Hetero‐Diels–Alder Cycloaddition with RAFT Polymers as Bioconjugation Platform

Abstract We introduce the bioconjugation of polymers synthesized by RAFT polymerization, bearing no specific functional end group, by means of hetero‐Diels–Alder cycloaddition through their inherent terminal thiocarbonylthio moiety with a diene‐modified model protein. Quantitative conjugation occurs over the course of a few hours, at ambient temperature and neutral pH, and in the absence of any catalyst. Our technology platform affords thermoresponsive bioconjugates, whose aggregation is solely controlled by the polymer chains.


S2
Dynamic light scattering (DLS) measurements were performed at 25 °C at an angle of 173° (backscattering mode) with a Zetasizer Nano S from Malvern using a 4mW He-Ne laser at 633 nm. Analysis of the data was carried out using the Nano DTS v.5.10 software. Protein-polymer conjugates were diluted to 0.1 mg/mL in PBS solution. Experiments were performed with 12 readouts of 3 independent measurements for each sample. The temperature-dependent aggregation experiment was performed by progressively heating the sample (increments of 1 °C), with 5 min allowed for stabilization at each step.
Matrix-assisted laser desorption ionization coupled to time-of-flight (MALDI-ToF) spectrometry. Mass spectra were acquired with a 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) in positive ion linear mode and a mass range of 60 000 to 80 000 Da. The laser intensity was set to 4700. The spectra obtained represent the average of laser shots taken by an automatic scheme measuring spectra over the whole spot. Sinapinic acid was used as matrix.
Circular dichroism (CD) measurements. CD spectra were measured using a Jasco J-815CD Spectrometer. Spectra were acquired at 0.2 mg mL -1 BSA concentration in water using 0.1 cm pathlength quartz cuvette. All CD spectra were recorded with a bandwidth of 1 nm at 1 nm increments and 10 s average time, over a wavelength range from 190 to 260 nm.

S5
Triethylene glycol methyl ether acrylate (mTEGA) mTEGA was synthesized using a procedure adapted from the literature. [4] Triethylene glycol monomethyl ether (3.0 g, 18.3 mmol, 1.0 eq.) was dissolved in DCM (20 mL). TEA (2.2 g, 20.7 mmol, 1.1 eq.) was added and the reaction flask kept in an ice bath. After 15 min, acryloyl chloride (2.0 g, 21.2 mmol, 1.2 eq.) was added dropwise over 30 min under N 2 atmosphere. After complete addition, the ice bath was removed and the reaction mixture was stirred overnight at RT. The triethylamine salt was filtered off and the solution was diluted with DCM. The product was isolated by sequential washing with aq. NaHCO 3 , water (3 × 50 mL), and brine (3 × 50 mL). The organic phase was dried over sodium sulfate. The solvent was removed under reduced pressure to yield mTEGA as a colorless oil (yield 66%).

Introduction of diene moieties in BSA
Bovine serum albumin (BSA) was taken as model protein to demonstrate the straightforward RAFT-HDA polymer-protein bioconjugation. As a first step, soluble diene-functionalized BSA, dBSA, was synthesized by direct coupling of DSS (27.8 µmol dissolved in 500 µL of DMSO) to the primary amino groups of a BSA (0.57 µmol in 5 mL of 30 mM sodium phosphate solution at pH 8.4). The reaction mixture was kept at 37 °C for 1 h. Thereafter, unreacted material was removed by filtering the reaction mixture through a 30 kDa MWCO membrane. The modified protein was further washed in sequential filtration steps with a total of 50 volumes of MilliQ water (five steps with 10 volumes of water). The coupling reaction was confirmed by MALDI-MS ( Figure S10), with an increase of m/z value of approx. 1200, which corresponds to the introduction of an average of approx. 6-7 diene units per BSA molecule. The dBSA solution was stored at -20 °C until its use.

RAFT end group stability in aqueous media
The stability of the RAFT end-group of PmTEGA2000 in water and in a range of buffers and pH values was checked by UV-Vis spectroscopy. PmTEGA2000 (0.25 mM) was dissolved in water (pH 6.8), PBS (10 mM sodium phosphate, 135 mM NaCl, 2.5 mM KCl, pH 7.4), sodium citrate (50 mM, pH 4.5), sodium acetate (50 mM, pH 5.6), sodium phosphate (50 mM, pH 6.0 and pH 8.1), Tris-HCl (50 mM, pH 8.1), and sodium bicarbonate (50 mM, pH 9.1). The absorbance of these solutions were immediately monitored at 327 nm for 180 min and plotted as % of the absorbance at t = 0. Experiments were performed in triplicate.
As observed in the Figure S12, the RAFT group is hydrolyzed faster at higher pH values. Moreover, Tris buffer seems detrimental for the stability of RAFT group compared to phosphate buffer at the same pH of 8.1. This effect might be explained by the presence of a primary amine group in Tris and the known susceptibility of the dithioester end-group towards primary amines. [5] Figure S11. Evolution of the absorbance at 327 nm of aqueous solutions of PmTEGA6000 in various buffer conditions.

RAFT-HDA test reaction
A first HDA test reaction was performed mixing PmTEGA6000 (0.05 µmol) with 1, 2, and 3 equivalents of 1 in phosphate buffer (50 mM, pH 6.0). DMSO (10%, v/v) was added to solubilize 1. The absorption of the RAFT group at 327 nm was used to monitor the reaction course. A solution of PmTEGA6000 without diene was used as control.

Protein-polymer bioconjugation
A typical RAFT polymer-protein HDA bioconjugation experiment was performed by mixing dBSA (70 µM) with a RAFT polymer (100 equivalents, 7 mM) in sodium phosphate buffer (30 mM, pH 6.0). The reaction was stirred overnight at room temperature. Unreacted material was removed by sequential filtration steps through a 30 KDa cutoff membrane. Protein-polymer conjugates were characterized by DLS and gel electrophoresis (SDS-PAGE).

Esterase assay
Glyceryl triacetate was used as substrate and phenol red as pH indicator. The hydrolytic activity of BSA leads to the release of acetic acid and to the subsequent decrease of pH, which can be monitored by the decrease of the absorbance at 560 nm, resulting from protonation of the phenol red dianion. Reactions were performed using a mixture of phenol red (1 mM), glyceryl acetate (5%, v/v), BSA (either free or conjugated version, 0.2 mg mL -1 ) in phosphate buffer (30 mM, pH 8.3). The absorbance at 560 nm was recorded by taking aliquots at different reaction times (from 0 to 30 min).