Bio‐orthogonal Fluorescent Labelling of Biopolymers through Inverse‐Electron‐Demand Diels–Alder Reactions

Abstract

Bio‐orthogonal labelling schemes based on inverse‐electron‐demand Diels–Alder (IEDDA) cycloaddition have attracted much attention in chemical biology recently. The appealing features of this reaction, such as the fast reaction kinetics, fully bio‐orthogonal nature and high selectivity, have helped chemical biologists gain deeper understanding of biochemical processes at the molecular level. Listing the components and discussing the possibilities and limitations of these reagents, we provide a recent snapshot of the field of IEDDA‐based biomolecular manipulation with special focus on fluorescent modulation approaches through the use of bio‐orthogonalized building blocks. At the end, we discuss challenges that need to be addressed for further developments in order to overcome recent limitations and to enable researchers to answer biomolecular questions in more detail.

Bistetrazine‐Cyanines as Double‐Clicking Fluorogenic Two‐Point Binder or Crosslinker Probes

“…[29] This approachr elies on the fact that the first, intermolecular step is the rate determining and the second step, regardlesso f being intra-or intermolecular, is much faster.T his is supported by the experimental observation that we were unable to isolate mono-reacted species and even with 0.5 equivalent BCN the double-reacted product was observed. [6] Probe 6 reacteda bout four times faster than 7 (k 2 = 2.6 10 3 vs.6 10 2 m À1 s À1 )w ith the peptide. Second-order rate constants turned out to be somewhat lower than that obtained for bismaleimides [29a] and are in good accordance with literature values measured for methyl-tetrazines.…”

“…[1][2][3][4][5][6] These improvementsw ere greatly facilitated by the development of probes capable of (site) specific tagging of biomolecules of interest. [1][2][3][4][5][6] These improvementsw ere greatly facilitated by the development of probes capable of (site) specific tagging of biomolecules of interest.…”

“…Severalt echniques are used to address these tasks;f or example, the use of fusionp roteins such as tailoredf luorescentp roteins (FPs),t ags (e.g.,H alo, SNAP,C ys 4 ), or small engineered enzymes (e.g.,l ipoic acid ligase). [13][14][15][16] There are several explanations in the literaturef or the quenching mechanism present in these systems.T on ame af ew,p hotoinduced electron transfer (PET), [13] through-bonde nergy transfer (TBET), [15] [6,16] or,i nc ase of azides, increased efficiency of internal conversion as ar esult of fast rotation and bending of the azide [14] (for detailedd iscussion of possible quenching mechanisms see Ref. Advances in genetic code expansion (GCE) methods by means of Amber codon suppressioni nc ombination with bioorthogonal chemistry have opened an ew horizon in the field of site-specific tagging schemes, enablings ingle aminoa cid incorporation at virtually anyp ositions of the POI with minimal perturbation.…”

Protein Spin Labeling with a Photocaged Nitroxide Using Diels-Alder Chemistry

“…[13] We present herein an ovel approach to address both ncAAmediated spin labeling and nitroxide stability ( Figure 1). Introductiono ft he novel label is achieved through the DA inv reaction [14] of a1,2,4,5-tetrazine with astrained alkyne (cyclooctyne, SCO) [15] or alkene (trans-cyclooctene, TCO). [16] This reaction forms the corresponding pyridazine and dihydropyridazine, respectively.Due to its excellent water compatibility,the DA inv reaction has proven to be suitable for abroad range of biochemical applications both in vitro and in vivo.…”

“…[16] This reaction forms the corresponding pyridazine and dihydropyridazine, respectively.Due to its excellent water compatibility,the DA inv reaction has proven to be suitable for abroad range of biochemical applications both in vitro and in vivo. [14,17] Strain-promoted DA inv cycloadditions have not been used for the SDSL of proteins so far (notably,r ecently an in vitro transcribed RNA segment was site-specifically labeled with at etrazine-nitroxyl moiety [18] ). Moreover,w ea imed to address nitroxide stability by using ap rotection strategy.S of ar,a lkylation, [19] silylation, [20] acylation, [21] and photoremovable protecting groups( PPGs) [22] have been demonstrated to protect nitroxides and to release them as needed.…”