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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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Diels–Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells
2016Co-Authors: Daniel S. Liu, Anupong Tangpeerachaikul, Ramajeyam Selvaraj, Joseph M Fox, Michael T. Taylor, 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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improved metabolic stability for 18f pet probes rapidly constructed via tetrazine trans cyclooctene ligation
Bioconjugate Chemistry, 2015Co-Authors: Ramajeyam Selvaraj, Li-peng Yap, Srinivasa R. Chintala, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, 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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Improved Metabolic Stability for 18F PET Probes Rapidly Constructed via Tetrazine Trans-Cyclooctene Ligation
2015Co-Authors: Ramajeyam Selvaraj, Li-peng Yap, Srinivasa R. Chintala, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Peter S Conti, Benjamin Giglio, 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 on product’s specific activity). The radiochemical purity of the 18F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that 18F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of 18F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels–Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in 18F-PET probe construction, providing nearly quantitative 18F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system
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conformationally strained trans cyclooctene with improved stability and excellent reactivity in tetrazine ligation
Chemical Science, 2014Co-Authors: Ampofo Darko, Olga Dmitrenko, Ryan A Mehl, Stephen Wallace, Melodie M Machovina, Jason W Chin, Joseph M FoxAbstract:Computation has guided the design of conformationally-strained dioxolane-fused Trans-Cyclooctene (d-TCO) derivatives that display excellent reactivity in the tetrazine ligation. A water soluble derivative of 3,6-dipyridyl-s-tetrazine reacts with d-TCO with a second order rate k2 366,000 (+/- 15,000) M-1s-1 at 25 °C in pure water. Furthermore, d-TCO derivatives can be prepared easily, are accessed through diastereoselective synthesis, and are typically crystalline bench-stable solids that are stable in aqueous solution, blood serum, or in the presence of thiols in buffered solution. GFP with a genetically encoded tetrazine-containing amino acid was site-specifically labelled in vivo by a d-TCO derivative. The fastest bioorthogonal reaction reported to date [k2 3,300,000 (+/- 40,000) M-1s-1 in H2O at 25 °C] is described herein with a cyclopropane-fused Trans-Cyclooctene. d-TCO derivatives display rates within an order of magnitude of these fastest Trans-Cyclooctene reagents, and also display enhanced stability and aqueous solubility.
Ramajeyam Selvaraj - One of the best experts on this subject based on the ideXlab platform.
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Diels–Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells
2016Co-Authors: Daniel S. Liu, Anupong Tangpeerachaikul, Ramajeyam Selvaraj, Joseph M Fox, Michael T. Taylor, 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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improved metabolic stability for 18f pet probes rapidly constructed via tetrazine trans cyclooctene ligation
Bioconjugate Chemistry, 2015Co-Authors: Ramajeyam Selvaraj, Li-peng Yap, Srinivasa R. Chintala, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, 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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Improved Metabolic Stability for 18F PET Probes Rapidly Constructed via Tetrazine Trans-Cyclooctene Ligation
2015Co-Authors: Ramajeyam Selvaraj, Li-peng Yap, Srinivasa R. Chintala, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Peter S Conti, Benjamin Giglio, 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 on product’s specific activity). The radiochemical purity of the 18F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that 18F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of 18F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels–Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in 18F-PET probe construction, providing nearly quantitative 18F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system
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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.
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, Li-peng Yap, Srinivasa R. Chintala, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, 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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Improved Metabolic Stability for 18F PET Probes Rapidly Constructed via Tetrazine Trans-Cyclooctene Ligation
2015Co-Authors: Ramajeyam Selvaraj, Li-peng Yap, Srinivasa R. Chintala, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Peter S Conti, Benjamin Giglio, 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 on product’s specific activity). The radiochemical purity of the 18F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that 18F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of 18F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels–Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in 18F-PET probe construction, providing nearly quantitative 18F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system
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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, Li-peng Yap, Shuanglong Liu, 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, Li-peng Yap, Shuanglong Liu, 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.
Peter S Conti - 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, Li-peng Yap, Srinivasa R. Chintala, Benjamin C Giglio, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, 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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Improved Metabolic Stability for 18F PET Probes Rapidly Constructed via Tetrazine Trans-Cyclooctene Ligation
2015Co-Authors: Ramajeyam Selvaraj, Li-peng Yap, Srinivasa R. Chintala, Shuanglong Liu, Hui Wang, Mengzhe Wang, Hong Yuan, Peter S Conti, Benjamin Giglio, 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 on product’s specific activity). The radiochemical purity of the 18F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that 18F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of 18F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels–Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in 18F-PET probe construction, providing nearly quantitative 18F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system
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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, Li-peng Yap, Shuanglong Liu, 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, Li-peng Yap, Shuanglong Liu, 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 18f labeled probes
ChemInform, 2011Co-Authors: Hancheng Cai, Melissa L Blackman, Peter S Conti, Matthew Hassink, Richard C D Brown, Joseph M FoxAbstract:A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 2,6-diaryl-tetrazines, such as (III), and a 18F-labeled Trans-Cyclooctene analogue of (II) is described.
Matthew Hassink - One of the best experts on this subject based on the ideXlab platform.
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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, Li-peng Yap, Shuanglong Liu, 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, Li-peng Yap, Shuanglong Liu, 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 18f labeled probes
ChemInform, 2011Co-Authors: Hancheng Cai, Melissa L Blackman, Peter S Conti, Matthew Hassink, Richard C D Brown, Joseph M FoxAbstract:A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 2,6-diaryl-tetrazines, such as (III), and a 18F-labeled Trans-Cyclooctene analogue of (II) is described.
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tetrazine trans cyclooctene ligation for the rapid construction of 18f labeled probes
Chemical Communications, 2010Co-Authors: Hancheng Cai, Melissa L Blackman, Peter S Conti, Matthew Hassink, Richard C D Brown, Joseph M FoxAbstract:A radiolabeling method for bioconjugation based on the Diels–Alder reaction between 3,6-diaryl-s-tetrazines and an 18F-labeled Trans-Cyclooctene is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations.
