Palladium-catalysed carbonylation of aryl diazonium salts to access bioconjugation-ready esters

Abstract

We report a palladium-catalyzed esterification protocol using aryl diazonium salts and N-hydroxysuccinimidyl (NHS) formate as a CO surrogate, avoiding the use of toxic CO gas. This mild and efficient method employs readily available starting materials and affords NHS esters suitable for late-stage functionalization and bioconjugation applications.

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N-Hydroxysuccinimidyl esters (NHS esters) are used as vital components in synthetic organic chemistry.¹ NHS esters are very stable and easy to handle. Furthermore, they can be readily transformed into amides, esters, and thioesters by functionalization with various amines, alcohols, and thiols under mild reaction conditions.² The traditional method for synthesizing NHS esters involves the reaction of carboxylic acids with coupling reagents such as DCC. However, this approach has several drawbacks, such as the formation of urea byproducts that complicate product purification and the inherent toxicity of the coupling agents. There are only a few reported methods for the synthesis of NHS active esters (Scheme 1). One approach involves the amidation of aromatic aldehydes or alcohols with NHS under oxidative conditions.³ In 2003, Wentland et al. reported an efficient method for preparing NHS esters via palladium-catalyzed carbonylation of aryl halides or triflates under a carbon monoxide (CO) atmosphere in the presence of NHS.⁴ More recently, Skrydstrup et al. elegantly demonstrated the synthesis of NHS esters through palladium-catalyzed carbonylation of (hetero)aromatic bromides, employing an ex situ CO generation strategy using two-chamber equipment.⁵

Scheme 1 Synthetic strategies towards redox active NHS esters.

One challenge associated with these approaches is the use of toxic CO gas. In 2015, Levacher et al. reported a Pd-catalyzed carbonylation method for synthesizing NHS esters from (hetero)aryl, alkenyl, and allyl halides.⁶ Although effective, this protocol involves the use of relatively expensive halide precursors. In 2022, Zhao et al. developed a Pd-catalyzed NHS ester synthesis via C–H thianthrenation, enabling access to diverse esters from aryl thianthrenium salts.⁷

In recent years, aryl diazonium salts have emerged as valuable building blocks in contemporary synthetic chemistry due to their economical synthesis, broad functional group compatibility and ability to undergo diverse transformations under mild conditions.⁸ These salts, typically prepared from aniline derivatives, have been exploited as powerful intermediates in various transformations, enabling the introduction of diverse functional groups and scaffolds.^9–15

Inspired by these favourable characteristics of aryl diazonium salts, we envisioned that aryl diazonium salts could serve as effective substrates for the synthesis of NHS esters under mild, CO-free conditions. Herein, we report a palladium-catalysed carbonylative strategy that utilizes aryl diazonium salts 1 and N-hydroxysuccinimidyl formate 2 to efficiently generate NHS esters.

Preliminary experiments were performed using 4-methoxy phenyl diazonium tetrafluoroborate as a model substrate. To our delight, the coupling reaction of 1a (1 equiv.) with 2 (2 equiv.) carried out in the presence of Pd(OAc)₂ (10 mol%), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos, 10 mol%), Ag₂CO₃ (0.5 equiv.) and Et₃N (1.2 equiv.) in THF at room temperature for 12 h, afforded the desired ester product 3a in 58% GC yield (Table 1, entry 2).

Table 1 Optimization of the reaction conditions^a

Entry	Variations from standard conditions	Yield^b (%)
a Standard conditions: 1a (0.45 mmol), 2 (1.35 mmol), Pd(OAc)2 (10 mol%), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (10 mol%), Ag2CO3 (0.5 equiv.) and Et3N (1.2 equiv.) in THF at room temperature for 12 h. b GC yield. c Isolated yield in the parentheses.
1	None	83 (81)^c
2	2 equiv. of 2	58
3	2-MeTHF/MeCN/toluene/DCE as solvents	79/70/58/69
4	Pd(PPh3)4/PdCl2 instead of Pd(OAc)2	50/42
5	No ligand	21
6	PPh3/dppf instead of Xantphos	71/24
7	No Ag-salt	Trace
8	AgOAc instead of Ag2CO3	46
9	60 °C instead of room temperature	85
10	Cs2CO3/DMAP instead of Et3N	54/3

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Encouraged by these results, we aimed to optimize the reaction conditions to improve the yield of the desired product. Interestingly, increasing the amount of 2 to 3 equiv. led to a significant enhancement in yield of 3a, from 58% to 83% (Table 1, entries 1 and 2). Next, we explored different solvents and found that 2-MeTHF provided a comparable yield of approximately 79% (Table 1, entry 3). The use of solvents like MeCN, toluene, DCE decreased the yield of the product (Table 1, entry 3). Substituting Pd(OAc)₂ with Pd(PPh₃)₄ or PdCl₂ had a detrimental effect on the reaction, leading to a reduced yield of the desired product (Table 1, entry 4). In the absence of ligand, the reaction showed a marked decrease in product yield (Table 1, entry 5), demonstrating the crucial role of the ligand in promoting the reaction. Ligands like PPh₃ and dppf, were tested, but they did not improve the yield (Table 1, entry 6). Without Ag(I), the reaction did not proceed at all, and the use of AgOAc was found to be less effective than Ag₂CO₃, as it resulted in a reduced yield (Table 1, entries 7 and 8). Increasing the temperature to 60 °C did not result in a significant improvement in yield, which indicates that elevated temperature had little effect on the reaction (Table 1, entry 9). Furthermore, when Cs₂CO₃ or DMAP were used as bases instead of Et₃N, the yields of the desired product 3 were considerably lower (Table 1, entry 10).

Motivated by these results, we next explored the substrate scope of the catalytic system using a variety of aryl diazonium salts (Scheme 2). Remarkably, an excellent yield (87%) was obtained for the para-dimethylamino substituted aryl diazonium salt (3b). Phenoxy and benzyloxy substituents at the para-position also produced the corresponding NHS esters in good yields (3c,3d).

Scheme 2 Substrate scope of different aryl diazonium salts.^{a a} Standard conditions: 1a (0.90 mmol), 2 (2.70 mmol), Pd(OAc)₂ (10 mol%), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (10 mol%), Ag₂CO₃ (0.5 equiv.) and Et₃N (1.2 equiv.) in THF at room temperature for 12 h. ^b The reaction mixture was heated at 60 °C for 12 h.

The diazonium salt derived from unsubstituted aniline gave the corresponding NHS ester in good yield (79% isolated) (3e). Methoxy-substituted substrates also reacted smoothly, regardless of the position on the ring, to afford the desired products (3f–3h) in moderate to excellent yields (Scheme 2).

1,3-Dioxolane and 1,4-dioxane containing aromatic rings furnished corresponding NHS esters in good yield (3i, 3j). It was noteworthy that aryl diazonium salts bearing halogens (F, Cl, Br) at the para-position as well as meta-position were well tolerated, and the halogen substituents remained intact under the reaction conditions (3k–3n). Our next step was to investigate the influence of alkyl substituents on the aromatic ring in the reaction. Diazonium salts with methyl, isopropyl and tert-butyl group at para-position of the phenyl ring generated the product smoothly with good yields (3o–3q). Furthermore, two or more methyl groups in the aromatic ring underwent the reaction to deliver corresponding NHS esters in very good yields (3r–3t). Interestingly, a phenolic –OH group remained unaffected in the reaction, affording the product 3u cleanly. But, the yield in this case was low probably due to some competitive, unwanted side reactions (Scheme 2).

We then examined the combined effect of having both alkyl and heteroatom-substitution in the aromatic ring of the aryl diazonium salts (3v–3x). In all three cases, moderate to good yields (59–74%) of the corresponding products were obtained. Good yield of the product was also observed when naphthyl diazonium salt was used as the coupling partner (3y). Diazonium salts containing a biphenyl system (3z) at the ortho-position also produced the corresponding NHS esters in good yield. Electron-withdrawing substituents like cyano, keto and ester groups at the para-position were well tolerated, resulting in the corresponding NHS esters smoothly (3aa–3ac). Notably, the diazonium salt bearing a para-azobenzene group gave a significantly lower yield (3ad), though the reason for this reduced efficiency remains unclear.

Moreover, this methodology is not limited to carbocyclic systems—we successfully extended it to heterocyclic diazonium salts derived from fluorene, carbazole and benzothiazole with products (3ae–3ag) obtained in good yields. However, aryl diazonium salts containing a nitro group failed to furnish the desired product. The structure of the product 3af was confirmed by X-ray crystallographic analysis.¹⁶ To evaluate the scalability of the protocol, a gram-scale synthesis was performed (Scheme 3). Reaction of 1.0 g of 1a with 1.9 g of 2 under the optimized conditions furnished the desired NHS ester 3a in 74% isolated yield (831 mg).

Scheme 3 Scale-up experiment of the aryl diazonium salt.

A key advantage of this carbonylation protocol is its broad functional group tolerance, which enables the synthesis of structurally diverse N-hydroxysuccinimide (NHS) ester derivatives. Owing to their electrophilic nature, NHS esters are widely employed in bioconjugation protocols for their rapid and selective reactivity with amine nucleophiles.^17,18 To further demonstrate the versatility and synthetic potential of these intermediates, we explored their application in the conjugation of biologically relevant molecular fragments (Scheme 4). We performed the bioconjugation of naturally occurring amino acid methyl esters derived from glycine, tyrosine, and valine with the NHS ester scaffold 3a in the presence of triethylamine (Et₃N) in anhydrous dichloromethane (DCM). The corresponding amide products 4–6 were obtained in good yields under mild reaction conditions without the requirement for harsh reagents, elevated temperatures, or the use of toxic additives (Scheme 4). This demonstrates the potential of NHS esters as promising building blocks for the modification of long peptides and proteins.

Scheme 4 Derivatization of NHS ester 3a.

Moreover, the widely used antipyretic agent paracetamol and the naturally occurring aromatic compound eugenol also reacted efficiently with 3a in the presence of sodium hydride (NaH, 60% dispersion in mineral oil) in tetrahydrofuran (THF) at ambient temperature, affording the corresponding esters 7 and 8 in high yields (Scheme 4).

We next sought to demonstrate the broader applicability of our methodology by synthesizing pharmaceutically active molecules (Scheme 5). Using the same reaction conditions optimized for amino acid bioconjugation (Scheme 4), we employed NHS ester derivative 3h in a reaction with a structurally relevant primary amine to obtain trimethobenzamide 9, a clinically approved antiemetic agent effective against nausea and vomiting.¹⁹ In a parallel transformation, NHS ester 3l was coupled with morpholinoethylamine to obtain moclobemide 10, a well-known reversible inhibitor of monoamine oxidase A (RIMA), widely prescribed for its antidepressant and anxiolytic effects.¹⁹ Collectively, these examples highlight the practical utility of our approach in constructing structurally diverse and functionally enriched molecules of pharmaceutical importance.

Scheme 5 Syntheses of two biologically relevant compounds.

Scheme 6 outlines a proposed mechanism consistent with our experimental observations and previous literature reports.^7,20 The reaction may be initiated by ligand-assisted reduction of Pd(II) to Pd(0) and oxidative addition of aryl diazonium salt 1 to Pd(0), forming the aryl–Pd(II) intermediate A. Ag(I) salts may promote the formation of aryl radicals from diazonium salts. These radicals are likely to accelerate oxidative addition to the Pd(0) species, and facilitates catalytic turnover.²¹ Simultaneously, NHS formate 2 undergoes base-mediated reaction to generate CO and NHS (2′). Insertion of CO into intermediate A yields the acyl–Pd(II) species B, which then undergoes anion exchange with NHS (2′) to form intermediate C. Finally, reductive elimination from C affords the desired NHS ester 3, completing the catalytic cycle (Scheme 6).

Scheme 6 Plausible mechanism.

In summary, we have developed a robust and versatile palladium-catalyzed carbonylation strategy for the synthesis of N-hydroxysuccinimide (NHS) esters using aryl diazonium salts and NHS formate as a benign CO surrogate. This method avoids the use of toxic carbon monoxide and operates under mild, operationally simple conditions with broad substrate scope and excellent functional group tolerance. The resulting NHS esters have been employed in bioconjugation with amino acids, small molecule drugs, and pharmacologically relevant amines, showcasing their potential as valuable intermediates for drug discovery and bioconjugate chemistry. Furthermore, this approach enables the efficient synthesis of clinically significant compounds such as trimethobenzamide and moclobemide. Ongoing efforts in our laboratory are focused on extending this methodology to the site-specific functionalization of complex biomolecules, including peptides, proteins, and nucleic acids.

JD thanks SERB (grant number: CRG/2021/004525) and ICMR (grant number: IIRPIG-2024-01-01128) for funding. This work was supported by DST for funding. PP thanks UGC-India for junior and senior research fellowships.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the ESI.†

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: General information, experimental details, characterization of compounds, NMR spectral data, crystallographic data. CCDC 2446092. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5cc02739j

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