1,3-Butadiynyl sulfide-based compact trialkyne platform molecule for sequential assembly of three azides

Abstract

A compact trialkyne platform with a silyl-protected 1,3-butadiynyl sulfide moiety and a terminal alkyne group has been developed for sequential regioselective transition metal-catalyzed triazole formation reactions with three azides. This method enabled the facile construction of a low-molecular-weight triazole library and the synthesis of middle-molecular-weight trifunctional probes for protein modification.

---

The sequential assembly of components into a platform molecule with multiple connectable groups is an efficient approach for the synthesis of a wide range of compounds. This strategy enables the concise construction of chemical libraries and the synthesis of multifunctional compounds. Particularly, methods employing sequential click reactions¹ such as azide–alkyne cycloadditions offer a reliable and efficient approach to multicomponent assembly.^2–5 In this context, our research group has developed a series of platform molecules with multiple clickable groups.³ For instance, we previously reported various platform molecules with three or four distinguishable azido groups.^3a,d These platform molecules enabled facile synthesis of multitriazoles via sequential reactions with terminal alkynes, strained cycloalkynes, and β-keto carbonyl compounds and successfully applied to the development of multifunctional probes for protein modification (Scheme 1A). Other groups reported different triclickable platform molecules with three distinguishable alkyne moieties (Scheme 1B and C).⁴ These trialkyne platform molecules facilitate the triple click assembly compared with our multiazido and other hetero-triclickable platform molecules⁵ because only azides are needed as reaction partners. However, these platform molecules require multistep synthesis procedures and are not suitable for the construction of libraries consisting of low-molecular-weight compounds owing to the large size of their core structures. Herein, we report a novel trialkyne platform molecule 1 with a minimum required structure, which was readily synthesizable enabling the sequential assembly of three azides (Scheme 1D). The electron-rich, strongly coordinating thioalkyne moiety of 1 was expected to react selectively by transition metal-catalyzed azide–thioalkyne cycloaddition before or after the copper-catalyzed azide–alkyne cycloaddition (CuAAC) at the terminal alkyne moiety.^6–8

Scheme 1 Distinguishable homo-triclickable platform molecules for the synthesis of tristriazoles via sequential click reactions.

1,3-Butadiynyl homopropargyl sulfide 1 was synthesized via a one-pot procedure from 1,4-bis(trimethylsilyl)-1,3-butadiyne (2); monolithiation of 2 with MeLi·LiBr,⁹ followed by treatment with thiosulfonate¹⁰3a afforded triyne 1 as a bench-stable oil (Scheme 2). This method was effective for preparing other simple alkyl 1,3-butadiynyl sulfides (Scheme S1, ESI†) except propargyl analog 4.¹¹

Scheme 2 Synthesis of 1,3-butadiynyl sulfides.

Although reactions of 1,3-butadiyne¹² or thioalkyne^6–8 with azides affording the corresponding triazoles were previously reported, the reaction between 1,3-butadiynyl sulfide and azides remained unexplored. Therefore, before using trialkyne 1, we investigated the reaction of 1,3-butadiynyl sulfide using 5a as a model substrate (Table 1). When the reaction of 5a with benzyl azide (6a) was conducted under the reported ruthenium-catalyzed azide–thioalkyne cycloaddition (RuAtAC) conditions,^6b triazole 7a was obtained as a sole product in 69% yield, indicating that the reaction occurred chemo- and regioselectively at the thioalkyne moiety (entry 1). The reaction with an iridium⁷ or rhodium⁸ complex resulted in lower yield (entries 2 and 3). Using the ruthenium complex with triphenylphosphine ligands instead of a COD ligand provided the best result, affording 7a in 87% yield (entry 5). This reaction could be conducted in various solvents, such as 1,4-dioxane, toluene, and water (entries 6–8). The optimized conditions was applicable to the reactions with various azides (Scheme S2, ESI†).

Table 1 Optimization of reaction conditions for triazole formation at the thioalkyne moiety of 1,3-butadiynyl sulfide 5a

Entry	Catalyst	Solvent	Yield (%)
a A trace amounts of the desilylprotonated derivative of 7a was detected.
1	Cp*RuCl(cod)	CH2Cl2	69^a
2	[IrCl(cod)]2	CH2Cl2	37
3	[RhCl(CO)2]2	CH2Cl2	30
4	[Ru(p-cym)Cl2]2	CH2Cl2	0
5	Cp*RuCl(PPh3)2	CH2Cl2	87
6	Cp*RuCl(PPh3)2	1,4-Dioxane	83
7	Cp*RuCl(PPh3)2	Toluene	80
8	Cp*RuCl(PPh3)2	H2O	74^a

---

Conducting the reaction of trialkyne platform 1 with azide 6a under the optimized RuAtAC conditions afforded the desired triazole 8 in 25% (¹H NMR yield). However, the formation of another triazole and bistriazole reacted at the terminal alkyne was also observed, which is consistent with the known reactivity of terminal alkynes with azides using a ruthenium catalyst.^6a Although the reaction using [IrCl(cod)]₂ selectively afforded 8, this approach required to use increased amounts of the catalyst to obtain 8 in a reasonable yield (Scheme 3A). Therefore, no further investigation was performed on this approach.

Scheme 3 Sequential assembly of three azides onto trialkyne 1. (A) First triazole formation via RuAtAC. (B) CuAAC with 1 followed by the second and third triazole formations in two ways.

Alternatively, since internal thioalkynes were reported inert under CuAAC conditions,^6b,8b,c we decided to conduct the first triazole formation at the terminal alkyne moiety of trialkyne 1. The reaction of 1 with azide 6a using a cationic copper catalyst afforded triazole 9a, indicating the selective reaction at the terminal alkyne (Scheme 3B). The second triazole formation of diyne 9a with 6a using the ruthenium catalyst proceeded chemo- and regioselectively at the thioalkyne moiety, and subsequent deprotection of the silyl group with TBAF afforded bistriazole 10aa. The remaining terminal alkyne was employed for the third triazole formation with 6avia CuAAC to afford tristriazole 11aaa. The second triazole formation of diyne 9a was also feasible at the silyl-protected alkyne moiety. CuAAC with 6a in the presence of TBAF resulted in desilylprotonation and subsequent click reaction in one-pot¹³ to yield bistriazole 12aa. For the third triazole formation at the thioalkyne moiety of 12aa, an iridium catalyst afforded a better result (79% yield of tristriazole 11aaa using 11 mol% of [IrCl(cod)]₂) than a ruthenium catalyst (34% yield using 10 mol% of Cp*RuCl(PPh₃)₂). These results demonstrate that trialkyne 1 serves as an efficient triclickable platform for the facile synthesis of tristriazoles.

In principle, various types of triazoles can be synthesized from trialkyne platform 1 simply by changing the azide in each step, enabling the facile construction of a low-molecular-weight compound library (Scheme 4A).^4c Theoretically, CuAAC/RuAtAC/deprotection/CuAAC and CuAAC/deprotection–CuAAC sequences can provide a chemical library consisting of 48 triazole derivatives that contain three types of monotriazoles 9, 18 types of bistriazoles 10 and 12, and 27 types of tristriazoles 11 only from four molecules, i.e., trialkyne 1 and three types of azides (Scheme 4B). To verify this concept, we synthesized 16 of these compounds using three azides 6g–6i bearing fluoro, ester, and Boc-protected amino groups, respectively, which are substructures commonly found in pharmaceuticals. The first triazole formation via CuAAC of 1 with azides 6g–6i afforded monotriazoles 9g–9i, respectively, in 91–93% yields. Subsequently, the second triazole formation via RuAtAC with 6g, 6h, or 6i, respectively, followed by desilylprotonation gave six bistriazoles 10 in 70–94% yields. The second triazole formation through the deprotection CuAAC sequences afforded isomeric bistriazole 12gh in 92% yield. Finally, the third cycloaddition via CuAAC with azides 6g–6i resulted in six tristriazoles 11 in 78–88% yields. The constructed compound library contains 16 triazoles with molecular weights between 335 and 641, which are preferable for drug candidates.¹⁴ Our method outperformed previously reported strategies in terms of the ease of the synthesis of trialkyne platform 1 and the compactness of the core structure of the resulting tristriazoles (Scheme S3, ESI†). Other advantage of our method is that the size of the compound library can be easily expanded by adopting several simple approaches, including (1) the use of a broader variety of azide compounds, (2) performing RuAAC at the alkyne moiety of bistriazoles to afford regioisomeric 1,5-triazoles, (3) oxidation of sulfide to sulfoxide or sulfone and subsequent transformation such as S_NAr reactions or cross-coupling reactions, and (4) reduction or other transformations at the alkyne moiety of mono- or bistriazoles.

Scheme 4 Construction of a triazole library using trialkyne 1 and three azides 6g–6i. ^a Deprotection of the trimethylsilyl group was performed using K₂CO₃ in EtOH to avoid ester hydrolysis. The total yields over two steps are shown.

We further demonstrated the utility of trialkyne platform 1 by preparing a trifunctional probe using azide modules including two HaloTag ligands 13_short and 13_long and two biotin derivatives 15_short and 15_long with different linker lengths, as well as tetraethylsulforhodamine derivative 14 (Scheme 5A). The CuAAC of trialkyne 1 with 13_short or 13_long, followed by RuAtAC with 14 and subsequent silyl deprotection, afforded fluorescent HaloTag ligands 17_short and 17_long. The CuAAC of 17_short and 17_long with biotin–azides 15_short and 15_long, respectively, proceeded smoothly to afford trifunctional molecules 18_short and 18_long with molecular weights of 1721 and 2419, respectively.

Scheme 5 Protein modification using trifunctional probes prepared from 1. (A) Synthesis of middle-molecular-weight multifunctional molecules from 1 and functional azide modules. (B) SDS-PAGE analysis of GST-fused HaloTag protein eluted from the resin after modification with multifunctional probes (lanes 2–5).

The synthesized multifunctional molecules were applied for protein modification (Scheme 5B). After adding each probe candidate 17 and 18 to a cell lysate containing a GST-fused HaloTag protein (59 kDa), an SDS-PAGE analysis was performed. The gels were analyzed by fluorescence detection, followed by a Western blot analysis with HRP-conjugated streptavidin or CBB-staining. As a result, the rhodamine-labeled HaloTag proteins were clearly detected in the fluorescence analysis in all experiments, indicating that the covalent formation between HaloTag protein and the ligands proceeded efficiently (lanes 2–5). Meanwhile, the protein labeled with 18_long was successfully detected in the Western blot analysis, whereas that with 18_short was not, indicating that the linker must possess a sufficient length for the streptavidin recognition of the biotinylated protein (lanes 4 and 5).^3a These results showcase that trialkyne 1 is also a useful platform for the development of effective multifunctional probes because it can expeditiously supply a small library of probe candidates.

In summary, we developed a compact triclickable platform molecule that enabled assembly of three azides via three sequential chemo- and regioselective triazole formation reactions. This method enabled not only the facile construction of a low-molecular-weight tristriazole library that is favorable for drug discovery purposes but also the synthesis of middle-molecular-weight multifunctional molecules that could be used as a dual-detectable probe for protein modification. Further studies on the application of this platform molecule for the synthesis of multifunctional molecules for biological applications are currently underway in our laboratory.

This work was supported by AMED under Grant Number JP24ama121043 (BINDS); JSPS KAKENHI Grant Numbers JP23H02091 and JP23K26784 (Scientific Research (B); T. H.), JP23K17911 (Challenging Research (Exploratory); T. H.), JP23K13853 (Early-Career Scientists; J. T.); JST, CREST under Grant Number JPMJCR22E3 (T. H.); the Cooperative Research Program of “Network Joint Research Center for Materials and Devices (MEXT)”; and the Cooperative Research Project of Research Center for Biomedical Engineering.

Data availability

All the data supporting this article have been included in ESI.†

Conflicts of interest

There are no conflicts to declare.

Notes and references

1. (a) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed., 2001, 40, 2004; (b) C. S. McKay and M. G. Finn, Chem. Biol., 2014, 21, 1075.
2. For a recent review of multiclickable platform molecules, see: H. Tanimoto and T. Tomohiro, Chem. Commun., 2024, 60, 12062 , and references cited therein.
3. For our recent examples of platform molecules, see: (a) S. Yoshida, K. Kanno, I. Kii, Y. Misawa, M. Hagiwara and T. Hosoya, Chem. Commun., 2018, 54, 3705; (b) S. Yoshida, T. Kuribara, H. Ito, T. Meguro, Y. Nishiyama, F. Karaki, Y. Hatakeyama, Y. Koike, I. Kii and T. Hosoya, Chem. Commun., 2019, 55, 3556; (c) T. Meguro, Y. Sakata, T. Morita, T. Hosoya and S. Yoshida, Chem. Commun., 2020, 56, 4720; (d) S. Yoshida, Y. Sakata, Y. Misawa, T. Morita, T. Kuribara, H. Ito, Y. Koike, I. Kii and T. Hosoya, Chem. Commun., 2021, 57, 899.
4. For examples of platform molecules with distinguishable three alkyne moieties, see: (a) P. R. Werkhoven, H. van de Langemheen, S. van der Waals, J. A. W. Kruijtzer and R. M. J. Liskamp, J. Pept. Sci., 2014, 20, 235; (b) V. Vaněk, J. Pícha, B. Fabre, M. Buděšínský, M. Lepšík and J. Jiráček, Eur. J. Org. Chem., 2015, 3689; (c) B. Fabre, J. Pícha, V. Vaněk, I. Selicharová, M. Chrudinová, M. Collinsová, L. Žáková, M. Buděšínský and J. Jiráček, ACS Comb. Sci., 2016, 18, 710.
5. For examples of other triclickable platform molecules, see: (a) I. E. Valverde, A. F. Delmas and V. Aucagne, Tetrahedron, 2009, 65, 7597; (b) B. Fabre, J. Pícha, V. Vaněk, M. Buděšínský and J. Jiráček, Molecules, 2015, 20, 19310; (c) A.-C. Knall, M. Hollauf, R. Saf and C. Slugovc, Org. Biomol. Chem., 2016, 14, 10576; (d) T. Yokoi, T. Ueda, H. Tanimoto, T. Morimoto and K. Kakiuchi, Chem. Commun., 2019, 55, 1891; (e) M. Pantin, J. Caillé, F. Boeda, L. Fontaine, M. S. M. Pearson-Long and P. Bertus, Eur. J. Org. Chem., 2019, 7359; (f) H. Takemura, G. Orimoto, A. Kobayashi, T. Hosoya and S. Yoshida, Org. Biomol. Chem., 2022, 20, 6007; (g) M. Hamada, G. Orimoto and S. Yoshida, Chem. Commun., 2024, 60, 7930.
6. For RuAtAC, see: (a) J. R. Johansson, T. Beke-Somfai, A. S. Stålsmeden and N. Kann, Chem. Rev., 2016, 116, 14726; (b) P. Destito, J. R. Couceiro, H. Faustino, F. López and J. L. Mascareñas, Angew. Chem., Int. Ed., 2017, 56, 10766; (c) A. Gutiérrez-González, P. Destito, J. R. Couceiro, C. Pérez-González, F. López and J. L. Mascareñas, Angew. Chem., Int. Ed., 2021, 60, 16059.
7. For IrAtAC, see: (a) S. Ding, G. Jia and J. Sun, Angew. Chem., Int. Ed., 2014, 53, 1877; (b) M. Li, N. Zheng, J. Li, Y. Zheng and W. Song, Green Chem., 2020, 22, 2394; (c) K. Sugiyama, Y. Sakata, T. Niwa, S. Yoshida and T. Hosoya, Chem. Commun., 2022, 58, 6235.
8. For RhAtAC, see: (a) J. Ma and S. Ding, Asian J. Org. Chem., 2020, 9, 1872; (b) W. Song, N. Zheng, M. Li, K. Dong, J. Li, K. Ullah and Y. Zheng, Org. Lett., 2018, 20, 6705; (c) W. Song, N. Zheng, M. Li, J. He, J. Li, K. Dong, K. Ullah and Y. Zheng, Adv. Synth. Catal., 2019, 361, 469.
9. C. Heinz and N. Cramer, J. Am. Chem. Soc., 2015, 137, 11278.
10. (a) S. Yoshida, Y. Sugimura, Y. Hazama, Y. Nishiyama, T. Yano, S. Shimizu and T. Hosoya, Chem. Commun., 2015, 51, 16613; (b) K. Kanemoto, S. Yoshida and T. Hosoya, Org. Lett., 2019, 21, 3172.
11. The formation of trialkyne 4 was observed, but it completely decomposed during isolation by silica-gel column chromatography. Although we could not identify the decomposed products, [3,3] sigmatropic rearrangement of similar alkynyl propargyl sulfides was previously reported. See: (a) S. Aoyagi, M. Hakoishi, M. Suzuki, Y. Nakanoya, K. Shimada and Y. Takikawa, Tetrahedron Lett., 2006, 47, 7763; (b) S. Aoyagi, K. Sugimura, N. Kanno, D. Watanabe, K. Shimada and Y. Takikawa, Chem. Lett., 2006, 35, 992.
12. (a) B. C. Doak, M. J. Scanlon and J. S. Simpson, Org. Lett., 2011, 13, 537; (b) V. Fiandanese, D. Bottalico, G. Marchese, A. Punzi and F. Capuzzolo, Tetrahedron, 2009, 65, 10573; (c) A. I. Govdi, N. A. Danilkina, A. V. Ponomarev and I. A. Balova, J. Org. Chem., 2019, 84, 1925.
13. (a) B. C. Englert, S. Bakbak and U. H. F. Bunz, Macromolecules, 2005, 38, 5868; (b) S. Maisonneuve, Q. Fang and J. Xie, Tetrahedron, 2008, 64, 8716.
14. (a) C. A. Lipinski, Drug Discovery Today, 2004, 1, 337; (b) M. Gu, S. Sun, Q. You and L. Wang, Molecules, 2023, 28, 7941.

---

Footnote

† Electronic supplementary information (ESI) available: General remarks, experimental procedures, characterization data of new compounds, references and NMR charts. See DOI: https://doi.org/10.1039/d4cc05205f

---