Application of azides in chemoselective amidation reactions

Abstract

Chemical methods that enable the synthesis and the site-selective modification of biomolecules offer great possibilities for studying their biological function and have found widespread use in chemical biology. Most often these methods employ chemoselective ligation reactions which feature mutually and uniquely reactive functional groups to enable the covalent coupling of unprotected biomolecules without interference of any other functional groups either present in the biomolecule to be coupled or in the environment in which the ligation should take place. Today, the repertoire of chemoselective ligation reactions is recognized as a valuable tool for the synthesis of complex bioconjugates and in particular larger peptides and proteins. The most sophisticated methods (Native Chemical Ligation/Expressed Protein Ligation) facilitate the aqueous coupling of unprotected peptide segments via a native amide bond. A restriction of these methods, however, is the need for a cysteine (mimic) at the site of ligation. To date, a general applicable method for the chemical synthesis of (poly)peptides featuring a chemoselective amidation reaction that is independent of the amino acid residue at the ligation site, is not yet available and would be highly desirable. On the other hand, chemoselective ligation reactions can also be used for the imaging of biomolecules, for example, via a two-step bioorthogonal chemical reporter strategy. In this context, the azide moiety is the most versatile bioorthogonal chemical reporter group available for introduction into biomolecules and subsequent conjugation to a suitably functionalized biophysical tag; for instance, via the copper(I)-catalyzed 1,3-dipolar cycloaddition (the paradigm of “click”-chemistry). However, the toxicity of the copper catalyst may involve serious drawbacks for biological applications. The high complexity of biomolecules combined with the wide range of applications, generates a continuing demand for the development of new azide-based chemoselective ligation reactions as a tool for bioconjugation. In this thesis, a “traceless” version of the so-called Staudinger ligation and the reaction of thio acids with azides were investigated for application as novel chemoselective amide ligation method for the chemical synthesis of peptides. It was found, however, that the chemoselective coupling of peptide segments by the Staudinger ligation method as well as the thio acid/azide amidation reaction, suffered from sluggish reaction kinetics giving rise to low yields. Conversely, the reaction between (peptide derived) thio acids and sulfonyl azides is highly efficient (also in aqueous solvents), chemoselective, it does not require the addition of any catalyst, and non-toxic nitrogen and sulfur are formed as the only side products. Additionally, the resulting N-acyl sulfonamide linkage is highly stable and non-immunogenic and renders special properties to the coupling product since it functions as a carboxylic acid isostere. The special properties of the thio acid/sulfonyl azide amidation resemble those of the “ideal click reaction” and have been applied as efficient loading and activation strategy for the N-acyl sulfonamide linker, to obtain peptide-based organo/hydrogelators and synthetic approaches toward bioconjugations, peptide dendrimers and prolyl dipeptide derived organocatalysts. From these examples it can be concluded that the thio acid/sulfonyl azide amidation reaction is a suitable method for the synthesis of peptide conjugates.