4-(Hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-D-mannofuranose

Abstract

Low molecular weight chitooligosaccharide with one 2,5-anhydro-d-mannofuranose unit at the reducing end (COSamf) was prepared by nitrous deamination of fully N-deacetylated chitosan. The functionalization of the amf unit by reductive amination with 4-(hexyloxy)aniline in presence of NaBH₃CN was achieved in high yield. The chemical structure of the targeted 4-(hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-d-mannofuranose was fully characterized by NMR spectroscopy, MALDI-TOF mass spectrometry and size-exclusion chromatography. This synthesis opens the way to a new generation of COSamf derivatives with potential amphiphilic properties.

Chitosan is a random linear polysaccharide of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) units linked by β-(1→4) glycosidic bonds. Chitosan is generally obtained by chemical or enzymatic N-deacetylation of chitin, the second most abundant naturally occurring polymer produced industrially from shells of crustaceans and squid pens [1,2,3,4]. Chitooligosaccharides (COS), also named chitosan or chitin oligomers, have recently received considerable attention as functional biomolecules with a wide range of applications in food, agriculture, medicine, pharmaceutics and cosmetics. COS take advantage of their various interesting physico-chemical and biological properties, including water-solubility, biocompatibility, antibacterial, antifungal and antitumoral activities [5,6,7,8,9]. In order to improve the scope of their properties, chemical modifications of COS have been investigated for a decade [10,11,12]. In this study, we described the synthesis of the 4-(hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-d-mannofuranose. The interest of this work is to take advantage of the reactivity of the aldehyde group of the 2,5-anhydro-d-mannofuranose (amf) unit present at the reducing end of COS obtained by nitrous deamination of chitosan, to generate original amphiphilic COS derivatives.

Results and Discussion

4-(hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-d-mannofuranose was efficiently synthesized from chitosan in a two-step procedure involving the reductive amination of chitooligosaccharide-2,5-anhydro-d-mannofuranose (COSamf, 1) with 4-(hexyloxy)aniline as illustrated in Scheme 1.

COSamf 1 was prepared by nitrous acid deamination of a fully N-deacetylated chitosan based on the method previously described by Tommeraas et al. [13]. Thus, the depolymerization of chitosan (DA 0%, Mw = 270 kg/mol; Mn = 115 kg/mol, Ð = 2.3) by NaNO₂ (GlcN/NaNO₂ molar ratio = 10) in aqueous acid solution at room temperature led to COSamf 1 in 67% mass yield after 24 h of reaction. The chemical structure of COSamf 1 was fully confirmed by ¹H and ¹³C-NMR spectroscopies, MALDI-TOF mass spectrometry and size-exclusion chromatography (see Supporting Information). Therefore it has been shown COSamf 1 is composed of a mixture of oligomers, with an average number of GlcN units into chains around 23.

The reductive amination of COSamf 1 with 4-(hexyloxy)aniline in presence of NaBH₃CN was carried out at 40 °C in buffer solution (pH 5.5) for 48 h, leading to the targeted 4-(hexyloxy)aniline-linked COSamf 2 in an excellent mass yield (92%). The chemical structure of the title compound was entirely characterized by ¹H and ¹³C-NMR spectroscopies thanks to two-dimensional NMR analyses, pointing out the coupling reaction between the aldehyde function of COSamf 1 and the amine group of the aniline residue. Thus, the presence of the corresponding CH₂-N covalent linkage was displayed at δ 3.50 ppm for methylene protons and 69.7 ppm for the methylene carbon, respectively in ¹H and ¹³C-NMR spectra. As confirmed by MALDI-TOF mass spectrometry (see Supporting Information), 4-(hexyloxy)aniline-linked COSamf 2 is composed of a mixture of oligomers, with an average number of GlcN units into chains, determined by both ¹H-NMR and SEC, equal to 23 as for COSamf 1.

Experimental

4-(Hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-d-mannofuranose 2: A fully N-deacetylated chitosan (2.1 g, 13 mmol GlcN unit) was solubilized in 1 L of water by addition of 11.5 mL HCl (37% w/w). A freshly prepared solution of NaNO₂ (1.3 mmol) was added and the reaction was allowed to proceed for 24 h at room temperature. The product was precipitated by addition of conc. NH₄OH, centrifuged (15 min, 11200 rpm), washed with distilled water until neutral pH, then freeze-dried leading to COSamf 1 (1.4 g, 67% mass yield) as a white powder. COSamf 1 (0.5 g, 0.14 mmol of amf unit) was then solubilized in 20 mL of ammonium acetate buffer (50 mM, pH 5.5). 271 mg of 4-(hexyloxy)aniline (1.4 mmol) in 10 mL ethanol and 88 mg of sodium cyanoborohydride (1.4 mmol) were added and the reaction was allowed to proceed for 48 h at 40 °C. The product was precipitated by addition of conc. NH₄OH, centrifuged (15 min, 11,200 rpm), washed with water/ethanol (50:50) then freeze-dried leading to 2 (460 mg, 92% mass yield) as a white powder. ¹H-NMR (300 MHz, D₂O, 298 °K): δ (ppm) 7.45 (d, J = 9.0 Hz, 2H, H aromatic), 7.15 (d, J = 9.0 Hz, 2H, H aromatic), 4.90–4.70 (m, 23H, H-1 GlcN), 4.32 (m, 1H, H-3 amf), 4.24 (m, 1H, H-5 amf), 4.18 (m, 1H, H-4 amf), 4.14 (m, 1H, H-2 amf), 4.08 (t, J = 6.6 Hz, 2H, CH₂O), 4.00–3.40 (m, H-3 to H-6 GlcN, H-6 amf, CH₂N), 3.18 (t, J = 8.9 Hz, 23H, H-2 GlcN), 1.75 (m, 2H, CH₂), 1,40 (m, 2H, CH₂), 1.30 (m, 4H, 2CH₂), 0.85 (t, J = 7.0 Hz, 3H, CH₃). ¹³C-NMR (125 MHz, D₂O, 298 °K): δ (ppm) 159.8 (CO aromatic), 127.3 (CN aromatic), 124.7 (2CH aromatic), 116.8 (2CH aromatic), 99.3 (C-1' GlcN), 98.1 (C-1 GlcN), 86.8 (C-4 amf), 83.1 (C-5 amf), 78.6 (C-2 amf), 77.9 (C-3 amf), 77.0 (C-5’ GlcN), 76.9 (C-4 GlcN), 75.3 (C-5 GlcN), 72.3 (C-3' GlcN), 70.6 (C-3 GlcN), 70.2 (C-4' GlcN), 69.7 (CH₂O), 61.9 (C-6 amf), 60.9 (C-6' GlcN), 60.6 (C-6 GlcN), 56.4 (C-2 GlcN), 56.1 (C-2' GlcN), 53.2 (CH₂N), 31.3 (CH₂), 28.7 (CH₂), 25.4 (CH₂), 22.5 (CH₂), 13.9 (CH₂). Note that C’ represents carbon atoms of the GlcN unit linked to the amf unit. MALDI-TOF MS: presence of a major peak at m/z 1650.5 attributed to HO-(GlcN)₈-C₁₈H₂₈NO₄ (m/z monoisotopic calcd for [C₆₆H₁₁₇O₃₇N₉Na]⁺ = 1650.7 mass units (Δ = 0.01%)). HRMS (ESI): calcd for C₆₆H₁₁₇O₃₇N₉Na: m/z 1650.7448; found 1650.7432 [M+Na]⁺ (difference = 1.6 ppm).