Focus on Thiol Reactivity

This last part of the introduction will deal with strategies that can be used with thiol groups of cysteine residues present in the protein of interest. Indeed, cysteines represent a convenient target for selective modification due to the strong nucleophilicity of the thiol sidechain (pK_a ≈ 8.2).^[187] Therefore, one of the most common strategies to introduce modifications is through cysteine alkylation with suitable electrophiles. However, the conditions need to be carefully applied in order to avoid the alkylation of other nucleophilic residues, such as lysines or hystidines.^[188]

Iodoacetamide 23 was the first electrophile used for the direct alkylation of cysteines, like the one of derivative 114, in 1935 (Figure 47).^[189] Since then, it has been mainly used in proteomic studies to label cysteines in cells and cellular lysates.[190, 191] In the field of protein-polymer conjugation, however, Michael acceptors, such as maleimide 47 and vinyl sulfone 115, are more often used for cysteine alkylation (Figure 47).^[192-196] These reactions are convenient since they occur in physiological conditions and the obtained products, 116-118, are stable over time.^[187] A major drawback is, however, represented by the possible reaction with amines of lysine residues.^[74]

Figure 47. Common reagents used for cysteine alkylation: iodoacetamide 23, maleimide 47 and vinyl sulfone derivative 115, to give, after reaction with general cysteine 114, conjugates 116-118, respectively. Figure adapted from reference.^[187]

A more chemoselective ligation involves the oxidation of thiols to disulfides.

Disulfides are known to be essential in protein stabilization, and indeed they naturally occur in their tertiary structure as well as are part of common post-translational modifications.^[197] They can be formed by oxidation of the two thiol groups,^[198] which can, however, damage the protein, or by disulfide exchange between the thiol and the disulfide,^[199] or between two disulfides.^[200] These latter cases call for activated disulfides which possess a good sulfur-containing leaving group, like in reagent 119 (Figure 48). This disulfide, 5,5-dithiobis(2-nitrobenzoate), is also known as DTNB or Ellman’s reagent since it was developed by Ellman in 1958,^[201] to quantify the thiol groups present in different human tissues. Since then, it has been widely used to measure protein thiol

content,^[187] as well as to form disulfide linkages to stabilize polymeric capsules for DNA delivery.^[202-203]

Caruso and coworkers have introduced DTNB 119 on thiol-containing polymer 120 to obtain disulfide 121 used for the conjugation to antibodies (Figure 48).^[202] Another commonly used activated derivative is thiopyridyl disulfide, such as the one contained in polymer 122 (Figure 48). Indeed, the disulfide in 122 was installed at the initiator level and, after polymerization, conjugated to BSA protein 99 which presents a free cysteine residue.^[204] The successful conjugation to afford 123 was monitored by detecting the absorption of the released sideproduct 124, as well as by detecting the increased size through gel electrophoresis.^[204]

Figure 48. Chemical ligation using disulfides. Disulfide exchange between a) DTNB 119 and polymer 120 to give 121 and b) polymer 122 and BSA protein 99 to give conjugate 123 while releasing 124. Figure adapted from reference.^[202,

204]

Another type of ligation that can be applied to cysteine-containing proteins is thiol-ene chemistry.^[159] Thioether linkages are formed between thiols and alkenes via either a radical-based mechanism or catalysed Michael additions.^[168,

205] The generation of thiyl radical species using initiators, light or heat, leads to addition to the alkene with the generation of a second radical. The reaction can propagate easily and, for this reason, is commonly used as a polymerization method.^[74] However, the generation of free thiyl radicals at the cysteine level of proteins can lead to unwanted reactions with other amino acid sidechains.

Moreover, the use of prolonged UV-irradiation or heating can lead to damage of the protein structure or loss of the biological function.^[168]

For these reasons, in the area of protein-polymer conjugation, the use of a radical-free conjugation strategy is more desirable. An interesting approach has been proposed by Haddleton and coworkers in 2009,^[206] where the water soluble commercially available tris(2-carboxyethyl) phospine (TCEP) 125 is used to first reduce the disulfide-bridge in protein 126 and then to catalyse the Michael addition between the free thiols of 127 and an alkene-containing PEG 128 (Figure 49). Using this approach, the bioactivity of the final doubly PEGylated protein 129 was retained.^[206] Although the thiol-ene reaction is considered part of the so-called “click” chemistries, sideproducts, such as dimers and disulfides are often observed.^[207]

Figure 49. Bifunctional use of TCEP 125 to first reduce the disulfide bridge of 126 to the thiolated 127, and then catalyse the thiol-ene reaction with alkene 128 to obtain doubly-substituted 129. Figure adapted from reference.^[206]

Finally, the reactions described in this chapter can be used to introduce the bioorthogonal handles discussed earlier using the less abundant thiols of cysteine residues of the protein sequence compared to other functional groups, such as the amines of lysines.^[159]

In conclusion, the conjugation of polymers to proteins can be advantageous to either enhance the properties of the protein, such as stability and serum half-life,[156-159, 169, 181, 184] or to enable its cell entry, for example using the attachment to the CPDs.[56, 58, 186]

Different strategies can be adopted to obtain protein-polymer conjugates according to the sequence of the protein, the aim of the conjugation, the approach used for polymer functionalization, etc. Only a few types of ligations, considered relevant for the understanding of this work, were described in this chapter.

However, many more examples can be found in the literature where efforts are made to improve present approaches as well as developing new ones.^{[158, 169]}

126

Chapter 2

OBJECTIVES

The objective of this thesis is to improve specific properties of the CPDs developed in the group. In particular, overcome certain drawbacks, which are characteristic of the best-performing polymers in hand, and develop strategies to incorporate new functionalities at both the sidechain and the terminator level.

The first goal is centered on establishing an efficient method for the sidechain functionalization of the CPDs. To achieve this, we envisioned the introduction of a dynamic covalent bond orthogonal to disulfides using a model fluorescent substrate to monitor the reaction. In case the characteristics of bioorthogonality, high yields and versatility that we are looking for cannot be fulfilled, efforts will then be oriented towards the use of a covalent approach.

The sidechain modification strategy developed in the first part will then be applied to introduce different functionalities on the CPDs bearing a comparable scaffold. The aim is to engage the new poly(disulfide)s in cellular uptake experiments to discern between the two mechanisms of entry of the CPDs, that is counterion- and thiol-mediated. In particular, the goal is to understand which pathway contributes the most to achieve efficient cell delivery.

In the second part, sidechain modification will be used to overcome a major limitation of the CPDs, i.e. to improve the solubility of the polymers in conditions of high concentration. Different solubilizing groups will be introduced, such as PEG chains or carbohydrates. The focus will be also oriented towards the carbohydrate-containing polymers with regard to their mechanism of entry. Indeed, the introduction of monosaccharide units will have the dual role

If indeed a new multifunctional uptake is established, then it will be applied for the delivery of larger cargos like proteins.

The aim of the last part is to modify the poly(disulfide)s at the terminator level. In order to achieve this, new reagents will be screened with the goal of increasing the incorporation yields of the terminator.

Finally, strategies will be investigated for the delivery of substrates bearing a cysteine residue. The goal is to develop a fast, selective and efficient method to incorporate new functionalities at the terminator level that can be applied to biomolecules, such as proteins.

Chapter 3