Globular Polymer Grafts Require a Critical Size for Efficient Molecular Sieving of Enzyme Substrates

Abstract

As eries of 2,2-bis(hydroxymethyl)propionic acid dendrons of generation 2t hrough 8h aving as trained cyclooctyne at the core and hydroxy groups at the periphery were prepared by ad ivergent method and used to functionalize azide-decorated a-chymotrypsin. The ability of the appended dendrons to selectively blocke nzyme activity (through am olecular sieving effect) was investigated using as mall molecule substrate (benzoyl-l-tyrosine p-nitroanilide), as well as two proteins of different size( casein and bovine serum albumin). Additionally,t he ability of dendrons to block complexation with ac hymotrypsin antagonist, a-antichymotrypsin, was investigated, and it was found that the dendron coating effectively prevented inhibition by this antagonist. We found that ac ritical generation is required to achieve efficient sieving with bis-MPAd endrons,w hich illustrates the importance of macromolecular architecture and sizeinthe shielding of proteins.

New Perspectives for Developing Therapeutic Bioconjugates of Metabolite-Depleting Enzymes: Lessons Learned Combating Glutamate Excitotoxicity

“…For instance, libraries of alpha-chymotrypsin and l -asparaginase modified with poly­(oligo­(ethylene glycol) methyl ether methacrylate) have shown that strong rejection of large molecules could be achieved with low loss of activity toward small molecule substrates. Other studies have shown similar results for proteins modified with dendronized polymers or dendrimers, respectively. , In fact, the work with dendrimers suggests that entanglement of adjacent polymer chains also plays a role on the effective pore size of the coatings, while other studies have elucidated a kinetic component to the phenomenon . Overall, this type of work provides motivation for exploring new polymer designs that minimally affect the activity of metabolite-depleting enzymes.…”

“…Several groups including ours have explored ways to mitigate loss of activity caused by polymer conjugation. While not specifically examined for glutamate-depleting enzymes, our group as well as those of Adronov and Russell, have shown than coatings made of branched, comb-shaped, and dendronized polymers are more easily permeated by small molecules than those made of linear mPEG. − Hence, enhanced circulation time and stability could be provided to metabolite-depleting enzymes such as l -asparaginase, while maintaining higher levels of activity. While the underlying mechanism explaining the semipermeability characteristics of these coatings has never been fully demonstrated, much evidence suggests a steric component to the phenomenon.…”

“…Other studies have shown similar results for proteins modified with dendronized polymers or dendrimers, respectively. 58,61 In fact, the work with dendrimers suggests that entanglement of adjacent polymer chains also plays a role on the effective pore size of the coatings, while other studies have elucidated a kinetic component to the phenomenon. 59 Overall, this type of work provides motivation for exploring new polymer designs that minimally affect the activity of metabolite-depleting enzymes.…”

Molecular Dynamics-Guided Design of a Functional Protein–ATRP Conjugate That Eliminates Protein–Protein Interactions

“…It is worth noting that the R g for aprotinin is only 1.1 nm. While it has been demonstrated that increasing polymer grafting density or attachment of several generation dendrimers can reduce permeation of smaller molecules (casein) and hinder permeation of larger molecules (BSA), 14,39 the data provided herein provide the first evidence, to our knowledge, that rationally designed nanoarmored protein−polymer conjugates can prevent protein−protein interactions while preserving biological activity. We next investigated the impact of protein−ATRP molecular sieves on the inhibition of CT by BBI (which was shown to be a competitive inhibitor) and α 1 anti-CT. Again, the dense comb-shaped polymers eliminated inhibition at 10fold molar excesses, and we had to use a 100-fold molar excess to observe just a 20% reduction in activity (Figure 4c and d).…”

Enzyme‐Polymer Conjugates for Tuning, Enhancing, and Expanding Biocatalytic Activity

“…Only substrates smaller than the cut‐off size set by the polymer conformation will be transformed [19] . Moreover, the distribution of the polymers and therefore, the size of the gap can be computationally simulated [47,48] . The bulkiness of the polymers can also be harnessed to control the degradation of enzymes on demand.…”