Fluorescent active radical tags for synchronized glycan quantification and characterization

Abstract

Compounds useful for quantification and / or characterization of glycans and other similar molecules are provided. Such compounds include a compound of formula (1a) or (1b) substituted with at least one TEMPO group or an analog thereof and a coupling site wherein X1, X2 and X may be one of NH, N-alkyl, O, NO, C = O, CH, N or CR9. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 348,795, filed on June 3, 2022, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention relates to fluorescent active free radical labels, including substituted compounds of formula (Ia) and formula (Ib):

[0004]

[0005] and methods of using these and related compounds.One such use is the quantification and characterization of glycans.

[0006] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes and to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein should not be construed as an admission that the reference is prior art to the present invention. Background Art

[0007] In recent decades, the study of glycans and related structures or glycomics has triggered significant thinking and research. A lot of attention has turned to the ability to quantify, distinguish and characterize glycans and their isomers. Unlike other biopolymer molecules such as peptides and nucleic acids, which involve subunits connected by a defined main chain composed of amide bonds and phosphodiester bonds, respectively, glycans can have monosaccharide subunits arranged in a branched manner through complex regiochemical and stereochemical connections. Since glycan structures are different and are described by their connection type, monosaccharide composition and overall configuration, many structural isomers and stereoisomers are possible. Unlike other biomolecules, glycans are involved in basic functions within life systems, such as major metabolism, signal transduction and structural effects. In addition, it is known that glycans change in structure and amount 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 developments in the field of glycomics may lead to the use of glycans as early biomarkers for certain human diseases in medical settings in the future.

[0008] Equally important is the analysis of glycosylation derived from biopharmaceuticals, which requires an analytical method to facilitate the characterization of glycans for monitoring and regulation purposes. Glycosylation of biopharmaceuticals is difficult to control because minor conditional differences in pH, cell line, dissolved oxygen, temperature, ammonia concentration, and manufacturing mode can affect glycosylation. Therefore, glycan characterization techniques are needed because the presence of certain glycosylation may affect safety, efficacy, half-life, immune response, and binding. For example, the monoclonal antibody drug cetuximab, which targets the epidermal growth factor receptor to inhibit the progression of colorectal cancer, is processed in the presence of gal-α(1→3)-gal, which causes allergic reactions in patients. Ultimately, the rapid development of improved and modern analytical techniques that can quantify and characterize glycans from a variety of sources across a wide range of industries is of significant benefit.

[0009] The study of glycans involves a variety of techniques, including high performance liquid chromatography (HPLC), ion mobility, electrophoresis, and nuclear magnetic resonance (NMR) spectroscopy. For glycan structural analysis, HPLC, ion mobility, and NMR spectroscopy require well-characterized glycan standards that must be sufficiently pure, which is challenging, time-consuming, and costly to obtain. In particular, NMR data are difficult to interpret because glycans involve many carbons and protons with similar chemical environments. Nevertheless, the use of HPLC for glycan quantification is the most powerful and optimal combination after glycan characterization by mass spectrometry. Electrospray ionization mass spectrometry is known for multiple dissociation techniques, minimal sample consumption, short acquisition time, high sensitivity, high mass accuracy, and high resolution. However, the ionization efficiency and fragmentation of free glycans by mass spectrometry are relatively poor compared to labeled glycans. Therefore, to further aid in the analysis of glycans, many research laboratories have adopted the development and use of labeling reagents. However, these labeling reagents cannot provide glycan quantification and characterization functions simultaneously and involve additional challenges such as poor chemical stability and limited analysis of a single class of glycans. In addition, to compensate for the presence of protein amines, some labeling reagents that recruit NHS-carbamate groups to achieve rapid labeling require large amounts of reagents that are up to 133 times the amount required to label glycans. Therefore, a new labeling reagent for improved glycan analysis is needed. Summary of the invention

[0010] The present invention provides compounds that can be used to quantify and / or characterize polysaccharides and other similar molecules. Such compounds include compounds of formula (Ia) or (Ib),

[0011]

[0012] It is substituted with at least one TEMPO group or its analog and a coupling site, wherein X 1 , X 2and X can be NH, N-alkyl, O, NO, C=O, CH, N or CR 9 one of the.

[0013] Other such compounds include those having the following structures:

[0014]

[0015] Where R 1 is the glycan labeling site and R 2 and A may independently be hydrogen or a substituent.

[0016] The present invention also relates to a method for quantifying and / or characterizing a glycan, comprising obtaining a compound and contacting the compound with the glycan. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described below are for illustration purposes only and are not intended to limit the scope of the present invention.

[0018] Figure 1 Depicted is an exemplary novel fluorescent tag (Glyc·RadiFluor) coupled with a radical mechanistic approach for glycan characterization and other important sites for glycan analysis by HILIC-ESI+MS.

[0019] Figure 2 The synthetic route of a new fluorescent tag (Glyc·RadiFluor) is depicted.

[0020] Figure 3 The present invention describes the synthesis of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. 1 HNMR spectrum.

[0021] Figure 4 The present invention describes the synthesis of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. 13 CNMR spectrum.

[0022] Figure 5 Depicted is the mass spectrum of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide, where this compound is detected at m / z 456.

[0023] Figure 6 Depicted is the MS of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide after being subjected to collision-induced dissociation to produce loss of TEMPO and residual fragment at m / z 300. 2 Spectrum.

[0024] Figure 7 The mass spectrum of the proton adduct of the conjugate detected as m / z 782 is depicted.

[0025] Figure 8 Depicted is the MS of the conjugate after being subjected to collision-induced dissociation to produce TEMPO loss and a residual fragment of m / z 626. 2 Spectrum.

[0026] Fig. 9 Depicted is the MS of the conjugate after being subjected to collision-induced dissociation to produce labeled lactose fragments. 3 Spectrum.

[0027] Fig.10 Depicted ThermoFisher Scientific TM NanoDrop TM The nM range of fluorescent labels ranged from 500 nM to 100 μM (NanoDrop TM ) Calibration curve of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide in fluorescence measurement. The excitation wavelength was set to 365±10 nm and the emission wavelength was set to 520 nm.

[0028] Fig.11 Depicted in nM range (Cary ) in the Agilence Cary Fluorescence measurements were performed on fluorescent labels with concentrations ranging from 1 nM to 1 μM. The excitation wavelength was set to 390 ± 5 nm, and the emission wavelength was set to 520 ± 5 nm.

[0029] Fig.12 Depicted is the overlay of fluorescence detection at 515 nm following excitation at 385 nm and reveals the separation of a mixture of six reagent-labeled analytes on two columns by ultra-performance liquid chromatography: (black) glycan BEH amide and (red) discovery C 18 .

[0030] Fig.13 Depicted is the total ion current spectrum resulting from heated electrospray ionization of analytes separated on a glycan BEH amide column.

[0031] Fig.14 Depicted is the mass spectrum at 8.53 minutes, which is consistent with Fig.13 The results shown in and reveal the expected m / z peak for reagent-labeled maltose; [M+H]+ =782.45 and [M+2H] 2+ =391.73.

[0032] Fig.15 Depicted is the mass spectrum at 11.31 minutes, which is consistent with Fig.13 The results shown in and reveal the expected m / z peak for reagent-labeled maltotriose; [M+H] + =944.51 and [M+2H] 2+ =472.76.

[0033] Fig.16 Depicted is the mass spectrum at 14.31 minutes, which is consistent with Fig.13 The results shown in and reveal the expected m / z peak for reagent-labeled maltotetraose; [M+H] + =1106.56 and [M+2H] 2+ =553.78.

[0034] Fig.17 Depicted is the mass spectrum at 16.96 minutes, which is consistent with Fig.13 The results shown in and reveal the expected m / z peak for reagent-labeled maltopentaose; [M+H] + =1268.61 and [M+2H] 2+ =634.81.

[0035] Fig.18 Depicted is a mass spectrum at 19.15 minutes that correlates with the results shown in FIG. 113 and reveals the expected m / z peak for reagent-labeled maltohexaose; [M+H] + =1430.67 and [M+2H] 2+ =715.84.

[0036] Fig.19 Depicted is the mass spectrum at 21.06 minutes, which is consistent with Fig.13 The results shown in and reveal the expected m / z peak for reagent-labeled maltoheptaose; [M+H] + =1592.72 and [M+2H] 2+ =796.87.

[0037] Fig. 20Depicted is a fluorescence chromatogram showing the separation of about 46 pmol of an injection of six GRF-derived maltose with variable lengths prepared in an equimolar mixture (about 7.7 pmol per analyte); the peaks are identified as follows: (1) maltose, (2) maltotriose, (3) maltotetraose, (4) maltopentaose, (5) maltohexaose, and (6) maltoheptaose. Based on peak integration (see Table 2), the average analyte amount was 7.7 ± 0.2 pmol.

[0038] Fig.21 The nomenclature of glycan fragment ions is depicted.

[0039] Fig. 22 A novel nomenclature for the fragment ions of the reducing-end open-chain isomers is depicted.

[0040] Figures 23A and 23B depict: (A) the fragmentation pattern of monoprotonated GRF-derivatized maltoheptaose, and (B) the HCD spectrum of monoprotonated GRF-derivatized maltoheptaose, wherein the NCE is 30 (arbitrary units); 0,2 The fragments between X and Z and Z-nH2O- x OH is associated. (In this figure and in other figures, PIS and IS are abbreviations for partially ionized site and ionized site, respectively.)

[0041] Figures 24A and 24B depict: (A) the fragmentation pattern of monomethylated GRF-derivatized maltoheptaose, and (B) the HCD spectrum of monomethylated GRF-derivatized maltoheptaose, wherein the NCE is 30 (arbitrary units).

[0042] Fig.25 Depicted is HILIC separation and fluorescence detection of a sample containing unknown amounts of the branched isobaric glycans lacto-N-difucohexaose I (LNDFH I; peak 2) and lacto-N-difucohexaose II (LNDFH II; peak 1) after labeling with GRF.

[0043] Fig.26A , 26B , 26C and 26D depict: (A) the fragmentation pattern of monoprotonated GRF-derivatized LNDFH II, (B) the HCD spectrum of monoprotonated GRF-derivatized LNDFH II, where the NCE is 28 (arbitrary units), (C) the fragmentation pattern of monoprotonated GRF-derivatized LNDFH I, and (D) the HCD spectrum of monoprotonated GRF-derivatized LNDFH I, where the NCE is 28 (arbitrary units).

[0044] Fig.27A , 27B, 27C and 27D depict: (A) fragmentation pattern of monomethylated GRF-derived LNDFH II, (B) HCD spectrum of monomethylated GRF-derived LNDFH II, where NCE is 32 (arbitrary units), (C) fragmentation pattern of monomethylated GRF-derived LNDFH I, and (D) HCD spectrum of monomethylated GRF-derived LNDFH I, where NCE is 32 (arbitrary units).

[0045] Fig.28 Depicted is the fluorescence chromatogram of a neutral GRF-derivatized dextran ladder separated by hydrophilic interaction liquid chromatography (HILIC).

[0046] Fig.29 Depicted is an example logarithmic plot of glucose unit values ​​versus retention time for a neutral GRF-derivatized dextran ladder specifically designed for HILIC-FLD analysis.

[0047] Fig.30 Depicted is a fluorescence chromatogram depicting the separation of GRF-derived RNase B high mannose N-glycans; the peaks are identified as follows: (1) Man5(GlcNAc)2, (2) Man6(GlcNAc)2, (3) Man7(GlcNAc)2, (4) Man8(GlcNAc)2, (5) Man9(GlcNAc)2.

[0048] Fig.31A and 31B Depicted are: (A) HCD spectrum of monoprotonated GRF-derivatized Man5(GlcNAc)2, where NCE is 25 (arbitrary units), and (B) HCD spectrum of monoprotonated GRF-derivatized Man5(GlcNAc)2, where NCE is 28 (arbitrary units).

[0049] Fig.32A and 32B Depicted are: (A) HCD spectrum of monoprotonated GRF-derivatized Man6(GlcNAc)2, where NCE is 25 (arbitrary units), and (B) HCD spectrum of monoprotonated GRF-derivatized Man6(GlcNAc)2, where NCE is 28 (arbitrary units). DETAILED DESCRIPTION

[0050] It should be understood that the terminology used herein is for the purpose of describing specific embodiments and is not intended to be limiting. Further, although any methods, devices and materials similar or equivalent to the methods, devices and materials described herein can be used in the practice or testing of the present invention, certain methods, devices and materials are now described.

[0051] Unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It should be further understood that the terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and in the present disclosure, and will not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0052] It should be understood that in describing the present invention, many techniques and steps are disclosed. Each of these techniques and steps has a separate benefit, and each can be used in combination with one or more techniques and steps, or in some cases, all other disclosed techniques can be used. Therefore, for the sake of clarity, this specification will avoid repeating every possible combination of individual steps in an unnecessary manner. However, the specification and claims should be read, and it should be understood that such combinations are fully within the scope of the present invention and claims.

[0053] The present disclosure is further illustrated by the following description, which should not be construed as limiting the scope or spirit of the present disclosure to the specific procedures described herein. It should be understood that descriptions are provided to illustrate certain embodiments, and it is not intended to limit the scope of the present disclosure thereby. It should be further understood that, without departing from the spirit of the present disclosure and / or the scope of the appended claims, various other embodiments, variations and equivalents thereof that can be thought of by those skilled in the art may be resorted to. For example, it is contemplated that each embodiment disclosed herein is applicable to each embodiment in other disclosed embodiments.

[0054] The present invention relates to methods and compositions for glycan analysis of complex solutions comprising proteins and cells in a biological sample.

[0055] Provided is a compound of formula (Ia) or (Ib),

[0056]

[0057] Its R 1 and A, and optionally substituted by R 2 replace,

[0058] in

[0059] is a single bond or a double bond;

[0060] if is a double bond, then:

[0061] X 1 is C, and X 2 is C or N, or

[0062] X 2 is C, and X 1 is N;

[0063] if is a single bond, then:

[0064] X 1 is NH, N-alkyl, O or NO, and X 2 is C=O, or

[0065] X 1 is NH, N-alkyl or NO, and X 2 is O, or

[0066] X 1 is C=O, and X 2 is NH, N-alkyl, O or NO, or

[0067] X 1 is O, and X 2 is NH, N-alkyl or NO;

[0068] X is CH, N or CR 9 ;

[0069] R 1 Selected from the group consisting of:

[0070] or optionally a derivative thereof;

[0071] Y 1 and Y 2 Each independently selected from the group consisting of:

[0072] H, O, OH, CH3, N3, CN, NH2, R 3 -(CH2) y CO(CH2) p CH3, R 3 -(CH2) y COH 、 R 3 -(CH2) y -NH2、N[(CH2) y (CH3)]2, R 3 -(CH2) y -N[(CH2) p CH3]2, COOH, R 3 -(CH2) y -COOH, R 3 -(CH2) y -COO(CH2) pCH3, R 3 -(CH2) y -CN、R 3 -(CH2) y -N3,

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Or u and v are 0, and Y 1 and Y 2 together forming a structure selected from the group consisting of:

[0080]

[0081] or Y 1 and / or Y 2 Acting as and independently selected from R 1 A linker connected to a group defined in the group, wherein the linker is selected from the group consisting of:

[0082]

[0083] The condition is that if Y 1 or Y 2 is a carbonyl group, then the other is not a carbonyl group,

[0084] It is a halogen;

[0085] Z is a counterion;

[0086] R 2 Selected from the group consisting of:

[0087]

[0088] A is selected from the group consisting of:

[0089]

[0090] R 3each 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;

[0091] R 4 , R 5 and R 6 Each is independently selected from the group consisting of: H, OH, O, CN, N3, COOH, alkyl, tert-butyl, sec-butyl, isobutyl, isopropyl, tetrahydropyran, alkylamino, alkylsulfonic acid, alkylphosphonic acid, R 3 -(CH2) y CO(CH2) p CH3, R 3 -(CH2) y COH 、 NH2、R 3 -(CH2) y -NH2、N[(CH2) y (CH3)]2, R 3 -(CH2) y -N[(CH2) p CH3]2, R 3 -(CH2) y -COOH, R 3 -(CH2) y -COO(CH2) p CH3, R 3 -(CH2) y -CN、R 3 -(CH2) y -N3, CH3,

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] or R 4 and R 5 and the nitrogen to which it is attached can together form an optionally substituted 5- to 8-membered saturated or partially unsaturated ring; or R 4and / or R 5 Acting as and independently selected from R 1 A linker connected to a group defined in the group, wherein the linker is selected from the group consisting of:

[0099]

[0100] x, q, t, v, s, u, m, n, r, w, y, p, p a and p b Each independently is an integer from 0 to 24;

[0101] R 7 It is a secondary amine, a tertiary amine or a reduced amine, an ether, or an alcohol;

[0102] R 8 is an alkylated or hydrogenated imide or oxygen;

[0103] R 9 yes

[0104]

[0105]

[0106] Any hydrogen atom can be replaced by a fluorine, deuterium or tritium atom.

[0107] Provided that if the structure is a compound of formula (Ib), then formula (Ib) is replaced by R 1 , R 2 and A substituted;

[0108] The condition is that if X1 is N and X2 is C, and R1 is And R 2 or one of A is H, then R 2 or each of A is Substituents other than .

[0109] In an embodiment, R 2 and A is divided by Other substituents.

[0110] In an embodiment, R 2 and A is divided by Substituents other than .

[0111] In an embodiment, R 2 and A is divided by and / or Substituents other than 2 Or when one of A is hydrogen.

[0112] In some embodiments, the compound is R 2In some embodiments, the compound is substituted with A. In a certain embodiment, the compound is substituted with R 2 In other embodiments, the compound is substituted with R 2 and A are substituted.

[0113] In one embodiment, R 1 , R 2 and A is not isocyanate or succinimidyl carbamate. In another embodiment, R 1 , R 2 and A is not isocyanate or its derivatives. In another embodiment, R 1 , R 2 and A is not succinimidyl carbamate or a derivative thereof.

[0114] In some embodiments, the compound has formula (Ic)

[0115]

[0116] In an embodiment, the compound has formula (Id), formula (Ie) or formula (If)

[0117]

[0118]

[0119] Where X 2 is NH, N-alkyl or NO.

[0120] In some embodiments, the compound is

[0121]

[0122] In some embodiments, the compound is

[0123]

[0124] In an embodiment, the compound is

[0125]

[0126] Provided is a compound, which is

[0127]

[0128] And X, A, R 1 and R 2 Defined as previously described.

[0129] In an embodiment, R1 is

[0130]

[0131] In an embodiment, R2 is

[0132]

[0133] In an embodiment, A is

[0134]

[0135] In an embodiment, X is CH or N.

[0136] In one embodiment, R 6 Each is independently selected from the group consisting of H, alkyl, tert-butyl, sec-butyl, isobutyl, isopropyl, alkylamino, alkylsulfonic acid or alkylphosphonic acid.

[0137] In an embodiment, the compound is a fluorescent tag.

[0138] In an embodiment, the compound is:

[0139]

[0140]

[0141] In an embodiment, x, q, t, v, s, u, m, n, r, w, y, p, p a and p b Each is independently an integer from 0 to 18, 0 to 12, 0 to 6, 0 to 4, 0 to 2 or 1 to 2.

[0142] In an embodiment, A is

[0143] Substituents other than 2 When it is hydrogen.

[0144] The counter ions include alkali metal lithium (Li + ), 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 protons (H + The counter ions further include alkaline earth metal beryllium (Be 2+ ), magnesium (Mg 2+), calcium (Ca 2+ ), Sr 2+ ), barium (Ba 2+ ) and radium (Ra 2+ ) or any transition metal in any of its various oxidation states (for example, but not limited to, Cu + and Cu 2+ ) and any composite ions formed therefrom. The counter ions may further include halides, fluorides (F - ), 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- ), 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 2- ) or any hypohalite (FO - or ClO - or BrO - or IO - or AtO - ), any rock salt (FO2 - or ClO2 - or BrO2 - or IO2 - or AtO2 - ) or any halogen acid salt (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 - In an embodiment, the counterion Z can be any ion with an opposite charge to the species presented. For example, if the species has a 2+ charge, the counterion Z can be any ion from any group with an opposite charge in the form of 2-.

[0145] A method for quantifying and / or characterizing glycans is provided, the method comprising:

[0146] a) obtaining the above compound;

[0147] b) contacting the compound with the glycan, thereby forming a labeled glycan.

[0148] In embodiments, the method further comprises dissociating the labeled glycans to form glycan fragments.

[0149] In some embodiments, the dissociation is collision induced dissociation (CID) or high energy collision dissociation (HCD).

[0150] In embodiments, the method further comprises quantifying and / or characterizing the glycans thereby.

[0151] In some embodiments, the glycan comprises a reducing terminus.

[0152] In some embodiments, the glycan is not an N-glycan.

[0153] In an embodiment, the method further comprises analyzing the labeled glycan or glycan fragments with an instrument that is a fluorometer, a mass spectrometer, or a liquid chromatography instrument, optionally and with an instrument capable of detecting fluorescence and / or absorbance.

[0154] The instrument may be an ultra-high performance liquid chromatograph (UPLC), a linear quadrupole ion trap (LTQ-XL) mass spectrometer, a QExactive orbitrap mass spectrometer or a liquid chromatography-mass spectrometer (LC-MS).

[0155] In some embodiments, the instrument is equipped with an electrospray ionization (ESI) or heated electrospray ionization (HESI) source and / or a fluorescence detector.

[0156] Provided is a compound for glycan detection or glycan quantification, or for preparing labeled glycans or glycan fragments.

[0157] In one aspect, the invention provides a method for glycan analysis of a sample, the method comprising the steps of contacting the glycan with a compound of the invention and scanning the analyte by mass spectrometry to detect and identify the presence of the glycan.

[0158] The present invention also provides for quantifying and / or characterizing samples containing compounds such as biomolecules, non-biological molecules, proteins, peptides or amino acids in the same manner as glycans. In some embodiments, these compounds have functional groups similar to the reducing ends of glycans.

[0159] In one embodiment, the sample comprises at least one protein solution. In one embodiment, the sample comprises at least one cell population.

[0160] In one exemplary method of the invention, a compound disclosed herein is contacted with a glycan of interest. In this step, the chemical reaction can form a covalent bond between the glycan of interest and the compound, thereby labeling the glycan. In another exemplary step of the invention, a liquid chromatography instrument equipped with a fluorescence detector is used to quantify the amount of glycans in a given sample by the fluorescence caused by the labeled glycans. Next, for example, a mass spectrometer is used to induce glycan fragmentation, which then provides data that can be used to characterize the glycans of interest.

[0161] definition

[0162] "Glycans" are sugars, such as oligosaccharides and polysaccharides, which can be monomers or polymers of sugar residues generally connected by glycosidic bonds. In some embodiments, the terms "glycans", "oligosaccharides" and "polysaccharides" can refer to the carbohydrate portion of a glycoconjugate (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 "glycans" includes homopolymers and heteropolymers of sugar residues. The term "glycans" also encompasses glycan components of glycoconjugates (e.g., glycoproteins, glycolipids, proteoglycans, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from glycoconjugates. Polysaccharides can include O-linked polysaccharides or N-linked polysaccharides. The O-linked polysaccharides have a structure in which the polysaccharides are connected to the side chains of amino acid residues serine (Ser) or threonine (Thr) in the protein through the -OH group contained in the amino acid side chain. O-linked polysaccharides are divided into one to eight types according to the core structure. The N-linked polysaccharides refer to polysaccharides connected to the nitrogen atom of the amide group in the side chain of the asparagine residue (Asn) of the protein. The N-linked polysaccharides include polysaccharides formed with mannose as a base point, and examples thereof include two-branched polysaccharides, three-branched polysaccharides and four-branched polysaccharides, etc. In addition, the N-linked polysaccharides can be divided into basic, high mannose, hybrid and complex types, etc. according to their structure.

[0163] As used herein, the term "alkyl" alone or in combination with other groups refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon group of one to twenty carbon atoms, one to sixteen carbon atoms, and one to ten carbon atoms in another embodiment. In some embodiments, the alkyl group is a lower alkyl group.

[0164] The term "lower alkyl" alone or in combination with other groups refers to a branched or straight chain alkyl group of one to nine carbon atoms, in one embodiment one to six carbon atoms, in another embodiment one to four carbon atoms, and in another embodiment four to six carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 3-methylbutyl, n-hexyl, 2-ethylbutyl, and the like.

[0165] As used herein, the term "alkoxy" means alkyl-O--; and "alkanoyl" means alkyl-CO--. Alkoxy substituents or alkoxy-containing substituents may be substituted, for example, with one or more alkyl or halide groups.

[0166] As used herein, the term "halogen" means fluoro, chloro, bromo, or iodo, or in some embodiments, fluoro, chloro, or bromo.

[0167] As used herein, "aralkyl" refers to an alkyl group substituted by at least one aryl group. Similarly, as used herein, "heteroaralkyl" refers to an alkyl group substituted by at least one heteroaryl group.

[0168] The term "cycloalkyl" refers to a monovalent monocarbocyclic group or polycarbocyclic group of three to ten, three to six carbon atoms in one embodiment. This term is further illustrated by groups such as the following: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl, indanyl, etc. In one embodiment, a "cycloalkyl" moiety can be optionally substituted by one, two, three or four substituents. Unless specifically specified otherwise, each substituent can be independently alkyl, alkoxy, halogen, amino, hydroxyl, aryl, heteroaryl or oxygen. The example of a cycloalkyl group includes but is not limited to the cyclopropyl optionally substituted, the cyclobutyl optionally substituted, the cyclopentyl optionally substituted, the cyclopentenyl optionally substituted, the cyclohexyl optionally substituted, the cyclohexenyl optionally substituted, the cycloheptyl optionally substituted, or the cycloalkyl moiety specifically illustrated herein.

[0169] The term "aryl" refers to an aromatic monocyclic or polycyclic group of 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, and the like.

[0170] Alkyl, lower alkyl, aryl and spiroalkyl can be substituted or unsubstituted. In addition, aralkyl and heteroaralkyl can also be substituted by substituents except aryl or heteroaryl. When substituted, generally there will be 1 to 4 substituents, for example. These substituents can optionally form a ring with the alkyl, lower alkyl or aryl to which they are connected. Substituents may include, for example, 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 alcohols (e.g., hydroxy, hydroxyalkyl, aryl(hydroxy)alkyl), ethers (e.g., alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl, in other embodiments, such as methoxy and ethoxy), aldehydes (e.g., formaldehyde), ketones (e.g., alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acids (e.g., carboxyl, carboxylalkyl), acid derivatives such as esters (e.g., alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amides (e.g., aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl), carbamates (e.g., alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono- or di-alkyl aminocarbonyloxy, arylaminocarbonyloxy) and urea (e.g. mono- or dialkylaminocarbonylamino or arylaminocarbonylamino); nitrogen-containing groups such as amines (e.g. amino, mono- or dialkylamino, aminoalkyl, mono- or dialkylaminoalkyl), 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 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, pyrrononyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, hexahydroazepine yl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, pyridopyridinyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl and carbolinyl).

[0171] The term "heteroaryl" refers to an aromatic monocyclic or polycyclic group of 5 to 12 atoms having at least one aromatic ring containing one, two or three ring heteroatoms selected from N, O and S, the remaining ring atoms being C. Examples of such groups include, but are not limited to, pyridyl, pyrazinyl, pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, oxazolyl, thiazolyl, and the like.

[0172] The heteroaryl groups described above may be independently substituted with one, two or three substituents. Substituents may include, for example, 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 alcohols (e.g., hydroxy, hydroxyalkyl, aryl(hydroxy)alkyl), ethers (e.g., alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl), aldehydes (e.g., formaldehyde), ketones (e.g., alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acids (e.g., carboxyl, carboxylalkyl), acid derivatives such as esters (e.g., alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amides (e.g., aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl), carbamates (e.g., alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono- or di-alkylaminocarbonyloxy, arylamino alkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, alkylsulfonylalkyl ... and heterocyclic groups containing one or more heteroatoms (e.g., thiophenyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyrrononyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, hexahydroazepine yl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, pyridopyridinyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl, benzothiazolyl and carbolinyl).

[0173] Where any group is referred to as optionally substituted, the group may be substituted or unsubstituted. Substitution may be by one or more of the specified substituents which may be the same or different. It will be appreciated that the number and nature of the substituents will be selected to avoid any sterically undesirable combinations.

[0174] When applied to any group, "optionally substituted" means that the group may be substituted, if desired, with one or more substituents, which may be the same or different. Examples of suitable substituents for "substituted" and "optionally substituted" moieties include halo, deuterated, C 1-6 Alkyl or C 1-3 Alkyl, hydroxyl, C 1-6 Alkoxy or C 1-3 Alkoxy, cyano, amino, nitro or SF5 (a known NO2 mimetic), aryl, heteroaryl, heterocyclic, C3-C6 cycloalkyl, C 1-3 Alkylamino, C 2-6 Alkenylamino, di-C 1-3 Alkylamino, C 1-3 Acylamino, di-C 1-3 Acylamino, carboxyl, C 1-3 Alkoxycarbonyl, carbamoyl, mono-C 1-3 Carbamoyl, di-C 1-3 Carbamoyl or wherein the alkyl part itself is halogenated in any of the above-mentioned.In the group containing Sauerstoffatom such as hydroxyl and alkoxyl group, Sauerstoffatom can be substituted by sulphur to form groups such as mercaptan (SH) and alkylthio (S-alkyl).Therefore, optional substituent comprises groups such as S-methyl.In alkylthio, sulphur atom can be further oxidized to form sulfoxide or sulfone, and therefore optional substituent comprises groups such as S (O)-alkyl and S (O)2-alkyl.

[0175] Thus, substituted groups include, for example, Cl, F, OMe, Me, COCH3, CONH2, NHC(O)CH(CH3)2, CO2CH2CH3, etc. In the case of aryl groups, the substitutions may be in the form of rings of adjacent carbon atoms in the aryl ring, for example, cyclic acetals such as O-CH2-O.

[0176] The optional substituent of any alkyl, alkenyl, alkynyl, alkoxy, alkylene or alkenylene described herein can be selected from C1-C3 alkoxy, halogen, hydroxyl, thiol, cyano, amino, amide, nitro and SF5, wherein alkoxy can be optionally substituted by halogen. Especially, the optional substituent can be selected from halogen, hydroxyl, thiol, cyano, amino, amide, nitro and SF5, more particularly fluorine or hydroxyl.

[0177] Typically, hydrogen is not considered a substituent. However, in selected embodiments, A and / or R 2 It's hydrogen.

[0178] Any range disclosed herein means that all percentiles, tens and integer unit quantities within the range are specifically disclosed as part of the present invention. Thus, for example, 0.01 to 50 means 0.02, 0.03 ... 0.09; 0.1; 0.2 ... 0.9; and 1, 2 ... 49 unit quantities are included as embodiments of the present invention.

[0179] In any disclosure herein, if a specific R group can be more than one functional group and the structure of the compound has more than one of the specific R groups, the R groups can each be a different functional group. For example, if the structure has two R 4 If one is hydrogen, then the other may be an alkyl. In a similar manner, each letter representing a number in a structure may be different in each case if it occurs multiple times in a single structure.

[0180] 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 (e.g., racemates), optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates, or mixtures of diastereomeric racemates. Optically active forms may be obtained, for example, by resolving racemates, by asymmetric synthesis, or asymmetric chromatography (chromatography using chiral adsorbents or eluents). The present invention encompasses all of these forms.

[0181] The compounds of the present invention can be prepared starting from commercially available starting materials and utilizing general synthetic techniques and procedures known to those skilled in the art.

[0182] A large number of dissociation techniques via MS have been noted, and the most recent ones are still under development. For example, collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) are well known to produce fragments via glycosidic bond cleavage. In contrast, ultraviolet photodissociation (UVPD) and high-energy collision dissociation (HCD) have previously been shown to produce more informative analyte fragments. Free radical-driven dissociation (RDD), electron capture dissociation (ECD), electron excited dissociation (EED), electron transfer dissociation (ETD), and electron escape dissociation (EDD), which are often grouped together as free radical-driven dissociation techniques, also show great potential for glycan structural analysis. Nevertheless, UVPD, MS n, ion mobility mass spectrometry and EDD remain the most effective in resolving mass spectra belonging to glycan isomers (especially stereoisomers, such as anomers and epimers). As shown in the following examples, by using MS n The combination of CID and reagents including free radical precursors allows for feasible, accurate and rapid glycan structural analysis. In addition, the gas phase ion / ion reaction that occurs in an improved mass spectrometer is demonstrated, showing the potential for future application in glycan structural analysis, and the mass spectrometer is capable of charge conversion of anionic analytes. Experiments are performed in the gas phase and condensed phase, where reagents using free radical precursors form complexes with selected analytes, followed by free radical-guided fragments. However, this method currently does not allow sensitive detection and quantification of glycans by fluorescence.

[0183] Recently, free radical activated glycan sequencing reagents (FRAGS) have been developed to react with and covalently link glycans at their respective reducing ends. FRAGS reagents contain free radical precursors (e.g., nitroxyl radical precursors such as 2,2,6,6-tetramethyl-1-piperidinyloxy or TEMPO) that, upon collision activation by labeled glycans, generate localized nascent free radicals that simultaneously induce predictable, diagnostic, and systemic glycan fragmentation. Furthermore, in addition to hydrazide or aminooxy labeling sites that react with the reducing ends of glycans, basic pyridyl sites for charge induction are recruited in FRAGS reagents. Previously, the first generation of FRAGS was methylated at the pyridine moiety (Me-FRAGS I) and covalently linked to isomeric oligosaccharides with subtle differences. Methylation induces localized charges that prevent gas-phase glycan rearrangements often observed in protonated species involving mobile charges. Subsequently, MS involving Me-FRAGS I was developed. 3Collision-induced dissociation (CID) spectra showed the ability to accurately and rapidly distinguish nine isomeric disaccharides and two isomeric tetrasaccharides. Although FRAGS-labeled glycans can be detected by liquid chromatography, they lack the fluorescent active moiety that would significantly improve the sensitivity of liquid chromatography detection and quantification by fluorescence. In addition, relative to the development of tags with free amino groups as coupling sites, the coupling efficiency and stability of hydrazide and aminooxy labeling sites are reduced. In particular, the hydrazide group has a strong interaction with sodium ions after reductive amination, thereby hindering the methylation rate of the fixed charge. In addition, an in-depth investigation of the tags currently on the market found that these tags are unable to simultaneously quantify and characterize glycans. In addition, some tags containing NHS-carbamate functional groups have poor stability in the presence of atmospheric humidity. Therefore, tags were developed that include: (1) a radical precursor that generates nascent radicals capable of inducing systematic and predictable glycan fragmentation; (2) an ionization site that allows for the generation of fixed charges, thereby increasing ionization and methylation efficiency; (3) a fluorophore that allows for sensitive quantification of labeled glycans; and (4) an amino coupling site that significantly increases the coupling efficiency between glycans (see Figure 1 ). This article describes these developed tags. Unlike many tags currently in use, these tags allow for simultaneous quantification and characterization of glycans and have enhanced chemical stability.

[0184] In fact, the compounds and methods of the present invention are capable of quantifying glycans through fluorescence functions and, unlike many prior art methods, are capable of characterizing glycans through mass spectrometry and free radical chemistry-induced fragmentation. In addition, the compounds and methods disclosed herein can be used for glycan coupling with any glycan having a reducing end. In contrast, many prior art methods are limited to N-glycans.

[0185] The compounds of the present invention are able to introduce TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) groups or their analogs into polysaccharides and similar molecules, which in turn allows free radical chemistry to occur. The TEMPO group is superior to other groups, such as the succinimide group, in many ways. For example, the succinimide group typically decomposes rapidly in the presence of atmospheric moisture, whereas the TEMPO group typically does not.

[0186] The present invention will be better understood by reference to the following experimental details, but one skilled in the art will readily appreciate that the specific experiments detailed are merely illustrative of the present invention as described in more detail in the claims below.

[0187] Example

[0188] Example 1

[0189] General Synthesis. The general synthesis of fluorescent tags begins with a cyclization reaction involving substituted benzene and olefins as shown in Scheme 1. The final product is obtained through a series of six steps.

[0190]

[0191] Scheme 1. Overview of the general synthesis of the fluorescent tags of the present invention.

[0192] 5-Methyl-7-nitro-2-quinolinecarboxylic acid ethyl ester (1)

[0193] In a clean, oven-baked flask equipped with a stirring rod, 1,2-dimethyl-3,5-dinitrobenzene (5mmol) was reacted with ethyl acrylate (2 equivalents) in a solution of cesium carbonate (3 equivalents) in anhydrous tetrahydrofuran (25mL) under an argon atmosphere. After reflux for 8 hours, the crude mixture was cooled and evaporated in vacuo, and the product was then extracted from the residue with dichloromethane. The collected product was purified by flash chromatography on silica gel using ethyl acetate and hexane. The target product (360.5mg, 28% yield) was obtained as a yellow solid.

[0194] 5-Bromomethyl-7-nitro-2-quinolinecarboxylic acid ethyl ester hydrobromide (2)

[0195] Into flame-dried flask, load 5-methyl-7-nitro-2-quinolinecarboxylic acid ethyl ester (1.385mmol), N-bromosuccinimide (1.25 equivalents), benzoyl peroxide (0.1 equivalent), and evacuate 30 minutes, then they are placed under argon atmosphere.Reactant is dissolved in anhydrous carbon tetrachloride (70mL) and refluxes 1.5 hours and stirs with photocatalyst (250W).After completing, make crude mixture cool to room temperature and purify with ethyl acetate and hexane (3:7) by silica gel flash chromatography.By adding hydrobromic acid, stir 30 minutes subsequently and evaporate solvent completely under reduced pressure, target product is converted into salt.

[0196] 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylic acid ethyl ester (3)

[0197] The 5-bromomethyl-7-nitro-2-quinolinecarboxylic acid ethyl ester hydrobromide (assuming 1.385mmol) from the previous step is dissolved in water and the solution is alkalized with sodium hydroxide until pH~7, thereby converting it back to the neutral form. The organic compound is extracted with benzene (60mL) and the collection is dried over anhydrous sodium sulfate. The dry benzene layer is transferred to a flame-dried flask and TEMPO (1.2 equivalents), Cu (OTf) 2 (0.1 equivalents), Nbpy (0.4 equivalents) and copper powder (1.2 equivalents) are degassed with argon for 30 minutes. Under stirring, the solution is refluxed under argon for 2 hours. After cooling to room temperature, the crude mixture is filtered through a short silica gel pad and eluted with ethyl acetate. The organic layer is transferred to a separatory funnel and washed with saturated NH4Cl, 1M NH4OH and brine. The organic layer is dried over anhydrous sodium sulfate and then purified by silica gel flash chromatography with ethyl acetate and hexane (1:5). The desired product was obtained as a white to off-white solid (144.8 mg, 25% overall yield from bromination followed by TEMPO coupling).

[0198] Ethyl 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylic acid (4)

[0199] 5-(TEMPO) methyl-7-nitro-2-quinolinecarboxylic acid ethyl ester (0.3485mmol) is added to a clean flask and dissolved with THF (10mL) and methanol (10mL). 2M KOH aqueous solution (20mL) is slowly added to the mixture. The reaction is continued under stirring at room temperature. After completion, the crude mixture is evaporated under reduced pressure to remove THF and methanol. 2M HCl aqueous solution is added to the crude mixture until pH ~ 7, then extracted five times with ethyl acetate and the organic layer is dried over anhydrous sodium sulfate. The organic layer is then purified by silica gel flash chromatography with methanol and DCM (20: 1). The target product (98.7mg, 73% yield) as a white solid is obtained.

[0200] N-[2-(Diethylamino)ethyl]-5-(TEMPO)methyl-7-nitro-2-quinolinecarboxamide (5)

[0201] Add ethyl 5-(TEMPO)methyl-7-nitro-2-quinolinecarboxylic acid (0.2543mmol) to an oven-baked flask and evacuate for 30 minutes, then dissolve in thionyl chloride (10mL). Make the reaction occur by stirring and refluxing for 2 hours. Then, thionyl chloride is evaporated in vacuum and the residue is immediately redissolved in anhydrous DCM (5mL). In a separate oven-baked flask, N-diethylethylenediamine (50 μL) and anhydrous trimethylamine (140 μL) are dissolved in anhydrous DCM (5mL). In 10 minutes, acyl chloride is added to this mixture in a dropwise manner. After stirring at room temperature for 2.5 hours, the mixture is diluted with 15mL DCM and washed once with saturated sodium bicarbonate aqueous solution. Then the water layer is extracted twice with DCM and the combined organic layer is dried over anhydrous sodium sulfate. Then the organic layer is purified by silica gel flash chromatography with methanol and DCM. The desired product was obtained as a white solid (100.8 mg, 82% yield).

[0202] 7-Amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide (6)

[0203] N-[2-(diethylamino) ethyl]-5-(TEMPO) methyl-7-nitro-2-quinolinecarboxamide (0.2076mmol) is added in a clean flask equipped with a stirring rod. Before adding 10% Pd / C (0.5 equivalent), the solid is dissolved in anhydrous ethanol (5mL). A hydrogen source is connected to the flask and the reaction is stirred at room temperature for 2.5 hours. After completion, the crude mixture is filtered through sand and diatomaceous earth, then eluted with ethanol. The organic layer is purified by silica gel flash chromatography with methanol and DCM, to obtain the final pure product (51.5mg, 54% yield) in yellow solid.

[0204]

[0205] Scheme 2: Proposed mechanism for quaternary amine fragmentation, resulting in mobile charges.

[0206] Mobile phase A: 50 mM ammonium formate, pH 4.4

[0207] Mobile phase B: LC-MS acetonitrile

[0208]

[0209] Table 1: Gradient for HILIC-FLD-ESI+MS analysis of labeled glycans.

[0210]

[0211] Table 2. Peak integration and peak mass of HILIC-FLD-ESI+MS analysis of labeled maltose.

[0212] Figure 3-7 The invention relates to nuclear magnetic resonance spectrum and mass spectrum of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide, which confirm the chemical structure of the compound.

[0213] Preparation of glycan samples with 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. A volume of 30 μL of methanol containing 20 mM fluorescent label (7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide) and 20 μL of water containing 1 mM glycan sample (e.g., lactose) were added to a PCR tube and evaporated in vacuo. 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 glycan labeling was completed, the mixture was evaporated in vacuo at 70°C. The residue was washed with HPLC grade acetone to extract unreacted reagents and sodium cyanoborohydride. The precipitate was dissolved in 18.2 megohm water containing 50% HPLC grade methanol before analysis by LC-MS.

[0214] The analysis was performed by mass spectrometry using a Thermo-Fisher Scientific TM Linear quadrupole ion trap (LTQ-XL) mass spectrometer (Thermo, San Jose, CA, USA). 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 gas nitrogen flow rate of 10 (arbitrary units), a spray voltage of 5.00 kV, a capillary voltage of 20-40 V, a capillary temperature of 275 ° C, and a tubular lens voltage of 50-200 V. The ion optical parameters were optimized by the autotune function in the LTQ-XL tuning program to maximize the signal intensity. Systematic glycan fragments were obtained by subjecting the ionized bioconjugates to collision-induced dissociation (CID) of 15 to 50 (arbitrary units).

[0215] Fluorescence assay. Fluorescent labels with a concentration range of 1 pM to 100 μM were prepared to evaluate the fluorescence function of the fluorescent labels. First, the 100 μM solution was scanned for absorbance to determine the potential excitation wavelength. Then the emission of each excitation wavelength was tested by Agilent CaryEclipse fluorescence spectrophotometer. The optimal excitation and emission wavelengths were selected to construct a preliminary calibration curve to evaluate any correlation between fluorescence and concentration. For instrument repeatability, another instrument (ThermoFisher Scientific TM NanoDrop TM ) was used to construct the preliminary calibration curve.

[0216] Figure 7-11 The results of this experiment are provided, which relate to the labeling of disaccharide samples. This experiment was conducted, which relates to labeling disaccharide samples of known molecular weight and chemical structure by using the present invention and established protocols. Considering that the m / z value of the present invention (m / z 456) has an expected increase relative to the m / z value (m / z 782) of the labeled polysaccharide by mass spectrometry analysis, the labeling is successful. The labeled disaccharide is then separated, and fragments are induced. The first fragment that appears is the expected loss of TEMPO, which will produce a loss of m / z 156. This initial fragment confirms that the polysaccharide has been labeled with compound 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. Further separation and fragmentation of the fragments (the m / z of the separated fragment is 626) reveal additional fragments of the covalently linked disaccharide. These fragments can be used to characterize the disaccharide.

[0217] UPLC-FLD and UPLC-MS analysis. All the following analyses were performed using a ThermoFisher Scientific TM Vanquish Flex TM UPLC Instruments and ThermoFisher Scientific TM QExactive Plus Oribtrap mass spectrometer. A mixture of six reagent-labeled glycans (maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) was prepared to a final concentration in the range of 10-20 μM. The mass spectrometer was used with Supelco Discovery C 18 A column (5 μm particle size, L 25 cm x ID 4.6 mm) and an XBridge Polysaccharide BEH Amide XP column (2.5 μm particle size, L 150 mm x ID 3.0 mm) were used to separate the analytes. 18The flow rates of the column and the polysaccharide BEH amide column were set to 0.750 ml / min and 0.400 ml / min, respectively, with column temperatures of 30.00°C and 60.00°C, respectively. 18 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 polysaccharide BEH amide column, mobile phase A consisted of 50 mM ammonium formate in water (pH 4.403), and mobile phase B consisted of LC-MS grade acetonitrile. The gradient started from 86% B and decreased to 50% B over 55 minutes. Fluorescence detection was performed with parameters set to excitation 385 nm, emission 515 nm, and detection sensitivity 5. Mass spectrometry detection was performed with parameters set to capillary voltage +3.50 kV, capillary temperature 320°C, sheath gas flow rate 50 (arbitrary units), and S lens RF level 50.0 (arbitrary units).

[0218] The results of this experiment are shown in Figure 12-19 In, the figure includes fluorescence experimental data. For any light emission, the excitation wavelengths of 270nm and 390nm are evaluated. Both excitation wavelengths cause emission at a wavelength of 520nm. Further experiments and discussions show that 390nm is a better excitation wavelength for fluorescence determination. Then a calibration curve was successfully constructed on two different fluorometers, where the linear fit between the nanomolar concentration and the micromolar concentration of the present invention was 0.9997.

[0219] in conclusion

[0220] In order to perform optimal mass spectrometry and liquid chromatography analysis and fluorescence detection on polysaccharides, the fluorescent reagent disclosed herein contains important chemical sites. As previously described, among these sites, fluorophore bases, basic sites (ionization / methylation sites), polysaccharide labeling sites (coupling sites) and free radical precursors are included, which are lacking in all commercially available labels. The fluorophore bases exhibit the fluorescence function required for polysaccharide detection and quantitative capabilities in a concentration-dependent manner. The purpose of utilizing fluorescence is to maximize the sensitivity of liquid chromatography detection and quantitative methods. The basic sites allow positive charges to be induced by protonation, ion-dipole interactions or methylation. The importance behind the recruitment of the basic sites is to allow ionization to occur during the mass spectrometry analysis of polysaccharides. The polysaccharide labeling site is the most basic feature included on the label because it allows reaction with the polysaccharide and then forms a covalent connection. Finally, the free radical precursor allows the formation of nascent free radicals during any of the dissociation techniques mentioned above. Such dissociation techniques include collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Ultimately, the free radicals react simultaneously with the glycans to produce glycan fragments that are reproducible, systematic, and efficient for glycan characterization, identification, and differentiation between glycans and their isomers.

[0221] Based on the data provided, the fluorescent tags disclosed herein show excellent correlation between fluorescence and concentrations as low as 1 nM. This correlation and similar results were also observed on another fluorescence spectrophotometer instrument. In addition, the basic sites on the fluorescent tags are effective for the ionization process during mass spectrometry. After labeling the glycan molecules, the fluorescent tags can induce systematic and repeatable fragments for glycan analysis. Therefore, containing the fluorophore base, the basic site, the glycan labeling site and the free radical precursor is completely the best combination for glycan labeling and analysis. By using liquid chromatography to separate glycans and glycan isomers, combining fluorescence detection and synergistic mass spectrometry to perform glycan and glycan isomer fragment analysis, and characterizing and identifying with this, the fluorescent tags will eventually provide powerful, new and modern analytical functions in the field of glycomics. Therefore, the evolution of new analytical techniques has promoted the development of relatively new fields, which have the potential to study glycans as biomarkers of human diseases.

[0222] Example 2

[0223] Materials. For the synthesis of the novel fluorescent tags, the starting material 1,2-dimethyl-3,5-dinitrobenzene was purchased from 1ClickChemistry (Kendall Park, NJ). All other chemicals used to synthesize the novel fluorescent tags 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). Lactose-N-difucose I (LNDFH I) and lactose-N-difucose 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 purification and analysis of samples described herein were HPLC grade and purchased from Fisher Scientific (Hampton, NH).

[0224] N-glycan deglycosylation of RNase B. Deglycosylation of N-glycans from RNase B was performed following the manufacturer's protocol. To ensure complete deglycosylation of the glycoprotein, the incubation step was extended to 14 hours. The resulting aqueous solution was then cooled to room temperature before purification by PGC SPE. The column was activated with acetonitrile and equilibrated with 5% acetonitrile before applying the sample. The column was then washed five times with 1 mL of water and the glycans were eluted and collected four times through a 0.2 μm nylon filter with 250 μL of 40% ACN and 0.1% formic acid. The eluate was evaporated in vacuo at 60°C before proceeding to the derivatization step.

[0225] Synthesis of 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. Briefly, 1,2-dimethyl-3,5-dinitrobenzene is cyclized with ethyl acrylate. Subsequently, the remaining methyl group is brominated and the product is converted to the hydrobromide form. After neutralization and extraction into an organic solvent for subsequent reactions, the intermediate undergoes a TEMPO coupling reaction. The resulting product is then subjected to a strong base to produce a carboxylic acid (referred to as a common precursor, such as Figure 2 ). The carboxylic acid is first converted to an acid chloride intermediate and then reacts with N,N-diethylethylenediamine via amide bond formation. Finally, the remaining nitro group is hydrogenated with hydrogen to produce the final product. Alternatively, an acidic analog of this structure can be obtained by directly hydrogenating the nitro group of the previously mentioned common precursor for analysis of (currently under investigation) glycans by negative ion mode mass spectrometry.

[0226] N-glycan and standardized glycan samples were prepared with 7-amino-N-[2-(diethylamino)ethyl]-5-(TEMPO)methyl-2-quinolinecarboxamide. A volume of 30 μL of methanol containing 20 mM labeling reagent and 20 μL of water containing 1 mM standardized glycans were added together and evaporated in vacuo. 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 hours. After the glycan labeling was completed, the mixture was evaporated by nitrogen degassing or evaporated in vacuo at 70°C. For a total of three replicates, the residue was vortexed and sonicated with 50 μL of HPLC grade acetone to extract unreacted reagents and sodium cyanoborohydride, centrifuged at 14 krpm for 5 minutes, and the supernatant containing free reagents was recovered for future desalting and purification. After final collection, the pellet containing labeled glycans was completely dried under ambient conditions for 10 min prior to LC-MS analysis.

[0227] Fluorescence Assay. A concentration range of 1 nM to 100 μM was prepared to evaluate the linearity of the fluorescent label. Initially, a 100 μM solution was subjected to an excitation scan with the emission mode set to zero order by an Agilence Cary Eclipse fluorescence spectrophotometer to determine the preliminary excitation wavelength value. Then an emission scan was performed for each excitation wavelength to determine the wavelength that matched the best fluorescence intensity. With the parameters set to 965V PMT detector voltage, 5nm excitation and emission slits, and 1.000 seconds averaging time, the optimal excitation and emission wavelengths at 280nm and 520nm were selected, respectively, to construct a concentration-dependent fluorescence graph to evaluate the correlation between fluorescence intensity and concentration.

[0228] UPLC-FLD and UPLC-MS analysis. All the following experiments were performed using a ThermoFisher Scientific Vanquish Flex UPLC instrument and a ThermoFisher Scientific QExactive Plus Oribtrap mass spectrometer equipped with a fluorescence detector. A mixture of six maltose 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 and LNDFH II analytes were mixed with unknown amounts and unknown total concentrations. In order to analyze the N-glycans from RNase B, a labeled N-glycan with a concentration of 74 pmol / μL was prepared. The labeled glycans were reconstructed in an initial state mobile phase consisting of 50 mM ammonium formate (pH 4.4) and LC-MS acetonitrile (14:86 v / v) and injected at 1.0 μL. An XBridge Glycan BEH Amide XP column (2.5 μm particle size, L 150 mm x ID 3.0 mm) was used to separate the fluorescent tag derivatized glycan analytes according to the gradient described in Table 1. The flow rate was set to 0.400 ml / min and the column temperature was 60.00°C. Fluorescence detection was performed with parameters set to excitation 280.0 nm, emission 520.0 nm, detection sensitivity 8 (arbitrary units), and scan rate 5.00 Hz. Mass spectrometry experiments were performed with parameters set to: capillary voltage +3.50 kV, capillary temperature 263°C, sheath gas flow rate 50 (arbitrary units), auxiliary gas heater temperature 425°C, auxiliary gas flow rate 13 (arbitrary units), purge gas flow rate 3 (arbitrary units), and S lens RF level 50.0 (arbitrary units). For the MS of the analytes during each run 2 Analysis was performed with an isolation window of 4.0 m / z at a resolution of 70,000 for each of the expected masses listed in the inclusion list. The HCD collision energy was varied in normalized collision energy (NCE) mode.

[0229] Results and discussion

[0230] A new fluorescent tag (Glyc·RadiFluor; abbreviated as GRF) containing a free radical precursor was synthesized to enhance the glycan characterization function, which not only promotes high fluorescence sensitivity, feasible methylation and mass spectrometry ionization, and improved chemical stability, but also eliminates the interconversion between multiple isomers at the reducing end of the glycan. Before performing additional experiments involving glycans, the fluorescence detection and quantification functions of GRF were first evaluated. After the optimal excitation and emission wavelengths were determined to be 280±5nm and 520±5nm, respectively, the linear relationship was further evaluated. The correlation between the fluorescence intensity and the GRF concentration in the range of 1nM to 1μM was plotted and linear regression analysis was performed ( Fig.10 ). The linear fit value of 0.9998 is interpreted as an excellent linearity between fluorescence intensity and GRF concentration. In addition, the linear relationship across the nanomolar concentration depicts the fluorescence detection and quantification sensitivity of GRF, which is suitable for the quantitative analysis of glycoprotein-derived glycans.

[0231] After confirming the fluorescence detection and quantification capabilities of GRF, a mixture of maltose with different sugar unit lengths was labeled by reductive amination and the resolution was evaluated on two columns that exhibited opposite separation methods. In short, the importance of using reductive amination for the labeling scheme is to eliminate the interconversion of α, β, and open chain isomers, which would otherwise produce additional peaks that complicate LC analysis. Furthermore, the experiments showed that the labeling ability of GRF during reductive amination was such that there was no significant difference in reaction yield when the reaction was run for two hours versus 14 hours. The columns initially used to separate the analytes involved C 18 stationary phase (data not shown) and ethylene bridged hybrid (BEH) amide (or HILIC) stationary phase, where reverse phase separation and normal phase separation techniques were used, respectively. As expected, when normal phase separation was used, the peak separation between the maltose was the best ( Fig. 20 The improvement in separation on the BEH amide column can be explained by the enhanced interaction between the maltose moiety of the analyte and the hydrophilic amide groups of the stationary phase. 18 The number of interactions of the stationary phase with the maltose moiety was reduced. In contrast, the interaction with the fluorescent tag moiety was much closer, which explains the poor separation of maltose. Therefore, due to the improved peak separation, the BEH amide column was used for all future experiments described in this article.

[0232] In early attempts at quantitative analysis of a mixture of maltose, the integrated values ​​of the determined peaks were not relatively equal. It was observed that labeled maltohexaose and labeled maltoheptaose had lower integrated values ​​relative to the shorter maltose. This problem was addressed by noting that the purity of the longer length standardized glycans was relatively lower than the purity of the shorter length standardized glycans. For example, maltoheptaose and maltohexaose were 65% or greater in purity, while maltotetraose was 95% or greater in purity. In response, 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 Fig. 20 The integration of the middle peaks returns relatively equal values. For the longer maltose, the peak width increases slightly, resulting in a shorter peak height. Nonetheless, the integration of the peaks is relatively similar to the integration associated with the early eluting maltose. After peak integration, the amount of each analyte is determined based on the known total injected amount of 46 pmol. By multiplying the ratio of the analyte peak area to the sum of the peak areas of all analytes by the known total molar amount, the result returns a value related to the molar amount of the individual analyte that was injected. For example, given that the peak area for maltose is 1.090×10 8 , and the total combined peak area of ​​all analytes was 6.397 × 10 8 , it can be inferred that 7.8 pmol of GRF-derived maltose was injected. Similar calculations can be performed for all other analytes derived from the same sample (see Table 2). Based on the results of these calculations, the average analyte injection was determined to be 7.7 ± 0.2 pmol, which is consistent with the expected equimolar amount of 7.7 pmol per analyte. In addition, for an amount of approximately 7.7 pmol per analyte, the normalization level was 2.43 × 10 7 , further depicting the high detection sensitivity and high quantitative sensitivity for glycans via emission of quinolyl fluorophores upon excitation. Finally, each maltose produces a single peak associated with the open chain isomer, rather than multiple peaks corresponding to the additional α and β isomers. This represents the efficiency of the reducing agent in reducing the resulting Schiff base during the labeling reaction, thereby producing a single isomer that can no longer be interconverted at the reducing end of the glycan. This, therefore, produces a single peak for each analyte, establishing a simpler and more feasible analytical method for detecting analytes and their corresponding isomers in any given glycan sample by LC.

[0233] After the unknown glycans are detected and quantified, it is also particularly important to characterize and identify the unknown glycans. In this example, the ability of the GRF to characterize the maltose mixture was evaluated by performing high energy collision dissociation (HCD) on each of the six analytes. As the HCD collision energy increases, TEMPO radical precursor fragments induce nascent but localized free radicals that react with glycans simultaneously, causing cross-ring and glycosidic bond cleavage. The abundance of these fragments varies not only by structure or stereochemistry, but also by the type of fragments. Therefore, valuable structural information can be obtained through different fragmentation pathways to identify and characterize a large number of glycans and their isomers. All product fragment ions described in this article are assigned based on the Domon and Costello nomenclature of glycan fragments ( Fig.21 ). Currently, there is no known nomenclature for the fragment ions corresponding to the reducing open-chain isomer (sugar unit 0). Therefore, a new nomenclature system was designed accordingly ( Fig. 22 ). As shown in Figures 23A and 23B, the monoprotonated GRF-derived maltoheptaose was subjected to an HCD normalized collision energy of 30 (arbitrary units) to fragment the TEMPO groups and generate free radicals. At the same time, highly unstable free radicals reacted and induced the cleavage of cross-rings and glycosidic bonds at each constituent sugar unit. These fragments include 0,2 X and 1,5 X-type ions (cross-ring fragments), and Y and Z-type ions (glycosidic bond cleavage).Other fragments (designated as n0 fragments) of C2H5O2 groups are also observed, and the other fragments are connected to the unique tertiary carbon at the reducing end.In addition, multiple losses of water are observed and are related to Z-type fragment ions, thereby forming double bonds along the CC bond subsequently.Although the fragments observed by the protonated species provide valuable information about the labeled maltoheptaose, a large amount of fragments can be acid-catalyzed by mobile charges, and polysaccharide rearrangement can occur.This polysaccharide rearrangement can produce misleading fragments, and the misleading fragments deviate from the fragments corresponding to the actual structure.The complete fragments of the ionization site seem to be catalyzed by newborn free radicals, which eventually cause quinolyl nitrogen to extract protons from the polysaccharide moiety.Due to the presence of multiple potential protonation sites, positive charge may have been stabilized on quinolyl nitrogen or amide nitrogen during ionization.

[0234] To eliminate any polysaccharide rearrangement and acid-catalyzed fragmentation, a fixed charge was introduced by reacting GRF-derivatized maltoheptaose with methyl iodide. Tertiary amines have a large affinity for nucleophiles and are selectively attacked by methyl cations to generate positively charged quaternary amines. As shown in Figures 24A and 24B, complete loss of ionization sites was no longer observed. Although, additional fragmentation was present with partial loss of ionization sites, it was still observed throughout the linear biopolymer. 0,2 X. 1,5 X, Y, and Z product ions.

[0235] After determining the optimal method conditions and instrumental parameters for peak separation and mass spectrometric characterization of maltose, the method was further evaluated on a mixture of branched isobaric isomers. Lactose-N-difucohexaose I (LNDFH I) and lactose-N-difucohexaose II (LNDFH II) are hexasaccharide isomers that differ only in the attachment position of a single fucose subunit ( Figures 26A-26D The fluorescence chromatogram shows the ability of HILIC chromatography to separate labeled LNDFHI and LNDFHII isomers ( Fig.25 When the normalized HCD energy is kept constant at 28 (arbitrary units), one of the main differences in the HCD spectra of the branched isomers is the relative abundance and type of fragments ( Figures 26A-26D ). For example, the HCD spectrum of labeled and protonated LNDFH II shows unique fragments, including 1,5 X 2α , Y 2α and Z 2α The product ions for labeled and protonated LNDFH I are absent. This is due in particular to the branched nature of the glycans, which results in the loss of four sugar residues for LNDFH I but three sugar residues for LNDFH II when fragmentation occurs at sugar unit 2 (or sugar unit 2α for LNDFH II). This can be seen by comparing the Y 2α This is further exemplified by the base peak associated with the LNDFH II 2α product ion, which has a higher mass-to-charge ratio than the base peak associated with the Y2 product ion of LNDFH I. Similarly, an example can be drawn from the methylated species, where the fragments associated with the LNDFH II 2α product ion are also unique at a normalized HCD energy of 32 (arbitrary units). For both protonated and methylated species, the abundance and type of fragments are relatively high for the LNDFH II isomer relative to the LNDFH I isomer.

[0236] To normalize the retention time of glycans, a dextran ladder standard solution was prepared and labeled with a novel labeling reagent. Dextran ladder standards are commonly used in glycan analysis to define the retention time of glycans in terms of glucose units. Figures 27A-27D As shown, the dextran ladder is separated and detected by fluorescence, and each peak is defined according to the number of glucose units. In a separate 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 by the labeled dextran ladder, the logarithm of the glucose unit and the retention time are plotted to construct a curve and obtain the equation ( Fig.28). The equation can then be used to convert the retention times of all glycan analytes obtained from future experiments into glucose units. By describing the glycan analytes according to glucose units, the retention times observed by a method can be compared with the retention times obtained by other methods. Since the retention time of the glycan analytes is relative to the retention time of the glucan ladder for a particular method, all glycans can be assigned a single glucose unit literature value. Ultimately, no matter which method is used to obtain the data, the glucose unit value can be used as a preliminary glycan characterization technique before a more extensive mass spectrometry free radical mediated characterization. In addition, the glycans are characterized by the glucose unit values ​​of the observed glycans, combined with free radical mediated characterization by mass spectrometry, to achieve more complex and more powerful glycan characterization techniques. For example, by utilizing the HILIC-FLD-MS method, it is observed that the theoretical probability of a pair of structurally or stereochemically different glycans with the same glucose unit value, the same accurate mass, and the same mass spectrometry fragmentation pattern is extremely low.

[0237] An experiment was performed involving the analysis of N-glycans from ribonuclease B (RNase B) where they were first enzymatically released from asparagine residues by peptide-N-glycosidase F (PNGase F). RNase B is often used as a positive control for endoglycosidases and is a high mannose glycoprotein with N-glycans represented by the structure Man 5-9 (GlcNAc)2. After enzymatic release, high-mannose N-glycans were labeled with a novel tag via reductive amination to eliminate the possibility of interconversion between reducing end isomers. Fig.30 As depicted by the fluorescence chromatogram of the labeled N-glycans in the , Man5(GlcNAc)2 has the highest abundance, followed by Man6(GlcNAc)2, and Man 7-9 (GlcNAc)2 was the least abundant, which is consistent with literature data obtained from glycoprotein manufacturers. Upon closer inspection of the HCD mass spectrum of Man5(GlcNAc)2, the fragments provided evidence behind the structural nature of the isomers. For example, in the case of the loss of four mannose subunits, Z 3α +Z 3β The existence of fragments can only be achieved through Fig.31A The structures shown were obtained instead of the corresponding linear isomers. In addition, the loss intensity of two mannose subunits and four mannose subunits was relatively lower than that of one mannose, three mannose, and five mannose subunits, which further provided evidence for the glycan structure. Fig.31AThe branched nature of the structure shown readily induces fragmentation of one, three, and five mannose subunits. In contrast, the loss of two and four mannose subunits is more challenging and thus induces multiple external residue losses by moving protons, which are multiple sugar fragments catalyzed by moving charges. In addition, the higher abundance of fragments corresponding to the loss of two mannose subunits relative to the abundance of fragments corresponding to the loss of four mannose subunits may be attributed to the number of possible fragmentation pathways. For example, since in the case of the loss of two mannose subunits 1,5 There are a total of six possible fragmentation pathways for the X product ion, so relative to the one associated with the loss of four mannose subunits 1,5 There are two possible fragmentation pathways for the X product ion, and the probability of producing this fragment becomes greater. Fig.31B As shown, it is a fragment of a labeled glycan that is methylated to generate a quaternary amine at the ionization site, and the only possible way to lose two mannose subunits (and similarly to lose four mannose subunits) is after partial fragmentation of the ionization site, which is proposed to generate a mobile proton (Scheme 2). Fig.31A This observation provides additional evidence supporting the isomeric structure and branching position of the mannose subunit of Man5(GlcNAc)2, as shown in FIG.

[0238] Similarly, the HCD mass spectrum of Man6(GlcNAc)2 provides evidence that the major isomer present on RNase B is the biantennary isomer, in which a monosaccharide extension occurs at the 3β unit of the Man5(GlcNAc)2 precursor ( Fig.32A ). In its protonated and labeled form ( Fig.32A ), HCD of Man6(GlcNAc)2 produced highly abundant fragments 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 produced product ions similar to protonation, and additional fragments were generated at the same time as the partial loss of the ionization site. Fig.32B ). Therefore, the proposed isomeric structures of Man6(GlcNAc)2 satisfy the fragmentation data, and vice versa.

[0239] in conclusion

[0240] For optimal LC-MS analysis and free radical-mediated glycan characterization with fluorescence detection, such biomolecules must be derivatized with labeling reagents that are first carefully defined by incorporating multiple important chemical sites. Among these sites, as previously described, are the fluorophore base, the ionization and methylation sites with high affinity for positively charged ions, the glycan labeling site that forms a stable covalent linkage, and the free radical precursor that promotes systematic fragmentation, which is commonly lacking in all commercially available tags. The fluorophore base exhibits the fluorescence function required for glycan detection and quantification capabilities in an amount-dependent manner. The purpose of utilizing fluorescence is to maximize the sensitivity of liquid chromatography detection and quantification methods. The basic site allows for the feasible induction of positive charges through protonation, ion-dipole interactions, or methylation. The importance behind recruiting the basic site is to allow ionization to occur during or prior to mass spectrometry analysis of glycans, thereby significantly improving the mass spectrometry signal-to-noise ratio. The glycan labeling site is the most essential feature included on the tag because it selectively reacts with a unique glycan reducing end. Finally, the radical precursor allows for the low-energy formation of nascent radicals by dissociation techniques such as high-energy collision dissociation (HCD) or collision-induced dissociation (CID). Ultimately, the radicals react simultaneously with the glycans to produce glycan fragments that are reproducible, systematic, predictable, diagnostic, and effective for glycan characterization, identification, and differentiation between glycans and their isomers. In summary, the rational recruitment of these chemical sites constructs a new type of tag that is optimal not only for quantification of low-concentration glycans (which is a typical feature of samples obtained from glycoproteins), but also for pairing sensitive MS detection with free radical-mediated glycan characterization functions. In addition, this new type of tag is designed to be chemically stable and lasts for significantly longer storage periods compared to reagents that recruit NHS-carbamate coupling sites.

[0241] Multiple polysaccharide samples with different attributes are labeled and analyzed by hydrophilic liquid chromatography-fluorescence detection-mass spectrometry (HILIC-FLD-MS) to comprehensively evaluate the function of the novel reagent. The maltose composed of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose is pre-mixed into an equimolar mixture before labeling and fluorescence quantification. Next, the longest linear maltose is subjected to high-energy collision dissociation to produce information-rich fragments through a free radical-mediated approach, wherein cross-ring fragments and glycosidic cleavage are observed in the entire linear biopolymer. In order to further evaluate the ability of the novel reagent to produce polysaccharide fragments of the structure of the distinguishable branched isomer, lactose-N-difucose I and lactose-N-difucose II are pre-mixed, labeled, and subjected to high-energy collision dissociation. After separating the labeled branched isomers by HILIC, the fragments of the two isomers reveal the difference in fragment abundance and type, which reflects their structure. Ultimately, the novel reagent successfully distinguished between two branched isomers based solely on the resulting product ions. The novel reagent was further evaluated by labeling N-glycans that were first enzymatically released from RNase B. The labeled N-glycans were separated by HILIC, detected by fluorescence, and accurately characterized by free radical-mediated characterization. By using HILIC for glycan separation, combined with fluorescence detection and tandem mass spectrometry for fragmentation induction, the novel reagent will ultimately provide powerful and modern analytical capabilities. The same analytical capabilities of the other reagents described in this article are considered. Therefore, the evolution of this new analytical technology is expected to enable further development of glycan analysis with the potential to study glycans as biomarkers of human disease and improve quality assurance assays for biopharmaceuticals.

[0242] ***

[0243] It should be understood that the present invention is not limited to the specific embodiments of the invention described above, as variations may be made thereto and still fall within the scope of the appended claims.

[0244] The present invention is further described by, but not limited to, the following numbered paragraphs:

[0245] 1) a compound of formula (Ia) or (Ib),

[0246]

[0247] Its R 1 and A, optionally substituted by R 2 replace,

[0248] in

[0249] is a single bond or a double bond;

[0250] if is a double bond, then:

[0251] X 1 is C, and X 2 is C or N, or

[0252] X 2 is C, and X 1 is N;

[0253] if is a single bond, then:

[0254] X 1 is NH, N-alkyl, O or NO, and X 2 is C=O, or

[0255] X 1 is NH, N-alkyl or NO, and X 2 is O, or

[0256] X 1 is C=O, and X 2 is NH, N-alkyl, O or NO, or

[0257] X 1 is O, and X 2 is NH, N-alkyl or NO;

[0258] X is CH, N or CR 9 ;

[0259] R 1 Selected from the group consisting of:

[0260]

[0261]

[0262] or its derivatives;

[0263] Y 1 and Y 2 Each independently selected from the group consisting of:

[0264] H, O, OH, CH3, N3, CN, NH2, carbonyl, R 3 -(CH2) y CO(CH2) p CH3, R 3 -(CH2) y COH, R 3 -(CH2) y -NH2、N[(CH2) y (CH3)]2, R3 -(CH2) y -N[(CH2) p CH3]2, COOH, R 3 -(CH2) y -COOH, R 3 -(CH2) y -COO(CH2) p CH3, R 3 -(CH2) y -CN、R 3 -(CH2) y -N3,

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] The condition is that if Y 1 or Y 2 is a carbonyl group, then the other is not a carbonyl group,

[0274] Or u and v are 0, and Y 1 and Y 2 together forming a structure selected from the group consisting of:

[0275]

[0276] or Y 1 and Y 2 Acting as and independently selected from R 1 A linker connected to a group defined in the group, wherein the linker is selected from the group consisting of:

[0277]

[0278] It is a halogen;

[0279] Z is a counterion;

[0280] R 2 Selected from the group consisting of:

[0281]

[0282] A is selected from the group consisting of:

[0283]

[0284] R 3 each 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;

[0285] R 4 , R 5 and R 6 Each is independently selected from the group consisting of: H, O, OH, carbonyl, CN, N3, COOH, alkyl, tert-butyl, sec-butyl, isobutyl, isopropyl, tetrahydropyran, alkylamino, alkylsulfonic acid, alkylphosphonic acid, R 3 -(CH2) y CO(CH2) p CH3, R 3 -(CH2) y COH 、 NH2、R 3 -(CH2) y -NH2、N[(CH2) y (CH3)]2, R 3 -(CH2) y -N[(CH2) p CH3]2, R 3 -(CH2) y -COOH, R 3 -(CH2) y -COO(CH2) p CH3, R 3 -(CH2) y -R 3 -(CH2) y -N3, CH3,

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293] or R 4 and R 5 and the nitrogen to which it is attached can together form an optionally substituted 5- to 8-membered saturated or partially unsaturated ring; or R 4 and R 5 Acting as and independently selected from R 1 A linker connected to a group defined in the group, wherein the linker is selected from the group consisting of:

[0294]

[0295] x, q, t, v, s, u, m, n, r, w, y, p, p a and p b Each independently is an integer from 0 to 24;

[0296] R 7 is a secondary amine, a tertiary amine, a reduced amine, an ether or an alcohol;

[0297] R 8 is an alkylated or hydrogenated imide or oxygen;

[0298] R 9 yes

[0299]

[0300]

[0301] any hydrogen atom in which may be replaced by a fluorine, deuterium or tritium atom;

[0302] Provided that if the structure is a compound of formula (Ib), then formula (Ib) is replaced by R 1 , R 2 and A substituted,

[0303] The condition is that if X1 is N and X2 is C, and R1 is And R 2 or one of A is H, then R 2 or A is not

[0304] 2) The compound according to paragraph 1, wherein R 2 replace.

[0305] 3) The compound according to paragraph 1, which has the formula (Ic)

[0306]

[0307] 4) The compound according to paragraph 1, which has formula (Id), formula (Ie) or formula (If)

[0308]

[0309] Where X 2 is NH, N-alkyl or NO.

[0310] 5) The compound according to paragraph 1, which is

[0311]

[0312]

[0313] 6) The compound according to paragraph 1, which is

[0314]

[0315] 7) The compound according to paragraph 1, which is

[0316]

[0317]

[0318] 8) A compound which is

[0319]

[0320] Among them, X, A, R 1 and R 2 As defined in paragraph 1. 9) The compound according to paragraph 1, wherein R1 is

[0321] or

[0322] Wherein R1 is selected from one of the following structures:

[0323] wherein R is independently selected from H, alkyl, alkylamino, ester, ether, OH, =O, COOH, NH2, alkylphosphonic acid, and halide.

[0324] 10) The compound according to paragraph 1, wherein R2 is

[0325]

[0326] 11) The compound according to paragraph 1, wherein A is NH2

[0327]

[0328] 12) The compound according to paragraph 5, wherein X is CH or N. 13) The compound according to paragraph 1, which is:

[0329]

[0330]

[0331] 14) A method for quantifying and / or characterizing glycans, the method comprising:

[0332] a) obtaining the compound according to paragraph 1;

[0333] b) contacting the compound with the glycan, thereby forming a labeled glycan.

[0334] 15) The method according to paragraph 14, further comprising dissociating the labeled glycans to form glycan fragments.

[0335] 16) The method according to paragraph 15, wherein the dissociation is collision induced dissociation (CID) or high energy collision dissociation (HCD).

[0336] 17) The method according to paragraph 14, further comprising analyzing the labeled glycans or glycan fragments using an instrument that is a fluorometer, a mass spectrometer or a liquid chromatograph, optionally and using an instrument that can detect fluorescence and / or absorbance.

[0337] 18) The method according to paragraph 14, wherein the glycan comprises a reducing terminus.

[0338] 19) The method according to paragraph 14, wherein the glycan is not an N-glycan.

[0339] 20) The method according to paragraph 14, wherein the instrument is an ultra-high performance liquid chromatograph (UPLC), a linear quadrupole ion trap (LTQ-XL) mass spectrometer, a Q Exactive orbital trap mass spectrometer or a liquid chromatography-mass spectrometer (LC-MS).

[0340] 21) The method according to paragraph 14, wherein the instrument is equipped with an electrospray ionization (ESI), heated electrospray ionization (HESI) source and / or a fluorescence detector.

[0341] 22) The compound according to paragraph 1, which is used for preparing labeled glycans or glycan fragments, or for glycan detection or glycan quantification.

[0342] It should be understood that the present invention is not limited to the specific embodiments of the invention described above, as variations may be made thereto and still fall within the scope of the appended claims.

Claims

1. A compound of formula (Ia) or (Ib), Its R 1 and A, optionally substituted by R 2 replace, in is a single bond or a double bond; if is a double bond, then: X 1 is C, and X 2 is C or N, or X 2 is C, and X 1 is N; if is a single bond, then: X 1 is NH, N-alkyl, O or NO, and X 2 It is C=O, or X 1 is NH, N-alkyl or NO, and X 2 is O, or X 1 is C=O, and X 2 is NH, N-alkyl, O or NO, or X 1 is O, and X 2 is NH, N-alkyl or NO; X is CH, N or CR 9 ; R 1 Selected from the group consisting of: or its derivatives; Y 1 and Y 2 Each independently selected from the group consisting of: H、O、OH、CH3、N3、CN、NH2、R 3 -(CH2) y CO(CH2) p CH3、R 3 -(CH2) y COH 、 R 3 -(CH2) y -NH2、N[(CH2) y (CH3)]2、R 3 -(CH2) y -N[(CH2) p CH3]2、COOH、R 3 -(CH2) y -COOH、R 3 -(CH2) y -COO(CH2) p CH3、R 3 -(CH2) y -CN、R 3 -(CH2) y -N3、 The condition is that if Y 1 or Y 2 is a carbonyl group, then the other is not a carbonyl group, Or u and v are 0, and Y 1 and Y 2 together forming a structure selected from the group consisting of: or Y 1 and Y 2 Acting as and independently selected from R 1 A linker connected to a group defined in the group, wherein the linker is selected from the group consisting of: It is a halogen; Z is a counterion; R 2 Selected from the group consisting of: A is selected from the group consisting of: R 3 each 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 Each is independently selected from the group consisting of: H, OH, O, CN, N3, COOH, alkyl, tert-butyl, sec-butyl, isobutyl, isopropyl, tetrahydropyran, alkylamino, alkylsulfonic acid, alkylphosphonic acid, R 3 -(CH2) y CO(CH2) p CH3, R 3 -(CH2) y COH、NH2、R 3 -(CH2) y -NH2、N[(CH2) y (CH3)]2, R 3 -(CH2) y -N[(CH2) p CH3]2, R 3 -(CH2) y -COOH, R 3 -(CH2) y -COO(CH2) p CH3, R 3 -(CH2) y -R 3 -(CH2) y -N3、CH3、 or R 4 and R 5 The nitrogen to which it is attached can together form an optionally substituted 5- to 8-membered saturated or partially unsaturated ring; Or R4 and R 5 Acting as and independently selected from R 1 A linker to which a group defined in 4 and R 5 together forming a structure selected from the group consisting of: 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 It is a secondary amine, a tertiary amine or a reduced amine, an ether, or an alcohol; R 8 is an alkylated or hydrogenated imide or oxygen; R 9 yes Any hydrogen atom may be replaced by a fluorine, deuterium or tritium atom; Provided that if the structure is a compound of formula (Ib), then formula (Ib) is replaced by R 1 , R 2 and A substituted; The condition is that if X1 is N and X2 is C, and R1 is And R 2 or one of A is H, then R 2 or A is not 2. The compound according to claim 1, wherein R 2 replace.

3. The compound according to claim 1, which has the formula (Ic) 4. The compound according to claim 1, which has formula (Id), formula (Ie) or formula (If) Where X 2 is NH, N-alkyl or NO.

5. The compound according to claim 1, which is 6. The compound according to claim 1, which is 7. The compound according to claim 1, which is 8. A compound which is Among them, X, A, R 1 and R 2 The definition as claimed in claim 1.

9. The compound according to claim 1, wherein R1 is selected from one of the following structures: wherein R is independently selected from H, alkyl, alkylamino, ester, ether, OH, =O, COOH, NH2, alkylphosphonic acid, and halide.

10. The compound according to claim 1, wherein R1 is 11. The compound according to claim 1, wherein R2 is 12. The compound according to claim 1, wherein R2 is 13. The compound according to claim 1, wherein A is NH2 14. The compound according to claim 1, wherein A is NH2 15. The compound of claim 5, wherein X is CH or N.

16. The compound according to claim 1, which is:

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

18. The method of claim 17, further comprising dissociating the labeled glycans to form glycan fragments.

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

20. The method of claim 17, further comprising analyzing the labeled glycans or glycan fragments with an instrument of a fluorimeter, a mass spectrometer, or a liquid chromatograph, optionally and with an instrument capable of detecting fluorescence and / or absorbance.

21. The method of claim 17, wherein the glycan comprises a reducing terminus.

22. The method of claim 17, wherein the glycan is not an N-glycan.

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

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

25. The compound according to claim 1, for use in preparing labeled glycans or glycan fragments, or for glycan detection and / or glycan quantification.

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