The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Neal K. Devaraj - One of the best experts on this subject based on the ideXlab platform.
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advances in Tetrazine bioorthogonal chemistry driven by the synthesis of novel Tetrazines and dienophiles
Accounts of Chemical Research, 2018Co-Authors: Neal K. DevarajAbstract:ConspectusBioorthogonal chemistry has found increased application in living systems over the past decade. In particular, Tetrazine bioorthogonal chemistry has become a powerful tool for imaging, detection, and diagnostic purposes, as reflected in the increased number of examples reported in the literature. The popularity of Tetrazine ligations are likely due to rapid and tunable kinetics, the existence of high quality fluorogenic probes, and the selectivity of reaction. In this Account, we summarize our recent efforts to advance Tetrazine bioorthogonal chemistry through improvements in synthetic methodology, with an emphasis on developing new routes to Tetrazines and expanding the range of useful dienophiles. These efforts have removed specific barriers that previously limited Tetrazine ligations and have broadened their potential applications.Among other advances, this Account describes how our group discovered new methodology for Tetrazine synthesis by developing a Lewis acid-promoted, one-pot method fo...
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bioorthogonal Tetrazine mediated transfer reactions facilitate reaction turnover in nucleic acid templated detection of microrna
Journal of the American Chemical Society, 2014Co-Authors: Brandon T Cisneros, Christian M Cole, Neal K. DevarajAbstract:Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic Tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic Tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates.
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synthesis and reactivity comparisons of 1 methyl 3 substituted cyclopropene mini tags for Tetrazine bioorthogonal reactions
Chemistry: A European Journal, 2014Co-Authors: Jolita Seckutė, Jun Yang, K. N. Houk, Yong Liang, Neal K. DevarajAbstract:Substituted cyclopropenes have recently attracted attention as stable "mini-tags" that are highly reactive dienophiles with the bioorthogonal Tetrazine functional group. Despite this interest, the synthesis of stable cyclopropenes is not trivial and their reactivity patterns are poorly understood. Here, the synthesis and comparison of the reactivity of a series of 1-methyl-3-substituted cyclopropenes with different functional handles is described. The rates at which the various substituted cyclopropenes undergo Diels-Alder cycloadditions with 1,2,4,5-Tetrazines were measured. Depending on the substituents, the rates of cycloadditions vary by over two orders of magnitude. The substituents also have a dramatic effect on aqueous stability. An outcome of these studies is the discovery of a novel 3-amidomethyl substituted methylcyclopropene tag that reacts twice as fast as the fastest previously disclosed 1-methyl-3-substituted cyclopropene while retaining excellent aqueous stability. Furthermore, this new cyclopropene is better suited for bioconjugation applications and this is demonstrated through using DNA templated Tetrazine ligations. The effect of Tetrazine structure on cyclopropene reaction rate was also studied. Surprisingly, 3-amidomethyl substituted methylcyclopropene reacts faster than trans-cyclooctenol with a sterically hindered and extremely stable tert-butyl substituted Tetrazine. Density functional theory calculations and the distortion/interaction analysis of activation energies provide insights into the origins of these reactivity differences and a guide to the development of future Tetrazine coupling partners. The newly disclosed cyclopropenes have kinetic and stability advantages compared to previously reported dienophiles and will be highly useful for applications in organic synthesis, bioorthogonal reactions, and materials science.
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expanding room for Tetrazine ligations in the in vivo chemistry toolbox
Current Opinion in Chemical Biology, 2013Co-Authors: Jolita Seckutė, Neal K. DevarajAbstract:There is tremendous interest in developing and refining methods to predictably perform chemical reactions within the framework of living systems. Here we review recent advances in applying Tetrazine cycloadditions to live cell and in vivo chemistry. We highlight new syntheses of the Tetrazine and dienophile precursors useful for in vivo studies. We briefly overview the use of this reaction in combination with unnatural amino acid technology and discuss applications involving the imaging of glycans on live cells. An emerging area is the use of Tetrazine ligations for the development of in vivo imaging probes such as those used for positron emission tomography. We summarize recent applications involving Tetrazine cycloadditions performed in live mice for pretargeted imaging of cancer cell biomarkers.
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live cell imaging of cyclopropene tags with fluorogenic Tetrazine cycloadditions
Angewandte Chemie, 2012Co-Authors: Jun Yang, Jolita Seckutė, Christian M Cole, Neal K. DevarajAbstract:There is growing interest in the use of inverse Diels–Alder Tetrazine cycloadditions as rapid catalyst-free bioorthogonal reactions.[1–3] Fluorogenic Tetrazines that increase in fluorescence after reaction with dienophiles are particularly useful for live-cell imaging applications.[4] Fluorogenic Tetrazines have been recently used for live-cell imaging of small molecules, biomolecules tagged enzymatically with dienophiles, and proteins modified by reactive unnatural amino acids.[4,5] Although fluorogenic Tetrazine probes hold great potential for intracellular imaging of small molecules, previous approaches are limited by requiring a large strained dienophile, such as trans-cyclooctene, cyclooctyne, or norbornene.[1,6] This situation is in contrast to Staudinger ligations or strain-promoted azide–cycloalkyne cycloadditions that utilize a small azide functional group.[7,8] This requirement has limited the use of Tetrazine reactions in methods that require tags with minimal steric impact or nominal effect on the partition ratio.[2] The development of smaller dienophile partners capable of reacting rapidly with Tetrazines would therefore represent a major advance. However, it has been unclear whether small dienophiles could be developed that react rapidly with Tetrazines while maintaining their stability. Herein, we demonstrate the applicability of methylcyclopropene tags as dienophiles for reaction with fluorogenic Tetrazines. Through systematic synthetic modifications we have optimized the stability, size, and reactivity of the cyclopropene scaffold. We have developed methylcyclopropene derivatives that react rapidly with Tetrazines while retaining their aqueous stability and small size. These cyclopropene handles elicit fluorescent responses from quenched Tetrazine dyes and are suitable for cellular imaging applications, which we demonstrate by imaging cyclopropene phospholipids distributed in live human breast cancer cells.
Ralph Weissleder - One of the best experts on this subject based on the ideXlab platform.
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unraveling Tetrazine triggered bioorthogonal elimination enables chemical tools for ultrafast release and universal cleavage
Journal of the American Chemical Society, 2018Co-Authors: Jonathan C T Carlson, Hannes Mikula, Ralph WeisslederAbstract:Recent developments in bond cleavage reactions have expanded the scope of bioorthogonal chemistry beyond click ligation and enabled new strategies for probe activation and therapeutic delivery. These applications, however, remain in their infancy, with further innovations needed to achieve the efficiency required for versatile and broadly useful tools in vivo. Amongst these chemistries, the Tetrazine/trans-cyclooctene click-to-release reaction has exemplary kinetics and adaptability but achieves only partial release and is incompletely understood, which has limited its application. Investigating the mechanistic features of this reaction’s performance, we discovered profound pH sensitivity, exploited it with acid-functionalized Tetrazines that both enhance and markedly accelerate release, and ultimately uncovered an unexpected dead-end isomer as the reason for poor release. Implementing facile methods to prevent formation of this dead end, we have achieved exceptional efficiency, with essentially complete ...
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building blocks for the construction of bioorthogonally reactive peptides via solid phase peptide synthesis
ChemistryOpen, 2014Co-Authors: Brian M. Zeglis, Jason S. Lewis, Ralph Weissleder, Fabien Emmetiere, Nagavarakishore Pillarsetty, Thomas ReinerAbstract:The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels–Alder copper-free click chemistry: Tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of Tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.
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bodipy Tetrazine derivatives as superbright bioorthogonal turn on probes
Angewandte Chemie, 2013Co-Authors: Jonathan C T Carlson, Scott A Hilderbrand, Labros G Meimetis, Ralph WeisslederAbstract:The fastest and the brightest: A new design that intimately connects Tetrazine to a BODIPY fluorophore enables exceptionally efficient energy transfer and quenching. Upon reaction of the Tetrazine, the brightness of the fluorophore increases more than a thousand-fold, a fluorogenic activation up to two orders of magnitude greater than previously described.
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reactive polymer enables efficient in vivo bioorthogonal chemistry
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Neal K. Devaraj, Greg M Thurber, Edmund J Keliher, Brett Marinelli, Ralph WeisslederAbstract:There has been intense interest in the development of selective bioorthogonal reactions or “click” chemistry that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concentration gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified Tetrazines (PMT) are a key enabler for in vivo bioorthogonal chemistry based on the very fast and catalyst-free [4 + 2] Tetrazine/trans-cyclooctene cycloaddition. Using fluorescent PMT for cellular resolution and 18F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.
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Modular Strategy for the Construction of Radiometalated Antibodies for Positron Emission Tomography Based on Inverse Electron Demand Diels–Alder Click Chemistry
Bioconjugate Chemistry, 2011Co-Authors: Brian M. Zeglis, Priya Mohindra, Gabriel I. Weissmann, Vadim Divilov, Scott A Hilderbrand, Ralph Weissleder, Jason S. LewisAbstract:A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-Tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes 64Cu and 89Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with Tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with 64Cu and 89Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7–5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the 64Cu-DOTA- and 89Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per a...
Joseph M Fox - One of the best experts on this subject based on the ideXlab platform.
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conformationally strained trans cyclooctene stco enables the rapid construction of 18 f pet probes via Tetrazine ligation
Theranostics, 2016Co-Authors: Mengzhe Wang, Hui Wang, Hong Yuan, Dennis Svatunek, Katarina Rohlfing, Yu Liu, Ben Giglio, Joseph M FoxAbstract:The bioorthogonal reaction between Tetrazines and trans-cyclooctenes is a method for the rapid construction of F-18 probes for PET imaging. Described here is a second generation (18)F-labeling system based on a conformationally strained trans-cyclooctene (sTCO)-a dienophile that is approximately 2 orders of magnitude more reactive than conventional TCO dienophiles. Starting from a readily prepared tosylate precursor, an (18)F labeled sTCO derivative ((18)F-sTCO) could be synthesized in 29.3 +/- 5.1% isolated yield and with high specific activity. Tetrazine ligation was carried out with a cyclic RGD-conjugate of a diphenyl-s-Tetrazine analogue (RGD-Tz) chosen from a diene class with an excellent combination of fast reactivity and stability both for the diene as well as the Diels-Alder adduct. For both the Tetrazine and the sTCO, mini-PEG spacers were included to enhance solubility and improve the in vivo distribution profile of the resulting probe. Extremely fast reactivity (up to 2.86 x 10(5) M(-1)s(-1) at 25 °C in water) has been observed in kinetic studies in the reaction of sTCO with diphenyl-s-Tetrazine derivatives. A kinetic study on sTCO diastereomers in 55:45 MeOH:water showed that the syn-diastereomer displayed slightly faster reactivity than the anti-diastereomer. An (18)F-sTCO conjugate with RGD-Tz demonstrated prominent and persistent tumor uptake in vivo with good tumor-to-background contrast. Unlike most radiolabeled RGD peptides, the tumor uptake of this PET agent increased from 5.3 +/- 0.2% ID/g at 1 h post injection (p.i.), to 8.9 +/- 0.5% ID/g at 4 h p.i., providing evidence for prolonged blood circulation. These findings suggest that Tetrazine ligations employing (18)F-sTCO should serve as a powerful and general platform for the rapid construction of peptide or protein derived PET agents.
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improved metabolic stability for 18f pet probes rapidly constructed via Tetrazine trans cyclooctene ligation
Bioconjugate Chemistry, 2015Co-Authors: Ramajeyam Selvaraj, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Srinivasa R Chintala, Li Peng Yap, Peter S Conti, Joseph M FoxAbstract:The fast kinetics and bioorthogonal nature of the Tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an 18F-labeling system based on a CF3-substituted diphenyl-s-Tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-Tetrazine derivative via EDC-mediated coupling. The resulting Tetrazine-RGD conjugate was combined with a 19F-labeled TCO derivative to give HPLC standards. The analogous 18F-labeled TCO derivative was combined with the diphenyl-s-Tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-Tetrazine-RGD combines with an 18F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of Tetrazine-RGD relative to the 18F-labeled TCO (concentration calculated based...
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Tetrazine trans cyclooctene ligation for the rapid construction of integrin αvβ3 targeted pet tracer based on a cyclic rgd peptide
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Ramajeyam Selvaraj, Shuanglong Liu, Li Peng Yap, Joseph M Fox, Matthew Hassink, Chiun Wei Huang, Ryan Park, Peter S ContiAbstract:Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the Tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the Tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a Tetrazine-RGD conjugate and 2 mCi (5 μM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the Tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.
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Tetrazine trans cyclooctene ligation for the rapid construction of integrin αvβ targeted pet tracer based on a cyclic rgd peptide
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Ramajeyam Selvaraj, Shuanglong Liu, Li Peng Yap, Joseph M Fox, Matthew Hassink, Chiun Wei Huang, Ryan Park, Peter S ContiAbstract:Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the Tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the Tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a Tetrazine-RGD conjugate and 2 mCi (5 μM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the Tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.
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design and synthesis of highly reactive dienophiles for the Tetrazine trans cyclooctene ligation
Journal of the American Chemical Society, 2011Co-Authors: Michael T. Taylor, Melissa L Blackman, Olga Dmitrenko, Joseph M FoxAbstract:Computation was used to design a trans-cyclooctene derivative that displays enhanced reactivity in the Tetrazine–trans-cycloctene ligation. The optimized derivative is an (E)-bicyclo[6.1.0]non-4-ene with a cis-ring fusion, in which the eight-membered ring is forced to adopt a highly strained ‘half-chair’ conformation. Toward 3,6-dipyridyl-s-Tetrazine in MeOH at 25 °C, the strained derivative is 19 and 27 times more reactive than the parent trans-cyclooctene and 4E-cyclooct-4-enol, respectively. Toward 3,6-diphenyl-s-Tetrazine in MeOH at 25 °C, the strained derivative is 160 times more reactive than the parent trans-cyclooctene.
Ramajeyam Selvaraj - One of the best experts on this subject based on the ideXlab platform.
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improved metabolic stability for 18f pet probes rapidly constructed via Tetrazine trans cyclooctene ligation
Bioconjugate Chemistry, 2015Co-Authors: Ramajeyam Selvaraj, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Srinivasa R Chintala, Li Peng Yap, Peter S Conti, Joseph M FoxAbstract:The fast kinetics and bioorthogonal nature of the Tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an 18F-labeling system based on a CF3-substituted diphenyl-s-Tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-Tetrazine derivative via EDC-mediated coupling. The resulting Tetrazine-RGD conjugate was combined with a 19F-labeled TCO derivative to give HPLC standards. The analogous 18F-labeled TCO derivative was combined with the diphenyl-s-Tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-Tetrazine-RGD combines with an 18F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of Tetrazine-RGD relative to the 18F-labeled TCO (concentration calculated based...
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efficient 18f labeling of cysteine containing peptides and proteins using Tetrazine trans cyclooctene ligation
Molecular Imaging, 2013Co-Authors: Shuanglong Liu, Ramajeyam Selvaraj, Li Peng Yap, Matthew HassinkAbstract:18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the Tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of Tetrazine-RGD (80-100 μM) or 15 μg of Tetrazine-VEGF (6.0 μM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the Tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.
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diels alder cycloaddition for fluorophore targeting to specific proteins inside living cells
Journal of the American Chemical Society, 2012Co-Authors: Anupong Tangpeerachaikul, Michael T. Taylor, Ramajeyam Selvaraj, Alice Y TingAbstract:The inverse-electron-demand Diels–Alder cycloaddition between trans-cyclooctenes and Tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a Tetrazine–fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels–Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.
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Tetrazine trans cyclooctene ligation for the rapid construction of integrin αvβ3 targeted pet tracer based on a cyclic rgd peptide
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Ramajeyam Selvaraj, Shuanglong Liu, Li Peng Yap, Joseph M Fox, Matthew Hassink, Chiun Wei Huang, Ryan Park, Peter S ContiAbstract:Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the Tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the Tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a Tetrazine-RGD conjugate and 2 mCi (5 μM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the Tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.
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Tetrazine trans cyclooctene ligation for the rapid construction of integrin αvβ targeted pet tracer based on a cyclic rgd peptide
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Ramajeyam Selvaraj, Shuanglong Liu, Li Peng Yap, Joseph M Fox, Matthew Hassink, Chiun Wei Huang, Ryan Park, Peter S ContiAbstract:Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the Tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the Tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a Tetrazine-RGD conjugate and 2 mCi (5 μM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the Tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.
Li Peng Yap - One of the best experts on this subject based on the ideXlab platform.
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improved metabolic stability for 18f pet probes rapidly constructed via Tetrazine trans cyclooctene ligation
Bioconjugate Chemistry, 2015Co-Authors: Ramajeyam Selvaraj, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Srinivasa R Chintala, Li Peng Yap, Peter S Conti, Joseph M FoxAbstract:The fast kinetics and bioorthogonal nature of the Tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an 18F-labeling system based on a CF3-substituted diphenyl-s-Tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-Tetrazine derivative via EDC-mediated coupling. The resulting Tetrazine-RGD conjugate was combined with a 19F-labeled TCO derivative to give HPLC standards. The analogous 18F-labeled TCO derivative was combined with the diphenyl-s-Tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-Tetrazine-RGD combines with an 18F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of Tetrazine-RGD relative to the 18F-labeled TCO (concentration calculated based...
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efficient 18f labeling of cysteine containing peptides and proteins using Tetrazine trans cyclooctene ligation
Molecular Imaging, 2013Co-Authors: Shuanglong Liu, Ramajeyam Selvaraj, Li Peng Yap, Matthew HassinkAbstract:18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the Tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of Tetrazine-RGD (80-100 μM) or 15 μg of Tetrazine-VEGF (6.0 μM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the Tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.
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Tetrazine trans cyclooctene ligation for the rapid construction of integrin αvβ3 targeted pet tracer based on a cyclic rgd peptide
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Ramajeyam Selvaraj, Shuanglong Liu, Li Peng Yap, Joseph M Fox, Matthew Hassink, Chiun Wei Huang, Ryan Park, Peter S ContiAbstract:Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the Tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the Tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a Tetrazine-RGD conjugate and 2 mCi (5 μM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the Tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.
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Tetrazine trans cyclooctene ligation for the rapid construction of integrin αvβ targeted pet tracer based on a cyclic rgd peptide
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Ramajeyam Selvaraj, Shuanglong Liu, Li Peng Yap, Joseph M Fox, Matthew Hassink, Chiun Wei Huang, Ryan Park, Peter S ContiAbstract:Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the Tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the Tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a Tetrazine-RGD conjugate and 2 mCi (5 μM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the Tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.
