Structural Analysis of Difructose Anhydrides (DFAs) Synthesized from Monosaccharides by Thermal Treatment

Akira Yamamori; Yusuke Takata; Eri Fukushi; Jun Kawabata; Hideki Okada; Naoki Kawazoe; Keiji Ueno; Shuichi Onodera; Norio Shiomi

doi:10.5458/jag.jag.JAG-2015_004

Abstract

Ten difructose anhydrides (DFAs) were the predominant products formed from the thermal treatment of equal amounts of D-glucose and D-fructose under melting conditions at 150°C for 60 min. The DFAs were isolated from the reaction mixture by carbon-Celite column chromatography and preparative high-performance liquid chromatography. The structures of the saccharides were confirmed by NMR measurements. We present the complete assignments of the ¹H- and ¹³C-NMR signals of two of these DFAs for the first time.

Abbreviations

COSY, 2D correlation spectroscopy; DFA, difructose anhydride; HMBC, heteronuclear multiple-bond correlation.

TEXT

Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two monosaccharides and are expected to have novel physiological functions owing to their unique structures and properties. DFAs promote the calcium absorption from the small intestinal.¹⁾ Therefore, DFAs is expected to have the prevent effect from the osteoporosis. DFAs are known to be formed by heating fructose, sucrose, inulin,²⁾ and levan,³⁾ or by reaction with an enzyme, such as yeast invertase or fructotransferase from microorganisms.⁴⁾ ⁵⁾ ⁶⁾ ⁷⁾ ⁸⁾ ⁹⁾

We have previously reported the structural analyses of a number of oligosaccharides, including β-D-fructopyranosyl-(2→6)-D-glucopyranose (β-Fp2→6G),¹⁰⁾ β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→3)-D-glucopyranose,¹¹⁾ β-D-fructopyranosyl-(2→6)-[β-D-glucopyranosyl-(1→3)]-D-glucopyranose,¹⁰⁾ β-D-fructopyranosyl-(2→6)-D-fructofuranose,¹²⁾ β-D-fructopyranosyl-(2→1)-β-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside,¹²⁾ and α-D-fructofuranosyl-(2→6)-D-glucopyranose (α-Ff2→6G)¹³⁾ from the fermented beverage of plant extracts. Two of the above saccharides, β-Fp2→6G and α-Ff2→6G, can be synthesized from D-glucose and D-fructose using a thermal melting treatment, and several characteristics of the saccharides have been reported.¹⁴⁾ ¹⁵⁾ DFAs can also be synthesized from D-glucose and D-fructose using the thermal treatment. In this paper, we confirmed the structures of ten DFAs formed during the thermal treatment of a mixture of D-glucose and D-fructose powders. In addition, we report the complete assignments of the ¹H- and ¹³C-NMR signals of two of these DFAs for the first time.

A powdered mixture of 30 g each, D-glucose and D-fructose (Sigma Chemical Co., St. Louis, USA) was carefully heated at 150°C for 60 min in an electric furnace. After cooling, the saccharide melt was dissolved in distilled water (250 mL). The saccharide solution (300 mL) was loaded onto a carbon-Celite column {4.8 × 36 cm [1:1; charcoal and Celite-535 (Wako Pure Chemical Industries, Osaka, Japan)]} and successively eluted with water (4.4 L) and 5% ethanol solution (10.0 L). Almost all the unreacted D-glucose and D-fructose was eluted with water (600 mL), followed by saccharides 1, 2, 3, and 4, also eluted with water (3.2 L). The water fraction containing the four saccharides was concentrated to 24.0 mL. Saccharides 5, 6, 7, 8, 9, and 10 were eluted with 5% ethanol solution (5.0 L). The ethanol fraction containing the six saccharides was concentrated to 12.0 mL. Subsequently, the concentrated fractions were purified at 20°C (50 times; 200 μL injection volume) or 35°C (50 times; 100 μL injection volume), using a preparative HPLC system (Tosoh Corp., Tokyo, Japan) equipped with an ODS-100V column (20 mm × 25 cm) and refractive index detector. Fractions were eluted with distilled water at 3.0 mL/min. The saccharides were further purified at 25°C with an analytical HPLC system (Tosoh) equipped with three ODS-100V columns (4.6 mm × 25 cm) connected by series and refractive index detector, eluting with distilled water at 0.4 mL/min (chromatograms shown in Figs. 1(A) and 1(B)). Finally, the purified saccharides 1‒10 (1, 44 mg; 2, 33 mg; 3, 52 mg; 4, 39 mg; 5, 169 mg; 6, 25 mg; 7, 10 mg; 8, 69 mg; 9, 206 mg; and 10, 2 mg) were obtained as white powders. The yields of saccharides 1‒10 were 0.073, 0.055, 0.086, 0.065, 0.282, 0.042, 0.017, 0.115, 0.343, and 0.003%, respectively.

Fig. 1.

Preparative HPLC of the DFA fractions eluted during column chromatography.

The solution of synthesized saccharides was loaded onto a carbon-Celite column and successively eluted with water (A) and 5% ethanol (B).

The structures of saccharides 1‒10 were confirmed using NMR spectroscopy (¹H at 500 MHz and ¹³C at 126 MHz). The saccharides were identified as: 1 (β-D-fructofuranose β-D-fructopyranose 2,1′:3,2′-dianhydride), 2 (β-D-fructofuranose β-D-fructofuranose 2,1′:3,2′-dianhydride; DFA II), 3 (α-D-fructofuranose β-D-fructofuranose 1,2′:2,3′-dianhydride; DFA III), 4 (α-D-fructofuranose β-D-fructofuranose 1,2′:2,1′-dianhydride; DFA I), 5 (α-D-fructofuranose β-D-fructopyranose 1,2′:2,1′-dianhydride), 6 (α-D-fructofuranose α-D-glucopyranose 1,1′:2,2′-dianhydride), 7 (α-D-fructofuranose α-D-fructopyranose 1,2′:2,1′-dianhydride), 8 (β-D-fructofuranose β-D-fructofuranose 1,2′:2,1′-dianhydride), 9 (α-D-fructofuranose α-D-fructofuranose 1,2′:2,1′-dianhydride), and 10 (β-D-fructofuranose β-D-fructopyranose 1,2′:2,1′-dianhydride). The structures of saccharide 1‒10 are shown in Fig. 2. The ¹H- and ¹³C-NMR spectral data are shown in Tables 1, 2, and 3. Although saccharides 1‒10 are known materials, the complete assignments of the ¹H- and ¹³C-NMR spectra of saccharides 1 and 10 were realized for the first time.

Fig. 2.

Structures of synthesized DFAs.

Saccharide 1 (β-D-fructofuranose β-D-fructopyranose 2,1′:3,2′-dianhydride), Saccharide 2 (β-D-fructofuranose β-D-fructofuranose 2,1′:3,2′-dianhydride; DFA II), Saccharide 3 (α-D-fructofuranose β-D-fructofuranose 1,2′:2,3′-dianhydride; DFA III), Saccharide 4 (α-D-fructofuranose β-D-fructofuranose 1,2′:2,1′-dianhydride; DFA I), Saccharide 5 (α-D-fructofuranose β-D-fructopyranose 1,2′:2,1′-dianhydride), Saccharide 6 (α-D-fructofuranose α-D-glucopyranose 1,1′:2,2′-dianhydride), Saccharide 7 (α-D-fructofuranose α-D-fructopyranose 1,2′:2,1′-dianhydride), Saccharide 8 (β-D-fructofuranose β-D-fructofuranose 1,2′:2,1′-dianhydride), Saccharide 9 (α-D-fructofuranose α-D-fructofuranose 1,2′:2,1′-dianhydride), and Saccharide 10 (β-D-fructofuranose β-D-fructopyranose 1,2′:2,1′-dianhydride).

Table 1.

¹H- and ¹³C-NMR spectral data (δ^a in ppm, J in Hz) for saccharides 1, 2, and 3.

^aThe chemical shifts of ¹H (δ_H) and ¹³C (δ_C) in ppm were respectively determined relative to the external standard of sodium [2,2,3,3-²H₄]-3-(trimethylsilyl) propanoate in D₂O (δ_H 0.00 ppm) and 1,4-dioxane (δ_C 67.40) in D₂O. ^bbr s, broad singlet. ^cm, multiplet.

Table 2.

¹H- and ¹³C-NMR spectral data (δ^a in ppm, J in Hz) for saccharides 4, 5, and 6.

Table 3.

¹H- and ¹³C-NMR spectral data (δ^a in ppm, J in Hz) for saccharides 7, 8, 9, and 10.

^a The chemical shifts of ¹H (δ_H) and ¹³C (δ_C) in ppm were respectively determined relative to the external standard of sodium [2,2,3,3-²H₄]-3-(trimethylsilyl) propanoate in D₂O (δ_H 0.00 ppm) and 1,4-dioxane (δ_C 67.40) in D₂O. ^b br s, broad singlet.

We characterized the structure of saccharide 1 based on the following observations with 2D NMR technique and the assignment of the chemical shifts. For one of the Fru residues, the COSY spectrum was assigned using the spin system from H3′ to H5′. The heteronuclear multiple-bond correlations (HMBCs) of C5′/H6′, C1′/H3′, and C2′/H6′ confirmed the assignment of these signals. This unit was assigned as βFru_p by the HMBCs of C2′/H6′, the δ_C chemical shifts, and J_(H,H) values. Fru5 of the other Fru residue was estimated from the δ_C chemical shifts. The HMBC_s of C5/H6, C4/H6, C4/H3, and C2/H3 in this Fru residue confirm the assignment of the signals (Fig. 3). The signal for C2 of βFru_f showed an inter-residual HMBC to H1 of βFru_p, whereas C2 of βFru_p did not show any HMBCs to protons of βFru_f. Therefore, the ¹³C-NMR spectrum of saccharide 1 in H₂O:D₂O (9:1) was compared with that in D₂O. The δ_C of C3 did not change, allowing us to estimate the C3‒C2′ connectivity. These results led to the assignment of 1 as β-D-fructofuranose β-D-fructopyranose 2,1′:3,2′-dianhydride.¹⁶⁾ All ¹H- and ¹³C-NMR signals were assigned as shown in Table 1.

Fig. 3.

Part of HSQC (a) and HMBC (b) spectra of saccharides 1 and 10.

Next, we analyzed saccharide 10. The COSY spectrum assigned the spin system of one of the Fru residues, from H3 to H5. The HMBCs of C6/H4 and C2/H5,H1 in this residue confirmed the assignment of these signals. This residue was assigned as αFru_f by the C2/H5 HMBC, the δ_C chemical shifts, and J_(H,H) values. For the other fructose residue, the COSY spectrum assigned the spin system from H3′ to H5′. The HMBCs of C1′/H3′ and C2′/H6′, H1′ in this residue also confirmed the assignment of these signals. This residue was assigned as βFru_p by the C2′/H5′ HMBC, the δ_C chemical shifts, and J_(H,H) values. The C2 of βFru_f and C2′ of βFru_p showed inter-residual HMBCs to H1′ of βFru_p and H1 of βFru_f, respectively (Fig. 3). These results led to the assignment of 10 as β-D-fructofuranose β-D-fructopyranose 1,2′:2,1′-dianhydride. All ¹H-and ¹³C-NMR signals were assigned as shown in Table 3.

In this study, ten DFAs were isolated from a caramelized mixture of D-glucose and D-fructose formed via thermal treatment; however, these saccharides may be produced in the same way from only fructose. We intend to investigate the possibility of forming these saccharides from fructose or other monosaccharides.

REFERRENCES

Corresponding author

Register with J-STAGE for free!