(E)-Cyclooct-4-en-1-yl (2,5-dioxopyrrolidin-1-yl) carbonate - CAS 1191901-33-3

(E)-Cyclooct-4-en-1-yl (2,5-dioxopyrrolidin-1-yl) carbonate is a linker widely used in antibody-drug conjugates (ADCs).

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Molecular Formula
C13H17NO5
Molecular Weight
267.28

(E)-Cyclooct-4-en-1-yl (2,5-dioxopyrrolidin-1-yl) carbonate

    • Specification
      • Purity
        95% rel-(1R-4E-pR)-equatorial
        Solubility
        Soluble
        Storage
        Please store the product under the recommended conditions in the Certificate of Analysis.
        Shipping
        Room temperature in continental US; may vary elsewhere.
        IUPAC Name
        [(4E)-cyclooct-4-en-1-yl] (2,5-dioxopyrrolidin-1-yl) carbonate
        Synonyms
        TCO-NHS ester; TCO-NHS; (E)-Cyclooct-4-en-1-yl (2,5-dioxopyrrolidin-1-yl) carbonate
    • Properties
      • InChI Key
        OUGQJOKGFAIFAQ-OWOJBTEDSA-N
        InChI
        InChI=1S/C13H17NO5/c15-11-8-9-12(16)14(11)19-13(17)18-10-6-4-2-1-3-5-7-10/h1-2,10H,3-9H2/b2-1+
        Canonical SMILES
        C1CC=CCCC(C1)OC(=O)ON2C(=O)CCC2=O
    • Reference Reading
      • 1. Comparison of analytical methods for antibody conjugates with application in nuclear imaging - Report from the trenches
        Irene V J Feiner, Beatrice Longo, Vanessa Gómez-Vallejo, Javier Calvo, Marion Chomet, Danielle J Vugts, Albert D Windhorst, Daniel Padro, Matteo Zanda, Luka Rejc, Jordi Llop Nucl Med Biol. 2021 Nov-Dec;102-103:24-33.doi: 10.1016/j.nucmedbio.2021.08.001.Epub 2021 Aug 18.
        Introduction:Monoclonal antibodies (mAbs) are widely used in nuclear imaging. Radiolabelling with positron emitting radionuclides, typically radiometals, requires the incorporation of a bifunctional chelator for the formation of the radiometal-mAb complex. Additionally, mAbs can be conjugated with small molecules capable to undergo bioorthogonal click reactions in vivo, enabling pre-targeting strategies. The determination of the number of functionalities attached to the mAb is critically important to ensure a good labelling yield or to guarantee pre-targeting efficacy. In this work, we compare three different analytical methods for the assessment of average functionalisation and heterogeneity of the conjugated mAbs. Methods:Two selected mAbs (Trastuzumab and Bevacizumab) were randomly conjugated through lysine residues with 3-10 equivalents p-isothiocyanatobenzyl-desferrioxamine (p-NCS-Bz-DFO) or 20-200 equivalents trans-cyclooctene-N-hydroxysuccinimide ester (TCO-NHS). The DFO- or TCO-to-mAb ratio were determined using three different methods: direct titration (radiometric for DFO-conjugated mAbs, photometric for TCO-conjugated mAbs), MALDI/TOF MS mass analysis (Matrix-Assisted Laser Desorption-Ionization/Time of Flight Mass Spectrometry), and UPLC/ESI-TOF MS mass analysis (Ultra High Performance Liquid Chromatography/Electrospray Ionization-Time of Flight Mass Spectrometry). Results:Radiometric and photometric titrations provided information on the average number of DFO and TCO functionalities per mAb respectively. MALDI/TOF MS provided equivalent results to those obtained by titration, although investigation of the heterogeneity of the resulting mixture was challenging and inaccurate. UPLC/ESI-TOF MS resulted in good peak resolution in the case of DFO-conjugated mAbs, where an accurate discrimination of the contribution of mono-, di- and tri-substituted mAbs could be achieved by mathematical fitting of the spectra. However, UPLC/ESI-TOF MS was unable to discriminate between different conjugates when the smaller TCO moiety was attached to the mAbs.Conclusions:The three techniques offered comparable results in terms of determining the average number of conjugates per mAb. Additionally, UPLC/ESI-TOF MS was able to shed a light on the heterogeneity of the resulting functionalised mAbs, especially in the case of DFO-conjugated mAbs. Finally, while using a single analytical method might not be a reliable way to determine the average functionalisation and assess the heterogeneity of the sample, a combination of these methods could substantially improve the characterization of mAb conjugates.
        2. A Pretargeted Imaging Strategy for Immune Checkpoint Ligand PD-L1 Expression in Tumor Based on Bioorthogonal Diels-Alder Click Chemistry
        Lin Qiu, Hui Tan, Qingyu Lin, Zhan Si, Wujian Mao, Tingting Wang, Zhequan Fu, Dengfeng Cheng, Hongcheng Shi Mol Imaging Biol. 2020 Aug;22(4):842-853.doi: 10.1007/s11307-019-01441-3.
        Purpose:The use of antibodies as tracers requires labeling with isotopes with long half-lives due to their slow pharmacokinetics, which creates prohibitively high radiation dose to non-target organs. Pretargeted methodology could avoid the high radiation exposure due to the slow pharmacokinetics of antibodies. In this investigation, we reported the development of a novel pretargeted single photon emission computed tomography (SPECT) imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating immune checkpoint ligand PD-L1 expression in tumor based on bioorthogonal Diels-Alder click chemistry.Procedures:The radioligand [99mTc]HYNIC-PEG11-Tz was achieved by the synthesis of a 6-hydrazinonicotinc acid (HYNIC) modified 1,2,4,5-tetrazine (Tz) and subsequently radiolabeled with technetium-99m (Tc-99m). The stability of [99mTc]HYNIC-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. Atezolizumab was modified with trans-cyclooctene (TCO). The [99mTc]HYNIC-PEG11-Tz and atezolizumab-TCO interaction was tested in vitro. Pretargeted H1975 cell immunoreactivity binding and saturation binding assays were evaluated. Pretargeted biodistribution and SPECT imaging experiments were performed in H1975 and A549 tumor-bearing modal mice to evaluate the PD-L1 expression level. Results:[99mTc]HYNIC-PEG11-Tz was successfully radiosynthesized with a specific activity of 9.25 MBq/μg and a radiochemical purity above 95 % as confirmed by reversed-phase HPLC (RP-HPLC). [99mTc]HYNIC-PEG11-Tz showed favorable stability in NS, PBS, and FBS and rapid blood clearance in mice. The atezolizumab was modified with TCO-NHS ester to produce a conjugate with an average 6.4 TCO moieties as confirmed by liquid chromatograph-mass spectrometer (LC-MS). Size exclusion HPLC revealed almost complete reaction between atezolizumab-TCO and [99mTc]HYNIC-PEG11-Tz in vitro, with the 1:1 Tz-to-mAb reaction providing a conversion yield of 88.65 ± 1.22 %. Pretargeted cell immunoreactivity binding and saturation binding assays showed high affinity to H1975 cells. After allowing 48 h for accumulation of atezolizumab-TCO in H1975 tumor, pretargeted in vivo biodistribution revealed high uptake of the radiotracer in the tumor with a tumor-to-muscle ratio of 27.51 and tumor-to-blood ratio of 1.91. Pretargeted SPECT imaging delineated the H1975 tumor clearly. Pretargeted biodistribution and SPECT imaging in control groups demonstrated a significantly reduced tracer accumulation in the A549 tumor.Conclusions:We have developed a HYNIC-modified Tz derivative, and the HYNIC-PEG11-Tz was labeled with Tc-99m with a high specific activity and radiochemical purity. [99mTc]HYNIC-PEG11-Tz reacted rapidly and almost completely towards atezolizumab-TCO in vitro with the 1:1 Tz-to-mAb reaction. SPECT imaging using the pretargeted strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) demonstrated high-contrast images for high PD-L1 expression H1975 tumor and a low background accumulation of the probe. The pretargeted imaging strategy is a powerful tool for evaluating PD-L1 expression in xenograft mice tumor models and a potential candidate for translational clinical application.
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