PPEylation of proteins: Synthesis, activity, and stability of myoglobin-polyphosphoester conjugates

Abstract

Protein-polymer conjugates are used to treat several diseases. PEGylation, i.e. the modification with poly(ethylene glycol) (PEG) is the currently used strategy. However, due to its non-biodegradability, the design of effective and degradable conjugates is of both academic and industry potential. We present the preparation and studies of the activity and stability of novel biodegradable myoglobin-polyphosphoester conjugates. Poly(ethyl ethylene phosphate) (PEEP) is a water-soluble polyphosphoester, which had been reported to be biocompatible and biodegradable. PEEP is a promising candidate as a degradable substitute for the "gold standard" PEG, which can cause long-term effects, as it is not degradable. PEEPylated conjugates with a variable degree of polymer grafting were synthesized, characterized (with online triple detection size exclusion chromatography, mass spectrometry, and gel electrophoresis), and compared with PEGylated analogs. We highlight differences in how the structure, the number, and the length of the polymer influence the properties of the conjugates. Overall, the analyses conducted (including activity assay, calorimetry, and fluorimetry measurements) show that the covalent attachment of the polymer does not irrevocably affect the protein's features under physiological conditions, suggesting the potential of this new class of polymers for the design of a new generation of fully degradable conjugates.

Thermodynamic stability of myoglobin-poly(ethylene glycol) bioconjugates: A calorimetric study

“…The comparison between the spectra of myoglobin, My-PEG (1) and My-PEG (2) recorded at room temperature showed only slight differences in the Soret band intensity with respect the reference. These slight differences, that regards a local effect on the heme group, were the same in the two conjugates and seemed not to compromise the functionality of the protein at room temperature according to previous results of activity assays that showed a high preservation of the protein activity [27].…”

“…The synthesis and characterization of the PEGylated samples My-PEG (1) and My-PEG (2) was performed following a procedure reported in detail in the literature for analogue conjugates [27]. Suffice here to say that for the two bioconjugates we obtained purity = 99 %, dispersity Đ = 1.051, number-average molar mass M n = 47.490 and average number of attached chains = 3.3 for My-PEG (1), and purity = 97.5 %, dispersity Đ= 1.008, number-average molar mass M n = 63.970 and average number of attached chains = 5.2 for My-PEG (2).…”

“…We may conclude that the destabilizing effect of the bioconjugation on this protein seems to follow the extent of the covalent modification, but do not alter the mechanism of thermal denaturation that remains a single step mechanism. Furthermore, taking into account the unaltered functionality of the proteins at room temperature [27], we may argue that the reduced thermodynamic stability of bioconjugated myoglobin, is mainly due to the altered solvent-surface interactions that maybe affect more the denatured state of the protein, that seems do not reach a complete random coil state because of the boundaries dictated by the bioconjugation, rather than to peculiar reorganizations of the native protein tertiary structure, that remains functional at room temperature. This scenario is further supported by the reversibility enhancement in the case of both bioconjugate systems.…”

In Vitro and In Vivo Studies of Biodegradability and Biocompatibility of Poly(εCL)-b-Poly(EtOEP)-Based Films

“…Ethylene phosphates, phosphonates and phosphoramidates with short alkyl substituents ( Scheme 1 a) represent promising cyclic substrates for the synthesis of copolymers with hydrophilic biodegradable blocks using catalytic ring-opening polymerization (ROP, Scheme 1 b) [ 5 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. 2-Ethoxy-1,3,2-dioxaphospholane 2-oxide (ethyl ethylene phosphate, EtOEP) is one of the most reliable monomers for the preparation of hydrophilic polyphosphoesters (PPEs) due to high reactivity and low tendency to form branched polymers [ 15 , 16 , 17 ]. Block copolymers poly(εCL)- b -poly(EtOEP) were synthesized previously using tin (II) octanoate as a catalyst [ 15 ].…”

Thermally Assisted Generation of Protein–Poly(ethylene sodium phosphate) Conjugates with High Mineral Affinity

“…7,11 As an alternative, Wurm and co-workers have proposed degradable protein–polyphosphoester (PPE) conjugates with a relatively high bioactivity retention to address such PEG shortcomings. 12,13 Nonetheless, protein–polymer conjugations require multistep preparations and purification. Noncovalent binding is an interesting alternative formation route involving electrostatic forces, hydrogen bondings, and hydrophobic attractions.…”