Site-directed polymer conjugation

Bontempo and Maynard first reported CRP directly from a protein. This technique, known as grafting from, involves the use of a protein macroinitiator for ATRP or macroCTA for RAFT polymerization. This strategy has several advantages. Purification is easier because only small molecules rather than polymer chains need to be removed. In addition, characterization can be simpler because the site of polymer attachment is predetermined by the attachment of the initiator or CTA fragment, which is easier to analyze. Finally, hydrophobic polymer conjugates are more readily prepared.However, some desired monomers are insoluble in the aqueous systems... 

Besides short ELPS, longer ELPs have also been conjugated to synthetic polymers. In one approach, Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry was applied. For this purpose, ELPs were functionalized with azides or alkynes via incorporation of azidohomoalanine and homopropargyl glycine, respectively, using residue-specific replacement of methionine in ELP via bacterial expression [133]. More recently, an alternative way to site-selectively introduce azides into ELPs was developed. Here, an aqueous diazotransfer reaction was performed directly onto ELP[V5L2G3-90] using imidazole-1-sulfonyl azide [134]. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

A special group of carrier-linked prodrugs are the site-specific chemical delivery systems [23], Macromolecular prodrugs are synthetic conjugates of drugs covalently bound (either directly or via a spacer) to proteins, polypeptides, polysaccharides, and other biodegradable polymers [24],... 

Fig. 10 Schematic representation of the two approaches mainly used in EzILAs. (a) The enzyme conjugate and the analyte compete for the selective binding sites of the polymer finally, a substrate is converted into a product that generates a chemical signal (e.g., fluorescence, absorbance, electrochemical) at a rate which is proportional to the amount of bound enzyme and hence to the concentration of analyte in the sample, (b) Direct assay where the analyte is the enzyme which is quantified by a coupled enzymatic reaction...

A critical criterion for emissive polymers for LEDs is their stability, which determines the device lifetime. The major problem is the susceptibility of extended conjugated systems to attack by oxygen and/or water, which cannot be totally excluded even by the best device encapsulation techniques [130], In PAVs, the most vulnerable sites are the vinylene moieties. These could be protected by direct attachment of electron-withdrawing groups to them, but this is not always possible and may have undesirable effects upon the emission color. Generally speaking the use of less electron-rich arylene units seems to be the best approach so that the aryl-substituted PPV 53 is stable enough to be used commercially in LEDs. This material is also one of the most defect-free PPVs known, which also... 

The simplest way to prepare a site-selective protein-reactive polymer via ATRP is through the use of a functionalized initiator. After the polymer is formed, it is employed either directly in the conjugation or after a simple deprotection step. A number of protein-reactive initiators for ATRP have been reported. ... 

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