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Title: Synthesis of polymethyl(methacrylate)-polystyrene-based diblock copolymer containing biotin for selective protein nanopatterning

Author(s): Anna Lagunas,* Barbara Sasso, Nicolas Tesson, Carme Cantos, Elena Martínez, Josep Samitier

Electronic Supplementary Material (ESI) for Polymer Chemistry.This journal is © The Royal Society of Chemistry 2015

Atomic force microscopy (AFM) images processing for calculation of Feret distance in nanodomains and for the calculation of the minimum interdomain distance

Domain characteristics can be estimated from topographic AFM images (Dimension 3100 AFM from Veeco Instruments equipped with silicon AFM tips from Budgetsensors with spring constant 40 N/m, and operated in tapping mode at room temperature in air) from manually selected image thresholds processed with Image J 1.44p freeware (Figure S1).

Figure S1. AFM image processing. (a) 3x3 µm AFM representative topographical image of P(PEGMA)-b-P(S-co-bioS) 3 spin-coated onto flat silicon surface. (b) Manually selected nanodomains in the corresponding 8-bit image. (c) Image threshold (from which, domain morphological parameters such as Feret distance can be obtained with Image J integrated plugin), and (d) minimum interdomain distance distribution obtained from nanodomain position processing.

Nanodomain position processing was conducted by customized MATLAB code for minimum interdomain distances calculation:

for i=1:length(particle_coordinates) min_distance=100000000; for j=1:length(particle_coordinates) if i~=j distance=sqrt((particle_coordinates(i,1)-particle_coordinates(j,1))^2+(particle_coordinates(i,2)-particle_coordinates(j,2))^2); if distance < min_distance min_distance = distance; end end end min_distances(i)=min_distance;end

Functionalization of styrene with biotin moiety through an ester bond without spacers

In order to introduce the biotin moiety into the final block copolymer, we prepared an analogue of styrene as outlined in Scheme S1 by adapting a synthetic procedure described in literature.[1] In a first attempt, the commercially available 4-vinylbenzyl chloride was reacted with biotin in a one-step reaction using potassium carbonate and tetrabutylammonium iodide (TBAI) as catalyst and biotin-styrene was obtained in 11% yield. Such a low yield is associated to the difficult separation of TBAI from the final product: after all the attempts of purifications such as chromatographic column and selective precipitations the product was still contaminated with traces of TBAI. Therefore, and because reaction proceed with high rate, we decide to suppress TBAI from reaction. This way, the product biotin-styrene was afforded in a 50% yield.

[1] M. K. W. Choi, P. H. Toy. Tetrahedron, 2003, 59, 7171-7176

To a 100 mL two necked flask equipped with magnetically stirring containing Biotin (4.15 g, 17 mmol), potassium carbonate (3.87 g, 28 mmol) and TBAI (0.52 g, 1.40 mmol) under N2 atmosphere, was added 28 mL anhydrous DMF before adding at room temperature 4-vinylbenzylchloride (2.13 g, 14 mmol). The resulting suspension was heated at 50-60 ºC for 24h. The disappearance of 4-vinylbenzylchloride was monitored by TLC (EtOAc/Hexane 10:90 - ). Then the final mixture was cooled to room temperature before filtrating over celite® and washed with DMF. The filtrate was evaporated to dryness under vacuum at 40ºC affording off-yellow residue. This crude was purified by column chromatography (column 18 cm x 5 cm charged with 113 g of silica – eluent solvent DCM/MeOH 95:5). After evaporation of the pure fractions, 2.5 g of the desired product was obtained as a pale yellow solid (Yield 50 %).

1H-NMR (DMSO-d6, δ in ppm): 7.47 (d, J = 8.2 Hz, 2H arom.), 7.33 (d, J = 8.2 Hz, 2H arom.), 6.73 (dd, J = 10.9 Hz, J = 17.6 Hz, 1H vinyl), 6.41 (s, 1H, NH), 6.35 (s, 1H, NH), 5.84 ((dd, J = 0.8 Hz, J = 17.6 Hz, 1H vinyl), 5.27 (dd, J = 0.8 Hz, J = 10.9 Hz, 1H vinyl), 5.07 (s, 2H, Ar-CH2-), 4.32-4.26 (m, 1H, CH-NH-CO), 4.14-4.09 (m, 1H, CH-NH-CO), 3.11-3.03 (m, 1H, S-CH), 2.81 (dd, J = 5.1 Hz, J = 12.5 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.35 (t, J = 7.4 Hz, 2H, C(O)-CH2-), 1.67-1.25 (m, 6H, 3xCH2)

Scheme S1. Synthesis of biotin-styrene.

Random copolymerization of styrene and biotin-styrene using P(PEGMA) as the macroRAFT agent with the aim of obtaining the fully functionalized P(PEGMA)-b-P(S-co-bioS) block copolymer

RAFT polymerization was chosen to synthesize a brush-type amphiphilic diblock copolymer containing the P(PEGMA) hydrophilic brushes and biotin-functionalized PS hydrophobic block. The overall synthetic path is depicted in Scheme S2. Fully functionalized P(PEGMA)-b-P(S-co-bioS) will be prepared by two consecutive RAFT polymerizations: step 1) synthesis of well-defined P(PEGMA) via RAFT of PEGMA OMe, using CDB as the RAFT agent and BPO as initiator as previously described, and step 2) synthesis of well-defined diblock copolymer P(PEGMA)-b-P(S-co-bioS) via RAFT random copolymerization of styrene and biotin-styrene, using the P(PEGMA) obtained in the first step as the macroRAFT agent.

Scheme S2. Random copolymerization of styrene and biotin-styrene using P(PEGMA) as macroRAFT agent.

According to Scheme S2, P(PEGMA) in toluene was reacted with a mixture of styrene and biotin-styrene (Table S1). We found that toluene is not a good solvent for this polymerization system (run EP33-1-155): the solubility of biotin-styrene is very low at ambient temperature and incomplete at 80 ºC, the reaction mixture tends to jellify although in a reversible way. Both 1H-NMR and GPC show that the chain extension does not take place at all: the recovered material at the end of reaction is a mixture of P(PEGMA) and biotin-modified styrene. The most logic hypothesis could be the premature death of the initiator due to the heterogeneity of reaction mixture at t = 0 min. and RT.In a second attempt, the initiator was added in a second step, after heating the reaction mixture at 80 ºC (run EP33-1-160). Again, no copolymerization did take place.

Table S1. Experimental conditions and results for the random copolymerization of styrene and biotin-modified styrene in the presence of P(PEGMA) as the macroRAFT agent at 80 ºC for 19 hours.

ENTRY P(PEGMA)CODE Mn PDI [M]0

a [M]0a/

[CTA]0b

[CTA]0b/

[BPO]0bioSc % Solvent Mn

d PDId f PSe

%

EP33-1-155

EP33-1-130B 37500 1.27 2,00 340 10 5 Toluenef 38300 1.30 0

EP33-1-160

EP33-1-130A 39700 1.29 2,00 340 10 5 Toluenef nd nd 0

EP33-1-166

EP33-1-130A 39700 1.29 2,00 340 10 5 DMFg

a) M = total monomer. In this specific case, it refers to the sum of styrene + biotin-styrene.b) CTA = chain transfer agent. In this specific case, it refers to P(PEGMA).c) Referred to styrene, i.e. 5% of biostyrene (bioS) corresponds to styrene:bioS 95:5 molar ratio.d) Mn and PDI from GPC results, calibrated with PMMA standards.e) From GPC results. Calculated from [(total Mn - P(PEGMA)'s Mn)/(total Mn)]*100.f) Toluene analytical grade was used as received.g) N,N-dimethylformamide (DMF) was dried over 3Å molecular sieves and stored under nitrogen.

Taking into account the solubility of the different starting products and the solvents compatible with the RAFT polymerization method led us to select DMF as the solvent for the synthesis of P(PEGMA)-b-P(S-co-bioS). Then, the reaction was repeated in the same experimental conditions as before but in dry DMF (run EP33-1-166). The 1H-NMR spectrum indicates that a block copolymer has been obtained. However, it could not be said whether the biotin moiety was introduced into the copolymer due to significant amounts of unreacted monomer that impede a correct characterization. The purification of such copolymer resulted impossible due to the scarce solubility of bioS in a vast majority of routinely employed solvents. Therefore, this synthetic approach was discarded.

Product characterization

Figure S2. CDB 1H-NMR (DMSO-d6, δ in ppm): 7.82-7.77 (m, 2H), 7.62-7.53 (m, 3H), 7.45-7.38 (m, 2H), 7.36-7.29 (m, 2H), 7.25-7.19 (m, 1H), 1.96 (s, 6H).

Figure S3. CDB High Pressure Liquid Chromatography (HPLC) (Agilent HP1100 with Column Luna C18 50 x 4,6 mm 3μm, Mobil phase: water:ACN (95:5) – 10 min – (0:100) – 10 min – (0:100) – 10 min – (0:100) – 15 min – (95:5) – Detector. 254 nm - Flux: 1 mL /min – Sample preparation: 2 mg/mL in CH2Cl2 - Injection: 10 μL): purity 96.3% a/a

Figure S4. P(PEGMA) 1 1H-NMR (Acetone-d6, δ in ppm): 7.95-7.89 (m, Raft end-group), 7.53-7.46 (m, Raft end-group), 7.42-7.35 (m, Raft end-group), 7.33-7.26 (m, Raft end-group), 4.14 (s, 2H, CO-OCH2), 3.75 (s, 2H, CO-O-C-CH2-O), 3.70-3.57 (m, 12H, -O-CH2-C), 3.52 (s, 2H, -CH2-O-CH3), 3.34 (s, 3H, O-CH3), 2.13-1.80 (m, 2H, CH2), 1.26-0.87 (m, 3H, CH3).

Figure S5. P(PEGMA) 1 GPC (column 2xPLgel 5 um MIXED-C 300x7.5 mm + 1xPLgel 5 um GUARD 50x7.5 mm at 30 ºC, Eluent THF 100%, Flux 1 mL/min, detector refractive index, Data analyzed using Agilent Chemstation software package, Molecular weight determined relative to narrow poly(methylmethacrylate) standards): Mn 43000 – PDI 1.33

Figure S6. P(PEGMA)-b-PS 1H-NMR (Acetone-d6, δ in ppm): 7.96-7.81 (m, Raft end-group), 7.64-7.52 (m, Raft end-group), 7.47-6.37 (m, 1.8 H, H arom. S), 4.15 (s, 2H, CO-O-CH2-), 3.74 (s, 2H, CO-O-C-CH2-O), 3.70-3.57 (m, 12H, -O-CH2-C), 3.52 (s, 2H, -CH2-O-CH3), 3.34 (s, 3H, O-CH3), 2.08-1.82 (m, 2H, CH2), 1.75-1.22 (m, 1H, CH2), 1.19-0.78 (m, 3H, CH3)

Figure S7. P(PEGMA)-b-PS GPC (column 2xPLgel 5 um MIXED-C 300x7.5 mm + 1xPLgel 5 um GUARD 50x7.5 mm at 30 ºC, Eluent THF 100%, Flux 1 mL/min, detector refractive index, Data analyzed using Agilent Chemstation software package, Molecular weight determined relative to narrow poly(methylmethacrylate) standards): Mn 53000 – PDI 1.45

Figure S8. P(PEGMA):P(S-co-VBC) 1H-NMR (Acetone-d6, δ in ppm): 7.94-7.82 (m, Raft end-group), 7.62-7.50 (m, Raft end-group), 7.44-6.39 (m, 2.3 H, H arom. S and VBC), 4.75-4.51 (m, 0.06 H, CH2-Cl) - 4.14 (s, 2H, CO-OCH2), 3.75 (s, 2H, CO-O-C-CH2-O), 3.70-3.57 (m, 12H, -O-CH2-C), 3.52 (s, 2H, -CH2-O-CH3), 3.34 (s, 3H, O-CH3), 2.13-1.82 (m, 2H, CH2), 1.76-1.22 (m, 0.7H, CH2), 1.24-0.87 (m, 3H, CH3).

Figure S9. P(PEGMA):P(S-co-VBC) GPC (column 2xPLgel 5 um MIXED-C 300x7.5 mm + 1xPLgel 5 um GUARD 50x7.5 mm at 30 ºC, Eluent THF 100%, Flux 1 mL/min, detector refractive index, Data analyzed using Agilent Chemstation software package, Molecular weight determined relative to narrow poly(methylmethacrylate) standards): Mn 56000 – PDI 1.41

Figure S10. P(PEGMA)-b-P(S-co-bioS) 1H-NMR (Acetone-d6, δ in ppm): 7.53-7.36 (m, 2.3H arom.), 5.21-4.92 (m, 0.05 H, CH2-O-CO-), 4.51-4.38 (m, 0.03H, CH-NH-CO), 4.14 (s, 2 H, CO-OCH2), 3.75 (s, 2H, CO-O-C-CH2-O), 3.70-3.57 (m, 14H, -O-CH2-C), 3.52 (s, 2H, -CH2-O-CH3), 3.34 (s, 3H, O-CH3), 2.74-2.62 (m, 0.03H, S-CH2-), 2.46-2.33 (m, 0.05H, -CH2-O-CO-CH2-), 2.06-1.77 (m, 2H, CH2), 1.77-1.24 (m, 1H, CH2), 1.22-0.75 (m, 3H, CH3) (Raft group not detected by 1H NMR)

Figure S11. P(PEGMA)-b-P(S-co-bioS) GPC (column 2xPLgel 5 um MIXED-C 300x7.5 mm + 1xPLgel 5 um GUARD 50x7.5 mm at 30 ºC, Eluent THF 100%, Flux 1 mL/min, detector refractive index, Data analyzed using Agilent Chemstation software package, Molecular weight determined relative to narrow poly(methylmethacrylate) standards): Mn 40000 – PDI 1.33

Figure S12. Comparison of P(PEGMA)-b-P(S-co-bioS) and P(PEGMA):P(S-co-VBC) 1H-NMR spectra (Acetone-d6, δ in ppm) showing how the signal corresponding to CH2-Cl from P(PEGMA):P(S-co-VBC) at 4.75-4.51 ppm disappears and new signals due to the presence of biotin appeared: δ = 2.46-2.33: O-C(O)-CH2; δ = 2.74-2.62: S-CH2; δ = 4.50-4.38: CH-NH.