Cationic Charged Helical Glycopolypeptide Using Ring Opening

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Cationic Charged Helical Glycopolypeptide using Ring Opening Polymerization Of 6-deoxy-6-Azido Glyco-N-carboxyanhydride Ashif Yasin Shaikh, Soumen Das, Debasis Pati, Vinta Dhaware, Sayam Sen Gupta, and Srinivas Hotha Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5009537 • Publication Date (Web): 14 Aug 2014 Downloaded from http://pubs.acs.org on August 25, 2014

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Cationic Charged Helical Glycopolypeptide using Ring Opening Polymerization Of 6-deoxy-6azido-Glyco-N-Carboxyanhydride Ashif Y. Shaikh,a, b† Soumen Das,a† Debasis Pati, a Vinita Dhaware, a Sayam Sen Gupta a* and Srinivas Hotha b* a

CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune-411 008 (INDIA).

b

Department of Chemistry, Indian Institute of Science Education and Research, Pune-411 008

(INDIA). †

Contributes equally to the manuscript [email protected] and [email protected]

ABSTRACT

Glycopolypeptides with a defined secondary structure are of significance in understanding biological phenomena. Synthetic glycopolypeptides, or polypeptides featuring pendant carbohydrate moieties, have been of particular interest to the field of tissue engineering and drug delivery. In this work, we have synthesized charged water-soluble glycopolypeptides that adopt a helical conformation in water. This was carried out by the synthesis of a glyco-N-

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carboxyanhydride (glyco-NCA) containing azide group at the sixth position of the carbohydrate ring. Subsequently, the NCA was polymerized to obtain azide-containing glycopolypeptides having good control over molecular weights and polydispersity index (PDI) in high yields. We were also able to control the incorporation of the azide group by synthesizing random coglycopolypeptide containing 6-deoxy-6-azido and regular 6-OAc functionalized glucose. This azide functionality allows for the easy attachment of a bioactive group, which could potentially enhance the biological activity of the glycopolypeptide. We were able to obtain water-soluble charged glycopolypeptides by both reducing the azide groups into amines and using CuAAC with propargylamine. These charged glycopolypeptides were shown to have a helical conformation in water.

Preliminary studies showed that these charged glycopolypeptides

showed good biocompatibility and were efficiently taken up by HepG2 cells. INTRODUCTION Glycosylated peptides and proteins occur widely in nature and are known to carry out important biological functions, such as mediation of cellular interaction and lubrication in eyes and joints.13

Synthetic polypeptides are envisaged to have secondary conformations such as α-helix, β-

sheets and random coils, which can be useful materials for biomedical application.4 Synthetic glycopolypeptides (GPPs) are currently being investigated as vehicles for drug delivery, scaffolds for tissue engineering and as templates for self-assembled structures;5-12 these can also function as tools for investigating carbohydrate-protein interactions.4,

13-15

The synthesis of

homogenous GPPs with defined architectures is still a daunting task, since their synthesis demands the exploitation of the chemistries of both peptides and saccharides. 16-21 The identified reagents and reaction conditions must be compatible with all the functional and protective groups present in amino acids and carbohydrates. Also, the envisioned synthetic strategy must be easy


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and amenable to the synthesis of GPPs in a modular fashion, so that a large number of them can be accessed. Two mutually complementary approaches have been developed recently to meet this challenge.22-30 First, GPPs with glycan periodicity and control were obtained by ring opening polymerization (ROP) of preformed glyco-N-carboxyanhydride (glyco-NCAs) with amine initiators.31-37 This strategy has been used to synthesize GPPs having 100% glycosylation with high efficiency.

4, 35

Alternatively, two orthogonally reacting functional groups (azide/alkyne or

thiol/ene, for example) can be identified and the polypeptide can be synthesized with one of them by NCA polymerization such that glycan moiety with the other can be grafted after the polymerization is completed. For example, azide-containing side chain polypeptides have been recently synthesized and were subsequently post-functionalized using alkyne-containing saccharides to yield GPPs.

24-30

Each method is unique and has its own advantages and

disadvantages. We envisioned GPPs with interesting properties if we combine the strategies described above, that is if we synthesize polypeptides containing 100% glycosylation and incorporate functionality that can be post-functionalized. For example, since GPPs are hydrophilic and uncharged, they do not enter mammalian cells easily. However, if some of the saccharide side chains are cationic, it might help these charged polypeptides to enter mammalian cells. Indeed, both natural and synthetic cationic polysaccharides have been used for gene delivery for decades as carbohydrates are thought to be involved in condensing DNA via hydrogen bonding.38 This, in turn, reduces the need of excess cationic charge and, hence, decreases the overall toxicity of the system. Further, the monomeric carbohydrate units have the potential to interact with specific cell receptors and may serve as vehicles for targeted drug delivery. However, most of the cationic polysaccharides that have been studied for gene delivery have a random coil structure.


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In contrast, these charged GPPs are expected to be in a helical conformation, since the cationic groups will be located far away from the peptide backbone (>11 bonds) and will be unable to disrupt the H-bonding of the polypeptide backbone necessary for the stability of the α-helix.

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The synthesis of cationic helical glycopolypeptides would thus allow us to explore the role of helicity in these polymers for gene delivery. In recent studies, cationic helical polypeptides have been synthesized and shown to be efficient agents of DNA transfection.40-48 In this manuscript, we report the synthesis of 6-deoxy-6-azido functionalized glyco-Lys-Ncarboxyanhydride and its subsequent ROP to form GPPs having a pendant azide group. Subsequently, the azide groups were converted into charged groups (-NH2) by either reduction of the azide or by using CuAAC to install charged side chains. We also report the synthesis of random co-glycopolypeptide containing 6-deoxy-6-azido functionalized glyco-Lys together with the regular 6-OAc protected glyco-Lys. Since both the NCA monomers are chemically similar, differing only in a distal position (6-OAc vs 6-N3), their rates of polymerization are expected to be similar and allow for the formation of truly random ‘clickable’ co-glycopolypeptides. Such a synthetic scheme therefore allows installation of cationic groups randomly over the length of the glycopolypeptide. We also demonstrate that the synthesized cationic GPPs retain their helicity in water and are taken up by HepG2 cancer cells. EXPERIMENTAL SECTION Materials and method A 9-BBN dimer was purchased from Sigma Aldrich. All other chemicals used were obtained from Merck, India. Diethyl ether, petroleum ether (60−80 °C), ethyl acetate, dichloromethane, tetrahydrofuran, and dioxane were purchased from Merck, India; dried by conventional methods


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and stored in the glove box. FT-IR spectra were recorded on Perkin-Elmer FT-IR spectrum GX instrument by making KBr pellets. Pellets were prepared by mixing 3.0 mg of sample with 97.0 mg of KBr. 1H NMR spectra were recorded on Bruker Spectrometers (200 MHz, 400 or 500 MHz).

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C NMR and DEPT spectra were recorded on Bruker Spectrometer (50, 100, or 125

MHz) and reported relative signals according to deuterated solvent used. HRMS data was recorded on Waters Synapt G-2 LC-MS. Gel permeation chromatography (GPC) was performed on a VISKOTEK TDA 305-040 Triple Detector array refractive index (RI), viscometer (VISC), low angle light scattering (LALS), right angle light scattering (RALS) GPC/SEC module. Using universal calibration absolute molecular weights were determined by using a dn/dc value of 0.059.49 Separations were achieved by three columns (T6000M, GENERAL MIXED ORG 300x7.8 MM) and one guard column (TGUARD, ORG GUARD COL 10x4.6 MM), 0.025 M LiBr in DMF as the eluent at 60 °C. GPC samples were prepared at concentrations of 5 mg/mL. A constant flow rate of 1 mL/min was maintained. System was calibrated by PMMA standards. HepG2 cell lines were purchased from ATCC and were maintained as per the manufacturer’s instructions. General procedure for the C-6 functionalized glyco-N-carboxyanhydrides

To a solution of 6-deoxy-6-azido-2,3,4-tri-O-acetyl-β-D-glucopyranoside-L-lysine (3a)/ 2,3,4,6tetra-O-acetyl-β-D-glucopyranoside-L-lysine (3b) (0.1 mmol) in freshly distilled anhydrous tetrahydrofuran (30 mL) was added a solution of triphosgene (0.05 mmol) in anhydrous tetrahydrofuran (5 mL) under argon atmosphere. α-Pinene (0.15 mmol) was subsequently added and the reaction mixture was heated to 50 °C for 1 h. It was then cooled to room temperature and then poured into dry hexane. The white precipitate of the N-carboxyanhydride was vacuum


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filtered quickly and reprecipitated (two times) by dissolving in ethyl acetate followed by addition of light petroleum. The resulting precipitate was filtered and dried under vacuum (Yield 80%). N-carboxyanhydride of 6-deoxy-6-azido-2,3,4-tri-O-acetyl-β-D-glucopyranoside-L-lysine (4a): 1

H NMR (400.13 MHz, CD2Cl2): δ (ppm) 1.42-1.50 (m, 2H), 1.55-1.61 (m, 2H), 1.78-1.85 (m,

2H), 2.00 (s, 3H), 2.02 (s, 3H), 2.06 (s, 3H), 3.24-3.37 (m, 4H), 3.75-3.80 (m, 1H), 4.09-4.12 (d, J = 15.1 Hz, 1H), 4.29-4.33 (m, 2H), 4.61-4.63 (d, J = 7.8 Hz, 1H), 5.00-5.05 (m, 2H), 5.25 (t, J =9.5 Hz, 1H), 6.62 (bs, 1H), 7.34 (bs, 1H). 13

C NMR (100.61 MHz, CD2Cl2): δ (ppm) 20.9 (2C), 21.2, 22.2, 28.8, 31.4, 38.4, 51.4, 58.1,

69.1, 69.7, 72.0, 72.4, 74.1, 100.9, 152.5, 169.4, 170.0, 170.4, 170.7, 170.9. N-carboxyanhydride of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside-L-lysine (4b):

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H NMR

(399.78 MHz, CDCl3): δ (ppm) 1.40-1.42 (m, 2H), 1.51-1.54 (m, 2H), 1.77-1.79 (m, 2H), 1.96 (s, 3H), 1.97 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 3.21-3.30 (m, 2H), 3.68-3.70 (m, 1H), 4.01-4.08 (t, J = 15.0 Hz, 1H), 4.20-4.27 (m, 3H), 4.47-4.49 (d, J = 7.8 Hz, 1H), 4.97-5.02 (m, 2H), 5.165.21 (t, J = 9.6 Hz, 1H), 6.56 (t, J = 5.5 Hz, 1H), 7.26 (bs, 1H). 13

C NMR (100.53 MHz, CDCl3): δ (ppm) 20.5, 20.6, 20.7, 20.9, 21.3, 28.2, 30.6, 37.7, 57.4,

61.6, 68.1, 68.6, 71.6, 72.0, 72.1, 100.5, 152.1, 169.0, 169.4, 170.1 (2C), 170.3, 170.7 General Procedure for the Synthesis of Glycopolypeptides To a solution of glyco-L-lysine NCA (100 mg/mL) in dry dioxane was treated with ‘proton sponge’ N,N′-tetramethyl napthalene (1.0 equiv to monomer; 1 M stock solution) as an additive and hexyl-amine (0.5 M stock solution) as the initiator inside the glove box. The progress of the polymerizations was monitored by FT-IR spectroscopy by comparing with the intensity of


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anhydride stretching at 1785 and 1858 cm−1 of the parent NCA. The reactions were generally complete within 36 h. Aliquots for GPC analysis were picked periodically until completion of polymerization. Finally, the solvent was removed under reduced pressure from the reaction mixture. The resulting residue was re-dissolved in dichloromethane and the polymer was precipitated by the addition of diethyl ether. The precipitated polymer was collected by centrifugation and dried to afford glycopolypeptides in greater than 90% yield as white powder. Polymer 5a: 1H NMR (399.78 MHz, CDCl3): δ (ppm) 0.85 (m, 3H), 1.22−1.85 (m, 6H), 2.00−2.14 (3s, 9H), 3.11−3.44 (m, 4H), 3.78 (m, 1H), 4.06-4.09 (m, 1H), 4.24−4.28 (m, 2H), 4.58−4.66 (m, 1H), 5.01 (m, 2H), 5.22 (m, 1H). 13


71.2, 72.0, 73.4, 100.4, 168.1, 168.2, 169.4, 169.7, 170.0. Polymer 6a: 1H NMR (399.78 MHz, CDCl3): δ (ppm) 0.86 (m, 3H), 1.24−1.87 (m, 6H), 2.00−2.17 (3s, 9H), 2.88−3.31 (m, 4H), 3.72 (m, 1H), 4.09-4.11 (m, 1H), 4.25−4.29 (m, 2H), 4.65 (m, 1H), 5.00-5.07 (m, 2H), 5.21−5.26 (m, 1H). 13


71.2, 72.0, 73.4, 100.4, 168.1, 168.2, 169.4, 169.7, 170.0. Polymer 7a: 1H NMR (399.78 MHz, CDCl3): δ (ppm) 0.85 (m, 3H), 1.23−1.85 (m, 6H), 1.99−2.06 (3s, 9H), 2.90−3.28 (m, 4H), 3.79 (m, 1H), 4.04-4.13 (m, 1H), 4.24−4.26 (m, 2H), 4.64 (m, 1H), 5.00-5.08 (m, 2H), 5.23−5.25 (m, 1H). 13


71.2, 72.0, 73.4, 100.4, 168.1, 168.2, 169.4, 169.7, 170.0.


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Deprotection Procedure for the Glycopolypeptides Hydrazine monohydrate (25 equivalent) was added to the solutions of all the acetyl-protected glycopolypeptides in THF:Methanol (4:1) (10 mg/mL) and the reactions were stirred for 7−8 h at room temperature. Acetone was added to quench the reactions; subsequently, the solvent was evaporated under reduced pressure. The solid residues were re-dissolved in deionized water and transferred to dialysis tubing (3.5 and 12 KDa molecular weight cut off according to polymer molecular weight). The samples were dialyzed against deionized water for three days; water was changed once every two hours for the first day, and then three times per day. Dialyzed polymers were lyophilized to yield glycopolypeptides as white fluffy solids (~ 90% yield). Polymer 5b: 1H NMR (399.78 MHz, D2O): δ (ppm) 0.83, 1.39−1.54, 1.86, 3.21, 3.35−3.40, 3.373.53, 3.97, 4.25, 4.47−4.48. 13

C NMR (100.53 MHz, D2O): δ (ppm) 20.1, 23.0, 27.9, 28.3, 29.7, 38.8, 50.9, 68.2, 70.5, 72.9,

75.4, 102.8, 171.3, 176.2. Polymer 6b: 1H NMR (399.78 MHz, D2O): δ (ppm) 0.83, 1.25−1.65, 1.75−2.10, 3.10−3.30, 3.35−3.40, 3.43-3.61, 3.91-4.05, 4.20−4.30, 4.45−4.52. Polymer 7b: 1H NMR (399.78 MHz, D2O): δ (ppm) 0.83, 1.25−1.65, 1.75−2.10, 3.10−3.30, 3.35−3.40, 3.43-3.61, 3.91-4.05, 4.20−4.30, 4.45−4.52. Circular Dichroism Measurements All the GPP solutions were filtered through 0.22 µm syringe filters. CD (190−250 nm) spectra of the GPPs (1.0-0.25 mg/mL in water or 10 mM phosphate buffer, pH: 7.0) were recorded by


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JASCO J-815 CD spectrophotometer) in a cuvette with 1 mm path length. All the spectra were recorded for an average of three scans and the spectra were reported as a function of molar ellipticity [θ] versus wavelength. The molar ellipticity was calculated using the standard formula, [θ] = (θ × 100 × Mw)/(C × l), where θ = experimental ellipticity in millidegrees, Mw = average molecular weight, C = concentration in mg/mL, and l = path length in cm. The % α helicity was calculated by using the formula: % α helicity = [(−[θ]222 nm + 3000)/ 39000]×100. Cell Viability Assay (MTT assay) HepG2 cells were seeded in a 96-well plate at a density of 1×104 cells/well and incubated for 18 h at 37 °C in MEM containing 10% FBS. The medium was replaced with serum-free MEM. Glycopolypeptide 7c and 7d (1.0 mg/mL in MEM) was then added to make up the final concentration of glycopolypeptide to 50, 40, 35, 30, 25, 20, 15, 10, 8, 6, 4 and 2 µg per well. Cells were further incubated for 4 h, and then the medium was replaced with MEM containing 10% FBS. After another 40 h incubation at 37 °C, the media was removed and 100 µL solution of MEM containing 10% FBS with filter sterilized MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) solution (0.45 mg/mL) was added into each well. After incubation at 37 °C for 4 h with MTT, the media was aspirated from the wells and 100 µL DMSO was added to dissolve insoluble formazan crystals. The absorbance was measured at 550 nm using a microtitre plate reader (Veroscan, Thermo Scientific) and the cell viability was calculated as a percentage relative to untreated control cells. Flow Cytometry analysis for the Cellular Uptake of Rhodamine-labelled glycopolypeptide HepG2 cells were plated in a 24-well plate (7.5×105 cells/well) under 5% CO2 atmosphere at 37 °C using MEM medium supplemented with 10% FBS for 24 hours. The cells were treated with a


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pre-determined amount of glycopolypeptide 7d and 7c (30 and 15 µg/mL; labelled with Rhodamine B; degree of labelling 2 mol%) under 5% CO2 atmosphere at 37 °C for two hours. The medium was then removed and the cells were washed twice with cold PBS, then digested with EDTA-trypsin and collected by centrifugation at 1000 rpm for 10 min at each time point and re-suspended in 400 µL of PBS buffer. Fluorescence histograms were recorded with a BD FACS Calibur flow cytometer and were analysed using Flowjo software. Minimum of 10,000 events were analysed to generate each histogram. The gate was arbitrarily set for the detection of red fluorescence (using PE filter). Cellular uptake study using Confocal Microscopy HepG2 cells were seeded on glass cover slips at a density of 50,000 per well in MEM containing 10% FBS and incubated for 18 hours. The media was then replaced with serum-free MEM. Rhodamine-B labeled glycopolypeptide (degree of labelling 2 mol%) 7c and 7d was prepared in serum free MEM and then added to make a final concentration of 30 and 15 µg/well and further incubated for 2 h. The cells were then washed thrice with cold PBS and fixed with 3.5% paraformaldehyde-PBS (pH 7.4) solution for 15 minutes. The fixative was removed and cells were washed again thrice with cold PBS solution and mounted on glass slides using mounting media containing DAPI to stain the nuclei. Images were acquired using CLSM. RESULTS AND DISCUSSION Synthesis of C-6 functionalized glycopolypeptides The synthesis of cationic glycopolypeptide was envisaged by first preparing a glycopolypeptide bearing an azido functional group on the side chain carbohydrate, which could be further


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modified post-polymerization. Reduction of azide to amine or formation of triazole using bifunctional alkyne-amine by CuAAC would be good alternatives for modifying the polymer to generate cationic glycopolypeptides. Therefore, synthesis of glyco-amino acid conjugate containing an azide group at the C-6 position of the monosaccharide was first targeted. Allyl 6deoxy-6-azido-2,3,4-tri-O-acetyl β-D-glucopyranoside was oxidized to the corresponding carboxylic acid 2c using NaIO4 and RuCl3 in acetonitrile (SI figure 1).50 The carboxylic acid 2c was directly used in the coupling reaction with 9-BBN conjugated lysine derivative 1. Interestingly, the 9-BBN group serves as the protective group for the amino acid and it can be removed under mild conditions without affecting the esters or N3 groups of glucose. The other protecting groups, which are routinely used in peptide chemistry, are not envisaged due to perceived problems in deprotection of them or non-compatibility of them in subsequent reaction conditions.

Scheme 1: Synthesis of 6-deoxy-6-azido functionalized glycopolypeptides.


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The coupling of carboxylic acid 2c and 9-BBN L-lysine complex (1) was done by using 1-ethyl3-(3 dimethylaminopropyl) carbodiimide) (EDCI) and hydroxybenzotriazole (HOBt) to obtain the corresponding amino acid glycoconjugate 3a. The resulting amide was characterized by 1H and 13C NMR analysis. Deprotection of 9-BBN moiety was achieved by stirring compound 3a in a mixture of chloroform and methanol at room temperature for 24 h. Resulting amino acid was treated with triphosgene and α-pinene in tetrahydrofuran at 50 °C to obtained 6-deoxy-6-azidoglyco-N-carboxyanhydride (4a) monomer.4,

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The overall yield of the NCA monomer from

commercially available allyl-2,4,5,6-tetra-O-acetyl-β-D-glucopyranoside is 37%. Formation of 6-deoxy-6-azido-glyco-N-carboxyanhydride was confirmed by FT-IR,

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H and

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C NMR

spectroscopic analysis. Ring opening polymerization (ROP) of 6-deoxy-6-azido-glyco-N-carboxyanhydride (4a) with n-hexylamine using a monomer:initiator ratio of 30:1 was carried out by following a methodology described before.4,37 Progress of the ROP reaction was monitored by disappearance of anhydrides stretch at 1785 cm-1 and 1858 cm-1 using FT-IR spectroscopy. After completion of polymerization, the resulting polypeptide 5a was purified by multiple precipitations using mixture of dichloromethane and diethyl ether. GPC of purified 5a (SI figure 2) displayed a narrow monomodal molecular weight distribution (PDI = 1.03) and the Mn was estimated to be 17.7×103 g/mol (SI Table 1). The degree of polymerization was estimated to be 34 from 1H NMR by comparing the relative intensity of the peak at 0.85 ppm due to characteristic proton present in the initiator (hexylamine; -CH3) with the proton peaks of the methyl group (CH3OCO) present in the acetate moiety of the carbohydrate (1.30 ppm). The degree of polymerization estimated from 1H NMR (DP = 34) matches fairly well with the DP estimated from GPC (DP = 36). Additionally, the molecular weight (Mn) of the azide-functionalized glycopolypeptide


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obtained was close to the expected molecular weight (SI Table 1) based on monomer: initiator ratio, thus implying that unwarranted side products were not formed during the course of the polymerization. FT-IR of the polymer 5a also displayed IR stretches at 2100 cm-1 characteristic for the organo azide group (SI figure 3). Similarly, azide-functionalized glycopolypeptide 6a and 7a were synthesized using monomer: initiator ratio of 40:1 and 50:1 respectively. Polymers 6a and 7a also displayed a narrow monomodal molecular weight distribution (SI figure 2, SI Table 1) and the degree of polymerization and Mn obtained by 1H NMR and GPC were found to be fairly close to the expected molecular weight based on monomer: initiator feed ratios. Circular dichrosim analysis of this glycopolypeptides in acetonitrile displayed peaks having minima at 208 nm and 222 nm that are indicative of the polypeptide having α-helical conformation (Figure 1A). The de-protection of acetate group of glycopolypeptides (5a, 6a and 7a) was performed using hydrazine hydrate in THF-MeOH system at RT followed by extensive dialysis against deionized water. Freeze-drying of the resultant solutions afforded GPPs 5b, 6b and 7b as a fluffy white solid. Water-soluble azide functionalized GPPs were characterized by 1H,

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C NMR and

FT-IR analysis. We then proceeded to explore if GPPs containing controlled amounts of 6-deoxy-6-azido functionalized glucose can be synthesized using our methodology. Controlling the mole fraction of side chain azide would be important for biomedical applications since the azide chains can be further functionalized with cell targeting agents using CuAAC. Further, the structural similarity of both monomers would most likely lead to the formation of a truly random coglycopolypeptide since their individual rates of ROP is expected to be similar. We therefore proceeded to synthesize a glycopolypeptide in which only 10 % and 20% of the side chains had 6-deoxy-6-azido functionalized glucose whereas the other contained normal acetyl protected


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glucose. This was done by performing random ring-opening co-polymerization of 6-deoxy-6azido-glyco-N-carboxyanhydride (4a) and 6-OAc-glyco-N-carboxyanhydride (4b) monomers at a feed ratio of 1:9 and 1:4 respectively using n-hexylamine as initiator. The progress of the polymerization was followed by FT-IR and the glycopolypeptide 8a and 9a were purified by multiple precipitations as has been described before. The Mn of 8a and 9a was calculated to be 16.2 and 16.6×103 g/mol respectively while the polydispersity of the GPPs were determined to be 1.08 using GPC (SI Table 1; SI figure 3). However we were unable to calculate the ratio of the monomer incorporation (ratio of 4a:4b) in the synthesized glycopolypeptide 8a and 9a from 1H NMR since the monomer 4a did not contain any resonance that could distinguish itself from 4b. We therefore proceeded to “click” phenylacetylene to 8a and 9a using CuAAC to generate unique resonances for the azide containing glyco-monomers in the GPPs 8d and 9d respectively (SI figure 5). The extent of click reaction was followed by FT-IR (disappearance of the resonance at 2100 cm-1 that is unique for organo-azide) and it was found to be almost quantitative (SI figure 6). Further the 1H NMR spectrum of the resultant GPPs isolated after click reaction displayed resonances in the aromatic region representing the triazole formation in addition to resonances characteristic for the glycopolypeptide (SI figure 7). The ratio of 6-deoxy-6-azido (4a) to 6-OAc (4b) monomer ratio was then determined by 1H NMR to be1.1:9 and 1.1:4 for the polymer 8d and 9d respectively (feed ratio was 1:9 and 1:4 for 8a and 9a respectively) (SI figure 7). Synthesis of Cationic glycopolypeptides Cationic GPPs were synthesized using post modification approach following two methods. In the first method, azido functionality of water soluble glycopolypeptide (5b, 6b and 7b) was converted to amine by treatment with excess of trimethylphosphine in THF:H2O (1:9). The


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progress of the reaction was monitored by the decrease of the FT-IR resonance of azide group at 2100 cm-1 and complete reduction to amine was observed in about 48 h (SI figure 8). Purification of the reduced glycopolypeptide (5c, 6c and 7c) was carried out by extraction of excess of trimethylphosphine (Toxic!) and other organic soluble by-products using ethyl acetate followed by extensive dialysis against deionised water for 48 h. The polymer 8b and 9b were also reduced to polymer 8c and 9c by following the same procedure. The amine groups are expected to be protonated at neutral pH thus rendering the resultant glycopolypeptide to be cationic at neutral pH. In the second approach, the azide group in the glycopolypeptide 7b was clicked with propargyl amine by CuAAC reaction to obtain polypeptide 7d. The completion of the reaction was confirmed by the disappearance of the azide stretching at 2100 cm-1 using FT-IR spectroscopy (SI figure 11). The resultant glycopolypeptide 7d was purified by performing extensive dialysis against EDTA (5 wt%) to remove the copper salts and then against deionised water. The cationic GPPs 7c and 7d were characterized by FT-IR, 1H and

13

C NMR spectroscopic analysis

(Supporting Information).


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Scheme 2: Synthesis of cationic glycopolypeptides. Secondary structural investigation of the glycopolypeptides Secondary structures of all the synthesized GPPs were studied using circular dichroism (CD) spectroscopy (SI Table 2). The CD spectra of 6-deoxy-6-azido acetyl protected GPPs (5a, 6a and 7a) were recorded in acetonitrile and displayed two minima at 208 nm and 222 nm that is characteristic of the helical conformation (Figure 1A). This is expected since GPPs that are built upon L-lysine backbone have been shown adopt an α-helical conformation.4 The water soluble azide functionalized glycopolypeptide 7b also displayed peaks having two distinct minima at 208 nm and 222 nm that are also indicative of the polypeptide having α-helical conformation (Figure 1B). The percentage helicity for polymer 7b (Mn=21.0×103 g/mol; DP = 56) was determined to be 85%. The CD spectra of 6b and 5b in water were also recorded to evaluate the effect of conformation upon decreasing molecular weight. For 6b (Mn=15.7×103 g/mol; DP = 42) and 5b (Mn=12.7×103 g/mol; DP = 34) the percentage helicity of was determined to be 64% and 52% respectively. Therefore, as has been shown for polypeptides,51 the percentage helicity of the azide functionalized water-soluble glycopolypeptides decreased with decreasing molecular weight. It is well known that incorporation of charged side-chains in the peptide backbone modulates the helicity due to electrostatic repulsions. For example, poly-L-lysine exists as a random coil at pH 7 (when the amines are charged) but become α-helical at higher pH where the side-chains are uncharged. Recently, Cheng et al. demonstrated that when the charge is moved further away from the peptide backbone, at some point the charge may have a negligible effect on the helicity of polypeptide with long and straight side chains. As a result, these charged polypeptides can


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simultaneously maintain the water solubility and stable helical structures. They also investigated that the charged moiety should place at least 11 carbon atoms away from the peptide back bone to retain the helicity in a polypeptide with charged side-chains.

39

Similarly, 6-amine

functionalized GPPs 5c, 6c and 7c that were formed upon reduction of 6-azide moiety of the GPPs 5b, 6b and 7b were expected to be cationic at pH 7 since the amine groups were expected to remain protonated at this pH (SI figure 12). The CD signal of the charged Polymer 7c (DP = 56) also displayed CD signal characteristic of an α-helix (Figure 1C). The percentage helicity of 7c was identical to its azide containing neutral precursor 7b (percentage helicity 85% and 83% respectively). Polypeptide 7d, which was synthesized by click reaction of propargylamine and 7b, also shows ~80% helicity (SI figure 14). Hence introduction of the charged amino group 12 atoms from the peptide backbone has no effect of the secondary conformation and this is in concurrence with what has been proposed earlier by Cheng et. al.39 Cationic glycopolypeptide 6c and 5c, having a shorter chain length (DP = 42 and 34), also adopts a helical conformation although its percentage helicity (53% and 36%) is slightly lesser than their azide containing neutral precursor 6b and 5b (64% and 52%). In fact, the CD spectrum of 5c (DP = 34) exhibited a strong minima at 202 nm (Figure 1C) which exhibits the lack of formation of a helical structure.52, 53 Finally, polymers 8c and 9c which have molecular weight similar to 5c (~30 mer) but have a lower positive charge on their side chain, also adopt a helical conformation but their percentage helicity is higher than 5c due to lower charge content on their side chain. These results indicate that the 11-carbon rule holds well for cationic glycopolypeptides having high molecular weight (~50 mer). However, for short cationic glycopolypeptides the helicity is severely compromised in comparison to their neutral analogs.


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Figure 1: Circular dichroism spectra of the glycopolypeptides (A) in acetonitrile, (B), (C) and (D) in water. Cell viability and Cellular Uptake Studies: Preliminary cell viability and cellular entry assays were conducted to ascertain whether the cationic glycopolypeptide 7c and 7d was able to enter mammalian cells. The cell viability assay of 7c and 7d were performed on liver cancer HePG2 cell lines shows >90% viability up to concentrations of 400 µg/mL (Figure 2).


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Figure 2: The cell viability of the charged glycopolypeptides 7c and 7d to the HePG2 cell lines.


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Figure 3: The cellular uptake of the RhB labelled charged glycopolypeptide to the HepG2 cell lines; A-C) cells treated with RhB labelled polymer 7d in 30 µg/mL; D-F) cells treated with RhB labelled polymer 7d in 15 µg/mL; G-I) cells treated with RhB labelled polymer 7c in 30 µg/mL. Finally, GPPs 7c and 7d were labelled by reaction of the N-terminal amine with Rhodamine B Isothiocyanate (2 mol% labelled; SI figure 13) and their cellular uptake study onto HepG2 cell line was studied using confocal microscopy (Figure 3) and FACS (Figure 4). Incubation of HepG2 cells with 30 µg/mL of 7c or 7d for 2 hrs showed >90% uptake of these glycopolypeptides onto the cells by FACS. When a lower concentration of 7d was added (15 µg/mL) the percentage uptake by cells reduced to 70%. The FACS studies were also


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corroborated by confocal microscopy where the fluorescence from the RhB labelled glycopolypeptide was detected. (Figure 3) The presence of positive charge on the GPPs would facilitate attachment to the cell membrane and further help in getting it internalised into the cytoplasm. The cellular uptake and biocompatibility of these GPPs show that they are promising materials for the purpose of gene delivery.

Figure 4: The cellular uptake of the charged glycopolypeptides 7c and 7d to the HePG2 cell lines by FACS measurement. CONCLUSIONS The facile syntheses of GPPs bearing 6-deoxy-6-azido groups in the carbohydrate side chain by ROP of 6-deoxy-6-azido-glyco-NCAs have been reported. This methodology also allows for the synthesis of random co-glycopolypeptides containing 6-deoxy-6-azido and regular 6-OAc functionalized glucose. The percentage of azide containing monomer incorporation into this random glycopolypeptides can be controlled by varying the amount of 6-deoxy-6-azido-glycoNCA. This azide functionality allows for the easy attachment of a bioactive group or fluorescent


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probe, which can enhance the biological activity of the glycopolypeptide. We were able to obtain for the first time water-soluble charged cationic glycopolypeptides by either converting the azide groups into amines by reduction or by using CuAAC with propargylamine. These charge glycopolypeptides were shown to have a helical conformation in water. Preliminary studies showed that these charged glycopolypeptides showed good biocompatibility and were efficiently up taken by HepG2 cells. Therefore, the biocompatibility and the cellular internalization ability of these glycopolypeptides make them promising biomaterials for the purpose of gene delivery. SUPPORTING INFORMATION Experimental procedures and spectral data for all NCA monomers, glycopolypeptides, FT-IR and NMR spectra of polypeptides. This information is available free of charge via Internet at http://pubs.acs.org/. REFERENCES (1) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev.2008, 109, 131-163. (2) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046-1051. (3) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., In. Ed.1998, 37, 2754-2794. (4) Pati, D.; Shaikh, A.; Das. S.; Nareddy, P. K.; Swamy, M. J.; Hotha, S.; Sen Gupta, S. Biomacromolecules, 2012, 13, 1287-1295. (5) Manning, D. D.; Hu, X.; Beck, P.; Kiessling, L. L. J. Am. Chem. Soc.1997, 119, 3161-3162. (6) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc.1994, 116, 12053-12054. (7) Prescher, J. A.; Bertozzi, C. R. Nat Chem Biol 2005, 1, 13-21. (8) Wang, Y.; Kiick, K. L. J. Am. Chem. Soc.2005, 127, 16392-16393.


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88x35mm (300 x 300 DPI)


Cationic Charged Helical Glycopolypeptide Using Ring Opening

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