Triazine-Modified 7-Deaza-2′-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2′-Deoxyuridines

Abstract

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels–Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2′-deoxyadenosine triphosphate and to the 5-position of 2′-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]­nonyne–rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2′-deoxyadenosine in comparison to the attachment at the 5-position of 2′-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2′-adenosines than conventional and available 2′-deoxyuridines.

Triazines: Syntheses and Inverse Electron-demand Diels–Alder Reactions

“…Although 119b was much more synthetically available, 119c was found to be better suited for DNA polymerase-based oligonucleotide preparations (Scheme 37b). 150 Furthermore, oligonucleotides 120b attached with two bioorthogonally reactive groups (1-methylcyclopropene and 1,2,4-triazine) could be obtained via a sequential DNA polymerases technique. Dual labeling with two different fluorescent dyes (BCN-rhodamine and tetrazine derivative 122a) was found to be not only bioorthogonal in general but also mutually orthogonal (Scheme 37c).…”

“…174 In contrast to the concerted mechanism for the conventional IEDDA reactions, this reaction was postulated to proceed via a stepwise pathway. In the presence of base, the nucleophilic addition of activated acetonitriles 148 or ketones 150 Although the normal IEDDA reactions of 1,2,3-triazines prefer to proceed via the C4/N1 (C6/N3) mode, the cycloaddition pattern could be tuned to C5/N2 mode in some cases. In 2014, Boger and co-workers disclosed the C5/N2mode IEDDA reaction for the synthesis of pyridazines (Scheme 48).…”

Postsynthetic Modifications of DNA and RNA by Means of Copper-Free Cycloadditions as Bioorthogonal Reactions

“…This property was used to create another level of orthogonality for nucleic acid labeling (Figure ). Our group managed to incorporate the 1,2,4-triazine-modified nucleotides 57 – 59 enzymatically into DNA and labeled them by BCN-modified dyes as reactive partners. , The dUTP-derived building block 58 was just hardly tolerated by the DNA polymerases, while the 7-deaza-2′-deoxyadenosine derivative 59 apparently was much better suited for enzymatic DNA preparation, making its tough synthetic accessibility worth it.…”

“…The 6-ethynyl-1,2,4triazine modification is much better tolerated by the KlenTaq DNA polymerase when attached to 7-position of the triphosphate of 7-deaza-2′-deoxyadenosine 59 than to the 5position of 2′-deoxyuridine 58 according to PAGE analysis of PCR products and real-time kinetic analysis of DNA polymerase activity during PEX on biosensors. 125 Such intriguing results were obtained also by PEX experiments using the 1-methylcyclopropene-modified building blocks 76 and 77 for a variety of different DNA polymerases (Hemo KlenTaq Vent (exo-) and Deep Vent (exo-). 158 The elongation products were labeled in vitro by 1,2,4,5-tetrazine-modified TAMRA dyes (90% yield after 2 h).…”

Bioorthogonal Ligations and Cleavages in Chemical Biology

“…[136] Recently, the method was successfully used in the DNA labeling. [137] In 2019, Boger's group reported the synthesis, characterization, and cycloaddition reactivity of a monocyclic aromatic 1,2,3,5-tetrazine and found that it showed good stability and rapid IEDDA rate with electron-rich or strain-containing dienophiles. [138] In addition, Murphy and Houk reported the cycloaddition reactions of tetrachlorocyclopentadiene ketal (142) with bicyclononyne (BCN) (85) or trans-cyclooctene (TCO) for bioorthogonal labeling (Figure 30).…”