nm À1 , suggesting the absence of b-sheet structures for the longer polypeptides. The primary signal (q*) and two other signals at 7.9 and 10.3 nm À1 , with relative positions 1 : 3 1/2 : 4 1/2 , is a typical indication of the formation of an a-helical secondary structure. These three signals represented the (10), (11), and (20) reections of two-dimensional hexagonally packed cylinders consisting of 18/5 a-helices with a cylinder distance of 1.16 nm.
The FTIR spectra and WAXD patterns provided the following information: for pyrene-DPA-PBLG(9) and pyrene-DPA-PBLG (6), with low average DPs (<19), both a-helical and b-sheet secondary structures were present; when the DP was greater than 19, however, the b-sheet structures disappeared and only a-helical structures were favored.

Solution UV-Vis and PL emission spectra
We studied the photophysical properties of pyrene-DPA-2NH 2 and pyrene-DPA-PBLG from their UV-Vis absorption and PL emission spectra recorded at a concentration of 10 À4 M in various solvents. Pyrene-DPA-2NH 2 has high solubility in MeOH, THF, DCM, DMF, and acetone; the pyrene-DPA-PBLG(n) polypeptides displayed high solubility in MeOH, THF, DMF, and DMSO, but low solubility in DCM and acetone. As illustrated in Fig. S3, † absorption maxima of pyrene-DPA-2NH 2 and pyrene-DPA-PBLG appeared in the regions 290-350 and 275-350 nm, representing the n-p* and p-p transitions of the conjugated pyrenyl and phenyl segments. In addition, an absorption band appeared in the region 375-500 nm for both pyrene-DPA-2NH 2 and pyrene-DPA-PBLG, arising from dimerization of the pyrene segments. Moreover, a strong solvent effect was evident in the absorption spectra of pyrene-DPA-2NH 2 a n refers to the total number of incorporated units in the two PBLG chains per pyrene-DPA-PBLG.
[ Fig. S3(A) †]. The absorbance maximum of pyrene-DPA-2NH 2 in THF, DCM, and DMF appeared at 313 nm; in acetone, the absorption maximum red-shied to 326 nm, due to the strong guest-host interactions between pyrene-DPA-2NH 2 and the acetone environment. In contrast, the absorption maximum of pyrene-DPA-2NH 2 blue-shied to 306 nm in MeOH because of the strong hydrogen bonding between the uorophore and MeOH molecules, thereby increasing the stability of pyrene-DPA-2NH 2 in the ground state. Interestingly, we observed that the intensity of the absorbance band at 427 nm of pyrene-DPA-PBLG in DMF increased upon decreasing the DP [ Fig. S3(B) †]: DPs of 24, 19, 9, and 6 provided absorbance intensities of 0.25, 0.44, 0.65, and 0.83 au, respectively. This behavior suggests that dimerization of the pyrene units in the short-chain polypeptides occurred more readily than that in the long-chain polypeptides.
In contrast to the absorption spectra, pyrene-DPA-2NH 2 exhibited almost no uorescence emission in MeOH, THF, DMF, or acetone, and little-improved emission in DCM, while all of the pyrene-DPA-PBLG polypeptides displayed strong uorescence emissions in MeOH, THF, DMF, and DMSO. For example, pyrene-DPA-2NH 2 has weak emission in DCM at a concentration of 10 À3 M, while pyrene-DPA-PBLG(5) had a 16fold stronger emission in THF at the same concentration (Fig. 7). This behavior is consistent with pyrene-DPA-2NH 2 possessing a exible DPA moiety, which can undergo dynamic intramolecular rotation (IR), thereby quickly quenching its excited states and resulting in the absence of luminescence (Fig. 7). Conversely, the strong emissions of the pyrene-DPA-PBLG polypeptides were due to the intramolecular rotations being restricted by the PBLG side chains. The blue shi of pyrene-DPA-PBLG (6) in comparison with pyrene-DPA-2NH 2 could be attributed to the hydrogen bond interaction between the oxygen atom of THF solvent and the N-H group of the polypeptide, which increased the stability of pyrene-DPA-PBLG(6) in the ground state. Additionally, this blue shi can also arise from the conversion of pyrene excimer from dynamic excimer within pyrene-DPA-2NH 2 into static excimer within pyrene-DPA-PBLG (6). As previously reported, pyrenes can be formed two kinds of excimers; dynamic and static excimers. 50 The rst one occurred when an excited-state molecule of pyrene formed a dimer with another ground-state molecule of pyrene, while the second one occurred when a pyrene dimer formed rstly in the ground state and then excited. Where, the hydrogen bonding between pyrene-DPA-PBLG(6) polypeptide chains induced pyrene molecules to come in close proximity to each other and form a dimer in the ground state. The images of the pyrene-DPA-PBLG polypeptides in Fig. 8 indicate that the uorescence color of a THF solution of each of these polypeptides was greenish yellow. This paper is, therefore, the rst to report the turning-on of the radiative pathway of a non- Paper uorescent molecule in the presence of PBLG chains, providing strongly emissive polypeptides; this approach could be used to tailor uorophore-containing polypeptides with varying functionalities for specic applications. Therefore, we performed further experiments to study the uorescence behavior of these kinds of polypeptides.
Pyrene compounds exhibit environmental solvatochromic effects, in which the relative maximum of the emission bands is strongly dependent on the solvent polarity. 51 Therefore, to study the solvatochromic behavior of our synthesized pyrene-DPA-PBLG polypeptides, we investigated their uorescence emissions in solvents of various polarities in which they could be dissolved, namely THF, MeOH, DMF, and DMSO. As illustrated in Fig. 9, the pyrene-DPA-PBLG polypeptides exhibited uorescence emission peaks at 542, 540, and 541 nm in MeOH, DMF, and DMSO, respectively. These emission peaks arose from p-p* transitions in the pyrenyl and phenyl units. The emissions of the pyrene-DPA-PBLG polypeptides underwent hypsochromic shis upon decreasing the solvent polarity; for example, the emission peaks of the polypeptides appeared at 519 nm in THF. We attribute this solvatochromic behavior to rapid amine-topyrene and amine-to-amide intramolecular charge-transfer (ICT) processes in the excited state, as well as the stability of the excited states in the high-polarity solvents. 52 In particular, the emission maximum of the pyrene-DPA-PBLG polypeptides was dependent upon the solvent polarity (i.e., solvatochromic behaviour). This behavior is typical of pyrene derivatives and TPA-containing compounds. 53 Moreover, as shown in Fig. 9A-D, all pyrene-DPA-PBLG(n) polypeptides with different degrees of polymerization have the same the solvatochromic behavior in  solutions which indicated that the a-helical and b-sheet secondary structures of our polypeptides did not affect the uorescence properties of polypeptides.

Aggregation-induced emission (AIE)
Luminescent materials having strong p-conjugated systems typically exhibit a strong emission in diluted solutions; in concentrated solutions or in the aggregate (solid) state, however, their uorescence emissions can be very weak. This ACQ behavior arises as a result of noncovalent intermolecular interactions (e.g., p-stacking). 54 To examine the AIE behavior of pyrene-DPA-2NH 2 and pyrene-DPA-PBLG(n), we evaluated their solution PL behavior using the concentration effect and solvent/ nonsolvent pairs. We investigated the concentration effect by measuring the uorescence emissions of pyrene-DPA-2NH 2 and pyrene-DPA-PBLG(6) at various concentrations in DCM and THF, respectively. As displayed in Fig. 10(A), the emission intensity of pyrene-DPA-2NH 2 decreased upon increasing the solution concentration. This behavior, termed "concentrationquenched emission," arose from strong face-to-face p-stacking interactions of neighboring pyrene units, leading to the formation of excimers, which were quickly quenched. Conversely, upon increasing the concentration of pyrene-DPA-PBLG(n) in solution from 10 À5 to 10 À3 M, the intensity of the emission increased gradually [ Fig. 10(B) and S4 †]. This behavior, termed "concentration-enhanced emission," can be ascribed to the AIE effect. On the whole, the concentration effect strongly suggested that the PL emission of pyrene-DPA-2NH 2 transformed from ACQ to AIE aer incorporation into the main chain of PBLG, and formed an AIE-active polypeptide. As previously reported, the common method to prepare AIE-active polypeptide 55 or polymer 56 is depended on polymerization  reaction in which AIE materials were utilized as initiators. On the contrary, in this study, we prepared AIE-active polypeptides through the ROP of BLG-NCA initiated with an ACQ material (pyrene-DPA-2NH 2 ). We further evaluated the AIE behavior of the pyrene-DPA-PBLG polypeptides by measuring their PL emissions in solvent/nonsolvent pairs (MeOH/toluene mixtures; Fig. 11). We selected MeOH as the solvent and toluene as the poor solvent, gradually aggregating the polypeptides by increasing the concentration of toluene. As displayed in Fig. 11, all of the diluted (10 À4 M) solutions of pyrene-DPA-PBLG(n) in MeOH exhibited weak PL emissions. Interestingly, the resultant intensity of the uorescence emission increased continuously upon increasing the concentration of toluene from 20 to 60 vol%, but decreased suddenly for each of the solutions at 80 vol% toluene. The PL emissions were enhanced in the solutions upon increasing the toluene content from 20 to 60 vol% because of aggregation, consistent with AIE behavior. We attribute the unexpected decrease in emission for the 80 vol% toluene solutions to precipitation of pyrene-DPA-PBLG(n) in solutions consisting mainly of the nonsolvent toluene. The precipitation of polypeptide aer increasing the nonsolvent content has been reported by Hong et al. 57 Consequently, the formed precipitates were isolated by centrifuging and then investigated by FTIR, which found to be pyrene-DPA-PBLG(n). Further, the uorescence of pyrene-DPA-PBLG(n) in the solid state was also investigated, as shown in Fig. 12. Pyrene-DPA-PBLG(n) polypeptides showed a massive PL emission peak at 528 nm which conrmed the AIE behavior of our polypeptides. These PL data conrmed that all of the pyrene-DPA-PBLG polypeptides exhibited AIE behavior, whereas pyrene-DPA-2NH 2 displayed ACQ behavior. Additionally, in comparison with PL data in Fig. 11(A)-(D) and 12, we can conrm that the pyrene-DPA-PBLG(24) and pyrene-DPA-PBLG (19) with only ahelical secondary structure and pyrene-DPA-PBLG(9) and pyrene-DPA-PBLG(6) with both a-helical and b-sheet secondary structures are AIE materials. Thus, the AIE behaviour of these polypeptides was not dependent upon the changing of secondary structure content.

Pyrene-DPA-PBLG/MWCNTs composites: fabrication and CNT dispersion
Because of strong p-stacking between CNTs, the dispersion of MWCNTs in solvents can be difficult. Many supramolecular complexes have, however, been reported as dispersing agents for MWCNTs, due to their benecial noncovalent interactions. 58 Polypeptides themselves are feeble dispersing agent for MWCNTs because the interactions between these species are weak. Nevertheless, we suspected that the incorporation of components (e.g., pyrene units) capable of strong p-stacking might increase the interactions between polypeptides and MWCNTs and, thus, increase the dispersion of MWCNTs. Accordingly, we examined our pyrene-DPA-PBLG polypeptides, which feature pyrene units in their backbones, as dispersing agents for MWCNTs. Fig. 13(A) displays the PL emission spectra of pyrene-DPA-PBLG(6) (chosen as an example polypeptide) and the pyrene-DPA-PBLG(6)/MWCNT complex in DMF solution aer excitation at 343 nm. Pyrene-DPA-PBLG(6) exhibited a strong PL emission at 540 nm, reecting the presence of the pyrene unit, while the pyrene-DPA-PBLG(6)/MWCNT complex displayed very weak PL emission. This behavior suggests that the pyrene units interacted strongly with the MWCNTs through p-stacking, leading to energy transfer from the light-emitted pyrene units to the MWCNTs and then quenching of the polypeptide's uorescence. 59 The presence of interactions between the pyrene-DPA-PBLG(n) polypeptides and the MWCNTs is further evident in Fig. 13(B)-(F), which display photographs of the pyrene-DPA-PBLG(24)/MWCNT, pyrene-DPA-PBLG(19)/ MWCNT, pyrene-DPA-PBLG(9)/MWCNT, and pyrene-DPA-PBLG(6)/MWCNT complex dispersions and pristine MWCNT dispersion, respectively, in DMF aer 24 h. As shown in Fig. 13(F), the pristine MWCNTs revealed a high degree of aggregation aer 24 h, which compatible with the reported nding. 60 In contrast, none of the pyrene-DPA-PBLG/MWCNT complex dispersions exhibited any obvious aggregation [ Fig. 13(B)-(E)]. Moreover, TEM imaging analysis conrmed the presence of stabilizing interactions between our synthesized polypeptides and the MWCNTs. Fig. 13(G)-(I) reveal that the pristine MWCNTs underwent a high degree of aggregation in  DMF, due to strong p-p interactions, whereas the pyrene-DPA-PBLG(19)/MWCNT complexes formed uniform dispersions of aggregated MWCNTs, stabilized through a series of noncovalent interactions. In brief, we conclude that our pyrene-DPA-PBLG polypeptides have the ability to disperse MWCNTs effectively-a property that might be useful for medicinal and manufacturing applications.

Conclusion
We have used a simple ROP to prepare a series of new polypeptides, each containing a di(4-aminophenyl)pyrenylamine luminophore, with various DPs. The chemical structures and DPs of the polypeptides were conrmed using FTIR, NMR, and MALDI-TOF spectroscopy. The polypeptides with DPs of less than 19 were mixtures of a-helical and b-sheet structures, while those with higher DPs featured only a-helical structures. Interestingly, PL data revealed that pyrene-DPA-2NH 2 has a weak uorescence emission, due to the strong IRs, which quickly quenched its excited states, while the emissions of the pyrene-DPA-PBLG polypeptides were 16-fold stronger, due to restriction of the IRs induced by the presence of the PBLG side chains. In addition, pyrene-DPA-2NH 2 is an ACQ compound, but it became a strongly AIE compound aer incorporation into the PBLG segments with rigid-rod conformations. Furthermore, pyrene-DPA-PBLG/MWCNT composites were highly dispersible in DMF as a result of noncovalent interactions between the pyrene units in the polypeptides and the MWCNT. Fluorescence spectroscopy and photographic and TEM images conrmed the dispersibility of our polypeptides and their p-p interactions with the MWCNTs. The development of such multifunctional polypeptides displaying AIE behavior and p-p interactions may lead to wider biomedical applicability. In addition, our new pyrene-DPA-PBLG/MWCNT could be used as carbon nanotube/uorescent peptide probe for sensitive detection of enzyme activity such as cancerrelated enzymes.

Conflicts of interest
There are no conicts to declare.