WO2025230475A1

WO2025230475A1 - Beta-silyl alkynoates: versatile reagents for biocompatible and selective amide bond formation

Info

Publication number: WO2025230475A1
Application number: PCT/SG2025/050298
Authority: WO
Inventors: Teck Peng Loh; Khokan CHOUDHURI
Original assignee: Nanyang Technological University
Current assignee: Nanyang Technological University
Priority date: 2024-05-02
Filing date: 2025-05-02
Publication date: 2025-11-06
Anticipated expiration: 2026-11-02

Abstract

The current invention relates to a method of forming an amide bond, the method comprising the steps of (a) providing a compound according to formula (I), where R₁ represents a linear alkyl group, an aromatic group or a heteroaromatic group, which linear alkyl group, aromatic group or heteroaromatic group are unsubstituted or are substituted by one or more electron withdrawing groups; R₂, R_2',and R_2'' are each independently selected from a branched C₃to C₄ alkyl group and an aromatic group; and (b) reacting the compound of formula I with a primary or secondary amine of formula II, where R₃ is an alkyl group, optionally bearing one or more substituents; and R₄ is H or an alkyl group; or R₃ and R₄ together with the nitrogen atom they are attached to form a carbocylic ring system having from 3 to 12 carbon atoms, optionally bearing one or more substituents, in an aqueous solution for a period of time to provide a compound of formula III, where R₂, R_2', R_2'', R₃ and R₄ are as defined hereinbefore.

Description

BETA-SILYL ALKYNOATES: VERSATILE REAGENTS FOR BIOCOMPATIBLE AND SELECTIVE AMIDE BOND FORMATION

Field of Invention

The current invention relates to methods of forming an amide bond.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Amide bonds are pivotal in organic synthesis, forming the backbone of proteins and a variety of synthetic polymers. These bonds are foundational in the structure of biomolecules and crucial in the development of innovative conjugated products such as antibody-drug conjugates (ADCs) (Bioconjugate Chem. 34, 1951-2000 (2023); Signal Transduct. Target. Ther. 7, 93 (2022)), insulin analogs (J. Am. Chem. Soc. 144, 23332-23339 (2022)), and ubiquitin conjugates (Chem. Rev. 118, 889-918 (2018)). It is estimated that around 25% of all FDA-approved pharmaceutical drugs contain an amide bond, underscoring its significance. Representative amide-containing marketed drugs include Penicillin, Metoclopramide, Atorvastatin, Lacosamide, Lasmiditin, Oseltamivir, Trimethobenzamide, etc. Additionally, considerable attention is devoted to amide-containing payloads, particularly in Antibody-Drug Conjugates (ADCs), which are designed for the selective targeting of cancer cells (FIG. 1 A). In consequence, a diverse array of synthetic methods (Bodanszky, M. Principles of Peptide Synthesis', Springer Berlin Heidelberg, 169 (1993); Chem. Soc. Rev. 43, 2714-2742 (2014); J. Am. Chem. Soc. 85, 2149-2154 (1963)) has been developed for amide bond formation (FIG. 2). 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 (J. Am. Chem. Soc. 77, 1067-1068 (1955)), phosphonium salts (J. Am. Chem. Soc. 91 , 5669-5671 (1969)), and aminium/uronium derivatives (J. Org. Chem. 66, 5245-5247 (2001)), have been developed and successfully brought into commercial use. In 1975, Bragg and Hou (Arch. Biochem. Biophys. 167, 31 1-321 (1975)) introduced NHS esters as reactive ends of homobifunctional crosslinkers which revolutionized the amide bond construction in peptides and proteins. Zhao and colleagues recently introduced ynamides (J. Am. Chem. Soc. 138, 13135-13138 (2016); J. Am. Chem. Soc. 143, 10374-10381 (2021 ); J. Am. Chem. Soc. 146, 4270 - 4280 (2024)) and allenone (Lowe, D.; A New Way to Synthesize Peptides. Science 2024) mediated coupling reactions between carboxylic acid and amine. Nevertheless, these methods frequently lack specificity towards 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 (Science 266, 776-779 (1994)) and Tam (Proc. Natl. Acad. Sci. U. S. A., 92, 1 485-12489 (1995)) for assembling amide bonds in peptides and proteins inspired by Wieland's observations (Liebigs Ann. Chem. 583, 129-149 (1953)). However, its applicability is limited to specific substrates only. Furthermore, traditional methods (Nat. Common. 14, 5013 (2023); Nat. Common. 1, 1 1732 (2016)) for peptide syntheses rely heavily on the legacy reagents and technologies developed in the 1950-1980s, which are reaching their inherent limitations including the potential risk of racemization, and non-biocompatible conditions. To address these issues, the American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) has emphasized the importance of developing catalytic or catalyst-free methods for amide bond formation as a key initiative in Green Chemistry (Green Chem. 9, 41 1-420 (2007); Green Chem. 22, 996-1018 (2020)). Innovations in this field have been notable. In 2007, Milstein (Science 317, 790-792 (2007)) and coworkers introduced a breakthrough with their ruthenium-catalyzed dehydrogenative coupling of amines and alcohols (Angew. Chem. Int. Ed., 50, 8917-8921 (2011 )). This was followed by Madsen (J. Am. Chem. Soc. 130, 17672-17673 (2008)) and his team, who expanded the scope of dehydrogenative coupling with an alternative catalyst system (J. Am. Chem. Soc. 143, 6792-6797 (2021)). Hall (Angew. Chem. Int. Ed. 47, 2876- 2879 (2008)) and colleagues recently presented a method for boronic acid-catalyzed amidation reactions using carboxylic acids (Sci. Adv. 3, e1701028 (2017)). Direct aminolysis of the readily available, simple, and stable ester with an amine could represent an attractive method for amide formations due to its enhanced atom economy and the accessibility of starting materials. However, this strategy suffers from the pre-requisite of C-0 bond activation of the esters by transition metal catalysts. Therefore, amide bond formation by using stable ester under biocompatible conditions remains a significant hurdle (Nat. Common. 8, 14878 (2017); Org. Lett. 16, 2018-2021 (2014); Angew. Chem. Int. Ed. 57, 12925-12929 (2018); Angew. Chem. Int. Ed. 55, 2810 (2016); J. Am. Chem. Soc. 127, 28, 10039-10044 (2005); Synthesis 44, 42-50 (2012); Org. Chem. Front. 10, 1817-1846 (2023)).

It is apparent that a major breakthrough is still needed to overcome the current challenges. As such, there is a need to address the current limitations by having a method for amide bond formation with broad compatibility with various primary and secondary amines (starting material) under catalyst-free conditions. Summary of Invention

The present invention aims to address the current limitations with the developed methods for amide bond formation with broad compatibility with various primary and secondary amines (starting material) under catalyst-free conditions.

The current invention introduces a novel method for amide bond formation that addresses several limitations of conventional approaches. The current invention introduces a pivotal |3- silyl alkynoate-based methodology for amide bond formation, marking a significant leap in organic chemistry and biotechnology. It utilizes the p-silyl alkynoate molecule, where the alkynyl group activates the ester for efficient amide formation, while the bulky TIPS (triisopropylsilane) group effectively prevents unwanted 1 ,4-addition reactions. It has been surprisingly found that methods of the current invention exhibits high chemoselectivity for amines, making the methods compatible with a wide range of substrates, including secondary amines, and targets the specific c-amino group of lysine among the native amino ester’s derivatives. Stereochemistry is retained during amide bond formation and TIPS group removal, thus allowing a versatile platform for post-synthesis modifications such as click reactions and peptide-drug conjugations. These advancements hold significant promise for pharmaceutical development and peptide engineering, opening new avenues for research applications.

Aspects and embodiments of the invention will now be described by reference to the following numbered clauses.

1 . A method of forming an amide bond, the method comprising the steps of:

(a) providing a compound according to formula I:

, where:

Ri represents a linear alkyl group, an aromatic group or a heteroaromatic group, which linear alkyl group, aromatic group or heteroaromatic group are unsubstituted or are substituted by one or more electron withdrawing groups; R₂, R₂’ and R₂- are each independently selected from a branched C₃ to C₄ alkyl group and an aromatic group; and

(b) reacting the compound of formula I with a primary or secondary amine of formula II:

, where:

R₃ is an alkyl group, optionally bearing one or more substituents; and

R₄ is H or an alkyl group; or

R₃ and R₄ together with the nitrogen atom they are attached to form a carbocylic ring system having from 3 to 12 carbon atoms, optionally bearing one or more substituents, in an aqueous solution for a period of time to provide a compound of formula III:

, where

R₂, RZ, R₂ , Rs and R₄ are as defined hereinbefore.

2. The method according to Clause 1 , wherein R₂, R₂’ and R₂- are each independently selected from a branched C₃ to C₄ alkyl group or phenyl.

3. The method according to Clause 2, wherein R₂, Rz and R₂” are each independently selected from Pr, ⁽Bu or phenyl.

4. The method according to Clause 3, wherein R₂, R₂ and R₂ are each Pr.

5. The method according to any one of the preceding clauses, wherein the aqueous solution is water or a buffer solution, optionally wherein one or both of the following apply: (i) the buffer solution is selected from one or more of the group consisting of a phosphate buffered saline solution, and a tris-HCI buffer solution; and

(ii) the buffer solution has a pH of from 5 to 9, such as from 6 to 8, such as about

7.

6. The method according to any one of the preceding clauses, wherein the aqueous solution further comprises an organic solvent that is selected from one or more of the group consisting of a polar aprotic solvent and an alcohol, optionally wherein the organic solvent is selected from one or more of the group of acetonitrile, DMSO and, more particularly, ethanol.

7. The method according to Clause 6, as dependent upon Clause 5, wherein when an organic solvent is present, it is present in an amount of from 1 :10 to 10:1 vol/vol relative to water or the buffer solution, such as from 1 :6 to 6:1 , such as about 1 :4.

8. The method according to any one of the preceding clauses, wherein the reaction of step (b) in Clause 1 is conducted at a temperature of from 0 to 100 °C, such as from 15 to 80 °C, such as from 25 to 50 °C, such as about 40 °C.

9. The method according to any one of the preceding clauses, wherein Ri is selected from a linear Ci to Ce alkyl group, phenyl or a heterocyclic group, where the phenyl or heterocyclic group is substituted by one or more substituents selected from nitro, Cl, F, and =0.

10. The method according to Clause 9, wherein Ri is selected from Me, Et, wiggly line represents the point of attachment to the rest of the molecule.

11 . The method according to any one of the preceding clauses, wherein the primary or secondary amine of formula II is an antibody, a small molecule, a drug molecule, an amino acid, a di-peptide, a tri-peptide, an oligopeptide, a polypeptide, or a protein. 12. The method according to any one of the preceding clauses, wherein the primary or secondary amine of formula II is selected from the group of:

lysine; (+)-dehydroabietylamine; amlodipine; primaquine; mexiletine; deacetyl linezolid; histamine; bovine serum albumin; myoglobin; lysozyme; and cytochrome C.

13. The method according to any one of the preceding clauses, wherein the compound of formula III is subjected to a deprotection reaction to provide a compound according to formula IV: where Rs and R4 are as defined in Clause 1 , optionally wherein the deprotection reaction uses Et₃N.3HF.

14. The method according to Clause 13, wherein the compound according to formula IV is used in one or more of:

(ai) a Sonogashira coupling reaction;

(aii) a Click reaction (e.g. with an azide);

(aiii) a C-N conjugation reaction;

(aiv) a partial reduction of the alkyne functional group to an alkene functional group; and (av) reaction with a primary thiol-containing compound to provide a cysteine derivative.

Drawings

FIG. 1 depicts the amide bonds in bioactive molecules.

FIG. 2 depicts the classical methods for amide bond formation. Limitations of the classical methods include (I) possible racemization of stereogenic center, (ii) requiring pre-activation of the ester group, (iii) non-discrimination of the amine group, (iv) usage of toxic coupling reagent, and (v) being not bio-compatible.

FIG. 3 depicts the current invention - biocompatible amide bond formation using /3-silyl alkynoates via 1 ,2 additions. Advantages of the current invention include (i) utilising metal-free biocompatible buffer medium, (ii) producing ethanol as the only byproduct, (iii) no observation of epimerization, (iv) selectivity for native £-amino ester, (v) region-selectivity of 1 ,2-addition, and (vi) containing a versatile handle.

FIG. 4 depicts the chemical reactivity of amino esters.

FIG. 5 depicts the application of the reaction in protein, drug, and peptide modification. FIG. 6A and FIG. 6B depict the inductively coupled plasma mass spectrometry (ICP-MS) plot for metal concentration in the reaction mixture.

FIG. 7 depicts the control experiment - nucleophilic competitive experiment. Reaction conditions: (1 ) H₂N-Bn (1.5 equiv.), HS-Bn (1.5 equiv.), PBS/EtOH (4:1 ), 40 °C, 48 h; (2) H₂N-Bn (1 .5 equiv.), HO-Bn (1 .5 equiv.), PBS/EtOH (4:1 ), 40 °C, 48 h; (3) HS-Bn (1 .5 equiv.), PBS/EtOH (4:1 ), 40°C, 48 h; (4) HO-Bn (1.5 equiv.), PBS/EtOH (4:1 ), 40 °C, 48 h. Reaction yield was determined from the crude reaction mixture by ¹ H nuclear magnetic resonance (NMR) using CH₂Br₂ as an internal standard. PBS: Phosphate buffered saline (pH 7.0).

FIG. 8 depicts the substrate scope of amine nucleophiles to /3-Silyl alkynoates. 1a (50 mg, 0.196 mmol), 2a (0.295 mmol, 1.5 equiv.), solvent (0.5 mL) for 48 hours; Isolated yield; ^aThe remaining starting materials were recovered. ^b PBS buffer (pH=7.0)/CH₃CN (4:1 ) was used.

FIG. 9 depicts the crystal structure of 3b.

FIG. 10 depicts the scope of the amine-containing bioactive molecule and drug modification. 1a (50 mg, 0.196 mmol), 2a (0.295 mmol, 1.5 equiv.), solvent (0.5 mL) for 48 hours; Isolated yield; ^a PBS buffer(pH=7.0)/CH₃CN (4:1 ) solvent was used; ^b PBS buffer(pH=7.0) was used as a solvent. ^cThe remaining starting materials were recovered.

FIG. 1 1 depicts the stereoselectivity assessment in peptides using ¹³C NMR spectrum for epimer of amine-conjugated product.

FIG. 12 depicts the ¹³C NMR stuck plot of 3am and 3am’ to determine the epimerization at the chiral centre.

FIG. 13 depicts the ¹³C NMR stuck plot of 5am and 5am’ to determine the epimerization at the chiral centre.

FIG. 14 depicts the application of /V-substituted propiolamide - (A) gram scale synthesis; and (B) product derivatization of /V-benzylpropiolamide.

FIG. 15 depicts the application in linker chemistry for peptide-drug conjugation (PDCs).

FIG. 16 depicts the selectivity comparison between activated esters and ethyl alkynoates. FIG. 17 depicts the specific amino ester selectivity.

FIG. 18 depicts the polypeptide modification with 1a.

FIG. 19 depicts the polypeptide modification with modified Lanreotide conjugates.

FIG. 20 depicts the high-resolution mass spectra (HRMS) spectra of modified H-Lys-Val-Ala- Trp-Phe-NH2 with 1a.

FIG. 21 A and FIG. 21 B depict the liquid chromatography-mass spectrometry (LC-MS) spectra for modified Lanreotide after treatment with TCEP.

FIG. 22 depicts the HRMS spectra of modified Lanreotide.

FIG. 23 depicts the liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra for reductive modification Lanreotide.

FIG. 24 depicts the LC-MS/MS spectra for H-Met-Lys-Leu-Val-Phe-Gly-ser-Ala-NH2 with 1a.

FIG. 25 depicts the HRMS spectra of modified H-Met-Lys-Leu-Val-Phe-Gly-ser-Ala-NH2 with 1a.

FIG. 26 depicts the high-performance liquid chromatography (HPLC) spectra to determine the conversion of 3au to 3aw in two steps.

FIG. 27 depicts the HRMS spectra of modified desilylated Lanreotide (3aw).

FIG. 28 depicts the HRMS spectra for desilylation/conjugation of modified Lanreotide with benzyl mercaptan (3ax).

FIG. 29 depicts the modification of (A) Bovine Serum Albumin (BSA), (B) Myoglobin, (C) Lysozyme, and (D) Cytochrome C protein using /3-silyl alkynoates.

FIG. 30 depicts the deconvoluted mass spectrum of unmodified Bovine Serum Albumin (BSA).

FIG. 31 depicts the deconvoluted mass spectrum of modified Bovine Serum Albumin (BSA). FIG. 32 depicts the relative abundance values of components from the modified Bovine Serum Albumin (BSA).

FIG. 33 depicts the tandem MS (mass spectroscopy) result for the modification of BSA with 1a (ZZG-1 D).

FIG. 34A and FIG. 34B depict the tandem MS analysis for Sequence: Sequence: ECCHGDLLECADDRADLAK, C2-Carbamidomethyl (57.02146 Da), C3-Carbamidomethyl (57.02146 Da), C10-Carbamidomethyl (57.02146 Da), K19-ZZG-1 D (51.99492 Da) Charge: +3, Monoisotopic m/z: 767.31744 Da (+0.05 mmu/+0.06 ppm), (M+H)⁺: 2299.93778 Da, RT: 37.9140 min, Identified with: Sequest HT (v1.17); XCorr:5.15, Ions matched by search engine: 0/0 Fragment match tolerance used for the search: 0.02 Da Fragments used for search: b; b-H₂O; b-NH₃; y; y-H₂O; y-NH₃ Protein references (1 ):- BSA.

FIG. 35A and FIG. 35B depict the tandem MS analysis for Sequence: VHKECCHGDLLECADDRADLAK, C5-Carbamidomethyl (57.02146 Da), C6- Carbamidomethyl (57.02146 Da), C13-Carbamidomethyl (57.02146 Da), K22-ZZG-1 D (51.99492 Da) Charge: +3, Monoisotopic m/z: 888.72595 Da (+1.13 mmu/+1 .27 ppm), (M+H)+: 2664.16330 Da, RT: 29.5013 min, Identified with: Sequest HT (v1.17); XCorr:7.46, Ions matched by search engine: 0/0 Fragment match tolerance used for search: 0.02 Da Fragments used for search: b; b-H₂O; b-NH₃; y; y-H₂O; y-NH₃; Protein references (1):- BSA.

FIG. 36A and FIG. 36B depict the tandem MS analysis for Sequence: VHKECCHGDLLECADDRADLAK, C5-Carbamidomethyl (57.02146 Da), C6- Carbamidomethyl (57.02146 Da), C13-Carbamidomethyl (57.02146 Da), K22-ZZG-1 D (51 .99492 Da) Charge: +4, Monoisotopic m/z: 666.79535 Da (-0.09 mmu/-0.14 ppm), MH+: 2664.15957 Da, RT: 29.4782 min, Identified with: Sequest HT (v1.17); XCorr:5.70, Ions matched by search engine: 0/0 Fragment match tolerance used for search: 0.02 Da Fragments used for search: b; b-H₂O; b-NH₃; y; y-H₂O; y-NH₃; Protein references (1 ):- BSA.

FIG. 37 depicts the deconvoluted mass spectrum of unmodified Myoglobin.

FIG. 38 depicts the relative abundance values of components from the unmodified Myoglobin.

FIG. 39 depicts the deconvoluted mass spectrum of modified Myoglobin with /3-silyl methyl alky noate (6). FIG. 40 depicts the relative abundance values of components from the modified Myoglobin with /3-silyl methyl alkynoate (6).

FIG. 41 depicts the tandem MS results for the modification of Myoglobin with /3-silyl methyl al kyn oates (6).

FIG. 42 depicts the deconvoluted mass spectrum of modified Myoglobin with /3-silyl ethyl alkynoate 1a.

FIG. 43 depicts the relative abundance values of components from the modified Myoglobin with /3-silyl ethyl alkynoate 1a.

FIG. 44A and FIG. 44B depict the tandem MS analysis for Sequence: KGHHEAELKPLAQSHATK, K1-ZZG-1 D (51.99492 Da) Charge: +4, Monoisotopic m/z: 509.27109 Da (+2.75 mmu/+5.39 ppm), (M+H)⁺: 2034.06252 Da, RT: 17.2201 min, Identified with: Sequest HT (v1.17); XCorr: 6.38, Ions matched by the search engine: 0/0 Fragment match tolerance used for search: 0.02 Da Fragments used for search: b; b-H₂O; b- NH₃; y; y-H₂O; y-NH₃ Protein references (1 ): - Myoglobin OS=Equus caballus OX=9796 GN=MB PE=1 SV=2

FIG. 45A and FIG. 45B depict the tandem MS analysis for Sequence: KGHHEAELKPLAQSHATK, K9-ZZG-1 D (51.99492 Da) Charge: +3, Monoisotopic m/z: 678.68726 Da (-1 .45 mmu/-2.13 ppm), (M+H)⁺: 2034.04721 Da, RT: 50.8106 min, Identified with: Sequest HT (v1 .17); XCorr:2.98, Ions matched by the search engine: 0/0 Fragment match tolerance used for the search: 0.02 Da Fragments used for the search: b; b-H₂O; b- NH₃; y; y-H₂O; y-NH₃; Protein references (1 ): Myoglobin OS=Equus caballus OX=9796 GN=MB PE=1 SV=2

FIG. 46A and FIG. 46B depict the tandem MS analysis for Sequence: Sequence: KGHHEAELKPLAQSHATK, K1 -ZZG-1 D (51 .99492 Da), K9-ZZG-1 D (51 .99492 Da) Charge: +4, Monoisotopic m/z: 522.27020 Da (+3.13 mmu/+6 ppm), (M+H)⁺: 2086.05898 Da, RT: 21 .6084 min, Identified with: Sequest HT (v1 .17); XCorr:2.89, Ions matched by search engine: 0/0 Fragment match tolerance used for the search: 0.02 Da Fragments used for search: b; b- H₂O; b-NH₃; y; y-H₂O; y-NH₃; Protein references (1 ): Myoglobin OS=Equus caballus OX=9796 GN=MB PE=1 SV=2 FIG. 47A and FIG. 47B depict the tandem MS analysis for Sequence: Sequence: KGHHEAELKPLAQSHATK, K1 -ZZG-1 D (51 .99492 Da), K9-ZZG-1 D (51 .99492 Da) Charge: +5, Monoisotopic m/z: 418.01755 Da (+2.44 mmu/+5.83 ppm), (M+H)+: 2086.05863 Da, RT: 16.4761 min, Identified with: Sequest HT (v1 .17); XCorr:3.78, Ions matched by the search engine: 0/0 Fragment match tolerance used for the search: 0.02 Da Fragments used for the search: b; b-H₂O; b-NH₃; y; y-H₂O; y-NH₃; Protein references (1 ):- Myoglobin OS=Equus caballus OX=9796 GN=MB PE=1 SV=2

FIG. 48 depicts the deconvoluted mass spectrum of unmodified Lysozyme.

FIG. 49 depicts the deconvoluted mass spectrum of modified Lysozyme with /3-silyl methyl alky noate (6).

FIG. 50 depicts the relative abundance values of components from the modified Lysozyme with 6.

FIG. 51 depicts the deconvoluted mass spectrum of modified Lysozyme with /3-silyl ethyl alkynoate (1a).

FIG. 52 depicts the relative abundance values of components from the modified Lysozyme with 1a.

FIG. 53 depicts the deconvoluted mass spectrum of unmodified Cytochrome C.

FIG. 54 depicts the deconvoluted mass spectrum of modified Cytochrome C with 1a.

FIG. 55 depicts the relative abundance values of components from the modified Cytochrome C.

Description

It has been surprisingly found that one or more problems identified above can be solved through the use of /3-silyl alkynoates to couple with amines under biocompatible conditions. Thus, in a first aspect of the invention, there is provided a method of forming an amide bond, the method comprising the steps of:

(a) providing a compound according to formula I:

, where:

Ri represents a linear alkyl group, an aromatic group or a heteroaromatic group, which linear alkyl group, aromatic group or heteroaromatic group are unsubstituted or are substituted by one or more electron withdrawing groups;

R₂, R₂’ and R₂- are each independently selected from a branched C₃ to C₄ alkyl group and an aromatic group; and

, where:

R₃ is an alkyl group, optionally bearing one or more substituents; and

R₄ is H or an alkyl group; or

, where

R₂, R₂’, Rr, Rs and R₄ are as defined hereinbefore. Without wishing to be bound by theory, it is believed that the bulky trialkylsilyl (e.g. triisopropylsilyl group) may help to suppress 1 ,4-addition reactions, while the alkynyl group may help to enhance the ester group's reactivity and directs nucleophiles toward forming the desired amide bond. Additionally, the subsequent removal of the silicon group without epimerization of the stereogenic center facilitates the use of the resulting amide in a diverse range of chemical reactions. As such, the current invention 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 introduces the ability to use the resulting products in the execution of click reactions, peptide-drug conjugates (PDCs), etc. which represent a significant advancement in the synthesis and functionalization of biomolecules under biocompatible conditions.

It is important to note that the method of the current invention is no particularly limited in terms of the starting materials used. A wide array of starting materials include many diverse functionalities present in Ri to F (particularly R₃ and R₄) are demonstrated in the examples section below. As such, it is believed that the methodology disclosed herein is a broadly applicable methodology for the formation of amide bonds.

The word “comprising” refers herein may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like. References herein (in any aspect or embodiment of the invention) to compounds of formula I, II and III includes references to such compounds perse, to tautomers of such compounds, as well as to salts or solvates of such compounds.

Examples of salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1 - hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et a/., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, acyclic or cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl)hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term “alkyl” refers to an acyclic group, it is preferably C1-10 alkyl and, more preferably, C1-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term “alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, Cs io (e.g. C5-7) cycloalkyl.

Unless otherwise specified herein, a “carbocyclic ring system” may be 3- to 14-membered, such as a 5- to 12-membered (e.g. 6- to 10-membered, such as a 6-membered or 10- membered), carbocyclic group that may be aromatic, fully saturated or partially unsaturated, which carbocyclic group may comprise one or two rings. Examples of carbocyclic ring systems that may be mentioned herein include, but are not limited to cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, phenyl, naphthyl, decalinyl, tetralinyl, bicyclo[4.2.0]octanyl, and 2, 3, 3a, 4, 5, 6, 7,7a- octahydro-1 H-indanyl. Particularly preferred carbocyclic groups include phenyl, cyclohexyl and naphthyl.

In certain embodiments of the invention, R2, Rz and R₂” may each be independently selected from a branched C3 to C4 alkyl group or phenyl. More particularly, R₂, Rz and R₂- may each be independently selected from 'Pr, ^rBu or phenyl. For example, R₂, R₂ and R₂” may each be 'Pr.

In certain embodiments that may be mentioned herein, the aqueous solution may be water or a buffer solution. For example, in certain embodiments that may be mentioned herein, one or both of the following may apply: (i) the buffer solution is selected from one or more of the group consisting of a phosphate buffered saline solution, and a tris-HCI buffer solution; and

In certain embodiments that may be mentioned herein, the aqueous solution may further comprise an organic solvent that is selected from one or more of the group consisting of a polar aprotic solvent and an alcohol. In particular embodiments that may be mentioned herein, the organic solvent may be selected from one or more of the group of acetonitrile, DMSO and, more particularly, ethanol.

In embodiments of the invention where the aqueous solution further comprises an organic solvent, the organic solvent may be present in an amount of from 1 :10 to 10:1 vol/vol relative to water or the buffer solution, such as from 1 :6 to 6:1 , such as about 1 :4.

The reaction of step (b) of the method disclosed herein may be conducted at any suitable temperature. For example, the reaction of step (b) may be conducted at a temperature of from 0 to 100 °C, such as from 15 to 80 °C, such as from 25 to 50 °C, such as about 40 °C.

In embodiments of the invention, Ri may be selected from a linear Ci to Ce alkyl group, phenyl or a heterocyclic group, where the phenyl or heterocyclic group is substituted by one or more substituents selected from nitro, Cl, F, and =0. In more particular embodiments of the invention, Ri may be selected from Me, Et,

, and , where the wiggly line represents the point of attachment to the rest of the molecule. In embodiments of the invention that may be mentioned herein, the primary or secondary amine of formula II may be an antibody, a small molecule, a drug molecule, an amino acid, a di-peptide, a tri-peptide, an oligopeptide, a polypeptide, and a protein.

In more particular embodiments of the invention, the primary or secondary amine of formula II may be selected from the group of:

EE

The method disclosed above may be used to manufacture further down-stream products. Thus, in further embodiments of the invention, the compound of formula III may be subjected to a deprotection reaction to provide a compound according to formula IV: where R3 and FU are as defined hereinbefore. Any suitable reagent to remove the silicon protecting group may be used. For example, the deprotection reaction may use EtsN.3HF, as detailed in the examples section below.

The compound according to formula IV may be used in one or more of:

(ai) a Sonogashira coupling reaction;

(aii) a Click reaction (e.g. with an azide);

(aiii) a C-N conjugation reaction;

Examples of these kinds of reactions are provided in the examples below.

Further aspects and embodiments of the invention are described in the following numbered statements.

1 . A reaction comprising: a) providing and mixing

(i) a /3-silyl alkynoate;

(ii) a primary and/or secondary amine;

(iii) a solvent; b) stirring the above mixture for a period of time at a temperature between 37 °C to 80 °C and obtaining an amide.

2. The reaction according to Statement 1 , wherein the silyl group in the /3-silyl alkynoate is a triisopropylsilyl group.

3. The reaction according to Statement 1 or 2, wherein the solvent is Phosphate buffer saline (PBS) (pH 7, was purchased from Alfa Aesar, which contains water (99.05% w), potassium dihydrogen phosphate monohydrate (0.90% w), and sodium hydroxide (0.05% w), or PBS mixed with organic solvent (PBS/organic solvent = 4/1 ).

4. The reaction according to Statement 3, wherein the organic solvent is tetrahydrofuran, acetonitrile, dimethylsulfoxide, dimethylformamide, or ethanol, in particular, the organic solvent is ethanol.

5. The reaction according to Statements 1 to 4, wherein the period of time is 24 hours to 60 hours, in particular, 48 hours.

As demonstrated in the examples section below, the current invention exhibits a selective affinity towards 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 towards the jB, y, and long-chain amino esters. It uniquely maintains the integrity of stereogenic centers in peptides and specifically targets the s-amine of lysine among native amino esters. However, it demonstrates resistance to the amine which is attached to the a- position of the electron-withdrawing groups such as esters or amides (FIG. 4). Expanding upon this observation, the current invention has demonstrated its applicability in the realms of peptide chemistry, protein engineering, and drug development (FIG. 5). Such developments have profound implications for biomedical research and biotechnology, marking a substantial leap forward in these fields.

Some advantages of the current invention over existing methods may include one or more of the following.

(1 ) A distinctive feature of the current invention 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 f-amine of lysine, crucial in peptide and protein engineering, while concurrently avoiding other native amino ester, significantly broadening the scope of this method.

(2) Furthermore, the current invention 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 the disclosed amide products opens new avenues for further functionalization, such as click reactions, Peptide-drug conjugates (PDCs), etc.

(3) Notably, the current invention exhibits exclusive reactivity with /3-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 significant 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.

Further aspects and embodiments of the invention will now be described by reference to the following non-limiting examples.

Examples

Materials

All the chemicals and solvents were purchased from commercial sources and used as received unless otherwise specified. All the reactions were carried out in an open atmosphere condition. Flash chromatography was performed using Merck 40-63 D 60A silica gel. Phosphate buffer solution (pH 7, Cat No. 38712) was purchased from Alfa Aesar, which contains water (99.05% w), potassium dihydrogen phosphate monohydrate (0.90% w), and sodium hydroxide (0.05% w). Lanreotide acetate (CAS: 2378114-72-6) was purchased from MedChem Express and used without further purification. Bovine Serum Albumin was purchased from the Sigma-Aldrich Catalog no. (A2153-10G), protein was used without further purification. Myoglobin protein (Equine skeletal) was purchased from the Sigma-Aldrich Catalog no. (M0630-250MG), protein was used without further purification. Lysozyme was purchased from the Sigma-Aldrich Catalog no. (L6876-1 G), protein was used without further purification. Cytochrome C was purchased from the MedChemExpress Catalog no. (HY - 125857), protein was used without further purification.

Characterisation Methods

Nuclear Magnetic Resonance (NMR) measurements

¹H, ¹³C, and ¹⁹F NMR spectra of the compounds were recorded on either Bruker 400 MHz, JEOL 400 MHz, and JEOL 500 MHz NMR spectrometer at 25 °C. The chemical shift values in ppm (6) were reported for the residual chloroform (7.26 for ¹H and 77.16 ppm for ¹³C) and DMSO (2.50 for ¹ H and 39.52 ppm for ¹³C).

High-Resolution Mass Spectra (HRMS)

High-resolution mass spectra (HRMS) were recorded on a Waters G2-XS Q-tof spectrometer with ESI mode unless otherwise stated. Electrospray Ionization Mass Spectrometry (ESI-MS)

For Example 1 1 : ESI-MS was performed on a linear quadrupole ion trap detector mass spectrometer (LTQ XL from Thermo Fisher Scientific) coupled to an Ultimate 3000 UPLC. Data were processed using X-Calibur software.

For Example 12: ESI-MS was performed on a linear quadrupole ion trap detector mass spectrometer (LTQ XL from Thermo-Fisher Scientific) coupled to Vanquish UHPLC (from Thermo-Fisher Scientific). Data were processed using Thermo BioPharma Finder 3.1 .

Example 1. Synthesis of (3-Silyl Alkynoates

The alkyne esters were prepared according to the previously reported literature (Nat. Common. 13, 380 (2022); Nat. Protoc. 2, 3247-3256 (2007); Nature 465, 1027-1032 (2010)) method with slight modifications.

General procedure A

To a stirred solution of ethynyltriisopropylsilane (1 ml, 4.45 mmol) in anhydrous THF (20 mL), ⁿ BuLi (2 M in cyclohexane, 2.23 mL, 4.45 mmol) was added at -40 °C under Argon atmosphere. The resulting mixture was stirred at the same temperature for 1 hour. Then the corresponding chloroformate (4.90 mmol) in anhydrous THF (5 mL) was added dropwise to the reaction mixture at -40 °C. The reaction mixture was stirred for 90 minutes at the same temperature, then it was warmed to room temperature and allowed to be stirred for another 90 minutes. After that, the mixture was quenched with NH₄CI (sat. aq, 20 mL). The organic layer was extracted with diethyl ether (3 x 10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. To afford the pure product, the crude reaction mixture was purified over silica gel column chromatography using ethyl acetate/hexane as eluent.

General procedure B

To a stirred solution of ethynyltriisopropylsilane (1 ml, 4.45 mmol) in anhydrous THF (15 ml), isopropyl magnesium chloride (2 M in THF, 2.68 mL, 5.35 mmol) was added dropwise to the solution while maintaining the internal temperature (10-15) °C. The mixture was stirred for 45 minutes at room temperature, and then ethyl chloroformate (5.35 mmol) was slowly added. The resulting solution was stirred for 12 hours at room temperature. The organic layer was extracted with diethyl ether (3 x 10 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. To afford the pure product, the crude reaction mixture was purified over silica gel column chromatography using ethyl acetate/hexane as eluent.

Step I: To a stirred solution of ethynyltriisopropylsilane (1 ml, 4.45 mmol) in anhydrous THE (40 ml), ⁿ BuLi (2 M in cyclohexane, 2.45 mL, 4.90 mmol) was added at -40 °C under Argon atmosphere and then the mixture was cooled to -78 °C and stirred for 1 hour. Then CO₂ gas was bubbled into the solution for 2 h. The reaction mixture was quenched by 2.0 M aqueous KHSO4 solution, warmed to rt, diluted with water, and extracted with ethyl acetate. The crude mixture was diluted with toluene poured onto a pad of SiO₂ and eluted with ethyl acetate. The filtrate was concentrated to afford triisopropylsilanylpropynoic acid which was further used for the next step.

Step II: To a stirred solution of triisopropylsilanylpropynoic acid (0.5 gm, 2.21 mmol) in DCM (10 ml), ^f BuOH (0.251 mL, 2.65 mmol), DCC (0.501 gm, 2.43 mmol) and DMAP (0.027 gm, 0.221 mmol) was added at 0 °C. Then the resulting mixture was stirred for 12 h at room temperature. After that, the mixture was filtered, the filtrate was diluted with DCM and washed with water. The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. To afford the pure product, the crude reaction mixture was purified over silica gel column chromatography using ethyl acetate/hexane as eluent.

Results and Discussions

The following p-silyl alkynoates were synthesised according one of the general procedures above.

Example 2. Addition of Amine to p-Silyl Alkynoates

To a 4 ml glass vial, 0.1 mL 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 minutes 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 anhydride sodium sulfate. The crude mixture was purified over silica gel column chromatography and 30% ethyl acetate/hexane was used as an eluent to afford the product (3a).

Example 3. Addition of Benzylamine to Ethyl 3-(Triisopropylsilyl)propiolate and Reaction Condition Optimization

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. For the reaction condition optimization, modifications were made to the general procedure disclosed in Example 2.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for the Determination of Metal Ions

O

Deionized water

^OEt

TIPS"' 48 h, 40 ‘C

2a 3a

General Procedure for ICP-MS Experiments

In a 4ml glass vial, 1a (60 mg, 0.236 mmol), and 2a (0.354 mmol) were taken in 0.8 mL Deionized water. Then the reaction mixture was vigorously stirred at 40 °C for 48 h. After that, a 0.3 mL aliquot of the reaction mixture was placed to evaluate ICP-MS.

The Agilent Technologies 7700 Series ICP-MS instrument have been used to determine the metal concentration in the reaction mixture.

Sample preparation: 0.3 mL aliquot of reaction mixture was diluted by adding 4.7 mL of 3% HNO3 matrix solution in water. Then, the metal concentration in the reaction mixture was recorded.

Results and Discussions

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 1-8 and 15-16). Notably, using a PBS buffer at pH 7 resulted in an excellent yield of 80% within 48 h, with the remaining substrate left unreacted (Table 1 , entry 4). Our experiments with organic solvents, including DMSO, CH₃CN, EtOH, THF, and others, yielded results that were less than satisfactory (Table 1 , entries 9-14). We expanded our studies by incorporating various organic solvents into a PBS buffer solution at pH 7 (Table 1 , entries 17-20). 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 20). Additionally, 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 21 ). However, it is crucial to note that while elevating the temperature to 80 °C, the reaction rate can be significantly hastened to complete it within 24 hours (Table 1 , entry 22). 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 23).

Table 1. Optimization of reaction conditions³.

Entry Solvent pH value Temperature (°C) 3a (%)^b

1 H₂O 7 37 56

2 Sodium acetate buffer 5.2 37 18

3 PBS buffer 6 37 30

4 PBS buffer 7 37 80

5 PBS buffer 8 37 54

6 PBS buffer 9 37 52

7 Tris-HCI buffer 8 37 40

8 Tris-HCI Buffer 9.22 37 37

9 CH₃CN — 37 17

10 DMSO — 37 39

1 1 DMF — 37 19

12 THF — 37 14

13 EtOH — 37 58

14 DCM — 37 18

15 H₂O/EtOH (4:1 ) 7 37 84

16 H₂O/ EtOH (2:1 ) — 37 62

17 PBS/CH₃CN 7 37 46

18 PBS/DMSO 7 37 48

19 PBS/DMF 7 37 30

20 PBS/EtOH 7 37 85

21 PBS/EtOH 7 40 92 (88)^c

22 PBS/EtOH 7 80 90^d

23 PBS/EtOH 7 40 86^e

24 PBS/EtOH 7 40 89^f ^a Condition: 1a (50 mg, 0.196 mmol), 2a (0.295 mmol, 1.5 equiv.), solvent (0.5 mL) for 48 hours; ^b yield was determined by ¹H NMR using CH₂Br₂ as an internal standard; ^c Isolated yield; ^d Reaction was conducted for 24 hours; ^e reaction was performed in a plastic vial. ^f used 1a was synthesized from the Grignard method. PBS buffer: Phosphate buffered saline; PBS/Organic solvent = 4/1 .

For completion, reaction time and temperature optimization have also been conducted and the results are shown in Table 2 and Table 3, respectively.

Table 2. Reaction time optimization.

Entry Time (Hours) Yield (%)^b

1 12 h 38

2 24 h 55

3 36 h 75

4 48 h 92 ^a: Ethyl 3-(triisopropylsilyl)propiolate 1a (50 mg, 0.196 mmol), benzylamine 2a (0.295 mmol, 1.5 equiv.), PBS buffer (pH=7.0)/EtOH (4:1 ) = 0.5 ml at 40 °C for the indicated time. ^b Yield was determined by ¹H NMR using CH₂Br₂ as an internal standard.

Table 3. Reaction temperature optimization.

Entry Temperature (°C) Time Yield (%)^b

1 25 °C 48 h 40

2 37 °C 48 h 85

3 40 °C 48 h 92 4 80 °C 24 h 90 ^a: Ethyl 3-(triisopropylsilyl)propiolate 1a (50 mg, 0.196 mmol), benzylamine 2a (0.295 mmol, 1.5 equiv.), PBS buffer (pH=7.0)/EtOH (4:1 ) = 0.5 ml. ^bYield was determined by ¹H NMR using CH₂Br₂ as an internal standard.

Subsequently, ICP-MS analysis confirmed that negligible metal presence in the reaction mixture was detected at parts per billion (PPB) levels (FIG. 6).

Example 4. Addition of Benzylamine to Various ^-Substituted Alkynyl Esters

Next presented herein is the systematical investigations on various /3-substituted alkynyl esters, examining their reactions with benzylamine (2a) under optimal conditions disclosed in Example 3.

Results and Discussions

It was observed that phenyl-substituted alkynyl esters produced both 1 ,2- and 1 ,4-addition products with 39% and 45% yields, respectively (Table 4, 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. (Table 4, entry 2). A notable change was evident when we employed the bulkier 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% (Table 4, entry 4).

Table 4. Regio-selectivity of the reaction using a different protecting group³

_R .............

1a 2a 3a (1 2-addition) 4a (1 ,4-addition)

Entry R-group 1 ,2-addition (%) ^b 1 ,4-addition (%) ^b

1 Ph 39 45

2 TMS^C 0 66

3 TES^C 12 55

4 TIPS 92 0 ^a: 1a (50 mg, 0.196 mmol), benzylamine 2a (0.295 mmol, 1.5 equiv.), PBS buffer (pH=7.0)/EtOH (4:1 ) = 0.5 ml. ^bYield was determined from the crude mixture by ¹ H NMR using CH₂Br₂ as an internal standard. ^cThe crude complex mixture may contain other possible byproducts.

Subsequently, we focused on assessing the effect of various ester groups on these reactions. The results, presented in the accompanying table in Table 5, showed that, regardless of the ester group variations, the reactions favored the formation of 1 ,2-addition products with commendable efficiency (Table 5, Entries 1-3). However, the substitution of the ester group with either a tert-butyl or an acid group failed to produce any product formation (Table 5, Entries 4-6). A significant 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 (Table 5, Entries 8-9). 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.

Table 5. Reactivity comparison with different ester groups³

Entry R¹-group R²-group Product Yield (%)^b

1 Me 86 %

4 *Bu No Reaction

5 H No Reaction

6 PhCH₂ H No Reaction

7 Ph Et Trace

8 Et No Reaction 9 Et No Reaction ^a: Ethyl 3-(triisopropylsilyl)propiolate 1a (50 mg, 0.196 mmol), benzylamine 2a (0.295 mmol, 1.5 equiv.), PBS buffer (pH=7.0)/EtOH (4:1 ) = 0.5 ml. ^b Yield was determined by ¹H NMR using CH₂Br₂ as an internal standard.

Example 5. Competitive Experiments with Different Nucleophiles

We then proceeded to conduct competitive experiments to assess the selectivity of the reaction towards amine in the presence of both benzyl thiol and alcohol under our optimized reaction conditions.

Results and Discussions

The selectivity of the reaction towards amine in the presence of both benzyl thiol and alcohol under our optimized reaction conditions is as outlined in FIG. 7

We were pleased to find that no C-S or C-0 bond formation occurred, with only products resulting from C-N bond formation observed. Notably, when we conducted separate reactions of /3-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 significant chemoselectivity of the reaction towards amines.

Example 6. Addition of Various Amines to Ethyl 3-(Triisopropylsilyl) Propiolate

With the optimized reaction conditions disclosed above, the substrate scope of amines in forming the amide bonds with Tl PS-conjugated ester 1a was explored.

Crystallographic Investigation

The compound 3b were crystallized by the slow evaporation of chloroform and hexane mixture (ca. 30%).

Results and Discussions

As depicted in FIG. 8, the experiments revealed that both linear and branched chain primary alkyl amines were effectively accommodated, resulting in the formation of adducts 3a-3ad with moderate to excellent yields. It is worth noting that, the single crystal XRD analysis of compound 3b (CCDC: 2310713) conclusively establishes the 1 ,2-addition reaction, providing robust evidence for its regiochemical arrangement. 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-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-3s with yields ranging from 95% - 72%. Moreover, the branch alkyl amine also demonstrated remarkable effectiveness in producing the corresponding products 3t-3v. The use of cyclohexyl amine produced products 3w with a notable yield of 98%. Interestingly, the reaction of amino alcohol with 1a was selective, preserving the hydroxy group intact in the adduct 3x-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 a-amino esters were used.

Compound 3b (CCDC 2310713)

Crystal structure of 3b (FIG. 9)

Empirical formula C20H31 NOSi

Formula weight 329.55 g/mol

Crystal system monoclinic

Crystal habit colourless block Space group P 1 21/c 1

Unit cell dimensions a =18.524(2) A a = 90° b = 12.5421(18) A p = 98.355(6)° c = 8.5118(10) A y = 90°

Volume 1956.6(4) 3

Z 4

Density (calculated) 1.1 19 g/cm³

Crystal size 0.060 x 0.200 x 0.220 mm

Final R indice [l>2sigma(l)] R1 = 0.0832, wR2 = 0.1926

R indices (all data) R1 = 0.1410, wR2 = 0.2309

To further evaluate the versatility of this reaction, we reacted /3-silyl alkynoates with various biomolecules and drugs containing free amines (FIG. 10). In an initial experiment, /V-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. Additionally, 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.

Example 7. Exploring Epimerisaton at Chiral Centres

Determining whether the method disclosed in the Examples above can be applied to peptides or proteins without causing epimerization at existing stereogenic centers is critical, as depicted in FIG. 1 1 , FIG. 12, and FIG. 13. This concern particularly applies during the amide bond formation step and the subsequent removal of the TIPS group.

Dipeptide Synthesis and Epimerization Experiment

General procedure

Step I: To a stirred solution of Boc-Lys(Z)-OH (0.5 gm, 1 .315 mmol) and L-Valine methyl ester hydrochloride (0.242 gm, 1.447 mmol) in dry CH2CI2 (10 ml), 1 -ethyl-3-(3-dimethyl- aminopropyl) carbodiimide (EDC) (1 .97 mmol), Hydroxybenzotriazole (HOBt) (1 .97 mmol) and A/,/V-Diisopropylethylamine (DIPEA) (2.631 mmol) was added at room temperature. The resulting solution was stirred for 12 hours. Then the mixture was diluted with CH2CI2 and washed with water. The combined organic was separated, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude mixture was purified over silica gel flash column chromatography to afford the product 27.

Step II: To a stirred solution of 27 (0.5 gm, 1.013 mmol) in methanol, 5 wt% of Pd-C was added in Hydrogen atmosphere (H₂ balloon). The resulting solution was stirred for 12 h at room temperature. Then the mixture was filtered through a pad of celite, the filtrate was dried to afford the pure product of 15.

Exploring Epimerization at Chiral Centres Following Biocompatible Amide Bond Formation

Reaction

General procedure. To a 4 ml glass vial, ethyl 3-(triisopropylsilyl)propiolate (1a, 60 mg, 0.236 mmol) and 15 (127 mg, 0.354 mmol) was taken. Then 0.5 mL of pH-neutral phosphate buffer was added. The resulting solution was vigorously stirred for 48 h 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, and hexane/ethyl acetate was used as an eluent to afford the pure product methyl A/2-(tert-butoxycarbonyl)-M6-(3- (triisopropylsilyl)propioloyl)-L-lysyl-L-valinate (3am).

Following the general procedure we have prepared the methyl A/2-(tert-butoxycarbonyl)-/\/6- (3-(triisopropylsilyl)propioloyl)-L-lysyl-L-valinate (3am) and methyl methyl A/2-(tert- butoxycarbonyl)-A/6-(3-(triisopropylsilyl)propioloyl)-L-lysyl-D-valinate (3am').

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 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.

Exploring Epimerization at Chiral Centers after desilylation of the alkynyl amide.

To a 10 mL round bottom flask, 3am (60 mg, 0.105 mmol) and triethylamine trihydrofluoride (5 equiv.) were taken in 2 mL THF at 0°C. Then the mixture was vigorously stirred for 12 hours at room temperature under an argon atmosphere. After that, the organic layer was washed with water, extracted with ethyl acetate, and dried over anhydrous sodium sulfate, concentrated under reduced pressure. The crude mixture was purified over silica gel column chromatography to afford the pure product 5am.

Following the general procedure, we have prepared the methyl A/2-(tert-butoxycarbonyl)-/V6- propioloyl-L-lysyl-L-valinate (5am) and methyl /\/2-(tert-butoxycarbonyl)-/V6-propioloyl-L-lysyl- D-valinate (5am’). Results and Discussions

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 EtsN.SHF reagent in THF at room temperature.

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. We made the stuck plot of ¹³C NMR spectra of 3am and 3am’ (FIG. 12), and 5am and 5am’ (FIG. 13). 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. 1 1 , FIG. 12, and FIG. 13). The spectra clearly show that no carbon atom peak is getting overlaps of these two compounds 3am and 3am’. From the spectra, it is concluded that no epimerization was observed at the chiral center of the silyl alkynyl amide products (3am, 3am’). From the spectra, it was visible that no carbon atom is getting overlaps of these two compounds 5am and 5am’. After the desilylation step, no epimerization was observed at the chiral canter of the desilylated alkynyl amide.

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.

Example 8. Gram-Scale Synthesis and Product Derivatisation

Gram-scale synthesis of 3a under the same reaction condition as the optimized one disclosed in Example 3 was performed. Product derivatisation of the product 5a (obtained from 3a) was also explored.

Step I: In a 50 mL round bottom flask, 1a (1 .27 gm, 5 mmol) and 2a (0.82 ml, 7.5 mmol) were taken in 3 mL ethanol. Then, the solution was stirred for 5 minutes to achieve a homogeneous solution. Subsequently, 12 mL of pH-neutral phosphate buffer was added, and the resulting mixture was vigorously stirred at 40°C for 48 hours. After that, the mixture was settled down in a cold place to complete the precipitation of the product. The mixture was filtered, and the residue was washed with water and dried under vacuum to afford the pure product of 3a (1.36 gm).

Step II: In a 50 mL round bottom flask, 3a (1 .36 gm, 4.3 mmol) and EtsN.3HF (5 equiv.) were taken in 15 ml THF at 0 °C in an argon atmosphere. The mixture was vigorously stirred for 12 hours at room temperature under an argon atmosphere. 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.

Biocompatible C-N Bond Formation Reaction

Procedure: To a 4 ml glass vial, 5a (50 mg, 0.314 mmol) and 16 (0.377 mmol) were taken. Then add 1 mL PBS buffer (pH=8.0) to the mixture. The resulting solution was stirred at 40 °C for 12 hours. After that, the mixture was diluted with ethyl acetate, washed with water, and dried over anhydrous sodium sulfate, concentrated under reduced pressure. The crude mixture was washed with cold hexane to afford the pure product 16a in 88% yield.

Sonogashira Coupling Reaction for the Synthesis of N-Benzyl-3-Phenylpropiolamide.

Procedure: In an oven-dried seal tube, 5a (60 mg, 0.377 mmol), 17 (0.566 mmol), Pd(PPh₃)₂Cl2 (0.018 mmol), and Cui (0.037 mmol) were taken in triethylamine (1 mL). The resulting solution was refluxed at 70 °C for 6 h. Then the mixture was diluted with ethyl acetate and washed with a brine solution, the organic layer was separated, dried over anhydrous NasSC , and concentrated under reduced pressure. The crude mixture was separated over silica gel flash column chromatography to afford pure 17a in 74% yield.

Synthesis of 5-lodo-1 ,2,3-Triazoles Compound via Click Reaction.

Used for ¹²⁵l Labeled Molecular Probes

Procedure: To a stirred solution of 5a (65 gm, 0.408 mmol) and 18 (0.45 mmol) in dry DMF, were added A/-iodosuccinimide (0.490 mmol), copper (I) Iodide (0.4 mmol) and triethylamine (0.082 mmol). The resulting solution was stirred at room temperature under an argon atmosphere for 4 h. After that, the mixture was diluted with ethyl acetate and washed with water, and the organic layer was separated and dried over anhydrous Na₂SO₄, concentrated under reduced pressure. The crude mixture was purified by silica gel flash column chromatography to afford 18a in 82% yield.

Procedure: To a Schlenk tube equipped with a magnetic stir bar 5a (70 mg, 0.440 mmol), Lindlar catalyst (10 wt.%) in methanol under H₂ atmosphere (H₂ Balloon). Then the reaction was stirred for 18 h at room temperature. After that, the mixture was filtered through a pad of celite, and the filtrate was dried to afford the pure product of 19a in 86% yield.

Results and Discussions

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. 14A). It is noteworthy that the M-propiolamide derivatives obtained from our method exhibit highly resourceful building blocks in organic synthesis Org. Biomol. Chem. 20, 2671-2680 (2022); J. Med. Chem. 60, 839-885 (2017); Org. Lett. 21 , 3392-3395 (2019)) (FIG. 14B). 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. Additionally, the alkyne group within these compounds (5a) can also be selectively reduced to the alkene derivative 19a using a Lindlar catalyst.

Example 9. Linker Application - Conjugation between Drug and Peptide

During the last decades, Peptide-drug conjugates (Curr. Med. Chem. 24, 3373 -3396, (2017)) (PDCs) have received significant attention as promising targeting therapies, akin to Antibodydrug conjugates (ADCs). These conjugates typically consist of monoclonal peptides, drug payloads, and cleavage/non-cleavage linkers. Capitalizing on the inherent versatility (Eur. J. Med. Chem. 183, 11 1687, (2019)) of A/-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 peptide-drug conjugates (PDCs) (FIG. 15).

Biocompatible C-S Bond Formation

Procedure: To a 4 ml glass vial, 5am (45 mg, 0.109 mmol) and 20 (0.1 .32 mmol) were taken in 0.4 mL ethanol. Subsequently, add 0.4 ml of water to the mixture. Then the resulting solution was stirred at 40 °C for 6 hours. After that, 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 to yield a pure product of 20a in 78%. Procedure: 5am (45 mg, 0.109 mmol) and 21 (0.1.32 mmol) were taken in a 4 mL glass vial. Subsequently, water: ethanol (1 :1 ) mixture of solvent 0.8 mL was added to the vial. Then the resulting solution was stirred at 40 °C for 6 hours. After that, 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 to yield a pure product of 21a in 84%.

Zidovudine Drug Modification

Procedure: In a Schlenk tube, 5am (45 mg, 0.109 mmol), Zidovudine (0.120 mmol), copper iodide (0.109 mmol), and triethylamine (0.109 mmol) were taken in dry DMF. The resulting solution was stirred at room temperature under an argon atmosphere for 4 hours. After that, the mixture was diluted with ethyl acetate and washed with water (4 times), the organic layer was separated and dried over anhydrous Na2SC>4, concentrated under reduced pressure. The crude mixture was purified over silica gel flash column chromatography to afford the pure product 22a in 61 % yield.

Results and Discussions

This approach harnesses the unique properties of /V-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, 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 /V-propiolamide derivatives as anchors for coupling with various biomolecules, leading to the production of valuable peptide-drug conjugates. Such versatility broadens the potential applications of this methodology in biochemical conjugation and drug development.

Example 10. Selectivity Comparison between Activated Ethyl Alkynoates and /3-Silyl Alkynoates

As previously mentioned, we were intrigued to find that cr-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 /3-silyl ethyl alkynoates regarding their specific reactivity toward amino esters (FIG. 16).

Reaction of Activated Ethyl Alkynoates with Amino Esters

General Procedure: To a 4 ml glass vial, 0.1 mL ethanol was added to the mixture of NHS alkynoates (23, 50 mg, 0.154 mmol) and the corresponding amino ester (2, 0.232 mmol). The mixture was stirred for 5 minutes to achieve a homogeneous solution. Subsequently, 0.4 mL of pH-neutral phosphate buffer was added, and the resulting mixture was vigorously stirred for 24 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 and hexane/ethyl acetate was used as an eluent to afford the corresponding product (3ao or 3ap).

Reaction of Ethyl Alkynoates with Amino Esters

3ar, 82% Trace C amino este; a -amino amide

General Procedure: To a 4 ml glass vial, 0.1 mL ethanol was added to the mixture of ethyl 3- (triisopropylsilyl) propiolate (1a, 50 mg, 0.196 mmol) and the corresponding amino ester (2, 0.295 mmol). The mixture was stirred for 5 minutes 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 and hexane/ethyl acetate was used as an eluent to afford product 3 with the corresponding yield.

Similarly, under standard conditions, we conducted the experiments between ethyl 3- (triisopropylsilyl) propiolate (1a) with a-amino amide. Notable, only a trace amount (<10%) of product formation was observed.

Results and Discussions

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. Due to their high reactivity, activated esters readily react with both a- and P-amino esters to form the corresponding amide products. For a comprehensive comparison, we synthesized p-nitrophenyl (23a), A/-hydroxysuccinimide (23b), and 2,4,5-trichlorophenyl (23c) silyl alkynoates. It is essential to highlight that all the activated alkynoates demonstrated equal reactivity towards both a- and /3-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 towards various amino esters revealed that a-amino esters (3ao) displayed negligible reactivity, while a-amino amides (3ao’) yielded only traces of product.

Conversely, /3-amino esters, /-amino esters, and 5-amino esters efficiently produced the desired products with yields of 50%, 80%, and 82%, respectively (FIG. 17). 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.

Example 11. Peptide Modification

Further exploration of the reactivity of /3-silyl alkynoates, we conducted experiments using lysine amide (3as), which contains two free amino groups (FIG. 18). Conjugation of modified Lanreotide with a benzyl mercaptan was also explored (FIG. 19).

Lysine amide 3as was prepared according to the protocol disclosed in Example 10.

To a 4 ml glass vial, 0.1 mL acetonitrile was added to the mixture of 3t (TEA salt) (3 mg, 3.9 pmol) and ethyl 3-(triisopropylsilyl)propiolate (1a, 30 equiv.). Subsequently, 0.5 mL of buffer solution (pH=10.0) was added, and the resulting mixture was vigorously stirred for 72 hours at 40°C. After that, 20 pL aliquot of the reaction mixture was evaluated by LC-MS to calculate the conjugation rate based on the consumption of the unmodified 3at. The conjugated species was obtained with one ligand modification, which was confirmed by ESI-MS (FIG. 20). The residue was purified by preparative HPLC to obtain the modified products 3at (1 .9 mg) in 58% of yield.

HPLC methods: HPLC column was performed using Shim-pack Scepter C18-120 (5 pm, 4.6 x 250 mm) columns with a mobile phase of water with 0.1% trifluoroacetic acid (A) and acetonitrile with 0.1% trifluoroacetic acid (B) at a flow rate of 1 .0 mL/min. The gradient used: solvent B (5-60)% for (0-10) min, then (60-90)% for (10-30) min, then 90% for (30-35) min, then (90-15)% for (35-36) min, then 15 for (36-45) min. A = 220 nm, Retention time (tp) = 15.784 min at 45 °C. Procedure for the Modification of Peptides Lanreotide Acetate with 1a

General Procedure: To a 4 ml glass vial, 0.1 mL acetonitrile was added to the mixture of Lanreotide acetate (5 mg, 4.3 pmol) and ethyl 3-(triisopropylsilyl)propiolate (1a, 30 equiv.). Subsequently, 0.5 mL of buffer solution (pH=10.0) was added, and the resulting mixture was vigorously stirred for 72 hours at 40°C. After that 20 pL aliquot of the reaction mixture was evaluated by LC-MS to calculate the conjugation rate based on the consumption of the unmodified lanreotide (FIG. 21). The conjugated species was obtained with one ligand modification, which was confirmed by ESI-MS (FIG. 22). The residue was purified by preparative HPLC to obtain the modified pure products (2.8 mg) as an off-white solid.

Procedure for the Reduction of Oxidative-modified Lanreotide

The regio-selectivity of the modification was analysed by LC-MS/MS after treatment with TCEP [(tris(2-carboxyethyl)phosphine)] (2.0 equiv.) for 30 min (FIG. 23).

HPLC methods: HPLC column was performed using Shim-pack Scepter C18-120 (5 pm, 10 x 250 mm) columns with a mobile phase of water with 0.1 % trifluoroacetic acid (A) and acetonitrile with 0.1% trifluoroacetic acid (B) at a flow rate of 2.5 mL/min. Gradient used Solvent B (70 - 90)% over 90 min. A = 220 nm, tn = 14.776 min.

Procedure for the Modification of Peptides H-Met-Lys-Leu-Val-Phe-Gly-ser-Ala-Nffa with 1a

To a 4 ml glass vial, 0.1 mL acetonitrile was added to the mixture of 3v (TFA salt) (3 mg, 3.1 pmol) and ethyl 3-(triisopropylsilyl)propiolate (1a, 30 equiv.). Subsequently, 0.5 mL of buffer solution (pH=10.0) was added, and the resulting mixture was vigorously stirred for 72 hours at 40°C. After that, 20 pL aliquot of the reaction mixture was evaluated by LC-MS to calculate the conjugation rate based on the consumption of the unmodified 3av (FIG. 24). The conjugated species was obtained with one ligand modification, which was confirmed by ESIMS (FIG. 25). The residue was purified by preparative HPLC to obtain the pure modified products 3av (1 .6 mg) in 50% of yield.

HPLC methods: HPLC column was performed using Shim-pack Scepter C18-120 (5 pm, 10 x 250 mm) columns with a mobile phase of water with 0.1 % trifluoroacetic acid (A) and acetonitrile with 0.1% trifluoroacetic acid (B) at a flow rate of 2.5 mL/min. The gradient used: solvent B (0-15)% for (0-2) min, then (15-95)% for (2-72) min, then 95% for (72-85) min, then (95-15)% for (85-87) min then 15% for (87-90) min; A = 220 nm, t« = 51 .47 min

De-silylation and Conjugation of Modified Lanreotide

To a 5 mL round-bottom flask, Modified Lanreotide (3au, 2.5 mg, 1 .91 pmol) was added to 0.5 mL of methanol. Subsequently, 3HF.EtsN (60 equiv.) was slowly added to the reaction mixture. The resulting mixture was vigorously stirred for 48 hours at room temperature. Afterward, an aliquot of the sample was analyzed using analytical HPLC to identify the product formation and confirm the complete conversion of 3au to 3aw (FIG. 26). Without purifying the crude mixture 3aw, we proceeded to the next step for C-S conjugation.

To a 10 mL round bottom flask, benzyl mercaptan (24, 50 equiv.) was added to the crude 3aw mixture, followed by the addition of MeOH: H2O (1 :1 ) solvent (1 mL). The resulting mixture was vigorously stirred for 36 hours at 40°C. An aliquot of the sample was then analyzed using analytical HPLC to identify the product formation and confirm the complete conversion of 3aw to 3ax (FIG. 26). To identify the products, we purified small amounts of 2aw and 3ax using analytical HPLC and recorded their HRMS spectra (FIG. 27 and FIG. 28).

HPLC Method for 3au, 3aw, and 3ax

HPLC column was performed using Shim-pack Scepter C18-120 (5 pm, 4.6 x 250 mm) columns with a mobile phase of water with 0.1% trifluoroacetic acid (A) and acetonitrile with 0.1 % trifluoroacetic acid (B) at a flow rate of 1.0 mL/min. The gradient used: solvent B (5- 60)% for (0-10) min, then (60-90)% for (10-30) min, then 90% for (30-35) min, then (90-15)% for (35-36) min, then 15 for (36-45) min. A = 220 nm, 45 °C.

Retention time (t_R) for 3au is = 18.98 min

Retention time (t_R) for 3av is = 11 .816 min

Retention time (t_R) for 3aw is = 12.612 min

Results and Discussions

The results demonstrated that only the side chain E-NH₂ of lysine reacted with the /3-silyl alkynoates, while the a-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.

Based on the controlled experiment (Table 5. and FIG. 17), a plausible mechanism for the amide bond formation reaction under biocompatible conditions has been rationalized. In /3- 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 s-amino group of lysine due to its higher nucleophilicity, while the limited nucleophilicity of the a-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. 18). Notably, polypeptides with the s-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 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 (TCEP) to break the S-S linkage, forming a linear peptide chain. LC- MS/MS analysis revealed that only the £-NH₂ group of lysine in Lanreotide was reacting by /3- silyl alkynoates, while other nucleophilic amino acids, such as threonine, cysteine, tryptophan, tyrosine, and the terminal amine, remained mostly untouched (FIG. 21 and FIG. 23).

The desilylation of modified Lanreotide could also be achieved using EtgN.SHF reagent in a polar protic methanol solvent at room temperature (FIG. 19). This step enabled further conjugation with a benzyl mercaptan, forming the desired conjugate product (3ax) (FIGs. 26- 28).

Example 12. Protein Modification

Leveraging this success, we extended the disclosed methodology to protein modification targeting lysine under biocompatible conditions, using bovine serum albumin (BSA, 90 pM) as a model protein (FIG. 29). To further demonstrate the versatility of our method, we also applied it to myoglobin protein from equine skeletal muscle, which contains 19 lysine residues. Additionally, we modified lysozyme and cytochrome C using /3-silyl alkynoates.

Bovine Serum Albumin Protein Modification

Bovine Serum Albumin was purchased from the Sigma-Aldrich Catalog no. (A2153-10G). Protein was used without further purification.

The sequence of Bovine Serum Albumin (BSA) (Nature 368, 836-838 (1994); Nature 480, 471-479 (201 1 )). BSA is composed of 583 amino acids and contains one free cysteine (Cys34) and 59 lysin.

DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGC EKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLC DEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETM REKVLTSSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECC HGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADF AEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACY STVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGK VGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPD ETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDK

CCAADDKEACFAVEGPKLVVSTQTALA.

Reaction Procedure of Bovine Serum Albumin with 1a

To a 4-mL reaction tube, Bovine Serum Albumin (BSA) (3 mg, 4.5x10^-5 mmol) was added with 1a (20 mg) in PBS buffer (pH=7.0) (0.5 mL) solvents. After stirring for 36 hours at 40 °C, a 20 pL aliquot of the reaction mixture was placed for evaluation by performing ESI-MS, and the diconjugated adduct with the loss of TIPS (triisopropylsilane) group was detected.

Sample Information for Tandem MS Analysis forBSA:To a 4-mL reaction tube, Bovine Serum Albumin (BSA) (3 mg, 4.5x1 O'⁵ mmol) was added with 1a (20 mg) in PBS buffer (pH=7.0) (0.5 mL) solvents. After stirring for 36 hours at 40 °C. After that, an aliquot amount of the sample was subjected to in-gel digestion and was digested by trypsin, identified by applying the nanoLC-MS/MS platform.

Method Information for Liquid Chromatography with Tandem Mass Spectroscopy: The peptides were separated and analyzed using a Vanquish Neo UHPLC System coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific, MA, USA). Separation was performed on an EASY-Spray 75 pm x 15 cm column packed with PepMap Neo C18 2 pm, 100 A (Thermo Fisher Scientific) using solvent A (0.1% formic acid) and solvent B (0.1 % formic acid in 80% ACN) at flow rate of 300 nL/min with a 60 min gradient. Peptides were then analyzed on an Orbitrap Exploris 480 apparatus with an EASY nanospray source (Thermo Fisher Scientific) at an electrospray potential of 2.0 kV. Raw data files were processed and searched using Proteome Discoverer 2.1 (Thermo Fisher Scientific). The Sequest algorithm was then used for data searching to identify proteins.

Dynamic Modifications:

1. Dynamic Modification: Oxidation / +15.995 Da (M)

2. Dynamic Modification: Phospho / +79.966 Da (S, T, Y)

3. Dynamic Modification: Carbamidomethyl/ +57.02146 Da (C) 3. Dynamic Modification: ZZG-1 D (K)/ +51 .99492 Da (K)

Myoglobin Protein Modification

Myoglobin protein (Equine skeletal) was purchased from the Sigma-Aldrich Catalog no. (M0630-250MG). Protein was used without further purification.

The sequence of Myoglobin consists of 154 amino acids and contains 19 lysin units.

MGLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASE DLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDF GADAQGAMTKALELFRNDIAAKYKELGFQG.

Reaction procedure of Myoglobin with p-Silyl Methyl Alkynoate 6 ,

(6, 200 equiv.)

To a 4-mL reaction tube, Myoglobin (6 mg, 0.35 pmol) was added with /3-silyl methyl alkynoate 6 (200 equiv.) in PBS buffer (pH=8.0) (0.5 mL) solvents. After stirring for 48 hours at 40 °C, a 20 pL aliquot of the reaction mixture was placed for evaluation by performing ESI-MS, and the two-fold conjugated adduct with the loss of TIP (triisopropylsilane) group was detected.

Reaction Procedure of Myoglobin with /3-Silyl Ethyl Alkynoate (1a)

(1a, 200 equiv.)

To a 4-mL reaction tube, Myoglobin (6 mg, 0.35 pmol) was added with 1a (200 equiv.) in PBS buffer (pH=8.0) (0.5 mL) solvents. After stirring for 48 hours at 40 °C, a 20 pL aliquot of the reaction mixture was placed for evaluation by performing ESI-MS, and the two-fold conjugated adduct with the loss of TIP (triisopropylsilane) group was detected. Sample information for tandem MS for Myoglobin: To a 4-mL reaction tube, Myoglobin (6 mg, 3.5 pmol) was added with 6 (200 equiv.) in PBS buffer (pH=8.0) (0.5 mL) solvents. Then the mixture was stirred for 48 hours at 40 °C. After that, an aliquot amount of the sample was subjected to in-gel digestion and was digested by trypsin, identified by applying the nanoLC- MS/MS platform.

Method Information for Liquid Chromatography with Tandem Mass Spectroscopy: The peptides were separated and analyzed using a Vanquish Neo UHPLC System coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific, MA, USA). Separation was performed on a EASY-Spray 75 m x 15 cm column packed with PepMap Neo C18 2 pm, 100 A (Thermo Fisher Scientific) using solvent A (0.1% formic acid) and solvent B (0.1 % formic acid in 80% ACN) at flow rate of 300 nL/min with a 60 min gradient. Peptides were then analyzed on an Orbitrap Exploris 480 apparatus with an EASY nanospray source (Thermo Fisher Scientific) at an electrospray potential of 2.0 kV. Raw data files were processed and searched using Proteome Discoverer 2.1 (Thermo Fisher Scientific). The Sequest algorithm was then used for data searching to identify proteins.

Dynamic Modifications:

1. Dynamic Modification: Oxidation / +15.995 Da (M)

2. Dynamic Modification: Phospho / +79.966 Da (S, T, Y)

3. Dynamic Modification: ZZG-1 D (K)/ +51 .99492 Da (K)

Lysozyme Modification

Lysozyme was purchased from the Sigma-Aldrich Catalog no. (L6876-1 G). Protein was used without further purification.

Sequence of Lysozyme: It consists of 147 amino acids and contains 7 lysine units.

MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQAT NRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNG MNAWVAWRNRCKGTDVQAWIRGCRL

Reaction Procedure for Lysozyme Modification with /3 -Silyl Methyl Alky noate (6)

To a 4-mL reaction tube, Lysozyme (6 mg, 0.42 pmol) was added with 6 (200 equiv.) in PBS buffer (pH=8.0) (0.5 mL) solvents. After stirring for 48 hours at 40 °C, a 20 pL aliquot of the reaction mixture was placed for evaluation by performing ESI-MS. A maximum three-fold conjugated adduct with the loss of the TIP (triisopropylsilane) group was detected.

Reaction Procedure for Lysozyme Modification with fi -Silyl Ethyl Alky noate (1a)

To a 4-mL reaction tube, Lysozyme (6 mg, 0.42 pmol) was added with 1a (200 equiv.) in PBS buffer (pH=8.0) (0.5 mL) solvents. After stirring for 48 hours at 40 °C, a 20 pL aliquot of the reaction mixture was placed for evaluation by performing ESI-MS. A maximum three-fold conjugated adduct with the loss of the TIP (triisopropylsilane) group was detected.

Cytochrome C Protein Modification

Cytochrome C was purchased from the MedChemExpress Catalog no. (HY - 125857). Protein was used without further purification.

Sequence of Cytochrome C: It consists of 1 17 amino acids and contains 19 lysin units.

MGDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIW GEDTLMEYLADVYEKMKDRNTHEEKYIPGTKMPMIFAGIKKKEERADLIAYLKKEE

Reaction procedure for Cytochrome C modification with 1a

To a 4-mL reaction tube, Cytochrome C (6 mg, 0.49 pmol) was added with 6 (200 equiv.) in PBS buffer (pH=8.0) (0.5 mL) solvents. After stirring for 48 hours at 40 °C, a 20 pL aliquot of the reaction mixture was placed for evaluation by performing ESI-MS. A maximum three-fold conjugated adduct with the loss of the TIPS (triisopropylsilane) group was detected.

Results and Discussion

The deconvoluted mass spectrum revealed that the molecular weight of unmodified BSA (66,387 Da, FIG. 30) shifted to 66,491 Da (FIG. 31 and FIG. 32), indicating the modification of BSA with two ligands and the release of the TIPS group (FIG. 29A). 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 (FIGs. 33-36).

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 /3-silyl methyl alkynoates 6 in PBS buffer (pH 8.0) resulted in conjugated products with a maximum of two-fold modification, as identified by LC-MS analysis (FIG. 29B). The deconvoluted mass spectrum showed that unmodified myoglobin (16,950 Da, FIGs. 37-38). shifted to 17,048 Da (FIGs. 39-40) for one-fold modification, representing myoglobin modified with one de-silylated ligand and the consumption of two sodium ions. For two-fold modification, LC-MS showed a peak at 17,102 Da (FIGs. 39-41 ).

Figures 42 depicts the deconvoluted mass spectrum of modified Myoglobin with /3-silyl ethyl alkynoate 1a. FIG. 43 depicts the relative abundance values of components from the modified Myoglobin with j(3-silyl ethyl alkynoate 1a (FIGs. 42-43). 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 (FIGs. 44-47). Inspired by these results, we also modified lysozyme and cytochrome C using /3-silyl alkynoates (FIG. 29C-D). Both proteins showed a maximum of three-fold modification after the removal of the TIPS group (FIGs. 48-55). Interestingly, both the ethyl and methyl alkynoates (1a, 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 the current invention for targeted protein modifications.

Claims

1 . A method of forming an amide bond, the method comprising the steps of:

(a) providing a compound according to formula I:

, where:

R2, R2' and R₂" are each independently selected from a branched C3 to C4 alkyl group and an aromatic group; and

H N— R₃ R/ II

, where:

R₃ is an alkyl group, optionally bearing one or more substituents; and

R₄ is H or an alkyl group; or

, where

R₂, R₂', R₂ ", R3 and R4 are as defined hereinbefore.

2. The method according to Claim 1 , wherein R₂, R₂’ and R₂- are each independently selected from a branched C3 to C4 alkyl group or phenyl.

3. The method according to Claim 2, wherein R₂, R₂' and R₂ are each independently selected from 'Pr, 'Bu or phenyl.

4. The method according to Claim 3, wherein R₂, R₂ and R₂ are each 'Pr.

5. The method according to any one of the preceding claims, wherein the aqueous solution is water or a buffer solution, optionally wherein one or both of the following apply:

(i) the buffer solution is selected from one or more of the group consisting of a phosphate buffered saline solution, and a tris-HCI buffer solution; and

(ii) the buffer solution has a pH of from 5 to 9, such as from 6 to 8, such as about 7.

6. The method according to any one of the preceding claims, wherein the aqueous solution further comprises an organic solvent that is selected from one or more of the group consisting of a polar aprotic solvent and an alcohol, optionally wherein the organic solvent is selected from one or more of the group of acetonitrile, DMSO and, more particularly, ethanol.

7. The method according to Claim 6, as dependent upon Claim 5, wherein when an organic solvent is present, it is present in an amount of from 1 :10 to 10:1 vol/vol relative to water or the buffer solution, such as from 1 :6 to 6:1 , such as about 1 :4.

8. The method according to any one of the preceding claims, wherein the reaction of step (b) in Claim 1 is conducted at a temperature of from 0 to 100 °C, such as from 15 to 80 °C, such as from 25 to 50 °C, such as about 40 °C.

9. The method according to any one of the preceding claims, wherein Ri is selected from a linear Ci to C₆ alkyl group, phenyl or a heterocyclic group, where the phenyl or heterocyclic group is substituted by one or more substituents selected from nitro, Cl, F, and =0.

10. The method according to Claim 9, wherein Ri is selected from Me, Et, wiggly line represents the point of attachment to the rest of the molecule.

11 . The method according to any one of the preceding claims, wherein the primary or secondary amine of formula II is an antibody, a small molecule, a drug molecule, an amino acid, a di-peptide, a tri-peptide, an oligopeptide, a polypeptide, or a protein.

12. The method according to any one of the preceding claims, wherein the primary or secondary amine of formula II is selected from the group of:

13. The method according to any one of the preceding claims, wherein the compound of formula III is subjected to a deprotection reaction to provide a compound according to formula IV: where R3 and F are as defined in Claim 1 , optionally wherein the deprotection reaction uses Et₃N.3HF.

14. The method according to Claim 13, wherein the compound according to formula IV is used in one or more of:

(ai) a Sonogashira coupling reaction; (aii) a Click reaction (e.g. with an azide);

(aiii) a C-N conjugation reaction;

(aiv) a partial reduction of the alkyne functional group to an alkene functional group; and

(av) reaction with a primary thiol-containing compound to provide a cysteine derivative.