Double‐Click Strategy Combining CuAAC and (Thia‐) Diels‐Alder Reactions; Application Toward Peptide Labeling

Abstract

We report an efficient double‐click strategy combining copper‐catalyzed azide–alkyne cycloaddition (CuAAC) with (thia‐)Diels–Alder (DA) reactions, enabled by newly designed heterobifunctional platforms bearing orthogonal clickable groups: alkyne–dithioester, alkyne–maleimide, or azide–diene. These platforms were evaluated through both one‐pot sequential protocols (CuAAC/DA or CuAAC/thia‐DA) and three‐component reactions (3CR), using model substrates with complementary functionalities. The use of a highly reactive s‐cis‐constrained exocyclic diene, enabled rapid cycloadditions with maleimide or phosphonodithioester dienophiles. All reactions were performed under mild, biocompatible conditions (37 °C, 1:1 H₂O/iPrOH, 1–2 hours), and most afforded the desired conjugates in good isolated yields. The methodology was further validated through the successful bioconjugation of three small peptides with either a fluorophore or biotin, demonstrating its efficiency, versatility, and compatibility with biologically relevant functional groups.

A double click‐strategy combining copper‐catalyzed azide‐alkyne cycloaddition with (thia‐)Diels‐Alder reactions involving a reactive s‐cis‐constrained diene is reported. Three different one‐pot protocols (i.e., sequential or three‐component reactions) are proposed and offer valuable guidance for future applications. The reactions are performed under mild conditions (37 °C in H₂O/iPrOH for 1‐2 hours) and can afford the desired bioconjugates in good overall isolated yields. Successfully applied on small peptides, the method demonstrates its efficiency and interest toward applications in chemical biology.

1. Introduction

Introduced by Sharpless in 2001,^{[ 1 ]} the concept of “click chemistry” is now seen as the “gold standard” method for coupling a wide variety of molecular units of interest, including imaging agents,^{[ 2 ]} nucleic acids,^{[ 3 ]} polymers,^{[ 4 ]} metal‐organic frameworks (MOFs),^{[ 5 ]} or even proteins in cellulo.^{[ 6 ]} This approach has enabled a broad spectrum of applications across diverse fields such as material chemistry,^{[ 7 ]} medicinal chemistry,^{[ 8 ]} or chemical biology.^{[ 9 ]} Building on these successful developments, researchers then focused on methodological studies regarding the orthogonality between various click reactions^{[ 10 ]} and multi‐click strategies. A particular area of interest has emerged around the design and synthesis of hetero‐multifunctional linkers or platforms that incorporate at least two distinct and orthogonal clickable functionalities.^{[ 11 ]} This innovative approach facilitates the synthesis of multi‐conjugates by reducing steric hindrance around the reactive site, and enables the rapid incorporation of linkers with specific physicochemical properties (i.e., length, lipophilicity, hydrosolubility) and/or reactivity. Several reviews have highlighted the progress and potential of the multi‐click strategy in the multifunctionalization of biomolecules^{[ 12 ]} and in polymer chemistry.^{[ 13 ]} As the name suggests, the double‐click strategy involves two successive click reactions using a (hetero)bifunctional reagent/platform and two appropriately functionalized partners, ultimately leading to the formation of a (bio)conjugate (Scheme 1) with reports as recent as 2025.^{[ 14 ]} Due to their versatility and good reaction conditions tolerance, well‐established cycloaddition click reactions, such as the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC), the strain‐promoted azide‐alkyne cycloaddition (SPAAC), and the inverse electron demand Diels–Alder reaction (IEDDA), are often employed in multi‐ and double‐click strategies. On the other hand, although some normal electron‐demand Diels‐Alder (NEDDA) reactions involving dienophiles such as maleimides,^{[ 15 ]} or heterodienophiles such as dithioesters^{[ 16} , ^{17 ]} have found some applications in both polymer chemistry and bioconjugation, they remain scarcely explored in the context of multi‐click strategies. This is likely due to the limited availability of hetero‐multifunctional linkers or platforms bearing the appropriate reactive functionalities, such as electron‐rich dienes or electron‐deficient dithioesters, within the current synthetic toolbox.

Scheme 1.

Double‐click strategy and selected examples of reported heterobifunctional platforms

Recently, our group developed an efficient method for the chemoselective labeling of fully deprotected peptides with fluorine‐18¹⁸ or fluorophores^{[ 17 ]} under mild conditions. This approach relies on a catalyst‐free hetero‐Diels‐Alder reaction (HDA) − more specifically a thia‐Diels‐Alder (thia‐DA) reaction − between an electron‐deficient thiocarbonyl heterodienophile (i.e., phosphonodithioester) attached to the peptide and a diene attached to the imaging tool. Notably, for radiofluorination toward in vivo positron emission tomography (PET) imaging, the short half‐life of the ¹⁸F‐radioisotope (t _1/2 = 109.8 minutes) necessitates a thia‐DA cycloaddition with rapid kinetics. To address this, we designed and synthesized a highly reactive s‐cis constrained exocyclic diene that meets the kinetic requirements of ¹⁸F‐chemistry, while remaining stable toward dimerization. However, the Diels‐Alder reaction between this exocyclic diene and maleimide has not yet been used in peptide labeling.

Herein, we describe the synthesis of several small heterobifunctional platforms and their evaluation in a double‐click approach combining CuAAC and NEDDA (either DA or thia‐DA) reactions (Scheme 2). The former was preferred over metal‐free click reactions (namely SPAAC or IEDDA) due to the amide‐like character and small size of the triazole formed, thus limiting steric hindrance around the peptide moiety. On the other hand, the latter includes cycloadditions between diene derivatives based on our exocyclic diene structure and either maleimides or phosphonodithioesters as dienophile partners. For each bifunctional platform, we examined the CuAAC/DA or CuAAC/thia‐DA orthogonality using both one‐pot sequential double‐click and three‐component reaction (3CR) strategies, employing model substrates. Finally, to highlight the efficacy of our approach, the most efficient strategies were applied for the preparation of three labeled small peptide conjugates.

Scheme 2.

Overview of our double‐click strategy and general structures of heterobifunctional platforms described in this work

2. Results and Discussion

The structures of the reaction partners and resulting products from this study are depicted in Figure 1. First, we designed and synthesized in a few steps (see Supporting Information) four small bifunctional platforms incorporating two distinct clickable functionalities: alkyne‐dithioester (BP1a and BP1b), alkyne‐maleimide (BP2), and azide‐diene (BP3). Platforms featuring azide‐maleimide and azide‐dithioester were set aside due to difficulties encountered for their synthesis.^{[ 19 ]} On the other hand, an alkyne‐diene platform was synthesized,^{[ 19 ]} however, it was excluded from this study as it underwent rapid intramolecular [4 + 2] cycloaddition particularly in the presence of a Cu(I)‐catalyst^{[ 20 ]} (i.e., CuAAC conditions), thus compromising its stability. Next, seven substrates (S1–S3, S4a,b, S5a,b) bearing the appropriate clickable functions (i.e., azide, alkyne, exocyclic diene, maleimide, and phosphonodithioformate) were selected. Substrate S1 was commercially available, while the others (S2–S5) were prepared following literature protocols or adapted procedures (see Supporting Information for details). The interest of using the phosphorylated alkyne S2 was considered in terms of facility in the process of analyzing the formation of the different cycloadducts by ³¹P NMR spectroscopy. The different P1–P4 products that we obtained are illustrated in Figure 1.

Figure 1.

Structures of bifunctional platforms (BP), substrates (S), and products (P)

Three strategies were evaluated, depending on whether the click reactions were performed one‐pot sequentially (NEDDA/CuAAC or CuAAC/NEDDA) or simultaneously in a three‐component reaction (3CR). In all cases it was possible to identify the starting materials, the intermediates, and the final products by HPLC and TLC monitoring.

All reactions were carried out under mild conditions, compatible with the intended applications on peptides, at 37 °C in a 1:1 mixture of water and isopropanol (H₂O/iPrOH). Stoichiometric amounts of copper sulfate (CuSO₄) and sodium ascorbate (NaAsc) were deliberately used to ensure high reaction rates for the CuAAC reaction, comparable to those observed for the thia‐DA cycloaddition. The corresponding results are summarized in Table 1.

Table 1.

Results obtained in the different one‐pot double‐click approaches^{[ a ]}

Entry	Double‐click approach	Partners/Sequence	Product	Yield [%]
1	CuAAC/thia‐DA	(S1 + BP1a) + S3	P1a	nd [b]
2	thia‐DA/ CuAAC	(S3 + BP1a) + S1		67
3	3CR	S1 + S3 + BP1a		nd [b]
4	thia‐DA/CuAAC	(S3 + BP1b) + S1	P1b	55
5	CuAAC/DA	(S1 + BP2) + S3	P2	39
6	DA/CuAAC	(S3 + BP2) + S1		61
7	3CR	S1 + S3 + BP2		60
8	CuAAC/DA	(S2 + BP3) + S4a	P3a	46
9	DA/CuAAC	(S4a + BP3) + S2		30
10	3CR	S2 + S4a + BP3		49
11	CuAAC/DA	(S2 + BP3) + S4b	P3b	50
12	DA/CuAAC	(S4b + BP3) + S2		56
13	3CR	S2 + S4b + BP3		54
14 [c]	3CR	S2 + BP3 + S5a	P4a	54
15	CuAAC/thia‐DA	(S2 + BP3) + S5b	P4b	65
16	thia‐DA/CuAAC	(S5b + BP3) + S2		63
17	3CR	S2 + S5b + BP3		43

^[a] Conditions (with 1 equiv BP, 1.2 equiv S). (i) DA or HDA: H₂O/iPrOH (1:1), 37 °C, 1 hour; (ii) CuAAC or 3CR: CuSO₄·H₂O (1 equiv), NaAsc (2 equiv), H₂O/iPrOH (1:1), 37 °C, 1 hour.

^[b] Not determined; the product was not isolated.

^[c] Modified conditions: CuSO₄·5H₂O (10 mol%), NaAsc (20 mol%), EtOH, 60 °C, 3 hours.

In a first experiment, the bifunctional alkyne‐dithioester platform BP1a, the azide partner S1, and the diene partner S3 were subjected to a double‐click CuAAC/thia‐DA sequence (entry 1).

Analysis of the ³¹P NMR spectrum of the crude reaction mixture revealed the complete disappearance of BP1a (signal at ‐4.4 ppm) prior to the addition of the diene S3, along with the appearance of several unidentified species (at least eight other visible signals).

A similar result was obtained in the 3CR version (entry 3), with complete consumption of BP1a after one hour and the detection of at least five of the same unidentified side products. However, in the 3CR experiment, thanks to the presence of diene S3 the thia‐DA reaction took place, and the expected product P1a could be spotted as a mixture of four regio‐/stereoisomers (signals at 19.7, 18.3, 17.9, and 17.7 ppm). Unfortunately, due to the complexity of the mixture, we were unable to isolate P1a in pure form.

We supposed that this result is due to the reactivity of the dithioester function of BP1a when exposed to the copper/ascorbate catalytic system. To assess this hypothesis, we treated BP1a alone with either CuSO₄·5H₂O/NaAsc (1 equiv./2 equiv. in H₂O/iPrOH) or CuI (1 equiv. in DMF) at 37 °C for one hour. In the presence of CuSO₄/NaAsc, complete degradation of BP1a was observed by ³¹P NMR, yielding the same set of side products as in the previous experiments. In contrast, BP1a remained intact under CuI conditions.

When the reaction sequence was reversed, starting with the thia‐DA followed by the CuAAC reaction, the desired conjugate P1a was successfully obtained as an inseparable mixture of four isomers (in a ratio of 17:14:29:40, measured by ³¹P NMR spectroscopy) and isolated in a satisfactory yield of 67% (entry 2).

To illustrate the versatility of the linker structure, we synthesized a second alkyne‐dithioester platform, BP1b, starting from cysteine ester (see Supporting Information). In this case, the phosphonodithioester moiety was placed on the amino group of cysteine, while the propargyl group was introduced on the sulfur atom. Reaction of BP1b with substrates S1 and S3 using the one‐pot thia‐DA/CuAAC double‐click sequence afforded the desired product P1b in 55% isolated yield (entry 4).

We then evaluated the efficiency of the alkyne‐maleimide platform BP2 for coupling the same partners S1 and S3 (entries 5–7). Regardless of the reaction sequence employed, the expected product P2 was obtained. While the CuAAC/thia‐DA sequence gave a moderate yield, higher yields (60–61%) were achieved using either the thia‐DA/CuAAC or the 3CR approach. Lower overall yields were observed in the case where the azide carboxylate S1 was used in the CuAAC as the first step (entry 5). This may be partially attributed to S1 which can be involved in other side reactions such as enolate addition or [3 + 2] cycloaddition on the electron‐poor double bond of the maleimide moiety.

The azide‐diene bifunctional reagent BP3 was used to couple S‐propargyl phosphorothioate S2 with maleimide derivatives S4a or S4b, affording products P3a and P3b, respectively. For P3a, all three methods led to moderate yields (30–49%, entries 8–10). This limited efficiency is likely due to the carboxylic acid function, which may chelate the copper catalyst, thereby slowing the CuAAC reaction and complicating purification. In practice, the crude product had to undergo several washes with ethylenediaminetetraacetic acid (EDTA) to remove residual copper, which also led to partial loss of the desired product.

To overcome this issue, we replaced the acid derivative S4a with its ethyl ester counterpart S4b. This modification led to a significant improvement, with P3b being isolated in higher yields (50–54%, entries 11–13). Notably, the Diels–Alder cycloaddition with maleimide occurred with complete endo selectivity, resulting in the exclusive formation of a single diastereomer for both P3a and P3b.

Finally, BP3 was also reacted with alkyne S2 and the dithioester partner S5b. The desired product P4b was obtained under all conditions tested, with yields of 65% and 63% for the sequential reactions (entries 15 and 16), and a slightly lower yield of 43% in the 3CR approach (entry 17). The presence of a phosphorus atom in the starting materials S2 and S5b, as well as two phosphorus atoms in the final product P4b, enabled straightforward monitoring of the reaction progress and analysis of the crude mixture by ³¹P NMR spectroscopy. For example, in the case of thia‐DA/CuAAC sequence, the spectrum of the crude displayed four distinct signals at 19.2, 18.0, 17.5, and 16.6 ppm, in a ratio of 25:9:11:55, corresponding to the phosphorus atom linked to the quaternary carbon‐stereocenter of the thia‐DA cycloadduct (obtained as an inseparable mixture of four regio‐/stereoisomers). Additionally, a broad signal, consisting in four overlapped peaks between 23.6 and 24.2 ppm, was assigned to the phosphorothioate function linked to the triazole moiety.

We also conducted an experiment involving BP3 and S2, this time using phosphonodithioester S5a bearing a methylsulfanyl substituent. Modified 3CR conditions were applied, using catalytic amounts of Cu(OAc)₂ and sodium ascorbate in ethanol at 60 °C for three hours (entry 14). These conditions improved the solubility of the dithioester and helped to minimize its degradation under CuAAC conditions. Similar to the results obtained for P4b, product P4a was formed as a mixture of four cycloadducts, as observed by ³¹P NMR spectroscopy (signals at 19.6, 18.2, 17.9, and 16.9 ppm in a 22:14:14:50 ratio, along with a broad signal at 23.5 ppm), and was isolated in 54% yield. Notably, in this case, two major isomers P4a‐F1 and P4a‐F2 (representing 22% and 50% of the mixture) were successfully separated by semi‐preparative reversed‐phase HPLC, enabling full structural characterization by NMR spectroscopy (Figure 2 and see Supporting Information). These compounds were identified as the 2,6‐ and 2,3‐regioisomers thanks to carbon‐phosphorus coupling constants. Moreover, the 2D‐NOESY experiments showed that both of them correspond to the endo cycloadducts, with the phosphoryl and methylene‐alkoxy substituents in syn orientation, consistent with the expected stereochemical outcome of the Diels–Alder reaction. The major P4a‐F2 isomer, characterized by two ³¹P NMR signals at 23.5 ppm (P─S) and 16.9 ppm (P─C), was assigned to the endo 2,6‐regioisomer while the second major isomer P4a‐F1, with ³¹P NMR signals at 23.5 (P‐S) and 19.6 (P‐C), was assigned to the endo 2,3‐regioisomer. To summarize, in this example, the thia‐DA cycloaddition proceeded with moderate 2,6‐/2,3‐regioselectivity (64:36) and good endo/exo stereoselectivity (72:28).

Figure 2.

Structures and NMR correlations of the two major P4a isomers

Overall, in this methodological part of the study, the desired products were obtained in most cases with isolated yields exceeding 50% over two steps, either through sequential one‐pot double‐click sequences or via three‐component reactions (3CR). This corresponds to a satisfactory average yield of approximately 75% per click reaction, highlighting the good synthetic potential of the developed strategy.

We next applied our bifunctional platforms in double‐click reactions for the labeling of three small peptides with either a squaraine fluorophore or a biotin moiety, two labels ubiquitously used in chemical biology for imaging and pulldown applications, respectively. As a first example, we focused on labeling a model tripeptide (Arg‐Tyr‐Leu‐NH₂) with a far‐red‐emitting fluorogenic squaraine fluorophore.^{[ 21 ]} The tripeptide was functionalized at its N‐terminus with a phosphonodithioester moiety, while the fluorophore was coupled to a propargyl amine (see Supporting Information). The resulting tripeptide‐dithioester S6 and squaraine‐alkyne S7 were then conjugated via platform BP3 using the thia‐DA/CuAAC sequence, affording the labeled tripeptide P5 in a satisfactory isolated yield of 49% (Scheme 3).

Scheme 3.

Synthesis of fluorophore labeled tripeptide (P5)

As a second application, we employed our method to introduce a biotin label onto a PSMA (prostate‐specific membrane antigen) pseudopeptide. To access the corresponding reaction partners S8 and S9, the PSMA ligand containing a KuE residue (KuE: lysine–urea–glutamate) was functionalized with a phosphonodithioester, while the carboxylic acid group of biotin was coupled to propargyl amine (see Supporting Information). These two partners were then conjugated using the bifunctional platform BP3 via the CuAAC/thia‐DA sequence, affording the desired PSMA–biotin bioconjugate P6, which was isolated in 53% yield (Scheme 4).

Scheme 4.

Synthesis of biotin labeled PSMA ligand (P6)

Finally, platform BP2 was employed in the CuAAC/DA approach for the functionalization of a tetrapeptide (Lys–Tyr–Lys(N₃)–Ala), with a biotin moiety at an internal position of the sequence (Scheme 5). The tetrapeptide–azide S10 was synthesized using a standard solid‐phase peptide synthesis (SPPS) strategy. The biotin–diene partner S11 was prepared by coupling the exocyclic dienol to biotin via an ester linkage. Using the maleimide–alkyne bifunctional platform BP2, the two partners were successfully conjugated, affording the desired biotinylated peptide P7 in a satisfactory isolated yield of 31%.

Scheme 5.

Synthesis of biotin labeled tetrapeptide (P7)

The isolated overall yields of approximately 50% for compounds P5 and P6 correspond to stepwise yields around 72%, which is considered acceptable given the structural complexity of these conjugates. The bioactive peptide P7 was obtained in pure form with an isolated yield of 31% (corresponding to stepwise yields of approximately 56%).

3. Conclusion

In conclusion, we have developed a double‐click strategy based on the orthogonality between CuAAC and NEDDA (DA or thia‐DA) reactions. To this end, we synthesized several small heterobifunctional platforms incorporating two distinct clickable functionalities: alkyne–dithioester, alkyne–maleimide, and azide–diene. Each platform was evaluated using one‐pot sequential double‐click sequences (CuAAC/DA or CuAAC/thia‐DA) as well as 3CR, with simple model substrates bearing complementary reactive groups (azide, alkyne, diene, maleimide, or phosphonodithioformate). The structures of dienic partners were based on a highly reactive s‐cis‐constrained exocyclic diene, enabling rapid [4 + 2] cycloadditions with maleimides or phosphonodithioesters as dienophiles. In most cases, the desired products were obtained in good isolated yields, regardless of the sequence employed. Importantly, the reactions proceeded under mild conditions, at 37 °C in a 1:1 water/isopropanol mixture, within two hours, making them fully compatible with bioconjugation applications. The most efficient protocols were then applied to the synthesis of labeled small peptide conjugates. Three selected peptides functionalized at either terminal or internal positions were successfully coupled with a fluorophore or biotin, yielding the expected conjugates in good yields and high purity. Notably, these double‐click reactions were compatible with reactive functional groups commonly found in peptides, including primary amines, guanidine, hydroxyl (from lysine, arginine, and tyrosine), and urea moieties.

These results demonstrate the efficiency and robustness of these orthogonal double‐click strategies while offering valuable guidance toward applications in bioconjugation and beyond.

Supporting Information

The authors have cited additional references within the Supporting Information.^{[ 18} , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ^{30 ]}

Author Contributions

Conceptualization (TM, DB, MG), Investigation (TM, CVW, LT, VLB, BRN), Resources (DB, MG), Writing Original Draft (TM, MG), Review & Editing (TM, NG, DB, MG), Supervision (TM, NG, DB, MG).

Conflict of Interest

There are no conflicts to declare.

Supporting information

Supporting Information

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.