Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry

Abstract

Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.

Inverse electron demand Diels–Alder (iEDDA)-initiated conjugation: a (high) potential click chemistry scheme

“…This allows us to use the inherently reactive thiol groups to attach more complex functionalities by applying 33 in combination with iEDDA click chemistry. Later approaches involving tetrazines and alkenes combined norbornenes for iEDDA and terminal alkynes for CuAAC using DNA 8 or block copolymers 42 as backbone. The respective ''cross-reactions'' (1,3-dipolar addition of azides to the norbornene double bond 38 and iEDDA between tetrazines and terminal alkynes) have been occasionally observed, yet under very harsh reaction conditions.…”

Conformationally strained trans-cyclooctene with improved stability and excellent reactivity in tetrazine ligation

“…With more reactive tetrazines under aqueous conditions, rates can exceed 10 6 M −1 s −1 , making s-TCO the most reactive dienophile reported to date for bioorthogonal applications. 44, 62, 64 With GFP derivative 2 , fluorogenic labelling by s-TCO takes place rapidly inside living bacteria (Scheme 1c). 58 …”

trans-Cyclooctene—a stable, voracious dienophile for bioorthogonal labeling

“…In 45:55 water:MeOH at 25° C, s-TCO 20 combines with 21 at a rate too fast to measure by stopped-flow kinetics (>200,000 M −1 s −1 ) [37 •• ]. Recently, the rate of s-TCO 18b and 14 was measured to be 380,000 M −1 s −1 (7:1 water:dioxane, 25 °C) [64]. The rate of an s-TCO derivative with 8 was measured to be 2,800,000 M −1 s −1 at 37 °C in PBS [61].…”