Readily Accessible Strained Difunctionalized trans‐Cyclooctenes with Fast Click and Release Capabilities

Abstract The click reaction between a functionalized trans‐cyclooctene (TCO) and a tetrazine (Tz) is a compelling method for bioorthogonal conjugation in combination with payload releasing capabilities. However, the synthesis of difunctionalized TCOs remains challenging. As a result, these compounds are poorly accessible, which impedes the development of novel applications. In this work, the scalable and accessible synthesis of a new bioorthogonal difunctionalized TCO is reported in only four single selective high yielding steps starting from commercially available compounds. The TCO‐Tz click reaction was assessed and revealed excellent kinetic rates and subsequently payload release was shown with various functionalized derivatives. Tetrazine triggered release of carbonate and carbamate payloads was demonstrated up to 100 % release efficiency and local drug release was shown in a cellular toxicity study which revealed a >20‐fold increase in cytotoxicity.


Introduction
Ever since the visionary publication of the Sharpless group about click chemistry, this field has grown significantly and has had a tremendous impact on drug discovery, bioconjugation and modern chemistry in general. [1,2] Many click reactions are bioorthogonal, a term first used by Bertozzi in 2003, indicating that functional groups are only mutually reactive and do not interfere with biochemical processes occurring in living systems. [3][4][5][6] Examples of bioorthogonal click reactions include the strain-promoted azide-alkyne cycloaddition (SPAAC), the Staudinger ligation and the strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOQC). [7][8][9] In the last decade, bioorthogonal chemistry has successfully been employed in both diagnostic (e. g. radiolabeling) [10,11] and therapeutic applications such as antibody-drug conjugates (ADCs) [12] and prodrugs. [13][14][15] The inverse-electron-demand Diels-Alder reaction (IEDDA), a reaction between an electron-rich dienophile and an electron-poor diene, has been applied for the ligation of trans-cyclooctenes (TCOs) and tetrazines (Tz). [16] This bioorthogonal ligation usually shows high reaction rates and makes in vivo bioconjugation possible at low concentrations, as demonstrated in numerous studies. [17][18][19][20] As a next step, in 2013 Versteegen et al. reported on the design and synthesis of TCO derivatives that upon reaction with a tetrazine eliminate a substituent at the allylic position of the TCO. This so-called click-to-release approach was applied to enable site-specific and traceless drug release upon TCO-tetrazine ligation. [21,22] The fast reaction kinetics and click and release capability of the TCO-tetrazine adduct opens up countless possibilities for novel applications in drug release and beyond. [23,24] However, the complex synthesis of TCOs -especially of the releasing ones -prevents the roll-out of this versatile technology towards novel applications.
In 2016 Rossin et al. reported the synthesis of the first difunctionalized cis-cyclooctene (CCO) with an allylic moiety in seven steps starting from cis,cis-1,5-cyclooctadiene (1,5-COD, Figure 1A). [17] The authors reported the formation of a mixture of the axial and equatorial difunctionalized TCO derivatives after isomerization with UV-light and a sensitizer as described by the Fox group ( Figure 1B). [25] Currently, the synthetic accessibility of the most commonly used difunctionalized TCOs with releasing properties is rather problematic, mainly due to low yielding steps and a lengthy overall synthesis. [17,24] Ideally, a TCO should have multiple orthogonal handles, a fast click reaction with a tetrazine and a rapid release functionality. In comparison with the difunctionalized TCO of Rossin et al., a shorter and higher yielding synthesis method of a TCO with similar properties is desired.
Herein, we present the straightforward synthesis of a strained and allylic functionalized TCO with two orthogonal handles in only four steps starting from readily available 1,5-COD ( Figure 1C). After separation, both the endo-and exo-diastereomers were diastereoselectively functionalized at the allylic position prior to the photochemical isomerization step of the cis-double bond. A recent continuous flow isomerization method including an aqueous extraction to separate the cisand trans-cyclooctynes, developed by Blanco et al. was employed in this work, facilitating the scalability of this pathway. [26] Functionalized derivatives of the TCO were evaluated for their releasing capabilities. This method represents a major step forward in the ease and efficiency of the synthesis of releasing TCOs.

Results and Discussion
In the design of new TCOs, we were inspired by our previous work on bicyclo[6.1.0]nonyne (BCN), where we demonstrated that the fusion of a cyclopropane with the cyclooctyne induced excellent conjugation rates and kinetics due to the increased ring strain. [8] Cyclopropanated CCOs have also been isomerized by the Fox group to obtain strained TCOs (sTCOs), revealing an enhanced reactivity towards tetrazines as a result of the fusion with the cyclopropane ring. [27,28] Our synthesis commenced with monocyclopropanation of 1,5-COD (1) by reaction with ethyl diazoacetate in the presence of a catalytic amount of copper(II) acetylacetonate under neat conditions to allow 1,5-COD recovery after completion. [29] This afforded a mixture of the exoand endo-diastereomers of 2 (Scheme 1) in a 2 : 1 ratio, respectively (61 % total yield), which were separated by silica gel chromatography. In our experience, substitution reactions on eight-membered rings have proven to be inefficient, therefore we envisioned that activating 2 with N-iodosuccinimide (NIS) in the presence of sodium acetate and acetic acid would incorporate both the iodide and acetate in a single step as in the work of Goto and co-workers. [30] We were pleased to find that this reaction, which proceeds through the corresponding iodiranium ion, leads to highly diastereoselective acetate addition resulting in the acetylated iodohydrin 3 in 83 % yield.
Subsequently, a dehydroiodination reaction was performed with 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) in toluene to afford difunctionalized CCO 4 in 89 % yield, presenting the Oac and CHCO 2 Et groups cis to each other. The stereochemical elucidation of 4 was carried out based on the single-crystal Xray diffraction ( Figure 2).
Compound 4 was then subsequently isomerized into the TCO derivative with UV irradiation at a wavelength of 254 nm in the presence of methyl benzoate (photosensitizer) in a continuous flow setup. [25,26] Selective removal of the TCO with aqueous silver nitrate via liquid-liquid extraction coupled to the photoflow system gave access to multigram amounts of the desired TCO. 14 With the isomerization a new stereoelement was introduced, giving rise to an inseparable mixture of the exo-  diastereomers 5 and 6. Fortunately, the products could be easily separated with silica gel chromatography after ethanolysis of the acetate with potassium carbonate in ethanol to furnish the corresponding hydroxylated TCOs 7 and 8, having the allylic hydroxyl group available for functionalization with a payload.
When the alkene of compound 4 is isomerized, the two planes C sp 3 -C sp 2 -H on both sides of the alkene rotate both clockand counterclockwise to form P-and M-cyclooctenes 5 and 6, respectively. [26,31] In these cases, the resulting TCOs adopt a halfchair-chair conformation where the acetoxy group occupies an axial position in the P-isomer 5 and an equatorial position in the M-isomer 6 (Scheme 2).
Next to the exo-derivatives, the endo-TCO analogues 13 and 14 were synthesized utilizing the same synthetic methodology (Scheme 1, endo-pathway). The iodohydrin step was performed on 2 and afforded 9 diastereoselectively in 82 % yield. The endo-TCO-precursor 10 was obtained after dehydroiodination with DBU and subsequently isomerized into the corresponding endo-TCOs 11 and 12 in a 1 : 1 ratio. Similar to the exo-pathway, we obtained an inseparable mixture of the equatorial and axial diastereomers 11 and 12. Unfortunately, after ethanolysis with potassium carbonate an inseparable mixture of the two diastereoisomers 13 and 14 was obtained.
In order to study the click and release kinetics various allylic functionalized TCO derivatives were synthesized. First, we aimed to monitor the click and release reactions with 19

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Research Article doi.org/10.1002/chem.202203375 methane to afford 15 and 16 in 43 and 32 % yield, respectively. Although the deacetylated endo-TCOs were obtained as a mixture of axial and equatorial diastereoisomers, we still studied the release capabilities. Therefore, the endo-PFP analogues 17 and 18 were synthesized in a similar fashion with DPFPC and DIPEA in an overall 33 % yield obtained as an inseparable mixture of the axial and equatorial derivatives (ratio 1 : 2.5 respectively).
Having the new allylic functionalized TCO derivatives in hand, we set out to investigate the click and release kinetics with 19 F NMR spectroscopy (Scheme 4). The functionalized TCOs 15, 16, 17 and 18 were dissolved in CDCl 3 , a solution of 3,6-di-2-pyridyl-1,2,4,5-tetrazine (DPTZ, 19) was added and a 19 F NMR spectrum was recorded every 48 seconds. In case of the axial-TCO 15, we observed complete release of the PFP moiety within 20 minutes. Further investigation of the click and release reactions with 15 revealed that at À 20°C solely the click conjugate was observed and no release of PFP was obtained. Only after elevating the temperature to 10°C, the PFP carbonate was released upon tautomerization from the conjugated product at such a rate that the intermediate tautomeric product could not be observed (see: Figure S8). The equatorial-TCO 16 clearly showed conjugation with tetrazine 19, but unfortunately no release of the PFP moiety. Instead, a stable tautomerized product with the intact PFP carbonate moiety was formed over time (Scheme 4). This rate is in line with previous studies showing that the equatorial isomer has a significant reactivity decrease because of the steric and possible electronic effects, as again confirmed in this study. [21,32] In case of the diastereomic mixture of the endo-derivatives 17 and 18, we observed a significantly slower click reaction under the same conditions than for the exo-TCOs. Similar to the exo-derivatives, the reaction was followed via 19 F NMR spectroscopy. The different axial and equatorial intermediates and PFP product had an isolated signal which was integrated over time.
The endo-axial isomer 17 showed only minor PFP release within 120 minutes and the equatorial isomer 18 primarily afforded the tautomeric product and no PFP release (see: Figure S9).
Since we observed minor release of PFP with the endo-TCO derivatives 17 and 18, we focused on the exo-isomers, also taking into account that the endo-TCO (13 and 14) was obtained as an inseparable diastereomeric mixture. Besides, the precursor was synthesized in an overall lower yield as compared to the exo-one and an excess of the exo-derivative was formed in the cyclopropanation step (2, 2 : 1 ratio). Conversely, we showed that the mixture of isomers in 2 can be isomerized solely into the exo-isomer with 1 M potassium tert-butoxide in THF and dry ethanol (data not shown). [33] Next, the click kinetics of the axial exo-TCO 7 were investigated upon reaction with a single equivalent of tetrazine 19 or 20. The second-order rate constants were determined in acetonitrile at room temperature. [21] For the click reaction of tetrazine 19 with 7 at 188 μM we found a K 2 = 594.0 M À 1 s À 1 � 12.38 and for tetrazine 20 K 2 = 0.8589 M À 1 s À 1 � 0.001, as also expected due to increased electron density of tetrazine 19 (see: Table 1). When the click reaction was carried out at 97 μM we encountered similar values. These results seem to demonstrate that our new TCO outperforms the currently available releasing difunctional TCOs in the literature in terms of click kinetics.
Next to the carbonate-linked PFP TCOs, we wanted to explore more stable carbamate linked derivatives, as these have already been reported in the literature to provide successful release. [22] We synthesized fluorescently caged exo-TCO derivatives by coupling 7-amino-4-methylcoumarin (AMC) via a carbamate linkage to the allylic alcohol on our TCO. With this approach we could directly asses the release properties of our system with straightforward fluorescence measurements, since the characteristic fluorescence of AMC can easily be quenched by modifying the free amine as also demonstrated in similar TCO-AMC release studies in the recent literature. [34,35] The best synthesis results for the fluorescent TCO derivatives 21 and 22 were obtained by first forming the isocyanate of AMC with triphosgene and DIPEA in toluene at reflux temperature. Subsequently, the TCO derivative with the free allylic hydroxyl 7 or 8 and 4-(dimethylamino)pyridine (DMAP) were dissolved in dichloromethane and the AMC-isocyanate was added. This afforded both the axial and equatorial exo-TCO derivatives 21 and 22 in 63 and 42 % yield, respectively.
Moreover, we also synthesized aliphatic carbamate derivatives of the exo-TCOs to further elucidate the scope of their release properties (Scheme 5). To this extent, we coupled a glycine or sarcosine derivative to the allylic alcohol of the TCO. These compounds were chosen with the work of Carlson and co-workers in mind, who showed that an unmethylated nitrogen -as in the glycine derivative -can result in formation of a dead-end product, while an N-methylated nitrogen as in sarcosine should prevent this. [36] All compounds were generated in a two-step synthesis, wherein hydroxy TCOs 7 and 8 were Scheme 4. 19 F NMR click reaction kinetics study of the axial-TCOs 15 and 17 and equatorial-TCOs 16 and 18 by reacting them with DPTZ (19) in CDCl 3 . TCO 15 revealed complete release of PFP upon the click reaction within 20 minutes. In case of 16, only the click conjugate and the tautomerized products were observed, but no release of PFP. In case of the diastereomic mixture of the endo-derivatives 17 and 18, minor release of PFP upon the click reaction of the axial isomer with 19 was observed, the equatorial primarily afforded a stable tautomeric product.

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Research Article doi.org/10.1002/chem.202203375 first activated with DPFPC, DIPEA and DMAP. In the second step, the amine was added in a basic environment to form the desired carbamate. The glycine derivatives 23 and 24 were obtained in 27 and 42 % yield and the sarcosine derivatives 25 and 26 in 31 and 25 %, respectively.
Next, we examined the elimination kinetics of the caged fluorescent TCO derivatives 21 and 22. Both derivatives were incubated in phosphate-buffered saline (PBS, pH = 7.4) and subsequently a 2.5-fold excess of either tetrazine 19 or 20 was added. The characteristic fluorescence of the released AMC (excitation 380 nm, emission 450 nm) was measured as a function over time in a plate reader. As positive control the maximum amount of possible released AMC was also measured, in this way the releasing efficiency could readily be calculated. As expected, and also observed in our 19 F NMR experiments, the equatorial-TCO derivative 22 furnished minimal release of the fluorescent AMC moiety with tetrazine 19 or 20 (< 3% in 22 h, Figure 3). Nonetheless, the axial analogue 21 afforded complete release of the fluorescent AMC within 23 h upon ligation with 20, after less than three hours already more than 50 % release was observed (k elim = 8.15 × 10 À 5 s À 1 ). As anticipated, the elimination reaction was heavily depending on the used tetrazine, as for tetrazine 19 the release was significantly slower (10 % within 23 h, k elim = 2.96 × 10 À 6 s À 1 ), as also confirmed in the recent literature. [35] Hereafter, tetrazine 20 was chosen to compare the four allylic carbamate TCO derivatives 23-26. The derivatives were dissolved in a mixture of deuterated methanol and PBS (1 : 1, v/ v) and hereafter 20 was added. 1 H NMR spectra were measured over a period of three hours. All derivatives revealed a complete click reaction with 20 within the first five minutes. The axial compounds 23 and 25 showed rapid release of the amino acid payloads (Figure 4, in red) accompanied by a respective decrease in the initial click conjugates (Figure 4, in blue). The respective equatorial derivatives 24 and 26 revealed less release and a more gradual decrease of the click conjugate. Compared

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Research Article doi.org/10.1002/chem.202203375 to their axial analogues, 38 % of the glycine equatorial isomer 24 and 23 % of the sarcosine equatorial isomer 26 released payload successfully.
Hereafter, the stability of the releasing axial TCO was investigated in both PBS and mouse serum at 37°C. The free carboxylic derivative of 7 dissolved in PBS showed no changes in the 1 H NMR spectrum after seven days (see: Figure S1). TCO 7 was stable in mouse serum up to several hours and showed no isomerization into the CCO analogue, however, slow degradation of 7 over time was observed (see: Figure S2).

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Research Article doi.org/10.1002/chem.202203375 After we observed that the new TCO gave satisfactory payload release, we decided to synthesize a drug-conjugated TCO that would release the drug upon tetrazine ligation. This was examined with the release of the chemotherapeutic drug doxorubicin in a cellular environment. [21] The doxorubicin conjugate 27 was synthesized from the 4-nitrophenyl activated TCO and purified with reversed-phase HPLC. The effectiveness of doxorubicin release was tested in HeLa cells through an EC 50 assay ( Figure 5). The cells were incubated for three days at 37°C with varying concentrations of the indicated compounds (tetrazine 20, doxorubicin, TCO-Dox (27), or TCO-Dox (27) + 10 equiv. tetrazine 20). Addition of 10 equivalents of tetrazine 20 increased the cytotoxicity of the doxorubicin TCO conjugate 27 with a 21-fold (75.9 � 12.08 nM vs. 1.654 � 0.418 μM) which indicates successful release of doxorubicin (Table 2). This change cannot be attributed to toxicity of the tetrazine, as its EC 50 was in the mid-micromolar range (29.81 � 5.21 μM). Together, these findings confirmed that conjugation to the TCO reduces doxorubicin's toxicity and that the drug can effectively be released upon tetrazine activation. Besides, HPLC analysis of the same click reaction in solution revealed successful and rapid release of doxorubicin from the conjugate (see: Figure S14).

Conclusion
In conclusion, we have developed a straightforward synthesis of a novel difunctionalized strained TCO with cargo-releasing capabilities in only four high yielding steps. The acetylated iodohydrin intermediate and the subsequent elimination reaction were key steps in this pathway to afford the TCOs in excellent yields. To our knowledge this is the shortest reported synthesis of difunctionalized TCOs with releasing properties allowing improved access to these click reagents for both diagnostic and therapeutic applications. Furthermore, because of the high yields and the continuous photoflow isomerization process our pathway is amenable for larger scale synthesis. The TCOs have two orthogonal handles so that derivatization with different moieties is possible, while the allylic alcohol enables release of a payload upon click reaction with a tetrazine derivative, as demonstrated with carbonate and carbamate linked cargo molecules, even in a cellular environment. Moreover, we identified a TCO that fully retains the allylic moiety upon the click reaction. This product may be valuable for nonreleasable applications requiring stable conjugates with orthogonal handles on the TCO. The broad range applicability of the TCO-tetrazine click and release system combined with this efficient synthetic accessibility will in our view strongly contribute to new in vitro and in vivo biomedical applications in the near future.

exo-TCOs 5 and 6:
A custom-made long-necked flask was charged with an aqueous solution of silver nitrate (7.00 g, 41.2 mmol) in water (100 mL). Next, a solution of 4 (3.50 g, 150 mmolar, 13.9 mmol) and methyl benzoate (3.84 g, 3.56 mL, 28.2 mmol) in deoxygenated heptane (160 mL) and MTBE (40 mL) was loaded into a UV irradiation setup (as described in Blanco-Ania et al.). [26] The continuous process ran for 16 h. Next, the biphasic reaction mixture was loaded into a separation funnel and the aqueous layer was collected. The organic layer was washed with water (100 mL). The combined aqueous layers were washed with heptane (100 mL