General, Divergent Platform for Diastereoselective Synthesis of trans‐Cyclooctenes with High Reactivity and Favorable Physiochemical Properties**

Abstract

trans-Cyclooctenes (TCOs) are essential partners for the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or in vivo experiments. Described is a new class of TCOs, 'a-TCOs', that is prepared in high yield via stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on large scale in two steps from 1,5-cyclooctadiene. Computational transition state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a trans-oxocene analog. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to washout more rapidly than other TCOs. File list (2) download file view on ChemRxiv aTCO paper Jan 30 JMF no watermark.pdf (3.24 MiB) download file view on ChemRxiv aTCO SI Jan 30 no water mark.pdf (3.60 MiB)

Uncovering the Key Role of Distortion in Bioorthogonal Tetrazine Tools That Defy the Reactivity/Stability Trade-Off

“…The inverse electron demand Diels–Alder (IEDDA)-initiated ligation of 1,2,4,5-tetrazines (Tz) with strained dienophiles represents a group of exceptionally fast bioorthogonal reactions. − In particular, trans –cyclooctene ( TCO ) derivatives − provide several orders of magnitude higher reactivity than other dienophiles such as cyclopropenes or norbornenes . In the rate-determining step, the tetrazine first reacts with the trans -cyclooctene in a [4 + 2]-cycloaddition reaction to form a bicyclic intermediate that rapidly undergoes cycloreversion to give dihydropyridazines as ligation products (Figure a).…”

Bioorthogonal Cleavage of Tetrazine-Caged Ethers and Esters Triggered by trans-Cyclooctene

“…Although the cleavage mechanism of allyl-functionalized TCO has been systematically studied and several strategies have been employed to improve release yield and kinetics, ,− the second-order rate constant ( k 2 ) of the first IEDDA cycloaddition step using allyl-substituted TCO is much lower than that achieved using conformationally strained or 4-axial TCO analogues. , The rate constant is one of the most critical parameters for the successful application of bioorthogonal cleavage reactions in living systems, especially in cutting-edge applications such as molecular imaging and release of antibody–drug conjugates. , Therefore, recent research has focused on developing new tetrazine chemistry that accelerates cleavage.…”

“…In addition, the k 2 value of the reaction between methyl-substituted tetrazine 1a and 4e-TCO was 156.6 ± 8.4 M –1 s –1 in 50% MeCN/PBS at room temperature, about 9-fold higher than the rate constant for the reaction with analogue 1b . The recently reported a-TCOs gave a k 2 of 454.5 ± 17.3 M –1 s –1 in 50% MeCN/PBS at room temperature, which is about 8.7-fold faster than the previously reported tetrazine cleavage strategy using allyl-functionalized TCO as the precursor (Figures S5–S7).…”

Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light

“…The high reactivity of 2a is expected due to the activating nature of the pyridyl and amide substituents, which may increase reactivity through a combination of electron withdrawing and ground-state destabilizing effects . As expected, faster reactivity was observed with soluble a-TCO and o-TCO derivatives, which reacted with 2a in PBS with rates of 1.46 × 10 6 M –1 s –1 and 1.13 × 10 6 M –1 s –1 . Under aqueous conditions, the reaction with the conformationally strained alkene s-TCO , was too rapid for us to measure using stopped flow spectroscopy (>10 7 M –1 s –1 ).…”

“…As displayed in Figure , HeLa cells were transfected with a green fluorescent protein HaloTag-GFP construct fused to a “protein of interest” (POI) that controls subcellular localization and then labeled by the small molecule DHTz-HaloTag . Subsequently, cells are treated with a photocatalyst and either a-TCO-TAMRA or o-TCO-TAMRA (Figure B), fluorescent conjugates of TCO derivatives that were chosen for their hydrophilicity and improved permeability relative to traditional TCO derivatives . Upon irradiation, conjugation is expected only in those cells that express the HaloTag fusion protein, and analysis by SDS–PAGE with fluorescent imaging was used to provide a measure of conjugation efficiency.…”