Thiol-specific silicon-containing biocompatible click reagent: beta-silyl alkynyl carbonyl compounds

Abstract

The current invention relates to a method of producing a compound of formula I; the method comprising reacting a compound of formula II; with a compound of formula III: HSR5 in a solvent comprising an aqueous phosphate buffer that has a pH of from 5 to 9, where R1 to R5 are as described in the description of this application.

Description

[0001] THIOL-SPECIFIC SILICON-CONTAINING BIOCOMPATIBLE CLICK REAGENT: BETASILYL ALKYNYL CARBONYL COMPOUNDS

[0002] Field of Invention

[0003] The current invention relates to the use of a (3-silyl alkynyl carbonyl compound in a selective click reaction to provide a thiol-containing product.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Site-specific chemical modification of proteins has been recognised as a versatile strategy for probing natural systems and creating novel therapeutic conjugates. Over the past two decades, a number of methodologies have been developed, which targeted primarily at lysine, N- terminus and cysteine residues due to their high nucleophilicities. Among them, labeling of the free thiol on cysteine residues is the most common for site-specific functionalization due to the less abundance (<2%) of cysteine in natural proteins and the relatively inert properties of disulfide bonds, avoiding the generation of mixture of random modified products (Green Synth. Catal. 3, 309-316 (2022)).

[0007] Conventional approaches to thiol conjugation rely on maleimides (Michael addition) and iodoacetamide (alkylation). For example, maleimides are used extensively as a thiol-targeted linker of antibody-drug conjugates (ADCs). However, limitations including the instability of maleimide-based conjugates in plasma and comparatively low chemoselectivity of iodoacetamide have stimulated the development of alternative cysteine modification methods. Recently, thiol-specific electrophiles 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 patterns such as arylation with aryl thioethers and aryl halides / triflates (Chem. Sci. 12, 5209-5215 (2021); Nature 526, 687-691 (2015)) and electrophilic alkynylation with dibenzothiophenium triflates and hypervalent iodonium reagents (Bioconjug. Chem. 32, 1570- 1575 (2021); J. Am. Chem. Soc. 135, 9620-9623 (2013)) have advantages of excellent chemoselectivity and product stability, complementing the existing conjugation methods. Despite the above-mentioned advances, the expectation of thiol click reactions under metal-free, biocompatible conditions and high atom economy has not yet been fully realized. Non- cleavable thiol specific reagents are necessary for cysteine-targeted covalent inhibitors, whereas only a few methods are available including allenamides (Angew. Chem. Int. Ed. 53, 7491-7494 (2014); Angew. Chem. Int. Ed. 59, 18054-18061 (2020)), perfluoroarenes (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)).

[0008] In recent years, thiol click reactions with alkynyl carbonyl compounds have gained considerable attention from the chemistry community (Chem. Rev. 119, 11110-11244 (2019); Acc. Chem. Res. 55, 2355-2369 (2022); Research 2018, 3152870 (2018)), especially in the areas of polymer chemistry (Chem. Rev. 121, 6744-6776 (2021)), fluorescent sensors (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, stoichiometric or catalytic organic bases are necessary for the generation of the reactive thiolate species. For instance, Dove et al. developed the regioselective addition of alkyl thiols to terminal propiolates modulated by bases and solvent polarity (Angew. Chem. Int. Ed. 52, 4132-4136 (2013)). In the field of bioconjugation, electron-deficient alkynes were first recognized as the thiol labeling reagent by Che and coworkers (FIG. 1) (Chem. Eur. J. 15, 3839-3850 (2009)). Despite the pioneering findings that enabled the reactions to proceed under the physiological conditions, this conjugation technology still has some limitations, such as instability of the vinyl-sulfide linker, possibility of double thiol addition (Angew. Chem. Int. Ed. 60, 15359-15364 (2021); Angew. Chem. Int. Ed. 59, 3609-3617 (2020)) and unsatisfactory E / Z ratio of the conjugates when coupled with terminal, aliphatic and aryl alkynyl carbonyl compounds (J. Environ. Chem. Eng. 4, 2004-2007 (2016)).

[0009] Summary of Invention

[0010] It has been surprisingly found that a silicon moiety can be used as a thiol-specific click reagent, allowing the generation of a thioether product. This product can undergo further downstream reactions to provide further useful products. The reaction is able to be conducted under physiological conditions, is highly atom-economic and additive-free, is thiol-specific and non- cleavable and enables for the introduction of lipophilic silicon handles, which may be of use as-is, or to enable further chemistry to be conducted.

[0011] Aspects and embodiments of the current invention will now be discussed by reference to the following numbered clauses. 1. A method of producing a compound of formula I: the method comprising reacting a compound of formula II: with a compound of formula III:

[0012] HSR5III in a solvent comprising an aqueous phosphate buffer that has a pH of from 5 to 9, wherein:

[0013] R1represents C1to C10alkyl, C2to C10alkenyl, C2to C10alkynyl, a C6to C12aromatic group, a 5-10 membered heterocyclic group, and a C3 to C12carbocyclic ring system, where each of the groups is unsubstituted or substituted by one or more groups selected from halo, C1to C3alkyl, CONR6aR6b, CO2R6c, ORM, a C6to C12aromatic group, a 5-10 membered heterocyclic group, and a C1to C12carbocyclic ring system, where the C1to C3alkyl, the C6to C12aromatic group, the 5-10 membered heterocyclic group and the C3to C12carbocyclic ring system are unsubstituted or substituted by one or more halo atoms; each R2to R4independently represents C1to C10alkyl or O(C1to C10alkyl), where each group is independently unsubstituted or substituted by one or more groups selected from C1to C3 alkyl, phenyl, and a 5-10 membered heterocyclic group, where said groups are unsubstituted or substituted by one or more of halo and OR7;

[0014] R5represents an organic moiety;

[0015] RBato R6drepresents C1to C10alkyl that is unsubstituted or substituted by one or more halo atoms; and

[0016] R7represents a C1to C10alkyl.

[0017] 2. The method according to Clause 1 , wherein the solvent further comprises acetonitrile, optionally wherein the acetonitrile is present in an amount of from 10 to 30% v / v, such as about 20% v / v.

[0018] 3. The method according to Clause 1 or Clause 2, wherein R1represents C1to C10alkyl, C2to C10alkenyl, a C6to C10aromatic group, a 5-10 membered heterocyclic group, and a C5to C6carbocyclic ring system, where each of the groups is unsubstituted or substituted by one or more groups selected from halo, C1to C3alkyl, CONR6aR6b, CO22R6c, OR8d, a C6to C10aromatic group, a 5-10 membered heterocyclic group, and a C5to Ca carbocyclic ring system, where the C1to C3the C6to C10aromatic group, the 5-10 membered heterocyclic group and the C5to C6carbocyclic ring system are unsubstituted or substituted by one or more halo atoms.

[0019] where the wavy line represents the point of attachment to the rest of the molecule.

[0020] 5. The method according to any one of the preceding clauses, wherein each of R2to R4is independently selected from one of the following:

[0021] Me, Et,iPr,tBu, and , where the wavy line represents the point of attachment to the rest of the molecule.

[0022] 6. The method according to any one of the preceding clauses, wherein R5is selected from one of the following:

[0023]

[0024] 7. The method according to any one of Clauses 1 to 5, wherein the compound of formula III is selected from bovine serum albumin, an amino acid containing a thiol substituent, an oligopeptide containing a thiol substituent, a peptide containing a thiol substituent, a mono-, di- or tri-saccharide containing a thiol substituent, or a drug molecule containing a thiol substituent, optionally wherein the compound of formula III is selected from N-Boc-glutathione- OMe, 1-thiol-D-glucose acetate, captopril, and mertansine.

[0025] 8. The method according to any one of the preceding clauses, wherein the pH of the phosphate buffer is from 6 to 8, optionally wherein the pH of the phosphate buffer is about 7.

[0026] Drawings

[0027] FIG. 1 depicts the overview of thiol click reactions with alkynyl carbonyl compounds. Ref: Dove et al. 2013 (Angew. Chem. Int. Ed. 52, 4132-4136 (2013)) and Che et al, 2009 (Chem. Eur. J. 15, 3839-3850 (2009))

[0028] FIG. 2 depicts thiol conjugation with alkynones containing silicon functionality. This powerful thiol-specific approach proceeds smoothly under biocompatible conditions (metal-free, additive-free, ambient temperature and neutral pH) in almost 100% atom economy. Advantages include (1) physiological conditions; (2) highly atom-economic and additive-free; (3) thiol-specific and non-cleavable; and (4) additional lipophilic silicon handles.

[0029] FIG. 3 depicts organosilanes in medicinal and material applications.

[0030] FIG. 4 depicts the reactivity of β-silyl alkynyl ester and amide with benzyl mercaptan 2a.

[0031] FIG. 5 depicts substrate scope of thiol nucleophiles to β-trimethylsilyl alkynones - scope of thiols.

[0032] FIG. 6 depicts substrate scope of thiol nucleophiles to β-trimethylsilyl alkynones - scope of aliphatic and aromatic alkynones.

[0033] FIG. 7 depicts effect of trialkyl silyl substituents and silyl ether - scope of silyl substituents.

[0034] FIG. 8 depicts reaction of silyl ether-tethered alkynone.

[0035] FIG. 9 depicts chemoselectivity of 1-phenyl-3-(trimethylsilyl)prop-2-yn-1-one 1a towards various nucleophiles: Reaction time is 180 min in most of experiments. Yield of 3a in the presence of the shown nucleophiles. The exception is the reaction in the presence of N-Cbz- Trp-OMe, which needed 12 hours for the full conversion of 1a.

[0036] FIG. 10 depicts stability of thiol conjugate 3a in the presence of endogenous molecules: Phosphate buffer (pH 7) was chosen as the solvent in most of experiments. The exception is dopamine hydrochloride, which was dissolved in 0.1 M NH4HCO3 buffer (pH 8).

[0037] FIG. 11 depicts stability of thiol conjugate 3a and reagent 1a under the acidic, pH neutral, basic and oxidative (10 mM H2O2) conditions: 3a (6.5 mg, 0.02 mmol) or 1a (4.0 mg, 0.02 mmol) was dissolved in 0.4 mL of the above-mentioned solvent, stirring for 24 hours at 37 °C. Conversions were determined by1H-NMR using CH2Br2 as the internal standard.

[0038] FIG. 12 depicts Stability experiment under bovine serum condition: (A) Stability evaluation of thiol conjugate 3a in serum over time; (B)1H-NMR spectrum at 48-hour incubation time (90% of 3a remaining in bovine serum). FIG. 13 depicts functionalization of thiol-containing biomolecules and drugs using β-silyl alkynones. 4h from N-Ac-Cys-OMe, 4i from 1 -thio-D-glucose acetates; 4j in pH 7 phosphate buffer from Captopril; 4k in pH 7 phosphate buffer from Mertansine.

[0039] FIG. 14 depicts derivatization of the thiol conjugate.

[0040] FIG. 15 depicts deconvoluted mass spectrum of unmodified BSA.

[0041] FIG. 16 depicts Modification results of BSA with TES-alkynone (1f) in phosphate buffer: +1 modified = singly modified protein.

[0042] FIG. 17 depicts deconvoluted mass spectrum of unmodified HSA.

[0043] FIG. 18 depicts modification results of HSA with TMS-alkynone (1a) in phosphate buffer: +1 modified = singly modified protein.

[0044] FIG. 19 depicts the Oak Ridge Thermal Ellipsoid Plot (ORTEP) of 3p.

[0045] FIG. 20 depicts the Oak Ridge Thermal Ellipsoid Plot (ORTEP) of 3t.

[0046] Description

[0047] It has been surprisingly found that a novel type of silicon-containing alkynone reagent can target free thiols in the presence of other nucleophiles. This powerful thiol-specific approach proceeds smoothly under biocompatible conditions (metal-free, additive-free, ambient temperature and neutral pH) in almost 100% atom economy. It is especially noteworthy that the reaction only works in a buffer solution, and affords a stable and mono-functionalized product with full retention of the silicon moiety. The compatibility of these silicon compounds allows this methodology to incorporate the more lipophilic silicon functionalities into the conjugate adducts. In addition to simple aliphatic and aromatic thiols, this biocompatible method is applicable to the labelling of thiol-containing amino acids, peptides and pharmaceuticals. This methodology allows for the use of new silicon-containing reagents, and also protein-based drugs that contain silicon moieties. Additionally, exploiting the inorganic properties of silicon may provide bridges between different disciplines for many applications.

[0048] Thus, in a first aspect of the invention, there is provided a method of producing a compound of formula I:

[0049] the method comprising reacting a compound of formula II: with a compound of formula III:

[0050] HSR5III in a solvent comprising an aqueous phosphate buffer that has a pH of from 5 to 9, wherein:

[0051] R1represents C1to C10alkyl, C2to C10alkenyl, C2to C10alkynyl, a C6to C12aromatic group, a 5-10 membered heterocyclic group, and a C3to C12carbocyclic ring system, where each of the groups is unsubstituted or substituted by one or more groups selected from halo, C1to C3alkyl, CONR6aR6b, CO2R6G, OR6d, a C6to C12aromatic group, a 5-10 membered heterocyclic group, and a C3to C12carbocyclic ring system, where the C1to C3alkyl, the C6to C12aromatic group, the 5-10 membered heterocyclic group and the C3to C12carbocyclic ring system are unsubstituted or substituted by one or more halo atoms; each R2to R4independently represents C1to C10alkyl or O(C1to C10alkyl), where each group is independently unsubstituted or substituted by one or more groups selected from C1to C3alkyl, phenyl, and a 5-10 membered heterocyclic group, where said groups are unsubstituted or substituted by one or more of halo and OR7; R5represents an organic moiety;

[0052] R6ato R6drepresents C1to C10alkyl that is unsubstituted or substituted by one or more halo atoms; and

[0053] R7represents a C1to C10alkyl.

[0054] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0055] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0056] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, and the like.

[0057] While the reaction can be run using only an aqueous phosphate buffer that has a pH of from 5 to 9 (e.g. from 6 to 8, such as about 7), it may also incorporate an organic solvent that is miscible with water. For example, in certain embodiments of the invention, the solvent may further comprise acetonitrile. In such embodiments, the acetonitrile may be present in any suitable amount. For example, the acetonitrile may be present in an amount of from 10 to 30% v / v, such as about 20% v / v.

[0058] As noted hereinbefore, it was unexpectedly found that an aqueous phosphate buffer (or using a phosphate salt, e.g. K3PO4at a concentration of 20 mol% in water) is required for the reaction disclosed herein to function. That is, the use of water alone or an organic solvent alone did not allow for the reaction to proceed. As such, a phosphate salt and / or a phosphate buffer are seen as an essential component of the method.

[0059] Compounds of formula I may contain double bonds and may thus exist as E (entgegen) and Z (zusammeri) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention. The reaction may preferentially provide the compound of formula I in an E-enriched state (as such, the compound of formula I is shown as the E-isomer). For example, in certain embodiments, the ratio of E- to Z- may be at least 55:45, such as 85:15, such as 90:10, such as 95:5, such as >95:5 mokmol.

[0060] In certain embodiments of the invention, R1may represent C1to C10alkyl, C2to C10alkenyl, a C2to C10aromatic group, a 5-10 membered heterocyclic group, and a C5to C5carbocyclic ring system, where each of the groups is unsubstituted or substituted by one or more groups selected from halo, C1to C3alkyl, CONR6aR6b, CO2R60, ORM, a C6to C10aromatic group, a 5-10 membered heterocyclic group, and a C5to C5carbocyclic ring system, where the C1to C3alkyl, the C6to C10aromatic group, the 5-10 membered heterocyclic group and the C5to C6carbocyclic ring system are unsubstituted or substituted by one or more halo atoms.

[0061] In more particular embodiments of the invention, R1may be selected from one of the following:

[0062] where the wavy line represents the point of attachment to the rest of the molecule.

[0063] In certain embodiments of the invention that may be mentioned herein, each of R2to R4may independently be selected from one of the following:

[0064] Me, Et,iPr,tBu, and , where the wavy line represents the point of attachment to the rest of the molecule.

[0065] In certain embodiments of the invention that may be mentioned herein, R5may be selected from one of the following:

[0066]

[0067] The current invention allows for a wide range of possible thiol-containing substrates, as is demonstrated by the examples hereinbelow. As such, the compound of formula III may be selected from almost any thiol-containing compound, including polymeric materials (e.g. synthetic polymers and natural polymers, such as proteins). For example, the compound of formula III may be selected from bovine serum albumin, an amino acid containing a thiol substituent, an oligopeptide containing a thiol substituent, a peptide containing a thiol substituent, a mono-, di- or tri-saccharide containing a thiol substituent, or a drug molecule containing a thiol substituent, optionally wherein the compound of formula III is selected from N-Boc-glutathione-OMe, 1-thiol-D-glucose acetate, captopril, and mertansine.

[0068] The incorporation of silicon may provide an additional handle to further modify the compounds of formula I, since organosilicon compounds are versatile building blocks and can act as removable protecting / directing groups in organic transformations. Owing to their unique physical, physiological and optoelectronic properties, silicon isosteres have a wide variety of applications in medicinal and material fields. Compared to its carbon isostere, the organosilicon molecule has enhanced lipophilicity, which allows it to pass through blood brain barrier. Representative silicon-containing marketed drugs include Amsilarotene, Silafluofen and Flusilazole. In addition, silole-based ^-conjugated compounds such as silicon-bridged stilbenes are promising electroluminescent materials. Furthermore, silyl ethers have been used to serve as biomedical materials including poly-siloxane and immolative linkers (e g. an immolative linker in the conjugate nanoparticle-Si(R)2-OCH2-Drug). The silyl moiety also enables organic functional groups to be immobilized onto an inorganic glass surface. The compounds of formula I are also a useful intermediate product that can be reacted in a number of different ways to generate additional down-stream products. For example, the compounds of formula I may be subjected to:

[0069] (a) reduction (e.g. LiAIH4in diethylether) to provide the alcohol;

[0070] (b) reduction (e.g. UAIH4 in diethylether) and silyl migration (LiOtBu, THF) to provide the silyl- protected alcohol;

[0071] (c) silyl removal (e.g. CsF in water and DMSO);

[0072] (d) oxidation of the sulfur (e.g. m-CPBA and NaHCO3in DCM) to provide the sulfoxide (if 1 equivalents of m-CPBA used) or sulfone (if 2.5 equivalent of m-CPBA used); and

[0073] (e) a cyclisation reaction (if there is a suitable group alpha- to the ketone moiety (e.g. a phenyl ring)); BCI3in DCM.

[0074] Examples of these reactions may be seen in FIG. 14 and in the examples below.

[0075] Further aspects and embodiments of the current invention will be described by the following non-limiting examples.

[0076] Examples

[0077] Materials

[0078] Unless noted otherwise, commercially available chemicals and solvents were used as received without purification. Phosphate buffer solution (pH 7, Catalogue No. 38712) was purchased from Alfa Aesar, which contains H2O (99.05% w), K3PO4H2O (0.90% w) and NaOH (0.05% w). Fetal bovine serum was purchased from Bidepharm (DR-Class, BD01839326). Albumin from bovine serum (BSA) was purchased from Sigma-Aldrich (catalog No.: A2153). Albumin from human serum (HSA) was purchased from Sigma-Aldrich (catalog No.: A3782). Both proteins are used without further purification.

[0079] Sequence of albumin from bovine serum (BSA): BSA is composed of 583 amino acids and contains one free cysteine (Cys34) and 59 lysines.

[0080] DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGC EKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLC DEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETM REKVLTSSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECC HGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADF AEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACY STVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGK VGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPD ETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDK CCAADDKEACFAVEGPKLVVSTQTALA

[0081] Sequence of albumin from human serum (HSA): HSA is composed of 585 amino acids and contains one free cysteine (Cys34) and 60 lysines.

[0082] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVD VMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMRADLPSLA ADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPH ECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFS ALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDF AAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0083] General Information for Purifications and Characterisations of Compounds

[0084] Flash Chromatography

[0085] Flash chromatography was performed using Merck 40-63D 60 A silica gel.

[0086] Nuclear Magnetic Resonance (NMR) Measurements

[0087] 1H,13C,19F and11B NMR spectra were acquired on Bruker 400 MHz NMR spectrometers in indicated deuterated solvents.1H NMR chemical shifts were recorded relative to residual protic solvents (CDCI3: 5 7.26, CD3CN: 5 1.94). Multiplicities were given as: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). The number of protons (n) for a given resonance was indicated by nH. Coupling constants were reported as a J value in Hz.13C NMR chemical shifts were recorded relative to solvent resonance (CDCI3: δ 77.16, CD3CN: 6 118.26 ± 0.02, 1.32 ± 0.02).18F NMR chemical shifts were uncorrected.

[0088] Optical Rotation Measurements

[0089] Optical rotation was measured using a JASCO P-1030 polarimeter equipped with a sodium vapor lamp at 589 nm and the concentration of samples was denoted as c.

[0090] High Resolution Mass Spectra (HRMS) Measurements High resolution mass spectra (HRMS) were recorded on a Waters G2-XS QT of spectrometer with ESI mode unless otherwise stated.

[0091] Example 1. Optimisation of Reaction Conditions

[0092] General Procedure for Solvent Screening

[0093] To a 4-mL reaction tube, 0.4 mL of solvent was added to the mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan 2a (17 pl_, 0.15 mmol). After vigorously stirred for 3 hours at 37 °C, the emulsion was extracted with diethyl ether three times. The combined organic layers were then evaporated under the reduced pressure. Afterwards, the crude product was subjected to1H-NMR to determine the conversion of 1a and the yield of 3a.

[0094] General Procedure for the Studies of Additives

[0095] To a 4-mL reaction tube, 0.4 ml_ of deionized water and the indicated amount of additive were sequentially added to the mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan 2a (17 μL, 0.15 mmol). After vigorously stirred for 3 hours at 37 °C, the emulsion was extracted with diethyl ether three times. The combined organic layers were then evaporated under the reduced pressure. Afterwards, the crude product was subjected to1H-NMR to determine the yield of 3a and 3a'.

[0096] (E)-3-(Benzylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3a)

[0097] The product 3a was isolated by flash chromatography (diethyl ether / hexanes 1 :70) as colorless oil. 29 mg, 88% yield, E / Z > 95:5.

[0098] 1H NMR (400 MHz, CDCI3): 6 7.86-7.84 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.46-7.32 (m, 7H), 7.21 (s, 1 H), 4.12 (s, 2H), 0.34 (s, 9H).

[0099] 13C NMR (100 MHz, CDCI3): 6 186.6, 168.6, 139.0, 135.1 , 132.3, 129.1, 129.0, 128.6, 128.4, 127.8, 123.4, 38.0, -0.2.

[0100] HRMS (ESI): Calcd for Ci9H23OSSi [M+H]+: 327.1239; found: 327.1234.

[0101] 3,3-Bis(benzylthio)-1-phenylpropan-1-one (3a1) [74896-60-9] 0.1 mmol scale for 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 the mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan 2a (17 pl_, 0.15 mmol). The product 3a' was isolated by flash chromatography (diethyl ether / hexanes 1:50) as colorless oil. 31 mg, 82% yield.

[0102] 1H NMR (400 MHz, CDCI3): 5 7.74 (d, J = 7.3 Hz, 2H), 7.54 (t, J = 7.4 Hz, 1 H), 7.42-7.38 (m, 2H), 7.35-7.28 (m, 8H), 7.24-7.20 (m, 2H), 4.33 (t, J = 7.0 Hz, 1 H), 3.85 (s, 4H), 3.31 (d, J = 7.0 Hz, 2H).

[0103] 13C NMR (100 MHz, CDCI3): δ 196.2, 138.0, 136.7, 133.4, 129.2, 128.7, 128.2, 127.2, 46.1 , 45.4, 35.5.

[0104] HRMS (ESI): Calcd for C23H22OS2Na [M+Na]+: 401.1010; found: 401.1013.

[0105] Results and Discussions

[0106] Our investigation began with the solvent screening and the influence of additives of the model reaction between 1-phenyl-3-(trimethylsilyl)prop-2-yn-1-one 1a and benzyl mercaptan 2a at 37 °C under metal-free conditions (Table 1).

[0107] Table 1. The solvent screening of the model reaction between 1-phenyl-3-(trimethylsilyl)prop- 2-yn-1-one 1a and benzyl mercaptan 2a at 37 °C under metal-free conditions. 120 mol% of additive when use.240 mol% of additive when use.3Potassium phosphate buffer: 0.9% w of K3PO4• H2O and 0.05% w of NaOH, pH 7 from Alfa Aesar.4The pH of potassium phosphate buffer was modulated by the addition of conjugate acid or base.

[0108] We discovered that neither organic solvents nor pure water provided the conjugated product and the starting material 1a was consumed only in trace amount (Table 1 Entries 1-6). When the catalytic organic base such as Et3N was added in the water-based reaction, the thioacetal side-product 3a' was obtained predominantly via the double Michael addition (Angew. Chem. Int. Ed. 52, 4132-4136 (2013)) (Table 1 Entry 7). Encouragingly, the reaction in aqueous solution with 20 mol% K3PO4afforded the vinyl sulfide 3a in moderate yield (65%, Table 1 Entry 8). We assumed that the influence of inorganic salt on the reaction outcome may be due to the formation of reactive thiolate ions (ACS Catal. 9, 9923-9952 (2019)). The reaction in aqueous solution with 40 mol% NaOH provided 3a in comparable yield. To evaluate whether the mono-thiolation of 1a was feasible under a milder and even physiological condition, we performed the reaction in phosphate buffers with the pH ranging from 6 to 8 (Table 1 Entries 10-12). Ultimately, pH 7 phosphate buffer was chosen as the optimal media owing to the high yield (88%) and satisfactory E / Z ratio (>95:5) of the desired product (Table 1 Entry 10).

[0109] Example 2. Reactivity of Other Alkynyl Carbonyl Electrophiles

[0110] Furthermore, we investigated the reactivity of other alkynyl carbonyl electrophiles under the optimized conditions (FIG. 4).

[0111] General Procedure for the Reactivity Studies

[0112] To a 4-mL reaction tube, 0.4 ml_ of phosphate buffer solution (pH 7) was added to the mixture of β-silyl alkynyl ester 1b (or amide 1c, 0.1 mmol) and benzyl mercaptan 2a (17 JLLL, 0.15 mmol). After vigorously stirred for 18 hours at 37 °C, the emulsion was extracted with diethyl ether three times. The combined organic layers were then evaporated under the reduced pressure. Afterwards, the crude product was subjected to1H-NMR to determine the E / Z ratio. The yields of 3b and 3c were calculated after the purification on silica using flash column chromatography.

[0113] Benzyl 3-(benzylthio)-3-(trimethylsilyl)acrylate (3b)

[0114] The product was isolated by flash chromatography (ethyl acetate / hexanes 1:20) as colorless oil. 20 mg, 56% yield, E / Z = 55:45.1H NMR (400 MHz, CDCI3): 6 7.40-7.29 (m, 10H), 6.17 (s, 1 H), 5.16 (s, 2H), 4.19 (s, 2H), 0.31 (s, 9H).

[0115] 13C NMR (100 MHz, CDCI3): 6 165.0, 163.5, 136.6, 136.3, 129.4, 128.72, 128.69, 128.68,

[0116] 128.3, 127.6, 121.2, 66.2, 38.9, 0.1.

[0117] HRMS (ESI): Calcd for C20H25O2SSi [M+H]+: 357.1345; found: 357.1352.

[0118] 3-(Benzylthio)-N,A / -dimethylacrylamide (3c)

[0119] The product was isolated by flash chromatography (ethyl acetate / hexanes 1:10) as colorless oil. 17 mg, 75% yield, E / Z = 50:50.

[0120] 1H NMR of mixture of E and Z isomers (400 MHz, CDCI3): 5 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, 1 H), 3.89 (s, 1 H), 2.99 (s, 3H), 2.97 (s, 3H).

[0121] 13C NMR of mixture of E and Z isomers (100 MHz, CDCI3): 5 167.0 and 165.3, 145.7 and

[0122] 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

[0123] 112.4, 39.8, 37.35 and 37.28, 35.9 and 35.4.

[0124] Results and Discussions β-Silyl alkynyl ester 1 b provided the thiol conjugate 3b in 56% yield. The desilylated Michael addition was detected as the competing pathway, which could account for the loss of material balance. The reaction of β-silyl alkynyl amide 1c was incompatible with silicon functionalities, since only the desilylated adduct 3c was obtained.

[0125] Example 3. Synthesis of β-Silyl Alkynone Substrates

[0126] Table 2 shows the β-silyl alkynones disclosed herein. Non-commercially available β-silyl alkynones were synthesized using the following procedures.

[0127] Table 2. The scope of β-silyl alkynones.

[0128]

[0129] Synthesis ofEthynyldiisopropyl((4-methoxybenzyl)oxy)siiane [1229230-93-6] anisyl alcohol

[0130] To a solution of chlorodiisopropylsilane (1.8 ml_, 10.5 mmol) in anhydrous THF (10 ml_) at 0 °C under nitrogen, 20 mL of ethynylmagnesium bromide (0.5 M in THF) was added dropwise. The reaction was vigorously stirred overnight at room temperature. Upon completion, the mixture was quenched with phosphate buffer (pH 7) and extracted with diethyl ether (Et2O). The combined organic layers were dried over sodium sulphate (Na2SO4) and concentrated using the rotary evaporator (25 °C, 200 mbar). The crude product was then purified by flash column chromatography on silica gel using pentanes as the eluent.

[0131] To an ice-cold suspension of N-bromosuccinimide (NBS) (1.33 g, 7.5 mmol) in 10 ml_ of anhydrous DCM, ethynyldiisopropylsilane (1.05 g, 7.5 mmol) was added dropwise at 0 °C under N2atmosphere. The reaction was kept on stirring for 3 hours at room temperature. 4- dimethylaminopyridine (DMAP) (91.6 mg, 0.75 mmol) and 5 ml_ of DCM were charged into another round-bottom flask under nitrogen, followed by the sequential addition of anisyl alcohol (930 piL, 7.5 mmol) and Et3N (3.1 ml_, 22.5 mmol). The aforementioned solution of bromo(ethynyl)diisopropylsilane was then added dropwise under the ice-cold condition. The reaction mixture was stirred overnight at room temperature. Upon completion, the mixture was quenched with phosphate buffer (pH 7) and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4and concentrated under the reduced pressure. The crude product was then purified by flash column chromatography on Florisil® (diethyl ether / hexanes 1:20). The resultant ethynyl dialkylsilyl ether was then used for synthesis of 3- (Diisopropyl((4-methoxybenzyl)oxy)silyl)-1-phenylprop-2-yn-1-one.

[0132] Ethynyldiisopropyl((4-methoxybenzyl)oxy)silane [1229230-93-6] (Org. Lett. 12, 2860- 2863 (2010))

[0133] 1H NMR (400 MHz, CDCI3): 5 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, 1 H), 1.17-1.07 (m, 14H).

[0134] To a solution of ethynyl trialkylsilane (6 mmol) in THF at -20 °C under nitrogen, n-BuLi (2 M in hexanes, 2.6 ml_) was added dropwise. The mixture was stirred for 15 min at -20 °C. The aldehyde (5 mmol) was then added and the temperature was gradually raised to room temperature. After stirring overnight, the reaction was quenched with saturated NH4CI solution and extracted with diethyl ether. The organic layers were combined, dried with Na2SO4and concentrated under the reduced pressure. The crude product was directly used in the next step. β-Silyl propargyl alcohol was added to a stirred suspension of activated manganese oxide (MnO2) (5 equiv) in DCM. The reaction was stirred overnight at room temperature. The resulting mixture was then filtered through celite and washed with DCM. The product was isolated by flash chromatography (hexanes / ethyl acetate) on silica. 1-Piperonyl-3-(trimethylsilyl)prop-2-yn-1-one

[0135] 1H NMR (400 MHz, CDCI3): 6 7.81 (d, J = 8.2 Hz, 1 H), 7.54 (s, 1 H), 6.88 (d, J = 8.2 Hz, 1 H),

[0136] 6.07 (s, 2H), 0.30 (s, 9H).

[0137] 13C NMR (100 MHz, CDCI3): 0 176.0, 153.1 , 148.3, 131.9, 127.6, 108.5, 108.2, 102.2, 101.0,

[0138] 99.9, -0.5.

[0139] HRMS (ESI): Calcd for CI3HI5O3SI [M+H]+: 247.0790; found: 247.0786.

[0140] 1-( / V-Tosyl-indol-3-yl)-3-(trimethylsilyl)prop-2-yn-1-one

[0141] 1H NMR (400 MHz, CDCI3): 6 8.37 (s, 1 H), 8.30 (d, J = 7.0 Hz, 1 H), 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).

[0142] 13C NMR (100 MHz, CDCI3): 6 171.7, 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.

[0143] HRMS (ESI): Calcd for C2iH22NO3SiS [M+H]+: 396.1090; found: 1085.

[0144] 1-(Cyclohex-3-en-1-yl)-3-(trimethylsilyl)prop-2-yn-1-one

[0145] 1H NMR (400 MHz, CDCI3): 0 5.70-5.64 (m, 2H), 2.67-2.60 (m, 1 H), 2.26-2.24 (m, 2H), 2.12- 2.03 (m, 3H), 1.68-1.63 (m, 1 H), 0.22 (s, 9H).

[0146] 13C NMR (100 MHz, CDCI3): 6 190.7, 126.9, 125.1 , 101.3, 98.9, 48.2, 26.5, 24.5, 24.4, -0.6.

[0147] HRMS (ESI): Calcd for Ci2Hi9OSi [M+H]+: 207.1205; found: 207.1202.

[0148] 5-(Methylthio)-1-(trimethylsilyl)pent-1-yn-3-one

[0149] 1H NMR (400 MHz, CDCI3): 6 2.86 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.11 (s, 3H),

[0150] 0.24 (s, 9H).

[0151] 13C NMR (100 MHz, CDCI3): 3 185.7, 101.7, 99.1 , 45.2, 28.0, 15.8, -0.7.

[0152] HRMS (ESI): Calcd for C9H17OS1S [M+H]+: 201.0769; found: 201.0766.

[0153] (S)-5,9-Dimethyl-1-(trimethylsilyl)dec-8-en-1-yn-3-one

[0154] 1H NMR (400 MHz, CDCI3): 0 5.08 (t, J = 8.1 Hz, 1 H), 2.55 (dd, J = 15.4, 5.8 Hz, 1 H), 2.36 (dd, J = 15.4, 8.1 Hz, 1 H), 2.16-2.08 (m, 1 H), 2.05-1.93 (m, 2H), 1.68 (s, 3H), 1.60 (s, 3H), 1.39-1.31 (m, 1 H), 1.28-1.18 (m, 1 H), 0.94 (d, J = 6.6 Hz, 3H), 0.24 (s, 9H).

[0155] 13C NMR (100 MHz, CDCI3): 6 188.0, 131.8, 124.3, 102.5, 97.6, 52.8, 36.9, 29.5, 25.9, 25.5, 19.8, 17.8, -0.6.

[0156] HRMS (ESI): Calcd for C15H27OS1 [M+H]+: 251.1831; found: 251.1827.

[0157] A / ,N-Dimethyl-4-[3-(triisopropylsilyl)propioloyl]benzamide

[0158] 1H NMR (400 MHz, CDCI3): 0 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, 21 H).

[0159] 13C NMR (100 MHz, CDCI3): 6 176.8, 170.5, 141.8, 137.4, 129.8, 127.4, 103.0, 99.1 , 39.5,

[0160] 35.4, 18.7, 11.2.

[0161] HRMS (ESI): Calcd for C2iH32NO2Si [M+H]+: 358.2202; found: 358.2205.

[0162] 3-(Diisopropyl((4-methoxybenzyl)oxy)silyl)-1-phenylprop-2-yn-1-one

[0163] 1H NMR (400 MHz, CDCI3): 6 8.13 (d, J = 7.6 Hz, 2H), 7.62 (t, J = 7.3 Hz, 1 H), 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).

[0164] 13C NMR (100 MHz, CDCI3): 0 177.5, 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.

[0165] HRMS (ESI): Calcd for C23H28O3SINa [M+Na]+: 403.1705; found: 403.1722.

[0166] Example 4. Substrate Scope Studies With the optimized conditions in hand, we then studied the substrate scope of thiols in conjugate addition with TMS-alkynone 1a (FIG. 5). Next, a variety of substituted alkynones were examined (FIG. 6). We also evaluated the effect of silyl substituents on the alkynone substrates (FIG. 7). β-silyl alkynones used herein were either obtained via commercial source, or prepared by following the protocol disclosed in Example 3.

[0167] General Procedure for Thiol Addition to ft-Silyl Alkynones

[0168] To a 4-mL reaction tube, 0.4 ml_ of phosphate buffer solution (pH 7) was added to the mixture of β-silyl alkynone (0.1 mmol) and thiol nucleophile (0.15 mmol). After vigorously stirred for 3 hours at 37 °C, the emulsion was extracted with diethyl ether three times. The combined organic layers were then evaporated under the reduced pressure. Afterwards, the crude product was subjected to1H-NMR to determine the E / Z ratio. Unless otherwise noted, E and Z isomers can be separated on silica by flash chromatography.

[0169] (E)-3-[(4-Chlorobenzyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3d)

[0170] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :70) as pale yellow oil. 32 mg, 88% yield, E / Z > 95:5.

[0171] 1H NMR (400 MHz, CDCI3): 6 7.75 (d, J = 7.9 Hz, 2H), 7.46-7.43 (m, 1 H), 7.38-7.34 (m, 2H), 7.26-7.17 (m, 4H), 7.07 (s, 1 H), 3.99 (s, 2H), 0.25 (s, 9H).

[0172] 13C NMR (100 MHz, CDCI3): 6 186.6, 168.1 , 138.9, 133.7, 132.4, 130.4, 129.2, 128.7, 128.3, 123.6, 37.2, -0.3.

[0173] HRMS (ESI): Calcd for Ci9H22CIOSSi [M+H]+: 361.0849; found: 361.0848.

[0174] (E)-3-[(Furan-2-ylmethyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3e)

[0175] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :70) as colorless oil. 23 mg, 73% yield, E / Z > 95:5.

[0176] 1H NMR (400 MHz, CDCI3): 6 7.91 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.5 Hz, 1 H), 7.47-7.43 (m, 2H), 7.41 (s, 1 H), 7.26 (s, 1 H), 6.37 (d, J = 2.9 Hz, 1 H), 6.32 (d, J = 3.1 Hz, 1H), 4.14 (s, 2H), 0.33 (s, 9H).13C NMR (100 MHz, CDCI3): 6 186.6, 167.5, 149.2, 142.6, 139.0, 132.4, 128.7, 128.4, 123.2, 111.0, 108.6, 30.0, -0.3.

[0177] HRMS (ESI): Calcd for Ci7H2iO2SSi [M+H]+: 317.1032; found: 317.1023.

[0178] (E)-3-[(2-Hydroxyethyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3f)

[0179] The product was isolated by flash chromatography (ethyl acetate / hexanes 1 :5) as yellow oil.

[0180] 20 mg, 71 % yield, E / Z > 95:5.

[0181] 1H NMR (400 MHz, CDCI3): 6 7.93 (d, J = 7.3 Hz, 2H), 7.53 (t, J = 7.2 Hz, 1 H), 7.48-7.44 (m, 2H), 7.20 (s, 1 H), 3.98 (t, J = 5.4 Hz, 2H), 3.13 (t, J = 6.0 Hz, 2H), 2.01 (s, 1H), 0.33 (s, 9H).

[0182] 13C NMR (100 MHz, CDCI3): 6 186.7, 167.9, 138.9, 132.4, 128.7, 128.4, 123.2, 60.0, 35.2, - 0.2.

[0183] HRMS (ESI): Calcd for Ci4H20O2SSiNa [M+Na]+: 303.0851; found: 303.0853. major minor

[0184] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :70) as colorless oil. 24 mg, 75% yield, E / Z = 80:20.

[0185] (E)-3-(Cyclohexylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3g, major isomer)

[0186] 1H NMR (400 MHz, CDCI3): 5 7.92 (d, J = 7.7 Hz, 2H), 7.53 (t, J = 7.2 Hz, 1 H), 7.48-7.44 (m, 2H), 7.18 (s, 1H), 3.33-3.28 (m, 1 H), 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).

[0187] 13C NMR (100 MHz, CDCI3): 6 186.5, 168.6, 139.4, 132.2, 128.7, 128.3, 122.4, 44.0, 32.1 , 26.1, 26.0, -0.1.

[0188] HRMS (ESI): Calcd for Ci8H27OSSi [M+H]+: 319.1552; found: 319.1544.

[0189] (Z)-3-(Cyclohexylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3g', minor isomer)

[0190] 1H NMR (400 MHz, CDCI3): 5 7.93 (d, J = 7.0 Hz, 2H), 7.51 (t, J = 7.3 Hz, 1 H), 7.46-7.42 (m, 2H), 7.19 (s, 1H), 3.27-3.20 (m, 1 H), 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).13C NMR (100 MHz, CDCI3): 6 188.7, 164.3, 138.9, 132.3, 128.6, 128.4, 125.8, 46.0, 34.5, 26.4, 25.5, 0.5.

[0191] HRMS (ESI): Calcd for C18H27OSSi [M+H]+: 319.1552; found: 319.1548.

[0192] (E)-1 -Phenyl-3-phenylthio-3-(trimethylsilyl)prop-2-en-1 -one (3h) [82632-32-4]

[0193] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :100) as yellow oil.

[0194] 29 mg, 93% yield, E / Z > 95:5.

[0195] 1H NMR (400 MHz, CDCI3): 6 7.64-7.62 (m, 2H), 7.54-7.46 (m, 5H), 7.44-7.42 (m, 1 H), 7.36-

[0196] 7.32 (m, 2H), 6.73 (s, 1H), 0.40 (s, 9H).

[0197] 13C NMR (100 MHz, CDCI3): 6 186.8, 169.5, 138.8, 135.5, 132.3, 131.0, 130.0, 129.9, 128.5, 128.2, 125.0, -0.3.

[0198] HRMS (ESI): Calcd for Ci8H2iOSSi [M+H]+: 313.1082; found: 313.1077.

[0199] (E)-1-Phenyl-3-(p-tolyl)thio-3-(trimethylsilyl)prop-2-en-1-one (3i)

[0200] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :70) as pale yellow oil. 30 mg, 92% yield, E / Z > 95:5.

[0201] 1H NMR (400 MHz, CDCI3): 6 7.64 (d, J = 7.6 Hz, 2H), 7.46-7.39 (m, 3H), 7.36-7.26 (m, 4H), 6.74 (s, 1 H), 2.42 (s, 3H), 0.39 (s, 9H).

[0202] 13C NMR (100 MHz, CDCI3): 6 186.9, 169.9, 140.1, 138.9, 135.3, 132.2, 130.8, 128.5, 128.3, 127.4, 124.9, 21.5, -0.3.

[0203] HRMS (ESI): Calcd for C19H23OSSi [M+H]+: 327.1239; found: 327.1246.

[0204] (E)-3-[(4-Methoxyphenyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3j)

[0205] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as yellow oil.

[0206] 31 mg, 90% yield, E / Z > 95:5.

[0207] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 3.87 (s, 3H), 0.39 (s, 9H).

[0208] 13C NMR (100 MHz, CDCI3): 6 186.8, 170.5, 161.0, 138.8, 136.9, 132.2, 128.5, 128.2, 124.8, 121.5, 115.6, 55.6, -0.3. HRMS (ESI): Calcd for CieH^CkSSi [M+H]+: 343.1188; found: 343.1179.

[0209] (E)-3-[(3-Methoxyphenyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3k)

[0210] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as yellow oil.

[0211] 30 mg, 88% yield, E / Z > 95:5.

[0212] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 7.01 (d, J = 8.3 Hz, 1 H), 6.81 (s, 1 H), 3.84 (s, 3H), 0.40 (s, 9H).

[0213] 13C NMR (100 MHz, CDCI3): 6 186.9, 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.

[0214] HRMS (ESI): Calcd for Ci9H23O2SSi [M+H]+: 343.1188; found: 343.1183.

[0215] (E)-3-[(2-Methoxyphenyl)thio]-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3I)

[0216] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 26 mg, 76% yield, E / Z > 95:5.

[0217] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 3.86 (s, 3H), 0.41 (s, 9H).

[0218] 13C NMR (100 MHz, CDCI3): 6 186.8, 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.

[0219] HRMS (ESI): Calcd for Ci^ChSSINa [M+Na]+: 365.1007; found: 365.1000.

[0220] (E)-3-(Naphthalen-2-ylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (3m)

[0221] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :70) as yellow oil.

[0222] 29 mg, 80% yield, E / Z > 95:5.

[0223] 1H NMR (400 MHz, CDCI3): 3 8.08 (s, 1 H), 7.96-7.87 (m, 3H), 7.61-7.56 (m, 5H), 7.40 (t, J =

[0224] 6.8 Hz, 1 H), 7.29-7.25 (m, 2H), 6.85 (s, 1 H), 0.44 (s, 9H).

[0225] 13C NMR (100 MHz, CDCI3): 6 187.0, 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.

[0226] HRMS (ESI): Calcd for C22H22OSSiNa [M+Na]+: 385.1058; found: 385.1053.

[0227] (E)-1 -Phenyl-3-(pyridin-4-ylthio)-3-(trimethylsilyl)prop-2-en-1 -one (3n)

[0228] 0.1 mmol scale for 18 hours. The product was isolated by flash chromatography (ethyl acetate / hexanes 1 :10) as white solid. 22 mg, 70% yield, E / Z > 95:5.

[0229] 1H NMR (400 MHz, CDCI3): 3 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, 1 H), 0.38 (s, 9H).

[0230] 13C NMR (100 MHz, CDCI3): 3 187.1 , 165.2, 151.1, 142.5, 138.2, 132.8, 128.8, 128.7, 128.3, 127.2, -0.3.

[0231] HRMS (ESI): Calcd for C17H20NOSSi [M+H]+: 314.1035; found: 314.1034.

[0232] (E)-1-Phenyl-3-(thiophen-2-ylthio)-3-(trimethylsilyl)prop-2-en-1-one (3o)

[0233] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as yellow oil. 30 mg, 94% yield, E / Z > 95:5.

[0234] 1H NMR (500 MHz, CDCI3): 3 7.70-7.68 (m, 2H), 7.64 (d, J = 5.3 Hz, 1 H), 7.47 (t, J = 7.4 Hz, 1H), 7.39-7.36 (m, 2H), 7.28 (d, J = 3.6 Hz, 1 H), 7.20 (dd, J = 5.3, 3.6 Hz, 1 H), 6.85 (s, 1H), 0.39 (s, 9H).

[0235] 13C NMR (125 MHz, CDCI3): 3 187.0, 169.5, 138.6, 137.1 , 132.8, 132.4, 128.7, 128.6, 128.5, 128.3, 126.0, -0.4.

[0236] HRMS (ESI): Calcd for C16H18OS2SiNa [M+Na]+: 341.0466; found: 341.0461.

[0237] (E)-3-(Benzylthio)-1 -[(4-methoxycarbonyl)phenyl]-3-(trimethylsilyl)prop-2-en-1 -one (3p)

[0238] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :20) as white solid (mp: 106-107 °C). 36 mg, 94% yield, E / Z > 95:5.

[0239] 1H NMR (400 MHz, CDCI3): 3 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, 1 H), 4.13 (s, 2H), 3.94 (s, 3H), 0.34 (s, 9H).

[0240] 13C NMR (100 MHz, CDCI3): 3 185.7, 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.

[0241] HRMS (ESI): Calcd for C21H25O3SSi [M+H]+: 385.1294; found: 385.1297.

[0242] (E)-3-(Benzylthio)-1-(4-fluorophenyl)-3-(trimethylsilyl)prop-2-en-1-one (3q)

[0243] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 24 mg, 70% yield, E / Z > 95:5.

[0244] 1H NMR (400 MHz, CDCI3): 5 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).

[0245] 13C NMR (100 MHz, CDCI3): 5 185.1 , 169.0, 165.3 (d, JCF= 253.2 Hz), 135.3 (d, JCF= 2.8 Hz), 135.1 , 130.9 (d, JCF= 9.0 Hz), 129.1 , 129.0, 127.8, 123.1 , 115.6 (d, JCF = 21.7 HZ), 38.0, -0.3.19F NMR (376.6 MHz, CDCI3): 5 -106.7.

[0246] HRMS (ESI): Calcd for Ci9H2iFOSSiNa [M+Na]+: 367.0964; found: 367.0959.

[0247] (E)-3-(Benzylthio)-1-(4-methoxyphenyl)-3-(trimethylsilyl)prop-2-en-1-one (3r)

[0248] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 31 mg, 88% yield, E / Z = 90:10.

[0249] 1H NMR (400 MHz, CDCI3): 6 7.84 (d, J = 8.8 Hz, 2H), 7.43-7.31 (m, 5H), 7.18 (s, 1 H), 6.91 (d, J = 8.8 Hz, 2H), 4.11 (s, 2H), 3.86 (s, 3H), 0.33 (s, 9H).

[0250] 13C NMR (100 MHz, CDCI3): 6 185.4, 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.

[0251] HRMS (ESI): Calcd for C20H25O2SSi [M+H]+: 357.1345; found: 357.1342.

[0252] (E)-3-(Benzylthio)-1 -(piperonyl)-3-(trimethylsilyl)prop-2-en-1 -one (3s)

[0253] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 33 mg, 89% yield, E / Z = 91 :9.

[0254] 1H NMR (400 MHz, CDCIs): 6 7.42-7.29 (m, 7H), 7.11 (s, 1 H), 6.81 (d, J = 8.1 Hz, 1 H), 6.02 (s, 2H), 4.10 (s, 2H), 0.32 (s, 9H).

[0255] 13C NMR (100 MHz, CDCI3): 6 185.0, 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.

[0256] HRMS (ESI): Calcd for C20H22O3SSiNa [M+Na]+: 393.0957; found: 393.0959.

[0257] (E)-3-(Benzylthio)-1-(N-tosylindol-3-yl)-3-(trimethylsilyl)prop-2-en-1-one (3t)

[0258] 0.1 mmol scale in the mix solvents (0.5 ml_, pH 7 Kpi buffer / MeCN = 4:1) for 12 hours. The product was isolated by flash chromatography (diethyl ether / hexanes 1 :20) as white solid (mp: 175-176 °C). 48 mg, 92% yield, E / Z > 95:5.

[0259] 1H NMR (400 MHz, CDCI3): 6 8.36 (d, J = 7.7 Hz, 1 H), 8.03 (s, 1 H), 7.95 (d, J = 7.5 Hz, 1 H), 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).

[0260] 13C NMR (100 MHz, CDCI3): 6 181.8, 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.

[0261] HRMS (ESI): Calcd for C28H30NO3S2Si [M+H]+: 520.1436; found: 520.1444.

[0262] (E)-3-(Benzylthio)-1-(cyclohex-3-en-1-yl)-3-(trimethylsilyl)prop-2-en-1-one (3u)

[0263] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :50) as colorless oil. 29 mg, 88% yield, E / Z = 86:14.

[0264] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 2.22-2.06 (m, 4H), 1.92-1.87 (m, 1 H), 1.62-1.54 (m, 1 H), 0.26 (s, 9H).

[0265] 13C NMR (100 MHz, CDCI3): 6 198.9, 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.

[0266] HRMS (ESI): Calcd for Ci9H27OSSi [M+H]+: 331.1552; found: 331.1543.

[0267] (E)-1-(Benzylthio)-5-phenyl-1-(trimethylsilyl)pent-1-en-3-one (3v)

[0268] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 26 mg, 73% yield, E / Z = 89:11.

[0269] 1H NMR (400 MHz, CDCI3): 6 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).13C NMR (100 MHz, CDCI3): 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.

[0270] HRMS (ESI): Calcd for C21H27OSSi [M+H]+: 355.1552; found: 355.1547.

[0271] (E)-1-(Benzylthio)-1-(trimethylsilyl)oct-1-en-3-one (3w)

[0272] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 27 mg, 83% yield, E / Z = 90:10.

[0273] 1H NMR (400 MHz, CDCI3): 6 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).

[0274] 13C NMR (100 MHz, CDCI3): 6 196.6, 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.

[0275] HRMS (ESI): Calcd for C18H28OSSiNa [M+Na]+: 343.1528; found: 343.1526.

[0276] (E)-1 -(Benzylthio)-5-(methylthio)-1 -(trimethylsilyl)pent-l -en-3-one (3x)

[0277] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :40) as colorless oil. 28 mg, 86% yield, E / Z = 91 :9.

[0278] 1H NMR (400 MHz, CDCI3): 6 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).

[0279] 13C NMR (100 MHz, CDCI3): 6 194.0, 167.6, 134.9, 129.2, 129.0, 127.8, 125.4, 42.7, 37.8, 28.9, 16.0, -0.5.

[0280] HRMS (ESI): Calcd for C16H25OS2Si [M+H]+: 325.1116; found: 325.1110.

[0281] The product was isolated by flash chromatography (diethyl ether / hexanes 1:100) as colorless oil. The aldehyde was synthesized by the Swern oxidation of (S)-|3-Citronellol (J. Am. Chem. Soc. 130, 2898-2899 (2008)). 29 mg, 76% yield, E / Z = 82:18.

[0282] (S,£)-1 -(Benzylthio)-5,9-dimethyl-1 -(trimethylsilyl)deca-l ,8-dien-3-one (3y, major isomer) [a]22D= +2.9° (c = 1.0, CHCh).

[0283] 1H NMR (400 MHz, CDCI3): 6 7.36-7.28 (m, 5H), 6.47 (s, 1H), 5.09 (t, J = 7.0 Hz, 1 H), 3.98 (s, 2H), 2.44 (dd, J = 14.6, 5.6 Hz, 1H), 2.21 (dd, J = 14.6, 8.4 Hz, 1 H), 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).

[0284] 13C NMR (100 MHz, CDCI3): 6 196.2, 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.

[0285] HRMS (ESI): Calcd for C22H34OSSiNa [M+Na]+: 397.1997; found: 397.1996.

[0286] (S,Z)-1-(Benzylthio)-5,9-dimethyl-1-(trimethylsilyl)deca-1,8-dien-3-one (3y', minor isomer)

[0287] [a]22D= -15.1° (c = 0.8, CHCI3).

[0288] 1H NMR (400 MHz, CDCI3): <5 7.30-7.26 (m, 5H), 6.50 (s, 1H), 5.08 (t, J = 5.8 Hz, 1 H), 4.14 (s, 2H), 2.46 (dd, J = 15.4, 5.7 Hz, 1H), 2.27 (dd, J = 15.4, 8.0 Hz, 1 H), 2.05-1.94 (m, 3H), 1.67 (s, 3H), 1.59 (s, 3H), 1.38-1.30 (m, 1 H), 1.24-1.15 (m, 1 H), 0.91 (d, J = 6.6 Hz, 3H), 0.35 (s, 9H).

[0289] 13C NMR (100 MHz, CDCI3): 6 198.4, 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.

[0290] HRMS (ESI): Calcd for C22H34OSSiNa [M+Na]+: 397.1997; found: 397.1994.

[0291] 0 SiMe2Ph

[0292] Ph AA SBn

[0293] (E)-3-(Benzylthio)-3-[dimethyl(phenyl)silyl]-1 -phenylprop-2-en-1 -one (4a)

[0294] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :50) as colorless oil. 29 mg, 75% yield, E / Z = 93:7.

[0295] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 4.12 (s, 2H), 0.61 (s, 6H).

[0296] 13C NMR (100 MHz, CDCI3): 5 186.2, 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.

[0297] HRMS (ESI): Calcd for C24H24OSSiNa [M+Na]+: 411.1215; found: 411.1209. major minor

[0298] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :50) as colorless oil. 26 mg, 72% yield, E / Z = 80:20. (E)-3-(Benzylthio)-3-[dimethyl(tert-butyl)silyl]-1-phenylprop-2-en-1-one (4b, major isomer)

[0299] 1H NMR (400 MHz, CDCI3): 6 7.76 (d, J = 8.1 Hz, 2H), 7.43-7.32 (m, 8H), 7.18 (s, 1 H), 4.13 (s, 2H), 1.05 (s, 9H), 0.29 (s, 6H).

[0300] 13C NMR (100 MHz, CDCI3): 6 186.9, 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.

[0301] HRMS (ESI): Calcd for C22H29OSSi [M+H]+: 369.1708; found: 369.1701.

[0302] (Z)-3-(Benzylthio)-3-[dimethyl(tert-butyl)silyl]-1-phenylprop-2-en-1-one (4b', minor isomer)

[0303] 1H NMR (400 MHz, CDCI3): 6 7.88 (d, J = 7.3 Hz, 2H), 7.54 (t, J = 7.2 Hz, 1 H), 7.48-7.44 (m, 2H), 7.21-7.17 (m, 5H), 7.07 (s, 1 H), 4.00 (s, 2H), 1.01 (s, 9H), 0.28 (s, 6H).

[0304] 13C NMR (100 MHz, CDCI3): 6 189.8, 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.

[0305] HRMS (ESI): Calcd for C22H29OSSi [M+H]+: 369.1708; found: 369.1703.

[0306] The product was isolated by flash chromatography (diethyl ether / hexanes 1 :50) as colorless oil. 32 mg, 87% yield, E / Z = 78:22.

[0307] (E)-3-(Benzylthio)-1-phenyl-3-(triethylsilyl)prop-2-en-1-one (4c, major isomer)

[0308] 1H NMR (400 MHz, CDCI3): 6 7.89 (d, J = 8.0 Hz, 2H), 7.51 (t, J = 7.2 Hz, 1 H), 7.44-7.31 (m, 7H), 7.20 (s, 1 H), 4.14 (s, 2H), 1.01-0.91 (m, 15H).

[0309] 13C NMR (100 MHz, CDCI3): 5 186.9, 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.

[0310] HRMS (ESI): Calcd for C22H29OSSi [M+H]+: 369.1708; found: 369.1705.

[0311] (Z)-3-(Benzylthio)-1-phenyl-3-(triethylsilyl)prop-2-en-1-one (4c', minor isomer)

[0312] 1H NMR (400 MHz, CDCI3): 6 7.89 (d, J = 7.0 Hz, 2H), 7.52 (t, J = 7.3 Hz, 1 H), 7.47-7.43 (m, 2H), 7.25-7.19 (m, 5H), 7.13 (s, 1 H), 4.10 (s, 2H), 1.05 (t, J= 8.0 Hz, 9H), 0.86 (q, J = 8.0 Hz, 6H).

[0313] 13C NMR (100 MHz, CDCI3): 3 188.9, 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.

[0314] HRMS (ESI): Calcd for C22H29OSSi [M+H]+: 369.1708; found: 369.1716. major minor

[0315] 0.1 mmol scale for 12 hours. The product was isolated by flash chromatography (diethyl ether / hexanes 1 :50) as colorless oil. 32 mg, 78% yield, E / Z = 57:43.

[0316] (E)-3-(Benzylthio)-1-phenyl-3-(triisopropylsilyl)prop-2-en-1-one (4d, major isomer)

[0317] 1H NMR (400 MHz, CDCI3): 6 7.71 (d, J = 7.1 Hz, 2H), 7.49 (t, J = 7.4 Hz, 1 H), 7.44-7.31 (m, 7H), 7.15 (s, 1 H), 4.16 (s, 2H), 1.67 (hept, J = 7.5 Hz, 3H), 1.16 (d, J = 7.5 Hz, 18H).

[0318] 13C NMR (100 MHz, CDCI3): 6 187.2, 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.

[0319] HRMS (ESI): Calcd for C25H35OSSI [M+H]+: 411.2178; found: 411.2177.

[0320] (Z)-3-(Benzylthio)-1-phenyl-3-(triisopropylsilyl)prop-2-en-1-one (4d', minor isomer)

[0321] 1H NMR (400 MHz, CDCI3): 6 7.91 (d, J = 7.1 Hz, 2H), 7.49 (t, J = 7.5 Hz, 1 H), 7.49-7.45 (m, 2H), 7.20-7.16 (m, 5H), 7.07 (s, 1 H), 3.96 (s, 2H), 1.40 (hept, J = 7.5 Hz, 3H), 1.16 (d, J = 7.5 Hz, 18H).

[0322] 13C NMR (100 MHz, CDCI3): 5 189.8, 156.4, 139.0, 136.6, 132.6, 129.8, 128.7, 128.6, 128.5, 127.4, 42.5, 18.8, 11.6. major isomer minor isomer

[0323] 0.1 mmol scale for 5 hours. The product was isolated by flash chromatography (ethyl acetate / hexanes 1 :2) as white solid. 35 mg, 73% yield, E / Z = 67:33.

[0324] (E)-4-[3-(Benzylthio)-3-(triisopropylsilyl)acryloyl]-M,N-dimethylbenzamide (4e, major isomer)

[0325] 1H NMR (400 MHz, CDCI3): 6 7.69 (d, J = 8.1 Hz, 2H), 7.44-7.31 (m, 7H), 7.10 (s, 1 H), 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).

[0326] 13C NMR (100 MHz, CDCI3): 6 186.4, 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.

[0327] HRMS (ESI): Calcd for C28H4oN02SSi [M+H]+: 482.2549; found: 482.2543. (Z)-4-[3-(Benzylthio)-3-(triisopropylsilyl)acryloyl]-N,N-dimethylbenzamide (4e', minor isomer)

[0328] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 3.97 (s, 2H), 3.14 (s, 3H), 2.98 (s, 3H), 1.40 (hept, J = 7.4 Hz, 3.

[0329] 13C NMR (100 MHz, CDCI3): 6 188.7, 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.

[0330] HRMS (ESI): Calcd for C28H4oN02SSi [M+H]+: 482.2549; found: 482.2542. (E)-3-(Benzylthio)-3-{diisopropyl[(4-methoxybenzyl)oxy]silyl}-1-phenylprop-2-en-1-one (4f)

[0331] 0.1 mmol scale for 24 h. To a 4-mL reaction tube, 0.4 ml_ of phosphate buffer solution (pH 7) was added to the mixture of alkynone silyl ether 1e (45.7 mg, 0.12 mmol) and benzyl mercaptan 2a (12 gL, 0.1 mmol). The product 4f was isolated on silica gel by flash chromatography (ethyl acetate / hexanes 1 :40) as yellow oil. 20 mg, 40% yield, E / Z > 95:5.

[0332] 1H NMR (400 MHz, CDCI3): 6 7.79 (d, J = 7.4 Hz, 2H), 7.50 (t, J = 7.6 Hz, 1 H), 7.41-7.37 (m, 2H), 7.30-7.28 (m, 4H), 7.21-7.18 (m, 4H), 6.87 (d, J = 8.6 Hz, 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).

[0333] 13C NMR (100 MHz, CDCI3): 6 188.8, 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.

[0334] HRMS (ESI): Calcd for C30H36O3SSiNa [M+Na]+: 527.2052; found: 527.2042.

[0335] 3,3-Bis(benzylthio)-3-{diisopropyl[(4-methoxybenzyl)oxy]silyl}-1-phenylpropan-1-one (4f)

[0336] 0.1 mmol scale for 24 h. To a 4-mL reaction tube, 0.4 mL of phosphate buffer solution (pH 7) was added to the mixture of alkynone silyl ether 1e (38.1 mg, 0.1 mmol) and benzyl mercaptan 2a (25 μL , 0.21 mmol). The product 4f' was isolated on silica gel by flash chromatography (ethyl acetate / hexanes 1 :40) as colorless oil. 26 mg, 41% yield.

[0337] 1H NMR (400 MHz, CDCI3): 5 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, 1 H), 4.54 (s, 2H), 3.90 (d, J = 13.1 Hz, 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).

[0338] 13C NMR (100 MHz, CDCI3): 3 158.8, 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.

[0339] HRMS (ESI): Calcd for C37H44O3S2SiNa [M+Na]+: 651.2399; found: 651.2396.

[0340] X-Ray Crystallography Analysis

[0341] Compound 3p and 3t were crystallized via slow evaporation of a solution in 0.5 ml_ of ethyl acetate and 2.5 mL of hexanes at room temperature.

[0342] Deposition number CCDC 2120673

[0343] ORTEP of 3p, thermal ellipsoids are shown at 50% probability (FIG. 19). Table 3 lists the X- ray data for deposition number CCDC 2120673.

[0344] Bond precisions C-C = 0.00 / 5 A Wavelength=O ■ 71073

[0345] Data completeness- 0. 996 Theta(max) = 27.500

[0346] R ( ref lections }= 0 , Q 871 ( 3262 ) wR2 ( reflections )= 9. 16?5 ( 4642 )

[0347] S = 1. 190 Spar= 240

[0348] Table 3

[0349] Deposition number CCDC 2120671 ORTEP of 3t, thermal ellipsoids are shown at 50% probability (FIG. 20). Table 4 lists X-ray data in respect of deposition number CCDC 2120671.

[0350] Table 4

[0351] Results and Discussions

[0352] Both aliphatic chains and benzyl rings on thiols were well tolerated, delivering adducts 3d-g in good efficiency. Notably, the reaction of 2-mercaptoethanol and 1a was selective, leaving the hydroxy group intact. Aryl thiols with different substituents on para-, meta- and ortho- positions of phenyl rings, afforded the corresponding products 3h-l with a single E-isomer in near quantitative conversion. This transformation was also applicable to thiols bearing 2- naphthylene and heteroarenes such as 4-pyridine and 2-thiophene (3m-o).

[0353] Reactions of both electron-rich and -deficient aromatic TMS-alkynones proceeded smoothly in pH neutral phosphate buffer at 37 °C (3p-t). Functionalities such as esters and indoles were compatible under the optimized conditions. The structure of 3p and 3t were also confirmed by X-ray crystallography analysis. Aliphatic alkynones were potent electrophiles, delivering thiol adducts in good E / Z ratio (3u-y). We found that β-silyl alkynone equipped with an unconjugated alkene moiety afforded the desired product 3u in high yield. Similarly, the reaction of (S)-citronellol-derived β-silyl alkynone underwent the thiol addition successfully with the full retention of the monoterpenoid structure. Unfortunately, substrates with conjugated alkenes led to complex mixtures of products due to the undistinguished reactivity between alkene and alkyne.

[0354] Next, we evaluated the effect of silyl substituents on the alkynone substrates (FIG. 7). Compared to methyl counterpart, the presence of phenyl and tert-butyl groups on the silicon center had almost no influence on the reactivity, despite the attenuated E / Z ratio (80:20) in the latter case. A comparable result was obtained when trimethyl-silane was replaced by triethylsilane. We noticed that the reaction of tri(isopropyl)-silane (TIPS) required prolonged time (18 h) for complete conversion due to the steric hindrance. Notably, an amide functionality on the aromatic ring of TIPS-alkynone was tolerated.

[0355] In addition to the above-mentioned trialkyl-silanes, we also explored the feasibility of the alkynyl silyl ether substrate (FIG. 8). The corresponding products, vinyl siloxanes, are labile in acidic media, which make them challenging for synthesis. However, this feature allows it to act as an immolative linker as the free OH-containing drug or biomolecule installed on the silyl ether handle can be readily released by the modulation of 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 the excess of benzyl mercaptan. Unexpectedly, the double-thiol addition was detected as the predominant pathway, which was marginal in the cases of trialkyl-silanes. When 1e was added in a slight excess (1.2 equiv), the desired vinyl siloxane 4f was obtained in modest yield (40%), along with the formation of desilylated side-product (4f').

[0356] Example 5. Reagent Chemoselectivity and Product Stability Experiments

[0357] To further demonstrate the potential of this methodology in bioconjugation, we paid attention to the reagent chemoselectivity and product stability.

[0358] General Procedure of Chemoselectivity Experiments

[0359] To a 4-mL reaction tube, 0.4 ml_ of phosphate buffer (pH 7) was added to the crude mixture of 1a (20.2 mg, 0.1 mmol) and benzyl mercaptan (14 μL, 0.12 mmol). Afterwards, the indicated amino acid derivative (0.1 mmol) was added to the stirring mixture. After stirring for 3 hours at 37 °C, the emulsion was extracted with ethyl acetate three times. The combined organic layers were then evaporated under the reduced pressure. Dibromomethane (7 μL, 0.1 mmol) was then added as the internal standard and dissolved in CDCI3. Subsequently, a1H-NMR (acquisition time = 25) was collected to evaluate the chemoselectivity of 1a in the presence of other nucleophiles.

[0360] Stability Experiment of Thiol Conjugate in the Presence of Endogenous Molecules

[0361] To a 4-mL reaction tube, the endogenous molecule (1 equiv) was added to 3a (6.5 mg, 0.02 mmol) in phosphate buffer solution (0.4 ml_). After stirring for 24 hours at 37 °C, the emulsion was extracted with ethyl acetate three times. The combined organic layers were then evaporated under the reduced pressure. Afterwards, dibromomethane (7 μL, 0.1 mmol) was added as the internal standard and dissolved in CDCI3. Subsequently, a1H-NMR (acquisition time = 25) was collected to evaluate the stability of the conjugate product in the presence of additives.

[0362] Stability Experiments of Thiol Conjugate under the Acidic, pH Neutral, Basic and Oxidative (10 mM H2O2) Conditions

[0363] To a 4-mL reaction tube, 3a (6.5 mg, 0.02 mmol) or 1a (4.0 mg, 0.02 mmol) was dissolved in 0.4 mL of buffer solutions in different pH and hydrogen peroxide solution. After stirring for 24 hours at 37 °C, the emulsion was extracted with ethyl acetate three times. The combined organic layers were then evaporated under the reduced pressure. Afterwards, dibromomethane (7 μL, 0.1 mmol) was added as the internal standard and dissolved in CDCI3. Subsequently, a1H-NMR (acquisition time = 25) was collected to evaluate the stability of 3a and 1a in different aqueous media.

[0364] Stability Experiment under Bovine Serum Condition

[0365] To a 4-mL reaction tube, 1 mL of fetal bovine serum (Bidepharm, DR-Class, BD01839326) was added to 3a (6.5 mg, 0.02 mmol). After stirring at rt for 24, 48 and 72 hours, the emulsion was transferred to 10-mL Eppendorf tube, added with MeCN (3 mL) and centrifuged for 2 min at 10,000 rpm. The supernatant was concentrated under the reduced pressure. Several 1 H- NMR were performed to measure the stability of 3a in serum using CH2Br2(7 [i L, 0.1 mmol) as the internal standard. Experiments were repeated for two runs.

[0366] Results and Discussions First, the stoichiometric benzylamine or amino acid derivatives were added in the reaction of benzyl mercaptan to examine the chemoselectivity of 1a. In all cases, the presence of other nucleophiles did not interfere the exclusive formation of the thiol conjugate, confirming the thiol-specific reactivity of this silicon-containing alkynone reagent (FIG. 9).

[0367] Next, we proceeded to study the stability of the purified thiol conjugate in the excess of amine or thiol nucleophiles. Unlike the results of terminal alkynones in previous reports28, we noticed that the thiol conjugate 3a was almost fully recovered in pH neutral buffer without decomposition or side reactions on the alkene moiety, which demonstrated the potential of β- trimethylsilyl alkynone 1a as a non-cleavable thiol linker (FIG. 10). When 3a was incubated with glutathione and some nucleophilic amino acids, we were delighted to observe that no retro process or side reaction took place over 24 h. Moreover, negligible degradation of thiol conjugate 3a was found in the presence of pH 7 buffer after stirring at 37 °C for one week. Comparably, both vinyl sulfide and silicon components in 3a were stable under the acidic (pH 4) and basic (pH 10) conditions. Nonetheless, there was noticeable decomposition of 3a when stirred with dopamine hydrochloride in pH 8 buffer for 1 day.

[0368] We also examined the stability of TMS-alkynone 1a (FIG. 11). Although this reagent exhibited excellent stability under acidic buffer and oxidative conditions, it decomposed completely in pH 7 and pH 10 buffers when incubated in the absence of nucleophilic reagents. At this point, it was not clear whether the silicon handles still remained intact when β-silyl alkynones reacted with the thiol-containing biomolecules. The possibility that C-Si bond in alkynone reagents decompose prior to the thiol-addition cannot be underestimated.

[0369] Thiol conjugate 3a displayed robust stability in bovine serum, as less than 20% deconjugation was detected over a period of 3 days (FIG. 12).

[0370] Example 6. Functionalisation of Thiol-containing Biomolecules and Drugs

[0371] Therefore, the labile TMS-alkynone was chosen to assess the feasibility of the reaction of biomolecules and drugs bearing free thiols (FIG. 13) by following the protocol disclosed in either in this Example or in Example 4.

[0372] Methyl A / 2-(tert-butoxycarbonyl)-W5-((R)-1-((2-methoxy-2-oxoethyl)amino)-1-oxo-3- (((E)-3-oxo-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl)thio)propan-2-yl)-L-glutaminate (4g)

[0373] 0.08 mmol scale in the mixed solvents (0.4 mL, pH 7 phosphate buffer / MeCN = 5:1) for 12 hours. The starting material, M-Boc-glutathione-OMe was synthesized based on the reported procedure (Bioorg. Med. Chem. 15, 1062-1066 (2007); Chem. Euro. J. 22, 11365-11370 (2016)). The product was isolated by flash chromatography (ethyl acetate / hexanes 2:1) as colorless oil. 44 mg, 86% yield, E / Z > 95:5.

[0374] [a]24D= -17.6° (c = 1.0, CHCI3).

[0375] 1H NMR (400 MHz, CDCI3): 6 8.03 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 6.8 Hz, 1 H), 7.47-7.43 (m, 2H), 7.35 (s, 1 H), 7.14 (s, 2H), 5.40 (d, J = 7.6 Hz, 1H), 4.77 (d, J = Q.2 Hz, 1 H), 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).

[0376] 13C NMR (100 MHz, CDCI3): 6 186.6, 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.

[0377] HRMS (ESI): Calcd for C29H43N3O9SSiNa [M+Na]+: 660.2387; found: 660.2388.

[0378] Methyl (£)-W-acetyl-S-(3-oxo-3-phenyl-1-(trimethylsilyl)prop-1-en-1-yl)-L-cysteinate (4h) 0.1 mmol scale in the mixed solvents (0.4 mL, pH 7 phosphate buffer / MeCN = 5:1) for 12 hours. The product was isolated by flash chromatography (ethyl acetate / hexanes 1 :1) as colorless oil. 31 mg, 82% yield, E / Z > 95:5.

[0379] [a]24D= -7.0° (c = 1.0, CHCI3).

[0380] 1H NMR (400 MHz, CDCIs): 6 7.99-7.97 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.49-7.45 (m, 2H), 7.23 (s, 1 H), 6.31 (d, J = 7.2 Hz, 1 H), 5.03-4.98 (m, 1 H), 3.78 (s, 3H), 3.47 (dd, J = 13.2, 5.2 Hz, 1 H), 3.31 (dd, J = 13.2, 5.2 Hz, 1 H), 2.02 (s, 3H), 0.31 (s, 9H).

[0381] 13C NMR (100 MHz, CDCI3): 3 186.6, 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.

[0382] HRMS (ESI): Calcd for Cis^sNC^SSiNa [M+Na]+: 402.1171; found: 402.1163.

[0383] (E)-1-{[3-Oxo-3-phenyl-1-(trirnethylsilyl)prop-1-en-1-yl]thio}-β-D-glucose tetraacetate (4i)

[0384] 0.1 mmol scale in the mixed solvents (0.4 ml_, pH 7 phosphate buffer / MeCN = 5:1) for 3 hours. The product was isolated by flash chromatography (ethyl acetate / hexanes 1:5) as white solid. 50 mg, 88% yield, E / Z = 83:17.

[0385] [a]22D = -71 .1 ° (c = 1.0, CHCb).

[0386] 1H NMR (400 MHz, CDCI3): 6 7.94 (d, J = 7.1 Hz, 2H), 7.56 (t, J = 7.3 Hz, 1 H), 7.50-7.46 (m, 2H), 7.42 (s, 1 H), 5.34-5.29 (m, 1H), 5.24-5.19 (m, 1 H), 5.14-5.10 (m, 1 H), 5.00 (d, J = 10.1 Hz, 1 H), 4.24 (dd, J = 12.4, 6.0 Hz, 1H), 4.16 (dd, J = 12.4, 2.3 Hz, 1 H), 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).

[0387] 13C NMR (100 MHz, CDCI3): 6 187.2, 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.

[0388] HRMS (ESI): Calcd for C26H34OioSSiNa [M+Na]+: 589.1540; found: 589.1547.

[0389] (S)-2-Methyl-3-{[(E)-3-oxo-3-phenyl-1-(trimethylsilyl)prop-1-en-1-dyl]thio} propanoyl-L- proline (4j)

[0390] 0.1 mmol scale for 12 hours. To a 4-mL reaction tube, sodium bicarbonate (16.8 mg, 0.2 mmol) was added to the 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_ of phosphate buffer solution (pH 7). The reaction mixture was acidified with glacial acetic acid and then extracted with ethyl acetate. The combined organic phase was evaporated under reduced pressure. The product was isolated by flash chromatography (ethyl acetate / hexanes / AcOH 66:33:1) as yellow oil. 28 mg, 67% yield, E / Z = 83:17. The two isomers cannot be separated on silica.

[0391] 1H NMR of a mixture of E and Z isomers (400 MHz, CDCI3): 5 7.94 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 6.8 Hz, 1 H), 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, 1 H), 3.05-2.80 (m, 2H), 2.37-2.33 (m, 1 H), 2.08-1.98 (m, 3H), 1.35-1.25 (m, 4H), 0.39 (s, 1.5H), 0.31 (s, 7.5H).13C NMR of a mixture of E and Z isomers (100 MHz, CDCI3): 3 188.6, 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. HRMS (ESI): Calcd for C2iH29NO4SSiNa [M+Na]+: 442.1484 found: 442.1480.

[0392] (14S, 16S,33S,2R,4R, 10E, 12Z, 14R)-86-Chloro-14-hydroxy-85,14-dimethoxy-33,2,7, 10- tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1 ,3)- benzenacyclotetradecaphane-10,12-dien-4-yl N-methyl-N-(3-(((E)-3-oxo-3-phenyl-1- (trimethylsilyl)prop-1-en-1-yl)thio)propanoyl)-L-alaninate (4k)

[0393] 0.05 mmol scale for 48 hours. To a 4-mL reaction tube, 1a (20.2 mg, 0.1 mmol) was added to a stirred suspension of Mertansine (36.9 mg, 0.05 mmol) in 0.4 ml_ of phosphate buffer solution (pH 7). The product was isolated by flash chromatography (ethyl acetate / hexanes 5:1) as white solid (mp: 166-167 °C). 22 mg, 46% yield, E / Z = 67:33. The two isomers cannot be separated on silica.

[0394] [O]22D = -75.7° (c = 1.0, CHCI3).

[0395] 1H NMR of a mixture of E and Z isomers (400 MHz, CDCI3): 6 7.91 (d, J = 7.4 Hz, 1.6H), 7.87 (d, J= 7.3 Hz, 0.4H), 7.56-7.50 (m, 1 H), 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, 1 H), 6.47-6.39 (m, 1 H), 6.22 (s, 1 H), 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, 1 H), 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, 1 H), 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 = 11.8 Hz, 1 H), 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).

[0396] 13C NMR of a mixture of E and Z isomers (100 MHz, CDCI3): 6 186.5, 170.7, 170.5, 168.9,

[0397] 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,

[0398] 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.

[0399] HRMS (ESI): Calcd for C47H62CIN30iiSSiNa [M+Na]+: 962.3461 found: 962.3466.

[0400] Results and Discussions A / -Protected cysteine methyl ester was first incubated with 1a for 12 h under the physiological conditions. The functionalization of this cysteine derivative was successful, producing the adduct 4h with the full retention of silicon handles. Acetonitrile (20% v / v relative to phosphate buffer) was added to ensure the good stirring during the reaction. Encouraged by this promising outcome, we further extended the methodology to the modification of glutathione, which contains a cysteine side chain. This tripeptide was smoothly converted to the monofunctionalized product 4g in a single isomer. The reaction of 1-thio-D-glucose acetates also worked well, demonstrating the latent application of this method as a facile entry point in the polysaccharide diversification. Moreover, the antihypertensive drug, Captopril, could undergo the thiol addition to TMS- alkynone in the presence of a free carboxylic acid, giving 4j in good yield. Incubation of Mertansine, a microtubule-targeted cytotoxin, with 2 equivalents of β-silyl alkynone 1a in the pH 7 phosphate buffer generated the monofunctionalized product 4k in moderate yield (46%).

[0401] Example 7. Derivatisation of the Thiol Conjugate

[0402] Subsequently, we demonstrated the synthetic versatility of the thiol conjugates (FIG. 14).

[0403] Selective 1,2-Reduction of Thiol Conjugate 3a

[0404] A 10-mL round bottom flask under argon in ice bath was charged with 3a (32.6 mg, 0.1 mmol) and Et2O (1 mL), followed by addition of LiAIH4(2.0 M in THF, 30 LIL, 0.06 mmol) at 0 °C. The temperature was warmed to room temperature gradually and stirred for 1 h. Saturated Na2SO4solution was added carefully at 0 °C to quench the reaction. After filtration with Et2O, the filtrate was concentrated on a rotary evaporator and then dried using the vacuum pump. Pure product was obtained as colourless oil without column chromatography. 26 mg, 80% yield.

[0405] (E)-3-(Benzylthio)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-ol (5a)

[0406] 1H NMR (400 MHz, CDCI3): 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, 1 H), 3.93 (d, J = 12.3 Hz, 1 H), 3.89 (d, J = 12.3 Hz, 1 H), 0.36 (s, 9H).

[0407] 13C NMR (100 MHz, CDCI3): 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.

[0408] HRMS (ESI): Calcd for C19H24OSSiNa [M+Na]+: 351.1215; found: 351.1212. Brook Arrangement to Afford Silyl Migration Product 5b

[0409] The 1 ,2-reduction of 3a (0.1 mmol) followed the above-mentioned procedure and the reduction intermediate 5a was directly used in the following step. To a solution of 5a in THF (0.6 mL) was added LiOt-Bu (100 μL, 1 M in THF) slowly at 0 °C under argon. After stirring for 12 h at room temperature, the mixture was quenched by water (5 mL) and extracted with Et20. The combined extracts were then dried over Na2SCO4filtered and concentrated under reduced pressure. The residue was isolated by flash chromatography (diethyl ether / hexanes 1 :50) as colorless oil. 20 mg, 61% yield.

[0410] (E)-{[3-(Benzylthio)-1-phenylallyl]oxy}trimethylsilane (5b)

[0411] 1H NMR (400 MHz, CDCI3): 6 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, 1 H), 5.17 (d, J = 6.3 Hz, 1 H), 3.90 (s, 2H), 0.09 (s, 9H).

[0412] 13C NMR (100 MHz, CDCI3): 6 143.4, 137.4, 131.8, 129.0, 128.7, 128.4, 127.3, 126.3, 124.3, 75.4, 37.1 , 0.3.

[0413] HRMS (ESI): Calcd for Ci9H25OSSI [M+H]+: 329.1395; found: 329.1403.

[0414] Desilylation of Thiol Conjugate 3a

[0415] To a solution of 3a (32.6 mg, 0.1 mmol) in DMSO (0.6 mL) were added CsF (16.7 mg, 0.11 mmol) and a drop of water. The mixture was stirred at 40 °C until 3a had been fully converted (by observing the absence of 3a when subjecting the mixture sample on a thin-layer chromatography (TLC) plate). The reaction mixture was quenched with water and extracted with Et20. The combined extracts were then dried over Na2SCO,4filtered and concentrated under reduced pressure. The E / Z ratio was determined to be 56:44 based on crude1H NMR. The residue was isolated by flash chromatography (ethyl acetate / hexanes 1 :20) as colorless oil. 23 mg, 90% yield.

[0416] (E)-3-(Benzylthio)-1-phenylprop-2-en-1-one [74896-61-0] (5c, major isomer)1H NMR (400 MHz, CDCI3): 3 7.93 (d, J = 14.8 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 7.2 Hz, 1 H), 7.46-7.29 (m, 7H), 6.94 (d, J = 14.8 Hz, 1 H), 4.14 (s, 2H).

[0417] 13C NMR (100 MHz, CDCI3): 3 187.2, 148.2, 138.2, 135.8, 132.6, 129.1, 128.9, 128.7, 128.5, 128.0, 119.3, 37.4.

[0418] HRMS (ESI): Calcd for CIBHISOS [M+H]+: 255.0844; found: 255.0835.

[0419] (Z)-3-(Benzylthio)-1-phenylprop-2-en-1-one [70061-48-2] (5c', minor isomer)

[0420] 1H NMR (400 MHz, CDCI3): 3 7.93 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz, 1 H), 7.46-7.42 (m, 2H), 7.39-7.28 (m, 6H), 7.05 (d, J = 9.8 Hz, 1 H), 4.01 (s, 2H).

[0421] 13C NMR (100 MHz, CDCI3): 3 189.3, 151.6, 138.1, 137.4, 132.5, 129.2, 129.0, 128.7, 128.1, 127.6, 117.1 , 40.7.

[0422] HRMS (ESI): Calcd for C16H15OS [M+H]+: 255.0844; found: 255.0832. 5d

[0423] To a mixture of 5a (97.8 mg, 0.3 mmol) and sodium bicarbonate (63 mg, 0.75 mmol) in DCM (3 ml_) was added m-CPBA (77 wt%, 168 mg, 0.75 mmol) at 0 °C. The reaction was stirred for 1 h at room temperature, before it was 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 isolated by flash chromatography (ethyl acetate / hexanes 1 :20) as white solid. 105 mg, 98% yield.

[0424] (E)-3-(Benzylsulfonyl)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (5d)

[0425] 1H NMR (400 MHz, CDCI3): 3 7.73 (s, 1H), 7.59-7.56 (m, 1 H), 7.46-7.38 (m, 9H), 4.33 (s, 2H), 0.34 (s, 9H).

[0426] 13C NMR (100 MHz, CDCI3): 5 192.4, 153.5, 152.1 , 135.5, 134.5, 131.2, 129.2, 129.03, 128.98, 128.9, 128.8, 61.5, 0.4.

[0427] HRMS (ESI): Calcd for Ci9H23O3SSi [M+H]+: 359.1137; found: 359.1130. de 5e

[0428] 3a 5e To a mixture of 5a (32.6 mg, 0.1 mmol) and sodium bicarbonate (8.4 mg, 0.1 mmol) in DCM (1 mL) was added m-CPBA (77 wt%, 27 mg, 0.12 mmol) at 0 °C. The reaction was stirred for 30 minutes at room temperature, before it was 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 isolated by flash chromatography (ethyl acetate / hexanes 1 :8) as colorless oil. 31 mg, 91% yield.

[0429] (E)-3-(Benzylsulfinyl)-1-phenyl-3-(trimethylsilyl)prop-2-en-1-one (5e)

[0430] 1H NMR (400 MHz, CDCI3): 6 7.80 (s, 1H), 7.78 (d, J = 7.4 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1 H), 7.44-7.34 (m, 5H), 7.25-7.22 (m, 2H), 4.31 (d, J= 13.1 Hz, 1 H), 3.66 (d, J = 13.1 Hz, 1H), 0.39 (s, 9H).

[0431] 13C NMR (100 MHz, CDCI3): 6 189.6, 164.6, 137.6, 137.1 , 133.7, 131.0, 129.1 , 129.0, 128.8, 128.65, 128.57, 59.4, -0.5.

[0432] HRMS (ESI): Calcd for CieH^CkSSi [M+H]+: 343.1188; found: 343.1183.

[0433] Nazarov Cyclisation of Thiol Conjugate 3a

[0434] Under nitrogen, to a solution of 5a (32.6 mg, 0.1 mmol) in 0.5 mL of dry DCM, BCI3(1.0 M solution in DCM, 120 μL, μμ 0LL.12 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature overnight, before pinacol (17.7 mg, 0.15 mmol) and Et3N (28 μL, 0.2 mmol) were added to quench the reaction. Saturated aqueous Na2CO3was then added. The aqueous layer was extracted with ether and the combined extracts were dried over Na2SC>4. The solvent was removed under reduced pressure and the residue was subjected to flash chromatography (ethyl acetate / hexanes 1 :20) as brown oil. 25 mg, 77% yield.

[0435] 3-(Benzylthio)-3-(trimethylsilyl)-2,3-dihydro-1H-inden-1-one (5f)

[0436] 1H NMR (400 MHz, CDCI3): 7 δ.73 (d, J = 7.7 Hz, 1 H), 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, 1 H), 3.27 (d, J = 12.0 Hz, 1 H), 3.02 (d, J = 20.0 Hz, 1 H), 2.86 (d, J = 20.0 Hz, 1 H), 0.04 (s, 9H).

[0437] 13C NMR (100 MHz, CDCI3): 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.

[0438] HRMS (ESI): Calcd for C19H23OSSi [M+H]+: 327.1239; found: 327.1234. Results and Discussions

[0439] Selective 1,2-reduction of 3a using 0.6 equiv of UAIH4 afforded the allylic alcohol 5a exclusively. Treatment of 3a with LiAH4followed by the addition of LiOt-Bu provided the silyl migration product 5b via Brook rearrangement. In the presence of CsF, C-Si bond of 3a was cleaved to give the desilylated products 5c and 5c'. Additionally, vinyl sulfide moiety in 3a readily underwent the quantitative oxidation, affording vinyl sulfone 5d and vinyl sulfoxide 5e respectively by alteration of m-CPBA’s stoichiometry. Notably, when 3a was treated with Lewis acid such as BCI3, 2,3-dihydroindenone 5f was produced in satisfactory yield via Nazarov cyclization, in which both the TMS group and thioether component remained intact.

[0440] Example 8. Protein Modification

[0441] We also attempted the modification of bovine serum albumin and human serum albumin, each of which contains one free cysteine residue.

[0442] Electrospray Ionization Mass Spectrometry (ESI-MS) Measurement

[0443] ESI-MS was performed on a linear quadrupole ion trap detector mass spectrometer (LTQ XL from ThermoFisher Scientific) coupled to Vanquish UHPLC (from ThermoFisher Scientific). Data were processed using Thermo BioPharma Finder 3.1.

[0444] General Procedure for Reactions of BSA with 1f unmodified BSA BSA-1f

[0445] To a 4-mL reaction tube, 1f (20 μL, 30 mM, 2 equiv.) in acetonitrile was added to 0.5 mM of bovine serum albumin (19.9 mg, 0.3 pmol) in phosphate buffer (pH 8, 0.6 mL). After vigorously stirring for 2 hours at 37 °C, a 50 μL aliquot of the reaction mixture was evaluated by performing ESI-MS and the mono-conjugated adduct containing silicon moiety was detected.

[0446] General Procedure for Reactions ofHSA with 1a To a 4-mL reaction tube, 1a (20 μL, 30 mM, 2 equiv.) in acetonitrile was added to 0.5 mM of human serum albumin (19.9 mg, 0.3 pmol) in phosphate buffer (0.6 mL). After vigorously stirring for 10 hours at 37 °C, a 50 μL aliquot of the reaction mixture was evaluated by performing ESI-MS and only desilylated mono-conjugated adduct was detected.

[0447] Results and DiscussionsTES-alkvnone 1f successfully added to the bovine serum albumin in phosphate buffer, providing the singly modified conjugate with the retention of the silicon moiety (FIG. 15 and FIG. 16).

[0448] The labelling of human serum albumin was achieved by TMS-alkynone 1a, affording the thiolselective product in pH 6 and 7 buffers, albeit with the decomposition of silicon component (FIG. 17 and FIG. 18).

Claims

Claims1. A method of producing a compound of formula I:the method comprising reacting a compound of formula II:with a compound of formula III:HSR5III in a solvent comprising an aqueous phosphate buffer that has a pH of from 5 to 9, wherein:R1represents C1to C10alkyl, C2to C10alkenyl, C2to C10alkynyl, a C6to C12aromatic group, a 5-10 membered heterocyclic group, and a C3to C12carbocyclic ring system, where each of the groups is unsubstituted or substituted by one or more groups selected from halo, C1to C3alkyl, CONR6aR6b, CO2R6C, OR8d, a C6to C12aromatic group, a 5-10 membered heterocyclic group, and a C3to C12carbocyclic ring system, where the C1to C3alkyl, the C6to C12aromatic group, the 5-10 membered heterocyclic group and the C3to C12carbocyclic ring system are unsubstituted or substituted by one or more halo atoms;each R2to R4independently represents C1to C10alkyl or O(C1to C10alkyl), where each group is independently unsubstituted or substituted by one or more groups selected from C1to C3alkyl, phenyl, and a 5-10 membered heterocyclic group, where said groups are unsubstituted or substituted by one or more of halo and OR7;R5represents an organic moiety;RBato R8drepresents C1to C10alkyl that is unsubstituted or substituted by one or more halo atoms; andR7represents a C1to C10alkyl.

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

3. The method according to Claim 1 or Claim 2, wherein R1represents C1to C10alkyl, C2to C10alkenyl, a C2to C10aromatic group, a 5-10 membered heterocyclic group, and a C5to C6carbocyclic ring system, where each of the groups is unsubstituted or substituted by one or more groups selected from halo, C1to C3alkyl, CONR6aR6b, CO2R6c, ORM, a C6to C10aromatic group, a 5-10 membered heterocyclic group, and a C5to C5carbocyclic ring system, where the C1to C3alkyl, the C6to C10aromatic group, the 5-10 membered heterocyclic group and the C5to C6carbocyclic ring system are unsubstituted or substituted by one or more halo atoms.where the wavy line represents the point of attachment to the rest of the molecule.

5. The method according to any one of the preceding claims, wherein each of R2to R4is independently selected from one of the following:Me, Et,iPr,tBu,tP and, dtBu , where the wavy line represents the point of attachment to the rest of the molecule.

6. The method according to any one of the preceding claims, wherein R5is selected from 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, an amino acid containing a thiol substituent, an oligopeptide containing a thiol substituent, a peptide containing a thiol substituent, a mono-, di- or tri-saccharide containing a thiol substituent, or a drug molecule containing a thiol substituent, optionally wherein the compound of formula III is selected from N-Boc-glutathione- OMe, 1-thiol-D-glucose acetate, captopril, and mertansine.

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

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