pH Responsive Supramolecular Core-Shell Protein Hybrids Christiane Seidler#, David Y.W. Ng*,#, Yuzhou Wu, Tanja Weil* Institute of Organic Chemistry III, Macromolecular and Organic Materials, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany Email: tanja.weil@uni-ulm.de # shared first authorship Supporting Information I. Materials and Methods (Chemical Synthesis) All solvents and reagents are bought from commercial sources (Merck, Sigma Aldrich, VWR, etc.) and are used directly without further purification. The extend of reaction is monitored by thin layer chromatography using Merck 60 F254 pre-coated silica on aluminium using appropriate staining (KMnO4) when needed. Column chromatography is carried out on Acros Organics silica gel 0.035 mm – 0.070 mm, 60 Å. 1H-NMR and 13 C-NMR spectra are recorded using Bruker DRX 400 MHz fourier transform spectrometer using deuterated solvents as mentioned. Chemical shifts are reported as parts per million referenced with respect to the residual solvent peak. Mass is determined using Shimadzu LC-MS 2020 (ESI). IR spectra are measured with FT-IR Bruker Vector 22. Synthesis of monomethyl ether salicylhydroxamate PEG2000 (SHA-PEG2000) 7 Supplementary Scheme 1. Synthesis of the monomethyl ether SHA-PEG2000 7. Synthesis of monomethyl ether PEG2000 tosylate 1 Poly(ethylene glycol) methyl ether with M = 2000 g/mol (PEG2000-ME, 2.00 g, 1.00 mmol) was dissolved in 10 mL pyridine. Toluene-4-sulfonyl chloride (1.91 g, 10.00 mmol, 10 eq) was added and the reaction was stirred overnight at room temperature. After 21 h the reaction solution was poured into 30 mL cold, distilled water and extracted 3 times with 30 mL dichloromethane. The combined organic layers were S2 washed with cold 6 M HCl-solution (2 times) and cold, distilled water (2 times). After drying with anhydrous magnesium sulfate, the solvent was removed under vacuum to obtain the tosylated PEG2000-ME (1) as white solid in 75 % yield. 1H-NMR (400 MHz, CDCl3) δ [ppm] = 7.80 (d, 2H, J = 8.3 Hz, HAr), 7.34 (d, 2H, J = 8.0 Hz, HAr), 4.15 (t, 2H, J = 4.8 Hz, CH2-OTos), 3.64 (m, O-CH2CH2), 3.38 (s, 3H, CH3-O-CH2CH2), 2.45 (s, 3H, CH3-Ph-O). 13C-NMR (100 MHz, CDCl3) δ [ppm] = 129.9, 128.06, 70.81, 70.63, 69.32, 68.74, 21.73. Synthesis of monomethyl ether PEG2000 azide 3 Tosylated PEG2000-ME 1 (0.40 g, 0.19 mmol) was dissolved in acetonitrile and sodium azide (0.15 g, 2.23 mmol, 12 eq) was added. The reaction mixture was refluxed overnight. After solvent removal and cooling down to room temperature, distilled water was added to the reaction solution and the aqueous solution was extracted 5 times with dichloromethane. The combined organic layers were dried with magnesium sulfate and the solvent was removed. The Azido-PEG2000-ME 3 was obtained by drying under vacuum as colourless solid in 84 % yield. 1H-NMR (400 MHz, CDCl3) δ [ppm] = 3.81 (t, 2H, J = 4.8 Hz, CH2-N3), 3.64 (m, O- CH2CH2), 3.38 (s, 3H, O-CH3). 13C-NMR (100 MHz, CDCl3) δ [ppm] = 72.06, 70.69, 70.17, 59.17, 50.81. IR (KBr) ν [cm-1] = 2889, 2109, 1968, 1633, 1470, 1345, 1282, 1113, 962, 842. Synthesis of trityloxy protected monomethyl ether PEG2000 salicylhydroxamate 5 Azido-PEG2000-ME 3 (0.15 g, 0.07 mmol) and ethynyl salicylhydroxamic acid i (0.05 g, 0.10 mmol, 1.3 eq) were dissolved in THF. Copper sulfate (0.02 g, 0.15 mmol, 2 eq) and sodium ascorbate (0.02 g, 0.10 mmol, 1.3 eq) were dissolved in distilled water and added to the reaction solution. The mixture was stirred overnight at room temperature. The solvent was removed and the crude product was purified by column chromatography (eluent: dichloromethane with 20 % methanol). After drying under vacuum, 5 was obtained as colourless solid in 76 % yield. S3 1H-NMR (400 MHz, CDCl3) δ [ppm] = 8.81 (br s, 1H, Ph-OH), 8.39 (s, 1H, NH-Ph), 7.60 (s, 1H, N-CH=C-N), 7.51 (dd, 6H, J = 8.0 Hz, J = 1.7 Hz, m-HAr of TrtO), 7.33 (m, 11H, o-HAr of TrtO, p-HAr of TrtO, HAr, CO-NH-O), 7.06 (m, 2H, HAr), 4.47 (t, 2H, J = 4.7 Hz, CH2-O), 3.79 (m, 4H, N-CH2CH2-O), 3.64 (m, CH2CH2-O), 3.37 (s, 3H, CH3-O), 3.08 (t, 2H, J = 6.4 Hz, CH2-CH2-CO-NH), 2.76 (t, 2H, J = 7.2 Hz, CH2-CH2CO-NH). 13C-NMR (100 MHz, CDCl3) δ [ppm] = 171.19, 161.16, 146.46, 143.93, 143.17, 141.82, 129.04, 128.84, 128.15, 128.02, 122.89, 110.50, 108.73, 107.61, 93.53, 72.03, 70.66, 70.50, 69.50, 59.14, 50.32, 36.70, 30.07, 21.25. Synthesis of monomethyl ether PEG2000 salicylhydroxamate 7 Trityloxy protected SHA-PEG2000-ME 5 (0.02 g, 8.00 µmol) was dissolved in 0.55 mL dichloromethane. Trifluoroacetic acid (TFA, 40 % v/v) and triisopropylsilane (5 % v/v) were added slowly and the reaction mixture was stirred at room temperature for 2 h. After removal of solvent and evaporation of TFA under vacuum, distilled water and toluene were added. The aqueous layer was washed two times with toluene and lyophilized afterwards to obtain the product 7 as white powder in 83 % yield. 1H-NMR (400 MHz, CDCl3) δ [ppm] = 12.07 (s, 1H, Ph-OH), 10.65 (s, 1H, CO-NH- OH), 8.79 (s, 1H, NH-Ph), 7.72 (s, 1H, CO-NH-OH), 7.63 (s, 1H, N-CH=C-N), 7.53 (d, 1H, J = 8.4 Hz, HAr), 7.22 (s, 1H, HAr), 7.15 (d, 1H, J = 8.4 Hz, HAr), 4.48 (t, 2H, J = 4.6 Hz, CH2-O), 3.80 (m, 4H, N-CH2CH2-O), 3.65 (m, CH2CH2-O), 3.38 (s, 3H, CH3O), 3.12 (t, 2H, J = 6.3 Hz, CH2-CH2-CO-NH), 2.80 (t, 2H, J = 6.4 Hz, CH2-CH2-CONH). S4 Synthesis of FITC labelled salicylhydroxamate PEG2000 (FITC-PEG2000-SHA) 8 Supplementary Scheme 2. Synthesis of FITC-PEG2000-SHA 8. Synthesis of monohydroxy PEG2000 tosylate 2 Firstly fresh silver oxide was prepared by adding a 85 °C hot solution of sodium hydroxide (0.19 g, 4.7 mmol) in 7 mL water to a also 85 °C hot solution of silver nitrate (0.80 g, 4.7 mmol) in 7 mL water. The resulting hot, brown suspension was quickly filtered and the filter cake was washed with hot water and methanol. The silver oxide was dried under vacuum to obtain it as a brown powder. Poly(ethylene glycol) with M = 2000 g/mol (PEG2000, 3.00 g, 1.50 mmol) was dissolved in dichloromethane and cooled down to 0 °C. Fresh silver oxide (0.52 g, 2.25 mmol, 1.5 eq), toluene-4-sulfonyl chloride (0.32 g, 1.65 mmol, 1.1 eq) and potassium iodide (0.05 g, 0.30 mmol, 0.3 eq) were added to the reaction solution. After 15 min. brown silver salt precipitated out of the reaction mixture. The suspension was allowed to warm up to room temperature and stirred overnight. To remove the solid, the reaction mixture was filtered through Celite using ethyl acetate as eluent. The solvent was reduced under vacuum and the crude product was further purified by column chromatography (eluent: dichloromethane/acetone – 3/2). After drying under vacuum, the product 2 was obtained as white solid in 75 % yield. S5 1H-NMR (400 MHz, CDCl3) δ [ppm] = 7.79 (d, 2H, J = 8.4 Hz, HAr), 7.34 (d, 2H, J = 8.4 Hz, HAr), 4.15 (t, 2H, J = 4.8 Hz, CH2-OTos), 3.64 (m, O-CH2CH2), 2.44 (s, 3H, CH3-Ph-O). 13C-NMR (100 MHz, CDCl3) δ [ppm] = 129.9, 128.06, 70.81, 70.63, 69.32, 68.74, 21.73. Synthesis of monohydroxy PEG2000 azide 4 Tosylated PEG2000 2 (1.70 g, 0.79 mmol) was dissolved in 15 mL acetonitrile and sodium azide (0.62 g, 9.47 mmol, 12 eq) was added. The reaction mixture was refluxed overnight. After solvent removal and cooling down to room temperature, distilled water was added to the reaction solution and the mixture was extracted 5 times with dichloromethane. The combined organic layers were dried with magnesium sulfate and the solvent was removed. The Azido-PEG2000 4 was obtained by drying under vacuum as colourless solid in 84 % yield. 1H-NMR (400 MHz, CDCl3) δ [ppm] = 3.81 (t, 2H, J = 4.8 Hz, CH2-N3), 3.64 (m, O- CH2CH2), 3.46 (t, 2H, J = 4.5 Hz, CH2-OH). 13C-NMR (100 MHz, CDCl3) δ [ppm] = 72.63, 70.61, 70.36, 70.09, 50.73. IR (KBr) ν [cm-1] = 2889, 2108, 1968, 1633, 1470, 1346, 1282, 1110. Synthesis of trityloxy protected monohydroxy PEG2000 salicylhydroxamate 6 Azido-PEG2000 4 (0.80 g, 0.40 mmol) and ethynyl salicylhydroxamic acid i (0.23 g, 0.47 mmol, 1.2 eq) were dissolved in THF. Copper sulfate (0.11 g, 0.71 mmol, 1.8 eq) and sodium ascorbate (0.09 g, 0.44 mmol, 1.1 eq) were dissolved in distilled water and added to the reaction solution. The mixture was stirred overnight at room temperature. The solvent was removed and the crude product was purified by column chromatography (eluent: dichloromethane with gradient of methanol from 10 % up to 20 %). After drying under vacuum, 6 was obtained as colourless solid in 76 % yield. 1H-NMR (400 MHz, CDCl3) δ [ppm] = 11.09 (s. 1H, OH), 8.96 (br s, 1H, Ph-OH), 8.64 (s, 1H, NH-Ph), 7.59 (s, 1H, N-CH=C-N), 7.49 (dd, 6H, J = 8.1 Hz, J = 1.7 Hz, mHAr of TrtO), 7.30 (m, 11H, o-HAr of TrtO, p-HAr of TrtO, HAr, CO-NH-O), 7.08 (m, 2H, HAr), 4.45 (t, 2H, J = 4.8 Hz, CH2-O), 3.79 (m, 4H, N-CH2CH2-O), 3.62 (m, S6 CH2CH2-O), 3.48 (m, 4H, CH2CH2-OH), 3.06 (t, 2H, J = 6.4 Hz, CH2-CH2-CO-NH), 2.74 (t, 2H, J = 7.2 Hz, CH2-CH2-CO-NH). 13C-NMR (100 MHz, CDCl3) δ [ppm] = 171.19, 161.16, 146.46, 143.93, 143.17, 141.82, 129.04, 128.84, 128.13, 128.02, 122.89, 110.50, 108.73, 107.61, 93.53, 72.03, 70.64, 70.50, 69.50, 50.32, 36.70, 30.07, 21.25. Synthesis of FITC labelled PEG2000 salicylhydroxamate 8 Trityloxy protected SHA-PEG2000-OH 6 (0.10 g, 40 µmol) and fluorescein isothiocyanate (ii, 17.0 mg, 44 µmol, 1.1 eq) were dissolved in 0.5 mL DMF. Triethylamine (11 µL, 8.0 mg, 79 µmol, 2 eq) was added to the reaction solution and the mixture was stirred for 2 d at room temperature in the dark. After solvent removal under vacuum, the crude product was purified by column chromatography using dichloromethane with 10 % methanol as eluent. A part of the obtained intermediate (20.0 mg, 7.0 µmol) was dissolved in 2 mL dichloromethane again and trifluoroacetic acid (TFA, 25 % v/v) and triisopropylsilane (13 % v/v) were added. After 3 h, the solvent and TFA was removed under vacuum. Distilled water was added and washed with toluene (2 times). The combined organic layers were extracted with distilled water and all aqueous layers were lyophilized to obtain a yellow, oily product (8) in 82 % yield. 1H-NMR (400 MHz, DMSO-d6) δ [ppm] = 12.42 (s, 1H, Ph-OH), 11.28 (s, 1H, NH-OH), 10.18 (s, 1H, Ph-OH), 10.11 (s, 1H, NH-Ph), 9.42 (s, 1H, CO-NH-OH), 8.04 (d, 1H, J = 1.7 Hz, HAr, FITC), 7.81 (m, 2H, HAr, FITC, N-CH=C-N), 7.58 (d, 1H, J = 8.5 Hz, HAr, SHA), 7.35 (d, 1H, J = 8.0 Hz, HAr, FITC), 7.29 (d, 1H, J = 1.9 Hz, HAr, SHA), 6.99 (dd, 1H, J = 9.4 Hz, J = 1.7 Hz, HAr, SHA), 6.68 (d, 2H, J = 2.1 Hz, HAr, FITC), 6.62 (s, 1H, HAr, FITC), 6.60 (s, 1H, HAr, FITC), 6.56 (d, 1H, J = 2.3 Hz, HAr, FITC), 6.54 (d, 1H, J = 2.4 Hz, HAr, FITC), 4.46 (t, 2H, J = 5.2 Hz, N-CH2CH2-O), 3.77 (t, 2H, J = 5.2 Hz, N- CH2CH2-O), 3.67 (t, 2H, J = 3.6 Hz, CH2-O-CS), 3.50 (m, CH2CH2-O), 2.92 (t, 2H, J = 6.9 Hz, CH2-CH2-CO-NH), 2.69 (t, 2H, J = 7.4 Hz, CH2-CH2-CO-NH). S7 Synthesis of NHS-ester activated 4-(carboxy)phenylboronic acid (NHS-PBA) 9 Supplementary Scheme 3. Synthesis of NHS-PBA 9. 4-(Carboxy)phenylboronic acid (2.00 g, 12.05 mmol) and N-hydroxysuccinimide (1.67 g, 14.46 mmol, 1.2 eq) were dissolved in 20 mL DMF. EDC*HCl (2.77 g, 14.46 mmol, 1.2 eq) and DMAP (0.18 g, 1.45 mmol, 0.12 eq) were added and the reaction solution was stirred overnight at room temperature. After solvent removal, a yellow oily raw product was purified using column chromatography (eluent: dichloromethane with 10 % methanol) and dried under vacuum. The product 9 was obtained as white solid in 72 % yield. 1H-NMR (400 MHz, DMSO-d6) δ [ppm] = 8.53 (s, 2H, BOH), 8.05 (d, 2H, J = 8.0 Hz, HAr), 8.00 (d, 2H, J = 7.8 Hz, HAr), 2.90 (s, 4H, CH2CON). 13C-NMR (100 MHz, DMSO-d6) δ [ppm] = 170.40, 162.10, 134.84, 134.22, 129.00, 128.85, 128.77, 125.57, 25.58. LC-MS (ESI) m/z = 572 [2M + 2Na]+, 309 [M + 2Na]+ mV(x1,000) Ch2 (254nm) 1,0 0,0 0,0 II. 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 min Materials and Methods (Protein Modification and Biological Studies) Human serum albumin (HSA) is purchased from Sigma-Aldrich and cytochrome c (CytC) from equine heart from Merck Millipore. All other chemicals/biologicals including alizarin red S, DMEM cell culture medium, fetal bovine serum and penicillin/streptomycin are purchased from multiple commercial sources (Merck, Alfa Aesar, Invitrogen etc.) and used directly without further purification. The grade of all water (H2O) used is MilliQ ultrapure grade. Ultrafiltration purification is performed using Vivaspin 6 ultrafiltration tubes (GE healthcare) with molecular weight cut-off S8 (MWCO) of 10 kDa (for CytC derivatives) and 30 kDa (for HSA derivatives). Fluorescence measurements are taken from Tecan M1000 Infinite microplate reader. Microscale thermophoresis measurements are performed using a Monolith NT.115 instrument and the data are analysed by NT.Analysis software version 1.5.41 (both from NanoTemper Technologies GmbH). Dynamic light scattering measurements are carried out using a Malvern Nanosizer instrument (Malvern Ltd, Malvern, UK). Luminescence intensities are measured from GloMax® Multi 96 well plate reader (Promega). Confocal laser scanning microscopy is conducted on Zeiss LSM 710, Observer Z.1 and processed using Zen software. Representative preparation of boronic acid modified proteins (HSA, CytC) The pre-activated boronic acid 9 (1.49 mg, 5.65 µmol, 14 eq) was dissolved in DMSO and added to cytochrome c (5.0 mg, 0.41 µmol, 1 eq), which was already dissolved in 0.5 mL NaHCO3 buffer (0.1 M, pH 8.5). The reaction was shaking overnight at room temperature and purified using Vivaspin 6 ultrafiltration tubes (10 kD MWCO) the next day. This was performed at 3500 rpm and 4 °C to obtain a concentrated, salt free protein solution, which was characterized by MALDI-TOF MS (see Supplementary Figure 1) and freeze dried for further use. Supplementary Figure 1. MALDI-TOF MS of boronic acid modified HSA (A) and CytC (B). Representative preparation of rhodamine labelled proteins (HSA, CytC) CytC-BA13 (boronic acid modified CytC with 13 functional groups on surface; 3.92 mg, 0.27 µmol) was dissolved in 0.5 mL NaHCO3 buffer (0.1 M, pH 8.5) and rhodamine isothiocyanate (29 µL, 10 mg/mL in DMSO, 0.27 µmol) was added to the S9 protein solution. The reaction mixture was shaken in the dark at 350 rpm overnight at room temperature. Purification was performed using Vivaspin 6 ultrafiltration tubes (10 kD MWCO) until no colour due to free dye was left in the filtrate. The concentrated protein solution was freeze dried to obtain salt free solid for further experiments. Representative Alizarin Red S fluorescence titration (HSA, CytC) CytC-BA13 (125 µL, 1 mg/mL in 100 mM phosphate buffer pH 7.4) was transferred into a UV transparent 96-well plate and subsequently added alizarin red S (10 µL, 10 mg/mL in DMSO). After incubation in an orbital shaker for 10 min, the fluorescence (λex = 495 nm, λem = 520-720 nm) was measured. PEG2000-SHA 7 (19.88 mg/mL in 100 mM phosphate buffer pH 7.4) was added in aliquots of 1 µL (corresponding to 1 mol. eq), mixing via orbital shaking for 2 min after each addition. For each addition step, a fluorescence scan was measured (λem, max = 600 nm). The addition of 7 was stopped, when no further emission changes were observed due to the end point of the alizarin red S – PEG 7 exchange. Microscale thermophoresis for binding affinity determination (HSA) FITC-PEG2000-SHA 8 was used as fluorescent binding partner and synthesized as described above. Dissolved in microscale thermophoresis (MST) buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 10 mM MgCl2), the concentration of FITC-PEG2000SHA 8 was adjusted to 400 nM for the MST measurements. Boronic acid modified HSA and native HSA (negative control) were chosen as ligands and a series of 14 1:1 dilutions for each sample was prepared using the identical buffer (MST buffer) producing ligand concentrations ranging from 19.6 nM to 160 µM. For thermophoresis, each ligand dilution was mixed with one volume of FITC-PEG2000-SHA 8, which leads to a final concentration of fluorescently labelled PEG 8 of 200 nM and final ligand concentrations ranging from 9.8 nM to 80 µM. After 10 min incubation at room temperature, followed by centrifugation at 13 000 x g for 5 min, approximately 8 µL of each solution was filled into Monolith NT Standard Treated Capillaries (NanoTemper Technologies GmbH). Thermophoresis was measured using a Monolith NT.115 instrument (NanoTemper Technologies GmbH) at an ambient temperature of 25 °C with 5 s/30 s/5 s laser off/on/off, respectively. Instrument parameters were adjusted to 30 % LED power and 20 % MST power. Data of three independently pipetted S10 measurements were analysed (NT.Analysis software version 1.5.41, NanoTemper Technologies GmbH) using the fluorescence signal. Preparation of SD test (boronic acid modified HSA, native HSA) The fluorescence signals of the 14 capillaries containing different mixtures of the two binding partners normally have to be around the same value (± 10 % deviation due to pipetting errors is acceptable, called random fluorescence change). For some investigated systems, one can observe ligand dependent fluorescence change upon binding, which describes even more precisely the binding event than the Thermophoresis + T-Jump data, which is usually used for analysis of the binding event by the NT.Analysis software. To find out whether the fluorescence change during the MST measurements were related to ligand-specific fluorescence change or randomly, NanoTemper Technologies GmbH suggests performing a SDS-denaturation test (SD test). One has to measure the fluorescence of all capillaries before and after this SD test via capillary scan by the Monolith NT.115 instrument and compare them. Ligandspecific fluorescence change upon binding is determined, when there is no fluorescence change (no binding) in the capillary scan observable anymore after addition of SD mix to thermophoresis samples. If there is still fluorescence change measured after performing SD test by the capillary scan, the fluorescence change is non ligand-specific. Carrying out the SD test, all the sample mixtures were centrifuged for 12 min with 13300 x g at room temperature. At the same time, 14 new tubes were filled with 9 µL of a SD mix solution (4 % SDS, 40 mM DTT). After centrifugation, 9 µL of the supernatant of the sample tubes was carefully removed and added to the SD mix solution in the 14 new tubes. After mixing well the two solutions, all SD test tubes were incubated 5 min at 95 °C during slightly shaking (around 400 rpm) to denature the samples. Briefly centrifugation of the tubes after heating was performed, to ensure that the solutions are at the bottom of the tube. The solutions were filled into Monolith NT Standard Treated Capillaries (NanoTemper Technologies GmbH) and a capillary scan was measured again. This protocol was fulfilled for the boronic acid modified HSA samples as well as for the negative control (native HSA). Comparison of Supplementary Figure 2 (capillary scan before performing SD test) and Supplementary Figure 3 (capillary scan after performing SD test) for the boronic acid modified HSA show ligand-specific fluorescence change S11 of the samples, why the fluorescence change was used for Kd determination (as suggested by NanoTemper Technologies GmbH). Supplementary Figure 2. Capillary scan for fluorescence intensity of FITC-PEG2000SHA mixed with different amounts of boronic acid modified HSA before performing SD test. Supplementary Figure 3. Capillary scan for fluorescence intensity of FITC-PEG2000SHA mixed with different amounts of boronic acid modified HSA after performing SD test. The SD test was also carried out for the native HSA samples (negative control system). Supplementary Figure 4 show the fluorescence intensity of the capillary scan before performing the SD test and Supplementary Figure 5 shows it afterwards. The slightly fluorescence changes were in both cases observable, which determines the changes in fluorescence signals as ligand-unspecific binding and underpin the usage of the fluorescence change for Kd determination for the boronic acid modified HSA – FITC- S12 PEG2000-SHA system (change in fluorescence for the negative control system was tested several times to avoid the changes coming from false sample handling). Supplementary Figure 4. Capillary scan for fluorescence intensity of FITC-PEG2000SHA mixed with different amounts of native HSA (negative control) before performing SD test. Supplementary Figure 5. Capillary scan for fluorescence intensity of FITC-PEG2000SHA mixed with different amounts of native HSA (negative control) after performing SD test. Dynamic light scattering (HSA, CytC) The hydrodynamic size distribution was characterized by dynamic light scattering (DLS). Native proteins (HSA, CytC) were measured as well as the boronic acid modified protein derivatives incubated with PEG2000-SHA 7 at pH 5.0 and 7.4 to demonstrate the pH-controllable polymer-assemblation via size increase at pH 7.4. The samples were prepared at 1 mg/mL concentrations in phosphate buffer solutions (100 mM filtered buffers) of the adequate pH value (native proteins in pH 7.4). S13 Therefore 25 eq PEG2000-SHA 7 for the HSA construct samples and 16 eq for the CytC containing samples were added to the protein solutions and all samples (native, modified) were incubated for 1 h with shaking (~ 350 rpm). The samples have been filtered through 0.2 µm syringe filters before measurement to avoid dust contamination. Autocorrelation functions were analysed by cumulants method to estimate hydrodynamic diameter. The hydrodynamic diameter distribution was presented as number distribution (see Supplementary Figure 6). Supplementary Figure 6. Overlay of DLS data of native CytC (green trace, continuous line), boronic acid modified CytC with PEG2000-SHA 7 at pH 5.0 (blue trace, dashed line) and pH 7.4 (red trace, dotted line). Fluorescence polarization (HSA, CytC) FITC-PEG2000-SHA 8 (30 µL, 1 mM in 100 mM phosphate buffer pH 7.4) was incubated with protein (native HSA and CytC, boronic acid modified HSA and CytC; 30 µL, 1 mM in 100 mM phosphate buffer pH 7.4) for 1 h. The concentrated solutions were diluted using 240 µL phosphate buffer (100 mM, pH 7.4) to obtain 300 µL solution. The mixtures were transferred into a UV-transparent 96-well plate for fluorescence polarization measurements (100 µL per well, triplicates). The G-factor (0.989) was calibrated using 100 nM fluorescein in 0.1 M NaOH (20 mP). The native proteins were used as controls and to define the initial values from which the molecular weight increase was observed. S14 Luminescent cell viability assay (CytC) A549 cells were seeded at 6500 cells/well in a half-area 96 well plate containing 50 µL of DMEM medium. Boronic acid modified CytC (0.5 mg, 35 nmol, 50 µM) was preincubated with SHA-PEG2000 7 (900 µM) in phosphate buffer (100 mM, pH 7.4) for 30 min. The complexed solutions were diluted with DMEM medium to afford CytC concentrations between 1 µM to 25 µM. After the cells were adhered for 24 h at 37 °C and 5% CO2, the medium in the wells was removed and the samples were added. Each experiment was done in triplicates. SHA-PEG2000 7 (1.8 mM) and native CytC (50 µM) were separately used as controls. The cells were incubated for 24 h at 37 °C, 5 % CO2 and subjected to CellTiter-Glo® luminescent cell viability kit (Promega) according to manufacturer´s protocol. Cellular uptake and localization (HSA, CytC) A549 cells were pre-cultured in high glucose DMEM medium fortified with 10 % fetal bovine serum, 1 % penicillin/streptomycin and seeded at 10,000 cells/well in a 8-well confocal microscopy chamber and left to adhere overnight at 37 °C, 5 % CO 2. Rhodamine labelled modified proteins (boronic acid modified HSA and CytC; 10 µM) were pre-incubated with SHA-PEG2000 7 (280 µM for HSA and 180 µM for CytC) in 15 µL phosphate buffer (100 mM, pH 7.4) for 30 min. The proteins were subsequently added to the confocal microscopy well plate containing DMEM medium to afford a final sample concentration of 0.5 µM. After incubation for 4 h at 37 °C and 5 % CO2, the medium containing the samples was removed and the cells washed (x3) with fresh DMEM medium before analysing with confocal laser scanning microscope. S15