Triazinium Ligation: Bioorthogonal Reaction of N1‐Alkyl 1,2,4‐Triazinium Salts**

Abstract

The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1‐alkylation of 1,2,4‐triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4‐triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1‐alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5‐tetrazines. Due to their high reactivity, stability, synthetic accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents.

Synthesis of C3-Substituted N1-tert-Butyl 1,2,4-Triazinium Salts via the Liebeskind–Srogl Reaction for Fluorogenic Labeling of Live Cells

“…In our previous work, we prepared tBu-triazinium salts from 1,2,4-triazines by tert-butylation with isobutylene in the presence of triflic acid (Figure 1A). 27 However, the substrate scope of this method is inherently restricted to compounds that survive acidic conditions. Moreover, overalkylation can pose a problem for compounds containing additional reactive nucleophilic sites.…”

“…In our previous study, we established that a C 5 -phenyl-substituted triazinium, with diethylaminocoumarin attached through a phenylmethylene amino linker at position C 3 , exhibits a 6.5-fold increase in fluorescence upon reaction with endo-BCN. 27 We discovered that substituting the phenyl group at C 5 with an electrondonating 4-methoxyphenyl yielded triazinium derivative Trz + Coum6, which exhibited a notable enhancement in fluorescence upon reaction with endo-BCN in phosphatebuffered saline (PBS). On the basis of these findings, we designed a second generation of fluorogenic Trz + Coum probes containing the dye attached to the triazinium core through the conjugated phenyl ring.…”

“…To meet this challenge, we recently introduced N1-alkyl triazinium salts (Trz + s). 27 These charged heterodienes react rapidly with strained alkynes in a process we call "triazinium ligation". Due to their high reactivity and excellent stability under biological conditions, the N1-tert-butylated triazinium salts are particularly valuable derivatives.…”

“…However, despite these achievements, the demand for more advanced bioorthogonal reactions and reagents remains. To meet this challenge, we recently introduced N 1-alkyl triazinium salts ( Trz + s ) . These charged heterodienes react rapidly with strained alkynes in a process we call “triazinium ligation”.…”

Catching up with tetrazines: coordination of Re(i) to 1,2,4-triazine facilitates an inverse electron demand Diels–Alder reaction with strained alkynes to a greater extent than in corresponding 1,2,4,5-tetrazines

“…6 Very recently Vrabel and co-workers introduced N1-alkyl-1,2,4-triazinium salts as an efficient diene system in reaction with BCN. 7 Metal coordination is another way to facilitate the IEDDA reaction, and it was shown that both tetrazines and triazines react much faster in IEDDA reactions if directly coordinated to a metal ions. For example, Lo and co-workers were first to demonstrate this effect in tetrazine Ir(III) complexes.…”