Smart PEGylation of Trypsin

Thermoresponsive oligo(ethylene glycol)-based copolymers were investigated for trypsin conjugation. These copolymers have been synthesized by atom transfer radical polymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) with oligo(ethylene glycol) methyl ether methacrylate (OEGMA475, Mn = 475 g·mol-1) at 60 °C in the presence of copper(I) chloride and 2,2'-bipyridyl. Two different ATRP initiators, containing succinimidyl ester moieties, were tested, namely, N-succinimidyl-2-bromopropionate and N-succinimidyl-2-bromoisobutyrate. In both cases, ATRP afforded well-defined polymers with a narrow molecular weight distribution and controlled chain-ends. However, the efficiency of initiation of the two initiators was lower than 1 and therefore the formed polymers exhibited a higher than expected mean degree of polymerization. Nevertheless, all types of polymers could be conjugated to trypsin. The conjugation reaction was performed in borax-HCl buffer. Sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. However, (co)polymers initiated by N-succinimidyl-2-bromopropionate led to the best conjugation results. The formed P(MEO2MA-co-OEGMA475)-trypsin conjugates were found to be thermoresponsive and moreover exhibited a higher enzymatic activity than unmodified trypsin

A "Click" strategy for tuning in situ the hydrophilic-hydrophobic balance of AB macrosurfactants

The self-organization of amphiphilic block copolymers in water strongly depends on their molecular structure, in particular on their hydrophilic-hydrophobic balance. Herein, a simple method for tuning the amphiphilicity of polymeric macrosurfactants in aqueous medium is proposed. This concept relies on the "click" ligation of an amphiphilic block copolymer (AB type) and a hydrophilic homopolymer (B type). In the present communication, the validity of this approach was examined with model polymers based on polystyrene (PS) and poly[oligo (ethylene glycol) acrylate] (POEGA) segments. A well-defined omega-azido functional diblock copolymer PS-b-POEGA and an alpha-alkyne functional homopolymer POEGA were prepared using atom transfer radical polymerization. These two polymers could be efficiently coupled to each other via copper-catalyzed azide-alkyne click chemistry in aqueous medium. Moreover, in this coupling strategy, an ester group was introduced at the junction between AB and B segments. This labile moiety may be "cut" by hydrolysis

PEGylated chromatography : efficient bioseparation on silica monoliths grafted with smart biocompatible polymers

Novel oligo(ethylene glycol)-based thermoresponsive stationary phases have been studied for the separation of bioanalytes Well-defined copolymers of (2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate were synthesized by atom-transfer radical polymerization in the presence of an N-succinimidyl-functionalized initiator. The reactive chain ends of these copolymers were then reacted with amino-functionalized silica monoliths. The formed composites were studied as chromatography materials. For instance, it was demonstrated that thermo responsive oligo(ethylene glycol)-based stationary phases allow rapid, efficient separation OF steroid and protein mixtures in pure water under isocratic high-performance liquid chromatographic elution

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

Conjugation-Induced Fluorescent Labeling of Proteins and Polymers Using Dithiomaleimides

Dithiomaleimides (DTMs) with alkyl substituents are shown to be a novel class of highly emissive fluorophores. Variable solubility and further functionalization can easily be tailored through the choice of N and S substituents. Inclusion of a DTM unit into a ROP/RAFT initiator or insertion into the disulfide bond of salmon calcitonin (sCT) demonstrates the utility for fluorescent labeling of polymers and proteins. Simultaneous PEGylation and fluorescent labeling of sCT is also demonstrated, using the DTM unit as both a linker and a fluorophore. It is anticipated that DTMs will offer an attractive alternative to commonly used bulky, planar fluorophores