Copper-Free Click Enabled Triazabutadiene for Bioorthogonal Protein Functionalization

Abstract

Aryl diazonium ions have long been used in bioconjugation due to their reactivity toward electron-rich aryl residues, such as tyrosine. However, their utility in biological systems has been restricted due to the requirement of harsh conditions for their generation in situ, as well as limited hydrolytic stability. Previous work describing a scaffold known as triazabutadiene (TBD) has shown the ability to protect aryl diazonium ions allowing for increased synthetic utility, as well as triggered release under biologically relevant conditions. Herein, we describe the synthesis and application of a novel TBD, capable of installation of a cyclooctyne on protein surfaces for later use of copper-free click reactions involving functional azides. The probe shows efficient protein labeling across a wide pH range that can be accomplished in a convenient and timely manner. Orthogonality of the cyclooctyne modification was showcased by labeling a model protein in the presence of hen egg proteins, using an azide-containing fluorophore. We further confirmed that the azobenzene modification can be cleaved using sodium dithionite treatment.

Direct Intracellular Delivery of Benzene Diazonium Ions As Observed by Increased Tyrosine Phosphorylation

“…Indeed, when working with variants of 2, the addition of the triazabutadiene into neutral water can be likened to adding freshly made aryl diazonium salts. 27 The reactivity of BDI suggests that it is likely able to react with membrane proteins and its small, positively charged nature suggests that it should also be able to pass through the cell's membrane. To understand the background reactivity of a nonguided probe, we sought to investigate the effect of simple addition of BDI to cells as a control (Figure 3a).…”

“…This was ideal because the BDI release is much faster than our phosphorylation assay and the timing of the release of the BDI was dependent upon the deprotection event that would liberate 2 in solution. Indeed, when working with variants of 2 , the addition of the triazabutadiene into neutral water can be likened to adding freshly made aryl diazonium salts . The reactivity of BDI suggests that it is likely able to react with membrane proteins and its small, positively charged nature suggests that it should also be able to pass through the cell’s membrane.…”

Chemical modification of proteins – challenges and trends at the start of the 2020s

“…It has been shown by Jewett and coworkers that this can be circumvented by temporarily masking the diazo moiety as a triazabutadiene which, at pH 7, releases the diazo ions at a controlled rate. 192 Alternatively, diazo ions can be released from triazenes via low-intensity UV-A irradiation. 193 Ene-like click reactions.…”

Site‐Selective Aryl Diazonium Installation onto Protein Surfaces at Neutral pH using a Maleimide‐Functionalized Triazabutadiene

“…Aryl diazonium cations will rapidly react with electron‐rich aryl motifs and secondary amine motifs, via either azo‐bond (Figure 1) [1] or triazene formation [2] under physiologically‐relevant conditions. This allows aryl diazonium cation labelling of canonical residues, such as tyrosine [1a–g,i,k,l,n] or histidine [1f–h] . Should an even more rapid and selective labelling be required, noncanonical residues such as 5‐hydroxytryptophan, [1i] 2‐amino‐3‐(6‐hydroxy‐2‐naphthyl)propanoic acid, [1j] or alkylated lysine motifs [2] can also be installed into proteins and subsequently be labelled with aryl diazonium cations.…”