N-Cyano sulfoximine-mediated thiazole ligation with N-terminal cysteine under mild aqueous conditions

Abstract

An N-terminal cysteine-selective click reaction employing N-cyano sulfoximines enables rapid thiazole formation under mild conditions. These three-dimensional, hydrophilic scaffolds offer high selectivity, tunable reactivity, and improved drug-like properties. The platform holds promise for bioorthogonal conjugation and ligand design in drug discovery applications.

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Efficient and selective chemical ligation methods have greatly advanced chemical biology and materials science.^1–3 Among these, click reactions such as the copper-catalyzed azide–alkyne cycloaddition (CuAAC)^4,5 and sulfur(VI) fluoride exchange (SuFEx) are powerful tools for modular synthesis. In particular, SuFEx using sulfuryl fluoride (SO₂F₂) affords stable linkages, though the resulting connectivities are typically limited to planar geometries.^6,7

The shift from two-dimensional (2D) to three-dimensional (3D) molecular design is emerging as a promising strategy in medicinal chemistry to enhance aqueous solubility without compromising permeability or efficacy. Adoption of non-planar molecular architectures provides an effective strategy to address solubility challenges in drug development (Fig. 1).^8–12

Fig. 1 The thiazolosulfoximine 3D linker identified in this study.

Thionyl tetrafluoride (SOF₄) enables the formation of tetrahedral iminosulfur oxydifluorides with two reactive S–F handles, offering precise spatial control and polyvalency for constructing 3D architectures in biomolecular engineering and materials science.^13–15 However, SuFEx chemistry remains limited by reagent availability, substrate scope, and scalability.^11,16

To establish a practical biomimetic approach, we selected readily accessible sulfoximines that enable the construction of three-dimensional molecular architectures.¹⁷ Bioisosteric replacement of sulfone or sulfonamide groups with sulfoximine moieties significantly enhances aqueous solubility and has facilitated the clinical progression of a lead compound.^18–20

N-Cyano sulfoximines^21–32 are particularly attractive due to their ease of synthesis (Fig. 2a), high aqueous solubility, three-dimensional molecular features, and the presence of a reactive cyano group amenable to click-type conjugation. Their potential as bioorthogonal platforms is further supported by the well-established reactivity of cyano groups—particularly cyanopyridines—with aminothiols.^33–42

Fig. 2 N-Cyano sulfoximine as a bioorthogonal conjugation handle.

Importantly, N-terminal cysteine, a naturally encoded amino acid, was deliberately selected as the reaction partner^43,44 to ensure that the product more closely resembles a naturally occurring structure rather than a purely synthetic one (Fig. 2b). This biomimetic strategy provides a distinct advantage by aligning chemical reactivity with biological relevance and compatibility.

To evaluate the reactivity, we first examined the model reaction of phenylmethyl sulfoximine 1a with cysteine. The desired click reaction proceeded smoothly to afford the corresponding thiazole 2a when the reaction was carried out in PBS (pH 7.4) at 37 °C in the presence of 10 equivalents of cysteine (entry 1, Table 1).⁴⁵

Table 1 Selectivity evaluation with other amino acids

Entry	Amino acids	Product
PBS (Phosphate Buffered Saline).a N-Cyano sulfoximine 1a was completely converted to the desired thiazole 2a, as confirmed by LC/MS analysis.b No reaction.
1
		2a
2		—^b
3		—^b
4		—^b

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No reaction occurred when N-cyano sulfoximine 1a was treated with serine, N-acetylcysteine, or glutathione (GSH), indicating high selectivity towards cysteine (entry 2 to 4, Table 1).

To modulate the reactivity of the nitrile group in the electrophilic N-cyano sulfoximine, various electron-withdrawing or electron-donating groups were introduced onto the phenyl ring. Additionally, heteroatoms were incorporated into the aromatic system to further tune the electron density.

The reactions of 1a and 1b were completed after 72 h and 25 h, respectively (entry 1 and 2, Table 2). For N-cyano sulfoximine 1b, which contains a heteroatom within the aromatic ring, a markedly shorter reaction time was observed (entry 2, Table 2).

Table 2 Reactivity modulation of the electrophilic N-cyano sulfoximines 1a–1d

Entry	N-Cyano sulfoximines	R	X	Reaction time (h)	Conversion (%) to thiazole 2^a
a Confirmed by LC/MS analysis. b After 49 hours, precipitation occurred.
1	1a	H	CH	72	2a (>99)
2	1b	H	N	25	2b (>99)
3	1c	OMe	CH	49	2c (9)^b
4	1d	NO2	CH	49	2d (47)^b

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For para-substituted N-cyano sulfoximines 1c and 1d, poor aqueous solubility prevented dissolution under standard buffer conditions; thus, 2.5% DMSO was added to achieve complete dissolution prior to the reaction. N-Cyano sulfoximine 1d, bearing an electron-withdrawing group, afforded the desired thiazole product 2d with a higher conversion ratio compared to 1c, which possesses an electron-donating group (entry 3 and 4, Table 2).

For comparison, the corresponding N-cyano sulfonamide 1ba was also subjected to the reaction. As shown in the X-ray crystal structures in Fig. 3, the sulfonamide 1ba adopts the 2D conformation, whereas the sulfoximine 1b exhibits the 3D structure. The sulfoximine 1b also demonstrated approximately 1.7-fold higher solubility. Furthermore, in the case of the sulfonamide 1ba, the desired thiazole product was not obtained upon reaction with cysteine.

Fig. 3 Comparison between N-cyano sulfonamide 1ba and sulfoximine 1b.⁴⁶

Based on previous findings indicating high reactivity and good solubility under buffer conditions, N-cyano sulfoximine 1b was selected to evaluate the effect of buffer pH on the reaction completion time for thiazole 2b formation.

Under acidic conditions (pH 4), no reaction was observed, whereas under mildly acidic conditions (pH 6), the reaction reached completion after 72 hours. Under neutral conditions (pH 7.4), the reaction of N-cyano sulfoximine 1b was complete after 24 h. Under basic conditions (pH 8.0–9.0), the reaction was found to reach completion within 7 hours. Interestingly, under strongly basic conditions (pH 10), the reaction was found to reach completion within just 1 hour (Fig. 4).

Fig. 4 Investigation of reaction time as a function of pH (N-cyano sulfoximine 1b to thiazole 2b).

To shorten the reaction completion time at neutral pH, the effect of varying the stoichiometric ratio between N-cyano sulfoximine 1b and cysteine was investigated. As illustrated in Table 3, an excess of the electrophile N-cyano sulfoximine 1b resulted in a faster reaction completion.⁴⁷

Table 3 Effect of stoichiometry on reaction rate

Entry	1b (mM)	Cysteine (mM)	2b (reaction completion time)^a
a This was confirmed by ^1H NMR analysis.
1	10	1	3 h
2	5	1	7.5 h
3	1	10	21 h

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Collectively, the results indicate that the use of 10 equivalents of N-cyano sulfoximine 1b and 1 equivalent of cysteine at neutral pH leads to complete conversion within 3 hours, representing the optimised condition.

A comparison of the predicted physicochemical properties between the reported azidophile⁴⁸ and the N-cysteinophile 1b developed in this study revealed that compound 1b exhibits significantly more hydrophilic characteristics.⁴⁹ The compound 1b was predicted to exhibit greater hydrophilicity than TMTHSI bearing a sulfoximine moiety, as reported by Liskamp and co-workers (Fig. 5).⁵⁰

Fig. 5 Prediction of the physicochemical properties of azidophiles and N-cysteinophile 1b.⁴⁹

To evaluate the practical applicability of the developed reaction, the transformation between N-cyano sulfoximine-derived methionine 1e and N-terminal cysteine was carried out. Gratifyingly, the reaction reached completion within 1.5 h under mild aqueous conditions (Fig. 6).⁵¹ This result is significant as it demonstrates the ability to link the two naturally occurring sulfur-containing amino acids, methionine and cysteine.

Fig. 6 Practical evaluation with N-cyano sulfoximine-derived methionine 1e.

To assess site selectivity under the optimized conditions, peptides bearing either N-terminal or internal cysteine residues were examined. Peptide 1 (CGKSRF) bearing an N-terminal cysteine readily underwent ligation with N-cyano sulfoximine 1b, affording the expected product after 28 h, as confirmed by LC-MS. In contrast, Peptide 2 (KSCGRF), containing an internal cysteine, showed no reaction even after 72 h. Notably, efficient ligation was also observed with the longer Peptide 3 (CGCGESGKSTIVKQMK), which features an N-terminal cysteine, completing the reaction within 3 h (Fig. 7).⁵³

Fig. 7 Reaction of 1b with cysteine-containing peptides.

In summary, we have developed a novel N-cyano sulfoximine-based click reaction that proceeds selectively with N-terminal cysteine to form thiazole linkages under mild, aqueous conditions. Systematic evaluation of structure–reactivity relationships revealed that electronic and solubility properties of the N-cyano sulfoximines significantly influence reaction kinetics. Among the tested analogs, compound 1b, featuring a heteroaryl moiety, exhibited enhanced reactivity and superior aqueous solubility. These findings highlight the potential of N-cyano sulfoximines as versatile and biocompatible electrophilic warheads for bioorthogonal conjugation. The practical utility of this method was further demonstrated by its successful application to a methionine-derived N-cyano sulfoximine substrate 1e. Notably, ligation occurred selectively at N-terminal cysteines, as shown by efficient conjugation of peptides 1 and 3, while no reaction was observed with the internal cysteine of peptide 2, highlighting the potential of this approach for bioconjugation and chemical biology applications.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5md00948k.

CCDC 2481304 and 2481305 contain the supplementary crystallographic data for this paper.^54a,b

Acknowledgements

This research was supported by the Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry and Energy, and Ministry of Health and Welfare (RS-2025-02223190, Republic of Korea) and by KRICT (KK2431-20 and KK2531-20).

Notes and references

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