Synthesis and Testing of a Novel Fluorescence Label for Carboxyls in Carbohydrates and Cellulosics

Abstract

A carboxyl-selective fluorescence label, 9H-fluoren-2-yl-diazomethane (FDAM, 6) for the detection of carboxyl groups specifically in polysaccharides and low-molecular weight carbohydrates has been developed. Attached in a pre-column derivatization step, the label allows detection of minute amounts of oxidized functionalities in oligosaccharides and cellulosics under the relatively harsh conditions of GPC separation in the DMAc/LiCl eluant and without any fluorescence interference with light scattering (MALLS) detection, so that carboxyl profiles of polysaccharides were obtained for the first time. Synthetic carbohydrate model compounds (1-3) were used to optimize the labeling reaction.

carboxyls - polysaccharides - uronic acids - fluorescence labeling - carbohydrates

References

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5

Auxiliary bases induced chain scission by β-elimination processes starting from carbonyls; generated hydrohalic acid caused chain cleavage at the glycosidic bonds.

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9H-Fluoren-2-yl-carboxaldehydrazone (1.04 g, 5.00 mmol) was dissolved in DMAc (approx. 13 mL) and stirred with MnO₂ (5.00 g) for 1.5 h at r.t. Excess oxidant and solids were removed by filtration through MgSO₄/Celite^® (w/w = 1:1), which was washed with DMAc to a filtrate volume of 40 mL. The resulting red solution of FDAM (6) was used for labeling, and can be stored in the dark at 4 °C for up to 7 d.

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Determined with an Agilent G1321A fluorescence detector.

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Example Procedure.
Methyl cellobiosiduronic acid (3, 64.1 mg, 0.17 mmol) dissolved in distilled H₂O (5 mL) was completely converted into the free acid by elution over ion exchange resin (H⁺-form, BioRad AG50 W-X8, 100-200 mesh) at r.t. After removal of the resin, the freeze-dried solution was dissolved in DMAc (4 mL) and treated with a freshly prepared solution of FDAM (6) in DMAc (40 mL, 0.125 M, 5.00 mmol) at 50 °C. After 10 min, TLC control (CHCl₃-MeOH, v/v = 10:3, R _f = 0.28) showed complete conversion, and glacial AcOH (1.5 mL) was added; parallel NMR experiments proved that consumption of 3 was complete after a few minutes. The mixture was dispersed on silica gel in a rotavapor at 70 °C and chromatographed on silica gel (CHCl₃-MeOH, v/v = 10:3) to afford 10 (80.4 mg, 85%, yield loss due to work-up and purification) as fawn crystals, mp 180-182 °C. ¹H NMR (DMSO-d ₆): δ = 7.91 (d, 2 H, ³ J = 7.9 Hz, H-4′′, H-5′′), 7.66 (s, 1 H, H-1′′), 7.60 (d, 1 H, ³ J = 7.2 Hz, H-8′′), 7.46 (d, 1 H, ³ J = 7.9 Hz, H-3′′), 7.42-7.40 (m, 1 H, H-6′′), 7.33 (ddd, 1 H, ³ J = 7.4 Hz, ⁴ J = 1.2 Hz, H-7′′), 5.35 (d, 1 H, ³ J = 5.1 Hz, OH-2), 5.29-5.16 (m, 2 H, O-CH₂-10′′), 5.05 (d, 1 H, ³ J = 4.5 Hz, OH-3′), 5.04 (d, 1 H, ³ J = 4.9 Hz, OH-2′), 4.99 (d, 1 H, ³ J = 5.3 Hz, OH-4′), 4.83 (d, 1 H, ³ J = 2.8 Hz, OH-3), 4.50 (t, 1 H, ³ J = 5.4 Hz, OH-6′), 4.29 (d, 1 H, ³ J = 7.8 Hz, H-1), 4.24 (d, 1 H, ³ J = 7.8 Hz, H-1′), 4.05 (d, 1 H, ³ J = 9.6 Hz, H-5), 3.94 (s, 2 H, CH₂-9′′), 3.74-3.69 (m, 1 H, H-6′), 3.61 (t, 1 H, ³ J = 9.2 Hz, H-4), 3.44-3.38 (m, 2 H, H-6′, H-3), 3.38 (s, 3 H, OCH₃-7), 3.21-3.14 (m, 2 H, H-5′, H-3′), 3.12-2.98 (m, 3 H, H-4′, H-2, H-2′). ¹³C NMR (CDCl₃): δ = 168.2 (C=O), 143.2, 143.2, 141.1, 140.6 (C-4a′′, C-4b′′, C-9a′′, C-9b′′), 133.9 (C-2′′), 127.4 (C-3′′), 126.9, 126.8 (C-6′′, C-7′′), 125.5 (C-1′′), 125.2 (C-8′′), 120.2, 119.8 (C-4′′, C-5′′), 104.0 (C-1), 102.9 (C-1′), 80.7 (C-4), 77.0, 76.5 (C-5′, C-3′), 73.8, 73.5 (C-5, C-3), 73.0, 72.8 (C-2, C-2′), 70.0 (C-4′), 66.9 (OCH₂-10′′), 61.1 (C-6′), 56.4 (OCH₃), 36.3 (CH₂-9′′). [α]_D ²⁰ -21 (c 0.5, DMSO).

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Synthesis and analytical data are reported elsewhere.

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General Procedure for the Determination of Carboxyls in Cellulosic Pulps by Heterogeneous Fluorescence Labeling.
Activated pulp, corresponding to 20 mg of dry pulp, was suspended in 0.1 M HCl and agitated for 20 s in a mixer. The pulp was washed with 0.1 M HCl, EtOH, DMAc, filtered off, transferred into a 4 mL vial, and suspended in 3 mL of DMAc. The FDAM solution (1 mL, approx. 0.125 M in DMAc) was added. The solution was agitated in a shaking bath for 7d at 40 °C. After derivatization, the pulp was filtered off, washed with DMAc, and transferred into a dry vial. Then, 1.6 mL of DMAc/LiCl (9%, m/v) were added, the sample was filtered through 0.45 µm filters after complete dissolution, and was analyzed by multi-detector GPC.