A specific bioconjugation of protein lysine residues and N-terminal via phthalimidine formation and a fluorogenic probe for 5-hydroxylysine

Abstract

Chemical protein modification has been used to monitor cellular events and to modify proteins. These modifications are achieved by bioconjugation, which covalently attach a chemical molecule onto the protein. These protein modifications include PEGylation, antibody-drug conjugation, fluorescent labeling, etc. Despite the vast range of applications of chemical protein modifications, scientists are facing many challenges in controlling the reactivity and selectivity of the modification. Native protein modification requires a chemoselective reaction under the physiological condition that preferentially reacts at one specific amino acid over the others in order to control the degree of modifications. Lysine residues are highly solvent accessed, with a good nucleophilic side chain functionality for chemical modification. In this thesis, a new chemical strategy using ortho-phthaldialdehyde (OPA), which is highly selective, stable, easy to manipulate, and has high reactivity towards lysine is reported.

The reaction of OPA and amines in organic solvents forming phthalimidines is well documented. However, such a reaction under the physiological condition with which to label and modify native proteins has not been explored previously. With a peptide model, we observed rapid lysine modification with OPA via phthalimidine formation in physiological buffers such as PBS, tricine, HEPES and as well as basic borate buffer. To demonstrate the vast applications, different functionalized OPA derivatives were chemically synthesized, which included different sizes of PEGs, alkyne and carboxylic acid group. These derivatives were tested with model peptides.

In the presence of other nucleophilic amino acid residues, such as serine, threonine, tyrosine and histidine, OPA selectively modified lysine, while classical NHS esters showed non-specific modifications. OPA also showed better modification efficiency, with a higher second order rate constant (7.49 M^(-1) s^(-1)) compared to that of NHS (1.27 M^(-1) s^(-1)-).

Proteins including cytochrome c, lysozyme, ribonuclease A, and myoglobin were successfully modified with OPA derivatives. Combining with an alkyne group, a fully functional heterobifunctional linker was synthesized and applied to bovine serum albumin (BSA). Protein immobilization on solid support was also demonstrated. PEGylation of L-asparaginase using OPA protocol showed increased stability against proteolytic degradation while maintaining high degree of activity.

In the next section, we turned our attention to the detection of 5-hydroxylysines. 5-hydroxylysines is naturally synthesized through a posttranslational modification of lysine. Although hydroxylysines are most widely known as a component of collagen, they also exist in “noncollagen” proteins such as mannose binding proteins, type I and II macrophage scavenger receptors. It is very likely that lysyl-5-hydroxylation as post-translational modification frequently happens in many other proteins. Thus, the biological significance of lysyl-5-hydroxylation requires extensive studies. Recently, our group has developed a new ligation strategy, named serine/threonine ligation (STL), which uses an N-terminal serine or threonine to mediate a chemoselective peptide ligation. 5,6-hydroxylamino group of 5- hydroxylysine has structural similarity with N-terminal serine and threonine. Thus, we designed and synthesized an umbelliferone based-fluorogenic probe, which could react with 5-hydroxylysine containing peptide through STL chemistry, generating a fluorescent product. The probe showed very high selectivity and could detect as low as 0.01 mM of hydroxylysine containing peptides in the system.