WO2025013907A1 - Use of spirooxindole oxirane derivative for modifying peptide and/or protein - Google Patents

Abstract

Provided are uses of spirooxindole oxirane derivatives represented by formula (I) for modifying peptides and/or proteins; a method of modifying peptides and/or proteins using the derivatives; agents for modifying peptides and/or proteins comprising the derivatives; peptides and/or proteins linked to the derivatives; pharmaceutical compositions comprising the peptides and/or proteins linked to the derivatives; kits for detecting biomarkers comprising the peptides and/or proteins linked to the derivatives; and the derivatives themselves. The definition of the substituents in the formula (I) are generally described in the specification.

Description

USE OF SPIROOXINDOLE OXIRANE DERIVATIVE FOR MODIFYING PEPTIDE AND/OR PROTEIN

The present disclosure relates to use of a spirooxindole oxirane compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof (hereinafter, they are also referred to collectively as “a spirooxindole oxirane derivative”) for modifying a peptide and/or a protein; a method of modifying a peptide and/or a protein using the spirooxindole oxirane derivative; an agent for modifying a peptide and/or a protein comprising the spirooxindole oxirane derivative; a peptide and/or a protein linked to the spirooxindole oxirane derivative; a pharmaceutical composition comprising the peptide and/or the protein linked to the spirooxindole oxirane derivative; a kit for detecting a biomarker substance comprising the peptide and/or the protein linked to the spirooxindole oxirane derivative; and the spirooxindole oxirane derivative itself.

Background

Chemical modification reactions of proteins and peptides are necessary for the synthesis of protein conjugates and peptide conjugates that are used as therapeutics, diagnostics, and tools for biomedical research (see, for example, NPLs 1-3). Traditional protein modification reactions include, for example, reactions at lysine amino groups with N-hydroxysuccinimide (NHS) ester derivatives (see, for example, NPL 1). Reactions with NHS derivatives often result in the formation of a mixture of products with different lysine positions modified. The NHS ester derivatives are also relatively unstable in aqueous buffers. Thus, the development of methods for selective modification at a single position or only a few positions within a protein is required.

Generally, there are fewer histidine residues than lysine residues on the surface of a protein. Previously, alkyl-substituted epoxide derivatives that are linked with ligands of the target proteins have been used for modification of histidine residue(s) of target proteins (see, for example, NPL 4).

NPL 1: Chemical Reviews, 2015, 115, 2174.
NPL 2: Biochemistry 2017, 56, 3863.
NPL 3: Nature Reviews Chemistry, 2019, 3, 147.
NPL 4: Journal of the American Chemical Society, 2003, 125, 8130.

Summary

However, the above-mentioned alkyl-substituted epoxide derivatives do not react with many proteins under aqueous buffered conditions. The development of reactions at histidine residues with modification molecules without the need of additional reagents or catalysts has lagged behind, and as such further development is required.

It is therefore an object of the present disclosure to provide a new method of modifying various peptides and proteins at fewer positions thereof using a spirooxindole oxirane derivative.

As a result of diligent investigation conducted with the aim of solving the problems set forth above, the inventors have found that a spirooxindole oxirane derivative modifies a peptide and/or a protein at histidine residue(s) and/or at the N-terminal amino group thereof, thereby completing the present disclosure.

Specifically, the present disclosure is as follows:
[1] Use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I) below for modifying a peptide and/or a protein:

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4.
[2] The use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof for modifying a peptide and/or a protein according to [1], wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
[3] The use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof for modifying a peptide and/or a protein according to [1] or [2], wherein R₂ comprises a linker.
[4] The use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof for modifying a peptide and/or a protein according to any one of [1] to [3], wherein the peptide is an antibody.
[5] A method of modifying a peptide and/or a protein
comprising:
contacting the peptide and/or the protein with a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I):

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4.
[6] The method of modifying a peptide and/or a protein according to [5], wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
[7] The method of modifying a peptide and/or a protein according to claim [5] or [6], wherein R₂ comprises a linker.
[8] The method of modifying a peptide and/or a protein according to any one of [5] to [7], wherein the peptide is an antibody.
[9] An agent for modifying a peptide and/or a protein comprising a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I) below for modifying a peptide and/or a protein:

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4.
[10] The agent for modifying a peptide and/or a protein according to [9], wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent
[11] The agent for modifying a peptide and/or a protein according to [9] or [10], wherein R₂ comprises a linker.
[12] The agent for modifying a peptide and/or a protein according to any one of [9] to [11], wherein the peptide is an antibody.
[13] A peptide and/or a protein, wherein one or more histidine residues and/or an N-terminal amino group of the peptide and/or the protein are linked to a substituent represented by formula (VI):

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4; and
* represents a binding site to the histidine residue(s) and/or the N-terminal amino group of the peptide and/or the protein.
[14] The peptide and/or the protein according to [13], wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
[15] The peptide and/or the protein according to [13] or [14], wherein R₂ comprises a linker.
[16] The peptide and/or the protein according to any one of [13] to [15], wherein the peptide is an antibody.
[17] A pharmaceutical composition comprising the peptide and/or the protein according to any one of [13] to [16], and a pharmaceutically acceptable additive.
[18] A kit for detecting at least one selected from the group consisting of an antigen, a hormone, a lipid, an enzyme, and a biomarker substance, comprising the peptide and/or the protein according to any one of [13] to [16].
[19] A compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I):

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4, and
wherein a compound represented by formula (1a):

is excluded.
[20] The compound or the racemate, the enantiomer, the diastereomer, the mixture thereof, or the pharmaceutically acceptable salt thereof according to [19], wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
[21] The compound or the racemate, the enantiomer, the diastereomer, the mixture thereof, or the pharmaceutically acceptable salt thereof according to [19] or [20], wherein R₂ comprises a linker.
[22] The compound or the racemate, the enantiomer, the diastereomer, the mixture thereof, or the pharmaceutically acceptable salt thereof according to any one of [19] to [21], wherein the peptide is an antibody.
(Advantageous Effects)

According to the present disclosure, it is possible to provide a new method of modifying various peptides and proteins at fewer positions thereof using a spirooxindole oxirane derivative.

In the accompanying drawing:
FIG.1: Figure 1 shows PDB structures of the proteins (a)-(k) modified with compound 1b and compound 1c. The positions described are those modified with (i.e. linked to) compound 1b and/or compound 1c. Protein names and their PDB codes are as follows: (a) lysozyme, 1dpx; (b) ubiquitin, 1ubq; (c) insulin, 6o17; (d) a-lactalbumin, 1f6r; (e) b-lactoglobulin, 3npo; (f) ribonuclease A, 1fs3; (g) cytochrome C, 1hrc; (h) myoglobin, 1npg; (i) a-chymotrypsin, 1yph; (j) a-chymotrypsinogen A, 1ex3; and (k) anti-CD20 antibody Fab, 6vja.

DETAILED DESCRIPTION

The presently disclosed techniques will be described in detail below.

< Definitions >
Listed below are definitions of various terms used to describe the present disclosure. These definitions apply to the terms as they are used throughout the specification and the appended claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

The singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, “a peptide” is understood to represent one or more peptides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

The terms “comprise” and “comprising” mean that other elements can also be present in addition to the defined elements presented. The use of “comprise” and “comprising” indicates inclusion rather than limitation.

The terms “consist of” and “consisting of” mean that any elements other than the defined elements are not present.

The term “alkyl” refers to a straight-chain or branched saturated hydrocarbon group. The alkyl preferably has from 1 to 12 carbon atoms (C_1-12 alkyl), more preferably from 1 to 8 carbon atoms (C_1-8 alkyl). Examples of the alkyl include, without limitation, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (e.g., n-hexyl, ethylhexyl), heptyl (e.g., n-heptyl), octyl (e.g., n-octyl), nonyl (e.g., n-nonyl), decyl (e.g., n-decyl), undecyl (e.g., n-undecyl), and dodecyl (e.g., n-dodecyl). Among the above, the alkyl is preferably methyl, ethyl, or propyl.

The term “alkenyl” refers to a straight-chain or branched unsaturated hydrocarbon group having one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). The alkenyl preferably has from 2 to 12 carbon atoms (C_2-12 alkenyl), more preferably from 2 to 8 carbon atoms (C_2-8 alkenyl). Examples of the alkenyl include, without limitation, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, octatrienyl, nonenyl, and decenyl. Among the above, the alkenyl is preferably ethenyl, 1-propenyl, or 1-butenyl.

The term “alkynyl” refers to a straight-chain or branched unsaturated hydrocarbon group having one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C_2-10 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). The alkynyl preferably has from 2 to 12 carbon atoms (C_2-12 alkynyl), more preferably from 2 to 8 carbon atoms (C_1-8 alkynyl). Examples of the alkynyl include, without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, pentynyl, hexynyl, pentynyl, octynyl, nonynyl, and decynyl. Among the above, the alkynyl is preferably ethynyl, 1-propynyl, or 1-butynyl.

The term “aryl” refers to a monocyclic or polycyclic group (e.g., bicyclic or tricyclic) having zero heteroatoms provided in the aromatic ring system. The aryl preferably has 6-20 ring carbon atoms in the aromatic ring system (“C_6-20 aryl”), and more preferably 6-14 ring carbon atoms in the aromatic ring system (“C_6-14 aryl”). Examples of the aryl include phenyl, naphthyl such as 1-naphthyl and 2-naphthyl, and anthracyl.

The term “heteroaryl” refers to a monocyclic or polycyclic group (e.g., bicyclic or tricyclic) having 1, 2, 3, or 4 heteroatoms selected from oxygen, nitrogen, sulfur, the remaining ring atoms being carbon provided in the aromatic ring system. The heteroaryl preferably has 5-20 ring atoms, and more preferably 5-14 ring atoms. Examples of the heteroaryl include, without limitation, furyl, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, pyrazolyl, pyridyl, pyridazinyl, indolyl, quinolyl, and isoquinolyl.

The term “alkoxy” is represented by “R-O- (wherein R is the above alkyl)”. Examples of the alkoxy include, without limitation, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

The term “amide” refers to a group containing a carbonyl linked to nitrogen. The term “carbonyl” refers to a group containing a carbon atom doubly bonded to an oxygen atom, represented by “C=O”. Examples of the amide include, without limitation, -C(=O)NH₂, -C(=O)NHCH₃, -C(=O)N(CH₃)₂, -C(=O)NHCH₂CH₃ and -C(=O)N(CH₂CH₃)₂.

The term “imide” refers to a group containing two carbonyl groups attached to a nitrogen atom. Examples of the imide include, without limitation, -CH₂C(=O)NHC(=O)CH₃, -CH₂C(=O)N(CH₃)C(=O)CH₃ and -C₂H₄C(=O)N(CH₃)C(=O)CH₃.

The term “ester” refers to a group wherein a carbonyl is adjacent to an ether linkage, represented by “-COOR (wherein R is the above alkyl)”.

The term “carbamate” refers to a group having a structure “-O(CO)NH₂”.

The term “cyano” refers to the -C≡N group.

The term “azide” refers to a -N₃ group.

The term “halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

The term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. Examples of a substitute include, without limitation, the above alkyl, the above aryl, the above heteroaryl, the above halogen, and a hydroxyl.

The term “functional label” refers to any detectable compound (or functional group), composition or particle that binds directly or indirectly to a molecule. The functional label may be detected using conventional methods such as photoluminescence, fluorescence, chemiluminescence, electrochemistry, mass spectrometry, chromatography, spectroscopy, colorimetry, radiography, microscopy, the enzyme-linked immunosorbent assay (ELISA) and the avidin-biotin complex (ABC) method. The detection of the detectable moiety may be achieved in the same way. Examples of the functional label include, without limitation, luminescent molecules, chemiluminescent molecules, fluorescent molecules (fluorophores), fluorescent quenchers, colored molecules, radioisotopes, optionally substituted PEG, biotin, avidin, streptavidin, Flag tags, myc tags, heavy metals, and enzymes (such as alkaline phosphatase, peroxidase, and luciferase). In case where a functional label is included in a substituent of a compound (for example, it is described that R₂ comprises a functional label), the term “functional label” means a functional label residue.

The term “biotin” refers to the compound biotin itself and analogues, derivatives and variants thereof. Thus, the term “biotin” includes biotin (cis-hexahydro-2-oxo-lH-thieno [3,4]imidazole-4-pentanoic acid) and any analogues, derivatives and variants thereof, including biotin-like compounds. Such compounds include, for example, biotin-e-N-lysine, biocytin hydrazide, amino or sulfhydryl derivatives of 2-iminobiotin and biotinyl-E-aminocaproic acid-N-hydroxysuccinimide ester, sulfosuccinimideiminobiotin, biotinbromoacetylhydrazide, p-diazobenzoyl biocytin, 3-(N- maleimidopropionyl)biocytin, desthiobiotin, and the like. The term “biotin” also comprises biotin variants that can specifically bind to one or more of a Rhizavidin, avidin, streptavidin, tamavidin moiety, or other avidin-like peptides.

The term “nanoparticle” refers to any particles having diameters in the nano size range, i.e., having diameters less than 1 micron.

The term “PEG” refers to a straight-chain or branched polyethylene glycol molecule and includes PEG derivatives such as PEG-amine, PEG-carboxyl, PEG-thiol, and PEG-azide. The PEG preferably has a molecular weight of 500 - 40,000 Da. The term “substituted PEG” refers to a PEG, wherein one of the terminal hydroxyl group is substituted with a substituent. In such a case where PEG is included in a substituent of a compound (for example, it is described that R₂ comprises a PEG), the term “PEG” means a PEG residue (for example, the remaining portion of PEG minus one terminal -OH group) and thus, it means that the compound is PEGylated or that the compound is a PEG conjugate.

The terms “protein” and “polypeptide” can be used interchangeably to refer to a polymer of amino acid residues linked by peptide bonds, and for the purposes of the present disclosure, have a typical minimum length of at least 25 amino acids. The term “protein” and “polypeptide” can encompass a multimeric protein, for example, a protein containing more than one domain or subunit.
The term “peptide” refers to a sequence of peptide bond-linked amino acids containing less than 25 amino acids, for example, between 2 amino acids and 25 amino acids in length.
Proteins and peptides can be composed of linearly arranged amino acids linked by peptide bonds, whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non-naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof greater than 25 amino acids are encompassed by the definition of protein. The terms also include polypeptides that have co- translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide -bond formation, glycosylation, acetylation, phosphorylation, lipidation, proteolytic cleavage (e.g., cleavage by metalloproteases), and the like. Furthermore, as used herein, a “polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate such as through site-directed mutagenesis. They can also be accidental such as through mutations of hosts that produce the proteins. Additionally errors due to PCR amplification or other recombinant DNA methods may occur.

The term “antigen” refers to any substance that prompts an immune response directed against the substance. An antigen can be a peptide, a polypeptide, a chemical or a moiety such as a carbohydrate.

The term “antibody” refers to any molecule that contains an antigen-binding site that binds immunospecifically to an antigen. Thus, the term antibody encompasses not only the entire antibody molecule but also antibody fragments or derivatives. Examples of antibody fragments include, without limitation, Fc, Fv, Fab, F(ab’)₂, Fab’, dsFv, scFv, sc(Fv)₂, and dual specificity antibodies.

The term “hormone” refers to any molecule which acts as a biochemical messenger that regulates physiological events in living organisms, and includes growth factors and cytokines.

The term “lipid” refers to any molecule that is insoluble in water but is soluble in an organic solvent. Examples of the lipid include, without limitation, fatty acids, triglycerides, phospholipids, cholesterol, steroids, and glycolipids.

The term “enzyme” refers to any protein capable of producing changes in a biological substance by catalytic action. Examples of the enzyme include, without limitation, kinases, dehydrogenases, oxidoreductases, GTPases, carboxyltransferases, acyltransferases, decarboxylases, transaminases, racemases, methyltransferases, formyltransferases, and alpha-ketodecarboxylases.

The term “virus” refers to whole virus also identified as virus particles and viral vector as well as virus like particles (VLPs) and viral proteins unless otherwise identified.

The term “biomarker substance” refers to any predictor that can be a characteristic indicator of a biological process, a biological event, and/or a pathological condition. The term “biomarker” encompasses both clinical and biological markers. A biomarker substance can be an antigen, a hormone, a lipid, an enzyme, a protein such as a membrane-binding protein, a metabolic product, or a nucleic acid (fragment) such as free DNA in blood.

The term “pharmaceutically acceptable” refers to an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use.
The term “pharmaceutically acceptable salts” refers to salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.
The term “pharmaceutically acceptable acid addition salt” refers to those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.
The term “pharmaceutically acceptable base addition salt” refers to those pharmaceutically acceptable salts formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, and polyamine resins.
The terms “pharmaceutically acceptable additive” refers to any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, excipients or lubricants used in formulating pharmaceutical products.

The term “pharmaceutically active compound” refers to any bioactive compound intended for administration to a mammal to prevent or treat a disease, a disorder or other undesirable condition.

The term “moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

The term “linker” refers to any moiety that connects two parts of a compound.

The terms “treat” or “treating” refer to recovery, amelioration, relaxation and/or delaying the progression of clinical symptoms of diseases or disorders.

< Use of spirooxindole oxirane derivatives >
The present disclosure is related to use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I) below for modifying a peptide and/or a protein:

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4.
Preferred ranges and examples of R₁ and R₂ in the formula (I) will be described in < Spirooxindole oxirane derivatives > below.

Spirooxindole oxirane derivatives have been used in the chemical synthesis of oxindole derivatives (see, for example, Advanced Synthesis & Catalysis 2016, 358, 172.; Organic Chemistry Frontiers, 2020, 7, 862.; and Green chemistry. 2017, 19, 2107.3). The use of a spirooxindole oxirane derivative for modifying a peptide and/or a protein is the first approach that the inventors have found. The epoxy ring on the spirooxindole seems to be key to good reactivity with a peptide and/or a protein.

A peptide and/or a protein is modified with a spirooxindole oxirane derivative represented by formula (I) as shown in Scheme 1.
Scheme 1:

A peptide and/or a protein is modified at one or more histidine residues and/or an N-terminal amino group thereof with a spirooxindole oxirane derivative represented by formula (I) without altering the catalytic site of the peptide or the protein as opposed to conventional methods such as NHS ester derivatives, which are known to modify multiple lysine amino groups. There are fewer histidine residues on the surface of a protein or peptide than lysine residues and there is only a single N-terminal amino group per polypeptide chain. Thus, reactions at histidine residues and/or at the N-terminal amino group result in more selective modification than conventional reactions with lysine residues.

The histidine residues located on or closer to the surface of a protein or peptide are more likely to be modified with the spirooxindole oxirane derivative. The number of histidine residues to be modified with the spirooxindole oxirane derivative may be from 1 to 3, 1 to 2, or only one per peptide or protein.

An LC-MS analysis can be used to determine the yields of modification products and a mass-mass analysis can be used to determine the modified position(s). More specifically, the LC-MS analysis and the mass-mass analysis can be conducted as described in the EXAMPLES section.

A peptide to be modified with the spirooxindole oxirane derivative is not particularly limited. The peptide may have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. The length of the peptide is preferably 3-20 amino acids, more preferably 4-17 amino acids, and even more preferably 5-10 amino acids.
A protein to be modified with the spirooxindole oxirane derivative is not particularly limited. Specific examples of the protein include, without limitation, lysozyme, ubiquitin, insulin, a-lactalbumin, b-lactoglobulin, ribonuclease A, cytochrome C, myoglobin, a-chymotrypsin, a-chymotrypsinogen A, or an antibody such as an anti-CD20 antibody Fab fragment.

< Method of modifying peptides and/or proteins >
The present disclosure is related to a method of modifying a peptide and/or a protein comprising contacting the peptide and/or the protein with a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I):

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4.
Preferred ranges and examples of R₁ and R₂ in the formula (I) will be described in < Spirooxindole oxirane derivatives > below.

A peptide and/or a protein is modified by the modification method as shown in Scheme 1.
Scheme 1:

The method of modifying a peptide and/or a protein using a spirooxindole oxirane derivative represented by formula (I) is stable in aqueous buffers and can be conducted without the need of special reagents or catalysts. Therefore, the method of modifying a peptide and/or a protein using the spirooxindole oxirane derivative of the present disclosure is simple and can be used for modifying a wider variety of peptides and proteins compared to conventional methods using alkyl-substituted epoxide derivatives that are linked with ligands of the target proteins.

In this modification method, a peptide and/or a protein is contacted with the spirooxindole oxirane derivative in an aqueous buffer to modify the peptide and/or the protein. In other words, the spirooxindole oxirane derivative and the peptide and/or the protein become linked. As the aqueous buffer, any conventionally used buffer for modifying a peptide and/or a protein such as DMF/Tris-HCl buffer can be used. The pH of the reaction solution should be set at between 1 and 11, preferably between 5 and 9, and more preferably 7.5. The reaction temperature should be kept between 25 °C and 50 °C, preferably between 30 °C and 40 °C. The reaction time should be sufficient to complete the reaction which may depend on scale, and may range from 10 to 50 hours, preferably 20 to 30 hours.

After the modification reaction, the products are preferably purified. The purification method is not particularly limited and a conventionally known method can be used.

The method of modifying a peptide and/or a protein using the spirooxindole oxirane derivative can, more specifically, be conducted by a method described in the EXAMPLES section.

A peptide and/or a protein is modified at one or more histidine residues and/or an N-terminal amino group thereof by the modifying method without altering the catalytic site of the peptide or the protein. This is preferable to conventional methods which use NHS ester derivatives as they are known to modify multiple lysine amino groups. As mentioned above, there are fewer histidine residues on the surface of a protein or peptide than lysine residues and there is only a single N-terminal amino group per polypeptide chain. Thus, reactions at histidine residues and/or at the N-terminal amino group result in more selective modification than conventional reactions with lysine residues.

The histidine residues located on or closer to the surface of a protein or peptide are more likely to be modified by the modifying method. The number of histidine residues to be modified by the modifying method may be from 1 to 3, 1 to 2, or only one per peptide or protein.

An LC-MS analysis can be used to determine the yields of modification products and a mass-mass analysis can be used to determine the modified position(s). More specifically, the LC-MS analysis and the mass-mass analysis can be conducted as described in the EXAMPLES section.

Specific examples of a peptide and/or a protein to be modified by the modifying method may be the same as those listed above.

< Agent for modifying peptides and/or proteins >
The present disclosure is related to an agent for modifying a peptide and/or a protein, comprising a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I) below:

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4.
Preferred ranges and examples of R₁ and R₂ in the formula (I) will be described in < Spirooxindole oxirane derivatives > below.

A peptide and/or a protein is modified using a modifying agent comprising a spirooxindole oxirane derivative represented by formula (I) as shown in Scheme 1.
Scheme 1:

In addition to a spirooxindole oxirane derivative represented by formula (I), the modifying agent may comprise a conventional additive used in the art for the preparation of modifying agents.

The reaction of modifying a peptide and/or a protein using a modifying agent comprising a spirooxindole oxirane derivative represented by formula (I) proceeds steadily in aqueous buffers without the need of special reagents or catalysts. Therefore, the modifying agent can be used for modifying a wider variety of peptides and proteins compared to alkyl-substituted epoxide derivatives that are linked with ligands of the target proteins.

A peptide and/or a protein is modified at one or more histidine residues and/or an N-terminal amino group thereof with the modifying agent without altering the catalytic site of the peptide or the protein. This is in contrast to conventional methods such as NHS ester derivatives, which are known to modify multiple lysine amino groups. As mentioned above, there are fewer histidine residues on the surface of a protein or peptide than lysine residues and there is only a single N-terminal amino group per polypeptide chain. Thus, reactions at histidine residues and/or at the N-terminal amino group result in more selective modification than conventional reactions with lysine residues.

The histidine residues located on or closer to the surface of a protein or peptide are more likely to be modified by the modifying agent. The number of histidine residues to be modified by the modifying agent may be from 1 to 3, 1 to 2, or only one per peptide or protein.

An LC-MS analysis can be used to determine the yields of modification products and a mass-mass analysis can be used to determine the modified position(s). More specifically, the LC-MS analysis and the mass-mass analysis can be conducted as described in the EXAMPLES section.

Specific examples of a peptide and/or a protein to be modified by the modifying method may be the same as those listed above.

< Spirooxindole oxirane derivatives >
As mentioned above, a spirooxindole oxirane derivative, which is used for modifying a peptide and/or a protein, may be compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I).

The subscript “m” is an integer from 0 to 4, preferably an integer from 0 to 2, and more preferably 0.

In case where “m” is an integer from 1 to 4, R₁ is each independently, a halogen atom, a hydroxyl group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group.

The alkyl group, the alkenyl group, the alkynyl group, the alkoxy group, the ester group, the amide group, the imide group, the carbamate group, the cyano group, the aryl group, and the heteroaryl group may be substituted with one or more selected from the group consisting of an alkyl, an aryl, a heteroaryl, a halogen atom, a hydroxyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, and a cyano group, and preferably the group consisting of an alkyl, an aryl, and a heteroaryl.

R₁ may be preferably an alkoxy group, more preferably a methoxy group. In some embodiments, the alkoxy group contributes to increase the reactivity of a spirooxindole derivative with a peptide and/or a protein.

R₂ is a moiety comprising a functional label or a pharmaceutically active compound. R₂ may be a functional label or a pharmaceutically active compound. R₂ may also comprise a linker that connects the nitrogen atom of the lactam amide of the spirooxindole oxirane derivative with, for example, a functional label or a pharmaceutically active compound. Accordingly, R₂ may be a moiety represented by -(X)-R³, wherein X is a single bond or a linker and R³ is a functional label or a pharmaceutically active compound. The linking of the nitrogen atom of the lactam amide of the spirooxindole oxirane derivative to a functional label or a pharmaceutically active compound can be achieved using conventional methods, including click chemistry, such as azide-alkyne cycloaddition and thiol-ene reactions.

Examples of a functional label include, without limitation, an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, and a fluorophore-substituted PEG.
The pharmaceutically active compound may be drugs and precursors, congeners, salts, complexes, analogs, and derivatives of said drugs. Examples of drugs include, without limitation, cancer-treating agents, immune disease-treating agents, autoimmune disease-treating agents, infectious disease-treating agents, inflammatory disease-treating agents. In some embodiments, cancer-treating agents include calicheamicin, monomethyl auristatin E, emtansine, exatecan, SN-38 and monomethyl auristatin F.

The linker can be appropriately determined depending on the functional label, the pharmaceutically active compound or another compound to be connected. Specific examples of the linker include, without limitation, a PEG, an alkyl chain (e.g., propyl, hexyl, dodecyl), an amino acid or peptide (e.g., Glycine, Gly-Gly, Gly-Gly-Gly), a disulfide linker, ethylene glycol, diethylene glycol, triethylene glycol, an alkyne linker, an azide linker, and an amide linker. Further examples of the linker include click adducts (e.g., a triazole moiety) that may be connected to a linker as described above (e.g. a PEG, and an alkyl chain), wherein the linker may be connected to a functional label, or a pharmaceutically active compound. PEGs used in the present disclosure include PEG derivatives suitable for connecting the PEG moiety to a functional label, including PEG-amine, PEG-carboxyl, PEG-thiol, and PEG-azide. In some embodiments, a linker represented by formula (II) is a preferred linker in the case where a functional label to be connected is, for example, a biotin or a fluorophore such as 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and dansyl chloride.

In the formula (II), ** represents a binding site bound to, for example, a functional label or a pharmaceutically active compound, and *** represents a binding site bound to the nitrogen atom of the lactam amide of the spirooxindole oxirane derivative.
The subscript “n” is an integer from 1 to 8, preferably an integer from 1 to 4, and more preferably 1 to 2.
In the formula (II), the PEG moiety can be replaced with an alkyl chain (e.g., propyl, hexyl, dodecyl), an amino acid or peptide (e.g., Glycine, Gly-Gly, Gly-Gly-Gly), a disulfide linker, ethylene glycol, diethylene glycol, triethylene glycol, an alkyne linker, an azide linker, and an amide linker.

Specific examples of R₂ include, without limitation, an ethynyl group or a moiety represented by formula (III), (IV) or (V):

In the formula (III), n is 1 or 2.
In other embodiments, a linker may include: Val-Cit, Val-Ala, Gly-Gly-Phe-Gly, Ala-Ala-Asn, glucuronic acid, a pyrophosphate ester, -(CH₂)y- and -(O-CH₂)y- (y is an integer from 1 to 10).

Specific examples of the spirooxindole oxirane derivative represented by the formula (I) includes, without limitation, compounds 1a to 1i.

The compounds represented by formula (I) can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, racemates, optically pure diastereioisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. According to the Cahn-Ingold-Prelog Convention, the asymmetric carbon atom can be of the "R" or "S" configuration.

(Preparation of spirooxindole oxirane derivatives)
The method of synthesizing a spirooxindole oxirane derivative represented by formula (I) is not particularly limited and a conventionally known method can be used.
For example, compound 1a can be synthesized from N-(2-propynyl)isatin by the procedure reported in Organic Chemistry Frontiers, 2020, 7, 862; compounds 1b and 1e can be synthesized from compound 1a by the azide-alkyne cycloaddition reaction procedure reported in Organic Letters, 2010, 12, 4952.

The method of synthesizing a spirooxindole oxirane derivative represented by formula (I) can, more specifically, be conducted by a method described in the EXAMPLES section.

< Modified peptides and/or proteins >
The present disclosure is related to a peptide and/or a protein, wherein one or more histidine residues and/or an N-terminal amino group of the peptide and/or the protein are linked to a substituent represented by formula (VI):

wherein
R₁ is each independently, a halogen atom, a hydroxyl group, and/or
the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
m is an integer from 0 to 4; and
* represents a binding site to the histidine residue(s) and/or the N-terminal amino group of the peptide and/or the protein.

Preferred ranges and examples of R₁ and R₂ may be the same as those listed above for R¹ of the general formula (I).

The peptide and/or the protein linked to the substituent represented by the formula (VI) can be prepared, for example, by the method of modifying a peptide and/or a protein using a spirooxindole oxirane derivative represented by formula (I) as mentioned above.

< Pharmaceutical composition >
The present disclosure is related to a pharmaceutical composition comprising the peptide and/or the protein linked to the substituent represented by formula (VI), and a pharmaceutically acceptable additive.

The pharmaceutically acceptable additive contained in the pharmaceutical composition can be appropriately determined depending on the purpose, use, method of use, etc., of the pharmaceutical composition for delivering the peptide and/or the protein, linked to the substituent represented by the formula (VI), to a target and reacting the peptide and/or the protein with the target. Specific examples of a pharmaceutically acceptable additive include, without limitation, a carrier, an excipient, a stabilizer, and an antioxidant.

A target disease or disorder of the pharmaceutical composition comprising the peptide and/or the protein linked to the substituent represented by the formula (VI) can be appropriately determined depending on the functional label or the pharmaceutically active compound contained in the pharmaceutical composition. Specific examples of a target disease include, without limitation, cancers, immune diseases, autoimmune diseases, infectious diseases, and inflammatory diseases.
The pharmaceutical composition can be used in the manufacture of a medicament for treating of a disease or disorder that can be treated with the peptide and/or the protein linked to the substituent represented by the formula (VI).

< Kit >
The present disclosure is related to a kit for detecting at least one of the following: antigens, hormones, lipids, enzymes, viruses, and other biomarker substances, comprising the peptide and/or the protein linked to the substituent represented by the formula (VI).

The kit can contain any additive in accordance with the purpose, use, method of use, etc., of the kit for delivering the peptide and/or the protein, linked to the substituent represented by the formula (VI), to a target and reacting the peptide and/or the protein with the target.

Specific examples of the purpose of the kit include, without limitation, to sense and diagnose a disease or disorder, or to determine a level of a disease. Specific examples of the disease include, without limitation, cancers, immune diseases, autoimmune diseases, infectious diseases, and inflammatory diseases.

Examples

The following provides more specific descriptions of the present disclosure based on examples.

Example 1 (Reactions of Compound 1a with Short Peptides)
Reactions of compound 1a with short peptides (AIKVF-NH₂, AIRVF-NH₂, LAFKVPEGDF-NH₂, AIHVF-NH₂, and YGGFL-OH) were examined. The results are shown in Table 1.

(Synthesis of Compound 1a)
Compound 1a was synthesized from N-(2-propynyl)isatin as shown in Scheme 2.
Scheme 2:

To a solution of trimethylsulphonium iodide (3.306 g, 16.2 mmol) in CH₃CN (50 mL), Cs₂CO₃ (5.270 g, 16.2 mmol) was added at room temperature (25 °C), and the mixture was stirred at 50 °C under N₂ for 1 h. To the mixture, a solution of N-(2-propynyl)isatin (1.50 g, 8.10 mmol) in CH₃CN (20 mL) was added dropwise over 10 min. The mixture was stirred at 50 °C for 16 h until the N-(2-propynyl)isatin was consumed (monitored by TLC). After being cooled, the mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (hexane/EtOAc = 8:2) to give compound 1a (1.35 g, 84%) as a pale yellow solid.

(Compound 1a)

The ¹H and ¹³C NMR data of compound 1a has been reported, for example, in Advanced Synthesis & Catalysis 2016, 358, 172.

(Procedure of reactions of compound 1a with short peptides)
A solution of short peptides in DMF (1.0 mM, 5 μL) was added to 20 mM Tris HCl buffer, pH 7.5 (90.0 μL) at room temperature (25 °C), and the mixture was vortexed for 10 s. To the mixture, a solution of compound 1a in DMF (50 mM, 5.0 μL) was added at the same temperature, and the mixture was vortexed for 10 s. The resulting mixture contained short peptides (50 μM) and compound 1a (2.5 mM) in 10% DMF/20 mM Tris HCl buffer, pH 7.5. The mixture was constantly shaken at 37 °C for 24 h. The solution (5 μL) was diluted with 0.1% formic acid in water (50 μL) and was used for the LC-MS analysis to determine the yields of the modification products and for the mass-mass analysis to determine the modified positions.
Regarding AIKVF-NH₂, the reactions were performed at different pH values (pH 7.5, 7.0, or 8.0).
The yields of the modified products were determined based on the ratios among the areas of the LC-MS peaks of the unmodified peptides and the modified peptides.

(Conditions for MS analysis of modified peptides)
Mass spectra were recorded via a Thermo Scientific Q Exactive Plus quadrupole-Orbitrap ESI ion trap mass spectrometer coupled with an UltiMate 3000 RSLC nano system.
LC and MS conditions were as follows:
HPLC column: Agilent ZORBAX 300SB-C18 column, particle size 3.5 μm, diameter 0.3mm, length 150 mm (Agilent 5064-8267).
LC settings: elution gradient program A = 0.1% HCOOH/H₂O, program B = 0.1% HCOOH/CH₃CN, column temperature 50 °C, flow rate 3.5 μL/min.

MS settings: bottom-up proteomics, m/z range 350-1500, scanning 17500.
Data analysis: Thermo xcalibur hunter, Proteome discoverer 2.0

Table 1. Reactions of Compound 1a with Short Peptides

mono = mono-modification product(s), di = di-modification product(s)

The structures of the short peptides used are as shown below. The positions enclosed in squares are those modified with (i.e. linked to) compound 1a.

As shown in Table 1, bond formation between the lysine side chain amino group and compound 1a was not detected in the MS/MS analyses (entries 1-3). The reaction of peptide AIKVF-NH₂ with compound 1a in 10% DMF/20 mM Tris-HCl buffer (pH 7.5) at 37 °C for 24 h afforded the mono-modification product in 93% yield based on the mass analysis, and no di-modification product was observed (entry 1). Reactions of other peptides were also performed under the same conditions (entries 5-7). For the histidine-containing peptide, the bond formations were observed at histidine and at the N-terminus (entry 6).

Example 2A (Synthesis of Compound 1b)
Compound 1b was synthesized from compound 1a as shown in Scheme 3 (an azide-alkyne cycloaddition reaction).
Scheme 3:

To a mixture of copper (I) thiophene-2-carboxylate (CuTC, 11 mg, 0.054 mmol) and compound 1a (108 mg, 0.54 mmol) in anhydrous CHCl₃ (7.0 mL), biotin-PEG3-azide (CAS No. 875770-34-6, 240 mg, 0.5 mmol) was added portionwise at 0 °C, and the mixture was stirred at the same temperature for 1 h and at room temperature (25 °C) for 16 h (monitored by TLC). To the mixture, aqueous NH₄Cl solution (20 mL) was added, and the mixture was extracted with EtOAc. Organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by flash column chromatography (neutral silica gel, CHCl₃/MeOH = 85:15) to give compound 1b (282 mg, 81%) as a pale yellow solid.

(Compound 1b)

¹H NMR (500 MHz, CDCl3): δ 7.77 (s, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.12-7.05 (m, 2H), 6.70-6.55 (m, 1H), 6.10 (s, 1H), 5.20 (brs, 1H), 5.11 (d, J = 15.5 Hz, 1H), 5.03 (d, J = 15.5 Hz, 1H) 4.53-4.46 (m, 3H), 4.30 (dd, J = 7.5 Hz, 5.0 Hz, 1H), 3.84 (t, J = 5.0 Hz, 2H), 3.75-3.30 (m, 14H), 3.16-3.10 (m, 1H), 2.89 (dd, J = 13.0 Hz, 5.0 Hz, 1H), 2.72 (d, J = 13.0 Hz, 1H), 2.26-2.13 (m, 2H), 1.80-1.60 (m, 4H), 1.50-1.35 (m, 2H). ¹³C NMR (125 MHz, CDCl₃): δ 173.2, 171.6, 163.5, 143.7, 142.2, 130.6, 124.0, 123.1, 122.3, 122.06, 122.05, 110.4, 70.5, 70.4, 70.0, 69.8, 69.2, 61.7, 60.1, 56.43, 56.42, 55.4, 54.3, 50.3, 40.5, 39.1, 35.84, 35.79, 28.05, 28.01, 25.5. ¹H NMR (500 MHz, CD₃OD): δ 8.03 (s, 1H), 7.38 (td, J = 8.0 Hz, 1.5 Hz, 1H), 7.20-7.16 (m, 2H), 7.11 (td, J = 8.0 Hz, 1.0 Hz, 1H), 5.09 (s, 2H), 4.57-4.53 (m, 2H), 4.47 (dd, J = 8.0 Hz, 5.0 Hz, 1H), 4.28 (dd, J = 8.0 Hz, 4.5 Hz, 1H), 3.88-3.83 (m, 2H), 3.57-3.30 (m, 14H), 3.21-3.15 (m, 1H), 2.90 (ddd, J = 13.0 Hz, 5.0 Hz, 3.0 Hz, 1H), 2.68 (dd, J = 13.0 Hz, 1.5 Hz, 1H), 2.19 (t, J = 7.5 Hz, 2H), 1.76-1.57(m, 6H), 1.49-1.36 (m, 2H). ¹³C NMR (125 MHz, CD₃OD): δ 176.1, 173.6, 166.1, 145.0, 143.5, 131.5, 125.6, 124.4, 124.2, 123.4, 111.2, 71.51, 71.48, 71.39, 71.2, 70.5, 70.3, 63.3, 61.6, 57.4, 57.0, 55.0, 51.5, 41.0, 40.3, 36.7, 36.5, 29.8, 29.5, 26.8. HRMS (ESI): m/z calcd for C₃₀H₄₂N₇O₇S ([M + H]⁺) 644.2861, found 644.2819.

Example 2B (Synthesis of Compound 1c)
Compound 1c was synthesized from compound 1a by the same method used for the synthesis of compound 1b but using biotin-PEG4-azide (CAS No. 1309649-57-7) instead of biotin-PEG3-azide.

To a mixture of copper (I) thiophene-2-carboxylate (CuTC,17 mg, 0.085 mmol) and compound 1a (170 mg, 0.85 mmol) in anhydrous CHCl₃ (8.0 mL), biotin-PEG4-azide (CAS No. 1309649-57-7, 416 mg, 0.85 mmol) was added portionwise at 0 °C, and the mixture was stirred at the same temperature for 1 h and at room temperature (25 °C) for 24 h (monitored by TLC). To the mixture, aqueous NH₄Cl solution (20 mL) was added, and the mixture was extracted with EtOAc. Organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by flash column chromatography (neutral silica gel, CHCl₃/MeOH = 82:18) to give compound 1c (435 mg, 75%) as a pale yellow solid.

(Compound 1c)

¹H NMR (400 MHz, CDCl₃): δ 7.78 (s, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.14-7.03 (m, 2H), 6.71-6.62 (m, 1H), 6.26 (brs, 1H), 5.34 (brs, 1H), 5.12 (d, J = 15.4 Hz, 1H), 5.06 (d, J = 15.4 Hz, 1H), 4.58- 4.45 (m, 1H), 4.38-4.34 (m, 1H), 3.90-3.82 (m, 2H), 3.80-3.35 (m, 18H), 3.20-3.10 (m, 1H), 2.91 (dd, J = 12.6 Hz, 4.0 Hz, 1H), 2.75 (d, J = 12.6 Hz, 1H), 2.30-2.15 (m, 2H), 1.82-1.50 (m, 4H), 1.55-1.35 (m, 2H), 1.26 (t, J = 6.0 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 173.2, 171.5, 163.6, 143.8, 130.6, 124.0, 123.1, 122.4, 122.0, 110.4, 70.53, 70.51, 70.43, 70.40, 70.35, 70.1, 69.9, 69.3, 61.7, 60.1, 56.4, 55.4, 54.3, 50.3, 40.5, 39.1, 35.9, 28.10, 28.06, 25.5. ¹H NMR (500 MHz, CD₃OD): δ 8.04 (s, 1H), 7.37 (td, J = 8.0 Hz, 1.0 Hz, 1H), 7.19-7.16 (m, 2H), 7.10 (td, J = 8.0 Hz, 1.0 Hz, 1H), 5.09 (s, 2H), 4.57-4.53 (m, 2H), 4.47 (dd, J = 8.0 Hz, 4.5 Hz, 1H), 4.29 (dd, J = 8.0 Hz, 4.5 Hz, 1H), 3.87-3.78 (m, 2H), 3.60-3.30 (m, 18H), 3.21-3.15 (m, 1H), 2.90 (ddd, J = 13.0 Hz, 4.5 Hz, 2.0 Hz, 1H), 2.69 (d, J = 13.0 Hz, 1H), 2.20 (t, J = 7.5 Hz, 2H), 1.76-1.52 (m 4H), 1.48-1.36 (m, 2H). ¹³C NMR (125 MHz, CD₃OD): δ 176.1, 173.6, 166.1, 145.0, 143.4, 131.5, 125.7, 124.4, 124.2, 123.4, 111.3, 71.51, 71.46, 71.44, 71.2, 70.5, 70.3, 63.3, 61.6, 57.4, 57.0, 55.0, 51.5, 41.1, 40.3, 36.7, 36.5, 29.8, 29.5, 26.9. HRMS (ESI): m/z calcd for C₃₂H₄₅N₇O₈SNa ([M + Na]⁺) 710.2943, found 710.2946.

Example 2C (Modification Reactions of Proteins with Compound 1b and with Compound 1c)
Compounds 1b and 1c were tested for modification reactions with proteins (lysozyme, ubiquitin, insulin, a-lactalbumin, b-lactoglobulin, ribonuclease A, cytochrome C, myoglobin, a-chymotrypsin, and achymotrypsinogen A) in aqueous buffers. The results are shown in Table 2.

(Procedure of modification reactions of proteins with compound 1b)
A solution of protein (1.0 mM in 20 mM Tris HCl buffer, pH 7.5, freshly prepared, 5.0 μL) was added to 20 mM Tris HCl buffer, pH 7.5 (90.0 μL), followed by compound 1b (50 mM in DMF, 5.0 μL) at room temperature (25 °C), and the mixture was vortexed for 10 s. The mixture contained protein (50 μM) and compound 1b (2.5 mM) in 5% DMF/20 mM Tris HCl buffer, pH 7.5. The mixture was constantly shaken at 37 °C for 24 h. The mixture was applied to PD Spin Trap G-25 gel filtration column device (Cytiva 28918004), which was washed with 20 mM Tris HCl buffer, pH 7.5 before the use, and the protein was recovered as a solution in 20 mM Tris HCl buffer, pH 7.5 (100 μL), according to the instruction of the device provided by the maker.
The solution (4.0 μL) was diluted with 0.1% formic acid in water (80 μL) and was used for the mass analysis to determine the yields of the modification products. Modified sites were determined by mass analyses including MS/MS analysis of trypsin-digested products.

(Procedure of modification reactions of proteins with compound 1c)
The same procedure as the modification with compound 1b was used but using compound 1c instead of compound 1b.

(Procedure of the trypsin digestion of the modified proteins that have cysteine residues)
To HCl buffer, pH 7.5, a solution of 100 mM Tris HCl, pH 7.8 with 6 M urea (20 μL) was added, and the mixture was incubated at 37 °C for 30 min. To the mixture, a solution of 0.2 M DTT in 100 mM Tris HCl, pH 7.8 (3.0 μL) was added, and the mixture was incubated at 37 °C for 1 h. To the mixture, a solution of 0.5 M iodoacetamide in 100 mM Tris HCl, pH 7.8 (4.0 μL) was added, and the mixture was incubated in the dark at 25 °C for 1 h to modify the thiol groups by carbamidomethyl groups. To the mixture, a solution of 0.2 M DTT in 100 mM Tris HCl, pH 7.8 (4.0 μL) was added to quench the unreacted iodoacetamide, and the mixture was incubated at 37 °C for 1 h. The mixture was diluted with water (LC-MS grade ultra-pure water, 130 μL). To the mixture, a suspension of trypsin in the resuspension buffer (Promega, sequencing grade modified trypsin, V5111, lyophilized, 20 μg in 50 mM acetic acid, 50 mM Tris HCl, 1 M CaCl₂, pH 7.6, 200 μL) (8.0 μL) was added, and the mixture was incubated at 37 °C for 24 h. The mixture was acidified with a solution of 1% trifluoroacetic acid/0.5% formic acid in water (300 μL) to adjust the pH to approximately 3 (verified by pH paper). This was applied to extraction disc (Empore C18(FF), 2215) and was eluted with 0.1% formic acid/H₂O-CH₃CN (4:6) (100 μL). This was used for the determination of the modification sites by mass analyses including MS/MS analysis.

(Procedure of the trypsin digestion of the modified proteins that do not have a cysteine residue)
To the modified sample that was purified by the gel filtration column device (50 μL in 20 mM Tris HCl buffer, pH 7.5), a solution of 100 mM Tris HCl, pH 7.8 with 6 M urea (20 μL) and t-BuOH (20 μL) was added, and the mixture was incubated at 37 °C for 30 min. The mixture was diluted with water (LC-MS grade ultra-pure water, 110 μL). To the solution, a suspension of trypsin in the resuspension buffer (Promega, sequencing grade modified trypsin, V5111, lyophilized, 20 μg in 50 mM acetic acid, 50 mM Tris HCl, 1 M CaCl₂, pH 7.6, 200 μL) (8.0 μL) was added, and the mixture was incubated at 37 °C for 24 h. The mixture was acidified with a solution of 1% trifluoroacetic acid/0.5% formic acid in water (300 μL) to adjust the pH to approximately 3 (verified by pH paper). This was applied to an extraction disc (Empore C18(FF), 2215) and was eluted with 0.1% formic acid/H₂O-CH₃CN (4:6) (100 μL). This was used for the determination of the modification sites by MS analyses including MS-MS analysis.

(Conditions for MS analysis of modified peptides)
Mass spectra were recorded using a Thermo Scientific Q Exactive Plus quadrupole-Orbitrap ESI ion trap mass spectrometer coupled with the UltiMate 3000 RSLC nano system.
LC and MS conditions are follows:
HPLC column: Agilent ZORBAX 300SB-C8 column, particle size 3.5 μm, diameter 0.3 mm, length 50 mm (Agilent 5065-4463).
LC settings: elution gradient program A = 0.1% HCOOH/H2O, program B = 0.1% HCOOH/CH₃CN, column temperature 50 °C, flow rate 10 μL/min.

MS settings: m/z range 700-2800, scanning 280000
Data analysis: Xcalibur and BioPharma Finder Software

(Conditions for MS analysis of trypsin-digested samples)
Mass spectra were recorded using a Thermo Scientific Q Exactive Plus quadrupole-Orbitrap ESI ion trap mass spectrometer coupled with a Dionex Ultimate 3000 RSLC nano UPLC system.
LC and MS conditions are follows:
HPLC column: Agilent ZORBAX 300SB-C18, particle size 3.5 μm, diameter 0.3 mm, length 150 mm (Agilent 5064-8267)
LC settings: elution gradient program: A = 0.1% HCOOH/H2O, B = 0.1% HCOOH/CH3CN column, temperature 50 °C, flow rate 3.5 μL/min

MS settings: top-down analysis, m/z range 350-1500, scanning 17500
Data analysis: Proteome Discoverer 2.0

Table 2. Modification Reactions of Proteins with Compound 1b and with Compound 1c

mono = mono-modification product(s), di = di-modification product(s), tri = tri-modification product(s)

As shown in Table 2, in many cases, the reactions resulted in histidine modification. Accessibility and/or reactivity (or pKa) of the histidine residues may determine whether or not reaction occurs. For example, a-lactalbumin has
three histidine residues, and only two were modified (entries 7 and 8). For cytochrome C and myoglobin, only certain histidine residues were modified, and the heme-liganded histidine residues were not modified (entries 13-16). Depending on the protein, the N-terminus and/or lysine side chain amino groups were also modified. For example, in the reaction of a-chymotrypsin, only the N-terminal amino group of chain A was modified (entries 17 and 18). The N-terminal amino groups of chains B and C were not modified, and neither were any histidine or lysine residues including the protease catalytic active site histidine. Ubiquitin has lysine residues critical for ubiquitylation and polyubiquitylation. In the reactions of ubiquitin, a single histidine was modified, and no modification at lysine residues was detected (entries 3 and 4). Notably, in the reaction of b-lactoglobulin, the histidine residues were modified, but the modification of the thiol of a cysteine residue was not detected (entries 9 and 10).

Example 2D (Modification Reactions of an Antibody Fab with Compound 1b and with Compound 1c)
Compounds 1b and 1c were tested for modification reactions with Fab of an anti-CD20 antibody. The results are shown in Table 3.

(anti-CD20 antibody Fab)
The genes of the L and H chains of the Fab of anti-CD20 antibody were chemically synthesized in fragments, assembled by PCR, and inserted into plasmid vectors. Plasmids encoding for the L-chain and the H-chain were cotransfected into 293 cells and the proteins were produced using an ExpiFectamine^TM 293 Transfection Kit (Thermo Fisher Scientific). The Fab proteins were purified with affinity chromatography using standard methods.

(Procedure of modification reaction of the Fab with compound 1b (2.5 mM))
To a solution of the Fab (30 μM in 20 mM Tris HCl buffer, pH 7.5, stored at 4 °C, 19.0 μL), compound 1b (50 mM in DMF, 1.0 μL) was added at room temperature (25 °C), and the resulting mixture was vortexed for 10 s. The resulting mixture contained anti-CD Fab (29 μM) and compound 1b (2.5 mM) in 5% DMF/20 mM Tris HCl buffer, pH 7.5. The mixture was constantly shaken at 37 °C for 24 h. The mixture was applied to an Amicon Ultra-0.5 mL 10K (Ultracel-10, 10,000 MW limit) centrifugal filter device (Millipore UFC5010) and washed with 20 mM Tris HCl buffer, pH 7.5 (3 x 0.5 mL) according to the instruction of the device provided by the maker to remove unreacted compound 1b, and the protein was recovered as a solution in 20 mM Tris HCl buffer, pH 7.5 (30 μL). The solution (1.0 μL) was diluted with 0.1% formic acid in water (70 μL) and was used for the mass analysis to determine the yields of the modification products. Modified sites were determined by mass analyses including MS/MS analysis of trypsin-digested products.

(Procedure of modification reaction of the Fab with compound 1b (1.5 mM))
The same procedure as the reaction with compound 1b (2.5 mM) was employed but using the Fab (30 μM in 20 mM Tris HCl buffer, pH 7.5, stored at 4 °C, 47.5 μL), compound 1b (50 mM in DMF, 1.5 μL), and DMF (1.0 μL) instead of the Fab (30 μM in 20 mM Tris HCl buffer, pH 7.5, stored at 4 °C, 19.0 μL) and compound 1b (50 mM in DMF, 1.0 μL). The resulting mixture contained the Fab (29 μM) and compound 1b (1.47 mM) in 5% DMF/20 mM Tris HCl buffer, pH 7.5.

(Procedure of modification reactions of the Fab with compound 1c)
The reactions were performed by the same procedure as the reactions of the Fab with compound 1b but using compound 1c instead of compound 1b.

(Procedure of determination of the modified positions: the trypsin digestion of the modified proteins)
To the modified sample that was treated by the Amicon Ultra-0.5 mL 10K (Ultracel-10, 10,000 MW limit) centrifugal filter device (26 μL in 20 mM Tris HCl buffer, pH 7.5), a solution of 100 mM Tris HCl, pH 7.8 with 6 M urea (20 μL) was added, and the mixture was incubated at 37 °C for 30 min. To the solution, a solution of 0.2 M DTT in 100 mM Tris HCl pH 7.8 (3.0 μL) was added, and the mixture was incubated at 37 °C for 1 h. To the solution, a solution of 0.5 M iodoacetamide in 100 mM Tris HCl, pH 7.8 (4.0 μL) was added, and the mixture was incubated in the dark at 25 °C for 1 h to modify the thiol groups by carbamidomethyl groups. To the solution, a solution of 0.2 M DTT in 100 mM Tris HCl, pH 7.8 (4.0 μL) was added to quench the unreacted iodoacetamide, and the mixture was incubated at 37 °C for 1 h. The mixture was diluted with water (LC-MS grade ultra-pure water, 155 μL). To the solution, a suspension of trypsin in the resuspension buffer (Promega, sequencing grade modified trypsin, V5111, lyophilized, 20 μg in 50 mM acetic acid, 50 mM Tris HCl, 1 M CaCl₂, pH 7.6, 200 μL) (8.0 μL) was added, and the mixture was incubated at 37 °C for 24 h. The mixture was acidified with a solution of 1% trifluoroacetic acid/0.5% formic acid in water (300 μL) to adjust the pH to approximately 3 (verified by pH paper). This was applied to an extraction disc (Empore C18(FF), 2215) and was eluted with 0.1% formic acid/H₂O-CH₃CN (4:6) (100 μL). This was used for the determination of the modification sites by mass analyses including MS/MS analysis.

(Conditions for MS analysis of modified Fab proteins)
Mass spectra were recorded using a Thermo Scientific Q Exactive Plus quadrupole-Orbitrap ESI ion trap mass spectrometer coupled with UltiMate 3000 RSLC nano system.
LC and MS conditions are follows:
HPLC column: MAbPac capillary reversed phase HPLC column, particle size 4 μm, diameter 0.15 mm, length 150 mm (Thermo Fisher 164947).
LC settings: elution gradient program A = 0.1% HCOOH/H2O, program B = 0.1% HCOOH/(CH3CN/H2O = 80:20), column temperature 70 °C, flow rate 2.0 μL/min.

MS settings: top-down analysis, m/z range 700-2800, scanning 17500.
Data analysis: Xcalibur and BioPharma Finder Software 2.0.

(Conditions for MS analysis of trypsin-digested samples)
Mass spectra were recorded using a Thermo Scientific Q Exactive Plus quadrupole-Orbitrap ESI ion trap mass spectrometer coupled with Dionex Ultimate 3000 RSLC nano UPLC system.
LC and MS conditions are follows:
HPLC column: Agilent ZORBAX 300SB-C18, particle size 3.5 μm, diameter 0.3 mm, length 150 mm (Agilent 5064-8267).
LC settings: elution gradient program: A = 0.1% HCOOH/H₂O, B = 0.1% HCOOH/CH₃CN, column temperature 50 °C, flow rate 3.5 μL/min.

MS settings: bottom-up proteomics, m/z range 350-1500, scanning 17500.
Data analysis: Proteome Discoverer 2.0.

The modification sites observed are shown in bold. All cysteine residues form S-S bonds in the folded structure (see FIG. 1(k)). The CDRs of this Fab defined in WO2013/004806A1 are underlined.
H chain
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHT
L chain
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Table 3. Modification Reactions of an antibody Fab with Compound 1b and with Compound 1c

mono = mono-modification product(s), di = di-modification product(s)

The Fab has four histidine residues in the H chain and three histidine residues in the L chain. The results shown in Table 3 indicate that only certain histidine residues were modified. No lysine or N-terminal modification was detected.

Example 3A (Synthesis of Compound 1f)
Compound 1f was synthesized from 5-methoxy-1-(2-propyn-1-yl)isatin as shown in Scheme 4.
Scheme 4:

To a solution of trimethylsulphonium iodide (1.673 g, 8.20 mmol) in CH₃CN (30 mL), Cs₂CO₃ (2.665 g, 8.20 mmol) was added at room temperature (25 °C), and the mixture was stirred at 50 °C under N₂ for 1 h. To the mixture, a solution of 5-methoxy-1-(2-propyn-1-yl)isatin (0.882 g, 4.10 mmol) in CH₃CN (10 mL) was added dropwise over 10 min. The mixture was stirred at 50 °C for 16 h until the 5-methoxy-1-(2-propyn-1-yl)isatin was consumed (monitored by TLC). After being cooled, the mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (hexane/EtOAc = 8:2) to give compound 1f (0.556 g, 67%) as a pale yellow solid.

(Compound 1f)

Example 3B (Synthesis of Compound 1g)
Compound 1g was synthesized from compound 1f as shown in Scheme 5 (an azide-alkyne cycloaddition reaction).
Scheme 5:

To a mixture of copper (I) thiophene-2-carboxylate (CuTC, 10 mg, 0.048 mmol) and compound 1f (110 mg, 0.48 mmol) in anhydrous CHCl₃ (7.0 mL), biotin-PEG3-azide (CAS No. 875770-34-6, 213 mg, 0.48 mmol) was added portionwise at 0 °C, and the mixture was stirred at the same temperature for 1 h and at room temperature (25 °C) for 16 h (monitored by TLC). To the mixture, aqueous NH₄Cl solution (20 mL) was added, and the mixture was extracted with EtOAc. Organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by flash column chromatography (neutral silica gel, CHCl₃/MeOH = 85:15) to give compound 1g (310 mg, 96%) as a pale yellow solid.

(Compound 1g)

Example 3C (Synthesis of Compound 1h)
Compound 1h was synthesized from 5-methyl-1-(2-propyn-1-yl)isatin as shown in Scheme 6.
Scheme 6:

To a solution of trimethylsulphonium iodide (1.640 g, 8.04 mmol) in CH₃CN (30 mL), Cs₂CO₃ (2.613 g, 8.04 mmol) was added at room temperature (25 °C), and the mixture was stirred at 50 °C under N₂ for 1 h. To the mixture, a solution of 5-methyl-1-(2-propyn-1-yl)isatin (0.800 g, 4.02 mmol) in CH₃CN (10 mL) was added dropwise over 10 min. The mixture was stirred at 50 °C for 16 h until the 5-methyl-1-(2-propyn-1-yl)isatin was consumed (monitored by TLC). After being cooled, the mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (hexane/EtOAc = 85:15) to give compound 1h (0.660 g, 77%) as a pale yellow solid.

(Compound 1h)

Example 3D (Synthesis of Compound 1i)
Compound 1i was synthesized from compound 1h as shown in Scheme 7 (an azide-alkyne cycloaddition reaction).
Scheme 7:

To a mixture of copper (I) thiophene-2-carboxylate (CuTC, 10 mg, 0.046 mmol) and compound 1h (100 mg, 0.46 mmol) in anhydrous CHCl₃ (7.0 mL), biotin-PEG3-azide (CAS No. 875770-34-6, 208 mg, 0.46 mmol) was added portionwise at 0 °C, and the mixture was stirred at the same temperature for 1 h and at room temperature (25 °C) for 16 h (monitored by TLC). To the mixture, aqueous NH₄Cl solution (20 mL) was added, and the mixture was extracted with EtOAc. Organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by flash column chromatography (neutral silica gel, CHCl₃/MeOH = 90:10) to give compound 1i (224 mg, 73%) as a pale yellow solid.

(Compound 1i)

Example 3E (Modification Reactions of Insulin with Compound 1b, 1g, or 1i)
Compounds 1b, 1g and 1i were tested for modification reaction with insulin in aqueous buffers. The results are shown in Tables 4-1 to 4-6.

(Procedure of modification reactions of insulin with compound 1b, 1g, or 1i)
A solution of insulin (1.0 mM in 20 mM Tris HCl buffer, pH 7.5, freshly prepared, 5.0 μL) was added to 20 mM Tris HCl buffer, pH 7.5 (90.0 μL), followed by compound 1b, 1g, or 1i (50 mM in DMF, 5.0 μL) at room temperature (25 °C), and the mixture was vortexed for 10 s. The mixture contained insulin (50 μM) and compound 1b, 1g, or 1i (2.5 mM) in 5% DMF/20 mM Tris HCl buffer, pH 7.5. The mixture was constantly shaken at 37 °C for 24 h. The mixture was applied to PD Spin Trap G-25 gel filtration column device (Cytiva 28918004), which was washed with 20 mM Tris HCl buffer, pH 7.5 before the use, and the protein was recovered as a solution in 20 mM Tris HCl buffer, pH 7.5 (100 μL), according to the instruction of the device provided by the maker.
The solution (4.0 μL) was diluted with 0.1% formic acid in water (80 μL) and was used for the mass analysis to determine the yields of the modification products.

The same reactions but at pH 7.0 instead of pH 7.5 and/or at 25 °C instead of 37 °C for the same or different time lengths were also performed.

(Conditions for MS analysis of modified insulin)
Mass spectra were recorded using a Thermo Scientific Q Exactive Plus quadrupole-Orbitrap ESI ion trap mass spectrometer coupled with the UltiMate 3000 RSLC nano system.
LC and MS conditions are follows:
HPLC column: Agilent ZORBAX 300SB-C8 column, particle size 3.5 μm, diameter 0.3 mm, length 50 mm (Agilent 5065-4463).
LC settings: elution gradient program A = 0.1% HCOOH/H2O, program B = 0.1% HCOOH/CH₃CN, column temperature 50 °C, flow rate 10 μL/min.

MS settings: m/z range 700-2800, scanning 280000
Data analysis: Xcalibur and BioPharma Finder Software

Tables 4-1 to 4-6. Modification Reactions of Insulin with Compound 1b, 1g, or 1i
Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

mono = mono-modification product(s), di = di-modification product(s), tri = tri-modification product(s)

As shown in Tables 4-1 to 4-6, compound 1g (5-methoxy-substituted compound) and compound 1i (methyl-substituted compound) both showed almost the same reactivity as compound 1b.

According to the present disclosure, a new method of modifying various peptides and proteins at fewer positions thereof using a spirooxindole oxirane derivative are provided and the method can contribute to the development of the pharmaceutical field such as antibody-drug conjugate therapies, as well as the development of sensing and diagnostic tools.

Claims (22)

1. Use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I) below for modifying a peptide and/or a protein:
   

   

   wherein
   R₁ is each independently, a halogen atom, a hydroxyl group, and/or
   the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
   R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
   m is an integer from 0 to 4.
   
2. The use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof for modifying a peptide and/or a protein according to claim 1, wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
   
3. The use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof for modifying a peptide and/or a protein according to claim 1, wherein R₂ comprises a linker.
   
4. The use of a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof for modifying a peptide and/or a protein according to claim 1, wherein the protein is an antibody.
   
5. A method of modifying a peptide and/or a protein
   comprising:
   contacting the peptide and/or the protein with a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I):
   

   

   wherein
   R₁ is each independently, a halogen atom, a hydroxyl group, and/or
   the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
   R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
   m is an integer from 0 to 4.
   
6. The method of modifying a peptide and/or a protein according to claim 5, wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
   
7. The method of modifying a peptide and/or a protein according to claim 5, wherein R₂ comprises a linker.
   
8. The method of modifying a peptide and/or a protein according to claim 5, wherein the protein is an antibody.
   
9. An agent for modifying a peptide and/or a protein, comprising a compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I) below:
   

   

   wherein
   R₁ is each independently, a halogen atom, a hydroxyl group, and/or
   the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
   R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
   m is an integer from 0 to 4.
   
10. The agent for modifying a peptide and/or a protein according to claim 9, wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
    
11. The agent for modifying a peptide and/or a protein according to claim 9, wherein R₂ comprises a linker.
    
12. The agent for modifying a peptide and/or a protein according to claim 9, wherein the protein is an antibody.
    
13. A peptide and/or a protein, wherein one or more histidine residues and/or an N-terminal amino group of the peptide and/or the protein are linked to a substituent represented by formula (VI):
    

    

    wherein
    R₁ is each independently, a halogen atom, a hydroxyl group, and/or
    the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
    R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
    m is an integer from 0 to 4; and
    * represents a binding site to the histidine residue(s) and/or the N-terminal amino group of the peptide and/or the protein.
    
14. The peptide and/or the protein according to claim 13, wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
    
15. The peptide and/or the protein according to claim 13, wherein R₂ comprises a linker.
    
16. The peptide and/or the protein according to claim 13, wherein the protein is an antibody.
    
17. A pharmaceutical composition comprising the peptide and/or the protein according to any one of claims 13-16, and a pharmaceutically acceptable additive.
    
18. A kit for detecting at least one selected from the group consisting of an antigen, a hormone, a lipid, an enzyme, a virus, and a biomarker substance, comprising the peptide and/or the protein according to any one of claims 13-16.
    
19. A compound or a racemate, an enantiomer, a diastereomer, a mixture thereof, or a pharmaceutically acceptable salt thereof represented by formula (I):
    

    

    wherein
    R₁ is each independently, a halogen atom, a hydroxyl group, and/or
    the R₁ is each independently an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an ester group, an amide group, an imide group, a carbamate group, a cyano group, an aryl group, or a heteroaryl group, wherein the group(s) are optionally substituted;
    R₂ is a moiety comprising a functional label or a pharmaceutically active compound; and
    m is an integer from 0 to 4, and
    wherein a compound represented by formula (1a):
    

    

    is excluded.
20. The compound or the racemate, the enantiomer, the diastereomer, the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 19, wherein R₂ comprises an optionally substituted alkynyl group, an optionally substituted azido group, an optionally substituted alkenyl group, an optionally substituted thiol group, an optionally substituted cyclooctynyl group, an optionally substituted tetrazinyl group, an optionally substituted trans-cyclooctenyl group, an optionally substituted epoxide group, an optionally substituted succinimidyl ester group, an optionally substituted isocyanate group, a biotin, a fluorophore, a nanoparticle, a PEG, an alkynyl group-substituted PEG, an azide-substituted PEG, a biotin-substituted PEG, a fluorophore-substituted PEG, a cancer-treating agent, an immune disease-treating agent, an autoimmune disease-treating agent, an infectious disease-treating agent, or an inflammatory disease-treating agent.
    
21. The compound or the racemate, the enantiomer, the diastereomer, the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 19, wherein R₂ comprises a linker.
    
22. The compound or the racemate, the enantiomer, the diastereomer, the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 19, wherein the protein is an antibody.

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