Photoredox protein modification

Abstract

The present invention relates to the photoredox-mediated functionalization of proteins with chemical groups via radical-generating C—C bond formation by using certain boronate and sulfone precursor compounds. The invention also relates to functionalized proteins that can be produced by this method, and to the certain boronate and sulfone precursor compounds themselves.

Description

[Technical Field]

[0001] Technical field This invention relates to the photoredox-mediated functionalization of proteins at chemical groups via radical generation and CC bond formation using specific boronate and sulfone precursor compounds. The invention also relates to the functionalized proteins that can be produced by this method, and to the specific boronate and sulfone precursor compounds themselves. [Background technology]

[0002] Background of the Invention Post-translational modifications (PTMs) greatly expand the structure and function of proteins in nature. In parallel, the emergence of synthetic protein functionalization strategies now allows for not only direct mimicry but also the creation of non-natural protein variants with diverse potential functions, ranging from drug delivery and tracking to imaging and cross-linking with partners. However, the range of functional groups that can be introduced through these modifications remains limited, especially in the case of reactive functional groups.

[0003] Methods utilizing the cellular translation mechanism offer several advantages in introducing selective modifications to proteins, but their scope and efficiency are limited. The resulting non-natural amino acid precursors may be degraded or unacceptable during biosynthesis, particularly in the case of amino acids with reactive side chains. Post-translational functionalization offers an alternative strategy that, when used in later stages, can potentially broaden its scope. In principle, its scope is limited only by the compatibility of the protein substrate, its environment, and the reaction conditions used.

[0004] As one version of post-translational functionalization, readily generated dehydroalanine (Dha) residues are used in proteins as single-occupied molecular orbital (SOMO) acceptors ("radical acceptors" or "SOMO-philes") that are highly reactive to several carbon radical species, thereby selectively generating β,γ-CC bonds and introducing new side chains in a "scarless / traceless" manner. However, the side chain / carbon radical precursors and the reagents that generate them (e.g., metals or BH4) - The incompatibility of single-electron transfer (SET) from 1 / 2 is currently limiting the scope of such techniques. Nevertheless, such isotropic 1e-chemistry has potential advantages over typical anisotropic 2e-reagents. Essential problems in biomolecular modification include: affinity for water; the need for "benign" properties; and low (or non-reactive) reactivity to numerous bioacids, amines, alcohols, and thiols (rapid 2e-reactants) present in many biological environments. On the other hand, water and natural proteins are low in reactivity to most carbon radicals. Therefore, by appropriately positioning SOMOphiles such as Dha, more general chemoselectivity and site selectivity can be enabled in certain 1e-chemical reactions.

[0005] Other methods exist for SET (and therefore, either oxidative or reductive carbon radical initiation). Catalytic protein methods offer clear advantages over conventional hyperstoichiometric methods that can lead to unwanted side reactions. Furthermore, when controlled with relatively benign and potentially tissue-penetrating triggers such as light, they allow for additional layers of control, such as temporal, spatial, and even kinetic control, to complement those with 1e-chemoselectivity. Photostimulated outer shell electron transfer (ET) has seen a resurgence in applications to small molecules. However, its use in site-selective biomolecular modification is more limited. Typical examples include peptides that sometimes require mixed organic solvents and / or are often limited to ET systems located at both ends of a redox "window," and it has been noted that side reactions occur. Moreover, reliance on specific precursor moieties that cannot be rearranged / prepositioned, such as α-C-carboxyl or β-CH, can lead to low site selectivity due to the limited and / or abundance of reaction sites. Thus, these methods have not yet reached their full potential in protein chemistry.

[0006] There is a need for a method of protein functionalization involving post-translational modification that is selective, reliable, and carries out with a moderate possibility of benign redox, while enabling the addition of reactive functional side chains. [Overview of the project]

[0007] Summary of the Invention The inventors unexpectedly discovered that by using specific radical precursors, such as boronic acid catechol ester derivatives and aryl sulfonyl fluorine derivatives, in the presence of a photocatalyst, radical-driven CC bond formation between functional side chains on proteins or peptides and SOMO acceptor residues becomes possible. This alteration of the CC side chain within an unaltered protein enables native and chemical post-translational modifications of proteins and peptides.

[0008] The method discovered by the inventors enables the generation of side-chain carbon radical precursors by photo-driven electron transfer, which allows for the formation of CC bonds without the need for harsh reaction conditions or organic solvents. Furthermore, by controlling the reaction redox, damage to proteins and peptides is minimized, enabling site-selective modification with good conversion rates. Specifically, the inventors discovered that in situ generation of easily oxidizable boronic acid catechol ester (BACED) derivatives generates RH2C· radicals and PTMs that can form native (βCH2-γCH2) bonds of intrinsic residues, while in situ enhancement of aryl sulfone fluorine derivatives and certain bromine fluorine derivatives with iron(II) generates RFXC· radicals (RF2C·, etc.), which can form equivalent (βCH2-γCXF) bonds with H→F labeling.

[0009] These reaction methods of the present invention can be carried out rapidly and with small amounts of reagents. Furthermore, these reactions are chemically tolerant, enabling the incorporation of an unprecedented range of functional groups into diverse protein scaffolds and sites. Chemoselective initiation can be applied in the presence of sensitive groups in the 13C radical precursor, making it possible to introduce previously incompatible side chains. This provides access to novel functions and reactivity of proteins. The novel methods described herein and the proteins / peptides produced thereby may find applications in many areas, such as (a) introduction of radical precursors for radical generation on isotropic proteins; (b) studying enzyme function using natural, unnatural, and "zero-size" labeled post-translational modified protein substrates by simultaneously sensing both chemoselectivity and stereoselectivity; and (c) generating access to general "alkylating agent proteins" with a spectrum of isotropic covalent bond-forming activity (reacting diversely with small molecules on the one hand, and selectively with protein targets on the other due to excellent mimicry). Thus, the novel reactions on the resulting proteins and post-translational access to chemical groups are useful in elucidating and creating protein functions.

[0010] Thus, the inventors demonstrated that the following triple combination: electron transfer at a benign and mild redox potential (i) using a low, quasi-stoichiometric amount of a “redox-compatible” side-chain functionalized C· radical precursor (ii) triggered by light of an appropriate luminous flux (iii) allows for the generation and utilization of off-protein and on-protein radicals, enabling protein modification via CC bond formation (see Figure 1). The resulting chemistry enables the introduction of unprecedented side chains with novel functional modes.

[0011] In a first embodiment, the present invention provides a method for functionalizing a protein or peptide with a functional side chain portion, wherein the protein or peptide comprises at least one single-occupied molecular orbital (SOMO) acceptor residue, the SOMO acceptor being a residue comprising a side chain containing an alkene group; the method is: (a) The oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in the radical precursor compound and in the photoactivated state. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide; The radical precursor compound is given by the following formula (II) or (III):

[0012] [ka]

[0013] (In the formula, R is a functional side chain portion that is bound to a protein or peptide via the -CFX- group when the compound of formula (II) is used, and via the -CH2- group when the compound of formula (III) is used; X is selected from the group consisting of hydrogen, fluorine, chlorine, -C(O)OH, and -C(O)NH2; A is an aryl or heteroaryl group which may be substituted with one or more R2 groups; j is 0, 1, 2 or 3; R1 and R2 are independently selected from halogen and C (1-6) esters and C (1-6) ethers substituted with one or more groups selected from the group consisting of C (1-6) alkyl); and When the compound of formula (II) is used as a radical precursor, step (a) further provides a method comprising contacting a protein or peptide with a source of Fe(II).

[0014] In a further aspect of the present invention described above, R is a group selected from (i) a medicament, a sugar, a polysaccharide, a peptide, a protein, a vaccine, an antibody, a nucleic acid, a virus, a labeled compound, a stabilized radical precursor, a biomolecule and a polymer, any of which may be linked via a linker group.

[0015] In a further aspect of the present invention described above, the linker is an alkyl group in which one or more non-adjacent carbon atoms may be substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-; polyethylene glycol and its analogs; saccharides; polysaccharides; polyglycine; polyamides; or a group L1 selected from a combination of two or more of these groups.

[0016] In a further aspect of the first embodiment described above, R is (ii) a functional group R F ; or one or more functional groups R linked via a linker group L2 F (wherein R F is - hydrogen, C 3-10 cycloalkyl, aryl or heteroaryl (the cycloalkyl, aryl and heteroaryl groups are unsubstituted or substituted with one or more groups selected from =O, =NRa, Y and (C 1-6 alkyl)-Y); or -C 2-6 alkenyl, C 2-6 alkynyl, halogen, hydroxy, -ORa , -SR a ,-S(O)R a -S(O)2R a , -OSO3R a , -NR a C(O)R b , -NR a CO2R b ,-NHC(O)NR a R b , -NHCNH2NR a R b , -NR a SO2R b , -N(SO2R a )2, -NHSO2NR a R b ,-OC(O)R a , -C(O)R a , -CO2R a -C(O)NR a R b , -C(O)(NHNH2), -ONH2, -C(O)N(OR a )R b -SO2NR a R b Or -SO(NR a )R b ;Cyano, Nitro, C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + A reactive group Y selected from; (In the formula: R a , R b and R c These are, independently, hydrogen and C 1-6 Alkyl, C 3-10 Represents cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl compounds. R a , R b and R c In this context, alkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl groups are either unsubstituted or halogen, hydroxyl, =O, -NH 2、 -SO3- and C 1-6 (substituted with one or more substituents selected from alkoxys); and L2 is an alkyl group in which one or more non-adjacent carbon atoms may be substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-; polyethylene glycol and its analogues; sugars; polysaccharides; polyglycine; polyamide; or selected from two or more combinations of these groups.

[0017] In a further embodiment, R is (ii) functional group R F ; or one or more functional groups R linked via a linker group L2 F (In the formula, R F ha:C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -OC(O)R a , -C(O)R a , -CO2R a -C(O)(NHNH2), -ONH2 and C 1-6 (is a reactive moiety selected from azidoalkyl); or R is a formula

[0018] [ka]

[0019] The reactive portion includes the reactive portion, where A is defined in claim 1; and the reactive portion

[0020] [ka]

[0021] The molecules may be linked via a linker group L2 (where L2 is an alkyl group in which one or more non-adjacent carbon atoms may be substituted with a group selected from NH, O, S, -C(O)NH-, or -NHC(O)-).

[0022] In a further embodiment, the reactive part is a halogen, C 1-6 Azid, C2-6 Alkinil,

[0023] [ka]

[0024] Selected from, preferably

[0025] [ka]

[0026] That is the case.

[0027] In a second embodiment, the present invention provides a method for functionalizing a protein or peptide containing at least one SOMO acceptor residue, as defined in the first embodiment, with a functional side chain moiety, the method being: (a) The oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in the form of a radical precursor compound, a source of Fe(II), and in a photoactivated state. ox ) to be brought into contact with a photocatalyst where the voltage is +1.2V or less; and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide; (the radical precursor compound is a group of the following formula (IV),

[0028] [ka]

[0029] (In the formula, R is a functional side chain portion that is bound to a protein or peptide via the group -CFX-; the group R is -COOR d and -CONR d R e (In the formula, R d is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, heterocyclyl, phenyl, benzyl, or heteroaryl (wherein R is the formula) dAlkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl groups in these compounds are either unsubstituted or halogen, hydroxyl, =O, -NH2, C 1-6 Alkoxy and -NHCOR e Represents (substituted with one or more substituents selected from); and R e is hydrogen or C 1-4 It is selected from (representing alkyl).

[0030] In a third embodiment, the present invention relates to a protein or peptide comprising at least one SOMO acceptor residue as defined in the first embodiment above.

[0031] [ka]

[0032] The present invention provides a method for functionalizing a functional side chain portion having the following characteristics: (a) The oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in the form of a radical precursor compound, a source of Fe(II), and in a photoactivated state. ox ) to be brought into contact with a photocatalyst where the voltage is +1.2V or less; and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide; (the radical precursor compound used has the following structure)

[0033] [ka]

[0034] (wherein the formula, bases A and X are defined in the first embodiment above)

[0035] In further embodiments of the above embodiments, if the functional side chain portion includes a reactive portion as defined above, the method may include reacting a peptide or protein via one of the reactive portions to link the functional side chain to a further molecule.

[0036] In a preferred embodiment, the further molecule is a medicine, sugar, polysaccharide, peptide, protein, vaccine, antibody, nucleic acid, virus, labeled compound, biomolecule or polymer.

[0037] In a further embodiment of any of the above embodiments, the SOMO acceptor residue is dehydroalanine.

[0038] In a further embodiment of the above embodiments, group A is phenyl, pyridinyl, pyrimidinyl, benzothiazolyl or pyrazinyl.

[0039] In a preferred embodiment of the above embodiments, group A is pyridinyl, pyrimidinyl or benzothiazolyl.

[0040] In a more preferred embodiment of the above embodiments, group A is 2-pyridinyl.

[0041] In a further embodiment of the above embodiments, group X is fluorine.

[0042] In a further embodiment of any of the above embodiments, the source of Fe(II) is iron(II) sulfate, FeOTf2, Fe(ClO4)2, FeF2 or (NH4)2Fe(SO4)2, preferably FeSO4·7H2O.

[0043] In a further embodiment of any of the above embodiments, the photocatalyst is a Ru(II) or Ir(II) based catalyst, preferably a Ru(II) catalyst.

[0044] In a more preferred embodiment of the above embodiments, the Ru(II) photocatalyst is Ru(bpy)3Cl2 or Ru(bpm)3Cl2.

[0045] In a further embodiment of any of the above embodiments, the light irradiation is in the region of 300 to 600 nm, preferably 400 to 500 nm, more preferably 430 to 470 nm.

[0046] In a further embodiment of the above embodiment, if the radical precursor compound is a compound of formula (III), the compound of formula (III) is produced in situ in step (a) by contacting a protein or polypeptide with a functionalized boron compound containing the -BCH2R moiety and a catechol derivative represented by the following formula (IIIB):

[0047] [ka]

[0048] (wherein R, R1, and j are defined in any of the embodiments described above).

[0049] In a fourth embodiment, the present invention relates to formula (IA):

[0050] [ka]

[0051] (wherein X is selected from hydrogen, fluorine, -COOH and -CONH2, preferably fluorine; R Z is hydrogen or methyl; and R provides a functionalized peptide or protein comprising at least one residue of (as defined in any of the embodiments above).

[0052] In a further embodiment of the above-described model, R is C 1-6 Haloalkyl, C 1-6 Azidoalkyl or

[0053] [ka]

[0054] That is the case.

[0055] In a further aspect of the above embodiment, the residue of formula (IA) is any one of the compounds listed in Examples 2a to 2ag.

[0056] In a further aspect of the above embodiment, X is fluorine.

[0057] In a fifth embodiment, the present invention provides a compound of formula (IB):

[0058]

Chemical formula

[0059] (wherein Ry is hydrogen or methyl; Rbac is C 1-6 alkyl (wherein the terminal carbon is substituted with at least one halogen), or Rbac is represented by the following formula

[0060]

Chemical formula

[0064] In a seventh embodiment, the present invention provides a method for covalently bonding the functionalized protein or peptide described in fourth above with a further protein or peptide, wherein the group R in the functionalized protein or peptide is

[0065] [ka]

[0066] (wherein A is defined in any of the above embodiments), and further proteins or peptides include a group that can react with a radical species to form a covalent bond.

[0067] In the eighth embodiment, the present invention provides compounds of the following formulas (II) or (III):

[0068] [ka]

[0069] (wherein A, X, R1, and j are defined in any of the embodiments described above). [Brief explanation of the drawing]

[0070] [Figure 1]Figure 1: The left side shows a schematic diagram of the method of the present invention, which provides a functionalized protein by reacting BACED (left) and pySOOF (right) derivatives with a Dha-containing residue. The upper right shows a portion of the diverse range of protein scaffolds and sites that can be functionalized using the method described herein. The lower right shows a portion of the diverse range of functional groups that can be bound to proteins or peptides by the method of the present invention. [Figure 2]Figure 2(a) shows the oxidation half-potential (Eox) spectra at the top, indicating catalytic compatibility with the protein's fundamental chemistry, such as related catalysts (catalysts 1-5) found in the literature, as well as the oxidation half-potentials of the BACED reagents, catechol and boron precursor compounds. Figure 2(b) shows the voltammetry response of 1 mM catechol and 12 mM phenethylboronic acid on GC in PBS, pH 7.10. Figure 2(c) shows a detailed reaction scheme for an example of the BACED reaction scheme described in Embodiment (ii) below (generating a Dha residue and functionalizing it with a specific side chain). Specifically, this scheme demonstrates low Eox activation (compared to other derivatives) of the BACED reagent's RCH2· radical (which subsequently reacts with Dha to introduce a side chain into the histone H protein) by [RuII] catalyst. Furthermore, LC-MS of the unchanged protein (see chromatogram and m / z on the right) shows the introduction of homohomophenylalanine (1h) into the histone H3 protein. Figure 2(d) shows a detailed reaction scheme for the pySOOF reaction example described in Embodiment (i) below (generating a Dha residue and functionalizing it with a specific side chain). Specifically, this scheme demonstrates the [RuII]-catalyzed activation of the pySOOF reagent to the RCF2· radical (which subsequently reacts with Dha in the protein to introduce a "zero-size" labeled side chain). The addition of [FeII] inhibits oxidation to the imine (and hydrate) by [RuII]*, achieving unprecedented efficiency (2–5 equivalents of the precursor), suggesting its important role as a reducing agent (readily available in biology) that quenches the alpha-C· radical adduct generated during the reaction. Untreated protein LC-MS shows that the introduction of difluoroethylglycine (DfeGly, 2a) into histone H3 protein was successful using [FeII] (see chromatogram and m / z in the upper right), and the conversion rate was improved compared to the reaction without iron (see lower center where unwanted byproducts were generated). [Figure 3]Figure 3 shows the reaction scheme for the isotropic and anisotropic reactivity on proteins by the introduction of radical precursors and electrophilic side chains. Figure 3(A) shows the use of the iodo-functionalized pySOOF derivative described in Embodiment (ia) of the method below. This scheme shows the reductive introduction of a pySOOF side chain on a protein, which is itself a protein radical precursor (highlighted). Both mono and difluoropySOOF side chains can be introduced by this method. The reagents and conditions used were: histone H3-Dha9 (66 μM), Iodo-pySOOF (2 eq), FeSO4·7H2O (20 eq), Ru(bpy)3Cl2 (0.4 eq), NH4OAc (500 mM, pH 6, 3 M GdnHCl), 50 W Blue LED, RT, 15 min. The untreated protein LC-MS is shown in the inset box in the lower right. After activation using standard mild conditions (see Figure 2), the resulting radicals on proteins enabled diverse further protein functionalization through various isotropic bond formation modes on different proteins. The radicals on proteins could be any of the following: polymerize with various radical acceptors via CC bond formation (right, top); CC trap with another Dha-containing protein to promote protein-protein crosslinking to form CC bonds (left, top); quench with the stable O radical nitroxide radical TEMPO to form CO bonds (left, middle); cleave and use of diserenide (SePh)2 to form C-Se bonds (left, bottom); or reduce to difluoroethylglycine (DfeGly) with additional Fe (overall CH bond formation) (right, middle). The reagents and conditions used were as follows: histone H3-pySOOF9 (66 μM), substrate (10-250 eq), FeSO4·7H2O (0-25 eq), Ru(bpy)3Cl2 (1-5 eq), NH4OAc (500 mM, pH 6, 3 M GdnHCl), 50 W Blue LED, RT, 15 minutes, see Example 4 for reaction details, residual Dha = 15179 Da. Figure 3(B) shows the use of alkyl-functionalized BACED halogenated as described in Embodiment (ii) of the method below.This scheme demonstrates oxidative introduction while preserving the C-halogen (C-Hal) bond. This introduces an electrophilic alkyl halide side chain on the protein (highlighted). The reagents and conditions used were as follows: histone H3-Dha9 (66 μM), alkylboronic acid pinacol ester (1000 eq), catechol (100 eq), Ru(bpm)3Cl2 (10 eq), NH4OAc (500 mM, pH 6, 3 M GdnHCl), 50 W Blue LED, RT, 1-3 hours). This provided a further reaction platform for isotropic bond formation modes on a variety of proteins. These alkyl halide electrophiles on proteins enabled higher concentrations and greater diversity of CP, CS, CN, and C-Hal bonding (see Example 3 for details, residual Dha = 15179 or 15180 Da) through substitution reactions with various low-molecular-weight P, S, N, and Hal nucleophiles (TCEP = tris(2-carboxyethyl)phosphine, βME = beta-mercaptoethanol). Furthermore, the ability to introduce various intrinsic reactive alkyl halide side chains in this method (e.g., chloro-(Cnl), bromo-(Bnl), iodo-(Inl)norleucine, untreated protein LC-MS, left, bottom) enabled proximity-driven protein-protein crosslinking with interaction partners (see Figure 4). [Figure 4]Figure 4 shows specific editing insertions of native, difluorolabeled, and electrophile-containing side chains into proteins. Such modifications provide insights into enzymes that perform post-translational modifications of proteins and can be used for binding with other proteins and enzymes. For example, the Sirt2 enzyme was shown to exhibit different deacylation rates for acetyllysine and benzoyllysine introduced onto histone eH3-K18 protein (as shown by LC-MS monitoring of untreated protein). The deacetylation reaction was also monitored directly and site-specifically by 19F-NMR via difluorotags on the CγF2 gamma carbon of the introduced Lys and AcLys side chains. Despite the four binding distances from the PTM site, the CγF2 labeling shows sufficient sensitivity to the chemical environment (δF perturbation), allowing for direct and simultaneous monitoring of Sirt2's chemoselectivity and stereoselectivity during processing. Figure 4(A) shows the functionalization of histone H3 with the BACED reagent according to embodiment (ii) / (iia) of the method described below, for use in the above enzyme studies. The reagents and conditions used for induction were as follows: histone H3-Dha9 (66 μM), alkylboronic acid pinacol ester (250 eq), catechol (100 eq), Ru(bpm)3Cl2 (10 eq), NH4OAc (500 mM, pH 6, 3 M GdnHCl), 50 W Blue LED, RT, 1 hour. Figure 4(B) shows the functionalization of histone H3 using the pySOOF type reagent described in embodiment (i) of the method described below, for use in the above enzyme study. The reagents and conditions used for induction were as follows: histone H3-Dha9 (66 μM), alkyl-pySOOF (50 eq), FeSO4·7H2O (50 eq), Ru(bpy)3Cl2 (2 eq), NH4OAc (500 mM, pH 6, 3 M GdnHCl), 50 W Blue LED, RT, 15 minutes, Met ox = 15838 Da. Figure 4(C) shows a general diagram of the ideal properties of the "alkylating agent protein". Reactions to be restricted or avoided are shown in the upper box, and desirable selective reactions are shown in the lower box.Figure 4(D) shows that the bridge between KDM4A and histone-eH3.1-Bhn4 / 9 / 27 (Bhn = bromohomonorleucine) traps the Zn-bound cysteine ​​of KDM4A near the active site. Coomassie-Blue-SDS-PAGE (bottom left), tryptic-LC-MS / MS (top right), and Zn(II)-release (bottom right) confirm the bridge between KDM4A and histone-eH3.1-Bhn9 (see also Figure 10c in ED) [Zn(II)-release rates: eH3-Bhn9 = 9.27±0.025 nM / min, eH3-WT = 0.09±0.006 nM / min, 1u-precursor = 0.805±0.010 nM / min, no compound = 0.87±0.028 nM / min, N=3 independent experiments. Plotted data are mean + / - standard deviation (N=3 technical replicates), p < 0.0001 [1-way ANOVA]. See also Figure 10 in ED for further alkylating agent protein experiments. Figure 4(E) shows incubation of the histone eH3.1-Bhn9 alkylating agent protein with HeLa nuclear lysate, capturing interaction partners via proximity-driven crosslinking. After enrichment via HA tag (on histone eH3.1), α-FLAG Western blot revealed multiple higher MW bands corresponding to the histone mass and the mass of the captured interaction partner. No higher MW bands were observed in the absence of Bhn. Figure 4(F) shows unprecedented Williamson COC bond ether formation in an intermolecular manner between H3 proteins (one Bhn4 bonded to the other hydroxyl) driven by effective moles, suggesting a transient dimer model for the function of KDM4A. [Figure 5] Figure 5 shows several functionalized protein residues successfully generated via the reaction methods described herein. The reagents and conditions used are provided in the Examples section. Figure 5(a) shows residues generated via the BACED reagent (Embodiment (ii) / (iia)). Figure 5(b) shows residues generated via the activated fluorinated radical precursor (Embodiment (i), (ia) and (ib)), which can be distinguished by containing at least one fluorine label on the γ carbon atom of the side chain. [Figure 6] Figure 6 shows the reaction schemes described in the various embodiments of the present invention as described in the examples. [Figure 7] Figure 7(A) shows the method for expressing maltose-binding protein in the presence of monoF-PySOOF-AA, as described in Example 8. Figure 7(B) shows: Top - SDS-Page gel of purified MBP. Bottom - MS analysis of the purified fraction showing the product and the contaminant PylRS. [Figure 8] Figure 8 shows the reaction schemes described in various embodiments of the present invention, as described in Example 9. [Figure 9] Figure 9 shows the reaction schemes described in various embodiments of the present invention, as described in Example 10.

[0071] Detailed statement The present invention provides a method for functionalizing a protein or peptide at a functional side chain portion, wherein the protein or peptide comprises at least one single-occupied molecular orbital (SOMO) acceptor residue, and the method is: (c) Contacting a protein or peptide with a specific radical precursor compound and photocatalyst having a functional group capable of binding to the protein or peptide; (d) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide;

[0072] SOMO acceptor residues are amino acid residues located in a peptide or protein and linked to one or two adjacent residues by peptide bonds. SOMO acceptor residues contain a group that is highly reactive to C radical species, and this group is a side chain having an alkene group. In some embodiments, SOMO acceptor residues are of the formula C 1-6 It may have an alkenyl side chain. Preferably, the C=C double bond is located at the end of the alkenyl group. In a preferred embodiment, the SOMO acceptor is dehydroalanine (Dha) or dehydrobutyrine (Dhb), preferably dehydroalanine.

[0073] The Dha residue can be introduced into the protein or peptide of interest by any suitable means, such as any of the means described in Chemical Sceince, Vol. 2, Number 9, Sept 2011, Pages 1617-1868, or Current Opinion in Chemical Biology, Vol. 46, Oct 2018, Pages 71-81.

[0074] The residue to be functionalized can be present at any suitable location in the protein or peptide chain.

[0075] Embodiment (i) Aryl sulfone fluoride derivative (ASOOF) In the first embodiment (i) of the above method, the radical precursor compound is a compound of formula (II), which is referred to herein as the ASOOF precursor:

[0076]

Chemical formula

[0077] In formula (II) above, R is a functional side chain moiety that is attached to the protein or peptide via the group -CFX-. A is an aryl or heteroaryl group that may be substituted with one or more R2 groups. Typically, A is unsubstituted or substituted with 1, 2 or 3 R2 groups, preferably A is unsubstituted or substituted with 1 or 2 R2 groups. Most preferably, A is unsubstituted. R2 is halogen and C (1-6) alkyl (unsubstituted or substituted with one or more groups selected from hydroxy, oxy, halogen, amino, carboxy, C (1-6) ester, and C (1-6) ether (e.g., 1, 2 or 3 groups, preferably 1 or 2 groups)). In some embodiments, R2 is C 1-4It is alkyl (unsubstituted or substituted with hydroxy, oxy, halogen, or amino). In a preferred embodiment, A is unsubstituted. In some embodiments, A is a six-membered ring. In preferred embodiments, A is phenyl, pyridinyl, pyrimidinyl, benzothiazolyl, or pyrazinyl, and more preferably A is pyridinyl, pyrimidinyl, or benzothiazolyl. In the most preferred embodiment, the compound of formula (II) is the compound of formula (IIA) described below.

[0078] [ka]

[0079] In the above formulas (II) and (IIA), X is selected from the list consisting of hydrogen, fluorine, chlorine, -COOH, and -CONH2, preferably fluorine or hydrogen, most preferably fluorine.

[0080] In the most preferred embodiment, the radical precursor compound is:

[0081] [ka]

[0082] This is referred to as "pySOOF" in this specification.

[0083] In a further embodiment, the radical precursor is

[0084] [ka]

[0085] That is the case.

[0086] In a further embodiment, the radical precursor compound is:

[0087] [ka]

[0088] (referred to as "BtSOOF" in this specification) or

[0089] [ka]

[0090] That is the case.

[0091] When using the compound of formula (II) as the radical precursor compound, the reaction composition must further include a source of Fe(II). Fe(II) acts to reduce the photocatalyst to an active form that can oxidize the radical precursor of formula (II) (for example, by reducing Ru(II) to Ru(I), as shown in Figure 2(d)). Furthermore, Fe(II) can act to reductively quench the radical protein / peptide intermediate produced by the initial reaction between the stabilized functional side-chain radical and the SOMO acceptor residue. This has the advantage of preventing oxidative quenching of the intermediate, which can result from an excess of oxidized photocatalysts, such as Ru(II) catalyst species, and lead to undesirable byproducts such as the formation of imines and hemiaminals (see Figure 2(d)).

[0092] The source of Fe(II) is not particularly limited. In preferred embodiments, the source of Fe(II) is iron(II) sulfate, iron(II) trifluoromethylsulfonate (FeOTf2), Fe(ClO4)2, FeF2, or (NH4)2Fe(SO4)2, preferably iron(II) sulfate, for example FeSO4·7H2O.

[0093] The amount of Fe(II) compound used is not particularly limited, but is typically 1 to 1000 equivalents, preferably 5 to 600 equivalents, more preferably 10 to 300 equivalents, and most preferably 25 to 250 equivalents, relative to the amount of protein substrate used.

[0094] The amount of the radical precursor compound used in this embodiment is not particularly limited, but is typically 0.1 to 1000 equivalents, preferably 0.5 to 250 equivalents, more preferably 0.5 to 50 equivalents, and most preferably 2 to 25 equivalents, relative to the amount of protein substrate used.

[0095] The reaction described in embodiment (i) can proceed according to the scheme shown in Figure 2(d). As can be seen, upon activation with a suitable beam of light, the photoexcited oxidation state of the photocatalyst (e.g., Ru(II) photocatalyst) is reductively quenched by Fe(II) to provide an active reduced species, e.g., (Ru(I)). This reduced species then reductively initiates the ASOOF precursor, yielding a stabilized RCFX· radical species, which then reacts via radical addition to the C=C double bond of a SOMO acceptor residue such as Dha, as shown below. The resulting radical on the α-carbon protein is then reduced via SET from iron(II) to form an enolate intermediate that is protonated under aqueous reaction conditions, ultimately yielding a functionalized protein / peptide.

[0096] Embodiment (ia) In a further special embodiment (ia), the same reaction conditions as in embodiment (i) are used, except that the group R in formula (II) is iodine instead of a side chain group for binding to a protein / peptide. Thus, the radical precursor compound is given by formula

[0097] [ka]

[0098] (wherein A and X are compounds as defined in embodiment (i) above.) In a preferred embodiment, A is pyridyl and X is fluorine such that the radical precursor is iodo-pySOOF.

[0099] Under the same reaction conditions as ASOOF described above, the reductively activated catalyst reductively activates the iodo radical precursor, forming the radicals shown below.

[0100] [ka]

[0101] This stabilized radical species is further reacted via radical addition to the C=C double bond of the SOMO acceptor residue via the same reaction pathway described above in the first aspect of this embodiment, in order to generate a protein / peptide that is functionalized with the ASOOF radical precursor side chain. This protein / peptide, functionalized at the ASOOF precursor side chain, may be activated via a photoredox catalyst and an iron(II) source using the same reaction conditions as in embodiment (i) to provide a stabilized radical on the protein that can be used for further binding to other species. Thus, this site allows for the functionalization of a variety of further proteins via an isotropic binding mechanism on various proteins (see Figure 3(A)).

[0102] Embodiment (iai) In further embodiments, the present invention provides a method for producing a protein / peptide containing residues including the ASOOF-functionalized side chain described in formula (IAi) below, by incorporating a synthetic amino acid according to formula (IIi) into the protein / peptide. This can be done, for example, using a genetic code extension technique as described in Example 8.

[0103] [ka]

[0104] (In formulas (IAi) and (IIi), A and X are defined in the above embodiments, and Lz may be substituted with one or more groups selected from halogen, hydroxyl, and amino.) 1-4It is an alkyl linker group. Preferably, Lz is methylene(-CH2-) or -CH(CH3)-. More preferably, Lz is methylene. Rt is hydrogen or a protecting group, preferably hydrogen or C 1-4 The group is alkyl, more preferably hydrogen or tert-butyl. Rs is hydrogen or a protecting group, more preferably hydrogen or tert-butoxycarbonyl (boc). In preferred embodiments, Rs and Rt are each hydrogen.

[0105] In preferred embodiments, Lz is methylene, X is hydrogen or fluorine, A is a heteroaryl selected from pyridinyl, pyrimidinyl, or benzothiazolyl, and Rs and Rt are both hydrogen or either boc and tert-butyl, respectively. More preferably, A is

[0106] [ka]

[0107] That is the case.

[0108] The protein / peptide functionalized in the ASOOF precursor side chain may be further activated / reacted as described in embodiment (ia) above.

[0109] Therefore, the present invention also provides the protein / peptide described in formula (IAi) above, and the synthetic amino acid described in formula (IIi) above. The present invention also provides salts of the compound of formula (III) above.

[0110] Embodiment (ib) In a further special embodiment (ib), the same reaction conditions as those used in embodiment (i) are used, except that the radical precursor compound of formula (IV) is used.

[0111] [ka]

[0112] R is a functional side chain portion that is bound to a protein or peptide via the -CF2- group.

[0113] Under the same reaction conditions as ASOOF described above, the reductively activated catalyst reductively activates the precursor, forming radicals as shown below.

[0114] [ka]

[0115] This stabilized radical species is further reacted via radical addition to the C=C double bond of the SOMO acceptor residue via the same reaction pathway described above in the first aspect of this embodiment, in order to generate a protein / peptide functionalized with side chain-CF2R.

[0116] Embodiment (ii) Boronic acid catechol-ester derivative (BACED) In a further embodiment (ii) of the above method, the radical precursor compound is the compound of formula (III), which is referred to herein as the BACED reagent:

[0117] [ka]

[0118] In formula (III), j is 0, 1, 2, or 3, typically j is 0, 1, or 2, preferably j is 0 or 1. In a preferred embodiment of embodiment (ii), the BACED reagent is the compound of formula (IIIA) shown below.

[0119] [ka]

[0120] In equation (IIIA), j is either 0 or 1. Each R1 in the above formula (III) or (IIIA) is independently selected from the group consisting of halogen and C (1-6) alkyl (unsubstituted or substituted with one or more groups selected from hydroxy, oxy, halo, amino, carboxy, C (1-6) ester and C (1-6) ether (e.g., 1, 2 or 3 groups, preferably 1 or 2 groups)). Preferably, the group R1 is C (1-4) alkyl (unsubstituted or substituted with one or two groups selected from hydroxy, halo, amino and carboxy). Most preferably, R1 is hydrogen, CH2CH2NH2 or CH2CH(NH2)COOH. R is a functional side chain moiety that is attached to a protein or peptide via the group -CH2-.

[0121] The BACED reagent should have an oxidation potential close to or lower than the oxidation potential (Eox) of the activated photocatalyst, since it is oxidized by the said catalyst during the reaction.

[0122] The BACED reagent may be generated in situ by adding a functionalized boron compound and a catechol derivative represented by the following formula (IIIB) to the reaction solution (wherein j and R1 are as defined above).

[0123]

Chemical formula

[0124] The functionalized boron compound may be any boron compound covalently bonded to a side chain (-CH2R) that binds to a protein or peptide, i.e., a boron compound containing a B-CH2-R unit. To form the active BACED reagent in situ, the boron compound is further preferable to be able to substitute the ligand in an aqueous environment. The boron component may be a boronic acid salt, a boronic acid, and / or a boronic acid ester. In one embodiment, the boron compound is of the formula [RCH2BQ3]V (where Q is independently a halogen, preferably chloro or fluoro, most preferably fluoro; and V is K + Li + na + or NH4 + The boron compound is a compound of any suitable counterion such as RCH2B(OR). In further embodiments, the boron compound is a compound of formula RCH2B(OR). f )2(R f The group is independently either hydrogen or C 1-6 Alkyl, or two R f The groups combine to form a linear or branched C chain, linking two oxygen atoms together to form a 4- to 7-membered ring with a boron atom bonded to an oxygen atom. 1-10 The compound is a compound that forms an alkyl chain. In preferred embodiments, the boron compound is RCH2BF3K, RCH2B(OH)2, or RCH2Bpin (where pin is a pinacolate group bonded to boron via two oxygen atoms).

[0125] The amount of boron compound used is not particularly limited, but is typically 5 to 1000 equivalents, preferably 10 to 600 equivalents, and more preferably 100 to 500 equivalents, relative to the amount of protein substrate used. The amount of catechol derivative (IIIB) added is not particularly limited, but is 0.02 equivalents or more relative to the boron compound. In one embodiment, the amount of catechol derivative is 0.02 equivalents or more and 1 equivalent or less relative to the amount of boron compound added to the reaction solution.

[0126] In embodiment (ii), the reaction can generally proceed according to the scheme shown in Figure 2(c). As can be seen, the photoexcitation catalytic state of the photocatalyst, for example Ru(II) * This process oxidatively initiates the BACED precursor to yield an RCH2· radical species, which then reacts via radical addition to the C=C double bond of a SOMO acceptor residue such as Dha, as shown in Figure 2(c). The resulting α-carbon radical on the protein is then reductively quenched via a reductive catalyst, e.g., SET from Ru(I), to form an enolate intermediate that is protonated under aqueous reaction conditions, ultimately yielding a functionalized protein / peptide.

[0127] Embodiment (iia) Boron Reagent In one embodiment, the present invention provides a method for functionalizing a protein or peptide with a functional side chain portion, wherein the protein or peptide comprises at least one single-occupied molecular orbital (SOMO) acceptor residue as described herein. The method is, (a) As described in the above embodiment, the oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in a functionalized boron compound, a catechol derivative of formula (IIIB), and in a photoactivated state. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide;

[0128] The functionalized boron compounds and catechol derivatives of formula (IIIB) are as defined in embodiment (ii) above.

[0129] While we do not wish to limit ourselves, it is currently understood that functionalized boron compounds of formula (IIIB) and catechol derivatives of formula (IIIB) generate the BACED reagent of formula (III) in situ during step (a). However, the present invention is not limited to the method by which the BACED reagent is formed (or detectable) during the reaction, and embodiments in which the BACED reagent is undetectable or not formed are also included within the scope of the present invention.

[0130] Reaction conditions The following are the reaction conditions for carrying out the method of the present invention. Unless otherwise specified, the embodiments described below relate to all embodiments of the method of the present invention, including methods in which the radical precursor compound is of formula (II), (IIA), (III), (IIIA), or (IV), and methods in which the reaction proceeds in the presence of a functionalized boron compound and a catechol derivative of formula (IIIB). An advantageous feature of the present invention is that the reaction can be carried out under mild redox conditions. Therefore, the photocatalyst used preferably has an oxidation half potential (Eox) in its photoactivated oxidation state when measured against a saturated calomel electrode. * The voltage is +1.2V or less, preferably +1.0V or less, and more preferably less than +1.0V.

[0131] The photocatalyst used, when measured against a saturated calomel electrode, preferably has a reduction half-potential (Ered) of -1.5V or less, more preferably -1.4V or less. To avoid any doubt, low reduction half-potentials as described herein are indicated by lower negative values ​​or higher positive values. Thus, a reduction half-potential of -1.4V is "less than" a reduction half-potential of -1.5V.

[0132] When using the method of embodiment (ii) / (iib), the oxidation half-potential (Eox) of the photoactivated photocatalyst is preferably 0.2 V or less lower than that of the radical precursor compound of formula (III) when measured against a saturated calomel electrode. Preferably, the Eox of the photocatalyst is greater than that of the radical precursor compound of formula (III) when measured against a saturated calomel electrode.

[0133] In some embodiments, the oxidation half-potential (Eox) of the radical precursor compound of formula (III) may be +1.2V or less, preferably +0.99V or less, more preferably 0.8V or less, and most preferably +0.5V or less, when measured against a saturated calomel electrode.

[0134] When using the method of embodiment (i), (ia), or (ib), the oxidation half-potential Eox in the photoactivated oxidation state of the photocatalyst is preferably +0.72V or higher.

[0135] When using the method of embodiment (i), (ia), or (ib), the reduction half-potential (Ered) of the photocatalyst is preferably 0.2 V or less lower than the Ered of the radical precursor compound of formula (II) / (IV) when measured against a saturated calomel electrode. Preferably, the Ered of the photocatalyst is greater than the Ered of the radical precursor compound of formula (II) / (IV) when measured against a saturated calomel electrode (i.e., it is a stronger reducing agent).

[0136] In some embodiments, the reduction half-potential (Ered) of the radical precursor compound of formula (II) / (IV) is -1.4V or less, preferably -1.2V or less, and more preferably -1.0V or less, when measured against a saturated calomel electrode.

[0137] The photocatalyst is preferably a Ru(II) or Ir(II)-based catalyst, and more preferably a Ru(II) catalyst. In a particularly preferred embodiment, the photocatalyst is Ru(bpy)3Cl2 or Ru(bpm)3Cl2.

[0138] The amount of photocatalyst used is not particularly limited, but may be substochiometric with respect to the amount of protein / peptide. In some embodiments, the amount of photocatalyst is 0.1 to 100 equivalents, preferably 0.1 to 10, and more preferably 0.25 to 1 equivalent, with respect to the amount of protein or peptide.

[0139] Appropriate light beam irradiation is used to activate a photocatalyst (e.g., Ru(II) photocatalyst), thereby initiating radical formation and coupling reactions. This has the advantage of allowing for temporal, spatial, and kinetic control of the reaction. Furthermore, the light can be benign, as it can penetrate tissue and not damage the sample (e.g., protein / peptide or tissue). The light irradiation is preferably visible light. In some embodiments, the light irradiation has wavelengths in the range of 300 nm to 600 nm, preferably 400 to 500 nm. In further embodiments, the light irradiation has wavelengths in the range of 430 to 470 nm.

[0140] The light intensity is not particularly limited, but in some embodiments, the light supplied to the reaction may be 0.1 to 1000 W, preferably 1 to 200 W, more preferably 1 to 100 W, and even more preferably 5 to 60 W. In a preferred embodiment, the light intensity supplied to the reaction is 45 to 55 W.

[0141] The use of photoactivation to initiate protein functionalization reactions described herein allows for precise spatiotemporal control of the reaction. Such time- and targetful use of potentially tissue-penetrating triggers can be used to modify and probe complex biological systems.

[0142] This reaction can be carried out without the need for harsh solvents that could damage proteins. The solvent used is preferably water.

[0143] The reaction is preferably carried out under anaerobic conditions to avoid unwanted oxidation reactions with radical intermediates.

[0144] The reaction can be advantageously carried out under mild pH conditions. Preferably, the reaction is carried out at a pH of 5.0 to 9.0. More preferably, the reaction is carried out at a pH of 5.5 to 8.5. In one embodiment, the reaction is carried out at a pH of 5.0 to 7.0. In a further embodiment, the reaction is carried out at a pH of 5.5 to 6.5.

[0145] The reaction mixture may optionally further contain one or more additional components, such as a buffer for adjusting the pH. In some embodiments, the buffer is selected from sodium phosphate buffer (NaPi), HEPES, FPBS, phosphate-buffered saline (PBS), NH4OAc, guanadinium chloride, and combinations thereof. Preferably, the buffer is a combination of NH4OAc and guanadinium chloride.

[0146] The reaction typically takes place at temperatures of 0 to 50°C, preferably 5 to 40°C, more preferably 10 to 30°C, and most preferably 15 to 25°C.

[0147] The present invention further enables a rapid reaction time. The reaction period is typically less than 4 hours, preferably less than 1 hour, more preferably less than 30 minutes, even more preferably less than 20 minutes, and most preferably less than 15 minutes.

[0148] Functional side chain moiety The following are functional side chain moieties that can be bound to proteins or peptides using the methods of the present invention. Unless otherwise specified, the following side chains can be added using any embodiment of the methods of the present invention, such as a method in which the radical precursor compound is of formula (II), (IIA), (III), (IIIA), or (IV), and a method in which the reaction proceeds in the presence of a functionalized boron compound and a catechol derivative of formula (IIIB).

[0149] Those skilled in the art will understand that functional side chain portions bound to proteins or peptides can provide a variety of functions, such as assisting in enzymatic or other biological studies, linking to specific payloads, and modulating their chemical properties.

[0150] This method enables the conjugation of a functional side chain moiety to a protein or peptide via a photomediated radical reaction under mild conditions. Generally, the group R will be conjugated to the protein / peptide via the group -CXF- when an ASOOF precursor is used (Embodiment (i), first aspect), via the group -CF2- when a precursor of formula (IV) is used, and via the group -CH2- when a boron-containing precursor is used (Embodiment (ii), (iia)). Those skilled in the art will understand that the method described herein is generally applicable and can be used even in the presence of a reactive group, and therefore there are no particular limitations on the group R that can be conjugated to a protein or peptide. Thus, the group conjugated to the protein or peptide may include any suitable chemical moiety useful for conjugation. This group may include, for example, a linker group containing a payload and / or a reactive functional group that can be conjugated to the payload via a further reaction. Such linkers, payloads, and reactive functional groups are well known in the field of protein conjugates.

[0151] In the first embodiment of the above embodiment, group R represents a payload optionally linked via a linker. The payload may be selected from the group consisting of pharmaceuticals, sugars, polysaccharides, peptides, proteins, vaccines, antibodies, nucleic acids (DNA, RNA), viruses, labeled compounds, stabilized radical precursors, biomolecules, and polymers, any of which may be linked via a linker group. In one embodiment, the payload is selected from pharmaceuticals, sugars, polysaccharides, peptides, proteins, antibodies, labeled compounds, stabilized radical precursors, and polymers. In further embodiments, the payload is selected from peptides, proteins, sugars, polysaccharides, labeled compounds, and polymers. Preferably, the payload is a sugar or a labeled compound. In a particular embodiment, the payload may be an amino acid, which may be either a natural or synthetic amino acid, and which may be covalently linked to its side chain.

[0152] The linker group L may be substantially any suitable polyvalent organic group, typically divalent or trivalent. In one embodiment, the linker group L may be an organic group having a molecular weight of 2000 or less, preferably 1500 or less, and more preferably 1000 or less. The linker may optionally contain polyethylene glycol (PEG) or a PEG analogue. Suitable PEG analogues are listed in Chemical Society Reviews, Vol. 47, Number 24, 21 Dec 2018, Pages 8971-9160. To avoid doubt, where the linker is described as "alkyl" or other related terms, it should be interpreted as encompassing a polyvalent group, such as a divalent "alkenyl" group.

[0153] In a preferred embodiment, the linker is: The group L1 is selected from groups selected from NH, O, S, -C(O)NH- or -NHC(O)-, in which one or more non-adjacent carbon atoms may be optionally substituted (i.e., exchanged); polyethylene glycol (PEG) and its analogues; sugars; polysaccharides; polyglycine; polyamide; and two or more combinations of these groups. In preferred embodiments, L1 may be an alkyl group in which one or more non-adjacent carbon atoms are substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-; selected from PEG, PEG analogues, polyamides, and two or more combinations of these groups. The alkyl group is typically C 1-20 It is alkyl, preferably C 1-10 Alkyl, more preferably C 1-6 It is alkyl. In a preferred embodiment, L1 is PEG or C where one, two, or three non-adjacent carbon atoms are substituted with groups selected from NH, O, S, -C(O)NH-, or -NHC(O)-. 1-20 It is alkyl. Preferably, L1 is C 1-10 Alkyl, more preferably C 1-6 It is alkyl.

[0154] Suitable polymers for bonding to the present invention include natural polymers such as polypeptides, polysaccharides, polynucleotides, and high molecular weight lipids, as well as synthetic polymers. Preferred polymers include PEG, PEG analogues, polyamides, polyacrylamides, polyacrylates, and RAFT (reversible addition-fragmentation chain transfer polymerization) generating polymers. Further preferred examples of polymers that can be bonded as payloads are described in Chemical Society Reviews, Vol. 47, Number 24, 21 Dec 2018, Pages 8971-9160. The polymers typically have a molecular weight of less than 10 kDa, preferably less than 5 kDa, more preferably less than 2 kDa, and most preferably less than 1 kDa. In preferred embodiments, the polymer is PEG, PEG analogues, polyacrylamide, or polyacrylate, and more preferably the polymer is PEG.

[0155] The payload bound to a protein or peptide may be a labeled compound, which is defined herein as a compound containing a labeling group that enables its detection in chemical and / or biological studies. Suitable labeling includes isotopic labeling, in which one or more atoms in the group are labeled with a specific isotope and can be detected by appropriate means such as NMR, mass spectrometry, and radiolabeling studies. Suitable labeling isotopes include deuterium, 19 F, 13 C and 15N is an example. Suitable labeling groups include biomolecules, sugars, and natural or synthetic amino acids labeled with one or more of the above isotopes at specific positions. Furthermore, the term labeling group is intended to encompass other payloads or side chain portions as described herein, labeled with specific isotopic labels as defined above. Other suitable labeling compounds include fluorophores and FRET reagents. Furthermore, suitable labeling compounds include compounds that can assist in the identification and / or isolation of the peptide of interest. In one embodiment, the labeling compound is a FLAG-tag or biotin. In a preferred embodiment, the labeling compound is biotin, which may be attached via its terminal carboxyl group, for example, in the form of an ester.

[0156] In one embodiment of the methods (i), (ia), (iai), and (ib) of the present invention and their products, the functional side chain portion R is 19 It is bound to proteins or peptides via fluorine-containing linking groups -CFX- or -CF2-. This group allows for monitoring of various reaction pathways via NMR, as demonstrated in Example 7. In further embodiments of the methods (i), (ia), (iai), and (ib) of the present invention and their products, the functional side chain portion R is 18 F-containing linking group -CFX- or -CF2- (i.e., one or both of the fluorine atoms bonded to the linking carbon atom) 18 It is bound to a protein or peptide via (which may be F). This group enables the labeling of the peptide / protein, which can allow monitoring of various reaction pathways, for example, as shown in Example 9. In some embodiments, in any of the compounds of formula (II), (IV), (IA), (IIi) and the iodine compound used as a radical precursor in embodiment (ia), one or both, preferably one, of the fluorine atoms bonded to the carbon adjacent to group R are 18 It is F.

[0157] A biomolecule or biological molecule is defined herein as a molecule present in an organism that is important for one or more biological processes. This term is intended to encompass organic low molecules typically having a molecular weight of less than 5 kDa, preferably less than 1.5 kDa, such as primary metabolites, secondary metabolites, and natural products used in important biological processes. This term includes endogenous and exogenous biomolecules, such as metabolites, vitamins, and other organic nutrients.

[0158] As used herein, a stabilized radical precursor refers to a functional group that can be used to generate radicals for further reactions, for example, by stimulation by light irradiation.

[0159] A suitable base is the formula

[0160] [ka]

[0161] Examples of the bases (wherein A and X are defined above with respect to embodiment (i)) are given.

[0162] As used herein, the term “pharmaceutical” refers to a chemical compound that has known biological effects on animals, such as humans. Typically, a drug is a chemical compound used to treat, prevent, or diagnose a disease. Preferred drugs are biologically active in that they produce a topical or systemic effect in animals, preferably mammals, and more preferably humans. Typically, drug molecules have a molecular weight of about 5 kDa or less. W It has a molecular weight of approximately 1.5 kDa or less. Preferably, the drug molecule has a molecular weight of approximately 1.5 kDa or less. W It holds.

[0163] A more complete, though not exhaustive, list of classes and specific drugs suitable for use in the present invention can be found, for example, in: (a) Pharmaceutical Substances: Syntheses, Patents, Applications, Axel Kleemann and Jurgen Engel (Thieme Medical Publishing, 1999) and (b) The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, ed. S Budavari et al. (CRC Press, 1996); the contents of these are incorporated herein by reference in their entirety.

[0164] As used herein, sugars include monosaccharides such as glucose, fructose, galactose, ribose, and deoxyribose, as well as disaccharides consisting of two monosaccharides linked by glycosidic bonds, such as sucrose, lactose, and maltose. As used herein, the term polysaccharide is intended to include macromolecules of three or more sugar molecules linked by glycosidic bonds, such as starch, cellulose, and chitin. As used herein, any sugar forming part of a sugar or polysaccharide may be a modified sugar, for example, in which the hydroxyl group of a natural sugar is replaced by a substituent. Acetyl groups, N-acetyl groups, and methyl groups are common examples of substituents. Alternatively, there may be no hydroxyl group, and it may be substituted with a hydrogen atom, for example. Thus, the sugars, sugars, and polysaccharides described herein may be unsubstituted or substituted with one or more, typically one or two, acetyl or N-acetyl groups. Thus, as used herein, the term sugar includes groups such as N-acetylglucosamine.

[0165] As used herein, the term "peptide" refers to a short chain of amino acid monomers linked by peptide (amide) bonds, either biologically generated or synthetically. Covalent chemical bonds are formed when the carboxyl group of one amino acid reacts with the amino group of another. The shortest peptides are dipeptides, consisting of two amino acids linked by a single peptide bond, followed by tripeptides, tetrapeptides, and so on. Polypeptides are continuous peptide chains containing multiple amino acids.

[0166] As used herein, the term “protein” refers to a biological molecule containing polymers of amino acid monomers, and is distinguished from peptides based on size, and can be understood, as an arbitrary criterion, to contain about 50 or more amino acids. Proteins consist of one or more polypeptides arranged to function biologically, and are often linked to ligands such as coenzymes or cofactors, or to other proteins, or other macromolecules (such as DNA or RNA), or to complex macromolecular assemblies.

[0167] In further embodiments of the present invention, R is a functional group R F ; or one or more functional groups R linked via linker groups of formula L2, typically one or two. F That is the case. R F teeth, - Hydrogen, C 3-10 Cycloalkyl, aryl, or heteroaryl (cycloalkyl, aryl, or heteroaryl groups are unsubstituted or =O, =NRa, Y, and (C 1-6 Substituted with one or more groups selected from alkyl)-Y; or -C 2-6 Alkenil, C 2-6 Alkynyl, halogen, hydroxy, -OR a , -SR a ,-S(O)R a -S(O)2R a , -OSO3R a , -NR a C(O)R b , -NR a CO2Rb ,-NHC(O)NR a R b , -NHCNH2NR a R b , -NR a SO2R b , -N(SO2R a )2, -NHSO2NR a R b ,-OC(O)R a , -C(O)R a , -CO2R a -C(O)NR a R b , -C(O)(NHNH2), -ONH2, -C(O)N(OR a )R b -SO2NR a R b Or -SO(NR a )R b ;Cyano, Nitro, C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + A reactive group Y selected from; (In the formula: R a , R b and R c These are, independently, hydrogen and C 1-6 Alkyl, C 3-10 R represents cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl compounds, where R is the dominant compound in the formula. a , R b and R c In this context, alkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl groups are either unsubstituted or halogen, hydroxyl, =O, -NH 2、 -SO3 - and C 1-6 (substituted with one or more substituents selected from alkoxys); and L2 is selected from alkyl groups in which one or more non-adjacent carbon atoms may be substituted (i.e., exchanged) with groups selected from NH, O, S, -C(O)NH- or -NHC(O)-; polyethylene glycol (PEG) and its analogues; sugars; polysaccharides; polyglycine; polyamide; or two or more combinations of these groups.

[0168] In preferred embodiments, L2 may be an alkyl group, PEG, PEG analogues, sugars, polyamides, and two or more combinations of these groups, in which one or more non-adjacent carbon atoms are substituted with a group selected from NH, O, S, -C(O)NH-, or -NHC(O)-. The alkyl group is typically C 1-20 Alkyl, preferably C 1-10 Alkyl, more preferably C 1-6 They are alkyl. Sugars are typically glucose, galactose, ribose, or deoxyribose.

[0169] In a preferred embodiment, L2 may be PEG, a sugar, or C where 1, 2, or 3 non-adjacent carbon atoms are substituted with groups selected from NH, O, S, -C(O)NH-, or -NHC(O)-. 1-20 Alkyl groups, or combinations of two or more of these groups.

[0170] In a preferred embodiment, L2 is PEG or a C1-20 alkyl group in which one, two, or three non-adjacent carbon atoms are substituted with groups selected from NH, O, S, -C(O)NH-, or -NHC(O)-.

[0171] In a further embodiment, L2 is C 1-10 It is alkyl, preferably C 1-6 It is alkyl.

[0172] In a particularly preferred embodiment, L2 is C such as methylene, ethylene, or propylene. 1-4 It is an alkyl group, preferably methylene or ethylene.

[0173] Typically, R is a functional group R F or base -L2-R F That is the case. In a further embodiment, R is -L2(R F )2. In some embodiments, R is an amino acid, and amino acids are covalently bonded via their side chains. In particular, R may have the structure described below, where Lz, Rs, and Rt are as defined in the above embodiment (iai).

[0174] [ka]

[0175] Typically, R F teeth, - Hydrogen, C 3-6 Cycloalkyl, phenyl, or pyridyl (wherein the formula, the cycloalkyl, phenyl, and pyridyl groups are unsubstituted or =O, =NRa, Y, and -(C) 1-6 Substituted with one or two groups selected from alkyl)-Y; or -C 2-6 Alkenil, C 2-6 Alkynyl, halogen, hydroxy, -OR a , -SR a ,-S(O)R a -S(O)2R a , -NR a C(O)R b ,-OC(O)R a , -C(O)R a , -CO2R a -C(O)NR a R b -C(O)(NHNH2), -ONH2, C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + The reactive group Y is selected from among them.

[0176] Preferably, R F teeth, - Hydrogen, cyclohexyl, phenyl; or -C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -S(O)2R a , -NR a C(O)R b ,-OC(O)R a , -C(O)R a , -CO2R a -C(O)NR a R b , C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + The reactive group Y is selected from among them.

[0177] In one embodiment, R F C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -S(O)2R a , -NR a C(O)R b ,-OC(O)R a , -C(O)R a , -CO2R a -C(O)NR a R b , C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + The reactive group Y is selected from the following. In one embodiment of this model, R F C 2-6 Alkenil, C 2-6 Alkynyl, halogen and C 1-6 The reactive group Y is selected from azidoalkyl groups.

[0178] In particular, R is a group Y or L2-Y (wherein L2 is C such as methylene, ethylene or propylene).1-4 Alkyl, preferably methylene or ethylene, and Y is C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -S(O)2R a , -NR a C(O)R b ,-OC(O)R a , -C(O)R a , -CO2R a -C(O)NR a R b , C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + Selected from, preferably Y is C 2-6 Alkenil, C 2-6 Alkynyl, halogen and C 1-6 (Selected from azidoalkyl)

[0179] Typically, R a , R b and R c These are, independently, hydrogen and C 1-6 Alkyl, 5-6 member heterocyclo, phenyl, benzyl, and 5-6 member heteroaryl, e.g., hydrogen, C 1-6 R represents alkyl, phenyl, benzyl, or pyridyl (wherein R is the formula). a , R b and R c In this context, alkyl, heterocyclo, phenyl, benzyl, and heteroaryl groups are either unsubstituted or halogen, hydroxyl, =O, -NH2, -SO3 - and C 1-6 (Substituted with one or more substituents selected from alkoxys). If present, group R a , R b and R c They may be the same or different. In one preferred embodiment, multiple R a , R b and R cWhen groups are bonded to the same Y portion, one of the groups is defined as described above, while the other R bonded to that portion is defined as follows: a , R b and R c The group consists of hydrogen and C 1-3 Selected from alkyl groups. In some embodiments, R a is hydrogen or C 1-4 Alkyl is also acceptable. In some embodiments, R b is hydrogen or C 1-4 Alkyl is also acceptable. In some embodiments, R c is hydrogen or C 1-3 Alkyl is also acceptable.

[0180] In a particular embodiment, R is a functional group R F ; or a functional group R linked via one or more, preferably one linker group L2 F And here R F ha: C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -OC(O)R a , -C(O)R a , -CO2R a -C(O)(NHNH2), -ONH2 and C 1-6 The reactive moiety Y is selected from azidoalkyl; or R is a formula

[0181] [ka]

[0182] (wherein the formula A is as defined above) includes the reactive part; and the reactive part

[0183] [ka]

[0184] The elements may be linked via a linker group L2. In a preferred embodiment of the above embodiment, L2 is an alkyl group in which one or more non-adjacent carbon atoms are substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-. In a more preferred embodiment of the above embodiment, L2 is a C such as methylene or ethylene. 1-4 It is alkyl. The reactive portion in the above embodiment is halogen, C 1-6 Azid, C 2-6 Alkinyl

[0185] [ka]

[0186] They may be selected from, preferably

[0187] [ka]

[0188] That is the case. The reactive portion in the above embodiment is preferably a halogen, C 1-6 Azid, C 2-6 Alkinyl and

[0189] [ka]

[0190] Selected from, preferably

[0191] [ka]

[0192] That is the case.

[0193] In any more preferred embodiment of the above embodiments, base L2-R F C 1-3Haloalkyl, preferably C 1-3 Iodoalkyl or C 1-3 It is a bromoalkyl. In one embodiment, the base R is -L2(R F In the case of )2, L2 is C 1-4 It is alkyl, and the first R F -CO2R a , the second R F -NR a R b or -NH-Boc (wherein Boc is the protecting group tert-butoxycarbonyl). Preferably, in the above embodiment, L2 is a C2 alkyl, and the first R F The base is -CO2H and the second R F It is NH2.

[0194] In one embodiment, R is the group -L2-Y, where Y is hydroxyl, -OR a , -NR a C(O)R b , -NR a R b and -(NR a R b R c ) + L2 is C 1-3 Alkyl, preferably methylene or ethylene.

[0195] In another embodiment, R is -SR a ,-S(O)R a -S(O)2R a , -C(O)R a , -CO2R a -C(O)NR a R b It is. a , R b and R c This is defined as described above.

[0196] In a preferred embodiment, the side chain R corresponds to that used in any of Examples 1a to 2ag. In further embodiments, the side chain is any group R that forms one of the native amino acids together with a -CH2-, -CXF-, or -CF2- linking group (from formula (III), (II), or (IV), respectively) and the residue to which it is bound (except that the γ carbon of the residue is substituted with one or two applicable fluorines). Functional group R F It may be bonded to any suitable location on the linker group, preferably at an end position such as a terminal carbon.

[0197] Embodiment (i) Functional side portion When using the method of embodiment (i), it has been found that using a specific halogen compound as group R can cause a side reaction in which a group other than R is added to the protein or peptide. Therefore, in a preferred embodiment of this embodiment where R is a halogen, it is fluorine.

[0198] When group X is hydrogen, R is preferably a group that can stabilize an intermediate in which the radical is located on an adjacent carbon. In a preferred embodiment of this embodiment, R is a halogen, hydroxyl, or -OR a , -SR a -SOR a , -SO2R a , -OSO3R a , -NR a COR b , -NR a CO2R b ,-NHCONR a R b , -NHCNH2NR a R b , -NR a SO2R b , -N(SO2R a )2, -NHSO2NR a R b , -OCOR a , -COR a , -CO2R a ,-CONR a R b , -CON(OR a )R b -SO2NR a Rb or -SO(NR a )R b Preferably, R is -CO2R a or -CONR a R b Most preferably, R is -COOH. base R a , R b and R c This is as defined above.

[0199] Embodiment (ib) Functional side portion When the radical precursor compound of formula (IV) is used (embodiment (ib)), R is -COOR d and -CONR d R e (In the formula, R d is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, heterocyclo, phenyl, benzyl, and heteroaryl (wherein R is used in the formula) d Alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are either unsubstituted or halogen, hydroxyl, =O, -NH2, C 1-6 Alkoxy and -NHCOR e Represents (which is substituted with one or more substituents selected from); and R e is hydrogen or C 1-4 Selected from alkyl (preferably representing hydrogen). Preferably, R d is hydrogen, C 1-6 Alkyl or 5- or 6-membered heterocycline (the alkyl or heterocycline group is unsubstituted or hydroxy, -NH2, C) 1-6 Alkoxy and -NHCOR e (Represents being replaced by one or more substituents selected from the above.) In one embodiment, R d This is hydrogen, or unsubstituted or hydroxyl, -NH2 and C 1-6 C substituted with one or two substituents selected from alkoxy 1-6 It is alkyl. In a more preferred embodiment, R is -C(O)OH, -CONH2, or -GlcNAc.

[0200] Embodiments (ii) and (iia) Functional side portion When the reaction method of embodiment (ii) or (iia) is used, R preferably includes a portion that stabilizes a radial intermediate, such as an adjacent electron-withdrawing group. In one embodiment of embodiments (ii) and (iia), R is a functional group R F ; or one or more functional groups R linked via linker groups of formula L2, typically one or two F That is the case. R F teeth -C 3-10 Cycloalkyl, heteroaryl (wherein the formula the cycloalkyl and heteroaryl groups are unsubstituted or =O, =NR) a , Y and (C 1-6 Substituted with one or more groups selected from alkyl)-Y; or -C 2-6 Alkinyl, halogen, -SR a ,-S(O)R a -S(O)2R a , -OSO3R a , -NR a C(O)R b , -NR a CO2R b ,-NHC(O)NR a R b , -NHCNH2NR a R b , -NR a SO2R b , -N(SO2R a )2, -NHSO2NR a R b ,-OC(O)R a , -CO2R a -C(O)NR a R b , -C(O)(NHNH2), -ONH2, -C(O)N(OR a )R b -SO2NR a Rb Or -SO(NR a )R b ;Cyano, Nitro, C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + The reactive group Y is selected from among them.

[0201] In this embodiment, the reactive group Y is a halogen, C 1-6 Azid, C 2-6 Alkinil,

[0202] [ka]

[0203] They may be selected from, preferably

[0204] [ka]

[0205] That is the case.

[0206] In this embodiment, R is C 1-6 Hello, preferably C 1-6 Bromo or C 1-6 It is iodine.

[0207] In the case of L2, R a , R b and R c This is as defined in any of the embodiments above.

[0208] In preferred embodiments of embodiments (ii) and (iia), R g but

[0209] [ka]

[0210] In this case, R can be methyl, tert-butyl, propeneyl, phenyl, or -C(O)R g isn't it.

[0211] In a further embodiment, R h C may be substituted with one or more hydroxyl groups. 1-6 Alkyl or C 2-6 In the case of alkenyls, R is -C(O)R h isn't it.

[0212] In embodiments (i), (ia), and (ib), the fluorine group present in the radical precursor compound acts as a stabilizing group.

[0213] Further reactions of the side chain In some embodiments, the functional side chain attached to a protein or peptide is a group that can undergo further reactions to modify it or to attach it to one or more further molecules. Accordingly, in embodiments, the present invention also provides the methods defined above for which a functional side chain attached to a protein reacts further to modify it or to attach it to further molecules.

[0214] As described above, the group R bound to a protein or peptide can be further reacted through any suitable reaction, for example, by linking them to one or more further molecules of interest. The further reaction is preferably a biocompatible reaction, i.e., a reaction that can be carried out with minimal damage to the protein or peptide, for example, a reaction that can be carried out under aqueous conditions without requiring excessive temperature. In a preferred embodiment, the group R comprises one or more of the above-described reactive sites that can be reacted through further linking reactions, as described below. Those skilled in the art will know of suitable linking reactions, such as standard "click chemistry" reactions, via azides, alkynyls, and reactive esters, such as NHS esters.

[0215] Further molecules that can bind to the reactive functional side chain portion of a functionalized protein / peptide include, but are not limited to, pharmaceuticals, sugars, polysaccharides, peptides, proteins, vaccines, antibodies, nucleic acids, viruses, labeled compounds, biomolecules, and / or polymers. In preferred embodiments, the further molecules are pharmaceuticals, sugars, peptides, proteins, antibodies, biomolecules, or polymers, and are preferably peptides, proteins, or polymers. These terms are defined above or below in relation to the functional side chain portion.

[0216] In one embodiment, if group R contains a suitable electrophile such as a halogen, it can react with a nucleophile, such as an off-protein nucleophile, via nucleophilic substitution by replacing a suitable leaving group such as a halogen. For example, if group R contains a halogen moiety, preferably a terminal halogen, it can generate a CS bond by reacting with a nucleophile such as a thiol (e.g., beta-mercaptoethanol); a CP bond by reacting with TCEP (tris(2-carboxyethyl)phosphine); or a CN bond (e.g., methylamine or N3 - The reaction may be carried out via appropriate chemistry (by reacting together with ). Alternatively, the electrophile containing group R may be reacted with an appropriate nucleophile on a further protein or peptide, such as a cysteine ​​or lysine side chain, to bind the protein or peptide to another protein or peptide. By adjusting the pH, the concentration of the off-protein nucleophile, and the choice of halogen, it is possible to selectively promote intermolecular nucleophilic substitution at the C-halogen bond while avoiding competing side reactions such as elimination or nucleophilic substitution within the protein.

[0217] In further embodiments, the halogen present on group R may be substituted with an alternative halogen group, such as I to Cl or Br to Cl, via a Finkelstein-type reaction. This may be done in addition to, or prior to, attaching group R to the desired further molecule, for example, via nucleophilic substitution.

[0218] In a further embodiment, the side chain itself is the base

[0219] [ka]

[0220] If it contains a stabilizing radical precursor such as, it may be activated using the reaction conditions described above, e.g., light irradiation, photocatalysis, and an iron(II) source, to provide a “protein-on” radical on the protein or peptide in question, as described in Example 4 (Figure 3). The protein-on radical may be further reacted with any suitable group containing a SOMO acceptor residue such as an alkene group. For example, to provide site-selective binding between a functionalized protein / peptide and a further protein / peptide, the protein-on radical may be reacted with a SOMO acceptor residue, e.g., an alkene group (e.g., C 1-6 It may be reacted with a protein or peptide having a side chain containing an alkene group, such as a further protein or peptide containing a Dha or Dhb residue.

[0221] Alternatively, to provide radical-initiated polymerization on a protein / peptide, a radical on the protein may be reacted with a suitable alkene containing monomer units.

[0222] For example, functionalized proteins or peptides have a general formula

[0223] [ka]

[0224] The monomer may be reacted with the monomer to provide a functionalized protein or peptide containing at least one functionalized residue of the following formula (IP).

[0225] [ka]

[0226] (wherein L is a linker group or bond as defined above; R z (wherein X is hydrogen or methyl, preferably hydrogen; and X is as defined above).

[0227] The monomers used are

[0228] [ka]

[0229] In this case, the base Rpol is

[0230] [ka]

[0231] (where q is typically 1 to 20, preferably 1 to 10, more preferably 1 to 5, most preferably 1, 2, or 3).

[0232] monomer

[0233] [ka]

[0234] If used, the base Rpol is instead

[0235] [ka]

[0236] That is the case.

[0237] The pendant groups Rpb and Rpc may, in some embodiments, be bonded together to form a ring. The polymer group Rpol may be terminated with any suitable group, such as hydrogen.

[0238] In one embodiment, the radical generated on the protein is given by formula,

[0239] [ka]

[0240] It may react with one or more monomers.

[0241] In an alternative embodiment, the radicals generated on the protein are hydroxy-TEMPO, or a diselenium compound of the formula Rh-SE-Rh (where each Rh is C), as shown in Figure 3. 1-6 Alkyl, C 1-6 Cycloalky, or C 1-6 The group Rpol may be reacted with a further radical-terminated group such as an aryl, preferably a phenyl. In such embodiments, the further functionalized protein or peptide produced is such that the group Rpol is Rrad (wherein Rrad is a radical-terminated group, -Se-R h ,or

[0242] [ka]

[0243] Except for being replaced by ), it may contain at least one functionalization residue as described in formula (IP) above.

[0244] If the method according to any one of claims 3 to 6 includes a functional side chain moiety comprising a reactive moiety as defined in any one of claims 4 to 6, the method further includes reacting a peptide or protein via one of the reactive moieties to link the functional side chain to a further molecule.

[0245] Functionalized proteins and peptides Further embodiments of the present invention relate to functionalized proteins or peptides produced by any of the methods described above.

[0246] The present invention also provides a functionalized protein or peptide containing a functionalized residue of the general formula (IA) shown below, which can be obtained from the method described in embodiment (i), (ia), or (ib) of the above method.

[0247] [ka]

[0248] The group Rz represents hydrogen or methyl. In a preferred embodiment, Rz represents hydrogen. R may be defined in any of the embodiments described above. In certain embodiments, R is a functional group R F ; or a functional group R linked via one or more, preferably one linker group L2 F (In the formula, R F (and L2 are as defined herein). Preferably, R F C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -OC(O)R a , -C(O)R a , -CO2R a -C(O)(NHNH2), -ONH2 and C 1-6 Azidoalkyl: is a reactive moiety selected from; or R is a formula

[0249] [ka]

[0250] (wherein A is defined above) comprises a reactive part; and reactive part

[0251] [ka]

[0252] The atoms may be linked via a linker group L2. Preferably, L2 is an alkyl group in which one or more non-adjacent carbon atoms are substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-. More preferably, L2 is a C such as methylene or ethylene. 1-4 It is alkyl.

[0253] In further embodiments, R may be a group resulting from the reaction of a functionalized side chain with a further molecule, as described in "Further Reactions of Side Chains" above. For example, R may be a group resulting from the generation of a radical on a protein by reaction with a further protein or peptide containing a SOMO acceptor residue, or a radical acceptor such as a monomer containing a radical acceptor group, following the activation of an ASOOF group on the protein.

[0254] In certain embodiments, R is linked either directly or via a linker group, preferably directly linked.

[0255] [ka]

[0256] (wherein A is as defined with respect to embodiment (i) above).

[0257] In a further embodiment, R is C 1-6 Haloalkyl, C 1-6 Azidoalkyl or

[0258] [ka]

[0259] That is the case.

[0260] In a preferred embodiment of the above embodiment, base R is

[0261] [ka]

[0262] Such proteins or peptides may be obtained, for example, by the method of embodiment (ia), i.e., by using an iodine-ASOOF radical precursor compound.

[0263] The group X in any of the above definitions may be selected from fluorine or hydrogen. In a preferred embodiment, X is fluorine.

[0264] The present invention provides a functionalized protein or peptide of the general formula (IB) shown below, which can be obtained by the method described in embodiment (ii) or (iia) of the above method.

[0265] The functionalized peptides described herein may be obtained by any suitable method as described above.

[0266] [ka]

[0267] Ry is either hydrogen or methyl. In a preferred embodiment, Ry represents hydrogen. Rbac is C 1-6 It is an alkyl group (wherein the formula the terminal carbon is substituted with at least one halogen). In a preferred embodiment, Rbac is C 1-4The alkyl group is (wherein the formula the terminal carbon is substituted with at least one halogen). In one embodiment of the above embodiment, the halogen is bromine or iodine.

[0268] In a further embodiment, Rbac is expressed by the following formula

[0269] [ka]

[0270] (wherein Z is a halogen). In one embodiment of the above embodiment, Z is bromine or iodine.

[0271] Covalent bonding of proteins / peptides For example, as described in the section above relating to further reactions of side chains, the functionalized proteins and peptides of the present invention may be further reacted to form covalent bonds with other proteins and peptides.

[0272] Therefore, in further embodiments, the present invention relates to a system in which the base R or Rbac is C 1-6 The present invention provides a method for covalently bonding a functionalized protein or peptide, such as one represented by formula (IA) or (IB) which is a haloalkyl, produced by any of the methods described above, to a further protein or peptide containing a group that can react with an alkyl halide to form a covalent bond.

[0273] The group that can react with the haloalkyl group may be a suitable nucleophilic group such as a hydroxyl, thiol, or amine group, as found in the side chains of various natural amino acids such as serine, cysteine, and lysine. In one preferred embodiment, the group that can react with the haloalkyl group is a thiol group of a cysteine ​​residue.

[0274] In a preferred embodiment of the above method, the functionalized protein or peptide and further proteins or peptides are "protein partners" that interact to form a protein-protein interface, bringing the halogenated alkyl group into close proximity to a group that can react with the halogenated alkyl group, when they are together in solution, optionally in the presence of further biological molecules such as enzymes and cofactors. This proximity allows a reaction to occur between the two groups, for example, by nucleophilic substitution. This proximity-driven reaction greatly increases the effective molar ratio of the groups relative to each other, enabling highly site-specific covalent bonding, as described in Examples 5 and 6.

[0275] In a particularly preferred embodiment, the protein-protein interface is a binding pocket in which one of a functionalized protein / peptide and a further protein / peptide is held in the binding pocket of the other protein / peptide. In a preferred embodiment of this embodiment, at least one of the proteins / peptides is an enzyme and the other is a substrate of the enzyme. The protein or peptide is preferably held in the binding pocket of the enzyme such that a reaction between an alkyl halide and a group reactable with the alkyl halide (e.g., a nucleophilic group) occurs at the active site of the enzyme. Preferably, the active site comprises one or more cysteine ​​residues configured to react with the alkyl halide.

[0276] Typically, in the above embodiment, the functionalized protein / peptide is a substrate having an alkyl halide at a position that will be held in a binding pocket of a further protein / peptide that is a substrate acceptor. If the binding pocket contains a nucleophilic group, particularly a thiol group of a cysteine ​​residue, the alkyl halide in the binding pocket will form a covalent bond with the cysteine ​​residue.

[0277] In a particular embodiment of the above embodiment, the enzyme or receptor protein / peptide is inhibited by the binding.

[0278] Therefore, the present invention provides a method for selectively introducing halogenated alkyl groups into proteins or peptides, such as enzyme substrates. The halogenated alkyl groups may be introduced at a position that allows them to enter the active site of the enzyme substrate. For example, a lysine residue involved in the binding interaction with the substrate can be modified by replacing it with a DHA residue, and then a halogenated alkyl group can be linked using the method of the present invention. The halogenated alkyl group thus introduced can then enter the binding pocket of the substrate and covalently bond with any nucleophilic group present in the binding pocket, such as a cysteine ​​residue, thereby inactivating the substrate (e.g., an enzyme). In this way, the method of the present invention can be used to selectively modify proteins / peptides and provide novel inhibitors.

[0279] In preferred embodiments of the above embodiments, the haloalkyl side chain R or Rbac on the functionalized protein or peptide is a bromoalkyl or iodoalkyl, preferably C 2-3 Bromoalkyl or C 2-3 iodoalkyls are, by industry, -CH2CH2BR, -CH2CH2I, -CH2CH2CH2BR, or -CH2CH2CH2I.

[0280] In further embodiments, the present invention provides a method for covalently bonding a functionalized protein or peptide described in formula (IA) above with a further protein or peptide (wherein the group R in the functionalized protein or peptide is

[0281] [ka]

[0282] (and further proteins or peptides include a group that can react with a radical species to form a covalent bond). Group A is as defined above with respect to embodiment (i). Functionalized proteins can be prepared by any suitable method as described in embodiment (ia) above.

[0283] This covalent bond may be formed, for example, by the application of light in the presence of a suitable photocatalyst and Fe(II) source, as described in detail in the above embodiments (e.g., Embodiment (i) and Example 4), or by the generation of radicals on the protein, as described in the above section on further reactions of the side chains. The radicals on the protein may then react with further proteins or peptides containing a group that can react with the radical species to form a covalent bond. Such a group that can react with the radical species may be an alkene group (e.g., C 1-6 Examples include SOMO acceptor residues such as alkene groups (e.g., C). Therefore, suitable further proteins or peptides include alkene groups (e.g., C). 1-6 It contains residues having an alkene side chain, preferably a side chain containing dha and / or dhb as described above.

[0284] In a preferred embodiment of this design, the further protein or peptide contains one or more dha residues.

[0285] In a preferred embodiment of the above embodiment, the functionalized protein or peptide of formula (IA) is such that Rz is hydrogen, X is fluorine, and A is a heteroaryl. In a more preferred embodiment, A is pyridinyl, pyrimidinyl, or benzothiazolyl, most preferably 2-pyridinyl.

[0286] In further embodiments, the present invention provides compounds of formula (II) or (III) as defined above.

[0287] In a further embodiment, the present invention provides the use of a compound of formula (II) or (III) as defined above in a method for functionalizing a protein. In a preferred embodiment of the above embodiment, the method is one of the protein functionalization methods using formula (II) or (III), respectively.

[0288] definition As used herein, the term "alkyl" refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated by the prefix. Therefore, the term "C 1-4 "Alkyl" refers to a linear saturated monovalent hydrocarbon radical with 1 to 4 carbon atoms or a branched saturated monovalent hydrocarbon radical with 3 or 4 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl). Preferably, the alkyl group is C 1-20 Alkyl alkyl groups, more C 1-12 Alkyl alkyl groups, and more preferably C 1-8 Alkyl alkyl groups, and most preferably C 1-4 It is an alkyl group. 1-6 "alkoxy", "C 1-6 "Ester", "C 1-6 Azidoalkyl, C 1-6 Derived expressions such as "ether" are interpreted accordingly.

[0289] As used herein, the term “alkenyl” refers to a linear or branched monovalent hydrocarbon radical having the number of carbon atoms indicated by the prefix and having at least one double bond. Thus, the term “C 2-6 "Alkenyl" refers to a linear monovalent hydrocarbon radical having 2 to 6 carbon atoms and at least one double bond, or a branched monovalent hydrocarbon radical having 3 to 6 carbon atoms and at least one double bond (e.g., ethenyl, propenyl, 1,3-butadienyl, (CH2)2CH=C(CH3)2, CH2CH=CHCH(CH3)2, etc.). Preferably, the alkenyl group is C 2-20 Alkenyl group, more preferably C 2-12 Alkenyl group, more preferably C 2-8 Alkenyl group, and most preferably C 2-4 It is an alkenyl group.

[0290] As used herein, the term “alkynyl” refers to a linear or branched monovalent hydrocarbon radical having the number of carbon atoms indicated by the prefix and having at least one triple bond. Thus, the term “C 2-6"Alkynyl" refers to a linear monovalent hydrocarbon radical having 2 to 6 carbon atoms and at least one triple bond, or a branched monovalent hydrocarbon radical having 4 to 6 carbon atoms and at least one triple bond (e.g., ethynyl, propynyl, etc.). Preferably, the alkynyl group is C 2-20 Alkynyl group, more preferably C 2-12 Alkynyl group, more preferably C 2-8 Alkynyl group, and most preferably C 2-4 It is an alkynyl group.

[0291] As used herein, the term “cycloalkyl” refers to a cyclic or bicyclic monovalent hydrocarbon radical having the number of carbon atoms indicated by the prefix. Cycloalkyls are typically saturated. Therefore, the term “C 3-10 "Cycloalkyl" may refer to, for example, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, or to bicyclo[3.1.0]hexanyl, bicyclo[4.1.0]heptanyl, and bicyclo[2.2.2]octanyl.

[0292] As used herein, the term "heterocyclyl" means a ring atom consisting of one or two N, O, or S(O) atoms. n The term heterocyclyl refers to a monovalent monocyclic or bicyclic group of 4 to 8 ring atoms, where n is an integer from 0 to 2, and the remaining ring atoms are carbon (C). Examples of heterocyclyls include, but are not limited to, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, piperazinyl, tetrahydropyranil, and thiomorpholinyl.

[0293] As used herein, the term “aryl” refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical (e.g., phenyl or naphthyl) with 6 to 10 ring atoms.

[0294] As used herein, the term “heteroaryl” refers to a monovalent monocyclic or bicyclic aromatic radical of 5 to 10 ring atoms, where one or more, preferably 1, 2, or 3 ring atoms are heteroatoms selected from N, O, or S, and the remaining ring atoms are carbon. Typical examples, but not limited to, include pyrrolyl, thienyl, thiazolyl, imidazolyl, furanyl, indolyl, iso-indolyl, oxazolyl, isoxazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazolyl, tetrazolyl, and preferably pyridinyl, pyrimidinyl, pyrazinyl, or pyridadinyl.

[0295] As used herein, the term "alkoxy" means -OR 9 Radical (R 9 (where is the alkyl group as defined above), for example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, tert-butoxy, etc. Preferably, the alkoxy group is C 1-20 Alkoxy group, more preferably C 1-12 Alkoxy group, more preferably C 1-8 Alkyl group, most preferably C 1-4 It is an alkoxy group.

[0296] As used herein, the term “halo” refers to fluoro, chloro, bromo, or iodine, preferably fluoro or chloro.

[0297] As used herein, the term polyethylene glycol is defined in formula

[0298] [ka]

[0299] (n is not particularly limited, but can be 1 to 500, preferably 1 to 200, more preferably 1 to 50, and one end is covalently bonded to a group such as a functionalized protein or peptide, and the other end is bonded to a hydrogen atom or further group) refers to a divalent radical polymer. In some embodiments, n is 1 to 10, typically 1 to 5, preferably 1 to 3.

[0300] As used herein, the term photocatalyst refers to a redox catalyst that increases its oxidative and / or reductive potential in response to stimulation by the emission of light of an appropriate beam, for example, by the excitation of electrons to higher energy levels. The oxidation half-potential as defined herein is measured relative to a saturated calomel electrode. The oxidation half-potential of a photocatalyst is the oxidation half-potential of the catalyst in its oxidation state, typically in its photoactivated state.

[0301] If the compounds and functional groups described herein have one or more chiral centers, they may exist as enantiomers accordingly. If the compounds used in the present invention have two or more chiral centers, they may further exist as diastereomers. The present invention is understood to extend to the use of all such enantiomers and diastereomers, as well as mixtures thereof in any proportion, including racemates. The formulas drawn below are intended to represent all individual stereoisomers and all possible mixtures thereof, unless otherwise noted or indicated. Furthermore, some of the compounds and groups described herein may exist as tautomers, such as keto (CH2C=O) ↔ enol (CH=CHOH) tautomers or amide (NHC=O) ↔ hydroxyimine (N=COH) tautomers. The formulas drawn below are intended to represent all individual tautomers and all possible mixtures thereof, unless otherwise noted or indicated.

[0302] Unless otherwise specified, each individual atom present in a group or formula as defined herein may actually exist in any form of its spontaneously occurring isotope, with the most abundant isotope being preferred. For example, each individual hydrogen atom present in a formula as defined herein may exist in any form of its spontaneously occurring isotope. 1 H, 2 H (deuterium) or 3 H (tritium) atoms, preferably 1 They may exist as H. Similarly, for example, each carbon atom present in any of the formulas depicted herein may exist as H. 12 C, 13 C or 14 C atom, preferably a C atom. 12 It may exist as a carbon atom.

[0303] If the compound used in the present invention supports an acidic moiety, such as a carboxyl group, the disclosure also includes suitable salts thereof, such as alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium salts; and salts formed with suitable organic ligands (e.g., quaternary ammonium salts).

[0304] If it is said that a portion may be substituted, it may be substituted, for example, by 0, 1, 2, or 3 groups. In some embodiments, it is substituted by 0, 1, or 2 groups, preferably 0 or 1 group.

[0305] When one group is bonded to another, for example, when a peptide, pharmaceutical, or sugar is bonded to a linker, they may be bonded by any suitable means known to those skilled in the art in the field of protein conjugation (such as esterification with a hydroxyl or carboxyl group on the molecule of interest).

[0306] As used herein, the term “amino acid” refers to any natural or synthetic amino acid, i.e., an organic compound containing carbon, hydrogen, oxygen, and nitrogen atoms and containing both amino (-NH2) and carboxylic acid (-COOH) functional groups. Typically, an amino acid is an α-, β-, γ-, or δ-amino acid. Preferably, the amino acid is one of the 22 naturally occurring proteinogenic α-amino acids. Alternatively, amino acids are synthetic amino acids, for example, α-amino-n-butyric acid, norvaline, norleucine, alloisoleucine, t-leucine, α-amino-n-heptanoic acid, pipecholic acid, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine, β-alanine, β-amino-n-butyric acid, β-aminoisobutyric acid, γ-aminobutyric acid, α-aminoisobutyric acid, isovaline, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N- The amino acids are selected from ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine, isoserine, α-hydroxy-γ-aminobutyric acid, homonorleucine, O-methyl-homoserine, O-ethyl-homoserine, selenium homocysteine, selenium methionine, selenium ethionine, carboxyglutamic acid, hydroxyproline, hypsin, pyroglutamic acid, aminoisobutyric acid, dehydroalanine, β-alanine, γ-aminobutyric acid, δ-aminolevulinic acid, 4-aminobenzoic acid, citrulline, 2,3-diaminopropanoic acid, and 3-aminopropanoic acid. Furthermore, the amino acids may be dehydroalanine, dehydrobutyrine, or synthesized dehydroalanine or dehydrobutyrine precursors. The stereocentered amino acids may exist as a single enantiomer or as a mixture of enantiomers (e.g., a racemic mixture). Preferably, when the amino acid is an α-amino acid, the amino acid has L-stereochemistry with respect to the stereoisomer center of the α-carbon.

[0307] All documents referenced herein are incorporated herein by reference.

[0308] Examples The following are examples illustrating the present invention. However, these examples are not intended to limit the scope of the present invention in any way.

[0309] Unless otherwise specified, parameters and values ​​are measured as defined in the following examples.

[0310] General method Unless otherwise specified, chemical reagents, culture media, and E. coli cell lines were obtained from commercially available suppliers (Sigma-Aldrich, Fluorochem, Carbosynth, VWR, Alfa Aesar, Fisher Scientific) and used without further purification. Sonication was performed using a Fisher Scientific Model 505 Sonic Dismembrator. Proteins were purified using an Akta FPLC System UPC-900 (GE Healthcare, UK). Gel electrophoresis was performed using Invitrogen NuPAGE 4-12% Bis-Tris gels, Novex MiniCell tanks, and a BioRad PowerPac controller. Western blotting was performed using a Thermo-Fisher iBlot gel transfer system. Antibodies were used according to the manufacturer's recommendations: anti-histone H3 (96C10)Mouse mAb for histone detection, Mouse monoclonal Anti-polyhistidine-Alkaline Phosphatase, Clone HIS-1 (Sigma, A5588) for KDM4A detection (6His tag), and Rabbit Anti-Mouse IgG (H+L)HRP conjugate (Promega, W4021) and Goat Anti-Mouse IgG H&L Alkaline Phosphatase (Abcam, ab97020) as secondary antibodies. Thin-layer chromatography was performed using Silica Gel 60 F254 plates (Merck) with 1-10% methanol in dichloromethane. Nuclear magnetic resonance spectra were recorded with a Bruker AVIII HD 400 nanobay (400MHz) spectrometer and analyzed with MestReNova11. Carbon nuclear magnetic resonance spectra were recorded using a Bruker DQX 400 (100 MHz) spectrometer. All 1H-NMR chemical shifts are cited in ppm using residual solvent as an internal standard for TMS (d6-acetone: 2.09 ppm).All 13C NMR chemical shifts are cited in ppm, using the central solvent peak as an internal standard for TMS (d6-DMSO 39.3 ppm). Coupling constants (J) are reported in Hertz (Hz). Infrared (IR) spectra were recorded with a Bruker Tensor 27 Fourier-Transform spectrophotometer. High-resolution low-molecular-weight mass spectra were recorded with a Micromass LCT (resolution = 5000 RWHM) using a rock spray source. Protein crystal structures were analyzed and displayed using MacPyMOL v.1.3 (Schrodinger, Inc.). Synthetic gene fragments (i.e., human histone eH3-FLAG-HA construct) were obtained from GeneArt Gene Synthesis (Thermo-Fisher). Nucleotide sequences were verified by Source Bioscience DNA Sanger sequencing services, based at the University of Oxford.

[0311] mass spectrometry Liquid chromatography / mass spectrometry (LC-MS / MS) was used to confirm site-selective post-translational protein editing and identify potential by-products. A general workflow for bottom-up LC-MS / MS analysis of post-translational edited proteins is described below. Samples were reduced (typically using TCEP or DTT) and alkylated (using iodine or chloroacetamide). Proteins were digested with proteases (trypsin, ArgC, LysC, AspN, elastase, etc.), and the resulting peptides were analyzed. Proteomics software such as PEAKS could be used to perform de-novo sequencing on the measured spectra or compare them to a database of protein sequences. Modifications were identified and manually validated.

[0312] Unaltered protein mass spectrometry Unaltered protein mass spectrometry was performed using a Waters Xevo G2-S QTof and Water Acquity UPLC combination. Separation was performed using a Thermo Proswift (250 mm x 4.6 mm x 5 μm) column with water + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) as the eluent systems, using a 10-minute linear gradient. Nitrogen was used as the desolvation gas for positive electrospray ionization (600 L / h). The operating voltages were 3000V for the capillary and 160V for the cone. Continuous calibration to leucine enkephalin standard solution was ensured by rock spray analysis.

[0313] Raw spectra containing multiple charged ion series were deconvolved using MassLynx(Waters) and its maximum entropy (MaxEnt1) deconvolution algorithm (Resolution 1.00 Da / channel, full width at half maximum: ion series / protein dependent, minimum intensity ratio: 33% left to right). Spectra were deconvolved between 10,000 and 20,000 Da for African clawed frog histone H3, between 10,000 and 25,000 Da for human histone eH3.1, between 5,000 and 15,000 Da for African clawed frog histone H4, between 10,000 and 30,000 Da for NPβ, between 30,000 and 50,000 Da for AcrA, and between 30,000 and 40,000 Da for PanC. Reaction conversion rates were calculated from the relative peak intensities of the deconvolved spectra. In histones, baseline (~10%) methionine oxidation frequently occurs during manufacturing, storage, and use, and these "+16 Da adducts" were aggregated into this starting material / product sum.

[0314] Tandem mass spectrometry Digestion in ArgC solution Variant 1: Denatured protein sample, no alkylation. Approximately 10 μg (20 μL) of desalted and denatured modified protein sample was placed in a 50 mM TEAB to a total volume of 100 μL and reduced with 10 mM TCEP at rt for 30 minutes. The sample was digested with Arg-C (1:20 w / w) in activation buffer (50 mM TEAB, 0.2 mM EDTA, 5 mM TCEP) at 37°C for 3 hours. The reaction was stopped by adding 10% FA to a final concentration of 0.5%. The sample was desalted with C18 (Oasis HLB 10 mg), dried with a speed-vac, and then resuspended in 5% FA and 5% DMSO.

[0315] Variant 2: Modified, alkylated Approximately 10 μg of modified protein sample was taken with 8 M urea in 50 mM TEAB containing 20 mM methylamine, brought to a total volume of 100 μL, and denatured at room temperature for 30 minutes. The sample was reduced with 10 mM TCEP at room temperature for 30 minutes and alkylated with 50 mM chloroacetamide at room temperature in the dark for 30 minutes. The sample was diluted with 1 M urea and digested with Arg-C (1:20 w / w) in activation buffer (50 mM TEAB, 0.2 mM EDTA, 5 mM TCEP) at 37°C for 4 hours over time.

[0316] The reaction was stopped by adding 10% FA to a final concentration of 0.5%. The sample was desalted with C18 (Oasis HLB 10 mg cartridge), dried with a speed-vac, and then resuspended in 5% FA and 5% DMSO.

[0317] Digestion in LysC solution Approximately 10 μg (20 μL) of desalted and denatured modified protein sample was taken in 8 M urea in 100 mM TEAB to a total volume of 100 μL. The sample was reduced with 10 mM TCEP at room temperature for 30 minutes and alkylated with 50 mM chloroacetamide at room temperature in the dark for 30 minutes. The solution was diluted with 6 M urea containing 50 mM TEAB and digested overnight at 37°C with LysC 1:20 (w / w). The sample was desalted with C18 (Oasis HLB 10 mg), dried with a speed-vac, and then resuspended in 5% FA 5% DMSO.

[0318] Digestion in trypsin, AspN, or elastase solution Approximately 10 μg (20 μL) of desalted and denatured modified protein sample was taken in 8 M urea in 100 mM TEAB to a total volume of 100 μL. The sample was reduced with 10 mM TCEP at room temperature for 30 minutes and alkylated with 50 mM chloroacetamide at room temperature for 30 minutes in the dark. The solution was diluted in 1 M urea containing 50 mM TEAB and digested with AspN, trypsin, or elastase 1:20 (w / w) at 37°C for 4 hours to overnight. The sample was desalted with C18 (Oasis HLB 10 mg), dried in a speed-vac, and then resuspended in 5% FA 5% DMSO.

[0319] Data collection Standard Data Acquisition - Q Exactive The obtained peptides were separated using a combination of Nanoflow reversed-phase liquid chromatography (UHPLC) Ultimate 3000 UHPLC system (Thermo Fisher Scientific) and a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fischer Scientific). The peptides were loaded onto a C18 PepMap100 pre-column (300 μm x 5 mm inner diameter, 3 μm C18 beads; Thermo Fisher Scientific) and separated using an in-house packed analytical column (ReproSil-Pur 120 C18-AQ, 1.9 μm, 120 Å, 75 μm x 50 cm inner diameter packed with Dr. Maisch GmbH). The separation of cross-linked peptides was performed at a flow rate of 200 nl / min, with a first-stage linear gradient from 15% to 35% of B for 30 minutes, and a second-stage gradient from 35% to 55% of B for another 15 minutes (A: 0.1% formic acid, B: 0.1% formic acid in acetonitrile). Raw data were acquired into the mass spectrometer in data-dependent mode. The system automatically switched from MS to higher-energy collision-induced dissociation MS / MS. Full scan spectra were acquired using Orbitrap [scan range 350–2000 m / z, resolution 70000, automatic gain control (AGC) target 3 × 10⁶, maximum injection time 50 ms]. After the MS scan, the top 10 strongest peaks were selected for HCD fragmentation at 30% of the normalized collision energy. The HCD spectrum was also acquired using Orbitrap (resolution 17500; AGC target 5×10⁴; maximum injection time 120 ms) to obtain the initial fixed mass at 180 m / z.

[0320] Data acquisition of cross-linked samples - Q Exactive Full scan spectra were acquired using Orbitrap [scan range 350–2000 m / z, resolution 70000, automatic gain control (AGC) target 3×10⁶, maximum injection time 100 ms]. After the MS scan, for HCD fragmentation, the top 10 strongest peaks were selected at 30% of the normalized collision energy, excluding 1+ and 2+ charged species. The HCD spectrum was also acquired using Orbitrap (resolution 17500; AGC target 5×10⁴; maximum injection time 120 ms, scan range 200–2000 m / z) for the first fixed mass at 180 m / z.

[0321] Data analysis (tandem mass spectrometry) Standard data analysis Investigations for identification and de-novo analysis were performed using Peaks version 8.5 (Bioinformatics Solutions Inc.). Raw MS files were investigated against a given protein sequence and contaminant list (generated from the MaxQuant contaminants database). Samples were further investigated with MaxQuant against the UniProt human database to confirm sample purity. The precursor mass tolerance was set to 10 ppm. The HCD fragment mass tolerance was set to 0.02 Da. Corresponding proteases were selected considering up to three incorrect cleavages and nonspecific cleavage at one end of the peptide. For elastases, nonspecific investigations were used. Oxidation (methionine), deamination (asparagine, glutamine), carbamide methylation (excluding cysteine-ArgC variant 1 digest), carbamylation (lysine, peptide N-terminus), amidation (C-terminus), and dehydroalanine (cysteine, -33.9887) were defined as variable modifications, similar to the sample-specific modifications shown in the table below. Up to four variable modifications were defined. A peptide-level FDR of 1% and de-novo ALC of 80 were applied. All spectra and identifications were manually validated. Isotope pattern analysis was performed manually using XCalibur Qual Browser 4.0.

[0322] Crosslinking mass spectrometry analysis The crosslinked samples were investigated using Peaks 8.5 as described above to confirm the presence of both proteins and verify the purity of the samples. The crosslinked samples were processed with the pLink 2.3.5 software package. Using the pConfig module, amino acid "B" with mass 205.01023 and composition H(12)C(7)N(1)O(1)Br(1) was defined. Note that the isotope pattern is irrelevant to the crosslinking analysis because HBr is removed. Linker 4BrBut was defined as linking alpha amino acid B to beta amino acid STYCHRKWDENQ with linker composition H(-1)Br(-1).

[0323] The mass tolerance for the first scan was set to 20 ppm, and the mass tolerance for the fragments was also set to 20 ppm. A 10 ppm mass filter was applied. E values ​​were calculated, and the global FDR was set to 1%. Trypsin (or LysC for H3-K4) was selected as a protease with up to three incorrect cleavages. Carbamide methylation (cysteine), oxidation (methionine), deaminolysis (asparagine, glutamine), carbamylation (lysine, peptide N-terminus), and amidation (C-terminus) were set as variable modifications. RAW files were searched against databases containing sequences of modified eH3 constructs and expressed KDM4. The resulting spectra were manually analyzed and validated using pLabel 2.3.5. As a rule of thumb, e values ​​higher than e-03 indicate promising identification, e values ​​higher than e-06 indicate reliable identification, and e values ​​higher than e-10 indicate very good identification.

[0324] Cyclic voltammetry Catechol and 4-bromobutylboronic acid were obtained from Sigma-Aldrich and used as received without further purification. All solutions were prepared at 25°C using ultrapure water with a resistivity of 18.2 MΩcm (Millipore) or higher, and thoroughly degassed with nitrogen (99.998%, BOC Gases plc) before use. A phosphate-buffered saline (PBS) solution (pH=6.0) consisting of 43.85 mM sodium phosphate monobase and 6.15 mM potassium phosphate dibase was used.

[0325] All voltammetry measurements were recorded using an Autolab PGSTAT30 computer-controlled potentiostat (Metrohm, Utrecht, The Netherlands). Experiments were conducted in a constant temperature (25.0 ± 0.3 °C) Faraday cage using a three-electrode setup. A glassy carbon macro electrode (3.0 mm diameter, CH Instrument) was used as the working electrode, a saturated calomel electrode (SCE) (SCE, ALS, distributed by BASi, Tokyo, Japan) as the reference electrode, and a graphite rod as the counter electrode. Before each voltammetry experiment, the working electrode surface was renewed by polishing with alumina slurry (Buehler Ltd, USA) in the order of 1.0 μm, 0.3 μm, and 0.05 μm sizes, followed by sonication in water and drying with nitrogen.

[0326] Comparison with HPLC analysis and LC-MS results All BACED and pySOOF reactions performed were monitored by LC-MS analysis of the crude reaction products, and chromatograms were constructed based on the total ion count detected by the mass spectrometer. In these cases, the ion series was generated by combining all spectra contained within the time frame encompassed by the protein peak in the chromatogram (see below; the maximum protein peak typically occurred around 4.50 minutes).

[0327] HPLC analysis was performed on a Shimadzu 2020 LC-MS instrument equipped with an LCM20AD pump and an SPD-20A UV / Vis operating at 220 nm and 280 nm, using a Phenomenex Jupiter C-4 (5 mm, 300 Å) 4.6 x 250 mm column operating at a flow rate of 1 mL min-1. The analysis was performed using mobile phases of 0.1 vol% TFA in water (solvent A) and 0.1 vol% TFA in MeCN (solvent B), with a linear gradient of 4 minutes at 0% B and 26 minutes from 0 to 100% B, as follows. Chromatograms recorded at 220 nm were analyzed using Shimadzu LabSolutions software.

[0328] 19 F-NMR research 19 F-NMR studies were performed following the general procedure below: FeSO4·7H2O (100 eq) was added to a glass vial (5 mL) and transferred to a glove box. Next, a fixed amount of Dha-tagged protein (1.5–4.6 mg, 0.5–1 mL, typical protein concentration 3–4.6 mg / mL), pySOOF reagent (5 eq [1M] in DMSO) and Ru(bpy)3Cl2 (2.5 eq in 10 μL of water) were added to the glass vial. The vial was then sealed with a plastic cap, transferred from the glove box, and irradiated with blue LED light (50W) for 15 minutes. The crude reaction mixture was treated with DTT (10 mg) or EDTA (5 mg), vortexed for 30 seconds, and purified using a PD MiniTrap G-25 column (GE Healthcare) followed by a PD MidiTrap G-25 column (GE Healthcare) (both columns were equilibrated with buffer in D2O according to a gravity protocol) to obtain fluorescently labeled protein. After concentrating the protein sample to 0.5 mL using a vivaspin column (MCW = 5000), the sample was then... 19 We are ready to record the F-NMR spectrum.

[0329] Histone formation We used bacterial expression plasmids encoding all the standard African clawed frog histones in the pET3 production vector. The genes for WT human histone eH3.1 (C-terminal FLAG-HA tag, C96A and C110A)24,25 were obtained from Thermofischer (GeneArt service) and cloned into pET3d expression plasmids with NcoI and BamHI restriction enzyme sites. Quickchange mutagenesis was performed according to the manufacturer's instructions (QuikChange II Site-Directed Mutagenesis Kit, Agilent) to produce the desired cysteine ​​variants. E. coli BL21(DE3)pLysS were transformed as appropriate and selected with chloramphenicol and ampicillin. Using single colonies, 5–20 mL starter cultures in LB broth were inoculated with the same antibiotics. 1% v / v starter cultures were inoculated into flasks containing 500 mL of 2xTY medium and grown at 37°C to OD600 = 0.4–0.8. Histone production was induced with 0.5 mM IPTG, allowed to proceed for 2 hours, then harvested and resuspended in 5x / weight "wash buffer" (50 mM Tris, pH 7.5, 100 mM NaCl, together with a protease inhibitor cocktail). The suspension was flash-frozen and stored at -80°C until thawed. Thawing was carried out by sonication in the presence of 1 mg of DNase at 40% amplitude for 5 x 30 seconds bursts. The sonicated material was centrifuged at 20 krpm at 4°C for 20 minutes. The supernatant was discarded, and the pellet was resuspended in 40 mL ["wash buffer" + 1% Triton-X detergent]. Sonication was repeated once at 40% amplitude for 30 seconds, and the suspension was centrifuged at 20 krpm for 10 minutes. The pellet was washed two more times in this manner, and then washed once with non-Triton-containing "wash buffer". 1 mL of DMSO was added to the pellet and roughly mixed with a spatula, and histone desolvation was assisted for 10 minutes. 10 mL of "unfolding buffer" (7 M Gdn-HCl, 20 mM Tris, pH 7.5, 10 mM DTT) was added, and the mixture was shaken at rt for 1 hour, after which the mixture was centrifuged at 20 krpm for 10 minutes at room temperature.The supernatant was loaded onto an S200 size exclusion column (GE Healthcare) pre-equilibrated with "SAU-100" buffer (7M urea, 20mM NaOAc, pH 5.2, 100mM NaCl, 1mM EDTA, 10mM DTT, 1mM benzamidine). Proteins were eluted with SAU-100 and analyzed by SDS-PAGE. Histone fractions were pooled and concentrated to 1–4 mL. Histones were further purified using cation exchange chromatography with a linear gradient of 0–100% SAU-1000 buffer ("SAU-100" with a final concentration of 1000 mM NaCl) (HiTrap SP 5 mL). The pure fractions were pooled, dialyzed against water (with 2 mM β-mercaptoethanol), and lyophilized.

[0330] AcrA Plasmid (pET24) was transformed into BL-21 (DE3) cells and plated onto kanamycin agar plates. Four 10 mL starter cultures (LB / kanamycin) of each plasmid were grown overnight at 37°C, then transferred to 500 mL of medium (LB / kanamycin). These cultures were grown at 37°C until OD600 = 0.6 to 0.8 (between 40 and 70 minutes), at which point the cells were induced with IPTG (1.25 mM) and incubated for a further 4 hours. The cells were then pelletized at 8 krpm for 10 minutes. The cell pellet was resuspended in buffer (50 mL (50 mM Tris, 100 mM NaCl, 10 mM imidazole, 1 mg / mL lysozyme, and 0.1 mg / mL DNAse)) and stirred on ice for 2 hours. Next, the pellet was sonicated (50% power, 30 seconds sonication, 1 minute rest, 4 times), and the resulting mixture was centrifuged (20 krpm, 45 minutes). The supernatant was purified using Ni-NTA resin (50 mL (50 mM Tris, 100 mM NaCl, 5 mM imidazole binding buffer) and 20 mL (50 mM Tris, 100 mM NaCl, 250 mM imidazole elution buffer)). The fraction containing the target protein (visualized by SDS-PAGE analysis) was dialyzed to 20 mL (50 mM Tris, 100 mM NaCl), and the concentration was analyzed.

[0331] Human sirtuin 2 (Sirt2) Human Sirt2 gene from pET6 plasmid was transformed into BL21-(DE3) cells and plated onto LB / agar / carbenicillin plates. Single colonies were picked into 5 mL of LB / carbenicillin and grown overnight at 37°C and 250 RPM. This starter culture was transferred to 4 x 500 mL of Superbroth medium and grown at 37°C and 250 RPM until OD 0.6 (2 hours). Expression was induced by adding IPTG stock to a final concentration of 0.3 mM, and incubated at 37°C and 250 RPM for 4 hours. Cells were pelletized at 8 kRPM and 9.6 kG (raverage) for 15 minutes, and the pellets were frozen at -80°C. The pellet was thawed on ice and resuspended in buffer (NaCl 500 mM, Tris 50 mM, Glycerol 5%, βME 5 mM, Imidazole 25 mM, one Roche cOmplete EDTA-free protease inhibitor cocktail tablet, pH 7.5, 10 mL). Cells were lysed on ice by sonication (30% amplitude, 2 seconds on, 2 seconds off for 5 minutes). Insoluble fractions were removed by centrifugation (25 kRPM 52 kG (average) for 1 hour), and the lysate was filtered through a 0.2 μm syringe filter and applied to an FPLC column. The proteins were first purified by passing them through a 1 mL ff-Histrap (A: NaCl 500 mM, Tris 50 mM, Glycerol 5%, βME 5 mM, Imidazole 25 mM pH 7.5, B: A + 225 mM Imidazole pH 7.5, 5 CV A 10 CV B step gradient) using 2D-FPLC. The fraction containing the desired protein (analyzed by SDS-PAGE) was concentrated to ~5 mL using 10 kDa GE vivaspin and passed through an s200 36 / 60 sec column in 150 mM NaCl, 25 mM Tris pH 8.0 buffer to obtain 50 mL of 0.1 mg / mL protein.

[0332] The following proteins were expressed and purified as described in the references below.

[0333] PanC: Dadova, J. et al. Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites. ACS Central Science 3, 1168-1173, doi:10.1021 / acscentsci.7b00341(2017)

[0334] cabLys3: Chen, Z.-L. et al. A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides. Nature Communications 10, 3404, doi:10.1038 / s41467-019-11337-z(2019).

[0335] NPβ-G2F-C61: Wright, T. H. et al. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science, aag1465, doi:10.1126 / science.aag1465 (2016).

[0336] Production of dehydroalanine XI histone H3-Dha9 - 10 mg of lyophilized Xl histone H3-C9 was added to denatured phosphate buffer (100 mM NaPi, pH 8, 3 M Gdn-HCl, 500 μL) and mixed until completely dissolved. DTT (30 mg) was added, and the mixture was shaken at rt and 500 rpm for 30 minutes to reduce the disulfide bonds. The mixture was then removed by desalting in 1 mL of the same buffer (PD Minitrap G25, GE Healthcare). The resulting protein concentration was measured (Nanodrop), and DBHDA (60 eq from freshly prepared 0.5 M DMSO stock) was immediately added. The mixture was then shaken at 25°C for 45 minutes, followed by 37°C for 2 hours (500 rpm). The protein was desalted as before to remove excess DBHDA, and the mixture was replaced with the desired buffer. Protein yield and concentration were measured by Nanodrop, and conversion rates were determined by LC-MS analysis.

[0337] Corresponding procedures were used for the formation of human histone eH3.1-Dha4 and human histone eH3.1-Dha9 from human histone eH3-C4, and for the formation of human histone eH3-C4, respectively.

[0338] The formation of AcrA-Dha123 was carried out as described in Wright, TH et al. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science, aag1465, doi:10.1126 / science.aag1465 (2016).

[0339] PanC-Cys44 / 47-PanC-Cys44 / 47 was buffer-exchanged from storage buffer to sodium phosphate buffer (100 mM, pH 8.0, 3M Gdn·HCl) using a PD MiniTrap G-25 column (GE Healthcare) equilibrated with sodium phosphate buffer (100 mM, pH 8.0, 3M Gdn·HCl), and a protein solution with a concentration of 2.56 mg / mL was obtained according to a gravity protocol. A fixed volume of 1 mL (29.2 nmol) was treated with methyl 2,5-dibromopentanoate (MDBP, 1 M in DMSO, 1.46 μmol) and shaken at 25°C and 500 rpm for 16 hours. Subsequently, excess alkylating reagent was removed using a PD MidiTrap G-25 column (GE Healthcare) equilibrated with ammonium acetate buffer (500 mM, pH 6.0, 3M Gdn·HCl), and a protein solution with a concentration of 1.56 mg / mL was obtained according to a gravity protocol. The conversion rate was determined by analyzing a fixed portion of the purified product by LC-MS.

[0340] A fixed amount of cabLys3-Cys104 in cabLys3-Dha104 PBS buffer (pH 7.4) was treated with DTT (4 mg) and incubated at 25°C for 30 minutes. Then, DTT was removed using a PD MidiTrap G-25 column (GE Healthcare) equilibrated with sodium phosphate buffer (50 mM, pH 8.0), and the solution was concentrated using a vivaspin column (MCW = 5000) according to a gravity protocol to obtain a crude protein solution of 0.9 mg / mL (0.5 mL). Next, DBHDA (0.5 M in DMSO, 14.25 μmol) was added to the protein solution, and the resulting reaction mixture was incubated at 37°C for 150 minutes. Purification was then performed using a PD MiniTrap G-25 column (GE Healthcare) equilibrated with ammonium acetate buffer (100 mM, pH 6.0) according to a gravity protocol. After concentrating the protein sample using a vivaspin column (MCW = 5000), a 0.5 mL stock solution of Dha-tagged cabLys3 was obtained at a protein concentration of 0.9 mg / mL.

[0341] Synthesis example The reagents and compounds used in the examples were synthesized according to the following method. These compounds 1 1H NMR and 13 13C NMR data was obtained and verified by comparing it with literature values.

[0342] 1-Allyl-2,3,5-tri-O-benzoyl-α-D-ribofuranose 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (10.0 g, 19.8 mmol) was added to an ice-cold mixture of allyltrimethylsilane (9.45 mL, 59.5 mmol) in 200 mL of acetonitrile, followed by dropwise addition of BF3·OEt2 (2.69 mL, 21.8 mmol). The reaction mixture was heated to rt over 4 hours, then diluted with saturated NaHCO3 aqueous solution and extracted with Et2O. The combined organic layer was dried over MgSO4, filtered, and concentrated. The oily residue was purified by column chromatography (SiO2, pentane:ethyl acetate (8:2)) to obtain 1-allyl-2,3,5-tri-O-benzoyl-α-D-ribofuranose (7.22 g, 14.9 mmol, 75%) as a green oil. C 29 H 26 O7 (486.5 g / mol).

[0343] 1-Allyl-α-D-ribofuranose To a stirred solution of 1-allyl-2,3,5-tri-O-benzoyl-α-D-ribofuranose (7.00 g, 14.3 mmol) in 50 mL of MeOH, NaOH (3.09 g, 57.2 mmol) was added under N2. The resulting reaction mixture was stirred for 1 hour, then cooled to 0°C and carefully neutralized with methanol solution of HCl (approximately 1 M). The crude mixture was concentrated under reduced pressure and purified by column chromatography (SiO2, ethyl acetate) to obtain 1-allyl-α-D-ribofuranose (2.10 g, 12.1 mmol, 85%) as a yellow oil. C8H 14 O₄ (174.2 g / mol).

[0344] 1-Allyl-2,3-isopropylidene-α-D-ribofuranose 1-Allyl-α-D-ribofuranose (2.00 g, 11.5 mmol) was added to a solution of p-toluenesulfonic acid monohydrate (9.72 g, 51.1 mmol) and triethyl orthoformate (12.1 mL, 72.6 mmol) in 200 mL of acetone. The reaction mixture was stirred overnight at rt. After neutralization with saturated Na2CO3 aqueous solution, the crude mixture was concentrated in a small amount of MeOH, and the product was crystallized from the solution at 0°C. The obtained product was filtered to obtain 1-allyl-2,3-isopropylidene-α-D-ribofuranose (1.50 g, 7.01 mmol, 60%) as a white solid. C9H 18 BN3O2 (211.1 g / mol).

[0345] 1-Allyl-2,3-isopropylidene-5-bromo-α-D-ribofuranose Under an inert atmosphere, CBr4 (1.55 g, 4.67 mmol) and polymer-bound PPh3 (1.23 g, 4.68 mmol) were added at 0°C to a solution of 1-allyl-2,3-isopropylidene-α-D-ribofuranose (0.50 g, 2.34 mmol) in dry CH2Cl2 (10 mL). The resulting reaction mixture was stirred overnight at room temperature. Next, the resin was filtered off, the organic layer was washed with water (2 x 10 mL), dried over Na2SO4, filtered off, and evaporated to dryness. The crude product was purified by column chromatography (SiO2, pentane:ethyl acetate (9:1)) to obtain 1-allyl-2,3-isopropylidene-5-bromo-α-D-ribofuranose (0.34 g, 1.17 mmol, 50%) as a yellow oil. C 11 H 17 BrO3 (277.2 g / mol).

[0346] 1-Allyl-2,3-isopropylidene-5-chloro-α-D-ribofuranose Under an inert atmosphere, 1-allyl-2,3-isopropylidene-α-D-ribofuranose (0.20 g, 0.93 mmol) and polymer-bound PPh3 (0.49 g, 1.87 mmol) were dissolved in CCl4 (10 mL), followed by the addition of imidazole (3 mg, 0.05 mmol). The resulting reaction mixture was heated under reflux overnight. Next, the reaction was quenched with ice-cold water, diluted with CH2Cl2, and filtered on Celite. After evaporating the solvent under reduced pressure, the crude product was purified by column chromatography (SiO2, pentane:ethyl acetate (9:1)) to obtain 1-allyl-2,3-isopropylidene-5-chloro-α-D-ribofuranose (0.17 g, 0.74 mmol, 78%) as a yellow oil. C 11 H 17 ClO3 (232.7 g / mol).

[0347] (4-(5-bromo-α-D-ribofuranose)butyl)boronic acid Under an inert atmosphere, BCl3 in CH2Cl2 (1 M, 0.71 mL, 0.71 mmol) was carefully added at -78°C to a mixture of 1-allyl-2,3-isopropylidene-5-bromo-α-D-ribofuranose (0.13 g, 0.48 mmol) and SiEt3H (91.3 μL, 0.57 mmol). The resulting suspension was stirred at this temperature for 30 minutes and then warmed overnight at rt. The HCl generated in situ induced deprotection of the acetonide group. The resulting mixture was diluted with water and Et2O, and the aqueous layer was extracted with Et2O. The combined organic layers were washed with saline and dried over MgSO4. After removing the solvent under reduced pressure, the crude product was purified by Prep HPLC using an RP XBridge Prep C18 column with a 0.25% NH4CO3 solution in water:CH3CN as the mobile phase, yielding (4-(5-bromo-α-D-ribofuranose)butyl)boronic acid (90.0 mg, 0.32 mmol, 67%) as a white solid. C8H 16 BBrO5 (282.9 g / mol).

[0348] (4-(5-chloro-α-D-ribofuranose)butyl)boronic acid Under an argon atmosphere, BCl3 in CH2Cl2 (1 M, 0.85 mL, 0.85 mmol) was carefully added at -78 °C to a mixture of 1-allyl-2,3-isopropylidene-5-chloro-α-D-ribofuranose (0.13 g, 0.57 mmol) and SiEt3H (108.7 μL, 0.681 mmol). The resulting suspension was stirred at this temperature for 30 minutes and then warmed overnight at rt. The resulting mixture was diluted with water and Et2O, and the aqueous layer was extracted with Et2O. The combined organic layers were washed with saline and dried over MgSO4. After removing the solvent under reduced pressure, the crude product was purified by Prep HPLC using an RP XBridge Prep C18 column with a 0.25% NH4CO3 solution in water:CH3CN as the mobile phase, yielding the pure compound (4-(5-chloro-α-D-ribofuranose)butyl)boronic acid (30.0 mg, 0.13 mmol, 23%) as a white solid. C8H 16 BClO5 (238.5 g / mol).

[0349] Peracetyl-β-D-GlcNAc Montmorillonite K-10 (24.0 g) was added in fractions over 10 minutes to an ice-cold, stirred suspension of D-GlcNAc (6.42 g, 29.0 mmol) in Ac2O (80 mL, 74.0 g, 725 mmol). The ice bath was removed, and the reaction mixture was stirred at this temperature for 24 hours. The reaction mixture was filtered through Celite, and the pad was washed with AcOEt until colorless. The combined filtrate was concentrated under reduced pressure. The orange residue was recrystallized twice from MeOH to obtain the title product as a white needle-like substance (2.39 g, 6.11 mmol, 131 °C, 19.5%). C 16 H 23 NO 10 (389.4 g / mol).

[0350] Peracetyl-iodoethyl-β-D-GlcNAc Under argon, ytterbium(II) triflate (240 mg, 0.387 mmol) was added to a solution of peracetyl β-D-GlcNAc (500 mg, 1.28 mmol) and 2-iodoethanol (400 μL, 882 mg, 5.12 mmol) in dry DCM (15 mL). The reaction mixture was heated under reflux overnight. Analysis by TLC (100% siRNA, sulfuric acid elution) indicated the completion of the reaction by complete consumption of the starting materials (16 hours) (Rf = 0.68). The reaction mixture was washed with Sat. Aq. NaHCO3 (3 x 30 mL) and concentrated. Purification by column chromatography (40% siRNA in petroleum ether, Rf = 0.33) yielded the title product as a colorless amorphous solid (524 mg, 1.04 mmol, 82%). C 16 H 24 INO9 (501.3 g / mol).

[0351] 1-Iodoethyl-β-D-GlcNAc To a solution of peracetyl 1-iodoethyl-β-D-GlcNAc (250 mg, 0.499 mmol) in dry methanol (5 mL), sodium methoxide (25%, 100 μL) in methanol was added and the mixture was stirred until TLC (100% siRNA, sulfuric acid development) showed the disappearance of the starting material and the appearance of a single spot, indicating the completion of the reaction (Rf 0.0). At completion (30 minutes), the reaction mixture was DOWEX H + The mixture was neutralized by adding [a certain substance], stirred for 5 minutes, filtered the reaction mixture, concentrated to 1 mL, washed with a silica plug (which had been thoroughly washed with methanol, water / isopropanol / ethyl acetate 1:2:5), and volatile components were removed under reduced pressure to obtain the title product as a white amorphous solid (153 mg, 0.409 mmol, 82%). C 16 H 24 INO9 (501.3 g / mol).

[0352] Peracetyl-2-chloro-α-D-GlcNAc A suspension of N-acetyl-D-glucosamine (25.0 g, 113 mmol) in acetyl chloride (50.0 mL, 55.0 g, 701 mmol) was sealed with Subasil and a balloon and stirred for 17 hours. At this point, TLC (100% siRNA, H2SO4 evolution) showed complete disappearance of the starting material (Rf 0.0) and the appearance of one major product (Rf 0.70) and one by-product (Rf 0.47). The crimson solution was diluted with DCM (500 mL), washed with saturated aqueous NaHCO3 (3 x 500 mL), dried over MgSO4, concentrated under reduced pressure (to 50.0 mL), and the crude product was precipitated with dry Et2O sodium (1.00 L) to obtain beige crystals. The product was purified by flash column chromatography (Pet ether / siRNA 40% -> 65% gradient elution) to obtain the title sample as a white amorphous solid (17.53 g, 47.9 mmol, 42%). C 14 H 20 ClNO8 (365.8 g / mol).

[0353] Peracetyl-1-azide-β-D-GlcNAc Peracetyl 2-chloro-α-D-GlcNAc (500 mg, 1.37 mmol) and tetrahydrogen sulfate in  (5 mL) and saturated aqueous NaHCO3 (5 mL) n Sodium azide (267 mg, 4.10 mmol) was added in fractions to a rapidly stirred solution of butylammonium (464 mg, 1.37 mmol). The reaction mixture was stirred for 1 hour, at which point TLC (100% siRNA, H2SO4 elution) analysis showed complete disappearance of the starting material (Rf 0.70) and appearance of a single product (Rf 0.52). The organic fraction was washed with saturated aqueous NaHCO3 (3 x 10 mL) and saturated NH4Cl (10 mL), and volatile components were removed under reduced pressure. The white amorphous solid was purified by flash column chromatography (Pet ether / siRNA 50% -> 80% gradient elution) to obtain the title product as a white amorphous solid (387 mg, 1.04 mmol, 76%). C 14 H20 N4O8 (372.3 g / mol).

[0354] Peracetyl-1-amino-β-D-GlcNAc Under Ar, NEt3 (0.9 mL, 0.653 g, 6.45 mmol) and 1,3-propanedithiol (0.6 mL, 0.648 g, 6.00 mmol) were sequentially added to a rapidly stirred solution of peracetyl 1-azido-β-D-GlcNAc (1.00 g, 2.69 mmol) in anhydrous methanol (16 mL). The effervescent reaction mixture was stirred at RT for 2 hours, at which point a large amount of white precipitate was observed floating in a colorless to pale yellow solution. TLC analysis (10% MeOH / CHCl3 anisaldehyde development) showed complete consumption of the starting material (Rf 0.72) and formation of a major spot (Rf 0.41). Methanol was removed under reduced pressure, the residue was dissolved in chloroform, loaded onto a short silica plug, washed with a large amount of chloroform, eluted with MeOH / CHCl3 (10%), the solvent was removed, and the glassy solid was stored overnight under high vacuum. The title sample was obtained as a colorless, glassy solid (0.74 g, 2.15 mmol, 80%). C 14 H 22 N2O8 (346.3 g / mol).

[0355] Peracetyl-1-(iodoacetamide)-β-D-GlcNAc Peracetyl 1-amino-β-D-GlcNAc (500 mg, 1.73 mmol) was added to a stirred solution of EEDQ (427 mg, 1.73 mmol) and iodoacetic acid (323 mg, 1.73 mmol) in THF (10 mL). The reaction mixture was stirred at room temperature for 24 hours, at which point TLC analysis (using 5% MeOH / CHCl3 elution, 254 nm, and anisaldehyde dip) showed significant product development (Rf = 0.38). The reaction mixture was concentrated on diatomaceous earth until dry (5.00 g), loaded onto a chromatography column pre-equilibrated with chloroform, and purified by column chromatography (0→10% MeOH / CHCl3 gradient elution) to obtain the title product as a white amorphous solid (410 mg, 951 μmol, 55%), which turned yellow upon continuous exposure to light. C 16 H 23 IN2O9 (514.3 g / mol).

[0356] 1-(iodoacetamide)-β-D-GlcNAc To a solution of peracetyl 1-(iodoacetamide)-β-D-GlcNAc (410 mg, 0.796 mmol) in methanol (8 mL), sodium methoxide (25%, 200 μL) in methanol was added and the mixture was stirred for 5 minutes. At this point, TLC (MeOH / CHCl330%, anisaldehyde) analysis showed the completion of the reaction by the disappearance of the starting material (Rf 0.9) and the appearance of one spot (Rf 0.45). DOWEX H was added to the reaction mixture. + (352 mg) was added to neutralize the mixture, and after stirring for 5 minutes, the reaction mixture was filtered and concentrated to dryness to obtain the title product as a white to pale orange amorphous solid (303 mg, 774 μmol, 98%), which turned brown upon prolonged exposure to light. C 10 H 17 IN2O6 (388.2 g / mol).

[0357] 2-(3-azidopropyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 3-bromopropylboronic acid pinacol ester (200 mg, 0.80 mmol), sodium azide (525 mg, 8.00 mmol), tetra-n-butylammonium bromide (130 mg, 0.40 mmol), water (2 mL), and ethyl acetate (2 mL) were added to a round-bottom flask. The resulting reaction solution was stirred at 85°C for 16 hours. After cooling to room temperature, water (10 mL) was added, and the resulting aqueous mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over MgSO4, concentrated under vacuum, and 12 g of RediSep R f CombiFlash R with silica gold column f The product (137 mg, 0.17 mmol, 81%) was purified by a flash chromatography system (gradient: 2 minutes 100% hexane followed by a linear gradient over 14 minutes to 50% petroleum ether:siRNA(95:5)) to obtain the product as a colorless liquid. C9H 18 BN3O2 (211.1 g / mol).

[0358] N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)benzamide Under a nitrogen atmosphere, 2-(3-azidopropyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (211 mg, 1.00 mmol) and chloroform (1 mL) were added to a round-bottom flask. Next, 2,6-lutidine (139 mg, 151 μL, 1.30 mmol) and thiobenzoic acid (276 mg, 2.00 mmol) were added to the reaction mixture and stirred at 55°C for 16 hours. The crude reaction mixture was then concentrated under vacuum and dissolved in ELISA (25 mL). The organic layer was washed with sodium bicarbonate solution (sat., 25 mL), water (25 mL), and saline solution (25 mL), dried over MgSO4, and concentrated under vacuum. The crude product was divided into 12 g RediSep R f CombiFlash R with silica gold column fThe product (50 mg, 0.17 mmol, 17%) was purified by a flash chromatography system (gradient: 2 minutes 100% hexane followed by a linear gradient over 14 minutes to 100% petroleum ether:siRNA (1:3)) to obtain the product as a white solid. C 16 H 24 BNO3 (289.2 g / mol).

[0359] N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)acetamide Under a nitrogen atmosphere, 2-(3-azidopropyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (211 mg, 1.00 mmol) and chloroform (1 mL) were added to a round-bottom flask. Next, 2,6-lutidine (139 mg, 151 μL, 1.30 mmol) and thioacetic acid (152 mg, 142 μL, 2.00 mmol) were added to the reaction mixture and stirred at 55°C for 16 hours. The crude reaction mixture was then concentrated under vacuum and dissolved in dimethyl acetate (25 mL). The organic layer was washed with sodium bicarbonate solution (sat., 25 mL), water (25 mL), and saline solution (25 mL), dried over MgSO4, and concentrated under vacuum. 12 g of the crude product was collected. f CombiFlash R with silica gold column f The product was purified by a flash chromatography system (gradient: linear gradient to 100% hexane for 2 minutes, then to 100% siRNA over 14 minutes, and finally to 100% siRNA over 6 minutes) to obtain the product as black oil (60 mg, 0.26 mmol, 26%). C 11 H 22 BNO3 (227.1 g / mol).

[0360] 2-(3-iodopropyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 500 mg, 2.00 mmol of 3-bromopropylboronic acid pinacol ester, 900 mg, 6.00 mmol of sodium iodide, and 5 mL of acetone were added to a round-bottom flask. The resulting reaction solution was stirred at 60°C for 16 hours. After cooling to room temperature, 25 mL of water was added, and the resulting aqueous mixture was extracted with HCl (3 x 25 mL). The combined organic layers were washed with aqueous sodium hyposulfite solution (sat. 2 x 25 mL), water (25 mL), and saline solution (25 mL), dried on MgSO4, concentrated under vacuum, and collected in a CombiFlash R 12 g RediSep Rf silica gold column (gradient: linear gradient from 100% hexane for 2 minutes, then to 100% petroleum ether: EtoAc (97:3) over 14 minutes). f The product was purified using a flash chromatography system and obtained as a colorless liquid (430 mg, 1.45 mmol, 73%). C9H 18 BIO2 (296.0 g / mol).

[0361] 3-Pinacol aminopropylboronic acid 3-azidopropylboronic acid pinacol ester (1.00 g, 4.70 mmol) was added to EtOH (15 mL), followed by 10% Pd-activated C (80 mg). The reaction mixture was bubbling with argon for 15 minutes before being purged with hydrogen for a further 15 minutes, and then allowed to stand for 24 hours of stirring under hydrogen balloon. The mixture was filtered through Celite and the solvent was removed under reduced pressure. The residue was washed with cold ether and filtered, leaving a white powder (310 mg, 1.68 mmol, 36%). C9H 20 BNO2 (185.1 g / mol).

[0362] 3-trimethylaminopropylboronic acid pinacol iodide 3-aminopropylboronic acid pinacol ester (150 mg, 810 μmol) was dissolved in MeOH (8 mL). 2 M LiOH (2.43 mL, 4.86 mmol) was added dropwise, followed by MeI (0.5 mL, 8.10 mmol), and the mixture was stirred at RT for 1.5 hours. The solvent was removed under reduced pressure, and the resulting white solid was extracted with acetonitrile to obtain the desired product in solution. After evaporation under reduced pressure, the solution was triturated with DCM, where the filtrate was evaporated and extracted with acetone to obtain a pale yellow oil (105 mg, 0.38 mmol, 47%). C6H 18 BINO2 (273.9 g / mol).

[0363] 2-acetylamino-N-benzyl-acrylamide To a stirred solution of 2-acetamidoacrylic acid (1.29 g, 10.0 mmol, 1.00 equiv.) and 4-methylmorpholine (1.21 mL, 11.0 mmol, 1.10 equiv.) in THF (100 mL), isobutyl chloroformate (1.43 mL, 11 mmol, 1.10 equiv.) and benzylamine (1.20 mL, 11.0 mmol, 1.10 equiv.) were added. The mixture was stirred at room temperature for 2 hours, then filtered and the solvent was evaporated. The residue was purified by flash chromatography (n-heptane / SiO2; 10-100% SiO2) to obtain the title compound as a white solid (1.62 g, 7.43 mmol, 74%). C 12 H 14 N2O2 (218.3 g / mol).

[0364] (2-Acetamide-3-(benzylamino)-3-oxopropyl)boronic acid To a stirred solution of 2-acetylamino-N-benzylacrylamide (100 mg, 0.46 mmol) in dry THF (5 mL), BH3·THF (1 M, 0.9 mL, 0.92 mmol) was added at 0°C. The mixture was stirred at 0°C for 10 minutes, then warmed to room temperature. The reaction mixture was stirred for 3 days, and 500 μL of water was added to quench it. The solvent was evaporated. The residual liquid was lyophilized and dissolved in H2O for purification. Purification was performed via preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50 x 250 mm, mobile phase: 0.25% NH4HCO3 aqueous solution, MeCN). After lyophilization, the title compound was obtained as a white solid (16.6 mg, 0.06 mmol, 14%). C 12 H 17 BN2O4 (264.0 g / mol).

[0365] BPin - Biotin Under argon, NEt3 (28 μL) was added to a solution of 3-aminopropylboronic acid pinacol ester (15 mg, 81 μmol) and active biotin ester (44 mg, 69 μmol) in anhydrous DCM. The reaction mixture was stirred overnight with rt and allowed to stand, then concentrated. The title product was obtained as a white solid (12 mg, 26%) by purification using flash column chromatography (CHCl3 / MeOH 0→10% gradient elution). C 30 H 55 BN4O9S (658.7 g / mol).

[0366] 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propanoic acid Trifluoroacetic acid (1.5 mL) was added to a solution of 2-tBu-ethylboronic acid pinacol ester (500 mg, 1.95 mmol) in CH2Cl2 (1.5 mL). After stirring the solution at room temperature for 2 hours, it was concentrated under a nitrogen stream and then azeotropically distilled from CH2Cl2 to quantitatively obtain the desired carboxylic acid as a viscous oil, which was used without further purification. C9H 17BO4 (200.0 g / mol).

[0367] 2-((1,1-difluoroethyl)sulfonyl)pyridine Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (193 mg, 1.00 mmol), THF (4 mL), and DMI (0.4 mL) were placed in a heat-gun-dried two-necked flask. The reaction mixture was then cooled to -78°C in an isopropanol / dry ice mixture, followed by the addition of methyl iodide (766 mg, 0.33 μL, 5.40 mmol), and the addition of LiHMDS (1 M in THF, 2.5 mL, 2.50 mmol) dropwise. After complete addition, the mixture was stirred at -78°C for 30 minutes. After quenching with aqueous ammonium chloride solution (sat., 5 mL), the resulting aqueous solution was extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over MgSO4, concentrated under vacuum, and the crude product was purified by column chromatography (SiO2, hexane:ethyl acetate (3:1), dx h. 3.5 x 13 cm) to obtain the product (123 mg, 0.59 mmol, 59%) as a yellow solid. C7H7F2N2O2S (207.2 g / mol).

[0368] tert-butyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)carbamate Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (1.04 g, 5.38 mmol), 3-Boc-1,2,3-oxathiazolidine 2,2-dioxide (1 g, 4.48 mmol), THF (20 mL), and DMI (2 mL) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -95°C in a methanol / liquid nitrogen mixture, followed by dropwise addition of LiHMDS (1 M in THF, 5.5 mL, 5.5 mmol). After complete addition, the mixture was stirred at -95°C. After 30 minutes, the reaction mixture was quenched by adding sulfuric acid (1 M, 20 mL), warmed to room temperature, and stirred for 3 hours. At 0°C, the reaction mixture was adjusted to an alkaline pH (>10) by adding aqueous NaOH solution (1 M), and the resulting aqueous mixture was extracted with ELISA (3 × 100 mL). The combined organic layers were washed with LiCl aqueous solution (sat., 20 mL) and saline solution (20 mL), dried on MgSO4, and concentrated under vacuum. Purification by comi yielded the product (630 mg, 1.88 mmol, 42%) as a yellow solid. C 13 H 18 F2N2O4S (336.4 g / mol).

[0369] Benzyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)carbamate Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (350 mg, 1.82 mmol), benzyl 1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (700 mg, 2.75 mmol), THF (7 mL), and DMI (0.7 mL) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixed solvent, and then LiHMDS (1 M in THF, 2.2 mL, 2.2 mmol) was added dropwise. After complete addition, the mixture was stirred at -78°C. After 30 minutes, the reaction mixture was quenched by adding sulfuric acid (1 M, 10 mL), warmed to room temperature, and stirred for 3 hours. At 0°C, the reaction mixture was adjusted to an alkaline pH (>10) by adding NaOH solution (1 M), and the resulting aqueous mixture was extracted with ELISA (3 × 50 mL). The combined organic layers were washed with LiCl aqueous solution (sat., 10 mL) and saline solution (10 mL), dried on MgSO4, and concentrated under vacuum. The crude product was then prepared using 4 g RediSep R. f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (linear gradient: 2 minutes to 100% petroleum ether, followed by 14 minutes to 100% siRNA) to obtain the product (290 mg, 0.79 mmol, 43%) as a white solid. C 16 H 16 F2N2O4S (370.4 g / mol).

[0370] 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminotrifluoroacetate Under a nitrogen atmosphere, tert-butyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)carbamate (230 mg, 0.69 mmol) and DCM (5 mL) were placed in a round-bottom flask. Next, the reaction mixture was cooled to 0°C in an ice bath, followed by dropwise addition of TFA (1.19 g, 800 μL, 10.5 mmol). After complete addition, the mixture was stirred at 0°C for 2 hours. Subsequently, the crude reaction mixture was concentrated under vacuum and dried under high vacuum to obtain the product (231 mg, 0.69 mmol, 100%) as a yellow solid. C 10 H 11 F5N2O4S (350.4 g / mol).

[0371] N-(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)acetamide Under a nitrogen atmosphere, 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminium trifluoroacetate (202 mg, 0.60 mmol), DCM (7 mL), and DIPEA (263 mg, 355 μL, 2.04 mmol) were placed in a heat-gun-dried two-necked flask, followed by dropwise addition of acetic anhydride (76.7 mg, 71 μL, 0.75 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under vacuum. Next, the crude mixture was dissolved in DCM (25 mL), and the resulting organic layer was washed with NaOH (2 M, 20 mL), HCl (1 M, 20 mL), and saline solution (20 mL), dried over MgSO4, and concentrated under vacuum. The crude product was then divided into 4 g RediSep R f CombiFlash R with silica gold column f The product was purified by a flash chromatography system (gradient: linear gradient to 100% petroleum ether over 2 minutes, then to 100% siRNA over 14 minutes, and finally to 100% siRNA over 5 minutes), and the product (110 mg, 0.40 mmol, 66%) was obtained as a pale yellow solid. C 10 H 12 F2N2O3S (278.3 g / mol).

[0372] tert-butyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)(methyl)carbamate Under a nitrogen atmosphere, tert-butyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)carbamate (241 mg, 0.72 mmol) and DMF (8 mL) were added to a heat-gun-dried two-necked flask. The reaction mixture was then cooled to 0°C in an ice / water bath, followed by the addition of MeI (204 mg, 90.0 μL, 1.44 mmol) and NaH (60% in mineral oil, 43 mg, 1.08 mmol). The resulting mixture was stirred at room temperature for 6 hours. The crude reaction mixture was quenched by the addition of water (25 mL), and the resulting aqueous mixture was extracted with RINKAN (3 x 25 mL). The combined organic layers were washed with water (25 mL) and saline (25 mL), dried over MgSO4, and concentrated under vacuum. The crude product was then concentrated using 4 g RediSep R f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 minutes to 100% petroleum ether, followed by a linear gradient over 14 minutes to 100% siRNA / petroleum ether (4:5)) to obtain the product (210 mg, 0.60 mmol, 83%) as yellow rubber. C 14 H 20 F2N2O4S (350.4 g / mol).

[0373] 3,3-Difluoro-N-methyl-3-(pyridine-2-ylsulfonyl)propane-1-aminium trifluoroacetate Tert-butyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)-propyl)(methyl)carbamate (175 mg, 0.50 mmol) and CH2Cl2 (5 mL) were placed in a round-bottom flask. Next, the reaction mixture was cooled in an ice bath, and then TFA (1.19 g, 0.80 mL, 10.5 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. The crude mixture was then concentrated and dried under vacuum to obtain the product (182 mg, 0.50 mmol, 100%) as a yellow oil. C11 H 13 F5N2O4S (364.3 g / mol):

[0374] 3,3-Difluoro-N,N-dimethyl-3-(pyridine-2-ylsulfonyl)propan-1-amine 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminium trifluoroacetate (70 mg, 0.20 mmol) and MeOH (2 mL) were added to a round-bottom flask. Next, formaldehyde (37 wt.% in H2O, 66.6 mg, 180 μL, 2.22 mmol) was added, and the resulting reaction mixture was stirred at room temperature for 10 minutes, after which sodium triacetoxyborohydride (179 mg, 0.84 mmol) was added. After stirring for a further 16 hours at room temperature, the reaction mixture was concentrated under vacuum. The crude product was divided into 4 g RediSep R f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 minutes to 100% CH2Cl2, then a linear gradient to 100% CH2Cl2 / MeOH (1:1) over 14 minutes), and the product (40 mg, 0.15 mmol, 76%) was obtained as a pale yellow liquid. C 10 H 14 F2N2O2S (264.3 g / mol).

[0375] 3,3-Difluoro-N,N,N-trimethyl-3-(pyridine-2-ylsulfonyl)propane-1-aminium 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminium trifluoroacetate (150 mg, 0.43 mmol), MeCN (2.7 mL), and MeOH (1.3 mL) were added to a round-bottom flask. Next, DIPEA (332 mg, 448 μL, 2.57 mmol) and MeI (609 mg, 267 μL, 4.29 mmol) were added, and the resulting reaction mixture was stirred at room temperature for 30 hours. The crude mixture was then concentrated and dried under vacuum. The obtained crude solid was triturated with a solution of chloroform and MeOH (10%), and the white solid was filtered off. The white solid was washed with a solution of chloroform and MeOH (10%), dried under vacuum, and the product (110 mg, 0.39 mmol, 92%) was obtained as a white solid. C 11 H 17 F2N2O2S (279.1 g / mol).

[0376] 2-((difluoro(methylthio)methyl)sulfonyl)pyridine Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (500 mg, 2.59 mmol), THF (10 mL), DMI (1 mL), and methyl S-methanethiosulfonate (488 mg, 368 μL, 3.90 mmol) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixed solvent, followed by dropwise addition of LiHMDS (1 M in THF, 3.2 mL, 3.20 mmol). After complete addition, the mixture was stirred at -78°C for 30 minutes. After quenching with ammonium chloride aqueous solution (sat., 10 mL), the resulting aqueous solution was extracted with ethyl acetate (3 x 25 mL). The combined organic layers were washed with LiCl aqueous solution (sat., 25 mL) and saline solution (25 mL), dried over MgSO4, and concentrated under vacuum. The crude product was concentrated using 12 g RediSep R f CombiFlash R with silica gold column fThe product (500 mg, 2.10 mmol, 81%) was purified using a flash chromatography system (gradient: 2 min 100% CHCl3 / heptane (1:1), then a linear gradient over 14 min to 100% CHCl3 / heptane / siRNA (3:3:1)) to obtain the product as a white solid. C7H7F2NO2S2 (239.3 g / mol).

[0377] 2-((difluoro(methylsulfinyl)methyl)sulfonyl)pyridine Under a nitrogen atmosphere, 2-((difluoro(methylthio)methyl)sulfonyl)pyridine (180 mg, 0.75 mmol) and CH2Cl2 (3 mL) were added to a heat-gun-dried round-bottom neck flask. The reaction mixture was then cooled to 0°C in an ice / water mixture, followed by dropwise addition of 3-chloroperbenzoic acid (≤77%, 186 mg, 0.82 mmol) in CH2Cl2 (1 mL). After complete addition, the mixture was stirred at room temperature for 16 hours. The crude mixture was concentrated under vacuum, dissolved in toluene (30 mL), and the organic layer was washed with aqueous NaHCO3 solution (sat., 2 x 30 mL), water (30 mL), and saline solution (30 mL). The mixture was dried over MgSO4 and concentrated under vacuum. The crude product was then mixed with 12 g of RediSep R f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 minutes to 100% hexane, followed by a linear gradient over 14 minutes to 100% petroleum ether / siRNA (4:5)) to obtain the product (110 mg, 0.43 mmol, 56%) as a colorless liquid. C7H7F2NO3S2 (255.3 g / mol).

[0378] 2-((difluoro(methylsulfonyl)methyl)sulfonyl)pyridine Under a nitrogen atmosphere, 2-((difluoro(methylthio)methyl)sulfonyl)pyridine (100 mg, 0.42 mmol), MeCN (2 mL), CH2Cl2 (1 mL), and water (3 mL) were added to a round-bottom flask. Next, the reaction mixture was cooled to 0°C with an ice water mixture, followed by the addition of sodium periodate (411 mg, 1.93 mmol) and RuCl3xH2O (1 mg), and the mixture was stirred for 16 hours. After dilution with water (30 mL), the resulting aqueous solution was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with water (25 mL) and saline solution (25 mL), dried on MgSO4, and concentrated under vacuum. The crude product was divided into 4 g RediSep R f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 min 100% petroleum ether, followed by a linear gradient over 12 min to 100% petroleum ether / siRNA (4:3)) to obtain the product (108 mg, 0.40 mmol, 95%) as a white solid. C7H7F2NO4S2 (271.3 g / mol).

[0379] 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propan-1-ol) and 3,3-difluoro-3-(pyridine-2-ylsulfonyl)-propylacetate Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (965 mg, 5.00 mmol), 1,3,2-dioxathiolane 2,2-dioxide (931 mg, 7.50 mmol), THF (20 mL), and DMI (2 mL) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixture, followed by dropwise addition of LiHMDS (1 M, 6.00 mL, 6.00 mmol in THF). After complete addition, the mixture was stirred at -78°C. After 30 minutes, the reaction mixture was quenched by adding aqueous ammonium acetate (1 M, 10 mL), warmed to room temperature, and stirred for 3 hours. At 0°C, the reaction mixture was adjusted to an alkaline pH (>10) by adding NaOH solution (1 M), and the resulting aqueous mixture was extracted with ELISA (3 × 50 mL). The combined organic layers were washed with LiCl aqueous solution (sat., 10 mL) and saline solution (10 mL), dried on MgSO4, and concentrated under vacuum. The crude product was then prepared using 24 g RediSep R. f CombiFlash R with silica gold column f The solution was purified using a flash chromatography system (gradient: linear gradient from 100% petroleum ether for 2 minutes, followed by 14 minutes of linear gradient to 100% siRNA) to obtain 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propan-1-ol (210 mg, 0.89 mmol, 18%) as a white solid and 3,3-difluoro-3-(pyridine-2-ylsulfonyl)-propyl acetate (400 mg, 1.43 mmol, 29%) as a colorless liquid. Analysis data for 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propan-1-ol): C8H9F2NO3S (237.2 g / mol). Analytical data for 3,3-difluoro-3-(pyridine-2-ylsulfonyl)-propylacetate: C 10 H 11 F2NO4S (279.3 g / mol).

[0380] 3,3-Difluoro-3-(pyridine-2-ylsulfonyl)propyl hydrogen sulfate Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (1.93 g, 10.0 mmol), 1,3,2-dioxathiolane 2,2-dioxide (1.86 g, 15.0 mmol), THF (40 mL), and DMI (4 mL) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixture, followed by dropwise addition of LiHMDS (1 M in THF, 12.0 mL, 12.0 mmol). After complete addition, the mixture was stirred at -78°C. After 30 minutes, the reaction mixture was quenched by adding formic acid in water (1%, 10 mL), warmed to room temperature, and concentrated under vacuum. The crude product was divided into 80 g RediSep R f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 min 100% CHCl3, then a linear gradient to 100% CHCl3 / MeOH (1:1) over 14 min), and the product (3.00 g, 9.46 mmol, 95%) was obtained as a yellow solid. C8H9F2NO6S2 (317.3 g / mol).

[0381] 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl 4-methylbenzenesulfonate 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl bisulfate (1.00 g, 3.16 mmol) and THF (24 mL) were placed in a round-bottom flask. After adding hydrochloric acid (37%, 1.60 mL), the reaction mixture was stirred at room temperature for 16 hours. Next, the crude mixture was cooled in an ice bath and quenched with aqueous NaHCO3 solution (sat., 30 mL). The resulting aqueous solution was extracted with toluene (3 x 25 mL), the combined organic layers were washed with saline solution (25 mL), dried on MgSO4, and concentrated under vacuum to obtain crude alcohol (745 mg, 3.14 mmol, 100%) as a yellow solid.

[0382] Crude alcohol was dissolved in CH2Cl2 (25 mL) and cooled in an ice bath. Triethylamine (850 mg, 617 μL, 6.10 mmol) and 4-toluenesulfonyl chloride (700 mg, 3.67 mmol) were added at 0°C, and the resulting reaction solution was stirred overnight in an ice bath. Next, aqueous hydrochloric acid (1 M, 30 mL) was added to quench the mixture, the aqueous layer was extracted with CH2Cl2 (3 x 25 mL), the combined organic layers were washed with water (30 mL) and saline solution (30 mL), dried on MgSO4, and concentrated under vacuum. The crude product was then prepared using 24 g RediSep R f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 mins to 100% petroleum ether, followed by a linear gradient over 14 mins to 100% siRNA / petroleum ether (3:2)) to obtain the product (825 mg, 2.09 mmol, 66%) as a white solid. C 15 H 15 F2NO5S2 (391.4 g / mol).

[0383] 2-((3-azido-1,1-difluoropropyl)sulfonyl)pyridine 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl 4-methylbenzenesulfonate (370 mg, 0.95 mmol), DMF (10 mL), and sodium azide (308 mg, 4.75 mmol) were added to a round-bottom flask. After stirring at 85°C for 3 hours, the reaction mixture was diluted with water (30 mL). The aqueous mixture was extracted with toluene (3 x 25 mL), the combined organic layers were washed with water (3 x 25 mL) and saline solution (2 x 25 mL), dried over MgSO4, and concentrated under vacuum to obtain the product (203 mg, 0.77 mmol, 82%) as a yellow liquid. C8H8F2N4O2S (262.2 g / mol).

[0384] 2-((1,1-difluoro-3-iodopropyl)sulfonyl)pyridine 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl 4-methylbenzenesulfonate (391 mg, 1.00 mmol), acetone (10 mL), and sodium iodide (749 mg, 5.00 mmol) were added to a round-bottom flask. After stirring at 60°C for 6 hours, the reaction mixture was concentrated and then diluted with water (30 mL). The aqueous mixture was extracted with toluene (3 x 25 mL), and the combined organic layer was washed with aqueous solution of Na₂S₂O₃ (sat., 2 x 25 mL), water (25 mL), and saline solution (25 mL). The mixture was dried over MgSO₄ and concentrated under vacuum to obtain the product (277 mg, 0.88 mmol, 88%) as a yellow liquid. C8H8F2INO2S (347.1 g / mol).

[0385] 2-((difluoroiodomethyl)sulfonyl)pyridine Under a nitrogen atmosphere, difluoromethyl 2-pyridylsulfone (290 mg, 1.50 mmol), THF (6 mL), and DMI (0.6 mL) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixture, followed by the addition of diiodoethane (1.06 g, 3.75 mmol), and then LiHMDS (1 M in THF, 3.75 mL, 3.75 mmol) was added dropwise. After complete addition, the mixture was stirred at -78°C for 30 minutes. After quenching with aqueous ammonium chloride solution (sat., 10 mL), the resulting aqueous solution was extracted with chloroform (3 x 25 mL). The combined organic layers were dried over MgSO4, concentrated under vacuum, and 12 g of the crude product was collected. f CombiFlash R with silica gold column f The product (223 mg, 0.70 mmol, 47%) was purified using a flash chromatography system (linear gradient: 2 minutes to 100% petroleum ether, followed by 14 minutes to 100% siRNA) to obtain a yellow solid. C6H4F2INO2S (319.1 g / mol).

[0386] 2-bromo-2,2-difluoroacetamide Bromodifluoroethyl acetate (1.58 g, 1.0 mL, 7.78 mmol) and methanol (5 mL) were placed in a round-bottom flask. The reaction mixture was then cooled to -15 °C in a sodium chloride / ice mixture, followed by the dropwise addition of ammonia (7N, 2.5 mL) in methanol. After stirring at room temperature for 48 hours, the crude mixture was concentrated and dried under vacuum to obtain the product (1.25 g, 92%) as a white solid. C2H2BrF2NO (173.9 g / mol).

[0387] 2-Bromo-2,2-difluoroacetate sodium Sodium hydroxide (300 mg, 7.71 mmol) and methanol (7 mL) were placed in a round-bottom flask. The reaction mixture was then cooled to 0°C in an ice bath, and ethyl bromodifluoroethyl acetate (1.58 g, 1.0 mL, 7.78 mmol) was added dropwise. After stirring at room temperature for 16 hours, the crude mixture was concentrated and dried under vacuum to obtain the product (1.30 g, 86%) as a white solid. C2BrF2Na (196.9 g / mol).

[0388] Ethyl 2-fluoro-2-(pyridine-2-ylsulfonyl) acetate Under a nitrogen atmosphere, 2-mercaptopyridine (1.50 g, 13.5 mmol) and ethanol (34 mL) were placed in a round-bottom flask using a heat gun. The reaction mixture was then cooled to 0°C in an ice / water bath, followed by the dropwise addition of triethylamine (1.38 g, 1.90 mL, 13.5 mmol). After stirring for 10 minutes, ethyl bromofluoroethyl acetate (2.50 g, 1.6 mL, 13.49 mmol) was added dropwise, and the resulting mixture was stirred at room temperature for 16 hours. The crude mixture was then quenched by adding aqueous hydrochloric acid (1 M, 50 mL), and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic layers were washed with saline solution (50 mL), dried on MgSO4, concentrated under vacuum, and the crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate (6:1), dxh: 6 x 9.5 cm) to obtain a sulfide precursor (2.81 g, 13.1 mmol, 97%) as a colorless oil. Sulfide precursor (1.00 g, 4.65 mmol), acetonitrile (6 mL), dichloromethane (6 mL), and water (15 mL) were added to a round-bottom flask. Next, sodium periodate (4.50 g, 21.4 mmol) and ruthenium chloride hydrate (3 mg) were added to the reaction mixture, and the resulting solution was stirred at room temperature for 16 hours. The crude mixture was then diluted with water (50 mL), and the aqueous mixture was extracted with ether (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried on MgSO4, concentrated under vacuum, and the crude product was purified by column chromatography (SiO2, dichloromethane:chloroform (20:1), d×h:6×12 cm) to obtain the product (1.05 g, 4.25 mmol, 91%) as a colorless oil. C9H 10 FNO4S (247.2 g / mol).

[0389] 2-Fluoro-2-(pyridine-2-ylsulfonyl)acetamide 490 mg (1.98 mmol) of ethyl 2-fluoro-2-(pyridine-2-ylsulfonyl) and 6 mL of ethanol were added to a round-bottom flask. The reaction mixture was then cooled to 0°C in an ice bath, followed by the dropwise addition of ammonia in methanol (7N, 4.00 mL). After stirring at room temperature for 30 minutes, the crude mixture was concentrated under vacuum. The resulting solid was then triturated with ethyl acetate / hexane (4:2, 6 mL), dried under vacuum, and the product (380 mg, 84%) was obtained as a white solid. C7H7FN2O3S (218.2 g / mol).

[0390] 2-Fluoro-2-(pyridine-2-ylsulfonyl)sodium acetate 450 mg, 1.82 mmol of ethyl 2-fluoro-2-(pyridine-2-ylsulfonyl), 8 mL of MeOH, and 8 mL of THF were placed in a round-bottom flask. Next, 1.9 mL of aqueous sodium hydroxide solution was added dropwise to the reaction mixture and stirred for 10 minutes. The crude mixture was concentrated and dried under vacuum to obtain the product (424 mg, 97%) as a white solid. C7H5FNNaO4S (241.2 g / mol).

[0391] 2-(ethylsulfonyl)pyridine Under a nitrogen atmosphere, 2-mercaptopyridine (3.1 g, 27.8 mmol), THF (56 mL), and MeCN (56 mL) were placed in a round-bottom flask using a heat gun. The reaction mixture was then cooled to 0°C in an ice / water bath, followed by dropwise addition of DBU (4.68 g, 4.60 mL, 30.8 mmol). After stirring for 5 minutes, ethyl iodide (6.50 g, 3.35 mL, 41.7 mmol) was added dropwise, and the resulting mixture was stirred at room temperature for 16 hours. The crude mixture was then diluted with water (200 mL), extracted with toluene (3 x 50 mL), and the combined organic layer was washed with water (50 mL), aqueous HCl (1 M, 50 mL), and brine (50 mL). The mixture was dried over MgSO4 and concentrated under vacuum to obtain crude sulfide (750 mg) as a yellow oil.

[0392] Crude sulfide (750 mg), acetonitrile (30 mL), dichloromethane (10 mL), and water (40 mL) were placed in a round-bottom flask and cooled to 0°C in an ice / water bath. Next, sodium periodate (5.30 g, 24.9 mmol) and ruthenium chloride hydrate (5 mg) were added to the reaction mixture, and the resulting solution was stirred at room temperature for 16 hours. Then, the crude mixture was diluted with water (40 mL), and the aqueous mixture was extracted with toluene (3 x 60 mL). The combined organic layers were washed with water (50 mL) and saline solution (50 mL), dried on MgSO4, concentrated under vacuum, and 24 g of the crude product was collected. f CombiFlash R with silica gold column f The product (430 mg, 2.51 mmol, 9%) was purified using a flash chromatography system (gradient: 2 minutes with 100% hexane, followed by a linear gradient over 14 minutes to 100% petroleum ether / siRNA (1:1)) to obtain a yellow oil. C7H9NO2S (171.2 g / mol).

[0393] 2-((1-fluoroethyl)sulfonyl)pyridine Under a nitrogen atmosphere, benzyl 1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (400 mg, 2.33 mmol) and THF (10 mL) were added to a heat-gun-dried two-necked flask. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixture, followed by the addition of NFSI (880 mg, 2.80 mmol), and then LiHMDS (1 M in THF, 2.5 mL, 2.5 mmol) was added dropwise. After complete addition, the mixture was stirred at -78°C for 90 minutes, and then at room temperature for another 90 minutes. The reaction mixture was then quenched by adding NH4Cl aqueous solution (sat., 20 mL) and extracted with RINKAN (3 x 25 mL). The combined organic layers were washed with NaHCO3 aqueous solution (sat., 30 mL), water (30 mL), and saline solution (30 mL), dried over MgSO4, and concentrated under vacuum. 4 g of the crude product was collected. fCombiFlash R with silica gold column f The product (138 mg, 0.73 mmol, 31%) was purified using a flash chromatography system (gradient: 2 minutes for 100% petroleum ether, then a linear gradient over 14 minutes to 100% petroleum ether / siRNA (5:4)) to obtain the product as a colorless liquid. C7H8FNO2S (189.2 g / mol).

[0394] 2,2-Difluoro-2-(pyridine-2-ylthio)ethyl acetate Cesium carbonate (23.5 g, 72.0 mmol) was placed in a round-bottom flask and heated three times under vacuum for 10 minutes using a heat gun. Next, DMF (340 ml), 2-mercaptopyridine (4.00 g, 36.0 mmol), and ethyl bromodifluoroethyl acetate (14.6 g, 9.23 mL, 72.0 mmol) were added under a nitrogen atmosphere, and the resulting mixture was stirred at room temperature for 18 hours. Subsequently, the reaction mixture was diluted with water (300 mL), and the aqueous mixture was extracted with phenylethylamine (3 x 200 mL). The combined organic layers were washed with water (100 mL) and saline solution (100 mL), dried on MgSO4, and concentrated under vacuum. 80 g of the crude product was collected. f CombiFlash R with silica gold column f The product (6.30 g, 27.0 mmol, 75%) was purified by flash chromatography (gradient: 2 min 100% petroleum ether, then linear gradient to 100% petroleum ether / SiO(5:1) over 14 min) to obtain the product as a yellow liquid. 80 g of the crude product was then mixed with RediSep R. f CombiFlash R with silica gold column f The product was purified using a flash chromatography system (gradient: 2 minutes to 100% petroleum ether, then a linear gradient over 14 minutes to 100% petroleum ether / siRNA (5:1)), and the product was obtained as a yellow liquid (6.30 g, 27.0 mmol, 75%). C9H9F2NO2S (233.2 g / mol).

[0395] 2,2-difluoro-2-(pyridine-2-ylthio)ethane-1-ol Under a nitrogen atmosphere, 2,2-difluoro-2-(pyridine-2-ylthio)ethyl acetate (3.00 g, 12.9 mmol), THF (7.5 mL), and EtOH (52.5 mL) were added to a heat-gun-dried round-bottom flask. The reaction mixture was then cooled to 0°C in an ice / water bath, followed by the addition of sodium borohydride (583 mg, 15.4 mmol), and the resulting mixture was stirred at 0°C for 1 hour. The crude mixture was then quenched by the addition of aqueous hydrochloric acid (1 M, 15 mL), and the solvent was removed under vacuum. The aqueous layer was extracted with dimethyl acetate (3 x 60 mL), the combined organic layers were washed with saline solution (50 mL), dried over MgSO4, and concentrated under vacuum to obtain the product (2.25 g, 11.8 mmol, 91%) as a yellow liquid. C7H7F2NOS (191.2 g / mol).

[0396] 2,2-difluoro-2-(pyridine-2-ylsulfonyl)ethane-1-ol Under a nitrogen atmosphere, 2,2-difluoro-2-(pyridine-2-ylthio)ethane-1-ol (2.25 g, 10.9 mmol) and CH2Cl2 (100 mL) were added to a heat-gun-dried round-bottom flask. Next, the reaction mixture was cooled to 0°C in an ice / water bath, followed by the addition of meta-chloroperoxybenzoic acid (4 x 1.55 g, 27.2 mmol) in small portions, and the mixture was stirred in a cooling bath for 16 hours. The crude mixture was then quenched by the addition of 0.5 M sodium hydroxide solution (120 mL), the aqueous layer was extracted with CH2Cl2 (3 x 100 mL), the combined organic layers were washed with water (100 mL) and saline solution (100 mL), dried over MgSO4, and concentrated under vacuum to obtain the product (2.25 g, 11.8 mmol, 91%) as a yellow liquid. 24 g of the crude product was then collected in RediSep R f CombiFlash R with silica gold column fThe product was purified by a flash chromatography system (gradient: 2 minutes for 100% petroleum ether, then a linear gradient over 12 minutes to 100% petroleum ether / siRNA (4:3)) to obtain the product (647 mg, 2.90 mmol, 27%) as pale yellow rubber. C7H7F2NO3S (223.2 g / mol).

[0397] 2,2-difluoro-2-(pyridine-2-ylthio)ethyl 4-methylbenzenesulfonate Under a nitrogen atmosphere, 2,2-difluoro-2-(pyridine-2-ylthio)ethane-1-ol (1.00 g, 5.23 mmol) and CH2Cl2 (20 mL) were added to a heat-gun-dried round-bottom flask. The reaction mixture was then cooled to 0°C in an ice / water bath, followed by the addition of triethylamine (794 mg, 1.09 mL, 7.85 mmol) and p-toluenesulfonyl chloride (1.50 g, 7.85 mmol). The resulting mixture was stirred in a cooling bath for 16 hours. The crude mixture was then quenched by adding aqueous hydrochloric acid (1 M, 30 mL), diluted with CH2Cl2 (30 mL), the organic layer was washed with saline solution (30 mL), dried over MgSO4, and concentrated under vacuum. 24 g of the crude product was collected. f CombiFlash R with silica gold column f The product (1.60 g, 4.64 mmol, 89%) was purified by a flash chromatography system (gradient: 2 minutes with 100% petroleum ether, then a linear gradient over 12 minutes to 100% petroleum ether / SiO(2:1)) to obtain the product as a yellow solid. C 14 H 13 F2NO3S2 (345.4 g / mol).

[0398] 2,2-Difluoro-2-(pyridine-2-ylsulfonyl)ethyl 4-methylbenzenesulfonate 2,2-difluoro-2-(pyridine-2-ylthio)ethyl 4-methylbenzenesulfonate (7 g, 20.3 mmol), acetonitrile (100 mL), dichloromethane (50 mL), and water (150 mL) were added to a round-bottom flask and cooled to 0°C in an ice / water bath. Then, sodium periodate (21 g, 98.2 mmol) and ruthenium chloride hydrate (20 mg) were added to the reaction mixture, and the resulting solution was stirred at room temperature for 16 hours. Subsequently, the crude mixture was diluted with water (200 mL), and the aqueous mixture was extracted with ELISA (3 x 250 mL). The combined organic layers were washed with water (100 mL) and saline solution (100 mL), dried on MgSO4, concentrated under vacuum, and 80 g of the crude product was collected. f CombiFlash R with silica gold column f The product (7.66 g, 20.3 mmol, 100%) was purified by a flash chromatography system (linear gradient: 2 minutes to 100% petroleum ether, followed by 14 minutes to 100% acetate) to obtain the product as a white solid. C 14 H 13 F2NO5S2 (377.4 g / mol).

[0399] 2-((2-azido-1,1-difluoroethyl)sulfonyl)pyridine Under a nitrogen atmosphere, 2,2-difluoro-2-(pyridine-2-ylsulfonyl)ethyl 4-methylbenzenesulfonate (1.51 g, 4.00 mmol), sodium azide (1.30 g, 20 mmol), and DMF (32 mL) were placed in a heat-gun-dried round-bottom flask. After stirring at 70°C for 133 hours, the reaction mixture was cooled to room temperature, diluted with water (70 mL), extracted with ELISA (3 x 70 mL), dried over MgSO4, and concentrated under vacuum. 40 g of the crude product was collected. RediSep R f CombiFlash R with silica gold column fThe product (650 mg, 2.62 mmol, 66%) was purified by a flash chromatography system (gradient: 2 minutes with 100% hexane, followed by a linear gradient over 14 minutes with 100% petroleum ether:siRNA (5:4)) to obtain a white solid. C7H6F2N4O2S (248.2 g / mol).

[0400] 2,2-difluoro-2-(pyridine-2-ylsulfonyl)ethane-1-amine Under a nitrogen atmosphere, 2-((2-azido-1,1-difluoroethyl)sulfonyl)pyridine (650 mg, 2.62 mmol) and MeOH (20 mL) were added to a heat-gun-dried round-bottom flask. Next, triethylamine (448 mg, 617 μL, 4.43 mmol) and 1,3-propanedithiol (826 mg, 890 μL, 7.63 mmol) were added to the reaction mixture and stirred at room temperature for 2 hours. After concentration under vacuum, 24 g of the crude product was added to RediSep R f CombiFlash R with silica gold column f The product (520 mg, 2.34 mmol, 89%) was purified by a flash chromatography system (gradient: 2 minutes with 100% hexane, followed by a linear gradient over 14 minutes to 80% CHCl3:MeOH (95:5)) to obtain the product as a pale yellow liquid. C7H8F2N2O2S (222.2 g / mol).

[0401] N-(2,2-difluoro-2-(pyridine-2-ylsulfonyl)ethyl)acetamide Under a nitrogen atmosphere, 2,2-difluoro-2-(pyridine-2-ylsulfonyl)ethane-1-amine (50 mg, 0.23 mmol), CH2Cl2 (1 mL), and DIPEA (101 mg, 136 μL, 0.78 mmol) were added to a heat-gun-dried round-bottom flask, followed by dropwise addition of acetic anhydride (29.5 mg, 27.2 μL, 0.29 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under vacuum. Next, the crude mixture was dissolved in DCM (25 mL), and the resulting organic layer was washed with NaOH (2 M, 10 mL), HCl (1 M, 10 mL), and saline solution (10 mL), dried over MgSO4, and concentrated under vacuum. 4 g of the crude product was collected. f CombiFlash R with silica gold column f The product (520 mg, 2.34 mmol, 89%) was purified by a flash chromatography system (linear gradient: 2 mins to 100% CHCl3, then 12 mins to 100% CHCl3:MeOH (95:5), and then 5 mins to 100% siRNA) to obtain the product as a pale yellow liquid. C9H 10 F2N2O3S (264.3 g / mol).

[0402] (2R,3S,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-bromo-2,2-difluoroacetamido)-tetrahydro-2H-pyran-3,4-diyldiaacetate Under a nitrogen atmosphere, (2R,3S,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-aminotetrahydro-2H-pyran-3,4-diyldiaacetate (400 mg, 1.15 mmol), bromodifluoroacetic acid (242 mg, 1.38 mmol), EEDQ (341 mg, 1.38 mmol), and THF (16 mL) were added to a heat-gun-dried round-bottom flask, and the resulting mixture was stirred at room temperature. After 16 hours, the crude product was concentrated under vacuum and 40 g of RediSep R was added. f CombiFlash R with silica gold column fThe product was purified by a flash chromatography system (gradient: 1.5 min 100% CHCl3, followed by a linear gradient over 15 min to 100% CHCl3 / MeOH (9:1)) to obtain the product (410 mg, 0.81 mmol, 71%) as a white solid. C 16 H 21 BrF2N2O9 (503.3 g / mol).

[0403] N-((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-2-bromo-2,2-difluoroacetamide Under a nitrogen atmosphere, (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-bromo-2,2-difluoroacetamido)-tetra-hydro-2H-pyran-3,4-diyldiaacetate (250 mg, 0.50 mmol) and MeOH (5 mL) were added to a heat-gun-dried round-bottom flask. Next, a solution of sodium methoxide (25%, 114 μL) was added, and the resulting reaction mixture was stirred at room temperature for 2 hours. After that, the crude mixture was heated to DOWEX H + The mixture was quenched by adding (100 mg) and stirred for 5 minutes. Finally, the reaction mixture was filtered and concentrated under vacuum to obtain the product (185 mg, 0.49 mmol, 98%) as a white solid. C 10 H 15 BrF2N2O6 (377.1 g / mol).

[0404] 2-((trifluoromethyl)sulfonyl)pyridine Pifurusul (2.60 mmol) and KHF2 (2 mg, 0.26 mmol, 10 mol%) in DMSO (4 mL) were added to a 25 mL round-bottom flask. TMSCF3 (384 μL, 2.60 mmol, 1.0 equiv) was added to this mixture. The reaction mixture was stirred for 30 minutes and extracted in toluene (20 mL). The organic fractions were combined, dried over MgSO4, filtered, and concentrated under vacuum to obtain the product as a pale yellow solid in high purity with a yield of 80%. C6H4F3NO2S (211.0 g / mol).

[0405] 2-((fluoromethyl)sulfonyl)pyridine To a solution of NaH (60%, wt%, 151 mg, 3.77 mmol, 1.05 equiv) and DMF (10 mL), pyridine-2-thiol (400 mg, 3.6 mmol, 1.0 equiv), dissolved in DMF (10 mL) at 0°C, was added dropwise under a stream of N2. Next, CH2FI (1.0 mL, 14.4 mmol, 4.0 equiv) (Note: CH2FI is volatile and highly toxic) was added dropwise over 30 minutes. The reaction was then slowly warmed to room temperature and stirred overnight for 12 hours. The reaction mixture was then quenched with H2O (50 mL) and extracted with Et2O (3 x 30 mL). The separated organic phase was then washed with saline solution (50 mL) and dried over MgSO4. The resulting solution was filtered and concentrated under vacuum to obtain crude 2-((fluoromethyl)thio)pyridine as a yellow oil. Next, this crude product was used without further purification in the following step. Crude 2-((fluoromethyl)thio)pyridine was added to a 50 mL round-bottom flask containing MeCN (10 mL), DCM (10 mL), and H2O (20 mL). Then, NaIO4 (3.0 g, 14.5 mmol) and RuCl3xH2O (3 mg) were added. The reaction was then continued until complete. 19 The reaction was monitored by 1F NMR. Upon completion, 10 mL of distilled H2O was added, and the resulting reaction mixture was extracted with Et2O (3 × 30 mL). The organic phase was then washed with saturated NaHCO3 (30 mL) and saline solution (30 mL). The solution was then filtered and dried under vacuum. The crude residue was then subjected to silica gel chromatography (pentane / ethylacetal, 3:1) to obtain 2-((fluoromethyl)sulfonyl)pyridine as a colorless solid. Yield 55% (2 or more steps). C6H6FNO2S (175.1 g / mol).

[0406] 2-((fluoroiodomethyl)sulfonyl)pyridine In a 100 mL pear-shaped Schlenk tube, 2-((fluoromethyl)sulfonyl)pyridine (0.5 g, 2.9 mmol) and iodine crystals (1.46 g, 11.5 mmol, 4.0 equiv) were added in 10 mL of degassed anhydrous DMF under nitrogen. To this mixture, tBuOK (1.1 g, 10 mmol, 3.5 equiv) in 10 mL of DMF was added at 5°C. The reaction mixture was warmed to room temperature and quenched with saturated ammonium chloride aqueous solution (10 mL) when complete consumption of the starting materials was observed. The product was then extracted in SiO (3 × 20 mL) and stirred with aqueous NaHSO3 (10 g in 100 mL of distilled water). 19 The complete conversion rate of the diiodation product was determined using 1F NMR (approximately 10 hours). The organic phase was then separated, washed with H2O (2 × 30 mL) and saline solution (1 × 30 mL), and dried over MgSO4. After filtration, the reaction mixture was concentrated under vacuum. The crude product was then subjected to column chromatography (siRNA / pentane, 1:3) to obtain 2-((fluoroiodomethyl)sulfonyl)pyridine as a white solid in 62% yield. C6H5FNIO2S (301.1 g / mol).

[0407] 2-Fluoro-1-(4-methoxyphenyl)-2-(pyridine-2-ylsulfonyl)ethane-1-one In a 100 mL pear-shaped Schlenk tube, under nitrogen at -78°C, LiHMDS (24 mL, 1.0 M, 24 mmol, 1.4 equiv) was added to a solution of 2-((fluoromethyl)sulfonyl)pyridine (3.0 g, 17.1 mmol, 1.0 equiv) and 4-methyl methoxybenzoate (4.3 g, 25.7 mmol, 1.5 equiv) in 50 mL of THF. The reaction mixture was then stirred at this temperature for 30 minutes. Next, HCl(aq) (3 M, 15 mL) was slowly added. The reaction mixture was then warmed to room temperature. The organic phase was extracted with SiO2 (2 × 100 mL) and then washed with distilled H2O (100 mL) and saline (100 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. Next, the crude product was purified by silica gel chromatography (siRNA / pentane, 1:3) to obtain 2-fluoro-1-(4-methoxyphenyl)-2-(pyridine-2-ylsulfonyl)ethane-1-one as a white solid (yield 81%). C 14 H 12 FNO4S (309.0 g / mol).

[0408] 2-((chlorofluoromethyl)sulfonyl)pyridine In a 25 mL pear-shaped Schlenk tube, 2-fluoro-1-(4-methoxyphenyl)-2-(pyridine-2-ylsulfonyl)ethane-1-one (154 mg, 0.5 mmol, 1.0 equiv) and NCS (89 mg, 0.66 mmol, 1.3 equiv) were added in DMF (5 mL) under nitrogen. The reaction mixture was cooled to -78°C. Next, LiHMDS (0.75 mL, 1.0 M in THF, 0.75 mmol, 1.5 equiv) was added dropwise over 10 minutes at -78°C. Then, NaOH (aq) (3 mL, 0.5 M) was added, and the reaction mixture was warmed to room temperature. The organic phase was extracted with ELISA (2 x 100 mL), followed by washing with distilled H2O (100 mL) and saline (100 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. Next, the crude product was purified by silica gel chromatography (SiO₂ / pentane, 1:3) to obtain 70% of the title compound as a colorless oil. C6H5ClFNO2S (209.6 g / mol).

[0409] py-SOOF Biotin Under argon, 29 μL of NEt3 was added to a solution of 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propan-1-amine hydrochloride (23 mg, 84 μmol) and active biotin ester (45 mg, 70 μmol) in anhydrous DCM. The reaction mixture was stirred overnight with rt and then concentrated. The product was purified by flash column chromatography (CHCl3 / MeOH 0→10% gradient elution) to obtain the title product as a white solid (25 mg, 50%). C 29 H 45 F2N5O9S2 (709.8 g / mol).

[0410] 2-((4-methoxybenzyl)sulfonyl)pyridine To a solution of 2-thiopyridine (1.11 g, 10 mmol) in MeCN (100 mL), PMB-Cl (1.62 mL, 12 mmol) was added, followed by the dropwise addition of NEt3 (2.09 mL, 15 mmol). The reaction mixture was stirred at room temperature for 2 hours, then diluted with H2O (150 mL) and neutralized to pH ~7 with 2M HCl. The mixture was then extracted with ELISA (3 × 100 mL), the combined organic matter was washed with saline solution (100 mL), dried, filtered, and concentrated under vacuum. The crude yellow oil was then used without further purification. The crude oil described above was dissolved in CH2Cl2 (30 mL) and cooled to 0°C. Next, mCPBA (4.5 g, 20 mmol) was added in small amounts. Then, the mixture was warmed to room temperature, stirred for 3 hours, quenched with saturated aqueous Na2S2O3 (20 mL) solution, and diluted with CH2Cl2 (70 mL). The organic phase was washed with saturated aqueous NaHCO3 (3 × 60 mL) and saline solution (70 mL), dried (MgSO4), filtered, and concentrated under vacuum. Next, the crude product was purified by flash chromatography (1:1 siRNA:petroleum ether) to obtain the desired pyridal sulfone as a white solid (1.51 g, 57% yield). C 13 H 13 NO3S (263.3 g / mol).

[0411] 2-((difluoro(4-methoxyphenyl)methyl)sulfonyl)pyridine To a solution of sulfone (526 mg, 2 mmol) and NFSI (1.58 g, 5 mmol) in THF (80 mL), a solution of NaHMDS (4.4 mL, 4.4 mmol, 1 M) in THF was added at -78°C. The mixture was stirred at this temperature for 2.5 hours, then warmed to room temperature and stirred for 1.5 hours. The reaction mixture was then cooled to 0°C, quenched with saturated aqueous NH4Cl (200 mL), and extracted with SiO2 (2 × 100 mL). The combined organic layer was then washed with saturated aqueous NaHCO3 (200 mL) and saturated aqueous NaCl (200 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (1:1 SiO2:petroleum ether) to obtain the desired thioether as a yellow oil (487 mg, 80%). C 13 H 11 F2NO3S (299.3 g / mol).

[0412] 2-(benzylthio)pyridine Under argon, PySH (2 g) was dissolved in 25 mL of anhydrous MeCN. Et3N (3.8 mL) was added to this solution. After 5 minutes, benzyl bromide (2.65 mL) was added dropwise over 5 minutes. After the starting materials were consumed (2 hours), the reaction was quenched with 1 M HCl. The mixture was partitioned between toluene and water, the aqueous phase was extracted three times with 20 mL of toluene, dried over MgSO4, and concentrated under vacuum. Half of the crude reaction product was purified by flash chromatography on silica using hexane / toluene up to 8%. 1.34 g of pure product (37% with respect to complete stoichiometry) was obtained. C 12 H 11 NS (201.3 g / mol).

[0413] 2-(benzylsulfonyl)pyridine 0.5 g of crude PySCH2Ph ​​was dissolved in 15 mL of MeCN and 12 mL of DCM. To this mixture, 20 mL of aqueous KIO4 (5.75 g) suspension and 6 mg of RuCl3xH2O were added, and the reaction mixture was stirred overnight in rt. The mixture was then partitioned between DCM and water and separated, and the aqueous phase was extracted three times with 15 mL of DCM. The combined organic fractions were filtered, dried over MgSO4, filtered through a silica plug, and evaporated to dryness to obtain 556 mg of a brownish solid. C 12 H 11 NO2S (233.3 g / mol).

[0414] 2-((difluoro(phenyl)methyl)sulfonyl)pyridine To a solution of sulfone (526 mg, 2 mmol) and NFSI (1.58 g, 5 mmol) in THF (80 mL), a solution of NaHMDS (4.4 mL, 4.4 mmol, 1 M) in THF was added at -78°C. The mixture was stirred at this temperature for 2.5 hours, then warmed to room temperature and stirred for 1.5 hours. The reaction mixture was cooled to 0°C, quenched with saturated aqueous NH4Cl (200 mL), and extracted with SiO2 (2 × 100 mL). The combined organic layer was then washed with saturated aqueous NaHCO3 (200 mL) and saturated aqueous NaCl (200 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (1:1 SiO2:petroleum ether) to obtain the desired thioether as a yellow oil (487 mg, 80%). C 12 H9F2NO2S (269.3 g / mol).

[0415] pySOOF-Arg Boc To a solution of amine (50 mg, 0.23 mmol) in CH2Cl2 (2.3 mL), Goodman's guanidination reagent (88 mg, 0.23 mmol) was added, followed by NEt3 (32 mL, 0.23 mmol). The mixture was stirred at room temperature for 3 days and then diluted with CH2Cl2 (10 mL). The organic layer was then washed with 0.5 M HCl (3 x 10 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (3:7 siRNA:petroleum ether) to obtain the desired protected guanidine as a white solid (83 mg, 77%). C 18 H 26 F2N4O6S (464.5 g / mol).

[0416] pySOOF-Arg To a solution of diBoc-guanidine (as described above) (35 mg, 0.075 mmol) in CH2Cl2 (1 mL), TFA (0.5 mL) was slowly added at 0°C. This solution was stirred while being heated to room temperature over 1.5 hours, and then stirred at room temperature for another 1.5 hours. Subsequently, it was concentrated under vacuum to obtain free guanidine as a pale yellow oil (30.2 mg, quant.) as the TFA salt. C8H 10 F2N4O2S (464.5 g / mol).

[0417] Catecolo-Ru(bpy)2 Catechol (30 mg, 0.27 mmol, 1.0 equiv) was dissolved in hot ethanol (2 mL) in a dry flask. KOH (32 mg, 0.54 mmol, 2.0 equiv) was added, followed by cis-bis(2,2'-bipyridine)dichlororuthenium(II) hydrate (110 mg, 0.21 mmol, 0.80 equiv). A Dimroth condenser (any type will do) was introduced into the flask, and the mixture was placed under Ar and refluxed overnight. The mixture was cooled to room temperature, and ferrocenium hexafluorophosphate (69 mg, 0.27 mmol, 1 equiv) was added to ensure complete formation of semiquinone. EtOH was removed, and the complex was precipitated with aqueous saturated KPF6. The solid was dried overnight to obtain 290 mg of a very deep red solid. Complete conversion was observed by mass spectrometry. This complex was further dissolved in acetonitrile and purified by elution with 5% saturated aqueous KPF6 / acetonitrile on a silica column (10 g silica) to obtain 32 mg (48%) of a deep red solid (almost black). The reaction was tracked by thin-layer chromatography (product Rf = 0.64 at 5% aq. KPF6 / MeCN); the reactants / products were red of different shades and could be confirmed visually. The desired product was confirmed by high-resolution mass spectrometry (calculated value 522.06243, observed value 522.06256).

[0418] General experimental protocols for BACED and pySOOF reactions All solutions were degassed in a glove box for at least 8 hours (<6 ppm O2). Clear glass vials with airtight caps (Cat. No VWRI548-3298, VWR) were used (Chromacol 300 μL fixed insertion vial, screw cap, Thermo Scientific for <100 μL; and 2 mL CLR RAM VIAL 9MM THD, 32009-1232, Novetech for ≥100 μL). The standard reaction consisted of mixing the selected Dha-containing protein with the desired reaction buffer in a glove box, and then sequentially adding the catalyst, additives, and chemical substrates from freshly prepared stocks in the buffer in the glove box. Most reaction optimization and chemical substrate screening reactions were performed in volumes of 50–200 μL at a final concentration of 1 mg / mL (66 μM) against the model protein substrate Xl histone H3-Dha9 (1 mg / mL) in denaturation buffer (500 mM NH4OAc, 3 M guanidia chloride, pH 6.0). All reagents were first transferred to a glove box (<6 ppm O2) and then prepared with stock solutions for the reaction. All reagents were water-soluble at their final concentration and did not require a co-solvent unless otherwise noted. After thoroughly mixing the reaction mixtures with a pipette, they were capped and removed from the glove box for irradiation. A 3W blue (approximately 450 nm) LED flashlight was positioned to uniformly irradiate up to 20 reaction vials at a time, or a variable-intensity photobox was used for up to 7 reactions at a time with a blue LED intensity range of 5–50W (each dialed to indicate intensity levels 1–10). At short reaction times (<20 minutes), no significant temperature increase was observed, but at longer reaction times (>20 minutes), the temperature could be controlled by submerging the reaction vial in a glass beaker filled with water at the desired temperature. Irradiation was carried out for the desired time, after which a fixed amount of the reactant was diluted 25-fold for mass spectrometry (2 μL in 48 μL of water + 0.1% formic acid), and the conversion rates relative to the total ion count of the starting materials, single addition, double addition, and observed side reactions were calculated.Using a PD SpinTrap G-25 (GE Healthcare) desalting column, the absorbance of the entire protein was tracked, and the protein recovery rate was generally above 85%, although this analysis was not performed for all conditions and substrates. The modified proteins did not degrade or develop new adducts even when stored in the freezer as a crude reaction mixture for several months, and in some cases, it was possible to continue the incomplete reaction simply by degassing the reaction mixture again and continuing irradiation.

[0419] Example 1 - BACED reaction The reactions according to embodiments (ii) and (iii) described herein were demonstrated using various substituents to functionalize exemplary Dha-containing proteins having a variety of different functional side chains.

[0420] All side chains introduced using the BACED reaction manifold (1a-1y, see Figure 5) were screened on the model protein substrate histone H3-Dha9. In many cases, multiple side chain precursor substrates could be used to yield the same side chain product; for example, potassium ethyltrifluoroborate and ethylboronic acid both yielded side chain product 1a. In these cases, all tested conditions yielding the same side chain product are described. Various different side chains were introduced to different histone variants, modification sites, or protein scaffolds. LC-MS / MS analysis was performed to confirm site-specific side chain introduction. The conversion rate was calculated as the ratio of total product to Dha starting material, based on the deconvolution intensity of the LC / MS spectrum. In some cases, minor undesirable products such as double additions and catechol adducts were present and expressed as a percentage of the total product. A 10% baseline cut was used as a rule when analyzing the intensity of the deconvolved spectrum. In some cases, small amounts of methionine oxidation occurred during preparation, storage, and use (+16 Da + / - 1 Da). These adducts were included in the total for the calculation of starting material and product.

[0421] 1a - In one example, ethyl was introduced into the protein substrate using the BACED reaction manifold according to Figure 6(A).

[0422] In a glove box, NH4OAc buffer (500 mM, pH 6, 3M Gdn-HCl, 90 μL) containing histone H3-Dha9 (100 μg, final concentration 1 mg / mL, 66 μM) was added to a glass HPLC vial. Ru(bpy)3Cl2 (1 μL, 10 eq, freshly prepared 66 mM stock in water), catechol (1 μL, 100 eq, freshly prepared 660 mM stock in water), and potassium ethyltrifluoroborate (10 μL, 500 eq, freshly prepared 330 mM stock in buffer) were added sequentially. The vial was then capped and removed from the glove box, and irradiated with blue LED light (50 W) for 20 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture by LC-MS (82% conversion rate).

[0423] Using the same method, several different groups were introduced into the protein substrate. The following table lists these further examples along with variations in reaction conditions. The resulting functionalized side chains are shown in Figure 5.

[0424] [Table 1-1]

[0425] [Table 1-2]

[0426] [Table 1-3]

[0427] [Table 1-4]

[0428] [Table 1-5]

[0429] [Table 1-6]

[0430] [Table 1-7]

[0431] [Table 1-8]

[0432] [Table 1-9]

[0433] [Table 1-10]

[0434] [Table 1-11]

[0435] [Table 1-12]

[0436] [Table 1-13]

[0437] Further examples of the BACED reaction are described below.

[0438] 1h implementation - Large scale Glass vials containing pre-weighed amounts of Ru(bpm)3Cl2 (1.8 mg, 2.8 μmol), catechol (3.1 mg, 28 μmol), and 4-bromobutylboronic acid (76 mg, 420 μmol) were transferred to a glove box (<6 ppm O2), and NH4OAc buffer (500 mM, pH 6, 3M Gdn-HCl, 2 mL) containing human histone H3-Dha9 (5 mg, final concentration 2.5 mg / mL, 140 μM) were added. After solubilizing the reagents with a short pipette missing, the vials were capped and removed from the glove box, and irradiated with blue LED light (50 W) for 1 hour. After the reaction, the solution was dialyzed three times against milliQ H2O (twice every 2 hours, once overnight, at 4°C), and the protein recovery rate (94%) was measured by nanodropping. The conversion rate was determined by analyzing a fixed portion of the mixture after dialysis using LC-MS.

[0439] AcrA-Dha123 was introduced in 1 hour. In a glove box, a glass HPLC vial was filled with fluorinated phosphate buffer (20 mM NaPi, 100 mM NaF, pH 7.4, 95 μL) containing AcrA-Dha123 (4 μM final concentration). Ru(bpy)3Cl2 (10 eq of freshly prepared 4 mM stock in 1 μL of water), catechol (50 eq of freshly prepared 20 mM stock in 1 μL of water), and potassium phenethyltrifluoroborate (500 eq of freshly prepared 40 mM stock in 5 μL of buffer) were added in succession. The vial was then capped, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS. After the reaction, the sample was desalted in the same buffer (PD Minitrap G25) to remove excess reagents, and analyzed by circular dichroism along with the relevant protein controls.

[0440] Introduced to NPβ-G2F-Dha61 for 1 hour. In a glove box, fluorinated phosphate buffer (20 mM NaPi, 100 mM NaF, pH 7.4, 95 μL) containing NPβ-G2F-M61Dha (40 μM final concentration) was added to a glass HPLC vial. Ru(bpy)3Cl2 (40 mM stock, freshly prepared in 1 μL of water, 10 eq), catechol (200 mM stock, freshly prepared in 1 μL of water, 50 eq), and potassium phenethyltrifluoroborate (400 mM stock, freshly prepared in 5 μL of buffer, 500 eq) were added in succession. The vial was then capped, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture by LC-MS. After the reaction, the sample was desalted in the same buffer (PD Minitrap G25) to remove excess reagents, and analyzed by circular dichroism along with the relevant protein controls.

[0441] 1h installation on PanC-Dha47 In a glove box, NH4OAc buffer (500 mM, pH 6, 3M Gdn·HCl, 95 μL) containing charged PanC-Dha47 (4 μM final concentration) was added to a glass HPLC vial. Ru(bpy)3Cl2 (10 eq of freshly prepared 4 mM stock in 1 μL of water), catechol (50 eq of freshly prepared 20 mM stock in 1 μL of water), and potassium phenethyltrifluoroborate (500 eq of freshly prepared 40 mM stock in 5 μL of buffer) were added in succession. The vial was then capped, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed portion of the crude mixture by LC-MS.

[0442] Example 2 - ASOOF, iodine-ASOOF, and difluorobromo precursor reaction The reactions according to the methods of embodiments (i), (ia), and (ib) described herein were demonstrated using various substituents to functionalize exemplary Dha-containing proteins having a variety of different functional side chains. A pySOOF reaction manifold was used as the exemplary ASOOF moiety according to embodiment (i).

[0443] All side chains introduced using the pySOOF reaction manifold (2a-2ag, see Figure 5) were screened with the model protein substrate histone H3-Dha9. Since the RC(O)CF2Br (embodiment (ib)) radical precursor follows the same mechanistic pathway, this example also includes substrate ranges derived from them. Various different side chains were introduced for different histone variants, modification sites, or protein scaffolds. Site-specific side-chain introduction was confirmed by LC-MS / MS analysis. All reactions defined as "large-scale" used >1 mg of protein Dha as a starting material, and yields were measured in Nanodrop after changing buffers to remove low molecular weight reaction components. All reactions were monitored by LC-MS. Conversion rates were calculated as the ratio of total product to Dha starting material, based on the deconvolution intensity of the LC / MS spectrum. In some cases, minor undesirable products such as double additions were present and expressed as a percentage of the total product. A 10% baseline cut was used as a rule when analyzing the intensity of the deconvolved spectrum. Often, small amounts of methionine oxidation occurred during preparation, storage, and use (+16 Da + / - 1 Da). These adducts were combined for the calculation of starting material and product.

[0444] 2a-In the first example, -CF2H was introduced into the protein substrate using the pySOOF reaction manifold shown in Figure 6(B).

[0445] In a glove box, a fixed amount of histone H3-Dha9 (100 μg, 6.59 nmol) was added to a glass HPLC vial containing FeSO4·7H2O (408 μg, 1.65 μmol), and the final protein concentration was diluted to 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding difluoromethyl 2-pyridylsulfone (13.2 nmol [0.02 M] in DMSO) and Ru(bpy)3Cl2 (16.48 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture by LC-MS (conversion rate 100%). Using the same method, numerous different groups were introduced into the protein substrate. The table below lists these further examples and describes any changes in reaction conditions. The resulting functionalized side chains are shown in Figure 5.

[0446] [Table 2-1]

[0447] [Table 2-2]

[0448] [Table 2-3]

[0449] [Table 2-4]

[0450] [Table 2-5]

[0451] [Table 2-6]

[0452] [Table 2-7]

[0453] [Table 2-8]

[0454] [Table 2-9]

[0455] [Table 2-10]

[0456] The pySOOF reaction was also carried out on a large scale for many starting materials using essentially the same method, except that the crude mixture was treated with EDTA (8 mg) and low molecular weight reagents were removed by buffer exchange using PD midiTrap G25. Subsequently, the protein concentration was measured via Nanodrop to obtain the yield. Examples are shown in the table below.

[0457] [Table 3-1]

[0458] [Table 3-2]

[0459] [Table 3-3]

[0460] The pySOOF reaction was also carried out on a large scale for many starting materials using essentially the same method, except that beta-mercaptoethanol was added at a concentration of 80 mM after the reaction. This reaction was observed to have a favorable effect in suppressing the excessive methionine oxidation often observed when working with FLAG-HA-tagged human histone eH3. Examples are shown in the table below.

[0461] [Table 4-1]

[0462] [Table 4-2]

[0463] [Table 4-3]

[0464] 2t - In further examples, the group -CF2C(O)NH2 was introduced into a protein substrate using the difluorobromoradical precursor of embodiment (ib) shown in Figure 6(C).

[0465] In a glove box, a fixed amount of histone H3-Dha9 (100 μg, 6.59 nmol) was added to a glass HPLC vial containing FeSO4·7H2O (408 μg, 1.65 μmol), and the mixture was diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding 2-bromo-2,2-difluoroacetamide (32.9 nmol in DMSO [0.02 M]) and Ru(bpy)3Cl2 (16.48 nmol in 2 μL of water), the vial was capped, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture by LC-MS. Using the same method, numerous different groups were introduced into the protein substrate. The table below lists these further examples and describes any changes in reaction conditions. The resulting functionalized side chains are shown in Figure 5.

[0466] [Table 5-1]

[0467] [Table 5-2]

[0468] 2ae - In further examples, the iodine-pySOOF radical precursor of embodiment (ia) shown in Figure 6(D) was used to introduce a monofluorinated pySOOF group into a protein substrate.

[0469] In a glove box, a fixed amount of histone H3-Dha9 (100 μg, 6.59 nmol) was added to a glass HPLC vial containing FeSO4·7H2O (408 μg, 1.65 μmol), and the mixture was diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding 2-((fluoroiodomethyl)sulfonyl)pyridine (65.9 nmol in DMSO [0.1M]) and Ru(bpy)3Cl2 (33 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture by LC-MS. The resulting functionalized side chains are shown in Figure 5.

[0470] [Table 6]

[0471] Example 3 - Anisotropic reaction on a protein As shown above, the method of the present invention, such as using a BACED reagent functionalized with alkyl halides, can functionalize proteins with highly reactive side chains such as alkyl halides. Such electrophilic side chains enable diverse and further functionalization, as shown in Figure 3(b).

[0472] The scheme in Figure 3(b) outlines the reaction scheme and LC / MS spectra for the photocatalytic introduction of bromonolleucine (Bnl) and iodonorleucine (Inl) into a boronate radical precursor. Stability studies of Bnl and Inl in a weakly acidic buffer revealed that the only reaction was the slow halogen exchange of Cl- ions to both I and Br, producing chloronorleucine (Cnl). Both Bnl and Inl exhibited similar reactivity to Cl- and reached complete conversion after several days. By manipulating the pH or substrate equivalent, unwanted hydroxyl group substitution or elimination side reactions can be avoided, and excellent conversions for forming CS, CP, and CN bonds from alkyl halide reactive handles on proteins can be obtained, as described later.

[0473] Formation of chloronorleucine (Cnl) at a mild pH The reaction products derived from the introduction of iodonorleucine (Inl) and bromonolleucine (Bnl), histone H3-Inl9 and H3-Bnl9, were buffered immediately after the reaction in phosphate buffer (100 mM NaPi, 3 M Gdn-HCl, pH 6) to test their long-term stability in weakly acidic buffer. Samples of each modification (100 μL, 10 μM histone H3-Inl9 or H3-Bnl9) were incubated at 37°C with shaking (600 rpm), and a fixed amount was taken from the crude reaction mixture for LC-MS analysis after 1, 16, 36, and 64 hours. Analysis showed that both H3-Inl9 and H3-Bnl9 underwent slow but almost complete conversion (~90%) to the product histone H3-Cnl9-containing chloronorleucine in nearly the same proportion, with little evidence of other side reactions at a significant level (Figure 3b).

[0474] Addition of βME to histone H3-Inl / Bnl9 The reaction products derived from the introduction of Inl and Bnl, histone H3-Inl9 and H3-Bnl9, were buffer-exchanged immediately after the reaction with phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10). Neat βME was added to both samples (25 mM, 100 μL total reaction volume, 10 μM histone H3-Inl9 or H3-Bnl9), and the samples were incubated at 37°C for 4 hours with shaking (600 rpm). A fixed amount was taken from the crude reaction mixture for LC-MS analysis. The analysis showed complete conversion of histone H3-Inl9 and H3-Bnl9 by βME substitution, consisting of H3-Cnl9 formation (chloronorleucine formation by halogen exchange) as the major product and minor product in both cases (Figure 3b).

[0475] Addition of TCEP to histone H3-Inl / Bnl9 The reaction products derived from the introduction of Inl and Bnl, histone H3-Inl9 and H3-Bnl9, were buffer-exchanged immediately after the reaction in phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10). TCEP (25 mM from 50 mM stock in buffer) was added to both samples (100 μL total reaction volume, 10 μM histone H3-Inl9 or H3-Bnl9), and the samples were incubated at 37°C for 12 hours with shaking (600 rpm). A fixed amount was taken from the crude reaction mixture for LC-MS analysis. Analysis showed complete conversion for histone H3-Inl9 and incomplete conversion for H3-Bnl9 due to TCEP substitution, consisting of H3-Cnl9 formation (chloronorleucine formation via halogen exchange) as the major product and minor product in both cases (Figure 3b).

[0476] Addition of azide to histone H3-Inl / Bnl9 The reaction products derived from the introduction of Inl and Bnl, histone H3-Inl9 and H3-Bnl9, were buffer-exchanged immediately after the reaction in phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10). Sodium azide (200 mM from 400 mM stock in buffer) was added to both samples (100 μL total reaction volume, 10 μM histone H3-Inl9 or H3-Bnl9), and the samples were incubated at 37°C for 12 hours with shaking (600 rpm). A fixed amount was taken from the crude reaction mixture for LC-MS analysis. Analysis showed complete conversion for histone H3-Inl9 and incomplete conversion for H3-Bnl9 due to azide substitution consisting of the major product in both cases and the formation of H3-Cnl9 as a minor product in the reaction with H3-Bnl9 (chloronorleucine formation by halogen exchange) (Figure 3b).

[0477] Addition of methylamine to histone H3-Inl / Bnl9 The reaction products derived from the introduction of Inl and Bnl, histone H3-Inl9 and H3-Bnl9, were buffer-exchanged immediately after the reaction in phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10). Methylamine (0.5 M from 1 M stock prepared in buffer from an aqueous methylamine source) was added to both samples (100 μL total reaction volume, 10 μM histone H3-Inl9 or H3-Bnl9), and the samples were incubated at 37°C for 12 hours with shaking (600 rpm). A fixed amount was taken from the crude reaction mixture for LC-MS analysis. Analysis showed moderate conversion to the desired modification for both H3-Inl9 and H3-Bnl9 by methylamine substitution, consisting of the major product in both cases but with significant amounts of by-products. The high pH required for the deprotonation of methylamine created significant competition with the side reactions discussed above and previously (Figure 3b), allowing only nearly molar equivalents of the reagent to be present for the addition of methylamine as the major product (Figure 3b).

[0478] Addition of dimethylamine to histone H3-Inl / Bnl9 The reaction products derived from the introduction of Inl and Bnl, histone H3-Inl9 and H3-Bnl9, were buffer-exchanged immediately after the reaction in phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10). Dimethylamine (0.5 M from 1 M stock prepared in buffer from the HCl salt) was added to both samples (100 μL total reaction volume, 10 μM histone H3-Inl9 or H3-Bnl9), and the samples were incubated at 37°C for 1 hour with shaking (600 rpm). A fixed volume was taken from the crude reaction mixture for LC-MS analysis. Analysis showed moderate conversion to the desired modification for both H3-Inl9 and H3-Bnl9 by dimethylamine substitution, consisting of the major product in both cases but with significant amounts of by-products. The high pH required for the deprotonation of dimethylamine created significant competition with the side reactions discussed above (Figure 3b), and the addition of methylamine as the major product was facilitated using nearly molar equivalents of the reagent (Figure 3b).

[0479] Addition of trimethylamine to histone H3-Inl / Bnl9 The reaction products derived from the introduction of Inl and Bnl, histone H3-Inl9 and H3-Bnl9, were buffer-exchanged immediately after the reaction in phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10). Trimethylamine (0.5 M from 1 M stock prepared in buffer from an aqueous source) was added to both samples (100 μL total reaction volume, 10 μM histone H3-Inl9 or H3-Bnl9), and the samples were incubated at 37°C for 1 hour with shaking (600 rpm). A fixed volume was taken from the crude reaction mixture for LC-MS analysis. Analysis showed excellent conversion to the desired modification for both H3-Inl9 and H3-Bnl9 by trimethylamine substitution, which consists of the major product in both cases, but with the formation of a small amount of H3-Cnl9 present in the H3-Bnl9 reaction (Figure 3b). Trimethylamine acts as an excellent nucleophile for both H3-Inl9 and H3-Bnl9 because side reactions are suppressed, and it works even better than mono- or dimethylamine substitution reactions.

[0480] As demonstrated in the example above, the flexibility (both structural and reactivity) of the incorporated halogen nucleophiles provided a novel anisotropic reaction platform on proteins for binding to extraprotein nucleophiles (Figure 3B). This effectively enabled a strategic reversal (umpolung) of the common but non-site-specific practice in the field of protein binding: targeting extraprotein nucleophiles with nucleophiles widely present in proteins (e.g., Cys, Lys). By adjusting pH, off-protein nucleophile concentration, and halogen selection, it was demonstrated that intermolecular nucleophilic substitution at C-Hal bonds could be selectively promoted while avoiding putative competitive side reactions of elimination and intraprotein nucleophilic substitution. In addition to the generation of CS bonds (with thiols, beta-mercaptoethanol, and BME), CP bonds (with phosphine TCEP), and CN bonds (with methyllysine PTM and various methylamines that make N3-donating Anl), it was even possible to further adjust Finkelstein-type nucleophilic reactivity by direct halogen exchange (Br→Cl or I→Cl).

[0481] Example 4 - Radical reaction on protein As shown above, the method of the present invention allows for the functionalization of proteins using radical precursor moieties (such as the ASOOF motif) on proteins. Such groups enable the functionalization of a wider variety of proteins, as shown in Figure 3(a). Below are various radical reactions on proteins that can be used to further functionalize proteins or peptides, for example, through on-site radical polymerization, reactions with further radical substituents, and protein-protein crosslinking.

[0482] General Protocol In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding radical acceptor reagent (10-200 eq, in DMSO [0.1M-0.5M] or water [0.1M-1M]), Ru(bpy)3Cl2 (2-5 eq in 2 μL water), and FeSO4·7H2O (0-100 eq in 4 μL water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS. This procedure is summarized in Figure 3a.

[0483] Converting pySOOF to DfeGly In a glove box, a fixed amount of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding Ru(bpy)3Cl2 (16.48 nmol in 2 μL of water) and FeSO4·7H2O (1.648 μmol in 4 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture by LC-MS.

[0484] Introduction of vinylboronic acid In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding vinylboronic acid pinacol ester (1.318 μmol [1M] in DMSO) and Ru(bpy)3Cl2 (16.48 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0485] Introduction of N-acetyldehydroalanine In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding N-acetyldehydroalanine (0.824 μmol [0.5M] in DMSO) and Ru(bpy)3Cl2 (26.36 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0486] TEMPO implementation In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding 4-hydroxy TEMPO (65.9 nmol [0.1M] in water), Ru(bpy)3Cl2 (13.18 nmol in 2 μL of water), and FeSO4·7H2O (164.8 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0487] Introduction of diphenyldiselenide In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After administration of diphenyl diselenide (131.8 nmol [0.1M] in DMSO), Ru(bpy)3Cl2 (26.36 nmol in 2 μL of water), and FeSO4·7H2O (164.8 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0488] Introduction of Boc-4-methylene-piperidine In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding Boc-4-methylene-piperidine (659 nmol [0.5M] in DMSO) and Ru(bpy)3Cl2 (32.95 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0489] Introduction of 3,4-butenediol In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding 3,4-butenediol (659 nmol [0.5M] in DMSO) and Ru(bpy)3Cl2 (32.95 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0490] Introduction of vinyl acetate In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding vinyl acetate (659 nmol [0.5M] in DMSO) and Ru(bpy)3Cl2 (32.95 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0491] Introduction of dimethylethylidene malonate In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding dimethylethylidene malonate (659 nmol in DMSO [0.5M]) and Ru(bpy)3Cl2 (32.95 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0492] Introduction of acrylamide In a glove box, a fixed volume of histone H3-pySOOF9 (100 μg, 6.59 nmol) was added to a glass HPLC vial and diluted to a final protein concentration of 1 mg / mL with NH4OAc (500 mM, pH 6, 3M Gdn·HCl). After adding acrylamide (131.8 nmol [0.1M] in DMSO), FeSO4·7H2O (164.8 nmol in 2 μL of water), and Ru(bpy)3Cl2 (32.95 nmol in 2 μL of water), the vial was sealed with a cap, removed from the glove box, and irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed volume of the crude mixture by LC-MS.

[0493] Furthermore, functionalization with dehydroalanine containing proteins. The histone H3 protein was functionalized with an additional dehydroalanine-containing protein, FLAG-labeled eH3-Dha9, as shown in Figure 6(E). This reaction was carried out under the same conditions as the radical reaction on the protein described above, using the specific reagents and conditions presented in the reaction scheme below. The resulting cross-linked protein-protein complex was confirmed by SDS-Gel electrophoresis (see Figure 3A).

[0494] Example 5 - KDM4A crosslinking to histone-containing Bhn The above initiation method allows for the insertion of various halogenated (chloro-, bromo-, and iodo-), potentially electrophilic side chains into proteins, such as proteins, with side chain lengths precisely matched to Lys. This highlights the remarkable chemoselectivity and efficiency of the present invention by using reagents that not only include moieties (alkyl halides) conventionally used for 2e-heteroalkylation of protein-based nucleophiles, but can also act as radical precursors via 1e-reductive initiation (see above); here, they were left untouched during 1e-radical introduction via CC bond formation. When aliphatic 4-bromobutyl-boronic acid (precursor of bromoalkyl side-chain bromohomonorleucine, Bhn, (1u)) was evaluated using cyclic voltammetry under catechol-enhanced conditions (1:12), an irreversible oxidation event corresponding to a half-potential Eox = +0.93V was observed. Notably, the absence of a reduction peak for C-Br activation and the lack of oxidation in the absence of catechol further confirmed the usefulness of the BACED reagent. This control of halogenated side chain insertion into the protein made it possible to design site-selective "protein alkylating agents" (Figure 4C). These remain essentially inactive under typical conditions of in vivo mixtures (Figure 4C), but when properly acting on the bound protein-protein interface (PPI) to "fit" snugly, their alkylation reactivity can be "inducibly" increased through solvent exclusion, effective molar count, and appropriate mimicry (Figure 4C). Such systems require a balance between electrophilic reactivity and native shape fidelity, enabling investigations of protein-protein interactions, such as enzyme substrate analysis.

[0495] Site-selective insertion of the smallest size alkyl halide side chains, such as bromonolleucine (Bnl), bromohomonorleucine (Bhn), or iodonorleucine (Inl), into proteins has been impossible until now (Figure 3B). Therefore, the method of the present invention opens up the possibility of more closely mimicking the binding of specific side chains (and thus site-specific crosslinking) to nucleophilic residues in interacting protein partners (Figure 4D). Thus, Bhn, Bnl, or Inl, by having the same simple alkyl side chain, represent almost direct (unextended) alkyl halide mimics of Lys (Figure 3A), enabling the potential probe of buried protein-protein interfaces where Lys may be present in wild-type proteins, without artifacts. This type of residue cannot be incorporated, for example, using complementary amber codon suppression. To validate this mimicry in a rigorous, buried (i.e., space-limited), transient (substrate-enzyme) strict PPI, bromohomonorleucine (Bhn, 1u) was introduced as a Lys bromination mimic to three sites of human histone isoform H3.1 (C-terminal FLAG-HA tagged, eH3.1) where Lys normally resides (sites 4, 9, and 27), creating eH3.1-Bhn4, eH3.1-Bhn9, and eH3.1-Bhn27, respectively. These "guided alkylating agent proteins" and WT controls were incubated with human histone Lys-demethylase KDM4A (N-terminal His-tagged, Figure 4D), a representative partner enzyme that processes and binds Lys residues. Coomassie staining and Western blotting showed crosslinking only with a mixture of KDM4A and Bhn-containing histone H3 protein, but not with WT histone H3 (Figure 4D). This "inducible" crosslinking property was confirmed by incubation with control proteins. None of the Bhn-containing histones showed evidence of crosslinking with either serum albumin (bovine, BSA, known as a Cys-rich control) or known nucleosome-binding partner histone H4 (which non-covalently forms H3 / H4 dimers and tetramers and is Cys-free), even after extended incubation periods and increased temperatures (2 hours at 37°C).In particular, the H3-H4 PPI is not accompanied by Lys4, Lys9, or Lys27,64, but only the H3·KDM4A PPI is. Despite the possibility of nonspecific reactions and / or binding of BSA or H4, the absence of these reactions suggests that crosslinking of our edited functionalized H3.1-Bhn variant requires a suitable PPI, such as that at KDM4A.

[0496] This apparent PPI-selective response was confirmed by MS / MS analysis (Figure 4D), revealing highly conserved crosslinking in KDM4A to two cysteine ​​molecules (Cys234 and Cys306) located in the Zn-binding domain within the critical H3-KDM4A PPI interface pocket (Figure 4C). Thus, Bhn, due to its structural similarity to Lys, is a representative mimetic of Lys behavior—when "edited" and inserted into the relevant Lys site in the protein, it jointly probes the same protein-protein interface as Lys without artifacts, thanks to the advantage of being a nearly direct (unextended) structural analogue of Lys. In this way, Bhn in the H3-Lys→Bhn "mutant" created the same "reach" to the same site as the corresponding H3-Lys wild-type protein in the limited PPI of the H3·KDM4A complex.

[0497] The reaction between the Bhn side chain and KDM4A in eH3.1-Bhn was directly and quantitatively evaluated by zinc release analysis (Figure 4D).

[0498] The ability of alkylating agent proteins to capture interaction partners was investigated using double-FLAG+HA-tagged histone eH3.1-Bhn9 immobilized on beads containing anti-HA-flag antibodies to promote the capture of interaction partners of eH3.1-Lys9 present in cells, and incubated (4 hours, 37C) with human cell (HeLa) nuclear lysates. Following such capture, Western blotting (anti-FLAG for selective detection of any eH3.1-adduct species) revealed several distinct eH3.1 adduct species found only in samples containing the alkylating agent protein histone eH3.1-Bhn9 (with a 1u side chain at site 9), while wild-type histone eH3.1 and histone-free controls showed nothing (Figure 4E).

[0499] The retained intrinsic reactivity of the eH3 protein with high concentrations of low-molecular-weight compounds; the failure of the reaction between KDM4A and low concentrations of low-molecular-weight side-chain reagents; and the successful reaction of eH3-Bhn with KDM4A at low levels (nM~μM) confirmed the origin of this novel “effective mole-driven” crosslinking reaction (again at the EM > 103 level).

[0500] General protocol for histone eH3-Bhn-KDM4A crosslinking Histone modifying enzyme KDM4A (2 μM) was mixed with either modified histone eH3.1-Bhn4 / 9 / 27 or WT control (4 μM) in HEPES buffer (50 mM, pH 7.4) and incubated at the specified temperature and time. The crosslinking reaction was quenched by adding 5X Laemmli buffer and analyzed by SDS-PAGE or Western blotting (see Figure 4E).

[0501] Details of antibodies for Western blot analysis DYKDDDDK(FLAG) tagged monoclonal antibody (eBioscience, catalog number 14-6681-82, clone FG4R, lot number 1981531, dilution 1:1,000), monoclonal anti-polyhydroxyalkali phosphatase (Sigma-Aldrich, catalog number A5588, clone HIS-1, lot number 085M4836V, dilution 1:2,000), histone H3 antibody (Cell Signaling Technology, catalog number 3638S, clone 96C10, lot number 10, 1:1,000), goat anti-mouse IgG H&L alkaline phosphatase (Sigma-Aldrich, catalog number A3562, polyclonal, lot number SLCB8722, dilution 1:10,000), anti-mouse IgG(H+L)HRP conjugate (Promega, catalog number W4021, polyclonal, lot number (0000306114, dilution 1:2,500). All antibodies were used as directed by the manufacturer.

[0502] Zn release assay The Zn release assay was performed using N-(6-nethoxy-8-quinolyl)-p-toluenesulfonamide (TSQ)(Enzo)Zn(II) fluorophores as described with some modifications 28 and 33. Briefly, the assay was carried out in a 384-well black μCLEAR® unbound plate (Grenier) using a BMG CLARIOstar (360ex / 490em) at 37°C with a reaction volume of 100 μL. The plate was shaken (5s, 700rpm) every 22 seconds for 270 cycles before reading each. The reaction consisted of 10 μM TSQ, 25 μM ebselen or 20 μM H3 K9Bhn / H3-wt / 4-bromobutylboronic acid, and an enzyme containing 2 μM KDM4A, all accompanied by 1.1% (v / v) DMSO in 50 mM HEPES (pH 7.5). After adding the compound and TSQ to the plate, KDM4A was added using a CLARIOstar injector to initiate the assay (Figure 4d). An internal calibration curve for ZnCl2 (0–2 μM) in 50 mM HEPES (pH 7.5) was included in each experiment to quantify the concentration of released Zn(II). Data were normalized by subtracting the enzyme-free control of the compound at each time point. The mean ± standard deviation (n=3 technical replicas) was plotted at each time point using GraphPad Prism 5.0, showing representative data from three biological replicas.

[0503] MS / MS data suggested that histone eH3-9Bhn cross-links to the Cys3-His Zn(II) binding site near the active site. The Zn(II) release rate was calculated from the slope of the linear regression plotted on the linear region of Zn(II) release (946 to 3982 s) plotted in GraphPad Prism 5.0. Time-dependent release of Zn(II) from KDM4A was observed when incubated with eH3-Bhn9 (9.270 ± 0.025 nM / min), but not with unmodified eH3 or 4-bromobutylboronic acid (Figure 4d). This contrasts with the rapid Zn(II) release rate of > 1663 nM / min observed when using ebselen, a small molecule inhibitor of Zn(II) chelation of KDM4A activity 28. This suggests that the release rate of Zn(II) depends on the bridging rate of H3-Bhn9 to KDM4A.

[0504] Example 6 - Effective Mole-Driven Crosslinking Reaction The enhancement of nucleophilicity due to the "effective molar number" at the protein-protein interface was further demonstrated by the unprecedented formation of Williamson-type (-COC-) ethers (Figure 4F). The second-order rate constant of this type of reaction has long been considered too low to effectively bridge protein-protein interactions at low concentrations (nM~μM) of protein (k app < 10 -4 Ms -1 Therefore, the formation of an ether bond (not within) between Cβ-O-CH2-Bhn4 suggests that the protein-protein interaction between one H3 protein and another was strongly enhanced by EM. This is thought to be due to the transient presence of H3·H3 dimers in the presence of KDM4A.

[0505] This demonstrates the potential of the present invention's method to functionalize proteins to include precisely mimicking residues such as Bhn that can capture transient intermediates, thereby providing information on novel hypothetical mechanism models.

[0506] Protein partner binding To further investigate the ability of such alkylating agent proteins to capture interaction partners, dual-FLAG+HA-tagged histone eH3.1-Bhn9 was immobilized on beads containing anti-HA-FLAG antibodies and incubated with human cell (HeLa) nuclear lysate (4 hours, 37°C) to promote the capture of interaction partners of eH3.1-Lys9 present in cells, as shown below, demonstrating the presence of various protein interaction partners.

[0507] Histone samples (20 μg or either human histone eH3.1-WT, human histone eH3.1-Bhn9, or a control without histones) were immobilized via their HA epitope tags onto anti-HA magnetic beads (Pierce 88836, 50 μL / sample, pre-equilibrated with the immobilization buffer) in HEPES buffer (50 mM, pH 7.5) at RT for 30 minutes. The beads were then incubated with HeLa nuclear lysate (250 μL, 0.5 mg / mL, 4 hours r, 37 °C, 600 rpm) to promote crosslinking. The HeLa nuclear lysate was prepared as previously described in section 2. After incubation, the beads were washed (500 μL HEPES buffer + 0.1% Tween20wx5, sdH2Ox1). Histone + interaction partners were eluted from the beads with glycine (0.1 M, pH 2.0, 100 μL, 10 min, 37 °C) and quenched with Tris buffer (1 M, pH 8.5, 15 μL). This elution and quenching was repeated with the beads. To analyze crosslinking and immunoprecipitation, all histone and lysate controls, as well as samples under all conditions of incubation, final wash, and elution, were checked via SDS-PAGE using either Coomassie blue staining or Western blotting with α-FLAG antibody (histone eH3 samples were tagged with the FLAG-HA epitope), detecting high MW bands corresponding to the mass of histone eH3.1 covalently crosslinked to unknown interaction partners (see Figures 4C and 4E).

[0508] Example 7 - Study of the reaction mechanism Inserting native and "zero-size" labeled reactive side chains into proteins provided further insights into the enzymes responsible for post-translational modifications.

[0509] Lys mimics (Figure 4) were tested by introducing acetyl-(AcLys / KAc, 1m) and benzoyllysine (BzLys / KBz, 1n) side chains, as well as H→F labeled side chain analogues K[γF2]Ac 2k and K[γF2] 2f, into protein precursors, respectively.

[0510] H3-K18Ac and H3-K18Bz were generated using the BACED reagent (Figure 4A). These proteins enabled time-course studies when incubating both histone H3-K18Ac and H3-K18Bz with Sirt2, confirming that the true Sirt2 activity in both acylated Lys is clearly due to Sirt2's strong substrate KAc > KBz selectivity (Figure 4A).

[0511] Furthermore, corresponding H→F labeled side-chain analogs such as K[γF2]Ac and K[γF2] side-chains 2k and 2f were also generated using the pySOOF reagent. In these systems, the centrally positioned γ-carbon-F2 label proved effective in enabling in situ reporting of the modification status of these side chains. Changes in the identity of the 18-position side chain of human H3.1 are related to the protein. 19The modification could be easily detected using 1H NMR (565 MHz) (which sensitively distinguished the identity of H3.1-K18 → H3.1-KAc18 despite the distance of 4 or 5 bonds from the γ-carbon-F2 label to the modification site, and thus probed the modification state (side chain 2f → 2k = δF -98.0 → -99.4, Figure 4B)). Changes in other side chains could similarly be distinguished at different sites within the same protein (e.g., H3.1-K9 → H3.1-KAc9 → H3.1-Kme39 (side chain 2f → 2k → 2j = δF -99.0 → -98.0 → -99.2) or H3.1-M27 (side chain 2x δF -74.8) or H3.1-E9 (side chain 2u δF -103.3)). Thus, the diverse range of available side chains allows this approach to explore in numerous additional directions, such as monitoring heteroatom changes (e.g., N→O, the "deaza-oxo" variant KOAc, H3.1-K18 → H3.1-KOAc18, side chain 2r, side chain 2k → 2r) ​​or more accurately assaying the side chain Met oxidation state (H3.1-M27 → H3.1-Mox27, side chain 2x → 2y → 2z).

[0512] Due to the insertion site selectivity and excellent sensitivity in "zero background" environments of this label, 19 Not only could we "read" the chemical shift of the F signal, but we could also determine its multiplicity through correlation simulation (Figure 4B). In this way, γ-F2 labeling was able to report both side-chain modification processing (KAc→K at the Nε site, 5 "lower" side chain bonds) and stereochemical processing (and therefore selectivity L vs D at the Cα site, 3 "upper" side chain bonds) simultaneously, thanks to highly sensitive CF2-diastereotopy. Similarly, this remarkable sensitivity across the entire side chain of residues enabled real-time in situ reporting of enzyme-mediated post-translational modifications on the protein. This suggests that Sirt2, an HDAC deacylate (despite its processing of modifications 6 bonds away), can report > 14 (ΔΔGφ > 6.6 kJ mol) -1It was revealed that it exhibits L / D selectivity.

[0513] Thus, the insertion of site-specific labels according to the present invention makes it possible to simultaneously determine both the substrate selectivity and stereoselectivity of post-translational modifying enzymes in unchanged proteins in real time, which was previously impossible. The sensitivity of γ-F2 labeling was applied to monitor the difference between folding and higher-order assembly states in a single protein. In this way, the use of H3-DfeGly9 makes it possible to directly monitor the entire stepwise process of histone octamer assembly, even at low submilligram levels, from unfolded H3 monomer → folded H3 monomer → (H3)2·(H4)2 heterotetramer to the complete (H3)2·(H4)2·(H2A)2·(H2B)2 heterooctamer.

[0514] 19 Reconstruction of octamers for F-NMR measurements Unfolded histone H3-DfeGly9 19 After 1F-NMR measurement, 2 mg of histone protein was buffer-exchanged in 1 mL of unfolding buffer (7 M Gdn-HCl, 10 mM Tris, 1 mM EDTA, 10 mM DTT, 1 mM benzamidine, pH 7) using a PD10 G-25 Minitrap, and then buffer-exchanged again in Tris buffer (150 mM NaCl, 10 mM Tris, 1 mM EDTA, 2 mM βME, pH 7.5). A final 50% D2O buffer was prepared with an equal volume of deuterated Tris buffer (same as above, but prepared with 100% D2O) and concentrated to 0.75 mL (Vivaspin 6, 5 kDa MWCO). This mixture was centrifuged (15000 rpm, 10 min, 4°C) to pelletize the precipitate, and the internal standard substance trifluoroethanol (0.001 μL) was added. The concentration was measured (Nanodrop, 2.0 mg / mL), folding was confirmed by circular dichroism (CD), and the sample was filtered into an NMR tube.

[0515] To reconstitute the histone H3-DfeGly9-H4 tetramer, modified histone H3 and histone H4 WT (1:1 molar ratio, 2.5 mg of histone H3-DfeGly9) were mixed in unfolding buffer (6 mL), incubated at RT for 30 minutes, and then dialyzed in refolding buffer (3 times per 1 L, 2 hours each, overnight). The resulting solution was centrifuged (15000 rpm, 10 min, 4°C) to pelletize the precipitate, measured the concentration (0.5 mg / mL, 1 mL), and purified via size exclusion (Superdex S75, pre-equilibriumized with 16 / 60 refolding buffer). The tetramer-containing fractions (visualized by SDS-PAGE analysis) were combined, concentrated, and resuspended in 50% deuterated refolding buffer (prepared with 1:1 H2O / D2O). Trifluoroethanol (0.1 μL per 1 mL) was added as an internal NMR standard. The final concentration was measured (Nanodrop, 2.5 mg / mL), folding was confirmed by circular dichroism (CD), and the sample was filtered into an NMR tube.

[0516] To reconstitute the histone H3-DfeGly9-H4-H2A-H2B octamer, all histones were dissolved in unfolding buffer (1:1:1.1:1.1 molar ratio, 25 nmol of denatured histone H3), incubated at RT for 30 minutes, and dialyzed in refolding buffer (3 x 1 L, every 2 hours, once, overnight). The resulting solution was centrifuged (15000 rpm, 10 min, 4°C) to pelletize the precipitate into 310 pellets, and purified via size exclusion as shown above. Fractions containing the H3F-H4-H2A-H2B octamer were collected and their concentrations measured (Nanodrop, 0.8 mg total), and internal standard trifluoroethanol was added (0.1 μL). The NMR sample was prepared and measured as shown above. After NMR, the octamer was analyzed by checking the correct folding by SDS-PAGE and CD.

[0517] Further synthesis examples Further compounds used in the examples were synthesized as follows. The reaction product was 1 H, 13 C and 19 Analysis and confirmation were performed using 1F NMR.

[0518] [ka]

[0519] To a solution of aspartic acid (5.0 g, 17.3 mmol) in HF (170 mL), isobutyl chloroformate (6.8 mL, 51.8 mmol), followed by iPr2NEt (4.5 mL, 25.95 mmol), was added at 0°C and the mixture was stirred at 0°C for 2 hours. NaBH4 (4.58 g, 121.3 mmol) was added in small amounts, followed by careful addition of H2O (40 mL) over approximately 30 minutes. The mixture was warmed to room temperature and then extracted with HCl (3 x 200 mL) quenched with saturated aqueous solution NH4Cl (300 mL). The combined organic layer was washed with saturated aqueous solution NaCl (300 mL), dried (MgSO4), filtered, and concentrated under vacuum to obtain its alcohol as a yellow oil. The crude product was then purified by flash chromatography (3:7, HCl:petroleum ether) to obtain the desired difluorosulfone as a colorless oil (4.3 g, 90% yield) AMG-1-48-A.

[0520] [ka]

[0521] To a solution of alcohol (2.0 g, 7.24 mmol) in CH2Cl2 (50 mL), triethylamine (2.5 mL, 18.15 mmol) was added at 0°C, followed by the slow addition of mesyl chloride (670 μL, 8.7 mmol). The mixture was stirred at this temperature for 30 minutes and then poured into saturated aqueous NaCl (150 mL). The aqueous phase was extracted with CH2Cl2 (3 x 100 mL), and the combined organic layers were dried (MgSO4), filtered, and concentrated under vacuum to obtain mesylate as a white needle-like substance. Crude mesylate solution in MeCN (50 mL) was mixed with 2-thiopyridine (968 mg, 8.71 mmol) and triethylamine (1.52 mL, 10.52 mmol). The reaction mixture was stirred for 72 hours, quenched with H2O (100 mL), and the pH was adjusted to ~7 with 1 M HCl. The aqueous phase was then extracted with ELISA (3 x 70 mL), the combined organic layers were dried (MgSO4), filtered, and concentrated under vacuum to obtain a yellow oil. To a crude thioether solution in CH2Cl2 (50 mL), mCPBA (3.56 g, 15.97 mmol, 77 wt%) was added at 0°C. The mixture was stirred at this temperature for 3 hours, quenched with 10% aqueous Na2S2O3 (100 mL), and extracted with CH2Cl2 (2 x 50 mL). The combined organic phase was washed with saturated aqueous NaHCO3 (3 x 150 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (7:13, siRNA:petroleum ether) to obtain the desired sulfone as a white solid (2.02 g, 69% yield from starting alcohol) AMG-1-64-A

[0522] [ka]

[0523] A solution of sulfone (1.25 g, 3.21 mmol) and NFSI (1.37 g, 4.27 mmol) in THF (45 mL) was added dropwise to a solution of NaHMDS in THF (7.49 mL, 1 M) at -78 °C. The solution was stirred at this temperature for 4.5 hours and quenched with saturated aqueous solution NH4Cl (150 mL). The aqueous phase was then extracted with SiO (3 x 100 mL), the organic phase was dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (5:95, SiO:CH2Cl2) to obtain the desired sulfone (~8% diF compound contamination) as a white solid (740 mg, 57% yield from starting alcohol) AMG-2-20-A AMG-3-05

[0524] [ka]

[0525] To a solution of mono-fluorosulfone (1.00 g, 2.4 mmol) in DCM (5 mL), TFA (5 mL) was added. The solution was stirred at RT for 3 hours and then concentrated under vacuum. The residue was reprocessed under the same conditions. After further concentration, the residue was dissolved in anhydrous MeOH (3 mL) and HCl (4 M, 1 mL) in dioxane was added. The solution was stirred for 15 minutes and concentrated under vacuum. This was repeated at least twice to obtain a white powder.

[0526] [ka]

[0527] To a solution of difluoromethylpyridylsulfone (500 mg, 2.6 mmol) in THF (10.4 mL), iodine (2.6 g, 10.36 mmol) was added at -35 °C, followed by KOtBu (1 M, 10.4 mL in THF). The reaction mixture was stirred at this temperature for 1 hour, at which point it was quenched with HCl (1 M, 20 mL). The aqueous phase was extracted with ELISA (2 x 30 mL), and the combined organic layers were washed with sat. aq. Na2S2O3 (50 mL) and saline (50 mL), then dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (3:7 pet. ether:DCM) to obtain the desired iodine-Hu as a white solid (418 mg, 51%).

[0528] [ka]

[0529] To a solution of Boc-Ser-OMe (2.68 g, 12 mmol) in MeCN (30 mL), Boc2O (5.87 g, 26 mmol) was added at 0°C, followed by DMAP (0.30 g, 2.4 mmol). The solution was stirred and gradually warmed to RT over 6 hours, after which DBU (0.18 mL, 1.2 mmol) was added. The mixture was stirred at RT for 16 hours and then concentrated under vacuum. The residue was then dissolved in HCl (150 mL), washed with HCl (1 M, 100 mL) and sat. aqueous NaHCO3 (100 mL), then dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (1:19 → 1:4, HCl:pet. ether) to obtain the desired Dha as a white solid (1.80 g, 50%) AMG-2-98.

[0530] [ka]

[0531] Dha (21 mg, 0.07), iodine-Hu (15 mg, 0.04), H-atomic source, e.g., Hansch ester (15.5 mg, 0.06), and photocat (0.01 eq) were placed in vials and transferred to a glove box. Next, DMSO / H2O (0.5 mL, 5:1) was added, the vials were sealed and removed from the glove box, and irradiated for 5 hours in either a photobox or a small multiwell plate. The reaction efficiency was first evaluated using TLC analysis, and the best reaction with a conversion rate of approximately 30-40% was used. 1 H-NMR and 19 F-NMR analysis was performed. Bt-AA

[0532] [ka]

[0533] To a solution of aspartic acid (4.80 g, 16.6 mmol) in THF (170 mL), isobutyl chloroformate (6.53 mL, 49.8 mmol) was added at 0°C, followed by iPr2NEt (4.32 mL, 24.9 mmol), and the mixture was stirred at 0°C for 3 hours. NaBH4 (4.40 g, 116.2 mmol) was added in small amounts, and H2O (38 mL) was added carefully over approximately 30 minutes. The mixture was then warmed to room temperature, diluted with HCl (3 x 300 mL, 0.4 M), washed with aqueous HCl (3 x 300 mL, 0.4 M) and saturated aqueous NaCl (300 mL), dried (Na2SO4), filtered, concentrated under vacuum, and a yellow oil was obtained as an alcohol, which was used without further purification. AMG-3-43

[0534] To a solution of alcohol XX (16.6 mmol) in CH2Cl2 (120 mL), triethylamine (5.78 mL, 41.5 mmol) was added at 0°C, followed by the slow addition of mesyl chloride (1.54 mL, 19.9 mmol). The mixture was stirred at this temperature for 30 minutes and poured into saturated aqueous NaCl (200 mL). The aqueous phase was extracted with CH2Cl2 (3 x 150 mL), the combined organic layers were dried (Na2SO4), filtered, and concentrated under vacuum to obtain mesylate as a white solid. To a crude mesylate solution in MeCN (120 mL), mercaptobenzothiazole (3.61 g, 21.58 mmol) and triethylamine (3.47 mL, 24.9 mmol) were added. After 16 hours, TLC showed the presence of SM, so excess K2CO3 (4.8 g, 34.8 mmol) was added, and the reaction was stirred for a further 72 hours. The reaction was diluted with siRNA (250 mL), washed with saturated aqueous NaHCO3 (2 x 200 mL), water (200 mL), aqueous HCl (3 x 200 mL, 0.5 M), and saturated aqueous NaCl (200 mL), dried (Na2SO4), filtered, and concentrated under vacuum to obtain a yellow oil. AMG-3-46

[0535] To a solution of crude thioether (13.42 mmol) in CH2Cl2 (150 mL), mCPBA (9.02 g, 40.26 mmol, 77 wt%) was added at 0°C. The mixture was stirred at this temperature for 5 hours, and then a portion of the mCPBA (2.0 g, 8.92 mmol, 77 wt%) was added, and the reaction was stirred for 16 hours. At this point, the reaction mixture was cooled to 0°C, quenched with 10% aqueous Na2S2O3 (200 mL), and diluted with CH2Cl2 (200 mL). The organic phase was washed with saturated aqueous NaHCO3 (5 x 400 mL) and saturated aqueous NaCl (300 mL), dried (Na2SO4), filtered, and concentrated under vacuum to obtain the sulfone as a yellow powder. The crude product was analyzed by MS and NMR. AMG-3-54 LRMS(ESI)479.0(M+Na + )

[0536] [ka]

[0537] To a solution of Bt-sulfone (410 mg, 0.90 mmol) in THF (5 mL), LiHMDS (2.70 mL, 2.70 mmol, 1 M in THF) was added dropwise at -78 °C. The solution was stirred at this temperature for 5 minutes, at which point NFSI (6.75 mL, 2.70 mmol, 0.4 M in THF) solution was added dropwise. The solution was stirred at this temperature for 30 minutes until TLC analysis showed consumption of SM. The reaction mixture was then quenched at -78 °C with saturated aqueous NH4Cl (10 mL) and Et2O (5 mL). The aqueous layer was then extracted with Et2O (2 x 15 mL), and the combined organic layers were washed with saturated aqueous NH4Cl (30 mL) and saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. Next, the crude product was purified by flash chromatography (DCM → 1:49 Et2O:DCM) to obtain the desired diF-Bt-AA as a white solid (219 mg, 49% yield from starting aspartic acid). AMG-3-55-A LRMS (ESI) 515.0 (M+Na + )

[0538] [ka]

[0539] A solution of diF-Bt sulfone (180 mg, 0.37 mmol) in TFA (4.5 mL) was mixed with water (0.5 mL). The solution was stirred at RT for 3 hours and then concentrated under vacuum. The residue was dissolved in anhydrous MeOH (3 mL) and HCl (4 M, 1 mL) in dioxane was added. The solution was stirred for 15 minutes and then concentrated under vacuum. This was repeated at least twice to obtain a white powder. AMG-3-56 or AMG-3-76-cr (no decomposition) LRMS (ESI)337.0 (M+H + )

[0540] [ka]

[0541] To a solution of Bt-sulfone (500 mg, 1.10 mmol) in THF (6 mL), LiHMDS (2.20 mL, 2.20 mmol, 1 M in THF) was added dropwise at -78 °C. The solution was stirred at this temperature for 5 minutes, at which point NFSI (3.57 mL, 1.43 mmol, 0.4 M in THF) solution was added dropwise. The solution was stirred at this temperature for 50 minutes, until TLC analysis showed consumption of SM. The reaction mixture was then quenched at -78 °C with saturated aqueous NH4Cl (10 mL) and Et2O (5 mL). The aqueous layer was then extracted with Et2O (2 x 15 mL), and the combined organic layers were then quenched with saturated aqueous NH4Cl (30 mL) and saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. Next, the crude product was purified by flash chromatography (DCM → 1:49 Et2O:DCM) to obtain the desired mono-F-Bt-AA as a white solid (258 mg, 49% yield). AMG-3-80-A

[0542] [ka]

[0543] A solution of diF-Bt sulfone (250 mg, 0.53 mmol) in TFA (4.5 mL) was mixed with water (0.5 mL). The solution was stirred at RT for 3 hours and then concentrated under vacuum. DCM was added and concentrated under vacuum. This process was repeated at least twice to obtain a white powder (210 mg, 95%). AMG-3-86 Fluoro-Lys

[0544] [ka]

[0545] To a solution of mercaptobenzothiazole (2.92 g, 17.4 mmol) in MeCN (100 mL), K2CO3 (4.80 g, 34.8 mmol), NaI (350 mg, 2.33 mmol), and 3-Boc-aminopropyl bromide (5.00 g, 20.9 mmol) were added. The reaction mixture was stirred for 16 hours, diluted with SiO2 (250 mL), washed with water (150 mL), saturated aqueous NH4Cl (150 mL), and saturated aqueous NaCl (150 mL), dried (Na2SO4), filtered, and concentrated under vacuum to obtain a yellow oil. To a solution of crude thioether (17.4 mmol) in CH2Cl2 (200 mL), mCPBA (11.7 g, 50.2 mmol, 77 wt%) was added at 0°C. The mixture was stirred at this temperature for 5 hours, and then a portion of mCPBA (3.0 g, 13.38 mmol, 77 wt%) was added, and the reaction was stirred for 16 hours. At this point, the reaction mixture was cooled to 0°C, quenched with 10% aqueous Na2S2O3 (200 mL), and diluted with CH2Cl2 (200 mL). The organic phase was washed with saturated aqueous NaHCO3 (5 x 400 mL) and saturated aqueous NaCl (300 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was then purified by flash chromatography (2:49 → 1:19 siRNA:DCM) to obtain the desired Bt-sulfone as a white solid (4.85 g, 78% yield over two steps). LRMS (ESI) 379.0 (M+Na+ ).

[0546] [ka]

[0547] LiHMDS (2.0 mL, 2.0 mmol, 1 M in THF) was added dropwise to a solution of sulfone (350 mg, 1.0 mmol) in THF (5 mL) at -78 °C. The solution was stirred at this temperature for 5 minutes, at which point a solution of NFSI (3.75 mL, 1.5 mmol, 0.4 M in THF) was added dropwise. The solution was stirred at this temperature for 30 minutes until TLC analysis showed consumption of SM. The reaction mixture was then quenched at -78 °C with saturated aqueous NH4Cl (15 mL) and Et2O (20 mL). The aqueous layer was then extracted with Et2O (2 x 20 mL), and the combined organic layers were washed with saturated aqueous NH4Cl (30 mL) and saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. Next, the crude product was purified by flash chromatography (DCM → 3:97 Et2O:DCM) to obtain the desired mono-F-sulfone as a white solid (150 mg, 40% yield). AMG-3-68A

[0548] [ka]

[0549] A solution of Boc amine (73 mg, 0.195 mmol) in DCM (2 mL) was mixed with 4 M HCl (0.48 mL, 1.95 mmol) in dioxane. The mixture was stirred at room temperature for 2 hours, and then concentrated under an N2 stream while co-evaporating by adding excess DCM. This quantitatively obtained the desired amine as a white solid hydrochloride. AMG-3-70-cr

[0550] [ka]

[0551] To a solution of sulfone (350 mg, 1.0 mmol) in THF (5 mL), LiHMDS (3.0 mL, 3.0 mmol, 1 M in THF) was added dropwise at -78 °C. The solution was stirred at this temperature for 5 minutes, at which point mNFSI (7.50 mL, 3.0 mmol, 0.4 M in THF) solution was added dropwise. The solution was stirred at this temperature for 30 minutes until TLC analysis showed consumption of SM. The reaction mixture was then quenched at -78 °C with saturated aqueous NH4Cl (15 mL) and Et2O (20 mL). The aqueous layer was then extracted with Et2O (2 x 20 mL), and the combined organic layers were washed with saturated aqueous NH4Cl (30 mL) and saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. Next, the crude product was purified by flash chromatography (DCM → 3:97 Et2O:DCM) to obtain the desired mono-F-sulfone as a white solid (200 mg, 51% yield). AMG-3-69A

[0552] [ka]

[0553] To a solution of Boc amine (80 mg, 0.204 mmol) in DCM (1 mL), TFA (200 μL) was added. The mixture was stirred at room temperature for 2 hours, and then concentrated by co-evaporating the excess DCM under a stream of N2 gas. This quantitatively obtained the desired amine as a TFA salt as a white solid. AMG-3-83 LRMS (ESI)(M+H + )293.0

[0554] [ka]

[0555] LiHMDS (3.0 mL, 3.0 mmol, 1 M in THF) was added dropwise to a solution of sulfone (356 mg, 1.0 mmol) in THF (5 mL) at -78 °C. The solution was stirred at this temperature for 5 minutes, at which point NBS solution (531 mg, 3.0 mmol) was added. The solution was stirred at this temperature for 45 minutes until TLC analysis showed consumption of SM. The reaction mixture was then quenched at -78 °C with saturated aqueous NH4Cl (15 mL) and Et2O (20 mL). The aqueous layer was then extracted with Et2O (2 x 20 mL), and the combined organic layers were washed with saturated aqueous NH4Cl (30 mL) and saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. Next, the crude product was purified by flash chromatography (DCM → 3:97 Â:DCM) to obtain the desired monoBr-sulfone as a white solid (245 mg, 56% yield). AMG-3-84-A LRMS (ESI)(M+H + ) 434.5, 436.5

[0556] Example 8 - Synthesis and incorporation of pySOOF amino acids into proteins The following synthetic amino acids were incorporated into a maltose-binding protein using the protocol described below and in Figure 7, according to the method of embodiment (iai) described herein.

[0557] [ka]

[0558] Plasmids containing pyroLys tRNA, a tRNA synthetase pair, and maltose-binding protein (MBP) were co-transformed into E. coli BL21(DE3) cells and then plated. Single colonies were used for expression, and amino acids were administered at OD 0.5, followed by induction of expression at OD = 0.8 cells using IPTG. Cells were then harvested after overnight expression.

[0559] Next, the crude protein was purified using Ni affinity chromatography. The desired protein, including non-natural amino acids, was isolated with reasonable purity. Analysis of the protein via MS using Xevo confirmed the desired protein incorporating PySOOF AA.

[0560] Example 9: Labeling of proteins using fluoro-Bt-sulfone Using a Bt-sulfone system, various Dha-containing proteins were functionalized with various substituents using the method of Embodiment (i) described herein. The reaction scheme is illustrated in Figures 8A-D. As can be seen, proteins were successfully labeled using both diF-Bt-AA and biotinylated Bt-sulfone. Furthermore, fluorinated lys analogs were generated on proteins using the Bt-sulfone system.

[0561] Example 10 Protein using fluoro-Bt-sulfone 18 F sign As mentioned above, 18 Proteins and peptides containing 1F radiolabeling can be prepared using the methods described herein. 18 The 1F-labeled protein is produced using the method of embodiment (i) above. In the first step, [ 18 F]KF / K 222 Using 18 A radical precursor compound was provided by a halogen exchange (halex) reaction with 2-(bromofluoromethyl)thio)pyridine without an adduct to introduce F, followed by oxidation to a sulfone reagent (see Figure 9).

[0562] The reaction shown in Figure 9A was carried out using the procedure described below. Activity (3.4 GBq dried [ 18 F]KF / K 222 To the full batch of ), the precursor solution (11.1 mg, 0.04 mmol in 0.5 mL MeCN) was added, and the solution was stirred at 110°C for 10 minutes. Next, 18The crude reaction product containing the fluorine-labeled compound was cooled and diluted with 4 mL of H2O. The mixture was then filtered through a C18 Plus cartridge (pre-treated with EtOH (10 mL) and H2O (10 mL)). NaIO4 (52 mg, 0.24 mmol) and RuCl in H2O (4 mL) 3· A solution containing xH2O (2 mg, 0.010 mmol) was passed through a C18+ cartridge with a 30-second pause every 1 mL. After complete addition, oxidation was performed by standing at room temperature for 5 minutes. Next, the crudely labeled sulfone reagent was eluted from the cartridge with 1.2 mL of MeCN, and then purified by semi-prep HPLC (55% MeCN in 25 mM ammonium formate buffer). Benzothiazole sulfone CH 18 The peak corresponding to FF was collected in a collection vial containing 20 mL of water for approximately 12.5 minutes (retention time). This solution was then passed through a C18 Plus cartridge (pre-treated with EtOH (10 mL) and H2O (10 mL)). The reagent was eluted from the cartridge and eluted into a reaction vial containing Et2O (~1.2 mL total volume). A certain amount of purification was performed. 18 Et2O solution containing F-sulfone reagent was dispensed into reaction vials so that the initiation activity for each protein labeling reaction was approximately 25-30 MBq. This solution was then concentrated under N2 supply until dry at rt. Next, a protein solution containing photocatalyst, iron, and DMSO was added under buffering conditions.

[0563] Histone NTEV R2Dha was reacted using the reaction conditions shown in the table below (RCY = radiochemical yield). 18 Functionalization was performed using fluorine-labeled BtSOOF (shown in Figure 9A).

[0564] [Table 7]

[0565] Desired 18 The formation of the 1F-labeled compound was confirmed by comparing its retention time with that of the low-temperature standard using RP-HPLC.

[0566] The reaction shown in Figure 9B was carried out using the following procedure. Activity (3.4 GBq dried [ 18 F]KF / K 222 To the full batch of ), the precursor solution (11.1 mg, 0.04 mmol in 0.5 mL MeCN) was added, and the solution was stirred at 110°C for 10 minutes. Next, 18 The crude reaction product containing the 1F-labeled compound was cooled and diluted with 4 mL of H2O. The mixture was then filtered through a C18 Plus cartridge (pre-treated with EtOH (10 mL) and H2O (10 mL)). NaIO4 (52 mg, 0.24 mmol) and RuCl in H2O (4 mL) 3· A solution containing xH2O (2 mg, 0.010 mmol) was passed through a C18+ cartridge in 1 mL increments, with a 30-second pause between additions. After complete addition, oxidation was performed by standing at room temperature for 5 minutes. Next, the crudely labeled sulfone reagent was eluted from the cartridge with 1.2 mL of MeCN, and then purified by semi-prep HPLC (55% MeCN in 25 mM ammonium formate buffer). Benzothiazole sulfone CH 18 The peak corresponding to FF was collected in a collection vial containing 20 mL of water for approximately 12.5 minutes (retention time on a Gemini column). This solution was then passed through a C18 Plus cartridge (pre-treated with EtOH (10 mL) and H2O (10 mL)). The reagents were eluted from the cartridge and into a reaction vial containing Et2O (~1.2 mL total volume). This solution was then concentrated under N2 supply until dry at rt. Next, a protein solution containing photocatalyst, iron, and DMSO was added under buffering conditions.

[0567] Histone NTEV R2Dha was reacted using the conditions shown in the table below. 18 Functionalization was performed using fluorine-labeled mono-BtSOOF (shown in Figure 9B).

[0568] [Table 8]

[0569] Protein purification The remaining18 The 15-labeled protein reaction mixture was loaded onto a PD MiniTrap G-25 (pre-equilibrated with HEPES (100 mM, pH 7.4)) and eluted with 800 μL of HEPES buffer. MS analysis showed minimal oxidation and the expected mass of Dha protein (16003 Da). Due to the higher molar activity of mono-BtSOOF compared to BtSOOF, background 19 No mass corresponding to the F-CH2F histone H3 was observed.

[0570] In Figure 9C, human histone EH3 K4Dha is reacted using the reaction conditions shown in the table below. 18 Functionalized with 1F-labeled mono-BtSOOF (see Figure 9C).

[0571] [Table 9]

[0572] Human histone H3 18 For 1F-labeling, good RCY was observed with only low levels of oxidation. As mentioned above, the corresponding 19 F-labeled proteins were not observed by MS. When mono-BTSOOF was used as a fluorination reagent for human histone H3, milder conditions, such as reduced light output, were used here to reduce the possibility of double addition that can occur under standard conditions (50W, 15 min).

[0573] In Figure 9D, neurofilament light chains (NfL Dha) were reacted using the reaction conditions shown in the table below. 18 F was functionalized with labeled mono-BtSOOF; see Figure 9D.

[0574] [Table 10]

[0575] RadioHPLC traces of the product were obtained, showing good RCY (32%) and favorable labeling of NfL.18 This is an improvement compared to the RCY (10%) obtained with F-BtSOOF. 18 The molar activity of F-mono-BtSOOF is, 18 It is more expensive than F-BtSOOF (difluoroalkylation reagent).

[0576] Example 11 - Biocompatibility of Zebrafish To investigate the biocompatibility of the reaction conditions, zebrafish (3 dpf, n = 25 for each condition) were anesthetized with tricaine and injected into the posterior inferior part of the head (~2 nL, reagent: 10 mM Tris pH 7.5). There were four injection conditions. (1) No-infusion control. This is a negative control and the baseline for survival observation. (2) The reaction conditions with the Dha-containing protein removed. This was done to reduce the possibility of histone toxicity in the event of larval death and to confirm the background reactivity of the reagent without the Dha substrate. (3) Complete reaction conditions. These are complete experimental conditions that include both Ru(bpy)3Cl2 and BtSOOF-biotin (as explained in the example above). (4) All reaction conditions except for blue light irradiation. This is to demonstrate that light is a necessary trigger for the reaction and that the product was not formed spontaneously or before microinjection.

[0577] After injection, the larvae were placed in a fresh petri dish of E3 medium, where they rapidly regained their motility. Under conditions requiring light irradiation, the petri dish was placed directly above a 50W blue LED in a photobox for 5 minutes. In all conditions, 5 of the 25 larvae were placed in a separate petri dish for observation of their survival. In all conditions, not a single larva died within 2 days after injection, demonstrating the excellent biocompatibility of the reagent.

Claims

1. A method for functionalizing a protein or peptide at a functional side chain portion, wherein the protein or peptide comprises at least one single-occupied molecular orbital (SOMO) acceptor residue, the SOMO acceptor being a residue comprising a side chain containing an alkene group; The method is: (a) The oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in the radical precursor compound and in the photoactivated state. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide; The radical precursor compound is represented by formula (II) or formula (III) below: 【Chemistry 1】 (In the formula, R is via the group -CFX- if the compound of formula (II) is used, or via the group -CH if the compound of formula (III) is used) 2 -A functional side chain portion that is bound to a protein or peptide via; X is hydrogen, fluorine, chlorine, -C(O)OH and -C(O)NH 2 Selected from the group consisting of; A has one or more R 2 It is an aryl or heteroaryl group, which may be substituted with a group; j is 0, 1, 2, or 3; R 1 and R 2 are each, independently, halogen and unsubstituted or hydroxy, oxy, halogen, amino, carboxy, C (1-6) ester and C (1-6) alkyl substituted with one or more groups selected from the group consisting of C (1-6) alkyl); and when the compound of formula (II) is used as a radical precursor, step (a) further comprises contacting a protein or peptide with a source of Fe(II).

2. The method according to claim 1, wherein R is a group selected from (i) pharmaceuticals, sugars, polysaccharides, peptides, proteins, vaccines, antibodies, nucleic acids, viruses, labeled compounds, stabilized radical precursors, biomolecules, and polymers, any of which may be linked via a linker group.

3. The method according to claim 2, wherein the linker is a group L1 selected from alkyl groups, polyethylene glycol and its analogues, saccharides, polysaccharides, polyglycine, polyamides, or two or more combinations thereof, in which one or more non-adjacent carbon atoms may be substituted with groups selected from NH, O, S, -C(O)NH- or -NHC(O)-.

4. R is (ii) functional group R F ; or one or more functional groups R linked via a linker group L2 F ; (wherein, R F but - Hydrogen, C 3-10 Cycloalkyl, aryl, or heteroaryl (wherein the formula the cycloalkyl, aryl, and heteroaryl groups are unsubstituted or =O, =NRa, Y, and (C 1-6 (substituted with one or more groups selected from alkyl)-Y; or -C 2-6 Alkenil, C 2-6 Alkynyl, halogen, hydroxy, -OR a , -SR a ,-S(O)R a , -S(O) 2 R a , -OSO 3 R a , -NR a C(O)R b , -NR a CO 2 R b ,-NHC(O)NR a R b , -NHCNH 2 NR a R b , -NR a SO 2 R b , -N(SO 2 R a ) 2 , -NHSO 2 NR a R b ,-OC(O)R a , -C(O)R a , -CO 2 R a -C(O)NR a R b -C(O)(NHNH 2 ), -ONH 2 , -C(O)N(OR a )R b , -SO 2 NR a R b Or -SO(NR a )R b ;Cyano, Nitro, C 1-6 Azidoalkyl, -NR a R b and -(NR a R b R c ) + A reactive group Y selected from; (In the formula: R a , R b and R c However, each independently, hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, hetero Cyclyl, phenyl, benzyl, and heteroaryl (wherein R is used in the formula) a , R b and R c In this context, alkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl groups are either unsubstituted or halogen, hydroxyl, =O, -NH 2、 -SO 3 - and C 1-6 From alkoxy Represents (which is substituted with one or more substituents of selection); and L2 may be an alkyl group in which one or more non-adjacent carbon atoms are substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-; polyethylene glycol and its analogues; sugars; polysaccharides; polyglycine; polyamide; or selected from two or more combinations of these groups. The method according to claim 1, which is (to be).

5. R is (ii) functional group R F ; or one or more functional groups R linked via a linker group L2 F (In the formula, R F ga:C 2-6 Alkenil, C 2-6 Alkynyl, halogen, -OC(O)R a , -C(O)R a , -CO 2 R a -C(O)(NHNH 2 ), -ONH 2 and C 1-6 (The reactive part is selected from azidoalkyl) R or R is an expression 【Chemistry 2】 The reactive portion includes (wherein A is defined in claim 1; and the reactive portion 【Transformation 3】 The method of claim 1 or claim 4, wherein the linker group L2 (where L2 is an alkyl group in which one or more non-adjacent carbon atoms may be substituted with a group selected from NH, O, S, -C(O)NH- or -NHC(O)-) is linked.

6. The reactive moiety is halogen, C 1-6 azide, C 2-6 alkynyl, 【Chemistry 4】 The method of claim 5, selected from among.

7. The reactive portion is 【Transformation 5】 The method according to claim 5.

8. A method for functionalizing a protein or peptide containing at least one SOMO acceptor residue as defined in claim 1 at a functional side chain moiety, the method being: (a) The oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in the form of a radical precursor compound, a source of Fe(II), and in the photoactivated state. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide; The radical precursor compound is a group of the following formula (IV) 【Transformation 6】 where R is a functional side chain moiety that is bound to a protein or peptide via the group -CFX-; and the group R is -COOR d and -CONR d R e where R d is hydrogen, C 1-6 alkyl, C 3-10 cycloalkyl, heterocyclyl, phenyl, benzyl or heteroaryl (wherein the alkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl groups in R d are unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, =O, -NH 2 , C 1-6 alkoxy and -NHCOR e ); and R e is hydrogen or C 1-4 alkyl) is selected from).

9. A protein or peptide comprising at least one SOMO acceptor residue as defined in claim 1 is structured. 【Transformation 7】 A method for functionalizing a functional side chain portion having the following characteristics: (a) The oxidation half potential (E) of a protein or peptide when measured against a saturated calomel electrode in the form of a radical precursor compound, a source of Fe(II), and in the photoactivated state. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Exposing the obtained composition to light irradiation in order to provide a functionalized protein or peptide; The radical precursor compound used has the following structure 【Transformation 8】 A method having (wherein bases A and X are defined in claim 1).

10. A method according to any one of claims 3 to 7, wherein the functional side chain portion includes a reactive portion as defined in claim 4 to 7, the method further comprising reacting a peptide or protein via one of the reactive portions to link the functional side chain to a further molecule.

11. The method according to claim 10, wherein the further molecule is a pharmaceutical, sugar, polysaccharide, peptide, protein, vaccine, antibody, nucleic acid, virus, labeled compound, biomolecule or polymer.

12. The method according to any one of claims 1 to 11, wherein the SOMO acceptor residue is dehydroalanine or dehydrobutyrine.

13. The method according to any one of claims 1 to 12, wherein the SOMO acceptor residue is dehydroalanine.

14. The method according to any one of claims 1 to 7 and 9 to 13, wherein group A is phenyl, pyridinyl, pyrimidinyl, benzothiazolyl, or pyrazinyl.

15. The method according to any one of claims 1 to 7 and 9 to 13, wherein group A is pyridinyl, pyrimidinyl, or benzothiazolyl.

16. The method according to claim 14 or 15, wherein group A is 2-pyridinyl.

17. The method according to any one of claims 1 to 7 and 9 to 16, wherein group X is fluorine.

18. The source of Fe(II) is iron(II) sulfate, FeOTf 2 Fe(ClO) 4 ) 2 FeF 2 or (NH 4 ) 2 Fe(SO 4 The method according to any one of claims 1 to 17, wherein )2.

19. The method according to any one of claims 1 to 17, wherein the source of Fe(II) is FeSO₄・7H₂O.

20. The method according to any one of claims 1 to 19, wherein the photocatalyst is a Ru(II) or Ir(II)-based catalyst.

21. The method according to any one of claims 1 to 19, wherein the photocatalyst is a Ru(II) catalyst.

22. Ru(II) photocatalyst, Ru(bpy) 3 Cl 2 Or Ru(bpm) 3 Cl 2 The method according to claim 20 or 21.

23. The method according to any one of claims 1 to 22, wherein the light irradiation is in the region of 300 to 600 nm.

24. The method according to any one of claims 1 to 22, wherein the light irradiation is in the region of 400 to 500 nm.

25. The method according to any one of claims 1 to 22, wherein the light irradiation is in the region of 430 to 470 nm.

26. The radical precursor compound is the compound of formula (III), and the compound of formula (III) in step (a) transforms the protein or polypeptide into a BCH-B 2 Functionalized boron compounds containing the R moiety, and the following formula (IIIB): 【Chemistry 9】 (In the formula, R, R 1 The method according to any one of claims 1 to 7 or 10 to 25, wherein the product is produced in situ by contacting a catechol derivative represented by (and j as defined in any one of claims 1 to 4).

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