Photoredox protein modification

Abstract

This invention provides a method for protein functionalization involving post-translational modification in a manner that is selective, reliable, and under the possibility of benign, moderate redox, enabling the addition of reactive functional side chains. [Solution] The present invention relates to photoredox-mediated functionalization of proteins at chemical groups via radical generation and CC bond formation using specific boronate and sulfone precursor compounds. The present invention also relates to functionalized proteins that can be produced by this method, and the specific 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 present invention also relates to functionalized proteins that can be produced by this method, and to 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. Consequently, with the emergence of synthetic protein functionalization strategies, it is now possible not only to directly mimic them but also to create non-natural protein variants with diverse potential functions, ranging from drug delivery and tracking to imaging and crosslinking with partners. However, the range of functional groups that can be introduced through these modifications is still 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, highly for some carbon radical species Because it is reactive, this selectively generates β,γ-CC bonds, resulting in "scarless / tray Dehydroalanine (Dha) residues, which are readily generated, are used in proteins as single-occupied molecular orbital (SOMO) acceptors ("radical acceptors" or "SOMO-philes") that can introduce new side chains in a "scarless / traceless" manner. However, side chain / carbon radical precursors and the reagents that generate them (e.g., metals or BH4) - The incompatibility of single-electron transfer (SET) from is currently limiting the scope of such technology. Nevertheless Some isotropic 1e-chemistry has potential advantages over typical anisotropic 2e-reagents. Essential issues in biomolecular modification include: affinity for water; the need for "benign" reactions; and the presence of numerous bioacids, amines, alcohols, and thiols (rapid 2e-reactants) in many biological environments. Examples include low (or no) reactivity to these substances. 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 selection can be achieved in certain 1e-chemical reactions. Selectivity can be made possible.

[0005] Other methods of SET (and therefore, either oxidative or reductive carbon radical initiation) Yes, catalytic protein methods offer clear advantages over conventional hyperstoichiometric methods that can cause unwanted side reactions. Furthermore, if controlled with relatively benign and potentially tissue-penetrating triggers such as light, they complement those with 1e-chemoselectivity in terms of time and space. This allows for additional layers of control, such as target and even kinetic control. 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 ET systems located at both ends of a redox "window," and it has been pointed out that side reactions occur. Furthermore, α-C-carboxyl and β-CH groups, etc. The reliance on a specific precursor moiety that cannot be re- / prepositioned limits and / or enriches the reaction site. This can lead to low site selectivity. Therefore, 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 have 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 bonding can be established between functional side chains on proteins or peptides and SOMO acceptor residues. We discovered that this makes it possible. This change in the CC side chain within the unchanged protein is a protein It 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 have shown 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 amplification of aryl sulfone fluorine derivatives and certain bromine fluorine derivatives with iron(II) is possible. We discovered that when this is done, RFXC· radicals (such as RF2C·) are generated, which can form equivalent (βCH2-γCXF) bonds with H→F labeling.

[0009] These reaction methods of the present invention can be carried out rapidly 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. They are initiated chemoselectively in the presence of sensitive groups in the 1C radical precursor. Because it can be applied, it makes it possible to introduce side chains that were previously incompatible. This provides access to new functions and reactivity of proteins. The novel methods described herein and the proteins / peptides produced therefrom are, for example, (a) isotropic (b) Introduction of radical precursors for radical generation on proteins; (b) Chemoselectivity and stereoselectivity By simultaneously sensing both sexes, we study enzyme function using natural, unnatural, and "zero-size" labeled post-translational modified protein substrates; (c) Isotropic covalent bond formation activity (on the other hand) This may lead to access to a general "alkylating agent protein" with a spectrum that reacts with a variety of small molecules and, on the other hand, selectively reacts with protein targets due to its excellent mimicry; and it may find applications in many areas such as [examples of applications]. Therefore, 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 have identified the following triple combination: a side-chain functionalized group C· that is "redox-compatible" to a low, quasi-stoichiometric amount of photocatalyst triggered by light of an appropriate luminous flux (iii). Electron transfer at a benign and gentle redox potential using a radical precursor (ii) (i) We demonstrated that the generation and utilization of off-protein and on-protein radicals enable protein modification via CC bond formation (see Figure 1). The resulting chemistry allows for 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 at a functional side chain portion, wherein the protein or peptide comprises at least one single-occupied molecular orbital (SOMO) acceptor residue, and the SOMO acceptor comprises a side chain containing an alkene group. The residues included; the method is: (a) A protein or peptide is saturated with a radical precursor compound in a photoactivated state. Oxidation half potential (E) measured against a Japanese calomel electrode ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Irradiate the obtained composition to provide a functionalized protein or peptide with light. Exposure to; including, The radical precursor compound is given by the following formula (II) or (III):

[0012] [ka]

[0013] (In the formula, R is the compound of formula (III) via the group -CFX- when the compound of formula (II) is used) When a substance is used, the functional side is bound to a protein or peptide via the -CH2- group. It is the chain part; 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 halogen and unsubstituted or hydroxy, oxy, halogen, amino, carboxy, C (1-6) Esters and C (1-6) One or more selected from ether C substituted with a group (1-6) Selected from the group consisting of alkyl groups; 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, 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, the linker is an alkyl which may be substituted with a group selected from one or more non-adjacent carbon atoms being NH , O, S, -C(O)NH- or -NHC(O)-; polyethylene glycol and its analogs; saccharides; polysaccharides; polyglycine; polyamide; or a group L1 selected from a combination of two or more of these groups.

[0016] In a further aspect of the first embodiment, 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, ar yl 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 alkenyl, C 2-6 alkynyl, halogen, hydroxy, -OR 2-6 , -SR a , -S(O)R a , -S( a 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 Cycloalkyl, hetero Represents cyclyl, 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 consists of one or more non-adjacent carbon atoms selected from NH, O, S, -C(O)NH-, or -NHC(O)-. Alkyl groups which may be substituted with the group being treated; polyethylene glycol and its analogues; sugars; polysaccharides; polyglycine; polyamides; or selected from two or more combinations of these groups. It is.

[0017] In a further embodiment, R is (ii) functional group R F ; or linked via linker base L2 One or more functional groups R 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, C 2-6 Alkinil,

[0023] [ka]

[0024] Selected from, preferably

[0025] [ka]

[0026] That is the case.

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

[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 Cycloa Lukir, heterocyclyl, phenyl, benzyl or heteroaryl (wherein R d The alkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and heteroaryl groups in are unsubstituted or halogen, hydroxy, =O, -NH2, C 1-6 alkoxy and -NHCOR e or substituted with one or more substituents selected from); and R e is hydrogen or C 1-4 alkyl).).

[0030] In a third embodiment, the present invention provides a method for functionalizing a protein or peptide comprising at least one SOMO acceptor residue defined in the first embodiment with a functional side chain moiety having the structure

[0031] [Chemical formula]

[0032] The method comprises:[[]] (a) contacting the protein or peptide with a radical precursor compound, a source of Fe(II) and a photocatalyst having an oxidation half potential (E ) measured against a saturated calomel electrode in the photoactivated state of +1.2 V or less; and ox (b) exposing the composition obtained to provide the functionalized protein or peptide to light irradiation ; comprising (the radical precursor compound used has the following structure

[0033] [Chemical formula]

[0034] (wherein the groups A and X are as 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, further molecules include pharmaceuticals, sugars, polysaccharides, peptides, proteins, vaccines, antibodies, nucleic acids, viruses, labeled compounds, biomolecules, or polymers.

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

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

[0039] In a preferred embodiment of the above embodiment, group A is pyridinyl, pyrimidinyl, or ben It is zothiazolyl.

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

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

[0042] In any further embodiment 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 any further embodiment 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 embodiment, 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 range 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, when the radical precursor compound is a compound of formula (III), the compound of formula (III) is generated in situ in step (a) by contacting a protein or polypeptide with a functionalized boron compound containing a -BCH2R moiety and a catechol derivative represented by the following formula (IIIB):

[0047]

Chemical formula

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

[0049] In the fourth embodiment, the present invention provides a functionalized peptide or protein comprising at least one residue of formula (IA):

[0050]

Chemical formula

[0051] (wherein X is selected from hydrogen, fluorine, -COOH and -CONH2, preferably fluorine; R is hydrogen or methyl; Z and R is defined in any of the above embodiments).

[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 embodiment of the above embodiment, the residue of formula (IA) is one of the compounds listed in Examples 2a to 2ag.

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

[0057] In a fifth embodiment, the present invention relates to formula (IB):

[0058] [ka]

[0059] (wherein Ry is hydrogen or methyl; Rbac is C 1-6 Alkyl (where the terminal carbon is substituted with at least one halogen) R or Rbac can be expressed by the following formula:

[0060] [ka]

[0061] The present invention provides a functionalized peptide or protein containing at least one residue of (wherein Z is a halogen).

[0062] In a sixth embodiment, the present invention provides a method for covalently bonding a functionalized protein or peptide described in the fourth or fifth embodiment with a further protein or peptide, wherein the group R or Rbac in the functionalized protein or peptide is C 1-6 Ha It is a rhalkyl group and contains a group that can react with an alkyl halide to form a covalent bond with a further protein or peptide.

[0063] In a further embodiment of the above embodiment, the functionalized protein or peptide is a substrate for a further protein or peptide, and the halogenated alkyl group is held in a binding pocket of the other protein or peptide in order to bring the halogenated alkyl group close to a group that can react with the halogenated alkyl group.

[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 contain groups that can react with radical species to form covalent bonds.

[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 located in peptides or proteins, and are adjacent to one or more other residues. It is an amino acid residue linked to two other residues by a peptide bond. The SOMO acceptor residue contains a group that is highly reactive to C radical species, and this group has an alkene group. It is a side chain. In some embodiments, the SOMO acceptor residue is of formula C 1-6 It may have an alkenyl side chain. Preferably, the C=C double bond is located at the terminal of the alkenyl group. In a preferred embodiment, the SOMO acceptor is dehydroalanine (Dha) or dehydro It is butyrine (Dhb), preferably dehydroalanine.

[0073] The Dha residue can be introduced into the target protein or peptide 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 position 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: [[ID=I5]]

[0076]

Chemical formula

[0077] In the above formula (II), R is a functional side chain moiety that is bonded to the protein or peptide via the group -CFX-. It is a functional side chain moiety. [[ID=3I]]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) [[ID=3I]]alkyl (unsubstituted or hydroxy, oxy, halogen, amino, carboxy, C (1-6) ester, and C (1-6)Selected from the group consisting of one or more groups selected from ethers (e.g., substituted with 1, 2, or 3 groups, preferably 1 or 2 groups). In some embodiments, R2 is C 1-4 It 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 a preferred embodiment, A is phenyl, pyridinyl, pyrimidinyl, benzothia Zolyl or pyrazinyl, more preferably A is pyridinyl, pyrimidinyl or It is benzothiazolyl. In the most preferred embodiment, the compound of formula (II) is the compound of formula (IIA) described below. That is the case.

[0078] [ka]

[0079] In equations (II) and (IIA) above, X is derived from hydrogen, fluorine, chlorine, -COOH, and -CONH2. A selection is made from the following list, 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, as shown in Figure 2(d), Ru (By reducing (II) to Ru(I)). Furthermore, Fe(II) is the stabilized functional side. It can act to reductively quench the radical protein / peptide intermediate produced by the initial reaction between the chain radical and the SOMO acceptor residue. This has the advantage of preventing oxidative quenching of the intermediate, which can occur due to an excess of oxidized photocatalysts, such as Ru(II) catalyst species, and can 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) proceeds according to the scheme shown in Figure 2(d). Yes, it is possible. As can be seen, when activated with the appropriate beam of light, the photoexcited oxidation state of the photocatalyst (e.g., Ru(II) photocatalyst) is reductively quenched by Fe(II), providing an active reduced species, e.g., (Ru(I)). This reduced species then reductively initiates the ASOOF precursor, resulting in a stabilized RCFX radical species, which then initiates SOMO-accelerating reactions such as Dha, as shown below. The reaction occurs via radical addition to the C=C double bond of the pter residue. The resulting α-carbon protein The qualitative radical is then reduced via SET from iron(II), prototyping under aqueous reaction conditions. An enolate intermediate is formed, and finally, a functionalized protein / peptide is obtained.

[0096] Embodiment (ia) In a further specific embodiment (ia), the group R in formula (II) is bound to a protein / peptide. The reaction conditions are the same as in embodiment (i) above, except that it is iodine and not a side chain group. Therefore, the radical precursor compound is given by formula

[0097] [ka]

[0098] (wherein A and X are compounds as defined in embodiment (i) above.) Preferred embodiment In this, the above radical precursor becomes iodo-pySOOF, where A is pyridyl and X is f It is simple.

[0099] Under the same reaction conditions as ASOOF described above, the reductively activated catalyst reduces the iodo radical precursor. It is activated and forms radicals as shown below.

[0100] [ka]

[0101] This stabilized radical species is functionalized in the side chain of the ASOOF radical precursor protein / pe To produce the plutide, the same reaction pathway as described above in the first aspect of this embodiment is used. Then, the C=C double bond of the SOMO acceptor residue is further reacted via radical addition. This protein / peptide, functionalized at the ASOOF precursor side chain, can lead to further species formation. To provide a radical on a stabilized protein that can be used for bonding, the same reaction conditions as in embodiment (i) are used via a photoredox catalyst and an iron(II) source. It may also be activated. Therefore, this site enables diverse and further protein functionalization via isotropic bond formation mechanisms on various proteins (see Figure 3(A)).

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

[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.) 1-4 a It is a lukyl linker group. Preferably, Lz is methylene(-CH2-) or -CH(CH3)-. Preferably, Lz is methylene. Rt is hydrogen or a protecting group, preferably hydrogen or C 1-4 Alkyl, more preferably hydrogen or tert-butyl. Rs is hydrogen or a protecting group, Preferably, it is hydrogen or tert-butoxycarbonyl (boc). In preferred embodiments... In this case, Rs and Rt are hydrogen atoms.

[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 radical precursor compound of formula (IV) is used, except The same reaction conditions used in embodiment (i) above are 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, and then... It forms radicals as shown.

[0114] [ka]

[0115] This stabilized radical species, via the same reaction pathway described above in the first aspect of this embodiment, generates a protein / peptide functionalized with side chain-CF2R, and is then used to access SOMO-accelerated proteins / peptides. Further reaction occurs via radical addition to the C=C double bond of the pter residue.

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

[0117]

Chemical formula

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

[0119]

Chemical formula

[0120] In formula (IIIA), j is 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 the protein or peptide via the group -CH2-.

[0121] The BACED reagent is oxidized by the catalyst during the reaction, thus activating the photocatalyst acid. It should have an oxidation half-potential close to or below the oxidation half-potential (Eox).

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

[0123] [ka]

[0124] Functionalized boron compounds are any boron compounds that are covalently bonded to a side chain (-CH2R) that is attached to a protein or peptide, i.e., boron compounds containing a B-CH2-R unit. Good. To form the active BACED reagent in situ, it is preferable that the boron compound can also be substituted with a ligand in an aqueous environment. The boron component is a boronic acid salt, boronic acid and It may also be a boronate ester. In one embodiment, the boron compound is Formula [RCH2BQ3]V (where Q is independently a halogen, preferably chloro or fluoro, most preferably Mashiku is fluoro; and V is K + Li + kaNa + or NH4 + Any appropriate counter to It is a compound of the formula RCH2B(OR). In further embodiments, the boron compound is a compound of the formula RCH2B(OR). f )2(R f The group is independently either hydrogen or C 1-6 Alkyl, or two R f The base together This results in a linear or branched carbon chain where two oxygen atoms are linked together to form a 4- to 7-membered ring with a boron atom to which an oxygen atom is bonded. 1-10It is a compound that forms an alkyl chain. In a particular embodiment, the boron compound is RCH2BF3K, RCH2B(OH)2, or RCH2Bpin(pin is (It 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. ru.

[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 oxidatively initiates the BACED precursor, yielding an RCH2 radical species, which then reacts via radical addition to the C=C double bond of SOMO acceptor residues such as Dha, as shown in Figure 2(c). The resulting α-carbon radical on the protein is then subjected to a reduction catalyst, such as SET from Ru(I). The enolate intermediate is reductively quenched via [a specific method] and protonated under aqueous reaction conditions to form an enolate intermediate, 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 embodiments, a protein or peptide is functionalized and boronated Compound, catechol derivative of formula (IIIB), and in the photoactivated state, against a saturated calomel electrode The oxidation half potential (E) measured when measured in this way. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Irradiate the obtained composition to provide a functionalized protein or peptide with light. Exposure to; including.

[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 be limiting, the functionalized boron compound of formula (IIIB) and the catechol derivative of formula (IIIB) generate the BACED reagent of formula (III) in situ during step (a). This is the current understanding. However, the present invention is that the BACED reagent is formed during the reaction ( Not limited to methods in which the BACED reagent is undetectable or formed Other embodiments 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 are methods in which the radical precursor compound is of formula (II), (IIA), (III), (IIIA), or (IV), and in which the reaction is carried out with a functionalized boron compound. The present invention relates to all embodiments of the method, including a method that proceeds in the presence of 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 is preferably such that, when measured against a saturated calomel electrode, its oxidation half-potential (Eox) in its photoactivated oxidation state is * +1.2V or higher Preferably, the voltage is +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 the Eox of the radical precursor compound of formula (III) when measured relative to a saturated calomel electrode. Preferably, the Eox of the photocatalyst is lower than the Eox of the saturated calomel electrode. In contrast, when measured, it is larger than Eox of the radical precursor compound in equation (III).

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

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

[0135] When using the method of embodiment (i), (ia), or (ib), the reduction half potential of the photocatalyst (Ered Preferably, the Ered of the photocatalyst is 0.2 V or less lower than that 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 that 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 is 0.1 The power may range from 1000W to 1000W, preferably 1 to 200W, more preferably 1 to 100W, and even more preferably 5 to 60W. In a preferred embodiment, the light intensity supplied to the reaction is 45 to 55W.

[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 contain one or more additional components, such as a buffer to adjust the pH. It may also contain the following. 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, Preferably, it is 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 attachment of a functional side chain moiety to a protein or peptide via a photomediated radical reaction under mild conditions. Generally, the group R is used via the group -CXF- when an ASOOF precursor is used (Embodiment (i), first aspect), and the precursor of formula (IV) is used. If a boron-containing precursor is used, it will be bound to the protein / peptide via the -CF2- group, and if a boron-containing precursor is used, it will be bound via the -CH2- group (Embodiments (ii), (iia)). Therefore, it is understood that the methods described herein are generally applicable and can be used even when reactive groups are present, and that there are no particular limitations on the group R that can be bound to a protein or peptide. Therefore, the group that binds to the protein or peptide may contain any suitable chemical moiety useful for binding. This group may, for example, include a linker group containing the payload and / or a reactive functional group that can bind to the payload via further reactions. 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: One or more non-adjacent carbon atoms selected from NH, O, S, -C(O)NH- or -NHC(O)- Alkyl compounds whose children may be optionally substituted (i.e., exchanged); polyethylene glycol (PEG) and its analogues; sugars; polysaccharides; polyglycine; polyamide; and The group L1 is selected from two or more combinations of these groups. In preferred embodiments, L1 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)-; selected from PEG, PEG analogues, polyamides, and two or more combinations of these groups. 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 in 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. A further preferred example of a polymer that can be bound as a load is described in Chemical Society Reviews, Vol. 47, Number 24, 21 Dec 2018, Pages 8971-9160. Examples include polymers typically have a density of less than 10 kDa, preferably less than 5 kDa, more preferably The polymer has a molecular weight of less than 2 kDa, most preferably less than 1 kDa. In preferred embodiments, the polymer is PEG, a PEG analogue, 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 labels include one or more atoms in the group being specific isotopes. It can be labeled and detected via appropriate means such as NMR, mass spectrometry, and radiolabeling studies. Isotope labeling is one example. Suitable labeling isotopes include deuterium, 19 F, 13 C and 15 N raised Appropriate labeling groups include biological samples labeled with one or more of the above isotopes at specific positions. Examples include molecules, sugars, and natural or synthetic amino acids. Furthermore, the term labeling group is intended to encompass other payloads or side chain portions, such as those described herein, labeled with specific isotopic labels as defined above. Other suitable labeling compounds include fluorophores and FRET reagents. Further suitable labeling compounds include those that can aid 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 (F may also be used) via protein or peptide It binds to the cydo. This group enables the labeling of peptides / proteins, which can allow for the monitoring of various reaction pathways, as shown in Example 9, for example. In some embodiments, compounds of formulas (II), (IV), (IA), (IIi) and embodiments In state (ia), in any of the iodine compounds used as radical precursors, one or both of the fluorine atoms bonded to the carbon adjacent to the group R, preferably one of them, 18 It is F.

[0157] In this specification, biomolecules or biological molecules are those that are important to one or more biological processes. This term is defined as a molecule present in an organism. It 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, including 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 this invention can be found, for example, in each of the following: (a) Pharmaceutical Substances: Syntheses, (b) 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 fully referenced. The body is incorporated into this specification.

[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 replaced by a hydrogen atom, for example. Therefore, the sugars, sugars, and polysaccharides described herein may be unsubstituted or have one or more, typically one or two, acetyl groups or The group may be substituted with an N-acetyl group. Therefore, 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 often contain coenzymes or cofactors. It is linked to Gand, or another protein, or other macromolecules (such as DNA or RNA), or to a complex macromolecular assembly.

[0167] In further embodiments of the present invention, R is a functional group R F ; or one or more typical It contains one or two functional groups R linked via linker groups of formula L2. F That is the case. R F teeth, - Hydrogen, C 3-10 Cycloalkyl, aryl, or heteroaryl (cycloalkyl, aryl) The reel or heteroaryl group is unsubstituted or =O, =NRa, Y and (C 1-6 Alkyl)-Y It is replaced by one or more elements selected from; 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 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 1-6Azidoalkyl, -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 Cycloalkyl, hetero R represents cyclyl, 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 Alkoxy (substituted with one or more substituents selected from; and L2 consists of one or more non-adjacent carbon atoms selected from NH, O, S, -C(O)NH-, or -NHC(O)-. Alkyl groups that may be substituted (i.e., exchanged) with the group being replaced; polyethylene glycol (PEG) and its analogues; sugars; polysaccharides; polyglycine; polyamide; or selected from two or more combinations of these groups.

[0168] In a preferred embodiment, L2 is selected from alkyl groups, PEGs, PEG analogs, sugars, polyamides, and two or more combinations thereof, in which one or more non-adjacent carbon atoms are substituted with groups selected from NH, O, S, -C(O)NH-, or -NHC(O)-. Typically, C 1-20 Alkyl, preferably C 1-10 Alkyl, more preferably C 1-6 They are alkyl. The 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 Alkyl ru.

[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 shared via their side chains. They are joined together. 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, C3-6 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 oh Call (NR) a R b R c ) + The reactive group Y is selected from the following. In one embodiment of this embodiment, 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 2-6Alkynyl, 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, for example, hydrogen , C 1-6 R represents alkyl, phenyl, benzyl, or pyridyl (wherein R is the formula). a , R b and R c Alkyl, heterocyclo, phenyl, benzyl and heteroalkyl The group is unsubstituted or halogen, hydroxyl, =O, -NH2, -SO3 - and C 1-6 Alkoxy (Substituted with one or more substituents selected from). If present, group R a , R b and R c is the same One or more may be different. In one preferred embodiment, multiple R a , R b and R c When two groups are bonded to the same Y portion, one of the groups is defined according to one of the above definitions. As is the case, other R's attached to that part 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 1-3Alkyl is also acceptable.

[0180] In certain embodiments, R is a functional group R F ; or one or more, preferably one linker Functional group R linked via 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 Y is a reactive moiety 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 1-3 Hello Walk Lu, 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, one Me no R F -CO2R a , the second R F -NR a R b or -NH-Boc(wherein Boc is a protecting group) -It is a butoxycarbonyl. Preferably, in the above embodiment, L2 is a C2 alkyl and the first R FThe 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, It is 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 the one 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), if a specific halogen compound is used as the group R, then R It has been found that an external group can cause a side reaction in which it is added to a protein or peptide. Therefore, in a preferred embodiment of this design 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 n, hydroxy, -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 R b or -SO(NR a )R b Preferably, R is -CO2R a Ma taha-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 Oh B-CONR d R e (In the formula, R d is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, heterocyclo( heterocyclo), phenyl, benzyl and heteroaryl (wherein R is used in the formula) d Alkyl and cy The chloroalkyl, heterocyclyl, aryl, and heteroaryl groups are unsubstituted or contain halogen, hydroxyl, =O, -NH2, or 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 (Representing alkyl, preferably hydrogen) Selected from. 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, cycloalkyl and heteroaryl The base is 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 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 ) + 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 Iodine That is the case.

[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 Al Kill 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 bound to a protein or peptide is further modified or attached to one or more additional molecules in order to modify it or to attach it to one or more additional molecules. It is a group that can receive a response. Therefore, in embodiments, the present invention also provides the method defined above, in which a functional side chain attached to a protein reacts further to modify it or to attach it to a further molecule.

[0214] As described above, the group R bound to a protein or peptide can be activated through any appropriate reaction. They can then be reacted further, for example, by binding them to one or more additional molecules of interest. This can be done. Further reactions are preferably biocompatible reactions, i.e., reactions that can be carried out with minimal damage to proteins or peptides, for example, reactions that can be carried out under aqueous conditions without requiring excessive temperature. In preferred embodiments, group R is described below. One of the above-mentioned reactive sites can be reacted via a further coupling reaction. It contains one or more of these. Those skilled in the art will know of azides, alkynyls and reactive esters, such as NHS esters. You should be familiar with appropriate coupling reactions, such as standard "click chemistry" reactions, that involve a pharmacokinetic.

[0215] Further molecules that can bind to the reactive functional side chain portion of functionalized proteins / peptides 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, further molecules may be drugs, sugars, peptides, proteins, antibodies, biomolecules, or polymers, 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 the group R contains a suitable electrophile such as a halogen, it is a halogen It can react with a nucleophile, such as an off-protein nucleophile, through nucleophilic substitution by replacing an appropriate leaving group such as n. For example, the group R is a halogen moiety, preferably terminal. If halogens are present, they produce CS bonds by reacting with nucleophiles such as thiols (e.g., beta-mercaptoethanol); or by reacting with TCEP (tris(2-carboxyethyl)phosphine) to produce CP bonds; or It generates CN bonds (e.g., methylamine or N3) - The reaction may be carried out via appropriate chemistry (by reacting together with the protein). Alternatively, an electrophile containing the group R may be used with the protein. Alternatively, the peptide may be reacted with a suitable nucleophile on a further protein or peptide, such as a cysteine ​​or lysine side chain, to bind it 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 intraprotein nucleophilic substitution.

[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. For example, in addition to attaching the group R to the target further molecule via nucleophilic substitution, or You may go before that.

[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 is reacted with the following to obtain at least one functionalized residue of formula (IP) below. The functionalized protein or peptide containing the functionalized protein or peptide may also be provided.

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

[0232] monomer

[0233] [ka]

[0234] If used, the base Rpol is instead

[0235] [ka]

[0236] That is the case.

[0237] In some embodiments, the pendant groups Rpb and Rpc are joined 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 generated radical on the protein is hydro, as shown in Figure 3. Xy-TEMPO, or diselenium compounds of the formula Rh-SE-Rh (where each Rh is C 1-6 Alkyl, C 1-6 Cycloalky, or C 1-6 Other than aryl, preferably phenyl, etc. It may be reacted with a radical-terminal group. In such embodiments, the further functionalized protein or peptide produced is a radical-terminal group Rpol with Rrad (wherein Rrad is a radical-terminal group, -Se-R h ,or

[0242] [ka]

[0243] Except for being replaced by the above formula (IP), at least one functionalization described in formula (IP) It may contain residues.

[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 can be obtained from the method described in embodiment (i), (ia), or (ib) of the above method. Functionalized proteins containing functionalized residues of the general formula (IA) shown below, It also provides peptides.

[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 functional groups 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 A azidoalkyl: A reactive moiety Y selected from R is an expression; or R is an expression

[0249] [ka]

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

[0251] [ka]

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

[0253] In a further embodiment, R is as described in “Further Reactions of Side Chains” above, It may also be a group resulting from the reaction of an activated side chain with a further molecule. For example, R is For example, following the activation of the ASOOF group on the protein, further activation of SOMO acceptor residues The group may also be a group resulting from the generation of radicals on a protein by reaction with a radical acceptor such as a protein or peptide, or a monomer containing a radical acceptor group.

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

[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 particular embodiment, X is fluorine.

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

[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 Alkyl (wherein the formula, the terminal carbon is substituted with at least one halogen) In a preferred embodiment, Rbac is C 1-4 The 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 Hello Walk The present invention provides a method for covalently bonding a functionalized protein or peptide, produced by any of the above methods, such as that described by formula (IA) or (IB), 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, optionally in the presence of further biological molecules such as enzymes and cofactors, when they are together in solution. This allows for reactions to occur via nuclear substitution. This proximity-driven reaction significantly increases the effective molar ratio of 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 such that one of the functionalized protein / peptide and further protein / peptides can interact with other protein / peptide This is a binding pocket that is held in the binding pocket of the peptide. In a preferred embodiment of this embodiment, at least one of the protein / peptide 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 the reaction between the halogenated alkyl group and a group that can react with the halogenated alkyl group (e.g., a nucleophilic group) occurs at the active site of the enzyme. Preferably, the active site contains one or more cysteine ​​residues configured to react with the halogenated alkyl group.

[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 enters the active site of the enzyme substrate. For example, a lysine residue involved in the binding interaction with the substrate may be modified by replacing it with a DHA residue, and then the halogenated alkyl group may be introduced using the method of the present invention. Kill groups can be linked. The halogenated alkyl group introduced in this way then enters the binding pocket of the substrate and covalently bonds 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, using the method of the present invention, proteins / peptides can be site-selectively modified to 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 a further embodiment, the present invention relates to a functionalized protein as described in formula (IA) above. This provides a method for covalently bonding a protein or peptide to a further protein or peptide (wherein the formula R is the group in the functionalized protein or peptide,

[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 Alkene side chain, preferably DHA as described above. It contains residues having side chains containing (and / or dhb).

[0284] In a preferred embodiment of this design, there may be one or more additional proteins or peptides. It contains the dha residue.

[0285] In a preferred embodiment of the above embodiment, a functionalized protein or pe of formula (IA) In the ptyde, 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. Preferred form of the above embodiment In this manner, the method uses protein functional groups of formula (II) or (III) as described above. It is one method of transformation.

[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, alkyl The base 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" is, for example, cyclopropyl This refers to cyclobutyl, cyclopentyl, or cyclohexyl, or bicyclo[3.1.0]hexanil, bicyclo[4.1.0]heptanil, and bicyclo[2.2.2]octanil, etc. That's fine.

[0292] As used herein, the term "heterocyclyl" means a ring atom consisting of one or two N, O, or S(O) atoms. n A heterocycline is 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 include pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, piperazinyl, tetrahydropyranil, and thiomorpholinyl, but are not limited to these.

[0293] As used herein, the term "aryl" refers to a monovalent monocyclic compound with 6 to 10 ring atoms. This refers to bicyclic aromatic hydrocarbon radicals (for example, phenyl or naphthyl).

[0294] As used herein, the term "heteroaryl" means one or more, preferably 1, 2, or A monovalent monocyclic or bicyclic aromatic radical consists of 5 to 10 ring atoms, where three ring atoms are heteroatoms selected from N, O, or S, and the remaining ring atoms are carbon. Examples include, but are not limited to, pyrrolyl, thienyl, thiazolyl, imidazolyl, furanyl, indolyl, iso-indolyl, oxazolyl, isoxazolyl, and benzothiazolyl. Examples include lyl, benzoxazolyl, quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazolyl, tetrazolyl, and the like, with pyridinyl, pyrimidinyl, pyrazinyl, or pyridadinyl being preferred.

[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 Alcocy C group, most preferably C 1-4 It is an alkoxy group.

[0296] As used herein, the term “halo” means fluoro, chloro, bromo, or iodine. Preferably, it refers to 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, These may exist as enantiomers accordingly. The compounds used in this invention may be two or more If they have a chiral center, they can also 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 depicted below are intended to represent all individual stereoisomers and all possible mixtures thereof, unless otherwise stated 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. Unless otherwise stated or indicated, the given formulas are intended to represent all individual tautomers and all possible mixtures thereof.

[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 part may be substituted, it may be substituted, for example, by 0, 1, 2, or 3 groups. In some embodiments, it may be 0, 1, or 2 groups, preferred Alternatively, it may be replaced by zero or one element.

[0305] When one group is bonded to another, for example, when a peptide, pharmaceutical, or sugar is bonded to a linker, they are 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). It can also be used in combination.

[0306] As used herein, the term “amino acid” means any natural or synthetic amino acid, i.e., containing carbon, hydrogen, oxygen and nitrogen atoms, and consisting of amino(-NH2) and carboxylic acid(-COOH) compounds. This refers to organic compounds that contain both functional groups. Typically, amino acids have α-, β-, and γ- or is a δ-amino acid. Preferably, the amino acid is one of 22 naturally occurring proteinogenic α-amino acids. Alternatively, the amino acid is a synthetic amino acid, such as α-amino-n-butyric acid, norvaline, norleucine, alloisoleucine, t-leucine, or α-amino-n-he Butanoic 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, iso-valine, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, N-methylβ-alanine, N-ethylβ-alanine, isoserine, α-Hydro Roxy-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 Selected from phosphorus, 2,3-diaminopropanoic acid, and 3-aminopropanoic acid. Furthermore, A The amino acid may be dehydroalanine, dehydrobutyrine, or a synthesized dehydroalanine or dehydrobutyrine precursor. The stereocentered amino acid may exist as a single enantiomer or as a mixture of enantiomers (e.g., a racemic mixture). Preferably, if the amino acid is an α-amino acid, the amino acid is It possesses 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 are obtained from commercially available suppliers (Sigma-Aldrich, Fluorochem, Carbosynth, VWR, Alfa Aesar, Fisher Scientific). The proteins were 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. Histone blotting was performed using a Thermo-Fisher iBlot gel transfer device. Antibodies used were as recommended by the manufacturer: anti-histone H3 (96C10) Mouse mAb for histone detection, Mouse monoclonal anti-polyhistidine-alkaline phasphatase, Clone HIS-1 (Sigma, A5588) for KDM4A detection (6His tag), and Rabbit Anti-Mouse IgG (H+L) HRP conjugate (Promega) as a secondary antibody. The following methods were used: (W4021) and Goat Anti-Mouse IgG H&L Alkaline Phosphatase (Abcam, ab97020). Thin-layer chromatography was performed using 1-10% methanol in dichloromethane. The analysis was performed using a Silica Gel 60 F254 plate (Merck). 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 were recorded using the residual solvent as an internal standard for TMS (d6-acetone: 2.09 ppm). The values ​​are cited in ppm using [a specific method / tool]. 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 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.). Synthesis The gene fragment (i.e., the human histone eH3-FLAG-HA construct) was obtained from GeneArt Gene Synthesis (Thermo-Fisher). The nucleotide sequence was obtained from Ox. This was confirmed by Source Bioscience DNA Sanger sequencing services, based at Ford University.

[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). The proteins were digested with proteases (trypsin, ArgC, LysC, AspN, elastase, etc.), and the resulting peptides were analyzed using proteomics software such as PEAKS. This involves performing de-novo sequencing on the measured spectra or analyzing them to determine which proteins are involved. The array can be compared with a database. Modifiers were identified and manually verified.

[0312] Unaltered protein mass spectrometry Unaltered protein mass spectrometry is performed using a combination of Waters Xevo G2-S QTof and Water Acquity UPLC. The procedure was carried out in conjunction with the analysis of the following components. 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 linear gradient for 10 minutes. 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] The 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). The spectra of African clawed frog histone H3 were deconvolved between 10,000 and 20,000 Da, human histone eH3.1 between 10,000 and 25,000 Da, African clawed frog histone H4 between 5,000 and 15,000 Da, NPβ between 10,000 and 30,000 Da, AcrA between 30,000 and 50,000 Da, and PanC between 30,000 and 40,000 Da. The reaction conversion rates were calculated from the relative peak intensities of the deconvoluted spectra. In histones, baseline (~10%) methionine oxidation often occurs during manufacturing, storage, and use, and these "+16 Da adducts" are... The results were summarized as the sum of the starting materials and the products.

[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 for 30 minutes at rt. The sample was then refueled with activation buffer (50 The reaction was carried out with Arg-C (1:20 w / w) in mM TEAB, 0.2 mM EDTA, and 5 mM TCEP at 37°C for 3 hours. The mixture was stopped at a final concentration of 0.5% after adding 10% FA. The samples were decontaminated with C18 (Oasis HLB 10 mg). After salting and drying in a speed vac, the mixture was resuspended in 5% FA 5% DMSO.

[0315] Variant 2: Modified, alkylated Approximately 10 μg of modified protein sample was taken in 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 then incubated in 10 mM TCEP at room temperature for 30 minutes. The sample was reduced and alkylated with 50 mM chloroacetamide at room temperature for 30 minutes in the dark. 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 hot.

[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 mixed with 5% FA and 5% DMSO. It was resuspended.

[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 in 10 mM TCEP at room temperature for 30 minutes, and then mixed with 50 mM CUBB. Alkylated with loroxetamide at room temperature in the dark for 30 minutes. The solution was then treated with 6M urea containing 50mM TEAB. Diluted to 1:20 (w / w) and digested overnight at 37°C. %. The sample was C18 (Oasis HLB 10 The solution was desalted (mg), dried using 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 in 10 mM TCEP at room temperature for 30 minutes, and then mixed with 50 mM CUBB. Alkylated with loroxetamide at room temperature for 30 minutes in the dark. The solution was then mixed with 1M urine containing 50mM TEAB. Dilute with water and inoculate with AspN, trypsin, or elastase 1:20 (w / w) at 37°C for 4 hours. The samples were digested overnight. %. The samples were 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 analyzed using nanoflow reversed-phase liquid chromatography (UHPLC) with an Ultimate 3000. Separation was performed using a combination of the Thermo Fisher Scientific System and the Thermo Fisher Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. Peptides were placed on a C18 PepMap100 pre-column (300 μm x 5 mm inner diameter, 3 μm C18 beads; Thermo Fisher Scientific). The samples were loaded 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). Cross-linked peptides were separated at 200 nl / min. The flow velocity is a linear gradient in the first stage from 15% to 35% of B for 30 minutes, and then from 35% to 55% of B. The second stage was carried out for another 15 minutes (A: 0.1% formic acid, B: 0.1% formic acid in acetonitrile). The raw data was acquired into the mass spectrometer in data-dependent mode. The system automatically switched from MS to higher-energy collision-induced dissociation MS / MS. A full scan spectrum was 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 most intense HCD fragmentation was observed. The top 10 peaks were selected 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) at 180 m / z for the initial fixed mass.

[0320] Data acquisition of crosslinked 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]. MS skim After the initial analysis, the top 10 strongest peaks were normalized for HCD fragmentation. 30% of the ghee was selected to exclude 1+ and 2+ charged species. The HCD spectrum was also decomposed using Orbitrap. The initial fixed mass was acquired at 180 m / z with a power of 17500; AGC target 5×10⁴; maximum injection time 120 ms, scan range 200~2000 m / z.

[0321] Data analysis (tandem mass spectrometry) Standard data analysis Identification and de-novo solutions were performed using Peaks version 8.5 (Bioinformatics Solutions Inc.). An investigation for analysis was conducted. Raw MS files were examined against the given protein sequences and contaminant list (generated from the MaxQuant contaminants database). To verify the purity of the sample, the pull was performed against the UniProt human database using MaxQuant Further investigations were conducted. The precursor mass tolerance was set to 10 ppm. The HCD fragment mass tolerance was set to 0.02 Da. The 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), deaminonation (asparagine, glutamine), carbamide methylation (excluding cysteine-ArgC variant 1 digest), carbamylation (lysyl methylation). The following modifications were defined as variable modifications, similar to the sample-specific modifications shown in the table below: (peptide N-terminus), amidation (C-terminus), and dehydroalanine (cysteine, -33.9887). A maximum of 4 variable modifications were defined. The settings were configured as follows: Peptide level FDR was 1%, and de-novo ALC was applied at 80. Pectols and identifications were manually verified. Isotope pattern analysis was performed manually using XCalibur Qual Browser 4.0.

[0322] Crosslinking mass spectrometry analysis The cross-linked sample was investigated with Peaks 8.5 as described above, and the presence of both proteins was confirmed. The purity of the sample was confirmed. The crosslinked sample was 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. The isotopic pattern is crosslinked because HBr is removed. Note that this is irrelevant to the analysis. Linker 4BrBut is 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 survey 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. The E-value was 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: carbamid methylation (cysteine), oxidation (methionine), deamination (asparagine, glutamine), carbamylation (lysine, peptide N-terminus), and amide. The modification (C-terminus) was set as a variable modification. The RAW file contains the modified eH3 construct. The sequence of expressed KDM4 was investigated against databases. The obtained spectra were manually analyzed and validated using pLabel 2.3.5. As a rule of thumb, e values ​​higher than e-03 are considered to be An e-value higher than e-06 indicates a promising identification, an e-value higher than e-06 indicates a reliable identification, and an e-value higher than e-10 indicates a very good identification.

[0324] Cyclic voltammetry Catechol and 4-bromobutylboronic acid were obtained from Sigma-Aldrich and further purified. I used it as received. All solutions were prepared at 25°C using ultrapure water with a resistivity of 18.2 MΩcm (Millipore) or higher, and thoroughly rinsed with nitrogen (99.998%, BOC Gases plc) before use. Degassed. A 43.85 mM sodium phosphate monobase and a 6.15 mM potassium phosphate dibase ri Acid-buffered saline (PBS) solution (pH=6.0).

[0325] All voltammetry measurements are performed using the Autolab PGSTAT30 computer-controlled potentiometer. The data was recorded using a Tat (Metrohm, Utrecht, The Netherlands) scanner. The experiment was 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 graphite as the counter electrode. Rods were used. Before each voltammetry experiment, 1.0 μm, 0.3 μm, and 0.05 μm rods were used. The working electrode surface was renewed by polishing with alumina slurry (Buehler Ltd, USA) in order of size, ultrasonic treatment 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, SPD-20A UV / Vis operating at 220nm and 280nm, and a Phenomenex Jupiter C-4 (5mm, 300Å) 4.6 x 250 meter operating at a flow rate of 1 mL min-1. The analysis was performed using a mm column. The analysis used mobile phases of 0.1 vol% TFA in water (solvent A) and 0.1 vol% TFA in MeCN (solvent B), employing a linear gradient: 4 minutes at 0% B, and 26 minutes from 0% to 100% B. The chromatogram recorded at 220 nm was obtained using Shimadzu LabSolutions software. Analysis was performed using A.

[0328] 19 F-NMR research 19 F-NMR studies were performed following the general procedure below: FeSO4·7H2O (100 eq) was placed in a glass vial (5 mL) and then 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 a glass vial. The vial was then sealed with a plastic cap. The samples were 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 the fluorescently labeled protein. The protein sample was concentrated to a volume of 0.5 mL using a vivaspin column (MCW = 5000), and then the sample was... 19We 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 washed with 5 times the amount / weight of "wash buffer" (50 mM). The Triton-X detergent was resuspended in Tris, pH 7.5, 100 mM NaCl (with a protease inhibitor cocktail). The suspension was flash-frozen and stored at -80°C until dissolved. Dissolution was carried out by sonication in the presence of 1 mg of DNase with 5 bursts of 30 seconds at 40% amplitude. 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]. The sonication was repeated once for 30 seconds at 40% amplitude, and the suspension was centrifuged at 20 krpm for 10 minutes. The pellet was washed two more times in this manner, and then the non-Triton-containing Washed once with "washing 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" Add (7M Gdn-HCl, 20mM Tris, pH 7.5, 10mM DTT), shake at rt for 1 hour, then mix The mixture was centrifuged at 20 krpm for 10 minutes at room temperature. The supernatant was pre-equilibrated with "SAU-100" buffer (7M urea, 20mM NaOAc, pH 5.2, 100mM NaCl, 1mM EDTA, 10mM DTT, 1mM benzamidine). The protein was loaded onto an S200 size exclusion column (GE Healthcare). The protein was eluted with SAU-100, analyzed by SDS-PAGE, and the histone fractions were pooled and concentrated to 1-4 mL. Cations were also analyzed. Histones were further purified using 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 fraction is pooled and mixed with water (with 2 mM β-mercaptoethanol). The tissue was dialyzed and freeze-dried.

[0330] AcrA Plasmid (pET24) was transformed into BL-21(DE3) cells and then placed on a kanamycin agar plate. The plasmids were plated. Four 10 mL starter cultures (LB / kanamycin) were used for each plasmid. The cells were grown overnight at 37°C, then transferred to 500 mL of medium (LB / kanamycin). This culture was grown at 37°C until the OD600 reached 0.6-0.8 (between 40-70 minutes), at which point the cells were induced with IPTG (1.25 mM) and incubated for a further 4 hours. The cells were then incubated at 8 krpm for 10 minutes. The cell pellet was prepared in a buffer (50 mL (50 mM Tris, 100 mM NaCl, 10 mM I)). The pellet was resuspended in midazole, 1 mg / mL lysozyme, and 0.1 mg / mL DNAse, and stirred on ice for 2 hours. The pellet was then sonicated (50% power, 30 seconds sonication, 1 minute rest, 4 times), The resulting mixture was treated by centrifugation (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 its concentration was analyzed.

[0331] Human sirtuin 2 (Sirt2) The human Sirt2 gene in the pET6 plasmid was transformed in BL21-(DE3) cells, and LB / agar / agar The cells were plated onto rubenicillin plates. Single colonies were picked in 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 the OD reached 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. The cells were pelleted at 8 kRPM and 9.6 kG (raverage) for 15 minutes. The pellets were frozen at -80°C. The pellets were 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). Ultrasonic thawing was performed on ice. Cells were lysed by wave treatment (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 0.2 μm silicate. The sample was filtered through a range filter and then applied to an FPLC column. The protein was first subjected to 2D-FPLC in 1 mL of 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 The solution was purified by passing it through a step gradient. 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, 1168-1 173, 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, TH et al. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science, aag1465, doi:10.1126 / science.aag1465 (2016).

[0336] Dehydroalanine Production XI histone H3-Dha9 - Lyophilized Xl histone H3-C9 (10 mg) 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 into 1 mL of the same buffer (PD Minitrap G25, GE Healthcare). The resulting protein concentration was measured (Nanodrop), and immediately afterwards, DBHDA (freshly prepared 0.5 M DMSO) was added. 60 eq) was added from the stock, then shaken (500 rpm) at 25°C for 45 minutes, and then at 37°C for 2 hours. As before, desalt the protein to remove excess DBHDA and replace it with the desired buffer. Protein yield and concentration were measured using 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 in sodium phosphate buffer (100 mM, pH 8.0, 3M). Storage buff using a PD MiniTrap G-25 column (GE Healthcare) equilibrated with Gdn·HCl Buffer replacement from [the previous step] to sodium phosphate buffer (100 mM, pH 8.0, 3M Gdn·HCl). Then, following the gravity protocol, a protein solution with a concentration of 2.56 mg / mL was obtained. The specified amount (29.2 nmol) was dissolved in methyl 2,5-dibromopentanoate (MDBP, 1 M in DMSO, 1.46 μmol). The sample was processed and shaken at 25°C and 500 rpm for 16 hours. Then, 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) to obtain a protein solution of 1.56 mg / mL according to a gravity protocol. The conversion rate was determined by analyzing a fixed portion of the purified product by LC-MS.

[0340] cabLys3-Dha104 - constant cabLys3-Cys104 in cabLys3-Dha104 PBS buffer (pH 7.4) The sample was treated with DTT (4 mg) and incubated at 25°C for 30 minutes. Then, it was equilibrated with sodium phosphate buffer (50 mM, pH 8.0) on a PD MidiTrap G-25 column (GE Healthcare). DTT is removed using and a vivaspin column (MCW = 5000) is used according to the gravity protocol. After concentration, a crude protein solution of 0.9 mg / mL (0.5 mL) was obtained. Next, DBHDA (0.5 M, 14.25 μmol in DMSO) was added to the protein solution, and the resulting reaction mixture was incubated at 37°C for 150 minutes. Purification was then carried out using a PD MiniTrap G-25 column (GE Healthcare) equilibrated with ammonium acetate buffer (100 mM, pH 6.0) according to the gravity protocol. After concentrating the protein sample using a vivaspin column (MCW = 5000), 0.5 mL of Dha-tagged cabLys3 was added. A stock solution was obtained with 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 subjected to column chromatography (SiO2, The solution was purified with 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. C29 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) in 200 mL of acetone, p-Tol The reaction mixture was added to a solution of ensulfonic acid monohydrate (9.72 g, 51.1 mmol) and triethyl orthoformate (12.1 mL, 72.6 mmol). The reaction mixture was stirred overnight at rt. In a saturated Na2CO3 aqueous solution... After neutralization, the crude mixture was concentrated in a small amount of MeOH, and the product was crystallized from the solution at 0°C. The resulting 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 dissolved in 1-allyl-2,3-isopropylidene-α-D-riboph in dry CH2Cl2 (10 mL) at 0°C. The mixture was added to a solution of lanose (0.50 g, 2.34 mmol). 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 on Na2SO4, and filtered. The product was separated and evaporated to dryness. The crude product was purified by column chromatography (SiO2, pentane:ethyl acetate (9:1)) and obtained 1-allyl-2,3-isopropylidene-5-bromo-α-D-ribofuran A solution (0.34 g, 1.17 mmol, 50%) was obtained 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), and 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)) and 1-allyl-2,3-isopropylidene-5-chloro-α-D-ribofuranose. (0.17 g, 0.74 mmol, 78%) was obtained 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 (1M, 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 solution 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) is carefully dissolved at -78 °C with 1-allyl-2,3-isopropylidene-5-chloro-α-D-ribofuranose (0.13 g, 0.57 mmol) The mixture was then added to SiEt3H (108.7 μL, 0.681 mmol). The resulting suspension was incubated at this temperature for 30 minutes. The mixture was stirred and then heated 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 solution and dried over MgSO4. Under reduced pressure, the solvent was dried. After removing the ions, 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, and the pure compound (4-(5-chloro-α-D-ribo Furanose-butyl-boronic acid (30.0 mg, 0.13 mmol, 23%) was obtained as a white solid. C8H 16 BClO5 (238.5 g / mol).

[0349] Peracetyl-β-D-GlcNAc Ice-cold, stirred suspension of D-GlcNAc (6.42 g, 29.0 mmol) in Ac2O (80 mL, 74.0 g, 725 mmol). Montmorillonite K-10 (24.0 g) was added in small portions over 10 minutes. The ice bath was removed, and the mixture was turned back. The reaction mixture was stirred at this temperature for 24 hours. The reaction mixture was filtered through Celite and the pad was fermented with AcOEt. Washed until colorless. The combined filtrate was concentrated under reduced pressure. The orange residue was extracted from MeOH. The product was recrystallized multiple times to obtain the title sample as a white, needle-shaped 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) showed that the reaction was complete (16 hours) due to the complete consumption of the starting materials (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 analysis by TLC (100% siRNA, sulfuric acid development) showed the disappearance of the starting material and the appearance of a single spot. The mixture was stirred until completion was indicated (Rf 0.0). At completion (30 minutes), the reaction mixture was DOWEX H + By adding Neutralize, stir for 5 minutes, filter the reaction mixture, concentrate to 1 mL, and plug with silica plug (methanol, After thoroughly washing with water / isopropanol / ethyl acetate (1:2:5), vaporize under reduced pressure. The active ingredients were removed 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, stirred for 17 hours, and at that point TLC (100) was performed. Analysis by % siRNA, H2SO4 evolution showed complete disappearance of the starting material (Rf 0.0) and one of the major products (Rf The presence of Rf 0.70 and one of the by-products (Rf 0.47) was observed. The crimson solution was diluted with DCM (500 mL), washed with saturated aqueous NaHCO3 (3 x 500 mL), dried on 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. Purification by Rush column chromatography (Pet ether / Depositphotos 40% -> 65% gradient elution). The title product was obtained 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 Butylammonium (464 mg, 1.37 mmol) Sodium azide (267 mg, 4.10 mmol) was added in small portions to the rapidly stirred solution. (Reaction mixture) The mixture was stirred for 1 hour, and at that point, TLC (100% siRNA, H2SO4 development) analysis was performed, indicating the complete formation of the starting material. The organic fraction showed complete disappearance (Rf 0.70) and the 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 to obtain a white amorphous solution. The 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 H 20 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) under argon. The effervescent reaction mixture was stirred at RT for 2 hours. At that 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, and the residue was dissolved in chloroform. This was then 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 Stirring of EEDQ (427 mg, 1.73 mmol) and iodoacetic acid (323 mg, 1.73 mmol) in THF (10 mL) Peracetyl 1-amino-β-D-GlcNAc (500 mg, 1.73 mmol) was added to the mixed solution. The reaction mixture was stirred at room temperature for 24 hours, at which point TLC (5% MeOH / CHCl3) development was performed at 254 nm and anisaldehyde Analysis using dehydration dip showed significant product separation (Rf = 0.38). The reaction mixture was concentrated on diatomaceous earth until dry (5.00 g), loaded onto a chromatography column pre-equilibriumized 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%). When exposed for an extended period, it turned yellow. 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), add sodium methoxide (25%, 200 μL) in methanol and steep for 5 minutes. After stirring, analysis by TLC (MeOH / CHCl330%, anisaldehyde) at that point indicated the end of the reaction, with 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 dry. In addition, the title product was obtained as a white to pale orange amorphous solid (303 mg, 774 μmol, 98%). When exposed to light for extended periods, it turned brown. C 10 H 17 IN2O6 (388.2 g / mol).

[0357] 2-(3-azidopropyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane Place 3-bromopropylboronate pinacol ester (200 mg, 0.80 mmol) in a round-bottom flask. 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. 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 mixed with ethyl acetate (3 x Extracted with 10 mL. The combined organic layers were dried on MgSO4 and concentrated under vacuum, then 12 g RediSep R f CombiFlash R with silica gold column f Flash chromatography system The product (137 mg, 0.17 mmol, 81%) was purified by a linear gradient (2 minutes from 100% hexane followed by 14 minutes from 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. The organic layer was dissolved in toluene (25 mL). The organic layer was washed with sodium bicarbonate solution (sat., 25 mL), water (25 mL), and saline solution (25 mL), dried on MgSO4, and concentrated under vacuum. The crude product was divided into 12 g RediSep R f CombiFlash R with silica gold column f Flash Chromatography The product was purified using a Fie 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 (50 mg, 0.17 mmol, 17%) 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 toluene (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. Crude product 12g RediSep R f CombiFlash R with silica gold column f Flash Chromatography The product was purified using a Raffy system (linear gradient: 2 minutes 100% hexane followed by 14 minutes 100% siRNA and 6 minutes 100% siRNA) 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 Place 3-bromopropylboronic acid pinacol ester (500 mg, 2.00 mmol) in a round-bottom flask. Sodium iodide (900 mg, 6.00 mmol) and acetone (5 mL) were added. The reaction was as follows: The solution was stirred at 60°C for 16 hours. After cooling to room temperature, water (25 mL) was added to obtain the aqueous mixture. The combined organic layer was extracted with toluene (3 x 25 mL). The combined organic layer was then treated with an aqueous solution of sodium hyposulfite (sat. 2 Wash with (25 mL x), water (25 mL), and saline solution (25 mL), dry on MgSO4, and concentrate under vacuum. And, a CombiFlash R with a 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 Flash chromatography The product was purified using a Graphy system and obtained as a colorless liquid (430 mg, 1.45 mmol, 73 mg). %). 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 Dissolve 3-aminopropylboronic acid pinacol ester (150 mg, 810 μmol) in MeOH (8 mL). The mixture was then mixed with 2 M LiOH (2.43 mL, 4.86 mmol) followed by MeI (0.5 mL, 8.10 mmol), and stirred at RT for 1.5 hours. The solvent was removed under reduced pressure, and the resulting white solid was extracted with acetonitrile. The desired product was then collected in solution. After evaporation under reduced pressure, it was triturated with DCM. The filtrate was then 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 evaporated. The residue was purified by flash chromatography (n-heptane / alkyl; 10-100% alkyl). The title compound was obtained 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 freeze-dried and dissolved in H2O for purification. Purification is preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50x250 mm, mobile phase: 0.25% The reaction was carried out via an aqueous solution of NH4HCO3 (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 2-tBu-ethylboronic acid pinacol ester (500 mg, 1.95 mmol) in CH2Cl2 (1.5 mL) Trifluoroacetic acid (1.5 mL) was added to the solution. After stirring the solution at room temperature for 2 hours, nitrogen gas was used. The solution was concentrated by flowing water, and then azeotropically removed from CH2Cl2 to quantitatively obtain the desired carboxylic acid as a viscous oil, which was used without further purification. C9H 17 BO4 (200.0 g / mol).

[0367] 2-((1,1-difluoroethyl)sulfonyl)pyridine Under a nitrogen atmosphere, heat-gun drying, difluoromethyl 2-ply, in a two-necked flask. Lysyl sulfone (193 mg, 1.00 mmol), THF (4 mL), and DMI (0.4 mL) were administered. Next, The reaction mixture was 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 LiHMDS (1 M in THF, 2.5 mL, 2.50 mmol). The mixture was added dropwise, and after complete addition, it was stirred at -78°C for 30 minutes. After quenching with ammonium chloride aqueous 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 collected by column chromatography (SiO2, The product was purified using 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 Difluoromethyl 2-pyridylsulfone is placed in a two-necked flask and dried with a heat gun under a nitrogen atmosphere. (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. 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 aqueous LiCl solution (sat., 20 mL) and saline solution (20 mL), dried on MgSO4, and concentrated under vacuum. The product (630 mg, 1.88 mmol, 42%) was purified with comi to obtain a yellow solid. C 13 H 18 F2N2O4S (336.4 g / mol).

[0369] Benzyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl)carbamate Difluoromethyl 2-pyridylsulfone is placed in a two-necked flask and dried with a heat gun under a nitrogen atmosphere. (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. 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). The resulting aqueous mixture was extracted with toluene (3 × 50 mL). The combined organic layers were washed with aqueous LiCl solution (sat., 10 mL) and saline solution (10 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 Flash chromatography system (gradient: linear gradient from 2 minutes to 100% petroleum ether, then over 14 minutes to 100% ethyl acetate) The product was purified using a gradient filtration system, and the product (290 mg, 0.79 mmol, 43%) was obtained as a white solid. C 16 H 16 F2N2O4S (370.4 g / mol).

[0370] 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminotrifluoroacetate te 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. I got it. 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. The crude mixture was then dissolved in DCM (25 mL), and the resulting organic layer was dissolved in NaOH (2 M, 20 mL) and HCl (1 M). Washed with (20 mL) and saline solution (20 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 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)carb Mate Under a nitrogen atmosphere, heat gun dry two-necked flask tert-butyl(3,3-difluoro-3- (Pyridine-2-ylsulfonyl)propyl)carbamate (241 mg, 0.72 mmol) and DMF (8 mL) were added. Next, the reaction mixture was 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), and 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 toluene (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 mixed with 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 Refluoroacetate In a round-bottom flask, tert-butyl(3,3-difluoro-3-(pyridine-2-ylsulfonyl)-prop (Methyl)carbamate (175 mg, 0.50 mmol) and CH2Cl2 (5 mL) were added. 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 allowed to cool to room temperature. The mixture was stirred overnight. The crude mixture was then concentrated and dried under vacuum to obtain the product (182 mg, 0.50 mmol, 100%) as yellow oil. C 11 H 13 F5N2O4S (364.3 g / mol):

[0374] 3,3-Difluoro-N,N-dimethyl-3-(pyridine-2-ylsulfonyl)propan-1-amine In a round-bottom flask, combine 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminyl Mutrifluoroacetate (70 mg, 0.20 mmol) and MeOH (2 mL) were added. Next, formaldehyde (37 wt.% in H2O, 66.6 mg, 180 μL, 2.22 mmol) was added, and the resulting reaction was... After stirring the mixture at room temperature for 10 minutes, sodium triacetoxyborohydride (179 mg, 0 0.84 mmol was added. After stirring at room temperature for a further 16 hours, the reaction mixture was concentrated under vacuum. The crude product was divided into 4g 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 In a round-bottom flask, combine 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propane-1-aminyl Mutrifluoroacetate (150 mg, 0.43 mmol), MeCN (2.7 mL), and MeOH (1.3 mL) It was administered. Next, DIPEA (332 mg, 448 μL, 2.57 mmol) and MeI (609 mg, 267 μL, 4.29 An mmol was 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 resulting 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%). The product was purified and dried under vacuum to obtain a white solid (110 mg, 0.39 mmol, 92%). C 11 H 17 F2N2O2S (279.1 g / mol).

[0376] 2-((difluoro(methylthio)methyl)sulfonyl)pyridine Under a nitrogen atmosphere, heat-dry the two-necked flask with difluoromethyl 2-pyridyl sulfone. 500 mg, 2.59 mmol of ammonium chloride, 10 mL of THF, 1 mL of DMI, and 488 mg, 368 μL, 3.90 mmol of methyl S-methanethiosulfonate were added. Next, the reaction mixture was cooled to -78°C in an isopropanol / dry ice mixed solvent, and then LiHMDS (1 M in THF, 3.2 mL, 3.20 mmol) was added dropwise. After complete addition, the mixture was stirred at -78°C for 30 minutes. After quenching with liquid (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 on MgSO4, and concentrated under vacuum. The crude product was then prepared using 12 g RediSep R f CombiFlash R with silica gold column f Flash chromatography system (gradient: 2 min, 100% CHCl3 / heptane) (1:1), then over 14 minutes, a linear gradient to 100% CHCl3 / heptane / SiO(3:3:1) The product (500 mg, 2.10 mmol, 81%) was purified using the solution and obtained 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 placed in a heat-gun-dried round-bottom neck flask. Next, the reaction was performed. The solution is cooled to 0°C in an ice / water mixture, and then 3-chloroperbenzoic acid in CH2Cl2 (1 mL) ( (≤77%, 186 mg, 0.82 mmol) was added dropwise, and after complete addition, the mixture was stirred at room temperature for 16 hours. The crude mixture was concentrated under vacuum and dissolved in toluene (30 mL), and the organic layer was dissolved in an aqueous solution of NaHCO3 (sat. 2 mL). Washed with (30 mL x), water (30 mL), and saline solution (30 mL), dried with MgSO4, and concentrated under vacuum. The crude product was divided into 12g RediSep R f CombiFlash R with silica gold column f flash Chromatography system (gradient: 2 minutes 100% hexane, then 14 minutes 100% petroleum) The product was purified using a linear gradient of 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 ice water, followed by the addition of sodium periodate (411 mg, 1.93 mmol) and RuCl3xH2O (1 mg), and the mixture was stirred for 16 hours. Diluted with water (30 mL). Next, 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 then 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, then a linear gradient over 12 min to 100% petroleum ether / siRNA (4:3)), and the product (108 mg, 0.40 mmol, 95%) was obtained as a white solid. I obtained it by doing so. 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, heat-dry a two-necked flask and add difluoromethyl 2-pyridylsulfate. 965 mg, 5.00 mmol of phosphate (Fon), 1,3,2-dioxathiolane 2,2-dioxide (931 mg, 7.50 mmol), THF (20 mL), and DMI (2 mL) were added. 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, 6.00 mL, 6.00 mmol). After complete addition, the mixture was stirred at -78°C. After 30 minutes, the reaction mixture was converted to aqueous acetate ammonium compound. Quenched by adding monoium (1M, 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 (1M), and the resulting aqueous mixture was extracted with HCl (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 divided into 24 g RediSep R f CombiFlash R with silica gold columnf Flash chromatography The solution was purified using a TEMM (gradient: linear gradient from 100% petroleum ether for 2 minutes, followed by 14 minutes of linear gradient to 100% ammonium ether) to obtain 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propan-1-ol) (210 mg, 0.89 mmol, 18%) as a white solid. Honyl)-propylacetate (400 mg, 1.43 mmol, 29%) was obtained 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 Difluoromethyl 2-pyridyl sulfonate is added to a two-necked flask and dried with a heat gun under a nitrogen atmosphere. (1.93 g, 10.0 mmol), 1,3,2-dioxathiolan 2,2-dioxide (1.86 g, 15.0 mmol) THF (40 mL) and DMI (4 mL) were added. Next, the reaction mixture was mixed with isopropanol / dry The mixture was cooled to -78°C in Iaice, and then LiHMDS (1M in THF, 12.0 mL, 12.0 mmol) was added dropwise. 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 portions. f CombiFlash R with silica gold column f Flash Chromatography System (gradient: 2 minutes for 100% CHCl3, then 14 minutes for linear conversion to 100% CHCl3 / MeOH (1:1)) The product was purified using a gradient filtration method, 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 to 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl hydrogen sulfate (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 prepared. The mixture was stirred at room temperature for 16 hours. Next, the crude mixture was cooled in an ice bath and quenched with an aqueous solution of NaHCO3 (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. At 0°C, triethylamine (850 mg, 617 μL, 6.10 mmol) and 4-toluenesulfonyl chloride (700 mg, 3.67 mmol) were used. Add the following and stir the resulting reaction solution overnight in an ice bath. Next, add hydrochloric acid aqueous solution (1M, 30 mL) In addition, the mixture was quenched, the aqueous layer was extracted with CH2Cl2 (3 x 25 mL), and the combined organic layer was diluted with water (30 Washed with (mL) and saline solution (30 mL), dried on MgSO4, and concentrated under vacuum. The crude product was divided into 24 g RediSep R f CombiFlash R with silica gold column f Flash chromatography The product was purified using a system (gradient: 2 mins to 100% petroleum ether, then a linear gradient over 14 mins to 100% siRNA / petroleum ether (3:2)), and the product (825 mg, 2.09 mmol, 66%) was obtained as a white solid. I acquired it physically. C 15 H 15 F2NO5S2 (391.4 g / mol).

[0383] 2-((3-azido-1,1-difluoropropyl)sulfonyl)pyridine In a round-bottom flask, add 3,3-difluoro-3-(pyridine-2-ylsulfonyl)propyl 4-methyl 370 mg, 0.95 mmol of sulfonate, 10 mL of DMF, and 308 mg, 4.75 mmol of sodium azide were added. After stirring at 85°C for 3 hours, the reaction mixture was diluted with water (30 mL). The aqueous mixture was extracted with HCl (3 x 25 mL), and the combined organic layer was diluted with water (3 x 25 mL) and saline (2 x 25 mL). Wash with (mL), dry on MgSO4, and concentrate under vacuum to obtain the product (203 mg, 0.77 mmol, 82%). It was obtained 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, heat-dry the two-necked flask with difluoromethyl 2-pyridyl sulfone. N (290 mg, 1.50 mmol), THF (6 mL), and DMI (0.6 mL) were added. Next, the reaction mixture was prepared. The 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 and concentrated under vacuum, and 12 g of the crude product was collected. f CombiFlash R with silica gold column f Flash chromatography system (gradient: 2 min, 100% petroleum ether) The solution was then purified over 14 minutes using a linear gradient to 100% siRNA to obtain the product (223 mg, 0.70 mmol, 47%) as a yellow solid. C6H4F2INO2S (319.1 g / mol).

[0386] 2-bromo-2,2-difluoroacetamide Place 1.58 g, 1.0 mL, 7.78 mmol of bromodifluoroethyl acetate and methanol in a round-bottom flask. 5 mL of ru was added. Next, the reaction mixture was cooled to -15 °C in a sodium chloride / ice mixture. The mixture was then turned over, and ammonia (7N, 2.5 mL) in methanol was added dropwise. 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. I obtained it. C2H2BrF2NO (173.9 g / mol).

[0387] 2-Bromo-2,2-difluoroacetate sodium Add sodium hydroxide (300 mg, 7.71 mmol) and methanol (7 mL) to a round-bottom flask. Next, the reaction mixture was cooled to 0°C in an ice bath, and then 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) was added to a round-bottom flask using a heat gun. ) and ethanol (34 mL) were added. Next, the reaction mixture was cooled to 0°C in an ice / water bath, and then triethylamine (1.38 g, 1.90 mL, 13.5 mmol) was added dropwise. After stirring for 10 minutes, ethyl bromofluoroethyl (2.50 g, 1.6 mL, 13.49 mmol) was added dropwise, and the resulting mixture was stirred at room temperature for 16 hours. Subsequently, the crude mixture was mixed by adding aqueous hydrochloric acid (1 M, 50 mL) Quenched, the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic layer was then extracted with saline solution (50 mL). The solution was washed with (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. Place sulfide precursor (1.00 g, 4.65 mmol), acetonitrile (6 mL), and dichloronitrile in a round-bottom flask. Lomethane (6 mL) and water (15 mL) were added. Next, sodium periodate (4.50 g, 21.4 (molecule) and ruthenium chloride hydrate (3 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 (50 mL), and the aqueous mixture was mixed with ether (3 x 50 Extraction was performed using (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, 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). The mixture was stirred at room temperature for 30 minutes. The crude mixture was then concentrated under vacuum. The resulting solid was then triturated with ethyl acetate / hexane (4:2, 6 mL) and dried under vacuum to obtain the product (380 mg, 84%) as a white solid. C7H7FN2O3S (218.2 g / mol).

[0390] 2-Fluoro-2-(pyridine-2-ylsulfonyl)sodium acetate 2-fluoro-2-(pyridine-2-ylsulfonyl)ethyl acetate (450 mg, 1.82 mmol), MeOH (8 mL), and THF (8 mL) were placed in a round-bottom flask. Next, 1.9 mL of sodium hydroxide aqueous solution was added dropwise to the reaction mixture and stirred for 10 minutes. The crude mixture was concentrated and then vacuum-fed. The product (424 mg, 97%) was dried under the sun to obtain 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. Next, the reaction mixture was 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. Subsequently, the crude mixture was diluted with water (200 mL), extracted with phenylethylamine (3 x 50 mL), and the combined organic layer was washed with water (50 mL), aqueous HCl (1 M, 50 mL), and saline solution (50 mL), dried on MgSO4, and then fermented. The mixture was concentrated under open air to obtain crude sulfide (750 mg) as yellow oil.

[0392] Crude sulfide (750 mg), acetonitrile (30 mL), and dichloromethane in a round-bottom flask. (10 mL) and water (40 mL) were added and cooled to 0°C in an ice / water bath. Next, sodium periodate was added. Adding um (5.30 g, 24.9 mmol) and ruthenium chloride hydrate (5 mg) to the reaction mixture, The prepared solution was stirred at room temperature for 16 hours. Then, the crude mixture was diluted with water (40 mL) to form an aqueous mixture. The product 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 the crude product was 24 g RediSep R f Silica Gold Color CombiFlash R equipped with f Flash chromatography system (gradient: 2 min 100% Xane is then refined over 14 minutes to 100% petroleum ether / HCl (1:1) with a linear gradient. The product (430 mg, 2.51 mmol, 9%) was obtained as 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 placed in 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. Subsequently, the reaction mixture was cooled. The mixture was quenched by adding NH4Cl aqueous solution (sat., 20 mL), and then eluted with siRNA (3 x 25 mL). Extraction was performed. The combined organic layers were then treated with NaHCO3 aqueous solution (sat., 30 mL), water (30 mL), and saline solution (30 mL). Washed, dried on MgSO4, and concentrated under vacuum. 4 g of the crude product was collected using RediSep R. f CombiFlash R with silica gold column fThe 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 Place cesium carbonate (23.5 g, 72.0 mmol) in a round-bottom flask and heat under vacuum for 10 minutes using a heat gun. The mixture was heated three times using [a specific method]. Next, under a nitrogen atmosphere, DMF (340 ml) and 2-mercaptopyridine (4.00 g) were added. Add 36.0 mmol of ethyl bromodifluoroethyl acetate (14.6 g, 9.23 mL, 72.0 mmol) and the resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was then diluted with water (300 mL), and the aqueous mixture was extracted with phenylethyl acetate (3 x 200 mL). The combined organic layers were washed with water (100 mL) and saline solution (100 mL), dried over MgSO4, and concentrated under vacuum. 80 g of the crude product was collected. f CombiFlash R with silica gold column f Flash chromatography system (gradient: 2 min) The product was purified by 100% petroleum ether, then by a linear gradient over 14 minutes to 100% petroleum ether / SiO(5:1), to obtain the product (6.30 g, 27.0 mmol, 75%) as a yellow liquid. 80 g of the crude product was then added to RediSep R. f CombiFlash R with silica gold column f Flash chromatography - The system refines it (gradient: 2 minutes for 100% petroleum ether, then 14 minutes for 100% petroleum aether) A linear gradient was applied to the ammonium compound (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, heat gun dry round-bottom flask and add 2,2-difluoro-2-(pyridine-2-yl) (e) Ethyl acetate (3.00 g, 12.9 mmol), THF (7.5 mL), and EtOH (52.5 mL) were added. Next The reaction mixture was 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 toluene (3 x 60 mL), and the combined organic layers were washed with brine (50 mL), dried on MgSO4, and then removed under vacuum. The solution was concentrated 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, heat gun dry round-bottom flask and add 2,2-difluoro-2-(pyridine-2-yl) (o) Ethane-1-ol (2.25 g, 10.9 mmol) and CH2Cl2 (100 mL) were added. Next, the reaction mixture was added. The mixture was cooled to 0°C in an ice / water bath, then met-chloroperoxybenzoic acid (4 x 1.55 g, 27.2 mmol) was added in small portions, and the mixture was stirred in a cooling bath for 16 hours. After that, the crude mixture was dissolved in sodium hydroxide. Quenched with thorium aqueous solution (0.5 M, 120 mL), the aqueous layer was extracted with CH2Cl2 (3 x 100 mL), the combined organic layer was washed with water (100 mL) and saline solution (100 mL), dried on 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 collected in RediSep R fCombiFlash R with silica gold column f Flash chromatography Stem (gradient: 2 minutes 100% petroleum ether, then 12 minutes 100% petroleum ether / SiO2) The product (647 mg, 2.90 mmol, 27%) was purified by a linear gradient (4:3) to obtain a pale yellow rubber. It was obtained as such. C7H7F2NO3S (223.2 g / mol).

[0397] 2,2-difluoro-2-(pyridine-2-ylthio)ethyl 4-methylbenzenesulfonate Under a nitrogen atmosphere, heat gun dry round-bottom flask and add 2,2-difluoro-2-(pyridine-2-yl) (o) Ethane-1-ol (1.00 g, 5.23 mmol) and CH2Cl2 (20 mL) were added. Next, the reaction mixture was 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 the addition of aqueous hydrochloric acid (1 M, 30 mL). Then, dilute with CH2Cl2 (30 mL), wash the organic layer with saline solution (30 mL), dry on MgSO4, and... Concentrated under open air. 24g of crude product was collected using RediSep R. f CombiFlash R with silica gold column f Flash chromatography system (gradient: 2 min 100% petroleum ether, then 12 min) The product was purified by a linear gradient of 100% petroleum ether / siRNA (2:1) to obtain the product (1.60 g, 4.64 mmol, 89%) 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-methylbenzene in a round-bottom flask. Sulfonate (7 g, 20.3 mmol), acetonitrile (100 mL), dichloromethane (50 mL) ), water (150 mL) was added and cooled to 0°C in an ice / water bath. Then sodium periodate was added. 21 g, 98.2 mmol of ruthenium chloride and 20 mg of ruthenium chloride 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 (200 mL), and the aqueous mixture was extracted with SiO2 (3 x 250 mL). The combined organic layers were then separated by water (100 mL) and saline solution (100 mL). Wash, dry on MgSO4, concentrate under vacuum, and take 80 g of the crude product. RediSep R f Silica Gold CombiFlash R with column f Flash chromatography system (gradient: 2 min 100 The product was purified by a linear gradient from % petroleum ether to 100% butyl over 14 minutes to obtain the product (7.66 g, 20.3 mmol, 100%) 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, heat-dry a round-bottom flask and add 2,2-difluoro-2-(pyridine-2-i) (Sulfonyl)ethyl 4-methylbenzenesulfonate (1.51 g, 4.00 mmol), sodium azide (1.30 g, 20 mmol), and DMF (32 mL) were added. After stirring at 70°C for 133 hours, the reaction mixture was cooled to room temperature, diluted with water (70 mL), extracted with SiO2 (3 x 70 mL), dried on MgSO4, and concentrated under vacuum. 40 g of the crude product was collected. RediSep R f CombiFlash R with silica gold column f Flash chromatography system (gradient: 2 mins, 100% hexane, so The mixture was then purified over 14 minutes using a linear gradient to 100% petroleum ether:siRNA (5:4) to obtain the product (650 mg, 2.62 mmol, 66%) as 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 placed in a heat gun-dried round-bottom flask. Next, triethyl Luamine (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, heat gun-dried round-bottom flask containing 2,2-difluoro-2-(pyridine-2-yl) Rufonyl)ethane-1-amine (50 mg, 0.23 mmol), CH2Cl2 (1 mL), and DIPEA (101 mg, 136 μL, 0.78 mmol) were added, 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). The product was purified, dried on MgSO4, and concentrated under vacuum. 4 g of the crude product was collected using RediSep R. f CombiFlash R with silica gold column f Flash chromatography system (gradient: 2 min 100 The solution was purified by a linear gradient of %CHCl3, then 100% CHCl3:MeOH (95:5) over 12 minutes, and then 100% siRNA over 5 minutes to obtain the product (520 mg, 2.34 mmol, 89%) 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-diyldiacetate 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 f centre The product (410 mg, 0.81 mmol, 71%) was purified using a Rush chromatography system (gradient: 1.5 min 100% CHCl3, then a linear gradient to 100% CHCl3 / MeOH (9:1) over 15 min), and the product was obtained. It was obtained 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 + Quench by adding (100 mg) for 5 minutes. The mixture was stirred. 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 15BrF2N2O6 (377.1 g / mol).

[0404] 2-((trifluoromethyl)sulfonyl)pyridine In a 25 mL round-bottom flask, add DMSO (4 mL) containing piflusul (2.60 mmol) and KHF2 (2 mg, 0.26 mmol). %) mmol, 10 mol%) was added. 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). Organic fraction The mixture was combined, dried on MgSO4, filtered, and concentrated in vacuum to obtain the product as a pale yellow solid. It was obtained with a purity and yield of 80%. C6H4F3NO2S (211.0 g / mol).

[0405] 2-((fluoromethyl)sulfonyl)pyridine In 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 dissolved under a stream of N2 gas. It was added dropwise. Next, CH2FI (1.0 mL, 14.4 mmol, 4.0 equiv) (Note: CH2FI is volatile) A highly toxic substance was added dropwise over 30 minutes. The reaction was then slowly warmed to room temperature for 12 hours. The mixture was stirred overnight. Next, the reaction mixture was 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 on MgSO4. The solution was filtered and concentrated under vacuum to obtain crude 2-((fluoromethyl)thio)pyridine as a yellow oil. This crude product was then used in the next step without further purification. 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). Subsequently, NaIO4 (3.0 g, 14.5 mmol) and RuCl3xH2O (3 mg) were added. The reaction was then continued until complete. 19 Monitored by F NMR Upon completion, 10 mL of distilled H2O was added, and the resulting reaction mixture was extracted with Et2O (3 × 30 mL). Next, the organic phase was washed with saturated NaHCO3 (30 mL) and saline solution (30 mL). Then the solution was filtered and dried under vacuum. Next, the crude residue was subjected to silica gel chromatography (pentane / siRNA, 3:1), and 2-((fluoromethyl)sulfonyl)pyridine was obtained as a colorless solid. It was obtained using this method. Yield: 55% (2 or more stages). C6H6FNO2S (175.1 g / mol).

[0406] 2-((fluoroiodomethyl)sulfonyl)pyridine In a 100 mL pear-shaped Schlenk tube, 2-((fluoromethyl fluoride) in 10 mL of degassed anhydrous DMF under nitrogen. (0.5 g, 2.9 mmol) and iodine crystals (1.46 g, 11.5 mmol, 4.0 equiv) were added. To this mixture, tBuOK (1.1 g, 10 mmol, 3.5 equiv) in DMF (10 mL) was added at 5°C. The reaction mixture was warmed to room temperature, and when complete consumption of the starting materials was observed, it was quenched with saturated ammonium chloride aqueous solution (10 mL). The product was then converted to SiO2 (3 × 20 mL). The sample was extracted and stirred with aqueous NaHSO3 (10g in 100mL of distilled water). 19 Using F NMR, diiod The complete conversion rate of the chemical product was determined (approximately 10 hours). Next, the organic phase was 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 (HCl / pentane, 1:3). The mixture was then subjected to a specific method to obtain 2-((fluoroiodomethyl)sulfonyl)pyridine as a white solid in a yield of 62%. 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 in THF, 24 mmol) was stored. The 1.4 equiv of ((fluoromethyl)sulfonyl)pyridine (3.0 g, 17.1 mmol, 1.0 equiv) and methyl 4-methoxybenzoate (4.3 g, 25.7 mmol, 1.5 equiv) solution in 50 mL of THF were added. 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 SiO (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 in vacuum. Next, the crude product was siliceous gelatinized. Purification by chromatography (siRNA / pentane, 1:3) yielded 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, under nitrogen, 2-fluoro-1-(4-methoxyphenyl phosphate in DMF (5 mL) (154 mg, 0.5 mmol, 1.0 equiv) and NCS (89 mg, 0.66 mmol, 1.3 equiv) were added. 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 SiO2 (2 x 100 mL), followed by washing with distilled H2O (100 mL) and saline solution (100 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. Next, the crude product was saturated. The title compound was purified by gel chromatography (SiO₂ / pentane, 1:3) and removed. 70% was obtained as color oil. C6H5ClFNO2S (209.6 g / mol).

[0409] py-SOOF Biotin Under argon, the dissolution 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. NEt3 (29 μL) was added to the solution. The reaction mixture was stirred overnight with rt and left to stand, then concentrated. The solution was purified by flash column chromatography (CHCl3 / MeOH 0-10% gradient elution). The product described in the title was then obtained 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 SiO2 (3 × 100 mL), and the combined organic matter was washed with saline solution (100 mL), dried, and (MgSO4). The oil was filtered and concentrated in a vacuum. The crude yellow oil was then used without further refinement. The crude oil described above was dissolved in CH2Cl2 (30 mL) and cooled to 0°C. Then, mCPBA (4.5 g, 20 mmol) was added in small amounts. Next, the mixture was warmed to room temperature and stirred for 3 hours, then 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 A solution of sulfone (526 mg, 2 mmol) and NFSI (1.58 g, 5 mmol) in THF (80 mL) is mixed with -78 At °C, a solution of NaHMDS (4.4 mL, 4.4 mmol, 1 M) in THF was added. 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 layers were 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 light, 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 HCl and water, the aqueous phase was extracted three times with 20 mL of HCl, dried over MgSO4, and concentrated under vacuum. Half of the crude reaction product was purified by flash chromatography on silica using up to 8% hexane / HCl. 1.34 g of pure A remarkable 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 with MgSO4, filtered through a silica plug, and evaporated to dryness. This yielded 556 mg of a brownish solid. C 12 H 11 NO2S (233.3 g / mol).

[0414] 2-((difluoro(phenyl)methyl)sulfonyl)pyridine A solution of sulfone (526 mg, 2 mmol) and NFSI (1.58 g, 5 mmol) in THF (80 mL) is mixed with -78 At °C, a solution of NaHMDS (4.4 mL, 4.4 mmol, 1 M) in THF was added. This 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 HCl (2 × 100 mL). Next, the combined organic layers were 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 HCl: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, then diluted with CH2Cl2 (10 mL). The organic layer was then washed with 0.5 M HCl (3 x 10 mL). The product was dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then flash-chromatographed. The guanidine was purified with HCl (3:7 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 In a solution of diBoc-guanidine (above) (35 mg, 0.075 mmol) in CH2Cl2 (1 mL), at 0°C... TFA (0.5 mL) was slowly added. 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. After that, it was concentrated under vacuum to obtain free guanidine. It was obtained as a pale yellow oil as a TFA salt (30.2 mg, quant.). C8H 10 F2N4O2S (464.5 g / mol).

[0417] Catecolo-Ru(bpy)2 Dissolve catechol (30 mg, 0.27 mmol, 1.0 equiv) 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). Insert a Dimroth capacitor (any kind will do), place it under Ar, and recirculate it overnight. The mixture was allowed to cool to room temperature, and ferrocenium hexafluorophosphate (69 mg) was added. 0.27 mmol (1 equiv) was added to ensure complete formation of semiquinone. EtOH was removed, and the complex was precipitated by adding aqueous saturated KPF6. The solid was dried overnight, and 290 mg of very dark red A colored solid was obtained. 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 visible. 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) (Chromacol 300 μL fixed insertion vials, screw cap, Thermo Scientific for <100 μL; and ≥100 μL) A 2 mL CLR RAM VIAL 9MM THD (32009-1232, Novetech) was used for μL. The standard reaction involves mixing the selected Dha-containing protein with the desired reaction buffer in a glove box. The process consisted of combining the components, then sequentially adding catalysts, additives, and chemical substrates from a newly prepared stock in a buffer in a glove box. Most of the reaction optimization and chemical substrate screening reactions were performed in denatured buffer (500 mM NH4OAc, 3 M guanidia chloride, pH 6.0). The reaction was carried out in volumes of 50–200 μL at a final concentration of 1 mg / mL (66 μM) using the model protein substrate Xl histone H3-Dha9 (1 mg / mL). All reagents were first transferred to a glove box (<6 ppm O2) and then prepared with stock solutions for the reaction. All reagents are water-soluble at the final concentration and do not require a co-solvent unless otherwise specified. After thoroughly mixing the reaction mixture with a pipette, the vials 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, ranging from 5–50W (each dialed to intensity 1–10). Up to seven reactions were used simultaneously within the blue LED intensity range (to read the readings). No significant temperature rise was observed for short reaction times (<20 minutes), but for longer reaction times (>20 minutes), the temperature could be controlled by submerging the reaction vials in a glass beaker filled with water at the desired temperature. Irradiation was performed 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. PD SpinTrap G-25 (GE Healthcare) Using a salt column, the absorbance of the entire protein was tracked, and the protein recovery rate was generally above 85%, but this analysis was not performed for all conditions and substrates. The protein did not deteriorate or develop new adducts even when stored in a freezer as a crude reaction mixture for several months. In some cases, it was possible to continue the incomplete reaction simply by degassing the reaction mixture again and continuing the irradiation.

[0419] Example 1 - BACED reaction The reactions according to the methods of embodiments (ii) and (iii) described herein may vary in various ways. Various substituents are used to functionalize exemplary Dha-containing proteins that have functional side chains. And it was proven.

[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. For example, ethyl triflu In many cases, multiple side-chain precursor substrates could be used to yield the same side-chain product, such as potassium oroborate and ethylboronic acid, both of which gave side-chain product 1a. In these cases, all tested conditions that yielded the same side-chain product are described. Various different side chains were introduced for 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 are present and are expressed as a percentage of the total product. When analyzing the intensity of an evolved spectrum, a 10% baseline is generally used. In-cut was used. In some cases, small amounts of methionine oxidation occurred during manufacturing, storage, and use (+16 Da + / - 1 Da). These adducts were used for calculations of the starting materials and products. It was adjusted to the calculation.

[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, histone H3-Dha9 (100 μg, final concentration 1 mg / mL, 66 μM) Add the NH4OAc buffer (500 mM, pH 6, 3 M Gdn-HCl, 90 μL) containing the solution to a glass HPLC vial. Ru(bpy)3Cl2 (1 μL of freshly prepared 66 mM stock in water, 10 eq), catechol ( After sequentially adding 1 μL (100 eq) of freshly prepared 660 mM stock in water and potassium ethyltrifluoroborate (10 μL (500 eq) of freshly prepared 330 mM stock in buffer), The aluminum was sealed with a cap, removed from the glove compartment, and irradiated with blue LED light (50W) for 20 minutes. The conversion rate was determined by analyzing a certain portion 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 Pre-weighed amounts of Ru(bpm)3Cl2 (1.8 mg, 2.8 μmol) and catechol (3.1 mg, 28 μmol) , and a glass vial containing 4-bromobutylboronic acid (76 mg, 420 μmol) glove The sample was transferred to a 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) was added. After solubilizing the reagent with a brief missing step, the vial was sealed with a cap, 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 was recovered by nanodropping (94%). The %) was measured. 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. Inside the glove box, a glass HPLC vial containing AcrA-Dha123 (4 μM final concentration) A phosphate buffer (20 mM NaPi, 100 mM NaF, pH 7.4, 95 μL) was added. Ru(bpy)3Cl2 (4 mM stock freshly prepared in 1 μL of water, 10 eq), catechol (in 1 μL of water) After successively adding a newly prepared 20 mM stock (50 eq) and potassium phenethyltrifluoroborate (a newly prepared 40 mM stock in 5 μL of buffer, 500 eq), the via Seal the bottle with the cap, remove it from the glove compartment, and shine a blue LED light (50 W) on it for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture using LC-MS. After the reaction, The sample is desalted in the same buffer (PD Minitrap G25) to remove excess reagents, and related Analysis was performed using circular dichroism along with 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 (freshly prepared 40 mM stock in 1 μL of water, 10 eq) and catechol (1 μL) were added. After successively adding a freshly prepared 200 mM stock (50 eq) in water and potassium phenethyltrifluoroborate (a freshly prepared 400 mM stock (500 eq) in 5 μL of buffer), Seal the vial with the cap, take it out of the glove box, and shine a blue LED light (50 W) for 15 minutes. The mixture was irradiated for several minutes. The conversion rate was determined by analyzing a fixed portion of the crude mixture using LC-MS. After the reaction, the sample is desalted in the same buffer (PD Minitrap G25) to remove excess reagent. The analysis was performed using circular dichroism along with related 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 (freshly prepared 4 mM stock in 1 μL of water, 10 eq), catechol (in 1 μL of water) After successively adding a newly prepared 20 mM stock (50 eq) and potassium phenethyltrifluoroborate (a newly prepared 40 mM stock in 5 μL of buffer, 500 eq), the via Seal the bottle with the cap, remove it from the glove compartment, and shine a blue LED light (50 W) on it for 15 minutes. The conversion rate was determined by analyzing a fixed portion of the crude mixture using 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 can be varied To functionalize exemplary Dha-containing proteins having different functional side chains, various configurations are used. This was demonstrated using a conversion group. A pySOOF reaction manifold was used as the exemplary ASOOF portion according to embodiment (i).

[0443] All side chains introduced using the pySOOF reaction manifold (2a-2ag, see Figure 5) were screened with histone H3-Dha9, a model protein substrate. Since the RC(O)CF2Br (embodiment (ib)) radical precursors follow the same mechanistic pathway, this example is derived from their substrates. This includes the surrounding area. 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 after changing the buffer to remove small molecular weight reaction components, the yield was measured using Nanodrop. All reactions were measured by LC-MS. The conversion rate was determined based on the intensity of deconvolution in the LC / MS spectrum. It was calculated as the ratio of product to Dha starting material. In some cases, minor processes such as double addition occurred. Undesirable products are present and expressed as a percentage of the total products. When analyzing the intensity of the deconvoluted spectrum, a 10% baseline is used as a general rule. Cuts were used. In many cases, small amounts of methionine oxidation occurred during manufacturing, storage, and use (+16 Da + / - 1 Da). These adducts were totaled for the calculation of the starting materials and products. They were combined.

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

[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 NH4OAc (500 mM, pH 6, 3M) was added. The final protein concentration was diluted to 1 mg / mL with Gdn·HCl. Difluoromethyl 2-pyridyl After adding rufon (13.2 nmol [0.02M] in DMSO) and Ru(bpy)3Cl2 (16.48 nmol in 2 μL of water), seal the vial with the cap, remove it from the glove box, and shine a blue LED light (50 W) on it for 15 minutes. Irradiation was performed. The conversion rate was determined by analyzing a fixed amount of the crude mixture using 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 the 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 crude mixture was treated with EDTA (8 mg), and the low molecular weight was reduced by buffer exchange using PD midiTrap G25. The pySOOF reaction was also carried out on a large scale for many starting materials using essentially the same method, except for the removal of reagents. The protein concentration was then 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 similar, except that beta-mercaptoethanol was added at a concentration of 80 mM after the reaction. This reaction was carried out on a large scale for many starting materials using essentially the same method. This reaction was observed to have a favorable effect in suppressing excessive methionine oxidation, which is 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 embodiments, the difluorobromolacum of embodiment (ib) described in Figure 6(C) The group -CF2C(O)NH2 was introduced into the protein substrate using a precursor.

[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 solution 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 [0.02M]) 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 illuminated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture using LC-MS. Using this 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 a further embodiment, the iodine-pySOOF radica of embodiment (ia) shown in Figure 6(D) Using a precursor, a monofluorinated pySOOF group was introduced into the 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 the addition of 2-((fluoroiodomethyl)sulfonyl)pyridine (65.9 nmol in DMSO [0.1M]) and Ru(bpy)3Cl2 (33 nmol in 2 μL of water), Seal the vial with the cap, remove it from the glove compartment, and shine a blue LED light (50 W) on it for 15 minutes. Intermittent irradiation was performed. The conversion rate was determined by analyzing a fixed portion of the crude mixture by LC-MS. The functionalized side chains are shown in Figure 5.

[0470] [Table 6]

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

[0472] The scheme in Figure 3(b) shows photocatalytic bromonoleation of boronate radical precursors. This paper outlines the reaction scheme and LC / MS spectra for the introduction of leucine (Bnl) and iodonorleucine (Inl). Stability studies of Bnl and Inl in weakly acidic buffers revealed that the only reaction is the slow halogen exchange of Cl- ions to both I and Br, producing chloronorleucine (Cnl). Both Bnl and Inl exhibit similar reactivity to Cl-. It demonstrated this, and a complete transformation was achieved a few days later. By manipulating the pH or substrate equivalent, unwanted hydroxyl group substitution or elimination side reactions can be avoided, and as described later, CS, CP, and CN bonds can be formed from alkyl halide reactive handles on the protein. Excellent transformations for forming a compound can be obtained.

[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 immediately buffered in phosphate buffer (100 mM NaPi, 3 M Gdn-HCl, pH 6) to test their long-term stability in weakly acidic buffer. Each modified sample (100 μL, 10 μM histone H3-Inl9 or H3-Bnl9) was shaken at 37°C (600 rpm). The mixture was incubated, 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 nearly complete conversion (~90%) to the product histone H3-Cnl9-containing chloronorleucine in nearly the same proportions, 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 immediately exchanged for phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10) after the reaction. 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). Crude reaction mixture A specific amount was taken from the mixture for LC-MS analysis. The analysis was performed on the major product in both cases. And in both cases, H3-Cnl9 formation as a minor product (chlorono by halogen exchange) Complete conversion was observed for both histone H3-Inl9 and H3-Bnl9 by βME substitution (composed of leucoin formation) (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 immediately exchanged for phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10) after the reaction. 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 volume was taken from the crude reaction mixture for LC-MS analysis. Analysis was performed on both samples. H3-Cnl9 formation as the major product in one case and the minor product in both cases (halo TCEP substitution, which consists of chloronorleucine formation via gene exchange, resulted in complete conversion of histone H3-Inl9 and incomplete conversion of H3-Bnl9 (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 immediately exchanged for phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10) after the reaction. 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 shaken (600 rpm) at 37°C for 12 hours. The mixture was incubated. A certain amount was taken from the crude reaction mixture for LC-MS analysis. The analysis revealed the major product and the minor product for the reaction with H3-Bnl9 in both cases. Azide substitution consisting of H3-Cnl9 formation (chloronorleucine formation by halogen exchange) results in a complete conversion for histone H3-Inl9 and an incomplete conversion for H3-Bnl9. This showed a significant shift (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 immediately exchanged for phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10) after the reaction. Methylamine (0.5 M from a 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. The mixture was incubated for 12 hours with shaking (600 rpm). A certain amount was taken from the crude reaction mixture for LC-MS analysis. The analysis consisted of major products in both cases. Methylamine substitution with a suitable amount of byproduct resulted in moderate conversion to the desired modification for both H3-Inl9 and H3-Bnl9. The high pH required for deprotonation of methylamine induced significant competition with the side reactions discussed above and previously (Figure 3b), allowing only nearly molar equivalents of 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 immediately exchanged for phosphate buffer (100 mM NaPi, 3 M Gdn·HCl, pH 10) after the reaction. 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 shaken at 37°C for 1 hour. The mixture was incubated at 600 rpm. A certain amount of the crude reaction mixture was analyzed by LC-MS. Samples were collected for this purpose. 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 a significant amount of by-products. The high pH required to deprotonate dimethylamine induced significant competition with the side reactions discussed above (Figure 3b), and facilitated the addition of methylamine as the major product using nearly molar equivalents of 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 mM Add μL of 10 μM histone H3-Inl9 or H3-Bnl9 to the total reaction volume and incubate the sample at 37°C for 1 The mixture was incubated with shaking (600 rpm) for a set period of time. A fixed amount was taken from the crude reaction mixture for LC-MS analysis. The analysis showed that in both cases, the major product consisted of trimethylamine substitution with the formation of H3-Cnl9, which is present in the H3-Bnl9 reaction, and H3-Inl9 and Both H3-Bnl9 showed excellent conversion to the desired modification (Figure 3b). Trimethylamine Since side reactions are suppressed, it acts as an excellent nucleophile for both H3-Inl9 and H3-Bnl9, and is even more effective than using mono- or dimethylamine substitution reactions.

[0480] As demonstrated in the example above, the flexibility (both structural and reactivity) of the incorporated halogen nucleophile 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, intermolecular nucleophilic substitution at C-Hal bonds could be selectively promoted, while elimination and tanning could be avoided. It was demonstrated that the presumed competitive side reactions of intraprotein nucleophile substitution can be avoided. CS Binding (with thiols, beta-mercaptoethanol, and BME), CP binding (with phosphine TCEP), CN binding (forming various methylamines that form methyllysine PTM and N3-donating Anl) In addition to the generation of (along with) the Finkelstein-type nucleophilic reactivity, it was even possible to further adjust the Finkelstein-type reactivity by the direct exchange of halogens (Br → Cl or I → Cl).

[0481] Example 4 - Radical reaction on protein As shown above, the present invention is used to obtain a radical precursor moiety on a protein (ASOOF Proteins can be functionalized using motifs, etc. Such groups are shown in Figure 3(a). As shown, this enables the functionalization of a wider variety of proteins. The following are examples of functionalization via on-site radical polymerization, reactions with further radical substituents, and protein-protein crosslinking. These are various radical reactions on proteins that can be used to further functionalize proteins or peptides.

[0482] General Protocol 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). 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) were added. Next, seal the vial with the cap, take it out of the glove compartment, and turn on the blue LED light (50 W). The mixture was irradiated for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture using LC-MS. This process 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 and removed from the glove box, and then subjected to blue LED light. The mixture was irradiated with light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture using LC-MS. It was decided.

[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 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 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 and placed in a glove box. The mixture was removed 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 using LC-MS.

[0486] TEMPO implementation 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 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 calculated as follows: The specific amount of the compound was determined by analyzing it using LC-MS.

[0487] Introduction of diphenyldiselenide 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 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... This was determined by analyzing a fixed amount of the crude mixture using 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 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 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 and removed from the glove box. The mixture was irradiated with blue LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture using LC-MS.

[0490] Introduction of vinyl acetate 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 vinyl acetate (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 and removed from the glove box. The mixture was irradiated with color LED light (50 W) for 15 minutes. The conversion rate was determined by analyzing a fixed amount of the crude mixture using LC-MS.

[0491] Introduction of dimethylethylidenemalonate 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 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 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 calculated as follows: The specific amount of the compound was determined by analyzing it using 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 is shown in the reaction scheme below. The reaction was carried out under the same conditions as described above for the radical reaction on the protein, using specific reagents and conditions. The resulting cross-linked protein-protein complexes were confirmed by SDS-Gel electrophoresis. (See Figure 3A).

[0494] Example 5 - KDM4A crosslinking to histone-containing Bhn The above initiation method allows for various halogenation (chloro-, bromo-, and iodo-), potentially electrophilic side chains, and proteins such as proteins with side chain lengths precisely matching Lys. It can be inserted into the quality. This is conventionally a 2e-heterozygous protein-based nucleophile. In addition to containing the part (alkyl halide) that has been used for killation, it also has a 1e-reductive initiation. (See above) The use of reagents that can also act as radical precursors highlights the remarkable chemoselectivity and efficiency of the process of the present invention; here, they were left untouched during the introduction of 1e-radicals by CC bond formation. Aliphatic 4-bromobutyl- Boronic acid (precursor of bromoalkyl side chain bromohomonorleucine, Bhn, (1u)) When evaluated using cyclic voltammetry under call enhancement conditions (1:12), An irreversible oxidation event corresponding to a half-potential Eox = +0.93V was observed. Notably, no reduction peak was observed in response to C-Br activation, and oxidation was not observed in the absence of catechol, further confirming the usefulness of the BACED reagent. Controlling insertion has 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 act appropriately on the bound protein-protein interface (PPI) to "fit snugly". When adapted, alkylation reactivity can be "inducibly" enhanced due to solvent exclusion, effective molar count, and the effectiveness of appropriate mimicry (Figure 4C). Such a system requires a balance between electrophilic reactivity and native shape fidelity, enabling investigations of protein-protein interactions, such as enzyme substrate analysis.

[0495] Site selection of the smallest alkyl halide side chains, such as bromonolleucine (Bnl), bromohomonorleucine (Bhn), or iodonorleucine (Inl), onto proteins. Selective insertion 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 to nucleophilic residues in interacting protein partners (and thus site-specific crosslinking) (Figure 4D). Thus, Bhn, Bnl, or Inl, by having the same simple alkyl side chain, represent nearly direct (unextended) alkyl halide mimics of Lys (Figure 3A), and the possibility of Lys being present in wild-type proteins. This allows for the potential probe of buried protein-protein interfaces 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-constrained) transient (substrate-enzyme) rigorous PPI, bromohomon Leleucine (Bhn, 1u) was introduced as a bromination mimic of Lys at three sites of the human histone isoform H3.1 (C-terminal FLAG-HA tag type, eH3.1) where Lys normally exists (sites 4, 9, and 27). eH3.1-Bhn4, eH3.1-Bhn9, and eH3.1-Bhn27 were fabricated, respectively. These "guided The alkylating agent protein and WT control are representative proteins that process and bind Lys residues. It was incubated with the partner enzyme human histone lys-demethylase KDM4A (N-terminal His-tagged, Figure 4D). Coomassie staining and Western blotting showed KDM4A and Bhn Crosslinking was observed only in mixtures with the contained histone H3 protein, but not with WT histone H3 (Figure 4D). This "inducible" property of crosslinking was confirmed by incubation with control proteins. Any of the Bhn-containing histones were found to be linked to serum albumin (bovine, BSA, known as a Cys-rich control) or known nucleosome-binding partner histones. Neither H4 (which non-covalently forms H3 / H4 dimers and tetramers and is Cys-free) showed evidence of crosslinking, even after extending the time and increasing the temperature (2 hours at 37°C). In particular, H3-H4 PPIs do not involve Lys4, Lys9, or Lys27,64, but only H3·KDM4A PPIs do. BSA or H4 Despite the possibility of nonspecific reactions and / or binding, these reactions are absent. This suggests that crosslinking of the functionalized H3.1-Bhn variant we edited requires an appropriate PPI, similar to that used in KDM4A.

[0496] This apparent PPI-selective reaction 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). Therefore, Bhn, based on the structural similarity of Lys, is nearly direct to Lys in its (non-extended) structural... Due to the advantages of its analogues, Bhn is a representative mimetic of Lys behavior—when "edited" and inserted into the site of the relevant Lys in a protein, it becomes the same protein as Lys—sharing the protein interface without artifacts. In this way, Bhn in the H3-Lys→Bhn "mutant" creates the same "reach" to the same site as the corresponding H3-Lys wild-type protein at a 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 their interaction partners was immobilized onto beads containing anti-HA-flag antibodies to facilitate the capture of interaction partners of eH3.1-Lys9 present in cells. The data was then examined using double-FLAG+HA-tagged histone eH3.1-Bhn9 incubated with human cell (HeLa) nuclear lysates (4 hours, 37°C). Following this capture, several were observed only in samples containing the alkylating protein histone eH3.1-Bhn9 (with a 1u side chain at site 9) by Western blotting (anti-FLAG for selective detection of any eH3.1- adduct species). Different eH3.1 adducts were identified, while wild-type histone eH3.1 and histone-deficient controls showed no such adducts (Figure 4E).

[0499] The inherent reactivity of eH3 protein with high concentrations of low molecular weight molecules; failure of reaction between KDM4A and low concentrations of low molecular weight side-chain reagents; and success of reaction between eH3-Bhn and KDM4A at low levels (nM~μM). From this reaction, we confirmed the origin of this new "effective mole-driven" crosslinking reaction (again, EM > 103). (At that level).

[0500] General protocol for histone eH3-Bhn-KDM4A crosslinking Histone modifying enzyme KDM4A (2 μM) was modified in HEPES buffer (50 mM, pH 7.4). Mix with either eH3.1-Bhn4 / 9 / 27 or WT control (4μM), and heat to the specified temperature and mark. The mixture was incubated for a set time. The crosslinking reaction was quenched by adding 5X Laemmli buffer and analyzed via 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, Loan FG4R, lot number 1981531, dilution 1:1,000), monoclonal anti-polyhydroxyalkali phosphatase (Sigma-Aldrich, catalog number A5588, clone HIS-1, lot number 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 according to the manufacturer's instructions.

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

[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 indicates that KDM4A activity 28 is a Zn(II) chelate small molecule inhibitor. This contrasts sharply with the rapid emission rate of Zn(II) > 1663 nM / min when using ebselen. 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 for this type of reaction has long been considered too low to effectively crosslink protein-protein interactions with 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. This suggests that the protein-protein interaction between one H3 protein and another was strongly enhanced by EM. This indicates the transient H3·H3 dimerization in the presence of KDM4A. It is thought to be due to its existence.

[0505] This includes residues that precisely mimic Bhn, which can capture transient intermediates. To this end, the present invention demonstrates the potential of a method that functionalizes proteins and provides information on new 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 sample (20 μg or human histone eH3.1-WT, human histone eH3.1-Bhn9, Either the histone-free control or the HA epitope tag was immobilized via the HA epitope tag onto anti-HA magnetic beads (Pierce 88836, 50 μL / sample pre-equilibrated with the buffer used for immobilization) in HEPES buffer (50 mM, pH 7.5) at RT for 30 minutes. The material was 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% Tween20 wx5, 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 stained Coomassie blue or α-FLAG antibody. Checks were performed via SDS-PAGE using either a Western blot with a body (histone eH3 samples are tagged with FLAG-HA epitope), and a high MW band corresponding to the mass of histone eH3.1 co-bridged to an unknown interaction partner was detected (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 Quality is the time taken when incubating both histone H3-K18Ac and H3-K18Bz with Sirt2. Course research was made possible, and true Sirt2 activity in both acylated Lys was clearly strengthened by Sirt2. We confirmed that the substrate KAc > KBz selectivity (Figure 4A).

[0511] Furthermore, using the pySOOF reagent, corresponding components such as K[γF2]Ac and K[γF2] side chains 2k and 2f are used. H→F labeled side-chain analogues were also generated. In these systems, the centrally located γ-carbon-F2 label proved effective in enabling in situ reporting of the modification state of these side chains. The change in the identity of the side chain at position 18 of human H3.1 is a protein 19 This could be easily detected using 1H NMR (565 MHz) (which sensitively identifies 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 change site, and therefore probes the modification state (side chain 2f → 2k = δF -98.0 → -99.4, Figure 4B)). Changes in other side chains could be detected similarly. They could be distinguished at different sites within the same protein (for example, 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, available Further diverse ranges of side chains allow this approach to, for example, heteroatom changes (e.g., N → O, "deaza-oxo" variant KOAc, H3.1-K18 → H3.1-KOAc18) This allows for exploration in numerous additional directions, such as monitoring the 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 F-type Not only could we "read" the chemical shift of Gunal, but we could also determine its multiplicity through correlation simulation (Figure 4B). In this way, the γ-F2 label was used to modify the side chain. Simultaneously with repositioning (KAc→K at the Nε site, 5 "lower" side chain bonds), high sensitivity CF2-dias Thanks to teleotopy, we were able to report both stereochemical processing (and therefore selectivity L vs D at the Cα site, and "upper" triple linkages on the side chain). Similarly, this remarkable sensitivity across the entire side chain of residues allows for real-time in situ reporting of enzyme-mediated post-translational modifications of proteins. This enabled a high-quality report. This indicates that Sirt2, an HDAC deacylate (despite its processing of 6 bond-detached modifications), has a mol content of > 14 (ΔΔGφ > 6.6 kJ mol). -1 ) L / D selection It became clear that it exhibits 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 is such that it can detect fall in single proteins. It was applied to monitor the difference between the ding and higher-order assembly states. Thus, using H3-DfeGly9 made it possible to directly monitor the complete stepwise process of histone octamer assembly, even at low submilligram levels, from unfolded H3 monomer → folded H3 monomer → (H3)2·(H4)2 heterotetramer to complete (H3)2·(H4)2·(H2A)2·(H2B)2 heterooctamer.

[0514] 19 Reconstruction of octamers for F-NMR measurements Unfolded histone H3-DfeGly9 19After F-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 buffered in Tris buffer (150 mM NaCl, 10 mM Tris, 1 mM EDTA, 2 mM βME, pH 7.5). - The buffer was replaced. A final 50% D2O buffer was prepared using 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 mixture 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 refolded in refolding buffer (1 L). The solution was dialyzed three times (2 hours each time, overnight). The resulting solution was centrifuged (15000 rpm, 10 minutes, 4°C). The precipitate was pelletized, its concentration was measured (0.5 mg / mL, 1 mL), and it was purified via Size Exclusion (Superdex S75, pre-equilibriumized with 16 / 60 refolding buffer). The mer-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 mixture was filtered into an NMR tube.

[0516] For the reconstruction of the histone H3-DfeGly9-H4-H2A-H2B octamer, all histones are annealed. The solution was dissolved in folding 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 minutes, 4°C) to form a 310 pellet of precipitate, which was purified via size exclusion as described above. Octadiene of H3F-H4-H2A-H2B Fractions containing the substance were collected and their concentrations were measured (Nanodrop, 0.8 mg total), and an internal standard was used. Fluoroethanol (0.1 μL) was added, and the NMR sample was prepared and measured as shown above. After NMR, the octamer was checked for correct folding by SDS-PAGE and CD. Further analysis was conducted.

[0517] Further synthesis examples Further compounds used in the examples were synthesized as follows. The reaction product was 1 H, 13 C oyo Beauty 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) was added at 0°C, followed by iPr2NEt (4.5 mL, 25.95 mmol), 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 H2O (40 mL) over approximately 30 minutes. The mixture was carefully added. The mixture was warmed to room temperature, then quenched with saturated aqueous solution NH4Cl (300 mL). The mixture was then extracted with HCl (3 x 200 mL). The combined organic layers were washed with saturated aqueous solution NaCl (300 mL). The mixture was dried (MgSO4), filtered, and concentrated in a vacuum to obtain the alcohol as a yellow oil. Crude The resulting product is then purified by flash chromatography (3:7, HCl:petroleum ether). The desired difluorosulfone was obtained 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, concentrated under vacuum, and whitened. Mesylate was obtained as a colored 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) and combined. The organic layer was 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 subjected to flash chromatography. -Refined with (7:13, SiO: petroleum ether), the desired sulfone was obtained 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. The mixture was mixed and quenched with saturated aqueous solution NH4Cl (150 mL). The aqueous phase was then extracted with SiO2 (3 x 100 mL), the organic phase was dried (MgSO4), filtered, and concentrated under vacuum. The crude product was then flash-chromatographed. Purified by Graph (5:95, Â:CH2Cl2) to obtain the desired sulfone (containing ~8% diF compound). AMG-2-20-A AMG-3-05 was obtained as a white solid (740 mg, 57% yield from starting alcohol).

[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, then concentrated under vacuum. The residue was reprocessed under the same conditions. Again After concentration, the residue is dissolved in anhydrous MeOH (3 mL), and HCl (4 M, 1 mL) from the dioxane is added. The solution was stirred for 15 minutes and concentrated in a 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] Boc-Ser-OMe (2.68 g, 12 mmol) solution in MeCN (30 mL) was mixed with Boc2O (5.87 g, 26 mmol) at 0°C, followed by DMAP (0.30 g, 2.4 mmol). The solution was stirred and gradually heated to RT over 6 hours. 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. The mixture was then washed with aqueous NaHCO3 (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was then flushed. Purification by sulfonometry (1:19 → 1:4, SiO:PET ether) yielded 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., Hantsch ester (15.5 mg) The vials were filled with 0.06 ml of 0.06 ml of 0.01 eq of photocat 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. Illuminate either the photobox or the small multi-well plate for 5 hours. 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 then poured into saturated aqueous NaCl (200 mL). The aqueous phase was obtained from CH2Cl2 (3 x The mixture was extracted with 150 mL of water, the combined organic layers were dried (Na2SO4), filtered, and concentrated under vacuum to obtain mesylate as a white solid. Crude mesylate solution in MeCN (120 mL) was mixed with mercaptobenzothiazole (3.61 g, 21.58 mmol) and triethylamine (3.47 mL, 24.9 mmol). After 16 hours, TLC revealed the presence of SM. As indicated, excess K2CO3 (4.8 g, 34.8 mmol) was added and the reaction mixture was stirred for a further 72 hours. The reaction mixture was diluted with HCl (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 filtered. It was concentrated in the air to obtain a yellow oil. AMG-3-46

[0535] In 150 mL of CH2Cl2, a solution of crude thioether (13.42 mmol) was dissolved in mCPBA (9.02 g, 40.26 mmol) at 0°C. 77% by weight was added. The mixture was stirred at this temperature for 5 hours, and then a portion of the mCPBA (2.0 g, 8.92%) was added. mmol, 77 wt%) was added, and the reaction mixture 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. A MG-3-54 LRMS(ESI)479.0(M+Na + )

[0536] [ka]

[0537] LiHMDS (2.70 mL, 2.70 mmol, 1 M in THF) was added dropwise to a solution of Bt-sulfone (410 mg, 0.90 mmol) in THF (5 mL) at -78 °C. The solution was stirred at this temperature for 5 minutes, and at this point NFSI (6.75 mL, 2.70 mmol) was added. A solution of 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 (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. The crude product was then 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-ALRMS (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] LiHMDS (2.20 mL, 2.20 mmol, 1 M in THF) was added dropwise to a Bt-sulfone (500 mg, 1.10 mmol) solution in THF (6 mL) at -78 °C. The solution was stirred at this temperature for 5 minutes, and at this point, NFSI (3.57 mL, 1.43 mmol, 0.4 M in THF) solution was added dropwise. The solution was left at this temperature for 50 minutes, and TLC analysis showed that SM was eliminated. The mixture was stirred until the reaction time was indicated. Next, the reaction mixture was heated at -78 °C in saturated aqueous solution NH4Cl (10 mL) and Et2O (5 mL). Quenched with L). Next, the aqueous layer was extracted with Et2O (2 x 15 mL), and then the combined organic layer was saturated with water. Quench with 30 mL of NH4Cl and 30 mL of saturated aqueous NaCl, dry (Na2SO4), filter, and remove under vacuum. The product was concentrated. The crude product was then 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 vacuum-sealed. Inside, it was concentrated 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 solution was cooled to 0°C, quenched with 10% aqueous Na2S2O3 (200 mL), and diluted with CH2Cl2 (200 mL). The organic phase was then converted to saturated aqueous solution. Wash with NaHCO3 (5 x 400 mL) and saturated aqueous NaCl (300 mL), dry (Na2SO4), filter, and vacuum. The solution was concentrated. Next, the crude product was purified by flash chromatography (2:49 → 1:19 SiO: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 solution NH4Cl (15 mL) and Et2O (20 mL). The mixture was then extracted with Et2O (2 x 20 mL), and the combined organic layer was washed with saturated aqueous NH4Cl (30 mL) and saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was then 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 heated at -78 °C with saturated aqueous solution NH4Cl (15 mL) and Et2O (20 mL). The mixture was then quenched. Next, the aqueous layer was 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. The crude product was then 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, then concentrated by co-evaporating excess DCM under a stream of N2 gas. This quantitatively yielded 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 solution NH4Cl (15 mL) and Et2O (20 mL). The aqueous layer was then extracted with Et2O (2 x 20 mL), and the combined organic layer was then saturated with saturated aqueous solution NH4Cl (30 mL). Washed with aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated under vacuum. Next, the crude product was... The desired monobranyl sulfone was obtained as a white solid by purification using flash chromatography (DCM → 3:97 Ã:DCM) (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 Using the protocol shown below and in Figure 7, according to the method of embodiment (iai) described herein Then, the following synthetic amino acids were incorporated into a maltose-binding protein.

[0557] [ka]

[0558] Plasmids containing pyroLys tRNA, tRNA synthetase pair, and maltose-binding protein (MBP) were co-transformed into E. coli BL21(DE3) cells and subsequently plated. Single colonies were used for expression, amino acids were administered at an OD of 0.5, and then expression was induced in cells with an OD of 0.8 using IPTG. Cells were then expressed overnight and harvested.

[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 2-((bromofluoromethicillin to introduce F) The reaction of thiopyridine with halogen exchange (halex) without adducts, followed by the sulfone reagent. A radical precursor compound was provided by oxidation to [a specific compound], see Figure 9.

[0562] The reaction shown in Figure 9A was carried out using the procedure described below. Activity (3.4 GBq dry [ 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 Contains F-labeled compound The crude reaction mixture 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, yielding Et2O (~1.2 mL total volume). It was eluted into a reaction vial containing [the substance]. A certain amount of purification was then performed. 18 Each Et2O solution containing F-sulfone reagent The reaction vials were dispensed so that the initiation activity of the protein labeling reaction was approximately 25-30 MBq. This solution was concentrated under an N2 supply until it dried at rt. Next, the photocatalyst, iron, and DMSO were buffered. The protein solution containing the protein was added under the specified 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 18The 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 dry [ 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 Contains F-labeled compound The crude reaction mixture was cooled and diluted with 4 mL of H2O. Next, the mixture was mixed in a C18 Plus cartridge (Et Filtered through OH (10 mL) and H2O (10 mL) (pre-treated). NaIO4 (52) in H2O (4 mL) (mg, 0.24 mmol) and RuCl 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 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 reagent was eluted from the cartridge and Et2O( The solution was eluted into a reaction vial containing approximately 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 F-labeled mono-BtSOOF (shown in Figure 9B) ) was functionalized.

[0568] [Table 8]

[0569] Protein purification The remaining 18 F-labeled protein reaction solution PD MiniTrap G-25 (prepared with HEPES (100 mM, pH 7.4)) The sample was loaded (for equilibration) and eluted with 800 μL of HEPES buffer. MS analysis showed minimal oxidation and the expected mass of Dha protein (16003 Da). Compared to BtSOOF, mono-BtSOOF has higher molar activity, so the 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 F-shaped sign -Functionalized with 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 protein is not observed by MS. Mono-BTSOOF When used as a fluorinating reagent for human histone H3, under standard conditions (50W, 15 minutes), this does not occur. To reduce the double addition, milder conditions were used here, such as lowering the light output.

[0573] In Figure 9D, neurofilament light chains (NfL Dha) were reacted using the reaction conditions shown in the table below.18 F 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 used. The patient was anesthetized with Ricaine and injected into the lower posterior part of the head (~2 nL, reagent: 10 mM Tris pH 7.5). Note There were four conditions for entry. (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 reduces the possibility of histone toxicity in the event of larval death and also ensures the background reactivity of the reagent without the Dha substrate. It was meant to be acknowledged. (3) Complete reaction conditions. Complete experimental conditions including both Ru(bpy)3Cl2 and BtSOOF-biotin. (As explained in the example above). (4) The entire reaction conditions with blue light irradiation removed. This is because light is a necessary trigger for the reaction. This is to prove 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. They were left for a while. Under all conditions, 5 of the 25 larvae were placed in a separate petri dish to ensure their survival. Observations were conducted. Under all conditions, not a single larva died within two 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 is a residue containing a side chain with an alkene group; The method is: (a) A protein or peptide is saturated with a radical precursor compound in a photoactivated state. The oxidation half-potential (E) measured against a Japanese calomel electrode. ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Irradiate the obtained composition to provide a functionalized protein or peptide with light. Exposure to; including, The radical precursor compound is represented by formula (II) or formula (III) below: 【Chemistry 1】 (In the formula, R is either via the -CFX- group if the compound of formula (II) is used, or via the group of formula (III)) When the compound is used, the group -CH 2 - A mechanism through which proteins or peptides are bound. It is the potential side chain portion; 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 However, independently, halogens and unsubstituted or hydroxy, oxy, halogen, amino, carboxy, C (1-6) Esters and C (1-6) One or more selected from ether C substituted with a group of (1-6) selected from the group consisting of alkyl); and formula (II If the compound of ) is used as a radical precursor, step (a) further involves the protein and This method involves contacting a 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 linker may be an alkyl group, polyethylene glycol and its analogues, saccharides, polysaccharides, polyglycine, polyamide, or a combination of two or more of these groups, where one or more non-adjacent carbon atoms are substituted with groups selected from NH, O, S, -C(O)NH-, or -NHC(O)-. The method according to claim 2, wherein the base L1 is used.

4. 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 but - Hydrogen, C 3-10 Cycloalkyl, aryl, or heteroaryl (wherein the formula, cycloalkyl The aryl and heteroaryl groups are either 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 consists of one or more non-adjacent carbon atoms selected from NH, O, S, -C(O)NH-, or -NHC(O)-. Alkyl groups which may be substituted with the group being treated; polyethylene glycol and its analogues; sugars; polysaccharides; polyglycine; polyamides; 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】 Selected from, preferably, 【Transformation 5】 The method according to claim 5.

7. A protein or pe comprising at least one SOMO acceptor residue as defined in claim 1. A method for functionalizing a butylene at its functional side chain portion, wherein the method is: (a) Protein or peptide, radical precursor compound, Fe(II) source and photoactivation The oxidation half-potential (E) measured against a saturated calomel electrode in this state ox ) to be brought into contact with a photocatalyst whose voltage is +1.2V or less, and (b) Irradiate the obtained composition to provide a functionalized protein or peptide with light. Exposure to; including, The radical precursor compound is a group of the following formula (IV) 【Transformation 6】 (wherein 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 (wherein R d is hydrogen, C 1-6 alkyl, C 3-10 cycloalkyl, heterocyclyl, phenyl, benzyl or heteroaryl (wherein , R d Alkyl, cycloalkyl, heterocyclyl, phenyl, benzyl, and The heteroaryl group is either unsubstituted or contains halogen, hydroxyl, =O, or -NH. 2 , C 1-6 Alcocy C and -NHCOR e (Substituted with one or more substituents selected from); and R e However, hydrogen also is C 1-4 A method in which (representing alkyl) is selected.

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

9. A method according to any one of claims 3 to 6, wherein the functional side chain portion includes a reactive portion as defined in claim 4 to 6, the method further comprises one of the reactive portions A method comprising reacting a peptide or protein to link a functional side chain to a further molecule.

10. The method according to claim 9, wherein the further molecule is a pharmaceutical, sugar, polysaccharide, peptide, protein, vaccine, antibody, nucleic acid, virus, labeled compound, biomolecule, or polymer.

11. The method according to any of the prior claims, wherein the SOMO acceptor residue is dehydroalanine.

12. Group A is phenyl, pyridinyl, pyrimidinyl, benzothiazolyl, or pyrazinyl The method according to any one of claims 1 to 6 and 8 to 11, preferably pyridinyl, pyrimidinyl, or benzothiazolyl.

13. The method according to claim 12, wherein group A is 2-pyridinyl.

14. The method according to any one of claims 1 to 6 and 8 to 11, wherein group X is fluorine.

15. The source of Fe(II) is iron(II) sulfate, FeOTf 2 Fe(ClO) 4 ) 2 FeF 2 or (NH 4 ) 2 Fe(SO 4 ) 2 , preferred For more information, see FeSO 4 7H 2 The method according to any of the prior claims, wherein O.

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

17. Ru(II) photocatalyst, Ru(bpy) 3 Cl 2 Or Ru(bpm) 3 Cl 2 The method according to claim 16.

18. The method according to any one of the preceding claims, wherein the light irradiation is in the region of 300 to 600 nm, preferably 400 to 500 nm, more preferably 430 to 470 nm.

19. The radical precursor compound is the compound of formula (III), and the compound of formula (III) is used in step (a). In this process, the protein or polypeptide is converted to BCH 2 Functionalized boron compounds containing the R moiety, and the following formula (IIIB): 【Chemistry 9】 (In the formula, R, R 1 and j is a categor represented by any one of claims 1 to 4) The following is a description of a product produced in situ by contacting a derivative, according to claims 1 to 6 or 9 to 18. The method described in any one of the items.

20. Formula (IA): 【Chemistry 10】 (wherein X is hydrogen, fluorine, -COOH and -CONH) 2 Selected from, preferably fluorine; R Z However, it is hydrogen or methyl; and A functionalized peptide or protein comprising at least one residue of (where R is defined in any one of claims 2 to 7).

21. R, C 1-6 Haloalkyl, C 1-6 Azidoalkyl or 【Chemistry 11】 The functionalized protein or peptide according to claim 20.

22. The functionalized protein or peptide according to claim 20, wherein the residue of formula (IA) is one of the compounds listed in Examples 2a to 2ag.

23. The functionalized protein according to any one of claims 20 to 22, wherein X is fluorine. Citrate or peptide.

24. Formula (IB): 【Chemistry 12】 (wherein Ry is hydrogen or methyl; Rbac is C 1-6 It is an alkyl group (where the terminal carbon is substituted with at least one halogen). Or, Rbac is the following: 【Chemistry 13】 A functionalized peptide or protein comprising at least one residue of (represented by the formula (where Z is a halogen)).

25. A functionalized protein or peptide according to any one of claims 21 to 24 a method for covalently bonding with a further protein or peptide, wherein the group R or Rbac in the functionalized protein or peptide is C 1-6 It is a haloalkyl and further protein A method comprising a group in which a chlorine or peptide can react with an alkyl halide to form a covalent bond.

26. The method according to claim 25, wherein a functionalized protein or peptide is a substrate for a further protein or peptide, and the halogenated alkyl group is held in the binding pocket of the other protein or peptide in order to bring the halogenated alkyl group close to a group that can react with the halogenated alkyl group.

27. A functionalized protein or peptide according to any one of claims 21 to 23 a method for covalently bonding with a further protein or peptide, wherein the group R in the functionalized protein or peptide is 【Chemistry 14】 (where A is defined in any one of claims 1, 12, and 13), and further protein A method comprising a group in which a substance or peptide can react with a radical species to form a covalent bond.

28. The following equation (II) or (III): 【Chemistry 15】 (In the formula, A, X, R 1 The compound as defined in claims 1 and 12-14, and R as defined in any one of claims 2-6.

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