Synthesis and Characterization of Glycopolymer-Polypeptide Triblock

Biomacromolecules 2004, 5, 224-231

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Synthesis and Characterization of Glycopolymer-Polypeptide Triblock Copolymers Chang-Ming Dong,† Xue-Long Sun,† Keith M. Faucher,† Robert P. Apkarian,‡ and Elliot L. Chaikof*,†,§ Laboratory for Biomolecular Materials Research, Departments of Surgery and Biomedical Engineering, Emory University School of Medicine, Atlanta, Georgia 30322, Integrated Microscopy & Microanalytical Facility, Emory University, Atlanta, Georgia 30322, and the School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332 Received September 11, 2003; Revised Manuscript Received October 15, 2003

Glycopolymer-polypeptide triblock copolymers of the structure, poly(L-alanine)-b-poly(2-acryloyloxyethyllactoside)-b-poly(L-alanine) (AGA), have been synthesized by sequential atom transfer radical polymerization (ATRP) and ring-opening polymerization (ROP). Controlled free radical polymerization of 2-O-acryloyloxyethoxyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (AEL) by ATRP with a dibromoxylene (DBX)/CuBr/bipy complex system was used to generate a central glycopolymer block. Telechelic glycopolymers with diamino end groups were obtained by end group transformation and subsequently used as macroinitiators for ROP of L-alanine N-carboxyanhydride monomers (Ala-NCA). Gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) spectroscopy analysis demonstrated that copolymer molecular weight and composition were controlled by both the molar ratios of the Ala-NCA monomer to macroinitiator and monomer conversion and exhibited a narrow distribution (Mw/Mn ) 1.06-1.26). FT-IR spectroscopy of triblock copolymers revealed that the ratio of R-helix/β-sheet increased with poly(L-alanine) block length. Of note, transmission electron microscopy (TEM) demonstrated that selected amphiphilic glycopolymer-polypeptide triblock copolymers self-assemble in aqueous solution to form nearly spherical aggregates of several hundreds nanometer in diameter. Significantly, the sequential application of ATRP and ROP techniques provides an effective method for producing triblock copolymers with a central glycopolymer block and flanking polypeptide blocks of defined architecture, controlled molecular weight, and low polydispersity. Introduction The design and synthesis of polymers with pendant saccharide residues (glycopolymers) have been motivated in part by the recognition that glycopolymers may potentiate multivalent protein-carbohydrate interactions.1-7 This observation has led to the development of a number of novel glycopolymers that hold significant potential in pharmacotherapy, tissue engineering, and molecular diagnostics.8-11 Thus, improved synthetic routes and control over glycopolymer architecture have become an important objective for a variety of biomedical applications. Glycopolymers exhibiting low fluctuations in both size and composition have been generated by cationic polymerization,12-15 ring-opening polymerization (ROP),16,17 ring-opening methathesis polymerization,18,19 cyanoxyl- and nitroxide-mediated radical polymerization,20-27 as well as transition-metal-catalyzed atom transfer radical polymerization (ATRP).28-32 * To whom correspondence should be addressed. Elliot L. Chaikof, M.D., Ph.D., 1639 Pierce Drive, Woodruff Memorial Research Building, Rm 5105, Emory University, Atlanta, GA 30322. Phone: (404) 727-8413. Fax: (404) 727-3660. E-mail: [email protected]. † Emory University School of Medicine. ‡ Emory University. § Georgia Institute of Technology.

In the development of controlled polymerization techniques for generating block copolymers, recent efforts have illustrated the potential versatility of combining both ATRP and ROP methodologies. For example, the preparation of block copolymers comprised of poly(caprolactone) and poly(methyl methacrylate) blocks by sequential ROP and ATRP has been described.33,34 More complex macromolecular architectures, such as dendritic starlike block copolymers and highly branched and graft copolymers, have also been reported.35-38 Indeed, Chen et al. have recently reported ABA and star amphiphilic block copolymers composed of galactosebearing glycopolymer.28 An important characteristic of homopolypeptides is their capacity to self-assemble into higher order structures by either hydrophobic, electrostatic, or hydrogen bond mediated interactions.39-41 Thus, if incorporated into amphiphilic copolymers as polypeptide blocks, micellar or network structures can be produced depending, in part, upon block architecture design. Important recent examples include the synthesis of amphiphilic polybutadiene-b-polyglutamic acid copolymers that have led to the development of stimuliresponsive vesicles,42,43 poly(ethylene oxide)-b-polypeptide copolymer micelles for drug delivery and gene therapy,44,45 self-assembling polypeptide-containing multiblock copoly-

10.1021/bm0343500 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/02/2003

Glycopolymer-Polypeptide Triblock Copolymers

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Scheme 1. Synthesis of Poly(L-alanine)-b-poly(2-acryloyloxyethyl-lactoside)-b-poly(L-alanine) Triblock Copolymer (AGA)a

a Reagents and conditions: (a) 2-hydroxy-ethyl acrylate, BF -Et O/CH Cl ; (b) CuBr, bipy/chlorobenzene, 100 °C; (c) (i) N-BOC-1, 4-diaminobutane, 3 2 2 2 TEA/THF, r.t.; (ii) TFA (20%, v/v)/ CHCl3, r.t; (d) Ala-NCA/DMF, r.t.; (e) NH2‚NH2‚H2O/DMSO, rt.

mers inspired from spider silk,46 and amphiphilic diblock and triblock polypeptides forming hydrogels.47,48 As an extension of these synthetic efforts, recent investigations have focused on the generation of glycopeptide mimics.49,50 For example, Okada et al.16,17,51,52 have synthesized glycopeptides by ring-opening polymerization of sugarsubstituted R-amino acid N-carboxyanhydride monomers, in which sugar residues are pendant to a polypeptide backbone. Similarly, Takasu et al.53 reported the synthesis of periodic glycopeptides by a step polycondensation method. However, to some extent, the secondary structure and the self-assembly behavior of polypeptide will be affected by the pendant sugar. Poly(L-alanine) naturally occurs in β-sheet crystalline domains and forms temporary cross-links in spider silk. Because of its natural tendency to readily aggregate, poly(L-alanine) constitutes excellent building blocks for selfassembled nanostructures in solid state and in solution.46 Moreover, poly(L-alanine) with different chain length usually self-assembles into β-sheet and/or R-helix structures.54 The objective of the work reported herein is to take advantage of two aspects, one in which glycopolymer may function as a multivalent ligand utilized for site-specific drug delivery, and the other in which poly(L-alanine) with the tunable secondary structures can be used to manipulate the size and shape of self-assembled nanostructures. Specifically, we have designed and synthesized a new class of amphiphilic glycopolymer-polypeptide triblock copolymer, poly(L-alanine)b-poly(2-acryloyloxyethyl-lactoside)-b-poly(L-alanine) (AGA), via sequential atom transfer radical polymerization (ATRP) and ring-opening polymerization (ROP), as shown in Scheme 1. Using an end group transformation method, amino terminated telechelic glycopolymers, initially produced by ATRP, were used as macroinitiators for the ring-opening polymerization of L-alanine-N-carboxyanhydride (AlaNCA). In this manner, a glycopolymer midblock was framed by polypeptide endblocks affording an amphiphilic glycopolymer-polypeptide triblock copolymer, which self-assembled in aqueous solution to form nearly spherical

aggregates with large sizes. To our knowledge, this is the first report to describe the sequential application of ATRP and ROP techniques to generate well-defined glycopolymerpolypeptide triblock copolymers as well as their potential to exhibit self-assembly behavior. Significantly, this approach provides an effective method for producing glycopolymerpolypeptide triblock copolymers of defined architecture, controlled molecular weight, and low polydispersity. Experimental Section Materials. All solvents and reagents of the highest grade were purchased from commercial sources and were used as received, unless otherwise noted. Anhydrous dimethylformamide (DMF), methylsulfoxide (DMSO), toluene, and tetrahydrofuran (THF) were used under an argon atmosphere. The Spectra/Por molecular porous membrane (Mw cutoff: 3500) was purchased from Spectrum Laboratories Inc. AlaNCA was synthesized, as detailed elsewhere,55 and recrystallized twice from THF/toluene (yield, 76%). 1H NMR (CDCl3), δppm: 6.4 (s, 1H), 4.45-4.38 (q, 1H), 1.58-1.56 (d, 3H). 13C NMR (CDCl3), δppm: 170.0, 152.3, 53.3, 17.6. HR-MS (EI): calcd for C4H5O3N, 115.0269; Found, 115.0267 M+. Methods. All reactions were performed in flame-dried glassware under an atmosphere of dry argon. The reaction medium solutions were evaporated under reduced pressure with a rotary evaporator, and the residue was chromatographed on a silica gel (230-400 mesh) column. Analytical thin-layer chromatography (TLC) was performed on Whatman silica gel aluminum backed plates of 250 mm thickness on which spots were visualized with UV light or charring the plate before and/or after dipping in a H2SO4-EtOH mixture. Mass spectra (MS/FAB) were obtained at an ionizing voltage of 70 eV. 1 H and 13C NMR spectra were recorded at room temperature with a Varian INOVA 400 (400 MHz) and Varian

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UNITY 600 (600 MHz) spectrometers, respectively. In all cases, the sample concentration was 10 mg/mL, and the appropriate deuterated solvent was used as internal standard. The gel permeation chromatography (GPC) equipment comprised a Waters model 510 HPLC pump, appropriate columns, and a Wyatt Technology Optilab 903 refractometer. The actual molar masses of the polymer samples were determined from the response of the DAWN EOS (Wyatt Technology) multiangle laser light-scattering (LLS) detector that was connected to the outlet of the SEC apparatus using Wyatt ASTRA 4.0 software for data analysis. Columns for glycopolymers consisted of three Styragel columns in sequence (HT2 + HT3 + HT4) using THF as the eluent at 1.0 mL/min and 25 °C. For protected triblock copolymers, DMSO was used as the eluent at 1.0 mL/min and 30 °C. For deprotected triblock copolymers, Waters Ultrahydrogel 250 and 2000 columns were used with deionized water as eluent containing 0.1 mol/L NaNO3 and 0.05 wt % sodium azide at a flow rate of 0.7 mL/min and 30 °C. Infrared spectra of test samples in powder form were acquired using a BioRad FTS-60 Fourier Transform Infrared (FT-IR) spectrometer equipped with a wide band MCT detector. Transmission electron microscopy (TEM) was performed using a JEOL 1210 TEM at a 90 kV accelerating voltage. Samples were deposited onto the surface of 200 mesh Formvar-carbon film-coated copper grids. Excess water was wicked away, and the grids were dried in a vacuum. The image contrast was enhanced by negative staining with uranyl acetate. Synthesis of 2-O-Acryloyloxyethyl-(2,3,4,6-tetra-Oacetyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-β-Dglucopyranoside (AEL). AEL was synthesized using a protocol similar to that described by our group and others.56 Briefly, BF3-etherate (3.20 mL, 25 mmol, 5 equiv) was added to a cooled (ice-water bath), stirred solution of β-lactose octaacetate (3.39 g, 5 mmol) and 2-hydroxy-ethyl acrylate (0.60 mL, 6 mmol, 1.2 equiv) in dichloromethane (20 mL). The reaction mixture was stirred for 2 h at 0 °C and then for an additional 16 h at room temperature under argon atmosphere. The mixture was diluted with chloroform (60 mL), washed with water (30 mL) and saturated aqueous sodium hydrogen carbonate (30 mL), and filtered. The filtrate was evaporated to give a residue, which was purified by column chromatography (SiO2) using acetone-n-hexane (1: 3) as eluent to afford the glycomonomer AEL (2.77 g, 75%). 1 H NMR (CDCl3), δppm: 6.40 (dd, 1H), 6.10 (m, 1H), 5.82 (dd, 1H), 5.32-4.80 (m, 5H), 4.56-3.60 (m, 13H), and 2.20-1.92 (m, 21H). 13C NMR (CDCl3), δppm: 170.5, 170.3, 170.2, 169.9, 169.7, 169.2, 169.1, 166.0, 131.5, 128.1, 100.9, 76.3, 72.8, 71.6, 71.1, 70.8, 69.2, 67.6, 66.7, 63.4, 62.4, 62.2, 62.1, 60.9, 20.8. MS/FAB, m/z: calcd for C31H42O20Li, 741.2429; found, 741.2418 [M + Li]+. Synthesis of a Protected Glycopolymer (G) with Dibromo End Groups via ATRP of AEL. The ATRP of AEL was carried out in chlorobenzene at a monomer concentration of 0.5 mol/L. In a typical experiment, Cu(I)Br, bipy, and a mixture of dibromo xylene (DBX) and AEL were weighed into a glass tube containing chlorobenzene and a stirring bar.

Dong et al.

The representative feed was DBX:CuBr:bipy ) 1:1:3 (molar ratio). The mixture was immediately purged with argon for 5 min and degassed 3 times by freeze-pump-thaw cycles. The tube was then sealed under vacuum and put into a preheated oil bath at 100 °C. After a prescribed time period, the tube was opened and the solution was diluted with THF. The resultant glycopolymer solution was precipitated by dropwise addition into a large excess of diethyl ether. The purified glycopolymer was dried in a vacuum, and the monomer conversion was determined gravimetrically. Synthesis of a Protected Glycopolymer (G-NH2) with Diamino End Groups. Bromine terminated glycopolymers were reacted with N-BOC-1,4-diaminobutane followed by deprotection in trifluoroacetic acid to yield a diaminoterminated glycopolymer. In a typical experiment, a glycopolymer with dibromo end groups (G10, 0.162 g, 0.02 mmol) was dissolved in 4 mL of THF. Triethylamine (30 equiv, 1.2 mmol, 0.17 mL) along with a 10-fold excess of N-BOC1,4-diaminobutane (0.4 mmol, 0.08 mL) were added under argon atmosphere. After stirring for 48 h at room temperature, the glycopolymer solution was precipitated by dropwise addition into a large excess of diethyl ether to yield 0.150 g of white power (yield, 93%). The product was dissolved in 4 mL of chloroform after which 1 mL of trifluoroacetic acid was added under argon atmosphere. After stirring for 12 h at room temperature, the solution was diluted with 70 mL of chloroform; washed with water (30 mL), saturated aqueous sodium hydrogen carbonate (30 mL), and water (30 mL); and filtered. The filtrate was evaporated to give a residue, which was dissolved in 4 mL of THF and dropwise precipitated into a large excess of diethyl ether to give the glycopolymer with diamino end groups (G10-NH2, 0.128 g; yield, 85%). Yields varied within 80-90% in the generation of all diamino-terminated glycopolymers. Synthesis of Poly(L-alanine)-b-poly(2-acryloyloxyethyl octaacetyllactoside)-b-poly(L-alanine) (AGA-Ac) triblock copolymers. Triblock copolymers were synthesized using a protected glycopolymer with diamino end groups as a macroinitiator of ring opening polymerization of Ala-NCA. In a representative reaction, a glycopolymer with a diamino end group (G10-NH2, 0.162 g, 0.02 mmol) was added to a 10 mL round-bottom flask equipped with a stirring bar. The flask was sealed with a rubber septum, and 2 mL of DMF was introduced via a syringe. The mixture was stirred until the solids dissolved and then degassed three times by freezepump-thaw cycles, after which a degassed solution of AlaNCA (0.232 g, 2.0 mmol) in 3 mL of DMF was added via a syringe under argon atmosphere. The mixture was allowed to stir at room temperature for 64 h. The reaction mixture was diluted with THF and then dropwise precipitated into a large excess of diethyl ether and filtered. The solids thus obtained were initially washed with 10 mL of THF to extract any unreacted macroinitiator and subsequently washed with 15 mL of DMSO to remove homopoly(L-alanine) that may have been generated by an unintended side reaction. The resultant triblock copolymer solution was precipitated by dropwise addition into a large excess of diethyl ether affording a white powder that was dried in a vacuum (A37G10A37-Ac, 0.331 g; yield, 84%). Copolymer yields and



Table 1. Syntheses of Glycopolymers with Dibromo End Groups (G) and the Related Glycopolymers with Diamino End Groups (G-NH2) via ATRP of AEL Monomer in Chlorobenzene at 100 °C with Conditions M ) AEL, I ) Initiator ) Dibromo-p-xylene, [M] ) 0.5 mol/L, [I]:[CuBr]:[Bipy] ) 1:1:3 entry

[M]/[I]

time (h)

conversion %

Mn,NMRb

Mn,thc

G8 G10 G10-NH2a G24 G24-NH2a G36 G36-NH2a G52 G52-NH2a

10 10 10 40 40 60 60 80 80

16 24

80 96

24

58

5440 8090 7840 17640 16170

30

62

36

65

5880 7350 7350 16910 16910 27200 27200 38220 38220

24990 35280

Mn,GPCd

MWDe

9310 8640 20640 19580 30530

1.24 1.20 1.29 1.28 1.19

38170 39700

1.35 1.36

a G10-NH , G24-NH , G36-NH , and G52-NH glycopolymers with diamino end groups were obtained from the precursor glycopolymers with dibromo 2 2 2 2 end groups, respectively. b Mn,NMR is determined by 1H NMR spectroscopy, n ) 4(b +c)/18a, in which n is the degree of polymerization of glycopolymer, a is the integral value of the initiator xylene residues, b + c represent the sum of the integral value of lactose, linker -OCH2CH2O- and methine proton of -CHBr. c Mn,th ) Mw,AEL × [M]/[I] × conversion %. d Mn,GPC is determined by GPC using LLS and RI as detectors, with THF as eluent at 25 °C. e MWD ) Mw/Mn.

Table 2. Syntheses of AGA-Ac Triblock Copolymers and the Related Deprotected Triblock Copolymers (AGA) via Ring-Opening Polymerization of Ala-NCA Using a Glycopolymer with Diamino End Groups as a Macroinitiator in DMF at Room Temperature entry

[M]/[I]c

yield %d

conversion %

Mn,NMRe

Mn,thf

Mn,GPCg

MWD

A10G10A10-Aca

30 30 100 300 30 100 100 50 50 100 100

91

73

84 65 94 81

73 57 66 54

1.27 1.23 1.38 1.41

46

29680

1.06

88

48

41340

9640 5970 12530 20230 19040 20320 13560 39870 24580 41630 26050

9550 6160 14260 24300

92

8770 6190 13340 21290 19340 21620 14560 39500

31860

1.26

A10G10A10b A37G10A37-Ac A93G10A93-Ac A12G24A12-Ac A28G24A28-Ac A28G24A28b A9G52A9-Ac A9G52A9b A22G52A22-Ac A22G52A22b

a The reaction time is 40 h, and all of the others are 64 h. b A10G10A10, A28G24A28, A9G52A9, and A22G52A22 triblock copolymers are obtained from the deacetylation of the precursors A10G10A10-Ac, A28G24A28-Ac, A9G52A9-Ac, and A22G52A22-Ac copolymers, respectively. c M ) AlaNCA, I ) macroinitiator. d Yield ) WAGA/(WG + WNCA). e Mn,NMR is determined by 1H NMR spectroscopy, 2m ) 4b/3a, in which 2m is the degree of polymerization of polypeptide, a is the integral value of the initiator xylene residues, b is the integral value of methyl (-CH3) groups attributed to poly(Lalanine). f Mn,th ) Mn,th (G) + Mw, NCA× [M]/[I] × conversion %. g Mn,GPC is determined by GPC using LLS and RI as detectors.

monomer conversions were determined gravimetrically and ranged between 65 and 94% and 46-73%, respectively Deacetylation of AGA-Ac Triblock Copolymers. In a characteristic experiment, 50.0 mg of A10G10A10-Ac copolymer was stirred in 10 mL of DMSO at room temperature for 4 h, followed by dropwise addition of 0.21 mL (4.40 mmol) of hydrazine monohydrate at 0 °C under argon atmosphere. After the mixture was vigorously stirred at room temperature for 24 h, 0.65 mL (8.90 mmol) of acetone was added to the solution with cooling at 0 °C. The mixture was then diluted with water and dialysis performed with a Spectra/Por molecular porous membrane (MWCO 3500). After lyophilization, 31.7 mg of A10G10A10 copolymer was obtained (yield, 90%). Yields varied between 90 and 95% for the deacetylation of AGA-Ac triblock copolymers. Results and Discussion Synthesis of a Protected Glycopolymer with Diamino End Groups (G-NH2) via ATRP. The ATRP of AEL ([M] ) 0.5 mol/L) was carried out under heterogeneous conditions using bipy as the ligand and DBX as a bifunctional initiator in conjunction with CuBr in chlorobenzene at 100 °C. The results of these investigations are summarized in Table 1.

Figure 1. 1H NMR spectrum of glycopolymer (G10) in CDCl3 (no. 2, Table 1).

Using multi-angle laser light scattering, absolute glycopolymer molecular weights (Mn,GPC) were consistent with predicted theoretical molecular weights (Mn,th) with associated narrow molecular weight distributions (Mw/Mn ) 1.19-1.35), where Mn,th ) [M]/[I] × MAEL × % conversion (see the Supporting Information). Moreover, Figure 1 illustrates that the integral ratio of 1H NMR peaks assigned to lactose residues, the spacer -OCH2CH2O-, and the terminal methine (-CHBr) proton at 5.42-3.40 ppm to the aromatic proton peaks of the dibromo xylene initiator at 7.20-6.90 ppm provided an additional means of calculating glycopoly-

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Figure 2. (A) 1H NMR spectrum of A10G10A10-Ac triblock copolymer) in DMSO-d6 (no. 1, Table 2). (B) 1H NMR spectrum of A10G10A10 triblock copolymer in D2O (no. 2, Table 2).

mer molecular weight (Mn,NMR). As noted in Table 1, Mn,NMR were in agreement with Mn,th and Mn,GPC. Significantly, the number average molecular weight (Mn) can be predicted by both the molar ratio of monomer to initiator ([M]/[I]) and monomer conversion (see the Supporting Information). All told, these data indicate that ATRP of the AEL monomer has some “living”/controlled characteristics yielding welldefined glycopolymers with dibromo end groups. As described elsewhere,57,58 dibromo end groups can be successfully transformed to diamino end groups by nucleophilic substitution using N-BOC-1,4-diaminobutane followed by deprotection in trifluoroacetic acid. 1H NMR spectroscopy confirmed that the efficiency of end group transformation was nearly quantitative, as demonstrated by the integral ratio of the proton peaks of BOC end groups to the aromatic proton peaks of dibromo xylene residues (see the Supporting Information). In addition, complete deprotection of the BOC group was confirmed by loss of its proton signal at 1.40 ppm. Purity of the diamino-terminated glycopolymer was verified by GPC with molecular weights and polydispersities similar to those of respective precursor polymers (Table 1; see the Supporting Information).

Synthesis of Acetylated Triblock Copolymers (AGAAc). Telechelic glycopolymers G-NH2 with diamino end groups were used as macroinitiators for ring-opening polymerization of Ala-NCA in DMF at room temperature. The molar ratio of Ala-NCA monomer to macroinitiator was varied from 30:1 to 300:1, and the results of the polymerization reactions are summarized in Table 2. Fully protected AGA-Ac triblock copolymers were insoluble in chloroform, THF, 1,4-dioxane, and DMF but were found to be soluble in DMSO, which was used as the GPC eluent. GPC traces of triblock copolymers (AGA-Ac) were unimodal. Peaks shifted toward higher molecular weight with increasing poly(L-alanine) block length, without the presence of detectable peaks indicative of residual macroinitiator or poly(L-alanine) homopolymer (see the Supporting Information). Copolymer molecular weights could also be determined by analysis of 1H NMR spectra and were consistent with those determined by GPC. For example, the 1H NMR spectrum of the A10G10A10-Ac triblock copolymer demonstrates that, in addition to the characteristic signals of the glycopolymer block, proton signals at 1.42-1.00 ppm could be assigned to the methyl groups of the poly(L-alanine) block (Figure



Figure 3.

13C

NMR spectrum of A10G10A10-Ac in DMSO-d6 (no. 1, Table 2).

2A). Thus, the calculated Mn,NMR of the glycopolymer and poly(L-alanine) blocks were 7350 and 1420, respectively. These values are in agreement with the calculated Mn,NMR of the original G10-NH2 macroinitiator (7,840), as well as Mn,th (1550) for the poly(L-alanine) block. Similar results were also obtained for diamino terminated glycopolymers of larger molecular weight (G24-NH2 and G52-NH2; Table 2). Significantly, our investigations confirmed that both of the molar ratios of Ala-NCA to macroinitiator and monomer conversion were predictive of poly(L-alanine) block length (see the Supporting Information). These results are similar to those reported for the ROP of γ-benzyl-L-glutamate N-carboxyanhydride when initiated by amine-terminated polystyrene, as well as the ROP of various amino acid NCA monomers initiated by 4-aminobenzoyl-terminated poly(caprolactone).59,60 Further confirmation of the AGA-Ac block structure was provided by 13C NMR and FT-IR spectroscopy with the A10G10A10-Ac copolymer providing a representative example of this analysis. 13C NMR reveals characteristic chemical shifts of carbonyls for R-helix and β-sheet structures of poly(L-alanine) at 176.4 and 171.8 ppm ((0.5 ppm), respectively.54 Notably, intermediate signals, suggestive of other nonblock copolymer structures, were not detected between the single carbonyl peak (171.6 ppm) of the poly(L-alanine) β-sheet dominant block and those carbonyl peaks (173.8, 170.2-169.0 ppm) assigned to the glycopolymer block (Figure 3). Further verification of the secondary molecular structure of the poly(L-alanine) block was established by FT-IR spectroscopy (Figure 4) in which characteristic peaks for R-helix and β-sheet structures are found at 1656 and 1630 cm-1, respectively.54 We observed that poly(L-alanine) exists exclusively in a β-sheet conformation within short blocks (m ) 10), whereas both β-sheet and R-helix structures are evident in blocks of larger sizes. Indeed, the spectra of triblock copolymers demonstrate a progression of secondary structures that is similar to that reported for pure poly(L-alanine) of varying molecular weights.54,61

Figure 4. FT-IR spectra of AmG10Am-Ac triblock copolymers (m ) 10, 37, and 93).

Deacetylation of AGA-Ac Triblock Copolymers. Deacetylation of the O-protecting acetyl groups of the lactose moiety was successfully achieved by treatment with hydrazine monohydrate in DMSO at room temperature. As an example, 1H NMR spectroscopy of A10G10A10 reveals that peaks assignable to the O-protecting acetyl groups at 1.90-2.10 ppm are entirely absent after deacetylation (Figure 2B), whereas those due to the protons of lactose residues and the pendant spacer arm are still present and upfield shift to 3.004.40 ppm. In addition, methine (CH) peaks at 2.40-2.10 ppm and methylene (CH2) peaks at 2.00-1.50 ppm that represent the backbone of the glycopolymer block remain visible. Likewise, FT-IR spectroscopy of A28G24A28 triblock copolymers obtained before and after O-deacetylation demonstrate the loss of the CdO absorption peak of the acetyl groups at 1751 cm-1 after O-deacetylation with the appearance of a broad absorption peak at 3272 cm-1, which corresponds to the presence of hydroxyl groups (Figure 5). Notably, the absorption peak at 1732 cm-1, which is ascribed to the ester bond that connects the pendant lactose units to the main chain of the copolymer, remains unchanged, and the secondary structure of the poly(L-alanine) block remains unaffected, which is suggested by the absorption peak at 1631 cm-1. The molecular weights and polydispersities of the deprotected AGA triblock copolymers as determined by GPC

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Figure 5. FT-IR spectra of deprotected A28G24A28 and A28G24A28Ac triblock copolymers.

and NMR are summarized in Table 2. As anticipated, the decrease in molecular weight of the deprotected copolymers AGA is fully accounted for by the removal of the acetyl groups with molecular weight distributions in the range of 1.06-1.26 ppm (see the Supporting Information). Accurate molecular weights of the AGA triblock copolymers could be determined from 1H NMR, which is in agreement with GPC determined and theoretical values. In summary, Oprotecting acetyl groups can be removed quantitatively from the glycopolymer midblock without affecting either polymer composition or the secondary structure of poly(L-alanine) endblocks. Thus, sequential ATRP and ROP provides a facile approach for the synthesis of a new class of amphiphilic glycopolymer-polypeptide triblock copolymers. Morphology of Glycopolymer-Polypeptide Triblock Copolymers in Aqueous Solution. The morphology of A9G52A9 and A22G52A22 copolymers in aqueous solution was examined in preliminary investigations by TEM. Figure 6 demonstrates the formation of spherical nanoparticles from both triblocks. Particle diameters of 200-700 nm were observed from 5 mg/mL solutions of A9G52A9, whereas particle diameters of 100-400 nm were noted upon increasing the copolymer concentration to 10 mg/mL. Similarly, the A22G52A22 copolymer (2.5 mg/mL) produced uniform spheres with diameters of 400-600 nm. These demonstrate that AGA triblock copolymers may prove to be useful for drug delivery applications in which site-specific delivery is mediated by multivalent carbohydrate-protein interactions. We anticipate that this class of glycopolymer-polypeptide triblock copolymer will establish a useful starting point for the generation of self-assembling carbohydrate-based biomaterials with greater control over nano- and microscale structure. Conclusions A new class of well-defined glycopolymer-polypeptide triblock copolymer was successfully synthesized by the combination of ATRP of a protected lactose bearing glycomonomer followed by ROP of Ala-NCA. The molecular weight and the unit composition of triblock copolymers were controlled both by the molar ratio of Ala-NCA monomer to glycopolymer macroinitiator and monomer conversion. The ratio of the R-helix/β-sheet secondary structure within the

Figure 6. TEM microphotographs of nearly spherical aggregates from amphiphilic glycoprotein triblock copolymer in water: (A) A9G52A9, 10 mg/mL; (B) A9G52A9, 5 mg/mL; (C) A22G52A22, 2.5 mg/mL.

poly(L-alanine) endblocks increased with block length. As anticipated, deacetylation of the protected glycopolymer midblock generated amphiphilic triblock copolymers comprised of a hydrophilic lactose-bearing central block flanked by hydrophobic poly(L-alanine) end-blocks, which selfassemble in aqueous solution to form nearly spherical submicron aggregates. Acknowledgment. This work was supported by grants from the NIH. The authors acknowledge the Emory University NMR and Mass Spectrometry Centers for their facilities. Supporting Information Available. GPC traces for G, G-NH2, and AGA triblock copolymers, and 1H NMR spectra


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BM0343500

Synthesis and Characterization of Glycopolymer-Polypeptide Triblock

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