Tags for carbohydrate analysis

Abstract

Fluorophore-containing compounds for use as fluorescent tags for the detection or analysis of carbohydrates are provided. Also described are linkers for the preparation of the fluorescent compounds, methods of preparing and using the fluorescent compounds, and carbohydrate conjugates formed from the compounds.

Description

Tags for carbohydrate analysis

[0001] The present invention relates to compounds comprising a fluorophore which find use as fluorescent tags for the detection or analysis of carbohydrates. The invention further relates to the preparation of the fluorescent tags from precursor compounds, methods of using the fluorescent tags, and conjugates formed from the tags.BACKGROUND

[0002] Chemical labeling of carbohydrate-containing substrates with fluorescent tags is known as a highly efficient approach for maximizing both sensitivity and selectivity for their analyses. Analysis of labelled carbohydrates may be conducted with chromatography or electrophoresis. Notably, to use tags in electrophoresis, the tags require one or more ionizable groups. There are very few known carbohydrate tags which are suitable for a high-performance electrophoresis. Specifically, fluorophores with net charges larger than -3 are commercially unavailable.

[0003] Gel electrophoresis in particular is the best method for separating proteins, carbohydrates, and biomolecules based on their charges and masses. A known limitation of gel electrophoresis is that the speed of separation is limited by the melting temperature of the gel as an increase in current also increases resistive heating. It is therefore desirable to improve efficiency in electrophoretic techniques by increasing the electrophoretic mobility of the fluorophore. The latter depends on the net charge of the molecule and on the number of ionizable groups, respectively.

[0004] The current library of fluorescent glycan tags is known to have poor variety in colour and emission wavelengths. This can make the distinction of carbohydrates labelled with different tags difficult. Common green emitting tags are 3-aminopyrene-1 ,6,8-trisulfonic acid (APTS), 8-aminonaphthalene-1 ,3,6-trisulfonic acid (ANTS) and 5-aminofluorescein. ANTS has a non-optimal maximum absorption, and 5-aminofluorescein no ionizable groups. In electrophoresis-based analyses with fluorescence detection APTS is used almost exclusively

[0005] Known blue-emitting dyes are 2-aminobenzoic acid and 2-aminobenzamide. Known yellow emitting tags include 2-aminoacridone, dansyl hydrazine and nitrobenzoxadiazolyl hydrazine, but these have no ionizable groups. The dye “Lucifer yellow (LY)” does have ionizable groups, but its extinction coefficient is low, and its absorbance is non-optimal for conventional lasers. 2-Aminoacridone also suffers from the same drawbacks.

[0006] Some modified pyrene-containing glycan tags with net charges larger than -3 were recently disclosed ([1] E. A. Savicheva, et al., Angew. Chem. Int. Ed. 2021 , 60, 3720; [2] E. A. Savicheva, et al., Angew. Chem. Int. Ed. 2020, 59, 5505; [3] W02020151804A1). However, they all have the core of APTS with exactly same positions of the substituents and of the glycan-reactive amino group. Therefore, a possible spectral shift relative to the parent fluorophore is still very limited. Also, the extinction coefficient remains the same as in APTS, and it is quite low compared to that in fluorophores of other classes, e.g., rhodamine or cyanine dyes. The same drawbacks are also evident in the acridine dyes with multiple negatively charged groups ([4] M. A. Fomin, et al., Anal. Chem., 2020 92 (7), 5329-5336). No attempts to modify or improve the glycan-reactive group in those pyrene compounds in order to facilitate and accelerate glycan labeling are disclosed. As seen from these sources, the labeling recipes with those modified dyes are lengthy, performed in two steps, andinvolve prolonged incubation and freeze-drying. Finally, pyrene- and acridone-containing dyes have an inherently broad fluorescence maximum, which makes the use of dye pairs problematic.

[0007] There remains a need for fluorescent tags for use in the detection and analysis of carbohydrates (glycans). In particular, there is a need for tags which provide one or more of the following benefits: high- yielding and irreversible labelling, fast and highly selective for glycans under mild conditions, with a simple workup and short reaction times; an extended and potentially unlimited spectral range and a broader range of instruments suitable for analysis; high fluorescence quantum yields; high extinction and fluorescence signal under excitation with conventional light sources; tailored electrophoretic mobility; and narrow fluorescence maxima to provide pairs of fluorophores with zero crosstalk. Particularly, there is a need for tags that improve and facilitate the labelling of glycan analytes.

[0008] The present invention has been devised with these issues in mind.BRIEF SUMMARY OF THE DISCLOSURE

[0009] According to a first aspect of the invention, there is provided a compound of general formula (I):wherein:Ring A is a 3-10 membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L2 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH-, -N(R2)(R3), -O-, -S-, - S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F,-N(R2)-, -N(R2R3), -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O- C(O)NH-, -O-C(O)NH2or Z;Z is an ionizable group;R1 is selected from H, 2-, 3-, or 4-aminophenyl, NH2, PG and NH-PG wherein PG is a protective group, or R1 is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl;R2 and R3 are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, -CH2-;FL comprises a fluorophore; and m is an integer of from 0 to 12, or a salt or hydrate thereof.

[0010] According to a second aspect of the invention, there is provided a conjugate formed from a compound of formula (I) and at least one carbohydrate moiety.

[0011] According to a third aspect of the invention, there is provided a compound of general formula (IV):wherein:Ring A is a 3- to 10-membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L5 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -NH2, -NH2+, -C(O)NH-, -C(S)NH-, -S(O)2NH-, - N(R2)(RS), -O-, -S-, -S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, -N(R2Rs), -NH2, -C(O)R2-, -CONH-, - CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;R1 is selected from H, NH2, 2-, 3-, or 4-nitrophenyl, PG and NH-PG wherein PG is a protective group, optionally wherein the protective group is selected from t-butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9- fluorenylmethoxycarbonyl (Fmoc);R2 and R3 are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-;Z is an ionizable group; m is an integer of from 0 to 12; andQ is selected from OH, H, H+, Br, Cl, F, I, CO2H, CO2LG, (CH2)jCO2LG , CO(CH2)jCO2LG and (CH2)jOCO2LG , wherein j is an integer of from 1 to 12, and LG is a leaving group selected from N- succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.

[0012] According to a fourth aspect of the invention, there is provided a method of preparing a compound of formula (I), the method comprising reacting a compound comprising a fluorophore with a compound of general formula (IV).

[0013] According to a fifth aspect of the invention, there is provided a method of forming a conjugate, the method comprising reacting a non-fluorescent organic dye with a compound of general formula (IV).

[0014] According to a sixth aspect of the invention, there is provided the use of a compound of formula (I) for the detection or analysis of an analyte comprising at least one carbohydrate moiety. Optionally, the carbohydrate moiety comprises a glycosylamine.

[0015] In a further aspect, the invention provides a method for detecting and / or analysing a carbohydrate that may be present in a sample, the method comprising: a) labeling the sample(s) (e.g. the sample(s) containing carbohydrate(s) of unknown composition(s) / structure(s)) and standard(s) (e.g. the standard(s) containing carbohydrate(s) of known composition(s) / structure(s)) with two or more spectrally different compounds, wherein at least one of the spectrally different compounds comprises a compound of Formula (I) according to claim 1 , or any one of claims 3 to 15 when dependent on claim 1 , so as to provide labelled sample(s) and labelled standard(s); b) mixing the labelled sample(s) and the labelled standard(s) to obtain a mixture; c) analysing the mixture using an electrokinetic separation method (e.g. CE or CGE) or a chromatographic separation method, so as to determine the migration times or the retention times of the labelled carbohydrates in the sample(s) and in those in the standard(s); and d) using the determined migration or retention times of the labelled standard(s) to detect and / or identify the carbohydrate(s) in the sample(s).

[0016] According to a further aspect of the invention, there is provided a kit comprising at least one compound of Formula (I) or a compound of Formula (IV), and instructions for use.

[0017] According to another aspect of the invention, there is provided a compound according to the formula:wherein Rh is

[0018] Embodiments of the invention will now be described by way of example and with reference to the accompanying figures in which:

[0019] Figures 1 a and 1 b show fluorescence spectra of maltotriose (MF3, an exemplary single glycan) labeled with APTS (3-Aminopyrene-1 ,6,8-trisulfonic acid, the predominantly used green-emitting glycan tag) and with Compound 3a (orange emitting). Spectra recorded at pH 8 (TEAB buffer). A zero crosstalk with the APTS detection channel is thus demonstrated. The fluorescence maximum of the conjugates is 516 and 580 nm, respectively. Another pair of maltotriose conjugates with zero crosstalk is also shown (Figure 1 b). Both labels belong to the current invention: Compound 9d (fl. max. 553 nm, yellow-emitting) and Compound 19 (fl. max. 666 nm, red-emitting). Differently to APTS, the fluorescence maxima of the new labels with linkers do not show noticeable shifts after their conjugation to glycans;

[0020] Figure 2 is a CGE-LIF electropherogram of an individual glycan -- maltotriose (MF3) labeled with Compound 3a. Obtained on a genetic analyser with multi-channel detection. An exceptional purity and a negligible crosstalk with the APTS detection channel (522 nm) are demonstrated;

[0021] Figure 3a is a chromatogram from a HPLC monitoring of a glycan labeling reaction with Compound 4a and maltotriose (MF3). The fluorescence detector set to 575 nm (emission) and 550 nm (excitation). The reductive amination reached the conversion of ca. 90 % in 1 hour at 45°C with exceptional purity. Data obtained on a C-18 column with aqueous TEAB buffer and acetonitrile as the mobile phase. Analysis load ~0.05 nmol of the label;

[0022] Figure 3b a chromatogram from a HPLC monitoring of a glycan labeling reaction with Compound 4k and maltotriose (MF3). The fluorescence detector set to 575 nm (emission) and 550 nm (excitation). The reductive amination reached the conversion of ca. 60%) in 1 hour at 45°C with exceptional purity. Data obtained on a C-18 column with aqueous TEAB buffer and acetonitrile as the mobile phase. Analysis load ~0.05 nmol of the label;

[0023] Figure 4 is a CGE-LIF electropherogram of a maltose ladder labelled with Compound 4a. Analysis performed with a genetic analyser and multi-channel detection. Resolution and distribution of the glycans in the sample are demonstrated. A post-derivatization cleanup removed the excess of the unreacted tag by means of a conventional HILIC-SPE method;

[0024] Figure 5 is a CGE-LIF electropherogram of a dextran ladder labelled with a red-emitting glycan tag (Compound 18 with a net charge of -6) prepared from a known rhodamine dye and linker L 3B). Excitation with an argon laser, detection at 655 nm;

[0025] Figure 6 is a CGE-LIF electropherogram of a red-emitting Compound 18-maltohexaose conjugate in picomolar amounts obtained on a genetic analyser with multi-channel detection. Excitation with an argon laser, emission detection at 655 nm., compound load ~ 50 pmol.

[0026] Figure 7 is a HILIC HPLC chromatogram of a dextran ladder 1000 labelled with Compound 3a. Obtained on a column with a TSKgel Amide-80 solid phase and acetonitrile - aqueous formate buffer as the mobile phase. Fluorescence detection at 575 nm;

[0027] Figure 8 is an electropherogram of the fingerprint region of human IgG N-glycans labelled with Compound 4k and analysed with CGE-LIF on an Applied Biosystems™ Genetic Analyser 3130XL. Excitation with a built-in argon laser at 488 nm (partly 514 nm). Emission detected at 575 nm;

[0028] Figure 9 is an electropherogram of the fingerprint region of human IgG N-glycans labelled with Compound 3a and analysed with CGE-LEDIF on a BiOptic™ Inc. QSep100™ bioanalyser. Excitation with a built-in LED light source at 525 -- 535 nm, emission detected in the range 590 -- 625 nm with a 590 nm longpass filter installed;

[0029] Figure 10 is a CGE-LIF electropherogram of a non-reductive coupling (condensation) of maltotriose (MF3) with a hydrazine-substituted Compound 1 i. Reaction conditions: 20% aq. acetic acid as solvent at room temperature overnight. Genetic analyser, argon laser, emission detection at 575 nm. A very selective labeling with high conversion is demonstrated with no need in additional reagents for reduction;

[0030] Figure 11 is a CGE-LIF electropherogram of a dextran ladder labelled with a red-emitting glycan tag (Compound 19 with a net charge of -5) prepared from the commercial dye Sulfo-Cy-5 and linker L 2B. Excitation with an argon laser, detection at 655 nm. The new label demonstrates a sufficiently high signal, convenient migration times, and a good resolution;

[0031] Figure 12 is an exemplary CGE-LIF electropherogram ofthe fingerprint region of human IgG N-glycans labelled separately with Compound 3a or APTS (3-Aminopyrene-1 ,6,8-trisulfonic acid), respectively. A two- channel detection performed with an Applied Biosystems™ Genetic Analyser 3130XL (see also FIG. 8). Emission detected at 575 and 522 nm. The new label demonstrates a very high signal, convenient migration times, and a good resolution;

[0032] Figure 13 is a set of CGE-LIF electropherograms of several elaborated glycans: 3'-FL = 3'- Fucosyllactose,6'-SL = 6'-Sialyllactose, LNnT = Lacto-N-neo-tetraose, Gly54 = Lewis A tetraose. Compound 4a taken as a label;

[0033] Figure 14 is a comparison of electropherograms with human IgG-derived / V-glycans labelled separately with APTS, Compound 4a or Compound 3a. The same amount of the substrate (20 pg of human IgG) was labelled under the same standard conditions with an excess of the dye. Signal intensities in the two detection channels (522 nm for APTS and 575 nm for 3a and 4a) were normalized to the noise in the respective detection channel and are expressed as the signal-to-noise-ratio (SNR). Labeling with the new compounds results in much higher signal intensities (expressed in SNR) compared to that with APTS.

[0034] Figure 15a is a chromatogram of a glycan labeling with a Compound 13a, which is non-fluorescent in basic media. Absorbance detector set to 575 nm. The labeling degree reached ca. 70% in 30 min. at 45°C. Data obtained on a C-18 column with aqueous TEAB buffer (pH = 8.5) and acetonitrile as a mobile phase. Analysis load 0.1 nmol of the label. Figure 15b shows the fluorescence spectra of Compound 13a, whose fluorescence is quenched in a basic medium (TEAB buffer).DefinitionsUnless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0035] Reference herein to a “compound of the invention” is a reference to any of the compounds disclosed herein including compounds of the Formulae (I), (Ila), (lib), (He), (Illa), (lllb), (IV), (V), (VI) or a compound described in any of the Examples, or a salt, solvate, salt of a solvate, tautomer or hydrate thereof.

[0036] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Alkyl is an uncyclized chain. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n- pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0037] An “alkenyl” group is one having one or more double bonds. Examples include, but are not limited to, vinyl, 2-propenyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1 ,4-pentadienyl), ethynyl, 1- and 3- propynyl, 3-butynyl, and the higher homologs and isomers.

[0038] An “alkynyl” group is one having one or more triple bonds.

[0039] The term “alkylene,” means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2CH2-. The term “alkenylene”, means, unless otherwise stated, a divalent radical derived from an alkene. The term “alkynylene”, means, unless otherwise stated, a divalent radical derived from an alkyne. The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. Non-limiting examples of aryl groups include phenyl and naphthyl.

[0040] The term “heterocyclyl”, “heterocyclic” or “heterocycle” includes a non-aromatic saturated or partially saturated ring systems. The heterocyclyl group may be a 3-7, for example, a 4, 5 or 6 membered non-aromatic cyclic or partially saturated group comprising 1 , 2 or 3 heteroatoms independently selected from O, S and N in the ring system (in other words 1 , 2 or 3 of the atoms forming the ring system are selected from O, S and N).

[0041] By partially saturated it is meant that the ring may comprise one or two double bonds. The double bond will typically be between two carbon atoms but may be between a carbon atom and a nitrogen atom. Examples of non-aromatic saturated ring systems include piperazinyl, piperidinyl, morpholino, pyrrolidinyl, or azetidinyl.

[0042] The term “heteroaryl” and “heteroaromatic” includes an aromatic mono- or bicyclic ring incorporating one or more (for example 1-4, particularly 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur. The ring or ring system has 4n + 2 electrons in a conjugated IT system where all atoms contributing to the conjugated IT system are in the same plane.

[0043] Examples of heteroaryl and heteroaromatic groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl or heteroaromatic group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10-membered bicyclic ring, for example a bicyclic structure formed from fused five and six membered rings or two fused six membered rings.Bicyclic heteroaryl groups can be vicinally fused, i.e., where the rings are linked to each other through two adjacent carbon and / or nitrogen atoms. Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically, the heteroaryl ring will contain up to 4, for example up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general, the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

[0044] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 1 H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl and imidazo[1 ,2-b][1 ,2,4]triazinyl. Examples of heteroaryl groups comprising at least one nitrogen in a ring position include pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,3,5-triazenyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl and pteridinyl.

[0045] “Heteroaryl” or “heteroaromatic” also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur. Partially aromatic heteroaryl bicyclic ring systems can be vicinally fused, i.e., where the rings are linked to each other through two adjacent carbon and / or nitrogen atoms. Examples of partially aromatic heteroaryl groups include for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-1 ,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro-benzo[1 ,4]dioxinyl, benzo[1 ,3]dioxolyl, 2,2-dioxo-1 ,3- dihydro-2-benzothienyl, 4,5,6,7-tetrahydrobenzofuranyl, indolinyl, 1 ,2,3,4-tetrahydro-1 ,8-naphthyridinyl, 1 ,2,3,4-tetrahydropyrido[2,3-b]pyrazinyl and 3,4-dihydro-2 / 7-pyrido[3,2-b][1 ,4]oxazinyl.

[0046] Examples of five-membered heteroaryl groups include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups. Examples of six-membered heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl.

[0047] Particular examples of bicyclic heteroaryl groups containing a six-membered ring fused to a fivemembered ring include but are not limited to benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl, pyrrolopyridine, and pyrazolopyridinyl groups.

[0048] Particular examples of bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl and pteridinyl groups. The term “heteroarylene” refers to a divalent heteroaromatic group, including any divalent group derived from a heteroaromatic group described herein.

[0049] The term “-C(O)-” or “oxo” as used herein, means an oxygen that is double bonded to a carbon atom.

[0050] The term "optionally substituted" includes either groups, structures, or molecules that are substituted and those that are not substituted.

[0051] It is to be understood that reference to a “heterocyclyl” refers to a divalent moiety derived from a heterocyclic group, for example one of the heterocyclyl groups defined herein. Illustrative heterocyclyl groups which may be present in the linker group L, include but are not limited to:

[0052] Where optional substituents are chosen from “one or more” groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups, which may be the same or different. For example, “one or more optional substituents” may refer to 1 or 2 or 3 substituents (e.g., 1 substituent or 2 substituents).

[0053] Where a moiety is substituted, it may be substituted at any point on the moiety where chemically possible and consistent with atomic valency requirements. The moiety may be substituted by one or more substituents, e.g., 1 , 2, 3 or 4 substituents; optionally there are 1 or 2 substituents on a group. Where there are two or more substituents, the substituents may be the same or different.

[0054] Substituents are only present at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without undue effort which substitutions are chemically possible and which are not.

[0055] The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention. In addition, the compounds disclosed herein may exist as resonance structures, tautomers, i.e., open and closed (lactone) forms in rhodamine dyes, and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted. Ionizable groups are depicted uncharged, except the zwitter-ionic parts of the structures.

[0056] A bond terminating in a “J'r' " or “ * ” represents that the bond is connected to another atom that is not shown in the structure. A bond terminating inside a cyclic structure and not terminating at an atom of the ring structure represents that the bond may be connected to any of the atoms in the ring structure where allowed by valency.

[0057] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0058] Features, integers, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims and abstract), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and abstract), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0059] Compounds described in this specification may be isotopically-labelled (or “radio-labelled”). Accordingly, one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of radionuclides that may be incorporated include2H (also written as “D” for deuterium),3H (also written as “T” for tritium),11C,13C,14C,15O,170,180,13N,15N,18F,36CI,35S and the like. The radionuclide that is used will depend on the specific application of that radio-labelled derivative. Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

[0060] As used herein, the term “fluorophore” has its usual meaning in the art, and refers to a compound that is or comprises a chemical moiety that is capable of re-emitting light upon light excitation such that it is fluorescent.

[0061] The term “carbohydrate” in the present invention refers to all substrates that contain at least one, e.g., single or multiple, carbohydrate moiety: glycans, particularly O-glycans and N-glycans, glycoconjugates, i.e., glycoproteins, glycosylamines, glycopeptides, proteoglycans, peptidoglycans, glycolipids, GPI-anchors and lipopolysaccharides. In some embodiments, the carbohydrate comprises a glycosylamine.COMPOUNDS

[0062] The invention provides a compound of general formula I:wherein:Ring A is a 3- to 10-membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L2 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH-, -N(R2)(R3), -O-, -S-, - S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, N(R2R3), -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;Z is an ionizable group;R1 is selected from H, NH2, 2-, 3-, or 4-aminophenyl, PG and NH-PG wherein PG is a protective group, or R1 is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl;R2and R3are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-;FL comprises a fluorophore; and m is an integer of from 0 to 12, or a salt or hydrate thereof.

[0063] Ring A is a 3- to 10-membered aryl or heteroaryl monocyclic or bicyclic ring. Suitable rings include any of the aryl or heteroaryl rings disclosed herein. In some embodiments, ring A is a 4- to 7-membered or a 5- to 6-membered aryl or heteroaryl monocyclic ring. In some embodiments, ring A is a 6-membered aryl or heteroaryl ring.

[0064] In some embodiments, ring A is selected from furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl (e.g. 1 ,3,5-triazinyl), benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, benzisoquinolinyl, pyridopyrazinyl and naphthalenyl. Preferably, ring A is phenyl or triazinyl (e.g. 1 ,3,5-triazinyl).

[0065] In some embodiments, ring A is phenyl.

[0066] In some embodiments, ring A is triazinyl (e.g. 1 ,3,5-triazinyl).

[0067] In some embodiments R1 is selected from H, NH2, 2-, 3-, or 4-aminophenyl, PG and NH-PG wherein PG is a protective group. In some embodiments R1 is H. In some embodiments R1 is NH2. In some embodiments R1 is 2-, 3-, or 4-aminophenyl. In some embodiments R1 is PG. In some embodiments R1 is NH- PG.

[0068] In some embodiments, R1 is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, / V-succinimidyl (NHS), sulfo- / V-succinimidyl,1 -benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.

[0069] In some embodiments PG is selected from the group consisting of t-butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9-fluorenylmethoxycarbonyl (Fmoc).

[0070] Li and L2 are independently absent or selected from: a bond (or absent), alkylene (e.g. C1-C12 alkylene), alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene (e.g. C1-C12 alkenylene), alkynylene (e.g. C1-C12 alkynylene), -C(O)-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH-, -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -NR2-, NR2R3-, -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O- C(O)NH-, -O-C(O)NH2or Z.

[0071] In some embodiments, Li is absent or is selected from a bond, alkylene (e.g. C1-C12, Ci-Cs, C1-C6 or C1-C4 alkylene, such as methylene or ethylene) and -N(R2)(R3). In some embodiments, Li is absent. In some embodiments, Li is a bond. In some embodiments, Li is NR2R3, optionally wherein R2 and R3 are each alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce or C1-C4 alkylene, such as methylene or ethylene), further optionally wherein R2 and R3 are each ethylene.

[0072] In some embodiments, Li is -N(R2)(Rs) wherein R2 is H and R3 is alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce alkylene), optionally tertiary alkylene.

[0073] Thus, in some embodiments, Li has the structure:wherein n is an integer of from 1 to 12, from 1 to 6, or from 1 to 4, e.g. 2.

[0074] In some embodiments, Li has the structure:wherein n is an integer of from 1 to 12, from 1 to 6, or from 1 to 4, e.g. 2.

[0075] In some embodiments, L2 is absent or is selected from: a bond, alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce or C1-C4 alkylene), heterocyclyl, -NH-, -C(O)-, -C(O)NH-, -C(S)NH-, -O-, and -S-, optionally wherein the alkylene or heterocyclyl is substituted with one or more substituents independently selected from -F, -NR2-, -NH2, - NR2R3-, -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O- C(O)NH2or Z.

[0076] In some embodiments, L2 is alkylene (e.g. C1-C12, Ci-Cs, C1-C6 or C1-C4 alkylene), optionally wherein the alkylene is substituted with one or more -NR2-. In such embodiments, each R2 may be independently selected from H, Ci-Ce alkyl (e.g. methyl) or Ci-Ce w-oxyalkyl, optionally oxyethyl (-(CH2)2OH). For example, each R2 may be methyl.

[0077] In some embodiments L2 is: -NH-, alkylene (e.g. C1-C12 alkylene), -C(O)-, -S(O)2-, -C(O)NH-, - S(O)2NH-, -S-, heterocyclyl or heteroarylene, wherein the heterocycle or heteroarylene contain at least one heteroatom independently selected from N, O and S, optionally wherein the heterocyclyl or heteroarylene is piperidyl, piperazinyl or triazinyl. In some embodiments the piperidyl, piperazinyl or triazinyl is substituted with one or more substituents selected from NR2, -NR2R3-, C(O)R2, and Z.

[0078] In some embodiments, L2 is triazinyl is substituted with one or more substituents selected from -NH-, Z and -NR2R3-, wherein R2 and R3 are each alkylene (e.g. C1-C12, Ci-Cs, C1-C6 or C1-C4 alkylene) substituted with Z.

[0079] In some embodiments, L2 is piperazinyl, optionally wherein the piperazinyl is substituted with -C(O)R2-, further optionally wherein R2 is alkylene (e.g. C1-C12, Ci-Cs, C1-C6 or C1-C4 alkylene).

[0080] R2 and R3 are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F. In some embodiments, R2 and R3 are not both H.

[0081] In some embodiments, R2 and R3 are independently selected from H and C1-C12 alkylene, with the proviso that R2 and R3 are not both H. In some embodiments, R2 and R3 are independently selected from Ci- 012 alkylene, e.g. Ci-Cs, C1-C6 or C1-C4 alkylene. In some embodiments, both R2 and R3 are C1-C4 alkylene, preferably ethylene.

[0082] In some embodiments, R2 is H and R3 is alkylene (e.g. C1-C12, Ci-Cs, C1-C6 alkylene), optionally wherein R3 is tertiary alkylene.

[0083] X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene (e.g. C1-C12 alkylene) and alkenylene (e.g. C1-C12 alkenylene). In some embodiments, X is a bond. In some embodiments, X is -NH-. In some embodiments, X is -C(O)-. In some embodiments, X is alkylene, such as C1-C12 alkylene, Ci-Cs alkylene, or C1-C6 alkylene. In some embodiments, X is methylene. In some embodiments, X is ethylene. In some embodiments, X is alkenylene, such as C1-C12 alkenylene, Ci-Cs alkenylene or C1-C6 alkenylene. In some embodiments, X is ethenylene. In some embodiments, X is -S(O)2-. In some embodiments, X is -S-.

[0084] In some embodiments, X and L2 are not both a bond (i.e. only one of X and L2 is a bond, or neither X nor l_2 is a bond).

[0085] Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-. In some embodiments, Y is absent. In some embodiments, Y is a bond. In some embodiments, Y is -S(O)2-.

[0086] Z is an ionizable group. In some embodiments, each Z is independently selected from -Cl, -NH2, -OH, -SO3H, -OSO3H, -SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2. As ionizable groups, sulfonic acid (SO3H)and phosphate (OP(O)(OH)2) groups are preferable, as they are charged in a wide range of pH and synthetically most feasible.

[0087] m is an integer of from 0 to 12. It may be that m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, m is 0, 1 , 2 or 3. In some embodiments m is zero. Thus, it may be that Z is absent. In some embodiments, m is 1 . In some embodiments, m is 2. In some embodiments, m is 3.

[0088] In some embodiments, group -Y-Li-Z has a structure selected from: -N((CH2)PSO3H)2; - N((CH2)pCO2H)2; -N((CH2)pOP(O)(OH)2)2; -SO3H; -CO2H; -S(O)2-N((CH2)pOP(O)(OH)2)2; -C(O)-N((CH2)POP(O)(OH)2)2; -S(O)2-N((CH2)PCO2H)2; -C(O)-N((CH2)pCO2H))2; -S(O)2-NHC(CH2OP(O)(OH)2)3 and - C(O)-NHC(CH2OP(O)(OH)2)3, wherein p is an integer of from 1 to 12, e.g. from 1 to 8, from 2 to 6 or from 3 to 4, optionally wherein p is 2.

[0089] In some embodiments, group X-L2 comprises or has a structure selected from:(i) a bond; (ii) -NH-; (iii) -(CH2)c-, wherein c is an integer of from 1 to 6 or from 2 to 4, optionally wherein c is 1 or 2; (iv) -C(O)NH-; (v) -NH-C(O)-NH-; (vi) -NH-C(S)-NH-; (vii) -S(O)2NH-; (viii) -S-;(ix) -S-[CH2]d-S-, wherein d is an integer of from 1 to 6 or from 2 to 4, optionally wherein d is 2 or 3;wherein a is 1 , 2 or 3 and b is 0, 1 , 2 or 3, optionally wherein a is 1 and b is 2;wherein R14 is:- Z, optionally wherein Z is Cl or OH; or- NR2R3, optionally wherein R2 and R3 are both C1-C4 alkylene substituted with Z, further optionally wherein Z is SO3H;wherein f is an integer of from 1 to 12 (e.g. from 1 to 8 or from 2 to 6), optionally wherein f is 1 or 2, and R2 are independently selected H, C1-C6 alkyl (e.g. methyl) or C1-C6 oxyalkyl, optionally oxyethyl (-(CH2)2OH). In some embodiments, f is 1 and each R2 is methyl;(xiii)wherein g is an integer of from 1 to 12 (e.g. from 1 to 8 or from 2 to 6), optionally wherein g is 1 or 2.

[0090] The FL moiety is or comprises a fluorophore. Many suitable fluorophores will be known to those skilled in the art. In some embodiments, FL is or comprises xanthene, carbopyronine, naphthalimide, coumarin, acridine, acridone (e.g. 2-aminoacridone), oxazine, benzoxazole, dipyrromethene, rhodamine (e. g., rhodamine B, rhodamine 110, rhodamine 101 , rhodamine 6G, TMR, Si- or Ge-rhodamine), fluorescein, triarylmethane, dipyrromethene, pyrene, APTS, ANTS, dansyl, 7-nitrobenzo-2-oxa-1 ,3-diazole, squaraine, Lucifer Yellow or a cyanine (e.g. C3-C9 cyanine).

[0091] In some embodiments, FL is not, or does not comprise, fluorescein.

[0092] In some embodiments of Formula (I): Ring A is phenyl; Y and Li are absent; m is 0 (i.e. Z is absent); L2 and X are independently selected from a bond, NH and CO; and R1 is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, N- succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.

[0093] In some embodiments of Formula (I), the compound has the structure:

[0094] In some embodiments, FL comprises or has a structure according to Formula (Ila):wherein:R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H, q1 , q2 and q3 are independently selected from 0, 1 , 2, 3 or 4; each l_4 is independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, -CO-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH- -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, - CO2H, -O-C(O)NH-, and -O-;R2 is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F, and OH;Y2 is selected from a bond, -NH-, -C(O)-, and -C(S)-; andZ is an ionizable group, as defined herein.

[0095] In some embodiments, FL comprises or has a structure according to Formula (lib):wherein: each R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN, and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H; q1 , q2 and q3 are independently selected from 0, 1 , 2, 3 or 4;each l_4 is independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, -CO-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH- -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, - CO2H, -O-C(O)NH-, and -O-;R2is selected from H, alkyl and oxyalkyl; each X2is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH; andZ is an ionizable group, as defined herein.

[0096] In some embodiments, FL has a structure according to Formula (Illa):wherein: each R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN, and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H; q3 is selected from 0, 1 , 2, 3 or 4; each L is independently selected from: a bond, alkylene (e.g. C1-C12 alkylene), alkylarylene, arylalkylene, arylene, alkenylene (e.g. C1-C12 alkenylene), alkynylene (e.g. C1-C12 alkynylene), -CO-, -NH-, -C(O)NH-, - C(S)NH-, -S(O)2NH- -N(R2)(RS), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, and -O-, wherein R2is selected from H, alkyl and oxyalkyl;each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH;Z is an ionizable group, as defined herein;Y2 is selected from a bond, -NH- -C(O)-, and -C(S)-;R10, R11, R12 and R13 are independently selected from H, alkyl or SOsH, wherein said alkyl group is optionally substituted with F or SOsH, or wherein R10 and Rn, and / or Ri2 and R13, together with the atoms to which they are attached, form a ring; and each r1 and r2 is independently selected from 0 and 1 .

[0097] In some embodiments of Formula (Ila), (lib) and (Illa) each Z is independently selected from SO3H, OSO3H, SO2NHCN, CO2H, OP(O)(OH)2and P(O)(OH)2. In some embodiments of Formula (Ila), (lib), (He) and (Illa) each Z is independently selected from SO3H and OP(O)(OH)2.

[0098] In some embodiments of Formula (Ila), (lib) and (Illa), each l_4 is independently selected from H and alkylene, such as C1-C12 alkylene, preferably C1-C4 alkylene (e.g. C2-C3 alkylene).

[0099] In some embodiments of Formula (Ila), (lib) and (Illa), each X2 is independently selected from H and Z.

[0100] In some embodiments of Formula (Ila) and (lib), q1 and q2 are independently selected from 0 and 1 . In some embodiments, q1 and q2 are both 0.

[0101] In embodiments of Formula (Ila), (lib) and (Illa), q3 is 0, 1 , 2, 3 or 4. In some embodiments, q3 is 0. In some embodiments, q3 is 1. In some embodiments, q3 is 2. In some embodiments, q3 is 3. In some embodiments, q3 is 2. In some embodiments, q3 is 4.

[0102] In some embodiments of Formula (Ila), (lib) and (Illa), Y2 is a bond. In some embodiments of Formula (Ila), (lib) and (Illa), Y2is -C(O)-.

[0103] In some embodiments of Formula (Ila), (lib) and (Illa), each R4 is selected from F, CO2H and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2.

[0104] In some embodiments of Formula (Ila), (lib) and (Illa), q3 is 1 and R4 is CO2H.

[0105] In some embodiments of Formula (Ila) and (Illa), q3 is 1 , R4 is CO2H and Y2 is a bond.

[0106] In some embodiments of Formula (Ila) and (Illa), q3 is 4, each R4 is selected from F, CO2H and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and Y2 is a bond.

[0107] In some embodiments of Formula (Ila) and (Illa), q3 is 0 and Y2 is -C(O)-.

[0108] In some embodiments of Formula (lib), q3 is 0.

[0109] In some embodiments of Formula (Ila), (lib) and (Illa), at least one R4 is C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl (e.g. C1-12 or Ci-6 alkyl), carbocycle, heterocycle (e.g. morpholine, piperidine, piperazine) or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from OH, CO2H and SO3H.

[0110] In some embodiments of Formula (Ila), (lib) and (Illa), R5 and Re are independently selected from H and alkyl (e.g. C1-12 alkyl, preferably C1-4 alkyl). For example, in some embodiments of Formula Illa, each R5 and Re is alkyl, preferably C1-4 alkyl, in particular methyl.

[0111] In some embodiments of Formula Illa, both r1 and r2 are 1.

[0112] In some embodiments of Formula Illa, R10, Rn, R12 and R13 are independently selected from H andC1-12 alkyl, or C1-6 alkyl. In some embodiments, R10, Rn, R12 and R13 are independently selected from H and C1-C4 alkyl, preferably methyl. In some embodiments, R10 and R13 are C1-C4 alkyl (e.g. methyl), and Rn and R12 are H.

[0113] In some embodiments, R10 and Rn, and / or R12 and R13, together with the atoms to which they are attached, form a ring. The ring may be a 5- or a 6-membered ring, preferably a 6-membered ring. In some embodiments, R10 and Rn, together with the atoms to which they are attached, form a ring, and Ri2 and R13, together with the atoms to which they are attached, also form a ring.

[0114] In some embodiments, FL comprises or consists of a moiety selected from structures a to u:

[0115] In some embodiments, FL comprises or is:

[0117] In some embodiments, FL is not:

[0119] In some embodiments, FL is not:

[0121] In some embodiments, FL is not:

[0122] In some embodiments, FL is not:

[0123] In some embodiments, FL is not:

[0124] In some embodiments, FL is not:

[0125] In some embodiments, FL is not:

[0126] In some embodiments, a compound of the invention has a structure selected from:

[0127] The principal approach of the invention is the installation of an aminoaryl or hydrazino aryl group or their hetero aryl analogs on fluorophores. The aminoaryl or hydrazino aryl group is glycan-reactive. The decoration of fluorophores with such a linker accelerates and facilitates glycan labeling to a very large extent, 1as seen in Table 2. The fluorophores can conveniently be chosen from a wide variety of dyes with required spectral properties and suitable functional groups. This gives glycan tags of a potentially unlimited spectral range, with tailored properties, and with no need in further chemical modifications. The attachment of an aminoaryl or hydrazino aryl moiety, optionally decorated with multiple ionizable groups, can be efficiently achieved by means of two synthetic strategies. Both strategies utilize custom-synthesized linkers and, as a result, give a compound of general Formula I.

[0128] The present invention thus provides a range of carbohydrate fluorescent tags which have tuneable net charge, m / z ratio and electrophoretic mobility.

[0129] The compounds of Formula (I) according to the invention advantageously have one or more (in some cases, all) of the following properties:1) high-yielding, fast and highly selective conjugation to glycans under mild conditions,2) the possibility of non-reductive conjugation, where the bleaching of the fluorophore is excluded3) extended spectral range,4) high fluorescence quantum yields,5) high extinction under excitation with conventional light sources,6) higher fluorescence signal as compared to the predominantly used labels,7) tailored electrophoretic mobility and8) narrow fluorescence maxima to provide pairs of fluorophore pairs with a zero crosstalk.

[0130] These features enable high-throughput and highly sensitive chromatographic and / or electrokinetic analyses of carbohydrates.

[0131] At the same time, the compounds of the invention provide a “pallet” of electrophoretically mobile carbohydrate-reactive fluorophores with limited or zero crosstalk with each other and / or with the predominantly used green-emitting tags, e.g. APTS.

[0132] Compounds of the invention advantageously can provide higher degrees of carbohydrate labeling, higher labeling velocity, higher extinction, higher fluorescence signal and / or higher net charges of the conjugates, as compared to those with conventional labels.

[0133] Conveniently, the compounds of the invention may be used for a wide variety of analytical methods, especially those based on chromatography and electrophoresis, in particular in multiplexed systems and / or multi-channel detection.

[0134] The invention further provides a conjugate formed from a compound of Formula (I) and a compound comprising at least one carbohydrate moiety.

[0135] Scheme 1 , below, provides an example of a conjugation product of a hydrazine-substituted fluorophore. Importantly, in this particular case, no reductive reagents are required, so possible bleaching of the fluorophore is completely avoided.Scheme 1 : Exemplary conjugation product of an individual glycan and a fluorophore decorated with a hydrazine-substituted linker. See also example in Figure 10.

[0136] The compound comprising at least one carbohydrate moiety may be a monosaccharide (e.g. xylose, arabinose, glucose, galactose, mannose, fructose), a disaccharide (e.g. lactose, sucrose, maltose), a homo- or hetero-oligosaccharide (e.g. a galactooligosaccharide (GOS), a fructooligosaccharide (FOS), a milk oligosaccharide (MOS)), fucose, N-acetylglucosamine, N-acetylgalactosamine, a homo- or heteropolysaccharide (e.g. amylose, amylopectin, cellulose, starch glycogen), or a glycoconjugate (e.g. a glycosaminoglycan (GAG), a glycosylamine, a glycoprotein, a glycopeptide, a proteoglycan, a peptidoglycan, a glycolipid, a GPI-anchor, or a lipopolysaccharide).

[0137] The present invention also provides a compound of general formula (IV):wherein:Ring A is a 3- to 10-membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L5 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -NH2, -NH2+, -C(O)NH-, -C(S)NH-, -S(O)2NH-, - N(R2)(RS), -O-, -S-, -S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, -NH2, NFV, N(R2Rs), -C(O)R2-, -CONH-, - CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;R1 is selected from H, NH2, 2-, 3-, and 4-nitrophenyl, PG and NH-PG wherein PG is a protective group;R2 and R3 are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-;Z is an ionizable group; m is an integer of from 0 to 12; andQ is selected from OH, H, H2+, Br, Cl, F, I , C(O)OH, C(O)O)LG, (CH2)jCO2LG , CO(CH2)jC(O)OLG or (CH2)jOC(O)OLG , wherein j is an integer of from 1 to 12, and LG is a leaving group selected from N- succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.

[0138] The compounds of Formula (IV) may be referred to herein as “linkers”. Advantageously, the linkers of this invention can be coupled to a wide variety of commercially available fluorophores. This provides fluorescent tags of a potentially unlimited spectral range, with tailored properties, and with no need for further chemical modifications.

[0139] In some embodiments of Formula (IV), ring A is selected from furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl (e.g. 1 ,3,5 triazinyl), benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, benzisoquinolinyl, pyridopyrazinyl and naphthlenyl.

[0140] Preferably, ring A is phenyl or triazinyl. In some embodiments, ring A is phenyl. In some embodiments, ring A is triazinyl (e.g. 1 ,3,5 triazinyl).

[0141] In some embodiments of Formula (IV), R1 is selected from H, NH2, 2-, 3-, or 4-nitrophenyl, PG and NH-PG wherein PG is a protective group. In some embodiments R1 is H. In some embodiments R1 is NH2. In some embodiments, R1 is 2-, 3-, or 4-nitrophenyl, In some embodiments R1 is PG. In some embodiments R1 is NH-PG.

[0142] In some embodiments of Formula (IV), PG is selected from the group consisting of t-butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9-fluorenylmethoxycarbonyl (Fmoc).

[0143] In some embodiments, R1 is NH-tBoc.

[0144] In some embodiments of Formula (IV), Li is selected from a bond, alkylene (e.g. C1-C12, Ci-Cs, C2-C6 or C3-C4 alkylene) and -N(R2)(R3). In some embodiments, Li is NR2R3 wherein R2 and R3 are each alkylene (e.g. C1-C12 alkylene), optionally ethylene. In some embodiments, Li is NR2R3 wherein R2 is H and R3 is alkylene, optionally tertiary alkylene.

[0145] In some embodiments of Formula (IV), Li has the structurewherein n is an integer of from 1 to 12 (e.g. from 1 to 6).

[0146] In some embodiments of Formula (IV), L5 is selected from a bond, alkylene (e.g. C1-C12, Ci-C3, C1-C6 or C1-C4 alkylene) and heterocyclyl (e.g. piperazinyl, piperidinyl or morpholinyl), optionally where the alkylene or heterocyclyl are substituted with one or more substituents independently selected from -F, -N(R2)-, -NH2, N(R2R3), -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O- C(O)NH2or Z.

[0147] In some embodiments, Q is H, and L5 is selected from piperazinyl, piperidinyl, alkylenediamine and Ci-Ce alkylene, optionally wherein the piperazinyl, piperidinyl, alkylenediamine or Ci-Ce alkylene is substituted with one or more substituents independently selected from S, N, NH, and NR2.

[0148] In some embodiments L5 is alkylene, such as C1-C12, C1-8 or C1-6 alkylene. Optionally, the alkylene is substituted with one or more substituents independently selected from -NH-, -O-, -S-, and -N(R2)-. In some embodiments, L5 is alkylene (e.g. C1-C4 alkylene or C2-C3alkylene) substituted with S. In some embodiments, l_5 is alkylene (e.g. C1-C4 alkylene or C1-C2 alkylene) substituted with one or more -N(R2)-. Each R2 may be independently selected from H, alkyl (e.g. Ci-Ce alkyl) and oxyalkyl.

[0149] In some embodiments, Ls is -N(CH3)-CH2-CH2-N(CH3)-.

[0150] In some embodiments, Ls is -CH2-CH2-CH2-S-.

[0151] In some embodiments Ls is heterocyclyl or heteroarylene, wherein the heterocycle or heteroarylene contains at least one heteroatom independently selected from N, O and S, optionally wherein the heterocyclyl or heterarylene is substituted with one or more substituents selected from NR2, -NR2R3-, C(O)R2, and Z. In some embodiments, the heterocyclyl or heterarylene is piperidyl, piperazinyl or triazinyl, optionally wherein the piperidyl, piperazinyl or triazinyl is substituted with one or more substituents selected from NR2, -NR2R3-, C(O)R2, and Z. In some embodiments, Ls is unsubstituted piperidyl. In some embodiments, Ls is unsubstituted piperazinyl. In some embodiments, Ls is unsubstituted triazinyl.

[0152] R2 and R3are independently selected from H, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F. In some embodiments, R2 and R3are not both H.

[0153] In some embodiments, R2 and R3are independently selected from C1-C12 alkylene, e.g. Ci-C3, C2-C6 or C3-C4alkylene. In some embodiments, both R2 and R3are C2-C6 alkylene, preferably ethylene.

[0154] In some embodiments of Formula (IV), X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, - SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene (e.g. C1-C12 alkylene) and alkenylene (e.g. C1-C12 alkenylene). In some embodiments, X is a bond. In some embodiments, X is -S-. In some embodiments, X is -NH-. In some embodiments, X is -C(O)-. In some embodiments, X is alkylene, such as C1-C12 alkylene, Ci-C3alkylene, or C2-C6 alkylene. In some embodiments, X is methylene. In some embodiments, X is ethylene. In someembodiments, X is alkenylene, such as C1-C12 alkenylene, Ci-Ca alkenylene or C2-C6 alkenylene. In some embodiments, X is ethenylene. In some embodiments, X is -S(O)2-.

[0155] In some embodiments of Formula (IV), X and L5 are not both a bond.

[0156] In some embodiments of Formula (IV), Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-. In some embodiments, Y is absent. In some embodiments, Y is a bond. In some embodiments, Y is -S(O)2-. In some embodiments, Y is -C(O)-.

[0157] In some embodiments of Formula (IV), each Z is independently selected from -Cl, -NH2, -OH, -SO3H, -OSO3H, -SO2NHCN, -CO2H, -OP(O)(OH)2 and -P(O)(OH)2. In some embodiments, Z is -Cl. In some embodiments, Z is -OH. In some embodiments, Z is -SO3H. In some embodiments, Z is -OP(O)(OH)2.

[0158] In embodiments of Formula (IV), m is an integer of from 0 to 12. It may be that m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, m is 0, 1 , 2 or 3. In some embodiments m is zero. Thus, it may be that Z is absent. In some embodiments, m is 1 . In some embodiments, m is 2. In some embodiments, m is 3.

[0159] In some embodiments of Formula (IV), group -Y-L1-Z has a structure selected from: -N((CH2)PSO3H)2; -N((CH2)pCO2H)2; -N((CH2)pOP(O)(OH)2)2; -SO3H; -CO2H; -S(O)2-N((CH2)pOP(O)(OH)2)2; -C(O)- N((CH2)POP(O)(OH)2)2; -S(O)2-N((CH2)PCO2H)2; -C(O)-N((CH2)pCO2H)2; -S(O)2-NHC((CH2OP(O)(OH)2)3 and - C(O)-NHC((CH2OP(O)(OH)2)3, wherein p is an integer of from 1 to 12, e.g. from 1 to 8, from 2 to 6 or from 3 to 4, optionally wherein p is 2.

[0160] In some embodiments of Formula (IV), Q is H. In some embodiments of Formula (IV), Q is Hal (e.g. Cl).

[0161] In some embodiments of Formula (IV), L5 is a bond, heterocyclyl or alkylene, optionally wherein said alkylene is substituted with -S-.

[0162] In some embodiments of Formula (IV), L5 is a bond.

[0163] In some embodiments of Formula (IV), L5 is heterocyclyl. The heterocyclyl may be piperazinyl, piperidinyl or morpholinyl. In some embodiments, L5 is piperazinyl.

[0164] In some embodiments of Formula (IV), the compound has a structure of general Formula (V):wherein: Li, L5, Q, X, Z and m are as defined above in relation to Formula (IV).

[0165] In some embodiments of Formula (V):Li is a bond or NR2R3, wherein R2 and R3 are independently selected from H and C2-C6 alkylene);Y is C(O) or S(O2);Z is OH, SH, SO2NHCN, SO3H or OP(O)(OH)2;X is a bond, -NH- or -S-; m is 1 , 2 or 3 l_5 is piperazinyl, piperidinyl, alkylenediamine, or C1-C6 alkylene (e.g. C1-C4 alkylene) substituted with one or more substituents independently selected from S and NR2, wherein R2 is independently C1-C6 alkyl (e.g. Ci- 02 alkyl); andQ is H.

[0166] In some embodiments, both R2 and R3 are C2-C6 alkylene, preferably wherein both R2 and R3 are ethylene.

[0167] In some embodiments, R2 is H and R3 is C2-C6 alkylene, optionally wherein R3 is a tertiary alkylene(e.g. tertiary butylene).

[0168] In some embodiments, the compound of Formula (V) has a structure selected from:

[0169] In some embodiments of Formula (IV), the compound has a structure of general Formula (VI):wherein:Hal is halogen (e.g. F, Cl, Br, I); andLi, Ri, Y, Z and m are as defined above in relation to Formula (IV).

[0170] In some embodiments of Formula (VI), Hal is Cl.

[0171] In some embodiments of Formula (VI), Ri is selected from H, NH2, 2-, 3-, or 4-nitrophenyl, PG and NH-PG wherein PG is a protective group. In some embodiments Ri is H. In some embodiments Ri is NH2. In some embodiments Ri is 2-, 3-, or 4-nitrophenyl (e.g. 3-nitrophenyl). In some embodiments Ri is PG. In some embodiments Ri is NH-PG.

[0172] In some embodiments of Formula (VI), PG is selected from the group consisting of t-butoxycarbonyl(t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9-fluorenylmethoxycarbonyl (Fmoc).

[0173] In some embodiments of Formula (VI), Ri is NH-tBoc.

[0174] In some embodiments of Formula (VI): (i) Y is a bond and Li is a bond; or(ii) Y is a bond and Li is -NR2R3-, wherein R2 and R3 are both alkylene, optionally ethylene.

[0175] In some embodiments of Formula (VI), -Y-Li-[Z]mhas a structure selected from:(i) -Cl; (ii) -OH; (iii) -N(CH2CH2SO3H)2; and (iv) -N(CH2CH2OP(O)(OH)2)2.

[0176] In some embodiments, the compound of Formula (VI) has a structure selected from:

[0177] In certain embodiments compounds of the invention include, for example, compounds of Formulae (I), (Ila), (lib), (He), (Illa), (lllb), (IV), (V), (VI), or a salt or hydrate thereof, wherein, unless otherwise stated, each of X, X2, R1, R2, R3, R , R5, Re, R7, Rs, R9, R10, R11, R12, R13, R14, Y, Y2, Y3, Z, L-i, L2, L3, L4, L5, m, q1 , q2, q3, FL, Q, W and PG, has any of the meanings defined hereinbefore. These statements are independent and interchangeable. In other words, any of the features described in any of the embodiments described herein may (where chemically allowable) be combined with the features described in one or more other embodiment. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the statements which describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter which is contemplated as forming part of the disclosure of this invention in this specification.PREPARATION OF COMPOUNDS

[0178] The invention also provides a method of preparing a compound of Formula (I), the method comprising reacting a fluorophore with a compound of Formula (IV), wherein the fluorophore comprises one of an electrophilic and a nucleophilic reactive centre, and group -L5-Q of Formula (IV) comprises the other of an electrophilic and a nucleophilic reactive centre.

[0179] In some embodiments, the nucleophilic reactive centre is selected from NH2, -NH- and SH.

[0180] In some embodiments, the electrophilic reactive centre comprises Hal, COHal, CO2H, CO2LG, (CH2)jCO2LG , CO(CH2)jCO2 LG or (CH2)jOCO2 LG .wherein j is an integer of from 1 to 12, Hal is halogen (e.g. Br, Cl, F or I), and LG is a leaving group selected from / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1- benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl and tetra- or pentafluorophenyl.

[0181] In some embodiments, the electrophilic reactive centre comprises Hal or COHal wherein Hal may be Br, Cl, F or I, preferably Cl.

[0182] In some embodiments, the method comprises reacting a compound of Formula (IV) with a fluorophore of Formula (lie):wherein:X2, L4, R4, R5, Re, q1 , q2 and q3 are as defined above in relation to Formula Ila; andY3 comprises a nucleophilic or an electrophilic reactive centre, as defined above.

[0183] In some embodiments, Y3 is NH2, or -C(G)CI.

[0184] In some embodiments, the method comprises reacting a compound of Formula (IV) with a fluorophore of Formula (I lib):wherein:X2, l_4, R4, R5, Re, R10, R11 , R12, R13, r1 , r2 and q3 are as defined above in relation to Formula Illa; and Y3 comprises a nucleophilic or electrophilic reactive centre, as defined above.

[0185] In some embodiments, Y3 is NH2, or -C(0)CI.

[0186] The method of preparing a compound of Formula (I) may comprise reacting the fluorophore with a compound of Formula (V) or (VI). In some embodiments, the method comprises reacting the fluorophore with a compound selected from L1A, L2A, L3A, L4A, L5A, L6A, L7A, L8A, L9A, L10A, L1 B, L2B, L3B, L4B, L5B, L6B and L7B.

[0187] In some embodiments, the method comprises reacting the fluorophore with the compound of Formula (IV), wherein the fluorophore comprises a nucleophilic reactive centre, and group -L5-Q of Formula (IV) comprises an electrophilic reactive centre. These embodiments may be referred to as “strategy A”. For example, the nucleophilic reactive centre may be NH2 and the electrophilic reactive centre may be Hal (e.g. Cl).

[0188] In some embodiments, the method comprises reacting:- a fluorophore of Formula (lie) or Formula (I II b) , wherein Y3 is NH2; with- a compound of Formula (IV), wherein L5 is a bond and Q is Hal (e.g. Br, Cl, F or I, preferably Cl).

[0189] In some embodiments, the method comprises reacting a fluorophore with a compound of Formula (VI), wherein the fluorophore comprises a nucleophilic reactive centre, and Hal of Formula (VI) comprises an electrophilic reactive centre.

[0190] In some embodiments, the method may further comprise removing a protecting group. Removing a protecting group may comprise carrying out reduction of a nitro group or deprotection of a hydrazine group. For example, in embodiments wherein the method comprises reacting a fluorophore with a compound selected from L5A, L6A, L7A, L8A, L9A and L10A, the method may further comprise reduction of the nitro group to an amine.

[0191] In some embodiments, the method comprises reacting:- a fluorophore of Formula (lie) or Formula (I II b) , wherein Y3 is NH2; with- a compound of Formula (VI), optionally wherein the compound of Formula (VI) is selected from:(i) L1A, L2A, L3A and, L4A, further optionally wherein the method further comprises cleavage of the t-Boc protective group; or(ii) L5A, L6A, L7A, L8A, L9A and L10A, further optionally wherein the method further comprises reduction of the nitro group to amine.

[0192] By way of example, the use of strategy A to prepare a compound of Formula (I) is shown in Scheme2 below. Further examples are provided in Schemes 5, 10 and 11 :Scheme 2: strategy A. *PG is a temporary protective group.

[0193] In alternative embodiments, the method comprising reacting the fluorophore with the compound of Formula (V), wherein the fluorophore comprises an electrophilic reactive centre, and group -L5-Q of Formula (V) comprises a nucleophilic reactive centre. These embodiments may be referred to as “strategy B”. For example, the nucleophilic reactive centre may be -NH and the electrophilic reactive centre may be -C(O)Hal and Hal is a halogen (e.g. Cl).

[0194] In some embodiments, the method comprises reacting:- a fluorophore of Formula (lie) or Formula (I II b) , wherein Y3 is C(O)Hal and Hal is a halogen (e.g. Cl); with- a compound of Formula (IV), wherein L5 is heterocyclyl (e.g. piperidinyl or piperazinyl) or L5 is alkylene substituted with S or N, and Q is H.

[0195] In some embodiments, the method comprises reacting:- a fluorophore of Formula (lie) or Formula (I I lb), wherein Y3 is C(O)Hal or C(O)OH and Hal is a halogen (e.g. Cl); with- a compound of Formula (V), optionally a compound selected from L1 B, L2B, L3B, L4B, L5B and L6B.

[0196] By way of example, the use of strategy B to prepare a compound of Formula (I) from the commercially available Rhodamine B acid chloride is shown in Scheme 3. Further examples are provided in Schemes 4 (Compound 13a) and 8 (Compounds 14a, c and 15 a,c).Scheme 3: strategy B. *PG is a temporary protective group. Alternatively, the nitro group is reduced to the amine after conjugation.

[0197] In a further aspect, there is provided a method of preparing a compound of Formula (I) comprising reacting an amino-substituted fluorophore with a nitro-substituted benzoic acid chloride, a nitro-substituted benzene sulfochloride, a nitro-substituted phenyl isocyanate or a nitro-substituted phenyl isothiocyanate. The method may further comprise hydrolysis of an extra chlorine-containing group. In some embodiments, the method comprises subsequent reduction of a nitro group to amine. In some embodiments the nitro-substituted benzoic acid chloride, the nitro-substituted benzene sulfochloride, the nitro-substituted phenyl isocyanate or the nitro-substituted phenyl isothiocyanate is selected from compounds L 11A to L 22A:

[0198] In some embodiments, the amino-substituted fluorophore is selected from Compounds 1a, 2a and9rh:

[0199] Such a method may be used to produce glycan labels exemplified e.g. by the following compounds:

[0200] In a further aspect, the invention provides a method of forming a conjugate, the method comprising reacting a non-fluorescent organic dye with a compound of general formula (IV).

[0201] In some embodiments, the non-fluorescent dye is selected from azo compounds, triarylmethanes, xanthenes, rhodamines, oxazines, polyenes, azomethines, anthraquinones, stilbenes, porphyrins, thiazines, naphthoquinones, alizarines, squaraines, naphthalimides, indigoid or thioindigoid dyes. In some embodiments, the method comprises reacting a non-fluorescent dye with a compound of Formula (V). In some embodiments, the method comprises reacting a non-fluorescent dye with a compound of Formula (VI).

[0202] The compounds of Formula (I) find utility in the analysis or detection of analytes, in particular analytes comprising at least one carbohydrate moiety.

[0203] Thus, in a further aspect the invention provides the use of a compound of Formula (I), or a conjugate formed by reacting a non-fluorescent organic dye with a compound of general formula (IV), in a method of analysing or detecting an analyte comprising at least one carbohydrate moiety.

[0204] The analyte comprising at least one carbohydrate moiety may be a monosaccharide (e.g. xylose, arabinose, glucose, galactose, mannose, fructose), a disaccharide (e.g. lactose, sucrose, maltose), a homo- or hetero-oligosaccharide (e.g. a galactooligosaccharide (GOS), a fructooligosaccharide (FOS), a milk oligosaccharide (MOS)), fucose, N-acetylglucosamine, N-acetylgalactosamine, a homo- or heteropolysaccharide (e.g. amylose, amylopectin, cellulose, starch glycogen), or glycoconjugate (e.g. a glycosaminoglycan (GAG), a glycosylamine, a glycoprotein, a glycopeptide, a proteoglycan, a peptidoglycan, a glycolipid, a GPI-anchor, or a lipopolysaccharide). In some embodiments, the analyte comprises a glycosylamine.

[0205] in some embodiments, the method is a chromatographic separation method.

[0206] In some embodiments, the method is an electrophoretic separation method. In some embodiments, the electrophoretic separation method is capillary electrophoresis (CE) or capillary gel electrophoresis (CGE), such as CGE with laser induced fluorescence detection (CGE-LIF).

[0207] In some embodiments, there is provided the use of the conjugate formed by reacting a non- fluorescent organic dye with a compound of general formula (IV) in a method of analysing or detecting an analyte comprising at least one carbohydrate moiety, wherein the method is an electrophoretic separation method. In some embodiments, the conjugate is used as a calibration standard.

[0208] In a further aspect, the invention provides a method for detecting and / or identifying one or more carbohydrates in a sample, the method comprising: a) labeling the sample(s) (e.g. sample(s) containing carbohydrate(s) of unknown composition(s) / structure(s)) and standard(s) (e.g. standard(s) containing carbohydrate(s) of known composition(s) / structure(s)) with two or more spectrally different compounds, wherein at least one of the spectrally different compounds comprises a compound of Formula (I) as defined herein, so as to provide labelled sample(s) and labelled standard(s); b) mixing the labelled sample(s) and the labelled standard(s) to obtain a mixture; c) analysing the mixture using an electrokinetic separation method (e.g. CE or CGE) or a chromatographic separation method, so as to determine the migration times or the retention times of the labelled carbohydrates in the sample(s) and in those in the standard(s); and d) using the determined migration or retention times of the labelled standard(s) to detect and / or identify the carbohydrate(s) in the sample(s).

[0209] In some embodiments, the standard comprises a mixture of carbohydrates of different sizes (e.g. a ladder).

[0210] In some embodiments, step (e) comprises aligning the determined migration or retention time(s) to migration or retention time indice(s) based on given standard migration or retention time indice(s) of thestandard; and comparing the migration / or retention time indice(s) of the carbohydrate(s) with standard migration / retention time indice(s) from a database.

[0211] In some embodiments, the compound of Formula (I) and the other compound(s) for labeling emit light at different wavelengths. It may be that the first compound emits light predominantly at a wavelength which is at least 50, at least 100 or at least 200 nm greater than, or less than, the emission wavelength of the second compound. For example, the carbohydrate present in the sample may be labelled with a first compound which emits light at a wavelength of X (e.g. 500 nm), while the standard is labelled with a second compound which emits light at a wavelength of X+50 (e.g. 550 nm). The labelled sample and the labelled standard may then be mixed and run down a capillary. The first and second compounds may then be excited (e.g. by light from a lamp or laser), causing the compounds to emit. The detector may be constructed so that the light emitted by the first compound can be recorded separately from the light emitted by the second compound. The compounds of the invention enable such a method to be carried out by providing glycan-reactive fluorophores with zero cross-talk. Optionally, one of the compounds is a conventional glycan label, e.g. APTS.

[0212] In a further aspect, the invention provides a kit comprising at least one compound of Formula (I) or a compound of Formula (IV), and instructions for use.

[0213] The kit may further comprise a chromatographic or electrophoretic separation apparatus. Optionally, the electrophoretic separation apparatus is CE or CGE apparatus, such as CGE-LIF apparatus. In some embodiments the CGE-LIF apparatus is a capillary DNA-sequencer. In some embodiments the chromatographic separation apparatus is a chromatography column.

[0214] The kit may further comprise one or more standards of known composition.EXAMPLESExample 1 : Compound preparation

[0215] The isomerically pure (regioselective) synthesis of amino rhodamine 1a is not trivial. It utilizes the directing influence of the nitro group in the ortho- position of 3-nitrophthalic anhydride. As a result, in the condensation of aminophenol precursor AP1 ((3, 3’-[(3-hydroxyphenyl)amino]bis-1 -propanesulfonic acid) with 3-nitrophtalic anhydride, a single rhodamine isomer is formed. The product has a nitro group in the orthoposition relative to the rhodamine carboxyl group, as shown in Scheme 4.

[0216] The directing influence of the nitro group in 3-nitrophthalic anhydride was neither explicitly mentioned nor explored with respect to the isomeric purity, even though some simple o / Yho-substituted nitro- and amino rhodamines are known (see, e.g., [5] I. Zhang, et al, RSC Adv., 2015, 5, 66416-66419; [6] Y. Wang, et al., Nat. Mater. 2019, 18, 1335-1342;

[0027] H. Kojima, et al, Anal. Chem. 2001 , 73, 1967-1973;). On the other hand, reactions of all other unsymmetrically substituted phthalic anhydrides (e.g., 4-nitrophthalic anhydride) with aminophenols always lead to a 1 :1 mixture of isomers (see, e.g., [7] M. V. Kvach, et al, Bioconjugate Chem., 2009, 20, 1673-1682.; H. Yu, Y. Xiao and H. Guo, Org. Lett. 2012, 14, 2014-2017). Also, the condensation that involves precursor AP1 with pre-installed sulfoalkyl groups requires a special approach (see Synthesis Description Section). The nitro group in Compound 1 b is then reduced to amine 1a by conventional methods. The exact same scheme is applied to the synthesis of amino rhodamine 9rh, as well.

[0217] The regioselective synthesis of amino rhodamine 2a (an isomer of 1a) utilizes the condensation of 2- formyl-5-nitrobenzoic acid with precursor AP1. The condensation is also followed by reduction of the nitro group.

[0218] An alternative regioselective synthesis of rhodamine-containing glycan tags is exemplified by amino-substituted rhodamine 13a (see Scheme 4). It utilizes a precursor 13b with active fluorine atoms and the known reaction of aromatic nucleophilic substitution in the meso- aromatic ring (see, e.g., [8] G. Yu. Mitronova, et al, J. of Org. Chem., 2015(2), 337-349). The rhodamine precursor 13b, although being a new compound, was obtained by a conventional condensation from AP1. As the tetrafluorophthalic anhydride is symmetrically substituted, only a single isomer is formed. Also importantly, the next step of the synthesis takes an advantage of the different reactivity of aryl amino and aryl thiol groups in 4-aminothiophenol. As a result, the protection of the amino groups is unnecessary, the synthesis is shorter, and the isolated reaction product 13a is already suitable for reductive amination of carbohydrates. The same applies to other exemplary rhodamine-containing tags: Compounds 14a, 14c (Scheme 8) and Compound 18 with a custom-made linker L3B (see syntheses description).Scheme 4. Isomerically pure syntheses of suitably-functionalized rhodamine dye precursors 1a, b, 2a, b and of a glycan label 13a,b.

[0219] Benzoylation of Compounds 1a and 2a with para- or meta- nitrobenzoylchlorides under Schotten- Baumann conditions in water followed by reduction to the amines gives way to Compounds 3a and 4a, as shown in Scheme 5. The fluorescence of the latter is unaffected by the pH factor in a wide range (pH 2—11), which is different to Compound 13a (see Figure 15). Apart from nitrobenzoyl chlorides, nitro-substituted benzene sulfonyl chlorides, nitrophenyl isothiocyanates and phenyl isocyanates can be used in the similar syntheses, as exemplified by Compounds 9 a,b,c,d, 10a,b,c, 4s and 4t.

[0220] Hydrazino aryl derivatives 3h and 4h can be prepared by acylation of compounds 1a and 2a usingFmoc-protected 3- or 4-hyrdazinobenzoic acids (see Scheme 5 and Syntheses Description Section).Scheme 5. Linker installation according to Strategy A: amino-benzoylation and hydrazino-benzoylation of amino rhodamines; for full structures of the compounds are provided herein.

[0221] Additionally, it is shown that amides of Compound 1a, particularly Compound 4b can undergo cyclization to form benzoxazinones (4 / 7-3, 1-benzoxazin-4-ones) and compound 4c, respectively, as shown in Scheme 6. The cyclization of 2-aminobenzoic acid amides is a known reaction (see, e.g., [9] G. M. Coppola in J. Het. Chem., 1999, 36(3), 563-588) which, however, has not been utilized for modification of organic dyes so far. The reduction of the nitro group leads to a carbohydrate-reactive Compound 4d, which, due to the cyclization of the carboxyl group, has a smaller net charge than 4a. Alternatively, the amino rhodamine 4a can be cyclized to 4d in presence of proper dehydrating agents. In a basic medium, the cyclic products are cleaved back to give Compounds 4a and 4b, respectively.Scheme 6. Reversible cyclization of the 2-amidobenzoyl moiety in a rhodamine dye. For preparation of Compounds 4a and 4b see Scheme 5.Scheme 7. Rhodamine-based glycan tags with aminobenzoyl moieties as embodiments of Strategy A. Also included are benzoxazinone derivatives (4c,d,m,n) with an extra fused heterocycle.

[0222] Red-emitting amino rhodamines with emission maxima of 600-660 nm were synthesized analogously to the dye 13a using precursors with additional fused rings. Following the same Strategy B, compounds 14a,c and 15a,c (see Scheme 8) can be obtained from a known red-emitting rhodamine precursors 14b and 15b. Remarkably, the absorbance and emission maxima of Compounds 14a and 14c are very much shifted to the red region relative to the conventional rhodamines with a free carboxyl group. Precursors 14b and 15b have two preinstalled sulfonic acid groups already, and the substitution reaction with thiols proceed quite selectively, as was demonstrated by specific examples 14a and 14c. Also, more ionizable group can be installed. The final products show an intense pH-independent fluorescence.Scheme 8. Model red-emitting carbohydrate tags 14a,c and 15a,c as simple embodiment of Strategy B.

[0223] The preferable but not necessarily required property of the compounds in the present invention is their reactivity towards carbohydrates or carbohydrate-containing analytes. Carbohydrate-containing analytes contain or form nucleophilic moieties that, in turn, react with nucleophile-reactive tags of the invention.

[0224] Isomerically pure nucleophile-reactive label with enhanced negative net charge can be prepared from compound 4a by conventional alkylation with chloroacetic acid at the NH2 site. This was followed by preparation of NHS ester at the terminal carboxyl group of the dye. The structure of the label is depicted in Scheme 9.Scheme 9. Exemplary nucleophile-reactive instant label with enhanced negative net charge and the corresponding free acid.

[0225] The synthesis of the phosphorylated Compound 7b was performed analogously to that of the sulfonated rhodamine 3a (Scheme 5). The synthesis involved the condensation of 2-formyl-5-nitrobenzoic acid with the 3-hydroxypropyl-substituted aminophenol precursor AP 3. The hydroxyl group gets acetylated in acetic acid medium and afterwards needs to be deprotected by an alkali. The resulting rhodamine dye 7a was isolated, phosphorylated at the hydroxyl sites, and then amino-benzoylated in two more steps, with reduction of the nitro compound, exactly as shown in Scheme 5 for Compound 3a. In the same fashion was performed the synthesis of Compounds 5a, b, only the precursor (AP 4) had only one 3-hydroxypropyl group. The synthesis of Compound 6b (an isomer of 5b) also involved the precursor AP 3 and is described in the Synthesis Description Section. The structures and syntheses of other phosphorylated dyes are also shown..Scheme 10. Synthesis of an exemplary phosphorylated label 7b with a net charge of -8.

[0226] Apart from nitro / amino-benzoylation, other reactions were performed on amino-substituted dye precursors 1a and 2a within the Strategy A framework. More specifically, sulfoamidation with 4-nitrobenzenesulfochloride or reacting with a custom-synthesized 3,5-dichlorotriazinyl precursors (L 1A and L 5A) were carried out, as exemplified in Scheme 11. The latter were obtained by a controlled and stepwise chlorine substitution in 1 ,3,5-trichlorotriazine with 4-nitroaniline and Boc-hydrazine, respectively. Triazinyl- substituted reactive dyes were prepared by means of a stepwise controlled nucleophilic substitution in cyanuric chloride (1 ,3,5-trichloro-s-triazine) under proper conditions. The process is well known for industrial “active” dyestuffs (see, e. g.,

[0010] Reactive triazine dyes, their preparation and use. US5420257A. BASF SE and

[0011] K. A. Kolmakov, J. Het. Chem., 2008, 45, 533-539). So were prepared, e.g., linkers L 1A and L 5A (See Scheme 11 for structures). The aromatic nitro compounds are reduced to the carbohydrate-reactive amines 9a, b and 11c, while the Boc-hydrazine-containing intermediate 1 h is deprotected in the final step to give Compound 1 i. For more derivatives (e. g., 9a, b, 11a, b,c and 12a, b,c) see Syntheses Description Section. The hydrazine-containing labels 1 i and 1j are of special importance, as they do not require reducing agents for labeling. Possible reductive bleaching of the fluorophores is thus avoided. At the same time, their molecular weights and the m / z ratios are low, which is especially favourable for electrokinetic-based methods (i.e. electrophoresis).Scheme 11. Syntheses of additional exemplary dyes according to Strategy A (see also Scheme 2).Custom-synthesized linkers with pre-installed ionizable groups and carbohydrate-reactive centres.

[0227] Synthesis of linkers with pre-installed ionizable groups start with 2-chloro-5-nitrobenzenesulfonic acid, a known compound that has a chlorine atom strongly activated by two electron donor groups. The syntheses utilize 1 ,4-pipreazine or 1 ,3-propane dithiol as bifunctional reagents and takes an advantage of the different reactivity at the nitrogen or sulfur atoms, respectively (see Scheme 12). This allows to obtain mono-N- substituted derivatives in high yields or, similarly, mono-thioethers. The aromatic halogen substitution reaction is followed by reduction of the nitro group. The latter can proceed in a one-pot fashion with an excess of the dithiol (a reducing agent) in case of linker L3. Importantly, both linkers have one highly reactive nucleophiliccentre that remains from the bifunctional reagent utilized (diamine or dithiol, respectively). The aromatic NH2 group is far less reactive towards electrophiles.Scheme 12. Synthesis of linkers (L 1 B, L 2B, L 3B) with pre-installed ionizable groups and two nucleophilic reaction centres.

[0228] To prepare a carbohydrate-reactive dye conjugate, a custom-synthesized linker (Scheme 12) needs to be reacted with a commercial or a custom-synthesized dye in its active form, particularly as an NHS ester or a dye with a reactive halogen (Scheme 3 and Syntheses Descriptions). This gave a set of linker-dye conjugates with a broad spectral “palette” and various net charges. Most of the specific compounds in the set are so shifted to the red that crosstalk with the APTS (predominantly used glycan label) detection channel (ca. 520 nm) is zero. Also, one can choose a pair of dyes with a zero crosstalk where both fall under the scope of Formula (I) according to the present invention (Figure 1).

[0229] The coupling reactions with commercial and custom-made active esters proceed in mild conditions with high yields, and products are usually easy to isolate, especially by means of HPLC. So do the reactions with dyes as acid chlorides (e.g., Rhodamine B chloride, while preparing Compounds 16 and 23) and tetrafluoro-substituted dye precursors (while preparing Compounds 13a, 14a, c, and 18 in Schemes 4 and 8). In some cases, it is preferable to utilize a linker with an aromatic nitro group and reduce the conjugation product to the amine. That was done in the preparation of Compounds 16, 17 and 23 because the aromatic amino group might react with acid chlorides and with coupling reagents, e.g., HATU.Abbreviations: HOAc - acetic acid, EtOAc - ethyl acetate, TFA - trifluoracetic acid, Et3N - triethylamine, DMF - / V, / V’-dimethylformamide, DMSO - dimethylsulfoxide, DCM - dichloromethane, TEAB - triethylammonium bicarbonate, TEAA - triethylammonium acetate, MeCN - acetonitrile, MeOH - methanol, EtOH - ethanol, HATU - O-(7-Aza Benzotriazol-1-yl)-A / ,A / ,A / ,A / -tetramethyl uronium-hexafluorophosphate, (a coupling reagent), CDI - 1 ,1-carbonyldiimidazole, Py - pyridine, HPLC - high-performance liquid chromatography, RT - room temperature, > - elevated temperature, aq. - aqueous.Aminophenol precursor AP1 ((3, 3’-[(3-hydroxyphenyl)amino]bis-1 -propanesulfonic acid).

[0230] Precursor AP 1 was prepared by sulfoalkylation of 3-aminophenol according to

[0012] N-h. Ho, R. Weissleder and C-H. Tung, Tetrahedron 2006, 62, 578-585. The compound was purified by means of an HPLC on a C-18 phase with a TEAB buffer and MeCN and used as a di-triethylammonium salt (2 x Et3N) with M = 555. The same procedure applies to the homolog AP 2, as well.Linkers L 1 A and L 2A.

[0231] To a stirred solution of cyanuric chloride (1 ,3,5-trichlorotriazine, 550 mg, 3 mmol) in MeCN (40 mL) solid Boc-hydrazine (f-butylcarbazate, 465 mg, 3.5 mmol, 1.17 equiv) and finely powdered K2HPO4 (potassium hydrogen phosphate, 1 g, 5.75 mmol, 1 .9 equiv) were added, and the stirring continued for 1 .5 h. The reaction mixture was filtered, the inorganic precipitate washed with MeCN (10 mL), and the combined solution rotary evaporated at RT until the product started to crystallize. An excess of water (50 mL) was added, and the flask was sonicated. The solid was filtered, washed with water and dried to give 840 mg (76%) of the dichlorosubstituted linker L 1A which was stored at -20°C and used as it was. MS (ESI+): mlz, % = 280 (100) [M+H]+; HRMS (C8H11CI2N5O2): 280,0361 (found M+H), 280,0368 (calc.).1H NMR (400 MHz, MeCN-d3): <5 = 1.42 (s, 9H, CH3BOC), 7.43 (s, 1 H, NH), 8.41 (s, 1 H, NH) ppm.13C NMR (102 MHz, MeCN-d3): 27.9 (CH3Boc), 81.8 (C Boc), 155.3 (CO), 168.8 (CN), 170.7 (CN), 171.7 (CN). Linkers L 2A and L 6A (see structure below) were prepared by reaction of the dichloro-substituted linkers L 1A and L 5A (see structure below), respectively, with the commercially available disodium salt of iminodiacetic acid (HN(CH2COONa)2 x H2O). The reaction was performed in presence of Et3N in aq. MeCN overnight, as described for L 3A, and the course of the reaction was monitored by HPLC. The products were isolated by means of HPLC under the same conditions with the yields of ca. 50% as 2 x Et3N salts. Purity and identity of the compounds was confirmed by LC / MS. HRMS (ESI-) of L 2A (C12H16CIN6O6): 375,0829 (found M-H), 375,0820 (calc.);1H NMR (400 MHz, D2O): <5 = 1.15 (t, 16 H, J = 7 Hz, CH3, 2Et3N), 1 .32 (s, 9H, CH3Boc), 3.07 (q, 12 H, J = 7 Hz, CH2, 2Et3N), 4.08 (s, 4H, CH2) ppm. HRMS (ESI-) of L 6A (CisHioCINeOe): 381 ,0348 (found M-H), 381 ,0350 (calc.).1H NMR (400 MHz,DMSO-d6): 6 = 1.15 (t, 16 H, J = 7 Hz, CH3, 2Et3N), 3.10 (q, 12 H, J = 7 Hz, CH2, 2Et3N), 4.11 (S, 4H, CH2), 7.76 (t, J = 8 Hz, 1 H), 8.09 (m, 1 H), 8.16 (m, 1 H), 8.75 (m, 1 H) ppm.Linker L 3A

[0232] 2,2 -Ethanolamine disulfonic acid disodium salt (2,2'-azanediyldiethanesulfonic acid disodium salt, NH(CH2CH2SO3Na)2was obtained from sodium vinylsulfonate and taurine (2-aminoethane sulfonic acid) upon boiling, as described in

[0013] by S, De, E. Groaz, et al., Org. Biomol. Chem., 2015, 13, 3950-3962. The crude product was extracted with a hot 90% aq. MeOH, the solution filtered, evaporated, and the residue recrystallized from MeOH / EtOAc to give ca. 50% of the required compound (2 x Na salt, M = 263).1H NMR (400 MHz, D2O): <5 = 1 .46 (s, 9H, CH3), 3.01 - 3.08 (m, 4H, CH2N), 3.14 - 3.22 (m, 4H, CH2SO3) ppm. This disodium salt (80 mg, 0.30 mmol, 1.4 equiv) was dissolved in 3 mL of a 50% vol. aq. MeCN containing Et3N (40 pl, 0.30 mmol, 1 .4 equiv). Linker L 1 A (60 mg, 0.21 mmol) was added to the solution, which was left stirring overnight and evaporated at t < 35°C. The target compound was isolated with a yield of ca. 50% (2 x Et3N salt, M = 678) by means of a gradient HPLC on a C-18 phase with aq. TEAB buffer and MeCN. Properties of linker L 3A: MS (ESI+): m / z, % = 475 (100) [M-H] ; HRMS (CI2H2ICIN6O8S2): 475,0479 (found M-H), 475,0473 (calc.).1H NMR (400 MHz, D2O): <5 = 1.15 (t, 18 H, J = 7 Hz, CH3, 2Et3N), 1.34 (s, 9H, CH3), 3.07 (q, 12 H, J = 7 Hz, CH2, 2Et3N), 3.16 - 3.20 (m, 4 H, CH2), 3.74 - 3.94 (m, 4 H, CH2SO3) ppm.13C NMR (102 MHz, D2O): 8.1 (CH3, Et3N), 27.4 (CH3BOC), 46.7 (CH2, Et3N), 83.0 (C Boc), 158.2 (CO), 164.0 (m, CN), 166.5 (CN), 168.6 (CN).Linker L 4A

[0233] 2,2-Ethanolamine diphosphoric acid “EDAP” NH(CH2CH2OPO3H)2was prepared according to

[0014] Xh. Liu, et al., Cellulose, 2018, 25, 6745-6758. This compound was reacted with L 1A in presence of Et3N in aq. MeCN as described for L 3A, and the product isolated by means of HPLC under the same conditions with the yield of ca. 40% (2 x Et3N salt, M = 710). MS (ESI-): m / z, % = 507 (100) [M-H]-; HRMS (CI2H23CIN6OIOP2): 507,0491 (found M-H), 507,0561 (calc.).1H NMR (400 MHz, D2O): 6 = 1 .12 (t, 18 H, J = 7 Hz, CH3, 2Et3N), 1.31 (s, 9H, CH3), 3.07 (q, 12 H, J = 7 Hz, CH2, 2Et3N), 3.28 (m, 4 H, CH2N), 3.82 (m, 4 H, CH2O) ppm.31P NMR (162 MHz, D2O): <5 = +0.40 ppm.Linkers L 5A, L 6A and L 7A

[0234] 1.150 g (6.25 mmol, 1.4 equiv) cyanuric chloride was dissolved in glacial HAc (15 mL) and a solution of 0.60 g (4.35 mmol) 3-nitroaniline in a mixture of HAc (17 mL) and water (17 mL) was added upon stirring, which continued for 1 h. The precipitate was separated, well-washed with water and dried to give 896 mg (72%) of the dichloro-substituted linker L 5A which was stored at +5°C and used as it was.1H NMR (400 MHz, acetone-d6): 6 = 7.72 (t, J = 8 Hz, 1 H), 8.05 (m, 1 H), 8.12 (m, 1 H), 8.73 (m, 1 H), ppm.13C NMR (102 MHz, acetone-d6): 116.5 (C), 120.1 (C), 127.7 (C), 131.0 (C), 139.2 (C), 149.4 (C), 165.4 (CN), 170.8 (CN), 171.5 (CN). Linker L 7A was prepared analogously to L 3A and isolated with the yield of 55%. Its purity and identitty was confirmed by1H NMR (400 MHz, D2O): <5 = 1.12 (t, 18 H, J = 7 Hz, CH3, 2Et3N), 3.10 (q, 12 H, J = 7 Hz, CH2, 2Et3N), 3.18 - 3.22 (m, 4 H, CH2), 3.76 - 3.99 (m, 4 H, CH2SO3), 7.76 (t, J = 8 Hz, 1 H), 8.27 (m, 1 H), 8.38 (m, 1 H), 8.79 (m, 1 H), ppm.

[0235] Linker L 8A (2-nitrophenyl derivative) was prepared following exact same scheme as for L 7A starting from 2-nitroaniline and isolated with the yield of ca. 40% over two steps. Its purity and identitty was confirmed by analytical methods.Linker precursor A 01

[0236] 2-Chloro-5-nitrobenzenesulfonic acid was obtained by conventional method that involved sulfonation of chlorobebzene. The product was recryctallized, dried and converted to the corresponding sulfochloride with SOCI2. For amidation (see Scheme 12) , the sulfochloride (1.50g) was dissolved in dry MeCN (20 mL) and added to a chilled and stirred solution of diethanolamine (1 g) and Na2CO3(1g) in water (20 mL). The stirring continued for 20 min at +5°C and then overnight at RT. Ethyl acetate (50 mL) was added, and the organic layer evaporated. Recrystallization from an ethyl acetate-cyclohexane mixture gave ca. 1.10 g (~50%) of the sulfamide A 01. MS (ESI-): m / z, % = 323 (100) [M-H]-; HRMS (CI0HI3CIN2O6S): 323,0101 (found M-H), 323,0105 (calc.).1H NMR (400 MHz, DMSO-d6): <5 = 3.40 - 3.44 (m, 4 H, CH2O), 3.48-3.52 (m, 4 H, CH2N), 4.75 (t, J = 5 Hz, 2 H, 2OH), 7.95 (d, J = 9 Hz, 1 H), 8.41 , 8.43 (dd, J = 3 and 9 Hz, 1 H), 8.68 (d, J = 3 Hz, 1 H) ppm.Phosphorylated linker precursor A 02

[0237] To a stirred solution of A 01 (220 mg, 0.68 mmol) and pyridine (0.21 mL, 2.6 mmol, 4.0 equiv) in dry dioxane (30 mL) a neat POCh (0.27 mL, 2.9 mmol, 4.3 equiv) was added at RT, the flask sealed, flushed with nitrogen, and left stirring for 30 min. Ice cold water (30 mL) and EtaN (6 mL) were added, the mixture was wellshaken and left stirring overnight. Then more water (30 mL) was added, the solution extracted 2 times with a mixture of EtOAc (20 mL) and cyclohexane (10 mL), the aq. phase acidified with HOAc (0.1 mL), filtered, and subjected to a gradient HPLC on a C-18 phase with a TEAA buffer (pH 4.5) and MeCN, so that the less polar cyclic side-product was separated. Repeated freeze-drying furnished 220 mg of A 02 (48%) as a colourless amorphous material (2 x EtaN salt with M = 686).

[0238] MS (ESI-): m / z, % = 483 (100) [M-H]-; HRMS (CIOHI5CIN2OI2P2S): 482,9412 (found M-H), 482,9431 (calc.).1H NMR (400 MHz, D2O): 6 = 1 .18 (t, J = 7 Hz, 27 H, CH3, 3 Et3N), 3.20 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.77 (m, 4 H, CH2O), 3.96 (m, 4 H, CH2N), 7.90 (d, J = 8 Hz, 1 H), 8.44, 8.46 (dd, J = 3 and 8 Hz, 1 H), 8.87 (m, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.10 ppm.Linkers L 1 B-NO2and L 1 B

[0239] 2-Chloro-5-nitrobenzenesulphonic acid (100 mg, 0.42 mmol) was reacted with 1 ,4-piperazine (anhydrous, 300 mg, 3.5 mmol, 8 equiv) in water (1 mL) in 1 h at 90°C. The excess of the piperazine was sublimated under reduced pressure as completely as possible, and the residue subjected to a gradient HPLC with aq. TEAB buffer and MeCN. That gave 72 mg of linker L IB-NO2 (60% theor.) as a yellow solid. MS (ESI-): m / z, % = 286 (100) [M-H]-; HRMS (C10H13N3O5S): 286,0493 (found M-H), 286,0498 (calc.).1H NMR (400 MHz, DMSO-d6): <5 = 3.25 (m, 4H, 2CH2), 3.67 (m, 4H, 2CH2), 7.10 (d, J = 9 Hz, 1 H), 8.08, 8.11 (dd, J = 3 and 9 Hz, 1 H), 8.63 (br. M, 1 H) 8.66 (d, J = 3 Hz, 1 H) ppm. The nitro compound L I B-NO2 was reduced with an aq. ammonium sulfide (30 - 60 equiv) solution at 95°C as described for Compound 1a and other similar precursors. The reaction mixture was treated in the same fashion. Linker L 1 B was isolated as a colourless solid with a yield of ca. 70% by means of a gradient HPLC as described above. MS (ESI-): m / z, % = 256 (100) [M-H]-; HRMS (C10H15N3O3S): 256,0750 (found M-H), 256,0756 (calc.).1H NMR (400 MHz, MeOH-d4): <5 = 3.13 (m, 4H, 2CH2), 3.33 (m, 4H, 2CH2), 6.77, 6.79 (dd, J = 3 and 9 Hz, 1 H), 7.08 (d, J = 9 Hz, 1 H), 7.34 (d, J = 3 Hz, 1 H) ppm.Linkers L 2B-NO2and L 2B

[0240] 1 ,4-Piperazine (anhydrous, 240 mg, 3 mmol) was added to a solution of the phosphorylated precursor A 02 (50 mg, 0.1 mmol, see Scheme 12) in water (0.5 ml), the solution was stirred overnight at 45°C, diluted with water (30 mL) and evaporated two times (with a fresh portion of water) as completely as possible at t < 40°C. The solid residue was subjected to a gradient HPLC. That gave 44 mg of linker L 2B-NO2 (60% theor.) as a pale-yellow solid (2 x Et3N salt with M = 736). MS (ESI-): m / z, % = 533 (90) [M-H]-; HRMS (C14H24N4O12P2S): 533,0511 (found M-H), 533,0508 (calc.).1H NMR (400 MHz, D2O): <5 = 1.17 (t, J = 7 Hz, 18H, CH3, 2 Et3N), 3.10 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.32 (m, 4H, 2CH2), 3.38 (m, 4H, 2CH2), 3.56 (t, J = 6 Hz, 4H, 2CH2), 3.75 (m, 4H, 2CH2), 7.60 (d, J = 9 Hz, 1 H), 8.37, 8.39 (dd, J = 3 and 9 Hz, 1 H), 8.61 (d, J = 3 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): <5 = +0.80 ppm. Reduction of the nitro compound: linker L 2B-NO2 (30 mg, 0.04 mmol, 2 x EtaN salt) was dissolved in water (6 mL) containing sodium sulfide hydrate (60% wt. Na2S, 120 mg) and NH4CI (60 mg). The solution was heated for 1 .5 h in a screw-cup test tube at 80°C, chilled to RT, acidified with HOAc (0.15 mL), diluted with and equal vol. of water, filtered, neutralized with 1 M TEAB (2 mL), evaporated and subjected to a gradient HPLC on C-18 phase with aq. TEAB buffer and MeCN. That gave 15 mg of linker L 2B (53% theor.) as a colourless amorphous solid (2 x EtaN salt with M = 706). MS (ESI-): m / z, % = 503 (100) [M-H]-; HRMS (C14H26N4O10P2S): 503,0761 (found M-H), 503,0767 (calc.).1H NMR (400 MHz, D2O): <5 = 1.21 (t, J = 7 Hz, 18H, CH3, 2 Et3N), 3.10 (m, 4H, 2CH2), 3.14 (q, J = 7 Hz, 12 H, CH2, 2Et3N), 3.33 (m, 4H, 2CH2), 3.59 (t, J = 6 Hz, 4H, CH2), 3.81 (m, 4H, 2CH2), 6.99, 7.01 (dd, J = 3 and 10 Hz, 1 H), 7.29 (d, J = 3 Hz, 1 H) 7.32 (d, J = 10 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.62 ppm.Linker L 6B

[0241] The open-chain linker L 6B was prepared (43% over two steps) from Compound A 02 using N,N’- dimethyl ethylenediamine instead of 1 ,4-piperazine and following the same procedures as for L 2B. MS (ESI-): m / z, % = 505 (100) [M-H]-; HRMS (C14H27N4O10P2S): 505,0919 (found M-H), 505,0923 (calc.).1H NMR (400 MHz, D2O): 6 = 1 .19 (t, J = 7 Hz, 18H, CH3, 2 Et3N), 2.49 (m, 3H, CH3), 2,94 (s, 3H, CH3), 3.08 (m, 2H, CH2), 3.15 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.36 (m, 2H, CH2), 3.59 (m, 4H, CH2), 3.81 (m, 4H, 2CH2), 6.97, 7.02 (m, 1 H), 7.30 (d, J = 3 Hz, 1 H) 7.33 (d, J = 10 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.57 ppm.Linker L 7B (amide-containing compound)

[0242] The title compound was prepared following the exactly same scheme, which was used for linker L 2B (sulfamide). The synthsis started from the commercially availabe 2-chloro-5-nitrobenzoic acid, which was amidated, the amide phosphorylated and then reduced to amine. The steps were performed under the same condidtions as for the sulfamide analog.1) SOCI22) diethanolamine, K2CO3 3) POCI3,Py4) 1 ,4- piperidine 5) (NH^S

[0243] The final product was isolated with a yield of some 55% (last step) by means of a gradient HPLC on C-18 phase with aq. TEAB buffer and MeCN. Analytical data for L 7B: MS (ESI-): mlz, % = 503 (100) [M-H]-; HRMS (C15H25N4O9P2): 467,1091 (found M-H), 467,1097 (calc.).1H NMR (400 MHz, D2O): <5 = 1 .21 (t, J = 7 Hz, 18H, CH3, 2 Et3N), 3.12 (m, 4H, 2CH2), 3.15 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.30 (m, 4H, 2CH2), 3.61 (m, 4H, CH2), 3.81 (m, 4H, 2CH2), 6.96, 7.10 (dd, J = 3 and 10 Hz, 1 H), 7.32 (d, J = 3 Hz, 1 H) 7.36 (d, J = 10 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): <5 = +0.44 ppm.Linker L 3B

[0244] The title compound was prepared in a one-pot fashion that involves aromatic nucleophilic substitution followed by reduction of the nitro group. Typically, the phosphorilated precuror A 02 (140 mg, 0.2 mmol) in EtOH (2 ml) was added to a mixture of a 1 mmol / L NaOH solution (11 mL, 11 mmol) and EtOH (9 mL) containing 1 ,3-propanedithiol (600 mg, 5.5 mmol) and stirred at RT for 15 min. The flask (free volume > 50%) was sealed with septum, flushed with nitrogen, and the stirring continued for 3h at 65°C. The cold solution was acidified with HOAc (1 .5 mL), diluted with water (75 mL) and extracted two times with a mixture of cyclohexane (50 mL) and EtOAc (10 mL). The aq. layer was filtered, concentrated, and subjected to a gradient HPLC on a C-18 phase with a triethylammonium acetate buffer and MeCN. Evaporation and repeated freeze-drying of pure fractions from water gave 110 mg of linker L 3B (75% theor.) as a colourless amorphous material (2 x Et3N salt with M = 728). MS (ESI-): m / z, % = 525 (100) [M-H]-; HRMS (CI3H24N2OIOP2S3): 524,9954 (found M-H), 524,9990 (calc.).1H NMR (400 MHz, D2O): <5 = 1 .15 (t, J = 7 Hz, 18 H, CH3, 2 Et3N), 1.81 (m, 2H, CH2), 2.53 (t, 2 H, J = 7 Hz, CH2S), 3.02 (m, 2H, CH2), 3.05 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.60 (m, 4 H, CH2O), 3.88 (m, 4 H, CH2N), 7.15 (d, J = 8 Hz, 1 H), 7.45, 7.47 (dd, J = 3 and 8 Hz, 1 H), 7.58 (d, J = 3 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.30 ppm.Linker L 4B

[0245] The synthesis of the custom-made linker L 4B involved the following: TRIS triphosphate (phosphorylated tris(hydroxymethyl)aminomethane) H2NC(CH2OPO3H2)3was prepared from anhydrous TRIS (tris(hydroxymethyl)aminomethane)) as described in

[0015] WO 2006 / 122793. The crude product was three times recrystallized from a mixture of EtOH and EtOAc and then sulfamidated with 2-chloro-5- nitrobenzenesulfonyl chloride as described in the prepration of the sulfamide linker precursor A 01.

[0246] The sulfamide L 4B-NO2-CI was isolated with some 30% yield as a Et3N salt by means of repeated preparative gradient HPLC on a C-18 phase with aq. TEAB buffer and MeCN.1H NMR (400 MHz, D2O): <5 = 1 .19 (t, J = 7 Hz, 54 H, CH3, 6 Et3N), 3.22 (q, J = 7 Hz, 36 H, CH2, 6 Et3N), 3.90 - 4.05 (m, 6 H, CH2O), 7.93 (d, J = 8 Hz, 1 H), 8.53 (m, 1 H), 8.96 (m, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.14 ppm.

[0247] Further reaction with 1 ,4-piperazine was performed as described for L 2B-NO2. The nitro compound L 4B-NO2 was isolated with a yield of ca. 70% by means of a gradient HPLC.1H NMR (400 MHz, D2O): 6 = 1 .20 (t, J = 7 Hz, 45H, CH3, 5 Et3N), 3.16 (q, J = 7 Hz, 30 H, CH2, 5 Et3N), 3.35 (m, 4H, 2CH2), 3.86 (m, 4H, 2CH2), 3.92 - 4.02 (m, 6 H, CH2O), 7.12 (m, 1 H), 7.47 (d, J = 3 Hz, 1 H) 7.52 (m, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.40 ppm.

[0248] The nitro group was subjected to reduction with ammonium sulfide and isolated as described for linker L 2B. That gave linker L 4B as a colourless amorphous solid with a yield of ca. 55% (4 x Et3N salt with M = 1004). MS (ESI-): m / z, % = 599 (100) [M-H]-; HRMS (Ci4H27N4Oi4P3S): 599,0382 (found M-H), 599,0379 (calc.).1H NMR (400 MHz, D2O): 6 = 1.21 (t, J = 7 Hz, 36H, CH3, 4 Et3N), 3.16 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.33 (m, 4H, 2CH2), 3.81 (m, 4H, 2CH2), 3.94 - 4.00 (m, 6 H, CH2O), 7.06 (m, 1 H), 7.36 (d, J = 3 Hz, 1 H) 7.41 (m, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.32 ppm.Linker L 5B

[0249] Linker L 5B was prepared by a one-pot synthesis that utilized 1 ,3-propanedithiol as a reducing agent, exactly as described for L 3B. The crude product was subjected to a repeated gradient HPLC on a C-18 phase with aq. TEAA buffer and MeCN. Evaporation and repeated freeze-drying of pure fractions from water gave linker L 5B with the yield of 47% as a colourless amorphous material (3 x Et3N salt with M = 924). MS (ESI-): m / z, % = 620 (90) [M-H]-; HRMS (Ci3H24N2Oi4P3S3): 620,9621 (found M-H), 620,9623 (calc.).1H NMR(400 MHz, D2O): 5 = 1.19 (t, J = 7 Hz, 27 H, CH3, 3 EbN), 1.81 (m, 2H, CH2), 2.53 (t, 2 H, J = 7 Hz, CH2S), 3.02 (m, 2H, CH2), 3.16 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.94 - 4.00 (m, 6 H, CH2O), 7.11 (d, J = 8 Hz, 1 H), 7.41 , 7.44 (dd, J = 3 and 8 Hz, 1 H), 7.55 (d, J = 3 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): <5 = +0.23 ppm.Compound 1a (amino rhodamine) and 1 b (nitro rhodamine)

[0250] A pressure-resistant vessel was loaded with water (10 mL) containing nitro-substituted Compound 1 b (50 mg, 0.04 mmol), sodium sulfide hydrate (> 60 wt. % Na2S, 330 mg, 2.5 mmol) and NH4CI (150 mg, 2.8 mmol). The vessel was sealed, and the content heated for 2.5 h at 95°C upon magnetic stirring. The solution was diluted with an equal volume of water, acidified with HOAc (0.3 mL), filtered and concentrated. The crude product was subjected to a gradient HPLC on a C-18 reversed phase with aq. TEAA and then with aq. TEAB buffer (A) and MeCN (B). Repeated freeze-drying gave ca. 40 mg (~80%) of the amino-substituted Compound 1a as a 4 x Et3N salt (M = 1237). MS (ESI-): m / z, % = 832 (90) [M-H]"; HRMS (C32H39N3OI5S4): 832.1102 (found M-H), 832,1186 (calc.). Amax. abs. = 549 nm. Amax. fl. = 578 nm. (0.02 M TEAA buffer, pH ~ 6). Non- fluorescent in basic media.1H NMR (400 MHz, Methanol-d4): 6 = 1.24 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.09 (m, 8 H, CH2), 2.89 (m, 8H, CH2N), 3.09 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.60 (m, 8H, CH2SO3), 6.29 (d, J = 8 Hz, 1 H), 6.69 (m, 2 H), 6.71 - 6.73 (m, 2 H), 6.80 - 6.82 (m, 3 H), 7.36 (t, J = 7.5 Hz, 1 H) ppm.

[0251] Compound 1 b is prepared as follows: Precursor AP 1 (di-triethylammonium salt, 70 mg, 0.126 mmol) and powdered 3-nitrophthalic anhydride (120 mg, 0.60 mmol, 5 equiv) were dissolved in glacial HOAc (4 mL) upon heating, and the solvent was removed. The solid residue was heated in a nitrogen-flushed flask for 3 h at 160°C, cooled, refluxed for 15 min. with propionic acid (5 mL) and left cooling to RT. The liquid was decanted from the solid, and the pure nitro rhodamine isolated by means of a gradient HPLC on a C-18 reversed phase with an aq. TEAA buffer and MeCN. Evaporation and repeated freeze-drying of the pure fractions gave 36 mg (42%) of Compound 1 b as a 5 x Et3N salt (M = 1368). MS (ESI-): m / z, % = 862 (90) [M-H]-; HRMS (C32H37N3Oi7S4): 862.0967 (found M-H), 862.0928 (calc.).1H NMR (400 MHz, D2O): 6 = 1.27 (t, J = 7 Hz, 45 H, CH3, 5 Et3N), 2.16 (m, 8 H, CH2), 3.02 (m, 8H, CH2N), 3.17 (q, J = 7 Hz, 30 H, CH2, 5 Et3N), 3.79 (m, 8H, CH2SO3), 7.07 (m, 4 H), 7.24 (d, J = 8 Hz, 2 H), 7.73 (d, J = 8 Hz, 1 H), 7.82 (t, J = 7.5 Hz, 1 H), 8.39 (d, J = 8 Hz, 1 H) ppm. Amax. abs. = 551 nm (1 M TEAB, pH ~ 8).Compound 1 i (a hydrazine-containing glycan tag for non-reductive labeling)

[0252] Amino-substituted Compound 1a (13 mg, 0.01 mmol, 4 x Et3N salt with M = 1237) was dissolved in a mixture of MeCN (4 mL) and water (1 mL). To this solution a solid linker L 1A (140 mg, 0.50 mmol, 50 equiv) and finely powdered potassium dihydrogen phosphate (KH2PO4, 136 mg, 1.0 mmol, 100 equiv) were added in one portion upon stirring, which then continued overnight.

[0253] The reaction mixture was diluted with water (25 mL) and extracted with EtOAc (50 mL). The organic layer was extracted with water (10 mL), the aq. layers combined, filtered, and concentrated at t < 25°C. The product was subjected to a gradient HPLC on C-18 phase with aq. TEAB buffer and MeCN. That gave ca. 12 mg (85%, 4 x EtsN salt) of the intermediate product 1 h, whose identity was confirmed by LC / MS analysis and NMR spectra. (Calc, for C40H49CIN8O17S4 (M-2H)272 537.0815, found 537.0842). The intermediate was dissolved in water (3 mL) containing TFA (0.6 mL), the solution heated for 1 h at 90°C and evaporated under reduced pressure. The deprotected and dechlorinated product (1 i) was isolated by means of repeated gradient HPLC on a C-18 phase with aq. TEAB buffer as A and MeCN as B. The homogeneous fractions were pooled and the HPLC was repeated to give 7.5 mg (55% over 2 steps) of Compound 1i as a 4 x EtsN salt with M = 1362. Properties: Amax. abs. = 551 nm., Amax. fl. = 579 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 957 (70) [M-H]", 478 (30) [M-2H]2; HRMS (C35H42N8O16S4): 957,1531 (found M-H), 957,1523 (calc.).1H NMR of 1 i (400 MHz, D2O): 5 = 1.11 (t, 36 H, J = 7 Hz, CH3, 4 Et3N), 2.02 (m, 8 H, CH2), 2.88 (m, 8H, CH2N), 3.04 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.63 (m, 8H, CH2SO3), 6.93 (m, 4 H), 7.06 (m, 1 H), 7.25 (m, 2 H), 7.50 (m, 1 H), 7.98 (m, 1 H) ppm.

[0254] Compound 1j (net charge -6) was prepared as follows: 2,2-ethanolamine disulfonic acid disodium salt NH(CH2CH2SO3Na)2 (15 equiv, see preparation of linker L 3A) and EtsN (2 equiv) was refluxed with Compound 1 h (1 equiv) in water until the reaction completed. The solution was evaporated, the residue dissolved in a 10 vol. % aq. TFA, heated 30 min at 80°C and evaporated again. The target compound, whose identity was confirmed by LC / MS analysis, was isolated as a 6 x EtsN salt with a yield of ca. 42% by means of a gradient HPLC on a C-18 phase with aq. TEAB buffer and MeCN. MS (ESI-): m / z, % = 1172 (50) [M-H]-, HRMS (C39H50N9O21S6): 1172,1451 (found M-H), 1172,1446 (calc.).Compound 1 k (a tetramethyl rhodamine derivative for non-reductive labeling).

[0255] / V, / V, / V, / V’-Tetramethyl-3’-amino rhodamine (prepared as described, e. g., in [6] by Wang, et al., Nat.Mater. 2019, 18, 1335-1342 and in [5] by I. Zhang, et al., RSC Adv., 2015, 5, 66416 -66419) was reacted asa ~5 wt.% solution in ethylene glycol with ~15 equiv of linker L 4A in presence of 4 equiv EtsN at 120°C. As the starting amino rhodamine had reacted (3 - 5 h), the solution was diluted with 10 vol. of water, acidified with CF3COOH (1 / 10 vol.) and heated again at 80°C for 30 min for deprotection. The target compound (1 k) was isolated by means of a repeated gradient HPLC with the yield of ca. 28% over two steps (as 2 x EtsN salt, M = 974).

[0256] Alternatively, the same compound (1 k) was obtained with better yields (~60%) when N,N,N’,N’- tetramethyl-3’-amino rhodamine was first reacted with dichloro-substituted triazine linker L1A (5 equiv) in presence of KH2PO4 (> 30 equiv) in aq. MeCN at RT, as described for Compound 1 i. The intermediate monochloro- substituted product 1 k-Boc-CI was isolated by means of HPLC and then reacted with ~ 15 equiv of NH(CH2CH2OPO3H)2 (“EDAP”, see preparation of linker L 4A) in a 70 % aq. MeCN at 90°C in presence of EtsN (20 equiv). As the reaction was complete, the mixture was evaporated, and the t-Boc group in the intermediate compound cleaved in an aq. 10% vol. TFA. Purity and identity were confirmed by LC / MS (ESI-) analyses. Amax. abs. = 549 nm. Amax. fl. = 581 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 772 (90) [M- H]-; HRMS (C31H37N9O11P2): 772,2048 (found M-H), 772,2010 (calc.).1H NMR of 1 k (400 MHz, D2O): <5 = 0.90 - 1.16 (br. m, 12H, 4 CH3, NMe) 1 .20 (t, J = 7 Hz, 18 H, CH3, 2 Et3N), 3.06 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.65 (m, 4 H, 2 CH2O), 3.96 (m, 4 H, 2 CH2N), 6.60 - 6.66 (m, 4 H), 6.86 (m, 2 H), 7.20 (m, 2 H), 7.66 (m, 1 H) ppm.31P NMR (162 MHz, D2O): <5 = +0.21 ppm.Compound 1t (a derivative of tetramethyl rhodamine and a custom-synthesized LinkerL 3B).

[0257] / V, / V, / V( / V-Tetramethyl-3’-amino rhodamine (see preparation of compound 1 k) was acetylated with chloroacetyl chloride (5 equiv) by a routine procedure in presence of 10 equiv EtsN in dry MeCN at +5°C, and the reaction product was isolated with a yield of 43% after purification over a regular silica gel column with DCM / MeOH (2 :1) as a mobile phase. The / V-chloroacetyl amino rhodamine derivative TMR-NHCOCH2CI was then reacted with the custom-made thiol-containing linker L 3B in 80% aq. MeCN in presence of ca. 4 equiv EtsN at 60°C to give the target compound with the yield of ca. 70%.

[0258] Compound 1t (2 x EtsN salt, M = 1169) was isolataed by means of a gradient HPLC on a C-18 phase with aq. TEAB buffer and MeCN. Amax. abs. = 552 nm. Amax. fl. = 580 nm (1 M TEAB, pH ~ 8). MS (ESI-): m / z, % = 967 (90) [M-H]-; HRMS (C39H47N5O14P2S3): 966,1656 (found M-H), 966,1679 (calc.).1H NMR (400 MHz, D2O): <5 = 0.90 - 1.14 (br. m, 12H, 4 CH3, NMe) 1.21 (t, J = 7 Hz, 18 H, CH3, 2 Et3N), 1 .81 (m, 2H, CH2), 2.53 (m 2 H, CH2S), 3.03 (m, 2H, CH2), 3.06 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.62 (m, 4 H, 2 CH2O), 3.93 (m, 4 H, 2 CH2N), 4.44 (s, 2 H, COCH2S), 6.64 - 6.70 (m, 4 H), 6.84 (m, 2 H), 7.17 (d, J = 8 Hz, 1 H), 7.48 (dd, J = 3 and 8 Hz, 1 H), 7.61 (d, J = 3 Hz, 1 H), 7.63 (m, 1 H), 7.91 (m, 1 H), 8.11 (m, 1 H) ppm.31P NMR (162 MHz, D2O): 5 = +0.16 ppm.Compounds 2a and 2b

[0259] Compound 2b (nitro rhodamine) was prepared as follows: a vial was loaded with the aminophenol precursor AP 1 (2 x EtsN salt, 120 mg, 0.21 mmol) in HOAc (2 mL), 2-formyl-5-nitrobenzoic acid (35 mg, 0.18 mmol; for preparation see

[0016] G. Mudd, et al., Methods and Applications in Florescence, 2015, 3, 045002), sealed and heated for 30h at 120°C upon stirring. Chloranil (25 mg, 0.1 mmol) was added, the solution was heated 30 min at 80°C, filtered and evaporated. The residue was dissolved in water (50 mL), the solution filtered, concentrated, and subjected to a gradient HPLC, as described for Compound 1a. That afforded ca. 60 mg of Compound 2b (24% theor.) as a 5 x Et3N salt (M = 1368). MS (ESI-): m / z, % = 862 (90) [M-H]-; HRMS (C32H37N3O17S4): 862.0944 (found M-H), 862.0928 (calc.) 1 H NMR (400 MHz, Methanol-d4): 6 = 1.26 (t, J = 7 Hz, 45 H, CH3, 5 Et3N), 2.17 (m, 8 H, CH2), 2.91 (m, 8 H, CH2N), 3.17 (q, J = 7 Hz, 30 H, CH2, 5 Et3N), 3.84 (m, 8H, CH2SO3), 7.19 - 7.22 (m, 6 H), 7.58 (d, J = 8 Hz, 1 H), 8.98 (s, 1 H) ppm. Amax. abs. = 549 nm (1 M TEAB).

[0260] Compound 2a (amino rhodamine) was prepared by reduction of 2b with sodium / ammonium sulfides and isolated as described for 1a with a yield of ca. 80% (as a 4 x EtsN salt). MS (ESI-): m / z, % = 832 (90) [M- H]-; HRMS (C32H39N3O15S4): 832.1134 (found M-H), 832,1186 (calc.). 1 H NMR (400 MHz, Methanol-d4): 6 = 1.24 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.16 (m, 8 H, CH2), 2.90 (m, 8 H, CH2N), 3.17 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.82 (m, 8H, CH2SO3), 6.92 - 6.93 (m, 1 H), 6.99 (s, 1 H), 7.01 (s, 1 H), 7.11 (m, 1 H), 7.18 (d, J = 8 Hz,1 H), 7.21 (d, J = 8 Hz, 1 H), 7.43 (m, 2 H), 7.45 (s, 1 H) ppm. Non-fluorescent in basic media. Amax. abs. = 550 nm. Amax. fl. = 582 nm (0.02 M TEAA, pH ~ 6).Free acid 1-COOH and nucleophile-reactive fluorescent label 1-NHS

[0261] Compound 4a with a free amino group was alkylated with bromoactetic acid in presence of a base, as described

[0017] for some simple aromatic amines by Y. C. Chen, Y. T. Kuo and T. H. Ho in Photochem. Photobiol. Sci., 2019, 18, 190-197. The title compound was isolated by means of preparative gradient HPLC on a C-18 phase with aq. TEAB buffer and MeCN with the yield of 36%.1H NMR of 1-COOH in D2O (400 MHz): <5 = 1.23 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.18 (m, 8 H, CH2), 3.00 (m, 8 H, CH2N), 3.15 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.69 (s, 2 H, CH2CO), 3.74 (m, 8 H, CH2SO3), 7.02 (m, 4 H), 7.09 (m, H), 7.10 (d, J = 8 Hz, 1 H), 7.33 (m, 5H), 7.72 (t, J = 8 Hz, 1 H), 8.48 (d, J = 8 Hz, 1 H) ppm. MS (ESI-): m / z, % = 1009 (80) [M-H]-; HRMS (C41H45N4O18S4): 1009,1631 (found M-H), 1009,1612 (calc.). The reactive NHS ( / V - hydroxysuccinimidyl) ester 1-NHS was prepared in aliquots by conventional method from the corresponding free acid, as described

[0018] by K. Kolmakov, et al., in Photochem. Photobiol. Sci., 2020, 19, 1677-1689. That involved reaction with an excess of DSC reagent ( / V, / V’-disuccinimidyl carbonate) in dry DMF and was followed by removal of the DMF and the small-molecule compounds with a suitable inert solvent (e.g., EtOAc).Compounds 3a,b,i,h and 4a,b,k,h

[0262] Nitro-substituted Compounds 3b and 4b were obtained via a Schotten-Baumann benzoylation of Compounds 1a and 2a, respectively, using the corresponding nitrobenzoic acid chlorides. The reaction involved the following: a solution of the amine substrate (Compounds 1a or 2a, 40 mg, 0.032 mmol as 4 x EtsN salts) and K2CO3 (140 mg, 1 mmol) in a mixture of water (20 mL) and MeCN (10 mL) was chilled to 0 - +5°C, and a freshly prepared solution of 3- or 4-nitrobenzoyl chloride (for 1a and 2a, respectively, 95 mg, 0.50 mmol) in dry MeCN (2 - 5 mL) was added upon stirring, which was continued for 1 h at RT. In case of 1a (the ortho-aminobenzoyl derivative), an additional amount of base (EtsN, 0.5 mL) was added to the solution, and it was stirred for additional hour. The nitro compounds 3b and 4b were isolated (as 4 x EtsN salts) with the yields of 80 - 90% by means of a gradient HPLC. Their purity and identity were confirmed by analytical methods.

[0263] Compounds 3a, 3i and 4a (see below) were prepared by reduction of the nitro-substituted Compounds 3b, 3j and 4b, respectively, as described for Compound 1a. These carbohydrate-reactive amines were isolated with yields of about 60 - 70% (as 4 x EtsN salts) by means of a preparative gradient HPLC on a C-18 column with a TEAB buffer. MS (ESI-): m / z, % = 832 (90) [M-H]-; HRMS (C39H42N4O18S4): 981 ,1284 (found M-H), 981 ,1299 (calc.).

[0264] 1 H NMR of the Compound 3a (400 MHz, D2O): 6 = 1.25 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.16 (m, 8 H, CH2), 3.06 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.70 (m, 8H, CH2SO3), 6.70 (d, J = 8 Hz, 2 H), 6.95 - 6.97 (m, 4 H), 7.26 (d, J = 8 Hz, 1 H), 7.31 (d, J = 8 Hz, 2 H), 7.48 (d, J = 8 Hz, 2 H), 7.64 (m, 1 H), 7.79 (m, 1 H) ppm. MS (ESI-): m / z, % = 951 (90) [M-H]"; HRMS (C39H42N4OI8S4): 951 ,1543 (found M-H), 951 ,1557 (calc.). Amax. abs. = 550 nm. Amax. fl. = 578 nm (1 M TEAB, pH ~ 8).

[0265] Compound 3i with an extra charged group (CO2H) was prepared from the precursor 2a following the exaclly same procedure as described for Compound 3b. For the benzoylation, a large excess of 5- nitroisophthalic acid dichloride was used. That gave the corresponding nitro Compound 3j (an intermediate) in a quantitative yield, which was reduced to the amine with sodium / ammonium sulfide, as described for 1a. The purity and identity of compound 3i confirmed by LC / MS analyses and HRMS spectrometry. Electrophoresis showed its enhanced mobility compared to Compounds 3a and 4a. Amax. abs. = 550 nm. Amax. fl. = 580 nm (1 M TEAB, pH ~ 8). MS (ESI-) of Compound 3i: m / z, % = 995 (90) [M-H]-; HRMS (C40H44N4O18S4): 995,1423 (found M-H), 995,1455 (calc.).

[0266] The hydrazine-substituted Compound 3h was obtained from Compound 2a and the commercially available Fmoc-4-hydrazinobenzoic acid, which was converted to the chloride by a conventional method utilizing oxalyl chloride in dioxane. In the final step of the synthesis the Fmoc protective group was removed, and the compound was used for non-reductive labeling of glycans. Amax. abs. = 552 nm. Amax. fl. = 582 nm (1 M TEAB, pH ~ 8). MS (ESI-): m / z, % = 995 (90) [M-H]-;HRMS (C39H45N5Oi6S4): 966,1631 (found M-H), 966,1666 (calc.)

[0267] 1 H NMR of the Compound 3h (400 MHz, DMSO-d6): 6 = 1 .10 (t, J = 7 Hz, 45 H, CH3, 5 Et3N), 1.83 (m, 8 H, CH2), 2.47 (m, 8 H, CH2N), 2.90 (q, J = 7 Hz, 30 H, CH2, 5 Et3N), 3.48 (m, 8 H, CH2SO3), 6.65 - 6.74 (m, 5 H), 7.15 (d, J = 8 Hz, 1 H), 7.30 (br. s. NH, H2O), 7.74 (t, J = 8 Hz, 1 H), 7.74 (t, J = 7.5 Hz, 1 H), 7.93 (t,J = 7.5 Hz, 1 H), 8.45 - 8.51 (m, 2H) 8.55 (d, J = 8 Hz, 1 H), 8.82 (m, 1 H) ppm. MS (ESI-): m / z, % = 832 (90) [M-H]-;HRMS (C39H42N4O18S4): 981 ,1272 (found M-H), 981 ,1299 (calc.).

[0268] The isomeric hydrazine-substituted Compound 4h was obtained in exactly same way utilizing the commercially available 3-hydrazinobenzoic acid. The latter was protected by a Fmoc group, converted to the acid chloride and reacted with precursor Compound 1a as described above for Compound 2a. The final deprotected product 4h was isolated by conventional methods and identified by means of LC / MS. Amax. abs. = 551 nm. Amax. fl. = 583 nm (1 M TEAB, pH ~ 8). MS (ESI-): m / z, % = 995 (90) [M-H]-; HRMS (C39H45N5O16S4): 966,1624 (found M-H), 966,1666 (calc.).

[0269] 1 H NMR of 4a in D2O (400 MHz): 6 = 1 .23 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.15 (m, 8 H, CH2), 3.02 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.74 (m, 8 H, CH2SO3), 6.99 (m, 4 H), 7.06 (m, 1 H), 7.15 (d, J = 8 Hz, 1 H), 7.30 (m, 5 H), 7.60 (t, J = 8 Hz, 1 H), 8.45 (d, J = 8 Hz, 1 H) ppm. MS (ESI-): m / z, % = 951 (90) [M-H]-; HRMS (C39H42N4O18S4): 951 ,1539 (found M-H), 951 ,1557 (calc.). Amax. abs. = 548 nm. Amax. fl. = 579 nm (1 M TEAB, pH ~ 8).

[0270] Compound 4k was prepared from the Precursor 1a following the exact same procedure as described for Compound 4a. In this case 2-nitrobenzoyl chloride was used for benzoylation. That gave the corresponding nitro compound in a quantitative yield, which was reduced to the amine with iron powder or another suitable reducing agent and isolated as described for Compound 4d with an extra heterocycle.

[0271] 1 H NMR of Compound 4k in D2O (400 MHz): 6 = 1 .15 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.07 (m, 8 H, CH2), 2.93 (m, 8 H, CH2N), 3.05 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.67 (m, 8 H, CH2SO3), 6.80 - 6.88 (m, 2 H), 6.95 (m, 4H), 7.08 (d, J = 8 Hz, 1 H), 7.27 - 7.34 (m, 3H), 7.55 (m, 2 H), 8.25 (d, J = 8 Hz, 1 H) ppm. MS (ESI-): m / z, % = 951 (80) [M-H]-; HRMS (C39H42N4O18S4): 951 ,1578 (found M-H), 951 ,1557 (calc.). Amax. abs. = 546 nm. Amax. fl. = 577 nm (1 M TEAB, pH ~ 8).Compounds 3I, 3m and 3n (rosamines)

[0272] Compound 3n (an exemplary rhodamine-based glycan label with the net charge of -3 and with no CO2H group). Rhodamine dye 3I with a nitro group was prepared with the isolated yield of ca. 18% according to the procedure described

[0019] for sulfonated rhodamine and so-called “rosamine” dyes by A. Chevalier, P.- Y. Renard, and A. Romieu, 2014, Chem. Eur. J., 20, 8330-8337. In this case, the synthesis involved a two- step condensation of 4-nitrobenzaldehyde with the sulfonated aminophenol Precursor AP 1 under acidic conditions.Compound 3I is an intermediate which was subjected to further steps. The nitro compound was reduced, and the amine 3m was benzoylated with 3-nitrobenzoyl chloride. The resulting compound was again reduced to the amine exactly as described for Compounds 3a and 4a and isolated by a preparative HPLC. Purity and identity of the final product 3n was confirmed by analytical methods.1H NMR of 3n in D2O (400 MHz): 6 = 1.21 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.15 (m, 8 H, CH2), 3.02 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.74 (m, 8 H, CH2SO3), 6.97 (m, 4 H), 7.06 (m, 1 H), 7.15 (d, J = 8 Hz, 1 H), 7.33 (m, 4H), 7.60 (d, J = 8 Hz, 2 H), 8.48 (d, J = 8 Hz, 2 H) ppm. Amax. abs. = 548 nm. Amax. fl. = 571 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 907 (90) [M-H]-; HRMS (C38H44N4Oi4S4): 907,1621 (found M-H), 907,1659 (calc.).Compound 4c.

[0273] To a solution of Compound 1a (15 mg, 0.012 mmol) in a mixture of DMF (3 mL) and pyridine (0.6 mL) 3-nitrobenzoyl chloride (20 mg, 0.010 mmol) was added, and the solution heated for 1 h at 100°C in a nitrogenflushed vessel.

[0274] The solvents were evaporated, and the residue was subjected to gradient HPLC on a C-18 phase with an aq. triethylammonium acetate buffer with pH 4.5, also containing free HOAc (A) and pure MeCN (B). Evaporation and repeated freeze-drying gave 12 mg (80%) of Compound 4c as a 3 x Et3N salt. Alternatively, the same product (4c) was obtained with a quantitative yield by heating a solution of Compound 4b in a mixture of HOAc and AC2O (4:1) at 110°C for 1 h. Bases cleave the cyclic compound (4c) back to its opened form (4b).Compounds 4d, 4g, and 4n

[0275] A solution of Compound 4c (10 mg, 0.008 mmol) in water (6 mL) containing HOAc (0.4 mL) and NH CI (30 mg) was vigorously stirred with iron powder (Fe°, 30 mg, 0.55 mmol) for 1--2 h. The solution was decantedand loaded onto a column with a C-18 reversed phase SiO2(8 g) and water. The column was eluted with aq. 1 % vol. HOAc and then with water. The coloured products were then eluted with 80% aq. MeOH containing 0.3% vol. HOAc, the solution filtered and evaporated. A gradient HPLC on a C-18 reversed phase with aq. TEAA buffer (A), also containing additional free HOAc, and MeCN (B) afforded 4.5 mg (45%) of Compound 4d as a a 3 x Et3N salt. All title compounds with an extra fused heterocyclic ring (4d, 4g, 4n) and 4j and 4p (see below) are only stable in acidic conditions, aqueous acetic acid incuding. In basic solutions the extra fused ring is cleaved.

[0276] Alternatively, Compound 4d was obtained by direct cyclization of the amine 4a taken as a 5 mg / mL solution in dry DMF, to which 20 equiv of CDI reagent and 10 equiv TFA (as a 5% vol. solution in DMF) was added. The mixture was left overnight, poured into ice-cold 5% aq. HOAc and subjected to a gradient HPLC in acidic (TEAA buffer), as described above. That gave ca. 60% of Compound 4d. Bases cleave the cyclic compound (4d) back to its opened form (4a).

[0277] 1H NMR of the cyclic nitro Compound 4c (400 MHz, DMSO-d6): <5 = 1 .15 (t, J = 7 Hz, 27 H, CH3, 3 Et3N), 1 .85 (m, 8 H, CH2), 2.48 (m, 8 H, CH2N), 3.07 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.52 (m, 8 H, CH2SO3), 6.75 - 6.80 (m, 5 H), 7.12 (d, J = 8 Hz, 1 H), 7.76 (t, J = 8 Hz, 1 H), 7.74 (t, J = 7.5 Hz, 1 H), 7.92 (t, J = 7.5 Hz, 1 H), 8.43 - 8.50 (m, 2H) 8.80 (s, 1 H), 9.06 (m, 1 H) ppm. MS (ESI-): m / z, % = 963 (90) [M-H]-; HRMS (C39H40N4Oi7S4): 963,1164 (found M-H), 963,1193 (calc.).

[0278] 1H NMR of the cyclic amino Compound 4d (400 MHz, DMSO-d6): <5 = 1 .16 (t, J = 7 Hz, 27 H, CH3, 3 Et3N), 1.83 (m, 8 H, CH2), 2.48 (m, 8 H, CH2N), 3.07 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.47 (m, 8 H, CH2SO3), 6.53 - 6.71 (m, 4 H), 7.06 (t, J = 8 Hz, 1 H), 7.74 (m, 2H) 8.35 (t, J = 8 Hz, 1 H), 7.92 (t, J = 7.5 Hz, 1 H), 8.58 (m, 1 H),8.94 (s, 1 H), 8.95 - 9.20 (m, 2H) ppm. MS (ESI-): m / z, % = 933 (90) [M-H]-; HRMS (C39H42N4OI5S4): 933,1468 (found M-H), 933,1451 (calc.). Amax. abs. = 561 nm. Amax. fl. = 582 nm (1 M TEAA, pH ~ 5).

[0279] Compound 4g and 4n were prepared following the same procedure as for 4d but utilizing commercially available 4-nitrophenylacetic acid and 2-nitrobenzoylchloride for the acylation of Precursor 1a. That was followed by reduction of the intermediate nitro compounds (see Schemes 6 and 9) and cyclization.

[0280] 1H NMR of the cyclic amino Compound 4n (400 MHz, DMSO-d6): <5 = 1 .16 (t, J = 7 Hz, 27 H, CH3, 3 Et3N), 1.85 (m, 8 H, CH2), 2.51 (m, 8 H, CH2N), 3.05 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.44 (m, 8 H, CH2SO3), 6.50 - 6.68 (m, 4 H), 7.05 (t, J = 8 Hz, 1 H), 7.71 (m, 2H) 8.41 (t, J = 8 Hz, 1 H), 7.92 (t, J = 7.5 Hz, 1 H), 8.64 (m, 1 H), 9.02 (m, 1 H), 8.99 - 9.22 (m, 2H) ppm. MS (ESI-): m / z, % = 933 (90) [M-H]-; HRMS (C39H42N4OI5S4): 933,1459 (found M-H), 933,1451 (calc.). Amax. abs. = 562 nm. Amax. fl. = 584 nm (1 M TEAA, pH ~ 5).

[0281] 1H NMR of the Compound 4g (400 MHz, D2O): 6 = 1 .25 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.16 (m, 8 H, CH2), 3.06 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.70 (m, 8H, CH2SO3), 4.12 (s, 2H CH2Ar) 6.70 (d, J = 8 Hz, 2 H), 6.95 - 6.97 (m, 4 H), 7.26 (d, J = 8 Hz, 1 H), 7.31 (d, J = 8 Hz, 2 H), 7.48 (d, J = 8 Hz, 2 H), 7.61 , 7.64 (m, 1 H), 7.79 (m, 1 H) ppm. HRMS (C4OH43N4OI5S4): 947,1608 (found M-H), 947,1688 (calc.). Amax. abs. = 560 nm. Amax. fl. = 582 nm (1 M TEAA, pH ~ 5).

[0282] The title compounds were prepared following the same procedure as for 4a but utilizing 5- nitroisophthalic acid dichloride and 4-nitrobenzoylchloride, respectively, for the acylation of precursor amine 1a. That gave Compounds 4f and 4o (see structures below) and was followed by reduction of the corresponding nitro compounds and cyclization into compounds 4j and 4p, respectively. The cyclization was performed as described for compound 4d. The isolation of involved a gradient HPLC on a C-18 phase with aq. TEAA buffer as A and MeCN as B. That gave the amines in ca. 30% yield over three steps. All the compounds with an extra fused ring (4 / 7-3, 1 -benzoxazin-4-one derivatives) are stable in slightly acidic media.

[0283] 1H NMR of 4j (400 MHz, DMSO-d6): <5 = 1 .16 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 1 .83 (m, 8 H, CH2), 2.48 (m, 8 H, CH2N), 3.07 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.47 (m, 8 H, CH2SO3), 6.53 - 6.71 (m, 4 H), 7.06 (m, 1 H), 7.74 (m, 2H) 8.35 (m, 1 H), 7.92 (t, J = 7.5 Hz, 1 H), 8.58 (m, 1 H), 8.94 (s, 1 H), 8.95 - 9.20 (m, 1 H) ppm. MS (ESI-): m / z, % = 977 (80) [M-H]-; HRMS (C4oH42N4Oi7S4): 977,1306 (found M-H), 977,1350 (calc.). Amax. abs. = 561 nm. Amax. fl. = 582 nm (1 M TEAA, pH ~ 5).

[0284] 1H NMR of Compound 4p (400 MHz, DMSO-d6): <5 = 1 .19 (t, J = 7 Hz, 27 H, CH3, 4 Et3N), 1.93 (m, 8 H, CH2), 2.52 (m, 8 H, CH2N), 3.10 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.50 (m, 8H, CH2SO3), 6.72 (d, J = 8 Hz, 2 H), 6.95 - 6.97 (m, 4 H), 7.29 (d, J = 8 Hz, 1 H), 7.31 (d, J = 8 Hz, 2 H), 7.48 (d, J = 8 Hz, 2 H), 7.61 , 7.64 (m, 1 H), 7.79 (m, 1 H) ppm. MS (ESI-): m / z, % = 951 (90) [M-H]-; HRMS (C39H42N4OI5S4): 933,1451 (foundM-H), 933,1446 (calc.). Amax. abs. = 560 nm. Amax. fl. = 581 nm (1 M TEAA, pH ~ 5).1H NMR of 4f (400 MHz, DMSO-d6): 6 = 1.17 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 1.83 (m, 8 H, CH2), 2.48 (m, 8 H, CH2N), 3.07 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.47 (m, 8 H, CH2SO3), 6.50 - 6.75 (m, 4 H), 7.06 (m, 1 H), 7.77 (m, 2H) 8.32 (m, 1 H), 7.95 (m, 1 H), 8.64 (m, 1 H), 8.94 (s, 1 H), 8.95 - 9.20 (m, 1 H) ppm. MS (ESI-) of 4f: m / z, % = 995 (90) [M-H]-; HRMS (C40H44N4O18S4): 995,1439 (found M-H), 995,1455 (calc.). MS (ESI-) of Compound 4o: m / z, % = 951 (90) [M-H]- HRMS (C39H42N4OI8S4): 951 ,1549 (found M-H), 951 ,1557 (calc.).Compounds 4s and 4t

[0285] Compound 4s was prepared by reaction of amine 1a with an excess (~ 20 equiv) of 2-nitrophenyl isocyanate in DMF at 130°C. After the reaction completed, the mixture was diluted with water, filtered, evaporated under reduced pressure, and subjected to a gradient HPLC as described for compound 4a. The intermediate o / Yho-nitro compound was reduced to the amine with iron powder or another suitable reducing agent and isolated as described for Compound 4d. That gave Compound 4s in a 40% yield over two steps. The synthesis of Compound 4t involved prolonged heating of amine 1a with 4-nitrophenyl isothiocyanate in ethylene glycol at 160°C, which was followed by isolation (HPLC) and reduction as described for Compound 4d.

[0286] 1H NMR of Compound 4s in D2O (400 MHz): <5 = 1 .16 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.05 (m, 8 H, CH2), 2.90 (m, 8 H, CH2N), 3.09 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.62 (m, 8 H, CH2SO3), 6.83 - 6.95 (m, 2 H), 7.02 (m, 4H), 7.12 (d, J = 8 Hz, 1 H), 7.31 - 7.42 (m, 3H), 7.61 (m, 2 H), 8.43 (d, J = 8 Hz, 1 H) ppm. MS (ESI-): m / z, % = 966 (70) [M-H]-; HRMS (C39H44N5Oi6S4): 966,1595 (found M-H), 966,1666 (calc.). Amax. abs. = 549 nm. Amax. fl. = 581 nm (1 M TEAB, pH ~ 8).1H NMR of Compound 4t in D2O (400 MHz): 6 = 1.14 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.08 (m, 8 H, CH2), 2.94 (m, 8 H, CH2N), 3.12 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.64 (m, 8 H, CH2SO3), 6.80 - 7.03 (m, 2 H), 7.10 (m, 4H), 7.16 (m, 1 H), 7.26 - 7.40 (m, 3H), 7.79 (m, 2 H), 8.61 (m, 2 H) ppm. MS (ESI-): m / z, % = 982 (50) [M-H]-; HRMS (C39H44N5OI5S5): 982,1440 (found M-H), 982,1437 (calc.). Amax. abs. = 552 nm. Amax. fl. = 584 nm (1 M TEAB, pH ~ 8).Compounds 4v and 4w

[0287] Compounds 4v and 4w were prepared from the amine precursor 1a in two steps as described for compounds 3a and 4a. Purity and identity of the compounds was confirmed by LC / MS analyses and HRMS spectrometry. The synthesis involved the commercially available 2-nitrophenyl acetic acid and 4-nitrophenyl cinnamic acid, respectively. The acids were converted to the chlorides by conventional procedure using thionyl chloride, and the crude products were used for benzoylation of 1a. That was followed by isolation and reduction of the intermediate nitro compounds to amines.1H NMR of Compound 4v in D2O (400 MHz): <5 = 1 .13 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.08 (m, 8 H, CH2), 2.91 (m, 8 H, CH2N), 3.09 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.70 (m, 8 H, CH2SO3), 3.78 (s, 2H, CH2), 6.82 - 6.90 (m, 2 H), 6.96 (m, 4H), 7.11 (d, J = 8 Hz, 1 H), 7.29 - 7.34 (m, 3H), 7.62 (m, 2 H), 8.21 (d, J = 8 Hz, 1 H) ppm. MS (ESI-): m / z, % = 965 (80) [M-H]"; HRMS (C40H46N4O16S4): 965,1709 (found M-H), 965,1713 (calc.). Amax. abs. = 547 nm. Amax. fl. = 578 nm (1 M TEAB, pH ~ 8).

[0288] 1H NMR of the Compound 4w (400 MHz, D2O): <5 = 1 .24 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.14 (m, 8 H, CH2), 3.06 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.68 (m, 8H, CH2SO3), 6.70 (d, J = 8 Hz, 2 H), 7.68(d, J = 16 Hz, 1 H, CH), 6.73 (d, J = 16 Hz, 1 H, CH), 6.95 - 6.97 (m, 4 H), 7.26 (d, J = 8 Hz, 1 H), 7.31 (d, J = 8 Hz, 2 H), 7.48 (d, J = 8 Hz, 2 H), 7.61 , 7.64 (m, 1 H), 7.79 (m, 1 H) ppm. MS (ESI-): m / z, % = 977 (90) [M-H]-; HRMS (C41 H46N4O16S4): 978,1778 (found M-H), 978,1792 (calc.). Amax. abs. = 551 nm. Amax. fl. = 578 nm (1 M TEAB, pH ~ 8).Phosphorylated rhodamine dyes 5b, 6b, 7b and 8

[0289] The synthesis of Compound 6b (ca. 15% in 6 steps, see below) involved the following: Ketone 1 was prepared from 3-aminophenol and 3-nitrophthalic anhydride according to the procedure described [6] by Y. Wang, et al., Nat. Mater. 2019, 18, 1335 -1342. The aminophenol precursor AP 3 was prepared by N- alkylation of 3-aminophenol with 3-bromopropanol by conventional methods, and then reacted with Ketone 1 under acidic conditions, as described in the reference above and shown in scheme below.

[0290] The condensation gave the key intermediate - a nitro-substituted rhodamine 6a-OAc-NC>2 with acetylated OH groups. After hydrolysis, compound 6a-OH-NO2 was was phosphorylated analogously to thePrecursor A 02. The nitro group was reduced to amine, and the amino rhodamine 6a was benzoylated. The resulting 3-nitrobezoyl derivative was reduced as it was done for, e.g., Compounds 3a and 4a.a - HOAc; b - NaOH; C - H+; d - (NH4)2S; e - POCI3, Py; f - 3-O2NPhCOCI

[0291] The synthesis of Compound 5b (yield ca. 18% in 5 steps) involved the following: the aminophenol Precursor AP 4 was obtained by / V-alkylation of the commercially available 3- / V-methylaminophenol with 3- bromopropanol by conventional methods and then condensed with Aldehyde 1 (2-formyl-5-nitrobenzoic acid) upon prolonged heating in acetic acid, as described for Compound 2a. The resulting nitro rhodamine with acetylated OH groups 5b-OAc-NO2 was isolated by means of column chromatography on regular silica gel, and afterwards the acetate groups were hydrolized in an alkaline medium.

[0292] The hydroxy-substituted compound was phosphorylated analogously to the Precursor A 02. The nitro compound was reduced, the amine 5a was nitro-benzoylated and the resulting compound reduced again, as described for Compound 3a. This gave Compound 5b, which is an isomer of 6b (see scheme above for structure). The two compounds are spectrally almost identical, and identical to Compounds 3a and 4a, aswell (see Table 1 for properties). The net charge and the m / z ratio of 5b and 6b are also the same. Purity and identity of Compounds 5a, b and 6a, b was confirmed by analytical methods.1H NMR of 5a (400 MHz, D2O): <5 = 1.14, 1 .17 (m, 6H, NMe) 1.20 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.09 (m, 4 H, CH2), 3.03 (m, 4 H, CH2N), 3.10 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.84 (m, 4 H, CH2OPO), 3.90 (m, 4 H, CH2N), 6.94 (m, 4 H), 7.15 (m, 1 H), 7.42 (m, 4H) ppm.31P NMR (162 MHz, D2O): 6 = +0.18 ppm. MS (ESI-): m / z, % = 648 (80) [M-H]-; HRMS (C28H32N3OIIP2): 648,1509 (found M-H), 648,1512 (calc.).1H NMR of 6a (400 MHz, D2O): 6 = 0.90 - 1 .14 (br. m, 6H, NMe) 1 .22 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.11 (m, 4 H, CH2), 3.03 (m, 4 H, CH2N), 3.07 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.72 (m, 4 H, CH2OPO), 3.93 (m, 4 H, CH2N), 7.04 (m, 4 H), 7.11 (m, 1 H), 7.49 (m, 4H) ppm.31P NMR (162 MHz, D2O): 6 = +0.13 ppm. MS (ESI-): m / z, % = 648 (80) [M-H]-; HRMS (C28H32N3OiiP2): 648,1519 (found M-H), 648,1512 (calc.).

[0293] 1H NMR of 5b (400 MHz, D2O): <5 = 1 .12, 1 .15 (m, 6H, NMe) 1 .20 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.09 (m, 4 H, CH2), 3.03 (m, 4 H, CH2N), 3.07 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.81 (m, 4 H, CH2OPO), 3.90 (m, 4 H, CH2N), 6.99 (m, 4 H), 7.13 (m, 1 H), 7.22 (d, J = 8 Hz, 1 H), 7.44 (m, 4H), 7.59 (s, 1 H), 8.53 (d, J = 8 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): <5 = +0.13 ppm. MS (ESI-): m / z, % = 767 (80) [M-H]-; HRMS (C35H38N4Oi2P2): 767,1889 (found M-H), 767,1883 (calc.). Amax. abs. = 549 nm. Amax. fl. = 580 nm (TEAB buffer, pH ~ 8).1H NMR of 6b (400 MHz, D2O): <5 = 0.90 - 1.14 (br. m, 6H, NMe) 1 .22 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.11 (m, 4 H, CH2), 3.03 (m, 4 H, CH2N), 3.07 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.72 (m, 4 H, CH2OPO), 3.93 (m, 4 H, CH2N), 7.00 (m, 4 H), 7.09 (m, 1 H), 7.17 (d, J = 8 Hz, 1 H), 7.44 (m, 4H), 7.59 (m, 1 H), 8.49 (d, J = 8 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.11 ppm. MS (ESI-): m / z, % = 767 (90) [M-H]-; HRMS (C35H38N4Oi2P2): 767,1877 (found M-H), 767,1883 (calc.).

[0294] Compound 7b with a net charge of -8 was synthesized starting from the aminophenol Precursor AP 3 and Aldehyde 1 (2-formyl-5-nitrobenzoic acid) as shown in Scheme 10. The condensation was followed by alkaline hydrolysis of the acetylated hydroxyl groups and their phosphorylation by conventional methods, particularly as described for Precursor A 02. In the final steps, the intermediate 7a was amino-benzoylated as described for Compounds 5b and 6b.

[0295] Purity and identity of 7b was confirmed by LC / MS analysis.1H NMR of 7b in D2O (400 MHz): <5 = 1.21 (t, J = 7 Hz, 72 H, CH3, 8 Et3N), 2.13 (m, 8 H, CH2), 3.01 (m, 8 H, CH2N), 3.17 (q, J = 7 Hz, 48 H, CH2, 8 Et3N), 3.76 (m, 8 H, CH2OPO), 7.03 (m, 4 H), 7.12 (m, 1 H), 7.17 - 7.21 (m, 2 H), 7.44 (m, 4H), 7.62 (t, J = 8 Hz, 1 H), 8.51 (m, Hz, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.11 ppm. MS (ESI-): m / z, % = 1015 (80) [M-H]-; HRMS (C39H4SN402OP4): 1015,1486 (found M-H), 1015,1434 (calc.). Amax. abs. = 551 nm. Amax. fl. = 581 nm (TEAB buffer, pH ~ 8). Data for compound 7a:1H NMR in D2O (400 MHz): 6 = 1 .21 (t, J = 7 Hz, 72 H, CH3, 8 Et3N), 2.13 (m, 8 H, CH2), 3.01 (m, 8 H, CH2N), 3.17 (q, J = 7 Hz, 48 H, CH2 8 Et3N), 3.76 (m, 8 H, CH2OPO),6.99 (m, 4 H), 7.10 (m, 1 H), 7.51 (m, 4H) ppm.31P NMR (162 MHz, D2O): 5 = +0.14 ppm. MS (ESI-): m / z, % = 896 (80) [M-H]-; HRMS (C32H42N3O19P4): 896,1359 (found M-H), 896,1363 (calc.).

[0296] The hydrazine-substituted Compound 8 was prepared from phosphorylated amine 6a exactly as desribed for Compound 1 i, also utilizing the triazine-containing linker L 1A. The reaction was followed by deprotection of the hydrazine group.1H NMR of 8 (400 MHz, D2O): <5 = 0.90 - 1.14 (br. m, 6H, NMe) 1 .22 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.14 (m, 4 H, CH2), 3.00 (m, 4 H, CH2N), 3.09 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.77 (m, 4 H, CH2OPO), 3.96 (m, 4 H, CH2N), 7.11 (m, 4 H), 7.21 (m, 1 H), 7.54 (m, 4H) ppm.31P NMR (162 MHz, D2O): 6 = +0.19 ppm. MS (ESI-): m / z, % = 648 (80) [M-H]-; HRMS (C31 H35N8O12P2): 773,1846 (found M-H), 773,1850 (calc.). Amax. abs. = 551 nm., Amax. fl. = 579 nm (TEAB buffer, pH ~ 8).Compounds 9a,b,c,d,e and 10a, b

[0297] The title sulfamides were prepared from the amine-containing Compounds 1a and 2a, respectively, on a ca. 5 mg scale. Purity and identity of the compounds was confirmed by LC / MS analyses and HRMS spectrometry. The synthesis involved the commercially available 3-and 4-nitrobenzene sulfonyl chlorides and 5-nitro-1 ,3-benzenedisulfonyl chloride prepared from the corresponding disulfonic acid. The latter was obtained according to

[0020] EP0013079A1. Typically, a sulfonyl or dusulfonyl chloride (15 equiv) was reacted over 1-2 h with the amino-substituted dye (1 equiv) in 50% aqueous MeCN (2 mL for 5 mg of the amine) containing NaHCCh (20 equiv) upon vigorous stirring at RT. The solution was filtered, evaporated, and subjected to a gradient HPLC on a C-18 phase with an aq. TEAB buffer as A and MeCN as B. That gave 40 - 60% of the corresponding nitro compounds. Their reduction with with aquoeous sodium / ammonium sulfide, as described for Compounds 1a and 2a, gave Compounds 9a, b, c and 10a, b, respectively, whose spectral properties are virtually identical to the aminobenzoyl analogues 3a and 4a.

[0298] Analogously were synthesized Compounds 9d and 9e, whose syntheses started form the aminophenol precursor AP 3, which has only one sulfonic acid group. The aminophenol was condensed with 3-nitrophthalic anhydride to give an analogue of the commercial dye Rhodamine 6G. The condensation and reduction of the nitro compound to the amine were performed as described for Compounds 1 b and 1a, respectively.

[0299] 1H NMR of Compound 9c (isomer of 9a and 10a) 400 MHz, D2O): 5 = 1 .21 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.18 (m, 8 H, CH2), 3.03 (m, 8 H, CH2N), 3.12 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.68 (m, 8H, CH2SO3), 6.74 (d, J = 8 Hz, 2 H), 6.90 - 7.12 (m, 2 H), 7.33 (m, 1 H), 7.38 (d, J = 8 Hz, 2 H), 7.51 (d, J = 8 Hz, 2 H), 7.65 - 7.69 (m, 2 H), 7.79 (m, 2 H) ppm. MS (ESI-): m / z, % = 987 (70) [M-H]-; HRMS (C38H44N4Oi7S5): 987,1209 (found M-H), 987,1217 (calc.). Amax. abs. = 547 nm. Amax. fl. = 574 nm (1 M TEAB, pH ~ 8). The purity and identity of Compounds 9a and 10a was confirmed by LC / MS analyses and HRMS spectrometry. Electrokinetic methods showed mobility consistent with the net charge of -4. Amax. abs. = 549 nm. Amax. fl. = 577 nm (1 M TEAB, pH ~ 8). MS (ESI-) of 9a: m / z, % = 987 (80) [M-H]-; HRMS (C38H44N4Oi7S5): 987,1201 (found M-H), 987,1217 (calc.); for the isomeric compound 10a M - H was found 987,1232.

[0300] Compounds 9b and 10 b with an extra SO3H group were analysed by the same methods, which confirmed their purity and identity: MS (ESI) of Compound 9b: m / z, % = 1067 (60) [M-H]-; HRMS (C38H44N402OS6): 1067,0784 (found M-H), 1067,0795 (calc.). Amax. abs. = 549 nm. Amax. fl. = 579 nm (1 M TEAB, pH ~ 8); Compound 10b, MS (ESI-): m / z, % = 1067 (70) [M-H]-; HRMS (C38H44N402oS6): 1067,0779 (found M-H), 1067,0795 (calc.). Amax. abs. = 549 nm. Amax. fl. = 579 nm (1 M TEAB, pH ~ 8). The electrophoretic mobility of the compounds is consistent with the presence of an extra ionizable group, as compared to Compounds 9a and 10a, which have the net charge of -4.

[0301] The synthesis of Compounds 9d and 9e (labels spectrally similar to the commercial dye Rhodamine 6G) started from the aminophenol precursor AP 3 with just one ionizable group, which was prepared by a conventional method from 3-aminophenol. The aminophenol precursor was condensed with 3-nitrophthalic anhydride to give a nitro-substituted rhodamine dye, which upon reduction gave the amino-substituted precursor 9rh. The condensation and reduction to the amine were performed as described for Compounds 1a and 1 b.

[0302] The amine precursor 9rh was benzoylated or sulfonated as described for the dyes 4a,4k,3i and 9a, b, respectively. The intermediate nitrophenyl-substituted compounds were reduced with iron powder and isolated as described for 4d. That gave compounds 9d and 9e with a net charge of -3 and absorbance / fluorescence maxima of 520 / 553 nm (see Table 1).1H NMR of Compound 9d (amide, as 3 x Et3N salt) in D2O (400 MHz): <5 = 1.19 (t, J = 7 Hz, 27 H, CH3, 3 Et3N), 2.16 (m, 4 H, CH2), 3.05 (m, 4 H, CH2N), 3.19 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.81 (m, 4 H, CH2SO3), 6.82 - 6.91 (m, 2 H), 6.95 (m, 4H), 7.27 - 7.34 (m, 3H), 7.37 (s, 1 H),7.60 (s, 1 H), 8.51 (s, 1 H) ppm. MS (ESI-): m / z, % = 751 (70) [M-H]-; HRMS (C34H32N4O12S2): 751 ,7546 (found M-H), 751 ,7595 (calc.). Amax. abs. = 520 nm. Amax. fl. = 553 nm (1 M TEAB, pH ~ 8). Compound 9e (sulfonamide) was identified by means of LC / MS. MS (ESI-): m / z, % = 823 (70) [M-H]-; HRMS (C32H32N4O14S4): 823,0717 (found M-H), 823,0720 (calc.). Amax. abs. = 522 nm. Amax. fl. = 555 nm (1 M TEAB, pH ~ 8).

[0303] The amino-substituted rhodamine dye 9rh with two SO3H groups was prepared as follows: acetic acid (4 mL) was added to a solid precursor AP 3 (140 mg, 0.6 mmol, prepared by a conventional method from 3- aminophenol), then 3-nitrophthalic anhydride (270 mg, 1.7 mmol) and EtsN (110 ml, 0.8 mmol) was added, and the mixture was heated until the solids had dissolved. Afterwards, the solvent removed under vacuum. The residue was heated 3h at 155°C under a nitrogen atmosphere and then refluxed 1 h upon stirring with propionic acid (10 mL) and, after decanting, with EtOAc (10 mL). The solid was dried, dissolved in water (15 mL) containing EtsN (0.5 mL), the solution filtered and evaporated. The crude product was subjected to a gradient HPLC on a C-18 phase with an aq. TEAB buffer as A and MeCN as B. The resulting nitro compound was reduced with aquoeous sodium / ammonium sulfide, as described for Compounds 1a and 2a, gave 37 mg (18% over two steps) of the amine 9rh as (as a 2 x EtsN salt).1H NMR of Compound 9rh (400 MHz, DMSO- d6): 5 = 1.09 (t, J = 7 Hz, 18 H, CH3, 2 Et3N), 1.89 (m, 4 H, CH2), 2.55 (m, 4H, CH2N), 2.98 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.39 (m, 4H, CH2SO3), 6.55 (d, J = 8 Hz, 1 H), 6.66 (m, 2 H), 7.64 (m, 1 H), 7.66 (m, 1 H), 8.10 (m, 3 H), 8.29 (m, 1 H) ppm. MS (ESI-): m / z, % = 588 (70) [M-H]-; HRMS (C26H27N3O9S2): 588,1164 (found M-H), 588,1110(calc.). Amax. abs. = 520 nm. Amax. fl. = 556 nm (1 M TEAB, pH ~ 8).Compounda 11a and 11c (triazine-containing labels)

[0304] The triazine-containing linker L 5A was prepared via a known procedure that involves reaction of an aromatic amine (in this case 3-nitroaniline) with cyanuric chloride in acetic acid (see

[0011] K. A. Kolmakov, J. Het. Chem., 2008, 45, 533-539 and the preparation of L 5A and L 7A). Afterwards, L 5A was reacted with amino-substituted Compound 1a analogously to the preparation of Compound 1 i. The reaction was performed in glacial HAc at RT with no base.

[0305] The syntheses are also depicted in Scheme 11. The monochloro-substituted intermediate with a nitro group (11a) was isolated by means of a gradient HPLC on a C-18 phase with an aq. TEAA buffer andafterwards reacted with 2,2-ethanolamine disulfonic acid disodium salt NH(CH2CH2SO3Na)2 at elevated temperature, as described for Compound 1 k. In the last step, the nitro group was reduced to the amine via a routine procedure (see preparation of 1a) with ammonium sulfide or another suitable reducing agent. Compound 11c with a net charge of -6 was isolated as a 6 x Et3N salt by means of a gradient HPLC with the yield of some 60% over to steps. Typically, Compound 1a (as 4 x Et3N salt) in HAc (6 mL) was stirred with L 5A (170 mg, 50 equiv) overnight. The solution diluted with an equal volume of MeCN containing 50 DI (0.35 mmol) Et3N and then with water (50 mL). The solution was filtered, extracted with EtOAc (100 mL), evaporated at t < 30°C and subjected to preparative HPLC, which gave some 70% of 11 a. Alternatively, the precursor Compound 1a is reacted with linker L 7A at elevated temperature in glacial HOAc, isolated, and then reduced. Properties of 11c: Amax. abs. = 552 nm. Amax. fl. = 583 nm (TEAB buffer, pH ~ 8). MS of (ESI-): mlz, % = 1248 (60) [M-H]-; HRMS (C45H55N9O21S6): 1248,1789 (found M-H), 1248,1759 (calc.). The exchange of the the chlorine atom in 11a to OH was performed in aqueous 20 vol. % TFA at 90°C. That was followed by evaporation and reduction of the crude nitro Compound 11a with ammonium sulfide at 95°C (see preparation of 1a), which gave Compound 11b in a 73% yield. MS of 11b (ESI-): mlz, % = 1033 (70) [M-H]-; HRMS (C41H45N8O16S4): 1033,1841 (found M-H), 1033,1836 (calc.).1H NMR of 11b in D2O (400 MHz): 6 = 1.21 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.15 (m, 8 H, CH2), 3.02 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.71 (m, 8 H, CH2SO3), 6.95 (m, 4 H), 7.11 (m, H), 7.17 (d, J = 8 Hz, 1 H), 7.33 (m, 5H), 7.62 (m, 1 H), 8.51 (d, J = 8 Hz, 1 H) ppm.Compounds 13a and 13b (red-emitting rhodamines with a large net negative charge)

[0306] Precursor AP1 (di-triethylammonium salt, 50 mg, 0.09 mmol) and tetrafluorophthalic anhydride (120 mg, 0.55 mmol) and propionic acid (1 mL) were heated upon magnetic stirring up to 155°C in a septum-sealed flask under a nitrogen purge. As the liquid evaporated and boiling ceased, the residue was kept for 3.5 h at this temperature, cooled to RT and refluxed for 5 min upon stirring with propionic acid (4 mL). The liquid was decanted from the solid, which was subjected to a gradient HPLC on a C-18 reversed phase with an aq. TEAA buffer as A and MeCN as B. Evaporation and repeated freeze-drying of the pure fractions from water furnished 21 mg of Compound 13b (36% theor. from 0.045 mmol expected) as a 4 x Et3N salt (M = 1294). Amax. abs. = 564 nm, Amax. fl. = 592 nm (TEAB buffer, pH ~ 8). MS (ESI-): mlz, % = 889 (90) [M-H]-; HRMS (C32H34F4N2OI5S4): 889,0731 (found M-H), 889,0700 (calc.).1H NMR (600 MHz, D2O): <5 = 1 .18 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.10 (m, 8 H, CH2), 2.95 (m, 8H, CH2), 3.09 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.73 (m, 8H, CH2SO3), 6.98 (d, J = 3 Hz, 2 H), 7.07, 7.09 (2m, 2H), 7.32 (d, J = 12 Hz, 2H) ppm.19F NMR (376.5 MHz, D2O): <5 = -155.7 (m, 1 F), -151 .7 (m, 1 F), -141 .8 (m, 1 F), -137.78 (m, 1 F) ppm.

[0307] 4-Aminothiophenol (20 mg, 0.16 mmol) in DMF (0.2 mL) was added to a chilled solution of Compound 13b (10 mg, 0.008 mmol, 4 x Et3N salt) and Et3N (60 pl, 0.4 mmol) in the same solvent (1 mL) at 0°C andstirred overnight at RT after an argon purge. The solution was diluted with water (30 mL), buffered with HOAc (150 pl), extracted with EtOAc (20 mL) and subjected to gradient HPLC on a C-18 phase. The major isomer was isolated to give 6 mg (55%) of Compound 13a (4 x EtsN salt, M = 1399). Amax. abs. = 571 nm, Amax. fl. = 595 nm (TEAA buffer, pH ~ 5).

[0308] MS (ESI-): m / z, % = 994 (90) [M-H]"; HRMS (C38H40F3N3O15S5): 994,0921 (found M-H), 994,0937 (calc.).1H NMR (600 MHz, D2O): <5 = 1 .20 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.11 (m, 8 H, CH2), 2.97 (m, 8H, CH2), 3.12 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.78 (m, 8H, CH2SO3), 6.59 (d, J = 8 Hz, 2H, 4-C6H4), 6.98 (d, J = 3 Hz, 2 H), 7.07, 7.09 (2m, 2H), 7.16 (d, J = 8 Hz, 2H, 4-C6H4), 7.32 (d, J = 12 Hz, 2H) ppm.19F NMR (376.5 MHz, D2O): <5 = -143.6 (m, 1 F), -131 .4 (m, 1 F), -121 .5 (m, 1 F), ppm.Red-emitting labels: Compounds 14a, c

[0309] As precursors, sulfonated rhodamine dye 14b were taken (see structure below and

[0021] K. Kolmakov, et al., Chem.-Eur. J., 2010, 16, 158-166) and reacted with 4-aminothiophenol (3 - 4 equiv) in DMF containing 10 vol. % H2O and ca. 20 equiv EtsN at 70°C overnight. The reaction mixtures were diluted with water, extracted with ethyl acetate, the aqueous solutions were concentrated and subjected to a gradient HPLC on C-18 phase with aq. TEAB buffer as A and MeCN as B. That gave Compound 14a with the yield of 80% (as 2 x EtsN salt).

[0310] 1 H NMR of 14a (400 MHz, DMSO-d6): 6 = 1 .12 (t, J = 7 Hz, 18 H, CH3, 2 EtsN), 1.44 / 1.54 (2S, 12 H, CH3), 1 .89 - 1.99 (m, 4 H, 2CH2), 2.87 (m, 4 H, CH2 + CH2N), 2.92 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.22 (m, 2 H, CH2SO3), 3.43 (m, 2 H, CH2SO3), 3.55 (m, 4H, CH2 + CH2N), 5.79 (s, 2 H), 6.55 (d, J = 8 Hz, 2H, 4-C6H4), 6.67 (s, 2 H), 7.18 (d, J = 8 Hz, 2H, 4-C6H4), 7.27 (s, 2 H, NH2) ppm;19F NMR (162 MHz, DMSO-d6): <5 = - 143.3 (s, 1 F), -126.9 (m, 1 F), 110.8 (m, 1 F) ppm. Amax. abs. = 624 nm. Amax. fl. = 645 nm (1 M TEAB). MS of 14a (ESI-): m / z, % = 906 (70) [M-H]"; HRMS (C^H^FsNsOgSs): 906,1822 (found M-H), 906,1801 (calc.). The substitution of the second fluorine in 14b with a -S(CH2)2SO3H group was performed in DMF conataining 20 equiv EtsN and ca. 10 equiv HS(CH2)2SC>3Na at 90°C overnight. Chromatographic purification gaveCompound 14c with the yield of ca 50% (as 3 x EtsN salt). Purity and identity of the compound were confirmed by LC / MS analyses and HRMS spectrometry. MS of 14c (ESI-): m / z, % = 1028 (60) HRMS(C46H44F2N3O12S5): 1028,1488 (found M-H), 1028,1492 (calc.). For 14a and 14c Amax. abs. = 625 - 626 nm, Amax. fl. - 650 nm. (1 M TEAB, pH ~ 8).Compound 16 (derivative of Rhodamine B)

[0311] Rhodamine B (hydrochloride salt, 50 mg, 0.10 mmol) was refluxed 2h in chlorobenzene (6 mL) containing POCh (0.1 mL) using an air reflux condenser and a CaCh-tube. The solution was evaporated on a rotary evaporator, and the procedure was repeated two more times with fresh potions of the solvent.

[0312] Compound L 2B-NO2 (20 mg, 0.027 mmol as a 2 x EtsN salt) was dissolved in a mixture of DMF (8 mL), water (0.8 mL) and EtsN (0.1 mL), the solution chilled to 0°C and added in one portion onto a freshly prepared solid Rhodamine B chloride with vigorous swirling. The chloride was pre-cooled to the same temperature under a nitrogen purge. The solution was left stirring overnight at RT, water (100 mL), DCM (100 mL) and MeCN (20 mL) were added to the solution, and the layers were separated after shaking. The deeply coloured aqueous layer was extracted with DCM (100 mL), filtered, concentrated, and subjected to a gradient HPLC on C-18 phase with 0.3% aq. EtsN as A and MeCN as B. Pure fractions were evaporated to give 22 mg of the intermediate Compound I6-NO2, whose identity was confirmed by LC-MS. The reduction of the nitro group was performed as follows: the product was dissolved in water (6 mL) containing sodium sulfide hydrate (60% wt. Na2S, 200 mg) and NH4CI (100 mg). The solution was heated for 2 h in a screw-cup test tube at 85°C upon stirring, chilled to RT, acidified with HOAc (0.20 mL) diluted with and equal volume of water and filtered (0.45 pm). The solution was basified with EtsN (0.5 mL), evaporated, and subjected to a gradient HPLC, which after freeze-drying, gave 13 mg of Compound 16 (~46% over two steps, as a mono-EtsN salt with M = 1029). Alternatively, Rhodamine B chloride was reacted directly with linker L 2B (taken in a three-fold excess) to give the target compound in one step with a lesser yield (~30%). Dye properties: Amax. abs. = 565 nm, Amax. fl. = 590 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 927 (90) [M-H]-; HRMS (C42H54N6O12P2S): 927,2926 (found M-H), 927,2917 (calc.).1H NMR (600 MHz, D2O): 6 = 0.83 - 1.20 (br. m, 12H, 4CH3, NEt) 1.23 (t, J = 7 Hz, 9H, CH3, Et3N), 3.10 (m, 4H, 2CH2), 3.13 (q, J = 7 Hz, 6 H, CH2, Et3N), 3.33 (m, 4H, 2CH2), 3.40 - 3.55 (br. m, 8H, 4CH2, NEt), 3.72 (m, 4H, 2CH2), 6.64 (m, 2H), 6.74 (br. m, 2H), 6.71 - 6.73 (m, 2H), 6.80 (m, 1 H), 6.96 (br. m, 1 H), 7.15 (d, J = 3 Hz, 1 H), 7.51 , 7.56 (2m, 2H), 7.37 (d, J = 9 Hz, 1 H), 7.79 (t, J = 10 Hz, 1 H), 7.84 (t, J = 10 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): 6 = +0.17 ppm.Compound 17 (derivative of Rhodamine B)

[0313] The rhodamine-containing precursor 17-Rh was prepared from aminophenol AP 1 and phthalic anhydride as described for Compound 1 b. The rhodamine was amidated with a linker L 1 B-NO2 in presenceof HATU reagent in DMF by a conventional procedure for a peptide-type coupling in dye chemistry

[0018] (K.Kolmakov, et al., Photochem. Photobiol. Sci., 2020, 19, 1677-1689).

[0314] The nitro-substituted Compound 17-NO2 was reduced with an iron powder (Fe°) in aq. HOAc acid as described for 4b to give the amino-substituted Compound 17 with the yield of ca. 40% over 2 steps. The products were isolated by means of a gradient HPLC with TEAB buffer and MeCN. The purity and identity the intermediate compounds were confirmed by LC / MS analyses. Analytical data for 17: Amax. abs. = 563 nm, Amax. fl. = 588 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 1056 (90) [M-H]-; HRMS (C42H51N5O17S5): 1056,1834 (found M-H), 1056,1805 (calc.).1H NMR (400 MHz, MeOH-d4): 6 = 1 .24 (t, J = 7 Hz, 27H, CH3, 3 Et3N), 2.09 (m, 8 H, CH2), 2.89 (m, 8H, CH2N), 3.09 (q, J = 7 Hz, 18 H, CH2, 3 Et3N), 3.60 (m, 8H, CH2SO3), 6.71 (m, 2 H), 6.69 (m, 2 H), 6.71 - 6.73 (m, 2 H), 6.79, 6.88 (dd, J = 3 and 9 Hz, 1 H), 6.82 - 6.84 (m, 3 H), 7.37 (d, J = 9 Hz, 1 H), 7.54 (m, 1 H), 7.76 (m, 1 H) ppm.Compound 18 (a red-emitting glycan tag with a large negative net charge)

[0315] The fluorinated rhodamine dye 14b with two sulfonate groups (13 mg, 0.015 mmol, for preparation see

[0021] K. Kolmakov, et al., Chem.-Eur. J., 2010, 16, 158-166) was combined with linker L 3B (25 mg, 0.036 mmol, 2.3 equiv) in DMF (0.8 mL) containing Et3N (50 pl, 23 equiv) in a 2 mL vial with a stirring bar. The vial was flushed with nitrogen, sealed and the solution stirred for 15 h at 70°C. The solution was diluted with water (10 mL) and subjected to a gradient HPLC on a C-18 phase with aq. TEAB buffer and MeCN. That gave 15 mg of Compound 18 (58% theor.) as a dark-blue amorphous solid (4 x Et3N salt with M = 1712). Amax. abs. = 626 nm, Amax. fl. = 653 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 1307 (90) [M-H]-; HRMS (C5iH57F3N4Oi9P2S5): 1307,1524 (found M-H), 1307,1570 (calc.).

[0316] 1 H NMR (600 MHz, D2O): <5 = 1.25 (t, 36 H, J = 7 Hz, CH3, 4Et3N), 1.47 / 1 .54 (2s, 12 H, CH3), 1.73 - 1.92 (m, 6 H, 3CH2), 2.56 (m, 2H, CH2), 2.75 (m, 2H, CH2) 2.58 - 3.13 (4m, 8 H, 4CH2), 3.17 (q, 24 H, J = 7 Hz, CH2, 4Et3N), 3.64 (m, 4H, 2OCH2), 3.71 (d, 2 H, J = 14 Hz, CH2SO3), 3.89 (d, 2 H, J = 14 Hz, CH2SO3), 4.02 (m, 4H, 2NCH2), 5.89 (s, 2 H), 6.28 (d, J = 9 Hz, 1 H), 6.75 (d, J = 9 Hz, 1 H), 7.27 (m, 3 H) ppm;19F NMR (376.5 MHz, D2O): <5 = -142.0 (s, 1 F), -123.29 (m, 1 F), 109.2 (s, 1 F) ppm.31P NMR (162 MHz, D2O): 6 = +0.14 ppm.Compounds 19, 20 and 21

[0317] Preparation of the linker-dye conjugates involved the active (NHS) esters of commercially available dyes. Acridine Orange NHS ester was synthesized by a conventional method, which involves alkylation of the parent dye with a halogenated carboxylic acid ester, which was followed by saponification and installation of an NHS moiety as described

[0022] by O. G. Kulyk, et al., Dyes and Pigments, 200, 2022, 110148. For the alkylation, ethyl 4-bromobutyrate was taken. Other active esters, i.e., 7-(Diethylamino)-coumarin-3-carboxylic acid NHS ester and Sulfo-Cyanine 5 NHS esters were taken as commercially available products and reacted with Linker L 3B following the standard recipe (see the section “Conjugation of commercially available dyes as active esters with custom-synthesized linkers”). The purity and identity of the linker-decorated glycan labels 19, 20 and 21 was confirmed by LC / MS analyses. The HRMS spectrometry data of these three dyes and their properties listed in Table 1. The synthesis of Compound 16 (a Rhodamine B derivative), for which the same Linker L 3B was utilized, is somewhat special and is described separately.

[0318] For the identification of the cyanine-containing Compound 19 the following1H NMR spectrum (400 MHz, D2O) was measured: <5 = 1 .21 (t, J = 7 Hz, 36H, CH3, 4 Et3N), 1.52 (m, 2H, CH2), 1 .75 (S, 12 H, 4 CH3), 1 .60 - 1.90 (m, 4H, 2CH2), 2.20 (t, J = 6 Hz, NCH2), 3.10 (m, 4H, 2CH2), 3.14 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.35 (m, 4H, 2CH2), 3.59 (t, J = 6 Hz, 2H, CH2), 3.64 (s, 3H, NCH3) 3.84 (m, 4H, 2CH2), 4.12 (m, 2H, CH2CO), 6.34 (m, 2H, 2CH), 6.70 (m, 1 H, CH), 7.09 (m, 1 H), 7.29 (m, 1 H) 7.30 (d, J = 9 Hz, 1 H), 7.35 (t, J = 8 Hz, 2H, 2CH), 7.90 (m, 4H, 4CH), 8.31 (t, J = 8 Hz, 2H, 2CH) ppm.31P NMR (162 MHz, D2O): <5 = +0.41 ppm.Sulfo-Cyanine 5NHS esterCompound 22 (a fluorescein derivative)

[0319] 5-Aminofluorescein was reacted with Linker L 3A at elevated temperature, as described for Compound 1k. That was followed by isolation and cleavage of the f-Boc group and isolation by means of a gradient HPLC with the yield of ca. 25% (2 x EtaN salt, M = 889).Alternatively, 5-aminofluorescein was first reacted with dichloro-substituted linker L 1A and then with 2,2- ethanolamine disulfonic acid disodium salt NH(CH2CH2SO3Na)2, as described for Compound 1j and isolatedby means of preparative HPLC. That was followed by deprotection of the hydrazine group analogously to Compounds 1i and 1 k. Purity and identity was confirmed by LC / MS (ESI-) analyses. Amax. abs. = 490 nm. Amax. fl. = 522 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 772 (100) [M-H]"; HRMS (C27H25N7O11S2): 686,0912 (found M-H), 686,0975 (calc.).1H NMR of 22 (400 MHz, DMSO-d6): <5 = 1 .19 (t, J = 7 Hz, 18 H, CH3, 2 Et3N), 3.08 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.16 - 3.20 (m, 4 H, CH2), 3.74 - 3.94 (m, 4 H, CH2SO3), 5.86 (br s, 2H), 6.55 (m, 4H), 6.90 (m, 3H), 7.04 (m, 1 H), 7.33 (m, 1 H).Compound 23 (a rhodamine dye with enhanced negative net charge)

[0320] Compound 23 was synthesized by reaction of a freshly prepared Rhodamine B chloride and the linker L B4-NO2. The reaction was performed as described for compound 16, and the intermediate nitro product was isolated and reduced in the same fashion. That gave Compound 23 with a yield of 43% in two steps (isolated as 2 x Et3N salt, M = 1210). Properties: Amax. abs. = 567 nm, Amax. fl. = 591 nm (TEAB buffer, pH ~ 8). MS (ESI-): m / z, % = 1007 (70) [M-H]-; HRMS (C42H55N6OI5P3S): 1007,2569 (found M-H), 1007,2581 (calc.).1H NMR (600 MHz, D2O): 6 = 0.85 - 1 .21 (br. m, 12H, 4CH3, NEt) 1.22 (t, J = 7 Hz, 18 H, CH3, 2 Et3N), 3.13 (q, J = 7 Hz, 12 H, CH2, 2 Et3N), 3.35 (m, 4H, 2CH2), 3.40 - 3.55 (br. m, 8H, 4CH2, NEt), 3.81 (m, 4H, 2CH2), 3.94 - 4.00 (m, 6 H, CH2O), 6.64 (m, 2H), 6.72 (br. m, 2H), 6.73 - 6.76 (m, 2H), 6.80 (m, 1 H), 6.96 (br. m, 1 H), 7.15 (d, J = 3 Hz, 1 H), 7.54, 7.66 (2m, 2H), 7.42 (d, J = 9 Hz, 1 H), 7.83 (t, J = 10 Hz, 1 H), 7.91 (t, J = 10 Hz, 1 H) ppm.31P NMR (162 MHz, D2O): <5 = +0.27 ppm.Compound 24 (a cyanine dye)

[0321] Compound 24 was prepared from the custom-made linker L3 A and the commercially available Sulfocyanine 5 amine as follows: 10 mg (0.015 mmol, 2 x Et3N salt, M = 678) of linker L 3A was dissolved in EtOH (1 mL) containing Et3N (5 mL) and reacted with the amine (32 mg, 0.045 mmol) overnight in a sealed vial under stirring at 90°C.

[0322] The solution was evaporated, dissolved in 5 vol. % aq. TFA and heated 30 min at 60°C. The solution was evaporated and subjected to a preparative gradient HPLC on a C-18 phase with aq. TEAB buffer as A and MeCN as B. The homogeneous fractions were pooled and freeze-dried to give 7 mg (30%) of Compound 24 (3 x Et3N salt, M = 1383). MS (ESI-): m / z, % = 967 (90) [M-H]"; HRMS (C45H64N10O13S4): 1079,3451 (found M-H), 1079,3459 (calc.).Example 1 : Conjugation of commercially available dyes as active esters with custom-synthesized linkers

[0323] Piperazine-based linkers, e. g., Linker L 2B were reacted with active (NHS) esters of commercially available dyes as follows: a 2% wt. stock solution of L 2B as a 2 x EtsN salt with M = 706 (isolated by means of HPLC and two times freeze-dried from water) in a mixture DMF - H2O (5:1) was prepared. To this solution 2 or more equiv EtsN was added, the solution was stored at +5°C and used as it is. In a vial containing 1 equiv of a solid dye NHS ester a chilled (~0°C) stock solution of 1 .5 - 3 equiv of L 2B in aq. 50 - 75% DMF was added, the mixture shaken and left overnight at RT. The reaction solution was diluted with 5 - 10 volumes of water, and the dye conjugates were isolated by means of a gradient HPLC on a C-18 reversed phase with aq. TEAB buffer or 0.3% vol. EtsN as A and MeCN as B. Most of the unmodified dyes can be extracted from a basified (~ 1 / 100 vol. EtsN) reaction solution with EtOAc or DCM beforehand. Also, with a large excess of a lipophilic dye NHS ester, the reaction can be performed such that the amine-containing

[0324] Llinker L 2B reacts completely, while the unreacted dye is extracted from aqueous solutions. Organic dyes in the form of acid chlorides, e. g., Rhodamine B chloride, can react with the aromatic amino group in linker L 2B too. Therefore, in case of Rhodamine B chloride, the corresponding nitro compound (2B-NO2) was utilized to obtain better yields. The benzoylation was followed by reduction to the amine (see above for Compound 16). Purity and identity of the dye-linker conjugates were confirmed by LC / MS analyses and HRMS spectrometry. Also, labeling tests with glycans were performed in each case. Importantly, the commercially available NHS esters of the fluorophores do not react with the aromatic amino group in the custom-made linkers, so its temporal protection becomes unnecessary. For the preparation of Compound 18, a red-emitting rhodamine with reactive aromatic halogen 14b and the thiol-substituted Linker L 3B were used. Similarly, commercial dyes in the form of maleimides or iodoacetamides can be conjugated to L 3B. Exemplary compound 24 was prepared by reacting the commercially available aminohexylamino derivative of sulfo-Cy5 with Linker L 3A. The conjugates were isolated by means of a preparative gradient HPLC. Purity and identity of the compounds was confirmed by LC / MS analyses. Table 1 shows spectral maxima, molecular weights, formulae, net charges, and the MS spectrometric (ESI-) data of the most representative dyes of the invention.Table 1 Principal properties of selected glycan labels of the invention: spectral maxima (TEAB buffer, pH = 8), molecular weights (MW), brutto molecular formulae, net charges (z) and m / z ratios. (* -- actual net charge of phosphorylated compounds might depend on the pH).Example 2: Glycan labeling

[0325] Reductive amination of exemplary substrates, e.g., maltotriose was performed using a modified protocol decribed in

[0023] (see L. R. Ruhaak, et al., Journal of Proteome Research, 2010 9 (12), 6655-6664) as follows: to a 10 - 20 mmol / L aq. solution of a fluorescent tag (1 equiv) were consequtively added citric acid (30 equiv, trihydrate, as a 3 M aq. solution) or acetic acid (50 - 100 equiv), a proper reducing agent (an amineborane, hydroxyboron or hydroxydiboron complex, 20 - 50 equiv, as a 0.5 - 4.0 M solution in DMSO or water), and a glycan (3 - 10 equiv, as a 5 - 10 % wt. aq. solution). The mixture was vortexed and incubated for 0.5 - 5h at 37 - 45°C. The course of reaction was monitored by HPLC with an absorbance UV / vis or fluorescence detector, as shown in Figure 3. When necessary, the products are isolated by means of a gradient HPLC on C-18 or hexyl-phenyl phase (for APTS and the prior art pyrene dyes, see Table 2) with aq. 0.03 M TEAB buffer as A and MeCN as B after the reaction solution is diluted and buffered to pH of 7 - 8. Purity and identity of the glycan conjugates was confirmed by LC / MS analyses and HRMS spectrometry. As an illustrative example, the analytical data for the conjugate of Compound 4a and maltotriose as a single glycan are presented (see also Figure 3a and the structure below).MS (ESI-): m / z, 1439 (20%) [M-H]-; HRMS (C57H75N4O31S4): 1439,3289 (found M-H), 1439,3298 (calc.), 719 (60%) [M-2H]2", 719,1109 (found M-2H / 2), 719,1100 (calc.).1H NMR of 4a-maltotriose in D2O (400 MHz): 6 = 1.21 (t, J = 7 Hz, 36 H, CH3, 4 Et3N), 2.14 (m, 8 H, CH2), 3.01 (m, 8 H, CH2N), 3.14 (q, J = 7 Hz, 24 H, CH2, 4 Et3N), 3.43 (m, ~4 H), 3.74 (m, 8 H, CH2SO3), 3.88 - 3.90 (m, ~6 H), 4.22, 4.41 (m, m, 2 H, CH2N), 5.37 (m, ~4 H), 5.22 (m, ~4 H), 4.65 (m, ~4 H), 6.99 (m, 4 H, Ar), 7.04 (m, 1 H, Ar), 7.12 (d, J = 8 Hz, 1 H, Ar), 7.33 (m, 5 H, Ar), 7.62 (t, J = 8 Hz, 1 H, Ar), 8.48 (d, J = 8 Hz, 1 H, Ar) ppm.

[0326] The same protocol was also used on a nanomolar scale for analyses of individual glycans and glycan mixtures, e.g., dextran ladders. The C(G)E-LIF analyses are presented in Figures 2, 4, 5, 6, 7, 8, 9, 11 and 13. All stock solutions were stored at -20°C. Usually, the labelling of complex glycan mixtures is performed with an excess (5 - 10 equiv) of a glycan tag (1 - 2 pl of a 20 mmol / L glycan tag solution) and the same volumes of reagent stock solutions). The labelling reaction was stopped by adding a concentrated (80%) aqueous acetonitrile (ca. 50 - 100 vol.). Post-derivatization cleanup removed the rest of the unreacted tag when it was necessary. It was performed by HILIC-SPE as follows: samples were applied on a filter plate well containing a bead slurry and incubated for 5 min at ambient temperature for binding. Samples were washed and eluted with suitable aqueous buffers. Finally, the substrates were analysed by means of xCGE-LIF using a genetic analyser equipped with capillary array filled with an appropriate polymer. The samples were electrokinetically injected and analysed by conventional methods. Figure 13 shows electropherograms of thelactose-based sugars Lewis A tetraose, Lacto-N-neo-tetraose, 3’Fucosyllacose and 6’-Sialyllactose, labelled with Compound 4a. These small sugars show a big difference in migration times and thus provide their easy distinction in complex mixtures.Table 2 Labeling degrees with maltotriose as an exemplary single glycan under mild conditions and in a short timescale and without evaporation: prior art compounds vs selected new labels. ‘Compound 1a is a dye precursor with no linker.

[0327] Table 2 shows the labeling degrees with maltotriose as exemplary single glycan under mild conditions. Two exemplary prior art glycan labels (see [1] E. A. Savicheva, et al., Angew. Chem. Int. Ed. 2021 , 60, 3720; [2] ibid., 2020, 59, 5505; and [3] Sulfonated 2(7)-aminoacridone and 1 -aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis. W02020151804A1) and the predominantly used label APTS were taken for comparison. Differently to the sources [1] and [2], neither evaporation nor freeze-drying was needed. Also, one does not need to perform the labelling stepwise or use strong acids (e.g., malonic acid). The two exemplary prior art compounds show very poor yields, far less compared to the labels of the present invention and even to APTS. This is despite the large excess of the glycan. Reductive amination was performed as described in Example 2. The experiments involved an excess (10 equiv) of the glycan and ca. 200 equiv of the reducing agent (amine-borane complex as a 20 wt.% solution in DMSO). Each reaction was performed in aqueous solution containing 25 - 30vol. % acetic acid and up to ca. 25 vol.% DMSO. The reaction time was set to 30 min, and the temperature to 45°C and 60°C, respectively. Data were obtained by means of HPLCanalyses on a reversed-phase hexyl-phenyl or C-18 column with aqueous TEAB buffer and acetonitrile as the mobile phase (see also Figures 3a, 3b and related descriptions). The fluorescent detector (except for precursor Compound 1a, for which an absorbance detector was used) was set to the emission maxima of each label. Analysis load ~0.05 nmol of the label. The conversion degrees were determined by measuring peak areas of the residual dye and product at isosbestic point (see Table 1 for spectral properties). Differently to APTS and other pyrene dyes (see sources

[0001] and [2]), the fluorescence maxima of the new labels with linkers do not show noticeable shifts after their conjugation to glycans.

[0328] Structures of the prior art compounds are disclosed in the references cited. Underthe same conditions, yet at a higher temperature or for a longer time, the labeling tests with an excess of the dye (5 and 10 equiv., respectively) were performed. In the experiments with an excess of the substrate, maltotriose was used as an aqueous (0.2M) stock solution. The labeling degree (conversion) of the given amount of glycan was estimated by means of HPLC basing on the amount of the dye that had reacted. Despite of the excess of the dye, the labeling degrees with the prior art compounds remain quite low even at a longer incubation time. Under the same conditions, all the selected labels of the invention demonstrate much higher labelling degrees. Particularly, despite the very low concentration of the glycan in the mixture and despite the short reaction time, > 60% of the glycan is labelled with Compounds 3a, 4a and 18. The results demonstrate that decoration of fluorophores with a carbohydrate-reactive linker greatly accelerates and facilitates glycan labeling.Example 3: Human immunoglobulin (IgG) analyses

[0329] N-Glycans of a commercially available human IgG were released with PNGase F and labelled with an excess (at least 4 equiv) of the dye. The released N-glycans were then resuspended in 1 pL MilliQ-water, then 1 pL 20 mmol / L aq. solution of a label (e. g., 3a), 1 pL 3,6 M aq. citric acid and 1 pL of a reductive reagent (amine-borane complex) as a solution in DMSO (4 M) was added. After incubation at 37 °C the reaction mixture was quenched with 100 pL 80% MeCN and treated with a HILIC-SPE phase to remove the excess of the label, as described in

[0024] R. Hennig, et al., in “ / V-Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF in Carbohydrate-Based Vaccines. Methods in Molecular Biology, 2015, 1331, 123-143, B. Lepenies (ed.) Humana Press, NY. See also

[0025] R. Hennig, et al., in ’’Towards personalized diagnostics via longitudinal study of the human plasma / V-glycome” in Biochim Biophys Acta, 2016, 1860 (8), 1728-1738. Exemplary fingerprint analyses are presented in Figures 8, 9, 12 and 14. In general, it is shown that the N-glycans of human IgG are well-resolved. The analyses with a laser excitation are presented in Figures 8, 12 and 14. A typical CGE- LEDIF analysis with an LED-induced excitation is exemplified in Figure 9. Importantly, Figure 9 shows that the tuneable spectral properties of the new compounds (see Figure 1 , Table 1 , and syntheses descriptions for spectral data) allow glycan analyses on the instruments with other light sources, e.g. LEDs. The latter are usually not able to excite the predominantly used glycan label APTS. The actual details, instruments, settings and brief conclusions are presented in the descriptions of the figures. In general, it is shown that the N-glycans of human IgG are well-resolved and can be detected on widely distributed CGE-LIF instruments with multichannel detection using conventional argon lasers, even though the latter do not perfectly match the excitation maxima of the exemplary compounds 3a and 4a. As shown in Figures 12 and 14, a good resolution of the N- glycan substrates is achieved at shorter or at comparable to APTS absolute migration times on the conventional CGE-LIF instruments. Also, Figure 9 shows that a good resolution is still achieved even at extremely short absolute migration times on an inexpensive CGE-LEDIF instrument with a shorter capillary. An analysis overlay in Figure 14 shows that labeling with the new compounds (3a and 4a taken as examples)yields much higher signal intensities than by labeling with APTS. In Figure 14 the signal was expressed as the signal-to-noise-ratio (SNR). Importantly, compounds 3a and 4a have zero crosstalk with APTS, which enables a two-channel detection (see Figure 1). The latter feature is crucial for the method of glycan analysis that utilizes two spectrally different lalbels in a sample and in a standard composition, as described in

[0026] WO2020151799A1.

[0330] The non-reductive labeling involved an incubation of a 10 - 20 mmol / L aq. solution of a hydrazinecontaining label with a glycan substrate (5 equiv, as a 5 - 10 % wt. aq. solution) in presence of acetc acid, whose concentration in the mixture was 5 - 50% vol, also in the form of an acetate buffer. Usually, the incubation was performed for 1 h at 37°C or overnight at RT. A typical electropherogram of an untreated reaction mixture with a single glycan is shown in Figure 10.REFERENCES1. E. A. Savicheva, et al., Angew. Chem. Int. Ed. 2021 , 60, 3720-3726.2. E. A. Savicheva, et al., Angew. Chem. Int. Ed. 2020, 59, 5505-5509.3. W02020151804A1 . Sulfonated 2(7)-aminoacridone and 1 -aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis.4. M. A. Fomin, et al., Anal. Chem., 2020 92 (7), 5329-5336..5. I. Zhang, et al., RSC Adv., 2015, 5, 66416-66419.6. Y. Wang, et al., Nat. Mater. 2019, 18, 1335-1342.7. M. V. Kvach, et al., Bioconjugate Chem., 2009, 20, 1673-1682.8. G. Yu. Mitronova, et al., Eur. J. Org. Chem., 2015(2), 337-349.9. G. M. Coppola in J. Het. Chem., 1999, 36(3), 563-588.10. US5420257A. Reactive triazine dyes, their preparation and use.11. K. A. Kolmakov, J. Het. Chem., 2008, 45, 533-539.12. N-h. Ho, R. Weissleder and C-H. Tung, Tetrahedron, 2006, 62, 578-585.13. S. De, et al., Org. Biomol. Chem., 2015, 13, 3950-3962.14. Xh. Liu, et al., Cellulose, 2018, 25, 6745-6758.15. WO 2006 / 122793. Copolymers base on phosphorous-containing monomers, methods for the production thereof and their use.16. G. Mudd, et al., Methods and Applications in Florescence, 2015, 3, 045002.17. Y. C. Chen, Y. T. Kuo and T. H. Ho, Photochem. Photobiol. Sci., 2019, 18, 190-197.18. K. Kolmakov, et al., Photochem. Photobiol. Sci., 2020, 19, 1677-1689.19. A. Chevalier, P.-Y. Renard, and A. Romieu, 2014, Chem. - Eur. J., 20, 8330-8337.20. EP0013079A1. Symm. phenyltris(sulfonylimino)tri-benzene sulfonic acids and salts, a method of inhibiting the complement system in a body fluid with such a compound, a method for the preparation of such compounds and a pharmaceutical composition comprising such a compound.21. K. Kolmakov, et al., Chem. - Eur. J., 2010, 16, 158-166.22. O. G. Kulyk ef a / ., Dyes and Pigments, 2022, 200, 110148.23. L. R. Ruhaak, et al., Journal of Proteome Research, 2010 9 (12), 6655-6664.24. R. Hennig, et al., in “ / V-Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF in Carbohydrate-Based Vaccines. Methods in Molecular Biology, 2015, 1331, 123-143, B. Lepenies (ed.) Humana Press, NY.25. R. Hennig, et al., in ’’Towards personalized diagnostics via longitudinal study of the human plasma N- glycome” in Biochim Biophys Acta, 2016, 1860 (8), 1728-1738.26. WO2020151799A1 Advanced methods for automated high-performance identification of carbohydrates and carbohydrate mixture composition patterns and systems therefore as well as methods for calibration of multi wavelength fluorescence detection systems therefore, based on new fluorescent dyes.27. H. Kojima, et al., Anal. Chem. 2001 , 73, 1967-1973.The invention may also be described by any of the following clauses:Clauses1 . A compound of general formula I:wherein:Ring A is a 3-10 membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L2 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH-, -N(R2)(R3), -O-, -S-, - S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, -N(R2R3), -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;Z is an ionizable group;R1 is selected from H, 2-, 3-, or 4-aminophenyl, NH2, PG, and NH-PG wherein PG is a protective group, or R1 is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl;R2 and R3are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, -CH2-;FL comprises a fluorophore; and m is an integer of from 0 to 12, or a salt or hydrate thereof.2. A compound of general formula (IV):wherein:Ring A is a 3- to 10-membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L5 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -NH2, -NH2+, -C(O)NH-, -C(S)NH-, -S(O)2NH-, - N(R2)(RS), -O-, -S-, -S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, -NH2, -N(R2Rs), -C(O)R2-, -CONH-, - CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;R1 is selected from H, NH2, 2-, 3-, or 4-nitrophenyl, PG and NH-PG wherein PG is a protective group optionally wherein the protective group is selected from t-butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9- fluorenylmethoxycarbonyl (Fmoc);R2 and R3 are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-;Z is an ionizable group; m is an integer of from 0 to 12; andQ is absent or selected from OH, H, Br, Cl, F, I, CO2H, CO2LG, (CH2)jCO2 LG , CO(CH2)jCO2LG and (CH2)jOCO2LG, wherein j is an integer of from 1 to 12, and LG is a leaving group selected from N- succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.3. The compound of clause 1 or clause 2, wherein ring A is a 5- to 6-membered aryl or heteroaryl ring.4. The compound of any one of clauses 1 to 3, wherein ring A is phenyl.5. The compound of any one of clauses 1 to 3, wherein ring A is triazinyl (e.g. 1 ,3,5-triazinyl).6. The compound of any preceding clause, wherein PG is selected from the group consisting of t- butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9-fluorenylmethoxycarbonyl (Fmoc).7. The compound of any preceding clause, wherein Li is absent or is selected from a bond, alkylene (e.g.C1-C12, Ci-Cs, or Ci-Ce alkylene) and -N(R2)(R3).8. The compound of any one of clauses 1 to 6, wherein Li is absent.9. The compound of any one of clauses 1 to 6, wherein Li is a bond.10. The compound of any one of clauses 1 to 6, wherein Li is alkylene, optionally C1-C12, Ci-Cs, or C1-C6 alkylene, further optionally wherein Li is methylene or ethylene.11 . The compound of any one of clauses 1 to 6, wherein Li is -N(R2)(Rs), optionally wherein R2 and R3 are each alkylene (e.g. C1-C12, Ci-Cs, C1-C6 or C1-C4 alkylene), further optionally wherein R2 and R3 are each ethylene.12. The compound of any one of clauses 1 to 6, wherein Li is NR2R3 wherein R2 is H and R3 is alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce alkylene), optionally wherein R3 is tertiary alkylene.13. The compound of clause 1 , or any one of clauses 3-12 when dependent on clause 1 , wherein L2 is absent or is selected from: a bond, alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce or C1-C4 alkylene), heterocyclyl, -NH-, - C(O)-, -C(O)NH-, -C(S)NH-, -O-, and -S-, optionally wherein the alkylene or heterocyclyl is substituted with one or more substituents independently selected from -F, -NR2-, -NH2, -NR2R3-, -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z.14. The compound of clause 13, wherein L2 is absent.15. The compound of clause 13, wherein L2 is a bond.16. The compound of clause 13, wherein L2 is -NH-.17. The compound of clause 13, wherein L2 is alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce or C1-C4 alkylene), optionally wherein L2 is methylene or ethylene, further optionally wherein the alkylene is substituted with one or more substituents independently selected from -S-, O, and N.18. The compound of clause 13, wherein L2 is -C(S)NH- or -C(O)NH-.19. The compound of clause 13, wherein L2 is a heterocyclyl or heteroarylene, optionally wherein the heterocyclyl or heteroarylene is piperidyl, piperazinyl or triazinyl, further optionally wherein the piperidyl, piperazinyl or triazinyl is substituted with one or more substituents selected from NR2, -NR2R3-, C(O)R2, and Z.20. The compound of clause 13, wherein L2 is triazinyl is substituted with one or more substituents selected from -NH-, Z and -NR2R3-, wherein R2 and R3 are each alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce or C1-C4 alkylene) substituted with Z.21 . The compound of clause 13, wherein L2 is piperazinyl, optionally wherein the piperazinyl is substituted with -C(O)R2-, further optionally wherein R2 is alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce or C1-C4 alkylene).22. The compound of any preceding clause, wherein R2 and R3 are independently selected from C1-C12 alkylene, e.g. Ci-Cs, Ci-Ce or C1-C4 alkylene.23. The compound of any preceding clause, wherein both R2 and R3 are C1-C4 alkylene, preferably ethylene.24. The compound of any one of clauses 1 to 22, wherein R2 is H and R3 is alkylene (e.g. C1-C12, Ci-Cs, Ci-Ce alkylene), optionally wherein R3 is tertiary alkylene.25. The compound of any preceding clause, wherein X is a bond.26. The compound of any one of clauses 1 to 24, wherein X is -C(O)-.27. The compound of any one of clauses 1 to 24, wherein X is -NH-.28. The compound of any one of clauses 1 to 24, wherein X is alkylene (e.g. C1-C12 alkylene, Ci-Cs alkylene, or Ci-Ce alkylene), optionally wherein X is methylene or ethylene.29. The compound of any one of clauses 1 to 24, wherein X is alkenylene (e.g. C1-C12, Ci-Cs, or Ci-Ce alkenylene), optionally wherein X is ethenylene.30. The compound of any one of clauses 1 to 24, wherein X is -S(O)2-.31 . The compound of any one of clauses 1 to 24, wherein X is -S-.32. The compound of any preceding clause, wherein X and L2 are not both a bond.33. The compound of any preceding clause, wherein Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-.34. The compound of any one of clauses 1 to 32, wherein Y is a bond.35. The compound of any one of clauses 1 to 32, wherein Y is -S(O)2-.36. The compound of any preceding clause, wherein each Z is independently selected from -SO3H, - OSO3H, -SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2.37. The compound of any preceding clause, wherein m is 0, 1 , 2, 3 or 4.38. The compound of any preceding clause, wherein group -Y-L1-Z has a structure selected from: - N((CH2)pSO3H)2; -N((CH2)pCO2H)2; -N((CH2)pOP(O)(OH)2)2; -SO3H; -CO2H; -S(O)2-N((CH2)pOP(O)(OH)2)2; - C(O)-N((CH2)pOP(O)(OH)2)2; -S(O)2-N((CH2)pCO2H)2; -C(O)-N((CH2)pCO2H)2; -S(O)2-NHC((CH2OP(O)(OH)2)3 and -C(O)-NHC((CH2OP(O)(OH)2)3, wherein p is an integer of from 1 to 12, e.g. from 1 to 8, from 2 to 6 or from 3 to 4, optionally wherein p is 2.39. The compound of any preceding clause, wherein group -X-L2 comprises or has a structure selected from: (i) a bond; (ii) -NH-; (iii) -(CH2)c-, wherein c is an integer of from 1 to 6 or from 2 to 4, optionally wherein c is 1 or 2; (iv) -C(O)NH-; (v) -NH-C(O)-NH-; (vi) -NH-C(S)-NH-; (vii) -S(O)2NH-; (viii) -S-; (ix) -S-[CH2]d-S-, wherein d is an integer of from 1 to 6 or from 2 to 4, optionally wherein d is 2 or 3;wherein a is 1 , 2 or 3 and b is 0, 1 , 2 or 3, optionally wherein a is 1 and b is 2;wherein R14 is:- Z, optionally wherein Z is Cl or OH; or- NR2R3, optionally wherein R2 and R3 are both C1-C4 alkylene substituted with Z, further optionally wherein Z is SO3H;(xii)wherein f is an integer of from 1 to 12 (e.g. from 1 to 8 or from 2 to 6), optionally wherein f is 1 or 2, and each R2 is independently selected from C1-C6 alkyl or C1-C6 oxyalkyl (e.g oxyethyl (-(CH2)2OH)), optionally wherein each R2 is methyl;(xiii)wherein g is an integer of from 1 to 12 (e.g. from 1 to 8 or from 2 to 6), optionally wherein g is 1 or 2.40. The compound of Formula (IV) of any one of clauses 2 to 39, wherein L5 is a bond, heterocyclyl (e.g. piperazinyl, piperidinyl or morpholinyl) or alkylene (e.g. C1-C12, Ci-Ca, C1-C6 or C1-C4 alkylene), optionally wherein said alkylene or heterocyclyl is substituted with one or more substituents independently selected from -NH-, -O-, -S- and -NR2-.41 . The compound of Formula (IV) of any one of clauses 2 to 39, wherein L5 is a bond.42. The compound of Formula (IV) of any one of clauses 2 to 40, wherein L5 is heterocyclyl (e.g. piperazinyl, piperidinyl or morpholinyl), optionally wherein L5 is piperazinyl.43. The compound of Formula (IV) of clause 40, wherein L5 is alkylene (e.g. C1-C4 alkylene or C1-C2 alkylene) substituted with one or more -N(R2)-, wherein each R2 is independently selected from H, C1-C6 alkyl and C1-C6 w-oxyalkyl, optionally oxyethyl (-(CH2)2OH), optionally wherein each R2 is methyl.44. The compound of Formula (IV) of clause 43, wherein L5 is -N(CH3)-CH2-CH2-N(CH3)-.45. The compound of Formula (IV) of clause 40, wherein L5 is -CH2-CH2-CH2-S-.46. The compound of Formula (IV) of any one of clauses 2 to 39, wherein L5 is heterocyclyl or heteroarylene, wherein the heterocycle or heteroarylene contains at least one heteroatom independently selected from N, O and S, optionally wherein the heterocyclyl or heterarylene is substituted with one or more substituents selected from -NR2, -NR2R3-, -C(O)R2, and -Z, further optionally wherein the heterocyclyl or heterarylene is piperidyl, piperazinyl or triazinyl47. The compound of Formula (IV) of any one of clauses 2 to 45, wherein Q is halogen (e.g. Cl).48. The compound of Formula (IV) of any one of clauses 2 to 45, wherein Q is H.49. The compound of Formula (IV) of any one of clauses 2 to 45, wherein the compound has a structure of general Formula (V):50. The compound of Formula (V) of clause 49, wherein:- Li is a bond or NR2R3, wherein R2 and R3 are independently selected from H and C2-C6 alkylene;- Y is S(O2) or C(O);- Z is OH, SH, SO2NHCN, SO3H or OP(O)(OH)2- X is a bond, -NH- or -S-;- m is 1 , 2 or 3;- 1_5 is piperazinyl, piperidinyl or Ci-Ce alkylene (e.g. C1-C4 alkylene) substituted with one or more substituents independently selected from S and NR2, wherein each R2 is independently C1-C6 alkyl (e.g. C1-C2 alkyl); and- Q is H.51 . The compound of Formula (V) of clause 49 or 50, wherein the compound has a structure selected52. The compound of Formula (IV) of any one of clauses 2 to 48, wherein the compound has a structure of general Formula (VI):wherein Hal is halogen (e.g. F, Cl, Br, I).53. The compound of Formula (VI) of clause 52, wherein Hal is Cl.54. The compound of Formula (VI) of clause 52 or clause 53, wherein R1 is selected from H, NH2, PG and NH-PG wherein PG is a protective group, optionally wherein PG is t-Boc.55. The compound of Formula (VI) of any one of clauses 52-54, wherein(i) Y is a bond and Li is a bond; or(ii) Y is a bond and Li is -NR2R3-, wherein R2 and R3 are both alkylene, optionally ethylene.56. The compound of Formula (VI) of any one of clauses 52-55, wherein group -Y-Li-[Z]mhas a structure selected from: (i) -Cl; (ii) -OH; (iii) -N(CH2CH2SO3H)2; and (iv) -N(CH2CH2OP(O)(OH)2)257. The compound of Formula (VI) of any one of clauses 52-56, wherein the compound has a structure selected from:58. The compound of any one of clauses 1 and 3-39, wherein FL comprises xanthene, coumarin, acridine, acridone (e.g. 2-aminoacridone), oxazine, benzoxazole, carbopyronine, naphthalimide, dipyrromethene, rhodamine (e.g., rhodamine B, rhodamine 110, rhodamine 101 , rhodamine 6G, TMR, Si- or Ge-rhodamine), fluorescein, triarylmethane, dipyrromethene, pyrene, APTS, ANTS, dansyl, 7-nitrobenzo-2-oxa-1 ,3-diazole, Lucifer Yellow or a cyanine (e.g. C3-C9 cyanine).59. The compound of any one of clauses 1 and 3-39, wherein FL is not fluorescein, or does not comprise fluorescein. 60. The compound of any one of clauses 1 and 3-39, wherein FL comprises a structure according toFormula (Ila):R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H, q1 , q2 and q3 are independently selected from 0, 1 , 2, 3 or 4; each l_4 is independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, -CO-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH- -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, - CO2H, -O-C(O)NH-, and -O-;R2 is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH;Y2 is selected from a bond, -NH-, -C(O)-, and -C(S)-; andZ is an ionizable group, optionally wherein each Z is independently selected from -SO3H, -OSO3H, - SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2.61. The compound of any one of clauses 1 and 3-39, wherein FL comprises a structure according to Formula (lib):wherein:each R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN, and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H; q1 , q2 and q3 are independently selected from 0, 1 , 2, 3 or 4; each l_4 is independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, -CO-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH- -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -CO2H, -O-C(O)NH-, and -O-;R2 is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH; andZ is an ionizable group, optionally wherein each Z is independently selected from -SO3H, -OSO3H, - SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2.62. The compound of any one of clauses 1 and 3-39, wherein FL comprises a structure according to Formula (Illa):each R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN, and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H; q3 is selected from 0, 1 , 2, 3 or 4; each L4 is independently selected from: a bond, alkylene (e.g. C1-C12 alkylene), alkylarylene, arylalkylene, arylene, alkenylene (e.g. C1-C12 alkenylene), alkynylene (e.g. C1-C12 alkynylene), -CO-, -NH-, -C(O)NH-, - C(S)NH-, -S(O)2NH- -N(R2)(RS), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, and -O-, wherein R2is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH;Z is an ionizable group, optionally wherein each Z is independently selected from -SO3H, -OSO3H, -SO2NHCN , -CO2H, -OP(O)(OH)2and -P(O)(OH)2;Y2 is selected from a bond, -NH- -C(O)-, and -C(S)-;R10, Rn, R12 and R13 are independently selected from H, alkyl or SOsH, wherein said alkyl group is optionally substituted with F or SO3H, or wherein R10 and R11, and / or R12 and R13, together with the atoms to which they are attached, form a ring; and each r1 and r2 is independently selected from 0 and 1 .63. The compound of any one of clauses 60-62, wherein each Z is independently selected from SO3H, OSO3H, SO2NHCN, CO2H, OP(O)(OH)2 and P(O)(OH)2, optionally wherein each Z is independently selected from SO3H and OP(O)(OH)2.64. The compound of any one of clauses 60-63, wherein each l_4 is independently selected from H and alkylene, such as C1-C12 alkylene, preferably C1-C4 alkylene (e.g. C2-C3 alkylene).65. The compound of any one of clauses 60-64, wherein each X2 is independently selected from H and Z.66. The compound of any one of clauses 60-65, wherein q1 and q2 are independently selected from 0 and 1 , optionally wherein q1 and q2 are both 0.67. The compound of any one of clauses 60-66, wherein Y2 is a bond.68. The compound of any one of clauses 60-66, wherein Y2 is -C(O)-.69. The compound of any one of clauses 60-68, wherein each R4 is selected from F, CO2H and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2.70. The compound of any one of clauses 60-69, wherein q3 is 1 and R4 is CO2H.71 . The compound of any one of clauses 60-69, wherein q3 is 0.72. The compound of any one of clauses 60-68 and 69-71 , wherein at least one R4 is C(O)NR?R8, whereinR7 and Rs are independently selected from H, alkyl (e.g. C1-12 or C1-6 alkyl), carbocycle, heterocycle (e.g. morpholine, piperidine, piperazine) or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from OH, CO2H and SO3H.73. The compound of any one of clauses 60-72, wherein R5 and Re are independently selected from H and alkyl (e.g. C1-12 alkyl, preferably C1-4 alkyl).74. The compound of any one of clauses 60-73, wherein each R5 and Re is alkyl, preferably C1-4 alkyl, in particular methyl.75. The compound of any one of clauses 60-74, wherein both r1 and r2 are 1 .76. The compound of any one of clauses 60-75, wherein R10, R11 , R12 and R13 are independently selected from H and C1-12 alkyl, or Ci-6 alkyl (e.g. methyl).77. The compound of any one of clauses 60-76, wherein R10 and R13 are C1-C4 alkyl (e.g. methyl), and R11 and R12 are H.78. The compound of any one of clauses 60-77, wherein R10 and Rn, and / or R12 and R13, together with the atoms to which they are attached, form a ring, optionally a 5- or a 6-membered ring, preferably a 6- membered ring.79. The compound of any one of clauses 60-78, wherein FL comprises or consists of a moiety selected from structures a to u80. The compound of any one of clauses 1 and 3-39, wherein FL is not one, some, or any of structures(a) to (i) or (r):81. The compound of any one of clauses 1 , 3-39 and 55-78, wherein the compound has a structure selected from:82. A conjugate formed from a compound of Formula (I) according to any one of clauses 1 , 3-39 and 58- 81 , and at least one carbohydrate moiety.83. The conjugate of clause 82, wherein the compound comprising at least one carbohydrate moiety is a monosaccharide (e.g. xylose, arabinose, glucose, galactose, mannose, fructose), a disaccharide (e.g. lactose, sucrose, maltose), a homo- or hetero-oligosaccharide (e.g. a galactooligosaccharide (GOS), a fructooligosaccharide (FOS), a milk oligosaccharide (MOS)), fucose, N-acetylglucosamine, N- acetylgalactosamine, a homo- or hetero-polysaccharide (e.g. amylose, amylopectin, cellulose, starch glycogen), or a glycoconjugate (e.g. a glycosaminoglycan (GAG), a glycosylamine, a glycoprotein, a glycopeptide, a proteoglycan, a peptidoglycan, a glycolipid, a GPI-anchor, or a lipopolysaccharide).84. A method of preparing a compound of Formula (I), the method comprising reacting a fluorophore with a compound of Formula (IV), wherein the fluorophore comprises one of an electrophilic and a nucleophilic reactive centre, and group -L5-Q of Formula (IV) comprises the other of an electrophilic and a nucleophilic reactive centre.85. The method of clause 84, the method comprising reacting a compound of Formula (IV) with awherein:X2, l_4, R4, R5, Re, q1 , q2 and q3 are as defined in clause 60; andY3 comprises a nucleophilic or an electrophilic reactive centre.86. The method of clause 84 or 85, the method comprising reacting a compound of Formula (IV) with a fluorophore of Formula (lllb):wherein:X2, L4, R , Re, Re, R10, R11 , R12, R13, r1 , r2 and q3 are as defined in relation to Formula (Illa) in clause 62; andY3 comprises a nucleophilic or electrophilic reactive centre.87. The method of any one of clauses 84-86, wherein the nucleophilic reactive centre is selected from NH2, -NH- and SH.88. The method of any one of clauses 84-87, wherein the electrophilic reactive centre is Hal (e.g. Br, Cl, F or I), CO2H, CO2LG, (CH2)jCC>2 LG, CO(CH2)jCO2 LG or (CH2)jOCO2LG, wherein j is an integer of from 1 to 12, and LG is a leaving group selected from / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl and tetra- or pentafluorophenyl.89. The method of clause 88, wherein the electrophilic reactive centre comprises Hal, COHal, CO2H, CO2LG, (CH2)jCO2 LG , CO(CH2)jCO2 LG or (CH2)jOCO2 LG .wherein j is an integer of from 1 to 12, Hal is halogen (e.g. Br, Cl, F or I), and LG is a leaving group selected from N-succinimidyl (NHS), sulfo-N- succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl and tetra- or pentafluorophenyl.90. The method of clause 89, wherein the electrophilic reactive centre comprises or is Hal or COHal, optionally wherein Hal is Cl.91 . The method of any one of clauses 84-90, wherein Y3 is NH2 or -C(O)CI.92. The method of any one of clauses 84-88, comprising reacting the fluorophore with a compound of Formula V (e.g. L1 B, L2B, L3B, L4B, L5B, L6B or L7B) or VI (e.g. L1A, L2A, L3A, L4A, L5A, L6A, L7A, L8A, L9A or L10A).93. The method of any one of clauses 84-92, comprising reacting the fluorophore with the compound of Formula (IV), wherein the fluorophore comprises a nucleophilic reactive centre (e.g. NH2), and group -L5-Q of Formula (IV) comprises an electrophilic reactive centre (e.g. Cl).94. The method of any one of clauses 84-93, comprising reacting:- a fluorophore of Formula (lie) or Formula (I II b) , wherein Y3 is NH2; with- a compound of Formula (IV), wherein L5 is a bond, and Q is Hal (e.g. Br, Cl, F or I, preferably Cl).95. The method of any one of clauses 84-94, comprising reacting a fluorophore of Formula (He) or Formula (II lb), wherein Y3 is NH2, with a compound of Formula (VI), optionally wherein the method further comprises removing a protecting group.96. The method of any one of clauses 84-95, wherein the compound of Formula (VI) is selected from:(i) L1A, L2A, L3A and L4A, optionally wherein the method further comprises cleavage of the t-Boc protective group; or(ii) L5A, L6A, L7A, L8A, L9A and L10A, optionally wherein the method further comprises reduction of the nitro group to amine.97. The method of any one of clauses 84-96, comprising reacting the fluorophore with the compound of Formula (IV), wherein the fluorophore comprises an electrophilic reactive centre (e.g. -C(O)CI), and group -L5- Q of Formula (IV) comprises a nucleophilic reactive centre (e.g. -NH).98. The method of clause 97, comprising reacting:- a fluorophore of Formula (lie) or Formula (I II b) , wherein Y3 is C(O)CI; with- a compound of Formula (IV), wherein L5 is heterocyclyl (e.g. piperidinyl or piperazinyl) or L5 is alkylene substituted with S or N, and Q is H.99. The method of clause 97, comprising reacting:- a fluorophore of Formula (He) or Formula (111 b), wherein Y3 is C(O)Hal or C(O)OH and Hal is halogen (e.g. Cl, Br, F or I), with- a compound of Formula (V), optionally a compound selected from L1 B, L2B, L3B, L4B, L5B, L6B and L7B100. A method of forming a conjugate, the method comprising reacting a non-fluorescent organic dye with a compound of general Formula (IV), optionally with a compound of Formula (V) or Formula (VI), further optionally wherein the non-fluorescent organic dye is selected from from azo compounds, triarylmethanes, xanthenes, rhodamines, oxazines, polyenes, azomethines, anthraquinones, stilbenes, porphyrins, thiazines, naphthoquinones, alizarines, squaraines, naphthalimides, indigoid or thioindigoid dyes.101. A conjugate obtainable by the method of clause 100.102. The use of a compound of Formula (I) according to any one of clauses 1 and 3-39 for the detection or analysis of an analyte comprising at least one carbohydrate moiety.103. The use of clause 102, wherein the analyte comprising at least one carbohydrate moiety is a monosaccharide (e.g. xylose, arabinose, glucose, galactose, mannose, fructose), a disaccharide (e.g. lactose, sucrose, maltose), a homo- or hetero-oligosaccharide (e.g. a galactooligosaccharide (GOS), a fructooligosaccharide (FOS), a milk oligosaccharide (MOS)), fucose, N-acetylglucosamine, N- acetylgalactosamine, a homo- or hetero-polysaccharide (e.g. amylose, amylopectin, cellulose, starch glycogen), or a glycoconjugate (e.g. a glycosaminoglycan (GAG), an N-glycan, a glycosylamine, a glycoprotein, a glycopeptide, a proteoglycan, a peptidoglycan, a glycolipid, a GPI-anchor, or a lipopolysaccharide).104. The use of any one of clauses 102-103, wherein the method is a chromatographic separation method.105. The use of any one of clauses 102-104, wherein the method is an electrophoretic separation method, optionally wherein the electrophoretic separation method is capillary electrophoresis (CE) or capillary gel electrophoresis (CGE), e.g. CGE with fluorescence induced by laser (CGE-LIF).106. The use of the conjugate of clause 100 in a method of analysing or detecting an analyte comprising at least one carbohydrate moiety, wherein the method is an electrophoretic separation method, optionally wherein the conjugate is used as a calibration standard.107. A method for detecting and / or analysing a carbohydrate that may be present in a sample, the method comprising: a) labeling the sample(s) (e.g. sample(s) containing carbohydrate(s) of unknown composition(s) / structure(s)) and standard(s) (e.g. standard(s) containing carbohydrate(s) of known composition(s) / structure(s)) with two or more spectrally different compounds, wherein at least one of the spectrally different compounds comprises a compound of Formula (I) according to claim 1 , or any one of claims 3 to 15 when dependent on claim 1 , so as to provide labelled sample(s) and labelled standard(s); b) mixing the labelled sample(s) and the labelled standard(s) to obtain a mixture; c) analysing the mixture using an electrokinetic separation method (e.g. CE or CGE) or a chromatographic separation method, so as to determine the migration times or the retention times of the labelled carbohydrates in the sample(s) and in those in the standard(s); and d) using the determined migration or retention times of the labelled standard(s) to detect and / or identify the carbohydrate(s) in the sample(s).108. A kit comprising at least one compound of Formula (I) according to any one of clauses 1 or 3-39, or a compound of Formula (IV) according to any one of clauses 2-57 (e.g. Formula (V) or Formula (VI)), and instructions for use.109. A compound according to the formula:wherein Rh is110. A method of preparing a compound of Formula (I), the method comprising reacting an aminosubstituted fluorophore with a nitro-substituted benzoic acid chloride, a nitro-substituted benzene sulfochloride, a nitro-substituted phenyl isocyanate or a nitro-substituted phenyl isothiocyanate, optionally comprising reduction of a nitro group to amine, further optionally wherein the nitro-substituted benzoic acid chloride, the nitro-substituted benzene sulfochloride, the nitro-substituted phenyl isocyanate or the nitro-substituted phenyl isothiocyanate is selected from compounds L11A to L22A:

Claims

1. Claims1 . A compound of general formula (I) or a salt, solvate or hydrate thereof:Z - L, - YI mwherein:Ring A is a 3-10 membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L2 are independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH-, -N(R2)(R3), -O-, -S-, - S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, -N(R2R3), -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;Z is an ionizable group, wherein each Z is independently selected from -SO3H, -OSO3H, -SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2;R1 is selected from H, 2-, 3-, or4-aminophenyl, NH2, PG, and NH-PG wherein PG is a protective group, wherein PG selected from t-butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9-fluorenylmethoxycarbonyl (Fmoc), or R1 is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl;R2 and R3are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, -CH2-; m is an integer of from 0 to 12; andFL comprises a fluorophore, wherein:(i) FL comprises a structure according to Formula (Ila):wherein:R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H, q1 , q2 and q3 are independently selected from 0, 1 , 2, 3 or 4; each l_4 is independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, -CO-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH- -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, - CO2H, -O-C(O)NH-, and -O-;R2 is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH;Y2 is selected from a bond, -NH-, -C(O)-, and -C(S)-; andZ is an ionizable group, optionally wherein each Z is independently selected from -SO3H, -OSO3H, - SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2; or(ii) FL comprises a structure according to Formula (lib):each R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN, and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H; q1 , q2 and q3 are independently selected from 0, 1 , 2, 3 or 4; each l_4 is independently selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, -CO-, -NH-, -C(O)NH-, -C(S)NH-, -S(O)2NH- -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -CO2H, -O-C(O)NH-, and -O-;R2 is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH; andZ is an ionizable group, optionally wherein each Z is independently selected from -SO3H, -OSO3H, - SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2; or(iii) FL comprises a structure according to Formula (Illa):each R4, R5 and Re are independently selected from: alkyl, alkenyl, alkynyl, Br, Cl, F, I, NO2, CO2H, SO3H, CN, and S(CH2)fSO3H, wherein f is an integer of from 1 to 6, optionally wherein f is 2, and C(O)NR?R8, wherein R7 and Rs are independently selected from H, alkyl, carbocycle, heterocycle or heteroaryl, wherein the alkyl, carbocycle, heterocycle or heteroaryl is optionally substituted with one or more substituents independently selected from F, OH, CO2H and SO3H; q3 is selected from 0, 1 , 2, 3 or 4; each L4 is independently selected from: a bond, alkylene (e.g. C1-C12 alkylene), alkylarylene, arylalkylene, arylene, alkenylene (e.g. C1-C12 alkenylene), alkynylene (e.g. C1-C12 alkynylene), -CO-, -NH-, -C(O)NH-, - C(S)NH-, -S(O)2NH- -N(R2)(RS), -O-, -S-, -S(O)2-, carbocyclyl, heterocyclyl or heteroarylene, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl is substituted with one or more substituents independently selected from -F, -NH-, -N(R2)-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, and -O-, wherein R2is selected from H, alkyl and oxyalkyl; each X2 is independently selected from Z, H, alkyl, alkylaryl, aryl, heteroaryl, carbocycle, heterocycle or heteroaryl, optionally wherein the alkyl, alkylaryl, aryl, heteroalkyl, carbocycle, heterocycle or heteroaryl is substituted with one or more substituents independently selected from F and OH;Z is an ionizable group, optionally wherein each Z is independently selected from -SO3H, -OSO3H, -SO2NHCN, -CO2H, -OP(O)(OH)2and -P(O)(OH)2;Y2 is selected from a bond, -NH-, -C(O)-, and -C(S)-;R10, R11, R12 and R13 are independently selected from H, alkyl or SO3H, wherein said alkyl group is optionally substituted with F or SO3H, or wherein R10 and Rn, and / or R12 and R13, together with the atoms to which they are attached, form a ring; and each r1 and r2 is independently selected from 0 and 1 ; or(iv) FL comprises a structure selected from:

2. A compound of general formula (IV), or a salt, solvate or hydrate thereof:wherein:Ring A is a 3- to 10-membered aryl or heteroaryl monocyclic or bicyclic ring;X is selected from: a bond, -NH-, -SO2-, -S-, -O-, -OCH2-, -SCH2-, -NHCH2-, -C(O)-, heterocyclyl, alkylene and alkenylene;Li and L5 are independently absent or selected from: a bond, alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, -C(O)-, -NH-, -NH2, -NH2+, -C(O)NH-, -C(S)NH-, -S(O)2NH-, -N(R2)(R3), -O-, -S-, -S(O)2-, carbocyclyl or heterocyclyl, optionally wherein said alkylene, alkylarylene, arylalkylene, arylene, heteroarylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl is substituted with one or more substituents independently selected from -F, -N(R2)-, -NH2, -N(R2R3), -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, -S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z;R1 is selected from H, NH2, 2-, 3-, or4-nitrophenyl, PG and NH-PG, wherein PG is a protective group, optionally wherein the protective group is selected from t-butoxycarbonyl (t-Boc), trifluoroacetyl (COCF3), benzyloxycarbonyl (Cbz), phthalimide (Phth), benzyl (Bn), benzylidene, benzylidenamine and 9- fluorenylmethoxycarbonyl (Fmoc);R2 and R3are independently selected from H, alkyl, alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl, optionally wherein said alkylene, alkyl ether, alkyl thioether, oxyalkyl, and thioalkyl is substituted with one or more substituents independently selected from -Z or -F;Y is absent or is selected from: a bond, -NH-, -S(O)2-, -C(O)-, and -CH2-;Z is an ionizable group, wherein each Z is independently selected from -SO3H, -OSO3H, -SO2NHCN, -CO2H, -OP(O)(OH)2 and -P(O)(OH)2; m is an integer of from 0 to 12; andQ is selected from OH, H, H2+, Br, Cl, F, I, -C(O)OH, C(O)OLG, (CH2)jC(O)OLG , CO(CH2)jC(O)OLG and (CH2)jOC(O)OLG, wherein j is an integer of from 1 to 12, and LG is a leaving group selected from N- succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.

3. The compound of claim 1 or claim 2, wherein ring A is a 5- to 6-membered aryl or heteroaryl ring, optionally wherein ring A is phenyl or triazinyl (e.g. 1 ,3,5-triazinyl).

4. The compound of any one of claims 1 to 3, wherein Li is absent or is selected from a bond, alkylene (e.g. C1-C12, Ci-C3, Ci-Ce or Ci-C4 alkylene) and -N(R2)(R3), optionally wherein R2 and R3are independently selected from H and C1-C12 alkylene (e.g. Ci-C3, Ci-Ce or Ci-C4 alkylene), further optionally wherein both R2 and R3are C1-C4 alkylene (e.g. ethylene).

5. The compound of claim 1 , or claim 3 or claim 4 when dependent on claim 1 , wherein Ls is absent or is selected from: a bond, alkylene (e.g. C1-C12, Ci-C3, Ci-Ce or C1-C4 alkylene), heterocyclyl, -NH-, -C(O)-, - C(O)NH-, -C(S)NH-, -O-, and -S-, optionally wherein the alkylene or heterocyclyl is substituted with one or more substituents independently selected from -F, -NR2-, -NR2R3-, -C(O)R2-, -CONH-, -CONH2, -C(O)-, -O-, - S-, -SO2, -S(O)-, -C(O)O-, -C(O)OH, -O-C(O)NH-, -O-C(O)NH2or Z.

6. The compound of any preceding claim, wherein X is selected from a bond, -NH-, -C(O)-, alkylene (e.g. Ci-C3, Ci-Ce or C1-C4 alkylene), alkenylene (e.g. C1-C12 alkenylene, Ci-C3alkenylene or Ci-Ce alkenylene), - S(O)2- and S.

7. The compound of any preceding claim, wherein Y is absent, a bond, C(O) or -S(O)2-.

8. The compound of any preceding claim, wherein m is 0, 1 , 2, 3 or 4.

9. The compound of claim 2, or any one of claims 3 to 8 when dependent on claim 2, wherein L5 is heterocyclyl (e.g. piperazinyl, piperidinyl or morpholinyl) or alkylene (e.g. C1-C12, Ci-Ca, C1-C6 or C1-C4 alkylene), optionally wherein said alkylene or heterocyclyl is substituted with one or more substituents independently selected from -NH-, -O-, -S- and -NR2-, optionally wherein each R2 is independently selected from H, C1-C6 alkyl (e.g. methyl) and C1-C6 w-oxyalkyl.

10. The compound of claim 2, or any one of claims 3 to 9 when dependent on claim 2, wherein Q is halogen (e.g. F, Cl, Br or I) or H.

11. The compound of claim 2, or any one of claims 3 to 10 when dependent on claim 2, wherein the compound has a structure of general Formula (V) or (VI):wherein Hal is halogen (e.g. F, Cl, Br or I).

12. The compound of claim 11 , wherein the compound has a structure selected from:

13. The compound of claim 1 , wherein FL comprises or consists of a moiety selected from:

14. The compound of claim 1 , or any one of claims 3 to 13 when dependent on claim 1 , wherein the compound has a structure selected from:

15. A conjugate formed from a compound of Formula (I) according to claim 1 , or any one of claims 3 to 15 hen dependent on claim 1 , and at least one carbohydrate moiety.

16. A method of preparing a compound of Formula (I), the method comprising reacting a fluorophore with a compound of Formula (IV), wherein the fluorophore comprises one of an electrophilic and a nucleophilic reactive centre, and group -L5-Q of Formula (IV) comprises the other of an electrophilic and a nucleophilic reactive centre, optionally wherein:(a) the method comprises reacting the fluorophore with a compound of Formula (V), wherein the fluorophore comprises an electrophilic reactive centre, and group -L5-Q of Formula (IV) comprises a nucleophilic reactive centre; or(b) the method comprises reacting the fluorophore with a compound of Formula (VI), wherein the fluorophore comprises a nucleophilic reactive centre, and Hal of Formula (VI) comprises an electrophilic reactive centre, optionally wherein the method further comprises reduction of a nitro group to amine or deprotection of a hydrazine group.

17. The method of claim 16, wherein the compound of Formula (VI) is selected from:optionally wherein the method further comprises cleavage of the t-Boc protective group; oroptionally wherein the method further comprises reduction of the nitro group to amine.

18. The use of a compound of Formula (I) according to claim 1 , or any one of claims 3 to 15 when dependent on claim 1 , for the detection or analysis of an analyte comprising at least one carbohydrate moiety, optionally wherein the carbohydrate moiety comprises a glycosylamine.

19. A method for detecting and / or analysing carbohydrate(s) that may be present in a sample, the method comprising: a) labelling the sample(s) (e.g. sample(s) containing carbohydrate(s) of unknown composition(s) / structure(s)) and standard(s) (e.g. standard(s) containing carbohydrate(s) of known composition(s) / structure(s)) with two or more spectrally different compounds, wherein at least one of the spectrally different compounds comprises a compound of Formula (I) according to claim 1 , or any one of claims 3 to 15 when dependent on claim 1 , so as to provide labelled sample(s) and labelled standard(s); b) mixing the labelled sample(s) and the labelled standard(s) to obtain a mixture; c) analysing the mixture using an electrokinetic separation method (e.g. CE or CGE) or a chromatographic separation method, so as to determine the migration times or the retention times of the labelled carbohydrates in the sample(s) and in those in the standard(s); and d) using the determined migration or retention times of the labelled standard(s) to detect and / or identify the carbohydrate(s) in the sample(s).

20. A method of preparing a compound of Formula (I), the method comprising reacting an aminosubstituted fluorophore with a nitro-substituted benzoic acid chloride, a nitro-substituted benzene sulfochloride, a nitro-substituted phenyl isocyanate or a nitro-substituted phenyl thioisocyanate, optionally wherein the method further comprises reduction of a nitro group to amine, further optionally wherein the nitro-substitutedbenzoic acid chloride, the nitro-substituted benzene sulfochloride, the nitro-substituted phenyl isocyanate or the nitro-substituted phenyl thioisocyanate is selected from compounds L11A to L22A:21 . A method of forming a conjugate, the method comprising reacting a non-fluorescent organic dye with a compound of general Formula (IV), optionally with a compound of Formula (V) or Formula (VI), further optionally wherein the non-fluorescent organic dye is selected from azo compounds, triarylmethanes, xanthenes, rhodamines, oxazines, polyenes, azomethines, anthraquinones, stilbenes, porphyrins, thiazines, naphthoquinones, alizarines, squaraines, naphthalimides, indigoid or thioindigoid dyes.

22. A conjugate of a non-fluorescent organic dye with a compound of general Formula (IV), optionally with a compound of Formula (V) or Formula (VI), further optionally wherein the non-fluorescent organic dye is selected from azo compounds, triarylmethanes, xanthenes, rhodamines, oxazines, polyenes, azomethines, anthraquinones, stilbenes, porphyrins, thiazines, naphthoquinones, alizarines, squaraines, naphthalimides, indigoid or thioindigoid dyes23. The compound of claim 1 , wherein:Ring A is phenyl;Y and Li are absent; m is 0 (i.e. Z is absent); l_2 and X are independently selected from a bond, NH and CO;Ri is selected from (CH2)jC(O)OR4, CO(CH2)jC(O)OR4 and (CH2)jOC(O)OR4, wherein j is an integer of from 1 to 12, and R4 is selected from H, / V-succinimidyl (NHS), sulfo- / V-succinimidyl, 1-benzotriazolyl, cyanomethyl, 2- or 4-nitrophenyl, pentachlorophenyl, tetrafluorophenyl and pentafluorophenyl.

24. The compound of claim 23, having the structure:

25. The compound of claim 23 or claim 24, wherein FL is

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