Rare 6-deoxy-d-altrose from the folk medicinal mushroom Lactarius akahatsu

A rare sugar, 6-deoxy-d-altrose, isolated from a polysaccharide extracted from an edible folk medicinal mushroom (Lactarius akahatsu) was identified using 1H and 13C-NMR including 2D-COSY and 2D-HSQC spectroscopy, and specific rotation. The 6-deoxy-sugar isolated from the acid hydrolysate of the polysaccharide extracted from L. akahatsu was involved in four anomeric isomers (αand β-pyranose, and α-and β-furanose) in aqueous solution due to mutarotation. Almost all of the signals from the 1D (1Hand 13C-) and 2D (COSY and HSQC)-NMR spectra of the 6-deoxy-sugar agreed with the data from the authentic 6-deoxy-daltrose. The specific rotation [α]589 of the 6-deoxy-sugar isolated from L. akahatsu was +17.6 0. Thus, the 6-deoxy-sugar isolated from Lactarius akahatsu was identified as 6-deoxy-d-altrose.


Introduction
In proceeding studies, we isolated a novel acetyl fucoidan from commercially marine cultured Cladosiphon okamuranus [1,2], and which has been patented [3]. The acetyl fucoidan has some biological activities, such as antitumor [4] and immuneenhancing [5]. Specifically, over-sulfated acetyl fucoidan, the sulfate content of which was 32.8%, showed significant antitumor activity [4]. The results suggested that the oversulfated acetyl fucoidan was applicable to an anticancer drug.
The fruiting bodies of mushrooms have been used as foodstuffs and folk medicine throughout the world since ancient times, especially in Japan and China [6,7]. Many attempts have been made to explore the use of mushrooms and their metabolites for the treatment of various human ailments [8,9]. Certain polysaccharides (β-glucans) from mushrooms have been applied as anti-tumor and immune-enhancing agents for clinical use in Japan [7,10]. Consequently, mushroom polysaccharides have drawn the attention of chemists and immune-biologists in recent years because they possess antitumor and immuneenhancing properties.
The fruiting bodies of a Lactarius lividatus (previously Lactarius hatsudake) have historical use as antitumor and antiviral agents in Japanese and Chinese folk medicine [11]. We identified a rare 6-deoxy-d-altrose from the mushroom, which is widespread in natural environments within South-East Asia, East Asia, North America, and Europe [12]. Our previous report detailed the first complete identification of a 6-deoxy-d-altrose in nature.
Lactarius akahatsu is widespread in natural environments throughout the world. In Okinawa Prefecture, Japan, its fruiting bodies have been eaten since ancient times alongside L. lividatus. The fruiting bodies of Lactarius akahatsu have also been used as folk medicine in Japan and China.
This paper describes identification of rare 6-deoxy-d-altrose from a polysaccharide isolated from L. akahatsu.

Materials and methods
The fresh fruiting bodies of L. akahatsu were collected in Onna Village, Okinawa, Japan in the February of 2009. The pilei of the fruiting bodies are 40-100 mm in diameter, and a light brownish orange in color. The lamellae and stipe are both orange. The fresh fruiting bodies were washed with distilled water and air dried at 40°C for 48 h before being ground into powder.
The fruiting bodies of L. akahatsu were suspended in ethanol (90%) or acetone, and stirred for 3 h to extract the pigment or lipid, respectively. The sample (20 g) was suspended in distilled water and stirred at 90°C for 3 h to extract the polysaccharide. The extract was then centrifuged at 23000 g for 20 min, and the supernatant was filtered through Celite 545 (Nakarai, Japan). The filtrate was precipitated by adding 2 volumes of ethanol, and the resulting solid was dried in vacuo [12].
The crude polysaccharide was dissolved in distilled water and 10% trichloroacetic acid was added to precipitate any protein. The solution was passed through Celite 545 and dialyzed at 4°C for 3 days. The dialysate was deionized by passage through a cation exchange column composed of Amberlite 120A H + (Organo, Japan). The solution was dialyzed against distilled water for 24 h at room temperature and subsequently lyophilized [12].

Synthesis of the authentic 6-deoxy-d-altrose
The authentic 6-deoxy-d-altrose was synthesized from d-mannose via the d-rhamnoside [14]. The selective 3-O-benzylation of the d-rhamnoside was accomplished using stannylation, in which an unprotected starting material was activated with dibutyltin oxide, benzylated in the presence of benzyl bromide and tetrabutylammonium iodide, and finally acetylated to afford the 3-O-benzylated derivative in 79% yield over two steps. The deprotection of benzyl group, which occurred without acetyl group migration, was accomplished in 98% yield by hydrogenolysis over palladium on carbon at atmospheric pressure. Next, the triflation was achieved using triflic anhydride and pyridine in dichloromethane to give the triflate, which was treated with tetrabutylammonium acetate in toluene to afford the expected 6-deoxy-d-altropyranoside in 71% over two steps. The 1 H NMR data of the compound confirmed that the desired structure with the signal at 5.17 (dd, 1 Hz, J 2,3 = 2.0 Hz, J 3,4 = 3.5 Hz, H-3) was synthesized. A dramatic change in the J 3,4 value from 9.6 Hz to 3.5 Hz indicated that the inversion of configuration had occurred at C-3. The oxidative removal of the p-methoxyphenyl group of the per-O-acetylated 6-deoxy-d-altropyranoside with ceric ammonium nitrate (CAN) afforded the hemiacetal in 68% yield, which was tentatively protected with a tetrahydropyranyl (THP) group to avoid any side reactions that may occur under the basic conditions in the next reaction. The removal of the acetyl groups under Zemplen conditions with subsequent acid hydrolysis of the THP group provided the desired free 6-deoxy-d-altrose in 78% over two steps.

Chemical procedures
The total carbohydrate content was determined with the phenol-sulfuric acid method using d-glucose as a standard [15]. The purified polysaccharide (70 mg) was dissolved in distilled water (20 mL) and sulfuric acid was added to reach a final concentration of 1.0 m. The mixture was subsequently heated to 100°C for 3 h. The hydrolysate was neutralized with BaCO 3 .
Ascending paper chromatography of the acid hydrolysate from the polysaccharide, as well as authentic d-glucose, d-galactose, d-mannose, d-xylose, l-arabinose, l-fucose, and l-rhamnose, was performed on filter paper (No. 50; Advantec, Japan) using an eluent composed of butanol-ethanol-water (4:1:5, by volumes). Spots composed of reducing sugars were stained by spraying with the aniline hydrogen phthalate reagent and subsequent heating at 105°C for 4 min.
The unknown sugar was separated by paper chromatography (Advantec Filter Paper No. 50; butanol-ethanol-water=4:1:5 as solvent). The band attributed to the unknown sugar (Spot 1: Rf 0.41) was cut off and extracted with distilled water at 4°C for 24 h. The extract was concentrated and freeze-dried.

High-performance anion exchange chromatography coupled with the pulse amperometric detector (HPAEC-PAD)
The monosaccharides in the hydrolysate of the polysaccharide were identified using a HPAEC (DX-500, Dionex Co., CA, USA), fitted with Carbopack PA1 column and a pulsed amperometric detector. The column was eluted at flow rate of 1 mL/min at 35°C with 10 mM NaOH.

Results and discussion
The isolated yield of the polysaccharide was estimated to be 1.3% (n=5), based on the dried weight. The polysaccharide contained 93.5% (W/W) of carbohydrate. The specific rotation [α] 589 of the polysaccharide (0.2%, W/V) was +23.8°, indicating that the α-configurations are predominated. Using high performance anion-exchange chromatography coupled with a pulse amperometric detector (HPAEC-PAD) to separate the hydrolysate of the polysaccharide, not shown in Figure, d-glalactose and d-glucose were identified in a molar ratio 1.0:3.0.
The 1 H-NMR spectrum of the polysaccharide collected at 500 MHz was shown in (Figure 1A). The four signals were observed in the anomeric region (δ 5.5−4.5) at 5.387, 5.119, 5.023 and 5.004 ppm, respectively. In addition, a major signal at 1.331 and 1.320 ppm was also observed, indicating that a 6-deoxy-sugar was integrated into the polysaccharide.
The 13 C-NMR spectrum of the polysaccharide was shown  in (Figure 1B). The three anomeric signals were observed at 103.17, 101.68, and 101.32 ppm, respectively. A methyl signal at 20.54 ppm was observed, indicating that a 6-deoxy-sugar was part of the polysaccharide.
Although the HPAEC-PAD data suggested that the polysaccharide consisted only of d-galactose and d-glusoe, an unknown 6-deoxy-sugar, which would have overlapped with the peak of d-galactose or d-glucose in the HPAEC-PAD data, was detected in the 1 H-and 13 C-NMR spectra. These results agreed with the data from the polysaccharide isolated from L. lividatus [12].
Paper chromatography of the acid hydrolysate of the polysaccharide was conducted and is displayed in (Supplement figure S1). Spot 1 (Rf, 0.41) was higher than the spot attributed to the standard l-rhamnosyl residue (0.37). Alhough d-glucose and d-galactose were detected with HPAEC-PAD, both sugars overlapped on the paper chromatogram (spot 2 and 3).
Using paper chromatography on a preparative scale, 6-deoxy-sugar (spot 1) was isolated from the mixture obtained by hydrolyzing the polysaccharide with acid and extracting the product with distilled water.
The 1 H-NMR of the isolated 6-deoxy-sugar was presented in (Figure 2A). As reported previously [12], the 6-deoxy-d-altrose existed in aqueous solution as a mixture of the α and β conformers of the pyranose and furanose forms. This phenomenon was also observed with 6-deoxy-l-altrose [16]. The spectrum of the 6-deoxy-sugar (Figure 2A . All of the signals agreed with the data collected from the authentic 6-deoxy-d-altrose, (not shown), as well as the sample of the 6-deoxy-d-altrose isolated from L. lividatus, previously reported [12].
The 2D COSY spectrum of the 6-deoxy-sugar was shown in (Figure 3). From the Figure,  Almost all of the signals for the 6-deoxy-sugar agreed with the data from the authentic 6-deoxy-d-altrose and the 6-deoxyd-altrose from L. lividatus [12]. The coupling constants for the 6-deoxy-sugar that were determined from COSY experiment were summarized in (Table 1). Almost all of the coupling constants for the 6-deoxy-sugar were identical with those of the authentic, (Supplement figure S2) and the 6-deoxy-daltrose from L. lividatus. The results of the COSY experiment and the analysis of the HSQC spectrum (Figure 4) helped to assign residue a of the C-1, C-2, C-3, C-4, C-5 and C-6 peaks in the 6-deoxy-sugar to δ 95.4, δ 77.0, δ 74.2, δ 84.6, δ 68.1 and δ 20.2 ppm, respectively. The other residues (b, c and d) were

Chemical shift (ppm)
Chemical shift (ppm) also assigned by a similar procedure and summarized in Table 1. Almost all of the signals of the 6-deoxy-sugar agreed with the signals of the authentic 6-deoxy-d-altrose, (Supplement figure S3), and the 6-deoxy-d-altrose isolated from L. lividatus [12]. Therefore, from the results, the 6-deoxysugar isolated from L. akahatsu was identified as 6-deoxyd or l-altrose.

Conclusions
In conclusion, by combining the results obtained in the current study with the data from a previous paper [12], four isomers of 6-deoxy-d-altrose (a, b, c and d) were identified as 6-deoxy-β-d-furanose (9.4%), 6-deoxy-α-d-furanose (16.8%),       The four isomers were designated as residues a, b, c, and d according to their decreasing proton chemical shifts. b Calculated from the height of signals of the anomeric protons in Figure 2.