Abstract: This work focuses on profiling N-linked glycans by capillary electrophoresis coupled to mass spectrometry using a novel fluorescent and mass spectrometry (MS) active derivatization tag. The label is based on 2-phenylpyridine bearing tertiary amine and hydrazide functionalities. It provides efficient labeling via hydrazone formation chemistry, promising fluorescence properties, and ionization efficiency in the positive ion MS mode. Electrophoretic analysis in a neutral-coated capillary allowed baseline separation of maltooligosaccharides with limits of detection in nanomolar concentrations. The developed labeling method was successfully applied to the analyses of N-linked glycans released from several glycoproteins such as bovine ribonuclease B, human immunoglobulin G, or chicken albumin.
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(4) 6-chloronicotinate methyl ester(7-1), 1.0 g, 5.85 mmol) was placed in THF (10 mL) and the temperature was dropped to 0 C.A solution of LiAlH4 (LAH, 244 mg, 6.44 mmol) in THF (10 mL) was obtained. After the dropwise addition, the reaction mixture was reacted at 20 C for 2 h.After completion of the reaction, it was quenched with NaOH solution (6.25 mol), then extracted twice with ethyl acetate (2×50 mL) and dried over anhydrous Na2SO4.Filter to dry to give a white solid6-chloro-3-methylhydroxypyridine(6-1), 780 mg, 5.59 mmol, yield 96%),Used directly in the next step.
95%
To a solution of methyl 6-chloronicotinate (0.60 g, 3.50 mmol) in THF (10 niL) at - 78 C was added a solution of DIBAL-H (1 M in toluene, 10.5 m-L, 10.5 mmol) and the reaction stirred from -78 0C to room temperature for 1 h. The reaction was diluted with a saturated aqueous solution of sodium potassium tartrate (25 mL) and CH2Cl2 (30 mL) and stirred vigorously overnight. The layers were separated and the aqueous layer was extracted with CH2Cl2 (2 x 15 mL). The combined organic extracts were dried (Na2SO4) and concentrated to afford the desired alcohol (0.48 g, 95%) as a white solid.
70%
With lithium aluminium tetrahydride; In tetrahydrofuran; at 0 - 20℃; for 3h;Inert atmosphere;
a) (6-chloropyridin-3-yl)methanol To a suspension of LiAIH4(1.5 g, 40 mmol) in THF(80 mL) was added methyl 6-chloronicotinate (1.56 g, 10 mmol) slowly at 0-5 Π under N2atmosphere. The reaction mixture was stirred at rt for 3h before it was quenched with 15% NaOH and water. The mixture was filtered through Celite and the cake was washed with EtOAc (100 ml_x3). Thefiltrate was washed with brine (200 mL), dried and concentrated to give the desired product as yellow oil (1 g, 70%). [LCMS: RtA = 0.88 min, m/z 144.2 [M+H]+].
70%
With lithium aluminium tetrahydride; In tetrahydrofuran; at 0 - 20℃; for 3h;Inert atmosphere;
a) (6-chloropyridin-3-yl)methanol To a suspension of LiAlH4 (1.5 g, 40 mmol) in THF (80 mL) was added methyl 6-chloronicotinate (1.56 g, 10 mmol) slowly at 0-5□ under N2 atmosphere. The reaction mixture was stirred at rt for 3 h before it was quenched with 15% NaOH and water. The mixture was filtered through Celite and the cake was washed with EtOAc (100 mL*3). The filtrate was washed with brine (200 mL), dried and concentrated to give the desired product as yellow oil (1 g, 70%). [LCMS: RtA=0.88 min, m/z 144.2 [M+H]+].
With sodium sulfate; In tetrahydrofuran;
Reference example 1. 6-chloropyridine-3-carboaldehyde 0.20g (1.17mmol) of methyl 6-chloronicotinate (Lancaster) was dissolved in 4ml of tetrahydrofuran anhydrous, and 0.045g (1.17mmol) of lithium aluminum hydride was added thereto under ice bath cooling, followed by stirring for 30 minutes. Sodium sulfate 10 hydrate was added to the reaction mixture until no foam appeared, and after stirring for 2 hours, filtration with Celite was performed. The filtrate was evaporated, and the residue was purified with silica gel column chromatography (ethyl acetate) to obtain 0.090g of 6-chloropyridine-3-methyl alcohol. 1H NMR (CDCl3, 300MHz) δ 3.79(1H, brs), 4.70 (2H, s), 7.31 (1H, d, J = 8.3Hz), 7.69 (1H, dd, J = 2.4, 8.3Hz), 8.28 (1H,d, J= 2.4Hz) mass spectrum EIMS, m/z:143(M+)
With 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione; at 25℃; for 12h;
General procedure: DBDMH (20 mmol) was added in portion wise to a mixture of 1b (5 mmol) andmethanol (30 ml). The reaction was kept at room temperature. After the mixture wasstirred for 12h, the methanol was vacuum evaporated. The residue was dissolved byMTBE (30 ml), washed with water (330 ml).The organic extracts was dried byanhydrous MgSO4, filtered, and concentrated under reduce pressure. The residue waspurified by column chromatography (silica gel: petroleum ether/ethyl acetate, 30:1) toafford the product as light yellow solid ( 92% yield).
methyl 6-[3-(trifluoromethoxy)phenoxy]pyridine-3-carboxylate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With potassium carbonate; In N,N-dimethyl-formamide; at 100℃; for 5h;
General procedure: A mixture of 2-chloro-5-(trifluoromethyl)pyridine (5.45 g,30 mmol), ethyl 3-hydroxybenzoate (5.5 g, 33 mmol), K2CO3(4.98 g, 36 mmol) and DMF (20 mL) was stirred at 100 C for 5 h.After cooling to room temperature, the reaction mixture wasdiluted with water and extracted with AcOEt. The organic layerwas washed with brine, dried over Na2SO4 and concentrated invacuo. The resulting residue was purified by silica gel chromatography(hexane-AcOEt) to give 10a
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