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.

Key words

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.

11

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.

12

Determined with an Agilent G1321A fluorescence detector.

13

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).

14

Synthesis and analytical data are reported elsewhere.

15

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.