Supporting information

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1 Glycopolymer Grafted Nanoparticles: Synthesis using RAFT Polymerization and Binding Study with Lectin S N Raju Kutcherlapati, Rambabu Koyilapu, Uma Maheswara Rao Boddu, Debparna Datta, Ramu Sridhar Perali, * Musti J. Swamy * and Tushar Jana * School of Chemistry University of Hyderabad Hyderabad, India Tel: (91) Fax: (91) tusharjana@uohyd.ac.in tjscuoh@gmail.com (* To whom correspondence should be addressed) Supporting information

2 Experimental methods Supporting information Scheme 1: Synthesis of 2,3,4,6-tetra-O-acetyl-α-D-mannopyranose (2) from penta-o-acetyl-α-d-mannopyranoside (1). 2,3,4,6-tetra-O-acetyl-α-D-mannopyranose (2) 1 To a solution of penta-o-acetyl-α-d-mannopyranoside 1 (22 g, mmol) in THF (220 ml) benzylamine (9.23 ml, mmoles) was added and the reaction mixture was stirred for 20 h at room temperature (25 0 C), after which the reaction did not seem to proceed further. The solvent was evaporated under reduced pressure and the crude material was taken into dichloromethane and washed progressively with HCl 1.0 M (75 ml x 2), sodium hydrogen carbonate (satd. aq., 75 ml) and deionised water (75 ml). The organic layer was dried over Na 2 SO 4 and concentrated leaving a yellow oil (28.24 g) that was subjected to column chromatography (EtOAc: petroleum ether, 1:4) yielded 2,3,4,6-tetra-O-acetyl-D-mannopyranose 2 (19.44 g, 99%), as a colour less gum. R f 0.4 (EtOAc: petroleum ether, 1:1). 1 H NMR (500 MHz, CDCl 3 ): δ = (dd, 1H, J = 3.0 Hz, J = 10.0 Hz), 5.30 (t, 1H, J = 10.0 Hz), 5.26 (dd, 1H, J = 2.0 Hz, 3.5 Hz), 5.24 (d, 1H, J = 2.0 Hz), (m, 2H), (m, 1H), 3.92 (bs, 1H), 2.16 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H). ppm. 13 C NMR (125 MHz, CDCl 3 ): δ = , 170.1, 169.8, 92.2, 70.1, 68.8, 68.5, 66.2, 62.6, 20.9, 20.7, 20.7, ppm. IR (neat): = 3457, 2956, 1742, 1433 cm -1. HRMS (ESI): Calcd. for C 14 H 20 O 10 NH 4 [M + NH 4 ] ; found S2

3 Supporting information Scheme 2: Synthesis of 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl trichloroacetimidate (3) from 2,3,4,6-tetra-O-acetyl-α-D-mannopyranose (2). 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl trichloroacetimidate (3) 1 To a solution of hemiacetal 2 (19 g, mmol) in anhydrous dichloromethane (750 ml) was added in 4 Å molecular sieves powder under N 2 and the mixture was stirred at room temperature for 10 min. To this, trichloroacetonitrile (16.41 ml, mmol) and DBU (1.05mL, 7.02 mmol) were added, respectively, and the stirring was continued. After two hour s the solution was filtered through Celite and concentrated and the obtained crude residue was purified by silica gel column chromatography (EtOAc: petroleum ether, 1:2) to give trichloroacetimidate 3 as a foam (24.74 g, 92%), R f 0.56 (EtOAc: petroleum ether, 1:1). 1 H NMR (400 MHz, CDCl 3 ): δ = 8.80 (s, 1H), 6.29 (d, 1H, J = 1.6 Hz), 5.48 (d, 1H, J = 1.6 Hz), (m, 2H), 4.29 (dd, 1H, J = 4.8 Hz, J = 12.0 Hz), (m, 2H), 2.21 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H). ppm. 13 C NMR (100 MHz, CDCl 3 ): δ = , 169.7, 169.6, 159.7, 94.5, 90.5, 71.2, 68.8, 67.8, 65.4, 62.0, 20.7, 20.6, 20.6, ppm. IR (neat): = 2956, 1742, 1680, 1427 cm -1. HRMS (ESI): Calcd. for C 16 H 20 Cl 3 NO 10 Na [M + Na] ; found S3

4 Supporting information Scheme 3: Synthesis of 2-O-(2', 3', 4', 6'-tetra-O-acetyl- -D-mannosyl) ethyl methacrylate (4) from 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl trichloroacetimidate (3). Synthesis of 2-O-(2', 3', 4', 6'-tetra-O-acetyl- -D-mannosyl) ethyl methacrylate (4) 2 A mixture of 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl trichloroacetimidate 3 (24 g, mmol), 2-hydroxyethyl methacrylate (HEMA) (7.94 ml, mmol), molecular sieves powder in anhydrous dichloromethane (160 ml) was stirred under nitrogen atmosphere at -18 C. To this, a solution of boron trifluoride ethyl etherate (6.13 ml, mmol, 1 eq, 0.4 M) in anhydrous dichlormethane (122.6 ml) was added drop wise and the reaction mixture was then stirred for two hours at the same temperature. Then, the solution was washed with distilled water (3 x 25 ml) and the organic layer was dried over anhydrous sodium sulphate and filtered. Evoperation of the solvent under reduced pressure followed by the purification of the obtained crude product by column chromatography (EtOAc: petroleum ether, 1:2) yielded (11.96g, 55%) compound 4 as a colourless solid. R f 0.5 (EtOAc: petroleum ether, 1:1). 1 H NMR (500 MHz, CDCl 3 ): δ = 6.14 (s, 1H), 5.61 (t, 1H, J = 1.5 Hz), 5.36 (dd, 1H, J = 3.0 Hz, J = 2.0 Hz), (m, 2H), 4.88 (d, 1H, J = 1.5 Hz), 4.35 (t, 2H, J = 4.5 Hz), 4.28 (dd, 1H, J = 5.5 Hz), 4.09 (dd, 1H, J = 2.0 Hz, J = 7.0 Hz), (m, 1H), (m, 1H), (m, 1H), 2.17 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H) ppm. 13 C NMR (125 MHz, CDCl 3 ): δ = , 169.8, 169.7, 167.1, 135.9, 126.0, 97.5, 69.4, 68.9, 68.6, 66.1, 65.9, 63.1, 62.4, 20.8, 20.7, 20.6, 20.6, 18.2 ppm. IR (neat): = 2961, 2925, 1748, 1717, 1634, 1448 cm -1. HRMS (ESI): Calcd. for C 20 H 28 O 12 NH 4 [M + NH 4 ] ; found S4

5 Synthesis of activated RAFT agent The synthesis of RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPDB) was carried out according to the methods in the literature. 3, 4 The activation of CPDB was carried out by using N-Hydroxysuccinamide (NHS) as described elsewere. 5 Briefly the method was as follows: CPDB (2.04 g, 7.3 mmol), NHS (0.84 g, 7.3 mmol) were dissolved in dry dichloromethane (15 ml) and then N,N'-dicyclohexylcarbodiimide (DCC, 1.51 g, 7.3 mmol) was added to the above reaction mixture under nitrogen atmosphere, stirred at room temperature for 18 h is the dark condition. The insoluble product was filtered, the remaining solution was concentrated and was purified by using silica column chromatography in 4:1 (v/v) n-hexane/ethyl acetate and removal of solvent gave product as red solid (2.28 g, 83% yield). This activated RAFT agent (CPDB) is abbreviated as CPDB-NHS. 1 H NMR (500 MHz, CDCl 3 ): δ (ppm.) 1.94 (s, 3 H), (m, 4 H), 2.64 (t, 4 H), 7.40 (m, 2 H), 7.56 (m, 1 H), 7.90 (m, 2 H). 13 C NMR (500 MHz, CDCl 3 ): δ (ppm) (PhC=S), (C=O), (C=O), 144.6, 133.7, 127.7, 126.3, (CN), 45.9, 34.6, 33.6, 28.9, Synthesis of silica nanoparticles (SiNPs) Well known StÖbber process was used to synthesize SiNP. 6 Ammonium hydroxide (10 ml) and ethanol (400 ml) were added to a 1000 ml round bottomed flask at room temperature. To this tetraethylorthosilicate (TEOS, 10 ml) and HPLC water (10 ml) were added dropwise under vigorous stirring. After stirring for 24 h at room temperature, the formed silica nanoparticles were isolated by using centrifugation at 10,000 rpm for 30 min. The sediments were redispersed in ethanol (one time) and water (3 times) followed by centrifugation at 10,000 rpm for 30 min, respectively. The obtained silica nanoparticles were dried under vacuum at 50 C for 48 h. S5

6 Synthesis of amine modified silica nanoparticles (SiNP-NH 2 ) The above synthesized SiNPs (3 g) was transferred into round bottom flask containing dry toluene (70 ml) and dispersed by using ultrasonication for 45 min, followed by the dropwise addition of (3-Aminopropyl)triethoxysilane (APTES, 200 μl, mmol) under vigorous stirring. After stirring for 24 h at 90 C, the reaction mixture was precipitated in hexane (300 ml). The amine modified SiNP (SiNP-NH 2 ) were isolated by using centrifugation at 7000 rpm for 30 min. The sediments were redispersed in acetone and centrifuged at 10,000 rpm for 15 min. and this purification cycle was repeated 3 times. The obtained amine modified silica nanoparticles (SiNP-NH 2 ) were dried under vacuum at 70 C for 48 h. The obtained yield was 2.85 g (95%). Characterization techniques 1 H and 13 C NMR spectroscopic analysis were performed using a Bruker AV 500 MHz NMR spectrometer at room temperature. In case of GP-g-SiNP, 10 mg of powdered samples was dissolved in CDCl 3 or D 2 O to perform the NMR experiment. Field emission scanning electron microscopy (FESEM) images were captured using Carl Zeiss Ultra-55 using EHT detector at 5 kv voltage. Sample preparation was done by dispersing GP-g-SiNP powder in water then drop casted on a glass plate, dried at room temperature and gold coated before imaging in FESEM. Transmission electron microscopy (TEM) studies were conducted on FEI (Technai Model No. 2083) TEM machine at an accelerating voltage of 200 kv. The samples were prepared by placing a drop of water dispersed glyconanoparticle powder on carbon coated copper (200 mesh) grids. Molecular weights (M. W.) and polydispersity index (PDI) of glycopolymers (which were obtained after cleaving the SiNPs from the GP-g-SiNP as described earlier) were S6

7 determined by Gel permeation chromatography (GPC) using polystyrene standards and eluted in DMAc at flow rate of 0.2 ml/min at 25 C on a GPC (Waters 515 HPLC) fitted with Waters 2414 refractive index detector and using Styragel HR 2 DMF column. Thermo gravimetric analysis (TGA) of GP-g-SiNP was carried out on TGA (TGA Q500, TA instruments, USA) from 30 to 800 o C with a scanning rate of 10 C/min in presence of nitrogen flow. Particle size measurements of GP-g-SiNP were performed using a Zetasizer Nano S90 (Malvern Instruments, Germany) operating at 4mW He-Ne laser with 633nm wavelength at room temperature. Fourier transform infrared (FT-IR) spectra were recorded on Nicolet 5700 FT-IR spectrometer with a resolution of 0.5 cm -1. All spectra were obtained with KBr pellets from dry solid samples and 64 scans were taken with background subtraction. S7

8 Fig. S1. 1 H NMR spectra of 1. S8

9 Fig. S2. 13 C NMR spectra of 1. S9

10 Fig. S3. 1 H NMR spectra of 2. S10

11 Fig. S4. 13 C NMR spectra of 2. S11

12 Fig. S5. 1 H NMR spectra of 3. S12

13 Fig. S6. 13 C NMR spectra of 3. S13

14 Fig. S7. 1 H NMR spectra of 4. S14

15 Fig. S8. 13 C NMR spectra of 4. S15

16 Fig. S9. 1 H NMR spectra of 5. S16

17 Fig. S C NMR spectra of 5. S17

18 Fig. S11. FE-SEM (left panel) and TEM images (right panel) of (a) bare SiNP, (b) SiNP-NH 2 and (c) SiNP-CPDB. S18

19 Fig. S C NMR spectra of SiNP-CPDB (A) and particle size measurements using light scattering of bare SiNP, SiNP-NH 2 and SiNP-CPDB. S19

20 Fig. S13 TGA thermogram showing the role of solvent for the synthesis of GP-g-SiNP. Refer to the Table 1 for sample identification. S20

21 Fig. S14 FE-SEM images (left panel) and TEM images (right panel) of GP-g-SiNP obtained after purification for different chain lengths prepared using water/ethanol (7:3) as a solvent. The description of sample identity (P1, P2 and P3) are described in Table 1. S21

22 References (1) Tosin, M.; Murphy, P. V. J. Org. Chem. 2005, 70, (2) Song, E.- H.; Manganiello, M. J.; Chow, Y.-H.; Ghosn, B.; Convertine, A. J.; Stayton, P. S.; Schnapp, L. M.; Ratner, D. M. Biomaterials 2012, 33, (3) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Macromolecules 2001, 34, (4) McCormick, C. L.; Lowe, A. B., Acc. Chem. Res. 2004, 37, (5) Warren, N. J.; Mykhaylyk, O. O.; Mahmood, D.; Ryan, A. J.; Armes, S. P. J. Am. Chem. Soc. 2014, 136, (6) Stöber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, S22