An Oxygen-Tolerant PET-RAFT Polymerization for Screening Structure-Activity Relationships

Abstract

The complexity of polymer-protein interactions makes rational design of the best polymer architecture for any given biointerface extremely challenging, and the high throughput synthesis and screening of polymers has emerged as an attractive alternative. A porphyrin-catalysed photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) polymerisation was adapted to enable high throughput synthesis of complex polymer architectures in dimethyl sulfoxide (DMSO) on low-volume well plates in the presence of air. The polymerisation system shows remarkable oxygen tolerance, and excellent control of functional 3- and 4-arm star polymers. We then apply this method to investigate the effect of polymer structure on protein binding, in this case to the lectin concanavalin A (ConA). Such an approach could be applied to screen the structure-activity relationships for any number of polymer-protein interactions.

Keywords: PET-RAFT; click chemistry; glycopolymers; oxygen tolerance; photomediated radical polymerization.

Figure 1.

a) Pseudo first-order kinetics and b) molecular weight evolution of DMA polymerisations using RAFT agent R1 and either 0.01 or 0.02 equiv ZnTPP relative to RAFT, in well plates with 300 μL and 40 μL volumes. Target DP = 200, [DMA] = 1m.

Figure 2.

a),b) Pseudo first-order kinetics of polymerisations with [DMA]/[RAFT]/[ZnTPP] = 200:1:0.01 at 1m DMA in a a) 96-well plate and b) an NIR cuvette, with and without removal of oxygen prior to polymerisation. MacroR1 = DP 15 DMA macroRAFT agent prepared from R1 and purified prior to chain extension. Arrows indicate the bubbling of air through the cuvette with a pipette after measurement. c),d) GPC molecular weight distributions from the chain extension experiment and final kinetic samples (c) as well as from five other acrylamides at full conversion (d).

Figure 3.

a),b) GPC molecular weight distributions of DMA/NHS polymer library polymerised at a monomer concentration of a) 0.5m, [DMA]/[NHS] = 9:1 (Supporting Information, Table S3) and b) 1m, [DMA]/[NHS] = 9:1 and 8:2 (Supporting Information, Table S5). c) FTIR on crude polymers from 0.5m polymerisation (Supporting Information, Table S6) after functionalization with DBCO-NH₂ and Man(OAc)-N₃ showing almost full functionalization. d) GPC molecular weight distributions of DP40 polymers before and after functionalization with DBCO-NH₂ and PEG₇-N₃ (Supporting Information, Table S4). All polymers containing NHS were reacted with butyl amine before running the GPCs.

Figure 4.

(a) Enzyme-linked lectin binding assay. Polymer is incubated with the lectin-HRP conjugate at varying concentrations. Free unbound Con-A-HRP adheres to a mannose-coated well plate, and the HRP developed to determine the fraction of Con-A bound to the polymer. b)–d) Binding curves for linear (b), 3 arm star (c), and 4 arm star (d) polymer series to ConA, showing the percentage of unbound lectin vs. polymer concentration. e) IC50 values extracted from the sigmoidal fit, normalised to mannose content showing the effect of size and structure on binding affinity. Error bars correspond to ±1 standard deviation from the mean in triplicate measurements. The IC50 for the linear and 4-arm polymers at DP 320 and 640 could not be determined as minimal inhibition was observed at the concentration range tested, but were found to be much greater than 2000 μm mannose.

Scheme 1.

Representation showing the ZnTPP polymerisation mechanism and RAFT agents used in the library design.