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

Selvaraj, Ramajeyam; Fox, Joseph M.

doi:10.1016/j.cbpa.2013.07.031

Cited by 192 publications

(147 citation statements)

References 71 publications

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“…In comparison, the diazaborine formation is faster than the azide-alkyne click chemistry or the alkyne-nitrone cycloaddition by 2–3 orders of magnitude. Although the tetrazine ligation can be fast, its reaction rate is quite sensitive to how the tetrazine moiety is linked to its biological target 36 and typically bulky diaryl tetrazine derivatives are needed to afford fast kinetics, 37 which are less ideal due to the outer membrane barrier.…”

Section: Discussionmentioning

confidence: 99%

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

2017

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Bioorthogonal conjugation chemistry has enabled the development of tools for the interrogation of complex biological systems. Although a number of bioorthogonal reactions have been documented in literature, they are less ideal for one or several reasons including slow kinetics, low stability of the conjugated product, requirement of toxic catalysts, and side reactions with unintended biomolecules. Herein we report a fast (>103 M−1s−1) and bioorthogonal conjugation reaction that joins semicarbazide to an aryl ketone or aldehyde with an ortho-boronic acid substituent. The boronic acid moiety greatly accelerates the initial formation of a semicarbazone conjugate, which rearranges into a stable diazaborine. The diazaborine formation can be performed in blood serum or cell lysates with minimal interference from biomolecules. We further demonstrate that application of this conjugation chemistry enables facile labeling of bacteria. A synthetic amino acid D-AB3, which presents a 2-acetylphenylboronic acid moiety as its side chain, was found to incorporate into several bacterial species through cell wall remodeling, with particularly high efficiency for Escherichia coli. Subsequent D-AB3 conjugation to a fluorophore-labeled semicarbazide allows robust detection of this bacterial pathogen in blood serum.

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Section: Discussionmentioning

confidence: 99%

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

2017

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“…11 These IEDDA reactions have second-order rate constants ranging from k 2 = 10 3 –10 6 M −1 s −1 , which are primarily driven by expulsion of N 2 and TCO strain relief following the cycloaddition 12,13 Furthermore, electron withdrawing substituents attached to the tetrazine confer increased reaction rates between the dienophile and diene. 14,15 Successful pretargeted in vivo nuclear imaging has been demonstrated in preclinical models using PET isotopes, including 11 C (t 1/2 20.3 minutes), 16,17 18 F (t 1/2 110 minutes) 18–20 and 64 Cu (t 1/2 12.7 hours), 4,21–24 as well as SPECT isotopes including 99m Tc (t 1/2 6 hours), 25–27 111 In (t 1/2 2.8 days) 28–30 and 177 Lu (t 1/2 6.7 days). 27,31–33 The recent success of IEDDA reactions for in vivo pretargeting and other chemical biology applications has been reviewed by several groups.…”

Section: Introductionmentioning

confidence: 99%

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Mandikian¹,

Rafidi²,

Adhikari³

et al. 2018

mAbs

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Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

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“…1,2 In recent years, the bioorthogonal reactions of strained alkenes with s -tetrazines have served as tools for rapid bioorthogonal labeling, with applications that extend to cell biology, 3–5 nuclear medicine 6–8 and materials science. 9–12 In particular, the bioorthogonal reactions of s -tetrazines with trans- cyclooctene (TCO) derivatives have been shown to be exceptionally rapid, with rate constants in excess of k 2 = 10 6 M −1 s −1 13,14. …”

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confidence: 99%

An efficient and mild oxidant for the synthesis of s-tetrazines

Selvaraj

Fox

2014

Tetrahedron Letters

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PhI(OAc)2 serves as a mild and effective oxidant for the synthesis of s-tetrazine derivatives— molecules of emerging significance to the field of bioorthogonal chemistry. This reagent serves as a complementary oxidant to harsher nitrous reagents. Use of PhI(OAc)2 improves the synthesis of 5-amino-di(pyridin-2-yl)-s-tetrazine, a molecule that has been broadly used for cellular imaging and nuclear medicine. The generality of PhI(OAc)2 as the oxidant for tetrazine synthesis is demonstrated for nine tetrazines in 75–98% yield.

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

Cited by 192 publications

References 71 publications

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

An efficient and mild oxidant for the synthesis of s-tetrazines

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