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Organic Chemistry
β-Silyl alkynoates: Versatile reagents for biocompatible and selective amide bond formation
β-Silyl alkynoates: Biocompatible and selective amide bond formation
https://orcid.org/0000-0002-6751-4028
Choudhuri Khokan Conceptualization Data curation Formal analysis Investigation Methodology Project administration Validation Writing - original draft Writing - review & editing 1 2
https://orcid.org/0000-0001-7872-4947
Zhang Zhenguo Conceptualization Formal analysis Investigation Methodology Validation 1 2
https://orcid.org/0000-0002-2936-337X
Loh Teck-Peng Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Visualization Writing - original draft Writing - review & editing 1 2 *
1 College of Advanced Interdisciplinary Science and Technology, Henan University of Technology, Zhengzhou 450001, China.
2 Division of Chemistry and Biological Chemistry, School of Chemistry Chemical Engineering & Biotechnology, Nanyang Technological University, 21 Nanyang Link, Singapore, 637371, Singapore.
* Corresponding author. Email: teckpeng@ntu.edu.sg
20 9 2024
18 9 2024
10 38 eadp754410 4 2024
13 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).
2024
The Authors
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The study introduces a previously unidentified method for amide bond formation that addresses several limitations of conventional approaches. It uses the β-silyl alkynoate molecule, where the alkynyl group activates the ester for efficient amide formation, while the bulky TIPS (triisopropylsilane) group prevents unwanted 1,4-addition reactions. This approach exhibits high chemoselectivity for amines, making the method compatible with a wide range of substrates, including secondary amines, and targets the specific ε-amino group of lysine among the native amino ester’s derivatives. It maintains stereochemistry during amide bond formation and TIPS group removal, allowing a versatile platform for postsynthesis modifications such as click reactions and peptide-drug conjugations. These advancements hold substantial promise for pharmaceutical development and peptide engineering, opening avenues for research applications.

An example of a catalyst-free biocompatible amide bond formation reaction using β-silyl alkynoates is reported.

http://dx.doi.org/10.13039/501100001348 Agency for Science, Technology and Research M21K2c0114 http://dx.doi.org/10.13039/501100001348 Agency for Science, Technology and Research M22K9b0049 http://dx.doi.org/10.13039/501100001475 Nanyang Technological University Distinguished University Professor grant
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pmcINTRODUCTION

Amide bonds are pivotal in organic synthesis (1–7), forming the backbone of proteins (8) and a variety of synthetic polymers (9). These bonds are foundational in the structure of biomolecules and crucial in the development of innovative conjugated products such as antibody-drug conjugates (ADCs) (10, 11), insulin analogs (12), and ubiquitin conjugates (13). It is estimated that around 25% of all Food and Drug Administration–approved pharmaceutical drugs contain an amide bond, underscoring its significance. Representative amide-containing marketed drugs include penicillin, metoclopramide, atorvastatin, etc. In addition, considerable attention is devoted to amide-containing payloads, particularly in ADCs, which are designed for the selective targeting of cancer cells (Fig. 1A). In consequence, a diverse array of synthetic methods (14–16) has been developed for amide bond formation (Fig. 1B). Among the various synthetic approaches, the direct coupling of carboxylic acids or alcohols with amines stands out as the most prevalent strategy, often aided by coupling reagents. In recent years, a multitude of coupling reagents, including carbodiimides (17), phosphonium salts (18), and aminium/uronium derivatives (19), have been developed and successfully brought into commercial use. In 1975, Bragg and Hou (20) introduced N-hydroxysuccinimide esters as reactive ends of homobifunctional cross-linkers, which revolutionized the amide bond construction in peptides and proteins. Zhao and colleagues recently introduced ynamide-mediated (21–23) and allenone-mediated (24) coupling reactions between carboxylic acid and amine. Nevertheless, these methods frequently lack specificity toward native amino acids and suffer from sub-stoichiometric efficiency, resulting in substantial waste and complicating the subsequent purification process to isolate the desired products. Another notable strategy for amide bond formation is native chemical ligation which was independently developed by Kent (25) and Tam (26) for assembling amide bonds in peptides and proteins inspired by Wieland’s observations (27). However, its applicability is limited to specific substrates only. Furthermore, traditional methods (28, 29) for peptide syntheses rely heavily on the legacy reagents and technologies developed in the 1950 to 1980s, which are reaching their inherent limitations including the potential risk of racemization, and nonbiocompatible conditions. To address these issues, the American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable has emphasized the importance of developing catalytic or catalyst-free methods for amide bond formation as a key initiative in Green Chemistry (30, 31). Innovations in this field have been notable. In 2007, Milstein (32) and co-workers introduced a breakthrough with their ruthenium-catalyzed dehydrogenative coupling of amines and alcohols (33). This was followed by Madsen (34) and his team, who expanded the scope of dehydrogenative coupling with an alternative catalyst system (35). Hall (36) and colleagues recently presented a method for boronic acid–catalyzed amidation reactions using carboxylic acids (37). Direct aminolysis of the readily available, simple, and stable ester with an amine could represent an attractive method for amide formations because of its enhanced atom economy and the accessibility of starting materials. However, this strategy suffers from the prerequisite of C─O bond activation of the esters by transition metal catalysts. Therefore, amide bond formation by using stable ester under biocompatible conditions remains a substantial hurdle (38–44).

Fig. 1. Background and reaction design.

(A) Amide bonds in bioactive molecules. (B) Classical methods for amide bond formation. (C) This work: Biocompatible amide bond formation using β-silyl alkynoates via 1,2 additions. (D) Chemical reactivity of amino esters. (E) Application of the reaction in protein, drug, and peptide modification.

Our approach to amide bond formation addresses these challenges by forgoing conventional reagents and using β-silyl alkynoates (45–47) to couple with amines under biocompatible conditions (Fig. 1C). We anticipate that the bulky triisopropylsilyl group will suppress the 1,4-addition reactions (48–50), while the alkynyl group enhances the ester group’s reactivity and directs the nucleophiles toward forming the desired amide bond. The strategic removal of the silicon group without epimerization of the stereogenic center facilitates the postmodification of a diverse range of chemical reactions. Our approach not only offers selective amide bond formation that preserves stereogenic centers but also leverages the terminal amide’s alkyne anchor as a Michael acceptor. This capability will introduce additional functionality such as the execution of click reactions (51), peptide-drug conjugates (PDCs) (52), etc., which represent a substantial advancement in the synthesis and functionalization of biomolecules under biocompatible conditions (53–55). Furthermore, our method exhibits a selective affinity toward alkynyl esters over other alkane and alkene esters and is compatible with a wide array of aliphatic primary and secondary amines. A distinct feature of the reaction is its selectivity toward the β, γ, and long-chain amino esters. It uniquely maintains the integrity of stereogenic centers in peptides and specifically targets the ε-amine of lysine among native amino esters. However, it demonstrates resistance to the amine that is attached to the α-position of the electron-withdrawing groups such as esters or amides (Fig. 1D). Expanding upon this observation, we have demonstrated the applicability of our methodology in the realms of peptide chemistry, protein engineering, and drug development (Fig. 1E). Such developments have profound implications for biomedical research and biotechnology, marking a substantial leap forward in these fields.

RESULTS AND DISCUSSION

In our continuous pursuit of developing a green method for amide bond formation, which is also suitable for amine conjugation in biological systems, we conducted an in-depth study of the reaction using ethyl 3-(triisopropylsilyl)propiolate (1a) with benzylamine (2a) under catalyst-free conditions, the results are summarized in Table 1. We performed this reaction in both aqueous media and a variety of buffer solutions at different pH levels while maintaining a constant temperature of 37°C (Table 1, entries 1 to 6). Notably, using a phosphate-buffered saline (PBS) at pH 7 resulted in an excellent yield of 80% within 48 hours, with the remaining substrate left unreacted (Table 1, entry 3). Our experiments with organic solvents, including dimethyl sulfoxide (DMSO), acetonitrile (CH3CN), ethanol (EtOH), tetrahydrofuran (THF), and others, yielded results that were less than satisfactory, refer to the Supplementary Materials. We expanded our studies by incorporating various organic solvents into a PBS buffer solution at pH 7 (Table 1, entries 7–9). A notable finding was that combining buffer with ethanol in a 4:1 ratio resulted in the most effective outcomes, achieving an 85% yield of the desired product within 48 hours (Table 1, entry 9). In addition, we observed an improvement in the reaction yield up to 92% for 3a with slight temperature increases, reaching an optimum at 40°C (Table 1, entry 10). However, it is crucial to note that while elevating the temperature to 80°C, the reaction rate can be substantially hastened to complete it within 24 hours (Table 1, entry 11). Nevertheless, we are continuing our investigation at 40°C under biocompatible conditions, motivated by our keen interest in applying this method within the realm of protein and peptide chemistry. To eliminate any possibility of catalytic interference from glass silica, we opted to perform the reaction in a plastic reaction vial, achieving an outstanding 82% yield of 3a (Table 1, entry 12). Subsequently, inductively coupled plasma mass spectrometry (ICP-MS) analysis confirmed that negligible metal presence in the reaction mixture was detected at parts pe r billion levels (details in the "ICP-MS for the determination of the metal ions" section in the Supplementary Materials).

Table 1. Optimization of reaction conditions.

Condition: 1a (50 mg, 0.196 mmol), 2a (0.295 mmol, 1.5 equiv.), solvent (0.5 mL) for 48 hours.

Entry	Solvent	pH value	Temperature (°C)	3a (%)*	
1	H2O	7	37	56	
2	PBS buffer	6	37	30	
3	PBS buffer	7	37	80	
4	PBS buffer	8	37	54	
5	Tris-HCl buffer	8	37	40	
6	H2O/EtOH (4:1)	7	37	84	
7	PBS/CH3CN	7	37	46	
8	PBS/DMSO	7	37	48	
9	PBS/EtOH	7	37	85	
10	PBS/EtOH	7	40	92 (88)†	
11	PBS/EtOH	7	80	90‡	
12	PBS/EtOH	7	40	86§	
*Yield was determined by 1H NMR using CH2Br2 as an internal standard.

†Isolated yield.

‡Reaction was conducted for 24 hours.

§Reaction was performed in a plastic vial. PBS buffer, phosphate-buffered saline; PBS/organic solvent = 4/1.

We next systematically investigated various β-substituted alkynyl esters, examining their reactions with benzylamine (2a) under optimal conditions. We observed that phenyl-substituted alkynyl esters produced both 1,2- and 1,4-addition products with 39 and 45% yields, respectively (Fig. 2A, entry 1). However, using of less bulky TMS-substituted alkynyl ester results in the formation of a desilylated 1,4-addition product in 66% yield (Fig. 2A, entry 2). A notable change was evident when we used the bulkier triisopropylsilane (TIPS)–substituted alkynyl ester, which shifted the reaction away from the 1,4-pathway, resulting in a predominant 1,2-addition product with a yield of 92% (Fig. 2A, entry 4). Subsequently, we focused on assessing the effect of various ester groups on these reactions. The results, presented in the accompanying table in Fig. 2B, showed that, regardless of the ester group variations, the reactions favored the formation of 1,2-addition products with commendable efficiency. However, the substitution of the ester group with either a tert-butyl or an acid group failed to produce any product formation (Fig. 2B, 9 to 11). A notable observation was found that the reactions of simple alkane or alkene esters did not yield the anticipated amide product formation, suggesting the pivotal role of the alkyne functionality in activating the ester carbonyl group for the 1,2-addition reaction (Fig. 2B, 13 and 14). This finding underscores the importance of the alkyne group in the reaction mechanism. Moreover, distinguishing between different ester compounds offers valuable insights for the functionalization of complex molecules containing multiple ester functionalities, paving the way for more tailored applications in chemical synthesis.

Fig. 2. Control experiments.

(A) Effect of β-substitution on chemoselective 1,2- versus 1,4-addition. (B) Comparison of ester reactivity. (C) Nucleophilic competitive experiment. Reaction yield was determined from the crude reaction mixture by 1H NMR using CH2Br2 as an internal standard. N.R, no reaction; PBS, phosphate-buffered saline (pH 7.0).

We then proceeded to conduct competitive experiments to assess the selectivity of the reaction toward amine in the presence of both benzyl thiol and alcohol under our optimized reaction conditions, as outlined in Fig. 2C. We were pleased to find that no C─S or C─O bond formation occurred, and only products resulting from C─N bond formation were observed. Notably, when we conducted separate reactions of β-silyl alkynoates with benzyl mercaptan and benzyl alcohol, no product formation was observed in either of the reactions. The results of these experiments emphasized the substantial chemoselectivity of the reaction toward amines.

Scope of substrates

With our optimized reaction conditions in hand, we explored the substrate scope of amines in forming the amide bonds with TIPS-conjugated ester 1a, as depicted in Fig. 3. Our experiments revealed that both linear and branched chain primary alkyl amines were effectively accommodated, resulting in the formation of adducts 3a to 3ad with moderate to excellent yields. The use of benzylic amines with various substituents at the ortho-, meta-, and para-positions on the phenyl rings led to the corresponding products 3a to 3i with excellent efficiency. Notably, the introduction of fluorine at the ortho position on the phenyl group yielded 3h with a moderate outcome. This transformation proved to be equally effective with heteroarenes, affording products 3j and 3k with yields of 48 and 55%, respectively. In contrast, reactions with linear chain alkyl amine effectively produce the corresponding products 3l to 3s with yields ranging from 95 to 72%. Moreover, the branch alkyl amine also demonstrated remarkable effectiveness in producing the corresponding products 3t to 3v.

Fig. 3. Substrate scope of amine nucleophiles to β-silyl alkynoates.

1a (50 mg, 0.196 mmol), 2 (0.295 mmol, 1.5 equiv.), and solvent (0.5 ml) for 48 hours. Isolated yield. aThe remaining starting materials were recovered (for details, see Supplementary Materials). bPBS buffer (pH = 7.0)/CH3CN (4:1) was used.

The use of cyclohexyl amine produced products 3w with a notable yield of 98%. The reaction of amino alcohol with 1a was selective, preserving the hydroxy group intact in the adduct 3x to 3ab. Secondary amines 2ac and 2ad also reacted favorably, producing products with 80 and 92% yields. However, this method failed to yield any amide product formation when aniline, a heteroaryl amine such as 3-aminopyridine, and α-amino esters were used. To further evaluate the versatility of this reaction, we reacted β-silyl alkynoates with various biomolecules and drugs containing free amines (Fig. 4). In an initial experiment, N-Boc-Lysine-tert-butyl ester was reacted with 1a under standard conditions, successfully functionalizing this lysine derivative to yield adduct 3ae with remarkable conversion. Encouraged by these results, we extended this approach to modify peptide and drug molecules containing free amines using 1a. For example, amlodipine, commonly used for treating hypertension, was modified to produce 3ag with an impressive 94% yield. In addition, drugs like dehydroabietylamine, deacetyl linezolid, mexiletine, primaquine, and histamine were efficiently modified, yielding their respective products with good to excellent efficiency. The functional linker Boc-Aminooxy-PEG2-C2-amine also reacted successfully with 1a, resulting in product 3ak with a commendable 48% yield.

Fig. 4. Scope of the amine-containing bioactive molecule and drug modification.

1a (50 mg, 0.196 mmol), 2 (0.295 mmol, 1.5 equiv.), and solvent (0.5 ml) for 48 hours. Isolated yield. aPBS buffer (pH = 7.0)/CH3CN (4:1) solvent was used. bPBS buffer (pH = 7.0) was used as a solvent. cThe remaining starting materials were recovered (for details, see Supplementary Materials).

Determining whether this method can be applied to peptides or proteins without causing epimerization at existing stereogenic centers is critical, as depicted in Fig. 5. This concern particularly applies during the amide bond formation step and the subsequent removal of the TIPS group. To evaluate this, we tested the method on both L-lysyl-L-valinate and L-lysyl-D-valinate derivatives of dipeptides 15 and 15′ and successfully obtained the desired amide products with yields of 81 and 78%, respectively. Subsequently, the removal of the TIPS group was efficiently achieved by using Et3N.3HF reagent in THF at room temperature. Nuclear magnetic resonance (NMR) analysis, including both 1H NMR and 13C NMR, confirmed that neither the amide bond formation nor the TIPS group removal led to any epimerization. The stacked plot of the 13C NMR spectrum revealed that none of the chiral carbon peaks overlapped with each other in either of the modified dipeptides 3am and 3am′ or the desilylated dipeptides 5am and 5am′ (Fig. 5). After successfully confirming the retention of configuration at the stereogenic centers throughout the processes of amide bond formation and the removal of the TIPS (triisopropylsilyl) group in the synthesized peptides, we proceeded to the next step to explore the potential synthetic applications of the alkyne anchor present in the amide products.

Fig. 5. Stereoselectivity assessment in peptides using 13C NMR spectrum for epimer of amine-conjugated product.

The gram-scale synthesis of 3a confirms the viability and practicality of the reaction, ensuring its potential for large-scale use in both research and industry (Fig. 6A). It is noteworthy that the N-propiolamide derivatives obtained from our method exhibit highly resourceful building blocks in organic synthesis (56–58) (Fig. 6B). The alkyne moiety of 5a readily undergoes reaction with amines under biocompatible conditions, yielding a C-N–conjugated adduct 16a with outstanding yield. Moreover, it effectively engages in Sonogashira coupling and azide-alkyne click reactions, yielding the desired products 17a and 18a with excellent efficiency. In addition, the alkyne group within these compounds (5a) can also be selectively reduced to the alkene derivative 19a using a Lindlar catalyst.

Fig. 6. Application of N-substituted propiolamide.

(A) Gram-scale synthesis. (B) Product derivatization of N-benzylpropiolamide (C) Application in linker chemistry for peptide-drug conjugation (PDCs).

During the last decades, peptide-drug conjugates (PDCs) (59) have received substantial attention as promising targeting therapies, akin to antibody-drug conjugates (ADCs). These conjugates typically consist of monoclonal peptides, drug payloads, and cleavage/noncleavage linkers. Capitalizing on the inherent versatility (60) of N-propiolamide derivatives, we carefully combine drug molecules with peptide (5am) using an alkyne anchor linker, making it simpler to blend them smoothly into the formation of PDCs (Fig. 6C). This approach harnesses the unique properties of N-propiolamide derivative to accurately join drugs with peptides, which could enhance the efficacy and specificity of targeted therapies. The addition of a mercaptan with 5am provides a versatile platform for synthesizing sulfur-containing drug conjugation (20a and 21a) under biocompatible conditions. Moreover, the alkyne group of 5am readily engages in copper-catalyzed azide-alkyne cycloaddition (click) reactions, enabling the conjugation of the Zidovudine drug 22a in 61% of yield. These findings underscore the efficacy of N-propiolamide derivatives as anchors for coupling with various biomolecules, leading to the production of valuable PDCs. Such versatility broadens the potential applications of this methodology in biochemical conjugation and drug development.

As previously mentioned, we were intrigued to find that α-amino esters did not yield the desired amide product with 1a, while linear alkyl amines did so with good efficiency. Therefore, we conducted a comparative study between the activated esters and β-silyl ethyl alkynoates regarding their specific reactivity toward amino esters (Fig. 7). Notably, most of the literature known activated esters are unstable under ambient biocompatibility conditions and are typically prepared in situ and used immediately for amidation reactions. Because of their high reactivity, activated esters readily react with both α- and β-amino esters to form the corresponding amide products. For a comprehensive comparison, we synthesized p-nitrophenyl (23a), N-hydroxysuccinimide (23b), and 2,4,5-trichlorophenyl (23c) silyl alkynoates. It is essential to highlight that all the activated alkynoates demonstrated equal reactivity toward both α- and β-amino esters, yielding the respective products with good to excellent efficiency. This observation highlights the lack of selectivity in the discrimination of the positions of the amine groups in amino ester derivatives. However, a systematic investigation into the reactivity of ethyl alkynoates toward various amino esters revealed that α-amino esters (3ao) displayed negligible reactivity, while α-amino amides (3ao′) yielded only traces of product. Conversely, β-amino esters, γ-amino esters, and δ-amino esters efficiently produced the desired products with yields of 50, 80, and 82%, respectively (Fig. 8A).

Fig. 7. Selectivity comparison between activated esters and ethyl alkynoates.

Fig. 8. Amino ester selectivity and polypeptide modification with 1a.

(A) Specific amino ester selectivity. (B) Polypeptide modification with 1a. (C) Modified lanreotide conjugates.

Further exploration of the reactivity of β-silyl alkynoates, we conducted experiments using lysine amide (3as), which contains two free amino groups (Fig. 8B). The results demonstrated that only the side chain ε-NH2 of lysine reacted with the β-silyl alkynoates, while the α-NH2 group remained unreacted. This indicates that the presence of an electron-withdrawing group adjacent to the amino group hinders the reaction. These findings highlight a notable selectivity for lysine conjugation over other native amino ester derivatives.

On the basis of the controlled experiment (Figs. 2B and 8A), a plausible mechanism for the amide bond formation reaction under biocompatible conditions has been rationalized. In β-silyl alkynoates, the sp-hybridized carbon of the alkyne moiety induces a partial positive charge at the ester’s carbonyl group by withdrawing electron density. This electron-deficient carbonyl carbon then becomes susceptible to nucleophilic attack by the amine, resulting in the formation of the amide product and the release of ethanol as a byproduct. The reaction favors the ε-amino group of lysine because of its higher nucleophilicity, while the limited nucleophilicity of the α-amino group in native amino esters hinders the desired outcome.

These initial findings encouraged us to expand our method to various polypeptide molecules containing different nucleophilic amino acid side chains (Fig. 8B). Notably, polypeptides with the ε-amine group of lysine underwent conjugation with 1a, while other nucleophilic groups within the amino acid chains and the terminal amino group were mostly unaffected, as confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of 3at. Consequently, the desired amide products 3at, 3au, and 3av were formed, achieving yields of 58%, 51%, and 50%, respectively. To verify the specific selectivity of lysine in cyclic peptides such as lanreotide, which contain multiple nucleophilic amino acid chains, we treated modified lanreotide with tris(2-carboxyethyl)phosphine to break the S─S linkage, forming a linear peptide chain. LC-MS/MS analysis revealed that only the ε-NH2 group of lysine in lanreotide was reacting by β-silyl alkynoates, while other nucleophilic amino acids, such as threonine, cysteine, tryptophan, tyrosine, and the terminal amine, remained mostly untouched. The desilylation of modified lanreotide could also be achieved using Et3N.3HF reagent in a polar protic methanol solvent at room temperature (Fig. 8C). This step enabled further conjugation with a benzyl mercaptan, forming the desired conjugated product (3ax).

Leveraging this success, we extended our methodology to protein modification (61) targeting lysine under biocompatible conditions, using bovine serum albumin (BSA, 90 μM) as a model protein (Fig. 9). The deconvoluted mass spectrum revealed that the molecular weight of unmodified BSA (66,387 Da) shifted to 66,491 Da, indicating the modification of BSA with two ligands and the release of the TIPS group. Furthermore, LC-MS/MS analysis identified all relevant peptide segments and modifications of a free lysine residue (K361) by a molecule (51.99492 Da) corresponding to 1a after the TIPS group fell off. To further demonstrate the versatility of our method, we applied it to myoglobin protein from equine skeletal muscle, which contains 19 lysine residues. Incubation with 200 equivalents of β-silyl methyl alkynoates in PBS buffer (pH 8.0) resulted in conjugated products with a maximum of twofold modification, as identified by LC-MS analysis. The deconvoluted mass spectrum showed that unmodified myoglobin (16,950 Da) shifted to 17,048 Da for onefold modification, representing myoglobin modified with one desilylated ligand and the consumption of two sodium ions. For twofold modification, LC-MS showed a peak at 17,102 Da. Further LC-MS/MS analysis confirmed all relevant peptide segments and the modification of free lysine residues (K80 and K88) by a molecule (51.99492 Da) corresponding to 1a after the deprotection of the TIPS group. Inspired by these results, we also modified lysozyme and cytochrome C using β-silyl alkynoates. Both proteins showed a maximum of threefold modification after the removal of the TIPS group. Both the ethyl and methyl alkynoates (1a and 6) produced the same modified biopolymer products with myoglobin and lysozyme after TIPS group deprotection. This may be due to the presence of a free carboxylic acid group, which induces TIPS group hydrolysis over prolonged reaction times. Despite a relatively sluggish reaction rate, our approach exhibited remarkable selectivity for lysine, while preserving the integrity of other amino groups within the protein. This level of specificity underscores the potential of our method for targeted protein modifications.

Fig. 9. Modification of bovine serum albumin, myoglobin, lysozyme, and cytochrome c protein using β-silyl alkynoates.

In conclusion, our study introduces a pivotal β-silyl alkynoate-based methodology for amide bond formation, marking a substantial leap in organic chemistry and biotechnology. This technique is characterized by the activation of the ester group through an alkynyl group and the effective suppression of the 1,4-addition pathway by a bulky TIPS group, ensuring both high selectivity and efficiency. A distinctive feature of our method is its broad compatibility with various amines, including secondary amines, and its unique ability to differentiate the amino group in native amino ester derivatives. This specificity proves especially beneficial in targeting the ε-amine of lysine, crucial in peptide and protein engineering, while concurrently avoiding other native amino ester, substantially broadening the scope of this method. Furthermore, our approach preserves the integrity of stereogenic centers in peptides throughout the amide bond formation and the TIPS group removal, an aspect vital for the pharmaceutical industry. The creation of a versatile alkynyl anchor functional group in our amide products opens avenues for further functionalization, such as click reactions, PDCs, etc. Notably, our method exhibits exclusive reactivity with β-silyl alkynoates, as evidenced by no reaction or poor selectivity with other ester compounds, and efficiently yields ethanol as the only byproduct, underscoring its green chemistry credentials. This substantial advancement overcomes the limitations of traditional amide formation techniques and offers immense potential for revolutionizing biomolecule synthesis and modification, thereby catalyzing progress in drug development and biotechnological applications.

MATERIALS AND METHODS

General procedure for amine addition to β-silyl alkynoates

To a 4-ml glass vial, 0.1 ml of ethanol was added to the mixture of ethyl 3-(triisopropylsilyl) propiolate (1a, 50 mg, 0.196 mmol) and benzylamine (2a, 0.295 mmol). The mixture was stirred for 5 min to achieve a homogeneous solution. Subsequently, 0.4 ml of pH-neutral phosphate buffer was added, and the resulting mixture was vigorously stirred for 48 hours at 40°C. The mixture was diluted with ethyl acetate, washed with water, and dried over anhydrous sodium sulfate. The crude mixture was purified over silica gel column chromatography using 30% ethyl acetate inhexane as an eluent to afford the desired product (3a).

General procedure for desilylation of propiolamide

To a 10-ml round-bottom flask, 3a (60 mg, 0.190 mmol) and triethylamine trihydrofluoride (5 equiv.) were taken in 2 ml of THF at 0°C under an argon atmosphere. Then the mixture was vigorously stirred for 12 hours at room temperature. After that, the mixture was washed with water, extracted with ethyl acetate, and dried over anhydrous sodium sulfate, concentrated under reduced pressure. The yield of 5a was calculated after purification of the crude mixture using silica gel flash column chromatography.

Acknowledgments

We thank Y. Li (Nanyang Technological University) for assistance with the x-ray structure analysis, W. Meng (Nanyang Technological University) for the mass analysis of proteins, and S. Mitra for the ICP-MS analysis.

Funding: We gratefully acknowledge the financial support from the Distinguished University Professor grant (Nanyang Technological University) and the Agency for Science, Technology and Research (A*STAR) under its MTC Individual Research Grant (M21K2c0114) and RIE2025 MTC Programmatic Fund (M22K9b0049).

Author contributions: T.-P.L. conceptualized and directed this research. K.C. contributed to the main experiments including condition optimization, substrate synthesis, scope evaluation, synthetic application, and peptide conjugation. Z.Z. performed part of the experiments including the application of the reaction in protein chemistry and revised the manuscript. K.C. designed the study and wrote the manuscript with final revisions by T.-P.L.

Competing interests: T.-P.L. and K.C. are inventors on a patent application (10202401292S) submitted by Nanyang Technological University that covers β-silyl alkynoates. The other author declares no competing interests.

Data and materials availability: Data relating to the characterization of products, general methods, optimization studies, and experimental procedures are available in the Supplementary Materials. Crystallographic data for compound q is available free of charge from the Cambridge Crystallographic Data Centre under reference number 2310713.

Supplementary Materials

The PDF file includes:

Supplementary Materials and Methods

Supplementary Text

Tables S1 to S5

Figs. S1 to S163

Legend for data S1

Other Supplementary Material for this manuscript includes the following:

Data S1
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