The application of click chemistry in chemical ligation and peptide modification

Connecting multiple peptide fragments to form an extended peptide chain is referred to as ligation. Click chemistry offers a convenient method for creating peptide-peptide linkages. By introducing an alkyne group to one peptide fragment and an N-terminal azide moiety to another, a triazole linker, akin to an amide bond, can be formed to unite the two peptide units.

Likewise, the generation of multimeric peptides can be achieved through the incorporation of orthogonal side chain protecting groups like ivDde or Aloc (for modifying Lys side chains). This process involves subsequent selective deprotection, introduction of an alkyne function, and the subsequent click reaction with N-terminal azide peptides. Numerous instances of peptide ligation have been documented, including:

The preparation of a clickable RGD peptide involved the reaction of the Lys side chain with azido acetic acid. This resulted in a peptide capable of being linked to another peptide fragment.
Additionally, the synthesis of a cell-permeable peptide therapeutic was accomplished by employing click chemistry. The alkynyl-modified peptide drug, prepared using cost-effective propargylamine or 1-(2-nitrophenyl)propargyl alcohol, was conjugated with nona-arginine modified with an azide group.
The synthesis of heterodimers containing neurotensin (8-13) was achieved through the click chemistry reaction of an alkyne-functionalized neurotensin with the azide group of a phosphorylated hexapeptide that binds to Plk1-PBR. The resulting oligopeptides, incorporating triazole linkers, exhibited self-dimerization in a head-to-tail manner, mimicking the natural dimerization pattern of the native peptides.

1. 1.PEPTIDE CYCLIZATION

Various methods for macrocyclization are accessible to enhance the clinical effectiveness and bioavailability of peptides. The click reaction has been harnessed in diverse cyclization approaches, including on-resin cyclization of disulfide-containing peptides either before or after the removal of side-chain protecting groups. Additionally, innovative heterodetic cyclopeptides, featuring a triazole bridge, have been synthesized through intramolecular side chain-to-side chain click reactions. Copper(I)-and Ruthenium(II)-mediated click cyclizations of tripeptides have been employed to generate vancomycin-inspired mimics. On-resin cyclization of peptide ligands targeting the vascular endothelial growth factor (VEGF) receptor-1 has also been explored.

In numerous instances, the click-mediated macrocyclization reactions have led to the formation of significant quantities of macrocyclic heterodimers, presenting opportunities for the synthesis of intricate peptide structures that would otherwise be challenging to achieve. A novel stapling approach for 3(10)-helical peptides, utilizing CuAAC click reaction in a model aminoisobutyric acid (Aib)-rich peptide, yielded a more ideal 3(10)-helix compared to its acyclic precursor.

2. 2.BIOCONJUGATION

Bioconjugation is the process of attaching synthetic molecules to biological targets or linking biomolecules together. In recent years, click chemistry has had a profound impact on bioconjugation. Arginine-rich TAT peptides, modified with a clickable azido group, can be conjugated to oligonucleotides, cytotoxic drugs, kinase inhibitors, etc., enabling enhanced cell penetration for therapeutic purposes. The CuAAC reaction has emerged as a potent chemical method for creating mimetics of glycopeptides and glycoproteins (neoglycopeptides and neoglycoproteins) with well-defined homogeneous structures. Complex cyclopeptide-centered multivalent glycoclusters have been synthesized using the Cu-catalyzed click reaction. Self-assembling peptide fibers have the ability to incorporate multiple clickable peptides in a non-covalent, stoichiometric manner without disrupting their structure or stability. These fibers can be conjugated to biotin, followed by streptavidin-nanogold particles or rhodamine, and visualized through electron and light microscopy. This approach facilitates the development of multi-component functionalized systems. The click reaction is particularly valuable for conjugating fluorescent molecules to peptides and proteins under mild conditions, representing a crucial application in the emerging fields of cell biology and functional proteomics.

3. 3.PEPTIDOMIMETICS DESIGN, SYNTHESIS AND DRUG DISCOVERY

The 1,2,3-triazole function formed through click chemistry exhibits a physicochemical similarity to an amide bond, owing to its relative planarity and substantial dipole moment. This triazole linkage has found widespread application in the realm of peptidomimetics. Notably, the triazole unit demonstrates resistance to enzymatic degradation, hydrolysis, and oxidation, making it an appealing moiety for substituting more labile linkers in biologically active compounds. The click reaction has been employed as a conjugation strategy in crafting intricate biomimetic architectures, wherein the triazole linkage replaces, and in certain instances serves as a surrogate for, peptide and phosphodiester bonds. The substitution of a peptide bond with a triazole unit can lead to the formation of intriguing structures with distinctive conformational characteristics. Triazole units, generated through the click reaction, can function as helical components, β-turn units, and modifiers of cis/trans-prolyl ratios. Moreover, these triazole units can effectively replace a peptide segment in HIV-1 protease inhibitors. Modified peptides, incorporating a triazole ring in the peptide backbone or attached to the side chain of a residue, represent promising candidates for the design of novel antimicrobial agents.

4. 4.RADIOLABELING AND IMAGING

The Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) proves to be an excellent ligation reaction for radiolabeling sensitive biomolecules. Alkyne or azide derivatives of radioisotope-containing compounds can be employed to label biomolecules, including folic acid, peptides, proteins, and glycopeptides. For instance, an 11C isotope label was introduced by converting [11C]-CH3 I into [11C]-CH3 N3 through nucleophilic substitution. The resulting azide was then reacted with an alkyne-modified peptide. In the case of 18F labeling for PET imaging, azidomethyl-4-[18F]-fluorobenzene was clicked onto a modified peptide. It is crucial to note a significant limitation of CuAAC: Chelators such as DOTA can form a complex with the catalyst, making the conjugation with such compounds more challenging to achieve.

Fig 1.COPPER-FREE CLICK REACTION USING CYCLOOCTYNE-BASED SUBSTRATES

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