Peptide conjugation via CuAAC 'click' chemistry

Abstract

The copper (I)-catalyzed alkyne azide 1,3-dipolar cycloaddition (CuAAC) or 'click' reaction, is a highly versatile reaction that can be performed under a variety of reaction conditions including various solvents, a wide pH and temperature range, and using different copper sources, with or without additional ligands or reducing agents. This reaction is highly selective and can be performed in the presence of other functional moieties. The flexibility and selectivity has resulted in growing interest in the application of CuAAC in various fields. In this review, we briefly describe the importance of the structural folding of peptides and proteins and how the 1,4-disubstituted triazole product of the CuAAC reaction is a suitable isoster for an amide bond. However the major focus of the review is the application of this reaction to produce peptide conjugates for tagging and targeting purpose, linkers for multifunctional biomacromolecules, and reporter ions for peptide and protein analysis.

Scheme 1

Prior thiol capture involving intramolecular O,N-acyl transfer reaction. (A) Activated thiol species (B) captured thiol fragment, and (C) after acyl transfer reaction, (D) desired peptide is formed.

Scheme 2

An example of NCL reaction of peptides containing cysteine or its derivatives. Reaction (A) intermolecular thioesterification; (B) intramolecular S➔N acyl transfer; (C) (optional) desulfurization of cysteine derivatives to cysteine residue.

Scheme 3

General reaction for CuAAC reaction producing a triazole ring.

Figure 1

From amino acids to protein. (A) free amino acids; (B) primary structure (peptide bonds); (C) secondary structure (α-helix or β-sheet); (D) tertiary structure (whole protein or subdomain protein); (E) quaternary structure (multiple domain protein, HIV Protease, Protein Data Bank (PDB) number = 1HSG) [57].

Figure 2

Triazole as amide bond bioisosters. Arrow ( ) represent hydrogen bonding sites. (PDB: 1HPV [59] and 1ZP8 [53]).

Figure 3

Examples of resin-bound peptidotriazoles constructs synthesized via CuAAC.

Figure 4

¹⁸F-labeled TOCA analogs for tumor imaging. CuAAC condition: pH 5 acetate buffer, DMF and acetonitrile (MeCN) (8:3:10) at 25 °C plus; CuSO₄ and NaAsc.

Figure 5

Structural comparison between; (A) CuAAC locked-DNA; (B) locked-DNA; and (C) unmodified DNA. Red structures highlight the DNA backbone.

Figure 6

(A) synthesis of azide modified Pam₂Cys via four steps: (i) piperidine, dichloromethane; (ii) imidazole-1-sulfonyl azide, potassium carbonate, methanol; (iii) palmatic acid (Pam), diisopropylcarboiimide, dimethylaminopyridine, tetrahydrofuran; (iv) trifluoroacetic acid; (B) CuAAC reaction of Pam₂Cys construct.

Figure 7

Multiple conjugation strategy using CuAAC approach; (A) dendritic, (B) linear, (C) cyclic, (D) cross-linked.

Figure 8

Example of multiple triazole scaffolds synthesized via CuAAC; (A) lipid core peptide (LCP), (B) dendron scaffold, and (C) polymeric dendrimer.

Figure 9

Cyclic peptides scaffold for CuAAC bioconjugation with PBA polymer: (A) two-arm cyclic peptide, (B) four-arm cyclic peptide.

Figure 10

(A) Amide backbone modification with triazole rings; (B) HIVPR inhibitor; (C) Triazolamer-based HIVPR inhibitors.

Figure 11

(A) General chemical structures of one and two triazole constructs. (B) Compound exhibited best somatostatin receptor binding experiment (IC₅₀). (C) Chemical structure of SRIF-28.

Figure 12

Head-to-tail CuAAC conjugation producing cyclodimer or cyclomonomer.

Figure 13

Formation of hydrogel based on multilinker conjugation via CuAAC.

Figure 14

3₁₀ helix side-chain-to-side-chain CuAAC cyclization.

Figure 15

Structural improvement via side chain triazole linker.

Figure 16

β-Hairpin structure of TP-1 peptide (in single letter amino acid code). (A) Native structure with two disulfide bonds. (B) Hairpin-like structure via triazole linkers. (C) Globule-like structure due to incorrect folding.

Figure 17

Gas phase fragmentation of triazole into reporter ion.