BioPerspectives

Jul 9, 2012

Click Chemistry for Peptide Synthesis

Synthesis and chemical modification of peptides are becoming popular with click chemistry techniques.

  • Click chemistry is a term introduced by Professors Valery V. Fokin and Nobel laureate K.B. Sharpless to describe chemistry tailored to generate substances quickly and reliably by joining small units together similar to the modular strategy adopted by Mother Nature. The term “click chemistry” implies that the reactions are highly efficient, wide in scope, product isolation is easy, stereospecific, simple to perform using inexpensive reagents, and can be conducted in benign solvents such as water. The copper-catalyzed variant of Huisgen azide-alkyne cycloaddition (CuAAC) fits the concept well and is one of the most popular prototype click reactions to date. Click chemistry is finding a number of applications in the areas of drug discovery, bioconjugation, and material science. It has been successfully utilized in the synthesis of peptides, particularly in peptide cyclization and modifications.

  • Chemistry

    CuAAC click reaction between an azide and alkyne takes place in presence of a Cu (I) catalyst under mild conditions, resulting in the formation of a triazole link connecting the two molecules. In peptide chemistry the increasing popularity of the CuAAC is largely a result of the unique properties of both azides and the resulting triazoles. Azide groups are easy to introduce, stable to water and oxidative conditions, and orthogonal to many functional groups in peptide synthesis. For applications in vitro and in vivo, azides are virtually absent from any naturally occurring species (bioorthogonal). Interestingly, the triazole moiety formed by click reaction has unique similarity to an amide bond. The relative planarity, strong dipole moments, and hydrogen bonding ability of triazole linkage make it as attractive as an amide bond with the added advantage that it is less prone to hydrolytic cleavage.

    Click chemistry provides a number of avenues for peptide synthesis and modifications and could be combined with other techniques to make complex structures and multicomponent functionalized systems with ease. For example, peptides can be converted postsynthetically to an azido derivative which can be clicked with an appropriate substrate containing a clickable alkynyl group or vice versa. Peptides can also be made by inter- and intramolecular click reactions using azide or alkyne containing amino acids or building blocks during peptide synthesis. Building blocks containing clickable moieties are instrumental in constructing side-chain modified peptides, interside-chain peptide chimera, peptide small molecule conjugates, and cyclic peptides. Solid phase resins modified with clickable groups can also be used for making clickable/modified peptides. Click chemistry is compatible with various protected amino acid side chains used in peptide synthesis.

    A number of reagents and building blocks can be utilized for peptide click chemistry. These include azido-amino acids (e.g. Fmoc- protected for solid phase synthesis), propargyl amino acids, PEG azide and alkynes (Maleimide-PEG3-Azide and acetylene PEG maleimide for pegylation), alkyne and azide containing chemical modification reagents (propargyl amine, 1-(2-nitrophenyl) propargyl alcohol, succinidyl-hex-5-ynoate, succinimidyl-4-azovalerate, pentynoic acid, 2-azido-3-methyl propanoic acid) and Diazo transfer reagents (imidazole-1-sulfonyl azide).

  • Synthesis of Cyclic Peptides

    A variety of macrocyclization methods are available to increase the clinical efficacy and bioavailability of peptide drugs. Cyclization stabilizes the peptide molecule by locking its conformation, thus increasing potency and in vivo half-life. Introduction of azide and alkyne moieties into structurally diverse peptide side chains, combined with on-resin macrocyclization conditions, is used to design structurally constrained peptides.

    Click reaction has been exploited in a number of different peptide cyclization reactions such as the cyclization of a disulfide-containing peptide on the resin with or without protecting groups on; the preparation of novel heterodetic cyclopeptides by an intramolecular side chain-to-side chain click reaction, forming a 1,4-disubstituted [1,2,3] triazolyl-containing bridge; cyclization of tripeptides for making vancomycin-inspired mimics; on-resin cyclization of peptide ligands of the vascular endothelial growth factor receptor 1, etc. Formation of macrocyclic heterodimers were observed in many cases in high yield during click-mediated macrocyclization reactions, opening up the prospects of synthesizing complex peptide structures, which are otherwise difficult to make. Side chain-to-side chain cyclization, e.g., by ring-closing olefin metathesis, known as stapling, is one approach to increase the biological activity of short peptides that has shown promise when applied to 3(10)- and α-helical peptide. A novel stapling methodology for 3(10)-helical peptides using CuAAC click reaction in a model aminoisobutyric acid (Aib) rich peptide resulted in a more ideal 3(10)-helix than its acyclic precursor.

  • Chemical Ligation of Peptides

    Joining two or more peptide fragments together to make a larger peptide chain is called ligation. Click chemistry can be conveniently utilized to make peptide–peptide linkages. A peptide fragment functionalized with an alkyne group could be ligated to another peptide with an N-terminal azide moiety resulting in a triazole linker (similar to an amide bond as explained earlier) holding two peptide units together. Similarly, multimeric peptides can be made by orthogonal side chain–protecting groups such as Ivdde or Aloc (e.g. side chain of Lys) followed by deprotection, attachment of an alkyne function and clicking with N-terminal azide peptides.

    Several examples of peptide ligation is available, such as: the synthesis of a clickable RGD peptide (made by reacting Lys side chain with azido acetic acid) that can be clicked to another peptide fragment; synthesis of cell-permeable peptide by ligating a therapeutic alkynyl-modified peptide (using inexpensive propargylamine or 1-(2-nitrophenyl) propargyl alcohol) with nona-arginine modified with an azide group; synthesis of neurotensin (8–13) containing heterodimers by clicking alkyne-neurotensin (made by reacting with succinidyl-hex-5-ynoate) with azide of a Plk1-PBR binding phosphorylated hexapeptide (made by reacting with succinimidyl-4-azidovalerate). Click triazole-based oligopeptides were also found to self-dimerize in a head-to-tail fashion.

  • Synthesis of Modified Peptides

    Modification of peptides by pegylation has been achieved by click chemistry. For example a lipopeptide was assembled on a solid phase resin followed by an on-resin pegylation reaction (using azido-peg) and cleavage of the pegylated peptide off the resin. Such molecules are ideally suited for functionalization of solid-supported lipid bilayers and liposomal drug delivery systems, and are particularly valuable in enzyme activation strategies. There is a tremendous potential for click chemistry for various chemical modification of peptides and proteins (e.g., attaching ligands, lipophilic or liphophobic groups, hydrophilic and hydrophobic linkers etc.) and a number of clickable substrates can be designed for this purpose.

    Arginine-rich TAT peptides (capable of penetrating plasma membrane directly) modified with clickable azido group can be conjugated to oligonucleotides, cytotoxic drugs, kinase inhibitors etc. to facilitate cell penetration for therapeutic applications. Alkyne or azido containing prosthetic groups of radioisotopes could be used for labeling modified peptides.

    In conclusion, click chemistry is a powerful technique for the synthesis and modification of peptides. In the future, the applications of click chemistry to peptides will grow drastically due to the potential of these molecules in drug development, diagnostics, cosmetics, and material science combined with the simplicity and efficiency of click reactions. It will be possible to overcome difficulties in making complex peptides employing this elegant chemistry.


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