JPH0259836B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel oligomannoside and a method for producing the same.
The present invention relates to a linear mannan having a bond and an α1-3 bond, and a method for producing the same.

Mannan is a polysaccharide that is widely distributed from higher plants to microorganisms. In recent years, research on their chemical structures has progressed, and structural formulas of several repeating units have been proposed. For example, yeast mannan and potato mannan are proteomannans that exist on the surface of cells (T. Nakajima, H. Sasaki,
M. Sato, K. Tamari and K. Matsuda, J.
(Biochem. (Tokyo) 82, 1657-1662) A complex branched structure is considered. Since these mannans exist on the surface layer, it is highly likely that they are also involved in recognition phenomena between organisms. Furthermore, antitumor activity has been reported for some mannans. The present inventors are conducting research on the synthesis of oligomannosides having accurate structures, with the aim of elucidating the correlation between these biological functions, antitumor activities, and mannan structure.

The present inventors have already reported a method for synthesizing 2,6- and 3,6-branched mannan structures (Japanese Patent Application Laid-Open No. 72996/1983).
JP 57-72997) and a method for synthesizing linear mannans having α1-2 bonds (JP 57-146797). The present invention has been completed through extensive research into the following method for synthesizing linear mannan found in Saccharomyces cerevisiae.

Man α1 → 3Man α1 (→2Man α1) → _o 2Man (n = 0 or 2) By sequentially reacting this with two types of monosaccharide sugar donors, a pentasaccharide linear mannan having an α1-2 bond and an α1-3 bond is synthesized. The present invention will be explained in detail below.

The present invention is based on the general formula (In the formula, R represents hydrogen or a benzyl group,
R ¹ , R ² , R ³ and R ⁴ represent hydrogen, a benzyl group or an acetyl group, and may be the same or different. ) and its production method. The above mannopentaose is expressed by the formula (1) (R represents a benzyl group) Compound (1) represented by and formula (2) (R is a benzyl group, Ac is an acetyl group, and X is a halogen atom) by reacting the compound (2) represented by the formula (3). Compound (3) represented by is obtained, and compound (4) obtained by deacetylating this and formula (6) (R ¹ , R ² , R ³ , R ⁴ are benzyl groups or acetyl groups, and X represents a halogen atom) It is obtained by reacting a compound represented by the following formula, deacetylating if necessary, and further debenzylating.

The trisaccharide receptor (1), which is the starting material of the present invention, can be obtained, for example, by the following steps. i.e., monosaccharide receptor compounds (7) (Bn represents a benzyl group) is a monosaccharide donor compound (8) A disaccharide compound (9) was obtained by reacting with The compound (9) is deacetylated to form a compound (10),
Furthermore, a sugar donor compound (8) is reacted to form a trisaccharide compound (11).
It can be obtained by deacetylating this.

The sugar donor compound (2) in the present invention can be obtained, for example, by the following process. α-D-mannose was treated with allyl alcohol/acetyl chloride to give allyl α-D-mannopyranoside (17), which was then treated with trityl chloride in pyridine to tritylate the 6-position (compound (18)). n-
After treatment with Bu ₃ Sn) ₂ O, Bu ₄ NCl and allyl bromide to allylate the 3-position (compound (19)), detritylation with acetic acid yielded compound (20),
The 2, 4, and 6 positions were benzylated by treatment with NaH/benzyl bromide in DMF (compound (21)), and then deallylated by treatment with PdCl ₂ /A _c ON _a in acetic acid (compound (22)). ), and further benzylated with Ac ₂ O/pyridine (compound (23)), followed by treatment with HCl gas in CH ₂ Cl ₂ to obtain hydride (2).

The other sugar donor compound in the present invention is represented by the general formula (6) Any protected sugar halide represented by can be used. As the halide, chloride, bromide, and iodide are preferred, and as the protecting group, acetyl group, benzyl group, etc. are generally used. One of R ¹ to R ⁴ is an acetyl group,
If the others are protected with a benzyl group, after deacetylating the pentasaccharide obtained by the sugar chain elongation reaction,
Furthermore, the sugar chain can be extended. Also, R ¹
If two of ~R ⁴ are protected with acetyl groups and the others with benzyl groups, oligosaccharides having branched sugar chains can be synthesized. Examples of compounds represented by general formula (6) include those in which R ¹ to R ⁴ are benzyl groups, those in which R ¹ to R ⁴ are acetyl groups, and R ¹
Compound (8) where is an acetyl group and R ² to ^{R 4} are benzyl groups, Compound (2) where R ² is an acetyl group and the others are benzyl groups, Compound (2) where R ⁴ is an acetyl group and the others are benzyl groups ( 24) etc. The above compound (8)
The manufacturing method is described in JP-A-57-146797.
Compound (24) is synthesized, for example, by the following steps. That is, the allyl 6-0-trityl-α
-D-Mannoside (18) was treated with NaH/benzyl bromide in DMF to benzylate the 2, 3, and 4 positions (compound (25)), detritylated with acetic acid (compound (26)), and acetic anhydride. Acetylation with (Ac ₂ O)/pyridine (compound (27)), and further in acetic acid,
Obtained by deallylation by treatment with PdCl ₂ /Ac ONa (compound (28)), followed by treatment with thionyl chloride in CH ₂ Cl ₂ .

Reacting compound (4) with compound (8) yields pentasaccharide compound (29), which is deacetylated to yield compound (30), and further debenzylated to yield compound (31). . In addition, when compound (4) is reacted with compound (24), pentasaccharide compound (32) is obtained, and when this is deacetylated, compound (33) is obtained, and when further debenzylated, compound (31) is obtained. can get.

The reaction between the trisaccharide acceptor (1) or the tetrasaccharide acceptor (4) and a halide such as the sugar donor compound (2), (8) or (24) can be performed using 1,2-dichloroethane, dichloromethane, chloroform, or nitromethane. , HgBr2 _, Hg ₍ CN) ₂ , _AgOSO2CF3 ,
This is carried out using a catalyst such as Ag ₂ CO ₃ , Ag ₂ O, AgClO ₄ or the like. At this time, it is preferable to add molecular sieve 4A to the reaction mixture for the purpose of removing acids such as HBr generated during the reaction.

The deacetylation reaction of compounds (3), (29) and (32) is
sodium methoxide, sodium ethoxide,
Using catalysts such as tertiary organic bases such as triethylamine, methanol, ethanol, n- and iso
- The process proceeds satisfactorily in propanol, water, or a mixed solvent thereof at a temperature of -30°C to 100°C for 0.5 to 30 hours.

The debenzylation reaction of compounds (4), (30), and (33) was performed by converting these compounds into water-ethanol, THF
-ethanol, ethanol, methanol, acetic acid,
This is carried out by dissolving in a solvent or mixed solvent such as THF-water, dioxane-water, DMF, etc., and hydrogenating at normal pressure or under pressure using Pd/C or the like as a catalyst. Appropriate reaction temperature is 0°C to 100°C and reaction time is approximately 1 to 100 hours.

In addition, the compound (2) obtained in the above step,
(3), (4), (18), (19)(20), (21), (22), (23), (24),
(25), (26), (27), (28), (29), (30), (31), (32)
and (33) are both novel compounds synthesized for the first time by the present inventors.

The above steps of the present invention will be schematically illustrated below.

(1) Trisaccharide acceptor + (2) Monosaccharide donor → (3) Tetrasaccharide deacetylation――――――→ (4)Debenzylation――――――→ (5) (4) Tetrasaccharide acceptor + (8) Monosaccharide donor → (29) Pentasaccharide deacetylation――――――→ (30) Debenzylation――――――→ (31) (4) Tetrasaccharide acceptor body + (24) Monosaccharide donor → (32) Pentasaccharide deacetylation---------→ (33) Debenzylation---------→ (31) The above-mentioned novel compound obtained by the present invention is useful as an intermediate in the synthesis of mannan having physiological activities such as antitumor properties, and as a reagent in elucidating the biological significance and function of the mannan.

The present invention will be explained in more detail with reference to Examples below, but these are not intended to limit the scope of the present invention in any way.

Note that the reference examples and examples shown below show the synthesis steps of the following compounds, respectively.

Reference Example Process 1 α-D-Mannose → (17) 2 (17) → (18) 3 (18) → (25) 4 (25) → (26) 5 (26) → (27) 6 (27 ) → (28) 7 (28) → (24) Example 1 (1) + (2) → (24) 2 (3) → (4) 3 (4) + (8) → (29) 4 ( 29) → (30) 5 (30) → (31) 6 (4) + (24) + (32) 7 (32) → (33) Reference example 1 Acetyl chloride in 500 ml of allyl alcohol
Add 25 ml and stir at room temperature for 1 hour after purging with nitrogen. After adding 100 g (0.55 M) of α-D mannose under ice-cooling and stirring at room temperature for 9 days, the Rf0.15 spot in the raw material almost disappeared on TLC (chloroform:methanol 2:1), and a new Rf0.57 spot appeared. appeared. After adding 100 ml of triethylamine, the mixture was stirred at room temperature for 30 minutes and the solvent was removed to obtain 172 g of a syrupy crude product (17).

Reference example 2 The entire amount of the obtained (17) was dissolved in 500 ml of pyridine,
After adding 180 g (0.65 M) of trityl chloride and stirring overnight at room temperature after purging with nitrogen, the spot near the origin of the raw material almost disappeared on TLC (toluene: ethyl acetate 2:1), and a new spot with Rf0.35 appeared. Ta.
Approximately 400 g of a syrupy substance obtained by removing the solvent was separated by flash chromatography using 500 g of silica gel (500 ml of toluene → toluene: ethyl acetate 2:1) to obtain 302 g of a powdery crude product (18).
I got it. (95% from α-D-mannose) [α] _D = +2.1° (C = 0.72, CHCl ₃ ) Reference example 3 (18) 2.21g (5mM) was dissolved in 100ml of DMF, and 50% NaH0 was added under ice cooling. Add .76 (33mM) and stir at room temperature for 1 hour. Furthermore, under ice cooling, benzyl bromide 2.7
ml dropwise and stirred at room temperature for 3 hours, the origin spot of the raw material disappears on TLC (toluene) and a new
A spot of Rf0.50 appeared. Under ice cooling, methanol
Add 10 ml and stir at room temperature for 30 minutes. After removing the solvent, add 50 ml of water and extract three times with 50 ml of ether.
After washing with water and saturated brine, dehydration with MgSO ₄ , the solvent was removed, and isolation was performed by flash chromatography (toluene) on 100 g of silica gel to obtain 3.0 g of a syrup-like substance (25). (80%) As C ₄₉ H ₄₈ O ₆ , C80.30 H 6.60 Measured value C80.46 H 6.56 [α] _D = +18.3° (C = 0.88, CHCl ₃ ) Reference example 4 (25) 25 g Dissolve in 400ml of acetic acid and add 100ml of water.
After stirring at 100°C for 45 minutes, the Rf0.50 spot of the raw material disappeared on TLC (toluene), and a new Rf0.10 spot appeared along with the Rf0.90 spot. After removing the solvent, a spot with an Rf of 0.10 was isolated by flash chromatography (toluene:ethyl acetate 4:1) using 300 g to obtain 15 g of a syrupy substance (26). (94%) As C ₃₀ H ₃₄ O ₆ , C 73.45 H 6.99 C 73.22 H 6.99 [α] _D = +37.0° (C = 0.45, CHCl ₃ ) Reference example 5 (26) 15 g was mixed with acetic anhydride: Pyridine 1 : When dissolved in 150 ml of a mixed solution of 2 and stirred overnight at room temperature, the Rf0.29 spot of the raw material disappeared on TLC (toluene: ethyl acetate 5:1), and a new single Rf0.56 spot appeared. . After removing the solvent, add 500ml of saturated sodium bicarbonate solution,
After extraction with 400 ml of chloroform three times, wash twice with water, dehydrate with MgSO ₄ , remove the solvent, and further extract with ethanol,
Azeotroped three times with toluene to obtain 16.5 g of syrupy substance (2
7) was obtained. (Quantitative) [α] _D = +8.4゜ (C = 1.2, CHCl ₃ ) As C ₃₂ H ₃₆ O ₇ , C 72.16 H 6.81 Measured value C 71.48 H 6.57 Reference example 6 (27) 5 g was added to 40 ml of acetic acid After dissolving 1.2 g of pdCl ₂ and adding 1.25 g of sodium acetate and 2 ml of water and stirring at room temperature for 3 days, the Rf0.66 spot of the raw material almost disappeared with TLC (toluene: ethyl acetate 5:1) and a new Rf0.12 was added.
A spot appeared. After passing through diatomaceous earth (FC), the solvent was removed, 300 ml of water was added, and the mixture was extracted three times with 300 ml of ethyl acetate. After washing with saturated sodium bicarbonate solution, water, and saturated saline, drying with MgSO ₄ and removing the solvent, silica gel 100
Flash chromatography of g (toluene:
Isolation with ethyl acetate (3.5:1) gave 4.1 g of syrup (28). (88%) [α] _D = +10.3 (C = 0.5, CHCl ₃ ) C ₂₉ H ₃₂ O ₇・2/3CH ₃ As COOH C 68.37 H 6.56 Measured value C 68.34 H 6.52 Reference example 7 (28) Dissolve 2g in 40ml dichloromethane
Add 0.1 ml of DMF and 4 ml of thionyl chloride and stir overnight. TLC (toluene: ethyl acetate 5:1)
Above, a new Rf0.71 spot appeared along with a small Rf0.16 spot from the raw material. After removing the solvent, flash chromatography (toluene:ethyl acetate 7:1) was performed on 10 g of silica gel to obtain 1.4 g of a syrupy substance (24). (68%) [α] _D = +59.3° (C = 0.77 CHCl ₃ ) Example 1 Molecular sieves in a 30ml brown two-necked flask
Add 2 g of 4A powder and stir at 180°C under reduced pressure for 6 hours.
AgOSO ₂ CF ₃ 200 dissolved in 10 ml of toluene after cooling to room temperature
mg and remove the solvent under reduced pressure at 50°C. Compound (1) 290mg dissolved in 15ml dichloroethane after nitrogen substitution
(0.208mM) and cooled to -15℃ (2) 110mg
(0.215mM1.03eq) was dissolved in 5ml of dichloroethane and injected dropwise. After stirring for 3 hours at room temperature, traces of (1) were removed on TLC (toluene:ethyl acetate 10:1).
Along with the Rf0.38 spot, a new Rf0.64 spot appeared. Cool to -15℃ and add 2 ml of dichloroethane.
After adding 20 mg (0.067 mM) of solution (2) and stirring overnight at room temperature, an almost single Rf0.64 spot appeared on TLC (same as above). After evaporation, the solvent was removed and the residue was isolated by flash chromatography on 20 g of silica gel to obtain 310 mg of syrup-like substance (3). (80%) As C ₁₁₇ H ₁₂₂ O ₂₂ , C 74.74 H 6.54 Measured value C 74.23 H 6.50 [α] _D = +20.0° (C = 0.39, CHCl ₃ ) Example 2 (3) 260 mg in THF 20 ml Dissolved, 0.01N
Add 30ml of NaOMe/MeOH and stir overnight at room temperature.
The Rf0.86 spot disappeared and a single Rf0.74 spot appeared. After neutralization with Amberlyst A-15,
The solvent was removed and isolated by flash chromatography on 5 g of silica gel (toluene:ethyl acetate 10:1) to give 210 mg of syrup (4).

As C ₁₁₅ H ₁₂₀ O ₂₁ , C 75.15 H 6.58 Measured value C 74.49 H 6.61 [α] _D = +12.8° (C = 0.6, CHCl ₃ ) Example 3 Molecular sieves in a 20 ml melt brown two-necked flask Add 1 g of 4A powder and stir at 190°C under reduced pressure for 6 hours. After cooling to room temperature, add 10ml of toluene solution.
Inject 60 mg of AgSO ₃ CF ₃ and remove the solvent under reduced pressure at 40°C. 105 mg of (4) dissolved in 10 ml of dichloroethane
After injecting (0.058mM), the mixture was cooled to -15°C, and 100mg (0.189mM) of (8) dissolved in 1ml of dichloroethane was injected dropwise. Further, after stirring overnight at room temperature, the Rf0.42 spot in (4) disappeared on TLC (toluene:ethyl acetate 10:1), and a new Rf0.49 spot appeared along with the Rf0.52 spot in (8). Ta. Dilute with 30 ml of dichloromethane, filter, remove the solvent, and perform flash chromatography on 10 g of silica gel (toluene: ethyl acetate 10:
1), yielding 77 mg of a syrupy substance (29). (58%) Example 4 Dissolve 77mg of (29) in 5ml of THF and add 0.1N NaOMe/
Add 5 ml of MeOH and stir at room temperature for 8 hours.
(29) on (toluene:ethyl acetate 5:1)
The Rf0.74 spot disappeared, and a new single Rf0.59 spot appeared. After neutralization with Amberlyst A-15, the solvent was removed and the mixture was isolated by flash chromatography on 15 g of silica gel to obtain 60 mg of a syrupy substance (30).

As C ₁₄₂ H ₁₄₅ O ₂₆ , C 75.11 H 6.57 Measured value C 75.11 H 6.57 [α] _D = +34.5° (C = 0.17 CHCl ₃ ) Example 5 Dissolve 55 mg of (30) in 10 ml of acetic acid and make 10% When 30 mg of PD-C was added and hydrogenated at 50°C for 6 hours, the raw material spot disappeared on TLC (butanol:acetic acid:water 2:1:1), and a new spot with Rf0.16 appeared. After passing through diatomaceous earth (FC), the layer was washed with water, and after removing the solvent along with the washing solution, 20g of Cephadex G25g (H ₂ O) was added.
17 mg of powdered material (31)
I got it.

[α] _D = +27.2゜ (C = 0.4, water) δ _C (D ₂ O): 102.48 ( ¹ J _CH 170.9Hz, C-1d, C-1e) 100.87 ( ¹ J _CH 169.7Hz, C- 1b, C-1e) 92.70 (C-1a) δ _H (D ₂ O at 63°): 5.351 (H-1a) (d, J=2Hz
) 5.273 (H-1b, H-1c) (d, J = 2Hz) 5.158 (H-1e) (d, J = 2Hz) 5.054 (H-1d) (d, J = 2Hz) Example 6 30ml Melted brown Put 1 g of molecular sieves 4A powder into a 2-necked flask and stir overnight at 190°C under reduced pressure. AgSO ₃ CF ₃ 150 dissolved in 2 ml of toluene after cooling to room temperature
mg and remove the solvent under reduced pressure at room temperature. Next, 250 mg (1.36 mM) of (4) dissolved in 10 ml of dichloroethane was injected, cooled to -15°, and (2) dissolved in 2 ml of dichloroethane was injected.
4) Inject 150mg (0.269mM) dropwise. 1 at room temperature
After stirring overnight, TLC (toluene: ethyl acetate 10:1)
Above, the Rf0.35 spot in (4) disappears, and the spot in (24)
Along with the Rf0.49 spot, Rf0.46 and Rf0.49 spots appeared at a concentration ratio of 1:4 as determined by eye. After diluting with 50 ml of dichloroethane and filtering, after removing the solvent, it was determined to have Rf0.46 by flash chromatography on 25 g of silica gel (toluene: ethyl acetate 12:1).
A mixture with Rf 0.49 was isolated as 350 mg of syrup (32).

Example 7 (32) Dissolve the entire amount in 10ml of THF and add 0.1N
After adding 10 ml of NaOMe/MeOH solution and stirring at room temperature for 3 hours, two spots of the raw material disappeared on TLC (toluene: ethyl acetate 10:1), and new Rf0.42 and
Two spots of 0.39 appeared with a concentration ratio of 4:1. p-
tlc (preparative-tlc) (toluene: ethyl acetate
10:1) and Rf 0.42 was isolated, yielding 150 mg of a syrupy substance (33).

As C ₁₄₂ H ₁₄₈ O ₂₆ , C 75.11 H 6.57 Measured value C 74.96 H 6.68 [α] _D = +26.0° (C = 1.99, CHCl ₃ )

Claims (1)

[Claims] 1. An oligomannoside represented by the following general formula. In the formula, R represents hydrogen or a benzyl group,
R ¹ , R ² , R ³ and R ⁴ represent hydrogen, a benzyl group or an acetyl group, and may be the same or different. 2 formulas (In the formula, R represents a benzyl group) Compounds and formulas represented by (R ¹ , R ² , R ³ , R ⁴ are an acetyl group or a benzyl group, and X is a halogen atom) are reacted, deacetylated and debenzylated as necessary, and the general formula (In the formula, R represents hydrogen or a benzyl group,
R ¹ , R ² , R ³ , and R ⁴ represent hydrogen, a benzyl group, or an acetyl group. 3 formulas (R represents a benzyl group) and a compound represented by the formula (R is a benzyl group, Ac is an acetyl group, and X is a halogen atom) by reacting a compound represented by the formula (R represents a benzyl group, Ac represents an acetyl group) A compound obtained by deacetylating this and a compound of the formula (R ¹ , R ² , R ³ , R ⁴ are an acetyl group or a benzyl group, and X is a halogen atom) are reacted, deacetylated and debenzylated as necessary, and the general formula (In the formula, R represents hydrogen or a benzyl group,
R ¹ , R ² , R ³ , and R ⁴ represent hydrogen, a benzyl group, or an acetyl group.