Exploring Tyrosine‐Triazolinedione (TAD) Reactions for the Selective Conjugation and Cross‐Linking of N‐Carboxyanhydride (NCA) Derived Synthetic Copolypeptides

Abstract

Highly efficient functionalization and cross-linking of polypeptides is achieved via tyrosine-triazolinedione (TAD) conjugation chemistry. The feasibility of the reaction is demonstrated by the reaction of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with tyrosine containing block copolymer poly(ethylene glycol)-Tyr as well as a statistical copolymer of tyrosine and lysine (poly(Lys -st-Tyr )) prepared form N-carboxyanhydride polymerization. Selective reaction of PTAD with the tyrosine units is obtained and verified by size exclusion chromatography and NMR spectroscopy. Moreover, two monofunctional and two difunctional TAD molecules are synthesized. It is found that their stability in the aqueous reaction media significantly varied. Under optimized reaction conditions selective functionalization and cross-linking, yielding polypeptide hydrogels, can be achieved. TAD-mediated conjugation can offer an interesting addition in the toolbox of selective (click-like) polypeptide conjugation methodologies as it does not require functional non-natural amino acids.

Gelatin-Based Hydrogels through Homobifunctional Triazolinediones Targeting Tyrosine Residues

“…Gelatin can be easily dissolved in aqueous solutions above 37 °C; however, TADs are reported to undergo degradation in water [50], despite a certain stability of some TADs in aqueous medium reported in bioconjugation reactions by PTAD [56] or in cross-linking reaction of a synthetic Lys-Tyr polypeptide in TRIS buffer solution by difunctional TAD 2 [53]. In our hands both 1 and 2 almost immediately decomposed in any type of aqueous environment, as clearly indicated by the disappearing of their characteristic fuchsia color in a few seconds (Figure 1b).…”

“…Triazolinedione ene-type chemistry recently emerged as a click-reaction for the bioconjugation to tyrosine residues, mediated by cyclic monofunctional diazodicarboxamides, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) [48,49,50,51]. Hetherobifunctional triazolinediones (TADs), have been used for the synthesis of DNA−protein conjugates [52], while homobifunctional TADs have been applied to the cross-linking of synthetic polypetides [53].…”

Bio-orthogonal triazolinedione (TAD) crosslinked protein nanocapsules affect protein adsorption and cell interaction

“…8,13 Herein we report the synthesis of protein nanocapsules that were crosslinked with a 4-phenyl-triazolinedione (TAD) derivative, which specifically reacts with amino acids with delocalized π-electron systems. [14][15][16][17] The ability of TAD to act as a strong dienophile in a Diels-Alder reaction has already been shown almost 60 years ago. 18 Recent studies performed by Du Prez and coworkers again showed its versatility in current applications in polymer science.…”

Crosslinking of Electrospun Fibres from Unsaturated Polyesters by Bis-Triazolinediones (TAD)

“…As the double bond is preserved in the Alder–ene reaction, further functionalisation can be undertaken utilising the shifted double bond. TADs have been used to modify unsaturated polymers [32], prepare films [33], produce hydrogels [34,35] and produce crosslinked nanostructures [36]. In one example, a bis-functional TAD was applied to unsaturated fatty acids to produce crosslinked films from vegetable oils [37,38].…”