Advanced methods and systems for automated high performance identification of carbohydrates and carbohydrate mixture composition patterns based on novel fluorescent dyes

Abstract

This invention relates to improved methods for identifying carbohydrate composition and determining compositional patterns of carbohydrate mixtures. Based on advanced internal standards, it utilizes novel fluorescent dyes combined with highly efficient separation techniques to determine accurate and highly reproducible migration and retention time indices. The highly efficient separation techniques include highly sensitive capillary (gel) electrophoresis or (ultra) high-performance liquid chromatography. It also relates to automated analytical methods for determining and / or identifying compositional patterns of carbohydrates and / or carbohydrate mixtures, using at least first and second fluorescent labels to respectively label migration / retention time aligned standards and samples or different samples, wherein at least one fluorescent dye is a compound as defined herein. This invention also relates to kits or systems for determining and / or identifying compositional patterns of carbohydrate mixtures, providing carbohydrate dye conjugates comprising dyes as defined herein for use in the methods of this invention, the dye having the following formula A or B:

Description

Technical Field

[0001] This invention relates to an improved (i.e., simplified / easier, more stable, and more reproducible) method for identifying carbohydrate composition (e.g., from complex carbohydrate mixtures) and determining compositional patterns (e.g., glycosylation patterns) of carbohydrate mixtures, based on advanced internal standards to determine accurate and highly reproducible migration and retention time indices using novel fluorescent dyes combined with high-performance separation techniques, such as capillary (gel) electrophoresis (C(G)E) or (ultra)high-performance liquid chromatography (U)HPLC) with highly sensitive detection (e.g., (laser-induced) fluorescence detection).

[0002] In a first aspect, the present invention relates to a method for automatically determining and / or identifying compositional patterns of carbohydrates and / or mixtures of carbohydrates, and a method for automated analysis of compositional patterns of mixtures of carbohydrates, said method being based on labeling migration / retention time alignment standards and samples or different samples respectively using at least first and second fluorescent labels, wherein at least one fluorescent dye is a compound as defined herein.

[0003] Furthermore, the present invention relates to a method for calibrating a multi-wavelength fluorescence detection system, as well as a calibration system or calibration standards, and describes new compounds suitable for calibration.

[0004] The present invention further relates to kits or systems for determining or identifying compositional patterns of carbohydrate mixtures, and kits or systems for determining and / or identifying compositional patterns of carbohydrate mixtures. Additionally, carbohydrate dye conjugates for use in the methods of the present invention are provided, comprising dyes as defined herein. Background Technology

[0005] The importance of glycosylation in many biological processes is widely accepted and has been discussed in the literature for decades. Glycosylation is a common and highly diverse post-translational modification of proteins in eukaryotic cells. Various cellular processes, including the deposition of carbohydrates on protein surfaces, have been described. The importance of glycans in protein stability, protein folding, and protease resistance has been confirmed in the literature. Furthermore, the roles of glycans in cell signaling, regulation, and development have been established in the field.

[0006] Carbohydrates are monosaccharides, such as xylose, arabinose, glucose, galactose, mannose, fructose, fucose, N-acetylglucosamine, and sialic acid; (homologous or heterologous)disaccharides, such as lactose, sucrose, maltose, and cellobiose; (homologous or heterologous) oligosaccharides, such as polysaccharides (e.g., N-glycans and O-glycans), galactooligosaccharides (GOS), fructooligosaccharides (FOS), lactooligosaccharides (MOS), or even the glycosyl groups of glycolipids; and polysaccharides, such as amylose, amyloid proteins, cellulose, glycogen, glycosaminoglycans, or chitin. Oligosaccharides and polysaccharides can be linear or (multi)branched.

[0007] Glycoconjugates are compounds in which a carbohydrate (glycosyl group) is linked to a non-carbohydrate moiety (glycoside). Typically, the aglycone is a protein or lipid; therefore, glycoconjugates are called glycoproteins or glycolipids, respectively. In a more general sense, glycoconjugates refer to carbohydrates covalently linked to any other chemical entity, including proteins, peptides, lipids, and even carbohydrates.

[0008] Glycoconjugates represent the most structurally and functionally diverse molecules in nature. They range from simple glycoconjugates consisting of a single nucleotide and a monosaccharide moiety to highly complex and multiglycosylated proteins. The most common carbohydrate moieties in glycoconjugates are concentrated on a few monosaccharides (including N-acetylglucosamine, N-acetylglucosamine, mannose, galactose, fucose, glucose, as well as xylose and sialic acid) and their modifications (including phosphorylated or sulfated modifications), exhibiting structural diversity potentially far greater than that of proteins or DNA.

[0009] This diversity stems from the presence of anomers and the ability of monosaccharides to branch and form different glycosyl bonds. Therefore, oligosaccharides with relatively short chain lengths can possess a large number of structural isomers. In contrast to protein biosynthesis, which is based on RNA as a template, the flow of information from the genome to the glycomolecular group is complex and not a template-driven process. For example, co-translation and post-translational modifications of proteins in glycan biosynthesis are based on enzymatic reactions. Due to glycan biosynthesis, the complexity and structural diversity of glycans increase dramatically. It is noteworthy that the terms "glycan" and "glycosyl group" are used synonymously, both referring to the carbohydrate portion of a glycoconjugate.

[0010] Furthermore, the terms glycan, oligosaccharide, and polysaccharide are used synonymously to refer to "a compound having a (moderate or large) number of monosaccharides linked by glycosidic bonds." In proteins, oligosaccharides are primarily linked to the protein backbone via N- (via Asn) or O- (via Ser or Thr) glycosidic bonds, while N-glycosylation represents a more common type in glycoproteins. Variations in glycosylation site occupancy (macroscopic heterogeneity), and variations in the complex sugar residues linked to a single glycosylation site (microscopic heterogeneity), result in a diverse set of protein glycoforms. These possess different physical and biochemical properties, leading to additional functional diversity in glycoproteins. For example, macroscopic and microscopic heterogeneity has been shown to influence protein properties when producing therapeutic proteins in mammalian cell cultures. For instance, the correlation between glycosylation profiles and monoclonal antibody therapeutic profiles has been documented. Notably, glycan structures, particularly N-glycan structures, also depend on various factors during the production process, such as substrate levels and other culture conditions. Therefore, glycoprotein production depends not only on the host cell's glycosylation mechanism but also on external parameters such as culture conditions and the extracellular environment. Other parameters affecting glycosylation in culture production include temperature, pH, aeration, substrate supply, or the accumulation of byproducts such as ammonia and lactate. For example, in the pharmaceutical field, glycosylation profiling is of particular interest because the glycosylation profiles of drugs must be determined for regulatory reasons.

[0011] Similarly, in the food and pharmaceutical industries, the beneficial effects of different types of glycoconjugates, namely their nutritional and / or biological effects, are attracting increasing attention. Today, complex mixtures of soluble but also oligomeric and / or polymeric carbohydrates, synthesized or obtained from natural sources such as plant, human, or animal milk, are used as nutritional supplements or pharmaceuticals. The presence of sialic acid or sialic acid derivatives, as well as monosaccharides with phosphate, sulfate, or carboxyl groups in these complex natural carbohydrates, further increases their complexity. Due to this complexity, prebiotic oligosaccharides or polysaccharides, such as neutral or acidic galactooligosaccharides, long-chain fructooligosaccharides, or (human)lacto oligosaccharides ((H)MOS), which may possess nutritional and / or biological effects, are attracting increasing interest from the food and pharmaceutical industries.

[0012] To elucidate the structural characteristics of glycosides (which refer to the entire set of free carbohydrates and glycoconjugates in the cell) produced under specific conditions, and to understand their functions and their inverse relationship with DNA and protein mechanisms, rapid, robust, and high-resolution analytical techniques are essential.

[0013] Extensive strategies and analytical techniques have been established for the analysis of glycoconjugates, including glycoproteins, glycopeptides, and released N- or O-glycans. For example, complex samples containing multiple different oligosaccharides can be separated using chromatographic or electrokinetic techniques. These techniques include chromatographic techniques such as size exclusion chromatography (SEC), hydrophilic interaction chromatography (HILIC), reversed-phase liquid chromatography (RPLC), and reversed-phase ion-pair chromatography (RPIPC), as well as porous graphitized carbon chromatography (PGC). Furthermore, structural data of complex molecules, including carbohydrates, derived from glycoconjugates are analyzed either by mass spectrometry (MS) or nuclear magnetic resonance spectroscopy (NMR), which are typically laborious and time-consuming techniques for sample preparation and data interpretation. For example, combinations of several techniques are often used, such as a combination of liquid chromatography (LC) with NMR or MS, or capillary electrophoresis (CE) with MS or NMR. Typically, glycosylation patterns, also identified as compositional patterns of carbohydrate mixtures, are obtained to identify characteristic properties of the glycosyl groups, such as retention or migration times. By comparing the data obtained from unknown samples with the measured parameters, unknown samples can be quickly screened and evaluated.

[0014] Each of these techniques has its advantages and disadvantages. Choosing one method for each given problem would be a time-consuming and laborious task. For example, NMR provides detailed structural information but is a relatively insensitive method (nmol) and cannot be used as a high-throughput method. MS is more sensitive than NMR (fmol). However, quantification can be difficult, and without addressing the linkages of monomeric sugar compounds, only non-specific structural information can be obtained. Both techniques require extensive sample preparation and separation of complex polysaccharide mixtures before the corresponding spectroscopic analysis can be performed. Furthermore, a team of skilled scientists is needed to ensure the proper functioning of both techniques.

[0015] Analytical methods based on electrokinetics and chromatographic separation are easier, cheaper, and therefore more common. The most common and often adulterated are chromatographic sugar analysis techniques, such as fluorescence detection hydrophilic interaction chromatography (HILIC-FLR) and fluorescence detection reversed-phase liquid chromatography (RPLC-FLR). They can be operated as high-performance liquid chromatography or ultra-high-performance liquid chromatography (HPLC or UHPLC), but so far only external standards (i.e., not in the same raceway and column as the sample, as internal standards are used) are used for retention time alignment, resulting in only limited (long-term) reproducibility (Kobata A, et al., Methods Enzymology 1987, 138, 84-94. Tomiya N, et al., Analytical Biochemistry 1988, 171, 73-90. Guile GR, et al., Analytical Biochemistry 1996, 240, 210-226).

[0016] Although separation techniques based on the principle of capillary electrophoresis, such as capillary gel electrophoresis, have previously been considered in the field for the separation of complex carbohydrates (e.g., Callewaert, N. et al., Glycobiology 2001, 11, 275-281, WO 01 / 92890; Callewaert, N. et al., Nat. Med. 2004, 10, 429-434; Hennig R. et al., Biochimica et Biophysica Acta-General Subjects 2016, 1860, 1728-1738; Ruhaak LR. et al., Journal of Proteome Research 2010, 9, 6655-6664, EP2112506 A1), there remains a persistent need for reliable and rapid systems that allow for automated high-throughput carbohydrate analysis.

[0017] Examples of electrokinetic separation techniques include capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). These techniques allow for high-resolution, rapid separation and quantification. For example, laser-induced fluorescence detection multiplex capillary gel electrophoresis (xCGE-LIF) has proven to be a particularly powerful tool for sugar analysis. One advantage of multiplex capillary array settings is that very high throughput analysis is possible due to the parallelization of separation. Another reason for using xCGE-LIF is due to the very high sensitivity of LIF detection. CGE is defined as "a special case of capillary sieving electrophoresis in which the capillary is filled with a cross-linked gel (polymer)."

[0018] The electrophoretic mobility of a compound depends on its mass-to-charge ratio, and when using, for example, CGE, it also depends on molecular shape due to the gel sieving effect. Typically, natural carbohydrates cannot be separated by their mass-to-charge ratio because, except for those containing charged residues such as sialic acid, glucuronic acid, sulfated, or phosphorylated moieties, they are mostly electroneutrally neutral. However, one problem with CE is the (long-term) reproducibility of migration time, as in CGE, due to gel aging present in the capillary. Therefore, its usability has been somewhat limited to date, even when using internal standards aligned to migration time (such as DNA base pair (bp) ladders with fluorescent tags emitting wavelengths different from dyes (such as APTS) used for carbohydrate samples), because although there are comparable mass-to-charge ratios (m / z), m and z are very different for bp alignment standards and carbohydrate samples (see EP2112506 A1). Therefore, the content and composition of the matrix (such as salt, solvent, gel, etc.) as well as temperature and time (which also cause changes in the matrix, such as due to gel aging) reduce reproducibility and thus reduce usability.

[0019] Since Sanger discovered chain termination methods for DNA sequencing in 1977, significant progress has been made in increasing sequencing throughput. The first improvement came in the mid-1980s with the replacement of radioactive labeling of DNA fragments with fluorescent dyes. By labeling each DNA base with a separate fluorescent dye (containing different excitation and emission wavelengths), all four reaction mixtures can be loaded into a single lane of a slab gel and analyzed simultaneously. A laser scanning system with filters enables wavelength-resolved detection of the fluorescence emission of all four dyes (representing all types of DNA bases). Converting these to digital signals paved the way for the development of automated DNA sequencing, such as the ABI PRISM 377 genetic analyzer.

[0020] In traditional slab gel electrophoresis systems, multiple samples are separated in a thin gel with multiple individual lanes. Unfortunately, because the separation rate is limited by the electric field strength, which cannot be increased due to the heat generated in the gel, it is difficult to increase the throughput. Furthermore, the detection rate is limited to one to a few seconds per data point.

[0021] To overcome this problem, capillary electrophoresis (CE) systems have been developed with several parallel capillaries (capillary arrays) with diameters of only 10-50 μm. Due to their large surface area / volume, better heat transfer is achieved, allowing for higher field strengths and faster separation. Optimized optics within these multi-capillary CE systems, with laser beams laterally aligned with the parallel capillaries, allow for the simultaneous excitation of all fluorescently labeled analytes within all capillaries. These laser-induced fluorescence (LIF) detections offer the lowest detection limits. During detection, the emitted fluorescence is filtered using a set of virtual filters (viewing windows), and the fluorescence signal is subsequently captured from a defined single channel (multi-wavelength detection) using a CCD camera.

[0022] Figure 32 : Detection mode of multi-capillary CE system with multi-wavelength detection.

[0023] Because the emission spectra of fluorescent dyes are always quite broad and overlapping (as shown in Scheme 1), a dummy filter needs to be calibrated. Therefore, the goal is not to collect the maximum emission, but to minimize the overlap of the emission profiles on the CCD array. However, spectral overlap still occurs to some extent, and there is always some crosstalk, as shown in Scheme 1 for intermediate fluorescent dyes.

[0024] For DNA sequencing, each of the four nucleotides is labeled with a fluorescent dye. During sequencing, the most prominent peak in each color channel is always selected to define the nucleotide. Spectral crosstalk is less important for DNA sequencing because there is no need to consider smaller crosstalk signals from adjacent dye channels.

[0025] Oligosaccharide analysis via multiplexed CE (xCE) systems requires entirely different requirements. Generally, an unknown sample labeled with one fluorescent dye is co-injected and co-separated with an alignment standard labeled with another fluorescent dye. This internal standard is then used to align the migration time of the unknown sample. Through this alignment, automated determination and / or identification of sample components becomes possible.

[0026] For accurate analysis, it is essential to eliminate spectral crosstalk between the two dye channels (between the unknown sample and the alignment standard). For example, the electrophoresis pattern of an unknown sample (a mixture of complex oligosaccharides) contains peaks with intensities varying by several orders of magnitude. Signals "leaking" from the alignment standard channel can introduce additional peaks, altering the composition of the unknown sample and thus increasing the analytical burden. Recalibrating the multi-channel CE system is crucial to eliminating crosstalk between dye channels.

[0027] Natural carbohydrates are difficult to detect using spectroscopic methods. Only UV light (ultraviolet light) with wavelengths below 200 nm is detectable. To overcome this limitation, released N-glycans are labeled with fluorescent tags before (chromatographic or electrokinetic) separation so that they can be well detected by UV, VIS, FLR, and LIF detectors.

[0028] Figure 1 The main steps of separation based on glycan analysis are shown. The procedure can be divided into the following steps: sample preparation, chromatographic or electrokinetic separation with fluorescence detection, and data evaluation. The labeling of glycans and the detection of labeled products are described in the art. The main reaction mechanism for reductive amination of fluorescently labeled carbohydrates is shown in Scheme 2.

[0029] Scheme 2 below shows the main reaction sequence of carbohydrate reductive amination (see N. Volpi, Capillary electrophoresis of carbohydrates. From monosaccharides to complex polysaccharides, Humana Press, New York, 2011, pp. 1-51).

[0030]

[0031] Option 2

[0032] The first step of reductive amination involves a nucleophilic addition reaction, in which the lone pair of electrons of the amine nitrogen attacks the electrophilic aldehyde carbon atom of the carbohydrate residue in the open-chain form (1b). Acid-catalyzed removal of water from intermediate 2 yields an imine (3a). Since the formation of the imine is reversible, the imine must be converted to a secondary amine (4) via irreversible acid-catalyzed reduction using a hydride source (the reducing agent in scheme 2). The nature of the reducing agent is important because only the imine ion 3b needs to be reduced, while the carbohydrate R... 2 CHO(1b) must remain unreactive to reduction (they only react with amines R, which are fluorescently labeled). 3 (NH2 reaction).

[0033] The reaction sequence described in Scheme 2 is based on the availability and sufficient reactivity of a specific reducing agent (borane) that does not react with aldehydes (or reduces them very slowly), but readily reduces imine ions under acidic conditions (3b). Weak or moderately strong acids, such as acetic acid (pKa = 4.76), malonic acid (pK1a = 2.83), or citric acid (pK1a = 3.13), are often used at pH 3–6 to achieve irreversible and rapid reduction reactions (KRAnumula, Anal. Biochem. 2006, 350, 1–23).

[0034] Therefore, the amine (R) used 3 NH2) must be a weak base (because in Scheme 2, only unprotonated amines react with aldehyde 1b). According to Scheme 2, in proteins, the nucleophilic nitrogen atoms of the aliphatic amino group of lysine, histidine, and arginine residues are protonated at pH 3–6 and do not react with carbohydrates. Therefore, only aromatic amines with relatively low pKa values ​​of 3–5 (these are the values ​​of conjugate acids) are needed, and they are widely used as analytical reagents for the reductive amination of natural glycans. Three commercially available aromatic amines are shown below, suitable for labeling glycans by reductive amination, chromatographic or electrokinetic separation of conjugates, and sensitive detection by fluorescence.

[0035]

[0036] Option 3

[0037] 3-Aminopyrene-1,6,8-trisulfonic acid (APTS), 2-aminobenzamide (2-AB), and 2-aminobenzoic acid (2-AA) are currently the most widely used reagents for carbohydrate labeling in CE (APTS) and LC (2-AB and 2-AA) base analysis. In particular, APTS, with its three strongly acidic residues (sulfonic acid groups), introduces three negative charges over a very wide pH range (pH>2) to enable flexible and robust analysis.

[0038]

[0039] Option 4

[0040] Alkoxyamino (Scheme 4a) and acylhydrazine (Scheme 4b) groups also provide convenient chemoselective methods for labeling carbohydrates. The acylhydrazine group reacts with the reducing end of the free carbohydrate to form products primarily in the form of cyclic β-anomes (see Scheme 4b). Reaction conditions range from acidic and superneutral to alkaline pH at high temperatures. For example, the typical acylhydrazine labeling reaction for fluorescein (see Scheme 3) can be carried out at 70°C and pH 7 for 1 hour.

[0041]

[0042] Option 5

[0043] Furthermore, reactive carbamate chemicals can be used for carbohydrate labeling, as shown in Scheme 5. For this labeling reaction, the carbohydrate needs to be present in its glycosylamine form (the released carbohydrate forms a glycoconjugate, such as an N-glycan released by the enzymatic release of peptide N-glycosidase F (PNGase F)). This reaction is non-specific because reactive carbamates can react with other available amines, such as proteins (the amino acid lysine). The typical reaction of N-hydroxysuccinimide (NHS) carbonate with glycosylamines takes only a few minutes at room temperature.

[0044] Because the reductive amination of carbohydrates is highly specific and complete, this reaction is currently the most widely used method for carbohydrate labeling.

[0045] After facultative purification (to remove proteins, excess electrolytes, excess dyes, labeling reagents, etc.), the labeled samples are injected separately into the chromatographic column and electrokinetic capillary for separation (see [link]). Figure 1 Due to their different properties (such as hydrophobicity, mass / charge, shape, etc.), carbohydrates reach the detector based on their characteristic retention time and migration time (see [link]). Figure 2-22 ).

[0046] When the labeled carbohydrate reaches the fluorescence detector, the covalently linked fluorescent dye is excited and the emission signal is detected.

[0047] Currently, glycan analysis is performed on commercial (U)HPLC systems with fluorescence detectors after labeling with, for example, 2-AB or 2-AA (see Scheme 3). However, “truly” high-throughput analysis of labeled glycans can only be performed on commercial multiplex CGE systems. These xCGE-LIF instruments include a multiplex capillary gel electrophoresis unit for separating charged analytes (such as APTS-labeled glycans), a laser, and a fluorescence detector.

[0048]

[0049] Scheme 6: Spectroscopic properties of APTS and its N-alkylated derivatives in aqueous buffer solutions (based on Z. Sharrett, et al., Org. Biomol. Chem. 2009, 7, 1461-1470 and RAEvangelista, MS. Liu, FT. A. Chem., Anal. Chem. 1995, 67, 2239-2245).

[0050] Other dyes besides APTS can be used as fluorescent labels for analysis based on isolated carbohydrates and their derivatives (e.g., dyes 2-AB, 2-AA, and fluorescent yellow, see Scheme 3 and the review by NVShilova and NVBovin, Russ. J. Bioorg. Chem. 2003, 29(4), 339-355). Further examples are acridinone dyes described in WO 2002 / 099424 A3 and WO 2009 / 112791 A2, but not 7-aminoacridinone-2-sulfonamide. WO 2012 / 027717 A1 describes a system comprising a functionally substituted 1,6,8-trisulfonamide-3-aminopyrene (an APTS derivative), an analyte reactive group, a cleavable anchor, and a porous solid phase. WO 2010 / 116142 A2 describes a variety of fluorophores and fluorescence-sensing compounds, including aminopyrene dyes. However, none of these dyes show or suggest superior spectral and electrophoretic properties, particularly as conjugates with carbohydrates, compared to APTS.

[0051] Techniques for the separation and analysis of carbohydrates, as well as analyses of glycosylation patterns, have been described in this field. For example, Callewaert N et al., Glycobiology 2001, 11, 275-281, WO 01 / 92890, Callewaert N et al., Nat. Med., 2004, 10, 429-439, or Khandurina et al., Electrophoresis, 2004, 25, 3122-2127, have confirmed methods for carbohydrate analysis. Domann et al., Practical Proteomics, 2007, 7, 70-76, have confirmed 2D HPLC analysis, mass spectrometry, and lectin affinity chromatography.

[0052] The inventors have described further developments in EP 2112506 A1 and US 2009 / 0288951 A1. The techniques described therein have been successfully applied.

[0053] However, a major drawback of evaluating glycan spectra is the limited availability of suitable dyes. That is, none of the currently known dyes are considered to have excellent spectroscopic or electrophoretic properties, especially as conjugates with carbohydrates, yet the current standard is the use of APTS.

[0054] Therefore, fluorescent dyes with improved performance are needed, such as higher electrophoretic mobility and / or higher brightness compared to APTS. These properties are particularly important for fluorescent tags used for carbohydrate analysis based on both electrokinetic and chromatographic separations, using fluorescence detection, to achieve higher performance. Furthermore, fluorescent dyes that can be used in combination with known dyes, including APTS, are required, allowing for the detection of two different colors in the same run and thus enabling internal alignment of migration and retention times. Summary of the Invention

[0055] The object of this invention is to provide a novel method for determining and / or identifying the compositional pattern profiling of carbohydrates and / or mixtures of carbohydrates. This method is based on the alignment of retention / migration times with internal standards and uses at least two different fluorescent dyes, allowing highly reproducible electrokinetic / chromatographic separation by subsequent fluorescence detection or laser-induced fluorescence detection. The carbohydrate sample and carbohydrate standards are labeled with at least two suitable fluorescent dyes emitting different wavelengths, which is essential for this internal migration / retention time alignment, enabling high long-term reproducibility and matrix / sample independence, as described below.

[0056] In a first aspect, a method for automatically determining and / or identifying compositional patterns of carbohydrates and / or mixtures of carbohydrates includes the following steps:

[0057] a) Obtain a sample containing at least one carbohydrate;

[0058] b) Label the carbohydrate with a first fluorescent marker;

[0059] c) Provide standards of known composition labeled with a second fluorescent marker;

[0060] d) Use electrokinetic / chromatographic separation techniques combined with fluorescence or laser-induced fluorescence detection to determine the migration / retention time of the carbohydrates and standards of known composition;

[0061] e) Align the migration / retention time with the migration / retention time index based on the given standard migration / retention time index of the standard;

[0062] f) Compare these migration / retention time indices of carbohydrates with standard migration / retention time indices from a database;

[0063] g) Identify or determine the compositional patterns of carbohydrates and / or mixtures of carbohydrates.

[0064] The standard composition is added to a sample containing unknown carbohydrates and / or mixtures of carbohydrates. The first and second fluorescent labels are distinct, and the first or second fluorescent label is a fluorescent dye having multiple ionizable and / or negatively charged groups, said fluorescent dye being selected from compounds of the following general formulas A and B:

[0065]

[0066] in,

[0067] R 1 R 2 R 3 R 4 R 5 They are independent of each other and can represent each other:

[0068] H, CH3, C2H5, straight-chain or branched C3-C 12 Preferably C3-C6, alkyl or perfluoroalkyl, phosphorylated alkyl (CH2) m P(O)(OH)2, wherein m = 1-12, preferably m = 2-6, has a straight-chain or branched alkyl chain, (CH2) n COOH, wherein n = 1-12, preferably n = 1-5, or (CH2) n COOR 6 Where n = 1-12, preferably n = 1-5, and R 6 It can be an alkyl group, especially C1-C6 alkyl, CH2CN, benzyl, fluorene-9-yl, polyhalogenated alkyl, polyhalogenated phenyl, such as tetrafluorophenyl or pentafluorophenyl, pentachlorophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, or other potentially nucleophilic leaving groups, alkyl sulfonic acid group ((CH2) n SO3H) or alkyl sulfate group ((CH2) n OSO3H), where n = 1-12, preferably n = 1-5, and any (CH2) n The alkyl chain in it can be straight or branched;

[0069] Hydroxyalkyl (CH2) m OH or thioalkyl (CH2) m SH, wherein m = 1-12, preferably m = 2-6, has a straight-chain or branched alkyl chain, and is a phosphorylated hydroxyalkyl group (CH2). m OP(O)(OH)2, wherein m = 1-12, preferably m = 2-6, has a straight-chain or branched alkyl chain; R1 Or R 2 One of the groups can be a carbonate or carbamate derivative (CH2). m OCOOR 7 or COOR 7 Where m = 1 - 12 and R 7 = Methyl, ethyl, tert-butyl, benzyl, fluorene-9-yl, CH2CN, N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazole, phenyl, substituted phenyl, such as 2- or 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluorophenyl, 2-pyridyl, 4-pyridyl, pyrimidin-4-yl;

[0070] (CH2) m NR a R b Where m = 1-12, preferably 2-6, and has a straight-chain or branched alkyl chain; R a R b Each is independent of the others and represents hydrogen and / or C1-C4 alkyl, hydroxyalkyl (CH2). m OH, where m = 2-6, has a straight-chain or branched alkyl chain, phosphorylated hydroxyalkyl (CH2). m OP(O)(OH)2, wherein m = 1-12, preferably 2-6, has a straight-chain or branched alkyl chain; alkyl azide (CH2) m N3, wherein m = 1-12, preferably 2-6, has a straight-chain or branched alkyl chain;

[0071] R 1 R 2 R 3 R 4 R 5 It may contain a terminal alkoxyamino group (CH2). m ONH2, wherein m = 1-12, preferably 2-6, has a straight-chain or branched alkyl chain;

[0072] (CH2) n CONHR 8 Where n = 1-12, preferably 1-5; R 8 =H, C1-C6 alkyl, (CH2) m N3, or (CH2) m -N-maleimide group ((CH2) m -N-maleimido), (CH2) m -NH-COCH2X (X = Br or I), where m = 1-12, preferably 2-6, and in (CH2) n (CH2) m and R 6It contains straight-chain or branched alkyl chains;

[0073] Group R 1 R 2 R 3 R 4 R 5 R is preferred 1 R 2 R 3 It can be represented by the primary amino group that forms arylhydrazine Ar-NHNH2, where Ar represents the dye residue of formula A, including the aryl amino group and the linker;

[0074] Hydroxyl group, preferably R 2 Or R 3 It is the hydroxyl group that forms the aryl hydroxylamine Ar-NH2OH, where Ar represents the dye residue of formula A, including the aryl amino group and the linker;

[0075] Furthermore, residue R 1 R 2 R 3 R 4 R 5 One of them can represent CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2 or CSNHC6H4-NH2, where C6H4 is 1,2-, 1,3- or 1,4-phenylene; COC5H3N-NH2 or CH2-C5H3N-NH2, where C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl or pyridine-3,5-diyl;

[0076] In addition, R 2 ---R 3 and / or (R) 4 ---R 5 It can form four-, five-, six-, or seven-membered rings, or have or not have a primary amino group (NH2) or a secondary amino group (NHR) attached to a carbon atom in the ring. a , where R a = A four-, five-, six-, or seven-membered ring of C1-C6 alkyl, hydroxyl (OH), or phosphorylated hydroxyl group -OP(O)(OH)2;

[0077] Optional, R 2 ---R 3 and / or (R) 4 ---R 5 It can form four-membered, five-membered, six-membered or seven-membered heterocycles, which also contain 1-3 heteroatoms, such as O, N or S;

[0078] Furthermore, R 1may represent an unsubstituted phenyl group, a phenyl group having one or several electron-donating substituents selected from the group consisting of OH, SH, NH2, NHR a , NR a R b , R a O, R a S, wherein R a and R b are independent of each other and may be a C1-C6 alkyl group having a straight-chain or branched-chain carbon chain, a phenyl group having one or several electron-accepting substituents selected from the group consisting of NO2, CN, COH, COOH, CH═CHCN, CH═C(CN)2, SO2R a , COR a , COOR a , CH═CHCOR a , CH═CHCOOR a , CONHR a , SO2NR a R b , CONR a R b , wherein R a and R b are independent of each other and may be H or a C1-C6 alkyl group having a straight-chain or branched-chain carbon chain; or R 1 may represent a heteroaromatic group.

[0079] The compounds of formula A may exist as salts, solvates and hydrates and may be used as salts, solvates and hydrates, preferably as salts with alkali metal cations including Na + , Li + , K + and organic ammonium;

[0080] provided that in all compounds of formula A above, under basic conditions, i.e., 7 < pH < 14, there are at least two, preferably at least 3, 4, 5 or 6 negatively charged groups, and these negatively charged groups represent residues of at least partially deprotonated ionizable groups selected from: SH, COOH, sulfonic acid residue SO3H, primary phosphoric acid group OP(O)(OH)2, secondary phosphoric acid group OP(O)(OH)R a , wherein R a ═C1-C4 alkyl or substituted C1-C4 alkyl, primary phosphonic acid group P(O)(OH)2, secondary phosphonic acid group OP(O)(OH)R a , wherein R a ═C1-C4 alkyl or substituted C1-C4 alkyl;

[0081]

[0082] Where R 1 and / or R 2 They are independent of each other and can represent each other:

[0083] H, CH3, C2H5, straight-chain or branched C3-C 12 Alkyl or perfluoroalkyl, or substituted C2-C 12 Alkyl group; specifically, (CH2) n COOR 3 Where n = 1-12, preferably 1-5, R 3 It can be H, alkyl, especially C1-C6, CH2CN, benzyl, 2- and 4-nitrophenyl, fluorene-9-yl, polyhalogenated alkyl, polyhalogenated phenyl, such as tetra- or pentafluorophenyl, pentachlorophenyl, N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazolyl or other potentially nucleophilic leaving groups, and (CH2) n The alkyl chain in the form can be straight or branched; and

[0084] R1-R2 can form a four-, five-, six-, or seven-membered non-aromatic carbon ring, which has an additional primary amino group NH2 or a secondary amino group NHR attached to one carbon atom of the ring. a , where R a = C1-C6 alkyl, or hydroxyl OH; optionally, R1---R2 can form a four-, five-, six-, or seven-membered non-aromatic heterocycle containing additional heteroatoms, such as O, N, or S;

[0085] Hydroxyalkyl (CH2) m OH, wherein m = 1-12, preferably 2-6, has a straight-chain or branched alkyl chain; R 1 Or R 2 One of the groups can be a carbonate or carbamate derivative (CH2). m OCOO R 4 or COOR 4 Where m = 1 - 12 and R 4 = Methyl, ethyl, 2-chloro-ethyl, N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazolyl, phenyl or substituted phenyl, such as 2- or 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2, 3, 5, 6-tetrafluorophenyl, 2-pyridyl or 4-pyridyl;

[0086] (CH2) m NR a R b Where m = 1-12, preferably 2-6, and has a straight-chain or branched alkyl chain; R a R bThey are independent of each other and can be H, or optionally substituted C1-C4 alkyl groups, particularly R. 1 Or R 2 One of the groups can be an azide group (CH2). m N3 where m = 2-6 and has a straight-chain or branched alkyl chain; R 1 Or R 2 One of them can be (CH2) of n = 1-12. n SO2NR 5 NH2, and substituent R 5 It can be composed of H, alkyl, hydroxyalkyl, or perfluoroalkyl groups (C1-C). 12 express;

[0087] R 1 Or R 2 One of the groups can be a primary amino group to form arylhydrazine Ar-NR. 6 NH2, where Ar is the entire pyrene residue in formula B, and R 6 =H or alkyl; R 1 Or R 2 One of the groups can be a hydroxyl group to form an aryl hydroxylamine Ar-NR. 7 OH, where Ar is the entire pyrene residue in formula B, and R 7 =H or alkyl;

[0088] R 1 Or R 2 One of the groups may contain a terminal alkoxyamino group (CH2) with n = 1-12. n ONH2 can be obtained through one or more alkylamines (CH2) in all possible combinations of m = 0-12. m NH or alkylamide (CH2) m CONH group linkage;

[0089] R 1 Or R 2 One of the groups can be CO(CH2). n COOR 8 Where n = 1-5, and has straight-chain or branched alkyl chains (CH2). n And R 8 Selected from H, straight-chain or branched C1-C6 alkyl, CH2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, N-succinimide;

[0090] Furthermore, R 1 Or R 2 One of them could be (CH2). n CONHR9 Where n = 1 - 5 and R 9 =H, C1-C6 alkyl, (CH2) m N3, (CH2) m -N-maleimide group, (CH2) m -NHCOCH2X (X = Br or I), where m = 2-6 and in (CH2) n and R 9 It contains straight-chain or branched alkyl chains;

[0091] Or R 1 Or R 2 One of them can represent CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2, or CSNHC6H4-NH2, where C6H4 is 1,2-, 1,3-, or 1,4-phenylene; COC5H3N-NH2 or CH2-C5H3N-NH2, where C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl, or pyridine-3,5-diyl; or R 1 Or R 2 One of them can be an alkyl azide (CH)N3 or an alkyne, especially propargyl;

[0092] The connector L contains at least one carbon atom and may contain alkyl, heteroalkyl, particularly alkoxy such as CH2OCH2, CH2CH2O, CH2CH2OCH2, alkylamino or dialkylamino, particularly diethanolamine or N-methyl(alkyl)monoethanolamine such as N(CH3)CH2CH2O- and N(CH2CH2O-)2, perfluoroalkyl such as mono- or polydifluoromethyl (CF2), olefin or alkyne moiety, in any combination, in any case, straight-chain or branched, with a length ranging from C1 to C2. 12 ;

[0093] The connector may also include a carbonyl (CH2CO, CF2CO) moiety;

[0094] X represents a solubilizing and / or ionizable anion-providing moiety, particularly consisting of or including moiety selected from the group consisting of hydroxyalkyl (CH2). n OH, thioalkyl ((CH2) n SH), carboxylalkyl ((CH2) n CO2H), alkyl sulfonic acid group ((CH2) n SO3H), alkyl sulfate group ((CH2) n OSO3H), alkyl phosphate group ((CH2)n OP(O)(OH)2) or phosphonic acid group ((CH2) n P(O)(OH)2), where n is an integer from 0 to 12, or an analogue thereof, in which one or more CH2 groups are replaced by CF2,

[0095] Furthermore, the anion-providing moieties can be linked via non-aromatic O-, N- and S-containing heterocycles, such as piperazines, pipecloines, or, alternatively, one of the groups X can carry any of the moieties listed above for group R 1 and R 2 listed, can also carry any of the types of bonds listed for group L, and are independent of the other substituents;

[0096] The compounds of formula B can exist as salts, solvates and hydrates and can be used as salts, solvates and hydrates, preferably as salts with alkali metal cations including Na + 、Li + 、K + ,NH4 + and organic ammonium or phosphonium cations.

[0097] In more specific embodiments, the fluorescent dye salts according to the invention can comprise a negatively charged acid group, particularly a sulfonic acid group and / or a phosphoric acid group, and counterions selected from inorganic or organic cations, preferably alkali metal cations, ammonium cations or cations of organic ammonium or phosphonium compounds (such as trialkylammonium cations), and / or can comprise a positively charged group or a charge transfer complex formed at the nitrogen site N(R1)R2 of the dye of general formula A-D, and counterions, particularly anions selected from strong mineral acids, organic acids or Lewis acids.

[0098] Provided that in all compounds represented by formula B, under basic conditions, i.e. 7 < pH < 14, there are three or six negatively charged groups in the residue X of formula B, and these negatively charged groups represent residues of at least partially deprotonated ionizable groups selected from: SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a 、where R a = C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)R a 、where R a = C1-C4 alkyl or substituted C1-C4 alkyl.

[0099] On the other hand, a method for automated analysis of the composition patterns of carbohydrate mixtures includes the following steps:

[0100] a) Provide a first sample containing a first unknown mixture of carbohydrates;

[0101] b) Label the carbohydrate mixture composition with a first fluorescent marker;

[0102] c) Provide a second sample comprising a mixture of second carbohydrates labeled with a second fluorescent marker, which may optionally be added to the first sample;

[0103] d) Using electrokinetic / chromatographic separation techniques combined with fluorescence or laser-induced fluorescence detection, generate an electrophoretic / chromatographic image of the carbohydrate mixture composition of the sample composition;

[0104] e) Analyze the similarities and / or differences between the carbohydrate mixture composition patterns of the first and second samples.

[0105] The first fluorescent label of the first sample is different from the second fluorescent label of the second sample, and at least one of the first fluorescent label and the second fluorescent label is a fluorescent dye of general formula A or B as defined above, similar to general formula C or D as defined below.

[0106] In a further aspect, a method for automated analysis of compositional patterns in carbohydrate mixtures includes the following steps:

[0107] a) Provide a sample containing the first carbohydrate mixture;

[0108] b) Label the carbohydrate mixture composition with a first fluorescent marker;

[0109] c) Provide a second sample labeled with a second fluorescent marker, which contains a second mixture of carbohydrates to be compared;

[0110] d) Using electrokinetic / chromatographic separation techniques combined with fluorescence or laser-induced fluorescence detection, generate an electrophoretic / chromatographic image of the carbohydrate mixture of the first and second sample compositions;

[0111] e) Compare the standard migration / retention time indices calculated from the electrophoresis / chromatograms of the first and second samples obtained;

[0112] f) Analyze the similarities and / or differences between the carbohydrate mixture composition patterns of the first and second samples, wherein the standard migration / retention times of the carbohydrates present in the samples are calculated based on internal standards of known composition labeled with a third fluorescent marker, and

[0113] One of the first and second fluorescent labels is a fluorescent dye as defined above, having a structure of general formula A or B, similar to general formula C or D as defined below.

[0114] In one embodiment of the above method for automated analysis of carbohydrate mixture composition patterns, the second carbohydrate mixture composition is a known carbohydrate mixture composition having a known pattern map.

[0115] This invention aims to provide a method for the determination and / or identification of carbohydrates, wherein a labeled sample to be analyzed, containing at least one carbohydrate, is combined with a standard composition added to the mixture of said unknown carbohydrates. The sample containing both the unknown carbohydrate (mixture) and the standard composition is labeled with a first fluorescent label and a second fluorescent label. At least one of the fluorescent labels is a novel fluorescent dye, such as general formula A or B as described herein, similar to general formula C or D as defined below.

[0116] In one embodiment of the invention, a single sample may contain at least two different analyte probes, namely, two different labeled carbohydrates or mixtures of carbohydrates other than a standard composition. That is, the novel fluorescent dyes described herein allow for the determination, analysis, or identification of different carbohydrates in a single sample in a single run. In particular, when first applying the calibration method of the multi-wavelength fluorescence detection system according to the invention, it is reasonable to use at least three or more, such as at least four, different fluorescent dyes (see Tables 2 and 3).

[0117] The novel fluorescent dye has multiple negatively charged residues and aromatic amino or hydrazide groups attached to the fluorophore, which can be excited by an argon ion laser, for example, in an ionized (deprotonated) form.

[0118] In other words, the dyes according to the invention allow for increased throughput and sensitivity. Embodiments using the novel dyes described herein include: one embodiment in which the sample to be analyzed contains two different analyte probes, one probe labeled with, for example, APTS, and the other probe labeled with the novel dye. Furthermore, standards labeled with the novel dye, such as carbohydrate standards or base pair standards, are provided. Another embodiment includes a sample containing three different probes, which are detected together with standards labeled with the novel dyes according to the invention. The three probes present in the sample include one APTS-labeled probe and two probes labeled with the dyes according to the invention, wherein the dyes are selected in a manner that they do not interfere with each other in the emission spectrum. Another embodiment involves a sample containing three probes, one probe labeled with APTS, the other probes labeled with two different novel dyes that differ in emission spectrum, and standards, which are also aligned standards labeled with the novel dyes. Another embodiment includes a sample containing four analyte probes, i.e., one probe is labeled with APTS, while the other three probes are labeled with different novel dyes, and standards (such as base pair standards).

[0119] The choice of dyes should minimize crosstalk between wavelengths. Suitable combinations are described below.

[0120] Labeling a sample with a dye as described herein to indicate the presence of carbohydrates in the probe being analyzed can improve sensitivity. The dyes described herein are advantageous for instrument spectral calibration and for adding an analyte or probe present in a sample. The sample can be analyzed using a single capillary. Therefore, the number of capillaries can be reduced, and sensitivity and alignment characteristics can be improved.

[0121] Furthermore, the sensitivity of samples labeled with the dye can be increased by shifting the excitation wavelength to a larger wavelength (redshift). Moreover, the dyes described herein exhibit better quantum yields compared to APTS, thus further enhancing sensitivity.

[0122] Furthermore, due to increased sensitivity and reduced inter-wavelength crosstalk, this method is more stable, reproducible, and accurate over the long term, and more independent of operating parameters, samples, sample matrices, instruments, operators, laboratories and locations, and time points. This is particularly true for capillary and gel aging. Differences between short-term, medium-term, and long-term runs can be compensated for by the described internal standards. Moreover, more precise alignment is possible based on the calibration method described herein and in conjunction with novel dyes. Therefore, capillary and column usage can be extended, overcoming aging issues that typically alter sample migration / retention times. Furthermore, the capillary / column itself can be modified (e.g., shortened, thus reducing analysis time) without altering the aligned migration / retention times.

[0123] Furthermore, samples can be run on the capillary using different instruments and under different operating parameters, such as temperature and voltage. This is demonstrated in the examples below. In summary, the new dye improves throughput and sensitivity and can also be used for internal alignment migration and retention time. The sugar analysis method based on electrokinetic and / or chromatographic separation described herein allows for the use of universal (carbohydrate-based) alignment standards, enabling the determination of aligned migration / retention times unaffected by environmental factors such as system, operator, matrix, etc.

[0124] Specifically, the dyes defined herein represent dyes whose emission maximum deviates significantly from that of APTS-labeled analogs. Therefore, it is possible to simultaneously detect two or even three different fluorescent dyes, with minimal interference between them. The fluorescent dyes described herein are typically dyes with multiple net negative charges, particularly high in phosphorylated derivatives with negative charges of -4 and -6, which, when conjugated with glycoconjugates, provide higher dye electrophoretic mobility compared to APTS glycoconjugates.

[0125] In this invention, the term "carbohydrate" refers to monosaccharides such as xylose, arabinose, glucose, galactose, mannose, fructose, fucose, N-acetylglucosamine, N-acetylglucosamine, and sialic acid; (homopolymer or heteropolymer) disaccharides such as lactose, sucrose, maltose, and cellobiose; (homopolymer or heteropolymer) oligosaccharides such as polysaccharides (e.g., N- and O-glycans), galactooligosaccharides (GOS), fructooligosaccharides (FOS), lactooligosaccharides (MOS), or even the glycosyl groups of glycolipids; and (homopolymer or heteropolymer) polysaccharides such as amylose, amylopectin, cellulose, glycogen, glycosaminoglycans (GAG), or chitin. Oligosaccharides and polysaccharides can be linear or branched.

[0126] The term “glycoconjugate” as used in this article refers to a compound containing a carbohydrate moiety. Examples of glycoconjugates include glycoproteins, glycopeptides, proteoglycans, peptidoglycans, glycolipids, GPI-anchors, and lipopolysaccharides.

[0127] As used herein, the term "carbohydrate mixture composition pattern analysis" refers to establishing a specific pattern of the composition of a detected carbohydrate mixture based on the quantity of different carbohydrates present in the mixture, the relative amounts of said carbohydrates present in the mixture, and the types of carbohydrates present in the mixture, and analyzing said pattern, for example, as a diagram or graphic, such as an electrophoresis plot or chromatogram, respectively. Thus, a fingerprint pattern is obtained, for example, in the form of aligned electrophoresis plots / chromatograms, graphics, or diagrams. For example, glycosylation pattern analysis based on fingerprint patterns falls within the scope of this term. In this regard, the term "fingerprint pattern" as used herein refers to aligned electrophoresis plots and / or chromatograms, diagrams, or graphics specific to carbohydrates or carbohydrate mixtures.

[0128] The terms "quantitative determination" or "quantitative analysis" refer to the relative and / or absolute quantification of carbohydrates. Relative quantification can be directly accomplished by the individual peak height of each compound, which is linearly related to its concentration (within the linear dynamic range of the FLR and / or LIF detectors). Relative quantification summarizes the ratio of each carbohydrate present in a composition or standard to another or additional carbohydrates. Furthermore, absolute (semi-)quantitative analysis is possible.

[0129] Internal carbohydrate standards of known composition, for example, can be a group of mono, di, tri, tetra, and / or pentamers, linear and / or branched up to 40 polymers (or more), elute / migrate across the entire range of the fingerprint spectrum of the carbohydrate sample to be analyzed, but are detected in another wavelength trace / channel because they are fluorescently labeled with a different tag than the carbohydrate sample emitting at another wavelength, and therefore do not appear in the sample trace / channel. Examples include:

[0130] a. A carbohydrate-based homopolymer comprising pentoses (such as xylose or arabinose), hexoses (such as glucose, galactose, or mannose), and hexosamines (such as glucosamine, galactosamine, N-acetylglucosamine, or N-acetylglucosamine) of length n = 1 to 40 (or higher), as well as glycosidic bonds in α1-2 (mannose oligosaccharides), α1-4 (such as maltose, starch), α1-5 (arabinoligosaccharides), α1-6 (such as dextran, pullulan, starch), α1-3 (such as dextran, pullulan), β1-3 (such as cellobiose glucose), β1-4 (such as cellulose, mannan, xylooligosaccharides, chitosan), and β1-6.

[0131] b. Heteropolymers, such as hemicellulose, arabinoxylan, arabinogalactan, and fructan.

[0132] cN-glycans

[0133] dO-glycans

[0134] e. Glycolipids

[0135] f. Lactoligosaccharides (MOS)

[0136] This invention represents a further development of the methods described in EP 2112506 A1, US 2009 / 0288951 A1, and their corresponding documents. In particular, using the novel dyes described herein, (internal) standards identical or similar to the samples can be used, since both are now carbohydrates, respectively mixtures of carbohydrates with the same or similar properties (e.g., size, mass, charge, hydrophilicity, hydrophobicity, etc.), and therefore exhibit the same or similar behavior with changes in the environment, such as different matrices (e.g., the content and composition of salts, solvents, gels, etc.), but also including temperature and time (which also cause changes in the matrix, e.g., due to gel aging). Therefore, highly reproducible and precisely aligned migration / retention times allow for highly reliable identification of carbohydrates via migration / retention time matching through respective databases containing carbohydrates and their respective aligned migration / retention times.

[0137] This allows for the identification of unknown carbohydrates and unknown glycosylation patterns with greater sensitivity and specificity. This is particularly true for complex carbohydrate formulations and glycosylation patterns.

[0138] As used herein, the term "substituted" generally refers to the presence of one or more substituents, particularly those selected from the group consisting of: straight-chain or branched alkyl groups, especially C1-C4 alkyl groups, such as methyl, ethyl, propyl, and butyl; isoalkyl groups, such as isopropyl and isobutyl(2-methylpropyl); secondary alkyl groups, such as sec-butyl(but-2-yl); and tertiary alkyl groups, such as tert-butyl(2-methylpropyl). Furthermore, the term "substituted" may also refer herein to an alkyl group having at least one deuterium, fluorine, chloro, or bromine atom substituted for a hydrogen atom, or methoxy, ethoxy, 2-(alkoxy)ethoxy (AlkOCH2CH2O), and more generally, the prior art Alk(OCH2CH2). n OCH2CH2- oligo(ethylene glycol) residues, where Alk = CH3, C2H5, C3H7, C4H 10 And n = 1 - 23.

[0139] As used herein, the term "aromatic heterocyclic group" or "heteroaromatic group" generally refers to an unsubstituted or substituted cyclic aromatic group (residue) having 5 to 10 ring atoms, wherein at least one ring atom is selected from sulfur, oxygen, and nitrogen; the group is attached to the remainder of the molecule through any one of the ring atoms. Representative but non-limiting examples are furanyl, thiophene, pyridinyl, pyrazinyl, pyrimidinyl, pyrroleyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, quinolinyl, and isoquinolinyl.

[0140] The compound of general formula A is an acridinone dye, and the compound of general formula B is a pyrene dye.

[0141] More specifically, according to IUPAC rules, compounds of general formula A are 7-aminoacridine-2-sulfonamides, while compounds of general formula B are 1-aminopyrene dyes with functionally substituted sulfonyl groups at positions 3, 6, and 8, namely (functionally substituted) 1,6,8-trisulfonyl-3-aminopyrene, as shown in the basic structural formulas of general formulas A and B in the schemes below.

[0142]

[0143] The novel fluorescent dyes of this invention exhibit many advantageous features:

[0144] • Aromatic amino groups (NH2), hydrazine (NRNH2), acylhydrazine (CONRNH2), hydroxylamine (NROH), reactive carbamate (NHCOOR), or alkoxyamino group (RONH2) are used for efficient and clean reductive amination or direct condensation with carbohydrates at, for example, pH 2-5; preferably, the aromatic amino group is a primary amino group, but it can also be a secondary amino group; see the diagram above for the structure.

[0145] • A significant net charge in the conjugate – in the range of -3 to -12, at a pH of at least 7 to 14.

[0146] • It has good solubility in aqueous media over a wide pH range;

[0147] • High brightness (this is the overall result of fluorescence quantum yield and extinction)

[0148] • Excellent stability of the dye core, for example, resistance to reduction by boron alkyl reagents.

[0149] • It can be excited by argon-ion lasers emitting at 488 and 514 nm, exhibiting perfect spectral matching and high fluorescence quantum yield.

[0150] Minimum emission at approximately 520nm

[0151] The dye can be purified to a purity of up to 99%.

[0152] The novel fluorescent tag of this invention even allows the detection of "heavy" polysaccharides with very long migration times. These "heavy" polysaccharides are difficult to detect by electrokinetics due to their long migration times and peak broadening, especially if APTS is used as the fluorescent tag.

[0153] More specific embodiments of the invention are described below.

[0154] In equation A above, NR 1 and / or N(R) 2 )R 3 Preferably, it contains a carbonyl group or a nucleophilic reactive group. R 1 R 2 and R 3 It can be represented by hydrogen, straight-chain or branched alkyl, hydroxyalkyl, or perfluoroalkyl. Substituent R 3 R 4 and R 5 Preferably, it contains solubilizing and / or anion-providing groups, especially hydroxyalkyl ((CH2) groups). n OH), thioalkyl ((CH2) n SH), carboxylalkyl ((CH2) n CO2H), alkyl sulfonic acid group ((CH2) n SO3H), alkyl sulfate group ((CH2) n OSO3H), alkyl phosphate group ((CH2) n OP(O)(OH)2) or alkylphosphonic acid group ((CH2) n P(O)(OH)2), where n is an integer from 1 to 12.

[0155] Alternatively, substituent R 1 R 2 R 3 R 4 and R 5 It can be formed from carboxylic acid residues (CH2). n COOH represents esters where n = 1-12, and their reactivity is ester (CH2). n COOR 6 As a nucleophilic reactive group. R 6 It can be hydrogen, alkyl (including tert-butyl), benzyl, fluorene-9-yl, polyhalogenated alkyl, CH2CN, polyhalogenated phenyl (e.g., tetrafluorophenyl or pentafluorophenyl, pentachlorophenyl), 2- and 4-nitrophenyl, N-succinimide, sulfonyl-N-succinimide, 1-oxobenzotriazolyl, or other potentially nucleophilic leaving groups. Alkyl chain (or main chain) (CH2) n It can be a straight chain or a branched chain.

[0156] Furthermore, the arylamino group (NR) in formula A 1 and NR 2 R 3 ) can be transmitted through (poly)methylene groups,

[0157] Carbonyl group, nitrogen- or sulfur-containing straight or branched chain head, especially (CH2). m CON(R 7 ), CO(CH2) m N(R 7 ), CO(CH2) m S(CH2) n (CH2) m S(CH2) n CO, CO(CH2) m SO2(CH2) n (CH2) m SO2(CH2) n CO, and combinations thereof, are attached to the reactive group of the analyte, or are attached as part of a nitrogen-free non-aromatic heterocyclic compound (e.g., piperazine, pipecoline, oxazoline); m and n are integers from 0 to 12 or 1 to 12. Substituent R 7 R can be derived from the above. 1 R 2 R 3 R 4 and R 5 Any of the listed functional groups are represented.

[0158] Substituent R in Formula A 1 R 2 and R 3 It can also be represented by a primary amine, and therefore includes carbonyl reactive arylhydrazines, (R 2 =H,R 1 Or R 3 =NH2 or R 1 =NH2,R 2 R 3 =alkyl, perfluoroalkyl, or alkyl), with partial conjugation or substitution by solubilizing and / or anions, listed under R 4 and R 5 Possible candidates, particularly: hydroxyalkyl (CH2) n OH, thioalkyl ((CH2) n SH), carboxylalkyl ((CH2) n CO2H), alkyl sulfonic acid group ((CH2) n SO3H), alkyl sulfate group ((CH2) n OSO3H), alkyl phosphate group ((CH2) n OP(O)(OH)2) or alkylphosphonic acid group ((CH2)n P(O)(OH)2), where n is an integer from 0 to 12 or from 1 to 12. Alternatively, hydrazine derivatives can be represented by sulfonylhydrazine, where R 4 =NH2,R 5 It is an alkyl, perfluoroalkyl, or alkyl modified with solubilizing and / or anionic providing groups of the above types.

[0159] Alternatively, the aromatic amino group (NR) in formula A 1 and / or NR 2 R 3 It can be indirectly linked to an acylhydrazine or alkylhydrazine moiety via a linker, thereby containing an acylhydrazine (ZCONHNH2) or a hydrazine (ZNHNH2), respectively. Here, Z represents the dye residue of formula A, including the aryl amino group and the linker. In particular, R 1 and R 2 It can be represented as (CH2) in the following way. m CON(R 7 ), CO(CH2) m N(R 7 ), CO(CH2) m S(CH2) n (CH2) m S(CH2) n CO, CO(CH2) m SO2(CH2) n (CH2) m SO2(CH2) n CO and its combinations; m and n are integers from 0 to 12. Substituent R 7 It can be generated by R 1 R 2 R 3 R 4 and R 5 Any functional groups indicated that these functional groups are listed above as R 1 -R 5 Candidate functional groups, especially: hydroxyalkyl (CH2) n OH, thioalkyl ((CH2) n SH), carboxylalkyl ((CH2) n CO2H), alkyl sulfonic acid group ((CH2) n SO3H), alkyl sulfate group ((CH2) n OSO3H), alkyl phosphate group ((CH2) n OP(O)(OH)2) or alkylphosphonic acid group ((CH2) n P(O)(OH)2), where n is an integer ranging from 0 to 12 or from 1 to 12. The connector can also be represented by a non-aromatic heterocycle containing O, N, and S (e.g., piperazine, piperoline).

[0160] In addition, R 1 R 2 and R 3 It can be represented by CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2 or CSNHC6H4-NH2, where C6H4 is 1,2-, 1,3- or 1,4-phenylene; or COC5H3N-NH2 or CH2-C5H3N-NH2, where C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl, or pyridine-3,5-diyl.

[0161] At variable position R 1 R 2 R 3 R 4 and R 5 The reactive group of the analyte can be represented by an aromatic or heterocyclic amine, a carboxylic acid, an ester of a carboxylic acid (e.g., an N-hydroxysuccinimide group or another amino reactive ester); or by an alkyl azide (CH2). n N3, alkynes (propynyl), amino-alkoxy groups (CH2) n ONH2, maleimide (C4H3NO2 with a nucleophilic double bond) or haloketone functional group (COCH2X; X = Cl, Br and I), and haloamide group (NRCOCH2X, R = H, C1-C6-alkyl, X = Cl, Br, I), directly or indirectly linked by a linker containing a carbonyl, amide, nitrogen, oxygen or sulfur group listed as a hydrazine derivative, where n = 1-12.

[0162] According to some more preferred embodiments of the present invention, the substituent R in formula A above 1 The definition is as follows:

[0163] R in formula A 1 Represents hydrogen, a lower alkyl group (C1-C4), an unsubstituted phenyl group, or a phenyl group having one or more electron-donating substituents selected from the group consisting of: OH, SH, NH2, NHR. a NR a R b R a O, R a S、OP(O)(OR a (OR) b ), where R a and R b They can be independent of each other and can be C1-C with straight chains or branches. 12Preferably, it is a C1-C6 alkyl group, and has one or more electron acceptors selected from the group consisting of: NO2, CN, COH, COOH, CH=CHCN, CH=C(CN)2, SO2R a SO3R a COR a COOR a CH = CHCOR a CH = CHCOOR a CONHR a SO2NR a R b CONR a R b 、P(O)(OR a (OR) b ), where R a and R b They can be independent of each other and can be H, or C1-C6 alkyl groups having straight or branched carbon chains; or, R 1 It can represent an aromatic heterocyclic group, especially 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, pyrimidin-4-yl, pyrimidin-2-yl, pyrimidin-5-yl, or other electron acceptor groups derived from aromatic heterocycles, such as 4-pyridyl-N-oxide, N-alkylpyridinium salt, or betaine, especially N-(ω-sulfoalkyl)-4-pyridinium, N-(ω-sulfoalkyl)-2-pyridinium, and N-(1-hydroxy-4,4,5,5-tetrafluoro-cyclopent-1-en-3-one-2-yl)-4-pyridinium. (N-(1-hydroxy-4,4,5,5-tetrafluoro-cyclopent-1-en-3-on-2-yl)-4-pyridinium), N-(1-hydroxy-4,4,5,5-tetrafluorocyclopent-1-en-3-one-2-yl)-2-pyridinium (N-(1-hydroxy-4,4,5,5-tetrafluorocyclopent-1-en-3-on-2-yl)-2-pyridinium).

[0164] In particular, R 1 It can represent a positively charged heterocyclic group derived from 2-pyridyl, 3-pyridyl, or 4-pyridyl precursors, having a 7-aminoacridine-2-sulfonamide skeleton and an alkylating agent (e.g., alkyl halide, alkyl sulfonate, alkyl trifluoromethanesulfonyl, 1,3-propanesulfonate lactone, 1,4-butanesulfonate lactone) or an electrophilic agent (e.g., perfluorocyclopentene).

[0165] Particularly preferred are compounds containing aminoacridones having one of the following formulas:

[0166]

[0167] In formula B, L is a divalent linker that connects the dye core to the solubilizing and / or ionizable portion and modulates the spectral characteristics.

[0168] Typically, its presence results in a significant bathofloric shift and bathochromic shift compared to APTS dye tags, while also providing better matching with commercially available 488nm lasers, where fragment L is absent and group X is a hydroxyl group.

[0169] The connector L contains at least one carbon atom or is composed of at least one carbon atom and may represent any combination of alkyl, heteroalkyl (e.g., alkoxy: CH2OCH2, CH2CH2O, CH2CH2OCH2), difluoromethyl (CF2), olefin, or alkyne moieties, in any case, straight-chain or branched, with a length of C1 to C12. The connector may also include a carbonyl group (CH2CO, CF2CO), and when L is an alkylamino or dialkylamino, examples are sulfonamides, particularly diethanolamine or N-methyl(alkyl)monoethanolamine moieties (i.e., N(CH3)CH2CH2O- and N(CH2CH2O-)2), which allows for further attachment to the solubilizing and / or ionizable moieties X. Certain embodiments of the invention represent combinations of moieties L and X according to formula (CH2)3OP(O)(OH)2 and N(CH3)(CH2)2OP(O)(OH)2. Thus, this type of sulfonamide has the general formula SO2NR. 3 R 4 , where R 3 and R 4 They are independent of each other and can represent any combination of hydrogen, alkyl, heteroalkyl (e.g., alkoxy: CH2OCH2, CH2CH2O, CH2CH2OCH2), difluoromethyl (CF2), straight or branched, with a length of C1 to C12 and a terminal OH group.

[0170] N(R) in formula B 1 )R 2 Preferably, it contains a carbonyl group or a nucleophilic reactive group. Substituent R 1 and R 2 They are independent of each other and can all be represented by hydrogen. One of them can be a straight-chain or branched alkyl (perfluoroalkyl) C1-C 12 Meanwhile, R 1 and R 2 One of them can be composed of a carboxylic acid residue (CH2). n COOH and their conventional or reactive esters (CH2) n COR5 This represents a group of residues, where n is an integer from 1 to 12. 5 It can be H, alkyl, (including butyl), benzyl, fluorene-9-yl, polyhalogenated alkyl, CH2CN, polyhalogenated phenyl (e.g., tetrafluoro or pentafluorophenyl, pentachlorophenyl), 2- and 4-nitrophenyl, N-sulfonylimide, sulfon-N-sulfonylimide, or other potentially nucleophilic leaving groups. Alkyl chain (or main chain) (CH2) n It can be linear or branched. In particular, the formula can be described as Z-NR. 1 (CH2) n COR 5 Z is the remaining molecule in formula B, which also includes groups L and X.

[0171] In addition, the nucleophilic reactive group COR 5 It can be attached to the arylamino group N(R) via the following method. 1 )R 2 : (poly)methylene, oxymethylene (CH2OCH2, CH2CH2OCH2, PEG) carbonyl, carbonate, urethane, nitrogen- or sulfur-containing branched or straight-chain head (spacer group), especially (CH2) m CON(R 6 ), CONH(CH2) n (CH2) m OCONH(CH2) n CO(CH2) n CO(O)NR 6 (CH2) m SO 2m N(R 6 ), CO(CH2) m S(CH2) n (CH2) m S(CH2) n CO, CO(CH2) m SO2(CH2) n (CH2) m SO2NR 6 and their combinations; m and n are integers from 0 to 12. Reactive group R 5 It can be linked via non-aromatic heterocycles containing O, N, and S (e.g., piperazine, piperidine, oxazoline). Substituent R 6 It can be composed of H, alkyl, hydroxyalkyl, or perfluoroalkyl C1-C 12 express.

[0172] Substituent R in Formula B 1 and R 2 One of them can be represented by a primary amine, thus including carbonyl reactive arylhydrazine (R1 =NH2,R 2 =alkyl, perfluoroalkyl) or represented by hydroxyl to form aryl oxime (ArNHOH). Alternatively, the alkyl hydrazine or oxime reactive moiety in formula B can be represented by the reactive group R listed above. 4 The connector is attached to the arylamino group N(R) 1 )R 2 When R has n = 1 - 12 1 Or R 2 =(CH2) n SO2NR 6 In the case of NH2, sulfonyl hydrazide constitutes a special case, while the substituent R 6 It can be composed of H, alkyl, hydroxyalkyl, or perfluoroalkyl C1-C 12 This indicates that the sulfonylamide (sulfonamide, sulfamide) group can also be connected to the reactive group R through the various connectors listed above. 3 R 4 and R 5 connect.

[0173] In addition, R 1 and R 2 It can be represented by CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2 or CSNHC6H4-NH2, where C6H4 is 1,2-, 1,3- or 1,4-phenylene; or COC5H3N-NH2 or CH2-C5H3N-NH2, where C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl, or pyridine-3,5-diyl.

[0174] Substituent R 1 and R 2 It can also be made from alkyl azide (CH2). n N3, alkynyl (propynyl), maleimide (C4H3NO2 with a nucleophilic double bond) or haloketone functional group (COCH2X; X = Cl, Br and I), directly linked or linked through a linker containing a carbonyl, amide, nitrogen or sulfur group listed as a hydrazine derivative; n = 1-12.

[0175] In Formula B, the X group represents a solubilizing and / or ionizable anion-providing substance, particularly one that provides enhanced electrophoretic mobility. Group X may include a hydroxyalkyl group (CH2). n OH, thioalkyl ((CH2) n SH), carboxylalkyl ((CH2) n CO2H), alkyl sulfonic acid group ((CH2) nSO3H), alkyl sulfate group ((CH2) n OSO3H), alkyl phosphate group ((CH2) n OP(O)(OH)2) or phosphonic acid group ((CH2) n P(O)(OH)2), where n is an integer from 0 to 12. Alternatively, the CH2 group can be replaced by CF2. The anion-donating part can also be connected by a non-aromatic heterocycle containing O, N, and S (e.g., piperazines, piperoliners). Alternatively, one of the groups X can be accompanied by a group R. 1 and R 2 Any carbonyl or nucleophilic active moiety listed may also carry any type of bond listed for group L, independently of other substituents. Compounds of formula B may exist and be used in the form of salts, which include all possible types of cations, preferably Na. + K + Li + Or trialkylammonium.

[0176] Fluorescent dyes of formula B can exist as salts, solvates, or hydrates, especially those with cations including Na. + K + Li + NH4 + Salts of organic ammonium or organic phosphonium cations.

[0177] According to one specific embodiment of the invention, the anionic group X, each time it appears in Formula B, can represent one to four groups SO3H attached to the linker group L, as in the term (SO3H) in Formula B as claimed in claim 3. n As shown in (n=1-4).

[0178] According to a specific embodiment of the present invention, the compound of structural formula B is an alkylsulfonyl derivative of formula C.

[0179]

[0180] in

[0181] R 1 and / or R 2 They are independent of each other and can represent:

[0182] H, CH3, C2H5, straight-chain or branched C3-C 12 Preferably C3-C6 alkyl, or substituted C2-C 12 Preferably C2-C6 alkyl groups; particularly, (CH2) n COOR 3 Where n = 1-12, preferably 1-5, R 3It can be H, CH2CN, 2- and 4-nitrophenyl, 2, 3, 5, 6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazolyl, and (CH2). n The alkyl chain in the form can be straight or branched; and

[0183] R 1 ---R 2 It can form quaternary, pentaneous, hexanal, or heptaneous non-aromatic carbon rings, with an additional primary amino group (NH2) or secondary amino group (NHR) attached to one carbon atom of the ring. a , where R a = C1-C6 alkyl, or hydroxyl OH; optionally, R 1 ---R 2 It can form four-, five-, six-, or seven-membered non-aromatic heterocycles, with additional heteroatoms such as O, N, or S included in the heterocycle; hydroxyalkyl (CH2) m OH, wherein m = 1-12, preferably 2-6, has a straight-chain or branched alkyl chain; R 1 Or R 2 One of the groups can be a carbonate or carbamate derivative, wherein R 1 Or R 2 One of the groups is (CH2). m OCOOR 4 or COOR 4 Where m = 1 - 12 and R 4 = Methyl, ethyl, 2-chloroethyl, N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazole, or substituted phenyl, such as 2- and 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluorophenyl, 2-pyridyl, or 4-pyridyl; (CH2) m NR a R b Where m = 1-12, preferably 2-6, and has a straight-chain or branched alkyl chain; R a R b They are independent of each other and can be H, or optionally substituted C1-C4 alkyl groups, particularly R. 1 Or R 2 One of the groups can be an alkyl azide group (CH2). m N3, where m = 2-6 and has a straight or branched alkyl chain;

[0184] R 1 Or R 2 One of the groups can be (CH2). n COOR 5Where n = 1-5, and alkyl chains (CH2) have straight or branched chains. n And R 5 Selected from H, straight-chain or branched C1-C6 alkyl, CH2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, sulfonyl-N-succinimide, N-succinimide, 1-oxybenzotriazolyl;

[0185] Furthermore, R 1 Or R 2 One of them could be (CH2). n CONHR 6 Where n = 1-12, preferably 1-5, and R 6 =H, C1-C6 alkyl, (CH2) m N3, (CH2) m -N-maleimide group, (CH2) m -NHCOCH2X (X = Br or I), where m = 2-6 and in (CH2) n and R 6 It contains straight-chain or branched alkyl chains; or R 1 Or R 2 One of them can represent CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2, or CSNHC6H4-NH2, where C6H4 is 1,2-, 1,3-, or 1,4-phenylene; or COC5H3N-NH2 or CH2-C5H3N-NH2, where C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl, or pyridine-3,5-diyl; in formula B, (CH2) is between residue X of SO2 fragment and (CH2) fragments, n = 1-5. n -CH2 connectors can represent straight-chain, branched, or cyclic groups with 2-6 carbon atoms;

[0186] X=SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a ,in

[0187] R a =Optionally substituted C1-C4 alkyl groups, P(O)(OH)2, P(O)(OH)R a Among them, R a =Optionally substituted C1-C4 alkyl groups;

[0188] Provided that in all compounds represented by formula C, under basic conditions, i.e., 7 < pH < 14, there are three or six negatively charged groups in residue X of formula B, and these negatively charged groups represent residues of at least partially deprotonated ionizable groups selected from: SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a , where R a = C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)R a , where R a = C1-C4 alkyl or substituted C1-C4.

[0189] According to a more specific embodiment of the present invention, the fluorescent dye of the present invention is represented by formula C or its salt, where X is SO3H each time it appears, and n is 1-12, preferably 1-6.

[0190] According to another specific embodiment of the present invention, the compound of formula B above is a sulfonamide derivative of formula D

[0191]

[0192] where

[0193] R 1 and / or R 2 are independent of each other and can represent H, CH3, C2H5, or straight-chain or branched, optionally substituted C3-C 12 , preferably C3-C6 alkyl; in particular, (CH2) n COOR 4 , where n = 1-12, preferably 1-5, and R 4 can be H, CH2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxobenzotriazolyl, and the alkyl chain in (CH2) n can be straight-chain or branched; and

[0194] R 1 ---R 2 can form a four-, five-, six- or seven-membered non-aromatic carbocyclic ring having an additional primary amino group NH2 attached to one carbon atom in the ring, a secondary amino group NHR a , where R a = optionally substituted C1-C6 alkyl, or hydroxyl OH; or optionally, R 1 ---R 2 can form a four-, five-, six- or seven-membered non-aromatic heterocyclic ring, with heteroatoms such as O, N or S included in the heterocyclic ring;

[0195] R 1 and / or R 2 It can also represent:

[0196] Hydroxyalkyl (CH2) m OH, wherein m = 1-12, preferably 2-6, has a straight or branched, optionally substituted alkyl chain; R 1 Or R 2 One of the groups can be a carbonate or carbamate derivative (CH2). m OCOOR 5 or COOR 5 Where m = 1 - 12 and R 5 = Methyl, ethyl, 2-chloroethyl, CH2CN, N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazolyl, phenyl or substituted phenyl, such as 2- and 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2, 3, 5, 6-tetrafluorophenyl, 2-pyridyl, 4-pyridyl;

[0197] (CH2) m NR a R b Where m = 1-12, preferably 2-6, and has a straight-chain or branched alkyl chain; R a R b They are independent of each other and represent hydrogen and / or optionally substituted C1-C4 alkyl groups;

[0198] (CH2) m N3, m = 1-12, preferably 2-6, has a straight-chain or branched alkyl chain;

[0199] (CH2) n CONHR 6 Where n = 1-12, preferably 1-5, R 6 =H, substituted or unsubstituted C1-C6 alkyl group, (CH2) m N3, (CH2) m -N-maleimide group, (CH2) m -NHCOCH2Y (Y = Br, I), where m = 1-12, preferably 2-6, in (CH2) n and R 6 It contains straight-chain or branched alkyl chains;

[0200] R 1 Or R 2 One of the groups can be a primary amino group to form arylhydrazine Ar-NR. 7 NH2, where in formula D, Ar is the entire pyrene residue, and R 7 =H or alkyl;

[0201] R 1 Or R 2 One of the groups can be a hydroxyl group to form aryl hydroxylamine Ar-NR 8 OH, where in formula D, Ar is the entire pyrene residue, and R 8 =H or alkyl;

[0202] R 1 Or R 2 One of the groups may contain a terminal alkoxyamino group (CH2) with n = 1-12. n ONH2, which can be expressed by one or more alkylamino groups (CH2) with m = 0-12. m NH, alkylamide group (CH2) m All possible combinations of CONH, alkyl ether or alkyl ester groups are linked;

[0203] Furthermore, R 1 Or R 2 It can represent CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2 or CSNHC6H4-NH2, where C6H4 is 1,2-, 1,3- or 1,4-phenylene; COC5H3N-NH2 or CH2-C5H3N-NH2, where C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl or pyridine-3,5-diyl;

[0204] R 3 =H, (CH2) q CH2X, C2H5, straight-chain or branched C3-C6 alkyl group, C m H 2m OR, where m = 2-6, has a straight-chain or branched alkane-diyl chain C m H 2m And R = H, CH3, C2H5, C3H7, CH3(CH2CH2O) k CH2CH2; where k = 1-12; and (CH2) q CH2 connectors can represent straight-chain, branched, or cyclic groups with 2-6 carbon atoms;

[0205] In formula D, n = 1-12, preferably 1-5 (CH2) between the sulfonamide fragment SO2N and residue X. n -CH2 connectors can represent straight-chain, branched, or cyclic groups with 2-6 carbon atoms;

[0206] X=SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a, where

[0207] R a = substituted or unsubstituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)R a , where R a = substituted or unsubstituted C1-C4 alkyl;

[0208] However, it is a condition that in all compounds represented by formula D, under alkaline conditions, i.e., 7 < pH < 14, there are three, six, nine or twelve negatively charged groups in the residue X of formula C, and these negatively charged groups represent residues of at least partially deprotonated ionizable groups selected from: SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a , where R a = C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)R a , where R a = C1-C4 or substituted C1-C4 alkyl.

[0209] According to a preferred embodiment of the present invention, the substituents R 1 and R 2 in the above formulas B, C and D are defined as follows:

[0210] R 1 and / or R 2 in formula B represents H, CH3, (CH2) n COOR 3 , where n = 1-4, and R 3 can be H, CH2CN, 2- or 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxobenzotriazole, and the alkyl chain in (CH2) n is straight; n = 1-12.

[0211] The compounds of formulas C and D can exist and be applied in the form of salts, and the salts include all possible types of cations, preferably Na + , K + or trialkylammonium cations.

[0212] Particularly preferred amino-pyrene compounds of the above general formulas B, C and D have one of the following general formulas:

[0213]

[0214] A preferred embodiment of the invention relates to compounds of the above formula AB or AD, wherein the negative charge is provided by several primary phosphate groups, particularly bis-O-phosphorylated 7-aminoacridin-2-sulfonamide (two phosphate groups), tri-O-phosphorylated 1,6,8-tris[(ω-hydroxyalkyl)sulfonyl]pyrene-3-amine (three phosphate groups), and 1,6,8-tris[ω-(hydroxyalkyl)sulfonamide]pyrene-3-amine. These compounds exhibit higher brightness and better electrophoretic mobility compared to APTS and have been successfully applied to labeling polysaccharides and analyzing conjugates by capillary gel electrophoresis (CGE) (detected by laser-induced fluorescence (LIF)).

[0215] Another preferred embodiment of the invention relates to a compound of formula B, C or D, wherein R 1 and / or R 2 Representatives: H, deuterium, alkyl or deuterated alkyl, particularly alkyl or deuterated alkyl having 1-12 C atoms, preferably 1-6 C atoms, wherein one, several or all H atoms of the alkyl group may be substituted with deuterium atoms, 4,6-dihalo-1,3,5-triazine (C3N3X2), wherein halogen X is preferably chlorine, 2-, 3- or 4-aminobenzoyl (COC6H4NH2), N-[(2-, N-[(3- or N-[(4-aminophenyl)ureido group (NHCONHC6H4NH2), N-[(2-, N-[(3- or N-[(4-aminophenyl)thioureido group (NHCSNHC6H4NH2) or linked carboxylic acid residues and their general formula reactive esters, with the general formula (CH2). m1 COOR 3 (CH2) m1 OCOOR 3 (CH2) n1 COOR 3 or (CO) m1 (CH2) m2 (CO) n1 (NH) n2 (CO) n3 (CH2) n4 COOR 3 , where the integers m1, m2 and n1, n2, n3, n4 are independently 1 to 12 and 0 to 12 respectively, where the chain (CH2) m / n It is straight-chain, branched, saturated, unsaturated, partially or fully deuterated, and / or included in carbon rings or heterocycles containing N, O or S, while R 3 It is an H, D or nucleophilic leaving group, preferably including but not limited to N-succinimide, sulfon-N-succinimide, 1-oxybenzotriazole, cyanomethyl, polyhalogenated alkyl, polyhalogenated phenyl, such as tetra- or pentafluorophenyl, 2- or 4-nitrophenyl.

[0216] The novel compounds of this invention have small molecular sizes and, in a preferred embodiment, provide a dramatically increased high negative net charge (z) (e.g., at least z = -4 for phosphorylated acridinones and at least z = -6 for phosphorylated pyrene dyes). These two requirements correspond to low hydrodynamic radii and low mass-to-charge ratios (m / z), respectively. Therefore, rapid separation with high speed and high analytical resolution can be achieved when performing electrokinetic measurements on these compounds and their corresponding labeled carbohydrates.

[0217] The negative charge is provided by the acidic group, which can be deprotonated in alkaline or even neutral media. Therefore, a phosphate group is preferred because the pK values ​​of the first and second acidic protons of the primary alkyl phosphate group (R-OPO3H2) are relatively high. a The values ​​are in the ranges of 1-2 and 6-7, respectively. Therefore, under alkaline conditions, a single phosphate group can introduce two negative charges into a buffer solution (e.g., above pH 8, the presence of R-OPO3). 2- To obtain a negative charge of -4, the connection of two phosphate groups is necessary, and so on. However, other acidic groups, especially acidic groups selected from group X as defined in the above general formula AB, are also suitable.

[0218] Generally, compounds of formula AB are suitable and advantageous for use as fluorescent labels for amino acids, peptides, proteins (including primary and secondary antibodies, single-domain antibodies, docetaxel, avidin, streptavidin and its modifications), aptamers, nucleotides, nucleic acids, toxins, lipids, carbohydrates (including 2-deoxy-2-glucosamine and other 2-deoxy-2-amino-aminopyranosides, polysaccharides, dextran), biotin and other small molecules (e.g., actin polymerization inducers (jasplakinolide) and their modifications).

[0219] Compounds 7-R (R = H, Me), 13a, 13b, 16, and 18 (see Scheme 7 below) have free hydroxyl groups, making them suitable as precursors for obtaining phosphorylated pyrene dyes of general formula B. Specifically, compound 7-R (R = H, Me) is phosphorylated to yield dye 8-R (R = H, Me). Compounds 13a, b, and 18 are similarly phosphorylated. Thus, for example, precursor dyes 13a and 13b both produce (after basic treatment of the reaction mixture) compound 15. Compound 16 has a free carboxyl group, which can be used as the reaction center for bioconjugates. Therefore, compound 16 represents the fluorescent labeling of amino acids, peptides, proteins (including primary and secondary antibodies, single-domain antibodies, docetaxel, avidin, streptavidin and their modifications), aptamers, modified nucleotides, amino-containing modified nucleic acids, toxins, lipids, carbohydrates (including 2-deoxy-2-glucosamine and other 2-deoxy-2-aminoaminopyranosides, modified biotin (e.g., biocytin) and other small molecules).

[0220]

[0221] Option 7

[0222] Exemplary aminopyrene compounds and their precursors of the present invention

[0223] Therefore, a closely related aspect of the present invention relates to the use of compounds of general structural formula AD as fluorescent reagents for conjugation with a wide range of analytes, wherein said conjugation includes the formation of at least one covalent chemical bond or at least one molecular complex with a chemical entity or substance, such as amines, carboxylic acids, aldehydes, alcohols, aromatic compounds, heterocycles, dyes, amino acids, amino acid residues, and any chemical entity, peptide, protein, carbohydrate, nucleic acid, toxin, and lipid.

[0224] The claimed compounds are applicable to and can be used in methods for fluorescent labeling and detection of target molecules. Typically, this method means reacting a compound according to any one of the above formulas A and D with a target molecule selected from the group consisting of: amino acids, peptides, proteins (including primary and secondary antibodies), single-domain antibodies, docetaxel, avidin, streptoavidin and its modifications, aptamers, (modified) nucleotides, (modified) nucleic acids, toxins, lipids, carbohydrates (including 2-deoxy-2-glucosamine and other 2-deoxy-2-aminoaminopyranosides, polysaccharides, dextran), (modified) biotin (e.g., biocytin), and other small molecules (e.g., actin polymerization inducers (jasplakinolide) and their modifications). After labeling, the labeled fluorescent derivatives are separated, detected, quantified, and / or isolated by chromatographic and / or electrokinetic techniques.

[0225] The inventors have discovered that chromatographic separation techniques (such as reversed-phase or hydrophilic interaction (U) HPLC), at all possible scales (from nanoscale to analytical scale and larger) and electrokinetic separation techniques (electrophoresis, gel electrophoresis, capillary electrophoresis, capillary gel electrophoresis, or capillary electrochromatography) – all with fluorescence or laser-induced fluorescence detection – are well-suited for the described improved methods for the automated, high-performance analysis, identification, and / or determination of carbohydrates and carbohydrate mixtures. In particular, the use of multiplex capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) allows for rapid yet robust and reliable analysis and identification of compositional patterns of carbohydrates and / or carbohydrate mixtures (e.g., glycosylation patterns of glycoproteins). The method according to the invention for glycoprotein analysis allows visualization of the composition of carbohydrate mixtures (e.g., glycan pools of glycoproteins), including structural analysis of carbohydrates, while eliminating the need for very expensive and complex equipment such as mass spectrometers or NMR (nuclear magnetic resonance) instruments. Capillary electrophoresis, particularly capillary gel electrophoresis, has been considered for the separation of complex carbohydrates due to its superior separation performance and efficiency compared to other separation techniques. However, this technique is not recommended in the art because it is said to have disadvantages, see, for example, Domann et al. or WO2006 / 114663. Nevertheless, when the method according to the invention is applied, xCGE-LIF technology allows for the high-performance, sensitive, and reliable determination and identification of carbohydrate structures. In particular, the use of capillary DNA sequencers (e.g., 4-capillary sequencers: 3100-Avant Genetic Analyzer, 3130 Genetic Analyzer, SeqStudio, and Spectrum Compact; 16-capillary sequencers: 3100 Genetic Analyzer and 3130xl Genetic Analyzer; 48-capillary sequencers: 3730 DNA Analyzer; 96-capillary sequencers: 3730×l DNA Analyzer from Applied Biosystems; 8-capillary sequencers: 3500 Genetic Analyzer; 24-capillary sequencers: 3500×l Genetic Analyzer and Promega Spectrum) allows for high performance of the methods according to the invention. The advanced / improved methods of the invention enable easier and more accurate characterization of variations in complex compositions of natural or synthetic carbohydrate mixtures, and, when comparing samples to known carbohydrate mixture compositions, directly characterize carbohydrate mixture compositional patterns (e.g., protein glycosylation patterns) through carbohydrate "fingerprint" alignment.

[0226] The method according to the invention is a further simplified and more robust yet still highly sensitive and reproducible method for sugar analysis, with high separation performance.

[0227] In particular, the combination of the aforementioned instrument with up to 96 parallel capillaries with the software / database tools included in this invention enables truly high-throughput automated analysis.

[0228] A further specific embodiment of this aspect relates to a method for fluorescently labeling carbohydrates with a dye of formula AD, the method comprising at least the following steps:

[0229] a) Prepare a 1-400 mM dye solution in a 0.5-4 M organic acidic aqueous solution, particularly a dye of formula 6-H, 6-Me, 8-H, 15, 23 or 23b as shown in claim 5;

[0230] b) Prepare a 0.05-3 M borane solution in DMSO, water, methanol, ethanol, diethylene glycol dimethyl ether, tetrahydrofuran, or a mixture of these solvents;

[0231] c) Mix the solutions prepared in steps a) and b) above with the carbohydrate-containing analyte solution in a reaction vessel;

[0232] d) Incubate the reaction mixture at 10-90℃ for 0.1-48 h;

[0233] e) Add water and a mixture of water-miscible organic solvents to the reaction mixture in a ratio of 1:10 to 10:1, and stir the contents of the reaction vessel to stop the reaction in step d) and dissolve the reaction products.

[0234] f) Optionally, cause the mixture produced in step e) to generate vortices; and

[0235] g) Optionally, the mixture produced in step f) can be subjected to electrophoresis.

[0236] More specifically, the organic solvent is selected from the group consisting of acetonitrile, ethanol, methanol, isopropanol, tetrahydrofuran, acetic acid, dioxane, sulfolane, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, nitromethane, hexamethylphosphortriamide, diethylene glycol dimethyl ether, and methyl cellosolve, with acetonitrile being the preferred organic solvent.

[0237] Furthermore, the present invention also includes carbohydrate-dye conjugates comprising fluorescent dyes according to formula AB or AD above.

[0238] More specifically, the dye in the conjugate, particularly the carbohydrate conjugate, is selected from compounds of formula 6-H, 6-Me, 8-H, 15, 23, 23b, as shown in Scheme 8 below.

[0239] Because of their reactive groups (aromatic amino (NH2), hydrazine (NRNH2), acylhydrazine (CONRNH2), hydroxylamine (NROH), reactive carbamate (NHCOOR) or alkoxyamino (RONH2), the compounds of formulas A to D above are suitable and advantageous for reductive amination or direct condensation reactions with suitable carbohydrates having aldehyde groups or amino groups in free or protected forms (e.g., as hemiacetals) (as shown in schemes 2-6 and 8).

[0240] Therefore, a closely related aspect of the present invention relates to such uses and methods for reductive amination or direct condensation, comprising reacting a compound of formula AD above with a suitable carbohydrate having an aldehyde or amino group in free form or as a hemiacetal, allowing sufficient time for selective reductive amination and chromatographic or electrokinetic separation of the labeled fluorescent derivative, followed by detection of the analyte by optical spectroscopic methods, including fluorescence detection and / or mass spectrometry. Example 8 provides an example of a dye-conjugate structure.

[0241] Compounds of formula AD and carbohydrate-dye conjugates containing such compounds are particularly suitable and advantageous for the spectral calibration of fluorescence detectors, especially for detectors that detect laser-induced fluorescence (LIF), as they are commonly used in C(G)E systems.

[0242]

[0243] Option 8

[0244] Spectral properties of new dyes

[0245] The spectral properties of the dyes are shown in Table 1 below.

[0246] Table 1. Spectroscopic properties of phosphorylated aminoacridones 6-H and 6-Me, sulfonamide pyrene 8-R (R = H, Me), alkylsulfonyl modified pyrene dyes 15, 16, 18, 23 and their precursors and related compounds: 19, 20 and dye APTS (structures shown in schemes 7-13).

[0247]

[0248] a - Absolute value of fluorescence quantum yield (unless otherwise specified); b - TEAB is an aqueous solution of Et3N*H2CO3 buffer, pH = 8-8.5; c - 375nm excitation; d - Relative value, with Rhodamine 6G as the reference dye, Φ fl =0.9; e- For mono-N-alkylated APTS derivatives, the maximum absorption (abs.) and excitation (emiss.) values ​​are 457 and 516 nm, respectively (ε ~ ​​19000 M). -1 cm -1f - In PBS buffer solution, excited at 515 nm; g - Obtained using fluorescein as a reference dye, in 0.1 M NaOH, excited at 496 nm, Φ fl =0.9; h- When switching from PBS (pH 7.4) to TEAB buffer (pH 8-8.5), no significant changes were observed in aminopyrene dyes, including APTS.

[0249]

[0250] Scheme 13. Phosphorylated fluorescent dyes 19 and 20, sulfonated dyes 23 and 23b, and APTS were used as reference compounds, which have different negative charges and different spectral properties.

[0251] The structural characteristics and data in Table 1 indicate that the diphosphorylated aminoacridones 6-H and 6-Me, and the triphosphorylated pyrene dyes 8-H, 8-Me, and 15 meet the criteria for fluorescent labeling as defined above. Furthermore, it needs to be demonstrated whether they can be used for the reductive amination of polysaccharides, and whether the emission of their conjugates interferes with the emission of polysaccharides labeled with APTS (structural and spectral data are shown in Schemes 7-12 and Table 1). For example, the m / z ratios of compounds 6-R (R = H, Me) are 134 and 138, respectively (m / z = 151 for APTS). They have several absorption maxima and emit orange light (two emission maxima are at 485 nm and 585 nm, with a relative intensity of approximately 1:2; see...). Figure 24A Although their absorption at 488 nm is relatively low, red emission is a significant feature, with a corresponding Stokes shift of approximately 160 nm. The absolute value of the fluorescence quantum yield of compound 6-R is 5-6%. Therefore, despite the relatively low brightness, even the red-emitting dye 6-R (and the brighter pyrene dyes 8-R and 15) represents a new tag that can be used to label glycans, including “heavy” and “exotic” glycans (which are currently undetectable due to the relatively low net charge (-3) of APTS and the low mobility of “heavy” carbohydrates modified with APTS tags). In fact, the phosphorylated dyes presented here provide better electrophoretic mobility for conjugates and reduce their migration time due to the presence of four negative charges and extremely low m / z ratios, thus revealing and highlighting large-volume and high-mass carbohydrates.

[0252] All pyrene dyes listed in Table 1 exhibit high fluorescence. Non-phosphorylated pyrene 7-R (R = H, Me), 13b, 16, and 18 allow for more accurate estimation of extinction coefficients. The longest wavelength extinction coefficients range from 18,000 to 23,000, with the maximum wavelength located between 465 and 507 nm. Therefore, argon-ion lasers readily produce fluorescence at 488 nm. Maximum emission values ​​were found in the 535–563 nm range, with consistently high fluorescence quantum yields (71–97%). Therefore, sulfonated 1-aminopyrene represents a brighter dye than 2-sulfonamide-7-aminoacridone. Brightness is proportional to the product of the extinction coefficient (at 488 nm) and the fluorescence quantum yield. We can assume this value is approximately 1500 × 0.06 = 90 for acridinone dyes and 20000 × 0.9 = 18000 for pyrene. This rough estimate means that trisulfonated 1-aminopyrene is about 200 times brighter than the dye of 2-sulfonamide-7-aminoacridone. This property makes the pyrene dye of the present invention superior to 2-sulfonamide-7-aminoacridone and APTS. If we assume that APTS has an extinction coefficient of 19000 at its maximum (457 nm) (Scheme 6), and that its absorption at 488 nm is typically about 35% of the maximum absorption at 457 nm, then we get a relative brightness of 6000 (assuming the same fluorescence yield). Therefore, the dye of the present invention is about 3 times brighter than APTS (when conjugated with glycans). The pyrene dyes of the present invention, especially compounds 8-H, 15, 23, and 23b, represent a new label that can be used to label glycans, including “heavy” and “exotic” glycans (which are currently undetectable due to the relatively low net charge (-3) and lower brightness of APTS).

[0253] To shift the emission band to the red spectral region, an N-methylated derivative, 8-Me, was prepared. This dye has an N-methylamino group, and therefore, the fluorophore it represents is very similar to the reductive amination product formed from the glycan and the parent dye 8-H (compared to compound 6 in scheme 9). The absorption maximum shifted to red (+37 nm; 8-H → 8-Me), but the emission maximum experienced a fluorine shift of “only” 19 nm (see Table 1). Therefore, the Stokes shift decreased from 79 nm to 61 nm.

[0254] Another tool is used to enhance the bathofloric and bathochromic red shifts of aromatic fluorescent dyes, provided they have electron donor and electron acceptor groups with a so-called "push-pull" electron interaction (direct polar conjugate). In the example of 1-aminopyrene dyes, the electron-donating group is fixed (it cannot enhance its electron-donating properties), but the electron-withdrawing groups at positions 3, 6, and 8 can be different. In particular, alkyl sulfone groups (R-SO2, present in compounds 13b, 15, 16, 18, 23, and 23b) have been shown to be stronger acceptors than sulfonamide groups (present in compounds 7-H, 7-Me, 8-H, 8-Me; see scheme 7). However, after preparing compounds 8-H and 15 and comparing their spectral properties in aqueous solution (Table 1), their red shifts were determined to be consistent with expectations at 12 nm, but the position and band shape of the emission maximum remained unchanged. The simplest explanation is based on the assumption that the monoamino group (as a donor) has "reached its limit" and cannot provide more electron density for a π-system modified by three very strong acceptor groups (however strong they may be). Fortunately, further (bathofloric shift and bathochromic shift) occurred during the reductive alkylation of the nitrogen atom (see Scheme 2) (compared to the spectral data of compounds 8-H and 8-Me discussed above), and the conjugate of compound 15 with the glycan exhibited bright light with no crosstalk to the APTS detection channel.

[0255] This invention is based on the separation and detection of carbohydrate mixtures (e.g., glycan pools), utilizing xCGE-LIF technology (e.g., using a capillary DNA sequencer) to generate a carbohydrate composition pattern fingerprint. By matching the internally normalized CGE-migration time of each individual compound in the sample mixture with a database, automated structural analysis of the separated carbohydrates is achieved. The claimed method allows for compositional analysis of carbohydrate mixtures from synthetic or naturally derived sources, such as glycosylation pattern analysis of glycoproteins. Advanced internal normalization of carbohydrate migration time and migration time index is based on using a series of internal carbohydrate standards similar to the sample but labeled with one or more novel fluorescent dyes, whose emission wavelengths differ from those of one or more tags on the sample. Internal carbohydrate standards of known composition (e.g., a set of mono, di, tri, tetra, and / or pentamers, linear and / or branched, up to 100-mers (or more)) elute / migrate across the entire fingerprint range of the carbohydrate sample to be analyzed, but are detected in another trace / channel because they are fluorescently labeled with different tags than the carbohydrate sample, thus emitting at a different wavelength and not appearing in the sample's trace. This advanced internal carbohydrate standard... These advanced internal carbohydrate standards, which elute / migrate across the entire migration / retention time range of the carbohydrate sample fingerprint but are detected in a different wavelength trace, can be used for highly accurate and reproducible "advanced" internal normalized migration / retention times. They are used to generate calibration curves that are highly accurate in terms of curvature / shape, y-intercept, and slope.

[0256] This improved migration time index determination allows for highly accurate and absolutely reproducible analysis of carbohydrates, independent of sample type and source, analysis time point, laboratory, instrument, and operator.

[0257] The combined use of the method and system also allows for the quantitative analysis of the composition of the carbohydrate mixture. Therefore, the method and system according to the invention represent a powerful tool for monitoring changes in the composition of carbohydrate mixtures, such as protein glycosylation patterns, without requiring complex structural investigations. For fluorescently labeled carbohydrates, LIF detection allows for detection limits as low as 10⁻¹⁸ moles.

[0258] The alignment standards required for each run may be present in a single sample or may be included in the carbohydrate sample to be analyzed.

[0259] One of the fluorescent markers used to label carbohydrates can be, for example, the fluorescent marker 8-amino-1,3,6-pyrene trisulfonic acid, also known as 9-aminopyrene-1,4,6-trisulfonic acid (APTS) or other preferred multi-charged fluorescent dyes, while the other fluorescent marker is one of the dyes of general formula A or B.

[0260] Qualitative and quantitative analysis can be performed in the presence of standards. Relative quantification can be easily accomplished by analyzing the individual peak heights of each compound, which are linearly related to concentration (within the linear dynamic range of the LIF detector).

[0261] This invention addresses the shortcomings of other known methods such as chromatography, mass spectrometry, and NMR in carbohydrate analysis. NMR and mass spectrometry represent time-consuming and labor-intensive techniques. Furthermore, these methods require expensive equipment. Moreover, most of these methods, such as NMR, cannot be scaled up to high-throughput methods. Mass spectrometry can achieve high sensitivity. However, configuration can be difficult, and non-specific structural information can only be obtained by locating the bonds of monomeric sugar compounds. HPLC is also quite sensitive depending on the detector and allows for quantification. However, as mentioned above, truly high-throughput analysis can only be achieved by extensive use of expensive high-performance liquid chromatography systems and solvents.

[0262] Other known techniques in the art are based on enzyme treatment, which is highly sensitive and can yield detailed structural information, but requires combination with other methods such as HPLC (high performance liquid chromatography), MS (mass spectrometry), and NMR (nuclear magnetic resonance). Further techniques known in the art involve lectins or monoclonal antibody affinity, which provide only preliminary data and not definitive structural information.

[0263] The method according to the invention allows for high-throughput identification of carbohydrate mixtures with unknown compositions, or high-throughput identification or analysis of compositional patterns of carbohydrate mixtures (e.g., glycosylation patterns of glycoproteins). In particular, the invention allows for the quantitative determination of the components of carbohydrate mixtures.

[0264] The method of the present invention can even rapidly and reliably measure the composition of complex mixtures, and thus can determine and / or identify the compositional patterns of carbohydrates and / or carbohydrate mixtures (e.g., glycosylation patterns), which are independent of the instrument used and are only related to the alignment migration time (migration time index).

[0265] This invention allows for applications in a variety of fields. For example, the method can be used to analyze the glycosylation of mammalian cell culture-derived molecules (such as recombinant proteins, antibodies, or viral or viral components, such as influenza A virus glycoproteins). Information regarding the glycosylation patterns of these compounds is particularly important for food and pharmaceutical applications. Starting with the separation of complex protein mixtures using 1D / 2D gel electrophoresis, the method of this invention can also be used for the glycan analysis of any other glycoconjugates. Furthermore, pre-purified glycoproteins, for example, captured by chromatography or affinity, can also be processed according to the method of this invention, replacing the gel separation and gel deglycosylation steps with in-solution deglycosylation, continuing after protein and enzyme precipitation. Finally, complex mixtures of synthetic or naturally derived soluble oligosaccharides and / or polysaccharides, which are important nutritional additives / alternatives today, or used in or as pharmaceuticals, can be analyzed.

[0266] Therefore, two types of analysis can be performed on carbohydrate mixtures. On the one hand, carbohydrate mixture composition pattern analysis, such as glycosylation pattern analysis, can be performed; on the other hand, carbohydrate identification based on matching carbohydrate migration time indices with database data is possible.

[0267] Therefore, the method according to the invention presents a wide range of potential applications, from production and / or quality control to early diagnosis of diseases that are developing, causing changes in glycosylation patterns, or caused by glycoproteins.

[0268] In particular, this method can be applied in medical diagnostics (such as chronic inflammation identification or early cancer diagnosis), where changes in protein glycosylation patterns are powerful indicators of disease. Changes in glycosylation patterns can be easily determined by comparing fingerprints obtained regarding the number, height, and migration time of peaks. Therefore, disease markers can be identified because they are described using similar proteomics methods. That is, similar to comparing an individual's proteome at continuous time points, an individual's glycome can be analyzed as an indicator of disease or to identify patients at risk.

[0269] In one embodiment, the method according to the invention is a method in which the fluorescent dye is a dye having the following formula C.

[0270]

[0271] In another embodiment, the fluorescent dye is a dye having the formula D.

[0272]

[0273] In a preferred embodiment, the compounds of formulas A to D are selected from...

[0274]

[0275] Or compounds of 7-R (R = H, Me), 13a, 13b, 16, and 18.

[0276]

[0277] On the other hand, the present invention relates to a method for calibrating a multi-wavelength fluorescence detection system, particularly a capillary gel electrophoresis system, having an acridinone- and / or pyrene-based fluorescent dye, which may selectively exist as a conjugate with a substrate moiety (including a carbonate compound), thereby the method comprising detecting at least one compound of formula A or B as defined in claim 1 (including compound C or D), and additional fluorescent dyes that allow for different wavelengths, preferably including at least one compound APTS, compound 19, or compound 20, as shown below.

[0278]

[0279] As demonstrated in the embodiments, calibrating the multi-wavelength fluorescence detection system with the dye improves the sensitivity of the instrument and allows for more independent implementation of the method according to the invention from the operator, the instrument, etc.

[0280] In particular, as discussed in further embodiments, system or instrument calibration increases sensitivity, thereby improving the applicability and usability of the method.

[0281] In embodiments of the calibration method according to the present invention, acridinones and / or pyrene dyes and combinations thereof used for spectral calibration are shown in Tables 2 and 3 of Examples 2 and 3, respectively.

[0282] Furthermore, a carbohydrate dye conjugate comprising the fluorescent dye according to the invention is disclosed for use in the method according to the invention. In one embodiment, the dye conjugate according to the invention is a dye selected from compounds of the following formula.

[0283]

[0284]

[0285] In a further aspect, calibration standards are provided. That is, for example, calibration standards effective in the calibration methods described herein are carbohydrate standards that include fluorescent dyes, said fluorescent dyes including at least one of fluorescent dyes according to formula A, B, C or D, which may be conjugated to carbohydrates, and optionally further comprising at least one compound 19 or 20.

[0286] Typical examples of calibration standards related to wavelength calibration methods are described.

[0287] In another aspect, the present invention relates to a standard composition comprising compounds labeled with a fluorescent dye according to formula A or B, particularly a fluorescent dye according to formula C or D, or different dyes of formulas A to D. In one embodiment, the standard composition consists of carbohydrates labeled with said dye; alternatively, the compound is a DNA base pair ladder label or a similar nucleic acid base standard. Furthermore, the dye is preferably at least one of 6-H, 6-Me, 8-R, 15, 13a, 13b, 16, 18, 23, and 23b. The standard composition can be used in the method according to the invention, particularly for comparing the migration / retention times of carbohydrates to be measured.

[0288] In addition, compounds of general formula 20 were also disclosed.

[0289]

[0290] In a further aspect, the present invention relates to a kit or system for determining and / or identifying compositional patterns of carbohydrate mixtures, the kit or system comprising a data processing unit having a non-transient memory containing a database containing alignment migration / retention times and / or alignment migration / retention time indices of carbohydrates, the migration / retention times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification by analysis of compositional patterns of carbohydrates and / or carbohydrate mixtures, comprising the following steps:

[0291] a) Obtain a sample containing at least one carbohydrate;

[0292] b) Label the carbohydrate with a first fluorescent marker;

[0293] c) Provide standards of known composition labeled with a second fluorescent label;

[0294] d) Determine the migration / retention times of the carbohydrates and standards of known composition as described herein, for example using capillary gel electrophoresis-laser-induced fluorescence;

[0295] e) Align the migration / retention time with the migration / retention time index based on the given standard migration / retention time index of the standard;

[0296] f) Compare the migration / retention time indices of these carbohydrates with the standard migration / retention time indices in the database;

[0297] g) Identify or determine the compositional patterns of carbohydrates and / or mixtures of carbohydrates.

[0298] In this process, a standard composition is added to a sample containing a mixture of unknown carbohydrates. The first fluorescent label and the second fluorescent label are different, wherein the first fluorescent label or the second fluorescent label is a fluorescent dye having multiple ionizable and / or negatively charged groups, which are selected from the group consisting of compounds of general formulas A to D.

[0299] On another front, the present invention relates to a kit or system for determining and / or identifying carbohydrate mixture composition pattern analysis, the kit or system comprising a data processing unit having a non-transient memory containing a database containing alignment migration / retention times and / or alignment migration / retention time indices of carbohydrates, the migration / retention times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification by carbohydrate and / or carbohydrate mixture composition pattern analysis, comprising the following steps:

[0300] a) Provide a sample containing a mixture of carbohydrates;

[0301] b) Label the carbohydrate mixture composition with a first fluorescent marker;

[0302] c) Provide a second sample labeled with a fluorescent marker, which has a known carbohydrate mixture composition pattern to be compared with;

[0303] d) As described in the methods disclosed herein, generate an electrophoretic map / chromatogram of the carbohydrate mixture of the first and second samples, for example using capillary (gel) electrophoresis-laser induced fluorescence or chromatography;

[0304] e) Compare the migration / retention time indices of the standards calculated from the electrophoresis / chromatograms of the first and second samples obtained;

[0305] f) Analyze the consistency and / or differences in the compositional patterns of the carbohydrate mixtures of the first and second samples, wherein the standard migration / retention time index of the carbohydrates present in the samples is calculated based on an internal standard of a known component labeled with a second fluorescent label, and wherein one of the first or second fluorescent labels is a fluorescent dye of general formula A or B according to the present invention.

[0306] Furthermore, the present invention relates to a kit or system for automated analysis of carbohydrate mixture composition patterns, the kit or system comprising a data processing unit having a non-transient memory containing a database containing calibration-aligned migration / retention times and / or alignment-aligned migration / retention time indices for carbohydrates, the migration times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification by analysis of carbohydrate and / or carbohydrate mixture composition patterns, comprising the following steps:

[0307] a) Provide a first sample containing a mixture of unknown carbohydrates;

[0308] b) Label the carbohydrate mixture composition with a first fluorescent marker;

[0309] c) Add a second sample, labeled with a second fluorescent marker, having a known carbohydrate mixture composition pattern, to the first sample;

[0310] d) Generate an electrophoretic or chromatographic image of the carbohydrate mixture of the sample using capillary (gel) electrophoresis-laser induced fluorescence or chromatography;

[0311] e) Analyze the similarities and / or differences in the carbohydrate mixture composition patterns of the first and second samples;

[0312] The first fluorescent marker of the first sample is different from the second fluorescent marker of the second sample, and at least one of the first fluorescent marker and the second fluorescent marker is a fluorescent dye of general formula A or B according to the present invention.

[0313] In one embodiment, the kit or system according to the invention further includes a capillary (gel) electrophoresis-laser-induced fluorescence device. For example, the device may be a capillary DNA sequencer known in the art.

[0314] In a further aspect, a carbohydrate dye conjugate comprising a fluorescent dye as defined herein conjugated with the carbohydrate described herein, for use in the method according to the invention.

[0315] In one embodiment, the carbohydrate dye conjugate is a conjugate in which the dye is selected from compounds of the following formula:

[0316]

[0317]

[0318] In some embodiments of the specific compounds described above, the dye exists as a carbohydrate dye conjugate, thereby identifying the carbohydrates bound to the dye.

[0319] The present invention will be further described through embodiments to illustrate the invention in more detail, but is not limited thereto. Attached Figure Description

[0320] Figure 1 - A workflow for carbohydrate analysis according to the present invention is provided.

[0321] Figure 2 - 19(I), 20(II), and 6-H labeled maltotriose (6-H) before (A) and after (B) spectral calibration of a specific calibration mixture of these four dyes on an xCGE-LIF instrument. a ;III) and APTS-labeled maltotetraose (APTS) a ;IV) spectral calibration mixture.

[0322] Figure 3 - On the xCGE-LIF instrument, 19, 20, 6-H a and APTS a 6-H labeled maltose ladder before (A) and after (B) spectral calibration. Before measurement, the VB9163 labeled maltose ladder in B was diluted in water at a 1:2 ratio. The described peaks are maltose at 13.2 min, maltotriose at 15.3 min, maltotetraose at 17.2 min, maltopentose at 19 min, maltohexaose at 20.8 min, maltoheptaose at 22.2 min, maltooctaose at 23.9 min, etc.

[0323] Figure 4 - 15-labeled maltotriose (15) before (A) and after (B) spectral calibration of a specific calibration mixture of five dyes on an xCGE-LIF instrument. a ;I), 19(I), 20(IV), 6-Me-labeled maltotriose (6-Me a ;V) ​​and APTS-labeled maltotetraose (APTS) a ) spectral calibration mixture.

[0324] Figure 5 - In xCGE-LIF instrument pair 15 a 19, 20, 6-Me a and APTS a APTS-labeled dextran ladder (APTS) before (A) and after (B) spectral calibration bThe peaks described are the dextran trimer at 14.1 minutes, the tetramer at 16.2 minutes, the pentamer at 18.3 minutes, the hexamer at 20.9 minutes, the heptamer at 23 minutes, and so on.

[0325] Figure 6 - In xCGE-LIF instrument pair 15 a 19, 20, 6-Me a and APTS a 15-labeled dextran ladder before (A) and after (B) spectral calibration (15 b The peaks described are the dextran trimer at 9.8 minutes, the tetramer at 11 minutes, the pentamer at 12 minutes, the hexamer at 13.1 minutes, the heptamer at 14.2 minutes, and so on.

[0326] Figure 7 - In xCGE-LIF instrument pair 15 a 19, 20, 6-Me a and APTS a 6-Me-labeled dextran ladder (6-Me) before (A) and after (B) spectral calibration b The peaks described are the dextran trimer at 14.9 minutes, the tetramer at 16.3 minutes, the pentamer at 18.2 minutes, the hexamer at 20.1 minutes, the heptamer at 22 minutes, and so on.

[0327] Figure 8 - In xCGE-LIF instrument pair 15 a 19, 20, 6-Me a and APTS a After spectral calibration, the APTS-labeled citrate plasma-derived N-glycan (522 nm trace), the 15-labeled carbohydrate standard (554 nm trace), and the 6-Me-labeled carbohydrate standard (575 nm trace) overlapped (see [reference]). Figure 7 The 522nm, 554nm, and 575nm channels now show spectral crosstalk with other channels, confirming successful spectral calibration.

[0328] Figure 9 - Electrophoresis patterns of different alignment standards. A - GeneScan 500LIZ size standard. B - Carbohydrate standard labeled with an acridinone-based fluorescent dye (6-Me). The labeled peaks were used to calculate the polynomial fit of the alignment procedure (see...). Figure 11 ).

[0329] Figure 10- Human citrate plasma-derived N-glycan fingerprints aligned with either a base-pair size standard (A) or a base-pair size standard (B) purified from orthogonal carbohydrate standards. The relative peak height ratio (PHP) is the signal intensity of the fingerprint relative to the sum of 15 selected peaks. Polymers 1 and 2 have different production dates / batches. Days 1-9 are calculated based on the number of days the polymers were at room temperature.

[0330] Figure 11 - In contrast to base pair size standards (A) or carbohydrate standards labeled with acridinium fluorescent dyes (6-Me) b (B) Aligned N-glycan fingerprints derived from human citrate plasma. The relative peak height ratio (PHP) is the signal intensity of the fingerprint relative to the sum of 15 selected peaks. Polymers 1 and 2 are POP7 polymers from different production dates. Days 1-9 were calculated as the number of days the POP7 polymers were at room temperature.

[0331] Figure 12 - Polynomial fitting of internal standards with different alignment procedures. A - Second-order polynomial fitting suitable for alignment with base pair size standards. 13 peaks are selected, such as... Figure 9 A - As shown. B - Suitable for second-order polynomial fitting aligned with base pair size standards, adjusted via a second alignment step, using four internal oligosaccharide peaks. C - Suitable for second-order polynomial fitting aligned with carbohydrate standards labeled with acridinone-based fluorescent dyes (6-Me). Sixteen peaks were selected, as shown... Figure 9 As shown in B.

[0332] Figure 13 - Electrophoresis diagrams of different alignment standards. A - Base pair size standard. B - Carbohydrate standard labeled with pyrene-based fluorescent dye (15). The labeled peaks were used to calculate the polynomial fit of the alignment procedure (see Figure 16).

[0333] Figure 14 -In the case of base pair size standard (A), base pair size standard + pyrene fluorescent dye labeled carbohydrate standard (B), or pyrene fluorescent dye (15) labeled carbohydrate standard (15) b (C) Aligned N-glycan fingerprints derived from human citrate plasma. The relative peak height ratio (PHP) is the signal intensity of the fingerprint relative to the sum of 15 selected peaks. Polymers 1 and 2 are POP7 polymers from different production dates. Days 1-9 are calculated as the number of days the POP7 polymers were at room temperature.

[0334] Figure 15 - In xCGE-LIF instrument pair 15 a 19, 20, 6-Me a and APTSa After spectral calibration, APTS-labeled citrate plasma-derived N-glycans (522 nm trace), labeled carbohydrate standards (554 nm trace), and base pair standards (655 nm trace) overlapped (see [reference]). Figure 7 The 522nm and 554nm channels now show spectral crosstalk with other channels, demonstrating successful spectral calibration. Small spectral crosstalk with the 595nm and 575nm channels can be observed in the base pair size standard containing the 655nm channel, because the 655nm channel was not spectrally calibrated for the bp dye.

[0335] Figure 16 - Polynomial fitting of internal standards with different alignment procedures. A - Second-order polynomial fitting suitable for alignment with base pair size standards. 13 peaks are selected, such as... Figure 13 A is shown. B - is suitable for second-order polynomial fitting to pyrene-based fluorescent dye (15) labeled carbohydrate standards. 22 peaks were selected, as shown... Figure 13 As shown in B

[0336] Figure 17 - The N-glycan fingerprints of APTS-labeled citrate plasma derivatives were measured using different instruments and overlapped with those of base-pair size standards (A), base-pair size standards + oligosaccharide realignment (B), base-pair size standards + carbohydrate standards labeled with pyrene fluorescent dye (23) realignment (C), or carbohydrate standards labeled with pyrene fluorescent dye (23) (D). The following instruments were used: 3130_1 – the first ABI DNA genetic analyzer 3130 (serial number: 21363-yyy) equipped with a 50cm four-capillary array; 3130_2 – the second ABI DNA genetic analyzer 3130 (serial number: 1521-yyy) equipped with a 50cm four-capillary array; 3130xl_1 – the first ABI DNA genetic analyzer 3130x1 (serial number: 19248-yyy) equipped with a 50cm 16-capillary array; 3130xl_2 – the second ABI DNA genetic analyzer 3130x1 (serial number: 1208-yyy) equipped with a 50cm 16-capillary array; 3500 – Thermo Scientific DNA analyzer 3500 (serial number: 21106-yyy) equipped with a 50cm eight-capillary array; and 3730 – ABI... The DNA Genetic Analyzer 3730 (serial number: 18124-yyy) is equipped with a 50cm 48-capillary array. All measurements were performed using POP7.

[0337] Figure 18- The N-glycan fingerprints of APTS-labeled citrate plasma derivatives were measured using different electric field strengths and overlapped with base-pair size standards (A) or carbohydrate standards labeled with pyrene fluorescent dye (23) (B). Measurements were performed using an ABI DNA genetic analyzer equipped with a 50 cm capillary array filled with glyXpop_fast at field strengths of 300 V / cm (“…” curve, 15 kV), 200 V / cm (“---” curve, 10 kV), or 100 V / cm (“–” curve, 5 kV).

[0338] Figure 19 -Measured at different operating temperatures, and the N-glycan fingerprints of APTS-labeled citrate plasma derivatives were overlaid with base-pair size standards (A) or carbohydrate standards labeled with pyrene fluorescent dye (23) (B). Measurements were performed using an ABI DNA genetic analyzer equipped with a 50 cm capillary array filled with POP7, at operating temperatures of 45 °C (“…” curve), 30 °C (“---” curve), or 18 °C (“–” curve).

[0339] Figure 20 -Measured with different capillary array lengths and overlapped with N-glycan fingerprints of APTS-labeled citrate plasma derivatives aligned with base pair size standards (A) or carbohydrate standards labeled with pyrene fluorescent dye (23) (B). Measurements were performed using an ABI DNA genetic analyzer equipped with a 50 cm capillary array (“…” curve), a 36 cm capillary array (“--” curve), or a 22 cm capillary array (“–” curve) filled with POP7.

[0340] Figure 21 - Overlapping N-glycan fingerprints of APTS-labeled citrate plasma derivatives measured with different separating polymers. Unaligned electrophoresis is shown in minutes (A), fingerprints aligned with base-pair size standards are shown in base pairs (B), and fingerprints aligned with pyrene fluorescent dye (23)-labeled carbohydrate standards are shown in oligosaccharide units (C). Measurements were performed using an ABI DNA Genetic Analyzer equipped with a 50 cm capillary array filled with POP7 (Thermo Scientific; black curve), nanoPOP7 (MCLAB; gray curve), nimaPOP7 (Nimagen; light gray curve), POP6 (Thermo Scientific; black "---" curve), or glyXpop_fast (experimental polymer from glyXera GmbH; black "..." curve).

[0341] Figure 22- Overlap of APTS-labeled human IgG-derived N-glycan fingerprints aligned with pyrene fluorescent dye (23)-labeled carbohydrate standards. Measurements were performed using an ABI DNA genetic analyzer equipped with a 50 cm capillary array and filled with POP7 polymer. Measurements were performed by re-injecting the same sample with polymer for durations D1–D52 (calculated as the number of days the POP7 polymer remained in the instrument at room temperature).

[0342] Figure 23 Emission spectra of dyes used in DNA sequencing (showing one of several possible groups), along with the corresponding set of virtual filters. 5-FAM: 5'-carboxyfluorescein; JOE: 2,7-dimethoxy-3,4-dichlorofluorescein 6'-carboxy isomer; NED is a brighter dye than TMR (structure unknown); it has absorption and emission maxima at 546 nm and 575 nm, respectively. ROX is rhodamine with two julonidine fragments incorporated into the xanthan fluorophore (and a 5'- or 6'-carboxyl group). During fluorescence sequencing, these (or similar) dyes provide traces of four colors; for example, blue represents cytosine, green represents adenine, red represents thymine, and yellow represents guanine.

[0343] Figure 24A Normalized absorption and emission spectra of phosphorylated aminoacridone dyes 6-H and 6-Me in aqueous triethylamine-bicarbonate buffer solution (pH 8) are shown.

[0344] Figure 24B Normalized absorption and emission spectra of triphosphorylated aminopyrene dyes 8-H and 15 in aqueous triethylamine-bicarbonate buffer solution (pH 8) are shown.

[0345] Figure 25 An overview of the electrophoretic patterns of two dyes is shown: aminopyrene triphosphate 8-H and APTS, with a maltose ladder labeled with APTS (background). Although the m / z ratio of 8-H (144) is lower than that of APTS (151), the retention time of 8-H is higher than that of APTS. In APTS, the charged group (sulfonic acid residue) is directly attached to the fluorophore. In 8-H, the presence of the N-methyl-N-(2-hydroxyethyl) linker increases the hydrodynamic ratio of the dye, which explains the higher retention time of the free dye 8-H.

[0346] Figure 26The image shows amplified peaks for 8-H and APTS. This figure was obtained through color calibration using a standard DNA sequencer. The “traditional” filter set has five color channels: 522nm (fluorescein, APTS), 554nm (e.g., VIC dyes or Rhodamine 6G), 575nm (e.g., NED dyes or TMR), 595nm (e.g., PET dyes or ROX), and 650nm (LIZ dye as an additional “fifth” color). Due to strong crosstalk with the APTS color channel (shown at the top of the figure), dye 8-H (likely its conjugate with a glycan) cannot be used with APTS in any analytical assay. The same applies to pyrene triphosphate dye 15 (compare). Figure 24B (The emission spectra of 8-H and 15 are shown). Therefore, a new color calibration is needed for the DNA sequencer to reduce or, as far as possible, eliminate crosstalk between APTS and emission channels caused by the 8-H and 15 pyrene triphosphate dyes.

[0347] Figure 27 The results show the spectral composition of maltotriose and dye 15(15) before spectral calibration. a Electrophoresis diagram of the reduced amination product obtained.

[0348] Figure 28 The same electrophoretic pattern of the reduced amination products obtained from maltotriose and dye 15 after spectral calibration is shown. Figure 27 ).

[0349] Figure 29A Figures B and C show electrophoretic plots of conjugates obtained from a mixture of carbohydrates “dextran 1000” (29A) and “dextran 5000 ladder” (29B) with dye 15; “1000” and “5000” correspond to the average molecular weight of the dextran oligomers. The time difference between peaks is approximately 1 minute. In the case of APTS, the time difference between peaks is approximately 2.3 minutes (see Figure B). Figure 25 (--- curve); the addition of glucose units leads to an increase in migration time that is roughly the same as that of maltose units. The smaller the time difference between peaks, the better (more support points for linearly aligned curve fitting).

[0350] Figure 30A Figures B and B show electrophoretic images of the conjugates (reductive amination products) obtained from maltotriose with dyes 6-H and 6-Me before spectral calibration. For both 6-H and 6-Me dyes, the crosstalk between the APTS channel (522 nm) and the "595 nm channel" (also applicable to 6-H and 6-Me) is quite small; less than 15 for dyes (…). Figure 27 For dye 6-H, the crosstalk is approximately 7.8%, and for dye 6-Me, it is approximately 3.4%. However, even small amounts of crosstalk between the standard channel and the observation channel are undesirable, as they can lead to false positives (for analytes not present).

[0351] Figure 31A Figures B and B show electrophoretic images of conjugates obtained from the “Dextran 1000” and “Dextran 5000” ladders with dye 6-Me after spectral calibration. Spectral calibration was based on the use of 6-H and 6-Me dyes conjugated with maltotriose (see Figures B and C, respectively). Figure 2 and Figure 4 Their spectral properties and the properties of their conjugates are very similar. There is no crosstalk between the APTS color channel (522nm) and the "new" 575nm channel. Detailed Implementation

[0352] General Materials and Methods

[0353] Reductive amination of carbohydrates

[0354] For the reductive amination of carbohydrates using the compounds of the present invention, for example, the prior art method of fluorescently labeling N-glycans with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) and reducing agent, as published by Hennig R, Rapp E et al. in Methods Molecular Biology in 2015, was used with minor modifications.

[0355] The initial procedure requires a moderately strong acid (such as citric acid monohydrate; CA) and solvents—dimethyl sulfoxide (DMSO), acetonitrile (ACN), and water (H₂O). The main steps involve preparing a 10–80 mM dye solution (solution A) in a 1.2–3.6 M aqueous CA solution and a boronyl reducing agent solution (solution B) in DMSO. Then, three equal volumes (1–4 μL) of solutions A and B and the sample (free carbohydrates or the carbohydrate portion of the released glycoconjugate) are mixed and incubated at 37 °C for 3–16 h. After reductive amination, an ACN-water mixture (80:20, v / v) is added. For example, if 2 μL of solution A, 2 μL of solution B, and 2 μL of analyte sample are used, 50 μL of aqueous ACN solution is added and mixed. This process provides a clear solution suitable for sugar analysis based on electrokinetic and / or chromatographic separation.

[0356] Acylhydrazine labeling

[0357] Using the compounds of this invention, hydrazide labeling was performed at 60-80°C and pH 6-8 for 1-6 hours. A 10-80 mM dye solution was mixed with an equal volume (1-4 μL) of the sample. After the reaction was complete, 50 μL of an ACN-water mixture (80:20, v / v) was added. The diluted labeling mixture was subjected to sugar analysis based on electrokinetic and / or chromatographic separation.

[0358] Reactive carbamate chemistry

[0359] The compounds of this invention were subjected to glycosylamine labeling with disuccinimide carbonate or NHS ester-assisted methods at room temperature for 10–60 min at slightly alkaline pH. Samples were purified using the HILIC-SPE method published by Hennig R, Rapp E et al., 2015. The purified samples were then subjected to sugar analysis based on electrokinetic and / or chromatographic separations.

[0360] Example 1 - Selected fluorescent dyes with a large negative net charge and desired spectral characteristics (see also Scheme 13 and Table 1)

[0361]

[0362] The red-emitting rhodamine dye having multiple ionizable groups of structure 20 is obtained by phosphorylation of the corresponding hydroxyl-substituted rhodamine precursor, and isolated similarly to compound 19 (another phosphorylated rhodamine dye, see schemes 6 and 11 above), which was previously described by K. Kolmakov et al. in Chem Eur. J. 2012, 18, 12986-12998 (see the properties and phosphorylation details of compound 7-H therein). The hydroxyl-substituted precursor of compound 20 is synthesized according to K. Kolmakov et al. (Chem Eur. Journal. 2013, 20, 146-157; see compound Et-14 therein). After phosphorylation, the ethyl ester group is saponified by a conventional procedure as described above.

[0363] The purity and identity of compound 20 were confirmed by the following analytical data: 1 H NMR(400MHz DMSO-d6): δ = 1.23 (s, 6H, CH3), 1.28 (s, 6H, CH3), 2.62 (s, 6H, NCH3), 4.21 (m, 4H, 2C H2), 5.70 (s, 2H), 6.76 (s, 2H), 7.16-7.30 (br.m, 4H), 8.55 (m, 1H), 8.36 (m, 1H)ppm. 13 C NMR (101MHz, DMSO-d6): δ=29.1(CH3), 34.2(CH3), 95.8(CH2), 118.2(CH), 121.7(C), 122.6(C), 125.5( CH), 127.3(CH), 127.4(CH), 128.0(CH), 129.8(CH), 133.9(C), 136, 8(C), 155.0(CO), 157.0(CO)ppm.

[0364] 1¹H NMR (400MHz, CD₃OD, 20 for Et₃N⁻ salt): δ = 1.12 (t, J = 7Hz, 9H, CH3 CH2), 1, 25 (t, J=7Hz, 27H, CH3 CH2), 1.52(s, 6H, CH3), 1.53(s, 6H, CH3), 3.11, 3.31(m, 24H, CH3 CH2 ), 3.18(s, 6H, NCH3), 3.61(m, 2H, CH2), 4.45(m, 2H, CH2), 6.03(s, 2H), 6.8( s, 2H), 6.9 (s, 2H), 7.28 (d, J=8Hz, 1H), 8.16 (d, J=8Hz, 1H), 8.66 (m, 1H)ppm. 31 P NMR (161.9 MHz): δ = -0,2 (DMSO-d6) and 0,63 (CD3OD) ppm (s, OP(O)(OH)2)).

[0365] HPLC: t R = 3.9 min (Kinetex EVO C-18 column, 0.02 M aq. Et3N (A) and 3% MeCN (B), isocratic flow rate 0.5 mL / min, detection wavelength 254 nm). TLC: R f =0.25 (silicone plate, MeCN / H2O 5:1 + 0.2% Et3N). HR-MS (ESI): C 35 H 35 N2O 13 P2 - ([MH) - Calculated value: 753.1614, Found value: 753.1672. UV-VIS (PBS buffer, pH = 7.4) max abs. = 582nm, λ max .fl.=609nm.

[0366] Example 2 - Spectral calibration of a multi-wavelength fluorescence detection system for four acridinone and pyrene-based fluorescent dyes described herein.

[0367] For the current example, the procedure exemplifies modified commercial DNA genetic analyzers 310, 3100, 3130(xl), 3730(xl), and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). However, depending on the detection mode, the recalibration described here is also possible for instruments from other manufacturers. The commercial genetic analyzer used contains a multiplex capillary gel electrophoresis (xCGE) unit with laser-induced fluorescence detection (LIF), which can (depending on the instrument and operating software) simultaneously detect up to six different fluorescence signals in different dye channels.

[0368] According to the manufacturer, the instrument's virtual filters can be calibrated according to various predefined dye sets, such as F, D (both available: four detection windows) or G5 (five detection windows). As the default spectral calibration for oligosaccharide analysis, the predefined dye set G5 [EP 2112506 B1, Ruhaak 2010, Reusch 2015, Feng 2017] is used. G5 is calibrated according to the dye set 6-Fam. TM (Recorded within the 522nm dye trace) (554nm), NED TM (575nm) (595nm) and Calibration was performed using the DS-33 matrix standard (655nm). This calibration allowed APTS-labeled oligosaccharides to be calibrated at 6-Fam levels. TM The dye trace was recorded within 522 nm, while the comparison standard GeneScan 500 LIZ was used. TM exist Recording was performed within the dye trace (655 nm). Unfortunately, using G5 spectral calibration, APTS produced signals in all other dye traces, such as... Figure 2 As shown in Figure A, APTS-labeled maltodextrose at 16.3 min. This large crosstalk is caused by APTS and 6-Fam TM The different spectral characteristics are the cause. To perform migration-time alignment without affecting the crosstalk signal from APTS, a GeneScan 500LIZ was used. TM (LIZ500) because LIZ records within the dye traces that are as far away from the emitted light from the APTS channel as possible.

[0369] To enable the use of alignment standards different from those of the LIZ500 and to reduce spectral crosstalk, the xCGE-LIF instrument was exemplarily used to calibrate a set of four dyes, including APTS and three novel dyes of this invention. Prior to spectral calibration, all the fluorescent dyes (their oligosaccharide derivatives, respectively) exhibited fluorescence signals in multiple dye traces / channels. Figure 2 A). In particular, 6-H-labeled carbohydrates exhibited large spectral crosstalk with all dye channels, such as Figure 2 Maltotriose in A and Figure 3 The maltose ladder in A is shown. Therefore, since the use of internal alignment standards requires complete absence of fluorescence signal from other dyes within the APTS channel (522 nm), it is impossible to use, for example, a 6-H labeled maltose ladder as an internal alignment standard without prior spectral calibration of the instrument. The xCGE-LIF instrument supports 19, 20, 6-H labeled maltotriose (6-H... a ) and APTS-labeled maltotetraose (APTS) a Spectral calibration can completely eliminate spectral crosstalk (see [reference]). Figure 2 B&3B).

[0370] Following spectral calibration on the xCGE-LIF instrument, the 6-H-labeled maltose ladder can be used for internal alignment with APTS-labeled carbohydrates. Therefore, co-injection of the 6-H-labeled maltose ladder with APTS-labeled carbohydrates detects the same sample background as the APTS-labeled carbohydrates. As a side effect, the better fit to the spectral calibration results in an increase in the signal intensity of the 6-H-labeled ladder. Figure 3 The signal intensity of the 6-H maltose peak increased 1.5-fold at 13.2 minutes (from approximately 2000 RFU to approximately 3000 RFU). At 16.3 minutes, the signal intensity... Figure 2 The peak IV of APTS can be observed a The same effect.

[0371] As shown in Table 2, the spectral calibration of a set of four fluorescent dyes by a multi-wavelength system may result in significant variations in the dyes of this invention.

[0372] Table 2: Spectral calibration of a set of four dyes using a multi-wavelength system.

[0373] The possibilities for using the 3100, 3130, 3130xL, 3730, 3730xL, 3500, and 3500xL instruments for four dye spectral calibrations are illustrated exemplarily. For spectral calibration, each trace requires the use of one fluorescent dye; doubling is not necessary. For example, to analyze APTS-labeled samples, the 522 nm spectral trace is used to calibrate an APTS-labeled carbohydrate (APTS). zSimultaneously, the spectral trace at 560 nm was used to calibrate one of the following dyes: 6-H, 6-Me, 6-H z 6-Me z , 8-H, 8-H z 15, 15 z 23, 23 z ; spectral trace of 575 nm is used for 20, 6-H, 6-Me, 6-H z Or 6-Me z The spectral trace at 607 nm is used for 19 or 20. One possible spectral calibration is APTS. z 15 z 6-Me z And 19. These spectral calibrations are capable of analyzing up to three samples (APTS-, 15-, and 6-Me-labeled, at spectral traces of 522 nm, 560 nm, and 575 nm) as well as base-pair-based internal alignment standards (at spectral trace of 607 nm).

[0374]

[0375] Exponent z = fluorescent dye - carbohydrate derivative → e.g., APTS z It can be an APTS-labeled maltotetraose (see...) Figure 2 ), or 15 z It can be 15-labeled maltotriose (used for) Figure 4 ).but z It can be any other carbohydrate, such as O-glycan, N-glycan, lactooligosaccharide, homopolymers (such as maltose, starch, cellulose, dextran) or heteropolymers (such as hemicellulose, arabinoxylan, glycosaminoglycan).

[0376] Example 3 - Spectral calibration of a multi-wavelength fluorescence detection system for a group of five acridinone and pyrene-based fluorescent dyes described herein.

[0377] For the current example, the procedure exemplifies the modified commercial DNA genetic analyzers 310, 3100, 3130(x1), 3730(x1), and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). However, depending on the detection mode, the recalibration described here is possible for instruments from other manufacturers. The commercial genetic analyzer used includes a multiplex capillary gel electrophoresis (xCGE) unit with laser-induced fluorescence detection (LIF), which can (depending on the instrument and operating software) simultaneously detect up to six different fluorescence signals in different dye channels.

[0378] The virtual filters of these instruments can be calibrated according to various predefined dye groups, such as E5, G5, or D. Therefore, dye groups E5 and G5 define five detection windows for five different fluorescent dyes, while dye group D defines four detection windows for four different fluorescent dyes. For oligosaccharide analysis, the predefined dye group G5 is used, according to the formula containing dye 6-Fam. TM (Recorded within the 522nm dye trajectory) (at 554nm), NED TM (at 575nm) (at 595nm) and Calibration was performed using the DS-33 matrix standard at 655 nm [EP 2112506 B1, Ruhaak 2010, Reusch 2015, Feng 2017], followed by the light emitted by the APTS-labeled oligosaccharide at the dye trace (Fam) at 522 nm. TM Recorded within the dye trace, aligned with the GeneScan 500 LIZ standard. TM The light emitted by the (LIZ 500) is recorded within the 655 nm dye trace. Because the instrument is not specifically calibrated for APTS dyes, the light emitted by APTS-labeled oligosaccharides enters several dye traces, such as... Figure 4 As shown in Figure A, peak V at 16.3 min represents APTS-labeled maltodextrose. Since the absence of spectral crosstalk between the two dye traces is crucial for accurate analysis, this large crosstalk needs to be reduced. Furthermore, in order to use oligosaccharide-based alignment standards labeled with the fluorescent dyes of this invention, such as 15, 6-H, 6-Me, 8-H, or 23, customized spectral calibration is required for these dyes.

[0379] For example, the xCGE-LIF instrument was spectrally calibrated for a group of five dyes, such as... Figure 4 As shown. Before spectral recalibration (for APTS and the four new dyes of this invention, which are their oligosaccharide derivatives respectively), large crosstalk can be observed in multiple dye traces / channels for all the fluorescent dyes used. Figure 4 A). In particular, the 15-labeled carbohydrate (peak I) and the 6-Me-labeled carbohydrate (peak IV) exhibited significant spectral crosstalk in all other dye traces, such as Figure 4 As shown in A, 6A, and 7A. Because the use of internal alignment standards requires complete absence of fluorescence signal within the APTS channel (522 nm), spectral calibration of the instrument is necessary. This is in contrast to the use of 19,15-labeled maltotriose (15... a ), 20, 6-Me-labeled maltotriose (6-Me a ) and APTS-labeled maltotetraose (APTS)a After spectral calibration, spectral crosstalk can be completely eliminated, such as Figure 4 As shown in B, 5B, 6B and 7B.

[0380] In addition, for dye derivative 15 a and 6-Me a The spectral calibration allows for the simultaneous comparison of alignment performance using two different carbohydrate-based standards, such as... Figure 8 As shown, there is no crosstalk between the 522nm (APTS), 554nm (15), and 575nm (6-Me) traces.

[0381] As shown in Table 3, the spectral calibration of a group of five fluorescent dyes by a multi-wavelength system may lead to significant variations in the dyes of this invention.

[0382] Table 3: Spectral calibration of a group of five dyes using a multi-wavelength system.

[0383] The possibilities for spectral calibration of the 3100, 3130, 3130xL, 3730, 3730xL, 3500, and 3500xL instruments with five dyes are illustrated exemplarily. For spectral calibration, each trace requires the use of one fluorescent dye, without doubling the amount. For example, to analyze APTS-labeled samples, the 522 nm spectral trajectory is used to calibrate APTS-labeled carbohydrates (APTS). z Simultaneously, the spectral trace at 554 nm was used to calibrate one of the following dyes: 8-H, 8-H z 15, 15 z 23 or 23 z ; spectral trace of 575nm is used for 6-H, 6-Me, 6-H z Or 6-Me z The spectral trace at 595 nm is used for 20, and the spectral trace at 655 nm is used for 19. For example, for APTS z ,twenty three z 6-Me z Spectral calibration of 1, 20, and 19 allows for the analysis of two samples (APTS- and 23-labeled, at spectral traces of 522 nm and 554 nm) as well as carbohydrate-based alignment standards (6-Me-labeled, at spectral trace of 575 nm) and / or base-pair-based internal alignment standards (at spectral trace of 655 nm).

[0384]

[0385] Exponent z = fluorescent dye - carbohydrate derivative → e.g., APTS z It can be an APTS-labeled maltotetraose (see...) Figure 2 ), or 15z It can be 15-labeled maltotriose (see...) Figure 4 ).but z It can be any other carbohydrate, such as O-glycan, N-glycan, lactooligosaccharide, homopolymers (such as maltose, starch, cellulose, dextran) or heteropolymers (such as hemicellulose, arabinoxylan, glycosaminoglycan).

[0386] Example 4 - Internal migration time alignment using acridinone fluorescent dye derivatives according to the present invention

[0387] Current examples include the use of modified commercial DNA genetic analyzers 310, 3100, 3130(xl), 3730(xl), and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). However, the carbohydrate-based calibration alignment standards presented in this paper can also be used in conjunction with (single or multiplex capillary) CE / CGE instruments or (U) HPLC instruments from other manufacturers. Generally, migration time alignment of DNA fragment sizes (e.g., for short tandem repeat (STR) or restriction fragment length polymorphism (RFLP) analysis in genomics) and migration time alignment of carbohydrates in CE / CGE and xCGE are currently achieved using base pair size standards, such as… Figure 9 This is exemplified in A (EP2112506 A1). For this purpose, the migration time of the unknown sample is compared to a co-injected base pair size standard. For oligonucleotides (DNA / RNA), this alignment of internal migration time with the co-injected base pair standard is highly reproducible because the sample background affects the migration time of both the unknown sample and the standard in the same way. The sample and standard are labeled with different fluorescent dyes, enabling simultaneous wavelength-resolved detection of both.

[0388] While the long-term alignment quality of unknown DNA fragments with DNA-based base pair size standards was excellent, the long-term alignment quality of oligosaccharides with base pair size standards was not as good. The alignment migration time of carbohydrates with base pair size standards showed some fluctuations over longer periods among different polymer batches (see [link to relevant documentation]). Figure 10 A). To improve alignment quality, an additional (second) orthogonal alignment step was introduced, using an increased cross-bracketing carbohydrate standard (US2009 / 028895A1), such as... Figure 10 As shown in B.

[0389] However, the second (orthogonal) alignment step also compensates for this long-term fluctuation in carbohydrates, but not completely. The poor long-term alignment ability is due to the different physicochemical properties of the base pair standards and the labeled carbohydrates. For example, a 360-base-pair long fragment ( Figure 9 Peak 10 in A contains 360 negatively charged nucleotides (deoxyribose + phosphate + nitrogenous bases) and a fluorescently labeled carbohydrate peak with a similar migration time. Figure 10 The peak at 360 base pairs in A contains only 10 (monosaccharides) with approximately three negative charges. Therefore, the relatively low-charged small molecule is compared to the high-charged large molecule. Alignment is possible due to their similar mass-to-charge ratios. However, changing the measurement conditions will have different effects on the two types of molecules. Therefore, the migration time of carbohydrates after base pair alignment is variable in the long run, such as... Figure 10 As shown in Figure A.

[0390] This invention enables the alignment of carbohydrate migration times using carbohydrate-based standard mixtures. A novel suite of fluorescent dyes has been developed to label oligosaccharide samples and / or these carbohydrate standards / mixtures. The newly developed fluorescent dyes exhibit different spectral characteristics compared to fluorescent dyes used for labeling unknown samples. This allows for the co-injection of fluorescently labeled samples with fluorescently labeled carbohydrate alignment standards, and the simultaneous detection of two analytes in different dye / wavelength traces, such as... Figure 8 As shown. Compared to base pair size standards, the new carbohydrate-based standards contain physicochemical properties that are close to / consistent with those of the samples. Besides similar mass-to-charge ratios, the carbohydrate-based size standards have similar absolute charge and mass to the carbohydrates in the samples. Figure 11 Compared to B, such as Figure 11 As shown in A, this greatly improves the long-term reproducibility of migration time alignment.

[0391] For the example given here, as described in Hennig et al. 2016, human citrate plasma N-glycans were analyzed by xCGE-LIF using the dyes described herein. Briefly, citrate plasma proteins were denatured and linearized. N-glycans were released by peptidyl N-glycosidase F (PNGase F) and labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). After HILIC-SPE purification, the N-glycans were analyzed using Applied... A genetic analyzer was used to analyze APTS-labeled N-glycans via multiplex capillary gel electrophoresis (xCGE-LIF) with laser-induced fluorescence detection. Regarding internal migration time alignment, APTS-labeled samples were compared with 6-Me-labeled carbohydrate-based alignment standards (6-Me...). b (Together injected, see) Figure 11A, or related to GeneScan TM 500LIZ TM Dye size standards (LIZ500) were co-injected, see Figure 11 B.

[0392] As described in Example 3, the instrument is for 15 a 19, 20, 6-Me a and APTS a Spectral calibration was performed. APTS samples were recorded at the 522 nm dye trace, 6-Me b The dye trace was recorded at 575 nm, and the LIZ500 trace was recorded at 655 nm. For migration time alignment of the LIZ500, 13 standard peaks were selected, such as... Figure 9 As shown in Figure A. The migration time alignment used a second-order calibration curve, as follows: Figure 12A As shown in (EP 2112506 A1). To improve migration time alignment (US 2009 / 028895 A1), four additional spiked-in cross-bracketing carbohydrate standard peaks were selected, and the second-order calibration curve was adjusted, as shown in... Figure 12B As shown. For only with 6-Me b For the migration time comparison, 16 standard peaks were selected, such as... Figure 9 As shown in B. The second-order calibration curve is calculated as follows: Figure 12C The calculations are shown and used for alignment.

[0393] As described in US 8,293,084, orthogonal adjustments to the LIZ500 alignment can improve the migration time effect on qi (see [reference]). Figure 12B This improvement can be achieved by using only the carbohydrate-based size standard 6-Me. b Further enhancement, such as Figure 12C As shown. Its excellent long-term reproducibility is as follows. Figure 11 As shown. Although citrate plasma N-glycans aligned with LIZ500 showed different migration times depending on the polymer batch and measurement date, they showed similar migration times compared to 6-Me b The alignment only showed near-perfect overlap. To evaluate this in more detail, 15 of the largest peaks in the aligned electrophoresis pattern (e.g., Figure 10 The results (shown in B and 11B) were used to calculate their root mean square error (RMSE), as shown in Table 4. Orthogonal second alignment (orthogonal double alignment) can reduce the RMSE by a factor of 4 (3.151% to 0.727%), only compared to 6-Me. b Alignment can reduce RMSE by nearly 10 times (3.151% to 0.359%). This means that only 6-Me... bMigration time alignment resulted in a 10-fold reduction in variation and a 10-fold improvement in precision. For monocharged N-glycans, the lowest RMSE of 0.236% was obtained. Furthermore, doubly charged and neutral N-glycans showed RMSDs of 0.391% and 0.357%, respectively, very close to those of monocharged glycans. Therefore, alignment standards based on (only) acridinone dye-labeled carbohydrates, such as 6-Me... b The reproducibility is best for neutral and low-charge oligosaccharides (which can be found on, for example, five human proteins (such as IgG) or recombinant monoclonal antibodies (mAbs) [Reusch 2015]), but they are also applicable to high-charge oligosaccharides. Due to this high precision and robustness of migration time, the method according to the invention is significantly improved and more widely applicable, independent of polymer age and batch, and it is possible to establish and use a database of corresponding peak annotations by migration time matching without requiring the additional orthogonal alignment step described in patent US 2009 / 028895 A1.

[0394] Table 4: Comparison of alignment precision for N-glycan alignment with LIZ500 base-pair ladder, alignment with LIZ500 base-pair ladder improved by additional cross-linking of carbohydrates, and alignment with carbohydrate standards labeled only with acridinone dye (6-Me). b Alignment. For Figure 10 For the sample shown, the root mean square error (RMSD) of citrate plasma N-glycan was calculated. Figure 10 B depicts 15 selected peaks. N-glycan groups contain peaks: 10–15 indicate neutral, 9–7 indicate single charge, 2–6 indicate double charge, and peak 1 indicates triple charge (for detailed notes on glycan peaks, see Hennig et al., 2016). In the base pairs used for LIZ 500 alignment, in the migration time units used for LIZ500+ cross-carbohydrate (oligosaccharide) realignment, and only 6-Me... b Aligned with carbohydrate (oligosaccharide) units, the absolute RMSD is given.

[0395]

[0396] Example 5 - Internal migration time alignment using pyrene fluorescent dye derivatives according to the present invention

[0397] Currently, by using the base pair size standard (EP 2112506 A1), alignment of DNA fragment size and carbohydrate migration time in CE / CGE and xCGE has been achieved, such as... Figure 13As exemplified in A. For this purpose, the migration time of the unknown sample is aligned with a co-injected base pair-sized standard. For oligonucleotides (DNA / RNA), this alignment with the migration time of the co-injected base pair standard is characterized by high reproducibility because the same sample background affects the migration time of both the sample and the standard in the same way. The sample and the standard are labeled with different fluorescent dyes, thereby enabling simultaneous wavelength-resolved detection of both.

[0398] While the long-term alignment quality of unknown DNA fragments with DNA-based base pair size standards was excellent, the long-term alignment quality of carbohydrates with base pair size standards was not as good. The alignment migration time of oligosaccharides with base pair size standards showed some fluctuations between different polymer batches over several days (see [link to original text]). Figure 14 A). To improve alignment quality, carbohydrate-based alignment standards are needed. Therefore, a novel suite of fluorescent dyes for carbohydrate labeling has been developed. These newly developed fluorescent dyes possess spectral characteristics different from APTS (for sample labeling) and LIZ (ROX-labeled base pair size standards). (Instrument pair 15) a 19, 20, 6-Me a and APTS a The spectral calibration (as described in Example 3) allows for the simultaneous detection of co-injected labeled carbohydrate samples and 15 labeled carbohydrate-based alignment standards (15... b ) and LIZ500 base pair standards, such as Figure 15 As shown. When the APTS-labeled sample was recorded at 522 nm, the 15-labeled carbohydrate standard and the LIZ500 base pair standard were simultaneously recorded at 554 nm and 655 nm, respectively. Therefore, the internal standards LIZ500 and 15... b All can be used for migration time alignment and can be directly compared with each other. For alignment with the LIZ500, 13 standard peaks were selected, such as... Figure 13 As shown in A. To be consistent with 15 b For migration time alignment, 22 peaks were selected (see...). Figure 13 B) covers a similar migration time range as a LIZ500 standard. As shown in Figure 16, a second-order polynomial fit was performed on the selected peaks. Migration time alignment was significantly improved by using a 15-labeled carbohydrate standard, as shown... Figure 14 As shown in B&C. Compared to base pair-based size standards, the new carbohydrate-based size standards contain the same physicochemical properties as the samples. Besides similar mass-to-charge ratios, carbohydrate-based size standards have similar absolute charges and similar absolute masses. Therefore, using carbohydrate-based standards (such as 15...) bThis makes the migration time alignment of carbohydrates (such as N-glycans, O-glycans, glycolipids, human milk oligosaccharides, glycosaminoglycans, and other oligosaccharides with reducing ends and / or glycosaminoglycan ends) more precise and reproducible.

[0399] In comparison with carbohydrate-based size standards 15 b Alignment can result in improved long-term reproducibility, such as... Figure 14 As shown in C. When aligned with the LIZ500 standard based on base pairs ( Figure 14 A) shows the different migration times of all peaks, depending on the polymer batch and measurement date, compared with the LIZ500 standard +15 based on base pairs. b The alignment shows improved alignment. Figure 14 B). Optimal results can be achieved by using 15 b Alignment is used to achieve a near-perfect overlap. Figure 14 C). For a more detailed evaluation, 15 of the largest peaks were selected from all samples, such as... Figure 14 As shown in C. The root mean square error (RMSE) of these 15 peaks in all measurements is calculated as shown in Table 5. Comparing the two alignments, 15 b The aligned RMSE value (percentage of average) was 0.627%, five times smaller than the 3.151% RMSE value after LIZ500 alignment. The minimum RMSE value for the tricharged N-glycan was 0.236%, indicating that 15 b Alignment yielded the highest reproducibility for high-charge oligosaccharides (which can be found, for example, on human or recombinant erythropoietin (rhEPO) (Meininger, 2016)), but they also applied to low-charge and / or neutral oligosaccharides. Therefore, by 15 b Alignment improves the precision and robustness of migration time, independent of polymer age and batch, and allows for the creation and use of oligosaccharide databases for peak annotation via migration time matching, without the need for additional alignment as described in US 2009 / 028895 A1.

[0400] Therefore, the method according to the invention has a highly precise and robust migration time, and can be applied much more widely, regardless of polymer age.

[0401] This improved alignment procedure can also be performed using five other oligosaccharide ladders, such as chitin, cellulose, maltose, amylopectin, and glycosaminoglycans, as well as using complex carbohydrates such as glycolipids, O-glycans, N-glycans, and lactooligosaccharides (e.g., lactose, lact-N-tetrasaccharide, lact-N-hexasaccharide, and their fucose and / or lactose extensions).

[0402] Table 5: N-glycans aligned with LIZ500 base pair ladders (LIZ 500 vs. LIZ 500), and base pair ladders improved by realignment with additional carbohydrates (LIZ 500+15). b (Comparison), and with carbohydrate standards labeled only with pyrene dye (15) (15) b The alignment precision was compared. For the samples shown in Figure 12, the root mean square error (RMSD) of citrate plasma N-glycans was calculated. Figure 12C Fifteen selected peaks were plotted. Peaks containing N-glycan groups are: 10–15 indicating neutral, 9–7 indicating single charge, 2–6 indicating double charge, and peak 1 indicating triple charge (see Hennig et al., 2016 for detailed annotations of glycan peaks). Absolute RMSDs are given, in base pair form for LIZ 500 alignment, or for LIZ 500 + 15. b and only 15 b Alignment, in units of carbohydrates (oligosaccharides).

[0403]

[0404] For this embodiment, as described in Hennig et al. 2016, human citrate plasma N-glycans were analyzed by xCGE-LIF using the dyes described herein. Briefly, citrate plasma proteins were denatured and linearized. N-glycans were released by peptidyl N-glycosidase F (PNGase F) and labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). After HILIC-SPE purification, the N-glycans were analyzed using Applied... The genetic analyzer analyzed APTS-labeled N-glycans using multiplex capillary gel electrophoresis (xCGE-LIF) with laser-induced fluorescence detection. As described in Example 3, the instrument analyzed 15 a 19, 20, 6-Me a and APTS a Perform spectral calibration.

[0405] Example 6 - Pyrene and / or acridinone-labeled carbohydrates as universal alignment standards

[0406] Current embodiments include the use of modified commercial DNA genetic analyzers 310, 3100, 3130(xl), 3730(xl), and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). However, the carbohydrate-based alignment standards presented herein can also be used in conjunction with CE / CGE and (U)HPLC (single-capillary or multi-capillary) instruments from other manufacturers.

[0407] Generally, migration time alignment of DNA fragments and carbohydrates in (x)CE / (x)CGE is currently achieved using base pair size standards (EP 2112506 A1). For this purpose, the migration times of unknown samples are aligned with co-injected base pair size standards. Good results have been shown when alignment is based on base pair size standards, but alignment of carbohydrate samples shows significant differences, as illustrated in Examples 2 and 3. This variation is even more pronounced when different standards are used:

[0408] ·instrument( Figure 17 (and Table 6)

[0409] • Experimental setup, such as field strength ( Figure 18 or operating temperature Figure 19 )

[0410] • Instrument parameters, such as capillary length ( Figure 20 ), polymer type (Figure 21), polymer usage time ( Figure 22 (and Table 6) and polymer batches (Table 6)

[0411] In this stress test, these parameters were modified, and the alignment procedures (base pairs versus carbohydrate standards) were compared. For all examples, the carbohydrate alignment procedure showed superior performance. Stable migration times were achieved for most variations, as shown in the examples with different capillary lengths. This means that by using the carbohydrate alignment procedure, a comprehensive carbohydrate database can be used, even if experimental settings, instrument parameters, or instruments are changed. This is not possible with base pair-based alignment standards.

[0412] Table 6: Comparison of alignment precision, for N-glycans aligned with the base-pair ladder LIZ500 (LIZ500 alignment), for LIZ500 base-pair ladder alignment improved by realignment with additional cross (b) carbohydrates (oligosaccharides (OS)) (LIZ500+bOS(=cross-oligosaccharides) alignment), and for carbohydrate standards labeled with additional acridinone dye (23) (23). c Improved LIZ500 base pair ladder alignment (LIZ500+23) c Alignment) and carbohydrate standards labeled only with acridinone dye (23) (23) c Alignment (only 23) c (Aligned). For example... Figure 12CAs shown, the root mean square error (RMSD) of citrate plasma N-glycans for 15 selected peaks was calculated. N-glycan groups include peaks: 10–15 represent neutral, 9–7 represent single charge, 2–6 represent double charge, and peak 1 represents triple charge (for detailed notes on glycan peaks, see Hennig et al., 2016). The calculations were performed in the base pairs used for LIZ500 alignment, in the migration time units used for LIZ500+ cross-carbohydrate realignment, and in the LIZ500+ 23... c and only 23 c The absolute RMSD is given for the only aligned carbohydrate unit. For instrumental comparison, [the following was used]. Figure 15 Data (six different instruments). For polymer batch alignment, citrate plasma N-glycans were measured within 3130 x 11 using four different POP7 polymer batches (batches: 1612560, 1701565, 1703117, and 1705571). For polymer aging comparison, citrate plasma N-glycans were measured within 3130 x 11 using fresh polymer (batch number: 1708574), freshly opened one-year-old polymer (batch number: 1411512), one-year-old opened polymer (batch number: 1411512), and five-year-old opened polymer (batch number: 1208456). Based on all comparison cases, RMSD values ​​could be reduced by a factor of 5 (from 10.697 to 2.172), and by a maximum of 7 (from 2.246 to 0.334).

[0413]

[0414] Example 7 - Recalibration of a DNA sequencer using the novel fluorescent acridinone and pyrene dye set according to the present invention

[0415] Commercial capillary electrophoresis systems may have a multi-wavelength detector, and therefore several color channels.

[0416] These systems contain so-called "virtual filters," in which software defines specific wavelength ranges for collecting fluorescence emissions from different dyes.

[0417] These regions are called virtual filters. Each of them is associated with a relatively narrow range of visible light emitted by only one type of dye. Figure 23 The main dataset from the DNA sequencer has four colored traces. Figure 23This corresponds to four nucleotides. In fact, any number of dummy filters can be used, as a filter is simply a software-specified location on a simple CCD array. Since the emission spectrum of a dye is always quite broad, a portion of it is recorded by dummy filters, rather than by filters designed to collect its maximum emission. Dyes in each group are selected in such a way that they have widely spaced emission maximums to minimize overlap of emission spectra on the CCD array. However, spectral overlap still occurs to some extent, and some crosstalk is always present. On the other hand, each position in a DNA sequence contains only one of the four nucleotides, and during sequencing, each nucleotide is detected in its "own" color channel. Therefore, the problem of crosstalk is far less significant for DNA sequencing than for glycan analysis, because the four lanes of DNA sequencing contain peaks of similar intensity, and only one color trace has a prominent peak at a given position.

[0418] Importantly, the emission of the APTS dye and its conjugate with the glycan always occurs in the channel with the shortest wavelength and is completely free of crosstalk with the reference channel; this is crucial. After APTS labeling, the electrophoresis pattern of the complex glycan mixture contains peaks with intensities varying by orders of magnitude. Therefore, the fluorescence signal in the APTS channel must be completely free from any emission “leakage” from the reference channel. The reference sample contains a mixture labeled with another fluorescent dye and is injected simultaneously with the analytical sample. The requirement of “complete” crosstalk between the observation channel (APTS dye or its alternative) and the reference channel seems easy to meet, but it is not, as both dyes must be excited by the same light source and their emission spectra must overlap. So far, in the 655 nm observation channel, the LIZ dye (attached to the “DNA ladder” and used as an internal alignment standard in glycan analysis) has been used as an additional color. To detect the LIZ dye, the virtual filter group G5 (including 6-Fam) was used on the ABI 3100 DNA sequencer (ABI User Manual). TM , NED TM , and This dye consists of a FRET pair of a donor dye and an acceptor dye. This combination (similar to dyes with very large Stokes shifts) provides no crosstalk because the donor dye is efficiently excited by green light, transferring energy to the acceptor, which emits only red light. However, FRET pairs with complete energy transfer, multiple negative charges, and an aromatic amino group are too complex to synthesize easily. Therefore, this invention provides fluorescent dyes with extended Stokes shifts. As an alternative to internal comparison standards, these dyes do not emit in the APTS (observation) channel.

[0419] To eliminate crosstalk with the APTS channel, it is necessary to recalibrate the commercial DNA sequencer (manufactured by Applied Biosystems) using a different set of fluorescent dyes. According to the manufacturer, any number of (various) virtual filters (observation windows) are available. Therefore, new detection channels can be specified. For example, the emission maximum values ​​of five arbitrary fluorescent dyes define five (new) detection windows (filters). To minimize crosstalk, the maximum absorption of the new reference dye must be more or less uniformly distributed in the range of 500 nm to 655 nm. The "crosstalk" (overlap) between emission colors on the CCD array is corrected by a matrix file in the software. This process is well-known and is referred to as "linear unmixing" (T. Zimmermann, et al., Methods Mol. Biol. 2014, 1075, 129-148).

[0420] The matrix file is generated by a separate "matrix" run, in which a reference dye or its derivative is separated into individual peaks by capillary electrophoresis, and its emission spectrum is recorded across the entire spectral range. The matrix file contains information about the emission light from a single dye input falling onto a specific filter (detected within a specific observation window). For each filter (detection window), the input of one dye is the largest, but there are also components from other dyes "contaminating" the entire signal passing through that particular filter.

[0421] exist Figure 25 The image shows a comparison of dyes 8-H (aminopyrene triphosphate) and APTS (aminopyrene trisulfate). The maltose ladder with added (spiked-in) APTS labeling (for both samples) provides a temporal orientation. Although the m / z ratio of 8-H (144) is lower than that of APTS (151), the retention time of 8-H is higher than that of APTS. In APTS, the charged group (sulfonic acid residue) is directly attached to the fluorophore. The presence of the N-methyl-N-(2-hydroxyethyl) linker in 8-H increases the hydrodynamic ratio of the dye, which explains the higher retention time of the free dye 8-H.

[0422] Figure 26 A magnified view of the 8-H and APTS peaks is shown. This image was obtained before spectral calibration. The 8-H peak is associated with the APTS color channel (522 nm). Figure 26 Due to strong crosstalk from the black dye in A, dye 8-H cannot be used with APTS in any analytical assay. Figure 27The same applies to the pyrene triphosphate dye 15 shown and the acridinone diphosphate dyes 6-Me and 6-H shown in Figure 30. Therefore, it is necessary to perform new color calibration on the DNA sequencer to reduce or, as far as possible, eliminate crosstalk between emission channels caused by APTS and pyrene triphosphate dyes 6-H, 6-Me, or 8-H and 15.

[0423] To this end, negatively charged fluorescent dyes 19, 20, 6-R, and 15 (see below) were selected and used in conjunction with APTS in a new combination for spectral calibration of the electrophoresis unit integrated into the DNA sequencing device. Using these dyes, a new matrix file can be generated and used to correct spectral overlap.

[0424]

[0425] Table 7 shows the properties of fluorescent dyes (including Rhodamine 19 and 20 (see K. Kolmakov, et al., Chem. Eur. J. 2012, 18, 12986-12998 and K. Kolmakov, et al., Chem. Eur. Journal. 2013, 20, 146-157.), 6-R, and 15) and their conjugates with oligosaccharides composed of maltose units. Notably, the retention time of the dye 8-H conjugate with maltose (13.1 min) is much shorter than that of the APTS derivative obtained from maltotetrasaccharide (16.5 min). Although the hydrodynamic ratios of dyes 8-H and 15 are greater than those of APTS, the presence of six negative charges in these dyes (relative to three in APTS) strongly increases their electrophoretic mobility in an electric field.

[0426] Table 7. Properties of fluorescent dyes 6-R, 15, 19, 20 and 23 used in the new combination for spectral calibration of the fluorescence detection unit for integration into DNA sequencing devices, together with APTS.

[0427]

[0428] a--N-alkylated dyes conjugated with carbohydrates and / or substituted with amino groups shift the absorption and emission bands by approximately 20 nm into the red spectral region (see Table 1). b--Retention (migration) time in the additional color channel where the dye exhibits maximum emission, as measured in a gel at pH 8. c--Conjugates of the 8-H dye exhibit large crosstalk between the 522 and 544 nm channels.

[0429] In fact, on the one hand, comparing the maximum emission values ​​of the color channels in Figure 24 and Table 7 yields very similar conclusions. Only the maximum emission value of the "575nm channel" shows a slight difference, while the difference for the "595nm channel" is even smaller. The term "575nm channel" is used to define this value. Figure 27 and 28 The new emission band is very wide. The emission maximum of the "new 595nm channel" is slightly redshifted (from 595nm to approximately 607nm). However, these minor differences are sufficient to completely eliminate any crosstalk.

[0430] To obtain the color traces depicted in Figure 29, five new virtual filters were set up in the DNA sequencer (Table 3). The shortest wavelength channel corresponds to all APTS conjugates (522 nm), the next corresponds to the emission maximum of the pyrene 15-maltotriose conjugate (554 nm; applicable to all conjugates of dye 15), "green" corresponds to all conjugates of acridinone dyes 6-H and 6-Me with reducing sugars (575 nm), and another corresponds to the emission maximum of free dye 20 (595 nm). Figure 4 And finally, the "red" channel (655nm) is selected based on the emission of dye 19; Figure 4 This selection eliminates any crosstalk between the APTS channel (522nm) and the 554nm channel, as well as between the APTS channel (522nm) and the 575nm (green) channel (see Figures 29 and 31).

[0431] Figure 29A Figures A and B show electrophoretic plots of conjugates obtained from a mixture of carbohydrates (“Dextran 1000” (A) and “Dextran 5000” (B) ladders) and dye 15; “1000” and “5000” correspond to the average molecular weight of the dextran oligomers. The time difference between peaks is approximately 1 minute. In the case of APTS, the time difference between peaks is approximately 2.3 minutes (see Figure 1). Figure 25 The addition of glucose units leads to an increase in migration time that is approximately the same as that of maltose units. If the fluorescent dye is used to generate a new mixture of internal standards, a smaller time difference between peaks is more advantageous.

[0432] Figure 30A Figures B and B show electrophoretic images of the conjugates (reductive amination products) obtained from maltotriose and dyes 6-H (A) and 6-Me (B) before color calibration. For both 6-H and 6-Me dyes, the crosstalk between the APTS channel (522 nm) and the "595 nm channel" (also applicable to 6-H and 6-Me) is quite small; less than 15 for dyes (…). Figure 27For dye 6-H, the crosstalk is approximately 7.8%, and for dye 6-Me- it is approximately 3.4%. However, even small amounts of crosstalk between the standard and observation channels are undesirable, as it can lead to false positives (for analytes not present).

[0433] Figure 31A Figures A and B show electrophoretic maps of conjugates obtained from the “Dextran 1000” (A) and “Dextran 5000” (B) steps and dye 6-Me after spectral calibration (see Example 3). The new color calibration is based on the use of dyes 6-H and 6-Me with maltotriose conjugates. Their spectral properties are very similar to those of their conjugates. There is no crosstalk between the APTS channel (522 nm) and the new “575 nm” channel.

[0434] For dyes 6-Me (and 6-H), the interpeak time difference is approximately 1.5 minutes, corresponding to the four negative charges on the dye residues. The right side of Figure 31 shows peaks with migration times up to 60 minutes or longer; these suggest that dyes 6-Me (and 6-H; data are similar and therefore not shown) may be better than APTS. Figure 25 ).

[0435] literature

[0436] Feng HT, et al., Electrophoresis (2017) 38, 1788-1799. doi: 10.1002 / elps.201600404. Epub 2017 May 11.

[0437] Hennig R, et al., Biochimica et Biophysica Acta-General Subjects 2016, 1860, 1728-1738.

[0438] Hennig R, et al., Methods Molecular Biology 2015, 1331, 123-143.

[0439] Meininger M, et al., Journal of Chromatography B 2016, 1012, 193-203.

[0440] Reusch D, et al., MAbs. 2015, 7, 167-179. doi: 10.4161 / 19420862.2014.986000.

[0441] Ruhaak LR, Hitoshi, Journal of Proteome Research 2010, 9, 6655-6664.

Claims

1. A method for automated determination and / or identification of carbohydrate and / or carbohydrate mixture composition pattern analysis comprising the steps of: a) obtaining a sample containing at least one carbohydrate; b) labeling said carbohydrate with a first fluorescent label; c) providing a standard of known composition labeled with a second fluorescent label; d) determining the migration / retention time of said carbohydrate and the standard of known composition using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection; e) aligning the migration / retention time with the migration / retention time index based on the given standard migration / retention time index of the standard; f) comparing the migration / retention time index of the carbohydrate with the standard migration / retention time index from a database; g) identifying or determining the carbohydrate and / or carbohydrate mixture composition pattern, wherein the standard composition is added to a sample containing an unknown carbohydrate and / or carbohydrate mixture composition, the first fluorescent label and the second fluorescent label are different, and wherein the first fluorescent label or the second fluorescent label is a fluorescent dye selected from the group consisting of the following compounds: wherein, n＝0-12 R 1 and / or R 2 are independent of each other and can be represented as: H, CH3, C2H5, straight-chain or branched C3-C 12 alkyl, or substituted C2-C 12 alkyl; or R 1 ---R 2 forming a quaternary, pentary, hexary or heptary non-aromatic carbocycle, which carbocycle bears an additional primary amino group NH2, secondary amino group NHR a , or tertiary amino group N(R a )2, connected to one carbon atom of the ring; = C1-C6 alkyl, or hydroxy OH; hydroxyalkyl (CH2) m OH, with m = 1-12, with a linear or branched alkyl chain; or R 1 ---R 2 form a four-, five-, six- or seven-membered non-aromatic heterocyclic ring, the additional heteroatom O, N or S being included in the heterocyclic ring; or R 1 or at least one of the R 2 groups is hydroxyalkyl (CH2) m OH, where m = 2-6, with a straight or branched alkyl chain; or R 1 or R 2 is (CH2) n COOR 3 where n = 1-12, R 3 = H, CH2CN, 2-nitrophenyl and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentafluorophenyl, pentachlorophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1- oxypbenzotriazole, and any alkyl chain in (CH2) n may be straight or branched; or R 1 or at least one of the R 2 groups is a carbonate or carbamate derivative (CH2) m OCOOR 4 or COOR 4 where m = 1-12 and R 4 = methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1- oxypbenzotriazole phenyl or substituted phenyl, or R 1 or R 2 at least one of the groups R m NR a R b wherein m = 1-12, with a straight or branched alkyl chain; R a , R b are independent of each other and can be H, or optionally substituted C1-C4 alkyl; or R 1 or R 2 is an alkyl azide group (CH2) m N3, where m = 2-6, and has a straight or branched alkyl chain; or R 1 or R 2 is (CH2) n COOR 5 where n = 1-5, with a linear or branched alkyl group (CH2) n R 5 is selected from H, a linear or branched C1-C6 alkyl group, CH2CN, 2-nitrophenyl and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, sulfo-N-succinimidyl, N-succinimidyl or 1-oxobenzotriazolyl; or R 1 or R 2 is (CH2) n CONHR 6 where n = 1-12, R 6 = H, C1-C6 alkyl, (CH2) m N3, (CH2) m -N-maleimide, (CH2) m -NHCOCH2X, where X = Br or I, m = 2-6, and (CH2) n and R 6 is a straight or branched alkyl chain; or R 1 or at least one of R 2 represents CH2-C6H4-NH2, COC6H4-NH2, CONHC6H4-NH2or CSNHC6H4-NH2, wherein C6H4is 1,2-, 1,3- or 1,4-phenylene, COC5H3N-NH2or CH2-C5H3N-NH2, wherein C5H3N is pyridine-2,4-diyl, pyridine-2,5-diyl, pyridine-2,6-diyl or pyridine-3,5-diyl; or R 1 or at least one of the groups R 2 is a primary amino group to form an arylhydrazine Ar-NR 7 NH2, wherein Ar is the entire pyrene residue in the formula C and R 7 = H or alkyl; or R 1 or at least one of the groups R 2 is hydroxyl to form an aryl hydroxylamine Ar-NR 8 OH, wherein Ar is the entire pyrene residue in formula C and R 8 = H or alkyl; or R 1 or one of the R 2 groups contains a terminal alkoxyamino (CH2) n group with n = 1-12, which is connected via one or more alkylamino (CH2) m groups in all possible combinations with m = 0-12; or NH or alkylamido (CH2) m groups; and CONH, alkyl ether or alkyl ester groups; and (CH2) between the SO2 fragment and the residue X in formula C n -CH2linker is a straight chain, branched or cyclic group having 2-6 carbon atoms, wherein n = 1-5; X = SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a , wherein R a = optionally substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)R a , wherein R a = optionally substituted C1-C4 alkyl; But the condition is that in all compounds represented by formula C, in basic conditions, i.e. 7 < pH < 14, there are three or six negative charge groups in the residue X, and these negative charge groups represent the residues of at least partially deprotonated ionizable groups selected from the group consisting of: SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)R a , wherein R a = C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)R a , wherein R a = C1-C4 alkyl or substituted C1-C4 and the compounds of formula C can exist and be used as salts, solvates and hydrates.

2. The method according to claim 1, wherein the standard of known composition is a standard base pair ladder and / or a known carbohydrate mixture composition.

3. The method of claim 1, wherein, R 1 and / or R 2 independently of one another and can be represented as linear or branched C3-C6alkyl, or substituted C2-C6alkyl.

4. The method of claim 1, wherein, R 1 and / or R 2 are independent of each other and can be represented as: (CH2) n COOR 3 where n = 1-5, R 3 = H, CH2CN, 2-nitrophenyl and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentafluorophenyl, pentachlorophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1- oxypbenzotriazole, and the alkyl chain in any (CH2) n may be straight or branched.

5. The method of claim 1, wherein, R 4 are 2-nitrophenyl and 4-nitrophenyl, pentachlorophenyl, pentaf luorophenyl, 2,3,5,6-tetrafluoro-phenyl, 2-pyridyl or 4-pyridyl.

6. The method of claim 1, wherein, R 1 or at least one of the groups R 2 is (CH2) m NR a R b with m = 2-6, having a straight-chain or branched alkyl chain, R a , R b are independent of each other and can be H, or optionally substituted C1-C4 alkyl.

7. The method of claim 1, wherein, The salts are salts with alkali metal cations including Na + , Li + , K + and organic ammonium or phosphonium cations.

8. The method of claim 1, wherein, R 1 and / or R 2 are independent of each other and can be represented as: (CH2) n CONHR 6 where n = 1-5, R 6 = H, C1-C6 alkyl, (CH2) m N3, (CH2) m -N-maleimide, (CH2) m -NHCOCH2X, X = Br, I, where m = 2-6, in (CH2) n and R 6 have straight or branched alkyl chains.

9. The method of claim 1, wherein, The method adds at least two orthogonal standards to the sample and orthogonally cross aligns based on the given standard migration / retention time index of the at least two orthogonal standards.

10. The method of claim 1, wherein, The sample contains a mixture of carbohydrates.

11. The method of claim 1, wherein, The sample is an extract of glycans and the method allows identification of the glycosylation pattern profile.

12. The method of claim 1, wherein, The method identifies the glycosylation pattern of a glycoprotein.

13. The method of claim 1, wherein, The constituents of the carbohydrate mixture are quantitatively determined.

14. The method of claim 1, wherein, The compound of formula C is at least one of 15, 23 or 23b:

15. The method of claim 1, wherein the compound of Formula C is selected from: or a compound of 13a, 13b, 16, 18, 23 and 23b or a salt thereof.

16. A method for automated carbohydrate mixture composition pattern analysis comprising the steps of: a) providing a first sample containing a first carbohydrate mixture composition; b) labeling said first carbohydrate mixture composition with a first fluorescent label; c) providing a second sample containing a second carbohydrate mixture composition, a second fluorescent label can optionally be added to the second sample; d) generating electropherograms / chromatograms of the carbohydrate mixture composition of the first and second sample using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection; e) analyzing the identity and / or differences between the carbohydrate mixture composition pattern profile of the first and second sample; wherein the first fluorescent label of the first sample is different from the second fluorescent label of the second sample, and wherein at least one of the first fluorescent label and the second fluorescent label is a fluorescent dye as defined in claim 1.

17. A method for automated analysis of carbohydrate mixture composition patterns according to claim 16, comprising the steps of: a) providing a first sample containing a first carbohydrate mixture composition; b) labeling said first carbohydrate mixture composition with a first fluorescent label; c) providing a second sample labeled with a second fluorescent label containing a second carbohydrate mixture composition to be compared; d) using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection to generate electropherograms / chromatograms of the carbohydrate mixture compositions of the first and second sample; e) comparing the standard migration / retention time indices calculated from the obtained electropherograms / chromatograms of the first and second sample; f) analyzing the identity and / or differences between the carbohydrate mixture composition pattern profiles of the first and second sample, wherein the standard migration / retention time indices of the carbohydrates present in the first and second sample are calculated based on an internal standard of known composition labeled with a third fluorescent label, and wherein one of the first or second fluorescent label is a fluorescent dye as defined in claim 1.

18. A kit for determining and / or identifying carbohydrate mixture composition patterns comprising (i) a data processing unit having a non-transitory memory, said memory containing a database containing aligned migration / retention times and / or aligned migration / retention time indices of carbohydrates, said migration / retention times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification of carbohydrate and / or carbohydrate mixture composition pattern analysis, including the steps of: a) obtaining a sample containing at least one carbohydrate; b) labeling said carbohydrate with a first fluorescent label; c) providing a standard of known composition labeled with a second fluorescent label; d) determining the migration / retention times of said carbohydrate and the standard of known composition using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection; e) aligning the migration / retention times with the migration / retention time indices according to the given standard migration / retention time indices of the standard; f) comparing the migration / retention time indices of these carbohydrates with the standard migration / retention time indices in the database; g) identifying or determining the carbohydrate and / or carbohydrate mixture composition pattern, wherein the standard composition is added to a sample containing an unknown carbohydrate mixture composition, the first fluorescent label and the second fluorescent label are different, wherein the first fluorescent label or the second fluorescent label is a fluorescent dye as defined in claim 1 formula C; and (ii) a fluorescent dye as defined in claim 1.

19. The kit according to claim 18, further comprising a capillary gel electrophoresis-laser induced fluorescence device.

20. The kit of claim 19, wherein wherein the capillary gel electrophoresis-laser induced fluorescence device is a capillary DNA sequencer. wherein the capillary gel electrophoresis-laser induced fluorescence device is a capillary DNA sequencer.

21. A kit for automated carbohydrate mixture composition pattern analysis comprising (i) a data processing unit having a non-transitory memory, said memory containing a database containing aligned migration / retention times and / or aligned migration / retention time indices of carbohydrates, said migration / retention times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification of carbohydrate and / or carbohydrate mixture composition pattern analysis, automated carbohydrate mixture composition pattern analysis comprising the following steps: a) providing a first sample containing an unknown carbohydrate mixture composition; b) labeling said carbohydrate mixture composition with a first fluorescent label; c) adding to said first sample a second sample labeled with a second fluorescent label having a known carbohydrate mixture composition pattern; d) generating electropherograms / chromatograms of the carbohydrate mixture composition of said first and second sample using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection; e) analyzing the identity and / or difference of the carbohydrate mixture composition pattern analysis of the first and second sample; wherein the first fluorescent label of the first sample is different from the second fluorescent label of the second sample, and wherein at least one of the first fluorescent label and the second fluorescent label is a fluorescent dye as defined in claim 1; and (ii) a fluorescent dye as defined in claim 1.

22. The kit according to claim 21, further comprising a capillary gel electrophoresis-laser induced fluorescence device.

23. The kit of claim 22, wherein wherein the capillary gel electrophoresis-laser induced fluorescence device is a capillary DNA sequencer.

24. A system for determining and / or identifying a carbohydrate mixture composition pattern comprising (i) a data processing unit having a non-transitory memory, said memory containing a database containing aligned migration / retention times and / or aligned migration / retention time indices of carbohydrates, said migration / retention times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification of carbohydrate and / or carbohydrate mixture composition pattern analysis, determining and / or identifying a carbohydrate mixture composition pattern comprising the following steps: a) obtaining a sample containing at least one carbohydrate; b) labeling said carbohydrate with a first fluorescent label; c) providing a standard of known composition labeled with a second fluorescent label; d) determining the migration / retention times of said carbohydrate and the standard of known composition using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection; e) aligning the migration / retention times with the migration / retention time indices according to the given standard migration / retention time indices of the standard; f) comparing the migration / retention time indices of these carbohydrates with the standard migration / retention time indices in the database; g) identifying or determining the carbohydrate and / or carbohydrate mixture composition pattern, wherein a standard composition is added to a sample containing an unknown carbohydrate mixture composition, the first fluorescent label and the second fluorescent label are different, wherein the first fluorescent label or the second fluorescent label is a fluorescent dye as defined in claim 1 formula C; and (ii) a fluorescent dye as defined in claim 1.

25. The system of claim 24, further comprising a capillary gel electrophoresis-laser induced fluorescence device.

26. A system for automated carbohydrate mixture composition pattern analysis comprising (i) a data processing unit having a non-transitory memory, said memory containing a database, said database containing aligned migration / retention times and / or aligned migration / retention time indices of carbohydrates, said migration / retention times and / or migration / retention time indices being obtained by automated determination and / or identification of carbohydrates, and / or identification of carbohydrate and / or carbohydrate mixture composition pattern analysis, automated carbohydrate mixture composition pattern analysis comprising the following steps: a) providing a first sample containing an unknown carbohydrate mixture composition; b) labeling said carbohydrate mixture composition with a first fluorescent label; c) adding to said first sample a second sample having a known carbohydrate mixture composition pattern labeled with a second fluorescent label; d) generating electropherograms / chromatograms of the carbohydrate mixture composition of said first and second sample using electrokinetic / chromatographic separation techniques in combination with fluorescence or laser induced fluorescence detection; e) analyzing the identity and / or differences of the carbohydrate mixture composition pattern analysis of the first and second sample; wherein the first fluorescent label of the first sample is different from the second fluorescent label of the second sample, and wherein at least one of the first fluorescent label and the second fluorescent label is a fluorescent dye as defined in claim 1 ; and (ii) a fluorescent dye as defined in claim 1.

27. The system of claim 26, further comprising a capillary gel electrophoresis-laser induced fluorescence device.

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