Fluorescent Active Free Radical Tags for Simultaneous Glycan Quantification and Characterization

Abstract

Provided are compounds useful for the quantification and / or characterization of glycans and other similar molecules. Such compounds are compounds of formula (Ia) or (Ib) substituted with at least a TEMPO group or an analog thereof and a coupling site, wherein X 1 , X 2 , and X can each be one of NH, N-alkyl, O, NO, C=O, CH, N or CR 9 . Also targeted is a method for quantifying and / or characterizing a glycan, the method comprising the step of obtaining a compound and the step of contacting the compound with the glycan.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 348,795, filed on June 3, 2022, the content of which is hereby incorporated by reference in its entirety into this specification.

[0002] Field of the Invention The present invention relates to fluorescently active free - radical tags comprising substituted compounds of formula (Ia) and formula (Ib):

Chem.

[0003] All published documents, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual published document, patent, patent application, or other reference were individually and specifically indicated to be incorporated by reference for all purposes. The citation of references in this specification should not be construed as an admission that they are prior art to the present invention.

Background Art

[0004] Background of the Invention Research on glycans and related structures, i.e., glycobiology, has provided significant insights and research over the past few decades. Considerable interest has been directed towards the ability to quantify, distinguish, and characterize glycans and their isomers. Unlike other biopolymer molecules, such as peptides and nucleic acids (which involve the linkage of subunits through defined backbones consisting of amide and phosphodiester bonds, respectively), glycans can have monosaccharide subunits arranged in a branched manner through complex regiochemical and stereochemical linkages. Since glycan structures are described differently depending on the type of connection, monosaccharide composition, and overall stereoconfiguration, many structural and stereoisomers can exist. Unlike other biomolecules, glycans are involved in essential functions in biological systems, such as metabolism, signaling, and structure. Moreover, glycans are known to differ in structure and quantity during the onset of many diseases, including but not limited to cancer metastasis, autoimmune diseases, genetic diseases, pathogen-host interactions, immune recognition, and Alzheimer's disease. Therefore, further progress in the field of glycobiology may lead to the future use of glycans as early biomarkers for certain human diseases in the medical field.

[0005] Analysis of glycosylation derived from biopharmaceuticals is also important, which requires analytical techniques that facilitate the characterization of glycans for monitoring and regulation purposes. Glycosylation of biopharmaceuticals is difficult to control because minor differences in conditions such as pH, cell line, dissolved oxygen, temperature, ammonia concentration, and manufacturing mode can affect glycosylation. As a result, glycan characterization techniques are needed because the presence of a particular glycosylation can affect safety, efficacy, half-life, immune response, and binding. For example, the monoclonal antibody drug cetuximab (which targets the epidermal growth factor receptor and inhibits the progression of colorectal cancer) was processed in the presence of gal-α(1→3)-gal, causing anaphylaxis in patients. Ultimately, it is significantly beneficial to rapidly develop improved modern analytical techniques that enable both the quantification and characterization of host-derived glycans, which are causative across numerous industries.

[0006] A number of techniques, including high-performance liquid chromatography (HPLC), ion mobility, electrophoresis, and nuclear magnetic resonance (NMR) spectroscopy, are involved in glycan research. In the structural analysis of glycans, HPLC, ion mobility, and NMR spectroscopy require well-characterized glycan standards, which must be sufficiently pure and are difficult to obtain and time-consuming and costly. In particular, NMR data are difficult to interpret because glycans contain many carbons and protons with similar chemical environments. Nevertheless, the use of HPLC in the quantification of glycans after glycan characterization by mass spectrometry is the most powerful and optimal combination. Electrospray ionization mass spectrometry has a proven track record in multiple dissociation techniques, minimal sample consumption, short acquisition times, high sensitivity, high mass accuracy, and high resolution. However, the ionization efficiency and fragmentation of free glycans by mass spectrometry are relatively insufficient compared to tagged glycans. Therefore, many laboratories have adopted the development and use of tagging reagents to further utilize glycans in analysis. However, these tagging reagents cannot simultaneously provide the ability to quantify and characterize glycans and are accompanied by further challenges such as insufficient chemical stability and limitations in the analysis of single-category glycans. Moreover, certain tagging reagents that mobilize NHS-carbamate groups for rapid tagging functional groups require 133 times the amount of reagent needed to label glycans for the purpose of compensating for the presence of proteinaceous amines. Therefore, there is a strong desire for new tagging reagents for the improvement of glycan analysis. Summary of the Invention Means for Solving the Problems

[0007] Summary of the Invention The present invention provides compounds useful for the quantification and / or characterization of glycans and other similar molecules. Such compounds are compounds of formula (Ia) or (Ib) substituted with at least a TEMPO group or an analog thereof and a coupling site: [Chemical] [wherein, X 1 , X 2 , and X can be one of NH, N-alkyl, O, NO, C=O, CH, N or CR 9 .

[0008] Other such compounds have the following structure: [Chemical] [Chemical] Compounds having wherein R 1 is a glycan labeling site, and R 2 and A can independently be hydrogen or a substituent.

[0009] Also, the present invention is directed to a method for quantifying and / or characterizing a glycan, comprising the steps of obtaining a compound and contacting the compound with the glycan.

[0010] The following drawings are for illustrative purposes only and are not intended to limit the scope of the present invention. [Brief Description of the Drawings]

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DETAILED DESCRIPTION OF THE INVENTION

[0043] DETAILED DESCRIPTION OF THE INVENTION It is understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Further, any methods, devices, and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but certain methods, devices, and materials are described herein.

[0044] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the relevant art and the present disclosure, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0045] In the description of the present invention, it is understood that several techniques and steps are disclosed. Each of these has its individual advantages and each can also be used in combination with one or more, or in some cases all, of the other techniques disclosed. Therefore, for clarity, this specification refrains from unnecessarily repeating all possible combinations of the individual steps. Nevertheless, this specification and the claims should be interpreted with the understanding that such combinations are fully within the scope of the present invention and the claims.

[0046] The present disclosure will be further described by the following explanations, which should not be construed as limiting the present disclosure to the specific explanations described herein by scope or spirit. The following explanations are provided to describe certain embodiments, and it is understood that there is no intention to limit the scope of the present disclosure thereby. It is further understood that various other embodiments, modifications, and equivalents may be used, and these may be suggested to those skilled in the art in themselves without departing from the spirit and / or scope of the appended claims. For example, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0047] The present invention relates to methods and compositions for the analysis of glycans with a complex solution containing proteins and cells in a biological sample.

[0048] R 1 and A, and optionally substituted with R 2 a compound of formula (Ia) or (Ib):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0049] In an embodiment, R 2 and A are [Chemical formula] substituents other than.

[0050] In an embodiment, R 2 and A are [Chemical formula] substituents other than.

[0051] In an embodiment, optionally when one of R 2 or A is hydrogen, R 2 and A are [Chemical formula] substituents other than.

[0052] In some embodiments, the above compound is substituted with R 2 . In some embodiments, the above compound is substituted with A. In some embodiments, the above compound is substituted with R 2 or A. In other embodiments, the above compound is substituted with R 2 and A.

[0053] In one embodiment, R 1 , R 2 , and A are neither isocyanate nor succidimidyl carbamate. In another embodiment, R 1 , R 2 , and A are neither isocyanate nor its derivative. In another embodiment, R 1 , R 2 , and A are neither succidimidyl carbamate nor its derivative.

[0054] In some embodiments, the above compound has the formula (Ic)

Chemical formula

[0055] In an embodiment, the above compound has the formula (Id), formula (Ie) or formula (If)

Chemical formula

[0056] In some embodiments, the above compound is

Chemical formula

[0057] In some embodiments, the above compound is

Chemical formula

[0058] In an embodiment, the above compound is

Chemical formula

[0059] [Chemical] a compound (wherein X, A, R 1 , and R 2 are defined as those listed above) is provided.

[0060] In an embodiment, R1 is [Chemical] as follows.

[0061] In an embodiment, R2 is [Chemical] as follows.

[0062] In an embodiment, A is [Chemical] or NH2.

[0063] In an embodiment, X is CH or N.

[0064] In one embodiment, R 6 is independently selected from the group consisting of H, alkyl, t-butyl, sec-butyl, isobutyl, isopropyl, alkylamino, alkylsulfonic acid, or alkylphosphonic acid.

[0065] In an embodiment, the above compound is a fluorescent tag.

[0066] In an embodiment, the above compound is [Chemical] [Chemical] as follows.

[0067] In the embodiment, x, q, t, v, s, u, m, n, r, w, y, p, p a , and p b are each independently an integer from 0 to 18, 0 to 12, 0 to 6, 0 to 4, 0 to 2, or 1 to 2.

[0068] In the embodiment, when R 2 is hydrogen, A is [Chemical formula] a substituent other than these.

[0069] The counter ion includes lithium (Li + ) of an alkali metal, sodium (Na + ), potassium (K + ), rubidium (Rb + ), cesium (Cs + ), and francium (Fr + ). In addition, the counter ion may also include substituted or unsubstituted ammonium (NH4 + or RNH3 + or R2NH2 + or R3NH + ) and proton (H + ). The counter ion includes beryllium (Be 2+ ) of an alkaline earth metal, magnesium (Mg 2+ ), calcium (Ca 2+ ), strontium (Sr 2+ ), barium (Ba 2+ ), and radium (Ra 2+ ), or any one of transition metals in any of various oxidation states (for example, Cu + and Cu 2+ but not limited to these), and any complex ions formed by these. The counter ion includes fluoride (F - ) of a halide, chloride (Cl - ), bromide (Br - ), iodide (I - ), and astatide (At - ) or any phosphate (H2PO4- or Li2PO4 - or Na2PO4 - or K2PO4 - or Rb2PO4 - or Cs2PO4 - or Fr2PO4 - or BePO4 - or MgPO4 - or CaPO4 - or SrPO4 - or BaPO4 - or RaPO4 - or HPO4 2- or LiPO4 2- or NaPO4 2- or KPO4 2- or RbPO4 2- or CsPO4 2- or FrPO4 2- or PO4 3- ) or any borate (H2BO3 - or Li2BO3 - or Na2BO3 - or K2BO3 - or Rb2BO3 - or Cs2BO3 - or Fr2BO3 - or BeBO3 - or MgBO3 - or CaBO3 - or SrBO3 - or BaBO3 - or RaBO3 - or HBO3 2- or LiBO3 2- or NaBO3 2- or KBO3 2- or RbBO3 2- or CsBO3 2- or FrBO3 2- or BO3 3- ) or any sulfate (HSO4 - or LiSO4 - or NaSO4 -or KSO4 - or RbSO4 - or CsSO4 - or FrSO4 - or SO4 2- ) or any sulfite (HSO3 - or LiSO3 - or NaSO3 - or KSO3 - or RbSO3 - or CsSO3 - or FrSO3 - or SO3 2- ) or any arsenate (H2AsO4 - or Li2AsO4 - or Na2AsO4 - or K2AsO4 - or Rb2AsO4 - or Cs2AsO4 - or Fr2AsO4 - or BeAsO4 - or MgAsO4 - or CaAsO4 - or SrAsO4 - or BaAsO4 - or RaAsO4 - or HAsO4 2- or LiAsO4 2- or NaAsO4 2- or KAsO4 2- or RbAsO4 2- or CsAsO4 2- or FrAsO4 2- or AsO4 3- ) or any carbonate (HCO3 - or LiCO3 - or NaCO3 - or KCO3 - or RbCO3 - or CsCO3 - or FrCO3 - or CO3 2- ) or any silicate (HSiO3 -or LiSiO3 - or NaSiO3 - or KSiO3 - or RbSiO3 - or CsSiO3 - or FrSiO3 - or SiO3 2- ) or any selenate (HSeO4 - or LiSeO4 - or NaSeO4 - or KSeO4 - or RbSeO4 - or CsSeO4 - or FrSeO4 - or SeO4 2- ) or any oxide (HO2 - or LiO2 - or NaO2 - or KO2 - or RbO2 - or CsO2 - or FrO2 - or O2 2- or HO - or LiO - or NaO - or KO - or RbO - or CsO - or FrO - or O 2- ) or any hypohalite (FO - or ClO - or BrO - or IO - or AtO - )、any halite (FO2 - or ClO2 - or BrO2 - or IO2 - or AtO2 - ) or any halate (FO3 - or ClO3 - or BrO3 - or IO3 - or AtO3 -)、any perhalate (FO4 - or ClO4 - or BrO4 - or IO4 - or AtO4 - ) or any acetate (CF3COO - or CCl3COO - or CBr3COO - or CI3COO - or CH3COO - ) or any sulfide (HS - or LiS - or NaS - or KS - or RbS - or CsS - or FrS - or S 2- )、formate (CHOO - )、cyanate (CN - )、nitrate (NO3 - )、nitrite (NO2 - ) may further be included. In embodiments, the counterion can be any ion of opposite charge to the species presented. For example, if the species has a 2+ charge, the counterion Z can be any ion derived from any group having an opposite charge of type 2-.

[0070] A method for quantifying and / or characterizing a glycan, comprising: a) obtaining the compounds listed above; b) contacting this compound with a glycan, thereby forming a labeled glycan; is provided.

[0071] In embodiments, the method further includes dissociating the labeled glycan to form glycan fragmentation.

[0072] In some embodiments, the dissociation is collision-induced dissociation (CID) or high-energy collision dissociation (HCD).

[0073] In an embodiment, the method further includes thereby quantifying and / or characterizing the glycan.

[0074] In some embodiments, the glycan includes a reducing end.

[0075] In some embodiments, the glycan is other than an N-glycan.

[0076] In an embodiment, the method further includes analyzing the labeled glycan or glycan fragmentation using an instrument that is a fluorometer, a mass spectrometer, or a liquid chromatography instrument, an instrument capable of detecting fluorescence and / or absorbance as needed.

[0077] The instrument can be ultra-high performance liquid chromatography (UPLC), a linear quadrupole ion trap (LTQ-XL) mass spectrometer, a Q Exactive Orbitrap mass spectrometer, or liquid chromatography-mass spectrometry (LC-MS).

[0078] In some embodiments, the instrument is equipped with an electrospray ionization (ESI) source or a heated electrospray ionization (HESI) source and / or a fluorescence detector.

[0079] Provide a compound for use in the detection of glycan or in the quantification of glycan, or for use in the preparation of labeled glycan or glycan fragmentation.

[0080] In one aspect, the present invention provides a method for glycan analysis of a sample, the method including contacting the glycan with a compound of the present invention and scanning the analyte by mass spectrometry to detect and identify the presence of the glycan.

[0081] Further, the present invention provides for the quantification and / or characterization of a sample containing a compound, such as a biomolecule, a non-biomolecule, a protein, a peptide or an amino acid, by the same method as the glycans thereof. In some embodiments, these compounds have a functional group similar to the reducing end of the glycan.

[0082] In one embodiment, the sample comprises at least one protein solution. In one embodiment, the sample comprises at least one cell population.

[0083] In one exemplary method of the present invention, the compounds disclosed herein are contacted with the glycans of interest. In this step, a chemical reaction may form a covalent bond between the glycan of interest and the compound, thereby labeling the glycan. In another exemplary step of the present invention, a liquid chromatography instrument equipped with a fluorescence detector is used to quantify the amount of glycan in a given sample by the fluorescence generated by the labeled glycan. Fragmentation is then induced on the glycan, for example, using a mass spectrometer, which then provides data that can be used for the characterization of the glycan of interest.

[0084] Definitions "Glycan" refers to sugars, such as oligosaccharides and polysaccharides, and can be monomers or polymers of sugar residues linked mostly by glycosidic bonds. In some embodiments, the terms "glycan", "oligosaccharide", and "polysaccharide" may refer to the carbohydrate portion of glycoconjugates (e.g., glycolipids, glycoproteins or proteoglycans). Glycans can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.). The term "glycan" includes homopolymers and heteropolymers of sugar residues. Also, the term "glycan" encompasses the glycan component of glycoconjugates (e.g., glycoproteins, glycolipids, proteoglycans, etc.). This term also includes free glycans, which are either cleaved or otherwise released from glycoconjugates and contain glycans. Glycans can include O-linked glycans or N-linked glycans. O-linked glycans have a structure in which the glycan is linked via an -OH group contained in the amino acid side chain to the side chain of the amino acid residues serine (Ser) or threonine (Thr) in the protein. O-linked glycans are classified into 1 to 8 types according to the core structure. N-linked glycans refer to glycans that bind to the nitrogen atom of the amide group of the side chain of the asparagine residue (Asn) of the protein. N-linked glycans include mannose used as a base point and glycans forming branched portions, examples of which include 2-branched glycans, 3-branched glycans, and 4-branched glycans, etc. Also, N-linked glycans can be classified into basic type, high-mannose type, hybrid type, and complex type, etc. according to their structure.

[0085] As used herein, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon radical having 1 to 20 carbon atoms, in one embodiment 1 to 16 carbon atoms, and in another embodiment 1 to 10 carbon atoms, either alone or in combination with other groups, which can be branched or straight-chain. In some embodiments, alkyl is lower alkyl.

[0086] The term "lower alkyl", alone or in combination with other groups, refers to a branched or straight-chain alkyl radical having 1 to 9 carbon atoms, in one embodiment 1 to 6 carbon atoms, in another embodiment 1 to 4 carbon atoms, and in a further embodiment 4 to 6 carbon atoms. This term is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, 3-methylbutyl, n-hexyl, 2-ethylbutyl, and the like.

[0087] As used herein, the term "alkoxy" means alkyl-O--, and "alkoyl" means alkyl-CO--. An alkoxy substituent or an alkoxy-containing substituent may be substituted, for example, by one or more alkyl or halo groups.

[0088] As used herein, the term "halogen" means a fluorine, chlorine, bromine or iodine radical, or in some embodiments, a fluorine, chlorine or bromine radical.

[0089] As used herein, "ara-alkyl" refers to an alkyl group substituted with at least one aryl group. Similarly, as used herein, the term "heteroara-alkyl" group refers to an alkyl group substituted with at least one heteroaryl group.

[0090] The term "cycloalkyl" refers to a monocyclic or polycyclic radical having 3 to 10 carbon atoms, and in one embodiment, 3 to 6 carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl, indanyl, etc. In one embodiment, the "cycloalkyl" moiety may be optionally substituted with 1, 2, 3 or 4 substituents. Each substituent may independently be alkyl, alkoxy, halogen, amino, hydroxyl, aryl, heteroaryl or oxygen, unless otherwise specifically indicated. Examples of the cycloalkyl moiety include, but are not limited to, optionally substituted cyclopropyl, optionally substituted cyclobutyl, optionally substituted cyclopentyl, optionally substituted cyclopentenyl, optionally substituted cyclohexyl, optionally substituted cyclohexylene, optionally substituted cycloheptyl, etc. or those specifically exemplified herein.

[0091] The term "aryl" refers to an aromatic monocyclic or polycyclic radical having 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, 1,2-dihydronaphthyl, indanyl, 1H-indenyl, etc.

[0092] An alkyl group, a lower alkyl group, an aryl group, and a spirocycloalkyl group may be substituted or unsubstituted. In addition, an ara-alkyl group and a heteroara-alkyl group may be substituted with substituents in addition to an aryl or heteroaryl group. When substituted, for example, 1 to 4 substituents generally exist. These substituents may, if necessary, form a ring with the alkyl group, lower alkyl group, or aryl group to which they are attached. Examples of substituents include carbon-containing groups such as alkyl, aryl, arylalkyl (e.g., substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen-containing groups such as haloalkyl (e.g., trifluoromethyl); oxygen-containing groups such as alcohol (e.g., hydroxyl, hydroxyalkyl, aryl(hydroxyl)alkyl), ether (e.g., alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl, in other embodiments, for example, methoxy and ethoxy), aldehyde (e.g., carboxyaldehyde), ketone (e.g., alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arycarbonylalkyl), acid (e.g., carboxy, carboxyalkyl), acid derivatives such as ester (e.g., alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amide (e.g., aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl), carbamate (e.g., alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono- or di-alkylaminocarbonyloxy, arylaminocarbonyloxy(arylminocarbonloxy), and urea (e.g., mono- or di-alkylaminocarbonylamino or arylaminocarbonylamino);Nitrogen-containing groups, such as amines (e.g., amino, mono- or di-alkylamino, aminoalkyl, mono- or di-alkylaminoalkyl), azides, nitriles (e.g., cyano, cyanoalkyl), nitro; sulfur-containing groups, such as thiols, thioethers, sulfoxides, and sulfones (e.g., alkylthio, alkylsulfinyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl); and heterocyclic groups containing one or more heteroatoms (e.g., thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, hexahydroazepinyl, piperazinyl, morpholinyl, thianaphthyl, benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolinyl, isoquinolinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl and carbolinyl) may be mentioned.;

[0093] The term "heteroaryl" refers to an aromatic monocyclic or polycyclic radical of 5 to 12 atoms having at least one aromatic ring and containing 1, 2 or 3 ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. Examples of such groups include, but are not limited to, pyridinyl, pyrazinyl, pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, oxazolyl, thiazolyl, etc.;

[0094] The above-mentioned heteroaryl group may be independently substituted with one, two or three substituents. Examples of the substituents include carbon-containing groups such as alkyl, aryl, arylalkyl (e.g., substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen-containing groups such as haloalkyl (e.g., trifluoromethyl); oxygen-containing groups such as alcohol (e.g., hydroxyl, hydroxyalkyl, aryl(hydroxyl)alkyl), ether (e.g., alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl), aldehyde (e.g., carboxaldehyde), ketone (e.g., alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acid (e.g., carboxy, carboxyalkyl), acid derivatives such as ester (e.g., alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amide (e.g., aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl), carbamate (e.g., alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono- or di-alkylaminocarbonyl oxy, arylaminocarbonyl oxy), and urea (e.g., mono- or di-alkylaminocarbonylamino or arylaminocarbonylamino); nitrogen-containing groups such as amine (e.g., amino, mono- or di-alkylamino, aminoalkyl, mono- or di-alkylaminoalkyl), azide, nitrile (e.g., cyano, cyanoalkyl), nitro; sulfur-containing groups such as thiol, thioether, sulfoxide, and sulfone (e.g., alkylthio, alkylsulfinyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl);and a heterocyclic group containing one or more heteroatoms (e.g., thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, hexahydroazepinyl, piperazinyl, morpholinyl, thianaphthyl, benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolinyl, isoquinolinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl, benzothiazoyl and carbolinyl) may be mentioned.;

[0095] When any group is referred to as being optionally substituted, the group may be substituted or unsubstituted. The substitution may be by one or more of the specified substituents, which may be the same or different. It is understood that the number and nature of the substituents are selected so as to avoid any combination that is sterically undesirable.

[0096] When "optionally substituted" is applied to any group, it means that, if desired, the group may be substituted by one or more substituents, which may be the same or different. Examples of substituents suitable for the "substituted" moiety and the "optionally substituted" moiety include halo, deuterio, C 1~6 alkyl or C 1~3 alkyl, hydroxy, C 1~6 alkoxy or C 1~3 alkoxy, cyano, amino, nitro or SF5 (a known NO2 mimic), aryl, heteroaryl, heterocyclyl, C3-C6 cycloalkyl, C 1~3 alkylamino, C 2~6Alkenylamino, di-C 1~3 Alkylamino, C 1~3 Acylamino, di-C 1~3 Acylamino, carboxy, C 1~3 Alkoxycarbonyl, carbamoyl, mono-C 1~3 Carbamoyl, di-C 1~3 Examples include any of the above where the carbamoyl or its hydrocarbyl moiety itself is substituted by halo. For groups containing an oxygen atom, such as hydroxy and alkoxy, the oxygen atom may be replaced by sulfur to form groups such as thio (SH) and thio-alkyl (S-alkyl). Accordingly, optional substituents as required include groups such as S-methyl. In the case of a thio-alkyl group, the sulfur atom may be further oxidized to form a sulfoxide or sulfone, and accordingly, optional substituents as required include groups such as S(O)-alkyl and S(O)2-alkyl.

[0097] Accordingly, examples of substituents include, for example, Cl, F, OMe, Me, COCH3, CONH2, NHC(O)CH(CH3)2, CO2CH2CH3, etc. In the case of an aryl group, the substituent may be in the form of a ring derived from adjacent carbon atoms within the aryl ring, such as a cyclic acetal, for example, O-CH2-O.

[0098] Optional substituents for any alkyl, alkenyl, alkynyl, alkoxy, alkylene or alkenylene group described herein may be selected from C1-C3 alkoxy, halogen, hydroxyl, thiol, cyano, amino, amide, nitro, and SF5, where the alkoxy may be optionally substituted with halogen. In particular, optional substituents may be selected from halogen, hydroxyl, thiol, cyano, amino, amide, nitro, and SF5, more preferably fluorine or hydroxyl.

[0099] Generally, hydrogen is not considered a substituent. However, in selected embodiments, A and / or R 2 is hydrogen.

[0100] Any range disclosed herein means that all second decimal places, first decimal places, and integer unit amounts within that range are disclosed in detail as part of the present invention. Thus, for example, 0.01 to 50 means that unit amounts of 0.02, 0.03 ··· 0.09; 0.1; 0.2 ··· 0.9; and 1, 2 ··· 49 are included as embodiments of the present invention.

[0101] In any of the disclosures herein, a particular R group may be more than one functional group, and when the structure of a compound has more than one of that particular R group, the R groups may be different functional groups respectively. For example, when the structure has two R 4 groups and one is hydrogen, the other may be an alkyl group. In a similar manner, when present multiple times in a single structure, each letter representing a number in the structure may be different in each case.

[0102] The compounds of the present invention may have one or more asymmetric carbon atoms and may exist in the form of optically pure enantiomers, mixtures of enantiomers, such as racemic compounds, etc., optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemic compounds or mixtures of diastereoisomeric racemic compounds. The optically active forms can be obtained, for example, by resolution of racemic compounds, by asymmetric synthesis or by asymmetric chromatography (chromatography using a chiral adsorbent or eluent). The present invention encompasses all of these forms.

[0103] The compounds of the present invention can be prepared starting from commercially available starting materials and using general synthetic techniques and procedures known to those skilled in the art.

[0104] There are a number of established dissociation techniques by MS, and the state-of-the-art techniques are also continuously being developed. As examples, in collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD), it is generally known that fragmentation is brought about by cleavage of glycosidic bonds. In comparison, ultraviolet photodissociation (UVPD) and high-energy collision dissociation (HCD) have been shown to result in more informative analyte fragmentation. Radical-driven dissociation (RDD), electron capture dissociation (ECD), electron excitation dissociation (EED), electron transfer dissociation (ETD), and electron detachment dissociation (EDD) are often grouped together as free radical-driven dissociation techniques, but have similarly demonstrated great potential for glycan structure analysis. Nevertheless, UVPD, MS n , ion mobility mass spectrometry, and EDD are still the most powerful in the discrimination of mass spectra corresponding to glycan isomers, particularly stereoisomers such as anomers and epimers. As shown in the following examples, MS n and CID can be combined with a reagent containing a free radical precursor to conveniently, accurately, and rapidly perform glycan structure analysis. In addition, gas-phase ion / ion reactions occurring within a modified mass spectrometer capable of charge reversal of anionic analytes have been demonstrated, showing potential for future applications to glycan structure analysis. Experiments have been carried out in both the gas phase and the condensed phase, where the reagent (which utilizes a free radical precursor) forms a complex with the selected analyte, allowing fragmentation induced by free radicals to occur subsequently. However, this approach currently does not enable the high-sensitivity detection and quantification of glycans to be performed by fluorescence.

[0105] Recently, free radical activation glycan sequencing reagents (FRAGS) have been developed that react with and covalently bond to each reducing end of glycans. FRAGS reagents recruit a free radical precursor (e.g., 2,2,6,6-tetramethyl-1-piperidinyloxy, i.e., TEMPO, among other nitroxyl free radical precursors), thereby generating a localized nascent free radical that simultaneously induces predictable, diagnostic, and systematic glycan fragmentation upon collision activation of the tagged glycan. Moreover, FRAGS reagents recruit a basic pyridyl site for charge induction in addition to a hydrazide or aminooxy labeling site that reacts with the glycan reducing end. To date, first-generation FRAGS were methylated in the pyridine moiety (Me-FRAGS I), covalently bound to oligosaccharide isomers, and showed little difference. Methylation hinders gas-phase glycan rearrangement, which is frequently observed in protonated species containing mobile charges, as it induces charge localization. Subsequently, MS including Me-FRAGS I 3The Conflict-Induced Dissociation (CID) spectrum demonstrated the ability to accurately and rapidly distinguish between nine disaccharide isomers and two tetrasaccharide isomers. FRAGS-labeled glycans are detectable by liquid chromatography, but these do not contain a fluorophore that substantially improves the sensitivity of liquid chromatography detection and quantification by fluorescence. Moreover, at the hydrazide-labeling site and the aminooxy-labeling site, the coupling efficiency and stability are reduced compared to the development of tags using the free amino group as the coupling site. In particular, the hydrazide group has a strong interaction with sodium ions after reductive amination, thus reducing the methylation rate in the generation of fixed charges. In addition, according to a detailed review of currently commercially available tags, they cannot simultaneously provide the quantification and characterization of glycans. Moreover, certain tags that mobilize the NHS-carbamate functional group have insufficient stability in the presence of atmospheric moisture. Accordingly, the inventors have developed tags that include the following sites: (1) a free radical precursor from which a nascent free radical capable of inducing systematic and predictable glycan fragmentation can be obtained, (2) an ionization site that enables the generation of fixed charges with enhanced ionization efficiency and methylation efficiency, (3) a fluorophore that enables highly sensitive quantification of tagged glycans, and (4) an amino coupling site that dramatically enhances the coupling efficiency between glycans (see Figure 1). These developed tags are described herein. These tags, unlike many currently in use, enable the simultaneous quantification and characterization of glycans and also enable enhanced chemical stability.

[0106] Indeed, the compounds and methods of the present invention enable the quantification of glycans by fluorescence energy, and, unlike many prior art methods, enable the characterization of glycans by mass spectrometry and the induction of fragmentation by free radical chemical reactions. In addition, the compounds and methods disclosed herein can be used for glycan conjugation with any glycan having a reducing end. In comparison, many prior art methods are limited to only N-glycans.

[0107] The compounds of the present invention can introduce a TEMPO (2,2,6,6 - tetramethylpiperidine - 1 - oxyl) group or an analog thereof into a glycan and a similar molecule, and then generate a free radical chemical reaction. The TEMPO group is superior to other groups, such as the succinimide group, in many aspects. For example, the succinimide group is generally rapidly decomposed in the presence of atmospheric moisture, while the TEMPO group is generally not rapidly decomposed in the presence of atmospheric moisture.

[0108] The present invention is better understood by referring to the following experimental details, but those skilled in the art can easily understand that the specific experiments described in detail are merely examples of the present invention, as will be more fully described in the claims that follow.

Example

[0109] Example 1

[0110] General synthesis. As shown in Scheme 1, the general synthesis of the fluorescent tag starts with a cyclization reaction involving a substituted benzene and an olefin. The final product was obtained by a series of six reactions.

Chemical formula

[0111] Ethyl 5 - methyl - 7 - nitro - 2 - quinolinecarboxylate (1)

[0112] Into a clean, oven - baked flask equipped with a stir bar, 1,2 - dimethyl - 3,5 - dinitrobenzene (5 mmol) was reacted under argon in a solution of cesium carbonate (3 equivalents) in ethyl acrylate (2 equivalents) and anhydrous tetrahydrofuran (25 mL). After refluxing for 8 hours, the crude mixture was cooled and evaporated under vacuum, and then the product was extracted from the residue with dichloromethane. The collected fractions were purified by silica gel flash chromatography using ethyl acetate and hexane. The desired product was obtained as a yellow solid (360.5 mg, yield 28%).

[0113] Ethyl 5-bromomethyl-7-nitro-2-quinolinecarboxylate hydrobromide (2)

[0114] Into a flame-dried flask, ethyl 5-methyl-7-nitro-2-quinolinecarboxylate (1.385 mmol), N-bromosuccinimide (1.25 equivalents), and benzoyl peroxide (0.1 equivalent) were added. After evacuating for 30 minutes, these were allowed to stand under an argon atmosphere. The reaction mixture was dissolved in anhydrous carbon tetrachloride (70 mL) and refluxed and stirred with a photocatalyst (250 W) for 1.5 hours. Upon completion, the crude mixture was cooled to room temperature and purified by silica gel flash chromatography using ethyl acetate and hexane (3:7). After adding hydrobromic acid, the mixture was stirred for 30 minutes, and the desired product was converted to a salt by completely evaporating the solvent under reduced pressure.

[0115] Ethyl 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylate (3)

[0116] The ethyl 5-bromomethyl-7-nitro-2-quinolinecarboxylate hydrobromide (assumed to be 1.385 mmol) from the previous step was converted back to the neutral form by dissolving the salt in water and basifying the solution with sodium hydroxide until the pH reached about 7. The organic compound was extracted with benzene (60 mL), and the collected material was dried over anhydrous sodium sulfate. After dissolving TEMPO (1.2 equiv), Cu(OTf)2 (0.1 equiv), Nbpy (0.4 equiv), and copper powder (1.2 equiv), the dried benzene layer was transferred to a flame-dried flask and degassed with argon for 30 minutes. The solution was refluxed under argon for 2 hours with stirring. After cooling to room temperature, the crude mixture was filtered through a short pad of silica gel and eluted with ethyl acetate. The organic layer was transferred to a separatory funnel and washed with saturated NH4Cl, 1 M NH4OH, and brine. After drying the organic layer over anhydrous sodium sulfate, it was purified by silica gel flash chromatography using ethyl acetate and hexane (1:5). The desired product was obtained as a white to off-white solid (144.8 mg, 25% overall yield for the TEMPO coupling after bromination).

[0117] Ethyl 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylic acid (4)

[0118] Ethyl 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylate (0.3485 mmol) was added to a clean flask and dissolved using THF (10 mL) and methanol (10 mL). 2 M aqueous KOH (20 mL) was slowly added to the mixture. The reaction was continued at room temperature with stirring. Upon completion, the crude mixture was evaporated under reduced pressure to remove THF and methanol. 2 M aqueous HCl was added to the crude mixture until the pH reached about 7, and then the mixture was extracted 5 times with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate. The organic layer was then purified by silica gel flash chromatography using methanol and DCM (20:1). The desired product was obtained as a white solid (98.7 mg, 73% yield).

[0119] N-[2-(Diethylamino)ethyl]-5-(TEMPO)methyl-7-nitro-2-quinolinecarboxamide (5)

[0120] Ethyl 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylate (0.2543 mmol) was added to an oven-dried flask, evacuated for 30 minutes, and then dissolved in thionyl chloride (10 mL). The reaction was allowed to proceed by refluxing for 2 hours with stirring. Thereafter, thionyl chloride was evaporated under vacuum, and the residue was immediately redissolved in anhydrous DCM (5 mL). In another oven-dried flask, N-diethylethylenediamine (50 μL) and anhydrous trimethylamine (140 μL) were dissolved in anhydrous DCM (5 mL). The acid chloride was added dropwise to this mixture over 10 minutes. After stirring at room temperature for 2.5 hours, the mixture was diluted with 15 mL of DCM and washed once with saturated aqueous sodium bicarbonate. The aqueous layer was then extracted twice with DCM, and the combined organic layers were dried over anhydrous sodium sulfate. The organic layer was then subjected to purification by silica gel flash chromatography using methanol and DCM. The desired product was obtained as a white solid (100.8 mg, 82% yield).

[0121] 7-Amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide (6)

[0122] N-[2-(Diethylamino)ethyl]-5-(TEMPO)methyl-7-nitro-2-quinolinecarboxamide (0.2076 mmol) was added to a clean flask equipped with a stir bar. The solid was dissolved in anhydrous ethanol (5 mL), and then 10% Pd / C (0.5 equiv) was added. A hydrogen gas source was attached to the flask, and the reaction was allowed to proceed with stirring at room temperature for 2.5 hours. Upon completion, the crude mixture was filtered through sand and celite and eluted with ethanol. The organic layer was purified by silica gel flash chromatography using methanol and DCM to give the final pure product as a yellow solid (51.5 mg, 54% yield).

Chemical Structure

Table 1

Table 2

[0123] Figures 3 to 7 relate to the nuclear magnetic resonance spectrum and mass spectrometry spectrum of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. These spectra confirm the chemical structure of this compound.

[0124] Preparation of a glycan sample using 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. 30 μL of a 20 mM fluorescent tag (7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide) in methanol and 20 μL of a 1 mM glycan sample (e.g., lactose) in water were added together into a PCR tube and evaporated under vacuum. The residue was redissolved in 50 mM sodium cyanoborohydride in DMSO and acetic acid (7:3 v / v). The mixture was incubated at 70 °C for 2 hours. After completion of the glycan labeling, the mixture was evaporated under vacuum at 70 °C. The residue was washed with HPLC-grade acetone to extract unreacted reagents and sodium cyanoborohydride. The precipitate was dissolved in 50% HPLC-grade methanol in 18.2 megohm water and then analyzed by LC-MS.

[0125] Analysis by mass spectrometry. Thermo-Fisher Scientific equipped with electrospray ionization (ESI) TMA linear quadrupole ion trap (LTQ-XL) mass spectrometer (Thermo, San Jose, CA, USA) was used. The sample was directly injected into the ESI source of the mass spectrometer at a flow rate of 10 μL / min. The parameters of the mass spectrometer included a sheath nitrogen gas flow rate of 10 (arbitrary unit), a spray voltage of 5.00 kV, a capillary voltage of 20 - 40 V, a capillary temperature of 275 °C, and a tube lens voltage of 50 - 200 V. The signal intensity was maximized by optimizing the ion optical parameters using the auto-tuning function of the LTQ-XL tuning program. Systematic glycan fragmentation was achieved by subjecting the ionized bioconjugate to collision-induced dissociation (CID) of 15 - 50 (arbitrary unit).

[0126] Fluorescence assay. A concentration range of 1 pM - 100 μM was prepared to evaluate the fluorescence ability of the fluorescent tag. An absorbance scan was first performed on a 100 μM solution to determine the potential excitation wavelength. Then, the emission at each excitation wavelength was examined using an Agilence Cary Eclipse fluorescence spectrophotometer. The optimal excitation and emission wavelengths were selected for the construction of a preliminary calibration curve to evaluate any correlation between fluorescence and concentration. The preliminary calibration curve was also created on another instrument (ThermoFisher Scientific TM NanoDrop TM ) for instrument reproducibility.

[0127] Figures 7-11 present the results of this experiment, including the labeling of disaccharide samples. This experiment was conducted, including the labeling of disaccharide samples of known molecular weights and chemical structures using the protocols of the present invention and established ones. By labeling, we successfully increased the m / z value of the present invention (m / z 456) to the labeled glycan (m / z 782) as expected by mass spectrometry. Subsequently, the labeled disaccharide was isolated and fragmentation was induced. The first fragmentation that occurred was the expected disappearance of TEMPO, which resulted in a decrease in m / z 156. This initial fragmentation verified that the glycan was labeled with compound 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. Further isolation and fragmentation of the fragments (the isolated fragment has m / z 626) revealed further fragmentation, including the covalently bound disaccharide. These fragmentations can be used to characterize the disaccharide.

[0128] UPLC-FLD and UPLC-MS analysis. ThermoFisher Scientific equipped with a fluorescence detector TM Vanquish Flex TM UPLC instrument and ThermoFisher Scientific TM The Q Exactive Plus Oribtrap mass spectrometer was used for all of the following analyses. Mixtures of six reagent-labeled glycans (maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) were each prepared to a final concentration within the range of 10-20 μM. Supelco Discovery C 18 column (particle size 5 μm, L 25 cm × I.D. 4.6 mm) and XBridge Glycan BEH Amide XP column (particle size 2.5 μm, L 150 mm × I.D. 3.0 mm) were used for the separation of the analytes. The flow rate was set at 0.750 mL / min for the C 18 column and 0.400 mL / min for the Glycan BEH Amide column, and the column temperature was C 18The column was set at 30.00 °C and the Glycan BEH Amide column was set at 60.00 °C. C 18 For the column, mobile phase A consisted of 99.9% 18.2 MΩ water and 0.1% formic acid, and mobile phase B consisted of 99.9% LC-MS grade acetonitrile and 0.1% formic acid. The gradient was maintained at 10% B for 5 minutes and then increased to 30% B over 55 minutes. For the Glycan BEH Amide column, mobile phase A consisted of 50 mM aqueous ammonium formate solution (pH 4.403), and mobile phase B consisted of LC-MS grade acetonitrile. The gradient started at 86% B and decreased to 50% B over 55 minutes. Fluorescence detection was performed with the parameters set at excitation 385 nm, emission 515 nm, and detection sensitivity 5. Mass spectrometry detection was performed with the parameters set at capillary voltage +3.50 kV, capillary temperature 320 °C, sheath gas flow rate 50 (arbitrary unit), and S lens RF level 50.0 (arbitrary unit).

[0129] The results of this experiment are shown in Figures 12 - 19, which include fluorescence experiment data. The emission of any light at excitation wavelengths 270 nm and 390 nm was evaluated. Both excitation wavelengths resulted in emission at wavelength 520 nm. Further experiments and considerations revealed that 390 nm is a better excitation wavelength for use in fluorescence assays. Subsequently, calibration curves were successfully created on two different fluorometers, and the linear fit between nanomolar and micromolar concentrations according to the present invention was 0.9997.

[0130] Conclusion For optimal mass spectrometry and liquid chromatography analysis of glycans, as well as fluorescence detection, the fluorescent reagents disclosed herein contain important chemical sites. Among these sites, as mentioned previously, are fluorophore bases, basic sites (ionization / methylation sites), glycan labeling sites (coupling sites), and free radical precursors, all of which are lacking in commercially available tags. The fluorophore base exhibits fluorescence energy required for the detection and quantification capabilities of glycans in a concentration-dependent manner. The purpose of using fluorescence is to maximize the sensitivity of detection and quantification methods by liquid chromatography. The basic site enables the induction of a positive charge either by protonation, ion-dipole interaction, or methylation. The importance behind mobilizing the basic site is to cause ionization during the analysis of glycans by mass spectrometry. The glycan labeling site is the most essential feature to include on the tag in order to react with the glycan and then enable the formation of a covalent bond. Finally, the free radical precursor enables the formation of nascent free radicals during any of the dissociation techniques mentioned previously. Such dissociation techniques include collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Ultimately, the free radicals are reacted with the glycans simultaneously to result in reproducible, systematic, and efficient fragmentation of the glycans for characterization, identification, and differentiation between glycans and their isomers.

[0131] Based on the data presented, the fluorescent tags disclosed herein exhibit an excellent correlation between fluorescence and concentration starting from a low level of about 1 nM. This correlation was also observed on another fluorescence spectrophotometer instrument, and similar results were obtained. Moreover, the basic sites on the fluorescent tags are efficient for the ionization process during analysis by mass spectrometry. The fluorescent tags are capable of inducing systematic and reproducible fragmentation in the analysis of glycans when tagging the glycan molecules. Thus, the recruitment of fluorophore bases, basic sites, glycan labeling sites, and free radical precursors represents an optimal combination overall for glycan tagging and analysis. By using liquid chromatography for the separation of glycans and glycan isomers in combination with fluorescence detection and tandem mass spectrometry for glycan and glycan isomer fragmentation and hence characterization and identification, the fluorescent tags ultimately provide a powerful, novel, and modern analytical capability within the field of glycobiology. Therefore, the evolution of new analytical techniques enables the development of a relatively new field with potential for research on glycans as human disease biomarkers.

[0132] Example 2 Materials. For the synthesis of the novel fluorescent tag, the starting material 1,2-dimethyl-3,5-dinitrobenzene was purchased from 1Click Chemistry (Kendall Park, NJ). All other chemicals used in the synthesis of the novel fluorescent tag were purchased from Sigma-Aldrich (St. Louis, MO, USA). Maltose, maltohexaose, maltoheptaose, and GPC-grade dextran ladder standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Maltotriose was purchased from Thermo Scientific (Waltham, MA). Maltotetraose and maltopentaose were obtained from Biosynth Carbosynth (Staad, Switzerland). Lacto-N-difucohexaose I (LNDFH I) and lacto-N-difucohexaose II (LNDFH II) were purchased from Dextra Laboratories (Reading, UK). Bovine pancreatic ribonuclease B (RNase B) and peptide-N-glycosidase F (PNGase F) were purchased from New England Biolabs (Ipswich, MA, USA). LC-MS grade ammonium formate was purchased from Sigma-Aldrich (St. Louis, MO, USA). All solvents used for the purification and analysis of the samples described herein were HPLC grade and were purchased from Fisher Scientific (Hampton, NH).

[0133] N-glycan deglycosylation of RNase B. For the deglycosylation of N-glycans by RNase B, the manufacturer's protocol was followed. To ensure complete deglycosylation of the glycoprotein, the incubation step was extended to 14 hours. Subsequently, the resulting aqueous solution was cooled to room temperature and then subjected to purification by PGC SPE. The cartridge was activated with acetonitrile and equilibrated with 5% acetonitrile, after which the sample was applied. Then, the cartridge was washed a total of 5 times with 1 mL of water, the glycan was eluted, and collected a total of 4 times using 250 μL of 40% ACN with 0.1% formic acid through a 0.2 μm nylon filter. After the eluate was evaporated at 60 °C under vacuum, the derivatization step was followed.

[0134] Synthesis of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. Briefly, 1,2-dimethyl-3,5-dinitrobenzene was subjected to a cyclization reaction with ethyl acrylate. Thereafter, the remaining methyl group was brominated and the product was converted to the hydrobromide salt form. After neutralization and extraction into the organic solvent used in the subsequent reaction, the intermediate was subjected to a TEMPO coupling reaction. Then, the resulting product was subjected to a strong base to obtain a carboxylic acid (referred to as a common precursor as shown in Figure 2). The carboxylic acid was first converted to an acid chloride intermediate and then reacted with N,N-diethylethylenediamine by forming an amide bond. Finally, the remaining nitro group was hydrogenated with hydrogen gas to obtain the final product. Alternatively, an acidic analog of this structure (currently under investigation) for the analysis of glycans by negative ion mode mass spectrometry can be obtained by directly hydrogenating the nitro group derived from the aforementioned common precursor.

[0135] Preparation of N-glycan and standardized glycan samples using 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. 30 μL of 20 mM tagging reagent in methanol and 20 μL of 1 mM standardized glycan in water were added together and evaporated under vacuum. The residue was redissolved in 50 mM sodium cyanoborohydride in anhydrous DMSO and glacial acetic acid (7:3 v / v), and the resulting mixture was incubated at 70 °C for 2 h. After completion of glycan labeling, the mixture was evaporated by nitrogen degassing or under vacuum at 70 °C. During a total of three repetitions, unreacted reagent and sodium cyanoborohydride were extracted by vortexing and sonication of the residue with 50 μL of HPLC-grade acetone and centrifuged at 14 krpm for 5 min. The supernatant containing the free reagent was reused for subsequent desalting and purification. After final collection, the pellet containing the tagged glycan was completely dried under ambient conditions for 10 min and then subjected to LC-MS analysis.

[0136] Fluorescence assay. A concentration range of 1 nM to 100 μM was prepared to evaluate the linearity of the fluorescent tag. An excitation scan in emission mode set to zero order was first performed on a 100 μM solution using an Agilence Cary Eclipse fluorescence spectrophotometer to determine the value of the pre-excitation wavelength. Each excitation wavelength was then subjected to an emission scan to determine the wavelength pair with optimal fluorescence intensity. With parameters set to a PMT detector voltage of 965 V, excitation and emission slits of 5 nm, and an averaging time of 1.000 s, an excitation wavelength of 280 nm and an emission wavelength of 520 nm, optimal for the creation of a concentration-dependent fluorescence plot, were selected to evaluate the correlation between fluorescence intensity and concentration.

[0137] UPLC-FLD and UPLC-MS analysis. A ThermoFisher Scientific Vanquish Flex UPLC instrument equipped with a fluorescence detector and a ThermoFisher Scientific Q Exactive Plus Oribtrap mass spectrometer were used in all of the following experiments. A mixture of six maltoses consisting of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose was labeled and diluted to prepare a solution of 46 pmol / μL. The labeled dextran ladder was prepared to a final concentration of 300 μM. The labeled LNDFH I analyte and LNDFH II analyte were mixed in an unknown amount with an unknown total concentration. For the analysis of N-glycans derived from RNase B, a tagged N-glycan with a concentration of 74 pmol / μL was prepared. The labeled glycan was reconstituted with an initial mobile phase consisting of 50 mM ammonium formate (pH 4.4) and LC-MS acetonitrile (14:86 v / v) and injected in 1.0 μL. An Xbridge Glycan BEH Amide XP column (particle size 2.5 μm, L 150 mm × I.D. 3.0 mm) was used for the separation of fluorescent tag derivatized glycan analytes with the gradient described in Table 1. The flow rate was set at 0.400 mL / min and the column temperature was set at 60.00 °C. Fluorescence detection was performed with the parameters set at excitation 280.0 nm, emission 520.0 nm, detection sensitivity 8 (arbitrary unit), and scan rate 5.00 Hz. The mass spectrometry experiment was performed with the parameters set at capillary voltage +3.50 kV, capillary temperature 263 °C, sheath gas flow rate 50 (arbitrary unit), auxiliary gas heater temperature 425 °C, auxiliary gas flow rate 13 (arbitrary unit), sweep gas flow rate 3 (arbitrary unit), and S lens RF level 50.0 (arbitrary unit). For the MS 2 analysis of analytes in each run, an isolation window of 4.0 m / z was executed at a resolution of 70,000 for each of the predicted masses listed in the inclusion list. The HCD collision energy was varied under the normalized collision energy (NCE) mode.

[0138] Results and Discussion By synthesizing a novel fluorescent tag that mobilizes free radical precursors (abbreviated as Glyc·RadiFluor; GRF), in addition to promoting high fluorescence sensitivity, achievable methylation and ionization by mass spectrometry, and improved chemical stability, it enhances the ability to characterize glycans and eliminates the interconversion at the reducing ends of glycans among multiple isomers. After initially evaluating the fluorescence detection and quantification capabilities of GRF, further experiments involving glycans were carried out. After determining the optimal excitation wavelength and emission wavelength to be 280 ± 5 nm and 520 ± 5 nm respectively, linearity was further evaluated. The correlation between fluorescence intensity and GRF concentration within the range of 1 nM and 1 μM was plotted and subjected to linear regression analysis (Figure 10). The linear fit value of 0.9998 is interpreted as excellent linearity between fluorescence intensity and GRF concentration. Moreover, the linearity through nanomolar concentrations depicts the fluorescence detection sensitivity and quantification sensitivity of GRF, which are suitable for the quantitative analysis of glycans derived from glycoproteins.

[0139] After confirming the fluorescence detection and quantification capabilities of GRF, a mixture of maltoses with variable sugar unit lengths was tagged by reductive amination, and the separation was evaluated for two columns showing opposite separation methods. Briefly, the importance of the use of reductive amination in the tagging protocol is to eliminate the interconversion of α-isomers, β-isomers, and open-chain isomers, which would otherwise complicate LC analysis by generating additional peaks. Moreover, experiments showed that there was no significant difference in the reaction yield due to the tagging ability of GRF during reductive amination when reacting for 2 hours versus 14 hours. The columns initially used for the separation of the analyte included both a C 18 stationary phase (data not shown) and an ethylene-bridged hybrid (BEH) amide (or HILIC) stationary phase (these stationary phases utilize reverse-phase separation technology and normal-phase separation technology respectively). As expected, peak separation between maltoses was optimal when using normal-phase separation (Figure 20). The improvement in separation by the BEH amide column can be explained by the increased interaction between the maltose moiety of the analyte and the hydrophilic amide groups from the stationary phase. Conversely, the hydrophobic C 18In the stationary phase, the number of interactions with the maltose moiety decreases. Instead, the interactions are closer with the fluorescent tag moiety, which explains that the separation of maltose is less sufficient. Therefore, due to the improvement of peak separation, the BEH amide column was used in all subsequent experiments described herein.

[0140] In previous attempts at quantitative analysis of the maltose mixture, it was determined that the integrated peak values were not relatively equal. It was observed that the integrated values of tagged maltohexaose and tagged maltoheptaose were smaller compared to the shorter maltoses. This problem was solved by noting that the purity of the longer-length standardized glycans was relatively lower than that of the shorter-length standardized glycans. As an example, the purity of maltoheptaose and maltohexaose was 65% or higher, while the purity of maltotetraose was 95% or higher. Accordingly, the concentrations of maltoheptaose and maltohexaose were increased during sample preparation to compensate for the decrease in purity. After subsequent quantitative analysis, it was observed that relatively equal values were returned by the integration of the peaks in Figure 20. For the longer maltoses, the peak width increased slightly, resulting in a smaller peak height. Nevertheless, the integration of the peaks was relatively similar to the values associated with the earlier-eluting maltoses. After peak integration, the amount of each analyte was determined based on the known total injection volume of 46 pmol. By multiplying the ratio of the analyte peak area to the sum of all analyte peak areas by the known total molar amount, the result returns a value related to the molar amount corresponding to the individual analyte injected. As an example, maltose had a peak area of 1.090×10 8 and the combined total peak area of all analytes was 6.397×10 8Assuming that it is so, it is estimated that 7.8 pmol of the GRF-derivatized maltose can be injected. Similar calculations are possible for all other analytes from the same sample (see Table 2). Based on the results of these calculations, the average analyte injection value was determined to be 7.7 ± 0.2 pmol. This is consistent with the equimolar amount of 7.7 pmol per analyte expected. Moreover, the normalization level of approximately 7.7 pmol per analyte of 2.43×10 7 by 7 further depicts the high detection sensitivity and quantification sensitivity of the glycans by the quinolinyl fluorophore luminescence upon excitation. Finally, each maltose results in a single peak associated with the open-chain isomer rather than multiple peaks corresponding to further α and β isomers. This represents the efficiency of the reducing agent in reducing the resulting Schiff base during the labeling reaction, thereby resulting in a single isomer that is no longer interconvertible even at the reducing end of the glycan. Thus, this results in a single peak for each analyte, and for the detection of analytes and their corresponding isomers in any given glycan sample, a simpler and more feasible analytical approach by LC is established.

[0141] After detecting and quantifying the unknown glycans, it is also particularly essential to characterize and identify them. In this example, the ability of GRF to characterize the maltose mixture was evaluated by subjecting each of the six analytes to high-energy collision dissociation (HCD). As the HCD collision energy was increased, the TEMPO free radical precursor fragmented to induce nascent and more localized free radicals that reacted simultaneously with the glycan to effect cross-ring cleavage and glycosidic bond cleavage. Not only did the abundance of these fragmentations differ based on structure or stereochemistry, but the type of fragmentation also differed. As a result, useful structural information can be obtained by means of various fragmentation pathways for identifying and characterizing large amounts of glycans and their isomers. All of the product fragmentation ions described herein are assigned based on the Domon and Costello nomenclature for glycan fragmentation (Figure 21). Currently, there is no known nomenclature for fragmentation ions corresponding to the reducing-end open-chain isomers (sugar unit 0). Therefore, a new nomenclature system was devised accordingly (Figure 22). As shown in Figures 23A and 23B, protonated GRF-derivatized maltoheptaose alone was subjected to an HCD normalized collision energy of 30 (arbitrary units) to fragment the TEMPO group and generate free radicals. At the same time, highly unstable free radicals reacted to induce cross-ring cleavage and glycosidic bond cleavage in each of the constituent sugar units. In particular, the fragmentation is cross-ring fragmentation, 0,2 type X ions and 1,5It includes X-type ions, as well as Y-type ions and Z-type ions which are glycosidic bond cleavages. Further fragmentation of the C2H5O2 group that binds only to the tertiary carbon at the reducing end is also observed (assigned as η0 fragmentation). In addition, multiple disappearances of water are observed, which associate with the Z-type fragment ions, thereby subsequently forming a double bond along the C-C bond. The fragmentation observed in the protonated species provides useful information about the tagged maltoheptaose, but many fragmentations may be acid-catalyzed by the mobile charge and glycan rearrangement can occur. Such glycan rearrangement results in misleading fragmentations that deviate from the fragmentations corresponding to the actual structure. The complete fragmentation of the ionization site seems to be catalyzed by the nascent free radical, which ultimately leads to the extraction of a proton from the glycan moiety by the quinolinyl nitrogen. Due to the existence of multiple possible sites of protonation, the positive charge may have been pre-stabilized during ionization either at the quinolinyl nitrogen or the amide nitrogen.

[0142] To eliminate any glycan rearrangement and acid-catalyzed fragmentation, a fixed charge is introduced by reacting the GRF-derivatized maltoheptaose with iodomethane. Tertiary amines have a high affinity for nucleophiles and are selectively attacked by the methyl cation to yield a positively charged quaternary amine. As shown in FIGS. 24A and 24B, the complete disappearance of the ionization site is no longer observed. However, there is further fragmentation accompanied by a partial disappearance of the ionization site. 0,2 X product ions 1,5 X product ions, Y product ions, and Z product ions are still observed throughout the linear biopolymer.

[0143] After determining the optimal method conditions and instrument usage parameters for the peak separation and mass spectrometric characterization of maltose, the method was further evaluated for a mixture of branched isomers. Lacto-N-difucohexaose I (LNDFH I) and Lacto-N-difucohexaose II (LNDFH II) are hexasaccharide isomers that differ only in the linkage position of a single fucose subunit (structures are shown in Figures 26A - 26D). Fluorescence chromatograms demonstrate the ability of HILIC chromatography in the separation of labeled LNDFH I and labeled LNDFH II isomers (Figure 25). When the normalized HCD energy is maintained constant at 28 (arbitrary units), one of the major differences in the HCD spectra of the branched isomers is the relative abundance and type of fragmentation (Figures 26A - 26D). As an example, the HCD spectrum of labeled and protonated LNDFH II contains 1,5 X 2α product ions, Y 2α product ions, and Z 2α unique fragmentation including product ions. This is due, in particular, to the branching nature of the glycan, such that upon fragmentation occurring at sugar unit 2 (or sugar unit 2α of LNDFH II), four sugar residues are lost in LNDFH I, while three sugar residues are lost in LNDFH II. This is further exemplified by comparing the reference peaks related to the 2α Y product ion of LNDFH II, which is in a higher state of mass-to-charge ratio than the reference peak related to the Y2 product ion of LNDFH I. Similarly, an example can also be derived from methylated species, and when the normalized HCD energy is 32 (arbitrary units), the fragmentation associated with the LNDFH II2α product ion is also unique. For both protonated and methylated species, the abundance and type of fragmentation for the LNDFH II isomer are relatively greater compared to the LNDFH I isomer.

[0144] To normalize the retention time of glycans, a dextran ladder standard solution was prepared and tagged with a novel tagging reagent. Dextran ladder standards are frequently used in glycan analysis to define the retention time of glycans in terms of glucose units. As shown in FIGS. 27A - 27D, the dextran ladder was separated, detected by fluorescence, and each peak was defined in terms of the number of glucose units. In another experiment not described herein, the mass - to - charge ratio of the analyte at each peak was further verified by mass spectrometry. Based on the data obtained with the tagged dextran ladder, a curve was created by plotting the logarithm of the glucose units against the retention time to obtain an equation (FIG. 28). The equation can then be used to convert the retention times of all glycan analytes obtained from subsequent experiments into glucose units. By describing glycan analytes in terms of glucose units, the retention times observed by one method can be compared with those obtained by other methods. In a particular method, since the retention times of glycan analytes are related to those of the dextran ladder, a single glucose - unit literature value can be assigned to all glycans. Finally, the glucose - unit values, despite using the method for data acquisition, can be used as a preliminary technique for glycan characterization and then used for more extensive free - radical - mediated characterization by mass spectrometry. In addition, the characterization of glycans by their observed glucose - unit values in relation to free - radical - mediated characterization by mass spectrometry enables a more complex and powerful technique for glycan characterization. As an example, by utilizing the HILIC - FLD - MS technique, the theoretical probability of observing a pair of structurally or stereochemically different glycans with the same glucose - unit value, the same exact mass, and the same mass spectrometric fragmentation pattern is extremely low.

[0145] Experiments involving the analysis of N-glycans derived from ribonuclease B (RNase B) were carried out. First, they were enzymatically released from asparagine residues by peptide-N-glycosidase F (PNGase F). RNase B is typically used as a positive control for endoglycosidases and is a high-mannose glycoprotein with an N-glycan consisting of the Man 5-9 (GlcNAc)2 structure. After enzymatic release, the high-mannose N-glycans were tagged by reductive amination reaction with a novel tag to eliminate the possibility of interconversion between reducing-end isomers. As depicted in the fluorescence chromatogram of the tagged N-glycans in Figure 30, the abundance of Man5(GlcNAc)2 was the highest, followed by Man6(GlcNAc)2, and the abundance of Man 7-9 (GlcNAc)2 was the lowest, which was consistent with the literature data obtained from glycoprotein manufacturers. During a more detailed examination of the HCD mass spectrum for Man5(GlcNAc)2, fragmentation provided evidence behind the nature of the isomeric structures. For example, the presence of Z 3α +Z 3β fragmentation under the loss of four mannose subunits could only be obtained by the structure shown in Figure 31A, rather than the linear isomeric counterpart. Additionally, the intensities of the loss of two mannose subunits and four mannose subunits were relatively lower than those of the loss of one mannose subunit, three mannose subunits, and five mannose subunits, which provided further evidence regarding the structure of the glycan. Due to the branched nature of the structure shown in Figure 31A, the fragmentation of one mannose subunit, three mannose subunits, and five mannose subunits is more likely to be induced. In contrast, the loss of two mannose subunits and four mannose subunits is more difficult and thus is induced by mobile protons, resulting in the loss of multiple external residue losses. This is multiple sugar fragmentations catalyzed by mobile charges. Moreover, the higher abundance of the fragmentation corresponding to the loss of two mannose subunits compared to the abundance corresponding to the loss of four mannose subunits may be due to the number of possible fragmentation pathways. For example, under the loss of two mannose subunits1,5 Since the X product ion has a total of six possible fragmentation pathways, the probability of such fragmentation is associated with the loss of four mannose subunits. 1,5 Compared to the total of two possible fragmentation pathways of the X product ion, it is higher. As shown in Figure 31B, which corresponds to the fragmentation of a labeled glycan methylated to produce a quaternary amine at the ionization site, the only possible pathways for the loss of two mannose subunits (and similarly for the loss of four mannose subunits) are after the partial fragmentation of the ionization site proposed to yield a mobile proton (Scheme 2). This finding provides further evidence to support the isomeric structure and branching position of the mannose subunits of Man5(GlcNAc)2, as shown in Figure 31A.

[0146] Similarly, the HCD mass spectrum of Man6(GlcNAc)2 provides evidence that the main isomer present on RNase B becomes bifurcated by the occurrence of a single sugar extension in the 3β unit derived from the Man5(GlcNAc)2 precursor (Figure 32A). In its protonated and labeled form (Figure 32A), HCD of Man6(GlcNAc)2 results in a highly extensive fragmentation, which is associated with the loss of one, two, three, and six mannose residues, but not with the loss of four and five mannose residues. Similarly, methylated and labeled Man6(GlcNAc)2 gives product ions similar to those of the protonated form by additional fragmentation occurring simultaneously with the partial loss of the ionization site (Figure 32B). Therefore, the proposed isomeric structure of Man6(GlcNAc)2 satisfies the fragmentation data, and vice versa.

[0147] Conclusion For optimal LC-MS analysis in relation to the fluorescent detection of glycans and the determination of free radical-mediated properties, the derivatization of such biomolecules must be carried out using tagging reagents, which are carefully defined initially by the incorporation of multiple important chemical sites. Among these sites are, as previously mentioned, fluorophore bases, ionization and methylation sites with high affinity for positively charged ions, glycan labeling sites that form stable covalent bonds, and free radical precursors that facilitate the occurrence of systematic fragmentation, all of which are lacking in commercially available tags collectively. The fluorophore bases show the fluorescence energy required for the detection and quantification capabilities of glycans in a quantity-dependent manner. The purpose of utilizing fluorescence is to maximize the sensitivity of detection and quantification methods by liquid chromatography. The basic sites enable the realization of positive charge induction by any of protonation, ion-dipole interaction, or methylation. The importance behind the mobilization of basic sites is to generate ionization during or prior to the analysis of glycans by mass spectrometry, thereby significantly enhancing the signal-to-noise ratio by mass spectrometry. The glycan labeling site is the most essential feature to include on the tag as it selectively reacts with the unique glycan reducing end. Finally, the free radical precursors enable the formation of low-energy nascent free radicals by dissociation techniques, such as high-energy collision dissociation (HCD) or collision-induced dissociation (CID). Ultimately, the free radicals react simultaneously with the glycans, resulting in reproducible, systematic, predictable, diagnostic, and efficient glycan fragmentation for the characterization, identification, and differentiation between glycans and their isomers. In summary, the rational mobilization of these chemical sites constructs not only a novel tag optimal for the quantification of glycans at typical low concentrations in samples obtained from glycoproteins but also the combination of high-sensitivity MS detection and the ability to determine free radical-mediated glycan properties. Moreover, this novel tag is designed to be chemically stable with a significantly longer shelf life than reagents that mobilize NHS-carbamate coupling sites.

[0148] A plurality of glycan samples with different properties were labeled and analyzed by hydrophilic liquid chromatography - fluorescence detection - mass spectrometry (HILIC - FLD - MS) to fully evaluate the ability of the new reagent. Maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltose consisting of maltoheptaose were pre - mixed into an equimolar mixture, and then labeling and fluorescence quantification were performed. Next, the longest linear maltose was subjected to high - energy collision dissociation to cause fragmentation rich in information through a free - radical - mediated pathway. Both of these cross - ring fragmentations and glycosidic cleavages were observed throughout the linear biopolymer. To further evaluate the ability of the new reagent in the generation of glycan fragmentation enabling the discrimination of the structures of branched isomers, lacto - N - difucosylhexao se I and lacto - N - difucosylhexao se II were pre - mixed, labeled, and subjected to high - energy collision dissociation. After separation of the labeled branched isomers by HILIC, differences in the abundance and type of fragmentation reflecting their structures became apparent due to fragmentation of both isomers. Finally, the new reagent successfully distinguished between the two branched isomers based on the product ions generated exclusively. The new reagent was further evaluated by first labeling the N - glycans enzymatically released from ribonuclease B. The labeled N - glycans were separated by HILIC, detected by fluorescence, and accurately characterized by free - radical - mediated characterization. Using HILIC for glycan separation in combination with fluorescence detection and tandem mass spectrometry for the induction of fragmentation results in a powerful and modern analytical ability finally provided by the new reagent. The same analytical ability is also contemplated for other reagents described herein. Therefore, with the evolution of this new analytical technique, further development in glycan analysis with the potential for research on glycans as human disease biomarkers, and improvement of quality - assurance assays for biopharmaceuticals are expected to be possible.

[0149] It should be understood that the present invention is not limited to the specific embodiments of the present invention described above, as variant forms of certain embodiments can be made and can be further included within the scope of the appended claims.

[0150] The present invention is further described, without limitation, by the following numbered paragraphs.

[0151] 1) R 1 and A, and optionally R 2 substituted compounds of formula (Ia) or (Ib): [Chemical formula] [wherein, [Chemical formula] is a single bond or a double bond, [Chemical formula] when is a double bond, X 1 is C, X 2 is C or N, or X 2 is C, X 1 is N, [Chemical formula] when is a single bond, X 1 is NH, N-alkyl, O or NO, X 2 is C=O, or X 1 is NH, N-alkyl or NO, X 2 is O, or X 1 is C=O, X 2 is NH, N-alkyl, O or NO, or X 1 is O, X 2 is NH, N-alkyl or NO, X is CH, N or CR 9 where, R 1 is [Chemical formula]

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0152] 2) R 2 The compound according to item 1, wherein it is replaced by

[0153] 3) Formula (Ic)

Chemical formula

[0154] 4) Formula (Id), formula (Ie) or formula (If)

Chemical formula

Chemical formula

[0155] 5)

Chemical formula

[0156] 6)

Chemical formula

[0157] 7)

Chemical formula

Chemical formula

[0158] 8)

Chemical formula

Chemical formula

[0159] 9) R1 is

Chemical formula

Chemical formula

[0160] 10) R2 is

Chemical formula

[0161] 11) A is NH2,

Chemical formula

[0162] 12) X is CH or N, the compound according to item 5.

[0163] 13)

Chemical formula

Chemical formula

[0164] 14) A method for quantifying and / or characterizing a glycan, a) obtaining the compound according to item 1; b) contacting the compound with the glycan, thereby forming a labeled glycan; A method comprising:

[0165] 15) The method according to item 14, further comprising dissociating the labeled glycan to form glycan fragmentation.

[0166] 16) The method according to item 15, wherein the dissociation is collision-induced dissociation (CID) or high-energy collision dissociation (HCD).

[0167] 17) The method according to item 14, further comprising analyzing the labeled glycan or glycan fragmentation using a device such as a fluorometer, a mass spectrometer, or a liquid chromatography device, and a device capable of detecting fluorescence and / or absorbance as needed.

[0168] 18) The method according to item 14, wherein the glycan contains a reducing end.

[0169] 19) The method according to item 14, wherein the glycan is other than an N-glycan.

[0170] 20) The method according to item 14, wherein the device is ultra-high performance liquid chromatography (UPLC), a linear quadrupole ion trap (LTQ-XL) mass spectrometer, a Q Exactive Orbitrap mass spectrometer, or liquid chromatography-mass spectrometry (LC-MS).

[0171] 21) The method according to item 14, wherein the device is equipped with an electrospray ionization (ESI) source, a heated electrospray ionization (HESI) source, and / or a fluorescence detector.

[0172] 22) The compound according to item 1 for use in the preparation of a labeled glycan or glycan fragmentation, or for use in the detection or quantification of a glycan.

[0173] It is to be understood that the invention is not limited to the specific embodiments of the invention described above, as variations of particular embodiments may be made and may still be within the scope of the appended claims.

Claims

1. R １ and replaced by A, and optionally R ２ a compound of formula (Ia) or (Ib) which is replaced by 【Chemical Formula 101】 wherein, 【Chemical Formula 102】 is a single bond or a double bond, 【Chemical Formula 103】 when is a double bond, X １ is C, X ２ is C or N, or X ２ is C, X １ is N, 【Chemical Formula 104】 when is a single bond, X １ is NH, N-alkyl, O or NO, X ２ is C=O, or X １ is NH, N-alkyl or NO, X ２ is O, or X １ is C=O, X ２ is NH, N-alkyl, O or NO, or X １ is O, X ２ is NH, N-alkyl or NO, X is CH, N or CR ９ wherein, R １ is 【Chemical Formula 105】 selected from the group consisting of or derivatives thereof, Y １ and Y ２ are H, O, OH, CH ３ , N ３ , CN, NH ２ , R ３ -(CH ２ ) ｙ CO(CH ２ ) ｐ CH ３ , R ３ -(CH ２ ) ｙ COH, R ３ -(CH ２ ) ｙ -NH ２ , N[(CH ２ ) ｙ (CH ３ )] ２ , R ３ -(CH ２ ) ｙ -N[(CH ２ ) ｐ CH ３ )] ２ , COOH, R ３ -(CH ２ ) ｙ , R ３ -(CH ２ ) ｙ -COO(CH ２ ) ｐ CH ３ , R ３ -(CH ２ ) ｙ , CN, R ３ -(CH ２ ) ｙ -N ３ , 【Chemical 106】 【Chemical 107】 【Chemical 108】 【Chemical 109】 【Chemical 110】 【Chemical 111】 【Chemical 112】 【Chemical 113】 are each independently selected from the group consisting of, provided that when Y １ or Y ２ is carbonyl, the other is not carbonyl, or u and v are 0, and Y １ and Y ２ together form 【Chemical Formula 114】 a structure selected from the group consisting of, or Y １ and Y ２ are R １ independently selected from the group defined in, and function as a linker for a group, and the linker is 【Chemical Formula 115】 selected from the group consisting of 【Chemical Formula 116】 is a halogen Z is a counterion R ２ is 【Chemical Formula 117】 selected from the group consisting of A is 【Chemical Formula 118】 selected from the group consisting of R ３ is independently selected from the group consisting of substituted nitrogen, oxygen, carbonyl, amide, ester, ether, urea, hydrazide, carbamate, carbonate, thiocarbonate, thiol, thiourea, sulfur, sulfoxide, and sulfone R ４ 、R ５ 、and R ６ are H, OH, O, CN, N ３ 、COOH, alkyl, t-butyl, sec-butyl, isobutyl, isopropyl, tetrahydropyran, alkylamino, alkylsulfonic acid, alkylphosphonic acid, R ３ -(CH ２ ) ｙ CO(CH ２ ) ｐ CH ３ 、R ３ -(CH ２ ) ｙ COH, NH ２ , R ３ -(CH ２ ) ｙ -NH ２ , N[(CH ２ ) ｙ (CH ３ )] ２ , R ３ (CH ２ ) ｙ -N[(CH ２ ) ｐ CH ３ ) ２ , R ３ (CH ２ ) ｙ -COOH, R ３ (CH ２ ) ｙ -COO(CH ２ ) ｐ CH ３ , R ３ (CH ２ ) ｙ -R ３ (CH ２ ) ｙ -N ３ , CH ３ , 【Chemical Formula 119】 【Chemical Formula 120】 【Chemical Formula 121】 【Chemical Formula 122】 【Chemical Formula 123】 【Chemical Formula 124】 【Chemical Formula 125】 【Chemical Formula 126】 are each independently selected from the group consisting of, or R ４ and R ５ may, together with the nitrogen to which they are attached, form a 5- to 8-membered saturated or partially unsaturated ring optionally substituted, or R4 and R ５ are R １functions as a linker for a group selected independently from the groups defined herein, or R ４ and R ５ together form a structure selected from the group consisting of [Chemical Formula 127] where x, q, t, v, s, u, m, n, r, w, y, p, p x, q, t, v, s, u, m, n, r, w, y, p, p ａ and p ｂ are each independently an integer from 0 to 24, R ７ is a secondary, tertiary or reduced amine, ether, alcohol, R ８ is an alkylated or hydrogenated imide or oxygen, R ９ is [Chemical Formula 128] and where any hydrogen atom may be replaced by a fluorine atom, deuterium atom, and tritium atom, provided that when the structure is a compound of formula (Ib), formula (Ib) is substituted with R １ R ２ and A, provided that X １ is N, X ２ is C, R １ is [Chemical Formula 129] and when one of R ２ or A is H, R ２ or A is not [Chemical Formula 130] A compound. Compound. Claim 2 The compound according to claim 1, wherein R ２ is substituted. Claim 3 Formula (Ic) [Chemical Formula 131] The compound according to claim 1, having

4. Formula (Id), formula (Ie) or formula (If) 【Chemical Formula 132】 【Chemical Formula 133】 The compound according to claim 1, having wherein X ２ is NH, N-alkyl or NO.

5. 【Chemical Formula 134】 The compound according to claim 1, which is

6. 【Chemical Formula 135】 The compound according to claim 1, which is

7. 【Chemical Formula 136】 【Chemical Formula 137】 The compound according to claim 1, which is

8. 【Chemical Formula 138】 【Chemical Formula 139】 The compound which is where X, A, R １ , and R ２ are defined as described in claim 1. compound.

9. R １ is one of the following structures 【Chemical Formula 140】 selected from where R is independently selected from H, alkyl, alkylamino, ester, ether, OH, =O, COOH, NH2, alkylphosphonic acid, and halide. The compound according to claim 1.

10. R １ is 【Chemical Formula 141】 The compound according to claim 1.

11. R ２ is 【Chemical Formula 142】 The compound according to claim 1.

12. R ２ is 【Chemical Formula 143】 The compound according to claim 1.

13. A is NH ２ , 【Chemical Formula 144】 The compound according to claim 1.

14. A is NH ２ , 【Chemical Formula 145】 The compound according to claim 1.

15. The compound according to claim 5, wherein X is CH or N.

16. 【Chemical Formula 146】 【Chemical Formula 147】 The compound according to claim 1.

17. A method for quantifying and / or characterizing a glycan, comprising: a) obtaining the compound according to claim 1; and b) contacting the compound with the glycan to thereby form a labeled glycan. A method comprising the steps of:

18. The method according to claim 17, further comprising dissociating the labeled glycan to form glycan fragmentation.

19. The method according to claim 18, wherein the dissociation is collision-induced dissociation (CID) or high-energy collision dissociation (HCD).

20. The method according to claim 17, further comprising analyzing the labeled glycan or glycan fragmentation using a fluorometer, a mass spectrometer, or an instrument such as a liquid chromatography instrument, and an instrument capable of detecting fluorescence and / or absorbance as necessary.

21. The method according to claim 17, wherein the glycan contains a reducing end.

22. The method according to claim 17, wherein the glycan is other than an N-glycan.

23. The method according to claim 17, wherein the instrument is an ultra performance liquid chromatography (UPLC), a linear quadrupole ion trap (LTQ-XL) mass spectrometer, a Q Exactive Orbitrap mass spectrometer, or a liquid chromatography-mass spectrometry (LC-MS).

24. The method according to claim 17, wherein the instrument is equipped with an electrospray ionization (ESI) source, a heated electrospray ionization (HESI) source, and / or a fluorescence detector.

25. The compound according to claim 1, for use in the preparation of a labeled glycan or glycan fragmentation, or for use in the detection and / or quantification of a glycan.

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