Maltopentaose-Conjugated CTA for RAFT Polymerization Generating

Oct 20, 2014 - Thus, these Mal5-hybrid amphiphilic BCP samples have a body centered cubic (BCC) phase morphology. The domain spacing (d) values of the...
0 downloads 8 Views 3MB Size
Download PDF
Article pubs.acs.org/Biomac

Maltopentaose-Conjugated CTA for RAFT Polymerization Generating Nanostructured Bioresource-Block Copolymer Daichi Togashi,† Issei Otsuka,‡,§ Redouane Borsali,‡,§ Koichi Takeda,† Kazushi Enomoto,† Seigou Kawaguchi,† and Atsushi Narumi*,† †

Department of Polymer Science and Engineering, Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa 992-8510, Japan ‡ Univ. Grenoble Alpes, CERMAV, F-38000 Grenoble, France § CNRS, CERMAV, F-38000 Grenoble, France S Supporting Information *

ABSTRACT: We now describe the synthesis of a new family of oligosaccharide-conjugated functional molecules, which act as chain transfer agents (CTAs) for the reversible addition−fragmentation chain transfer (RAFT) polymerization. The synthesis was started from the catalyst-free direct N-glycosyl reaction of 5-azidopentylamine onto maltopentaose (Mal5) in dry methanol at room temperature and subsequent N-protected reaction with acetic anhydride, producing a stable oligosaccharide-building block, such as Mal5 with an azidopentyl group (Mal5-N3). The azido group was hydrogenated using platinum dioxide (PtO2) as a catalyst to give Mal5 with aminopentyl group (Mal5-NH2), which was then reacted with CTA molecules bearing activated ester moieties. These reactions produced Mal5-modified macro-CTAs (Mal5-CTAs, 1), which were used for the RAFT polymerizations of styrene (St) and methyl methacrylate (MMA) in DMF. The polymerizations were performed using the [M]0/[1]0 values ranging from 50 to 600, affording the Mal5-hybrid amphiphilic block copolymers (BCPs), such as Mal5-polystyrene (2) and Mal5-poly(methyl methacrylate) (3), with a quantitative end-functionality and the controlled molecular weights between 4310 and 20 300 g mol−1. The small-angle X-ray scattering (SAXS) measurements were accomplished for 2 and 3 to ensure their abilities to form phase separated structures in their bulk states with the increasing temperatures from 30 to 190 °C. The featured results were observed for 2 (ϕMal5 = 0.14) and 3 (ϕMal5 = 0.16) at temperatures above 100 °C, where ϕMal5 denotes the volume fraction of the Mal5 unit in the BCP sample. For both BCP samples, the primary scattering peaks q* were clearly observed together with the higher-ordered scattering peaks √2q* and √3q*. Thus, these Mal5hybrid amphiphilic BCP samples have a body centered cubic (BCC) phase morphology. The domain spacing (d) values of the BCC morphology for 2 (ϕMal5 = 0.14) and 3 (ϕMal5 = 0.16) were 10.4 and 9.55 nm, respectively, which were determined using Bragg’s relation (d = 2π/q*). The present RAFT agents were shown to eventually provide the phase separated structural polymeric materials in which 5.4 nm bioresource-spherical domains were periodically arrayed at the interval of about 10 nm.

■

INTRODUCTION

Monosaccharides, disaccharides, and oligosaccharides are also categorized as saccharide resources and have been effectively utilized as building blocks for biofunctional materials with the development of conjugation chemistry;33−55 for example, malto-oligosaccharides (Maln) have been hybridized with polymers.33−35,37,40−44,47−55 This application would be benefited by the fact that Maln are molecules readily soluble in water, which differs from their analogues with longer chains such as amylose. Furthermore, different from the mixtures of the oligosaccharides known as dextrin, Maln’s act as welldefined segments as for the hydrophilic property in the

Saccharides have been extensively utilized as the sources of polymeric materials showing biofunctions,1−12 whereas they have played another very important role as raw molecules to prepare organic/polymeric materials, which will contribute to the development of a sustainable chemistry. The use of polysaccharides is a promising method; as examples, the modifications of cellulose, chitin, chitosan, dextran, and amylose, which numerously exist in unspecific places in the earth, have been studied with applications for medical,13−16 optical,17,18 and chemical19−23 materials and so forth.24−32 Furthermore, recent topics include nanostructured papers that have been prepared from cellulose-based nanofibers, exhibiting an ultrahigh optical transparency and ultrahigh haze, for use in solar cell devices.32 © XXXX American Chemical Society

Received: September 4, 2014 Revised: October 15, 2014

A

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Scheme 1. Synthesis of Mal5-Building Blocks through the N-Glycosylation Reaction of 5-Azidopentylamine onto Reducing End for Mal5 and Subsequent Reducing Reaction of Azido Group

Scheme 2. Synthesis of Mal5-CTAs (1) through the Condensation Reactions between Mal5-NH2 and Two Types of CTA Molecules Containing NHS-Activated Ester Groups (4)

Scheme 3. Synthesis of Mal5-Hybrid Amphiphilic BCPs (2 and 3) through the RAFT Polymerization of St and MMA Using Mal5-CTAs (1)

sized, and they eventually produced Maln-hybrid amphiphilic BCPs, while the characterizations for the construction of the nanophase separated structures have not been described. On the other hand, Maln-conjugated chain-transfer agents (MalnCTAs) used for the reversible addition−fragmentation chain transfer (RAFT) polymerization for the purpose of preparing hybrid amphiphilic BCPs have not been demonstrated due to the difficulty in the synthetic aspects related to the selective introduction of CTA-moieties into Maln, the necessities of a tedious protection/deprotection process, the possibility of a side reaction such as hydrolysis of the CTA-moieties, and so forth. Thus, we aimed to develop new routes to easily prepare the maltopentaose-conjugated CTA (Mal5-CTA). Schemes 1 and 2 depict the synthesis of the Mal5-CTAs (1), including the reaction in which the reducing end of Mal5 is selectively replaced by the n-pentyl amino group (Mal5-NH2). We report that Mal5-NH2 is an efficient building block for obtaining the target Mal5-CTAs without any protection/deprotection steps. The resulting Mal5-CTAs are used for the RAFT polymerizations of styrene (St) and methyl methacrylate (MMA) to produce Mal5-PSt (2) and Mal5-PMMA (3), respectively, as

conjugated materials because they possess definite numbers of hydroxyl groups. As examples, maltopentaose (Mal5), maltohexaose (Mal6), and maltoheptaose (Mal7) contain 17, 20, and 23 hydroxyl groups, respectively. A powerful application of such Maln-resources includes their use as building blocks for nanotemplates, which meets the requirement for the development of sustainable next-generation block copolymer (BCP) lithography. 48 Based on the literature,47,48,50,52−55 one can cite that several types of Malnhybrid amphiphilic BCPs have been prepared by the controlled/living polymerization combined with click chemistry including the Cu(I)-catalyzed azide−alkyne cycloaddition.56,57 Aissou et al. reported for the first time that the Mal7polystyrene (PSt) produced nanoscale periodic structures around 12 nm in both the bulk and thin film states.47 Another rational method to prepare Maln-hybrid amphiphilic BCPs, which were earlier reported, should be the “initiator method” based on the controlled radical polymerization (CRP) techniques. Maln-conjugated initiators for the CRPs, such as the nitroxide-mediated radical polymerization (NMP)41 and atom transfer radical polymerization (ATRP),33 were syntheB

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(Tosoh Co., Japan) with Mw’s = 775, 422, 186, 114, 44.1, 16.7, 8.30, 5.12, 2.36, 0.87, 0.50, and 0.11 kg mol−1. The Mn,SEC and Mw/Mn of Mal5-PMMA (3) were also determined by the RI based on linear PMMA standards (Polymer Laboratories Ltd., U.K.) with Mw’s = 1250, 659, 300, 139, 60.2, 30.7, 20.6, 10.3, 4.90, 2.68, 1.30, 0.63, and 0.11 kg mol−1. Type 2 is as follows: The weight-average molecular weight (Mw,MALS) of 3b-III was determined by using the same SEC instruments as those for Type 1 using DMF containing 0.01 M LiBr as the eluent at the flow rate of 1.0 mL min−1 and room temperature of 25 °C. This SEC was equipped with three columns [Shodex OHpak SB-802.5 HQ (size, 8.0 mm × 300 mm; average beads size, 6 μm; exclusion limit, 10 kg mol−1 estimated from pullulan) and two Shodex KD-806 M (size, 8.0 mm × 300 mm; average beads size, 10 μm; exclusion limit, 20 × 103 kg mol−1 estimated from poly(ethylene glycol))], a multiangle laser light scattering (MALS) detector (DAWN DSP, wavelength = 632.8 nm, Wyatt Technology Co.), an RI detector (RI-71, Showadenko Co., Japan), and a UV detector (UV-8000, Tosoh Co., Japan). N-Acetyl-N-(5′-azido)pentyl-[(O-α-D-glucopyranosyl)-(1→ 4)]4-β-D-glucopyranoside (Mal5-N3). Mal5 (0.201 g, 0.243 mmol) and 5-azidopentylamine (0.171 g, 1.33 mmol) were dissolved in dry MeOH (ca. 5 mL), and the mixture was vigorously stirred at 40 °C for 72 h. The reaction was monitored by TLC (silica gel 60 F254, CH3CN/ H2O = 13:7). After complete disappearance of the spot due to Mal5 (Rf = 0.42), the reaction mixture was poured into dry CH2Cl2 (150 mL). The formed precipitates were collected by filtration using a 0.45 μm pore-sized PTFE membrane filter and washed with a mixture of 1:3 dry MeOH/dry CH2Cl2 (50 mL). A solution of Ac2O (2.16 g, 21.2 mmol) in dry MeOH (26 mL) was added to the obtained solid, and the resulting mixture was stirred overnight at room temperature. After confirming complete solubilization of the solid, the mixture was evaporated to dryness. The residue was redissolved in H2O (5 mL) and freeze-dried to give the target Mal5-N3 as a white solid (0.158 g, 66.4%). Rf = 0.79 (silica gel 60 F254, CH3CN/H2O = 13:7). 1H NMR (400 MHz, DMSO-d6 in the presence of a small amount of D2O, ppm): δ 5.31 and 4.74 (2 × d, J = 9.6 and 9.2 Hz, 1H, H-1Mal1, rotamers), 5.07 and 5.04 (2 × d, J = 3.6 Hz, 4H, H-1Mal2‑Mal5, rotamers), 3.73−3.23 (m, 32H, H-2, 3, 4, 5, 6Mal1‑Mal5 and −CH2N3), 3.10 (t, J = 9.4 Hz, 2H, −NAcCH2−), 2.08−2.01 (m, 3H, −NCOCH3, rotamers), 1.59−1.24 (m, 6H, −CH2−). 13C NMR (100 MHz, D2O, ppm): δ 176.2, 175.2, 99.9, 99.6, 86.9, 77.2, 76.7, 73.3, 72.9, 71.6, 70.1, 69.4, 60.6, 55.5, 51.1, 39.3, 29.2, 27.8, 23.5, 21.9, 21.5. FT-IR (KBr) v (cm−1): 3700−3000, 2933, 2349, 2100, 1724, 1635, 1439, 1369, 1257, 1138, 1030. ESI-TOF MS (m/z) calcd for [M + Na]+, 1003.38; found, 1003.50. [α]D = +117.5° (c 0.5, DMF). N-Acetyl-N-(5′-amino)pentyl-[(O-α-D-glucopyranosyl)-(1→ 4)]4-β-D-glucopyranoside (Mal5-NH2). Mal5-N3 (0.141 g, 0.141 mmol) was dissolved in dry MeOH (36 mL), and then PtO2 (50.0 mg, 0.220 mmol) was added to the mixture under a nitrogen atmosphere. The hydrogen gas was bubbled in the reaction mixture at room temperature. After 24 h, the starting material was consumed as judged by the TLC (silica gel 60 F254, CH3CN/H2O = 13:7). The mixture was filtered to remove any remaining metal sources using a 0.10 μm membrane filter and evaporated to dryness. The resulting solid was redissolved in a small amount of H2O. The aqueous solution was freeze-dried to obtain the target Mal5-NH2 as a white solid (0.128 g, 93.2%). Rf = 0.03 (silica gel 60 F254, CH3CN/H2O = 13:7). 1H NMR (400 MHz, DMSO-d6 in the presence of a small amount of D2O, ppm): δ 5.31 and 4.74 (2 × d, J = 9.6 and 8.4 Hz, 1H, H-1Mal1, rotamers), 5.11−5.03 (m, 4H, H-1Mal2‑Mal5), 3.70−3.21 (m, 30H, H-2, 3, 4, 5, 6Mal1‑Mal5), 3.09 (t, J = 9.0 Hz, 2H, −NAcCH2−), 2.75 (t, J = 7.4 Hz, 2H, −CH2NH2), 2.08−2.01 (m, 3H, −NCOCH3, rotamers), 1.60−1.24 (m, 6H, −CH2−). 13C NMR (100 MHz, CD3OD, ppm): δ 174.8, 173.4, 102.8, 88.9, 81.3, 80.7, 78.9, 75.0, 74.3, 73.8, 73.4, 71.6, 62.8, 62.4, 62.2, 41.0, 40.1, 29.9, 29.4, 29.1, 28.9, 25.0, 24.8, 24.3, 22.6, 22.3. FT-IR (KBr) v (cm−1): 3700−3000, 2931, 2357, 1635, 1443, 1138, 1030. ESI-TOF MS (m/z) calcd for [M + H]+, 955.39; found, 955.52. [α]D = +113.7° (c 0.5, DMF). N-Acetyl-N-[5′-[4-cyano-4′-{(2-phenyl-1-thioxo)thio}pentanamide]]pentyl-[(O-α-D-glucopyranosyl)-(1→4)]4-β-D-glu-

shown in Scheme 3. The ability of the resulting Mal5-hybrid amphiphilic BCPs to build nanophase separated structures with smaller feature sizes in the bulk states is examined based on the small-angle X-ray scattering (SAXS) measurement.

■

EXPERIMENTAL SECTION

Materials. Maltopentaose (Mal5, 98.7%) was kindly supplied by Hayashibara Biochemical Laboratories Inc., Japan, and used as received. 4-Cyano-4′-{(2-phenyl-1-thioxo)thio}pentanoic succinimido (4a),58 bis(dodecylsulfanylthiocarbonyl)disulfide,59 4,4′-azobis(4-cyanopentanoic succinimide) (ACPS),58 and 5-azidopentylamine60 were prepared according to the literature. Dry DMF, dry MeOH, and dry CH2Cl2 were prepared by the distillations of the commercially available solvents using drying agents of CaH2, Mg, and anhydrous CaCl2, respectively. Acetic anhydride (Ac2O, >97.0%), styrene (St, >99.0%), and methyl methacrylate (MMA, >98.0%) were purchased from Kanto Chemical Co., Japan, and distilled over CaH2 prior to use. 2,2′-Azobis(isobutyronitrile) (AIBN, Wako Pure Chemical Industries Japan, 98.0%) was used after recrystallization from MeOH. Silica gel 60 F254 (TLC plates), silica gel 60 RP-18 F254s (TLC plates), silica gel 60N (spherical, neutral pH, 63-210 μm), and silica gel 120 RP-18 (spherical, 40−50 μm) were purchased from Kanto Chemical Co., Japan. Deionized water (electrical conductivity = 18.3 MΩ cm) was prepared by using Advantec Aquarius GSH-5000 and Millipore MilliQ Jr. instruments. Other chemicals were obtained from commercial sources and used as received unless otherwise stated. Instruments. The 1H and 13C NMR spectra were recorded using a JNM-ECX400 instrument (JEOL Ltd., Japan). The Fourier-transform infrared (FT-IR) spectra were recorded using an FT-720 spectrometer (HORIBA Ltd., Japan). The electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) measurements were performed by using a JMS-T100LC AccuTOF LC instrument (JEOL Ltd., Japan). The purities (%) of the Mal5-CTAs (1) were determined by HPLC [Agilent 1200 system equipped with a XDB C-18 column (size: 4.6 mm × 150 mm, average beads size: 5 μm), a refractive index (RI) detector, and an ultraviolet (UV) detector] at 40 °C in CH3CN/H2O (2/3) or MeOH with 0.8 mL min−1. Preparative size exclusion chromatography (SEC) was performed at 25 °C in THF (3.5 mL min−1) using an LC-9201 system (JAI Co., Ltd., Japan) equipped with two columns [Shodex HF-2002 (size, 20.0 mm × 500 mm; average beads size, 6 μm; exclusion limit, 5.0 × 103 kg mol−1) and HF-2003 (size, 20.0 mm × 500 mm; average beads size, 6 μm; exclusion limit, 70 × 103 kg mol−1)] and a UV-3740 detector (JAI Co., Ltd., Japan). The specific refractive index increment (dn/dc) values of 3b-III were measured in DMF containing 0.01 M LiBr at 25 °C using a DRM1021 as a differential refractometer (Otsuka Electronics Co., Ltd., Japan) at the wavelength of 632.8 nm. Specific rotations ([α]D, c 0.5) were measured in DMF using a DIP-1000 digital polarimeter (JASCO Co., Japan) at 25 °C. The SAXS measurements of the BCP samples were performed using a NANO-viewer (Rigaku Co., Japan). The thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements of the BCP samples were carried out using the TGA4000 and DSC4000 instruments (PerkinElmer Japan Co., Ltd.). Molecular Weight Analysis. The characterizations for the molecular weights of the resulting BCP samples were performed by two types of size exclusion chromatography (SEC) instruments in this study. Type 1 is as follows: The number-average molecular weight (Mn,SEC) and the polydispersity index (Mw/Mn) of the resulting BCP samples were determined by SEC [pump, PU-2080 Plus (JASCO Co., Japan); degasser, DG-2080-53 (JASCO Co., Japan); column oven, CO-2060 Plus (JASCO Co., Japan); temperature of the column oven, 40 °C] using THF as the eluent at the flow rate of 1.0 mL min−1 and room temperature of 25 °C. This SEC apparatus was equipped with columns [two Tosoh TSK-gel Multipore HXL-M (size, 7.8 mm × 300 mm; average beads size, 5 μm; exclusion limit, 1.0 × 103 kg mol−1)], an RI detector (RI-2031 Plus, JASCO Co., Japan), and a UV detector (UV-2075 plus, JASCO Co., Japan). The Mn,SEC and Mw/Mn of Mal5PSt (2) were determined by the RI based on linear PSt standards C

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(t, J = 6.8 Hz, 3H, −S(CH2)11CH3). 13C NMR (100 MHz, CD3OD, ppm): δ 219.4, 175.2, 174.6, 173.2, 120.2, 102.7, 88.7, 81.2, 80.8, 78.8, 74.8, 73.7, 71.4, 62.3, 48.1, 47.9, 40.4, 37.7, 35.6, 35.1, 33.1, 32.2, 30.7, 30.5, 30.2, 29.9, 29.4, 29.0, 25.4, 25.0, 23.8, 22.3, 14.6. FT-IR (KBr) v (cm−1): 3700−3000, 2925, 2854, 1646, 1457, 1375, 1153, 1079, 1029. ESI-TOF MS (m/z) calcd for [M + Na]+, 1362.55; found, 1362.60. [α]D = +84.0° (c 0.5, DMF). RAFT Polymerization. A representative example is as follows: St (0.188 g, 1.81 mmol) and 1b (48.2 mg, 36.0 μmol) were added to the DMF solution (93 μL) containing AIBN (2.96 mg, 18.0 μmol). The mixture was placed in dry glass ampule and then degassed by three freeze−evacuate−thaw cycles. After flame-sealed under vacuum, the ampule was allowed to stand at 65 °C for 24 h ([St]0/[1b]0/[AIBN]0 = 50/1/0.5, [St]0 = 6.00 mol L−1, total volume = 300 μL). The polymerization was stopped by rapid cooling with liquid nitrogen. The characterizations of the original sample were performed by gas chromatography (GC) and size exclusion chromatography (SEC) to determine the monomer conversion of 95%, number-average molecular weight (Mn,SEC) of 4890 g mol−1, and molecular weight distribution (Mw/Mn) of 1.24. The original sample was injected into a preparative SEC apparatus, producing a polymeric product 2b-I as a pale yellow powder (0.151 g, 63.1%). The 1H NMR measurement of 2b-I in pyridine-d5 with small amounts of D2O was carried out in order to calculate the number-average molecular weight (Mn,NMR). Mn,SEC = 4890 g mol−1, Mn,NMR = 7070 g mol−1, Mw/Mn = 1.24. [α]D = +13.9° (c 0.5, DMF). 1H NMR (400 MHz, pyridine-d5 in the presence of a small amount of D2O, ppm): δ 7.50−7.08 (br, aromatic, (m) and (p)), 7.08−6.57 (br, aromatic, (o)), 5.95−5.82 (m, 4H, H-1Mal2‑Mal5), 5.35 (d, J = 8.8 Hz, 1H, H-1Mal1), 4.73−3.90 (m, 30H, H-2, 3, 4, 5, 6Mal1‑Mal5), 3.43−3.30 (br, 4H, −NAcCH2−, −CH2NHCO−), 2.70− 2.06 (br, −CHPh−: (PSt backbone)), 2.06−1.40 (br, −CH2−: (PSt backbone)), 1.40−1.12 (br, −CH 2 −), 0.92−0.83 (br, −S(CH2)11CH3). FT-IR (KBr) v (cm−1): 3640−3100, 3082, 3059, 3025, 2922, 2850, 1950, 1870, 1800, 1750, 1640, 1600, 1493, 1450, 1370, 1153, 1073, 1029. Small Angle X-ray Scattering (SAXS) Measurements. The SAXS experiments were performed at various temperatures (from 30 to 190 °C) using a NANO-Viewer. The wavelength of the X-ray was 1.54 Å. The scattering intensities were recorded using a High-Speed 2D X-ray detector, DECTRIS 100k PILATUS (DECTRIS Ltd., Switzerland). The scattered vector (q) was calibrated using silver behenate. The resulting BCP powders were placed in 2.0 mm diameter glass capillaries and then flame-sealed. These samples were placed in sample holders equipped with an integrated heating system. The sample temperature was controlled by stepwise heating at a 10 °C interval (heating rate = 5 °C min−1) and kept at that temperature for 10 min before the measurements.

copyranoside (1a). 4-Cyano-4′-{(2-phenyl-1-thioxo)thio}pentanoic succinimide (4a, 0.190 g, 0.503 mmol) and Mal5-NH2 (0.401 g, 0.419 mmol) were dissolved in dry DMF, and the mixture was stirred at 40 °C. The reaction was monitored by TLC (silica gel 60 F254, CH3CN/ H2O = 13:7). After 48 h, the spot due to the starting materials (4a: Rf = 0.93, Mal5-NH2: Rf = 0.03) disappeared, while the one due to the product appeared by the UV light irradiation (Rf = 0.54). The mixture was evaporated to dryness, and the residue was purified with octadecylsilyl modified (ODS) silica gel column chromatography (silica gel 120 RP-18, CH3CN/H2O = 2:3) to give the target Mal5CTA (1a) as a pale pink solid (0.171 g, 33.6%). Rf = 0.48 (silica gel 60 RP-18 F254s, CH3CN/H2O = 2:3). 1H NMR (400 MHz, pyridine-d5 in the presence of a small amount of D2O, ppm): δ 8.44 (d, J = 7.2 Hz, 1H, −NHCO−), 7.90 (d, J = 7.8 Hz, 2H, aromatic (o)), 7.72 (t, J = 7.2 Hz, 1H, aromatic (p)), 7.52 (dd, J = 7.2 and 7.8 Hz, 2H, aromatic (m)), 6.20 and 5.42 (2 × d, J = 9.2 and 8.8 Hz, 1H, H-1Mal1, rotamers), 5.87−5.68 (m, 4H, H-1Mal2‑Mal5), 4.67−3.90 (m, 30H, H-2, 3, 4, 5, 6Mal1‑Mal5), 3.77−3.31 (m, 4H, −NAcCH2− and −CH2NHCO−), 2.90−2.75 (m, 4H, −NHCO(CH2)2−), 2.54−2.46 (m, 3H, −C(CN)CH3), 2.06 (s, 3H, −NCOCH3), 1.99−1.39 (m, 6H, −CH2−). 13C NMR (100 MHz, CD3OD, ppm): δ 224.8, 174.5, 173.2, 145.8, 134.3, 129.7, 127.6, 119.7, 102.5, 81.1, 78.7, 74.8, 74.0, 73.6, 73.1, 71.5, 71.2, 62.5, 62.0, 47.4, 40.3, 34.9, 32.1, 29.7, 29.4, 25.4, 24.2, 22.3. FT-IR (KBr) v (cm−1): 3700−3000, 2933, 1655, 1572, 1557, 1541, 1456, 1153, 1082, 1030. ESI-TOF MS (m/z) calcd for [M + Na]+, 1238.48; found, 1238.53. [α]D = +93.1° (c 0.5, DMF). 4-Cyano-4′-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic Succinimide (4b). 4,4′-Azobis(4-cyanopentanoic succinimide) (ACPS, 1.42 g, 2.70 mmol) and bis(dodecylsulfanylthiocarbonyl)disulfide (1.10 g, 1.80 mmol) were dissolved in EtOAc (ca. 140 mL). The mixture was degassed by three freeze−evacuate−thaw cycles to remove the oxygen and then refluxed under a nitrogen atmosphere for 34 h. The reaction was monitored by TLC (silica gel 60 F254, CHCl3/EtOAc = 10:1). After disappearance of the spot due to the disulfide (Rf = 0.90), the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (silica gel 60N, CHCl3/EtOAc = 10:1) to give the target product as a yellow solid (1.36 g, 68.4%). Rf = 0.47 (silica gel 60 F254, CHCl3/EtOAc = 10:1). 1H NMR (400 MHz, CDCl3, ppm): δ 3.27 (t, 2H, J = 8.4 Hz, −SCH2CH2−), 2.91−2.41 (m, 8H, −OCO(CH2)2−, −CO(CH2)2CO−), 1.82 (s, 3H, −C(CN)CH3), 1.63 (quint, 2H, J = 8.0 Hz, −SCH2CH2−), 1.41−1.16 (m, 18H, −CH2−), 0.81 (t, 3H, J = 6.6 Hz, −CH3). 13C NMR (100 MHz, CDCl3, ppm): δ 216.5, 168.8, 167.0, 118.6, 45.9, 37.1, 33.1, 31.9, 29.5, 29.3, 28.9, 27.6, 26.8, 25.5, 24.7, 22.6, 14.1. FT-IR (KBr) v (cm−1): 2915, 2850, 1822, 1737, 1471, 1201, 1076. ESI-TOF HRMS (m/z) calcd for [M + Na]+, 523.1735; found, 523.1753. N-Acetyl-N-[5′-{4-cyano-4′-((dodecylsulfanylthiocarbonyl)sulfanyl)pentanamide}]pentyl-[(O-α-D-glucopyranosyl)-(1→ 4)]4-β-D-glucopyranoside (1b). 4-Cyano-4′[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic succinimide (4b, 0.203 g, 0.405 mmol) and Mal5-NH2 (0.384 g, 0.402 mmol) were dissolved in dry DMF and stirred for 48 h at 40 °C. The reaction was monitored by TLC (silica gel 60 F254, CH3CN/H2O = 13:7). The spot due to the starting materials (4b: Rf = 0.97, Mal5-NH2: Rf = 0.03) decreased, while that due to the product (Rf = 0.76) increased. After the reaction almost completely finished, the mixture was evaporated to remove the solvent. The residue was redissolved in MeOH and filtered using a 0.45 μm pore-sized PTFE membrane filter to remove the unreacted 4b. After removing the solvent, the product was redissolved in the eluent and purified by ODS silica gel column chromatography (silica gel 120 RP-18, MeOH/H2O = 10:1) to give the target Mal5CTA (1b) as a pale yellow solid (0.281 g, 51.7%). Rf = 0.33 (silica gel 60 RP-18 F254s, MeOH/H2O = 10:1). 1H NMR (400 MHz, pyridined5 in the presence of a small amount of D2O, ppm): δ 6.37 and 5.36 (2 × d, J = 9.6 and 8.8 Hz, 1H, H-1Mal1, rotamers), 5.96−5.77 (m, 4H, H1Mal2‑Mal5), 4.76−3.94 (m, 30H, H-2, 3, 4, 5, 6Mal1‑Mal5), 3.78−3.31 (m, 4H, −NAcCH2−, −CH2NHCO−, and −SCH2−), 2.96−2.63 (m, 4H, −NHCO(CH2)2−), 2.40 and 2.17 (2 × s, 3H, −NCOCH3, rotamers), 1.92 (s, 3H, −C(CN)CH3), 1.63−1.15 (m and br, 26H, −CH2−), 0.88

■

RESULTS AND DISCUSSION

Synthesis and Characterization of Mal5 Building Blocks. The development of a versatile synthetic route for the oligosaccharide building block has been a key for this study. For such a purpose, we aimed to develop a direct Nglycosylation reaction onto the malto-oligosaccharide to produce the building block with one azido group. Maltopentaose (Mal5) was reacted with 5-azidopentylamine in dry MeOH, followed by the treatment with Ac2O to give a product (Scheme 1). The 1H NMR spectrum of the product exhibited the signals of the methylene and methine protons due to the Mal5 unit (5.1−5.0 and 3.8−3.2 ppm) together with the ones of the methylene protons due to azidopentyl moieties (1.7−1.2 ppm) (Figure SI-1, Supporting Information). Furthermore, the characteristic signals due to the N-acetyl (NAc) protons (2.1 ppm) and those due to the anomeric proton linked to the NAc group (5.3 and 4.7 ppm) appeared. The FT-IR spectrum of the product showed the stretching vibration derived from the azido group at 2100 cm−1 (Figure SI-2). The electrospray ionization D

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 1. 1H NMR spectra in pyridine-d5/D2O of (a) 1b and (b) the product obtained through the RAFT polymerization of St using 1b with the condition of [St]0/[1b]0/[AIBN]0 = 50/1/0.5.

mass spectrum (ESI-MS) exhibited the main strong peak at m/ z = 1003.50, which was consistent with the calculated [M + Na]+ value for the target compound of 1003.38. These results indicated that the product was assignable to the target Mal5 with an azidopentyl group (Mal5-N3). The Mal5-N3 was further characterized by the SEC measurements equipped with an RI detector in an aqueous solution containing 0.05 M NaNO3 (Figure SI-3). The main peak at the retention time of 31 min was due to Mal5-N3. Also, a small peak derived from the unreacted Mal5 was observed at the retention time of 28 min. It might be mentioned that the molecular weight of Mal5-N3 is higher than that of Mal5, however, the retention time of Mal5N3 was later than that of Mal5. This result might be attributable to the hydrophobic interaction between the introduced pentylene chains for Mal5-N3 and the PSt gel. The important result from this measurement is that the purity of Mal5-N3 was the high value of 93% (Figure SI-3). Sugars containing azido-groups are useful as the molecular building blocks in conjunction with the Cu (I)-catalyzed azide− alkyne cycloaddition (CuAAC) affording the glyco-conjugated materials.61−63 To date, such molecules have been prepared by the glycosylation reaction of azido-containing alcohols onto the protected sugars using Lewis acids.61 As the representative methods starting from unprotected sugars, the direct azidation using the PPh3/CBr4/NaN3 system or the Mitsunobu conditions with hydrazoic acid have been developed.64−70 However, it was difficult to apply these methods to the modification of the higher oligosaccharides due to the possibility of the cleavages of their multiple O-glycoside linkages. On the other hand, there have been few reports on the direct modifications of unprotected oligosaccharides, except that the one-pot and regioselective modifications of the reducing end for oligosaccharides by the azido group has been achieved using 2-chloro-1,3-dimethylimidazolinium chloride (DMC) in aqueous media, followed by chromatographic purifications.70 Hence, the present method would become a new and convenient technique to produce oligosaccharides containing azido groups without any additives or catalysts, protection/deprotection processes, or chromatographic purifi-

cations. The azido compound is also known to be important as the precursor of amine derivatives.65,66,71 In this study, in order to apply not the CuAAC method but a conventional amidation route, Mal5-N3 was treated with hydrogen in the presence of platinum dioxide (PtO2) to produce another important building block such as Mal5-NH2. The product of the reaction was assigned to target Mal5-NH2 by the FT-IR and 1H NMR measurements (Figures SI-2 and SI-4, respectively). Synthesis and Characterization of Mal5-CTAs (1). Mal5NH2 was reacted with two types of CTA molecules containing NHS-activated ester moieties, such as 4-cyano-4′-{(2-phenyl-1thioxo)thio}pentanoic succinimide (4a) and 4-cyano-4′[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic succinimide (4b), as shown in Scheme 2. The respective reactions proceeded under mild conditions without any additives producing the target dithiobenzoate type Mal5-CTA (1a) and trithiocarbonate type Mal5-CTA (1b). The characterizations for 1b will now be described. Figure 1a shows the 1H NMR spectrum of 1b in pyridine-d5/D2O. The characteristic signals of the methylene and methine protons due to the Mal5 unit (k at 6.0−5.8 ppm and l, m, n, o, and p at 4.8−3.9 ppm) appeared together with the ones of the methylene and methyl protons derived from the alkyl chains in the trithiocarbonate derivative (a at 0.9 ppm and b at 1.6−1.1 ppm). Also, the characteristic signals due to the NAc protons (i at 2.4 and 2.2 ppm) and those due to the anomeric proton linked to the NAc group (j at 6.4 and 5.4 ppm) were confirmed. The ESI-MS exhibited the main strong peak at m/z = 1362.60, which fairly agreed with the calculated [M + Na]+ value for the target compound of 1362.55. The analysis using reversed phase high performance liquid chromatography (HPLC) equipped with an ODS column, an RI detector, and a UV detector was performed in order to determine the purity of 1b. The purity of 1b was above 99%, which was based on the comparison between the peak area of the UV chromatogram due to 1b and that due to the unmodified CTA (4b or its hydrolyzed product) (Figure SI5d). The dithiobenzoate type Mal5-CTA (1a) was synthesized using a procedure similar to that for 1b, and its 1H NMR spectrum is shown in Figure SI-6. Figure SI-5b indicates that E

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

the purity of 1a was >99%. Consequently, we developed easy routes to afford two types of Mal5-CTAs (1) with high purities required for the subsequent RAFT polymerization stage. As representative studies on the preparations of glyco-CTA, Stenzel et al. reported that the trithiocarbonate-CTA moieties were introduced into the modified-cellulose, glucose, and βcyclodextrin via ester linkages.45,72 However, the main objective of their studies might not be the end-functionalization of the synthetic polymer chains. In addition, Bathfield et al. reported that the dithiobenzoate derivative bearing the isopropylideneprotected galactose was synthesized,58 and Akeroyd et al. reported that a xanthate derivative with protected Mal7 was synthesized via the CuAAC, while they did not demonstrate the deprotections for the sugar-moieties.73 Compared to these previous studies, the Mal5-CTAs prepared in this study are a new class in terms of the fact that they are made up of not a monosaccharide, but an oligosaccharide and also that the 17 hydroxyl groups they contain are in the free forms (not protected). Another characteristic property for the Mal5-CTAs would be their solubilities. The Mal5-CTAs showed a high water solubility, though they are nonionic molecules. They are also soluble in polar organic solvents including CH3OH, DMF, and DMSO, while they exhibit poor solubilities in toluene, CHCl3, and 1,4-dioxane as summarized in Table SI-1. Interestingly, 1b gave a clear solution in THF in spite of bearing 17 hydroxyl groups in the Mal5 units due to the presence of dodecyl chains. RAFT Polymerization of St and MMA Using Mal5-CTAs (1). The polymerizations of vinyl monomers, such as St and MMA, were performed using 1a and 1b as the CTAs (Scheme 3). We first describe the St/1b system. The polymerizations were conducted with AIBN as the initiator in DMF using the conditions of the [St]0 of 6.00 mol L−1and the [St]0/[1b]0/ [AIBN]0 of 200/1/0.5. Figure 2 shows the typical SEC traces of

Figure 3. Plots of conversions of St (●) and ln([St]0/[St]t) (○) for the polymerization of St using 1b ([St]0/[1b]0/[AIBN]0 = 200/1/0.5) as a function of polymerization time.

Figure 4. Plots of Mn and Mw/Mn for the products obtained through the polymerizations of St using 1b ([St]0/[1b]0/[AIBN]0 = 200/1/ 0.5) as a function of conversion.

concentration at the predetermined polymerization time. The plot linearly increased, indicating that the concentration of the active species in the polymer chain-end remained constant throughout the polymerization. Figure 4 shows the plots of the Mn,SEC and Mw/Mn values determined by the SEC measurements as a function of the monomer conversion. The Mn,SEC values linearly increased, while the Mw/Mn remained relatively low at around 1.3. These results indicated that the St/1b polymerization system proceeded in a living fashion. We should note that the Mn,SEC values were estimated lower than the calculated molecular weight values (Mn,calcd). This would be possibly due to the fact that the Mal5 units should influence the diffusion of the samples in the eluents of THF, which was supported by the result that the Mn,SEC value of 1b was 390 g mol−1 (determined by RI based on linear PSt standards). The polymerization was carried out using the [St]0/[1b]0 of 50/1 for 24 h and the resulting mixture in which the conversion of St reached 95% (Table 1) was purified by preparative SEC to isolate the polymeric product 2b-I (Mn,SEC = 4890 g mol−1 and Mw/Mn =1.24). Figure 1b shows the 1H NMR spectrum of 2b-I

Figure 2. SEC traces of 1b and the polymerization mixtures obtained through the RAFT polymerizations of St for 1.5, 3, 5, 9, and 24 h using 1b ([St]0/[1b]0/[AIBN]0 = 200/1/0.5).

1b and the mixtures after the polymerizations for 1.5, 3, 5, 9, and 24 h. The respective traces exhibited a unimodal and symmetrical peak, which shifted to the high molecular weight regions with the increasing polymerization times. The conversions of St at the respective polymerization stages determined by the GC measurements are plotted in Figure 3. The conversions increased to 60% after the induction period of ca. 1 h. Figure 3 also displays the plots of the rates of the monomer consumption, ln([St]0/[St]t)s, as a function of the polymerization times, where [St]t represents the monomer F

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Table 1. RAFT Polymerization of St Using Mal5-CTAsa Mal5-CTA

[St]0/[Mal5-CTA]0

conv (%)c

1a 1a 1a 1a 1a 1b 1b 1b 1b

50/1 100/1 200/1b 300/1b 400/1b 50/1 100/1 200/1 600/1

70 61 41 43 44 95 82 63 33

BCP sample Mal5-PSt Mal5-PSt Mal5-PSt Mal5-PSt Mal5-PSt Mal5-PSt Mal5-PSt Mal5-PSt Mal5-PSt

Mn,calcd (g mol−1)d

Mn,SEC (g mol−1)e

Mw/Mne

[α]D (deg)f

4850 7530 9840 14 600 19 400 6290 9880 14 500 22 200

5210 8340 9160 15 100 18 100 4890 7860 11 000 20 300

1.33 1.27 1.35 1.33 1.27 1.24 1.27 1.26 1.31

+18.9 +10.1 +7.4 +5.4 +5.4 +13.9 +9.2 +6.8 +3.5

2a-I 2a-II 2a-III 2a-IV 2a-V 2b-I 2b-II 2b-III 2b-IV

a Polymerizations were performed in DMF using AIBN as an initiator for 24 h at 65 °C using the [St]0 of 6.00 mol L−1 and the [Mal5-CTA]0/ [AIBN]0 of 1/0.5. bPolymerizations were performed using the [St]0 of 5.50 mol L−1. cDetermined by GC. dMn,calcd = [St]0/[Mal5-CTA]0 × conv × MWst + MWCTA. eDetermined by SEC in THF using linear PSt standards with an RI detector. fMeasured in DMF at 25 °C (c 0.5).

Table 2. RAFT Polymerization of MMA Using Mal5-CTAsa Mal5-CTA

[MMA]0/[Mal5-CTA]0

conv (%)b

1a 1a 1a 1b 1b 1b 1b

50/1 100/1 200/1 50/1 100/1 200/1 300/1

100 100 100 100 100 99 98

BCP sample Mal5-PMMA Mal5-PMMA Mal5-PMMA Mal5-PMMA Mal5-PMMA Mal5-PMMA Mal5-PMMA

3a-I 3a-II 3a-III 3b-I 3b-II 3b-III 3b-IV

Mn,calcd (g mol−1)c

Mn,SEC (g mol−1)d

Mw/Mnd

6220 11 200 21 200 6350 11 400 21 200 30 800

5340 10 100 16 400 4310 9830 15 900e 12 900

1.16 1.15 1.41 1.28 1.23 1.25 1.65

Polymerizations were performed in DMF using AIBN as an initiator for 24 h at 65 °C using the [MMA]0 of 6.00 mol L−1 and the [Mal5-CTA]0/ [AIBN]0 of 1/0.5. bDetermined by GC. cMn,calcd = [MMA]0/[Mal5-CTA]0 × conv × MWMMA + MWCTA. dDetermined by SEC in THF using linear PMMA standards with an RI detector. eMn,MALS = 21 100 g mol−1 (Mn,MALS = Mw,MALS × Mn/Mw, where the Mw,MALS was determined by SEC-MALS in DMF containing 0.01 M LiBr using the dn/dc value of 0.0631 mL g−1). a

CTA]0 ratio of 600/1. The [α]D values of 2 ranged from +3.5 o to +18.9 o. These values were plotted as a function of their weight fractions of the Mal5-CTA unit in 2, W1s, which were calculated on the basis of Mn,SEC (Figure SI-7). The dashed lines represent the [α]D values, which were expected from the [α]D of +93.1° and +84.0° for 1a and 1b, respectively. The plots for 2a and 2b were in good agreement with the respective dashed lines, indicating that the Mal5 unit quantitatively conjugated into the resulting polymer chain ends. This result also proved that the decompositions of the Mal5 moieties, such as cleavages of the O-glycoside linkages, did not occur under our polymerization conditions. The system was expanded to the polymerization of MMA. Table 2 summaries the polymerizations of MMA with Mal5CTAs for 24 h using the [MMA]0/[1]0 ratios ranging from 50/ 1 to 300/1. MMA was almost quantitatively consumed to produce the Mal5-hybrid amphiphilic BCPs (Mal5-PMMA 3) with the Mn,SEC between 4310 and 16 400 g mol−1. The Mw/Mn values ranged from 1.16 to 1.65. As judged from the comparisons between the Mn,SEC and Mn,calcd values and also the Mw/Mn values, the MMA/1a system with the [MMA]0/ [1a]0 ratios of 50/1 and 100/1 produced Mal5-PMMAs 3a with well-defined structures. Similarly, the MMA/1b system with the [MMA]0/[1b]0 ratios of 50/1, 100/1, and 200/1 provided Mal5-PMMAs 3b with well-defined structures. As examples, 3aI (Mn,SEC = 5340 g mol−1 and Mw/Mn = 1.16) and 3b-III (Mn,SEC = 15 900 g mol−1 and Mw/Mn = 1.25) were obtained. We should mention here that the Mn,SEC values were lower than the Mn,calcd values and the difference was larger for 3 as compared to that for 2. Hence, we determined the molecular weight of 3b-III by SEC-MALS in DMF containing 0.01 M LiBr. The weight-average molecular weight determined by SEC-MALS (Mw,MALS) was 26 400 g mol−1, which was divided

in pyridine-d5 in the presence of a small amount of D2O. The intense signals due to the protons of PSt, such as the aromatic ones (3, 4, and 5 at 7.5−6.6 ppm) and methine and methylene ones (1 and 2 at 2.7−1.4 ppm) were observed. The signals derived from the CTA-unit clearly appeared (assignments were already mentioned in the CTA-synthesis section). The ratio of the peak areas due to the β-anomeric protons in the Mal5 unit (k) and those due to the methyl protons in the dodecylthiocarbonate unit (a) for 2b-I was in accordance with that for 1b. The Mn,NMR of 2b-I was 7070 g mol−1, which was calculated from the peak areas due to the β-anomeric protons in the Mal5 unit (k) and those due to the aromatic protons in the PSt unit (3) in Figure 1b. This value was similar to the Mn,calcd value of 6290 g mol−1. These results showed that 2b-I was assigned to the target Mal5-hybrid amphiphilic BCPs, whose main chain was composed of PSt (Mal5-PSt). The controlled polymerization of St proceeded at the ω-chain-ends, keeping the αchain-ends with the Mal5 unit not cleaved. 2b-I also showed the specific rotation ([α]D) of +13.9° (Table 1), supporting that the 1b unit was almost quantitatively introduced into 2b-I, which will be discussed later. The St/1a system showed results similar to those for the St/1b system. Table 1 summarizes the results for the RAFT polymerizations of St with Mal5-CTAs for 24 h using diverse [St]0/ [Mal5-CTA]0 ratios ranging from 50/1 to 600/1. The conversions of St were dependent on the [St]0/[Mal5-CTA]0 ratios and ranged from 33% to 95% to produce Mal5-PSt (2) with the Mn,SEC ranging from 4890 g mol−1 to 20 300 g mol−1. The Mn,SEC values of 2 were in good agreement with the Mn,calcd values. The Mw/Mn values of 2 were relatively low, between 1.24 and 1.35. As one example, Mal5-PSt 2b-IV (Mn,calcd = 22 200 g mol−1, Mn,SEC = 20 300 g mol−1, and Mw/Mn =1.31) was obtained through the St/1b system using the [St]0/[Mal5G

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 5. Variation in SAXS profiles of 2b-I (left) and 3a-I (right) with the increasing temperature ranging from 30 to 190 °C.

by the Mw/Mn of 1.25. This gave the number-average molecular weight determined by SEC-MALS (Mn,MALS) of 21 100 g mol−1 and this value well agreed with the Mn,calcd one of 21 200 g mol−1 (see footnote e in Table 2). Consequently, the Mal5hybrid amphiphilic BCPs, such as Mal5-PSt (2) and Mal5PMMA (3), could be obtained through the RAFT polymerizations with Mal5-CTAs (1). Characterization of nanostructured BCP in the bulk state. The SAXS measurements were demonstrated for Mal5PSt (2) and Mal5-PMMA (3) to ensure their self-organized abilities to build up phase separated structures. We performed the measurements of the BCP samples in their bulk states with the increasing temperatures from 30 to 190 °C. Figure 5 shows the SAXS profiles of 2b-I and 3a-I, in which characteristic results were observed. For both BCP samples, only primary scattering peaks due to q* appeared at the temperatures up to 100 °C. On the other hand, upon heating, higher-ordered scattering peaks progressively appeared at q values ranging from 0.07 to 0.12 Å−1. Some long-range periodic structures might start to be constructed in this temperature range. The featured results were observed at a temperature above 100 °C, in which the higher-ordered scattering peaks corresponding to √2q* and √3q* clearly appeared. Thus, 2b-I and 3a-I constructed the morphology of the body centered cubic (BCC) phase. Figure 6 exhibits the differential scanning calorimetry (DSC) profiles of the BCP samples. The glass-transition temperatures (Tg’s) of 2b-I and 3a-I were 80.6 and 98.9 °C, respectively. Hence, the morphology changes above 100 °C would be attributable to the Tg’s of the respective polymer main chains. Furthermore, the intensities for the peaks located at √2q* and √3q* were enhanced above 140 °C, which might be due to the Tg of 137 °C for the Mal5 unit.74 We now describe the sizes of the BCC phase separated structures. Table 3 summaries the molecular parameters and morphological characteristics of the BCP samples (2b-I and 3aI). The volume fractions of the Mal5 units (ϕMal5) were 0.14 and 0.16 for 2b-I and 3a-I, respectively, which were calculated using the Mn,NMR values of the resulting BCPs and also the densities for the segments of Mal5 (ρMal5), PSt (ρPSt),75 and PMMA (ρPMMA).76 These ϕMal5 values also supported the fact that the morphology of the structures were assigned to the

Figure 6. DSC traces of the 2b-I (solid line) and 3a-I (dashed line).

BCC phase when the mean-field phase diagram of the symmetric diblock copolymer system77 was adopted to the present system. The domain spacing (d) values of the BCC morphology were calculated from the following Bragg’s relation using the scattered vectors (q) at q*. 2π d= q*

q=

4π sin θ λ

The d values of the BCC phase formed by 2b-I and 3a-I were 10.4 and 9.55 nm, respectively. These were slightly lower than those of Mal7-PSt of 10−12 nm, which were reported elsewhere.47,55 Thus, the present BCPs prepared by the RAFT polymerizations have the potentials of being utilized in lithographic applications or in flexible electronic applications. The radii of the bioresource-spherical domains in the BCC phase consisting of the Mal5 unit (rSAXS) were estimated by the following equation using the ϕMal5 and d values. rSAXS = H

d 3 3ϕMal5 2 π dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Table 3. Summary of Molecular Parameters and Morphological Characteristics BCP sample Mal5-PSt 2b-I Mal5-PMMA 3a-I

Mn,NMR (g mol−1)a 7070 6790

Mw/Mn b

1.24 1.16c

ϕMal5d

morphologye

d (nm)ef

rSAXS (nm)eg

0.14 0.16

BCC phase BCC phase

10.4 9.55

2.7 2.6

a Determined by 1H NMR spectra in pyridine-d5/D2O. bDetermined by SEC in THF using linear PSt standards with an RI detector. cDetermined by SEC in THF using linear PMMA standards with an RI detector. dCalculated using the values of Mn,NMR, ρMal5 = 0.923 g cm−3, ρPSt = 1.13 g cm−3,75 and ρPMMA = 1.26 g cm−3.76 eDetermined by SAXS measurements. fDomain spacing. gRadii of a spherical domain.

The rSAXS values were 2.7 and 2.6 nm for 2b-I and 3a-I, respectively, which are in very good agreement with the calculated values determined by its chemical structure for a Mal5 unit (rcalcd = ca. 2.45 nm) as illustrated in Figure SI-8. This result would suggest that very small spherical domains composed of bioresource-molecules were constructed by the present BCPs. This would be attributed to the high Flory− Huggins interaction parameter, that is, the χ parameter, between the bioresource-blocks and synthetic polymer chains, such as the PSt and PMMA segments, due to their amphiphilic properties and rod-like nature of the Mal5 unit.47,48,50,52,53,55 Figure 5 shows another featured result for our systems, in which the scattering peaks at √2q* and √3q* disappeared upon heating at about 190 °C. Figure 7 shows the TGA profile

Figure 8. Illustration of the self-organization of BCP samples in their bulk states building up nanoscale BCC morphology.

group, producing our target biohybrid molecules to act as the chain transfer agents (CTAs), i.e., Mal5-conjugated CTAs. The reversible addition−fragmentation chain transfer (RAFT) polymerizations with the Mal5-CTAs were demonstrated, which produced Mal5-hybrid amphiphillic block copolymers (BCPs) with well-defined structures, in which the Mal5 moieties were conjugated into the α-chain end of the polymer main chains without any cleavage of their multiple O-glycoside linkages. The resultant Mal5-hybrid amphiphillic BCPs showed self-organized properties to build up the nanoscale periodic structures such as a body centered cubic (BCC) morphology in their solid states. We have proved that the present method based on the RAFT polymerization using Mal5-CTAs is a promising method to produce polymeric materials with high functions by utilizing natural resources, which would open the door leading to a sustainable chemistry.

Figure 7. TGA curve of 1b.

of 1b, in which the thermal degradations for the spherical domains composed of Mal5 units started from ca. 190 °C. These results suggested that the BCC morphology was destroyed by increasing temperature above 190 °C, eventually forming a disordered phase. Consequently, the BCP samples 2b-I and 3a-I obtained through the RAFT polymerizations of St and MMA using Mal5-CTAs were shown to form a nanoscale BCC morphology during heating from 120 to 180 °C as summarized in Figure 8.

■

■

ASSOCIATED CONTENT

S Supporting Information *

CONCLUSIONS A versatile synthetic route for a bioresource-building block utilized for conjugation chemistry, such as maltopentaose (Mal5)-N3, has been developed. This route needed Mal5 and 5azidopentylamine as the starting materials, whereas it did not require any catalysts, protection/deprotection processes, and chromatographic purifications. In this study, further modification of Mal5-N3 was demonstrated to produce another useful building block, such as Mal5-NH2, which favored the condensation reaction with acid derivatives. The availability of Mal5-NH2 was shown by the reactions with dithiobenzoate and/or trithiocarbonate derivatives bearing an activated-ester

1 H NMR spectra and IR spectra of Mal5-N3 and Mal5-NH2, SEC traces of Mal5-N3 and Mal5, HPLC traces of 1, 1H NMR spectrum of 1a, solubility of 1, plots of specific rotations for 2 as a function of W1s, and supporting illustration for the phase separated structures formed by 2b-I. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/fax: +81-(0)238-263829. I

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Notes

(26) Seufert, M.; Schaub, M.; Wenz, G.; Wegner, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 340−343. (27) Bernard, J.; Save, M.; Arathoon, B.; Charleux, B. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 2845−2857. (28) Ryno, L. M.; Reese, C.; Tolan, M.; O’Brien, J.; Short, G.; Sorriano, G.; Nettleton, J.; Fulton, K.; Iovine, P. M. Biomacromolecules 2014, 15, 2944−2951. (29) Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.; Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.; Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T. J. Mater. Sci. 2010, 45, 1−33. (30) Isogai, A.; Saito, T.; Fukuzumi, H. Nanoscale 2011, 3, 71−85. (31) Ifuku, S.; Saimoto, H. Nanoscale 2012, 4, 3308−3318. (32) Fang, Z. Q.; Zhu, H. L.; Yuan, Y. B.; Ha, D.; Zhu, S. Z.; Preston, C.; Chen, Q. X.; Li, Y. Y.; Han, X. G.; Lee, S.; Chen, G.; Li, T.; Munday, J.; Huang, J. S.; Hu, L. B. Nano Lett. 2014, 14, 765−773. (33) Haddleton, D. M.; Ohno, K. Biomacromolecules 2000, 1, 152− 156. (34) Narumi, A.; Kaga, H.; Kawasaki, K.; Taniguchi, Y.; Satoh, T.; Kakuchi, T. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 4061−4067. (35) Narumi, A.; Matsuda, T.; Kaga, H.; Satoh, T.; Kakuchi, T. Polymer J. 2001, 33, 939−945. (36) Narumi, A.; Matsuda, T.; Kaga, H.; Satoh, T.; Kakuchi, T. Polymer 2002, 43, 4835−4840. (37) Narumi, A.; Satoh, T.; Kaga, H.; Kakuchi, T. Macromolecules 2002, 35, 699−705. (38) Kakuchi, T.; Narumi, A.; Matsuda, T.; Miura, Y.; Sugimoto, N.; Satoh, T.; Kaga, H. Macromolecules 2003, 36, 3914−3920. (39) Narumi, A.; Yamane, S.; Miura, Y.; Kaga, H.; Satoh, T.; Kakuchi, T. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 4373−4381. (40) Narumi, A.; Kaga, H.; Miura, Y.; Otsuka, I.; Satoh, T.; Kaneko, N.; Kakuchi, T. Biomacromolecules 2006, 7, 1496−1501. (41) Narumi, A.; Miura, Y.; Otsuka, I.; Yamane, S.; Kitajyo, Y.; Satoh, T.; Hirao, A.; Kaneko, N.; Kaga, H.; Kakuchi, T. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 4864−4879. (42) Narumi, A.; Otsuka, I.; Matsuda, T.; Miura, Y.; Satoh, T.; Kaneko, N.; Kaga, H.; Kakuchi, T. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 3978−3985. (43) Otsuka, I.; Fuchise, K.; Halila, S.; Fort, S.; Aissou, K.; PignotPaintrand, I.; Chen, Y. G.; Narumi, A.; Kakuchi, T.; Borsali, R. Langmuir 2010, 26, 2325−2332. (44) Loos, K.; Müller, A. H. E. Biomacromolecules 2002, 3, 368−373. (45) Stenzel, M. H.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 4498−4512. (46) Giacomelli, C.; Schmidt, V.; Putaux, J. L.; Narumi, A.; Kakuchi, T.; Borsali, R. Biomacromolecules 2009, 10, 449−453. (47) Aissou, K.; Otsuka, I.; Rochas, C.; Fort, S.; Halila, S.; Borsali, R. Langmuir 2011, 27, 4098−4103. (48) Cushen, J. D.; Otsuka, I.; Bates, C. M.; Halila, S.; Fort, S.; Rochas, C.; Easley, J. A.; Rausch, E. L.; Thio, A.; Borsali, R.; Willson, C. G.; Ellison, C. J. ACS Nano 2012, 6, 3424−3433. (49) Modolon, S. D.; Otsuka, I.; Fort, S.; Minatti, E.; Borsali, R.; Halila, S. Biomacromolecules 2012, 13, 1129−1135. (50) Otsuka, I.; Isono, T.; Rochas, C.; Halila, S.; Fort, S.; Satoh, T.; Kakuchi, T.; Borsali, R. ACS Macro Lett. 2012, 1, 1379−1382. (51) Otsuka, I.; Travelet, C.; Halila, S.; Fort, S.; Pignot-Paintrand, I.; Narumi, A.; Borsali, R. Biomacromolecules 2012, 13, 1458−1465. (52) Isono, T.; Otsuka, I.; Kondo, Y.; Halila, S.; Fort, S.; Rochas, C.; Satoh, T.; Borsali, R.; Kakuchi, T. Macromolecules 2013, 46, 1461− 1469. (53) Isono, T.; Otsuka, I.; Suemasa, D.; Rochas, C.; Satoh, T.; Borsali, R.; Kakuchi, T. Macromolecules 2013, 46, 8932−8940. (54) Otsuka, I.; Osaka, M.; Sakai, Y.; Travelet, C.; Putaux, J. L.; Borsali, R. Langmuir 2013, 29, 15224−15230. (55) Otsuka, I.; Tallegas, S.; Sakai, Y.; Rochas, C.; Halila, S.; Fort, S.; Bsiesy, A.; Baron, T.; Borsali, R. Nanoscale 2013, 5, 2637−2641. (56) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596−2599.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge Hayashibara Biochemical Laboratories Inc., Japan, for providing maltopentaose (Mal5). The authors thank Dr. Y. Jinbo for his help with the SAXS measurements. The authors thank Prof. O. Haba for the HPLC measurement. This study was partly supported by JSPS KAKENHI 25350550. The authors acknowledge support from CNRS, the PolyNat Carnot Institute, and LabEx ARCANE (ANR-11-LABX-0003-01). The authors thank the Japan Society for the Promotion of Science (JSPS) − Ministère des Affaires Etrangères et Européennes (MAEE) joint project (SAKURA program) during 2011−2013.

■

REFERENCES

(1) Kobayashi, K.; Sumitomo, H.; Kobayashi, A.; Akaike, T. J. Macromol. Sci., Chem. 1988, A25, 655−667. (2) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585−588. (3) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053−12054. (4) Nakamae, K.; Miyata, T.; Ootsuki, N.; Okamura, M.; Kinomura, K. Macromol. Chem. Phys. 1994, 195, 2663−2675. (5) Sun, X. L.; Faucher, K. M.; Houston, M.; Grande, D.; Chaikof, E. L. J. Am. Chem. Soc. 2002, 124, 7258−7259. (6) Sun, X. L.; Grande, D.; Baskaran, S.; Hanson, S. R.; Chaikof, E. L. Biomacromolecules 2002, 3, 1065−1070. (7) Miyagawa, A.; Watanabe, M.; Igai, K.; Kasuya, M. C. Z.; Natori, Y.; Nishikawa, K.; Hatanaka, K. Biomaterials 2006, 27, 3304−3311. (8) Sato, H.; Miura, Y.; Saito, N.; Kobayashi, K.; Takai, O. Biomacromolecules 2007, 8, 753−756. (9) Seto, H.; Ogata, Y.; Murakami, T.; Hoshino, Y.; Miura, Y. ACS Appl. Mater. Interfaces 2012, 4, 411−417. (10) Seto, H.; Kamba, S.; Kondo, T.; Hasegawa, M.; Nashima, S.; Ehara, Y.; Ogawa, Y.; Hoshino, Y.; Miura, Y. ACS Appl. Mater. Interfaces 2014, 6, 13234−13241. (11) Min, E. H.; Ting, S. R. S.; Billon, L.; Stenzel, M. H. J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 3440−3455. (12) Ahmed, M.; Lai, B. F. L.; Kizhakkedathu, J. N.; Narain, R. Bioconjugate Chem. 2012, 23, 1050−1058. (13) Kamide, K.; Okajima, K.; Matsui, T.; Ohnishi, M.; Kobayashi, H. Polymer J. 1983, 15, 309−321. (14) Amako, K.; Shimodori, S.; Imoto, T.; Miake, S.; Umeda, A. Appl. Environ. Microbiol. 1987, 53, 603−605. (15) Murata, J.; Saiki, I.; Makabe, T.; Tsuta, Y.; Tokura, S.; Azuma, I. Cancer Res. 1991, 51, 22−26. (16) Watanabe, K.; Saiki, I.; Matsumoto, Y.; Azuma, I.; Seo, H.; Okuyama, H.; Uraki, Y.; Miura, Y.; Tokura, S. Carbohydr. Polym. 1992, 17, 29−37. (17) Gray, D. G. Faraday Discuss. 1985, 79, 257−264. (18) Shimamoto, S.; Gray, D. G. Chem. Mater. 1998, 10, 1720−1726. (19) Muzzarelli, R. A. A.; Isolati, A. Water, Air, Soil Pollut. 1971, 1, 65−71. (20) Kawabata, Y.; Tanaka, M.; Ogata, I. Chem. Lett. 1976, 1213− 1214. (21) Ichida, A.; Shibata, T.; Okamoto, I.; Yuki, Y.; Namikoshi, H.; Toga, Y. Chromatographia 1984, 19, 280−284. (22) Okamoto, Y.; Kawashima, M.; Hatada, K. J. Am. Chem. Soc. 1984, 106, 5357−5359. (23) Yamashita, M.; Naoi, M.; Imoto, H.; Oshikawa, T. Bull. Chem. Soc. Jpn. 1989, 62, 942−944. (24) Uragami, T.; Ohsumi, Y.; Sugihara, M. Polymer 1981, 22, 1155− 1156. (25) Miyamoto, T.; Long, M.; Donkai, N. Macromol. Symp. 1995, 99, 141−147. J

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(57) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057−3064. (58) Bathfield, M.; D’Agosto, F.; Spitz, R.; Charreyre, M. T.; Delair, T. J. Am. Chem. Soc. 2006, 128, 2546−2547. (59) Moad, G.; Chong, Y. K.; Postma, A.; Rizzardo, E.; Thang, S. H. Polymer 2005, 46, 8458−8468. (60) Srinivasan, R.; Tan, L. P.; Wu, H.; Yang, P. Y.; Kalesh, K. A.; Yao, S. Q. Org. Biomol. Chem. 2009, 7, 1821−1828. (61) Huang, C. Y.; Thayer, D. A.; Chang, A. Y.; Best, M. D.; Hoffmann, J.; Head, S.; Wong, C. H. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15−20. (62) Dedola, S.; Nepogodiev, S. A.; Field, R. A. Org. Biomol. Chem. 2007, 5, 1006−1017. (63) Murphy, P. V. Eur. J. Org. Chem. 2007, 4177−4187. (64) Hanessian, S.; Ducharme, D.; Massé, R.; Capmau, M. L. Carbohydr. Res. 1978, 63, 265−269. (65) Blanco, J. L. J.; Fernández, J. M. G.; Gadelle, A.; Defaye, J. Carbohydr. Res. 1997, 303, 367−372. (66) Györgydeák, Z.; Tiem, J. Adv. Carbohydr. Chem. Biochem. 2006, 60, 103−182. (67) Gouin, S. G.; Kovensky, J. Tetrahedron Lett. 2007, 48, 2875− 2879. (68) Gudmundsdottir, A. V.; Nitz, M. Org. Lett. 2008, 10, 3461− 3463. (69) Besset, C.; Chambert, S.; Fenet, B.; Queneau, Y. Tetrahedron Lett. 2009, 50, 7043−7047. (70) Tanaka, T.; Nagai, H.; Noguchi, M.; Kobayashi, A.; Shoda, S. Chem. Commun. 2009, 3378−3379. (71) Wen, S.; Guo, Z. Org. Lett. 2001, 3, 3773−3776. (72) Stenzel, M. H.; Davis, T. P.; Fane, A. G. J. Mater. Chem. 2003, 13, 2090−2097. (73) Akeroyd, N.; Pfukwa, R.; Klumperman, B. Macromolecules 2009, 42, 3014−3018. (74) Imamura, K.; Sakaura, K.; Ohyama, K.; Fukushima, A.; Imanaka, H.; Sakiyama, T.; Nakanishi, K. J. Phys. Chem. B 2006, 110, 15094− 15099. (75) Natta, G.; Corradini, P. Makromol. Chem. 1955, 16, 77−80. (76) Kusanagi, H.; Tadokoro, H.; Chatani, Y. Macromolecules 1976, 9, 531−532. (77) Matsen, M. W.; Bates, F. S. Macromolecules 1996, 29, 1091− 1098.

K

dx.doi.org/10.1021/bm501314f | Biomacromolecules XXXX, XXX, XXX−XXX