Thiol-specific silicon-containing biocompatible click reagent: beta-silyalkynylcarbonyl compound

Abstract

The present invention relates to methods of producing compounds of formula I; the method comprises mixing a compound of formula II; the present invention relates to a process for the preparation of a compound of Formula III: HSR5 wherein R1 to R5 are as described in the specification of the application, in a solvent comprising an aqueous phosphate buffer having a pH of 5 to 9.

Description

Technical Field

[0001] This invention relates to the use of β-silyl alkynyl carbonyl compounds in selective click reactions to provide thiol-containing products. Background Technology

[0002] The listing or discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are part of the prior art or common general knowledge.

[0003] Site-specific chemical modification of proteins has been recognized as a versatile strategy for exploring natural systems and creating novel therapeutic conjugates. Over the past two decades, numerous methods have been developed, primarily targeting lysine, N -Terminal and cysteine ​​residues are preferred due to their high nucleophilicity. Among these, free thiol labeling on cysteine ​​residues is most common for site-specific functionalization because cysteine ​​abundance is low in native proteins (<2%), and disulfide bonds have relatively inert properties, avoiding the formation of a mixture of randomly modified products. Green Synth. Catal. 3, 309-316 (2022)).

[0004] Traditional methods for thiol conjugation rely on maleimide (Michael addition) and iodoacetamide (alkylation). For example, maleimide is widely used as a thiol-targeting linker for antibody-drug conjugates (ADCs). However, limitations (including the instability of maleimide-based conjugates in plasma and the relatively low chemoselectivity of iodoacetamide) have spurred the development of alternative cysteine ​​modification methods. Recently, thiol-specific electrophilic agents have been developed (… Org. Lett. 22, 2038-2043 (2020); Commun. Chem. 2, 93-102 (2019); J. Am. Chem. Soc. 144,10396-10406 ​​(2022); J. Am. Chem. Soc. 139, 6146-6151 (2017); Angew. Chem. Int. Ed. 57, 11598-11602 (2018)). New reaction modes, such as the arylation of aryl sulfides and aryl halides / trifluoromethanesulfonates ( Chem. Sci. 12, 5209-5215 (2021); Nature 526, 687-691 (2015)) and electrophilic alkynylation of dibenzothiophene trifluoromethanesulfonate and high-valent iodine reagents ( Bioconjug. Chem. 32, 1570-1575 (2021); J. Am. Chem. Soc.135, 9620-9623 (2013)), with its excellent chemoselectivity and product stability, complements existing conjugation methods. Despite the aforementioned progress, the expectation of metal-free, biocompatible conditions and high atom economy for thiol click reactions has not yet been fully realized. Uncleavable thiol-specific reagents are essential for cysteine-targeting covalent inhibitors, and only a few methods are available, including allenamides ( Angew. Chem. Int. Ed. 53, 7491-7494 (2014); Angew. Chem. Int. Ed. 59, 18054-18061 (2020)), perfluoroaromatics ( J. Am. Chem. Soc. 135, 5946-5949 (2013); Nat. Chem. 8, 120-128 (2016)) and carbonyl vinyl compounds ( Org. Lett. 20, 6526-6529 (2018); Org. Lett. 5, 1967–1970 (2003); J. Am. Chem. Soc. 120, 10994-10995 (1998)).

[0005] In recent years, the click reaction of thiols with alkynyl carbonyl compounds has attracted widespread attention in the chemical community. Chem. Rev. 119, 11110-11244 (2019); Acc. Chem. Res. 55, 2355-2369 (2022); Research 2018, 3152870 (2018)), particularly in polymer chemistry ( Chem. Rev. 121, 6744-6776 (2021)), fluorescence sensor ( J. Am. Chem. Soc. 135, 6338-6344 (2013)) and thiophene synthesis ( Org. Lett. 20, 198-200 (2018); Tetrahedron Lett. 56, 5386-5389 (2015)). In most cases, stoichiometry or catalytic organic bases are necessary for the formation of reactive thiolates. For example, Dove et al. developed a method for the regioselective addition of alkyl thiols to terminal propynyl esters by modulating the polarity of the base and solvent. Angew. Chem. Int. Ed. 52, 4132-4136 (2013)). In the field of bioconjugation, Che and colleagues were the first to identify electron-deficient alkynes as thiol labeling reagents ( Figure 1 ()( Chem. Eur. J.15, 3839-3850 (2009)). Although these groundbreaking findings enable the reaction to proceed under physiological conditions, this conjugation technique still has some limitations, such as the instability of the vinyl-sulfide junction and the possibility of dithiol addition ( Angew. Chem. Int. Ed. 60, 15359-15364 (2021); Angew. Chem. Int. Ed. 59, 3609-3617 (2020)), and when coupled with terminal aliphatic and arylalkynyl carbonyl compounds, the E / Z ratio of the conjugate is not ideal ( J. Environ. Chem. Eng. 4, 2004-2007 (2016)). Summary of the Invention

[0006] Surprisingly, the silicon moiety was found to function as a thiol-specific click reagent, allowing the formation of thioether products. These products can then undergo further downstream reactions to yield more useful derivatives. The reaction is physiologically feasible, highly atom-economical, additive-free, thiol-specific, and non-cleavable, and allows for the introduction of lipophilic silicon handles that can be used directly or for further chemical reactions.

[0007] Various aspects and embodiments of the invention will now be discussed with reference to the following numbered clauses.

[0008] 1. A method for producing a compound of formula I:

[0009] The method includes using a compound of formula II:

[0010] Compounds of Formula III: HSR 5 III The reaction is carried out in a solvent comprising an aqueous phosphate buffer solution with a pH of 5 to 9, wherein: R 1 Indicates C1 to C 10 Alkyl, C2 to C 10 alkenyl, C2 to C 10 alkynyl group, C6 to C 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 A carbocyclic system wherein each of the groups is either unsubstituted or substituted by one or more groups selected from the following. Halogen, C1 to C3 alkyl, CONR 6a R 6b CO2R 6cOR 6d C6 to C 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 Carbocyclic system, wherein C1 to C3 alkyl groups, C6 to C4 alkyl groups, and C5 alkyl groups are all C1 to C3 alkyl groups. 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 The carbon ring system is either unsubstituted or substituted by one or more halogen atoms; Each R 2 To R 4 Independently represent C1 to C 10 Alkyl or O (C1 to C1) 10 Alkyl groups, wherein each group is independently unsubstituted or substituted by one or more groups selected from C1 to C3 alkyl, phenyl, and 5-10 membered heterocyclic groups, wherein the unsubstituted groups are substituted or substituted by halogens and OR 7 One or more of them are replaced; R 5 Indicates the organic part; R 6a To R 6d This refers to C1 to C1 atoms that are either unsubstituted or substituted by one or more halogen atoms. 10 Alkyl; and R 7 Indicates C1 to C 10 alkyl.

[0011] 2. The method according to Clause 1, wherein the solvent further comprises acetonitrile, optionally wherein the acetonitrile is present in an amount of 10 to 30% v / v, such as about 20% v / v.

[0012] 3. The method described according to Clause 1 or Clause 2, wherein R 1 Indicates C1 to C 10 Alkyl, C2 to C 10 alkenyl, C6 to C 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5-C6 carbocyclic systems, wherein each of the groups is unsubstituted or substituted by one or more groups selected from the following. Halogen, C1 to C3 alkyl, CONR 6a R 6b CO2R 6c OR 6d C6 to C 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5 to C6 carbide ring systems, wherein C1 to C3 alkyl groups, C6 to C6 carbide rings, and C5 to C6 carbide rings are present. 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5 to C6 carbon ring systems are either unsubstituted or substituted by one or more halogen atoms.

[0013] 4. The method according to Clause 3, wherein R 1 Choose one of the following:

[0014] The wavy lines represent the attachment points to the rest of the molecule.

[0015] 5. The method according to any one of the preceding clauses, wherein R 2 To R 4 Each of the following is selected independently: Me, Et, i Pr、 t Buhe The wavy line represents the attachment point to the rest of the molecule.

[0016] 6. The method according to any one of the preceding clauses, wherein R 5 Choose one of the following:

[0017] 7. The method according to any one of clauses 1 to 5, wherein the compound of formula III is selected from bovine serum albumin, amino acids containing thiol substituents, oligopeptides containing thiol substituents, peptides containing thiol substituents, monosaccharides, disaccharides or trisaccharides containing thiol substituents, or pharmaceutical molecules containing thiol substituents, optionally wherein the compound of formula III is selected from N-Boc-glutathione-OMe, 1-mercapto-D-glucose acetate, captopril, and mertansine.

[0018] 8. The method according to any one of the preceding clauses, wherein the pH of the phosphate buffer is 6 to 8, optionally wherein the pH of the phosphate buffer is about 7. Attached Figure Description

[0019] Figure 1 An overview of the click reactions of thiols with alkynyl carbonyl compounds is described. See: Dove et al., 2013. Angew. Chem. Int. Ed. 52, 4132-4136 (2013) and Che et al., 2009 ( Chem. Eur. J. 15,3839-3850 (2009)) Figure 2The conjugation of thiols with acetones containing silicon functional groups was described. This robust thiol-specific method was successfully performed with near 100% atom economy under biocompatible conditions (metal-free, additive-free, ambient temperature, and neutral pH). Advantages include (1) physiological conditions; (2) high atom economy and additive-free; (3) thiol specificity and indestructibility; and (4) additional lipophilic silicon handle.

[0020] Figure 3 Organosilanes in pharmaceutical and materials applications are described.

[0021] Figure 4 The reactivity of β-silyl alkynyl esters and amides with benzyl thiol 2a was described.

[0022] Figure 5 The substrate range of thiol nucleophiles for β-trimethylsilylacetyne is described—the range of thiols.

[0023] Figure 6 The substrate range of thiol nucleophiles for β-trimethylsilylacetyne is described—both aliphatic and aromatic acetyne ranges.

[0024] Figure 7 The effects of trialkylsilyl substituents and silyl ethers are described—the range of silyl substituents.

[0025] Figure 8 The reaction of ketal acetylacetonates with silyl ether chains is described.

[0026] Figure 9 The chemoselectivity of 1-phenyl-3-(trimethylsilyl)prop-2-yn-1-one 1a to various nucleophiles was described: in most experiments, the reaction time was 180 min. 3a was produced in the presence of the indicated nucleophiles. Exceptions were observed in… N- The reaction in the presence of Cbz-Trp-OMe requires 12 hours for complete conversion of 1a.

[0027] Figure 10 The stability of thiol conjugate 3a in the presence of endogenous molecules was described: phosphate buffer (pH 7) was chosen as the solvent in most experiments. Except for dopamine hydrochloride, which was dissolved in 0.1 M NH₄HCO₃ buffer (pH 8).

[0028] Figure 11 The stability of thiol conjugate 3a and reagent 1a under acidic, neutral, basic, and oxidative (10 mM H₂O₂) conditions was characterized: 3a (6.5 mg, 0.02 mmol) or 1a (4.0 mg, 0.02 mmol) was dissolved in 0.4 mL of the above solvent and stirred at 37 °C for 24 hours. CH₂Br₂ was used as an internal standard. 1Conversion rate was determined by H-NMR.

[0029] Figure 12 depicts the stability experiment under bovine serum conditions: (A) Evaluation of the stability of thiol conjugate 3a in serum over time; (B) Stability after 48 hours of incubation. 1 H-NMR spectroscopy (90% of 3a is retained in bovine serum).

[0030] Figure 13 The functionalization of thiol-containing biomolecules and drugs using β-silylacetylenes is described. (4h from) N- Ac-Cys-OMe ;4i is from 1-thio-D-glucose acetate; 4j in pH 7 phosphate buffer is from captopril; 4k in pH 7 phosphate buffer is from maytansine.

[0031] Figure 14 The derivatization of thiol conjugates was described.

[0032] Figure 15 The unconvolution mass spectra of unmodified BSA were depicted.

[0033] Figure 16 The results of BSA modification using TES-acetylene (1f) in phosphate buffer were described: +1 modified = monomodified protein.

[0034] Figure 17 The unconvolution mass spectra of the unmodified HSA were depicted.

[0035] Figure 18 The results of modifying HSA with TMS-acetylene (1a) in phosphate buffer are depicted: +1 modified = monomodified protein.

[0036] Figure 19 The Oak Ridge Thermal Ellipsoid Plot (ORTEP) was depicted at 3p.

[0037] Figure 20 The Oak Ridge thermal ellipsoid (ORTEP) at 3t was depicted. Detailed Implementation

[0038] Surprisingly, novel silylacetylenes have been found to target free thiols in the presence of other nucleophiles. This robust thiol-specific method proceeds smoothly with near 100% atom economy under biocompatible conditions (metal-free, additive-free, ambient temperature, and neutral pH). Of particular note is that the reaction is carried out solely in buffer solutions and provides stable monofunctionalized products while completely preserving the silicon moiety. The compatibility of these silicon compounds allows for the incorporation of more lipophilic silicon functional groups into conjugated adducts. In addition to simple aliphatic and aromatic thiols, this biocompatible method is also applicable to the labeling of thiol-containing amino acids, peptides, and drugs. This method allows for the use of novel silicon-containing reagents, as well as protein-based drugs incorporating silicon moieties. Furthermore, leveraging the inorganic properties of silicon can provide a bridge between different disciplines for many applications.

[0039] Therefore, in a first aspect of the invention, a method for producing a compound of formula I is provided:

[0040] The method includes using a compound of formula II:

[0041] Compounds of Formula III: HSR 5 III The reaction is carried out in a solvent comprising an aqueous phosphate buffer solution with a pH of 5 to 9, wherein: R 1 Indicates C1 to C 10 Alkyl, C2 to C 10 alkenyl, C2 to C 10 alkynyl group, C6 to C 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 A carbocyclic system wherein each of the groups is either unsubstituted or substituted by one or more groups selected from the following. Halogen, C1 to C3 alkyl, CONR 6a R 6b CO2R 6c OR 6d C6 to C 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 Carbocyclic system, wherein C1 to C3 alkyl groups, C6 to C4 alkyl groups, and C5 alkyl groups are all C1 to C3 alkyl groups. 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 The carbon ring system is either unsubstituted or substituted by one or more halogen atoms; Each R 2 To R 4 Independently represent C1 to C10 Alkyl or O (C1 to C1) 10 Alkyl groups, wherein each group is independently unsubstituted or substituted by one or more groups selected from C1 to C3 alkyl, phenyl, and 5-10 membered heterocyclic groups, wherein the unsubstituted groups are substituted or substituted by halogens and OR 7 One or more of them are replaced; R 5 Indicates the organic part; R 6a To R 6d This refers to C1 to C1 atoms that are either unsubstituted or substituted by one or more halogen atoms. 10 Alkyl; and R 7 Indicates C1 to C 10 alkyl.

[0042] In the embodiments herein, the word "comprising" can be interpreted as requiring the mentioned features but not limiting the presence of other features. Alternatively, the word "comprising" can also refer to the situation where only the listed components / features are intended to be present (e.g., the word "comprising" can be replaced by the phrases "consisting of" or "substantially consisting of"). It is explicitly contemplated that both the broader and narrower interpretations can apply to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms can be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0043] The phrase “consistent with…” and its pseudonyms can be interpreted in this text as referring to materials that may contain small amounts of impurities. For example, the material can be greater than or equal to 90% purity, such as greater than 95% purity, such as greater than 97% purity, such as greater than 99% purity, such as greater than 99.9% purity, such as greater than 99.99% purity, such as greater than 99.999% purity, such as 100% purity.

[0044] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, references to “composition” include mixtures of two or more such compositions, etc.

[0045] While the reaction can be carried out using only an aqueous phosphate buffer solution with a pH of 5 to 9 (e.g., 6 to 8, such as about 7), it can also be incorporated with an organic solvent miscible with water. For example, in some embodiments of the invention, the solvent may further comprise acetonitrile. In such embodiments, acetonitrile may be present in any suitable amount. For example, acetonitrile may be present in amounts from 10 to 30% v / v, such as about 20% v / v.

[0046] As previously mentioned, it was unexpectedly discovered that an aqueous phosphate buffer (or the use of phosphates, such as K3PO4 at a concentration of 20 mol% in water) is essential for the reactions disclosed herein to function. That is, the reactions cannot proceed using water alone or organic solvents alone. Therefore, phosphates and / or phosphate buffers are considered an important component of this method.

[0047] Compounds of Formula I can contain double bonds, and therefore can be used as compounds surrounding individual double bonds. E (opposite side) and Z (Same-side) geometric isomers exist. All such isomers and mixtures thereof are included within the scope of this invention. The reaction can preferentially provide a rich... E The state of the compound of formula I (therefore, the compound of formula I is shown as) E -Isomers). For example, in some implementations, E -and Z The ratio can be at least 55:45, such as 85:15, such as 90:10, such as 95:5, such as >95:5 mol:mol.

[0048] In some embodiments of the present invention, R 1 It can represent C1 to C 10 Alkyl, C2 to C 10 alkenyl, C6 to C 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5-C6 carbocyclic systems, wherein each of these groups is either unsubstituted or substituted by one or more groups selected from the following. Halogen, C1 to C3 alkyl, CONR 6a R 6b CO2R 6c OR 6d C6 to C 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5 to C6 carbide ring systems, wherein C1 to C3 alkyl groups, C6 to C6 carbide rings, and C5 to C6 carbide rings are present. 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5 to C6 carbon ring systems are either unsubstituted or substituted by one or more halogen atoms.

[0049] In a more specific embodiment of the present invention, R 1 You may choose one of the following:

[0050] The wavy lines represent the attachment points to the rest of the molecule.

[0051] In some embodiments of the invention that may be mentioned herein, R 2 To R 4Each of the following can be independently selected: Me, Et, i Pr、 t Buhe The wavy line represents the attachment point to the rest of the molecule.

[0052] In some embodiments of the invention that may be mentioned herein, R 5 You may choose one of the following:

[0053] This invention allows for the use of a wide range of possible thiol-containing substrates, as illustrated in the examples below. Therefore, compounds of Formula III can be selected from virtually any thiol-containing compound, including polymeric materials (e.g., synthetic and natural polymers, such as proteins). For example, compounds of Formula III can be selected from bovine serum albumin, amino acids containing thiol substituents, oligopeptides containing thiol substituents, peptides containing thiol substituents, monosaccharides, disaccharides, or trisaccharides containing thiol substituents, or pharmaceutical molecules containing thiol substituents, optionally wherein the compounds of Formula III are selected from N-Boc-glutathione-OMe, 1-mercapto-D-glucose acetate, captopril, and mertansine.

[0054] Silicon incorporation provides an alternative pathway for further modification of Formula I compounds, as organosilicon compounds are multifunctional building blocks and can act as removable protecting / directing groups in organic transformations. Silicon isosteres have wide applications in the pharmaceutical and materials fields due to their unique physical, physiological, and optoelectronic properties. Compared to carbon isosteres, organosilicon molecules exhibit enhanced lipophilicity, enabling them to cross the blood-brain barrier. Representative commercially available silicon-containing drugs include amsilarotene, silafluofen, and flusilazole. Furthermore, π-conjugated compounds based on silole (such as silicon-bridged stilbene) are also promising electroluminescent materials. In addition, silyl ethers have been used as biomedical materials, including polysiloxanes and immolative linkers (e.g., immolative linkers in conjugated nanoparticles -Si(R)2-OCH2- drugs). The silicon moiety also allows for the immobilization of organic functional groups on inorganic glass surfaces.

[0055] Compounds of Formula I are also useful intermediates that can be reacted in a variety of different ways to generate additional downstream products. For example, compounds of Formula I can be reacted as follows: (a) Reduction (e.g., LiAlH4 in diethyl ether) to provide an alcohol; (b) Reduction (e.g., LiAlH4 in diethyl ether) and silicon migration (LiO) t Bu, THF), to provide alcohols for silicon-based protection; (c) Removal of silicon-based materials (e.g., CsF in water and DMSO); (d) Sulfur oxides (e.g., in DCM) m -CPBA and NaHCO3) to provide sulfoxide (if using 1 equivalent) m -CPBA) or sulfone (if using 2.5 equivalent) m -CPBA); and (e) Cyclization reaction (if the ketone moiety has a suitable α-group (e.g., benzene ring)); BCl3 in DCM.

[0056] Examples of these reactions can be found in Figure 14 And the following examples.

[0057] Further aspects and embodiments of the present invention will be described through the following non-limiting examples.

[0058] Example Material Unless otherwise specified, commercially available chemicals and solvents were used directly without purification. Phosphate buffer solution (pH 7, catalog number 38712) was purchased from Alfa Aesar and contained H2O (99.05% w), KH2PO4·H2O (0.90% w), and NaOH (0.05% w). Fetal bovine serum was purchased from Bidepharm (DR-Class, BD01839326). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (catalog number: A2153). Human serum albumin (HSA) was purchased from Sigma-Aldrich (catalog number: A3782). Neither protein required further purification before use.

[0059] The sequence of bovine serum albumin (BSA): BSA consists of 583 amino acids, including one free cysteine ​​(Cys34) and 59 lysines.

[0060] DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRH PYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLTSSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAE VEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCC TKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA The sequence of human serum albumin (HSA): HSA consists of 585 amino acids and contains one free cysteine ​​(Cys34) and 60 lysines.

[0061] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMRADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL General information on compound purification and characterization rapid chromatography Rapid chromatography was performed using Merck 40-63D 60 Å silica gel.

[0062] Nuclear magnetic resonance (NMR) measurement Acquired in a specified deuterated solvent using a Bruker 400 MHz NMR spectrometer. 1 H, 13 C 19 F and 11 B NMR spectra. Recording relative to the residual proton solvent. 1 ¹H NMR chemical shifts (CDCl₃: δ 7.26, CD₃CN: δ 1.94). Multiplicity is expressed as: s (single peak), d (double peak), t (triple peak), q (quadruple peak), and m (multiple peak). The number of protons (n) for a given resonance is denoted by nH. The coupling constant is reported as... J Values, in Hz. Recorded relative to solvent resonance. 13C NMR chemical shifts (CDCl3: δ77.16, CD3CN: δ 118.26 ± 0.02, 1.32 ± 0.02). 19 The F NMR chemical shifts were not corrected.

[0063] Optical rotation measurement Optical rotation was measured at 589 nm using a JASCO P-1030 polarimeter equipped with a sodium vapor lamp, and the concentration of the sample is expressed as follows: c .

[0064] High-resolution mass spectrometry (HRMS) measurement Unless otherwise stated, high-resolution mass spectrometry (HRMS) was recorded on a Waters G2-XS QT spectrometer with ESI mode.

[0065] Example 1. Optimization of reaction conditions General procedure for solvent screening To a 4-mL reaction tube, 0.4 mL of solvent was added to a mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan 2a (17 µL, 0.15 mmol). After vigorous stirring at 37°C for 3 hours, the emulsion was extracted three times with diethyl ether. The combined organic layers were then evaporated under reduced pressure. The crude product was then subjected to further analysis. 1 H-NMR was used to determine the conversion rate of 1a and the yield of 3a.

[0066] General procedures for additive research To a 4-mL reaction tube, 0.4 mL of deionized water and an indicated amount of additive were sequentially added to a mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan 2a (17 µL, 0.15 mmol). After vigorous stirring at 37°C for 3 hours, the emulsion was extracted three times with diethyl ether. The combined organic layers were then evaporated under reduced pressure. The crude product was then subjected to further processing. 1 H-NMR was used to determine the yields of 3a and 3a'.

[0067]

[0068] ( E 3-(benzylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3a) Product 3a was separated by rapid chromatography (diethyl ether / hexane 1:70) as a colorless oil. 29 mg, 88% yield, E / Z > 95:5.

[0069] 1H NMR (400 MHz, CDCl3): δ 7.86-7.84 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.46-7.32 (m, 7H), 7.21 (s, 1H), 4.12 (s, 2H), 0.34 (s, 9H).

[0070] 13 C NMR (100 MHz, CDCl3): δ 186.6, 168.6, 139.0, 135.1, 132.3, 129.1,129.0, 128.6, 128.4, 127.8, 123.4, 38.0, -0.2.

[0071] HRMS (ESI): C 19 H 23 OSSi [M+H] + Calculated value: 327.1239; Measured value: 327.1234.

[0072]

[0073] 3,3-Bis(benzylthio)-1-phenylprop-1-one (3a')[74896-60-9] 0.1 mmol scale, duration 3 hours. To a 4-mL reaction tube, 0.4 mL of deionized water and trimethylamine (Et3N) (3 µL, 0.02 mmol) were sequentially added to a mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan 2a (17 µL, 0.15 mmol). Product 3a' was separated by rapid chromatography (diethyl ether / hexane 1:50) as a colorless oil. 31 mg, 82% yield.

[0074] 1 H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 7.3 Hz, 2H), 7.54 (t, J = 7.4 Hz,1H), 7.42-7.38 (m, 2H), 7.35-7.28 (m, 8H), 7.24-7.20 (m, 2H), 4.33 (t, J = 7.0Hz, 1H), 3.85 (s, 4H), 3.31 (d, J = 7.0 Hz, 2H).

[0075] 13C NMR (100 MHz, CDCl3): δ 196.2, 138.0, 136.7, 133.4, 129.2, 128.7,128.2, 127.2, 46.1, 45.4, 35.5.

[0076] HRMS (ESI): C 23 H 22 OS2Na [M+Na] + Calculated value: 401.1010; Measured value: 401.1013.

[0077] Results and Discussion Our study began with the effects of solvent screening and additives on the model reaction between 1-phenyl-3-(trimethylsilyl)prop-2-yn-1-one 1a and benzyl thiol 2a at 37 °C under metal-free conditions (Table 1).

[0078] Table 1. Solvent screening for the model reaction between 1-phenyl-3-(trimethylsilyl)prop-2-yn-1-one 1a and benzyl thiol 2a at 37 °C under metal-free conditions.

[0079]

[0080]

[0081] 1 The additive used is 20 mol%. 2 The additive used is 40 mol. 3 Potassium phosphate buffer: 0.9% w KH2PO4•H2O and 0.05% w NaOH, pH 7, from Alfa Aesar. 4 The pH of potassium phosphate buffer can be adjusted by adding conjugated acids or bases.

[0082] We found that neither organic solvents nor pure water provided the conjugated product, and starting material 1a was consumed in trace amounts (Table 1, entries 1-6). When a catalytic organic base such as Et3N was added to a water-based reaction, the thioacetal byproduct 3a' was mainly obtained via a bisMichael addition reaction. Angew. Chem. Int. Ed. 52, 4132-4136 (2013) (Table 1, entry 7). Encouragingly, the reaction in an aqueous solution containing 20 mol% K3PO4 yielded a moderate yield of vinyl sulfide 3a (65%, Table 1, entry 8). We hypothesize that the effect of the inorganic salt on the reaction outcome may be due to the formation of reactive thiolate ions ( ACS Catal.9, 9923-9952 (2019)). The reaction, carried out in an aqueous solution containing 40 mol% NaOH, provided 3a in comparable yields. To evaluate the feasibility of 1a monothiolization under milder or even physiological conditions, we carried out the reaction in phosphate buffers ranging from pH 6 to 8 (Table 1 entries 10-12). Ultimately, pH 7 phosphate buffer was selected as the optimal medium due to the high yield of the desired product (88%) and a satisfactory E / Z ratio (>95:5) (Table 1 entry 10).

[0083] Example 2. Reactivity of other alkynyl carbonyl electrophilic reagents In addition, we investigated the reactivity of other alkynyl carbonyl electrophilic reagents under optimized conditions. Figure 4 ).

[0084] General procedures for reactivity studies To a 4-mL reaction tube, 0.4 mL of phosphate buffer (pH 7) was added to a mixture of β-silyl alkynyl ester 1b (or amide 1c, 0.1 mmol) and benzyl mercaptan 2a (17 µL, 0.15 mmol). After vigorous stirring at 37°C for 18 hours, the emulsion was extracted three times with diethyl ether. The combined organic layers were then evaporated under reduced pressure. The crude product was then subjected to further processing. 1 ¹H-NMR was used to determine the E / Z ratio. The yields of 3b and 3c were calculated after purification on silica gel using rapid column chromatography.

[0085]

[0086] 3-(benzylthio)-3-(trimethylsilyl) benzyl acrylate (3b) The product was separated by rapid chromatography (ethyl acetate / hexane 1:20) and was a colorless oil. 20 mg, 56% yield, E / Z = 55:45.

[0087] 1 H NMR (400 MHz, CDCl3): δ 7.40-7.29 (m, 10H), 6.17 (s, 1H), 5.16 (s,2H), 4.19 (s, 2H), 0.31 (s, 9H).

[0088] 13 C NMR (100 MHz, CDCl3): δ 165.0, 163.5, 136.6, 136.3, 129.4, 128.72, 128.69, 128.68, 128.3, 127.6, 121.2, 66.2, 38.9, 0.1.

[0089] HRMS (ESI): C 20 H 25 O2SSi [M+H] + Calculated value: 357.1345; Measured value: 357.1352.

[0090]

[0091] 3-(benzylthio)- N , N -Dimethacrylamide (3c) The product was separated by rapid chromatography (ethyl acetate / hexane 1:10) and was a colorless oil. 17 mg, 75% yield, E / Z = 50:50.

[0092] E and Z Isomer mixtures 1 H NMR (400 MHz, CDCl3): δ 7.62 (d, J = 14.7 Hz, 0.5H),7.36-7.24 (m, 5H), 6.90 (d, J = 10.0 Hz, 0.5H), 6.23 (d, J = 14.7 Hz, 0.5H), 6.15(d, J = 10.0 Hz, 0.5H), 4.02 (s, 1H), 3.89 (s, 1H), 2.99 (s, 3H), 2.97 (s, 3H).

[0093] E and Z Isomer mixtures 13 C NMR (100 MHz, CDCl3): δ 167.0 and 165.3, 145.7 and 143.3, 138.0 and 136.5, 129.2 and 128.9, 128.82 and 128.77, 127.7 and 127.3, 114.3 and 112.4, 39.8, 37.35 and 37.28, 35.9 and 35.4.

[0094] Results and Discussion β-Silylalkynyl ester 1b provides thiol conjugate 3b in 56% yield. Desilication Michael addition was detected as a competing pathway, which could explain the loss of mass equilibrium. The reaction of β-silylalkynyl amide 1c is incompatible with silicon functional groups, as only desilication adduct 3c is obtained.

[0095] Example 3. Synthesis of β-silylacetylenes substrate Table 2 shows the β-silylacetylenes disclosed herein. Non-commercially available β-silylacetylenes were synthesized using the following procedure.

[0096] Table 2. Range of β-silylacetylenes.

[0097]

[0098] Synthesis of ethynyl diisopropyl ((4-methoxybenzyl)oxy)silane [1229230-93-6]

[0099] Under nitrogen atmosphere, 20 mL of ethynyl magnesium bromide (0.5 M in THF) was added dropwise to a solution of diisopropylsilane (1.8 mL, 10.5 mmol) in anhydrous THF (10 mL) at 0 °C. The reaction was stirred vigorously overnight at room temperature. After completion, the mixture was quenched with phosphate buffer (pH 7) and extracted with diethyl ether (Et₂O). The combined organic layers were dried over sodium sulfate (Na₂SO₄) and concentrated using a rotary evaporator (25 °C, 200 mbar). The crude product was then purified by rapid column chromatography on silica gel using pentane as the eluent.

[0100] Under a nitrogen atmosphere, ethynyl diisopropylsilane (1.05 g, 7.5 mmol) was added dropwise to an ice-cold suspension of N-bromosuccinimide (NBS) (1.33 g, 7.5 mmol) in 10 mL of anhydrous DCM at 0 °C. The reaction was stirred at room temperature for 3 hours. Under nitrogen, 4-dimethylaminopyridine (DMAP) (91.6 mg, 0.75 mmol) and 5 mL of DCM were transferred to another round-bottom flask, followed by the sequential addition of anisole (930 µL, 7.5 mmol) and Et3N (3.1 mL, 22.5 mmol). Then, the above-mentioned bromo(ethynyl) diisopropylsilane solution was added dropwise under ice-cold conditions. The reaction mixture was stirred overnight at room temperature. After completion, the mixture was quenched with phosphate buffer (pH 7) and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was then passed through Florisil. ® The ether was purified by rapid column chromatography (diethyl ether / hexane 1:20). The resulting ethynyl dialkyl silyl ether was then used to synthesize 3-(diisopropyl((4-methoxybenzyl)oxy)silyl)-1-phenylprop-2-yn-1-one.

[0101] Ethynyl diisopropyl((4-methoxybenzyl)oxy)silane [1229230-93-6] Org. Lett. 12,2860-2863 (2010)) 1H NMR (400 MHz, CDCl3): δ 7.28 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 4.81 (s, 2H), 3.80 (s, 3H), 2.47 (s, 1H), 1.17-1.07 (m, 14H).

[0102] General procedure for the synthesis of β-silylacetylenes

[0103] Under nitrogen atmosphere, acetyltrialkylsilane (6 mmol) was added dropwise to a solution of acetylenetrialkylsilane in THF at -20°C. n -BuLi (2 M, 2.6 mL in hexane). The mixture was stirred at -20 °C for 15 min. Then, the aldehyde (5 mmol) was added, and the temperature was gradually raised to room temperature. After stirring overnight, the reaction was quenched with saturated NH4Cl solution and extracted with diethyl ether. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was used directly in the next step.

[0104] β-Silylpropyne alcohol was added to a stirred suspension of activated manganese oxide (MnO2) (5 equivalents) in DCM. The reaction was stirred overnight at room temperature. The resulting mixture was then filtered through diatomaceous earth and washed with DCM. The product was separated by rapid chromatography (hexane / ethyl acetate) on silica gel.

[0105]

[0106] 1-Piperido-3-(trimethylsilyl)prop-2-yn-1-one 1 H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 8.2 Hz, 1H), 7.54 (s, 1H), 6.88(d, J = 8.2 Hz, 1H), 6.07 (s, 2H), 0.30 (s, 9H).

[0107] 13 C NMR (100 MHz, CDCl3): δ 176.0, 153.1, 148.3, 131.9, 127.6, 108.5,108.2, 102.2, 101.0, 99.9, -0.5.

[0108] HRMS (ESI): C 13 H15 O3Si [M+H] + Calculated value: 247.0790; Measured value: 247.0786.

[0109]

[0110] 1-( N -Toluenesulfonyl-indol-3-yl)-3-(trimethylsilyl)prop-2-yn-1-one 1 H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 8.30 (d, J = 7.0 Hz, 1H), 7.91(d, J = 7.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.40-7.34 (m, 2H), 7.32-7.29 (m, 2H), 2.38 (s, 3H), 0.35 (s, 9H).

[0111] 13 C NMR (100 MHz, CDCl3): δ 171.7, 146.3, 135.6, 135.2, 134.5, 130.5,127.4, 126.9, 126.2, 125.2, 123.0, 122.6, 113.3, 101.4, 97.2, 21.8, -0.5.

[0112] HRMS (ESI): C 21 H 22 NO3SiS [M+H] + Calculated value: 396.1090; Measured value: 1085.

[0113]

[0114] 1-(cyclohexyl-3-en-1-yl)-3-(trimethylsilyl)prop-2-yn-1-one 1 H NMR (400 MHz, CDCl3): δ 5.70-5.64 (m, 2H), 2.67-2.60 (m, 1H), 2.26-2.24 (m, 2H), 2.12-2.03 (m, 3H), 1.68-1.63 (m, 1H), 0.22 (s, 9H).

[0115] 13C NMR (100 MHz, CDCl3): δ 190.7, 126.9, 125.1, 101.3, 98.9, 48.2, 26.5, 24.5, 24.4, -0.6.

[0116] HRMS (ESI): C 12 H 19 OSi [M+H] + Calculated value: 207.1205; Measured value: 207.1202.

[0117]

[0118] 5-(methylthio)-1-(trimethylsilyl)pent-1-yn-3-one 1 H NMR (400 MHz, CDCl3): δ 2.86 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.11 (s, 3H), 0.24 (s, 9H).

[0119] 13 C NMR (100 MHz, CDCl3): δ 185.7, 101.7, 99.1, 45.2, 28.0, 15.8, -0.7.

[0120] HRMS (ESI): C9H 17 OSiS [M+H] + Calculated value: 201.0769; Measured value: 201.0766.

[0121]

[0122] ( S 5,9-Dimethyl-1-(trimethylsilyl)dec-8-en-1-yn-3-one 1 H NMR (400 MHz, CDCl3): δ 5.08 (t, J = 8.1 Hz, 1H), 2.55 (dd, J = 15.4, 5.8 Hz, 1H), 2.36 (dd, J= 15.4, 8.1 Hz, 1H), 2.16-2.08 (m, 1H), 2.05-1.93 (m,2H), 1.68 (s, 3H), 1.60 (s, 3H), 1.39-1.31 (m, 1H), 1.28-1.18 (m, 1H), 0.94(d, J = 6.6 Hz, 3H), 0.24 (s, 9H).

[0123] 13 C NMR (100 MHz, CDCl3): δ 188.0, 131.8, 124.3, 102.5, 97.6, 52.8, 36.9, 29.5, 25.9, 25.5, 19.8, 17.8, -0.6.

[0124] HRMS (ESI): C 15 H 27 OSi [M+H] + Calculated value: 251.1831; Measured value: 251.1827.

[0125]

[0126] N , N -Dimethyl-4-[3-(triisopropylsilyl)propynyl]benzamide 1 H NMR (400 MHz, CDCl3): δ 8.20 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 7.9 Hz, 2H), 3.13 (s, 3H), 2.96 (s, 3H), 1.22-1.15 (m, 21H).

[0127] 13 C NMR (100 MHz, CDCl3): δ 176.8, 170.5, 141.8, 137.4, 129.8, 127.4,103.0, 99.1, 39.5, 35.4, 18.7, 11.2.

[0128] HRMS (ESI): C 21 H 32 NO2Si [M+H] + Calculated value: 358.2202; Measured value: 358.2205.

[0129]

[0130] 3-(diisopropyl((4-methoxybenzyl)oxy)silyl)-1-phenylprop-2-yn-1-one 1 H NMR (400 MHz, CDCl3): δ 8.13 (d, J = 7.6 Hz, 2H), 7.62 (t, J = 7.3 Hz,1H), 7.50-7.46 (m, 2H), 7.31 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 4.89 (s, 2H), 3.79 (s, 3H), 1.15-1.12 (m, 12H), 1.09-1.03 (m, 2H).

[0131] 13 C NMR (100 MHz, CDCl3): δ 177.5, 159.1, 136.7, 134.4, 132.6, 129.8,128.8, 128.2, 113.9, 102.4, 95.1, 66.5, 55.4, 17.3, 17.2, 13.3.

[0132] HRMS (ESI): C 23 H 28 O3SiNa [M+Na] + Calculated value: 403.1705; Measured value: 403.1722.

[0133] Example 4. Substrate Range Study After mastering the optimized conditions, we investigated the substrate range for the conjugation addition of thiols to TMS-acetylene 1a. Figure 5 Next, various substituted acetylenes were examined. Figure 6 We also evaluated the effect of silicon substituents on acetylacetonate substrates. Figure 7 The β-silylacetyl ketones used in this article can be obtained from commercial sources or prepared according to the method disclosed in Example 3.

[0134] General procedure for the addition of thiols to β-silylacetylenes To a 4 mL reaction tube, 0.4 mL of phosphate buffer (pH 7) was added to a mixture of β-silylacetylinone (0.1 mmol) and thiol nucleophile (0.15 mmol). After vigorous stirring at 37 °C for 3 hours, the emulsion was extracted three times with diethyl ether. The combined organic layers were then evaporated under reduced pressure. The crude product was then subjected to further processing. 1 H-NMR is used to determine the E / Z ratio. Unless otherwise specified, the E and Z isomers can be separated on silica gel by rapid chromatography.

[0135]

[0136] ( E )-3-[(4-chlorobenzyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3d) The product was separated by rapid chromatography (diethyl ether / hexane 1:70), and was a pale yellow oil. 32 mg, 88% yield, E / Z > 95:5.

[0137] 1 H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.9 Hz, 2H), 7.46-7.43 (m, 1H), 7.38-7.34 (m, 2H), 7.26-7.17 (m, 4H), 7.07 (s, 1H), 3.99 (s, 2H), 0.25 (s, 9H).

[0138] 13 C NMR (100 MHz, CDCl3): δ 186.6, 168.1, 138.9, 133.7, 132.4, 130.4,129.2, 128.7, 128.3, 123.6, 37.2, -0.3.

[0139] HRMS (ESI): C 19 H 22 ClOSSi [M+H] + Calculated value: 361.0849; Measured value: 361.0848.

[0140]

[0141] ( E )-3-[(furan-2-ylmethyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3e) The product was separated by rapid chromatography (diethyl ether / hexane 1:70) and was a colorless oil. 23 mg, 73% yield, E / Z > 95:5.

[0142] 1 H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.5 Hz,1H), 7.47-7.43 (m, 2H), 7.41 (s, 1H), 7.26 (s, 1H), 6.37 (d, J = 2.9 Hz, 1H), 6.32 (d, J = 3.1 Hz, 1H), 4.14 (s, 2H), 0.33 (s, 9H).

[0143] 13 C NMR (100 MHz, CDCl3): δ 186.6, 167.5, 149.2, 142.6, 139.0, 132.4,128.7, 128.4, 123.2, 111.0, 108.6, 30.0, -0.3.

[0144] HRMS (ESI): C 17 H 21 O2SSi [M+H] + Calculated value: 317.1032; Measured value: 317.1023.

[0145]

[0146] ( E )-3-[(2-hydroxyethyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3f) The product was separated by rapid chromatography (ethyl acetate / hexane 1:5), and was a yellow oil. 20 mg, 71% yield, E / Z > 95:5.

[0147] 1 H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 7.3 Hz, 2H), 7.53 (t, J = 7.2 Hz,1H), 7.48-7.44 (m, 2H), 7.20 (s, 1H), 3.98 (t, J = 5.4 Hz, 2H), 3.13 (t,J = 6.0Hz, 2H), 2.01 (s, 1H), 0.33 (s, 9H).

[0148] 13 C NMR (100 MHz, CDCl3): δ 186.7, 167.9, 138.9, 132.4, 128.7, 128.4,123.2, 60.0, 35.2, -0.2.

[0149] HRMS (ESI): C 14 H 20 O2SSiNa [M+Na] + Calculated value: 303.0851; Measured value: 303.0853.

[0150]

[0151] The product was separated by rapid chromatography (diethyl ether / hexane 1:70) and was a colorless oil. 24 mg, 75% yield, E / Z = 80:20.

[0152] ( E 3-(cyclohexylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3g, major isomer) 1 H NMR (400 MHz, CDCl3): δ 7.92 (d, J = 7.7 Hz, 2H), 7.53 (t, J = 7.2 Hz,1H), 7.48-7.44 (m, 2H), 7.18 (s, 1H), 3.33-3.28 (m, 1H), 2.12-2.08 (m, 2H),1.85-1.81 (m, 2H), 1.70-1.66 (m, 1H), 1.54-1.34 (m, 5H), 0.32 (s, 9H).

[0153] 13 C NMR (100 MHz, CDCl3): δ 186.5, 168.6, 139.4, 132.2, 128.7, 128.3,122.4, 44.0, 32.1, 26.1, 26.0, -0.1.

[0154] HRMS (ESI): C 18 H 27 OSSi [M+H] +Calculated value: 319.1552; Measured value: 319.1544.

[0155] ( Z 3-(cyclohexylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3g', minor isomer) 1 H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 7.0 Hz, 2H), 7.51 (t, J = 7.3 Hz,1H), 7.46-7.42 (m, 2H), 7.19 (s, 1H), 3.27-3.20 (m, 1H), 1.97-1.94 (m, 2H),1.78-1.74 (m, 2H), 1.62-1.58 (m, 1H), 1.42-1.23 (m, 5H), 0.37 (s, 9H).

[0156] 13 C NMR (100 MHz, CDCl3): δ 188.7, 164.3, 138.9, 132.3, 128.6, 128.4,125.8, 46.0, 34.5, 26.4, 25.5, 0.5.

[0157] HRMS (ESI): C 18 H 27 OSSi [M+H] + Calculated value: 319.1552; Measured value: 319.1548.

[0158]

[0159] ( E )-1-Phenylacetyl-3-(trimethylsilyl)prop-2-en-1-one (3h) [82632-32-4] The product was separated by rapid chromatography (diethyl ether / hexane 1:100), and was a yellow oil. 29 mg, 93% yield, E / Z > 95:5.

[0160] 1 H NMR (400 MHz, CDCl3): δ 7.64-7.62 (m, 2H), 7.54-7.46 (m, 5H), 7.44-7.42 (m, 1H), 7.36-7.32 (m, 2H), 6.73 (s, 1H), 0.40 (s, 9H).

[0161] 13 C NMR (100 MHz, CDCl3): δ 186.8, 169.5, 138.8, 135.5, 132.3, 131.0,130.0, 129.9, 128.5, 128.2, 125.0, -0.3.

[0162] HRMS (ESI): C 18 H 21 OSSi [M+H] + Calculated value: 313.1082; Measured value: 313.1077.

[0163]

[0164] ( E )-1-Phenylacet-3-(p-Tolyl)thio-3-(trimethylsilyl)prop-2-en-1-one (3i) The product was separated by rapid chromatography (diethyl ether / hexane 1:70), and was a pale yellow oil. 30 mg, 92% yield, E / Z > 95:5.

[0165] 1 H NMR (400 MHz, CDCl3): δ 7.64 (d, J = 7.6 Hz, 2H), 7.46-7.39 (m, 3H), 7.36-7.26 (m, 4H), 6.74 (s, 1H), 2.42 (s, 3H), 0.39 (s, 9H).

[0166] 13 C NMR (100 MHz, CDCl3): δ 186.9, 169.9, 140.1, 138.9, 135.3, 132.2,130.8, 128.5, 128.3, 127.4, 124.9, 21.5, -0.3.

[0167] HRMS (ESI): C 19 H 23 OSSi [M+H] + Calculated value: 327.1239; Measured value: 327.1246.

[0168]

[0169] ( E )-3-[(4-methoxyphenyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3j) The product was separated by rapid chromatography (diethyl ether / hexane 1:40), and was a yellow oil. 31 mg, 90% yield, E / Z > 95:5.

[0170] 1 H NMR (400 MHz, CDCl3): δ 7.65 (d, J = 7.1 Hz, 2H), 7.45-7.42 (m, 3H), 7.37-7.33 (m, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.73 (s, 1H), 3.87 (s, 3H), 0.39 (s, 9H).

[0171] 13 C NMR (100 MHz, CDCl3): δ 186.8, 170.5, 161.0, 138.8, 136.9, 132.2,128.5, 128.2, 124.8, 121.5, 115.6, 55.6, -0.3.

[0172] HRMS (ESI): C 19 H 23 O2SSi [M+H] + Calculated value: 343.1188; Measured value: 343.1179.

[0173]

[0174] ( E )-3-[(3-methoxyphenyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3k) The product was separated by rapid chromatography (diethyl ether / hexane 1:40), and was a yellow oil. 30 mg, 88% yield, E / Z > 95:5.

[0175] 1 H NMR (400 MHz, CDCl3): δ 7.66 (d, J = 7.1 Hz, 2H), 7.48-7.33 (m, 4H), 7.15 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 3.84 (s, 3H), 0.40 (s, 9H).

[0176] 13 C NMR (100 MHz, CDCl3): δ 186.9, 169.1, 160.7, 138.8, 132.3, 132.0,130.8, 128.6, 128.3, 127.6, 125.0, 119.9, 116.2, 55.6, -0.3.

[0177] HRMS (ESI): C 19 H 23 O2SSi [M+H] + Calculated value: 343.1188; Measured value: 343.1183.

[0178]

[0179] ( E )-3-[(2-methoxyphenyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3l) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 26 mg, 76% yield, E / Z > 95:5.

[0180] 1 H NMR (400 MHz, CDCl3): δ 7.65 (d, J = 7.9 Hz, 2H), 7.54-7.42 (m, 3H), 7.36-7.32 (m, 2H), 7.08-7.02 (m, 2H), 6.65 (s, 1H), 3.86 (s, 3H), 0.41 (s, 9H).

[0181] 13 C NMR (100 MHz, CDCl3): δ 186.8, 167.6, 159.9, 139.0, 137.3, 132.2,132.1, 128.5, 128.2, 124.7, 121.9, 118.7, 111.8, 56.2, -0.3.

[0182] HRMS (ESI): C 19 H 22 O2SSiNa [M+Na] + Calculated value: 365.1007; Measured value: 365.1000.

[0183]

[0184] ( E)-3-(naphthyl-2-thio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3m) The product was separated by rapid chromatography (diethyl ether / hexane 1:70), and was a yellow oil. 29 mg, 80% yield, E / Z > 95:5.

[0185] 1 H NMR (400 MHz, CDCl3): δ 8.08 (s, 1H), 7.96-7.87 (m, 3H), 7.61-7.56(m, 5H), 7.40 (t, J = 6.8 Hz, 1H), 7.29-7.25 (m, 2H), 6.85 (s, 1H), 0.44 (s, 9H).

[0186] 13 C NMR (100 MHz, CDCl3): δ 187.0, 169.1, 138.6, 135.2, 134.2, 133.6,132.2, 131.3, 129.6, 128.5, 128.4, 128.2, 128.14, 128.06, 127.5, 126.9,125.4, -0.2.

[0187] HRMS (ESI): C 22 H 22 OSSiNa [M+Na] + Calculated value: 385.1058; Measured value: 385.1053.

[0188]

[0189] ( E )-1-Phenylacet-3-(pyridin-4-ylthio)-3-(trimethylsilyl)prop-2-en-1-one (3n) 0.1 mmol scale, duration 18 hours. The product was separated by rapid chromatography (ethyl acetate / hexane 1:10) as a white solid. 22 mg, 70% yield, E / Z > 95:5.

[0190] 1 H NMR (400 MHz, CDCl3): δ 8.71 (d, J = 5.1 Hz, 2H), 7.67 (d, J= 7.9 Hz, 2H), 7.50-7.44 (m, 3H), 7.39-7.35 (m, 2H), 6.92 (s, 1H), 0.38 (s, 9H).

[0191] 13 C NMR (100 MHz, CDCl3): δ 187.1, 165.2, 151.1, 142.5, 138.2, 132.8,128.8, 128.7, 128.3, 127.2, -0.3.

[0192] HRMS (ESI): C 17 H 20 NOSSi [M+H] + Calculated value: 314.1035; Measured value: 314.1034.

[0193]

[0194] ( E )-1-Phenylacet-3-(thiophene-2-ylthio)-3-(trimethylsilyl)prop-2-en-1-one (3o) The product was separated by rapid chromatography (diethyl ether / hexane 1:40), and was a yellow oil. 30 mg, 94% yield, E / Z > 95:5.

[0195] 1 H NMR (500 MHz, CDCl3): δ 7.70-7.68 (m, 2H), 7.64 (d, J = 5.3 Hz, 1H), 7.47 (t, J = 7.4 Hz, 1H), 7.39-7.36 (m, 2H), 7.28 (d, J = 3.6 Hz, 1H), 7.20 (dd, J = 5.3, 3.6 Hz, 1H), 6.85 (s, 1H), 0.39 (s, 9H).

[0196] 13 C NMR (125 MHz, CDCl3): δ 187.0, 169.5, 138.6, 137.1, 132.8, 132.4,128.7, 128.6, 128.5, 128.3, 126.0, -0.4.

[0197] HRMS (ESI): C 16 H18 OS2SiNa [M+Na] + Calculated value: 341.0466; Measured value: 341.0461.

[0198]

[0199] ( E )-3-(benzylthio)-1-[(4-methoxycarbonyl)phenyl]-3-(trimethylsilyl)prop-2-en-1-one (3p) The product was separated by rapid chromatography (diethyl ether / hexane 1:20) as a white solid (mp: 106-107℃). 36 mg, 94% yield, E / Z > 95:5.

[0200] 1 H NMR (400 MHz, CDCl3): δ 8.08 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.2 Hz,2H), 7.42-7.32 (m, 5H), 7.16 (s, 1H), 4.13 (s, 2H), 3.94 (s, 3H), 0.34 (s,9H).

[0201] 13 C NMR (100 MHz, CDCl3): δ 185.7, 170.5, 166.5, 142.6, 135.0, 133.1,129.8, 129.0, 128.2, 127.9, 123.0, 52.5, 38.1, -0.4.

[0202] HRMS (ESI): C 21 H 25 O3SSi [M+H] + Calculated value: 385.1294; Measured value: 385.1297.

[0203]

[0204] ( E )-3-(benzylthio)-1-(4-fluorophenyl)-3-(trimethylsilyl)prop-2-en-1-one (3q) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 24 mg, 70% yield, E / Z > 95:5.

[0205] 1H NMR (400 MHz, CDCl3): δ 7.86-7.82 (m, 2H), 7.42-7.32 (m, 5H), 7.14-7.07 (m, 3H), 4.11 (s, 2H), 0.33 (s, 9H).

[0206] 13 C NMR (100 MHz, CDCl3): δ 185.1, 169.0, 165.3 (d, J CF = 253.2 Hz), 135.3 (d, J CF = 2.8 Hz), 135.1, 130.9 (d, J CF = 9.0 Hz), 129.1, 129.0, 127.8,123.1, 115.6 (d, J CF = 21.7 Hz), 38.0, -0.3.

[0207] 19 F NMR (376.6 MHz, CDCl3): δ -106.7.

[0208] HRMS (ESI): C 19 H 21 FOSSiNa [M+Na] + Calculated value: 367.0964; Measured value: 367.0959.

[0209]

[0210] ( E )-3-(benzylthio)-1-(4-methoxyphenyl)-3-(trimethylsilyl)prop-2-en-1-one (3r) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 31 mg, 88% yield, E / Z = 90:10.

[0211] 1 H NMR (400 MHz, CDCl3): δ 7.84 (d, J = 8.8 Hz, 2H), 7.43-7.31 (m, 5H),7.18 (s, 1H), 6.91 (d, J= 8.8 Hz, 2H), 4.11 (s, 2H), 3.86 (s, 3H), 0.33 (s, 9H).

[0212] 13 C NMR (100 MHz, CDCl3): δ 185.4, 167.0, 163.0, 135.3, 131.7, 130.6,129.1, 129.0, 127.7, 123.6, 113.8, 55.6, 37.9, -0.1.

[0213] HRMS (ESI): C 20 H 25 O2SSi [M+H] + Calculated value: 357.1345; Measured value: 357.1342.

[0214]

[0215] ( E )-3-(benzylthio)-1-(piperyl)-3-(trimethylsilyl)prop-2-en-1-one (3s) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 33 mg, 89% yield, E / Z = 91:9.

[0216] 1 H NMR (400 MHz, CDCl3): δ 7.42-7.29 (m, 7H), 7.11 (s, 1H), 6.81 (d, J =8.1 Hz, 1H), 6.02 (s, 2H), 4.10 (s, 2H), 0.32 (s, 9H).

[0217] 13 C NMR (100 MHz, CDCl3): δ 185.0, 167.5, 151.2, 148.2, 135.2, 133.6,129.1, 129.0, 127.8, 124.2, 123.4, 108.4, 107.9, 101.8, 37.9, -0.2.

[0218] HRMS (ESI): C 20 H 22 O3SSiNa [M+Na] + Calculated value: 393.0957; Measured value: 393.0959.

[0219]

[0220] ( E )-3-(benzylthio)-1-( N (-Toluenesulfonylindol-3-yl)-3-(trimethylsilyl)prop-2-en-1-one (3t) The product was prepared at a scale of 0.1 mmol in a mixed solvent (0.5 mL, pH 7 Kpi buffer / MeCN = 4:1) for 12 hours. The product was separated by rapid chromatography (diethyl ether / hexane 1:20) as a white solid (mp: 175–176 °C). 48 mg, 92% yield, E / Z > 95:5.

[0221] 1 H NMR (400 MHz, CDCl3): δ 8.36 (d, J = 7.7 Hz, 1H), 8.03 (s, 1H), 7.95(d, J = 7.5 Hz, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.53-7.46 (m, 4H), 7.40-7.28 (m,5H), 7.04 (s, 1H), 4.20 (s, 2H), 2.39 (s, 3H), 0.38 (s, 9H).

[0222] 13 C NMR (100 MHz, CDCl3): δ 181.8, 166.5, 145.9, 135.4, 135.0, 134.8,130.5, 130.3, 129.2, 128.9, 128.4, 127.8, 127.2, 125.7, 124.7, 124.4, 123.4,122.7, 113.2, 38.0, 21.8, -0.2.

[0223] HRMS (ESI): C 28 H 30 NO3S2Si [M+H] + Calculated value: 520.1436; Measured value: 520.1444.

[0224]

[0225] ( E 3-(benzylthio)-1-(cyclohex-3-en-1-yl)-3-(trimethylsilyl)prop-2-en-1-one (3u) The product was separated by rapid chromatography (diethyl ether / hexane 1:50) and was a colorless oil. 29 mg, 88% yield, E / Z = 86:14.

[0226] 1 H NMR (400 MHz, CDCl3): δ 7.36-7.27 (m, 5H), 6.56 (s, 1H), 5.73-5.67(m, 2H), 4.00 (s, 2H), 2.66-2.59 (m, 1H), 2.22-2.06 (m, 4H), 1.92-1.87 (m,1H), 1.62-1.54 (m, 1H), 0.26 (s, 9H).

[0227] 13 C NMR (100 MHz, CDCl3): δ 198.9, 166.4, 135.1, 129.1, 128.9, 127.7,126.8, 125.8, 125.4, 46.3, 37.8, 27.3, 25.2, 24.9, -0.4.

[0228] HRMS (ESI): C 19 H 27 OSSi [M+H] + Calculated value: 331.1552; Measured value: 331.1543.

[0229]

[0230] ( E )-1-(benzylthio)-5-phenyl-1-(trimethylsilyl)pent-1-en-3-one (3v) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 26 mg, 73% yield, E / Z = 89:11.

[0231] 1 H NMR (400 MHz, CDCl3): δ 7.35-7.32 (m, 4H), 7.30-7.27 (m, 3H), 7.21-7.18 (m, 3H), 6.44 (s, 1H), 3.92 (s, 2H), 2.93 (t, J = 7.7 Hz, 2H), 2.77 (t, J =7.7 Hz, 2H), 0.26 (s, 9H).

[0232] 13C NMR (100 MHz, CDCl3): δ 195.1, 166.8, 141.4, 135.0, 129.2, 128.9, 128.6, 128.5, 127.8, 126.2, 125.8, 44.6, 37.7, 30.7, -0.4.

[0233] HRMS (ESI): C 21 H 27 OSSi [M+H] + Calculated value: 355.1552; Measured value: 355.1547.

[0234]

[0235] ( E )-1-(benzylthio)-1-(trimethylsilyl)oct-1-en-3-one (3w) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 27 mg, 83% yield, E / Z = 90:10.

[0236] 1 H NMR (400 MHz, CDCl3): δ 7.36-7.29 (m, 5H), 6.49 (s, 1H), 3.98 (s,2H), 2.43 (t, J = 7.4 Hz, 2H), 1.63-1.56 (m, 2H), 1.37-1.25 (m, 4H), 0.89 (t, J =6.8 Hz, 3H), 0.26 (s, 9H).

[0237] 13 C NMR (100 MHz, CDCl3): δ 196.6, 166.0, 135.1, 129.2, 128.9, 127.7, 126.0, 43.1, 37.7, 31.6, 24.5, 22.6, 14.1, -0.4.

[0238] HRMS (ESI): C 18 H 28 OSSiNa [M+Na] + Calculated value: 343.1528; Measured value: 343.1526.

[0239]

[0240] ( E)-1-(benzylthio)-5-(methylthio)-1-(trimethylsilyl)pent-1-en-3-one (3x) The product was separated by rapid chromatography (diethyl ether / hexane 1:40) and was a colorless oil. 28 mg, 86% yield, E / Z = 91:9.

[0241] 1 H NMR (400 MHz, CDCl3): δ 7.36-7.28 (m, 5H), 6.48 (s, 1H), 3.99 (s,2H), 2.77-2.73 (m, 4H), 2.12 (s, 3H), 0.26 (s, 9H).

[0242] 13 C NMR (100 MHz, CDCl3): δ 194.0, 167.6, 134.9, 129.2, 129.0, 127.8,125.4, 42.7, 37.8, 28.9, 16.0, -0.5.

[0243] HRMS (ESI): C 16 H 25 OS2Si [M+H] + Calculated value: 325.1116; Measured value: 325.1110.

[0244]

[0245] The product was separated by rapid chromatography (diethyl ether / hexane 1:100), and was a colorless oil. This aldehyde was separated by (…). S Swern oxidation synthesis of β-citronellol J. Am. Chem. Soc. 130, 2898-2899 (2008)). 29 mg, 76% yield, E / Z=82:18.

[0246] ( S , E 1-(benzylthio)-5,9-dimethyl-1-(trimethylsilyl)dec-1,8-dien-3-one (3y, major isomer) [α] 22 D = +2.9 ( c = 1.0, CHCl3).

[0247] 1H NMR (400 MHz, CDCl3): δ 7.36-7.28 (m, 5H), 6.47 (s, 1H), 5.09 (t, J =7.0 Hz, 1H), 3.98 (s, 2H), 2.44 (dd, J = 14.6, 5.6 Hz, 1H), 2.21 (dd, J = 14.6,8.4 Hz, 1H), 2.02-1.94 (m, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.35-1.19 (m,2H), 0.91 (d, J = 6.6 Hz, 3H), 0.27 (s, 9H).

[0248] 13 C NMR (100 MHz, CDCl3): δ 196.2, 166.3, 135.1, 131.6, 129.2, 128.9,127.8, 126.4, 124.6, 50.6, 37.8, 37.3, 30.1, 25.9, 25.6, 19.9, 17.8, -0.4.

[0249] HRMS (ESI): C 22 H 34 OSSiNa [M+Na] + Calculated value: 397.1997; Measured value: 397.1996.

[0250] ( S , Z )-1-(benzylthio)-5,9-dimethyl-1-(trimethylsilyl)dec-1,8-dien-3-one (3y', minor isomer) [α] 22 D = -15.1 ( c = 0.8, CHCl3).

[0251] 1 H NMR (400 MHz, CDCl3): δ 7.30-7.26 (m, 5H), 6.50 (s, 1H), 5.08 (t, J =5.8 Hz, 1H), 4.14 (s, 2H), 2.46 (dd, J = 15.4, 5.7 Hz, 1H), 2.27 (dd,J = 15.4,8.0 Hz, 1H), 2.05-1.94 (m, 3H), 1.67 (s, 3H), 1.59 (s, 3H), 1.38-1.30 (m,1H), 1.24-1.15 (m, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.35 (s, 9H).

[0252] 13 C NMR (100 MHz, CDCl3): δ 198.4, 162.0, 136.6, 131.5, 129.4, 128.72,128.67, 127.6, 124.6, 50.9, 39.4, 37.2, 29.7, 25.8, 25.7, 20.0, 17.8, 0.6.

[0253] HRMS (ESI): C 22 H 34 OSSiNa [M+Na] + Calculated value: 397.1997; Measured value: 397.1994.

[0254]

[0255] ( E )-3-(benzylthio)-3-[dimethyl(phenyl)silyl]-1-phenylprop-2-en-1-one (4a) The product was separated by rapid chromatography (diethyl ether / hexane 1:50) and was a colorless oil. 29 mg, 75% yield, E / Z = 93:7.

[0256] 1 H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.0 Hz, 2H), 7.67-7.65 (m, 2H),7.49 (t, J = 7.0 Hz, 1H), 7.41-7.30 (m, 10H), 7.26 (s, 1H), 4.12 (s, 2H), 0.61 (s, 6H).

[0257] 13C NMR (100 MHz, CDCl3): δ 186.2, 166.3, 138.8, 138.0, 135.2, 134.4,132.3, 129.05, 128.98, 128.5, 128.3, 127.8, 127.5, 124.4, 38.4, -1.5.

[0258] HRMS (ESI): C 24 H 24 OSSiNa [M+Na] + Calculated value: 411.1215; Measured value: 411.1209.

[0259]

[0260] The product was separated by rapid chromatography (diethyl ether / hexane 1:50) and was a colorless oil. 26 mg, 72% yield, E / Z = 80:20.

[0261] ( E )-3-(benzylthio)-3-[dimethyl(tert-butyl)silyl]-1-phenylprop-2-en-1-one (4b, major isomer) 1 H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.1 Hz, 2H), 7.43-7.32 (m, 8H), 7.18 (s, 1H), 4.13 (s, 2H), 1.05 (s, 9H), 0.29 (s, 6H).

[0262] 13 C NMR (100 MHz, CDCl3): δ 186.9, 164.0, 139.2, 135.4, 132.2, 129.1, 129.0, 128.5, 128.4, 127.7, 126.1, 38.0, 28.4, 19.0, -2.6.

[0263] HRMS (ESI): C 22 H 29 OSSi [M+H] + Calculated value: 369.1708; Measured value: 369.1701.

[0264] ( Z )-3-(benzylthio)-3-[dimethyl(tert-butyl)silyl]-1-phenylprop-2-en-1-one (4b', minor isomer) 1 H NMR (400 MHz, CDCl3): δ 7.88 (d, J = 7.3 Hz, 2H), 7.54 (t, J = 7.2 Hz, 1H), 7.48-7.44 (m, 2H), 7.21-7.17 (m, 5H), 7.07 (s, 1H), 4.00 (s, 2H), 1.01 (s, 9H), 0.28 (s, 6H).

[0265] 13 C NMR (100 MHz, CDCl3): δ 189.8, 158.7, 138.9, 136.4, 132.6, 129.7, 128.7, 128.6, 128.5, 128.4, 127.4, 41.9, 27.1, 17.6, -4.8.

[0266] HRMS (ESI): C 22 H 29 OSSi [M+H] + Calculated value: 369.1708; Measured value: 369.1703.

[0267]

[0268] The product was separated by rapid chromatography (diethyl ether / hexane 1:50) and was a colorless oil. 32 mg, 87% yield, E / Z = 78:22.

[0269] ( E 3-(benzylthio)-1-phenyl-3-(triethylsilyl)prop-2-en-1-one (4c, major isomer) 1 H NMR (400 MHz, CDCl3): δ 7.89 (d, J = 8.0 Hz, 2H), 7.51 (t, J = 7.2 Hz, 1H), 7.44-7.31 (m, 7H), 7.20 (s, 1H), 4.14 (s, 2H), 1.01-0.91 (m, 15H).

[0270] 13C NMR (100 MHz, CDCl3): δ 186.9, 165.6, 139.1, 135.2, 132.2, 129.04, 128.96, 128.6, 128.4, 127.7, 124.7, 37.9, 8.00, 3.6.

[0271] HRMS (ESI): C 22 H 29 OSSi [M+H] + Calculated value: 369.1708; Measured value: 369.1705.

[0272] ( Z )-3-(benzylthio)-1-phenyl-3-(triethylsilyl)prop-2-en-1-one (4c', minor isomer) 1 H NMR (400 MHz, CDCl3): δ 7.89 (d, J = 7.0 Hz, 2H), 7.52 (t, J = 7.3 Hz,1H), 7.47-7.43 (m, 2H), 7.25-7.19 (m, 5H), 7.13 (s, 1H), 4.10 (s, 2H), 1.05(t, J = 8.0 Hz, 9H), 0.86 (q, J = 8.0 Hz, 6H).

[0273] 13 C NMR (100 MHz, CDCl3): δ 188.9, 160.6, 138.9, 136.5, 132.4, 129.6,128.7, 128.6, 128.5, 127.5, 127.0, 40.2, 7.5, 4.3.

[0274] HRMS (ESI): C 22 H 29 OSSi [M+H] + Calculated value: 369.1708; Measured value: 369.1716.

[0275]

[0276] 0.1 mmol scale, duration 12 hours. The product was separated by rapid chromatography (diethyl ether / hexane 1:50) as a colorless oil. 32 mg, 78% yield, E / Z = 57:43.

[0277] ( E 3-(benzylthio)-1-phenyl-3-(triisopropylsilyl)prop-2-en-1-one (4d, major isomer) 1 H NMR (400 MHz, CDCl3): δ 7.71 (d, J = 7.1 Hz, 2H), 7.49 (t, J = 7.4 Hz,1H), 7.44-7.31 (m, 7H), 7.15 (s, 1H), 4.16 (s, 2H), 1.67 (hept, J = 7.5 Hz, 3H), 1.16 (d, J = 7.5 Hz, 18H).

[0278] 13 C NMR (100 MHz, CDCl3): δ 187.2, 164.1, 139.3, 135.3, 132.2, 128.99,128.97, 128.6, 128.4, 127.7, 126.1, 38.0, 19.4, 12.8.

[0279] HRMS (ESI): C 25 H 35 OSSi [M+H] + Calculated value: 411.2178; Measured value: 411.2177.

[0280] ( Z )-3-(benzylthio)-1-phenyl-3-(triisopropylsilyl)prop-2-en-1-one (4d', minor isomer) 1 H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 7.1 Hz, 2H), 7.49 (t, J = 7.5 Hz,1H), 7.49-7.45 (m, 2H), 7.20-7.16 (m, 5H), 7.07 (s, 1H), 3.96 (s, 2H), 1.40(hept, J = 7.5 Hz, 3H), 1.16 (d, J = 7.5 Hz, 18H).

[0281] 13C NMR (100 MHz, CDCl3): δ 189.8, 156.4, 139.0, 136.6, 132.6, 129.8,128.7, 128.6, 128.5, 127.4, 42.5, 18.8, 11.6.

[0282]

[0283] 0.1 mmol scale, duration 5 h. The product was separated by rapid chromatography (ethyl acetate / hexane 1:2) as a white solid. 35 mg, 73% yield, E / Z = 67:33.

[0284] ( E )-4-[3-(benzylthio)-3-(triisopropylsilyl)acryloyl]- N , N -Dimethylbenzamide (4e, major isomer) 1 H NMR (400 MHz, CDCl3): δ 7.69 (d, J = 8.1 Hz, 2H), 7.44-7.31 (m, 7H), 7.10 (s, 1H), 4.16 (s, 2H), 3.12 (s, 3H), 2.95 (s, 3H), 1.66 (hept, J = 7.5 Hz, 3H), 1.15 (d, J = 7.5 Hz, 18H).

[0285] 13 C NMR (100 MHz, CDCl3): δ 186.4, 170.9, 165.2, 140.0, 139.8, 135.2,129.0, 128.9, 128.4, 127.7, 127.2, 125.8, 39.6, 38.0, 35.5, 19.4, 12.7.

[0286] HRMS (ESI): C 28 H 40 NO2SSi [M+H] + Calculated value: 482.2549; Measured value: 482.2543.

[0287] ( Z )-4-[3-(benzylthio)-3-(triisopropylsilyl)acryloyl]- N , N-Dimethylbenzamide (4e', minor isomer) 1 H NMR (400 MHz, CDCl3): δ 7.90 (d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.2 Hz,2H), 7.19-7.16 (m, 5H), 7.04 (s, 1H), 3.97 (s, 2H), 3.14 (s, 3H), 2.98 (s,3H), 1.40 (hept, J = 7.4 Hz, 3H), 1.15 (d, J = 7.4 Hz, 18H).

[0288] 13 C NMR (100 MHz, CDCl3): δ 188.7, 170.8, 158.0, 140.1, 139.8, 136.4,129.8, 128.6, 128.5, 128.0, 127.43, 127.38, 42.6, 39.6, 35.5, 18.8, 11.6.

[0289] HRMS (ESI): C 28 H 40 NO2SSi [M+H] + Calculated value: 482.2549; Measured value: 482.2542.

[0290]

[0291] ( E )-3-(benzylthio)-3-{diisopropyl[(4-methoxybenzyl)oxy]silyl}-1-phenylprop-2-en-1-one (4f) 0.1 mmol scale, duration 24 h. Add 0.4 mL of phosphate buffer (pH 7) to a mixture of acetylacetone silyl ether 1e (45.7 mg, 0.12 mmol) and benzyl mercaptan 2a (12 µL, 0.1 mmol) to a 4 mL reaction tube. Separate product 4f onto silica gel by rapid chromatography (ethyl acetate / hexane 1:40) as a yellow oil. 20 mg, 40% yield, E / Z > 95:5.

[0292] 1 H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 7.4 Hz, 2H), 7.50 (t,J = 7.6 Hz,1H), 7.41-7.37 (m, 2H), 7.30-7.28 (m, 4H), 7.21-7.18 (m, 4H), 6.87 (d, J = 8.6Hz, 2H), 4.91 (s, 2H), 4.13 (s, 2H), 3.79 (s, 3H), 1.36 (hept, J = 7.3 Hz, 2H), 1.20 (d, J = 7.3 Hz, 12H).

[0293] 13 C NMR (100 MHz, CDCl3): δ 188.8, 159.1, 158.4, 138.8, 136.6, 132.7,132.4, 129.6, 128.62, 128.58, 128.5, 127.7, 127.4, 127.3, 114.0, 65.8, 55.4, 40.4, 18.0, 17.7, 13.6.

[0294] HRMS (ESI): C 30 H 36 O3SSiNa [M+Na] + Calculated value: 527.2052; Measured value: 527.2042.

[0295]

[0296] 3,3-Bis(benzylthio)-3-{diisopropyl[(4-methoxybenzyl)oxy]silyl}-1-phenylprop-1-one (4f') 0.1 mmol scale, duration 24 h. Add 0.4 mL of phosphate buffer (pH 7) to a mixture of acetylacetone silyl ether 1e (38.1 mg, 0.1 mmol) and benzyl mercaptan 2a (25 µL, 0.21 mmol) to a 4 mL reaction tube. Separate product 4f' on silica gel by rapid chromatography (ethyl acetate / hexane 1:40) as a colorless oil. 26 mg, 41% yield.

[0297] 1 H NMR (400 MHz, CDCl3): δ 7.37 (d, J = 5.8 Hz, 2H), 7.32 (d, J = 7.1 Hz,4H), 7.28-7.20 (m, 9H), 7.08 (d,J = 8.4 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 5.11(d, J = 10.6 Hz, 1H), 4.95 (d, J = 10.6 Hz, 1H), 4.54 (s, 2H), 3.90 (d, J = 13.1Hz, 2H), 3.80 (d, J = 13.3 Hz, 2H), 3.77 (s, 3H), 0.91-0.89 (m, 12H), 0.86-0.83 (m, 2H).

[0298] 13 C NMR (100 MHz, CDCl3): δ 158.8, 150.9, 138.5, 138.3, 132.9, 129.2,128.6, 128.5, 128.2, 127.5, 127.0, 126.5, 113.7, 108.9, 64.7, 55.4, 45.4, 35.4, 17.4, 17.3, 12.8.

[0299] HRMS (ESI): C 37 H 44 O3S2SiNa [M+Na] + Calculated value: 651.2399; Measured value: 651.2396.

[0300] X-ray crystallography analysis Compounds 3p and 3t were crystallized at room temperature by slow evaporation of a solution in 0.5 mL ethyl acetate and 2.5 mL hexane.

[0301] Storage number CCDC 2120673 3p of ORTEP, thermal ellipsoids are displayed with a 50% probability ( Figure 19 Table 3 lists the X-ray data for storage number CCDC 2120673.

[0302]

[0303] Table 3 Storage number CCDC 2120671 3t of ORTEP, thermal ellipsoids show a 50% probability ( Figure 20 Table 4 lists the X-ray data for storage number CCDC 2120671.

[0304]

[0305] Table 4 Results and Discussion Both the aliphatic chain and benzyl ring on the thiol exhibit good tolerance, efficiently delivering the adduct 3d-g. Notably, the reaction of 2-mercaptoethanol with 1a is selective, preserving the intact hydroxyl group. Aryl thiols with different substituents at the para, meta, and ortho positions on the benzene ring are obtained with near-quantitative conversion to single hydroxyl groups. E -The corresponding product of the isomer is 3h-l. This transformation also applies to thiols with 2-naphthalene and heteroaromatic hydrocarbons such as 4-pyridine and 2-thiophene (3m-o).

[0306] The reactions of both electron-rich and electron-deficient aromatic TMS-acetylenes proceeded smoothly at 37 °C in pH-neutral phosphate buffer (3p-t). Under optimized conditions, functional groups such as esters and indoles were compatible. The structures of 3p and 3t were also confirmed by X-ray crystallography. Aliphatic acetylenes are potent electrophiles, delivering thiol adducts with a good E / Z ratio (3u-y). We found that β-silylacetylenes with non-conjugated olefin moieties could give the desired product 3u in high yield. Similarly, ( S The reaction of β-silyl acetylacetons derived from citronellol successfully proceeded to thiol addition with complete preservation of the monoterpene structure. Unfortunately, substrates with conjugated alkenes resulted in complex mixtures of products due to the lack of significant reactivity between the alkene and alkyne.

[0307] Next, we evaluated the effect of silicon-based substituents on the acetylacetonate substrate. Figure 7 The presence of phenyl and tert-butyl groups on the silicon center had almost no effect on reactivity compared to the methyl counterpart, although the E / Z ratio decreased (80:20) in the latter case. Comparable results were obtained when trimethylsilane was replaced with triethylsilane. We note that the reaction of tris(isopropyl)silane (TIPS) required a relatively long time (18 h) for complete conversion due to steric hindrance. Notably, the amide functional group on the aromatic ring of the TIPS-acetylenone was tolerated.

[0308] In addition to the aforementioned trialkyl-silanes, we also explored the feasibility of alkynyl silyl ether substrates. Figure 8 The corresponding products, vinylsiloxanes, are unstable in acidic media, making their synthesis challenging. However, this characteristic allows them to act as self-cleaving junctions, as free OH-containing drugs or biomolecules mounted on the silyl ether handle can be readily released by adjusting the pH. J. Am. Chem. Soc. 135, 13330-13333 (2013); Tetrahedron Lett.38, 5771-5774 (1997); J. Am. Chem. Soc. 118, 8765-8766 (1996)). Initially, the reaction of silyl ether 1e was attempted in excess benzyl mercaptan. Unexpectedly, dithiol addition was detected as the dominant pathway, while in the case of trialkyl-silanes, this was a marginal pathway. When a slight excess (1.2 equivalents) of 1e was added, the desired vinylsiloxane 4f could be obtained in a moderate yield (40%), while a desilylation byproduct (4f') was formed.

[0309] Example 5. Reagent chemical selectivity and product stability experiment To further demonstrate the potential of this method in biological conjugation, we focused on reagent chemoselectivity and product stability.

[0310] General Procedure for Chemical Selectivity Experiments To a 4-mL reaction tube, 0.4 mL of phosphate buffer (pH 7) was added to a crude mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan (14 µL, 0.12 mmol). Then, the indicated amino acid derivative (0.1 mmol) was added to the stirred mixture. After stirring at 37°C for 3 hours, the emulsion was extracted three times with ethyl acetate. The combined organic layers were then evaporated under reduced pressure. Dibromomethane (7 µL, 0.1 mmol) was then added as an internal standard and dissolved in CDCl3. The emulsion was subsequently collected. 1 H-NMR (acquisition time = 25) was used to evaluate the chemoselectivity of 1a in the presence of other nucleophiles.

[0311] Stability test of thiol conjugates in the presence of endogenous molecules To a 4-mL reaction tube, the endogenous molecule (1 equivalent) was added to 3a (6.5 mg, 0.02 mmol) in 0.4 mL of phosphate buffer. After stirring at 37°C for 24 hours, the emulsion was extracted three times with ethyl acetate. The combined organic layers were then evaporated under reduced pressure. Dibromomethane (7 µL, 0.1 mmol) was then added as an internal standard and dissolved in CDCl3. Subsequently, the emulsion was collected. 1 H-NMR (collection time = 25) was used to evaluate the stability of the conjugate product in the presence of additives.

[0312] Thiol conjugates in acidic, neutral, basic and oxidizing (10 mM H2O) conditions 2 O 2 Stability experiment under the following conditions To 4 mL reaction tubes, 3a (6.5 mg, 0.02 mmol) or 1a (4.0 mg, 0.02 mmol) was dissolved in 0.4 mL of buffer solutions of different pH and hydrogen peroxide solutions. After stirring at 37 °C for 24 hours, the emulsion was extracted three times with ethyl acetate. The combined organic layers were then evaporated under reduced pressure. Dibromomethane (7 µL, 0.1 mmol) was then added as an internal standard and dissolved in CDCl3. Subsequently, the emulsion was collected. 1 H-NMR (sampling time = 25) was used to evaluate the stability of 3a and 1a in different aqueous media.

[0313] Stability test under bovine serum conditions Add 1 mL of fetal bovine serum (Bidepharm, DR-Class, BD01839326) to 3a (6.5 mg, 0.02 mmol) in a 4 mL reaction tube. After stirring at rt for 24, 48, and 72 hours, transfer the emulsion to a 10 mL Eppendorf tube, add 3 mL of MeCN, and centrifuge at 10,000 rpm for 2 min. Concentrate the supernatant under reduced pressure. Perform several repetitions using CH2Br2 (7 µL, 0.1 mmol) as an internal standard. 1 ¹H-NMR was used to measure the stability of 3a in serum. The experiment was repeated twice.

[0314] Results and Discussion First, stoichiometric amounts of benzylamine or amino acid derivatives were added to the reaction of benzyl thiol to examine the chemoselectivity of 1a. In all cases, the presence of other nucleophiles did not interfere with the exclusive formation of the thiol conjugate, confirming the thiol-specific reactivity of this silynyl ketone reagent. Figure 9 ).

[0315] Next, we continued to investigate the stability of the purified thiol conjugates in excess amines or thiol nucleophiles. This is consistent with the results previously reported for terminal acetylacetonates. 28 Unlike other compounds, we noted that thiol conjugate 3a was almost completely recovered in a pH-neutral buffer, while the olefin moiety did not undergo decomposition or side reactions, demonstrating the potential of β-trimethylsilylacetyl ketone 1a as an uncleavable thiol linker. Figure 10When 3a was incubated with glutathione and some nucleophilic amino acids, we were pleased to observe no reverse process or side reactions within 24 h. Furthermore, after stirring at 37°C for one week, the degradation of the thiol conjugate 3a was found to be negligible in the presence of a pH 7 buffer. In contrast, the vinyl sulfide and silicon components in 3a were stable under both acidic (pH 4) and alkaline (pH 10) conditions. Nevertheless, when 3a was stirred with dopamine hydrochloride in a pH 8 buffer for one day, significant decomposition of 3a was observed.

[0316] We also examined the stability of TMS-acetylene 1a. Figure 11 Although the reagent exhibits excellent stability in acidic buffers and under oxidizing conditions, it completely decomposes in pH 7 and pH 10 buffers when incubated in the absence of a nucleophile. It is currently unclear whether the silicon handle remains intact when β-silylacetylenes react with thiol-containing biomolecules. The possibility that the C-Si bond in the acetylene reagent decomposes prior to thiol addition should not be underestimated.

[0317] Thiol conjugate 3a exhibited strong stability in bovine serum, with less than 20% of disconjugation detected over a 3-day period (Figure 12).

[0318] Example 6. Functionalization of Thiol-Containing Biomolecules and Drugs Therefore, by following the scheme disclosed in this embodiment or Example 4, the feasibility of selecting unstable TMS-acetylenes to evaluate the reaction between biomolecules and drugs containing free thiols is assessed. Figure 13 ).

[0319]

[0320] N 2-(tert-Butoxycarbonyl)- N 5-(( R )-1-((2-methoxy-2-oxylideneethyl)amino)-1-oxylidene-3-((( E )-3-oxoylidene-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl)thio)prop-2-yl)- L - methyl glutamate (4g) The concentration was 0.08 mmol in a mixed solvent (0.4 mL, pH 7 phosphate buffer / MeCN = 5:1) over 12 hours. This was based on a previously reported procedure ( Bioorg. Med. Chem. 15, 1062-1066 (2007); Chem. Euro. J. 22,11365-11370 (2016)) Synthetic starting materials N-Boc-glutathione-OMe. The product was separated by rapid chromatography (ethyl acetate / hexane 2:1) as a colorless oil. 44 mg, 86% yield, E / Z > 95:5.

[0321] [α] 24 D = -17.6 ( c = 1.0, CHCl3).

[0322] 1 H NMR (400 MHz, CDCl3): δ 8.03 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 6.8 Hz,1H), 7.47-7.43 (m, 2H), 7.35 (s, 1H), 7.14 (s, 2H), 5.40 (d, J = 7.6 Hz, 1H), 4.77 (d, J = 6.2 Hz, 1H), 4.39-4.33 (m, 1H), 4.13-3.98 (m, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 3.40-3.30 (m, 2H), 2.38-2.33 (m, 2H), 2.30-2.15 (m, 1H), 1.97-1.84 (m, 2H), 1.40 (s, 9H), 0.29 (s, 9H).

[0323] 13 C NMR (100 MHz, CDCl3): δ 186.6, 172.9, 172.5, 170.2, 169.9, 166.3,156.1, 138.5, 132.5, 128.6, 123.6, 80.5, 60.6, 52.6, 52.5, 51.6, 41.5, 33.1,32.3, 29.2, 28.4, 14.3, -0.2.

[0324] HRMS (ESI): C 29 H 43 N3O9SSiNa [M+Na] + Calculated value: 660.2387; Measured value: 660.2388.

[0325]

[0326] ( E)- N -acetyl- S -(3-oxoylide-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl)- L -Cysteine ​​methyl ester (4h) The product was prepared at a scale of 0.1 mmol in a mixed solvent (0.4 mL, pH 7 phosphate buffer / MeCN = 5:1) for 12 hours. The product was separated by rapid chromatography (ethyl acetate / hexane 1:1) as a colorless oil. 31 mg, 82% yield, E / Z > 95:5.

[0327] [α] 24 D = -7.0 ( c = 1.0, CHCl3).

[0328] 1 H NMR (400 MHz, CDCl3): δ 7.99-7.97 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H),7.49-7.45 (m, 2H), 7.23 (s, 1H), 6.31 (d, J = 7.2 Hz, 1H), 5.03-4.98 (m, 1H), 3.78 (s, 3H), 3.47 (dd, J = 13.2, 5.2 Hz, 1H), 3.31 (dd, J = 13.2, 5.2 Hz, 1H), 2.02 (s, 3H), 0.31 (s, 9H).

[0329] 13 C NMR (100 MHz, CDCl3): δ 186.6, 170.8, 170.1, 166.7, 138.6, 132.6,128.7, 128.5, 123.5, 53.2, 51.0, 34.0, 23.2, -0.2.

[0330] HRMS (ESI): C 18 H 25 NO4SSiNa [M+Na] + Calculated value: 402.1171; Measured value: 402.1163.

[0331]

[0332] ( E)-1-{[3-oxoylide-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl]thio}-β-D-glucose tetraacetate (4i) The product was prepared at a scale of 0.1 mmol in a mixed solvent (0.4 mL, pH 7 phosphate buffer / MeCN = 5:1) for 3 hours. The product was separated by rapid chromatography (ethyl acetate / hexane 1:5) as a white solid. 50 mg, 88% yield, E / Z = 83:17.

[0333] [α] 22 D = -71.1 ( c = 1.0, CHCl3).

[0334] 1 H NMR (400 MHz, CDCl3): δ 7.94 (d, J = 7.1 Hz, 2H), 7.56 (t, J = 7.3 Hz,1H), 7.50-7.46 (m, 2H), 7.42 (s, 1H), 5.34-5.29 (m, 1H), 5.24-5.19 (m, 1H),5.14-5.10 (m, 1H), 5.00 (d, J = 10.1 Hz, 1H), 4.24 (dd, J = 12.4, 6.0 Hz, 1H), 4.16 (dd, J = 12.4, 2.3 Hz, 1H), 3.93-3.88 (m, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.80 (s, 3H), 0.30 (s, 9H).

[0335] 13 C NMR (100 MHz, CDCl3): δ 187.2, 170.7, 170.3, 169.5, 169.3, 164.1,138.6, 132.8, 128.9, 128.4, 125.9, 82.8, 76.2, 73.9, 69.4, 68.4, 62.6, 20.7,20.5, -0.4.

[0336] HRMS (ESI): C 26 H 34 O 10 SSiNa [M+Na]+ Calculated value: 589.1540; Measured value: 589.1547.

[0337]

[0338] ( S )-2-methyl-3-{[( E )-3-oxoylidene-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl]thio}propionyl- L -Proline (4j) 0.1 mmol scale, duration 12 h. Sodium bicarbonate (16.8 mg, 0.2 mmol) was added to a mixture of 1-phenyl-3-(trimethylsilyl)prop-2-yn-1-one 1a (20.2 mg, 0.1 mmol) and captopril (32.6 mg, 0.15 mmol) in 0.4 mL phosphate buffer (pH 7) to a 4 mL reaction tube. The reaction mixture was acidified with glacial acetic acid and then extracted with ethyl acetate. The combined organic phases were evaporated under reduced pressure. The product was separated by rapid chromatography (ethyl acetate / hexane / AcOH 66:33:1) as a yellow oil. 28 mg, 67% yield, E / Z = 83:17. The two isomers could not be separated on silica gel.

[0339] [α] 22 D = -44.8 ( c = 0.7, CHCl3).

[0340] E and Z Isomer mixtures 1 H NMR (400 MHz, CDCl3): δ 7.94 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 6.8 Hz, 1H), 7.49 -7.46 (m, 2H), 7.25 (s, 0.17H), 7.14 (s, 0.83H), 4.62-4.58 (m, 1H), 3.60-3.56 (m, 2H), 3.48-3.22 (m, 1H), 3.05-2.80 (m, 2H), 2.37-2.33 (m, 1H), 2.08-1.98 (m, 3H), 1.35-1.25 (m, 4H), 0.39 (s, 1.5H), 0.31 (s, 7.5H).

[0341] E andZ Isomer mixtures 13 C NMR (100 MHz, CDCl3): δ 188.6, 186.6, 167.7, 138.8 and 138.6, 132.6, 128.8, 128.4, 128.3, 123.2, 48.0, 36.8, 35.4, 29.8, 27.9, 25.0, 18.0, 0.7 and -0.2.

[0342] HRMS (ESI): C 21 H 29 NO4SSiNa [M+Na] + Calculated value: 442.1484 Measured value: 442.1480.

[0343]

[0344] ( 14S , 16S , 33S , 2R , 4R , 10E , 12Z , 14R )-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxane-7-aza-1(6,4)-oxazinane-3(2,3)-epoxypropylene-8(1,3)-benzenecyclotetradecane-10,12-diene-4-yl N -methyl- N -(3-((( E )-3-oxoylidene-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl)thio)propionyl)- L -Alanine ester (4kJ) 0.05 mmol scale, duration 48 hours. Add 1a (20.2 mg, 0.1 mmol) to a stirred suspension of maytansine (36.9 mg, 0.05 mmol) in 0.4 mL phosphate buffer (pH 7) to a 4 mL reaction tube. Separate the product by rapid chromatography (ethyl acetate / hexane 5:1) as a white solid (mp: 166-167 °C). 22 mg, 46% yield, E / Z = 67:33. The two isomers cannot be separated on silica gel.

[0345] [α] 22 D = -75.7 ( c = 1.0, CHCl3).

[0346] E and Z Isomer mixtures 1 H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 7.4 Hz, 1.6H), 7.87 (d, J = 7.3 Hz, 0.4H), 7.56-7.50 (m, 1H), 7.47-7.43 (m, 2H), 7.20 (s,0.2H), 7.10 (s, 0.8H), 6.76-6.71 (m, 2H), 6.64 (s, 1H), 6.47-6.39 (m, 1H),6.22 (s, 1H), 5.62 (dd, J = 15.2, 9.1 Hz, 1H), 5.49-5.44 (m, 0.8H), 5.40-5.34(m, 0.2H), 4.75 (d, J = 9.5 Hz, 1H), 4.27 (t, J = 11.0 Hz, 1H), 3.95 (s, 2.4H), 3.91 (s, 0.6H), 3.68 (d, J = 12.6 Hz, 1H), 3.50 (d, J = 8.8 Hz, 1H), 3.36 (s,3H), 3.24-3.01 (m, 8H), 2.85-2.83 (m, 4H), 2.65-2.56 (m, 2H), 2.18 (d, J = 11.8Hz, 1H), 1.56-1.45 (m, 3H), 1.32-1.23 (m, 9H), 0.79 (s, 3H), 0.34 (s, 1.8H), 0.21 (s, 7.2H).

[0347] E and Z Isomer mixtures 13C NMR (100 MHz, CDCl3): δ 186.5, 170.7, 170.5, 168.9, 167.9, 156.1, 152.3, 142.3, 141.0, 139.7, 138.8, 133.5, 132.5, 128.7, 128.4, 127.6, 125.3, 122.9, 122.4, 119.0, 113.4, 88.6, 81.0, 78.4 and 78.3, 74.2, 67.4, 60.0, 56.8, 56.7, 52.6, 46.7, 39.0, 36.2, 35.6, 32.5, 31.5, 30.8, 27.3, 15.7, 14.7, 13.5, 12.2, 0.8 and -0.3.

[0348] HRMS (ESI): C 47 H 62 ClN3O 11 SSiNa [M+Na] + Calculated value: 962.3461 Measured value: 962.3466.

[0349] Results and Discussion First, N - Protected cysteine ​​methyl ester was incubated with 1a under physiological conditions for 12 h. Functionalization of the cysteine ​​derivative was successful, yielding an adduct 4h with complete retention of the silanine handle. Acetonitrile (20% v / v relative to phosphate buffer) was added to ensure good stirring during the reaction. Encouraged by this promising outcome, we further extended the method to the modification of glutathione containing a cysteine ​​side chain. This tripeptide was successfully converted to a monofunctionalized product 4g, a single isomer. The reaction of 1-thio-D-glucose acetate also proceeded well, demonstrating the potential application of this method as a convenient entry point for polysaccharide diversification. Furthermore, the antihypertensive drug captopril could be thiolated with a TMS-acetylene in the presence of free carboxylic acid to give 4j in good yield. Incubation of the microtubule-targeting cytotoxic maytansin with 2 equivalents of β-silylacetylene 1a in pH 7 phosphate buffer yielded a monofunctionalized product 4k in a moderate yield (46%).

[0350] Example 7. Derivatization of thiol conjugates Subsequently, we demonstrated the synthetic multifunctionality of thiol conjugates. Figure 14 ).

[0351] Selective 1,2-reduction of thiol conjugate 3a

[0352] In an ice bath under argon atmosphere, 3a (32.6 mg, 0.1 mmol) and Et₂O (1 mL) were transferred into a 10-mL round-bottom flask, followed by the addition of LiAlH₄ (2.0 M, 30 µL, 0.06 mmol in THF) at 0 °C. The mixture was gradually warmed to room temperature and stirred for 1 h. A saturated Na₂SO₄ solution was carefully added at 0 °C to quench the reaction. After filtration with Et₂O, the filtrate was concentrated on a rotary evaporator and then dried under vacuum. The pure product, a colorless oil, was obtained without column chromatography. 26 mg, 80% yield.

[0353] ( E 3-(benzylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-ol (5a) 1 H NMR (400 MHz, CDCl3): δ 7.39-7.37 (m, 4H), 7.35-7.27 (m, 7H), 5.96(d, J = 9.6 Hz, 1H), 5.52 (d, J = 9.6 Hz, 1H), 3.93 (d, J = 12.3 Hz, 1H), 3.89 (d, J = 12.3 Hz, 1H), 0.36 (s, 9H).

[0354] 13 C NMR (100 MHz, CDCl3): δ 143.2, 140.6, 136.2, 134.5, 129.2, 128.6,127.6, 127.3, 126.1, 72.7, 36.7, 0.6.

[0355] HRMS (ESI): C 19 H 24 OSSiNa [M+Na] + Calculated value: 351.1215; Measured value: 351.1212.

[0356] Brook alignment provides silicon-based migration products 5b

[0357] The 1,2-reduction of 3a (0.1 mmol) was carried out according to the above steps, and the reduction intermediate 5a was directly used in the next step. LiO was slowly added to a solution of 5a in THF (0.6 mL) under argon atmosphere at 0°C. t-Bu (100 µL, 1 M in THF). After stirring at room temperature for 12 h, the mixture was quenched with water (5 mL) and extracted with Et2O. The combined extracts were then dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by rapid chromatography (diethyl ether / hexane 1:50) as a colorless oil. 20 mg, 61% yield.

[0358] (E)-{[3-(benzylthio)-1-phenylallyl]oxy}trimethylsilane (5b) 1 H NMR (400 MHz, CDCl3): δ 7.33-7.30 (m, 7H), 7.28-7.25 (m, 3H), 6.23(d, J = 15.0 Hz, 1H), 5.73 (dd, J = 15.0, 6.3 Hz, 1H), 5.17 (d, J = 6.3 Hz, 1H), 3.90 (s, 2H), 0.09 (s, 9H).

[0359] 13 C NMR (100 MHz, CDCl3): δ 143.4, 137.4, 131.8, 129.0, 128.7, 128.4,127.3, 126.3, 124.3, 75.4, 37.1, 0.3.

[0360] HRMS (ESI): C 19 H 25 OSSi [M+H] + Calculated value: 329.1395; Measured value: 329.1403. Desilication of thiol conjugate 3a

[0361] To a solution of 3a (32.6 mg, 0.1 mmol) in DMSO (0.6 mL), CsF (16.7 mg, 0.11 mmol) and one drop of water were added. The mixture was stirred at 40 °C until 3a was completely converted (by observation that 3a was absent when the mixture sample was placed on a thin-layer chromatography (TLC) plate). The reaction mixture was quenched with water and extracted with Et₂O. The combined extracts were then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Based on crude... 1 ¹H NMR determined the E / Z ratio to be 56:44. The residue was separated by rapid chromatography (ethyl acetate / hexane 1:20) as a colorless oil. 23 mg, 90% yield.

[0362] ( E )-3-(benzylthio)-1-phenylprop-2-en-1-one [74896-61-0] (5c, major isomer) 1 H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 14.8 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 7.2 Hz, 1H), 7.46-7.29 (m, 7H), 6.94 (d, J = 14.8 Hz, 1H), 4.14(s, 2H).

[0363] 13 C NMR (100 MHz, CDCl3): δ 187.2, 148.2, 138.2, 135.8, 132.6, 129.1,128.9, 128.7, 128.5, 128.0, 119.3, 37.4.

[0364] HRMS (ESI): C 16 H 15 OS [M+H] + Calculated value: 255.0844; Measured value: 255.0835.

[0365] ( Z )-3-(benzylthio)-1-phenylprop-2-en-1-one [70061-48-2] (5c', minor isomer) 1 H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz,1H), 7.46-7.42 (m, 2H), 7.39-7.28 (m, 6H), 7.05 (d, J = 9.8 Hz, 1H), 4.01 (s, 2H).

[0366] 13C NMR (100 MHz, CDCl3): δ 189.3, 151.6, 138.1, 137.4, 132.5, 129.2, 129.0, 128.7, 128.1, 127.6, 117.1, 40.7.

[0367] HRMS (ESI): C 16 H 15 OS [M+H] + Calculated value: 255.0844; Measured value: 255.0832.

[0368] The oxidation of thiol conjugate 3a yields vinyl sulfone 5d.

[0369] At 0°C, 5a (97.8 mg, 0.3 mmol) and sodium bicarbonate (63 mg, 0.75 mmol) were added to a mixture in DCM (3 mL). m -CPBA (77 wt%, 168 mg, 0.75 mmol). The reaction was stirred at room temperature for 1 h, then quenched with sodium thiosulfate solution. The aqueous layer was extracted with DCM. The combined organic layers were then dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was separated by rapid chromatography (ethyl acetate / hexane 1:20) as a white solid. 105 mg, 98% yield.

[0370] ( E )-3-(benzylsulfonyl)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (5d) 1 H NMR (400 MHz, CDCl3): δ 7.73 (s, 1H), 7.59-7.56 (m, 1H), 7.46-7.38(m, 9H), 4.33 (s, 2H), 0.34 (s, 9H).

[0371] 13 C NMR (100 MHz, CDCl3): δ 192.4, 153.5, 152.1, 135.5, 134.5, 131.2, 129.2, 129.03, 128.98, 128.9, 128.8, 61.5, 0.4.

[0372] HRMS (ESI): C 19 H 23 O3SSi [M+H] +Calculated value: 359.1137; Measured value: 359.1130.

[0373] The oxidation of thiol conjugate 3a yields vinyl sulfoxide 5e.

[0374] At 0°C, 5a (32.6 mg, 0.1 mmol) and sodium bicarbonate (8.4 mg, 0.1 mmol) were added to a mixture of DCM (1 mL). m -CPBA (77 wt%, 27 mg, 0.12 mmol). The reaction was stirred at room temperature for 30 min, then quenched with sodium thiosulfate solution. The aqueous layer was extracted with DCM. The combined organic layers were then dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was separated by rapid chromatography (ethyl acetate / hexane 1:8) as a colorless oil. 31 mg, 91% yield.

[0375] ( E )-3-(benzylsulfinyl)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (5e) 1 H NMR (400 MHz, CDCl3): δ 7.80 (s, 1H), 7.78 (d, J = 7.4 Hz, 2H), 7.56(t, J = 7.4 Hz, 1H), 7.44-7.34 (m, 5H), 7.25-7.22 (m, 2H), 4.31 (d, J = 13.1 Hz, 1H), 3.66 (d, J = 13.1 Hz, 1H), 0.39 (s, 9H).

[0376] 13 C NMR (100 MHz, CDCl3): δ 189.6, 164.6, 137.6, 137.1, 133.7, 131.0,129.1, 129.0, 128.8, 128.65, 128.57, 59.4, -0.5.

[0377] HRMS (ESI): C 19 H 23 O2SSi [M+H] + Calculated value: 343.1188; Measured value: 343.1183.

[0378] Nazarov cyclization of thiol conjugate 3a

[0379] Under nitrogen atmosphere and at 0 °C, BCl3 (1.0 M solution in DCM, 120 µL, 0.12 mmol) was added dropwise to a solution of 5a (32.6 mg, 0.1 mmol) in 0.5 mL of dry DCM. The resulting mixture was stirred overnight at room temperature, and then pinacol (17.7 mg, 0.15 mmol) and Et3N (28 µL, 0.2 mmol) were added to quench the reaction. A saturated aqueous solution of Na2CO3 was then added. The aqueous layer was extracted with ether, and the combined extracts were dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was subjected to rapid chromatography (ethyl acetate / hexane 1:20) as a brown oil. 25 mg, 77% yield.

[0380] 3-(benzylthio)-3-(trimethylsilyl)-2,3-dihydro-1 H 1-Indene (5f) 1 H NMR (400 MHz, CDCl3): δ 7.73 (d, J = 7.7 Hz, 1H), 7.68-7.63 (m, 2H),7.35 (t, J = 7.7 Hz, 1H), 7.21-7.16 (m, 3H), 7.05 (d, J = 6.4 Hz, 2H), 3.44 (d, J =12.0 Hz, 1H), 3.27 (d, J = 12.0 Hz, 1H), 3.02 (d, J = 20.0 Hz, 1H), 2.86 (d, J =20.0 Hz, 1H), 0.04 (s, 9H).

[0381] 13 C NMR (100 MHz, CDCl3): δ 204.2, 158.0, 137.4, 137.0, 135.2, 129.1,128.6, 127.2, 127.0, 126.1, 123.6, 47.9, 42.4, 32.8, -3.6.

[0382] HRMS (ESI): C 19 H 23 OSSi [M+H] + Calculated value: 327.1239; Measured value: 327.1234.

[0383] Results and Discussion Selective 1,2-reduction of 3a with 0.6 equivalents of LiAlH4 yielded only allyl alcohol 5a. 3a was treated with LiAlH4 followed by the addition of LiO. t -Bu, via the Brook rearrangement, provides silicon-based migration product 5b. In the presence of CsF, the C-Si bond of 3a is cleaved, yielding desilication products 5c and 5c'. Furthermore, the vinyl sulfide moiety in 3a readily undergoes quantitative oxidation, by altering... m - CPBA stoichiometry yielded vinyl sulfone 5d and vinyl sulfoxide 5e, respectively. Notably, when 3a was treated with a Lewis acid (such as BCl3), 2,3-dihydroindanone 5f was generated in satisfactory yields via Nazarov cyclization, in which the TMS group and thioether component remained intact.

[0384] Example 8. Protein Modification We also attempted to modify bovine serum albumin and human serum albumin, each containing a free cysteine ​​residue.

[0385] Electrospray ionization mass spectrometry (ESI-MS) measurement ESI-MS was performed on a linear quadrupole ion trap detector mass spectrometer (LTQ XL from ThermoFisher Scientific) coupled to a Vanquish UHPLC (from ThermoFisher Scientific). Data were processed using ThermoBioPharma Finder 3.1.

[0386] General procedure for the reaction of BSA with 1f

[0387] To a 4-mL reaction tube, 1f (20 µL, 30 mM, 2 equivalents) of acetonitrile was added to 0.5 mM bovine serum albumin (19.9 mg, 0.3 µmol) in phosphate buffer (pH 8, 0.6 mL). After vigorous stirring at 37°C for 2 hours, an aliquot of 50 µL of the reaction mixture was evaluated by ESI-MS, and a monoconjugated adduct containing the silicon moiety was detected.

[0388] General procedure for the reaction of HSA with 1a To a 4-mL reaction tube, 20 µL (30 mM, 2 equivalents) of acetonitrile 1a was added to 0.5 mM human serum albumin (19.9 mg, 0.3 µmol) in phosphate buffer (0.6 mL). After vigorous stirring at 37 °C for 10 hours, a 50 µL aliquot of the reaction mixture was evaluated by ESI-MS, and only desilication monoconjugated adducts were detected.

[0389] Results and Discussion TES-acetylene 1f was successfully added to bovine serum albumin in phosphate buffer, providing a separately modified conjugate that retains the silicon moiety. Figure 15 and Figure 16 ).

[0390] Human serum albumin was labeled using TMS-acetylene 1a, yielding thiol-selective products in pH 6 and 7 buffers, despite the presence of silicon component decomposition. Figure 17 and Figure 18 ).

Claims

1. A method for producing a compound of formula I: The method includes using a compound of formula II: Compounds of Formula III: HSR 5 III The reaction is carried out in a solvent comprising an aqueous phosphate buffer solution with a pH of 5 to 9, wherein: R 1 Indicates C1 to C 10 Alkyl, C2 to C 10 alkenyl, C2 to C 10 alkynyl group, C6 to C 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 A carbocyclic system wherein each of the groups is either unsubstituted or substituted by one or more groups selected from the following. Halogen, C1 to C3 alkyl, CONR 6a R 6b CO2R 6c OR 6d C6 to C 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 Carbocyclic system, wherein C1 to C3 alkyl groups, C6 to C4 alkyl groups, and C5 alkyl groups are all C1 to C3 alkyl groups. 12 Aromatic groups, 5-10 membered heterocyclic groups and C3 to C4 groups 12 The carbon ring system is either unsubstituted or substituted by one or more halogen atoms; Each R 2 To R 4 Independently represent C1 to C 10 Alkyl or O (C1 to C1) 10 Alkyl groups, wherein each group is independently unsubstituted or substituted by one or more groups selected from C1 to C3 alkyl, phenyl, and 5-10 membered heterocyclic groups, wherein the unsubstituted groups are substituted or substituted by halogens and OR 7 One or more of them are replaced; R 5 Indicates the organic part; R 6a To R 6d This refers to C1 to C1 atoms that are either unsubstituted or substituted by one or more halogen atoms. 10 Alkyl; and R 7 Indicates C1 to C 10 alkyl.

2. The method according to claim 1, wherein, The solvent further comprises acetonitrile, optionally wherein, The acetonitrile is present in amounts ranging from 10% to 30% v / v, such as about 20% v / v.

3. The method according to claim 1 or claim 2, wherein, R 1 Indicates C1 to C 10 Alkyl, C2 to C 10 alkenyl, C6 to C 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5-C6 carbocyclic systems, wherein each of the groups is unsubstituted or substituted by one or more groups selected from the following. Halogen, C1 to C3 alkyl, CONR 6a R 6b CO2R 6c OR 6d C6 to C 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5 to C6 carbide ring systems, wherein C1 to C3 alkyl groups, C6 to C6 carbide rings, and C5 to C6 carbide rings are present. 10 Aromatic groups, 5-10 membered heterocyclic groups, and C5 to C6 carbon ring systems are either unsubstituted or substituted by one or more halogen atoms.

4. The method according to claim 3, wherein, R 1 Choose one of the following: The wavy lines represent the attachment points to the rest of the molecule.

5. The method according to any one of the preceding claims, wherein, R 2 To R 4 Each of the following is selected independently: Me, Et, i Pr, t Buhe The wavy line represents the attachment point to the rest of the molecule.

6. The method according to any one of the preceding claims, wherein, R 5 Choose one of the following: 。 7. The method according to any one of claims 1 to 5, wherein, The compound of Formula III is selected from bovine serum albumin, amino acids containing thiol substituents, oligopeptides containing thiol substituents, peptides containing thiol substituents, monosaccharides, disaccharides or trisaccharides containing thiol substituents, or pharmaceutical molecules containing thiol substituents, optionally wherein the compound of Formula III is selected from N-Boc-glutathione-OMe, 1-mercapto-D-glucose acetate, captopril and maytansine.

8. The method according to any one of the preceding claims, wherein, The pH of the phosphate buffer solution is from 6 to 8, optionally wherein the pH of the phosphate buffer solution is about 7.

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