Single-Chain Folding of Diblock Copolymers Driven by Orthogonal H

Aug 19, 2014 - (1, 10) Typically, two approaches are followed to induce single-chain folding of .... Abbreviations used below in the description of th...
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Single-Chain Folding of Diblock Copolymers Driven by Orthogonal H‑Donor and Acceptor Units Ozcan Altintas,†,‡ Peter Krolla-Sidenstein,§ Hartmut Gliemann,§ and Christopher Barner-Kowollik†,‡,* †

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76128 Karlsruhe, Germany ‡ Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany § Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany S Supporting Information *

ABSTRACT: We report the precision single-chain folding of narrow dispersity diblock copolymers via pairwise orthogonal multiple hydrogen bonding motifs and single chain selected point folding. Well-defined linear polystyrene (PS) and poly(n-butyl acrylate) (PnBA) carrying complementary recognition units have been synthesized via activators regenerated by electron transfer/atom transfer radical polymerization (ARGET ATRP) utilizing functional initiators yielding molecular weights of Mn,SEC = 10900 Da, Đ = 1.09 and Mn,SEC = 3900 Da, Đ = 1.10, respectively. The orthogonal hydrogen bonding recognition motifs were incorporated into the polymer chain ends of the respective building blocks (to yield an eight shaped single chain folded polymers). Diblock copolymer formation was achieved via the Cu(I) catalyzed azide−alkyne cycloaddition (CuAAC) reaction, while the single-chain folding of the prepared linear diblock copolymer−at low concentrations−was driven by orthogonal multiple hydrogen bonds via three-point thymine−diaminopyridine and six-point cyanuric acid−Hamilton wedge self-association. The self-folding process was followed by proton nuclear magnetic resonance (1H NMR) spectroscopy focused on the respective recognition pairs at low temperature. In addition, the single-chain folding of the diblock copolymer was analyzed by dynamic light scattering (DLS) and concentration dependent diffusion ordered NMR spectroscopy (DOSY) as well as atomic force microscopy (AFM), providing a limiting concentration for self-folding (in dichloromethane at ambient temperature) of close to 10 mg mL−1.

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INTRODUCTION Several types of macromolecules exist in nature, including proteins, nucleic acids, and polysaccharides, which are a constant source of inspiration for the development of new materials. One of the most important driving forces for synthetic macromolecular design is the emulation of natural processes and the design of chemical reaction sequences that are inspired by nature.1−5 Undoubtedly, reversible self-folding processes are nature’s approach for exerting control over the three-dimensional constitution of large molecules.6,7 Proteins are an important class of biological macromolecules present in all organisms. To be able to perform their biological function, proteins typically undergo folding into one or more specific spatial conformations leading to complex secondary structures such as α-helices or β-sheets, which can in turn self-organize into tertiary and quaternary structures.8 In most cases, these folded structures are stabilized by a number of noncovalent interactions including intramolecular hydrogen-bonding, aromatic-stacking, and hydrophobic interactions.9 Additional secondary interactions which can be found in the self-folding process of proteins (α-helices) are based on single polypeptide chains (random coil) via intramolecular hydrogen bonds. © 2014 American Chemical Society

Single-chain folding of synthetic macromolecules is a fast moving and innovative field in polymer science, constituting a promising pathway toward artificial, adaptative and smart single-chain polymer nanodevices.1,10 Typically, two approaches are followed to induce single-chain folding of synthetic macromolecules: (i) The collapse of the chain through interactions of functionalities within the side groups (repeating unit folding) or (ii) by positioning self-assembly points at specific places along the polymer chain (selective point folding).1 Biomacromolecules typically carry several hydrogen donor/acceptor systems, which lead to the singlechain folding of the primary structure into the local substructures enabling the biomacromolecules to fulfill a certain function.8 Recently, several examples have been provided in the field of controlled folding of synthetic polymers through covalent,11−15 dynamic covalent,16,17 or noncovalent bonds.18−25 Such single-chain synthetic polymers−when carrying specific functionalities−may be reversibly folded into Received: June 8, 2014 Revised: July 28, 2014 Published: August 19, 2014 5877

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Scheme 1. Single-Chain Eight-Shaped Folding of a Diblock Copolymer (PS-b-PnBA) via Two Orthogonal H-Bonding Entities

(AFM). Scheme 1 represents the overall architecture of the target molecule (in a simplified fashion) as well as its selffolding action.40

well-defined unimolecular structures akin to the folding action of biomacromolecules. Well-defined polymers carrying complementary recognition motifs based on hydrogen bonds at preselected position can be generated by a combination of controlled/living radical polymerization (CLRP) techniques26 such as atom transfer radical polymerization (ATRP)27 as well as modular ligation chemistries28−30 such as Cu(I) catalyzed azide−alkyne cycloaddition (CuAAC).31 These techniques allow for the preparation of single-chain self-folded polymers with sizes below 20 nm, which are required for potential applications in cellular delivery systems.32 More recently, we and others reported several examples of the single-chain folding of well-defined linear polymers carrying complementary recognition motifs at preselected positions within the polymer backbone or random copolymers through multiple hydrogen bonds, which emulatein a simple fashionthe self-folding behavior of natural biomacromolecules.33−36 However, no reports that address the one point single-chain folding of diblock copolymers as well as 8-shape folded structures featuring complementary hydrogen bonding recognition motifs exist. Block copolymers present an important polymer class, mainly based on their unique properties in solution and the solid state, which are a consequence of their molecular structure.37 In the context of self-folding polymer systems, block copolymers can be employed to introduce flexible and rigid domains into the lateral chain thus possibly allowing to tune and affect their folding behavior.38 Thus, in the present contribution, we unambiguously evidence the single chain folding of the diblock copolymer structure by devising a widely applicable synthetic technology platform on the example of a well-defined polystyrene-b-poly(n-butyl acrylate) diblock copolymer (a rigid-flexible diblock system), featuring two pairs of mutually orthogonal hydrogen bonding motifs (cyanuric acid−Hamilton wedge and thymine−diaminopyridine) at well-defined points within the polymer chains. In addition to providing a synthetic technology platform for self-folding block copolymer design, we investigate whether there is an effect of the molecular elasticity on the single chain folding behavior in the polystyrene/poly(nbutyl acrylate) system in an effort to understand the self-folding of block copolymer systems. To achieve the above synthetic aim, we employ activators regenerated by electron transfer (ARGET)39 ATRP allowing for a reduction of the amount of copper catalyst as well as CuAAc. Subsequently, we demonstrate the single chain folding of the diblock copolymer through complementary hydrogen bonding motifs into a confined geometry. The resulting single-chain self-folded polymers are characterized in-depth by proton nuclear magnetic resonance (1H NMR) spectroscopy (including a low temperature NMR study), dynamic light scattering (DLS) analyses, concentration dependent diffusion-ordered NMR spectroscopy (DOSY) as well as atomic force microscopy

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EXPERIMENTAL SECTION

Materials. Styrene (Sigma-Aldrich) and n-butyl acrylate were passed through a column of basic alumina (Acros) prior to use and subsequently stored at −19 °C. 6-Bromohexanol (97%, ABCR GmbH and Co. KG), 11-bromoundecanoic acid (99%, Aldrich), ethylenediaminetetraacetic acid disodium salt (EDTA) (99%, Acros), 2,2dimethoxypropane (99%, Acros), p-toluenesulfonic acid monohydrate (99%, Aldrich), 2,2-bis(hydroxymethyl)propionic acid (99%, Aldrich), cyanuric acid (CA) (99%, ABCR GmbH and Co. KG), 4dimethylamino pyridine (DMAP) (99%, Acros), N,N-dicyclohexylcarbodiimide (DCC) (99%, Acros), N,N-dimethylformamide extra dry (DMF) (99.8%, Acros), tetrahydrofuran extra dry (THF) (99.8%, Acros), sodium azide (99.8%, Acros), α-bromo isobutyric acid (98%, Aldrich), thymine (99.9%. Aldrich), 5-bromovaleryl chloride (97%. Aldrich), hydrochloric acid (37%, Carl Roth GmbH and Co. KG), benzoyl chloride (99%, Aldrich), oxalyl chloride (99%, Aldrich), sodium hydroxide (98%, Carl Roth GmbH and Co. KG), 5hydroxyisophthalic acid (97%, Aldrich), sulfuric acid (95%, Carl Roth GmbH and Co. KG), propargyl alcohol (99%, Acros), 3,3dimethylbutyryl chloride (99%, Aldrich), 2,6-diaminopyridine (98%, Aldrich), triethylamine (99.7%, ABCR GmbH and Co. KG), CuBr (99.9%, Acros) N,N,N′,N″,N″-pentamethyldiethyltriamine (PMDETA) (99.9%, Merck), cupric sulfate pentahydrate (99.5%, Aldrich), (+)-sodium L-ascorbate (98%, Aldrich) and succinic anhydride (97%, Aldrich) were used as received. Acetone, ethyl acetate, dichloromethane, cyclohexane, toluene, methanol, and tetrahydrofuran were purchased as analytical grade (Aldrich) and used as received. Characterization. Size Exclusion Chromatography (SEC). SEC measurements were performed on a Polymer Laboratories PL-GPC 50 Plus Integrated System, comprising an autosampler, a PLgel 5 μm bead-size guard column (50 × 7.5 mm) followed by three PLgel 5 μm Mixed-C and one PLgel 3 μm Mixed-E columns (300 × 7.5 mm) and a differential refractive index detector using THF as the eluent at 35 °C with a flow rate of 1 mL min−1. The SEC system was calibrated using linear poly(styrene) (PS) standards ranging from 476 to 2.5 × 106 g mol−1. Calculation of the molecular weight proceeded via the Mark−Houwink parameters, i.e., K = 14.1 × 10−5 dL g−1, α = 0.70 (PS).41a For poly(n-butyl acrylate) the following Mark−Houwink parameters were employed for universal calibration, i.e., K = 12.2 × 10−5 dL g−1, α = 0.70.41b Electrospray Ionization-Mass Spectrometry (ESI−MS). Mass spectra were recorded on an LXQ mass spectrometer (ThermoFisher Scientific, San Jose, CA) equipped with an atmospheric pressure ionization source operating in the nebulizer assisted electrospray mode. The instrument was calibrated in the m/z range 195−1822 using a standard containing caffeine, Met-Arg-Phe-Ala acetate (MRFA) and a mixture of fluorinated phosphazenes (Ultramark 1621) (all from Aldrich). A constant spray voltage of 3.5 kV and a dimensionless sheath gas of 8 and a sweep gas flow rate of 2 were applied. The capillary voltage, the tube lens offset voltage, and the capillary temperature were set to 60 V, 120 V and 275 °C, respectively. Fourier Transform Infrared Spectroscopy (FT-IR). Solid-state Fourier transform infrared spectra were recorded with an attenuated 5878

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total reflectance unit (Bruker, Golden Gate) coupled to a Bruker Vertex 80 Fourier-transform spectrometer, equipped with a tungsten halogen lamp, a KBr beam splitter, and DTGS detector. Nuclear Magnetic Resonance (NMR) Spectroscopy. The structures of the synthesized compounds were confirmed via 1H- and 13C NMR spectroscopy using a Bruker AM 400 MHz spectrometer for hydrogen nuclei and 100 MHz for carbon nuclei. Samples were dissolved in CDCl3, DMSO-d6 or CD2Cl2 (for the self-assembly study). The δ-scale was referenced with tetramethylsilane (δ = 0.00) as internal standard. Abbreviations used below in the description of the materials’ syntheses include singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), broad multiplet (bm), and unresolved multiplet (m). Diffusion Ordered Spectroscopy (DOSY) NMR. DOSY experiments were performed on a 400 MHz Bruker Avance III spectrometer equipped with a broadband 1H decoupling probe (PABBO) using an Eddy current compensated bipolar gradient pulse sequence (BPLED) at a temperature of 298 K. Proton pulse lengths were determined to be 10.12 μs and bipolar gradients of δ = 1.5−2.5 ms (depending on the diffusion behavior of the measured sample) length were incremented from G = 1 G/cm to 49 G/cm in 32 steps. Eight scans with 12 k complex data points were recorded for each increment with 8 dummy scans per experiment, leading to an overall experiment time of 17 min and 58 s per sample. The diffusion delay Δ was set to 100 ms. Processing was achieved using Topspin 3.1 with a Dynamics Center 2.0.4. After zero filling to 24k points and apodization using an exponential window function with an additional line width of 0.1 Hz, 1D increment spectra were Fourier transformed and the signal decay due to gradients was fitted using

f (G) = I0e(−γH

HQ:NSC18/No Al cantilever (MikroMasch, Lady's Island, USA) was used in intermittent contact mode (AC-Mode), at 1 mm scan size, 0.5 Hz scan rate and a resolution of 512 or 384 scan lines and points. Synthesis. Propargyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate (1),42 N1,N3-bis(6-(3,3-dimethyl-butanamido)pyridin-2-yl)-5hydroxyisophthalamide (3), 43 prop-2-yn-1-yl 3-((2-bromo-2methylpropanoyl)oxy)-2-(hydroxymethyl)-2-methylpropanoate (6),42 11-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)undecanoic acid (7),43 N-(6-aminopyridin-2-yl)-3,3-dimethylbutanamide (10),23 prop-2-yn-1-yl 4-chloro-4-oxobutanoate (11)43 and 6-(2,4,6-trioxo1,3,5-triazinan-1-yl)hexyl 2-bromo-2-methylpropanoate (13)23 were synthesized according to literature procedures. Synthesis of prop-2-yn-1-yl 3-((5-bromopentyl)oxy)-2-(hydroxymethyl)-2-methyl-propa-noate (2). 5-Bromopentanoyl chloride (2.03 mL, 15.1 mmol) was dissolved in 10 mL of dry THF and added dropwise to a solution of 1 (2.6 g, 15.1 mmol) and triethylamine (2.1 mL, 15.1 mmol) in 30 mL of dry THF and the mixture was stirred for 1 h at 0 °C. Subsequently, the solution was warmed to ambient temperature and stirred for 12 h. The precipitate formed during the reaction was filtered off and the solvent was subsequently removed under reduced pressure. The mixture was diluted with 200 mL of ethyl acetate, and the mixture was extracted two times with 50 mL of a saturated aqueous solution of sodium hydrogen carbonate. The organic phase was dried over sodium sulfate, filtered off and the solvent was subsequently removed under reduced pressure. The crude product was purified via column chromatography on silica gel eluting with ethyl acetate/n-hexane (1/4) to remove the side product (5bromopentanoyl chloride also reacted with both hydroxyl functions of 1), eluting with ethyl acetate/n-hexane (1/1) to obtain product 2 as pale yellow liquid (2.15 g, 45%). 1H NMR (400 MHz, CDCl3): δ (ppm) 4.74 (d, J = 2.5 Hz, 2H), 4.38−4.20 (q, 2H), 3.71−3.69 (d, 2H), 3.41 (t, J = 6.4 Hz, 2H), 2.49 (t, J = 2.5 Hz, 1H), 2.38 (t, J = 7.1 Hz, 2H), 1.98−1.68 (m, 4H), 1.25 (s, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 173.55, 173.15, 77.22, 75.23, 65.67, 64.92, 52.52, 48.33, 33.12, 32.90, 31.89, 23.41, 17.40. Synthesis of 2-(Hydroxymethyl)-2-methyl-3-oxo-3-(prop-2-yn-1yloxy)propyl 5-(3,5-Bis((6-(3,3-dimethylbutanamido)pyridin-2-yl)carbamoyl)phenoxy)pentanoate (4). 2 (1 g, 2.98 mmol), 3 (1.11 g, 1.98 mmol), potassium carbonate (1.24 g, 8.94 mmol), and 10 mL of DMF was stirred at ambient temperature for 24 h under an argon atmosphere. The solution was diluted with ethyl acetate and subsequently extracted twice with water. The combined organic phases were dried over Na2SO4, filtered, and evaporated. The crude product was purified via column chromatography on silica gel, eluting with ethyl acetate/dichloromethane (1/10) to remove the residual unreacted compound 2, eluting with ethyl acetate/dichloromethane (1/1) to obtain product 4 as a white solid (1.48 g, yield: 92%). 1H NMR (400 MHz, DMSO): δ 10.49 (s, 1H), 10.04 (s, 1H), 8.13 (s, 1H), 7.92−7.67 (m, 8H), 5.05 (t, J = 5.2 Hz, 1H), 4.71 (d, J = 2.4 Hz, 2H), 4.25−4.05 (m, 4H), 3.58−3.48 (m, 3H), 2.43 (t, J = 7.2 Hz, 2H), 2.32 (s, 4H), 1.77 (bm, 4H), 1.13 (s, 3H), 1.03 (s, 18H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 172.81, 172.47, 170.89, 164.88, 162.45, 158.60, 150.52, 150.03, 139.96, 135.53, 119.69, 117.12, 110.64, 109.96, 78.29, 77.58, 67.73, 65.36, 63.77, 52.00, 49.04, 48.13, 35.74, 33.03, 30.86, 29.55, 27.90, 21.07, 16.93. Synthesis of 2-(((5-(3,5-Bis((6-(3,3-dimethylbutanamido)pyridin2-yl)carbamoyl)phenoxy)pentanoyl)oxy)methyl)-2-methyl-3-oxo-3(prop-2-yn-1-yloxy)propyl 5-Bromopentanoate (5). 5-Bromopentanoyl chloride (0.33 mL, 2.46 mmol) was dissolved in 10 mL of dry THF and added dropwise to a solution of 4 (1 g, 1.23 mmol), 4dimethylaminopyridine (DMAP) (0.030 g, 0.25 mmol), and triethylamine (0.34 mL, 2.46 mmol) in 30 mL of dry THF at 0 °C and the solution was subsequently stirred at ambient temperature for 16 h. The precipitate formed during the reaction was filtered off and the solvent was removed under reduced pressure. The crude product was purified by column chromatography with dichloromethane/ethyl acetate (4:1) giving a white solid 5 after the removal of solvent (1.13 g, yield 94%). 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 10.45 (s, 2H), 10.00 (s, 2H), 8.09 (s, 1H), 7.94−7.54 (m, 8H), 4.70 (d, J = 2.4 Hz, 2H), 4.26− 4.03 (bm, 6H), 3.56 (t, J = 2.4 Hz, 1H), 3.49 (t, J = 6.5 Hz, 2H), 2.40−

G δ (Δ− δ ))D 3

2 2 2

with the proton gyromagnetic ratio γH and the full signal intensity I0. The resulting diffusion coefficients (D) of the polymer signals and the solvent are the result of the fitting procedure (see the Appendix to the Supporting Information). The size of the plotted points exceeds the values of the standard deviations of the fitting procedure, so no additional error bars are plotted. Dynamic Light Scattering (DLS). The solutions were prepared by dissolving the polymer in DCM at an appropriate concentration and then taking a part of this master solution to perform a series of concentration by dilution. After allowing the equilibrium to be established (around 30 min) the solutions were filtered via a 0.2 μL filter. Hydrodynamic diameters where determined with dynamic light scattering (Nicomp 380 DLS spectrometer from Particle Sizing Systems, Santa Barbara, CA, laser diode: 90 mW, 658 nm). The measurements were performed in automatic mode and evaluated with a standard Gaussian and an advanced evaluation method, the latter proceeding via an inverse Laplace algorithmen to analyze for multimodal distributions. The values provided in the study are the number-weighted average values. All measurements were determined at 90° to the incident beam. The associated autocorrelation functions can be found in the Supporting Information. Atomic Force Microscopy (AFM): Sample preparation. Mica discs, double-sided carbon tabs and metal specimen used for particle sample preparation are purchased from TED PELLA, Inc., Redding, USA. 14 mm mica discs were freshly cleaved in dichloromethane and separately stored in dichloromethane overnight, before being used as substrates for particle adsorption. 10 mL of particle suspension (8 mg mL−1 in DCM) were spread on a N2 dried substrate by a constant N2 gas flow immediately after deposition. To prevent electrical charge effects during the scanning process, the samples were placed on double-sided carbon tabs prefixed on a 10 mm metal specimen disc prior to force microscopy. Samples were dried for 30 min at 333 K and stored, protected from light, under vacuum until AFM analysis. Performing AFM. Atomic force microscopy was performed on an MFP-3D-BIO AFM (Asylum Research, Santa Barbara, USA) equipped with an electric sample holder and a standard cantilever holder. The electric sample holder was used to ground sample charges by contacting the mica sample surface with a conductive clamp. A silicone 5879

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2.28 (bm, 8H), 1.81−1.76 (bm, 4H), 1.12 (s, 3H), 1.02 (s, 18H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 172.38, 172.08, 170.89, 164.88, 150.51, 150.03, 139.94, 135.50, 119.60, 117.11, 110.63, 109.95, 77.85, 68.11, 64.85, 52.44, 49.04, 46.01, 40.64, 40.04, 39.73, 39.52, 39.31, 39.10, 38.89, 34.44, 33.30, 32.31, 31.38, 30.85, 29.55, 28.95, 28.82, 28.74, 28.65, 28.57, 28.37, 25.45, 24.33, 17.14. ESI−MS (M + Na)+ C48H61BrN6O11: theoretical, 999.35; experimental, 999.50. Synthesis of Prop-2-yn-1-yl 3-((2-Bromo-2-methylpropanoyl)oxy)-2-methyl-2-(((5-(5-meth-yl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)pentyl)oxy)methyl)propanoate (8). 7 (0.5 g, 1.61 mmol) was dissolved in 2 mL of dry DMF. 6 (0.620 g, 1.93 mmol) and DMAP (0.04 g, 0.32 mmol) were dissolved in 5 mL of dry DCM and subsequently added to the mixture. N,N′-dicyclohexylcarbodiimide (DCC) (0.5 g, 2.4 mmol) was dissolved in 3 mL of dry DCM and then added to the solution. The reaction was carried out at ambient temperature for 24 h. Solids were filtered off, the filtrate was concentrated and the crude product was purified by column chromatography on silica gel eluting with ethyl acetate/n-hexane (1/ 2) to give the product 8 as a pale yellow liquid (0.56 g, 55%). 1H NMR (400 MHz, DMSO): δ 11.18 (s, 1H), 7.52 (s, 1H), 4.75 (s, 2H), 4.26 (s, 2H), 4.19 (s, 2H), 3.59 (dd, J = 12.2, 4.7 Hz, 3H), 2.29 (t, J = 7.2 Hz, 2H), 1.88 (s, 6H), 1.75 (s, 3H), 1.61−1.43 (m, 4H), 1.24 (s, 15H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 172.32, 172.03, 171.56, 164.17, 150.75, 141.32, 108.25, 77.84, 77.74, 64.84, 64.75, 52.38, 47.02, 45.95, 34.40, 33.23, 32.24, 31.32, 28.73, 28.68, 28.52, 28.50, 28.33, 28.27, 25.70, 24.25, 22.89, 17.08, 11.80. ESI−MS (M + Na)+ C28H41BrN2O8: theoretical, 635.19; experimental, 635.33. Synthesis of α-(Alkyne/Thy) Functional Polystyrene (9). n-Butyl acrylate (12 mL, 83 mmol), CuBr2 (11.6 mg, 0.05 mmol), and Me6TREN (13.3 μL, 0.05 mmol) were dissolved in anisole (1 mL) in a Schlenk flask and subsequently purged with argon for 30 min. Sn(EH)2 (101.3 mg, 0.25 mmol) and 8 (306 mg, 0.5 mmol) were each dissolved in anisole (2 mL) in separate round-bottom flasks and purged with argon for 10 min. After removing oxygen, the initiator and reducing agent were transferred to the reaction flask via a cannula. The reaction mixture was subsequently placed into an oil bath tempered at 60 °C for 40 min and then the flask was cooled to ambient temperature in a water bath and opened to the atmosphere. The copper catalyst was removed by passing the solution over a short column of neutral alumina oxide. The purified polymer was obtained by 2-fold precipitation into cold methanol/water mixture (4:1, v/v) (100 mL). 1H NMR (400 MHz, CDCl3): δ (ppm) δ 8.06 (1H, NH of Thy), 6.97 (1H, CCH of Thy), 4.78 (2H, HCCH2−), 4.03 (CH2CH2OCO of PnBA), 2.29 (CH2CH2OCO of PnBA). Mn,NMR = 4200 Da, Mn,SEC = 3900 Da, and Đ = 1.09. Synthesis of Prop-2-yn-1-yl 4-((6-(3,3-Dimethylbutanamido)pyridin-2-yl)amino)-4-oxobutanoate (12). 11 (3.13 g, 18 mmol) was dissolved in 40 mL of dry THF and added dropwise to a solution of 10 (2.5 g, 12 mmol) and triethylamine (2.5 mL, 34 mmol) in 30 mL of dry THF at 0 °C. The solution was stirred at ambient temperature for 16 h. The precipitate formed during the reaction was filtered off and the solvent was subsequently removed under reduced pressure. The mixture was diluted with 200 mL of dichloromethane, and the mixture was extracted two times with 50 mL of a saturated aqueous solution of sodium hydrogen carbonate. The organic phase was dried over sodium sulfate, filtered off and the solvent was subsequently removed under reduced pressure. The crude product was purified via column chromatography on silica gel eluting with ethyl acetate/ dichloromethane (1/4) to obtain product 12 as white solid (2.73 g, 62%). 1H NMR (400 MHz, DMSO): δ 10.15 (s, 1H), 9.90 (s, 1H), 7.91−7.46 (m, 3H), 4.69 (d, J = 2.0 Hz, 2H), 3.54 (s, 1H), 2.71 (t, J = 6.1 Hz, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.30 (s, 2H), 1.01 (s, 9H). 13C NMR (101 MHz, DMSO): δ 172.18, 171.33, 171.08, 150.71, 140.30, 109.57, 109.43, 78.93, 78.13, 52.19, 49.50, 31.34, 31.10, 30.02, 28.70. ESI−MS (M + Na)+ C18H23N3O4: theoretical, 368.16; experimental, 368.25 Synthesis of α-CA-ω-Azide Functional Polystyrene (14b). Styrene (6 mL, 52 mmol), CuBr2 (5.9 mg, 0.026 mmol), and Me6TREN (7 μL, 0.026 mmol) were dissolved in anisole (5 mL) in a Schlenk flask and subsequently purged with argon for 30 min. 13 (98.7 mg, 0.26

mmol) and Sn(EH)2 (53 mg, 0.13 mmol) were each dissolved in anisole (1.5 mL) in separate round-bottom flasks and purged with argon for 10 min. After removing oxygen, the initiator and reducing agent were transferred to the reaction flask via a cannula. The reaction mixture was subsequently placed into an oil bath tempered at 90 °C for 24 h and the flask was next cooled to ambient temperature in a water bath and opened to the atmosphere. The copper catalyst was removed by passing the solution over a short column of neutral alumina oxide. The purified polymer (14a) was obtained by 2-fold precipitation into cold methanol (200 mL). Previously obtained CAPS-Br (14a) (2 g, 0.18 mmol) was dissolved in DMF (10 mL) and sodium azide (0.12 g, 1.8 mmol) was added. The reaction mixture was stirred for 24 h at ambient temperature. The reaction mixture was diluted with ethyl acetate and washed with distilled water. The organic layer was dried with Na2SO4, filtered and the solvent was removed in vacuum. The polymer was dried for 24 h under high vacuum and isolated as a white solid (14b) (1.8 g).44 Mn,SEC = 10900 Da, Đ = 1.09. 1 H NMR (400 MHz, CD2Cl2): δ (ppm) 7.8 (2H, NH of CA), 7.01− 6.39 (5H, ArH of PS), 3.92 (2H, CH-N3), 3.81 (2H, CH2CH2N), 3.52 (2H, CH2CH2OCO), 1.78−1.18 (aliphatic protons of PS). Synthesis of α-CA-ω-(HW and Azide) Functional Polystyrene (15). 14 (0.65 g, 0.056 mmol), 5 (0.082 g, 0.085 mmol), copper(II) sulfate pentahydrate (0.028 g, 0.11 mmol), and sodium ascorbate (0.023 g, 0.11 mmol) were dissolved in DMF (5 mL). The resulting mixture was stirred at ambient temperature for 24 h and subsequently diluted by the addition of CH2Cl2 and extracted with 5% EDTA solution to remove the copper (a catalyst also complexed by the recognition units). The organic phase was dried over Na2SO4, concentrated, and subsequently precipitated two times into 80 mL of cold methanol. The polymer was dried for 24 h under high vacuum resulting in a white powder (15a) (0.66 g). The end group transformation from bromine to azide was accomplished following the above procedure for 14 from 15a to 15b. 1H NMR (400 MHz, CDCl3) δ (ppm) 8.15−7.71 (aromatic protons of HW), 7.06−6.61 (aromatic protons of PS), 5.17−5.02 (3H, adjacent protons of triazole ring), 4.14−4.06 (4H, ester protons of 5), 3.22 (2H, −CH2N3), 2.33 (4H, −(CH2− (CH3)3)2), 1.83−1.16 (aliphatic protons of PS), 1.09 (18H, -(CH2− (CH3)3)2). Mn,SEC = 11500 Da, Đ = 1.04. Synthesis of CA-PS-HW-Thy-PnBA-Azide (16). 15 (0.62 g, 0.054 mmol), 9 (0.34 g, 0.081 mmol), copper(II) sulfate pentahydrate (67 mg, 0.27 mmol), and sodium ascorbate (54 mg, 0.27 mmol) were dissolved in DMF (10 mL). The resulting mixture was stirred at ambient temperature for 24 h. Subsequently, an azide-functionalized resin (200 mg) was added to remove the excess of 9 and the reaction mixture was stirred for a further 14 h. The mixture was diluted with THF, the resin was filtered off and the solvent was removed under reduced pressure. The bromide functionality was converted to an azide group as described above for 14b. The crude polymer was purified and dried as described above for 14b to give 16b as white solid (750 mg). 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.15−7.71 (aromatic protons of HW), 7.06−6.61 (aromatic protons of PS), 5.21−5.05 (5H, adjacent protons of triazole ring), 4.28 (2H, adjacent protons of triazole ring), 4.03 (CH2CH2OCO of PnBA), 2.33 (4H, −(CH2− (CH3)3)2), 2.27 (CH2CH2OCO of PnBA), 1.83−1.16 (aliphatic protons of PS), 1.09 (18H, −(CH2−(CH3)3)2). Mn,SEC = 15000 Da, Đ = 1.11. Synthesis of Diblock Polymer with Dual Complementary Motifs (17). 15 (0.52 g, 0.035 mmol), 12 (0.25 mg, 0.07 mmol), copper(II) sulfate pentahydrate (0.027 g, 0.105 mmol) and sodium ascorbate (0.21 g, 0.105 mmol) were dissolved in DMF (5 mL). The resulting mixture was stirred at ambient temperature for 24 h. The crude polymer was purified and dried as described above for 14a to give 17 as a white solid (750 mg). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.15−7.71 (aromatic protons of HW), 7.06−6.61 (aromatic protons of PS), 5.21−5.05 (5H, adjacent protons of triazole ring), 4.28 (2H, adjacent protons of triazole ring), 4.03 (CH2CH2OCO of PnBA), 2.49 (4H, COCH 2 CH 2 CO) 2.33 (4H −(CH 2 −(CH 3 ) 3 ) 2 ), 2.27 (CH2CH2OCO of PnBA), 1.83−1.16 (aliphatic protons of PS), 1.09 (18H, −(CH2−(CH3)3)2). Mn,SEC = 15400 Da, Đ = 1.11 5880

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Scheme 2. Synthetic Strategy for the Preparation of the Building Blocks and the Complementary Recognitions Motifsa

a Key: (a) 5-bromopentanoyl chloride, TEA, THF, room temperature; (b) K2CO3, 3, DMF, room temperature; (c) 5-bromopentanoyl chloride, TEA, DMAP, THF, room temperature; (d) 7, DCC, DMAP, DCM, DMF, room temperature; (e) n-butyl acrylate, CuBr2, Me6TREN, Sn(EH)2, anisole, 60 °C; (f) TEA, THF, room temperature

■

RESULTS AND DISCUSSION Single-chain folding technology is a relatively new and dynamic field of polymer science. In the current contribution, a block copolymer carrying two complementary recognition motifs at defined positions within the chain is synthesized by a combination of advanced polymer synthetic technologies, i.e. ARGET and CuAAC. The prepared block copolymers can fold into a three-dimensional structure able to mimic−on a simplistic level−the folding/unfolding processes of naturally occurring large biomolecules. In the following section, the synthesis of the dual self-folding system block copolymer will be discussed in detail. The synthetic section is followed by the in-depth investigation of the single chain self-folding process as well as a critical comparison to existing systems. Preparation of a Linear Polymer Carrying Binding Motifs at Well-defined Positions. In the following section, the preparation and characterization of the linear homopolymers, diblock copolymers and corresponding complementary

recognition motifs are discussed (see Scheme 2 and Scheme 3). First, an esterification reaction was carried out between 1 and 5bromovaleryl chloride in the presence of TEA as reagent and THF as solvent. The 1H NMR spectrum of 2 indicates that new characteristic proton resonances for the CCH2OCO and CH2CH2Br functions are observed at 4.38 and 3.41 ppm, respectively (refer to the Supporting Information, Figures S1 and S2). Subsequently, an etherification reaction was readily accomplished in the presence of K2CO3 in DMF, which was confirmed by 1H NMR spectroscopy indicating the proton resonance associated with the amides of 4 at 10.47 and 10.03 ppm (refer to the Supporting Information, Figures S3 and S4). Next, a Hamilton wedge compound carrying a bromine and an alkyne functionality was synthesized via esterification of compound 4 with 5-bromovaleryl chloride in the presence of TEA as reagent, DMAP as catalysts and THF as solvent. The structure of compound 5 was confirmed via 1H NMR, 13C 5881

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Scheme 3. Synthetic Strategy for the Preparation of the Diblock Copolymer with Orthogonal Complementary Motifs alongside the Polymer Chaina

Key: (a) Styrene, CuBr2, Me6TREN, Sn(EH)2, anisole, 90 °C; (b) NaN3, DMF, room temperature; (c) 5, CuSOx·5H2O, sodium ascorbate, DMF, room temperature; (d) 9, CuSOx·5H2O, sodium ascorbate, DMF, room temperature; (e) 12, CuSOx·5H2O, sodium ascorbate, DMF, room temperature.

a

NMR and ESI−MS (refer to the Supporting Information, Figure S5, S6, S7 and Table S1). The related ATRP initiator carrying thymine and alkyne functionalities for n-butyl acrylate polymerization was synthesized via an esterification reaction between 6 and 7 in the presence of DCC as reagent, DMAP as catalyst and DCM/ DMF as solvents. The 1H NMR spectrum of 8 indicates that the proton resonance corresponding to COOH has disappeared and new characteristic proton signals for HCCCH2OCO and C(CH3)Br are observed at 4.73 and 1.88 ppm (refer to the Supporting Information, Figure S8). Its structure and purity was additionally determined by 13C NMR and ESI−MS (refer to the Supporting Information, Figures S9 and S10 and Table S2). The ARGET ATRP of n-butyl acrylate to polymer 8 was performed at 60 °C in the presence of anisole, affording a narrow monomodal molecular weight distribution (see Figure 1). The chemical structure of the obtained polymer was additionally verified via 1H NMR spectroscopy (refer to the Supporting Information, Figure S11). The molecular weight of the final α-(alkyne/thymine) functional PnBA was determined by comparing the 1H NMR integration values of the protons from the polymer chains (COOCH2CH2−) at 4.03 ppm with

Figure 1. SEC traces of the precursor and target polymers. The structural images of the related polymers can be found in Schemes 2 and 3. 9 (Mn,SEC = 3900 Da, Đ = 1.09), 14 (Mn,SEC = 10900 Da, Đ = 1.10), 15 (Mn,SEC = 11500 Da, Đ = 1.10), 16 (Mn,SEC = 15000 Da, Đ = 1.11), 17 (Mn,SEC = 15400 Da, Đ = 1.11).

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The targeted diblock copolymer was modularly synthesized using the polymer with the azide functionality carrying CA/ HW recognition motifs (15) and the polymer with the alkyne functionality and featuring the Thy motif (9) (see Scheme 3). To drive the reaction to completion, a slight excess of alkyne functional PnBA was used. After 24 h of reaction, the residual alkyne groups were reacted by addition of azide functional Merrifield resin overnight and thus the excess of alkyne polymer could readily be removed by filtration. Formation of the PS-b-PnBA diblock copolymer (16) was determined by a complete shift in the SEC trace toward higher molecular weight, as can be seen in Figure 1. Additionally, no increase of Đ was observed for the formed diblock copolymer. The 1H NMR spectrum of 16 evidences that the resonances associated with CH2N3 have completely disappeared at 3.21 ppm and new resonances associated with the CH2 next to the triazole ring and CH2CH2OCO of the PnBA repeating unit are observed at 4.31 ppm and 4.03, respectively, suggesting that the CuAAC reaction between 9 and 15 was quantitative (refer to the Supporting Information, Figure S17). Further evidence for the diblock copolymer formation was obtained from the FT-IR spectra, where the band at 2090 cm−1, corresponding to the azide functionality, completely disappeared. The same methodology was applied to convert the ω-bromide end group of the polymer into an azide using sodium azide in DMF, yielding an ω-azide functionalized diblock copolymer. Copper catalyzed azide−alkyne conjugation chemistry was subsequently employed to couple 16 and compound 12 in the same fashion as 15 and 16 to afford the corresponding diblock copolymer with orthogonal complementary recognition motifs. The SEC traces (see Figure 1) indicate a slight shift to the higher molecular weight region (from 15000 to 154000 Da) and the 1H NMR spectrum confirmed that new proton resonances for C OCH2CH2CO were observed at 2.49 ppm (refer to the Supporting Information, Figure S17). The SEC and 1H NMR spectrum underpin the introduction of the small molecule 12 at the polymer chain-end. Single-Chain Folding of the Diblock Copolymer via Orthogonal Multiple Hydrogen Bonds. The dynamic nature of single-chain polymers in their folded and unfolded state makes them challenging to be characterized using conventional polymer analysis techniques.45 A combination of several techniques for the characterization of noncovalent interactions can provide convincing evidence of the single-chain folding of synthetic polymers. Therefore, the H-bonding directed single-chain folding of linear polymers is efficiently characterized by several (concentration dependent) NMR spectroscopic techniques, allowing the study of dynamic interactions in a wide range of concentrations and temperatures. On the other hand, dynamic light scattering is critically important and allows following the size distributions of singlechain polymers in variable solvents and at variable concentrations. Furthermore, atomic force microscopy (AFM) is a regularly employed technique used to visualize the single-chain folding of a linear conformation. In the present study, the formation of the single-chain folded structures was monitored by 1H proton and DOSY NMR spectroscopy, DLS, and AFM. For temperature based 1H NMR studies, a solution of 17 was prepared in dichloromethane at a concentration of 6.5 mg mL−1 and left for 1 h at ambient temperature to allow for the self-assembly. Subsequently, a 1H NMR spectrum was recorded at 25 °C as shown in Figure S19 in the Supporting Information. It is evident from Figure S19 that at the temperature of 25 °C

the CH2 protons of the alkyne end-group at 4.73 ppm (Mn,NMR = 4200 Da). The NMR deduced number-average molecular weight is in good agreement with SEC based value of 3900 g mol−1 (based on a universal calibration procedure employing the MHKS parameters for poly(n-butyl acrylate). It should be noted that the monomer conversion was kept low to avoid undesired side-reactions, e.g. radical transfer to the alkynemoiety. Furthermore, an alkyne functional diaminopyridine (DAP) molecule was synthesized via an amidation reaction between 10 and 11 in the presence of TEA as reagent and THF as solvent. The product was analyzed via 1H NMR, 13C NMR, and ESI−MS (refer to the Supporting Information, Figures S12, S13, and S14 and Table S3). Styrene was polymerized via ARGET ATRP in the presence of a CuBr2/Me6TREN catalyst system in anisole utilizing a cyanuric acid (CA) bearing initiator. After ARGET ATRP polymerization, the bromide end groups present in the polymers were converted to azide moieties by nucleophilic substitution using sodium azide in DMF, yielding α-CA ω-azide functionalized polystyrene (14b). A complete upfield shift of the methine protons adjacent to the end groups in the 1H NMR spectrum (refer to the Supporting Information, Figure S15) and the appearance of signals in the FT-IR spectra close to 2090 cm−1 (see Figure 2), indicate the presence of the azide

Figure 2. FT-IR spectra of 14, 15, 16, and 17. a refers to the associated precursor polymer before the azide transformation.

function. The number-average molecular weight, Mn, of 14 was determined via SEC (molecular weight reported relative to PS standards) indicating unimodal molecular weight distributions with a low Đ (polydispersity). The Hamilton wedge-functionalized polymer (15) was subsequently synthesized via copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC) of the azide chain-end functionalized polymer with an alkyne functional Hamilton wedge compound (5) (Scheme 2). The successful ligation can be followed by the shift of the full molecular weight distribution determined by SEC (see Figure 1) as well as new proton resonances associated with the CH2 and CH next to the triazole ring observed at 5.21 ppm and the new resonance corresponding to the Hamilton wedge moiety at 8.24−7.67 ppm in the 1H NMR (refer to Supporting Information, Figure S16). The transformation of the bromine end-group to the azide chain-end functionality was successfully confirmed by 1H NMR spectroscopy indicating the shift of the proton resonance at 3.34 ppm of the CH2-Br to 3.21 as CH2-N3 (refer to Supporting Information, Figure S16) and the appearance of azide signals in FT-IR spectra (see Figure 2). 5883

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the imide protons of CA are in a coalescence regime, where the signals of the bound NH protons are very broad or cannot be detected anymore.34 However, the 1H NMR spectrum reveals a strong shift of the amide protons of the Hamilton wedge to lower field resonance signals at 9.33 and 9.76 ppm. Furthermore, a new resonance appears at 9.17 ppm corresponding to the NH proton of the thymine unit in the self-assembly motif. At low temperatures, the association− dissociation exchange is relatively slow on the NMR time scale and the interactions are much stronger between the recognition units. Therefore, the solution of polymer 17 was cooled to −30 °C and the 1H NMR spectra indicate a significant downfield shift of the resonances associated with the protons that undergo self-assembly at 13.52 ppm for CA, at 10.04 and 9.61 for HW, and at 9.97 ppm for Thy, thus evidencing that supramolecular assembly is operational for all hydrogen recognition units (see Figure 3). It can be concluded that, by lowering the temperature, the thermodynamic equilibrium is shifted toward the self-folded species.

Figure 4. Hydrodynamic diameter, Dh, distributions of polymer 17 recorded at a 90° angle in THF and DCM at 25 °C. The concentration of polymer 17 was kept constant at 6.5 mg mL−1. Typical autocorrelation functions for all DLS measurements can be found in Figures S23, S24, S25, and S26 in the Supporting Information.

reference−a polystyrenes standard (Mn,SEC = 17400 Da; Đ = 1.04) was employed in DLS measurements to determine a size distribution in THF and DCM at 6.5 mg mL−1 (refer to the Supporting Information, Figure S20). The Dh of the PS standards was determined as close to 5.6 nm in DCM and 5.8 nm in THF. The Dh of the nonfunctional polymer displays basically no change in the size distribution when switching the solvent. Furthermore, as even more stringent control experiment, a linear polystyrene-b-poly(n-butyl acrylate) (PS-bPnBA) (featuring a similar block length ratio as polymer 17, refer to Figure S21 in the Supporting Information), which does not contain any complementary binding motifs, was assessed in the two solvents. The DLS results (refer to Figure S22 in the Supporting Information) unambiguously evidence that there is no significant difference of the size distributions in the two solvents, thus implying that the size change for polymer 17 when going from DCM to THF is associated with the folding action of the system. While the above data suggest that self-folding indeed occurs, a quantitative measure for the concentration at which selffolding occurs, csingle fold, needs to be established. Diffusionordered spectroscopy (DOSY) NMR provides a method of molecular size determination via the measurement of diffusion coefficients by the NMR signal decay within a defined gradient spin echo.47 DOSY NMR has been applied for the characterization of supramolecular polymers in solution as diffusion coefficients decrease with increasing size of the polymers,48−51 yet never to self-folding single chain H-bonding systems via selective point folding. Thus, especially concentration dependent DOSY NMR can provide information whether the formation of the hydrogen bond structure results in intramolecular single-chain folding or intermolecular multiple block copolymer formation. Initially, DOSY NMR experiments on polymer 17 (Mn,SEC= 15400, Đ = 1.11) were performed at 298 K in dichloromethane-d2 at a concentration of 104 mg mL−1 and the diffusion coefficient was determined as being close to 2.95 × 10−11 m2 s−1. In order to identify the onset of the single chain folding regime, a series of concentrations of 17 was prepared (by subsequent dilutions by a factor of 2) and the diffusion coefficient determined. The on-set of single chain

Figure 3. 1H NMR spectrum of the single chain self-folding of polymer 17 in dichloromethane-d2 at −30 °C showing the resonances associated with the bound imide protons of cyanuric acid (CA, a) and thymine (Thy, d) as well as the resonances of the amide protons of the Hamilton wedge (HW, b and c). The concentration of polymer 17 was kept constant at 6.5 mg mL−1.

The disassembly of the complementary recognition units on the polymer chains can be achieved by adding a cosolvent or pure polar solvent that perturbs the hydrogen bond formation46 such as THF, DMSO or methanol. Dynamic light scattering has developed into a powerful and versatile tool for estimating the size distribution of small particles or polymers in solution, effective for particles in the size range of a few nanometers up to several micrometers. The size distribution of the species formed by the self-assembly and disassembly in THF and DCM was determined using DLS relative to a polystyrene standard. Therefore, we performed DLS measurements on solutions of 17 in THF at 6.5 mg mL−1. The Dh of 17 in the THF solution was determined to be close to 8.1 nm. The related size distributions are shown in Figure 4. As the Dh of 17 was determined as 5.9 nm in DCM at 6.5 mg mL−1, a clear reduction in the average size from 8.1 nm in THF to 5.9 nm in DCM was observed. Since 1H NMR spectroscopy clearly shows the formation of self-assembly between complementary motifs, this is consistent with a collapse of the polymer chain as a result of H-bond formation. The observed reduction in size distribution is in good agreement with those reported earlier using these complementary motifs.34 In addition−as a 5884

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5.3 nm (3.25 mg mL−1), respectively (see Figure 5, right-hand y-axis). Importantly, an assessment of the self-diffusion coefficient of deuterated dichloromethane solution as a function of the concentration of polymer 17 indicates that the diffusion coefficient is almost not affected by the increasing viscosity of the solution (refer to Figure S27 in the Supporting Information). Furthermore, polymer 17 was characterized by DLS at four concentrations (3.25, 6.5, 13, and 26 mg mL−1) as a direct comparison to the DOSY NMR derived hydrodynamic diameter values. The Dh of the polymers in these solutions were measured to be close to 6.0, 5.9, 7.6, and 12.2 nm, respectively (refer to Figure S28 in the Supporting Information). The average Dh of polymer 17 increases with concentration, in excellent agreement with the DOSY measurements, indicating that higher concentrations favor the formation of interchain assemblies of 17 (refer to Figure 5). As the concentration is reduced, the diameter of 17 is observed to progressively decrease eventually reaching a limiting value, thus suggesting that more compact single-chain self-assembled cyclic structures are formed. In addition, the concentration dependent hydrodynamic diameters of polymer 17 determined by DLS and DOSY experiments are in excellent agreement. Figure 5 summarizes the DOSY and DLS results as well as graphically presents the determination of the limiting concentration for self-folding. In addition, the unfolding action of polymer 17 when going from DCM to THF was monitored by DOSY (refer to Figure S29 in the Supporting Information), giving the same trend as the DLS measurements: The polymer unfolds in THF. Atomic force microscopy (AFM) can be helpful for imaging nanostructures including those generated from with individual macromolecules53 such as single-chain folding systems.20 In addition to the solution analysis, we thus attempted to visualize the self-collapsed block copolymer chains. Therefore, polymer 17 was adsorbed onto freshly cleaved mica discs from diluted dichloromethane solutions (0.1 μg L−1). Height AFM map images of the cast surfaces with a scan size of 1 × 1 μm are depicted in Figure 6. The original mica surface and the cast polymer 17 are depicted in Figure 6, parts a and b, respectively. Because of the inherent dispersity of polymer 17although relatively small−one can expect that the generated single chain assemblies feature a certain size distribution. Inspection of Figure 6c shows that the observed species feature a height of approximately 2 nm and an average diameter of 51 nm which are similar in size to other reports for single-chain folding systems. For example, Meijer and co-workers reported54 the formation of single-chain polymeric nanoparticles formed via noncovalent driven repeat unit folding (Mn,SEC = 15 300 Da, Đ = 1.31) and studied the particles via AFM, finding a particle height of close to 4.2 nm on average with an average diameter of 46.5 nm. Recently, our research group also reported12 height measurements for single-chain folding polymeric nanoparticles constructed by covalent cross-linking (Mn,SEC = 20 200 Da, Đ = 1.56) identifying an (average) of approximately 3 nm. A direct comparison with the diameters observed via DLS in solution is challenging as certainly different diameters will be observed in the solid state vs solution. In addition, the AFM measurement can lead to images which−compared to the real diameter− show an increased width of the single-chain folded species due to the effect of AFM tip convolution.55 The diameters extracted from AFM images are known to yield larger values larger than those observed in solution in DLS.12,54 While the Dh of

folding−as opposed to intermolecular association−should be associated with a constant diffusion coefficient, i.e. Rh, from a given concentration onward. The further determined diffusion coefficients of polymer 17 in the concentration series read 6.56 × 10−11 m2 s−1 (52 mg mL−1), 1.05 × 10−10 m2 s−1 (26 mg mL−1), 1.86 × 10−10 m2 s−1 (13 mg mL−1), 1.96 × 10−10 m2 s−1 (6.5 mg mL−1) and 1.98 × 10−10 m2 s−1 (3.25 mg mL−1), respectively and are depicted in Figure 5. Inspection of Figure 5

Figure 5. Diffusion coefficient (D, left y-axis, closed circles) and hydrodynamic diameter (Dh, right y-axis, open circles) of polymer 17 as a function of concentration (3.25, 6.5, 13, 26, 52, 104 mg mL−1) in CD2Cl2 determined via DOSY NMR as well as the hydrodynamic diameter (Dh, right y-axis) of polymer 17 as a function of concentration (3.25, 6.5, 26, mg mL−1) in DCM (open triangles) and (6.5 mg mL−1) in THF (closed square) determined via DLS. The concentration below which single chain folding occurs, csingle fold, is close to 10 mg mL−1.

indicates that from a concentration of 10 mg mL−1 onward−the single folding threshold concentration csingle fold − the diffusion coefficient becomes invariant to concentration, thus supporting the formation of a compact single-chain self-assembled cyclic structure. Furthermore, by measuring the diffusion coefficient of polymer species via DOSY measurements, one may obtain information on the size of the polymers in solution. The obtained diffusion coefficients were further employed to calculate the hydrodynamic diameter of the related polymers via the Stokes−Einstein equation Rh =

kT 6πηD

(1)

where Rh (hydrodynamic diameter, Dh = 2Rh) is the hydrodynamic radius of the polymer coil in meters, k is the Boltzmann constant (1.380 × 10−23 J K−1), T is the temperature in Kelvin (K), η is the viscosity of the solvent in Pascal seconds and D is the diffusion coefficient. The diffusion coefficient of the polymer 17 in dicholoromethane-d2 at 298 K at a concentration of 104 mg mL−1 reads 2.95 × 10−11 m2 s−1 and the viscosity of the solvent reads 0.413 mPa·s.52 Applying the Stokes−Einstein eq 1, the hydrodynamic diameter (DhDOSY) of the polymer 17 is calculated to be 35.9 nm, again showing the formation of higher order self-assembled aggregates. The DhDOSY of polymer 17 at the various other concentrations was determined to be close to 16.2 nm (52 mg mL−1), 10.1 nm (26 mg mL−1), 5.7 nm (13 mg mL−1), 5.4 nm (6.5 mg mL−1), and 5885

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Figure 6. AFM topography images of (a) a freshly cleaved mica-surface and (b) a sample containing polymer 17 cast from a 0.1 μg L−1 solution in DCM. The red line in the topography image depicts which particles have been analyzed in part c, representing a height cross section of two of the particles determined by AFM.

have been employed (our initial homopolymer example featured Mn,SEC = 15400 Da; Đ = 1.12).34 While their individual synthesis as well as their self-folding into single chain nanoparticles has been unambiguously evidenced, it is worthwhile to compare their mean diameters in the folded and unfolded state. It must, however, be noted that the geometries prepared in both studies is somewhat different: Herein, we self-assemble orthogonally into an eight shaped geometry with the focal folding points in the same position (the central line cross over in the figure eight geometry), whereas in the previous homopolymer design the eight was achieved by forming a circular structure with one binding motif pair, while the focal point was generated by tying the chains together in the a midchain position of the circle with the other orthogonal pair. The design difference has synthetic reasons: In the previous study,34 sequential chain extension was employed as a method to obtain the linear block homopolymer. However, this method has limitations such as the decrease in chain-end fidelity with increasing number of polymer blocks and a modular approach has inherent advantages such a concomitant reduction in dispersity of the conjugate with every ligation step. The DLS studies indicate that there is a significant difference in terms of the mean diameters in the folded (approximately Dh = 5.9 and 8.1 nm) and the unfolded state (approximately Dh = 8.1 and 10.1 nm), respectively, for the two systems. In addition, the 1 H NMR studies demonstrate that there is a slight but significant difference in the chemical shift of the NH protons of the thymine moiety. The interactions between the thymine and

polymer 17 was also determined as 7.6 nm via DLS in DCM, the AFM study indicate approximately 51 nm as the average size, a values clearly in excess of the solution number. Nevertheless, the data could be congruent with the formation of single-chain folded species and are in good agreement with earlier reported values of polymeric nanoparticle, especially when compared to earlier reports, where the single chain nanoparticles are prepared from similar molecular weight polymers (Mn,SEC = 17 300 Da, Đ = 1.29).56 Moreover, no large and undefined aggregates can be observed in the AFM micrographs. However, it is clear that the AFM images should not be overinterpreted and can only serves as an additional qualitative indication for nanoparticle formation as they are in agreement with AFM studies on earlier reported single chain nanoparticle systems. Finally, it is instructive to compare the current diblock copolymer self-folding system to the homopolymer system described in a previous study (Mn,SEC = 15400 Da; Đ = 1.12).34 In the current study we establish a general strategy for the synthesis of diblock self-folding systems featuring pairwise orthogonal moieties, whereas in the previous study, we demonstrated the orthogonal nature of the employed hydrogen binding motifs and subsequently the self-folding action of a single polymer chain based on their orthogonal interactions. In both studies, DLS and 1H NMR have been employed to characterize both self-folding process. The final polymers carrying the complementary motifs at the preselected positions and in both studies polymers of similar overall molecular weight 5886

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Table 1. Comparison between the Homopolymer34 and the Current Diblock Copolymer Systems in Terms of Their Hydrodynamic Diameter, Dh, as Determined via DLS as well as the Chemical Shifts Observed during 1H NMR Experiments of the NH Proton Resonancesa polymer

folded state Dh (DLS)/nm

non-folded state Dh (DLS)/nm

homopolymer diblock copolymer

8.2 5.9

10.1 8.1

chemical shift, CA/ppm chemical shift, HW/ppm 13.40 13.52

10.09; 9.56 10.04; 9.61

chemical shift, Thy/ppm 9.01 9.97

a

The Dh values were determined in DCM (folded state) and THF (non-folded state) and the NMR chemical shifts are reported for CA, HW, and Thy.

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diaminopyridine motifs take place in the flexible part of the single-chain folding system constituted by the poly(n-butyl acrylate) segment. Such an affect could be due to the chain flexibility of poly(n-butyl acrylate) (see Table 1), which in turn affects the chemical surrounding and thus exchange processes of the imaged protons.

ASSOCIATED CONTENT

S Supporting Information *

1 H NMR, 13C NMR, and ESI−MS spectra of the following compounds: 2, 4, 5, 8, 9, 12, 11, and 14, 1H NMR spectra of the polymers 14a, 14b, 15a, 15b, 16a, 16b, and 17, and mean hydrodynamic diameters of the polystyrene standard and the reference block copolymer in DCM and THF as well as primary DOSY data. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSIONS We report a viable modular synthetic strategy for the design of a well-defined block copolymer system featuring pairwise orthogonal hydrogen bonding motifs−a cyanuric/acid-Hamilton wedge and thymine/diaminopyridine couple −which allows for the defined folding of the block copolymer into a predefined geometry. The homopolymers, i.e. cyanuric acid functional polystyrene and thymine functional poly(n-butyl acrylate) have been prepared via activators regenerated by electron transfer/ atom transfer radical polymerization (ARGET ATRP) utilizing the respective functional initiators. A Hamilton wedge functionality was attached to the ω-position of the polystyrene via a Cu(I) catalyzed azide−alkyne cycloaddition (CuAAC) reaction. The diblock copolymer was modularly synthesized via a CuAAC reaction using polystyrene with an azide functionality carrying CA/HW recognition motifs and the poly(n-butyl acrylate) with alkyne functionality and Thy moiety. A CuAAC reaction was subsequently employed to insert the diaminopyrinde recognition motif into the diblock copolymer. All small molecules carrying complementary recognition motifs were analyzed by 1H NMR, 13C NMR, and ESI−MS. The welldefined precursor macromolecules were characterized via both 1 H NMR and SEC techniques. The folding process of the diblock copolymer was monitored by proton nuclear magnetic resonance (1H NMR) spectroscopy between complementary two recognition pairs at low temperatures. The single chain folding of the diblock copolymer was further evidenced by dynamic light scattering (DLS) and−importantly−concentration dependent diffusion ordered NMR spectroscopy (DOSY) as well as atomic force microscopy (AFM). In particular, the concentration dependent DOSY analysis demonstrated that there exists a threshold concentration, csingle fold, at which single chain collapse is observed in contrast to intermolecular aggregation. Importantly, the DLS and DOSY results yield complementary results for the compactness of the folded state. The folded structure hydrodynamic diameter is close to 6 nm in size (in dichloromethane), while it expands to over 8 nm (in THF) in its nonfolded configuration. A related diblock copolymer system consisting of a styrene and butyl acrylate block with approximately the same block length ratio, yet no Hbonding functionalities, showed no diameter change upon switching the solvent. It thus appears that changes in the segment type used in the construction of the self-folding system has a significant influence on the folded state.

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AUTHOR INFORMATION

Corresponding Author

*(C.B.-K.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.B.-K. is grateful for continued support from the Karlsruhe Institute of Technology (KIT) in the context of the Helmholtz BIF program and the Ministry of Science and Arts of the State of Baden-Württemberg. The authors are additionally grateful to David Schulze-Suenninghausen (KIT) and Prof. Burkhard Luy (KIT) for fruitful discussions regarding DOSY measurements as well as Kim K. Oehlenschlaeger (KIT) for the on-line NMR temperature dependent measurement.

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