Dataset Information

Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via Cyclopropenone-Caged Bicyclononynes.

ABSTRACT: Inverse electron-demand Diels-Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light-triggered version of iEDDAC (photo-iEDDAC) is presented that confers spatio-temporal control to bioorthogonal labeling in?vitro and in?cellulo. A cyclopropenone-caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo-DMBO) was designed that is unreactive towards tetrazines before light-activation, but engages in iEDDAC after irradiation at 365?nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site-specific incorporation of tetrazine-containing amino acids into proteins in living cells. In?situ light activation of photo-DMBO conjugates allows labeling of tetrazine-modified proteins in living E.?coli. This allows proteins in living cells to be modified in a spatio-temporally controlled manner and may be extended to photo-induced and site-specific protein labeling in animals.

SUBMITTER: Mayer SV

PROVIDER: S-EPMC6856800 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Publications

Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via Cyclopropenone-Caged Bicyclononynes.

Mayer Susanne V SV Murnauer Anton A von Wrisberg Marie-Kristin MK Jokisch Marie-Lena ML Lang Kathrin K

Angewandte Chemie (International ed. in English) 20190924 44

Inverse electron-demand Diels-Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light-triggered version of iEDDAC (photo-iEDDAC) is presented that confers spatio-temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone-caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo-DMBO) was designed that is unreactive towards tetrazines before light-activation, but enga ...[more]

PMID: 31476269

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Project description:Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18F (t½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel's Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [18F]AlF-NOTA moiety with [18F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[18F]fluoronicotinyl-PEG4-methyltetrazine to provide [18F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 ([18F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB) also were synthesized. Radiochemical purity, affinity (KD) and immunoreactive fraction of [18F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [18F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[125I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [18F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[125I]SGMIB-5F7 as well as those observed for the 18F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [18F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [18F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.

Dataset Information

Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via Cyclopropenone-Caged Bicyclononynes.

Publications

Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via Cyclopropenone-Caged Bicyclononynes.

Similar Datasets

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