Photoresponsive Supramolecular Architectures Based on Polypeptide

Article pubs.acs.org/Macromolecules

Photoresponsive Supramolecular Architectures Based on Polypeptide Hybrids Daniela Mazzier,† Marco Maran,† Omar Polo Perucchin,† Marco Crisma,*,‡ Mirco Zerbetto,† Valerio Causin,† Claudio Toniolo,†,‡ and Alessandro Moretto*,†,‡ †

Department of Chemical Sciences, University of Padova, 35131 Padova, Italy Institute of Biomolecular Chemistry, Padova Unit, CNR, 35131 Padova, Italy

‡

S Supporting Information *

ABSTRACT: Self-aggregation has recently emerged as an efficient tool for the production of well-ordered supramolecular structures at the nanometric scale. In this framework, peptides offer important advantages as building blocks because of their biocompatibility and 3D-structural/functional diversities. The chemical diversity of peptides may be further expanded by use of noncoded amino acids. In the present work, we focused our attention on two known photoswitchable azobenzene-containing α-amino acids and used them as initiators for the reversible modulation of the cis/trans conformational states of two poly(γ-benzyl-L-glutamate)-based hybrid molecules with either C2 or C3 symmetry. The microscopic photoresponsive self-assembly of these compounds was examined in detail. Moreover, these hybrids were exploited in the construction of macroscopic supramolecular architectures via the electrospinning technique. Finally, after appropriate thiol functionalization, we fabricated and characterized dimeric and trimeric gold nanoparticle/polypeptide hybrid systems.

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INTRODUCTION The construction of “smart” supramolecular architectures with stimulus-responsive behavior is gaining interest1,2 due to their potential exploitation in areas such as drug delivery,3,4 controlled release,5 and nanoreactor applications.6 Among the various external stimuli, e.g. pH, solvent, or temperature changes, light is particularly attractive as it can be controlled/utilized cleanly, easily, and rapidly.7 A common method used for obtaining photocontrol on the 3D structures of molecules is the incorporation of photoresponsive moieties. In this connection, many molecules have been designed with different locations of the responsive groups. Azobenzene is one of the most widely used photoswitch chromophores, as it can isomerize rapidly, reversibly, and with high quantum yield upon irradiation between the cis and trans forms (Scheme 1, lower part), originating geometrical and conformational changes in the molecule.8−10 In recent years, the syntheses of block copolymers with peptide domains and polypeptides functionalized with photoresponsive moieties have been extensively explored.11−13 Polypeptides are attractive not only for their ability to self-assemble into ordered structures but also for the considerable chemical diversity they can offer. Beyond the 20 canonical amino acids, a variety of nonprotein or synthetic amino acids are readily accessible from commercial sources, and others can be designed and synthesized to tailor specific properties. Since in the past decade substantial progress has been made in the synthesis of polypeptides via N-carboxyanhydrides (NCAs),14,15 novel photoactive peptide-based materials have been easily prepared by © XXXX American Chemical Society

ring-opening polymerization (ROP) of NCAs, and new applications have been discovered and explored.16 Poly(γ-benzyl-L-glutamate) (PBLG) is a polypeptide that adopts a highly stable α-helical secondary structure in common organic solvents and even in the solid state.17,18 The unidirectional alignment of the intramolecular hydrogen bonds that stabilize the α-helix, parallel to its main axis, promotes the formation of a macroscopic dipole in this rodlike polymer.19 It is worth recalling that PBLG and related α-helical poly (α-amino acids) are considered to have the highest singlemolecule electric dipole among all organic compounds.20,21 Under appropriate conditions PBLG displays liquid-crystal behavior.22,23 Apart from the ability to assemble in a variety of supramolecular structures24,25 at nanoscale dimensions, another unique characteristic of rod−polypeptide segments such as PBLG is the possibility to endow various functionalities with photophysical and electrochemical properties to the supramolecular materials.26−28 The present work focuses on the study of the effect of geometry (cis/trans isomerization) on the response behavior of azobenzene-functionalized PBLG microstructures. To this aim, we exploited either bifunctional (Scheme 1, upper part) or trifunctional (Figure 1, left part) photochromic amine initiators for the preparation of PBLG-based hybrids with C2 or C3 type symmetry, respectively. These compounds were studied in Received: August 4, 2014 Revised: October 16, 2014

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dx.doi.org/10.1021/ma501601r | Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of H-bis-azo-L-Phe-OMe (5), C2 Symmetrya

a

(top) Reagents and conditions: (i) Boc2O, TEA, 1:1 CH3CN/H2O, rt, 18 h; (ii) Pd/C, H2, MeOH, rt, 40 min; (iii) Zn, NH4Cl, 5:1 EtOH/H2O, rt, 2 h; (iv) FeCl3, 5:1 EtOH/H2O, 0 °C, 30 min; (v) AcOH, rt, 24 h; (vi) 50% TFA in CH2Cl2, rt, 40 min. (bottom) Schematic representation of the light-driven, reversible photoisomerization process occurring for compound 5. High-Resolution Mass Spectroscopy. Mass spectra were obtained by electrospray ionization (ESI) on a Perseptive Biosystem Mariner ESI-ToF 5220 spectrometer. Data were collected in the positive mode. High-Performance Liquid Chromatography. The HPLC measurements were performed on an Agilent 1200 apparatus equipped with a UV detector at 226 nm. Conditions: Phenomenex C18 (100 Å) (stationary phase), 5−80% B, 25 min, 1 mL/min (eluants: A = 9:1 H2O/CH3CN, 0.05% TFA; B = 1:9 H2O/CH3CN, 0.05% TFA). Ultraviolet−Visible Absorption. UV−vis absorption spectra were recorded using a Shimadzu model UV-2501 PC spectrophotometer. A 1 cm path length quartz cell was used. Electronic Circular Dicroism. ECD measurements were carried out at room temperature using a Jasco J-715 spectropolarimeter. A fused quartz cell of 1 mm path length (Hellma) was used. Dynamic Light Scattering. DLS measurements were carried out at 25 °C on a Malvern Zetasizer Nano-S instrument using a He−Ne laser (633 nm) and a scattering angle of 176°. Transmission Electron Microscopy. Samples were analyzed on a Jeol 300 PX TEM instrument. A glow discharged carbon coated grid was floated on a small drop of the nanosphere suspension and excess was removed by #50 hardened Whatman filterpaper. Scanning Electron Microscopy. A Carl Zeiss Merlin field emission SEM operating at 5 kV accelerating voltage was used. A small drop of the nanosphere suspension was placed on a microscope glass coverslip and allowed to dry overnight. Size Exclusion Chromatography. SEC analyses were performed on an Agilent 1260 Infinity system equipped with 1260 isopump, 1260 TCC, 1260 VWD VL, 1260 RID, Phenogel 5 μm linear/mixed guard column (30 × 4.6 mm), followed by Phenomenex Phenogel 5 μm 104 Å (300 × 4.6 mm) column working at 60 °C. DMF was used as eluant at a flow rate of 1 mL/min. Before SEC analysis, the samples were filtered through a 0.2 μm PTFE filter (15 mm, Phenomenex).

terms of microscopic photoresponsive self-aggregation and as precursors for the fabrication of macroscopic materials via the electrospinning technique. The preparation and characterization of dimeric and trimeric gold nanoparticle/polypeptide hybrids are also described.

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EXPERIMENTAL SECTION

Materials. γ-Benzyl-L-glutamate (BLG), p-nitrobenzylamine, and 4-(dimethylamino)pyridine (DMAP) were obtained from Fluka. 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC·HCl) was purchased from GL Biochem (Shanghai). Triphosgene, di-tert-butyl dicarbonate (Boc2O), triethylamine (TEA), p-aminobenzoic acid, 2,2′-dimethoxy-2-phenylacetophenone (DMPA), β-mercaptoethanol, p-nitro-L-phenylalanine methyl ester hydrochloride [H-L-Phe(pNO2)-OMe·HCl], 2,4,6-triallyloxy-1,3,5-triazine, acetic acid (AcOH), trifluoroacetic acid (TFA), and α-pinene were obtained from Sigma-Aldrich. The deuterated solvents CDCl3 and DMSO (dimethyl-d6 sulfoxide) were purchased from Euriso-Top. Catalyst 10% Pd/C was obtained from Acros-Janssen. All other chemicals and solvents are Sigma-Aldrich, Fluka, or Acros products and were used as provided (without further purification). General Methods. Nuclear Magnetic Resonance. 1H and 13C NMR spectra were recorded at room temperature on a Bruker AC-200 (200 MHz) instrument using TMS (tetramethylsilane) as the internal reference. The multiplicity of a signal is indicated as s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad. Chemical shifts (δ) are expressed in ppm. Fourier Transform Infrared Absorption. FT-IR absorption spectra in KBr disk were recorded with a PerkinElmer 1720X spectrophotometer. The v maxima values for the main absorption bands are given. B


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Figure 1. Synthesis of initiator (11), C3 symmetry. (left) Reagents and conditions: (i) Boc2O, TEA, 1:1 CH3CN/H2O, rt, 18 h; (ii) Zn, NH4Cl, 5:1 EtOH/H2O, rt, 2 h; (iii) FeCl3, 5:1 EtOH/H2O, 0 °C, 40 min; (iv) AcOH, p-aminobenzoic acid, rt, 48 h; (v) 10% mol DMPA, 2-mercaptoethanol, hv = 365 nm, 40 min; (vi) EDC, DMAP, DMF, rt, 18 h; (vii) 50% TFA in CH2Cl2, rt, 2 h. (right) (A) Schematic representation of the light-driven, reversible photoisomerization process occurring for 11. (B) HPLC profile of a few-second-irradiated (at 365 nm) solution of 11, which revealed formation of all (four) of its possible isomers. The MWs were calculated according to a calibration using polystyrene standards. Microstructure Preparation. Typically, 20 mg of polymer was dissolved in 10 mL of a DMF/THF 3:7 (v/v) solvent mixture. This solution was put into a cellulose dialysis tubing with a MW cutoff of 12 kDa (flat width 35 mm, Sigma). The dialysis process was carried out against deionized water for 48 h. Electrospinning of PBLG Fibers. Three solutions at different concentrations (4, 8, and 12 wt %) were prepared by dissolving PBLG in dicloroacetic acid at 50 °C. A 2 mL SGE syringe with stainless steel needle was used as an electrode. Syringes with 8 mL of each concentration were loaded in a syringe pump (Genei Plus Syringe pump, Kent Scientific) to control the flow rate of the polypeptide solutions. Electrospinning was performed at ambient conditions with a constant applied voltage of +8 kV at needle and −5 kV at collector with the flow rate set at 0.01 mL/min to maintain a constant size of droplet at the tip of the syringe needle. A circular aluminum plate was used as a collector. The tip-to-collector distance was kept at 15 cm. Electrospinning was performed for about 2 h to obtain a nonwoven, continuous fiber mat. All fibers were dried prior to use for the photostimuli experiments. Synthesis of Initiators. a. H-bis-azo- L -Phe-OMe (5), C 2 Symmetry Initiator (Scheme 1, Upper Part).29,30 Boc-L-Phe(pNO2)OMe (1). H-L-Phe(pNO2)-OMe·HCl (4.9 g, 18.8 mmol) was dissolved in an 1:1 CH3CN/H2O solvent mixture (20 mL), and TEA was added to pH = 9. Then, Boc2O (5.50 g, 25.2 mmol), dissolved in CH3CN (10 mL), was added. The mixture was stirred at rt for 18 h. The mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (ethyl acetate). The organic layer was washed with 5% KHSO4(aq), 5% NaHCO3(aq), and brine. After drying over Na2SO4,

the solvent was removed under reduced pressure, and the compound was obtained as a solid (5.9 g, 97%). FT-IR absorption: υ̅ 3358, 2985, 1732, 1689, 1524, 1346 cm−1. HRMS (ESI+): m/z calcd for C15H20N2O6 324.1321; found 325.3298 [M + H]+. 1H NMR (200 MHz, CDCl3): δ (ppm) 1.40 (s, 9H), 3.11 (dd, J = 13.7, 6.2 Hz, 1H), 3.27 (dd, J = 13.7, 6.2 Hz 1H), 3.73 (s, 3H), 4.58−4.68 (m, 1H), 5.06 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 8.7 Hz, 2H), 8.6 (d, J = 8.7 Hz, 2H). Boc-L-Phe(pNH2)-OMe (2).29,30 Boc-L-Phe(pNO2)-OMe (2.40 g, 7.4 mmol) was dissolved in MeOH (50 mL), and N2 was bubbled in the solution for 10 min. Then, Pd/C (300 mg) was added, and the mixture was stirred at rt under an atmospheric pressure of H2 gas for 1 h. The catalyst was removed by filtration through Celite. The solvent was evaporated, and the residue was purified via flash chromatography (eluant: 1:1 EtOAc/hexane). The product was recovered as a solid (2.1 g, 96%). FT-IR absorption: υ̅ 3397, 3374, 3232, 1741, 1690, 1515 cm−1. HRMS (ESI+): m/z calcd. for C15H22N2O4 294.1579; found 295.1732 [M + H]+. 1H NMR (200 MHz, CDCl3): δ (ppm) 1.40 (s, 9H), 2.96 (d, J = 5.7 Hz, 2H), 3.44 (s br, 2H), 3.70 (s, 3H), 4.45−4.55 (m, 1H), 4.95 (d, J = 7.9 Hz, 1H); 6.61 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H). Boc-L-Phe(pNO)-OMe (3).29,30 Boc-L-Phe(pNO2)-OMe (1.07 g, 3.3 mmol) and NH4Cl (790 mg, 14.8 mmol) were dissolved in a 5:1 EtOH/H2O (42 mL) solvent mixture. Then, Zn dust (600 mg, 9.2 mmol) was added. The resulting suspension was stirred at rt for 2 h, then filtered through Celite, and washed with a cold mixture of 5:1 EtOH/H2O (40 mL) to remove the excess of Zn. The filtrate was cooled to 0 °C, and a cold solution of FeCl3·6H2O (6.33 g, 23.4 mmol) in 5:1 EtOH/H2O (35 mL) was added. The mixture was stirred at 0 °C for 30 min, diluted with brine, and extracted with EtOAc. C


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Scheme 2. Synthesis of PBLG Hybrid with C2 Symmetry (12)

dissolved in 5:1 EtOH/H2O (25 mL). Then, Zn dust (940 mg, 14.4 mmol) was added, and the obtained suspension was stirred at rt for 2 h. The suspension was filtered through Celite, the filtrate was cooled to 0 °C, and a cold solution of FeCl3·6H2O (6.33 g, 23.4 mmol) in 5:1 EtOH/H2O (35 mL) was added. The mixture was stirred at 0 °C for 30 min, diluted with brine, and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was evaporated, and the crude was rapidly purified via flash chromatography (eluant: 100:1 CH2Cl2/MeOH), affording compound 7 as a green solid (880 mg), which was immediately used for the next synthetic step. This compound was dissolved in glacial AcOH (10 mL), and a solution of p-aminobenzoic acid (0.746 g, 5.4 mmol) in glacial AcOH (5 mL) was added. After 48 h under stirring at rt, the mixture was concentrated and diluted with EtOAc. The organic solution was washed with 5% NaHCO3(aq), 5% KHSO4(aq), and brine. After drying over MgSO4, the filtrate was concentrated and the crude purified via flash chromatography (eluant: CH2Cl2/EtOAc, increasing the solvent mixture polarity from 9:1 to 1:1). The compound was obtained as an orange solid after precipitation from EtOAc/petroleum ether (370 mg, 28%). FT-IR absorption: υ̅ 3343, 2980, 1682, 1507, 1426, 1168 cm−1. HRMS (ESI+): m/z calcd for C19H21N3O4 355.1532; found 356.1691 [M + H]+. 1H NMR (200 MHz, DMSO, d6): δ (ppm) 1.41 (s, 9H), 4.24 (d, J = 6 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.88−7.97 (m, 4H), 8.14 (d, J = 8.4 Hz, 2H), 13.18 (s, 1H). Compound 9. 2,4,6-Triallyloxy-1,3,5-triazine (5.03 g, 20.2 mmol), 2-mercaptoethanol (8.66 g, 110.8 mmol), and DMPA (2,2-dimethoxy2-phenylacetophenone) (376 mg, 1.5 mmol) were mixed in a beaker and stirred under irradiation at 365 nm with a UV lamp for 40 min. The mixture was diluted with MeOH (10 mL), and Et2O was added until the precipitation of an oily compound took place. The supernatant was removed and the oily compound was washed twice with Et2O. Compound 9 was recovered as a colorless oil (8.2 g, 84%). FT-IR absorption: 3389, 2920, 1567, 1417, 1332, 1140 cm−1. HRMS (ESI+): m/z calcd for C18H33N3O6S3 483.1531; found 484.1626 [M + H]+. 1H NMR (200 MHz, CDCl3): δ (ppm) 2.00−2.13 (m, 6H), 2.30 (s br, 3H), 2.65−2.75 (m, 12H), 3.72 (t, J = 6.2 Hz, 6H), 4.49 (t, J = 6.2 Hz, 6H). Compound 10. To a solution of compound 8 (320 mg, 0.91 mmol), compound 9 (120 mg, 0.25 mmol), and DMAP (36 mg, 0.29 mmol) in 10 mL of anhydrous DMF cooled to 0 °C, EDC·HCl (190 mg, 0.97 mmol) was added, and the reaction mixture was stirred at rt for 48 h. The solvent was removed under reduced pressure, the residue dissolved in CH2Cl2 and washed with 5% KHSO4(aq) and brine. After

The combined organic phases were washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure, and the residue was purified rapidly via flash chromatography (eluant: 30:1 CH2Cl2/EtOH). Compound 3 was obtained as a green oil (910 mg, 89%), which was immediately used for the next synthetic step. Boc-Bis-Azo-L-Phe-OMe (4).29,30 A solution of freshly prepared compound 2 (1.0 g, 3.4 mmol) in glacial AcOH (15 mL) was added to a solution of compound 3 (1.15 g, 3.7 mmol) in glacial AcOH (15 mL). The mixture was stirred at rt for 24 h. The solvent was evaporated, and the crude product was purified via flash chromatography (eluant: 50:1 CH2Cl2/EtOH). After precipitation from EtOAc/ hexane, compound 4 was recovered as an orange solid (830 mg, 43%). FT-IR absorption: υ̅ 3356, 1752, 1735, 1688, 1520 cm−1. HRMS (ESI+): m/z calcd for 584.2846; found 585.2832 [M + H]+. 1H NMR (200 MHz, CDCl3): δ (ppm) 1.42 (s, 18H), 3.14−3.20 (m, 4H), 3.73 (s, 6H), 4.58−4.68 (m, 2H), 5.03 (d, J = 7.27 Hz, 2H), 7.27 (d, J = 8.3 Hz, 4H), 7.84 (d, J = 8.3 Hz, 4H). H-Bis-Azo-L-Phe-OMe (5). The removal of the Boc protecting group was obtained by treating Boc-bis-azo-L-Phe-OMe (175 mg, 0.29 mmol) with an 1:1 TFA/CH2Cl2 mixture (10 mL) at rt under stirring for 40 min. Then, the solvent was evaporated, and the residue suspended in water and lyophilized. Product 5 was obtained as an orange solid (170 mg, 98%). FT-IR absorption: υ̅ 3425, 3007, 1748, 1674 cm−1. HRMS (ESI+): m/z calcd for 384.1797; found 385.2035 [M + H]+. 1H NMR (200 MHz, DMSO, d6): δ (ppm) 3.21 (d, J = 5.3 Hz, 4H), 3.70 (s, 6H), 4.42 (t, 2H), 7.46 (d, J = 8.4 Hz, 4H), 7.86 (d, J = 8.4 Hz, 4H), 8.59 (s br, 4H). 13C NMR (50 MHz, DMSO-d6) δ: 169.41, 151.22, 138.51, 130.66, 122.84, 53.05, 52.81, 35.82. b. Compound (11), C3 Symmetry Initiator (Figure 1, Left Part). Boc-p-Nitrobenzylamine (6). Solid Boc2O (4.5 g, 20.8 mmol) was added to a solution of p-nitrobenzylamine (2 g, 10.6 mmol) and TEA (2 mL, 14.4 mmol) in CH2Cl2 (45 mL) cooled to 0 °C. The mixture was stirred at rt for 18 h. Then, the mixture was diluted with CH2Cl2 and washed with 5% KHSO4(aq) and brine. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. After precipitation from CH2Cl2/petroleum ether, compound 6 was recovered as a solid (1.8 g, 67%). FT-IR absorption: υ̅ 3325, 2984, 2935, 2914, 1688, 1520, 1165 cm−1. HRMS (ESI+): m/z calcd for C12H16N2O4 252.1110; found 197.0652 [M-tBu + H]+. 1H NMR (200 MHz, CDCl3): δ (ppm) 1.46 (s, 9H), 4.40 (d, J = 6.2 Hz, 2H), 4.98 (s br, 1H), 7.44 (d, J = 8.7 Hz, 2H), 8.19 (d, J = 8.7 Hz, 2H). Boc-4-(aminomethyl)phenylazobenzoic Acid (8).31 Boc-p-nitrobenzylamine (1 g, 4 mmol) and NH4Cl (690 mg, 13 mmol) were D


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Scheme 3. Synthesis of PBLG Hybrid with C3 Symmetry (13)

drying over MgSO4, the filtrate was concentrated and the crude was purified via flash chromatography (eluant: CH2Cl2/MeOH, increasing the solvent mixture polarity from 60:1 to 40:1). Compound 10 was recovered as an orange solid (270 mg, 70%). FT-IR absorption: υ̅ 3335, 2975, 1716, 1684, 1513, 1167 cm−1. HRMS (ESI+): m/z calcd for C75H90N12O15S3 1494.5810; found 1495.6822 [M + H]+. 1H NMR (200 MHz, CDCl3): δ (ppm) 1.48 (s, 27H), 2.01−2.20 (m, 6H), 2.78 (t, J = 7.1 Hz, 6H), 2.90 (t, J = 7.1 Hz, 6H), 4.39 (s, 6H), 4.46−4.53 (m, 12H), 4.95 (s br, 3H), 7.43 (d, J = 8.4 Hz, 6H), 7.89−7.85 (m, 12H), 8.17 (d, J = 8.6 Hz, 6H). Compound 11. The removal of the Boc protecting group was obtained by treating compound 10 (61 mg, 0.041 mmol) with an 1:1 TFA/CH2Cl2 mixture (4 mL) at rt under stirring for 2 h. The solvent mixture was evaporated; the residue was suspended in water and lyophilized. Product 11 was obtained as an orange solid (47 mg, 96%). FT-IR absorption: υ̅ 3423, 2955, 1715, 1561, 1271 cm−1. HRMS (ESI +): m/z calcd for C60H66N12O9S3 1194.4237; found 1195.5364 [M + H]+. 1H NMR (200 MHz, DMSO, d6): δ (ppm) 1.95−2.01 (m, 6H), 2.67−2.74 (m, 6H), 2.89−2.85 (m, 6H), 4.17 (s, 6H), 4.34−4.48 (m, 12H), 7.70 (m, 6H), 7.98 (m, 12H), 8.13−8.17 (m, 6H), 8.41 (s, 12H). 13C NMR (50 MHz, DMSO-d6): δ (ppm) 172.52, 164.92, 154.38, 151.69, 149.90, 138.19, 131.66, 130.58, 130.01, 122.98, 122.75, 66.34, 64.17, 59.30, 41.88, 32.56, 29.67, 28.17, 27.64. PBLG Derivatives. γ-Benzyl-L-glutamate N-Carboxyanhydride (BLG-NCA).32 BLG (5.05 g, 21.3 mmol) and α-pinene (6.64 g, 48.7 mmol) were dissolved in EtOAc (70 mL) in a three-neck flask and heated under reflux. Triphosgene (4.24 g, 14.2 mmol) was dissolved in EtOAc (25 mL) and added slowly with a dropping funnel once the reflux started. After 4 h of reaction, heating was interrupted and most of the solvent was evaporated under reduced pressure. The compound was precipitated by addition of petroleum ether, and the recovered solid was recrystallized twice, subsequently filtered, and washed with petroleum ether. BLG-NCA was recovered as a solid (4.6 g, 84%). 1 H NMR (200 MHz, CDCl3): δ (ppm) 7.35 (s, 5H), 6.61 (s, 1H), 5.14 (s, 2H), 4.37 (t, J = 6 Hz, 1H), 2.59 (t, J = 6.8 Hz, 2H), 2.36− 2.02 (m, 2H). C2 Symmetry PBLG Hybrid (12) (Scheme 2). BLG-NCA (1.10 g, 4.2 mmol) was dissolved in dry 1,4-dioxane (2 mL) under an N2 atmosphere, and the solution was cooled to 0 °C. Then, a solution of compound 5 (5 mg, 0.013 mmol) in 2:1 1,4-dioxane/DMF (600 μL)

solvent mixture was added, and the mixture was stirred at 25 °C for 48 h. The mixture was diluted with 1,4-dioxane, and the polypeptide hybrid was precipitated by adding MeOH. The obtained solid was filtered, washed with MeOH and Et2O, and dried in vacuo. The polypeptide hybrid was recovered as a light yellow solid (680 mg, 62%). SEC MW: 63 500 (PDI 1.16). FT-IR absorption: υ̅ 3295, 3003, 2951, 1734, 1653, 1546, 1163 cm−1. C3 Symmetry PBLG Hybrid (13) (Scheme 3). BLG-NCA (1.00 g, 3.81 mmol) was dissolved in dry 1,4-dioxane (2.3 mL) under an N2 atmosphere, and the solution was cooled to 0 °C. Then, a solution of compound 11 (12 mg, 0.010 mmol) in DMF (1 mL) was added, and the mixture was stirred at 25 °C for 72 h. The mixture was diluted with 1,4-dioxane, and the polypeptide hybrid was precipitated by adding MeOH. The obtained solid was filtered, washed with MeOH and Et2O, and dried in vacuo. The polypeptide hybrid was recovered as a light yellow solid (750 mg, 76%). SEC MW: 46 800 (PDI 1.56). FT-IR absorption: υ̅ 3331, 3064, 2940, 1734, 1653, 1545, 1161 cm−1. This synthesis was repeated under similar conditions by using a higher monomer/initiator ratio (500:1) and a prolonged reaction time (5 days). The polypeptide hybrid was recovered as a light yellow solid (550 mg, 76%). SEC MW: 154 000 (PDI 1.66). FT-IR absorption: υ̅ 3329, 3062, 2941, 1732, 1651, 1542, 1161 cm−1. Gold Nanoparticles−PBLG Hybrids. Thiol Derivatives. Trt (trityl)-β-mercaptopropionic acid (120 mg, 0.34 mmol) and HOAt (7-aza-1-hydroxy-1,2,3-benzotriazole) (48 mg, 0.34 mmol) were dissolved in 3 mL of anhydrous DMF and cooled to 0 °C. Then, EDC·HCl (66 mg, 0.34 mmol) was added. The mixture was stirred for 15 min at 0 °C. The C2 symmetry polypeptide hybrid (12) (150 mg, 2.36 μmol) or the C3 symmetry polypeptide hybrid (13) (110 mg, 2.36 μmol) was dissolved in 2 mL of DMF and added with TEA (100 μL, 0.72 mmol) to the solution of the active ester. After stirring the solution at rt for 48 h, the polypeptide hybrids were precipitated by adding MeOH, then filtered, and washed several times with MeOH. In both cases, the polypeptide hybrids were obtained in almost quantitative yield after drying. Gold Nanoparticles. Gold nanoparticles (GNP) (13 nm size) were prepared using the standard reduction of tetrachloroauric(III) acid (HAuCl4·3H2O) with trisodium citrate bis-hydrate.33 Typically, 10 mL of 38.8 mM sodium citrate solution was rapidly added to a boiling water solution of HAuCl4·3H2O (1.0 mM, 100 mL) under vigorous E


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Figure 2. (A) Schematic representation of the cis/trans photoisomerization for the C2 symmetry PBLG hybrid (12). (B) UV−vis absorption spectra of the cis and trans isomers of the C2 symmetry PBLG hybrid in THF solution (two cycles of irradiation are shown). (C) SEC analysis which shows the different elution profiles occurring for the cis and the trans forms of the C2 symmetry PBLG hybrid. (D) ECD spectra of the cis and trans isomers of the C2 symmetry PBLG hybrid in 1,1,1,3,3,3-hexafluoroisopropanol solution (HFIP) (the cis red trace was slightly shifted up for clarity).

and β-mercaptoethanol. Condensation of compounds 8 and 9 using the EDC/DMAP C-activation, followed by Bocdeprotection, gave the trifunctional amine initiator (11). In Figure 1A (right), the four isomers expected from the photoisomerization of (11) are shown. Indeed, four species could be detected by HPLC after 40 s of irradiation at 365 nm (Figure 1B, right). A more prolonged irradiation (≥2 min) at 365 nm is able to reduce the population of the all-trans form to less than 7%. Back-conversion to about 70% population of the all-trans form (and 0% of the all-cis isomer; the remaining 30% population being distributed between the trans−cis−cis and trans−trans−cis isomers) was achieved by 30 min irradiation at 420 nm (Figures S4 and S5). Again, 1H NMR and UV−vis absorption spectra after two cycles of irradiation at 365 and 420 nm (Figures S6 and S7) clearly support the reversibility of the process. The PBLG hybrids with C2 and C3 symmetries were obtained via NCA-ROP using compounds 5 and 11, respectively, as the initiators. BLG-NCA was synthesized via phosgenation of BLG following a reported protocol.31 The polymerizations using BLGNCA were performed in 1,4-dioxane or DMF. The solid PBLG hybrids, obtained after precipitation with MeOH, were characterized by SEC analysis. We obtained a MW distribution of 63 500 Da, PDI (polydispersity index) 1.16 for the C2 symmetry PBLG hybrid. Two different polymerization conditions used for the preparation of the C3 symmetry PBLG

stirring, and the mixture was kept boiling for an additional 10 min. Then, the mixture was allowed to cool to rt while stirring for an additional 15 min. After their syntheses, red GNP colloids (1.25 × 10−8 mol/L, assuming a density for gold colloids identical to that of gold in bulk) were kept in the dark at 5 °C. Citrate−Thiol Exchange. After various attempts, the citrate−thiol exchange experiments were performed as follows. To a 100 mL of 1.25 × 10−8 mol/L water solution of GNP, a DMF solution of the C2 symmetry polypeptide hybrid (12) (20 mL, 0.01 μmol) or C3 symmetry polypeptide hybrid (13) (20 mL, 1 nmol) was slowly added using a syringe pump system, under vigorously stirring, for 2−6 h. TEM images were recorded after each preparation.

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RESULTS AND DISCUSSION The bis-aminoester (5) (initiator of the C2 symmetry) was prepared through a modification of a reported procedure.29,30 The synthesis involved the condensation of two L-Phe derivatives, bearing an amino (2) and a nitroso (3) moiety, respectively, as para-substituents (Scheme 1). The HPLC profiles, monitoring the time-dependent photoinduced trans/cis isomerization of 5 along with the 1H NMR and UV−vis absorption spectra after two cycles of irradiation at 365 and 420 nm, are reported in the Supporting Information (Figures S1−S3). The synthesis of the initiator with C3 symmetry (11) initially involved the preparation of the azo compound 8 (Figure 1, left). The C3-platform (9) was easily obtained via thiol−ene “click” chemistry starting from the tris-alkene core 2,4,6-triallyloxy-1,3,5-triazine F


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Figure 3. Upper part: schematic representation of the thiol-functionalized C2 symmetry PBLG hybrid and the corresponding dimeric GNP/PBLG hybrid system. (A) TEM images of single and monodisperse (12 nm), citrate passivated GNP. (B) TEM images of multimers of the GNP/PBLG hybrid system, obtained by using high concentration and fast addition experimental conditions. (C, D) TEM images of the dimeric GNP/PBLG hybrid system, obtained by using more diluted and slow addition experimental conditions. (E, F) TEM images showing details of the dimeric GNP/ PBLG hybrid system, obtained by two different experiments.

characterized by a longer elution time, indicative of a more compact 3D shape, with respect to the trans form. Conversely, as expected, cis/trans isomerization does not affect the polypeptide secondary structure of the PBLG hybrid, the ECD spectra of which are almost identical for both isomers and indicative of an overwhelmingly prevailing α-helical conformation (Figure 2D). We decided to exploit the C2 symmetry of this PBLG hybrid for the construction of a more complex, dimeric GNP/ polypeptide hybrid systems. To this aim, both N-termini of the PBLG hybrid were functionalized with β-mercaptopropionic acid (Figure 3, upper part) following a protocol previously reported by some of us.26 Monodisperse, citrate-passivated GNP (diameter 13 nm, Figure 3A) were prepared33 and subsequently subjected to a thiol/citrate exchange reaction with the bis-βmercaptopropionic acid functionalized PBLG hybrid. By running this experiment under appropriate diluted conditions, we were able to detect by TEM the formation of dimeric species. Specifically, a fast addition of a DMF solution of the thiolfunctionalized PBLG hybrid to an aqueous GNP solution promoted the formation of GNP/PBLG hybrid multimers (Figure 3B), while a slow addition, combined with a high dilution, allowed the onset of dimeric units (Figure 3C−F). The self-assembly properties of the C2 symmetry PBLG hybrid were investigated in aqueous environment.16 Starting

hybrid afforded polypeptides characterized by a MW distribution of 46 800 Da, PDI 1.56, and 154 000 Da, PDI 1.66, respectively. The reliability of the MW distributions of the C2 and the lower MW C3 symmetry PBLG hybrids was checked by cleaving each of them at the level of the azo group(s) through reaction with sodium hydrosulfite.34 SEC analysis of the linear fragments derived from the cleavage of the C2 symmetry PBLG hybrid gave a MW of 31 500 Da (PDI: 1.21), nearly 1/2 the MW of the original material. Similarly, the fragments obtained from the 46 800 Da C3 symmetry PBLG hybrid gave a MW distribution of 13 000 Da (PDI: 1.18), a value not far from 1/3 the MW of the original PBLG hybrid. C2 Symmetry PBLG Hybrid. The C2 symmetry PBLG hybrid (12) was studied in terms of photoresponsive behavior (Figure 2A). After UV irradiation at λ = 365 nm of a PBLG hybrid solution in THF, isomerization of the azobenzene moiety from trans-to-cis was observed, as indicated by the decrease of the π−π* transition band at 328 nm and the concomitant increase of the n−π* transition band at 440 nm (Figure 2B). This process was found to be reversible for at least four cycles of repeated irradiation at 365 and 420 nm. Two cycles of irradiation are shown in Figure 2B. Interestingly, the cis and trans isomers can be differentiated by SEC analysis (Figure 2C). Specifically, the cis form is G


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Figure 4. TEM (A) and SEM (B) images of the self-assembled structures obtained from the C2 symmetry PBLG hybrid in aqueous suspension. (C) Tentative, vesicle-like model of the self-assembled structures. (D−G) TEM images of the microstructure transitions occurring after different irradiation times under UV light at λ = 365 nm (20, 40, 60, and 80 s, respectively).

We recorded the fluorescence spectra of a diluted water suspension of the spherical aggregates prior and after exposure to UV light at λ = 365 nm (Figure 5A). The UV-induced collapse of the aggregates (demonstrated above) is accompanied by a dramatic enhancement in the CF fluorescence. This result strongly supports the view that our spherical aggregates of the C2 symmetry PBLG hybrid are indeed vesicles, hosting in their inner cavity CF molecules at relatively high concentration. This phenomenon gives rise to a low fluorescence emission owing to the self-quenching effect. Upon disruption of the vesicles, diluted CF is released into the medium, fully restoring its strong fluorescence. As shown in Figure 5B, the maximum fluorescence intensity is reached after 6 min irradiation at 365 nm. This time is much longer than the 20−80 s required for the collapse of the empty vesicles reported in Figure 4. Such a discrepancy might be rationalized by observing that CF absorbs part of the irradiating light. Disruption/destabilization of the vesicles, leading to CF release within a few minutes, can be also achieved by addition to the aqueous suspension of either MeOH (which acts as a precipitant) or a diluted NaOH solution which neutralizes the positive charges on the vesicle surface that are instrumental in preventing the aggregation of this colloidal form (Figure 5B). C3 Symmetry PBLG Hybrid. The strategy described above for the preparation of the dimeric GNP/PBLG hybrid was also exploited for the synthesis of a trimeric system starting from the C3 symmetry PBLG hybrid (Figure 6, left). The TEM images reported in Figure 6, panels A−F, from samples obtained by six independent preparations in which the thiol/citrate exchange was performed at very high dilution, clearly show the formation of GNP/PBLG hybrid trimers with C3 symmetry. It has to be noted that for both the dimeric and trimeric nanoparticle hybrid systems, the distances between nanoparticles within the assemblies appear from the TEM images (Figures 3 and 6) to be much shorter than those expected on the basis of the polypeptide lengths. This observation may suggest that GNPs could be attached to the polypeptide side rather than to the thiol-functionalized ends. However, in control experiments carried out using the nonthiol functionalized C2 and C3 symmetry PBLG hybrids, no formation of GNP dimers and

from a solution of this hybrid in an organic solvent mixture (20 mg in 10 mL of 3:7 DMF/THF v/v), a milk-like suspension was obtained after dialysis against ultrapure water for 48 h (membrane cutoff, 12 kDa). A few drops of this suspension examined by TEM (without uranyl-acetate treatment) and SEM revealed formation of aggregates which display polydisperse spherical morphologies with diameters of 100− 500 nm (Figure 4A,B). The relative high and positive value of the Z-potential function (+30 mV)35 (related to the degree of electrostatic repulsion between adjacent, similarly charged particles) obtained from these experiments provided evidence for the presence of electrostatic net charges, due to the Nterminal, protonated amino groups, present at least in the outer layer of these microstructures. Moreover, a high Z-potential value is often associated with double-layer systems, such as vesicles. Indeed, our rod-like PBLG hybrid, largely hydrophobic but carrying protonated amino groups at both termini, might give rise to a vesicle-like self-assembly, as shown in Figure 4C. Considering that this hybrid has an average MW of 63 500, which corresponds to about 280 BLG units, and that the elevation per residue in the α-helix is 0.15 nm,36 the wall thickness of the vesicles might be of the order of 40 nm. Upon UV irradiation (λ = 365 nm) of the suspension, a rapid and progressive collapse of these ordered structures was observed (Figure 4D−G). In all probability, the change in the 3D geometry which occurs as a consequence of the trans-tocis azobenzene isomerization, even if taking place for a fraction of the polypeptide hybrid molecules, is able to destabilize the self-assembled microstructures. Evidence for the vesicular nature of the self-assembled structures was obtained by the carboxyfluorescein (CF)entrapped technique.37 The self-assembly process described above was repeated in the presence of CF in the starting organic medium. After dialysis, the resulting suspension was submitted to SEC, thus allowing removal of the nonentrapped CF and isolation of a red-orange colored main fraction. This fraction consisted of spherical self-assembled structures, homogeneous in size (200 nm) according to TEM and DLS analyses (Figure 5, upper part, and Supporting Information, Figure S8, respectively). H


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Figure 5. Upper part: TEM image of purified (by gel-exclusion chromatography) spherical aggregates of the C2 symmetry PBLG hybrid formed in the presence of CF. Lower part: (A) Comparison of the fluorescence emission spectra of the CF-encapsulated aggregates before (red line) and after (black line) UV irradiation at 365 nm. (B) Time evolution of the release of CF from the spherical aggregates as a result of either UV irradiation (365 nm) or denaturation by addition of 10% MeOH (v/v) or 5% 0.1 M NaOH (v/v).

Figure 6. Left: schematic representation of the trimeric GNP/PBLG hybrid system based on the thiol-functionalized C3 symmetry PBLG hybrid. (A−F) TEM images showing details of the trimeric GNP/PBLG hybrid system (from samples obtained by six different experiments at very high dilution).

trimers, respectively, could be detected. Tentatively, the short interparticle distances may be ascribed to the solid-state level at which TEM analyses are carried out, i.e., in the absence of solvation. Covalently linked GNPs may experience enhanced cohesion forces as the result of the drying process, possibly

strong enough to overcome the stiffness of the polypeptide chains connecting them. We also investigated the self-assembly properties of the C3 symmetry PBLG hybrid (average MW 46 800) in aqueous environment. The microstructures formed starting from an I


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Figure 7. Upper part: TEM (center) and SEM (right) images of the self-assembled structures obtained from the C3 symmetry PBLG hybrid (left) in aqueous suspension. Central part, left: TEM images of the microstructures after UV irradiation (λ = 365 nm) for different times (A, no irradiation; B, 5 min; C and D, 10 min; E, 20 min; F, 30 min). Central part, right: schematic representation of the light-induced microstructure reorganization. Lower part: SEM images showing the microstructures before (left) and after (right) 30 min UV irradiation.

variation of morphology could be detected even after prolonged UV irradiation (Supporting Information, Figure S9). We assume that in this case the noncovalent interactions among the polypeptide chains would be strong enough to prevent a conformational change to take place as a result of the photoswitch process. Therefore, inspired by a recent publication on electrospun α-helical PBLG fibers38 and considering the characteristics of our C3 symmetry PBLG hybrid (it has a MW comparable to that reported in ref 38), we decided to process our high-MW C3 symmetry PBLG hybrid via the electrospinning technique. The characterization of the resulting material is illustrated in Figure 8. The densely packed fibrillar network detected by SEM (Figure 8A) corresponds, to the macroscopic level, to a solid layer (Figure 8B). We tested the photoresponsive behavior of this solid layer under UV irradiation. The fibers, originally remarkably homogeneous in shape, tend to progressively “swell” into larger domains after 30−60 min of irradiation (Figure 8C,D). A total collapse of the fiber morphology was achieved after 200 min UV irradiation (Figure 8E). Interestingly, application of the electrospinning technique to the C2 symmetry PBLG hybrid also led to the formation of a fibrillar network. In this case, however, the fiber morphology did not change even after prolonged UV irradiation (up to 3 h). In all probability, the packing of polypeptide chains within the fibers

organic solution upon dialysis against water are characterized by an oval shape and dimensions of 100−400 nm (Figure 7, top). After UV irradiation for increasing times up to 30 min, we observed the reorganization of the microstructures into smaller aggregates as detected by TEM analyses (Figure 7A−F). In particular, a partial disassembly of the oval aggregates was detected after 5 min irradiation, whereas prolonged irradiation (up to 20−30 min) led to the formation of smaller organizations of spherical nature (20−40 nm) as shown by SEM experiments (Figure 7, lower part). Since each molecule of the initial aggregates contains three azobenzene units in the trans disposition, in all probability the disassembly is related to a partial conversion to the cis form. The reorganization into smaller microstructures that takes place upon prolonged irradiation is quite surprising. A deeper understanding of the entire process will require additional investigations, possibly including a computational approach. In any case, the oval aggregates are not of vesicular nature, since their formation in the presence of CF did not lead to encapsulation of the dye. Finally, we investigated the photoresponsive behavior of a C3 symmetry PBLG hybrid characterized by a much higher MW (154 000 Da). In this case, the self-assembly process obtained starting from an organic solution upon dialysis against water led again to the formation of oval microstructures. At variance with the lower MW polypeptide, however, for this architecture no J


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Figure 8. (A) SEM image of the fibers obtained from the high-MW C3 symmetry PBLG hybrid after processing by the electrospinning technique. (B) Image showing the macroscopic material collected by electrospinning. (C−E) SEM images of the microstructure transition occurring at different irradiation time at λ = 365 nm (30, 60, and 200 min, respectively).

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of this linearly organized material is tighter than in the case of the C3 symmetry PBLG hybrid, the molecules of the latter being characterized by three arms that can give rise to a 3D network.

ASSOCIATED CONTENT

S Supporting Information *

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Figures S1−S3: compound 5 (photoswitch behavior); Figures S4−S7: compound 11 (photoswitch behavior); Figure S8: DLS of CF vesicles from C2 symmetry PBLG; Figures S9−S11: C3 symmetry PBLG characterizations; Figures S12−S14: FT-IR absorption spectra of polypeptides; Figures S15−S26: 1H NMR spectra of the compounds reported in this work. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSIONS We took advantage from two known azobenzene-based amino acids as polymerization initiators for the construction of “smart” supramolecular systems endowed with photoswitchable behavior. The two starting materials are an Nα-protected derivative of the achiral Moroder’s (4-aminomethyl)phenylazobenzoic acid (8)31 and the Sewald/Cativiela’s azo-bridged di-α-aminodiester 4,4′-diazenediyldi-L-Phe (5).29,30 In particular, the NCA polymerization methodology, using the BLG heterocyclic derivative as substrate, was utilized to prepare two polypeptide hybrid molecules characterized by either C2 or C3 symmetry. In the first case, the bis-initiator was the di-α-amino-diester (5) itself, while in the second case the tris-initiator was the threebranched compound 11 with an 1,3,5-triazine central core. Moreover, both polypeptide hybrids were doubly or triply modified at their N-termini upon treatment with excess β-mercaptopropionic acid. This appropriate functionalization allowed us to covalently link them to gold nanoparticles. These azobenzene-containing compounds and materials were reversibly trans-to-cis photoisomerized, and the micro- and macroscopic changes in their 3D structures, self-aggregation properties, and overall characteristics were carefully analyzed by chromatography and UV−vis absorption/CD spectroscopies as well as by TEM/SEM nanomaterial techniques. In particular, the results reported here clearly indicate that the morphology of the self-aggregated systems can be finely tuned by a geometric (trans vs cis) modification of one (or more) azobenzene units of the building blocks. As the observed phenomena are hierarchical processes, conformational variations of a single azobenzene amino acid component propagate to the final 3D structures and macroscopic properties. The novel polypeptide hybrids presented in this paper are promising materials to expand the repertoire of photocontrolled releasers of biologically relevant molecules.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.C.). *E-mail: [email protected] (A.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the University of Padova (grant PRAT A. M. C91 J11003560001) and MIUR, Ministero dell’Istruzione, Università e Ricerca, (grant FIRB 2012) is gratefully acknowledged. A.M. thanks Dr. F. Caicci for assistance with TEM data recording.

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Photoresponsive Supramolecular Architectures Based on Polypeptide

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