Logo JG-Universität MainzProf. Dr. Axel Müller


PhD Thesis

Novel Precursors for Polymer-Protein Conjugate Synthesis via Reversible Addition-Fragmentation Chain Transfer Polymerization

Christine Breiner (01/2003-01/2003)

Support: Axel H. E. Müller


Polymeric precursors for the synthesis of polymer-protein conjugates were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. These precursors include the stimuli-responsive polymers poly(N-isopropylacrylamide) and poly(acrylic acid) as well as amine-reactive polymers, such as poly(2-vinyl-4,4-dimethyl-5-oxazolone). Chain transfer agent structures had to be adjusted to the individual monomers and new transfer agents were synthesized for polymerization in a controlled manner.

The polymers obtained by RAFT polymerization represent macromolecular chain transfer agents that can be used to synthesize block copolymers by further monomer addition. Stimuli-responsive block copolymers consisting of poly(N-isopropylacrylamide) and poly(acrylic acid) or poly(2-vinyl-4,4-dimethyl-5-oxazolone) blocks, respectively, were synthesized by RAFT for the first time for use in protein/drug conjugation.

The presence of dithiocarbonyl endgroups in the polymers enabled their hydrolysis to sulfhydryl-terminated polymers. Sulfhydryl-terminated poly(N-isopropylacrylamide), PNIPAAm, and poly(N-isopropylacrylamide)-block-poly(acrylic acid), PNIPAAm-b-PAA, were used for conjugation to the protein streptavidin.

The RAFT polymerization of N-isopropylacrylamide with two different chain transfer agents, namely benzyl 1-pyrrolecarbodithioate and cumyl 1-pyrrolecarbodithioate, yielded polymers with narrow molecular weight distributions as well as Mn values that were in good agreement with the calculated ones. A comparison between the Mn values determined from gel permeation chromatography, GPC, and the values from MALDI-TOF mass spectrometry showed that the molecular weights obtained from GPC using polystyrene standards were considerably higher. A relation between log Mn,MALDI and log Mn,GPC was established, which permitted construction of a calibration curve for PNIPAAm polymers.

In-situ Fourier-transform near-infrared spectroscopy was applied for the reliable determination of monomer conversions and it indicated living characteristics. Both polymerization processes showed an induction period that seems to be correlated with a retardation in rate, where the induction time is higher for the cumyl chain transfer agent as compared to the benzyl chain transfer agent of the same concentration. The induction periods decrease with decreasing transfer agent concentration and were explained in terms of the different stabilities of the respective radicals that add to monomer in the reinitiation step. The more stable cumyl radical adds slower than the benzyl radical.

Both UV spectroscopy and MALDI-TOF mass spectrometry confirm the presence of the expected dithiocarbamate endgroups. MALDI-TOF characterization of the polymer samples showed the transfer agent endgroups together with some initiator-derived polymers. Endgroups that seemed to originate from disproportionation or transfer were the result of fragmentation under MALDI conditions as was shown by a post source decay analysis and MALDI-TOF characterization of the hydrolyzed polymer.

Poly(N-isopropylacrylamide), PNIPAAm, was further investigated in terms of its lower critical solution temperature, LCST, and it was shown that its cloud point increases with increasing molecular weight as the hydrophobic endgroups lower the LCST until it reaches 32 °C for high-molecular weight PNIPAAm.

Dithiocarbamate-terminated PNIPAAm obtained from RAFT polymerization with cumyl and benzyl chain transfer agent, respectively, was hydrolyzed under basic conditions in order to obtain sulfhydryl-terminated PNIPAAm for subsequent conjugation to model compounds and streptavidin. Formation of these endgroups was probed using several techniques, including MALDI-TOF analysis.

With amine-reactive diacetone acrylamide, 2-vinyl-4,4-dimethyl-5-oxazolone and N-hydroxysuccinimide methacrylate, new monomers were polymerized via RAFT in a controlled manner. Poly(diacetone acrylamide) and poly(2-vinyl-4,4-dimethyl-5-oxazolone) showed low polydispersities and good control over molecular weight, where poly(N-hydroxysuccinimide methacrylate) displayed relatively high polydispersities despite the controlled polymerization evident from the monomodal GPC traces. These amine-reactive polymers were subsequently used for successful conjugation to the primary amino group of the model peptide glycine-leucine.

The RAFT polymerization of tert-butyl acrylate and acrylic acid gave polymers with low polydispersities when using suitable chain transfer agents. Successful, direct polymerization of acrylic acid without protection group chemistry demonstrated the potential of this technique, tolerating virtually any monomer functionality.

For poly(N-isopropylacrylamide)-block-poly(acrylic acid), PNIPAAm-b-PAA, it was demonstrated that hydrogen bonding between N-isopropylacrylamide and acrylic acid units influences strongly its behavior in both the solid state and in solution. The block copolymers form micelles in aqueous solutions in dependence of pH and temperature. Cloud point measurements indicated the formation of larger aggregates at pH 4.5 and temperatures above LCST, whereas micelles formed at pH 5-7 and temperatures above LCST. At pH 5.6 and 50 °C, only micelles were found, whereas, at lower temperatures, larger aggregates and micelles coexist. Formation of larger aggregates by hydrogen bonding interactions was revealed by IR and Raman spectroscopy as well as by cryogenic transmission electron microscopy and dynamic light scattering.

Differential scanning calorimetry yielded glass transition temperatures of PNIPAAm-b-PAA that were well above the transition temperatures of the homopolymers, demonstrating molecular interactions between the acrylic acid and the N-isopropylacrylamide blocks.

For poly(N-isopropylacrylamide)-block-poly(2-vinyl-4,4-dimethyl-5-oxazolone), PNIPAAm-b-PVO, an increase of LCST with respect to the homopolymer poly(N-isopropylacrylamide) was found that was ascribed to the hydrophilic poly(2-vinyl-4,4-dimethyl-5-oxazolone) block. Differential scanning calorimetry showed complete mixing of the two blocks in the solid phase.

Conjugation of sulfhydryl-terminated PNIPAAm to thiol disulfide exchange reagents and maleimides was probed for later conjugation to proteins. Evaluation of the different cross-linking systems resulted in the choice of maleimides as cross-linkers for subsequent conjugation to the protein streptavidin.

Sulfhydryl-terminated PNIPAAm-b-PAA was conjugated to the streptavidin mutant S139C using a bismaleimide cross-linker and also direct conjugation via disulfide linkage. Both conjugations were successful and proceeded with more than 50 % conversion.

Conjugation of PNIPAAm and PNIPAAm-b-PAA was also achieved by non-covalent attachment of the biotinylated polymers to wild-type streptavidin. Conjugates of wild-type streptavidin with biotinylated PNIPAAm-b-PAA were found to remain dissolved at temperatures above LCST even at very low pH values, which was in contrast to the observed precipitation of the unconjugated block copolymer at pH ≤ 4.5. Conjugates of wild-type streptavidin with biotinylated PNIPAAm of different molecular weights formed aggregates in aqueous solutions above LCST and a dependence of aggregate size on the size of the polymer was found.

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