NOVEL FLUORESCENCE DYES AND NOVEL ANALYTICAL PROCESSES FOR GLYCAN ANALYSIS

Abstract

The invention relates to a fluorescent marker according to the following formula I

##STR00001##

wherein Fluo is selected from the group consisting of substituted or non-substituted C10-C30 acridone, aminoacridine or pyrene, L is a linker and the moiety X is either a —SO.sub.2— or a —PO(OH)— moiety. Furthermore, the present invention relates to the use of the fluorescent markers of the invention, in particular also as a standard in glycan analysis, conjugates of fluorescent markers and glycans, as well as a kit-of-parts containing the fluorescent markers of the invention.

Claims

1. A fluorescent marker according to the following formula I ##STR00024## wherein R.sub.1=—H or C1-C6 Alkyl; R.sub.2=—H or —PO(OH).sub.2 X=—SO.sub.2— or —PO(OH)—; L=—(CH.sub.2).sub.n—CHR.sub.3—CH.sub.2—, n=1, 2, 3 and R.sub.3=—H or —(CH.sub.2)—OPO(OH).sub.2; wherein Fluo is selected from the group consisting of substituted or non-substituted C10-C30 acridones, aminoacridines or pyrenes.

2. The fluorescent marker according to claim 1, wherein Fluo is selected from the group consisting of the fluorophores according to the formula II-IV ##STR00025## wherein R.sub.4, R.sub.5=each independently of one another —OPO(OH).sub.2 or —OH; R.sub.6, R.sub.7=each independently of one another —SO.sub.2—CH.sub.2—CHR.sub.2—(CH.sub.2).sub.q—R.sub.4 or —PO(OH)CH.sub.2—CHR.sub.2—(CH.sub.2).sub.q—R.sub.4 and q=1, 2, 3; o, p=each independently of one another 1, 2, 3; and the Fluo according to the formula II-IV can be linked at each possible binding site to NH and X.

3. The fluorescent marker according to claim 1, wherein the fluorophore Fluo corresponds to the formula V-VII ##STR00026## wherein the fluorophore can be linked via the curved bonds to NH and X.

4. The fluorescent marker according to claim 1, wherein X=—SO.sub.2—.

5. The fluorescent marker according to claim 1, wherein in the linker L R.sub.3=—(CH.sub.2)—OPO(OH).sub.2.

6. The fluorescent marker according to claim 1, wherein in the fluorophore according to the formula III or VI R.sub.4 and R.sub.5 each are —OPO(OH).sub.2.

7. The fluorescent marker according to claim 1, wherein in the Fluorophore according to the formula IV and VII R.sub.6 and R.sub.7 are each —PO(OH)—CH.sub.2—CHR.sub.2—(CH.sub.2)—OPO(OH).sub.2.

8. A use of a fluorescent marker according to claim 1 as a standard in a glycan analysis.

9. A conjugate of a fluorescent marker and a glycan, wherein the Glycan is covalently bound to at least one fluorescent marker according to claim 1.

10. The conjugate according to claim 9, wherein the glycan is selected from the group consisting of mono-, oligo-, polysaccharides, glycoproteins, glycolipids, proteoglycans or mixtures thereof.

11. A kit-of-parts at least comprising one or more container comprising one or more solvents and one or more container each containing a fluorescence marker according to claim 1 or conjugates according to claim 9.

Description

DRAWINGS

[0043] Further advantages and advantageous embodiments of the objects according to the invention are illustrated by the figures and explained below. It should be noted that the figures are descriptive only and are not intended to limit the invention in any way.

[0044] The figures show:

[0045] FIG. 1 A synthetic route for a phosphorylated acridone fluorescent marker according to compound 1;

[0046] FIG. 2 Absorption/emission spectra of example compound 1 (in 0.05 M TEAB buffer with excitation at 488 nm) as an isolated fluorescent label (-) and as a glucose conjugate ( - - - ) in the wavelength range between 250 and 800 nm.

DETAILED DESCRIPTION

[0047] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0048] FIG. 1 shows a possible synthetic route for obtaining compound 1. The individual synthesis steps and reaction conditions are described further below in the example section.

[0049] FIG. 2 shows the absorption and emission properties of a fluorescent label according to the invention and of a conjugate of a fluorescent label and a simple sugar. Shown are the normalized absorption and emission spectra of 7-[(3-phosphopropyl)sulfonyl]-2-amino-acridin-9(10H)-one (1) and its glucose conjugate. The absorbance characteristics were measured in 0.05 M TEAB buffer (triethylammonium bicarbonate buffer) at pH 8.0. Excitation was performed at 488 nm. Fluorescence emission can be clearly seen with a maximum around 585 nm. Due to the formation of the conjugate, the emission maximum is only slightly shifted to higher wavelengths and lies at approx. 590 nm. The spectroscopic properties of the fluorescent marker make it particularly suitable for glycan analysis. The fluorescent marker can be used alone or, due to its emission maximum, simultaneously in combination with a prior art fluorescent marker. Due to the different emission windows, two fluorescence markers can be detected independently of each other.

Examples

[0050] Synthesis of the Example Compounds

[0051] A possible synthesis route to obtain the compound (1) is shown in FIG. 1.

(I) 5-Nitro-N-phenyl-anthranilic acid (4)

[0052] ##STR00010##

[0053] 2-chloro-5-nitrobenzoic acid (7.5 g, 38 mmol) are dissolved in 50 mL n-butanol at 40° C. Ani-line (7.7 mL, 83 mmol, 2.2 eq.) and DIEA (82.5 mmol, 14.4 mL, 2.2 eq.) are added. The mixture is stirred under reflux (120° C.) for 3 days. After cooling to room temperature, the volatile components are evaporated off in vacuo. A brown oil is obtained. The residue is transferred to a vigorously stirred mixture of crushed ice and hydrochloric acid (1 M, 100 mL). The precipitated product is filtered off, washed with water and dried in air. The crude product is recrystallized in acetic acid (180 mL), filtered off and washed with diethyl ether. After drying in vacuo, compound (4) (6.37 g, 24.7 mmol, 66%) is obtained as a yellow residue.

[0054] MS (ESI): m/z (positive mode)=259.1 [M+H].sup.+, 281.1 [M+Na].sup.+.

[0055] HR-MS (ESI): C13H10N2O4: Calc. [M+H]+ 259.0713, found 259.0714, [M+Na]+: Calc. 281.0533, found: 281.0535.

[0056] .sup.1H-NMR (400 MHz, DMF-d.sub.7): δ [ppm]=14.42 (s, 1H, COOH), 10.53 (s, 1H, NH), 8.87 (d, J=2.8 Hz, 1H, 6-H), 8.25 (dd, J=9.4, 2.8 Hz, 1H, 4-H), 7.56-7.49 (m, 2H, 3′-H), 7.47-7.42 (m, 2H, 2′-H), 7.32 (m, 1H, 4′-H), 7.26 (d, J=9.4 Hz, 1H, 3-H),

[0057] .sup.13C-NMR (101 MHz, DMF-d.sub.7): δ [ppm]=170.2, 154.0, 139.7, 138.2, 131.0, 130.4, 129.7, 126.9, 125.2, 114.4, 112.3,

[0058] TLC: R.sub.f=0.54 (MeOH/H.sub.2O, 80/20)

(II) 2-Nitroacridin-9(10H)-one (5)

[0059] ##STR00011##

[0060] Compound 4 (0.51 g, 1.96 mmol) is dissolved in POCl.sub.3 (5 mL) and the reaction mixture is stirred under reflux (125° C.) for 3 hours. The POCl.sub.3 is evaporated in vacuo and crushed ice (50 g) and hydrochloric acid (1 M, 100 mL) are added to the residue. The mixture is boiled under reflux for one hour until a yellow product precipitates. The precipitate is filtered, washed with water, cold methanol and diethyl ether and then dried in air. A pure 2-nitroacridin-9(10H)-one (0.40 g, 1.66 mmol, 85%) is obtained as a light yellow solid.

[0061] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=12.37 (s, 1H, NH), 8.99 (d, J=2.7 Hz, 1H, 1-H), 8.49 (dd, J=9.2, 2.7 Hz, 1H, 3-H), 8.26 (dd, J=8.1, 1.5 Hz, 1H, 8-H), 7.84 (ddd, J=8.4, 7.0 Hz, 1H, 7-H), 7.69 (d, J=9.2 Hz, 1H, 4-H), 7.61 (dd, J=8.4, 1.5 Hz, 1H, 5-H), 7.39 (ddd, J=8.1, 7.0 Hz, 1H, 6-H),

[0062] HPLC: t.sub.R=9.87 min (ACN/H.sub.2O+0.1% TFA; 30/70.fwdarw.10/0 in 12 min; detection at 254 nm)

(III) 7-Bromo-2-nitroacridin-9(10H)-one (6)

[0063] ##STR00012##

[0064] Compound 5 (0.4 g, 1.6 mmol) is suspended in nitrobenzene (40 mL). Bromine (140 μL, 2.5 mmol, 1.5 eq.) is added and the reaction mixture is stirred for 18 h at 120° C. After cooling to 0° C., the precipitated precipitate is filtered off and washed with diethyl ether. Further diethyl ether (100 mL) is added to the filtrate and the mixture is cooled to 0° C. for one hour. The precipitate formed is filtered off and washed with diethyl ether. The combined solids are dried in vacuo to give compound 6 (442 mg, 1.38 mmol, 86%) as a yellow solid.

[0065] MS (ESI): m/z (positive mode): 341.0 [M+Na].sup.+, 660.9 [2M+Na].sup.+, 978.9 [3M+Na].sup.+,

[0066] HR-MS (ESI): C.sub.13H.sub.7N.sub.2O.sub.3: calc. [M+H].sup.+: 318.9713, found: 318.9719, [M+Na].sup.+: calc. 340.9532, found: 340.9539.

[0067] .sup.1H-NMR (400 MHz, DMF-d.sub.7): δ [ppm]=12.66 (s, 1H, NH), 9.08 (d, J=2.7 Hz, 1H, 1-H), 8.54 (dd, J=9.2, 2.7 Hz, 1H, 3-H), 8.39 (d, J=2.4 Hz, 1H, 8-H), 7.99 (dd, J=8.8, 2.4 Hz, 1H, 6-H), 7.81 (d, J=9.2 Hz, 1H, 4-H), 7.70 (d, J=8.8 Hz, 1H, 5-H),

[0068] .sup.13C-NMR (101 MHz, DMSO-d.sub.4): δ [ppm]=176.8, 146.0, 142.7, 141.2, 138.2, 129.4, 128.8, 124.2, 123.7, 121.8, 120.8, 120.5, 116.3;

[0069] HPLC: t.sub.R=12.32 min (ACN/H.sub.2O+0.1% TFA; 30/70.fwdarw.100/0 in 12 min; detection at 254 nm)

(IV) 7-[(3-hydroxypropyl)thio]-2-nitroacridin-9(10H)-one (7)

[0070] ##STR00013##

[0071] Compound 6 (500 mg, 1.57 mmol) is added to a suspension of K.sub.2CO.sub.3 (433 mg, 2 eq.) in DMF (40 ml). 3-Mercapto-1-propanol (216 mg, 2.35 mmol, 1.5 eq.) and triethylamine (150 μL) were added. Argon is passed through the solution for 10 min and then xantphos (308 mg, 530 μmol, 0.34 eq.) and Pd.sub.2(dba).sub.3 (402 mg, 430 μmol, 0.28 eq.) are added. The reaction mixture is stirred for 23 h at 105° C. under argon atmosphere until complete conversion (HPLC). The solvent is evaporated off under reduced pressure, and the residue is dissolved in ACN (+10% TFA) and loaded onto a Celite. Flash chromatography (Reveleris (SiO.sub.2) 40 g cartridge, ACN/DCM, ACN: 8%.fwdarw.66%) yields pure product 7 (229 mg, 0.69 mmol, 44%).

[0072] MS (ESI): m/z (positive mode): 331.1 [M+H].sup.+, 353.1 [M+Na].sup.+, 683.1 [2M+Na].sup.+, 1113.2 [3M+Na].sup.+.

[0073] HR-MS (ESI): C.sub.16H.sub.14N.sub.2O.sub.4S: calc. [M+H].sup.+: 331.0747, found: 331.0751, [M+Na].sup.+: calc. 353.0566, gefunden: 353.0566.

[0074] .sup.1H-NMR (400 MHz, DMF-d.sub.7): δ [ppm]=12.40 (s, 1H, NH), 8.95 (d, J=2.7 Hz, 1H, 1-H), 8.45 (dd, J=9.2, 2.7 Hz, 1H, 3-H), 8.08 (d, J=2.3 Hz, 1H, 8-H), 7.78 (dd, J=8.7, 2.3 Hz, 1H, 6-H), 7.65 (d, J=9.2 Hz, 1H, 4-H), 7.56 (d, J=8.7 Hz, 1H, 5-H), 3.52 (t, J=6.1 Hz, 2H, 3′-H), 3.13-3.01 (m, 2H, 1′-H), 1.83-1.66 (m, 2H, 2′-H).

[0075] .sup.13C-NMR (101 MHz, DMSO-d.sub.6): δ [ppm]=176.2, 144.9, 141.4, 139.4, 135.6, 131.6, 127.4, 125.4, 123.3, 121.8, 119.9, 119.4, 109.9, 60.0, 32.5, 30.3.

[0076] HPLC: t.sub.R=9.67 min (ACN/H.sub.2O+0.1% TFA; 30/70.fwdarw.100/0 in 12 min; detection at 254 nm)

(V) 7-[(3-Hydroxypropyl)sulfonyl]-2-nitroacridin-9(10H)-one (8)

[0077] ##STR00014##

[0078] Compound 7 (255 mg, 0.77 mmol) is suspended in a mixture of acetic acid (10 mL) and water (2.4 mL). Na.sub.2WO.sub.4 2H.sub.2O (63 mg, 0.19 mmol, 0.25 eq) is added and the suspension is cooled down to 0° C. Hydrogen peroxide (10 mL, 50% (v/v)) is then added over 10 minutes. The suspension is warmed to room temperature and stirred for an additional 5 h with dissolution of the solid. After complete reaction of the reactant (HPLC), the reaction mixture is freeze-dried overnight. The crude product is dissolved in DMF (0.5 mL) and purified using a Biotage SNAP Ultra 25 g cartridge. Flash chromatography (ACN/DCM, ACN 10%.fwdarw.100%) results in 8 (179 mg, 0.49 mmol, 64%) as an orange solid.

[0079] MS (ESI): m/z (positive mode): 363.1 [M+H].sup.+, 385.1 [M+Na].sup.+, 747.1 [2M+Na].sup.+, 1109.2 [3M+Na].sup.+.

[0080] HR-MS (ESI): C.sub.16H.sub.14N.sub.2O.sub.6S: calc. [M+H].sup.+: 363.0645, found: 363.0650, [M+Na].sup.+: calc. 385.0465, found: 385.0465.

[0081] .sup.1H-NMR (400 MHz, DMF-d.sub.7): δ [ppm]=12.86 (s, 1H, NH), 9.06 (d, J=2.7 Hz, 1H, 1-H), 8.79 (d, J=2.1 Hz, 1H, 8-H), 8.58 (dd, J=9.2, 2.7 Hz, 1H, 3-H), 8.26 (dd, J=8.7, 2.1 Hz, 6-H), 7.89 (d, J=8.7 Hz, 1H, 5-H), 7.84 (d, J=9.2 Hz, 1H, 4-H), 4.74 (bs, 1H, OH), 3.60 (t, J=6.2 Hz, 2H, 1′-H), 3.56-3.41 (m, 2H, 3′-H), 1.93-1.81 (m, 2H, 2′-H).

[0082] .sup.13C-NMR (101 MHz, DMF-d.sub.7): δ [ppm]=177.5, 146.1, 145.15, 143.2, 134.5, 133.6, 129.2, 128.7, 124.0, 121.6, 121.5, 120.8, 120.8, 60.4, 53.9, 27.6.

[0083] HPLC: t.sub.R=7.68 min (ACN/H.sub.2O+0.1% TFA; 30/704100/0 in 12 min; detection at 254 nm)

(VI) 7-[(3-hydroxypropyl)sulfonyl]-2-aminoacridin-9(10H)-one (2)

[0084] ##STR00015##

[0085] Pd/C (10%, in oxidized form, 8 mg) is suspended in 2-propanol (5 mL) in an argon atmosphere. Compound 8 (20 mg, 55 μmol) is added and a hydrogenate atmosphere is established. The suspension is stirred at room temperature overnight with partial dissolution of the solid. The yellow fluorescent solution is filtered over Celite to remove the Pd/C and the solid is washed with 2-propanol. The solvent is then removed under reduced pressure. The pure compound 2 is obtained (HPLC, 12 mg, 37 μmol, 68%).

[0086] MS (ESI): m/z (positive mode): 333.1 [M+H].sup.+, 355.1 [M+Na].sup.+, 665.2 [2M+H].sup.+, 687.2 [2M+Na].sup.+.

[0087] HR-MS (ESI): C.sub.16H.sub.16N.sub.2O.sub.4S: calc. [M+H].sup.+: 333.0904, found: 333.0911, [M+Na].sup.+: calc. 355.0723, found: 355.0730.

[0088] .sup.1H-NMR (400 MHz, DMSO-d.sub.4): δ [ppm]=12.03 (s, 1H, NH), 8.62 (d, J=2.2 Hz, 1H, 1-H), 7.98 (dd, J=8.8, 2.2 Hz, 1H, 3-H), 7.65 (d, J=8.8 Hz, 1H, 4-H), 7.46-7.37 (m, 2H, 6-H, 8-H), 7.19 (dd, J=8.8, 2.7 Hz, 1H, 5-H), 5.39 (s, 2H, NH.sub.2), 4.61 (t, J=5.3 Hz, 1H, OH), 3.40 (q, J=5.9 Hz, 2H, 3′-H), 3.35-3.27 (m, 1H, 1′-H), 1.78-1.60 (m, 2H, 2′-H).

[0089] .sup.13C-NMR (101 MHz, DMSO-d.sub.6): δ [ppm]=175.8, 144.6, 142.5, 132.4, 129.9, 129.4, 127.9, 123.8, 122.5, 118.7, 118.6, 117.9, 105.9, 58.7, 52.5, 26.2.

[0090] HPLC: t.sub.R=4.2 min (ACN/H.sub.2O+0.05 M TEAB pH˜8; 90/10.fwdarw.450/0 in 12 min; detection at 254 nm)

(VII) 7-[[3-(O,O-di-tert-butylphosphate)propyl]sulfonyl]-2-nitroacridin-9(10H)-one (9)

[0091] ##STR00016##

[0092] Compound 8 (58 mg, 162 μmol) is dissolved in DMF (1 mL) under argon atmosphere in a dry flask. 1H-tetrazole (39 mg, 0.561 mmol, 4 eq.) is dissolved in DMF (1 mL) and added together with di-tert-butyl N,N-diisopropylphosphoramidite (203 μL, d=0.88 g/mL, 179 mg, 0.646 mmol, 4 eq.). The reaction mixture is stirred at 40° C. for 1.5 h. After cooling to room temperature, mCPBA (139 mg, 0.805 mmol, 5 eq.) dissolved in DCM (1 mL) is added. The reaction mixture is stirred for 1 h and HPLC indicates complete conversion of the starting material. The solvents are removed under reduced pressure. The residue is dissolved in DMF (300 μL) and placed in a preparative HPLC (ACN/H.sub.2O+0.1% TFA 40/60.fwdarw.70/30, in 20 min). Pure compound 9 (59 mg, 106 μmol, 65%) is obtained.

[0093] MS (ESI): m/z (positive mode): 577.1 [M+Na].sup.+, 1131.3 [2M+Na].sup.+.

[0094] HR-MS (ESI): C.sub.24H.sub.31N.sub.2O.sub.9S: calc. [M+H].sup.+: 555.1561, found: 555.1565, [M+Na].sup.+: calc. 577.1380, found: 577.1380.

[0095] .sup.1H-NMR (400 MHz, DMF-d.sub.7): δ [ppm]=12.95 (s, 1H, NH), 9.05 (d, J=2.7 Hz, 1H, 1-H), 8.79 (d, J=2.2 Hz, 1H, 8-H), 8.57 (dd, J=9.2, 2.7 Hz, 1H, 3-H), 8.27 (dd, J=8.8, 2.2 Hz, 1H, 6-H), 7.90 (d, J=8.8 Hz, 1H, 5-H), 7.85 (d, J=9.2 Hz, 1H, 4-H), 4.06 (m, 2H, 3′-H), 3.60-3.41 (m, 2H, 1′-H), 2.12-2.00 (m, 2H, 2′-H), 1.41 (s, 18H, .sup.tBu).

[0096] .sup.13C-NMR (101 MHz, DMF-d.sub.7): δ [ppm]=177.2, 145.9, 145.0, 143.0, 133.8, 133.3, 129.0, 128.6, 123.7, 121.4, 121.3, 120.7, 120.5, 82.5, 82.5, 65.4, 53.1, 30.0, 30.0, 25.1.

[0097] .sup.31P-NMR (162 MHz, DMF-d.sub.7) δ [ppm]=−9.78.

[0098] HPLC: t.sub.R=11.62 min (ACN/H.sub.2O+0.1% TFA; 30/704100/0 in 12 min; detection at 254 nm)

(IIX) 7-[(3-phosphopropyl)sulfonyl]-2-aminoacridin-9(10H)-one (1)

[0099] ##STR00017##

[0100] Pd/C (10%, in oxidized form, 11 mg) is suspended in 2-propanol (3 mL) in an argon atmosphere. Compound 9 (24 mg, 43 μmol) is added and a hydrogenate atmosphere is established. The suspension is stirred at room temperature for 21 h with partial dissolution of the solid. The solution is filtered over Celite to remove the Pd/C and the solvent is evaporated off. HPLC indicates complete conversion of the reactant. The crude product is suspended in water (5 ml) and cooled to 0° C. TFA (10 mL) is then added dropwise. Warm the reaction mixture to room temperature and stir for 3 h. HPLC shows complete deprotection of the phosphate. The reaction mixture is freeze-dried overnight and then dissolved in aq. TEAB buffer (pH 8). The crude product is purified via preparative HPLC (ACN/H.sub.2O (0.05% v/v TEAB buffer pH 8, 5/95%.fwdarw.27/73%, in 10 min, t.sub.R=8.3 min). The triethylammonium salt (1.5 eq.) of compound 1 is obtained as a brown solid (13 mg, 31 μmol, 86%).

[0101] MS (ESI): m/z (positive mode): 413.1 [M+H].sup.+, 435.0 (M+H).sup.+, 457.0 [M+2Na].sup.+,

[0102] HR-MS (ESI): C.sub.16H.sub.17N.sub.2O.sub.7PS: calc. [M+H].sup.+: 413.0572, found: 413.0567, [M+Na].sup.+: calc. 435.0386, found: 435.0393.

[0103] .sup.1H-NMR (400 MHz, D.sub.2O): δ [ppm]=8.37 (d, J=2.1 Hz, 1H, 1-H), 7.83 (dd, J=8.9, 2.1 Hz, 1H, 3-H), 7.21 (d, J=8.9 Hz, 1H, 4-H), 7.06-6.95 (m, 2H, 6-H, 8-H), 6.91 (d, J=8.7 Hz, 1H, 5-H), 3.82 (q, J=5.9 Hz, 2H, 1′-H), 3.65-3.42 (m, 2H, 3′-H), 3.15 (q, J=7.4 Hz, 10H, NEt.sub.3) 2.08-1.95 (m, 2H, 2′-H), 1.24 (t, J=7.4 Hz 0.15H, NEt.sub.3),

[0104] .sup.13C-NMR (100 MHz, D.sub.2O): δ [ppm]=177.3, 142.2, 141.9, 133.9, 129.7, 128.7, 127.9, 126.1, 120.7, 118.9, 118.6, 117.3, 107.8, 61.9, 52.9, 46.6 (NEt.sub.3), 23.9, 8.2 (NEt.sub.3).

[0105] .sup.31P-NMR (162 MHz, D.sub.2O): δ [ppm]=3.76.

(IX) Synthesis of the Glucose-Conjugates

[0106] Synthesis of the Compound 1-GLU

##STR00018##

[0107] Compound 17 (3 mg, 7 μmol) is dissolved in a malonic acid solution (0.36 mL, 1 M in DMSO). Aqueous glucose solution (72 μL, 0.1 M, 1.1 eq.) and pic-BH.sub.3 (87 μL, 1 M, 12 eq., in DMSO) are added. The reaction mixture is incubated at 35° C. for 18 h on a shaker. After addition of H.sub.2O/ACN (3:1), the reaction mixture is freeze-dried. The crude product is purified via preparative HPLC (ACN/H.sub.2O+0.1% TFA: 2/98.fwdarw.30/70, in 10 min). Pure 1-Glu is obtained as an orange solid (2.1 mg, 3 μmol, 33%).

[0108] MS (ESI): m/z (negative mode)=575.1 [M].sup.−

[0109] Synthesis of the Compound 2-GLU

##STR00019##

[0110] Compound 2 (3.35 mg, 10 μmol) is dissolved in a malonic acid solution (0.4 mL, 1 M in DMSO). Aqueous glucose solution (90 μL, 0.1 M, 1.1 eq.) and pic-BH.sub.3 (108 μL, 1 M, 12 eq., in DMSO) are added. The reaction mixture is incubated at 35° C. for 18 h on a shaker. After addition of H.sub.2O/ACN (3:1), the reaction mixture is freeze-dried. The crude product is purified via preparative HPLC (ACN/H.sub.2O+0.1% TFA: 2/98.fwdarw.30/70, in 10 min). Pure 2-Glu is obtained as an orange solid (2 mg, 4 μmol, 40%).

[0111] MS (ESI): m/z (negative mode)=495.2 [M].sup.−

[0112] HR-MS (ESI): C.sub.22H.sub.28N.sub.2O.sub.9S: calc. [M+H].sup.+: 497.1600, found: 497.1588.

[0113] Photophysical Properties of Fluorescent Markers

[0114] The spectroscopic properties are shown by way of example in the following two tables using compounds 1 and 2.

[0115] For compound 1, the following properties are obtained as a function of the solvent:

TABLE-US-00001 TABLE 1 [00020] Solvent MEOH TEAB (pH 8) Absorption, nm (ε, M.sup.−1 .Math. cm.sup.−1) 428 (2530) 423 (2750) Emission (nm) 567 583 Absolute quantum yield φ f, % 11.5 (420 nm) 5.3 (420 nm) (Excitation wavelength) Lifetime (τ, ns) 10.1 3.1 Stokes-Shift (nm) 139 157 Stokes-Shift (10.sup.3 cm.sup.−1) 73 000 64 000

[0116] The fluorescence marker is characterized by high quantum yields even in different solvents and shows emission at wavelengths that differ from the wavelengths of conventional fluorescence markers, especially in glycan analysis. It is therefore possible to work in parallel with two different fluorescence markers. Thus, sample and reference can be labeled differently and simultaneously detected in different wavelength ranges. In this way, a pre- or post-run with a separate molecular weight reference can be omitted.

[0117] Compound 1 can also be characterized by the following parameters:

[0118] MS(ESI): m/z (positive mode): 413.1 [M+H].sup.+, 435.0 (M+H).sup.+, 457.0 [M+2Na].sup.+

[0119] HR-MS (ESI): C16H17N2O7PS: calc. [M+H].sup.+: 413.0572, found: 413.0567, [M+Na].sup.+: calc. 435.0386, found: 435.0393.

[0120] .sup.1H-NMR (400 MHz, D.sub.2O): δ [ppm]=8.37 (d, J=2.1 Hz, .sup.1H, 1-H), 7.83 (dd, J=8.9, 2.1 Hz, .sup.1H, 3-H), 7.21 (d, J=8.9 Hz, .sup.1H, 4-H), 7.06-6.95 (m, 2H, 6-H, 8-H), 6.91 (d, J=8.7 Hz, .sup.1H, 5-H), 3.82 (q, J=5.9 Hz, 2H, 1′-H), 3.65-3.42 (m, 2H, 3′-H), 3.15 (q, J=7.4 Hz, 10H, NEt.sub.3) 2.08-1.95 (m, 2H, 2′-H), 1.24 (t, J=7.4 Hz 0.15H, NEt.sub.3).

[0121] .sup.13C-NMR (100 MHz, D.sub.2O): δ [ppm]=177.3, 142.2, 141.9, 133.9, 129.7, 128.7, 127.9, 126.1, 120.7, 118.9, 118.6, 117.3, 107.8, 61.9, 52.9, 46.6 (NEt.sub.3), 23.9, 8.2 (NEt.sub.3).

[0122] .sup.31P-NMR (162 MHz, D.sub.2O): δ [ppm]=3.76.

[0123] For compound 2, the following properties are obtained as a function of the solvent:

TABLE-US-00002 TABLE 2 [00021] Solvent MEOH TEAB (pH 8) Absorption, nm (ε, M.sup.−1 .Math. cm.sup.−1) 427 (2200) 423 (2940) Emission (nm) 567 588 Absolute quantum yield φ f, % 17 (420) 5.3 (4520) (Excitation wavelength) Lifetime (τ, ns) 9.7 2.8 Stokes-Shift (nm) 138 165 Stokes-Shift (10.sup.3 cm.sup.−1) 72 000 61 000

[0124] The fluorescent marker is characterized by high quantum yields in different solvents and shows emission at wavelengths that differ from the wavelengths of conventional fluorescent markers, especially in glycan analysis. It is therefore possible to work in parallel with two different fluorescence markers. The sample and reference can thus be labeled differently and detected simultaneously in different wavelength ranges. In this way, a pre- or post-run with a separate molecular weight reference can be omitted.

[0125] Compound 2 can also be characterized by the following parameters:

[0126] MS (ESI): m/z (positive mode): 333.1 [M+H].sup.+, 355.1 [M+Na].sup.+, 665.2 [2M+H].sup.+, 687.2 [2M+Na].sup.+.

[0127] HR-MS (ESI): C16H16N2O4S: calc. [M+H].sup.+: 333.0904, found: 333.0911, [M+Na].sup.+: calc. 355.0723, found: 355.0730.

[0128] .sup.1H-NMR (400 MHz, DMSO-d 6): δ [ppm]=12.03 (s, 1H, NH), 8.62 (d, J=2.2 Hz, 1H, 1-H), 7.98 (dd, J=8.8, 2.2 Hz, 1H, 3-H), 7.65 (d, J=8.8 Hz, 1H, 4-H), 7.46-7.37 (m, 2H, 6-H, 8-H), 7.19 (dd, J=8.8, 2.7 Hz, 1H, 5-H), 5.39 (s, 2H, NH 2), 4.61 (t, J=5.3 Hz, 1H, OH), 3.40 (q, J=5.9 Hz, 2H, 3′-H), 3.35-3.27 (m, 1H, 1′-H), 1.78-1.60 (m, 2H, 2′-H),

[0129] .sup.13C-NMR (101 MHz, DMSO-d.sub.6): δ [ppm]=175.8, 144.6, 142.5, 132.4, 129.9, 129.4, 127.9, 123.8, 122.5, 118.7, 118.6, 117.9, 105.9, 58.7, 52.5, 26.2.

[0130] HPLC: tr=4.2 min (ACN/H.sub.2O+0.05 M TEAB pH˜ 8; 90/10.fwdarw.50/0 in 12 min; detection at 254 nm)

[0131] Photophysical Properties of Fluorescent Marker Conjugates

[0132] The spectroscopic properties of the fluorescent marker conjugates with a glucose molecule are shown as examples in the following two tables.

[0133] The following properties are obtained for the conjugate of compound 1:

TABLE-US-00003 TABLE 3 [00022] Solvent TEAB (pH 8) Absorption, nm 429 (2750) (ε, M.sup.−1 .Math. cm.sup.−1) Emission (nm) 593 Absolute quantum yield φ f, 5.3 (430 nm) % (Excitation wavelength) Lifetime (τ, ns) 3.4 Stokes-Shift (nm) 164 Stokes-Shift (10.sup.3 cm.sup.−1) 61 000

[0134] The fluorescent marker is characterized by high quantum yields in different solvents and shows emission at wavelengths that differ from the wavelengths of conventional fluorescent markers, especially in glycan analysis. It is therefore possible to work in parallel with two different fluorescence markers. The sample and reference can thus be labeled differently and detected simultaneously in different wavelength ranges. In this way, a pre- or post-run with a separate molecular weight reference can be omitted. It should also be noted that the spectroscopic properties of the fluorescent label change only slightly due to conjugate formation. This is an indication of a stable spectroscopic system.

[0135] The following properties are obtained for the conjugate of compound 2:

TABLE-US-00004 TABLE 4 [00023] Solvent TEAB (pH 8) Absorption, nm (ε, M.sup.−1 .Math. cm.sup.−1) 433 (2490) Emission (nm) 597 Absolute quantum yield φ f, % 4.5 (430 nm) (excl. wavelength) lifetime (τ, ns) 3.4 Stokes-Shift (nm) 164 Stokes-Shift (10.sup.3 cm.sup.−1) 61 000

[0136] The fluorescent marker is characterized by high quantum yields in different solvents and shows emission at wavelengths that differ from the wavelengths of conventional fluorescent markers, especially in glycan analysis. It is therefore possible to work in parallel with two different fluorescence markers. The sample and reference can thus be labeled differently and detected simultaneously in different wavelength ranges. In this way, a pre- or post-run with a separate molecular weight reference can be omitted. It should also be noted that the spectroscopic properties of the fluorescent label change only slightly due to conjugate formation. This is an indication of a stable spectroscopic system.

[0137] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are inter-changeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.