Trans-cyclooctenes Research Articles

HPMA-Based Nanoparticles for Fast, Bioorthogonal iEDDA Ligation.

Fast and bioorthogonally reacting nanoparticles are attractive tools for biomedical applications such as tumor pretargeting. In this study, we designed an amphiphilic block copolymer system based on HPMA using different strategies to introduce the highly reactive click units 1,2,4,5-tetrazines (Tz) either at the chain end (Tz-CTA) or statistical into the hydrophobic block. This reactive group undergoes a rapid, bioorthogonal inverse electron-demand Diels–Alder reaction (iEDDA) with trans-cyclooctenes (TCO). Subsequently, this polymer platform was used for the preparation of different Tz-covered nanoparticles, such as micelles and colloids. Thereby it was found that the reactivity of the polymeric micelles is comparable to that of the low molar mass tetrazines. The core-cross-linked micelles can be successfully conjugated at rather low concentrations to large biomacromolecules like antibodies, not only in physiological buffer, but also in human blood plasma, which was confirmed by fluorescence correlation spectroscopy (FCS).

Studies on the stability and stabilization of trans-cyclooctenes through radical inhibition and silver (I) metal complexation

Conformationally strained trans-cyclooctenes (TCOs) engage in bioorthogonal reactions with tetrazines with second order rate constants that can exceed 106 M−1s−1. The goal of this study was to provide insight into the stability of TCO reagents and to develop methods for stabilizing TCO reagents for long-term storage. The radical inhibitor Trolox suppresses TCO isomerization under high thiol concentrations and TCO shelf-life can be greatly extended by protecting them as stable Ag(I) metal complexes. 1H NMR studies show that Ag-complexation is thermodynamically favorable but the kinetics of dissociation are very rapid, and TCO•AgNO3 complexes are immediately dissociated upon addition of NaCl which is present in high concentration in cell media. The AgNO3 complex of a highly reactive s-TCO-TAMRA conjugate was shown to label a protein-tetrazine conjugate in live cells with faster kinetics and similar labeling yield relative to a ‘traditional’ TCO-TAMRA conjugate.

Rapid Production of trans‐Cyclooctenes in Continuous Flow

AbstractA new method for rapid larger‐scale production of trans‐cyclooctenes (TCOs) in continuous flow was developed: Up to 2.2 g/h of specific TCOs can be produced by using this procedure. This method utilizes the classical photoisomerization of cis‐cyclooctenes (CCOs) for the synthesis of TCOs but substitutes the conventionally used silver nitrate impregnated silica gel column for a liquid−liquid extraction module. In addition, this method provides the possibility of external addition of a substrate to scale up and speed up the production of TCOs because the concentration of the CCO is kept constant. This method was applied for the synthesis of several commonly used TCOs.

DOTA-tetrazine probes with modified linkers for tumor pretargeting

IntroductionPretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels–Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO–antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes. MethodsFour new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum. The in vivo biodistribution profile and tumor-targeting potential of the probes were evaluated in LS174T tumor-bearing mice pretargeted with TCO–antibody conjugates using non-pretargeted mice as control. ResultsRadiolabeling of all probes proceeded in high yields providing the 177Lu-labeled tetrazines in >95% radiochemical purity without any further purification. In mouse serum, half-lives of the probes varied between 8 and 13 h, with the exception of the most lipophilic probe, [177Lu]1b, with a serum half-life of less than 1 h. This probe also showed the fastest blood clearance (t1/2 = 5.4 min), more than 2-fold faster than PEG-linked probes [177Lu]3 and [177Lu]4, and even 3-fold faster than the other small probes without the PEG-linker, [177Lu]1a and [177Lu]2. In the pretargeting experiments, tumor uptake of the lead probe [177Lu]4 (~6 %ID/g) was most closely approached by [177Lu]2, followed by [177Lu]3 and [177Lu]1a. While all the smaller and more lipophilic probes suffered from increased liver uptake, the PEG-linked probe [177Lu]3 with its additional negative charge surprisingly showed the highest kidney uptake among all of the probes. ConclusionThe in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [177Lu]Lu-DOTA-PEG11-Tz ([177Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.

Efficient low-cost preparation oftrans-cyclooctenes using a simplified flow setup for photoisomerization.

Bioorthogonal ligations have emerged as highly versatile chemical tools for biomedical research. The exceptionally fast reaction between 1,2,4,5-tetrazines and trans-cyclooctenes (TCOs), also known as tetrazine ligation, is frequently used in this regard. Growing numbers of applications for the tetrazine ligation led to an increased demand for TCO compounds, whose commercial availability is still very limited. Reported photochemical procedures for the preparation of TCOs using flow chemistry are straightforward and high yielding but require expensive equipment. Within this contribution, we present the construction and characterization of a low-cost flow photoreactor assembled from readily accessible components. Syntheses of all commonly used trans-cyclooctene derivatives were successfully carried out using the described system. We are convinced that the presented system for photoisomerization will promote access to bioorthogonally reactive TCO derivatives.Graphical abstractElectronic supplementary materialThe online version of this article (doi:10.1007/s00706-016-1668-z) contains supplementary material, which is available to authorized users.

Thiiranation of 2′-Adamantylidene-9-benzonorbornenylidene andcis- andtrans-Cyclooctenes with 4,4′-Dithiodimorpholine in Acetic Anhydride

Thiiranation of 2'-adamantylidene-9-benzonorbomenylidene (1) with 4,4'-dithiodimorpholine (5) in Ac 2 O at -15°C gave thiiranes 2 and 3 without the decomposition of the thermodynamically less stable 3. cis- and trans-Cyclooctenes underwent thiiranation with retention of their stereochemistry.