Mathew W. Jones

Polymeric Dibromomaleimides As Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation Agents

A series of dibromomaleimides have been shown to be very efficacious at insertion into peptidic disulfide bonds. This conjugation proceeds with a stoichiometric balance of reagents in buffered solutions in less than 15 min to give discrete products while maintaining the disulfide bridge and thus peptide conformation. The insertion is initiated by disulfide reduction using a water-soluble phosphine, tris(2-carboxyethyl)phosphine (TCEP) which allows for subsequent substitution of the two maleimide bromides by the generated thiols. Reaction of salmon calcitonin (sCT) with 2,3-dibromomaleimide (1.1 excess) in the presence of TCEP (1.1 equiv) in aqueous solution at pH 6.2 gives complete production of a single conjugate which requires no workup. A linear methoxy poly(ethylene glycol) (PEG) was functionalized via a Mitsunobu reaction and used for the successful site-specific and rapid pegylation of sCT. This reaction occurs in 15 min with a small stoichiometry excess of the pegylating agent to give insertion at the disulfide with HPLC showing a single product and MALDI-ToF confirming conjugation. Attempts to use the group in a functional ATRP polymerization initiator led to polymerization inhibition. Thus, in order to prepare a range of functional polymers an indirect route was chosen via both azide and aniline functional initiators which were converted to 2,3-dibromomaleimides via appropriate reactions. For example, the azide functional polymer was reacted via a Huisgen CuAAC click reaction to an alkyne functional 2,3-dibromomaleimide. This new reagent allowed for the synthesis of conjugates of sCT with comb polymers derived from PEG methacrylic monomers which in addition gave appropriate cloud points. This reaction represents a highly efficient polymer conjugation method which circumvents problems of purification which normally arise from having to use large excesses of the conjugate. In addition, the tertiary structure of the peptide is efficiently maintained.

Tunable thermo-responsive polymer–protein conjugates via a combination of nucleophilic thiol–ene “click” and SET-LRP

Herein we report the synthesis of a protein macroinitiator in a one-pot strategy using phosphine-mediated thiol–ene “click”, the macroinitiator was used to polymerise ethylene glycol containing monomers to yield polymer–protein conjugates with tunable thermoresponsive behaviour.

Synthesis and modification of thermoresponsive poly(oligo(ethylene glycol) methacrylate) via catalytic chain transfer polymerization and thiol–ene Michael addition

Various poly(oligo(ethylene glycol) methyl ether methacrylate)s (POEGMEMAs) have been prepared by Catalytic Chain Transfer Polymerization (CCTP) using a range of OEGMEMA monomers (molecular weight from 180 to 1100 g mol−1). The chain transfer constants of bis(boron difluorodimethylglyoximate) cobalt(II) (CoBF) were determined and are reported for each monomer. The copolymerization of POEGMEMA (Mn = 475 g mol−1) with diethylene glycol methyl ether methacrylate (DEGMEMA) yielded thermoresponsive polymers. The lower critical solution temperatures (LCSTs) of the polymer chains can be tuned by the copolymer composition over the range 30 °C to 95 °C. In addition, the presence of the vinylic end-group, characteristic of CCT polymerization, provided further scope for post-synthetic modification via thiol–ene click chemistry, through nucleophilic Michael addition with various functional thiol compounds such as 2-mercaptoethanol, 3-mercaptopropionic acid, benzyl mercaptan and 1-dodecanethiol. The thiol–ene reaction was rigorously tested, optimized and characterized in this study in terms of solvents and most importantly the choice of the catalyst: dimethyl phenyl phosphine, tertiary amine or hexylamine. The optimum conditions reported allow near-quantitative functionalization of these macromonomers without significant side reactions. The effect of the end-group on the LCST has also been investigated, as well as thermal stability temperature of the copolymers.

Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts

Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditions-pH, stoichiometry, and chemical structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG(2000) to sCT did not affect the peptides ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca(2+)] plasma levels by mPEG(2000)-sCT conjugate in rat animal models.

Probing Bacterial-Toxin Inhibition with Synthetic Glycopolymers Prepared by Tandem Post-Polymerization Modification: Role of Linker Length and Carbohydrate Density†

Probing the depths: A tandem post-polymerization modification strategy was used to systematically probe the multivalent inhibition of a bacterial toxin as a function of linker length (see scheme), carbohydrate density, and glycopolymer chain length. Guided by structural-biology information, the binding-pocket depth of the toxin was probed and used as a means to specifically improve inhibition of the toxin by the glycopolymer.

Thermoresponsive behaviour of poly[(oligo(ethyleneglycol methacrylate)]s and their protein conjugates: importance of concentration and solvent system

Thermoresponsive poly[oligo(ethyleneglycol) methacrylate]s with a variety of different oligo(ethyleneglycol) graft lengths were synthesised by reversible-addition fragmentation chain transfer (RAFT) polymerisation. The lower critical solution temperature (LCST) behaviour of these polymers was evaluated as a function of the polymer concentration and the concentration of dissolved solutes, in order to understand their applicability for in vitro and in vivo applications. It was observed that in the relevant dilute (<1 mg mL−1) concentration range the observed LCSTs increased by approximately 6 °C compared to higher concentrations. This was confirmed by complimentary dynamic light scattering and differential scanning calorimetry measurements. The impact of biological solutions on the LCST was determined using bovine blood plasma, which resulted in observed LCSTs lower than what is found in traditional buffer or pure aqueous solutions. Finally, a well-defined polymer–protein conjugate was synthesised by ‘grafting from’ using single-electron transfer (SET) polymerisation. This model polymer–protein therapeutic also displayed similar concentration dependant behaviour, highlighting the importance of testing novel ‘smart’ materials and conjugates at both relevant concentration ranges and in appropriate solvent systems in order to use them in biotechnological applications.

Glycopolymers with secondary binding motifs mimic glycan branching and display bacterial lectin selectivity in addition to affinity

The application of synthetic glycopolymers to anti-adhesive therapies has so far been limited by their lack of lectin specificity. Here we employ a macromolecular engineering approach to mimic glycan architecture. A new, 3-step tandem post-polymerisation methodology was developed which afforded precise control over both chain length and carbohydrate (galactose)-polymer backbone linker distance. This route also allowed a secondary binding (branched) motif to be introduced onto the linker, increasing specificity and affinity towards bacterial toxins without the need for extensive carbohydrate or organic chemistry. Sequential variation of this motif was found to dramatically alter both the affinity and the specificity of the glycopolymers towards two lectins, CTx and PNA, by up to 20-fold either via direct binding, or increased steric constraints. Using this method, a glycopolymer that showed increased specificity towards CTx was identified.

Terminal functional glycopolymers via a combination of catalytic chain transfer polymerisation (CCTP) followed by three consecutive click reactions

The combination of catalytic chain transfer polymerisation (CCTP) with both thiol-ene and copper catalysed alkyne azide coupling (CuAAC) click chemistry has been employed to give a new route to functional glycopolymers for applications in bioconjugation and biological targeting. Ring opening of poly(glycidyl methacrylate) with sodium azide and subsequent reaction with alkyne functional carbohydrates, as prepared by Fischer glycosylation, has been exploited. This combination of a range of efficient chemistry gives a route to multi gram quantities of glycopolymers avoiding the need for living radical polymerisation chemistry.

A facile route to end-functionalised polymers synthesised by SET-LRP via a one-pot reduction/thiol-ene Michael-type addition

We report the facile synthesis of well defined, disulfide containing polymers via SET-LRP. A one-pot reduction/conjugation reaction enables post polymerisation modification with functional (meth)acrylates and acrylamides.

Poly(azlactone)s: versatile scaffolds for tandem post-polymerisation modification and glycopolymer synthesis

The synthesis of polymers with latent reactivity suitable for ‘click’ type modifications in a tandem post-polymerisation modification process starting with poly(azlactone) precursors is investigated. Poly(azlactones), obtained by copper(I) mediated radical polymerisation, were functionalised in a one-pot process with amines bearing functional groups which are incompatible with controlled radical polymerisation: alkynes, alkenes, furfuryl and phenol. The reaction is quantitative and 100% atom efficient presenting an efficient route to clickable scaffolds without the need for protecting group chemistry. Additionally, the poly(azlactones) were exploited to obtain synthetic glycopolymers. The ring opening procedure introduces a 5-atom spacer between glycan and backbone, which provides improved access to carbohydrate-binding proteins with deep binding pockets, such as the cholera toxin, for anti-adhesion applications.